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NANOPHARMACEUTICALS
EXPECTATIONS AND REALITIES OF
MULTIFUNCTIONAL DRUG
DELIVERY SYSTEMS
VOLUME 1
Edited by

RANJITA SHEGOKAR, PhD

Capnomed GmbH, Zimmern, Germany
Elsevier
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 Contributors
Hend Abd-Allah Department of Pharmaceutics and
Industrial Pharmacy, Faculty of Pharmacy, Ain
Shams University, Egypt
Mona Abdel-Mottaleb Department of Pharmaceu-
tics and Industrial Pharmacy, Faculty of Pharmacy,
Ain Shams University, Egypt; PEPITE EA4267,
Univ. Bourgogne Franche-Comté, Besançon, France
Annis Catur Adi Faculty of Health, University of
Airlangga, Surabaya, Indonesia
Mukta Agrawal Rungta College of Pharmaceutical
Sciences and Research, Bhilai, Chhattisgarh, India
Amit Alexander Rungta College of Pharmaceutical
Sciences and Research, Bhilai, Chhattisgarh, India
Mahavir Bhupal Chougule Translational Bio-
pharma Engineering Nanodelivery Research Labo-
ratory, Department of Pharmaceutics and Drug
Delivery, School of Pharmacy, University of Missis-
sippi, University, MS, United States; Pii Center for
Pharmaceutical Technology, Research Institute of
Pharmaceutical Sciences, University of Mississippi,
University, MS, United States; National Center
for Natural Products Research, Research Institute
of Pharmaceutical Sciences, University of Missis-
sippi, University, MS, United States
Juan Bueno Research Center of Bioprospecting
and Biotechnology for Biodiversity Foundation
(BIOLABB), Armenia, Quindío, Colombia
J.R. Campos Department of Pharmaceutical Tech-
nology, Faculty of Pharmacy, University of Coim-
bra (FFUC), P

olo das Ciências da Sa

ude, Coimbra,
Portugal
Anne Marie Clark CAS, a Division of the American
Chemical Society, Columbus, OH, United States
Diana Diaz-Arévalo Molecular Biology and Immu-
nology Department, Fundaci

on Instituto de Inmu-
nología de Colombia-FIDIC, School of Medicine
vii
and Health Sciences, Universidad del Rosario,
Bogot

a, DC, Colombia
Riham I. El-Gogary Department of Pharmaceutics
and Industrial Pharmacy, Faculty of Pharmacy,
Ain Shams University, Egypt
N.B. Jadav Centre for Pharmaceutical Engineering
Sciences, Faculty of Life Sciences, University of
Bradford, Bradford, United Kingdom
AnCelka B. Kovacevi
c Department of Pharmaceu-
tical Technology, Institute of Pharmacy, Faculty of
Biological Sciences, Friedrich-Schiller University
Jena, Jena, Germany
Atsarina Larasati Research Center for Nanosciences
and Nanotechnology, Bandung Institute of Tech-
nology, Bandung, Indonesia
Peter Mattei CAS, a Division of the American
Chemical Society, Columbus, OH, United States
Maha Nasr Department of Pharmaceutics and In-
dustrial Pharmacy, Faculty of Pharmacy, Ain
Shams University, Egypt
A. Paradkar Centre for Pharmaceutical Engineering
Sciences, Faculty of Life Sciences, University of
Bradford, Bradford, United Kingdom
Heni Rachmawati School of Pharmacy, Bandung
Institute of Technology, Bandung, Indonesia;
Research Center for Nanosciences and Nanotech-
nology, Bandung Institute of Technology, Bandung,
Indonesia
Kobra Rostamizadeh Zanjan Pharmaceutical Nano-
technology Research Center, Zanjan University of
Medical Sciences, Zanjan, Iran; Center for Pharma-
ceutical Biotechnology and Nanomedicine, North-
eastern University, Boston, MA, United States
A. Santini Department of Pharmacy, University of
Napoli “Federico II”, Napoli, Italy
viii Contributors
Shailendra Saraf University Institute of Pharmacy,
Pt. Ravishankar Shukla University, Raipur, Chhat-
tisgarh, India
Swarnlata Saraf University Institute of Pharmacy,
Pt. Ravishankar Shukla University, Raipur, Chhat-
tisgarh, India
P. Severino Universidade Tiradentes (Unit), Aracaju,
Sergipe, Brazil; Instituto de Tecnologia e Pesquisa,
Laborat
orio de Nanotecnologia e Nanomedicina
(LNMed), Aracaju, Sergipe, Brazil; Tiradentes
Institute, Dorchester, United States
Ranjita Shegokar Capnomed GmbH, Zimmern,
Germany
A.M. Silva School of Biology and Environment, Uni-
versity of Tr
as-os-Montes e Alto Douro (UTAD), Vila
Real, Portugal; Centre for Research and Technology
of Agro-Environmental and Biological Sciences
(CITAB), University of Tr
as-os-Montes e Alto Douro
(UTAD), Vila Real, Portugal
S.B. Souto Department of Endocrinology, S. Jo~ ao
Hospital, Alameda Prof. Hern^ani Monteiro, Porto,
Portugal
E.B. Souto Department of Pharmaceutical Technol-
ogy, Faculty of Pharmacy, University of Coimbra
(FFUC), P
olo das Ciências da Sa
ude, Coimbra,
Portugal; CEB - Centre of Biological Engineering,
University of Minho, Braga, Portugal
Asur Srinivasan CAS, a Division of the American
Chemical Society, Columbus, OH, United States
Amanda Starling-Windhof CAS, a Division of the
American Chemical Society, Columbus, OH,
United States
Tina Tomeo CAS, a Division of the American
Chemical Society, Columbus, OH, United States
Vladimir P. Torchilin Center for Pharmaceutical
Biotechnology and Nanomedicine, Northeastern
University, Boston, MA, United States
Mingtao Zeng Center of Emphasis in Infectious Dis-
eases, Department of Molecular and Translational
Medicine, Paul L. Foster School of Medicine, Texas
Tech University Health Sciences Center El Paso, El
Paso, TX, United States
 Preface

The book series titled Expectations and Real-
ities of Multifunctional Drug Delivery Systems
covers several important topics on drug-delivery
systems, regulatory requirements, clinical studies,
intellectual properties trends, new advances,
manufacturing challenges, etc. written by leading
industry and academic experts. Overall, the
chapters published in this series reflect the broad-
ness of nanopharmaceuticals, microparticles,
other drug carriers and the importance of the
respective quality, regulatory, clinical, GMP scale
up, and regulatory registration aspects.
This series is destined to fill the knowledge
gap through information sharing and with orga-
nized research compilation between diverse
areas of pharma, medicine, clinical, regulatory
practices, and academics.
Expectations and Realities of Multifunctional Drug
Delivery Systems is divided into four volumes:
Volume 1: Nanopharmaceuticals
Volume 2: Delivery of Drugs
Volume 3: Drug Delivery Trends
Volume 4: Drug Delivery Aspects
The specific objectives of this book series are
to:
(1) provide a platform to discuss opportunities
and challenges in development of
nanomedicine and other drug-delivery
systems;
(2) discuss current and future market trends;
(3) facilitate insight sharing within various
areas of expertise; and
(4) establish collaborations between academic
scientists, and industrial and clinical
researchers.
Innovative cutting-edge developments in
micro-nanotechnology offer new ways of pre-
venting and treating diseases like cancer, ma-
laria, HIV/AIDS, tuberculosis, and many more.
The application of micro-nanoparticles in drug
delivery, diagnostics, and imaging is vast.
Hence, Volume 1: Nanopharmaceuticals, in
the book series mainly reviews advances in
drug delivery area via targeted therapy with
improved drug efficiency at a lower dose, trans-
portation of the drug across physiological bar-
riers as well as reduced drug-related toxicity.
One of the contributions by Campos et al.
(Chapter 1) discusses the influence of physico-
chemical factors affecting long-term stability,
release and toxicological profiles of solid lipid
nanoparticles. This chapter also reviews the
importance of composition and administration
routes studied for lipid nanocarrier systems.
In another manuscript by Rachmawati et al.
(Chapter 2), the authors highlight the current
status of drug delivery development for herbal
bioactives. Along with various mucosal bio-
carriers, the authors describe biokinetic and
clinical translation challenges with herbal deliv-
ery and limitations with regulatory procedures.
In this chapter herbal nanocarriers like lipid
nanoparticles, nanosuspensions etc. are
discussed in detail.

