一氧化氮(Nitric oxide, NO)是一種小分子氣體，廣泛分佈於各組織中，且 具有多種不同的功能。其中一氧化氮的抗纖維化(Antifibrotic)機制可抑制肌成 纖維細胞(myofibroblasts)分泌膠原蛋白 I(collagen I)，以及促使肌成纖維細胞 進行細胞凋亡(apoptosis)程序，相當具有作為腎纖維化治療藥物的潛力。
臨床上採用傳統中醫及西醫療法治療纖維化的過程繁複且痛苦，成效亦相當 有限，因此我們選用了兩種不同的酸鹼應答高分子材料設計及開發出兩種不同的 酸鹼應答奈米載體，並包覆一氧化氮供體(Nitric oxide donor, NO donor)作為核 心治療藥物，同時利用纖維化環境受發炎環境影響而呈酸性有利於奈米載體崩解 而釋放藥物之特性，進行一氧化氮於腎臟纖維化之短期化學治療的應用與評估。

y organism, carrying multifunction. For the antifibrotic role of Nitric oxide, it can suppress myofibroblasts from accumulating and secreting collagen I, preventing extracellular matrix form deposing, and inducing myofibroblasts go under apoptosis process. For this purpose, there is highly potential of treating renal fibrosis basic on the using of nitric oxide.
Clinically, the common strategy for renal fibrosis therapy takes long time and uncomfortable. Also, the effect is limited. In order to study and enhance the treatment effect, here we designed three types of nanocarrier that encapsulate nitric oxide donor to treat renal fibrosis and two of them feature pH-sensitive polymers. By applying the pH-sensitive nanocarriers to fibrosis and endosome environment which have lower pH value surrondings than normal tissue, the pH-sensitive nanocarriers can easily be degraded and release drug.

誌謝........................................................................................................................ I 摘要....................................................................................................................... II
Abstract ................................................................................................................ III 總目錄..................................................................................................................IV
圖目錄..................................................................................................................IX 縮寫......................................................................................................................XI
第一章、
第二章、
2.1
研究動機與目的............................................................................ 1
文獻探討........................................................................................ 3 腎纖維化.......................................................................................................... 3
2.1.1 腎纖維化病變...................................................................3 2.1.2 單側尿路梗阻腎纖維化模型...........................................6
IV
2.1.3 腎纖維化環境的酸鹼值...................................................8 2.1.4 治療腎纖維化的手段.......................................................9
2.2 一氧化氮........................................................................................................ 10
2.2.1 腎纖維化中的一氧化氮.................................................10
2.2.2 一氧化氮與抗纖維化.....................................................10
2.2.3 一氧化氮與發炎............................................................. 11
2.2.4 一氧化氮供體-雙亞硝基鐵錯合物 DNICs ................ 12
2.3 藥物遞送........................................................................................................ 13
2.3.1 以奈米載體遞送一氧化氮供體以治療疾病.................13
2.3.2 腎臟藥物遞送.................................................................14
2.3.3 酸鹼應答高分子 PDPA..................................................16
第三章、 材料與方法.................................................................................. 18
3.1 實驗細胞.........................................................................18
3.2 實驗材料.........................................................................18

3.3 實驗動物.........................................................................19
3.4 動物治療研究.................................................................19
3.5 製備 PEG-PLGA 奈米粒子 ........................................... 19
3.6 製備 PEG-PDPA 奈米粒子 ............................................ 20
3.7 製備 PEG-PBA 奈米粒子 .............................................. 21
3.8 動態雷射光散射儀及界達表面電位.............................21
3.9 穿透式電子顯微鏡(Transmission Electron Microscope， TEM) ............................................................................22
3.10 藥物包覆率.....................................................................22
3.11 藥物釋放速率.................................................................23
3.12 RRE-SEt 藥物穩定性測試.............................................24
3.13 細胞存活率分析.............................................................25
3.14 蘇木精-伊紅染色(Hematoxylin and Eosin Staining， H&E Staining)..............................................................25
3.15 馬森三色染色(Masson’s Trichrome Staining，MTS) ...................................................................................... 25
VI

