Keywords

1 Introduction

Since the discovery of DNA’s structure, its functions, and gene transfer applications have gotten a lot of interest, from cell transfection to the manufacture of transgenic animals by getting transgenic embryos to gene therapy. Several studies were conducted, as follows:

  • Changing the gene code and looking into the roles of genes.

  • Transfer of DNA to the organism or its cells (transgenesis).

  • Gene therapy is used to treat gene mutations or deficiencies.

  • Therapeutics are produced using transgenic prokaryotes and eukaryotes, particularly pharmaceutical animals such as goats and cattle (bio-pharming of therapeutics).

  • Model laboratory animals are created to study genetic illnesses caused by genetic damage or mutations.

  • Farm animals, particularly pigs, are being studied for use as a tissue bank for human transplants (xenotransplantation).

In these investigations, many successful outcomes have been obtained, as well as innovative methodologies. Apart from in vitro experiments, in vivo applications have also been carried out, but due to some unsolved problems and restrictions or difficulties, such as targeting of gene carrier particles, undesirable acute or late side effects of genes, and their carrier systems, obstacles relating to human applications have yet to be overcome. Transfection (gene transfer) is a process in which a gene is transferred to the nucleus of another cell and implanted in its DNA. Many approaches and protocols for transgenesis applications have been created by researchers. The gene is transferred to the tissue/cells using a variety of ways.

  • Electroporation.

  • Direct injection of genes into the nucleus or pronucleus.

  • Using viral vectors to transfer genes.

  • Non-viral vectors are used to deliver genes.

Because the electroporation technique is only utilized in cell suspension, it has limited use. Furthermore, microinjection of the gene directly into the nucleus/pronucleus necessitates specialized and costly equipment, and this technique can be utilized in cell culture systems and after egg fertilization. The reliability of viral vectors in vivo experiments is still an open question, and the results of these studies are generally poor. As a result, researchers created a novel targeted gene carrier system, and non-viral gene carrier systems have become more commonly used in transgenesis and gene therapy applications. However, these methods have issues such as difficulty in targeting the gene carrier system, early cytoplasmic enzymatic activity degradation of the particles and the gene in the cells, and non-viral agent cytotoxicity. Currently, research is focused on resolving these issues. By delivering genetic material to the patient, gene therapy has been utilized to prevent genetic abnormalities. With this technique, the patient's therapy, which is the regeneration of damaged biological activities or the restoration of homeostasis, is carried out at the molecular level. The basic goal is to overcome biological barriers that prevent therapeutic genes from reaching the desired location. Preclinical and clinical gene therapy research has advanced considerably in the last 15 years. Gene therapy has recently gained popularity as a promising treatment option for genetic illnesses, cancer, cardiovascular disease, and viral infection. Gene therapy not only tries to treat diseases but also to transfer recombinant genetic material to the nucleus, where gene expression, which activates or deactivates protein synthesis, occurs. It is clear that well-targeted, non-toxic gene carrier mechanisms are required to transport the gene to the nucleus. Chitosan is a non-viral gene carrier that is commonly used in gene therapy.

