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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5615_Библиотеки_им_академика_М_И_Перельмана.pdf
14 Reversible PEGylation ofNanocarriers
Fig. 14.5 Temperature responsive nanoparticles based on PEGylated polyaspartamide derivative
for drug delivery. Adopted from (Zhang and Jiang 2019) under Creative Commons Attribution (CC
BY) license (http://creativecommons.org/licenses/by/4.0/) Copyright 2019
397
Rieger Jutta etal. reported this approach, and by using this, simple reversible
addition-fragmentation chain-transfer polymerization (RAFT)-mediated aqueous
dispersion polymerization process has successfully prepared PEGylated thermoresponsive micelles and coreshell nanogels such as double hydrophilic (poly (ethylene oxide)-b-poly (N, N-dimethyl acrylamide)). Excellent control over the aqueous
dispersion polymerization of N, N-diethyl acrylamide (DEAAm) was achieved with
the development of PEO-b-PDMAAm reversible chain transfer agents, which have
a tunable length of the poly (N, N dimethyl acrylamide) (PDMAAm) segment.
These agents were used in the radical polymerization of DEAAm in the presence of
a crosslinker, resulting in thermoresponsive gel particles in one step.
The researchers found that a critical minimum length of the PDMAAm segment
was necessary to obtain nanometric particles, which decreased with increasing
length. This approach is a straightforward method for preparing PEO-coated nanogels with a limited number of reactants and high solids contents (Rieger etal. 2009).
Polymers like poly (N-isopropyl acrylamide) (PNIPAM) is a temperature-sensitive
polymer that can undergo a phase transition around body temperature. This property
can be utilized to create PEGylated drug carriers where PEGylation is reversible
upon changes in temperature.
One example of a temperature-responsive PEGylated polyaspartamide derivative
is mPEG-PAAHP, which was synthesized by the click reaction. This derivative was
characterized and conrmed using FTIR and 1H NMR spectroscopy (Zhang and
Jiang 2019). In drug delivery, mPEG-PAAHP has shown potential as a promising
398
M. Mishra et al.
system due to its apparent biocompatibility and temperature-responsive properties
(Zhang and Jiang 2019).
Another study investigated the effect of PEGylation on the stability of thermoresponsive nanogels. It was observed that in the absence of a PEG coating, the
nanogels aggregated at elevated temperatures (Motlaq etal. 2018). However, the
addition of a PEG coating improved the heat responsiveness of the polymers, requiring a lower thermal dose to trigger drug release or gelation (Amin etal. 2022).
Overall, temperature-responsive PEGylation is a promising approach that can
enhance the performance of drug delivery systems and tissue engineering materials.
Developing temperature-responsive PEGylated drugs faces several challenges.
One of the main challenges is the potential loss of activity and heterogeneity of
PEGylated therapeutic proteins, which can affect their conformation, electrostatic
binding, and hydrophobicity, leading to a reduction in binding afnity and biological activity. Additionally, the existence of anti-PEG antibodies and vacuoles related
to PEGs can limit the extensive use of PEGylation in some cases (Zhang and Jiang
2019). Another challenge is the difculty in achieving optimal drug release kinetics,
as the temperature-responsive properties of the materials can be affected by various
factors, such as the molecular weight and concentration of the PEGylated polymers
(Amin etal. 2022). Furthermore, the stability of thermo-responsive nanogels can be
affected by the absence of PEG coating, leading to aggregation at elevated temperatures (Motlaq et al. 2018). These factors need to be carefully considered when
developing temperature-responsive pegylated drugs to ensure their efcacy
and safety.
In summary, temperature-responsive PEGylation is a promising approach in
drug delivery and tissue engineering, as it allows for the development of materials
that exhibit controlled drug release or gelation in response to temperature changes.
This strategy can improve the biocompatibility and stability of thermo-responsive
materials, making them more effective for various applications.
14.2.6 Light-Responsive PEGylation
PEG chains are reversibly linked to and disengaged from medications or carriers in
light-responsive reversible PEGylation, an inventive approach to drug delivery systems. This tactic makes use of photosensitive compounds, which are frequently
added to PEG chains and which alter structurally when exposed to particular light
wavelengths. This change initiates a reversible transformation of the PEGylation
status, inuencing the carrier’s or drug’s interaction with the environment. Lightresponsive reversible PEGylation offers a viable path towards the creation of
dynamic and exible drug delivery systems with improved therapeutic precision by
enabling precise and spatiotemporal control over drug release and bioavailability.
