臺灣博碩士論文加值系統

高分子奈米微胞為具親水與疏水鏈段組成核-殼結構，其中疏水核層能有效攜載疏水性抗癌藥物。高分子奈米微胞可根據腫瘤組織微環境設計成具環境應答性。因此當具環境應答性之高分子奈米微胞依被動標的方式累積在腫瘤組織時，可因應答作用促使奈米微胞釋放藥物，達到局部釋放的效果。本研究旨為開發一具活性氧化物(reactive oxygen species；ROS)與穀胱苷肽(glutathione；GSH)雙重應答性高分子奈米微胞，其中奈米微胞核層為具ROS應答之硫化基團與具GSH應答之雙硫鍵基團所構成。當進入腫瘤組織時，由於因ROS與GSH過度表現使奈米微胞因氧化還原作用變形而釋放藥物，進而毒殺癌細胞。
本研究已成功合成含硫化基與雙硫鍵之雙親性高分子mPEG-b-poly(diethyl sulfide-alt-cystamine)n(mPEG-b-poly(DES-alt-cys)n)，亦成功製備出大小約為200 nm之奈米微胞，並可作為疏水性抗癌藥物-喜樹鹼之藥物載體，其載藥率約為50%。在模擬腫瘤氧化還原環境中，喜樹鹼釋放量於48小時可達100%。根據細胞毒性與細胞週期分析結果顯示，攜載喜樹鹼高分子奈米微胞影響人類大腸癌細胞之細胞週期並有相當良好的毒殺效果。藉由共軛焦顯微鏡可觀察到攜載喜樹鹼高分子奈米微胞被攝入大腸癌細胞內並釋放藥物。動物全身分布影像顯示載喜樹鹼奈米微胞能有效累積在腫瘤組織，並在腫瘤抑制中有良好的抑制效果。綜合上述體內、體外之結果，相信本研究開發之具氧化還原雙重應答高分子奈米微胞具有癌症治療之潛能。

Micelles have core-shell structure in which the hydrophobic core provides the basis for the hydrophobic anticancer drugs. Polymeric micelles could be designed for environment-sensitivity based on the external stimuli in tumors. The environment-sensitive micelles could therefore accumulate in tumor by passive targeting and release drug specifically in tumors. In this study, we developed ROS and GSH dual redox-responsive polymeric micelles in which the core layers were composed of sulfide groups and disulfide bond groups. When dual-redox-responsive polymeric micelles are transported into tumor tissue, they would deform and release drug in response to high levels of intracellular ROS and GSH, leading to cytotoxicity.
An amphiphilic copolymer, mPEG-b-poly(diethyl sulfide-alt-cystamine)n (mPEG-b-poly(DES-alt-cys)n), containing sulfide group and disulfide bond was successfully synthesized. Micelles with 200 nm diameter were prepared by solvent exchange method. The anticancer drug, camptothecin(CPT) was incorporated into micelles with a high drug content of 50 w.t.%. After 48h, CPT molecules were completely released in the redox environment. According to the results of in vitro cytotoxicity study and cell cycle analysis, CPT-loaded micelles showed high toxicity for HCT116 colon cancer cells. The images observed by CLSM confirmed the internalization of the polymeric micelles and CPT release behaviors. The in vivo biodistribution study indicated that CPT-loaded micelles exhibited high accumulation in tumor. The tumor inhibition test demonstrated that the CPT-loaded micelles showed good therapeutic effect compared to the CPT molecules. These results suggest that dual-redox-responsive polymeric micelles have promising potential as CPT carriers for cancer therapy.

