癌症治療是生物醫學領域的重要研究方向，由於受限於癌細胞種類及抗癌藥物的特性，以往在癌症的化療和放射治療上有一定的難度，然而，近年來學術界已有不少結合奈米粒子藥物表面改質的研究，包括有效加強目前抗癌藥物的治療效果及降低副作用等。本論文使用生物相容性佳的奈米及高分子生醫材料進行實驗，結合了標靶(targeting)和磁導引(magnetic guiding)的特性，開發出多功能用途的奈米殼核膠囊，並佐以雙重藥物釋放的功能，用於加強癌症治療，以期降低化療副作用及提升療效。
論文第一部分為磁性奈米殼核膠囊(Nanocapsule)的合成與表面特性對癌細胞與動物腫瘤累積的研究。首先，以熱裂解法合成高產量的奈米氧化鐵：經由控制升溫速率可得到不同粒徑的產物，在實驗最佳化條件下合成出的氧化鐵約為10奈米，可穩定分布於油相溶液中，藉由磁性分析得知該奈米粒子為超順磁性奈米氧化鐵(Superparamagnetic Iron Oxide Nanoparticles, SPION)。磁性奈米殼核膠囊(Nanocapsule)的製備乃以聚乙二醇(Poly vinyl alcohol，PVA)作為介面活性劑，利用雙乳化步驟將油相氧化鐵形成奈米殼層結構並包覆油相藥物紫杉醇(Paclitaxel，PTX) 及經環狀醣類Beta-cyclodextrin (CD)修飾的水相紫杉醇(PTX-CD)。在合成過程中，比較只有PVA及含PVA修飾PEG-PLGA後的載體特性，發現使用分子量25,000 g/mol的PVA皆可以形成中空球結構，而利用PEG-PLGA合成的載體表面形貌較為平滑，此外，由電子顯微鏡(SEM, TEM)、X光繞射分析儀及超導量子干涉儀可以得知奈米殼核膠囊為一具有超順磁性的中空球體，且粒徑分布範圍在150到250奈米間，可被磁場吸引並穩定分布於水相中。而細胞毒殺實驗結果顯示，修飾PEG-PLGA後的奈米殼核膠囊有較佳的藥物釋放量及細胞毒殺特性，根據過去的文獻推測其原因為PEG及PLGA高分子都具有較佳的生物相容性，且親水端的PEG分子可引導藥物載體較易經由受體媒介式胞吞作用(receptor-mediated endocytosis)進入癌細胞中。而鍵結形成PEG-PLGA後，親水端的PEG可以延長藥物載體在細胞及生物體內的循環，親油端的PLGA則可以穩定載體的殼核結構使之不易在循環過程中降解，增加了藥物載體在體內循環的時間，進而累積較多的磁性奈米殼核膠囊至腫瘤位置。論文第二部分為加強奈米殼核膠囊的標靶功能，將Beta-cyclodextrin (CD)經由化學修飾上具細胞表面蛋白CD44標靶特性的玻尿酸(Hyaruic acid，HA)，並包覆油相紫杉醇於Beta-cyclodextrin中，使之形成水相且有標靶功能的藥物分子(HA-CD-PTX)。最後將改質完成的藥物同樣以乳化方式形成PVA/PEG-PLGA的奈米殼核膠囊，進行藥物釋放及標靶功能的測試，並檢測其物理性質與改質前無差異。表面改質根據文獻採化學合成及官能基置換的方法，分別將前驅物Beta-cyclodextrin接上硫醇基形成CD-SH、玻尿酸接上戊酸形成HA-pen，再經由反應後即可完成鍵結，在核磁共振儀及XPS電子能譜儀分析後可以推斷鍵結成功，且具有細胞毒殺效果。動物實驗方面則可以有效經由高週波磁場生熱及藥物標靶釋放後，抑制裸鼠多型性膠質母細胞瘤RG2的生長。
本論文開發出以奈米氧化鐵及PVA、PEG-PLGA高分子為基質的奈米殼核膠囊，可同時包覆水相及油相的抗癌藥物，外加磁場導引至腫瘤位置並以EPR效應經腫瘤細胞的微血管累積至細胞表面，藉由磁場生熱及胞吞作用而降解，可使水相及油相藥物依序釋放。水相藥物釋放後可經標靶作用深入腫瘤細胞，同時磁場生熱溫度達攝氏43度以上，可經由物理作用有效毒殺癌細胞、抑制腫瘤生長，達到化療與熱療的效果。

