碩士 / 國立中央大學 / 化學工程與材料工程學系 / 102 / Gene carrier design is essentially critical on the gene therapy. One of efficient gene carriers for gene delivery is branched polyethylenimine (B-PEI) with molecular weight of 25 KDa. B-PEI with higher buffering capacity facilitates the gene escape from endosome, this is “proton sponge” hypothesis. The containing of tertiary amine in B-PEI plays an important role on higher transfection efficiency. Unfortunately, the primary amine of B-PEI has been examined to cause the necrosis of cell, and the cytotoxicity limits its clinical application. In this study, we synthesized a series of B-PEI derivatives with various content of tertiary amine to correlate the proton sponge efficiency to gene transfection efficiency. Through the Michael addition reaction, the primary amine would be modified to secondary amine. The complexes by mixing different ratios of B-PEI derivatives and DNA plasmid were incubated with the HEK-293T cell for 4 hr. After two days, the gene transfection efficiencies of B-PEI derivatives were determined by luciferase assay and BCA protein assay. Besides, the pH sensitive fluorophore-labeled DNA was used to determine the proton sponge efficiency of B-PEI derivatives. The results showed that the gene transfection efficiency is not directly related to the proton sponge efficiency. We should consider both the internalization of complexes and proton sponge efficiency for gene transfection. Based on our finding, we determined a window to tune the complex composition to optimize the transfection efficiency of B-PEI derivatives. Consequently, the complex with higher transfection efficiency and lower cytotoxicity can be approached from this investigation.
| Identifer | oai:union.ndltd.org:TW/102NCU05063093 |
| Date | January 2014 |
| Creators | Yi-yeuh Hsu, 許意悅 |
| Contributors | Wei-wen Hu, 胡威文 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 65 |
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