大腸直腸癌腹腔轉移 (peritoneal carcinomatosis colorectal cancer, pcCRC) 是最為棘手且難以有效治療的癌症轉移 (metastasis) 病狀之一，主要因為腫瘤於腹腔內不斷反覆地進行剝落、種植與增生。目前較為有效的治療方式為腫瘤減量手術(cytoreductive surgery) 合併術中腹膜內高溫化學治療 (hyperthermic intraperitoneal perioperative chemotherapy, HIPC)，但諸多使用限制和致死率高，只適用於極少數的pcCRC 病患。為了克服傳統靜脈注射化療藥物對腹腔轉移腫瘤累積及治療效率窘境，本研究擬開發出針對pcCRC 具有雙重標靶功能的奈米微粒傳遞系統。在此使用生物可降解材料poly(lactic-co-glycolic acid) (PLGA) 建構奈米粒子疏水核心，以喜樹鹼 (camptothecin) 衍生化療藥物SN38 利用疏水作用力裝載於PLGA 核心中，並在表面上修飾所合成的N-acetyl histidine modified D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS) (Histidine-TPGS) 和folate-functionalized TPGS (Folate-TPGS)，可賦予奈米載體具有標靶至葉酸受器 (folate-receptor) 過量表現的癌細胞與可於腫瘤微酸環境中轉換奈米微粒表面電性以增加藥物於腫瘤內累積。研究中使用奈米沉澱法所製備出之載藥奈米微粒可具有粒徑均一、直徑約160 nm與包覆率可達70 %以上的性質。帶有Histidine-TPGS與Folate-TPGS之載藥奈米粒子於表面電位測量上成功證實其於微酸環境中的電性轉正之能力；於細胞吞噬實驗中，亦證實具有電性轉換能力之奈米微粒相較於控制組於微酸環境中被癌細胞攝取數量有明顯提升。此外，具葉酸標靶性的奈米微粒可標靶累積至高葉酸受器表現的癌細胞株，並大幅提升藥物於癌細胞內之濃度。於動物腫瘤抑制實驗結果亦可於經給予腹腔腫瘤小鼠雙標靶載藥奈米微粒治療組別上發現雙重標靶性確實可以有效累積於腫瘤處，幫助化療藥物抑制腹腔腫瘤生長。綜觀以上成果，本研究成功開發出針對治療pcCRC之雙標靶奈米載體傳輸系統，並成功驗證其抑制腫瘤之功效，相信本研究能提供一新方法來有效改善化療藥物對病灶的累積效率，使藥物能發揮出最佳治療效果，提高病患治療後的存活期。

Peritoneal carcinomatosis colorectal cancer (pcCRC) has become one of the most difficult metastatic tumors in clinical treatments. The difficulty of treatments is caused largely by the detachment of the cancer cells from original tumors on peritoneum, which then further invade into other normal tissues repeatedly. Promising therapeutic approaches are still in great and urgent demand. Therefore, to improve the efficacy of cancer treatment, sufficient accumulation of therapeutic agents at tumor sites plays a crucial role. Herein, a smart dual target-responsive nanoparticle (NP) system not only capable to switch NP surface charges within tumor microenvironment for enhancing tumor cell uptake but also to selectively target cancer cells by folic acid residues was developed and employed for delivery of the anticancer drug, SN38. The biodegradable copolymer, poly(lactic acid-co-glycolic acid) (PLGA), was adopteded as the major material together with SN38 in NP formation while the surfaces were concomitantly decorated with N-acetyl histidine modified D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS) (His-TPGS) and folate-functionalized TPGS (Folate-TPGS) to improve the colloidal stability of NPs and reduce NP uptake by other organs. The SN38-loaded nanoparticles showed a mean particle size of ca 160 nm with a mono-model size distribution and a good stability in DMEM over 7 days. The zeta potential characterization showed that the SN38-loaded nanoparticles coated with Histidine-TPGS were positively charged in weak acidic environment (pH ≤ 6.5) via protonation of the histidine/imidazole moieties that can enhance their affinity with negatively charged cell membranes in tumor. The in vitro cellular uptake data also indicated that the coating of SN38-loaded nanoparticles with Histidine-TPGS can efficiently promote cellular drug uptake 2-4 folds upon the decrease in pH mimicking tumor microenvironment. While SN38-loaded nanoparticles were coated with Folate-TPGS, the cancer cell line overexpressing the folate-receptor can enhance the cellular drug uptake 2 to 3 folds. The ex vivo biodistribution suggest that the dual-targeting SN38-loaded nanoparticles can efficiently accumulation in tumor and in vivo tumor inhibition data showed that the dual-targeting SN38-loaded nanoparticles can efficiently inhibit the proliferation of pcCRC by the MCI score evaluation. Based on the results, this study provides a promising strategy for the development of dual targeted nanotherapeutics for effective chemotherapy treatment of pcCRC.

