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研究生： 黃奕豪
Yi-Hao Huang
論文名稱： 非水溶性藍藻蛋白-聚乙二醇單甲基醚接枝物之特性探討及其在藥物包覆上之應用
Characterization of (insoluble cyanophycin)- (polyethylene glycol methyl ether) conjugates and its application in drug encapsulation
指導教授： 曾文祺
Wen-Chi Tseng
口試委員: 林析右
Shi-Yow Lin
方翠筠
Tsuei-Yun Fang
學位類別： 碩士
Master
系所名稱： 工程學院 - 化學工程系
Department of Chemical Engineering
論文出版年： 2017
畢業學年度： 105
語文別： 中文
論文頁數： 115
中文關鍵詞： 非水溶性藍藻蛋白 、聚乙二醇單甲基醚 、溫度敏感性高分子 、藥物包覆
外文關鍵詞： water insoluble cyanophycin, polyethylene glycol methyl ether, thermo-responsive polymer, drug encapsulation
相關次數： 點閱：355  下載：0 
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  •  上一筆

    • 藍藻蛋白(cyanophycin granule polypeptide, CGP)，主要由天門冬胺酸(aspartic acid)為主鏈，精氨酸(arginine)為側鏈，是一種非核醣體合成的蛋白質。利用基因重組菌所生產的藍藻蛋白，側鏈上部分的精氨酸會被賴氨酸 (lysine)所取代，而賴胺酸上的一級胺則容易進行化學修飾。藍藻蛋白屬於溫度敏感性高分子，在水溶液中具有最高臨界溶解溫度(upper critical solution temperature, UCST)的相轉移行為。但是非水溶性藍藻蛋白在中性環境下幾乎無法溶解，因此在應用上受到較多限制。
    本研究嘗試將同樣具有生物相容性且對水溶解度極佳的聚乙二醇單甲基醚 (polyethylene glycol methyl ether, mPEG)以化學修飾的方式與非水溶性藍藻蛋白做接枝反應，再以紅外線光譜儀鑑定接枝後的官能基，並以TNBSA比色法確認其接枝的程度。
    接枝聚乙二醇單甲基醚後，使得非水溶性藍藻蛋白的溶解度提升。接枝物於中性環境之緩衝溶液中，相轉移溫度落在40℃附近。本研究探討在生理條件下不同接枝率、濃度、酸鹼值以及接枝不同分子量的聚乙二醇單甲基醚對於相轉移溫度的影響。並且利用穿透式電子顯微鏡 (TEM)與動態光散射分析儀 (DLS)觀察粒徑大小。
    最後嘗試利用非水溶性藍藻蛋白接枝聚乙二醇單甲基醚與阿黴素 (doxorubicin)做藥物包覆，利用改變藥物與載體的比例，及利用不同方式進行包覆，比較彼此間的包覆效率。實驗結果顯示，使用藥物與載體比例為1 : 3，並利用直接降溫法做包覆，其包覆效率可達40 %。由於非水溶性藍藻蛋白接枝聚乙二醇單甲基醚為溫度敏感性高分子，因此在釋放期間提升釋放時的溫度，其釋放率可由25%提升至最終釋放率為95%，表示利用溫度的高低可控制藥物釋放的程度，而此特性有利於應用在藥物傳遞上。

    Cyanophycin granule polypeptide is a non-ribosomal polypeptide, consisting of aspartic acid as a backbone with arginine and lysine as the side chains. When cyanophycins are produced from genetic recombinant strains, lysine can replace some of the arginine on the side chain. The primary amine on lysine provides a moderate condition for chemical modification. Cyanophycin has been shown to be a thermo-responsive polymer, which has an upper critical solution temperature in the aqueous solution. However, the water insoluble cyanophycin can hardly be dissolved in neutral aqueous solution, which might limit its applications.
    In this study, we used polyethylene glycol methyl ether (mPEG) to modify insoluble cyanophycin. After reaction, the functional groups of the (insoluble cyanophycin) - (mPEG) conjugates were analyzed by Fourier transform infrared spectroscopy. The grafting degrees of the conjugates were determined by the TNBSA colorimetrically.
