通過腺嘌呤和脲嘧啶之互補性氫鍵作用力來開發超分子藥物傳載系統

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研究生：	鍾艾 Ai Chung
論文名稱：	通過腺嘌呤和脲嘧啶之互補性氫鍵作用力來開發超分子藥物傳載系統 Development of Supramolecular Drug Delivery System Through the Complementary Hydrogen Bonding Interaction of Adenine and Uracil
指導教授：	郭東昊 Dong-Hau Kuo 鄭智嘉 Chih-Chia Cheng
口試委員:	郭東昊 Dong-Hau Kuo 鄭智嘉 Chih-Chia Cheng 楊銘乾 Ming-Chien Yang
學位類別：	碩士 Master
系所名稱：	工程學院 - 材料科學與工程系 Department of Materials Science and Engineering
論文出版年：	2021
畢業學年度：	109
語文別：	中文
論文頁數：	140
中文關鍵詞：	超分子藥物傳載系統、羅丹明6G 、腺嘌呤、脲嘧啶、BU-PPG
外文關鍵詞：	supramolecular drug delivery system, R6G, adenine, uracil, BU-PPG
相關次數：	點閱：293 下載：0
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上一筆

結合高螢光和穩定性的藥物傳載系統在癌症治療中仍具有很高的挑戰性。在此論文中，我們成功開發了一新型基於羅丹明的超分子藥物（A-R6G），該藥物包含多重氫鍵的腺嘌呤部分，其展現出高疏水性和獨特的物理特性，包含優異的螢光特性、特異的自組裝行為及高度細胞毒性。通過麥可加成與脲嘧啶反應的聚丙二醇二丙烯酸甲酯，創建其 A-R6G 的互補性夥伴，即含有雙官能脲嘧啶末端基的水溶性超分子微胞（BU-PPG）。由於通過腺嘌呤和脲嘧啶之間具有互補性氫鍵作用力的特性，疏水性的 A-R6G 和親水性的 BU-PPG 能夠形成複合體，進而在水溶液中形成具高度穩定包載 A-R6G 的 BU-PPG 微胞。所得包載 A-R6G 的微胞具有極高的 A-R6G 包載量（高達 84％）、出色的 A-R6G 包封性及在富含血清的培養基中的穩定性、可控的 pH /溫度響應性藥物釋放能力。有趣的是，體外細胞毒性研究證實，包載 A-R6G 的 BU-PPG 微胞對癌細胞表現出選擇性的細胞毒性，且不會傷害正常細胞，這可歸因於在癌細胞的微酸環境下，微胞中的互補性氫鍵脲嘧啶-腺嘌呤二聚體迅速解離，隨後在細胞內釋放 A-R6G，從而在癌細胞中產生高選擇性的生物毒性作用。更重要的是，螢光影像和流式細胞儀分析清楚地表明，包載 A-R6G 的 BU-PPG 微胞透過內吞途徑進入癌細胞中並有效誘導細胞凋亡，但抑制其正常細胞的內化並導致高度生物相容性。因此，新開發的互補性藥物傳遞系統是增強癌症化學療法安全性和有效性的可行策略及潛在的途徑。

The combination of both high fluorescence and stable drug delivery system for cancer therapy remains highly challenging. In this thesis, we successfully developed a novel rhodamine-based supramolecular drug （A-R6G） containing multiple hydrogen-bonded adenine moiety that exhibit high hydrophobicity and unique physical properties including excellent fluorescence features, as well as intriguing self-assembly behavior and high cytotoxicity. A complementary partner to A-R6G, water-soluble supramolecular micelles （BU-PPG） containing difunctional uracil-containing end groups, was created by Michael addition of poly（propylene glycol） diacrylate reacted with uracil. Due to the nature of the molecular recognition between the adenine and uracil moieties through complementary hydrogen bonding interactions, hydrophobic A-R6G and hydrophilic BU-PPG are able to form complexes that can form highly stable A-R6G-loaded BU-PPG micelles in aqueous solution. The resulting A-R6G-loaded micelles possess extremely ultra-high A-R6G-loading capacity （up to 84%）, excellent A-R6G-encapsulation retention and stability in serum-rich medium, as well as well-controlled pH/temperature-responsive drug release ability. Interestingly, in vitro cytotoxicity studies confirmed that A-R6G-loaded BU-PPG micelles exhibit selective cytotoxicity in cancer cells, without harming normal cells, which is attributed to the rapid dissociation of the complementary hydrogen-bonded uracil-adenine dimers within the micelles at mildly acidic intracellular pH of cancer cells, followed by intracellular release of A-R6G, thus resulting in high selective cytotoxic effect in cancer cells. More importantly, fluorescence images and flow cytometric analysis clearly demonstrated that A-R6G-loaded BU-PPG micelles selectively entered cancer cells through endocytic pathways and efficiently induced apoptotic cell death, but inhibited internalization by normal cells and resulted in high biocompatibility against normal cells. Thus, this newly-developed complementary drug delivery system could be a viable strategy and potential route for enhancing the safety and efficacy of cancer chemotherapy.

