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Methods andProtocols fortheSynthesis
ofPEGylated Pharmaceutical
Nanocarriers
Ala’AdnanAli, RakeshKumarTekade,
andRandaS.H.Mansour
Abstract
This chapter focuses on topics related to synthesizing PEGylated nanocarriers and
their characterization. The types of poly(ethylene glycol) (PEG) derivatives and con-
formation in addition to PEGylation techniques are detailed. Moreover, the methods
employed for synthesizing PEGylated nanocarriers are categorized and discussed in
detail. The methods explained are classied into self-assembly from PEG-containing
molecules by either nanoprecipitation or emulsication and surface modication of
nanocarriers with PEG chain covalently and noncovalently. Indirect assessments of
surface PEGylation efciency using particle size, surface hydrophilicity, zeta poten-
tial, etc. are emphasized, in addition to other methods used for direct assessments,
including colorimetric methods, chromatographic quantication methods, UV and
uorescence spectroscopy, and more. Finally, alternative polymers to PEG are briey
illustrated, as well as the challenges of nanocarriers PEGylation.
2
Keywords
PEGylated nanocarriers · Nanoprecipitation · Zeta potential · Polymers · Solvent
evaporation · Solvent diffusion · Chemical conjugation · Physical adsorption ·
Mushroom conformation · Brush conformation
A. A. Ali (*)
Faculty of Pharmacy, University of Jordan, Amman, Jordan
R. K. Tekade
National Institute of Pharmaceutical Education and Research (NIPER) Ahmedabad, An
Institute of National Importance, Government of India, Department of Pharmaceuticals,
Ministry of Chemicals and Fertilizers, Palaj, Opp. Air force station, Gandhinagar,
Gujarat, India
R. S. H. Mansour
Faculty of Pharmacy, Zarqa University, Zarqa, Jordan
29
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A. A. Ali et al.
Abbreviations
APTES Amino propyl triethoxy silane
APTMS Amino propyl trimethoxy silane
CDCl3 Deuterated chloroform
Chln Cholane
Chol Cholesterol
CMC Critical micelle concentration
D2O Deuterated water
DLS Dynamic light scattering
DMAP 4-Dimethylaminopyridin
DMSO Dimethyl sulfoxide
DSPE Distearoyl-sn-glycero-3-phosphoethanolamine
EDC N′-ethylcarbodiimide
ELSD Evaporative light scattering detector
FT-IR Fourier transform-infrared spectroscopy
G4-PAMAM Generation 4 poly(amidoamine)
HIC Hydrophobic interaction chromatography
HPLC High-pressure liquid chromatography
LC Liquid chromatography
mPEG Methoxy-poly(ethylene glycol)
MS Mass spectrometry
NA Nucleic acid
NHS N-hydroxysuccinimide
NMR Nuclear magnetic resonance
PCL Poly(ε-caprolactone)
PCS Photon correlation spectroscopy
PEG Poly(ethylene glycol)
PEI Poly(ethylene imine)
PEO-PPO-PEO Polyethylene oxide-b-polypropylene oxide-b-polyethylene oxide
PGA Poly(glutamic acid)
PGs Poly(glycerols)
PHDCA Poly(hexadecyl cyanoacrylate)
PHEMA Poly(2-hydroxyethyl methacrylate)
PHPMA Poly(hydroxypropyl methacrylate)
PLA Poly(lactic acid)
PLGA Poly(lactide-co-glycolide)
PLL Poly(-lysine)
POX Poly(oxazolines)
PPG Poly(propylene glycol)
PSA Polysebacic acid
pSLN PEGylatec solid lipid nanoparticles
PVL Poly(δ-valerolactone)
PVP Poly(vinylpyrrolidone)
RH Hydrodynamic radius”
2 Methods andProtocols fortheSynthesis ofPEGylated Pharmaceutical Nanocarriers
31
RI Refractive index
SEM Scanning electronic microscopy
SLN Solid lipid nanoparticles
SM Sphingomyelin
TEA Triethanolamine
TEM Transmission electron microscopy
THF Tetrahydrofuran
UV Ultra-violet
XPS X-ray photoelectron spectroscopy
2.1 Introduction
Several hydrophilic polymers have been utilized to coat the surface of nanocarriers,
such as polysaccharides, polyacrylamide, poly(glycerols), poly(vinyl alcohol),
poly(oxazolines), poly(ethylene glycol) (PEG), and PEG-containing co-polymers.
