Sorafenib作為現行肝癌（hepatocellular carcinoma, HCC）治療一線用藥，除了抑制腫瘤生長外，還能夠減少腫瘤中的血管新生。然而sorafenib平均只能讓病患延緩三個月的壽命，研究發現其療效可能受限於減少血管新生後，伴隨造成的腫瘤缺氧性（hypoxia）嚴重化，引發HIF1訊號機制導致腫瘤惡化。因此本實驗室開發NanoMnSor作為新型多功能藥物遞送載體，搭載肝腫瘤專一性標靶SP94以遞送sorafenib與二氧化錳。已經證實NanoMnSor能夠催化過氧化氫（H2O2）產生氧氣，舒緩缺氧性以提升sorafenib的抗癌效果，模擬在腫瘤微環境中的反應，並且減少缺氧引發的癌細胞上皮間質轉換（epithelial-mesenchymal transition, EMT），降低癌細胞的擴散能力，同時可作為MRI診斷的追蹤劑。本研究延續此成果，在小鼠肝腫瘤模型進行試驗，發現NanoMnSor能夠同時舒緩腫瘤內缺氧性與抑制血管新生，二氧化錳與H2O2反應不只會產生氧氣，尤其透過Fenton reaction產生氫氧自由基（hydroxyl radicals），促進巨噬細胞分化為M1型態，有助於抵抗腫瘤生長與刺激免疫發生。經由調節腫瘤內免疫抑制性，提升T細胞浸潤腫瘤、顯著減少腫瘤生長與促進細胞凋亡，並且偕同免疫療法進行複合性治療，發現NanoMnSor有助於提升免疫治療的療效，相較於單純使用PD-1抗體或是細胞疫苗。我們更進一步地，設計能夠共同遞送二氧化錳與蛋白質藥物的MnSilk NP，二氧化錳調節腫瘤微環境，幫助細胞激素或抗體的作用能力，利用蠶絲水膠包覆蛋白質藥物，作為穩定釋放以及保護的效果。我們發現MnSilk NP同樣具有將巨噬細胞分化為M1型態的能力，並且蠶絲水膠的參與不影響二氧化錳的作用。結合免疫治療，MnSilk NP共同遞送PD-1抗體，在小鼠胰臟癌模型進行測試，初步得知有抑制腫瘤內TAM與抑制腫瘤生長的效果。

Sorafenib has become a first-line drug for hepatocellular carcinoma (HCC). However, it can only extend patients' life expectancy by three months. Studies have found that the limitation may come from the reduction of angiogenesis, leading to hypoxia which triggers the HIF1 signaling to cause tumor deterioration. Therefore, we developed NanoMnSor as a new multi-functional drug delivery vehicle, equipped with SP94, a specific target for liver tumors, to deliver sorafenib and manganese dioxide (MnO2). Results confirmed that NanoMnSor catalyzed hydrogen peroxide (H2O2) to produce oxygen, soothing hypoxia and reducing the epithelial-mesenchymal transition (EMT). It enhanced sorafenib therapeutic effect and could be used as a contrast agent for MRI diagnosis. In this study, we applied NanoMnSor in a mouse HCC model, exhibiting hypoxia relief and angiogenesis inhibition simultaneously. Reaction of MnO2 and H2O2 produced not only oxygen, but especially hydroxyl radicals via Fenton reaction, promoting macrophages differentiation into the M1 type for tumor resistant and immune stimulation. By the help of modulating immunosuppressive tumor microenvironment, NanoMnSor treatment increased T cells infiltration, significantly reduced tumor growth and promoted apoptosis. Moreover, NanoMnSor increased immune- therapeutic effect significantly in combination with PD-1 antibody or whole cell vaccine. Besides, we developed a MnO2 and protein drugs co-deliver platform, MnSilk NPs. MnO2 regulated the immunity of TME supporting the function of cytokines or antibodies, and silk hydrogel was used to stable release and protect protein drugs. We found that MnSilk NP also had the ability to differentiate macrophages into the M1 type. Combination with immunotherapy by co-delivers PD-1 antibody inhibited tumor growth and decreased TAM in a mouse pancreatic cancer model.

