臺灣博碩士論文加值系統

臨床上Mitomycin C具有廣效性的抗癌活性，也是常用在第一線治療癌症的化療藥物，但是Mitomycin C的半衰期短，很快被體內排除，且治療區間 (Therapeutic Index, TI)狹窄，易造成很大的毒性，因此限制了Mitomycin C的治療效果。本研究中，我們使用對pH值具有敏感性的磷脂質 (DOPE)和半琥珀酸酯 (CHEMS)，以及具有標靶功能的DSPE-PEG2000-folate製備得到酸鹼敏感型之微脂粒，並評估其物化特性，並藉由在腫瘤組織中的弱酸性環境，以及標靶效果，在特定的腫瘤組織釋放藥物。微脂粒處方比例為DOPE/CHEMS/DSPE-PEG2000-folate=6/4/0.01 (Molar ratio)，其粒徑大小 (Size) 144.5±2.8 nm，粒徑分散性 (Polydispersity index) 0.343±0.047，表面電位 (Zeta potential) -66.7±0.3 mV，包埋率 (Encapsulation efficiency) 66.5±0.1%。藉由癌細胞來探討微脂粒處方的毒殺功能，其結果顯示微脂粒具有細胞毒殺效果，IC50為8.75 &;#61549;M。在動物實驗中，微脂粒處方經靜脈注射投與給健康大鼠，並與溶液劑做比較，觀察大鼠體內的藥物動力學參數情形，得知Mitomycin C溶液劑和微脂粒處方的半衰期分別為1.35±0.15和1.60±0.04 小時，並由老鼠血液生化檢測評估微脂粒處方之副作用。

Mitomycin C is a potent anticancer drug. But the severe side effect like cumulative myelosuppression and nephrotoxicity limit its therapeutic efficacy. Moreover, the clinical use of Mitomycin C is significantly hindered due to the rapid elimination from the body. The half-life of Mitomycin C is about 23~78 minutes. The purpose of this study was to formulate the pH-sensitive liposomes for cancer therapy. DOPE, CHEMS and DSPE-PEG2000-folate were used as materials to prepare pH-sensitive liposomes. The physicochemical characteristics of pH-sensitive liposomes : particle size 144.5±2.8 nm, polydispersity index 0.343±0.047, zeta potential -66.7±0.3 mV and entrapment efficiency 66.5±0.1%. In vitro drug release was modestly prolonged and presented good pH sensitivity. In cytotoxicity assays, the IC50 of pH-sensitive liposomes was 8.75 &;#61549;M. The half-life of pH-sensitive liposomes and Mitomycin C solution were 1.35±0.15 and 1.60±0.04 hours. Besides, the blood toxicity observations of rats would be revealed.

目錄.... I
圖目錄.. III
表目錄...V
摘要.... VI
Abstract....... VII
第一章 緒論...... 1
ㄧ、 研究背景.... 1
二、 藥物介紹.... 3
三、 微脂粒...... 4
四、 研究目的.... 22
第二章 材料與儀器設備..... 23
ㄧ、 藥品與化學試劑.......23
二、 細胞培養.... 24
三、 微脂粒製備材料....... 24
四、 儀器設備.... 25
第三章 實驗方法.. 27
ㄧ、 分析方法之建立...... 27
二、 Mitomycin C溶液劑生體分佈試驗...35
三、 評估Mitomycin C在不同pH值下之安定性...37
四、 微脂粒劑型設計及其製備方法.... 38
五、 微脂粒之粒徑大小、粒徑分散性及表面電位測量...... 41
六、 微脂粒之包埋率....... 42
七、 微脂粒處方之穿透式電子顯微鏡觀察....... 43
八、 微脂粒處方之體外釋放試驗...... 43
九、 微脂粒之血漿和血清穩定性...... 44
十、 微脂粒處方之安定性評估........44
十一、 生物活性評估....... 45
十二、 Mitomycin C微脂粒處方生體分佈試驗... 49
十三、 老鼠血液生化檢測... 49
十四、 統計分析.. .49
第三章 結果與討論...50
ㄧ、 分析方法之建立...... 50
二、 Mitomycin C溶液劑生體分佈試驗...62
三、 Mitomycin C在不同pH值下之安定性...... 66
四、 處方設計.... 68
五、 微脂粒穿透式電子顯微鏡觀察.... 75
六、 Mitomycin C體外釋放試驗....76
七、 微脂粒處方之血漿和血清穩定性...78
八、 微脂粒處方之安定性試驗........79
九、 細胞存活率試驗....... 82
十、 流式細胞儀之細胞吞噬實驗...... 84
十一、 微脂粒處方生體分佈試驗......86
十二、 Mitomycin C溶液劑和微脂粒處方之藥物動力學參數比較...88
十三、 Mitomycin C溶液劑和微脂粒處方之組織分布......91
十四、 老鼠血液生化檢測....92
第四章 結論.......95
第五章 參考文獻 96

1. http://www.hpa.gov.tw/Bhpnet/Web/Index/Index.aspx.
