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2 Methods andProtocols fortheSynthesis ofPEGylated Pharmaceutical Nanocarriers
37
Fig. 2.5 Methods of PEGylation of polymeric nanocarriers
2.2.1.1 Self-Assembly fromPEG-Containing Molecules
To prepare PEGylated nanocarriers using the self-assembly method, PEG is linked/
bound with hydrophobic molecules such as lipids or hydrophobic polymers to create amphiphilic moieties. After that, it proceeds by two basic approaches: nanoprecipitation (also called solvent diffusion) or emulsication (also called solvent
evaporation or nanoemulsion) (Huckaby and Lai 2018; Hussain etal. 2019).
It is anticipated that PEG segments will segregate completely toward the water
while the hydrophobic part of the polymer will be retained in the particle core.
However, some of the PEG segments can also be retained in the particle core, thus
diminishing PEG concentration at the nanocarrier surface (Vila etal. 2004). This
phenomenon can be attributed to several reasons, such as entanglements, loss of
small polymeric chains in the water phase, physical processes related to nanocarrier
preparation (for example, diffusion of solvent and water, viscosity differences, and
polymer–polymer interactions.), solidication processes, or the existence of aqueous cavities within the particle (Rabanel etal. 2014).
Nanoprecipitation (Solvent Diffusion)
Many PEGylated nanocarriers have been prepared by the nanoprecipitation method
(Kolishetti etal. 2010; Gu etal. 2008). The nanoprecipitation method has several
advantages, including it is a one-step process, an environmentally friendly approach,
38
A. A. Ali et al.
and it does not require high-energy input or high-shear forces, so it is a low-energy
process.
The nanoprecipitation method involves two steps, as shown in Fig.2.6. Firstly,
PEG-containing moieties are dissolved in a water-miscible organic solvent (such as
acetonitrile) with drug(s). Secondly, dispersing the organic phase gradually into the
aqueous phase with mixing. This will lead to the partitioning of the hydrophilic
PEG chains toward the aqueous phase at the nanoparticle surface, and the hydrophobic segments will separate away from the aqueous phase to form the core of the
nanoparticle (Hussain etal. 2019; Huckaby and Lai 2018).
The assembly of the polymer occurs in three steps (Bovone etal. 2022):
1. Dissolving the block co-polymers in an excellent solvent (organic solvent).
2. Mixing with water to lower the strength of the organic solvent. This change trig-
gers the assembly of block co-polymer into dynamic aggregates that grow
in size.
3. Increasing the water fraction, which results in growth arrest. This occurs at a
critical solvent strength, creating kinetically trapped core−shell nanocarriers.
4. Different parameters affect the size of nanocarriers prepared by this method
including the mixing time and the solvent type. A study reported the effect of
using different solvents on the size of poly(ethylene glycol)-block-polylactide
(PEG-b-PLA) nanocarriers. The smallest nanocarriers were formed with dimethylformamide (DMF), followed by acetone, acetonitrile, tetrahydrofuran (THF),
and dimethyl sulfoxide (DMSO) (Bovone etal. 2022).
Emulsification (Solvent Evaporation or Nanoemulsion)
The emulsication method involves several steps, which require emulsier/s and/or
a high-pressure homogenizer or sonicator. On the other hand, drug loading capacity
is higher using the emulsication method compared to the nanoprecipitation method.
The PEG-linked moieties/polymers are dissolved with drugs in a waterimmiscible organic solvent. This organic solvent is dispersed gently into an aqueous
Fig. 2.6 Schematic representation of preparing PEGylated nanocarriers by the nanoprecipitation method
2 Methods andProtocols fortheSynthesis ofPEGylated Pharmaceutical Nanocarriers
39
solvent with the assistance of emulsiers and/or by high-energy homogenization or
sonication. Then the organic solvent is removed by evaporation, letting the hydrophilic PEG chains partition toward the oil/water interface of the nanocarrier surface
(Fig.2.7) (Xu etal. 2015; Huckaby and Lai 2018).
