索拉菲尼，一種多重酪胺酸酶抑制劑，是臨床上用以治療具高度血管化的肝癌組織(HCC)之抗血管新生療法用藥；近年來其不僅表現臨床療效不佳，還造成嚴重的肝癌復發情形。索拉菲尼治療將誘導腫瘤組織的缺氧情況進而活化「第四型半胱氨酸─X─半胱氨酸受器」(CXC4)與其配體「基質細胞衍生因子─1α 」(SDF-1α)之結合，引導肝癌組織朝向促腫瘤發展之微環境，使之對抗血管新生療法產生抗藥性(Resistance)。此研究中，我們研發出一種具標靶CXCR4且攜載索拉菲尼之脂質包覆PLGA奈米粒子；奈米粒子表面以CXCR4 拮抗劑─AMD3100進行修飾，透過全身循環方式將索拉菲尼輸送至肝癌組織內並提高肝癌組織對索拉菲尼的敏感性。此研究於in vitro 與 in vivo 實驗結果皆呈現標靶CXCR4之奈米粒子能有效率地將索拉菲尼輸送至肝癌細胞以及人類臍靜脈內皮細胞(HUVECs)並達到顯著地細胞毒殺以及抗血管新生效果。於動物實驗結果，儘管 SDF-1α 的表現量會受到具標靶CXCR4且同時攜載索拉菲尼之奈米粒子治療而造成組織缺氧之情形提高，修飾 AMD3100於奈米粒子的表面上能夠阻斷 SDF-1α與CXCR4 鍵結，進而導致腫瘤相關巨噬細胞(TAMs)的浸潤情況減少、抗血管新生效果提升、抑制腫瘤生長以及延長具有原位肝癌模型小鼠之總存活期(Overall survival)，相較於控制組之結果。總結，本研究結果顯示出具有高度臨床潛力的多功能奈米粒子不僅能透過標靶方式輸送抗血管新生藥物至肝癌組織中，還改善腫瘤微環境以及克服肝癌組織對索拉菲尼的抗藥性，進而達到最有效的肝癌治療方法。

Sorafenib, a multikinase inhibitor, has been used as an anti-angiogenic agent against highly vascular hepatocellular carcinoma (HCC) – yet associated with only moderate therapeutic effect and the high incidence of HCC recurrence. We have shown intratumoral hypoxia induced by sorafenib activated C-X-C receptor type 4 (CXCR4)/stromal-derived factor 1α (SDF-1α) axis, resulting in polarization toward a tumor-promoting microenvironment and resistance to anti-angiogenic therapy in HCC. Herein, we formulated sorafenib in CXCR4-targeted lipid-coated poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) modified with a CXCR4 antagonist, AMD3100 to systemically deliver sorafenib into HCC and sensitize HCC to sorafenib treatment. We demonstrated that CXCR4-targeted NPs efficiently delivered sorafenib into HCCs and human umbilical vein endothelial cells (HUVECs) to achieve cytotoxicity and anti-angiogenic effect in vitro and in vivo. Despite the increased expression of SDF-1α upon the persistent hypoxia induced by sorafenib-loaded CXCR4-targeted NPs, AMD3100 attached to the NPs can block CXCR4/SDF-1α, leading to the reduced infiltration of tumor-associated macrophages, enhanced anti-angiogenic effect, a delay in tumor progression and increased overall survival in the orthotopic HCC model compared to other control groups. In conclusion, our results highlight the clinical potential of CXCR4-targeted NPs for delivering sorafenib and overcoming acquired drug resistance in liver cancer.

