317

Part IV

Applications
 319

11

Surface Modification of Nanoparticles: Methods and

Applications
Gopikrishna Moku 1,# , Vijayagopal Raman Gopalsamuthiram 2,# ,
Thomas R. Hoye 2 , and Jayanth Panyam 1
1
University of Minnesota, Department of Pharmaceutics, College of Pharmacy, 308 Harvard St SE,
Minneapolis, MN 55455, USA
2 University of Minnesota, Department of Chemistry, College of Science and Engineering, 207 Plesant St SE,

Minneapolis, MN 55455, USA

11.1 Introduction
The therapeutic potential of a drug molecule is dependent on its availability at the
target site at the requisite amount and for the required duration. In addition, it
is important to minimize the exposure of the drug to non-target tissues to avoid
potential side effects. It is estimated that greater than 70% of newly discovered
small molecules are hydrophobic and have poor aqueous solubility, limiting their
ability to be transported by blood and other body fluids [1, 2]. In addition, some
drugs undergo rapid clearance and thus have short half-life and residence time.
Delivering a drug to the right place, at the right concentration, and for the right
period of time is, thus, a challenge. Incorporation of the drug in a suitable delivery
system can overcome some of these challenges.
Drug delivery systems improve drug efficacy and safety by modifying the phar-
macokinetic properties (distribution, absorption, distribution, and elimination)
of the drug [3]. Over the past two decades, there has been an intense focus on
the use of carriers that are on the order of ∼100 nm in diameter for drug delivery.
This particle size range enables systemic administration because the smallest
blood capillaries are 10–20 μm in diameter. Further, carriers in this size range
could be used for targeted delivery of different types of therapeutic payloads to
specific organs and tissues [4]. Nano-delivery systems with different architec-
tures have been developed, including lipid nanoparticles, micelles, dendrimers,
polymeric conjugates, solid-lipid nanoparticles, and inorganic nanoparticles
[5]. Such systems have shown promising pre-clinical activity in various diseases
such as AIDS, cancer, malaria, diabetes, and tuberculosis [6–10], and some of
these have, in fact, been approved for human use [11]. This chapter will discuss
the various polymers used in the formulation of nanoparticles, fabrication and

#
Equal contribution.

Surface Modification of Polymers: Methods and Applications, First Edition.

Edited by Jean Pinson and Damien Thiry.
© 2020 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2020 by Wiley-VCH Verlag GmbH & Co. KGaA.
320 11 Surface Modification of Nanoparticles: Methods and Applications

characterization techniques, surface modification methods, and the different

types of targeting ligands that have been evaluated.

11.2 Polymers Used in the Preparation of Nanoparticles

Various materials are available for the fabrication of nanoparticles including poly-
mers, lipids, inorganic compounds (silica, silicate), and metals (gold, silver, iron).
Nature has also designed nanosize particles, specifically viruses, that have been
co-opted for tissue-specific gene delivery [12]. Due to their stability, drug load-
ing capacity, and tunable properties, polymers have specific advantages as a drug
carrier. Biodegradable polymers are more advantageous than nonbiodegradable
materials for drug delivery applications because of the need for easy removal
of the carrier after drug release [13]. The selection of polymer for fabricating
nanoparticles depends on the desired size and surface characteristics of the
particle as well as the nature of the drug or active ingredient. Physicochemical
properties of the polymer determine the fabrication process to be employed.

11.3 Common Biodegradable Polymers

for Nanoparticle Fabrication
Table 11.1 lists some of the polymers commonly used in the fabrication of
nanoparticles. Natural polymers tend to be hydrophilic, while synthetic poly-
mers can be hydrophobic or hydrophilic. A brief description of each of these
polymers is provided in the following text. Structural features of these polymers
are shown in Figure 11.1.

11.3.1 Albumin
Albumin is a natural transport protein that delivers vitamins, minerals, and other
hydrophobic compounds such as steroids to various tissues. This natural trans-
port function, its ability to internalize into different cell types, and multiple drug
binding sites provide the rationale for its use in drug delivery. Importantly, albu-
min is constituted by a single polypeptide chain of 585 amino acids and contains
a low amount of methionine and tryptophan and a large amount of glutamic acid,
cysteine, lysine, aspartic acid, and arginine. Another major advantage of albumin
in drug delivery is that the therapeutic drug of interest can be easily attached
covalently or non-covalently. Albumin is an endogenous protein and hence is
highly biocompatible. In addition, it has functional groups that can be used to
®
bind different ligands and complex drugs (e.g. paclitaxel in Abraxane , insulin
® ®
detemir Levemir , GLP-1 in Victoza ) [14, 15].

11.3.2 Alginate
Alginate, a naturally occurring anionic polysaccharide of α-l-guluronic acid
and β-d-mannuronic acid repeating units linked by a 1 → 4 linkage, is widely
used for pharmaceutical applications. It is biodegradable, nontoxic, inexpensive,
 11.3 Common Biodegradable Polymers for Nanoparticle Fabrication 321

Table 11.1 Examples of polymers used for preparation of nanoparticles in drug delivery.

Nature of the
polymer Name of the polymer Advantages Disadvantages

Natural Albumin Easily available Structural

Alginate Generally complexity
Chitosan nontoxic Batch-to-batch
Gelatin Biodegradable variations
Synthetic Polylactide (PLA) Biocompatibility Generally
Poly(lactide-co-glycolide) (PLGA) more
Possibility of expensive than
Poly(ε-caprolactone) (PCL)
sustained natural
Poly(malic acid) (PMLA) polymers
release
Polyacrylamide (PAM)
Poly(isobutyl cyanoacrylate) (PIBCA) Large diversity Can be
Poly(isohexyl cyanoacrylate) (PIHCA) in functional polydispersed,
Poly(n-butyl cyanoacrylate) (PBCA) groups depending on
the synthetic
Poly(acrylate) and poly(methacrylate)
Properties can method
(Eudragits)
be tuned
Poly(vinyl alcohol) (PVA)
Polyethylene glycol (PEG)
Poly(lactide)–polyethylene glycol
(PLA-PEG)
Poly(ε-caprolactone)–polyethylene
glycol (PCL-PEG)
Poly(lactide-co-glycolide)–polyethylene
glycol (PLGA-PEG)
Tween 20 and Tween 80
Dextran

and readily available and has been found to be a mucoadhesive, biocompatible,

and non-immunogenic material. Specifically, the simple aqueous-based gel
formulation of alginate in the presence of divalent cations such as Ca2+ has been
used in the preparation of alginate delivery systems [16].

