Chitosan and Its Modifications: Are They Possible Vehicles for Gene Therapy?

[Extract] In gene therapy, the most important step is how to effectively deliver the therapeutic gene to the target cells or organ. At present, there are two methods, which are those using a viral and a non-viral vector system. The most common viral vectors that have been used include retroviruses, herpes simplex viruses, lentiviruses, adenoviruses and adeno-associated viruses (Oligino et al., 2000). The advantages of most viral vectors are high transfection efficiency and fast transcription of the foreign material inserted in the viral genome. However, a number of disadvantages have also been described, such as poor target-specificity, low capacity to incorporate foreign deoxyribonucleic acid (DNA) sequence to their genome (Mansouri et al., 2004), toxic and inflammatory effects, wild– type mutations, potential oncogenic effects (Lee et al., 1998), and, in particular, unwanted immune responses. In clinical trials of gene replacement therapy using viral vectors, significant adverse effects have been reported, including a fatal inflammatory response associated with adenoviral vector (Raper et al., 2003), and the development of acute leukaemia in recipients of ex-vivo, adenoviral vector-transduced hematopoietic cells (Woods et al., 2006). Intravenous adenoviral vector has also resulted in high liver toxicity due to uptake by hepatocytes or Kupffer cells of the liver reticular endothelial system, immediately following systemic administration.


Introduction
In gene therapy, the most important step is how to effectively deliver the therapeutic gene to the target cells or organ. At present, there are two methods, which are those using a viral and a non-viral vector system. The most common viral vectors that have been used include retroviruses, herpes simplex viruses, lentiviruses, adenoviruses and adenoassociated viruses (Oligino et al., 2000). The advantages of most viral vectors are high transfection efficiency and fast transcription of the foreign material inserted in the viral genome. However, a number of disadvantages have also been described, such as poor target-specificity, low capacity to incorporate foreign deoxyribonucleic acid (DNA) sequence to their genome (Mansouri et al., 2004), toxic and inflammatory effects, wildtype mutations, potential oncogenic effects (Lee et al., 1998), and, in particular, unwanted immune responses. In clinical trials of gene replacement therapy using viral vectors, significant adverse effects have been reported, including a fatal inflammatory response associated with adenoviral vector (Raper et al., 2003), and the development of acute leukaemia in recipients of ex-vivo, adenoviral vector-transduced hematopoietic cells (Woods et al., 2006). Intravenous adenoviral vector has also resulted in high liver toxicity due to uptake by hepatocytes or Kupffer cells of the liver reticular endothelial system, immediately following systemic administration.

Non-viral vectors for gene delivery
Due to the limitations and disadvantages of using viral vectors, there has been an ongoing search for an efficient safe vector for gene therapy, which has lead to the development of non-viral gene therapy. They have some advantages over viral methods, including simple large scale production and relatively low host immunogenicity. Previously, low levels of transfection and expression of the gene limited the usefulness of non-viral methods. However, recent advances in vector technology have been useful in yielding molecules and techniques with transfection efficiencies approaching or surpassing those of viral vectors. Table 1 provides examples of the main non-viral methods of gene delivery.
www.intechopen.com Deacetylation of chitin can be performed by boiling chitin from crab or shrimp shells in sodium hydroxide after decolourisation with potassium permanganate (Van Der Lubben et al., 2001). Chitosan is a co-polymer of glucosamine and N-acetyl-D-glucosamine. When the number of N-acetylglucosamine units exceeds 50%, the biopolymer is called chitin; the term 'chitosan' is used to describe an N-acetyl-glucosamine unit content less than 50%. The chemical structure of chitosan is given in Figure 3.

Low toxicity of chitosan
Chitosan has low toxicity with an LD 50 (lethal dose for 50% of test population) level in the same dose as sugar or salt (Arai, 1968). Toxicity tests reported the LD 50 of chitosan in mice exceeded 16 g/kg. The molecular mass has minimal effect on cell viability, while the degree of deacetylation (DDA) of the polymer has greater effect on its toxicity (Richardson et al., 1999). DDA also affects the solubility, hydrophobicity and its ability to interact electrostatically with polyanions by affecting the number of protonatable amine groups of chitosan. Chitosan nanoparticles with lower DDA showed lower toxicity in vitro (Huang et al., 2004).

