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9 PEGylated Nanocarrier System forNucleic Acid Delivery
277
conformational changes, electrostatic binding, hydrophobicity, are changed. These
physiological and physical modications let the therapeutic ingredient stay in the
body for longer. Also, it can alter the distribution and absorption patterns and impact
how well the medicinal component binds to cell receptors. Making a molecule
heavier through PEGylation can confer numerous signicant pharmacological
advantages over the unaltered version. These advantages include improved drug
solubility, decreased dosage frequency with potential for reduced toxicity without
sacricing efcacy, extended circulation life, and higher drug absorption. PEGylated
versions might also qualify for patent protection due to their enhanced stability and
defense against proteolytic degradation (Guo etal. 2022).
This chapter will explain PEGylated nanocarrier methods for delivering nucleic
acids, including their design, synthesis, and applications. The advantages and limitations of PEGylation and the factors affecting PEGylated nanocarrier systems’ efciency and safety will be discussed. Finally, the current and prospects for nucleic
acid therapeutics of PEGylated nanocarrier systems will be outlined.
9.2 Advantage ofPEGylation
The developments in nanotechnology over the past several decades could signicantly affect the eld of drug delivery and diagnostic imaging. Although they have
also been developed as controlled release systems for water-soluble drugs, colloidal
drug delivery systems (CDDS) are most frequently used to deliver highly lipophilic
drugs and drugs that are unstable in biological environments (for example, proteins,
nucleic acids, and peptides). Polymeric nanoparticles, liposomes (self-assembled
lipid bilayers), micelles (self-assembled amphiphilic molecules), and dendrimers
are examples of solid lipid nanoparticles (SLNs, dispersions of solid lipids) (repeatedly branched spherical polymers) are examples of systems that are typically
included in CDDS (Fig.9.1). These nanostructures provide several benets over
traditional delivery methods for many medications (Howard etal. 2008a, b).
First, their small size and targeting skills enable them to deliver substances to
specic locations, increasing local concentrations, and lowering systemic toxicity.
The treatment of neurological illnesses, which is hampered by the difculty of
many medications to enter the BBB (BLOOD BRAIN BARRIER), and cancer therapy, in which toxic side effects are frequently manifested, are two signicant therapeutic areas where this may have an impact. Moreover, these nanostructures might
shield medicines from enzymatic deterioration.
Lastly, it has been demonstrated that nanocarriers generally have low toxicity
levels, high stability levels, and increased medication solubility. They could also be
functionalized to create release systems that are time- or space-controlled. The same
is true for delivering radionuclidic and contrast-enhancing drugs using nano carrivers to improve diagnostic imaging.
Nanocarriers are removed from the bloodstream with the opsonization of the
particles and subsequent absorption of the RES by cells. Nevertheless, interactions
with proteins adsorbed on the surface of the nanoparticles appear necessary for
278
Fig. 9.1 PEGylation process
A. Tiwari et al.
Fig. 9.2 Colloidal delivery systems
macrophage absorption of nanocarriers in most species proteins called opsonin
(Fig.9.2).
Colloidal drug delivery systems are nanoscale carriers that improve drug solubility, stability, and bioavailability. These systems include liposomes, micelles,
nanoparticles, and nanoemulsions, which encapsulate or bind drugs for targeted
delivery. By protecting drugs from degradation, enabling controlled release, and
enhancing site-specic accumulation, colloidal systems reduce side effects and
increase therapeutic efcacy. They are widely used in cancer therapy, gene delivery,
and the treatment of infectious and inammatory diseases. The small solid spheres
represent the hydrophilic head groups of amphiphilic molecules, and the long chains
are lipophilic tails depending on their size, charge, stiffness, or hydrophobicity,
including complement protein C3b, immunoglobulins G and M, bronectin,
C-reactive protein, −2-glycoprotein, and apolipoproteins. They need to be made
less likely to be recognized by these opsonin tropical substitutes for conventional
distribution methods. The most popular technique for reducing protein adsorption
and introducing “stealthiness” is PEGylation. A number of explanations have been
9 PEGylated Nanocarrier System forNucleic Acid Delivery
Fig. 9.3 Possible mechanisms governingPEG’s ability to stop protein adsorption. Adapted from
Creative Commons Attribution (CC BY) © 2019 license (http://creativecommons.org/licenses/
by/4.0/). (Howard etal. 2008a, b)
279
put out to explain why PEG makes nanocarriers more stealthy, including reduced
surface charge, hydrophilicity, molecular exibility, and non- immunogenicity
(Howard etal. 2008a, b).
