臺灣博碩士論文加值系統

在此研究中，P(HPMA-co-MABH)與pyridoxal hydrocloride藉由hydrazone鍵結接枝，合成出P(HPMA-co-MA-Hyd-VB6)。在酸性環境，pyridoxal會使的高分子帶正電，可以與帶負電的siRNA藉由靜電吸引力形成緊密核層，並於使用微脂粒結構包覆核層，形成具保護與載藥作用之奈米載體。當微脂粒與高分子複合系統被細胞利用吞噬作用進入細胞後，在吞噬小體可以藉由高分子的緩衝效果逃離，並於細胞質中性環境將siRNA與Dox釋放以進行基因干擾與化療效用。複合型微脂粒同時包覆了：siRNA、Dox與含有維生素B6之高分子可以有效地抑制腫瘤細胞增生並且抑制腫瘤細胞中蛋白質FAK之表現，以達到基因-化療藥物共治療之效果。

In our study, we successfully synthesized P(HPMA-co-MA-Hyd-VB6) polymers, whcich were made from P(HPMA-co-MABH) polymers and pyridoxal hydrochloride with hydrazone linkages. In the acidic surroumdings, pyridoxal provided positive charges on P(HPMA-co-MA-Hyd-VB6) polymers to interact with negative charged siRNA to form polyplex. Then polyplex liposome structure was used to encapsulate and protect the polyplex. In addition, chemotherapy drug, Doxorubicin was also loaded inside the liposomes. When the liposomes were internalized into cancer cells, P(HPMA-co-MA-Hyd-VB6) polymers could trigger the proton sponge effect for inducing endosomal escape; therefore, siRNA and dox could be released to cytosol for gene silencing and cancer chemotherapy, respectively. Liposomes encapsulated with Dox, siRNA, pyridoxal conjugated polymer could not only inhibit cancer cell proliferation, but inhibit FAK protein expression for cancer-gene co-therapy.

中文摘要…………I
Abstract…………II
目錄………………III
圖目錄……………VI
表目錄……………IX
第一章、 研究背景與動機 1
第二章、 文獻探討 3
2-1 腫瘤治療與腫瘤微環境 3
2-2 微脂粒於癌症治療之應用 8
2-3.1 微脂粒的發展 8
2-3.2 微脂粒之結構與特性 9
2-3.3 微脂粒進入腫瘤細胞模式 13
2-3 化療藥物Doxorubicin與維生素B6佐劑 14
2-4 基因治療與限制 17
2-4.1 RNA干擾作用機制 17
2-4.2 siRNA在癌症治療上的限制 18
2-5 基因載體 21
2-5.1 無機基因載體遞送 21
2-5.2 高分子載體遞送 22
2-5.3 微脂粒載體遞送 22
2-5.4 微脂粒與高分子複合系統系統遞送 23
2-6 腫瘤轉移之影響細胞凋亡 23
第三章、 實驗方法 26
3-1 實驗材料與方法 26
3-1.1 實驗藥品 26
3-1.2 實驗儀器與裝置 30
3-1.3 聚乙二醇修飾與改質 31
3-1.4 單體MABH之合成與鑑定 32
3-1.5 含維生素B6高分子之合成與鑑定 33
3-1.6 含維生素B6高分子siRNA吸附行為 35
3-1.7 含維生素B6高分子酸鹼應答行為 36
3-1.8 複合型微脂粒之製備與分析 36
3-1.9 複合型微脂粒之穩定性與藥物洩漏行為 38
3-1.10 複合型微脂粒之酸鹼與溫度應答性 39
3-1.11 複合型微脂粒之細胞毒性測試 40
3-1.12 複合型微脂粒之內吞與細胞內藥物釋放效果 41
3-1.13 複合型微脂粒對於細胞之FAK蛋白質抑制-西方點墨法 42
第四章、 結果與討論 45
4-1 聚乙二醇修飾與改質 45
4-2 單體MABH之合成與鑑定 51
4-3 含維生素B6高分子之合成與鑑定 54
4-4 含維生素B6高分子siRNA吸附行為 64
4-5 含維生素B6高分子之酸鹼應答特性 69
4-6 複合型微脂粒之製備與分析 71
4-7 複合型微脂粒之穩定性與藥物洩漏行為 72
4-8 複合型微脂粒之酸鹼與溫度應答性 75
4-9 複合型微脂粒之細胞毒性測試 78
4-10 複合型微脂粒之內吞與細胞內藥物釋放效果 81
4-11 複合型微脂粒對於細胞之FAK蛋白質抑制-西方點墨法 84
第五章、 結論 85
第六章、 參考資料 86

