Supramolecules in Drug Discovery and Drug Delivery

Methods in
Molecular Biology 2207
Thomas Mavromoustakos
Andreas G. Tzakos
Serdar Durdagi Editors
Supramolecules
in Drug Discovery
and Drug Delivery
Methods and Protocols
Methods in M o l e c u l a r B i o lo g y
Series Editor
John M. Walker
School of Life and Medical Sciences
University of Hertfordshire
Hatfield, Hertfordshire, UK
For further volumes:

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Supramolecules in Drug
Discovery and Drug Delivery
Methods and Protocols
Edited by
Thomas Mavromoustakos
Department of Chemistry, National and Kapodistrian University of Athens, Zografou, Greece
Andreas G. Tzakos
Department of Chemistry, Section of Organic Chemistry and Biochemistry, University of Ioannina,
Ioannina, Greece
Serdar Durdagi
Computational Biology and Molecular Simulations Laboratory, Department of Biophysics,
School of Medicine, Bahcesehir University, Istanbul, Turkey
Editors
Thomas Mavromoustakos Andreas G. Tzakos
Department of Chemistry Department of Chemistry
National and Kapodistrian University of Athens Section of Organic Chemistry and Biochemistry
Zografou, Greece University of Ioannina
Ioannina, Greece
Serdar Durdagi
Computational Biology and Molecular
Simulations Laboratory
Department of Biophysics, School of Medicine
Bahcesehir University
Istanbul, Turkey
ISSN 1064-3745 ISSN 1940-6029 (electronic)

Methods in Molecular Biology
ISBN 978-1-0716-0919-4 ISBN 978-1-0716-0920-0 (eBook)
https://doi.org/10.1007/978-1-0716-0920-0
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Preface
Amidst the sadness of endless mediocrity, which suffocates us everywhere, I

am comforted that somewhere, in an arch, some stubborn people are strug-
gling to neutralize corruption.—“The Little Epsilon”, Odysseus Elytis
This drug delivery systems volume is dedicated to drugs that are unable to reach the desired
pharmaceutical drug target as a result of several limitations, mainly due to the lack of selec-
tivity, poor targeting capacity, or undesired pharmacokinetic profile. The utilization of
proper “delivery vehicles” for such drugs can tailor their physical chemical properties and
enhance their efficacy, retaining their structures intact.
Techniques for preparing such systems are described in Chapters 1–3. Drug delivery
platforms are outlined in Chapters 4–13. Such platforms include cyclodextrins, micelles,
liposomes, polymeric, and nanotubes. These nanotechnology systems are studied using dif-
ferent biophysical techniques such as DSC, ITC, solid and liquid NMR spectroscopy, and
electrochemistry (Chapters 14–23).
All accumulated chapters aim toward one target: to provide readers with critical infor-
mation to accomplish (a) the synthesis of nanosystems, thus supramolecular entities com-
plexing with drugs; (b) their characterization; as well as (c) studying their physical-chemical
interactions that govern their stability and properties. Experimental details and notes not
found in the scientific literature to cover these three aspects of drug delivery systems are
provided by experts in the field. This volume is thus very useful for all scientists who study
or plan to study such systems.
We wish to thank all authors whose effort provided manuscripts that overcome “the
sadness of endless mediocrity” and build a foundation of solid knowledge on this growing
field. Rational design is restrained not only to novel structures but also to old ones which
decorate themselves with supramolecules that convert their disadvantages to advantages.
We also wish to thank Prof. John Walker for his valuable impact and all personnel of Springer
who “are struggling to neutralize corruption.”
Zografou, Greece Thomas Mavromoustakos

Ioannina, Greece Andreas G. Tzakos
Istanbul, Turkey Serdar Durdagi
v
Contents
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix
1 Application of Neutralization and Freeze-Drying Technique

for the Preparation of the Beneficial in Drug Delivery
2-Hydroxypropyl-β-Cyclodextrin Complexes with Bioactive Molecules�� 1
Eirini Christodoulou, Dimitrios Ntountaniotis, Georgios Leonis,
Thomas Mavromoustakos, and Georgia Valsami
2 Functionalized Carbon Nanohorns as Drug Delivery Platforms �� 13
Anastasios Stergiou and Nikos Tagmatarchis
3 Ultrasonics-Assisted Effective Isolation and Characterization
of Exosomes from Whole Organs�� 25
Burak Derkus and Emel Emregul
4 Aggregate Determination by Permeation Technique�� 35
Phennapha Saokham and Thorsteinn Loftsson
5 Study of Candesartan Cilexetil: 2-Hydroxypropyl-β-Cyclodextrin
Interactions: A Computational Approach Using Steered
Molecular Dynamics Simulations�� 45
Sofia Kiriakidi and Thomas Mavromoustakos
6 Drug Delivery: Hydrophobic Drug Encapsulation into Amphiphilic Block
Copolymer Micelles�� 71
Angeliki Chroni, Varvara Chrysostomou, Athanasios Skandalis,
and Stergios Pispas
7 Multisensitive Polymeric Nanocontainers as Drug Delivery Systems:
Biological Evaluation�� 85
Maria Theodosiou, Theodora Koutsikou, and Eleni K. Efthimiadou
8 Drug Incorporation in the Drug Delivery System of Micelles�� 99
Evangelia Soumelidou, Simona Golič Grdadolnik,
and Thomas Mavromoustakos
9 Molecular Dynamics Protocols for the Study of Cyclodextrin
Drug Delivery Systems�� 109
Georgios Leonis, Dimitrios Ntountaniotis, Eirini Christodoulou,
10 Drug Delivery Through Multifunctional Polypeptidic Hydrogels�� 127
Hermis Iatrou, Panagiota G. Fragouli, Dimitra Stavroulaki,
and Barbara Athanasiou
11 Polymersomes from Hybrids-Polypeptides for Drug Delivery Applications�� 139
Hermis Iatrou, Panagiota G. Fragouli, Dimitris Skourtis,
and Ioanna Stavropoulou
vii
viii Contents
12 Drug Delivery Systems Based on Modified Polysaccharides:

Synthesis and Characterization�� 151
Aikaterini-Foteini Metaxa, Eleni Vrontaki, Eleni K. Efthimiadou,
13 Differential Scanning Calorimetry (DSC) on Sartan/Cyclodextrin
Delivery Formulations�� 163
Nikolaos Naziris, Maria Chountoulesi, Dimitrios Ntountaniotis,
Thomas Mavromoustakos, and Costas Demetzos
14 Encapsulation of Small Drugs in a Supramolecule Enhances Solubility,
Stability, and Therapeutic Efficacy Against Glioblastoma Multiforme �� 175
Antonis D. Tsiailanis, Alexander Renziehausen, Serdar Karakurt,
Tim Crook, Nelofer Syed, and Andreas G. Tzakos
15 Unveiling the Thermodynamic Aspects of Drug-Cyclodextrin
Interactions Through Isothermal Titration Calorimetry�� 187
Maria V. Chatziathanasiadou, Thomas Mavromoustakos,
and Andreas G. Tzakos
16 Antitumor Efficacy of Ceranib-2 with Nano-Formulation of PEG
and Rosin Esters �� 199
Ali Ben Taleb, Selcan Karakuş, Ezgi Tan, Merve Ilgar, Özlem Kutlu,
Devrim Gözüaçık, Hatice Mehtap Kutlu, and Ayben Kilislioğlu
17 Association of the Thermodynamics with the Functionality
of Thermoresponsive Chimeric Nanosystems�� 221
Nikolaos Naziris, Athanasios Skandalis, Thomas Mavromoustakos,
Stergios Pispas, and Costas Demetzos
18 2D DOSY NMR: A Valuable Tool to Confirm the Complexation
in Drug Delivery Systems�� 235
Christos M. Chatzigiannis, Sofia Kiriakidi, Andreas G. Tzakos,
19 Drug-Encapsulated Cyclodextrin Nanosponges �� 247
Maria Tannous, Fabrizio Caldera, Gjylije Hoti, Umberto Dianzani,
Roberta Cavalli, and Francesco Trotta
20 Electrochemistry Investigation of Drugs Encapsulated in Cyclodextrins�� 285
Romana Sokolová and Ilaria Degano
21 A Differential Scanning Calorimetry (DSC) Experimental Protocol
for Evaluating the Modified Thermotropic Behavior of Liposomes
with Incorporated Guest Molecules�� 299
Maria Chountoulesi, Nikolaos Naziris, Thomas Mavromoustakos,
and Costas Demetzos
22 Applications of NMR in Drug:Cyclodextrin Complexes�� 313
Dimitrios Ntountaniotis, Georgios Leonis, Eirini Christodoulou,
23 Construction of Peptide-Drug Conjugates for Selective Targeting
of Malignant Tumor Cells �� 327
Eirinaios I. Vrettos and Andreas G. Tzakos
Index ��339
Contributors
Barbara Athanasiou • Department of Chemistry, Industrial Chemistry Laboratory,

National and Kapodistrian University of Athens, Zografou, Greece
Fabrizio Caldera • Dipartimento di Chimica, Università di Torino, Torino, Italy
Roberta Cavalli • Dipartimento di Scienza e Tecnologia del Farmaco, Università di
Torino, Torino, Italy
Maria V. Chatziathanasiadou • Section of Organic Chemistry and Biochemistry,
Department of Chemistry, University of Ioannina, Ioannina, Greece
Christos Μ. Chatzigiannis • Section of Organic Chemistry and Biochemistry,
Department of Chemistry, University of Ioannina, Ioannina, Greece
Maria Chountoulesi • Section of Pharmaceutical Technology, Department of Pharmacy,
School of Health Sciences, National and Kapodistrian University of Athens, Zografou,
Greece
Eirini Christodoulou • Laboratory of Biopharmaceutics-Pharmacokinetics, Department
of Pharmacy, School of Health Sciences, National and Kapodistrian University of Athens,
Zografou, Greece; Department of Chemistry, National and Kapodistrian University of
Athens, Zografou, Greece
Angeliki Chroni • Theoretical and Physical Chemistry Institute, National Hellenic
Research Foundation, Athens, Greece
Varvara Chrysostomou • Theoretical and Physical Chemistry Institute, National
Hellenic Research Foundation, Athens, Greece
Tim Crook • Department of Oncology, St Luke’s Cancer Centre, Royal Surrey County
Hospital, Guildford, UK
Ilaria Degano • Department of Chemistry and Industrial Chemistry, University of Pisa,
Pisa, Italy
Costas Demetzos • Section of Pharmaceutical Technology, Department of Pharmacy,
Greece
Burak Derkus • Chemistry Department, Faculty of Science, Ankara University, Ankara,
Turkey
Umberto Dianzani • Dipartimento di Scienze della Salute, Università del Piemonte
Orientale, Torino, Italy
Eleni K. Efthimiadou • Inorganic Chemistry Laboratory, Chemistry Department,
National and Kapodistrian University of Athens, Zografou, Greece; NCSR
“Demokritos”, Sol-Gel Laboratory, Institute of Nanoscience and Nanotechnology, Agia
Paraskevi, Attikis, Greece
Emel Emregul • Chemistry Department, Science Faculty, Ankara University, Ankara,
Turkey
Panagiota G. Fragouli • Laboratory of Dyeing, Finishing, Dyestuffs and Advanced
Polymers, DIDPE, University of West Attica, Athens, Greece
ix
x Contributors
Devrim Gözüaçık • Koç University Hospital, School of Medicine and Koç University
Research Center for Translational Medicine (KUTTAM), Koç University, Zeytinburnu,
Istanbul, Turkey
Simona Golič Grdadolnik • Laboratory for Molecular Structural Dynamics, National
Institute of Chemistry, Ljubljana, Slovenia
Gjylije Hoti • Dipartimento di Chimica, Università di Torino, Torino, Italy
Hermis Iatrou • Department of Chemistry, Industrial Chemistry Laboratory, National
and Kapodistrian University of Athens, Zografou, Greece
Merve Ilgar • Department of Chemistry, Faculty of Engineering, Istanbul University-
Cerrahpasa, Istanbul, Turkey
Serdar Karakurt • Department of Biochemistry, Faculty of Science, Selcuk University,
Konya, Turkey
Selcan Karakuş • Department of Bio and Nanotechnology, Faculty of Engineering,
Istanbul University-Cerrahpasa, Avcilar, Istanbul, Turkey; Department of Chemistry,
Faculty of Engineering, Istanbul University-Cerrahpasa, Avcilar, Istanbul, Turkey
Ayben Kilislioğlu • Department of Bio and Nanotechnology, Faculty of Engineering,
Istanbul University-Cerrahpasa, Avcilar, Istanbul, Turkey; Department of Chemistry,
Faculty of Engineering, Istanbul University-Cerrahpasa, Avcilar, Istanbul, Turkey
Sofia Kiriakidi • Department of Chemistry, National and Kapodistrian University of
Theodora Koutsikou • Inorganic Chemistry Laboratory, Chemistry Department,
National and Kapodistrian University of Athens, Zografou, Greece; NCSR
“Demokritos”, Sol-Gel Laboratory, Institute of Nanoscience and Nanotechnology, Agia
Paraskevi, Attikis, Greece
Hatice Mehtap Kutlu • Department of Biology, Faculty of Science, Eskişehir Technical
University, Eskişehir, Turkey
Özlem Kutlu • Nanotechnology Research and Application Center (SUNUM), Sabanci
University, Istanbul, Turkey
Georgios Leonis • Department of Chemistry, National and Kapodistrian University of
Thorsteinn Loftsson • Faculty of Pharmaceutical Sciences, University of Iceland,
Reykjavik, Iceland
Thomas Mavromoustakos • Department of Chemistry, National and Kapodistrian
University of Athens, Zografou, Greece
Aikaterini-Foteini Metaxa • Sol–Gel Laboratory, Institute of Nanoscience &
Nanotechnology, Agia Paraskevi, Attikis, Greece
Nikolaos Naziris • Section of Pharmaceutical Technology, Department of Pharmacy,
Greece
Dimitrios Ntountaniotis • Department of Chemistry, National and Kapodistrian
Stergios Pispas • Theoretical and Physical Chemistry Institute, National Hellenic
Alexander Renziehausen • John Fulcher Neuro-Oncology Laboratory, Imperial College
London, Hammersmith Hospital, London, UK
Phennapha Saokham • College of Pharmacy, Rangsit University, Pathum Thani,
Thailand
Contributors xi
Athanasios Skandalis • Theoretical and Physical Chemistry Institute, National Hellenic

Romana Sokolová • J. Heyrovský Institute of Physical Chemistry of the Czech Academy of
Sciences, Prague, Czech Republic
Evangelia Soumelidou • Department of Chemistry, National and Kapodistrian
Dimitra Stavroulaki • Department of Chemistry, Industrial Chemistry Laboratory,
Anastasios Stergiou • Theoretical and Physical Chemistry Institute, National Hellenic
Nelofer Syed • John Fulcher Neuro-Oncology Laboratory, Imperial College London,
Hammersmith Hospital, London, UK
Nikos Tagmatarchis • Theoretical and Physical Chemistry Institute, National Hellenic
Ali Ben Taleb • Department of Bio and Nanotechnology, Faculty of Engineering, Istanbul
University-Cerrahpasa, Istanbul, Turkey
Ezgi Tan • Department of Chemistry, Faculty of Engineering, Istanbul University-
Cerrahpasa, Avcilar, Istanbul, Turkey
Maria Tannous • Dipartimento di Chimica, Università di Torino, Torino, Italy;
Department of Chemistry, University of Balamand, Tripoli, Lebanon
Maria Theodosiou • Inorganic Chemistry Laboratory, Chemistry Department, National
and Kapodistrian University of Athens, Zografou, Greece; NCSR “Demokritos”, Sol-Gel
Laboratory, Institute of Nanoscience and Nanotechnology, Agia Paraskevi, Attikis,
Greece
Francesco Trotta • Dipartimento di Chimica, Università di Torino, Torino, Italy
Antonis D. Tsiailanis • Section of Organic Chemistry and Biochemistry, Department of
Chemistry, University of Ioannina, Ioannina, Greece
Andreas G. Tzakos • Section of Organic Chemistry and Biochemistry, Department of
Chemistry, University of Ioannina, Ioannina, Greece
Georgia Valsami • Laboratory of Biopharmaceutics-Pharmacokinetics, Department of
Pharmacy, School of Health Sciences, National and Kapodistrian University of Athens,
Zografou, Greece
Eirinaios I. Vrettos • Laboratory of Organic Chemistry, Department of Chemistry,
University of Ioannina, Ioannina, Greece
Eleni Vrontaki • Division of Pharmaceutical Chemistry, Department of Pharmacy,
Chapter 1
Application of Neutralization and Freeze-Drying Technique

for the Preparation of the Beneficial in Drug Delivery
2-Hydroxypropyl-β-Cyclodextrin Complexes with Bioactive
Molecules
Eirini Christodoulou, Dimitrios Ntountaniotis, Georgios Leonis,
Thomas Mavromoustakos, and Georgia Valsami
Abstract
Bioavailability of active substances is of great importance for the formulation of a drug product, as it actu-
ally reflects drug absorption and achievement of the optimum pharmacological effect. A great number of
chemical compounds with excellent pharmacological properties possess low solubility and permeability
values, ending in low bioavailability in the human body after administration (especially after per os admin-
istration). CDs are oligosaccharides that possess biological properties similar to their linear counterparts,
but some of their physicochemical properties differ. They are enhancing bioavailability and solving prob-
lems of absorption for poorly soluble lipophilic drugs by forming water-soluble inclusion complexes. For
this reason, they are widely used in drug delivery systems (Carrier et al. J Control Release 123:78–99,
2007; Kurkov and Loftsson, Int J Pharm 453:167–80, 2013). The main purpose of this chapter is to show
a protocol for the preparation of drug:CDcomplex delivery systems.
Key words Cyclodextrins, 2-Hydroxypropyl-β-CD, Methyl-β-CD, Bioavailability, Lyophilization,

Inclusion complex
1 Introduction
Cyclodextrins (CDs) are a-(1,4)-linked cyclic oligosaccharides

containing glucopyranose units. The glucopyranose units possess
chair conformation, due to which CDs are not cylindrical, and
exhibit torus-like shape (truncated cone), in which the primary
hydroxyl groups are present on narrow edge and secondary
hydroxyl groups are on the wider edge of the cone in the outer
surface. In the interior of the CD molecule, skeletal carbons with
hydrogen atoms and oxygen bridges are present leading to hydro-
philic outer surface and lipophilic central cavity of CD [1–3].
Thomas Mavromoustakos et al. (eds.), Supramolecules in Drug Discovery and Drug Delivery: Methods and Protocols,
Methods in Molecular Biology, vol. 2207, https://doi.org/10.1007/978-1-0716-0920-0_1,
1
2 Eirini Christodoulou et al.
One of the most remarkable features of CDs is their tendency

to form solid inclusion complexes with a wide variety of solid, liq-
uid and gaseous compounds through the process of molecular
complexation. The formed complexes are called host–guest type of
complexes. Such type of complexes involves a guest molecule
(drug) which is properly fitted into the lipophilic cavity of a host
(CD molecule) [4]. There is no breakage or formation of covalent
bonds during the formation of an inclusion complex
[5, 6]. When the CD is added to an aqueous solution, the polar
water molecules fit into the lipophilic cavity of the CD, but these
are immediately replaced by the more favored guest molecule,
which is less polar than the water molecules [7]. The main driving
force behind this complex formation is the replacement of high-
enthalpy water molecules with the guest moiety which adopts van
der Waals interactions, hydrogen bondings and charge transfer
interactions [6]. The ratio in which host and guest molecules bind
together is generally 1:1 [5], even though in many cases complexes
at different molecular ratios are formed. The equilibrium reaction
of complexation is shown below:
Drug free + CDfree  Drug : CDcomplex
The binding strength of the formed complex depends on the abil-

ity of the guest molecule to bind suitably to the host in order to
form a stable complex. The formation of an inclusion complex
depends upon the following factors:
(a) Size of the CD to the size of the guest molecule or some main
functional groups within the guest moiety: If the guest is of
inappropriate size, it will not fit properly into the host cavity.
(b) Thermodynamic interactions between the various components
of the system (CD, guest, solvent; [4]).
(c) Structure of added substituent to the CD derivative.
(d) Location of substituent within the CD molecule.
(e) Number of substituents per CD molecule.
CDs play an important role in the chemical stability of drugs. The
assumptions made after studying the interaction between CDs and
labile compounds are as follows [8]:
(a) CDs retard or accelerate degradation
(b) CDs have no effect on reactivity
The most commonly used methods for the preparation of drug-
cyclodextrin inclusion complexes include the following [4, 9–13]:
(a) Coprecipitation
It is the most commonly used method of complex prepara-
tion in laboratory scale. It is simple and easy to apply, with no
exceptional laboratory equipment needed. The necessary
Neutralization and Freeze-Drying Technique to Prepare Drug: CD Complexes 3
amount of cyclodextrin is dissolved in water and the guest

active ingredient, dissolved in an organic solvent, is gradually
added in the solution under continuous stirring. The solution
is cooled and complex precipitate as a final product is formed,
collected washed with another solvent or water and dried.
(b) Slurry complexation
In order to prepare the complex, it is not necessary for the
cyclodextrin to be completely dissolved. Cyclodextrin can be
added to water at a concentration of 50–60% w/v in solid
form and then stirred. The aqueous phase will be saturated
with cyclodextrin in solution. Guest molecules will complex
with the cyclodextrin in solution and, as the cyclodextrin com-
plex saturates the water phase, the complex will be crystallized
or precipitate out of the aqueous phase. The cyclodextrin crys-
tals will be dissolved and continue to saturate the aqueous
phase to form the complex and precipitate or crystallize out of
the aqueous phase. The complex can be collected in the same
manner as with the coprecipitation method.
(c) Kneading method: paste complexation
A small amount of water is added to the cyclodextrin along
with the active ingredient in order to form a paste, using a
mortar and pestle or (on a large scale) using a kneader. The
time required is dependent on the guest molecule. The result-
ing complex can be dried directly or washed with a small
amount of water to remove the non-complexed particles that
are adsorbed on the cyclodextrin surface and collected by fil-
tration or centrifugation to be dried under vacuum. Pastes will
sometimes get dry forming a hard mass instead of a fine pow-
der. This is dependent on the guest molecule and the amount
of water used in the paste.
(d) Damp mixing and heating in a sealed container
The guest and cyclodextrin molecules are thoroughly mixed
and after adsorbing a definite amount of water vapor are placed
in a sealed container. The amount of water used in the method
can range between the amount of water of hydration in the
cyclodextrin and added guest to up to 20–25% water on a dry
basis. The sealed container and its contents are heated to about
100 °C and then the contents are removed and dried. The
amount of water added, the degree of mixing and the heating
time have to be optimized for each guest.
(e) Extrusion
Extrusion is a variation of the heating and mixing method
and is a continuous system. Cyclodextrin, guest molecule and
water can be premixed or mixed as added to the extruder.
Degree of mixing and amount of heating and time can be con-
trolled in the barrel of the extruder. Depending upon the
amount of water, the extruded complex may dry as it cools or

the complex may be placed in an oven to dry.
(f) Dry mixing
Some guest molecules can be complexed by simply adding them to
the cyclodextrin and mixing. This works best with oils or liquid
guest molecules. The amount of mixing time required is vari-
able and depends on the guest. Generally, this method is per-
formed at ambient temperature and is an alternative to the paste
method.
In the following section a detailed guide is given for the prepara-
tion of solid-state inclusion complexes of a number of poorly
water-soluble lipophilic drug molecules with 2-hydroxypropyl-β-
cyclodextrin (2-HP-β-CD). The drug molecules of interest are
irbesartan, losartan, candesartan and candesartan cilexetil, as well
as five bioactive molecules of plant origin, namely caffeic acid, ros-
marinic acid, silibinin, curcumin, and quercetin.
Irbesartan (IRB), losartan (LOS), and candesartan (CAN) or
candesartan cilexetil as prodrug (CC) belong to the family of
angiotensin II receptor blockers and are used to treat high blood
pressure, while beyond their ability to lower blood pressure, they
also confer cardiovascular and renal protective effects [14–16].
IRB and LOS belong to class II of the Biopharmaceutics
Classification System, BCS [17, 18], meaning that their poor aque-
ous solubility is the rate-limiting step for drug absorption. CAN is
classified as a BCS Class IV drug, meaning that it shows both low
solubility and low permeability in vitro. CC is the prodrug form of
the active substance CAN and is classified as a BCS Class II drug
(low solubility) [16]. As a result, enhancement of the aqueous sol-
ubility of all three drug molecules can lead to improved oral
bioavailability.
Caffeic acid (CA) is an antioxidant molecule both in vitro and
in vivo [19]. Caffeic acid also shows immunomodulatory and anti-
inflammatory activities [20, 21]. Chemically, rosmarinic acid (RA)
is a caffeic acid ester of 3-(3,4-dihydroxyphenyl)-lactic acid and
can act as a prodrug for caffeic acid. CA is poorly soluble only in
hot water whereas RA is insoluble in water and soluble only in
organic solvents, not biologically compatible with the human
organism. As a result, the inclusion of both molecules in 2-HP-β-CD
is extremely useful in order to enhance oral bioavailability.
Silibinin (SLB) is the main active component of the flavonoid
mixture silymarin extracted from the plant Silybum marianum
(milk thistle) and is mainly known for its hepatoprotective proper-
ties. More specifically, there is some clinical evidence for the use of
SLB as a supportive element in alcoholic and child grade “A” liver
cirrhosis [22]. SLB is one of the most characteristic examples of
poorly water-soluble biomolecules, as its bioavailability is extremely
low when administered orally.
Curcumin (CRM) is a bright yellow chemical compound pro-

duced by Curcuma longa plants. It is the principal curcuminoid of
turmeric (Curcuma longa), a member of the ginger family,
Zingiberaceae. CRM has a surprisingly wide range of beneficial
properties, including anti-inflammatory, antioxidant, chemopre-
ventive and chemotherapeutic activity, but its low aqueous solubil-
ity, physical instability and low bioavailability make it difficult to
study [23].
Quercetin, (QUE) a plant flavonol from the flavonoid group
of polyphenols, is found in many fruits, vegetables, leaves and
grains; red onions and kale are common foods containing appre-
ciable content of QUE [24]. It has a bitter flavor and is used as an
ingredient in dietary supplements, beverages and foods. Due to its
antioxidant properties, QUE has been considered as a potent mol-
ecule against cancer, while recently it is considered protective agent
against early inflammatory stages of Alzheimer’s disease. Its low
solubility, and thus limited bioavailability, is the primary reason for
formulating the molecule into a 2-HP-β-CD inclusion complex.
2 Materials
1. Use purified water and analytical grade guest molecules.

2. Store all guest molecules at room temperature (unless indicated
otherwise).
3. Prepare ammonium hydroxide solution by diluting 20 mL of
ammonia solution (25% v/v concentrated solution) to a final
volume of 100 mL with purified water and mix gently. The con-
centration of the obtained NH4OH solution is 5% v/v. This
solution is used for all preparations described below.
4. Use 2-HP-β-CD as the host molecule and drug carrier for solu-
bility amelioration through a neutralization and freeze-drying
procedure in the following preparations.
3 Methods
3.1 Steps to Follow 1. Accurately weigh 60 mg of IRB and 408 mg of 2-HP-β-CD

for the Preparation and transfer them in a 100 mL beaker.
of Solid-State 2. Suspend with 50 mL of water.
Irbesartan–2-HP-β-CD
3. Add small amounts of ammonium hydroxide solution 5% v/v
Inclusion Complex
under continuous stirring and pH monitoring until complete
[25, 26]
dissolution. The optimum pH value is approximately 10.5 for
the complexation to take place (see Notes 1–3).
4. When complete dissolution is achieved (visual observation to
obtain clear colorless solution) fix the volume at 60 mL with
purified water.
5. The resulting solution at a molar ratio of 1:2 (IRB:2-HP-β-CD)

(see Notes 4–6) is thereafter frozen at −80 °C and freeze-dried
[15] to remove the water and obtain the white solid irbesartan–
2-HP-β-CD lyophilized product (see Note 7).
3.2 Steps to Follow 1. Accurately weigh 3445.6 mg of 2-HP-β-CD.

for the Preparation 2. Transfer in a glass vessel with 300 mL of purified water and set
of Solid-State LOS–2- under magnetic stirring.
HP-β-CD Inclusion
3. When the cyclodextrin is completely dissolved, add 500 mg of
Complex
LOS potassium (accurately weighed) in the vessel and continue
the magnetic stirring (LOS:CD molar ratio 1:2, see Notes 4–6).
4. Adjust the pH at approximately 10.5 with ammonium hydrox-
ide solution 5% v/v to facilitate the reaction of inclusion com-
plex formation (see Notes 1–3).
5. When the mixture turns to a clear and colorless solution, fix the
final volume at 500 mL with purified water.
6. Immediately freeze the solution at −80 °C for lyophilization to
follow and obtain the white solid LOS-2-HP-β-CD inclusion
complex (see Note 7).
3.3 Steps to Follow 1. Accurately weigh 30 mg of CAN or CC and 198.85 mg or

for the Preparation 143.37 mg of 2-HP-β-CD, respectively.
of Solid-State CAN–2- 2. Mix the weighed amounts of CAN (or CC) and 2-HP-β-CD in
HP-β-CD and CC–2- a glass beaker along with 20 mL of purified water.
HP-β-CD Inclusion
3. Add small amounts of the ammonium hydroxide solution 5%
Complexes [27]
v/v under continuous magnetic stirring.
4. Adjust pH at approximately 10.5 (see Notes 1–3).
5. Adjust the final volume at 30 mL with purified water and mix
thoroughly.
6. Freeze the resulting solution immediately after preparation at
−80 °C (see Note 7).
7. Freeze-dry the iced preparation in a lyophilizer (e.g., Kryodos-50
model Telstar) to obtain a white amorphous solid.
3.4 Steps to Follow 1. Weigh accurately 60 mg of CA and 0.964 g of 2-HP-β-CD.

for the Preparation 2. Transfer the weighed amounts of CA and 2-HP-β-CD in a
of Solid-State RA–2- 100 mL beaker and suspend with 50 mL of water.
HP-β-CD and CA–2-
3. Add small amounts of ammonium hydroxide 5% v/v aqueous
HP-β-CD Inclusion
solution and adjust the pH at approximately 9–10 under mag-
Complexes netic stirring (see Notes 1–3).
3.4.1 CA–2-HP-b-CD 4. Continue magnetic stirring under pH monitoring until a clear
Inclusion Complex solution is obtained.
5. Fix the final volume at 60 mL with purified water.
6. The resulting solution has a 1:2 molar ratio of CA:2-HP-β-CD

(see Notes 4–6).
7. Immediately transfer this clear colorless solution at a freezer at
−80 °C (see Note 7).
8. Freeze-dry the resulting iced product to obtain a white fluffy
solid-state CA–2-HP-β-CD lyophilized product.
3.4.2 RA–2-HP-b-CD 1. Accurately weigh 120 mg of RA and 964 mg of 2-HP-β-CD

Inclusion Complex and transfer in a 200 mL beaker.
2. Suspend with 100 mL of purified water.
3. Add small amounts of 5% v/v ammonium hydroxide solution
under magnetic stirring and pH monitoring (see Notes 1–3).
4. Keep pH at approximately 9-10.
5. After complete dissolution of the solids, fix the final volume at
120 mL with purified water.
6. Immediately transfer the resulting clear colorless solution (at
RA:2-HP-β-CD molar ratio of 1:2, see Notes 4–6) at a freezer
at −80 °C (see Note 7).
7. When it turns iced, freeze-dry to a lyophilizer to obtain the final
solid-state RA–2-HP-β-CD lyophilized product.
3.5 Steps to Follow 1. Accurately weigh 300 mg of SLB and 1860 mg of 2-HP-β-CD.
for the Preparation 2. Transfer the weighed amounts of SLB and 2-HP-β-CD in a
of Solid State SLB–2- glass vessel and suspend with 200 mL of purified water under
HP-β-CD Inclusion magnetic stirring.
Complex [28]
3. Add small amounts of 5% v/v ammonium hydroxide solution
under continuous stirring and pH monitoring (pH should be
adjusted at approximately 10–10.5, see Notes 1–3).
4. After complete dissolution a slight pink clear solution should be
obtained.
5. Fix the final volume of the solution with purified water at
300 mL.
6. Freeze the resulting clear, pink solution (at a 1:2 SLB:2-HP-
β-CD molar ratio, see Notes 4–6) at −80 °C (see Note 7).
7. Freeze-dry the iced product to obtain the solid-state inclusion
complex of SLB–2-HP-β-CD.
3.6 Steps to Follow 1. 150 mg of CRM and 1197 mg 2-HP-β-CD, accurately weighed,
for the Preparation should be mixed in a glass vessel containing 120 mL of purified
of Solid-State CRM–2- water (see Notes 4–6).
HP-β-CD Inclusion 2. Then, add small amounts of 5% v/v ammonium hydroxide
Complex solution to adjust the pH at approximately 10–10.5, under con-
tinuous stirring and pH monitoring (see Notes 1–3).
3. After complete dissolution a yellow solution is obtained.

4. Fix the final volume of the obtained solution at 150 mL with
purified water.
5. Immediately freeze this solution at −80 °C (see Note 7).
6. Freeze-dry the iced product to obtain a yellow solid state of the
CRM–2-HP-β-CD inclusion complex.
3.7 Steps to Follow 1. For the preparation of QUE–2-HP-β-CD aqueous solutions for
for the Preparation freeze-drying in a molar ratio of 1:2, mix 30 mg of QUE and
of Solid-State QUE–2- 306 mg of 2-HP-β-CD, accurately weighed in a 50 mL beaker
HP-β-CD and QUE– (see Notes 4–6).
Me-β-CD Inclusion 2. Suspend the mixture of solids with 20 mL of purified water.
Complexes [29–31] 3. The pH value should be adjusted at approximately 9–9.5 with
3.7.1 QUE–2-HP-β-CD the help of ammonium hydroxide solution (5% v/v) under con-
Inclusion Complex [29, 30] tinuous magnetic stirring (see Notes 1–3).
4. After complete dissolution fix the volume of the obtained solu-
tion (1:2 QUE:CD molar ratio) to 60 mL.
5. Freeze the obtained orange-colored solution at −80 °C (see
Note 7).
6. Freeze-dry the final iced product to remove water and obtain
the final solid-state yellowish QUE–2-HP-β-CD inclusion
complex.
3.7.2 QUE–Me-β-CD 1. For the preparation of QUE–Me-β-CD aqueous solution for

Inclusion Complex [31] freeze-drying in a molar ratio of 1:1, mix 2200 mg of Me-β-CD
and 500 mg of QUE, accurately weighed, into a glass vessel (see
Notes 4–6).
2. Suspend the mixture of solids with 400 mL of purified water.
3. The pH value should be adjusted at approximately 9–9.5 with
the help of ammonium hydroxide solution (5% v/v) (see Notes
1–3).
4. After complete dissolution, fix the volume of the obtained solu-
tion (1:1 QUE:CD molar ratio) to 500 mL.
5. Freeze the obtained orange-colored solution at −80 °C (see
Note 7).
6. Freeze-dry the final iced product to remove water and obtain
the yellow solid-state QUE–Me-β-CD inclusion complex.
4 Notes
1. The neutralization method [13] is applied prior to lyophiliza-

tion (freeze-drying) to facilitate drug dissolution.
2. pH monitoring is important during the preparation of the
drug-CD aqueous solution, to maintain up to 10.5 and avoid
CD decomposition.
3. Note that all drug (bioactive) molecules described in this chap-

ter are acidic in nature and therefore neutralization takes place
at an alkaline pH between 9 and 10.5.
4. For the preparation of inclusion complexes in solid state using
the neutralization and freeze-drying technique, aqueous solu-
tions of drug and CD should be prepared at the optimum molar
ratio for freeze-drying to follow.
5. In the described examples the optimum drug:CD molar ratio is
1:2 for the preparation of the aqueous solutions.
6. Aqueous solutions of drug and CD at a different drug:CD
molar ratio than 1:2 can also be used if needed. For example:
(a) Prepare SLB–2-HP-β-CD aqueous solutions for freeze-
drying in molar ratios of 1:1 and 1:4 following the steps
described in Subheading 3.5 but weigh accurately 930 mg
and 3720 mg of 2-HP-β-CD, respectively, and mix with
300 mg of SLB at a final volume of 300 mL [28].
(b) For the preparation of CAN− or CC−2-HP-β-CD aque-
ous solutions for freeze-drying in a molar ratio of 1:1 fol-
low the procedure described in Subheading 3.3 but weigh
accurately 99.28 mg or 71.69 mg of 2-HP-β-CD and mix
with 30 mg of CAN or CC, respectively (accurately
weighed), at a final volume of 30 mL.
7. Freeze the prepare aqueous solution immediately at ≤−70 °C,
to avoid layering during freezing.
5 Conclusions
The neutralization and freeze-drying (or lyophilization) technique

can be successfully applied for the preparation of drug:CD inclu-
sion complexes [25–31]. All the abovementioned example com-
plexes prepared were confirmed to be adequately complexed with
CD through NMR and thermal analysis procedures in order to
evaluate the strength of inclusion and possible molecular structure,
while in all cases in vitro bioactivity was confirmed [25–31]. In
parallel, in vitro solubility and dissolution experiments were con-
ducted to compare the complex developed against either the pure
drug or the reference marketed products and evaluate their possi-
ble bioavailability when orally administered [32–34].
Acknowledgments
This work was financially supported by the European Union and

Greek national funds through the program “Support for
Researchers with Emphasis on Young Researchers” (call code:
EDBM34, K.Ε. 14995, Preparation and Study of Novel Forms of
Administration of Pharmaceutical Molecules with the aim to

improve their pharmacological properties, PI Prof Th.
Mavromoustakos).
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Chapter 2
Functionalized Carbon Nanohorns as Drug Delivery

Platforms
Anastasios Stergiou and Nikos Tagmatarchis
Abstract
Carbon nanohorns (CNHs) resembling a single-layered graphene sheet wrapped in a conical shape can be
chemically modified in order to immobilize, carry, and release biologically active molecules. Here, we
describe the major routes for the preparation of CNH-based drug delivery platforms, via covalent coupling
and encapsulation, proficient to facilitate the design of sophisticated drug nanocarriers.
Key words Carbon nanohorns, Functionalization, Encapsulation, Hybrids, Biomaterials, Drug

delivery
1 Introduction
Drug delivery system technology is considered as a major area of

research and innovation on disease treatment. As a result, a pleth-
ora of delivery platforms have been developed and tested for their
efficacy, with some traditional formulations long being on the mar-
ket, others only recently approved, while numerous being under
screening and investigation. In addition, the flexibility in biomate-
rial synthesis and the extensive knowledge on self-assembly mecha-
nisms compel researchers to combine simple structures and/or
nanomaterials to generate more sophisticated drug nanocarriers.
Carbon nanohorns (CNHs), within the larger carbon allotrope
family and composed of sp2 hybridized carbon atoms, possess a
conical structure 2–5 nm in diameter and 40–50 nm in length.
Notably, CNHs aggregate in larger spherical superstructures of
around 100 nm [1] and can be economically produced in indus-
trial quantities without requiring the involvement of any toxic
metal catalyst. The latter is a significant advantage and key differ-
ence of CNHs as compared to carbon nanotubes [2]. In addition,
the dissimilarity in shape and size of CNHs as compared to the
micrometer-long single-walled carbon nanotubes affects their
properties and applications. However, CNHs lack solubility in
13
14 Anastasios Stergiou and Nikos Tagmatarchis
aqueous media, which is critical for manipulation and processing

for the envisaged applications. To overcome that handicap, chemi-
cal functionalization of CNHs is imperative and to this end a pleth-
ora of strategies have been developed broadly based on the covalent
addition of organic species onto the outer skeleton of CNHs [3–
17], as well as on the non-covalent/supramolecular interactions
through electrostatic association [18–20] with charged species
and/or π − π stacking forces [21–23] with planar aromatic mole-
cules. In addition, oxidation of the conical tips of CNHs, introduc-
ing carboxylic units suitable for further chemical alteration, is
another widely employed methodology for effectively modifying
CNHs [18, 24–33]. A typical process for the oxidation of CNHs
involves heat treatment in the presence of oxygen, while the light-
assisted oxidation of CNHs in the presence of hydrogen peroxide
is comparatively a much milder technique [33, 34]. Hence without
surprise, a variety of biomolecules were attached through
carbodiimide-mediated condensation reactions on oxidized CNHs.
For example, bovine serum albumin (BSA) was covalently incorpo-
rated at the carboxylic units of light-assisted oxidized CNHs. The
BSA-CNH material formed homogeneous dispersions into phos-
phate buffer saline, allowing incorporation inside mammalian cells
through endocytosis [33].
A significant benefit of CNHs as compared to carbon nano-
tubes is that they do not have the high aspect ratio issues related to
toxicity observed in longer nanotubes. Furthermore, the irregular
structure of CNHs permits the opening of nanowindows at the
tips and sidewalls of CNHs, from which incorporation and release
of bioactive molecules are feasible and easier. Hence, CNHs as a
highly pure and easily available material have been successfully
tested as drug carriers [35–38].
Summarizing, the capability for large-scale production of
CNHs accompanied by their handy chemical manipulation, as
illustrated in Fig. 1, makes this unique carbon nanostructure a
promising biocompatible drug delivery platform.
2 Materials
2.1 Preparation 1. A chamber equipped with a high-power CO2 laser source

of CNHs (wavelength 10.6 μm, maximum power 5 kW, variable pulse
duration from 10 ms to continuous illumination, and beam
Fig. 1 (continued) at the edge of the conical tips of CNHs, which can then be converted to the corresponding
acyl chlorides for subsequent coupling with hydroxyl- or amine-terminated drugs/biomolecules. Right: Harsh
oxidative treatment of CNHs, followed by reduction of the introduced oxygen functionalities, generates pores at
the edge of the cone and onto the sidewalls of CNHs, thereby enabling the release of the drugs/biomolecules
encapsulated by nanoprecipitation into CNHs
Functionalized Carbon Nanohorns 15
Fig. 1 Illustration of the three general methods for the preparation of CNH-based drug/biomolecule delivery
systems. Left: Pristine CNHs are modified with a BOC-aniline derivative via diazonium chemistry, followed by
cleavage of the BOC-protecting group and covalent coupling of the free amine terminus with carboxylic acid-
terminated drugs/biomolecules. Middle: Sequential oxidation of CNHs introduces carboxylic acid functionalities
diameter of 10 mm and a plastic-resin reaction chamber

30 × 30 × 25 cm3) equipped with a vacuum pumping system,
with inlet and outlet gas valves, gas pressure and flow control-
lers, and a ZnSe lens system for adjusting the beam intensity.
2. A 50 mm long graphite target rod with a 30 mm diameter and
purity 99.99%, with no catalytic metal inclusions.
3. Cylindrical filters, 50 mm in diameter and 150 mm long.
2.2 Drying Solvents 1. Tetrahydrofuran (THF).

2. Dichloromethane (DCM).
3. Nitrogen gas.
4. Metal sodium.
5. Benzophenone.
6. Calcium chloride.
2.3 Synthesis 1. p-Nitrobenzoyl chloride.

of BOC-Aniline 2. tert-Butyl 2-(2-(2 aminoethoxy)ethoxy)-ethylcarbamate.
Derivative
3. NaOH pellets.
4. Anhydrous Na2SO4.
5. Pt/C 10 wt%.
6. H2 gas.
7. Celite®.
2.4 Synthesis 1. CNHs powder.

of BOC-CNHs 2. BOC-aniline derivative.
and the Corresponding
3. Isoamyl nitrite.
NH2-CNHs
4. 1,2-Dichlorobenzene.
5. N2 gas.
6. Acetonitrile.
7. Dimethyl formamide.
8. Dichloromethane.
9. HCl gas.
10. Polytetrafluoroethylene (PTFE) membrane filters with pore
size of 0.2 μm.
11. Glass apparatus for vacuum filtration.
12. Vacuum pump.
13. A microwave reactor up to 300 W power (optional—see Note 3).
2.5 Oxidation 1. CNH powder.

of CNHs 2. A high-temperature (600 °C) pressurized furnace equipped
with gas inlet.
3. O2 gas.
4. 30% w/w H2O2.
5. Halogen lamp 500 W.
6. Deionized water.
7. Methanol.
8. PTFE membrane filters with pore size of 0.2 μm.
2.6 Conjugation 1. Oxidized CNHs.

of Drugs/Biomolecules 2. Thionyl chloride.
3. Dry tetrahydrofuran.
4. Amine (or hydroxyl)-terminated drug/biomolecule.
2.7 Nanomesh CNHs 1. CNH powder.

2. A high-temperature (1200 °C) furnace with gas inlet.
3. O2 gas.
4. H2 gas.
2.8 Encapsulation 1. Nanomesh CNHs.

of Drugs/Biomolecules 2. Drugs/biomolecules.
3. Dimethyl formamide.
3 Methods
All experiments are carried out under inert atmosphere conditions

unless otherwise stated. Solvents, chemicals and CNHs are used as
received unless otherwise stated.
3.1 Preparation The CNHs are produced in a plastic chamber by CO2 laser abla-
of CNHs tion of graphite in an Ar (760 Torr) atmosphere at room tempera-
ture. The inside of the chamber is evacuated, Ar gas is introduced
and flowed through it, while the gas pressure is kept constant, typi-
cally at 760 Torr. The gas flow rate is 40 L/min, which is required
to move the produced CNHs immediately from the reaction cham-
ber to the collection filter. The graphite target rod is located in the
middle of the reaction chamber. The graphite rod is rotated around
its axis at 6 rpm, and advanced along its axis so that a fresh target
is continually exposed to the laser beam. The rod is illuminated by
the laser beam vertically at its cylinder-wall surface. All laser ablation
experiments are conducted at room temperature, although the
actual target temperature rises during the ablation. Carbonaceous
products are collected by cylindrical filters located in a pumping
line between the reaction chamber and the vacuum pump. Each
filter can collect up to 500 mg of CNHs produced before filtering
efficiency is deteriorated.
3.2 Procedures 1. Tetrahydrofuran (THF): THF (100 mL) is placed in a round-

for Drying Solvents bottom flask, followed by the addition of metal sodium flakes
(3–4 flakes of 2 × 2 cm2) soaked in oil. Caution: Handle metal
sodium lumps and flakes with special care. Do not wash the
covering oil and contact of metal sodium with atmosphere
moisture must be avoided. Then add 2–3 flakes of benzophe-
none (1 × 1 cm2), acting as indicator. Equip the flask with a
condenser and a collection flask. Boil the mixture gently under
nitrogen atmosphere until the solution turns deep blue in color.
Always check that there is sufficient amount of metal sodium in
the solution. If the mixture has a greenish color, then add 1–2
more flakes of benzophenone. Then collect the dry THF sol-
vent and transfer to the reaction flask under nitrogen.
2. Dichloromethane (DCM): DCM is placed in a round-bottom
flask, followed by the addition of 10–20% powder w/w of
anhydrous calcium chloride. Let the mixture stand overnight.
Equip the system with a condenser and a collection flask. Boil
the mixture gently, under nitrogen atmosphere, for 2 h. Then
collect dry DCM and transfer it to the reaction flask under
nitrogen.
3.3 Covalent 1. Add pristine CNHs (5 mg) and 1,2-dichlorobenzene, oDCB,

Incorporation (10 mL), in a round-bottom flask under nitrogen atmosphere
of Amine Moieties and bath-sonicate for 10 min (see Note 1).
on CNHs 2. Dissolve the tert-butoxy carbamate (BOC)-protected aniline
derivative (2.6 mmol) in acetonitrile (5 mL) (see Note 2).
3. Add the solution of the BOC-protected aniline derivative in
the reaction mixture.
4. Add isoamyl nitrite (4.0 mmol) and stir the reaction mixture
at 60 °C for 18 h (see Note 3).
5. After cooling down to room temperature, dilute the reaction
mixture with N,N-dimethylformamide, DMF (30 mL).
6. Filter the reaction mixture over a polytetrafluoroethylene
(PTFE) membrane filter with pore size of 0.2 μm (see Note 4).
7. Wash the solid residue obtained on top of the PTFE filter with
DMF and dichloromethane (50 mL) (see Note 5).
8. Recover the BOC-modified CNHs as powder and store it at
room temperature in the dark.
9. Disperse the BOC-modified CNHs (5 mg) in dry dichloro-
methane (10 mL).
10. Bubble gaseous HCl for 30 s in the dispersion of the BOC-

modified CNHs (see Note 6).
11. Stir the reaction mixture under an inert atmosphere of nitro-
gen for 12 h.
12. Evaporate the solvent under reduced pressure (see Note 7).
13. Add fresh dichloromethane (10 mL), and filter the mixture
over a PTFE membrane filter with pore size of 0.2 μm.
DMF and dichloromethane (50 mL).
15. Recover the amine-modified CNHs as powder and store it at
room temperature in the dark.
3.4 Incorporation 1. Treat pristine CNHs (50 mg) with molecular oxygen at
of Carboxylic Acid 0.1 MPa for 10 min at 580° C.
Moieties on CNHs 2. Transfer the oxidized CNHs in a round-bottom flask and add
30% w/w solution of H2O2 (60 mL).
3. Apply light irradiation and heat the reaction mixture at 120° C
for 3 h (see Note 8).
4. Filter the dispersion of the CNHs through a PTFE membrane
filter with pore size of 0.2 μm.
a large amount of deionized water and methanol (50 mL).
6. Recover the carboxylic acid-modified CNHs as powder and
store it at room temperature in the dark.
3.5 Conjugation 1. Treat the carboxylic acid-modified CNHs (50 mg) with thio-
of Drugs/Biomolecules nyl chloride, SOCl2 (50 mL), at 70° C for 8 h under nitrogen
on Pre-modified CNHs atmosphere (see Note 9).
2. Evaporate the SOCl2 under reduced pressure. Then, add dry
THF (20 mL), bath-sonicate the mixture for 5 min, and then
evaporate the solvent to dryness. Repeat the latter twice and
keep the dry solid residue (acyl chloride-modified CNHs)
under nitrogen atmosphere (see Note 10).
3. Add dry THF (10 mL) to the acyl chloride-modified CNHs
and keep it under nitrogen atmosphere. Dissolve the hydroxyl-
or amino-terminated drug/biomolecule (100 mg) in dry
THF (20 mL) and add dropwise to the dispersion of the acyl
chloride-modified CNHs. Stir all the mixture at room tem-
perature for 24 h.
4. Filter the dispersion of the drug/biomolecule-functionalized
CNHs through a PTFE membrane filter with pore size of
0.2 μm.
THF and dichloromethane (50 mL) to remove any physi-
sorbed drug/biomolecule (see Note 11).
6. Recover the drug/biomolecule-functionalized CNHs as pow-
der and store it at room temperature in the dark.
3.6 Encapsulation 1. Treat pristine CNHs (50 mg) in oxygen gas at 570–580 °C for
of Drugs/Biomolecules 15 min to create holes on their walls. Then, heat the oxidized
Within CNHs material at 1200° C under hydrogen atmosphere to remove the
oxygen functionalities attached to the hole edges (see Note 12).
2. Dissolve the drug/biomolecule (400 mg) in DMF (100 mL)
in a glass container and add the as-prepared meshed CNHs
(50 mg). Bath-sonicate the mixture for 30 min. Subject the
mixture to slow evaporation of the DMF with the aid of dry
air over 5 s (see Note 13).
3. Collect the black material (CNHs filled with the drug/bio-
molecule) from the bottom of the container.
4. Wash the obtained CNHs filled with the drug/biomolecule
on top of a PTFE membrane filter with DMF and dichloro-
methane (50 mL) to remove any physisorbed drug/biomole-
cule (see Note 14).
5. Recover the drug/biomolecule encapsulated within CNH
material as powder and store it at room temperature in the dark.
4 Notes
1. Bath sonication allows dispersion of insoluble CNHs and facil-

itates reaction. Add the solvent in small portions to wet the
solid. CNH is a very light powder; thus fast incorporation of
the solvent may produce airstream inside the flask pushing the
light powder out.
2. The BOC-protected aniline derivative, namely tert-butyl
2-(2-(2-(4-aminobenzamido)ethoxy)ethoxy) ethylcarbamate,
is prepared according to the following procedure: a solution of
p-nitrobenzoyl chloride (464 mg, 2.5 mmol) in DCM (30 mL)
was added to a cold solution (ice bath) of tert-butyl
2-(2-(2-aminoethoxy)ethoxy)ethylcarbamate (500 mg,
2.0 mmol) in 0.2 N NaOH solution (50 mL). The reaction
mixture was stirred vigorously at 0° C for 3 h. The organic
layer was then separated, washed successively with 0.5 N
NaOH and water and then dried over anhydrous Na2SO4.
Evaporation of the solvent under reduced pressure affords the
nitro-derivative as a white solid with 85% yield. The aforemen-
tioned nitro-derivative (170 mg, 0.427 mmol) was dissolved
in dry ethanol (20 mL) and Pt/C 10 wt% (18 mg) was added.
Tip: Degassing the mixture with the aid of bath sonication and
a vacuum pump, followed by flushing with hydrogen gas, will
enhance the hydrogen adsorption in the porous catalyst. The
reaction mixture was stirred under hydrogen atmosphere for
24 h. The catalyst was removed by filtration on Celite®, the
filter pad was washed excessively with DCM and the filtrate
was evaporated under reduced pressure to furnish the BOC-
protected aniline derivative in 91% yield. Tip: It is suggested
to prepare fresh BOC-protected aniline derivatives.
3. Alternatively, microwave irradiation can be employed for
reducing reaction time and amount of solvents, especially
when reaction is performed at larger scale. In a typical proce-
dure, add pristine CNHs (30 mg), BOC aniline derivative
(350 mg, 0.95 mmol), and oDCB (2 mL) in a glass vial under
an inert nitrogen atmosphere and bath-sonicate for 15 min.
Then, add isoamyl nitrite (5.7 mmol), seal the vial with a sep-
tum cap, heat at 150 °C, and apply microwave irradiation with
100 Watts for 60 min.
4. Filtration over PTFE membrane filter with the particular
0.2 μm or smaller pore size allows retaining covalently func-
tionalized CNHs on top of the filter. If PTFE filter with bigger
pore size is employed, modified CNHs shall pass through the
filter or block/chock the pores.
5. Washing modified CNHs onto the PTFE filter allows to com-
pletely remove organic material physisorbed onto the surface
of CNHs. Furthermore, examination of the filtrate via UV-Vis
allows monitoring the purification.
6. Removal of the BOC-protecting group with gaseous HCl
yields a cleaner material as compared to the one obtained upon
the corresponding cleavage of BOC with trifluoroacetic acid
(TFA). Caution: Work with gaseous HCl only in a well-
ventilated fume hood.
7. During the evaporation of the solvent the dissolved HCl gas
will also be released.
8. A conventional 500 W halogen (or Xe) lamp is sufficient.
9. This treatment will convert the carboxylic acids to the corre-
sponding acyl chlorides allowing the subsequent reaction of
the modified CNHs with hydroxyl- and amino-terminated
drugs/biomolecules.
10. The objective is to remove the traces of SOCl2. The use of dry
solvent and nitrogen atmosphere is essential for the stability of
the acyl chlorides, since they are susceptible to hydrolysis by
traces of moisture. Caution: Work with SOCl2 only in a well-
ventilated fume hood. Contact of SOCl2 with moisture results
in hydrolysis affording SOx and HCl gas. Do not store the acyl
chloride-modified CNHs and use them instantly.
11. Examination of the filtrate via UV-Vis allows monitoring the

purification of the solid material.
12. The objective is to create pores across the sidewalls of CNHs
allowing the release of the drug/biomolecule via diffusion
from the interior of the CNH to the target.
13. The selection of the solvent is related to the property of DMF
to stabilize well the CNHs. Further, the drug/biomolecule
must be well dissolved prior to the addition of the opened
CNHs. Therefore, if the mentioned concentration (4 mg/
mL) of the drug/biomolecule is not achievable, increase the
amount of the required solvent. The method is based on the
slow evaporation of the solvent inducing the aggregation of
the drug/biomolecule in the interior of CNHs.
14. Examination of the filtrate via UV-Vis allows monitoring the
purification of the solid material.
5 Conclusions
Although the synthesis of CNHs requires advanced infrastructure,

the overall procedure is simple and straightforward, since no post-
treatment or purification is needed. Alternatively, CNHs can be
purchased in the market. As presented in detail, the synthesis of
CNH-based nanocarriers exploits simple methods adopted from
organic chemistry and chemistry of materials and the required puri-
fication/isolation is handy and capable of scale-up. It should be
underlined that for all CNH-based materials complementary char-
acterization is necessary in order to evaluate the success of the
described functionalization procedures. Namely, spectroscopic
(UV-Vis, Raman, ATR-IR), thermogravimetric (TGA), and micros-
copy (AFM, SEM, TEM) techniques are mandatory to verify the
generation of added functionalities or the presence of covalently
grafted drugs/biomolecules, the incorporation of holes on the side-
walls of CNHs, the presence of drugs/biomolecules into the cavity
of CNHs, etc. Concluding, the research on CNH-based delivery
platforms is based on simple chemical protocols and requires well-
equipped laboratories for synthesis and characterization and most
importantly interdisciplinary research collaborations in order to
realize CNHs as nanocarriers in living cells and organisms.
Acknowledgments
Partial support by the project “National Infrastructure in

Nanotechnology, Advanced Materials and Micro-/
Nanoelectronics” (MIS 5002772) which is implemented under
the “Reinforcement of the Research and Innovation
Infrastructures,” funded by the Operational Program

“Competitiveness, Entrepreneurship and Innovation” (NSRF
2014–2020) and co-financed by Greece and the European Union
(European Regional Development Fund), is acknowledged.
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Chapter 3
Ultrasonics-Assisted Effective Isolation

and Characterization of Exosomes from Whole Organs
Burak Derkus and Emel Emregul
Abstract
Exosomes, natural and nanovesicular structures surrounded by a lipid membrane, tend to be secreted
toward extracellular environments by almost all cell types. Late studies have shown them to be effective in
several complex biological processes like cancer development and metastasis, immune system regulation,
cellular signal transduction, stem cell differentiation, and regeneration of damaged tissues. Although there
are many studies dealing with the role of exosomes in the aforementioned fields, the mechanisms remained
largely unknown. There is therefore a need for further study on exosome isolation from different sources.
While researchers mostly use serum, plasma, urine, and cell culture media as a source for exosome isola-
tion, there are no studies dealing with direct isolation of exosomes from whole organs in literature. In this
study, we propose a protocol for effective isolation of exosomes from whole organs. Mouse brain, heart,
and liver were chosen as the sources of exosomes in this study. Isolated exosomes were successfully charac-
terized with BCA test, western blot, transmission electron microscopy and ELISA.
Key words Extracellular vesicles, Nanovesicles, Exosomes, Isolation, Ultrasonication
1 Introduction
Exosomes, which are released by almost all kinds of cells to the

extracellular environment, are lipid-involved natural nanovesicles
with a diameter of 50–150 nm. Although they were evaluated as
cellular wastes in the early studies, their potential of use in diagno-
sis and treatment of diseases was recognized after 2000s. Owing to
their rich microRNA (miRNA) content, which is 60 times higher
than regular biological fluids, exosomes today are utilized in diag-
nostics [1], cancer treatment [2], drug delivery [3] and stem cell-
based technologies [4]. Exosomes are mainly isolated from cell
culture wastes [5], plasma [6], serum [7], urine [8], and semen
[9]. While they are mostly and practically isolated from cell culture-
conditioned media (CM), it is thought that exosomal content
might be unsteady due to genotypic and phenotypic differences
25
26 Burak Derkus and Emel Emregul
between cell lines [10]. Moreover, the cellular content does not
fully reflect the molecular composition of tissues. Since the exo-
somes isolated from secondary cell lines do not fully reflect the
properties of the primary cells or original tissue [11], it is difficult
to judge the molecular contents of exosomes. It is important to
know the exact molecular content of the exosomes during secre-
tion from the tissue so that the diagnostic and therapeutic
approaches can be properly examined. In addition, in order to fully
illuminate the mechanisms of exosomes in cellular communication,
attempted in various publications [12], isolation from natural envi-
ronments namely living tissues and organs would be preferred
instead of cell culture CM. However, it can be seen in the literature
that there are only a couple studies dealing with the isolation of
exosomes from whole tissues/organs [13]. This study mainly
focuses on the effective isolation of exosomes from heart, brain
and liver. Ultrasonication was for the first time applied in this study
for exosome isolation that loosened the extracellular space of
organs and enabled us to perform a more efficient isolation pro-
cess. Bicinchoninic acid (BCA) and enzyme-linked immunosor-
bent assay (ELISA) tests (calibration graphs have been presented in
Fig. 1) revealed high protein contents (Table 1) indicating the
presence of exosomes. The calculated protein content varied
between 2.7 and 4.1 mg.mL−1, and the exosome count was found
to be 108–109 particles, which was quite good for downstream
analyses. Exosomal RNA, one of the most important elements of
exosomes, seemed suitable (Table 1) for cDNA synthesis and for
performing a reverse transcriptase-quantitative polymerase chain
reaction (RT-qPCR) or transcriptomics study.
Western blot images revealed the existence of CD63 and CD81
for brain- and heart-derived exosomes, whereas only CD81 expres-
sion was seen in the liver-derived exosomes (Fig. 2). Considering
the fact that the presence of one of the three (CD63 CD81 and
Alix) exosomal markers is sufficient for confirming the exosomes,
Fig. 1 Calibration graph for BCA test (a), and CD63 ELISA assay (b)
Ultrasonics-Assisted Effective Isolation and Characterization of Exosomes from Whole… 27
Table 1
Protein content, particle count and RNA content of isolated exosomes
BCA, Protein conc., ELISA CD63, Number of CD63+ RNA content,

O.D. μg.mL−1 O.D. exosomes ng.μL−1
Brain 3093 3781 0.195 2.6 × 109 45.5
exosomes
Heart 2217 2663 0.099 6.0 × 108 6.3
exosomes
Liver 3349 4107 0.305 4.9 × 109 143.0
exosomes
Fig. 2 Western blot analysis of CD63 and CD81 exosomal markers for brain-,
heart- and liver-derived exosomes
regarding the blotting results, we can conclude that the suggested

methodology is appropriate for exosome isolation from tissues and
organs.
Finally, the transmission electron microscope (TEM) images in
Fig. 3 for the brain (A)-, heart (B)-, and liver (C)-derived exo-
somes further demonstrated the success of the ultrasonication-
assisted isolation protocol. The exosomes obtained had a diameter
between 50 and 150 nm. The blue arrows seen in the figure clearly
show the stained membranes proving exosomal character.
2 Materials
2.1 Reagents Used 1. Freshly removed mouse brain, heart and liver. One-mouse hemi
for Exosome Isolation brain was used for exosome isolation.
2. 70% Ethanol.
3. Sulfuric acid (H2SO4).
4. Sterile scissors, tweezers/forceps, and razor blade for dissecting
and mincing the organs.
Fig. 3 Transmission electron microscopy images of brain (A)-, heart (B)- and liver (C)-derived exosomes
5. Sterile and cold phosphate-buffered saline (1× PBS, pH 7.4).

6. 50 mL Conical tubes.
7. Polyethylene glycol 4000 (PEG 4000).
8. 0.2 μm Filter adapters with surfactant-free cellulose acetate
membrane and 10 mL plunge syringes.
2.2 Reagents Used 1. 1× RIPA lysis buffer (1× lysis buffer, 200 mM PMSF, inhibitor
for the cocktail, and sodium orthovanadate) (Santa Cruz sc-24948).
Characterization 2. BCA kit (Thermo Scientific 23227).
of Isolated Exosomes
3. Flat-bottom 96-well microplate and adhesive covers.
2.2.1 Exosomal Protein 4. CD63 and CD81 ELISA kits (System Bioscience EXOEL-
Quantification CD63A-1 and EXOEL-CD81A-1).
2.2.2 Western Blot 1. Acrylamide/bis-acrylamide, 30% solution (Sigma).

Analysis 2. Tris (pH 8.8, 0.5 M).
3. Tris (pH 6.8, 0.5 M).

4. 10% Sodium dodecyl sulfate (SDS, Sigma).
5. 10% Ammonium persulfate (APS, AppliChem), freshly
prepared.
6. Tetramethylethylenediamine (TEMED, Merck).
7. Isopropanol.
8. 2× Laemmli buffer (0.125 mM Tris–HCl, 4% SDS, 20% glyc-
erol, 0.04% bromophenol blue, 10% β-mercaptoethanol,
pH 6.8).
9. 5× Running buffer (125 mM Tris base, 0.96 M glycine, 0.5%
SDS, pH 8.3).
10. 5× Transfer buffer (250 mM Tris base, 192 mM glycine, 0.1%
SDS, pH 9.2).
11. Tris-buffered saline with Tween 20 (TBST) buffer (20 mM
Tris pH 7.5, 150 mM NaCl, 0.1% Tween 20).
12. 0.45 μm Polyvinylidene fluoride membrane (PVDF)
(Immobilon, Millipore).
13. Methanol for hydrophilization of membrane.
14. Blocking buffer (5% nonfat milk powder) in TBS-T.
15. Monoclonal primary antibodies anti-CD63 (Abnova
MAB0931) and anti-CD81 (Abnova MAB6435) in TBS-T
(1:2000).
16. Anti-rabbit IgG secondary antibody in PBS (1:5000) (System
Bioscience).
17. ECL Western Blotting Substrate and SuperSignal West Femto
Substrate (Thermo Scientific) for luminescence-based
imaging.
2.2.3 Transmission 1. 4% Paraformaldehyde (Santa Cruz).

Electron Microscopy
2. 1% Glutaraldehyde, EM grade (Sigma).
Imaging
3. 0.5% Uranyl acetate, pH 4.0 (Polysciences).
4. PBS kept at 4 °C.
5. 200 mesh Formvar carbon-coated EM grids (Electron
Microscopy Sciences, FCF300).
6. Parafilm.
7. Clean forceps.
8. Whatman filter paper.
9. 0.22-μm-pore-sized syringe filter.
2.2.4 RNA Isolation 1. Promega SV total RNA isolation kit.

2. 60% Ethanol.
3 Methods
3.1 Isolation 1. Sacrifice a mouse sticking to the rules constructed by your ani-
of Exosomes mal care committee.
from Brain, Heart, 2. Remove the brain, heart and liver, and be sure that the organs
and Liver are clean of hair and other wastes. Olfactory bulbs of brain are
removed and the brain is divided into two hemi brains.
3. The sacrificed organs are washed with sterile and cold PBS,
transferred to separate tubes containing 15 mL sterile PBS and
then ultrasonicated for 3 min under 750 W power and 20 kHz
frequency ultrasonication conditions (Sonics VCX 750) in
order to dissociate the tissue and release the extracellular vesi-
cles (see Notes 1 and 2).
4. Centrifuge the tissue homogenates at 300 × g, 2000 × g and
13,000 × g for 15 min at 4 °C, respectively, and remove the cel-
lular debris, microvesicles and apoptotic bodies.
5. Transfer 10 mL of the supernatant into 50 mL tubes and add
10 mL 20% PEG 4000 solution.
6. Incubate the tissue homogenate-PEG mixture overnight at
4 °C.
7. Centrifuge the mixture at 13,000 × g at 4 °C and discard the
supernatant without disturbing the exosome pellet.
8. Resuspend the pellet in 100 μL PBS kept at 4 °C and store at
−20 °C for further use.
3.2 Protein Quantification of the exosomal protein content is performed fol-

Quantification lowing the manufacturer’s instruction. Briefly:
of Exosomes Using
1. Exosomes are diluted 1:2 in PBS.
BCA Test
2. Exosome lysate is obtained by adding an equal volume of RIPA
buffer including protease inhibitors.
3. Pipette 25 μL of each bovine serum albumin (BSA) standard
with concentrations between 12.5 μg.mL−1 and 2.5 mg.mL−1
and organ-derived exosome samples into a 96-well microplate.
The procedure is carried out in triplicate.
4. Add 200 μL of the working reagent into each well and shake
the plate on a platform shaker for 30 s.
5. Cover the plate and incubate at 37 °C for 30 min.
6. Measure the absorbance at 562 nm on a spectrophotometer
(Perkin Elmer 1420 Multilabel Counter).
3.3 Exosome Particle Determination of exosome particle count is performed following

Counting with CD63 the manufacturer’s instruction of CD63 ExoElisa Kit (System
ELISA Kit Bioscience). Briefly:
1. Add 50 μL of freshly prepared protein standards (108–

1.35 × 1010 exosome.mL−1) and exosome samples to the appro-
priate well of the microtiter plate.
2. Incubate the plate at 37 °C for 2 h.
3. Wash the plate three times for 5 min each with 100 μL wash
buffer.
4. Add 50 μL of 1:100 diluted CD-63 primary antibody to each
well and incubate while shaking at room temperature for 1 h.
5. Wash the plate three times for 5 min each with 100 μL wash
buffer.
6. Add 50 μL of 1:5000 diluted secondary antibody to each well
and incubate with shaking at room temperature for 1 h.
7. Add 50 μL of supersensitive TMB ELISA substrate and incu-
bate with shaking at room temperature for 15–45 min.
8. Add 50 μL of stop buffer and read the absorbance values at
450 nm immediately (Lambda Scan Instrument).
3.4 Western Blot 1. Add 2× Laemmli buffer to the exosome suspensions (18 μg),
Analysis and then boil the samples at 96 °C for 3 min.
2. Centrifuge the exosomal protein solutions at 13,000 × g for
3 min; take the supernatant into clean microtubes.
3. Load 25 μL of each sample into each well (5–12% gel system is
used) and then run proteins at 100 V, 35–40 mA, for about 2 h
(Bio-Rad Wet/Tank Blotting System).
4. Transfer the proteins electrophoretically (100 V, 400 mA) for
1 h onto immobilon PVDF membrane.
5. Block the membrane with 5% nonfat milk powder in TBS-T for
1 h on a platform shaker.
6. Incubate the membrane with CD63 and CD81 antibodies
overnight at 4 °C.
7. Following the washing step with TBS-T, incubate the mem-
brane with secondary antibody diluted in PBS for 1 h at 4 °C.
8. After washing with PBS, the membrane is treated with ECL
substrate and imaged on an Odyssey imaging system.
3.5 Preparation 1. The exosome suspension is treated with 4% paraformaldehyde

of TEM Samples for fixation.
2. Deposit 10 μL of the fixed exosomes onto Formvar carbon-
coated grids and wait for 20 min for adequate absorption.
3. Put 100 μL of PBS onto a piece of parafilm and place the grid
on PBS drop with a clean forceps. Repeat the washing step
three times.
4. Remove the excess water using filter paper.

