臺灣博碩士論文加值系統

本研究利用環氧乙烷(ethylene oxide, EO)滅菌使硫辛酸修飾聚賴氨酸(lipoic acid grafted poly(L-lysine), PLL-g-Lipo)生成分子鏈之間的雙硫交聯結構，進而能夠吸水膨脹成聚胜肽水膠，而聚胜肽/二氧化矽複合水膠則是在聚胜肽水膠中加入甲氧基矽烷(tetramethyl orthosilicate, TMOS)作為二氧化矽前驅物製備而得。由於鏈與鏈間形成了雙硫鍵結(disulfide bond)與高分子鏈本身的纏繞行為(Entanglement)，使得PLL-g-Lipo在很低的濃度就能夠成膠，透過掃描電子顯微鏡（scanning electron microscope, SEM）的觀察可發現其具有膜狀的網絡結構。聚胜肽與聚胜肽/二氧化矽複合水膠之力學性質會因高分子鏈長、硫辛酸接枝率以及水膠之組成(有機物與無機物的含量)而有所不同。在藥物包覆與釋放的實驗中發現，釋放速率可透過調整溶液之酸鹼值，以及加入穀胱甘肽(glutathione, GSH)製造出還原環境來控制，代表此種水膠具有酸鹼與還原雙重應答的性質。研究結果亦顯示出，二氧化矽的沉積不但可以穩定水膠的網絡結構、降低水膠之細胞毒性，也可以改變水膠的力學性質與藥物釋放行為。藉由適當的調控便可使此種水膠具有一定的穩定性與藥物釋放速率，因此具有作為藥物載體之潛力。

In this research, the preparation of dual stimuli-sensitive, biocompatible lipoic acid-modified poly(L-lysine) (PLL-g-Lipo) hydrogels/nanogels by chemical cross-link and polypeptide/silica hybrid hydrogels by depositing silica in the gel matrix. By ethylene oxide sterilization, hydrogels were prepared through the formation of inter-/intramolecular disulfide cross-link. Their mechanical properties and gelation were dictated by the amphiphilic nature and degree of inter-/intramolecular disulfide cross-link, which were influenced by the chain conformation. Upon changing the solution pH from neutral to acidic condition and/or cleaving disulfide bonds, these hydrogels showed redox- and pH-sensitive properties as demonstrated by the accelerated drug release. Silica deposition can stabilize the gel network and tune their mechanical properties as well as their payload release and colloidal properties.

總目錄
第一章 緒論 1
第二章 文獻回顧 3
2.1 聚胜肽 3
2.1.1 胺基酸的簡介 3
2.1.2 肽的簡介 4
2.1.3 聚胜肽的構型 5
2.2 水膠與其應答種類 6
2.2.1 水膠的簡介 6
2.2.2 水膠的應答行為 6
2.3 有機與無機複合材料 9
2.4 環氧乙烷滅菌 9
第三章 實驗方法與步驟 11
3.1 實驗藥品 11
3.2 實驗儀器與原理 12
3.2.1 凝膠滲透層析儀 12
3.2.2 核磁共振光譜儀 14
3.2.3 紫外線/可見光光譜儀 14
3.2.4 傅立葉轉換紅外線光譜儀 15
3.2.5 掃描式電子顯微鏡 16
3.2.6 流變儀 16
3.2.7 熱重分析儀 17
3.2.8 酶標儀 17
3.3 實驗方法 18
3.3.1 聚賴氨酸之合成 18
3.3.2 以硫辛酸於聚賴氨酸之側鏈進行修飾 20
3.3.3 利用環氧乙烷交聯硫辛酸修飾聚賴氨酸 22
3.3.4 製備聚胜肽水膠及聚胜肽/二氧化矽複合水膠 22
3.3.5 藥物包覆與釋放 22
3.3.6 細胞毒性測試 23
第四章 結果與討論 24
4.1 材料的性質與鑑定 24
4.1.1 聚賴氨酸之鏈長鑑定 24
4.1.2 硫辛酸之修飾比例鑑定 25
4.1.3 硫辛醯基與聚胜肽水膠 27
4.1.4 冷凍乾燥聚胜肽水膠之二級結構 28
4.1.5 凍乾聚胜肽水膠與複合水膠之表面型態 33
4.1.6 聚胜肽水膠與複合水膠之力學性質分析 36
4.1.7 聚胜肽水膠與複合水膠之細胞毒性測試 38
4.2 透過藥物包覆與釋放觀察水膠之酸鹼與還原應答 39
第五章 結論 41
第六章 參考文獻 42