2 Chitosan as a Gene Carrier

The carrier system that delivers a gene for gene expression is called a vector. Vectors are mainly divided into two groups: non-viral vectors and viral vectors. Effective transfection and gene expression of viral vector therapy genes are of great clinical importance for gene therapy. Due to its structure, the virus performs gene transfection very effectively. Due to this property of viral vectors, the required carrier system is preserved and improved. Recently, viral vectors with different genomic characteristics such as retrovirus, adenovirus, adeno-associated virus, herpesvirus, and poxvirus have been commonly used for the efficient provision of gene transport capacity and gene expression. Retroviruses are the most popular of these viral vectors because of their highest gene transfection efficiency and highest expression of therapeutic genes. While these functions emphasize the importance of safe RNA and DNA viral carrier systems, the same report reveals the difficulty of using clinical viral vectors [1, 2]. For example, adenovirus provides the highest gene expression and can infect dividing and non-dividing cells, but it elicits an immune response through viral protein and transient gene expression [3,4,5]. Similarly, retroviruses facilitate the manipulation of the viral genome. Although easy to combine with DNA, their advantages are difficult to target, complex combinations within the genome, and instability [5]. The first clinical study deals with viral vector compliance and reliability. There are numerous viral vector systems that have been tested in ex vivo and in vivo studies. In recent years, studies have focused on virus targeting, cell type expression, and time of expression to enhance existing effects [5]. Researchers prefer viral vectors for their effectiveness in transfection studies, but the use of these vectors has characteristics such as cytotoxicity, immunosensitivity to viral antigens, and possible viral combinations. Is limited because it is low. Because of these negative features, researchers have been drawn to non-viral vectors. Today, gene therapy is clinically effective in treating many illnesses. Since 1989, when gene therapy was first introduced, this approach has been used in more than 3000 patients with approximately 600 interventions, but no gene therapy product raises toxicity concerns. In addition, there are significant usage differences between viral and non-viral vectors. Viral vectors are used in about 75% of clinical treatment cases, while non-viral vectors are used in less than 25% [6, 7]. Some highlights of the gene transfer application are shown in chronological order in Fig. 5.1 [8,9,10,11,12,13,14,15]. Due to these instabilities, researchers not only compared viral vectors with non-viral vectors but also sought to understand the potential and limitations of non-viral systems. In many of these studies, the researchers of gene therapy and pharmaceutic technologies recognize that viral vectors are merged with the genome and the cell is mutated, so cancer occurs in vivo. Besides that, the immune response is activated, and that’s why the treatment becomes more difficult [7, 16]. Especially, although the numerous non-viral vectors are synthesized and their features designed, these systems are not effective enough for gene transfer, so there are not any commercial products. The non-viral vectors are divided into two groups lipophilic vectors and polymeric vectors.

Fig. 5.1
A timeline of events from 1962 to 1996 traces six important milestones in the development of gene transfer applications.

The Continuous growth of gene transfer applications

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Non-viral vectors containing cationic liposomes are commonly used before and during clinical trials, but an important part of non-viral lipophilic vectors has the same toxicity as viral vectors. And although there are clinical problems, the main advantage of these polymers is that they can create various modified cations in the structure of the polymer to enhance their potency. As a result, plasmid DNA is released in a controlled manner, increasing stability to enzymes in the blood such as nucleases, reducing non-specific uptake, regulating interactions with cells and plasma molecules, and simultaneously eliminating the immunizing of the system. These modifications are applied to improve the release properties of the polymer, allowing the genetic material to be released in a controlled manner at the target site [17]. On the other hand, non-viral gene transfer systems with different polymer structures are safe and economical gene transfer systems with improved various synthetic vectors, some of which are commercially available. In addition, non-viral vectors are not as efficient as viral vectors and generally solve as some of the cationic carrier systems used with anionic genes to generate ion complexes capable of exhibiting cytotoxic effects [18, 19]. The common goal of these studies is the synthesis of polymer carrier systems that are as effective and less toxic as viral vectors.

3 Application of Polymers in Gene Delivery

3.1 Artificial Polymers

There are several non-viral gene delivery systems for transferring genetic material to the cell nucleus. The non-viral gene delivery system consists of polymers and lipids or liposomes. Cationic polymers have many advantages for use in gene delivery such as low toxicity and immune response, being easy to handle, and being stable [20, 21]. However, some issues need to be resolved, including toxicity, reduced transfection efficiency, and lack of biodegradability. These properties of the polymer need to be modified in different ways. These biodegradable non-viral polymer gene delivery systems are called transgenic polymers. Transgenic polymers can be divided into two groups: natural transgenic polymers and synthetic transgenic polymers. Synthetic transgenic polymers are generally preferred for gene delivery because of their ease of modification. According to Amiji [6], synthetic transgenic polymers include non-biodegradable transgenic synthetic polymers [polyethylenimine (PEI), polyethylene glycol (PEG) conjugates, etc.], biodegradable transgenic synthetic polymers (poly-β-aminoesters, polyamido amines, poly-imidazoles, etc.), polyethylene oxide/polypropylene oxide copolymers and polymeric polyethylene oxide, polyalkylcyanoacrylate nanomicrospheres. However, the main drawbacks of these polymers are their high toxicity. The main reasons for toxicity are the polymer skeleton and the density and distribution of positive charges along with the molecular weight. PEI's with a molecular weight of 22–25 kDa are used for gene therapy because of their reduced cytotoxicity and high transfection efficiency [22, 23]. Other non-biodegradable transgenic polymers are used to deliver genetic material such as DNA, oligonucleotides, and small interfering RNAs. Some of these polymers are poly[2-(dimethylamino) ethyl methacrylate] (PDMAEMA), poly[2-(dimethylamino) ethyl acrylate] (PDMAEA), and N-vinyl pyrrolidone. They show high transfection efficiency like PEI, but due to their negative properties that reduce stability in blood, high interaction with serum components, etc., these polymers are PEG and poly[N-(2-hydroxypropyl methacrylamide)] (pHPMA) is bound [24]. PEG and pHPMA materials mask the instability of nanoparticles in serum due to the presence of nanoparticles in the hydrophilic layer on the polyplex. However, the PEG and pHPMA groups interfere with complex: DNA complex formation, thereby reducing the efficiency of polyplex transfection. Researchers are working on improving various ways to solve these problems [24,25,26].