5. Transfer the grids onto 1% glutaraldehyde drop placed on
parafilm and leave the exosomes for further fixation for a period
of 5 min.
6. Repeat the washing step three times and remove the excess
water using filter paper.
7. Put the grids onto 0.5% uranyl acetate drops and incubate for
10 min for adequate staining (see Note 3).
8. Observe the samples under the electron microscope (Joel USA
JEM 2100-F).
3.6 RNA Isolation 1. Put 175 μL RNA lysis buffer into an Eppendorf tube in which
and Quantification 30 mg of exosome suspension is situated.
2. Following homogenization by vortexing, pipetting is done for
efficient lysis.
3. Add 350 μL of RNA dilution buffer to 175 μL of lysate. Mix
by inverting the tube 3–4 times. Place in a heating block at
70 °C for 3 min.
4. Centrifuge at 13.000 × g for 10 min at 20–25 °C.
5. Transfer the lysate into a clean tube and add 200 μL 95% etha-
nol. Mix by pipetting 3–4 times.
6. Transfer the mixture to the spin column assembly and centri-
fuge at 13,000 × g for 1 min.
7. Discard the liquid in the collection tube and add 600 μL of
RNA wash solution to the spin column assembly. Centrifuge
at 13,000 × g for 1 min.
8. Apply 50 μL of the DNase incubation mix onto the column
membrane and incubate for 15 min at room temperature. Add
200 μL of DNase stop solution and centrifuge at 13,000 × g
for 1 min.
9. Add 600 μL of wash solution and centrifuge at 13,000 × g for
1 min.
10. Discard the liquid in the collection tube and add 250 μL RNA
wash solution into the column. Centrifuge at 13,000 × g for
2 min.
11. Add 100 μL of nuclease-free water into the column fitted with
an elution tube and centrifuge for 1 min.
12. Quantify the amount of exosomal RNA using a Nanodrop
(Thermo Fisher) by dropping 5 μL of RNA solution onto the
pedestal (see Note 4).
4 Notes
1. The ultrasonic probe should be washed with H2SO4, ultrapure

water and ethanol, respectively.
2. The ultrasonication step should be carried out on ice in order to
prevent heating of the tissues due to high-frequency
sonication.
3. Uranyl acetate solution should be filtered from 0.22 μm pore-
sized syringe filter in order to prevent accumulation of large or
aggregated particles onto the grid.
4. A260/280 ratio for RNA quantification should be around 2,
indicating the purity of RNA. If this ratio is over/lower, a
protein-caused impurity is a question that requires an extensive
purification step.
Acknowledgments
This work has been co-financed by the European Union and Greek
national funds through the program “Support for Researchers
with Emphasis on Young Researchers” (call code: EDBM34, ΚΕ
14995) and under the research title “Preparation and study of
innovative forms of administration of pharmaceutical molecules
targeting at improved pharmacological properties.”
References
1. Melo SA, Luecke LB, Kahlert C et al (2015) terization reveals a distinct microRNA signa-
Glypican-1 identifies cancer exosomes and ture in long duration type 1 diabetes. Sci Rep
detects early pancreatic cancer. Nature 7(1):5998
523:177–182 7. Helwa I, Cai J, Drewry MD et al (2017) A
2. Malla B, Zaugg K, Vassella E et al (2017) comparative study of serum exosome isola-
Exosomes and exosomal microRNAs in pros- tion using differential ultracentrifugation
tate cancer radiation therapy. Int J Radiat and three commercial reagents. PLoS One
Oncol Biol Phys 98(5):982–995 12(1):e0170628
3. Agrawal AK, Aqil F, Jeyabalan J et al (2017) 8. Miranda KC, Bond DT, Levin JZ et al (2014)
Milk-derived exosomes for oral delivery of Massively parallel sequencing of human uri-
paclitaxel. Nanomedicine 13(5):1627–1636 nary exosome/microvesicle RNA reveals a
4. Chen B, Li Q, Zhao B et al (2017) Stem cell- predominance of non-coding RNA. PLoS One
derived extracellular vesicles as a novel poten- 9(5):e96094
tial therapeutic tool for tissue repair. Stem 9. Vojtech L, Woo S, Hughes S et al (2014)
Cells Transl Med 6:1753–1758. https://doi. Exosomes in human semen carry a distinctive
org/10.1002/sctm.16-0477 repertoire of small non-coding RNAs with
5. Leblanc P, Arellano-Anaya ZE, Bernard E et al potential regulatory functions. Nucleic Acids
(2017) Isolation of exosomes and microvesicles Res 42(11):7290–7304
from cell culture systems to study prion trans- 10. Perkins EJ, Bao W, Guan X et al (2006)
mission. Methods Mol Biol 1545:153–176 Comparison of transcriptional responses in
6. Garcia-Contreras M, Shah SH, Tamayo A liver tissue and primary hepatocyte cell cultures
et al (2017) Plasma-derived exosome charac- after exposure to hexahydro-1, 3, 5-trinitro-1,
3, 5-triazine. BMC Bioinformatics 7(Suppl 12. Derkus B, Emregul KC, Emregul E (2017) A
4):S22 new approach in stem cell research-exosomes:
11. Katayama S, Skoog T, Jouhilahti EM et al their mechanism of action via cellular path-
(2015) Gene expression analysis of skin grafts ways. Cell Biol Int 41(5):466–475
and cultured keratinocytes using synthetic 13. Pérez-González R, Gauthier SA, Kumar A et al
RNA normalization reveals insights into differ- (2017) Method for isolation of extracellular
entiation and growth control. BMC Genomics vesicles and characterization of exosomes from
16:476 brain extracellular space. Methods Mol Biol
1545:139–151
Chapter 4
Aggregate Determination by Permeation Technique

Phennapha Saokham and Thorsteinn Loftsson
Abstract
Permeation technique is used to study molecular aggregation in aqueous solutions including formation of
cyclodextrin guest/host aggregates. Since only guest molecules, host molecules and guest/host aggre-
gates that are smaller than the pore size of a given semipermeable membrane are able to permeate through
the membrane, negative deviation of permeation profiles indicates formation of guest/host aggregates or
self-aggregates. This chapter describes how the method is used to detect formation of nano-sized aggre-
gates and to determine the critical aggregation concentration (cac) from permeation profiles of a guest
molecule.
Key words Inclusion complexes, Critical aggregation concentration (cac), Permeation, Aggregates
1 Introduction
Clear aqueous solutions can contain molecular aggregates with

diameter below the wavelength of visible light. Frequently these
aggregates and clusters are transient; that is, they are constantly
being formed and dissembled [1]. Thus, it can be difficult to detect
them through conventional methods like dynamic light scattering
(DLS). Molecular membrane permeation is a passive diffusion pro-
cess as described by Fick’s law [2]. Herein, we demonstrate how
the permeation technique through semipermeable membranes is
used to study the formation of transient guest/host aggregates
(e.g., drug/cyclodextrin complex aggregates) and clusters (i.e.,
loosely connected molecular structures). This method is based on
permeation of aggregating molecules through semipermeable
membranes of different molecular weight cutoffs (MWCOs) [3–
9]. Donor solutions (i.e., the test solutions) containing various
concentrations of, for example, guest/host aggregates or self-
aggregates are placed in the donor chamber of Franz diffusion cells
(Fig. 1) [3–8] or miniature dialysis devices such as micro-
equilibrium dialysis device (Fig. 2) [10] or cuplike MINI dialysis
35
36 Phennapha Saokham and Thorsteinn Loftsson
Fig. 1 Jacketed (left) and unjacketed (right) Franz diffusion cells consist of donor and receptor compartments
separated by semipermeable membrane
Fig. 2 A 96-well micro-equilibrium dialysis device which places semipermeable membrane between two
Teflon bars to form donor and receptor compartments
device (Fig. 3) [9, 11]. The donor and receptor compartments are
separated by a single-layer semipermeable membrane. In general,
the studies are performed at room temperature. After fluxes from
various MWCO membranes have been obtained, plots of fluxes
against sampling time (i.e., permeation profiles) are designed. A
negative deviation of permeation profile for Fick’s first law pro-
vides quantitative analysis of formation of nano-sized aggregates
that are unable to permeate through a given MWCO semiperme-
Aggregate Determination by Permeation Technique 37
Fig. 3 A cuplike MINI dialysis device attached with specific MWCO semiperme-
able membrane and 1.5 mL Eppendorf tube as receptor compartment
able membrane [3–5] and the critical aggregation concentration

(i.e., the lowest concentration of host molecule that forms guest/
host aggregates and clusters) is determined [6–9, 11].
2 Materials
2.1 Tested Solutions Prepare saturated guest molecule (e.g., drug) in various concentra-
or Donor Solutions: tions of host molecule (e.g., cyclodextrin) using ultrapure water,
Saturated Guest/Host purifying deionized water with 18 MΩ-cm at 25 °C and analytical
Aggregate Solution grade reagents. Add excess amount of guest to aqueous solution of
host molecule (see Note 1). Then heat, sonicate or autoclave sus-
pensions to promote aggregate formation. Continue to add small
amount of the guest until precipitation is observed after equilibra-
tion. Equilibrate at room temperature (or some other tempera-
ture) for 3–7 days (see Note 2) under constant agitation using, for
example, orbital laboratory shaker. After equilibrium, filtrate the
suspensions through 0.45 μm cellulose acetate or comparable
membrane filter. Analyze the amount of guest permeating through
given membrane by an appropriate analytical method such as high-
performance liquid chromatography (HPLC).
2.2 Self-Aggregate Prepare aqueous solutions containing various concentrations of

or Cluster Solutions aggregating molecules (e.g., drug or cyclodextrin) using ultrapure
water and analytical grade reagents. Filtrate through 0.45 μm cel-
lulose acetate or comparable membrane filter and analyze the
amount of compound permeating through membrane by an
appropriate analytical method.
2.3 Receptor Prepare aqueous solutions containing identical composition as sat-

Solutions urated guest/host solution without guest compound or medium
in case of cluster solutions. Filtrate through 0.45 μm cellulose ace-
tate membrane filter or sonicate to deaerate prior to use.
2.4 Semipermeable Cut single-layer semipermeable cellulose ester membrane to a suit-

Membrane able size to cover the diffusion area of the Franz cells or dialysis
devices. Soak in degassed receptor solution for at least 6 h or over-
night. Membrane must be in equilibrium with the receptor solu-
tion prior to the test. Do not let membrane become dry.
3 Methods
3.1 Permeation 1. Mount receptor compartments of vertical Franz diffusion cells

Studies on stage magnetic stirring apparatus or suitable supportive
apparatus to hold each cell in place. Point the sampling ports
3.1.1 Franz
at an angle out to the side so samples can be easily collected.
Diffusion Cells
2. Place a magnetic stirring bar in each cell. Ensure that suitable
size of magnetic stirring bars is used.
3. Pre-fill each receptor compartments with deaerated receptor
solution until the cell is about 90% filled (see Note 3).
4. Start magnetic stirring apparatus at constant speed (see Note
4) to ensure that there are no bubbles in the cell especially on
the glass wall of receptor compartment. If bubbles are noticed,
run the stirrer at high speed for a few seconds or use a sam-
pling needle to release bubbles.
5. Continue to fill receptor compartment (with receptor solution)
until there is a positive meniscus covering the top of the cell.
6. Carefully mount a single-layer-soaked membrane using a pair
of tweezers (see Note 5). Inspect underneath the mounted
membrane for bubbles. Always ensure that the stirring is off
before placing the soaked membrane on the cells.
7. Assemble donor and receptor compartments, pinch together
with pinch clamp and ensure that there is no leakage. Soaked
membrane must completely cover the diffusion area of cell
and the two compartments aligned.
8. Start the stirrers and pipette tested solution into donor com-
partment. Start the timer for the experiment and cover donor
compartment with adhesive seal film (see Note 6). It is recom-
mended to run each test solution at least triplicate.
9. At the sampling time point (see Note 7), withdraw an appropriate
volume of receptor medium from close to the bottom of receptor
compartment through sampling port using syringe connected
with suitable length needle. Replace immediately with an equal
volume of degassed receptor solution. Confirm that there are no
bubbles underneath membrane during sampling and refilling.
10. Analyze the content of guest compound in sampling solutions
with an appropriate analytical method (e.g., HPLC).
3.1.2 Micro-Equilibrium 1. Place perpendicularly Teflon bar with two connecting rods at
Dialysis Device each edge of the bar.
2. Place precut soaked membrane on the Teflon bar. Ensure that
membrane below the top edge of the bar and the lower edge of
membrane covers the bottom of all wells.
3. Repeat layering soaked membrane and Teflon bar until the
device is fully assembled (see Note 8 and Fig. 2). Flatten mem-
brane before the next Teflon bar is put in place.
4. Insert the fully assembled Teflon block into the base of dialysis
device and then tighten them.
5. Immediately add receptor solution to the dialysis side of the
wells, assigned as receptor compartment, to prevent dehydra-
tion of soaked membranes. Ensure that no bubbles or leakage is
noticed.
6. Add equal volume of test solutions to the other side of the dial-
ysis well (as donor compartment) using appropriate pipetting
device (see Note 9) and start timer.
7. Cover the top surface of dialysis device with an adhesive sealing
film to prevent evaporation.
8. At equilibrium time (see Note 10), discard solutions from
receptor compartment to analyze the amount of aggregating
molecule with an appropriate analytical method.
3.1.3 Cuplike MINI 1. Add 1.2 mL of receptor medium to 1.5 mL conical tube and set
Dialysis Device aside.
2. Load ultrapure water into dialysis device and observe for at least
5 min to moisture membrane and check the integrity of device.
If droplets are noticed from across the membrane, leakage
occurs and the device should not be used.
3. Decant ultrapure water and shake dialysis device to completely
remove water. Do not touch the wetted membrane with
ungloved hands. Once the membrane is wet, do not let it
become dry.
4. Immediately pipette a 0.5 mL of tested solution and then cap
the dialysis device to prevent evaporation.
5. Slowly place the filled dialysis device into conical tube contain-
ing receptor (i.e., dialysis cell). Ensure that the membrane con-
tacts with receptor solution and does not introduce any
bubbles.
6. Place individually dialysis cell on microcentrifuge tube rack or
suitable supportive apparatus.
7. Shake gently on an orbital shaker (i.e., 100 rpm). Experiment is
performed at room temperature (unless indicated otherwise).
8. At sampling time, remove dialysis device and collect sample

from conical tube.
9. Analyze the amount of interested molecule permeating through
membrane with an appropriate analysis method.
3.1.4 Determination 1. Plot the amount of permeated compound over sampling time
of Permeation Profiles point and calculate the steady-state flux (J) following the
equation
dq
J=
A × dt
dq
where is the slope of the linear section of the amount of the
dt
guest in the receptor compartment (q) versus time (t) (see Note 11)
and A is the surface area of mounted membrane.
2. Plot steady-state flux of guest compound against concentration
of host compound for permeation profile (see sample in Fig. 4)
or steady-state flux vs. initial concentration of interested mole-
cule, in case of clusters.
3.2 Evaluation 1. Perform permeation profile of each MWCO of membrane

of Guest/Host separately.
Aggregate Profiles 2. Calculate the fraction of aggregates (fA) (see Note 12), aggre-
gates with molecular weight larger than pore size of membrane
and unable to penetrate through that membrane from perme-
ation profile, according to the following equation:
J exp
fA = 1−
J theo
where Jexp is the experimental flux and Jtheo is the theoretical flux
when the free guest compounds and aggregates are able to per-
meate through the membrane.
3. Analyze the aggregation process presented as aggregation pop-
ulation (fD) and defined as
f D = f Ai − f Aj
where i and j are incremental MWCO values and i < j.

Relationship between aggregation population and concentra-
tion of host molecule of each MWCO of defined membrane can
be illustrated as area plot (see sample in Fig. 5).
3.3 Evaluation 1. Perform permeation profile of desired MWCOs of membrane.

of Critical Aggregation 2. Draw the two straight lines fitted to the steep and close to the
Concentration (cac) horizontal part (see sample in Fig. 6). The critical aggregation
concentration (cac) is the concentration of host molecule at the
interaction point.
Fig. 4 Permeation profile of guest/host aggregates
Fig. 5 Aggregation profile of guest/host aggregates
Fig. 6 Graphic determination of critical aggregation concentration (cac)

4 Notes
1. Aqueous host (e.g., cyclodextrin) solution can be a mixture of

host and other molecules that may not disturb the formation of
guest/host aggregates, for example ophthalmic vehicle consist-
ing of host molecule and pharmaceutical excipients (e.g., poly-
mer, preservative, antioxidant, buffer salts, and osmotic agent)
that may not participate in the aggregate formation [12–14].
2. Equilibration period should be sufficient to reach solubility
equilibrium and depends on, for example, the physicochemical
stability of the guest molecule.
3. For jacketed vertical Franz diffusion cells, temperature should
be controlled by a bath circulation which is connected to indi-
vidual cell. The bath circulator flows through each cell to
maintain cell temperature at about 25 °C (or some other tem-
perature). Prior to the test, allow for few minutes to obtain
stable temperature in Franz diffusion cells.
4. The stirring speed should not interfere with the unstirred layer
or create bubble underneath mounted membrane, for exam-
ple, 300 rpm.
5. Confirm that soaked membrane has no visible damage before
mounting.
6. Verify the absence of leakage especially between donor and
receptor compartments. Mounted membrane should not be
pierced or torn off with a pipette tip.
7. Sampling time points and intervals are determined based on
the solubility of guest compound and sensitivity of the analysis
method. It is recommended to have at least five sampling
points and that the study duration does not exceed 6 h.
8. MWCOs of inserted membranes can be varied to suit the
experimental requirements. All Teflon bars must be assembled
prior to loading the Teflon block into the base.
9. Volume of each side can be varied from 25 to 150 μL.
10. The dialysis block can be incubated at desired temperature
and/or placed on an orbital or reciprocating shaker to facili-
tate equilibration. Equilibrium time depends on the solubility
of guest compound and sensitivity of the analytical method. It
is important to ensure that equilibrium conditions have been
achieved and study time should not exceed 6 h.
11. In case of miniature dialysis devices, calculate the steady-state
q dq
flux using instead of .
t dt
12. The fraction guest in aggregate ranges from 0 to 1, indicating
that all aggregates in tested solution are able or unable to per-
meate through the membrane, respectively.
Acknowledgments
References
1. Loftsson T, Saokham P, Sá Couto AR (2019) 8. Sá Couto AR, Ryzhakov A, Loftsson T
Self-association of cyclodextrins and cyclo- (2018) Self-assembly of α-cyclodextrin and
dextrin complexes in aqueous solutions. Int J β-cyclodextrin: identification and develop-
Pharm 560:228–234 ment of analytical techniques. J Pharm Sci
2. He X (2009) Chapter 18. Integration of physi- 107(8):2208–2215
cal, chemical, mechanical, and biopharma- 9. Saokham P, Sá Couto A, Ryzhakov A et al
ceutical properties in solid oral dosage form (2016) The self-assemble of natural cyclo-
development. In: Qiu Y et al (eds) Developing dextrins in aqueous solutions: application of
solid oral dosage forms. Academic Press, San miniature permeation studies for critical aggre-
Diego, CA gation concentration (cac) determinations. Int
3. Jansook P, Kurkov SV, Loftsson T (2010) J Pharm 505(1–2):187–193
Cyclodextrins as solubilizers: forma- 10. Stappaerts J, Do Thi T, Dominguez-Vega E
tion of complex aggregates. J Pharm Sci et al (2017) The impact of guest compounds
99(2):719–729 on cyclodextrin aggregation behavior: a series
4. Messner M, Kurkov SV, Brewster ME of structurally related parabens. Int J Pharm
et al (2011) Self-assembly of cyclodex- 529(1):442–450
trin complexes: aggregation of hydrocorti- 11. Saokham P, Do TT, Van den Mooter
sone/cyclodextrin complexes. Int J Pharm G et al (2018) Inclusion complexes
407(1–2):174–183 of p- hydroxybenzoic acid esters and
5. Messner M, Kurkov SV, Palazón MM et al γ-cyclodextrin. J Incl Phenom Macrocycl
(2011) Self-assembly of cyclodextrin com- Chem 90(1):111–122
plexes: effect of temperature, agitation and 12. Jansook P, Loftsson T (2009) CDs as solubiliz-
media composition on aggregation. Int J ers: effects of excipients and competing drugs.
Pharm 419(1):322–328 Int J Pharm 379(1):32–40
6. Sá Couto AR, Ryzhakov A, Loftsson T (2018) 13. Jansook P, Ritthidej GC, Ueda H et al (2010)
2-Hydroxypropyl-β-cyclodextrin aggregates: γCD/HPγCD mixtures as solubilizer: solid-
identification and development of analyti- state characterization and sample dexametha-
cal techniques. Materials (Basel, Switzerland) sone eye drop suspension. J Pharm Pharm Sci
11(10):1971 13(3):336–350
7. Saokham P, Loftsson T (2015) A new 14. Muankaew C, Jansook P, Loftsson T (2017)
approach for quantitative determination of Evaluation of γ-cyclodextrin effect on perme-
γ-cyclodextrin in aqueous solutions: applica- ation of lipophilic drugs: application of cello-
tion in aggregate determinations and solubil- phane/fused octanol membrane. Pharm Dev
ity in hydrocortisone/γ-cyclodextrin inclusion Technol 22(4):562–570
complex. J Pharm Sci 104(11):3925–3933
Chapter 5
Study of Candesartan Cilexetil: 2-Hydroxypropyl-β-

Cyclodextrin Interactions: A Computational Approach Using
Steered Molecular Dynamics Simulations
Sofia Kiriakidi and Thomas Mavromoustakos
Abstract
Cyclodextrins (CDs) are widely used in the pharmaceutical industry as transporters of lipophilic drugs due
to their amphiphilic nature. The detailed study of the molecular interactions of drug complexes with CDs
is very important in designing the best formulation for the transportation of lipophilic drugs. The drug:
CD binding should be strong enough so that the complex is stable in the aquatic environment of the
extracellular fluids, but also not very strong in order for the drug to be capable for being released in the
proximity of the target receptor. In a recent study, we investigated the ΔG of binding between the com-
mercially available, nontoxic 2-hydroxypropyl-β-cyclodextrin (2-HP-B-CD) and the antihypertensive drug
candesartan cilexetil (CC), with the use of steered (or biased) molecular dynamics (sMD) and umbrella
sampling method. This chapter describes comprehensively how to perform sMD and umbrella sampling in
order to calculate the ΔGbinding of CC to the 2-HP-B-CD.
Key words Steered MD, ΔGbinding, Umbrella sampling, Biased MD, Candesartan, Hypertension,
Lipophilicity, Drug transporter
1 Introduction
Candesartan cilexetil (CC, commercial name Atacand) is a pro-

drug, which is rapidly converted to the active drug, candesartan
(CAN), by ester hydrolysis during absorption from the gastrointes-
tinal area of the human body. Due to the highly lipophilic nature
of CC, the drug’s bioavailability is only 15% after oral administra-
tion [1]. The use of cyclodextrins (CDs) as drug carriers is investi-
gated in order to increase CAN’s bioavailability [2, 3], by increasing
its aqueous solubility before interacting with the membrane recep-
tor system, as depicted in Fig. 1. In our recent study [4], we inves-
tigated the ΔG of binding between the commercially available,
nontoxic 2-hydroxypropyl-β-cyclodextrin (2-HP-B-CD) [5, 6]
and the antihypertensive drug candesartan cilexetil (CC), with the
45
46 Sofia Kiriakidi and Thomas Mavromoustakos
Fig. 1 Schematic representation of a CD molecule acting as a drug transporter for a lipophilic drug molecule.
CD’s internal lipophilic cavity acts as a hospitable pocket for the drug, while its external hydrophilic surface
facilitates transportation in an aqueous environment
use of steered (or biased) molecular dynamics (sMD) and umbrella

sampling method. In this work, the details of the interactions
(energetics) of such complexation are studied with the use of
steered molecular dynamics (sMD) and umbrella sampling simula-
tions. The use of these methods can lead to the investigation of the
Gibbs free energy changes (ΔGs) as a function of a certain reaction
coordinate. In this specific case, the ΔG of binding between CC
and 2-hydroxypropyl-β-cyclodextrin (2-HP-B-CD) (denoted from
now on as CC:2-HP-B-CD complex) will be estimated as a func-
tion of the intramolecular distances of the centers of mass (COMs)
of the two species. In this way, the release energy of CC will be
computed (being the opposite of the complexation energy). This
method is based upon the implementation of an external potential
upon the system, which will force it to follow a specific “route”
upon the reaction coordinate [7]. Thereby, a better and more
complete sampling is achieved, even in regions of the phase space
that represent high-energy states (such as transition states), which
would not have been sampled with the use of classical unbiased
MD. The latter is based upon the ergodic hypothesis of statistical
thermodynamics, according to which every state in the phase space
is energetically equal and thus equiprobable (thus the system is in
equilibrium). In systems where the ergodic hypothesis does not
apply (e.g., a case where a drug:CD complex should overcome a
barrier in order for the drug to be released), unbiased MD cannot
provide with accurate results unless it is run for long enough time
(which would be too long for an average academic workstation),
since high-energy states are very rarely sampled. This is why such
phenomena are called “rare events”; due to their high-energy states
it is very rare that the phase space where they reside is sampled.
Thus, in order to study such phenomena, the system needs to be
forced to go through these high-energy states by imposing a per-
Study of Candesartan Cilexetil: 2-Hydroxypropyl-β-Cyclodextrin Interactions… 47
Fig. 2 Unbiased molecular dynamics is depicted in yellow: the system is trapped

at a local minimum. Steered molecular dynamics is depicted in blue: an external
potential can drag the system up to a maximum in order to explore other minima
in the potential energy surface (PES)
turbation to the system’s Hamiltonian according to the following

relation:
V ′ (r N ) = V (r N ) + W (r N )
where W(rN) is a weighting function which ensures that all possible

states will be sampled even if they are away from equilibrium. This
procedure is schematically depicted in Fig. 2. After the completion
of sMD, where the system is dragged along a reaction coordinate,
several successive unbiased MD simulations are performed in sev-
eral intervals of the reaction coordinate, where the system is let to
relax and reach equilibrium. The procedure of dividing the system
in successive “windows” where equilibrium dynamics are taking
place is illustrated in Fig. 3. Therefore, the equilibrium Gibbs free
energy is computed through the weighted histogram analysis
method (WHAM) [7]. The ΔGbinding is defined as the difference
between the maximum of the potential of mean force (PMF) func-
tion, which corresponds to the transition state and the minimum
which corresponds to the equilibrium state. A sign of equilibrium
is that the PMF function reaches a plateau after reaching its mini-
mum value.
2 Software
1. Maestro:
Download the latest free Maestro version and register for
academic use: https://www.schrodinger.com/freemaestro.
2. UCSF Chimera:
Reaction Coordinate
configuration
window
energy
function for
each
window
Fig. 3 The umbrella sampling procedure is illustrated: (Top) The sMD procedure drags the drug molecule
(orange polygon) along the reaction coordinate. (Middle) The pulling trajectory is divided into some windows
where the system is left to relax and equilibrium dynamics takes place. (Bottom) The energy function of each
window results in a histogram. Subsequent histograms must sufficiently overlap in order to acquire the poten-
tial of mean force (PMF) curve for the procedure under study
Download the latest UCSF Chimera release for your operat-

ing system form https://www.cgl.ucsf.edu/chimera/. Follow
the installation instructions.
3. Swiss Param:
Visit the Swiss Param server website in order to create your
molecule topologies at http://www.swissparam.ch/ (further
directions are in Subheading 3).
4. Gromacs:
Install the latest Gromacs release at your Linux workstation
or check for the available modules at your HPC server, http://
www.gromacs.org/.
5. VMD:
Download and install the latest VMD release for your operating
system, https://www.ks.uiuc.edu/Development/Download/
download.cgi?PackageName=VMD.
3 Methods
All the MD simulations were carried out using the Gromacs/2018.1

simulation package and the workflow below is adjusted for this
specific software [8]. Similar workflows can be used for different
software; however specific steps should be altered. The tutorial is

designed for working in a Linux machine.
3.1 System 1. You may download the desired molecules (CC and 2-HP-B-CD)
Preparation from chemical databases such as http://www.chemspider.com/
or alternatively use a chemical modeling software such as
3.1.1 Manually Create Schrodinger’s Maestro 3D builder [9] in order to create the 3D
the Drug: CD Complex structures of the drug molecule CC and the host molecule
2-HP-B-CD. If you decide to use chemspider.com search for
“candesartan cilexetil” and
“2-hydroxypropyl-beta-cyclodextrin.”
2. Open UCSF Chimera [10] or any chemical modeling software
in order to manually put the drug molecule inside the CD’s
cavity.
3. In Chimera click File → Open, choose the CC molecule that
you downloaded from a database or created with a modeling
software and save it on your workstation.
4. Do the same for 2-HP-B-CD.
5. Now, both molecules should be visible in the Chimera window.
Select CC by Ctrl+Left Click on one atom of the CC molecule.
Press the up arrow (↑) in order to expand the selection to the
whole molecule.
6. Click Tools → Movement → Movement Mouse Mode. In the
Movement Mouse Mode dialog box choose “Move Selection”
and minimize the dialog box.
7. Now move the molecule by pressing the middle mouse button
until you put it in the CD’s cavity.
8. Go again in the Movement Mouse Mode that you previously
minimized and choose “Normal.” Now the whole system can
be rotated by clicking the left mouse button. Always check
every perspective by rotating around in order to visualize that
the molecule is indeed inside the cavity. Do not mind if any
clashes are present since the structure will be minimized later.
9. In order to deselect click Select → Clear Selection.
3.1.2 Minimize Structure The crude complex structure needs to be energetically minimized
by performing a geometry optimization.
1. In the Chimera environment, click Tools → Structure
Editing → Minimize Structure. Keep the default settings and
click minimize and OK in all dialog boxes that appear. After a
while (depending on your machine’s capacity) the minimiza-
tion will be complete and a “Finished” indication will appear in
the bottom-left Chimera window. Save the final complex struc-
ture in .pdb format by pressing File → Save pdb and save it as
complex.pdb. In the “Save multiple models in” box choose “a

single file.”
2. In order to save the separate molecular structures select CC as
described before (CTRL+ click on one atom and press the up
arrow to expand selection) and then click Actions → Atoms/
Bonds → Delete. Save the 2-HP-B-CD structure as 2-HP-
BCD.pdb and one more time as 2-HP-B-CD.mol2.
3. Close the Chimera window and then reopen it. Press
File → Open and open the complex.pdb file that you previously
saved. In a similar manner, select and delete the 2-HP-B-CD
structure. Now save the CC structure both in .pdb and .mol2
format. For a more thorough procedure see Note 1.
3.1.3 Create Visit the SwissParam server [11] in order to create topologies for
the Topology Files for Both CC and 2-HP-B-CD using the Merck molecular force field
Drug and Host (MMFF) [12] (see Note 2).
1. Click browse and upload each separate molecule’s structure in
.mol2 format. There is no need to create topology for the com-
plex, just for the two separate molecules. Press submit Query.
After a short while (approximately 1 min) click on the link pro-
vided in the website in order to download the files that were
created by the server.
2. Extract the file from the downloaded zipped folder and copy
the gromacs topology file for each molecule (file extension .itp)
in your working directory.
3. In a text editor, open CC.itp (or however the SwissParam server
named your topology file for the CC molecule). Cut all lines
before [moleculetype]. Create a blank text document, name it
atomCC.itp, and paste all the information that you cut from the
topology file before. These specific steps were added in order to
avoid the frequent gromacs error “Invalid order for the direc-
tive [molecule type]”; see Note 3 for further information.
Repeat for the 2-HP-B-CD.itp file.
4. In this simulation, 2-HP-B-CD will be restrained and the rele-
vant drug molecule CC will be pulled out of the CD’s cavity. In
order to keep the host molecule restrained, open the 2-HP-
BCD.itp file in a text editor and paste the following lines in the
bottom:
#ifdef POSRES_B
#include "posreCYCL.itp"
#endif
(If you are using the vi editor, in order to edit the file press the let-
ter “i,” paste the lines and then press ESC. In order to save the
file and quit press “:wq” and press enter.
3.1.4 Create the In your Linux terminal, use the relevant commands to load the
Gromacs Input Files Gromacs software (e.g., ./gromacsxxxx.x or module load gro-
macs/xxxx.x if you are running on an HPC server).
1. For the study of CC:2-HP-B-CD complex, after loading gro-
macs, go to your working directory (where all the files that you
have created reside) and use the following terminal
commands:
gmx_d editconf –f CC.pdb –o CC.gro
gmx_d editconf –f 2-HP-B-CD.pdb –o 2-HP-B-CD.
gro
The command gmx_d calls the gromacs software to operate
in double precision while the module editconf converts the .pdb
format to the appropriate gromacs input format .gro. Now two
separate input files have been created.
2. In order to create an input file for the CC:2-HP-B-CD com-
plex, copy the 2-HP-B-CD.gro to a new file and name it com-
plex.gro using the following terminal commands:
cp 2-HP-B-CD.gro complex.gro
3. Open CC.gro in a text editor by pressing:
vi CC.gro
The first two lines are the title and the number of atoms;
keep the number of atoms in mind. In the third line and first
column you will see the name that has been given to your mol-
ecule. In case the molecule reads 1LIG (or exactly the same as
the 2-HP-B-CD molecule) change its molecule name. To do
so, if you are using vi press the following:
:%s/LIG/CC and press Enter
in order to name the molecule CC (or use any name you
like).
Ignore the first two lines and copy the rest, which are the
molecule’s coordinates.
In order to avoid any future errors, make sure that the same
name for the molecule is used in the .gro file, the .itp file (in
sections [ moleculetype ] and [ atoms ]/resid ) and topol.top
(the topol.top will be described in the next step).
4. Open the complex.gro file and paste the coordinates that you
just copied, before the last line which is the cell’s vectors. Make
sure that everything is aligned properly on the right. After copy-
ing the coordinates of the drug molecule in the host’s input file,
correct the number of coordinates in the second line of the .gro
file, by adding up the number of coordinates copied. For exam-
ple the number 217 for 2-HP-B-CD should be changed to 296
after having copied the coordinates for the 79 atoms of CC.
3.1.5 Manually Create Gromacs can automatically create topology files using the pdb-
the Topology File 2gmx module, which is a very powerful tool when dealing with
for the Run proteins. However, in this case where only heteromolecules are
present in the simulation, the topology file can be created
manually.
1. Create a text file and name it topol.top. A model text file is
available in the online version and presented in Fig. 4. The pre-
ferred force field for this simulation is charmm36 [13].
2. Copy the lines presented in Fig. 4 or download the available
topol.top file from the online version.
3. It is very important that the names of the drug molecules in the
last section of topol.top, [molecules], are exactly the same as in
the section [molecule type] of the CC.itp and 2-HP-B-CD.itp
files. If they are not, change them appropriately.
3.1.6 Create Next step is the creation of the simulation box with the editconf
the Simulation Box module. Special care should be taken in order to create a box long
enough, so that the dragged molecule does not interact with the
periodic image of the host molecule.
1. Use the following terminal command:
gmx_d editconf -f complex.gro -o newbox.
gro -center 2.181 2.4775 3.280 -box 6.560
4.362 12
In such a way, a long enough box is created and the complex
is centered at 2.181 2.4775 3.280. The drug molecule will be
pulled along the long dimension at a distance no greater than
half the dimension. This is of great importance since gromacs
calculates distances while taking periodicity into account. If half
of the pulling dimension is exceeded (i.e., 6 nm in this case) the
reference distance becomes the periodic distance which greatly
affects the results.
2. Always check in a visualization software (e.g., VMD [14] where
the simulation box can be visualized) that the complex is ori-
ented appropriately and that the long dimension is aligned with
your pulling direction, as depicted in Fig. 5.
3. In order to recreate Fig. 5 and in case vmd is downloaded and
installed in your workstation, in your working directory press:
vmd newbox.gro
4. In the VMD window press Extensions → Tk Console. In the Tk
Console environment press:
pbc box
and the simulation box will be depicted.
Fig. 4 A model topology file for the drug:CD system
3.1.7 Solvate the System In this case, only neutral species are involved, so a simple water
solvation is adequate.
1. Use the following terminal command:
Fig. 5 A screenshot of the pulling simulation box. The box must be long enough at the pulling dimension (z) so
that the reference distance for pulling does not become the periodic distance
gmx_d solvate -cp newbox.gro -cs spc216.gro -o

solv.gro -p topol.top
In case a charged species is involved, placing of appropriate
counterions is needed in order to have a neutral net charge;
please refer to Note 4.
3.2 Energy The solvated system should now be energy minimized before pro-
Minimization ceeding with the dynamics simulation. In order to do so, an energy
minimization protocol should be used. The energy minimization
3.2.1 Create Input protocol will be provided to the grompp module of gromacs suite
in order to produce a portable binary run input file of .tpr format.
The .tpr file contains information with regard to the starting struc-
ture of the simulation, as well as the molecular topology and all the
simulation parameters. The simulation parameters are described in
the protocol file em.mdp. An example protocol file (based on the
tutorials provided by Assist. Prof. Justin Lemkul at http://www.
mdtutorials.com/gmx/ but modified in order to be compatible
with the use of charmm36 force field (see Note 5)) is available in
the online version and presented in Fig. 6. The file contains self-
explanatory comments at each line.
1. The terminal command to be used in order to produce the
binary input file in accordance with the minimization protocol
is the following:
gmx_d grompp -f em.mdp -c solv.gro -p topol.
top -o em.tpr
3.2.2 Run the Energy In order to run the simulation, the mdrun module of the gromacs
Minimization Simulation suite should be used, which is its main computational chemistry
engine.
Fig. 6 A model protocol file for the energy minimization process
1. The terminal command is the following:

gmx_d mdrun –s em.tpr -deffnm em
The above option should be altered if it is run on a super-
computing system using several processors in parallel by using
the gmx_d_mpi command instead.
3.3 Equilibration The system will be first equilibrated in the canonical ensemble
under constant volume and temperature.
3.3.1 NVT Ensemble
Equilibration The input for the dynamics simulation will be created in a similar
Create Input fashion as described before for the minimization process. The
module grompp will be called and it will produce a binary input file
(nvt.tpr) after reading the relevant protocol file (nvt.mdp). An
example NVT equilibration protocol is available in the online ver-
sion and presented in Fig. 7.
Create Input
First, create an index file where you will group the 2-HP-B and CC
molecules together. In the terminal press:
gmx_d make_ndx –f em.gro –o index.ndx
Press the numbers of the molecules you want to group together
by pressing, e.g.,
2 | 3
If 2-HP-B-CD is 2 and CC is 3, then press q. In case the spe-
cies at your system are numbered differently, choose the appropri-
ate number to group CC and 2-HP-B-CD together.
Fig. 7 A model protocol file for the equilibration process in the canonical ensemble (NVT)
The terminal command to be used in order to produce the

binary input file in accordance with the minimization protocol is
the following:
gmx_d grompp -f nvt.mdp -c em.gro –n index.
ndx –r em.gro -p topol.top -o nvt.tpr
Run the Equilibration In a similar way, call the mdrun module of gromacs in order to run
Simulation the simulation using the following command:
gmx_d mdrun –s nvt.tpr -deffnm nvt
3.3.2 NPT Ensemble
Upon the completion of the NVT equilibration, the system will be
Equilibration
further equilibrated in the isothermal-isobaric ensemble, i.e., under
constant pressure and temperature. The procedure is similar as dis-
cussed for the canonical example but different equilibration proto-
cols will be used. The NPT equilibration protocol is available in the
online version and presented in Fig. 8.
Create Input The terminal command to be used in order to produce the binary
input file in accordance with the minimization protocol is the
following:
gmx_d grompp -f npt.mdp –n index.ndx -c nvt.
gro –r nvt.gro -p topol.top -o npt.tpr
Run the Equilibration Run the simulation using the following command:
Simulation
gmx_d mdrun –s npt.tpr -deffnm npt
3.4 Steered The next step after having properly equilibrated the system is to
Molecular Dynamics perform the sMD simulation. The CC molecule will be pulled out
Simulation of the 2-HP-B-CD’s cavity, by applying an external potential that
perturbs the system’s Hamiltonian as described previously.
3.4.1 Create Input It is very important to create a pulling protocol that reflects the
for the Pulling Simulation specific pulling action that is intended. Special care should be taken
in order to select the direction of your reaction coordinate. In this
specific case, we will pull the drug molecule along the z-direction
which is aligned with the cavity’s opening as illustrated in Fig. 9.
The pulling protocol is available in the online version and pre-
sented in Fig. 10. The total pulling simulation will be 500 ps and
snapshots will be saved every 1 ps. Although the protocol files are
commented with self-explanatory comments and most of it is simi-
lar to the protocols discussed below, some of its parts will be dis-
cussed further. In particular, the section that is responsible for the
pulling is the following:
; Pull code
pull = yes
pull_ncoords = 1 ; only one reaction coor-
dinate
pull_ngroups = 2 ; two groups defining one
reaction coordinate
pull_group1_name = CC
Fig. 8 A model protocol file for the equilibration process in the isothermal-isobaric
ensemble (NPT)
Fig. 9 The CAN:2-HP-B-CD complex. The CAN molecule (green highlight) will be
pulled out of the cavity through CD’s wide rim
pull_group2_name = CD
pull_coord1_type = umbrella ; harmonic po-
tential
pull_coord1_geometry = distance ; simple
distance increase
pull_coord1_dim = N N Y
pull_coord1_groups = 1 2
pull_coord1_start = yes ; define initial COM
distance > 0
pull_coord1_rate = 0.01 ; 0.01 nm per ps =
10 nm per ns
pull_coord1_k = 1000 ; kJ mol^-1 nm^-2
In the above code we ask the program to perform a pulling
simulation (pull=yes) and we impose one reaction coordinate. We
define two different groups (CC and 2-HP-B-CD in this case) by
imposing a harmonic potential (pull_coord1_type=umbrella). We
choose to pull as per distance (different options such as direction
are available; for further alternatives see Note 6). The next code
line (pull_coord1_dim= N N Y) specifies that the pulling will take
place only to the z-direction with N standing for No and Y stand-
ing for Yes. The reaction coordinate connects groups 1 and 2 and
the first COM distance is the reference distance for the first frame.
Fig. 10 A model protocol file for the pulling process in the isothermal-isobaric ensemble (NPT)
The simulation will take place with a pulling rate of 0.01 nm/ps
(meaning that during the 500 ns of simulation, the drug will be
pulled for 5 nm) and the force constant will be 1000 kl/mol.
1. Create the binary input file using the following command:
gmx_d grompp -f md_pull.mdp –n index.ndx -c

npt.gro -p topol.top -r npt.gro -t npt.cpt -o
pull.tpr
3.4.2 Run the Pulling The simulation will be run using a similar command, but in this
Simulation case the –px and –pf commands will be used additionally, in order
to save the pull COM coordinates and forces:
gmx_d mdrun -deffnm pull -pf pullf.xvg -px
pullx.xvg
3.4.3 Collect Frames Now a series of configurations will be extracted from the trajectory
in order to use some of them as the starting configuration for the
umbrella sampling procedure, as it is schematically illustrated in
Fig. 3. In order to extract the frames from the pulling trajectory (a
file will have been created and named as pull.xtc), use the trjconv
module of gromacs. When prompted, choose to save the whole
system. The terminal command for the frame extraction is
gmx_d trjconv -s pull.tpr -f pull.xtc -o
conf.gro –sep
A series of coordinate files (conf0.gro, conf1.gro, etc.) will be
produced, corresponding to each of the frames saved in the con-
tinuous pulling simulation. The COM distance between CC and
2-HP-B-CD will be calculated by the distance module of gromacs.
To iteratively call the module distance on all of these frames that
were generated, use the bash script (get_distances.sh) provided by
Assist. Prof. Lemkul at his website (http://www.mdtutorials.
com/gmx/umbrella/05_pull.html) and change it as follows. First
comment out or delete the line that starts with “echo 0” since it
contains the command that you just did, i.e., separate the trajec-
tory in frames. Then, alter the number of iterations (for ((i=0;
i<[number of generated conf#.gro here]))) and the name of the
groups (CC and CD in this case). Press the following in the com-
mand line:
./get_distances.sh
This script will generate a file named “summary_distances.dat”
with a list of all the conf#.gro files and the corresponding COM
distance of the groups of interest.
3.4.4 Choose In order to select the conf#.gro files that will serve as starting con-
the Starting Configurations figurations for the umbrella sampling simulation, you need to
decide the COM spacing. Usually a COM spacing of 0.1 nm is
adequate, but in case needed, more configurations can be added

later. Open the summary_distances.dat file and make note of the
number of each conf#.gro file that is separated by 0.1 nm. In this
way, you will create a list of 40–50 starting configurations, depend-
ing on your system. There is no need to be extremely precise with
the spacing. What is important is to have a good overlap between
the separate windows (i.e., the independent simulations for the
umbrella sampling procedure), as it will be explained more thor-
oughly in the next sections. Only after completing the umbrella
sampling simulations will you be able to check if the overlap is
adequate, so at first a guess on the spacing must be made.
3.5 Umbrella Before proceeding with the separate unbiased MD simulations, a

Sampling short equilibration will take place for each of the starting configu-
3.5.1 Equilibration rations. If the chosen starting configurations were, e.g., the follow-
ing (the numbers are just indicative):
1. conf0.gro
2. conf10.gro
3. conf31.gro
4. conf80.gro
…
50. conf500.gro
the grompp module will be used in order to create 50 binary
input files for the NPT equilibration of each of the starting win-
dows. Note that these conf#.gro files are indicative and in your
simulation might differ, because they depend on the exact pose of
the drug molecule inside 2-HP-B-CD’s cavity. The NPT equilibra-
tion protocol is available in the online version and presented in
Fig. 11.
1. The terminal command for creating the equilibration input is
the following:
gmx_d grompp -f npt_umbrella.mdp -c conf0.
gro -p topol.top -r conf0.gro -n index.ndx -o
npt1.tpr
...
gmx_d grompp -f npt_umbrella.mdp -c conf500.
gro -p topol.top -r conf500.gro -n index.
ndx -o npt50.tpr
2. Then, each equilibration simulation should be run indepen-
dently using the mdrun module as follows:
gmx_d mdrun -deffnm npt1
...
gmx_d mdrun -deffnm npt50
Fig. 11 A model protocol file for the equilibration process in the isothermal-isobaric ensemble (NPT) for the
umbrella sampling simulation
3.5.2 Umbrella After the completion of equilibration, the production run of each
Sampling Simulations of the unbiased MD simulation should be started. Again, in a simi-
lar fashion, a binary input file will be created using the grompp
module and a suitable protocol file. The MD protocol is available
in the online version and presented in Fig. 12.
1. The terminal commands for the input files are:
gmx_d grompp -f md_umbrella.mdp -c npt1.gro -t
npt1.cpt -p topol.top -r npt1.gro -n
index.ndx -o umbrella1.tpr
...
gmx_d grompp -f md_umbrella.mdp -c npt50.
gro -t npt50.cpt -p topol.top -r npt50.gro -n
index.ndx -o umbrella20.tpr
This protocol will create input for a 10 ns simulation for each
window. For demonstration issues this duration is adequate but
in case more accurate energy results are needed and depending
on your system, longer simulations might be necessary. After
having created the .tpr files run the simulations using the fol-
lowing terminal commands:
gmx mdrun -deffnm umbrella0

...
gmx mdrun -deffnm umbrella50
3.6 Analysis The ΔGbind of CC and CAN to 2-HP-B-CD can be estimated by

the potential of mean force (PMF) plot which is extracted from the
umbrella sampling procedure. The PMF of each simulation win-
dow will be calculated and plotted against the reaction coordinate,
which, in this case, is the COM distance. Given that the PMF plot
converges to a constant value at long distances, the difference
between the highest and lowest energy values corresponds to the
ΔGbind between the studied drug molecule and 2-HP-B-CD. In
order to calculate the PMF for each simulation window, the
weighted histogram analysis method (WHAM) will be used, which
is implemented in the WHAM module of gromacs.
3.6.1 Create Input The input to WHAM module consists of two files, one containing
the binary input of each simulation window (*.tpr files) and the
other containing the forces that the external potential imposes
onto the drug molecule that is dragged outside the CD’s cavity
(*pullf.xvg files).
1. Create a new document in a text processor (e.g., press this on
your terminal: vi tpr-files.dat) and make a list with all the .tpr
files in ascending order:
Fig. 12 A model protocol file for the molecular dynamics in the isothermal-isobaric ensemble (NPT) for the
umbrella sampling simulation
umbrella1.tpr
umbrella2.tpr
…
umbrella50.tpr
Name this file tpr-files.dat.
2. Create another text file where all the force files will be noted in
ascending order:
umbrella1_pullf.xvg
umbrella2_pullf.xvg
…
umbrella50_pullf.xvg
and name this file pullf-files.dat.
3.6.2 Run the WHAM In order to conduct the WHAM analysis use the following termi-
Analysis nal command:
gmx_d wham -it tpr-files.dat -if pullf-files.
dat -o profile.xvg –hist histo.xvg -unit
kCal -bs-method b-his -nBootstrap 100 -bsres
The –it and –if options provide the WHAM module with the tpr
and force files, respectively. The output option (−o) provides the out-
put file profile.xvg which contains the PMF plot for the process under
study, while the –hist provides the histo.xvg file which contains the
histograms of the process. The histogram analysis ensures that the
sampling around a reaction coordinate is adequate. An indication of
adequate sampling is a good overlap between the histograms of each
consequent window, as illustrated in Fig. 13. The –unit option defines
the unit for the energy value (in this case, the chemically relevant unit
of kcal/mol is preferred). The –bs method option is used in order to
define the error analysis method. In this case, we provide the key-
word b-his in order to use the Bayesian bootstrap method for our
error analysis. With this method, complete histograms are considered
as independent data points, and the bootstrap is carried out by assign-
ing random weights to the histograms. The –nBootstrap provides the
number of bootstraps to be used for the error analysis, in this case
100. For further details on the statistical analysis, please refer to refer-
ence [7]. The –bsres option provides the output of the Bootstrap
analysis which is a .xvg file containing the PMF plot along with the
standard deviations in energy.
Following these steps, the umbrella sampling simulation will
be completed. The results for CC:2-HP-B-CD complex are pre-
sented and briefly discussed in Fig. 14.
Umbrella histograms
CC:2-HP-B-CYCL
30000
25000
20000
count
15000
10000
5000
0
0 1 2 3 4 5
ξ (nm)
Fig. 13 Computed histograms after WHAM analysis of the umbrella sampling simulations. A good overlap
between subsequent histograms indicates a good sampling which will produce converged energy plots
Umbrella potential
CC:2-HP-B-CYCL
20
15
E (kcal mol-1)
10
0
0 1 2 3 4 5
ξ (nm)
Fig. 14 PMF plots for the CC:2-HP-B-CD complex. The ΔGbinding is calculated by the difference between the
highest and lowest energy values, given that the PMF plot converges to a plateau in long distances. In this case
the ΔGbinding is 10.22 kcal/mol
Fig. 15 A model protocol file for the ion-placing simulation
4 Notes
1. For a more thorough procedure, instead of manually creating

the complex, the drug molecule could be docked into CD’s
cavity using special docking software, where different docking
poses could be ranked energetically. However, the approach
described in this tutorial is accurate enough for the scope of this
chapter.
2. The MMFF was used for the topologies of the drug molecules,
since it is a compatible force field with charmm36 (a built-in
force field in gromacs) that was used in order to treat the rest of
the molecules, in this case, the water molecules. Other force field
combinations can be used but you should always check the com-
patibility between them; otherwise large errors might occur.
3. The different directives of .top and .itp files should share a com-
mon order. For further details about this error visit gromacs
manual at http://www.gromacs.org/Documentation/
Errors#Invalid_order_for_directive_xxx.
4. In this specific system, both drug molecules are neutral.
However, in case you might use this tutorial for a charged spe-
cies, or even for different protonation states of CAN, addition
of ions is needed. In order to add ions, first create the binary
input file ions.tpr using the ions.mdp protocol which is avail-
able in the online version and presented in Fig. 15. The termi-
nal commands that should be used in order to create the binary
input is the following:
gmx_d grompp -f ions.mdp -c solv.gro -p
topol.top -o ions.tpr
Then the genion module should be used in order to actually
place the ions:
gmx_d genion -s ions.tpr -o solv_ions.gro -p

topol.top -pname NA -nname CL -neutral -conc
0.115
The -pname option defines the type of the cation which in
this case is sodium (Na) while the –nname option defines the
type of the anion which in this case is chlorine (Cl). Option –
neutral asks genion to place ions until the system is neutralized
while –conc defines the additional salt concentration where in
this case the physiological salt concentration of 0.115 was cho-
sen. After having completed the ion placement, continue with
the energy minimization but in the terminal command, change
solv.gro with solv_ions.gro as follows:
gmx_d grompp -f em.mdp -c solv_ions.gro -p
topol.top -o em.tpr
5. Each force field requires different parametrization in the rele-
vant protocol file. For further details you may visit the gromacs
manual at http://www.gromacs.org/Documentation/
Terminology/Force_Fields/CHARMM.
6. Depending on the reaction coordinate you wish to study, you
might want to pull a group of your system in different ways.
The alternatives provided in the gromacs pull code are position,
direction, and direction-periodic. For further details and more
thorough theoretical background visit gromacs manual at
http://manual.gromacs.org/documentation/2019-rc1/
reference-manual/special/pulling.html.
Acknowledgments
Special acknowledgment should be given to Assistant Prof. Justin

Lemkul for providing the comprehensive umbrella sampling tuto-
rial available at www.mdtutorials.com/gmx/umbrella/index.html.
Special thanks to Mrs. Eleni Chontzopoulou for the extensive
tests to the comprehensibility of this protocol. This work was sup-
ported by the Hellenic Foundation for Research and Innovation
(HFRI) and the General Secretariat for Research and Technology
(GSRT), under the HFRI PhD Fellowship grant (GA. no. 14551).
The molecular dynamics simulations of this research work were
supported by computational time granted from the Greek Research
and Technology Network (GRNET) in the National HPC facil-
ity—ARIS—under project ID MDAT1R and from the ARIS
VI-SEEM HPC facility—under project ID AT1R. The research
leading to these results has been co-funded by the European
Commission under the H2020 Research Infrastructures contract
no. 675121 (project VI-SEEM).
This work has been co-financed by the European Union and

EDBM34, ΚΕ 14995) and under the research title “Preparation
and study of innovative forms of administration of pharmaceutical
molecules targeting at improved pharmacological properties.”
References
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review on cyclodextrin-based carriers for deliv- 9. 20192, SR Maestro (2019) Schrödinger. LLC,
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Comput 6(12):3713–3720
Chapter 6
Drug Delivery: Hydrophobic Drug Encapsulation into

Amphiphilic Block Copolymer Micelles
Angeliki Chroni, Varvara Chrysostomou, Athanasios Skandalis,
and Stergios Pispas
Abstract
Drug encapsulation into amphiphilic block copolymer micelles aims to increase drug solubility and mini-
mize drug degradation upon administration, avoid undesirable side effects and ameliorate drug bioavail-
ability. Drug encapsulation methodologies including thin-film hydration method and organic cosolvent
method are described in this chapter. Often, it is desirable to determine the most efficient solubilization
protocol leading to functional drug delivery nanovehicles in each case. The encapsulation of curcumin into
PEO-b-PPO-b-PEO (Pluronic F-127) polymeric micelles through thin-film hydration method presents
the most promising results. Indomethacin can be loaded successfully into the hydrophobic cores of PEO-
b-PCL amphiphilic block copolymer micelles following both encapsulation protocols.
Key words Drug delivery, Block copolymers, Curcumin, Indomethacin, Encapsulation processes,
Thin-film hydration, Organic solvent evaporation
1 Introduction
Polymer therapeutics paved the road for further innovations in the

field of drug delivery systems, by providing chemical stability,
improved drug solubilization, and controlled release of the drug
[1]. Hydrophobic drugs can be physically entrapped in the core of
biodegradable, biocompatible, amphiphilic block copolymer
micelles through encapsulation processes that exploit the self-
assembly properties and solubilization of block copolymers in for-
mulating efficient therapeutic delivery systems [2, 3]. Poly(ethylene
oxide)-b-poly(propylene oxide)-b-poly(ethylene oxide) (PEO-b-
PPO-b-PEO) block copolymer micelles have proved to be effective
nanocarriers for hydrophobic drugs, as they enhance the transport
of drugs across the blood-brain and intestinal barriers [4–7].
Various studies have verified the effective use of poly(ethylene
71
72 Angeliki Chroni et al.
oxide)-b-poly(ε-caprolactone) (PEO-b-PCL) block copolymers as

ideal drug delivery vehicles of hydrophobic drugs [8–10].
Over the last few years, curcumin has attracted a lot of research
interest, as it exhibits low cytotoxicity and potential anticancer
effects as proved by experiments in animals and human cancer cell
lines [11, 12]. It is characterized as an antiproliferative, anti-
inflammatory, antitumor and antioxidant polyphenol-type hydro-
phobic drug [13]. On the other hand, indomethacin is a
nonsteroidal anti-inflammatory and analgesic hydrophobic drug,
commonly used for various therapeutic applications related to
rheumatic disease (arthritis) [14]. Along with its antipyretic prop-
erties, recent studies focused on its potential chemoprotective
effects against tumors [15]. However, most of the clinical applica-
tions of hydrophobic drugs are restricted mainly due to their lack
of solubility in water, poor bioavailability and high prevalence of
their side effects [16].
In this chapter, two different encapsulation protocols, that of
thin-film hydration and the one involving the use of an organic
cosolvent, for encapsulating the above-mentioned hydrophobic
drugs into amphiphilic block copolymer micelles are described in
detail. The chemical structures of copolymers and drugs utilized
are illustrated in Figs. 1 and 2. According to the thin-film protocol
both copolymer and drug are firstly dissolved in a common organic
solvent to appropriate quantities. Rotary evaporator is used for the
efficient and gentle removal of the organic solvent (with minimal
heating) and a solid film composed of hydrophobic drug and
copolymer is created. Then the mixed film is hydrated with the
appropriate aqueous medium resulting in a dispersion of block
copolymer micelles encapsulating the drug. In the cosolvent
Fig. 1 Chemical structure of (a) PEO-b-PPO-b-PEO (Pluronic F-127) triblock terpolymer and (b) curcumin
Fig. 2 Chemical structure of (a) PEO-b-PCL diblock copolymer and (b) indomethacin
Drug Delivery: Hydrophobic Drug Encapsulation into Amphiphilic Block… 73
rotocol copolymer and drug are first dissolved in a common

p
organic solvent to appropriate quantities. The final mixture of
polymer and drug is added all at once to a predefined volume of
filtered Milli-Q water under vigorous stirring and removal of
organic solvent by evaporation follows. Some guidelines regarding
the physicochemical characterization of the micelle/drug formula-
tions using a gamut of techniques are also given.
The incorporation of curcumin into Pluronic F-127 micelles
has proved to be more effective through the thin-film hydration
protocol than through organic cosolvent mixing/evaporation pro-
cess, primarily in terms of achieving high encapsulation efficiencies
[17–19]. Both encapsulation protocols are also used in creating
indomethacin-loaded PEO-b-PCL micelles in order to compare
these protocols and determine the safest and more efficient one for
developing a drug delivery nanovehicle. The difference between
the two protocols utilized, depends on when the organic solvent
evaporation takes place.
2 Materials
Pluronic F-127 (Mw = 12.600 g.mol−1) from Sigma-Aldrich,

poly(ethylene oxide)-b-poly(ε-caprolactone) (PEO-b-PCL with
30% PCL, Mw = 7500 g.mol−1, synthesized in house by ring-
opening polymerization and using PEO from Sigma Aldrich,
M w = 5000 g.mol −1), curcumin (keto form) from Merck,
indomethacin (IND) from Fluka, tetrahydrofuran (THF,
99.9%), acetone (>99.5%), Milli-Q H2O.
Prepare all solutions using Milli-Q H2O (alternatively water for
injection can be used) and analytical grade reagents. Prepare and
store all reagents at room temperature (unless indicated otherwise).
3 Methods
Carry out all procedures at room temperature unless otherwise

specified. In the case of curcumin, experiments for the encapsula-
tion of 50% and 100% w/w drug relative to the hydrophobic core
were attempted, while in the case of indomethacin experiments for
the encapsulation of 20%, 50%, and 100% w/w drug relative to the
hydrophobic core were attempted (see Note 1).
3.1 Solution 1. In a glass vial weigh 10 mg Pluronic F-127 and add 1 mL

of Pluronic F-127 acetone. Wait for the polymer to dissolve (as inspected
with 50% Curcumin visually).
Encapsulated (Organic 2. In a second vial, weigh 1.5 mg curcumin and add 200 μL
Cosolvent Protocol) acetone. Wait for the drug to dissolve (as inspected visually).
3. Mix the Pluronic F-127 solution with the curcumin solution

in one vial.
4. In a pre-weighed vial, add 10 mL filtered Milli-Q water and a
magnetic stirrer, place it over a hot plate, and turn on the
stirring as much as needed in order to see a vortex.
5. Inject fast the mixed Pluronic F-127/curcumin acetone solu-
tion in the stirred water and wait until the solution becomes
clear (approximately 5–10 min).
6. Transfer the solution in a 100 mL round-bottom flask.
7. Place the flask in a rotary evaporator, set the water bath
temperature at 40 °C and the stirring at 600 rpm.
8. Leave the flask in the rotary evaporator until the whole amount
of acetone is evaporated.
9. When acetone is evaporated, remove the flask and put the
solution back in the vial.
10. Weigh the solution in a pre-weighed vial. The volume must be
10 mL in order to achieve a concentration of 1 × 10−3g/mL
(see Note 2). If it is less, it means that part of the water was
also evaporated during the evaporation step.
11. Fill out with filtered Milli-Q H2O till 10 mL final volume
accordingly (see Note 3).
3.2 Solution of 1. In a glass vial weigh 10 mg Pluronic F-127 and add 3 mL

Pluronic F-127 acetone. Wait for the polymer to dissolve (as inspected
with 50% Curcumin visually).
Encapsulated 2. In a second vial, weigh 1.5 mg curcumin and add 200 μL
(Thin-Film Protocol) acetone. Wait for the drug to dissolve (as inspected visually).
3. Mix the Pluronic F-127 solution with the curcumin solution
in a vial.
5. Place the flask in a rotary evaporator, set the water bath tem-
perature at 40 °C and the stirring at 600 rpm.
of acetone is evaporated and a thin film of polymer and cur-
cumin will be formed around the inner part of the flask.
7. When the thin film is formed, wait for 20 more minutes before
removing the flask from the rotary evaporator in order to
make sure that there is no acetone left.
8. Remove the flask and add 10 mL filtered Milli-Q H2O.
9. Gently shake the flask with your hand until the entire thin film
is dissolved.
10. Transfer the solution to a clean vial.
3.3 Solution of PEO- 1. Weigh 10 mg PEO-b-PCL in a vial and add 3 mL THF. Wait
b-PCL with 20% IND for the polymer to dissolve (as inspected visually).
Encapsulated (Organic 2. Weigh 0.6 mg IND in another vial and add 200 μL THF. Wait
Solvent Protocol) for the drug to dissolve (as inspected visually).
3. Mix IND solution with PEO-b-PCL solution.
4. In a pre-weighed vial add 10 mL filtered Milli-Q H2O and a
magnetic stirrer. Put the vial over a hot plate and turn on the
stirring as much as needed in order to see a vortex (see Note 4).
5. Inject fast the PEO-b-PCL/IND mixture in the stirring water
using a syringe and wait until the solution becomes clear
(appx. 5–10 min) (see Note 5).
of THF is evaporated.
9. When THF is evaporated, remove the flask and put the solu-
tion back in the vial.
10. Weigh the solution. The volume must be 10 mL. If it is less, it
means that part of the water was also evaporated during the
evaporation.
11. Fill out with filtered Milli-Q H2O till 10 mL final volume.
3.4 Solutions 1. In a glass vial weigh 10 mg PEO-b-PCL and add 3 mL THF.

of PEO-b-PCL 2. In a second vial, weigh 0.6 mg IND and add 200 μL THF.
with 20% IND
3. Mix the PEO-b-PCL solution with the solution of IND.
Encapsulated
(Thin-Film Protocol) 4. Transfer the solution in a 50 mL round-bottom flask.
of THF is evaporated and a thin film of polymer and IND will
be formed around the inner part of the flask.
7. When the thin film is formed, wait for 20 more minutes before
removing the flask from the evaporator in order to ensure that
there is no THF left.
8. Remove the flask and add 10 mL filtered Milli-Q H2O.
9. Gently shake the flask with your hand until the entire thin film
is dissolved.
10. Transfer the solution to a clean vial.
3.5 UV-Vis UV-Vis spectroscopy is used for the confirmation of the successful
Spectroscopy encapsulation of the drugs into the polymeric micelles. Since it is
known that the copolymers do not absorb in the UV-Vis region, the
wavelength of each peak observed from the drug/copolymer solu-
tions is referred to as the drug characteristic UV-Vis absorption.
UV-Vis measurements were performed using a Perkin Elmer
(Lambda 19) UV-Vis-NIR spectrophotometer (Waltham, MA,
USA). The wavelength of the measurements ranged from 200 to
800 nm. It should be noted that absorption observed in all cases
comes from the encapsulated drugs and not from the copolymers.
1. Select the desired measurement settings.
2. Perform a reference measurement without sample.
3. In a quartz cuvette (H × D × W: 48 mm × 12.5 mm × 12.5 mm,
from Sigma-Aldrich) add 3 mL of the solution and place it in
the instrument.
4. Perform the measurement.
5. We used 200 μL of the initial solution diluted with 2.8 mL
H2O (see Note 6).
6. The Pluronic F-127 solution does not show absorption in the
UV spectrum. On the contrary, the Pluronic F-127/curcumin
solutions show the characteristic peaks of curcumin at approxi-
mately 420 nm. This observation, together with the absence
of any precipitation, proves the successful encapsulation of
curcumin in the polymeric micelles (Fig. 3a).
7. Again, there are no peaks observed for the PEO-b-PCL micellar
solution in the UV-Vis region before the encapsulation of indo-
methacin. On the contrary, after the encapsulation of indo-
methacin with both protocols, the characteristic peak of
indomethacin can be observed at approximately 260 nm, imply-
ing the existence of the hydrophobic drug in polymeric micelles
(Fig. 3b). Solutions containing the micelle-encapsulated drugs
show no hint of precipitation.
3.6 Fourier Attenuated total reflection-Fourier transform infrared (ATR-

Transform Infrared FTIR) spectroscopy is used for the identification of the block
Spectroscopy copolymers/drug chemical structure, as well as for the confirma-
(ATR-FTIR) tion of the successful drug loading into the hydrophobic cores of
the micelles (appearance of new absorption peaks that correspond
to the characteristic functional groups of the drug).
Fourier transform infrared spectroscopy (FTIR) measurements
are performed in order to examine drug encapsulation into the
polymeric micelles core, using a Fourier transform instrument
(Bruker Equinox 55), equipped with a single-bounce attenuated
total reflectance (ATR) diamond accessory (Dura-Samp1IR II by
SensIR Technologies).
Fig. 3 UV-Vis spectra of (a) Pluronic F-127 and Pluronic F-127/curcumin solutions. The characteristic absorp-
tion peak of curcumin at 420 nm indicates its successful encapsulation in the polymeric micelles and (b)
PEO-b-PCL/20% indomethacin prepared by thin-film and organic solvent protocols. The observation of the
characteristic peak of indomethacin at 260 nm confirms the successful drug loading into the hydrophobic core
of the block copolymer
1. Select the preferable measurement options, e.g., spectral range

from 5000 to 500 cm−1, wavenumber resolution of 4 cm−1 and
number of scans 64.
2. Firstly, take a background before sample measurement.
3. Using a micropipette, add 20 μL of polymer solution directly
on the surface of the flat diamond crystal.
4. Using nitrogen gas flow, the polymer solution is purged at
100 ml/min flow rate to evaporate the solvent and the record-
ing of the spectrum starts simultaneously.
5. FTIR-ATR spectra are recorded in real-time mode until
solvent evaporation occurs and a polymeric thin film is formed
on the diamond’s flat surface.
6. Remove the formed thin film from the surface of the flat dia-
mond crystal by cleaning carefully the surface with pure H2O.
7. Perform again a new measurement without the deposition of
sample to clarify that the surface is clean as the obtained ATR-
FTIR spectrum indicates.
8. For the measurements of polymeric micelles with the encapsu-
lated drug, add 20 μL solution directly on the surface of the
flat diamond crystal.
9. Follow steps 4–8 and repeat the procedure with the same
measurement conditions.
10. The spectra obtained by ATR-FTIR spectroscopy certify the
expected chemical structure of the block copolymers and the
successful encapsulation of the drug into polymeric micelles
(see Note 7).
11. The analysis of Pluronic F-127 ATR-FTIR spectrum is accom-

plished as illustrated in Fig. 4a. It contains characteristic bands
of C-H stretching vibrations at 2882 cm−1, C-H bending
vibrations at 1468 cm−1, and one strong characteristic band at
1114 cm−1 due to the C-O-C stretching vibration of the ether
bonds. Spectral fingerprints are the same for all Pluronic F-127
samples prepared with thin-film or organic solvent protocol.
12. In the case of Pluronic F-127 with encapsulated curcumin, the
ATR-FTIR spectrum in Fig. 4a depicts the appearance of new
absorption peaks at 1627 cm−1 due to aromatic moiety C=C
stretching vibrations at 1586 cm−1 corresponding to benzene
ring stretching vibrations and at 1516 cm−1 due to C=O and
C=C stretching vibrations (see Note 8) [20].
13. In the case of PEO-b-PCL polymeric micelles, the ATR-FTIR
spectrum is presented in Fig. 4b. One of the characteristic
absorption peaks of PEO-b-PCL appears at 2885 cm−1 which
corresponds to C-H stretching vibrations. The peak at
1722 cm−1 is attributed to the C=O stretching vibration, while
the peak at 1468 cm−1 indicates the C-H bending vibrations.
Finally, the peak at 1110 cm−1 corresponds to C-O-C stretch-
ing vibrations.
14. The ATR-FTIR spectrum in Fig. 4b shows the PEO-b-
PCL polymeric micelles with encapsulated indomethacin. In
this case, there is a contribution of PEO-b-PCL and
indomethacin in the absorption of characteristic peaks, such as
at 2885 cm−1, at 1722 cm−1, at 1468 cm−1, and at 1110 cm−1.
The appearance of new characteristic peaks is hardly observed.
However, there are new peaks recorded at 1686 cm−1 due to
Fig. 4 ATR-FTIR spectra of (a) Pluronic F-127 and Pluronic F-127/ 50% curcumin, (b) PEO-b-PCL and PEO-b-
PCL/20% indomethacin. All samples were prepared by thin-film protocol. The appearance of new characteristic
absorption peaks confirms the existence of the model drug in the polymer-drug mixed solutions
the amide group of IND, as well as at 1595 cm−1 ascribed to

C-C stretching modes of the aromatic rings. Finally, the peak
at 760 cm−1 may be attributed to C-Cl stretching modes [21].
The spectral fingerprint is the same for all samples prepared
with both protocols. Concluding, the appearance of new peaks
indicates the existence of indomethacin in the formulations.
The peaks are hardly observed due to the low percentage of
the encapsulated drug (20%).
3.7 Dynamic Light Dynamic light scattering (DLS) measurements were conducted
Scattering (DLS) before and after the encapsulation of the drugs into the polymeric
micelles in order to determine possible differences between the
two encapsulation protocols regarding the scattering intensity,
size, and polydispersity index values of the empty/loaded micelles.
Size is one of the most important parameters/characteristics of
nanocarriers used in drug delivery [1, 2].
DLS measurements were conducted on an ALV/CGS-3 com-
pact goniometer system (ALVGmbH), equipped with an ALV
5000/EPP multi-τ digital correlator with 288 channels and an
ALV/LSE-5003 light scattering electronics unit for stepper motor
drive and limit switch control. A JDS Uniphase 22 mW He-Ne
laser (λ = 632.8 nm) was used as the light source. All solutions
were measured five times at each angle and the average was
taken. The solutions were filtered through 0.45 μm hydrophilic
PVDF filters (Millex-LCR from Millipore) before measurements.
The angular range for the measurements was 30–150°. Obtained
correlation functions were analyzed by the cumulant method and
the CONTIN software. The size data and figures shown below are
from measurements at 90° (see Note 9).
1. Adjust a 0.45 μm filter to a plastic syringe and add 4 mL of the
solution (see Note 10).
2. Take a RIA test tube (dimension: diameter 10 mm, height
75 mm) and remove the dust from its interior using rigorous N2
gas flow.
3. In the dust-free test tube, put 1 ml of filtered solution and shake
it in order to remove possible impurities or remaining dust in
the test tube. Repeat this procedure one more time.
4. Add 1 mL of the solution in the test tube and place it in the
instrument for measurement.
5. Apply the above-mentioned settings in the measurement setup.
6. Measure the polymeric solutions both before and after the
encapsulation of the hydrophobic drugs.
7. Size distribution graphs from CONTIN analysis for Pluronic
F-127 solutions prepared with organic solvent protocol show
the existence of a single peak before and after the encapsulation
of curcumin. The size of the micelles is ca. 7 nm before the

encapsulation and ca. 13 nm after the encapsulation of cur-
cumin. However, it seems that in the presence of curcumin the
size distributions become more narrow (Fig. 5a). The micelles
prepared with the thin-film protocol are also monomodal and
with the addition of curcumin the size distribution becomes
more narrow and much more symmetrical. The size increases
from ca. 21 nm before the encapsulation of curcumin to ca.
31 nm after the encapsulation.
As far as size distributions of PEO-b-PCL solutions are concerned,
it seems that there are no significant changes in the size of the
micelles after the encapsulation of indomethacin when the thin-
film protocol is utilized. Yet, the existence of the drug seems to
increase the polydispersity index of the nanoparticles in solution
(Fig. 5b).
Taking into consideration all the results, it seems that each proto-
col used plays an important role regarding the size and polydis-
persity of polymeric nanocarriers obtained (see Note 11).
4 Notes
1. Higher amounts of curcumin and indomethacin encapsulation

(ca. 100%) led to precipitation phenomena, when the drug/
copolymer solutions were added in the aqueous media. These
observations indicate that larger amount of the hydrophobic
drug results in colloidally unstable solutions/formulations.
Fig. 5 Size distribution graphs from CONTIN analysis for (a) Pluronic F-127 and Pluronic F-127/curcumin aque-
ous solutions prepared by the organic solvent protocol and (b) PEO-b-PCL and PEO-b-PCL/indomethacin aque-
ous solutions prepared using the thin-film protocol. In all cases, there are significant changes in the
hydrodynamic radii and size polydispersities before and after the encapsulation. All measurements were per-
formed at pH = 7 and 90°
2. For reasons of comparability, all final solutions prepared by

thin-film hydration and organic solvent evaporation protocols
had the same polymer concentration (1x10−3 g.mL−1).
3. In all cases, Milli-Q water was filtered before each dissolution
to ensure its clearance from dust and bacteria.
4. In the case of organic cosolvent protocol, when placing the
vial of filtered Milli-Q water on the hot plate, the stirring
speed should be enough to develop a vortex. This allows for
efficient and rapid mixing of the solutions resulting in more
stable and finely dispersed nanoparticles.
5. After the injection of drug/copolymer solution into the vial
containing the filtered Milli-Q water, the solution mixture
turned turbid due to the mixing of the solvents used and the
formation of drug-loaded micelles.
6. The absorbance of solutions in the UV-Vis range should be
below or equal to 1.0 in order for the Beer-Lambert law to be
applicable. If not, the solutions must be diluted as much as
needed in order for their absorbance to be within this range.
7. Baselines of all ATR-FTIR spectra were corrected after the
measurement by subtracting the baseline in air.
8. From the ATR-FTIR spectrum of Pluronic F-127 with encap-
sulated curcumin the existence of a peak at 3312 cm−1 corre-
sponding to O-H stretching is observed (Fig. 4a). This
particular peak is attributed to internal moisture of the
sample.
9. The size distributions obtained from DLS are from measure-
ments at 90°, 25 °C, and pH = 7.
10. It is recommended that syringes should not have Luer Lock in
order to minimize the risk of contamination.
11. It must be noted that the solutions prepared via the thin-film
protocol present better colloidal stability since precipitation
was observed in the solutions prepared by the organic solvent
method after several hours or days depending on the copoly-
mer/drug mixture.
Acknowledgments
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200814293
Chapter 7
Multisensitive Polymeric Nanocontainers as Drug Delivery

