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2 Methods andProtocols fortheSynthesis ofPEGylated Pharmaceutical Nanocarriers
nanoparticle surface and carboxylate functionalities to conjugate oxaliplatin to the
gold nanocarriers (Brown etal. 2010).
PEGylation of magnetic iron oxide nanocarriers is frequently done via a combination of PEG–silane due to the very high binding afnity of the silane group (SiH4)
with the surface of oxide nanocarriers, Fig.2.15 shows an example of preparing
PEGylated iron oxide nanocarriers. Two ways are mainly used for preparing
PEGylated magnetic iron oxide nanocarriers:
1. Coating the nanocarriers rst with a silane group via APTMS (aminopropyl tri-
methoxy silane) or APTES (aminopropyl tri ethoxy silane) followed by functionalizing the terminal amine group with a carboxy terminated PEG.
2. Producing PEG–silane rst and then reacting with magnetic iron oxide nanocar-
riers to get PEGylated magnetic iron oxide nanocarriers in one step with silane
as the primary shell and PEG forming the external shell (Larsen etal. 2009).
PEGylation of silicon nanocarriers can be performed by hydrosilylation of the
Si_H terminated silicon nanocarriers with alkene-terminated PEG using chloroplatinic acid as a catalyst (Mangeney etal. 2002).
47
2.3 Assessments ofSurface PEGylation Efficiency
2.3.1 Indirect Assessment (Qualitative Assessment)
To evaluate the existence of PEG on a particle surface, many indirect, qualitative
measurements are used based on the changes in the physical or chemical properties
upon PEGylation. Indirect assessment includes monitoring the particle size, measuring zeta potential (ζ), the surface measurements of hydrophilicity on the
PEGylated nanocarrier, and microscopic techniques (Rabanel etal. 2014).
2.3.1.1 Particle Size
Several methods are available to measure particle diameters. The most used
method is dynamic light scattering (DLS), also called photon correlation spectroscopy (PCS). The measured particle size depends on the core size of the nanocarrier and any attached or loosely bound polymer or solvent/ion molecules to the
particle surface (Fig.2.16). The measured particle size by DLS is expressed as a
“hydrodynamic radius” (RH). Additionally, it offers values comparable to size
values obtained by techniques such as transmission electron microscopy (TEM)
(Rabanel etal. 2014).
Typically, attaching PEG on a nanocarrier surface increases the nanocarrier
diameter. Consequently, measuring the hydrodynamic diameters of nanocarrier
before and after PEG grafting has been used to evaluate the degree of surface
PEGylation, wherein the diameter increases as the PEG layer thickness increases,
particularly in the brush regime (Fig.2.16). Both the PEG molecular weight and
coverage-density determine the PEG layer thickness, and it usually takes a value
between 1 and 10nm (Thierry and Griesser 2012; Gaumet etal. 2008).
48
Fig. 2.16 Core diameter
of the nongrafted
nanocarrier vs.
hydrodynamic diameter
PEGylated nanocarrier. (a)
Nongrafted nanocarrier;
(b) PEGylated nanocarrier
with mushroom
conformation; and (c)
PEGylated nanocarrier
with brush conformation.
((Rabanel etal. 2014) with
permission)
A. A. Ali et al.
For example, the particle size of the silica nanocarrier before and after attaching
PEG 5kD revealed differences in diameter consistent with the radius of gyration of
PEG (Thierry and Griesser 2012). Similarly, Redhead etal. attach PEG by physical
adsorption to the surface of PLGA nanocarrier by incubating them with Poloxamine
908®. Which causes an increase in mean particle size after physisorption but with
low values (4–6nm layer thickness) (Redhead etal. 2001).
The DLS technique has the following limitations:
1. Only spherical nanocarriers, monodisperse (narrow width of distribution) parti-
cles with sizes below 1μm can be analyzed (Gaumet etal. 2008). Other methods, such as microscopy, should be used for nanocarriers with different shapes
(Gaumet etal. 2008; Thierry and Griesser 2012).
