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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5615_Библиотеки_им_академика_М_И_Перельмана.pdf
PEGylated Nanocarriers forDrug
Delivery Applications
SalomeA.Chime andMumuniA.Momoh
Abstract
Polyethylene glycol (PEG), also called macrogol, is a polyether made up of ethoxy
units often obtained by polymerizing the ring opening of ethylene oxide. PEG
polymers are generally linear and have chemically active hydroxyl groups, facili-
tating conjugate formation with other functional groups. Thus, biomolecules and
nanocarriers can form conjugates with PEG in a process called PEGylation. It is an
approach employed by others to enhance the efciency of drugs, vaccines, and
gene delivery, and it is often utilized for cell and tissue targeting. It is a process of
modication of the surfaces of particles, which usually occurs by adsorption,
entrapment, or grafting of known polymer chain lengths of PEG.Incorporating
PEG polymers into various nanocarriers improves the pharmacokinetic prole of
drugs in the nanocarriers. PEGylation also enhances the diffusion of nanocarriers
drug delivery systems across different biological systems, including the mucosal
systems, extracellular spaces, and the brain. It also improves the physicochemical
proles of varying drug delivery systems and improves their biocompatibility by
facilitating drug solubility and reducing their toxicity. PEGylated formulations
have elongated residence in the body, enhanced drug stability, reduced metabolic
enzymes-induced degradation, and minimized protein elimination. In this work,
the process of PEGylation of different nanocarriers including lipid-based and poly-
meric nanoparticles, liposomes, and dendrimers will be explored. The applications
of PEGylated nanocarriers in drug delivery will also be exhaustively discussed.
4
S. A. Chime (*)
Department of Pharmaceutical Technology and Industrial Pharmacy, University of Nigeria,
Nsukka, Nigeria
e-mail: salome.chime@unn.edu.ng
M. A. Momoh
Department of Pharmaceutics, University of Nigeria, Nsukka, Nigeria
107
108
Keywords
S. A. Chime and M. A. Momoh
Drug delivery · Generally regarded as safe · Methods of PEGylation
4.1 Introduction
A lot of active pharmaceutical agents require specialized formulation technologies
and strategies to target them to the particular organ of the body where the drug’s
effect is mainly needed. There are also signicant challenges in controlling the drug
release, biodistribution, pharmacokinetic proles, and circulation time of drugs.
Hence, different drug delivery systems (DDS) and strategies have been developed to
solve this problem, including polymeric and lipid-based nanoparticles, hydrogels,
and surface-functionalized nanoparticles (Liu etal. 2016). Surface modication of
drug carrier systems with polyethylene glycol (PEG) has been used to control nonspecic and specic reactions of drugs and some signicant components of blood
and avoid opsonization (Vllasaliu etal. 2014; Abuchowski etal. 1977).
PEG is a “stealth” polymer generally employed in drug delivery (DD) due to its
record of nontoxicity in humans. They were classied by the FDA as Generally
Regarded as Safe (GRAS) (Jain and Manoj 2010); hence, they are safe and have
received approval from the FDA for systemic applications (Haris 1992). Polyethylene
glycol (PEG), often called macrogol, is a polyether consisting of ethoxy units
obtained by polymerizing the ring opening of ethylene oxide. It is a hydrophilic
polyether diol that is nonionic linear with outstanding biocompatibility and excellent biological and physiochemical characteristics such as nontoxicity, good solubility in aqueous as well as organic solvent, the absence of immunogenicity, and
antigenicity. The neutral nature of PEG polymers emanates from the weak hydrogen
bond acids of the hydroxyl end groups and ether linkages that are mainly weakly
basic, forming their backbone. The low-grade PEGs are viscous colorless liquids,
while the high molecular weight grades (2000–5000) are waxy. Polymers of PEG
are traditionally often linear and have chemically active hydroxyl groups facilitating
conjugate formation with various functional groups. Hence, nanocarriers and biomolecules can conjugate with PEG in a process called PEGylation process (Jain and
Manoj 2010). PEG polymers enhance the delivery of therapeutics agents and have
been employed to deliver drug-entrapped micro and nanocarriers (Vllasaliu
etal. 2014).