ix
x Preface
Chapter 3 by Rostamizadeh et al. describes
the development of polymeric micelles for multi-
ple drug delivery in oncology. The co-loading of
two or more drugs is possible using polymeric
micelles. The authors describe the types of
polymers employed, preparation methods, and
characterization techniques for such carrier
systems. Furthermore, wider applications like
chemotherapeutic delivery, stimuli-responsive
drug delivery, and targeted-drug delivery via
such carriers is discussed in detail. On the other
hand, hyaluronic acid nanoparticles are being
widely explored in nanomedicines. The promising
nanocarriers to deliver drugs in conjugated, self-
assembled, or in nanocomplex form are discussed
in the book chapter by Nasr et al. in Chapter 4. The
authors confirm that the current research
shows impressive research findings in areas like
osteoarthritis, tissue engineering, cancer target-
ing, theranostic applications, and so on, which
are under further exploration by industry.
The work by Jadav and Paradkar (Chapter 5)
is aimed at discussing widely studied drug
delivery systems in academics and in industry,
i.e. solid dispersions. Various aspects like classi-
fication of solid dispersions, their formulation
optimization, processing and physicochemical
characterization are reviewed in this chapter.
Chapter 6 by Bueno highlights the impor-
tance of understanding nanotoxicity at early
stages of development. Although nanocarriers
have shown promising results in delivering
drugs at target sites or locations, the accumula-
tion of nanoparticles at cellular and tissue
levels in excess causes toxicity. Current literature
contains very limited information on this topic.
This chapter reviews various aspects of nanotox-
icity and provides information on key concepts
for evaluation of the toxicity.
The topic presented by Diaz-Arévalo et al.
(Chapter 7) describes the systemic review of
nanoparticles-based vaccine development. Initial
results of nanocarriers like liposomes, virus like
particles, metallic and nonmetallic particles, and
polymeric nanoparticles in vaccine therapy are
promising although some limitations like stability
and cytotoxicity needs to be overcome.
Chapter 8 by Kovacevic discusses delivery of
poorly soluble and low-permeable drugs via
lipid nanocarriers. An overview of available
mechanistic studies in model and in real cell
membranes for better understanding of cell
internalization processes is provided.
The chapter by Alexander et al. (Chapter 9)
reviews approaches for effective brain drug deliv-
ery using nasal mucosa. This route can deliver
drugs effectively at target sites with improved
therapeutic performance of drugs. In addition,
the authors explain limitations of this drug deliv-
ery route and regulatory market approval
challenges.
Starling-Windhof et al. (Chapter 10) address
industry and technology trends in the intellec-
tual property (IP) landscape of pharmaceutical
drug delivery over 3 decades. It is fascinating
to see the global picture; it makes scientists
aware of the trends and special interests of
specific geographical regions or markets.
This chapter provides information on IP trends
in oral, topical, and parenteral drug delivery
area. Guidance on emerging trends and
IP-monitoring strategies are also presented.
In summary, I am sure this book volume and
the complete book series will provide you great
insights in areas of micro-nanomedicines, drug
delivery sciences, new trends, and regulatory
aspects.
O. Farokhzad, R. Langer, and the National
Cancer Institute are gratefully acknowledged for
the book cover image, which represents the po-
tential of nanopharmaceuticals in targeting drug
molecules. This photograph presented on cover
page captures interactions of surface-modified
polymeric nanoparticles with prostate cancer
cellsdit is an ideal example for drug targeting.
All the efforts of experts, scientists, and
authors are highly acknowledged for sharing their
knowledge, ideas, and insights about the topic.
Ranjita Shegokar, PhD
Editor
 C H A P T E R

1
Solid lipid nanoparticles (SLN):
prediction of toxicity, metabolism, fate
and physicochemical properties
J.R. Campos1, P. Severino2,3,4, A. Santini5, A.M. Silva6,7,
Ranjita Shegokar8, S.B. Souto9, E.B. Souto1,10
1
Department of Pharmaceutical Technology, Faculty of Pharmacy, University of Coimbra (FFUC), P
olo
ude, Coimbra, Portugal; 2Universidade Tiradentes (Unit), Aracaju, Sergipe, Brazil;
das Ci^encias da Sa

3
Instituto de Tecnologia e Pesquisa, Laborat
orio de Nanotecnologia e Nanomedicina (LNMed), Aracaju,
Sergipe, Brazil; 4Tiradentes Institute, Dorchester, United States; 5Department of Pharmacy, University of
Napoli “Federico II”, Napoli, Italy; 6School of Biology and Environment, University of Tr
as-os-Montes e
Alto Douro (UTAD), Vila Real, Portugal; 7Centre for Research and Technology of Agro-Environmental
and Biological Sciences (CITAB), University of Tr
as-os-Montes e Alto Douro (UTAD), Vila Real,
Portugal; 8Capnomed GmbH, Zimmern, Germany; 9Department of Endocrinology, S. Jo~ao Hospital,
Alameda Prof. Hern^ani Monteiro, Porto, Portugal; 10CEB - Centre of Biological Engineering,
University of Minho, Braga, Portugal

1. Introduction formulations [2]. Various controlled drug deliv-

Solid lipid nanoparticles (SLNs) gained
greater attention as a drug delivery system
when in 1991 M€ uller developed them [1]. This
promising drug carrier system is at the interface
in the preexisting lipid systems (emulsions and
liposomes) and polymeric nanoparticle systems.
Lipid nanoparticles, known as SLNs or nano-
structured lipid carriers (NLCs), have specific
features of structure and composition, showing
benefits in comparison to conventional
ery systems like polymer-based controlled-
release systems, hydrogels, as well as nano-
and microparticles have been introduced in
recent years in order to improve solubility, sta-
bility and bioavailability of poorly water-soluble
drugs. In this context, lipid nanoparticles offer
attractive and ideal properties for drug or gene
delivery. These particles (either composed of
solid lipids only in SLNs, or of a blend of solid
and liquid lipids in NLCs) stabilized with surfac-
tants have the advantages of other colloidal

