臺灣博碩士論文加值系統

目的

台灣尿毒症病患約10%接受腹膜透析治療，但是患者於後期常因為腹膜增厚、纖維化、血管新生等引起腹膜特性之改變而導致被迫中斷此種治療。其原因主要歸咎於反覆腹膜感染以及傳統之腹膜透析液不符合生理性(酸性、高乳酸值、高滲透壓、高濃度葡萄糖及過度醣化終產物等)所引起對腹膜引起傷害刺激所致。而這上述病理機轉中上皮細胞間質轉化 (epithelial-mesenchymal transition) 被認為占有重要角色。近年來我們實驗室和他人研究都發現維生素D對於上述所說腹膜透析相關之腹膜傷害及纖維化具有療效。遺憾的是其缺點為維生素D用於腹膜纖維化的有效治療劑量會造成高血鈣症與血管鈣化等副作用，這也將限制了維生素D於臨床治療上的應用。而目前已知奈米技術是在分子尺度上之工程材料系統科學，將其應用於醫藥稱之為奈米醫藥科技，以奈米粒子達到藥品傳輸亦是其應用之一。這些奈米藥物載體可透過增強滲透性和滯留作用達到優先聚集在病理部位之效果，因此它們非常適合於標靶治療的特性與應用。近年來有關奈米藥物載體如日方升，有許多奈米藥物已允許上市或是臨床實驗階段，因此我們希望利用其特質進一步探討透過奈米藥物載體加強維生素D對於腹膜透析治療的效果及減少副作用的可行性。

實驗設計

首先我們利用1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N- [amino-(polyethylene glycol)2000] (DSPE-PEG), L-a-phosphatidylcholine (PC) 做為奈米粒子載體的基本材料以及我們所要包覆的 Calcitriol (1α,25(OH)2D3) 維生素D，來建構出可攜帶維生素D的奈米藥物載體。接著我們測試維生素D奈米粒子對人類腹膜上皮細胞隨著時間進程的反應以及最適濃度，利用Transforming growth factor beta 1 (TGF-β1) 誘發腹膜細胞產生EMT的實驗，確認我們所製作出來的奈米藥物是有效且安全的。最後，將此奈米藥物外層接上抗腹膜細胞抗體，透過腹腔注射進入小鼠腹腔，觀察藥物能在腹腔滯留的時間，及是否能減少上述之不必要副作用的產生。

結果

我們製作出一種能攜帶維生素D且可專一標靶在腹膜細胞的奈米藥物載體，能直接給予腹膜細胞維生素D，進而可避免腹腔注射維生素D時所造成的高濃度維生素D對於小鼠的傷害。而直到目前為止，我們團隊也建構出了大小約小於200nm且可包覆維生素D的奈米藥物載體。經過細胞毒性、吞噬，TGF-β1誘發EMT之細胞實驗的測試，這些帶有維生素D的奈米粒子隨著時間的進程可被人類腹膜上皮細胞株所攝入，且無細胞毒性。而在動物實驗當中，奈米粒子也能透過外接抗體進而停滯於腹腔較長一段時間，且能減緩高劑量注射維生素D所導致之高血鈣等副作用。

結論

奈米科技說明了奈米粒子對於作為藥物的載體是非常具有可行性的，而透過我們目前的實驗結果說明了包覆維生素D的奈米粒子是穩定可行且安全的。因此，我們認為利用包覆維生素D的奈米粒子載體以避免腹腔注射維生素D所導致的系統性副作用是有臨床運用的可行性。