For example, Prawatborisut etal. prepared shellac-PEG (SHPEG) nanocarriers
by the miniemulsion solvent evaporation method. Firstly, at room temperature,
SHPEG was dissolved in a variety of tetrahydrofuran (THF) and they used a
chloroform:THF mixture (2.3:1 w:w). The solution was mixed then with water with
stirring at room temperature, followed by ultrasonication in an ice bath. Next, a
rotary evaporator was used to evaporate chloroform and THF (Prawatborisut
etal. 2019).
Nanocarriers composed of a biodegradable diblock co-polymer of polysebacic
acid and polyethylene glycol (PSA-PEG) have been prepared by self-assembly
using both nanoprecipitation and emulsication methods (Tang etal. 2009). The
surface PEG coating density for the PLGA-PEG nanocarriers was higher in nanocarriers prepared by emulsication (~100% of incorporated PEG found on nanocarrier surface compared to ~89% using nanoprecipitation) (Xu etal. 2015) in addition
to a higher level of drug-loading capacity (Hrkach etal. 2012). Despite those advantages, drawbacks of this method include high shear and high energy requirements,
which can be impractical for sensitive drugs and biologics (Karnik etal. 2008).
2.2.1.2 Surface Modification ofNanocarriers withPEG Chains
PEGs can be attached to the surface of preformed nanocarriers. By using this technique, the PEG chains will present totally at the surface instead of being embedded
within the particle core. However, the main limitation of this method is the low
density of PEG on the surface of the nanocarrier which is inadequate to reach the
brush conformation mainly due to the steric hindrance of adjacent bulky PEG
chains. Moreover, a purication step is required to remove the excess of nonadsorbed or unreacted PEG chains. This is a challenging step, especially in the case of
physical adsorption due to the weak interaction and the risk of desorption (Rabanel
etal. 2014).
As shown in Fig.2.8, PEGylation of nanocarriers via surface modication can be
done by (1) direct PEGylation, where the PEG molecules are physically adsorbed
Fig. 2.7 Schematic representation of preparing PEGylated nanocarriers by the emulsication method
40
A. A. Ali et al.
Fig. 2.8 Different strategies for PEGylation of nanocarriers via surface modication. ((Karakoti
etal. 2011) with permission)
on the nanocarrier surface or (2) chemical conjugation via covalent bonds of either
monofunctional PEG or bifunctional PEG molecules (Huckaby and Lai 2018).
Physical Adsorption Strategy
Physical adsorption is a simple, direct technique for PEG coating based on noncovalent association either by electrostatic interactions with charged particle surfaces
(Vandevondele etal. 2003) or by hydrophobic interactions (Redhead etal. 2001;
Huckaby and Lai 2018); nevertheless, those interactions are feeble which results in
the desorption of PEGs from the surfaces of nanocarriers invivo thus limiting the
efcacy of this technique (Pulkkinen etal. 2008; Cu and Saltzman 2009).
PEG coating by physical adsorption is done by simply incubating pre-formed
nanocarriers with an aqueous solution of PEG-containing molecules and allowing
the PEGs to associate with the nanocarrier surface (Huckaby and Lai 2018).
An illustrative example of physical adsorption is the adsorption of Pluronics onto
the nanocarrier’s surfaces. Pluronics is a triblock co-polymer of polyethylene oxideb- polypropylene oxide-b-polyethylene oxide (PEO-PPO-PEO) that can be adsorbed
onto the hydrophobic nanocarrier’s surfaces via hydrophobic interaction with the
PPO segment (Yang etal. 2011, 2014). Another example is the hydrophobic interaction of the poly(propylene glycol) (PPG) chain of poloxamers (nonionic block
2 Methods andProtocols fortheSynthesis ofPEGylated Pharmaceutical Nanocarriers
41
co- polymers of (PPG) surrounded by two hydrophilic chains of PEG) with the
nanocarrier surface (Storm etal. 1995). In addition, the PEG coating can also be
generated based on electrostatic interactions such as the coating of negatively
charged polylactic-co-glycolic acid (PLGA) nanocarriers with positively charged
PEG block polymers such as PEG-poly(ethylene imine) PEI and PEG- poly(lysine) PLL (Wang etal. 2010).