Among all the polymers, PEG and PEG-copolymer are the most commonly used
and recognized as the gold standard. PEGylated nanocarriers have several advantages, such as avoidance of the nonspecic recognition by serum proteins, cells,
tissues, and organs, reducing toxicity, and extending circulation time (Moghimi and
Szebeni 2003; Vonarbourg etal. 2006).Therefore, PEGylated nanocarriers can be
considered valid new pharmaceutical entities compared with their non-PEGylated
counterparts.
PEGylation refers to attaching or coating the nanocarrier surface with PEG via
adsorption, grafting, or entrapment methods (Howard etal. 2008). In recent few
years, a considerable amount of research has been directed toward PEGylation
chemistry, allowing the researcher to improve the PEGylation strategies from a
“random” to a “site-specic” PEGylation. In addition, PEGylation is currently
focused on using bifunctional PEG chains, which react from one side with the particle surface and on the other side with a targeting agent.
The objectives of this chapter are to provide the readers with the methods and
protocols of PEGylation, the tools to assess PEG surface quantitatively and qualitatively, and the main alternative polymers to PEG.
2.1.1 Physical andChemical Properties ofPEG
PEG is a synthetic polymer of ethylene glycol (HO-CH2-CH2-OH) that is available
in different molecular weights ranging from hundreds to several thousands of
Daltons (Chen etal. 2005). PEGs have good water solubility that decreases with
increases in chain lengths(Sanchez Armengol etal. 2022). PEGs with low molecular weight (less than 800Daltons) are highly soluble in water and are liquids at room
temperature (Chen etal. 2005).
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A. A. Ali et al.
Different useful features make PEG one of the most popular polymers for modifying nanocarriers. PEG is soluble in both water and many organic solvents due to
the presence of both hydrophilic and hydrophobic groups in its structure (Pasut and
Veronese 2012). Moreover, the PEG molecule has high exibility due to the absence
of a bulky substituent group on the PEG backbone (Mohapatra etal. 2019). Another
feature of PEG is its distinctive stability against decomposition by acids, bases,
moderately high temperatures, and hydrogen peroxide, oxidation, and reduction
(Chen etal. 2005). In addition, PEG can be functionalized by selectively oxidizing
the terminal OH group with various terminal end groups or by attaching large
ligands, such as biomolecules (Karakoti etal. 2011).
The synthesis of PEG (Fig.2.1) involves a condensation polymerization where
ethylene is reacted with aluminum oxide to obtain oxirane. Then, the polymerization of diethylene glycol as the starting molecule with oxirane takes place at 130°C
in the presence of alkaline catalysts, as it eliminates water, subsequently the reaction is terminated by adding an acid (Li and Kao 2003).
2.1.2 Uses ofPEG
PEG has been widely used in biomedical applications, especially in pharmaceutics, due to its high solubility in aqueous media, good tolerance, biocompatibility,
FDA- approved, and negligible interference with the drug release (Knop
etal. 2010).
A breakthrough in PEGylation was the modication of albumin and catalase by
Abuchowski in 1977, which decreased the immunogenicity of the proteins and
increased their circulating time in blood (Abuchowski et al. 1977). The rst
PEGylated nanocarrier introduced in 1995in the US market was Doxil®, a formulation of PEGylated liposomes of doxorubicin (Barenholz 2012). Later, various FDAapproved PEGylated products have emerged like cytokines (interferon-α2a and
interferon-α2b), enzymes (bovine adenosine deaminase, Lasparginase, and urate
oxidase), hormones (epoetin-β), antibodies and their fragments, other organic molecules (pegvisomant and pegatinib), and others are in clinical trials (Pasut and
Veronese 2009). In addition to enzymes and proteins, PEGylation has been widely
utilized in the modication of nanocarriers delivery systems, including polymeric
nanocarriers (Ebrahimnejad etal. 2011), liposomes (Allen etal. 1995), solid lipid
nanoparticles (SLN) (Yuan etal. 2013), and micelles (Lee etal. 2011). This results
in improving their invivo stability and solubility, increasing their accumulation at
the target site, and reducing their clearance rate from circulation (Torchilin 1998;
Maruyama 2002).
Fig. 2.1 Synthesis of PEG. ((Sanchez Armengol etal. 2022) with permission) . ©Copyright 2022.