11.3.2.1 Chitosan
Chitosan (CS) is a modified natural cationic polysaccharide prepared by chemical
deacetylation of chitin, the second most abundant natural biopolymer after cellu-
lose, and is derived from crustacean shells [17]. The primary amino groups in the
polymer backbone of CS provide positive charge to the polymer. Because of its
mucoadhesivity, CS has been regarded as a potential carrier for oral drug delivery.
Another important feature of CS is its metabolic degradation in the body. While
small molecular weight CS can be eliminated renally, large molecular weight CS
can be degraded by endogenous enzymes. The rate and extent of degradation
depends on the molecular weight and degree of acetylation of the polymer. CS has
many potential applications including for drug delivery through the oral, nasal,
transdermal, parenteral, vaginal, cervical, and rectal routes [18].
322 11 Surface Modification of Nanoparticles: Methods and Applications

O O O OH
O O O
O H H OH H OH
HO O
O n n OH n
n mO n n
PLA PLGA PEG PGA PVA PAA
O

O O O O
O O O
O O O O
n n O O

m n
PCL PHBV PBAT

N O O S
O O
H
H n
n
(a) PVP PSF

CN
O
N
O
n n O
R O O O
O O
n
14
PCA
(b) [Polycyanoacrylates] PHDCA PVMMA

Figure 11.1 Some of the polymers most commonly used in nanoparticle preparation.
(a) Commercially available. (b) Non-commercial.

11.3.3 Gelatin
Gelatin is a natural, biocompatible, biodegradable, and multifunctional protein
for use in controlled drug release. It is a polyampholyte having both cationic
and anionic groups as well as hydrophobic groups, and it can be obtained from
acid/alkaline/enzymatic hydrolysis of collagen. The gelatin molecule chain con-
tains ∼13% lysine and arginine (imparts positive charges); ∼12% glutamic and
aspartic acid (provides negative charges); ∼11% leucine, isoleucine, methionine,
and valine (imparts hydrophobicity) amino acids; and ∼64% glycine, proline, and
hydroxyproline amino acids. Commercially, gelatin is available as both cationic
(gelatin type A, isoelectric point (pI) 7–9) or anionic (gelatin type B, pI 4.8–5)
protein without the necessity for additional functionalization [19].

11.3.4 Poly(lactide-co-glycolide) (PLGA) and Polylactide (PLA)

Poly(lactide-co-glycolide) (PLGA) and polylactide (PLA) polyester polymers have
been extensively studied for drug delivery. They are widely used because they
undergo hydrolysis in the body and produce biologically compatible monomers
lactic acid and glycolic acid, which can be further metabolized via the citric acid
cycle. The degradation of PLGA and PLA is an autocatalytic process, in which
acidic degradation products generated in the interior of the carrier accelerate the
degradation reaction. The drug release from PLGA and PLA matrices depends
on both drug diffusion through the polymer matrix and the polymer degradation
rate. A wide spectrum of PLGA polymers with different molecular weights and
 11.4 Fabrication of Nanoparticles 323

lactide-to-glycolide weight ratios, which determine the biodegradation and drug

release rates, are available commercially. Polymers with higher molecular weight
usually exhibit lower degradation rates compared with lower molecular weight
polymers. The Food and Drug Administration (FDA) and European Medicine
Agency (EMA) have approved the use of PLGA and PLA in humans [20].

11.3.5 Poly-𝛆-caprolactone (PCL)

Poly-ε-caprolactone (PCL) is another synthetic aliphatic polyester that has been
investigated extensively for use in controlled drug delivery systems [21, 22]. It is a
biocompatible, biodegradable, and hydrophobic polymer suitable for drug deliv-
ery applications. It can form compatible blends with other polymers. Owing to
its slow biodegradation, it is ideally suited for long-term delivery, extending over
a period of more than one year.

11.4 Fabrication of Nanoparticles

Techniques used for the fabrication of nanoparticles can be broadly classified
based on whether a preformed polymer is used or the polymer is produced in
situ with the concomitant formation of nanoparticles.
This approach has been extensively studied for preparation of nanoparticles
of various sizes. Some common techniques that utilize preformed polymer
include:
(i) Emulsification–solvent evaporation [23]
(ii) Emulsification–solvent diffusion [24]
(iii) Salting out [24]
(iv) Nanoprecipitation [25]
(v) Dialysis [26]
(vi) Supercritical fluid technology [27]
Surfactants are used to stabilize nanoparticles, and various surfactants can be
used for this purpose. Surfactants serve as a stabilizer during the manufacturing
process and during nanoparticle use by offering a protective coating around the
nanoparticle and preventing aggregation. The most common surfactants used in
this process (Figure 11.2) include polysorbate 20, sodium cholate, cetyltrimethy-
lammonium bromide (CTAB), didodecyldimethylammonium bromide (DDAB),
and polyvinyl alcohol (PVA). In some instances, amphiphilic block copolymers
such as poly(lactide-co-glycolide)–polyethylene glycol (PLGA-PEG) or PEG-PLA
have been used based on their surface activity.
This is achieved through polymerization of monomers into a polymer that
encapsulates a drug during the fabrication process. This is typically done via two
approaches: (i) emulsion polymerization [28] or (ii) interfacial polymerization
[29]. A key limitation of these methods is that the monomers used are often
nonbiodegradable and can result in non-biocompatible by-products. Extensive
purification is often needed to remove monomers or initiators to afford an
324 11 Surface Modification of Nanoparticles: Methods and Applications

O
O
O
w
OH Br
O
OH x N
O O
z y OH n
HO CTAB
Polysorbate 20 PVA
OH

O
Stearic acid

O
OH OH
ONa n
Brij-35
H H O
H H N
HO OH
H
Sodium cholate DDAB

O
S
O O
O Sodium dodecyl sulfate (SDS)
O O
H O
n O
O O
H O OH
O
x y z
TPGS Pluronic F-68

Figure 11.2 Surfactants commonly used in nanoparticle preparation.

acceptable pharmaceutical formulation. Recently, some newer methods have

been reported for the preparation of nanoparticles. These include high frequency
thermal plasma methods [30], spray and microemulsion methods, laser ablation
method [31], flash creation and mechanochemical bonding methods, and spray
pyrolysis [32].

11.5 Linker Chemistry for Attaching Ligands

on Polymeric Nanoparticles
To develop an effective polymer-based targeted drug delivery system, it is critical
to immobilize one or more functionalities/ligands on the surface of nanoparticles
(Figure 11.3). To achieve this, it is necessary to chemically modify the surface of
nanoparticles with a suitable linker to introduce targeting moieties. In addition,
the targeting ligand must have a functional group that can be used for conjuga-
tion. A wide range of conjugation approaches have been studied, and the specific
method used depends on the nanoparticle preparation technique, surface chem-
istry, and the functional group available in the ligands. Polymeric nanoparticle
surface modification methods can be broadly divided in two categories.
 11.5 Linker Chemistry for Attaching Ligands on Polymeric Nanoparticles 325

Antibody

PEG
H O
e
rid
O cha
n H ac
lys
Po

Mannose
Drug-loaded Surface functionality Drug-loaded id
polymer nanoparticle polymer nanoparticle Lip

Biotin

er
tam
Ap
Folic acid

l
dio

e
tid
p
ra

Pe
Est
Figure 11.3 Graphical representation of surface-functionalized polymeric nanoparticles
loaded with drugs (incorporated within the matrix of the polymer), targeting molecules
(e.g. antibodies, peptides, aptamers, and small molecular ligands) for targeted drug delivery.
Left: drug-loaded nanoparticle; right: drug-loaded nanoparticle with surface functionalization.

