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9 PEGylated Nanocarrier System forNucleic Acid Delivery
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proteins from adhering. Surfaces of PEGylated particles may be accomplished by straightforward or adsorption by covalently attaching PEG to functional groups that have been activated on the particle’s exterior. Moreover, PEG molecules have been modied to accomplish the controlled release of medicinal chemicals trapped in them and enhance their absorption by certain targets (such as cancers). Various characterization approaches have been developed due to the increase in PEGylation, and mathematical modeling is now being used more frequently to direct formula­tion development. This study provides an overview of PEGylation theories, PEGylation techniques, PEGylation particle characterization, PEGylation-related mathematical modeling, and how PEGylation can improve nanocarrier drug deliv­ery systems to give an overview of the direction that nanocarrier PEGylation and nanomedicine will take the existing accomplishments and shortcomings of PEGylation are assessed (Sur etal. 2019a, b).
Targeted and customized drug delivery systems were developed to address the drawbacks of traditional dosage forms. A revolutionary drug delivery technology was in great demand at the time. One such method of targeted drug delivery is nanoparticles. Colloidal nanoparticles that deliver medications site specically. Nanoparticles come in many forms, but polymeric nanoparticles are unique. One of the subjects being investigated the most at the moment is polymeric nanoparticles. Their main benets include better therapeutic efcacy, longer clearance times, less toxicity, and greater control over size. Dendrimers, polymeric micelles, ligand­based nanoparticles, PEGylated nanoparticles, and other types of polymer-based nanoparticles are only a few examples(Sur etal. 2019a, b).
9.7 Challenges withNanocarriers forRNA Delivery
An essential biological macromolecule in nature, RNA conveys genetic information and directs the creation of proteins. It is a single-strand transcript created using one DNA strand as a template and can be used to construct and modify the general struc­ture of RNA nanocarriers. The double helix structure’s restrictions can be overcome by RNA nanotechnology, which can create a wide range of structures and many kinds of circular-structured modules. Many researchers have become interested in using RNA interference processes brought on by small-molecule RNA in the ther­apy of tumors in recent years. Some enzymes and antisense siRNA generate a com­plex that increases the quantity of double-stranded RNA the enzyme produces until the mRNA is relocated while simultaneously degrading similar single-stranded RNA.For tumor therapy, specic dsRNA may be delivered into cells using RNA­based vectors, which are more effective, more exact, and quicker than conventional approaches to target genes selectively. They may be adapted to different require­ments. Gene silencing, drug delivery, and biological imaging rely on RNA self­assembly and RNA decoration on nanoparticles (NPs). To overcome the drawbacks of chemotherapy, researchers have developed techniques for making nanocarriers with high selectivity and low cytotoxicity. To address this problem, scientists have combined RNA with polyethylene glycol, inorganic NPs, glue bundles, carbon
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nanospheres, peptides, proteins, and other compounds to make RNA composite nanomaterials. High selectivity, stability, and resistance to deterioration are all fea­tures of these nanomaterials. The addition of RNA overcomes the limitations of conventional NPs (Xue etal. 2015; Veronese and Pasut 2005).
A. Tiwari et al.
9.7.1 Nanocarriers forRNA Delivery Based onLipid
andtheDifficulties
Considering that lipid-based nanocarriers are favorable for the transport of RNA, several issues need to be xed before this class of delivery devices performs opti­mally invivo or the clinical situation. Several issues with the lipid nanocarriers, which are ostensibly safe, result in subpar toxicity proles of drugs (Xue etal. 2015).
9.7.1.1 Cationic Lipid Toxicology
To increase RNA encapsulation and stability, cationic lipids are employed in RNAi therapies; however, they also have serious toxicity problems. A lipoplex made of cationic lipid molecules can irritate an exposed cell at low concentrations, leading to cytoplasm vacuolization, decreased mitoses, and cell shrinkage. Cell lysis and necrosis may start when the lipoplex level is high enough. The interaction of the cationic groups with biological enzymes like protein kinase C may potentially result in cell damage. Several genes involved in cell apoptosis were discovered to be affected by oligofectamine, which raised the probability of early apoptotic cell death (Xue etal. 2015; Veronese and Pasut 2005).
9.7.1.2 RNA Nanocarriers Based onLipids
The production of tumor necrosis factor, interferon 6, interleukin 6, and 12, leuko­penia and thrombocytopenia, and mouse mortality have all been linked to lipoplexes as systemic toxins. More labile, biodegradable linkers may be utilized to lessen toxicity; however, doing so may affect stability and transfection effectiveness (Xue etal. 2015; Veronese and Pasut 2005).
