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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5373_Библиотеки_им_академика_М_И_Перельмана.pdf
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- •PEGylated Nanocarriers in Medicine and Pharmacy
- •Preface
- •Contents
- •Editors and Contributors
- •About the Editors
- •Contributors
- •1.1.3.1 Passive Targeting Agent
- •1.1.3.2 Solubility Enhancers
- •1. PEGylated Pharmaceutical Nanocarriers
- •1.1 PEGylation
- •1.1.1 PEG Characteristics
- •1.2 PEGylation Determination
- •1.2.2.1 Thermal Gravimetric Analysis (TGA)
- •1.2.2.2 Nuclear Magnetic Resonance (NMR)
- •1.2.2.4 X-Ray Photoelectron Spectroscopy
- •1.3.1 Nanoparticulate System
- •1.3.1.1 Solid Lipid Nanoparticles
- •1.3.1.2 Nanostructured Lipid Carriers (NLCs)
- •1.3.1.3 Polymeric Nanoparticles
- •1.3.2 Metal Nanoparticles
- •1.3.2.1 Silver Nanoparticles
- •1.3.2.2 Gold Nanoparticles
- •1.3.2.3 Titanium Dioxide Nanoparticles
- •1.3.2.4 Copper Nanoparticles
- •1.3.3 Vesicular Systems
- •1.3.3.1 Liposomes
- •1.3.3.2 Niosomes
- •1.3.3.3 Ethosomes
- •1.4.1 Cancer
- •1.4.2 Gene Delivery
- •1.4.3 Diagnostics Imaging
- •1.4.4 Vaccines
- •1.4.5 Rheumatoid Arthritis
- •1.4.6 Hemophilia
- •1.4.7 Pain Therapy
- •1.4.8 Diabetes
- •1.4.9 Others
- •1.6 Conclusion
- •References
- •2.1 Introduction
- •Nanoprecipitation (Solvent Diffusion)
- •Emulsification (Solvent Evaporation or Nanoemulsion)
- •Physical Adsorption Strategy
- •2.2.2.1 Pre-Insertion PEGylation
- •2.2.2.2 Post-Insertion PEGylation
- •2.3.1 Indirect Assessment (Qualitative Assessment)
- •2.3.1.1 Particle Size
- •2.3.1.2 Zeta Potential
- •2.3.1.3 Surface Hydrophilicity
- •2.3.1.4 Microscopic Techniques
- •2.3.1.5 Fourier Transform-Infrared Spectroscopy (FT-IR)
- •2.3.2 Direct Assessment (Quantitative Assessment)
- •2.3.2.1 Colorimetric Methods
- •2.3.2.2 Chromatographic Methods
- •2.3.2.4 Nuclear Magnetic Resonance (NMR)
- •2.3.2.5 X-Ray Photoelectron Spectroscopy (XPS)
- •References
- •3.1 Introduction
- •3.2 Characterization Techniques
- •3.3 Infrared Spectroscopy
- •3.4 Raman Spectroscopy
- •3.5 X-Ray Photoelectron Spectroscopy
- •3.6 Nuclear Magnetic Resonance
- •3.7 Energy-Dispersive X-Ray Spectroscopy
- •3.8 Mass Spectroscopy (MS)
- •3.9 Thermogravimetric Analysis
- •3.10 Differential Scanning Calorimetry
- •3.11 Atomic Force Microscopy
- •3.12 Scanning Electron Microscopy
- •3.13 Transmission Electron Microscopy
- •3.14 Conclusion
- •References
- •4.1 Introduction
- •4.3.1 Nanoparticles PEGylation
- •4.3.2 Polyplexes (PP) PEGylation
- •4.5.1 Systemic Drug Delivery
- •4.5.2 Nonsystemic Drug Delivery
- •4.5.2.3 PEGylated Intravaginal Nanocarriers
- •4.5.2.6 Vaccines Entrapped PEGylated Nanocarriers
- •4.6.2 PEG Molecular Weight (MW)
- •4.7 PEGylated Nanocarriers Products
- •4.8.3 Disadvantageous Physicochemical Properties
- •4.8.5 Limited RES Evasion Capacity
- •4.9 Conclusion
- •References
- •5.1 Introduction
- •5.1.2 PEG Solubility Characteristics
- •5.2 Water-Soluble PEGylated Small Molecule Drugs
- •5.3 Soluble PEGylated Proteins/Enzymes
- •5.3.2 Organic Solvent–Soluble PEGylated Proteins/Enzymes
- •5.4 Water-Soluble PEGylated Drug Nanocarriers
- •5.4.1 Water-Soluble PEGylated Silicon Nanocarriers