總目錄
中文摘要 i
英文摘要 ii
總目錄 iv
圖目錄 vii
表目錄 x
第 1 章 研究動機與目的 1
第 2 章 文獻回顧 3
2.1 喜樹鹼之基本介紹 3
2.2 活性氧化物之基本概念 4
2.3 穀胱甘肽之基本概念 5
2.4 癌細胞之活性氧化物調控 6
2.5 癌細胞之穀胱甘肽調控 7
2.6 環境應答性藥物載體之介紹 7
2.6.1 具氧化應答性藥物載體 7
2.6.2 具還原應答性藥物載體 10
2.6.3 具氧化還原雙重應答性藥物載體 13
第 3 章 實驗材料與方法 15
3.1 藥品 15
3.2 實驗儀器與裝置 16
3.3 具氧化還原雙重應答高分子之製備 17
3.3.1 Poly(ethylene glycol) methyl ether之末端改質 17
3.3.2 bis(4-nitrophenyl) diethyl sulfide之合成 20
3.3.3 mPEG-b-poly(diethyl sulfide -alt- cystamine)n之合成 20
3.4 高分子奈米微胞之製備 21
3.5 高分子奈米微胞之基本性質分析 21
3.5.1 粒徑分析 21
3.5.2 界面電位分析 22
3.5.3 微觀結構之分析 22
3.6 臨界微胞濃度之鑑定 22
3.7 穩定性測試 23
3.7.1 粒徑變化之觀測 23
3.7.2 微觀結構變化之觀測 23
3.8 氧化還原應答性測試 23
3.8.1 粒徑變化之觀測 23
3.8.2 微觀結構變化之觀測 24
3.9 高分子奈米微胞包覆抗癌藥物-喜樹鹼 24
3.10 喜樹鹼之檢量線製備 24
3.11 載藥率與有效包覆率分析 24
3.12 喜樹鹼之內酯環鑑定 25
3.13 藥物釋放分析 25
3.13.1 藥物洩漏分析 25
3.13.2 氧化還原環境之藥物釋放分析 26
3.14 高分子奈米微胞生物體外試驗 26
3.14.1 培養基製備 26
3.14.2 高分子奈米微胞之材料毒性測試 26
3.14.3 喜樹鹼與攜載喜樹鹼高分子奈米微胞之毒性測試 27
3.14.4 攜載喜樹鹼高分子奈米微胞之細胞內吞作用 28
3.14.5 攜載喜樹鹼高分子奈米微胞對細胞週期之影響 29
3.15 攜載喜樹鹼高分子奈米微胞之動物活體觀察 30
3.15.1 攜載喜樹鹼高分子奈米微胞之動物器官累積分布 30
3.15.2 攜載喜樹鹼高分子奈米微胞之腫瘤抑制效果 30
3.15.3 攜載喜樹鹼高分子奈米微胞之肝、腎毒性之影響 31
3.16 統計方法 31
第 4 章 結果與討論 32
4.1 具氧化還原雙重應答性高分子之製備 32
4.2 高分子奈米微胞之基本性質 41
4.3 臨界微胞濃度 42
4.4 穩定性與應答性測試 43
4.5 攜載喜樹鹼高分子奈米微胞之基本性質測定 47
4.6 喜樹鹼之藥物活性測試 47
4.7 藥物釋放 48
4.8 細胞毒性 51
4.9 攜載喜樹鹼高分子奈米微胞之細胞內吞作用 56
4.10 細胞週期 58
4.11 攜載喜樹鹼高分子奈米微胞之動物全身累積分布 60
4.12 攜載喜樹鹼高分子奈米微胞之腫瘤抑制效果 62
4.13 攜載喜樹鹼高分子奈米微胞之肝、腎毒性之影響 65
第 5 章 結論 67
參考文獻 69

