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2 Methods andProtocols fortheSynthesis ofPEGylated Pharmaceutical Nanocarriers
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67
Topographic Properties ofPEGylated
Nanocarriers
AprameyaGaneshPrasad, ShivamOtavi, MahimaMishra,
andRakeshKumarTekade
Abstract
Nanoparticles have been widely used as vectors for bioactive cargo due to their
high therapeutic index. Despite their attractiveness for safe and efcacious
drug delivery, nanoparticles are known to suffer from rapid systemic clearance.
PEGylation, conjugation of the nanocarrier with polyethylene glycol (PEG)
chains, is one of the strategies that has been developed to combat this chal-
lenge. PEGylation confers steric stability to the nanoparticle through a “stealth”
coating which masks the core of the nanoparticle thereby evading a response
from the immune and reticuloendothelial systems. PEG chains are also critical
to ensure the stability of the nanoparticles as they prevent aggregation in col-
loidal suspensions. Due to the crucial role they play in developing an optimal
formulation, it is imperative to establish robust analytical tools to characterize
the surface topography and morphology of PEGylated nanocarriers. Surface
analytical techniques are frequently modied to enable characterization at a
nanoscale. This chapter describes the major characterization tools we have at
our disposal to effectively analyze critical surface attributes. Examples of each
technique are also provided to demonstrate their application, benets, and
drawbacks.
3
A. G. Prasad (*)
Department of Chemical and Biomolecular Engineering, Johns Hopkins University,
Baltimore, MD, USA
S. Otavi · M. Mishra · 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
69
70
Keywords
A. G. Prasad et al.
PEGylation · PEG chain · Steric stability · Characterization · Topography
3.1 Introduction
Nanoparticles are widely used as carriers for a variety of payloads such as small
molecule drugs, oligonucleotides, and diagnostic agents. Their attractiveness in the
realm of drug delivery systems can be attributed to their superior physicochemical
properties including high drug loading efciencies, enhanced transport across vascular membranes, and tunable uptake and internalization in the case of targeted
delivery (Mitchell etal. 2021; Shi etal. 2021). Nanocarriers have thus been exploited
to treat multiple disease indications through a variety of dosage forms and drug
product presentations. A primary challenge that is associated with nanoparticles in
blood circulation is their poor pharmacokinetic prole (Zhu etal. 2022; Zelepukin
etal. 2020). Factors such as size, charge distribution, and surface functionalization
can trigger an immune response that results in rapid clearance from circulation
(Alexis etal. 2008; Zolnik etal. 2010; Hooftman and O'Neill 2022).
Polyethylene glycol (PEG) is a nontoxic, hydrophilic polymer that has been
extensively used in the eld of nanomedicine. It was approved by the Food and
Drug Administration (FDA) in 1990 and has since been used in several commercially available pharmaceutical formulations (Suk etal. 2016; Swierczewska etal.
2015). When coated on the surface of nanoparticles, PEG prevents the adsorption of
serum proteins and masks charges, if any, thereby modulating the biodistribution
and pharmacokinetics of the drug. Therefore, PEGylation is crucial to prevent clearance by the reticuloendothelial system (RES) (Jokerst etal. 2011; Mohamed etal.
2019). In addition to reducing the immunogenicity of nanocarriers, PEG can some-
times also be used for controlled or sustained-release applications (Stras etal. 2020;
Peleg-Shulman etal. 2004; Prasad etal. 2021). PEG also prevents aggregation of
nanoparticles and is therefore used as a stabilizer in nanomedicine (Larson etal.
2012). A lot of research has focused on modifying nanocarriers with PEG in differ-
ent ways—through covalent and noncovalent grafting, chemical conjugation, and
sometimes as a constituent of a copolymer matrix (Rabanel etal. 2014). It is therefore of critical importance to be able to characterize the efciency of the preparation
methods, conrm successful PEG conjugation, and monitor the stability of the conjugated nanocarriers over time. After systemic administration, the interaction of
PEG chains with the physiological environment may also need to be assessed to
predict the invivo efcacy of such nanocarriers.
Though nanoparticles are routinely characterized using several analytical tools,
there are limited methods that can investigate the surface topography of PEGylated
nanocarriers (Mourdikoudis etal. 2018; Baer etal. 2010; Joudeh and Linke 2022).
