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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5373_Библиотеки_им_академика_М_И_Перельмана.pdf
14 Reversible PEGylation ofNanocarriers
387
One of the recommended techniques for improving nanocarrier biocompatibility
is PEGylation, which is accomplished by adsorbing, grafting, or entrapping poly
(ethylene glycol) (PEG) onto surfaces improves stealth qualities are conferred by
this technique, which surpasses other chemicals’ ability to inhibit RES absorption.
“Stealth” property mentioned in the context of nanocarrier’s surfaces, diminishes
their recognition or uptake by the cellular immune system, leading to longer circulation time, reduced dosage and frequency, and superior site-selective delivery of
drugs (Howard etal. 2008).
14.1.3 Reversible PEGylation andIts Significance
The selection of a PEGylation strategy depends on various factors, such as the target
parameter, entity function, and structure (drug molecule or colloidal carrier).
Permanent modication via stable covalent binding of PEG strands to the agent or
particle surface and releasable PEGylation via the use of specially designed linkers
responsive to hydrolysis, enzymes, or certain stimuli are the two most investigated
approaches. But the primary barrier to PEGylation is the inactivation that frequently
occurs when a PEG chain covalently attaches to a protein, particularly peptide medications (Shechter etal. 2005).
A useful delivery platform is provided by reversible PEG modication, which
permits control over pharmacological action and active agent regeneration. The
conjugate created with this method becomes a “prodrug,” slowly and spontaneously
hydrolyzing under physiological conditions to release the native active peptide or
protein drug from the inert conjugation over an extended period of time (Shechter
etal. 2005). In a recent study, researchers used polyamidoamine dendrimers that
have been loaded with doxorubicin and PEGylated with both acid-insensitive succinic linkage and acid-sensitive cis-aconityl linkage (Zhu etal. 2010).
Another research group employed this phenomenon to enhance the circulatory
lifespan of peptide and protein drugs. A novel PEG-IFNR2 conjugate, PEG40FMS- IFNR2, was developed. This conjugate, formed through a slowly hydrolyzable bond, allows for the regeneration of native interferon R2 (IFNR2) under
physiological conditions (Fig.14.1). The invitro regeneration rate indicated a halflife of 65hours. The pharmacokinetic prole, simulated numerically, demonstrated
prolonged invivo maintenance and active-native IFNR2 regeneration, offering an
advantage over current formulations in terms of accessibility to peripheral tissues.
Utilizing this reversible PEGylation event has various benets, some of which were
already covered (Peleg-Shulman etal. 2004; Rieger etal. 2009).
Drug delivery techniques such as reversible PEGylation are essential because
they enable the temporary attachment of PEG chains to peptides and proteins, controlled release of the active agent, maintenance of the therapeutic activity of the
drug while prolonging its half-life in circulation, and improvement of the exibility
and adaptability of nanomedicines (Shechter etal. 2005).
388
Fig. 14.1 Diagrammatic
representation showing
PEGylation and reversible
PEGylation
M. Mishra et al.
14.2 Reversible PEGylation Strategies
Reversible PEGylation allows for precise control over the PEGylation state, offering advantages in drug delivery, protein engineering, and bioconjugation. Reversible
PEGylation is a novel technology that allows the release of native proteins, such as
interferon-alpha 2 (IFN-alpha 2), over a prolonged period invivo (Peleg-Shulman
etal. 2004). PEGylation, or the covalent attachment of polyethylene glycol (PEG)
chains, is a technique used to prolong the action of therapeutic proteins in the body.
However, PEGylated peptides and proteins are often inactive due to their shortcirculatory half-life (Peleg-Shulman etal. 2004). Reversible PEGylation aims to
overcome the limitations of traditional PEGylation by using a combination of PEG
and other components, such as 2-sulfo-9-uorenylmethoxycarbonyl (FMS), to create a slowly hydrolyzable bond between the PEG and the protein (Peleg-Shulman
etal. 2004). This allows the protein to retain its activity and be released gradually in
the body, providing a sustained therapeutic effect.