ABSTRACT
PURPOSE
Peritoneal dialysis (PD) is an established replacement therapy of end stage renal disease (ESRD). Unfortunately, the limitation of long-term PD is peritoneal fibrosis, resulting in technique failure. Epithelial-mesenchymal transition (EMT) of mesothelial cells (MCs) is the process of initiate and reversible during peritoneal fibrosis and sequential extracellular matrix (ECM) accumulation also is the key change. Recently, we have found that vitamin D to be one novel potential therapy in PD related peritoneal damage. But the most limitation in animal studies is that, therapeutic dose of vitamin D can induce vessel calcification and hypercalcemia and this defect will limit its applicability. Nanomedicine, the application of nanotechnology to medicine, enables the development of nanoparticle (NP) therapeutic carriers. These drug carriers can passively target to specific site to enhance permeability and retention effect and there are plenty of NP platforms have been approved by the Food and Drug Administration (FDA) or tested in clinical trials. Therefore, they are ideally suited for the delivery of specific drug as target therapy. For this reason, we’ll provide the hypothesis that site target specific nanoparticle delivery system can reinforce vitamin D therapeutic effect on peritoneal fibrosis and alleviate its side effect.
EXPERIMENTAL DESIGN
First we used 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N- [amino-(polyethylene glycol)2000] (DSPE-PEG), L-a-phosphatidylcholine (PC) and Calcitriol (1α,25(OH)2D3) to construct nanoparticles (D-NPs) which will package with 1α,25(OH)2D3 as a delivery system. To confirm the function and safety of D-NPs, we treated primary human MCs with TGF-b1 which could induce EMT and were reversed by D-NPs. Final, we conjugated antibodies (Glycoprotein M6A, GPM6A) to D-NPs. These particles (Ab-D-NPs) were implanted to mice by intraperitoneal (IP) injection and stagnated in the abdominal cavity and then alleviated the side effect that induced by high-dose vitamin D intraperitoneal injection.
RESULTS
We created the vitamin D nanoparticle delivery system, and then provided a possibility that low concentration of the vitamin D with high-efficiency therapy could alleviate the side effect of vitamin D through intraperitoneal injection in mice. As yet, we have constructed drug delivery vehicles with a sub-200 nm size which were loaded with vitamin D and released with a slow manner. These vitamin D nanoparticles were taken up by human peritoneal mesothelial cell line (HMrSV5 cells) in the time course, and were safety for HMrSV5 cells and MCs by the XTT assay. Whereas, fluorescence encapsulated NPs conjugated antibodies allow tissue specific fluorescent imaging and calcitriol encapsulated NPs conjugated antibodies could alleviate the side effect of intraperitoneal vitamin D injection in vivo.
CONCLUSIONS
Nanotechnology have shown that nanoparticles have a great potential as drug carriers, and our finding reveal that vitamin D loaded particles are feasible and safe to deliver vitamin D to the peritoneum and to alleviate hypercalcemia in a mouse model in vivo. Thus, the effect of nanoparticles vitamin D delivery systems in the prevention of intraperitoneal vitamin D injection may be a potential strategy in future.

CONTENTS
ABSTRACT i
摘要 iv
誌謝 vii
CONTENTS ix
List of Tables xii
List of Figures xiii
ABBREVIATIONS 1
CHAPTER 1. INTRODUCTION 4
1.1 Peritoneum damage in long-term peritoneal dialysis. 4
1.2 Pathologic significance of EMT of MCs in peritoneum structure and functional changes. 5
1.3 1a,25(OH)2D3 protective effect on peritoneum damage. 6
1.4 Nanoparticles as drug delivery systems 7
1.5 Nanomedicines for peritoneal mesothelial cells 7
CHAPTER 2. MATERIALS AND METHODS 9
2.1 Synthesis of the vitamin D3 loaded nanoparticles. 9
2.2 Synthesis of the rhodamine 6G (R6G) loaded nanoparticles. 10
2.3 Nanoparticles conjugate with glycoprotein M6A antibody. 10
2.4 General procedure for the quantification of vitamin D loading. 11
2.5 General procedure for determining the vitamin D release profile. 11
2.6 Determination of size distribution of nanoparticles by dynamic light scattering (DLS). 12
2.7 Transmission Electron Microscopy (TEM) of nanoparticles. 12
2.8 Human primary mesothelial cells. 13
2.9 Cell viability assay. 13
2.10 In vitro study design. 14
2.11 Western blotting for E-cadherin and Snail. 15
2.12 Nanoparticles conjugate with glycoprotein M6A antibody observed by Xenogen IVISR Spectrum Noninvasive Quantitative Molecular Imaging System (IVIS). 15
2.13 Intraperitoneal vitamin D injection model experimental protocol. 16
CHAPTER 3. RESULTS 17
3.1 Synthesis and characterization of vitamin D3 nanoparticles. 17
3.2 Release kinetic profile of vitamin D from nanoparticles 18
3.3 In vitro cytotoxicity and cellular uptake of NPs. 19
3.4 D-NPs can debilitate the TGF-b1-induced EMT in Vitro. 20
3.5 Ab-R6G-NPs, antibody conjugated NPs can allow specific tissue fluorescent imaging in vivo. 21
3.6 Ab-D-NPs can ameliorate the side effect of vitamin D from intraperitoneal injection in the mice model. 22
CHAPTER 4. DISCUSSION 23
TABLES 26
FIGURES 28
APPENDIX 52