3.2 Natural Polymers

Cationic polymers (polycations) are one of the most commonly used carrier systems in molecular gene transfer systems. The polyelectrolyte complex (PEC) obtained by the interaction of DNA and polycations protects DNA from enzymes such as DNAse. In addition, transgenic polycations interact with serum DNA due to their cationic properties at physiological pH.PEC systems are easier to manufacture and have a lower immune response than viral vectors, but researchers are pursuing further research due to negative properties such as biodegradability problems and reduced transfection efficiency. The main transgenic polycations are PEI, poly(L-lysine), dendrimers, gelatin, and chitosan, a natural cationic polysaccharide. As mentioned above, one of these polycations, chitosan, is natural, biodegradable, biocompatible, non-toxic, and does not contain negative charges in the PEC system [6], so it is used in transfection studies. It is attracting the attention of researchers. Natural polymers commonly used in gene delivery are poly (amino acids) such as poly-L-lysine (PLL), polyornithine, polyarginine, chitosan, dextran, collagen, gelatin, and their modified derivatives. PLLs and other polys (amino acids) are important polymers for use in gene delivery systems due to their biodegradability, but these polymers are highly toxic [22]. Other polycations, namely, dextran [27], collagen [28], gelatin [29, 30], and their modified derivatives are used in gene delivery systems, but researchers have observed sufficient transfection efficiency so did not do it. Therefore, chitosan has been widely used in many studies due to its characteristic properties.

4 General Characteristics of Chitosan

Chitosan is a linear polysaccharide composed of glucosamine and N-acetyl-glucosamine units bound by β (1–4) glycosidic bonds and a partially deacetylated product of the natural polysaccharide chitin. Chitin, a biopolymer, is the most abundant organic compound in nature and is an important component of the exoskeleton of animals, mainly found in the shells of crustaceans such as crabs, shrimp, and krill. Since chitosan is an N-deacetylated derivative of chitin, the degree of acetylation determines whether the biopolymer is chitin or chitosan. When the degree of deacetylation of chitin, which is the content of glucosamine, exceeds about 50%, it dissolves in acidic aqueous solutions such as acetic acid, lactic acid, hydrochloric acid, and aspartic acid, and is called chitosan [31, 32]. Chitosan does not dissolve at basic pH values. Chitosan exhibits varying degrees of solubility in dilute aqueous media, depending on the free amine content of the chain. The molar ratio of acetylated amine groups to deacetylated amine groups in chitosan also determines the sensitivity or biodegradability of the enzyme. Chitosan is an inexpensive, biocompatible, animal/human biodegradable, non-toxic cationic polymer. In addition to these properties, it exhibits other excellent biological properties such as immunological, antibacterial, and wound healing activity. Chitosan degradation products are also non-toxic, non-immunogenic, and non-carcinogenic. The chemical modification of chitosan that results in a variety of derivatives is easy to apply. A variety of possible modification reactions can be applied, including nitration, phosphorylation, sulfation, thiolation, acylation, hydroxyalkylation, graft polymerization, amination, and combinations of chitosan derivatives with cyclodextrin. In particular, the physical, mechanical, chemical, bioactive properties, and commercial availability of chitosan make chitosan a very attractive biomaterial in biomedicine. Therefore, since the nineteenth century [33,34,35], chitosan derivatives have found widespread use in many areas, including biotechnology (especially biomedicine) and environmental applications. For example, a future and important use of chitosan in biomedicine for gene delivery. Chitosan has a high positive charge density due to the D-glucosamine unit in the structure. It exhibits polycationic properties at acidic and neutral pH. The amine group of chitosan is protonated and chitosan forms a PEC with negatively charged DNA [6, 36, 37]. Currently, positively charged groups (amino groups) and negatively charged ones, nucleic acids that form stable complexes or biological membranes and in vivo targets (e.g., PEI or polyamide amine dendrimers) due to their low toxicity and immunogenicity [33, 34]. Therefore, chitosan and its derivatives have recently been recognized as safe and efficient cationic carriers for gene delivery [6, 33, 34, 38,39,40,41].