9.7.1.3 Concerns withPEGylation
Especially in those lipid carriers of RNA, lipid materials naturally interact ef­ciently with cell surfaces in a non-specic manner. While creating lipid nanocarriers for nucleic acids, PEGylation (surface coating with polyethylene glycol groups) is a crucial common procedure, although it has downsides. The RNA-loaded nanocar­riers must efciently enter the target cells by endocytosis for RNAi-mediated gene silencing to be effective, and the RNA payload must leave the endosomal compart­ment and enter the cytoplasm. The “accelerated blood clearance (ABC) phenome­non” refers to the ability of empty PEGylated liposomes given orally to speed up the clearance of subsequent injections of liposomes (Xue etal. 2013). The generation of anti-PEG IgM, following activation of the complement system, and rapid capture were responsible for this event made by Kupffer cells from liposomes. When stabi­lized plasmid lipid particles were administered in many doses, it was similarly
9 PEGylated Nanocarrier System forNucleic Acid Delivery
discovered that the targeted tumor had less expression, and the liver had more expression in the later doses (Xue etal. 2015; Veronese and Pasut 2005).
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9.7.2 Strategies toOvercome theChallenges
Several strategies have been explored to maintain the benets of lipid-based RNA nanocarriers while addressing their drawbacks (Xue et al. 2015, Veronese and Pasut 2005).
9.7.2.1 Modification ofLipids
Cationic lipids mainly cause the toxicity of lipid-based RNA nanocarriers, and effective engineering and alteration of the lipid molecule structure can enhance transfection efciency. A well-known example is DOTAP, which has a hydrophobic hydrocarbon backbone, a linker region, and a cationic head. It has been investigated how to increase transfection effectiveness without increasing toxicity by mixing multiple alkyl chains into the same lipid, employing an asymmetrical backbone, maximizing alkyl chain length, and leveraging steroid hydrophobic domains. One should use caution when selecting the “least hazardous” cationic lipids for invivo or clinical purposes because most of the reported lipid toxicity knowledge is based on invitro experiments (Xue etal. 2015; Veronese and Pasut 2005).
9.7.2.2 Utilization ofLipids Not Catatonic
Non-cationic lipids are often used in RNA nanocarriers, although they may decrease the RNA’s stability, encapsulation, and transfection efcacy. To boost many proper­ties, such as endosomal escape and physical stability, and decrease toxicity, neutral or anionic lipids can be added. Yet, these lipids might not always lessen toxicity. For instance, DOTAP coupled with cholesterol in a nanosystem killed less non-specic cells than DOTAP alone. Lipid mixtures must be carefully considered if developing a less dangerous nanocarrier is the primary objective (Xue etal. 2015; Veronese and Pasut 2005).
9.7.2.3 Nano Formulations ofRNA withHigh Potency
Researchers developed nano formulations that could suppress gene expression at extremely low siRNA dosage levels by combinatorial production and screening of lipid-like materials. In other research, replacing DOTAP with the newly created cationic lipid DLinMC3DMA in the lipid nanoparticles signicantly increased the potency of siRNA.From 10mg siRNA/kg body weight to 0.005mg, the IC50 was decreased. The authors hypothesized that the improvement is caused by employing ionizable cationic lipids with enhanced endosomal bilayer destabilizing properties and improved pKa values (Xue etal. 2015; Veronese and Pasut 2005).
9.7.2.4 Hybrid Lipid andPolymeric Materials Nanocarrier Integration
While polymeric materials often have strong afnities for giant molecules like RNA, lipid-based nanoparticles are skilled at managing drug release rates and have
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good biocompatibility and immunogenicity. Hybrid nanoparticles combining lipids and polymers have been produced to enhance RNA encapsulation and delivery, lessen cellular toxicity, and achieve high cell transfection efciencies. Recently, Ewe and Aigne (2014) created lip polyplexes for RNA transport. Inhalation delivery of lip polyplexes containing siRNA and DNA is possible (Xue etal. 2015, Veronese and Pasut 2005).
