臺灣博碩士論文加值系統

本研究使用擁有兩個末端位置碳碳雙鍵之雙馬來醯亞胺(BMI) 與巴比妥酸(BTA)上之四個活性氫進行聚合反應形成STOBA溶液，再添加不同比例之L –半胱胺酸 (L-Cysteine) 進行親水性改質，形成親水性之Cys-STOBA 樣品。
研究使用BMI/BTA莫爾比3/1與2/1，分別添加不同10% Cys與5% Cys合成樣品，在藥物釋放動力學實驗中，親水性之Cys-STOBA 樣品皆在低pH值的環境下DOX有最高的釋放比例，並隨著緩衝溶液的pH值上升DOX的釋放比例跟著下降，固定BMI/BTA莫爾比情況下，添加10%Cys在相同pH值環境下都比添加5%釋放比例高；固定Cysteine情況下，莫爾比3/1比莫爾比2/1的釋放比例高。
用Cys-STOBA 樣品以HeLa為細胞做細胞毒系測試中所有樣品存活率皆和控制組差不多，因此判斷為無細胞毒性；用包覆DOX的Cys-STOBA 樣品做細胞毒系測試跟純DOX的比較，發現此載體有延緩DOX釋放的效果，最後以包覆DOX的Cys-STOBA 樣品做細胞吞噬測試發現一樣有延緩DOX釋放的結果。

In this research, the N,N'-bismaleimide-4,4'-diphenylmethane (BMI) with two terminal carbon-carbon double bonds was used to polymerize with four active hydrogens on barbituric acid (BTA) to a STOBA solution and add different ratio of L-Cysteine to modify STOBA solution. Therefore, Cys-STOBA became a hydrophilic structure.
This research used different BMI/BTA molar ratio 3/1、2/1 and add 10% Cys、5% Cys to synthesize samples. In the drug release kinetics experiments, all Cys-STOBA samples have the highest release ratio of DOX at low pH value. As the pH value of the buffer solution rises, the release ratio of DOX decreases. In the case of fixed BMI/BTA molar ratio, 10% Cys has a higher release ratio than 5% Cys；In the case of fixed Cys, molar ratio 3/1 has a higher release ratio than molar ratio 2/1.
The survival rate of all samples in the cytotoxicity experiment is similar to the control group, so it is judged to be non-cytotoxic；In the cytotoxicity experiment, Comparing the Cys - STOBA loading DOX to free DOX. This carrier is found to delay the release of DOX；In the cell uptake assay experiment. It have the same results as the cytotoxicity experiment.

摘要 I
Abstract II
致謝 III
目錄 V
圖目錄 IX
表目錄 XI
第一章 緒論 1
1.1 前言 1
1.2 研究動機 2
第二章 文獻回顧 3
2.1 雙馬來醯亞胺(BMI) 3
2.2 巴比妥酸(Barbituric acid，BTA) 4
2.3 雙馬來醯亞胺聚合反應 5
2.3.1 麥克加成反應 (Michael addition) 6
2.3.2 自由基加成反應 (Free radical addition ) 8
2.4 L –半胱胺酸 ( L-Cysteine ) 10
2.5 微膠體粒子(microgel particles) 與水膠 (hydrogel) 13
2.5.1 微膠體粒子定義與分類 13
2.5.2 微膠體粒子的製備 14
2.5.3 微膠體粒子特性及應用 15
2.5.4 水膠定義 16
2.5.5 水膠類型 17
2.6 膠體粒子表面電荷來源 20
2.7 電雙層 (The Electric Double Layer) 理論 21
2.7.1 DLVO 理論 22
2.7.2 立體空間效應 (Steric hindrance effect) 24
第三章 實驗藥品、儀器與方法 25
3.1 實驗藥品 25
3.1.1 合成實驗 25
3.1.2 配置緩衝液(PBS)之藥品 26
3.1.3 配置藥物釋放緩衝液之藥品 27
3.1.4 配置培養基之藥品 28
3.1.5 MTT試劑 29
3.1.6 DOX (阿黴素) 30
3.1.7 螢光染色之藥品 30
3.2 實驗儀器 32
3.3 實驗方法與流程 34
3.3.1 實驗內容 34
3.3.2 實驗步驟 35
3.3.3 實驗流程圖 37
3.3.4 Doxorubicin 體外釋放實驗 38
3.4 體外細胞實驗 40
3.4.1 細胞來源 40
3.4.2 細胞繼代與培養 41
3.4.3 培養基配置 41
3.4.4 磷酸鹽緩衝液(phosphate-buffered saline, PBS)配置 43
3.4.5 Trypsin-EDTA配置 45
3.4.6 細胞冷凍保存 45
3.4.7 細胞解凍及培養 46
3.4.8 實驗操作 47
3.4.9 細胞計數 48
3.4.10 細胞毒性測試 (MTT assay) 50
3.4.11 細胞吞噬測試(cell uptake assay)實驗步驟 54
第四章 結果與討論 56
4.1 藥物釋放動力學 56
4.1.1 BMI/BTA莫爾比3/1 56
4.1.2 BMI/BTA莫爾比3/1的DLE與DLC 59
4.1.3 BMI/BTA莫爾比2/1 59
4.1.4 BMI/BTA莫爾比2/1的DLE與DLC 62
4.2 體外細胞實驗 63
4.2.1 BMI/BTA莫爾比3/1 without DOX 63
4.2.2 BMI/BTA莫爾比2/1 without DOX 65
4.2.3 BMI/BTA莫爾比3/1 loading DOX 67
4.2.4 BMI/BTA莫爾比2/1 loading DOX 69
4.3 細胞吞噬測試(cell uptake assay) 72
第五章 結論 75
參考文獻 77
附錄 83

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