ABSTRACT I
中文摘要 III
致謝 V
一、研究動機 1
二、文獻回顧 3
2.1 大腸直腸癌 3
2.2化學治療 5
2.3 奈米藥物載體傳遞系統 6
2.4 腫瘤微環境 8
2.4.1 Enhanced permeation and retention (EPR) effect 9
2.4.2 腫瘤微環境pH值 10
2.5 靶向標靶腫瘤細胞 12
2.5.1 Folate receptor-mediated endocytosis pathway 13
2.6 D-α-tocopheryl polyethylene glycol succinate (Vitamin E TPGS) 14
2.7 Histidine 與 Imidazole contain polymer 於藥物傳遞系統之應用 15
2.8化療藥物 7-Ethyl-10-hydroxy-camptothecin (SN38)介紹 16
三、實驗方法與步驟 18
3.1 高分子合成與性質鑑定 18
3.1.1 無水有機溶劑之製備 18
3.1.2 N-Acetyl-Histidine-TPGS合成 18
3.1.3 N-Acetyl-Histidine-TPGS高分子之組成鑑定 19
3.1.4 Folate-TPGS合成 19
3.1.5 Folate-TPGS高分子之組成鑑定 20
3.2 奈米微粒製備與性質分析 20
3.2.1 載藥奈米微粒的製備 20
3.2.2 奈米微粒粒徑與表面電荷分析 21
3.2.2.1 奈米微粒型態分析 21
3.2.2.2 奈米微粒粒徑分析 22
3.2.2.3 奈米微粒電荷分析 23
3.2.3 奈米微粒粒表面葉酸分析 23
3.2.4 載藥奈米微粒穩定度測試 23
3.2.5 奈米微粒藥物裝載效率測定 24
3.2.6 載藥奈米微粒體外(in vitro)藥物釋放分析 24
3.3 體外細胞實驗 25
3.3.1 細胞來源及適合之培養環境 25
3.3.2 配置細胞培養液與磷酸鹽緩衝溶液 25
3.3.3 細胞繼代 26
3.3.4 細胞計數 27
3.3.5 模擬腫瘤微酸環境之細胞培養液pH值調整 27
3.3.6 細胞胞內奈米粒子吞噬含量分析 28
3.3.6.1 細胞胞內SN38吞噬含量分析 28
3.3.6.2 螢光顯微鏡觀察 29
3.3.7 細胞凋亡分析 30
3.3.8 細胞毒性分析 31
3.4 動物實驗 32
3.4.1 動物來源 32
3.4.2 腹腔轉移腫瘤模型建立 32
3.4.3 動物體內奈米載體累積分佈 32
3.4.4腹腔轉移腫瘤抑制生長評估 33
3.4.5動物犧牲與腫瘤組織包埋 33
3.4.6組織切片脫蠟及抗原回復 34
3.4.7組織切片Hematoxylin and eosin (H&E)染色 34
3.4.8 腫瘤組織切片免疫螢光染色 34
3.4.9 生物毒性分析 35
3.5 數據統計 35
四、結果與討論 36
4.1 高分子鑑定 36
4.1.1 Histidine-TPGS 組成分析鑑定 36
4.1.2 Folate-TPGS 組成分析鑑定 37
4.2 奈米微粒性質分析 37
4.2.1 奈米微粒性質分析 37
4.2.2 奈米微粒穩定度分析 41
4.2.3 奈米微粒體外藥物釋放 42
4.3 細胞實驗 43
4.3.1 微酸環境下細胞吞噬評估 43
4.3.2 葉酸靶向細胞吞噬評估 45
4.3.3 細胞毒性分析 49
4.3.4 細胞凋亡分析 50
4.4動物實驗 53
4.4.1 腹腔轉移腫瘤模型建立 53
4.4.2 動物體內奈米載體累積分佈 54
4.4.3 腹腔轉移腫瘤抑制生長評估 57
4.4.4組織切片觀察 60
4.4.5生物毒性分析 66
五、結論 68
六、參考文獻 69