5 Factors Affecting Gene Transfer in Chitosan Delivery System

Many delivery systems have been developed to maximize transfection efficiency and minimize side effects. For this purpose, Kasyua and Karudahave been used in vivo, versatile payload acceptability, low toxicity or non-toxicity, low immunogenicity, stealth, active targeting, proper size, and proper surface charge. It provided information on the optimized properties of these carrier systems for efficient cellular penetration: activity, intracellular targeting mechanism and high productivity [42]. Bhavsar and Amiji pointed out other properties like reactivity, biocompatibility, non-heat resistance, impurities, availability in medicinal grade, load capacity, permeability, swelling, viscoelasticity, and local environment sensitivity [43]. As defined above, we have looked at these two classifications for rectification of the non-viral vectors compared to viral vectors. Size and charge density are very playing a crucial role in vitro and in vivo overall performance of polymeric gene delivery systems. The polymeric gene delivery systems have a positive charge which is complex with the DNA having a negative charge. These cationic densities boom the encapsulation performance and enhance the uptake of DNA into the cells through the interplay of the negatively charged cell membrane [18, 44,45,46].

5.1 Surface Charge and Zeta Potential

The polymeric gene delivery systems that are complexed with DNA are called polyplex. The positive charge density of the polymer increases the DNA encapsulation efficiency and stability as well as immunity. Therefore, in various studies, PEG or the same other molecule forms a complex with the polymer carrier to provide charge balance, and these modifications make these carriers safe [47, 48]. Charges are also important for cell penetration. Cationic nanoparticles enter cells via endocytosis. Positively charged particles react with the negatively charged sugar coating on the outside of the cell membrane (on the extracellular polymer material on the surface of the cell membrane) and are taken up by cells by various intracellular mechanisms. When particles (which did not invade cells by passive and active transport) are taken up by endocytosis, multiple acidic groups cause endosome destabilization at low pKa values, resulting in a proton sponge effect. The polymer is then protected by moving protons to the endosome and increasing the ion charge density until the endosome becomes unstable [10]. From the formation of polymer support-DNA complexes to cell invasion and gene transfer to the nucleus, the required surface charge is called the zeta potential [49, 50]. Expressed as colloidal stability of particles distributed in a liquid (usually water) and when an electric field is applied to the liquid, it moves to the negative or positive electrode according to the surface charge ratio. As is well-known, in the case of colloidal systems, when a net surface charge is formed, a reverse charge begins to be generated in the outer layer, thereby forming an electric double layer. The innermost layer that surrounds the opposite layer for each surface charge of a particle is called the star layer. Each particle acts as a single entity consisting of this bilayer whose positive charge is equal to its negative charge. The potential difference between this field and the surface charge of the particle is called the zeta potential (ζ). The unit is millivolts (mV) and is measured with a zeta meter [51]. Particles are stable below −30 mV and above +30 mV. For stable nanoparticles used in gene delivery applications, the average zeta potential is up to +30 mV. The ionization of the terminal groups of the non-viral polymer carrier depends on the degree of surface ionization proportional to the pH of the dispersion. For zwitterionic particles, the surface charge is positive at low pH and the surface charge is negative at high pH. These charges equilibrate at a zero point called the isoelectric point [51].