Nanopharmaceuticals
https://doi.org/10.1016/B978-0-12-817778-5.00001-4 1 © 2020 Elsevier Inc. All rights reserved.
2 1. Solid lipid nanoparticles (SLN)
particles (polymeric nanoparticles, fat emul-
sions, and liposomes) by overcoming their limi-
tations [3,4]. Taking account their polymeric
and lipid raw materials, several modifications
of drug delivery systems have been proposed
to increase the bioavailability of loaded drugs.
SLNs are made of a solid lipid matrix and a sur-
factant layer and they can load poorly water-
soluble drugs, delivering them at defined rates
and with improved bioavailability [5]. These
colloidal drug delivery systems protect the
drug against chemical degradation and modify
its release profile since the drug is entrapped in
the solid lipid matrix [6,7]. These nanoparticles
of spherical shape have a mean size of
40e1000 nm [8,9]. The lipid matrix is composed
of a solid lipid (or a mixture of solid and liquid
lipids) in a 0.1%e30% (w/w) concentration
dispersed in aqueous medium, and their stability
is ensured by the presence of a surfactant in a
0.5%e5% (w/w) concentration [8] and can be
used for lipophilic or hydrophilic drugs [9]. Tri-
glycerides (tristearin), sterols (cholesterol), par-
tial glycids (glyceryl monostearate), fatty acids
(stearic acid), as well as waxes (cetylpalmitate)
are especially used as lipids in the SLNs. In these
systems emulsifiers and polymers are used as
stabilizers in order to avoid aggregation of the
particles. Examples of stabilizers are bile salts
(e.g., taurodeoxycholate), lecithins, and copoly-
mers of polyoxyethylene and polyoxypropylene
(Poloxamer) [10].
It is clear that lipid nanocarriers are ideal for
sensitive bioactive compounds. SLNs exhibit
biocompatibility, matrix with lipophilic nature
protecting active compounds of chemical degra-
dation, drug targeting, controlled release profile,
and high drug payload [9,12]. Moreover, they
are suitable for industrial production mainly
because they are easy to scale up, are stable under
sterilization conditions, and they have the advan-
tage of being non-toxic or of very low toxicity,
because of their composition in Generally
Recognized as Safe (GRAS) excipients [4,6].
SLNs are biodegradable (fulfilling the require-
ments of preclinical safety) and are also stable in
blood, with prolonged lifetime in the bloodstream
[13e15]. In addition, compared to liposomes,
SLNs exhibit high encapsulation efficiency, stabil-
ity against light and oxygen, do not need organic
solvents for their preparation, and have high
drug-loading capacity (mainly for lipophilic com-
pounds) [12,15,16]. On the other hand, SLNs have
also limitations, mainly attributed to the risk of
polymorphic transitions (from a to b0 , and from
b0 to b) which causes stability challenges during
administration or storage, resulting in drug
expulsion from the particles and eventual particle
size increase [6,10,17]. These disadvantages are
related to the crystallization behavior and lipid
matrix’s polymorphic transitions, which depend
on the type of lipids used for the production of
SLNs [6]. There are various methods described
in the literature to produce SLNs based on
solidified emulsion technologies: high shear ho-
mogenization and ultrasound, high pressure
(hot and cold) homogenization, oil/water (O/
W) and water/oil/water (W/O/W) microemul-
sions, as well as solvent evaporation [10].
These techniques interfere with various character-
istics of the particles, mainly in morphology. Ac-
cording to the literature, the most commonly
applied methods are those that use high pressure
homogenizers (HPH). M€ uller and Luck devel-
oped the HPH technique (European Patent No.
0605497) for obtaining nanoemulsions for large-
scale parenteral nutrition [10]. There are diverse
kinds of equipment with various sizes, prices as
well as different capacities. The different equip-
ments work by pulling the liquid in high pressure
(100e2000 bar) through a narrow piston (nano-
meter scale), which is accelerated over a small
distance at a high speed (over 1000 km/h). This
fluid is subjected to high stress, disrupting the
macroscopic oil droplets by cavitation forces
and thus generating the nanodroplets [10]. In
 2. Toxicity profiling
the hot process, the hot preemulsion is passed
through the hot homogenizer to obtain nanoe-
mulsions, which are then cooled down in order
to solidify and crystallize the hot inner liquid
phase to obtain SLNs. In the cold process, the
drug is firstly ground milled in a mortar mill
with the solid lipid at room temperature, and
then the obtained powder is dispersed in an
aqueous surfactant solution, which is then sub-
jected to HPH. The influence of the type of
homogenizer, pressure, and number of cycles
employed, and the temperature used to obtain
the ideal particle size, have been intensively stud-
ied. Depending on the type of lipid, it is possible
to use lipid concentrations above 40% and obtain
the particle size distribution in a low range
(polydispersity index <0.2) [18]. To obtain SLNs
from microemulsions, Gasco and collaborators
developed a technique that has been modified
by numerous researchers. In this technique,
SLNs are produced by diluting a hot oil-in-water
(O/W) microemulsion in high volume of cold-
water (0e4o C). The internal phase of this microe-
mulsion is composed of low-melting lipids and,
when in contact with the cold water, they suffer
crystallization and form SLNs [18e20]. The type
of lipids used to make the microemulsion, the
preparation parameters (stirring and tempera-
ture), rate of microemulsion’s addition in the
cold water, volume of water, and the technique
used to remove the excess water, all affect the
characteristics of obtained nanoparticles [18].
This technique is therefore difficult to scale up.
Marengo has developed a device that processes
100 mL of microemulsion and can produce
SLNs of mean size below 100 nm [20].
The biomedical applications of lipid nanopar-
ticles are manyfold. Indeed, they can be used as
drug and gene carriers, and as contrast agents
for imaging analysis [21]. In recent years,
different administration routes (e.g., oral, paren-
teral, dermal, pulmonary, rectal, ocular) have
3
been explored for drug delivery using lipid
nanoparticles. Components of lipid nanopar-
ticles (lipids and surfactants) determine the
product quality, its physicochemical properties,
as well as the administration route [4] (Table
1.1). In this chapter, the concept behind the
SLNs and their physicochemical properties,
pharmacokinetics, and biopharmaceutics and
their toxicological testing are discussed.
2. Toxicity profiling
SLNs are known to be stable in aqueous
dispersion, allows the encapsulation of hydro-
philic and lipophilic drugs, are adaptable to
several administration routes, can modify the
release profile and avoid their adverse effects
(protecting the drug from undesirable interac-
tions or directing it to its target) [30]. However,
their toxicological profile has to be very well
characterized in vitro before any pre-clinical
and clinical studies [31]. There is a relationship
between the size of the particles and their
toxicity, as the lower the size, the higher the sur-
face area and the higher the reactivity. A pre-
requisite to be marketable is the GRAS status
of the excipients of SLNs [32], but additional
nanotoxicological studies are needed to allow
the understanding of the effect of nanoparticles
in the body [14,33,34]. Nanotoxicology helps in
identifying the SLN formulations to be selected
for preclinical studies by evaluating their safety,
tolerance, and cytotoxicity.
Preclinical toxicological studies allow to
determine the concentration of substances that
cause toxic effects, and allow identification of
target organs predisposed to these effects. Pre-
clinical safety tests require the appropriate selec-
tion of the animal species, age, physiological
status, the administration route, dosage form as
well as the treatment regime. Preclinical
TABLE 1.1 Commonly used lipids in the composition of SLNs/NLCs.

Therapeutic
Name Chemical structure Surfactant System Drug application References

Tristearin Dihexadecyl SLNs e Pulmonary [22]

phosphate delivery
PEGs SLNs e Oral delivery [23]

Glyceryl Mixture of Tween SLNs e e [24]

monostearate 80 and Span 80

Stearic acid Omega-3 PUFA SLNs Resveratrol Oral [25]

delivery
Cystamine SLNs e e [26]
Tween 80 NLCs e e [27]
Cholesterol Pluronic F-68 NLCs Simvastatin Oral [28]
delivery

Vitamin E O D-a-tocopheryl NLCs Rapamycin Ocular [29]