摘要 i
Abstract ii
致謝 iii
圖目錄 vi
表目錄 vii
縮寫表 viii
第一章、研究動機與目的 1
一、研究動機 1
二、研究目的 2
第二章、文獻探討 4
2.1 肝腫瘤（HCC）與sorafenib 4
2.2 腫瘤缺氧性 7
2.2.1 腫瘤缺氧性與sorafenib抗藥性 8
2.2.2 腫瘤缺氧性與免疫抑制性 9
2.3 二氧化錳改善腫瘤缺氧性 10
2.4 二氧化錳與Fenton reaction 11
2.5 胰臟導管腺癌（PDAC） 12
2.6免疫療法 13
2.7 奈米載體遞送藥物之優勢 15
第三章、實驗材料與方法 17
3.1 所用材料與儀器 17
3.1.1 材料 17
3.1.2 儀器 17
3.2 細胞培養 18
3.3 實驗動物 18
3.4 動物實驗時程 18
3.5 骨髓分化巨噬細胞(BMDM)之純化與培養 19
3.6 製備NanoMn nanoparticles 20
3.7 製備MnSilk PD-1 Abs nanoparticles 21
3.8 奈米載體定性分析 22
3.9 qPCR分析 22
3.10 氫氧自由基(hydroxyl radicals)的檢測 24
3.11 免疫組織化學染色法（IHC, Immunohistochemistry） 24
3.12 TUNEL assay細胞凋亡分析 25
3.13 流式細胞儀 25
3.14 製備細胞疫苗（whole cell vaccine） 25
3.15 數據計算與統計方式 26
第四章、實驗結果與討論 27
4.1 利用in vivo肝癌模型證明NanoMn改善腫瘤缺氧性 27
4.2 NanoMn藉由Fenton reaction改善巨噬細胞之免疫抑制性 29
4.3 NanoMnSor有助於T cell浸潤及抑制肝腫瘤 36
4.4 NanoMnSor有助於提升免疫療法之抗癌效果 37
4.5 製備裝載二氧化錳與蠶絲水膠之奈米粒子與定性分析 39
4.6 in vitro MnSilk NPs對巨噬細胞之分化影響 41
4.7 in vivo胰臟癌模型測試MnSilk PD-1 Abs NPs之抗癌效果 43
第五章、結論 45
第六章、探討與未來展望 47
第七章、參考文獻 50

1. Liu, L., et al., Sorafenib Blocks the RAF/MEK/ERK Pathway, Inhibits Tumor Angiogenesis, and Induces Tumor Cell Apoptosis in Hepatocellular Carcinoma Model PLC/PRF/5. Cancer Research, 2006. 66(24): p. 11851.
2. Blanchet, B., et al., Toxicity of sorafenib: clinical and molecular aspects. Expert Opinion on Drug Safety, 2010. 9(2): p. 275-287.
3. Zhu, Y.-j., et al., New knowledge of the mechanisms of sorafenib resistance in liver cancer. Acta Pharmacologica Sinica, 2017. 38(5): p. 614-622.
4. Méndez-Blanco, C., et al., Sorafenib resistance in hepatocarcinoma: role of hypoxia-inducible factors. Experimental & molecular medicine, 2018. 50(10): p. 1-9.
5. Zanganeh, S., et al., Iron oxide nanoparticles inhibit tumour growth by inducing pro-inflammatory macrophage polarization in tumour tissues. Nature nanotechnology, 2016. 11(11): p. 986-994.
6. Chumakov, A., V. Batalova, and Y. Slizhov, Electro-Fenton-like reactions of transition metal ions with electrogenerated hydrogen peroxide. AIP Conference Proceedings, 2016. 1772(1): p. 040004.
7. Prieto-Domínguez, N., et al., Melatonin enhances sorafenib actions in human hepatocarcinoma cells by inhibiting mTORC1/p70S6K/HIF-1α and hypoxia-mediated mitophagy. Oncotarget, 2017. 8(53): p. 91402-91414.
