以褐藻酸鈉調控聚電解質多層膜中DNA的吸附與釋放行為

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研究生：	鄒翔龍 Shiang-lung Tsou
論文名稱：	以褐藻酸鈉調控聚電解質多層膜中DNA的吸附與釋放行為 The modulation of DNA adsorption and release from polyelectrolyte multilayer using sodium alginate.
指導教授：	胡威文 Wei-wen Hu
口試委員:
學位類別：	碩士 Master
系所名稱：	工學院 - 化學工程與材料工程學系 Department of Chemical & Materials Engineering
畢業學年度：	100
語文別：	中文
論文頁數：	79
中文關鍵詞：	褐藻酸鈉、聚乙烯亞胺、疊層組裝
外文關鍵詞：	layer by layer, alginate, polyethyleneimine
相關次數：	點閱：21 下載：0
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褐藻酸鈉(Sodium alginate, ALG)是一個帶有負電的生物可降解高分子，在同一個環境中會與同帶負電的DNA去競爭正電高分子。在文獻中曾提到將褐藻酸鈉/DNA/聚乙烯亞胺(Polyethyleneimine, PEI)三種成分複合而成奈米粒子，藉由競爭來降低DNA與聚乙烯亞胺間的相互關係，並因此提高了基因轉染的效果。由於疊層組裝(Layer-by-Layer)系統對於細胞轉染表現還有增進的空間，所以在本篇研究中，我們想要利用褐藻酸鈉、DNA與聚乙烯亞胺三成分進行疊層組裝疊層組裝，形成(DNA/PEI/ALG/PEI)的模型，探討其物化性質並觀察是否也同樣能提升對細胞的轉染表現。從接觸角與紅外光譜來看我們知道三成分確實有吸附於材料表面。接著，在紫外光度計與石英微量天平的量測觀察中發現隨著褐藻酸鈉層濃度的提升(0.004, 0.02, 0.1, 0.5 g/l)，對於疊層組裝中DNA的吸附與釋放效果也會跟著提高，相較於由DNA與聚乙烯亞胺製備的疊層組裝在DNA吸附量來說可以提升到兩倍以上並且DNA釋放量提高50%左右，並且更深入去由大小與複合程度探討多層膜釋放出來粒子型態。結果顯示，在褐藻酸鈉濃度為0.1 g/l製備的疊層組裝能得到最佳的細胞轉染表現。

Sodium alginate(ALG) is a biodegradable polyelectrolyte with negative charges, which may compete the positive polyelectrolyte with anionic DNA. Therefore, some reports indicated that alginate can interact with DNA/polycation and form a triple component complex. This particle is able to enhance the gene transfectrion efficiency due to electrostatic neutralization of the complex. Due to this property, we want to complex the three component with layer-by-layer(LbL) to enhance the transfection ability. So, in this study, we use different alginate concentration(0.004, 0.02, 0.1, 0.5 g/l) create the (DNA/PEI/ALG/PEI)4 LbL model and investigate the properties of DNA changes during the multilayer system measuring by ATR-FTIR, contact angle, quartz crystal microbalance (QCM), UV-vis, DLS and eletrophoresis gel. We observe there is two-fold DNA release at the 0.1g/l alginate assembly condition and the DNA adsorption increase with increasing alginate concentration. Also, the particle size which release from the multilayer increase too. At least, we use the multilayer to transfect the NIH/3T3 cells. Observing the transfction efficiency at 0.1g/l of (DNA/PEI/ALG/PEI)4 multilayer enhanced up to three times compare to (DNA/PEI)4 .