This strategy is in line with the increasing interest in creating intelligent and
adaptable medication delivery systems to enhance patient outcomes. Articial
membranes that react to several stimuli at once can imitate biological channels and
pumps. Customized conical nanopores with combined light- and pH-responsive
14 Reversible PEGylation ofNanocarriers
399
features by grafting PEG-spiropyrans. According to studies on ionic transport, a
nanopore opens in reaction to UV light and shuts in response to visible light. The
gating property of the nanopore is facilitated by PEG-spiropyran self-assembly,
which is also affected by the solvent. Water inhibits the photoswitchable molecule
in its zwitterionic state, but conformational switching is encouraged by ethanol. The
pH can regulate the selectivity of nanopores under UV radiation; an acidic pH is
selective for anion transport, while a neutral pH is favorable for cation transport.
Using this method, adaptive systems that resemble biological pumps and channels can be designed (Ma etal. 2018). PEGylation increases the hydrophilicity of
nanoparticles, reduces immune clearance, and prolongs circulation time (Son etal.
2019). Light-responsive properties can be achieved by incorporating photosensitive
moieties into the polymeric structures of hydrogels (Xing etal. 2022).
Light-responsive hydrogels have been developed for various applications,
such as:
1. Controlled drug delivery: Light-responsive hydrogels can be used to release
drugs in a controlled manner, allowing for more precise drug delivery and
reduced side effects (Xing etal. 2022).
2. Biophotonic imaging: Light-responsive nanoparticles can be used for biopho-
tonic imaging, which enables real-time monitoring of drug release and distribution within the body (Son etal. 2019).
3. Targeted therapy: Light-responsive nanoparticles can be coated with PEG to
enhance their hydrophilicity and reduce immune clearance, allowing for more
selective targeting and improved drug delivery (Zhu etal. 2019).
4. Selective targeting and reduced side effects: The combination of PEGylation
and light-responsiveness enables more selective targeting and reduced side
effects, as it allows for precise drug delivery and release (Xing etal. 2022).
5. Improved biocompatibility and reduced immune clearance: Surface
PEGylation of nanoparticles enhances their hydrophilicity, reduces immune
clearance, and prolongs circulation time, leading to improved biocompatibility
(Xing etal. 2022).
These advantages make light responsive PEGylation a promising strategy for
controlled drug delivery, targeted therapy, and improved biomedical applications.
Light responsive drug delivery system built through covalent incorporation of specic light-sensitive chemical groups with the aim to locally release cargo from the
delivery system (Lehner and Hunziker 2012).
Some examples of light-responsive nanoparticles used in drug delivery include:
(a) Spiropyran-based nanoparticles: A nanoparticulate drug delivery system
comprising spiropyran and PEGylated lipid has been developed (Fig.14.6),
where the light-sensitive switch enables particles to uoresce and release drugs
inside cells when exposed to light (Xing etal. 2022).
400
Fig. 14.6 Spiropyran modied gold nanoparticles. Adapted with permission from Shiraishi etal.
(2014), Copyright 2014, Elsevier
M. Mishra et al.
(b) Photochemical reaction-based drug release: Light-responsive nanoparticles
have been used for photochemical reaction-based drug release, where the
release of drugs is triggered by light exposure.
(c) Gold nanoparticles: Gold nanoparticles have been explored for ocular drug
delivery through photothermal reactions. These nanoparticles can rapidly
absorb energy from various light sources and release energy as heat, which can
be used to trigger drug release (Abdelmohsen etal. 2023).
(d) Light-responsive hydrogels: Light-responsive hydrogels have been developed
for controlled drug delivery, using light as a powerful trigger for controlled drug
delivery systems. These systems can provide excellent manipulation of therapeutic agents in chemical and biological stimuli (Xing etal. 2022).
