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研究生： 施宗甫
論文名稱： 開發具磁性與酸鹼應答性複合高分子液胞作為腫瘤標的藥物傳遞及MR影像顯影之診斷治療奈米平台
Magnetic and pH-Responsive Complex Polymeric Vesicles as a Theranostic Nanoplatform for Tumor-Targeting Drug Delivery and Enhanced MR Imaging
指導教授： 邱信程
口試委員: 王麗芳
張建文
學位類別： 碩士
Master
系所名稱： 原子科學院 - 生醫工程與環境科學系
Department of Biomedical Engineering and Environmental Sciences
論文出版年： 2012
畢業學年度： 100
語文別： 中文
論文頁數： 66
中文關鍵詞： 高分子液胞 、藥物刺激控制釋放 、腫瘤標的
外文關鍵詞： polymersomes, stimuli-triggered drug release, tumor-targeting
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    • 為了在醫學上達到有效診斷與治療的效用，本研究開發出具磁性與酸鹼應答性高分子複合液胞作為攜帶治療藥劑和MR影像顯影劑之傳遞載體。於pH 5.0含有citrate-SPIO的水相中藉由高分子PAAc-co-DSA的自組裝行為形成奈米級SPIO-loaded 高分子液胞(ca. 110 nm)。DOX分子以靜電作用力吸附於液胞表面富含AAc鏈段的區域；其包覆效率約為70 %。為了提高結構的穩定性和具有主動腫瘤標的能力，藉由靜電作用力將高分子chitosan和γ-PGA(OSu)-g-PEG-folate以layer-by-layer的方式吸附於液胞表面，最後形成FA-conjugated SPIO/DOX-loaded高分子複合液胞(ca. 220 nm)。於MR影像顯影方面，SPIO-loaded 複合液胞具有較高的橫向鬆弛率r2 (i.e., 255 Fe mM-1 S-1 vs 183.4 Fe mM-1 S-1 of Resovist)相較於商品化顯影劑Resovist。在藥物釋放行為方面主要和環境的pH值有關；於10小時左右，來自SPIO/DOX-loaded複合液胞之藥物分子DOX的累積釋放量於pH 4.7和pH 7.4分別為70 %和20 %。另一方面，利用CLSM和流式細胞儀的分析結果得知表面修飾葉酸分子之高分子複合液胞有助於細胞(HeLa cells)以folate receptor-mediated的吞噬路徑進行uptake。於細胞(HeLa cells)毒性的結果顯示，FA-conjugated SPIO/DOX-loaded高分子複合液胞對HeLa cells的毒殺效果比folate-free SPIO/DOX-loaded高分子複合液胞來的好。最後，此多功能FA-conjugated SPIO/DOX-loaded高分子複合液胞具腫瘤標的藥物傳遞、藥物控制釋放能力及MR 影像顯影功能，可作為腫瘤標的藥物傳遞及MR影像診斷奈米平台。

    In this study, a multifunctional and pH-responsive polymeric complex vesicle as an anticancer drug and MRI contrast agent carrier has been developed for cancer theranostics. The superparamagnetic iron oxide (SPIO)-loaded polymeric vesicles (ca. 110 nm) were formed by self-assembly of the poly(acrylic acid-co-disterin acrylate) (P(AAc-co-DSA)) in the SPIO aqueous solution (pH 5.0) via sonication technique while the SPIO was simultaneously encapulated into the interior aqueous compartment of the vesicles. The anticancer drug, doxorubicin (DOX), was then loaded within ionized AAc-rich regions of the membrane of polymer vesicle via electrostatic interaction between the ionized AAc units and postively charged DOX molecules. To improve the stability of the resulting polymer vesicles, chitosan and folate-polyethylene glycol-grafted poly(glutamic acid) (PGA-g-PEG(FA)) were sequentially deposited on the outermost surfaces of the DOX/SPIO-loaded polymer vesicles. The loading efficiency of DOX was ca. 70 %. The in vitro drug release behavior of SPIO/DOX-loaded complex vesicles shows a significant increased drug release at pH 4.7 (70%) and a severely retarded drug release at pH 7.4 (20%). This indicates that the loaded DOX molecules can be effectivitly released in the acid environment of tumor (e.g. endosomes or lysosomes). Compared to commercial MRI contrast agent, Resovist, the relaxivity r2 value of SPIO/DOX-loaded polymeric complex vesicles was much higher than Resovist. Results from flow cytometry and confocal laser scanning microscopy (CLSM) analysis showed that folate-conjugated vesicles exhibited higher than folat-free vesicles which also led to higher cytotoxicity. Thus, these DOX/SPIO-loaded folate-conjugated polymer complex vesicles can provide pH-controlled drug release, as well as enhanced MRI contrast, thereby mtargeted cancer therapy and diagnosis possible.

