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研究生(外文):Chung-Wei Kao
論文名稱(外文):Combining Magnetic Nanoparticles with Peptides Derived from Monocyte Hemoattractant Protein-1 as the Targeting Tool for Atherosclerosis
指導教授(外文):Jiashing Yu
外文關鍵詞:iron oxide magnetic nanoparticlemonocyteMCP-1atherosclerosis
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單核球趨化蛋白-1 (MCP-1)具有誘導單核球趨化和激活單核細胞的雙重功能,在人體各器官的疾病中均有表達,並在患部吸引單核球的聚集與作用,促使單核球穿過內皮細胞進入血管壁,單核球也因此分化為巨噬細胞和泡沫細胞,形成動脈粥樣硬化。本實驗主要在研究氧化鐵奈米粒子,並在表面接有一段單核球趨化蛋白-1的氨基酸序列,利用此胺基酸序列讓氧化鐵奈米粒子對單核球有專一性鍵結,做為動脈粥樣硬化前期的顯影工具。
細胞實驗中利用不同種細胞測試可觀察到,接有MCP-1胺基酸序列的氧化鐵奈米粒子對單核球有專一性鍵結,而動物實驗中則使用apolipoprotein E (ApoE)基因缺陷小鼠,並餵食四周的高血脂食物以促進動脈粥樣硬化的產生作為實驗組,從小鼠尾靜脈注射氧化鐵奈米粒子,並利用magnetic resonance imaging (MRI)、in vivo imaging system (IVIS)顯影觀察氧化鐵奈米粒子的循環分布,發現在單核球聚集的器官或部位中,都有氧化鐵奈米粒子的聚集,尤其在主動脈的分布較其他組別明顯,最後利用油紅染動脈硬化組織和普魯士藍染氧化鐵奈米粒子,皆能證明接有MCP-1胺基酸序列的氧化鐵奈米粒子具有動脈粥樣硬化標定的能力。
Commonly, atherosclerosis is a multifactorial inflammatory disease that would progress silently for long period and widely accepted as the main cause of cardiovascular diseases. To prevent atherosclerotic plaques generating, imaging early molecular markers and quantifying the extent of disease progression are the effective ways in biomedical engneering. During the inflammation, circulating monocytes leave the bloodstream and migrate into incipient lipid accumulation in the artery wall, following conditioning by local growth factors and proinflammatory cytokines. Therefore, monocyte accumulation in the arterial wall can be observed in fatty streaks, rupture-prone plaques, and experimental atherosclerosis. Targeting monocytes as a strategy to diagnose atherosclerotic lesions could yield a molecular imaging tool that would detect the early-stage atherosclerosis, quantify the extent of plaque progression.
The objective of the work is to research monocyte-targeting iron oxide magnetic nanoparticles (MNPs), which are incorporated with the peptides derived from the chemokine receptor CCR2-binding motif of monocyte chemoattractant protein-1 (MCP-1) as diagnostic tools for potential atherosclerosis. In this study, MCP-1-motif MNPs had specific affinity to monocytes using in vitro fluorescence imaging. In addition, with MNPs injection in ApoE knock-out mice (ApoE KO mice), the well-characterized animal model of atherosclerosis, MNPs were found in specific organism or regions which had monocyte accumulation, especially the aorta of atherosclerosis model mice, through the in vivo imaging system (IVIS) imaging and magnetic resonance imaging (MRI). We also perform Oil Red O staining and Prussian Blue staining to confirm the specific affinity of MCP-1-motif MNPs.