These light-responsive nanoparticles have been utilized in various drug delivery
applications, including chemotherapy, immunotherapy, photodynamic therapy,
gene therapy, wound healing, and ocular drug delivery. The use of light as a trigger
for drug release offers precise control over the delivery of therapeutic agents,
improving patient compliance and convenience.
Light-responsive PEGylation has some potential disadvantages, including:
(i) Immunogenicity: PEGylation can lead to hypersensitivity indirectly by side
products formed during synthesis, and the existence of anti-PEG can cause
vacuoles related to PEGs (Zhang etal. 2014).
(ii) Disadvantages on liposomes: PEGylation can impose certain disadvantages
on liposomes, especially for the delivery of genes and nucleic acids in anticancer therapy (Mishra etal. 2016).
However, these disadvantages are not specic to light-responsive PEGylation
and can be addressed by optimizing the modication degree and molecular weight
14 Reversible PEGylation ofNanocarriers
of PEG, as well as designing new PEG reagents for site-directed PEGylation modication (Zhang etal. 2014).
401
14.3 Characterization ofReversible PEGylation
14.3.1 Analytical Techniques
Various analytical techniques are used to characterize reversible PEGylation in drug
delivery systems. These techniques help researchers understand the nature of
PEGylation, monitor changes, and optimize the design of drug carriers. Common
techniques include Dynamic Light Scattering (DLS), Nuclear Magnetic Resonance
(NMR), High-Performance Liquid Chromatography (HPLC), Fourier Transform
Infrared Spectroscopy (FTIR), UV-Visible Spectroscopy, Mass Spectrometry (MS),
Fluorescence Spectroscopy, and Zeta Potential Measurement. DLS measures particle size distribution, providing information about changes in hydrodynamic diameter of PEGylated nanoparticles. NMR spectroscopy is useful for studying the
structure and composition of PEGylated compounds, identifying PEG chains, and
determining their attachment sites.
HPLC separates and quanties components in mixtures, analyzing PEGylated
drugs and determining the degree of modication. FTIR helps identify chemical
bonds and functional groups, conrming PEG on drug carriers and understanding
the nature of bonds formed during PEGylation (Zalipsky etal. 2007). MS determines the molecular weight of PEGylated compounds, conrms PEG chain attachment, and assesses reversibility. Fluorescence spectroscopy studies uorescence
properties of labeled compounds, providing information about changes in the
microenvironment of uorophores. Zeta potential measurement evaluates the surface charge of nanoparticles, providing insights into the stability and surface characteristics of PEGylated carriers (Shechter et al. 2005). NMR is a ngerprint
technique for the characterization of these systems. Also (Rieger etal. 2009) mentioned in their report that chemical structures of SAD-PEG-SAD and CAD-PEGCAD which are reversible PEGylated complexes conrmed by proton nuclear
magnetic resonance.
14.3.1.1 Qualitative Characterization ofReversible PEGylation
Zeta Potential
Zeta potential can be determined based on the DLS principle. DLS measures the
molecular radii of the samples and hence can be used to estimate the molecular
weight of PEGylated proteins. It can also differentiate between linear and branched
PEGs. The DLS method is popularly used to study the interactions in monoPEGylated proteins.
The researchers have formulated the pH-reversible lipoplexes, and upon checked
for the zeta potential, they found that the surface charge changes signicantly when
the amount of PEG incorporated into the liposomes is increased. The zeta potential
402
HICRatio =Elution volume of sample /Elutionvolumeofreference
M. Mishra et al.
without PEG coating was +27mV; after incorporation of only 5% PEG, the zeta
potential was changed to +11mV.High surface charge on the liposomes might help
in interacting with the receptors, but can decrease the stability due to aggregation
(Nie etal. 2011). Fella etal. have made amine-reactive polyplexes. The polyplexes
had high positive zeta potential (+25mV). When the polyplexes were partly shielded
with PEG moiety, zeta potential changed to +15mV.But when the polyplexes are
shielded with 30-fold higher concentration of PEG, the zeta potential was found to
be below +5mV (Fella etal. 2008).
Hydrophobic Interaction Chromatography (HIC)
HIC comparatively measures the hydrophilicity and lipophilicity of the particle. In
this technique, two washes are provided, in which the rst wash selectively washes
the hydrophilic constituents away, whereas the second wash is responsible for elimination of comparatively more hydrophobic particles.