O n polyethylene glycol delivery
O
R O succinate (TPGS)
O O
 3. Physicochemical properties
toxicological evaluation of a new compound can
provide information about acute, subacute, sub-
chronic, and chronic toxicity. Before any preclin-
ical study, cytotoxicity assessment is carried out
in cell culture, also allowing the study of the
interaction between nanoparticles and cells.
The use of primary cells (isolated directly from
the animals) provides more realistic toxicological
results. On the other hand, in vivo studies eval-
uate the organism as a whole. Therefore, in vivo
studies are relevant to determine the location
and concentration of the drug in the tissues, and
systemic toxicity [35]. Systems with large quan-
tities of surfactants (e.g., nanoemulsions) have
higher risk of exhibiting cytotoxicity, since these
agents interact with cell membranes composed
of phospholipids. Likewise, their production re-
quires the use of organic solvents, which may
also increase the risk of toxicological events if
poorly removed. Although precise determina-
tion of the toxicity can only be quantified
in vivo, there are several in vitro toxicological
tests that provide preliminary information
[10,14,36]. In vitro studies have shown that
SLNs are acceptable at concentrations <1 mg/
mL (total lipids), and with particle diameter
>500 nm can be less tolerated, which can be
explained by their aggregation. It was also
shown that the stabilized formulations
composed of several surfactants are less biocom-
patible in comparison to those based on one sur-
factant only. For polysorbate 80 and poloxamer
188, two surfactants mostly used in SLNs formu-
lations, enough evidence has been found to
determine their safety [14]. It is clear that the
knowledge of the toxicological profile of any ma-
terial and the biocompatibility of the drug deliv-
ery systems are crucial for the implementation of
drug therapies, but the information of
Amount ðmgÞ of loaded drug determined experimentaly
EEð%Þ ¼
Theoretical amount of drug ðmgÞ in formulation
5
nanoparticle-based drug therapies available is
still very limited [8].
3. Physicochemical properties
The characterization of SLNs usually re-
quires the determination of the particle size
and zeta potential, morphological evaluation,
determination of the loading capacity and
encapsulation efficiency, kinetics of drug
release, as well as the nanoparticles over
time and storage temperature.
3.1 Encapsulation parameters
Determination of the amount of drug associ-
ated with the nanoparticles is a crucial parameter
for the characterization of SLNs. This task is
however difficult because of the small size of
the particles, which compromises the separation
of the free drug fraction from the associated frac-
tion. Ultracentrifugation is the most commonly
used separation procedure, after which the
non-loaded drug is quantified in the superna-
tant. From the difference between total drug
and the drug found in the supernatant, the
drug concentration associated with nanostruc-
tures is calculated [37,38]. Ultrafiltration can be
coupled to the process of ultracentrifugation. In
this approach, a membrane (100 kDa) is used to
separate the aqueous phase from the nanopar-
ticles. Although the free drug concentration in
this technique is determined in the ultrafiltrate,
the drug fraction associated with the nanostruc-
tures is also found by the difference between the
total and free concentrations [39]. Encapsulation
efficiency (EE%) and loading capacity (LC%) are
determined using the following equations [40]:
 100
6 1. Solid lipid nanoparticles (SLN)
Amount ðmgÞ of loaded drug determined experimentaly
LCð%Þ ¼
Theoretical amount of lipid ðmgÞ in formulation
3.2 Particle size
Size and polydispersity index are parameters
that indicate the stability of the nanoparticles.
An optimized SLN formulation should exhibit
a mean particle size less than 1 mm, together
with a small polydispersity index. Several pa-
rameters affect the particle size and polydisper-
sity, e.g., composition of the formulation
(mixture of surfactants, lipid structural proper-
ties, and also incorporated drug), methods and
conditions of the production (time, temperature,
stirring velocity, pressure, etc) [41]. There is a
relationship between the proportion of surfac-
tant/lipid and the particle size, i.e., the greater
the concentration of surfactant, the smaller the
particle size [42]. The temperature used in the
HPH technique is another parameter that inter-
feres with the particle size. In the production of
SLNs and NLCs, the lipid phase is heated up
to a temperature higher than the melting point
of the solid lipid. The final step is the cooling
down of the systems so that the lipid recrystal-
lizes to solidify the lipid droplets [43]. Homoge-
nization at low temperatures (i.e., below the
melting point of the solid lipid) favors the in-
crease of particle size, but temperatures near
the melting point improve the homogenization
and lower particle sizes can be obtained. Simi-
larly, the hot HPH procedure produces particles
with lower mean size and polydispersity than
the cold procedure [41]. Generally, the particle
size increases when lipids of long fatty acid
chains are employed. The use of mixtures of
short- and long-chain fatty acids may therefore
reduce the mean particle size and polydispersity
index of SLNs/NLCs [4,44].
 100
3.3 Zeta potential
Zeta potential is another parameter used to
evaluate the long-term stability of the particles
and its assessment is instrumental for the physi-
cochemical characterization of the nanoparticles'
dispersion. When determining the zeta potential,
it is possible to understand the interactions
between the particle and the drug. This param-
eter shows the surface electrical charge of the
particles, which is modified by changes in the
interface with the dispersing medium since there
is a dissociation of functional groups on the par-
ticle' surface and adsorption of ionic species of
the aqueous dispersion medium [39]. The mea-
surement of this parameter allows to clarify the
stability of the formulation during its storage
time. The higher the zeta potential, the higher
the electrostatic repulsion between the particles,
resulting in reduction of the risk of particles'
aggregation. According to literature, a zeta
potential higher than the 30 mV modulus gua-
rantees the stability of SLNs [45]. However, there
are SLN formulations with zeta potential below
|30 mV| that remain stable over time due to
the stereochemical stability offered by surfac-
tants [46]. Changing a formulation by varying
the concentration of its components, allows
understanding which of those whether
contribute to the stereochemical stability or to
the electrostatic stability [47]. Differential scan-
ning calorimetry, based on the study of the peaks
of SLN formulation recorded on the thermo-
gram, allows assessing if the drug is loaded
within the particles and eventually its contribu-
tion to the surface electrical charge of SLNs [48].
 3. Physicochemical properties
3.4 Particle morphology
To learn about the shape and size of nanopar-
ticles, scanning (SEM) and transmission electron
microscopy (TEM) are commonly used [50].
Atomic force microscopy (ATM) is another tech-
nique that gives information with high resolu-
tion in three dimensions at the nanometer scale
(even surface details at the atomic level), and is
also used to understand the surface morphology
of nanoparticles [39].
3.5 Differential scanning calorimetry
The physical and chemical changes of a sam-
ple can be measured as a function of its temper-
ature; differential scanning calorimetry (DSC)
quantifies the loss or heat gain resulting from
these changes. There are two types of DSC in-
struments: (1) the power compensate DSC,
which is made of two separate ovens; and (2)
the heat-flux DSC, which has only an oven that
heats up the reference and sample pans. The
sample and reference receive the heat through
the sample pan, which is placed in a disc that
is the main source of heat. Both differential
heat flow and sample temperature are moni-
tored, and the calorimetric sensitivity is main-
tained by the software linearization of the cell
calibration [52]. The endothermic processes, i.e.,
those that absorb heat, include fusion (melting),
boiling, sublimation, vaporization, desolvation,
as well as solid-solid transitions. The crucial
exothermic process, i.e., the one that releases en-
ergy, is crystallization. These analyzes can be
used to identify materials, investigate their pu-
rity, polymorphism or solvation, analyze quanti-
tative and qualitative degradation, aging, glass
transition temperature, as well as their affinity
to other substances. DSC is useful to obtain
information about the degree of crystallinity of
the lipid matrix and polymorphic behaviour. In
7
DSC analysis, a melting point depression is usu-
ally observed when transforming the bulk lipid
into lipid nanoparticles [48,52].
3.6 Stability of formulations and release
profile
The stability of the loaded drug within lipid
nanoparticles is dependent on the chemical
composition of the lipid matrix (e.g., type of
lipid, surfactant) and production procedure.
Upon storage, triglycerides undergo polymor-
phic changes that may result in drug expulsion
from the lipid nanoparticles [53]. Surfactants
also play a relevant role on the crystallization
behavior of the lipid nanoparticles, i.e.,
whether the recrystallization of the particles
occurs onto their surface or within the lipid
core [54]. The long-term stability of lipid nano-
particles over storage is therefore dependent
on the lipid and surfactant composition of the
particles and of the production procedure
[55]. The presence of imperfections in the lipid
matrix offers higher capacity to accommodate
drug molecules, improving the loading capac-
ity of the particles [19,56]. To achieve a modi-
fied release profile of the loaded drug, the
particles should however retain the drug dur-
ing storage until their administration. To
increase the encapsulation efficiency, the use
of mixed lipids (having fatty acids with
different liquid and solid chain lengths) is usu-
ally recommended. These blends create small
liquid reservoirs inside the particles e as hap-
pens in the NLCs e delaying the polymorphic
changes over storage [18, 53, 57]. The encapsu-
lation efficiency of lipophilic drugs is also
higher in SLNs and NLCs than hydrophilic
drugs. To load these latter in lipid matrices,
other strategies are needed such as the devel-
opment of insoluble conjugates between the
8 1. Solid lipid nanoparticles (SLN)
lipid and the drug by a covalent bonding [58,
59]. Hydrophilic drugs might show higher
risk to be partitioned to the aqueous phase dur-
ing the production of SLNs and create a drug-
enriched shell model. Indeed, upon cooling of
the dispersions, the lipid solidifies first, crystal-
lizes and forms the cores in which the hydro-
philic drug will be precipitating onto their
surface. This type of SLNs (drug-enriched shell
model) does not exhibit a modified release pro-
file but rather a fast release attributed to the
presence of drug onto the surface of the
SLNs. For drugs that solidify first (e.g., of
melting point higher than that of the solid
lipid) or for lipophilic drugs, a drug enriched
core model can be produced, which exhibits a
modified release profile [17]. Typical methods
used for assessment of the in vitro release are
the dialysis and the static or dynamic Franz
diffusion methods. The assays can be designed
so that isotonicity and pH value can be
adjusted to mimic the intended administration
route, and can also include the effect of protein
adsorption, plasma compatibility, whole blood
compatibility and sterilisation. It is known that
in a physiological environment, proteins can
bind the surfaces of nanoparticles, forming a
nanoparticleeprotein complex that influences
the biological response. Nanoparticles can be
incubated with bulk serum, plasma and also
with solutions of individual proteins, in order
to evaluate which physical properties are
responsible for the protein binding onto their
surface [60], such as the type of polymer used
to stabilize the particles [61,62]. Freeze-drying
may also be used to enhance the long-term
stability of SLNs and NLCs. Lipid
nanoparticles can be transformed in a dry
product to improve their physicochemical
stability [63].
4. Administration routes and drug
bioavailability
Pharmaceutical nanotechnology comes up as a
strategy to improve the bioavailability of poorly-
water soluble drugs, enhancing their therapeutic
effectiveness and reducing the risk of adverse re-
actions [64e67]. SLNs have been extensively
exploited as an interesting approach to improve
the drug’s bioavailability in particular those of
