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研究生： 歐大煒
Ta-wei Ou
論文名稱： 利用離子型高分子之電荷交聯穩定微脂粒
Investigation of high stable pegylate-liposome based on polyelectrolytic crosslinking
指導教授： 陳暉
Hui Chen
薛敬和
Ging-Ho Hsiue
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： 98
語文別： 中文
論文頁數： 104
中文關鍵詞： 微脂粒 、交聯劑 、酸鹼應答
外文關鍵詞： crosslinking agent, Liposome.pH sensitive
相關次數： 點閱：17  下載：0 
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    • 癌症是由控制細胞生長增殖機制的失常而引起，並且會局部侵入週遭正常組織，甚至經由體內循環系統或淋巴系統轉移到身體其它部份，使得癌症成為已開發國家中主要死亡原因之一。因此，研發有效率的抗癌藥物或藥物傳輸系統是當今醫療科學領域的首要目標之一。
    本研究主要係設計與合成具有酸鹼應答、生物相容性及生物可降解性之新穎二團聯共聚物mPEG- P(HPMA-co -MAAc)-Chol，以及具有免疫隱蔽性之交聯劑PEG-Lys，與磷脂質(DPPC)共同製備具良好生物相容性之新穎高分子電性結合以達交聯複合型奈米微胞，以作為抗癌藥物Doxorubicin之藥物載體，並進而探討此新劑型於癌症治療之效用。
    研究首先合成不同比例之二團聯共聚物，並探討摻混不同組成重量比於磷脂質下對於複合型微胞粒徑之影響，更進一步添加不同比例之交聯劑。由實驗結果顯示於不比例下皆能製備出粒徑大小介於50~80nm且有良好之粒徑均一性之複合型微胞。經由上述研究之最佳條件下，複合型奈米微胞導入Cy5.5與葉酸(Folate)以作為癌症組織之「辨識」與「顯影」功能，用以觀測癌細胞之毒殺與胞飲行為，以及動物實驗之微胞分佈與癌症治療情形。
    經由體外藥物釋放模擬實驗證實複合型藥物微胞於中性環境pH7.4下可以穩定地將藥物包覆。而在酸性環境pH5.0與pH4.5下微胞結構對環境產生應答，微胞藉由MAAc與mPEG間之氫鍵作用力使微胞擠壓進而釋放藥物，其藥物Dox約於5個小時釋放量達到80%。在材料細胞毒性研究中可知，複合型奈米微胞由於結構組成皆為FDA 核可，故毒性較低。而藥物毒殺人類子宮頸癌細胞（HeLa cells）實驗可知，具有主動標的功能之複合型奈米藥物微胞經由主動標的官能基與癌細胞細胞膜接受器結合，再經由receptor mediated endocytosis進入癌細胞達到較高毒殺效果。於動物實驗下，觀察實驗鼠腫瘤之治療情形。實驗顯示含有葉酸之50%P50MAAc 與50%P50MAAc-1 PEG- Lys複合型奈米藥物微胞具有高度穩定性與專一性，能有效抑制腫瘤之生長，且擁有較低之副反應，不造成正常細胞之毒殺。
    綜合本研究所得之結果，複合型奈米藥物微胞不論於細胞毒性研究或動物實驗均證實極具癌症治療效果。未來可運用微胞具標識腫瘤組織的功能，診斷癌症組織之分佈位置，早期於癌細胞擴散前追蹤與治療，有效達到抗癌效果，促進人類醫療福祉，對癌症治療醫學上作出重大貢獻。

    This study presents a novel and biocompatibility lipo-polymer complex which was constructed from DPPC phospholipid, a diblock copolymer (mPEG-P(HPMA-co-MAAc)-Chol) and a crosslinking agent PEG-Lysine. In this study, particle sizes and particular distribution were discussed first to evaluate the affection of the mole ratio of diblock copolymer and crosslinking agent on particles. Then, anticancer drug, Dox (doxorubicin-HCl) was encapsulated into lipo-polymer complex for evaluating anticancer efficiency. Additionally, folate and Cy5.5 molecules were introduced into lipo-polymer complex for cancer targeting and bio-distribution imaging. From the results, the optimal particle size of lipo-polymer complex was around 50~80 nm. Drug loading efficiency was about 20 wt%.
    To understand the future application of lipo-polymer complex in cancer therapy, drug release behavior, cell cytotoxicity, bio-distribution, and antitumor activity of drug-loaded lipo-polymer complex were evaluated. For drug release test, drug-loaded lipo-polymer complex was stabilized on pH7.4 and rapidly released of drug under pH5 condition due to hydrogen bond occurrence between MAAc and mPEG. For cell cytotoxicity, empty lipo-polymer complex showed a low cytotoxicity and drug-loaded lipo-polymer complex exhibited specific targeting for Hela cells. For bio-distribution evaluation, drug-loaded lipo-polymer complex observed high accumulation in tumor by optical imaging (Near IR).
    Finally, tumor–bearing nude mice were used to evaluate the antitumor activity by drug-loaded lipo-polymer complex and commercial lipoDox. The result indicated drug-loaded lipo-polymer complex increased mouse survival and tumor growth inhibition.