For example, a study developed a PEG-IFNalpha2 conjugate, PEG (40)-FMSIFNalpha2, which was capable of regenerating native interferon alpha 2 at a slow
14 Reversible PEGylation ofNanocarriers
389
rate under physiological conditions. The invitro rate of regeneration of native interferon was estimated to have a half-life of 65hours, and the active IFNalpha2 levels
peaked at 50h, with substantial levels still being detected 200hours after administration (Peleg-Shulman etal. 2004).
Another example is the use of a supramolecular polymer-based transformable
material, PEG-NH2-PRX, which is a mixing-type PEGylation material that provides amino groups to interact with protein drugs on demand (Utatsu etal. 2021).
This material has been shown to efciently form complexes with proteins and
improve their stability compared to other PEGylation materials (Utatsu etal. 2021).
In summary, reversible PEGylation is a promising approach to improve the therapeutic efcacy of protein drugs by releasing native proteins in a controlled manner,
overcoming the limitations of traditional PEGylation (Utatsu etal. 2021).
14.2.1 Reversible PEGylation Chemistry
The most basic linker design for releasable PEGylation could have an exposed ester
bond between the drug and the polymer. However, simplicity in design does not
deliver the needed customized release kinetics required for drug release control and
personalized pharmacokinetics (Filpula and Zhao 2008). Furthermore, molecules
frequently lack a functional hydroxyl or carboxyl group for ester formation. Many
biologically active compounds have additional functional groups, such as amines
and thiols, which necessitate more complex linker architectures. The present stage
of prociency in linker design for releasable PEGylation is a result of a recent
focused study on prodrug chemistry (Filpula and Zhao 2008).
14.2.2 Linkers forReversible PEGylation
PEG chains can be temporarily attached to biomolecules like proteins or peptides
with the help of reversible PEGylation linkers. These linkers then permit the PEG
chains to be cleaved or detached under specic circumstances. These linkers’ chemistry can vary, but they frequently contain functional groups or cleavable bonds that
react to variations in pH, temperature, or other external conditions. The chemistry
of a few popular varieties of reversible PEGylation linkers is shown in Fig.14.2.
Aromatic and aliphatic linkers are two broad categories of chemical linkers or
spacers used in various applications, including molecular biology, drug design, and
materials science.
14.2.2.1 Aromatic Linkers
Contains one or more aromatic rings in their structure which are characterized by a
closed loop of alternating single and double bonds, such as benzene rings. Aromatic
linkers often provide rigidity to the molecule due to the conjugated pi-electron system in the aromatic ring. They are generally planar and can participate in pi-pi
stacking interactions with other aromatic molecules. They can have distinct
390
Fig. 14.2 Attributes of releasable PEGylation. Diagram of key elements in rPEGylation that
include a trigger segment that initiates the linker degradation pathway and the release of the original protein as veried by multiple analytical methods
M. Mishra et al.
electronic properties and may be involved in electronic or photophysical processes
(Fig.14.3).
14.2.2.2 Aliphatic Linkers
They are characterized by open-chain structures, lacking the closed-loop of alternating single and double bonds found in aromatic rings. Aliphatic linkers often contain saturated carbon-carbon bonds, such as in alkyl chains. Aliphatic linkers are
typically exible and less rigid compared to aromatic linkers. They do not have the
same pi-pi stacking interactions that aromatic rings can form and are often used to
increase the exibility and mobility of attached functional groups (Fig.14.3).
14.2.3 Cleavage Linkers
Cleavable linkers in reversible PEGylation are used to attach PEG to a therapeutic
molecule, such as a protein or peptide, and enable its release under specic conditions (Gupta etal. 2019). These linkers allow for the temporary attachment of PEG,
which can be cleaved under certain biological conditions, leading to the release of
the active molecule. Examples of cleavable linkers include enzyme-cleavable ester
or amide bonds, disulde bonds, and other specic chemical moieties that can be
cleaved under physiological conditions. Reversible PEGylation using cleavable
linkers has been studied for its potential in drug delivery and cancer therapy (Gupta
etal. 2019).