class II and IV of the biopharmaceutical classifica-
tion system (BCS) [64, 67]. Indeed, due to their
lipid composition, they may act as absorption en-
hancers [67]. On the other hand, surfactants sur-
rounding the particles besides ensuring their
steric stability in aqueous dispersion, they induce
specific surface-chemical properties and may also
modulate the biopharmaceutical profile. For the
selection of the best surfactant, several parameters
have to be taken into account e.g., hydrophilic-
lipophilic balance (HLB) values, their effect on
the lipid polymorphism and on the particle size.
The HLB values for the stabilization of oil-in-
water dispersions vary between 8e18 [68]. The
right choice of the surfactant minimizes the
risk of production of particles’ aggregates which
may compromise the stability of the dispersion
in vitro and its performance in vivo [45].
4.1 Topical and dermal routes
The administration of drugs through the skin
may contribute to increase the drug’s bioavail-
ability as it overcomes the first-pass meta-
bolism. This administration route reduces the
in inter/intra-patient variation and increases
patient compliance. However, it is also associ-
ated with interactions of drug and/or excipients
with skin that may cause irritation [69]. The
 4. Administration routes and drug bioavailability
loading of drugs within lipid nanoparticles can
minimize the risk of skin irritation and aller-
genic reactions, by preventing direct contact
between the drug and the skin and by control-
ling the drug release through the skin
[14,70e72]. Besides, as they are composed of
biocompatible and physiological lipids of
GRAS status, SLNs and NLCs exhibit low risk
of acute and/or chronic toxicity [10]. Most of
the surfactants used in the production of SLNs
and NLCs are already used in topical pharma-
ceutical or cosmetic formulations. To further
reduce the risk of irritation, the surfactants
should be non-ionic, while polyethoxylated sur-
factants should also be avoided
[14,42,49,73e75]. There is evidence that most
of the PEG-free surfactants have shown to stabi-
lize lipid nanoparticles without the need of
cosurfactants. In addition, when compared to
the PEG-containing counterparts, the PEG-free
surfactants have been shown to require less con-
centration of surfactant to obtain small and uni-
form particle size [76]. The methods used to
evaluate the skin irritation include typical in
vitro test methods based on the reconstructed
human epidermis (RhE) and also in vivo animal
tests. In vivo experiments are more useful but
their cost, tight regulation, and ethical issues to-
wards the promotion of reduction, reuse, and
recycling imply the need to develop improved
in vitro tests [77]. New strategies to decrease
skin irritation are based on the use of controlled
release systems, i.e., creating a gradual drug de-
livery that prevents the accumulation of high
concentrations of drug in the skin, which are
usually responsible for this skin irritation, and
also increase drug deposition in the piloseba-
ceous unit, which reduces the dose frequency
and risk of adverse events [78]. Lipid nanopar-
ticles have additional advantages as they can
prevent and even reduce skin irritation by the
reinforcement and repair of the stratum
9
corneum lipid film. Indeed, these nanoparticles
create a protective lipid film onto the skin upon
their topical application, promoting skin hydra-
tion [76].
4.2 Ocular delivery
For the treatment of eye diseases, direct ocular
instillation is the most accepted approach by pa-
tients. Lipid nanoparticles have also gained inter-
est as drug carriers for this administration route,
due to their biocompatibility with the ocular tis-
sues, mucoadhesiveness and modified-release
profile [79e81]. Conventional ophthalmic solu-
tions have low precorneal retention time. Lipid
nanoparticles increase the retention time of ocular
drugs, improving their bioavailability [82]. To be
suitable for ocular instillation, lipid nanoparticles
should be of small particle size to avoid blurred
vision and discomfort [83]. To avoid damage of
the corneal tissues, inflammation and immuno-
logic reactions, the formulations also need to
exhibit sterility, isotonicity and pH between
7e9. Toxicity of the formulations that could alter
the corneal epithelial integrity or disrupt the tis-
sue, resulting in deficient drug delivery into eye
(which is not their aim) also need to be consid-
ered [84]. The Draize rabbit eye test is routinely
used to evaluate eye irritation. This test was
developed with the aim to predict human eye irri-
tation of pharmaceutic and cosmetic products. Its
drawbacks are the ethical issues associated with
the use of animals, its costs and the number of
variables of the test [85].
4.3 Oral administration
Oral delivery is painless and is easy for self-
administration. This administration route has
high patient compliance and is appropriate for
outpatients. All these advantages make it the
most accepted drug administration route [86].
10 1. Solid lipid nanoparticles (SLN)
However, the gastrointestinal tract has chemical
and enzymatic barriers that limit the effective-
ness of oral drug delivery, and also show low
permeability for several drugs [87]. By opti-
mizing the formulations, it is possible to amelio-
rate their efficiency and bioavailability,
promoting the therapeutic potency and reducing
side effects. Lipid nanoparticles may be used as
absorption enhancers through the gastrointes-
tinal tract to improve the oral bioavailability of
several drugs [88]. SLNs developed for oral
administration have been shown to enhance
and control drug delivery, mainly due to the spe-
cific characteristics of the surface modification,
increased permeation of the gastrointestinal
tract, as well as resistance against degradation.
The solid state of the matrix can protect chemi-
cally unstable drugs and promote the drug resi-
dence onto the site of absorption. SLNs have
shown low cytotoxicity against mammalian cells
and high tolerance in vivo. They can be further
formulated in classical dosage forms e.g., cap-
sules, tablets or pellets for oral administration
[89, 90]. To improve the formulation stability,
SLN conversion into powders for further pro-
cessing into solid dosage forms have also been
proposed. Shegokar et al. investigated the possi-
bility of enhancing the long-term stability of
nanoparticles with freeze-drying studies, con-
verting SLNs in a dry product and observing
that have good size distribution, which also
proves that SLNs are a suitable drug delivery
system [63]. There is still a demand to design
and develop new SLNs to obtain a viable release
profile, which depends on the drugs selected for
these formulations as different drugs have
different physicochemical characteristics and
also different interactions with nanoparticles.
The release profile from solid dosage forms
needs to be critically controlled; a burst drug
release can be linked to toxicity problems
whereas a slow drug release that can cause
inefficient activity in treating the diseases [91].
Many oral SLN tests in cell-based and animal
studies are described in the literature, however,
their clinical trials are still limited due to their
cost, as well as the unknown side effects (they
have to be investigated first). From the commer-
cial point of view, to be viable the nanoparticles
have to show 5-fold-improved oral bioavail-
ability or other convincing benefits [91].
4.4 Parenteral administration
Until now, typical SLN components have not
yet been used in parenteral formulations, except
medium-chain triglycerides (MCTs) and poly-
sorbate 80 [92]. Usually, the final suspension
for injection contains solid particles, which
means that its safety profile will have to be
confirmed. It is known that the parenteral
dispersion should not show any solid particles
visible to the naked eye. The admissible limits
of solid particles of size below 50, 25, and
10 mm are not yet defined, and are still open to
discussion, and there is no available regulation
of particles with size below 1 mm. SLNs have
shown intensity with a diameter below 1 mm
and, in some cases, below 200 nm, but it is neces-
sary to take into account that they have a ten-
dency to aggregate over time (especially when
exposed to high ionic strength environment or
proteins) [14,93,94]. The key parameters to be
evaluated for parenteral application include un-
derstanding of plasma drug profile, fate of nano-
particles, and acute and chronic dose toxicity.
Some studies have been published on bare and
surface-coated nanoparticles for parenteral
application, lab to large scale, targeting, toxicity
studies, solid product conversion, phagocytic
uptake studies, cytotoxicity studies, as well as
organ distribution studies. For example, Shego-
kar et al. evaluated the potential of lipid nano-
particles for active delivery of an antiretroviral
 5. Conclusions
drug to lymphatic tissues. SLNs were developed
taking account of various physicochemical
parameters, e.g., appearance, particle size, poly-
dispersity index, as well as zeta potential.
Authors investigated the targeting potential of
these SLNs through ex vivo cellular uptake
studies, showing an enhanced uptake in compar-
ison to pure drug, and the lymphatic drug levels
and organ distribution studies also showed the
efficiency of these nanoparticles for prolonged
residence. The study demonstrated that these
lipid carriers can be used for effective and tar-
geted drug delivery, enhance the therapeutic
safety and decrease collateral effects [95,96].
4.5 Nasal and pulmonary delivery
Lipid nanoparticles are also a new approach
for controlled drug delivery to lungs. There are
various studies of SLN formulations with the
aim of administration by inhalation. Many that
have been developed for delivery of antifungal
and antimicrobial drugs have proved efficacy
in vivo. Nasal administration is commonly used
to deliver drugs to the central nervous system
(CNS) in a noninvasive way, thereby providing
higher patients' compliance. SLNs for intranasal
administration are gaining more attention lately
but there are still few reports about their safety;
adverse effects have been observed in
laboratory animals, probably due to
encapsulated drugs, inappropriate SLN doses
used for these studies, or faulty selection of
excipients [14].
5. Conclusions
SLNs and NLCs are the most studied lipid-
based drug delivery systems, having potential
to deliver drugs and also nutrients for several
administration routes due to their biocompati-
bility, low toxicity, high-loading capacity, slow
release rate, and high stability. They are in devel-
opment as drug carriers for administration by
11
various routes, including dermal, ocular, and
oral, with the dermal route the safest. Lipid
nanoparticles are composed of biocompatible
and biodegradable lipids, with a melting point
above 40 C to ensure solid status at room and
also at body temperatures. SLN production is
based on the incorporation of the drug in the
melted lipid and then mixed with the aqueous
surfactant solution, and they can be made by
high energy techniques (e.g., ultrasound
methods and supercritical fluid technologies) or
low-energy techniques (e.g., solvent emulsifica-
tion evaporation, coacervation, microemulsion
technique, and phase-inversion temperature
method). Lipid nanoparticles protect the drug
against chemical degradation and achieve
controlled drug release, once the drug is entrap-
ped in a biocompatible lipid core surrounded by
a surfactant at the outer surface. Melt tempera-
ture and crystallinity index as well as the selec-
tion of excipients and SLN dose are their most
important features. They need to be evaluated
for final product stability, their thermal
behavior during storage, as well as their safety.
Nanoparticles have physicochemical properties
that give them exceptional biological activity,
with their toxicological profile dependent on
these properties, mainly particle size and size
distribution, as well as zeta potential. This is
crucial in toxicological studies because it allows
assessing their toxic effects, identifying routes
of exposure as well as predicting the risks of
their synthesis or use. The potential toxicity
and the biocompatibility of drug delivery for-
mulations are crucial for the implementation
of drug therapies. Although the toxicity mecha-
nisms of nanoparticles have been well studied,
the available information about nanoparticle-
based drug therapies is still very limited. In
summary, the development of SLN and NLC
formulations continues to grow, with many pat-
ents created worldwide. Toxicological testing
documents that lipid nanoparticles are safe
drug carriers for the various administration
routes.
12 1. Solid lipid nanoparticles (SLN)