Chemical Conjugation ofPEG Molecules
A preferred method for PEGylation of nanocarriers is by chemical conjugation due
to the formation of a stable PEG coating (Hussain etal. 2019). Several types of
bonds can be formed between PEG and the nanocarrier surface by chemical conjugation, such as:
1. Amide bond: involves a carbodiimide coupling reaction between a carboxyl-
modied nanocarrier with amine-modied PEG (Meng etal. 2004; Suh et al.
2007; Lai etal. 2007, 2010; Wang etal. 2008; Nance etal. 2012) or between a
carboxyl-modied PEG with amine-modied nanocarriers. In addition, stable
amide bonds can be formed by the reaction between N-hydroxysuccinimide
ester (NHS ester)-modied PEG with primary amines (Kim etal. 2013; Moser
etal. 2015).
2. Thioester bond: formed by reacting thiols with modied PEG yielding thioester
bonds (Matsumoto etal. 2014).
3. Alkyne/azide coupling reaction: performed with or without the presence of cop-
per catalyst (Cavalli etal. 2006; Kumar etal. 2010; O’Mahony etal. 2012; Breed
etal. 2009).
4. Avidin–biotin complex: represents the strongest noncovalent, high afnity, pro-
tein–ligand interactions. Biotinylated PEG is reacted with avidin-coated nanocarriers to form stable coatings on nanocarrier surfaces (Cu and Saltzman 2009;
Park etal. 2009; Jazayeri etal. 2016; Friedman etal. 2013).
For example, generation 4 poly(amidoamine) dendrimers (G4 PAMAM) are
nanoscale macromolecules, highly branched, with several amine groups on the surface.
PEGylated G4 PAMAM dendrimers were prepared by conjugating PEG on the surface
of G4-NH2 dendrimers via an amide bond. For this, hydroxy-terminated PEG was modied into succinyl PEG, having a free carboxylic group using succinic anhydride. Then
using a carbodiimide reaction, the modied PEG was conjugated into G4-NH2 dendrimers. The synthesis procedure for preparing PEGylated G4 dendrimers is represented
in Fig.2.9 (Jangid etal. 2022; Yang etal. 2004; Singh etal. 2008).
2.2.2 PEGylation ofLiposomes
The FDA has approved numerous drugs based on PEGylated liposomes that are
available in the market. Such as PEGylated liposomes of doxorubicin (Hensley
etal. 2001; Wibroe etal. 2016), vincristine (Sarris etal. 2000), and daunorubicin
(Allen and Cullis 2013).
42
Fig. 2.9 Scheme representing the synthesis of PEGylated G4 dendrimer. In step one, the hydroxyl
(–OH) group of PEG4K was modied into a carboxylic group (–COOH) by reacting it with succinic anhydride and 4-dimethylaminopyridin (DMAP). In step two, the –COOH group was activated by dissolving mPEG4K-COOH, N′-ethyl carbodiimide (EDC), and N-hydroxysuccinimide
(NHS) in anhydrous dimethyl sulfoxide (DMSO). Next, adding amine-terminated PAMAM G4
dendrimers and triethanolamine (TEA) to the above reaction mixture. ((Jangid etal. 2022) with
permission)
A. A. Ali et al.
Liposome PEGylation is performed either by mixing PEG–lipid conjugates with
a mixture of lipids at a xed ratio before liposome formation (pre-insertion method)
or by mixing PEG–lipids with preformed liposomes (post-insertion method)
(Nosova etal. 2019; Amoozgar and Yeo 2012).
2.2.2.1 Pre-Insertion PEGylation
Usually, PEGylated liposomes are prepared by the pre-insertion method. Liposomal
particles are formed by mixing cationic, anionic, or neutral lipids with PEG lipids,
followed by the drug-loading step (Kapoor and Burgess 2012; Bouxsein etal. 2007).
