**5.1 Use of chitosan nanoparticles**

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 (Masotti et al., 2009).

### **5.2 Use of siRNA loaded chitosan nanoparticles**

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

Chitosan and Its Modifications: Are They Possible Vehicles for Gene Therapy? 447

This system is of interest to numerous researchers, as well as many pharmaceutical companies, since the efficient siRNA delivery system will have clinical therapeutic impact in gene therapy (Mao et al., 2010; Park et al., 2010; Rudzinski & Aminabhavi, 2010). However,

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

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

> Target organ/ Disease

Expected route for fetal gene therapy

Expected route for diseased blood vessel

Expected route for pulmonary gene therapy

Expected route for multiple localized disease

Prostate cancer

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

wall

Study design Reference

Mouse Zhu et al.,

Mouse Mohri et al.,

2010

al., 2010

2010

Han et al., 2010

Feng et al., 2010

Yang et al., 2008; Jang et al., in press

Mouse (murine)

Lung cancer Mouse Okamoto et

Mouse models of melanoma and breast cancers

Mouse: xenograft model

further investigations are needed, especially *in vivo* experiments/clinical trials.

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

summarised in Table 3.

Route of transmission

Utero gene transfer (injection in amniotic sacs)

Local gene delivery via endovascular

stent

Local: inhalation

Local: inhalation

Local: localized hydrogel by intra–tumoural injection

Local: intratumoural injection

therapy

need for more studies in animals, including clinical trials.

Form of chitosan complexes

Chitosan-DNA (reporter gene)

Chitosan-DNA coated with dodecylated in endovascular

Chitosan-DNA (interferon-beta gene) complexes

Spray –freeze dry chitosan-DNA

Chitosan-SiRNA

Chitosan-SiRNA design to down regulate RXFP1 expression

stent

powder

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

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 on either end (Alper, 2006).

The siRNA strands guide the RISCs to complementary RNA molecules leading to cleavage and destroy the target RNA (Manjunath & Dykxhoorn, 2010). The mechanism of RNAi is described in Figure 5.

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 interference); mRNA=messenger RNA

This system is of interest to numerous researchers, as well as many pharmaceutical companies, since the efficient siRNA delivery system will have clinical therapeutic impact in gene therapy (Mao et al., 2010; Park et al., 2010; Rudzinski & Aminabhavi, 2010). However, further investigations are needed, especially *in vivo* experiments/clinical trials.