8. Long, K.B., A.I. Collier, and G.L. Beatty, Macrophages: Key orchestrators of a tumor microenvironment defined by therapeutic resistance. Mol Immunol, 2019. 110: p. 3-12.
9. Jo, Y.-Y., et al., Accelerated biodegradation of silk sutures through matrix metalloproteinase activation by incorporating 4-hexylresorcinol. Scientific reports, 2017. 7: p. 42441-42441.
10. Hannah Ritchie, M.R. Causes of Death. 2019; Available from: https://ourworldindata.org/causes-of-death#.
11. Rawla, P., et al., Update in global trends and aetiology of hepatocellular carcinoma. Contemporary oncology (Poznan, Poland), 2018. 22(3): p. 141-150.
12. Perz, J.F., et al., The contributions of hepatitis B virus and hepatitis C virus infections to cirrhosis and primary liver cancer worldwide. Journal of Hepatology, 2006. 45(4): p. 529-538.
13. Cancer Registry Annual Report, 2017, Taiwan. (Taiwan, 2020).
14. Chen, H.-J., et al., Understanding the inflammation-cancer transformation in the development of primary liver cancer. Hepatoma Research, 2018. 4: p. 29.
15. Villanueva, A., Hepatocellular Carcinoma. New England Journal of Medicine, 2019. 380(15): p. 1450-1462.
16. Llovet, J.M., et al., Sorafenib in Advanced Hepatocellular Carcinoma. New England Journal of Medicine, 2008. 359(4): p. 378-390.
17. Cheng, A.-L., et al., 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): p. 25-34.
18. Tseng, H.-W., Targeted Agents for the Treatment of Melanoma: An Overview. 2013.
19. Wilhelm, S., et al., Discovery and development of sorafenib: a multikinase inhibitor for treating cancer. Nature Reviews Drug Discovery, 2006. 5(10): p. 835-844.
20. Nobre, A.R., et al., The Different Routes to Metastasis via Hypoxia-Regulated Programs. Trends in Cell Biology, 2018. 28(11): p. 941-956.
21. Wilson, G.K., D.A. Tennant, and J.A. McKeating, Hypoxia inducible factors in liver disease and hepatocellular carcinoma: current understanding and future directions. J Hepatol, 2014. 61(6): p. 1397-406.
22. Nobre, A.R., et al., The Different Routes to Metastasis via Hypoxia-Regulated Programs. Trends Cell Biol, 2018. 28(11): p. 941-956.
23. Sormendi, S. and B.J.T.C.R. Wielockx, HIF-pathway proteins: central regulators of tumor immunology. 2016, 2016: p. S1503-S1508.
24. Yang, Z. and R. Poon, Vascular Changes in Hepatocellular Carcinoma. Anatomical record (Hoboken, N.J. : 2007), 2008. 291: p. 721-34.
25. Chatterjee, S., B. Behnam Azad, and S. Nimmagadda, Chapter Two - The Intricate Role of CXCR4 in Cancer, in Advances in Cancer Research, M.G. Pomper and P.B. Fisher, Editors. 2014, Academic Press. p. 31-82.
26. Qiu, Y., et al., Reversal of sorafenib resistance in hepatocellular carcinoma: epigenetically regulated disruption of 14-3-3η/hypoxia-inducible factor-1α. Cell Death Discovery, 2019. 5(1): p. 120.
27. Liang, Y., et al., Hypoxia-mediated sorafenib resistance can be overcome by EF24 through Von Hippel-Lindau tumor suppressor-dependent HIF-1α inhibition in hepatocellular carcinoma. Hepatology, 2013. 57(5): p. 1847-1857.
28. van Dalen, F.J., et al., Molecular Repolarisation of Tumour-Associated Macrophages. Molecules, 2018. 24(1).
29. Mantovani, A. and P. Allavena, The interaction of anticancer therapies with tumor-associated macrophages. 2015. 212(4): p. 435-445.
30. Doedens, A.L., et al., Hypoxia-inducible factors enhance the effector responses of CD8+ T cells to persistent antigen. Nature Immunology, 2013. 14: p. 1173.