The “HIC Ratio” is calculated as follows:
The lower HIC ratio indicates the presence of hydrophobic particles and higher
value indicates hydrophilic particles.
Researchers have used the hydrophobic interaction chromatography in the purication of mono-PEGylated antiepidermal growth factor receptors (EGFR). For the
selective capture, a weak cation exchanger was used. Upon increasing the size of
PEG from 5 to 30kDa, the retention on HIC was increased. HIC resulted in the suppressed aggregation of the protein and allowed good resolution with high purity and
product recovery. The purity was found to be increased to 98% (Moosmann
etal. 2012).
Near Infrared (NIR) Spectroscopy
The NIR spectroscopic technique coupled with Principal Component Analysis
(PCA) can also give the information about the hydrophilic/lipophilic balance of the
compound. PCA is the statistical technique which measures the variance of the
given set of data (Bista and Bruch 2008). NIR spectroscopy shows the upconversion
spectra when the PEGylated nanoparticles were subjected to NIR laser with 980nm
diode excitation. This upconversion of the nanoparticles is because of the use of
PEG moiety has been employed in the biolabeling in the biological window. Zhang
etal. have prepared the pH-reversible PEG conjugate and the methods to characterize it, the absorbance was measured by UV-VIS-NIR spectrometer to determine the
activity of PEGylated protein. The NIR data were used for the relative activity of the
proteins added in various formulations (Zhang etal. 2020).
Fourier Transform-Infrared Spectroscopy (FT-IR)
In FTIR spectra, the presence of the PEGylation can be seen by the presence of CH2
and C-O-C peaks. This method is effectively used for various kinds of metallic as
well as polymeric nanoparticles. The PEGylation of the nanoparticles can be
14 Reversible PEGylation ofNanocarriers
403
conrmed by the FTIR analysis. The characteristic peak for PEG moiety were
observed at wavelength of 2884cm−1 (stretching vibration) and 1468cm−1 (bending
vibration). Massoumi etal. have developed the pH-reversible polymeric nanocapsules and the FTIR spectra of neat PEG showed the stretching vibration of C-O at
1108 cm−1, bending vibration of -CH2- groups at 1467 cm−1 and aliphatic C-H
shows the stretching vibration at 2950–2800cm−1. The hydroxyl or adsorbed water
shows the vibration band at 3490cm−1 (Massoumi etal. 2020).
13C-NMR
In NMR, the decrease in the CH and CH2 peaks indicates the PEGylation of the
nanoparticles. NMR spectroscopy is mostly used for the PEGylated proteins, in
which the PEGylation leads to alteration in the intensity of peaks. Herald’s group
performed the solid state 13C-NMR, and the spectrum clearly showed the resonance
of methyl group at 17ppm and OCH2 groups shown 70.3 ppm intensity, which
clearly indicates that the groups were not resolved clearly. In the characterization of
the diblock copolymer of chitosan-PEG, when the 13C-NMR spectra of the proposed
diblock polymer was compared with the spectra of chitosan alone, one broad peak
(68.6ppm) and four narrow peaks (59.0, 63.4, 64.1, and 173ppm) were found to be
the characteristic peaks due to the presence of PEG micromere (Ganji and
Abdekhodaie 2008).
Mass Spectrometry
The specialized instrumentation employing Liquid Metal Ion Gun (LMIG) and
single- stage reectron analyzer is used for the qualitative determination of the
PEGylation in the given sample (Stigsnaes etal. 2007). Furthermore, TOF- SIMS
data are obtained and interpreted with software. The fragment ions C2H2O2 and
C2H5O+ indicate the presence of PEG moiety. MALDI-TOF mass spectrometry
was used by scientists to identify the sites of PEGylation of eluted fraction. They
have PEGylated various proteins. In the results, ve distinct peaks were obtained at
interval of 44Da. The graph also gave the unreacted free biotin-PEG molecules.