誌謝 i
摘要 iii
Chapter 1 Introduction 1
1.1 Atherosclerosis 1
1.1.1 Chemokine 2
1.1.2 Monocyte chemoattractant protein-1 3
1.1.3 Targeting Strategy 4
1.2 Nanoparticles 5
1.2.1 Magnetic Nanoparticles 6
1.2.2 Core-Shell Structure 7
1.2.3 Peptide and Fluorescence 9
1.3 Noninvasive Imaging 12
1.4 Research Frame Work 14
Chapter 2 Materials and Methods 21
2.1 Materials 21
2.2 Equipment 22
2.3 Solution Formula 23
2.3.1 Phosphate Buffered Saline Solution (PBS), pH 7.4 23
2.3.2 DMEM-HG Culture Medium for WEHI 274.1 Monocytes 23
2.3.3 DMEM-HG Culture Medium for 3T3 Cells 23
2.3.4 DMEM-HG/F-12 Culture Medium for hASCs 24
2.3.5 MTT Assay Working Solution 24
2.3.6 Cyanine 5 Staining Solution 25
2.3.7 Oil Red O Staining Solution 25
2.3.8 Prussian Blue Staining Solution 25
2.4 Methods 26
2.4.1 Characterization 26 Scanning Electron Microscope (SEM) and Transmission Electron Microscope (TEM) 26 Particle Size and Zeta Potential Analyzer 26 X-ray Photoelectron Spectroscope (XPS) and Magnetometer 26
2.4.2 in vitro Study 27 Cell Culture of WEHI 274.1 Monocytes 27 Cell Culture of 3T3 Cells and hASCs 27 MTT Assay 28 Live/Dead Assay 29 Nanoparticles Affinity Test 30
2.4.3 in vivo Study 31 Animal Model 31 Nuclear Magnetic Resonance Imaging (MRI) 32 Non-invasion in vivo Imaging System (IVIS) 32 Histology Staining 33
2.4.4 Statistical Analysis 33
Chapter 3 Results and Discussions 34
3.1 Characterization of Iron Oxide MNPs 34
3.1.1 Structural Property of Iron Oxide MNPs 34
3.1.2 Composition of Iron Oxide MNPs 36
3.1.3 Magnetic Measurements 38
3.2 Cytotoxicity and in vitro Study 43
3.2.1 Cytotoxicity and Cell Viability of MCP-1-motif MNPs 43
3.2.2 Specific Affinity of MCP-1-motif MNPs 44
3.3 in vivo Study, Imaging and Histology 50
3.3.1 Nuclear Magnetic Resonance Imaging (MRI) 50
3.3.2 Non-invasion in vivo Imaging System (IVIS) 52
3.3.3 Histology 54
Chapter 4 Conclusions 65
Chapter 5 Future Work 67
1.Woollard, K.J. and F. Geissmann, Monocytes in atherosclerosis: subsets and functions. Nature reviews. Cardiology, 2010. 7(2): p. 77-86.
2.Moss, J.W.E. and D.P. Ramji, Cytokines: Roles in atherosclerosis disease progression and potential therapeutic targets. Future medicinal chemistry, 2016. 8(11): p. 1317-1330.
3.Libby, P., P.M. Ridker, and A. Maseri, Inflammation and Atherosclerosis. Circulation, 2002. 105(9): p. 1135-1143.
4.Mlinar, L.B., et al., Active targeting of early and mid-stage atherosclerotic plaques using self-assembled peptide amphiphile micelles. Biomaterials, 2014. 35(30): p. 8678-8686.
5.Deshmane, S.L., et al., Monocyte chemoattractant protein-1 (MCP-1): an overview. J Interferon Cytokine Res, 2009. 29(6): p. 313-26.
6.Zernecke, A. and C. Weber, Chemokines in Atherosclerosis. Proceedings Resumed, 2014. 34(4): p. 742-750.
7.Libby, P., Current Concepts of the Pathogenesis of the Acute Coronary Syndromes. Circulation, 2001. 104(3): p. 365-372.
8.Szmitko, P.E., et al., New Markers of Inflammation and Endothelial Cell Activation. Part I, 2003. 108(16): p. 1917-1923.
9.Chung, E.J., et al., Monocyte-Targeting Supramolecular Micellar Assemblies: A Molecular Diagnostic Tool for Atherosclerosis. Advanced healthcare materials, 2015. 4(3): p. 367-376.
10.Schwartz, C.J., et al., The pathogenesis of atherosclerosis: an overview. Clin Cardiol, 1991. 14(2 Suppl 1): p. I1-16.
