根據文獻紀錄目前約有三十多種人類胜肽或蛋白質聚集形成類澱粉蛋白纖維，而此些類澱粉蛋白纖維存在又與人類疾病關係密切，可惜的是目前並沒有治癒類澱粉蛋白變性病的方法，因此抑制類澱粉蛋白聚集成為最重要的治療策略。人類降鈣素是由32個胺基酸所組成的激素肽，透過甲狀腺濾泡旁細胞(亦稱C細胞)分泌，主要功能為調節體內鈣離子濃度與維持骨骼結構，因此可應用於治療骨相關疾病。然而，人類降鈣素因聚集速度較快易於形成類澱粉蛋白纖維導致降低其生物利用度及治療活性。目前選擇用聚集速度較慢的鮭魚降鈣素做成的鼻噴劑來當作治療藥物，但其與人類降鈣素的胺基酸序列差異甚大，導致具有免疫反應相關問題，而能有效抑制人類降鈣素形成類澱粉蛋白聚集並維持其治療活性成為最重要的研究課題。磁性奈米材料因具有較高生物相容性、低毒性、獨特的磁性以及其他等優點，因此於各個領域皆廣泛地發展成為具有潛力的材料，此外亦有些許的研究是關於磁性奈米材料抑制類澱粉蛋白纖維的探討，並且發現此奈米材料抑制人類降鈣素聚集的文獻甚少。
本研究中，我們透過化學共沉澱法製備出氧化鐵奈米粒子，並且利用不同方法(如共價鍵與吸附)將小分子包覆於氧化鐵奈米粒子的表面，再觀察其對於人類降鈣素形成類澱粉蛋白纖維的影響。經由硫磺素-T動力學和穿透式電子顯微鏡證明Dopamine-Fe3O4及Dihydrocaffeic acid@Fe3O4這兩種材料皆能有效抑制人類降鈣素聚集以及可降解其類澱粉蛋白纖維，透過此些實驗結果，我們期望能再以人類降鈣素為有效成份的劑型開發中，尋求可用來穩定人類降鈣素的藥物賦形劑添加物。

According to pervious research literature, it has been discovered that more than thirty species of human peptides or proteins would aggregate to form amyloid fibrils and associated with some human diseases. Unfortunately, there is no cure for amyloid diseases now, it is important to develop therapeutic strategies which can be used to inhibit amyloid aggregation. Human calcitonin (hCT) is a hormone peptide which contains thirty-two amino acids and it is secreted by parafollicular cells (also known as C cells) in the human body. In principle, this hormone can regulate the concentration of calcium in human body and also can be used to treat bone-related diseases. However, hCT aggregates quickly in aqueous solution and forms amyloid fibril which would reduce bioavailability and therapeutic activity of the peptide. The currently strategy is use salmon calcitonin (sCT) in a nasal spray as a treatment drug which has less aggregation propensity then human calcitonin. However, the sequence of sCT is quite different to that of hCT and sometimes will lead to immune response problems, it is important to prevent hCT aggregation and maintain its therapeutic activity. Magnetic nanomaterials have been widely developed as potential materials for various fields due to their high biocompatibility, low toxicity, and unique magnetic properties and easy to functionalize. So far, only a few studies utilized magnetic nanomaterials to treat other amyloidogenic proteins, but not for hCT.
In this study, we prepared iron oxide nanoparticles by chemical co-precipitation by use different methods such as covalent bonding and adsorption to coating small molecules on the surface of iron oxide nanoparticles. Later, we tested their effects on hCT aggregation. By ThT kinetic assay and images which collected from transmission electron microscope, we found that both Dopamine-Fe3O4 and Dihydrocaffeic acid@Fe3O4 can inhibit hCT amyloid formation and dissociate preformed hCT amyloid fibrils. It appears to be one of the most promising ways to stabilizes hCT in solution condition.

1. Chiti, F. and C.M. Dobson, Protein misfolding, amyloid formation, and human disease: a summary of progress over the last decade. Annual review of biochemistry, 2017. 86: p. 27-68.
2. Yadav, K., et al., Protein misfolding diseases and therapeutic approaches. Current Protein and Peptide Science, 2019. 20(12): p. 1226-1245.
3. Hazenberg, B.P., Amyloidosis: a clinical overview. Rheumatic Disease Clinics, 2013. 39(2): p. 323-345.
4. Pilkington, E.H., et al., Effects of protein corona on IAPP amyloid aggregation, fibril remodelling, and cytotoxicity. Scientific reports, 2017. 7(1): p. 1-13.
5. Schmitz, O., B. Brock, and J. Rungby, Amylin agonists: a novel approach in the treatment of diabetes. Diabetes, 2004. 53(suppl 3): p. S233-S238.
6. Hashimoto, M., et al., Role of protein aggregation in mitochondrial dysfunction and neurodegeneration in Alzheimer’s and Parkinson’s diseases. Neuromolecular medicine, 2003. 4(1): p. 21-35.
