磷脂質對硬脂基化的Indolicidin的 自組裝與基因輸送的影響

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林彥妘(Yen-Yun LIN) 查詢紙本館藏

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化學工程與材料工程學系

論文名稱

磷脂質對硬脂基化的Indolicidin的自組裝與基因輸送的影響
(The Effects of Phospholipids on the Self-Assembly and Gene Delivery of Stearylated Indolicidin)

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摘要(中)

Indolicidin (IL)的C端硬脂基化所形成的兩親性ILs在水溶液中能自組裝，而將ILs混入輔助脂質二油酰磷脂酰乙醇胺 (DOPE)可促進基因輸送效果。為了瞭解輔助脂質與ILs之間的關係，我們除了DOPE另外比較加入了頭基有三甲基的二棕櫚酰磷脂酰膽鹼(DOPC)及尾基為飽和長碳鏈的1,2-二硬脂醯-sn-甘油-3-磷酸乙醇胺(DSPE)組別，將這三種脂質分別以莫耳比1:0.3與ILs進行混合後並名為ILs0.3E、ILs0.3C及ILs0.3S，以分別探討脂質的頭基大小和長碳鏈飽和度對於自組裝結構、基因輸送及轉染等之效果。由穿透式電子顯微鏡 (TEM)觀察到ILs呈現樹枝柱狀結構，ILs0.3E及ILs0.3C後則會自組裝變成球狀結構，ILs0.3S則同時具備球狀及柱狀結構。動態光散射儀 (DLS)結果證實到ILs可藉由加入輔助脂質縮小載體的粒徑。以小角度X光散射儀 (SAXS)進行擬合分析，推測ILs是以核殼柱狀的反膠束結構形成，並聚集成柱狀結構。ILs加入輔助脂質後則會形成囊胞，其中ILs0.3E及ILs0.3C組的厚度相似，但DSPE因具備飽和長碳鏈尾基，故其分子間有較強的作用力，造成ILs0.3S所得到的囊胞，兩種分子不易均勻分散彼此，使結構較為鬆散且膜厚增加。而此相容性不佳的現象也導致部分ILs仍保持原先自我的組裝結構，使TEM結果中能觀察到結構共存。以溴化乙錠(EtBr)進行電泳及包覆分析，ILs因為加入輔助脂質後可以形成小粒徑的囊胞，因此在低濃度時即可完整DNA。由流式細胞儀結果發現，ILs0.3E與ILs0.3C可以提高細胞攝取率，但ILs0.3S與ILs 的細胞攝取率相當。在轉染實驗中，ILs0.3E與ILs0.3C轉基因表現均高於ILs，而ILs0.3S沒有促進的效果。但其中ILs0.3E的表現明顯優於ILs0.3C。我們以螢光標定載體及DNA，並以雷射共軛焦顯微鏡去觀察其輸送途徑。所有的載體都能以胞吞的方式攝入，然而ILs0.3E比其他組別多了膜融合的途徑。相較於胞吞的DNA須自內體逃脫，若以膜融合途徑則可直接進入細胞並表現轉基因，最終使得ILs0.3E表現最佳的轉染效率。透過本研究我們發現胜肽加入輔助脂質後，不僅會改變自組裝結構，且可以促進基因裝載及細胞攝取，甚至還會影響輸送途徑及轉染的效率，這些結果顯示輔助脂質的添加對於胜肽基因載體具有關鍵的控制效果。

摘要(英)