- •5.4.2 Water-Soluble PEGylated Carbon Nanotubes
- •5.4.4 Water-Soluble PEGylated Dendrimers
- •5.4.5 Water-Soluble PEGylated Polymeric Micelles
- •5.5 Hydrated or Hydrophilic PEGylated Drug Nanocarriers
- •5.5.1 Hydrated PEGylated Lipid Nanocarriers
- •5.5.2 Hydrophilic PEG-Coated Zein Nanocarriers
- •References
- •5.6.4.1 PEG Chain Length/Molecular Weight
- •6.1 Introduction
- •Increased Solubility
- •Improved Stability
- •Reduced Immunogenicity
- •Enhanced Circulation Time
- •Heterogeneity
- •6.3.1 Enhancing Immune Responses
- •6.3.2 Suppressing Immune Responses
- •6.3.3 Immune Evasion
- •6.4.1 Strategies to Overcome Immunological Barriers
- •6.4.1.1 PEGylation
- •6.4.1.2 Cell Membranes
- •6.4.1.3 Carbohydrates
- •6.4.1.4 Proteins
- •6.6.1 Cancer Therapy
- •6.6.2 Gene Therapy
- •6.6.3 Immunotherapy
- •6.6.4 Central Nervous System (CNS) Drug Delivery
- •6.6.5 Pulmonary Drug Delivery
- •6.6.6 Ocular Drug Delivery
- •6.6.7 Cardiovascular (CVS) Drug Delivery
- •6.8 Conclusion
- •References
- •7.1 Introduction
- •7.3 Nanocarrier-Based Targeted Drug Delivery
- •7.4.1 Covalent Approach
- •7.4.2 Non-covalent Approach
- •7.4.2.1 PEGylation Via Monovalent Interactions
- •High-Affinity Host-Guest Interactions
- •7.4.2.2 PEGylation Via Multivalent Interactions
- •PEGylated Block Copolymers
- •PEGylated Graft Copolymers
- •Polyelectrolyte Complex-Based Systems
- •Non-ionic Interaction-Based Systems
- •PEGylated Dendritic Copolymers
- •PEGylated Copolymers Utilizing Mobile Side Groups
- •7.5 Various Targeting Strategies
- •7.5.1 Active Targeting
- •7.5.2 Passive Targeting
- •7.5.2.1 PEG Dilemma
- •7.7.1 Brain Disorders
- •7.7.2 Pulmonary Disorders
- •7.7.3 Cancer
- •7.7.4 Inflammatory Disorders
- •7.7.5 Bone Disorders
- •7.7.6 Blood Disorders
- •7.8 Stimuli-Sensitive Nanocarriers
- •7.8.1 External-Responsive Nanocarriers
- •7.8.1.1 Ultrasound-Responsive PEGylated Nanocarriers
- •7.8.1.2 Thermal-Responsive PEGylated Nanocarriers
- •7.8.1.3 Magnetic Responsive PEGylated Nanocarriers
- •7.8.2 Internal-Responsive Nanocarriers
- •7.8.2.1 pH-Responsive Systems
- •7.8.2.2 Redox-Responsive Systems
- •7.8.2.3 Enzyme-Responsive Systems
- •7.8.2.4 Hypoxia-Responsive Systems
- •7.8.3 Multimodal Responsive Nanocarriers
- •7.9 Conclusion
- •References
- •8.1 Introduction
- •8.3.1 PEGylated Liposome
- •8.3.2 PEGylated Micelles
- •8.3.3 PEGylated Nanogels
- •8.3.4 PEGylated Inorganic Nanoparticles
- •8.3.5 PEGylated Polymeric Nanoparticles
- •8.4.1 Cancer
- •8.4.1.1 Breast Cancer
- •8.4.1.2 Lung Cancer
- •8.4.1.3 Colon Cancer
- •8.4.1.4 Brain Cancer
- •8.4.2 Autoimmune Diseases
- •8.4.3 Inflammatory Disorders
- •8.4.4 Cardiovascular Diseases
- •8.4.5 Ocular Diseases
- •8.5 Conclusion
- •References
- •9.1 Introduction
- •9.4.1.1 Amino Acid Modifications
- •9.4.1.3 Cysteine Thiol Residue Conjugation
- •9.4.2 Releasable PEGs
- •9.7.1.1 Cationic Lipid Toxicology
- •9.8 RNA Lipid Nanoparticle
- •9.13 Conclusion
- •References
- •10.2.1 PEGylated Nanocarriers
- •10.2.1.1 Polymeric NPs
- •10.2.1.2 Liposomes
- •10.2.1.3 Dendrimers
- •10.2.1.4 Polymeric Micelles
- •10.6 Conclusion
- •References
- •11.1 Introduction
- •11.1.2 Factors Influencing PPDs’ Short-Term Efficiency
- •11.2 What Is PEGylation?