Systems: Biological Evaluation
Maria Theodosiou, Theodora Koutsikou, and Eleni K. Efthimiadou
Abstract
This chapter focuses on the in vitro biological evaluation of multisensitive nanocontainers as drug delivery
systems for cancer treatment. Cancer tissues possess some unique characteristics such as increased tempera-
ture due to inflammation, thermal vulnerability (40–45 °C), low cellular pH, and redox instabilities. The
employment of polymers bearing pH, thermo, and/or redox sensitivities in the synthesis of hollow poly-
meric nanostructures has led to the formulation of a variety of drug delivery vehicles that are capable of
targeted delivery and trigger specific drug release. The cavity in the structure allows for the encapsulation
of anticancer drugs as well as other moieties with anticancer activity, like iron oxide magnetic nanoparti-
cles. The drug loading and release capability of the nanocontainers is evaluated prior to biological studies
in order to determine the concentration of the drug in the structure. The in vitro assessment includes
cytotoxicity studies, quantitatively through the colorimetric MTT assay as well as qualitatively via the
scratch-wound healing assay, on both cancer and healthy cell lines. The cellular localization of the studied
drug-loaded and unloaded nanocontainers is determined through confocal fluorescence microscopy.
Key words Polymeric nanocontainers, Nanomedicine, MTT assay, Scratch-wound healing assay,
Multisensitive drug delivery systems, Triggered drug release
1 Introduction
Nanomedicine is the field of medicine where organic, inorganic,

and/or hybrid materials at the nanoscale (nanomaterials) can be
used in therapeutic and diagnostic (theranostic) applications for
various diseases [1]. Great focus has been given in the application
of nanomaterials as site-specific and sustainable drug delivery sys-
tems (DDS) [2, 3].
One of the main nanomaterials in DDS research are the stim-
uli-“sensitive” or -“responsive” polymers [4]. Polymers, depend-
ing on their physicochemical characteristics, display structural
changes upon endogenous or exogenous triggers and can be clas-
sified accordingly into three main categories: (i) pH-sensitive poly-
mers incorporating weak acidic or basic ionic functional groups
which respond to endogenous tissue pH variations [5], (ii) ther-
85
86 Maria Theodosiou et al.
mosensitive polymers that exhibit sol-gel transitions in response to

both endogenous and exogenous thermal fluctuations [6], and (iii)
redox-sensitive polymers commonly containing labile disulfide
linkages that respond to endogenous redox alterations [7]. Other
emerging types of responsive polymers for targeted drug delivery
have also been reported and can be separated based on the origin
of stimuli. For internally triggered DDS, biologically, enzyme- and
inflammation-responsive polymers have been developed while
externally triggered DDS include polymers that are photo, ultra-
sound, magnetically, or electrically sensitive [8]. Recent trends in
polymer DDS account for the combination of two or more of the
aforementioned characteristics in one polymeric structure which
can result in a dual or multisensitive polymer, thus leading to a
multifunctional material that can act in a more holistic approach
for treatment of the targeted disease [9]. The morphology of these
kinds of polymers at the nanoscale can result in various moieties
like polymersomes, nanocontainers, hydrogels, polymeric micelles,
nanospheres, and dendritic nanocarriers [10].
The use of stimuli-responsive polymers as DDS is based on
their ability to interact with a drug, bind covalently or electrostati-
cally, and release it upon specific trigger, which depends on the
type of the targeted disease [11]. For example, in cancer theranos-
tics the rationale behind the design of a responsive polymeric
nanomaterial is based on the physiology of cancer tissues, acidic
environment, thermal liability (40–45 °C), redox instabilities,
specificity of growth factors, and rise of the enhanced permeation
and retention (EPR) effect [12]. Bearing this into consideration by
loading an anticancer drug in a single- or multisensitive polymeric
nanocarrier which is further functionalized with targeting agents
for cancer cells, prolonged circulation of the drug, selective recog-
nition, and targeted release of the drug can be achieved. In this
way chemotherapy becomes more efficient and less toxic for the
patient [13]. Toniolo et al. synthesized a triple-sensitive (pH,
thermo, redox) polymeric nanocontainer, loaded with the model
anticancer drug daunorubicin hydrochloride. These nanocarriers
exhibited a drug-loading capacity and an encapsulation efficiency
of 85% and 68% approximately whereas the drug release profile was
significantly enhanced at acidic pH, increased temperature, and
presence of glutathione [14].
Inorganic nanomaterials can also be incorporated in the struc-
ture of polymeric nanocarriers. For the purpose of this study, iron
oxide nanoparticles are presented. Iron oxide nanoparticles are
superparamagnetic (mNPs) and thus under the application of an
alternating magnetic field they dissipate thermal energy due to
Néel and Brown relaxations. One of the most studied applications
of mNPs in cancer treatment is through magnetic hyperthermia
treatment [15]. Polymeric nanocontainers containing iron oxide
nanoparticles (mNCs) can be used in magnetic hyperthermia and
cause a synergistic effect of temperature increase in the tumor area
Multisensitive Polymeric Nanocontainers as Drug Delivery Systems: Biological Evaluation 87
that can inherently lead to regression as well as promote the release

of anticancer drugs loaded in the mNCs. Efthimiadou et al. fabri-
cated pH-responsive hollow microspheres by using emulsion
polymerization in a two-step procedure and further modified their
surface with iron oxide superparamagnetic nanoparticles (mNPs).
The mNPs added an extra functionality to the microspheres that
could be used as a bifunctional DDS for controlled drug release in
combination with magnetic hyperthermia. The inner cavity formed
during shell fabrication aims in the accommodation of guest mol-
ecules like the drug doxorubicin hydrochloride and iron oxide
nanoparticles. Microspheres’ drug-release behavior was evaluated
under different pH conditions as well as under magnetic hyper-
thermia, which induced controlled release [16–18].
Herein we present the basic strategies of testing the drug load-
ing and release behavior of a polymeric nanostructure as well as the
in vitro biological evaluation protocols followed to assess their
impact on cancer theranostics.
2 Materials
2.1 Evaluation All solutions must be prepared in double-distilled water at room

of Drug Loading temperature and stored as indicated.
and Release
1. Phosphate-buffered solution (PBS) 0.1 M, pH 7.4: Mix
2.1.1 Buffer Solutions
40.5 mL of solution A, 0.2 M sodium phosphate and dibasic
[19–21]
dihydrate (Na2HPO4•2H2O), with 9.5 mL of solution B,
sodium phosphate, monobasic, and monohydrate
(NaH2PO4•H2O), and dilute to 100 mL. Store at 4 °C.
2. Citrate buffer solution (CBS) 0.1 M, pH 4.6: Mix 44.5 mL of
solution A, 0.1 M citric acid monohydrate (C6H8O7•H2O),
with 55.5 mL of solution B, 0.1 M trisodium citrate and dihy-
drate (C6H5O7Na3•2H2O). Store at 4 °C.
2.1.2 Drug-Loading Suspend the desired amount of nanocontainers in PBS with the
Suspensions anthracycline model drug in a ratio of 1:1 (w/w) under gentle
agitation for 24–72 h depending on the interaction between the
selected drug and the nanocontainers. Detailed procedure is found
in Subheading 3.1.1.
2.1.3 Drug-Release Drug-loaded nanocontainers are injected in semipermeable cellu-

Suspensions lose membrane dialysis bag with a cutoff molecular weight that
allows the drug to be diffused.
2.2 Biological The growth medium is high-glucose Dulbecco’s modified Eagle

Evaluation medium (DMEM), supplemented with 10% heat-inactivated fetal
bovine serum (FBS), 2 mM l-glutamine, and antibiotics
2.2.1 Culture Media
(100 units/mL penicillin and 100 mg/mL streptomycin).
2.2.2 MTT Solution Dissolve 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium

Preparation bromide (MTT) in PBS at a concentration of 1 mg/mL by using
vortexing or sonication and filtration. Store the solution at −20 °C
for 4 months and at −4 °C for few days. MTT is photosensitive so
use foil to protect it.
2.2.3 Other Reagents 1. Dimethyl sulfoxide (DMSO).

and Materials
2. Dulbecco’s phosphate-buffered saline (sterile PBS, 10 mM).
3. Sonicator.
4. Cellulose membrane dialysis bag of specific molecular weight
cutoff.
5. Magnetic hyperthermia apparatus.
6. EDTA-trypsin.
7. L-Poly lysine.
8. p-Formaldehyde.
9. Antiphotobleacher: b-Mercaptoethanol.
10. 96-Well plates, 6-well plates.
11. UV-vis microplate reader.
12. UV-vis spectrophotometer.
13. Glass coverslips.
14. Hematocytometer.
3 Methods
3.1 Drug Loading Tumoral tissues exhibit pH, thermal, and redox instabilities which
and Release are taken into consideration when designing a polymeric nanocon-
tainer (NCs) as a DDS. In more detail, cancer cells proliferate in a
more acidic environment; they are very sensitive to temperatures
between 40 and 45 °C and they tend to thrive in a hypoxic environ-
ment. These three basic characteristics can be exploited by fabricat-
ing a model triple-sensitive polymeric nanocontainer. For example,
as a temperature-sensitive polymer PNIPAm is commonly used and
has a lower critical solution temperature (LCST) of 32 °C where it
shows a reversible volume-phase transition. Another commonly
used polymer is methacrylic acid (MAA) which is pH sensitive and
under acidic environment it is fully protonated, facilitating the
release of an electrostatically bound drug and cellular uptake
through adsorptive endocytosis. Finally, polymers containing gluta-
thione labile bonds like N,N′-(disulfanediylbis(ethane-2,1-diyl))
bis(2-methylacrylamide) (DSBMA) are employed in order to allow
site-specific cleavage in the reductive tumoral environment.
Nanocontainers synthesized with these kind of polymers can serve
as a model platform for intracellular release of anticancer drugs and
of enhanced therapeutic efficacy due to their stimulus-sensitive

nature. Above their volume-phase transition temperatures they
shrink; in acidic pH they are protonated and at high intracellular
glutathione (GSH) levels they suffer from disulfide bond collapse.
Finally, multisensitive polymeric nanocontainers modified with iron
oxide nanoparticles (mNCs) possess an extra sensitivity related to
heat in the concept of inducing cancer cell apoptosis and enhance-
ment of drug release under the application of magnetic hyperther-
mia. Combination of the aforementioned materials promotes drug
release and in general targeted intracellular drug delivery.
3.1.1 Drug Loading 1. Create a standard curve at pH 7.4 based on the concentration-
in Nanocontainers dependent UV-vis absorption peak of the employed model
drug (e.g., doxorubicin hydrochloride is at 480 nm) (see
Note 1).
2. In a container of known mass, disperse 5 mg of hollow NCs or
mNCs in 5 mL of phosphate-buffered solution at pH 7.4
(PBS) (see Note 2).
3. Add 5 mg of drug and sonicate for 5 min at 25 °C.
4. Stir for 24–72 h under gentle agitation at 25 °C.
5. Centrifuge the mixture in order to remove the unloaded drug.
Resuspend the material by vortexing and centrifuge at
6080 × g until supernatant is clear or measured absorption is
close to zero.
6. Measure the absorption of centrifugations supernatants con-
taining the unloaded drug.
7. Freeze-dry, weight, and store the precipitate at 25 °C for a few
days until the release study.
8. Determine the amount of loaded drug via standard curve
methodology.
9. Calculate the loading content using the equations below:
Weight of the drug in NCs
Loading capacity % 100
Total Weight of the NCs
Weight of the drug in NCs

Encapsulation efficiency % 100
Weight of the feeding drug
3.1.2 Drug Release Generally in vitro release studies are performed at 37 °C (physio-
from Nanocontainers logical temperature), though in some cases testing is performed at
elevated temperatures for exploring and characterizing drug release
using a variety of dosage forms. The most renowned and versatile
method of assessing drug release from nano-sized dosage forms is
the dialysis method. Using a dialysis membrane, which is perme-
able by the desired drug, physical separation will take place through
diffusion. The protocol for this is as follows:
1. Create a standard curve at pH 7.4 and 4.6 based on the

concentration-
dependent UV-vis absorption peak of the
employed model drug (e.g., doxorubicin hydrochloride is at
480 nm).
2. Redisperse × mg of loaded NCs in water.
3. Introduce the dispersion into the dialysis bag and seal it.
4. Place the dialysis bag in a vessel containing the desired buffer
solution (pH 7.4 and/or 4.6) at different temperatures.
5. Remove aliquots of 0.5 mL at different time points and add
0.5 mL to maintain the total volume.
6. Measure characteristic absorption wavelength of released drug
at each aliquot removed.
7. Quantify the release percentage by the standard curve equa-
tion created in step 1.
8. Investigate the mechanism of release according to Korsmeyer–
Peppas equation:
R % t kt n

R%(t) is the percent of drug released at time t, while k and n are

the kinetics constant and the release exponent, respectively.
3.2 Hyperthermia In order to assess the optimum frequency related to the appropri-
Measurements ate concentration for in vitro and in vivo experiments, first the
mNCs are tested in aqueous solutions ex vitro.
1. Create 1 mL dispersions of consecutive concentrations of
mNCs in ddH2O in glass vials of the same dimensions.
2. Insert glass vials in the coil of the magnetic hyperthermia
apparatus (see Note 3).
3. Apply alternating magnetic field of various frequencies for
30 min.
4. Record temperature fluctuations.
The optimum sample and frequency to be used for further bio-
logical evaluation should have a thermal response plateau between
40 and 45 °C at the shortest time interval.
3.3 Cytotoxicity Comparative studies should be performed where the tested sample
and Biocompatibility should include NCs and/or mNCs, drug-loaded NCs, and/or
Evaluation drug-loaded mNCs and pure drug. The concentrations at which all
the samples will be evaluated are tested in a range around the IC50
of the pure drug used for drug loading in order to assess the nov-
elty of the DDS compared to the free drug.
3.3.1 Cell Culture In order to perform an in vitro biological evaluation proper human
cell lines should be cultured according to American Tissue Type
Collection (ATTC) guidelines. Cells are commonly stored in liq-

uid nitrogen (−195.8 °C) in special vials and the thawing process
is as follows:
1. Prepare water bath at 37 °C.
2. Submerge the vials for 2–3 min and gently agitate.
3. Disperse them in growth medium.
4. Transfer to cell culture flask.
5. Incubate until it becomes confluent.
The incubation environmental conditions should be at 37 °C
in a humidified atmosphere with 5% CO2 supply.
The cryo-storage medium is FBS and it is common to be
enriched by DMSO which is used in order to avoid the formation
of ice crystals during freezing that would be detrimental for the
cells. In this case the growth medium where cells were incubated
should be removed as soon as they start to grow in order to avoid
contamination from DMSO.
When cell cultures reach confluency on the first passage the
cells can be detached and used for the biological evaluations dis-
cussed below. The detachment protocol involves the following
steps:
1. Remove supernatant growth medium.
2. Gentle rinse with PBS of a volume enough to cover the flask.
3. Remove PBS.
4. Add trypsin of known volume just enough to cover the flask.
5. Incubate for a time depending on the specification of each cell
line to achieve detachment.
Caution! Do not exceed the time limit as trypsin might cause
cell damage.
6. Agitate the flask gently.
7. Add triple volume of growth medium based on the added
trypsin volume and cell suspension.
8. Count alive cells with hematocytometer according to pub-
lished protocol (see Note 4).
9. Create dilution to reach the desired cellular population for the
applied assay.
For most assays a confluency of 70 or 80% is required after a
24-h incubation.
10. Seed the cell-containing growth medium of known popula-
tion in the appropriate plate for the experiment.
3.3.2 MTT Cell ΜΤΤ assay is a colorimetric methodology which is based on the
Proliferation Assay reduction of yellow tetrazolium to violet formazan crystals for
assessing quantitative cell viability when the cells are incubated
with materials, drugs, small molecules, nanoparticles, proteins, and
metal complexes. The assay is based on enzymatic reduction of

3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide
(MTT) to MTT-formazan which is catalyzed by mitochondrial
succinate dehydrogenase. MTT assay is dependent on mitochon-
drial respiration and indirectly assesses the cellular energy capacity
of a cell [22]. Cytotoxicity of the NCs can be evaluated by the
MTT assay on different human cell lines.
In order to perform the MTT assay (Fig. 1) the protocol is as
follows:
Following step 8 of the cell culture procedure (Subheading
3.3.1) described previously,
1. Seed 100 μL of 8 × 103 cells in each well in 96-well plate, flat-
bottomed microplates.
2. Incubate the cells 24 h before the experiment.
3. Disperse the NCs/mNCs, free drug, and loaded NCs/mNCs
in ddH2O or growth medium as stock solution.
4. Create serial dilutions of comparable concentration for each
sample in growth medium of at least six different
concentrations.
5. After 24 h (70% confluency), remove supernatant growth
medium.
6. Remove cellular debris by adding and quickly but gently
removing 100 μL PBS.
7. Add 100 μL of each concentration in row triplicate wells as
well as 3 columns of control wells in each plate containing
pure growth medium.
8. Incubate the cells for different time intervals (e.g., 4, 24, 48,
and 72 h).
9. Remove the supernatant and wash twice with PBS.
10. Replace with 100 μL MTT solution.
11. Incubate for 4 h, or until the formazan crystals have formed.
Fig. 1 Schematic representation of MTT assay procedure

12. Remove MTT solution.

13. Solubilize the formazan crystals by the addition of 100 μL
DMSO or isopropanol.
14. Record the absorbance using a UV-vis microplate reader at a
specific wavelength (see Note 5).
15. Average control wells’ absorption readings and calculate stan-
dard deviation.
16. Average the treated triplicate wells’ absorption readings and
standard deviation (SD).
17. Subtract control average from each treated average absorption
value.
18. Convert to percentile values that express cellular proliferation,
aka cell viability (see Note 6).
19. Plot the % viability (y-axis) versus the tested sample concentra-
tions (x-axis) in a column graph expressing the SD with error
bars.
3.3.3 In Vitro Cytotoxicity The induced cytotoxicity when cells are treated with mNCs drug
Studies Under loaded or unloaded can be assessed by performing the MTT assay
Hyperthermia after the application of magnetic hyperthermia in treated cell sam-
ples. According to our protocol the steps are briefly described
below:
1. Treat the appropriate number of cells with mnps or magnetic
NCs in desired concentrations.
2. Incubate the cells for 24 h (see Subheading 3.2, step 2).
3. Wash with PBS twice to remove the non-internalized
material.
4. Add the appropriate amount of trypsin to suspend the cells.
5. Suspend cells to growth medium in the appropriate concentra-
tion in sterile tube.
6. Treat the solution with alternating magnetic field for 30 min
in agreement to hyperthermia measurements ex vitro.
7. Seed the treated cells of different concentrations of drug-
loaded and -unloaded mNCs or mNPs in a 96-well plate.
8. Apply MTT assay (see Subheading 3.2, step 2).
9. In this way induced cytotoxicity from the treated samples can
be indirectly calculated from the % viability.
3.4 Scratch-Wound The scratch-wound healing assay [23] is performed usually in

Healing 6-,12-, or 24-well plates with plastic or glass slides in each well,
where the cells are seeded in the appropriate growth medium
(Fig. 2). The same number of cells should be seeded (3 × 10^5
cell/mL) in each well in order to have comparable cell culture
monolayers. As soon as confluency is reached (approx. 24 h) the
assay can begin:
Fig. 2 Schematic illustration of the scratch by a tip and compound treatment for the scratch-wound healing
assay
1. Remove supernatant from each well.

2. Add a small portion of PBS to remove cellular debris, then
gently shake the plate by hand, and remove it with a pipette.
When removing medium or PBS with a pipette do not touch
the formed cellular monolayer to avoid any unwanted scratches
that will impede the assay.
3. Create the scratch.
There are two methods that can be followed:
One is by hand with a sterilized tip of a pipette which is the
cost-effective way. Apply medium pressure as overpressing
the tip might harm the slide surface. Try to create consis-
tent scratches with gaps of the same width in all wells.
The other is by adding an insert before the cell seeding and
removing it after cells have reached confluency. Different
shapes and sizes of inserts are commercially available like
linear or circular. Inserts guarantee reproducibility of the
wound.
4. Wash again with PBS to remove the extracted cells.
5. Add growth medium with and without the tested substances.
Always use one well in each plate as control (plain growth
medium). The other wells should be treated with growth
medium containing the final product and separately all the
added moieties like drugs, targeting agents, and
nanoparticles.
6. Observe and acquire image under microscope at time point
zero (t = 0 s/min/h/days). This should be the 0% gap width
coverage or 100% gap width.
7. Continue the observations and image acquisitions at the
desired time intervals, e.g., at 24, 48, and 72 h, depending on
the tested substances.
8. You can conduct image analysis through different software
where the gap width at each time interval can be calculated as
the cells migrate or proliferate to close the gap.
9. You can perform quantitative analysis to calculate the percent-

age of wound closure by the equation [24]
Wound closure% At 0h At xh / At 0h 100,
where A is the wound area at different time points t = 0 h and xh.
3.5 Imaging via Confocal fluorescent microscopy can be employed in order to

Fluorescent Confocal identify the localization of the tested samples and interpret their
Microscopy interaction with the cells. The standard protocol used for this is
illustrated in Fig. 3. In more detail:
1. Cultivate the cells in a 6-well plate containing coverslips with
poly-l-lysine to improve adhesion.
2. Incubate for 24 h.
3. Remove growth medium.
4. Wash with PBS.
5. Add growth medium containing the sample.
6. Incubate for a desired amount of time.
7. Remove the growth medium.
8. Wash with PBS.
9. Add 4% p-formaldehyde for 8 min to fixate the cells on the
coverslips.
10. Wash them again with PBS.
11. If necessary, add appropriate a fluorescent dye to do co-
localization (DAPI blue fluorescent molecule that stains the
nucleus).
If the tested samples are autofluorescent it might not be needed
to add another dye.
12. Mount the cells by adding an anti-bleaching compound.
If the tested samples are autofluorescent and need to be tested
for the efficacy of their fluorescence this step can be omitted.
13. Observe under confocal fluorescent microscope.
Fig. 3 Preparation of samples for confocal fluorescent microscopy experiment

4 Notes
1. Standard curve preparation: Measure the absorbance of the

desired drug in different concentrations at its characteristic
concentration-dependent absorption wavelength. Then plot
the absorbance values and create a linear equation. This equa-
tion will be used to determine the concentration of the loaded
and released amount of drug during the treatment. Standard
curve should be prepared for all tested pH values.
2. The vials commonly used in loading and release studies are
polypropylene Eppendorf or Falcon tubes depending on the
sample volume being used.
3. There are many different types of magnetic hyperthermia
apparatus commercially available. In general, they consist of a
coil—or a set of coils of different dimensions—connected to a
monitor that allows changing the value of the applied mag-
netic field and/or the frequency. The sample is placed in the
center of the coil to ensure homogeneous magnetization of
the sample. The temperature is monitored and recorded
through various apparatus like electronic thermometers, fiber-
optic temperature detectors, and thermal cameras.
4. In order to determine the number of alive cells in the suspen-
sion and be able to seed the appropriate population required
to perform any of the biological assays, alive cells should be
counted with a hematocytometer after treatment with trypan
blue. It is a standard procedure which has already been pub-
lished by Springer Protocols [25].
5. The absorption measurement and recording of the MTT plate
can be in the range of 500–600 nm depending on the solvent
and reference filter should be over 600 nm. Control wells
should give values close to zero (± 0.1).
6. Treatment with biocompatible NCs shows higher absorption
values indicating increased viability whereas drug-loaded NCs
show lower absorption values indicating decreased viability.
Acknowledgments
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Chapter 8
Drug Incorporation in the Drug Delivery System of Micelles

Evangelia Soumelidou, Simona Golič Grdadolnik,
Abstract
Micelles is a system frequently used for drug delivery. Drugs are incorporated and protected in micelles
before being delivered. Nuclear magnetic resonance is a suitable technique to detect the localization and
incorporation of drugs into the micelle system. Free radicals are used to further facilitate the probing of
the interactions between drug and micelles. This information is critical because drug-micelle interactions
determine how easily the drug will be released from micelles and therefore how easily will be delivered to
the target.
Key words Micelles, Captopril, NMR, 5-DSA spin label, Sodium dodecyl sulfate (SDS)
1 Introduction
It is well known that conventional drugs possess many shortcomings

in clinical applications such as inactivity in drug delivery, toxic
metabolization, and high hydrophobicity. Nanopharmaceutics has
been developed for optimizing the drug delivery to the target and
addressing the above mentioned shortcomings. Various vehicles are
well known to be developed to transfer the drugs selectively and
control their release. Micelles are among the loading systems used
especially for delivering drugs and controlling their release. In this
matrix-loading system, the drug is embedded in the nanocarrier
micelle and its release is determined by the degradation of the carrier
or diffusion [1]. In our study micelles are composed by the surfac-
tant sodium dodecyl sulfate (SDS) which is an amphiphilic molecule
containing hydrophobic segment [CD3(CD2)11] and hydrophilic
SO3−Na+. When its concentration exceeds the critical micelle con-
centration (CMC) it agglomerates in D2O because its solubility
becomes very low. The hydrophobic tails of SDS squeeze together
in the core in an attempt to minimize their contacts with water. The
hydrophilic heads from the other aspect are exposed to water in
order to decrease the system energy and provide stable forms [2].
99
100 Evangelia Soumelidou et al.
Captopril (1-(2S)-3-mercapto-2-methyl-propionyl]-1-proline)
is an angiotensin-converting enzyme (ACE) inhibitor that has
been extensively used for the treatment of hypertension and con-
gestion heart failure. According to the drug bank https://www.
drugbank.ca/drugs/DB01197 it is water soluble (4.52 mg/mL)
with logP to range between 0.34 and 1.02 based on the sources
and becomes unstable as the pH becomes greater than 1.2. This
fact decreases the therapeutic effect of captopril [3].
For this reason there have been important advances in the area
of pharmananotechnology and the controlled release of drugs,
destined to circumvent many limitations of conventional therapies
for the treatment of diseases such as hyperlipidemia, hypertension,
myocardial infarction, stroke, and thrombosis [4]. Captopril,
according to Biopharmaceutical Classification System (BCS), is a
class II drug, with high solubility but poor permeability. Thus, it is
bioconjugated with a light subunit of Agaricus bisporus mushroom
tyrosinase, a drug carrier, and for oral delivery [5]. Biodegradable
hydrogels for its controlled delivery are used [6]. Optimization of
self-nanoemulsifying orodispersible films (SNEODF) of captopril
for hypertension was studied [7]. Captopril was coated with mag-
netic nanoparticles (MNPs) as a new dual-mode agent for simulta-
neous MRI contrast and drug delivery system [8]. Gastro-retentive
captopril-loaded alginate beads were prepared by an ionotropic
gelation method using sodium alginate in combination with natu-
ral gums containing galactomannans (Senna tora, seed gum, guar
gum, and locust bean gum) in the presence of calcium chloride.
The objective of this work is to develop successful formulation of
gastro-retentive mucoadhesive alginate beads of captopril with
galactomannan [9]. Captopril-polyethyleneimine (CP) containing
low-molecular-weight polyethyleneimine and antiangiogenesis
drug captopril conjugated via an amide bond was fabricated to
modify gold nanoparticles and complex with siRNA to construct
siRNA/CP/GNP complexes for the co-delivery of drug and
siRNA in antiangiogenesis breast cancer therapy [10]. Captopril
was engulfed in a cyclodextrin-based nanosponge for studying as a
potential delivery system [11]. Due to its narrow absorption win-
dow, captopril has to be administered to the upper parts of the
intestine in order to maintain sustained therapeutic levels. Thus, it
was examined if this could be achieved by gastro-retentive dosage
form (GRDF) which consists of a drug-loaded bilayer polymeric
film, folded into a hard gelatin capsule [12]. Systematic studies
were achieved with captopril-loaded polyester fiber mats [13] and
poly(L-lactic acid/captopril) composite monofiber membranes
prepared by electrospinning and in order to increase its delivery
[14]. The intercalation of captopril (CP) into the interlayers of
montmorillonite (MMT) affords an intestine-selective drug
delivery system [15]. Methocel and Eudragit RS were used in
captopril-
loaded microspheres as release-controlling factors to
Drug Incorporation in Micelles 101
evaluate its release kinetic models [16]. Silica-captopril formula-

tions were tested and found to be attractive controlled-release sys-
tems, potentially offering better bioavailability and other benefits,
compared to the commercial formulation [17, 18].
It is noted by Wang et al. [1] that there is a lack of a compre-
hensive understanding of the compositions or structural character-
istics of nanocarriers and this leads to limited designed ideas and
selectivity and makes it difficult to integrate advantages and achieve
optimal design. This notion triggered our interest to study the
interactions of captopril in SDS micelles using high-resolution 1H
NMR spectroscopy and to provide some evidence on its molecular
interactions. High-resolution NMR spectroscopy is the most suit-
able and powerful technique to prove at molecular level the inter-
actions of nanocarriers with drugs. 5-DOXYL-stearic acid (5-DSA)
free radical was used to further facilitate the probing of the interac-
tions between the quest molecule captopril and SDS micelles
(Fig. 1). 5-DSA spin label is a nitroxide radical localized near the
head of the stearate and induces relaxation of the NMR signals in
the proximity of the micelle surface. It was added to the NMR
solutions in a ratio of 1:4 with respect to the captopril [19].
In specific, the aim of this chapter is to provide experimental
details and notions on the procedure used, as well as to explain the
useful information that can be derived.
2 Materials
1. Deuterated solvents SDS-d25 (Fig. 1), D2O, and CD3OD are

obtained pure 99%+.
2. Vials for the mixing of the drug with CHCl3 were of appropri-
ate diameter and height to accept ca 4 mL solvent.
3. NMR tubes must be appropriate for the magnetic field used.
The experiments are performed in 600 MHz Varian Innova
instrument but of course can be run in any higher or lower
magnetic field NMR spectrometer.
4. Use 5-DSA as a probe in SDS micelles.
5. Mix 125.41 mg of dry SDS-d25 lipid with 2.20 mg captopril.
Fig. 1 Structures of captopril, SDS-d25, and 5-DOXYL-stearic acid

3 Methods
1. Mix 2.5 mg of SDS in 1 mL CD3OD/D2O and sonicate it for

15 min (see Note 1) in order to form micelles.
2. SDS concentration (400 mM) must exceed the critical micelle
concentration of 8.2 mM in order to ensure micelle formation
and the applied molar ratio of 5 mM captopril/400 mM SDS-
D25 (1:80) must be used to establish the immersion of at least
one drug molecule in the micellar aggregate, assuming an SDS
aggregation number of at least 60 monomers. The sample is
then transported to a 600 μL NMR tube.
3. Use increasing quantities of 5-DSA in the sample in order to
examine its concentration-dependent effects. Every addition
contains 5 μL and after four additions, the molar ratio of drug
to the spin label is 1:1 (see Note 2).
4. NMR data were collected on a Varian INOVA 600 MHz spec-
trometer (Slovenian NMR Centre at National Institute of
Chemistry) using pulse sequences and phase cycling routines
provided in spectrometer library of pulse programs (see Note
3). The 1H spectral width was set to 5679.8 Hz according to
the range of 1H resonances of captopril.
5. The ROESY experiment was recorded with 4096 data points in
t2, 238 complex points in t1, 32 scans, a relaxation delay of
1.5 s, and a 4 kHz spin-locking field strength. A mixing time of
150 ms was used according to our previous studies in SDS
micelle environment [19–22] (see Note 4).
6. Experiments were run right after the preparation and at stable
temperature (see Notes 5 and 6).
4 Results and Discussion
In Fig. 2 is shown the 1H NMR spectrum of captopril in SDS-d25/

CD3OD/D2O.
One approach for structure identification of captopril-observed
resonance peaks is of course to search if similar or identical experi-
ment is performed in the literature. This will save time and accelerate
the search in understanding the effects of captopril in SDS-d25. 1H
NMR results for captopril in D2O are reported by Casy and Dewar
[19] and are described in Table 1. Based on the D2O-reported
results the 1H NMR chemical shifts obtained with D2O/CΗ3ΟD/
SDS-d25 sample are easily elucidated and are shown in Table 1.
Fig. 2 1H NMR spectrum of captopril in SDS-d25/CD3OD/D2O obtained at 600 MHz and 25 °C
Table 1
1
H NMR chemical shifts of captopril in D2O/CΗ3ΟD/SDS-d25 obtained at 600 MHz and 25 °C
Chemical shift δ(ppm) Chemical shift δ(ppm) in Number of

Protons in D2O [REF] D2O/CΗ3ΟD/SDS-d25 protons Multiplicity
2′-Me M 1.14 1.21 3 d (J = 6.7 Hz)a
m 1.10 1.19 d(J = 6.82 Hz)b
3–4 1.95–2.05 and 1.90–2.13 and 2.25–2.45 4 m
2.25–2.35
2′ 3.01–3.08 3.01–3.08 1 m
3′ 2.45–2.7 2.58–2.80 2 m
5 M 3.62–3.80 M 3.74–3.88 2 m
m 3.62–3.38 m 3.51–3.68
2 M 4.42 M 4.51 dd (J = 8.7, 4.1)a
m 4.61 m 4.68 dd = (J = 2.18, 2.15)b
Refer to the sample captopril in D2O
a
Refer to the sample captopril in D2O/CΗ3ΟD/SDS-d25

b
Important features of the spectrum are the appearance of a

major (M) and minor (m) conformation according to the equilib-
rium shown below (Fig. 3). The 2D ROESY experiment is shown
in Fig. 4 and a model of the captopril which can explain all the
Fig. 3 Captopril exists in two forms. The major and minor. These forms are clearly
depicted in the 1H NMR spectrum both in D2O and in D2O/CΗ3ΟD/SDS-d25. The
protons adjacent to amide bond due to the different surrounding environment
clearly show different chemical shifts
Fig. 4 Spatial correlations of captopril in D2O/CΗ3ΟD/SDS-d25 performed at 600 MHz and 37° C
Fig. 5 This model satisfies the two critical ROEs 2′-5 and 2–5. The average dis-
tances of these two critical ROEs are 2.49 Å and 3.82 Å
ROE data is shown in Fig. 5. The most critical ROE observed is

between the protons 5 and 2′ which defines the relative orientation
of the ring with respect to the chain of the major conformer.
The gradual addition of 5-DSA in the D2O/CΗ3ΟD/SDS-d25
sample is shown in Fig. 6. There are two major characteristics of
the spectra obtained after the four subsequent additions of DSA.
(a) The chemical shifts due to the captopril are not affected and (b)
all the peaks decrease in intensity and are broadened which results
in losing the multiplicity information. For example, after the sec-
ond addition of DSA, the peaks resonated at 1.21 ppm and
1.19 ppm merge only to the one at 1.21 ppm. Thus, the informa-
tion for the minor conformer depicted at 1.19 ppm is lost. Clearly,
the results show that 5-DSA affects significantly all peaks of capto-
pril. This means that DSA which is localized in intermediate polar-
ity region (interface) of SDS is in spatial vicinity with captopril.
Thus, captopril is localized in the interface of SDS. In Fig. 7, a
cartoon is presented in which are shown the interactions of capto-
pril with SDS-d25 micelles.
5 Notes
1. During the sonication heat is generated, so the sample should

be in an ice bath; otherwise there is a danger of breaking the
glass vial.
2. Spectral precaution of the sonicator tip is given in order not to
touch the bottom or sides of the vial.
Fig. 6 Gradual addition of DSA in D2O/CΗ3ΟD/SDS-d25 sample. (a) No addition. (b) Addition of 5 μL DSA (1:1 DSA/
captopril molar ratio). (c) Addition of another 5 μL DSA (2:1 DSA/captopril molar ratio). (c) Addition of another 5 μL
DSA (3:1 DSA/captopril molar ratio). (c) Addition of another 5 μL DSA (4:1 DSA/captopril molar ratio)
Fig. 7 The picture depicts the interactions of captopril with SDS-d25 micelles. The carboxylate group of capto-
pril orients to the positively charged sodium while the rest of the molecule is localized in the intermediate polar
and hydrophobic region (interface) in order to maximize its interactions. This scheme explains the reason that
captopril is affected significantly by the spin label 5-DOXYL-stearic acid as it is in its spatial vicinity
3. All companies that sell spectrometers generally have a library of

pulse programs and stored default parameter sets for common
1D and 2D NMR experiments.
4. There is no special precaution for the handling of free radicals,
which are spin labels and used for investigating structural
changes in lipids.
5. Although micelles are relatively stable, the noise of the spec-
trum is deteriorated if sample is not fresh.
6. It is essential the NMR spectrometer room temperature be sta-
ble during the experiment as the quality of the spectrum is
affected by temperature instability.
Acknowledgments
targeting at improved pharmacological properties.” SGG acknowl-
edges support from the Slovenian Research Agency (Grant No.
J1-8145).
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Chapter 9
Molecular Dynamics Protocols for the Study

of Cyclodextrin Drug Delivery Systems
Georgios Leonis, Dimitrios Ntountaniotis, Eirini Christodoulou,
Abstract
Hypertension treatment is a current therapeutic priority as there is a constantly increasing part of the
population that suffers from this risk factor, which may lead to cardiovascular and encephalic episodes and
eventually to death. A number of marketed medicines consist of active ingredients that may be relatively
potent; however, there is plenty of room to enhance their pharmacological profile and therapeutic index
by improving specific physicochemical properties. In this work, we focus on a class of blood pressure regu-
lators, called sartans, and we present the computational scheme for the pharmacological improvement of
irbesartan (IRB) as a representative example. IRB has been shown to exert increased pharmacological
action compared with other sartans, but it appears to be highly lipophilic and violates Lipinski rule (MLogP
>4.15). To circumvent this drawback, proper hydrophilic molecules, such as cyclodextrins, can be used as
drug carriers. This chapter describes the combinatory use of computational methods, namely molecular
docking, quantum mechanics, molecular dynamics, and free energy calculations, to study the interactions
and the energetic contributions that govern the IRB:cyclodextrin association. We provide a detailed com-
putational protocol, which aims to assist the improvement of the pharmacological properties of sartans.
This protocol can also be applied to any other drug molecule with diminished hydrophilic character.
Key words Sartans, Irbesartan, 2-Hydroxypropyl-β-cyclodextrin, Blood pressure regulation,

Hypertension, Molecular modeling, Molecular dynamics, Pharmacological enhancement
1 Introduction
The pharmaceutical industry aims at the development of effective

medications with the lowest safety risk. However, this is not an easy
task, since efficacy is often compromised for diminished side effects.
Several marketed medicines contain active compounds, which pos-
sess specific physicochemical properties that deviate from an opti-
mal pharmacological profile. The main reasons for poor drug
performance are the low solubility and permeability, fast metabo-
lism and excretion, and also undesired side effects [1, 2]. It is noted
109
110 Georgios Leonis et al.
that at least 30–35% of marketed drugs suffer from low solubility

[3].
Sartans are a class of chemical compounds [angiotensin II type
1 receptor blockers (ARBs)] that hinder the detrimental hyperten-
sive effects of angiotensin II at the AT1 receptor in a pathological
state [4]. Despite their proven beneficial action against hyperten-
sion, sartans are characterized by high lipophilicity and violate the
Lipinski rule (MLogP >4.15). This feature negatively impacts the
absorbance, tissue penetration, and bioavailability of these
molecules.
To improve the pharmacological properties of such chemicals,
one may incorporate them into the structures of hydrophilic mol-
ecules, which form inclusion complexes with the drug and may act
as carriers for selective drug release to the active target. Cyclodextrins
have been shown to constitute proper transport systems for sar-
tans, as they can easily accommodate these molecules into their
binding region, thus inducing favorable interactions within the
inclusion complex [5].
Computational techniques are widely used for the study of
biomolecular systems, mainly because they combine reliable pre-
dictions with high speed at a minimal cost. Computational chem-
istry has been successfully applied to the study of proteins, nucleic
acids, lipid bilayers, and transport systems and in the drug design
process. Therefore, it is evident that such approaches can offer sig-
nificant help in the rational design of new drug formulations
[6–10].
In this work, we present the detailed computational methodol-
ogy for:
1. Modeling the inclusion of sartans into cyclodextrins
2. Monitoring and analyzing the conformational evolution of the
complexes
3. Predicting the energetic properties of the complexes
In particular, we apply computational methods [i.e., molecular
docking, quantum chemistry (QM), molecular dynamics (MD),
and binding free energy calculations] for the study of conforma-
tional properties and interactions between drug irbesartan (IRB)
and hydrophilic 2-hydroxypropyl-β-cyclodextrin (2-HP-β-CD) as
a representative example (Fig. 1). We note that the same method-
ology can also be applied to any other drug:cyclodextrin system.
Besides its effectiveness on blood pressure regulation, IRB exerts
poly-pharmacologic actions against other conditions, such as dia-
betes and cancer, and it can be considered superior to other sartans
[11–13]. However, IRB deviates from an excellent pharmaceutical
profile as it presents minimal hydrophilicity and low bioavailability
(approx. 65–70%) [14].
Molecular Dynamics and Drug Complexation with Cyclodextrins 111
Fig. 1 The structures of drug irbesartan (IRB) and 2-hydroxypropyl-β-cyclodextrin (2-HP-β-CD) used in this
work. Hydrogen atoms of IRB are not shown for simplicity. The structure of 2-HP-β-CD is presented as a
surface
2 Materials
Within this protocol, the following platforms, software suites, and

programs were used:
1. The Cambridge Structural Database (CSD): A compound
library, which contains >1 million crystal structures of organic
small molecules (free access). The CSD was used to retrieve the
initial structure of IRB (https://www.ccdc.cam.ac.uk/solu-
tions/csd-system/components/csd/).
2. PubChem (U.S. National Library of Medicine): A database of
chemical structures, which also contains biological activity
information (free access). PubChem was used for 2-HP-β-CD
structure retrieval (https://pubchem.ncbi.nlm.nih.gov/com-
pound/HP-beta-CD).
3. ArgusLab: [15] Software for molecular docking applications
(free of charge). ArgusLab was used for the inclusion of IRB
into 2-HP-β-CD (http://www.arguslab.com/arguslab.com/
ArgusLab.html).
4. AMBER 16: [16] A suite, which contains a number of pro-
grams for computational applications to biomolecular systems
(license fee required). AMBER was used to perform MD simu-
lations and molecular mechanics Poisson-Boltzmann surface
area (MM–PBSA) free energy calculations for IRB:2-β-CD
complexes. We note that AMBER operates on a Linux/Unix
environment. The unfamiliar user may consult relevant Unix
tutorials and can acquire AMBER documentation and instruc-
tions through http://ambermd.org/.
5. Gaussian 09: [17] A program for electronic structure calcula-

tions (license fee required). Gaussian was used to perform IRB
optimization and RESP charge derivation for IRB and
2-HP-β-CD (https://gaussian.com/).
6. UCSF CHIMERA 1.12: [18] A program for visualization and
analysis of biomolecules (free of charge). CHIMERA was used
for structure visualization, and figure generation (https://
www.cgl.ucsf.edu/chimera/).
3 Methods
3.1 Molecular Obtain the crystal structures of IRB and 2-HP-β-CD from the
Modeling CSD and PubChem, respectively. The reference codes correspond
to CCDC: 130127 for IRB and PubChem CID: 14049689 for
3.1.1 Structure Retrieval
2-HP-β-CD. Save the structures in PDB formats (IRB.pdb and
2-HP-β-CD.pdb).
3.1.2 Molecular Docking Molecular docking of IRB into 2-HP-β-CD: IRB can be included
Calculations into the cavity of 2-HP-β-CD in two different orientations. The
first orientation considers the tetrazole moiety of IRB near the HP
end of 2-HP-β-CD, while the opposite orientation associates the
butyl alkyl chain of IRB with the HP group (Fig. 2). The optimal
placement of IRB in its two orientations into 2-HP-β-CD and the
binding strength was predicted with ArgusLab. It is noted that any
other suitable docking software can also be applied. For the sake of
simplicity, hereafter we may occasionally refer only to one
orientation.
Open the main panel in ArgusLab and perform the following
steps:
1. Open molecule (browse and upload ligand).
2. The name of selected ligand (IRB.pdb) appears on the left-side
panel. Click on the cross to expand; on the extended menu click
Residues and then Misc. Next, select the molecule that appears
(i.e., 1 MOL) and right-click. On the new menu, select Make a
Ligand Group from this Residue.
3. Repeat the procedure for the receptor: open molecule in the
main panel and browse to upload 2-HP-β-CD.pdb.
4. Repeat step 2 and after clicking Misc, select all 2-HP-β-CD
subunits that appear. On the new menu, select Make a Group
from this Residue. Next select Binding Site.
5. In the main window, press Calculation → Dock a Ligand. A
new menu appears with a list of docking parameters. Most of
the default selections will suffice for a rough estimation. The
user should carefully experiment and choose among various
Fig. 2 The two possible orientations of IRB in 2-HP-β-CD as obtained by molecular docking calculations.
2-HP-β-CD is depicted as a transparent surface. The HP groups of 2-HP-β-CD are at the bottom. Hydrogen
atoms of IRB are not shown for simplicity
options that closely depend on the system under study. However,

based on personal experience, we indicate the following modi-
fications for optimal performance.
6. Binding Site Bounding Box: Box sizes that range between 15
and 25Å (depending on the system’s size) are preferable.
Docking Engine: GADock may be preferred. The other options
are generally safe to be left as default.
7. In the same menu, click Advanced and increase Max. Generations
to 10,000. Press OK.
8. In the main Docking menu press Start to begin the docking
calculation.
9. After the calculation is over, you are provided with a docking
score (in kcal/mol) and a PDB structure of the complex in its
most favorable conformation (save it as complex.pdb).
The two optimally docked conformations of IRB:2-HP-β-CD
complexes are next prepared for MD simulations.
3.2 Structure A general scheme of IRB, 2-HP-β-CD, and IRB:2-HP-β-CD com-

Preparation plex treatment is presented in Fig. 3. More detailed instructions
for Molecular are provided below.
Dynamics IRB preparation: The structure of IRB is geometrically opti-
mized at the HF/6-31G* level of theory with Gaussian.
1. Convert IRB file format from PDB to com with the
ANTECHAMBER subroutine of AMBER for subsequent
Gaussian calculations. Install and compile (i) ANTECHAMBER
Fig. 3 Schematic workflow for systems’ preparation for MD simulations
from the AmberTools program (free of charge) and (ii) AMBER

program; then run the command:
antechamber –i IRB.pdb –fi pdb –o IRB.com –fo gcrt
Note: For the required calculations, the use of AMBER and
Gaussian is not restrictive; other appropriate molecular model-
ing and QM packages may be used as well.
2. Install and compile Gaussian; run Gaussian (on Linux/Unix
environment) for geometry optimization of IRB and for elec-
trostatic potential (ESP) generation:
g09 IRB.com > IRB.log
The input file (IRB.com) for Gaussian calculations is shown
in Note 1. Output IRB.log contains the optimized structure of
IRB and information regarding ESP.
Note: Most of the output files produced by Gaussian and
AMBER regarding this case study are very large and therefore
are not presented here.
3. Generate charges according to the restrained electrostatic
potential (RESP) method with ANTECHAMBER. The geom-
etry of IRB and the RESP charges are included in the IRB.mol2
output file:
antechamber –i IRB.log –fi gout –o IRB.mol2 –fo
mol2 –c resp
4. Generate missing parameters for IRB with the parmchk routine
of ANTECHAMBER:
parmchk –i IRB.mol2 –f mol2 –o IRB.frcmod
Missing parameters are included in the IRB.frcmod output

file.
5. Assign force field and construct a parameter library for IRB
with the tLEaP module of AMBER. The general AMBER force
field (GAFF) was used to assign force field parameters to IRB
[19]:
(i) Open tLEaP program:

tleap
(ii) Load force field:

source leaprc.gaff
(iii)
Load mol2 file for IRB:

MOL = loadmol2 IRB.mol2
(iv) Load missing parameter file for IRB:

loadamberparams IRB.frcmod
(v) Save library for IRB:

saveoff MOL IRB.lib
The generated output IRB.lib and the IRB.frcmod will be used
later for complex construction.
2-HP-β-CD preparation: RESP charges for all atoms of
2-HP-β-CD, missing parameters (2-HP-β-CD.frcmod), and
library files only for the 2-HP part of cyclodextrin (2-HP.lib) are
generated similarly as in steps 1–5 above during IRB preparation.
AMBER employs a well-tested force field for the treatment of sug-
ars (i.e., cyclodextrin); however, modified structures cannot be
supported. Therefore, one may treat the 2-HP part of 2-HP-β-CD
as a small molecule with GAFF and has to manually generate a
library file (2-HP.lib) as commented above.
IRB:2-HP-β-CD complex preparation: Construct topology and
coordinate files, which will be used as inputs for MD simulations.
The force field GLYCAM 06 [20] is used to treat the CD part of
2-HP-β-CD while GAFF is used for the 2-HP part. The TIP3P
water model is employed for the treatment of solvation [21].
(i) Open tLEaP program:
tleap
(ii) Load force fields:
source leaprc.gaff
source leaprc.GLYCAM_06j-1
(iii) Assign explicit model for water molecules:
source leaprc.water.tip3p
(iv) Load missing parameters file for IRB and 2-HP
loadamberparams IRB.frcmod
loadamberparams 2-HP-β-CD.frcmod
(v) Load library files for IRB and 2-HP:
loadoff IRB.lib
loadoff 2-HP.lib
(vi) Load IRB:2-HP-β-CD complex file:
a = loadpdb complex.pdb
(vii) Assign pre-calculated RESP atomic charges to 2-HP-β-CD
(see Note 2).
(viii) Add a truncated octahedral box containing TIP3P water
molecules around the complex. The edge of the periodic box
is set to be at least 16 Å away from each complex atom:
solvateoct a TIP3PBOX 16
(ix) Save topology (complexparm.top), coordinates (complex-
parm.crd), and PDB (complex_final.pdb) files of the
complex:
saveamberparm a complexparm.top complex
parm.crd
savepdb a complex_final.pdb
3.3 Molecular The detailed preparatory steps for MD and the actual production
Dynamics Simulation MD run are described below. The MD calculations are performed
of the Complex with the GPU version of PMEMD [22] from AMBER 16.
3.3.1 Energy
Complexes are minimized in three stages for 10,000 cycles each.
Minimization
Minimization is performed with the steepest descent method for
the first 5000 steps and the conjugate gradient algorithm follows
for the next 5000 steps. The first step considers the complex practi-
cally fixed with the application of a harmonic force constant of
500 kcal mol−1 Å−2, thus allowing the structures of the water mol-
ecules to relax. During the second step, the restraint was reduced
to 10 kcal mol−1 Å−2, and finally all atoms were totally unrestrained
to move. A nonbonded cutoff of 10.0 Å is applied under constant
volume. The three input files are provided and explained in Note
3. An example of the command to perform minimization is
srun /amber16/bin/pmemd -O -i Min1.in -o com-
plex_min1.out -p complexparm.top -c complex-
parm.crd -r complex_min1.rst
Note the use of complexparm.top/crd files, which were gener-
ated from Subheading. 3.2; the complex_min1.rst file will be used
as the input for the second stage of minimization and so on. Output
file complex_min1.out contains the energy information for every
step of the run.
3.3.2 Heating The next procedure involves the gradual heating of the complexes
from 0 to 310 K using the Langevin thermostat [23] for tempera-
ture regulation. The simulation is performed under constant vol-
ume for 400 ps and the collision frequency is set at 2 ps−1. Positional
restraints of 10 kcal mol−1 Å−2 were applied to the atoms of the
complex. The algorithm SHAKE is enabled to keep hydrogen
atoms at their equilibrium position and a 2 fs time step was used
[24]. The corresponding input file is given in Note 4. Execute the
following command for heating MD run:
srun /amber16/bin/pmemd.cuda_SPFP -O -i Heat.
in -o complex_heat.out -p complexparm.top -c
complex_min3.rst -r complex_heat.rst -x com-
plex_heat.nc
Note the use of complex_min3.rst from the minimization pro-
cess as a restart file for heating; the complex_heat.rst file will be
used as the input for the next simulation step. The complex_heat.
nc file contains the trajectory generated by the run.
3.3.3 Density Equilibration of the complexes was performed under constant

Equilibration pressure in two steps of 400 ps each. In the first step, constraints of
10 kcal mol−1 Å−2 were applied to the solute, and all restraints were
removed in the last step. The two input files are provided in Note
5. The corresponding command lines are
Step1: srun /amber16/bin/pmemd.cuda_SPFP -O -i Density.in -o
complex_density.out -p complexparm.top -c complex_heat.rst -r
complex_density.rst -x complex_density.nc
Step2: srun /amber16/bin/pmemd.cuda_SPFP -O -i Eq.in -o com-
plex_eq.out -p complexparm.top -c complex_density.rst -r com-
plex_eq.rst -x complex_eq.nc
3.3.4 MD Production This is the final step for the generation of the MD trajectory for
Simulation further analysis. Two unrestrained, constant-pressure MD simula-
tions for IRB in the two binding orientations are performed at
310 K for 3 μs each. Additional details of the simulation are pro-
vided in Table 1 and the corresponding input file is shown in Note
6. Execute the following command:
srun /amber16/bin/pmemd.cuda_SPFP -O -i MD.in -o
complex_md.out -p complexparm.top -c complex_
eq.rst -r complex_md.rst -x complex_md.nc
3.4 Energetic The resulting trajectories are subjected to MM–PBSA analysis for
Analysis with the the estimation of the Gibbs free energy based on enthalpy and
MM–PBSA Method entropy contributions.
Enthalpy estimation: The enthalpy of binding can be predicted
with application of the MM–PBSA or the molecular mechanics–
Table 1
Simulation parameters for production of MD simulations of IRB:2-HP-
β-CD complexes
Parameters Value
Total simulation time 3 μs
Time step 2 fs
Periodic boundaries Yes (constant pressure)
Pressure scaling Isotropic position scaling
Pressure relaxation time 2.0 ps
Nonbonded cutoff 10.0
Restrained atoms None
Bonds constrained Hydrogens involving
(SHAKE)
Temperature control Langevin thermostat
Collision frequency 2.0 ps−1
Average temperature 310 K
generalized born surface area (MM–GBSA) method; [25–27]

details on the underlying theory can be found in other publications
[28, 29]. The input (mmpbsa_enthalpy.in) is presented in Note 7.
Execute the following Python script in AMBER:
/amber16/bin/MMPBSA.py -O -i mmpbsa_enthalpy.
in -o RESULTS_MMPBSA.dat -sp complexparm.
top -cp complex_only.parm.top -rp receptor_only.
parm.top -lp ligand_only.parm.top -y complex_
md.nc
Note that the topology (complexparm.top) and trajectory
(complex_md.nc) files from Subheading 3.3 are used as inputs for
the MM–PBSA calculations. Additional input files are the com-
plex_only.parm.top, receptor_only.parm.top, and l igand_only.
parm.top, which are topology files for the complex, 2-HB-β-CD,
and IRB, respectively, and do not include any water molecules.
The RESULTS_MMPBSA.dat file contains the enthalpy estima-
tion in kcal/mol and also provides individual energy components,
such as electrostatic, van der Waals, and nonpolar contributions.
Entropy estimation: The entropy estimation is obtained with
normal mode analysis. The execution is the same as for the enthalpy
calculations, with the only difference being the use of input
mmpbsa_entropy.in instead of mmpbsa_enthalpy.in (see differences
in Note 7).
3.5 Conformational Conformational analysis: Analysis of the resulting trajectories is

Analysis performed with the cpptraj module [30] of AMBER. The cpptraj
functionality is able to calculate a variety of properties, such as dis-
tances, angles, torsions, RMSD, hydrogen bonds, fluctuations,
J-coupling, surface areas, radii of gyration, average structures, dif-
fusion, and radial distribution functions, among many others.
Here, we provide a representative example of an input (ptraj.in),
which includes distance, RMSD, and fluctuation calculations
(see Note 8).
Execute in AMBER: cpptraj complexparm.top ptraj.in
Output files are not included in the above command line as
they are generated from cpptraj after they have been included in
the ptraj.in file.
4 Notes
1. A simplified Gaussian input file for the geometry optimization

and ESP generation of IRB (IRB.com) is shown below:
IRB.com
--Link1--
%chk=molecule
#HF/6-31G* SCF=tight Test Pop=MK iop(6/33=2)
iop(6/42=6) opt nosymm
remark line goes here
0 1
##Author’s note: The initial X- Y-
Z coordinates
of IRB follow here (not shown).
2. The RESP charges for 2-HP-β-CD are manually assigned to
each atom in tLEaP. The following command is an example for
charge assignment to the oxygen atom, which is labeled “O2”
and belongs to the first residue (HP group) of 2-HP-β-CD:
set a.1.O2 charge −0.631696
Similar command lines should be included in tLEaP for each
2-HP-β-CD atom and its respective RESP charge.
3. Input files for complex minimization with AMBER. Next to
each command, a short explanation is offered in italics:
Min1.in
Minimization of system keeping complex fixed
&cntrl
imin=1, Perform minimization
maxcyc=10000, The total number of cycles per
formed
ncyc=5000, First 5000 cycles with steepest de-
scent before switching to conjugate gradient
cut=10.0, Cutoff for nonbonded interactions
ntb=1, Constant volume
ntr=1, Obtain a restart file

/
Keep complex fixed
500.0 Positional restraints for the solute
(residues 1 to 15 of 2-HP-β-CD)
RES 1 15
END
END
Min2.in
Minimization of system keeping complex fixed
&cntrl
imin=1, maxcyc=5000, ncyc=5000,
cut=10.0, ntb=1, ntr=1,
/
Keep complex fixed
10.0
RES 1 15
END
END
Min3.in
Minimization of system with complex unre-
strained
&cntrl
imin=1, maxcyc=10000, ncyc=5000,
cut=10.0, ntb=1, ntr=0,
/
Note: The input files demonstrate the basic commands of
each procedure and they should be merely considered as simpli-
fied examples. The command choices and their values may vary
and depend on the particular system under study. Also, for sim-
plicity, some less important keywords were omitted.
4. Input file for complex heating with AMBER:
Heat.in
Equilibration with restraints on complex
&cntrl
imin=0, irest=0,
cut=10.0, ntb=1, ntr=1,
ntc=2, ntf=2, SHAKE algorithm applied
tempi=0, temp0=310.0, Target temperature
ntt=3, Langevin thermostat
gamma_ln=2.0, Collision frequency
nstlim=200000, # of MD steps
dt=0.002, time-step
/
Keep complex fixed

10.0
RES 1 15
END
END
/
Keep complex fixed
10.0
RES 1 15
END
END
5. Input files for density equilibration with AMBER:
Density.in
Molecular Dynamics on whole system
&cntrl
imin=0, irest=1, cut=10.0,
ntb=2, pres0=1.0, Constant pressure
ntp=1, taup=2.0, Pressure relaxation time (in
ps)
ntc=2, ntf=2,
tempi=310.0, temp0=310.0, ntt=3, gamma_ln=2.0,
nstlim=200000, dt=0.002,
/
Keep complex fixed
10.0
RES 1 15
END
END
Eq.in
Molecular Dynamics on whole system
&cntrl
imin=0, irest=1,
cut=10.0, ntb=2, pres0=1.0, ntp=1, taup=2.0,
ntr=0,
ntc=2, ntf=2,
tempi=310.0, temp0=310.0,
ntt=3, gamma_ln=2.0,
nstlim=200000, dt=0.002,
/end
6. Input file for production MD run with AMBER:
MD.in
Molecular Dynamics on whole system for 3 μs
&cntrl
imin=0, irest=1,
cut=10.0, ntb=2, pres0=1.0, ntp=1, taup=2.0,

ntr=0,
ntc=2, ntf=2,
tempi=310.0, temp0=310.0,
ntt=3, gamma_ln=2.0,
nstlim=1500000000, dt=0.002,
/end
7. Input files for MM–PBSA enthalpy and entropy calculations:
mmpbsa_enthalpy.in
Input file for running PB and GB
&general
endframe=1000, startframe=1, interval=10,Calculate from
trajectory frame 1 to 1000 every 10
/
&gb Perform MM–GBSA calculation
igb=5, Use a modified GB model[31]
saltcon=0.100 Salt concentration (in M)
/
&pb Perform MM–PBSA calculation
istrng=0.100, Ionic strength (in M)
/
mmpbsa_entropy.in
Input file for running entropy calculations using NMode
&general
endframe=1000,
/
&nmode Perform normal mode analysis
nmstartframe=100, nmendframe=1000, nminterval=10,
nmode_igb=1, Default GB model [32]
nmode_istrng=0.1, Ionic strength (in M)
/
8. Example of a simple input file for conformational analysis with
cpptraj:
ptraj.in
trajin complex_md.nc Read and analyze each
frame of MD trajectory
distance :2@O1 :3@H2 out distance_2- 3_complex.
out Calculate the distance between oxygen atom
labeled O1 in residue 2 and hydrogen atom la-
beled H2 in residue 3. Report the results for
every frame in output file named distance_2-3_
complex.out
rms first mass out rms_CD.out :1-14 Calculate
RMSD for all 2-HP-β-CD residues (1 to 14) and
report the results in file rms_CD.out
rms first mass out rms_IRB.out :15 Calculate

RMSD for all atoms of IRB (residue 15) and re-
port the results in file rms_IRB.out
atomicfluct out fluct_CD.out :1-
14 Calculate RMS
fluctuations for the 2-HP-β-CD residues
Acknowledgments
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Chapter 10
Drug Delivery Through Multifunctional Polypeptidic

Hydrogels
Hermis Iatrou, Panagiota G. Fragouli, Dimitra Stavroulaki,
and Barbara Athanasiou
Abstract
Over the last two decades, remarkable progress has been made to the discovery of novel drugs as well as
their delivery systems for the treatment of cancer, the major challenge in medicine. Pharmaceutical scien-
tists are trying to shift from traditional to novel drug delivery systems by applying nanotechnology and, in
particular, polymeric carriers to medicine. In complex diseases, very sophisticated nanocarriers should be
designed to encapsulate a significant quantity of drugs and bypass biological barriers with minimum cargo
loss to effectively and directly deliver the encapsulated drug to the desired pathological site. One of the
most promising classes of polymeric materials for drug delivery applications is polypeptides, combining the
properties of the traditional polymers with the 3D structure of natural proteins, i.e., a-helices and β-sheets.
In this chapter, we present the recent progress in the synthesis of polymers that form hydrogels in aqueous
solutions, based on polypeptides prepared through ring-opening polymerization of N-carboxy anhydrides
and which have been loaded with anticancer drugs and studied for their functionality. Advancements in
drug design and improvement of multifunctional nanocarriers from the combination of well-defined mac-
romolecular architectures and smart materials are the future for the successful treatment of numerous
lethal diseases.
Key words Polypeptides, Ring-opening polymerization, Hydrogels, pH- and enzyme stimuli-
responsive, Pancreatic cancer, Gemcitabine
1 Introduction
A novel, multifunctional hydrogel that exhibits a unique set of

properties for the effective treatment of pancreatic cancer (PC) is
presented. The material is comprised of a pentablock terpolypep-
tide of the type PLys-b-(PHIS-co-PBLG)-PLys-b-(PHIS-co-
PBLG)-b-PLys which is a noncytotoxic polymer [1]. It can be
implanted via the least invasive route and selectively delivers gem-
citabine to efficiently treat PC. Mixing the novel terpolypeptide
with an aqueous solution of gemcitabine within a syringe results in
the facile formation of a hydrogel that has the ability to become
127
128 Hermis Iatrou et al.
Fig. 1 Illustration of chemical treatment of pancreatic cancer: hydrogel with

encapsulated drug is implanted in least invasive way and melts only in the vicin-
ity of cancerous tissue due to lower pH, leading to targeted and directional deliv-
ery of PLys-b-(PHIS-co-PBLG)-PLys-b-(PHIS-co-PBLG)-b-PLys
liquid under the shear rate of the plunger. Upon injection in the
vicinity of cancer tissue, it immediately reforms into a hydrogel due
to the unique combination of its macromolecular architecture and
secondary structure. Because of its pH responsiveness, the hydro-
gel only melts close to PC; thus, the drug can be delivered direc-
tionally toward the cancerous rather than healthy tissues in a
targeted, controlled, and sustained manner (Fig. 1).
2 Materials
1. Boc-HIS(Trt)−OH (>99%).
2. Triphosgene (99%).
3. Triethylamine (>99%) (see Note 1).
4. Thionyl chloride (>99%).
5. l-Lysine (>99%).
6. γ-Benzyl-l-glutamate.
7. H-Leu-OH (>99%).
8. Diethyl ether.
9. Fluorescamine.
10. Trypsin.
11. Tetrahydrofuran (THF) (dried, max 0.005% water) (see Note 2).
12. Dichloromethane.
13. Trifluoroacetic acid (TFA) (>99%).
14. Ethyl acetate (>99.5%) (see Note 3).
Nanostructured Hydrogels for Controlled Drug Delivery 129
15. Hexane (>99%) (see Note 4).

