琥珀酐酸改質幾丁聚醣以增加水溶性及轉染效能

Succinated Chitosan as Gene Carrier for Improved Chitosan Solubility and Gene Transfection

杜凱雯(Elsie Toh @ Toh Khai Woon)

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(2009年)

摘要

幾丁聚醣 (Chitosan, CHI) 優異的生物相容性、生物可降解性、生物活性以及低生物毒性，為其展開了在保健食品、醫藥用品、食品加工、化妝品、紡織、環保、農業、化學工業、生物醫學材料等各方面的應用。因此在本研究中，我們利用NaBO3製備出低毒性、且被證實具有基因轉染潛力的低分子量幾丁聚醣，並將其與琥珀酐酸 (Succinic Anhydride, succ) 反應得到水溶性較好的CHI-succ作為基因載體。

由於幾丁聚醣結構上1° amine的pKa = 6.5，在酸性環境下可經由amine的質子化 (Protonation) 而帶有正電荷，因此可與帶有負電荷的DNA透過靜電作用力結合形成奈米複合體 (Polyplex)。由1H-NMR圖譜中得知幾丁聚醣的去乙醯化程度 (Degree of Deacetylation, D.D %) 約為 86~89 %；而在CHI:succ不同重量比下succ的取代度 (Degree of Substitution, D.S %) 分別為5 %、10 % 及20 % 。在IR分析和X-光繞射光譜儀方面，經succ取代後可觀察到COOH及Amide的形成；CHI的晶格特性峰則隨succ取代度增加而減弱。此外，由酸鹼滴定結果顯示succ的修飾並不會改變CHI的緩衝能力。

隨後我們利用不同的重量比 (weight ratio, w/w) 製備CHI-succ / DNA複合體與CHI / DNA複合體做比較，進行電泳膠片、粒徑大小、介面電位 (ζ-Potential) 以及穿透式電子顯微鏡 (TEM) 分析複合體所包覆質體DNA的效能。從實驗數據顯示在w/w = 20時DNA可較妥善地被包覆，且所有複合體的粒徑大小與介面電位約為110~140 nm及± 1~5 mV。在細胞實驗中，所有測試的複合體都不具有嚴重的細胞毒性，而轉染效果則以CHI-succ 5% 及CHI-succ 10% 的基因表現較佳，顯示我們成功地改善CHI與DNA包覆能力過於緻密的問題。

關鍵字

幾丁聚醣 ； 琥珀酐酸 ； 基因治療 ； 基因傳遞 ； 基因轉染

並列摘要

Chitosan, CHI, is a natural cationic polysaccharide with good biocompatibility, biodegradability, bioactivity, low cytotoxicity and non- allergenicity. These properties make CHI a good candidate for multiple applications in industries such as a food additive agent or a fat blocker, cosmetics, textiles, agriculture, chemical additives, biomedical devices, biomedicine and biomaterials. Recently, CHI is also reported for its potential as both drug and gene carrier. Therefore, in developing a novel gene carrier, three CHI-succ(s) of different degrees of substitution were synthesized from a low molecular weight CHI prepared from the hydrolysis by NaBO3.

CHI can be protonated in acidic condition (pH < 7) because its amino group has a pKa value of 6.5. This character facilitates CHI to form a complex with anionic DNA through electrostatic interactions, which is named as polyplex. From 1H-NMR spectrum, the degrees of deacetylation (D.D %) of hydrolyzed CHI(s) were around 86~89% while the degrees of substitution (D.S %) of succinic anhydride in three CHI-succ(s) were 5 %, 10 % and 20 %. After the substitution of succinic anhydrides, both COOH and amide absorption peak were observed in the IR spectra. The degree of crystallinity structure of CHI was decreased with increased succ substitution but the buffering capacity and pKa values were similar to CHI.

Next, various weight ratios (w/w) of CHI / DNA and CHI-succ / DNA polyplexes were prepared for gel retardation, particle size, ζ-potential and TEM analysis. Results showed that the plasmid DNA is readily entrapped at w/w = 20, and the sizes and ζ-potentials were around 110~140 nm and ± 1~5 mV. Finally in the cellular studies, all polymers and polyplexes exhibited low cytotoxity in HEK 293T cell lines. Both CHI-succ 5% and CHI-succ 10% showed improved transfection efficiency compared to unmodified CHI, indicating a looser but proper binding ability for CHI-based nucleic acid delivery system.

並列關鍵字

Chitosan ； Succinic Anhydride ； Gene Therapy ； Gene Delivery ； Gene Transfection

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