2. Several factors limit the sensitivity of DLS.Minor differences in the layer thick-
ness may not be detected when measuring particle sizes larger than this range
(>100–200nm). Conversely, little differences can be seen for smaller particles
(<20nm), with a constant polydispersity index (PDI) before and after grafting
(Thierry and Griesser 2012; Gaumet etal. 2008).
2 Methods andProtocols fortheSynthesis ofPEGylated Pharmaceutical Nanocarriers
49
3. Several factors can signicantly affect the measurements, such as viscosity, pH,
temperature, the release of surfactants from the particle surface, salt concentration changes, and nanocarrier concentration, which should be controlled to preserve similar conditions (Hackey and Clogston 2010).
4. Only recommended if the PEG layer is added after the formation of the nanocar-
rier (Rabanel etal. 2014).
2.3.1.2 Zeta Potential
Measuring the zeta potential to assess the PEGylation efciency is a simple, widely
used indirect technique. It has been applied to various types of nanocarriers, including liposomes (Webb etal. 1998; Dadashzadeh etal. 2008), polymeric nanocarriers
(Gref etal. 2000; Dunn etal. 1994; Shi etal. 2006; Li etal. 2001; Craparo etal.
2006; Peracchia etal. 1998), solid lipid nanoparticles (SLNs) (Bocca etal. 1998),
nanocapsules (Mosqueira etal. 1999; Loch-Neckel etal. 2007), gold nanocarriers
(Owens etal. 2007), nano complexes (Neu etal. 2007), magnetic nanocarriers (Jain
etal. 2005), and core–shell nanocarriers (Zahr etal. 2006).
An alteration in the ζ potential upon the adsorption of PEGylated macromolecules on a different type of particles has been reported (Redhead etal. 2001; Stolnik
etal. 1994; Suma etal. 2012; Poon etal. 2011). A reduction in the ζ potential occurs
by increasing the surface coverage density of PEG chains due to an increase in the
PEG layer thickness (Meng etal. 2004). However, by increasing PEG chain lengths,
comparable results could be obtained. Craparo etal. reported a reduction in the
surface charge of poly(hydroxy ethyl aspartamide methacrylate) (PHM) nanocarriers as the amount of PEG2000 used was increased, as shown in Table2.1 (Craparo
etal. 2006). In another study, the absolute value of the zeta potential for PEGylated
solid lipid nanoparticles (pSLN) was reduced from about 20 to 15mV by increasing
the amount of PEG (Yuan etal. 2013).
However, the zeta potential method has several limitations including:
1. It is challenging to have quantitative correlations between PEG coverage density
and ζ. In most studies, a plateau of the zeta potential value is reached quickly at
a low PEG content (Gref etal. 2000). Rahme etal. showed a non-linear relationship between PEG molecular weight and the ζ potential of gold particles, with a
plateau (a zeta potential minimum) at around 20kD PEG (Rahme etal. 2013).
Table 2.1 Mean diameter (nm) and the zeta potential for PEGylated poly(hydroxyethylaspartamide
methacrylated) nanocarrier
Extent of PEGylation
0 147.0
15 184.2
30 189.6
50 237.5
A decrease in the zeta potential of nanocarriers in water is noticed as the extent of PEGylation
increases (Craparo etal. 2006)
a
Expressed as % of PHM-PEG2000 co-polymer weight on the total co-polymer amount
a
Mean diameter (nm)
Zeta potential (mV)
−49.10±6.05
−19.63±5.72
−16.45±4.32
−15.86±4.65
50
A. A. Ali et al.
On the other hand, Meng etal. reported a linear relationship between PEG surface concentration and ζ (Meng etal. 2004).
2. The zeta potential value depends on other factors, not only the PEG layer, such
as pH, buffer type, and salt concentration, so it is impractical to compare results
obtained from different environments (Ebbesen etal. 2013).
3. No correlations between the zeta potential and PEG grafting are evident in some
cases, such as when PEG is being utilized to coat an already neutral surface. For
example, Webb etal. noted no difference between the surface charges of liposomes of egg sphingomyelin/cholesterol (SM/chol) and PEGylated ones, as near
zero potential was measured for both (Webb etal. 1998). In addition, PEGylation
of PLA or PLGA nanocarriers has revealed a weak effect on the highly negative
zeta potential despite adding a signicant amount of PEG (Duncanson et al.