1. Siegel, R.L., et al., Colorectal cancer statistics, 2017. CA Cancer J Clin, 2017. 67(3): p. 177-193.
2. Brenner, H., M. Kloor, and C.P. Pox, Colorectal cancer. Lancet, 2014. 383(9927): p. 1490-1502.
3. Wang, C.C. and J. Li, An update on chemotherapy of colorectal liver metastases. World J Gastroenterol, 2012. 18(1): p. 25-33.
4. Sugarbaker, P.H., Cytoreductive surgery plus hyperthermic perioperative chemotherapy for selected patients with peritoneal metastases from colorectal cancer: a new standard of care or an experimental approach? Gastroenterol Res Pract, 2012. 2012: p. 309417.
5. Glehen, O., et al., Cytoreductive surgery combined with perioperative intraperitoneal chemotherapy for the management of peritoneal carcinomatosis from colorectal cancer: a multi-institutional study. J Clin Oncol, 2004. 22(16): p. 3284-3292.
6. Ceelen, W.P. and M.E. Bracke, Peritoneal minimal residual disease in colorectal cancer: mechanisms, prevention, and treatment. Lancet Oncol, 2009. 10(1): p. 72-79.
7. Franko, J., et al., Treatment of colorectal peritoneal carcinomatosis with systemic chemotherapy: a pooled analysis of north central cancer treatment group phase III trials N9741 and N9841. J Clin Oncol, 2012. 30(3): p. 263-267.
8. Kuijpers, A.M., et al., Perioperative systemic chemotherapy in peritoneal carcinomatosis of lymph node positive colorectal cancer treated with cytoreductive surgery and hyperthermic intraperitoneal chemotherapy. Ann Oncol, 2014. 25(4): p. 864-869.
9. Elias, D., et al., Curative treatment of peritoneal carcinomatosis arising from colorectal cancer by complete resection and intraperitoneal chemotherapy. Cancer, 2001. 92(1): p. 71-76.
10. Elias, D., et al., Peritoneal colorectal carcinomatosis treated with surgery and perioperative intraperitoneal chemotherapy: retrospective analysis of 523 patients from a multicentric French study. J Clin Oncol, 2010. 28(1): p. 63-68.
11. Elias, D., et al., Role of hyperthermic intraoperative peritoneal chemotherapy in the management of peritoneal metastases. Eur J Cancer, 2014. 50(2): p. 332-340.
12. Thorn, C.F., et al., Doxorubicin pathways: pharmacodynamics and adverse effects. Pharmacogenet Genomics, 2011. 21(7): p. 440-446.
13. Schiff, P.B., J. Fant, and S.B. Horwitz, Promotion of microtubule assembly in vitro by taxol. Nature, 1979. 277(5698): p. 665-667.
14. Gill, S., R.R. Thomas, and R.M. Goldberg, Review article: colorectal cancer chemotherapy. Aliment Pharmacol Ther, 2003. 18(7): p. 683-692.
15. Parker, W.B. and Y.C. Cheng, Metabolism and mechanism of action of 5-fluorouracil. Pharmacol Ther, 1990. 48(3): p. 381-395.
16. Jackman, A.L., R. Kimbell, and H.E. Ford, Combination of raltitrexed with other cytotoxic agents: rationale and preclinical observations. Eur J Cancer, 1999. 35 Suppl 1: p. S3-8.
17. Graham, J., M. Mushin, and P. Kirkpatrick, Oxaliplatin. Nat Rev Drug Discov, 2004. 3(1): p. 11-12.
18. Xu, Y. and M.A. Villalona-Calero, Irinotecan: mechanisms of tumor resistance and novel strategies for modulating its activity. Ann Oncol, 2002. 13(12): p. 1841-1851.
19. Chen, X., et al., Quantification of irinotecan, SN38, and SN38G in human and porcine plasma by ultra high-performance liquid chromatography-tandem mass spectrometry and its application to hepatic chemoembolization. J Pharm Biomed Anal, 2012. 62: p. 140-148.