1.Mehrotra, R., et al., Similar outcomes with hemodialysis and peritoneal dialysis in patients with end-stage renal disease. Arch Intern Med, 2011. 171(2): p. 110-8.
2.Fenton, S.S., et al., Hemodialysis versus peritoneal dialysis: a comparison of adjusted mortality rates. Am J Kidney Dis, 1997. 30(3): p. 334-42.
3.Huang, C.C., K.F. Cheng, and H.D. Wu, Survival analysis: comparing peritoneal dialysis and hemodialysis in Taiwan. Perit Dial Int, 2008. 28 Suppl 3: p. S15-20.
4.Vonesh, E.F., et al., Mortality studies comparing peritoneal dialysis and hemodialysis: what do they tell us? Kidney Int Suppl, 2006(103): p. S3-11.
5.Dennis Fry, M., Peritoneal Dialysis in ESRD Patients_ Case planning and techniques for this cost-effective method of renal replacement therapy. Endovascular Today, 2012.
6.Kawaguchi, Y., et al., Issues affecting the longevity of the continuous peritoneal dialysis therapy. Kidney Int Suppl, 1997. 62: p. S105-7.
7.Davies, S.J., et al., What really happens to people on long-term peritoneal dialysis? Kidney Int, 1998. 54(6): p. 2207-17.
8.Han, S.H., et al., Improving outcome of CAPD: twenty-five years' experience in a single Korean center. Perit Dial Int, 2007. 27(4): p. 432-40.
9.Nakamoto, H., Y. Kawaguchi, and H. Suzuki, Is technique survival on peritoneal dialysis better in Japan? Perit Dial Int, 2006. 26(2): p. 136-43.
10.Schaefer, F., et al., Current practice of peritoneal dialysis in children: results of a longitudinal survey. Mid European Pediatric Peritoneal Dialysis Study Group (MEPPS). Perit Dial Int, 1999. 19 Suppl 2: p. S445-9.
11.Woodrow, G., J.H. Turney, and A.M. Brownjohn, Technique failure in peritoneal dialysis and its impact on patient survival. Perit Dial Int, 1997. 17(4): p. 360-4.
12.Davies, S.J., et al., Peritoneal glucose exposure and changes in membrane solute transport with time on peritoneal dialysis. J Am Soc Nephrol, 2001. 12(5): p. 1046-51.
13.Krediet, R.T., The peritoneal membrane in chronic peritoneal dialysis. Kidney Int, 1999. 55(1): p. 341-56.
14.Lee, Y.C., et al., Vitamin D can ameliorate chlorhexidine gluconate-induced peritoneal fibrosis and functional deterioration through the inhibition of epithelial-to-mesenchymal transition of mesothelial cells. Biomed Res Int, 2015. 2015: p. 595030.
15.Yanez-Mo, M., et al., Peritoneal dialysis and epithelial-to-mesenchymal transition of mesothelial cells. N Engl J Med, 2003. 348(5): p. 403-13.
16.Zheng, Z., et al., Peritoneal dialysis solutions disturb the balance of apoptosis and proliferation of peritoneal cells in chronic dialysis model. Adv Perit Dial, 2001. 17: p. 53-7.
17.Holmes, C.J. and D. Faict, Peritoneal dialysis solution biocompatibility: definitions and evaluation strategies. Kidney Int Suppl, 2003(88): p. S50-6.
18.Lee, Y.C., et al., Shorter daily dwelling time in peritoneal dialysis attenuates the epithelial-to-mesenchymal transition of mesothelial cells. BMC Nephrol, 2014. 15: p. 35.
19.Aroeira, L.S., et al., Epithelial to mesenchymal transition and peritoneal membrane failure in peritoneal dialysis patients: pathologic significance and potential therapeutic interventions. J Am Soc Nephrol, 2007. 18(7): p. 2004-13.