Abbreviations [3] Al-Qushawi A, et al. Preparation and characterization

of three tilmicosin-loaded lipid nanoparticles: physico-
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3 Rs Replace, reduce, and refine
Iran J Pharm Res 2016;15(4):663e76.
7AAD 7-amino-actinomycin D
[4] S
anchez-L
opez E, et al. Lipid nanoparticles (SLN,
ATM Atomic force microscopy
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AUC Area under the curve
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BCS Biopharmaceutical classification system
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BHT Butylhydroxytoluene
C):58e69.
CNS Central nervous system
[5] Doktorovova S, Souto EB, Silva AM. Hansen solubility
DMSO Dimethyl sulfoxide
parameters (HSP) for prescreening formulation of solid
DNA Deoxyribonucleic acid
lipid nanoparticles (SLN): in vitro testing of curcumin-
DPPH 2,2-DIPHENYL-1-PICRYLHYDRAZYL
loaded SLN in MCF-7 and BT-474 cell lines. Pharm Dev
DSC Differential Scanning Calorimetry
Technol 2017:1e10.
EE% Encapsulation efficiency
[6] Chantaburanan T, et al. Effect of binary solid lipid ma-
FBS Fetal bovine serum
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GRAS Generally recognized as safe
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HIV Human immunodeficiency virus
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HLB Hydrophilic-lipophilic balance
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HPH High-pressure homogenization
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HPLC High-performance liquid chromatography
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TEM Transmission electron microscopy
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PBS Phosphate-buffered saline
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Acknowledgments [12] Seyed Yagoubi A, et al. Preparation, characterization
The authors acknowledge the financial support received from and evaluation of physicochemical properties of
Portuguese Science and Technology Foundation (FCT/MCT) phycocyanin-loaded solid lipid nanoparticles and
and from European Funds (PRODER/COMPETE) under the nanostructured lipid carriers. J Food Meas Char 2017;
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 C H A P T E R

2
Role of nanocarriers and their surface
modification in targeting delivery of
bioactive compounds
Heni Rachmawati1,2, Atsarina Larasati2, Annis Catur Adi3,
Ranjita Shegokar4
1
School of Pharmacy, Bandung Institute of Technology, Bandung, Indonesia; 2Research Center for
Nanosciences and Nanotechnology, Bandung Institute of Technology, Bandung, Indonesia; 3Faculty of
Health, University of Airlangga, Surabaya, Indonesia; 4Capnomed GmbH, Zimmern, Germany

1. Bioactive compounds as promising are listed in various databases [1]. The

therapeutic agents
Apart from their typical other uses, plants are
a potential source for medicinal purposes. The
exploration of therapeutic functions of
substances present in plants has been done since
long before prehistoric age. The practical reasons
of traditional ways of medicine using plant
materials for a wide range of human ailments
are increase in population, insufficient drug
supplies, prohibitive cost of treatments, adverse
effects of several synthetic drugs, and develop-
ment of resistance to currently used agents for
communicable diseases.
According to the World Health Organization,
around 80% of people worldwide depend on
medicinal plants, in some cases for their primary
health care, and around 21,000 plant species
show potential benefits for human health and
continuing interest in exploring health benefits
of plants is evidence of the safety or minimal
adverse events of herbal medicines, a part of
the nature sync belief. A few of the species that
contain important antioxidants to cure a wide
range of diseases and have proven safe for
long-term use are described in this chapter,
including Curcuma sp, Zingiber sp, Silybum maria-
num L, Gnetum gnemon, and Physalis angulate.
1.1 Curcuma sp
Curcuma genus is classified under the family
Zingiberaceae. The genus consists of more than
80 species [2]. They grow abundantly in Asia,
including Southeast Asia, China, India, New
Guinea, and Northern Australia [3]. All curcuma
sp have similar flower spikes that arise from the
top of the pseudo stem or sometimes on a