20. Khodabandehloo, H., H. Zahednasab, and A. Ashrafi Hafez, Nanocarriers Usage for Drug Delivery in Cancer Therapy. Iran J Cancer Prev, 2016. 9(2): p. e3966.
21. Quail, D.F. and J.A. Joyce, Microenvironmental regulation of tumor progression and metastasis. Nat Med, 2013. 19(11): p. 1423-1437.
22. Tredan, O., et al., Drug resistance and the solid tumor microenvironment. J Natl Cancer Inst, 2007. 99(19): p. 1441-1454.
23. Maeda, H., et al., Tumor vascular permeability and the EPR effect in macromolecular therapeutics: a review. J Control Release, 2000. 65(1-2): p. 271-284.
24. Peer, D., et al., Nanocarriers as an emerging platform for cancer therapy. Nat Nanotechnol, 2007. 2(12): p. 751-760.
25. Stockhofe, K., et al., Radiolabeling of Nanoparticles and Polymers for PET Imaging. Pharmaceuticals (Basel), 2014. 7(4): p. 392-418.
26. Danhier, F., O. Feron, and V. Preat, To exploit the tumor microenvironment: Passive and active tumor targeting of nanocarriers for anti-cancer drug delivery. J Control Release, 2010. 148(2): p. 135-146.
27. Kobayashi, H., R. Watanabe, and P.L. Choyke, Improving conventional enhanced permeability and retention (EPR) effects; what is the appropriate target? Theranostics, 2013. 4(1): p. 81-89.
28. Erickson, J.W. and R.A. Cerione, Glutaminase: a hot spot for regulation of cancer cell metabolism? Oncotarget, 2010. 1(8): p. 734-740.
29. Brahimi-Horn, M.C. and J. Pouyssegur, Oxygen, a source of life and stress. FEBS Lett, 2007. 581(19): p. 3582-3591.
30. Sudimack, J. and R.J. Lee, Targeted drug delivery via the folate receptor. Adv Drug Deliv Rev, 2000. 41(2): p. 147-162.
31. Wang, S. and P.S. Low, Folate-mediated targeting of antineoplastic drugs, imaging agents, and nucleic acids to cancer cells. J Control Release, 1998. 53(1-3): p. 39-48.
32. Lu, Y.J. and P.S. Low, Folate-mediated delivery of macromolecular anticancer therapeutic agents. Advanced Drug Delivery Reviews, 2012. 64: p. 342-352.
33. Werner, M.E., et al., Folate-targeted nanoparticle delivery of chemo- and radiotherapeutics for the treatment of ovarian cancer peritoneal metastasis. Biomaterials, 2011. 32(33): p. 8548-8554.
34. Lu, P.L., et al., Multifunctional hollow nanoparticles based on graft-diblock copolymers for doxorubicin delivery. Biomaterials, 2011. 32(8): p. 2213-2221.
35. Li, H., et al., Targeting human clonogenic acute myelogenous leukemia cells via folate conjugated liposomes combined with receptor modulation by all-trans retinoic acid. International Journal of Pharmaceutics, 2010. 402(1-2): p. 57-63.
36. Zhang, Z., S. Tan, and S.S. Feng, Vitamin E TPGS as a molecular biomaterial for drug delivery. Biomaterials, 2012. 33(19): p. 4889-906.
37. Szakacs, G., et al., Targeting multidrug resistance in cancer. Nat Rev Drug Discov, 2006. 5(3): p. 219-234.
38. Collnot, E.M., et al., Mechanism of inhibition of P-glycoprotein mediated efflux by vitamin E TPGS: influence on ATPase activity and membrane fluidity. Mol Pharm, 2007. 4(3): p. 465-474.
39. 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. Mol Pharm, 2010. 7(3): p. 642-651.
40. Mi, Y., Y. Liu, and S.S. Feng, Formulation of Docetaxel by folic acid-conjugated d-alpha-tocopheryl polyethylene glycol succinate 2000 (Vitamin E TPGS(2k)) micelles for targeted and synergistic chemotherapy. Biomaterials, 2011. 32(16): p. 4058-4066.