11.Taub, D.D., Chemokine-leukocyte interactions. The voodoo that they do so well. Cytokine Growth Factor Rev, 1996. 7(4): p. 355-76.
12.Lewis, D.R., et al., Polymer-based therapeutics: nanoassemblies and nanoparticles for management of atherosclerosis. Wiley Interdisciplinary Reviews: Nanomedicine and Nanobiotechnology, 2011. 3(4): p. 400-420.
13.Khodabandehlou, K., et al., Targeting cell adhesion molecules with nanoparticles using in vivo and flow-based in vitro models of atherosclerosis. Exp Biol Med (Maywood), 2017. 242(8): p. 799-812.
14.Pan, H., et al., Programmable nanoparticle functionalization for in vivo targeting. The FASEB Journal, 2013. 27(1): p. 255-264.
15.Richards, D.A., A. Maruani, and V. Chudasama, Antibody fragments as nanoparticle targeting ligands: a step in the right direction. Chemical Science, 2017. 8(1): p. 63-77.
16.Zhang, J., et al., Detection and treatment of atherosclerosis using nanoparticles. Wiley Interdiscip Rev Nanomed Nanobiotechnol, 2017. 9(1).
17.Kim, Y., et al., Probing nanoparticle translocation across the permeable endothelium in experimental atherosclerosis. Proceedings of the National Academy of Sciences, 2014. 111(3): p. 1078-1083.
18.Lobatto, M.E., et al., Perspectives and opportunities for nanomedicine in the management of atherosclerosis. Nat Rev Drug Discov, 2011. 10(11): p. 835-852.
19.Kolhatkar, A.G., et al., Tuning the Magnetic Properties of Nanoparticles. International Journal of Molecular Sciences, 2013. 14(8): p. 15977-16009.
20.Lu, A.H., E.L. Salabas, and F. Schuth, Magnetic nanoparticles: synthesis, protection, functionalization, and application. Angew Chem Int Ed Engl, 2007. 46(8): p. 1222-44.
21.Popescu, R.C., E. Andronescu, and A.M. Grumezescu, In vivo evaluation of Fe(3)O(4) nanoparticles. Rom J Morphol Embryol, 2014. 55(3 Suppl): p. 1013-8.
22.Bietenbeck, M., et al., Remote magnetic targeting of iron oxide nanoparticles for cardiovascular diagnosis and therapeutic drug delivery: where are we now? Int J Nanomedicine, 2016. 11: p. 3191-203.
23.Estévez, M., et al., Novel wear resistant and low toxicity dental obturation materials. Materials Letters, 2007. 61(14): p. 3025-3029.
24.Mahmoudi, M., et al., Superparamagnetic iron oxide nanoparticles (SPIONs): development, surface modification and applications in chemotherapy. Adv Drug Deliv Rev, 2011. 63(1-2): p. 24-46.
25.Jadhav, N.V., et al., Synthesis of oleic acid functionalized Fe3O4 magnetic nanoparticles and studying their interaction with tumor cells for potential hyperthermia applications. Colloids Surf B Biointerfaces, 2013. 108: p. 158-68.
26.Chen, L., et al., Facile synthesis and magnetic properties of monodisperse Fe3O4/silica nanocomposite microspheres with embedded structures via a direct solution-based route. Journal of Alloys and Compounds, 2010. 497(1–2): p. 221-227.
27.Yang, J., et al., A comprehensive study on the synthesis and paramagnetic properties of PEG-coated Fe3O4 nanoparticles. Applied Surface Science, 2014. 303: p. 425-432.
28.Lüdtke-Buzug, K., et al., Preparation and Characterization of Dextran-Covered Fe3O4 Nanoparticles for Magnetic Particle Imaging, in 4th European Conference of the International Federation for Medical and Biological Engineering: ECIFMBE 2008 23–27 November 2008 Antwerp, Belgium, J. Vander Sloten, et al., Editors. 2009, Springer Berlin Heidelberg: Berlin, Heidelberg. p. 2343-2346.