Chemical conjugation allows for stable incorporation of the targeting ligand on

the surface of nanoparticles. However, this requires the presence of a functional
group on the surface that can subsequently be used to attach the targeting ligand.
Various functional groups have been immobilized onto the surface of polymeric
nanoparticles including carboxylic acids, amines, thiols, alcohols, maleimides,
azides, aldehydes, and alkynes. Some of these covalent conjugation methods are
summarized in Table 11.2.
Formation of an amide bond is a widely used method to conjugate targeting lig-
ands onto the surface of polymeric nanoparticles. Formation of the amide takes
place in two steps. In the first, carboxylic acid group(s) present on either the
polymer nanoparticles or the conjugating ligands are activated using 1-ethyl-3-
(3-dimethylaminopropyl)carbodiimide (EDC) hydrochloride or dicyclohexylcar-
bodiimide (DCC). Since EDC is readily soluble in water, it is often preferred over
DCC. Frequently, N-hydroxysuccinimide (NHS) or sulfo-NHS is used along with
EDC to stabilize the highly active intermediate generated via EDC and increase
the efficiency of the coupling reaction. In the following step, primary amine(s)
present in either the conjugating ligand or the polymeric nanoparticles react with
the activated ester to form stable amide bonds between the conjugating ligand
and nanoparticles. The main advantage with this conjugation reaction is that it
does not require any initial modification of the targeting ligand, which can result
in loss of activity. In a recent study, Feng et al. modified polymeric nanoparti-
cles by trans-activator of transcription (TAT) peptide or anti-EpCAM antibody
using EDC-NHS reaction [33]. Bare particles had greater negative ζ potential due
to the higher number of carboxylic acid groups compared with TAT/antibody
conjugated particles. Moreover, bare nanoparticles showed greater mobility in
gel compared with TAT/antibody conjugated nanoparticles. In another study,
polymeric nanoparticles with carboxylic acid surface groups were modified with
 326 11 Surface Modification of Nanoparticles: Methods and Applications

Table 11.2 Schematic representation of covalent conjugation methods.

Type of Stability under

covalent physiological
conjugation Linkage Reaction scheme conditions

Acid/amine Amide O O Highly stable

L
bond NP
OH + H2N
L
EDC NH
NP
DCC

Maleimide/ Thio–ether O Highly stable

O
thiol bond HS S
NP N +
L NP N L

O
O

Thiol/thiol Disulfide NP SH + HS L NP Cleavable

S S L
under
reducing
conditions
Hydrazide/ Hydrazone O O O Highly stable
NH2 N
N
aldehyde NP H + H L NP
NH
L

Azide/alkyne Triazole N N Highly stable

NP + N N+
(click ring N– L NP N
L
chemistry)

NP, polymeric nanoparticle; L, ligand.

a targeting antibody (anti-CD63) using EDC/NHS coupling. Anti-CD63 antibod-

ies were detected on the nanoparticle surface by a secondary anti-IgG1 antibody
against the fragment crystallizable (Fc) region of the targeting antibody [34].
Thio–ether bond formation is another popular covalent conjugation method
in which a maleimide group reacts with a thiol to form a stable thio–ether
bond. Typically, amine groups in the targeting ligands are converted into
free thiol/activated sulfhydryl group using Traut’s reagent (2-iminothiolane),
N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), or N-succinimidyl-S-
acetylthioacetate (SATA), followed by reduction with the reducing agent dithio-
threitol (DTT)/tris(2-carboxyethyl)phosphine (TCEP) hydrochloride. In the
second step, the thiol group in the targeting ligand is reacted with maleimide
groups on the surface of nanoparticles. This reaction is quick and highly efficient
and occurs in aqueous solutions under mild conditions. This bond is stable for
24 hours even under reducing conditions [35]. The main disadvantage with this
bond is that conversion of amines in the targeting ligand into free thiols may
lead to loss in activity. In addition, side reactions such as intermolecular rear-
rangement or formation of disulfides makes this reaction less selective toward
the thio–ether bond formation in aqueous solutions. Our group successfully
conjugated imino-thiolated anti-CD133 antibody to maleimide functionalized
PLGA nanoparticles. First, amines in the anti-CD133 antibody were converted
into free thiols using 2-iminothiolane. In the next step, iminothiolated antibody
was added to maleimide functionalized PLGA nanoparticles. The conjugation
 11.5 Linker Chemistry for Attaching Ligands on Polymeric Nanoparticles 327

of anti-CD133 antibody was confirmed by observing bands corresponding to

heavy and light chains in Western blotting [36].
The disulfide bond formation occurs by the conjugation of thiol groups
present on nanoparticles and thiol groups in the targeting ligands. In the case of
antibodies, free thiol groups can be formed by reducing disulfide linkages with
DTT. Free thiols can be introduced in targeting ligands by allowing it to react
with Traut’s reagent/SPDP/SATA. The main disadvantage with this bond is its
instability in presence of reducing agents, which hinders its use for in vivo appli-
cations. In addition, competing disulfide formation between two of the same
thiol (homo-coupling) can lower the selectivity of the desired cross-coupling
reaction.
Due to its high conjugation efficiency, click reactions represent a promising
conjugation strategy for the immobilization of biological ligands on to the surface
of polymer nanoparticles. A hallmark of a click reaction is that it is highly specific
in the presence of many other types of common functionality and in aqueous
solutions and the products are stable. In 2002, the Sharpless and Meldal groups
independently developed the copper(Cu-I)-catalyzed alkyne-azide cycloaddition
(CuAAC). For some drug delivery applications, copper-free cycloaddition
reactions are desired over copper-catalyzed reactions. Copper ions interact with
biomolecules and can cause toxicity. Recently, Layek et al. applied click chemistry
in vivo to deliver dibenzocyclooctyne (DBCO) functionalized, paclitaxel-loaded
PLGA nanoparticles for cancer therapy [37]. They developed a two-step tumor
targeting strategy based on mesenchymal stem cells (MSCs), which actively
traffic tumors. First, they expressed non-natural azide groups on the surface
of MSCs using glycoengineering protocols. These glycoengineered MSCs were
intravenously administered into lung tumor bearing mice. In response to
inflammatory signals, these glycoengineered MSCs actively trafficked to tumor
sites. In the second step, intravenous administration of DBCO functionalized
PLGA nanoparticles resulted in their accumulation in tumor tissues that were
pre-targeted with MSCs.

