本研究目標為開發兼具基因治療與診斷功能之奈米複合材料，研究內容為開發兩種不同設計之高分子/超順磁氧化鐵奈米粒子 (polymer/SPION) 載體，並研究其作為磁性導引基因傳遞的效果。第一部分的研究為將生物可降解陽離子聚合物 (SPEI) 以逐層修飾的方法包覆於SPION表面，所得SPEI/SPION粒徑分布約150nm，接著利用靜電作用力將質體DNA (plasmid DNA, pDNA) 包覆於其外，形成SPEI/SPION/pDNA基因載體，由核酸膠體電泳分析結果可知此奈米載體對pDNA有良好的保護效果，而普魯士藍染色結果初步判定SPEI/SPION可被細胞攝入，體外細胞基因傳遞結果證實SPEI/SPION在HEK-293T細胞與MCF7細胞中分別可達到90%與50%的轉染效率，值得一提的是在此載體系統於含有血清的狀況仍保有良好基因傳遞效果以及低細胞毒性，此外，外加磁場可更進一步提升SPEI/SPION/pDNA的基因轉染效率。第二部分的研究中，我們以共沉澱法合成的水溶性SPION，於其表面鍵結小分子化合物 (2-(pyridyldithio)-ethylamine, PDS)，以獲得SPION-PDS，並利用傅立葉轉換光譜儀的圖譜確認PDS官能團。此載體與含有硫醇基的聚乙烯亞胺(PEI800)鍵結後，PEI的親水性質使SPION-ss-PEI800在水中的維持良好的分散性，其平均粒徑約為150nm，並具有高密度正電性，使之可作為攜帶核酸分子的載體，此部分研究中我們選擇以小片段干擾RNA (small interfering RNA, siRNA) 進行基因傳遞，針對細胞內特定基因表現進行抑制，細胞實驗結果已證實可有效針對綠色螢光蛋白 (eGFP) 與血管內皮新生因子 (VEGF) 兩種基因表現進行抑制，經由ELISA方式分析細胞分泌出的VEGF含量，證實以SPION-ss-PEI800 /VEGF siRNA可有效抑制細胞VEGF的表現，並於轉染後的24小時達最大抑制率 (75%)，隨後持續回升，但在42小時仍可達50% 的抑制率；相對而言，控制組 (scramble siRNA) 於轉染後24及42小時，其VEGF表現量僅分別減少14%與8%，此結果證實SPION-ss-PEI800/VEGF siRNA可專一性地針對VEGF造成基因沉默，且其具有可忽略的細胞毒性 (細胞存活率達100%)。綜合以上所述，本研究所獲得之兩種載體皆於基因治療上極具潛力，因此未來研究除了以此載體應用進行動物實驗外，也將嘗試鍵結其它含硫醇基的生物活性分子，進一步開發其於生物醫學應用的潛力。

In this study, two new types of theranostic polymer/superparamagnetic iron oxide nanoparticle (SPION) nanocomplexes were designed and studied for their gene delivery capability. In the first part of this study, SPION was synthesized by thermal decomposition and used to prepare SPEI/SPION (~150nm in diameter) via layer-by-layer deposition process. Via the electrostatic interactions, SPEI/SPION complexed with plasmid DNA (pDNA) and protected it from DNAse degradation, as analyzed using gel electrophoresis. Efficient cellular uptake of SPEI/SPION/pDNA was confirmed by prussian blue staining of SPION. Efficient in vitro transfection efficiency of SPEI/SPION/pDNA was observed using fluorescent reported gene on two mammalian cell lines. The transfection efficiency (72 hr post-transfection) was up to 90% and 50% in 293T and MCF7 cells respectively under serum-free transfection condition. It is worth of mentioning that SPEI/SPION/pDNA maintained efficient gene delivery capability even in serum-containing transfection condition. Additionally, external magnetic field was found to further promote the gene delivery efficiency. In second part of this study, water-soluble SPION was synthesized by co-precipitation method and subsequently conjugated with pyridyl disulfide-containing linker (2-(pyridyldithio)-ethylamine hydrochloride, PDS). The presence of PDS in the resultant hydrophobic SPION-PDS was confirmed by FTIR spectrum. SPION-ss-PEI800 was then prepared by PEI800-SH conjugation onto the surface of SPION-PDS. The as-synthesized SPION-ss-PEI800 possess small size (150 nm in diameter), well dispersion in water and capable of effectively interacting with small interfering RNA (siRNA). The in vitro transfection results show that SPION-ss-PEI800 could deliver siRNA into cytoplasm and successfully knock down the expression of enhanced green fluorescent protein (eGFP) or vascular endothelial cell growth factor (VEGF) gene. 24 hr after transfection, the most significant VEGF gene silencing was observed (74.5% knock down) using ELISA assay. 42 hr after transfection, the level of VEGF remained low (50.0% knock down). In contrast, the gene silencing effect by the control treatment (scramble siRNA) was not significant (14% and 8% VEGF knockdown for 24 or 42 hr post-transfection respectively) as expected. The results verified the specificity of VEGF gene knockdown by SPION-ss-PEI800/siRNA. In the future, we will explore the potential of using SPION-PDS as a universal carrier system for thiol-containing molecules conjugation and the biomedical applications.

