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研究生:陳永安
研究生(外文):Yung Ann Chen
論文名稱:異丙基丙烯胺/丙烯酸共聚物[P(NIPAAm-co-AAc)]作為眼用藥物釋放載體之研究
論文名稱(外文):Studies of intravitreal carries for sustained released of ophthalmic drugs by P(NIPAAm-co-AAc)
指導教授:蔡曉雯蔡曉雯引用關係
指導教授(外文):S. W. Tsai
學位類別:碩士
校院名稱:長庚大學
系所名稱:生化與生醫工程研究所
學門:工程學門
學類:化學工程學類
論文種類:學術論文
論文出版年:2008
畢業學年度:96
論文頁數:71
中文關鍵詞:老年黃斑部病變溫感性水膠釋放趨勢
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中文摘要

老年性視網膜黃斑部病變(AMD)為一種眼部產生新生血管所造成的疾病,已經有許多方法用於改善病人的病情,目前臨床上,較常使用的治療方式為於玻璃體腔內注射抗血管新生藥物-Avastin,Avastin為一種對於VEGF分子具有拮抗作用的單株抗體。然而直接於玻璃體腔內注射Avastin,藥物可作用的半衰期相當短,因此,此研究主要目的為製備一可持續釋放Avastin的水膠載體-P(NIPAAm-co-AAc),並評估載體釋放特性。
藉由合成過程中加入不同莫爾數比的N-isopropylacrylamide (NIPAAm)與Acrylic acid (AAc)可製備出多種的共聚物。由實驗結果顯示,這些共聚物均可在三分鐘內完全凝膠,將這些共聚物與不同分子量的蛋白質混合後,探討其釋放趨勢。由體外試驗結果得知,實驗中所使用的各種不同分子量的蛋白質,P(NIPAAm)水膠相較於P(NIPAAm-co-AAc)水膠均具有明顯burst release的現象。此外,於P(NIPAAm)與P(NIPAAm-co-AAc)水膠載體的釋放過程中,牛血清白蛋白、γ-球蛋白與Avastin的釋放量均出超過75%,且在1200分鐘達釋放平衡。
P(NIPAAm-co-AAc)水膠系統不僅不會改變Avastin投藥方式,延長Avastin於體內的半衰期,可在未來療性試劑的傳遞上,提供選擇。
Abstract
Age-related macular degeneration (AMD) is one of the angiogenesis ocular diseases. Numerous methods have been developed to prevent the patients losing of vision. In clinical, one of the treating methods is intravitreal administration of anti-VEGF (vascular endothelial growth factor) antibody, Avastin that is a full-length monoclonal antibody against the VEGF molecule. However, Avastin is hinder by the short half-life when using intravitreal injection directly. Thus, the purpose of this study is to fabrication a sustained release carrier of Avastin by P(NIPAAm-co-AAc) hydrogel and characterization.
Various copolymer of P(NIPAAm-co-AAc) were prepared by polymerization with various molar ratio of N-isopropylacrylamide (NIPAAm) to acrylic acid (AAc). Experimental results inducated all of these hydrogels could completely gelation less than 3 minutes. Mixture of copolymer and various molecular weight proteins to investigate the release profiles. From the in vitro studies, the P(NIPAAm) hydrogel was found to display significant burst release compared P(NIPAAm-co-AAc) regardless proteins used. And the release amount of bovine serum albumin, γ-globulin and Avastin could reach more than 75% in both of P(NIPAAm) and P(NIPAAm-co-AAc) hydrogels within 1200 minutes.
The hydrogel system does not alter the administration method of Avastin, but also could elongation the half-time of Avastin, and may offer a mean of delivering other therapeutic agents in the future.
目錄

指導教授推薦書
口試委員審定書
授權書 iii
誌謝 iv
中文摘要 v
Abstract vii
第一章 緒論 1
1-1 前言 1
1-2 研究背景 2
1-2-1 溫感性水膠簡介 3
1-2-2 老年性視網膜黃斑部病變(AMD)介紹 7
1-3 研究動機與目的 12

