Non-viral Gene Therapy - Intracellular Delivery: Fundamentals and Applications - page 642

polyplexes, whereas the transfection efficiency of DNA/DOTAP complexes was
decreased. Conjugating tetragalactose to TM-CS conferred the vector with the ability
of specific gene delivery hepatocytes, taking advantage of the receptor-mediated endo-
cytosis (Murata et al. 1997 ). To further increase the transfection activity of TM-CS,
Rojanarata et al. have synthesized a quaternized N-(4-N,N-dimethylaminobenzyl)
chitosan (TM-Bz-CS), in which hydrophobic N,N-dimethylaminobenzyl was intro-
duced. Transfection of pDNA encoding GFP in human hepatoma cell lines (Huh7
cells) showed the higher efficiency in the case of TM-Bz-CS formulated polyplexes,
in comparison to those of TM-CS and CS. The improved gene transfection was due
to the promoted interaction by hydrophobic N,N-dimethylaminobenzyl, and the
increased water solubility by N-quaternization (Rojanarata et al. 2008 ). Unfortunately,
quaternization of CS may increase its cytotoxicity, to some extent. This higher toxicity
of TM-CS could be reduced by PEGylation (Germershaus et al. 2008 ).
CS has been modified by various ligands that enable specific binding to the
receptors of the cells and enhance the transfection efficiency. Galactose, mannose,
transferrin, and folate are frequently used ligands for modification. Kim et al. con-
jugated lactobionic acid bearing galactose group to CS (GA-CS) for hepatocytes
specificity (Kim et al. 2004b ). Cytotoxicity study in HepG2 and HeLa confirmed
the extremely low cytotoxicity of GA-CS. The transfection efficiency of GA-CS/
DNA complexes in HepG2, which has asialoglycoprotein receptors, was higher
than that in HeLa without the receptors. This ligand-mediated increase in the trans-
fection activity of modified CS in target cells has been also observed by other
researchers (Hashimoto et al. 2006 ). Mannose ligand was covalently linked to CS
to synthesize vectors that can target antigen presenting cells (APCs) via receptor-
mediated endocytosis (Kim et al. 2006c ). In addition, transferrin and folated were
conjugated to CS for targeting tumor cells such as HEK293, HeLa and MCF-7 cells
(Mao et al. 2001 ; Lee et al. 2006 ).
To improve the buffering capacity and interactions with cell surfaces, and reduce the
aggregation of CS based polyplexes, CS has been modified with various hydropho-
bic moieties including deoxycholic acid, stearic acid, and alkyl chains. Lee et al.
synthesized deoxycholic acid modified CS (DA-CS) by an EDC-mediated coupling
reaction (Lee et al. 1998 ). DA-CS could self-assemble into nanoparticles of
162 nm, which could efficiently mediate DNA transfection in COS-1 cells. Precise
control over the size and structure of DA-CS nano-assemblies could be imple-
mented by depolymerizing CS with subsequent DA modification (Kim et al.
2001b ). The transfection efficiency of these nano-structured assemblies in COS-1
cells was significantly influenced by their size and structure. Compared with the
unmodified CS, the transfection activity of the stearic acid and alkyl modified CS
was strongly enhanced (Hu et al. 2006 ; Liu et al. 2003 ).

3.7.2 Dextran Derivatives

Due to its well-demonstrated safety in vivo , chemically modified dextrans have
been broadly investigated for drug and gene delivery. For instance, diethylamino-
ethyl-dextran (DEAE-dextran) has been a commonly used gene vector for decades