- •11.3.1 Random PEGylation
- •11.3.2 Site-Specific PEGylation
- •11.3.2.1 Amine Conjugation
- •11.3.2.2 Cysteine Conjugation
- •11.4.1 Binding Affinity
- •11.4.2 Altered Biological Activity
- •11.4.3 Physicochemical Modifications
- •11.4.4 PEG Size
- •11.4.5 PEG Structure
- •11.6 PK Profiling
- •11.9 FDA-Approved PEGylated Products
- •11.11 Conclusion
- •References
- •12.1 Introduction
- •12.1.2 Current Market Scenario
- •12.2.1 PEGylated Iron Oxide Nanoparticles
- •References
- •13.1 Introduction
- •13.2.1 PEGylated Lipid-Based NPs
- •13.2.2 PEGylated Polymeric Nanoparticles
- •13.2.3 PEGylated Metal-Based Nanoparticles
- •13.2.4 Multifunctional PEGylated Nanocarriers
- •13.2.5 Targeted PEGylated Nanocarriers
- •13.3.1 Surface Modification Chemistry
- •13.3.2 Polymer Chemistry
- •13.3.4 Characterization Techniques
- •13.4.1 Longer Circulation Time
- •13.4.2 Enhanced Cellular Uptake
- •13.4.3 Controlled Drug Release
- •13.5.1 Cancer Theragnostic
- •13.5.2 Cardiovascular Theragnostic
- •13.7.2 Prolonged Circulation Time
- •13.7.3 Improved Drug Delivery
- •13.7.4 Diagnostic Functionality
- •13.8 Technical Challenges
- •13.8.4 Limited Clinical Validation
- •13.10 Conclusion
- •References
- •14.1 Introduction
- •14.2 Reversible PEGylation Strategies
- •14.2.1 Reversible PEGylation Chemistry
- •14.2.2.1 Aromatic Linkers
- •14.2.2.2 Aliphatic Linkers
- •14.2.3 Cleavage Linkers
- •14.2.3.1 Hydrolyzable Linkers
- •14.2.3.2 Enzymatically Cleavable Linkers
- •14.2.4 pH-Responsive PEGylation
- •14.2.4.1 Proteasome Inhibitor MG132
- •14.2.5 Temperature-Responsive PEGylation
- •14.2.6 Light-Responsive PEGylation
- •14.3.1 Analytical Techniques
- •Zeta Potential
- •Hydrophobic Interaction Chromatography (HIC)
- •Near Infrared (NIR) Spectroscopy
- •Fourier Transform-Infrared Spectroscopy (FT-IR)
- •13C-NMR
- •Mass Spectrometry
- •High-Performance Liquid Chromatography (HPLC)
- •Calorimetry
- •X-Ray Photoelectron Spectroscopy (XPS)
- •Nuclear Magnetic Resonance (NMR)
- •TGA-DSC
- •14.3.2.1 Protein Adsorption
- •14.3.2.2 Cellular Association
- •14.3.2.5 Bioactivity Assay
- •14.3.2.6 Enzyme-Linked Immunosorbent Assay (ELISA)
- •14.3.2.7 Sandwich ELISA
- •14.3.2.8 Anti-PEG ELISA
- •14.3.3.1 In Vivo Blood Circulation Half-Life
- •14.3.3.2 Radiolabeling
- •14.4.1 Therapeutic Applications
- •14.4.1.1 Anticancer Activity
- •14.4.1.2 Antibiotic Administration
- •14.4.1.3 Enzyme-Replacement Therapy
- •14.4.1.4 Red Blood Cell Substitution
- •14.4.1.5 Oxygen Toxicity Diseases
- •14.4.2 Pharmaceutical Applications
- •14.4.2.1 PEGylated Liposomes
- •14.4.2.2 PEGylated Proteins
- •14.4.2.3 Targeted Delivery
- •14.5.1 Design Complexity
- •14.5.3 Biological Environment Stability
- •14.5.4 Trigger Selection
- •14.5.5 Immunogenicity
- •14.5.6 Scale-up Difficulties
- •14.5.8 Cost
- •14.6 Conclusion
- •References
- •15. Stimuli-Responsive PEGylated Nanocarriers
- •15.1 Introduction