31. Barsoum, I.B., et al., A Mechanism of Hypoxia-Mediated Escape from Adaptive Immunity in Cancer Cells. Cancer Research, 2014. 74(3): p. 665.
32. Facciabene, A., et al., Tumour hypoxia promotes tolerance and angiogenesis via CCL28 and Treg cells. Nature, 2011. 475(7355): p. 226-230.
33. Lin, C.C., et al., The efficacy of a novel vaccine approach using tumor cells that ectopically express a codon-optimized murine GM-CSF in a murine tumor model. Vaccine, 2016. 34(1): p. 134-41.
34. Wang, L., et al., Fluorescent DNA Probing Nanoscale MnO2: Adsorption, Dissolution by Thiol, and Nanozyme Activity. Langmuir, 2018. 34(9): p. 3094-3101.
35. Zhang, X., et al., Gold Cube-in-Cube Based Oxygen Nanogenerator: A Theranostic Nanoplatform for Modulating Tumor Microenvironment for Precise Chemo-Phototherapy and Multimodal Imaging. ACS Nano, 2019. 13(5): p. 5306-5325.
36. Hu, D., et al., Oxygen-generating Hybrid Polymeric Nanoparticles with Encapsulated Doxorubicin and Chlorin e6 for Trimodal Imaging-Guided Combined Chemo-Photodynamic Therapy. Theranostics, 2018. 8(6): p. 1558-1574.
37. Yang, G., et al., Hollow MnO2 as a tumor-microenvironment-responsive biodegradable nano-platform for combination therapy favoring antitumor immune responses. Nat Commun, 2017. 8(1): p. 902.
38. Fenton, H.J.H., LXXIII.—Oxidation of tartaric acid in presence of iron. Journal of the Chemical Society, Transactions, 1894. 65(0): p. 899-910.
39. Wang, J.L. and L.J. Xu, Advanced Oxidation Processes for Wastewater Treatment: Formation of Hydroxyl Radical and Application. Critical Reviews in Environmental Science and Technology, 2012. 42(3): p. 251-325.
40. Gapstur, S.M., et al., Association of Alcohol Intake With Pancreatic Cancer Mortality in Never Smokers. Archives of Internal Medicine, 2011. 171(5): p. 444-451.
41. Pelucchi, C., et al., Smoking and Body Mass Index and Survival in Pancreatic Cancer Patients. Pancreas, 2014. 43(1).
42. Olson, S.H., et al., Allergies, obesity, other risk factors and survival from pancreatic cancer. International Journal of Cancer, 2010. 127(10): p. 2412-2419.
43. Siegel, R.L., K.D. Miller, and A. Jemal, Cancer statistics, 2018. CA: A Cancer Journal for Clinicians, 2018. 68(1): p. 7-30.
44. Boldrini, L., et al., Online adaptive magnetic resonance guided radiotherapy for pancreatic cancer: state of the art, pearls and pitfalls. Radiation Oncology, 2019. 14(1): p. 71.
45. Pu, N., W. Lou, and J. Yu, PD-1 immunotherapy in pancreatic cancer. Journal of Pancreatology, 2019. 2(1): p. 6-10.
46. Weniger, M., K.C. Honselmann, and A.S. Liss, The Extracellular Matrix and Pancreatic Cancer: A Complex Relationship. Cancers, 2018. 10(9): p. 316.
47. Hilmi, M., L. Bartholin, and C. Neuzillet, Immune therapies in pancreatic ductal adenocarcinoma: Where are we now? World journal of gastroenterology, 2018. 24(20): p. 2137-2151.
48. Fan, J.-q., et al., Current advances and outlooks in immunotherapy for pancreatic ductal adenocarcinoma. Molecular Cancer, 2020. 19(1): p. 32.
49. Lei, F., et al., Combination Therapies and Drug Delivery Platforms in Combating Pancreatic Cancer. Journal of Pharmacology and Experimental Therapeutics, 2019. 370(3): p. 682.