20.Thiery, J.P. and J.P. Sleeman, Complex networks orchestrate epithelial-mesenchymal transitions. Nat Rev Mol Cell Biol, 2006. 7(2): p. 131-42.
21.Kalluri, R. and R.A. Weinberg, The basics of epithelial-mesenchymal transition. J Clin Invest, 2009. 119(6): p. 1420-8.
22.Perl, A.K., et al., A causal role for E-cadherin in the transition from adenoma to carcinoma. Nature, 1998. 392(6672): p. 190-3.
23.Takeichi, M., Morphogenetic roles of classic cadherins. Curr Opin Cell Biol, 1995. 7(5): p. 619-27.
24.Yang, L., et al., Effect of 1,25(OH)(2)D(3) on rat peritoneal mesothelial cells treated with high glucose plus lipopolysaccharide. Cell Immunol, 2011. 271(1): p. 173-9.
25.Coronel, F., et al., Changes in peritoneal membrane permeability and proteinuria in patients on peritoneal dialysis after treatment with paricalcitol - a preliminary study. Clin Nephrol, 2012. 78(2): p. 93-9.
26.Hirose, M., et al., 22-Oxacalcitriol prevents progression of peritoneal fibrosis in a mouse model. Perit Dial Int, 2013. 33(2): p. 132-42.
27.Kang, S.H., et al., Paricalcitol ameliorates epithelial-to-mesenchymal transition in the peritoneal mesothelium. Nephron Exp Nephrol, 2014. 126(1): p. 1-7.
28.Lee, C.J., et al., Calcitriol decreases TGF-beta1 and angiotensin II production and protects against chlorhexide digluconate-induced liver peritoneal fibrosis in rats. Cytokine, 2014. 65(1): p. 105-18.
29.Gonzalez-Mateo, G.T., et al., Paricalcitol reduces peritoneal fibrosis in mice through the activation of regulatory T cells and reduction in IL-17 production. PLoS One, 2014. 9(10): p. e108477.
30.Wilczewska, A.Z., et al., Nanoparticles as drug delivery systems. Pharmacol Rep, 2012. 64(5): p. 1020-37.
31.Patil, S., et al., Synthesis, characterization and in vitro evaluation of novel vitamin D3 nanoparticles as a versatile platform for drug delivery in cancer therapy. Journal of Materials Chemistry B, 2013. 1(42): p. 5742.
32.Nevozhay, D., et al., [Current status of research on conjugates and related drug delivery systems in the treatment of cancer and other diseases]. Postepy Hig Med Dosw (Online), 2007. 61: p. 350-60.
33.Perez-Martinez, F.C., et al., Barriers to non-viral vector-mediated gene delivery in the nervous system. Pharm Res, 2011. 28(8): p. 1843-58.
34.Ortega, A., et al., Antimicrobial evaluation of quaternary ammonium polyethyleneimine nanoparticles against clinical isolates of pathogenic bacteria. IET Nanobiotechnol, 2015. 9(6): p. 342-8.
35.Santamaria, B., et al., A nanoconjugate Apaf-1 inhibitor protects mesothelial cells from cytokine-induced injury. PLoS One, 2009. 4(8): p. e6634.
36.Yoshizawa, H., et al., TGF-beta(1)-siRNA delivery with nanoparticles inhibits peritoneal fibrosis. Gene Ther, 2015. 22(4): p. 333-40.
37.Almouazen, E., et al., Nano-encapsulation of vitamin D3 active metabolites for application in chemotherapy: formulation study and in vitro evaluation. Pharm Res, 2013. 30(4): p. 1137-46.
38.Kamaly, N., et al., Nanomedicines for renal disease: current status and future applications. Nat Rev Nephrol, 2016. 12(12): p. 738-753.
