臺灣博碩士論文加值系統

本研究中將具生物可降解性之疏水性單體聚合製備成星狀疏水性高分子，簡稱starPCL後，以陽離子親水性單體聚合形成星狀兩性共聚物，簡稱starPCL-pDMA。所合成之共聚物產物藉由核磁共振儀 (1H-NMR)以及膠體滲透層析儀 (Gel permeation chromatography, GPC) 分析其物理及化學特性。starPCL-pDMA摻入含有PEG鍊段之兩性共聚物製備形成混摻型奈米微胞後，利用動態光散射分析儀 (Dynamic light scattering, DLS) 以及穿透式電子顯微鏡 (Transmission electron microscope, TEM) 分析其粒徑大小及界面電位。本實驗亦分別針對未混摻及混摻含有PEG之兩共聚物之奈米微胞對於質體DNA之包覆能力、細胞毒性以及細胞轉染效果進行分析比較混摻前後奈米微胞之間的差異。

In this study, amphiphilic star-shaped cationic copolymers were synthesized of hydrophobic monomer and hydrophilic cationic monomer. The synthesized polymer was determined by 1H-NMR spectroscopy and gel permeation chromatography (GPC). The synthesized star-shaped cationic copolymer micelles and hybrid micelles contained PEG are formed by oil-in-water solvent evaporation method. The polyplex binding ability, transfection efficiency and cytotoxicity of micelles are all compared by gel electrophoresis performance, luciferase assay and MTT assay.

第一章 緒論 1
1-1 前言 1
1-2 研究動機及目的 2
第二章 文獻回顧 5
2-1 基因治療 5
2-2 病毒式基因載體 (Viral Gene Vector) 7
2-3 非病毒式基因載體 (Non-Viral Gene Vector) 10
2-4 實驗載體之設計 16
2-4.1 聚甲基丙烯酸-2-二甲基胺基乙酯 (PDMA)之使用 16
2-4.2 疏水性高分子的導入 16
2-4.3 星狀高分子 18
2-4.4 聚乙二醇 (Polyethylene Glycol, PEG) 的混摻 19
第三章 實驗方法 22
3-1 實驗試劑 22
3-2 實驗儀器 24
3-3 實驗步驟 27
3-3.1 星狀共聚物的製備 27
3-3.1.1 starPCL的製備 27
3-3.1.2 starPCL-Br的製備 28
3-3.1.3 starPCL-pDMA的製備 29
3-3.2 starPCL-pDMA與starPCL-pDMA/mPEG-PCL微胞之製備 30
3-3.3 奈米微胞與DNA錯合物之製備 30
3-4 合成材料之分析方法 32
3-4.1 核磁共振光譜儀 (1H-NMR) 的測定 32
3-4.2 膠體滲透層析儀 (GPC) 的測定 32
3-4.3 奈米微胞之粒徑大小與表面電位測定 32
3-4.4 穿透式顯微鏡 (TEM) 33
3-4.5 臨界微胞濃度 (Critical Micelle Concentration, CMC) 之分析 33
3-4.6 奈米微胞錯合物之電泳分析 33
3-5 奈米微粒之體外 (in vitro) 試驗 34
3-5.1 載體之細胞毒性測試 34
3-5.2 載體與DNA錯合物之細胞轉染實驗 (Transfection) 34
3-5.2.1 以螢光DNA (pEGFP-C1) 觀察轉染效能 34

3-5.2.2 以冷光DNA (pGL3-Control) 之RLU (Relative Luciferase Unit)定量轉染效能 35
第四章 結果與討論 37
4-1 材料特性分析 37
4-1.1 1H-NMR測定產物結構及聚合度 37
4-1.2膠體滲透層析儀 (GPC) 分析 40
4-2 奈米微胞之性質分析 43
4-2.1 臨界微胞濃度 (Critical micelle concentration, CMC) 之測定 43
4-2.2 奈米微胞粒徑大小及表面電位測定 44
4-2.3 奈米微胞之TEM影像觀察 47
4-2.4 奈米微胞與DNA錯合物之分析 47
4-2.4.2 微胞與DNA錯合物之TEM影像觀察 52
4-2.4.3 微胞與DNA錯合物之電泳分析 52
4-3 體外實驗(In Vitro) 55
4-3.1 載體之毒性測試 55
4-3.2 載體之基因轉染測試 56
4-3.2.1 無血清環境下螢光DNA(pEGFP-C1)之轉染 56
4-3.2.2 含血清環境下螢光DNA(pEGFP-C1)之轉染 58
4-3.2.3 載體對於冷光DNA(PGL-3)之轉染定量分析 59
第五章 結論 61
參考文獻 63

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