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研究生: 陳俊安
Jun-An Chen
論文名稱: 以聚醚胺改質雙馬來醯亞胺/巴比妥酸 藥物傳遞之應用
N,N’-Bismaleimide-4,4-Diphenylmethane / Barbituric Acid modified by Jeffamine for Drug Delivery
指導教授: 陳崇賢
Chorng-Shyan Chern
口試委員: 鄭智嘉
Chih-Chia Cheng
邱信程
Hsin-Cheng Chiu
許榮木
Jung-Mu Hsu
學位類別: 碩士
Master
系所名稱: 工程學院 - 化學工程系
Department of Chemical Engineering
論文出版年: 2018
畢業學年度: 106
語文別: 中文
論文頁數: 103
中文關鍵詞: 聚醚胺巴比妥酸雙馬來醯亞胺藥物傳遞
外文關鍵詞: N,N’-Bismaleimide-4,4-Diphenylmethane, Barbituric Acid, Jeffamine, Drug Delivery
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  • 本研究以雙馬來醯亞胺(N,N’-bismaleimide-4,4’-diphenylmethane, BMI) 與巴比妥酸 (Barbituric acid, BTA) 調整BMI/BTA莫爾比為2/1及3/1進行聚合反應,形成STOBA 溶液,再使用Jeffamine M-1000進行改質,分別添加20%JF、30%JF及50%JF,最後形成親水性之STOBA-JF樣品。
    因本研究所合成之STOBA-JF具有酸鹼應答性,其在低pH值時, BTA提供的負電荷會愈少,粒徑上升;在高pH值時,JF的立體障礙使粒徑維持不變,故樣品包覆DOX後於低pH值時會因靜電作用力的減弱,而能釋放較多比例之DOX藥物。
    而使用HeLa細胞進行細胞毒性測試中所有未包覆DOX之樣品皆無明顯之細胞毒性;但若將樣品包覆DOX則在高濃度下有明顯的細胞毒性,證明具有毒殺癌細胞的功用。
    另外於細胞吞噬實驗則能進一步了解包覆DOX之樣品因靜電作用力而吸附於細胞周圍,逐漸被細胞吞噬,隨共同培養之時間增加,細胞數明顯減少,因此此一具有酸鹼應答特性之STOBA-JF樣品有極大的潛力應用於藥物載體。


    This work studied the polymerization reaction of N,N-bismaleimide-4,4-diphenylmethane (BMI) with Barbituric acid (BTA). The fisrt step was polymerized to STOBA solution by different BMI/BTA molar ratio. Second, using different proportions of Jeffamine M-1000 to modify STOBA solution so that the STOBA-JF became hydrophilic structure.
    At low pH value, BTA would provide less negative charges so that the particles size would increase;at high pH value, the particles size remain the same due to Jeffamine provided steric stabilization. Because of the electrostatic force reduce, STOBA-JF loaded DOX would release more at low pH value.
    Since all samples were non cytotoxicity in the HeLa cell cytotoxic experiment;however, all loaded DOX samples have obviously cytotoxicity in high concentration. Furthermore, the cell uptake experiment also told us that the loaded DOX samples would absorb in cell surface and be uptake slowly. As the time of co-cultivation increased, the cell density decrease significantly. As a result, these pH-responsive samples have great potential in the applications of drug carrier.

    摘要 I Abstract II 致謝 III 目錄 IV 圖目錄 VIII 表目錄 XI 第一章 緒論 1 1.1 前言 1 1.2 研究動機 3 第二章 文獻回顧 4 2.1 雙馬來醯亞胺 4 2.2 巴比妥酸及其衍生物 5 2.2.1 巴比妥酸 (Barbituric acid,BTA) 5 2.3 雙馬來醯亞胺與巴比妥酸聚合反應 7 2.3.1 麥克加成反應 (Michael addition) 7 2.3.2 自由基加成反應 (Free radical addition ) 8 2.4 Jeffamine polyetheramines ( M-1000 ) 9 2.5 微膠體粒子(microgel particles) 與水膠 (hydrogel) 11 2.5.1 微膠體粒子定義與分類 11 2.5.2 微膠體粒子的製備 11 2.5.3 微膠體粒子特性及應用 12 2.5.4 水膠定義 13 2.5.5 水膠分類 13 2.6 膠體粒子 16 2.6.1 電雙層 (The Electric Double Layer) 理論 16 2.6.2 DLVO 理論 17 2.6.3 立體障礙效應 (Steric hindrance effect) 19 第三章 實驗藥品、儀器與方法 21 3.1 實驗藥品 21 3.1.1 合成實驗 21 3.1.2 配置緩衝液之藥品 (PBS) 23 3.1.3 配置培養基之藥品 24 3.1.4 配置藥物釋放緩衝溶液之藥品 26 3.1.5 MTT試劑 26 3.1.5 DOX (阿黴素) 27 3.1.6 螢光染色之藥品 27 3.2 實驗儀器 29 3.3 實驗方法與流程 30 3.3.1 實驗內容 30 3.3.2 實驗步驟 31 3.3.3 實驗流程圖 33 3.3.4 Doxorubicin 體外釋放實驗 34 3.4體外細胞實驗 36 3.4.1 細胞來源 36 3.4.2 細胞繼代與培養 37 3.4.3 培養基配置 37 3.4.4 磷酸鹽緩衝液(phosphate-buffered saline, PBS)配置 40 3.4.5 Trypsin-EDTA配置 41 3.4.6 細胞冷凍保存 41 3.4.7 細胞解凍及培養 42 3.4.8 實驗操作 42 3.4.9 細胞計數 43 3.4.10 細胞毒性測試 (MTT assay) 45 3.4.11 細胞吞噬測試 (Cell uptake assay) 49 第四章 結果與討論 51 4.1 藥物釋放動力學 51 4.1.1 Doxorubicin 藥物釋放實驗 51 4.2 體外細胞實驗 61 4.2.1 STOBA-JF w/o DOX之細胞毒性測試 61 4.2.2 STOBA-JF w/ DOX之細胞毒性測試 66 4.2.3 細胞吞噬測試 (Cell uptake assay) 73 第五章 結論 78 第六章 參考文獻 80 附錄 一 86

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