A. Tiwari et al.

9.8 RNA Lipid Nanoparticle

Lipid nanoparticles have been shown to be the most effective RNA nanocarriers so far. Several approaches have been included to solve various problems with RNA delivery, such as restricted invivo circulation, ineffective transfection, and intoler­able toxicity. It is made up of cationic lipids, PEG-conjugated lipids, and neutral lipids such as cholesterol and 1,2-distearoylsn-glycero-3-phosphocholine. It is an electron-dense complex structure with a solid core that differs from liposomes in terms of its physicochemical characteristics. A thorough evaluation of LNP perfor­mance invivo and clinical settings revealed no appreciable complement activation, release of pro-inammatory cytokines, delayed coagulation, or other alterations in hematological parameters. No evidence of hepatotoxicity was seen in the phase I clinical study employing LNP delivering siRNA against apolipoprotein B, although u-like symptoms are probably related to the immunostimulant. One of the partici­pants given the highest treatment dosage level showed signs of a high siRNA pay­load. Thus, the manufacturer (Tekmira) chose to end the experiment early. LNPs have great promise for delivering RNA.More toxicology data, particularly those pertaining to immunogenicity, are required to support the therapeutic utility of these medicines (Xue etal. 2015, Veronese and Pasut 2005).
9.9 Clinical Trials forNucleic Acid Delivery ofNanoparticles
Many clinical trials and experiments have been conducted to assess the security and effectiveness of nanoparticle-based nucleic acid delivery. Examples of clinical trials with citations are as follows:
1. Phase I/II trial of RNAi treatment targeting VEGF and KSP in patients with
advanced solid tumors: In this study, the effectiveness and safety of an RNAi­based therapy administered via nanoparticles in patients with advanced solid tumors were assessed. The research showed that the treatment was both safe and well-tolerated, and there was proof of therapeutic efcacy.
2. Age-related macular degeneration: Phase I trial of siRNA targeting the RTP801
gene: This study assessed the effectiveness and safety of a siRNA-based treat­ment administered via nanoparticles in individuals with age-related macular degeneration. The study showed that the treatment was secure and well-tolerated and that biological activity was present.
9 PEGylated Nanocarrier System forNucleic Acid Delivery
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3. Human anti-TNF mRNA given through polymer in patients with rheumatoid
disease: In this study, individuals with rheumatoid arthritis received an mRNA­based treatment that was administered via nanoparticles. The therapy’s safety and effectiveness were assessed. The research showed that the treatment was both safe and well-tolerated, and there was proof of therapeutic efcacy.
4. Phase I investigation evaluating the safety and effectiveness of an RNAi-based
treatment administered utilizing nanoparticles to patients with hematological malignancies. This trial focused on the M2 isoform of pyruvate kinase. The research showed that the treatment was both safe and well-tolerated, and there was proof of therapeutic efcacy.
5. Phase I research of an mRNA vaccine for the Zika virus based on lipid nanopar-
ticles: In this study, healthy people were given the vaccine to assess its immuno­genicity and safety. The study proved that the vaccination was secure and well-tolerated and that immune responses were present.
9.10 Merits andProspects ofaNanocarrier System
These clinical trials describe the efciency of nucleic acid delivery using nanopar­ticles for various therapeutic applications.
1. Nanocarrier systems, also known as nanoparticles, are microscopic particles that
can be designed to transport drugs, genes, or other therapeutic agents to specic targets in the body. These systems have the potential to promote drug effectiveness and lessen side effects, leading to better patient outcomes. Here are some of the merits and prospects of nanocarrier systems, supported by relevant citations.
2. Improved drug delivery: Nanocarriers can encapsulate drugs and transport them
to specic tissues or cells, improving drug efcacy and reducing toxicity. For example, liposomes have been employed to deliver doxorubicin to cancer cells, resulting in better therapeutic outcomes and fewer side effects (Wang etal. 2019).
3. Targeted therapy: Target cell receptors can be bound explicitly to using nanocar-
riers functionalized with targeting ligands, such as antibodies or peptides. This might increase the precision and potency of medicine delivery. For instance, siRNA was delivered to tumor cells via a targeted nanocarrier technology, which enhanced survival in animal models and inhibited tumor development (Shi etal. 2017).
4. Increased bioavailability: Drugs’ bioavailability can be increased by using nano-
carriers to shield them from the body’s natural processes of breakdown and clearance. For instance, polymeric nanoparticles have been utilized to improve the oral bioavailability of medications with low solubility, like curcumin (Li etal. 2019).
5. Regulated release: Drugs can be released from nanocarrier systems in a con-
trolled way, for as in reaction to a particular trigger (for instance, temperature, pH, or enzymes). The pharmacokinetics may be enhanced, and toxicity may be decreased. A pH-sensitive nanoparticle system, for example, was created to
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deliver anticancer medications specically to tumor cells, improving treatment efcacy and minimizing adverse effects (Jhaveri and Torchilin 2014).
