陽離子型聚酯材料合成、改質及基因傳輸之研究

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標題:	陽離子型聚酯材料合成、改質及基因傳輸之研究 Synthesis and Modification of Cationic Polyester and Studies on Gene Delivery
作者:	張凱翔 Kui-Hsiang Chang
貢獻者:	蕭明達嘉南藥理科技大學：生物科技研究所
關鍵字:	細胞毒性緩衝能力自組裝共聚合聚摻合可降解性聚胺酯顆粒大小基因傳輸載體轉染 buffering capacities cytotoxicity self-assembled polyblend transfection gene delivery vector degradable poly(β-ester amine)s particle size
日期:	2006
上傳時間:	2008-11-24 17:01:48 (UTC+8)
摘要:	成左滌穧]治療都依賴傳遞載體系統，一個良好的基因載體有二個最重要的條件有效的轉染和無細胞毒性。本研究主要目的在發展新的質體DNA載體系統，新的水解性聚胺酯並評估其個種性質。這些聚酯由雙烯化合物單體和帶有胺基的單體經由Michael-type共軛加成反應，以高產率獲得。PE-AI顯示能有效縮合質體DNA形成奈米大小 (~86-192nm)並帶有正電荷的複合體。在質量比7/1(高分子/DNA)時PE-DP能結合DNA，形成大約1000奈米的大小，且帶正電的複合體，但無法符合細胞內胞飲作用的大小。為了提升陽離子型聚酯之轉染效率和粒徑大小，我們採用共聚合和聚摻合二個方法改善其結構特性。 (一)、我們合成了聚(胺基甲酸酯-酯) (PUE)，此共聚合物含有胺基甲酸酯及酯的官能基於主鏈，在側鏈則含有三級胺。由動力光學散射儀測定，發現此種陽離子型高分子PUE可與DNA形成奈米級的複合體，粒徑大小為80-115 nm且帶正電(和DNA之重量比分別為2/1 (polymer/DNA))。水解實驗中可得到PUE水解半生期為20小時，從緩衝實驗可得知此高分子具有不錯的緩衝效率。最後利用COS-7細胞株做毒性測試並與PEI做比較，得到較低毒性的結果。 (二)、聚酯衍生物 (PE-DP20)和短鍵PEI (分子量800)之新陽離子型高分子摻合作為基因載體，以50/1、40/1、30/1、20/1、10/1、5/1 和1/1高分子摻合組成比例製備和研究，使用動力光學散射儀(DLS)和酸鹼儀研究陽離子型高分子摻合物／DNA複合體特性，由DLS結果顯示當進一步的增加聚合體的濃度時顆粒的大小也隨之等比例的下降，粒徑大小的確與顆粒內的基質組成有相關性。二種方法均都能幫助聚胺酯與DNA自組裝成奈米大小複合體，聚合物/DNA複合體直徑少於200奈米以下，並比一般使用於DNA高分子載體PEI，更有效的減少細胞毒性在試驗中。 The success of gene therapy is largely dependent on the delivery vector system. Efficient transfection and nontoxicity are two of the most important requirements of an ideal gene delivery vector. The main objective of the present work was the development of new carrier systems for the delivery of plasmid DNA. Novel degradable poly(β-ester amine)s were evaluated as variety properties. These polyesters were obtained in high yields through a Michael-type conjugate addition of diacrylate monomers with amine monomers. Among them, PE-AI was shown to effectively condense plasmid DNA into nano-sized (~86-192nm) and positively charged complexes. PE-DP were able to combine with DNA and yield complexes with positive charge of size around 1000 nm at a mass ratio of 7/1 (polymer/DNA), without the size required for cellular endocytosis. To improve the transfection efficiency and particle size of cationic poly(β-ester amine)s, we need the following two distinguished methods, copolymerization and polyblend, to modify the structural characteristics of polymer. (A) We synthesized poly (urethane-co-ester) (PUE) bearing urethane and ester linkages in the backbone and tertiary amines in the side chain. Poly (urethane-co-ester) (PUE), readily self-assembled with the plasmid DNA (ρCMV-βgal) in HEPES buffer, were characterized by dynamic light scattering, zeta-potential. The results revealed that PUE was able to combine with DNA and yield complexes with positive charge of size around 80-115 nm at a mass ratio (W/W) of 2/1. The degradation studies indicated that the half-life of PUE in HEPES buffer is 20 hours. Titration studies were performed to determine the buffering capacities of the polymers. In addition, the COS-7 cell viabilities in the presences of PUE and PEI were studies. (B) A new cationic polymer was prepared through the blend of PE-DP20 and short chain PEI (Mw 800) and investigated in solution at 50/1, 40/1, 30/1, 20/1，10/1，5/1 and 1/1 polymer blend compositions. The characteristics of cationic polyblend/DNA complexes have been investigated using dynamic light scattering (DLS) and pH meter. The DLS results show that with the further increase of polymer mass content, the particle size of complexes proportionally decreased. In addition, the particle size was dependent on the composition of the paricle matrix. Two methods readily helped poly(β-ester amine)s readily self-assembled with plasmid DNA into nano-sized polymer/DNA complexes less than 200 nm diameter and significantly less cytotoxicity than commonly used DNA delivery polymer, poly(ethylene imine)(PEI) in vitro.
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