摘要 ......................................................................................................... I
Abstract .................................................................................................. II
目錄 ....................................................................................................... III
圖目錄 ................................................................................................... VI
第一章 序論 ........................................................................................ 1
1-1 背景 ........................................................................................ 1
1-2 實驗目的 ................................................................................ 2
第二章 文獻回顧 .......................................................................... 3
2-1 組織工程 ................................................................................ 3
2-2 生長因子種類......................................................................... 5
2-3 應用於基因釋放的生醫材料 ................................................. 6
2-3-1 聚乙烯亞胺 .................................................................. 7
2-3-2 褐藻酸鹽 ...................................................................... 9
2-4 控制釋放系統的載體 ........................................................... 11
2-5 疊層組裝 .............................................................................. 15
2-5-1 疊層組裝之原理 ......................................................... 15
2-5-2 疊層組裝之製備 ......................................................... 16
2-5-3 疊層組裝之應用 ......................................................... 17
IV
2-6 紫外光光譜儀....................................................................... 21
2-6-1 紫外光光譜儀原理 ..................................................... 21
2-7 螢光光度計 .......................................................................... 23
2-7-1 螢光光度計原理 ......................................................... 23
2-8 石英微量天平....................................................................... 25
2-8-1 石英微量天平檢測原理 ............................................. 25
第三章 實驗方法 ........................................................................ 27
3-1 實驗架構 .............................................................................. 27
3-2 實驗藥品與儀器 ................................................................... 28
3-2-1 藥品 ............................................................................ 28
3-2-2 儀器 ............................................................................ 29
3-3 試藥製備 .............................................................................. 30
3-3-1 DNA cloning ............................................................... 30
3-3-2 陽離子水溶液............................................................. 34
3-3-3 陰離子水溶液............................................................. 34
3-3-4 DMEM ........................................................................ 34
3-3-5 FBS ............................................................................. 34
3-3-6 DMEM with 10% FBS ................................................ 35
3-3-7 電泳膠片 .................................................................... 35
3-4 LbL製備 .............................................................................. 36
3-5 NIH/3t3細胞轉染實驗 ......................................................... 37
V
3-5-1 空白組製備 ................................................................ 37
3-5-2 Spiked alginate組 ....................................................... 37
3-5-3 夾層alginate 組 ......................................................... 37
3-6 材料化學表面分析 ............................................................... 38
3-6-1 ATR-FTIR 全光譜 ..................................................... 38
3-6-2 接觸角分析 ................................................................ 39
3-6-3 吸附實驗 .................................................................... 40
3-6-4 釋放實驗 .................................................................... 42
3-6-5 粒徑分布分析............................................................. 43
3-6-6 電泳量測DNA包覆程度 .......................................... 43
第四章 結果與討論 .................................................................... 44
4-1 外加褐藻酸鈉分析 ............................................................... 44
4-2 疊層組裝的材料分析 ........................................................... 46
4-2-1 接觸角分析 ................................................................ 46
4-2-2 紅外光譜儀分析 ......................................................... 47
4-3 褐藻酸鈉水溶液濃度對疊層組裝中DNA吸附量與釋放量的影響 ............................................................................................ 49
4-3-1 吸附 ............................................................................ 49
4-3-2 釋放 ............................................................................ 54
VI
4-4 由不同濃度褐藻酸鈉所製備的(DNA/PEI/ALG/PEI)疊層組裝轉染分析 .................................................................................... 55
4-5 從疊層組裝系統釋放出來的粒子分析 ................................ 57
4-5-1 包覆程度分析............................................................. 57
4-5-2 粒徑大小分析............................................................. 58
第五章 結論 ...................................................................................... 60
第六章 參考文獻 .............................................................................. 74
圖目錄
圖1. 分枝狀聚乙烯亞胺結構 ............................................................ 7
圖2. 質子海綿假說模型 .................................................................... 8
圖3. 褐藻酸鹽類結構 ........................................................................ 9
圖4. 細胞在不同固定褐藻酸鈉與聚乙烯亞胺與不同質量DNA(eGFP)合成的奈米粒子，對細胞轉染強度的表現 ..................... 11
圖5. 不同類型基因載體(a)陽離子脂質體，(b)陽離子高分子 ....... 13
圖6. 疊層組裝製備方式 .................................................................. 16
圖7. 玻片上做疊層組裝對COS-7細胞轉染情形 .......................... 20
圖8. 不同時間點疊層組裝中SPT7與eGFP質體DNA轉染細胞的情形 ....................................................................................................... 20
VII
圖9. 分子軌域躍遷 .......................................................................... 22
圖10. 電子躍遷圖 .............................................................................. 24
圖11. 自身纏繞的高分子以及存在於高分子內被遮蔽的官能基 .... 52
圖12. 褐藻酸鈉與DNA負電荷高分子存在於疊層組裝表面對於聚乙烯亞胺吸附的可能情況 .................................................................... 53
圖13. 細胞培養於(DNA/PEI)5的多層膜，並外加不同質量褐藻酸鈉，以螢光強度分析GFP表現 ........................................................... 62
圖14. 不同褐藻酸鈉濃度製備(DNA/PEI/ALG/PEI)4去轉染細胞的結果-螢光強度分析GFP表現 ............................................................. 63
圖15. PEI之紅外光譜圖 ................................................................... 64
圖16. DNA之紅外光譜圖 ................................................................ 65
圖17. ALG之紅外光譜圖 ................................................................. 66
圖18. (ALG/PEI)之疊層組裝與PEI及ALG純材料對照之紅外光譜圖 ................................................................................................. 67
圖19. (DNA/PEI/ALG/PEI)之疊層組裝與(ALG/PEI)及DNA純材料對照之紅外光譜圖 ............................................................................... 67
圖20. (DNA/PEI/ALG/PEI)4疊層組裝接觸角分析 ......................... 68
圖21. (DNA/PEI/ALG/PEI)4不同ALG濃度下DNA吸附行為 ..... 68
圖22. (DNA/PEI/ALG/PEI)4 不同ALG濃度下DNA釋放行為 .... 69
VIII
圖23. (DNA/PEI/alg/PEI)4 不同ALG濃度下DNA釋放比例 ....... 69
圖24. QCM實驗，(DNA/PEI/ALG/PEI)疊層組裝之 raw data ....... 70
圖25. QCM實驗，(DNA/PEI)疊層組裝之 raw data ....................... 70
圖26. QCM實驗，(DNA/PEI/ALG/PEI)與(DNA/PEI)疊層方式，DNA的吸附量 ............................................................................................... 71
圖27. QCM實驗，疊層組裝中每層PEI吸附量 ............................. 71
圖28. QCM實驗，(DNA/PEI/ALG/PEI)疊層組裝中各層ALG的吸附量。 ................................................................................................. 72
圖29. 不同條件疊層組裝釋放後之複合體粒徑大小 ....................... 72
圖30. 不同條件疊層組裝釋放後之複合體包覆程度電泳分析 ........ 73

                                

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