**4. Chitosan as a vector for gene therapy**

Cationic polymers, such as chitosan, are promising candidates for DNA transport in nonviral delivery systems (Kean & Thanou, 2010; Tong et al., 2009). Chitosan, a linear polysaccharide composed of randomly distributed β-(1-4)-linked D-glucosamine (deacetylated unit) and N-acetyl-D-glucosamine (acetylated unit), and has once been considered as an attractive gene transfer candidate for its superior biocompatibility, superior biodegradability and low cell toxicity. In recent years, with more researching methods involved, a more accurate and subtle view on the process of the entry of chitosan/DNA complexes into the cell nucleus has been developed. The enabling characteristics of Chitosan-DNA nanoparticles include biocompatibility, multiple ligand affinity, and a capacity of taking up large DNA fragments, while remaining small in size (Techaarpornkul et al., 2010). Chitosan and its derivatives, as favorable non-viral vectors involved in plasmid DNA delivery, have attracted attention in the field of siRNA delivery *in vitro* and *in* vivo (Andersen et al., 2009; Howard et al., 2009). Chitosan was once believed to be less effective than most other non-viral vectors because of its low stability and buffering capacity. However, recent technological advances in the chemical modification of chitosan have instituted improvements of its transfection efficiency without disturbing its biocompatibility and biodegradability. It has been demonstrated that transfection level is closely related to the molecular weight of polymers (Godbey et al., 1999). Chitosan (10-150 kDa), with a specific degree of deacetylation, allows maximum transgenic expression *in vitro*  (Lavertu et al., 2006). Another strategy for improving transfection is to take advantage of the mechanism of ligand-mediated uptake by cells to promote targeting and internalization, enhancing transfection efficiency. Ligand-mediated transfection has been shown to facilitate DNA internalization into cells via membrane receptors both *in vitro* and *in vivo.* Cell-specific ligand modification such as galactose, transferrin, folate and mannose can also effectively enhance the specificity of transfection through receptor-mediated endocytosis. Galactose ligand modification has been used to target HepG2 cells through the interaction with asialoglycoprotein receptors (ASGP-R) (Gao et al., 2003). A transferrin receptor is found on many mammalian cells, therefore it can be used as a universal ligand (Dautry-Varsat, 1986). Folate is not only over-expressed on macrophage surfaces, but is also over-expressed on many human cancer cell surfaces (Lee et al., 2006). Antigen presenting cells (APCs), the ideal targets of DNA vaccine, such as macrophages and immature dendritic cells are the target cells of mannose ligand (Kim et al., 2006). The specificity of these modifications can be demonstrated through ligand competitive inhibition experiments.

Chitosan-DNA/siRNA Nanoparticles for Gene Therapy 461

deacetylation in alkaline medium leads to the depolimerization (Domard & Rinaudo, 1983; Tolaimate et al., 2000). However it has been reported that chitin extracted from squid pens can be hydrolyzed under conditions that it allows obtaining chitosans of high molecular

Fig. 1. Chemical structure of chitin and chitosan. In the 2-amino-2-deoxy-d-glucopyranose

The homopolymer is a weak base with a pKa value of the D-glucosamine residue of about 6.3 and is therefore insoluble at neutral and alkaline pH values. In acidic mediums, the amine groups will be positively charged, conferring to the polysaccharide a high charge density. As in all polyelectrolytes, the dissociation constant of chitosan is not constant, but depends on the degree of dissociation at which it is determined. The pka value can be

Pka = pH + log [(1-α)/α] = pK0 –εΔψ(α)/kT Where Δψ is the difference in electrostatic potential between the surface of the polyion and the reference, α is the degree of dissociation, k is Boltzman's constant, T is the temperature and ε is the electron charge. Extrapolation of the pKa values to α = 1, where the polymer is uncharged and hence the electrostatic potential becomes zero, makes possible the value of the intrinsic dissociation constant of the ionizable groups, pK0, to be determined. The value obtained does not depend of the degree of *N*-acetylation, whereas the pKa value is dependent on this parameter, since the electrostatic potential will be varied depending of amount of the free amino groups. The pK0 value is called the intrinsic pKa of the chitosan. However chitosans of low molecular weight having degrees of deacetylation higher than 0.4 are also easily soluble in weakly acidic solvents such as

The physicochemical behavior in aqueous solution is highly dependent of pH and degree of acetylation and has received more attention only recently. Bertha et al. working on chitosans from 95 to 175kDa have recently determined the radius of gyration of chitosan (RG) (Bertha et al., 1998a; 2002b). The RG is an alternative measure of the size of the polymer chain and it can be measured by light scattering measurements. RG express the square mean radius of each one of the elements of the chain measured from its center of gravity. The study established the relationship between the molecular weight and radius of gyration (RG*)* of chitosan in aqueous solution, and the author indicated that chitosan behaved more like a Gaussian coil instead of the worm-like chain model found in common polyelectrolytes. At the same time the presence of *N*- acetyl groups on the chitosan backbone imparts hydrophobic properties. Schatz et al. (Schatz et al., 2003) have studied homogeneous series of chitosans with different degrees of acetylation and almost the same degree of

ring is shown the commonly used numbering for the carbon atoms.

calculated using the Katchalsky's equation (Roberts, 1992).

acetic acid and formic acid (Lee et al., 1995).

weight ( Tolaimate et al., 2003).

The low stability, low buffering capacity and low cell-specificity have also hindered its clinical applications. However, as a nature resource-based polysaccharide, chitosan has more functional groups that can be chemically modified than other cationic polymers, thus has many more potential chemical derivatives to overcome the deficiencies. Chitosan has been experimentally modified using hydrophilic, hydrophobic, pH-sensitive, thermosensitive and cell-specific ligand groups for enhancement of transfection efficiency (Ishii et al., 2001). The degree of deacetylation (DDA) and the molecular weight (MW) of chitosan or its derivatives, can affect the ultimate transfection efficiency. Most chitosan/DNA complexes are highly deacetylated (above 80%), because chitosan with a high degree of deacetylation exhibits an increased DNA binding efficacy (Kiang et al., 2004). Through chain entanglement, chitosan with a higher MW (longer chain length) can become more readily enmeshed with free DNA, once the initial electrostatic interaction has occurred. But it will also delay the disassociation of chitosan and DNA (Huang et al., 2005). Consequently, low MW chitosan requires a higher charge ratio to stably condense DNA for the same DDA, and a lower DDA requires a higher charge ratio to stably condense DNA at equal MW (Lavertu et al., 2006). The charge ratio for minimum complexation can be determined by agarose gel electrophoresis.
