博碩士論文 109223052 詳細資訊



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姓名 吳其杭(Chi-Hung Wu)  查詢紙本館藏   畢業系所 化學學系
論文名稱 錨定脂質飽和度對引信響應釋放的胜肽微脂體釋放的影響
(The Effect of Anchoring Lipid Saturation on Triggered Release Liposome)
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摘要(中) 錨定脂質 (Anchored lipid) 是一種功能化脂質，可以將生物分子或有機分子固定在脂質雙層膜上，用來增加微脂體功能（例如：將多肽接在微脂體表面上用來導向或引信響應破膜釋放藥物）。然而，錨定脂質雖科學上被試用在微脂體藥物上來耦合生物或有機分子（市面上藥用微脂體主要配方幾乎都是飽和脂肪，因為可提供較高的機械強度），但在其選擇方面仍缺乏足夠的描述。例如錨定脂質有飽和跟不飽和的版本，該如何選擇?本研究使用抗菌胜肽 Magainin 2 並通過硫醇跟馬來酰亞胺 thiol-maleimide Michael addition將多肽與飽和或不飽和錨定脂質連接，以將多肽錨定到微脂體上，形成了胜肽微脂體。我們將阿黴素封裝在胜肽微脂體中，通過觀察阿黴素的釋放比例來推測不同錨定脂質對微脂體上多肽破膜活性影響。我們發現，在使用不飽和錨定脂質（如 18:1 PE MCC）時，藥物的釋放率接近100%，而在使用飽和錨定脂質（如 18:0 PE MCC）時，藥物的釋放率幾乎為零。為了進一步證明是錨定脂質的不飽和度而不是微脂體本身不飽和度的影響，我們在含有飽和錨定脂質的胜肽微脂體中添加了相等濃度非反應性不飽和的脂質（如18:1 DOPC）。我們發現，微脂體仍然不會釋放藥物，這證明了不是不飽和脂質使得微脂體機械強度變弱而導致引信響應微脂體釋放成功，而是Magainin 2多肽耦合至不飽和錨定脂質後，其破膜能力才能發揮作用，這代表應該有其他的原因。通過圓二色光譜 (CD) 分析，我們還發現，含有 Magainin 2-18:1 脂肽的脂質體具有較高比例的 α-螺旋結構，而 Magainin 2-18:0 脂肽則顯示出較低比例的α-螺旋結構。這可能解釋了不同飽和度的脂肽對微脂體釋放的影響不同。為了比較飽和與不飽和的脂質在膜上的作用差異，我們利用巨型單層囊泡 (Giant unilamellar vesicles, GUVs) 來進行視覺上（螢光）觀察是否是由於脂肪飽和度造成微脂體脂肪相分離，或者是多肽耦合錨定脂質後與其他脂質產生相分離（phase separation）而導致多肽有效聚集進而破膜。由於GUVs的平均直徑達到10微米以上，因此只需在囊泡形成過程中摻入適當的螢光團，即可進行觀察膜的情況。觀察結果顯示，飽和錨定脂質摻入微脂體未出現相分離現象，多肽與飽和錨定脂質共價連結也未出現相分離現象。不飽和錨定脂質摻入微脂體也未出現相分離現象，但抗菌胜肽Magainin 2與不飽和錨定脂質共價連結後，會出現脂肪相分離的現象，說明了此系統中脂肪的相分離對錨定的多肽破膜，有極度的重要性。這些結果有助於我們在藥物輸送系統和膜蛋白功能重建系統中選擇適當的錨定脂質。
摘要(英) Anchoring lipids are functionalized lipids that can immobilize biomolecules or organic molecules on lipid membranes. They are used to enhance the functionality of liposomes, such as conjugating peptides to the surface for targeting or triggering drug release. Commercially available liposomal drugs primarily utilize formulations with saturated lipids due to their higher mechanical strength. However, when choosing between saturated and unsaturated versions of anchoring lipids, there is a lack of guidelines. In this study, we used the antimicrobial peptide Magainin 2 to conjugate to saturated or unsaturated anchoring lipids using thiol-maleimide Michael addition chemistry, which allowed us to anchor the peptide to liposomes and form peptidyl liposomes. We encapsulated doxorubicin in the peptidyl liposomes and observed the drug release to estimate the effect of different anchoring lipids on the membrane-disrupting activity. We found that when using unsaturated anchoring lipids, the drug release rate was close to 100%, whereas when using saturated anchoring lipids, the drug release rate was nearly zero. To further demonstrate that the effect is due to the unsaturation of the anchoring lipids rather than the unsaturation of the liposomes themselves, we added an equal concentration of non-reactive unsaturated lipid to the peptide liposomes containing saturated anchoring lipids. We found that the liposomes still did not release the drug, which