11.5.1 Hydrazone Bond Formation

Acid cleavable, hydrazone bonds are readily formed between a hydrazide group
and an aldehyde group. In general, targeting ligands do not contain aldehyde
functional groups. The hydroxyl groups present in targeting ligands can be
oxidized to aldehydes in the presence of various oxidizing agents [38]. In a recent
study, Liu et al. developed a dual pH-responsive multifunctional polymeric
nanoparticle system based on poly(l-histidine) (PHIS) and hyaluronic acid (HA)
[39]. They first prepared resiquimod (R848, a TLR7/8 agonist) loaded PHIS
(PHIS/R848) nanocores. Next, a polymeric prodrug HA-doxorubicin (DOX)
was prepared through hydrazone bond linkage. Finally, the polymer prodrug
HA-DOX was coated on the surface of PHIS/R848 nanocores to form HA-DOX/
PHIS/R848 nanoparticles. These multifunctional nanoparticles significantly
inhibited tumor growth by regulating both tumor immunity and killing tumor
cells in a 4T1 murine tumor model.
328 11 Surface Modification of Nanoparticles: Methods and Applications

11.5.2 Non-covalent Attachment

Non-covalent interactions are also used to attach targeting ligands to polymeric
nanoparticles. In general, this technique does not require any pre-modification
of targeting ligand or nanoparticle because the immobilization of ligand
onto the surface of the nanoparticles occurs through simple physical contact.
Non-covalent immobilization depends on attractive forces between the surface
of nanoparticles and the targeting ligand, including electrostatic interactions,
hydrogen bonding, hydrophobic interactions, and van der Waal’s interac-
tions. For example, tetrameric glycoprotein avidin (67 kDa) and streptavidin
(60 kDa) form a strong, non-covalent complex with the small molecule biotin
(244 Da), with a dissociation constant K d around 10−14 M [40]. Due to strong
irreversible binding, this noncovalent interaction has been used to functionalize
nanoparticles with targeting ligands. Recently, Angsantikul et al. developed
gastric epithelial cell membrane-coated PLGA nanoparticles loaded with
clarithromycin antibiotic by simply mixing PLGA nanoparticles and the cell
membrane [41]. These biomimetic nanoparticles retained the original surface
antigens of the source cells and showed preferential adhesion and retention with
Helicobacter pylori bacteria. These membrane-coated nanoparticles have shown
increased therapeutic efficacy compared to free drug in a mouse model of H.
pylori infection.
On the other hand, physical interactions are not specific, more labile, and
less reproducible compared to covalent conjugation methods. Importantly, it is
difficult to control the orientation of the targeting ligand, which in turn can affect
activity. For example, electrostatic attractions mainly depend on the surface
charge of nanoparticles and the targeting ligands. In the case of antibodies, their
orientation on the surface of nanoparticles relies on the position of charged
groups in the tertiary structure of the proteins. It also depends on the pH of
solution and the isoelectric point (pI) of the antibody and nanoparticle. If the
pH of the solution changes, the antibody may detach from the surface of the
nanoparticles. In addition to electrostatic interactions, hydrophobic interactions
can also cause a change in the tertiary structure of the protein, resulting in loss
of activity [42].
An interesting approach that seeks to eliminate the inefficiency and complex-
ity associated with the previously mentioned modification processes is based on
a dopamine polymerization technique. The product polydopamine (PDA) has
a unique ability to deposit on polymeric nanoparticles upon simple immersion
of nanoparticles in an aqueous dopamine solution buffered to pH 8–8.5. Subse-
quently, amine or thiol-containing molecules can be conjugated onto the PDA
layer via Michael addition or Schiff-base reactions [43].

11.6 Surface-functionalized Polymeric Nanoparticles

for Drug Delivery Applications
Polymeric nanoparticles offer an attractive platform for the delivery of thera-
peutic agents due to their unique physical properties that stem from a size range
 11.6 Surface-functionalized Polymeric Nanoparticles for Drug Delivery Applications 329

that is comparable to and compatible with biomolecular and cellular systems.

However, the nature of the nanoparticle surface determines its interaction with
the biological environment. Thus, the goal of nanoparticle surface functional-
ization is to affect these interactions so as to increase the circulation half-life
and minimize off-target drug exposure. This section discusses some examples
of nanoparticle surface functionalization and the important criteria to consider
during the fabrication process. In addition, the importance of surface chemistry
and potential drug delivery applications are described. Surface functionalization
strategies can be categorized based on the nature of the ligand that is being
conjugated:
(i) Functionalization with macromolecules such as polysaccharides, lipids,
peptides, antibodies, and nucleic acids (aptamers).
(ii) Functionalization with small molecule ligands such as mono or oligosaccha-
rides, steroids, and vitamins.
(iii) Functionalization with hydrophilic polymers.
Owing to their specific affinity for target sites, macromolecules such as polysac-
charides, lipids, peptides, and nucleic acids (aptamers) are widely used as recog-
nition ligands. In general, biomolecules can be coated on polymeric nanoparticles
via chemical bond formation between biomolecules and the substrate surface
either directly, through a spacer, or by noncovalent physisorption.

11.6.1 Polysaccharides
Polysaccharides have the advantages of biocompatibility, ease of availability, and
well-established modification schemes [44]. Polysaccharides such as dextran,
CS, HA, and heparin can provide steric protection against protein adsorption
and macrophage uptake and are recognized as stealth-coating materials [35].
Additionally, several studies have demonstrated the active targeting properties
of polysaccharides such as HA [45], CS [46], and chondroitin sulfate [44].
Some examples of polysaccharide-coated polymeric nanoparticles and their
applications are summarized in Table 11.3.

11.6.2 Lipids
Lipid-coated polymeric nanoparticles provide a range of advantages in drug
delivery, including a broad range of flexible strategies and ease of surface
engineering, extended circulation half-life, reduced cytotoxicity, and better
target specificity [66]. Natural phospholipids such as phosphatidylglycerol,
phosphatidylcholine, phosphatidylinositol, phosphatidylserine, phosphatidic
acid, and phosphatidylethanolamine and their synthetic counterparts {e.g. N-[1-
(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl-sulfate (DOTAP),
1,2-dipalmitoyl-3-trimethylammonium-propane (DPTAP), 1,2-distearoylphos-
phatidylethanolamine (DSPE), N-(methylpolyoxyethylene oxycarbonyl)-1,2-
distearoyl-sn-glycero-3-phosphoethanolamine (DSPE-PEG), 1,2-dipalmitoyl-sn-
glycero-3-phosphocholine (DPPC), 1,2-dilauroyl-sn-glycero-3-phosphocholine
(DLPC)} are often used to coat the surface of polymeric nanoparticles [67].
Table 11.3 Recent examples of biomolecule conjugated polymeric nanoparticles and their therapeutic uses.

