**8. Chitosan-siRNA delivery system**

siRNA silencing technology is exploited in a wide range of biological studies, but has also become one of the most challenging therapeutic strategies. However, because of its poor delivery and susceptibility to nuclease degradation, siRNA-based approaches need a protective delivery system. A variety of polymer formulations have been proposed in the literature as potential carriers (De Fougerolles 2008; Gary et al., 2007; Zhang et al., 2007). Polymer molecular weight, change density, N/P ratio (ratio of protonatable polymer amine groups to nucleic acid phosphate groups) and ionic strength of the medium can affect electrostatic binding between siRNA and cationic polymers. Research over the years has revealed that chitosan is one of the desirable polymeric carriers of siRNA because of its natural biocompatibility, biodegradability, nontoxicity, and high nuclease resistance. The effects of different chitosan (114-kDa or more)-siRNA complexes on transfection activity have been observed previously (Katas et al., 2008; Katas & Alpar, 2006; Liu et al., 2007; Rojanarata et al., 2008). Higher MW and DDA are desirable characteristics for the formation of chitosan nanoparticles, as higher MW chitosan molecules are long and flexible while higher DDA enhances its electrostatic interaction with siRNAs, thus synergically reducing the size of complexes and increasing their stability (Liu et al., 2007). A high charge ratio also enhances the stability of complexes because the loosely bound excess chitosan on the outer surface of nanoparticles can promote binding and uptake across anionic cell surfaces and also provide subsequent protection against siRNA degradation within endosome compartments (Liu et al., 2007). The method of complexation also affects the gene-silencing

such as deoxycholic acid modification (Lee et al., 1998), or 5β-cholanic acid modification (Yoo et al., 2005), can attenuate the electrostatic attractions between cationic polymers and anionic DNA. It is actually a contradiction between the stability and dissociation ability of complexes. A temperature-sensitive modification of poly(N-isopropylacrylamide) (PNIPAAm) can control the dissociation of PNVLCS (N-isopropylacrylamide/vinyl laurate copolymer with chitosan) complexes with DNA by a temporary reduction in the culture

The cytoplasm, a mesh-like network of microfilaments and microtubules, will limit the diffusion of complexes or DNA about 500-1000-fold. Adenovirus particles naturally bind to dynein and are actively transported towards the nuclear pore complexes once they are inside the cytoplasm. Prior to entry into the nucleus, the viruses dissociate into smaller structures and use their attached transport factors such as importins or karyopherins which have nuclear localization signals (NLS) to recognize the nuclear pore complex (NPC) (Whittaker & Helenius, 1998). Justin Hanes et al. used a new method called multiple particles tracking (MPT) to quantify the intracellular transport of non-viral DNA nanocarriers. They found that PEI/DNA complexes can accumulate in the perinuclear area through a subdiffusive transport, which is a combination of diffusive transport and active transport. This discovery is a dispute to the common belief that non-viral vectors go through the cell cytoplasm in a slow random way. Further investigation showed that actively transported complexes of PEI/DNA are in endosomes undergoing motor protein-driven movement guided by microtubules or physically associated with the motor proteins themselves (Suh et al., 2003). As to chitosan and its derivatives, however, few studies have examined how they pass through the highly structured cytoplasm and eventually enter into

siRNA silencing technology is exploited in a wide range of biological studies, but has also become one of the most challenging therapeutic strategies. However, because of its poor delivery and susceptibility to nuclease degradation, siRNA-based approaches need a protective delivery system. A variety of polymer formulations have been proposed in the literature as potential carriers (De Fougerolles 2008; Gary et al., 2007; Zhang et al., 2007). Polymer molecular weight, change density, N/P ratio (ratio of protonatable polymer amine groups to nucleic acid phosphate groups) and ionic strength of the medium can affect electrostatic binding between siRNA and cationic polymers. Research over the years has revealed that chitosan is one of the desirable polymeric carriers of siRNA because of its natural biocompatibility, biodegradability, nontoxicity, and high nuclease resistance. The effects of different chitosan (114-kDa or more)-siRNA complexes on transfection activity have been observed previously (Katas et al., 2008; Katas & Alpar, 2006; Liu et al., 2007; Rojanarata et al., 2008). Higher MW and DDA are desirable characteristics for the formation of chitosan nanoparticles, as higher MW chitosan molecules are long and flexible while higher DDA enhances its electrostatic interaction with siRNAs, thus synergically reducing the size of complexes and increasing their stability (Liu et al., 2007). A high charge ratio also enhances the stability of complexes because the loosely bound excess chitosan on the outer surface of nanoparticles can promote binding and uptake across anionic cell surfaces and also provide subsequent protection against siRNA degradation within endosome compartments (Liu et al., 2007). The method of complexation also affects the gene-silencing

temperature to 20℃ (Sun et al., 2005)

**8. Chitosan-siRNA delivery system** 

the nucleus.

activity of chitosan/siRNA complexes. Haliza Katas et al. studied the difference between simple complexation, ionic gelation (siRNA entrapment) and adsorption of siRNA onto the surface of preformed chitosan nanoparticles. Ionic gelation gave the strongest stability and the most efficient gene-silencing activity among the three methods tested. For the involvement of tripolyphosphate (TPP) ions during the complexation of ionic gelation, pH became one of the factors that mostly affected the gene-silencing activity. The decrease of pH resulted in a reduction in the charge number of TPP, which subsequently led to the need for more TPP ions for cross-linking of the chitosan by electrostatic forces (Katas & Alpar, 2006). Rojanarata et al. reported that chitosan-thiamine pyrophosphate (TPP)-mediated siRNA enhanced green fluorescent protein (EGFP) gene silencing efficiency depended on the molecular weight and weight ratio of chitosan and siRNA. The chitosan-TPP-siRNA complex with the lowest molecular weight of chitosan (20 kDa) at a weight ratio of 80 showed the strongest inhibition of gene expression (Rojanarata et al., 2008). A novel study of chitosan/siRNA nanoparticles with fluorescent quantum dots was taken to silence HER2/neu and achieved desirable silencing effects (Tahara et al., 2008; Tan et al., 2007). In the field of controlled release, chitosan coating PLGA nanospheres with a high loading efficiency of siRNAs were found to reduce the initial burst of nucleic acid release and to prolong release at later stages, without changing the release pattern (Tahara et al., 2008). Kenneth A. Howard found that the chitosan-based system had the ability for endosome escape through the proton sponge mechanism, because the endosomolytic agent chloroquine did not increase the effect of RNA interference (Howard et al., 2006). In terms of *in vivo* administration of chitosan/siRNAs complexes, only a few studies are available. Nasal administration to silence EGFP expression of the endothelial cells distributed in the bronchioles of transgenic EGFP mouse model has been successfully achieved without showing any adverse effects (Howard et al., 2006). Cross-linking of hyaluronan and chitosan has proven to have a higher efficiency of transfection in ocular tissue over unmodified chitosan (de la Fuente et al., 2008).