PEGylation enhances the efciency of drugs, vaccines, and gene delivery and is
often employed to target cells and tissues (Vllasaliu etal. 2014). It is a process of
modication of the surfaces of particles, which often occurs through the adsorption,
entrapment, or grafting of known polymer chain lengths of PEG. Incorporating
PEG polymers into various nanocarriers is a functional method to enhance the pharmacokinetic prole of drugs in the nanocarriers. PEGylation also enhances the diffusion of nanocarriers drug delivery systems across different biological systems,
including the mucosal systems, extracellular spaces, and the brain. It also enhances
various drug delivery systems’ biocompatibility proles and physicochemical properties by facilitating drug solubility and reducing their toxicity. PEGylated
4 PEGylated Nanocarriers forDrug Delivery Applications
109
formulations have improved drug stability, prolonged invivo body residence, minimized toxicity, reduced degeneration by metabolic enzymes, and reduced protein
elimination. PEGylation has been used to maximally improve the pharmacokinetic
prole of different novel drug delivery nanoformulations. It also enhances the
nanoparticles’ stability giving it improved supercial energy with tendencies of
aggregation and improved diffusion via biological systems, viz. mucosal cells,
brain, and extracellular spaces (Vllasaliu etal. 2014). In this work, the process of
PEGylation of different nanocarriers, including lipid-based and polymeric nanoparticles, liposomes, and dendrimers, will be explored. Also, the applications of
PEGylated nanocarriers in drug delivery will be exhaustively discussed.
4.2 PEGylated Nanocarriers andDrug Delivery Benefits
Nanotechnology has advanced tremendously over the years and has made huge successes in drug delivery and nanomedicines. Nanocarriers include both polymeric
and lipid nanoparticles, viz. solid lipid nanoparticles (SLNs), nanostructured lipid
carriers (NLC), liposomes, micelles, and dendrimers, amon others. These nanocarriers have numerous advantages over conventional delivery systems due to their
small size and targeting capabilities, which provide site-specic delivery and
increased local concentrations, decreasing systemic toxicity (Howard etal. 2008;
Owens and Peppas 2006; Sahoo and Labhasetwar 2003; Mumper et al. 2003).
Nanocarriers can inuence the drug release prole of most drugs and are used for
specic targeting of active pharmaceutical ingredients (APIs) to signicant organs
of the body, for example, brain (Howard etal. 2008). Drug targeting is vital in cancer chemotherapy, to circumvent several toxic effects of drugs, and in the treatment
of disorders of neurons, which are limited by the inability of many drugs to cross the
blood–brain barrier (Howard etal. 2008; Torchilin 2007; Francis etal. 1996; Calv
etal. 2001; Sinha etal. 2006). Nanocarriers also protect drugs from enzymatic degradation, have low levels of toxicity, and have high stability. They also result in
improved drug solubility with good potential to be functionalized to yield controlled
release systems (Chime etal. 2014, 2022; Yildirimer etal. 2011; Ou et al. 2018;
Yang etal. 2018; Pinheiro et al. 2021). However, nanocarriers suffer from many
disadvantages despite the huge successes recorded in the eld of drug delivery,
including issues of stability and drug leakage, reticuloendothelial system blood
clearance (RES uptake), hemolytic toxicity, immunogenicity, and hydrophobicity.
These disadvantages could be improved upon by nanocarriers PEGylation (Gajbhiye
etal. 2020).