5.2 Particle Size

In 1860, nanoparticle technology was born from nanoscale or nanometer (nm) scale materials. Currently, the nanoscale concept is described as a material with a particle size of 1–1000 nm. The scope of nanotechnology lies in the manufacture of nanoscale materials with various properties and the study of these properties. While chemistry, physics, molecular biology, and materials science are related to nanotechnology, many areas of science are beginning to associate nanotechnology with the development of technological methods [52, 53]. The evolution of nanotechnology is not limited to the position of atoms in the structure of materials as they are transported from one location to another. Moreover, nanomaterials, which have a high surface area due to their nanosize, are synthesized with exclusive properties throughout their controlled size. Therefore, nanomaterials are widely used in many fields such as electronic equipment, automobiles, military, medical, manufacturing of conductive and semi-conductive materials, ceramics, surface coating materials, ink manufacturing, and so on. This technological revolution is seen by scientists as the starting point for more development over the next 10–15 years. The concept of nanotechnology in gene therapy began in 1930 with the discovery of the intracellular nanoscale structure. Today, nanotechnology approaches in gene therapy are known for developing nanoparticle systems below 200 nm. The size of the polyplex is important for functionality. The diameter limit of the polyplex is about 10 nm at the first uptake in the liver. In addition, the upper limit size of the polyplex should be less than 200 nm. The size of the polyplex is changed by changing the DNA: polymer ratio [54, 55]. In particular, factors that influence the transfection efficiency of the chitosan-DNA complex are the degree of deacetylation and the molecular weight of chitosan, pH, serum, chitosan charge ratio to DNA, and viscosity, and cell type [6, 38, 56, 57]. Transfection efficiency and DNA loading capacity increase with the degree of deacetylation, and extracellular DNA protection and intracellular DNA release increase with molecular weight. The optimum pH for transfection media is 6.8–7.0 [56].

5.3 Chitosan Modification Reactions

Chitosan is insoluble in physiological pH and has low transfection efficiency, so modification studies are needed. For this purpose, several modification reactions are performed on chitosan such as modified with PEG or glycol [58], synthesized quaternized chitosan [59], low molecular weight chitosan [60], and reducing or thiolated chitosan (Fig. 5.2) [61]. Generally, PEG, glycol, or pHPMA is used to mask the instability of nanoparticles in serum and the formation of the hydrophilic layer onto the polyplex. The introduction of grafted PEG units onto the galactosylated chitosan was investigated by Parket al. which increases stability in water and cellpermeability [62]. Mao et al. have been studied grafted methoxyPEG (mPEG) units of different molecular weights onto the trimethylchitosan [63] to produce modified chitosan such as PEG-aldehyde [64, 65], PEGcarboxylicacid [66, 67], PEG-carbonate [68], PEG-iodide [69], PEG-epoxide [70], PEG-diacrylate [71], PEG-NHS ester [72], and PEG-sulfonate [73,74,75]. The introduction of colloidal stabilities of polyplexes with a pHPMAlinker was studied by Luten et al. [76] which enhanced the stability in serum for in vitro transfection with low cytotoxicity. Later on, a lot of studies based on this concept have been carried out to date [72, 77,78,79,80,81,82,83,84]. This modification is preferred over the others because of improvement in transfection efficiency and solubility in water. Numerous quaternized chitosans such as N-(4-pyridinylmethyl)chitosans [38], N-trimethylated chitosan oligomers [85], methylated N-(4-N,N-dimethylaminobenzyl) chitosan [86], octadecyl quaternized carboxymethyl chitosans [87], PEGgraft-quaternized chitosan [88], and other low molecular weight chitosan have been studied in non-viral gene delivery. Richardson et al. studied the effect of molecular weight of chitosan on the cytotoxicity, complexation with DNA, and relation to the protection of DNA from nuclease degradation. He found that the low molecular weight of chitosan is more effective than poly(L-lysine) for complexation with DNA, and there is no cytotoxic effect for use in gene delivery [89]. Various research articles related to the modification reactions of the low molecular weight chitosans appeared in the literature [90,91,92,93,94,95,96,97,98,99,100,101,102]. Reducing or thiolated chitosan is commonly used to dissociate DNA and vectors. In addition, modifications containing ester bonds [103,104,105] or biological macromolecules [106, 107] such as heparin and proteoglycans are used but not generally preferred by the researchers. The disulfide bonds are delivered to the gene switch through diverse strategies. One of those strategies is the cationic ligands, which can be coiled at the polymer segments with the disulfide bonds, then DNA is bonded through the electrostatic interaction, and so DNA is added into the cytosol via the dissociation of disulfide bonds from the polyplex. Alternatively, other methods are the formation of cross-linking points via disulfide bonds and the reaction with disulfide bonds via polymer segments. These bonds present in the polymer segment not only release DNA but also reduce cytotoxicity through the degradation of small molecule components [24, 25]. Lee et al. stated the thiol modification of chitosan for sustained gene transport. In this study, the thiolated chitosan/DNA nanocomplexes exhibited significantly stepped forward gene transport in vitro and in vivo via way of means of the oxidation of thiol groups to crosslink the thiolated chitosan [108]. Numerous reducible chitosan research had been stated so far [109,110,111,112,113].

