本研究旨在開發能針對腫瘤組織經放射線照射後大量產生活性氧物質(reactive oxygen species, ROS) 進行應答以釋放化療藥物的智慧型奈米藥物載體系統，用以輔助放射治療並抑制放射治療後之復發情形。
本研究成功開發出對活性氧物質具應答能力之高分子poly(thiodiethylene adipate) (PSDEA) 與其三團聯衍伸物Polyethylene glycol - poly(thiodiethylene adipate) - Polyethylene glycol (PEG-PSDEA-PEG)，並利用此高分子為材料製備出裝載化療藥物的奈米粒子。此高分子透過thiodiethylene glycol內硫醚 (sulfides) 在高氧化應力 (oxidative stress) 環境中會被氧化為亞碸 (sulfoxide) 之特性，賦予其疏水/親水性間轉換 (hydrophobic/hydrophilic transition) 能力。一旦構築奈米粒子的高分子被氧化轉變為親水特性時，即會導致奈米粒子瓦解並釋放出所載化療藥物。此奈米粒子直徑大小約為109 nm，並具良好化療藥物SN38裝載效率 (11 wt%)。體外載體氧化應答能力評估顯示，經過氧化後的奈米粒子形態會產生膨潤至瓦解之現象，且藥物累積釋放量經氧化後亦會明顯提升。在體外細胞實驗上證實此氧化應答性奈米粒子會被放射線 (X-ray) 照射所產生的ROS破壞而釋出藥物，造成細胞內部累積較多的藥物分子，導致BNL 1MEA.7R.1癌細胞凋亡。而在腫瘤生長抑制結果指出，當給予具應答性奈米粒子於腫瘤內並施予放射線照射後確實有最佳的抑制效果，證明載體可受放射線照射觸發化療藥物釋放並能成功結合化療與放射治療，強化治療效果並抑制腫瘤復發情形。綜合上述成果，本研究所開發之搭載SN38的氧化應答奈米粒子，具有深厚潛力對於現今放射線療法有很好的輔助功能，利用放療與化學複合治療，以期達成抑制癌細胞生長與復發的最大治療成效。

This work aims to develop a novel nanoparticle delivery system capable of liberating therapeutic payloads in response to the dramatic increase of the external reactive oxygen species (ROS) level upon radiation therapy for improving the overall anti-cancer efficacy. Herein, the ROS-responsive Poly(thiodiethylene adipate) have been successfully prepared by stepwise polymerization approach and employed as the major materials in developing the novel nanotherapeutics. Through the ROS-mediated oxidation reaction, the non-polar sulfide groups on the main chains of polyesters are chemically converted to the polar sulfoxide group, thus resulting in the hydrophobic-hydrophilic transition of the ROS-sensitive polymers. This inevitably leads to the structural disruption of nanotherapeutics comprising mainly the ROS-sensitive polyesters upon the irradiation and thus activates drug liberation for chemotherapy in combination with the radiation treatment against carcinoma. The nanotherapeutics with a particle size of ca109 nm in diameter demonstrate an excellent chemodrug confinement of the camptothecin analog, SN38. In vitro evaluation of oxidative responsive ability indicate the nanotherapeutics were oxidized under the exposure to ROS and, leading to the swelling and disassembly of the micelles, increasing significantly the amount of SN38 released from nanotherapeutics. In vitro cellular uptake data indicate that nanoparticles with X-ray irradiation-pretreatment show the efficiently promoted cellular uptake. Furthermore, cytotoxic analysis demonstrates that the viability of murine HCC cells (BNL 1MEA.7R.1) co-incubated with the X-ray irradiation-pretreated nanoparticles was reduced proportionally with the increase of the SN38 dose, while that of cancer cells incubated with the nanoparticles in the absence of ROS activation remained relatively high, indicating the strong dependence of drug release from the smart nanoparticles on the production of ROS. Importantly, in vivo tumor growth inhibition study strongly illustrates that the incorporation of radiation therapy and its induced chemotherapy with the smart nanoparticles not only exhibit potent antitumor efficacy but also inhibit tumor recurrence. Based on the above results, this study provides a promising strategy for the development of ROS responsive drug delivery nanotherapeutics for tumor therapy.

