近年來，生醫科技與奈米科技的快速發展，提升了在生醫治療上創新及多功能性平台的快速開發。設計磷脂質包覆的環境應答型奈米載體應用於癌症治療，在靶向傳輸及藥物控制釋放上具新穎性及研究潛力。本研究中，將具有二硫鍵 (disulfide bond) 的 3- (2-Pyridyldithio) propionyl hydrazide (PDPH) 透過腙鍵 (hydrazone bond) 與 Dox 鍵結而成的 pDox 前驅藥物分子，共價鍵結於檸檬酸鈉 (citrate) 還原而成的 13 nm 金奈米粒子上 (pDox-Au NPs)，此一藥物設計具有酸鹼應答的概念，可以在細胞內酸性胞器環境進行藥物釋放。接著，將對於人類急性白血病 T 細胞 (CCRF-CEM cells, CCL-119 T-cell, human acute lymphoblastic leukemia) 上的 protein tyrosine kinase 7 (PTK7) 具有辨識性的適體 (aptamer) T30-sgc8c，修飾於金奈米粒子上 (sgc8c/pDox-Au NPs)，再將載體以仿生材料脂質進行包覆，脂質組成為 1：1 的二肉豆蔻醯磷脂醯膽鹼 (1,2-dimyristoyl-sn-glycero-3-phosphocholine, DMPC) 與 1-十四醯-2-羥基卵磷脂 (1-myristoyl-2-hydroxy-sn-glycero-3-phosphocholine, MHPC)。磷脂質包覆的金奈米藥物載體 (AL:sgc8c/pDox-Au NPs) 透過動態光散射分析儀 (Dynamic light scattering, DLS)、穿透式電子顯微鏡 (Transmission electron microscope, TEM) 及 DiI 螢光探針進行鑑定。奈米藥物載體與 CCRF-CEM 的專一性作用則透過流式細胞儀 (Flow cytometric analyses)、石墨爐原子吸收光譜儀 (Graphite furnace absorption spectrometry)、螢光顯微鏡 (Fluorescence microscopy)、暗視野顯微鏡 (Dark field microscopy)、共軛焦螢光顯微鏡 (Confocal fluorescence microscope) 及細胞存活率進行分析。整體而言，以非共價性脂質包覆的金奈米藥物載體成功被開發，而酸鹼應答及適體的結合也使藥物能針對目標細胞進行更精準的控制釋放，應用於癌症治療中更具優勢。

In recent years, progress on biotechnology and nanotechnology has facilitated a rapid development of innovative multifunctional platforms in applications of biomedical therapy. The design of environmental-responsive nanocarriers on the basis of lipid-encapsulated nanoparticles is particularly promising for targeted delivery and controlled drug release in cancer research. In this report, we describe the synthesis of doxorubicin (Dox) conjugated 13-nm gold nanoparticles (pDox-Au NPs), which exhibited a pH-responsive drug release profile, followed by the modification of multiple aptamers, T30-sgc8c, with the capability to recognize protein tyrosine kinase 7 (PTK7) on leukemia human T cell lymphoblast-like (CCRF-CEM) cell line. Furthermore, lipid-encapsulation was achieved by trapping the nanoparticles, sgc8c/pDox-Au NPs within the biocompatible assembled lipid, which constituted 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1-myristoyl-2-hydroxy -sn-glycero-3-phosphocholine (MHPC). The lipid layer of AL:sgc8c/pDox-Au NPs, was characterized by dynamic light scattering (DLS), transmission electron microscopy (TEM) and DiI probe. Flow cytometric, atomic absorption spectroscopic, fluorescence microscopic, dark field microscopic, confocal microscopic and MTT assay also describe the specific interactions between AL:sgc8c/pDox-Au NPs and CCRF-CEM cells. Overall, a gold-based drug nanocarrier with nonthiol containing phospholipid was successfully established to improve the therapeutic efficacy in tumor cells. The pH-responsiveness was furthermore, highly promising for precise drug releasing in targeted drug delivery.

總目錄
摘要 I
Abstract II
致謝 III
總目錄 IV
圖目錄 VII
表目錄 IX
第一章　緒論 1
1.1 奈米材料與生醫應用 1
1.1.1 奈米材料簡介 1
1.1.2 金奈米粒子的發展及其優勢 2
1.1.3 分子探針結合奈米藥物載體於標靶治療之應用 6
1.1.4 磷脂質包覆奈米粒子的發展及其優勢 10
1.1.5 觸發式的藥物釋放 14
1.1.5.1 化學性刺激─酸鹼應答 15
1.1.5.2 物理性刺激─光照 18
1.2 癌症與治療 20
1.2.1 癌症 20
1.2.2 藥物治療 20
1.2.3 前驅藥物 21
1.2.4 奈米載體與細胞及生物體的作用 23
1.3 研究動機與目的 27
第二章　實驗材料與方法 29
2.1 實驗藥品與儀器 29
2.1.1 實驗藥品 29
2.1.2 緩衝溶液配置 31
2.1.3 細胞培養與操作 32
2.1.4 儀器 33
2.2 pDox 的合成與特性鑑定 34
2.2.1 pDox 的合成 34
2.2.2 pDox 的特性鑑定 34
2.2.3 pDox 的酸鹼應答特性 35
2.2.4 pDox 的毒性測試 35
2.3 奈米藥物載體的合成與特性鑑定 36
2.3.1 金奈米粒子的合成 36
2.3.2 將 pDox 以及適體修飾於金奈米粒子的表面 37
2.3.3 包覆磷脂質於金奈米粒子表面 38
2.3.4 鑑定包覆磷脂質的金奈米粒子 38
2.3.5 磷脂質奈米藥物載體的酸鹼應答測試 39
2.3.6 不同表面修飾的奈米藥物載體的製備、鑑定與酸鹼應答測試 39
2.3.7 不同表面修飾的奈米藥物載體與細胞的作用情形 40
2.4 藥物載體與目標細胞 CCRF-CEM 的作用 42
2.4.1 利用螢光標記法測定載體對目標細胞的選擇性 42
2.4.2 測定目標細胞吞噬奈米載體的能力 43
2.4.3 螢光顯微鏡及暗視野顯微鏡影像 44
2.4.4 細胞存活率分析 45
第三章　實驗結果與討論 47
3.1 pDox 的特性 47
3.1.1 pDox 的合成與特性鑑定 47
3.1.2 pDox 的酸鹼應答特性 48
3.2 奈米藥物載體的特性鑑定 49
3.2.1 奈米藥物載體之光學特性與飽和藥物承載量 49
3.2.2 奈米藥物載體之粒徑大小和 Zeta 電位分析 50
3.2.3 DiI 螢光標定奈米載體上的磷脂質 50
3.2.4 奈米藥物載體的酸鹼應答特性 51
3.2.5 不同表面修飾對於奈米藥物載體特性影響 51
3.3 奈米藥物載體與目標細胞 CCRF-CEM 的作用 52
3.3.1 奈米藥物載體的標靶性 52
3.3.2 共軛焦顯微鏡影像 54
3.3.3 奈米藥物載體對癌細胞的毒性作用 54
第四章　結論 55
圖表說明 56
參考文獻 71

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