**5. Chitosan and gene therapy**

444 Non-Viral Gene Therapy

lipofectamineTM. 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

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 lipofectamineTM 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

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

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

or primary cell lines. However, this factor requires further investigation.

preferentially taken up by the liver, followed by the spleen and the lungs.

ratio of the complexes.

**4.2.2 pH and degree of deacetylation (DDA)** 

ratio, which may have to be adjusted.

**4.3 Improving transfection efficiency** 

**4.2.3 Cell line dependency** 

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.