2007; Tobio etal. 1998).
2.3.1.3 Surface Hydrophilicity
Several methods can be used to assess the surface hydrophilicity of nanocarriers,
where PEGylated nanocarriers show higher surface hydrophilicity compared to
non-PEGylated nanocarriers.
One of those methods is the Rose Bengal method (Doktorovova etal. 2012).
Figure2.17 represents the surface hydrophobicity of PEGylated zein nanocarriers
using the Rose Bengal test as a function of the PEG-to-zein ratio. As the amount of
Fig. 2.17 Surface hydrophobicity of zein nanocarriers as a function of the PEG-to-zein ratio.
((Reboredo etal. 2021) with permission)
=´
2 Methods andProtocols fortheSynthesis ofPEGylated Pharmaceutical Nanocarriers
51
PEG used during the coating process increases, the surface hydrophobicity decreases
(Reboredo etal. 2021).
Another method used is comparing the HIC (hydrophobic interaction chromatography) ratios for PEGylated nanocarriers with non-PEGylated ones. This method
is based on the measurement of the surface hydrophobicity/hydrophilicity of a particle by washing the particles present in a column twice. The more hydrophilic
particles will be eluted in the rst wash, while the more hydrophobic particles will
be eluted in the second wash. Then HIC ratio is calculated using Eq. (2.1).
HICratio
AUCelutionpeak
AUC wash peak
100
(2.1)
More hydrophilic particles exhibit a larger HIC ratio. So the higher the degree of
PEGylation, the higher the HIC ratio. For illustration, Peracchia etal. utilized this
method to conrm the PEGylation of poly(hexadecyl cyanoacrylate) (PHDCA)
nanocarriers (Peracchia etal. 1998). Nevertheless, this method has some limitations. It is not quantitative, it only applies to particles with a highly hydrophobic
surface, and it also cannot be used alone to expect invivo behavior of a nanocarrier
(Howard etal. 2008).
2.3.1.4 Microscopic Techniques
A possible way to examine PEGylated nanocarrier is by using microscopic techniques, such as scanning electron microscopy (SEM) and electronic transmission
microscopy (TEM). They have been used quite often to characterize the morphology of nanocarriers (Howard etal. 2008) and measure the size of the core of the
particles (Bonevich and Haller 2010; Sitterberg et al. 2010; Roe et al. 2004)
(Fig.2.16). However, they are not commonly applied as a characterization technique for PEGylation (Howard et al. 2008) since most polymeric layers on
Fig. 2.18 TEM and SEM micrographs of diblock and star-shaped copolymer micelle-like nanocarriers. ((Jie etal. 2005) with permission)
52
Fig. 2.19 FTIR spectra of
zein, PEG 35,000, lysine,
bare zein nanocarriers, and
PEG-coated zein
nanocarriers. Straight lines
correspond to 1637 and
1521/cm stretching
vibration bands. The
dashed line corresponds to
the 1093/cm band
((Reboredo etal. 2021)
with permission)
A. A. Ali et al.
nanocarrier are not easily visible as they collapse in vacuum and have low electronic
density (Sitterberg etal. 2010; Roe etal. 2004). Generally, staining procedures can
be used to characterize the PEGylation, such as negative staining of PEGylated
micelle-like nanocarriers with phosphotungstic acid that results in a bright hydrophobic core surrounded by a gray hydrophilic shell (Fig.2.18) (Jie etal. 2005).
2.3.1.5 Fourier Transform-Infrared Spectroscopy (FT-IR)
Fourier transform-infrared spectroscopy (FT-IR) is a qualitative method used to
assess the PEGylation of nanocarriers (Howard etal. 2008). PEGylation is generally
conrmed by the following absorption bands:
• The stretching vibration of C–O–C (1143/cm).
• The vibration of CH2 groups (1465/cm).
• The C–O vibration of the OH end group of PEG (1093/cm).
Figure 2.19 demonstrates the FTIR spectra of the different nanocarriers and the
raw materials utilized in their preparation. The PEGylation of the nanocarrier is
conrmed by the presence of PEG vibration bands (1465, 1143, and 1058/cm) in the
spectra of PEGylated nanocarriers. In addition, it is worth mentioning that as the
PEG-to-zein ratio increases, the size of these signals increases (Reboredo etal. 2021).