Published by Informa UK Limited, trading as Taylor & Francis)
2 Methods andProtocols fortheSynthesis ofPEGylated Pharmaceutical Nanocarriers
33
2.1.3 Types ofPEG Derivatives
Diverse types of PEG derivatives with differences in shapes, functionality, and
molecular weights affect the option of a suitable PEG moiety for therapeutic applications (Mohapatra etal. 2019).
2.1.3.1 According totheShape ofthePolymer
According to the shape of the polymer, PEGs are classied into (Fig.2.2):
(a) Linear PEG derivatives: they are the simplest agents, having one reactive func-
tional group that aids in conjugation, and the other end is a methoxy group or a
targeting molecule to make the PEG monofunctional. Also, bifunctional PEGs
that have two functional groups (refers to Sect. 2.1.3.2) (Mohapatra etal. 2019).
(b) Y-shaped PEG derivatives: two linear PEG derivatives are linked to active
groups (Santos etal. 2018), they are characterized by higher invivo stability
against physiological conditions like pH, temperature, and enzymatic environment (Veronese etal. 1997).
(c) Forked PEG derivatives: they pose multi-proximal reactive groups at one or
both ends of a linear PEG chain (Veronese etal. 1997).
Fig. 2.2 Different-shaped PEGs. (a) Linear PEG. (b) Bifunctional PEG. (c) Y-shaped PEG. (d)
Multi-arm PEG. (e) Fork-shaped PEG; providing at one PEG chain end multi-proximal reactive
groups. (f) Fork-shaped PEG; providing at both PEG chains end multi-proximal reactive groups.
( ) represents functional groups of PEG derivatives
34
A. A. Ali et al.
(d) Multi-arm PEG derivatives: they are star-like structures with multi-hydroxyl or
functional groups. Thus, the number of active sites and molecular weight are
higher (Kim etal. 2016).
2.1.3.2 According toFunctionality
PEG functionalization of nanocarriers can enhance the targeting, controlling the
release of drugs from colloidal nanostructures (Howard etal. 2008), and increasing
the invivo circulation half-life (Steenpaß etal. 2006). The structure of PEG possesses two equivalent hydroxyl groups in which a variety of reactive functional
groups or bioactive species can be attached by covalent coupling (Gajbhiye
etal. 2007).
According to functionality, PEGs are classied into monofunctional PEGs and
bifunctional PEGs. In monofunctional PEGs, one of the hydroxyl groups is replaced
with a reactive functional group (Turecek etal. 2016). Examples of those functional
groups are bromo, amino, carboxymethyl, succinimido succinate, tosylate, mesylate, aldehyde, octadesylamine, monopalmitate, stearyl oxy, or methoxy (Harris 1985).
To illustrate, Steenpa etal. prepared monofunctional soy sterol-PEG1300 ethers
by attaching a tresyl group to the end of the PEG chain. Then the PEG chain was
attached to bovine serum albumin, and the sterol–PEG–protein formed was inserted
into the outer liposome monolayer using the post-insertion method. This resulted in
an increasing invivo circulation half-life of those particles (Steenpaß etal. 2006).
On the other hand, bifunctional PEGs have two reactive functional groups. They
are usually used for cross-linking and conjugation of drugs, targeting ligands, dyes,
and proteins.
Bifunctional PEGs could be homo-biofunctionalized or heterobifunctional. The
homo-functionalized PEGs contain two similar functional groups, and the heterobifunctional PEGs include two different groups. An example of a linear heterobifunctional PEG is thiol-PEG-coumarin for functionalizing gold nanoparticle surfaces
(Fig.2.3) (Fu etal. 2004).
2.1.4 PEG Conformations ontheNanocarrier Surface
PEG forms a exible layer on the surface of nanocarriers (Heald etal. 2002), blocking the adsorption of opsonins (proteins) by steric hindrance (Drobek etal. 2005)
and their consequent uptake by phagocytic cells (Storm etal. 1995). The PEG-chain
Fig. 2.3 Schematic representation of the functionalization of gold nanocarrier with coumarin
PEG-thiol. ((Fu etal. 2004) with permission)
2 Methods andProtocols fortheSynthesis ofPEGylated Pharmaceutical Nanocarriers
35
conformation on the particle surface is often described as a mushroom or a brush
model (Amoozgar and Yeo 2012).
When the surface PEG density is low, it results in the mushroom state conguration. As the PEG density increases, it results in the brush state conguration (Tirosh
etal. 1998) as in Fig.2.4.
