参聚離子型基因載體之研發及其在基因傳遞上之應用

A ternary polyion complex vector system for gene transfer based on Branched-Poly(ethylenimine) & Poly(2-ethyl-2-oxazoline)- Poly(methacrylic acid)

宋岳哲(HASH(0x1aca2cc0))

指導教授 ： 薛敬和

國立清華大學/工學院/化學工程學系/碩士(2008年)

摘要

本研究設計一具備複合型結構之高分子基因載體，利用具生物相容性之PEOz (poly(2-ethyl-2-oxazoline)與於中性環境下帶負電性之PMAA (polymethacrylic acid)合成雙團聯共聚物PEOz-b-PMAA,(poly (2-ethyl-2-oxazoline)-block-poly(methacrylic acid))，再進一步藉由電性吸引方式與表面帶正電性之Branched-Poly(ethylenimine)/ DNA (B-PEI/DNA)聚複合體前驅物結合，形成具有複合結構的非病毒型基因載體。親水性高分子PEOz裸露於載體外殼，以提高載體於血液循環中之安定性，降低載體本身之細胞毒性，並利用內層B-PEI之高轉染效率來達到改善基因傳輸之效率。
本研究首先分析不同重量比B-PEI/DNA之粒徑大小與界面電位，從中選取粒徑穩定同時表面具正電之微胞，再與PEOz-PMAA藉由電性相吸方式形成新型態之複合體。研究結果顯示B-PEI/DNA微胞之粒徑約在150nm左右，界面電位在30~35 mV，而加入PEOz-PMAA後之複合型微胞粒徑則在200~250nm間，且其界面電位則在0∼10mV之間，顯見PEOz可有效遮蔽B-PEI之正電性。在材料之細胞毒性測試實驗中，PEOz-PMAA相較於B-PEI具有相當低的細胞毒性，當B-PEI以濃度100μg/ml與細胞培養1天後，細胞存活率趨近於0，而同濃度下PEOz-PMAA則有80%以上存活率的表現。而在微胞之細胞毒性測試實驗中，比較B-PEI/DNA之複合體微胞與外層附加PEOz-PMAA之多層結構微胞兩者之細胞存活率，結果發現在同樣條件下，複合結構微胞可顯著的改善B-PEI/DNA微胞之細胞毒性。
此外複合體型態的觀察中，我們利用TEM影像比較B-PEI/DNA微胞與多層結構微胞，結果發現多層複合結構微胞具有相當明顯之殼核結構，並在pH 5之環境下觀測得質體之釋放。而比較各式複合型微胞之轉染效果，實驗結果顯示複合結構微胞之轉染效果相較於B-PEI/DNA雖略幅下降，然而仍具備相當優秀之轉染效率。研究並進一步藉由共軛焦顯微鏡CLSM同時觀察B-PEI/DNA與複合結構微胞在不同時間下細胞吞噬的情形，結果發現複合型微胞於3hr時開始有微量累積於細胞內，並於6hr後有明顯累積。
整體而言，利用PEOz-PMAA與B-PEI/DNA所結合之複合結構微胞可大幅提升B-PEI/DNA聚複合體之生物相容性，且表現出相當程度的轉染與細胞吞噬效率，因此為一具有相當潛力的非病毒型基因載體。

關鍵字

基因載體 ； 聚乙烯亞胺 ； 三聚離子型複合體 ； 陽離子型高分子 ； 聚(N-丙基乙烯亞胺) ； 聚甲基丙烯酸

並列摘要

The purpose of this study was to prepare polymeric gene carriers based on PEOz-b-PMAA, (poly(2-ethyl-oxazoline)- block-poly -methacrylic acid) and Branched-Poly (ethylenimine), (B-PEI). PMAA were partially dissociation under neuteral pH and were with negative charge, so that they could attach to the positive charge surface of B-PEI/DNA polyplex. The newly formed polyplex were biocompatible by the hydrophilic segment, PEOz and could enhance the stability of the polyplex in vivo.
We prepared B-PEI/DNA polyplex by varying weight ratio of B-PEI/DNA, till the polyplex were provide with stable particle size and highly positive charge. And the B-PEI/DNA polyplex were then formed a bilayer structure polyplex with PEOz-b-PMAA. The experiment results showed that the particle size of B-PEI/DNA polyplex were about 150 nm, and the bilayer polyplex were about 200~250nm. While B-PEI/DNA polyplex carried posive surface charge with zeta potential of 30mV, the PEOz-b-PMAA could covered up the positive charge of B-PEI when bilayer structure polyplex were prepared. Data of material cytotoxicity showed that B-PEI revealed extremely toxicity to Hela cells under 100μg/ml concentration, and the cells viability approached 0, and PEOz-b-PMAA showed 80% viability under the same conceration. Then We compared the viability of B-PEI/DNA and PEOz-b-PMAA /B-PEI/DNA, and the later kept higher viability then the former .
TEM observation showed that PEOz-PMAA/B-PEI/DNA polyplex formed core-shell structure, and the polyplex in pH 5 would collapse and release plasmid.The results of transfection effects indicated that the bilayer structure reduce the efficiency slightly, and RLU/unit weight of the bilayer gene carrier were about 1/10~1/100 of B-PEI/DNA polyplex. By CLSM observation, we observed celluptake of B-PEI/DNA polyplex and bilayer structure polyplex, the result revealed that the bilayer structure polyplex started to accumulate after 3hrs incubation and accumulated a significant amount after 6hrs.
In conclusion, the polyplex formed with PEOz-PMAA covered on B-PEI/DNA could improved the cytotoxicity of B-PEI/DNA. The newly ternary polyplex presented a well transfection efficiency and cell uptake efficiency, so the ternary polyplex constitute a useful approach for further design of gene carriers.

並列關鍵字

gene carrier ； Polyethyleneimine ； ternary polyanion ； cation polymer ； poly(2-ethyl-oxazoline) ； polymethacrylic acid

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