Nanoparticles (NPs) have the potential to deliver therapeutic payloads to various
tissues and organs of the body, prolonging the blood circulation time and partitioning into target organs (Suk etal. 2016). However, the administration of these NPs by
systemic drug delivery has been limited by the inuence of the mononuclear phagocyte system (MPS). The MPS includes the granulocytes, blood monocytes, dendritic cells, and tissue-resident macrophages in the liver, spleen, and lymph nodes in
charge of the clearing process and the decomposition of the exogenous materials in
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S. A. Chime and M. A. Momoh
the bloodstream (Moghimi etal. 2001). Endothelia cells in MPS-associated organs
mostly contain fenestration, which helps to screen circulating bodies based on particle size. Hence, NPs of about 100nm penetrate the endothelial fenestrae in the
spleen, liver, and lymph nodes (Alexis etal. 2008; Braet etal. 2007). MPS serves as
a defense mechanism protecting the body against pathogens and also eliminates
drug-loaded NPs rapidly from the blood. Therefore, NPs present in the bloodstream
are detected and entrapped by opsonins, which are serum proteins and complement
compounds such as bronectin, immunoglobulins, and apolipoproteins (Vonarbourg
etal. 2006), a process called opsonization. NPs are more responsive to phagocytosis
via MPS cells, hence, the need for PEGylation. Opsonization of NPs occurs mainly
by hydrogen bond interactions and hydrophobic and electrostatic interactions (Yoon
etal. 1998; Roser etal. 1998; Walkey etal. 2012; Gessner et al. 2000). However,
NPs may directly be captured by opsonin-independent scavenger receptors macrophages, which recognize mostly repeating patterns (Monopoli etal. 2012; Liu and
Liu 1996). Also, numerous other types of serum proteins readily associate with NPs
circulating within the bloodstream, yielding protein corona on the NPs’ surface that
improves NPs uptake by endothelial cells (Liu and Liu 1996; Tenzer etal. 2013). It
is worth noting that protein absorption minimizes the time of circulation of functionalized NPs and also weakens their targeting abilities (Salvati et al. 2013).
Generally, nonfunctionalized NPs are removed within 10min after systemic administration from the bloodstream, no matter their composition (Suk et al. 2016;
Moghimi etal. 2001; Salvati etal. 2013).
PEGylation technology is one of the most successful approaches used clinically
to improve the pharmacokinetic properties of drug-loaded NPs and minimize their
immunogenicity (Marwa et al. 2020; Veronese and Mero 2008). PEGylation of
nanocarriers improved the pharmacokinetics of drugs as they help to prevent the
opsonins of nanocarriers, leading to an increase in molecular weight (MW) of nanocarriers and particle size, leading to changes in the properties of nanocarriers. The
density and MW of PEG chains can alter the circulation time of nanocarriers as well
as affect the biocompatibility, size, charge, and interaction with macrophages
(Gajbhiye etal. 2020). Complement proteins, immunoglobulin, and bronectin are
the major opsonin proteins. However, dominant opsonin depends on the specic
properties of the nanocarriers, with the majority exhibiting improved adsorption of
many types of proteins (Buyens etal. 2012; Andriyanov et al. 2017). Hence, for
these nanocarriers to function as improved alternatives to the existing DDS, they
should not be readily recognized by the opsonins. PEGylation is one of the simplest
and most reliable methods of decreasing protein adsorption and imparting “stealthiness” to nanocarriers (Howard et al. 2008). The mechanisms by which PEG
improves the stealthiness of nanocarriers include the reduction in hydrophobicity
and surface charges of nanocarriers, thereby decreasing the attractive forces between
opsonin proteins and nanocarriers. Therefore, PEGylation increases the plasma
half-life of different drugs, viz. enzymes, proteins, and small molecular drugs,
achieved mainly by circumventing their clearance from circulation by opsonins,
leading to an enhanced therapeutic index (Marwa et al. 2020; Veronese and
Mero 2008).
4 PEGylated Nanocarriers forDrug Delivery Applications
111
Apart from improving the pharmacokinetics of drugs, PEGylation is also important in the mucosal delivery of drugs. Mucosal delivery of drug-entrapped nanocarriers is based on their ability to target the cell internalization pathways of epithelial
cells (Vllasaliu etal. 2014). The epithelial cells and mucosa present barriers in the
mucosal surface that affect or impede the passage of nanocarriers (Vllasaliu etal.