 
圖目錄
圖2.1胺基酸結構示意圖 3
圖 2.2 由胺基酸序列發展至四級結構 5
圖 3.1 GPC圖譜，紅線為折射率分析、綠線為粒徑分析、藍線為黏度分析 13
圖 3.2合成聚賴氨酸之流程圖 18
圖 3.3 Nε-Z-L-Lysine NCAs的合成 19
圖3.4 EDC/NHS活化羧酸之反應機制 21
圖3.5 lipoic acid接枝於PLL側鏈之流程圖 21
圖4.1以PLL90-g-Lipo0.1的1H NMR圖譜為例，lipoic acid接枝比例的計算 26
圖4.2以短鏈PLL的1H NMR圖譜為例，lipoic acid接枝比例增加則硫辛醯基的峰值也增加。(a) PLL50、(b) PLL50-g-Lipo0.1、(c) PLL50-g-Lipo0.2 26
圖4.3利用UV-vis測量EO滅菌前後硫辛醯基訊號之差異 27
圖4.4加入過量GSH造成鏈與鏈之間的雙硫鍵結斷裂，使得水膠的網絡結構被破壞而崩解 28
圖4.5不同lipoic acid接枝率、不同鏈長的PLL-g-Lipo IR圖譜，分別為(a) PLL180-g-Lipo0.2，(b) PLL180-g-Lipo0.1，(c) PLL50-g-Lipo0.2，(d) PLL50-g-Lipo0.1，(e) PLL90-g-Lipo0.1，樣品皆經EO滅菌處理 29
圖4.6 未經EO殺菌處理的 (a) PLL90-g-Lipo0.1 (b) PLL180-g-Lipo0.1 (c) PLL180-g-Lipo0.2的CD圖譜，主要為random coil並且帶有少許的-helix 30
圖4.7未經EO殺菌處理，但放置三個月以上的(a) PLL180-g-Lipo0.1，(b) PLL90-g-Lipo0.1的IR圖譜，顯示出放置一段時間後，鏈長較長者發生部分自我重新排列的情況，較短者則無 30
圖4.8簡化的乙氧基矽烷縮合反應 31
圖4.9不同TMOS加入量的IR圖譜，水膠濃度為3.5 wt%：(a) 0 v/v%，(b) 5 v/v%，(c) 10 v/v%，(d) 15 v/v%，(e) 20 v/v% 32
圖4.10不同TMOS加入量的TGA圖譜，水膠濃度為3.5 wt%：(a) 0 v/v%，(b) 5 v/v%，(c) 10 v/v%，(d) 15 v/v%，(e) 20 v/v% 33
圖4.11 EO滅菌過的PLL-g-Lipo能吸水，體積膨脹數倍而成膠 33
圖4.12凍乾後水膠的SEM圖：(a) PLL180-g-Lipo0.2，(b) PLL180-g-Lipo0.1，(c) PLL50-g-Lipo0.2，(d) PLL50-g-Lipo0.1，(e) PLL90-g-Lipo0.1，水膠濃度為3.5 wt% 34
圖4.13加入TMOS (a-b) 5 v/v%，(c-d) 10 v/v%，(e-f) 15 v/v%，(g-h) 20 v/v% 的PLL90-g-Lipo0.1凍乾水膠SEM圖，水膠濃度為3.5 wt% 35
圖4.14(a) 四種不同鏈長與接枝率的PLL-g-Lipo的儲存模數G’(實心符號)與損失模數G’’(空心符號)對應力作圖的變化趨勢，水膠濃度為3.5 wt%，掃描頻率為100 rad/s：(▲) PLL50-g-Lipo0.1，(●) PLL50-g-Lipo0.2，(◆) PLL90-g-Lipo0.1 以及 (■) PLL180-g-Lipo0.2；(b) 對頻率作圖，應力為1% 36
圖4.15(a) 加入不同TMOS體積比例的PLL90-g-Lipo0.1的儲存模數G’(實心符號)與損失模數G’’(空心符號)對應力作圖的變化趨勢，水膠濃度為3.5 wt% ，掃描頻率為100徑/秒：(◆) 0 v/v%，(●) 5 v/v%，(■) 10 v/v% 及 (▲) 20 v/v%；(b) 對頻率作圖，應力為1% 37
圖4.17 藥物釋放總量與時間關係圖，Hybrid代表加入5 v/v% TMOS，Polypeptide代表不含TMOS 40

 
表目錄
表2 1列舉部分常見的胺基酸 4
表4 1 PZLL的分子量與PDI 24
表4 2不同鏈長與接枝比例之樣品名稱對照表 25
表4 3以Scheffe作為事後檢定來判斷樣品之毒性 39

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