**3.1 Molecular structure of chitosan**

Chitosan (poly[β-(1-4)-2-amino-2-deoxy-D-glucopyranose]) is a deacetylation product of chitin (see Figure 1), a high Mw natural polymer found in the shells of marine crustaceans, such as shrimps (see Figure 2), as well as various insects, the internal structures of other invertebrates, and in the cell walls of fungi. It also provides an avenue for recycling of marine shellfish waste, which can now be "mined" for chitin and chitosan (Hayes et al., 2008).

Fig. 1. Chemical structure of Chitin

Gene gun

Most non-viral vectors have no limitation in DNA size for packaging and they have the possibility of modification with ligands for tissue- or cell-specific targeting with low commercial cost and high reproducibility. Among these carriers, cationic lipids (lipoplexes) and cationic polymers (polycations) are primarily used, especially in *in vitro* gene transfection. Lipoplexes can form micelles or liposomes, which are multilayered structures, where the DNA is sandwiched between the cationic lipids. The lipoplexes present some problems due to their low physiological stability, reproducibility, and their toxicity of polar and hydrophobic moiety containing structure. *In vivo*, the intravenous administration of cationic lipid/DNA complexes presented significant problems, as these reagents can be quite toxic. On the other hand, polycations are more stable than lipoplexes and can protect DNA against nuclease degradation (Gao & Huang, 1996). Their structures also show more variability and versatility, including the possibility of incorporation of target-specific cellular receptors. Thus, modifications to these polymers, such as molecular weight (Mw), geometry (linear verses branched) and ligand attachment, can be easily undertaken successfully (Kim et al., 2007; Pack et al*.*, 2005). Furthermore, they can compact DNA molecules to a relatively small particle size. However, the efficiency of gene delivery by both complexes is still relatively low, when compared to viral vectors. Polyethylenimine (PEI) is a cationic polymer that has been used for non-viral gene transfection for some time, but due to

At present, chitosan is the most prominent of the non-viral carriers being investigated. The biomaterial, chitosan, has interested many researchers around the world, particularly in relation to its ability to be a gene delivery vehicle or the ability to modify this biopolymer for the gene delivery vehicle. This is because of its properties of biodegradability, biocompatibility, and low toxicity, and because it can be modified for increasing transfection

Chitosan (poly[β-(1-4)-2-amino-2-deoxy-D-glucopyranose]) is a deacetylation product of chitin (see Figure 1), a high Mw natural polymer found in the shells of marine crustaceans, such as shrimps (see Figure 2), as well as various insects, the internal structures of other invertebrates, and in the cell walls of fungi. It also provides an avenue for recycling of marine shellfish waste, which can now be "mined" for chitin and chitosan (Hayes et

Lipoplexes and polyplexes

Direct methods Injection of naked DNA

Non-viral method Examples

Physical methods Electoporation

 Sonoporation Magnetofection Chemical methods Oligonucleotides

Dendrimers

its toxicity and the variable results, it has not been widely accepted.

efficiency, as well as for targeting gene delivery development.

**3. Chitosan as a non-viral carrier** 

**3.1 Molecular structure of chitosan** 

al., 2008).

Table 1. Examples of non-viral methods of gene delivery

Fig. 2. Commercial chitosan is derived from the shells of shrimp and other sea crustaceans, including the Alaskan pink shrimp, pictured here (US National Oceanic and Atmosheric Administration, 2011)

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.

Chitosan and Its Modifications: Are They Possible Vehicles for Gene Therapy? 443

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

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,

There are many factors that affect transfection efficiency. These include Mw, DDA, DNA

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

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;

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

complexes' charge ratio, pH and particle sizes, as well as the type of cell lines used.

**4. Chitosan and gene delivery** 

degradation before entering the nucleus.

**4.1 Transfection efficiency of chitosan**

**4.2 Factors affecting transfection efficiency**

2010; Mao et al., 2010).

**4.2.1 Molecular weight (Mw)** 

(see review of Mao et al., 2010).

Supaprutsakul et al., 2010).

Fig. 3. Chemical structure of chitosan. It is a linear polysaccharide composed of randomly distributed β-(1-4)-linked D-glucosamine (deacetylated unit) and N-acetyl-D-glucosamine (acetylated unit)

#### **3.2 Low toxicity of chitosan**

Chitosan has low toxicity with an LD50 (lethal dose for 50% of test population) level in the same dose as sugar or salt (Arai, 1968). Toxicity tests reported the LD50 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).

#### **3.3 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. Park et al., 2010).


Table 2. Some applications of chitosan