The C-terminus of Indolicidin (IL) was stearylated to form amphiphilic ILs, which can self-assemble in an aqueous environment. A helper lipid, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), has been combined with ILs to promote gene delivery. To elucidate the promotion effects of DOPE, we additionally investigated 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), which owns a trimethylated head group, and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), which owns saturated hydrocarbon tails. These 3 lipids were individually mixed with ILs in a molar ratio of 0.3:1 and denoted as ILs0.3E, ILs0.3C, and ILs0.3S, respectively. By this, we may evaluate the structure effects of head groups and tail saturation on self-assembly structure, gene delivery, and transfection efficiency. The transmission electron microscopy (TEM) results showed that ILs exhibited dendritic cylinder structures, whereas ILs0.3E and ILs0.3C formed spheres. Interestingly, ILs0.3S demonstrated both cylinder and sphere structures. The results of dynamic light scattering (DLS) indicated that incorporating helper lipids reduced the size of self-assembled carriers. Structures inferred from TEM images were evaluated by small-angle X-ray scattering (SAXS) fitting. The results indicated that ILs formed reverse micelles in core-shell cylindrical structures and aggregated to each other as bundles. The incorporation of helper lipids resulted in vesicle formation. Among them, ILs0.3E and ILs0.3C exhibited similar membrane thickness, whereas ILs0.3S vesicles demonstrated thicker membrane thickness. We speculated that stronger intermolecular forces between DSPE molecules due to their saturated hydrocarbon chain hindered their dispersion with ILs molecules in vesicle membranes. Some ILs cylinders were also retained without the addition of DSPE, so two different structures appeared in TEM images. Ethidium bromide (EtBr) displacement and electrophoresis analysis showed that smaller vesicles formed due to the incorporation of helper lipids demonstrated superior DNA complexation capacity even at low concentrations. Flow cytometry examination showed that ILs0.3E and ILs0.3C exhibited higher cellular uptake efficiency than those of ILs0.3S and ILs, and thus these two groups also demonstrated higher transgene expression. Interestingly, although the internalization efficiency of ILs0.3E and ILs0.3C were comparable, the transfection efficiency of ILs0.3E was higher than that of ILs0.3C. Therefore, we used fluorescent labeling to track DNA and carrier molecules during transfection, which were analyzed by confocal microscopy to determine their transportation pathways. All carriers can be internalized through endocytosis, but only ILs0.3E possessed an additional membrane fusion pathway. Different from endocytic DNA, which has to be released from endosomes, the fusion pathway allows DNA to directly enter cytosol for transgene expression, and thus ILs0.3E demonstrated the best transfection efficiency. Through this study, we demonstrated that peptides with helper lipid incorporation not only altered self-assembly structure but also promoted gene encapsulation and cell uptake, which eventually regulated transportation pathway and determined the transfection efficiency. These results provided useful information for peptide-lipid combo-vector development.

關鍵字(中)

★ 脂肽複合物
★ 輔助脂質
★ 雙親性胜肽
★ 基因輸送

關鍵字(英)

★ Indolicidin
★ Gene delivery
★ peptide
★ helper lipid

論文目次

摘要 i
Abstract iii
致謝 v
圖目錄 x
表目錄 xii
第一章緒論 1
1-1 研究背景 1
1-2 研究動機 3
1-3 實驗設計 6
第二章文獻回顧 7
2-1 基因治療 7
2-2 胜肽基因載體 8
2-2-1 細胞穿膜胜肽 (Cell Penetrating Peptides, CPPs) 8
2-2-2 細胞穿膜胜肽運輸途徑 10
2-2-3 Indolicidin (IL) 12
2-2-4 胜肽的硬脂基化改質 12
2-3 脂質 (Lipids) 14
2-3-1脂質體 (Liposomes) 15
2-3-2脂質體運輸途徑 16
2-3-3 輔助脂質(Helper lipid) 18
2-3-4 脂質/胜肽複合物 21
第三章實驗藥品、儀器與方法 23
3-1 實驗材料 23
3-1-1 質體DNA 23
3-1-2 胜肽(Peptide) 24
3-1-3 細胞培養藥品 25
3-1-4 分析藥品 26
3-2 實驗儀器 28
3-3 實驗方法 30
3-3-1 溶液配置 30
3-3-2 胜肽/脂質(Peptide/Lipid) 奈米粒子的stock solution製備 34
3-3-3 胜肽/脂質/螢光頭基脂質 (Peptide/Lipid/TF-PE)奈米粒子製備 36
3-3-4 質體DNA純化 38
3-3-5 HEK-293T細胞培養 38
3-3-6 載體及載體/DNA性質分析 42
3-3-7 包覆率測定 49
3-3-8 載體進入細胞之效率分析(Flow cytometer) 56
3-3-9 載體進入細胞之轉染途徑分析雷射共軛焦顯微鏡 (Confocal microscopy) 59
3-3-10 SAXS 數據處理 62
第四章結果與討論 73
4-1 自組裝奈米結構物性測量 73
4-1-1 奈米粒子結構與粒徑大小 73
4-1-2 奈米粒子的粒徑分布 76
4-1-3 表面電位 (Zeta potential) 78
4-1-4 小角X光散射 (Small angle-X ray scattering) 79
4-2 自組裝奈米粒子與DNA的複合結構 90
4-2-1 載體/DNA複合物的結構與粒徑大小 90
4-2-2 載體/DNA複合物的粒徑分布 92
4-2-3 載體/DNA複合物的表面電位 94
4-2-4 小角X光散射 (Small angle-X ray scattering) 95
4-2-5 載體/DNA包覆率測試 102
4-3 載體細胞攝取效果 106
4-3-1 流式細胞儀分析(Flow cytometry) 106
4-3-2 轉染效率分析 108
4-3-3 載體進入途徑 (雷射共軛焦顯微鏡) 110
4-4輔助脂質對於IL轉染的效應 114
第五章結論 116
第六章參考資料 119

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指導教授

胡威文(Wei-Wen Hu)

審核日期

2024-6-14

推文