Standard characterization tools may not provide sufcient resolution to probe the
surface at the nanoscale level (Takechi-Haraya etal. 2022). Modications to conventional methods and a combination of techniques may sometimes be necessary to
3 Topographic Properties ofPEGylated Nanocarriers
71
determine the topographical parameters precisely and accurately. Here, we discuss
multiple characterization techniques spanning microscopic, spectroscopic, calorimetric, and gravimetric analyses. Principles of each technique are discussed along
with the topographical information that can be obtained from each. Surface structure, composition, and morphological characterization are also considered due to
their interdependence on topography. Sample preparation methods and limitations
of each technique are also discussed to guide the selection of appropriate methods.
Discussion of each technique is also accompanied by examples of their application
in the characterization of PEGylated nanocarriers.
3.2 Characterization Techniques
3.2.1 Topographical Characterization Methods
forPEGylated Nanocarriers
Information
Technique
Infrared
spectroscopy
Raman
spectroscopy
XPS/ESCA Surface
NMR Molecular
EDS/EDX Elemental
available Limitations
Surface
composition
Ligand-binding
Chemical
structure
Crystallographic
orientation
Conformation
composition
Electronic
structure
Chemical state
Ligand-binding
Particle size
(<~10nm)
structure
Phase information
Ligand density
and arrangement
Atomic
composition
composition
Nonquantitative
Low sensitivity
Nonquantitative
Low sensitivity
Dry state
Specialized
equipment
Highly trained
personnel required
Poor lateral
resolution
Need for particle
resuspension in
D2O or
lyophilization
Complicated data
interpretation due
to surface
contamination
References
Coelho etal. (2015), Yoncheva
etal. (2005) and Retout etal.
(2019)
Katuwavila etal. (2020), León
etal. (2017) and Levin etal. (2006)
Ebbesen etal. (2013), Rabanel
etal. (2015), Sant etal. (2008),
Essa etal. (2010), Gref etal.
(2001), Damodaran etal. (2010)
and Gref etal. (1994)
Coelho etal. (2015), Yoncheva
etal. (2005), Retout etal. (2019),
Ebbesen etal. (2013), Rabanel
etal. (2015), Essa etal. (2010),
Vila etal. (2004), Conte etal.
(2019), Lu etal. (2019), Xu etal.
(2015), Chen etal. (2016), Essa
etal. (2011), Garcia-Fuentes etal.
(2004) and Vernooij etal. (1999)
Ebadi etal. (2019), Karim and
Chowdhury (2022), Iranpour etal.
(2021) and Maghsoudi etal. (2023)
72
Information
Technique
Mass
spectroscopy
TGA Mass
DSC Phase transition
AFM Particle shape and
SEM Morphology High
TEM Morphology
available Limitations
Elemental
composition
Molecular
orientation and
conformation
Size and size
distribution
Composition
Molecular
interactions
size
Morphology
Texture and
roughness
Structure
Size and size
distribution
Highly trained
personnel required
Dry state
Destructive
analysis
Large sample
requirement
Destructive
analysis
High
instrumentation
cost
Long scan time
Potential damage to
the sample from
probe
instrumentation
cost
Tedious sample
preparation
Limited spatial
resolution
High
instrumentation
cost
Tedious sample
preparation
Dry state
A. G. Prasad et al.
References
Hinterwirth etal. (2013), Sebby
and Manseld (2015), Ara etal.
(2014)
Sebby and Manseld (2015),
Butterworth etal. (2001),
Manseld etal. (2014) and
Maccarini etal. (2010)
Rabanel etal. (2015) and Essa etal.
(2010)
Sant etal. (2008), Essa etal.
(2010), Essa etal. (2011), Sacchetti
etal. (2013) and Backmann etal.
(2010)
Yoncheva etal. (2005), Ebbesen
etal. (2013) and Gref etal. (2001)
Coelho etal. (2015), Rabanel etal.
(2015), Gref etal. (2001), Vila
etal. (2004), Xu etal. (2015), Chen
etal. (2016), Maccarini etal.
(2010), Pelaz etal. (2015), Walkey
etal. (2012) and Allard-Vannier
etal. (2017)
3.3 Infrared Spectroscopy
In infrared (IR) spectroscopy, the molecules on the surface are exposed to an infrared light source, and absorption in the near-infrared region (0.7–2.5μm) creates
changes in dipole moments. A polychromatic source is useful to generate a spectrum over the full infrared range, whereas a monochromatic laser source is more
suitable to study vibrational resonance at a specic wavelength. The absorption or
transmission spectra are measured by a dispersive spectrometer where individual
spectral bands correspond to specic covalent bonds and functional groups. Through
the information about bonds and intermolecular interactions, the spectra can establish the chemical identity and molecular structure of the surface.