圖目錄
圖1-1、實驗概念圖 2
圖2-1、腫瘤微環境示意圖 3
圖2-2、Angiogenic switch示意圖 4
圖2-3、腫瘤擴張之示意圖 5
圖2-4、奈米藥物透過被動或是主動累積在腫瘤組織示意圖 6
圖2-5、載體於系統循環時影響因素示意圖 7
圖2-6、微脂粒之演進示意圖 8
圖2-7、磷脂質組成與微脂粒型態 10
圖2-8、微脂粒分類 10
圖2-9、微脂粒聚集與融合示意圖 12
圖2-10、微脂粒藥物傳遞路徑示意圖 14
圖2-11、抗癌藥物Doxorubicin抗癌作用機制 15
圖2-12、維生素B6代謝與癌症之關係 17
圖2-13、RNA干擾於真核細胞作用示意圖 18
圖2-14、基因治療應用限制示意圖 20
圖2-15、常見基因載體種類 21
圖2-16、FAK調控細胞遷移與侵犯機制示意圖 25
圖 3-1、mPEG5000-COOH之改質步驟 31
圖 3-2、mPEG5000-cholesterol之合成 32
圖 3-3、自由基單體MABH之合成 33
圖 3-4、含維生素B6高分子之合成步驟 35
圖 4-1、mPEG5000-COOH之1H NMR圖譜 46
圖 4-2、mPEG5000-COOH之FT-IR圖譜 47
圖 4-3、mPEG5000-cholesterol之1H NMR圖譜 49
圖 4-4、mPEG5000-cholesterol之FT-IR圖譜 50
圖 4-5、自由基單體MABH之1H NMR圖譜 52
圖 4-6、自由基單體MABH之FT-IR圖譜 53
圖 4-7、高分子P(HPMA-co-MABH)之1H NMR圖譜 55
圖 4-8、高分子P(HPMA-co-MA-Hyd-VB6)之1H NMR圖譜 57
圖 4-9、高分子P(HPMA-co-MA-Hyd-VB6)之FT-IR圖譜 58
圖 4-10、高分子P(HEMA-co-MA-Hyd-VB6)之1H NMR圖譜 59
圖 4-11、高分子P(HEMA-co-MA-Hyd-VB6)之FT-IR圖譜 60
圖 4-12、高分子P(MAAc-co-MA-Hyd-VB6)之1H NMR圖譜 61
圖 4-13、高分子P(MAAc-co-MA-Hyd-VB6)之FT-IR圖譜 62
圖 4-15、高分子P(HPMA-co-MA-Hyd-VB6)與P(HEMA-co-MA-Hyd-VB6)於中性環境吸附siRNA行為 65
圖 4-16、高分子P(MAAc-co-MA-Hyd-VB6)於酸性環境短時間吸附siRNA行為 66
圖 4-17、高分子P(MAAc-co-MA-Hyd-VB6)於酸性環境長時間吸附siRNA行為 66
圖 4-18、各組P(HPMA)高分子於酸性環境短時間吸附siRNA行為 68
圖 4-19、各組P(HPMA)高分子於酸性環境長時間吸附siRNA行為 69
圖4-20、含維生素B6高分子溶液之滴定圖 70
圖4-21、含維生素B6高分子溶液滴定型態變化 70
圖4-21、三種不同微脂粒於中性環境之TEM圖 72
圖4-22、微脂粒於生理環境在不同溫度下之穩定性 73
圖4-23、複合型微脂粒於不同pH值應答六小時後之TEM圖像 74
圖4-24、微脂粒於室溫生理酸鹼值之藥物洩漏行為 74
圖4-25、不同微脂粒於(A)酸性與(B)生理酸鹼值長時間釋放行為比較 76
圖4-26、不同微脂粒於(A)酸性與(B)生理酸鹼值短時間釋放行為比較 77
圖4-27、維生素B6與PHPMA-10000-45之高分子對於人類乳癌細胞MCF-7之存活率 79
圖4-29、Dox抗癌藥物於人類乳癌細胞MCF-7內之釋放效果 82
圖4-30、siRNA-FAM於人類乳癌細胞MCF-7內之釋放效果 83
圖4-31、複合型微脂粒siRNA對於人類乳癌細胞MCF-7之FAK蛋白質抑制效果-西方點墨法 84
 
表目錄
表2-1、微脂粒於臨床之應用 11
表4-1、各高分子組成與性質分析 63
表4-2、各種微脂粒之性質探討 71

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