2 Methods andProtocols fortheSynthesis ofPEGylated Pharmaceutical Nanocarriers
53
2.3.2 Direct Assessment (Quantitative Assessment)
2.3.2.1 Colorimetric Methods
Colorimetric assays have been utilized to quantify and localize PEG chains (Shi
etal. 2006; Bazile etal. 1995). In the iodine/potassium iodide colorimetric assay or
Baleux’s assay, a reaction between iodine and PEG produces a blue coordination
complex which is quantied via spectrophotometry (Baleux 1972). By comparing
the concentration of PEG quantied before and after alkaline hydrolysis of nanocarriers, it is possible to determine if PEG is located on the particle’s surface (Howard
etal. 2008). Based on the measured size of the nanocarriers, an estimation of PEG
coverage density and distance between adjacent chains can be done assuming a
homogeneous surface (Howard etal. 2008).
An additional calorimetric method is the aqueous ferro-thiocyanate assay. It
detects the PEG conjugate or nonionic PEGylated surfactant by creating a colored
complex when PEG partitions in an organic phase (Cheng etal. 2012; Nag etal.
1996; Al-Hanbali etal. 2007). In both methods degrading of PEG from the nanocar-
rier surface is required since only PEG in the solution can be measured. Moreover,
in these methods, fresh solutions and precise measurement time points are required
to obtain reliable data due to the limited sensitivity and a slow decline of absorbance
over time (Budijono etal. 2010).
2.3.2.2 Chromatographic Methods
Chromatographic methods have been developed to detect free and un-grafted PEG
chains using HPLC coupled with refractive index (RI) or viscosimetric detectors,
evaporative light scattering detector (ELSD), mass spectrometry (MS), rather than
UV, as PEGs lack a chromophore (Auriola etal. 1993).
An illustrative example is the quantication of free PEGs (nonattached to the gelatin nanocarrier) that separate from nanocarriers via asymmetric ow eld-ow fractionation (AF4) (Fraunhofer and Winter 2004). Then HPLC coupled to a refractive
index (RI) detector is used to detect and quantify free PEG in the efuent (Zillies etal.
2007). The amount of PEG grafted on nanocarriers is calculated as the difference
between the initial PEG quantity and the free PEG quantied by HPLC (Zillies etal.
2007). RI detectors have low sensitivity, unstable baseline, long equilibration time,
and high limit of detection due to the sensitivity of RI to impurities, temperature, and
ow. On the other hand, quantication methods using ELSD and LC–MS detectors
have higher sensitivity than RI and with less interference (Nair etal. 2006).
2.3.2.3 UV andFluorescence Spectroscopy
The amount of PEG can be quantied via UV and uorescence spectroscopy using
different strategies and via coupling of PEG with a chromophore or uorophore
(Rabanel etal. 2014). Two strategies of PEG quantication by uorescence or UV
spectroscopy are (Fig.2.20) the grafting of uorescent PEG conjugates and the
labeling of grafted PEG chains.
In the rst strategy, the nanocarriers are PEGylated using uorescent PEG conjugates such as uorescein-PEG 5kD (Perry et al. 2012). Then the bound PEG
54
A. A. Ali et al.
Fig. 2.20 Methods of PEG quantication by uorescence or UV spectroscopy. (a) Grafting of
uorescently labeled PEG and (b) labeling of grafted PEG chains with a uorescent or UV marker
molecules are quantied by uorescence spectroscopy. However, the problem with
this approach is that conjugating the uorescent tag to PEG could affect its physical
and chemical properties, affecting chain conformation, grafting yield, and eventually surface coverage density (Rabanel etal. 2014).
The second strategy (labeling of grafted PEG chains) requires the presence of a
reactive group at the end of the PEG chain to attach a uorescent dye or specic
ligand. The quantitative results are related to the yield and the rate of coupling. An
example is the Kaiser test (primary amine test), which is based on the reaction of the
amine group (NH2) of bound PEG with ninhydrin to produce a deep blue color
(Kaiser etal. 1970).