Fig. 5.2
A diagram illustrates the structure of chitosan-modified nanoparticles in D N A. The target group and P E G groups are highlighted.

Scheme for representation of chitosan-modified nanoparticles

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6 Application of Chitosan-Based Nanocomposites in Gene Therapy

Chitosan is the most studied natural macromolecule for gene delivery. It has a positive charge, binds to negatively charged cell membranes with high affinity, and forms a complex with DNA via electrostatic interaction [22, 114]. Mumper et al. [15, 115] have introduced firstly the role of chitosan in gene therapy. Afterward, in 1996–1997, Murata et al. synthesized the chitosan gene carrier systems having galactose residues for the transportation of the DNA molecule [116, 117]. Although gene transport mechanisms of cationic nanoparticles had now no longer been absolutely understood in those years, numerous reports related to the chitosan gene carrier systems were reported in the literature. Polymeric gene transport applications are quite tough to classify. In well-known, classification is performed further to the polymer type as natural and artificial polymeric gene transport structures. However, researchers understood that this type does not explain all the gene delivery applications so, in place of making the classifications, they realized that the gene shipping mechanisms want to be understood in aggregate with polymer-DNA out of the mobile to the occurrence of the related proteins into the mobile nucleus. Despite the fact that these mechanisms have now not been completely defined yet, the modification reactions are done in step with the diagnosed mechanisms. Wong et al. described seven essential steps for the transport of a gene to the nucleus: (1) healing genes packaging; (2) entry to the cellular; (3) endolysosomal getaway; (4) the impact of DNA/provider system release; (5) progression throughout the cytoplasm and transition into the nucleus; (6) gene expression; and (7) biocompatibility [24]. Gene packaging strategies are very critical for gene delivery structures. For the prevention of the same charge impact on the cell membrane resulting from the phosphate corporations of DNA, the condensation of the cumbersome shape of DNA, and the safety of DNA from the degradation extracellularly or intracellularly, researchers stepped forward with two packaging strategies for chitosan gene transport: (1) electrostatic interplay and a couple of encapsulation [24, 118, 119]. Usually, electrostatic interaction strategies are desired for the chitosan gene transport systems. Erbacher et al. used the electrostatic interplay methods for growing chitosan/DNA complexes and they located that this complicated required some feature modifications for strong and small complexes [107]. Chitosan has amino businesses that are protonated in impartial pH and have interaction with DNA spontaneously. Due to those features of chitosan, Lee et al. studied the hydrophobically modified chitosan complexes with plasmid DNA to put together self-aggregated nanoparticles in aqueous media with adjusted pH [120]. Aside from this examination, there are various articles reported relating to those capabilities of chitosan [121,122,123,124,125,126,127,128]. Especially because the encapsulation strategies are explored, the researchers have not generally favored the electrostatic interaction techniques. The encapsulation methods are used for the protection of genes from enzymatic degradation and offer the controlled launch of DNA via the biodegradable groups of chitosan [24]. Buddy et al. classified the techniques of obtaining the encapsulated chitosan-based totally on the nanoparticle systems; those are covalently connected nanoparticles, ionically pass-linked nanoparticles, and desolvated nanoparticles [129]. The covalently pass-related method is carried out with a chemical cross-linking agent, including glutaraldehyde, for cross-linking of chitosan [130]. The ionically cross-connected nanoparticles method or ionotropic gelation approach is the most famous technique for the use of polyanions, such as tripolyphosphate, for forming cross-connected chitosan [131,132,133,134]. In the desolvated nanoparticles approach, the desolvating agent is used for the precipitation of chitosan to extract water from chitosan polymeric chains. The complex coacervation approach is one of the desolvated strategies [129]. Bozkır and Saka studied the complicated formation of chitosan and plasmid DNA with the use of complicated coacervation and solvent evaporation techniques. In addition, they investigated the crucial parameters which include encapsulation efficiency, molecular weight, and deacetylation degree of chitosan [135]. The restrictions of the encapsulation techniques are that the polymeric provider systems are uncovered the natural solvents and high temperatures, which disrupt the genetic materials, much less encapsulation efficiency, much less DNA biocompatibility because of inadequate launch from the polymeric carrier structures, and the degradation of DNA due to the hydrolysis of ester bonds in low pH [24]. The polymer-DNA complexes as polyplexes come upon the primary barrier on the mobile, and it is far referred to as the plasma membrane. Polyplexes do not undergo passive diffusion because the transition membrane’s pores and canals are very constrained dimensionally. Various strategies have been employed to overcome bodily obstacles. Endocytic uptake of the molecules, which are not handed from the cell membrane via the easy diffusion or active delivery, is passed essentially from the mobile membrane