Conjugation
Class Nanoparticle Functionalization chemistry Drug Therapeutic use References

Polysaccharides PLGA Dextran Amine/acid Ifosfamide Osteosarcoma [47]

PLGA Chitosan/alginate Electrostatic Doxorubicin Cancer therapy [48]
interactions
Chitosan Alginate Dual cross-linker Naringenin Diabetes mellitus [49]
Poly(l-histidine) Hyaluronic acid Hydrazone R848/ Cancer therapy [39]
doxorubicin
PLGA Chitosan Electrostatic Tobramycin Cystic fibrosis [50]
interactions
Lipids PLGA Soybean lecithin Self-assembly Silymarin Nonalcoholic fatty [51]
liver disease (NAFLD)
PLGA DSPE-PEG2000 Self-assembly Docetaxel Cancer therapy [52]
PCL DSPE-PEG2000-NH-Fucose Self-assembly Methotrexate/ Cancer therapy [9]
aceclofenac
PLGA Gastric epithelial cell Physical Clarithromycin Helicobacter pylori [41]
membrane adsorption
PLGA DOPC/DOTAP/DSPE-PEG Self-assembly Sirolimus Restenosis [53]
Aptamers PLGA-PEG-COOH CD133 Acid/amine Propranolol Hemangioma [54]
BSA AS1411 Acid/amine Doxorubicin Cancer therapy [55]
PLGA/Chitosan 5TR1 Electrostatic Epirubicin Cancer therapy [56]
interactions
PLGA-PEG-COOH A10 Acid/amine Docetaxel Cancer therapy [57]
PLGA-COOH A10-3.2 Acid/amine Paclitaxel Cancer therapy [58]
Peptides PLGA Tet1 Acid/amine Nattokinase Alzheimer’s disease [59]
PLGA-PEG-COOH cNGR Acid/amine Docetaxel Cancer therapy [60]
PLGA-PEG-Maleimide iRGD Thio–ether bond Rosiglitazone/ Obesity [61]
PEGE2
PVDF CBO-P11 Click reaction NA Angiogenesis [62]
PFT-PS/PEG-COOH TAT Acid/amine Fluorescent dye Cancer cell imaging/ [33]
photodynamic
therapy
Antibodies Polystyrene Anti-CD63 Acid/amine and NA NA [34]
physical
adsorption
PEGylated Anti-Aβ1-42 Biotin-streptavidin Fluorescent dye Alzheimer’s disease [63]
P(HDCA-co-MePEGCA)
PLGA/PLA-PEG-Maleimide Anti-CD133 Thio–ether bond Paclitaxel Cancer therapy [36]
PLGA KIM-1 Acid/amine Resveratrol Chronic kidney [64]
disease
Multi-block copolymer Trastuzumab/folic acid Acid/amine Doxorubicin Cancer therapy [65]
PFT-PS/PEG-COOH Anti-EpCAM Acid/amine Fluorescent dye Cancer cell imaging/ [33]
photodynamic
therapy
332 11 Surface Modification of Nanoparticles: Methods and Applications

Due to the amphiphilic nature of the phospholipids, they can form membrane
mimetic structures on nanoparticles. Hydrophobic interactions and electrostatic
attraction are the major chemical forces responsible for the lipid self-assembly
process on polymeric nanoparticle surfaces [67]. A variation of this approach is
the use of membranes from various cells including red blood cells, neutrophils,
and T cells to coat the nanoparticle surface. For example, Zhang et al. prepared
neutrophil membrane-coated nanoparticles by fusing neutrophil membrane
onto polymeric nanoparticle cores. These nanoparticles inherit the antigenic
exterior and associated membrane functions of the source cells. In addition,
these nanoparticles neutralized proinflammatory cytokines, suppressed synovial
inflammation, targeted deep into the cartilage matrix, and provided strong
chondroprotection against joint damage [68].

11.6.3 Aptamers
Aptamers are single-stranded (ss) short oligonucleotides (6–30 kDa) that can
interact with cellular targets such as nucleic acids, transmembrane proteins, or
sugars with high affinity and selectivity by acquiring defined three-dimensional
secondary and tertiary structures [69]. Aptamers are synthetically prepared
from an initial library containing 1013 –1016 random ssDNA or ssRNA sequences
through an in vitro selection process termed systematic evolution of ligands by
exponential enrichment (SELEX) [70]. In this cell-based method, first a library
of oligonucleotides is incubated with the target of interest, and the ones that
have higher affinity are enriched and purified in the following steps. Aptamers
have distinct advantages over traditional antibodies, including ease of isolation,
smaller size, higher ratio of target accumulation, lack of immunogenicity, and
higher in vivo stability [70]. Importantly, aptamers do not have an Fc region,
which interacts with soluble Fc receptors or Fc receptors expressed on immune
cells and other certain type of cells. This obviates undesired interactions follow-
ing systemic administration, which may result in immune stimulation or other
unforeseen side effects. Also, for the treatment of solid tumors, an antibody’s
high molecular weight hinders its ability to penetrate deep into a tumor. The
molecular weight of an aptamer is usually in the range of 6–30 kDa, much
smaller than that of an antibody (∼150 kDa), which often leads to better tumor
uptake kinetics. In addition, an aptamer has greater stability than a protein in
biological fluids and lower production costs. However, the lack of an Fc region
limits their circulation half-life compared with that of an antibody, although this
is not a major impediment when using the aptamer as a targeting ligand.

11.6.4 Antibodies
Antibodies are attractive as targeting ligands because of their excellent target
specificity and affinity. The conjugation of polymer nanoparticles with antibodies
can impart high target recognition capability to nanoparticles. However, stable
conjugation of the antibody to a nanoparticle surface in the correct orientation
while avoiding aggregate formation is crucial for successful functionalization.
To conjugate the antibody covalently to nanoparticles, a suitable functional
 11.6 Surface-functionalized Polymeric Nanoparticles for Drug Delivery Applications 333

group must be introduced onto the nanoparticle surface and into the antibody
molecule. Amino (lysine), carboxy (glutamic acid and aspartic acid), and
sulfhydryl (cysteine) are the most widely used functional groups in antibodies.
Physical adsorption of antibodies on the surface of nanoparticles has been
investigated as well. In a recent study, Tonigold et al. demonstrated that the
pre-adsorption of antibodies on the surface of polymer nanoparticles resulted
in efficient targeting of nanocarrier compared with nanoparticles that were
chemically attached to the antibody [34]. In that study, binding affinity of the
antibody chemically conjugated to nanoparticles was significantly affected by
protein corona formed when nanoparticles were introduced in a biological fluid.
Physically adsorbed antibodies, on the other hand, remained functional and were
not affected by the protein corona. Monoclonal antibodies (mAb) targeting var-
ious markers and receptors present on cancer cells including human epidermal
growth factor receptor-2 (HER2), αvβ3 integrin, prostate-specific membrane
antigen (PSMA), and CD20 antigen on B-cell lymphomas have been used
for nanoparticle functionalization. However, the presence of full-length mAbs
(∼150 kDa) can affect the tumor penetration of the nanoparticle and also result in
increased macrophage uptake via Fc receptor (FcR) identification. To minimize
these issues, antibody fragments such as single-chain variable fragment (scFv)
and antigen-binding fragment (Fab) have been investigated as targeting ligands.