41. Muthu, M.S., et al., Theranostic liposomes of TPGS coating for targeted co-delivery of docetaxel and quantum dots. Biomaterials, 2012. 33(12): p. 3494-3501.
42. Guo, Y., et al., The applications of Vitamin E TPGS in drug delivery. Eur J Pharm Sci, 2013. 49(2): p. 175-186.
43. Zeng, X., et al., Cholic acid-functionalized nanoparticles of star-shaped PLGA-vitamin E TPGS copolymer for docetaxel delivery to cervical cancer. Biomaterials, 2013. 34(25): p. 6058-6067.
44. Lee, E.S., et al., Poly(L-histidine)-PEG block copolymer micelles and pH-induced destabilization. J Control Release, 2003. 90(3): p. 363-374.
45. Benjaminsen, R.V., et al., The possible "proton sponge " effect of polyethylenimine (PEI) does not include change in lysosomal pH. Mol Ther, 2013. 21(1): p. 149-157.
46. Qiu, L., et al., Self-assembled pH-responsive hyaluronic acid-g-poly((L)-histidine) copolymer micelles for targeted intracellular delivery of doxorubicin. Acta Biomater, 2014. 10(5): p. 2024-2035.
47. Pang, X., et al., pH-responsive polymer-drug conjugates: Design and progress. J Control Release, 2016. 222: p. 116-129.
48. Zhao, Y., et al., Tumor-specific pH-responsive peptide-modified pH-sensitive liposomes containing doxorubicin for enhancing glioma targeting and anti-tumor activity. J Control Release, 2016. 222: p. 56-66.
49. Li, S., et al., pH-responsive biocompatible fluorescent polymer nanoparticles based on phenylboronic acid for intracellular imaging and drug delivery. Nanoscale, 2014. 6(22): p. 13701-13709.
50. Edgcomb, S.P. and K.P. Murphy, Variability in the pKa of histidine side-chains correlates with burial within proteins. Proteins, 2002. 49(1): p. 1-6.
51. He, C., et al., Effects of particle size and surface charge on cellular uptake and biodistribution of polymeric nanoparticles. Biomaterials, 2010. 31(13): p. 3657-3666.
52. Xu, G., et al., Human carboxylesterase 2 is commonly expressed in tumor tissue and is correlated with activation of irinotecan. Clinical Cancer Research, 2002. 8(8): p. 2605-2611.
53. Bala, V., et al., Prodrug and nanomedicine approaches for the delivery of the camptothecin analogue SN38. J Control Release, 2013. 172(1): p. 48-61.
54. Kawato, Y., et al., Intracellular roles of SN-38, a metabolite of the camptothecin derivative CPT-11, in the antitumor effect of CPT-11. Cancer Res, 1991. 51(16): p. 4187-4191.
55. Pommier, Y., Topoisomerase I inhibitors: camptothecins and beyond. Nat Rev Cancer, 2006. 6(10): p. 789-802.
56. Hurley, L.H., DNA and its associated processes as targets for cancer therapy. Nature Reviews Cancer, 2002. 2(3): p. 188-200.
57. Liu, Y., et al., Comparison of two self-assembled macromolecular prodrug micelles with different conjugate positions of SN38 for enhancing antitumor activity. International Journal of Nanomedicine, 2015. 10: p. 2295-2311.
58. Vankayala, R., et al., A general strategy to achieve ultra-high gene transfection efficiency using lipid-nanoparticle composites. Biomaterials, 2014. 35(28): p. 8261-8272.
59. Varshosaz, J., et al., Uptake of Etoposide in CT-26 Cells of Colorectal Cancer Using Folate Targeted Dextran Stearate Polymeric Micelles. Biomed Research International, 2014.
60. Saul, J.M., et al., Controlled targeting of liposomal doxorubicin via the folate receptor in vitro. Journal of Controlled Release, 2003. 92(1-2): p. 49-67.
61. Leamon, C.P. and P.S. Low, Delivery of Macromolecules into Living Cells - a Method That Exploits Folate Receptor Endocytosis. Proceedings of the National Academy of Sciences of the United States of America, 1991. 88(13): p. 5572-5576.
62. Wallin, A., et al., Anticancer effect of SN-38 on colon cancer cell lines with different metastatic potential. Oncology Reports, 2008. 19(6): p. 1493-1498.