第二章 實驗藥品與儀器 13
2-1 實驗藥品 13
2-2 儀器設備 14

第三章 實驗方法 15
3-1共聚物製備與凝膠情形測試 15
3-2 1H-NMR圖譜鑑定AAc的接枝情形 16
3-3以酸鹼滴定方式定量AAc的接枝率 16
3-4共聚物之細胞毒性測試 16
3-5藥物釋放試驗 17
3-6 Avastin 抑制血管內皮細胞生長測試 18
3-7 Avastin 對於內皮細胞生長測試 18
3-8載體於生物體內凝膠情形測試 19

第四章 結果與討論 24
4-1 共聚物製備與凝膠情形測試 24
4-2 共聚合物之1H -NMR的測定 25
4-3 共聚合物之酸鹼滴定測試 25
4-4 共聚合物之細胞毒性測試 26
4-5 藥物釋放的探討 27
4-6 Avastin 抑制血管內皮細胞生長測試 30
4-7 Avastin 對於內皮細胞生長測試 31
4-8 載體於生物體內凝膠情形測試 32

第五章 結論 53

第六章 參考文獻 55

圖目錄
圖3-1 NIPAAm與AAc共聚合反應化學式 20
圖3-2於1H-液態核磁共振光譜圖(溶劑:D2O) 21
圖3-3不同莫爾數比的共聚合物包覆藥物後置於37度的生理食鹽水之中 22
圖3-4以紐西蘭白兔為實驗動物,於右眼注入藥物與載體的混合溶液,於左眼注入生理食眼水手術過程 23
圖4-1不同莫爾數比的共聚合物溶於D2O的NMR之1H圖譜 34
圖4-2以萃取法對pure NIPAAm的共聚合物進行細胞毒性測試於光學顯微鏡下觀察 35
圖4-3以0.1N的NaOH滴定不同莫爾數比的共聚合物 36
圖4-4 BSA於P(NIPAAm-co-AAc) hydrogel (NIPAAm:AAc= 98:2, 60 mg/ml)中的釋放情形 37
圖4-5 BSA於P(NIPAAm) hydrogel (60 mg/ml)中的釋放情形 38
圖4-6 γ-globulin於P(NIPAAm-co-AAc) hydrogel (NIPAAm:AAc= 98:2, 60 mg/ml)中的釋放情形 39
圖4-7 γ-globulin於P(NIPAAm) hydrogel (60 mg/ml)中的釋放情形 40
圖4-8 Avastin於P(NIPAAm-co-AAc) hydrogel (NIPAAm:AAc= 98:2, 60 mg/ml)中的釋放情形 41
圖4-9 Avastin於P(NIPAAm) hydrogel (60 mg/ml)中的釋放情形 42
圖4-10 比較以不同莫爾分率的P(NIPAAm-co-AAc) copolymer對BSA的釋放情形 43
圖4-11 比較不同藥物於P(NIPAAm-co-AAc) hydrogel (NIPAAm:AAc= 98:2,60 mg/ml)中的釋放情形 44
圖4-12 比較不同藥物於P(NIPAAm) hydrogel(60 mg/ml)中的釋放情形(BSA、γ-globulin與Avastin) 45
圖4-13 比較以不同莫爾分率的P(NIPAAm-co-AAc) copolymer對BSA釋放情形的差異 46
圖4-14 比較以不同莫爾分率的P(NIPAAm-co-AAc) copolymer對γ-globulin釋放情形的差異 47
圖4-15 比較以不同莫爾分率的P(NIPAAm-co-AAc) copolymer對Avastin釋放情形的差異 48
圖4-16 以Avastin進行抑制HUVEC生長情形測試 59
圖4-17 探討Avastin對於ARPE-19生長的影響 50
圖4-18 將藥物與載體的混合溶液以注射的方式注入白兔眼內,觀察載體於生物體內凝膠情形測試 51
圖4-19 以藥物與載體混合溶液注入白兔眼內,由外觀察載體於生物體內凝膠情形測試 52

表目錄

表1-1 各類溫感性水膠的低臨界溶液溫度(LCST) 4
表1-2 各類抗血管新生因子 11
表4-1 37℃下不同條件水膠之凝膠情形整理 33
第六章 參考文獻
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