16. Dimethylformamide (DMF) (99.9+%, special grade for pep-
tide synthesis with less than 50 ppm of active impurities) (see
Note 5) is the polymerization solvent.
17. 1,6-Diaminohexane (98%) is the initiator of the polymeriza-
tion (see Note 6).
3 Methods
3.1 Instrumentation 1. Use size-exclusion chromatography (SEC) to determine the

Mn and Mw/Mn values. Perform the analysis using a SEC
equipment composed of 600 high-pressure liquid chromato-
graphic pump, Ultrastyragel columns, a differential refractom-
eter detector, and a precision PD light scattering detector at
60 °C featuring two detectors at 15° and 90° (TALLS). Use a
DMF solution containing 0.1 N LiBr as an eluent at a rate of
1 mL/min, operating at 60 °C.
2. Perform nuclear magnetic resonance spectroscopy (1H NMR),
300 MHz. Take the spectra of the N-carboxyanhydrides
(NCAs) in CDCl3 at room temperature.
3. Perform Fourier transform infrared (FTIR) spectroscopy mea-
surements in KBr pellets at room temperature, in the range of
450–4000 cm−1.
4. Perform circular dichroism with an instrument containing
thermo-stabilizing system. Use cell of 1 mm Quartz Suprasil.
The aqueous solution concentration of the PBLG/PHIS
polymer with a ratio of the monomeric units 50/50 is
3.3 × 10−4 g/mL, while the aqueous solution concentration of
the PBLG/PHIS polymer with a ratio of the monomeric units
70/30 at every pH is 2.5 × 10−4 g/mL. Perform the adjust-
ment of pH through addition of diluted HCl or NaOH.
5. Perform scanning electron microscopy (SEM)-EDS of the
hydrogels using carbon grids. Prepare the samples first by
quick deep freezing of a small amount of the hydrogels with
liquid nitrogen, followed by freeze-drying to remove water.
Make sputter coating of the samples with gold prior to the
measurements.
6. Perform enzymatic degradation-fluorescence measurements
using 10 M multimode microplate reader and 96-well black
microplates. To measure the enzymatic degradation of the
hydrogel by either trypsin or leucine aminopeptidase (LAP),
mix 2 mg of hydrogel with 0.1 nM trypsin or 100 nM LAP in
PBS and incubate at 37 °C for 1 or 24 h, respectively. At the
end of the incubation, mix a sample of 10 μL from the reac-
tion with 90 μL of a solution of fluorescamine containing
1 mg/mL fluorescamine in acetonitrile, 15 μL of 0.1 M borate

buffer at pH 8.0, and 145 μL of Milli-Q water. After 5 min,
measure the fluorescence using excitation at 405 nm and emis-
sion at 485 nm.
3.2 Synthesis Perform the synthesis using high-vacuum techniques [2]. The
of Monomers purity of the NCA monomers is crucial for their successful living
polymerization utilizing ROP through primary amine difunctional
initiator and confirms by FTIR along with 1H NMR spectroscopy
analysis [3].
3.2.1 Synthesis
of ε-tert-Butoxycarbonyl-l- 1. Add Nα,Nε-Di-(tert-butoxycarbonyl)-l-lysine into a flask,
Lysine N-Carboxy place it on the vacuum line, and pump overnight.
Anhydride (Νε BOC-l-LYS
2. Distill purified ethyl acetate, followed by argon insertion, in
NCA)
order to reach atmospheric pressure and by the addition of
triphosgene. Leave the mixture to react for 10 min.
3. Dilute triethylamine in dry ethyl acetate, add it dropwise, and
immerse the solution in an ice-water bath for 6 h.
4. Filter the precipitate, in order to remove the HCl salt of trieth-
ylamine, and immerse the clear solution in an ice bath.
5. Extract the NCA repeatedly with Milli-Q water, until neutral
pH of the aqueous phase is achieved.
6. Recrystallize the purified NCA three times under high vacuum
in a custom-made apparatus, with ethyl acetate/hexane
(1/5 v/v) pair at −20 °C. The yield is 60%.
3.2.2 Synthesis 1. Add in a 500 mL round-bottom flask 20 g (40.2 mmol) of

of Nim-trityl-l-Histidine-N- Boc-His(Trt)-OH and dry overnight under high vacuum.
Carboxy Anhydride 2. Distill 150 mL of THF in the flask, giving a clear yellowish
(Trt-HIS-NCA) [4] solution.
3. In an ice bath place the reaction flask, filled with argon.
4. Dilute 3.25 mL (44.2 mmol) of thionyl chloride in 20 mL of
THF and add dropwise in a period of 10 min.
5. After 2 h, pour the solution in 2 L of cold (Et)2O with precipi-
tation of Trt-His-NCA.HCl as the major product.
6. Finally, filter the solid (glass sintered filter 3) and then transfer
it to a round-bottom flask of 500 mL. Drying in HV gives
17.2 g.
7. Recrystallize the above solid mixture, containing the HCl salt,
free anhydride, and the initial substrate by distilling 300 mL of
ethyl acetate under HV.
8. Remove the round-bottom flask of 500 mL, containing the
suspension, from HV and place it into a water bath, at 45 °C
for 1 h, resulting in dissolution.
9. Cool the solution to 0 °C with an ice bath and form Trt-His

NCA.HCl as a precipitate which is isolated as the only product
after filtration.
10. Transfer the NCA salt to another flask of 500 mL and dry
overnight under HV (12.5 g = 28 mmol).
11. Subsequently, distill 200 mL of EtAc into the flask; remove
the flask from HV, fill with Ar, and place to an ice bath.
12. Add slowly dropwise under vigorous stirring (duration of
additional 1 h), at 0 °C, 3.5 mL (28 mmol) of the stoichio-
metric amount of triethylamine dissolved in 50 mL of the
same solvent.
13. Filter off the resulted triethylamine hydrochloride and pour
the filtrate into 1.5 L of non-solvent hexane in order to recrys-
tallize the Trt-His-NCA.
14. Make a second recrystallization with a mixture of solvent/
non-solvent EtAc/hexane (1:5) and isolate by filtration the
white solid precipitate Trt-His-NCA.
15. Finally, dry Trt-HIS-NCA under HV overnight and transfer it
into glove box resulting in 11.05 g (27 mmol, 67% yield).
3.2.3 Synthesis 1. Suspend γ-benzyl-l-glutamate in dry ethyl acetate followed by

of γ-Benzyl-l-Glutamate addition of triphosgene.
N-Carboxy Anhydride 2. Heat the mixture at 70 °C until the solution becomes clear,
(BLG-NCA) indicating the formation of the NCA.
3. Distill off the solvent in the vacuum line, and distill fresh dry
ethyl acetate in the flask, to dissolve the crude NCA, followed
by removal of the solvent by distillation.
4. Repeat twice this procedure in order to remove the excess
phosgene that sublimes under high vacuum.
5. Remove the unreacted species, such as free amino acids along
with the HCl salts of the amino acids produced during the
synthesis, by extraction with an alkali solution in water.
6. Purify further the resulted BLG-NCA by three recrystalliza-
tions from dry ethyl acetate/hexane (1/5 v/v) under high
vacuum at −20 °C, leading to a 65% yield of the BLG-NCA
formation.
3.2.4 Synthesis 1. Add in a flame-dried 1 L two-neck round-bottom flask 15 g

of l-Leucine N-Carboxy (114 mmol) of l-leucine and degas under high vacuum
Anhydride (LEU-NCA) overnight.
2. Distill 250 mL of highly dry THF; remove the flask from the
vacuum line and brink to atmospheric pressure by the careful
addition of dry argon. The flask is equipped with a condenser
and an inlet for Ar streaming.
3. Add under stirring 29.6 mL (183 mmol, 1.6 eq.) of purified
(+) (−) limonene and allow the suspension to warm under vig-
orous stirring at 50–55 °C. At this time, add 13.6 g (46 mmol,
1.2/3 eq.) of triphosgene. Allow the reaction mixture to stir
at this temperature until a clear dark orange solution is formed
(after 1–2 h).
4. Stir the clear solution for another 1 h under Ar flow and trans-
fer the solution by filtration to the crystallization apparatus.
Attach the apparatus to the vacuum line and pump out the
solvent to dryness. Then, distill a small amount of highly dry
THF (~20 mL) capable to dissolve the solid monomer and a
clear solution is formed.
5. Slowly distill, under vigorous stirring, n-hexane (~500 mL) in
order for the NCA to precipitate in the form of a fine powder.
Keep the apparatus at −20 °C overnight.
6. Next day, filter off the orange supernatant and dry the solid in
vacuo for 1 h.
7. Perform three additional crystallizations and finally dissolve
the white solid product in dry THF and cannula transfer it in
a sealed flask. Attach the flask to the vacuum line, remove the
solvent, and dry overnight the final product.
8. Finally, move the flask to the glove box and weigh solid yield-
ing 16.6 g (95 mmol) of Leu-NCA (83%).
3.3 Synthetic 1. Add an α,ω-hexamethylenediamine solution in DMF, in a

Approach of solution of ε-tert-butoxycarbonyl-l-lysine N-carboxy anhy-
Polypeptides PLys-b- dride in DMF (Lys-NCA) (see Note 7).
(PHIS-co-PBLG (or 2. After completion of polymerization (middle block), dissolve in
PLEU))-b-PLys-b- DMF appropriate amounts of Nim-trityl-protected l-histidine
(PHIS-co-PBLG (or N-carboxy anhydride (HIS-NCA) and γ-benzyl-l-glutamate
PLEU))-b-PLys (See N-carboxy anhydride (BLG-NCA) (molar ratio 1:1) and add
Fig. 2) to the reaction flask. Allow the polymerization to continue for
6 days (two hydrophobic blocks).
3. Add then Lys-NCA, followed by precipitation, in diethyl ether
after the completion of the polymerization (see Note 8).
4. Mix the deprotected pentablock with Milli-Q water and dia-
lyze (membrane molecular weight cutoff 3500 Da) against
dilute HCl solution (pH = 3) for 2 days, dilute NaOH solu-
tion (pH = 8) for 2 days, and DI water for 2 days (for each step
change the water every 12 h).
Fig. 2 Schematic illustration of pentablock terpolypeptide of the type PLys-b-

(PHIS-co-PBLG (or PLEU))-b-PLys-b-(PHIS-co-PBLG (or PLEU))-b-PLys
5. Finally, lyophilize the solution to obtain the final pentablock

terpolypeptide (Fig. 3) (see Note 9) as a white powder (see
Note 10).
3.4 Formation of 1. Prepare pentablock terpolypeptide hydrogels very simply by

Hydrogels adding Milli-Q water in a vial containing the solid
polypeptide.
2. After 2–3 h, the hydrogel is ready without requiring any other
treatment. The water swells the polypeptide, which keeps the
general shape of the initial solid.
3.5 Formation 1. Prepare pentablock terpolypeptide hydrogels loaded with

of Hydrogels Loaded gemcitabine (Hydrogem) by first dissolving the appropriate
with Gemcitabine [5, 6] amount of gemcitabine in pyrogen-free Milli-Q water
(gemcitabine:polypeptide ratio 1:1 (w/w)), at 35 °C, followed
by addition of the warm gemcitabine solution to the polypep-
tide. Keep the temperature at 35 °C until complete swelling of
the polypeptide and formation of homogeneous hydrogel,
about 3–4 h (see Note 11).
2. The hydrogel is stable at room temperature for several days,
while the same gemcitabine solution in Milli-Q water precipi-
tates after 0.5 h when the temperature is lowered to 20 °C.
4 Notes
1. Dry triethylamine over calcium hydride for 1 day and then

distill and store in the vacuum line over sodium. The appropri-
ate quantity needed is freshly distilled in a vacuum line.
Fig. 3 Reactions used for the synthesis of polypeptide PLys-b-(PHIS-co-PBLG)-b-PLys-b-(PHIS-co-PBLG)

-b-PLys
2. Perform the purification of tetrahydrofuran using standard

high-vacuum techniques. THF is refluxed over Na under N2
atmosphere in the presence of benzophenone, until a bright
deep purple color is attained, before being collected in a
round-bottom flask containing fresh finely grounded CaH2.
Connect this flask to the vacuum line; degas the solvent and
distill in a flask containing a Na/K (1/3 by weight) alloy. The
bright blue color, which develops after stirring for some time,
indicates that the solvent is free from impurities.
3. Ethyl acetate is fractionally distilled over phosphorus
pentoxide.
4. Hexane is fractionally distilled over sodium.
5. Purify dimethylformamide by short-path fractional distillation
under vacuum in a custom-made apparatus. Use always the
middle fraction.
6. Distill fractionally 1,6-diaminohexane under high vacuum and
treat it with sodium at room temperature for 1 day. Then sub-
sequently distill it into precalibrated ampoules with break
seals. Dilute it then with DMF to the appropriate concentra-
tion in a sealed apparatus equipped with precalibrated
ampoules and keep away from light.
7. Synthesize the polypeptides using high-vacuum techniques.
Design the polymerization reactors to have volumes at least
three times larger than that of the CO2 generated by each
polymerization. Carry out polymerizations with
1,6-diaminohexane as the initiator.
8. The selective cleavage of trityl and tert-butoxycarbonyl groups
from poly (l-histidine) and poly(l-lysine) segments, respec-
tively, is achieved by treating with trifluoroacetic acid (TFA),
followed by addition of triethyl silane. The solution is precipi-
tated in diethyl ether, and the white solid is filtered and dried.
9. These terpolypeptides are composed of one middle hydro-
philic block (PLys), two hydrophobic blocks poly(l-histidine-
co-γ-benzyl-l-glutamate (or l-leucine)), followed by two
hydrophilic blocks of PLys, each one connected at the outer
sides of the hydrophobic blocks. The middle hydrophilic block
of poly(l-lysine) should have a high molecular weight to form
strong hydrogels at very low concentrations, that is, 3.33%
(w/w) in water.
10. A similar procedure is followed for the synthesis of the terpoly-
peptides exhibiting poly(l-leucine) (PLEU) instead of PBLG.
11. To ensure the formation of a homogeneous mixture, the
hydrogel is inserted into a syringe and passed between two
syringes multiple times using a syringe connector, prior to the
in vivo tests.
5 Results
Over the last 40 years, impressive advancements toward the discov-

ery of novel drugs as well as responsive multifunctional drug deliv-
ery systems (DDS) have been reported. As a consequence, out of
more than 200 different cancers, some highly lethal cancers are
now chronic diseases. Unfortunately, only limited progress has
been made for some specific forms of cancer such as PC. For this
complex disease, very sophisticated carriers should be designed to
bypass biological barriers with minimum cargo loss, and effective
and selective delivery to the desired pathological site, as demon-
strated by the material described in this work.
An amphiphilic pentablock terpolypeptide was developed by
optimizing the macromolecular architecture and composition as
well as the 3D structure of the hydrophobic blocks. The final pent-
ablock terpolypeptide exhibits a unique combination of properties
in the same molecule, which has not been achieved so far.
Simply mixing the polypeptide and gemcitabine dissolved in water
results in the facile formation of an injectable and quickly self-healing
hydrogel which forms in situ and can be injected in the least invasive way
close to cancer tissue. These properties rely on the secondary structure of
the hydrophobic part of the amphiphilic terpolypeptide. After implanta-
tion, the hydrogel becomes liquid only close to the cancer tissue, mainly
due to the lower pH of the pathological site, thus releasing the drug only
in the vicinity of cancer tissue.
The ability of these polypeptides to form hydrogels by adding
Milli-Q water depended on two parameters: their molecular and
compositional homogeneity and the PHIS/PBLG ratio. It was
found that the pentablocks should be well defined with a high
degree of molecular and compositional homogeneity in order to
form strong hydrogels. If the polydispersity was high by intention-
ally mixing two different polypeptides, or by intentionally omitting
one block, they either formed very weak hydrogels at higher poly-
peptide concentrations or did not gel at all.
This obliges the directional release of the drug toward the can-
cer rather than healthy tissue, as shown by in vivo experiments. It
was found that the delivery of only 40% of gemcitabine in one dose
directed by the hydrogel could slow down the development of the
cancer tissue to the same extent with the delivery of 100% of pure
gemcitabine in two doses, the typical chemotherapy used so far in
clinics. Therefore, we achieved the same or slightly better decelera-
tion of tumor growth with fewer drugs and less number of doses
due to the guided delivery through the Hydrogem. The hydrogel
also responds to enzymes, rendering it biodegradable, thus not
requiring removal through resection following drug delivery.
Regarding the PHis/PBLG ratio, PHis 100 and PHis 70
formed a weak hydrogel at 20 °C, while its strength was highly
temperature dependent, and at 37 °C it was transformed into liq-
uid. PHis 50 and PHis 30 formed strong hydrogels depending on
the polypeptide concentration and the pH of water provided that

at pH = 6.6 the polymer was transformed into liquid. A well-
ordered 3D structure of interconnected nanofibers and thin
nanosheets was maintained by the terpolypeptide PHis 50 at
pH = 7.4, while at pH = 6.5 the structure was akin to collapsed
thick sheets with interconnecting thick fibers and larger voids,
resembling a liquid-like structure. The presence of the α-helix con-
formation that the increased BLG content gives to the terpolypep-
tide is critical for the hydrogel formation.
The synthesized material is modular since its properties can be
modified by its molecular characteristics to deliver other drugs,
rendering it very useful for a variety of biological applications such
as bone regeneration and catheters for treatment of coronary artery
disease. We intend to improve the hydrogel effectiveness by incor-
porating more stimuli, such as temperature and redox, to increase
selectivity for targeting cancer cells. We believe that the synergy of
material and pharmaceutical scientists, biologists, and clinical
oncologists is imperative to produce efficient DDS that possess
advanced properties and required functionalities to fight cancer.
This work is a testimony of this important function.
Acknowledgments
References
1. Bilalis P, Skoulas D, Karatzas A, Marakis J, Controlled polymerization of histidine and syn-

Stamogiannos A, Tsimblouli C, Sereti E, thesis of well-defined stimuli responsive poly-
Stratikos E, Dimas K, Vlassopoulos D, Iatrou mers. Elucidation of the structure–aggregation
H (2018) Self-healing pH- and enzyme relationship of this highly multifunctional mate-
stimuli-responsive hydrogels for targeted deliv- rial. Polym Chem 5:6256–6278
ery of gemcitabine to treat pancreatic cancer. 5. Altunbas A, Pochan DJ (2012) Peptide-based
Biomacromolecules 19:3840–3852 and polypeptide-based hydrogels for drug
2. Hadjichristidis N, Iatrou H, Pispas S, Pitsikalis delivery and tissue engineering. Top Curr Chem
M (2000) Anionic polymerization: high vac- 310:135–167
uum techniques. J Polymer Sci Part A-Polymer 6. Huang J, Hastings CL, Duffy GP, Kelly
Chem 38:3211–3234 HM, Raeburn J, Adams DJ, Heise A (2013)
3. Aliferis T, Iatrou H, Hadjichristidis N (2004) Supramolecular hydrogels with reverse thermal
Living polypeptides. Biomacromolecules gelation properties from (oligo)tyrosine con-
5:1653–1656 taining block copolymers. Biomacromolecules
4. Mavrogiorgis D, Bilalis P, Karatzas A, Skoulas 14:200–206
D, Fotinogiannopoulou G, Iatrou H (2014)
Chapter 11
Polymersomes from Hybrids-Polypeptides for Drug

Delivery Applications
Hermis Iatrou, Panagiota G. Fragouli, Dimitris Skourtis,
and Ioanna Stavropoulou
Abstract
Recently, the explosion of progress of materials at the nanoscale level has paved the way for a new category
of healthcare technologies termed nanomedicine. Nanomedicine involves materials at the nanometer level
for products that can improve the currently used technologies for biomedical applications. While tradi-
tional therapeutics have allowed for limited control of their distribution in the body and clearing times,
engineering at the nanoscale level has allowed for significant advances in biocompatibility, biodistribution,
and pharmacokinetics. Among all materials, polymers have dominated the nanomedicine world, due to
their ability to manipulate their properties by combining different materials in a wide variety of macromo-
lecular architectures. The development of novel polymeric materials is guided by the goal of improving
patient survival and quality of life by increasing the bioavailability of drug to the site of disease, targeting
delivery to the pathological tissues, increasing drug solubility, and minimizing systemic side effects.
Polymersomes (vesicles) are the only type of polymeric nanocarriers that can physically encapsulate at the
same nanoparticle hydrophilic drugs in their aqueous interior and/or hydrophobic agents within their
lamellar membranes. Polymersomes have been shown to possess superior biomaterial properties compared
to liposomes, including greater stability and storage capabilities, as well as prolonged circulation time.
Key words Nanoparticles, Nanotechnology, Pancreatic cancer, Polymersomes, Polypeptides, Ring-

opening polymerization
1 Introduction
Well-defined amphiphilic polymers of the ABA and ABC type are

synthesized, where A is poly (l-lysine hydrochloride) (PLL), B is
poly(γ-benzyl-(d7) l-glutamate) (PBLG(-d7)), and C is
poly(ethylene oxide) (PEO) [1] (Fig. 1). Both polymers form
polymersomes in water. The polymersomes are loaded with doxo-
rubicin or paclitaxel. It is found that in the ABC, due to asymmetry
of the two hydrophilic blocks, PEO is always on the outer periph-
ery and the dimensions of the vesicles are smaller. The release of
the vesicles is temperature and pH dependent. In vivo, the empty
139
Fig. 1 Reactions used for the synthesis of the amphiphilic triblock-co-polypeptides poly(l-lysine hydroc-
hloride)-b-poly(γ-benzyl-d7-l-glutamate)-b-poly(l-lysine hydrochloride)
vesicles are not toxic. In vitro activity of the loaded vesicles against
human pancreatic cancer cell lines reveals comparable activity to
Myocet for the ABA loaded with doxorubicin, while lower activity
is observed for the ABC.
2 Materials
1. γ-Benzyl-l-glutamate (>99%).
2. l-Lysine (>99%).
3. Poly(ethylene oxide) end-functionalized monoamine used as
monofunctional macroinitiator.
4. N,N-dimethylformamide (DMF) (99.9+%, special grade for
peptide synthesis with less than 50 ppm of active impurities) is
the polymerization solvent (see Note 1).
5. 1,6-Diaminohexane (99.9%) serves as the initiator for the tri-
block copolypeptide (see Note 2).
6. Benzene (99%, thiophen free grade) (see Note 3).
7. Triphosgene (99%) was used as purchased.
Nanostructured Polymersomes for Controlled Drug Delivery 141
8. Triethylamine (Et3N >99%) (see Note 4).

9. Purification of tetrahydrofuran (THF; dried, max 0.005%
water) was performed using standard high-vacuum techniques
and dried over sodium potassium alloy.
10. Ethyl acetate (>99.5%) was fractionally distilled over phospho-
rous pentoxide.
11. Hexane (>99%, Merck) was fractionally distilled over sodium
and subsequently distilled over n-BuLi.
12. Solid Dox (98%) and Pacl (98%) were used as purchased (see
Note 5).
3 Methods
3.1 Instrumentation 1. Use size-exclusion chromatography (SEC) to determine the Mn

and Mw/Mn values. Use two SEC systems, one in order to
obtain the molecular weights of the protected polymers and the
other for the determination of the amount of Pacl contained in
the vesicles. The first system is composed of a 600 high-pressure
liquid chromatographic pump, columns for high temperatures,
a differential refractometer detector, and a two-angle (15°, 19°)
light scattering detector at 60 °C. A 0.1 N LiBr solution in
DMF is used as an eluent at a rate of 1 mL.min−1. The second
system is composed of a 600 high-pressure liquid chromato-
graphic pump, columns, and a differential refractometer detec-
tor at 25 °C. The system runs using a mixture of CHCl3/Et3N
as a mobile phase in a ratio of 95/5 (v/v).
2. Achieve the 1H NMR spectra of the polymers and NCAs in
CDCl3 at room temperature, and that of the triblock polymers
in dimethyl sulfoxide (DMSO).
3. Carry out Fourier transform infrared measurements (FT-IR) in
KBr pellets at room temperature, in the range of 450–4000 cm−1.
Use less than 1 mg of samples for each spectrum.
4. Perform UV spectroscopy from 190 to 500 nm, at room tem-
perature with cells requiring 120 mL.
5. Conduct dynamic light scattering measurements with a system
composed of a goniometer with a stepper motor controller, a
variable power Ar+ laser operating at 690 nm and with 10 mW
power, a temperature control unit, and a pump/filtering unit.
Analyze correlation functions by the cumulant method and the
Contin software. The correlation function is collected at 90°.
Perform all measurements in isotonic Tris buffer (0.010 M) at
pH = 7.4. Measure in the concentration range between 5 × 10−5
and 1 × 10−6 g.mL−1.
6. Measure the electrophoretic mobility of the empty and drug-

loaded vesicle dispersion. All the measurements are at least the
average of three runs performed at 25 °C. Carry out all mea-
surements in isotonic (0.150 M NaCl) Tris buffer (0.010 M) at
pH = 7.4.
7. Cell cultures: Human pancreatic adenocarcinoma cell lines
AsPC1 and BxPC-3, obtained from the American Type Culture
Collection. Propagate the cells in medium containing 5% fetal
calf serum and antibiotics (penicillin, streptomycin) in 5% CO2
atmosphere in a 37 °C incubator.
3.2 Synthesis Perform the synthesis using high-vacuum techniques [2]. The
of Monomers purity of the NCA monomers is crucial for their successful living
polymerization utilizing ROP through primary amine difunctional
initiator and is confirmed by FTIR along with 1H NMR spectros-
copy analysis [3].
3.2.1 Synthesis
of ε-tert-Butoxycarbonyl-l- 1. Add Nα,Nε-Di-(tert-butoxycarbonyl)-l-lysine into a flask, place
Lysine N-Carboxy it on the vacuum line, and pump overnight.
Anhydride, (Νε BOC-l-LYS
2. Distill purified ethyl acetate, followed by argon insertion in
NCA)
order to reach atmospheric pressure and by the addition of tri-
phosgene. Leave the mixture to react for 10 min.
3. Dilute triethylamine in dry ethyl acetate, add it dropwise, and
immerse the solution in an ice-water bath for 6 h.
4. Filter the precipitate, in order to remove the HCl salt of trieth-
ylamine, and immerse the clear solution in an ice bath.
5. Extract the NCA repeatedly with Milli-Q water, until neutral
pH of the aqueous phase is achieved.
6. Recrystallize the purified NCA three times under high vacuum
in a custom-made apparatus, with ethyl acetate/hexane
(1/5 v/v) pair at −20 °C. The yield is 60%.
3.2.2 Synthesis
of γ-Benzyl-l-Glutamate 1. Suspend γ-benzyl-l-glutamate in dry ethyl acetate followed by
N-Carboxy Anhydride addition of triphosgene.
(BLG-NCA)
2. Heat the mixture at 70 °C until the solution becomes clear,
indicating the formation of the NCA.
3. Distill off the solvent in the vacuum line, and distill fresh dry
ethyl acetate in the flask, to dissolve the crude NCA, followed
by removal of the solvent by distillation.
4. Repeat twice this procedure in order to remove the excess phos-
gene that sublimes under high vacuum.
5. Remove the unreacted species, such as free amino acids along
with the HCl salts of the amino acids produced during the syn-
thesis, by extraction with an alkali solution in water.
6. Purify further the resulted BLG-NCA by three recrystalliza-

tions from dry ethyl acetate/hexane (1/5 v/v) under high
vacuum at −20 °C, leading to a 65% yield of the BLG-NCA
formation.
3.3 Synthesis 1. Dissolve the copolypeptides in a mixture of trifluoroacetic acid

of the Triblock and dichloromethane (TFA/DCM 1:1, v/v) containing a few
Copolypeptide mL of anisole, for 1.5 h (see Note 6).
PLL-b-PBLG-d7-b-PLL 2. Distill TFA and DCM, and remove the low-molecular-weight
salts and reagents, using dialysis in aqueous HCl (pH = 5.0) (see
Note 7).
3.4 Synthesis The reactions used for the synthesis of the hybrid triblock terpoly-
of the Novel Triblock mer PEO-b-PBLG-b-PLL are shown in Fig. 2.
Copolypeptide
1. Pump to dryness in the HV line the amino end monofunctional
PEO-b-PBLG-b- PLL
PEO (1.0 g, 0.112 mmol of C-NH2 groups) (see Note 8).
2. Distill purified benzene (100 mL) (see Note 9).
3. Distill off the solvent and leave the polymer to dry overnight.
4. Distill DMF (10 mL) to dissolve the polymer.
5. Filtrate the solution in an ampoule (see Note 10).
6. Pump in the high-vacuum line to dryness for 1 day, 1.27 g
BLG-NCA (4.86 mmol).
7. Dissolve the monomer in 20 mL of freshly distilled DMF (see
Note 11).
8. Rupture the glass magnet of the macroinitiator ampoule,
allowing the ring-opening polymerization of BLG-NCA to
occur under vigorous stirring for 2 days, with occasional
degassing.
9. Remove an aliquot of the solution after the completion of the
polymerization for characterization of the PEO-b-PBLG
diblock copolymer.
10. Finally, add via cannula 1.22 g (4.50 mmol) of Boc-L-lysine-
NCA solution in DMF (see Note 12).
11. Transfer the PEO-b-PBLG solution in the Ar/vacuum line,
and leave under vigorous stirring for 3 days, with occasional
degassing, ultimately yielding PEO-b-PBLG-b-PBocLL (see
Note 13).
12. In order to obtain the PEO-b-PBLG-b-PLL: Dissolve 2.95 g
of the terpolymer in 15 mL of DCM.
13. Add TFA (DCM/TFA = 1/1 (v/v), ca. 10% w/w polymer
concentration) and leave it to react for 3 h.
14. Distill off the excess organic acid along with the solvent in the
HV line.
Fig. 2 Reactions used for the synthesis of the amphiphilic triblock hybrid terpolymer poly(ethylene oxide)-b-
poly(γ-benzyl-l-glutamate)-b-poly(l-lysine hydrochloride)
15. Dissolve the polymer in Milli-Q water in the presence of HCl,

to reach a pH = 5.0.
16. Dialyze six times against Milli-Q water with pH = 5.0 (see
Note 14).
17. Freeze-dry the final product.
Fig. 3 Schematic representation of the vesicles formed with the encapsulated

drugs and the curvature of the interphase. The lower asymmetry in the hydro-
philic blocks results in larger dimensions of the vesicles and lower curvature of
the interphase
3.5 Drug Loading 1. Dissolve 10 mg of the triblocks in 4 mL of DMSO (see Note 15).
(See Fig. 3) [4, 5] 2. Add 5 mg of Dox (HCl salt) after obtaining a clear solution (in
the case of PEO-b-PBLG-b-PLL) or as lightly turbid solution
3.5.1 Doxorubicin (in the case of PLL-b-PBLG-b-PLL).
Loading
3. Leave for half an hour to be dissolved.
4. Place the solution in a dialysis bag (see Note 16).
5. Dialyze against isotonic Tris buffer at pH = 7.4 (0.150 M NaCl,
0.010 M Tris) or isotonic carbonate buffer at pH = 10.5.
6. Take out the dialysis bag at pH = 10.5, to a 2 L solution of Tris
buffer at pH = 7.4 (see Note 17).
3.5.2 Pacl Loading 1. Dissolve 10 mg of polymer in 4 mL of DMSO (see Note 18).

2. Add 2 mg of Pacl, and leave for 1 h to be dissolved.
3. Place the solution in a dialysis bag (see Note 19).
4. Dialyze against isotonic Tris buffer at pH = 7.4 (0.150 M NaCl,

0.010 M Tris).
5. Place a small stir bar in the dialysis bag and vigorously stir dur-
ing the exchange of the solutions (600 rpm).
6. Remove excess of insoluble Pacl in the buffer by centrifugation
at 112 × g at 20 °C for 10 min.
7. Separate the supernatant very carefully from the solid (Pacl).
8. Repeat the centrifugation three times (see Note 20).
4 Notes
1. N,N-dimethylformamide is further purified by short-path

fractional distillation under high vacuum in custom-made
apparatus. Use always the middle fraction.
2. 1,6-Diaminohexane is a highly hygroscopic compound, leave
it to dry over a sodium mirror for 24 h, then dilute with puri-
fied DMF, subdivide into ampoules, and store under high
vacuum (HV) at room temperature.
3. Benzene is purified by stirring over concentrated sulfuric acid
for a week. It is subsequently washed with an aqueous solution
of NaOH and then with water many times until it becomes
neutral and finally dry with CaCl2. The dry material is then
transferred into a round-bottom flask containing fresh finely
grounded CaH2 and a magnetic stirring bar, and is attached to
the vacuum line and degassed. Leave the flask for reaction of
CaH2 with moisture overnight, degas again, and distill in a
calibrated cylinder containing n-BuLi and styrene.
4. Triethylamine is dried over calcium hydride for 1 day and then
distilled and stored in the vacuum line over sodium. The
appropriate quantity needed is freshly distilled in a vacuum
line prior to use.
5. For in vitro and in vivo experiments use the injectable forms of
Dox (adriamycin 2 mg.mL−1) and Pacl (taxol 6 mg.mL−1).
6. Since the benzyl groups are deuterated, the composition of
the protected copolypeptides cannot be obtained by 1H NMR
spectroscopy, but is verified using UV spectroscopy in DMF,
since the PBLG-d7 block absorbs at 267 nm. Anisole is used
as a scavenger of the tert-butyl cations. It is well established
that Boc and benzyl groups can be orthogonally deprotected:
the former under acidic and the latter under basic conditions.
Confirm the complete removal of the Boc groups by 1H NMR
spectroscopy.
7. The Boc groups are selectively deprotected.
8. Use a custom-made 100 mL glass apparatus (equipped with a
filter), which has been previously flame dried several times.
9. Benzene is used in order to azeotropically remove traces of

water.
10. The solution in the ampoules will be used as the macroinitiator.
11. The dissolution of the monomer takes place in another spe-
cially designed custom made glass apparatus containing the
macroinitiator equipped with a breakseal.
12. Custom-made 100 mL glass reactor.
13. The triblock copolymer is precipitated in cold ether and dried
in vacuo. The amount of polymer obtained is 2.95 g (96%).
14. Adjust the pH by adding drops of concentrated HCl, 1 day
each. The amount of polymer obtained is 2.40 g (88%).
15. The polymer PEO-b-PBLG-b-PLL loaded with Pacl will be
indicated as PEOPacl, the same polymer loaded with Dox as
PEODox, while the PLL-b-PBLG-d7-b-PLL loaded with Dox
will be indicated as PLLDox (Fig. 4). Load Dox at different
pH, i.e., pH = 7.4 and pH = 10.5, and at different initial
concentrations.
16. Spectrapor, molecular weight cutoff (MWCO): 3500 Da, 30 °C.
17. Initially deprotonate the Dox (hydrochloride), to become
hydrophobic Dox free base. This would then be condensed in
the hydrophobic block and finally be dissolved in the interior
of the vesicle. Vesicle concentration obtained is 1.67 mg.mL−1.
Dox loading will be given through the loading efficiency (%)
(mass of Dox in vesicles/Dox mass in the initial solution) and
loading content (%) (mass of Dox in vesicles/polymer mass).
18. Pacl is loaded at pH = 7.4 at 20 °C and at different
concentrations.
19. Spectrapor MWCO 3500 Da, 20 °C.
Fig. 4 The synthesized polymers PEO-b-PBLG-b-PLL and

PLL-b-PBLG-d7-b-PLL
20. Obtain the optimum conditions for the centrifugation by SEC

analysis of the supernatant and the solid (free Pacl) in the sys-
tem running CHCl3/Et3N. Under these conditions only free
Pacl is precipitated leaving the vesicles with the encapsulated
Pacl in solution. The amount of Pacl in the vesicles is obtained
by SEC analysis, using an appropriate set of columns in order
to separate the polymer from the drug. Quantification is per-
formed by setting calibration curves of Pacl in CHCl3/Et3N,
where the drug is soluble.
5 Results
5.1 Synthesis and A special set of size-exclusion columns was used in order to charac-
Characterization of the terize the polymer and determine the concentration of Pacl in
Polymers and polymersomes. As Pacl is a rather big molecule, compared to other
Determination of the side species of the solvent, it can be clearly separated and quanti-
Concentration of Pacl fied, by making a calibration curve. The polymer can also be quan-
in Polymersomes [6] tified, since it can be separated from Pacl that has a much smaller
hydrodynamic volume. It is also obvious that Pacl has smaller poly-
dispersity compared to the polymeric material.
5.2 Loading The highest concentration achieved for Dox was 0.89 mg Dox
and Stability mL−1 at pH = 7.4. The highest Dox loading efficiency achieved was
33.0% and loading content of 19.5%. The PLLDox vesicles
remained stable in storage for at least 12 h in a 10 vol% fetal calf
serum at 37 °C, while the PEODox vesicles in a 10 vol% fetal calf
serum at 37 °C were stable only for 2 h.
The highest Pacl concentration achieved was 0.4 mg Pacl
mL−1. The highest Pacl loading efficiency achieved was 25.0% and
a loading content of 13.0%. The PEOPacl nanoparticles remained
stable over storage at 37 °C for at least 12 h containing 10 vol%
fetal calf serum.
5.3 Polymersome In all polymersomes formed by the PEO-b-PBLG-b-PLL (blank

Characteristics and loaded), the zeta potential measured was between 3.2 and
5.4 mV, revealing that PEO is always in the outer periphery and
PLL is in the interior of the polymersome, since if PLL was in the
outer periphery, the zeta potential would be positive and much
higher. The dimensions of the nanoparticles were determined by
DLS, with an average diameter ranging from 100 to 300 nm.
5.4 In Vitro Release The release is temperature and pH dependent. In case of PEOPacl,
and Activity the release is higher at higher pH values and temperatures. In case
of Dox-loaded polymers, the release increases at lower pH values
and higher temperatures. In all cases the release profiles show a
rather quick release in the beginning and a slower release after
15–20 h that continued for more than 3 days, revealing a slow
sustainable (controlled) drug release.
In cultures where polymers loaded with Dox were added,

slight sediments were observed 24 h upon their addition at the
highest concentration tested. However, cultures with lower con-
centrations of the Dox-loaded polymers as well as those where
Pacl-loaded polymers were added were clear of sediments. Pacl-
loaded polymers (i.e., PEOPacl) were found to be the most active
among the three loaded polymers tested. From the two Dox-
loaded polymers, PEODox-loaded polymer (PEODox) was found
to be the less active as it showed only marginal antiproliferative
activity against BxPC-3 cells. The PLL polymer (i.e., PLLDox)
exhibited higher activity, which in fact was very close to that of
Myocet in regard to its antiproliferative and cytostatic activity. The
significantly lower activity of the PEODox might be due to the
lower stability that these nanoparticles showed.
5.5 In Vivo Empty PEO and PLL polymers were also tested for acute toxicity
Toxicity Study in immunocompromised (SCID) mice. Both polymersomes were
administered intraperitoneally in a single injection to the animals at
the following doses: 200, 133, 100, and 67 mg.kg−1. Animals were
subsequently weighted and observed for a period of 7 days for
signs of toxicity or changes in their routine. The only side effect
observed during this period was a light sedation starting 5 min and
progressing until 15 min after the administration of the 200 mg.kg−1
dose for both PEO-b-PBLG-b-PLL and PLL-b-PBLG-d7-b-PLL
polymers. All animals recovered 24 h later and no further sign of
toxicity was recorded until the end of the observation period.
6 Conclusions
Comparing the ABA triblock copolypeptide and ABC terpolymer,

both formed polymersomes in water, due to the macromolecular
architecture of the polymers along with the use of a rod middle
block.
It was found that the hydrophilic block with the higher volume
fraction in the ABC architecture remains in the outer periphery of
the formed vesicle. Therefore, it is possible to select between two
different hydrophilic blocks to be at the outer periphery just by
choosing their molecular characteristics. In case one of these is
PEO, it is preferred to be in the shell due to its properties.
Positively charged nanoparticles are incorporated faster into
the cell due to the presence of negatively charged species on the
cell membranes. This would result in higher accumulation of drug
within the cells for the ABA copolypeptide rather than the ABC,
and consequently higher activity. It is expected to further elucidate
the structure-delivery relationship in order to achieve more effi-
cient complex structures in terms of activity and selective delivery
of multiple anticancer drugs and genes.
Acknowledgments
References
1. Iatrou H, Dimas K, Gkikas M, Tsimblouli C, 4. Chécot F, Brûlet A, Oberdisse J, Gnanou Y,
Sofianopoulou S (2014) Polymersomes from Mondain-Monval O, Lecommandoux S (2005)
polypeptide containing triblock co- and terpoly- Structure of polypeptide-based diblock copoly-
mers for drug delivery against pancreatic cancer: mers in solution: stimuli-responsive vesicles and
asymmetry of the external hydrophilic blocks. micelles. Langmuir 21:4308–4315
Macromol Biosci 14:1222–1238 5. Matsumura Y, Kataoka K (2009) Preclinical
2. Hadjichristidis N, Iatrou H, Pispas S, Pitsikalis and clinical studies of anticancer agent-
M (2000) Anionic polymerization: high vac- incorporating polymer micelles. Cancer Sci
uum techniques. J Polymer Sci Part A-Polymer 100:572–579
Chem 38:3211–3234 6. Disher DE, Ahmed F (2006) Polymersomes.
3. Aliferis T, Iatrou H, Hadjichristidis N (2004) Living Annu Rev Biomed Eng 8:323–341
polypeptides. Biomacromolecules 5:1653–1656
Chapter 12
Drug Delivery Systems Based on Modified

Polysaccharides: Synthesis and Characterization
Aikaterini-Foteini Metaxa, Eleni Vrontaki, Eleni K. Efthimiadou,
Abstract
Common chemotherapeutic drugs exhibit no specificity for cancer cells and destroy simultaneously healthy
cells exhibiting high toxicity and reduced efficacy. The use of nanotechnology, especially of drug delivery
systems to the size of the nanoscale, provides rational drug design solutions. Such nanomaterials may have
a range of desired characteristics (lack of toxicity, response to certain characteristics of the cancer cells,
antimicrobial properties, specific activity, etc.) in order to achieve targeted cancer therapy. In this chapter,
polymeric systems with core-shell structure are synthesized, characterized, and studied as potent drug
delivery devices for targeted cancer therapy. These polymeric systems are based on natural polysaccharides
like cellulose, chitosan, and their derivatives, in combination with synthetic polymer. Polymethylmethacrylate
(PMMA) nanospheres are used as a core in order to coat the surface with multiple layers of polysaccharides
via layer-by-layer deposition. This design is advantageous due to the use of water as the appropriate sol-
vent. Fabricated polymeric carriers are characterized structurally by AT-IR spectroscopy and morphologi-
cally by transmission (TEM) and scanning electron microscopy (SEM). Finally, daunorubicin, an anticancer
agent, was encapsulated as a drug model into the carriers.
Key words Drug delivery systems, Polysaccharides, Chitosan, Cellulose, Drug vehicles
1 Introduction
In recent years, scientific research has focused on investigating new

systems for early diagnosis and therapy against different types of
cancer. Although a broad variety of chemotherapeutic agents are
developed, these suffer from a lack of specificity [1]. Due to this,
the chemotherapeutic agents along with the tumor cells destroy
also the healthy cells and therefore exhibit high toxicity [2]. To
reduce the toxicity, dose administration is minimized and at the
same time their activity and their effectiveness are reduced [3].
These problems can be solved with the use of drug delivery sys-
tems at the nanoscale, aimed directly at the pathogenic area [4, 5].
151
152 Aikaterini-Foteini Metaxa et al.
Natural polysaccharides and their derivatives represent a group

of polymers widely used in pharmaceuticals and biomedicine for
the controlled release of drugs. Polysaccharides have several advan-
tages over synthetic biopolymers to be used as a raw material for
the synthesis of new drug delivery systems. These materials are
nontoxic, and have good biocompatibility and low production
cost. Furthermore, polysaccharides are used for nanoparticle coat-
ing and are attractive candidates for biomedical applications
because of their biocompatibility and biodegradability. In addition,
the nanoparticles coated with polysaccharides are not recognized
by the phagocytic system. Since polysaccharides are hydrophilic
polymers, they induce system stability in the bloodstream and they
can lengthen the life span in the body and therefore increase the
absorption of encapsulated drugs. Thus, a combined use of poly-
saccharides with synthetic water-soluble polymers leads to the pro-
duction of materials with improved biochemical and mechanical
properties [6–9].
With about 1011 tons of cellulose growing and disappearing
annually, cellulose is the most common organic polymer in earth, a
poly-dispersed linear homopolymer consisting of region- and
enantioselective β-1,4 glycosidic-linked D-glucose units. In our
work, we use two derivatives of cellulose: cellulose succinate (CS)
and hydroxypropyl cellulose (HPC). CS is a pH-sensitive polymer
that is synthesized in solid phase at 398 K, without the use of sol-
vents, only by using the melting point of succinic anhydride [10,
11]. HPC is a nonionic biodegradable polysaccharide, derivative of
cellulose which is a thermosensitive polymer, with a low critical
solution temperature in water (LCST) observed at 41 °C [12].
Chitosan is compatible with the biological tissues and does not
cause allergic reactions. It is biodegradable as it is cleaved by
enzymes in harmless products (amino sugar) which are completely
absorbed by the human body without causing side effects [13].
Chitosan possesses pharmacological properties such as hypocholes-
terolemic action, enhances healing of wounds, helps in the treat-
ment of stomach ulcer, and has antimicrobial action [14, 15].
In the last years, searching the literature it can be realized that
there is an increase of publications related to drug delivery systems
based on polysaccharides. However, only few of the publications
are focusing on systems based on cellulose. In addition these pub-
lications do not focus particularly on pharmaceutical formulations
for specific diseases with in vitro and/or in vivo applications.
2 Materials
1. Polysaccharides: Powdered cellulose, MW = 78,000–

98,000 g/mol, was purchased from Riedel de Haen Ag Seelze—
Hannover. The medium molecular weight chitosan,
Design of Drug Delivery Systems Based on Polysaccharides 153
deacetylated, and hydroxypropyl cellulose MW = 370,000 g/

mol were purchased from Aldrich. Methyl methacrylate (99%
MMA) was purchased from Aldrich and purified by distillation
before use. Succinic anhydride was purchased from Sigma-
Aldrich and purified with recrystallization. Daunorubicin
hydrochloride (DNR) was provided by Pharmacia & Upjohn
and used as received.
2. Solvents: Deionized and distilled water was used in all
processes.
3 Methods
3.1 Synthesis The synthetic procedure of the PMMA@HPC@CS@CH micro-

of PMMA@HPC@CS@ spheres consists of four steps: (a) synthesis of PMMA nanospheres
CH Nanospheres (NCs) (NCs), (b) synthesis of PMMA@HPC NCs, (c) synthesis of
PMMA@HPC@CS NCs, and (d) synthesis of PMMA@HPC@CS@
CH NCs (see Note 1, Fig. 1).
Initially, PMMA nanospheres are synthesized by random radi-
cal emulsion polymerization of methyl methacrylate:
1. In a round-bottom flask, add:
(a) 28 mL H2O (see Note 2).
(b) 3 mL Methyl methacrylate (MMA) (see Note 3).
(c) 0,015 g KPS initiator (K2S2O8, t1/2 = 8 h at 70 °C).
2. Leave the reaction overnight for completion.
3. Centrifuge the milky emulsion for nanospheres’ isolation (see
Note 4).
Continuously, PMMA nanospheres are used as core for the
polysaccharides’ coating by sequential deposition technique of
cortices (layer-by-layer deposition).
Forming of the first layer by deposition of hydroxypropyl cellu-
lose (HPC):
4. Dissolve 0.4 g of hydroxypropyl cellulose (HPC) in 50 mL
acidic solution (see Note 5).
5. Add and disperse 0.4 g of PMMA spheres in 50 mL aqueous
solution (see Note 6).
6. Leave the mixture under stirring for 24 h at 50 °C.
7. Centrifuge the emulsion (see Note 7).
Forming of the second layer by succinic acid-modified cellulose
(CS, see Note 8):
8. Dissolve 0.1 g of cellulose succinate in 50 mL of basic solution
(see Note 6).
9. Add and disperse 0.4 g of HPC-coated spheres in 50 mL of
acidic aqueous solution (see Note 5).
MMA Core MMA@HPC
HPC CS
MMA
HPC
CS
CHIT
EDC/NHS
DAUNORUBICIN
CHIT
crosss lin
cro linkk
Fig. 1 Synthetic scheme of the present study
10. Add the suspended microspheres to the aqueous solution of

cellulose.
11. Leave the mixture under stirring at room temperature for
24 h.
Forming of the third layer by chitosan deposition (CS) (see
Note 10):
13. Dissolve 0.05 g of chitosan in 25 mL aqueous acidic solution
(see Note 5).
14. Add and disperse 0.4 g of modified spheres in 50 mL of basic
aqueous solution (see Note 6).
15. Add the dispersion to the aqueous solution of chitosan.
16. Leave the mixture under continuous stirring for 24 h at room
temperature.
Aiming at cross-linking the three layers are deposited on vehi-
cle’s surface:
18. Disperse 0.4 g of coated PMMA@HPC@CS@CH spheres in
2 mL water.
19. Add aqua solution of EDC (10 mg, 0.06 mmol) and NHS
(5 mg, 0.04 mmol) in the resulting emulsion.
20. Leave the mixture for 30 min under vigorous stirring.
21. Centrifuge the final mixture (see Note 9) and purify after sus-
pended in water.
3.2 Drug Drug encapsulation or drug loading is the process of incorporating

Encapsulation the pharmaceutical compound into a polymeric network or cap-
sule. The release of the drug in a controlled manner is the inverse
process by which the drug molecules leave the solid phase of the
polymeric network. Loading and drug release are closely related
processes that depend on the physicochemical properties of the
polymeric network, the physicochemical properties of the drug,
and the environmental impact [16].
The drug can be trapped in the system by intermolecular inter-
actions with the polymeric network, such as hydrogen bonds, ionic
interactions, and intramolecular dipole-dipole interactions. It can
also be trapped on the surface by adsorption. In most drug delivery
systems, encapsulation occurs through different mechanisms at the
same time [16].
In the present study, the anticancer drug daunorubicin was
used as a standard pharmaceutical compound (Fig. 1).
1. In a beaker disperse 10.0 mg of synthesized nanospheres in
10 mL of isotonic solution (0.9 wt% NaCl).
2. Add 4 mg of daunorubicin hydrochloride, DNR.
3. Leave the mixture to stir gently for 48 h at 25 °C.
4. Centrifuge (see Note 11) and keep the isolated supernatant to
determine the amount of daunorubicin not entrapped in the
spheres through standard curve method (Scheme 1, see Note
12). UV–vis absorption spectra in the wavelength range of
200–800 nm were obtained on a Jasco V-650 spectrometer,
with UV–vis at 480 nm spectrometer. An ultrasonic bath was
used for sonication.
Using the reference curve (Fig. 2), the amount of drug trapped
can be calculated. The absorbance of isolated supernatant (1 mL)
was A = 2.38, so we can calculate the concentration of daunorubi-
cin from the equation y = 0.0157x + 0.0619 (y = A and x = c (μg/
mL)). The concentration of the non-loaded drug was 147.65 μg/
mL, and as we know the total volume of the supernatant solution,
we can easily calculate the mass of the non-loaded
(10 mL × 147.65 μg = 1.4765 mg) and loaded drug (4 mg –
1.4765 mg = 2.5235 mg). Also, the encapsulation efficiency and
the ability to capture it from the microspheres (loading capacity)
are calculated according to the following equations (see Table 1):
Weight of the loaded drug in vehicles

Encapsulation capacity ( % ) = ×100
Total weight of the vehicles

Weight of the loaded drug in vehicles
Encapsulation efficiency ( % ) = ×100
Weight of the starting amount of drug

3,5
3 y = 0,0157x + 0,0619
R2 = 0,9958
2, 5
A 2
1, 5
0, 5
0
0 50 100 150 200 250
c(μg/ml)
Fig. 2 The reference curve
Table 1
Drug encapsulation results of the present study
Weight of vehicles (mg) 10

Absorbance (A) 2.38
Concentration of drug (c) at supernatant (μg/mL) 147.65
Non-loaded drug (mg) 1.4765
Starting amount of drug (mg) 4
Loaded drug (mg) 2.5235
Encapsulation efficiency (%) 63%
Encapsulation capacity (%) 25%
3.3 Characterization The fabricated vehicles can be characterized structurally by FT-IR

of Vehicles spectroscopy and morphologically by scanning (SEM) and trans-
mission electron microscopy (TEM) techniques (see Notes 12–15).
Microscopy is one of the most powerful techniques and can
provide valuable information about the size, shape, and morphol-
ogy of nanoparticles. Electron microscopy provides high-resolution
images and is the only technique that provides reliable information
about the shape on this scale. However, scientists need to ensure
that enough particles are examined to have a statistically valid rep-
resentation of the size and shape distribution. This can be very
difficult and time consuming and may require the analysis of the
image of literally thousands of individual particles. However, scien-
tists need to ensure that enough particles are examined to have a
statistically valid representation of the size and shape distribution.

This can be very difficult and time consuming and may require the
analysis of the image of literally thousands of individual particles.
There are many commercially automated image analysis systems
and software packages used for this purpose. The quality of the
images presented in these systems is critical to their performance.
It should also be noted that electron microscopy normally provides
two-dimensional images, so care must be taken to avoid bias [17,
18].
3.3.1 SEM Preparation 1. Convert microspheres into powder.

2. Press a small amount of powder on a conductive adhesive tape
and mount on a holder.
3. For conventional imaging in the SEM, the samples must be
electrically conductive, at least on the surface, to prevent the
accumulation of electrostatic charge. Nonconductive materials
are usually coated with electrically conductive material depos-
ited on the sample. The conductive material used as a coating
was gold.
4. The sample is irradiated with a high-energy electron beam. The
surface characteristics of the nanoparticles are illustrated by the
secondary electrons emitted from the surface of the sample.
5. Take images after every stage of the procedure to ensure the
deposition of each layer.
SEM images of microspheres in different stages of synthesis are
presented in Fig. 3.
3.3.2 TEM Preparation

The principle of transmission electron microscopy is based on prin-
ciples different from SEM, although it often provides the same
results.
1. The microspheres are dispersed in an appropriate solvent and
deposited on thin films.
2. The specimen characteristics are represented by a thin electron
beam that permeates the sample and interacts with it.
TEM images of PMMA@HPC@CS@CH microspheres are pre-
sented in Fig. 4.
3.3.3 AT-IR Preparation 1. Convert microspheres into powder.

2. Place a small amount of powder on the instrument and
measure.
3. Take samples for each stage of the synthesis and compare the
differences in spectra.
The most important peaks of FT-IR spectra at various stages of
the synthesis are summarized in Table 2.
Fig. 3 SEM images of microspheres in different stages of synthesis. A. PMMA spheres’ diameter ranges at
200 ± 15 nm with low polydispersity. B. PMMA@HPC spheres’ diameter is increased confirming the success-
ful deposition. C. PMMA@HPC@CS microspheres’ diameter is increased to 300 ± 30 nm. D. PMMA@HPC@
CS@CH microspheres’ diameter is increased to 350–370 nm
Fig. 4 TEM images of PMMA@HPC@CS@CH microspheres. The core of polymethyl methacrylate is distin-
guished with dark color and the deposited layers of polysaccharides are distinguished with a light gray layer.
Typical configurations of spheres are due to the intermolecular hydrogen bonds that grow between the spheres.
It is also observed that the coating is relatively uneven and varies forming core-shell morphology
Table 2
AT-IR characteristic peaks of synthesized multi-sensitive microspheres
Wavenumber (cm−1) Vibration

Synthesis of PMMA@HPC@CS microspheres
3669 C-H bond of hydroxypropyl
cellulose’s propyl group
2980, 2893 Aliphatic bonds C-H
1726 (sharp peak) -C=O of methyl methacrylate
and succinic acid segment of
modified cellulose
1059 C-O-C pyranose ring (see Note
16)
892 β-Glycosidic bonds between
the structural units of the
cellulose
Synthesis of PMMA@HPC@CS@CH microspheres
1640 N-H bond of chitosan
After cross-linking
1631 -C=O of N-H (see Note 17)
4 Notes
1. This composition is advantageous in comparison to the litera-

ture, due to the fact that in all synthetic steps, water is used as
solvent, avoiding the use of organic solvents.
2. Warm to 70 °C for 30 min with vigorous stirring in a nitrogen
flow.
3. Leave the mixture for 30 min.
4. The emulsion is centrifuged by various cycles of resuspension
in water (3 × 8000 rpm (7491 × g) for 5 min).
5. Aqueous solution of HCl, pH = 3.
6. Aqueous solution of NaOH, pH = 10.
7. The resulting emulsion is purified via various resuspension
cycles in water (4 × 10,000 rpm (11704 × g) for 5 min).
8. The intermediate layer is selected to be formed by use of mod-
ified cellulose which functions as a cross-linker for stabilization
purposes between the other two layers. This stabilization has
been performed by cross-linking between the modified cellu-
lose and hydroxypropyl cellulose via ester bonds and between
chitosan through amide bond formation.
9. The resulting emulsion is centrifuged and resuspended in

water for purification (3 × 10,000 rpm (11704 × g) for 5 min).
10. The last coating aims at improving the microsphere-protein
interactions.
11. Centrifuge for 5 min at 10,000 rpm.
12. The curve is constructed by absorption measurements of stan-
dard solutions of known concentration, since according to the
Lambert-Beer law, A = ε × b × c, the concentration of drug is
proportional to absorption.
13. SEM and TEM images are obtained on a FEI Inspect micro-
scope with W (tungsten) filament operating at 25 kV and a
FEI CM20 microscope operating at 200 kV, respectively.
14. The spectra are scanned over the range of 4000–500 cm−1.
UV–vis absorption spectra in the wavelength range of 200–
800 nm were obtained on a Jasco V-650 spectrometer, with
UV–vis at 480 nm spectrometer.
15. An ultrasonic bath was used for sonication (Elma Sonic, S.
30H).
16. Structural units of cellulose.
17. The appearance of the peak at 1631 cm−1 due to the absorp-
tion of the carbonyl amide bond and the absence of the peak
at 3669 cm−1 due to vibration of O-H group of HPC is con-
firmed by the successful cross-linking. The absence of peak
attributed to hydroxyl groups of hydroxypropyl cellulose and
carboxylic acids of the CS confirms the ester bond formation.
References
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Chapter 13
Differential Scanning Calorimetry (DSC) on Sartan/

Cyclodextrin Delivery Formulations
Nikolaos Naziris, Maria Chountoulesi, Dimitrios Ntountaniotis,
Thomas Mavromoustakos, and Costas Demetzos
Abstract
Differential scanning calorimetry (DSC) is a widely utilized method for the interactions of drug molecules
with drug delivery systems (DDSs). Herein is described a protocol for studying the interactions and
entrapment efficiency of the prototype sartan losartan and the polydynamic, structurally similar irbesartan
inside the nontoxic 2-hydroxypropyl-β-cyclodextrin (2-HP-β-CD). The thermal scan properties of both
sartan molecules have been studied when physically mixed or complexed with the cyclodextrin. The ther-
mograms indeed showed significant differences between the mixtures and complexes, establishing DSC as
a valuable method to characterize the state of the drugs in these pharmaceutical formulations.
Key words Differential scanning calorimetry, Irbesartan, Losartan, 2-Hydroxypropyl-β-cyclodextrin,

Mixing, Lyophilization, Interactions, Complexation
1 Introduction
Thermal analysis (TA) techniques are widely used in order to study

solid, semisolid, or liquid substances. Some of the commonly stud-
ied materials are foods, electronic materials, polymers, organic or
inorganic compounds, biological organisms, and pharmaceuticals
[1]. In pharmaceutical sciences, the most frequently used TA
method is differential scanning calorimetry (DSC), where the heat
flow rate difference between a reference and a sample material is
measured. It is primarily applied on crystalline solids, solid disper-
sions, and polymeric dosage forms, for the characterization of
polymorphic forms, study of the effects of lyophilization, as well as
kinetics of various phenomena, such as decomposition and acceler-
ated aging [2–4].
There is the heat-flux DSC and the power-compensation DSC,
the main difference of the two being that the first uses one furnace
heater for both samples, applying the same temperature to both
163
164 Nikolaos Naziris et al.
Fig. 1 Heat-flux DSC
sample and reference, while the second uses two individual heaters,
allowing it to measure the change in power or energy that is
required for sample and reference to have the same temperature
[5]. In the herein described work, the heat-flux type was utilized
for the analysis of the cyclodextrin:sartan formulations (Fig. 1).
Concerning pharmaceutical development, thermal analysis
techniques facilitate the study of phase transitions and changes in
the heat capacity of pharmaceutical formulations, including drug
delivery systems (DDSs), such as cyclodextrins (CDs). That way,
information about the purity, polymorphism and interactions of
final pharmaceutical forms, as well as properties of packaging mate-
rials, is provided, giving the opportunity for assessment of their
lifetime physical stability. For this reason, pharmaceutical thermal
analysis is a useful tool for the drug development process, ensuring
the physical stability of the final pharmaceutical product, by study-
ing the bioactive molecules, excipients, as well as compatibility
between them [6, 7].
DSC can be utilized for the analysis of thermal phenomena in
materials and biomaterials, like melting, crystallization, glass tran-
sition, evaporation, decomposition, and dehydration. A typical
DSC thermogram or thermal scan of an amorphous solid is given
in Fig. 2 [8].
DSC has been extensively utilized for the analysis and study of
drug-cyclodextrin complexes. In particular, these include
hydroxypropyl-β-cyclodextrin (HP-β-CD) or 2-HP-β-CD com-
plexes with thalidomide, paracetamol, or meclizine HCl, where the
thermal analysis of free forms, physical mixture, and complex
between the drug and the CD allows for the evaluation of the asso-
ciation between them [9–11]. The method of complexation, as
well as the physical state of each component, affects their degree of
association. For example, it has been demonstrated that the physi-
cal mixing/grinding or the preparation of complex through the
kneading, freeze-drying, or coprecipitation method may all yield
different degrees of interaction, which are reflected on the DSC
DSC on Sartan/Cyclodextrin Delivery Formulations 165
Fig. 2 Thermogram of an amorphous compound showing glass transition (Tg), crystallization (Tc), melting (Tm)
and degradation
profiles [10, 11]. Other DSC studies on drug-CD complexes

include methyl-β-cyclodextrin (M-β-CD) with lidocaine and
2-HP-β-CD with oxicams [12, 13]. In all these cases, each compo-
nent and each complex or mixture were analyzed and through
comparison, an estimation of the nature and degree of interactions
was obtainable. DSC generally provides an insight into the system
thermodynamics, which complements classic pharmaceutical for-
mulation tests, such as phase solubility, dissolution studies, and in
vitro permeability.
Sartans and specifically irbesartan and losartan have been char-
acterized by DSC analysis [14–17]. In addition, the complex
between valsartan and β-cyclodextrin (β-CD) was prepared by vari-
ous methods, i.e. solid dispersion, freeze-drying, kneading and
physical mixture, and studied by DSC [18]. The reduction of crys-
tallinity of the system is an indication of the complexation between
the two materials and is reflected on the observed peaks in the
calorimetric profile, where the melting transition of the drug mol-
ecule is diminished or vanished.
2 Materials
The materials described below are referred to the DSC analysis of

sartan-2-HP-β-CD mixtures or complexes or the components
alone. Herein, complexes are formed by either dissolving and

suspending the molecules in aqueous medium and freeze-drying
or physically mixing raw materials and lyophilized materials in all
possible combinations. Lyophilization is applied on frozen solu-
tions of the materials in purified water. All the final samples that are
utilized for DSC analysis, whether they are single materials or mix-
tures or complexes, are in dry powder form.
2.1 Lyophilization 1. Losartan potassium (Mw = 461.01).

of Raw Materials 2. 2-HP-β-CD (Mw = 1460 (average)).
3. Purified water.
4. NH3 5 N.
2.2 Complex 1. Irbesartan (Mw = 422.91) or losartan potassium.

of Sartan 2. 2-HP-β-CD.
with 2-HP-β-CD
3. Purified water.
4. NH3 5 N.
2.3 Differential 1. DSC aluminum pans with O-ring (40 μL).

Scanning Calorimetry 2. Pure indium (Tm = 156.6 °C).
(DSC)
3. Samples:
∙∙ Irbesartan or losartan raw material
∙∙ Lyophilized losartan
∙∙ 2-HP-β-CD raw material
∙∙ Lyophilized 2-HP-β-CD
∙∙ Raw material physical mixture
∙∙ Lyophilized form mixture (see Note 1)
∙∙ Raw material 2-HP-β-CD mixture with lyophilized losartan
∙∙ Lyophilized 2-HP-β-CD mixture with raw material losartan
∙∙ Complex of sartan with 2-HP-β-CD (see Note 2)
3 Methods
Heat-flux DSC is herein applied on samples containing the cyclo-

dextrin 2-HP-β-CD and irbesartan or losartan in various states. The
samples that are subjected to DSC analysis contain the raw material
forms of the cyclodextrin and sartans; their lyophilized forms,
except for irbesartan, which cannot be lyophilized; the lyophilized
complexes between the cyclodextrin and the drugs; the mixtures of
the raw materials; the mixtures of the lyophilized forms, whenever
possible; as well as all possible mixture combinations. Lyophilization
was applied on the raw materials of cyclodextrin and losartan, in

order to analyze them alone or in mixtures, as well as on a com-
plexed form between the cyclodextrin and each sartan.
3.1 Lyophilization 1. Dissolve 2-HP-β-CD and losartan potassium in purified water

of Raw Materials separately, both at a concentration of 8 mg mL−1.
2. Freeze the solutions at −80 °C overnight (see Note 3).
3. Lyophilize samples with any appropriate lyophilizer, under the
following conditions: condenser temperature −50 °C; vacuum
8.2 × 10−2 mb; and duration 24 h.
4. Store the lyophilized powders at 4 °C.
3.2 Complexation 1. Transfer 6783.62 mg of 2-HP-β-CD in a glass vessel with

of the Sartans 800 mL of purified water and set under magnetic stirring.
with 2-HP-β-CD 2. When the cyclodextrin is totally dissolved, add irbesartan or
losartan in the vessel and continue magnetic stirring (see Note
4). The molar ratio between cyclodextrin and either drug is
3.6:1 and usually the drug used is ca 1000 mg.
3. Adjust pH at approximately 10.5 with NH3 5 N and the reac-
tion of enclosure takes place.
4. When the solution is clear, fix the final volume at 1000 mL with
purified water and immediately freeze the solution of the com-
plex at −80 °C for lyophilization to follow.
3.3 DSC Sample 1. Prepare each sample for DSC analysis by weighting roughly
Preparation 3 mg of dry powder that contains one or more components
inside a 40 μL aluminum crucible with O-ring (see Note 5).
2. Seal each crucible by using a sealing press, in order to make a
hermetic pan, and leave it to rest for a 15-min period, in order
to achieve equilibration of the sample (see Note 6).
3.4 DSC Analysis 1. Obtain the DSC thermogram of each sample by utilizing any
appropriate DSC calorimeter.
2. Calibrate the calorimeter with pure indium (Tm = 156.6 °C)
before analyses, by applying a single heating cycle and checking
the characteristic transition temperature Tm and enthalpy change
ΔH to be within specifications.
3. Include for each analysis a 5-min isotherm at 10 °C (see Note 7)
and a heating process from 10 °C to 230 °C for irbesartan-
containing samples and 10 °C to 300 °C for losartan-containing
samples (see Note 8), at a heating rate of 10 °C min−1 (see Note
9), under constant nitrogen gas flow rate of 50 mL min−1 (see
Note 10).
3.5 DSC Diagram 1. Analyze the obtained calorimetric data by using the software
and Thermodynamic installed in the thermometer.
Parameter Extraction 2. Normalize each analysis per total sample weight (see Note 11).
3. Choose the desired thermodynamic parameters for each ther-
modynamic phenomenon, i.e. endothermic or exothermic.
These are the characteristic transition temperatures Tonset and T,
enthalpy change ΔH, and width at half peak height of the Cp
profiles ΔT1/2 (see Note 12).
4. During peak integration, manually set the baseline (see Notes
13–15).
3.6 DSC Profile The profiles of all samples containing 2-HP-β-CD and irbesartan
Analysis of the or losartan are presented in Fig. 3.
Cyclodextrin:Sartan The following observations can be made on the
Systems thermograms:
∙∙ 2-HP-β-CD and its lyophilized form exhibited a melting tran-
sition at around 170 °C, with the first being more crystalline
than the second, resulting in a sharper peak and slightly higher
transition enthalpy (Fig. 3a, b) [12]. The two drugs gave tran-
sition peaks at around 185 °C and 270 °C for irbesartan and
losartan, respectively, with these values being very close to the
bibliography (Fig. 3c, d). In addition, a pre-transition was
recorded for losartan [14, 16, 17]. The lyophilized form of
losartan led to the appearance of another form of the molecule
that melts at 165 °C (Fig. 3e).
∙∙ The nature of the raw material physical mixture between
2-HP-β-CD and losartan was very different from that with
irbesartan (Fig. 3f, g). Though the molar ratio of
cyclodextrin:sartan was the same in the two cases (3.6:1), the
mixture with losartan led to an endothermic peak at 175 °C,
suggesting the existence of crystalline cyclodextrin in the mix-
ture and the loss of crystallinity of losartan, due to intermo-
lecular interactions. On the contrary, for irbesartan, there was
a low enthalpy peak at 170 °C and another at 200 °C, the first
owed to the cyclodextrin and the second to the drug, which
suggests that part of the drug has not been complexed and
only a small amount of cyclodextrin is in the crystalline state.
∙∙ The physical mixture of the drugs with lyophilized 2-HP-β-CD
led to similar profiles, where a small peak was observed for
irbesartan at 180 °C and for losartan at 240 °C. For losartan,
this should be attributed to the drug; however, in the case of
irbesartan, it could be due to the cyclodextrin, since the melt-
ing peaks of the two are very close.
∙∙ The complex of 2-HP-β-CD with irbesartan or losartan
resulted in different DSC heating profiles. For the first com-
Fig. 3 The DSC thermograms of (a) 2-HP-β-CD raw material, (b) 2-HP-β-CD lyophilized, (c) irbesartan raw
material, (d) losartan raw material, (e) losartan lyophilized, (f) mixture of irbesartan raw material with
2-HP-β-CD raw material, (g) mixture of losartan raw material with 2-HP-β-CD raw material, (h) mixture of
irbesartan raw material with 2-HP-β-CD lyophilized, (i) mixture of losartan raw material with 2-HP-β-CD
lyophilized, (j) mixture of losartan lyophilized with 2-HP-β-CD raw material, (k) mixture of losartan lyophilized
with 2-HP-β-CD lyophilized, (l) lyophilized complex of irbesartan with 2-HP-β-CD, and (m) lyophilized complex
of losartan with 2-HP-β-CD
plex, it led to a wide peak that started from 20 °C and ended

at 240 °C, while for the second, it also absorbed energy from
low temperatures; however, it gave an endothermic peak at
170 °C that resembles the one of lyophilized 2-HP-β-CD. This
suggests that an excess of crystalline cyclodextrin exists after
complexation in the case of losartan.
From all these observations, we can assume that the amount of
2-HP-β-CD necessary to complex losartan may be lower than 3.6
molecules for every molecule of drug, while for irbesartan, it seems
that it is ideal; however, the physical mixture process does not yield
100% complexation.
∙∙ The physical mixture of lyophilized losartan with raw material
2-HP-β-CD led to interaction between the two molecules and
their existence in amorphous state.
∙∙ Finally, the physical mixture of lyophilized losartan with lyoph-
ilized 2-HP-β-CD was thermodynamically very close to their
complex, which indicates the high degree of interactions
between the molecules by utilizing these methods.
4 Notes
1. The raw materials are first subjected to lyophilization and then

physically mixed, in order to be analyzed. Lyophilization is
only possible for losartan in its salt form.
2. The complex is formed by dissolving the molecules and then
freeze-drying.
3. For losartan, this can be achieved because of the drug being
available in salt form with potassium, while this does not apply
for irbesartan.
4. The process of stirring inside the cyclodextrin solution aims to
increase the insertion of the drugs inside the cavity and lead to
complexation.
5. Single-component samples are neat 2-HP-β-CD, irbesartan,
losartan, or their lyophilized forms. Samples containing mix-
ture of raw materials, their lyophilized forms, or combinations
of those are prepared by weighting raw material or lyophilized
2-HP-β-CD and raw material or lyophilized sartan, in 3.6:1
molar ratio, for a total of approximately 3 mg.
Cyclodextrin:sartan complex preparations have been described
previously.
6. Equilibration is considered necessary for samples that have
been previously hydrated with aqueous media; however, the
same applies for solids, because of the pressure applied during
the crucible sealing.
7. The isotherm at the analysis starting point is necessary to
ensure that the sample is at equilibrium.
8. Typically, the analysis temperature range depends on the melt-
ing points and the various thermodynamic alterations, endo-
thermic or exothermic, that are expected for every component
that is analyzed. As a result, the range should be the same for
all samples, in order to be able to assess all types of interactions
between the components. Based on the bibliography, the
expected melting points for irbesartan and losartan, respec-
tively, are 185 °C and 270 °C, while 2-HP-β-CD has been
reported to melt at a broad range, from around 20 °C to
150 °C [9, 14, 15].
9. A high heating rate may not allow for certain kinetic phenom-
ena to appear and as a result should not be too high. Based on
the bibliography, 10 °C min−1 is appropriate for
cyclodextrin:sartan system analysis [9, 12, 15, 16].
10. Constant pressure is essential in this type of calorimetry, in
order to monitor the heat capacity of the sample under con-
stant pressure (Cp).
11. Each analysis may be normalized per total sample, per cyclo-
dextrin, or per sartan mass, depending on which DSC peak is
studied. For example, in a physical mixture of 2-HP-β-CD and
irbesartan, a certain amount of drug is probably not com-
plexed and will melt because of its crystalline state. In cases of
complexes and mixtures, certain peaks may reflect one mate-
rial or both materials in their complexed form and as a result
should be analyzed appropriately (Fig. 4).
12. A lot of other parameters that concern the calorimetric profiles
are available to extract; however, these are the most utilized in
the bibliography [11, 14, 16].
13. The baseline may vary each time it is set. For this reason, three
different attempts should be made, in order to extract a statis-
tical sample for the enthalpy change ΔH. This does not apply
on other thermodynamic parameters that are more accurate.
14. During analysis, the baseline might deviate from its original
horizontal, due to kinetically slow endothermic or exothermic
phenomena or because of the measurement itself. In case we
wish to include this deviation during peak integration, we have
to set the baseline accordingly. In this way, comparisons
between peaks and areas that contain these peaks are attain-
able. An example is given below (Fig. 5).
15. The enthalpy change ΔH is considered negative for an endo-
thermic process and positive for an exothermic process.
Acknowledgments
We gratefully thank Prof. G. Valsami and E. Christodoulou for

their help to prepare the complexes. This work has been co-
financed by the European Union and Greek national funds through
the program “Support for Researchers with Emphasis on Young
Researchers” (call code: EDBM34, ΚΕ 14995) and under the
research title “Preparation and study of innovative forms of admin-
istration of pharmaceutical molecules targeting at improved phar-
macological properties.”
Fig. 4 The DSC profiles of (a) physical mixture of irbesartan raw material with
2-HP-β-CD raw material, (b) irbesartan raw material with 2-HP-β-CD lyophi-
lized, and (c) complex of irbesartan with 2-HP-β-CD
Fig. 5 In this example of 2-HP-β-CD lyophilized, by setting the baseline of analysis at different points, the
integration will vary, including wider areas of endothermic or exothermic processes
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orimetry (DSC): a tool to study the thermal 14. Soma D, Attari Z, Reddy MS et al (2017)
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(2011) Preparation, characterization, and Losartan potassium loaded bioadhesive micro-
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MP et al (2019) Interaction of pseudo- Molecules 15:4067–4084
Chapter 14
Encapsulation of Small Drugs in a Supramolecule

Enhances Solubility, Stability, and Therapeutic Efficacy
Against Glioblastoma Multiforme
Antonis D. Tsiailanis, Alexander Renziehausen, Serdar Karakurt,
Tim Crook, Nelofer Syed, and Andreas G. Tzakos
Abstract
Cancer occupies a high rank in the global morbidity and mortality scale with glioblastoma multiforme
(GBM) accounting for almost 80% of all primary tumors of the brain. Despite the increasing availability of
targeted and immunotherapeutic agents, chemotherapy still plays an important role in the treatment of
neoplastic diseases. Limitations to the effective use of chemotherapy such as low aqueous solubility and
high toxicity have directed the scientific community’s interest to the development of new therapeutic
agents with enhanced efficacy and limited toxicity. Supramolecular chemistry has offered an alternative way
on the design and development of new therapeutic agents as a result of their unique properties.
Supramolecules can be used as drug carriers since their cavities can host a wide range of small drugs and
surpass in this way the drawbacks of current therapeutic agents. Herein, we present the principles that
should be followed for the encapsulation of small drugs in supramolecules with enhanced physicochemical
properties and increased efficacy against glioblastoma multiforme.
Key words Glioblastoma multiforme, Temozolomide, Supramolecule, p-sulfonatocalix[4]arene,

Encapsulation, 1H-NMR spectroscopy, Mass spectrometry, Liquid chromatography, LC-MS/MS
1 Introduction
Cancer is one of the leading global causes of morbidity and mortal-

ity. Although targeted and immunotherapeutic agents are increas-
ingly available, systemic chemotherapy continues to have an
important role in the management of the neoplastic disease.
Despite obvious benefits of chemotherapy, its utility is frequently
limited, particularly in metastatic disease, by innate and/or
acquired drug resistance (the latter an inevitable consequence of
tumor heterogeneity) and by toxicity of drugs [1]. Further limita-
tions to the effective use of chemotherapy arise due to poor aque-
ous solubility, instability, and low drug-loading capacity. The ability
175
176 Antonis D. Tsiailanis et al.
to enhance efficacy and reduce toxicity, i.e., to increase the

therapeutic index of anticancer drugs, would have a significant
impact on facilitating the optimal use of chemotherapy and serving
as an important driver in improving clinical outcomes [2, 3]. A
large pool of bioactive compounds and approved drugs has evolved
from multiple medicinal chemistry projects in the pharma industry
and academia. Supramolecular chemistry has served as a useful tool
in biomedical applications [4, 5]. Due to their unique properties,
supramolecules can be used as alternative drug carriers, based on
their ability to encapsulate small drugs in their cavity. The encapsu-
lation of small molecules into the cavity is the result of noncovalent
interactions, such as van der Waals interaction, hydrogen bonding,
and hydrophobic interactions. According to recent literature, mac-
romolecules such as cucurbit[n]urils, cyclodextrins, calix[n]arenes,
and their derivatives have drawn the attention of the scientific com-
munity, since they can surpass problems that current cytotoxic
agents present [6, 7].
Recently, our group developed a new formulation that demon-
strated enhanced activity against glioblastoma (GBM), the most
common and aggressive primary brain tumor in adults, by encap-
sulation of temozolomide (TMZ) into a supramolecular host [8].
TMZ is an alkylating agent used as first-line chemotherapy for
GBM [9, 10]. However, under slightly alkaline conditions it is rap-
idly hydrolyzed to MTIC, which in turn rapidly degrades to the
methyl diazonium cation and the metabolite 5-amino-imidazole-
4-carboxamide (AIC), resulting in reduced passage across the BBB
and therefore reduced clinical efficacy [11, 12] (Fig. 1). Hence,
high doses of TMZ are administrated to achieve therapeutic con-
centrations in the central nervous system (CNS), resulting in
numerous side effects including myelosuppression.
To abrogate these issues, we used p-sulfonatocalix[4]arene
(PSC4) nanocapsule as host. PSC4 contains a hydrophilic external
surface and a hydrophobic cavity that can entrap small drugs inside.
PSC4 has been widely used as host due to its high aqueous solubil-
ity and low toxicity [13, 14]. The encapsulation of TMZ in PSC4
greatly improved the stability of TMZ and its efficacy against GBM
in vitro and in vivo (Scheme 1).
Here, we summarize the principles that influence the develop-
ment of new formulations and, in particular, the encapsulation of
small molecules into PSC4. We describe the protocols that have
been developed for the determination of the encapsulated drug
and the stability of the complex. After the synthesis of the formu-
late, LC-MS/MS plasma stability assays were conducted in mice to
further explore the stability profile of TMZ@PSC4 in vivo. Finally,
the cytotoxicity of the analog was evaluated in vitro in patient-
derived primary lines that express MGMT and are normally highly
resistant to TMZ and in a mouse orthotopic model.
Encapsulation of Small Drugs in a Supramolecule Enhances Solubility, Stability… 177
Fig. 1 Reaction scheme for the base-catalyzed decomposition of TMZ to its active metabolite MTIC and then
to 5-amino-imidazole-4-carboxamide (AIC) and methyl diazonium cation
O NH2
O
N N
N N
N HN
N N N
N
H TMZ@PSC4
NH2
O
MTIC
Temozolomide
O
NH2
N N O
N
N
O N N
HN
N NH2 N N
O H
NH N
Temozolomide MTIC
N
N NH2
O
N
NH
s
osi
N N N
o pt H
Ap
Scheme 1 Schematic representation of the degradation of TMZ to 5-(3-methyl-triazen-1-yl) imidazole-4-car-

boxamide (MTIC) outside of the cell and the mechanism of stabilization of TMZ through complexation with
PSC4
2 Materials
2.1 Synthesis 1. Temozolomide (T2577) and p-sulfonatocalix[4]arene (55523)

and Characterization were purchased from Sigma-Aldrich.
of Encapsulated TMZ 2. Methanol and water (both LC-MS grade).
3. PBS buffer (10 mM, pH 7.0): Both interactants should be dis-

solved in the same buffer.
4. Deuterated dimethyl sulfoxide 99.8% (DMSO-d6) and deute-
rium oxide 99.9% (D2O).
5. Bruker 400 MHz Avance spectrometer equipped with a
z-gradient unit (Bruker BioSpin, Rheinstetten, Germany): The
NMR system is controlled by the software TopSpin 3.1.
2.2 UV-Vis 1. PBS buffer (10 mM, pH 7.0), water (double distilled),
Spectroscopy methanol.
2. 1 mL Cuvettes.
3. Water bath.
4. 37 °C Shaker and incubator.
5. UV-Vis spectrometer (slit = 1, speed 240 nm/min).
2.3 Liquid 1. Theophylline (internal standard).