Figure2.10 represents the formation of PEGylated lipoplexes using this method,
where nucleic acid (NA) is added after forming PEGylated liposomes (Halder etal.
2006; Whitehead etal. 2014).
The traditional anchoring PEG–lipids used for liposomal PEGylation
(Fig. 2.11) are monomethoxy-poly(ethylene glycol) (mPEG) conjugated to
phospholipids such as 1,2-distearoyl-sn-glycero-3-phosphoethanolamine
(DSPE) [DSPE-mPEG], where mPEG-OH is chemically conjugated by urethane linkage (−HN−COO−) to the terminal amino group of the lipid head
(Lavan etal. 2002). Others are mPEG- ceramide and mPEG-cholesterol. Those
PEG–lipids vary in their lipophilic domain, fatty acid type, and PEG chain
length (Fu etal. 2004).
2 Methods andProtocols fortheSynthesis ofPEGylated Pharmaceutical Nanocarriers
Fig. 2.10 Scheme represents the preparation of the PEGylated lipoplex by the pre-insertion
method. ((Nosova etal. 2019) with permission)
43
Fig. 2.11 Chemical structure of mPEG–lipid derivatives used for liposome coating. (a′) Linear
mPEG45-DSPE (L-mPEG45-DSPE); (a″) linear mPEG114-DSPE (L-mPEG114-DSPE); (b)
branched mPEG114-DSPE [B-(mPEG114)2-DSPE]; (c) linear mPEG114-cholesterol
(L-mPEG114-Chol); (d) linear mPEG114-cholane (L-mPEG114-Chln). ((Mastrotto etal. 2020)
with permission)
Fig. 2.12 Scheme representing the preparation of the PEGylated lipoplex by the post-insertion
method. ((Nosova etal. 2019) with permission)
Using the pre-insertion method, a low PEG density on the periphery can be
obtained (approximately 1%) (Fehring etal. 2014). Besides that, PEG chains will
exist on both the external and internal parts of the liposome. As a result, drug loading can be affected due to the PEG moieties present inside the liposome. However,
using the post-insertion method, PEG can exist only at the outer liposome surface
(Nosova etal. 2019).
44
A. A. Ali et al.
2.2.2.2 Post-Insertion PEGylation
The post-insertion method adds PEG–lipid solution or PEG micelles to preformed
liposomes after the drug loading step. Subsequently, a purication step is done to
remove unreacted PEG derivatives via centrifugation and ltration. Figure2.12
shows an example of the post-insertion method preparation for a PEGylated lipoplex (Nosova etal. 2019).
Using this technique, stable PEGylated liposomes are formed with a high level
of PEGylation (more than 5% PEG) (Uster etal. 1996). Moreover, PEG presents
only at the outer surface of the liposomal membrane; this will result in higher encapsulation rates than PEGylated liposomes prepared using the pre-insertion method
(Awasthi etal. 2004; Visser etal. 2005).
The type of bonding between the PEG derivative and the liposome surface can
vary. For example, chemical bonding can be formed between PEG with a terminal
azide group and a hydrophobic alkyne-ended anchor built into the lipid bilayer by a
“click” reaction.
As illustrated in Fig. 2.13, a high linear correlation is reported between the
amounts of PEG–lipid solutions added to liposomes for PEGylation and the actual
amount of modied PEG–lipid (Nakamura etal. 2012).
2.2.3 PEGylation ofMicelles
Polymeric micelles are formed by self-assembling diblock and triblock amphiphilic co-polymers, comprising both hydrophobic and hydrophilic segments at concentrations above the critical micelle concentration (CMC). PEG is employed as
the hydrophilic segment of those co-polymers. When co-polymers are dispersed
in aqueous media with hydrophobic drugs, they will assemble into nano-sized
spherical micelles (Fig.2.14), where the core involves the hydrophobic segments
Fig. 2.13 The modied
PEG–lipid amount as a
function of applied
PEG–lipid amount. For
PEGylation of the
liposomes, different
amounts of PEG–lipid
solution were added, and
after PEGylation, the
unmodied PEG–lipids
were removed using gel
permeation
chromatography.