中文摘要
Abstract
總目錄
圖片目錄
縮寫表示
章節一、文獻回顧
1.1 肝癌以及其治療方針
1.2 治療肝癌化學療法用藥─雷莎瓦與其抗藥性
1.3 肝癌組織對組織缺氧的回應
1.4 CXCR4拮抗劑 ─ AMD3100之應用
1.5 奈米粒子克服困境
1.6 研究目標
章節二、材料與方法
2.1 實驗材料
2.2 細胞培養
2.3 實驗動物培養與小鼠原位肝癌模型之建立
2.4 奈米粒子的製備
2.5 奈米粒子的特性測試
2.6 細胞活性測試
2.7 細胞吞噬測試
2.8 藥物釋放測試
2.9 藥物動力學與癌症模式老鼠之治療
2.10 組織切片製作
2.11 免疫螢光染色實驗
2.12 細胞凋亡分析之TUNEL染色方法
2.13 蘇木精─伊紅染色
2.14 資料統計與分析
章節三、實驗結果與討論
3.1 研發標靶CXCR4之脂質包覆PLGA奈米粒子以及其特性
3.2 標靶CXCR4之脂質包覆PLGA奈米粒子之in vitro細胞吞噬以及細胞毒性的測試
3.3 In vivo活體內藥物動力學以及標靶CXCR4之脂質包覆PLGA奈米粒子於腫瘤組織的累積
3.4 標靶CXCR4之脂質包覆PLGA奈米粒子輸送索拉菲尼至肝癌組織以及腫瘤微環境的改善
3.5 標靶CXCR4之PLGA奈米粒子輸送索拉菲尼與阻斷CXCR4來達成原位肝腫瘤生長抑制與遠端轉移之抑制並且提高原位肝癌模型小鼠總存活期
章節四、結果與討論
章節五、結論
章節六、圖片與表格
章節五、參考文獻

1. Siegel R, Ma J, Zou Z, Jemal A. Cancer statistics, 2014. CA: a cancer journal for clinicians 2014;64(1):9-29.
2. USA NCIot. Five year survival rate of patients with liver cancer (http://seer.cancer.gov/statfacts/html/livibd.html). National Cancer Institute of the USA 2015.
3. 中華民國衛生福利部國民健康署. 中華民國102年國民健康署之肝癌患者人數統計 (http://www.mohw.gov.tw/MOHW_Upload/doc/1040414%E8%AA%AA%E6%98%8E%E7%A8%BF%E9%99%84%E4%BB%B6_0049007002.pdf). 2011.
4. 中華民國衛生福利部國民健康署. 中華民國102年國民健康署之癌症死亡率 (http://health99.hpa.gov.tw/TXT/PreciousLifeZone/print.aspx?TopIcNo=846&DS=1-life). 2011.
5. Bishayee A. The Inflammation and Liver Cancer. In: Aggarwal BB, Sung B, Gupta SC, editors. Inflammation and Cancer. Volume 816, Advances in Experimental Medicine and Biology: Springer Basel; 2014. p 401-435.
6. Llovet JM. Updated treatment approach to hepatocellular carcinoma. Journal of Gastroenterology 2005;40(3):225-235.
7. Llovet JM, Ricci S, Mazzaferro V, Hilgard P, Gane E, Blanc JF, de Oliveira AC, Santoro A, Raoul JL, Forner A and others. Sorafenib in advanced hepatocellular carcinoma. N Engl J Med 2008;359(4):378-90.
8. Semela D, Dufour J-F. Angiogenesis and hepatocellular carcinoma. Journal of hepatology 2004;41(5):864-880.
9. Wang Y, Gao J, Zhang D, Zhang J, Ma J, Jiang H. New insights into the antifibrotic effects of sorafenib on hepatic stellate cells and liver fibrosis. Journal of hepatology 2010;53(1):132-144.
10. Hasskarl J. Sorafenib: targeting multiple tyrosine kinases in cancer. Small Molecules in Oncology: Springer; 2014. p 145-164.
11. Cheng A-L, Kang Y-K, Chen Z, Tsao C-J, Qin S, Kim JS, Luo R, Feng J, Ye S, Yang T-S. Efficacy and safety of sorafenib in patients in the Asia-Pacific region with advanced hepatocellular carcinoma: a phase III randomised, double-blind, placebo-controlled trial. The lancet oncology 2009;10(1):25-34.
12. Carmeliet P, Jain RK. Molecular mechanisms and clinical applications of angiogenesis. Nature 2011;473(7347):298-307.
13. Jain RK. Normalizing tumor microenvironment to treat cancer: bench to bedside to biomarkers. J Clin Oncol 2013;31(17):2205-18.