Chromatography 2. Regenerated cellulose membrane syringe filters 0.2 μm.
and Mass
3. Cellulose nitrate filters, 0.2 μm pore size, for mobile-phase
Spectrometry
filtering.
4. Acetonitrile, water, and formic acid (LC-MS grade).
5. Mobile phase A: Water containing 0.1% (v/v) formic acid.
6. Mobile phase B: Acetonitrile containing 0.1% (v/v) formic acid.
7. C18 column 100 mm × 2.1 mm, 2.6 um, with ProGuard col-
umn 2.1 mm.
8. Triple-quadrupole mass spectrometer coupled to ultrahigh-
performance liquid chromatography (Bruker’s EVOQ Elite
ER-UHPLC).
2.4 In Vitro Assays 1. Patient-derived primary GBM cell cultures (GBM31, GBM59,
of TMZ@PSC4 Activity GBM77) were established from fresh tumor tissue obtained
from first surgical debulking or stereotactic biopsies at Charing
Cross Hospital as described previously.
2. Tissue samples were provided by the Imperial College
Healthcare NHS Trust Tissue Bank, which is supported by the
National Institute for Health Research (NIHR) Biomedical
Research Centre based at Imperial College Healthcare NHS
Trust and Imperial College London.
3 Methods
3.1 Synthesis, 1. Dissolve p-sulfonatocalix[4]arene in phosphate buffer pH 7.0

Characterization, and (10 μM).
Quantification of the 2. Dilute TMZ in MeOH.
Encapsulated TMZ
3. Add dropwise TMZ in the aqueous solution and magnetically
3.1.1 Synthesis stir at room temperature for 1–2 h (see Note 1).
and Purification 4. Remove MeOH under low pressure to precipitate the unencap-
of Nanocapsule sulated drug.
5. Filter contents through a nylon filter with 0.45 μm pore size.
6. In order to obtain the complex evaporate aqueous phase under
high vacuum.
Fig. 2 1H-NMR spectra of PSC4 (green), TMZ (blue), and TMZ@PSC4 complex (red). The significant alteration
observed in the chemical shift of the Hb proton of the imidazotetrazine ring of TMZ in the NMR spectrum of the
TMZ@calix complex concerning the native TMZ pinpoints that the imidazotetrazine ring of TMZ is incorporated
deep inside the hydrophobic cavity of calix
Fig. 3 Calibration curve for the determination of TMZ by UV-Vis
3.1.2 Characterization
of the Nanocapsule 1. Perform the characterization of compounds via 1H-NMR and
13
C-NMR spectroscopy.
2. Dissolve 2 mg of each compound in 500 uL D2O in an
Eppendorf.
3. Transfer samples to 5 mm NMR tubes.
4. Load the sample into the Bruker AV-400 spectrometer.
5. Lock, tune, and match the sample according to the manufac-
turer’s guidelines (Fig. 2).
3.1.3 Quantification 1. Dilute TMZ in a proper volume of MeOH to obtain the desired
of Encapsulated TMZ Using concentration (seeNote 2).
UV-Vis Spectroscopy
2. Prepare aliquots of stock solutions from the initial stock of
Preparation TMZ (5–30 μM).
of the Calibration Curve 3. Scan samples by UV-Vis spectrophotometer in the range of
Using UV-Vis Spectroscopy 200–450 nm, using MeOH as blank.
4. Analyze samples for their respective absorbance at 330 nm λmax.
5. Plot the calibration curve (seeNote 3) (Fig. 3).
Quantification 1. Dissolve samples in double-distilled H2O at 100 μM and incu-
of Encapsulated Drug bate under shaking at 25 ± 0.1 οC at 600 rpm.
Using UV-Vis Spectroscopy
2. Centrifuge samples for 5 min and filter off the precipitated
TMZ through RC syringe filters of 0.2 μm pore size.
3. Scan the solution in UV region (200–440 nm) in triplicates.
Fig. 4 Stability of TMZ and TMZ@PSC4 at pH 7.1 and 37 °C as monitored by

UV–Vis. TMZ is rapidly transformed to AIC while TMZ@PSC4 is stable for at least
6h
4. Calculate the drug content of the sample using the standard

calibration curve.
3.2 Determination To validate the hypothesis that encapsulations could enhance the
of Chemical Stability stability of parent drug, time-dependent studies of the degradation
of TMZ and ΤΜΖ@ of parent drug have to be conducted. The stability studies should
PSC4 in Buffer Solutions be performed in aqueous buffer solutions under various pH condi-
Using UV-Vis, 1H-NMR, tions according to the FDA and European Medicines Agency
and LC-MS/MS (EMA) guidelines [15]. Stability could be monitored in a time-
dependent manner utilizing various analytical techniques such as
UV-Vis, NMR, and mass spectrometry.
3.2.1 Determination 1. Dilute TMZ@PSC4 in phosphate buffer (pH 7.1) to a final vol-
of Chemical Stability ume of 3 mL (100 μM).
of ΤΜΖ@PSC4 by UV-Vis
2. Incubate at 37 οC in a shaking bath.
3. Remove the samples from the bath at time intervals of 0, 2, 4,
6, and 8 h.
4. Transfer the samples to cuvette for analysis.
5. Conduct the same experiments for TMZ in order to compare
the stability of TMZ@PSC4 and TMZ.
6. Study all samples in triplicate (Fig. 4).
Fig. 5 1H-NMR spectra of the time-dependent degradation of native TMZ and TMZ@PSC4 to MTIC at deuter-
ated phosphate buffer in D2O (10 μm). The highlighted gray peak at 7.23 ppm represents the 7-H proton of the
MTIC form. The highlighted peaks at 3.52 ppm represent the 1-H proton of the MTIC form and its adduct
3.2.2 Determination 1. Dissolve TMZ and complex in 500 μL phosphate buffer

of Chemical Stability pH 7.1 in D2O.
of ΤΜΖ@PSC4 Using
2. Add tetramethylsilane (TMS) as internal standard.
1
H-NMR
3. Incubate samples under shaking at 37 °C ± 0.1.
6, 8, 16, and 24 h.
5. Transfer samples to 5 mm NMR tubes to analyze.
6. Load the sample into the Bruker AV-400 spectrometer.
7. Lock, tune, and match the sample according to the manufac-
turer’s guidelines (Fig. 5).
3.2.3 Determination 1. Prepare a stock solution of each compound at a concentration

of Chemical Stability of 1 mg/mL (seeNote 4).
of ΤΜΖ@PSC4 Using
2. Dilute stock solutions at a concentration of 500 ng/mL for
Liquid Chromatography
direct infusion in the MS (seeNote 5).
and Mass Spectrometry
3. Use the mass spectrometry software to calculate the most prev-
Method Development alent daughter ions of the parent compounds.
and Optimization 4. Dilute stock solutions at an appropriate concentration (1 μM)
for LC-MS/MS analysis.
5. Vortex-mix and filter the samples with 0.2 μm RC syringe
filters.
6. Transfer samples to LC-MS vials, load them onto auto-sampler,
and perform LC-MS runs to determine the optimal chromato-
graphic conditions (seeNote 6).
Fig. 6 Representative chromatogram of (a) TMZ and internal standard along with the optimal transitions and
most abundant daughter ion of (b) TMZ and (c) theophylline
7. Maintain the column and auto-sampler temperature stable (see-

Note 7).
8. On the same chromatographic runs, optimize the various ESI
parameters of mass spectrometer like spray voltage, heated
probe temperature, gas, and nebulizer.
9. Build up the method in mass spectrometer software, utilizing
multiple reaction monitoring (MRM) modes for the detection
and quantification of the compounds (Fig. 6).
Buffer Chemical
Stability Assay 1. Add 10 μL of TMZ@PSC4 in 280 μL hydrochloric acid
(pH 2.1) and incubate at 37 °C in a shaking bath.
6, 8, 16, and 24 h.
3. Add 10 μL of IS, vortex-mix, and transfer to LC-MS vials for
analysis.
4. Follow the same procedure for the samples incubated in ammo-
nium formate (pH 4.5) and phosphate buffer (pH 7.1).
5. Conduct the same experiments for TMZ in order to compare
the stability of TMZ@PSC4 and TMZ.
6. Study all samples in triplicate and plot the % fraction remaining
of parent TMZ against incubation time.
Fig. 7 The degradation rate of TMZ and TMZ@PSC4 in mice plasma. TMZ@PSC4
is more stable 4 h post-dosage in comparison to TMZ
3.3 Cell Culture Established GBM cell lines (U87, 8MG) and primary cultures were
cultured in DMEM or DMEM/F12, respectively, supplemented
with 10% FBS and maintained at 37 °C in a 5% CO2-humidified
incubator. Established lines were purchased from ATCC and pri-
mary cells were generated in-house from fresh brain tumor tissue
as described previously by Renziehausen et al. [8].
3.4 In Vitro 1. Seed the cells in 96-well plates at 2 × 103 cells per well in their
Cytotoxicity: respective culture medium supplemented with 2% FBS.
Sulforhodamine B 2. Treat 24 h post-plating cells with TMZ, PSC4, a physical mix-
(SRB) and Cell ture of TMZ and PSC4, or TMZ@PSC4 complex in culture
Counting Kit 8 (CCK8) medium supplemented with 2% FBS.
Assays
3. To determine the IC50 of native TMZ, treat cells with 2, 4, 8,
16, 32, 64, 128, 256, and 512 μM TMZ and analyze for prolif-
eration using SRB assay 9 days posttreatment as previously
described [16].
4. For experiments comparing the efficacy of the PSC4 complex
with native TMZ, use doses just below the IC50 of native TMZ
(5 μM and 10 μM for U87 and 8MG, respectively; 100 μM and
200 μM for the primary lines).
5. Calculate and use the equivalent equimolar concentrations of
the complex.
3.5 In Vivo 1. Separate C57BL/6 female mice 6 weeks old into two groups
Pharmacokinetic (n = 3) and inject them intraperitoneally either with a single
Analysis dose of TMZ or a single dose of TMZ@PSC4 complex.
2. Collect the blood by cardiac puncture before treatment (day 0)
and then at 0.5, 2, and 4 h posttreatment in tubes containing
heparin.
3. Centrifuge blood samples at 2862 × g for 10 min to separate the
plasma.
4. Acidify plasma (pH < 4) with phosphoric acid 85%.
5. Store all samples at −80 °C until further processing.
6. Prepare samples for analysis by adding 200 μL IS solution and
200 μL of 10 mM pH 3.5 ammonium fοrmate buffer to 100 μL
of acidified mouse plasma and precipitate this mixture with a
100 mM 1:1 methanol:zinc sulfate solution.
7. Vortex-mix for 1 min and centrifuge at 21,885 × g for 15 min.
8. Transfer the supernatant in glass vials and quantify TMZ by
LC-MS/MS (Fig. 7).
4 Notes
1. The pH of the solution must be stable on slightly basic condi-

tions during the reaction.
2. The solution was further sonicated for 15 min to obtain a clear
solution.
3. r2 must be over 0.99 to have an accurate calibration curve.
4. The pH of the stock solution of TMZ should be acidified to
stabilize TMZ.
5. Further dilution of stock solutions should be made with an
appropriate solvent that is compatible with the mass
spectrometer.
6. Various factors should be examined including type of elution
(gradient or isocratic), column properties, mobile-phase ratios,
etc. to achieve reliable and consistent peak shapes for all
compounds.
7. This is important to avoid sensitivity and repeatability issues
caused by temperature fluctuations.
Acknowledgments
This work was supported by the Hellenic Foundation for Research

and Innovation (H.F.R.I.) under the “First Call for
H.F.R.I. Research Projects to support Faculty members and
Researchers and the procurement of high-cost research equipment
grant” (Project Number:991, acronym PROTECT to AGT). We
would like to acknowledge Evgenios K. Stylos for his suggested
comments and corrections to this chapter.
References
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Chapter 15
Unveiling the Thermodynamic Aspects of Drug-

Cyclodextrin Interactions Through Isothermal Titration
Calorimetry
Maria V. Chatziathanasiadou, Thomas Mavromoustakos,
and Andreas G. Tzakos
Abstract
Due to their low toxicity and high aqueous solubility, cyclodextrins have emerged as a distinctive class of
supramolecules with wide application in the pharmaceutical and food industry. Their ability to improve the
water solubility, stability and pharmacokinetic profile of small molecules has established them as a rich
toolkit for drug formulation. In order to improve the physicochemical characteristics and the pharmacoki-
netic profile of a drug through cyclodextrin inclusion, the proper cyclodextrin type has to be selected
among the existing great variety consisting of both natural and synthetic variants. The selection of the most
proper cyclodextrin variant comes after drug-cyclodextrin screening studies targeting the characterization
of the complex formation and evaluation of the affinity and interaction forces participating in the complex-
ation. Numerous analytical, spectroscopic, separation and electrochemical techniques have been applied to
elucidate the interaction profile in a cyclodextrin-drug complex. Herein, we describe the application of
Isothermal Titration Calorimetry (ITC) on cyclodextrin-drug complexes that enables the charting of the
binding affinity and the thermodynamic profile of the inclusion complexes. We focus on the experimental
design and present technical tips of the ITC application. To better illustrate the technique’s rationale, the
interaction between 2-hydroxypropyl-β-cyclodextrin (HP-β-CD) and the antihypertensive drug losartan is
investigated.
Key words Isothermal titration calorimetry, 2-hydroxypropyl-β-cyclodextrin, Losartan, Inclusion

complex, Enthalpy, Entropy
1 Introduction
Cyclodextrins are cyclic oligosaccharides widely used in the phar-

maceutical industry for the inclusion of drugs suffered by reduced
stability and solubility to name some [1] and in the food industry
as additives [2]. Their cone-shaped structure composed of gluco-
pyranose units forms a distinctive supramolecule with a hydrophilic
outer surface and a lipophilic inner cavity able to host small mole-
187
188 Maria V. Chatziathanasiadou et al.
cules. According to the European Medicines Agency (EMA),

cyclodextrins are used as excipients in several marketed drugs
mainly to increase the aqueous solubility, bioavailability and stabil-
ity of the active substance [1]. Another advantage of these supra-
molecules is their low toxicity allowing their administration in high
doses. For example, HP-β-CD is not toxic when administered daily
at doses lower than 16 g [3]. The above characteristics make cyclo-
dextrins promising drug carriers and this is evident by the increas-
ing number of cyclodextrin-drug formulations presented in
literature and applied in the pharmaceutical industry [4–6].
Their application in drug encapsulation can sight toward dif-
ferent routes of administration including oral, parenteral, ocular,
dermal and nasal [1]. However, not every class of cyclodextrin is
suitable for the incorporation and delivery of a drug as their physi-
cochemical properties, contingent on their structural characteris-
tics, can affect the biopharmaceutical behavior and efficacy of
the drug. In terms of delivery for example, cyclodextrins with low
aqueous solubility like β-CD and methylated β-CDs are not being
utilized for parenteral drug administration due to their high toxic-
ity [7]. In terms of inclusion, factors like the size [8], type of
derivatization, and degree of substitution (DS) of the cyclodextrin
are of great importance in the complex formation with a guest
molecule [7]. The inclusion of a small molecule into the cyclodex-
trin core not only depends on the cyclodextrin type but also on the
guest’s physicochemical properties and other conditions such as
the solvent [9], pH [10–12] and temperature [7]. Thus, it is cru-
cial to explore each potential host-guest system individually in
order to define the driving forces of interaction.
Various analytical and spectroscopic techniques have been
recruited to characterize cyclodextrin complexes like surface plas-
mon resonance [13], nuclear magnetic resonance spectroscopy
[14, 15], UV–Vis and fluorescence spectroscopy [16, 17], mass
spectrometry [18], capillary electrophoresis [19], liquid chroma-
tography [20], cyclic voltammetry [21], and calorimetry [22, 23].
This chapter is focused on the ITC, a widely used technique to
chart such interactions, since it can estimate with high precision
both the binding affinity and the thermodynamic parameters of the
cyclodextrin complexes.
ITC is based on the determination of the heat changes that
occur during a binding process [24]. Briefly, the calorimeter con-
sists of two parts. The first is an adiabatic jacket bearing a sample
cell, where one of the two interactants is placed and usually—but
not necessarily—is the molecule with the highest molecular weight
and a reference cell which is loaded with buffer or water. The sec-
ond part is an automated syringe containing the other interactant
(ligand). The instrument aims to keep the temperature difference
between the two cells stable and close to zero (ΔΤ ≈ 0) and this is
succeeded by constantly providing power to the system. This
Unveiling the Thermodynamic Aspects of Drug-Cyclodextrin Interactions… 189
power is called differential power (DP) and when no titrations are

being conducted it is stable representing the experiment’s baseline.
Upon the ligand injection into the sample cell and providing the
condition that the two molecules are able to interact, heat will be
absorbed or released leading to an increased temperature differ-
ence between the two cells. The calorimeter “translates” this tem-
perature fluctuation as the less (in case of an exothermic reaction
where heat is released) or the additional (in case of an endothermic
reaction where heat is absorbed) power that must be provided in
order to keep the temperature difference constant and estimates
the heat as change in the enthalpy. Following the binding process
and as the molecule in the sample cell is getting saturated, the heat
fluctuations are shorter, reaching a final state where the small iso-
therms represent the heat changes due to the ligand dilution inside
the cell and not the heat of the interaction. By measuring the heat
differences, the calorimeter estimates the change in enthalpy (ΔH)
and the association constant (Kα). Providing that there are two
interactants and one binding site available, Kα is given by the fol-
lowing equation:
Kα =
[ AB ] (1)
[ A ][B ]
where [A] and [B] are the concentrations of the two unbound
interactants and [AB] is the concentration of the complex. The
changes in entropy (ΔS) and Gibbs free energy (ΔG) are not esti-
mated directly but through the established thermodynamic
equations:
∆G = ∆Η − T ∆S (2)
and
∆G = −RT ln K α (3)
where R is the gas constant (1.98722 cal/mol K) and T is the

absolute temperature in Kelvin. These parameters are pivotal for
the determination of an interaction, since the enthalpy change
reflects the participation of electrostatic forces, hydrogen bonding
and van der Waals interactions and entropy change depicts if hydro-
phobic interactions are involved [25, 26].
The cyclodextrin-drug inclusion complexes do not include
covalent bonds and overall the complexation is delineated by
hydrogen bonds, van der Walls forces, electrostatic interactions
(mainly ion-dipole and dipole-dipole interactions [27]) and hydro-
phobic interactions. The extent in which each kind of forces influ-
ences the cyclodextrin-guest interaction has been thoroughly
investigated and discussed in the literature [27, 28]. Generally,
most of the cyclodextrin-guest interactions are enthalpy driven,
while entropy may be favorable or not [28]. The van der Walls
interactions seem to play a significant role in the complexation [27,
28]. Regarding the affinity, in their majority the cyclodextrin-guest
interactions are weak (10–2000 M−1) [7], thus facilitating the
release of the guest molecule.
Herein, the study of HP-β-CD–losartan potassium inclusion
complex is investigated by ITC. The guest, losartan potassium, is a
known antihypertensive drug that blocks the angiotensin II recep-
tor, though its efficacy as chemotherapeutic agent is, also, under
investigation with promising results [29–32]. Losartan potassium
displays high water solubility and low permeability and suffers from
low oral bioavailability (33%) [33]. Furthermore, it exhibits a short
half-life, approximately 2 h [33]. To overcome these disadvantages
many formulations have been conducted in order to improve its
pharmacokinetic profile [32, 34–36]. HP-β-CD is a hydroxypropyl
derivative of the natural cyclodextrin β-CD consisting of seven
d-glucopyranose units. HP-β-CD has been selected since the
attached hydroxypropyl groups offer high aqueous solubility com-
pared to the lipophilic β-CD and make it non-toxic. Thus,
HP-β-CD is a widely applicable cyclodextrin as it has been used in
numerous formulations [15, 16, 18, 37].
In this work, the thermodynamics and the affinity of the inclu-
sion of losartan potassium into the HP-β-CD are evaluated via
ITC. Through this experimental process, the experimental design,
the ITC practice, and the data interpretation are also discussed.
2 Materials
2.1 Samples 1. HP-β-CD (MW ≈ 1540 g/mol, 1.0 molar substitution dextrin)
(see Note 1).
2. Losartan potassium (MW = 461.007 g/mol).
3. PBS buffer (10 mM, pH = 7): Both interactants have to be dis-
solved in the same buffer (see Note 2).
4. Water (LC-MS grade) for the reference cell loading.
2.2 Instrumentation 1. Degassing station to remove air bubbles from the samples.
2. Isothermal titration calorimeter.
3. Precision glass syringes for sample loading.
4. pH meter.
3 Methods
3.1 Experimental 1. If the calorimeter software provides an application for experi-

Design mental design and the binding constant of the studied complex-
ation is known, the user can estimate the concentrations of both

host and guest molecules in order to achieve an adequate C
value (1–1000). The C value is an ITC parameter defined as
[ A ]T
C= = K α ·[ Α ]Τ
Kd
where [A]T is the total concentration of the molecule placed in

the sample cell and given the fact that the stoichiometry is equal to
1. However, because low association constants are usually encoun-
tered between cyclodextrins and their guest molecules, low C val-
ues commonly occur (C < 1) and the final isothermal curve cannot
be perfectly sigmoidal.
2. As it is reported above, the binding affinity between a cyclodex-
trin and a small molecule in most cases is low. Thus, for a calo-
rimeter to detect the heat change during the interaction, high
concentrations (in the level of mM) of the two compounds are
required.
3. According to the basic experimental principles of ITC, the
compound with the lowest concentration is placed into the
sample cell and the other is loaded in the syringe. The sample
cell can be loaded with either the small molecule or the cyclo-
dextrin. However, because in most cases the encapsulation into
a cyclodextrin aims to increase the small molecule’s aqueous
solubility, the cyclodextrin presents higher solubility in aqueous
solutions than the small molecule and thus it is more flexible to
achieve higher concentrations. A good start is to load the sam-
ple cell with 0.5–1 mM of small molecule solution and the
syringe with cyclodextrin solution 10–25 times higher in respect
to the small molecule’s concentration. According to the out-
come, the experimental parameters can be altered in order to
optimize the sigmoidal profile of the curve. As losartan potas-
sium exhibits adequate aqueous solubility, it is feasible to load it
in the syringe. After the trial of different concentration ranges,
we ended up on using 1 mM of HP-β-CD in the sample cell and
25 mM of losartan potassium in the syringe.
4. The user has to apply different concentrations of the two inter-
actants in order to obtain adequate heat amounts during the
injections and a well-fitted curve that will approach the sigmoi-
dal shape and present a low Chi2 value.
3.2 Sample 1. Dissolve each compound in the proper volume of PBS buffer in
Preparation order to succeed the desired concentration (see Note 3).
and Loading 2. Degas the samples for at least 10 min to remove air bubbles
(see Note 4).
3. Before loading the sample and the reference cell, make sure that
they are clean (see Note 5).
4. Load the reference cell with H2O (see Note 6).
5. Load the HP-β-CD (1 mM) into the sample cell using a glass
syringe trying to avoid air bubble formation (see Note 7).
Approximately, 250 μL of sample solution is needed.
6. Load losartan (25 mM) in the titration syringe. The instrument
will automatically load and degas the drug solution. A volume
of 70 μL is enough for the syringe loading.
7. Put the automated syringe into the sample cell (see Note 8).
8. Follow the same procedure to run a control experiment by
titrating 25 mM of losartan potassium into buffer (see Note 9).
3.3 Run Parameters 1. Set temperature at 25 °C (see Note 10).

2. Set the DP to 6 μcal s−1 (see Note 11).
3. Set the number of injections to 17 (see Note 12)
4. Set the volume of the first titration to 0.4 μL and the duration
at 0.8 s.
5. Set the volume of the remaining 16 injections to 2 μL and their
duration to 0.8 s.
6. Set the spacing between the injections to 200 s (see Note 13).
7. Set the filter period to 3 s.
8. A moderate stirring is sufficient for the mixing of a cyclodextrin-
guest system.
9. Check the high feedback mode to elicit higher sensitivity.
3.4 Data Analysis 1. Process the raw data of each experiment (Fig. 1, upper panels of
a and b) in order to integrate the area of each injection. Obtain
the plot that provides the area of each injection as kcal per mole
of injectant against the molar ratio of the two interactants at
each time point (Fig. 1, lower panels of a and b).
2. When losartan is injected into the HP-β-CD or the buffer solu-
tion, endothermic events are observed in both cases. However,
the absorbed heat in the HP-β-CD–losartan interaction is much
less than the one that is required for the losartan’s dilution into
the buffer solution (see Note 14).
3. Subtract the data of the control experiment from the data of the
HP-β-CD–losartan interaction (Fig. 2). After the subtraction,
the negative enthalpy change that originates from the interac-
tion is revealed indicating that the inclusion of losartan into the
HP-β-CD cavity is an exothermic process.
4. Exclude the first injection from the data points (see Note 15).
Fig. 1 Raw data of (a) titration of losartan (25 mM) to HP-β-CD (1 mM) and (b) titration of losartan (25 mM) to
buffer at 298 K. The upper panel displays the isotherms of the interaction and the lower panel represents the
integrated area of each injection normalized per mol of injectant and plotted against the molar ratio of the
interactants. Both molecules were dissolved in PBS buffer (pH 7.0)
Fig. 2 The integrated areas of the losartan’s titration into HP-β-CD (lower line) and losartan’s titration into buf-
fer (upper line) plotted together before subtraction
Fig. 3 Nonlinear regression fitted curve (red line) following the one binding site model of the interaction
between losartan and HP-β-CD. The black squares represent the injection heats of the interaction, normalized
per mol of losartan, as they occurred after the subtraction of the control experiment and are plotted against the
molar ratio of losartan to HP-β-CD
5. Fit the curve according to the one set of site model (see Note
16). The best fitting is emerged when the Chi2 gets stabilized
(Fig. 3).
6. The Ka, the N, and the thermodynamic parameters (ΔH, ΔS)
are given directly from the software. Estimate ΔG through Eq.
(3). As it is presented in Table 1, the inclusion of losartan is
spontaneous and it is not favored by hydrophobic interactions.
The Ka suggests a low-affinity interaction as it commonly exists
in CD-guest complexes [7].
4 Notes
1. Beware of the molar substitution grade of the HP-β-CD as the

degree of substitution may affect the cyclodextrin-guest inter-
action [38].
2. The pH of the CD and the drug solutions must be the same;
otherwise it may lead to false results. Thus, it is recommended
to use a buffer for their dilution.
3. The concentrations of the samples must be accurate as they are
defined by the user in the experimental settings of the soft-
Table 1
Thermodynamic parameters and binding constant of the losartan-HP-β-CD interaction
T (K) N Ka (M−1) ΔΗ (cal/mol) ΔS (cal/mol/deg) ΔG (cal/mol)

298 0.757 820 ± 21.4 −4333 ± 179.6 −1.2 −3975
ware. Based on these and the occurred enthalpy change dur-

ing the experimental procedure, the calorimeter estimates the
Ka value and the stoichiometry. Erroneous sample concentra-
tions lead to invalid results and bad fitting.
4. The removal of air bubbles from the samples is crucial in the
calorimeter application as their presence can affect the mea-
surement of the heat (absorbed or released) leading to poor
experimental outcome and also can cause difficulties in the
manual loading of the samples into the cell. Depending on the
instrumentation, the lower the volume of the sample cell, the
greater the noise effect that can be caused by air bubbles.
5. Typically, the cells are rinsed thoroughly with water. A more
drastic cleaning is achieved with detergents like Contrad 70
and Decon 90 or with 12% w/v NaOH.
6. The reference cell is usually loaded with water or buffer when
the tested samples are dissolved in a specific buffer. In most
cases water is sufficient as a control. According to most manu-
facturers the reference cell content has to be renewed once a
week, to avoid microbial growth. Before each run, the user has
to confirm that the reference cell is properly loaded (no air
bubbles, no low water level) as in the opposite situation the
DP can be significantly affected (lower or higher), producing
unstable baseline and consequently poor results.
7. After the sample loading into the cell, the calorimeter thermo-
stats the cell to succeed the defined temperature. Thus, it is
recommended not to start immediately the run but wait until
it gets stabilized.
8. The syringe insertion into the sample solution may destabilize
its temperature. Thus, it is recommended to wait a few min-
utes (5 min are enough) before starting the experiment.
9. A control experiment, using the same ligand concentration, is
required for the estimation of the heat amount that is not
originated from the small molecule–cyclodextrin interaction.
For example, this heat may occur due to the cyclodextrin dilu-
tion into the sample cell. A titration of the supramolecule into
the water-loaded sample cell will provide the measurement of
this “noise” and the heat amount can be subtracted from the
main experiment.
10. Generally, a range of 2–80 °C can be applied in the calorime-

ter. This feature is convenient for the estimation of the heat
capacity (ΔCp) of a cyclodextrin-guest system as it can be
achieved by conducting the same experiment under different
temperature conditions.
11. For an unknown interaction, a DP equal to 5 or 6 μcal s−1 is
recommended. However, if the titration curves cross the mea-
surement range of the instrument, the user can change the DP
value.
12. A small number of injections of 10–20 is ample to derive reli-
able results. More injections do not necessarily lead to more
accurate results [24].
13. The time spacing between the injections offers time to the
instrument to calibrate the ΔΤ between the sample and the
reference cell so the signal will return back to the baseline
(DP).
14. The endothermic process observed upon the titration of losar-
tan is due to the fact that the drug forms aggregates which are
separated upon dilution into the sample cell’s content [39].
15. During the first titration, the heat measurement may not be
accurate. Thus, the initial titration is pilot, injecting a smaller
volume of the ligand into the sample cell compared to the rest
titrations (in order to not waste sample) and is excluded from
the data analysis.
16. If the stoichiometry of an interaction is known, the one set of
site model may be directly applied. In other case, additional
fitting models are provided like the two sets of site model, the
competitive binding, and the dissociation model.
Acknowledgments
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Chapter 16
Antitumor Efficacy of Ceranib-2 with Nano-Formulation

of PEG and Rosin Esters
Ali Ben Taleb, Selcan Karakuş, Ezgi Tan, Merve Ilgar, Özlem Kutlu,
Devrim Gözüaçık, Hatice Mehtap Kutlu, and Ayben Kilislioğlu
Abstract
Ceranib-2 is a recently discovered, poorly water-soluble potent ceramidase inhibitor, with the ability to
suppress cancer cell proliferation and delay tumor growth. However, its poor water solubility and weak
cellular bioavailability hinder its use as a therapeutic agent for cancer. PEGylated rosin esters are an excel-
lent platform as a natural polymer for drug delivery applications, especially for controlling drug release due
to their degradability, biocompatibility, capability to improve solubility, and pharmacokinetics of potent
drugs. In this study, stable aqueous amphiphilic submicron-sized PEG400-rosin ester-ceranib-2 (PREC-2)
particles, ranging between 100 and 350 nm in a 1:1 mixture, were successfully synthesized by solvent
evaporation mediated by sonication.
Conclusion: Stable aqueous PEGylated rosin ester nanocarriers might present a significant solution to
improve solubility, pharmacokinetic, and bioavailability of ceranib-2, and hold promises for use as an anti-
cancer adjacent drug after further investigations.
Key words Ceranib-2, Rosin ester, PEGylation, Nanoencapsulation, Anticancer
1 Introduction
Cancers are a major public healthcare problem worldwide and the

second leading cause of death in Europe and North America [1,
2]. The International Agency for Research on Cancer (IARC)
reported that the global cancer burden is estimated to rise to 21.5
million new cases and 11.500 million deaths by 2025 [3, 4].
Recently, significant success has been made in developing new
safe, less toxic anticancer drugs. Nonetheless, despite these suc-
cesses, many newly discovered potent anticancer agents fail to be
developed as promising clinical anticancer drugs due to several
physiochemical and biological reasons [5]. Therefore, efforts are
ongoing to identify novel efficient nonconventional drug delivery
system to achieve optimum therapeutic effects with minimal side
199
200 Ali Ben Taleb et al.
effects. In traditional pharmaceutical approaches, drug delivery

systems were usually formulated using active anticancer drugs to
address clinical and pharmacological issues related to anticancer
drug properties, without changes in the fundamental pharmacoki-
netic properties of the active pharmaceutical ingredient, while the
major pharmaceutical challenges associated with newly discovered
potent anticancer drugs were poor aqueous solubility, cellular per-
meability, and low cellular drug bioavailability [6, 7].
Sphingolipids are substantial elements of human cell mem-
branes. Currently, more than 300 sphingolipids (SPL) have been
detected. Sphingolipid metabolites, in particular, ceramide,
ceramide-1-phosphate (C1P), and sphingosine-1-phosphate (S1P),
are lipid agents which play the role of controlling sets of cellular
functions; this later contains cell growth, survival, migration,
immune cell trafficking, angiogenesis, apoptosis, autophagy, and
cancer [8, 9]. As a matter of fact, the generation of ceramide and
sphingosine is induced by chemotherapy, radiation, and/or oxida-
tive stress, and these sphingolipids mediate cell death, senescence,
and/or cell cycle arrest. Nevertheless, metabolic conversion of
sphingosine-1-phosphate (S1P) to ceramide (Cer), sphingomyelin,
or glucosylceramide has anti-apoptotic and pro-survival roles [10].
Sphingolipids (SPL) are ubiquitous molecules essential for the
maintenance and development of living organisms. They are not
evenly distributed along the membrane but grouped as lipid micro-
domains called rafts. It has been thought for a long time that SPLs
have only a structural role. However, now it has been known that
they are playing a role in receptors and second messengers involved
in major functions of the cellular life and many genetic diseases
(sphingolipidoses) which are explained as a dysfunction of their
metabolism [11].
Recently, the outcomes of various studies have shown the
importance of total cell sphingosine lipids (in the phosphorylated
form) which play an essential role in cell death or cell survival mes-
sage [12]. These studies have showed that different pathways of
the apoptosis occurrence, involving the internal, external, and
common pathway, will be quickly activated and the cell will soon
be directed to the planned death if this balance changes in any way
toward increasing total cellular ceramide [13]. On the contrary,
the segmentation of ceramides in the cytoplasm by increasing the
activity of diverse isoforms of ceramidase enzymes leads to a rise in
the levels of intracellular sphingosine [14]. Sphingosine, by medi-
ating environmental evolution factors and excitant cytokines of
proliferation and division, is exposed to the action of the
sphingosine-1-kinase. The phosphorylated sphingosine (through
the GPCR receptor) directly and indirectly catalyzes the contribu-
tory factors in cell division, such as PI3K and PLC [15]. It has
been shown that TNF stimulates apoptosis in tumor cells by raising
the output of ceramide; thus, ceramide does not need p53 [16].
Antitumor Efficacy of Ceranib-2 with Nano-Formulation of PEG and Rosin Esters 201
Moreover, adding the exogenous ceramide stimulates programmed

cell death and ceramide generation is an essential controller of
apoptosis [17, 18].
Ceramide and sphingosine-1-phosphate (S1P) have long been
suggested to have opposing missions in the regulation of cell fate
[19]. Although ceramide has been related to cell growth arrest and
apoptosis, S1P induces cell growth and survival [8]. Studies have
recently suggested that ceramide and S1P have as well opposing
impacts on autophagy [20]. The rise of ceramide enhances the
assemblage of Beclin-1 and the suppression of AKT phosphoryla-
tion that causes autophagic cell death [21]. The association of the
multi-kinase inhibitor sorafenib with histone deacetylase inhibitors
also raises ceramide levels notably by the promotion of de novo
synthesis. Increased ceramide synthesis has been implicated in
endoplasmic reticulum (ER) stress, autophagy, and apoptosis [22].
Additionally, exogenous dihydroceramide, an intermediate in the
biosynthesis of ceramide, or its promotion resulted from the fen-
retinide, which stimulates serine palmitoyl-transferase and inhibits
the desaturase, also inducing autophagy [23].
The factors enhancing or preventing ceramide operators and
sphingolipid-metabolizing enzymes have recently been regarded as
novel pharmacological goals for controlling apoptosis [24]. Other
cellular procedures which depend on these messengers have been
the subject of discussion of different biological studies [25].
Therefore, these data support the hypothesis that the alteration of
the metabolic activity of ceramidase and increased ceramide levels
are the key role in the induction of cancer cell apoptosis or tumor
suppression [26].
1.1 Sphingolipid In the following data analysis ceramide takes a focal position in
Metabolism biosynthesis and catabolism and as a precursor of complex sphin-
and Cancer golipids, currently thereby considered as a metabolic hub of sphin-
golipid metabolism. Ceramide can be produced by three different
pathways: salvage, degradation or hydrolytic, and de novo biosyn-
thesis pathway. On the contrary, there has been increasing evidence
that lipid mediators such as ceramide and sphingosine-1-phosphate
(S1P) have crucial roles in various human cancers [27], involving
breast [28] and prostate cancer [29]. S1P is generated inside the
cancer cells and exported out of the cells where it regulates many
functions in tumor microenvironment by binding to specific G
protein-coupled receptors expressed either in cancer cells or in
host cells, known as the “inside-out” signaling of S1P. In addition,
it has demonstrated that S1P levels are high not only in tumors,
but also in the tumor microenvironment. However, studies further
explained that high S1P production by tumor is linked to lymph
node metastasis, showing that S1P enhances cancer metastasis by
influencing tumor microenvironment in human breast cancer.
Thus, it was of great interest to examine the ceramide levels of
Fig. 1 Chemical structure of ceranib-2
cancer, peri-tumor, normal breast tissue, and interstitial fluid (that

is a component of tumor microenvironment), because ceramide
has not been as comprehensively studied and may have important
relationships with cancer.
1.2 Ceranib-2 Recently, several novel potent ceramidase inhibitors have been
as Potent Ceramidase reported as effective anticancer drugs [30–32]. In 2011, Draper
Inhibitor et al. reported ceranib-2 (C2) as a novel potent, small-molecule
ceramidase inhibitor (Fig. 1). These molecules have the ability to
inhibit human CDase activity, which leads to elevated exogenous
ceramide levels and decreased levels of sphingosine and S1P in a
cell line-based assay, subsequently being shown to inhibit prolifera-
tion of ovarian cancer cell line and induce cell cycle arrest alone or
with other chemotherapeutic agents. In addition, C-2 delays tumor
growth in an in vivo tumor model without hematologic suppres-
sion or signs of cellular toxicity usually associated with other
ceramidase inhibitors [33].
However, the exact mechanism of C2’s action has not been
fully elucidated. Lately, several in vivo data obtained support the
selection of ceranib-2 as a promising single or adjuvant drug in dif-
ferent cancer cell models, such as breast cancer [33], lung cancer
[34], prostate cancer [35], and colon cancer [36]. Taken together,
ceranib-2 is considered as a promising, clinical anticancer drug.
1.3 Therapeutic Although intensive in vitro and in vivo experiments have shown
Limitation the potency of C-2 as a CDase inhibitor [37], C-2 like most CDase
of Ceranib-2 inhibitors is characterized with long-chain alkyl moieties, and it has
as Anticancer Drug some pharmacokinetic and pharmacodynamic limitations when it
is delivered using a conventional drug delivery system. These limi-
tations might be due to its low aqueous solubility (hydrophobic-
ity), poor cellular bioavailability, and plasma protein binding
affinity [38]. These challenges hinder their development as novel
therapeutic agents for cancer treatment. Thus, there is a need for
novel pharmaceutical drug formulation and drug delivery system
to meet the potential of C-2 as a promising anticancer drug.
1.4 Nanostructure Nanotechnology is an emerging area gaining increased attention in

Material in Anticancer many applications with a great capability to resolve problems asso-
Drug Delivery System ciated with many daily applications. Nanotechnology as a science
refers to the understanding, manipulation, and development of
material at the nanometer scale, where unique physical and chemi-
cal properties can serve different applications, as well as society
[39]. Moreover, nanoparticle drug delivery systems are a very
promising technology, especially for anticancer drugs due to their
ability to reduce side effects, high specificity to cancer cells, and
capability of improving drug solubility and cellular accessibility
[40]. Furthermore, this technology enables precision targeting
and surface modification, and has the potential to enhance the
drug’s pharmacokinetics and pharmacodynamics, as well as con-
trolled sustained drug release at the site of the disease [41].
Recently, research has focused on the use of nanostructure materi-
als for drug delivery carriers to increase therapeutic effect of many
anticancer drugs [42]. In general, nanoparticle drug delivery sys-
tems can be classified into two groups, inorganic and organic
nanoparticles.
Organic nanostructure-based drug delivery systems are highly
promising for the development of efficient anticancer drug sys-
tems. They have been extensively investigated, and their success
has been demonstrated in many clinical applications, with many in
different phases of clinical trials [43]. They are beneficial in the
design of many efficient anticancer drug formulations, especially
those poorly soluble drugs, due to their benefits such as improved
solubility, stability, safety, biodegradability, biocompatibility, and
controlled drug release [44]. Moreover, organic polymeric
nanoparticles have the ability to alter their composition, surface
properties, and shape, which all lead to improved overall properties
[45]. To date, several synthetic and natural polymeric nanoparticle-
based drug delivery systems have been developed (Table 1) and are
available commercially [46].
The most widely investigated synthetic polymers are poly
(lactic-
co-glycolic) acid (PLGA) copolymer, polycaprolactone
(PCL), polylactide (PL), and polyacrylates (PA). Meanwhile, sev-
eral natural polymers such as chitosan, alginate, and albumin have
been extensively explored for many drugs [47]. Recently, two types
have been used for the formulation of anticancer drug including
nanosized spheres and nanosized capsules depending on their
composition. However, particle aggregations, physical properties,
and drug dose uniformity are the main limitations of using poly-
meric or organic nanostructure drug delivery systems [48].
1.5 Polymeric Despite the expansion in anticancer drug research, the number of
Nanoparticle Drug novel anticancer drugs that reach the pharmaceutical market has
Delivery Systems dramatically decreased, with some being dropped from the devel-
Table 1
Commercial nanostructure drug brands available in the market
Drug Brand name Manufacturer Uses or indication

Dexamethasone Limethasone Mitsubishi Pharmaceuticals Anti-inflammatory
Propofol Diprivan AstraZeneca Anesthetic
Vitamins Vitalipid Fresenius Supplements
Diazepam Diazemuls Kabi Pharmacia Sedative
Clevidipine Cleviprex The Medicines Company Ca channel blocker
opment pipeline because their pharmacological and therapeutic

effects are impeded by limited cellular bioavailability, low aqueous
solubility, lack of targeting, and rapid blood clearance. However,
several strategies have been proposed to overcome these challenges
[49]. Polymeric nanoparticle technology has been extensively used
for many applications, especially in pharmaceutical formulation
[50]. This method allows small bioactive molecules to be enclosed
within an inert biocompatible core; as a result, they can enhance
pharmacokinetics, cellular bioavailability, and controlled drug
release, and have the capability to modify their surface enabling
them to reach the target location [51]. However, particle size, par-
ticle shape, size distribution, and polydispersity index (PDI) still
play an important role in determining their cellular uptake and
therapeutic effects [52]. Several polymeric nanosized drug delivery
system preparation techniques are currently available, which basi-
cally depend on the physiochemical properties of the drug and
polymer material used [53].
1.6 Rosin Ester Nanostructure drug carriers such as polymeric nanospheres and
Nanoparticles in Drug polymeric nanoparticles hold great promise, especially for poor
Formulation and low water-insoluble anticancer drugs. They play an important
role in many pharmaceutical applications due to their capability for
further surface modification accessibility, ability to reduce adverse
side effects, and ability to enhance pharmacokinetics [54]. Various
natural polymers have been extensively investigated and approved
for advanced drug delivery system due to their unique properties,
such as abundance, biocompatibility, and biodegradability [52].
Rosin is a natural nonvolatile resin extracted from different species
of pine tree (Fig. 2). However, natural rosin usually contains a mix-
ture of rosin acid (90% rosin acids) compounds, such as tricyclic
diterpene carboxylic acids (abietic/pimaric), which possess two
chemical reactive centers, double bonds, and a carboxyl group, and
other nonacidic components with characteristic hydrophobic
hydrophenanthrene rings which make them excellent for film
forming [55].
Fig. 2 Showing rosin resin as raw material (a) chemical structure of rosin (b)
1.7 Improving Rosin derivatives are reported to have interesting biological fea-
the Structure tures when they are used as a drug delivery system due to their
capability to cross the blood-brain barrier, which makes it an excel-
lent drug carrier for several central nerve system diseases [56].
Esters of rosin and polymers are also reported to have good film
coating, especially for enteric tablets because of their delayed-
release profile [57]. Interestingly, rosin ester nanoparticle drug
delivery structure has been shown to be an excellent candidate for
several pharmaceutical applications, such as matrix material in
sustained-release tablets due to their biocompatibility, biodegrad-
ability, and antibacterial activity [58]. Moreover, transdermal drug
patches containing rosin matrix and polyvinyl pyrrolidone (PVP)
have been shown to improve pharmacokinetics and pharmacody-
namics, prolonging drug release from the drug matrix [59].
Interestingly, besides all the mentioned properties of rosin deriva-
tives as film coating, controlled sustained release, and biodegrad-
ability, rosin derivatives have significant antitumor activity
attributed to dehydroabietic acid, the major component of gum
[60]. However, rosin ester material is highly hydrophobic and
chemically dissolves only in organic solvents. Therefore, using
rosin material as a drug delivery system without surface modifica-
tion might be limited by rapid elimination of the drug from the
blood circulation.
1.8 PEGylation PEGylation is a well-known method used in several biotechnology

and pharmaceutical applications including drug coating, gene
therapy, and multifunctional nanoparticle drug formulations [61].
PEGylation as a technique was first introduced by Davis et al. in
1970, and can be defined as the attachment of one or more poly-
ethylene glycol (PEG) chains to the surface of drug or carrier mol-
ecules, aiming to improve solubility and pharmacokinetic and
pharmacodynamic properties of the bioactive drug ingredient
[62]. PEG is a water-soluble synthetic biocompatible polymer
which has been extensively used for various pharmaceuticals due to

its ability to overcome pharmaceutical formulation challenges,
such as water solubility, surface charge, systemic toxicity, short
half-life, and drug plasma protein binding. However, despite the
benefits of PEGylation technology, there are some limitations
which may arise during the formulation process [63] such as exten-
sive PEGylation that may lead to increased viscosity and reduced
surface charges, subsequently decreasing binding affinity between
the drug molecule and cells [64]. Thus, the optimum surface mod-
ification by PEGylation of NPs in a drug delivery system is an
important parameter to achieve the right balance between drug
molecule diffusion and prolonged drug clearance time [65].
1.9 Preparation Polymeric nanoparticles (NPs) have attracted much interest in

Methods many applications, particularly in anticancer drug delivery systems
for Nanoparticle Drug due to their ability to overcome toxicity, a controlled and release
Delivery System profile, and improved drug pharmacokinetic and pharmacody-
namic properties. Polymeric NPs are particulate dispersions or
solid particles in the submicron range, between 10 and 1000 nm in
size. Several methods have been developed to prepare polymeric
NPs for drug delivery; these methods rely on the direct synthesis
from preformed polymer material or polymerization. Current
techniques allow the preparation of nanoemulsions with an uni-
form and submicron particle size, which may be scaled up to pro-
duce large quantities [66]. However, these methods include solvent
evaporation, nanoprecipitation, emulsification/solvent diffusion,
salting out, dialysis, and supercritical fluid technology.
1.10 Solvent Several laboratory-scale preparation methods have been developed

Evaporation to prepare polymeric nanoparticle drug delivery systems classified
according to the macromolecules of polymer used or polymeriza-
tion method used in the preparation of NPs [67]. Solvent evapora-
tion was the first method developed to prepare polymeric
nanoparticles (Fig. 3). This method includes two steps and begins
by preparation of an emulsion using a high-speed homogenizer or
ultrasonication method, followed by evaporation of the solvent by
a continuous magnetic stirrer at room temperature. The solid
nanoparticle suspension is then obtained by centrifugation.
Recently, a few organic volatile solvents were used to overcome the
high toxicity profile of solvents [68].
1.11 Ultrasonic The use of high-energy-intensity ultrasound is an effective tool in

Cavitation several industries, providing a versatile method for the preparation
of a nanostructure drug delivery system [69]. The effects of ultra-
sound arise from the physical phenomena named acoustic cavita-
tion, where nanosized particles are formed, grow, and collapse
under the influence of sound [70]. In pharmaceutical application,
this technique can be used to produce new material suitable for
Organic phase with the drug
Ultrasonication
Solvent evaporation
Aqueuos phase Characterization

with stabilizer
Fig. 3 Schematic diagram showing a preparation method of nanoparticle drug delivery system using solvent
evaporation mediated by ultrasonic cavitation
sensitive drug and active ingredient without bulk high tempera-

ture, high pressure, or long reaction time [71]. In addition, this
technique has several advantages such as easy operation, low cost,
good energy efficiency, good dispersion stability, and low risk of
microbial contamination. The application of sonochemistry-
mediated particle size reduction and synthesis of nanosized parti-
cles with uniform size distribution and high stability have become
increasingly important [72].
Recently, ultrasound cavitation-assisted nanoencapsulation has
attracted more attention in many applications, including the design
of novel drug delivery systems, due to their properties such as sim-
plicity, reproductivity, and low energy requirements. The synthesis
of nanocapsules using cavitation or an ultrasound approach is very
efficient and flexible to control several variables, such as size and
size distribution [73]. Various pulsed horn-based ultrasonic sys-
tems have been developed and introduced to sonochemistry and in
the design of drug delivery systems, which provide more efficient
particle reduction. This system allows ultrasonic waves to be
directed onto the electrode surface, where the horn and electrodes
are immersed in the solution with the ultrasound horn emitter
(Fig. 4). For efficient particle size reduction synthesis, several
parameters should be considered during ultrasonic cavitation pro-
cess; these parameters include temperature, density, pulse time,
and pulse intensity [74]. At the present time, and to the best of our
knowledge, no other studies have investigated the effect of poly-
meric nanostructure form based on PEGylated rosin derivatives to
improve the solubility, cellular bioavailability, and pharmacokinet-
ics of C-2. This study aims to develop a novel efficient, stable aque-
ous nanostructure drug delivery system for ceranib-2 (C-2), with
nanoparticles ranging in size between 10 and 350 nm using rosin
Fig. 4 Pulsed horn ultrasonic cavitation
ester as the core and PEG as the external shield to address the chal-
lenges of conventional pharmaceutical formulations by optimizing
the pharmacokinetic and cellular bioavailability of ceranib-2.
2 Materials and Methods
Unless otherwise stated, all chemicals and materials used should be

pharmaceutical grade or pure, without further purification.
2.1 Synthesis 1. Resin ester powder.

of PREC-2 NPs 2. Polyethylene glycol (PEG) 400.
3. Ceranib-2.
4. Sodium acetate.
5. Ethyl acetate.
6. Dimethyl sulfoxide (DMSO).
7. Buffer solution.
2.2 Cell Culture 1. Dukes’ type B colorectal adenocarcinoma (SW480) cell line.
2. Cervix adenocarcinoma (HeLa) cell line.
3. Dulbecco’s modified Eagle medium (DMEM).
4. 10% (v/v) Fetal bovine serum (FBS).
5. 10 mM 4-(2-Hydroxyethyl)-1-piperazine-ethanesulfonic acid.
6. 2 mM L-glutamine.
7. 100 U/ml penicillin.
8. 100 U/ml streptomycin.
9. 1 N HCL.
10. 1 N NAOH.
11. 3.7 g/l Sodium bicarbonate (49.3 mL) per liter of DMEM.
2.3 In Vitro Release 1. Cellulose membrane dialysis tube.

Assessment 2. Receptor solution (sodium acetate buffer pH 5.5).
2.4 Preparation MTT 1. Dissolve 500 mg MTT powder in 10 mL PBS.

Assay Stock Solution 2. Stir the mixture with a magnetic stirrer for approximately 1 h
in the dark.
3. Filter sterilize solution with a 0.22 mm Millipore filter.
4. Store in 10 mL aliquots at −20 °C.
2.5 Equipment 1. Plate shaker.

and Instruments 2. Pipettes, 0.001–1 mL single channel and 0.01–0.3
multichannel.
3. Class 2B safety cabinet.
4. Benchtop centrifuge.
5. Microplate reader (ELISA reader).
6. Incubator with 5% O2 suitable for drug experiments.
7. Microplate 96 well.
8. Circulating hot water bath.
9. UV/VIS spectrophotometer.
10. Graduated glass container.
11. Ultrasonic homogenizers supplied with pulsed horn.
12. pH meter.
13. Glassware.
14. Refrigerator.
15. 15 Hot-plate stirrer.
2.6 Statistical 1. Assess the statistical significance of differences between groups

Analysis by two-tailed Student’s t-test.
2. Present the data as means of ±S.D. of four independent experi-
ments. Values with p < 0.05 were considered as significant.
3 Methods
3.1 Preparation 1. In 50 mL glass beaker, dissolve 1 mg of ceranib-2, and 1 mg

of Dispersion Phase of rosin ester powder in 0.1 mL of dimethyl sulfoxide (DMOS).
2. Place the mixture in 50 mL beaker.
3. Make up to 50 mL with ethyl acetate till fill line.
4. Mix the mixture using a stirrer at 1500 rpm for 10 min at
room temperature, before the addition of an appropriate vol-
ume of ethyl acetate till fill line.
3.2 Preparation 1. Dissolve 0.5 g of polyethylene glycol (PEG 400) in 77.6 mL

of Continuous Phase of deionized water in a 50 mL glass beaker at room tempera-
ture using a stirrer.
2. Make up to 50 mL with ethyl acetate till fill line.
3. Store at room temperature.
3.3 Synthesis 1. Place the contentious-phase beaker under sonication trans-

of PREC-2 NPs ducer horn (see Note 1).
2. Turn power on the ultrasonic generator device (see Note 2).
3. Set up sonication-independent parameter variables such as
amplitude and time, here samples 20 kHz, for 20 min, at room
temperature (see Note 3).
4. Dropwise and using a Pasteur pipette add the previously pre-
pared dispersion-phase mixture into the continuous phase
while sonicating the mixture for 20 min (see Note 4).
5. Remove excess ethyl acetate residues from the emulsion by
leaving the sample to evaporate for 6 h at 40 °C using hot
plate (Fig. 5).
6. Store samples at −4 °C until submitted for freeze-drying or
for further characterization.
3.4 Preparation 1. Prepare 800 mL of distilled water in 1 L glass container.

of Physiological 2. Using laboratory balance, weight 7.719 g sodium acetate and
Reception Solution add it to the 800 mL of water prepared previously.
for In Vitro Release
3. Add 0.353 g of acetic acid to the solution.
Experiment
4. Using pH meter adjust pH using HCL or NAOH.
5. Make up the volume to 1 L with distilled water.
3.5 In Vitro Drug- 1. Using scissors, cut appropriate length of cellulose membrane
Release Profile dialysis tube suitable for 5 mL of sample.
of PREC-2 NPs 2. Place a 2 mL of PREC-2 NPs into the membrane using pipette,
and close it tightly from both ends (Fig. 6).
Fig. 5 Experiment setup of PREC-2 NP synthesis using ultrasonic cavitation
Fig. 6 Illustration showing experiment setup of in vitro drug release using dialysis membrane for nanoparticle
drug delivery system
Fig. 7 In vitro drug release experiment steps of ceranib-2 from PREC-2 NPs. (a) Immerse of membrane tube
containing 2 mL of PREC-2 NPs in receptor solution. (b) Place the container containing membrane inside glass
bottle to prevent damage. (c) Keep both bottles in continuously circulating hot water
3. Immerse the filled membrane in a 100 mL graduated plastic

bottle with a cap containing preprepared receptor solution at
37 ± 0.5 °C, and PBS pH 5.5 (Fig. 7).
4. Place the system in a continuously circulating hot water bath
for 2 weeks at 37 ± 0.5 °C (see Note 5).
5. Set up the calibration curve by plotting the absorbance of sev-
eral dilutions of ceranib-2 as shown in Fig. 8.
6. Determine the concentration of drug released from the mem-
brane into the receptor solution at specific interval time for
2 weeks by withdrawing 3 mL from the middle of the receptor
solution and replace the quantity withdrawn with fresh
medium (Fig. 9).
7. Analyze samples using a UV/VIS spectrometer.
1,6
1,4
1,2
1,0
Absorbance
0,8
0,6
0,4
0,2
0,0
0,000 0,005 0,010 0,015 0,020 0,025

Concentration (mg/mL)
Fig. 8 PREC-2 NPs showing calibration curve (abs vs. conc.) obtained by UV/visible spectrophotometer
12
10
8
Drug Release (%)
0 100 200 300 400

t (hour)
Fig. 9 Release profiles of ceranib-2 from PRE nanoparticles
8. Record the absorption measurements after each interval time

at a wavelength of 337 nm (see Note 6).
3.6 Preparation 1. Clean the working place in the cabinet with alcohol every time
of DMEM Culture to prevent contamination.
Media for Cell Line 2. Place sterilized bottles and laboratory bottle-top filter under
hood.
3. Measure out the final volume required (900 mL) of pure water
into the 1000 mL beaker.
4. Measure out the rest of the volume (100 mL) into 150 mL
beaker.
5. Add DMEM powder slowly to the 1000 mL beaker while stir-
ring gently. Stir until it is dissolved.
6. Rinse the original DMEM package with a small amount of
pure water from the 150 mL beaker.
7. Add this to the solution and any remaining pure water in step 4.
8. Add 3.7 g of sodium bicarbonate to the solution.
9. Stir with mechanical homogenizer until it is dissolved (see
Note 7).
10. While stirring, adjust pH of solution using 1 N HCl and 1 N
NaOH until it reaches the desired pH (7.4).
11. Sterilize the prepared solution (DMEM) by sterile filtering
using a bottle-top filter (in 0.22 μm pore size) into 2500 mL
sterile bottles in the hood.
12. Label the container with the name of media, sterility, and date
of preparation.
13. Store the prepared media in the refrigerator.
3.7 Determination To evaluate the anticancer activity effect of ceranib-2 in polymeric

of the Efficacy nanoform, the in vitro MTT assay was performed using SW480
and Therapeutic Effect and Hela cancer cell line incubated with PREC-2 NPs for 24 h.
of PREC-2 NPs DMSO was used as a carrier in the negative control (CNT), and
etoposide (50 μM) was used as the positive control. The results are
presented in Tables 2 and 3, and Fig. 10.
1. Seed Dukes’ type B colorectal adenocarcinoma (SW480) and
cervix adenocarcinoma (HeLa) cells in a 96-well flat-bottom
microtiter plate at a density of 1 × 104 cells/well in 100 μL
DMEM supplemented with 10% FBS (see Note 8).
2. Treat cells with various concentrations of the PREC-2 for 24 h
at 37 °C in a CO2 incubator and 95% relative humidity (see
Notes 9 and 10).
3. After 24 h of incubation, replace with a fresh medium.
4. Add 10 μL of MTT working solution (5 mg/mL in phosphate
buffer solution) to each well and incubate the plate for 4 h at
37 °C in a CO2 incubator (see Note 11).
5. Aspirate the medium, and solubilize the formed formazan
crystals by adding 50 μL of DMSO per well for 30 min at
37 °C in a CO2 incubator (see Note 12).
6. Finally, quantify the intensity of the dissolved formazan crys-
tals (purple color) using the ELISA plate reader at 540 nm (see
Note 13).
7. Schematic presentation of assay format is given in Fig. 10.
Table 2
Relationship between ETO, ceranib-2 (C-2), PRE NPs, PREC-2, doses, and
% cell viability of cervical adenocarcinoma (Hela) cell line
Viability
Sample no. Treatment Dose (μM) (%)
S1 CNT (DMOS) 100 100
S2 ETO 50 60
S3 Pure ceranib-2 10 32
S7 PRE NPs 0.66 78
S8 PRE NPs 0.33 89
S9 PRE NPs 0.151 81
S10 PREC-2 NPs 0.66 56
S11 PREC-2 NPs 0.33 80
S12 PREC-2 NPs 0.151 85
Table 3
The relationship between ETO, ceranib-2 (C-2), PRE NPs, PREC-2, doses,
and % cell viability of Dukes’ type B colorectal adenocarcinoma cell line
(SW480)
Viability
Sample no. Treatment Dose (μM) (%)
S1 CNT (DMOS) 100 100
S2 ETO 50 60
S7 PRE NPs 0.66 78
S8 PRE NPs 0.33 80
S9 PRE NPs 0.151 82
S10 PREC-2 NPs 0.66 55
S11 PREC-2 NPs 0.33 80
S12 PREC-2 NPs 0.151 84
SW480
110
100 ns
90
80
70
%VIABILITY
60
50
40
30
20
10
0
CNT ETO 10 uM 25 uM 50 uM 100 uM 1:500 1:1000 1:2000 1:500 1:1000 1:2000
Ceranib Ceranib Ceranib Ceranib RE RE RE RE.Cer2 RE.Cer2 RE.Cer2
HELA
ns
ns
110
100
90
80
70
%VIABILITY
60
50
40
30
20
10
0
CNT ETO 10 uM 25 uM 50 uM 100 uM 1:500 1:1000 1:2000 1:500 1:1000 1:2000
Ceranib Ceranib Ceranib Ceranib RE RE RE RE.Cer2 RE.Cer2 RE.Cer2
Fig. 10 The effect of ceranib-2 on cancer cells. SW480 and HeLa cells
4 Notes
1. Avoid any contact of the glass container with the ultrasonic

probe to prevent any damage to glass beaker carrying
solution.
2. Wear hearing protectors before starting to use the device and
try to use a soundproof box to reduce sounds during process.
3. Before starting, make sure that you fix the transducer probe to
the booster horn and tighten it.
4. During sonication, transducer will generate heat; therefore
sensitive drugs should be continuously kept into an ice bath to
prevent excessive heating.
5. Circulation water bath used in this experiment is aimed to
minimize unstirring water layer effects.
6. The absorption measurements were recorded at room tem-

perature and after 10-min incubation time.
7. Do not heat; water temperature should be kept between 15
and 20 °C.
8. The cell concentration that is plated may vary for different cell
lines. For SW480 and HeLa cell lines, we use concentrations
in the range of 500 to 2000 cells/well, respectively.
9. For more reliable results the experiment should be done in
triplicate and in duplicate in case of primary cells and when a
range of drug concentrations are being tested.
10. Drugs should be added into the 96-well plates and stored at
−20 °C in advance of the experiments.
11. Shake the plates for 5 min on a plate shaker by slowly increas-
ing the shaking speed to a maximum of 900 shakes/min. Then
incubate the plate for another 4–6 h at 37 °C in a CO2 incuba-
tor, depending on the cell type.
12. The cell plate has to rest for 10 min before measuring.
13. The percentage of living cells can be determined: The average
optical density of the blank control wells (without cells and if
the drug has no specific optical density without drug as well)
is subtracted from the average optical density of the control
wells (cells but no drugs) and the wells containing the drugs.
Acknowledgments
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Chapter 17
Association of the Thermodynamics with the Functionality

of Thermoresponsive Chimeric Nanosystems
Nikolaos Naziris, Athanasios Skandalis, Thomas Mavromoustakos,
Stergios Pispas, and Costas Demetzos
Abstract
Stimuli-responsive nanosystems are an emerging technology in the field of therapy and are very promising
for various applications, including targeted drug delivery. In this chapter, our scope is to integrate two
different methodologies, namely differential scanning calorimetry (DSC) and dynamic light scattering
(DLS), in order to rationally approach the functional behavior of thermoresponsive chimeric/mixed lipo-
somes and interpret their thermoresponsiveness on a thermodynamic basis. In particular, chimeric bilayers
comprised of the phospholipid 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and two different-
in-
composition thermoresponsive amphiphilic block copolymers poly(N-isopropylacrylamide)-b-
poly(lauryl acrylate) (PNIPAM-b-PLA) 1 or 2 were built by a conventional evaporation technique,
followed by DSC, and chimeric liposomes of DPPC and PNIPAM-b-PLA 1 were developed and studied
by DLS, after preparation and after a simple heating protocol. The results from both methodologies indi-
cate the composition- and concentration-dependent lyotropic effect of the foreign copolymer molecule on
the properties and functionality of the lipidic membrane.
Key words Differential scanning calorimetry, Dynamic light scattering, Chimeric nanosystems,
Phospholipid, Thermoresponsive amphiphilic block copolymers
1 Introduction
Thermoresponsive nanosystems have gained considerable atten-

tion over the last few years, with applications in various fields of
therapy, including drug delivery, diagnosis of diseases, as well as
promising field of theranostics. Stimuli-responsive nanoparticles
are developed by incorporating functional biomaterials, such as
certain polymers, inside classic nanoplatforms, such as liposomes,
in order to build chimeric/mixed nanosystems, which belong to
the class of advanced drug delivery nanosystems (aDDnSs) [1–3].
In this approach, targeted and controlled drug delivery is achieved,
where the nanocarrier functionality is activated by endogenous or
221
exogenous stimuli, including pH and temperature fluctuations.

The concept for targeted and controlled drug delivery suggests the
release of the incorporated therapeutic moiety, e.g., the drug mol-
ecule, in a site- and time-specific manner. The result is the increased
bioavailability of drugs, their accumulation in diseased tissues
instead of healthy ones, and therefore increased efficacy and safety
[4, 5].
A thermoresponsive macromolecule that has drawn attention
in biomedical applications is poly(N-isopropylacrylamide)
(PNIPAM). This polymer has the distinct property of reversible
phase transition from water soluble to water insoluble, when
heated above a certain temperature value (32 °C) that is known as
the lower critical solution temperature (LCST). The transition is
also called coil-to-globule transition, since the polymer changes its
conformation, from hydrated and extended to dehydrated and
shrinked [6]. The LCST of PNIPAM and other thermoresponsive
materials can be tailored close to the physiological temperature
(37 °C), which makes them very promising candidates for thera-
peutic and other human-related bioapplications [7, 8].
PNIPAM has been combined with liposomal technology, lead-
ing to thermoresponsive chimeric nanosystems. One of the
approaches involves attaching PNIPAM to another polymer that is
hydrophobic, producing an amphiphilic diblock copolymer and
incorporating it inside lipidic bilayers [9, 10]. This integration has
in turn been studied on a thermodynamic and physicochemical
basis, which aims to delineate the thermodynamic and biophysical
properties of these combinatorial platforms, with the final aim of
predicting their stability and functionality for drug delivery appli-
cations [11–13].
2 Materials
The materials described below concern the preparation of thermo-

responsive chimeric/mixed bilayers, by utilizing a phospholipid
and two different thermoresponsive amphiphilic block copolymers;
the DSC analysis of the samples in 40 μL aluminum pans, hydrated
with phosphate-buffered saline (PBS) medium; the development
of thermoresponsive chimeric/mixed liposomes, by utilizing the
same phospholipid and one of the two copolymers that presented
the most promising functional properties; and finally the use of
dynamic light scattering for measurement of their physicochemical
properties, for which dilution was achieved with HPLC-grade
H2O.
2.1 Preparation 1. DPPC (Mw = 734.039) (Fig. 1A).

of Thermoresponsive
Chimeric Bilayers
Thermodynamics and Functionality of Thermoresponsive Chimeric Nanosystems 223
Fig. 1 Molecular structures of +++ (A) DPPC and (B) PNIPAM-b-PLA 1 (with n:m weight ratio 34:64) or 2 (with
n:m weight ratio 50:50)
2. PNIPAM-b-PLA 1 (66–34% w/w, Mw = 18,000) (see Notes

1–3) (Fig. 1B).
3. PNIPAM-b-PLA 2 (50–50% w/w, Mw = 6,400).
4. Chloroform.
2.2 Differential 1. DSC aluminum pans (40 μL).

Scanning Calorimetry 2. Pure indium (Tm = 156.6 °C).
(DSC)
3. Bilayer samples/multilamellar vesicles (MLVs) (see Note 4):
(a) DPPC.
(b) DPPC:PNIPAM-b-PLA 1 9:0.02 (see Note 5).
(c) DPPC:PNIPAM-b-PLA 1 9:0.05.
(d) DPPC:PNIPAM-b-PLA 1 9:0.1.
(e) DPPC:PNIPAM-b-PLA 1 9:0.2.
(f) DPPC:PNIPAM-b-PLA 1 9:0.5.
(g) DPPC:PNIPAM-b-PLA 1 9:1.
(h) PNIPAM-b-PLA 1.
(i) DPPC:PNIPAM-b-PLA 2 9:0.02.
(j) DPPC:PNIPAM-b-PLA 2 9:0.05.
(k) DPPC:PNIPAM-b-PLA 2 9:0.1.
(l) DPPC:PNIPAM-b-PLA 2 9:0.2.
(m) DPPC:PNIPAM-b-PLA 2 9:0.5.
(n) DPPC:PNIPAM-b-PLA 2 9:1.
(o) PNIPAM-b-PLA 2.
4. PBS medium (pH = 7.4) (see Note 6).
2.3 Preparation 1. DPPC.

of Thermoresponsive 2. PNIPAM-b-PLA 1.
Chimeric Liposomes
3. Chloroform.
4. PBS medium (pH = 7.4).
2.4 Dynamic Light 1. Polystyrene cuvettes (4.5 mL).