摘要...................................................1
英文摘要................................................3
目錄...................................................4
圖索引.................................................9
表索引.................................................11
第一章、緒論
1.1 前言...............................................12
1.2 實驗動機與實驗簡述...................................12

第二章、文獻回顧
2.1基因治療.............................................17
2.1.1質體 (plasmid DNA, pDNA........................19
2.1.2小片段干擾RNA (small interfering RNA, siRNA)....20
2.2基因載體.............................................21
2.2.1病毒載體........................................22
2.2.2 非病毒載體.....................................22
2.3聚乙烯亞胺簡介 (Polyethylenimine, PEI)................23
2.4超順磁性氧化鐵奈米粒子 (Superparamagnetic iron oxide, SPION)................................................24
2.5 2-(2-吡啶基二硫基)乙胺鹽酸鹽 (2-(pyridyldithio)-ethylamine hydrochloride).........................................26
2.6血管內皮新生因子 (VEGF)................................26

第三章、實驗方法與材料
3.1實驗材料.............................................28
3.2實驗材料製備與分析方法
3.2.1熱裂解氧化鐵奈米粒子之製備..........................28
3.2.2生物可降解性聚乙烯亞胺之合成........................29
3.2.3奈米粒子表面之配基交換.............................29
3.2.4共沉澱法氧化鐵奈米粒子之製備........................30
3.2.5 PDS-HCl 之合成與鑑定............................30
3.2.6 SPION-PDS合成..................................30
3.2.7 SPION-PDS 官能基分析............................31
3.2.8 PEI800-SH與 SPION-PDS 鍵結反應..................31
3.2.9 奈米粒子粒徑分析.................................32
3.2.10 磁滯曲線測量...................................32
3.2.11 膠體電泳分析(gel retardation assay)............32
3.2.12 DNAse 保護性測試...............................33
3.3 細胞實驗方法與分析
3.3.1細胞培養.........................................33
3.3.2體外核酸轉染實驗..................................33
3.3.3普魯士藍染色......................................34
3.3.4細胞存活度測試....................................34
3.3.4 VEGF 含量偵測......................................35

第四章、結果與討論
第一部分、開發生物可降解高分子/氧化鐵奈米粒子複合體做為腫瘤基因治療工具
4.1SPEI/SPION性質探討...................................36
4.2SPEI/SPION 載體粒徑分析...............................36
4.3SPION磁滯曲線探討.....................................37
4.4SPEI/SPION/nucleic acid載體包覆狀況以及穩定性測試........38
4.4.1 膠體電泳結果....................................38
4.4.2 SPEI/SPION/ Nucleic acid 在不同溶劑中之粒徑分析...38
4.4.3 DNAse 保護性測試................................41
4.5細胞攝入SPEI/SPION/nucleic acid的效率與毒性分析..........42
4.5.1轉染效率測試......................................42
4.5.2毒性分析.........................................44
4.5.3細胞毒性與轉染效果比較.............................45

第二部分、多功能性SPION-PDS物質攜帶載體系統
4.6 SPION-PDS 性質探討...................................45
4.7 SPION粒徑分布........................................45
4.8 SPION磁滯曲線的探討...................................46
4.9 PDS-HCl合成鑑定......................................47
4.10 SPION-PDS 粒徑分析..................................47
4.11 SPION-PDS FTIR圖譜量測..............................49
4.12 SPION-ss-PEI800粒徑分布.............................50
4.13 分析SPION-ss-PEI800 /nucleic acid 包覆狀況以及穩定性...51
4.13.1 SPION-ss-PEI800 /nucleic acid 膠體電泳分析.....51
4.13.2 SPION-ss-PEI800/siRNA 粒徑探討................52
4.14細胞攝入SPION-ss-PEI800/nucleic acid的效率與毒性分析....54
4.14.1轉染效率測試....................................54
4.14.2細胞存活率測試..................................55
4.14.3細胞毒性與轉染效果比較............................56
4.14.4 In vitro VEGFa 表現抑制.......................56

第五章、結論...............................................58

參考文獻..................................................59

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