11.6.5 Peptides
Short tissue-homing peptides offer the advantages of high stability, reduced
immunogenicity, and ease of synthesis and conjugation to nanoparticles.
However, the design of a small peptide molecule that fits into a usually shallow
and hydrophobic binding pocket on the target can be challenging. These homing
peptides are typically identified by phage display technology [71], which is a
screening tool that allows the selection of peptide sequences against specific
target tissues. Various peptides have been used as targeting ligands and can be
divided into two groups: cell-penetrating peptides (CPPs) and cell-targeting
peptides (CTPs). CPPs have the ability to enter cells, and they have been
employed in cellular delivery of biologically active therapeutics [72]. Among the
various CPPs, TAT peptide (∼1.5 kDa) is a well-known cationic CPP sequence
derived from the human immunodeficiency virus 1 (HIV-1) protein [14]. It
enhances cellular uptake of nanoparticles by interacting with proteoglycans of
the cell membrane. Unlike CPPs, CTPs interact in a cell- or tissue-specific man-
ner. Arginine–glycine–aspartic acid (RGD)-containing peptides (435–784 Da),
which interact specifically with αvβ3 integrin overexpressed on cancer cells and
tumor microvasculature endothelial cells, is an example of a CTP [73].
Because small molecule ligands can recognize certain markers or receptors
present on the surface of target cells, a wide variety of ligands have been
incorporated onto polymeric nanoparticle surfaces, allowing them to be used in
intracellular drug delivery [74]. Functionalization of nanoparticles with a small
molecule ligand has been shown to increase cellular uptake of nanoparticles
via receptor-mediated endocytosis, resulting in enhanced drug delivery and
therapeutic efficacy [75]. The major advantages of using a small molecule as
334 11 Surface Modification of Nanoparticles: Methods and Applications

the targeting ligand over macromolecules is their stability, ease of conjugation

with nanoparticles, and low cost. The drawbacks associated with the use of
small molecules as targeting ligands include the lack of an efficient approach to
identify/develop such ligands, and their lower specificity and affinity for surface
receptors on the target cells.
In general, rapidly dividing cancer cells require large amounts of certain vita-
mins, such as vitamin B9 (folic acid), vitamin B12, and vitamin H (biotin); there-
fore, the receptors involved in vitamin uptake are overexpressed on the surfaces
of cancer cells. Folic acid has been investigated extensively as a targeting ligand
for clinical applications. Folic acid is a high affinity ligand of endogenous folate
receptor, which is frequently overexpressed in many types of human cancer cells.
Several reports have been demonstrated that folic acid-functionalized polymeric
nanoparticles can be actively internalized via receptor-mediated endocytosis
resulting in enhanced therapeutic efficacy in various tumor models [76]. Our
group designed and developed folate and biotin functionalized PLGA/PLA-PEG
nanoparticles for tumor targeted drug and nucleic acid delivery [77].
Oligosaccharide–lectin interactions have been exploited for the targeting of
carbohydrate ligands to site-specific target receptors. Due to the low affinity of
mono/oligosaccharides with cell surface receptors, multiple carbohydrate ligands
are often required to achieve the required binding strength. One well-known
example uses galactose or galactose-mimics (e.g. N-acetylgalactosamine
(NAcGal)) as ligands to asialoglycoprotein receptor (ASGPR), an endocytic
cell surface lectin receptor primarily expressed on the sinusoidal surface of
hepatocytes [78]. The mannose receptor (CD206 or MR) is a 175 kDa C-type
lectin receptor present on cell surface of macrophages and immature dendritic
cells. Carbohydrates such as mannose, fucose, and N-acetyl glucosamine were
used to enhance the binding and uptake of the polymeric nanoparticles by
macrophages or dendritic cells [79].
Estradiol is a steroid hormone that can specifically bind to estrogen receptors
(ER) overexpressed on a variety of cancer types including ovarian, breast,
and endometrial cancers. Being an endogenous molecule, estradiol is also
biocompatible and non-immunogenic, which makes it a promising ligand for
targeted cancer therapy. Estradiol-functionalized polymeric nanoparticles have
been developed for the efficient tumor targeting and improved therapeutic
outcomes [80].
Some selected recent examples that demonstrate the utilization of small
molecule ligands for targeted delivery of polymeric nanoparticles are summa-
rized in Table 11.4.

11.6.5.1 Polyethylene Glycol (PEG)

An important characteristic of an effective targeted delivery system is its
ability to stay in systemic circulation for prolonged periods of time. Phagocytic
uptake by macrophages is the primary mechanism of particle clearance from
systemic circulation. Surface functionalization of nanoparticles with hydrophilic
polymers such as PEG (2–20 kDa) can increase the circulation half-life of
polymeric nanoparticles [88]. Surface modification with hydrophilic polymers
Table 11.4 Recent examples of small molecule ligand-functionalized polymeric nanoparticles and their therapeutic uses.

Conjugation
Nanoparticle Functionalization chemistry Drug Therapeutic use References

Poly(methacrylic Biotin Acid/amine Doxorubicin/ Cancer therapy [81]

acid-co-histidine/ imiquimod
doxorubicin/biotin
PLGA/polydopamine Folic acid/RGD Michael addition Doxorubicin Cancer therapy [78]
PLGA-PEG-Dopamine Dopamine Acid/amine FK506 Type 1 Diabetes [82]
Chitosan Folic acid Acid/amine Temozolomide (TMZ) Cancer therapy [83]
PLGA-PEG-estradiol 17β-estradiol Acid/amine Docetaxel (DTX) Cancer therapy [80]
Chitosan Mannose Reductive amination Curcumin Visceral leishmaniosis [84]
PLGA Monosaccharides 2-(2-Aminoethoxy) NA NA [79]
ethanol linker
Poly-(ε-caprolactone)/ DCL (pseudomimetic Acid/amine Epillgallocathechin- Cancer therapy [85]
PLGA-PEG-COOH dipeptide) 3-gallate (EGCG)
PLGA/PLA-PEG-DBCO DBCO Click reaction Paclitaxel Cancer therapy [37]
Chitosan Cysteine Acid/amine Amoxicillin Helicobacter pylori [86]
PLMB Carborane Acid/alcohol NA Cancer therapy [87]
Chitosan-TPE Galactose/dopamine Michael addition Tetraphenylethene Cancer therapy [78]
336 11 Surface Modification of Nanoparticles: Methods and Applications

creates a steric barrier and delays plasma protein adsorption or “opsonization,”

which is a critical first step in macrophage-mediated uptake of nanoparticles
[88]. This suppression of nonspecific interactions with the blood components
leads to reduced blood clearance of polymeric nanoparticles (“stealth effect”).
Furthermore, PEG increases the stability of polymeric nanoparticles during
storage and in aqueous dispersions by reducing the tendency of particles to
aggregate (steric stabilization) [89].
Although PEGylation is effective in prolonging polymeric nanoparticle blood
circulation time after intravenous administration, its presence on the surface of
a nanoparticle can reduce the nanoparticle interaction with target cells. This is
referred to as the “PEG dilemma.” [90] This issue can be overcome by function-
alizing the nanoparticles with cell- or receptor-specific ligands (in addition to
PEG), which further increase the nanoparticle interactions with target cells. The
presence of PEG can also result in the generation of anti-PEG antibodies and in
hypersensitivity reactions in some patients. In addition to PEG, other synthetic
hydrophilic polymers such as polyvinyl pyrrolidone (PVP), polyaminoacids,
poloxamers, and PVA have been employed as stealth coatings [88].