摘要    I
Abstract    II
致謝    IV
目錄    V
表目錄    X
圖目錄    XI
第一章 緒論    1
1 研究背景    1
2 研究動機    2
第二章 文獻回顧    3
1 超分子化學 （Supramolecular Chemistry）    3
2 分子自組裝 （Self-Assembly）    4
3 氫鍵 （Hydrogen Bond）    6
3.1 雙氫鍵 （Double hydrogen bond）    7
3.2 三重氫鍵 （Triple hydrogen bond）    8
4 羅丹明6G （Rhodamine 6G）    11
5 腺嘌呤 （Adenine）    17
6 BU-PPG    22
7 文獻回顧總結    24
第三章 實驗材料與方法    25
1 實驗設計    25
2 實驗材料    26
2.1 實驗藥品    26
2.2 實驗溶劑    29
2.3 細胞實驗材料    31
3 實驗設備與參數    34
3.1 旋轉蒸發儀 （Rotary Evaporator）    34
3.2 傅立葉轉換紅外光譜 （Fourier transform infrared spectroscopy）    34
3.3 紫外光可見光光譜儀 （UV/Vis Spectrophotometer）    34
3.4 光致發光光譜儀 （Photoluminescence Spectroscopy）    35
3.5 熱重分析儀 （Thermogravimetric Analysis）    35
3.6 二氧化碳培養箱（CO2 incubator）    36
3.7 離心機 （Centrifuge）    36
3.8 試管震盪器 （Vortex Mixer）    36
3.9 酵素免疫分析測讀儀 （ELISA Reader）    36
3.10 螢光顯微鏡 （Fluorescence Microscope）    37
3.11 流式細胞儀 （Flow Cytometry）    37
3.12液態超導核磁共振儀 （Nuclear Magnetic Resonance spectroscopy ）    37
3.13 動態光散射粒徑分析儀 （Dynamic Light Scattering）    38
3.14 原子力顯微鏡 （atomic force microscope）    38
3.15 旋轉式塗佈機 （Spin Coater）    39
3.16 掃描式電子顯微鏡 （Scanning Electron Microscope）    39
3.17 元素分析儀 （Elemental Analyzer）    40
3.18 高解析質譜儀 （Mass Spectrometry）    40
3.19 桌上型酸鹼度測定計 （pH meter）    41
4 實驗合成步驟    42
4.1 合成AMA    42
4.2 合成AMAC3NH2    43
4.3 合成A-R6G    44
4.4 合成BU-PPG    45
5 樣品製備    46
5.1 微胞製備    46
5.2 藥物微胞製備    46
5.3 藥物濃度檢量線    46
5.4 包覆率    46
5.5 體外藥物釋放模擬    47
5.6 微胞穩定性測試    47
5.7 奈米微胞刺激響應測試    47
6 細胞生物性製備    48
6.1 Hela細胞培養基    48
6.2 3T3細胞培養基    48
6.3 細胞解凍培養    48
6.4 細胞繼代    48
6.5染色與數細胞    49
6.6 細胞生物毒性測試    49
6.7 螢光顯微鏡    49
6.8 製備Annexin-binding buffer    50
6.9 製備Ghost Dye™ Red 780    50
6.10 流式細胞儀（Flow cytometer）    50
第四章 結果與討論    52
1 AMA材料鑑定    53
1.1 AMA透過傅里葉轉換紅外光譜（FTIR）材料鑑定    53
1.2 AMA透過核磁共振儀（1H和13C NMR）材料鑑定    54
1.3 AMA透過元素分析儀（EA）材料鑑定    55
1.4 AMA透過高解析質譜儀（MS）材料鑑定    56
2 AMAC3NH2材料鑑定    57
2.1 AMAC3NH2透過傅里葉轉換紅外光譜（FTIR）材料鑑定    57
2.2 AMAC3NH2透過核磁共振儀（1H和13C NMR）材料鑑定    58
2.3 AMAC3NH2透過元素分析儀（EA）材料鑑定    59
2.4 AMAC3NH2透過高解析質譜儀（MS）材料鑑定    60
3 A-R6G材料鑑定    61
3.1 A-R6G透過傅里葉轉換紅外光譜（FTIR）材料鑑定    61
3.2 A-R6G透過核磁共振儀（1H和13C NMR）材料鑑定    62
3.3 A-R6G透過元素分析儀（EA）材料鑑定    65
3.4 A-R6G透過高解析質譜儀（MS）材料鑑定    65
3.5 A-R6G透過核磁共振儀（1H NMR）觀察氫鍵位置    66
4 BU-PPG材料鑑定    68
4.1 BU-PPG透過傅里葉轉換紅外光譜（FTIR）材料鑑定    68
4.2 BU-PPG透過核磁共振儀（1H NMR）材料鑑定    69
4.3 BU-PPG透過凝膠滲透層析儀（GPC）材料鑑定    70
5 材料性質分析    71
5.1 熱重分析儀 （TGA）    71
5.2 溶解度測試    73
5.3 A-R6G/BU-PPG透過核磁共振儀（1H NMR）觀察氫鍵作用力    75
5.4 可見光/紫外光分光光譜儀 （UV/VIS Spectophotometer）與光致發光光譜儀 （PL）    79
5.5 奈米粒徑及介面電位量測儀 （DLS & Zeta Potential）    83
5.6掃描電子顯微鏡SEM、原子力顯微鏡AFM    84
6 藥物微胞分析    86
6.1 藥物包覆率與粒徑分布    86
6.2 掃描式電子顯微鏡 （SEM）、原子力顯微鏡 （AFM）    89
6.3 奈米藥物微胞穩定性分析    91
6.4 藥物奈米微胞刺激響應    92
6.5 體外藥物釋放    95
7 細胞實驗分析    98
7.1 細胞毒性測試 （Cytotoxicity test）    98
7.2 細胞螢光顯微鏡分析 （CLSM）    102
7.3 細胞攝取實驗    107
7.4 細胞凋亡實驗    111
第五章 結論    114
第六章 未來展望    116
第七章 參考文獻    117

                                

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