14. Semenza GL. Hypoxia-inducible factors in physiology and medicine. Cell 2012;148(3):399-408.
15. Huang Y, Goel S, Duda DG, Fukumura D, Jain RK. Vascular normalization as an emerging strategy to enhance cancer immunotherapy. Cancer Res 2013;73(10):2943-8.
16. Huang Y, Snuderl M, Jain RK. Polarization of tumor-associated macrophages: a novel strategy for vascular normalization and antitumor immunity. Cancer Cell 2011;19(1):1-2.
17. Motz GT, Coukos G. Deciphering and Reversing Tumor Immune Suppression. Immunity 2013;39(1):61-73.
18. Wilson WR, Hay MP. Targeting hypoxia in cancer therapy. Nat Rev Cancer 2011;11(6):393-410.
19. Burger M, Glodek A, Hartmann T, Schmitt-Gräff A, Silberstein LE, Fujii N, Kipps TJ, Burger JA. Functional expression of CXCR4 (CD184) on small-cell lung cancer cells mediates migration, integrin activation, and adhesion to stromal cells. Oncogene 2003;22(50):8093-8101.
20. Li YM, Pan Y, Wei Y, Cheng X, Zhou BP, Tan M, Zhou X, Xia W, Hortobagyi GN, Yu D. Upregulation of CXCR4 is essential for HER2-mediated tumor metastasis. Cancer cell 2004;6(5):459-469.
21. Chen Y, Huang Y, Reiberger T, Duyverman AM, Huang P, Samuel R, Hiddingh L, Roberge S, Koppel C, Lauwers GY and others. Differential effects of sorafenib on liver versus tumor fibrosis mediated by stromal-derived factor 1 alpha/C-X-C receptor type 4 axis and myeloid differentiation antigen-positive myeloid cell infiltration in mice. Hepatology 2014;59(4):1435-47.
22. Chen Y, Ramjiawan RR, Reiberger T, Ng MR, Hato T, Huang Y, Ochiai H, Kitahara S, Unan EC, Reddy TP and others. CXCR4 inhibition in tumor microenvironment facilitates anti-PD-1 immunotherapy in sorafenib-treated HCC in mice. Hepatology 2014.
23. Duda DG, Kozin SV, Kirkpatrick ND, Xu L, Fukumura D, Jain RK. CXCL12 (SDF1α)-CXCR4/CXCR7 pathway inhibition: an emerging sensitizer for anticancer therapies? Clinical Cancer Research 2011;17(8):2074-2080.
24. Farazi PA, DePinho RA. Hepatocellular carcinoma pathogenesis: from genes to environment. Nature Reviews Cancer 2006;6(9):674-687.
25. Friand V, Haddad O, Papy-Garcia D, Hlawaty H, Vassy R, Hamma-Kourbali Y, Perret G-Y, Courty J, Baleux F, Oudar O. Glycosaminoglycan mimetics inhibit SDF-1/CXCL12-mediated migration and invasion of human hepatoma cells. Glycobiology 2009;19(12):1511-1524.
26. Schimanski C, Bahre R, Gockel I, Müller A, Frerichs K, Hörner V, Teufel A, Simiantonaki N, Biesterfeld S, Wehler T. Dissemination of hepatocellular carcinoma is mediated via chemokine receptor CXCR4. British journal of cancer 2006;95(2):210-217.
27. Teicher BA, Fricker SP. CXCL12 (SDF-1)/CXCR4 pathway in cancer. Clinical Cancer Research 2010;16(11):2927-2931.
28. Force T, Krause DS, Van Etten RA. Molecular mechanisms of cardiotoxicity of tyrosine kinase inhibition. Nature Reviews Cancer 2007;7(5):332-344.
29. Duckles S, Irvine C. Handbook of experimental pharmacology. 1993.
30. Peterlin BM, Trono D. Hide, shield and strike back: how HIV-infected cells avoid immune eradication. Nature Reviews Immunology 2003;3(2):97-107.
31. De M, Ghosh PS, Rotello VM. Applications of nanoparticles in biology. Advanced Materials 2008;20(22):4225-4241.
32. Brigger I, Dubernet C, Couvreur P. Nanoparticles in cancer therapy and diagnosis. Advanced drug delivery reviews 2002;54(5):631-651.