PEG has been proposed as a way to reduce the interfacial energy needed for
protein binding, albeit has not been thoroughly investigated. According to Jeon
etal., PEG chains have an extended shape when they are free in solution due to their
hydrophilicity and exibility. A repulsive force is produced by this transition to a
higher energy shape, which can inhibit protein interaction. Several researchers say
exhibiting long-circulating properties involves drawing in the suitable proteins
rather than preventing protein adsorption. Several investigations by Moghimi and
Patel provide credence to the presence of opsonin which is particular to the liver and
spleen and may result in a changed distribution prole. Two serum components
were shown to be permanently linked to poloxamine 908 (four PEG polymers coupled to four polys (propylene gly-phthalcol) in early experiments (PPG); the polyester all joined by an ethylene diamine moiety (Fig.9.3).
9.3 PEGylated Nanocarriers asTherapeutic Delivery System
PEGylation, which involves applying poly (ethylene glycol) (PEG) to a surface
through adsorption, grafting, or entrapment techniques, has emerged as the preferred technique for enhancing nanocarriers’ ability to be biocompatible with
increased stealth. The features that PEG-coated surfaces convey, such as their
280
A. Tiwari et al.
resistance to the reticuloendothelial system (RES) uptake, have not been replicated
by other substances (Veronese and Mero 2008a, b). Nonetheless, there is currently
little general agreement on several related issues. Scientists interested in using this
technology frequently nd themselves with more questions than answers because of
the vast differences in systems, components, procedures, and accompanying ndings. Secondly, PEG seems to have contradictory characteristics, acting, depending
on the circumstance, as both a mucoadhesive polymer and a barrier to protein
adsorption. Second, while numerous research studies have been conducted to clarify how PEG endows nanocarriers with long-circulating properties, further work is
still required to guide its application. Most PEGylation research of several PEGylated
systems was compared to non-PEGylated systems. Because each situation is unique,
it can be challenging to transform the data is transformed into valuable knowledge.
9.4 Development ofPEGylated Nanocarrier
In the 1970s, they were swiftly followed by several investigations and advancements made by other teams, as listed in Table9.1. The US FDA has cleared PEG, a
non-toxic, non-immunogenic polymer polyethylene glycol, for internal usage. The
researchers took advantage of PEG’s unique features. The benet of PEG is that it
has a wide range of molecular weight species with low polydispersity, exceptional
exibility, excellent solubility in both aqueous and organic environments, high
hydration that increases its hydrodynamic volume, and favorable biological properties. 431 PEG-covalently bound compounds obtain each of these characteristics..
Since a water cloud surrounds the polymer, proteins conjugated with PEG become
more soluble and resistant due to their increased size (Fig. 9.1), and antibodies,
proteolytic enzymes, and cells are ultra-ltered less slowly by kidneys (Veronese
and Mero 2008a, b). Proteins are delicate molecules that are easily denatured and
inactivated. Finding a binding chemistry gentle enough not to harm the protein was
the rst challenge to attain success with PEGylation, which involves joining PEG to
proteins. There is a chemical process for gently converting the terminal hydroxyl
group of the PEG into one that combines with the leftovers of protein amino acids.
The amino groups in proteins have been primarily exploited for conjugation because
they are always present, commonly found near the protein surface, and are accessible to the solvent. Cysteine thiol residues are also very benecial when present
because they permit site-specic conjugation and are infrequently seen in proteins;
moreover, it is possible that these alterations were proven to be essential for maintaining the protein’s biological function (Veronese and Mero 2008a, b).
Developing a less polydisperse product species with a specic structure—the
branching form being the most efcient—was another important aspect of enhancing PEG properties. It is evident in this regard that the evolution of PEGylation
chemistry corresponds to the use and efciency of biological treatments (Table9.1).
PEGylation has contributed to the success of various therapeutic proteins and oligonucleotides, including some items that have become blockbusters (Table 9.1).