Applications of chitosan
Chitosan is a biodegradable polymer used in various industrial, biomedical and pharmaceutical applications due to its biocompatibility and the slow release of active molecules. Table 2 summarizes some of these broader applications. The novel properties of chitosan make it a versatile biomaterial for cell therapy, tissue engineering and gene therapy (Sui et al., 2006). Chitosan has a positive charge and hydrophilic character at an acidic pH. It is a continuum of primary aliphatic amine that can be protonated by acids; the pKa of the chitosan amine groups being around 6.3-6.5 (Kumar et al., 2004). The cationic amino groups on the C2 position of the repeating glucopyranose units of chitosan can interact electrostatically with the anionic groups (usually carboxylic acid groups) of other polyions to form polyelectrolyte complexes (Hamman, 2010). Many different polyanions from a natural origin (e.g. alginate, chondrotin sulfate or dextran sulphate) or from a synthetic orgin [e.g. poly(acrylic acid), polyphosphoric acid, or poly(L-lactide)] have been used to form polyelectrolyte complexes with chitosan, in order to provide the required physicochemical properties for design of specific drug delivery system, as well as specific target gene delivery (J. H. .

Usage
Examples of applications Agricultural and horticultural Natural biocontrol and elicitor Water process engineering Part of filtration process Biomedical and pharmaceutical Hemostatic agents Wound healing Tissue engineering Drug delivery Gene therapy

Chitosan and gene delivery
Chitosan has been broadly studied as a promising non-viral vector for gene delivery (Bowman & Leong, 2006). This cationic polysaccharide can bind DNA between the positive charges of its amino groups and the negative charges of the phosphate groups of the DNA backbone in order to form nano-or microparticles. The interaction between chitosan and nucleic acids is electrostatic. The charge interaction is sufficiently strong that chitosan-DNA or small interfering ribonucleic acid (SiRNA) complex does not dissociate until it has entered the cell. Moreover, chitosan also protected nucleic acids from enzymatic degradation before entering the nucleus. Tong et al. (2009) describes seven steps that should be overcome before the expression of exogenous DNA. They are complexation, in vivo administration, endocytosis, escape from endolysosome, release of DNA, trafficking through cytoplasm and finally importation of DNA into the nucleus. The transfection efficiency of chitosan itself is; however, relatively low, when compared to lipoplex or other methods. But this aminopolysaccharide can be modified for ease of DNA delivery, as well as for target gene delivery, which currently attracts many researchers to use chitosan and its modifications for gene delivery. Chitosan can be modified by ligand conjugation, such as transferrin-, folate-(folate and transferrin are over expressed in cancer cells), mannose-(target dendritic cells in tumor) and galactose (target Kupffer cells of the liver) conjugated chitosan, which can improve transfection efficiency of the targeted cells via receptor-mediated endocytosis (Duceppe & Tabrizian, 2010;Mao et al., 2010).

Factors affecting transfection efficiency
There are many factors that affect transfection efficiency. These include Mw, DDA, DNA complexes' charge ratio, pH and particle sizes, as well as the type of cell lines used.