indicates that it is not the unsaturated lipids weakening the mechanical strength of the liposomes and causing successful triggering of drug release. Instead, it is the membrane-disrupting ability of the synergistic effect of Magainin 2 peptide conjugated to unsaturated anchoring lipids that plays a role. This suggests that there should be more than simple peptide-membrane interaction involved. Through circular dichroism (CD) analysis, we also observed that liposomes containing Magainin 2-18:1 lipopeptide exhibited a higher extent of α-helical structure, while Magainin 2-18:0 lipopeptide showed a lower extent of α-helical structure. This further shows that peptides interact with membrane differently when using anchoring lipid with/without saturation. To compare the differences in the effects of anchoring lipid saturation on the membrane, we used Giant Unilamellar Vesicles (GUVs) for visual (fluorescent) observation to determine whether the lipid phase separation in liposomes is caused by general lipid saturation or the coupling of the peptide to anchoring lipids leading to phase separation and effective peptide aggregation for membrane disruption. Since GUVs have an average diameter of over 10 micrometers, the observation of the membrane′s condition can be conducted by adding appropriate fluorescent probes during the vesicle formation process. The observation results showed that only when Magainin 2 was conjugated to unsaturated anchoring lipids, lipid phase separation occurred. This indicates the crucial importance of lipid phase separation in this system for the membrane-disrupting activity of the anchored peptide. These findings contribute to the selection of appropriate anchoring lipids in drug delivery systems and membrane protein functional reconstruction systems.
關鍵字(中) ★ 微脂體
★ 錨定脂質
★ 巨型單層囊泡		 關鍵字(英) ★ liposome
★ Anchoring Lipid
★ GUV
論文目次 目錄
中文摘要 I
Abstract III
目錄 VI
圖目錄 IX
符號說明 XIV
一、 緒論 1
1-1 前言 1
1-2 微脂體 1
1-3 微脂體膜的物理化學 5
1-4 抗菌胜肽 9
1-5 錨定脂質 11
1-6 不同飽和度的錨定脂質對引信多肽微脂體的影響 13
1-7 實驗設計 15
二、 實驗部分 18
2-1微脂體合成、定性與定量 18
2-1-1 錨定脂質的合成 18
2-1-2 微脂體合成實驗步驟 20
2-1-3 巨大單層囊泡合成 21
2-1-4 微脂體磷脂質濃度的定量 22
2-2 胜肽合成、定性與定量 23
2-2-1 胜肽合成 23
2-2-2 螢光胜肽合成 24
2-2-3 高效能液相層析法 26
2-2-4 質譜法 27
2-2-5 以胺基酸定量進行胜肽定量 28
2-3 圓二色光譜 29
2-4 胜肽微脂體 31
2-4-1 胜肽微脂體合成 31
2-4-2 胜肽微脂體藥物釋放分析 31
2-4-3 光誘導的胜肽微脂體釋放 32
2-4-4 胜肽微脂體的冷凍電子顯微鏡成像 32
2-5 利用巨大單層囊泡觀測脂質相分離 33
2-5-1 倒立螢光顯微鏡分析 33
2-5-2 共軛焦顯微成像技術分析 34
三、結果與討論 37
3-1 胜肽微脂體粒徑及界面電位分析 37
3-2 不同飽和度錨定脂肪共價連結胜肽後的圓二色性光譜分析 41
3-3 冷凍電子顯微鏡影像分析 46
3-4 脂質不飽和度對胜肽不同表面取代量誘發微脂體釋放的影響 51
3-5 脂質不飽和度對紫外光誘發胜肽微脂體藥物釋放量分析 53
3-6 脂質不飽和度對螢光標記脂肪和螢光標記胜肽的分布（觀察相分離） 56
四、總結 62
參考資料 64
附錄 67
附錄一、含不同飽和度錨定脂質微脂體的包覆藥物分析 67
附錄二、標記螢光的破膜活性多肽之破膜活性 68
附錄三、微脂體之共軛焦顯微鏡圖像 69
附錄四、錨定脂質PE MAL的探討 71
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指導教授 李賢明 謝發坤(Hsien-Ming Lee Fa-Kuen Shieh) 審核日期 2023-7-28
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