Scattering (DLS) 2. Liposomal samples/small unilamellar vesicles (SUVs) (see Note
and Heating Protocol 7):
(a) DPPC.
(b) DPPC:PNIPAM-b-PLA 1 9:0.02.
(c) DPPC:PNIPAM-b-PLA 1 9:0.05.
(d) DPPC:PNIPAM-b-PLA 1 9:0.1.
3. HPLC-grade H2O.
3 Methods
The methods described herein are a conventional evaporation

technique to prepare thermoresponsive chimeric bilayers, DSC
sample preparation, sample analysis, extraction and analysis of the
results, thin-film hydration method for developing thermorespon-
sive chimeric liposomes or liposomes in general, physicochemical
characterization of the latter through dynamic light scattering, and
a simple heating protocol to test the thermoresponsive behavior of
the developed nanoparticles. The results from the DSC analysis
and the physicochemical characterization are presented and
discussed.
3.1 Preparation 1. Dissolve the desired amount of DPPC and each one of the chi-
of Thermoresponsive meric nanosystems DPPC:PNIPAM-b-PLA 1 or 2 at 9:0.02,
Chimeric Bilayers 9:0.05, 9:0.1, 9:0.2, 9:0.5, and 9:1 molar ratios in chloroform
(see Note 8).
2. Evaporate the solvent under vacuum and heat conditions
(−1 bar and 40 °C), using a rotary evaporator.
3. Maintain the formed dry lipid films under these conditions for
30 min (see Note 9).
4. Place the dry lipid films in a desiccator, for at least 24 h, in order
to remove possible traces of solvent.
3.2 DSC Sample 1. Prepare each sample for DSC analysis by weighting 3 mg of dry
Preparation powder of lipidic or chimeric nanosystem inside a 40 μL alumi-
num crucible and hydrating it with 20 μL PBS medium (see
Notes 10).
2. Seal each crucible by using a sealing press, in order to make a
hermetic pan, and leave it to rest for a 15-min period, in order
to achieve equilibration of the sample (see Note 11).
3.3 DSC Analysis 1. Calibrate the calorimeter with pure indium (Tm = 156.6 °C)
before analyses, by applying a single heating cycle, and check
the characteristic transition temperature Tm and enthalpy change
ΔH to be within specifications.
2. Obtain the DSC thermogram of each sample by utilizing any
appropriate DSC calorimeter.
3. Include for each analysis a 10-min isotherm at 20 °C (see Note
12), two heating-cooling cycles between 20 and 60 °C, and a
final heating process from 20 °C to 60 °C (see Notes 13 and
14), at a scanning rate of 5 °C min−1 (see Note 15), under con-
stant nitrogen gas flow rate of 50 mL min−1 (see Note 16).
3.4 DSC Profile 1. Analyze the obtained calorimetric data by using the software
and Thermodynamic installed in the calorimeter.
Parameter Extraction 2. Select the desired heating or cooling cycle to analyze (see Note
17). In this case, choose the first and second heating scan, as
well as the first cooling scan (see Note 18).
3. Normalize each sample analysis per weight/moles of total sam-
ple, lipid, or polymer (see Note 19).
4. Choose the desired thermodynamic parameters for each ther-
modynamic phenomenon, i.e., endothermic or exothermic.
These are the characteristic transition temperatures Tonset and T,
enthalpy change ΔH, and width at half peak height of the Cp
profiles ΔT1/2.
5. During peak integration, manually set the baseline carefully.
3.5 DSC Profile The profiles of all samples containing DPPC bilayers with incorpo-
Analysis of the rated thermoresponsive amphiphilic block copolymers PNIPAM-
Thermoresponsive b-PLA 1 or 2 are presented in Fig. 2.
Chimeric Systems The following observations can be made on the
thermograms:
∙∙ Incorporation occurs and the copolymers alter the thermo-
tropic behavior of DPPC bilayers in a concentration- and
composition-dependent effect, where increasing amounts of
the same polymer lead to main transition temperature Tm
(41.8 °C) elevation and ΔΗm reduction during the first heating
cycle. As a result, the lyotropic effect of the polymers is evident
and the higher Tm values are associated with a new metastable
functional/thermoresponsive phase, which is created on the
membrane when the PLA segment is incorporated inside the
membrane, with the PNIPAM groups extending outside to
form a hydrophilic corona.
Fig. 2 The DSC thermograms of DPPC:PNIPAM-b-PLA 1 (A) first and (B) second DSC heating cycles for systems
a. DPPC and DPPC:PNIPAM-b-PLA 1, b. 9:0.02, c. 9:0.05, d. 9:0.1, e. 9:0.2, f. 9:0.5, g. 9:1, and h. PNIPAM-b-
PLA 1 and of DPPC:PNIPAM-b-PLA 2 (C) first and (D) second DSC heating cycles for systems a. DPPC and
DPPC:PNIPAM-b-PLA 2, b. 9:0.02, c. 9:0.05, d. 9:0.1, e. 9:0.2, f. 9:0.5, g. 9:1, and h. PNIPAM-b-PLA 2
∙∙ The PNIPAM groups interact with the phospholipid head

groups, which leads to the gradual elimination of the second-
ary pretransition peak (Ts, 36.6 °C).
∙∙ During the first heating process, the polymer chains respond
and rearrange inside the membranes, also leading to mem-
brane disruption, redistribution, or detachment of the hydro-
phobic anchor and formation of domains or self-assembly of
each copolymer to new types of structures. These phenomena
are more obvious after the second heating scan, where the
membrane disruption is reflected on the reduced ΔΗm values
compared to the first scan, the redistribution/detachment
from the reinstatement of the Tm, and the reappearance of the
pretransition in the case of PNIPAM-b-PLA 1 and the domain
formation/new structure formation from the appearance of a
new transition at 28 °C, which is very close to the LCST of
PNIPAM and is related to the thermoresponsive behavior of
the polymer.
∙∙ PNIPAM-b-PLA 1 is more efficient in rendering the DPPC

membranes thermoresponsive, due to its higher molecular
weight, but also due to its more hydrophilic composition. This
is evident from the alterations in the thermodynamic behavior
of DPPC from the first to the second heating scan, where the
newly formed phase leads to functional behavior and new phe-
nomena; phases appear after its transition, especially at high
polymer concentrations (see Note 20).
3.6 Preparation 1. Dissolve the desired amount of DPPC and each one of the chi-
of Thermoresponsive meric nanosystem DPPC:PNIPAM-b-PLA 1 at 9:0.02, 9:0.05,
Chimeric Liposomes and 9:0.1 molar ratios in chloroform.
Through the Thin-Film 2. Transfer each solution into a round flask, connected to a rotary
Hydration Method evaporator.
3. Evaporate the solvent under vacuum and heat conditions
(−1 bar and 40 °C).
4. Maintain the formed dry lipid films under these conditions for
30 min.
5. Place the dry lipid films in a desiccator, for at least 24 h, in order
to remove possible traces of solvent.
6. Hydrate the dry lipid films with PBS (pH = 7.4), for a final total
biomaterial concentration of 5 mg mL−1, and slowly stir for 1 h
in a water bath, above the phase transition temperature of the
containing phospholipid (45 °C) (see Notes 21–23).
7. Subject the resultant suspensions to two 5-min sonication cycles
(amplitude 70%, cycle 0.5 s), interrupted by a 5-min resting
period, by using a probe sonicator (see Note 24).
8. Allow the formed chimeric systems to anneal for 30 min (see
Note 25).
3.7 Dynamic Light 1. Dilute aliquots of the prepared chimeric systems 30-fold in
Scattering (DLS) HPLC-grade water (see Notes 26 and 27).
2. Measure the size (hydrodynamic diameter, Dh) and size distri-
bution (polydispersity index, PDI) with a photon correlation
spectrometer, at a detection angle of 90° and at 25 °C.
3. Analyze the measurements by the CONTIN method (see Note
28).
3.8 Heating of the 1. Place samples DPPC and DPPC:PNIPAM-b-PLA 1 9:0.02,

Thermoresponsive 9:0.05, and 9:0.1 at 45 °C for 30 min (see Note 29).
Chimeric Liposomes 2. Remove them from high temperature and allow them to reach
room temperature (see Note 30).
3. Measure the size (hydrodynamic diameter, Dh) and size distri-
bution (polydispersity index, PDI) with a photon correlation
spectrometer, with DLS, at a detection angle of 90° and at

25 °C.
4. Analyze the measurements by the CONTIN method.
3.9 Physicochemical The physicochemical properties of DPPC liposomes with incorpo-

Properties of the rated thermoresponsive amphiphilic block copolymer PNIPAM-b-
Thermoresponsive PLA 1 in different molar ratios after preparation, as well as after
Chimeric Liposomes heating, are presented in Fig. 3.
A Size
1200.0
1000.0
800.0
Dh (nm)
600.0
400.0
200.0
0.0
DPPC 9:0.02 9:0.05 9:0.1
Molar Ratio
B Polydispersity
1.000
0.800
0.600
PDI
0.400
0.200
0.000
DPPC 9:0.02 9:0.05 9:0.1
Molar Ratio
C
Thermoresponsive Liposomes Agglomeration
Fig. 3 Physicochemical properties, regarding (A) size and (B) polydispersity, of DPPC:PNIPAM-b-PLA 1 chimeric
liposomes after preparation (blue bars) and after heating at 45 °C (red bars) and (C) proposed mechanism of
the thermoresponsive behavior of chimeric liposomes
The following observations can be made on the diagrams:

∙∙ The initial particle size of DPPC:PNIPAM-b-PLA 1 chimeric
liposomes is lower than conventional DPPC, regardless of the
amount of polymer incorporated, and the polydispersity is
higher.
∙∙ After heating the liposomes, DPPC ones do not respond to the
stimulus on either the level of particle size or the size distribu-
tion. On the other hand, the chimeric liposomes with incorpo-
rated polymer respond to the increased temperature condition,
confirming their thermoresponsive behavior and functionality.
∙∙ The size increases after heating in a polymer-concentration-
dependent manner, as predicted by DSC experiments, while
the polydispersity reaches maximum for all chimeric nanosys-
tems. The results are accompanied by agglomerate formation,
which is macroscopically visible and increased for higher
amounts of polymer. These agglomerates are formed at high
temperature (45 °C) and more specifically above the thermo-
responsive functional phase that was recorded for the chimeric
nanosystems and are ostensibly reversible after reaching room
temperature again, but depending on the polymer amount,
membrane disruption and fusion phenomena occur that in
turn alter the liposomal properties.
∙∙ This behavior is owed to the responsive nature of PNIPAM,
which is hydrophilic below LCST and more hydrophobic
above it, resulting in the so-called coil-to-globule transition
and the transition of the polymer chains from an extended to a
shrinked conformation, leading to thermodynamically favor-
able hydrophobic interactions between the chimeric nanopar-
ticles. This transition affects the liposomal membranes because
of the incorporated PLA chain, but also because the membrane
surface is exposed and liposomes are more prone to fusion and
aggregation.
4 Notes
1. The copolymers were synthesized via reversible addition-

fragmentation chain-transfer (RAFT) polymerization method-
ology and further characterized by size-exclusion
chromatography (SEC) and nuclear magnetic resonance
spectroscopy (NMR). More details are provided in a previous
work [14].
2. The weight-average molecular weights Mw of PLA and
PNIPAM blocks were 5800 and 12,200 (18,000 total) for
PNIPAM-b-PLA 1 and 3900 and 2500 (6,400 total) for
PNIPAM-b-PLA 2, respectively. Polydispersity index (Mw/Mn)

of the copolymers was 1.55 and 1.16, respectively.
3. PLA is a hydrophobic polymer, while PNIPAM is hydrophilic
below and becomes hydrophobic above the LCST value [6,
15].
4. For DSC analysis, dry bilayers that were hydrated with low
amount of aqueous medium were utilized, resulting in MLV
formation, in order to have high concentration and strong
DSC signal.
5. Each ratio of 9:0.02, 9:0.05, 9:0.1, 9:0.2, 9:0.5, and 9:1
stands for phospholipid:copolymer molar ratio.
6. The PBS medium simulates the pH, osmolarity, and ion con-
centrations of the human physiological environment.
7. For liposomal development, the dry lipid films were hydrated
with more amount of aqueous medium, which resulted in the
proper concentration for liposomes to self-assemble and pro-
vide SUVs after size reduction.
8. Phospholipids and the particular diblock copolymer are solu-
ble in chloroform [16].
9. Most of the organic solvent evaporates rapidly under these
conditions; however, the formed dry films are maintained as
such for a short period of time.
10. The bilayers are considered as fully hydrated and the concen-
tration of the final system is 150 mg mL−1.
11. Equilibration is necessary for samples that have been previ-
ously hydrated with aqueous media.
12. The isotherm at the analysis starting point is necessary to
ensure that the sample is at equilibrium. For a lyotropic liquid
crystalline system like lipidic membranes, it is very important
to ensure that it is in equilibrium state before analysis.
13. The analysis temperature range should include all membrane
thermodynamic phenomena of interest that might be affected
by the insertion of the polymers. In addition, the LCST of the
PNIPAM was taken into account.
14. Three heating scans are necessary to ensure the reproducibility
of the thermodynamic phenomena, as well as the equilibrium
of the studied system.
15. 5 °C min−1 is a generally accepted heating or cooling rate for
lipidic bilayers [11].
16. Constant pressure is essential in this type of calorimetry, in
order to monitor the heat capacity of the sample under con-
stant pressure (Cp).
17. In general, the cycle after equilibrium of the system has been
achieved is selected for analysis in the case of membranes.
However, when thermoresponsive phenomena are involved,
which are expected to be nonreversible in most cases of drug
delivery nanosystems, all individual cycles could hold essential
information on the behavior of these systems.
18. The second heating scan is identical with the third and the first
cooling scan is identical to the second.
19. Each analysis may be normalized per total sample weight, per
lipid weight/moles, or per polymer weight/moles. In the case
of membranes, normalization usually is done per lipid.
However, phenomena that are of polymeric nature, such as
the LCST, should be normalized per polymer.
20. The new phase that is created by incorporating the copolymers
inside the lipidic membrane is characterized as nonequilib-
rium/metastable and nonreversible-phase thermoresponsive
functional phase.
21. The total biomaterial concentration of 5 mg mL−1 includes the
phospholipid and copolymer.
22. Hydration is carried out at temperatures where the phospho-
lipids are in their liquid crystalline state, which is of increased
mobility and allows for self-assembly in vesicles. Herein, poly-
mer insertion affects the main transition temperature and as a
result hydration temperature must be higher than for DPPC.
23. The nanosystems should be checked for any aggregation phe-
nomena during the early steps in hydration and vortexed ade-
quately in such a case, in order to resuspend the particles.
24. Probe sonication leads to high energy input to the sample and
this leads to very high temperatures, which are expected to set
off the thermoresponsive behavior of the chimeric nanosys-
tems. However, since no agglomeration occurs during this
procedure and in addition the thermoresponsive behavior was
detected later during physicochemical characterization, it is
concluded that this does not happen and probe sonication
facilitates the polymer incorporation inside the membrane.
25. Liposomal formulation is left to achieve an equilibrium state
after any size reduction method, especially if this includes high
energy input.
26. Samples must be diluted before measurement, in order to have
the appropriate intensity of scattered light during DLS mea-
surements. This varies, depending on the instrument utilized,
but generally a value of 300–500 KCps is acceptable.
27. HPLC-grade or deionized H2O is used as diluent, in order to
avoid any ingredients that might interfere with the measure-
ment. In certain cases, buffers may be preferable to dilute a
sample and measure its properties, but those might affect the
measurement quality.
28. The CONTIN method is appropriate for the analysis of poly-
disperse systems.
29. The samples are exposed to a temperature value that exceeds
the transition temperature of the thermoresponsive functional
phase.
30. The equilibration of samples back to room temperature is nec-
essary to evaluate the final effect of the thermoresponsive
polymer on the liposomal membrane.
Acknowledgments
The research work was supported by the Hellenic Foundation for

Research and Innovation (HFRI) and the General Secretariat for
Research and Technology (GSRT), under the HFRI PhD
Fellowship grant (GA. no. 392).
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Chapter 18
2D DOSY NMR: A Valuable Tool to Confirm the Complexation

in Drug Delivery Systems
Christos M. Chatzigiannis, Sofia Kiriakidi, Andreas G. Tzakos,
Abstract
Many bioactive substances face the problem of limited bioavailability, mainly due to low aqueous solubility
and poor metabolic stability. Their complexation with drug delivery systems offers a more optimum phar-
macological profile. Some of these drug delivery systems that have promising potential form complexes
with bioactive compounds such as cyclodextrins and calixarenes. The monitoring of the success and the
type of the complexation are of great importance and two-dimensional diffusion-ordered NMR spectros-
copy (2D DOSY) is a valuable tool for the studying of these complexes and described as “NMR chroma-
tography.” Herein we report the procedure for the complexation of the natural product quercetin in
2-hydroxypropyl-β-cyclodextrin and the anticancer drug temozolomide in p-sulfonatocalix[4]arene and
the determination of the complexation with 2D DOSY spectroscopy.
Key words Cyclodextrins, 2-Hydroxypropyl-β-cyclodextrin, Quercetin, Temozolomide, p-

Sulfonatocalix[4]arene, Complex, Bioavailability, Diffusion-ordered NMR spectroscopy, 2D DOSY,
NMR spectroscopy
1 Introduction
Quercetin (QUE) is a flavonoid with low aqueous solubility

responsible for its poor absorption through the gastrointestinal
tract after oral administration. Besides, its extensive metabolism
through enzymatic processes such as glucuronidation, sulfation,
and methylation that take place mainly in the liver and the small
intestine leads to the formation of metabolites, characterized by
less bioactivity. During intravenous injection, quercetin may cause
local toxicity and other side effects that limit the administered
dose. To circumvent these problems is encapsulated in cyclodex-
trins [1–4] (Fig. 1).
Cyclodextrins and especially 2-hydroxypropyl-β-cyclodextrin
(HP-β-CD) (Fig. 1) have been widely used to protect drugs against
conjugation and metabolic inactivation as well as to enhance the
235
236 Christos M. Chatzigiannis et al.
Fig. 1 Structure of QUE and one isomer of HP-β-CD (DS = 7)
aqueous solubility and hence to advance the oral bioavailability

[5–7]. HP-β-CD is an isomer mixture with a different degree of
hydroxyl-propylation substitution (DS) [8–10] (Fig. 1).
Temozolomide (TMZ) is established as a first-line chemother-
apeutic agent for the treatment of primary brain tumors and brain
metastases and especially glioblastoma (GBM). GBM represents
the most common and aggressive malignant primary brain tumor
[11]. TMZ can cross the blood-brain barrier (BBB) and belongs to
the group of prodrugs as it transforms to its active form, the
metabolite 5-(3-methyltriazen-1-yl) imidazole-4-carboxamide
(MTIC), which is an alkylating agent of the N-7 or O-6 position
of guanine residues of the DNA [12, 13]. Due to this process DNA
damage is caused which leads from inhibition of DNA replication
to finally cell cycle arrest. This methylation of the DNA residues
can be repaired by O6-methylguanine-DNA methyltransferase
(MGMT), limiting TMZ’s activity to approximately 60% making
GBM tumors resistant. Despite this acquired resistance of GBM
tumors, TMZ faces more obstacles which diminish its clinical
potential [13, 14]. One of those is its rapid hydrolysis to slightly
2D DOSY NMR: A Valuable Tool to Confirm the Complexation in Drug Delivery Systems 237
Fig. 2 (a) The chemical structures of temozolomide and p-sulfonatocalix[4]arene. (b) The chemical degradation
of temozolomide
alkaline pH conditions to the MTIC form, and this active alkylat-

ing agent quickly degrades to the methyl diazonium cation and the
metabolite 5-amino-imidazole-4-carboxamide (AIC) (Fig. 2).
In contrast to TMZ, MTIC possesses poor BBB penetration
and depletes cellular uptake. So, the therapeutic efficacy of MTIC
relies on the stability of TMZ and its delivery through the blood-
brain barrier. Unfortunately, due to these drawbacks, only approxi-
mately 20% of the administered amount of TMZ is detected inside
the cerebrospinal fluid at peak concentrations. Therefore high
doses of TMZ are required to achieve the desirable antitumor
effect, which leads to severe side effects [15, 16].
Calixarenes belong to the family of supramolecular macrocy-

cles and are cyclic oligomers obtained by condensation of suitable
p-functionalized phenols with formaldehyde, usually allowing for
the synthesis of the well-known small calixarenes (including up to
eight phenolic subunits). They have been mostly used in drug
encapsulations to improve the therapeutic efficacy of a drug, to
reduce the cytotoxicity in healthy tissues, to improve the pharma-
cokinetic profile of a drug, and to improve the bioavailability by
enhancing the aqueous solubility and slowing down the degrada-
tion rate of a therapeutic agent [16]. The main reason that these
supramolecular carriers have earned attention in medicinal chemis-
try is the chemical space they offer by introducing to their core
easily functional groups. The most popular types of calixarenes
consist of four, six, or eight subunits (Fig. 2) [17].
2D DOSY experiment is an accurate, noninvasive, molecular
diffusion measurement experiment useful for biofluids, complex
chemical mixtures, and multicomponent solutions. In the 2D
DOSY experiment the chemical shift is shown along the detected
F2 axis (x-axis) and the diffusion coefficient is along the other F1
axis (y-axis). Although in some cases we will be referred to
BRUKER NMR spectrometer, the instructions can be generalized
and applied for all NMR spectrometers (Fig. 3).
The goal of the 2D DOSY experiment is to separate species
spectroscopically (not physically) present in a mixture of compounds.
For this reason, 2D DOSY is also termed “NMR chromatography.”
For acquiring better results, diffusion coefficients of the components
of the mixture are desirable to differ significantly [18].
2D DOSY experiment is an accurate, noninvasive, molecular
diffusion measurement experiment useful for biofluids, complex
chemical mixtures, and multicomponent solutions. In the 2D
Fig. 3 A 2D DOSY experiment consists of two axes. On the F1 is plotted the diffusion coefficient and on the F2
axis the chemical shift
DOSY experiment the chemical shift is shown along the detected

F2 axis (x-axis) and diffusion coefficient is along the other F1 axis
(y-axis). Although in some cases we will be referred to BRUKER
NMR spectrometer, the instructions can be generalized and applied
for all NMR spectrometers (Fig. 3).
The goal of the 2D DOSY experiment is to separate species
spectroscopically (not physically) present in a mixture of compounds.
For this reason 2D DOSY is also termed “NMR chromatography.”
For acquiring better results the diffusion coefficients of the compo-
nents of the mixture are desirable to differ significantly [12].
One interesting experiment for checking the formation of the
complex between an organic molecule and supramolecule is the
2D DOSY (diffusion-ordered spectroscopy). In this chapter we
provide details for obtaining a decent spectrum in which the useful
parameter diffusion coefficient can be calculated.
Some terms are provided to understand the physics behind the
experiment. The experiment is based on the application of gradi-
ents. Let us consider spatially homogeneous B0 to be oriented in
the z-direction, and ω is the same throughout the sample. In addi-
tion to B0 there is a spatially dependent magnetic field gradient g
(T m−1):
ωeff (n,r ) = n (ω0 + γ ( g .r ) ) , (1)
where g is defined by the grad of the gradient field component

parallel to B0, i.e.,
∂Bz ∂Bz ∂Bz
g = ∇B 0 = i+ j+ k, (2)
∂x ∂y ∂z
where i, j, and k are unit vectors of the laboratory frame of refer-

ence. The important point is that if a homogeneous gradient of
known magnitude is imposed throughout the sample, the Larmor
frequency becomes a spatial label with respect to the direction of
the gradient.
The pulse sequence for the most frequently used ledbpgp2S
experiment is given below (which is installed inside the Bruker
NMR spectrometer library) (Fig. 4).
δ is the duration of the gradient pulse. Δ is the delay that takes
place during the diffusion and g is the gradient strength (ramped
in a series of experiments). Important parameters that must be
adjusted during the experiment are the diffusion delay (d20 in
Bruker instruments) and gradient duration (p30 in Bruker instru-
ments). In the pulse sequence bpg stands for bipolar gradients.
These gradients modify the known LED gradient pulse sequence
replaced by two pulses of different polarity separated by a 180°
pulse. This replacement offers two advantages: (a) eddy currents
are reduced to a minimum and (b) the effective gradient output is
double. Ledbgp is useful for relatively low diffusion coefficients
Fig. 4 Pulse sequence for a 2D DOSY experiment
and requires large gradients. It is the choice for many DOSY

experiments.
The intensity of the signal depends on Δ, δ, and g and is given
by the formula
 δ
I ( Mn ) = Io ( Mo ) e −∆ g 2δ 2  ∆ −  , (3)
 3
where I is the observed intensity or magnetization (Mn), Io is the

unattenuated reference signal intensity or magnetization (Mo),
and γ is the gyromagnetic ratio. If dipolar gradients are used for
dephasing and rephasing a correction for the time τ between those
bipolar gradients is inserted [12]:
 δ τ
I ( Mn ) = Io ( Mo ) e −∆ g 2δ 2  ∆ − −  .
 3 2
2 Materials
Quercetin, temozolomide, p-sulfonatocalix [4]arene, and

HP-β-CD can be obtained by any source and must be pure 99 + %.
3 Methods
3.1 Preparation 1. Weigh 0.030 g of QUE and 0.306 g of HP-β-CD accurately,

of the Complex transfer them in a 50 mL beaker, and suspend with 20 mL of
3.1.1 Preparation
water.
of the Que-HP-β-CD 2. Add small amounts of ammonium hydroxide under continuous
Complex stirring and monitor pH until complete dissolution.
3. Adjust pH to a value between 9 and 10.
4. Freeze the resulting solution at a molar ratio of 1:2 at −80 °C
and freeze-dry to get the lyophilized product. Temozolomide
and p-sulfonatocalix[4]arene can be obtained by any source and
must be pure 99+%.
3.1.2 Preparation 1. Dissolve 10 mg (1 eq) of p-sulfonatocalix[4]arene in 3 mL

of the TMZ-Calixarene phosphate buffer pH 7.0 (10 mM).
Complex 2. Dilute 4 mg (1.5 eq) of TMZ in 300 μL MeOH.
3. Mix the solutions of TMZ and calixarene and magnetically stir
at room temperature for 1 h.
4. Filter the homogenous solution through nylon filter with
0.45 mm pore size.
5. Evaporate MeOH under reduced pressure and lyophilize the
aqueous phase giving the temozolomide-calixarene complex.
3.2 NMR At the beginning you obtain a 1H NMR experiment for the com-
Spectroscopy plex. The following steps are used:
3.2.1 1
H NMR Spectrum 1. Lock the sample in D2Ο (see Note 1) where the complex is dis-
solved at a constant temperature (see Note 2) and a higher gas
flow than the normal (i.e., 400 L/h) (see Note 3).
2. Tune and match probe head.
3. Optimize the shim values as well as the receiver gain.
4. Obtain a conventional 1D spectrum using a pulsed field gradi-
ent unit capable of producing magnetic field pulse gradients in
the z-direction of 53 G cm−1 (Fig. 5).
5. Calibrate the 90° (1H) pulse.
6. Use a sufficient number of scans to obtain a decent signal/noise
(see Note 4).
A B Temozolomide-p-sulfonato-Calixarene Calix CH2-H

Free Quercetin 5'
OH
complex
Quercetin-Hp-β-CD 6'
4' TMZ CH3-H
8 3' Calix Aromatic-H
HO 9
0
1'
7 2 OH
2' TMZ Imidazole-H
10
6 4 3
5 OH O
2' H OH O N N
6tH Temozolomide(TMZ) N
5'H
8H N
6'H N CH3-H
NH3
O
Imidazole-H Temerolomide
HO3S SO3H SO3H SO3H
Hp-β-CD H p-sulfonato-Calixarene
(Calix) CH2-H
Aromatic - H OH OH OH OH
p-Sulfomato-Colix[4]arene
7.5 6.9 6.3 5.7 5.1

'H NMR chemical shift (ppm) 8 6 4 [PPM]
'H NMR chemical shift (ppm)
Fig. 5 (a) 1H NMR region between 5.0 and 7.8 ppm for free quercetin (red) and its complex (black). Two crucial
observations can be realized. First, the complex contains some new peaks attributed to the presence of
HP-β-CD and second the chemical shifts of the aromatic peaks are shifted downfield. (b) 1H NMR region
between 3 and 10 ppm for free temozolomide (red), p-sulfonatocalixarene (black), and its complex (green).
Two important observations can be realized; both are regarding the shifts of temozolomide’s imidazole-H and
the methyl group which are both downshifted
7. Measure T1 of your sample. The relaxation delay should be

5×T1 with respect to the largest relaxation measurement. T1
values are calculated from 2 to 3.5 s after they have been deter-
mined by the inversion recovery time pulse.
8. Spin your sample (see Note 5).
3.2.2 2D DOSY NMR The second step is the establishment of the intensities of the sec-
Spectrum ond spectrum that are only 2–5% relative to the first one. The steps
to achieve this are the following:
1. Run the first spectrum with gpz6 = 2 and the second with
gpz6 = 95. This means that the intensity of the peaks on the
second spectrum is only 5% relative to the first one. The mag-
netization falls according to Fig. 6.
2. Check the ratio of the intensities of the two spectra. If it is not
proper it means that the above curve is not followed and decays
with either slower or faster fashion (Fig. 6).
3. If you are not achieving the proper ratio, change p30 and d20
parameters accordingly until you achieve it. Start first with p30
and then with d20. The range of values you can have to achieve
your aim is as follows:
d20 ≤ 2500 ms
p30 ≤ 5 ms
4. Collect 16 BPPLED spectra with 16 K data and set the eddy
current delay (Te) to 5 ms. A 2D DOSY for the quercetin com-
plex with HP-β-CD is shown in Fig. 7 and a 2D DOSY for the
temozolomide calix[4]arene is shown in Fig. 8 (see Note 6).
Fig. 6 Magnetization falls during the various defined experiments on the 2D DOSY experiments (top). Faster or
slower decay of magnetization which is not desired (bottom)
Fig. 7 2D DOSY of quercetin complexed with HP-β-CD

Fig. 8 2D DOSY of temozolomide complexed with p-sulfonatocalix[4]arene
As it can be observed, on the F1 axis are shown the D values

and on F2 the chemical shifts of the complex. It is apparent that
the free HP-β-CD has higher D than the complex as it is expected
to be more mobile.
Specifically, the diffusion coefficient of free HP-β-CD is found
to be 3.55 × 10−10 m2s−1 and that of the complex of 3.09 × 10−10 m2s−1
indicating the formation of a complex. The comparison of the 1H
NMR spectra of free QUE and complex QUE–HP-β-CD also
indicates the formation of the complex as it is clearly showed the
targeted shifting of the aromatic protons of QUE (Fig. 5). As the
protons 6 and 8 of QUE demonstrate the most intense shifting it
turns out to be the protons with the stronger interaction.
In order to further provide an application of this technique to
another formulation of a drug we illustrate also the case of temo-
zolomide complexed with p-sulfonatocalix[4]arene. The diffusion
coefficient of free p-sulfonatocalix[4]arene is found to be
3.371 × e−10 m2s−1 while for the free temozolomide the diffusion
coefficient is 7.997 × e−10 m2s−1. The complex has a diffusion coef-
ficient of 2.997 × e−10 m2s−1 indicating its formation but there is
also another signal with a diffusion coefficient of 4.786 × e−10 m2s−1
which probably means that there are two kinds of complexation.
The comparison of the 1H NMR spectra of free TMZ and complex
TMZ-calix also indicates the formation of the complex as it is
clearly showed by the targeted shifting of the protons of TMZ
(Fig. 8). As the methyl group protons of TMZ demonstrate the

most intense shifting it turns out to be the protons with the stron-
ger interaction.
4 Notes
1. Select a solvent with a high viscosity. More viscous materials will

be less likely to create convection. Use solutions such as DMSO
or D2O when possible.
2. The experiment should be run at constant temperature. An
accurate temperature calibration with standards methanol or
glycol is necessary. When NMR diffusion experiments are per-
formed at temperature different from ambient temperature,
temperature gradients due to probe design can cause thermal
convection and therefore significantly affect the signal ampli-
tude. This can skew the diffusion rate constants. The influence
of the convection can be seen in distortions in decay profiles. In
such cases NMR tubes with smaller diameter are preferable
because they impose larger temperature gradient for a convec-
tion within the sample.
3. Gas flow should be steady (and higher than standard, i.e.,
525 L/h) during the performance of the experiment to ensure
better dispersion.
4. Use sufficient number of dummy scans.
5. Especially the spinning is recommended for isotropic solution.
Spinning can help stabilize forces that may create the convective
flow. Alternatively, specialized sequences can be used to com-
pensate for the convection influences.
6. If you are a Bruker user you can process the data in two ways,
either by calculating the diffusion coefficient through the spec-
trum or by using the dynamics subroutine where you get a
report with a diffusion coefficient written.
Acknowledgments
References
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insights. Angew Chem Int Ed 44(4):520–554
Chapter 19
Drug-Encapsulated Cyclodextrin Nanosponges

Maria Tannous, Fabrizio Caldera, Gjylije Hoti, Umberto Dianzani,
Roberta Cavalli, and Francesco Trotta
Abstract
To date, a number of nanocarriers, either inorganic or organic, have been developed to improve the deliv-
ery and therapeutic efficacy of various drugs. Drug delivery systems have attempted to overcome the
undesirable pharmacokinetic problems encountered. Among the various nanomaterials that have been
designed as potential nanocarriers, cyclodextrin-based polymers are of particular interest in this review.
Cyclodextrins (CD) are a class of cyclic glucopyranose oligomers, obtained from starch by enzymatic
action, with a characteristic toroidal shape that forms a truncated cone-shaped lipophilic cavity. The main
common native cyclodextrins are named α, β, and γ which comprise six, seven, and eight glucopyranose
units, respectively. Cyclodextrins have the capability to include compounds whose size and polarity are
compatible with those of their cavity.
Cyclodextrin-based cross-linked polymers, often referred to as “cyclodextrin nanosponges” (CDNSs),
attract great attention from researchers for solving major bioavailability problems such as inadequate solu-
bility, poor dissolution rate, and limited stability of some agents, as well as increasing their effectiveness and
decreasing unwanted side effects.
Registered patents about this novel system in various fields, different pharmaceutical applications, and
classes of drugs encapsulated by CDNSs are detailed. The features outlined make CDNSs a promising
platform for the development of innovative and advanced delivery systems.
Key words Cyclodextrin, Nanosponges, Encapsulation, Anticancer, Antiviral, Antibacterial, Gas

delivery, Miscellaneous
1 Introduction
Nanosponges (NSs) are versatile biocompatible cross-linked poly-

mers that greatly expand the performances of their parent cyclo-
dextrin (CD). The three native CDs used are α, β, and γ, with 6, 7,
and 8 glucopyranose units, respectively [1]. These natural deriva-
tives of starch molecules are cyclic oligosaccharides in a truncated
cone structure arrangement of glucose molecules with an inner
lipophilic cavity and hydrophilic exterior [2, 3]. The common
247
248 Maria Tannous et al.
characteristic of NSs is the presence of nanoscale pores giving them

particular properties owing to very high inclusion stability con-
stants and marked encapsulation capacity [4–9].
The synthesis of cyclodextrin nanosponges (CDNSs) can be
modulated by varying the reaction conditions to better the applica-
tions selected in several scientific and technological fields including
chemistry, agriculture, environment, food, cosmetics, etc. (patents
[10–14]) consequently used to improve the aqueous solubility of
poorly water-soluble molecules, protect degradable substances,
and design innovative drug carriers owing to their outstanding
encapsulation properties and low toxicity, being safe in acute and
repeated selected doses after oral administration to rats [15].
Hitherto, the main area of investigation concerns the pharmaceuti-
cal and biomedical scope in which CDNSs have been mostly
employed as drug delivery systems [16–18].
2 Encapsulated Cyclodextrin Nanosponges
In the past decade, nanosponges have been extensively character-

ized to ascertain pharmaceutical applications. A recent EU com-
mission report focused on the use of NSs as a promising and
innovative carrier for drug delivery. Researchers have summarized
and reviewed the state of the art of NS in drug delivery till today,
including applications carved out for cancer therapeutics [9, 17,
19–21]. CDNSs can improve wetting and solubility of molecules
that have very poor aqueous solubility to overcome one of the big-
gest limitations of anticancer drugs. These drugs can be molecu-
larly dispersed within the matrix structure to avoid their
crystallization. CDNSs encapsulating anticancer drugs are pro-
vided below [8, 18, 22].
2.1 Cyclodextrin Paclitaxel (Taxol®) is a versatile small-molecule anticancer diterpe-

Nanosponges noid derived from the bark of the Pacific yew tree (Taxus brevifo-
for Anticancer Drugs lia). Orally administered paclitaxel presents a major therapeutic
problem because of low bioavailability due to poor solubility
(0.5 mg/L) and the first-pass metabolism that occurs in the liver
and intestine. Paclitaxel is used for the treatment of head and neck
cancer, small- and non-small cell lung cancer, and bladder cancer
but the dosage form available for clinical administration requests
the use of excipients (cremophor EL, polyethoxylated castor oil,
and dehydrated alcohol). The use of nanosponges to deliver pacli-
taxel can be an alternative to classical formulations [23].
β-Cyclodextrin nanosponges were synthesized and paclitaxel
inclusion complexes were prepared. Nanosponge complexes were
evaluated using DSC, FTIR, and NMR techniques for confirma-
tion of inclusion complex formation between paclitaxel and nano-
sponges [24]. Particle size and morphology were estimated using
Drug-Encapsulated Cyclodextrin Nanosponges 249
SEM, TEM, and dynamic light scattering techniques where the

average particle size was found out to be lower than 500 nm spher-
ical in shape. Nanosponges dissolved and encapsulated paclitaxel
up to 2 mg/mL [23]. The paclitaxel-loaded nanosponges formed
a water-stable colloidal system avoiding the recrystallization of
paclitaxel. The in vitro release studies showed an almost complete
release in 2 h without initial burst effect. Delivery of paclitaxel via
nanosponges increased the amount of paclitaxel entering cancer
cells and lowered paclitaxel IC50, therefore enhancing its pharma-
cological effect [23]. Cytotoxic efficacy was determined against
MCF-7 cells and paclitaxel nanosponge’s complex was found to be
cytotoxic and more effective against this cell line [25].
After oral administration to rats, paclitaxel-loaded nanosponges
showed a relative oral bioavailability RB% of 256; the area under
the plasma concentration time curve was significantly increased
(approximately threefold) in comparison to the control group
(p < 0.05); the plasma concentration of paclitaxel was determined
using liquid chromatography. These results indicated a promising
new formulation with a safe profile being non-hemolytic and non-
cytotoxic able to enhance the oral bioavailability of paclitaxel while
avoiding the use of cremophor EL [26].
Camptothecin (CAM), a plant alkaloid isolated from the
Chinese tree Camptotheca, shows a wide spectrum of anticancer
activities. Its therapeutic use is limited by poor aqueous solubility
and rapid inactivation due to the opening of the lactone ring at the
physiological pH yielding the carboxylate form and did not reach
clinical use because of its low solubility, high degradability, and
serious side effects. β-Cyclodextrin nanosponges have been
demonstrated to be able to increase the solubility of lipophilic
compounds, protect them from degradation, and control their
release [27].
In early studies, complexes of CAM with β-cyclodextrin NS are
obtained with different cross-linking ratios (1:2, 1:4, and 1:8 on a
molar basis with the cross-linker). NS formulations of crystalline
and paracrystalline were prepared and characterized. Formulations
protected the lactone ring of CAM after their incubation in physi-
ological conditions at 37 °C for 24 h with 80% w/w of intact lac-
tone ring when compared to only around 20% w/w of plain
CAM. Crystallinity of CAM decreased after loading, zeta poten-
tials were high for stable colloidal nanosuspensions, and F1:4 for-
mulation had the most optimal loading percentage (37%). In vitro
studies indicated a slow and prolonged CAM release over a period
of 24 h. Cytotoxicity studies showed that the CAM formulations
were more cytotoxic than plain CAM on HT-29 cells after 24 h of
incubation [27].
Later, β-cyclodextrin nanosponges were found to overcome
CAM chemical disadvantages and improve the in vitro antitumor
efficacy in the androgen refractory models of prostate cancer
DU145 and PC-3 and the androgen-sensitive model

LNCaP. Camptothecin-loaded nanosponges (CAM-NSs) inhib-
ited topoisomerase I activity, and induced DNA damage and cell
cycle arrest more effectively than CAM. Annexin V/propidium
iodide staining showed an induction of cell death at low concentra-
tions that were not effective for CAM but CAM-NSs still exerted
higher antiproliferative activity than CAM. Results demonstrated
the higher antitumor effectiveness of CAM-NSs compared to
CAM in prostate cancer cells [28].
Moreover, the increased antiangiogenic activity of CAM-NSs,
together with the increased antiproliferative activity, may explain
the strikingly different antitumor effectiveness of CAM-NSs and
free CAM. This difference may be ascribed to the deterred hydro-
lysis of the active drug and to CAM-NS accumulation in the tumor
due to the EPR effect dependent on the leaky vasculature and poor
lymphatic drainage of the tumor microenvironment. In vivo exper-
iments notably could not detect the potentially increased anti-
metastatic activity of CAM-NSs due to the inhibitory activity on
tumor cell adhesion [29].
Further studies showed enhanced cytotoxic effect of CAM-
NSs in anaplastic thyroid cancer (ATC) on orthotropic xenograft
tumors. Results showed that CAM-NSs inhibited the growth of
ATC cell lines both in vitro and in vivo to a higher extent than free
CAM, and also inhibited tumor cell adhesion to endothelial cells
and migration which suggest effectiveness to inhibit tumor meta-
static dissemination, supporting relevance use of CAM-NSs to
deliver anticancer drugs in vivo [30].
Tamoxifen is a low-dose therapeutic agent belonging to a class
of drugs called selective estrogen receptor modulators which have
both estrogenic and antiestrogenic effects, used for the treatment
of both early and advanced ER+ (estrogen receptor positive) breast
cancer in pre- and postmenopausal women and typically given to
patients for long periods of time [31]. Tamoxifen’s low water solu-
bility limits its therapeutic application and calls for a stable nano-
carrier which controls the rate of drug release. Tamoxifen
nanosponge complexes (TNC) from β-cyclodextrin NS cross-
linked by carbonyldiimidazole (ratios 1:2, 1:4, and 1:8) were
obtained by freeze-drying method. Differential scanning calorim-
etry, Fourier transformed infrared spectroscopy, and X-ray powder
diffraction studies confirmed tamoxifen complexation within
NS. The area under the curve (AUC) and maximum concentration
(Cmax) of TNC formulation (1236.4 ± 16.12 μg h/mL h,
421.156 ± 0.91 μg/mL) after gastric intubation were higher (1.4-
fold) than plain drug. Cytotoxic studies on MCF-7 cells showed
that TNC formulation was more cytotoxic than plain tamoxifen
after 24 and 48 h of incubation [31].
Temozolomide (TMZ), second-generation imidazotetrazine
prodrug that undergoes spontaneous conversion under physiologi-
cal conditions to the active alkylating agent MTIC, has d emonstrated

schedule-dependent antitumor activity against a variety of malig-
nancies, including glioma, metastatic melanoma, and other diffi-
cult-to-treat cancers [32]. β-Cyclodextrin and diphenyl carbonate
as starting materials for NS were synthesized to fabricate a novel
drug delivery system for the treatment of brain tumors. Successful
loading of TMZ in prepared NS was confirmed using solution state
interaction studies wherein shift in wavelength of TMZ with
increasing concentration of NS indicated masking of hydrophobic
groups. In vitro release profile studies showed slow release of
TMZ. FTIR and NMR confirmed complexation. DSC and XRD
studies showed masking of TMZ crystallinity after loading into
prepared NSs. Activity of developed NS was assessed on U-373
glioma cell line by performing SRB assay, cell viability studies per-
formed demonstrated cytotoxicity activity similar to that of pure
drug, and morphological investigations of the cells revealed degen-
eration/distortion of the cell structure [33].
Curcumin is a polyphenolic compound, naturally occurring in
turmeric spice, having potential application in tumor treatment but
has limited therapeutic utility because of its poor aqueous solubil-
ity at acidic and neutral pH, while it is soluble in alkali media [34].
Cyclodextrin-based nanosponges (NS) were obtained with
dimethyl carbonate as a cross-linker. The complex of curcumin
with NS (CrNS) was able to increase the therapeutic utility and
improve the solubility of curcumin by encapsulating it within the
internal cavity characterized for FTIR, XRD, and DSC studies.
The particle size of CrNS was found to be 487.3 nm with mini-
mum polydispersity index of 0.476 and zeta potential of −27 mV
indicated formation of a stable colloidal nanosuspension. CrNS has
shown more solubilization efficiency (20.89 μg/mL) in compari-
son with plain curcumin (0.4 μg/mL) and β-CD NS (5.88 μg/
mL). In vitro drug release of curcumin was controlled over a pro-
longed period of time; hemolysis study showed that the complex
was non-hemolytic. CrNS sample showed only a slight reduction
in cytotoxicity against MCF-7 cells indicating no change in molec-
ular structure of curcumin in CrNS formulation [34].
Sorafenib (SFN), class II drug, is an oral multikinase inhibitor
approved for the treatment of patients with advanced renal cell
carcinoma or unresectable hepatocellular carcinoma. SFN displays
antitumor activity across a variety of tumor types, including renal,
hepatocellular, breast, thyroid, and colorectal carcinomas. SFN is
water soluble at a rate of 9.86 ng/mL in a neutral pH environment
and its oral bioavailability is low (about 8.5%) due to its low aque-
ous solubility [35].
Inclusion complexes of β-CyD polymer and oligomer with
SFN have been studied as drug carriers. Water-soluble polymer
pCyD (92 kDa, 70% of CyDs, 57 CyD units) was purchased from
CyClolab and CyD oligomer (oCyD, 12 kDa, 65% of CyD, 7CyD
units) was synthesized by cross-linking with epichlorohydrin (EPI).

In vivo tissue toxicity of SFN and its soluble complexes indicated
that CyD polymer/SFN complexes have lower in vivo toxicity than
SFN alone. In particular, oCyD/SFN showed the lowest toxicity
in the lung, where SFN alone showed a strong toxicity under
experimental conditions. Toxicological data was also confirmed by
a lower general toxicity of oCyD/SFN in comparison to SFN or
pCyD/SFN. Moreover, both the polymer/SFN complexes showed
almost no liver toxicity as compared to SFN. Overall results sug-
gest that CyD polymers could provide a new formulation strategy
for the delivery of SFN in order to increase its bioavailability and
reduce its systemic toxicity [36].
ICOS is a T-cell co-stimulatory molecule involved in T-cell
function. It binds B7h expressed by several cell types including
tumor cells. ICOS interaction may modulate the spread of cancer
metastases, suggesting the novel use of ICOS-Fc as an immuno-
modulatory drug. In melanoma, treatment with anti-CTLA-4 Ab
induces an increase of ICOS+ effector T cells, indicating that the
ICOS/B7h pathway is required for antitumor responses. However,
in vivo administration of free ICOS-Fc solution induces only anti-
metastasis effects, without inhibiting the local growth of B16 cells
injected subcutaneously in mice [37].
ICOS-Fc was incorporated in β-cyclodextrin nanosponges
(NS), with the aim of increasing its delivery to the tumor, enabling
an anticancer effect. In vivo experiments were performed using the
B16 melanoma model of transplantable tumors. C57BL/6 mice
were injected subcutaneously with 105 B16-F10 cells and treated
with an i.v. injection of either the mouse ICOS-Fc, ICOS-Fc
loaded in NS, or empty NS as control. ICOS-Fc was loaded into
the NS with a good encapsulation efficiency. A slow and prolonged
in vitro release kinetics of ICOS-Fc from the nanoformulation was
observed. The loaded nanoformulation was stable for more than
6 months, protecting ICOS-Fc from degradation. In vivo experi-
ments showed that i.v. injection of ICOS-Fc loaded in NS remark-
ably inhibited either the metastasis formation or the growth of
established subcutaneous B16 tumors. Interestingly, the delivery
of ICOS-Fc with NS is crucial for its therapeutic effectiveness. This
result showed that the combination of ICOS with a nanocarrier
can enhance its antitumor response [38].
New generations of stimuli-responsive materials were pro-
posed as novel drug delivery systems for the time-controlled release
of drugs targeted to specific cells or organs by changing their con-
formation or chemical structure under an external stimulus, for
example, light, temperature [39] or pH [40], or biochemical stim-
uli, such as the presence of cellular antioxidant glutathione (GSH).
Chemoresistant tumor cells are reported to possess high levels of
GSH; its concentration inside cells (2–20 mm) is about 1000 times
higher than in plasma (2–20 mm) [41].
Trotta et al. reported a one-step synthesis of a new class of

GSH-responsive nanosponges (GSH-NSs) that are able to host
and to release anticancer drugs in the presence of GSH at concen-
trations similar to those found in chemoresistant cancer cells. The
synthesis was achieved in high yield by reacting commercially avail-
able and inexpensive 2-hydroxyethyl disulfide in the presence of
β-cyclodextrin and a suitable amount of the cross-linking agent
pyromellitic dianhydrides. The reaction was fast and complete in
few minutes at room temperature. GSH-NSs with different disul-
fide bridge content were easily obtained by varying the amount of
2-hydroxyethyl disulfide in the reaction mixture. Simple Soxhlet
extraction with acetone for few hours gave clean GSH-NSs [42].
Doxorubicin was chosen as a drug model to test the loading
capacities of GSH-NS and the responsiveness to GSH at intracel-
lular concentrations, particularly as unmodified β-cyclodextrin
does not form inclusion complexes with doxorubicin. GSH-NS
(B) was selected and underwent further formulation studies. The
doxorubicin interaction with GSH-NSs was confirmed by DSC;
absence of the drug melting peak indicates that it is molecularly
dispersed within the matrix, consequently not able to crystallize.
GSH-NSs retain some of the relevant properties of unmodified
nanosponges, such as ease of internalization in cells (Fig. 1). In
vitro studies of the release of doxorubicin from GSH-NS were per-
formed in the absence and in the presence of GSH at three differ-
ent concentrations (10, 25, 50 mM). Results indicated that
doxorubicin release was enhanced in the presence of GSH, which
induced the rupture and reduction of the disulfide bridges to thiol
groups. Indeed, the in vitro release kinetics were markedly influ-
enced by the disulfide group content in the nanosponge. The bio-
logical effect of GSH-NS was evaluated on three cell lines HCT-15,
HepG-2, and A2780, having the GSH concentration in the order
HepG-2 > HCT15 > A2780. Results suggest that GSH-NSs not
only are internalized faster than doxorubicin alone, but seem to
protect the drug from inactivation or extrusion, thus rendering the
low drug doses more effective for a longer time. Furthermore,
doxorubicin-loaded GSH-NSs displayed more apparent effects
than the free drug in cell lines having increased concentrations of
GSH [42].
Doxorubicin-loaded (Dox-GSH-NS) high effectiveness with
respect to Dox has been confirmed by in vivo experiments, per-
formed in mice transplanted with DU145 cells. Not only were they
more effective than Dox in reducing tumor growth, in terms of
tumor weight, but also Dox-GSH-NS highly reduced the number
of proliferating Ki67-positive cells, which were about 9% of total
cells in Dox-GSH-NS-treated mice, whereas this percentage
increased to 22% in Dox-treated mice and 46% in control mice.
The biodistribution of Dox-GSH-NS demonstrated that heart
accumulation of Dox-GSH-NS was significantly lower than that
Fig. 1 Internalization of coumarin 6-loaded GSH-NSs by HCT-15 colon cancer cells. The image was taken
15 min after the addition on a Zeiss LS510 confocal laser microscope (488 nm excitation laser band, 505–
530 nm band-pass emission filters) [1]
observed in Dox-treated animals, and the experiments performed

on rat cardiomyocytes, using same doses, showed that the toxicity
of these two formulations was similar. All these characteristics sug-
gest that GSH-NSs can be a suitable drug delivery carrier for future
applications in cancer therapy [43].
Natural compounds with therapeutic activity are exploited for
cancer treatment as they are biocompatible and have well-
characterized functions. Various plant-derived bioactive com-
pounds have been shown to inhibit cancer cell growth and
survival.
Strigolactones (SLs) are carotenoid-derived plant hormones
that are synthesized by plant roots and released into the rhizo-
sphere. SLs have a four-ring structure consisting of a tricyclic lac-
tone (ABC rings) linked to a methyl butenolide (D ring) through
an enol ether bridge, and they are indispensable for the establish-
ment of arbuscular mycorrhizae. Two SL analogs, namely MEB55
and ST362, could induce G2/M cell cycle arrest and apoptosis in
a variety of human cancer cell lines in vitro and inhibited growth of
breast cancer stem cell-enriched mammospheres and human breast
cancer xenograft tumors in vivo; however these compounds have
low aqueous solubility and stability at physiological pH [44].
GSH/pH-NS previously designed to deliver anticancer drugs
to cells with high GSH levels could enable the targeted delivery
and controlled release of SL analogs (MEB55 and ST362) in pros-
tate cancer cells, thereby enhancing the therapeutic efficacy. The
MEB55-loaded GSH/pH-NS demonstrated greater inhibition of

DU145 cell (high GSH content) viability in MTT assays compared
to free MEB55. In contrast, they had a lower effect on the viability
of PC-3 cells (low GSH content). LDH assays revealed that treat-
ment with the GSH/pH-NS resulted in cell death, which was
dependent on the intracellular GSH content. Treatment with free
SLs also induced cell death in PC-3 cells, which was likely related
to oxidative stress. Limited LDH release was observed in the other
cell types 24 h after treatment. Annexin-V staining indicated that
the DU145 cells underwent apoptotic cell death, and colony for-
mation assays confirmed the dependence of the cellular responses
on the intracellular GSH content. The incorporation of two SLs
into GSH/pH-NS enhanced their cytotoxic effects on prostate
cancer cells indicating that GSH/pH-NS are dual-stimuli-
responsive nanocarriers that may favor the selectively controlled
release of SLs in target cancer cells [45].
Ailanthone is a natural active compound isolated from the
Ailanthus altissima tree, which has been shown to possess an in
vitro growth inhibitory effect against several cancer cell lines.
Advanced bladder cancer is a common disease characterized by a
frequent onset of resistance to cisplatin-based therapy. It has been
demonstrated on bladder cancer cells, and the cisplatin resistance
(CDDP) is accompanied by an increase in Nrf2 protein expression
which contributes to conferring CDDP resistance. Indeed, Nrf2
overexpression is associated with clinically relevant CDDP resis-
tance in bladder cancer patients [46]. Increasing evidence has
demonstrated the involvement of YAP, a key component of the
Hippo tumor-suppressor pathway, in chemoresistance of several
types of cancers. Ailanthone is able to inhibit proliferation in resis-
tant cells. Incorporating it in similar nanocarriers can improve the
anticancer therapeutic activity [47].
Erlotinib belongs to BCS II class drugs approved initially for
non-small cell lung cancer, and is an epidermal growth factor
receptor (EGFR) tyrosine kinase inhibitor. Its potential in the
treatment of various other cancers such as breast cancer, locally
advanced or metastatic non-small cell lung cancer, ovarian cancer,
glioma, head and neck cancer, and colorectal cancer has also been
demonstrated. Systemic and uncontrolled administration of erlo-
tinib hydrochloride (ERL) is associated with severe toxicity [48].
One study utilized previously synthesized and characterized
NS based on the reaction between β-cyclodextrin monomer and
CDI cross-linker. The solubility of ERL was optimum in 1:4 w/w
proportions of drug and NS; solubility of all complexes ERL-NS
was significantly higher (p < 0.01) than pure ERL (3.16 ± 0.73 μg/
mL). ERL-NS was found to be more cytotoxic against both MIA
PaCa-2 and PANC-1 cells compared to free ERL. When incubated
with MIA PaCa-2 and PANC-1 pancreatic cells, IC50 values of
ERL-NS in each time frame (24 h, 48 h, and 72 h) were significantly
higher than free ERL, further validated by the apoptosis assay. The
relative bioavailability of the complex was approximately 200% in
comparison to pure ERL, additionally confirmed by the enhanced
bioavailability of ERL-NS, in agreement of in vitro dissolution
studies. Therefore inclusion complex of nanosponge with ERL is a
successful approach to increase its solubility and bioavailability
resulting in a reduction in dose and dose-related side effects [49].
Another study exploited the novel stimuli-responsive nano-
sponges first introduced by Trotta et al. [42] for the targeted deliv-
ery and extended release of ERL in lung cancer (Fig. 2). ERL-loaded
nanosponges (ERL-NS) exhibited in vitro-extended drug release,
directly proportional to the external GSH concentration
(76.89 ± 0.1% release at 168 h), thus demonstrating tumor target-
ing. Additionally, they showed enhanced in vitro cytotoxicity and
97.5% inhibition in tumor growth on administering ERL-NS when
compared to plain ERL (48% inhibition). Cell cytotoxicity study
was evaluated on human lung cancer A549 cell lines. Biodistribution
and in vivo tumor growth inhibition studies revealed drug release
to the cancerous cell, thus preventing unnecessary drug exposure
[50].
2.2 Cyclodextrin Acetyl salicylic acid (ASA) is a nonsteroidal anti-inflammatory and

Nanosponges antipyretic drug which acts by inhibition of prostaglandin synthesis
for Anti- and is used to relieve the pain. ASA is rapidly hydrolyzed in plasma
inflammatory Drugs to salicylic acid with a half-life of 15–20 min and has a direct irri-
tant effect on gastric mucosa due to inhibition of prostaglandins
causing ulceration, epigastric distress, and hemorrhage. Controlled-
release formulation of ASA would reduce the frequency of admin-
istration and the undesired side effects and improve the patient
compliance [51].
β-Cyclodextrin cross-linked with pyromellitic dianhydride NS
was able to complex efficiently with ASA and to release it slowly in
physiological media. These formulations possess nanosize and
spherical shape and display significantly better encapsulation and
stability. The encapsulation efficiency of complex between β-CD
and ASA might depend on different ways of inclusion complex
formation:
–– ASA could be hosted inside the CD cavity from the wider side
of the cone-shaped β-CD molecule with the lipophilic benzene
ring going in first and the acetyl group partly standing out of
the cavity.
–– ASA could go into CD cavity from wider side with the acetyl
group going in first and benzene ring partly standing out.
–– ASA could go into CD cavity from the narrow side with ben-
zene ring going in first and acetyl group partly standing out.
o o
βCD + HO SS OH + o o
o o
2-Hydroxy ethyl
β-Cyclodextrin Pyromellitic dianhydride
disulphide
RT,
TEA
o
o OH
o
HO S
o S
o o o
βCD
OH
o
o
o o OH
o
o
o o
o OH
GHS-NS
o o
HO
o
Erlotinib GHS-NS
Tumor plasma membrane Tumor
Lungs
NS reached at
Cyctoplasm
tumor site
Nucleus
Drug release
Inhibits DNA synthesis

and cell profileration
Fig. 2 Schematic diagram of ETB-GHS-NS synthesis, release, and its activity on tumor cells [2]
–– ASA could go into the CD cavity from narrow side with the
acetyl group going in first and benzene ring partly standing
out.
Either way it is worth noting that the encapsulation efficiency
between ASA and β-CD formulation was more in precipitation
method compared to ground mixture [52].
Ibuprofen (IBU) is a nonsteroidal anti-inflammatory drug
(NSAID) widely available over the counter (OTC) used to relieve
acute pain resulting from various causes, including headache.
Several well-designed clinical studies have demonstrated the effi-
cacy of OTC IBU in the treatment of tension-type headache, den-
tal pain, sore throat, and postpartum episiotomy pain. Ibuprofen
sodium fast-acting salt formulation dissolves more rapidly than
conventional ibuprofen [53].
Mele et al. published a series of studies focused on the diffu-
sion of solutes, including ibuprofen, through CD polymer hydro-
gels [54–56].
β-Cyclodextrin nanosponges (CDNS) obtained by cross-
linking with ethylenediaminetetraacetic acid dianhydride (EDTA)
at two different molar ratios CDNS/EDTA 1:4 and 1:8 (Fig. 3)
were investigated. The solid-state products were prepared via
freeze-drying of the hydrogels obtained by swelling the nano-
sponge with aqueous solutions of ibuprofen sodium salt (IP).
The entrapment of IP was achieved by swelling the two poly-
mers with a 0.27 M solution of IP in D2O (deuterium oxide), lead-
ing to colorless, homogeneous hydrogels loaded with IP. The
molecular environment and the transport properties of IP in the
hydrogels were studied by high-resolution magic angle spinning
spectroscopy (HRMAS NMR). The diffusion properties of IP in
CDNS can be modulated by suitable polymer synthesis; this find-
ing opens the possibility to design drug delivery systems with pre-
dictable and desired drug release properties [55]. Further
investigations on the structural changes of the host CDNS material
as well as the drug chemical and structural modifications in the
polymer network in the solid state were done relying on two differ-
ent methodological approaches: solid-state NMR spectra sup-
ported by powder X-ray diffraction (PXRD) data [56].
Flurbiprofen is a weakly acidic nonsteroidal anti-inflammatory
drug showing local gastrointestinal side effects, which are attrib-
uted partly to the insoluble drug particle adhesion to the gastric
mucosa, leading to high local concentrations.
β-CD carbonate nanosponges have been studied as drug deliv-
ery systems for flurbiprofen. As a result of the flurbiprofen com-
plexation in the NS, the aqueous solubility of the drug was
particularly increased, up to 15 wt%. The strong interaction
between the NS and flurbiprofen was also confirmed by a slow-
Fig. 3 Effect of the increasing amount of cross-linker with respect to CD (expressed here as mol of cross-linker
per mol of CD) on CDNS structure. The cross-linking degree increased up to 1:6; then further excess of EDTA
causes branching of CD units rather than further cross-linking [3]
release kinetics. Indeed, less than 10% of the loaded amount of

drug was released during the first 2 h [2].
Meloxicam is a nonsteroidal anti-inflammatory drug that acts
by inhibiting the synthesis of prostaglandins and has a limited
aqueous solubility of 9.4 μg/mL useful for therapeutic i ntervention
in the management of neurogenic and inflammatory pain [57].
Inclusion complexes of meloxicam with β-cyclodextrin and
β-cyclodextrin-based pyromellitic nanosponges were prepared to
enhance the solubility and stability of meloxicam and prolong its
release using different methods:
–– Physical mixing (P): β-Cyclodextrin and meloxicam complexes
were prepared at a molar ratio of 1:1 via trituration at ambient
temperature for 30 min.
–– Kneading (K): β-Cyclodextrin and meloxicam were triturated
in an equimolar ratio (1:1) in the presence of PEG 400 con-
tinually kneaded for 30 min and then dried at room
temperature.
–– Sonication (N): Meloxicam was added to a dispersion of nano-
sponges in DCM at a ratio of 1:1 (w/w) and then sonicated
for 24 h at room temperature.
The encapsulation efficiencies of the methods were found to
follow the order: P < K < N. The effective encapsulation of meloxi-
cam in β-CD and in the β-CD NSs might have been due to the van
der Waals force of attraction between the aromatic ring protons of
the drug and the inner protons of β-CD [58].
2.3 Cyclodextrin Viral diseases affect billions of people each year worldwide causing
Nanosponges millions of deaths. Despite the accumulation of a large body of
for Antiviral Drugs knowledge about the replicative and pathogenetic mechanisms of
many human viral pathogens, approved antiviral drugs are available
only for a restricted array of viruses. A number of nanocarriers,

either inorganic or organic, have been developed to improve the
delivery and therapeutic efficacy of antiviral medicines and
cyclodextrin- based polymers represent a promising strategy to
improve antiviral treatments [59].
Acyclovir (ACV) was the first antiviral to be licensed for the
treatment of HSV infections, and it is the drug of choice for the
treatment of epidermal, ocular, or systemic herpetic infections.
ACV is slightly soluble in water and has a short plasma half-life, its
absorption from gastrointestinal tract is slow and incomplete, and
oral bioavailability reaches to a max of 30%. As a consequence,
higher doses are prescribed, resulting in systemic toxicity and
adverse reactions. ACV bioavailability is low and highly variable,
associated with low retention at the vaginal mucosa and poor
patient compliance, and requires frequent administrations [60].
Novel polymeric nanoparticles based on a β-cyclodextrin-
poly(4-acryloylmorpholine) mono-conjugate (β-CD-PACM), a
tadpole-shaped polymer in which the β-CD ring is the hydrophilic
head and the PACM chain, the amphiphilic tail, were prepared by
the solvent injection technique. The antiviral activity of acyclovir
loaded into β-CD-PACM nanoparticles against two clinical isolates
of HSV-1 was evaluated and found to be remarkably superior com-
pared with that of both the free drug and a soluble β-CD-PACM
complex. It was also found that the nanoparticles are internalized
in cells and are located in the perinuclear compartment [61].
In an alternative study, new Carb-NS were purposely prepared
as the carrier for acyclovir. Carb-NS were obtained by reacting suc-
cinic anhydride on preformed NS in DMSO at 90 °C for 3 h. The
solid NS was recovered by filtration and washed with a large
amount of water. Fluorescent Carb-NS were also synthesized for
cellular trafficking studies (Fig. 4). For this purpose, preformed NS
were added to a fluorescein isothiocyanate solution in DMSO and
incubated at 90 °C for 3 h. The filtered, washed, and dried product
was reacted with the succinic anhydride to obtain fluorescent
Carb-NS [62]. The drug loading percentage for Carb-NS was 69%
(w/w), ascribed to the presence of the acid groups in the Carb-NS
structure acting as further sites for acyclovir electrostatic interac-
tions besides the cyclodextrin cavities. The antiviral activity was
performed using monolayers of Vero cells infected with a clinical
isolate of HSV-1. The dose–response curve obtained demonstrates
that the antiviral potency of the acyclovir-loaded Carb-NS was
higher than that of free acyclovir. The percent enhancement of cel-
lular uptake of acyclovir formulations in Vero cells compared to
that of the plain drug was approximately more than 200% for
Carb-NS. Carb-NS showed enhanced drug loading and more pro-
longed release kinetics in comparison with traditional NS [61].
Considering the biocompatibility of nanosponges, the develop-
Fig. 4 Cell uptake of fluorescent Carb-NS. Vero cells were incubated with the formulation for the times indicated
and then analyzed by confocal laser scanning microscopy without fixation. The upper panels show the fluores-
cence images while the lower panels show fluorescence images merged with phase-contrast images [4]
ment of acyclovir-loaded NS formulations holds great potential for

their use in various administration routes [62].
Rilpivirine (RPV) is a BCS class II drug used for the treatment
of HIV infection. The drug has low aqueous solubility (0.0166 mg/
mL) and dissolution rate leading to low bioavailability (32%).
Binary and ternary complexes of RPV with β-CD, HP-β-CD,
β-CD nanosponges, and tocopherol polyethylene glycol succinate
were prepared, characterized, and compared by phase solubility,
saturation solubility in different media, in vitro dissolution, and in
vivo pharmacokinetic studies. Solubility studies indicated that both
binary and tertiary complexes were stable compared to RPV, and
the relative bioavailability of RPV was enhanced nearly two- to
threefold for ternary complexes. The increase in solubility and dis-
solution rate can explain the increased bioavailability for binary
and ternary complex nanosponges [63].
Efavirenz (EFA), a BCS class II drug, is a non-nucleoside
reverse transcriptase inhibitor which is chronically prescribed for
HIV patients. Carbonate β-CD NS was used to enhance the low
solubility and dissolution of EFA. Binary and ternary complexes

were prepared with EFA, NS, and PVP K30 for comparative rea-
sons. Results obtained confirmed inclusion complexation and the
stability constant for ternary complex (1997 lit/mole) indicates
stable complex formation. Saturation solubility was 17-fold higher
with ternary complex in distilled water and about 4-fold in
simulated gastric fluid. In vitro dissolution was improved threefold
with ternary complex. Ternary nanosponge complexes were found
to have twofold increase in oral bioavailability of efavirenz as com-
pared to plain drug [64].
Imiquimod (IMQ) is an antiproliferative medication, which
enhances both the innate and cellular immune responses by pro-
ducing proinflammatory cytokines. IMQ can induce a cell-
mediated immune response and increase collagen breakdown. In
addition, IMQ can modify the expression of genes associated with
apoptosis and is clinically used for the treatment of neoplastic skin
diseases. IMQ is one of the emerging options for HS (hypertrophic
scar) prevention and therapy. β-CD nanosponges cross-linked with
pyromellitic dianhydrides (IMQ-CDNS) loaded with IMQ were
prepared and their antiproliferative action on HS fibroblasts was
evaluated. CDNS are soft and swellable and form stable nanohy-
drogel structure on contact with water, making them very suitable
for topical applications. The percentage of water uptake increases
with the pH of the external medium and reaches the best swelling
properties at pH values over 6 (mimics the pH of the skin in HS)
due to the presence of dissociated carboxylic groups of the pyro-
mellitic acid network [65].
CDNS can incorporate and store the drug to a good extent.
IMQ loaded in CDNS can be released with prolonged- and
controlled-release kinetics. Loaded nanosponge IMQ–NS could
act as a drug reservoir dispersed in the hydrogel, able to slowly
deliver IMQ through the skin, favoring dermal accumulation. This
behavior could allow for a reduction in the number of applications,
enhancing patient compliance. Moreover, a decrease in the irritat-
ing effect on skin might be obtained. These results might pave the
way to develop an innovative topical nanomedicine formulation for
the prevention and treatment of hypertrophic scars (Fig. 5) [66].
2.4 Cyclodextrin Many diseases and infections can be transmitted from one indi-
Nanosponges vidual to another during sexual contact (sexually transmitted dis-
for Antifungal Drugs eases, STDs). These diseases are caused by a myriad of pathogens
such as bacteria, viruses, parasites, and fungi. In immunocompro-
mised patients, they can cause mucosal, skin, or systemic infections
which are conventionally treated with antifungal agents such as
topically or orally administered azole drugs. However, prolonged
usage of such antifungal agents, such as fluconazole, can eventually
lead to reduced susceptibility and clinical response [67]. Econazole
nitrate (EN) is a commercially available imidazole antifungal used
Control
0h 24h 48h
IMQ (5 μg/ml)
NS
IMQ-NS (5 μg/ml)
IMQ-NS (25 μg/ml)
Fig. 5 Inhibitory effect of IMQ–NS in a scratch-wound assay. The figure shows micrographs of the extent of
closure obtained under control conditions compared to those with free IMQ (5 μg/mL), NS, or IMQ–NS (5 and
25 μg/mL) after 24- and 48-h treatment. The fibroblasts were synchronized for 2 h, wounded, and treated as
indicated, and phase-contrast microscopy pictures were taken of the wounded area (scale bar 100 μm) [5]
topically to relieve the symptoms of superficial candidiasis, derma-

tophytosis, pityriasis versicolor, and skin infections. Polymeric
nanosponges were an alternative carrier for EN that improved
retention onto the skin, through topical hydrogel formulation
[68]. Another example is nelfinavir mesylate (NFV), a BCS class II
protease inhibitor with low bioavailability, used to treat human
immunodeficiency virus (HIV) infections. CDNS showed the abil-
ity to enhance the solubility of NFV [69].
Itraconazole is a broad-spectrum triazole antifungal agent. It is
a BCS class II drug that has a limited dissolution rate and poor oral
bioavailability. Hydrophilic polymers have been used in the past for

enhancing the complexation efficiency of β-CD. With a similar
rationale copolyvidonum was used to enhance the complexation
efficiency of a β-CD nanosponge. Three formulations were pre-
pared by solid dispersion technique to incorporate drug in the
nanosponge: two binary systems of drug-nanosponge and
drug-
copolyvidonum and one ternary system of drug–nano-
sponge–copolyvidonum. The solubility was enhanced over 55-fold
in the case of ternary complex and over 27-fold in the case of
binary complex drug-nanosponge. PXRD studies showed maxi-
mum amorphousness of the drug in the case of ternary complex.
This can explain the enhanced dissolution and solubility of the itra-
conazole dispersed in the ternary complex. Thus, the bioavailabil-
ity of itraconazole can be expected to be more compared to plain
drug [70].
Clotrimazole (CTZ) is a BCS class II drug having a limited
therapeutic potential because of its poor aqueous solubility and
relatively short half-life. Hydroxypropyl β-cyclodextrin (HP-β-CD)
nanosponges were prepared using dimethyl carbonate as a cross-
linker and suitably gelled. They have been used to enhance the
solubility and efficacy of CTZ by forming a complex with NSs.
Nine formulations were prepared based on a 32 full factorial design
using different Pluronic F-127: Pluronic F-68 ratios, characterized
and assessed for in vitro release, in vitro bioadhesion, in vivo anti-
fungal activity, and in vivo irritation using female Wistar albino
rats. The optimized CTZ-NS in situ gel (F-10) demonstrated pro-
longed drug release up to 15 h (considerably longer than that of
the conventional in situ gel). The CTZ-NS gel was nonirritant in
vivo and showed higher in vivo antifungal activity and in vitro bio-
adhesion than did the conventional in situ gel. These results signi-
fied the promising applicability of the formulated CTZ-NS gel as a
novel delivery system for the local treatment of vaginal candidiasis
and other similar infections [71].
2.5 Cyclodextrin Quercetin is a flavonoid widely distributed in vegetables and fruits

Nanosponges and exhibits strong antioxidant activity, but the poor solubility and
for Antioxidants stability of quercetin limit its function and application. It has been
shown to have a variety of biological activities and pharmacological
actions, such as anti-oxidation, anti-inflammation, anticancer, anti-
platelet aggregation, antianemic action, and antianaphylaxis effects.
However, pharmaceutically, quercetin is a challenging molecule to
be delivered due to its poor solubility 7.7 μg/mL in water, <2%
bioavailability in humans, low hydrophilicity with logP value of
1.81, gastrointestinal instability, extensive first-pass metabolism,
and minimal absorption in gastrointestinal track [72]. An improved
oral formulation of quercetin is required for better bioavailability
and higher efficacy of quercetin. Cyclodextrin-based nanosponges
were prepared using diphenyl carbonate for the cross-linking.
Inclusion complexes with quercetin were obtained by freeze-

drying method. Results indicated that the NS degree of cross-
linking has a significant influence on the dissolution of the
encapsulated quercetin. The NSs with lower degree of cross-linking
released 91% of the drug within 45 min, whereas the NSs with
higher degree of cross-linking exhibited sustained-release kinetics
up to 24 h. Nanosponges showed higher dissolution in vitro than
commercial quercetin alone, which could translate into an increased
bioavailability in vivo. This would lead to considerable dose reduc-
tion for patients [73].
Gamma-oryzanol (GO) is a natural antioxidant extracted from
rice bran oil, usually employed to stabilize food and pharmaceuti-
cal raw materials. Its usefulness, however, is limited by its fast deg-
radation. One proposed approach to increase the stability and
effectiveness of antioxidants is based on the inclusion in supramo-
lecular structures. β-CD nanosponges have been chosen for their
ability to encapsulate a great variety of substances, to decrease their
side effects and to protect them from degradation. The inclusion
of GO in complex was prepared in 1:1 w/w ratio and characterized
by membrane diffusion runs. The photodegradation of encapsu-
lated GO upon UVA and UVB irradiation was found to be slowed
down. The antioxidant effectiveness of the inclusion complex,
assessed in vitro on porcine ear skin, revealed accumulation of GO
when entrapped in the host structure, and presented a significant
anti-lipoperoxidative activity. The formation of NS inclusion com-
plex with GO provides an indication that it may have potential as a
carrier for topically active substances [74].
Babchi oil (BO) is an important essential oil, used in several
traditional medicines due to its antioxidative properties. Yet the
volatile nature of BO, poor stability, and solubility restrict its phar-
maceutical applications. Encapsulation of BO in β-CD nano-
sponges was also used to overcome these limitations. Spectral
analysis ascertained the formation of inclusion complexes.
Solubilization efficiency of BO was checked in distilled water and
found to be enhanced 4.95 times with optimized β-CD nano-
sponges. The encapsulation of BO resulted in an increase of solu-
bility, photostability, and safety of this oil [75].
Coriander essential oil (CEO) has shown a broad spectrum of
antimicrobial activity ranging from Gram-positive to Gram-
negative bacteria, yeasts, and molds. Recently, CEO was incorpo-
rated in NSs prepared from α-CD and a mixture of HP-β-C and
β-CD. These CD-NSs displayed the ability to provide a controlled
oil release while inhibiting bacterial growth, thus representing a
potential strategy to overcome the poor efficacy of current antimi-
crobial food packaging [76].
2.6 Cyclodextrin
Nanosponges Rutin, phloridzin, and chlorogenic acid are some of the most
for Polyphenols important characteristic polyphenols found in apples and their by-
products (cider, apple juice, apple pomace, etc.). Despite their

antioxidant power, their low stability under light or heating condi-
tions restricts the use of this kind of molecules as nutraceuticals.
Encapsulation of polyphenols might be a solution to deal with this
issue. β-CD nanosponges, prepared using hexamethylene diisocya-
nate (HMDI-NS) and 1,1′-carbonyldiimidazole (CDI-NS) as
cross-linkers and synthesized with different molar ratios
(β-CD:cross-linker), were used for the encapsulation of rutin,
phloridzin, and chlorogenic acid. Physical characterizations of the
solid products obtained confirmed that they were not physical mix-
tures, but inclusion complexes. The highest encapsulation effi-
ciency for rutin (83.7%) was obtained by using CDI-NS(1:3).
However, HMDI-NS(1:8) showed the best results for the smaller
molecules as phloridzin and chlorogenic acid (87.2% and 77.5%,
respectively). In vitro dissolution studies of encapsulated polyphe-
nols showed that rutin and phloridzin were better dissolved in
ethanol, while chlorogenic acid was better dissolved in water. Thus
polyphenols from apple and its by-products could be encapsulated
to enhance their bioavailability [77].
Resveratrol is a polyphenolic phytoalexin that is found, free
and conjugated, in high concentrations in grape juice, peanuts,
mulberries, and other plant extracts. Resveratrol has been used for
decades in medicine for the treatment of many diseases like inflam-
mation, cardiovascular diseases, skin infections, chemopreventive
activity, gonorrhea, hyperlipidemia, and fever [78]. Hyper-cross-
linked NSs of β-CD and CDI (at varying molar ratios, i.e., 1:2, 1:4,
1:8) have been used to increase the solubility, stability, and perme-
ation of resveratrol. In vitro release and stability of NS-resveratrol
complex were increased. Cytotoxicity studies on HCPC-I cell
showed that resveratrol formulations were more cytotoxic than
plain resveratrol. Permeation studies showed good permeation of
the complex in pigskin. Accumulation studies in rabbit mucosa
showed better accumulation of complex than plain resveratrol sig-
nifying that resveratrol NS formulation can be used for buccal
delivery and topical application [79].
2.7 Cyclodextrin Norfloxacin (NFX) is a fluoroquinolone antibiotic that has emerged