29.Xu, J.K., et al., Bio and nanomaterials based on Fe3O4. Molecules, 2014. 19(12): p. 21506-28.
30.Bautista, M.C., et al., Comparative study of ferrofluids based on dextran-coated iron oxide and metal nanoparticles for contrast agents in magnetic resonance imaging. Nanotechnology, 2004. 15(4): p. S154.
31.Carmen Bautista, M., et al., Surface characterisation of dextran-coated iron oxide nanoparticles prepared by laser pyrolysis and coprecipitation. Journal of Magnetism and Magnetic Materials, 2005. 293(1): p. 20-27.
32.Hong, R.Y., et al., Synthesis, characterization and MRI application of dextran-coated Fe3O4 magnetic nanoparticles. Biochemical Engineering Journal, 2008. 42(3): p. 290-300.
33.Yu, Y. and Y. Chau, One-Step “Click” Method for Generating Vinyl Sulfone Groups on Hydroxyl-Containing Water-Soluble Polymers. Biomacromolecules, 2012. 13(3): p. 937-942.
34.Steitz, S.A., et al., Mapping of MCP-1 functional domains by peptide analysis and site-directed mutagenesis. FEBS Letters, 1998. 430(3): p. 158-164.
35.Valente, A.J., et al., Characterization of monocyte chemotactic protein-1 binding to human monocytes. Biochem Biophys Res Commun, 1991. 176(1): p. 309-14.
36.Pan, L., et al., Nuclear-targeted drug delivery of TAT peptide-conjugated monodisperse mesoporous silica nanoparticles. J Am Chem Soc, 2012. 134(13): p. 5722-5.
37.Hauser, A.K., K.W. Anderson, and J.Z. Hilt, Peptide conjugated magnetic nanoparticles for magnetically mediated energy delivery to lung cancer cells. Nanomedicine (Lond), 2016. 11(14): p. 1769-85.
38.Fan, L., et al., Negatively charged AuNP modified with monoclonal antibody against novel tumor antigen FAT1 for tumor targeting. Journal of Experimental & Clinical Cancer Research : CR, 2015. 34(1): p. 103.
39.Liu, L., et al., Self-assembled nanoparticles based on the c(RGDfk) peptide for the delivery of siRNA targeting the VEGFR2 gene for tumor therapy. Int J Nanomedicine, 2014. 9: p. 3509-26.
40.Tarkin, J.M., et al., Imaging Atherosclerosis. Circulation Research, 2016. 118(4): p. 750-769.
41.Schellenberger, E., et al., Protease-specific nanosensors for magnetic resonance imaging. Bioconjug Chem, 2008. 19(12): p. 2440-5.
42.Olson, E.S., et al., Activatable cell penetrating peptides linked to nanoparticles as dual probes for in vivo fluorescence and MR imaging of proteases. Proc Natl Acad Sci U S A, 2010. 107(9): p. 4311-6.
43.Makowski, M.R., et al., Noninvasive Assessment of Atherosclerotic Plaque Progression in ApoE−/− Mice Using Susceptibility Gradient Mapping. Circulation: Cardiovascular Imaging, 2011. 4(3): p. 295-303.
44.Moon, H., et al., Noninvasive assessment of myocardial inflammation by cardiovascular magnetic resonance in a rat model of experimental autoimmune myocarditis. Circulation, 2012. 125(21): p. 2603-12.
45.Nakashima, Y., et al., ApoE-deficient mice develop lesions of all phases of atherosclerosis throughout the arterial tree. Arterioscler Thromb, 1994. 14(1): p. 133-40.
46.Li, Y., et al., Progression of atherosclerosis in ApoE-knockout mice fed on a high-fat diet. Eur Rev Med Pharmacol Sci, 2016. 20(18): p. 3863-3867.
47.Swirski, F.K., et al., Monocyte accumulation in mouse atherogenesis is progressive and proportional to extent of disease. Proc Natl Acad Sci U S A, 2006. 103(27): p. 10340-5.