11.7 Characterization of Surface-modified

Nanoparticles
Characterization of nanoparticles for size, morphology, and surface charge
has been done using various microscopic techniques including scanning elec-
tron microscopy (SEM), and transmission electron microscopy (TEM). These
techniques have also been used to understand the variation in the physical
properties of particles after surface modification. The average particle diameter,
their size distribution, and charge affect the physical stability and their in vivo
distribution. Electron microscopy techniques are very useful in ascertaining the
overall shape of polymeric nanoparticles, parameters that influence their safety
and biodistribution. For example, disc-shaped particles distribute more to the
lungs than to the liver. The surface charge of nanoparticles affects the physical
stability and redispersibility of the polymer dispersion as well as their in vivo
performance. Many techniques have been used to study the surface modification
of nanoparticles.

11.7.1 Particle Size

Particle size distribution is an important parameter that affects the biological
performance of nanoparticles. Further, particle size affects the drug release rate,
which is a key performance parameter. Smaller particles offer larger surface area
for a given volume. On the other hand, smaller particles also tend to aggregate
during storage. Hence, there is often an optimal size that balances biological per-
formance with stability [91]. Polymer degradation can also be affected by the par-
ticle size. There are several tools for determining nanoparticle size as discussed
in the following text.
 11.7 Characterization of Surface-modified Nanoparticles 337

11.7.2 Dynamic Light Scattering (DLS)

The most popular and convenient method of determining particle size is photon-
correlation spectroscopy (PCS) or dynamic light scattering (DLS). DLS has been
widely used to determine the size of Brownian nanoparticles in colloidal
suspensions in the nano- and submicron ranges. Wang et al. reported the
preparation of a zwitterionic polymer capsuled protein-based nanogel by
in situ free radical polymerization on the surface of an acryloylated bovine
serum albumin (BSA) protein with 2-methacryloxyethyl phosphorylcholine
using N,N ′ -methylene bis(acrylamide) (BIS) and N,N,N ′ ,N ′ -tetramethylene
ethylenediamine (Scheme 11.1) [92]. Ammonium persulfate was used to initiate
the radical polymerization. Dialysis was then used to obtain the BSA nanogel,
which was surface modified by using EDC and N-hydroxy succinimide to
enable conjugation to the TAT peptide (Scheme 11.1). The size of the resulting
nanoparticles was observed to increase after surface modification using TAT
peptide from 5.24 nm for blank BSA to 19.46 nm for acrylic BSA to 22 nm for
TAT-5-BSA (Figure 11.4). Varying levels of conjugation was achieved by varying
the equivalents of TAT peptide used in the conjugation step.
O
O
O N O
BIS

MPC + AA

BSA

O O
O O P O N+
MPC
O–

O H H
N N
AA BIS
OH O O

GRKKRRQRRRPP-CH3O

Scheme 11.1 Synthesis of a zwitterionic polymer capsuled protein nanogel. Source: Wang
et al. 2017 [92]. https://creativecommons.org/licenses/by/3.0/.

11.7.3 Scanning Electron Microscopy (SEM)

In a typical scanning electron microscope, a focused electron beam is scanned
over a surface to create an image. The electrons in the beam interact with the
sample, producing signals that are often used to assess surface topography and
composition. Thus, SEM allows morphological examination with direct visual-
ization of the surface.
338 11 Surface Modification of Nanoparticles: Methods and Applications

Native BSA (5.24 ± 1.00 nm)

30 TBSA (7.45 ± 0.78 nm)
nBSA (18.33 ± 6.80 nm)
Acrylic nBSA (19.46 ± 5.35 nm)
TAT-1-nBSA (21.29 ± 4.66 nm)
TAT-5-nBSA (22.00 ± 3.83 nm)
TAT-10-nBSA (24.31 ± 4.42 nm)
Intensity (%)

15

0
1 10 100 1000
Hydrodynamic diameter (nm)

Figure 11.4 DLS image and sizes of BSA, TBSA, nBSA, acrylic nBSA TAT-1-nBSA, TAT-5-nBSA,
and TAT-10-nBSA nanoparticles. Source: Wang et al. 2017 [92].
https://creativecommons.org/licenses/by/3.0/

Prior to analysis by SEM, dry nanoparticles are mounted on a sample holder,

followed by coating with a conductive metal, such as gold, using a sputter coater.
The sample is then scanned with a focused fine beam of electrons and, upon
emission of secondary electrons from the sample surface, certain surface char-
acteristics of the sample are revealed. The limitations of this method are that
nanoparticles must be able to withstand vacuum and the electron beam-induced
damage. Often, the mean particle size obtained by SEM is compared with that
from DLS measurements, thereby providing additional experimental evidence
for evaluation of that important parameter.
As an example, surface-modified biodegradable polymeric nanoparticles
from PEG-PLGA was used to deliver GSE24.2 peptide intracellularly to cells
for the treatment of dyskeratosis congenita and other telomerase disorders by
Egusquiaguirre et al. [93] The GSE24.2 peptide was produced from Rosetta
2-gami cells by treatment with pGATEVGSE24.2 and lysates. PLGA and
PEG-PLGA nanoparticles were surface modified with GSE24.2 peptide by a
water-in-oil-in-water double emulsion solvent evaporation method. A variety
of CPPs were prepared by sequential deprotection, coupling, and deprotec-
tion using an Fmoc/Boc/Alloc solid-phase approach on a MBHA resin [94].
Cationic polymeric nanoparticles were prepared from PEG-PLGA nanopar-
ticles containing the GSE24.2 peptide either by treatment with dextran in
the aqueous phase or with polyethyleneimine in the organic phase. The CPPs
were then conjugated with the cationic polymeric nanoparticles containing the
GSE24.2 peptide using EDC/sulfo NHS chemistry. SEM analysis of the particles
before and after conjugation with CPPs showed uniform spherical morphology
(Figure 11.5).
 11.7 Characterization of Surface-modified Nanoparticles 339

(a) (b) (c)

Figure 11.5 SEM images of (a) blank PEG PLGA NPs, (b) PEG PLGA NPs with GSE24.2
(c) surface-modified PEG PLGA NPs containing GSE 24.2 with CPP. Source: Egusquiaguirre et al.
2015 [93]. Reproduced with permission of Elsevier.