- •15.2 External Stimuli-Responsive Systems
- •15.2.1 Thermoresponsive Systems
- •15.2.2 Magnetically Responsive Systems
- •15.2.3 Ultrasound-Triggered Drug Delivery
- •15.2.4 Light-Triggered Drug Delivery
- •15.2.5 Electroresponsive Systems
- •15.3 Internal Stimuli-Responsive Systems
- •15.3.1 pH-Responsive Systems
- •15.3.2 Redox-Responsive Systems
- •15.3.3 Enzyme-Responsive Systems
- •15.3.4 Self-Regulated Systems
- •15.4.3 Multistimuli Responsive Systems
- •15.7 Conclusion
- •References
- •16.1 Introduction
- •16.3 PEGylated Products
- •16.3.1 PEGylated Liposomes
- •16.3.2 PEGylated G-CSF
- •16.3.3 PEGylated Proteins
- •16.3.4 PEGylated Nanoparticles
- •16.5.1 Poly(Zwitterions)
- •16.5.2 Poly(Glycerols)
- •16.5.3 Poly(Amino Acids)
- •16.5.4 Poly(Oxazolines)
- •16.5.6 Poly(Vinylpyrrolidones)
- •16.5.8 Polypeptides
- •16.5.9 Carbohydrate-Based Systems
- •16.5.10 Hydrophilic Polymers
- •16.5.11 Non-PEGylated Nanoparticles
- •16.6 Future Prospects
- •16.7 Conclusion
- •References

9 PEGylated Nanocarrier System forNucleic Acid Delivery
287
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
modied 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 etal. 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 specically.
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 benets include better therapeutic efcacy, 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 etal. 2019a, b).
9.7 Challenges withNanocarriers forRNA 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, specic 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

288
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 etal. 2015; Veronese and Pasut 2005).
A. Tiwari et al.
9.7.1 Nanocarriers forRNA Delivery Based onLipid
andtheDifficulties
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 invivo or the clinical situation. Several issues with the lipid nanocarriers,
which are ostensibly safe, result in subpar toxicity proles of drugs (Xue etal. 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 etal. 2015; Veronese and Pasut 2005).
9.7.1.2 RNA Nanocarriers Based onLipids
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
etal. 2015; Veronese and Pasut 2005).
9.7.1.3 Concerns withPEGylation
Especially in those lipid carriers of RNA, lipid materials naturally interact efciently with cell surfaces in a non-specic 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 efciently 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 etal. 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 forNucleic Acid Delivery
discovered that the targeted tumor had less expression, and the liver had more
expression in the later doses (Xue etal. 2015; Veronese and Pasut 2005).
289
9.7.2 Strategies toOvercome theChallenges
Several strategies have been explored to maintain the benets of lipid-based RNA
nanocarriers while addressing their drawbacks (Xue et al. 2015, Veronese and
Pasut 2005).