2014; Vllasaliu etal. 2011). Also, proteases at the mucosal surface function as bar-
riers preventing the availability of nanocarrier-delivered protein drugs, inuencing
their absorption (Lee 1990). Epithelial tight junctions also limit the passage of macromolecules greater than 1000Da, therefore preventing most nanocarriers from traversing the paracellular space (Illum 2000). PEGylated nanocarriers in mucosal
drug delivery have been reported to have a major breakthrough in nanocarrier diffusion across the mucosal surfaces (Ensign etal. 2013; Cu and Saltzman 2009; Wang
etal. 2008). NPs diffusion is inuenced and may be limited by the PEG MW and
the density used in the coating; also, coating some PEGs with short lengths at high
density improves the diffusion of NPs through the mucosa in combination with
shape, size, and the kind of NPs (Vllasaliu etal. 2014; Cu and Saltzman 2009; Wang
etal. 2008; Shi et al. 2021).
4.3 Methods ofPEGylation ofNanocarriers
PEGylation signicantly alters the surface properties of nanocarriers; most nanocarriers present hydrophobic surfaces, which may be difcult to bind PEG, leading
to low conjugation efciency (Tobio etal. 2000). Several methods are adopted in
achieving PEGylation, including the physical or chemical adsorption, grafting, and
the incorporation of PEG chain (Jain and Manoj 2010; Li etal. 2001; Calvo etal.
2001; Gref etal. 1995, 2000). The major limitation in simple adsorption is the dis-
placement of the PEG layers of coating invivo; therefore, the PEGylation of nanocarriers by the covalent method is preferred. Derivatization and activation of PEG is
a vital step in synthesizing the PEGylated system. Chemical derivatization of the
end groups of PEG, which is often an essential rst step in the preparation of bioconjugates, yields stable products (Jain and Manoj 2010). The methods involved in
nanocarriers PEGylation are discussed below.
4.3.1 Nanoparticles PEGylation
There are three methods of applying PEG coating onto the surface of nanoparticles:
physical adsorption, chemical conjugation, and molecular self-assembly.
Physical Adsorption PEG adheres physically to nanoparticles by physical adsorp-
tion, including hydrophobic and electrostatic interaction. Coating of the surface of
nanoparticles with PEG is physical adsorption and is the most common method of
PEGylation. This process is simple and takes place under easily controlled conditions. This method is primarily suitable when low-density PEG and PEG derivatives
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S. A. Chime and M. A. Momoh
and substratum have good adsorption potentials (Tobio etal. 2000). In this method,
the charged or hydrophobic groups of the nanomaterial adsorb to PEG based on
electrostatic adsorption or hydrophobic ability. Physical adsorption, however, suffers signicant limitations due to low adsorption strength, sometimes causing
detachment of PEG chains from the surface of NPs (Tobio etal. 2000; Kaur et al.
2008; Xiao etal. 2013).
Chemical Conjugation In chemical conjugation, stable chemical bonds are
formed when PEG is securely grafted on the NPs’ surface. PEGylated nanoparticles
can be formed by covalent coupling to prevent PEG separation or desorption from
the surface of nanocarriers. This involves a chemical reaction between the exposed
NPs surface and end groups of PEG having minimal chemical interactions, yielding
rm bonds between PEG and NPs (Tami etal. 2004). In this method, PEG is present
only at the NPs’ surface, resulting in the presence of PEG in the core of the nanoparticles (Tobio etal. 2000). Hence, this could ensure that the shelf-life of PEGylated
products is prolonged longer than non-PEGylated products. However, surface graft
density could be a problem while trying to attain application requirements due to
issues of active surface for PEG grafting, and there may be batch-to-batch variations
in the graft ratio due to differences in reaction rate and space hindrances (Rabanel
etal. 2014; Liu etal. 2018).
Molecular Self-Assembly The self-assembly method utilizes the hydrophilic and
amphiphilic nature of PEG; hence, when PEG is combined with hydrophobic polymers and lipids molecules, they self-assemble with their molecules. Two basic
methods employed in formulating PEGylated nanoparticles by self-assembly are
the nanoprecipitation method, also known as solvent diffusion, and the emulsication method (also called nanoemulsion or solvent evaporation). The molecular selfassembly method of PEGylation of nanoparticles occurs mainly by nanoprecipitation,
also called solvent diffusion. Self-assembly could also be formed by emulsication/
solvent evaporation. Spherical nanoparticles form by self-assembly by amphiphilic
polymers in the presence of water without other compounds. In this method, hydrophilic PEGs bind to hydrophobic lipids or polymers (Serra etal. 2006). The nanoparticles form a hydrophobic core, while PEG coatings make the layers outside it
(Tobio etal. 2000).