33. Maeda H, Wu J, Sawa T, Matsumura Y, Hori K. Tumor vascular permeability and the EPR effect in macromolecular therapeutics: a review. Journal of controlled release 2000;65(1):271-284.
34. Matsumura Y, Maeda H. A new concept for macromolecular therapeutics in cancer chemotherapy: mechanism of tumoritropic accumulation of proteins and the antitumor agent smancs. Cancer research 1986;46(12 Part 1):6387-6392.
35. Peer D, Karp JM, Hong S, Farokhzad OC, Margalit R, Langer R. Nanocarriers as an emerging platform for cancer therapy. Nature nanotechnology 2007;2(12):751-760.
36. Ulery BD, Nair LS, Laurencin CT. Biomedical applications of biodegradable polymers. Journal of polymer science Part B: polymer physics 2011;49(12):832-864.
37. Acharya S, Sahoo SK. PLGA nanoparticles containing various anticancer agents and tumour delivery by EPR effect. Advanced drug delivery reviews 2011;63(3):170-183.
38. Avgoustakis K, Beletsi A, Panagi Z, Klepetsanis P, Karydas AG, Ithakissios DS. PLGA–mPEG nanoparticles of cisplatin: in vitro nanoparticle degradation, in vitro drug release and in vivo drug residence in blood properties. Journal of Controlled Release 2002;79(1–3):123-135.
39. Danhier F, Ansorena E, Silva JM, Coco R, Le Breton A, Préat V. PLGA-based nanoparticles: an overview of biomedical applications. Journal of controlled release 2012;161(2):505-522.
40. Feng S-S, Mu L, Win KY, Huang G. Nanoparticles of biodegradable polymers for clinical administration of paclitaxel. Current medicinal chemistry 2004;11(4):413-424.
41. Fonseca C, Simoes S, Gaspar R. Paclitaxel-loaded PLGA nanoparticles: preparation, physicochemical characterization and in vitro anti-tumoral activity. Journal of Controlled Release 2002;83(2):273-286.
42. Jain RA. The manufacturing techniques of various drug loaded biodegradable poly (lactide-< i> co-glycolide)(PLGA) devices. Biomaterials 2000;21(23):2475-2490.
43. Kumari A, Yadav SK, Yadav SC. Biodegradable polymeric nanoparticles based drug delivery systems. Colloids and Surfaces B: Biointerfaces 2010;75(1):1-18.
44. Mu L, Feng S. A novel controlled release formulation for the anticancer drug paclitaxel (Taxol®): PLGA nanoparticles containing vitamin E TPGS. Journal of controlled release 2003;86(1):33-48.
45. Mu L, Feng S-S. PLGA/TPGS nanoparticles for controlled release of paclitaxel: effects of the emulsifier and drug loading ratio. Pharmaceutical research 2003;20(11):1864-1872.
46. Yoo HS, Lee KH, Oh JE, Park TG. In vitro and in vivo anti-tumor activities of nanoparticles based on doxorubicin–PLGA conjugates. Journal of Controlled Release 2000;68(3):419-431.
47. Pinto-Alphandary H, Andremont A, Couvreur P. Targeted delivery of antibiotics using liposomes and nanoparticles: research and applications. International journal of antimicrobial agents 2000;13(3):155-168.
48. Samad A, Sultana Y, Aqil M. Liposomal drug delivery systems: an update review. Current drug delivery 2007;4(4):297-305.
49. Torchilin VP. Recent advances with liposomes as pharmaceutical carriers. Nature reviews Drug discovery 2005;4(2):145-160.
50. Vyas S, Rai S, Paliwal R, Gupta PN, Khatri K, Goyal AK, Vaidya B. Solid lipid nanoparticles (SLNs) as a rising tool in drug delivery science: One step up in nanotechnology. Current Nanoscience 2008;4(1):30-44.
51. Zhang L, Chan JM, Gu FX, Rhee J-W, Wang AZ, Radovic-Moreno AF, Alexis F, Langer R, Farokhzad OC. Self-assembled lipid− polymer hybrid nanoparticles: a robust drug delivery platform. ACS nano 2008;2(8):1696-1702.
52. Salvador-Morales C, Zhang L, Langer R, Farokhzad OC. Immunocompatibility properties of lipid–polymer hybrid nanoparticles with heterogeneous surface functional groups. Biomaterials 2009;30(12):2231-2240.