Nanosponges as one of the preferred agents in the treatment of urinary tract
for Antibacterial Drug infections that belongs to biopharmaceutical classification system
BCS class IV drugs [80]. NFX is captured by uptake transporters
but it inhibits them instead of being carried through the entero-
cyte. NFX is consumed by the uptake transporters, thus reducing
the drug content able to permeate in the gut lumen; thus oral
bioavailability is a crucial factor for NFX. In this context, nano-
sponges were designed to overcome this biopharmaceutical prob-
lem. β-CD was used as building block and diphenyl carbonate as
cross-linker agent; the molar proportion CD:cross-linker 1:2 was
chosen due to its higher encapsulation efficiency (80%). NFX-
loaded NS exhibited higher passage of NFX in comparison to drug

alone by using chamber method. The NS formulation also revealed
a mucoadhesive property improving the antibiotic activity in an in
vivo sepsis model [81].
2.8 Cyclodextrin DEET (N,N-diethyl-m-toluamide) currently is one of the most

Nanosponges effective insect repellents. It is available worldwide today in a vari-
for Anti-Zika ety of formulations including aerosols, creams, lotions, sprays, gels,
sticks, and wipes at concentrations ranging from 5% to 100% [82].
DEET shows repellence against mosquitoes, bugs, and ticks [82].
Zika is a mosquito-borne virus and to date no commercial vaccine
is available; hence, preventive measures, such as spurting insect
repellents frequently, should be taken to avoid the virus infection.
Encapsulation of DEET prolongs its persistence and decreases its
undesirable absorption by skin, as DEET may induce irritation
when applied directly on skin. Having a rigid and compact struc-
ture, deriving from the low molecular weight of the cross-linking
bridges, CDI-NSs were selected as encapsulating agents to limit
the diffusion and loss of entrapped DEET molecules. Polyester
fabrics were then functionalized by dispersion of DEET-loaded NS
particles among the textile fibers. CDI-NSs preloaded with DEET
(5:1 molar ratio of β-CD:DEET) showed a considerable amount of
DEET on the fabric (20%) due to strong interactions between
DEET and NSs [83].
2.9 Molecularly Molecularly imprinted polymers (MIPs) are three-dimensional

Imprinted Cyclodextrin nanostructures synthesized in the presence of a template molecule,
Nanosponges which is subsequently removed by thorough washing. Therefore,
for Parkinson’s MIPs contain highly selective sites able to recognize and adsorb
Disease the specific template molecule that was used for the imprinting.
MIPs are used over a wide range of applications, such as separa-
tion, purification, chemical sensors, and catalysis. MIP-based sen-
sors are less expensive and more resistant to chemicals, elevated
temperatures, and pressure than biological systems.
Recently, biomimetic systems based on CD MIPs for the eval-
uation of blood glucose levels have been developed [84]. The pres-
ence of CDs within an imprinted polymer network can improve
the performance of the MIP due to their peculiar structure and the
ability to form inclusion complexes.
Molecularly imprinted NSs, prepared using a drug as a tem-
plate agent, can be employed as innovative drug delivery systems
to achieve prolonged- and sustained-release kinetics, thanks to the
optimal interactions between the drug molecules and the sur-
rounding structure. In this case, the template molecule is not
removed after the synthesis reaction, but it is kept inside until the
final application. An example of molecularly imprinted NSs for the
delivery of L-DOPA has been recently described by Trotta et al.
L-DOPA [(S)-2-amino-3-(3,4-dihydroxyphenyl) propanoic

acid] is the gold standard for the treatment of Parkinson’s disease
(PD), which afflicts approximately 0.3% of the entire population,
causing symptoms such as tremors, rigidity, bradykinesia, and aki-
nesia. L-DOPA acts as a prodrug that is absorbed in the small
intestine and carried to the brain by the bloodstream. Since the
plasmatic concentration of L-DOPA tends to fluctuate over time,
drug delivery systems capable of releasing L-DOPA with stable and
prolonged kinetics are necessary to prevent on-off effects and thus
to improve the patients’ quality of life. L-DOPA-imprinted poly-
carbonate β-CD-based NSs were obtained by cross-linking β-CD
with CDI in the presence of highly reactive and unstable L-DOPA
molecules [85]. The stereochemistry of the β-CD/L-DOPA com-
plex was analyzed by the ROESY experiment (Fig. 6). The recorded
data are consistent with the inclusion of the aromatic moiety of
L-DOPA in the lipophilic cavity of the β-CD; hence the protective
action of β-CD on L-DOPA during the synthesis reactions through
the formation of inclusion complexes can be hypothesized. Finally,
in vitro release experiments were performed. L-DOPA was released
in its active form by the molecularly imprinted NSs with prolonged
and controlled kinetics over the first 48 h [85].
2.10 Cyclodextrin Telmisartan (TEL) belongs to BCS class II drugs. It is an antihy-

Nanosponges pertensive medication which belongs to angiotensin II receptor
for Hypertension (AT1) antagonists. Therapy with this drug offers a good quality of
Medication life for hypertensive patients; however its dissolution rate limits its
bioavailability. The aqueous solubility of TEL is estimated to be
9.9 μg/mL at neutral pH. To enhance the solubility and solve bio-
availability problems of TEL, it was incorporated by solvent evapo-
ration method into β-CD carbonate NSs. Phase solubility studies
were carried out, and β-CD complex of TEL was compared with
plain TEL and NS complexes of TEL. It was found that the solu-
bility of TEL was increased by 8.53-fold in distilled water, 3.35-
fold in 0.1 N HCl, and 4.66-fold in phosphate buffer pH 6.8 by
incorporating NaHCO3 in drug–NS complex than TEL alone.
NaHCO3 (ternary component alkalizer) in the NS-based complex
synergistically enhanced the dissolution of TEL by modulating
microenvironmental pH and by promoting the amorphization of
the drug [86].
Nifedipine acts as an oral calcium channel-blocking agent
employed in the treatment of hypertension and angina pectoris.
Although the oral route for nifedipine is extensively accepted, it is
coupled with contraindicative manifestations such as gastrointesti-
nal (GI) disturbance, first-pass metabolism, and less oral bioavail-
ability because of its short biological half-life with significant
fluctuations in plasma concentrations and possible photodegrada-
tion [87].
HO O H 6 OH
a H 4 5 HO
H2N b O HO 1H
2 5.6
3 1
c e OH
H O 3 4
d 7 H 2
OH
2
OH dec b b
F1
b (ρρη)
b 3.0 HO
4 O
2 3.5
5.6
3 a
4.0 e a
d
c H2N
4.5 b
1 5.0 5.6 H3 c
a e
3
5.5 d
6.0 OH
c 6.5 H5 HO
e
d
7.0
(a) 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 (b)
F2 (ρρη)
Fig. 6 (a) ROESY experiment results. (b) Schematic representation of β-CD interaction with L-DOPA [6]
Cyclodextrin ground nanosponges are widely used to augment

the solubility of the lipophilic drugs; the oral solubility of nifedip-
ine was enhanced by incorporating into β-CD and diphenyl car-
bonate NS. The fabricated batch displayed enhanced oral
bioavailability (Cmax 0.02 μg/mL for test and 0.055 μg/mL for
control formulation), while the area under the moment curve
(AUMC) and AUC were 4.8 μg h2/mL and 0.056 μg h/mL,
respectively, with a mean residence time of 8.57 h. Stability studies
rendered nifedipine-laden NS stable in terms of particle size, drug
entrapment, and drug release. Hence, it can be concluded that
nifedipine-laden β-CD NS bids a potential drug delivery system for
oral delivery of the active molecule [88].
2.11 Cyclodextrin Gabapentin (GBP) is a BCS class III drug used as a primary drug
Nanosponges therapy for the treatment of partial seizures in pediatric as well as
for Anti-seizure geriatric patients. The bioavailability of GBP decreases with
increase in dose due to its absorption via saturable transport sys-
tem and also due to other substrates following the same system.
The bitter taste is another problem associated with oral adminis-
tration of this drug. A controlled-release formulation could
potentially minimize the saturation uptake transporter, in that
way that enhances absorption and avoids any potential side
effects. The dry powder suspension of GBP-loaded β-CD NSs
was used as a sustained-release carrier; desired controlled-release
profile for 12 h and insignificant drug leaching were observed in
reconstituted suspension during storage for 7 days at 45 C/75%
RH. NSs effectively masked the taste of gabapentin and released
retarding polymers as ethyl cellulose and Eudragit RS-100 pro-
vided further coating. In vivo studies showed increase in bioavail-

ability of controlled-release suspension by 24.09% as compared
to pure GBP [89].
2.12 Cyclodextrin Melatonin is a hormone found in animals, plants, and microbes. It

Nanosponges has shown a sleep quality improvement in patients suffering from
for Sleep Disorder sleep diseases, with this molecule being functionally important in
the nocturnal regulation of sleep and thermoregulation. Along
other medical and healthcare properties, melatonin is also known
to exhibit antioxidant properties against the deleterious effects of
reactive oxygen and nitrogen species [90].
A suitable system of transferring the active ingredient to the
skin should be developed in order to fulfill the bioactive function
of melatonin. A bioactive functional fabric was prepared through
cotton fiber functionalization with β-CD carbonate-NSs and then
loaded with melatonin in water/ethanol suspension. The loading
efficiency of melatonin in the nanoporous structure was estimated
in 8 wt%. The in vitro release study with the Franz cell equipment
evidenced that melatonin release rate from the functionalized fab-
ric can be described by a zero-order law. This finding might be the
starting point for the development of biofunctional fabrics storing
multiple doses of melatonin to be released over time [91].
2.13 Cyclodextrin Lansoprazole is a proton pump inhibitor. It is a lipophilic, weak

Nanosponges base with chemo- and thermosensitive nature which is rapidly
for Gastric Reflux degraded and discolored when exposed to acidic media. It is clini-
cally used in the treatment of gastroesophageal reflux disease
Physical
β-cyclodextrin complex
method
Nanosuspension
+
Polymer
condensation
+ method
Lansoprazole
β-cyclodextrin
Pyromellitic
dianhydride
Nanosuspension
Nanosponges complex
Fig. 7 The mechanism of complexation by physical method and polymer condensation method, respec-
tively [7]
(GERD), gastric and duodenal ulcers, and Zollinger–Ellison syn-

drome [92]. Physicochemical investigation and comparative char-
acterization of nanosuspensions of lansoprazole complexed with
β-CD and β-CD-based NS were performed. Inclusion complexes
of lansoprazole with β-CD and NS were prepared by physical
method and polymer condensation method as shown below
(Fig. 7) [93].
β-CD-based NS and nanosuspensions complexed efficiently
with lansoprazole, thus improving its solubility and stability. The
solubilization efficiency of pyromellitate bridged β-CD NS was
more than 40% compared to CD nanosuspensions. This nanosus-
pension displayed a high encapsulation efficiency and stability pro-
tecting the drug from acidic degradation in stomach, a promising
carrier for nanoparticulate drug delivery in gastric ulcer.
2.14 Cyclodextrin Gases play an important role in medicine, either for diagnostic or
Nanosponges for Gas for treatment purposes. It is sometime difficult to deliver oxygen in
Delivery appropriate form and dosage in clinical practice. The deficiency of
adequate oxygen supply, named hypoxia, is related to various
pathologies, from inflammation to cancer. The design of delivery
systems providing oxygen for these cases is therefore necessary.
CDs can store gases in their cavity via molecular encapsulation.
The amount of gas complexation varies generally between 0.3 and
1.2 mol gas/mole β-CD. Cross-linked NSs denote superior com-
plexation abilities toward many molecules in comparison with pris-
tine β-CD and preliminary results on complexation abilities of NS
in respect to 1-methylcyclopropene, carbon dioxide, and oxygen
were reported [94].
The ability of NS to reversibly bind compounds, even in the
gas phase, through physisorption mechanisms, might lead to the
development of a new technology for the efficient storage of sig-
nificant amounts of gas in surprisingly small volumes, without the
need for high pressure and low temperatures, thus avoiding the
risks and energy loss associated with the current use of compressed
or liquefied gases [95].
Nanosponges might be a suitable carrier for oxygen topical
delivery. The application of US on NS aqueous suspension pro-
duced an oxygen permeation increase of about 192 ± 2% after
15 min with an initial peak of the gas permeated. The topical appli-
cation was improved through a new formulation using the combi-
nation of oxygen-encapsulating NS and Pluronic F127 hydrogel
was developed. The presence of US increases the oxygen release of
89.7 ± 2% after 30 min. The NS gel formed a regular sustained
oxygen release in the presence and absence of US and acted as
oxygen reservoir [96].
Improved nanosponge formulations for oxygen delivery were
developed. For this purpose, native α-CD, a soluble α-CD poly-
mer, and an insoluble α-CD NS were studied for the in vitro oxy-
gen release determination. Later, the three different formulations

were tested at 0.2, 2, and 20 μg/mL to ascertain their capability to
reduce cell mortality during hypoxia and reoxygenation (H/R) in
vitro protocols. H9c2, a cardiomyoblast cell line, was exposed to
normoxia (20% O2) or hypoxia (5% CO2 and 95% N2). The d ifferent
formulations, applied before hypoxia, induced a significant reduc-
tion in cell mortality (in a range of 15–30%) when compared to
samples devoid of oxygen. Moreover, their application at the
beginning of reoxygenation induced a considerable reduction in
cell death (12–20%). α-CD NS showed a marked efficacy in con-
trolled oxygenation, which suggests an interesting potential for
future medical application of polymer systems for myocardial
infarction (MI) treatment [97].
2.15 Cyclodextrin Repaglinide is an oral hypoglycemic agent that belongs to the

Nanosponges short-acting insulin secretagogues, which act by binding to β cells
for Antihyperglycemic of the pancreas to stimulate insulin release. The side effects of repa-
Agent glinide may be due its acid–base ampholyte character (pKa1 = 4.2;
pKa2 = 6.0) with low water solubility (34 μg/mL at 37 °C) and
high lipophilicity (log P = 3.97) [98]. Earlier studies focused on
the possibilities to improve pharmacokinetic characteristics of repa-
glinide using β-CD and several substituted derivatives. Polymeric
nanostructures derived from sulfobutylether β-cyclodextrin (SBE
β-CD) are less investigated because of the characteristic high
degree of substitution which results in less possibilities to bond the
cyclodextrin toroid. β-CD-NS and SBE β-CD-NS were synthe-
sized (1:10 CD/cross-linker ratio). These NS and their complexes
with repaglinide (5:1 NS:repaglinide) were characterized and solu-
bility was studied following Higuchi and Connors method, which
provided higher solubilization of repaglinide. Though its loading
capacity is significantly higher, the lower solubilization effect of
SBE β-CD-NS is probably due to a steric hindrance. The high
degree of substitution of SBE β-CD allows a small number of
intermolecular bonds and a less reticulated structure (i.e., lower
entrapment of the guest molecules) [99].
2.16 Cyclodextrin Atorvastatin calcium (AC) is a synthetic drug, assigned to class II

Nanosponges with high permeability and low aqueous solubility (log p¼ 5.39)
for Calcium Delivery that result in poor dissolution in the biological fluid and hence
limited oral bioavailability. AC can selectively and competitively
inhibit hydroxymethyl glutaryl-coenzyme A (HMG-CoA) reduc-
tase enzyme that catalyzes the conversion of HMG-CoA to meva-
lonate, an early and a rate-limiting step in the biosynthesis of
cholesterol; thus AC plays an important role in treatment and pre-
vention of hyperlipidemia-associated diseases [100].
CD-based NSs cross-linked with carbonyldiimidazole (ratios
1:2, 1:4, and 1:8) developed by solvent method were used to form
AC-NS complexes to improve oral absorption and bioavailability
of AC. The prepared AC-NS showed particle size ranging from

408.7 ± 12.9 to 423 ± 15.9 nm while zeta potential values varied
from 21.7 ± 0.90 to 22.7 ± 0.85 mV. The loading capacity varied
from 17.9 ± 1.21 to 34.1 ± 1.16%. DSC, FT–IR, and PXRD stud-
ies confirmed the complexation of AC with NS and amorphous
state of the drug in the complex. AC-NS displayed a biphasic
release pattern with increase in the dissolution rate of AC as com-
pared to plain AC. Oral administration of AC-NS (1:4 w/w,
drug:NS) to rats led to 2.13-fold increase in the bioavailability as
compared to AC suspension. Pharmacodynamics studies in rats
with fatty liver revealed significant reduction (p < .05) in total cho-
lesterol, triglyceride, LDL-C, and increased level of beneficial
HDL-C along with improvement in the associated liver steatosis as
confirmed through photomicrographs of liver sections. Thus,
complexation is a viable approach for improving oral bioavailability
and in vivo performance of AC [101].
Calcium carbonate is most widely used as calcium source. It is
also used as an antacid, in the treatment of osteoporosis and in the
management of hyperphosphatemia in renal failure. The major
part of calcium absorbed is accumulated in the skeleton [102].
Hyperphosphatemia can be caused by hypoparathyroidism due to
the lack of parathyroid hormone inhibiting the renal reabsorption
of phosphate. Commercially available phosphate binders for the
treatment of hyperphosphatemia produce toxicity like bone disease
and aluminum dementia. Nanosponges acted as reservoirs for cal-
cium carbonate. Enteric-coated controlled-release calcium carbon-
ate nanosponges efficiently bound free phosphate ions and
consequently offered an alternative to treat hyperphosphatemia
[103].
2.17 Cyclodextrin Therapeutically relevant proteins such as antibodies, cytokines,

Nanosponges growth factors, and enzymes are playing an increasing role in the
for Peptides treatment of viral, malignant, and autoimmune diseases. However,
and Steroids the development and successful application of therapeutic proteins
are often limited by several drawbacks. The encapsulation of pro-
teins within polymeric carriers can potentially overcome physiolog-
ical barriers. It can control the release of actives to specific cells,
protect them against degradation, and ensure their transport
[104]. CDNSs have been used as carriers for biocatalysts, and in
the delivery and release of enzymes, proteins, vaccines, and anti-
bodies [105, 106]. Polyamidoamine nanosponges (PAA-NS) have
been synthesized by cross-linking β-CD with either
2,2-bisacrylamidoacetic acid or polyamidoamine segments deriv-
ing from 2,2-bisacrylamidoacetic acid and 2-methylpiperazine.
The potential of PAA nanosponges were evaluated using bovine
serum albumin (BSA) as model molecule. BSA was successfully
incorporated in the two swellable PAA-NS and in vitro studies
showed that the BSA was released from the PAA-NS with
prolonged- release kinetics. Moreover, BSA encapsulation in

PAA-NS protected and stabilized the protein during storage [107].
Carboxylic CDNSs were functionalized with an anti-IgG anti-
body and loaded with horseradish peroxidase, an enzyme com-
monly used as a label in biosensor assays. The possibility to use
these modified CDNSs as a signal enhancement tool in enzyme-
linked colorimetric and electrochemical assays was evaluated using
a sandwich format comprising immobilized gliadin as the capture
molecule, a target anti-gliadin antibody, and an anti-IgG antibody.
This type of assay has been recognized as a diagnostic and thera-
peutic tool in celiac disease since levels of anti-gliadin antibodies in
serum are used for monitoring celiac patients’ compliance with a
gluten-free diet. CDNSs offer the advantage of one-step multi-
gram preparation and ease of bioconjugation under mild condi-
tions and the signal amplification obtained in the presence of
loaded horseradish peroxidase indicates that these types of nano-
bioconjugates are promising candidates in the development of
ultrasensitive biosensors (Fig. 8) [108].
Catechol 1,2-dioxygenases are iron-containing enzymes, a
precursor of the industrially important compound adipic acid. In
order to construct a bioreactor able to produce muconic acid with
high efficiency and operational stability, the enzyme catechol
1,2-dioxygenase was selected as it does not require any external
cofactor for catalysis. A small-scale bioreactor was constructed and
immobilized on β-CD-NS cross-linked with carbonate groups.
This support was chosen for its low cost and its ability to offer dif-
ferent types of interactions with the enzyme. The activity profiles at
different pH and temperatures showed a shift of the optimal pH
from 8.5, for the free protein, to 9.5, for the immobilized protein
and, similarly, a shift in optimal temperature from 30 °C to
50 °C. The activity was retained by the immobilized enzyme (with
high efficiency for 70 days) and toward other substrates [109].
Surface-active carbonyl diimidazole cross-linked β-CD-NSs
with and without CaCO3 and CMC were prepared and impreg-
nated by lysozyme due to their ability to adsorb protein via electro-
static interactions. Upon lysozyme impregnation, zeta potentials of
nanosponges were sufficiently increased suggesting stable formula-
tions by preventing aggregation. In vitro release studies showed
controlled release of lysozyme and calcium over a period of 24 h.
Lysozyme-impregnated nanosponge was a smart formulation to
deliver lysozyme in a conformationally stable structure and thera-
peutically active form for antimicrobial action and calcium in hypo-
calcemia condition at a controlled rate [110].
Penetratin, a cell-penetrating peptide, was site-specifically
modified with a bis-β-cyclodextrin group. Insulin-loaded nano-
complexes were prepared by self-assembly using penetratin or its
bis-β-cyclodextrin-modified derivative (P-bis-CD). Different cel-
lular internalization mechanisms were observed for the two nano-
O O
(OH)7 HOOC COOH
O O
O O
β-CD
NH3
(OH)14 HOOC COOH
CDNS
b,c
CDNS-Ab-HRP CDNS-Ab
Fig. 8 Preparation of CDNS-Ab-HRP: (a) EDC/NHS (15 min, 4 °C), anti-IgG antibody (2 h, 4 °C), (b) ethanolamine
(1 h, 4 °C), and (c) HRP (62 h, 4 °C, pH 6.5) [8]
complexes. In diabetic rats, intestinal administration of P-bis-CD

nanocomplexes resulted in a prominent hypoglycemic effect which
lasted for 6 h with maximum inhibitory rate at 60%. The relative
pharmacological availability and bioavailability of P-bis-CD nano-
complexes were 10.6% and 7.1%, which were 3.0-fold and 2.3-fold
higher than those of penetratin nanocomplexes, respectively.
Results demonstrated that P-bis-CD was a promising epithelium
permeation enhancer for insulin and suggested that the chemical
modification of cell penetration peptides was a feasible strategy to
enhance their potential [111].
Cationic amphiphilic cyclodextrin (click propyl-amine cyclo-
dextrin) as the primary complexing agent in nanoparticles (NPs)
developed entrapping insulin glulisine for intestinal administra-
tion. NPs exhibited colloidal stability in intestinal-biorelevant
media (SIF, supplemented-SIF 1% (w/v) and FaSSIF-V2) for up
to 4 h. Proteolysis studies indicated that the NPs conferred protec-
tion to the entrapped insulin relative to free insulin. In vivo rat
jejunum instillation studies demonstrated that the NPs mediated
systemic insulin absorption, accompanied by a decrease in blood
glucose levels. The relative bioavailability of the instilled insulin
(50 IU/kg) from the NP was 5.5% compared to subcutaneous
administration of insulin solution (1 IU/kg). The pharmacody-
namic and pharmacokinetic data indicate that this amphiphilic
cyclodextrin formulation may have potential for further research as
an oral insulin dosage form [112].
Dexamethasone (DEX), a type of corticosteroid, is extensively
used as an analgesic and an anti-inflammatory agent. Due to its
poor solubility and hydrophobic nature, it causes problems in its

efficient clinical usage. For instance in the ocular applications of
DEX, low residence time of formulations in the eye results in only
about 5% drug penetration and thus a need for frequent adminis-
tration arises. A NS carrier-based system may serve to increase the
residence time in the eye and release the drug in a controlled man-
ner, thereby also reducing the associated side effects [113]. Three
NSs were prepared by cross-linking β-CD and diphenyl carbonate
(DFC) at different β-CD:DFC molar ratios (1:2, 1:4, and 1:8) and
loading DEX through incubation method followed by lyophiliza-
tion of the drug-loaded NSs.
NS formulations were checked for safety in excised bovine cor-
neas. For a formulation to be suitable for ocular delivery it must
have uniform particle size (Fig. 9). The most desirable formulation
NS (1:4) showed a denser core as compared to other types. This
could pave a platform for improving the permeability of various
other drugs for ocular ailments, combining encapsulation abilities,
safety, and colloidal properties together with the adhesive effect to
obtain superior dosage forms [114].
2.18 Cyclodextrin NSs obtained by cross-linking with pyromellitic dianhydride can

Nanosponges swell to a very great extent and form a hydrated gel. β-Pyromellitic
with Photosensitizing/ NS with high degree of swelling was exploited to form gels or
Photooxidation Agents cream-gel for diclofenac (DIC) delivery to the skin. In addition,
the NS was used to solubilize and stabilize the photosensitizing
agent benzoporphyrin derivative monoacid ring A (BPDMA) and
the light-sensitive all-trans retinoic acid (atRA).
The effect of the pyromellitic NS concentration on the viscos-
ity of the corresponding water dispersions was preliminarily
assessed. At increasing NS concentrations, viscosity increased up to
1200 cps at 2% NS concentration and then progressively decreased.
As viscosity obtained was not high enough for a gel suitable for
application to the skin, the NS gel was reinforced with a 1%
Carbopol 940 (CB). DIC was incorporated in the gels by dissolu-
tion in water. pH of the gels was adjusted at 7.0 with triethanol-
amine. Stability of the drug in the formulations was evaluated by
thermal aging and cycle test. DIC transport through porcine ear
skin (selected on the basis of its demonstrated similarity with
human skin) was later assessed on Franz-type diffusion cell area of
0.785 cm2. Extraction technique was validated by placing known
amounts of DIC in contact with tape, epidermis, and derma, and
the drug has been extracted. A recovery yield higher than 98% was
obtained. After 24 h, the permeation experiment showed that the
amount of DIC found in the stratum corneum and epidermis was
the highest for the NS–CB gel as compared with the other formu-
lations. As a result, the NS showed the ability to modulate drug
transport through the skin in addition to the ability to improve
Fig. 9 The corneal cells of bovine remained intact after NS 1:4 treatment (magnification 10×) [9]
solubilization of lipophilic drugs in water-containing semisolid for-

mulations and to ameliorate drug photostability [115].
The interaction between β-CD-NSs and UV stabilizers in the
photooxidation of polypropylene has been investigated. For this
aim, two different kinds of stabilizers (2-hydroxy-4(octyloxy)-ben-
zophenone and triphenyl phosphite, TPP) have been mixed with a
β-CD-NS, and the synergistic effect of these two species has been
assessed in order to understand the possible chemical or physical
interactions occurring between them and the influence of each
component on the oxidation of the polymer matrix. Photooxidation
of NS and NS/stabilizer-based PP composites was recorded via
FTIR spectra. Oxidation induction time (OIT) and the subsequent
rate of photooxidation collected for formulations under study were
measured from absorbance vs. irradiation time plots. The main
results were collected: (1) a significant reduction of the oxidation
induction time in the presence of the β-CD-NS alone; (2) a remark-
able OIT increase when the NS is combined with 2-hydroxy-
4(octyloxy)-benzophenone; (3) a possible synergistic effect
between the two species, probably due to some chemical interac-
tions; and (4) a low OIT when TPP is employed in combination
with the NS, which turned out to be less efficient than TPP alone
[116].
2.19 Cyclodextrin The encapsulation of caffeine (Caf) inside CDNS hydrogels was
Nanosponges systematically investigated by using UV Raman scattering experi-
for Miscellaneous ments. The UV Raman spectra of the hydrogels, analyzed as a
Uses function of temperature, concentration of the guest molecule
loaded in the gel phase, and pH, enable the proposal of a molecu-
lar picture of loading. The loading of Caf in NS hydrogels tends to
favor access of the water solvent to the more hydrophobic portions
of the polymer matrix, which is in turn reflected in a marked
increase in the solvation of the whole system. The results of this
work appear to be of interest with respect to the design of new
possible strategies for controlling the diffusion/release of bioactive
molecules inside hydrogel networks to give new molecular insights
into complex phenomena affecting the hydrogel phase [117].
Organic/inorganic hybrid filters based on dendritic and cyclo-
dextrin nanosponges which are completely insoluble in water have
the property of encapsulating organic pollutants from water [118].
Ceramic porous filters impregnated with these compounds were
tested for the effective purification of water, by continuous filtra-
tion experiments, employing a variety of water pollutants. It has
been established that polycyclic aromatic hydrocarbons (PAHs)
can be removed very efficiently (more than 95%), and final concen-
trations of several ppb (μg/L) are easily obtained. Representatives
of the pollutant group of trihalogen methanes (THMs), monoaro-
matic hydrocarbons (BTX), and pesticides (simazine) can also be
removed (>80%), although the filters are saturated considerably
faster in these cases. For the efficient absorption of the more polar
substances employing hybrid organic/inorganic filter modules,
increased impregnation percentage and adequate contact time
between the alkylated compounds and the polluted water are
required. The latter can be achieved by low flow rates, increased
length of the ceramic membranes, or, alternatively, recirculation of
the polluted water. By careful selection of the suitable materials
and processing parameters the application of these composite fil-
ters may be extended and adopted for specific cases of water puri-
fication [119].
New water-soluble hyper-branched β-CD polymer was synthe-
tized and characterized. The synthetic route proposed seems to be
quite independent from the reaction time. The new polymer exhib-
its an exceptionally high solubility in water, DMF, and DMSO
while it is insoluble in low polar solvents and at very low pH. The
polymer shows good complexing properties toward organic mole-
cules. In addition, it strongly interacts with several metal cations
able to precipitate in the presence of Cu2+, Fe3+, Ba2+, Cd2+, Pb2+,
and Sn2+, by forming metal–organic complexes showing some
selectivity toward certain metal cations and indirect evidence of
polyelectrolyte behavior [120].
3 Conclusion
Cyclodextrin-based nanosponges constitute an atypical class of

biocompatible delivery system that allows a smooth transformation
from conventional delivery to a versatile delivery system due to

presence of flexible cross-linked polymers, and fulfils the aforemen-
tioned required characteristics. The required particle size, viscosity,
and release rate can be attained by controlling the polymer-to-
cross-linker ratio. Nanosponge-based delivery systems also address
the solubility dilemma that is associated with newly developed
drug entities, and protect the active moieties from degradation.
Various dosage forms of the same drug can be formulated as
desired, and the side effects associated with conventional formula-
tions can be overcome by advanced approaches like stimuli-sensitive
nanosponges and tumor targeting. To sum, cyclodextrin-based
nanosponges with multitude number of beneficial attributes can
contribute as a promising tool for effective and efficient drug deliv-
ery, and can be endorsed as an advanced carrier in the field of drug
delivery and nanotherapeutics.
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Chapter 20
Electrochemistry Investigation of Drugs Encapsulated

in Cyclodextrins
Romana Sokolová and Ilaria Degano
Abstract
The biological electron transfer reactions play an important role in the bioactivity of drugs; thus, the
knowledge of their electrochemical behavior is crucial. The formation of radicals during oxidation or
reduction, the presence of short-living intermediates, the determination of reaction mechanisms involving
electron and proton transfers, all contribute to the comprehension of drug activities and the determination
of their mode of action and their metabolites. In addition, if a drug is encapsulated in the cyclodextrin
cavity, its electrochemical properties can change compared to a free drug molecule. Here we describe the
combination of cyclic voltammetry, UV–Vis spectroelectrochemistry, GC-MS, HPLC-DAD, and
HPLC-MS/MS as techniques for evaluating the oxidation mechanism of a drug encapsulated in the cavity
of a cyclodextrin. The cavity of cyclodextrin plays a significant role in increasing the stability of the encap-
sulated products; therefore the identification of oxidation intermediates as semiquinone and benzofura-
none derivatives of quercetin is possible in these conditions. The differences in oxidation potentials of the
bioactive flavonol quercetin and its cyclodextrin complex relating to its antioxidant activity and the oxida-
tion mechanism are herein discussed.
Key words Drug oxidation, Drug–cyclodextrin complex, Electron transfer, Stability of intermediates,
Oxidation mechanism, Chromatography, Mass spectrometry, Cyclic voltammetry,
Spectroelectrochemistry
1 Introduction
Electrochemical methodologies have been successfully used to

explain correlations between chemical structure, oxidation poten-
tial, and biological activity of electroactive species [1]. For instance,
the antioxidant properties of bioactive compounds, allowing them
to prevent the formation of reactive oxygen species (ROS) often
generated at sites of inflammation and injury, or to neutralize free
radicals and minimize the oxidative stress resulting from a variety
of insults [2, 3], are closely related to their redox properties and in
particular to their ability to act as electron donors, i.e., to act as
reducing agents. Such properties can be deduced from the oxida-
285
286 Romana Sokolová and Ilaria Degano
tion potential of the studied compound in certain media, which are

usually chosen to fit with physiological conditions (i.e., buffer at
pH 7.4) [1, 4].
Polyphenolic compounds act as good antioxidants and radical
scavengers [5] and their antioxidant activity can be estimated by
electrochemical measurement. In particular, the cleavage of one
electron from a polyphenol (a radical cation is formed) can be fol-
lowed by a proton cleavage from phenolic group, yielding a radical
[6] which can be highly reactive toward environment and can
quench potentially harmful free radicals. Intermolecular hydrogen
bonds are of significant importance for the stability of the radical
and anionic species [7].
Electrochemical properties are generally measured using one
pair of electrodes or three-electrode setup, where the redox pro-
cesses take place at the electrode–solution interface. The obtained
electroanalytical signal is related to the concentration of electroac-
tive species at the electrode surface. The electrochemical voltam-
metric signal and its detailed examination can be used to determine
the mechanism of formation of the first reduction or oxidation
intermediates and to help solve the complicated redox mechanisms
of the studied compounds. Cyclic voltammograms are usually
recorded at different scan rates to obtain information about the
process at the electrode. The analysis of peak currents in depen-
dence on the rate of polarization is important to assess whether the
redox process is controlled by diffusion or by adsorption. A signifi-
cant increase in the scan rate can result in the appearance of new
redox peaks in the forward or backward scans, due to a product
formed at the electrode [8, 9]. When diffusion-controlled condi-
tions for Nernstian behavior are fulfilled, the electrochemical sig-
nals recorded at different scan rates and concentrations yield data
on the influence of coupled chemical reaction (C) on the measured
currents. Since chemical reactions directly affect the concentration
of the electroactive species available at the electrode surface, the
interpretation of the cyclic voltammograms recorded in different
conditions (e.g., at different pH values) gives unique information
about the reaction scheme. In particular, the analysis of oxidation
waves allows us to distinguish between reaction schemes entailing
EC (electron transfer followed by chemical reaction), CE (the
chemical reaction precedes the electron transfer), ECE, EE, ECEC,
etc., as well as disclose oxidation or reduction coupling with cata-
lytic processes [10, 11]. For instance, the shift of peak potential in
solutions at different pH values means that protons participate in
redox process [12, 13].
It was shown that metabolic oxidation of some drugs may be
successfully simulated by electrochemical methods because the
main biotransformation of drugs is based on reduction and oxida-
tion processes [14–16]. For instance, Guaiquil et al. found the
same oxidation products of vitamin C found by previous electro-
Electrochemistry Investigation of Drugs Encapsulated in Cyclodextrins 287
chemical studies as biological metabolites transported by rat

cardiomyocytes [2]. Moreover, studies concerning the metabolic
products of flavonoid quercetin processed by rat colonic flora fit
the decomposition pathways found in the recent studies of flavonol
quercetin oxidation mechanism [17].
The complete elucidation of the oxidation or reduction mech-
anism requires the use of analytical separation techniques and
online spectroelectrochemical methods [18, 19]. In situ UV–Vis
spectroelectrochemistry is an efficient technique giving informa-
tion about the changes in absorption spectra during electrolysis at
controlled potential. Spectroelectrochemical measurements are
performed in optically transparent thin-layer electrochemical cells
[20, 21]. Additionally, in situ FTIR spectroelectrochemistry can
characterize reactions occurring at the electrode surfaces by moni-
toring the change in absorbance of vibrations belonging directly to
the functional groups participating in the redox process. The con-
version of compounds to reduced or oxidized molecules shows a
clear isosbestic point in absorption spectra and thus this technique
successfully points out the presence of short-lived intermediates
and occurring subsequent reactions. Based on the principles men-
tioned above, we recently disclosed the difference in the oxidation
mechanisms of flavonols (as quercetin, fisetin, rhamnazin, and
rhamnetin) and flavanones (taxifolin) or flavones (luteolin) [13,
21–24]. The oxidation of flavonols (containing in their chemical
structure one hydroxyl group at C3 position and a double bond
between C2 and C3 atoms) leads to the formation of a benzofura-
none derivative, through the 2e−/2H+ oxidation of catechol group
at ring B and subsequent nucleophilic addition of water. The
absorption spectrum of the benzofuranone derivative was detected
by UV–Vis spectroelectrochemical oxidation and also by chemical
homogeneous oxidation by atmospheric oxygen. GC-MS and
HPLC-MS/MS analyses and data interpretation confirmed its for-
mation. Neither taxifolin (lacking the double bond between C2
and C3 atoms) nor luteolin (lacking the hydroxyl group at position
C3) did not follow this oxidation pathway, and hydroxylated mol-
ecules were found as their oxidation products.
Significantly, the principles and analytical strategies mentioned
above are valid also for the study of electroactive drugs encapsu-
lated in the cavity of a cyclodextrin molecule. Cyclodextrins (CDs)
are supramolecules, which form truncated cones and can form
complexes with a variety of guest molecules, depending on the size
of their hydrophobic cavity and possible chemical modification of
their rims. The external of the cyclodextrin is hydrophilic; thus
encapsulation may enhance the solubility in water and thus the
bioavailability of the drug [25]. These supramolecular complexes
are also widely studied by electrochemistry [26].
Several possible behaviors can be observed when performing
electroanalytical experiments on CD complexes. The CD-drug
interaction and the oxidation behavior of the complex can be

studied by adding CD solution at different concentrations to a
starting solution of the free drug. The interpretation of the data
must take into account that cyclodextrin can be adsorbed on the
surface of the electrode depending on solution composition; thus
Nernstian diffusion controlled waves and adsorptive pre-waves or
post-waves must be carefully assigned in cyclic voltammograms
before evaluating reaction mechanism [8].
Keeping this in mind, two main behaviors can be observed,
based on redox properties and stability of the complex:
1.1 The CD-Drug Subsequently, the CD-oxidized drug complex may dissociate,
Complex Is Oxidized depending on its stability. The products of oxidation can differ
at the Surface depending on the geometry of the cavity inside the CD macromol-
of Electrode ecule and relative position of the drug inside it. Some electroactive
and the Formation sites of the drug molecule can be hindered in the CD cavity and
of a Complex Between become unavailable to the surface of the electrode. If cyclodextrin
CD and Oxidated Drug as a host works as a proton donor to the guest molecule, and this
Is Observed proton donation is involved in a redox process, the oxidation path-
way of the complex can be different from that on the free molecule
[27]. CD can also allow reduction of the drug compound when
protonation is needed [28], or stabilize the electrogenerated radi-
cal in the cavity [29]. If electroactive sites of the drug molecule are
not hindered by the cavity, the oxidation process can result in the
same products as those obtained for the free molecule. This was
the case of a quercetin-2HP-β-CD inclusion complex, where quer-
cetin retained the same oxidation profile as observed for its native
state (Fig. 1) [30]. Moreover, some intermediates, which are dif-
ficult to identify due to their short life or fast-following reactions in
solution, can be stabilized in the hydrophobic cyclodextrin cavity.
This was observed for a semiquinone derivative of quercetin, which
was only identified in CD complexes [30].
1.2 The CD-Drug In this case, the oxidation of the complex requires higher potential
Complex Dissociates than that of the free drug, since the energy is partially consumed
and Afterwards for the cleavage of the complex. In this situation, a CE reaction
the Free Drug scheme best describes the oxidation of CD-drug complex.
Molecule Is Oxidized Moreover, the height of the oxidation wave can be lower in the
at the Surface presence of CD compared to the height of a free drug, due to the
of Electrode considerably lower diffusion coefficient of the complex compared
to that of the free drug. The difference of oxidation potentials
between CD-drug complex (E0)complex and free drug (E0)free can be
used to determine the stability constant of the complex according
to Eq. (1) [10, 31, 32]:
( E 0 )complex − ( E 0 )free = RT
nF
ln
D
D∗
−
RT
nF
ln KS −
RT
nF
ln [CD]
(1)
Fig. 1 Geometry of the 2HP-β-CD-quercetin complex chemical structure and position of frontier orbitals of
quercetin calculated using DFT (Spartan’14)
where Ks is the stability constant of the complex, [CD] is the con-

centration of CD-drug complex, and D and D* stand for the dif-
fusion coefficient of the free drug and CD-drug complex,
respectively.
2 Materials
The solutions are prepared with ultrapure deionized water with a

maximum resistivity of 18 MΩ cm, which was obtained by means
of a Milli-Q RG purification system (Millipore Co.). All reagents
are analytical grade. All buffer solutions are stored at 4 °C in the
dark and warmed at 25 °C before use. KCl solutions are stored at
room temperature. Solutions of drugs are freshly prepared before
the experimental work in solvents previously degassed by argon.
2.1 Reagents 1. Quercetin, β-cyclodextrin, quercetin-2-hydroxypropyl-β-

cyclodextrin (quercetin-2HP-β-CD complex, prepared as
described in detail in [33] using a freeze-drying procedure).
2. 0.1 M KCl in water.
3. Britton-Robinson buffers (BRB) at different pH values
(pH 4.0–11.0) prepared using an acidic stock solution (sepa-
rately prepared, containing 0.04 M H3PO4, CH3COOH,

H3BO3) and 0.2 M NaOH.
4. Ethanol (96%) for preparation of aqueous ethanolic solutions of
less soluble drugs (40% v/v).
2.2 Cyclic 1. Working electrode: Glassy carbon or platinum electrode of

Voltammetry diameter 0.7–2 mm.
2. Ag/AgCl/1 M KCl reference electrode separated from exam-
ined solution by a salt bridge.
3. Auxiliary electrode: Pt net.
4. The electrochemical cell with an inlet and outlet for argon or
nitrogen: In case of ethanolic solutions, argon is first bubbled
through the reservoir of ethanol to prevent the evaporation of
ethanol from the examined solution.
2.3 Electrolysis 1. The electrolytic cell with separated anodic and cathodic parts
and inlet/outlet of inert gas: In case of ethanolic solution,
argon is first bubbled through the reservoir of ethanol to pre-
vent the evaporation of ethanol from examined solution.
2. Working electrode: Glassy carbon or platinum electrode with a
large surface.
3. Ag/AgCl/1 M KCl reference electrode separated from solu-
tion by a salt bridge.
4. Auxiliary electrode: Carbon electrode present in the cathodic
compartment of the electrolytic cell (when oxidative generation
of products is measured).
2.4 UV–Vis 1. Optically transparent spectroelectrochemical cell (more details

Spectro- available in [20]).
electrochemistry 2. 0.5–1 mL Hamilton syringe with needle.
3. Tetrabutylammonium hexafluorophosphate in acetonitrile used
as supporting electrolyte for electrochemical cleaning of the
cell.
2.5 Gas 1. GC-MS parameters: Separation on a DB-5MS chemically

Chromatography bonded fused silica capillary column with stationary phase 5%
phenyl-95% methylpolysiloxane, and of dimensions 0.25 mm
i.d., 0.1 μm film thickness, 25 m and 30 m length. Gas chro-
matographic conditions as follows: initial temperature 57 °C,
2 min isothermal, then ramped at 10 °C/min up to 200 °C,
3 min isothermal, then ramped at 20 °C/min up to 300 °C,
and then isothermal for 20 min. Carrier gas He (purity
99.9995%), at a constant flow rate of 1.2 mL/min. Injection
volume 2 μL, splitless mode at 280 °C. Mass spectrometric
acquisition on a single quadrupole, electronic impact ionization
(70 eV), ion source temperature 230 °C, scan range m/z
50–800 (positive mode), and interface temperature 280 °C.
2. Internal standards: 2,4-Dihydroxybenzophenone (10 μg/g
solution in isopropanol), internal standard for derivatization
(IS1); hexadecane (100 μg/g solution in iso-octane), internal
standard for injection (IS2).
3. Derivatization agent: N,O-bis(trimethylsilyl)trifluoroacetamide
(BSTFA) containing 1% trimethylchlorosilane. Derivatization
takes place in ethyl acetate (AcOEt).
2.6 HPLC-DAD 1. Eluents for HPLC: Water (eluent A) and acetonitrile (eluent
Chromatography B), both HPLC grade. Mobile-phase modifier: Trifluoroacetic
acid (TFA) 0.1% in both eluents, or formic acid (FA) 0.1–1% in
both eluents.
2. Gradient for HPLC: Starting with 85–90% eluent A, hold for
2–5 min, then increase %B until 90–100% with a linear
gradient.
3. Flow rate: 0.2–1 mL/min, depending on the dimensions of the
column and of the stationary-phase particle diameter.
4. DAD: Acquisition in the 200–650 nm range.
5. Injection volume: 5–20 μL.
2.7 HPLC-MS/MS 1. Eluents for HPLC: Water (eluent A) and acetonitrile (eluent
Chromatography B), both LC-MS grade. Mobile-phase modifier: Formic acid
(FA) 0.1–1% in both eluents.
2. Gradient for HPLC: Starting with 85–90% eluent A, hold for
2–5 min, then increase %B until 90–100% with a linear
gradient.
3. Flow rate: 0.2–0.4 mL/min, depending on the dimensions of
the column and of the stationary-phase particle diameter.
4. MS and tandem MS: Ionization in electrospray ion source in
negative mode, acquisition in the 100–1700 m/z range by a
ToF analyzer for both MS and tandem acquisitions. Application
of 30–50 V in the collision cell for the collision-induced
dissociation.
5. Injection volume: 1–5 μL.
3 Methods
3.1 Cyclic 1. Insert the solution of the supporting electrolyte (0.1 M KCl or
Voltammetry BRB buffer) into the electrochemical cell, and in the salt bridge
of the reference electrode.
2. Mount all the electrodes on the cell. Manually polish the sur-
face of the working electrode with alumina (Al2O3) water sus-
pension on DP-Nap (Struers) prior to recording each cyclic
voltammogram.
3. Bubble the solution by argon or nitrogen to remove oxygen
before each measurement.
4. Record the cyclic voltammogram of the blank solution and then
transfer the solution of the target compound to the cell.
5. Record cyclic voltammograms at different scan rates and differ-
ent values of concentration.
6. Analyze the shape of cyclic voltammograms in Origin or Excel
software and use values of Ip and Ep to determine the electrode
processes (see Notes 1 and 2).
7. Figure 2 shows that Ep of quercetin-2HP-β-CD complex in
solution at different pH values differs, signifying that protons
participate in the oxidation (see Note 3). The dependence of
Ep/pH indicates the ratio between the number of participating
protons and electrons [21, 23].
3.2 The Generation 1. Prepare the solutions of the drug and of the CD-drug complex
of Oxidation Products in anoxic conditions, by bubbling argon in all vials and solvent
Electrochemically used for their preparation.
2. Transfer the blank solution to the spectroelectrochemical cell
3.2.1 In Situ UV–Vis
and record the blank, against which absorption spectra will be
Spectroelectrochemistry
recorded. Remove the blank solution from the cell.
3. Transfer the target solution to the spectroelectrochemical cell
by a syringe under inert atmosphere.
4. Set up the potentiostat to perform cyclic voltammetry experi-
ments with a scan rate of 5 mV/s, or to perform chronoam-
perometry for 300-s time.
5. In order to monitor the spectral changes during the electron
transfer, set the spectrophotometer for kinetics measurement to
record spectra every 3 s, sufficient to recognize the redox inter-
mediates and products.
6. Start simultaneously the application of potential by the poten-
tiostat and the monitoring of absorption spectra by the spectro-
photometer. Plot register data in Origin or Excel software
(Fig. 3).
7. Clean first the surface of the platinum working electrode
mounted on the cell by washing it with solvents (water, ethanol,
acetone, acetonitrile) and afterwards by electrochemical clean-
ing (see Note 4).
Fig. 2 Representative cyclic voltammograms of 2HP-β-CD-quercetin complex in BRB at different pH and free
molecule of quercetin at pH 8.8. Scan rate was 0.1 V/s, concentration of compounds 0.18 mM
3.2.2 The Generation 1. Insert the solution of electrolyte 0.1 M KCl or BRB buffer into
of Oxidation Products by the electrochemical cell, and in the salt bridge of reference elec-
Potential Controlled trode. Fill the cathodic space of the electrolytical cell with
Coulometry (Exhaustive degassed electrolyte solution.
Electrolysis) 2. Mount all the electrodes on the cell. Record the cyclic voltam-
mogram of the blank solution and of the target compound.
Polish the surface of the working electrode mechanically by
hand with alumina water suspension on DP-Nap prior to every
measurement of CV as mentioned above.
3. Bubble the solution by argon or nitrogen to remove any
remaining oxygen prior to every CV measurement.
4. Install in the cell a working electrode with a large surface (GC
or carbon paste electrode). Bubble again the solution by argon
and stir by an electromagnetic stirrer.
5. Transfer an aliquot of 100 μL of the solution by syringe to a
suitable vial and directly inject in HPLC-DAD or HPLC-MS/
MS systems (see below). The use of rubber septa allows the
transfer of the solution under anoxic conditions.
Fig. 3 UV–Vis spectroelectrochemistry of β-CD-quercetin complex during electrolysis, showing the absorption
spectrum of semiquinone intermediate increase and decrease, and the absorption increase at 294 nm due to
benzofuranone product
6. The electrolysis starts and the chronoamperometric curve

(current vs. time) is recorded to monitor the process.
7. Collect the aliquots of electrolyzed solution during and at the
end of the electrolysis at different time intervals, correspond-
ing to the number of electrons involved in the process z = 1,
z = 2, etc., which can be calculated accordingly to Faraday’s
laws (see Note 5). Directly transfer the aliquots by syringe to
suitable vials and directly inject in the HPLC-DAD or
HPLC-MS/MS systems to analyze the products formed by
oxidative or reductive electrolysis.
8. Collect samples during and after electrolysis and store at
−18 °C for further analysis using chromatographic techniques
as GC–MS.
9. Stop electrolysis when current values reach a plateau. Calculate
the number of electrons participating in the redox process
from the charge consumed during the electrolysis using
Faraday’s laws (see Note 5).
10. Mount the working electrode used for cyclic voltammetry on
the cell. Record the cyclic voltammogram of the solution after
the electrolysis and compare with that of drug solution before
the electrolysis.
3.3 Identification 1. Evaporate an aliquot of the sample containing quercetin and its
of Products possible electrolysis products in the presence or absence of
cyclodextrin (Fig. 4).
3.3.1 GC-MS
2. Add 10 μL of 2,4-dihydroxybenzophenone (solution in isopro-
Chromatography
panol; internal standard IS1) to the extract and evaporate.
3. Derivatization procedure: Add 30 μL of the derivatization

agent BSTFA in 50 μL of AcOEt to the dried sample. The reac-
tion takes place at 60 °C for 30 min into closed glass vials
inserted in a water bath. Just before injection, add 10 μL of
hexadecane (solution in iso-octane; internal standard IS2) and
150 μL of AcOEt.
3.3.2 HPLC-DAD 1. Possibly you may need to dilute the aliquot of the sample con-
Chromatography taining quercetin and its possible electrolysis products with
water.
2. Insert the sample in autosampler vials and inject a suitable
amount in the chromatographic system automatically.
3. Acquire the chromatogram in the 200–650 nm range.
3.3.3 HPLC-MS/MS 1. Possibly you may need to dilute the aliquot of the sample con-
Chromatography taining quercetin and its possible electrolysis products with
water.
2. Insert the sample in autosampler vials and inject a suitable
amount in the chromatographic system automatically.
3. Acquire the chromatogram in unsupervised, untargeted tan-
dem mass spectrometric acquisition mode.
4 Notes
1. The linear dependence of Ip on the square root of scan rate is

characteristic for a diffusion-controlled electrode process.
2. Figure 2 shows that cyclic voltammograms of quercetin-2HP-
β-CD complex and quercetin do not differ neither in Ep nor in
Ip indicating that all electroactive sites of quercetin encapsulated
in the cavity are available for oxidation.
3. The oxidation waves belonging to the oxidation of non-
dissociated drug complexed by cyclodextrin, its dissociated
form, and dianionic form are labeled in cyclic voltammograms
recorded at different pH.
4. Electrochemical cleaning of the cell: The cell is washed by ace-
tonitrile; then, 0.1 M tetrabutylammonium h exafluorophosphate
in acetonitrile is inserted into the cell. A potential of −1.9 V is
applied for 100 s. The cell is then washed carefully by acetoni-
trile and acetone.
5. The charge Q in coulombs consumed during the electrolysis
will give information about the number of electrons participat-
ing in the redox process. It is calculated using Eq. (2):
Q = mzF / M (2)
Fig. 4 Gas chromatogram of solution after the oxidative electrolysis of quercetin in the presence (A) and
absence (B) of β-CD. Quercetin and intermediates of oxidation were stabilized in cyclodextrin cavity while the
solution of free quercetin was completely oxidized
where m is the weight of complex in g, z is the number of partici-

pating electrons, F is Faradaic constant 96,485 C/mol, and M
is the molecular mass of complex in g/mol.
Acknowledgments
Czech Science Foundation (project no. 19-03160S) is

acknowledged.
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Chapter 21
A Differential Scanning Calorimetry (DSC) Experimental

Protocol for Evaluating the Modified Thermotropic
Behavior of Liposomes with Incorporated Guest Molecules
Maria Chountoulesi, Nikolaos Naziris, Thomas Mavromoustakos,
and Costas Demetzos
Abstract
Differential scanning calorimetry (DSC) is a well-established technique, suitable to monitor the interac-
tions that may take place among the drug delivery systems of liposomes and the potential bioactive mole-
cules that are incorporated inside them. Moreover, the DSC technique is considered to be a useful tool to
characterize the thermal behavior of lipidic bilayers in the absence and presence of drugs and to highlight
parameters, such as the cooperativity between the lipids and the guest molecules (i.e. drugs, polymers,
dendrimers), providing also a prediction of the behavior of potential future drug delivery liposomal plat-
forms. In this study, a protocol for DSC measurements on liposomal systems with incorporated guest
molecules is described.
Key words Differential scanning calorimetry, Liposomes, Lipid bilayers, Guest molecules, Chimeric,
Thermal behavior
1 Introduction
Vesicular phospholipid bilayers are easily prepared and have been

proven to be very useful models toward the simulation of cell
membranes and the performance of biophysical experiments,
regarding the effects of the incorporation of guest molecules into
membranes. The aforementioned guest molecules can be drugs,
bioactive agents, or biocompatible molecules such as polymers and
dendrimers [1–5]. DSC is characterized as a fast, diagnostic, and
relatively inexpensive technique that is able to highlight the ther-
motropic properties of lipidic membranes in the presence of incor-
porated guest molecules, screen the interactions that are taking
place between them, and investigate the potential effects that may
be caused by the incorporation of additives [6]. Furthermore, the
299
300 Maria Chountoulesi et al.
combination of DSC with other methods, such as molecular

dynamic simulations, Raman spectroscopy, and X-ray diffraction,
can reveal valuable information, such as the conformational
changes of the molecule inserted in the core of the lipidic core
[1–5, 7, 8]. Apart from the model lipid bilayers, the DSC tech-
nique is considered to be a useful method for the thermal evalua-
tion of drug delivery nanosystems, consisted of lipid bilayers, such
as liposomes. DSC provides significant information, such as mate-
rial transitions and metastable phases that are in strict correlation
with the functionality of nanosystems, as well as with their drug-
release kinetic profile and as a result with the pharmacokinetics and
bioavailability of their content drugs. DSC can play an important
role in the design and formulation process of liposomal drug deliv-
ery systems and subsequently the production and scale-up process
of safe and effective nanomedicines, eventually increasing their
translational bench-to-clinic efficacy [9, 10].
When liposomal nanosystems are studied with the DSC tech-
nique, apart from their incorporated drug content, there is also a
great variety of available biomaterials that can be incorporated into
the liposomal formulation as “guest molecules” and affect the
thermotropic behavior of the liposomal lipidic bilayer [11–15].
Specifically, in order for advanced chimeric liposomal systems to be
prepared, phospholipids are combined with other different-in-
nature biomaterials, such as block copolymers, or even whole dif-
ferent nanosystems, such as dendrimers or nanoparticles, are
loaded into liposomes [16]. Chimeric liposomes are classified as
innovative, next-generation nanosystems with promising therapeu-
tic applications. DSC may provide answers on crucial queries
regarding their properties, such as the cooperativity of the various-
in-nature incorporated materials, which is a key parameter, strictly
correlated with their functionality.
The herein presented DSC protocol report has been obtained
through a careful study of the literature referring to lipid bilayers or
liposomes with incorporated small molecules, such as drugs [1–5,
7, 8, 17, 18], as well as to advanced chimeric liposomal bilayers and
systems that incorporate a variety of different guest biomaterials,
such as polymers, dendrimers, and carbon nanotubes [11–15, 19].
2 Materials
2.1 Preparation of Prepare the different liposomal formulations by using the thin-film
Liposomes hydration method (see Note 1) as follows:
with Incorporated
1. Weight carefully the appropriate amounts of the chosen lipid
Guest Molecules
and the examined guest molecule, in order to achieve the
desired lipid:guest molecule molar ratio.
DSC on Liposomes and Bilayers Incorporating Drugs and Biomaterials 301
2. Dissolve the weighted amounts of the lipid and the examined

guest molecule in organic solvent, for example chloroform or
chloroform/methanol (9:1 v/v) (see Note 2), transfer into a
round flask, and connect it to a rotary evaporator.
3. Apply vacuum, until a mixed thin film is formed by slow removal
of the solvent at 50 °C for 2 h.
4. The mixed lipidic films should be maintained under vacuum for
at least 24 h in a desiccator to remove traces of the solvent.
5. Hydrate the mixed films in water solvent (see Note 3), by slowly
stirring for 1 h in a water bath and by heating above the phase
transition of the used lipid. The concentration of the lipid
should be 30 mg mL−1, in order to achieve liposomal bilayers.
In the cases of lipidic bilayers with incorporated guest mole-
cules, follow only steps 1–4.
6. The resultant formulations (multilamellar vesicles, MLVs) can
be subjected to sonication cycles before their use, until unila-
mellar vesicles (SUVs) be achieved (see Note 4). After the size
reduction, liposomes should be allowed to anneal for 30 min
before their use.
3 Methods
3.1 Differential 1. Use sealed stainless steel crucibles (for example aluminum) with
Scanning Calorimetry O-ring as sample holders, as well as a crucible with water or buf-
(DSC) Measurements fer as reference.
2. Prepare the samples into the crucibles:
(a) In the case of pre-prepared liposomes with incorporated
guest molecules: taking into consideration the lipid con-
centration of the liposomal dispersion (30 mg mL−1), use
directly the appropriate volume of the dispersion, in order
to achieve in the crucible a final quantity of 4–7 mg lipid.
Seal the crucible.
(b) In the case of lipidic bilayers with incorporated guest mol-
ecules: weight 4–7 mg of the dry lipidic bilayer directly in
the crucible and hydrate/disperse it with the chosen
hydration medium, in the appropriate concentration
(see Notes 3, 5 and 6). Seal the crucible and subsequently
vortex for 5 min, so MLVs can be achieved. The sample
should be prepared 30 min before the DSC measurement,
in order to be efficiently equilibrated before the
measurement.
3. Calibrate the DSC instrument. Pure indium with main transi-
tion temperature (Tm) at 156.6 °C is usually used as a standard
sample for the calibration of the instrument.
4. Choose the appropriate temperature scanning range and the

scanning rate (see Notes 7 and 8).
5. Prior to measurements the crucibles should be subjected to a
temperature over the transition of the used lipid, in order to
ensure efficient equilibration.
6. Before each heating cycle, the samples should be equilibrated at
a constant temperature (isotherm cycle at the starting tempera-
ture for 5–10 min) (see Note 9).
7. At least two heating-cooling cycles should be carried out, in
order to ensure good reproducibility of the data and obtain
identical thermograms (see Note 10).
8. Analyze the obtained calorimetric data [characteristic transition
temperatures Tonset,m/s and Tm/s (°C), enthalpy changes ΔHm/s
(kJ/mol) (see Note 11), and widths at half-peak height of the Cp
Fig. 1 Typical DSC thermogram, describing the characteristic thermodynamic parameters provided by the DSC
technique. Adapted from Ref. [9]. The same parameters apply for the proceeding of small endothermic event
between 45 and 50 °C
profiles ΔT1/2,m/s (°C) (see Note 12) of both the main transition
(m) and the pretransition (s) of the lipid] (Fig. 1), by using the
respective software of the DSC instrumentation (see Note 13).
9. Correlate the obtained calorimetric data with the guest molecule-

lipid interactions (for example the effect on the mobility of the
polar groups or the acyl chains of the lipids), the potential per-
turbation of the guest molecule in the membrane or not, and
the resultant cooperativity of the whole system. Use this infor-
mation to get details of the thermal effects of the drug mole-
cules in the lipid bilayers. These thermal effects may also be
related to its release profile and its ADME (absorption, distri-
bution, metabolism, and excretion) properties (see Note 14).
4 Notes
1. The thin-film hydration method is a technique capable of pro-

ducing self-assembled liposomal nanostructures by lipids hav-
ing a high phase transition temperature [11].
2. In the cases of hydrophilic and amphiphilic guest molecules,
such as dendrimers or amphiphilic block copolymers, dissolve
the lipid-molecule mixture in chloroform/methanol (9:1 v/v)
[11], while in the cases of molecules with higher grade of
hydrophobicity dissolve them in chloroform only [4, 5, 17].
3. Examples of usually used water solvents are the HPLC-grade
water and the phosphate buffer saline (PBS). The choice of
each solvent as a hydration medium depends on the preferred
pH value and/or the ionic strength. The DSC measurement of
the same sample can be repeated in different hydration/disper-
sion media in order to evaluate the effect of the above men-
tioned parameters on the thermotropic behavior. For example,
Kyrili et al. [20] used both PBS with pH = 7.4 and citrate buf-
fer with pH = 4.0 as hydration media, in order to investigate
the effect of the environmental pH on the thermotropic behav-
ior of hydrogenated soy phosphatidylcholine (HSPC) bilayers
with incorporated pH-responsive block copolymer.
4. The protocol of the sample preparation is found to strictly affect
the obtained thermograms. More analytically, depending on the
different preparation protocol, vehicles with different sizes can
be acquired, which may alter the thermodynamic parameters.
For example, Chiu et al. [21] described that SUVs can produce
a lower resolution peak than MLVs, accompanied with a reduc-
tion in cooperativity due to the smaller radius of SUVs over
MLVs, resulting in a less ordered orientation, which increases
the free motion of the hydrocarbon chains. This decrease in
cooperativity, reflected by a slightly broader endothermic peak,
is also due to SUV fusion to large unilamellar vesicles (LUVs)

upon heating, resulting in size inhomogeneity.
5. The grade of hydration can play a crucial role at the calorimet-
ric results. Most frequently, the lipidic bilayers are fully
hydrated [4, 5, 8, 18–20] that corresponds to 50 wt% hydra-
tion medium or 0.28 mmol of water and 7 × 10−3 mmol of
phospholipid or water:phospholipid 40:1 molar ratio [22]. In
the case of using the lipid bilayers as dry material, being sealed
into the crucibles without hydration medium, but also with no
precaution taken of preventing hydration, then this sample can
be called “partially hydrated sample” (this sample is found to
absorb 2.1% w/w water) and presents significant differences at
the obtained DSC data [22]. For example, in the case of
DPPC bilayers, the partially hydrated sample presented
absence of pretransition, due to the different organization of
the polar head groups in DPPC molecules after heating and
cooling cycles, while the fully hydrated sample presented the
reversible pretransition before its main transition [22].
6. In the case of hydrophilic guests, these type of molecules can
also be dissolved into the aqueous hydration medium, rather
than be incorporated into the membrane at the stage of bilayer
preparation. Furthermore, in the case of studying the perturba-
tion ability of a guest molecule, it is preferred to carry on DSC
measurements on two types of bilayers, prepared by two differ-
ent preparation protocols (the drug is provided outside during
the hydration step or included inside during the preparation of
the bilayer [17]), in order to evaluate the impact of the method
of incorporation inside the membranes. For example, Liossi
et al. [17] prepared bilayers with incorporated irbesartan drug
in pure form or complexed with HP-β-CD cyclodextrin (as
described in Subheading 2.1) on the one hand and on the other
hand the irbesartan–HP-β-CD complex was also added to the
aqueous phase of readily formed lipidic MLV dispersions.
7. The scanning temperature range should certainly include the
transition phenomena (main transition and pretransition) of
the used lipid. More specifically, it should be used at a tempera-
ture starting about 20 °C prior to the transition temperature of
the phospholipid employed and ending at about 20 °C above
the transition temperature of the phospholipid employed [23].
8. The choice of scan rate is a critical parameter that should be
carefully investigated before each experimental procedure.
Sometimes, trying different scanning rates is required in order
to select the most appropriate one that is able to reveal all the
secondary transitions that are taking place. After all, the
presence of foreign molecules is supposed to alter the calori-
metric profile of the lipid at an unknown manner. A scanning
rate of 2.5–5.0 °C min−1 is usually used in the cases of lipo-
somes or lipidic bilayers with incorporated foreign molecules

or other materials [4, 5, 11, 19].
9. This preliminary isotherm scan, running at temperatures
before the unfolding transition begins, minimizes baseline
artifacts that can be induced by the thermal shock involved in
the loading of the sample or reference cells [24].
10. Repeating the scanning cycles is necessary, in order to investi-
gate the presence or the absence of the reversibility of the phe-
nomena. Usually, the test for reversibility is to perform two
DSC scans and check that the second scan gives all of the
endotherm observed in the first scan [25].
11. ΔΗ reflects the total heat energy uptake by the sample under-
going the transition, depending on the amount of sample
present in the active volume of the DSC cell, and should be
normalized according to the sample size [24].
12. The ΔΤ1/2 reflects the sharpness of the curve and is correlated
with the cooperativity between the different materials of the
bilayer. In the presence of foreign molecules, an increase of ΔΤ1/2
possibly implies decrease of the cooperativity.
13. The second heating and cooling run are taken into account as
presenting results.
14. Three representative literature case studies, including exam-
ples of drugs, cyclodextrin complexes, polymers and den-
drimers incorporated into lipidic bilayers, have been chosen, in
order to clarify how the obtained DSC data can be explained:
(a) In the first presented example, Liossi et al. [17] used DSC
technique in order to investigate the interactions of irbe-
sartan drug and irbesartan–2-hydroxypropyl-β-
cyclodextrin (HP-β-CD) complex with dipalmitoyl
phosphatidylcholine (DPPC) lipidic bilayers by applying
two different preparation protocols (see Note 6). The DSC
results are presented in Fig. 2 and Table 1. Analytically, the
presence of irbesartan drug alone resulted in a reduction
of the Tm, narrowing of the transition width of the main
phase transition, and abolishment of the pre-transition, while
it did not affect ΔΗm considerably. These data indicate a
strong effect on the head-group region of lipid bilayers.
Contrariwise in the case of HP-β-CD incorporation, both
the pre-transition and the main transition are lowered by a
few degrees, indicating a smaller perturbation and a
decrease of the packing density of the lipids, but not so
intensively. In combination with SAXS and solid-state
NMR spectroscopy results, DSC results showed that the
HP-β-CD sits on the liposomal surface. In addition, the
pure drug exerted significant differences on the membrane
internalization compared to the encapsulated drug into
the HP-β-CD, as reflected by the different calorimetric
data and the combinational biophysical experiments, pro-
Fig. 2 DSC scans of (a) pure DPPC bilayers; (b) DPPC/irbesartan bilayers (80:20);
(c) DPPC/HP-β-CD bilayers (80:20); (d) DPPC MLV dispersion with [irbesartan/
HP-β-CD] added (80:20); and (e) DPPC/[irbesartan/HP-β-CD] bilayers (80:20).
Adapted from Ref. [17]
Table 1
Thermal parameters for different DPPC samples studied
Pre-transition state Transition state
Tm
Samples ΔΗ (J/g) Tpre (°C) ΔΗ (J/g) (°C)
DPPC 6.3 35 41.8 41.2
DPPC/IRB (80:20) – – 40.8 39.7
DPPC/HP-β-CD (80:20) 3.9 33 34.0 39.3
DPPC/[IRB–HP-β-CD] – – 38.2 39.3
complex added (80:20)
DPPC/[IRB–HP-β-CD] – – 41.0 39.8
(80:20)
Adapted from Ref. [17]
viding significant information for the establishment of

ADME profile of the studied drug.
(b) In the second presented example, Pippa et al. [12] devel-
oped thermoresponsive chimeric liposomes by incorporat-
ing the two different forms of the thermoresponsive
amphiphilic homopolymer and more specifically C12H25-
poly(N-isopropylacrylamide)-COOH (C12H25-PNIPAM-
COOH) into DPPC liposomes, while the hydrophobic
drug indomethacin was also incorporated.
a
f.
e.
d.
Heat flow/mW, endothermic
c.
b.
a.
22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56
Temperature / ºC
b f.
e.
d.
c.
Heat flow/mW, endothermic
b.
a.
24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56
Temperature / ºC
Fig. 3 (A) DSC heating scans of DPPC:PNIPAM 1: (a) 9:0, (b) 9:0.1, (c) 9:0.5, (d) 9:1, (e) 9:2, and (f) 9:3 molar
ratio chimeric liposomes. (B) DSC heating scans of DPPC:PNIPAM 2: (a) 9:0, (b) 9:0.1, (c) 9:0.5, (d) 9:1, (e)
9:2, and (f) 9:3 molar ratio chimeric liposomes. The limits for the calculation of thermotropic parameters are
from 25 °C to 45 °C. Adapted from Ref. [12]
s long as the DSC results (Fig. 3), the incorporation of

A
the first form of polymer (called PNIPAM 1) (20 kDa) in
DPPC liposomes affected only the specific enthalpy ΔHm of
the main transition, reflecting an alteration at the mobility
of the lipid polar head groups. The main transition tem-
perature Tm was slightly reduced, while the system
cooperativity was reduced (increase of ΔΤ1/2) at a
concentration-dependent manner. Moreover, the incorpo-

ration of highest polymer amounts caused the appearance
of new peaks, indicating the creation of new metastable
phases, as a result of lateral phase separation into PNIPAM
1-rich and PNIPAM 1-poor domains. Taking into account
the lack of sharp transitions and the LCST of the poly-
meric guest, authors concluded that the PNIPAM 1 is not
uniformly distributed in the DPPC liposomal membranes.
Taking into consideration the amphiphilic character of the
polymeric guest and the hysteresis on cooling (of ~2 °C)
that was observed, the authors suggested the formation of
an interdigitated phase. The incorporation of the second
form of polymer (called PNIPAM 2) (7 kDa) affected the
thermotropic behavior of the liposomal bilayers in a differ-
ent way, indicating that the characteristics of the guest,
such as its composition and its molecular weight, can play
a key role in the thermal behavior of the final liposomal
system. As long as the loading of the indomethacin, the
presence of the aforementioned metastable phases due to
the PNIPAM-1, as revealed by DSC, was found to act as a
promoter, resulting in a burst drug release profile, due to
the inhomogeneous drug distribution inside the chimeric
liposomal membrane.
(c) In the last presented example, Ionov et al. [26] incorpo-
rated cationic phosphorus-containing dendrimers (CPDs)
into liposomes. This category of dendrimers can provide
special surface properties that are strictly associated with
the pharmacokinetic profile of the drug delivery system.
More specifically, they used DSC in order to investigate
the thermotropic behavior of chimeric liposomes, when
generation 3 (G3) and generation 4 (G4) CPDs were
incorporated into uncharged DMPC and negatively
charged DMPC:DPPG membranes. Authors stated that
the information obtained from this DSC study can be use-
ful toward the rational design of new drug carriers com-
bining liposomal and dendrimeric technology. Regarding
the results from DMPC membrane (Fig. 4), both CPDs
caused concentration-dependent alterations of the pretran-
sition specific enthalpy ΔΗm, while the other calorimetric
parameters of the pretransition remained unaltered, reflect-
ing interactions of the positively charged dendrimer end
groups with the polar head groups of the phospholipid
membranes but not with the lipidic chains.
I e
Heat flow, Endotherm

c
10 12 14 16 18 20 22 24 26 28 30 32 ºC
II e
d
Heat flow, Endotherm
Vh= 2oC/min
10 12 14 16 18 20 22 24 26 28 30 32 ºC
Fig. 4 DSC thermograms of fully hydrated DMPC lipid bilayers with varying
amounts (a) 0%, (b) 2, (c) 5%, (d) 10%, (e) 20% (molar) of CPDs, I—G3; II—
G4. Adapted from Ref. [26]
ontrariwise, in negatively charged DMPC:DPPG mem-

