**6. Limitations in the use of chitosan**

446 Non-Viral Gene Therapy

4), assemble into endoribonuclease containing complexes known as RNA-induced silencing

Fig. 4. Schematic representation of a siRNA molecule. SiRNA have a well-defined structure: a short (usually 21-nucleotide-long) double-strand of RNA with 2-nucleotide 3' overhangs

The siRNA strands guide the RISCs to complementary RNA molecules leading to cleavage

Fig. 5. Mechanism of RNAi (Hood, 2004). dsRNA=double stranded RNA; shRNA=small hairpin RNA (sequence of RNA that can be used to silence gene expression via RNA

and destroy the target RNA (Manjunath & Dykxhoorn, 2010).

The mechanism of RNAi is described in Figure 5.

interference); mRNA=messenger RNA

complexes (RISCs).

on either end (Alper, 2006).

There are some limitations in the use of chitosan for non-viral gene therapy. Firstly, there is a lack of knowledge of the pharmacokinetics of chitosan-nucleic acid complexes during uptake inside the body. When chitosan-DNA nanoparticles enter the body, they were quickly removed from the blood and deposited on different organs. Administration of larger nanoparticles results in a substantial increase of the particles in the lung with a subsequent decrease in the liver, indicating a strong dependence of the tissue distribution on particle size (Liu, 2007). However, more information on this topic is required. Secondly, there is a need for more studies in animals, including clinical trials.

Most of the studies in the past few years about chitosan and gene therapy continue to use an *in vitro* model; however, more studies have been performed using mouse model, as summarised in Table 3.


Table 3. Summary of current animal experiments using chitosan as non-viral for gene therapy

Chitosan and Its Modifications: Are They Possible Vehicles for Gene Therapy? 449

An efficient gene delivery system is very important for gene therapy. Currently, the most efficient of these systems is a viral vector, which usually yields a transfection efficiency of more than 90%. However, by using a viral vector for gene therapy, there is a concern about the host versus vector immunological response, mutation and oncogenic effects; hence the need to develop a non-viral vector. There are many non-viral vectors; the high efficient one is cationic lipid, which gives high transfection efficiency, especially in tissue culture or *in vitro* conditions. *In vivo*, the intravenous administration of cationic lipid/DNA complexes presented significant problems, as these reagents can be quite toxic. PEI is another non-viral transfection material that has been used for some time, but due to its toxicity and the

Chitosan (poly[β-(1-4)-2-amino-2deoxy-D-glucopyranose]), a nontoxic biodegradable biopolymer, has been broadly studied as a promising non-viral vector for gene delivery. This cationic polysaccharide has been produced by partial deacetylation of chitin, a naturally polymer from crustacean shells. However, the transfection efficiency of chitosan itself is not efficient enough and depends on many factors such as Mw, DDA, DNA complexes charge ratio, pH and particle sizes, as well as the type of cells. There have been many attempts to modify chitosan in order to improve transfection efficiency. Some studies have revealed that low Mw chitosan, especially the product of oxidative depolymerisation from higher Mw chitosan with NaNO2, had low cytotoxicity and improved solubility properties, as well as having potential for gene delivery both *in vitro* and *in vivo*. However, some studies have reported decreased transfection efficiency with

There have been other attempts modifying the chemical structure of chitosan. These have included introducing a hydrophilic group, such as coupling dextran, as well as incorporating poly (vinyl pyrolidone) into the galactosylated chitosan, which can reduce the aggregation of particles and increase transfection efficiency. Some studies have also using hydrophobic modification of chitosan, such as deoxycholic acid-modified chitosan, in order to increase transfection efficiency through enhancement of complex interaction with cells and cellular uptake of the particles. Chitosan can be modified by conjugation of chitosan-DNA complexes with ligands to target specific cell surface receptors, but these attempts have had variable results. Many factors may have to be considered for improving transfection efficiency of chitosan, not just 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 ratio may have to

The design criteria of the effective vector for non-viral gene therapy should also consider cost–effectiveness in synthesis and purification steps, serum stability and efficient packaging of large amount of the vector-nucleic acid complexes. Moreover, the route of administration of this vector to the target cells or tumour lesion, high transfection efficiency, specific target gene delivery should also be considered. Once the complexes enter the target cells, they have to escape from enzyme degradation. The complexes then release the therapeutic gene/ nucleic acid to the target organelle, such as DNA, which has to enter the nucleus, while siRNA functions in cytoplasm. This release has to occur without too many difficulties, which means that the bonding between the vectors and nucleic acid should not be too strong. Most importantly, these non-viral vectors have to be

**7. Summary** 

lower Mw chitosan.

be adjusted.

variable results, it has not been widely accepted.

Lastly, the route of transmission and target gene delivery are the major factors which contribute to the success in gene therapy, which still requires further investigation. Table 4 summarises some attempts to modify the chitosan-nucleic acid complexes for target gene therapy.


Table 4. Some modifications of chitosan for target gene therapy