Molecular weight (Mw)
High Mw chitosan can bind DNA tightly, which is due to the high number of positive charge of amino groups, but binding DNA tightly may give low transfection efficiency, due to not releasing the DNA to the nucleus after endocytosis to the cell. The Mw of chitosan also influences the size of the chitosan-DNA complexes, as the higher sizes of chitosan-DNA complexes can affect the cellular uptake. These factors lead to transfection efficiency (see review of Mao et al., 2010). If the N/P ratio, which is the molar ratio between the amino groups of chitosan and the phosphate groups of DNA, was fixed, then the higher the Mw , the larger the chitosan-DNA complexes diameter (MacLaughlin et al., 1998). However, there have been differing conclusions proffered between the Mw of chitosan and transfection efficiency. Some studies have reported of high transfection efficiency with high Mw chitosan (Huang et al., 2005;Kiang et al., 2004;MacLaughlin et al., 1998). Other studies have reported that low Mw chitosan has better transfection efficiency (Koping-Hoggard et al., 2004;Lavertu et al., 2006;Supaprutsakul et al., 2010). MacLaughlin et.al. (1998) synthesised depolymerised chitosan oligomers with a Mw from 7 -92, but the transfection efficiency was much lower than at the higher Mw of 102 and 230 kDa, respectively, and being about 1000 times lower in transfection efficiency compared to lipofectamine TM . Haung et al. (2005) also found a decreased A549 cellular uptake with the decreasing Mw or DDA of chitosan and a N/P ratio of 6 was used in that study. But the study of Supaprutsahul et al. (2010) revealed much higher transfection efficiency with the depolymerised chitosan at Mw ~16 kDa (or Mn ~ 6.5). This may be because of the different chitosan/DNA ratio, as the previous study used low N/P ratio, while Supaprutsakul et al. (2010) used chitosan/plasmid at an N/P ratio of about 56:1, which meant than a much higher amount of chitosan was used for the lower Mw. This was consistent with the study of Romøren et al. (2003), who found that low Mw chitosan was beneficial at the higher charge ratio of the complexes.

pH and degree of deacetylation (DDA)
The study of Lavertu et al. (2006) also found that the low Mw chitosan, which had a numeric average Mw (Mn) of about 10 and 80% DDA at N/P ratio 10:1, gave higher transfection efficiency at the same level as lipofectamine TM at pH 6.5. However, the very low Mw (1.9-7.7 kDa) chitosans with high DDA were found to form aggregates easily, even at very low charge ratios (Morris et al., 2008), and this might lower the transfection. However, the depolymerised LW chitosan in this study had only 54% DDA, which may reduce the problem of particles aggregation and, after cells uptake the chitosan-DNA nanoparticles, the DNA may be released from the nanoparticles more easily, as DNA binding efficacy was reduced as DDA was decreased (Kiang et al., 2004). Hence, many factors may have to be considered for improving transfection efficiency of chitosan, not only the ligand binding, but also the method of binding or conjugation, the size and morphology of the particles, the aggregation of the complexes, and especially the chitosan itself, as Mw, DDA and charge ratio, which may have to be adjusted.

Cell line dependency
Another factor, which may affect transfection efficiency of chitosan, is cell line dependency. Higher mitotic cell lines, such as cancer cells, usually have higher transfection efficiencies that lower proliferative rates of the cell line. This may be related to differences in cell physiology affecting the internalisation mechanism and subsequent internal trafficking of the vectors (Douglas et al., 2008). It has also been found that dividing cells have higher transfection ability compared to quiescent cells (Brunner et al., 2000) and higher levels of gene expression have been observed just before or during mitosis (Mortimer et al., 1999). This may explain why the immortalised cell line, with higher mitotic activity, has higher transfection ability than normal or primary cell lines. However, this factor requires further investigation.

Improving transfection efficiency
There have also some attempts to modify the chemical structure of chitosan to improve transfection efficiency, which have involved hydrophilic and hydrophobic modifications. The main purpose of hydrophilic modification of chitosan is to increase solubility and reduce sensitivity of chitosan-DNA complexes to pH, as well as reduce the chitosan-DNA complexes aggregation, which may improve transfection efficiency. The hydrophilic chitosan modification includes quaternised chitosan (Thanou et al., 2002), PEGylated (covalent attachment of polyethylene glycol polymer chains to another molecule) chitosan (Jiang et al., 2006) and low Mw soluble chitosan (Ercelen et al., 2006). Interestingly, Brannon-Peppas & Blanchette (2004) found that particles with more hydrophobic surfaces were also preferentially taken up by the liver, followed by the spleen and the lungs.
Hydrophobic modifications of chitosan have been performed in many studies. The main objectives of these modifications were increasing transfection efficiency by modulating complex interactions with cells, especially in the complexes' adsorption on the cell surfaces and cell uptake (Kurisawa et al., 2000). Some hydrophobic units also help in the dissociation between the chitosan DNA complexes to release DNA to enter the nucleus after cellular uptake, as well as protecting it from enzymatic degradation and facilitating intra cellular pDNA (plasmid DNA) association, which can enhance transfection efficiency. These hydrophobic modifications included deoxycholic chitosan, N-alkylated chitosan, thiolated chitosan and hybrid chitosan (Mao et al., 2010). The combination of hydrophilic and hydrophobic modification of chitosan structure has been another interesting area that looks highly promising for the development of high transfection efficiency in a non-viral vector, using chitosan as a core structure.