6. Multipurpose platforms: Nanocarrier systems may be designed to perform vari-
ous tasks, including imaging and treatment. Iron oxide nanoparticles, for instance, have been employed as both MRI contrast agents and medication deliv­ery systems (Sun etal. 2018).
In conclusion, nanocarrier systems have the potential to boost bioavailability, permit targeted and controlled drug release, and improve drug delivery. They may also be designed to perform various tasks, giving them exible platforms for imag­ing and treatment.
A. Tiwari et al.
9.11 The Creation ofNucleic Acid-Based Nanocarriers
andTheir Use inNanobiology Delivery Systems
In nanobiology, nucleic acid-based nanocarriers like DNA and RNA have been thor­oughly investigated for their potential as medication delivery methods. These nano­carriers are capable of cellular absorption, targeted distribution to certain cells or tissues, and protection from degradation for therapeutic compounds. Below are some examples of current studies on the creation and use of nucleic acid-based nanocarriers.
1. DNA origami nanocarriers: DNA is used as a structural material in the DNA
origami method, which produces very accurate nanoscale forms. These struc­tures can be loaded with medicinal agents and functionalized with targeted ligand molecules for targeted drug delivery. For example, Li etal. (2018a, b) constructed DNA origami nanocarriers that were functionalized with folate for targeted delivery to cancer cells and loaded with doxorubicin for chemotherapy.
2. RNA nanocarriers: RNA molecules can also be used as nanocarriers and have
the advantage of being easier to synthesize and modify than DNA.For example, Guo etal. constructed RNA nanocarriers that were functionalized with targeting ligands and loaded with siRNA for gene silencing. Another study by Zhou etal. (2018) used RNA nanocarriers to deliver CRISPR-Cas9 gene editing machinery to target cells.
3. Hybrid DNA/RNA nanocarriers: Hybrid nanocarriers that combine the advan-
tages of DNA and RNA have also been developed. For example, Liu etal. con­structed hybrid DNA/RNA nanocarriers that were functionalized with targeting ligands and loaded with siRNA for gene silencing.
4. Aptamer-conjugated nanocarriers: Aptamers are short nucleic acid sequences
with a high afnity and selectivity for binding to particular targets. To allow targeted distribution to certain cells or tissues, they can be coupled to nanocarri­ers. For instance, Liu etal. developed aptamer-conjugated DNA nanocarriers to deliver doxorubicin to prostate cancer cells precisely.
9 PEGylated Nanocarrier System forNucleic Acid Delivery
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Overall, nucleic acid-based nanocarriers have great potential for drug delivery in nanobiology, and continued research in this area is expected to lead to new and improved therapies for a range of diseases.
9.12 PEGylated Nanocarrier System forNucleic Acid Delivery
forAnti-PEG IgM Production
Plasmid DNA (pDNA) and small interfering RNA (siRNA), for example, must be delivered systemically, and this needs carriers that are safe, efcient, and can over­come the pharmacokinetic restrictions of nucleic acids. The potential for producing nucleic acid-containing lipoplexes using an efcient cationic liposome is enormous (Abu Lila and Ishida 2019a, b).
In the delivery technique of gene therapy, most lipoplexes are PEG-treated for invivo stability and extended circulation (PEGylation). Nevertheless, when given to the same animal again at predened intervals, PEGylated liposomes lost their long­circulating properties. This unexpected and undesirable occurrence is known as the accelerated blood clearance (ABC) phenomenon. PEGylated liposomes’ initial dose resulted in anti-PEG IgM, which has been discovered as a signicant cause of ABC (Abu Lila and Ishida 2019a, b).

9.13 Conclusion

PEGylated nanocarrier systems are, in summary, promising techniques for deliver­ing nucleic acids. Enhancing the stability, biocompatibility, and pharmacokinetics of nanoparticles with PEG can result in more effective drug administration with lower toxicity. PEGylated nanocarriers can also overcome additional biological bar­riers that prevent the transfer of nucleic acids, such as endosomal escape and cel­lular absorption. Despite these benets, more investigation is required to improve the design and formulation of PEGylated nanocarriers for the distribution of nucleic acids and to comprehend their interactions with biological systems. In general, PEGylated nanocarrier systems show promise as a method for creating therapeutic nucleic acids that are both secure and efcient.

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A. Tiwari et al.