Abstract 9
摘要 11
致謝 12
一、研究動機 13
二、文獻回顧 15
2.1 腫瘤微環境 15
2.1.1 放療後腫瘤微環境與腫瘤復發機制 16
2.2惡性腫瘤 18
2.3癌症治療 20
2.3.1化學治療 20
2.3.1.1化療藥物 7-Ethyl-10-hydroxy-camptothecin (SN38)介紹 21
2.3.2放射線治療 22
2.3.2.1放射線增敏劑(radiosensitizer) 24
2.4奈米載體概況 27
2.4.1奈米藥物載體傳遞系統 27
2.4.2高分子微粒 29
2.4.3氧化應答於藥物傳遞系統的應用 31
2.4.4放射線療法於藥物傳遞系統的應用 34
三、實驗方法與步驟 36
3.1 高分子合成與鑑定 36
3.1.1 Poly(thiodiethylene adipate) (PSDEA) 和 Poly(hexamethylene adipate) (PHDEA) 合成 36
3.1.2 PEG-PSDEA-PEG 和 PEG-PHDEA-PEG合成 37
3.1.3 高分子氧化應答特性測試 37
3.1.4 高分子生物可降解測試 38
3.2 奈米微粒製備與性質探討 38
3.2.1 奈米微粒製備 38
3.2.2 塑造活性氧物質環境 (ROS treatment) 39
3.2.3 奈米微粒的粒徑分布 39
3.2.4 奈米微粒的藥物裝載量 40
3.2.5 奈米微粒膠體穩定性與冷凍乾燥保存測試 41
3.2.6 穿透式電子顯微鏡影像 41
3.2.7 奈米微粒體外藥物釋放實驗與分析 41
3.3 體外細胞實驗 42
3.3.1 細胞來源及適合之培養環境 42
3.3.2 配置細胞培養液與磷酸鹽緩衝溶液 42
3.3.3 細胞繼代 43
3.3.4 細胞計數 43
3.3.5 螢光顯微鏡觀察 44
3.3.6 細胞毒性分析 44
3.3.7 合併治療之細胞毒性分析 45
3.4 動物實驗 46
3.4.1 動物來源 46
3.4.2 腫瘤模型建立 46
3.4.3腫瘤抑制生長評估 46
3.4.4動物犧牲與腫瘤組織包埋 47
3.4.5組織切片 47
3.4.6腫瘤組織切片Hematoxylin and eosin (H&E)染色 48
3.4.7腫瘤組織切片免疫螢光染色 48
3.5 數據統計 49
四、結果與討論 50
4.1 高分子合成與鑑定 50
4.1.1 Poly(thiodiethylene adipate) (PSDEA) 與Poly(hexamethylene adipate) (PHDEA) 合成與組成鑑定 50
4.1.2 PEG-PSDEA-PEG 和 PEG-PHDEA-PEG合成與組成鑑定 51
4.2 高分子氧化應答能力 53
4.3 高分子生物可降解性 54
4.4 奈米粒子特性分析 55
4.4.1 奈米微粒性質分析 55
4.4.2 奈米微粒穩定度分析 57
4.4.3 奈米微粒氧化應答能力評估 58
4.4.4 奈米微粒體外藥物釋放 63
4.5 細胞實驗 65
4.5.1 奈米粒子於ROS treatment後細胞吞噬評估 65
4.5.2奈米粒子於ROS treatment後細胞毒性分析 66
4.5.3放射線結合化療之細胞毒性分析 69
4.6 動物實驗 70
4.6.1腫瘤抑制生長評估 70
4.6.2腫瘤組織切片觀察 73
五、結論 76
六、參考文獻 77

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