蔡偉博臺灣大學：化學工程學研究所陳英豪Chen, Ying-HaoYing-HaoChen2007-11-262018-06-282007-11-262018-06-282007http://ntur.lib.ntu.edu.tw//handle/246246/52084聚電解質多層膜由相反電荷的聚電解質逐層吸附於基材上而形成，其優點為能夠製備不同功能的表面，且可應用於不同地形的基材上，十分適合應用於生醫材料之表面改質。細胞與多層膜表面的交互作用可能會受到某些參數的影響，例如層數、膜製備條件等。為了避免細胞貼附在生醫器材上，而導致不必要的臨床併發症，聚氧乙烯(Polyethylene glycol)為抑制貼附的生醫材料，因為它本身具有親水和高度水和性的特性來阻止蛋白質吸附和細胞貼附。本研究目的是結合多層膜和PEG形成表面布滿PEG的抗吸附表面，觀察PEG接枝於多層膜上，對於L929細胞貼附、形態及生長之影響。 Poly(ethylene imine) (PEI)/Poly(acrylic acid) (PAA)為聚電解質多層膜正負電聚合物，最上層吸附PEG-g-PEI，接枝比例不同的PEG-g-PEI為不同層數之多層膜的最上層，評估L929細胞數量與型態貼附材料表面的抑制效果。本研究發現隨著接枝PEG比例越高的PEG-g-PEI於不同膜層數中最上層可以有效抑制細胞貼附，細胞型態跟PEI和TCPS表面有很大的差異性。隨著層數的增加，高比例的PEG-g-PEI的細胞貼附量會有些微上升，細胞貼附型態上以圓形為主。高接枝比例的PEG-g-PEI多層膜浸泡一跟兩週PBS可以提供穩定性且可以達到更好的抑制細胞貼附的效果。Layer-by-layer (LBL) deposition of polyelectrolyte multilayer films is an extremely versatile and useful technique for surface modification of medical devices. Several parameters in preparation of polyelectrolyte multilayer films, such as the number of layers and assembling conditions, might influence the interactions between surface and biological system. In order to reduce cell adhesion on medical devices which may cause undesired clinical complications, polyethylene glycol (PEG) has been considered a non-adhesive biomaterial because it possesses hydrophilic and heavily hydrated character to resist protein adsorption and cell adhesion. The aim of this work was to combine these two advantages, LBL and PEG, together to built up anti-adhesive polyelectrolyte multilayer films (PEMs) to cover the surface with PEG. The L929 cell was seeded onto peglyated PEMs, and cell adhesion, morphology and growth was studied. We established a layer-by-layer polyelectrolyte multilayer membrane based on poly(ethylene imine) /poly(acrylic acid)(PEI/PAA) with PEG-g-PEI as the topmost layers. Different graft ratios of PEG-g-PEI were synthesized to be different topmost layers to evaluate the efficacy of PEMs on inhibiting L929 cell adhesion and spreading by observing the morphology of L929 cell on the surface. We found that the top layer of pegylated PEI decreased cell adhesion, and L929 cell adhesion and morphology had great difference on the surface with high PEG graft ratios compared to the original TCPS substrate. High graft ratio of PEG-g-PEI increased cell adhesion amounts slightly with increasing number of layers, however cell morphology was still round shape. The high grafted ratio of peglyated PEMs immersed in PBS could stable during two weeks. The cell adhesion on peglyated PEMs of immersed PBS had better inhibition than PEMs without immersed PBS.目錄 誌謝 I 摘要 III Abstract V 目錄 VII 圖目錄 IX 表目錄 XII 英文縮寫與符號說明 XIII 中英對照 XV 第一章 緒論 1 1.1 研究背景 1 1.2 聚電解質多層膜介紹 2 1.2-1 聚電解質多層膜堆疊 2 1.2-2 影響聚電解質多層膜製備因素 3 1.2-3 QCM量測聚電直多層膜性質 6 1.2-4 聚電解質多層膜之生物相容性 7 1.3 PEG (Polyethylene glycol) 介紹 9 1.3-1 PEG接枝反應 9 1.3-2 PEG的血液與生物相容性 10 1.3-3 帶電性高分子接枝PEG 12 1.3-4 PEG-g-PEI介紹 12 1.4 研究動機 13 1.5 研究架構 14 1.6 研究目的 14 第二章 實驗藥品、儀器與方法 27 2.1 實驗藥品 27 2.1-1 PEG-g-PEI反應與多層膜製備 27 2.1-2 QCM晶片 27 2.1-3 細胞培養 27 2.2 實驗儀器 28 2.3 實驗耗材 29 2.4 溶液配置 29 2.5 實驗方法 30 2.5-1 PEG與Cyanuric chloride反應 30 2.5-2 PEI與mPEG-cyanuric chloride反應步驟 31 2.5-3 PEI與mPEG-cyanuric chloride氫譜鑑定 32 2.5-4 聚電解質多層膜製備 32 2.5-5 聚電解質多層膜分析 33 2.5-5-1 接觸角分析 33 2.5-5-2 石英微量天平 34 2.5-6 蛋白質吸附 37 2.5-7 細胞培養 37 2.5-8 細胞數測定 38 2.5-9 細胞貼附面積分析 38 2.5-10 統計分析軟體 38 第三章 實驗結果 45 3.1 PEG-g-PEI反應之鑑定 45 3.1-1 CmPEG純化 45 3.1-2 PEG-g-PEI鑑定 46 3.2 聚電解質多層膜分析 47 3.3 PEG-g-PEI對於蛋白質抑制作用 48 3.4 多層膜對於細胞抑制作用 49 3.5 多層膜穩定性測試 52 第四章 討論 73 第五章 結論與未來展望 83 5.1 結論 83 5.2 未來展望 85 參考文獻 87 附錄 912079939 bytesapplication/pdfen-US聚電解質多層膜聚乙二醇聚乙烯亞胺polyelectrolyte multilayer filmspolyethylene glycolpolyethylene iminethesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/52084/1/ntu-96-R94524016-1.pdf