3.16 免疫組織染色(Immunofluorescence staining) .........26
3.17 西方墨點法分析(Western Blot Analysis).................27
3.18 組織分布研究.................................................................27
第四章、 結果.............................................................................................. 29
4.1 開發包覆 SEt 之奈米載體並觀察其粒徑大小及界面電
位.....................................................................................29
4.2 包覆 RRE-SEt 之 PEG-PLGA、PEG-PDPA 及 PEG-PBA
奈米載體之藥物穩定性測試及體外細胞毒性測試.....32
4.3 包覆 C6 之 PEG-PLGA、PEG-PDPA 及 PEG-PBA 奈米
載體之藥物釋放速率測試.............................................35
4.4 PEG-PDPA 奈米載體之藥物動力學測試 ..................... 37
4.5 利用包覆 SEt 之奈米載體進行體外實驗有效抑制肌成 纖維細胞表現.................................................................38
4.6 利用包覆 SEt 之奈米載體治療單側尿路梗阻引發之腎 纖維化小鼠模型有效改善腎纖維化.............................41
第五章、 結論.............................................................................................. 51

第六章、 討論與未來展望.......................................................................... 53
6.1 奈米載體與一氧化氮.....................................................53
6.2 腎纖維化治療的探討.....................................................54
6.3 體外實驗.........................................................................55
6.4 體內實驗.........................................................................56
第七章、 參考文獻...................................................................................... 58

1 Levey, A. S. & Coresh, J. Chronic kidney disease. Lancet 379, 165-180, doi:10.1016/S0140-6736(11)60178-5 (2012).
2 Efstratiadis, G., Divani, M., Katsioulis, E. & Vergoulas, G. Renal fibrosis. Hippokratia 13, 224-229 (2009).
3 LeBleu, V. S. et al. Origin and function of myofibroblasts in kidney fibrosis. Nat Med 19, 1047-1053, doi:10.1038/nm.3218 (2013).
4 Wynn, T. A. & Ramalingam, T. R. Mechanisms of fibrosis: therapeutic translation for fibrotic disease. Nat Med 18, 1028-1040, doi:10.1038/nm.2807 (2012).
5 Potenta, S., Zeisberg, E. & Kalluri, R. The role of endothelial-to-mesenchymal transition in cancer progression. Br J Cancer 99, 1375-1379, doi:10.1038/sj.bjc.6604662 (2008).
6 Zeisberg, E. M., Potenta, S. E., Sugimoto, H., Zeisberg, M. & Kalluri, R. Fibroblasts in kidney fibrosis emerge via endothelial-to-mesenchymal transition. J Am Soc Nephrol 19, 2282-2287, doi:10.1681/ASN.2008050513 (2008).
7 Yang, H. C., Zuo, Y. & Fogo, A. B. Models of chronic kidney disease. Drug Discov Today Dis Models 7, 13-19, doi:10.1016/j.ddmod.2010.08.002 (2010).
8 Chevalier, R. L., Forbes, M. S. & Thornhill, B. A. Ureteral obstruction as a model of renal interstitial fibrosis and obstructive nephropathy. Kidney Int 75, 1145-1152, doi:10.1038/ki.2009.86 (2009).
9 Birbrair, A. et al. Type-1 pericytes accumulate after tissue injury and produce collagen in an organ-dependent manner. Stem Cell Res Ther 5, 122, doi:10.1186/scrt512 (2014).
10 Lee, G. H. et al. An acidic pH environment increases cell death and pro- inflammatory cytokine release in osteoblasts: the involvement of BAX inhibitor-1. Int