    第一章、緒論 9 1-1 前言 9 1-2 實驗動機與實驗簡述 9 第二章、文獻回顧 10 2-1高分子液胞(polymeric vesicles) 10 2-1-1雙性高分子其親水鏈段重量分率(f)對聚集形態的影響 11 2-1-2刺激應答型高分子液胞 (stimuli-responsive polymersomes) 12 2-2多功能型高分子液胞/微胞具治療與診斷功能上的應用 14 2-3以天然高分子作為生醫材料之應用 18 2-3-1聚麩胺酸 (γ-PGA)簡介 18 2-3-2幾丁聚醣 (chitosan) 簡介 19 2-4以葉酸(folic acid)作為標的官能基的特點 20 2-5利用聚丙烯酸(acrylic acid)結合抗癌藥物(DOX)的應用 21 第三章、實驗部分 23 3-1 儀器原理 23 3-1-1 動態光散射粒徑分析儀 (Dynamic Light Scattering, DLS) 23 3-1-2 穿透式電子顯微鏡(Transmission Electron Microscopy ,TEM) 24 3-2 實驗方法 25 3-2-1 實驗藥品 25 3-2-2藥物及氧化鐵粒子包覆量之定量 26 3-2-3 體外釋放測試 (In vitro release test) 26 3-2-4細胞吞噬表現探討 26 3-2-4-1 利用流式細胞儀(flow cytometry) 26 3-2-4-2 利用雷射共軛焦顯微鏡(confocal laser scanning microscopy) 27 3-2-5細胞培養方面 27 3-2-5-1 配置DMEM細胞培養液 27 3-2-5-2人類子宮頸癌細胞培養 (HeLa cells) 27 3-2-5-3 細胞繼代 27 3-2-6細胞毒性測試 28 3-2-7 親水性順磁奈米氧化鐵粒子(superparamagnetic iron oxide, SPIO)之製備 28 3-2-8 MR phantom (T2 relaxation rate)量測參數設定 28 3-2-9 Poly（acrylic acid-co-disteain acrylate）高分子合成 29 3-2-9-1前置作業 29 3-2-9-2 單體N-acroyloxysuccinimide (NAS) 之合成 29 3-2-9-3 Poly(NAS)之合成 30 3-2-9-4 Poly(NAS-co- DSA)之合成 30 3-2-10 γ-PGA-g-PEG-folate 高分子合成 31 3-2-10-1 γ-PGA-OSu之製備 31 3-2-10-2 folate-cysteamide的合成 33 3-2-10-3 γ-PGA(OSu)-g-PEG-folate的合成 34 3-3具磁性與酸鹼應答性高分子複合液胞製備流程示意圖 35 3-3-1 包覆順磁氧化鐵(SPIOs)之高分子液胞的製備 35 3-3-2 SPIO/DOX-loaded 奈米級高分子複合液胞的製備 36 第四章、結果與討論 37 4-1 高分子液胞性質探討 37 4-2 葉酸(folate)分子於高分子γ-PGA-g-PEG-folate上的定性 38 4-3 SPIO/DOX-loaded高分子複合液胞性質探討 40 4-4 探討交聯前後高分子複合液胞於 phosphate-buffered saline (PBS)下的穩定度 43 4-5 交聯高分子複合液胞於 fetal bovine serum (FBS)下的穩定度 45 4-6 SPIO/DOX-loaded polymeric complex vesicles於不同酸鹼環境進行體外釋放測試 46 4-7 釋放機制和粒徑變化探討 48 4-8 利用Flow cytometry進行癌細胞(HeLa cells)吞噬(uptake)探討 50 4-9 SPIO/DOX-loaded 高分子複合液胞之DOX、SPIO 包覆效率 54 4-10 高分子複合液胞於穿透式電子顯微鏡下的型態分析 55 4-11 SPIO/DOX-loaded 複合液胞橫向鬆弛(transverse relaxivities, r2)速率探討 57 4-12 In vitro phantom MRI 58 4-13 In vivo phantom MRI 58 4-14利用雷射共軛焦顯微鏡(CLSM)針對複合液胞於細胞內分佈累積情形進行探討 60 4-15 癌細胞毒性探討 61 結論 63 參考文獻 64

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