The “mushroom” conguration has meager surface coverage, leading to large
areas for protein binding. On the contrary, the “brush” conguration is characterized by a very high surface coverage, which provides a higher anti-opsonization
effect resulting in more effective binding, prolonging the circulation time of nanocarriers compared to the former (Photos etal. 2003).
The optimal PEG density varies by system. For example, for poly(lactic acid)
(PLA) nanocarriers (Sheng etal. 2009) or poly(lactide-co-glycolide) (PLGA) nanocarriers 10wt.% PEG density was reported as optimal regarding particle dispersibility and stealth effect (Beletsi etal. 2005). However, Gref etal. considered that
the optimal PEG surface density for PLA, PLGA, and polycaprolactone nanocarriers was 5wt.% and that any further increase in the levels of PEG content will not
result in further reduction in protein adsorption (Gref etal. 2000).
In liposomes, a mushroom-like shape is obtained using <5% PEG, mushroom- or
brush-like shape is obtained using 5–15% PEG, while >15% PEG gives a brush-like
shape. Hence, the higher the molar PEG–lipid/lipid composition ratio, the higher
the surface density (Nosova etal. 2019).
2.1.5 Types ofPEGylation Techniques
The key step of PEGylation is a conjugation of the PEG chains by different spacers
or linkers with nanocarriers like polymeric nanocarriers, micelles, liposomes, or
inorganic nanocarriers. Numerous derivatives of PEG (i.e., linear PEG and Y-shaped
PEG) are used in the PEGylation techniques (Zacchigna etal. 2011; Chen et al.
2011). There are three types of PEGylation techniques: rst, second, and third
generations.
Fig. 2.4 PEG-chain conformations on the nanocarrier surface, ( ) represent PEG chains
36
In rst-generation PEGylation, linear and low molecular weight PEG is conjugated with a reactive group with hydroxyl groups like carbonates, chlorides, and
anhydrides (Damodaran and Fee 2010). This type of PEGylation results in nonspecic random conjugations and is inefcient for protein conjugation. In secondgeneration PEGylation, branched structured PEG derivatives are used, which are
more site-specic because more functional groups like esters, aldehydes, and
amides are available (Damodaran and Fee 2010). In addition, branched PEGylation
is more effective in diminishing immunogenicity and increasing the circulation
time than linear PEG.On the other hand, it might affect the biomolecules’ activities; therefore, the third generation is developed to conserve drug bioactivity
(Monfardini et al. 1995). In third-generation PEGylation branched, Y-shaped
structures revealed a reduction in viscosity and lack of organ accumulation
(Singh 2015).
A. A. Ali et al.
2.2 Methods andProtocols fortheSynthesis
ofPegylated Nanocarriers
Due to variations in the chemical makeup, the methods and protocols used for the
PEGylation of nanocarriers can be classied under polymeric nanocarriers, liposomes, micelles, and inorganic nanocarriers.
2.2.1 PEGylation ofPolymeric Nanocarriers
The chemistry of PEGylation of polymeric nanocarriers cannot be dened under
a single heading, as that of proteins, due to high variance in the availability of
functional groups and mode of preparation. PEGylation of a nanocarrier surface
can be achieved in different ways (Fig.2.5), including the preparation of nanocarriers by self-assembly with block co-polymers of PEG and hydrophobic
polymers (Gref etal. 1994). Otherwise, it can achieved by the addition of PEGs
on preformed nanocarriers by physical adsorption or chemical conjugation of
functional PEGs to the available surface reactive groups (Owens and
Peppas 2006).
The main drawback of physical adsorption methods is the risk of desorption, the
removal of the coating layer invivo, and consequent loss in pharmacokinetic outcomes that the polymer should provide (Neal etal. 1998). To overcome the desorption, PEGylated nanocarriers are prepared by self-assembly using a modied
derivative of polymers by choosing a proper method of nanoparticle preparation
(emulsication and precipitation) in which the hydrophobic portions of PEG chains
embedded inside the matrix of nanocarriers and hydrophilic portions orient toward
the aqueous phase (Yoncheva etal. 2005). However, this does not ensure that PEG
chains are localized only on the surface of nanocarriers, as PEGs may retain within
the particle core (Vila etal. 2004). Therefore, the chemical conjugation method for
PEGylation of nanocarriers’ surface is preferred.