39.Williams, R.M., E.A. Jaimes, and D.A. Heller, Nanomedicines for kidney diseases. Kidney Int, 2016. 90(4): p. 740-5.
40.Harris, J.M. and R.B. Chess, Effect of pegylation on pharmaceuticals. Nat Rev Drug Discov, 2003. 2(3): p. 214-21.
41.Choi, K.Y., et al., Theranostic nanoplatforms for simultaneous cancer imaging and therapy: current approaches and future perspectives. Nanoscale, 2012. 4(2): p. 330-42.
42.Cheng, Z., et al., Multifunctional nanoparticles: cost versus benefit of adding targeting and imaging capabilities. Science, 2012. 338(6109): p. 903-10.
43.Rongjie FU, J.L.a.Y.W., Fat-Soluble Vitamins Analysis on an Agilent ZORBAX Eclipse PAH Polymeric C18 Bonded Column. Agilent Technologies, 2010. 5990-5342EN.
44.Diaz, C., et al., Ex vivo proliferation of mesothelial cells directly obtained from peritoneal effluent: its relationship with peritoneal antecedents and functional parameters. Adv Perit Dial, 1998. 14: p. 19-24.
45.Li, C., et al., Twist overexpression promoted epithelial-to-mesenchymal transition of human peritoneal mesothelial cells under high glucose. Nephrol Dial Transplant, 2012. 27(11): p. 4119-24.
46.Cano, A., et al., The transcription factor snail controls epithelial-mesenchymal transitions by repressing E-cadherin expression. Nat Cell Biol, 2000. 2(2): p. 76-83.
47.Wang, J., et al., 20-hydroxyvitamin D(3) inhibits proliferation of cancer cells with high efficacy while being non-toxic. Anticancer Res, 2012. 32(3): p. 739-46.
48.Zhu, F., et al., Preventive effect of Notch signaling inhibition by a gamma-secretase inhibitor on peritoneal dialysis fluid-induced peritoneal fibrosis in rats. Am J Pathol, 2010. 176(2): p. 650-9.
49.Abbasi, A., et al., Stability of vitamin D(3) encapsulated in nanoparticles of whey protein isolate. Food Chem, 2014. 143: p. 379-83.
50.Sengupta, P., et al., Cholesterol-tethered platinum II-based supramolecular nanoparticle increases antitumor efficacy and reduces nephrotoxicity. Proc Natl Acad Sci U S A, 2012. 109(28): p. 11294-9.
51.Davis, M.E., Z.G. Chen, and D.M. Shin, Nanoparticle therapeutics: an emerging treatment modality for cancer. Nat Rev Drug Discov, 2008. 7(9): p. 771-82.
52.Petros, R.A. and J.M. DeSimone, Strategies in the design of nanoparticles for therapeutic applications. Nat Rev Drug Discov, 2010. 9(8): p. 615-27.
53.Yameen, B., et al., Insight into nanoparticle cellular uptake and intracellular targeting. J Control Release, 2014. 190: p. 485-99.
54.Okamoto, R., et al., Inecalcitol, an analog of 1alpha,25(OH)(2) D(3) , induces growth arrest of androgen-dependent prostate cancer cells. Int J Cancer, 2012. 130(10): p. 2464-73.
55.Beer, T.M. and A. Myrthue, Calcitriol in cancer treatment: from the lab to the clinic. Mol Cancer Ther, 2004. 3(3): p. 373-81.
56.Wang, R., et al., Application of poly(ethylene glycol)-distearoylphosphatidylethanolamine (PEG-DSPE) block copolymers and their derivatives as nanomaterials in drug delivery. Int J Nanomedicine, 2012. 7: p. 4185-98.
57.Ramalho, M.J., et al., PLGA nanoparticles as a platform for vitamin D-based cancer therapy. Beilstein J Nanotechnol, 2015. 6: p. 1306-18.