    Grafting mPEG was found to enhance the solubility of insoluble cyanophycin. The phase transition temperatures of the conjugates were around 40℃. The effects of grafting degrees, conjugate concentrations, pH, and molecular weights of mPEG on the phase transition temperature were examined. The particle sizes of conjugates were measured by transmission electron microscopy and dynamic light scattering.
    Finally, we used (insoluble cyanophycin) - (mPEG) conjugates to encapsulate doxorubicin, and investigated the encapsulation efficiency by changing the ratios of drug to polymer as well as the methods of encapsulation. The results showed that using an instant cooling method at a drug/polymer ratio of 1/3, the encapsulation efficiency could be 40%. Because the (insoluble cyanophycin) - (mPEG) conjugates were thermoresponsive, we also tried to raise the temperature during the release. The results showed that the release increased from 25 % to a final 95 %, indicating that temperature can control drug release, and showing the potential to be used in drug delivery.

    中文摘要 I Abstract III 誌謝 V 目錄 VI 圖目錄 XII 表目錄 XVII 第一章 緒論 1 第二章 文獻回顧 3 2.1 藍藻蛋白 3 2.1.1藍藻蛋白簡介 3 2.1.2藍藻蛋白之結構 3 2.1.3利用基因重組方式生產藍藻蛋白 4 2.1.4藍藻蛋白的應用 6 2.2 聚乙二醇 7 2.2.1聚乙二醇簡介 7 2.2.2聚乙二醇化 9 2.2.3聚乙二醇化之應用 10 2.3 敏感性高分子 12 2.3.1溫度敏感性高分子 12 2.3.2溫度敏感性高分子之物理特性 13 2.3.2.1 霧點 13 2.3.2.2溫度敏感性高分子之線團狀-球狀轉換 14 2.3.2.3遲滯現象 15 2.3.3溫度敏感性高分子之應用 15 2.3.3.1 藥物傳遞 15 2.3.3.2 基因傳遞 16 2.3.3.3 組織工程 17 2.3.4酸鹼敏感性高分子 18 2.3.5臨界聚集濃度 20 第三章 實驗材料與方法 21 3.1實驗材料 21 3.2.1菌株培養 21 3.2.2實驗藥品 21 3.2藥品配製 23 3.2.1藍藻蛋白之生產-菌株培養 23 3.2.1.1 Ampicillin/Chloramphenicol二合一抗生素 23 3.2.1.2 Luria-Bertani medium agar plate (LB medium agar plate) 23 3.2.1.3 Luria-Bertani medium (LB medium) 24 3.2.1.4 Terrific Broth medium (TB medium) 24 3.2.1.5 Isopropyl β-D-1-thiogalactopyranoside (IPTG) 24 3.2.2 SDS-PAGE 25 3.2.2.1 10% SDS (sodium dodecyl sulfate) 25 3.2.2.2 20% APS (ammonium persulfate) 25 3.2.2.3 1 M Tris-HCl buffer ( pH=6.8 ) 25 3.2.2.4 1.5M Tris-HCl buffer ( pH=8.8 ) 