50. Zhang, N. and M.J. Bevan, CD8(+) T cells: foot soldiers of the immune system. Immunity, 2011. 35(2): p. 161-168.
51. Oleinika, K., et al., Suppression, subversion and escape: the role of regulatory T cells in cancer progression. 2013. 171(1): p. 36-45.
52. Gabrilovich, D.I. and S. Nagaraj, Myeloid-derived suppressor cells as regulators of the immune system. Nature Reviews Immunology, 2009. 9(3): p. 162-174.
53. Pico de Coaña, Y., A. Choudhury, and R. Kiessling, Checkpoint blockade for cancer therapy: revitalizing a suppressed immune system. Trends in Molecular Medicine, 2015. 21(8): p. 482-491.
54. Sambi, M., L. Bagheri, and M.R. Szewczuk, Current Challenges in Cancer Immunotherapy: Multimodal Approaches to Improve Efficacy and Patient Response Rates. J Oncol, 2019. 2019: p. 4508794.
55. O'Donnell, J.S., et al., Resistance to PD1/PDL1 checkpoint inhibition. Cancer Treatment Reviews, 2017. 52: p. 71-81.
56. Hernández-Pedro, N.Y., et al., Application of Nanoparticles on Diagnosis and Therapy in Gliomas. BioMed Research International, 2013. 2013: p. 351031.
57. Baetke, S.C., T. Lammers, and F. Kiessling, Applications of nanoparticles for diagnosis and therapy of cancer. The British journal of radiology, 2015. 88(1054): p. 20150207-20150207.
58. Naahidi, S., et al., Biocompatibility of engineered nanoparticles for drug delivery. Journal of Controlled Release, 2013. 166(2): p. 182-194.
59. Wang, X., et al., Sonication-induced gelation of silk fibroin for cell encapsulation. Biomaterials, 2008. 29(8): p. 1054-1064.
60. Zhang, X., et al., Production of silk sericin/silk fibroin blend nanofibers. Nanoscale Research Letters, 2011. 6(1): p. 510.
61. DeFrates, K., et al., Protein polymer-based nanoparticles: fabrication and medical applications. International journal of molecular sciences, 2018. 19(6): p. 1717.
62. Cao, Y., et al., Drug release from core-shell PVA/silk fibroin nanoparticles fabricated by one-step electrospraying. Scientific reports, 2017. 7(1): p. 11913.
63. Matsumoto, A., et al., Mechanisms of Silk Fibroin Sol−Gel Transitions. The Journal of Physical Chemistry B, 2006. 110(43): p. 21630-21638.
64. Whitehead, K.A., R. Langer, and D.G. Anderson, Knocking down barriers: advances in siRNA delivery. Nature Reviews Drug Discovery, 2009. 8(2): p. 129-138.
65. Zhao, Y., et al., Losartan treatment enhances chemotherapy efficacy and reduces ascites in ovarian cancer models by normalizing the tumor stroma. Proceedings of the National Academy of Sciences, 2019. 116(6): p. 2210.
66. Marim, F.M., et al., A method for generation of bone marrow-derived macrophages from cryopreserved mouse bone marrow cells. PloS one, 2010. 5(12): p. e15263-e15263.
67. Li, J., Y. Yang, and L. Huang, Calcium phosphate nanoparticles with an asymmetric lipid bilayer coating for siRNA delivery to the tumor. J Control Release, 2012. 158(1): p. 108-14.
68. Xu, Y., et al., The mechanism and efficiency of MnO2 activated persulfate process coupled with electrolysis. Science of The Total Environment, 2017. 609: p. 644-654.
69. Lin, Y., J. Xu, and H. Lan, Tumor-associated macrophages in tumor metastasis: biological roles and clinical therapeutic applications. Journal of Hematology & Oncology, 2019. 12(1): p. 76.
70. Yang, Q., et al., The role of tumor-associated macrophages (TAMs) in tumor progression and relevant advance in targeted therapy. Acta Pharmaceutica Sinica B, 2020.
71. Yu, T.W., et al., Novel GM-CSF-based vaccines: One small step in GM-CSF gene optimization, one giant leap for human vaccines. Hum Vaccin Immunother, 2016. 12(12): p. 3020-3028.