Delivery of oligonucleotide to specific cells and maintenance of its biological function are important for nucleic acid therapy. The objective of this paper is to demonstrate that galactosylated low molecular weight chitosan (gal-LMWC) is a safe and effective vector of antisense oligonucleotide (ASO) and plasmid DNA for the hepatocyte targeting delivery. Gal-LMWC has been successfully prepared and MTT cytotoxic assay shows that cytotoxicity of gal-LMWC is lower than that of high molecular weight chitosan (HMWC) and low molecular weight chitosan (LMWC) in HepG2 cells. Using a complex coacervation process, gal-LMWC can form stable nano-complexes with plasmid DNA or with ASO by the electrostatic interaction. The morphometrics, particle size, and the zeta potential of gal-LMWC/ASO complexes and gal-LMWC/plasmid DNA complexes are very similar. The transfection efficiency by using gal-LMWC vector is significantly higher than that of naked DNA or naked ASO in HepG2 cells. Transfection efficiency of gal-LMWC/ASO complexes and gal-LMWC/plasmid DNA complexes depends on the molar ratio of the positive chitosan amino group and the negative DNA phosphate group (N/P ratio) strongly. Inhibition experiments confirm that the enhanced transfection efficiency is due to the ASGR mediated endocytosis of the gal-LMWC/ASO complexes or gal-LMWC/DNA complexes. These results suggest that gal-LMWC can be used in gene therapy to improve the transfection efficiency in vitro and in vivo.

Cartilage defects as a result of disease or injury have a very limited ability to heal spontaneously. Recently, tissue engineering and local therapeutic gene delivery systems have been paid much attention in the cartilage natural healing process. Gene-activated matrix (GAM) blends these two strategies, serving as local bioreactor with therapeutic agents expression and also providing a structural template to fill the lesion defects for cell adhesion, proliferation and synthesis of extracellular matrix (ECM). In the current study, we used chitosangelatin complex as biomaterials to fabricate three-dimensional scaffolds and plasmid DNA were entrapped in the scaffolds encoding transforming growth factor-β1 (TGF-β1), which has been proposed as a promoter of cartilage regeneration for its effect on the synthesis of matrix molecules and cell proliferation. The plasmid DNA incorporated in the scaffolds showed a burst release in the first week and a sustained release for the other 2 weeks. The gene transfectd into chondrocytes expresses TGF-β1 protein stably in 3 weeks. The histological and immunohistochemical results confirmed that the primary chondrocytes cultured into the chitosan-gelatin scaffold maintained round and owned characters of high secretion of specific ECM. From this study, it can be concluded that this gene-activated chitosan-gelatins matrix has a potential in the application of cartilage defects regeneration.

Surface functionalization of biodegradable poly-l-lactic acid(PLLA) was achievedby plasma coupling reaction of chitosan. The structure of modified PLLA surfaces was characterized by contact angle measurements and X-ray photoelectron spectroscopy. Two cell lines, L929 (mouse fibroblasts) andL02 (human hepatocytes), were cultured on the modified PLLA surface. It was found that cells cultured on this film could hardly spread and tend to become round, and the film was demonstrated to be a poorly adhering substrate. However, cells grown on this substrate can proliferate at almost the same speed as cultured on a glass surface. These results suggest that the new substrate can be used to control the morphology of cells, and has potential applications in tissue engineering. It may be helpful in understanding the mechanism of the switch between cell phases of growth and differentiation, which is necessary for the design of tissue regeneration biomaterials.

Chitosan has the potential for DNA complexation and is useful as a non-viral vector for gene delivery. Highly purified low molecular weight chitosan (LMWC) was prepared. Lactobionic acid (LA) bearing galactose group was coupled with LMWC for liver-specificity. A series of galactosylated-LMWC (gal-LMWC) samples covering a range of galactose group contents were prepared. The chitosan/DNA complexes were obtained using a complex coacervation process. Gal-LMWCs were used to transfer pSV-_-galactosidase reporter gene into human hepatocellular carcinoma cell (HepG2), L-02, SMMC-7721, and human cervix adenocarcinoma cell line (HeLa) cell lines in vitro. Transfection efficiency of gal-LMWCs was evaluated by _-galactosidase assay and compared with those of lipofectin, calcium phosphate (CaP), high molecular weigh chitosan (HMWC) and LMWC. Gal-LMWC/DNA complex shows a very efficient cell selective transfection to hepatocyte. The transfection efficiency of gal-LMWCs increased with the improvement of the galactosylation degree. Cytotoxicity of gal-LMWCwas determined by 3-(4,5-dimethylthiazd-2-yl)-2,5-diphenyltentrazolium bromide (MTT) assay and the results show that the modified chitosan has relatively low cytotoxicity, giving the evidence that the modified chitosan vector has the potential to be used as a safe gene-delivery system.

To study the electrostatic interaction between two ionic polymer grafted surfaces in aqueous media, an atomic force microscopic tip surface was modified by graft polymerization of a cationic monomer, dimethylaminoethyl methacrylate (DMAEMA), after being coated with a thin layer of cyanoacrylate polymer. A poly(ethylene terephthalate) (PET) film which was the countersurface of the modified tip for the measurement of atomic force microscopy was also modified by surface graft polymerization of DMAEMA and an anionic monomer, acrylic acid (AAc) . An appreciable adhesive force was observed in water between the DMAEMA-grafted tip and the AAc grafted PET surface, while a repulsive interaction was noticed between the DMAEMA-grafted tip and the DMAEMA-grafted PET film. Addition of KCl to the medium for the atomic force measurement exhibited a remarkable reduction in the adhesive interaction. These interactions were attributed to the Coulombic force between the grafted ionic polymer chains.