Nanopharmaceuticals
https://doi.org/10.1016/B978-0-12-817778-5.00002-6 17 © 2020 Elsevier Inc. All rights reserved.
18 2. Role of nanocarriers and their surface modification in targeting delivery of bioactive compounds

separate stem directly from the rhizome. They

differ mainly in the inner part of rhizomes,
which vary in color, i.e., white, cream, yellow,
orange, blue, bluish-green, and black [3]. In the
family of curcuma, particularly the rhizomes
show economic potential both for food and med-
icine. For many years, the rhizomes of curcuma
have been used for food additives, spice, and
condiments. More importantly, the potential
therapeutic value of curcuma’s rhizomes are
reported elsewhere to cure various pathological
conditions, including cancer [4e11]. Various
species of Curcuma have been studied and
reported such as Curcuma amada Roxb, Curcuma
aromatica, Curcuma caesia Roxb, Curcuma longa,
Curcuma manga, Curcuma purpurascens, Curcuma
xanthorriza, and Curcuma zedoria. Plants
belonging to the genus Curcuma are gaining
importance worldwide and are an interesting
topic for investigation and exploration. The
plants contain bioactive molecules with various
biological activities such as antiinflammation,
antimicrobe, anti hypercholesterolemia, anti-
rheumatic, antivirus, antifibrosis, antivenomous,
antihepatotoxicity, antidiabetes, anticancer, and
gastroprotector (Fig. 2.1).
The common phytoconstituents found in Cur- FIGURE 2.1 Chemical structure of xanthorrhizol (1), cur-
cuma are phenols, flavonoids, alkaloids, terpe- cumin (2), demethoxycurcumin (3), bisdemethoxycurcumin (4).
noids, tannins, saponins, steroids, glycosides.
Among other phytoconstituents in Curcuma, Ginger originated in Southeast Asia. It has
xanthorrizhol and curcuminoid are two impor- been cultivated for thousands of years as a spice
tant substances showing similar bioactivities as well as for medicinal purposes in countries
and many health-promoting benefits, are being like India, China, Nigeria, Indonesia,
widely explored for therapeutic purposes [12]. Bangladesh, Thailand, Philippines, and Jamaica.
It is also grown in Australia, Fiji, Brazil, Sierra
Leone, Japan, United Kingdom, United States,
and Saudi Arabia. Ginger is an herbaceous rhizo-
1.2 Zingiber officinale matous perennial, reaching up to 90 cm in height
Ginger, scientifically known as Zingiber offici- under cultivation. It is widely used in a variety of
nale Roscoe, belonging to family Zingiberaceae, foods because of its nutritional value and
is one of the most important plants with several flavoring compounds. Ginger rhizomes are a
medicinal, nutritional, and ethnomedical values, rich source of carbohydrates, vitamins, minerals,
and hence explored extensively all over the and iron. Phytochemical analysis describes the
world as a spice, flavoring agent, and herbal content of ginger rhizome with a variety of phar-
medicine [13]. macological effects. Z. officinale is reported to
 1. Bioactive compounds as promising therapeutic agents 19
contain essential oils, phenolic compounds, silydianin, and silychristin. Silibinin is the most
flavonoids, carbohydrates, proteins, alkaloids, active of the three, and is largely responsible
glycosides, saponins, steroids, terpenoids, and for the hepatoprotective benefits attributed to
tannins as the major phytochemical groups silymarin (Fig. 2.3).
[13,14]. Ginger possesses its characteristic organ-
oleptic properties due to two classes of constitu-
ents: the odor and the flavor, determined by the 1.4 Gnetum gnemon
constituents of its steam-volatile oil. The volatile Gnetum gnemon L. (Gg) or melinjo is a plant
oil consists mainly of the mono- and sesquiter- growing abundantly in tropical areas such as
penes; camphene, b-phellandrene, curcumene, Southeast Asia. This plant belongs to the genus
cineole, geranyl acetate, terphineol, terpenes, of Gnetum (Gnetaceae family) [21,22]. The plant
borneol, geraniol, limonene, b-elemene, is a small- to medium-sized evergreen tree with
zingiberol, linalool, a-zingiberene, b-sesquiphel- nearly conical crown and a stature of 10e15 m.
landrene, b-bisabolene, zingiberenol, and a- Zin- The stem is well branched and possesses a cylin-
giberol are the principal aroma-contributing drical bole and a diameter up to 40 cm. Leaves
components of ginger rhizome [14] (Fig. 2.2). are petiolate, ovate-oblong, or elliptical,
10e20 cm long and 4e7 cm broad, reticulately
veined, glabrous and shiny, dark green, apex
1.3 Silybum marianum L acute to subacuminate, margins entire, base
Silybum marianum (L.) Gaertn, whose com- acute and phylotaxy opposite; young leaves are
mon name is milk thistle, is an edible plant reddish purple. Inflorescences are borne on
belonging to the Asteraceae family [15,16]. Milk young shoots and older branches.
thistle is a native of the Mediterranean region
and has also spread to East Asia, Europe,
Australia, and the Americas. Since the first cen-
tury, milk thistle has been used medicinally
[17e20]. Its flowers, leaves, and roots have
been used in the European diet as vegetable,
and its achene is used as a coffee. It is considered
as a spinach substitute. In 1968, a flavonolignan
complex in milk thistle fruit was identified and
isolated. The major bioactive substituent present
in the plant is the flavonoid complex silymarin,
which is about 80% of the extract. This complex
was found to be responsible for the medicinal
effects of the silymarin complex and is made
up of three parts: silibinin (also called silybin),