3 Topographic Properties ofPEGylated Nanocarriers
73
Fourier transform infrared (FTIR) spectroscopy is more suitable for the midinfrared (2.5–25μm) and far-infrared (25–1000μm) regions. In this technique, different wavelengths are scanned using an interferometer. Total light energy detected
at each position of the mounted mirror is converted to an energy spectrum by applying Fourier transformation across the wavelengths. FTIR can be used to deduce not
only the surface composition but also to conrm and investigate ligand-binding
density and structural arrangements as in the case of PEGylated and other functionalized nanocarriers.
When it is not possible to obtain the spectrum by transmission, attenuated total
reectance (ATR)-FTIR provides an improved sensitivity at the interface compared
to the FTIR technique. Here, a crystal is placed next to the sample in the direction
of incident light. Using the phenomena of total internal reection, the molecules in
the sample absorb energy from the standing evanescent wave thereby resulting in an
attenuated reected beam. Atomic force microscopy (AFM) can be coupled with IR
spectroscopy to include imaging capabilities. This technique is also called
photothermal- induced resonance (PTIR) and provides a better resolution at the
nanoscale (Dazzi et al. 2005). Upon illumination by a laser IR source, the heat
energy in the molecules results in thermal expansion. Such local spatial changes on
the surface are detected by the AFM cantilever at high sensitivity. A lateral resolution of as low as ~10nm is possible using this technique (Mathurin etal. 2018).
Sample preparation methods depend on the nature of the sample. Neat liquid
samples can be directly measured by placing a drop between sandwiched salt plates.
Solutions of liquids may also be used by accounting for IR absorption of the solvent. Aqueous samples are not suitable because water dissolves the potassium bromide (KBr) and sodium chloride (NaCl) plates that are typically used for dissolved
solid and liquid samples, respectively. Water and alcohols, like methanol and ethanol, also strongly absorb in the infrared region thereby masking critical bands in the
sample. Thin-lm liquid cells can also be used for solution-phase IR spectroscopy.
Solid samples can be dissolved in a solvent as described above. If using a solvent, it
is important to avoid volatile liquids as they can evaporate while the spectrum is
being acquired. However, it is typical to use a “Nujol mill” or “KBr press” for solid
samples. Nujol is a viscous hydrocarbon liquid in which ne solid particles are dispersed. Alternatively, the sample can be mixed with solid KBr and this mixture is
ground and pressed in a die. For ATR analysis, liquid and solid samples are placed
in a trough crystal and at crystal, respectively.
Even though IR spectroscopy is convenient, it only provides for a qualitative
analysis. The technique is not as sensitive as other spectroscopic techniques such as
mass spectroscopy. There is a limited choice of solid substrates that can be used to
enhance the sensitivity. In the majority of cases, the sample needs to be in a dried
state which is achieved by dehydration or the use of deuterium oxide. This restricts
the ability to discern the surface elemental analysis of dispersed nanocarriers in
their native aqueous state. AFM-IR suffers from some of the same limitations associated with AFM including the need to dilute and immobilize samples.
74
A. G. Prasad et al.
3.3.1 Application inPEGylated Nanocarriers
Silvia Coelho etal. used FTIR spectroscopy in conjunction with NMR spectroscopy
to study PEGylated gold nanoparticles designed for drug delivery to tumors. The
nanoparticles were encapsulated with Bortezomib and FTIR transmission spectra
were analyzed to investigate the extent of inclusion and interaction of the drug with
the PEGylated nanocarrier. Figure3.1 shows the spectra of PEGylated nanoparticles with (2) and without (1) the drug. The common PEG functional group resulted
in vibrations at 1091 and 1117/cm (C–O–C asymmetric group in the PEG chain),
and 1710/cm (C=O group in the PEG layer) for both variants. However, the spectra
for drug-loaded nanoparticles showed a shift in the stretching of the C=O group
(1628/cm) and missing 1250/cm resonance. Based on this observation, the authors
deduced electrostatic attraction between the positively charged Bortezomib and
negatively charged PEGylated gold nanoparticle surface. This hypothesis was corroborated by a substantial change in the zeta potential of the nanoparticles loaded
with the drug (Coelho etal. 2015). This demonstrates the benet of utilizing this
technique to investigate interactions between the drug and PEG, thereby shedding
light on the localization of encapsulated cargo at the surface of nanocarriers.
