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研究生： 湯偉鉦
論文名稱： γ-聚麩胺酸與聚乳酸組成之梳狀接枝共聚合物於藥物傳輸之研究
The Study of Comb-Like Copolymer γ-Poly (Glutamic Acid)-Poly L-Lactide for Drug Delivery
指導教授： 朱一民
口試委員:
學位類別： 碩士
Master
系所名稱： 工學院 - 化學工程學系
Department of Chemical Engineering
論文出版年： 2008
畢業學年度： 96
語文別： 中文
論文頁數： 84
中文關鍵詞： γ-聚麩胺酸 、梳狀共聚物 、高分子微胞 、藥物載體 、細胞毒性 、太平洋紫杉醇
外文關鍵詞： γ-poly glutamic acid, comb-like, drug carrier, amphiphilic, graft-copolymer, cytoxicity
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    • 近年來高分子微胞藥物載體在藥理學上的開發與應用備受期待，如何能讓微胞更穩定於體內循環並緩釋藥物，是現今重要課題之一。
    為了降低高分子的臨界微胞濃度，以提升其在人體內穩定度，並且讓enhanced permeability and retention effect發揮最大功能，著手嘗試以新穎天然高分子γ-聚麩胺酸為親水主鏈，以及通過美國食品藥物管理局（Food and Drug Administration，FDA）認可的生醫高分子聚乳酸為疏水側鏈，合成具有較高分子量的梳狀兩性高分子共聚合物（comb-like amphiphilic copolymer）。
    實驗中利用苯甲醇開環L-lactide合成了具單邊芳香烃取代基團的PLA，並透過質子化方式使γ-PGA在dimethyl sulfoxide中溶解度增加，再利用carbondiimide-based的常溫酯化反應以one-step reation成功合成γ-PGLA接枝共聚物（graft copolymer）。製備微胞是利用類似溶劑揮發法，以有機相tetrahydrofuran與水相1%葡萄糖水溶液共存方式使兩性高分子在介面自組裝（self-assembly），成功製備成直徑約110nm~150nm之奈米高分子微胞，當親疏水鏈段比值為1/2時可形成最小粒徑。
    電子顯微鏡下成功證明微胞的球體結構，影像中可觀察到粒子大小均勻分布在150nm左右；臨界微胞濃度分析結果顯示，當疏水鏈段總分子量增大時，臨界微胞濃度也隨之降低，且各親疏水鏈段比值之臨界微胞濃度與傳統團聯共聚物相比，均有較低的趨勢，這使得梳狀高分子在體內可能形成較穩定的微胞結構，而有利於提升藥物載體在體內之循環時間，並達到標的的功能。
    以形成最佳粒徑的γ-PGLA12嘗試包覆疏水抗癌藥物paclitaxel，在不同的初始投藥量包覆效率均可達到90％，包覆藥物量最高可達6%（drug/polymer）；不同含藥量的微胞完整釋藥時間均約兩天，且藥物釋放百分比可達90％以上，唯藥物在初期驟釋的現象仍具有改善空間；細胞毒性測試結果顯示材料具有良好生物相容性，在高濃度（500ppm）下與3T3 cell培養兩天仍可維持80％的存活率，且在包覆抗癌藥物paclitaxel後能在一天內有效毒殺細胞。

    Drug carriers are composed of polymeric micelle have great therapeutic potential. One problem need to be solved is how to make this kind of carriers have long-circulation and controlled-release ability. In order to reduce critical micelle concentration (CMC) to lift in vivo stability of drug carriers, and bring the enhanced permeability and retention effect to full play, a novel comb-like amphiphilic copolymer which is composed of γ-poly (glutamic acid)-graft-poly lactide (γ-PGLA) synthesized in this study successfully.
    In this research, we used carbodiimide-based reaction to graft aromatic-end poly lactide onto the protonated γ-poly-(glutamic acid). The micellar solution prepared by a pseudo-solvent-evaporation method, and the particle size of polymeric micelle was between 110nm~150nm with narrow distribution. Additionally, we found that the smallest micelle was formed when hydrophilic/hydrophobic ratio equals to 1/2. The spherical structure of micelle was identified by AFM and SEM images.
    For convenient storage and micellar stability, we used glucose instead of poly vinyl alcohol which has slight toxicity as the lyoprotectant to lyophilize and preserve the micelle. From fluorescence spectra, we found that the CMC would reduced with higher hydrophobic ratio of copolymers. Furthermore, γ-PGLA copolymers have lower CMCs than conventional block-copolymers in each hydrophilic/hydrophobic ratio； this result indicated that the comb-like copolymers can be expected to form more stable micelle.
    Paclitaxel-encapsulation results revealed that drug/polymer ratio can be considerably raised if we removed organic solvents by rotary evaporator rapidly. The result of in-vitro release study indicated the faster releasing rate in higher paclitaxel-loading micelle, this may result from that encapsulated paclitaxel will interfere with core stability of micelle. Finally, the in-vitro cytoxicity test demonstrated that this new material can be a non-toxic and efficient carrier for paclitaxel, and the paclitaxel-loading micelle was more sensitive to inhibit hela cells than 3T3 cells.