Chitosan and gene therapy
One of the significant applications of chitosan is in its application to gene therapy. It has a number of benefits. It has non-toxic, biodegradable and biocompatible with high cationic charge potential; protects DNA from degradation by nucleases; and has high yield transfection efficiency (Sui et al., 2006). Genetic material (DNA and RNA) has been explored for use as a treatment of genetic abnormalities or deficiencies, which is described as gene therapy. Gene therapy functions by transferring healthy genetic material or nucleic acid constructs, such as ribozymes, antisense molecules, decoy oligodeoxy nucleotides (ODNs), DNAzymes and siRNA, into diseased cells in an attempt to achieve a therapeutic effect that results in restoration of protein production, which was absent or deficient due to the preexisting genetic disorder (Tan et al., 2009). But using small nucleic acid, such as DNAzymes and siRNA, has some limitations, since they are rapidly degraded in plasma and cellular cytoplasm and cannot passively diffuse through cellular membrane, which is due to the strong anionic charge of the phosphate backbone and the consequent electrostatic repulsion from the anionic cell membrane surface as well as limited size of cellular entrance. So, these small nucleic acids encapsulated with chitosan nanoparticles can reduce the limitations of these small nucleic acids.

Use of chitosan nanoparticles
The development of chitosan and its modification for non-viral gene delivery is also a target for gene therapy. This is because chitosan nanoparticles have a low toxicity and are taken up by endosomes allowing the DNA or nucleic acid to overcome the permeability barrier posed by epithelium and also to protect against enzymatic degradation. There are some studies that have attempted to use chitosan for cancer therapy. Chitosan itself was able to demonstrate growth inhibitory effects on cancer cells and has apoptosis effect on bladder tumour cells via caspase-3 activation (Tan et al., 2009). The various manufacturing processes for chitosan nano-/micro-particles/spheres (nanofabrication) has been described elsewhere (Masotti et al., 2009).

Use of siRNA loaded chitosan nanoparticles
In current developments in chitosan for gene therapy, there is an attempt to develop siRNA loaded chitosan nanoparticles to silence the target gene. This method can silence the gene by means of RNA interfering (RNAi). SiRNAs, usually containing 20-25 base pairs (see Figure  4), assemble into endoribonuclease containing complexes known as RNA-induced silencing complexes (RISCs). Fig. 4. Schematic representation of a siRNA molecule. SiRNA have a well-defined structure: a short (usually 21-nucleotide-long) double-strand of RNA with 2-nucleotide 3' overhangs on either end (Alper, 2006).
The siRNA strands guide the RISCs to complementary RNA molecules leading to cleavage and destroy the target RNA (Manjunath & Dykxhoorn, 2010). The mechanism of RNAi is described in Figure 5.  (Hood, 2004). dsRNA=double stranded RNA; shRNA=small hairpin RNA (sequence of RNA that can be used to silence gene expression via RNA interference); mRNA=messenger RNA T h i s s y s t e m i s o f i n t e r e s t t o n u m e r o u s r e searchers, as well as many pharmaceutical companies, since the efficient siRNA delivery system will have clinical therapeutic impact in gene therapy (Mao et al., 2010;Rudzinski & Aminabhavi, 2010). However, further investigations are needed, especially in vivo experiments/clinical trials.