2. Malhotra, V. and M.C. Perry, Classical chemotherapy: mechanisms, toxicities and the therapeutic window. Cancer Biol Ther, 2003. 2(4 Suppl 1): p. S2-4.
3. Tomasz, M., Mitomycin C: small, fast and deadly (but very selective). Chem Biol, 1995. 2(9): p. 575-9.
4. Propper, D.J., Levitt, N. C., O''Byrne, K., Talbot, D. C., Ganesan, T. S., Thompson, C. H., Rajagopalan, B., Littlewood, T. J., Dixon, R. M. and Harris, A. L., Phase II study of the oxygen saturation curve left shifting agent BW12C in combination with the hypoxia activated drug mitomycin C in advanced colorectal cancer. Br J Cancer, 2000. 82(11): p. 1776-82.
5. Gabizon, A. and Martin, F., Therapeutic efficacy of a lipid-based prodrug of mitomycin C in pegylated liposomes: studies with human gastro-entero-pancreatic ectopic tumor models. J Control Release, 2012. 160(2): p. 245-53.
6. Gabizon, A., Tzemach, D., Horowitz, A. T., Shmeeda, H., Yeh, J. and Zalipsky, S., Reduced toxicity and superior therapeutic activity of a mitomycin C lipid-based prodrug incorporated in pegylated liposomes. Clin Cancer Res, 2006. 12(6): p. 1913-20.
7. Peer, D. and R. Margalit, Loading mitomycin C inside long circulating hyaluronan targeted nano-liposomes increases its antitumor activity in three mice tumor models. Int J Cancer, 2004. 108(5): p. 780-9.
8. Shuhendler, A.J., Cheung, R. Y., Manias, J., Connor, A., Rauth, A. M., Wu, X. Y., A novel doxorubicin-mitomycin C co-encapsulated nanoparticle formulation exhibits anti-cancer synergy in multidrug resistant human breast cancer cells. Breast Cancer Res Treat, 2010. 119(2): p. 255-69.
9. http://www.ktgh.com.tw/.
10. http://ezproxy.kmu.edu.tw:2068/micromedex2/librarian.
11. http://www.sigmaaldrich.com.
12. Vemuri, S. and C.T. Rhodes, Preparation and characterization of liposomes as therapeutic delivery systems: a review. Pharm Acta Helv, 1995. 70(2): p. 95-111.
13. Sharma, A. and U.S. Sharma, Liposomes in drug delivery: progress and limitations. International Journal of Pharmaceutics, 1997. 154(2): p. 123-140.
14. Liu, X. and G. Huang, Formation strategies, mechanism of intracellular delivery and potential clinical applications of pH-sensitive liposomes. Asian Journal of Pharmaceutical Sciences, 2013. 8(6): p. 319-328.
15. Jesorka, A. and O. Orwar, Liposomes: technologies and analytical applications. Annu Rev Anal Chem (Palo Alto Calif), 2008. 1: p. 801-32.
16. Jones, M.N., The surface properties of phospholipid liposome systems and their characterisation. Adv Colloid Interface Sci, 1995. 54: p. 93-128.
17. Mashaghi, S., Jadidi, T., Koenderink, G., and Mashaghi, A.,Lipid nanotechnology. Int J Mol Sci, 2013. 14(2): p. 4242-82.
18. http://www.avantilipids.com/.
19. Ohvo-Rekila, H., Ramstedt, B., Leppimaki, P., and Slotte, J. P., Cholesterol interactions with phospholipids in membranes. Prog Lipid Res, 2002. 41(1): p. 66-97.
