利用組胺酸-金屬配位和非共價鍵作用力調控膠原蛋白模擬胜肽自組裝及藥物包覆之探討

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研究生：	郭哲維 Kuo, Chei-Wei
論文名稱：	利用組胺酸-金屬配位和非共價鍵作用力調控膠原蛋白模擬胜肽自組裝及藥物包覆之探討 Modulating the Self-Assembly of Collagen-Mimetic Peptides via Histidine-Metal Coordination and Noncolvalent Interactions, and Study of Their Drug Encapsulation
指導教授：	洪嘉呈 Horng, Jia-Cherng
口試委員:	魯才德 Lu, Tsai-Te 許馨云 Hsu, Hsin-Yun
學位類別：	碩士 Master
系所名稱：	理學院 - 化學系 Department of Chemistry
論文出版年：	2022
畢業學年度：	110
語文別：	中文
論文頁數：	87
中文關鍵詞：	聚脯胺酸、胜肽、自組裝、組胺酸-金屬配位、膠原蛋白模擬胜肽、藥物包覆
外文關鍵詞：	Polyproline II, Peptide, Self-assembly, Histidine-metal coordination, Collagen mimetic peptides, Drug encapsulation
相關次數：	點閱：2 下載：0
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膠原蛋白是哺乳動物體內含量最豐富的蛋白質，並且已經被廣泛應用在生醫材料上。因此在醫材設計領域，尋找快速且高效的方法來將短鏈的膠原蛋白模擬胜肽自組裝成高階結構是非常重要的。在本研究中，我們使用了組胺酸(His)及金屬配位作用力、cation-π作用力以及疏水作用力來誘導膠原蛋白模擬胜肽自組裝成大型的結構，目的在於找出上述提到的三個作用力對於自組裝的影響。此外，我們同時也要找出His數目及位置對於自組裝結構的形貌及速度的影響。我們使用圓二色光譜儀來測量我們設計的膠原蛋白模擬胜肽的熱穩定性，並使用紫外-可見光光譜儀測量濁度來確認組裝速度，最後使用電子顯微鏡來拍攝自組裝的形貌。結果顯示，胜肽端點有His-金屬配位作用力的胜肽自組裝速度將會快於端點使用cation-π作用力的胜肽。至於形貌方面，胜肽兩端沒有進行His置換的胜肽可以形成較為有序且花狀的結構，但如果有更強的疏水作用力同時作用，則會因為疏水作用力的主導而形成有序的球狀結構。綜上所述，His的位置及數量還有疏水作用力都可以用於調控胜肽自組裝的形貌。由於胜肽可以形成大型自組裝結構，我們認為可以應用於藥物包覆方面。所以我們使用共聚焦顯微鏡確認藥物(FITC-Dexatran)可以鑲嵌於這些超分子結構上，接著我們使用微盤分析儀來監測藥物釋放的情形並發現愈不穩定的結構可以釋放出愈多的藥物，期望這些結果對於將來的膠原蛋白相關之材料研究能有所幫助。

Collagen is highly abundant in mammals and has been widely used in biomedical materials. Searching for a quick and effective way to assemble short collagen mimetic peptides (CMPs) into higher-order structures has emerged as an important and attractive research topic in biomaterial design. In this study, we utilized histidine (His) -metal interactions, cation-π interactions and hydrophobic interactions to promote the self-assembly of CMPs into large-scale constructs. We aimed to examine the different effects between ligand-metal interactions, cation-π interactions and hydrophobic interaction on the head-to-tail assembly of CMPs. Besides, we also investigated the impact of the number of His residue within a CMP on the assembly speed and structural morphology. We used circular dichroism (CD) to measure the thermal stability of the designed CMPs, turbidity measurements to monitor the assembly rate, and electron microscopy (EM) to characterize the assembled structures. The results showed that the His-metal interactions at the peptide end induced a much faster rate than cation-π interactions to form assemblies. As to morphology, the CMPs without His-metal interactions at the peptide termini could generate a more ordered and flower-like structure. But with stronger hydrophobic interaction, the CMPs could also generate ordered and spherical structure even there are His residues at the termini. Together, we demonstrated that the number and location of His residue within a CMP and the hydrophobic interaction could modulate the morphology of self-assemblies. Since the CMPs can form supramolecular structures, we suggested that these CMPs could be capable to encapsulate the drug. We used confocal to verify that the drug (FITC-Dextran) can be embedded in the supramolecular structure. We further used the ELISA reader to monitor the drug release and observed that the drug release increased as the stability of CMPs decreased. The results may be useful and helpful for the future development of collagen-related materials.

