Polyethylene imines（PEI）接枝幾丁聚醣（Chitosan）作為DNA載體之評估

Polyethylene imines（PEI）grafted chitosan as a non-viral gene vector

彭郁翔(Yu-Shiang Peng)

指導教授 ： 王麗芳

高雄醫學大學/生命科學院/醫藥暨應用化學研究所/碩士(2006年)

摘要

近年來利用聚電解質錯合物來作為基因載體已經被廣泛的研究。本研究利用 Polyethylene imines （PEI，MW=600） 化學改質製備 PEI 接枝幾丁聚醣（PEI-g-CHI）作為非病毒型基因載體，期望增加幾丁聚醣（Chitosan，CHI，MW=330K）轉染（transfection）的效能。我們使用新型的方法利用 ethylene glycol diglycidyl ether (EX-810)上的環氧基分別接枝在 PEI 及 CHI 的胺基上，並以 NMR 來鑑定 PEI 接上 CHI 的接枝率。
我們使用 MTT 方法並以人類腎臟細胞（293T cell）為模型來測試細胞毒性；實驗結果顯示 PEI-g-CHI 和 CHI 在低濃度時皆不具有生物毒性，但在高濃度（1mg/ml）時 PEI-g-CHI 具有較低的生物毒性；然而 PEI-g-CHI 和 CHI 的生物毒性皆較 PEI（MW=25K）及 LipofectamineTM2000 低。
之後我們將 PEI、 PEI-g-CHI 及 CHI 與質體 DNA 在不同的N/P（NH3+/PO4－）ratios 下形成複合體後，測量其粒徑大小（Particle size）、界面電位（Zeta potential）及穩定度（Stability efficiency），在粒徑測試結果上其粒子大小分布範圍－PEI/DNA（154-229 nm）、PEI-g-CHI/DNA（197-265 nm）及 CHI/DNA（185-294 nm）； PEI/DNA複合體其平均粒徑小於其他兩個複合體，而 PEI-g-CHI/DNA 複合體的平均粒徑小於 CHI/DNA 複合體；PEI-g-CHI/DNA 複合體的界面電位並沒有隨著不同的N/P ratios而增加。在細胞轉染測試，PEI-g-CHI/DNA 複合體在相同的重量比下，明顯較 CHI/DNA 複合體轉染的效能來的優異。

關鍵字

幾丁聚醣 ； 基因載體 ； 轉染

並列摘要

Polyelectrolyte complexes have been widely studied as gene carriers in recent years. Polyethylene imines (PEI, MW=600) was grafted onto chitosan (PEI-g-CHI) as a non-viral gene carrier in order to improve the inherent transfection efficiency of chitosan (CHI, MW=330K). In this study, we demonstrated a novel method to conjugate the amine groups between CHI and PEI through the epoxide rings of ethylene glycol diglycidyl ether (EX-810). We characterize the degree of substitution of PEI onto CHI by NMR. The cytotoxicity determined by MTT assay showed that the cell viability of PEI-g-CHI is compatible to CHI at the low concentrations using human kidney 293T cells, but a lower cytotoxicity at the high concentration of 1 mg/ml. However, the cytotoxicity of CHI and PEI-g-CHI was much lower than that of PEI (MW=25K) and LipofectamineTM2000. After the formation of polyplexes between PEI-g-CHI and plasmid DNA, we measured the particle size, zeta potential, and stability of the polyplexes at different N/P ratios. The mean particle sizes of PEI/DNA, PEI-g-CHI/DNA, and CHI/DNA were from 154 to 229, 197 to 265, and 185 to 294 nm, varying with N/P ratios, respectively. The mean particle size of the PEI/DNA polyplexes was smaller than that of other two polyplexes. Though the mean particle size of the PEI-g-CHI/DNA polyplexes was smaller than that of the CHI/DNA polyplexes, the zeta potential did not increase in the PEI-g-CHI/DNA polyplexes at different N/P ratios. When compared the transfection efficacy using the same weight ratios of polymers to plasmid DNA, PEI-g-CHI showed a better transfection efficacy than CHI (330K).

並列關鍵字

Chitosan ； Gene vector ； Transfection

參考文獻

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