20. G&;#243;mez-Hens, A. and J.M. Fern&;#225;ndez-Romero, Analytical methods for the control of liposomal delivery systems. TrAC Trends in Analytical Chemistry, 2006. 25(2): p. 167-178.
21. Mozafari, M.R., Johnson, C., Hatziantoniou, S. and Demetzos, C., Nanoliposomes and their applications in food nanotechnology. J Liposome Res, 2008. 18(4): p. 309-27.
22. Akbarzadeh, A., Rezaei-Sadabady, R., Davaran, S., Joo, S. W., Zarghami, N., Hanifehpour, Y., Samiei, M., Kouhi, M. and Nejati-Koshki, K., Liposome: classification, preparation, and applications. Nanoscale Res Lett, 2013. 8(1): p. 102.
23. Torchilin, V.P., Recent advances with liposomes as pharmaceutical carriers. Nat Rev Drug Discov, 2005. 4(2): p. 145-60.
24. Simoes, S., Moreira, J. N., Fonseca, C., Duzgunes, N. and Lima, M. C. , On the formulation of pH-sensitive liposomes with long circulation times. Adv Drug Deliv Rev, 2004. 56(7): p. 947-65.
25. Leite, E.A., Souza, C. M., Carvalho-Junior, A. D., Coelho, L. G., Lana, A. M., Cassali, G. D. and Oliveira, M. C., Encapsulation of cisplatin in long-circulating and pH-sensitive liposomes improves its antitumor effect and reduces acute toxicity. Int J Nanomedicine, 2012. 7: p. 5259-69.
26. Immordino, M.L., F. Dosio, and L. Cattel, Stealth liposomes: review of the basic science, rationale, and clinical applications, existing and potential. International Journal of Nanomedicine, 2006. 1(3): p. 297-315.
27. Gabizon, A. and F. Martin, Polyethylene glycol-coated (pegylated) liposomal doxorubicin. Rationale for use in solid tumours. Drugs, 1997. 54 Suppl 4: p. 15-21.
28. Scherphof, G.L., Dijkstra, J., Spanjer, H. H., Derksen, J. T. and Roerdink, F. H., Uptake and intracellular processing of targeted and nontargeted liposomes by rat Kupffer cells in vivo and in vitro. Ann N Y Acad Sci, 1985. 446: p. 368-84.
29. Allen, T.M., Hansen, C., Martin, F., Redemann, C. and Yau-Young, A., Liposomes containing synthetic lipid derivatives of poly(ethylene glycol) show prolonged circulation half-lives in vivo. Biochim Biophys Acta, 1991. 1066(1): p. 29-36.
30. https://zh.wikipedia.org.
31. Weitman, S.D., Lark, R. H., Coney, L. R., Fort, D. W., Frasca, V., Zurawski, V. R., and Kamen, B. A., Distribution of the folate receptor GP38 in normal and malignant cell lines and tissues. Cancer Res, 1992. 52(12): p. 3396-401.
32. Low, P.S., W.A. Henne, and D.D. Doorneweerd, Discovery and development of folic-acid-based receptor targeting for imaging and therapy of cancer and inflammatory diseases. Acc Chem Res, 2008. 41(1): p. 120-9.
33. Wu, J., Q. Liu, and R.J. Lee, A folate receptor-targeted liposomal formulation for paclitaxel. International Journal of Pharmaceutics, 2006. 316(1–2): p. 148-153.
34. Wu, M., W. Gunning, and M. Ratnam, Expression of folate receptor type alpha in relation to cell type, malignancy, and differentiation in ovary, uterus, and cervix. Cancer Epidemiol Biomarkers Prev, 1999. 8(9): p. 775-82.
35. Nakashima-Matsushita, N., Homma, T., Yu, S., Matsuda, T., Sunahara, N., Nakamura, T., Tsukano, M., Ratnam, M. and Matsuyama, T., Selective expression of folate receptor beta and its possible role in methotrexate transport in synovial macrophages from patients with rheumatoid arthritis. Arthritis Rheum, 1999. 42(8): p. 1609-16.
36. Sudimack, J.J., Guo, W., Tjarks, W. and Lee, R. J., A novel pH-sensitive liposome formulation containing oleyl alcohol. Biochim Biophys Acta, 2002. 1564(1): p. 31-7.