11.7.4 Transmission Electron Microscopy (TEM)

TEM is complementary to SEM; similar insight to nanoparticle properties
can be ascertained, although the principle is slightly different from that of
SEM. Because the key requirement in TEM is for the sample to be ultrathin
to enable electron transmittance, the sample preparation time and procedure
can be longer. Usually, the nanoparticle dispersion is typically deposited onto
support grids or films and either stained using a negative staining agent such as
phosphotungstic acid or uranyl acetate or by plastic embedding onto the surface
to enable the dispersion to withstand vacuum. Another approach is to flash
freeze an aqueous dispersion of nanoparticles, which results in embedding of the
sample in vitrified water. The surface properties of the sample are obtained when
a beam of electrons is transmitted through the ultrathin sample, interacting
with the sample as it passes through. An example application of TEM is its
use to characterize quercetin PLGA nanoparticles coated with BSA-bound
Adriamycin (ADR) [95]. In this case, the PLGA nanoparticles with quercetin
were prepared by an emulsion solvent evaporation method. ADR bound to BSA
(BSA-ADR) was independently prepared. PLGA nanoparticles were then added
to the BSA-ADR complex and sonicated. Incubation and ultracentrifugation
resulted in the formation of BSA-ADR-coated nanoparticles. TEM showed
the presence of three distinct layers, interpreted as one being the drug in the
core of nanoparticles, the second being the polymer encapsulating the core, and
the third being the BSA-ADR coating on the surface (Figure 11.6).

11.7.5 Surface Charge

The intensity and nature of the surface charge of nanoparticles determines
their interaction with the biological environment. The colloidal stability of
nanoparticles is determined by their ζ potential [96], which can be defined as
the potential difference that exists between the bulk solvent and the stationary
layer of solvent attached to the particle. The measurement of ζ potential enables
one to assess the storage stability of colloidal dispersion and predict the surface
hydrophobicity. High ζ potential values, either positive or negative, increases
stability by minimizing aggregation of particles. ζ Potential can also provide
information regarding the nature of material encapsulated within the particles or
340 11 Surface Modification of Nanoparticles: Methods and Applications

Figure 11.6 Transmission electron

microscope image of a
nanoformulation showing polymer
capsule, encapsulated quercetin, and
surface modification.

200 nm
CRNN (CU)

coated onto the surface. For example, polyquercetin (pQCT) nanoparticles were
synthesized by oxidative self-polymerization of quercetin under basic conditions
using sodium periodate [97]. PEGylation of the nanoparticles was done in bicine
buffer at pH 9 and using mPEG5K-NH2 . pQCT@PEG NPs were then loaded with
DOX as a model drug and as a fluorescent probe. The ζ potential of the nanopar-
ticles was measured in PBS at pH 7.4. QCT NPs exhibited a negative potential
of −14.9 ± 3.0 mV. This value significantly decreased upon PEGylation and after
encapsulation of DOX, providing further analytical evidence to substantiate
surface modification of pQCT NPs with PEG after DOX incorporation.

11.7.6 Surface Hydrophobicity

Techniques used to understand surface hydrophobicity include methods such
as hydrophobic interaction chromatography, biphasic partitioning, adsorption
of probes, and contact angle measurements. Amongst these, X-ray photon
correlation spectroscopy (XPS or XPCS) is unique in that it permits identi-
fication of specific functional groups on the surface of nanoparticles [98]. A
random diffraction or “speckle” pattern is produced when the coherent light
beam scatters light upon interaction with a disordered system. The observed
pattern is related to the precise spatial arrangement of the disordered scatterers
and can be interpreted as the instantaneous diffraction pattern of the disordered
system. If one uses a noncoherent light beam, instead of the speckle pattern,
an average over “many” speckle patterns is observed. In an XPCS experiment,
a “movie” is built by consecutive collection of a sufficient quantity of those
instantaneous images. The quercetin nanoparticles discussed earlier [95] were
also characterized by XPS spectra. The C/O ratio decreased from polyquercetin
nanoparticles upon PEGylation, which further decreased upon encapsulation of
DOX (Table 11.5). High resolution XPS spectra also showed increased nitrogen
(N1s) signals compared with the unmodified nanoparticles, indicating the
surface functionalization of the nanoparticles with mPEG-NH2 (Figure 11.7).
 11.7 Characterization of Surface-modified Nanoparticles 341

Table 11.5 Elemental composition of unmodified and modified polyquercetin nanoparticles

from X-ray photon correlation spectroscopy high resolution scans of C1s, O1s, and N1s regions.

NP C (%) O (%) N (%) C/O

pQCT 68.0 ± 3.3 31.6 ± 3.4 0.3 ± 0.2 2.15

pQCT@PEG 64.2 ± 2.9 34.7 ± 2.9 1.2 ± 0.1 1.85
pQCT@PEG@DOX 61.0 ± 0.4 37.4 ± 0.8 1.6 ± 0.4 1.63

Source: Sunoqrot et al. 2018 [97]. Reproduced with permission of RSC.

5 × 105 pQCT@PEG@DOX

405 400 395

4 × 105
Intensity (cps)

3 × 105 pQCT@PEG

2 × 105 405 400 395

1 × 105 pQCT

0
1200 1100 1000 900 800 700 600 500 400 300 200 100 0 405 400 395

(a) Binding energy (eV) (b)

Figure 11.7 (a) X-ray photon correlation spectroscopy for polyquercetin, PEGylated
nanoparticles of polyquercetin and nanoparticles loaded with doxorubicin. (b) N1s scans for
each sample showing increase in % N content. Source: Sunoqrot et al. 2018 [97]. Reproduced
with permission of RSC.

11.7.7 Fourier Transform IR (FTIR) Spectroscopy

FTIR spectroscopy has also been used as a tool to understand surface modifica-
tion of nanoparticles. An example of IR as a tool to examine nanoparticle surface
modification was done by Nivedh et al. [99] CS-PEG-PLGA nanoparticles were
surface modified with rabies viral antigen. For this purpose, the PEG-PLGA
polymer was first synthesized and the rabies viral antigen was incorporated
into the particles by emulsion solvent evaporation method. CS coating was
added by mixing CS with nanoparticles in the presence of acetic acid. The
resultant CS-PEG-PLGA rabies viral antigen nanoparticles were functional-
ized with biocompatible materials such as starch, acacia, and ovalbumin. To
confirm the surface functionalization, the infrared spectrum was recorded
for blank CS-PEG-PLGA nanoparticles and compared with those from the
polymer-treated particles. The changes in the peak positions for nanoparticles
after surface modification with rabies viral antigen with respect to the parent
unmodified CS-PEG-PLGA nanoparticles (CS NP) were noted. Significant
peak broadening across the entire regions in the case of CS-PEG-PLGA rabies
attenuated viral antigen nanoparticles with various biocompatible materials in
342 11 Surface Modification of Nanoparticles: Methods and Applications

comparison to the controls suggested the presence of viral antigen on the surface
of the nanoparticles.

11.8 Summary/Conclusion
Significant advances continue to be made for functionalizing the surface of
nanoparticles for specific therapeutic applications. Critical to the success of
surface functionalization are the methods used to functionalize the surface, the
choice of the linking agent, and the analytical methods used to characterize
such nanoparticles. Ease of synthesis and industrial scalability are significant
challenges and further research is needed to address these limitations.

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