**4.2 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.

### **4.2.1 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

Chitosan and Its Modifications: Are They Possible Vehicles for Gene Therapy? 445

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,

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

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

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

using chitosan as a core structure.

**5. Chitosan and gene therapy** 

these small nucleic acids.

(Masotti et al., 2009).

**5.1 Use of chitosan nanoparticles** 

**5.2 Use of siRNA loaded chitosan nanoparticles** 

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 ratio of the complexes.