FIGURE 2.3 Chemical structure of silibinin (1), silydianin

FIGURE 2.2 Chemical structure of zingiberol. (2), and silychristin (3).
20 2. Role of nanocarriers and their surface modification in targeting delivery of bioactive compounds
G. gnemon is one of the most studied wild
plants. The plants from the family of Gnetaceae
have been used as traditional medicines for
many years. Several bioactive phytoconstituents
present in G. gnemon, such as saponins, tannins,
and Melinjo seeds contain abundant resveratrol
(stilbenoid), mainly in the form of dimers (gnetin
C). Gnetin C has been explored for more than 10
years, principally by Japanese scientists. Well-
documented clinical data is already available
and even more studies are continuing to emerge
[21e24]. It has been reported that stilbenoids
from melinjo showed strong antioxidant activity
leading to various biological effects such as anti-
inflammatory, antiaging, antihyperuricemia,
antimicrobe, anticardiovascular, and anticancer
[25] (Fig. 2.4).
1.5 Physalis angulata
Physalis angulata L. is an annual herb indige-
nous to tropical areas like Africa, Asia, and
South America including the Amazon [26]. The
plant grows up to 1 m high, plant parts are
smooth and glabrescent, it bears small, yellow-
colored flowers, and produces small, light
yellowish- orange, edible fruit sometimes
FIGURE 2.4 Chemical structure of resveratrol (1) and
gnetin C (2).
referred to as cape gooseberry or cut leaf ground
cherry. Various phytochemicals including flavo-
noids, alkaloids, and plant steroids known as
physalins (A and B), with anolides and secoste-
roids, which have never been reported before,
are present in this plant [27e30]. P. angulata L.
has been explored in recent clinical research,
with preliminary evidence demonstrating it as
an effective immune stimulant, toxic to
numerous types of cancer like leukemia, and
shown to have antimicrobial properties (Fig. 2.5).
2. Complexity of bioactive compounds
Most bioactive substances isolated from
plants are either in the form of crude extract,
fraction, and single compound show poor
bioavailability, hence have low-therapeutic
outcome, resulting from the challenging physi-
cochemical properties, instability issues, or
extensive in vivo response. Bioavailability and
solubility are key issues that have emerged
as top technical concerns in drug formulation
and delivery even for bioactive compounds.
FIGURE 2.5 Chemical structure of physalin A (1) and
physalin B (2).
 3. Biological barriers
In addition, stability of the active agents both
physically and chemically also contribute signif-
icantly during formulation development. The
main challenges faced by pharmaceutical com-
panies in drug formulation as described in
many reports [31e36] are safety (c.79%), appro-
priate therapeutic and delivery profiles (61%),
and bioavailability (57%). In oral solid dosage
forms, the top three formulation challenges
are bioavailability (41%), stability (37%), and
solubility (35%).
The fact that bioactive compounds are chal-
lenging during formulation and delivery has
been described in several reports [31e36].
As discussed in the first part of introduction,
several potential bioactive compounds exhibit a
lack of pharmaceutical properties like low solu-
bility and permeability, sensitivity toward
external factors (humidity, light, and tempera-
ture), as well as presystemic degradation upon
entering the body. All these challenges have
led to unsuccessful therapy in the clinic using
bioactive components.
3. Biological barriers
A major challenge in the drug delivery area is
how to transport the active agents across the bio-
logical system entering the blood circulation and
reaching the target of action to demonstrate the
biological effects. Several biological barriers
include the bloodebrain barrier (BBB), the small
intestine, nasal, skin, and the mucosa. Biological
barriers are designed naturally with the aim to
protect the organism from foreign materials
that can damage homeostasis and physiologic
function and eventually can threaten the
FIGURE 2.6 The physical barrier illustration of drug absorption: (A) lining endothelial cells, (B) stratum corneum,
(C) lining epithelial cells, (D) mucosal membrane.
21
organism’s survival. Different types of biological
barriers are described next to present the critical
parameters on the therapeutic failures as well as
to find the appropriate strategies or solutions by
which a bioactive can be delivered successfully
in vivo to exert desired clinical effects.
3.1 Physical barriers
For most therapeutics, reaching the targets of
action require penetration across tissues and/or
entry within cells. The design of strategies to
control the transport of therapeutic compounds
through these physiological barriers has become
an imperative and a challenging need in the
quest for better therapeutics. The physical bar-
riers for drugs entering systemic circulation are
the membrane, a biological architecture of a
membrane border, like a single-layer or multi-
layer cell lining. This also includes lining of
endothelial cells of blood vessels, stratum
corneum of skin, lining of epithelial cells of
various mucosa, BBB, blood ocular barrier, and
the mucus covering the epithelial mucosa
(Fig. 2.6).
3.2 Biochemical barriers
The biochemical barriers that can reduce the
therapeutic function of a drug are the metabo-
lizing enzymes, transporters, and efflux pumps.
Drug-metabolizing enzymes are present in
almost all parts of the body where the drug is
passing through, such as gastrointestinal tract,
respiratory system, ocular mucosa, in the blood
circulation, and other entry points of drugs to
the systemic compartment.
22 2. Role of nanocarriers and their surface modification in targeting delivery of bioactive compounds
Drug transporters are the proteins that are
present in many organs (liver, lung, kidney,
intestine, brain, skin, blood vessels, and others).
Naturally, the proteins play important roles for
traffic between organs and elimination of drugs
and foreign compounds. Their function some-
how is beneficial, but they may also display
deleterious effects that do not allow drugs to
enter the target organ to show their effects.
In the BBB, drug transporters and drug metabo-
lizing enzymes are also present to control the
access to the brain and local concentration of
endobiotics and xenobiotics.
Efflux pumps demonstrate resistance to cyto-
toxic drugs, hence also act as a barrier for drug
delivery. By their nature, efflux pumps are trans-
port proteins involved in the extrusion of toxic
substrates from cells into the external environ-
ment. These membrane proteins function as a
pump that can decrease the intracellular accu-
mulation of drug, leading to the ineffective
drug therapy. For better drug delivery, the key
to understanding how these pumps operate
involves the determination of the structures of
representative pumps and the elucidation of
the conformational changes that accompany
drug translocation.
4. Nanocarrier: a strategy to overcome
biological barriers
A primary reason for drug failing to demon-
strate its effect is the biology underlying the mo-
lecular-, cellular-, and tissue-level barriers,
which makes the delivery process extremely
complex. Therefore, to bypass the limitations
regarding the biology of conventional drug
delivery, it might be best to improve on the
concepts and approaches that are efficient and
effective [37,38].
One way to overcome barrier challenges
could be formation of effective drug delivery.
Nanoparticles, due to their smaller size and
various other properties like surface coating,
type of matrix used, and timely delivery, can
help to deliver a drug at a target site. The suc-
cessful outcome of the nanocarrier to help the
bioactive compounds show their effect upon
reaching the target site of action is also deter-
mined by the in vivo behavior of the nanocarrier,
in particular in the phase of biological membrane
passage to reach the systemic circulation, during
traveling in the circulation to reach the target site
and after reaching the target.
Various nanoparticles are being studied for
delivery of synthetic as well as bioactive drugs.
In this section, different drug delivery systems
explored for bioactive drug delivery are dis-
cussed (Table 2.1).
Different types of nanocarrier systems are
developed based on the characteristic of the
bioactive compounds as well as the aim of their
delivery target. Table 2.2 presents various nano-
systems along with their method of preparation.
4.1 Lipid-based nanocarrier systems
Considering the complex in vivo barrier sys-
tem in which the drugs are protected to be able
to survive for therapeutic presentation, various
strategies in the area of formulation and delivery
either to target the drug at specific site or to con-
trol the drug release have recently been reported
and described [31e36]. The former focuses more
on the use of the excipients, the composition, and
the manufacturing process, while the later em-
phasizes the drug-carrier constructs. The
following discussion reviews the typically used
drug delivery systems in bioactive delivery that
are effective in overcoming the biological bar-
riers, thereby improving the drug therapeutic in-
dex. In this category, we selected liposomes,
nanoemulsions, and lipid nanoparticles; other
forms of nanoparticles are still under research
and have not yet reached the market like the
above ones, hence we decided to discuss only
the relevant ones.
 4. Nanocarrier: a strategy to overcome biological barriers 23
TABLE 2.1 Bioactive-loaded nanocarrier systems and the potential medical promise.

Challenges in Added value of

Class Category Compound Desired functionality formulation nanoemulsions

Flavonoid Flavanols EGCG, catechin, Free-radical scavenging, Catechins generally - Increased

epicatechin anticancer, decreasing exhibit high water storage
cholesterol level in solubility. Partition in the stability of
blood, preventing lipophilic core is easily
arterial sclerosis, improved by adding degradable
thrombosis, heart 1-dodecanol catechins
attacks, reducing
fat and sugar uptake
Flavonols Quercetin, Free-radical scavenging, The scarce solubility of - Improved
kaempferol, fighting the effects of most flavonols in oil phase thermo- and
myricetin aging and inflammation, requires the addition of photostability
downregulating or amphiphilic molecules in - Enhanced
suppressing the lipophilic core delivery
inflammatory pathways
and functions

Flavones Apigenin, Antimutagenic, Supersaturated flavones - Improved

luteolin, rutin, antiinvasive, and in oil phases easily form physical
tangeretin antiproliferative agent crystals, requiring the stability
addition of compounds, - Bioaccessibility
such as soy protein
isolates, to slow down the
crystallization process
Flavanones Naringenin, Antiinflammatory, Flavanones exhibit poor - Increased
hesperidin anticarcinogenic, water solubility. Oil and release of
hepatoprotective, and emulsifier are added to compounds
antilipid peroxidation ensure maximum loading - Improved the
and stability in the bioavailability
nanoemulsion
Isoflavones Daidzein, Protection against Incorporation in oil Improved the
genistein, hormone-related droplets should be permeation
puerarin disorders, menopausal promoted by suitable through epidermal
symptoms, heart disease, emulsifiers (i.e., lecithin) layers
and osteoporosis
Nonflavonoids Hydroxybenzoic Gallic acid, Antiproliferative and Complexation with Increased
acids ellagic acid, antioxidant phospholipids is bioaccessibility,
p-hydroxybenzoic required to increase reduced
acid physical stability in interaction with
nanoemulsions the food matrix
Hydroxycinnamic Cinnamic acid, Free-radical scavenging, The solubility of some Improved
acids coumaric acid, preventing cell damage phenolic acids is very biological activity,
ferulic acid, by ultraviolet light, low in aqueous permeability, the
caffeic acid antiaging, decreasing solutions, but they can absence of
blood glucose easily be adsorbed at cytotoxic effect
oil/water interface of
nanoemulsions

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