7. Chong, F.P., et al., Tau proteins and tauopathies in Alzheimer’s disease. Cellular and molecular neurobiology, 2018. 38(5): p. 965-980.
8. Tyedmers, J., A. Mogk, and B. Bukau, Cellular strategies for controlling protein aggregation. Nature reviews Molecular cell biology, 2010. 11(11): p. 777-788.
9. Fowler, D.M., et al., Functional amyloid–from bacteria to humans. Trends in biochemical sciences, 2007. 32(5): p. 217-224.
10. Xue, W.-F., S.W. Homans, and S.E. Radford, Systematic analysis of nucleation-dependent polymerization reveals new insights into the mechanism of amyloid self-assembly. Proceedings of the National Academy of Sciences, 2008. 105(26): p. 8926-8931.
11. Ban, T., et al., Direct observation of amyloid fibril growth monitored by thioflavin T fluorescence. Journal of Biological Chemistry, 2003. 278(19): p. 16462-16465.
12. Kumar, S. and J. Walter, Phosphorylation of amyloid beta (Aβ) peptides–A trigger for formation of toxic aggregates in Alzheimer's disease. Aging (Albany NY), 2011. 3(8): p. 803.
13. Xi, W.-H. and G.-H. Wei, Amyloid-β peptide aggregation and the influence of carbon nanoparticles. Chinese Physics B, 2015. 25(1): p. 018704.
14. Pryor, N.E., M.A. Moss, and C.N. Hestekin, Unraveling the early events of amyloid-β protein (Aβ) aggregation: techniques for the determination of Aβ aggregate size. International Journal of Molecular Sciences, 2012. 13(3): p. 3038-3072.
15. Copp, D.H., et al., Evidence for calcitonin—a new hormone from the parathyroid that lowers blood calcium. Endocrinology, 1962. 70(5): p. 638-649.
16. Hirsch, P.F., E.F. Voelkel, and P.L. Munson, Thyrocalcitonin: hypocalcemic hypophosphatemic principle of the thyroid gland. Science, 1964. 146(3642): p. 412-413.
17. Pearse, A.G.E., The cytochemistry of the thyroid C cells and their relationship to calcitonin. Proceedings of the Royal Society of London. Series B. Biological Sciences, 1966. 164(996): p. 478-487.
18. Muff, R., W. Born, and J.A. Fischer, Calcitonin, calcitonin gene-related peptide, adrenomedullin and amylin: homologous peptides, separate receptors and overlapping biological actions. European journal of endocrinology, 1995. 133(1): p. 17-20.
19. Naot, D. and J. Cornish, The role of peptides and receptors of the calcitonin family in the regulation of bone metabolism. Bone, 2008. 43(5): p. 813-818.
20. Khosla, S. and B.L. Riggs, Pathophysiology of age-related bone loss and osteoporosis. Endocrinology and Metabolism Clinics, 2005. 34(4): p. 1015-1030.
21. HINES, S.E., Paget's disease of bone: A new philosophy of treatment. Patient Care, 1999. 33(20): p. 40-40.
22. Carney, S., Calcitonin and human renal calcium and electrolyte transport. Mineral and electrolyte metabolism, 1997. 23(1): p. 43-47.
23. Cochran, M., et al., Renal effects of calcitonin. Br Med J, 1970. 1(5689): p. 135-137.
24. Sexton, P.M., et al., Localization and characterization of renal calcitonin receptors by in vitro autoradiography. Kidney international, 1987. 32(6): p. 862-868.
25. Stenbeck, G. Formation and function of the ruffled border in osteoclasts.Seminars in cell & developmental biology. 2002. Elsevier.
26. Swaminathan, R., J. Ker, and A. Care, Calcitonin and intestinal calcium absorption. Journal of Endocrinology, 1974. 61(1): p. 83-94.
27. Chesnut, C., et al., Salmon calcitonin: a review of current and future therapeutic indications. Osteoporosis international, 2008. 19(4): p. 479-491.
28. Chesnut III, C.H., et al., A randomized trial of nasal spray salmon calcitonin in postmenopausal women with established osteoporosis: the prevent recurrence of osteoporotic fractures study. The American journal of medicine, 2000. 109(4): p. 267-276.
29. Feletti, C. and V. Bonomini, Effect of calcitonin on bone lesions in chronic dialysis patients. Nephron, 1979. 24(2): p. 85-88.
30. Pun, K. and L. Chan, Analgesic effect of intranasal salmon calcitonin in the treatment of osteoporotic vertebral fractures. Clinical therapeutics, 1989. 11(2): p. 205-209.
31. Cudd, A., et al., Enhanced potency of human calcitonin when fibrillation is avoided. Journal of pharmaceutical sciences, 1995. 84(6): p. 717-719.
32. Arvinte, T., A. Cudd, and A. Drake, The structure and mechanism of formation of human calcitonin fibrils. Journal of Biological Chemistry, 1993. 268(9): p. 6415-6422.