Nanoprecipitation often yields long-circulating PEGylated nanoparticles that
could be given via intravenous route (Huang etal. 2000; Gu etal. 2008). It is a step
procedure during formulation that does not require high shear. Here, drugs and PEG
are dissolved in an organic solvent that mixes well with water, such as acetonitrile,
tetrahydrofuran, and acetone. This organic phase gradually transfers into the aqueous phase with or without that may contain surfactants (Suk etal. 2016). Changing
the organic phase may be used to control the PEG density and the particle size of
NPs (Karnik etal. 2008). The limitation of this method is that the PEG and drugs
4 PEGylated Nanocarriers forDrug Delivery Applications
113
must dissolve in the same solvent mixture. Hence, formulation challenges exist with
poorly soluble and unstable drugs or bioactive (Suk etal. 2016). Also, the nanoprecipitation method yields low drug loading. It has the limitation of poor mixing,
which may lead to incomplete self-assembly, causing the PEG chains to remain in
the core of the NP buried intact instead of partitioning to the surface of the NPs (Suk
etal. 2016; Xu etal. 2015).
The emulsication method of NPs PEGylation involves the application of adequate emulsiers into the aqueous phase and mixing the molecules containing PEG
dissolved in organic phase water-immiscible solvent into this aqueous phase with
proper mixing and sonication. After that, the organic solvent is evaporated gradually, causing the hydrophilic PEG chains to partition toward the oil/water interface
in the emulsion droplets (Suk et al. 2016). Partitioning of the chains of PEG is
allowed to the NP surface fully due to the relatively slow solidication of the emulsion droplets (Hrkach etal. 2012). The emulsication method also yields higher
drug loading than the nanoprecipitation method (Torchilin and Weissig 2003) and
can easily be scaled up. Also, different types of emulsion structures of NP could be
formulated (Xu etal. 2015). However, this method’s limitations include the uses of
high energy and shear, which could denature sensitive APIs. There may be a burst
release of drugs due to the accumulation of APIs at the water–particle interface,
causing burst release due to the evaporation of the organic solvent from the droplets
of emulsions (Xu etal. 2015). Different types of NPs could be PEGylated using
self-assembly and adsorption methods, including polyplexes, PEGylated liposomes
or polymersomes, solid lipid nanoparticles, and nanostructured lipid carriers, among
others (Suk etal. 2016; Petersen etal. 2002a).
4.3.2 Polyplexes (PP) PEGylation
Polyplexes (PP) are core-shell formulations/structures formed by the condensation
of polycation and PEG or PEG and lipid or with plasmid DNA.Here, the driving
force for self-assembly is the electrostatic reaction in the cationic groups of the
PEG-containing molecules and phosphate groups of the DNA.PEG could interact
covalently with a region of the cationic block co-polymers or could be grafted,
forming comb-like structures after interactions with the cationic regions (Suk etal.
2014, 2016; Nakamura etal. 2012).
4.3.3 PEGylation ofLiposomes (Polymersomes)
Liposomes could be easily PEGylated by preparing PEG-conjugated lipid and mixing the PEGylated lipid along with other lipids during the formulation processes
(Jain and Manoj 2010). Conjugates of PEG-lipid could penetrate into pre-formed
liposomes. Here, the lipid tail (hydrophobic) is attracted and penetrates the lipid
bilayer. This pre-insertion needs minimal PEG-lipid conjugate to achieve the same
surface PEGylation as the pre-insertion and have a more prolonged half-life in the
114
bloodstream (Suk etal. 2016; Uster etal. 1996). Also, the amount of PEG-lipids
added should be less than the critical micellar concentration. PEG-lipid solutions
are slowly incorporated in the lipids, temperatures should be close to the lipid melting temperature to circumvent the formation of micelles (Torchilin 2005). In the
post-insertion method, the bilayer of the liposomes’ outer surface could be altered
rather than in a self-assembly technique that allows the insertion of PEG randomly
into the core of liposomes. Post-conjugation could also be used to prepare PEGylated
liposomes, usually for targeting ligands (Gajbhiye etal. 2007).