    摘要 i Abstract iii 目錄 v 第一章、研究動機 1 第二章、 文獻回顧 4 2-1 高分子奈米微胞 4 2-2 奈米載體之分類 6 2-2-1 傳統性的奈米粒子 6 2-2-2 免疫隱蔽性的奈米微胞 7 2-2-3 生物可降解型奈米微胞 8 2-2-4 溫度應答型微胞 11 2-2-5 酸鹼應答型微胞 11 2-2-6 具癌細胞辨識性奈米微胞 14 2-2-7 具螢光顯影型微胞 15 2-3 複合型奈米微胞 18 2-3-1 高分子與高分子系統 19 2-3-2 高分子與微脂粒系統 24 2-4 奈米藥物載體在癌症治療之傳遞模式 35 2-5 高分子材料之特性及其應用 39 2-5-1 poly(ethylene glycol)之性質與應用 39 2-5-2 Poly(HPMA)之性質與應用 41 第三章、實驗方法 42 3-1 實驗藥品 42 3-2 實驗儀器與裝置 43 3-3 名詞對照 44 3-4 酯化反應催化劑DPTS之合成 44 3-5 Macroinitiator mPEG2-ABCPA之合成 45 3-6 膽固醇末端改質之合成 46 3-6-1 Cholesterol-COOH之合成 46 3-6-2 Cholesterol-NHS ester之合成 47 3-7 具酸鹼應答之二團聯共聚物之合成 47 3-7-1 mPEG-P(HPMA-co-MAAc)-NH2 二團聯共聚物之合成 47 3-7-2 mPEG-P(HPMA-co-MAAc)-Chol 二團聯共聚物之合成 48 3-8 PEG-Lysine之合成 49 3-9 兩團聯共聚合物Cy5.5-PEG-P(HPMA-co-MAAc)-Chol、 50 3-9-1 Boc-NH-PEG-P(HPMA-co-MAAc)-Chol 50 3-9-2 NH2-PEG-P(HPMA-co-MAAc)-Chol之合成 51 3-9-3 Folate-NHS ester之合成 52 3-9-4 Cy5.5-PEG-P(HPMA-co-MAAc)-Chol之合成 53 3-9-5 Folate-PEG-P(HPMA-co-MAAc)-Chol之合成 54 3-10 共聚合物之結構鑑定與分析 55 3-11 高分子與微脂粒之複合型奈米微胞之製備 56 3-12 高分子與微脂粒之複合型奈米微胞之鑑定與性質分析 56 3-13 高分子與微脂粒之複合型奈米微胞酸鹼應答行為測試 57 3-14 高分子與微脂粒之複合型奈米微胞之藥物包覆 58 3-15 高分子與微脂粒之複合型奈米微胞之藥物包覆量及性質分析 58 3-16 高分子與微脂粒之複合型奈米微胞之體外藥物釋放模擬分析 59 3-17 高分子與微脂粒之複合型奈米微胞之細胞存活率與細胞毒殺分析 59 3-18 高分子與微脂粒之複合型奈米微胞在動物體內之活體觀察 62 第四章、實驗結果與討論 64 4-1 酯化反應催化劑DPTS之合成與鑑定 64 4-2 巨起始劑mPEG2-ABCPA之合成與鑑定 66 4-3 Cholesterol-NHS ester之合成與鑑定 69 4-4 酸鹼應答之二團聯共聚mPEG-P(HPMA-co-MAAc)-Chol 70 4-5 PEG-Lysine 之製備與鑑定 72 4-6 二團聯共聚物Cy5.5-PEG-P(HPMA-co-MAAc)-Chol與Folate-PEG-P(HPMA-co-MAAc)-Chol之製備與鑑定 73 4-7 高分子與微脂粒之複合型奈米微胞之製備及最適化之條件探討 75 4-8 高分子與微脂粒之複合型奈米微胞之安定性分析 77 4-9 複合型奈米微胞之界面電位及酸鹼應答行為分析 79 4-10 複合型奈米微胞之DOX抗癌藥物包覆分析 82 4-11 複合型奈米微胞之核殼型態分析 85 4-12 複合型奈米微胞之體外藥物釋放模擬 87 4-13 複合型奈米微胞之體外細胞材料毒性及藥物毒殺實驗 88 4-14 複合型奈米微胞之動物體內治療情形 91 4-15 癌症顯影之複合型奈米微胞之動物體內微胞分佈情形 93 第五章、結論 95 第六章、參考文獻 99

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