((Nakamura etal. 2012)
with permission)
2 Methods andProtocols fortheSynthesis ofPEGylated Pharmaceutical Nanocarriers
Fig. 2.14 Polymeric micelles conformation made by triblock and diblock amphiphilic pegylated
polyesters. PLA poly(-lactide), PVL poly(δ-valerolactone), PCL poly(ε-caprolactone), PEG4000
poly(ethylene glycol) with molecular weight ∼4000Da, PEG10,000 poly(ethylene glycol) with
molecular weight ∼10,000 Da, MPEG methoxy poly(ethylene glycol). ((Lin etal. 2010) with
permission)
45
and the drug. In contrast, the hydrophilic segments form the shell (Aliabadi
etal. 2008).
Polymeric micelles are more stable than surfactant micelles because they have a
lower CMC.In other words, polymeric micelles are more able to maintain their
core–shell conformation after dilution with a bulk volume of blood in circulation
(Lin etal. 2010).
Several methods are available to prepare polymeric micelles, mainly depending
on the degree of hydrophilicity of the copolymer. The direct dissolution method is
used for the hydrophilic copolymer, in which the micelles are formed directly after
being dissolved in water above its critical micelle concentration (Zhang etal. 2009).
Another method is the lm rehydration method, wherein a volatile solvent is used
to dissolve the co-polymers and drugs, followed by solvent evaporation to form a
membrane, next adding water or buffer solution with stirring to dissolve the copolymer membranes to form the micelles (Zhan etal. 2010).
An example of the copolymer used to form polymeric micelles is the PEG-PLGA
block copolymer. PEG serves as a hydrophilic corona and PLGA acts as a hydrophobic core. In which hydrophobic drugs such as paclitaxel can be incorporated into
the particle core (Bockstaller etal. 2005; Grubbs 2005; Vaia and Maguire 2007;
Pozzo and Walker 2008; Ohshima 2016).
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A. A. Ali et al.
Nishiyama et al. prepared micelles by self-assembling block co-polymers
composed of PEG and polyaspartate loaded with cisplatin (Nishiyama et al.
2003). In another approach, Bae etal. developed a novel technique for the con-
jugation of doxorubicin with PEG-b-poly(aspartic acid) diblock co-polymers, in
which doxorubicin was linked to co-polymer by a pH-responsive hydrazine
linkage, which allowed its release in the acidic tumor microenvironment (Bae
and Kataoka 2009).
2.2.4 PEGylation ofInorganic Nanocarriers
Inorganic nanocarriers made of calcium phosphate, gold, silica, and iron oxide can
be prepared, resulting in nanocarriers with uniform size with easy surface functionalization (Karakoti etal. 2011). Inorganic nanocarriers have low stability and could
be toxic in biological systems. However, surface PEGylation can improve their biological stability and biocompatibility.
PEGylation methods for gold nanocarriers have been frequently done using thiol
(SH) terminated PEGs since the strong binding of thiol groups to gold (SAu bond
energy=47kcal/mol) (Fig.2.3). Accordingly, the stability of gold colloids increases
against aggregation in different buffers/mediums and at high ionic concentrations.
Short-chain low molecular weight PEG (below 5000Da) is often used to offer
enough surface coverage to cover the surface of nanocarriers completely.
Monofunctional PEG-SH can be used to render the surface of gold nanocarrier passive when no other surface ligands are required (Karakoti etal. 2011). On the other
hand, Brown etal. use heterobifunctional PEG with a thiol end coated on a naked
Fig. 2.15 Preparation of PEGylated magnetic iron oxide nanocarriers. In the rst step, iron ions
are precipitated under basic conditions in the presence of oleic acid, producing oleic-acid-coated
nanocarriers. In the second step, performed in toluene, oleic acid is replaced with a silane group
using ligand exchange with silane-PEG, resulting in PEGylated magnetic iron oxide nanocarriers
MNPs. ((Larsen etal. 2009) with permission)