37. Hilgenbrink, A.R. and P.S. Low, Folate receptor-mediated drug targeting: from therapeutics to diagnostics. J Pharm Sci, 2005. 94(10): p. 2135-46.
38. Lu, Y., Ding, N., Yang, C., Huang, L., Liu, J. and Xiang, G., Preparation and in vitro evaluation of a folate-linked liposomal curcumin formulation. J Liposome Res, 2012. 22(2): p. 110-9.
39. Meyer, F., Ridwelski, K., Gebauer, T., Grote, R., Martens-Lobenhoffer, J. and Lippert, H., Pharmacokinetics of the antineoplastic drug mitomycin C in regional chemotherapy using the aortic stop flow technique in advanced pancreatic carcinoma. Chemotherapy, 2005. 51(1): p. 1-8.
40. Lee, R.J. and L. Huang, Folate-targeted, anionic liposome-entrapped polylysine-condensed DNA for tumor cell-specific gene transfer. J Biol Chem, 1996. 271(14): p. 8481-7.
41. Ishida, T., Kirchmeier, M. J., Moase, E. H., Zalipsky, S. and Allen, T. M., Targeted delivery and triggered release of liposomal doxorubicin enhances cytotoxicity against human B lymphoma cells. Biochim Biophys Acta, 2001. 1515(2): p. 144-58.
42. Li, Y., Wu, H., Yang, X., Jia, M., Li, Y., Huang, Y., Lin, J., Wu, S., and Hou, Z., Mitomycin C-soybean phosphatidylcholine complex-loaded self-assembled PEG-lipid-PLA hybrid nanoparticles for targeted drug delivery and dual-controlled drug release. Mol Pharm, 2014. 11(8): p. 2915-27.
43. Ye, P., Zhang, W., Yang, T., Lu, Y., Lu, M., Gai, Y., Ma, X. and Xiang, G., Folate receptor-targeted liposomes enhanced the antitumor potency of imatinib through the combination of active targeting and molecular targeting. Int J Nanomedicine, 2014. 9: p. 2167-78.
44. http://www.haiyunzl.com/meitu/pic.
45. Zhou, Q.M., Wang, X. F., Liu, X. J., Zhang, H., Lu, Y. Y. and Su, S. B., Curcumin enhanced antiproliferative effect of mitomycin C in human breast cancer MCF-7 cells in vitro and in vivo. Acta Pharmacol Sin, 2011. 32(11): p. 1402-10.
46. http://www.enzolifesciences.com/ALX-850-039/cell-counting-kit-8.
47. Beijnen, J.H. and W.J.M. Underberg, Degradation of mitomycin C in acidic solution. International Journal of Pharmaceutics, 1985. 24(2–3): p. 219-229.
48. Sabeti, B., Noordin, M. I., Mohd, S., Hashim, R. and Dahlan, A., Development and characterization of liposomal doxorubicin hydrochloride with palm oil. 2014. 2014: p. 765426.
49. Shi, G., Guo, W., StepHenson, S. M. and Lee, R. J., Efficient intracellular drug and gene delivery using folate receptor-targeted pH-sensitive liposomes composed of cationic/anionic lipid combinations. J Control Release, 2002. 80(1-3): p. 309-19.
50. Thakur, R., A. Das, and A. Chakraborty, Interaction of human serum albumin with liposomes of saturated and unsaturated lipids with different phase transition temperatures: a spectroscopic investigation by membrane probe PRODAN. RSC Advances, 2014. 4(28): p. 14335.
51. Alexis, F., Pridgen, E., Molnar, L. K. and Farokhzad, O. C., Factors affecting the clearance and biodistribution of polymeric nanoparticles. Mol Pharm, 2008. 5(4): p. 505-15.
52. Muhonen, T.T., Wiklund, T. A., Blomqvist, C. P. and Pyrhonen, S. O., Unexpected prolonged myelosuppression after mitomycin, mitoxantrone and methotrexate. Eur J Cancer, 1992. 28a(12): p. 1974-6.
53. Verwey, J., J. de Vries, and H.M. Pinedo, Mitomycin C-induced renal toxicity, a dose-dependent side effect? Eur J Cancer Clin Oncol, 1987. 23(2): p. 195-9.
54. http://www.kmuh.org.tw/www/clireser/29.htm.