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.

1. https://www.who.int/zh/news-room/fact-sheets/detail/cancer.
2. https://www.hpa.gov.tw/Pages/Detail.aspx?nodeid=615&pid=112.
3. 中華民國105年癌症登記報告.
4. Mishra, B. and S. Chaurasia, Design of novel chemotherapeutic delivery systems for colon cancer therapy based on oral polymeric nanoparticles. Ther Deliv, 2017. 8(1): p. 29-47.
5. Banerjee, A., et al., Strategies for targeted drug delivery in treatment of colon cancer: current trends and future perspectives. Drug Discovery Today, 2017. 22(8): p. 1224-1232.
6. Siegel, R.L., et al., Colorectal cancer statistics, 2017. CA: A Cancer Journal for Clinicians, 2017. 67(3): p. 177-193.
7. http://thepoopstick.com/2016/01/03/stages-of-colorectal-cancer/.
8. Quail, D.F. and J.A. Joyce, Microenvironmental regulation of tumor progression and metastasis. Nature medicine, 2013. 19(11): p. 1423-1437.
9. Korkaya, H., S. Liu, and M.S. Wicha, Breast cancer stem cells, cytokine networks, and the tumor microenvironment. The Journal of clinical investigation, 2011. 121(10): p. 3804-3809.
10. Trédan, O., et al., Drug Resistance and the Solid Tumor Microenvironment. JNCI: Journal of the National Cancer Institute, 2007. 99(19): p. 1441-1454.
11. Maeda, H., et al., Tumor vascular permeability and the EPR effect in macromolecular therapeutics: a review. Journal of Controlled Release, 2000. 65(1): p. 271-284.
12. Peer, D., et al., Nanocarriers as an emerging platform for cancer therapy. Nature Nanotechnology, 2007. 2: p. 751.
13. Danhier, F., O. Feron, and V. Préat, To exploit the tumor microenvironment: Passive and active tumor targeting of nanocarriers for anti-cancer drug delivery. Journal of Controlled Release, 2010. 148(2): p. 135-146.
14. Sun, T., et al., Engineered Nanoparticles for Drug Delivery in Cancer Therapy. Angewandte Chemie International Edition, 2014. 53(46): p. 12320-12364.
15. Manfredi, J.J. and S.B. Horwitz, Taxol: an antimitotic agent with a new mechanism of action. Pharmacology & Therapeutics, 1984. 25(1): p. 83-125.
16. Parker, W.B. and Y.C. Cheng, Metabolism and mechanism of action of 5-fluorouracil. Pharmacology & Therapeutics, 1990. 48(3): p. 381-395.
17. Thorn, C.F., et al., Doxorubicin pathways: pharmacodynamics and adverse effects. Pharmacogenetics and genomics, 2011. 21(7): p. 440-446.
18. Srikantan, S., et al., Translational control of TOP2A influences doxorubicin efficacy. Molecular and cellular biology, 2011. 31(18): p. 3790-3801.
19. Gatenby, R.A. and R.J. Gillies, Why do cancers have high aerobic glycolysis? Nature Reviews Cancer, 2004. 4(11): p. 891-899.
20. Zhang, W., et al., Targeting Tumor Metabolism for Cancer Treatment: Is Pyruvate Dehydrogenase Kinases (PDKs) a Viable Anticancer Target? International journal of biological sciences, 2015. 11(12): p. 1390-1400.
21. Chen, W.-H., et al., Overcoming the Heat Endurance of Tumor Cells by Interfering with the Anaerobic Glycolysis Metabolism for Improved Photothermal Therapy. ACS Nano, 2017. 11(2): p. 1419-1431.
22. Zhang, R., et al., Glucose & oxygen exhausting liposomes for combined cancer starvation and hypoxia-activated therapy. Biomaterials, 2018. 162: p. 123-131.
23. Kim, T.J., et al., Combined anti-angiogenic therapy against VEGF and integrin alphaVbeta3 in an orthotopic model of ovarian cancer. Cancer biology & therapy, 2009. 8(23): p. 2263-2272.
24. Wang, J., et al., Selective depletion of tumor neovasculature by microbubble destruction with appropriate ultrasound pressure. International journal of cancer, 2015. 137(10): p. 2478-2491.
25. Rodríguez-Enríquez, S., et al., Kinetics of transport and phosphorylation of glucose in cancer cells. Journal of Cellular Physiology, 2009. 221(3): p. 552-559.
26. Mueckler, M. and B. Thorens, The SLC2 (GLUT) family of membrane transporters. Molecular Aspects of Medicine, 2013. 34(2): p. 121-138.
27. Pati, S., et al., Antitumorigenic effects of HIV protease inhibitor ritonavir: inhibition of Kaposi sarcoma. Blood, 2002. 99(10): p. 3771.
28. Sgadari, C., et al., Use of HIV protease inhibitors to block Kaposi's sarcoma and tumour growth. The Lancet Oncology, 2003. 4(9): p. 537-547.
29. Hresko, R.C. and P.W. Hruz, HIV Protease Inhibitors Act as Competitive Inhibitors of the Cytoplasmic Glucose Binding Site of GLUTs with Differing Affinities for GLUT1 and GLUT4. PLOS ONE, 2011. 6(9): p. e25237.
30. McBrayer, S.K., et al., Multiple myeloma exhibits novel dependence on GLUT4, GLUT8, and GLUT11: implications for glucose transporter-directed therapy. Blood, 2012. 119(20): p. 4686-4697.