    Keywords: graft-copolymer, comb-like, amphiphilic, drug carrier, CMC, in-vitro release, cytoxicity

    目錄 I 圖目錄 VI 表目錄 VIII 摘要 1 Abstract 3 研究動機與目的 5 第一章 文獻回顧 7 1.1高分子治療學【Polymer therapeutics】 7 1.2 生醫材料【Biomaterials】 9 1.3 生物可分解高分子【Biodegradable polymers】 10 1.3.1 天然生物可分解高分子 10 1.3.2 合成類生物可分解高分子 11 1.3.3 影響生物可分解高分子降解速率之因素9 14 1.4 高分子藥物傳輸系統【Drug delivery system】12 16 1.4.1高分子微胞 18 1.4.2 高分子微胞製備與包藥方式 20 1.4.3 藥物標的方式 21 1.4.4 影響高分子微胞穩定性之因素16 24 1.4.5 細胞吞噬藥物載體的機制 25 1.4.6 控制藥物釋放速率的優點與方式 26 1.5 γ-聚麩胺酸簡介【γ-Poly (glutamic acid)：γ-PGA】 28 1.5.1 γ-聚麩胺酸與α-聚麩胺酸 28 1.5.2 γ-聚麩胺酸之醱酵生產機制19 28 1.5.3 γ-聚麩胺酸的化學結構特性19 29 1.6 抗癌藥物太平洋紫杉醇（Paclitaxel） 30 1.7 MTT assay29 31 第二章 實驗設計與用品 33 2.1 實驗設計 33 2.1 實驗藥品 34 2.2 實驗儀器 36 第三章 實驗方法與步驟 38 3.1 將γ-PGA-Na+酸化成γ-PGA-H+ 38 3.2 合成單邊芳香烃取代的PLA（bz-PLA） 40 3.3 合成酯化反應催化劑DPTS 42 3.4 梳狀高分子共聚物 γ-PGLA之合成 43 3.5 高分子的結構與物性分析 45 3.5.1 結構鑑定 45 3.5.2 物性分析 45 3.6 以高分子微胞包覆抗癌藥物paclitaxel 45 3.6.1 微胞製備方式 46 3.6.2 粒徑分析 46 3.6.3 微胞表面結構分析 46 3.6.4 抗凍劑選擇 47 3.6.5 Paclitaxel濃度分析與檢量線 47 3.6.6 包覆效率與包覆量 47 3.6.7 In- vitro藥物釋放模擬 48 3.6.8 電子顯微鏡影像 48 3.7 測量臨界微胞濃度 49 3.7.1 配製pyrene水溶液 49 3.7.2 配製高分子與pyrene混合水溶液 49 3.7.3 臨界微胞濃度判定 50 3.8 材料細胞毒性測試 50 3.8.1 細胞培養 50 3.8.2 細胞存活率（cell viability ％） 50 第四章 結果與討論 52 4.1 將γ-PGA-Na+酸化成γ-PGA-H+ 52 4.1.1 γ-PGA-Na+與γ-PGA-H+分子量比較 52 4.1.2 γ-PGA-Na+與γ-PGA-H+氫原子核磁共振光譜比較 52 4.1.3 γ-PGA-Na+與γ-PGA-H+傅立葉紅外線光譜比較 54 4.2 合成單邊芳香烃取代的PLA（bz-PLA） 55 4.2.1 bz-PLA的氫原子核磁共振光譜分析結果 55 4.2.2 bz-PLA的分子量分析結果 56 4.2.3 bz-PLA的傅立葉紅外線光譜分析結果 57 4.3 合成酯化反應催化劑DPTS 58 4.3.1 DPTS與DMAP、PTSA之氫原子核磁共振光譜比較 58 4.3.2 DPTS與DMAP、PTSA的熔點比較 59 4.4 梳狀高分子共聚物 γ-PGLA之合成 60 4.4.1 γ-PGA、bz-PLA、γ-PGLA氫原子核磁共振光譜比較 61 4.4.2 γ-PGA、bz-PLA、γ-PGLA之FT-IR圖譜 63 4.5 製備高分子微胞 65 4.5.1 微胞粒徑分析 65 4.5.2 微胞表面結構分析 65 4.5.3 抗凍劑選擇 66 4.5.4 Paclitaxel的包覆效率與包覆量 68 4.5.5 In-vitro藥物釋放模擬 69 4.5.6 電子顯微鏡 72 4.6 測量臨界微胞濃度 73 4.7 材料細胞毒性測試 76 第五章 結論與未來展望 79 第六章 參考文獻 82 附錄一 英文縮寫對照表 i 附錄二 單位符號對照表 iii 附錄三 HPLC檢量線 iv

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