9.7.2.1 Modification ofLipids
Cationic lipids mainly cause the toxicity of lipid-based RNA nanocarriers, and
effective engineering and alteration of the lipid molecule structure can enhance
transfection efciency. 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 invivo
or clinical purposes because most of the reported lipid toxicity knowledge is based
on invitro experiments (Xue etal. 2015; Veronese and Pasut 2005).
9.7.2.2 Utilization ofLipids Not Catatonic
Non-cationic lipids are often used in RNA nanocarriers, although they may decrease
the RNA’s stability, encapsulation, and transfection efcacy. 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-specic
cells than DOTAP alone. Lipid mixtures must be carefully considered if developing
a less dangerous nanocarrier is the primary objective (Xue etal. 2015; Veronese and
Pasut 2005).
9.7.2.3 Nano Formulations ofRNA withHigh 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 signicantly increased the
potency of siRNA.From 10mg siRNA/kg body weight to 0.005mg, 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 etal. 2015; Veronese and Pasut 2005).
9.7.2.4 Hybrid Lipid andPolymeric Materials Nanocarrier Integration
While polymeric materials often have strong afnities for giant molecules like
RNA, lipid-based nanoparticles are skilled at managing drug release rates and have

290
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 efciencies. Recently,
Ewe and Aigne (2014) created lip polyplexes for RNA transport. Inhalation delivery
of lip polyplexes containing siRNA and DNA is possible (Xue etal. 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 invivo 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 invivo and clinical settings revealed no appreciable complement activation,
release of pro-inammatory 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 etal. 2015, Veronese and Pasut 2005).
9.9 Clinical Trials forNucleic Acid Delivery ofNanoparticles
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 efcacy.
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 forNucleic Acid Delivery
291
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 efcacy.
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 efcacy.
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 andProspects ofaNanocarrier System
These clinical trials describe the efciency 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 specic
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 specic tissues or cells, improving drug efcacy 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 etal. 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
etal. 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
etal. 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

292
deliver anticancer medications specically to tumor cells, improving treatment
efcacy 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 etal. 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 ofNucleic Acid-Based Nanocarriers
andTheir Use inNanobiology 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 etal. (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 etal. constructed RNA nanocarriers that were functionalized with targeting
ligands and loaded with siRNA for gene silencing. Another study by Zhou etal.
(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 etal. 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 afnity 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 etal. developed aptamer-conjugated DNA nanocarriers to
deliver doxorubicin to prostate cancer cells precisely.

9 PEGylated Nanocarrier System forNucleic Acid Delivery
293
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 forNucleic Acid Delivery
forAnti-PEG IgM Production
Plasmid DNA (pDNA) and small interfering RNA (siRNA), for example, must be
delivered systemically, and this needs carriers that are safe, efcient, and can overcome the pharmacokinetic restrictions of nucleic acids. The potential for producing
nucleic acid-containing lipoplexes using an efcient cationic liposome is enormous
(Abu Lila and Ishida 2019a, b).
In the delivery technique of gene therapy, most lipoplexes are PEG-treated for
invivo stability and extended circulation (PEGylation). Nevertheless, when given to
the same animal again at predened 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 signicant 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 benets, 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 efcient.
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A. Tiwari et al.

PEGylated Nanocarriers forProtein
andPeptide Delivery
TejasGirishAgnihotri, VasuPeddinti,
ShyamSudhakarGomte, BiswajitRout,
andAakanchhaJain
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 efciency of therapeutic proteins by protecting them from proteolytic
enzymes. The other advantages include site-specic 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
295

296
Keywords
T. G. Agnihotri et al.
Proteins · Peptides · PEGylation · Nanocarriers · Physicochemical properties
10.1 Introduction: Proteins andPeptides
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 specic physiological properties, proteins and peptides held a special place in the biopharmaceutical market rendering them one of the most effective drug deliveries (Jain etal. 2013). Therapeutic
peptides usually consist of amino acids of molecular weight ranging from
500–5000Da, which have been arranged in a specic order (Henninot etal. 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 purication 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 etal. 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 etal. 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 500Da (Oberli etal. 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 difcult 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 modication of peptides could
offer a plausible solution to solve the aforementioned challenges, PEGylation, being
one of them (Bruno etal. 2014). This chapter will emphasize the role of PEGylation
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