中文摘要    i
Abstract    ii
目錄    iii
圖目錄    vi
表目錄    x
第一章 緒論    1
1-1 膠原蛋白    1
1-1-1 膠原蛋白的結構    2
1-1-2 脯胺酸(Pro)和羥脯胺酸(Hyp)穩定膠原蛋白的能力    3
1-1-3 膠原蛋白中的作用力    4
1-1-4 膠原蛋白模擬胜肽單一置換穩定性探討    5
1-2 膠原蛋白的自組裝(self-assembly)    6
1-3 生物系統中的金屬    10
1-3-1 生物系統中的重要元素    10
1-3-2 生物系統中的配位基    12
1-3-3 金屬誘發膠原蛋白自組裝    13
1-3-4 膠原蛋白自組裝的應用    17
1-4 研究動機    20
第二章 實驗部分    21
2-1 實驗儀器    21
2-2 實驗藥品    23
2-3 合成Fmoc-Pro-Hyp-Gly-OH    25
2-3-1 合成Boc-Hyp-OH(1)    25
2-3-2 合成Boc-Hyp-Gly-OBn(2)    26
2-3-3 合成Fmoc-Pro-Hyp-Gly-OBn(3)    26
2-3-4 合成Fmoc-Pro-Hyp-Gly-OH(4)    27
2-4 合成Fmoc-Pro-Pro-Gly-OH    28
2-4-1 合成Boc-Pro-Gly-OBn(1)    28
2-4-2 合成Fmoc-Pro-Pro-Gly-OBn(2)    29
2-4-3 合成Fmoc-Pro-Pro-Gly-OH(3)    30
2-5 固相胜肽合成法(Solid phase Peptide Synthesis, SPPS)    31
2-5-1 醯胺化反應(amidation)    34
2-5-2 去保護(deprotection)    35
2-5-3 活化(activation)    36
2-5-4 耦合(coupling)    37
2-5-5 切除(cleavage)    37
2-6 胜肽的合成    38
2-6-1 利用微波合成儀合成    38
2-6-2 手動合成法    38
2-6-3 胜肽的切除與純化    39
2-6-4 鑑定胜肽純度    39
2-7 圓二色光譜儀(Circular Dichroism Spectrometer, CD)    40
2-8 掃描電子顯微鏡(Scanning Electron Microscopy, SEM)    43
2-9 金屬誘發膠原蛋白胜肽自組裝實驗    44
2-9-1 圓二色光譜儀量測    44
2-9-2 UV-VIS光譜儀量測濁度實驗(Turbidity)    44
2-9-3 動態光散射實驗(Dynamic Light Scattering, DLS)    44
2-9-4 掃描電子顯微鏡實驗(Scanning Electron Microscopy, SEM)    45
2-9-5 穿透式電子顯微鏡(Transmission electron microscopy, TEM)    45
2-9-6 共聚焦顯微鏡影像(Confocal Microscope System & TIRF, CMS)    45
2-9-7 藥物釋放實驗    46
2-10 圓二色光譜-變溫實驗數據處理    46
2-11 濁度實驗之數據處理    48
第三章 結果與討論    49
3-1 胜肽設計    49
3-2 Far-UV CD光譜    50
3-3 變溫實驗測量    51
3-4 濁度實驗    53
3-5 穿透式電子顯微鏡影像實驗    57
3-6 掃描式電子顯微鏡影像實驗    62
3-7 共聚焦顯微鏡影像實驗    73
3-8 藥物釋放實驗    75
第四章 結論    76
參考文獻    78
附錄    82
一、1H NMR圖譜    82
(1) Fmoc-POG-OH    82
(2) Fmoc-PPG-OH    83
二、胜肽純度    84
三、使用UV-Vis測量藥物釋放    87
                                

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