Self-assembly process (pre-insertion) is another technique for preparing polymersomes. Here, liposomes are prepared by thin lm hydration with an aqueous
medium (Petersen etal. 2002a), and PEGylated lipids could be added during the
self-assembly process, which will be inserted into the bilayer. At the same time, the
hydrophilic PEG chains will extend toward the aqueous phase. Partitioning of the
PEG molecules in the inner and outer layers of liposomes lipid bilayer could happen. To PEG in the core, which could affect drug loading, the PEG can be added by
post-conjugation or post-insertion methods (Suk etal. 2016). Monitored PEGylation
of liposome surface could circumvent liposome aggregation, while a large quantity
of PEG deposit on the surface has a limitation of compromising liposome formation
and stability (Suk etal. 2016).
S. A. Chime and M. A. Momoh
4.3.4 PEGylation ofDendrimers
PEGylation of dendrimers can be attained based on the orientation of the chains of
PEG needed in the dendrimers. Here, PEGylated dendrimers having PEG chains
conjugated to their periphery could simply be developed (Jain and Manoj 2010).
Also, dendrimers with inner PEG core could be developed. This creates more distance between branching units of the dendrimers, preventing the dumping of the
drug in the dendritic inner core. PEGylated dendrimers with branching monomer
units of PEG may also be formulated (Jain and Manoj 2010). Drug-conjugated
PEGylated dendrimers are another strategy for developing this formulation. Here,
PEG chains come between the dendrimers and the drug, forming steric hindrance
over the dendrimer, resulting in controlled drug release (Dunn etal. 1994).
4.4 Characterization ofPEGylation
4.4.1 Qualitative Assessments ofPEGylation
The qualitative assessment of PEGylated NPs is done using the methods discussed below:
Zeta Potential This method is the simplest method of assessing the quality of the
PEGylated nanoformulations used to determine the surface charge of NPs (Howard
etal. 2008). The surface coating of PEG and the PEGylating agent can inuence the
=×
4 PEGylated Nanocarriers forDrug Delivery Applications
115
zeta potential of the NPs. The zeta potential of NPs may also affect the liver uptake.
Liver uptake was found to decrease when the zeta potential neared zero (Craparo
et al. 2006). Also, the surface charge of PEGylated NP may decrease with an
increase in PEG as reported by some researchers (Churae and Nikologorskaja
1991). However, there is difculty in determining the quantitative amount of PEG
on the surface of NP (Howard etal. 2008). The thickness of the hydrodynamic layer
around the NP could be calculated from the zeta potential (Suzawa and Shirahama
1991; Webb etal. 1998). There is also difculty in measuring the zeta potential in
NPs with neutral surfaces and low surface charges (Peracchia etal. 1998).
The comparison of hydrophobic interaction chromatography (HIC) ratios: This
method involves the measurement of the surface hydrophobicity/hydrophilicity of
NPs (Howard etal. 2008). Here, NPs are transferred into a column and washed
twice. The rst wash will elute the hydrophilic particles more, while the second is
to elute more hydrophobic particles. Calculation of the HIC ratio is carried out from
the peaks of their area under the curve (AUC) thus:
HICratio
AUCelutionpeak
AUCwashpeak
100
(4.1)
Here, the more hydrophilic particles show a greater HIC ratio, hence giving
information on the PEG localization on the NPs’ surface and the amount of
PEGylation (Howard etal. 2008; Brigger etal. 2000). However, this method is inadequate for the quantitative evaluation of PEGylated NPs and not for use in particles
without hydrophobic surfaces (Howard etal. 2008).