The main drawback of the UV/uorescence approach is the need to prepare
labeled particles. Hence, the quantitative measurements rely on the yield of the
labeling reaction, so they may not be representative of the real particles. On the
other hand, NMR and XPS analyses do not need those modications (Rabanel
etal. 2014).
2 Methods andProtocols fortheSynthesis ofPEGylated Pharmaceutical Nanocarriers
55
Fig. 2.21 1H NMR spectra of PEG-PLA branched multiblock co-polymer (PLA attached to three
blocks of PEG) suspended in CDCl3, and 1H NMR spectra of nanocarriers prepared with the same
polymer by emulsication-solvent evaporation and suspended in D2O. ((Rabanel etal. 2014) with
permission)
2.3.2.4 Nuclear Magnetic Resonance (NMR)
NMR can assess the degree of PEGylation without any post-modication. It simply
depends on the presence or absence of specic chemical moieties. HrKach etal.
characterize qualitatively the PEGylation of nanocarriers made of the PEG-PLA
diblock. The PEGylation of nanocarrier was conrmed by the presence of a signal
in 1H NMR compared to free PEG in deuterated water (D2O). At the same time, the
polymeric solid inner core made of hydrophobic PLA segments gave no NMR signals (Fig.2.21) (Hrkach etal. 1997). In addition, PEG coating efcacy (% of total
PEG found on the surface) of PLA-PEG nanocarriers can be calculated as the ratio
of surface PEG acquired in D2O analysis to the total PEG obtained from nanocarriers dissolved in deuterated chloroform (CDCl3) (Sheng etal. 2009).
56
A. A. Ali et al.
2.3.2.5 X-Ray Photoelectron Spectroscopy (XPS)
X-ray photoelectron spectroscopy (XPS) is a technique used to characterize the
chemical composition of surfaces and allows for some quantication (Briggs 1990;
Briggs and Grant 2003). It is mainly used to identify the relative percentage of the
different chemical states of an atom; therefore, the chemical functionalities that
exist at the nanocarrier surface. The presence of PEG on the surface can be conrmed by comparing spectra before and after PEGylation (Howard etal. 2008). For
example, suppose the particle has no ether compounds. In that case, the appearance
of an ether bond specic to PEG (C–O–C peak) and the signal integration are related
to PEG concentration (Brindley etal. 1995).
Table 2.2 A list of potential alternatives of PEG
Polymers Comments
Poly(glycerols) (PGs) • Could be hyperbranched or linear, with a high degree of
functionalization due to multiple hydroxyl groups (Siegers
etal. 2004)
• Have low immunogenicity and good biocompatibility
• Hyperbranched PGs are more highly resistant to oxidation or
thermal stress than PEG and demonstrate long plasma
half-lives (Siegers etal. 2004)
• Application to liposomes was demonstrated (Hofmann etal.
2010)
• It tends to accumulate in the kidneys and liver, limiting their
use (Abbina and Parambath 2018)
Poly(oxazolines) (POX) • Soluble in both hydrophilic and hydrophobic solvents (Abbina
and Parambath 2018)
• Less prone to oxidation reactions, unlike PEG with no
bioaccumulation (Khutoryanskiy 2018)
• Thermo-sensitiveness (Abbina and Parambath 2018)
• Difcult, costly synthesis (Khutoryanskiy 2018; Hadjesfandiari
and Parambath 2018)
• Studies are still to be done to prove their effectiveness as an
alternative to PEG (Pytela etal. 1989)
Poly(acrylamide) and
poly(methacrylamide)
• Such as poly(hydroxypropyl methacrylate) (PHPMA) and
poly(2-hydroxyethyl methacrylate) (PHEMA) are nonionic
polymers that are widely used in biomedical applications
(Nunvářová etal. 2019)
• Have low immunogenicity, biocompatibility, and a prolonged
circulation time. However, the highly toxic monomers limit
their use as PEG alternatives (Abbina and Parambath 2018)
(Hadjesfandiari and Parambath 2018)
• PHPMA shows excellent efcacy in preclinical studies as a
carrier for chemotherapeutic drugs and has already entered
clinical trials (Abbina and Parambath 2018)
(continued)