From the molecular weights of the fragments, it can be inferred that which site is
utilized for the PEGylation among the possible seven amino groups. Higashi etal.
have PEGylated bromelain, and the MALDI TOF spectrum was obtained showing
specic peaks at 23,500m/z values. In case of modied bromelain for reversible
PEGylation approach, the peak was obtained at an m/z value of 24,400, suggesting
the effective PEGylation of the parent moiety has been occurred (Higashi etal. 2020).
14.3.1.2 Quantitative Characterization ofReversible PEGylation
High-Performance Liquid Chromatography (HPLC)
HPLC can be coupled with techniques such as refractive index detector, evaporative
light scattering detector, or mass spectrometry for the effective estimation of the
degree of PEGylation. Simple HPLC methodology employing UV detector often
fails due to the absence of chromophore in the PEG moiety. To obtain enhanced
hypotensive effect, etal. have conjugated atrial natriuretic peptide (ANP) with PEG
404
M. Mishra et al.
moiety. Furthermore, the HPLC analysis was performed using reverse phase-18 column and analyzed at the wavelength of 220nm. The HPLC analysis was found
useful to eliminate the free atrial natriuretic peptide from the prepared ANP-PEG
conjugates and serves as the better means of purication of the PEG conjugates
(Nesher etal. 2008).
Calorimetry
For the calorimetric quantication of the PEGylation, rst the alkaline hydrolysis of
nanoparticles is done and the concentration and localization of PEG chains is determined. For perfectly spherical nanoparticles, the surface density of PEG and the
distance between the PEG chains can also be determined effectively. The calorimetric analysis of PEG conjugates in mainly employed in the case of the macromolecules. Researchers have carried out the calorimetric study of the conjugates of the
PEG with myoglobin moiety and upon application of the thermodynamic models,
the thermal denaturation of the conjugates is examined. If the denaturation has
occurred, a single step state is observed. It is proposed from the calorimetric data
that the exothermic processes occurring do not contribute largely to the starting of
the thermodynamic curve (Pelosi etal. 2019).
X-Ray Photoelectron Spectroscopy (XPS)
This technique basically provides the elemental composition of the surface of
nanoparticle. The extent of PEGylation caused can be assessed by comparing the
spectrum obtained before and after PEGylation. This method is most useful in case
of the metallic nanoparticles, but possess some limitations in case of polymeric
nanoparticles. When various substrates were PEGylated with catechol-grafted PEG,
XPS analysis of the PEGylated substrates shown the differences in the atomic composition of the surface. Quantitative analysis by using XPS revealed the immobilization of the PEG molecules. After PEGylation, the signals for Si in silicon have
been decreased in intensity. For qualitative examination, it was evident that the signals for the substrate were decreased signicantly after modication with PEGcatechol; however, the signals arising due to catechol and PEG were increased. The
relationship between the XPS spectra values and the amount of PEG added can be
established for the accurate determination of the PEGylation. Zhang et al. have
examined different nanodiamond samples to identify the elements found in the
nanodiamonds. XPS scan was performed in the range of 0–1100eV.XPS studies
revealed the presence of C, N, and O atoms (Zhang etal. 2012).
Nuclear Magnetic Resonance (NMR)
Recently, the NMR analysis is also found to be an effective methodology for estimating the PEGylation. In this, the integrals of 1H NMR PEG peak are compared
with the peak of some standard compound to get an idea about the quantity of PEG
attached to the surface of nanoparticles. The scientists have used the NMR spectroscopy to determine the degree of PEGylation of the complex macromolecules by
using the NMR spectroscopy. The proton NMR spectroscopy of proteins was
14 Reversible PEGylation ofNanocarriers
405
carried out to determine the number of methoxy PEG chains grafted to a protein,
also known as the degree of PEGylation.
The proton NMR spectroscopy overcomes the biases caused to the chromatographic methods used in the determination of the degree of PEGylation. Spectroscopy
was performed at 300MHz for protons. For this, the biconjugate solution was transferred to the NMR tube, and also, DMSO was added as an internal standard, upon
carrying out the NMR spectroscopy, it was found that the singlet at 3.69ppm was
observed for ethylene groups of PEG and a singlet 2.71ppm peak was observed for
methyl groups of DMSO (Utatsu etal. 2021).