J Biochem Cell Biol 43, 1305-1317, doi:10.1016/j.biocel.2011.05.004 (2011).
11 Tian, S. et al. HMGB1 exacerbates renal tubulointerstitial fibrosis through facilitating M1 macrophage phenotype at the early stage of obstructive injury. Am J Physiol Renal Physiol 308, F69-75, doi:10.1152/ajprenal.00484.2014 (2015).
12 Tampe, D. & Zeisberg, M. Potential approaches to reverse or repair renal fibrosis. Nat Rev Nephrol 10, 226-237, doi:10.1038/nrneph.2014.14 (2014).
13 Boor, P. & Floege, J. Renal allograft fibrosis: biology and therapeutic targets. Am J Transplant 15, 863-886, doi:10.1111/ajt.13180 (2015).
14 Klinkhammer, B. M., Goldschmeding, R., Floege, J. & Boor, P. Treatment of Renal Fibrosis-Turning Challenges into Opportunities. Adv Chronic Kidney Dis 24, 117-129, doi:10.1053/j.ackd.2016.11.002 (2017).
15 Sun, D. et al. Effects of nitric oxide on renal interstitial fibrosis in rats with unilateral ureteral obstruction. Life Sci 90, 900-909, doi:10.1016/j.lfs.2012.04.018 (2012).
16 Gonzalez-Cadavid, N. F. & Rajfer, J. Treatment of Peyronie's disease with PDE5 inhibitors: an antifibrotic strategy. Nat Rev Urol 7, 215-221, doi:10.1038/nrurol.2010.24 (2010).
17 Ferrini, M. G. et al. Antifibrotic role of inducible nitric oxide synthase. Nitric Oxide 6, 283-294, doi:10.1006/niox.2001.0421 (2002).
18 Sharma, J. N., Al-Omran, A. & Parvathy, S. S. Role of nitric oxide in inflammatory diseases. Inflammopharmacology 15, 252-259, doi:10.1007/s10787-007-0013-x (2007).
19 Turesin, F., del Soldato, P. & Wallace, J. L. Enhanced anti-inflammatory potency of a nitric oxide-releasing prednisolone derivative in the rat. Br J Pharmacol 139, 966- 972, doi:10.1038/sj.bjp.0705324 (2003).
20 Duong, H. T. et al. The use of nanoparticles to deliver nitric oxide to hepatic stellate cells for treating liver fibrosis and portal hypertension. Small 11, 2291-2304,

doi:10.1002/smll.201402870 (2015).
21 Wu, S. C. et al. Water-Soluble Dinitrosyl Iron Complex (DNIC): a Nitric Oxide Vehicle Triggering Cancer Cell Death via Apoptosis. Inorg Chem 55, 9383-9392, doi:10.1021/acs.inorgchem.6b01562 (2016).
22 Maeda, H., Nakamura, H. & Fang, J. The EPR effect for macromolecular drug delivery to solid tumors: Improvement of tumor uptake, lowering of systemic toxicity, and distinct tumor imaging in vivo. Adv Drug Deliv Rev 65, 71-79, doi:10.1016/j.addr.2012.10.002 (2013).
23 Bruni, R. et al. Ultrasmall polymeric nanocarriers for drug delivery to podocytes in kidney glomerulus. J Control Release 255, 94-107, doi:10.1016/j.jconrel.2017.04.005 (2017).
24 Haraldsson, B., Nystrom, J. & Deen, W. M. Properties of the glomerular barrier and mechanisms of proteinuria. Physiol Rev 88, 451-487, doi:10.1152/physrev.00055.2006 (2008).
25 Qiao, H. et al. Kidney-specific drug delivery system for renal fibrosis based on coordination-driven assembly of catechol-derived chitosan. Biomaterials 35, 7157- 7171, doi:10.1016/j.biomaterials.2014.04.106 (2014).
26 Guo, L. et al. Targeted delivery of celastrol to mesangial cells is effective against mesangioproliferative glomerulonephritis. Nat Commun 8, 878, doi:10.1038/s41467- 017-00834-8 (2017).
27 Giacomelli, F. C. et al. pH-triggered block copolymer micelles based on a pH- responsive PDPA (poly[2-(diisopropylamino)ethyl methacrylate])inner core and a PEO (poly(ethylene oxide)) outer shell as a potential tool for the cancer therapy. Soft Matter 7, 9316-9325 (2011).
28 Dehaini, D. et al. Ultra-small lipid-polymer hybrid nanoparticles for tumor- penetrating drug delivery. Nanoscale 8, 14411-14419, doi:10.1039/c6nr04091h (2016).
29 Cai, H. et al. Enhanced local bioavailability of single or compound drugs delivery 60

to the inner ear through application of PLGA nanoparticles via round window administration. Int J Nanomedicine 9, 5591-5601, doi:10.2147/IJN.S72555 (2014).
30 Zhang, Z. & Feng, S. S. The drug encapsulation efficiency, in vitro drug release, cellular uptake and cytotoxicity of paclitaxel-loaded poly(lactide)-tocopheryl polyethylene glycol succinate nanoparticles. Biomaterials 27, 4025-4033, doi:10.1016/j.biomaterials.2006.03.006 (2006).
31 Gao, D. Y. et al. CXCR4-targeted lipid-coated PLGA nanoparticles deliver sorafenib and overcome acquired drug resistance in liver cancer. Biomaterials 67, 194- 203, doi:10.1016/j.biomaterials.2015.07.035 (2015).