本研究之目的為開發奈米脂質微粒，分別用以搭載化療藥物阿黴素 (DOX) 及作為飢餓治療藥物的利托那韋 (RTV) 與氯奎寧 (CQ) ，對於大腸癌細胞CT26.WT進化學/饑餓合併治療。首先利用二次乳化法製備無搭載藥物、搭載DOX、搭載RTV與CQ的固態脂質奈米微粒 (分別為SLNs, DSLNs, RCSLNs) 。DSLNs之粒徑為77.29 ± 0.74 nm，藥物裝載量為4.55 ± 0.63 wt %；RCSLNs之粒徑為68.06 ± 2.15 nm，RTV及CQ的含量分別為4.64 ± 0.55 wt %及5.84 ± 0.47 wt %，此載體之粒徑符合EPR effect所適合的尺寸大小，將能夠有效的累積至腫瘤區。利用掃描式雷射共軛焦螢光顯微鏡 (CLSM) 證明載體經由胞吞作用 (endocytosis) 進入CT26.WT細胞的胞內體 (endosome) 及溶酶體 (lysosome) ，並於溶酶體內釋放出藥物，即可對於細胞進行治療。透過細胞毒性測試可以證明在同樣濃度的藥物之下，有藥物載體包覆可以達到更好的毒殺效果；並以血糖機測量細胞培養液中葡萄糖濃度的變化證實RTV可以抑制葡萄糖轉運蛋白的細胞膜內domain，減少CT26.WT細胞對於葡萄糖的攝取；另外，也以細胞流式儀分析細胞內的LC3B蛋白質表現量，證實CQ可以抑制自噬作用過程中自噬溶小體的形成，阻斷自噬作用的進行，使癌細胞無法透過自噬作用獲得能量，避免自噬作用影響飢餓治療的療效。最後也利用MTT assay測噬細胞毒性，證實合併治療相較於單獨化療或是單獨飢餓治療，都具有較佳的療效。

The aim of this work is to develop the solid lipid nanoparticles (SLNs) utilized as the drug delivery system for the chemo/starvation combination therapy against colorectal cancer. In this study, doxorubicin (DOX)-loaded SLNs (DSLNs) and ritonavir (RTV)/chloroquine (CQ)-loaded SLNs (RCSLNs) were prepared by double emulsion method. The DSLNs had a particle size of 77.29 ± 0.74 nm. The loading content of DOX was 4.55 ± 0.63 wt %. The RCSLNs had a particle size of 68.06 ± 2.15 nm. The loading content of RTV and CQ was 4.64 ± 0.55 wt % and 5.84 ± 0.47 wt %, respectively. As the particle size of DSLNs and RCSLNs was appropriate for the EPR effect, DSLNs and RCSLNs would accumulate to the tumor regions effectively. The images of confocal laser scanning microscopy (CLSM) showed that colon cancer cells CT26.WT would uptake DSLNs and RCSLNs by endocytosis. Once DSLNs and RCSLNs were uptaken by the cells, they were then degraded in lysosomes and the drug DOX, RTV, and CQ were released. In the MTT assay, it was proved that drug-loaded SLNs had a higher cellular toxicity even if the drug concentration was the same. To verify the starvation therapy were caused by RTV, the change of glucose concentration of cell culture medium RPMI was measured by blood sugar machine. According to the results, we made sure that RTV could block the cytosomal domain of glucose transporters and stop cells from uptaking glucose. Also, the flow cytometry was used to analyze the expression of LC3B in the cell and proved that CQ did inhibit the formation of autolysosome (autophagolysosome) and the following autophagy steps, which avoid cancer cells from getting energy by themselves. We also tested the cellular toxicity of chemotherapy alone, starvation therapy alone, and chemo/starvation combination therapy by MTT assay. The results showed that chemo/starvation combination therapy could be a better treatment comparing to chemotherapy alone and starvation therapy alone.

一、研究動機 13
二、文獻探討 15
2.1 惡性腫瘤 15
2.1.1 大腸直腸癌 15
2.1.2 腫瘤微環境 16
2.1.3 Enhanced permeation and retention (EPR) effect 18
2.2癌症治療策略與藥物介紹 19
2.2.1 化學治療 (Chemotherapy) 19
2.2.2 化療藥物阿黴素 (Doxorubicin) 20
2.2.3 飢餓治療 (Starvation therapy) 22
2.2.4 飢餓治療藥物利托那韋 (Ritonavir) 23
2.2.5 自噬作用 (Autophagy) 25
2.2.6 抑制自噬作用藥物氯奎寧 (Chloroquine) 26
2.3 奈米微粒藥物載體傳遞系統用於癌症治療 27
2.3.1 固態脂質奈米微粒 28
2.3.2 D-α-tocopheryl polyethylene glycol succinate (Vitamin E TPGS) 29
三、實驗方法與步驟 31
3.1 CQ萃取 31
3.2 奈米微粒製備與性質探討 31
3.2.1 固態脂質奈米微粒(SLNs)製備 31
3.2.2 固態脂質奈米微粒粒徑分析 32
3.2.3 固態脂質奈米微粒表面電荷分析 33
3.2.4 固態脂質奈米微粒藥物裝載量 35
3.2.5 體外模擬生理環境之固態脂質奈米微粒粒徑監測 36
3.2.6 體外模擬生理環境下藥物釋放分析 36
3.3 體外細胞實驗 37
3.3.1 細胞來源及適合培養之環境 37
3.3.2 細胞培養液及磷酸鹽緩衝溶液之配置 37
3.3.3 細胞繼代 38
3.3.4 細胞計數 39
3.3.5 細胞對奈米微粒的吞噬情形分析 39
3.3.6掃描式雷射共軛焦螢光顯微鏡(CLSM)觀察細胞對奈米微粒的吞噬情形 40
3.3.7 細胞毒性分析 41
3.3.8 RTV抑制葡萄糖攝取分析 42
3.3.9 CQ抑制自噬作用分析 42
3.3.10 化學/飢餓合併治療 43
3.5 數據統計 44
四、結果與討論 45
4.1 奈米微粒特性分析 45
4.1.1 固態脂質奈米微粒特性分析 45
4.1.2 固態脂質奈米微粒在模擬生理環境之粒子穩定性 47
4.1.3 奈米微粒藥物釋放評估 49
4.2 細胞實驗 50
4.2.1 細胞對奈米微粒吞噬評估 50
4.2.2掃描式雷射共軛焦螢光顯微鏡 (CLSM) 觀察細胞對奈米微粒的吞噬情形 52
4.2.3 固態脂質奈米微粒之細胞毒性分析 55
4.2.4 RTV抑制葡萄糖攝取分析 58
4.2.5 CQ抑制自噬作用分析 59
4.2.6 化學/飢餓合併治療 64
五、結論 67
六、參考文獻 68

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