Limitations in the use of chitosan
There are some limitations in the use of chitosan for non-viral gene therapy. Firstly, there is a lack of knowledge of the pharmacokinetics of chitosan-nucleic acid complexes during uptake inside the body. When chitosan-DNA nanoparticles enter the body, they were quickly removed from the blood and deposited on different organs. Administration of larger nanoparticles results in a substantial increase of the particles in the lung with a subsequent decrease in the liver, indicating a strong dependence of the tissue distribution on particle size (Liu, 2007). However, more information on this topic is required. Secondly, there is a need for more studies in animals, including clinical trials.  Table 3.

Summary
An efficient gene delivery system is very important for gene therapy. Currently, the most efficient of these systems is a viral vector, which usually yields a transfection efficiency of more than 90%. However, by using a viral vector for gene therapy, there is a concern about the host versus vector immunological response, mutation and oncogenic effects; hence the need to develop a non-viral vector. There are many non-viral vectors; the high efficient one is cationic lipid, which gives high transfection efficiency, especially in tissue culture or in vitro conditions. In vivo, the intravenous administration of cationic lipid/DNA complexes presented significant problems, as these reagents can be quite toxic. PEI is another non-viral transfection material that has been used for some time, but due to its toxicity and the variable results, it has not been widely accepted. Chitosan (poly[β-(1-4)-2-amino-2deoxy-D-glucopyranose]), a nontoxic biodegradable biopolymer, has been broadly studied as a promising non-viral vector for gene delivery. This cationic polysaccharide has been produced by partial deacetylation of chitin, a naturally polymer from crustacean shells. However, the transfection efficiency of chitosan itself is not efficient enough and depends on many factors such as Mw, DDA, DNA complexes charge ratio, pH and particle sizes, as well as the type of cells. There have been many attempts to modify chitosan in order to improve transfection efficiency. Some studies have revealed that low Mw chitosan, especially the product of oxidative d e p o l y m e r i s a t i o n f r o m h i g h e r M w c h i t o s a n w i t h N a N O 2 , had low cytotoxicity and improved solubility properties, as well as having potential for gene delivery both in vitro and in vivo. However, some studies have reported decreased transfection efficiency with lower Mw chitosan. There have been other attempts modifying the chemical structure of chitosan. These have included introducing a hydrophilic group, such as coupling dextran, as well as incorporating poly (vinyl pyrolidone) into the galactosylated chitosan, which can reduce the aggregation of particles and increase transfection efficiency. Some studies have also using hydrophobic modification of chitosan, such as deoxycholic acid-modified chitosan, in order to increase transfection efficiency through enhancement of complex interaction with cells and cellular uptake of the particles. Chitosan can be modified by conjugation of chitosan-DNA complexes with ligands to target specific cell surface receptors, but these a t t e m p t s h a v e h a d v a r i a b l e r e s u l t s . M a n y f a c t o r s m a y h a v e t o b e c o n s i d e r e d f o r improving transfection efficiency of chitosan, not just ligand binding, but also the method of binding or conjugation, the size and morphology of the particles, the aggregation of the complexes, and especially the chitosan itself, as Mw, DDA and charge ratio may have to be adjusted. The design criteria of the effective vector for non-viral gene therapy should also consider cost-effectiveness in synthesis and purification steps, serum stability and efficient packaging of large amount of the vector-nucleic acid complexes. Moreover, the route of administration of this vector to the target cells or tumour lesion, high transfection efficiency, specific target gene delivery should also be considered. Once the complexes enter the target cells, they have to escape from enzyme degradation. The complexes then release the therapeutic gene/ nucleic acid to the target organelle, such as DNA, which has to enter the nucleus, while siRNA functions in cytoplasm. This release has to occur without too many difficulties, which means that the bonding between the vectors and nucleic acid should not be too strong. Most importantly, these non-viral vectors have to be safe enough for the patient, including being non toxic to the host body, non-immunogenic and non pathogenic. Chitosan is now one of the candidate biomaterials for selection as an effective non-viral vector for gene therapy, especially as it is safe, cheap and easy to modify.