Moreover, because conjugated proteins dissolve in some organic solvents, they can
9 PEGylated Nanocarrier System forNucleic Acid Delivery
Table 9.1 Background TO PEGylations
Decade
In 2000 era Used in
From 1990
to 2000 era
In
1980–1990
era
In 1970–80 PEG-
AA amino acids, NHS N-hydroxy succinimide, OPSS ortho-pyridyl disulde, PEG polyethylene glycol
PEGs General observation Application
enzymatic
coupling
reactions,
Coupling for
disuldes,
Continuous
PEGs, PEGs star
PEGs as
branched,
NHS-PEGs,
OPSS-PEGs
Aldehyde-PEGs,
carbonyl
imidazole PEGs,
Succinimidyl
carbonate- PEGs
etc.
succinimidyl
succinate
PEG-chloro
triazine
More strict regulatory
requirements, detailed
chemical and biological
characterization of
conjugates, and
combination of generic
engineering and
PEGylation in the design
and discovery of new
drugs.
PEGlyted drug selectivity
and marketing improved.
Absence of diols results
in reduced polydispersed
site-specic
conjugations.
Lack of selectivity in the
immunogenic autocratic
starting material,
extremely polydisperse
peg
PEGylation of
non-protein drugs,
oligonucleotide
PEGylation 3, and
cell PEGylation.
Hormones,
anticancer drug
targeting,
cytokines
Substitution
treatment of
enzymes.
Study
investigations,
biocatalyst
enzymatic
alterations.
281
Reference
Veronese
and Mero
(2008a, b)
be employed as new biocatalysts because PEG is an amphiphilic polymer (both
hydrophilic and hydrophobic). What makes this scenario intriguing are the unexpected catalytic roles of lipase or chymotrypsin in synthetic processes. A PEGprotein conjugate must consider several unique variables. In the following sections,
several aspects are discussed with typical illustrations of polymer-modied proteins. To obtain more detailed documentation, the reader is suggested to study several recent evaluations (Suk etal. 2016).
9.4.1 Chemistry ofPEGylation
A substance, usually a medication or a protein, is regulated by covalently adding
PEG. Extending the molecule’s circulation time in the body enhances its stability,
solubility, and therapeutic efcacy. The process of PEGylation contains the reaction
of a reactive group on a PEG molecule on the target molecule, generally an amine,
thiol, or carboxylic acid. The PEG molecule is typically activated with a functional
group like succinimidyl ester or maleimide. PEG-maleimide conjugation to a cysteine residue on a protein illustrates a PEGylation reaction. The maleimide functional
282
Fig. 9.4 Schematic representation ofprotein with bound polyethylene glycol
A. Tiwari et al.
group on PEG and the thiol group on cysteine interact in this reaction to generate a
thioether bond, which results in a strong covalent connection between the protein
and PEG. The conjugation of PEG-succinimidyl ester to an amine group on a
medicinal molecule is another illustration of PEGylation chemistry. The primary
amine group on the drug molecule and the succinimidyl ester functional group on
PEG combine in this reaction to produce an amide bond (Veronese and Mero 2008a,
b) (Fig.9.4).A protein with bound polyethylene glycol is shown schematically. The
ether-oxygen groups of the polymer work along with the surrounding circles to
simulate a cloud of water. This schematic depicts a protein conjugated with polyethylene glycol (PEG), where PEG chains are attached to the protein surface. The
ether-oxygen groups of the PEG are represented, along with surrounding circles,
simulating a hydrated “cloud” of water molecules. This hydration layer stabilizes
the protein, reduces immunogenicity, and improves solubility, making PEGylation
a valuable technique in drug development and therapeutic protein delivery.
9.4.1.1 Amino Acid Modifications
PEGylation is a common method for enhancing the pharmacokinetics and biodistribution of nanocarriers such as liposomes, nanoparticles, and micelles. In PEGylation,
which entails covalently gluing PEG chains to the surface of the nanocarrier, PEG
(polyethylene glycol) is a biocompatible and hydrophilic polymer that is frequently
utilized (Veronese and Mero 2008a, b).
Amino groups are another functional group that can be used for modifying nanocarriers. Amino groups can be introduced onto the surface of nanocarriers by using
amine-containing molecules, such as polyamines or amino acids, during the preparation of the nanocarrier. Once the amino groups are present on the upper surface of
the nanocarrier, they can be used for the covalent conjugation of PEG chains or
other molecules.
9 PEGylated Nanocarrier System forNucleic Acid Delivery
283
Collecting amino groups to the overhead of nanocarriers can provide several
advantages over traditional PEGylation. For example, amino groups can be used to
attach targeting ligands, such as peptides or antibodies, to the surface of the nanocarrier. This allows targeting the delivery of the nanocarrier to particular cells or
tissues, improving the therapeutic efcacy and reducing off-target effects.