Nowadays effective anticancer drug delivery therapy can be reached by designing a targeting strategy with high stability during long circulation in the body and efficiently uptake by the target tumor cancer cells then degraded and decomposed easily in the cell to ensure the release of anticancer drug. This study reports a nanocapsule which is a nanoscale drug carrier formed by SPION (Superparamagnetic Iron Oxide Nanoparticles), PVA (Polyvinyl alcohol) and PEG-PLGA (Poly(ethylene glycol) methyl ether-block-poly(lactide-co-glycolide)), loaded with two phases of the antitumor drug PTX (Paclitaxel) which is wrapped with and without CD (beta-cyclodextrin) to change between water and oil phase.
The special characteristic of SPION provides the drug delivery carrier well targeting and thermal function to accumulate specifically in tumor cell site and kill tumor cells by heating up to 43oC with applied magnet filed. PVA and PEG-PLGA help the drug carrier form a stable hollow sphere shape, which is capable of carrying both oil phase (SPION and PTX) and water phase (SPION and PTX in CD) in a mono device with multiple functions and fit the size (150-250nm) to aggregate the drug carrier in tumor site via EPR effect. The combination of water-soluble CD and paclitaxel confers higher stability in the circulation system, which efficiently delivers paclitaxel into the targeted tumor cells and release paclitaxel within the cell by degradation. In further part, the water phase drug (PTX in CD) is loaded with HA (hyaluronic acid), a CD44 targeting function ligand, which drives the drug to the surface of tumor cell and induce the endocytosis to enter the cell.
This nanoscale drug carrier delivery system exhibits significant antitumor activity in cancer cell lines of Hela (human epithelial cervix adenocarcinoma), MCF-7 (human epithelial breast adenocarcinoma), MCF-7/ADR (human epithelial breast adenocarcinoma with multidrug resistance) and RG2 (rat glioblastoma); as well as having good repression in tumor growth of the BALB/c female nude mice model with RG2 tumor. This strategy established in this study provides knowledge for the stable development of advanced anticancer drug delivery for chemotherapy and hyperthermia.

致謝 i
摘要 ii
Abstract iv
Table of Contents vi
List of Figures viii
List of Tables xii
List of Schemes xiii
Abbreviations xiv
Chapter 1 Introduction 1
Chapter 2 Literature Review and Theory 4
2.1 Nowadays Tactic of Drug Delivery Materials 4
2.1.1 Introduction of Stimuli Responsive Drug Delivery Systems 5
2.1.2 Polymer Application in Stimuli Responsive Drug Delivery Systems 7
2.2 PEG and PLGA Application for Polymeric Nanoparticle 9
2.3 Anticancer Drug – Paclitaxel 10
2.4 Motivation 10
Chapter 3 Experimental Procedures 12
3.1 Materials and Reagent 12
3.2 Apparatus 14
3.3 Synthesis of Superparamagnetic Iron Oxide Nanoparticles 16
3.4 Synthesis of PVA based Nanocapsule 17
3.4.1 Synthesis of PVA Nanocapsule by single emulsion 17
3.4.2 Drug loading of PVA Nanocapsule by double emulsion 17
3.5 Synthesis of PEG-PLGA based Nanocapsule by Copolymer Modification 18
3.5.1 Synthesis of PEG-PLGA Nanocapsule by single emulsion 18
3.5.2 Drug loading of PEG-PLGA Nanocapsule by double emulsion 19
3.6 Characterizations 20
3.7 Magnetic Thermal Heating Effect of Nanocapsules 21
3.8 Drug Loading Efficiency and Encapsulation Efficiency 21
3.9 Drug Release 22
3.10 In Vitro Experiment 22
3.10.1 Cell Culture 22
3.10.2 Cellular Uptake 23
3.10.3 Cell Compatibility and Cytotoxicity 24
3.10.4 Flow Cytometry 25
3.11 In vivo Experiment 25
Chapter 4 Results and Discussion 27
4.1 Characterizations of Superparamagnetic Iron Oxide Nanoparticles 27
4.2 Characterizations of PVA based Nanocapsules 31
4.3 Characterizations of PEG-PLGA Based Nanocapsules 34
4.4 Comparison of PVA and PEG-PLGA Based Nanocapsules 38
4.5 Thermal Studies of Selected PVA and PEG-PLGA Based Nanocapsules 42
4.6 Drug Release 44
4.7 In Vitro Experiment 46
4.7.1 Thermal Properties 46
4.7.2 Cellular Uptake Efficiency 47
4.7.3 Flow Cytometry 49
4.7.4 Cell compatibility and Cytotoxicity 51
4.8 In Vivo Experiment 52
Chapter 5 Targeting Ligand Modification 58
5.1 Synthesis process of HA-CD-PTX 58
5.2 Characterization of Nanocapsule with HA-CD-PTX 60
5.3 Characterization of HA-CD-PTX 61
5.4 Cell Cytotoxicity of Nanocapsule with HA-CD-PTX 65
5.5 In Vivo Study of Nanocapsule with HA-CD-PTX 67
Chapter 6 Conclusions 70
Chapter 7 Future Expectation 72
References 73

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