TGA-DSC
The combination of Thermogravimetric analysis (TGA) and Differential scanning
calorimetry (DSC) can be used to determine the amount of PEG grafted on the
nanocarrier system and can be interpreted as comparison of the amount of PEG
grafted on the surface to the total weight of the nanocarrier system. The samples are
vacuum dried prior to analysis. Zhang etal. have prepared the nanodiamonds, and
the TGA analysis have shown that the percent reduction in the weight of OH and
COOH was 0.4% and 3.1%, respectively, which suggests that the functional groups
are further attached to the surface of nanodiamond after oxidation. The TGA data
also revealed that the thermal decomposition temperature of PEG is about 400°C
and after 500°C, no PEG was remained on the surface (Zhang etal. 2012).
The scientists have PEGylated BSA protein and examined the thermal events by
using DSC.DSC gives the maximum heat capacity of the sample. The key nding
of the experiment that the apparent folding temperature, i.e., T
PEGylation, but not affected by the molecular weight of PEG.The apparent T
was lower for PEGylated BSA than for intact BSA, The apparent T
was affected by
max
was found to
max
max
be 82°C for intact BSA protein and was 2°C lower for the conjugate of PEG with
BSA (Paolino etal. 2017).
14.3.2 In Vitro Characterization ofReversible PEGylation
In vitro methods are generally used for the exact determination of complement consumption, protein adsorption, macrophage uptake, and also for the release rate of
the drug substance and its stability. Table 14.1 contains some of the important
invitro techniques along with their applications.
14.3.2.1 Protein Adsorption
For estimation of adsorption of proteins on the nanoparticle surface, the nanoparticles are diluted and vortexed. The size of nanoparticles is obtained in different sera
by nanoparticle tracking analysis (NTA). For determination of collagen-bound
nanoparticles, the confocal laser scanning images can be acquired after the polymerization of collagen, and the diffusion coefcient can then be determined by using
Raster image correlation spectroscopy (RICS) (Filpula and Zhao 2008).
406
Table 14.1 In vitro characterization of reversible PEGylation
In vitro technique Application
1. Electrophoretic methods, Electron Microscopy, Gel
Permeation Chromatography (GPC)
2. Surface PEO density Determination of complement
3. Cell uptake studies Uptake of PEGylated nano-carriers
4. Release studies Extent of PEGylation required
Study of PEGylated proteins and
structure of proteins
consumption
by RES
M. Mishra et al.
14.3.2.2 Cellular Association
The cellular association can be determined by using ow cytometry. The particular
kind of cells are rst cultured in the suitable culture media. The cells are then seeded
on 12-well plates, 100,000 cells per well of the plate. Once the log phase of the cellular growth is attained, the cells are incubated with PEGylated nanocarrier system
and kept for 3h. Then, the cells were detached and analyzed with ow cytometry.
The cellular uptake is determined by the uorescent intensity measurement of the
nanoparticles relative to the uorescent intensity obtained in the untreated population of cells (Filpula and Zhao 2008).
14.3.2.3 Cellular Effects andTarget Affinity
Upon the exposure to the PEGylated and non-PEGylated proteins, the short-term
and long-term cellular events are monitored by microelectronic cell sensor array
(MCSA). These measurements give increase in the mass over the time and allow
easy calculation of doubling time of the given population. Specically, the results
are checked for the presence of any kind of acute, chronic, or cell proliferation
effects (Hamidi etal. 2006).
14.3.2.4 Retention ofBiological Effect
The biological effect of PEGylation should be maintained for sufcient period of
time for the successful development of suitable drug candidate. As PEGylation
maintains the drug concentrations for comparatively longer times in the body, sometimes, a little decrease in the biological activities does not appear to be troublesome.
Using surface plasmon resonance (SPR), the binding afnities of proteins and the
PEG chains can be easily determined. Generally, PEGylation leads to little or no
loss of afnity for the biological site (Tao etal. 2009).
14.3.2.5 Bioactivity Assay
These assays can be used for the quantitative determination of the PEGylated molecules in serum samples. PEGylated enzymes are more popularly tested by this
approach as many detectable substrates are available for the enzymes.
Pharmacokinetic properties of PEGylated nanocarriers can be determined by the
cytopathic effect assay (CPE) (Tao etal. 2009).