C
branes (Fig. 5) there was an increase of ΔT1/2, implying a
reduction of the cooperativity of the materials due to the
reduction of the membrane rigidity, which was more
intense in the presence of CPD G3 rather than CPD G4.
Although a reduction of cooperativity had taken place, no
phase separation was observed even at high CPD concen-
tration. The observed concentration-dependent increase
of the fluidity of the DMPC:DPPG bilayer can be corre-
lated with the negative charge of the bilayer, due to stron-
ger dendrimer–lipid interaction via electrostatic forces.
_
d
Heat flow, Endothem

b
a
10 12 14 16 18 20 22 24 26 28 30 32 ºC
=
d
Heat flow, Endothem
c
b
a
Vh = 2°C/min
10 12 14 16 18 20 22 24 26 28 30 32 ºC
Fig. 5 DSC thermograms of fully hydrated DMPC/DPPG (97:3 molar ratio) lipid
bilayers with varying amounts: (a) 0%, (b) 2, (c) 5%, (d) 10%, (e) 20% (molar)
of CPDs, I—G3; II—G4. Adapted from Ref. [26]
Acknowledgments
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Chapter 22
Applications of NMR in Drug:Cyclodextrin Complexes

Dimitrios Ntountaniotis, Georgios Leonis, Eirini Christodoulou,
Abstract
NMR spectroscopy is an effective technique, applicable for studying bioactive materials or drug delivery
systems in order to obtain comprehensive details related to structural and dynamic characteristics at atomic
resolution. The applications of NMR spectroscopy have been increased considerably as a result of the
combination of advancement in technological NMR instrumentation and scientific knowledge. This chap-
ter is dedicated to highlight the applications of NMR spectroscopy in drug:cyclodextrin complexes using
both liquid- and solid-state NMR spectroscopy.
Key words Nuclear magnetic resonance, NMR spectroscopy for liquids and solids, Cyclodextrins,
Structural properties, Drug delivery systems
1 Introduction
Nuclear magnetic resonance (NMR) spectroscopy has been a key

weapon in the armamentarium of the scientific society and industry
in order to shed light on structural details and dynamic character-
istics at atomic resolution of various bioactive systems and particu-
larly drug delivery systems [1]. NMR is an efficacious tool, which
enables scientists to obtain compelling evidence on crucial ques-
tions of molecular biology [2, 3]. Researchers make full use of
variable NMR experiments and derive benefit from a variety of
valuable information of molecular structure and molecular interac-
tions. In this context, NMR works as a compass to orientate scien-
tists in the maze of a complex set of information of a biological
system. A common utility of NMR is to provide all the appropriate
data to achieve a molecular structure’s elucidation. This procedure
is a result of a combinatorial approach of NMR experiments and is
often a quite intricate project. Another example of NMR applica-
tion is to validate ligand binding or to distinguish and classify
ligands in mixtures of test compounds [1]. Hence, it is apparent
313
314 Dimitrios Ntountaniotis et al.
that each NMR technique is applied with the view of resolving a

variety of distinct biological issues.
In an attempt to approach and comprehend different issues
related to drug:cyclodextrin (CD) complexation, a wide range of
NMR techniques for liquids and solids have been applied. Of these
two, NMR for liquids is the most often applied, while solid-state
(ss) NMR is the latest technologically evolving.
High-resolution NMR for liquids: One significant feature of
liquid NMR spectroscopy, and specifically 1H NMR spectroscopy,
is that it can provide quantitative information through the mea-
surement of the intensities of the peaks that compose the NMR
spectrum [4–6]. For this reason, NMR spectroscopy has been used
across a great number of studies in order to obtain quantitative
information for several pharmaceutical systems [6–9].
In addition, liquid NMR spectroscopy provides the appropri-
ate environment to study CDs, since the driving force for drug:CD
complexation is of solvophobic nature [10]. Nuclear Overhauser
effect (NOE) has been used as a key weapon to record quantitative
space correlations [11–13]. Thus, NOE can provide valuable infor-
mation about conformational details of complexes. Hydrogen
bonding (intramolecular as well as intermolecular) in CD com-
plexes can also be observed by NMR experiments [10, 14, 15].
NMR chemical shift titration is another suitable method to
obtain information on the association constants and stoichiometry
of the CD inclusion complexes as well as conformations of com-
plexes [16–19]. Chiral discrimination of pharmaceutical com-
pounds can be achieved using cyclodextrins and conclusions can be
derived from liquid NMR spectroscopy [20, 21]. The upshot of
this application is that a drug containing a chiral center can be
inserted into a cyclodextrin, to generate diastereoisomers that are
detectable by NMR spectroscopy.
An interesting conclusion (derived also by NMR) about Z or
E configuration was reported in case of indomethacin. This mole-
cule is found to be present in an E/Z mixture (whose stereoiso-
mers are of amide type). The stereoisomers interchange rapidly.
After complexation, NMR NOE experiments as well as molecular
mechanics calculations showed a preference to Z configuration
[16, 22].
Isotope labeling strategy (15N, 13C, 2H) is applicable mainly for
shedding light on the protein-ligand interactions. An example of
the utility of this strategy was reported in solution NMR studies of
a 42 KDa Escherichia coli maltose-binding protein/β-cyclodextrin
complex [23]. The hard task of reporting weak intermolecular
NOEs among many strong intramolecular ones is thus facilitated
by the isotope labeling.
NMR spectroscopy for solids: ssNMR is applicable in insoluble
molecules and finds wide scope of applications in molecular b iology.
NMR Techniques Applied to Drug: Cyclodextrin Complexation 315
Labeling experiments with 13C, 15N, 2H, and 19F enables scientists
to obtain valuable structural information.
Magic-angle spinning (MAS), oriented-sample NMR (OS
NMR), and cross polarization (CP) methods have been applied in
many studies [24–28].
Structural information via 2H NMR spectroscopy can be
obtained from membrane lipids with 2H-labeled acyl chains or
polar groups [29]. This idea is applicable also in order to study
drug:CD complexes inside liposomes. The results can be studied
combinatorially with electron crystallography and X-ray experi-
ments [29].
2D ssNMR experiments offer information for comprehending
details related to structural and dynamic characteristics. Vogt and
Strohmeier (2012) applied many 2D ssNMR for analysis of inclu-
sion in drug:cyclodextrin complexes [30]. For example, 2D
1
H − 19F CP-HETCOR experiment can be employed in order to
investigate close association between β-CD and the drug diflunisal
which has fluorine in its structure [30]. 2D 1H − 13C CP-HETCOR
is a similar experiment, but the heteronucleus is carbon and this
fact reduces the sensitivity and consequently longer acquisition
times are necessary [30]. The case of 31P nucleus is analogous to
19
F, as it offers enhanced NMR sensitivity [30].
Cyclodextrins (CDs) are a family of cyclic oligosaccharides,
and consist of five or more α-1,4-linked glycosidic bonds. Examples
of such molecules are α-CD (six α-1,4-linked glycosidic bonds),
β-CD (seven α-1,4-linked glycosidic bonds), and γ-CD (eight
α-1,4-linked glycosidic bonds). Their shape is toroidal (or cone
shaped), due to the absence of free rotation of the bonds linking
the glucose units. Each glucose unit has three hydroxyl groups,
two secondary connected at carbons 2 and 3 of the glucose unit
and one primary connected at carbon 6 (Fig. 1). Thus, a total
number of 21 hydroxyl groups are present in β-CD and their pres-
ence is mainly responsible for CD water solubility. The primary
hydroxyl groups are located in the narrow rim of the cone, while
secondary hydroxyl groups are located in the wide rim. On the
other hand, the interior of cyclodextrins is relatively hydrophobic
due to the presence of ether oxygens at the C-4 and hydrogens
attached at carbons C-3 and C-5, thus creating a cavity for the
entrapment of hydrophobic molecules. α-Cyclodextrin may typi-
cally complex low-molecular-weight molecules or compounds with
aliphatic side chains. β-Cyclodextrin can complex aromatic and
heterocyclic molecules.
CDs are used extensively for drug delivery since they can
entrap pharmaceutical molecules and protect them. For example, it
is well known that peptides suffer from several disadvantages like
chemical and enzymatic instability, poor absorption through bio-
logical membranes, rapid plasma clearance, and peculiar dose-
response curves. Cyclodextrins are used to eliminate the
31
12 11 10 9 8 7 6
H3C CH2 CH2 CH2 CH2 CH2 CH2 30 12 13 22 21
29
11 14 17
5CH2 2H
20
1
N
OSO3NA CH2 CH2 CH2 CH2 2
5 16 15 18 19
3N 24 N 23
1 2 3 4 4 O
9 HN N
6 25 27
sodium dodecyl sulfate N
26
7 8
Irbesartan
OH
HO
OH
OH HO
OH OH
≡
HO
HO
OH H OCH2CHCH2
HO
OH 4 6 H O
5
OH OH
O 2
HO 3 1 11
OH
H OH
OH
OH
OH
OH HO H O
7
OH
OH
2-HP-β-CD
O
N+ 1”’ 2” 4” 6” 8” 10” 12” 14” 16”
O O 1” 3” 5” 7” 9” 11” 13” 15”
2”’
O P O O 16’
1’ 3’ 5’ 7’ 9’ 11’ 13’ 15’
2 2’ 4’ 6’ 8’ 10’ 12’ 14’
O 1 3
O
DPPC
Fig. 1 SDS, irbesartan, 2-HP-β-CD, and DPPC
abovementioned problems of peptides. The pharmaceutical inter-

est of CDs extends also to enhance the solubility, chemical stability,
and bioavailability of poorly soluble drugs; to reduce the toxicity;
and to control the rate of release of some drugs. 2-Hydroxypropyl-
β-cyclodextrin (2-HP-β-CD—Fig. 1) has demonstrated improved
water solubility and is a biocompatible substance (more toxicologi-
cally favorable).
Micellar systems can solubilize poorly soluble drugs.
Consequently, it is evident that they increase bioavailability.
Therefore, micelles are used for drug delivery. Sodium dodecyl sul-
fate (SDS—Fig. 1) has a hydrocarbon tail and an anionic head
group. So SDS has amphiphilic properties, which allow this mole-

cule to form micelles. SDS is the most widely employed micelle-
forming detergent for laboratory procedures [31]. Charged
detergents (including SDS) are very useful in NMR studies, where
features of these micelles such as small size are advantageous [31].
2 Materials
2.1 General Practical 1. All samples for liquid NMR spectroscopy should be sufficiently
Aspects soluble in the respective deuterated solvent.
2. Usually, 5 mm tubes are used for the preparation of liquid NMR
samples. For different NMR instruments (e.g., 400, 500,
600 MHz), NMR tubes of different quality are selected as well.
In case the sample dissolves glass, a polytetrafluoroethylene
(PTFE) insert or a Kel-F (a fluorochemical product) tube is
used.
3. The researcher should take into consideration that in some
cases the cap may contaminate the sample. The cap could release
materials (for example when CDCl3 or DMSO-d6 is used). The
researcher should avoid this possibility by closing the tube with
Teflon tape and afterwards with the tube’s cap [32].
4. In case the sample is air sensitive, a vacuum or an inert atmo-
sphere is required. Special NMR tubes are commercially avail-
able. Rarely the removal of oxygen is necessary, so the sample
should be prepared under argon [32].
5. Samples for ssNMR spectroscopy are placed into rotors made
by ZrO2. The most common types of rotors are (a) 80 μL for
solids and (b) 50 and 12 μL for semisolids [32]. For the last
two, special inserts (made from Teflon or Kel-F) are used. The
usage of inserts is necessary, if the sample is soft or contains a
liquid.
6. Researchers should avoid contamination of the sample with skin
lipids (oils), which results in NMR signals under magic-angle
spinning (MAS) experiments.
2.2 Materials All materials for liquid NMR experiments were stored at 4 °C.
for Liquid NMR
1. D2Ο (99%+).
Experiments
2. Sodium dodecyl-d25 sulfate was 98% [CD3(CD2)11OSO3Na]
pure (Fig. 1).
2.3 Materials All materials for ssNMR experiments were stored at 4 °C.
for ssNMR
1. Irbesartan (IRB—Fig. 1) was kindly provided by Prof.
Experiments
M. Koupparis (National and Kapodistrian University of Athens).
2. La-dipalmitoyl-phosphatidylcholine >99% (TLC) (DPPC—Fig.

1).
3. Deuterium-depleted water (99.99 atom% 16O).
4. 2-HP-β-CD (average Mw ~1460).
3 Methods
3.1 Investigation 1. Obtain 1H NMR spectra of the samples: (a) Irbesartan dissolved
of the Structural in D2O; (b) 2-HP-β-CD dissolved in D2O D2O; (c) irbesartan
Properties dissolved in SDS micelles; (d) IRB complexed with 2-HP-β-ΟΗ
of Irbesartan and dissolved in D2O; (e) IRB complexed with 2-HP-β-ΟΗ and
and Irbesartan–2- dissolved in SDS micelles. Pulse sequences are provided in spec-
Hydroxypropyl-β- trometer libraries.
Cyclodextrin Complex 2. Dissolve 9.8 mg of the complex irbesartan–2-hydroxypropyl-β-
in Micelles cyclodextrin in D2O in order to obtain NMR spectra. Τhe pulse
sequences included in the libraries of pulse programs were used
in order to acquire 1D 1H and 13C NMR spectra.
3. For the preparation of 5 mM of irbesartan in 400 mM SDS-
d25/D2O, subject the mixture to sonication in order to provide
a transparent solution.
4. The preparation of the complex irbesartan–2-hydroxypropyl-β-
cyclodextrin is carried out during a freeze-drying procedure as
it is described in [33]. Specifically, the neutralization method is
applied for the preparation of IRB-2-HP-β-CD aqueous solu-
tions for freeze-drying in a molar ratio (irbesartan/2-HP-
β-CD) 1:2. Transfer 408 mg of 2-HP-β-CD and 60 mg of
irbesartan in a 100 μL beaker and suspend them with 50 mL of
water. Subsequently, add small amounts of ammonium hydrox-
ide under stirring, while pH is monitored until complete dis-
solution and pH adjustment to a value of 9–10. Then, freeze
the resulting solution at −80 °C and freeze-dry using a
Kryodos-50 model Telstar lyophilizer.
3.2 The Application 1. Fully hydrate DPPC to form multilamellar bilayers.

of ssNMR 2. Record a 13C cross polarization/magic-angle spinning (13C
Spectroscopy to Study CP/MAS) solid-state NMR spectra on a 600 MHz spectrom-
Drug:Membrane eter equipped with a 3.2 mm HX MAS probe. Pack approxi-
Interactions mately 20 mg of each sample tightly into a zirconia rotor and
spin at 5 kHz. The duration of the CP block in the CP/MAS
experiment should be 5 ms, repetition delay between scans
should be 2 s, and the number of scans 400. The 13C chemical
shift axis should be referenced to as tetramethylsilane.
For the sample DPPC/HP-β-CD (x = 0.20), record both
13
C CP/MAS and MAS spectra at a sample rotation frequency
of 15 kHz. Record the spectra on a 600 MHz spectrometer
equipped with a 4 mm HX MAS probe. Pack approximately

50 mg of the sample tightly into a zirconia rotor. Apply cross
polarization for 13C excitation using an 80% linear ramp. During
13
C detection, 100 kHz Spinal-64 proton decoupling should be
applied [34–36]. The number of scans in the experiment should
be 1000.
3. Dissolve weighted amounts of dry lipid and IRB powder in
chloroform in order to prepare DPPC and IRB stock solutions.
The drug concentration should be 20 mol%. Prepare the mix-
tures by appropriate amounts of drug in a pure or complexed
form with 2-HP-β-CD. Evaporate the organic DPPC/IRB or
DPPC/complex IRB–2-HP-β-CD at room temperature under
a gentle stream of nitrogen and thereafter place them under
vacuum for 12 h and consequently a thin lipid film at the bot-
tom of glass vials should be formed. The obtained mixtures
should be then fully hydrated (50% w/w deuterium-depleted
water) and this procedure will result in multilamellar vesicles
(MLVs). Alternatively, add complexed IRB–2-HP-β-CD com-
pounds to the aqueous phase of readily formed DPPC MLV
dispersions.
4. Perform 13C CP/MAS spectra at three temperatures (25, 35,
45 °C) to cover all mesomorphic states of DPPC bilayers.
4 Notes
4.1 Notes Related 1. Irbesartan’s alkyl chemical shifts are not modified significantly
to Subheading 3.1 when it is complexed in D2O while in micelles a clear and sig-
nificant downfield chemical shift is observed. Interestingly, the
quintet at ca 1.43 ppm is better resolved when the complex is in
micelles than in D2O. The same trend is observed with cyclo-
pentane ring. The differences in the aromatic region are very
interesting. Downfield, but also upfield, shifts are observed in
the micelle environment compared to that of D2O.
2. (a) The complexation of IRB with 2-HP-β-CD induces some
chemical shift changes to IRB. (b) The micelle environment
induces many chemical shift changes to IRB. (c) Protons 6a,
6b, 7a, 7b, 8a, 8b, 9a, 9b, 19, 20, 21, 22, and 28 are affected
most when IRB is complexed with 2-HP-β-CD and (d) when
the complex is transferred to SDS environment most of the
chemical shifts are affected significantly (Table 1, Figs. 2 and 3).
The 1H NMR spectra of (a) irbesartan dissolved in D2O; (b)
2-HP-β-CD dissolved in D2O; (c) irbesartan dissolved in SDS
micelles; (d) IRB complexed with 2-HP-β-CD and dissolved in
D2O; and (e) IRB complexed with 2-HP-β-CD in SDS micelles
are presented in Figs. 2 and 3.
Table 1
Chemical shifts of irbesartan alone or complexed with 2-HP-β-CD in D2O and in SDS micelles
IRB in SDS Complex IRB-2-HP- Complex IRB-2-HP-

Hydrogen IRB in D2O [ppm micelles [ppm β-CD in D2O [ppm β-CD in SDS micelles
atoms (multiplicity)] (multiplicity)] (multiplicity)] [ppm (multiplicity)]
31 0.78 (triplet) 0.84 (triplet) 0.76 (triplet) 0.86 (triplet)
30 1.25 (sextet) 1.32 (sextet) 1.23 (sextet) 1.32 (sextet)
29 1.43 (pentet) 1.52 (pentet) 1.41 (pentet) 1.54 (pentet)
6a, 6b 1.82 1.91 1.91–1.96 1.97
9a, 9b 1.93–1.99 1.97–2.01 (multiplet and (multiplet and
(multiplet and (multiplet and multiplet) multiplet)
multiplet) multiplet)
7a,7b 1.93–1.99 1.91, 2.03–2.06 1.77–2.02 (multiplet) 1.80, 1.98 (multiplet)
(multiplet) (multiplet)
8a, 8b 1.93–1.99 2.03–2.06 1.77–2.02 (multiplet) 1.80, 1.98 (multiplet)
(multiplet) (multiplet)
10 4.78 (singlet) 4.84 (triplet) 4.76 (singlet) 4.77 (singlet)
12/16 7.10 (doublet) 7.10 (doublet) 7.09 or 7.14 7.12 or 7.13 (doublet)
(doublet)
13/15 7.10 (doublet) 7.16 (doublet) 7.09, or 7.14 7.12 or 7.13 (doublet)
(doublet)
22 7.53–7.55 7.41 (doublet) 7.48 (multiplet) 7.39 (doublet)
(multiplet)
20 7.53–7.55 7.52 (triplet) 7.49 (multiplet) 7.46 (triplet)
(multiplet)
19 7.59 (doublet) 7.84 (doublet) 7.62 (doublet) 7.78 (doublet)
4.2 Notes Related 1. The peaks of 2-HP-β-CD are barely observable. The absence of
to Subheading 3.2 cross polarization between 2-HP-β-CD and phospholipid
(Figs. 4 and 5) DPPC presumably shows that the former molecule approaches
the surface of the lipid bilayers and that the distances between
the 1H and 13C nuclei of the two species are thus sufficiently
large or that the dynamics of the 2-HP-β-CD is sufficiently fast,
so that the 1H–13C (residual) dipolar interaction is practically
negligible. 13C MAS experiments lead to the conclusion that
this preposition as the peaks attributed to 2-HP-β-CD is emi-
nent at low temperatures.
2. The presence of the drug in the lipid bilayers does not modify
significantly DPPC’s carbon chemical shifts.
Fig. 2 Part of 1H NMR spectra (0.7–2.5 ppm) of (a) irbesartan dissolved in D2O,
(b) 2-HP-β-CD dissolved in D2O, (c) irbesartan dissolved in SDS micelles, (d) IRB
complexed with 2-HP-β-CD and dissolved in D2O, (e) IRB complexed with
2-HP-β-CD in SDS micelles
3. The observation of 13C NMR spectrum of the drug is a proof

that the butyl chain of irbesartan and its cyclopentyl ring are
embedded in the more flexible hydrophobic core of DPPC
bilayers.
4. After irbesartan complex is inserted into DPPC bilayers, signals
of the drug are very broad (CP experiments). On the contrary,
2-HP-β-CD is not observed giving the information that this
CD is localized on the surface of DPPC bilayers.
Fig. 3 Part of 1H NMR spectra (3.3–7.9 ppm) of (a) irbesartan dissolved in D2O,
(b) 2-HP-β-CD dissolved in D2O, (c) irbesartan dissolved in SDS micelles, (d)
IRB complexed with 2-HP-β-CD and dissolved in D2O, (e) IRB complexed with
2-HP-β-CD in SDS micelles
Acknowledgments
C2*** C1*** (CH2)*10,(CH2)**10

DPPC C2
C1
N(CH3)3
C15*,C15**
c1’,c1” x2 X4 C14*,C14** C3*,C3**
C3
C2*,C2* C16*,C16**
DPPC/irbesartan
80:20
Cirb430
25 20
Cirb4
Cirb31
CHP6
70 60
DPPC/2-HP-β-CD
80:20
DPPC/ complex irbesartan-2-HP-β-CD

80:20
DPPC/ complex irbesartan-2-HP-β-CD(added from outside)

80:20
190 170 150 130 90 80 70 60 50 40 30 20 10 (ppm)
Fig. 4 13C CP/MAS NMR spectra of five samples recorded at 45 °C
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Chapter 23
Construction of Peptide-Drug Conjugates for Selective

Targeting of Malignant Tumor Cells
Eirinaios I. Vrettos and Andreas G. Tzakos
Abstract
Cancer constitutes a major threat to humanity, while its incidence and mortality rates are increasing rapidly
worldwide. To tackle cancer, numerous strategies have been exploited, including the development of pep-
tide–drug conjugates (PDCs), which are considered an appealing approach to selectively populate malig-
nant tumors with toxic substances. The general architecture of a PDC usually includes three parts: the
tumor-targeting peptide, the cytotoxic drug, and the biodegradable linker. Due to the fact that peptides
possess fast renal clearance, affecting the bioavailability of the PDC, a nanodrug formation concept can be
exploited to ameliorate this pitfall. Herein, we present methodologies to develop PDCs, along with certain
basic principles governing such constructs. In addition, we highlight possible problems that may appear
during the synthesis of PDCs, as also solutions to overcome them.
Key words Cancer, Drug delivery, Bioconjugates, Peptides, Peptide-drug conjugates (PDCs),
Targeted drug delivery
1 Introduction
Cancer is a rapidly increasing global threat and the rate of appear-

ance is continuously growing. The 12.7 million cancer cases and
7.6 million deaths in 2008 have mounted up to 18.1 million cases
and 9.6 million deaths in 2018 [1, 2]. The most common types of
cancer are lung, breast, colorectal, and prostate, contributing to
12.3%, 12.3%, 10.6%, and 7.5% of the total number of new cases in
2018, respectively [1]. These statistics indicate that the current
therapeutics in oncology are not effective against the evolving
complexity of cancer, often due to their unspecific toxicity, reduced
solubility, permeability, and drug resistance of cancer cells.
Current cancer treatments usually include a combination of
surgery, radiation, immunotherapy, and chemotherapy.
Chemotherapeutic drugs utilized for this purpose are toxic in order
to kill cancer cells, inevitably affecting the healthy cells and there-
327
328 Eirinaios I. Vrettos and Andreas G. Tzakos
fore deteriorating the patient’s health. For instance, gemcitabine,

an anticancer agent used against a variety of solid tumors even in
the latter stages, shows higher toxicity for healthy cells, after long-
term administration, due to the drug resistance that cancer cells
develop [3]. An appealing strategy to surpass this hurdle is by
offering to chemotherapeutic drugs selective delivery to malignant
cells (Fig. 1).
Moreover, chemotherapy is often ineffective due to the unfa-
vorable ADME (absorption, distribution, metabolism, excretion)
properties of the utilized drugs, associated with their poor water
solubility, low bioavailability, and rapid metabolic inactivation.
Therefore, it is of interest to invest in enhancing the biological
profile of existing cytotoxic drugs by transforming them into tar-
geted chemotherapeutics. Along these lines, drug delivery formu-
lations that could improve drug potency and be specifically tailored
for every type of cancer are of ultimate interest.
Peptide-drug conjugates (PDCs) could serve as ideal candi-
dates for this purpose, as they fulfill the requested features.
Different PDCs can be constructed for each type of cancer and
conform with each patient’s needs (personalized medicines), due
to the fact that plentiful combinations can be used. The usual
building blocks of a PDC include the cytotoxic agent, the tumor-
targeting peptide, and the linker tethering them (Fig. 2). The effi-
cacy of a PDC is mainly associated with the cytotoxicity of the
utilized drug and the targeting efficacy of the peptide. The drug
should be highly potent, with a known mechanism of metabolism
and action, and should possess conjugatable functional groups.
The tumor-homing peptide should bind selectively and with high
Fig. 1 Conventional chemotherapy versus targeted chemotherapy. Black color = solid malignant tumor;
green = conventional untargeted cytotoxic agent; blue = targeted cytotoxic agent. Reprinted from [4] with
permission
Construction of Peptide-Drug Conjugates for Selective Targeting of Malignant Tumor Cells 329
Fig. 2 The general architecture of a peptide-drug conjugate
affinity to a certain receptor, overexpressed or uniquely expressed

on the surface of the targeted cells. Also, the peptide should pos-
sess conjugatable groups (usually lysine and cysteine), but the con-
jugation site should be carefully selected since it could lead even to
the total abolishment of its binding affinity/selectivity to the cer-
tain receptor. The linker should be selected rationally as it plays a
crucial role in the properties of the final assembly, especially in the
stability of the conjugate and the release rate of the drug. Notably,
an improper linker could demolish the overall activity of the
bioconjugate.
Peptides are considered as an inextricable part of the arma-
mentarium against cancer as they display high affinity and selectiv-
ity for certain receptors overexpressed on malignant tumor cells,
they possess low inherent toxicity, and they are facile to synthesize.
However, peptides also display a very short half-life during the
blood circulation mainly due to excretion by the kidneys, because
of their small size, and to a lesser extent caused by their susceptibil-
ity to proteases. As a result, PDCs display low bioavailability, ren-
dering them inefficient against cancer.
An intriguing approach for extending the half-lives of PDCs is
by conjugating them to nanoparticles [5]. For instance, gold
nanoparticles (AuNPs) have been utilized for enhancing the half-
life of certain PDCs from ~10 min, when administered alone, up to
~20 h when administrated as conjugates on AuNPS, while retain-
ing their cytotoxicity profiles [6]. In addition, certain PDCs may
self-assemble into supramolecular structures like nanofibers, nano-
tubes, and hydrogels [7], offering two functions: They can act as
nanocarriers to selectively deliver the drug to malignant tumor
cells and/or can enhance the pharmacokinetic properties of the
utilized drug [8].
Although the common syntheses of PDCs are conducted in a
rapid and facile manner, various problems may arise. The most
usual ones appear during the synthesis and/or the purification of
the tumor-homing peptide as summarized below:
1. The peptide might be difficult to synthesize, especially when

the target peptide consists of more than 30 amino acids. The
interactions of the side chains of the amino acids cause prob-
lems during the synthesis, as the new amino acids to be
attached require higher reaction times, and the final peptide is
usually impure. An effective way to surpass this hurdle is by
shortening the sequence and/or substituting the hydrophobic
residues. The aforementioned problem is usually encountered
during the manual solid-phase peptide synthesis; in some cases
it can be addressed by utilizing microwave-assisted synthesis.
2. The synthesized peptide might possess low aqueous solubility,
which renders its purification extremely hard. Insolubility
issues can be surpassed by shortening the sequence through
elimination of hydrophobic residues or by lengthening the
sequence through the addition of polar amino acids. Another
effective way to face the low solubility is by altering the C-/N-
terminus and/or substituting specific hydrophobic residues.
3. During the conjugation between the peptide and the drug or
the linker, some problems may arise, derived from the utilized
coupling reagents. For instance, HATU is known to react with
the N-terminus of the peptide and recently, its participation in
the formation of side products on the side chains of specific
peptide residues has been described [9].
In this chapter, we provide the procedure to assemble a peptide-
drug conjugate consisting of D-Lys6-GnRH (peptide) and gem-
citabine (anticancer agent), tethered via various bonds (ester,
amide, carbamate and oxime) derived from the utilization of differ-
ent linkers (succinyl, carbamate, and PEG-aminooxy). The utilized
peptide (D-Lys6-GnRH-II) is gonadotropin-releasing hormone
that binds selectively on type II GnRH-receptor (GnRH-R), which
is overexpressed in various cancer types including prostate, lung,
and breast [10]. D-Lys6-GnRH possesses a lysine that can be uti-
lized for orthogonal coupling in liquid phase with the linker and
consequently the drug. Gemcitabine belongs to the antimetabolite
family of anticancer agents and is active against various solid malignant
tumors including ovarian, prostate, lung, breast, and pancreatic.
Gemcitabine possesses three possible conjugation sites: (1) a
primary –OH that is the site of intracellular phosphorylation lead-
ing to the active metabolites difluorodeoxycytidine diphosphate
(dFdCDP) and difluorodeoxycytidine triphosphate (dFdCTP), (2)
a secondary –OH, and (3) a primary –NH2 which mediates its met-
abolic inactivation toward 2′,2′-difluorodeoxyuridine (dFdU) via
the cytidine deaminase [11]. Herein, it will be described the syn-
thetic procedure concerning the primary or the secondary –OH,
where different linkers can be incorporated.
We present the synthesis of three PDCs with linkers consisting

of different lengths, bonds, and stability (they include various mix-
tures between ester, amide, carbamate, and oxime bonds), focus-
ing on the description of the synthetic procedures to obtain them.
2 Materials and Equipment
Materials used are commercially available and of the best quality,

unless otherwise stated.
2.1 Synthesis 1. D-Lys6-GnRH (synthesized via the classical solid-phase pep-

of PDCs tide synthesis on Rink amide resin, as previously described
2.1.1 Synthesis of PDCs
[12]) (see Note 1).
Using Succinic Acid 2. Gemcitabine base (gemcitabine is Boc-protected on the –NH2
as Linker (Ester and the secondary –OH prior to usage, as previously described
and Amide Bonds) [13]) (see Note 1).
3. O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium
hexafluorophosphate (HATU).
4. 1-hydroxybenzotriazole (HOBt).
5. Succinic anhydride.
6. Triisopropylsilane (TIS).
7. N,N-diisopropylethylamine (DIPEA).
8. Trifluoroacetic acid (TFA).
9. Anhydrous N,N-dimethylformamide (DMF).
10. Anhydrous dichloromethane (CH2Cl2).
2.1.2 Synthesis of PDCs 1. D-Lys6-GnRH (synthesized via the classical solid-phase pep-
Using Carbamate Bond tide synthesis on Rink amide resin, as previously described
in the Linker [12]).
2. Gemcitabine base (gemcitabine is Boc-protected on the –NH2
and the secondary –OH prior to usage, as previously described
[13]).
3. Bis(4-nitrophenyl)carbonate.
8. Anhydrous acetonitrile.
2.1.3 Synthesis of PDCs 1. D-Lys6-GnRH (synthesized via the classical step-by-step solid-
Using Aminooxy-PEG4- phase peptide synthesis on Rink amide resin, as previously
CH2CO2H as Linker (Amide described [12]) (see Note 1).
and Bond)
2. Gemcitabine base (gemcitabine is Boc-protected on the –NH2

and the primary –OH prior to usage, as previously described
[13]) (see Note 1).
3. 1-hydroxybenzotriazole (HOBt).
4. O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium
hexafluorophosphate (HATU).
5. t-Boc-Aminooxy-PEG4-CH2CO2H.
6. 4-dimethylaminopyridine (DMAP).
10. Fmoc-Ser(tBu)-OH.
11. Piperidine.
12. Imidazole.
13. Sodium periodate (NaIO4).
14. Ethylene glycol.
16. Anhydrous acetonitrile.
2.2 Purification 1. Column chromatography performed using SiO2.

and Characterization 2. Thin-layer chromatography (TLC) plates, silica gel coated
of Intermediates with fluorescent indicator F254 (see Note 2).
and Final Conjugates
3. Dichloromethane, analytical grade.
2.2.1 Column 4. Methanol, analytical grade.
Purification
5. Acetone, analytical grade.
2.2.2 RP-HPLC
Purification 1. HPLC solvent delivery system with binary gradient capability
and a UV detector.
2. Reversed-phase Jupiter® C18 column (25 cm × 21.2 mm) at a
flow rate of 20 mL/min and 10 μm particle size as stationary
phase.
3. Sample filters, 0.2 μm pore size.
4. Acetonitrile, water, and trifluoroacetic acid (TFA), HPLC
grade.
5. Mobile phase A: TFA 0.1% (v/v) in water.
6. Mobile phase B: TFA 0.1% (v/v) in acetonitrile.
2.2.3 Characterization 1. Acetonitrile, water, and formic acid (LC-MS grade).

with Mass Spectrometry
2. Mass spectrometer instrumentation with C18 column
and/or NMR Spectroscopy
100 mm × 2.1 mm, 2.6 μm, with ProGuard column 2.1 mm.
3. Mobile phase A: Water containing 0.1% (v/v) formic acid.
4. Mobile phase B: Acetonitrile containing 0.1% (v/v) formic

acid.
5. NMR spectrometer for 1H and 13C spectroscopy and the cor-
responding software.
6. DMSO-d6 (>99.8%) (see Note 3).
3 Methods
3.1 Synthesis of PDCs 1. Dissolve Boc-protected gemcitabine (8 eq) and succinic anhy-
dride (1 eq) in CH2Cl2.
3.1.1 Synthesis of PDCs
Using Succinic Acid 2. Add DIPEA (10 eq) at 0 °C.
as Linker (Ester and Amide 3. Stir the reaction for 12 h at room temperature.
Bonds) (Scheme 1) 4. Distill the solvent under reduced pressure.
Preparation 5. Purify the residue by RP-HPLC to receive gemcitabine-linker
of Gemcitabine-Linker (diBoc-Gemcitabine-Hemisuccinate).
(diBoc-Gemcitabine-
6. Characterize the compound with 1H/13C NMR spectroscopy
Hemisuccinate)
and mass spectrometry.
Preparation of the Final 1. Dissolve the gemcitabine linker (1 eq), HATU (1 eq) in anhy-
Conjugate (Gemcitabine- drous DMF under inert atmosphere (see Note 4).
Hemisuccinate-D- 2. Add DIPEA (2 eq) at 0 °C.
Lys6- GnRH)
3. After 10 min, add D-Lys6-GnRH (1 eq) dissolved in anhy-
drous DMF dropwise and stir the reaction for 12 h at room
temperature.
4. Distill the solvent.
5. Dissolve the residue in TFA-H2O-TIS (95:2.5:2.5) and stir for
30 min (see Note 5).
7. Purify the residue with RP-HPLC and lyophilize the peak to
receive the final conjugate in pure form.
8. Characterize the compound with mass spectrometry (see
Note 6).
3.1.2 Synthesis of PDCs 1. Dissolve Boc-protected gemcitabine (1 eq) in anhydrous

Using Carbamate Bond acetonitrile under an inert atmosphere.
in the Linker (Scheme 2)
2. Add DIPEA (40 eq) at 0 °C.
Preparation 3. After 5 min, add a solution of bis(4-nitrophenyl)carbonate
of Gemcitabine-Linker (8 eq) in anhydrous acetonitrile dropwise and stir at room
(diBoc-Gemcitabine-Bis temperature for 4 h.
(4-Nitrophenyl)Carbonate)
Scheme 1 Synthesis of PDCs using carbamate bond as linker. Reagents and conditions: (a) Succinic anhy-
dride, DIPEA, CH2Cl2, rt., 12 h; (b) D-Lys6-GnRH, HATU, DIPEA, DMF, rt., 12 h; (c)TFA-H2O-TIS (95:2.5:2.5), rt.,
30 min
5. Purify the residue via column chromatography (eluent:

CH2Cl2/acetone 9/1) to afford gemcitabine linker (diBoc-
gemcitabine-bis(4-nitrophenyl)carbonate).
6. Characterize with 1H and 13
C NMR spectroscopy and mass
spectrometry.
Preparation of the Final 1. Dissolve D-Lys6-GnRH (1 eq) in anhydrous DMF under inert
Conjugate (Gemcitabine- atmosphere.
carbamate-D-Lys6-GnRH)
2. Add DIPEA (3 eq) at 0 °C dropwise.
3. After 5 min, add gemcitabine-linker (1 eq) dissolved in anhy-
drous DMF dropwise and stir the reaction for 12 h at room
temperature.
5. Dissolve the residue in TFA-H2O-TIS (95:2.5:2.5) and stir for
12 h (see Note 5).
7. Purify the residue with RP-HPLC and lyophilize the peak to
receive the final conjugate in pure form.
Note 6).
3.1.3 Synthesis of PDC 1. Dissolve Boc-protected gemcitabine (1 eq), t-Boc-Aminooxy-

Using Aminooxy-PEG4- PEG4-CH2CO2H (1.5 eq), DCC (1.5 eq), and DMAP
CH2CO2H as Linker (Amide (0.5 eq) in anhydrous dichloromethane under inert
and Oxime Bond) atmosphere.
(Scheme 3)
2. Add DIPEA (4 eq) at 0 °C and stir the reaction for the required
Preparation time at room temperature (monitor the reaction progress with
of Gemcitabine-Linker TLC).
(diBoc-Gemcitabine-Boc- 3. Distill the solvent.
Aminooxy-PEG4-CH2CO2H)
4. Purify the residue with column chromatography (eluent:
CH2Cl2/acetone 9/1) to afford gemcitabine-linker
(diBoc-Gemcitabine-Boc-Aminooxy-PEG4-CH2CO2H).
Scheme 2 Synthesis of PDCs using carbamate bond in the linker. Reagents and conditions: (a) Bis(4-
nitrophenyl)carbonate, DIPEA, acetonitrile, rt., 6 h; (b) D-Lys6-GnRH, DIPEA, DMF, rt., 12 h; (c) TFA/H2O/TIS
(9.5/0.25/0.25, v/v), rt., 12 h
5. Characterize the compound with 1H and 13C NMR spectros-

copy and mass spectrometry.
Attachment of Aldehyde 1. Dissolve Fmoc-Ser(tBu)-OH (1.5 eq), HATU (1.5 eq), and
Group on D-Lys6-GnRH, HOBt (1.5 eq) in anhydrous DMF under inert atmosphere.
as previously reported [14],
2. Add DIPEA (5 eq) at 0 °C.
and described below
(D-Lys6-GnRH aldehyde) 3. After 5 min, add D-Lys6-GnRH (1 eq) dissolved in anhydrous
DMF dropwise and stir the reaction for the required time at
room temperature (monitor the reaction progress with TLC).
5. Wash the residue with acetonitrile to afford
Fmoc-Ser(tBu)-D-Lys -GnRH.
6
6. Dissolve Fmoc-Ser(tBu)-D-Lys6-GnRH in 20% piperidine in

DMF at 0 °C and stir at room temperature for the required
time (monitor the reaction progress with TLC).
7. Distill the solvent and wash the residue with acetonitrile to
afford Ser(tBu)-D-Lys6-GnRH.
8. Dissolve Ser(tBu)-D-Lys6-GnRH in TFA/TIS/H2O
(95/2.5/2.5, v/v) and stir at room temperature for the
required time (monitor the reaction progress with TLC).
9. Distill the solvent, and wash the residue with acetonitrile.
10. Purify the compound with RP-HPLC and lyophilize the frac-
tion to afford Ser-D-Lys6-GnRH.
11. Dissolve Ser-D-Lys6-GnRH (1 eq) in H2O.
12. Add imidazole (5 eq) and sodium periodate (1.2 eq) and stir
at room temperature for 5–10 min.
13. Quench the reaction with ethylene glycol (2 eq).
14. Purify the mixture with RP-HPLC and lyophilize the fraction
to afford D-Lys6-GnRH aldehyde in pure form.
Note 6).
Scheme 3 Synthesis of PDCs using Aminooxy-PEG4-CH2CO2H as linker (amide and oxime bond). Reagents
and conditions: (a) DCC, DMAP, DIPEA, CH2Cl2, rt., 12 h; (b) CH2Cl2:TFA 95:5, rt.; (c) D-Lys6-GnRH aldehyde, appro-
priate conditions, 1-24 h, rt.
Preparation of the Final 1. Dissolve Gemcitabine-Linker (diBoc-Gemcitabine-Boc-

Conjugate (Gemcitabine- Aminooxy-PEG4-CH2CO2H) (1 eq) in CH2Cl2/TFA (95/5)
PEG4CH2CO2H-D-Lys6- at 0 °C and stir the resulting mixture at room temperature for
GnRH) the required time (monitor the reaction progress with TLC).
3. Dissolve the residue in the appropriate solvent system depend-
ing on the substrates, including 0.2 M NaOAc buffer (pH
3-5), or 0.2 M NH4OAc buffer (pH 4-5), or 0.5 M Phosphate
buffer/acetonitrile 3:1 (pH 4.5) (see Note 7).
4. Add D-Lys6-GnRH aldehyde (1 eq) and stir at room tempera-
ture for 1–24 h.
5. Lyophilize the reaction to afford the final conjugate in crude
form and then purify with RP-HPLC.
6. Collect, lyophilize and characterize the final conjugate with
mass spectrometry (see Note 6).
4 Notes
1. In the specific projects we used gemcitabine as the anticancer

agent and D-Lys6-GnRH as the tumor-homing peptide. All
the reactions can be performed with other drug and tumor-
homing peptide that have the necessary conjugatable groups.
2. TLC plates are used for two purposes: to assist the column
purification and also to monitor the reactions. The reaction
times mentioned in the synthetic part are based on progress
according to TLC.
3. In the specific reactions we used DMSO-d6 to characterize the

purity of the compounds due to solubility. In other cases,
other deuterated solvents can be used as well.
4. HATU should be used in a ratio of 1:1 with the aminoacid
since excess quantity can form unwanted side products [9].
5. TFA cleavage cocktail is used to cleave the Boc-groups of
gemcitabine. If another drug is used that is not Boc-protected,
this step is not required.
6. If more peaks appear during HPLC purification, collect and
lyophilize all and then characterize all starting from the major
one, until you identify the expected product.
7. Usually the addition of DMSO or aniline-based catalysts are
mandatory for the reaction to proceed.
Acknowledgments
This research was co-financed by Greece and the European Union

(European Social Fund- ESF) through the Operational Program
“Human Resources Development, Education and Lifelong
Learning 2014–2020” in the context of the project “Strengthening
Human Resources Research Potential via Doctorate Research -
second Cycle” (MIS 5000432). The research work was supported
by the Hellenic Foundation for Research and Innovation (H.F.R.I.)
under the “First Call for H.F.R.I. Research Projects to support
Faculty members and Researchers and the procurement of high-
cost research equipment grant” (Project Number: 991, acronym
PROTECT).
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Index
A Biomolecules�� 4, 14–15, 17, 19–22, 112

Biopharmaceutical classification system (BCS)�� 4, 100,
Affinity�� 188, 190, 191, 194, 202, 206, 329 255, 261, 263, 264, 266, 268, 269
Agglomeration��231 Block copolymers�� 71, 72, 76, 77, 300, 303
Aggregations��22, 37, 40, 41, 102, 203, 229, 231, 264, 274 Blood-brain barrier (BBB)�� 236, 237
Amorphous state�� 170, 273 Blood pressure regulators��110
Amphiphilic block copolymers�� 71–81, 222, 225, 228, 303 Bovine serum albumin (BSA)��14, 30, 273, 274
Amphiphilic diblock copolymer��222 Brain exosomes��27
Amphiphilic triblock hybrid terpolymer��144
Angiotensin converting enzyme (ACE)��100 C
Angiotensin II type 1 receptor blockers��110
Caffeic acid (CA)��4
Aniline derivatives�� 18, 20, 21
Calixarenes��238
Antibacterial�� 205, 266–267
Cancer��5, 25, 72, 86–89, 100, 110,
Anticancer��72, 214, 248, 249, 252, 255
127, 128, 136, 137, 140, 151, 175, 199–203, 214,
Anticancer agents��199, 328, 330, 336
216, 248–250, 252–256, 271, 327–330
Anticancer drugs��86–88, 149, 176, 199, 200,
Cancer cells��72, 86, 88, 89, 137, 201–203,
202–204, 206, 248, 250, 253, 254
214, 216, 249, 250, 253–255, 327, 328
Antifungal��262–264
Candesartan (CAN)��4, 6, 9, 45–69
Anti-hypertensive drugs�� 45, 190
Candesartan cilexetil�� 4, 45–69
Anti-inflammatory�� 4, 5, 72, 204, 256, 258, 259, 275
Captopril��100–106
Antioxidants��4, 5, 42, 72, 252, 264–266,
Carbon nanohorns��13–22
270, 285, 286 13
C cross polarization/magic-angle spinning
Anti-seizure��269
(13C CP/MAS)�� 318, 319, 323
Antiviral��259–262
Cell cultures�� 25, 26, 90–93, 142, 178
Anti-zika��267
Cell growth�� 200, 201, 254
Aqueous solutions�� 2, 6, 8, 9, 35, 37, 80, 90,
Ceramidase inhibitors��202
127, 129, 146, 153, 154, 159, 179, 191, 258
Ceranib-2 (C-2)��199–217
Association constant (Kα)��189
Characteristic transition temperatures��167, 168, 225, 302
Asymmetry�� 139, 145
Characterizations�� 22, 25–33, 143, 148,
AT1 receptor��110
151–160, 163, 180, 210, 266, 271, 332–333
Attenuated total reflection-Fourier transform infrared
Chemical shift changes��319
(ATR-FTIR)�� 76–78, 81
Chemical stability�� 2, 71, 316
B Chimeric nanosystems��221–232
Circular dichroism��129
γ-benzyl-l-glutamate�� 128, 131, 140, 142, 144 CO2 laser�� 14, 17
Bilayers�� 100, 222–224, 230, 299–301, Complexation�� 2, 3, 5, 46, 164, 165, 167,
303–306, 308, 309, 318 169, 170, 177, 189–191, 235–245, 250, 251, 258,
Bioactive molecules��1–9, 14, 164, 278 262, 264, 270, 271, 273, 314, 319
Bioavailability�� 4, 5, 9, 45, 72, 101, 110, Complexation energy��46
188, 190, 200, 202, 204, 207, 208, 222, 236, 238, Complexes�� 1–9, 35, 46, 49–52, 59, 66–68, 92,
248, 249, 251, 252, 256, 260–266, 268–270, 272, 100, 102, 110, 111, 113, 115–119, 136, 149,
273, 275, 287, 300, 316, 328, 329 164–172, 176, 179, 182, 184, 185, 188, 189, 194,
Biocompatibility��152, 203–205, 260 201, 238, 239, 241, 242, 244, 248, 249, 251, 252,
Biodegradable��71, 100, 136, 152 255, 256, 259–262, 264–266, 268, 272, 273, 278,
Biomaterials��13, 164, 221, 227, 231, 300 287–289, 292–296, 304–306, 313–322
Methods in Molecular Biology, vol. 2207, https://doi.org/10.1007/978-1-0716-0920-0
339
340 ISndex
upramolecules in Drug Discovery and Drug Delivery: Methods and Protocols
Compositional homogeneity��136 1,2-dipalmitoyl-sn-glycero-3-phosphocholine

Compositions�� 26, 37, 101, 136, 146, (DPPC)�� 222–224, 227–229, 231,
159, 203, 227, 288, 308 304–308, 316, 318–320
Compounds�� 2, 5, 37, 38, 40, 42, 94, Dispersion phase��210
95, 109–111, 146, 155, 163, 165, 176, 180, 182, Dispersions�� 14, 19, 20, 72, 90, 142, 154,
183, 185, 191, 204, 238, 239, 249, 251, 254, 255, 163, 165, 206, 207, 245, 259, 264, 267, 276, 301,
271, 274, 278, 285–288, 292, 293, 313–315, 319, 303, 304, 306, 319
333–335, 337 Doxorubicin��87, 89, 139, 140, 145, 253
Computational chemistry�� 54, 110 5-doxyl-stearic acid (5-DSA)��101, 102, 105, 106
Computational protocol��45–69 DPPC bilayers�� 225, 304, 306, 319, 321
Concentrations��3, 5, 22, 30, 35, 37, Drug
40, 41, 69, 74, 81, 88, 90, 92, 93, 96, 99, 102, 129, absorption��4, 45, 76–78, 89, 90, 96,
135–137, 141, 143, 147–149, 155, 156, 160, 167, 100, 160, 315, 328
176, 180, 182, 184, 189, 191, 194, 195, 212, 214, CD complexes, 288
217, 227, 230, 231, 237, 249–253, 256, 258, delivery........................................1–9, 13–22, 25, 71–81,
266–268, 276–278, 286, 288, 289, 292, 293, 301, 85–96, 99, 100, 109–123, 139–149, 151, 152, 155,
309, 319 164, 188, 203–204, 206, 207, 221, 222, 231,
Confocal��95–96, 254, 261 235–245, 248, 252, 254, 258, 267, 268, 271, 279,
Conjugation�� 17, 19–20, 235, 329, 330 300, 313, 315, 316, 328
Constant pressure�� 57, 117, 118, 171, 230 encapsulations............................................238, 248, 271
Controlled drug release�� 87, 203, 204 formulations.......................13, 73, 79, 80, 100, 101, 110,
Cooperativity�� 300, 303, 305, 307, 309 188, 190, 202–205, 244, 276, 328
Co-precipitation�� 2, 3, 164 loading, 76, 77, 86, 87, 90, 99, 155, 175, 192, 260
Covalent coupling��14–15 localization................................................................. 95
Critical micelle concentration (CMC)�� 99, 102, 274 molecules............................... 4, 5, 37, 46, 48–52, 57, 62,
Crystalline state�� 168, 171, 231 64, 68, 87, 99, 102, 155, 165, 204–206, 222, 267,
Curcumin (CRM)��4, 5, 7, 72–74, 76–78, 80, 251 269, 287, 288, 299, 300, 303, 315, 317
Cyclodextrin (CD)��1–4, 6, 8, 9, 35, 37, membrane interactions............................................. 318
42, 46, 49, 50, 53, 59, 61, 64, 68, 109–123, 164, Drug delivery system (DDS)��13, 85–88, 90,
167–171, 188, 190, 191, 194, 195, 247–279, 99–107, 136, 137, 199, 202–207, 211, 251, 269, 308
287–289, 294–296, 304, 305, 313–322 Dry mixing ��4
Cyclodextrin inclusion��2 Dynamic light scattering (DLS)�� 35, 79, 81, 141,
Cytotoxicity��72, 90, 92, 93, 176, 184, 148, 222, 224, 227, 228, 231, 249
238, 249, 251, 256, 266, 328, 329
E
D
Electron microscopy��28, 29, 156, 157
Damp mixing and heating��3 Electrostatic association��14
Degree of substitution (DS)��188, 194, 236, 272 Electrostatic interactions�� 189, 260, 274
Dendrimers��299, 300, 303, 305, 308, 309 Encapsulated curcumin�� 78, 81
Deuterated solvents�� 101, 317, 337 Encapsulation of indomethacin�� 76, 80
Diagnosis�� 25, 151, 221 Encapsulation processes��71
Dialysis�� 35–40, 42, 87–90, 143, 145, 146, Encapsulation protocols�� 72, 73, 79
206, 209–211 Encapsulations�� 20, 71–81, 86, 155, 156,
1,6-Diaminohexane��129, 135, 140, 146 175–185, 188, 191, 248, 252, 256, 258, 259,
Differential power (DP)��189, 192, 195, 196 265–267, 271, 273, 274, 276, 277
Differential scanning calorimetry (DSC)�� 163–172, Endocytosis�� 14, 88
222–227, 229, 230, 248, 250, 251, 253, 273, Endothermic peak�� 168, 169, 303
299–309 Endothermic process�� 171, 196
Diffusion coefficients��238, 239, 244, 245, 288, 289 Energetics��46, 110, 117–118
Diffusion- ordered NMR spectroscopy��239 Enhancement of the aqueous solubility��4
Difunctional initiator�� 130, 142 Enthalpy�� 2, 117, 118, 122, 168, 189, 307, 308
3D structure�� 136, 137 Enthalpy changes�� 167, 168, 171, 189, 192,
Dipalmitoyl-phosphatidylcholine (DPPC)�� 222–229, 195, 225, 302
231, 304–308, 316, 318–321, 323 Entrapment efficiency�� 258, 269, 272, 315, 324
Supramolecules in Drug Discovery and Drug Delivery: Methods and Protocols
341
Index
Entropy�� 117, 118, 122, 189, 190 Hydrophobic interactions��176, 189, 194, 229
Equilibrium state�� 47, 230, 231 2-hydroxypropyl-β-cyclodextrin
Exosome during secretion from the tissue��26 (2-HP-β-CD) �� 4, 110, 111
Exosome from whole organs��25–33 Hyperlipidemia associated diseases��272
Exosome isolation��26–28 Hypertension��100, 110, 268–269
Exosomes��25–33 Hyperthermia�� 86–90, 93, 96
Exothermic processes�� 171, 173, 192
External potential��46, 47, 57, 64 I
Extracellular vesicles��30 Imaging��29, 31, 95, 157
Extra sensitivity��89 Improvement of the pharmacological
Extrusion�� 3, 253 properties��10, 70, 110, 152
In vitro�� 4, 9, 87, 89, 90, 140, 146,
F
148–149, 152, 165, 176, 202, 209–214, 249–256,
Fluorescence��95, 130, 188, 261 261, 262, 264–266, 268, 270–274
Fourier transform infrared spectroscopy��76 In vivo�� 4, 90, 135, 136, 139, 146, 149,
Free energy calculations�� 110, 111 152, 176, 202, 250, 252–254, 256, 261, 264, 265,
Freeze-drying�� 1–9, 164–166, 170, 258, 267, 270, 273, 275
289, 318 In vivo pharmacokinetic analysis��185
Functionalities�� 14–15, 20, 22, 87, 119, Inclusion complexes��2, 4–9, 110,
137, 221–232, 300 189, 190, 248, 251, 253, 256, 259, 265–268, 271,
Functionalization�� 14, 22, 270 288, 314
Functional phase�� 229, 231, 232 Indomethacin�� 72, 73, 76–80, 306,
308, 314
G Inorganic��85, 86, 163, 203, 260, 278
Gas delivery��271–272 Interactions��2, 14, 45–69, 87, 95, 101, 105, 106,
Gastric reflux��270–271 110, 155, 160, 164, 165, 168, 170, 176, 187–196,
Gemcitabine�� 127, 133, 136, 328, 330–337 244, 245, 251–253, 258, 267, 269, 274, 277, 288,
Gibbs free energy (ΔG)��46, 47, 117, 189, 194, 195 299, 303, 305, 308, 309, 314, 318–320, 330
Glioblastoma multiforme��175–185 Interdigitation��308
Glove box�� 131, 132 Interfaces��105, 106, 286, 291
Glutathione��86, 88, 89, 252 Irbesartan (IRB)�� 4–6, 110–119, 165–172,
Graphite�� 16, 17 304–306, 316–322
Gromacs�� 48, 50–52, 54, 57, 61, 64, 68, 69 Irbesartan2-HP-β-CD association��110
Isothermal Titration Calorimetry (ITC)�� 188, 190, 191
H Isotherms�� 167, 170, 189, 193, 225, 230, 302, 305
Half peak height�� 168, 225, 302 K

Heart exosomes��27
Heat capacity��164, 171, 196, 230 Kinetic phenomena��171
Heat-flux DSC�� 163, 164, 166 Kneading method��3
Heating rate�� 167, 171
L
Heating scans�� 225–227, 230, 231, 307
High energy states��46 LCST�� 152, 308
High vacuum techniques��130, 135, 141, 142 Lipid bilayers��110, 300, 303–305, 309, 310, 320
1H-NMR spectroscopy�� 101, 142, 146 Lipophilic drugs��4, 46, 269, 277
Host-guest interactions�� 2, 188 Lipophilicity/Hydrophilicity�� 110, 264, 272
Host-guest complex��2 Liposomes�� 221–224, 227–230, 299–310, 315
Hybrids��85, 139–149, 278 Liver exosomes��27
Hydrogels�� 86, 100, 127–137, 258, 262, 263, Losartan (LOS)�� 4, 6, 165–170, 192–194, 196
271, 277, 278, 329 Losartan potassium��166, 167, 190–192
Hydrogen bonds�� 119, 155, 158, 189, 286 Lower critical solution temperature
Hydrophilic blocks��135, 139, 145, 149 (LCST)��88, 222, 226, 229–231
Hydrophobic cores��73, 76, 77, 321 Lyophilization�� 6, 8, 9, 163, 166, 167, 170, 276
Hydrophobic drugs�� 71–81, 306 Lyotropic effect��225
342 ISndex
M NMR chromatography�� 238, 239

NMR spectroscopy�� 101, 130, 241–245, 305,
Macroinitiator�� 140, 143, 147 314, 315, 317, 332–335
Macromolecular architecture�� 128, 136, 149 Non-toxic��190
Main transition temperature��225, 231, 301, 307 Non-reversible Phase��231
Major (M) and minor (m) conformation��103 Nuclear magnetic resonance (NMR)�� 9, 129,
Melting temperature��152, 164, 165, 168, 170, 253 178–182, 188, 229, 313–321, 333
Metastable phases�� 300, 308 Nuclear Overhauser effect (NOE)��314
Methyl-β-cyclodextrin (M-β-CD)��165
MGMT expression�� 176, 236 O
Micellar systems��316
Organic solvent evaporation�� 73, 81
Mice plasma��184
Organic solvent method��81
Micelles��71–81, 99–107, 316–322
Oxidation�� 14–17, 277, 285–288, 292–296
MicroRNA (miRNA)��25
Microscope�� 32, 94, 95, 160, 254 P
Microwave irradiation��21
Milli-Q water��130, 132, 133, 136, 142, 144 Paclitaxel�� 139, 248, 249
Mixed nanosystems��221 Pancreatic cancer (PC)�� 127, 128, 140
Modifications�� 113, 203–206, 258, 275, 287 Parkinson’s disease (PD)��267–268
Molar ratios�� 6–9, 102, 106, 132, 167, Paste complexation��3
168, 170, 192–194, 224, 227, 228, 230, 241, 258, PEGylation��205–206
259, 266, 267, 276, 300, 304, 307, 310, 318 Pentablock terpolypeptide�� 127, 133, 136
Molecular docking��110–113 Peptides��129, 140, 273–276, 315, 316,
Molecular dynamics (MD)�� 45–69, 109–123 328–331, 336
Molecular homogeneity��136 Perturbation�� 46–47, 303–305
Molecular interactions�� 101, 313 pH�� 5–9, 28, 29, 80, 81, 85–90, 96,
Molecular Mechanics Poisson-Boltzmann 128–130, 132, 136, 137, 139, 141––148, 152, 159,
Surface Area (MM–PBSA) method�� 111, 117, 167, 178, 179, 181–183, 185, 190, 193, 194, 209,
118, 122 210, 212, 214, 222, 223, 227, 230, 237, 241, 249,
Molecular modeling��112–114 251, 252, 254, 255, 262, 268, 274–276, 278, 286,
Molecular structures�� 9, 35, 50, 251, 313 289, 292, 293, 295, 303, 318, 336
MTT assays�� 92, 93, 209, 214, 255 Pharmaceutical applications��204–206, 248, 265
Multi-component solutions��238 Pharmaceutical formulations��152, 164, 165, 204, 206, 208
Multifunctional drug delivery systems��136 Pharmaceutical systems��314
Pharmacological actions��264
N Phase transitions�� 88, 89, 164, 222, 227, 301, 303, 305
Phospholipids�� 222, 226, 227, 230, 231,
Nanocarriers�� 13, 22, 71, 79, 86, 99, 101, 221,
299, 300, 304, 308, 320
250, 252, 255, 260
Physical mixtures�� 164–166, 168, 170–172, 184, 266
Nanoencapsulation��207
Physicochemical characterization�� 73, 224, 231
Nanoparticles (NPs)��80, 81, 86, 87, 89, 91,
Physicochemical properties�� 109, 155, 188, 222, 228–229
94, 100, 148, 149, 152, 156, 157, 203–208,
Physisorption��271
210–215, 221, 224, 229, 260, 275, 300, 329
Plasma stability��176
Nanospheres��86, 153, 155, 204
Pluronic F-127��72–74, 76–81, 264
Nanosponges (NSs)�� 100, 247–279
Poly caprolacton (PLC)��200
Nanosystems�� 221, 231, 300
Poly(ethylene oxide) (PEO)��71–73, 88, 100,
Nanotechnology�� 22, 203
139, 140, 143, 148, 149
Nanotherapeutics��279
Poly(ethylene oxide)-b-poly(ε-caprolactone)
Nanovesicles��25
(PEO-b-PCL)��72, 73, 75–78, 80
Natural products��25, 100, 152, 190, 203, 204,
Poly(ethylene oxide)-b-poly(propylene oxide)-b-
247, 251, 254, 265
poly(ethylene oxide)
N-carboxy Anhydride�� 130–132, 142
(PEO-b-PPO-b-PEO)�� 71, 72
Neutralization method�� 8, 318
Poly(lactic-co-glycolic) acid (PLGA) copolymer��203
Nim–trityl-l-histidine��130
Poly(lauryl acrylate) (PLA)�� 225, 229, 230
Nitroxide radical��101
Poly(N-isopropylacrylamide) (PNIPAM)��222
Supramolecules in Drug Discovery and Drug Delivery: Methods and Protocols
343
Index
Poly(N-isopropylacrylamide)-b-poly(lauryl acrylate) Size exclusion chromatography (SEC)�� 129, 141, 148
(PNIPAM-b-PLA)��223–230 Sleep disorders��270
Polyacrylates (PA)��203 Slurry complexation��3
Polyalacticle (PL)��203 Sodium dodecyl sulfate
Polydispersities��79, 80, 136, 148, 204, 227–230 (SDS)��29, 99, 101, 102, 105, 316–322
Polyethylene glycol (PEG)�� 28, 30, 199–217, 259, 261 Solid state NMR and solution state NMR�� 258, 305, 318
Polymer therapeutics��71 Solubility�� 4, 5, 9, 13, 42, 45, 72, 99, 100, 109,
Polymeric micelles�� 76–79, 86 110, 165, 175–185, 187, 188, 190, 191, 200,
Polymeric nanocarriers�� 80, 86 202–205, 207, 235, 236, 238, 248, 249, 251,
Polymerizations��73, 87, 129, 130, 132, 135, 254–256, 258, 259, 261–266, 268, 269, 271, 272,
140, 142, 143, 153, 229 276, 278, 279, 287, 316, 327, 330, 337
Polymers�� 42, 73–75, 77, 81, 85, 86, 88, Solvent evaporation��77, 206, 207, 268
127, 129, 137, 139, 141, 143–145, 147–149, 152, Solvents��4, 17, 21, 152
163, 203–206, 221, 222, 225–227, 229–232, 247, Sonication�� 20, 33, 88, 105, 155, 160, 210,
251, 252, 258, 260, 264, 267, 269–272, 277–279, 216, 227, 231, 259, 301, 318
299, 300, 305, 307, 308 Spectroscopic�� 22, 188
Polymersomes��139–149 Sphingolipids (SPL)��200–202
Polypeptides�� 132–133, 135–137 Spin labels��101, 102, 106, 107
Polyphenols�� 5, 265, 266 Stabilizers��277
Poorly water-soluble molecules��248 Steered MD��45–69
Precipitation��37, 76, 80, 81, 130, 132, 258 Steroids��273–276
Pretransition��226, 303, 304, 308 Stimuli responsive�� 221, 252
Proliferation�� 91–93, 184, 200, 202, 255 Stoichiometry��191, 195, 196, 314
p-sulfonatocalix[4]arene�� 176, 177, 179, 237, Structural properties��318
241, 242, 244 Supramolecular carriers��238
PTFE membrane filters�� 17, 19–21 Supramolecular chemistry��176
Synthesis of polypeptides��134
Q
T
Quantum mechanics��110
Quercetin (QUE)��4, 5, 8, 235, 236, 241–244, Targeted Drug Delivery��86
264, 265, 287–289, 293–296 Temozolomide (TMZ)�� 176, 177, 179–185, 236,
237, 241, 242, 244, 250, 251
R Temperatures�� 4, 5, 16–20, 31, 32, 36, 37, 39,
Recrystallizations�� 131, 143, 249 42, 55, 57, 73–75, 86–90, 96, 102, 107, 117, 118,
Redox�� 86, 88, 137, 285–288, 292, 294, 295 129, 132, 133, 135–137, 139, 141, 146, 148, 152,
Release�� 14–15, 22, 30, 38, 46, 48, 71, 154, 163, 164, 167, 169, 170, 179, 183, 185, 188,
86–90, 96, 99–101, 110, 136, 139, 148–149, 152, 189, 192, 195, 196, 206, 207, 210, 217, 222, 227,
155, 190, 203, 205, 206, 209–213, 222, 249–260, 229–232, 241, 245, 252, 253, 259, 267, 271, 274,
264–266, 268–274, 276, 278, 279, 303, 308, 316, 277, 289–291, 302–305, 319, 320, 333–336
317, 329 ε-tert-butoxycarbonyl-l-lysine��130, 132, 135, 142
Ring opening polymerization�� 73, 143 Theranostics�� 85–87, 221
Rosin esters��199–217 Therapies��100, 151, 205, 221, 254, 255,
Rosmarinic acid (RA)�� 4, 7 262, 268, 269
Thermal analysis (TA)�� 9, 163, 164
S Thermo��28, 29, 32, 86
Thermodynamic parameters�� 168, 171, 188, 194,
Sartans�� 110, 164–171
195, 225, 302, 303
Scanning electron microscopy
Thermodynamics��2, 46, 165, 168, 170, 187–196,
(SEM)�� 129, 156–158, 160, 249
221–232
Scratch�� 93, 94, 163
Thermograms��164, 165, 167–169, 225, 302,
Secondary structure�� 128, 136
303, 309, 310
Self-assembly properties��71
Thermoresponsive�� 221–232, 306
Self-healing hydrogel��136
Thermotropic behavior�� 225, 299–310
Silibinin (SLB)�� 4, 7, 9
Thin film hydration��72, 73, 81, 224, 227, 300, 303
Size distributions�� 79–81, 204, 207, 227, 229
344 ISndex
Thin-film hydration method V

(TFHM)��224, 227, 300, 303
Thin film protocol�� 72, 74, 75, 78, 80, 81 Van der Walls forces�� 189, 259
Transporters�� 46, 266, 269 Vesicles��139–142, 145, 147–149, 223, 224,
Transport systems�� 110, 269 231, 301, 319
Triple quadrupole mass spectrometry��178
W
Two-dimensional diffusion-ordered NMR spectroscopy
(2D DOSY)��235–245 Water�� 2–8, 17, 19, 20, 32, 33, 37, 39, 53, 68,
72–75, 81, 87, 90, 91, 99, 100, 115, 116, 118,
U 128–133, 135–137, 139, 141, 142, 146, 147, 149,
U87, 8MG��184 152–154, 159, 160, 166, 167, 177, 178, 188, 190,
Ultra High Performance Liquid 195, 209, 210, 212–214, 216, 217, 222, 227, 241,
Chromatography��178 251, 260, 262, 264–266, 268, 270, 276, 278, 287,
Ultrasonication�� 26, 30, 33 289, 291–293, 295, 301, 303, 304, 318, 319, 332
Ultrasonic cavitation�� 206–208, 211 Water solubility�� 190, 206, 250, 272, 315, 316, 328
Umbrella sampling��46, 48, 61–67, 69 Wound closure��95
UV-Vis spectroscopy��76 Wound healing��94

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Methods in

Molecular Biology 2207

For further volumes:

ISSN 1064-3745 ISSN 1940-6029 (electronic)

© Springer Science+Business Media, LLC, part of Springer Nature 2021

Amidst the sadness of endless mediocrity, which suffocates us everywhere, I

Zografou, Greece Thomas Mavromoustakos

1 Application of Neutralization and Freeze-Drying Technique

12 Drug Delivery Systems Based on Modified Polysaccharides:

Barbara Athanasiou • Department of Chemistry, Industrial Chemistry Laboratory,

Athanasios Skandalis • Theoretical and Physical Chemistry Institute, National Hellenic

Application of Neutralization and Freeze-Drying Technique

Key words Cyclodextrins, 2-Hydroxypropyl-β-CD, Methyl-β-CD, Bioavailability, Lyophilization,

Cyclodextrins (CDs) are a-(1,4)-linked cyclic oligosaccharides

One of the most remarkable features of CDs is their tendency

The binding strength of the formed complex depends on the abil-

amount of cyclodextrin is dissolved in water and the guest

amount of water, the extruded complex may dry as it cools or

Curcumin (CRM) is a bright yellow chemical compound pro-

1. Use purified water and analytical grade guest molecules.

3.1 Steps to Follow 1. Accurately weigh 60 mg of IRB and 408 mg of 2-HP-β-CD

5. The resulting solution at a molar ratio of 1:2 (IRB:2-HP-β-CD)

3.2 Steps to Follow 1. Accurately weigh 3445.6 mg of 2-HP-β-CD.

3.3 Steps to Follow 1. Accurately weigh 30 mg of CAN or CC and 198.85 mg or

3.4 Steps to Follow 1. Weigh accurately 60 mg of CA and 0.964 g of 2-HP-β-CD.

6. The resulting solution has a 1:2 molar ratio of CA:2-HP-β-CD

3.4.2 RA–2-HP-b-CD 1. Accurately weigh 120 mg of RA and 964 mg of 2-HP-β-CD

3. After complete dissolution a yellow solution is obtained.

3.7.2 QUE–Me-β-CD 1. For the preparation of QUE–Me-β-CD aqueous solution for

1. The neutralization method [13] is applied prior to lyophiliza-

3. Note that all drug (bioactive) molecules described in this chap-

The neutralization and freeze-drying (or lyophilization) technique

This work was financially supported by the European Union and

Administration of Pharmaceutical Molecules with the aim to

hsmc.gr/wp-content/uploads/2019/02/ (2-hydroxypropyl)-cyclodextrin complex. Int J

Functionalized Carbon Nanohorns as Drug Delivery

Key words Carbon nanohorns, Functionalization, Encapsulation, Hybrids, Biomaterials, Drug

Drug delivery system technology is considered as a major area of

aqueous media, which is critical for manipulation and processing

2.1 Preparation 1. A chamber equipped with a high-power CO2 laser source

diameter of 10 mm and a plastic-resin reaction chamber

2.2 Drying Solvents 1. Tetrahydrofuran (THF).

2.3 Synthesis 1. p-Nitrobenzoyl chloride.

2.4 Synthesis 1. CNHs powder.

2.5 Oxidation 1. CNH powder.

2.6 Conjugation 1. Oxidized CNHs.

2.7 Nanomesh CNHs 1. CNH powder.

2.8 Encapsulation 1. Nanomesh CNHs.

All experiments are carried out under inert atmosphere conditions

3.2 Procedures 1. Tetrahydrofuran (THF): THF (100 mL) is placed in a round-­

3.3 Covalent 1. Add pristine CNHs (5 mg) and 1,2-dichlorobenzene, oDCB,

10. Bubble gaseous HCl for 30 s in the dispersion of the BOC-­

1. Bath sonication allows dispersion of insoluble CNHs and facil-

11. Examination of the filtrate via UV-Vis allows monitoring the

Although the synthesis of CNHs requires advanced infrastructure,

Partial support by the project “National Infrastructure in

Infrastructures,” funded by the Operational Program

1. Karousis I, Martinez IS, Ewels CP, tionalization of carbon nanohorns by high-­

Zografou, Greece Thomas Mavromoustakos

1 Application of Neutralization and Freeze-Drying Technique

12 Drug Delivery Systems Based on Modified Polysaccharides:

3.2 Procedures 1. Tetrahydrofuran (THF): THF (100 mL) is placed in a round-

10. Bubble gaseous HCl for 30 s in the dispersion of the BOC-

1. Karousis I, Martinez IS, Ewels CP, tionalization of carbon nanohorns by high-

Study of Candesartan Cilexetil: 2-Hydroxypropyl-β-

rotocol copolymer and drug are first dissolved in a common

3.6 Fourier Attenuated total reflection-Fourier transform infrared (ATR-