through 3 approaches: (1) phagocytosis; (2) pinocytosis; (3) receptor-mediated endocytosis. In general, the particles which are bigger than 250 nm pass via phagocytosis, and the smaller ones skip through endocytosis on the mobile membrane [136]. Polyplexes are uptaken in the mobile by means of receptor-mediated endocytosis. The focus on the agent in the provider structures is used for specific uptake of the gene in a mobile as for reaching endocytosis. The endogenous ligands, inclusive of folate and transferrin, are widely utilized in phrases of growing the biocompabilities and transfection efficiencies. However, the exogenous ligands have very constrained utilization in terms of generated immune reaction because of their overseas structures [24, 78, 114, 137,138,139,140,141,142,143,144,145]. The endolysosomal getaway of chitosan polyplexes is explained while after the endocytic uptake of polyplexes, they go back to the mobile floor, which is facilitated by means of lysosomes, intracell organelle, etc. This concept is expressed with the pKa value of polycations, which is stricken by a trade-in buffer capability. For chitosan polyplexes, which have a pKa value of approximately 6.5, the amino organizations are protonated within the cellular cytoplasm, but this function is applied for the endolysosomal break out of chitosan polyplexes [146]. In line with the effectiveness of molecular weight and degree of deacetylation, Huang et al. studied the transfection efficiency of chitosan. They said that chitosan has 2.5 times higher proton absorption ability than PLL [147]. However, Höggard et al. studied the connection between ultrapure chitosan and PEI and studied their characteristics. Consistent with their experimental consequences, the ultrapure chitosan does now not offer a sponge effect due to its primary amine groups as compared with PEI. In addition, its buffering capacity is lower than PEI on the acid endosomal pH interval of 4.5–5.5 [148]. due to these disadvantages, researchers have carried out a few modification reactions on chitosan. Moreira et al. studied to improve the transfection performance of chitosan with the aid of promoting the endosomal break out potential and buffer capability of chitosan polyplexes. For this cause, the chitosan backbone is modified with imidazole moieties with a view to escape endolysosomal degradation, much like PEI [149]. In another study, Chang et al. modified chitosan with histidine as buffering potential of histidine might help the escape of DNA inside the endosomal pH variety [150]. Comparable research was finished with the aid of others as well [151,152,153,154]. The most important steps for the transport of a gene to the nucleus are DNA/carrier device dissociation, cytosolic carrier, launch into the nucleus, and gene expression. The maximum crucial step for chitosan carrier systems is the DNA/vector dissociation among those primary steps because green gene transfer is completed with a minimum retention time of non-protective DNA in the cytosol. Efforts had been made to enhance the modification of the chitosan backbone, and charge reduction or modification of chitosan is done by modifying it with thermoresponsive groups, ester bonds, or disulfide bonds (reducible polymers). Among them, the most critical and typically desired strategies are modification with thermoresponsive groups and disulfide bonds. The thermoresponsive polymers are transformed to reversible frizz-circular form relying upon the temperature. Thus, the degree of DNA condensation is decided by the change in temperature. The frizz phase has the flexible, hydrophilic, the long-wide chain conformation, whereas the circular form has the collapse, hydrophobic, small stretched conformation. If the carrier system is circular up to the transition temperature, and frizz forms below the transition temperature, this transition temperature is called lower critical solution temperature (LCST) [155]. In fact, the thermoresponsive provider device that has an LCST value underneath the frame temperature is used for the condensation DNA with stretching form into the cell. In this regard, poly(N-isopropylacrylamide) (p-NIPAM), with an LCST value of 32 °C, is extensively utilized in transfection studies. This polymer offers excessive transfection efficiency, endosomal escape, cationic character, and hydrophobicity and NIPAM has been used for chitosan modification in many studies [156,157,158,159]. The thiolated polymers are commonly desired in gene transfer systems as a promising tool [160]. The disulfide linkages are modified to shape the gene carrier systems by the usage of various strategies; (1) electrostatic interactions, (2) reversible cross-linking, and (3) direct affiliation of the disulfide linkages at the polymer backbone. As cited above, those linkages preserve the polymer structure solid inside the cytosol, launch DNA into the cytosol, and furthermore the cytotoxicity of the carrier system is decreased with the dissociation of the carrier system to lower molecular weight components [24, 25, 160]. The thiolated chitosan carrier systems that enable gene transfection efficiency were developed by Schmitz et al. [161]. Jia et al. advanced a redox-responsive chitosan carrier system by using the PEG, PEI, and disulfide bonds for greater effective gene transfection in HeLa cells [162]. Targeted gene delivery is a significant step in chitosan carrier systems for obtaining selective and enhanced gene delivery to the target site. By using the in vivo approaches, the chitosan polyplex is administered to the body, it must be targeted to the specific sites as shown in Fig. 5.3.

