**2. 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.

Chitosan and Its Modifications: Are They Possible Vehicles for Gene Therapy? 441

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

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

Fig. 1. Chemical structure of Chitin

Administration, 2011)

chemical structure of chitosan is given in Figure 3.


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

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