48.Gajda, M., et al., Combined orcein and martius scarlet blue (OMSB) staining for qualitative and quantitative analyses of atherosclerotic plaques in brachiocephalic arteries in apoE/LDLR(−/−) mice. Histochemistry and Cell Biology, 2017. 147(6): p. 671-681.
49.den Adel, B., et al., Histological validation of iron-oxide and gadolinium based MRI contrast agents in experimental atherosclerosis: the do''s and don''t''s. Atherosclerosis, 2012. 225(2): p. 274-80.
50.Sharifi, S., et al., Superparamagnetic iron oxide nanoparticles for in vivo molecular and cellular imaging. Contrast Media & Molecular Imaging, 2015. 10(5): p. 329-355.
51.Mosmann, T., Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods, 1983. 65(1-2): p. 55-63.
52.Gerlier, D. and N. Thomasset, Use of MTT colorimetric assay to measure cell activation. J Immunol Methods, 1986. 94(1-2): p. 57-63.
53.van Meerloo, J., G.J. Kaspers, and J. Cloos, Cell sensitivity assays: the MTT assay. Methods Mol Biol, 2011. 731: p. 237-45.
54.Wen, S., et al., In vivo MRI detection of carotid atherosclerotic lesions and kidney inflammation in ApoE-deficient mice by using LOX-1 targeted iron nanoparticles. Nanomedicine, 2014. 10(3): p. 639-49.
55.Wu, S.C., et al., Bispecific Antibody Conjugated Manganese-Based Magnetic Engineered Iron Oxide for Imaging of HER2/neu- and EGFR-Expressing Tumors. Theranostics, 2016. 6(1): p. 118-30.
56.Koiwaya, H., et al., Augmented neovascularization with magnetized endothelial progenitor cells in rats with hind-limb ischemia. J Mol Cell Cardiol, 2011. 51(1): p. 33-40.
57.Liu, D., et al., Specific targeting of nasopharyngeal carcinoma cell line CNE1 by C225-conjugated ultrasmall superparamagnetic iron oxide particles with magnetic resonance imaging. Acta Biochim Biophys Sin (Shanghai), 2011. 43(4): p. 301-6.
58.Andres-Manzano, M.J., V. Andres, and B. Dorado, Oil Red O and Hematoxylin and Eosin Staining for Quantification of Atherosclerosis Burden in Mouse Aorta and Aortic Root. Methods Mol Biol, 2015. 1339: p. 85-99.
59.Mohanta, S., et al., Aorta Atherosclerosis Lesion Analysis in Hyperlipidemic Mice. Bio-protocol, 2016. 6(11): p. e1833.
60.Khalkhali, M., et al., The impact of polymer coatings on magnetite nanoparticles performance as MRI contrast agents: a comparative study. DARU Journal of Pharmaceutical Sciences, 2015. 23(1): p. 45.
61.Adhikari, U. and S. Scheiner, Preferred Configurations of Peptide–Peptide Interactions. The Journal of Physical Chemistry A, 2013. 117(2): p. 489-496.
62.Vallee, A., V. Humblot, and C.-M. Pradier, Peptide Interactions with Metal and Oxide Surfaces. Accounts of Chemical Research, 2010. 43(10): p. 1297-1306.
63.Bhattacharjee, S., DLS and zeta potential – What they are and what they are not? Journal of Controlled Release, 2016. 235: p. 337-351.
64.Li, G., et al., Three-dimensional porous carbon composites containing high sulfur nanoparticle content for high-performance lithium–sulfur batteries. Nature Communications, 2016. 7: p. 10601.
65.Liu, Y., et al., Nanoparticle layer deposition for highly controlled multilayer formation based on high- coverage monolayers of nanoparticles. Thin solid films, 2016. 598: p. 16-24.
66.Zhao, M., et al., Effect of nitrogen atomic percentage on N(+)-bombarded MWCNTs in cytocompatibility and hemocompatibility. Nanoscale Research Letters, 2014. 9(1): p. 142-142.
