為了改善癌症治療的功效，提高治療藥劑累積於腫瘤極為重要。為此，發展具增強腫瘤滲透與細胞吞噬之智慧型表面電荷轉換奈米給藥傳輸系統，此研究採用具特殊功能化材料N-acetyl histidine modified D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS) (簡稱, Histidine-TPGS)與生物可降解之poly(lactic-co-glycolic acid) (PLGA)為材料，於水溶液中利用疏水聚集形成穩定奈米微粒，並同時裝載癌症化療藥物doxorubicin (DOX)與光熱藥劑indocyanine green (ICG)。由物理表徵與體外細胞吞噬數據顯示Histidine-TPGS結合於載藥微粒表面，在微酸的環境下，微粒表面的imidazole質子化產生正電荷，促進與細胞膜上負電荷產生靜電作用力，有效地提高藥物被細胞吞噬的程度。更重要的，於活體影像與體內分佈結果顯示，具表面電荷翻轉能力的載藥奈米微粒，可以像似”奈米子彈”快速並大量累積於腫瘤部位，藉由粒徑較小、趨近電中性的表面，不僅能促進被癌細胞內吞的程度，且也能增加被腫瘤相關巨噬細胞吞噬的機會，進一步深層滲透至缺氧區。此外，腫瘤生長抑制研究結果明顯表眀，以ICG作為影像導引光熱治療，不僅造成TRAMP-C1癌細胞嚴重傷害，更促使ICG/DOX-loaded奈米微粒於腫瘤組織滲透，提高DOX化療對於抗腫瘤的功效。綜合上述結果，本研究創造ICG/DOX-loaded的奈米微粒表現出無限的潛力，利用光熱與化學複合治療，以期達成抑制癌細胞生長與復發的最大效益。

In order to improve the efficacy of cancer treatment, the sufficient accumulation of therapeutic agents in tumor sites is extremely required. To this end, a smart surface charge alterable nanovehicle capable of enhancing tumor penetration and cellular uptake was developed and employed to deliver indocyanine green (ICG) and doxorubicin (DOX) molecules. By the coating of amphiphilic N-acetyl histidine modified D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS) (Hitidine-TPGS) on the surface of hydrophobic core composed of poly(lactic-co-glycolic acid) (PLGA) segments, the functionalized nanocarriers were attained. The physiochemical characterization and in vitro cellular uptake data indicate that the ICG/DOX-loaded nanoparticles with Histidine-TPGS coating show the efficiently promoted cellular uptake in weak acidic environment through the increased surface positive charges by protonation of more histidine moieties able to enhance their affinity to negatively charged cell membrane. Importantly, the findings of in vivo and ex vivo biodistribution suggest that the surface charge switchable ICG/DOX-loaded nanoparticles acted as a “nanobullet” are able to rapidly and intensively deposit in solid tumor, and effectively penetrate into deep tumor hypoxia by their small size, nearly neutrally-charged surface and promoted internalization by TRAMP-C1 cancer cells and tumor-associated macrophages. Furthermore, the in vivo tumor growth inhibition study strongly demonstrates that the imaging-guided ICG-based photo-thermotherapy not only largely damage TRAMP-C1 cancer cells but also significantly boost the penetration of ICG/DOX-loaded nanoparticles into tumor tissue, thus promoting DOX chemotherapy to realize the potent antitumor efficacy. Based on the above results, the created ICG/DOX-loaded nanobullets exhibit great potential to enhance the cancer treatment by the photothermo/chemo combinational therapy.

ABSTRACT................................................15
中文摘要.................................................17
致謝.....................................................18
一、文獻回顧..............................................19
1-1 惡性腫瘤.............................................19
1-2 腫瘤微環境介紹........................................21
1-3 腫瘤缺氧區...........................................24
1-4 腫瘤相關巨噬細胞......................................26
1-5 腫瘤缺氧區趨向巨噬細胞................................27
1-6 腫瘤治療.............................................28
1-7 腫瘤光熱治療之介紹....................................30
1-8 Enhanced permeation and retention (EPR) effect......32
1-9 奈米藥物載體傳遞系統...................................34
1-10 化學治療藥物-Doxorubicin (DOX)介紹...................35
1-11 光熱治療藥物-Indocyanine green (ICG)介紹.............36
1-12 D-α-tocopheryl polyethylene glycol succinate (Vitamin E TPGS)...................................................38
1-13 Histidine 與 Imidazole contain polymer 於藥物傳遞系統之應用......................................................39
二、研究動機.............................................41
三、實驗方法與步驟 ........................................44
3-1 高分子合成與鑑定......................................44
3-1-1 二甲基亞碸(DMSO)的除水..............................44
3-1-2 聚乙二醇琥珀酸酯修飾N-Acetyl-L-Histidne.............44
3-2 奈米微粒製備與性質探討 ................................45
3-2-1 奈米微粒製備.......................................45
3-2-2 奈米微粒的粒徑分布與表面電荷分析......................46
3-2-3 奈米微粒的藥物裝載量................................48
3-2-4 奈米微粒膠體及光學穩定性.............................48
3-2-5 穿透式電子顯微鏡影像................................49
3-2-6 奈米微粒體外藥物釋放實驗與分析.......................49
3-2-7 奈米微粒經近紅外光雷射照射升溫實驗....................50
3-3 體外細胞實驗.........................................51
3-3-1 細胞來源及適合之培養環境............................51
3-3-2 配置細胞培養液與磷酸鹽緩衝溶液.......................51
3-3-3 細胞繼代..........................................52
3-3-4 細胞計數..........................................53
3-3-5 模擬腫瘤微酸環境之細胞培養液pH值調整.................53
3-3-6 流式細胞儀分析.....................................53
3-3-7 細胞胞內DOX吞噬含量分析.............................54
3-3-8 細胞胞內ICG吞噬含量分析.............................54
3-3-9 小鼠巨噬細胞於ICG吞噬含量分析........................55
3-3-10 螢光顯微鏡觀察.....................................55
3-3-11 細胞毒性分析.......................................56
3-4 動物實驗..............................................58
3-4-1 動物來源...........................................58
3-4-2 腫瘤模型建立........................................58
3-4-3 動物體內藥物累積分佈.................................58
3-4-4腫瘤抑制生長評估......................................59
3-4-5 動物犧牲與腫瘤組織包埋................................60
3-4-6 組織切片............................................60
3-4-7 腫瘤組織切片Hematoxylin and eosin (H&E)染色..........60
3-4-8 組織切片免疫螢光染色.................................61
3-5 數據統計..............................................62
四、結果與討論.............................................63
4-1 高分子鑑定與物化分析....................................63
4-1-1 Histidine-TPGS 組分析成鑑定..........................63
4-2 奈米粒子特性分析.......................................64
4-2-1 奈米微粒性質分析.....................................64
4-2-2 載藥奈米微粒光學及粒子穩定性..........................67
4-2-3 近紅外光雷射照射升溫實驗..............................69
4-3細胞實驗...............................................76
4-3-1 微酸環境下細胞吞噬評估...............................76
4-3-2 細胞毒性分析.........................................83
4-4動物實驗...............................................85
4-4-1 動物體內藥物累積分佈..................................85
4-4-2 腫瘤抑制生長評估.....................................87
4-4-2 腫瘤組織切片觀察.....................................92
4-4-3 巨噬細胞RAW 264.7吞噬載藥微粒的程度分析...............100
五、結論..................................................101
六、參考文獻..............................................102

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