Fig. 5.3
An illustration of the processes that take place when chitosan is administered in specific areas of the body along with D N A.

Reproduced from M. Junaid Dar et. al. with permission from Elsevier 2019

Schematic Pathway for Chitosan nanoparticles in targeted gene delivery.

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Numerous reports are reported in the literature relating to targeting these specific sites, such as tumors [163], liver [164, 165], lung [166, 167], and brain [168, 169]. In most of these studies, chitosan is conjugated with the protein, transferrin, peptide, antibody, etc. [170]. A peptide functionalized chitosan-DNA nanoparticles were reported by Talvitie et al. for cellular targeting which is targeted to the required cell receptors in a specific and time-dependent manner [171]. In addition, Wang et al. synthesized the pH-sensitive gene delivery system for cancer cell-targeting which improved gene delivery by the introduction of pDNA nanocomplexes in the core and a pH-sensitive anionic polymer folic acid-modified PEG tethered carboxylated chitosan coating on the surface [172]. Numerous studies focused on targeted chitosan carrier systems have been reported in the literature [62, 173,174,175,176,177,178,179,180]. Currently, a combination of both the viral vectors and chitosan is used for efficient and permanent transfection [181]. Lameiro et al. coupled the adenovirus into the chitosan microparticle for mucosal vaccination. The main reason behind this study was to defend viruses, lower the immune response, and prolonged release. However, there are some boundaries including the difficulty of controlled release, loss of viral activity, and less loading efficiency. More studies are needed to triumph over these shortcomings [181, 182].

7 Conclusion

The idea of gene delivery was introduced in 1963, and viral vectors have been most effectively utilized in the gene therapy area. Because early 2000, these vectors were in the main abandoned because of the adverse side effects of viral-based gene therapies. Researchers have targeted the synthesis and applications of non-viral vectors, which are lipophilic or polymer-primarily based gene delivery systems. Cationic polymers are desired as a non-viral vector inside the field of gene transport. Chitosan and chitosan derivatives are commonly desired by the other cationic polymers due to their superior properties. Chitosan and chitosan derivatives are especially biodegradable and biocompatible polysaccharides. These are chemically versatile for undergoing varieties of reactions having different physicochemical properties which were tuned through modification having lower cytotoxicity, and high transfection properties. They are also called effective DNA-condensing agents and provide protection against DNAase-degradation. In connection with this fact that polymer-based gene delivery systems are yet to gain a massive presence in medical trials. Chitosan and its derivatives have been utilized in gene delivery studies after numerous modifications. A number of in vitro and in vivo studies confirm that chitosan and its derivatives are suitable and promising materials for efficient non-viral gene and DNA vaccine delivery. It is evident that chitosan and its derivatives are strong candidates to be used as the most preferred non-viral vector for gene delivery clinical trials in the future.