In addition, amino groups can be used for pH-sensitive drug release from the
nanocarrier. By attaching pH-sensitive moieties, the medicine can be released in
reaction to pH variations thanks to the amino groups on the surface of the nanocarrier, such as in the tumor’s acidic environment (Veronese and Mero 2008a, b).
Overall, adding amino groups to nanocarriers creates a exible framework for
creating pH-sensitive and tailored drug delivery systems (Suk etal. 2016).
9.4.1.2 Conjugation ofHistidine totheImidazole Group
The pKa value of histidine’s secondary amine group is lower than that of its primary
amine. A properly activated PEG can be conjugated at this level with a pH close to
neutral. Nevertheless, because the acetate-histidine link is unstable, the conjugate
functions like a prodrug and releases the free protein over time, as with
Peginterferon-2b (Veronese and Mero 2008a, b).
9.4.1.3 Cysteine Thiol Residue Conjugation
PEG-cysteine reagents can create stable thiol-ethers or disuldes, such as maleimide
and vinyl sulfone (e.g., PEG-pyridyl disulde), to conjugate proteins. The latter
results in the production of a protein-PEG conjugate that may be a bifunctional
thiol-reactive PEG that reacts with a substance produced under reducing circumstances and might also be present to build a new body. Free cysteine residues stable
bridges with a polymer attached are highly uncommon because of this amino acid
conjugation to the carboxylic group. When present, arginine often participates in
catalysis or the formation of disulde bridges. Proteins employed for conjugation
contain it. Granulocyte colony-stimulating factor (also known as sugars CSF or Ghydroxyl residues) is one instance where the conjugal and a free cysteine are partly
accessible. Direct coupling of PEG-NH2 to these would cause the amines’ natural
protein to respond to activated protein carboxylic ion that is only conceivable after
partially reversible denaturation of the groups is not feasible. Alternatively, several
examples exist of the protein itself or another nearby protein molecule having cysteine inserted by genetic engineering at a valuable spot of the protein sequence connections within or between molecules.
The research of Zalipsky and Meno-Rudolph led to an innovative approach that
involved using PEG hydrazide, an amino donor that is reactive at low pH.Just
recently, a cunning plan was developed to take advantage, although no practical use
has been discovered to date. The cysteines are harmed when disulde bridges are
present. There are not many instances of arginine-reactive PEGs; these are mainly
based on PEG with a bifunctional thiol-reactive PEG (reported in proprietary literature only) reacts with a substance produced under reducing circumstances and
might also be present to produce a new body. Free cysteine residues stable bridges
with a polymer attached are highly uncommon because of this amino acid (Veronese
and Mero 2008a, b) (Fig.9.5).
284
Fig. 9.5 Schematic illustrates protein modication strategies targeting cysteine residues, including disulde bond formation, thiol-ene and thiol-yne reactions, maleimide conjugation, and oxidative modications. These approaches allow site-specic protein labeling, stabilization, and
conjugation with drugs or probes. Applications in biomedicine and biotechnology include targeted
drug delivery, protein engineering, biosensor development, and therapeutic antibody modication,
enhancing protein stability, functionality, and therapeutic efcacy.© 2021 by the authors. Licensee
MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and
conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/
licenses/by/4.0/) (Pessatti etal. 2021).
A. Tiwari et al.
9.4.1.4 Conjugation toArginine, Carboxylic Groups, andSugar or
Hydroxyl Residues
As they would react with the amine’s natural protein, as in the case of CSF, where a
free cysteine is partially exposed, direct binding of PEG-NH2 to activated protein
carboxylation takes place only after partially reversible denaturation of the groups.
On the other hand, the protein itself or a nearby protein molecule can produce multiple examples. Genetic engineering is used to insert cysteine into a useful location
in the protein sequence connections within or between molecules. The research of
Zalipsky and Meno-Rudolph led to an innovative approach that involved using PEG
hydrazide, an amino donor that is reactive at low pH.How a creative technique was
just recently developed to take advantage, although there has yet to be any practical
use. The cysteines are harmed by disulde bridges when they are present. There are
not many instances of arginine-reactive PEGs (reported in private literature only,
where free cysteine is rst produced via a reduction process); they are often based
on PEG with vicinal dicarboxylic groups, albeit the approach has not been successful due to a lack of selectivity.
