Characterization of Sacchachitosan and Comparison of Gene Transfection Efficiency with Various Sources of Chitosan

• Main Author: 劉慧如
• Other Authors: Ming-Thau Hsu
• Format: Others
• Language: zh-TW
• Published: 2002
• Online Access: http://ndltd.ncl.edu.tw/handle/86730604497051748943

碩士 === 台北醫學院 === 生物醫學材料研究所碩士班 === 90 === Several methods for nonviral gene therapy are being developed based on principles of advanced drug delivery, and development of an efficient method for introducing a therapeutic gene into target cells in vivo is the key issue in treating genetic and acquired disease by the gene therapy. With regard to nonviral vectors, major attention is paid to cationic polymers which are able to condense large DNA into smaller structures as well as to mask the negative DNA charges, necessary for transfecting cells. Chitosan, a natural polysaccharide which is partially deacetylated glucosamine, is being widely used as a pharmaceutical excipient so far as to apply on gene delivery system. Commercial chitosan all are obtained from crustacean shells. In view of mimic main structure, a new biomaterial for gene delivery vector, sacchachitosan, which is purified from Ganoderma cell wall being abundant of chitin, was examined. Following the purification processes, the most products were able to be manufactured under deacetylation condition in 50 % NaOH solution with heating at 130°C for 2h (yield about 13.6 %). Structural analysis by TLC revealed that these products mainly contained the same structural units of N-acetylglucosamine and glucosamine as commercial products. Observation from electrophoresis, sacchachitosan condensed in a less extent with DNA (< chitosan:DNA 1:1) than that with commercial chitosan (> chitosan:DNA 1:4). Co-culturing chitosan-DNA complexes with HeLa cells, autofluorescence performance through fluorescent microscopy was observed indicating transfection was achieved. According to the FASCan measurement, the chitosan-DNA complexes with different brands of commercial chitosan showed apparent transfection efficiency, whereas sacchachitosan-DNA complexes showed less significant but still detectable expression of transfection.