Retout and co-authors developed gold nanoparticles coated with PEG utilizing
different chemistries. The PEG ligands were functionalized with a thiol group at one
end, and a methoxy, carboxylate or alkyne group at the other end. The PEG layers
formed by coating the nanoparticles with varying proportions of the two PEG
ligands were systematically analyzed using IR spectroscopy. As shown in Fig.3.2,
all variants showed the characteristic C–O–C asymmetric stretching band at 1100/
cm. All samples with the carboxylate group in Fig.3.2a gave a similar spectrum
independent of the composition except at 1580/cm where a signicant absorption
Fig. 3.1 FTIR transmission spectra of (2) Bortezomib-loaded and (1) empty PEGylated gold
nanoparticles. (Figure adapted with permission from Silvia Coelho etal. (2015) Copyright © 2015,
Royal Chemical Society)
3 Topographic Properties ofPEGylated Nanocarriers
Fig. 3.2 Normalized IR absorbance spectra of gold nanoparticles functionalized with (a)
HS-PEG-CH3/HS-PEG-COOH and (b) HS-PEG-CH3/HS-PEG-C ≡ CH. (Figure adapted with
permission from Retout et al. (2019) Copyright ©2019 Elsevier)
75
band was seen only for pure carboxylate-PEG functionalized nanoparticles. For the
variants functionalized with the alkyne group shown in Fig.3.2b, there was no char-
acteristic signal seen in any of the samples. However, the intensity of the absorption
band at 1680/cm was seen to be signicantly higher for alkyne functionalized
nanoparticles compared to pure thiol-PEG functionalized nanoparticles. This difference was attributed to the presence of citrate ions on the surface of nanoparticles
pointing to a low grafting density of this functional group (Retout etal. 2019). This
study illustrates how IR spectroscopy can be effectively used as a tool to discern
differences in the degree of grafting density of various PEG groups.
Krassimiri Yoncheva et al. designed PEGylated nanoparticles made of
poly(methyl vinyl ether-co-maleic anhydride) (PVM/MA). To characterize the way
PEG was attached to the nanoparticles, IR spectroscopy was used. Figure3.3 depicts
the IR spectra obtained for PVM/MA nanoparticles before and after PEGylation. In
addition to the peaks common to PVM/MA, the PEGylated nanoparticles also
showed a peak at 1730–1740/cm which corresponds to the carbonyl group. The
authors attributed this to potential ester bonds that may have formed as a result of
the PEG-coupling reaction (Yoncheva et al. 2005). This report underscores the
importance of infrared spectroscopy in conrming the identity of PEGylated
nanocarriers.
76
Fig. 3.3 IR spectra of PEG 2000, PEG 2000-NP, and PVM/MA.The arrow shows the new peak
appeared in the spectrum of PEGylated nanoparticles missing in the spectrum of PVM/MA. (Figure
adapted with permission from Yoncheva et al. (2005) Copyright © 2005 Elsevier)
A. G. Prasad et al.
3.4 Raman Spectroscopy
Raman spectroscopy is a vibrational spectroscopic technique that works based on
the principle of inelastic scattering of light upon irradiation of a monochromatic
laser source. When a material is irradiated, part of the incident energy is elastically
scattered at the same wavelength and energy. There is also a small fraction of incident energy that gets scattered at a different wavelength (inelastic scattering). This
difference in energy arising due to the shift in wavelength is known as the “Raman
shift” and the magnitude of this shift is unique to a molecule and its constituent
bonds. The shift in wavelength can also be regarded as the vibrational energy of the
irradiated molecule. As the shift tends to be typically very small, there is a need to
use a monochromatic laser light source.
The irradiation causes perturbations in the electric dipole of the molecule thereby
changing its electrical eld. The resulting molecular vibrations known as photons
and phonons can be recorded and the Raman intensity is plotted versus the frequency of the shift to obtain the spectrum. Sharp bands in the spectrum correspond
to unique functional groups and this can be used to ascertain the identity, chemical
structure, and crystallographic orientation of the irradiated material. Raman spectroscopy not only provides insights into the covalent bonds, intra and intermolecular
interactions but also valuable information about interfacial interactions polymer
chain conformation. The main advantage of using this technique is that the presence
of water in the sample does not affect the spectra in any way.