圖目錄
圖　1-1. 具氧化還原雙重應答高分子奈米微胞於腫瘤組織之藥物釋放示意圖 2
圖　2-1. 活性氧於腫瘤內之惡性循環…………………………………...5
圖　2-2. 硼酸脂基團之氧化機制 9
圖　2-3. 硫代縮酮基團之氧化機制 9
圖　2-4. 硫化基團之氧化機制 10
圖　2-5. 以雙硫鍵與雙碳鍵鍵結之雙親性高分子 11
圖　2-6. 聚乙二醇可拆除式之基因載體示意圖 11
圖　2-7. 硫化鎘奈米粒子封頂之多孔性二氧化矽奈米微球 12
圖　2-8. 具雙硫鍵交聯之奈米微胞 12
圖　2-9. 高分子奈米粒子之藥物釋放機制 13
圖　2-10. 雙硒鍵奈米微胞之氧化還原應答示意圖 14
圖　3-1. mPEG-COOH合成示意圖 18
圖　3-2. mPEG-NHS ester合成示意圖 19
圖　3-3. mPEG-cystamine合成示意圖 19
圖　3-4. bis(4-nitrophenyl) diethyl sulfide合成示意圖 20
圖　3-5. mPEG-b-poly(DES-alt-cys)n合成示意圖 21
圖　3-6. 載藥率與有效包覆率計算公式 25
圖　4-1. mPEG-COOH之1H-NMR光譜圖 33
圖　4-2. mPEG-COOH之FT-IR光譜圖 33
圖　4-3. mPEG-NHS ester之1H-NMR光譜圖 34
圖　4-4. mPEG-NHS ester之FT-IR光譜圖 35
圖　4-5. mPEG-cystamine之1H-NMR光譜圖 36
圖　4-6. mPEG-cystamine之FT-IR光譜圖 36
圖　4-7. bis(4-nitrophenyl) diethyl sulfide之1H-NMR光譜圖 37
圖　4-8. bis(4-nitrophenyl) diethyl sulfide之FT-IR光譜圖 38
圖　4-9. mPEG-b-poly(DES-alt-cys)n之1H-NMR光譜圖 39
圖　4-10. mPEG-b-poly(DES-alt-cys)n之FT-IR光譜圖 40
圖　4-11. mPEG-b-poly(DES-alt-cys)n之GPC圖譜 40
圖　4-12. P27奈米微胞之TEM影像 42
圖　4-13. mPEG-b-poly(DES-alt-cys)27之臨界微胞濃度 43
圖　4-14. P27奈米微胞於pH.7.4 PBS與37oC之穩定度 45
圖　4-15. P27奈米微胞保存於不同溫度下之穩定度 45
圖　4-16. P27奈米微胞於不同氧化還原環境之48小時TEM影像 46
圖　4-17. P27奈米微胞於不同氧化還原環境之應答 46
圖　4-18. 攜載喜樹鹼高分子奈米微胞於不同藥物濃度下之性質 47
圖　4-19. 攜載喜樹鹼高分子奈米微胞之HPLC圖譜 48
圖　4-20. 模擬生物體內氧化還原環境之藥物釋放情形 50
圖　4-21. 蛋白質環境之藥物洩漏 50
圖　4-22. 奈米微胞對小鼠纖維母細胞L929之材料毒性測試 52
圖　4-23. 抗癌藥物喜樹鹼與攜載喜樹鹼高分子奈米微胞對小鼠纖維母細胞L929之細胞毒殺效果 52
圖　4-24. 抗癌藥物喜樹鹼與攜載喜樹鹼高分子奈米微胞對人類肺腺癌A549之細胞毒殺效果 53
圖　4-25. 抗癌藥物喜樹鹼與攜載喜樹鹼高分子奈米微胞對人類非小細胞肺癌H1299之細胞毒殺效果 54
圖　4-26. 抗癌藥物喜樹鹼與攜載喜樹鹼高分子奈米微胞對大腸癌細胞HCT116之細胞毒殺效果 55
圖　4-27. 標定FITC之攜載喜樹鹼高分子奈米微胞於腫瘤細胞氧化環境之共軛焦顯微鏡影像 57
圖　4-28. 標定Alexa 660之攜載喜樹鹼高分子奈米微胞於腫瘤細胞酸性胞器之共軛焦顯微鏡影像 58
圖　4-29. 奈米微胞、抗癌藥物喜樹鹼與攜載喜樹鹼高分子奈米微胞於3、6小時對大腸癌細胞HCT116之細胞週期影響 59
圖　4-30. 細胞週期之量化結果 60
圖　4-31. 非侵入式影像系統之全身藥物分布 61
圖　4-32. 攜載喜樹鹼高分子奈米微胞於24小時之器官累積情形 62
圖　4-33. 攜載喜樹鹼高分子奈米微胞於24小時之各器官相對螢光強 度 62
圖　4-34. 腫瘤抑制效果 64
圖　4-35. 裸鼠之肝功能指標與腎功能指標 66

表目錄
表格 1. 不同鏈長之mPEG-b-poly(DES-alt-cys)n 41
表格 2. 不同鏈長mPEG-b-poly(DES-alt-cys)n之奈米微胞 42

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