Near-Infrared (NIR) Spectroscopy Principal component analysis (PCA) in combination with NIR can also be used to evaluate the hydrophilic and hydrophobic
balance of PEGylated nanoparticles to conrm PEGylation (Hu etal. 2006).
Fourier Transform-Infrared Spectroscopy (FTIR), Microscopy, and 13CNMR The presence of the absorption bands of C–O–C and CH2 in PEGylated NPs
is qualitatively determined by FTIR, and it is one of the qualitative conrmations of
the presence of PEG in NPs (Howard etal. 2008; Arima etal. 2008; Jie etal. 2005).
NMR also reveals that some specic chemical groups conrm PEGylation. The
decrease in CH and CH2 peaks from a 13C-NMR spectrum has been used to verify
PEGylation (Arima etal. 2008).
Microscopic techniques have been used to characterize PEGylation. Transmission
electron microscopy (TEM) is applied in studying the morphological properties of
nanocarriers, but it has not commonly been applied as a characterization technique
for PEGylation (Howard etal. 2008). However, upon staining PEGylated micellelike nanoparticles with phosphotungstic acid, a bright hydrophobic core surrounded
by a gray hydrophilic shell was obtained (Khosravi-Darani etal. 2007). Atomic
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S. A. Chime and M. A. Momoh
force microscopy (AFM), scanning tunneling microscopy, and scanning probe
microscopy are all applied for surface imaging of NPs (Xia etal. 2012).
4.4.2 Quantification ofPEG Surface Density
PEGylation usually affects the properties of nanocarriers, including the hydrodynamic diameter, protein-binding capability, hydrophilicity, and surface charge
(Howard etal. 2008; Suk etal. 2016). Hence, the quantitative and qualitative measurement of PEGylated is essential. The methods that could be used for quantitative
determination of PEGylated NPs surface include:
TGA (Thermogravimetric Analysis) TGA measures the content of PEG and the
difference in content pre- and post-PEGylation as a result of thermal denaturation.
However, it has limitations because of inorganic materials and the need for a signicant amount of samples (Suk etal. 2016). The interaction between functionalized
PEG and terminal groups, viz. -SH,-NH2, etc. labeled with detectable dyes for absorbance readings can be employed in measuring the PEG that is unreacted in the supernatant. Hence, content PEG grafted on the surface of NPs can be indirectly calculated
(Suk etal. 2016; Walkey etal. 2012; Perry etal. 2012; Valencia etal. 2011). The PEG
and ligand densities, respectively, could be quantied by measuring the amount of
ligand grafted to PEG on the NP surface (Suk etal. 2016; Garcia- Fuentes etal. 2004).
NMR (Nuclear Magnetic Resonance) NMR is utilized in the study of the structural conformations of nanoparticle components (Zabaleta et al. 2007). The new
NMR pulse sequences have been used to increase diffusion-ordered NMR spectroscopy sensitivity to identify slow diffusion molecules with typical NMR probes
(Howard etal. 2008). The quantity of PEG on the surface of the nanocarrier could
be resolved here by comparison between the integrals of the 1H NMR (δ=3.6ppm)
PEG peak and the peak of an internal standard (benzylsulfonic sodium salt). The
density of PEG coatings is determined with reference to the SA (surface area) and
the content of surface PEG.Hence, new NMR techniques could be used for the
quantication and characterization of PEGylated NPs (Howard et al. 2008).
Therefore, the NMR technique could be employed in quantitatively and qualitatively assessing the PEG density on NPs surfaces (PEG peak typically observed at
~3.65ppm) (Howard etal. 2008; Suk etal. 2016).
HPLC Analysis and Anti-PEG Antibody Binding The amount of PEG deposited
on NPs’ surface can be quantied using the HPLC method (Howard etal. 2008;
Nair etal. 2006). Chromatography can be employed to quantify the PEG amount
and does not require conjugation or dye modication (Suk etal. 2016). However,
this method cannot assess PEG localization within the NPs, and they also have low
sensitivity to contaminants, unstable baselines, and high sensitivity limits when the
refractive index (RI) detectors are used (Suk etal. 2016; Chuang etal. 2010). Anti-