14 Reversible PEGylation ofNanocarriers
391
Fig. 14.3 Chemistry of releasable linkers for PEGylation. Adapted with permission from (Filpula
and Zhao 2008), Copyright 2007. (a) Prodrug designs of aromatic (BE: RNL or TML) and aliphatic (bicin) linkers include NHS leaving group, a trigger group for initiation of elimination reactions, and a linker segment attached to a spacer or directly to PEG.The linker segment may be
rationally or empirically designed to provide steric hindrance or additional nucleophilic sites such
that the release kinetics of the linker are controllable. (b) Reaction of an activated PEG of the BE
linker type with a protein amine to form the bioconjugate. In plasma, the initial ester cleavage in
the trigger element rst discharges the PEG and complete release of the linker element from protein occurs rapidly. (c) Chemical basis of the bicin series of releasable linkers. Bis-N-2hydroxyethylglycinamide (1) is subject to hydrolytic assistance from both hydroxyethyl side
chains and cyclizes to a morpholinolactone (2), which is rapidly hydrolyzed to the acid, bicin (4).
(d) Reaction of an activated PEG of the bicin linker type (1) with a protein amine to form the bioconjugate (2). In vivo, the acetyl group is rapidly lost, while the elimination reaction to release the
PEG segment occurs at a moderate and controllable rate with the nal release of the original protein predicted to occur via an intramolecular cyclization event
Cleavable linkers in PEGylation offer a versatile approach to controlling drug
release kinetics, allowing for targeted and controlled release of the therapeutic molecule under specic biological conditions.
392
M. Mishra et al.
14.2.3.1 Hydrolyzable Linkers
These linkers are designed to be cleaved by hydrolysis under specic conditions,
such as changes in pH or enzymatic activity. They enable the release of the PEG
from the therapeutic molecule under controlled biological environments.
Examples of hydrolyzable linkers in PEGylation include:
1. Hydrolytically labile ester linkers: These linkers are designed to be cleaved by
hydrolysis under specic conditions, such as changes in pH or enzymatic activity. They enable the release of the PEG from the therapeutic molecule under
controlled biological environments.
2. Carbonate linkers: A hydrolyzable carbonate linker can connect the drug pay-
load to the PEG.This connection breaks under acidic or basic conditions, allowing for controlled drug release.
3. Succinimide-based linkers: These linkers contain a succinimide ring that can
undergo a hydrolysis reaction when the linker-payload combination reacts with
cysteine residues on other proteins. This reaction can open the succinimide ring,
allowing the linker to undergo an elimination pathway and become completely
stable. Seattle Genetics’ new generation of linkers incorporate a primary amine
adjacent to the maleimide, which promotes a much faster hydrolysis reaction.
4. Glucuronic acid-based linkers: Some hydrolyzable linkers replace the
maleimide group, where the linker is cleaved when it enters the cancer cell, with
a hydrophilic glucuronic acid moiety. Lysosomal compartments of cells have
β-glucuronidase enzymes that can remove the glucuronic acid, which can
improve the ADC’s pharmacokinetic properties.
These hydrolyzable linkers in PEGylation offer a versatile approach to controlling drug release kinetics, allowing for targeted and controlled release of the therapeutic molecule under specic biological conditions.
14.2.3.2 Enzymatically Cleavable Linkers
These linkers are designed to be cleaved by specic enzymes, allowing for targeted
drug release at particular sites in the body. For example, protease-sensitive linkers
can be used to achieve controlled release of the therapeutic molecule in response to
enzymatic activity (Ghosal etal. 2021).
Examples of enzymatically cleavable linkers in PEGylation include:
1. Dipeptide-based linkers: Val-Cit and Phe-Lys are examples of dipeptide-based
linkers that can be used in enzymatically cleavable PEGylation. These linkers
can be cleaved by specic enzymes, allowing for controlled drug release
(Mccombs and Owen 2015).
2. PAB (p-amino benzyl alcohol): The dipeptide valine-citrulline combined with
PAB is a popular enzymatic cleavage sequence. PAB is a self-immolative linker
that can release the free drug when it is cleaved by enzymes (Mccombs and
Owen 2015).
