本研究目的為開發增加腫瘤組織累積能力及功能性高分子脂質載藥奈米粒子，可於腫瘤微環境中轉換表面電性增加藥物累積於腫瘤，同時攜帶氧氣強化藥物於腫瘤缺氧區的治療效果，並施予近紅外雷射結合光熱與光動力的雙重治療抑制腫瘤細胞生長。
本研究透過常見的酯質分子二棕櫚酰磷脂酰膽鹼 (DPPC) 作為主體包覆疏水液滴PFOB (攜帶氧氣) 於中心，利用膽固醇 (Cholesterol) 穩固酯質載體結構，並透過疏水作用力包覆光熱藥物 (IR-780) 及光動力藥物 (mTHPC) 兩種藥物於疏水介面，透過表面修飾合成具酸鹼應答的N-Acetyl-Histidine modified D-α-tocopheryl poly(ethylene glycol) succinate (NAcHis-TPGS) 幫助載體於水溶液分散。研究中透過薄膜水合法 (thin film hydration method) 及一次奈米乳化的方式製備出直徑約為250 nm及藥物包覆率70%以上的載藥奈米微粒。在載體物化特性鑑定實驗之中，帶有Histidine的NAcHis-TPGS製作出的奈米微粒成功證實具有表面電位於微酸環境轉正電之功能，同時包覆PFOB的載體藉由溶氧測定實驗證明其攜氧能力。於細胞實驗中，證實於微酸環境下電性轉換能力有效增加細胞攝取載體的數量，並於缺氧環境下，攜帶氧氣的奈米微粒能有效提升光治療對於細胞毒殺的效果。於動物腫瘤抑制實驗結果亦可以發現，具酸鹼應答標靶載體能大幅提升藥物載體累積於腫瘤組織的效果，並利用近紅外光照射所產生光熱效應，促使氧氣於腫瘤缺氧區釋放，進一步強化光動力治療的效果，於小鼠皮下腫瘤抑制實驗中，透過結合光熱及光動力的合併治療，能夠有效抑制小鼠腫瘤生長。綜合上述成果，本研究成功開發具標靶及功能性奈米光熱/光動力治療傳輸系統於前列腺癌治療上應用。

In order to enhance tumor uptake and therapeutic efficacy of photothermal/dynamic treatments of prostate cancer, a functionalized nanoparticle-based therapy system comprising perfluorooctyl bromide (PFOB) enclosed with the lipid membranes assembling from dipalmitoylphosphati dylcholine (DPPC), cholesterol and N-acetyl histidine modified D-a-tocopheryl polyethylene glycol succinate (NAcHis-TPGS) is developed in this work (PFOB@IMHNPs). The nanotherapeutics with switchable surface charges in response to tumor extracellular acidity (pHe) were capable of selectively co-delivering photosensitizers, IR-780 for photothermal therapy (PTT) and mTHPC for photodynamic therapy (PDT). Moreover, pronounced cytotoxicity by NIR-triggered PDT in hypoxia can be achieved due to the excellent oxygen carrying capacity of PFOB within NPs. As a result, the ex vivo imaging of IMHNPs and PFOB@IMHNPs in tumor-bearing mice showed enhanced tumor accumulation alongside excellent hyperthermia effect on tumor ablation. Consequently, in vivo tumor growth inhibition study shows that the incorporation of photothermal and photodynamic therapy with the smart nanoparticles exhibit effective antitumor efficacy. Based on the above results, this study provides a great potential strategy for the development of photothermal/phototdynamic drug delivery nanotherapeutics for tumor therapy.

Abstract I
摘要 II
目錄 i
圖目錄 v
表目錄 viii
一、研究動機 1
二、文獻回顧 3
2.1 惡性腫瘤 3
2.2 腫瘤微環境 3
2.2.1 Enhanced Permeation and Retention effect (EPR effect) 5
2.2.2 腫瘤微環境 pH 值 6
2.2.3 腫瘤缺氧區域 7
2.3 奈米微粒現況 8
2.3.1 奈米藥物載體傳遞系統 8
2.3.2 奈米乳化液體 9
2.4 D-α-tocopheryl poly(ethylene glycol) succinate (TPGS)簡介 10
2.5 Histamine與Imidazole contain polymer 於藥物傳遞系統之應用 11
2.6 光敏感藥物 (Photosensitizer) 及光治療 (Phototherapy) 12
2.6.1 IR-780 12
2.6.2 5,10,15,20-tetrakis(3-hydroxyphenyl) chlorin (mTHPC) 13
2.6.3 光熱治療 (Photothermal therapy) 13
2.6.4 光動力治療 (Photodynamic therapy) 14
2.7 全氟碳化合物（PFCs） 14
2.7.1 Perfluorooctylbromide (PFOB) 15
三、實驗方法與步驟 17
3.1 高分子合成與性質鑑定 17
3.1.1 無水有機溶液之製備 17
3.1.2 N-Acetyl-Histidine-TPGS合成 17
3.1.3 N-Acetyl-Histidine-TPGS高分子之組成鑑定 18
3.2 奈米微粒製備與性質分析 18
3.2.1 載藥奈米微粒的製備 18
3.2.2奈米微粒的粒徑分布 19
3.2.3 載藥奈米微粒氧氣釋放 21
3.2.4 奈米微粒的藥物裝載量 22
3.2.5搭載光熱藥物IR-780之奈米微粒升溫能力評估 22
3.2.6 活性氧物質生成測定(ROS treatment) 23
3.3 體外細胞實驗 23
3.3.1細胞來源 23
3.3.2 細胞繼代 24
3.3.3細胞計數 24
3.3.4細胞對奈米微粒吞噬情形分析 24
3.3.5 共軛焦雷射掃描螢光顯微鏡(CLSM)觀察細胞內ROS生成情形 26
3.3.6近紅外光雷射作用下之細胞毒性分析 27
3.3.7 Hoechst 33342/PI雙重染色分析 29
3.4動物實驗 29
3.4.1動物來源 29
3.4.2 腫瘤模型建立 29
3.4.3 腫瘤內載體累積時間分布 30
3.4.4 動物體內奈米微粒累積分布 30
3.4.5腫瘤抑制評估 30
3.4.6 動物犧牲與組織包埋 31
3.4.7 組織切片 31
3.4.8 組織切片免疫螢光染色 31
3.5 數據統計 32
四、結果與討論 33
4.1 材料鑑定與分析 33
4.1.1 NAcHis-TPGS 組成分析鑑定 33
4.2 奈米微粒性質分析 34
4.2.1 奈米微粒大小及電性分析 34
4.2.2 奈米微粒穩定度分析 36
4.2.3 奈米微粒之表面電荷轉換分析 37
4.2.4 奈米微粒包覆藥物穩定性分析 38
4.2.5 奈米微粒的升溫能力測試 41
4.2.6 奈米微粒攜帶氧氣的分析 41
4.2.7 奈米微粒產生ROS的能力評估 43
4.3 細胞實驗 45
4.3.1 奈米微粒生物相容性測試 45
4.3.2 奈米微粒之細胞吞噬評估 45
4.3.3 奈米微粒經NIR treatment熱治療後的分析 48
4.3.4 奈米微粒於不同環境下NIR treatment後細胞毒性分析 49
4.3.5 奈米微粒ROS產生能力測試 51
4.4動物實驗 53
4.4.1 藥物載體於動物體內累積分布 53
4.4.2 腫瘤區升溫能力測試 53
4.4.3 經熱治療後腫瘤缺氧區表現評估 55
4.4.4 腫瘤抑制評估 56
六、參考資料 63

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