53. Liu Y, Li K, Pan J, Liu B, Feng S-S. Folic acid conjugated nanoparticles of mixed lipid monolayer shell and biodegradable polymer core for targeted delivery of Docetaxel. Biomaterials 2010;31(2):330-338.
54. Cheow WS, Hadinoto K. Factors affecting drug encapsulation and stability of lipid–polymer hybrid nanoparticles. Colloids and Surfaces B: Biointerfaces 2011;85(2):214-220.
55. Tofilon PJ, Basic I, Milas L. Prediction of in vivo tumor response to chemotherapeutic agents by the in vitro sister chromatid exchange assay. Cancer research 1985;45(5):2025-2030.
56. Kim W, Seong J, Oh HJ, Koom WS, Choi K-J, Yun C-O. A novel combination treatment of armed oncolytic adenovirus expressing IL-12 and GM-CSF with radiotherapy in murine hepatocarcinoma. Journal of radiation research 2011;52(5):646-654.
57. Wang G, Yu B, Wu Y, Huang B, Yuan Y, Liu CS. Controlled preparation and antitumor efficacy of vitamin E TPGS-functionalized PLGA nanoparticles for delivery of paclitaxel. International journal of pharmaceutics 2013;446(1):24-33.
58. Capece D, Fischietti M, Verzella D, Gaggiano A, Cicciarelli G, Tessitore A, Zazzeroni F, Alesse E. The inflammatory microenvironment in hepatocellular carcinoma: a pivotal role for tumor-associated macrophages. Biomed Res Int 2013;2013:187204.
59. Zhou D, Huang C, Kong L, Li J. Novel therapeutic target of hepatocellular carcinoma by manipulation of macrophage colony-stimulating factor/tumor-associated macrophages axis in tumor microenvironment. Hepatol Res 2014;44(10):E318-9.
60. Liu Y, Feng L, Liu T, Zhang L, Yao Y, Yu D, Wang L, Zhang N. Multifunctional pH-sensitive polymeric nanoparticles for theranostics evaluated experimentally in cancer. Nanoscale 2014;6(6):3231-3242.
61. Xu Y, Chenna V, Hu C, Sun H-X, Khan M, Bai H, Yang X-R, Zhu Q-F, Sun Y-F, Maitra A. Polymeric nanoparticle-encapsulated hedgehog pathway inhibitor HPI-1 (NanoHHI) inhibits systemic metastases in an orthotopic model of human hepatocellular carcinoma. Clinical Cancer Research 2012;18(5):1291-1302.
62. Epstein FH, Luster AD. Chemokines—chemotactic cytokines that mediate inflammation. New England Journal of Medicine 1998;338(7):436-445.
63. Orimo A, Weinberg RA. Stromal fibroblasts in cancer: a novel tumor-promoting cell type. Cell cycle 2006;5(15):1597-1601.
64. De Clercq E. Inhibition of HIV infection by bicyclams, highly potent and specific CXCR4 antagonists. Molecular pharmacology 2000;57(5):833-839.
65. Debnath B, Xu S, Grande F, Garofalo A, Neamati N. Small molecule inhibitors of CXCR4. Theranostics 2013;3(1):47.
66. Wang X-q, Fan J-m, Liu Y-o, Zhao B, Jia Z-r, Zhang Q. Bioavailability and pharmacokinetics of sorafenib suspension, nanoparticles and nanomatrix for oral administration to rat. International journal of pharmaceutics 2011;419(1):339-346.
67. 拜耳集團(Bayer). Nexavar(蕾莎瓦)之作用機制 (http://www.nexavar.com/scripts/pages/en/home/images/moa-tumor-cells.png).
68. Busillo JM, Benovic JL. Regulation of CXCR4 signaling. Biochimica et Biophysica Acta (BBA)-Biomembranes 2007;1768(4):952-963.
69. Kim HS, Ham HO, Son YJ, Messersmith PB, Yoo HS. Electrospun catechol-modified poly (ethyleneglycol) nanofibrous mesh for anti-fouling properties. Journal of Materials Chemistry B 2013;1(32):3940-3949.