博碩士論文 107223007 詳細資訊




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姓名 鄭仁華(Ren-Hua Jheng)  查詢紙本館藏   畢業系所 化學學系
論文名稱 藉由點擊化學製備穀胱甘肽控制藥物釋放之胜肽微脂體
(Using Click Conjugation to Generate Glutathione Responsive Peptidyl Liposome)
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摘要(中) 穀胱甘肽是生物體內一種相當重要的小分子硫醇,其能扮演抗氧化劑來調節細胞的氧化還原壓力之外,同時也參與多項的生理代謝反應。再者,由於大部分腫瘤組織中穀胱甘肽的濃度皆為正常組織的數倍,此濃度的差異對於治療癌症的奈米載體而言,被視為一個良好的引信訊號來促使藥物釋放。過去有許多以穀胱甘肽為引信的微脂體藥物陸續被開發,其中大多數的研究皆以利用具有還原應答能力的非天然性磷脂質來作為微脂體的主要脂質組成,但此種磷脂質可能造成生物相容性上的相關疑慮。在本研究中,我們利用自然界的膜活性胜肽為基礎,成功開發了以穀胱甘肽為引信的破膜胜肽,並且此狀態下的破膜活性會被胜肽序列上的遮蔽區塊抑制。由於此多肽有硫醇敏感設計,與傳統多肽與微脂體的硫醇與丁二醯亞胺反應不相容,因此需開發新的多肽與微脂體共軛聯結方式。透過張力促使的疊氮-炔烴的環加成反應,我們成功地將此胜肽修飾於微脂體的表面上,也成功地促使引信穀胱甘肽可以控制微脂體的藥物釋放。從多肽的還原測試實驗中,可以發現此引信響應的胜肽可對於穀胱甘肽具有引信響應的能力,並同時轉變成具有高破膜活性的多肽型式。在藥物釋放方面,引信響應的多肽微脂體與腫瘤濃度下的穀胱甘肽作用後,其會造成較高比例藥物阿黴素的釋放。最後,我們藉由分析胜肽的圓二色光譜,發現此胜肽在與引信作用後,會造成胜肽兩親媒性二級結構上的增加,進而影響微脂體的藥物釋放。
摘要(英) Reduced glutathione (GSH) is an important low-molecular-weight physiological thiol that can be serve as antioxidant for balancing the redox environment of cells and adjusts many significant biological metabolisms. Because of the concentration of glutathione in cancerous tissue is higher than normal level, it can be used as a good triggering signal for nanocarrier to release drug. As a result, a lot of liposomal drugs that can be triggered release by glutathione have been developed in the past decade. However, most liposomes mentioned above are composed of thiol-responsive “unnatural” phospholipids, which have potential safety issue. In this study, we intend to develop a GSH-responsive peptidyl liposome which is more biocompatible. The designed thio-responsive peptide is not compatible with conventional thiol-maleimide chemistry to conjugate onto liposome, and new cross-linking chemistry is needed. We successfully developed peptide-to-liposome conjugation by strain-promote azide-alkyne cycloaddition. We also successfully induce the reductive cleavage of the GSH-responsive peptide on liposome and trigger liposome release. Finally, through the circular dichroism analysis, we discovered the correlation between amphiphilic helicity of liposomal peptide and liposome release.
關鍵字(中) ★ 穀胱甘肽
★ 膜活性多肽
★ 引信釋放
★ 以穀胱甘肽為引信響應的多肽微脂體
關鍵字(英) ★ glutathione
★ membrane active peptide
★ triggered release
★ GSH-responsive peptidyl liposome
論文目次 中文摘要 i
ABSTRACT ii
誌謝 iii
目錄 iv
圖目錄 vii
表目錄 xi
符號說明 xii
一、 緒論(INTRODUCTION) 1
1-1 前言 1
1-2 微脂體 1
1-3 微脂體藥物的包覆與釋放 3
1-4 穀胱甘肽 6
1-5 以穀胱甘肽為引信響應的反應8
1-6 膜活性胜肽 12
1-7 胜肽微脂體的合成方法 15
1-8 實驗動機與目的 16
二、 實驗部分 (EXPERIMENTAL SECTION) 17
2-1 微脂體合成、定性與定量 17
2-1-1微脂體磷脂質濃度的定量 18
2-1-2藥物包覆率 19
2-1-3動態光散射分析 20
2-2 胜肽合成、定性與定量 20
2-2-1胜肽合成 20
2-2-2高效能液相層析法及質譜法 23
2-2-3胺基酸定量分析 24
2-2-4引信響應胜肽的還原反應測試26
2-2-5圓二色光譜 26
2-3 胜肽微脂體合成、定性與定量 27
2-3-1後嵌入法 27
2-3-2胜肽與微脂體交聯反應-CuAAC28
2-3-3胜肽與微脂體交聯反應-SPAAC29
2-4 胜肽微脂體藥物釋放分析 30
2-4-1藥物引信釋放區間 30
2-4-2胜肽微脂體試管內藥物釋放 30
三、 實驗結果與討論 (RESULT and DISCUSSION) 33
3-1 引信響應膜活性胜肽的設計及合成 33
3-2 胜肽的定性及定量 39
3-3 穀胱甘肽對引信響應胜肽的還原反應測試 41
3-4 胜肽微脂體製備方式的最佳化 45
3-4-1後嵌入法修飾藥物微脂體 46
3-4-2銅催化的疊氮-炔烴環加成反應 48
3-4-3張力促進的疊氮-炔烴環加成反應 50
3-5 胜肽微脂體引信釋放區間的評估 52
3-6 以穀胱甘肽為引信控制多肽微脂體試管內的藥物釋放 56
3-6-1不同比例的DSPE-PEG2000對引信釋放的行為影響 56
3-6-2不同比例的錨定脂質對引信釋放的行為影響 59
3-6-3胜肽與微脂體交聯反應時間對引信釋放的行為影響 61
3-7 胜肽微脂體的定性 65
3-8 膜活性多肽之二級結構與多肽微脂體藥物釋放關係的探討 67
四、 結論 (CONCLUSION) 70
參考文獻 (REFERENCE) 71
附錄 (Supporting Information) 80
附錄一、 脂肽/疊氮化胜肽之合成轉換率分析 80
附錄二、Imidazole-1-sulfonyl Azide Hydrochloride之合成 84
附錄三、脂肽的還原反應測試實驗結果 84
附錄四、膜活性脂肽對於DOPC微脂體的嵌入效率分析 89
附錄五、胜肽/脂肽的HPLC圖譜與質譜圖 91
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指導教授 謝發坤 李賢明(Fa-Kuen Shieh Hsien-Ming Lee) 審核日期 2021-9-1
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