9.4.1.5 Conjugation ofGlutamine Residues
While the high glutamine amide and a large number of diol impurities in the samples cannot be precisely reacted chemically, conjugation may be carried out enzymatically by polydispersity, the presence in the polymer of distinct species of
transglutaminases, either specic or non-specic. Heterogeneity in the nished
product was caused by these two elements: a component found in all living things
and obtained from microbial sources. In the rst instance, covalent protein collects
9 PEGylated Nanocarrier System forNucleic Acid Delivery
285
and combines with the transamidase process between the protein’s PEG-NH2,
which acts as the nucleophile donor and glutamine. The following mass is diverse.
It took not only a specication, very high mass polymer (30–40kDa). Shearwater
sequence around the glutamine resides but also the sale of a pure, low polydisperse,
highly selective PEGylation, as we just discovered—polymers, Inc.—to x this
issue later, at the end of the 1990s a connection between the site of a certain conjugation and the exibility of the protein chain. APEG conjugation of glutamines to
the lysine group can also be catalyzed by the same enzyme. Pharmaceutical grade
PEG should have a polydispersity of between 1.01 and 1.1 for products with a 5kDa
to 50kDa molecular weight (Veronese and Mero 2008a, b). Keep in mind that the
value is 1.00 for a monodisperse product.
9.4.2 Releasable PEGs
Releasable PEG nanocarriers are a nanoscale drug delivery system that utilizes
polyethylene glycol (PEG) chains to increase the circulation time of the drug in the
body and protect it from immune system recognition. These nanocarriers are
designed to release the drug payload at a specic target site, such as a tumor, in a
controlled and sustained manner.
One approach to achieving this controlled release is to use stimuli-responsive
PEGs that change their properties, such as solubility or molecular weight, in reaction to certain triggers, such as pH changes, temperature changes, or the presence of
enzymes. The drug payload may be released from the nanocarrier due to this alteration in characteristics.
For example, tumors have a lower pH than healthy tissues, and their acidic environment can cause PEGs to release the therapeutic payload. Similarly, heat sensitivity is created to deliver the pharmacological payload in response to increased
temperature found in inamed or cancerous tissues (Veronese and Mero 2008a, b).
Releasable PEGs nanocarriers have improved drug solubility and stability, longer circulation time, less toxicity, and focused drug administration, which are only
a few benets over conventional drug delivery methods. These systems could
increase the effectiveness and safety of numerous drugs, including chemotherapeutic agents, hormones, and gene therapies.
9.5 Nanocarrier Mechanism ofDelivery toNucleic Acid
Nanocarriers are delivery vehicles that can deliver various types of cargo, including
nucleic acids, to particular cells or tissues inside the body. The mechanism of delivery of nanocarriers to nucleic acids involves several steps:
Nanocarrier uptake: The target cells must take up the nanocarriers to deliver their
cargo. This can occur through a variety of mechanisms, including endocytosis,
phagocytosis, or receptor-mediated uptake.
Endosomal escape: Once the nanocarriers are inside the cell, they are typically
sequestered within endosomes. To reach the nucleus, the nanocarriers must go away
286
Fig. 9.6 Nanocarrier mechanism of delivery to nucleic acid:Nanocarriers deliver nucleic acids by
encapsulating or binding them in protective structures, safeguarding them from degradation. They
enter target cells via endocytosis or membrane fusion. Once inside, nanocarriers release the nucleic
acids, which travel to the nucleus or cytoplasm. Common nanocarriers include lipid nanoparticles,
polymers, and dendrimers, designed to improve stability, targeting specicity, and transfection
efciency for therapeutic applications such as gene therapy or RNA interference
A. Tiwari et al.
from the endosome and enter the cytoplasm. This can be achieved through various
mechanisms, such as the proton sponge effect, photochemical internalization, or
other pH-sensitive strategies.
Nuclear localization: Finally, the nanocarriers must be able to reach the nucleus
and deliver their cargo to the nucleic acids within. This can be achieved through
nuclear localization signals or other targeting strategies.
Once the nanocarriers have delivered their cargo to the nucleic acids, they can be
used for various purposes, including gene therapy (RNA interference). The choice
of nanocarrier and delivery mechanism will depend on the specic application and
the properties of the target cells and nucleic acids(Fig. 9.6).
9.6 Release ofDrug fromPEGylated Nanocarriers
The most popular technique for giving drug nanocarriers stealth characteristics is
now “PEGylation.” The neutrality, hydrophilicity, exibility, and ability to hydrate
the PEG moiety in nanoparticles generate a steric barrier that prevents opsonin