25 3.2.2.5 4% SDS-PAGE (上層膠) 25 3.2.2.6 12% SDS-PAGE (下層膠) 25 3.2.2.7 15% SDS-PAGE (下層膠) 26 3.2.2.8 Protein loading buffer 26 3.2.2.9 Protein running buffer (10X) 26 3.2.2.10 Protein staining buffer 26 3.2.2.11 Protein de-staining buffer 26 3.2.2.12 Protein preserving buffer 26 3.2.3非水溶性藍藻蛋白與聚乙二醇單甲基醚接枝之製備 27 3.2.3.1 0.1 M citrate phosphate buffer, pH=3.0 27 3.2.3.2 0.1 M Reaction buffer 27 3.2.3.3 0.01 % TNBSA 27 3.2.4非水溶性藍藻蛋白與聚乙二醇單甲基醚接枝物相轉變分析 27 3.2.4.1 1X PBS (phosphate-buffer saline), pH=7.4 27 3.2.4.2 0.1 M citrate-phosphate buffer, pH=3.0、pH=4.0、pH=5.5 27 3.2.4.3 0.1 M borate buffer, pH=9.0、pH=10.0 28 3.3實驗儀器 28 3.4實驗步驟 30 3.4.1 利用搖瓶的方式製備藍藻蛋白 30 3.4.1.1 將菌株畫盤於LB medium aplate 30 3.4.1.2 將菌株轉養至2 mL LB medium 30 3.4.1.3 將菌株轉養至60 mL LB medium 30 3.4.1.4 將菌株轉養至150 mL TB medium 30 3.4.1.5 誘導 31 3.4.1.6 收菌 31 3.4.2 利用醱酵槽製備藍藻蛋白 31 3.4.2.1將菌株轉養至180 mL LB medium 31 3.4.2.2將菌株轉養至10L 醱酵槽 31 3.4.3 藍藻蛋白之純化 32 3.4.3.1泡菌 32 3.4.3.2破菌 32 3.4.3.3水溶性藍藻蛋白純化 32 3.4.3.4非水溶性藍藻蛋白純化 33 3.4.4 SDS-PAGE 33 3.4.4.1鑄膠 33 3.4.4.2 樣品配製 33 3.4.4.3 跑膠 34 3.4.5非水溶性藍藻蛋白與聚乙二醇單甲基醚接枝之製備 34 3.4.5.1聚乙二醇單甲基醚(mPEG500)的改質 34 3.4.5.2聚乙二醇單甲基醚(mPEG2000)的改質 35 3.4.5.3非水溶性藍藻蛋白與聚乙二醇單甲基醚接枝反應 36 3.4.6非水溶性藍藻蛋白與聚乙二醇單甲基醚接枝物官能基鑑定 39 3.4.7非水溶性藍藻蛋白與聚乙二醇單甲基醚接枝程度鑑定 40 3.4.8非水溶性藍藻蛋白與聚乙二醇單甲基醚接枝物相轉變分析 42 3.4.9非水溶性藍藻蛋白與聚乙二醇單甲基醚接枝物粒徑分析 43 3.4.9.1非水溶性藍藻蛋白-聚乙二醇單甲基醚水合直徑測量 43 3.4.9.2穿透式電子顯微鏡觀察 44 3.4.10非水溶性藍藻蛋白與聚乙二醇單甲基醚接枝物之藥物包覆 45 3.4.10.1 藥物包覆 45 3.4.10.1-1不同的藥物/載體比例進行包覆 46 3.4.10.1-2不同方式進行藥物包覆 47 3.4.10.1-3 以螢光分光光度計做藥物包覆率之測量 49 3.4.10.2 包覆藥物之體外釋放及釋放率測量 50 第四章 結果與討論 51 4.1非水溶性藍藻蛋白-聚乙二醇單甲基醚接枝物官能基鑑定 51 4.2非水溶性藍藻蛋白-聚乙二醇單甲基醚接枝程度鑑定 53 4.3非水溶性藍藻蛋白與聚乙二醇單甲基醚接枝物相轉變分析 56 4.3.1非水溶性藍藻蛋白與聚乙二醇單甲基醚接枝物霧點測試 56 4.3.2酸鹼值影響UCST之行為 63 4.3.3遲滯現象之探討 68 4.4非水溶性藍藻蛋白與聚乙二醇單甲基醚接枝物粒徑分析 79 4.4.1非水溶性藍藻蛋白-聚乙二醇單甲基醚水合直徑測量 79 4.4.2非水溶性藍藻蛋白-聚乙二醇單甲基醚在TEM下之粒徑觀察 82 4.5非水溶性藍藻蛋白與聚乙二醇單甲基醚接枝物之藥物包覆 84 4.5.1藥物包覆 84 4.5.1.1不同的藥物/載體比例進行包覆 85 4.5.1.2不同方式進行藥物包覆 86 4.5.2藥物釋放 87 4.5.2.1不同包覆方式的藥物釋放測試 87 4.5.2.2利用溫度控制藥物釋放 90 結論 93 附錄 95 參考文獻 111

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