31. Puente, C., R.C. Hendrickson, and X. Jiang, Nutrient-regulated Phosphorylation of ATG13 Inhibits Starvation-induced Autophagy. The Journal of biological chemistry, 2016. 291(11): p. 6026-6035.
32. Danieli, A. and S. Martens, p62-mediated phase separation at the intersection of the ubiquitin-proteasome system and autophagy. Journal of Cell Science, 2018. 131(19): p. jcs214304.
33. Bristol, M.L., et al., Dual functions of autophagy in the response of breast tumor cells to radiation: cytoprotective autophagy with radiation alone and cytotoxic autophagy in radiosensitization by vitamin D 3. Autophagy, 2012. 8(5): p. 739-753.
34. Apel, A., et al., Blocked Autophagy Sensitizes Resistant Carcinoma Cells to Radiation Therapy. Cancer Research, 2008. 68(5): p. 1485.
35. Carew, J.S., et al., Lucanthone is a novel inhibitor of autophagy that induces cathepsin D-mediated apoptosis. The Journal of biological chemistry, 2011. 286(8): p. 6602-6613.
36. Solomon, V.R. and H. Lee, Chloroquine and its analogs: A new promise of an old drug for effective and safe cancer therapies. European Journal of Pharmacology, 2009. 625(1): p. 220-233.
37. Shintani, T. and D.J. Klionsky, Autophagy in Health and Disease: A Double-Edged Sword. Science, 2004. 306(5698): p. 990.
38. Patel, S., et al., Vorinostat and hydroxychloroquine improve immunity and inhibit autophagy in metastatic colorectal cancer. Oncotarget, 2016. 7(37): p. 59087-59097.
39. Cook, K.L., et al., Hydroxychloroquine inhibits autophagy to potentiate antiestrogen responsiveness in ER+ breast cancer. Clinical cancer research : an official journal of the American Association for Cancer Research, 2014. 20(12): p. 3222-3232.
40. Cook, K.L., et al., Chloroquine Inhibits Autophagy to Potentiate Antiestrogen Responsiveness in ER⁺ Breast Cancer. Clinical Cancer Research, 2014. 20(12): p. 3222.
41. Khodabandehloo, H., H. Zahednasab, and A. Ashrafi Hafez, Nanocarriers Usage for Drug Delivery in Cancer Therapy. Iranian journal of cancer prevention, 2016. 9(2): p. e3966-e3966.
42. Zhang, Z., S. Tan, and S.-S. Feng, Vitamin E TPGS as a molecular biomaterial for drug delivery. Biomaterials, 2012. 33(19): p. 4889-4906.
43. Szakács, G., et al., Targeting multidrug resistance in cancer. Nature Reviews Drug Discovery, 2006. 5(3): p. 219-234.
44. Collnot, E.-M., et al., Vitamin E TPGS P-Glycoprotein Inhibition Mechanism: Influence on Conformational Flexibility, Intracellular ATP Levels, and Role of Time and Site of Access. Molecular Pharmaceutics, 2010. 7(3): p. 642-651.
45. Guo, Y., et al., The applications of Vitamin E TPGS in drug delivery. European Journal of Pharmaceutical Sciences, 2013. 49(2): p. 175-186.
46. Yeh, Y.-C., et al., Fabrication of multiresponsive bioactive nanocapsules through orthogonal self-assembly. Angewandte Chemie (International ed. in English), 2014. 53(20): p. 5137-5141.
47. Corzo-Martínez, M., et al., Effect of ultra-high pressure homogenization on the interaction between bovine casein micelles and ritonavir. Pharmaceutical research, 2015. 32(3): p. 1055-1071.
48. Chen, H.-H., et al., pH-Responsive therapeutic solid lipid nanoparticles for reducing P-glycoprotein-mediated drug efflux of multidrug resistant cancer cells. International journal of nanomedicine, 2015. 10: p. 5035-5048.
49. Ogawara, K.-i., et al., In vivo anti-tumor effect of PEG liposomal doxorubicin (DOX) in DOX-resistant tumor-bearing mice: Involvement of cytotoxic effect on vascular endothelial cells. Journal of Controlled Release, 2009. 133(1): p. 4-10.
50. Dintaman, J.M. and J.A. Silverman, Inhibition of P-Glycoprotein by D-α-Tocopheryl Polyethylene Glycol 1000 Succinate (TPGS). Pharmaceutical Research, 1999. 16(10): p. 1550-1556.
51. Racoma, I.O., et al., Thymoquinone inhibits autophagy and induces cathepsin-mediated, caspase-independent cell death in glioblastoma cells. PloS one, 2013. 8(9): p. e72882-e72882.
52. Lefort, S., et al., Inhibition of autophagy as a new means of improving chemotherapy efficiency in high-LC3B triple-negative breast cancers. Autophagy, 2014. 10(12): p. 2122-2142.
53. Satyavarapu, E.M., et al., Autophagy-independent induction of LC3B through oxidative stress reveals its non-canonical role in anoikis of ovarian cancer cells. Cell Death & Disease, 2018. 9(10): p. 934.
54. Zha, B.S., et al., HIV protease inhibitors disrupt lipid metabolism by activating endoplasmic reticulum stress and inhibiting autophagy activity in adipocytes. PloS one, 2013. 8(3): p. e59514-e59514.