14 Reversible PEGylation ofNanocarriers
393
3. Peptide linkers: Creative Biolabs offer a class of enzymatically cleavable pep-
tide linkers for conjugating antibodies and drugs. These linkers can be designed
to be cleaved by specic enzymes, allowing for controlled drug release (Mccombs
and Owen 2015).
4. β-glucuronide linkers: These linkers incorporate a hydrophilic sugar group that
can be cleaved by β-glucuronidase. Once the sugar is cleaved from the phenolic
backbone, self-immolation of the PAB group releases the free drug.
These enzymatically cleavable linkers in PEGylation offer a versatile approach
to controlling drug release kinetics, allowing for targeted and controlled release of
the therapeutic molecule under specic biological conditions (Mccombs and
Owen 2015).
14.2.4 pH-Responsive PEGylation
It is a technique used in drug delivery systems to target specic cells or tissues. It
involves the use of PEG chains that are attached to nanoparticles and can be designed
to be pH sensitive (Rustad etal. 2022). PEGylation, which reverses after a therapeutic agent reaches the target cell, is an attractive feature for drug, protein, or nucleic
acid delivery. PEG has been widely used to enhance the circulation time of nanocarriers in the bloodstream, improving drug delivery efciency (Sun et al. 2015a).
However, its nonspecic binding can hinder effective drug release at the target site.
It also appears that the covalent binding of PEG to proteins results in steric interference, which shields conjugates from proteolysis and lowers the pace at which they
are removed from the bloodstream by renal ltration and intracellular absorption
(Tsubery etal. 2004). The pH-responsive reversible PEGylation is a groundbreaking advancement in drug delivery systems, allowing nanocarriers to be coated with
PEG chains that selectively cleave in response to changes in pH levels. This allows
for precise drug release at the desired location, reducing the risk of premature
release.
The pH-responsiveness of these systems arises from the protonation of specic
groups, leading to the destabilization of the nanoparticles and the release of the
encapsulated drug (Fig.14.4). Research has shown that pH-responsive PEGylation
can affect release kinetics and cellular uptake in cancer cells, particularly in glioblastoma cells. pH-sensitive liposomes with cleavable PEGylation have been
designed to target tumor cells, exhibiting a signicantly charge shift and highly
efcient phagocytosis by tumor cells while retaining long blood circulation time.
Therefore, pH-responsive PEGylation represents a promising strategy for targeted
drug delivery, especially in the context of cancer therapy (Ghosh and Dey 2020).
This method has applications in cancer therapy and inammatory diseases,
enhancing treatment efcacy while minimizing side effects (Nie etal. 2011). This
innovative approach represents a signicant step towards personalized medicine,
paving the way for more effective and patient-friendly treatments. These merits are
in agreement with the reports of Sun etal. They synthesized acid-sensitive PEGylated
394
M. Mishra et al.
Fig. 14.4 Diagrammatic representation showing reversible PEGylation strategies and its merit
14 Reversible PEGylation ofNanocarriers
395
DOX, which enhanced tumor accumulation and accelerated intracellular drug
release in acidic conditions. This pH-responsive reversible DOX demonstrated prolonged circulation time in blood, enhanced tumor tissue accumulation, and accelerated intracellular drug release, suggesting potential for clinical chemotherapy of
various malignancies. The study also showed no damage to the body (Sun
etal. 2015b).
Another group of researchers use this phenomenon, they use PEG aldehydecarboxypyridylhydrazone, N-hydroxysuccinimide esters (PEG-HZN-NHS) and
synthesized for bioreversible surface shielding of DNA polyplexes. Both monofunctional and bifunctional mPEG-HZN-NHS were used, with mPEG-HZN-NHS
shielded particles remaining shielded at pH7.4. Luciferase gene transfections with
epidermal growth factor (EGF)-containing polyplexes showed up to 16-fold
enhancement in gene expression with reversibly shielded polyplexes compared to
stably shielded polyplexes. Reversibly shielded polyplexes also mediated an
enhanced tumor-specic invivo transgene expression (Fella etal. 2008).
pH-responsive PEGylated nanocarriers offer several advantages in drug delivery,
including:
1. Targeted drug delivery: pH-responsive nanocarriers can be designed to release
drugs specically in the acidic environment of tumors, leading to targeted drug
delivery and reduced side effects (Alsawaftah etal. 2022).