PEGylated Nanocarriers forProtein andPeptide Delivery
TejasGirishAgnihotri, VasuPeddinti, ShyamSudhakarGomte, BiswajitRout, andAakanchhaJain
Abstract
PEGylation is the biochemical process of modifying bioactive molecules with
polyethylene glycol (PEG), which endows proteins/peptides, antibodies, and
vesicles with several desirable properties that are used for the therapy. Protein
and Peptide drug delivery systems are considered novel drug delivery systems
and have immense therapeutic potential. As a result, using proteins and peptides
as therapeutic agents is thought to be a promising strategy for treating a variety
of diseases. However, the main limitation with protein and peptide drugs is poor
permeation across intestinal mucous barriers and they tend to get rapidly
degraded and cleared from the bloodstream. The PEGylation process increases
the efciency of therapeutic proteins by protecting them from proteolytic
enzymes. The other advantages include site-specic drug delivery, increased cir-
culation time in the blood, decreased immunogenicity, and prevention or
decreased uptake by the reticuloendothelial system. PEGylation of such drugs
results in improved physicochemical properties such as an increase in hydrophi-
licity, size, and molecular weight, changes in conformation, and steric hindrance
of intermolecular interactions. This book chapter discusses about protein and
peptide drug delivery and their importance in drug delivery, along with
PEGylation and its importance, types of PEGylated nanocarriers, the importance
of PEGylation in protein and peptide drug delivery, and characterization of
PEGylated nanocarriers. The limitations and strategies to overcome the limita-
tions of PEGylated nanocarriers have also been discussed at length.
10
T. G. Agnihotri · V. Peddinti · S. S. Gomte · B. Rout · A. Jain (*) Department of Pharmaceutics, National Institute of Pharmaceutical Education and Research (NIPER)-Ahmedabad, Gandhinagar, Gujarat, India e-mail: aakanchha.jain@niperahm.res.in
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Keywords
T. G. Agnihotri et al.
Proteins · Peptides · PEGylation · Nanocarriers · Physicochemical properties
10.1 Introduction: Proteins andPeptides
Proteins and peptides, the building blocks of life have been implicated in various cel­lular functions such as gene expression, regulation, enzymatic activity, signal trans­duction, cell apoptosis, immunity, etc. They have also been known to take part in a myriad of diseases like cancer, hypertension, metabolic disorders, and neurological disorders to name a few. As a result, they form an indispensable part of the therapeutic regimen to counteract those disorders. Owing to their specic physiological proper­ties, proteins and peptides held a special place in the biopharmaceutical market ren­dering them one of the most effective drug deliveries (Jain etal. 2013). Therapeutic peptides usually consist of amino acids of molecular weight ranging from 500–5000Da, which have been arranged in a specic order (Henninot etal. 2018; Wang et al. 2022). The synthesis of insulin in 1921 paved the way for the further development of peptides, which culminated in the approval of more than 80 therapeu­tic peptides globally. With the advancement of protein purication and synthesis, structural elucidation, genetic sequencing, and biotechnological tools, peptide devel­opment has taken a giant stride from the 1950s to the 1990s. During this period, apart from natural peptides, synthetic vasopressin, oxytocin, and estrogen have emerged. With the arrival of the twenty-rst century, peptide drug development ushered into a new phase with rapid progress in structural biology, recombinant biologics, and new synthetic and analytic tools considerably speeding up the process. Since 2000, 33 non-insulin peptide medicines have been authorized globally (Wang etal. 2022).
Proteins and peptides are not generally recommended for oral administration for the fear of instability in the gastrointestinal tract (GIT). The size and hydrophilicity of peptides and proteins further complicate the process of absorption leading to low bioavailability (Deb etal. 2019; Goldberg and Gomez-orellana 2003). Other routes of administration also are not devoid of any side effects and accompanied by aller­gic reactions, scarring, pain, and infection for intravenous delivery. The transdermal delivery also poses absorption challenges owing to the presence of a skin barrier and impeding the entry of especially hydrophilic drugs having a molecular weight greater than 500Da (Oberli etal. 2016).
Proteins and peptides are also prone to metabolic reactivity and usually have low bioavailability. Further, peptide oral bioavailability is restricted by a breakdown in the GI tract as well as their inability to pass the epithelial barrier. These medications often have high molecular weight peptides, poor lipophilicity, and charged func­tional groups, which make them difcult to absorb. Other factors including greater metabolism, protease degradation, opsonization, conformational changes, non­covalent complexation with plasma proteins, etc. could amount to low bioavailabil­ity of therapeutic peptides and proteins. Structural modication of peptides could offer a plausible solution to solve the aforementioned challenges, PEGylation, being one of them (Bruno etal. 2014). This chapter will emphasize the role of PEGylation