**4. Chitosan and gene delivery**

442 Non-Viral Gene Therapy

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

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

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

> 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

nanoparticles with lower DDA showed lower toxicity *in vitro* (Huang et al., 2004).

(acetylated unit)

**3.2 Low toxicity of chitosan** 

**3.3 Applications of chitosan** 

target gene delivery (J. H. Park et al., 2010).

Table 2. Some applications of chitosan

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.