67.Liu, Y., et al., Efficient and durable hydrogen evolution electrocatalyst based on nonmetallic nitrogen doped hexagonal carbon. Scientific Reports, 2014. 4: p. 6843.
68.Gharbi, A., et al., Surface functionalization by covalent immobilization of an innovative carvacrol derivative to avoid fungal biofilm formation. AMB Express, 2015. 5: p. 9.
69.Wang, Q., et al., Fluorescent carbon dots as an efficient siRNA nanocarrier for its interference therapy in gastric cancer cells. Journal of Nanobiotechnology, 2014. 12: p. 58.
70.Liang, P.C., et al., Doxorubicin-modified magnetic nanoparticles as a drug delivery system for magnetic resonance imaging-monitoring magnet-enhancing tumor chemotherapy. Int J Nanomedicine, 2016. 11: p. 2021-37.
71.Bumb, A., et al., Synthesis and characterization of ultra-small superparamagnetic iron oxide nanoparticles thinly coated with silica. Nanotechnology, 2008. 19(33): p. 335601-335601.
72.Ho, D., X. Sun, and S. Sun, Monodisperse Magnetic Nanoparticles for Theranostic Applications. Accounts of chemical research, 2011. 44(10): p. 875-882.
73.Mohapatra, J., et al., Surface controlled synthesis of MFe2O4 (M = Mn, Fe, Co, Ni and Zn) nanoparticles and their magnetic characteristics. CrystEngComm, 2013. 15(3): p. 524-532.
74.Yao, Y., et al., In Vivo Imaging of Macrophages during the Early-Stages of Abdominal Aortic Aneurysm Using High Resolution MRI in ApoE(−/−) Mice. PLoS ONE, 2012. 7(3): p. e33523.
75.Kitagawa, T., et al., RGD targeting of human ferritin iron oxide nanoparticles enhances in vivo MRI of vascular inflammation and angiogenesis in experimental carotid disease and abdominal aortic aneurysm. Journal of Magnetic Resonance Imaging, 2017. 45(4): p. 1144-1153.
76.Salinas, B., et al., Surface-Functionalized Nanoparticles by Olefin Metathesis: A Chemoselective Approach for In Vivo Characterization of Atherosclerosis Plaque. Chemistry – A European Journal, 2015. 21(29): p. 10450-10456.
77.Braidwood, L., et al., Potent efficacy signals from systemically administered oncolytic herpes simplex virus (HSV1716) in hepatocellular carcinoma xenograft models. Journal of Hepatocellular Carcinoma, 2014. 1: p. 149-161.
78.Varna, M., et al., In vivo Distribution of Inorganic Nanoparticles in Preclinical Models. Journal of Biomaterials and Nanobiotechnology, 2012. Vol.03No.02: p. 11.
79.Longmire, M., P.L. Choyke, and H. Kobayashi, Clearance Properties of Nano-sized Particles and Molecules as Imaging Agents: Considerations and Caveats. Nanomedicine (London, England), 2008. 3(5): p. 703-717.
80.Ali, A., et al., Synthesis, characterization, applications, and challenges of iron oxide nanoparticles. Nanotechnology, Science and Applications, 2016. 9: p. 49-67.
81.Liao, N., et al., Poly (dopamine) coated superparamagnetic iron oxide nanocluster for noninvasive labeling, tracking, and targeted delivery of adipose tissue-derived stem cells. Scientific Reports, 2016. 6: p. 18746.
82.Langheinrich, A.C., et al., Atherosclerosis, inflammation and lipoprotein glomerulopathy in kidneys of apoE(-/-)/LDL(-/- )double knockout mice. BMC Nephrology, 2010. 11: p. 18-18.
83.Yoo, M.K., et al., Folate-PEG-superparamagnetic iron oxide nanoparticles for lung cancer imaging. Acta Biomater, 2012. 8(8): p. 3005-13.
84.Lee, B.-S., et al., Simvastatin and Losartan Differentially and Synergistically Inhibit Atherosclerosis in Apolipoprotein E(-/-) Mice. Korean Circulation Journal, 2012. 42(8): p. 543-550.
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