2. Improved drug stability: PEGylation can improve the stability of drugs, pro-
tecting them from degradation and premature release (Li etal. 2022).
3. Increased circulation time: PEGylation can increase the circulation time of
nanocarriers in the bloodstream, allowing for prolonged drug exposure and
improved therapeutic efcacy (Karimi etal. 2016; Alsawaftah etal. 2022).
4. Controlled drug release: pH-responsive nanocarriers can be designed to release
drugs in a controlled manner, allowing for sustained drug release and improved
therapeutic outcomes (Alsawaftah etal. 2022).
However, there are also limitations to consider, such as reduced intracellular
uptake of PEGylated nanoparticles and immunogenicity of PEG.Despite these limitations, pH-responsive PEGylated nanocarriers remain a promising strategy for
targeted drug delivery, especially in the context of cancer therapy.
There are several drugs that have been successfully delivered using pH- responsive
PEGylated nanocarriers. Some examples include:
(a) Paclitaxel: Acetal-linked PEGylated paclitaxel prodrugs have been developed
to form free-paclitaxel-loaded pH-responsive micelles with high drug loading
capacity and improved drug delivery (Li etal. 2022).
(b) Doxorubicin: pH-sensitive liposomes with a PEGylated pH-responsive poly
(amino acid) (PAA) corona have been developed to release encapsulated doxorubicin in a pH-responsive manner, showing potential for tumor-specic drug
delivery (Karimi etal. 2016).
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M. Mishra et al.
14.2.4.1 Proteasome Inhibitor MG132
Micelles composed of poly (ethylene glycol)-poly-aspartate copolymer with 45nm
particle size have been developed to deliver proteasome inhibitor MG132, showing
low invivo toxicity of micelles than free MG132 and prolonged circulation of drugloaded micelles in the bloodstream (Karimi etal. 2016).
These examples demonstrate the potential of pH-responsive pegylated nanocarriers in delivering drugs for cancer therapy and other applications. pH-responsive
PEGylation has been explored in various drug delivery systems, such as liposomes
and poly (lactic-co-glycolic acid) (PLGA) nanoparticles, for cancer therapy. Here
are some examples:
1. pH-responsive liposomes with cleavable PEGylation: These liposomes exhibit
a signicant charge shift and highly efcient phagocytosis by tumor cells while
retaining long blood circulation time.
2. pH-sensitive liposomes with a PEGylated pH-responsive poly (amino acid)
(PAA) corona: These liposomes display a high colloidal stability in blood and a
pH-responsive release of encapsulated doxorubicin both invitro and invivo. The
invitro doxorubicin release showed a half-life of 3.4h at pH7.4, which increased
to 21.6h at pH5.0. The invivo antitumor efcacy was evaluated in mice bearing
4 T1 luciferase-labeled breast cancer, showing the potential of these pHresponsive liposomes for tumor-specic drug delivery.
3. pH-responsive PEGylation of PLGA nanoparticles: The effect of pH on the
stability of PLGA nanoparticles with respect to the pH range of 7.4 (pH of cytoplasm) to 6.5 (pH of lysosome) was investigated. The study found that the pH of
the medium does not affect the stability of the formulation, suggesting that the
pH of the medium does not inuence the degradation of the polymer (Hu
etal. 2020).
These examples demonstrate the potential of pH-responsive PEGylation in cancer drug delivery, offering improved targeted delivery and reduced side effects.
14.2.5 Temperature-Responsive PEGylation
A clever tactic in drug delivery systems is temperature-responsive reversible
PEGylation, in which medications or carriers are linked to PEG chains, which are
then attached and detached in response to variations in temperature (Fig.14.4). This
technique enables PEGylation to be modulated reversibly, enabling a dynamic
response to temperature changes. PEG chains cover the drug or carrier at lower
temperatures and separate at higher temperatures, which may improve drug release
and interactions with biological systems shown in Fig. 14.5. This reversible
PEGylation that is temperature-responsive enhances the exibility and effectiveness
of drug delivery systems by providing a customized response to the physiological
environment for better therapeutic results.