合成分支型聚乙烯亞胺衍生物之基因傳送載體以促進質子海綿效應與增加細胞攝取基因之量

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姓名

許意悅(Yi-yeuh Hsu) 查詢紙本館藏

畢業系所

化學工程與材料工程學系

論文名稱

合成分支型聚乙烯亞胺衍生物之基因傳送載體以促進質子海綿效應與增加細胞攝取基因之量
(Implication of Proton Sponge Effect and Cellular Uptake of gene on Gene Delivery by Branched Polyethylenimine Derivatives)

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摘要(中)

分支型聚乙烯亞胺(branched polyethylenimine，b-PEI)是由一、二、三級胺以比例1:1.3:1組成的聚陽離子型高分子，其常用來當作基因載體。由於b-PEI的三級胺之pKa等於6-7，所以當基因與載體以胞飲(endocytosis)作用進到核內體(endosome)後，會因三級胺的質子化，使得基因更容易逃脫核內體，以利於基因轉染程序的進行，這種效應即為質子海綿效應(proton sponge effect)。而b-PEI雖有高基因轉染效率，但高細胞毒性也限制了b-PEI在臨床的應用，由於一級胺是造成細胞毒性的主因，所以我們利用麥可加成(Michael addition)反應，將具有三級胺的N-[(3-二甲氨基)丙基]丙烯酰胺(N-[3-(Dimethylamino)propyl]acrylamide)接枝於b-PEI的一級胺上成為P-D，希望P-D能增進proton sponge effect且同時降低細胞毒性。除此之外，因四級胺不會增進proton sponge effect，所以我們也接枝帶有四級胺的(3-丙烯酰氨丙基)三甲基氯化铵((3-Acrylamidopropyl)trimethylammonium Chloride)於PEI上形成P-T，進而與P-D比較。結果顯示利用P-D與P-T的載體傳送帶有冷光酵素的基因質體進入HEK-293T細胞後，細胞毒性都比b-PEI低，但轉染效率並未高於b-PEI，這是因為P-D有增進proton sponge effect的效果，但基因胞飲量卻低於PEI和P-T。而當我們以1:1的比例混合的P-D/P-T當作載體時，結果發現細胞毒性降低且轉染效率略高於b-PEI，這意味著增加含有三級胺的P-D可以藉由proton sponge effect幫助基因逃脫endosome，而P-T的添加可以增進基因進入細胞的量，這結果對於設計更佳的PEI衍生物而言是一重大發現。

摘要(英)

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.

關鍵字(中)

★ 基因傳遞
★ 基因轉染
★ 聚乙烯亞胺
★ 海綿質子效應
★ 逃脫核內體

關鍵字(英)

★ gene delivery
★ gene transfection
★ polyethylenimine
★ proton sponge effect
★ endosomal escape

論文目次

中文摘要......................................................................................................................... i
英文摘要........................................................................................................................ ii
誌謝.............................................................................................................................. iii
目錄............................................................................................................................... iv
圖目錄.......................................................................................................................... vii
表目錄........................................................................................................................... ix
第一章緒論........................................................................................................ 1
1.1 研究動機................................................................................................ 1
1.2 研究目的................................................................................................ 2
第二章文獻回顧................................................................................................ 3
2.1 基因治療................................................................................................ 3
2.1.1 何謂基因........................................................................................ 3
2.1.2 如何基因治療................................................................................ 3
2.1.3 基因治療之案例與風險................................................................ 4
2.2 基因傳遞................................................................................................ 5
2.2.1 載體進入細胞之途徑.................................................................... 5
2.2.1.1 內吞作用................................................................................ 5
2.2.1.2 直接穿膜................................................................................ 6
2.2.2 基因載體........................................................................................ 7
2.2.2.1 病毒型載體............................................................................ 7
2.2.2.2 非病毒型載體........................................................................ 7
2.3 PEI及其衍生物進行基因傳遞........................................................... 10
2.3.1 結構與功能之關係...................................................................... 10
2.3.2 基因傳遞機制.............................................................................. 13
2.3.3 PEI作為載體的缺點................................................................... 17
2.3.4 PEI作為載體的運用................................................................... 17
第三章實驗藥品、設備及實驗方法.............................................................. 18
3.1 實驗藥品.............................................................................................. 18
3.1.1 質體 DNA ...................................................................... 18
3.1.2 細胞................................................................................. 18
3.1.3 藥品................................................................................. 18
3.2 實驗設備.............................................................................................. 20
3.3 實驗方法.............................................................................................. 20
3.3.1 b-PEI衍生物之合成 ................................................................... 20
3.3.2 複合體之製備.............................................................................. 21
3.3.3 基因載體與複合體之粒徑大小與表面電位.............................. 21
3.3.4 酸鹼滴定實驗.............................................................................. 21
3.3.5 溴化乙錠嵌入檢測法.................................................................. 21
3.3.6 膠體電泳之檢測.......................................................................... 22
3.3.7 細胞培養...................................................................................... 22
3.3.8 基因轉染...................................................................................... 23
3.3.9 流式細胞術.................................................................................. 24
第四章比較不同PEI衍生物載體對基因轉染效率的影響.......................... 26
4.1 PEI及其衍生物之物性與化性的分析............................................... 27
4.1.1 PEI衍生物之合成與鑑定........................................................... 27
4.1.2 PEI衍生物之粒徑大小與表面電位........................................... 31
4.1.3 PEI衍生物之緩衝能力............................................................... 33
4.1.4 基因載體之粒徑大小與表面電荷結果分析.............................. 34
4.1.5 基因載體與質體DNA之包覆穩定性分析 ............................... 37
4.1.6 基因載體與質體DNA之結合能力分析 ................................... 39
4.2 不同基因載體對基因傳遞機制的影響.............................................. 40
4.2.1 不同基因載體對細胞攝取量的影響.......................................... 40
4.2.2 不同基因載體對逃脫內體的影響.............................................. 42
4.3 基因載體與細胞活性、轉染效率的關係.......................................... 43
4.3.1 單一種b-PEI衍生物作為載體.................................................. 43
4.3.2 混合不同b-PEI衍生物作為載體.............................................. 45
第五章結論...................................................................................................... 48
參考文獻...................................................................................................................... 49

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指導教授

胡威文(Wei-wen Hu)

審核日期

2014-8-6

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