33. Jha, N.N., et al., Comparison of α-synuclein fibril inhibition by four different amyloid inhibitors. ACS chemical neuroscience, 2017. 8(12): p. 2722-2733.
34. Huang, R., et al., NMR characterization of monomeric and oligomeric conformations of human calcitonin and its interaction with EGCG. Journal of molecular biology, 2012. 416(1): p. 108-120.
35. Guo, C., et al., Inhibitory effects of magnolol and honokiol on human calcitonin aggregation. Scientific reports, 2015. 5(1): p. 1-11.
36. Lantz, R., et al., Flavonoids with Vicinal Hydroxyl Groups Inhibit Human Calcitonin Amyloid Formation. Chemistry–A European Journal, 2020. 26(57): p. 13063-13071.
37. Freeman, M., A. Arrott, and J. Watson, Magnetism in medicine. Journal of Applied Physics, 1960. 31(5): p. S404-S405.
38. Su, C.-H. and F.-Y. Cheng, In vitro and in vivo applications of alginate/iron oxide nanocomposites for theranostic molecular imaging in a brain tumor model. RSC advances, 2015. 5(109): p. 90061-90064.
39. Mornet, S., et al., Magnetic nanoparticle design for medical applications. Progress in Solid State Chemistry, 2006. 34(2-4): p. 237-247.
40. Jain, T.K., et al., Iron oxide nanoparticles for sustained delivery of anticancer agents. Molecular pharmaceutics, 2005. 2(3): p. 194-205.
41. Akbarzadeh, A., M. Samiei, and S. Davaran, Magnetic nanoparticles: preparation, physical properties, and applications in biomedicine. Nanoscale research letters, 2012. 7(1): p. 1-13.
42. Noqta, O.A., et al., Recent advances in iron oxide nanoparticles (IONPs): synthesis and surface modification for biomedical applications. Journal of Superconductivity and Novel Magnetism, 2019. 32(4): p. 779-795.
43. Wahajuddin, S.A., Superparamagnetic iron oxide nanoparticles: magnetic nanoplatforms as drug carriers. International journal of nanomedicine, 2012. 7: p. 3445.
44. Mahmoudi, M., et al., Superparamagnetic iron oxide nanoparticles (SPIONs): development, surface modification and applications in chemotherapy. Advanced drug delivery reviews, 2011. 63(1-2): p. 24-46.
45. Karlsson, H.L., et al., Copper oxide nanoparticles are highly toxic: a comparison between metal oxide nanoparticles and carbon nanotubes. Chemical research in toxicology, 2008. 21(9): p. 1726-1732.
46. Laurent, S., et al., Magnetic iron oxide nanoparticles: synthesis, stabilization, vectorization, physicochemical characterizations, and biological applications. Chemical reviews, 2008. 108(6): p. 2064-2110.
47. Ajinkya, N., et al., Magnetic Iron Oxide Nanoparticle (IONP) Synthesis to Applications: Present and Future. Materials, 2020. 13(20): p. 4644.
48. Maity, D., et al., Synthesis of magnetite nanoparticles via a solvent-free thermal decomposition route. Journal of Magnetism and Magnetic Materials, 2009. 321(9): p. 1256-1259.
49. Chen, D., S. Ni, and Z. Chen, Synthesis of Fe3O4 nanoparticles by wet milling iron powder in a planetary ball mill. China Particuology, 2007. 5(5): p. 357-358.
50. Bednarikova, Z., et al., Effect of nanoparticles coated with different modifications of dextran on lysozyme amyloid aggregation. Journal of Magnetism and Magnetic Materials, 2019. 473: p. 1-6.
51. Bellova, A., et al., Effect of Fe3O4 magnetic nanoparticles on lysozyme amyloid aggregation. Nanotechnology, 2010. 21(6): p. 065103.
52. Javdani, N., et al., Effect of superparamagnetic nanoparticles coated with various electric charges on α-synuclein and β-amyloid proteins fibrillation process. International journal of nanomedicine, 2019. 14: p. 799.
53. Mochizuki, M., et al., Regioselective formation of multiple disulfide bonds with the aid of postsynthetic S-tritylation. Organic letters, 2015. 17(9): p. 2202-2205.
54. 陳昱傑, 具多巴胺與五胜肽 DFNKF 修飾的中孔洞氧化矽奈米粒子對人類降鈣素聚集之影響. 2021.
55. Shieh, D.-B., et al., Aqueous dispersions of magnetite nanoparticles with NH3+ surfaces for magnetic manipulations of biomolecules and MRI contrast agents. Biomaterials, 2005. 26(34): p. 7183-7191.
56. Zhang, L., et al., Efficient purification of His-tagged protein by superparamagnetic Fe3O4/Au–ANTA–Co2+ nanoparticles. Materials Science and Engineering: C, 2013. 33(4): p. 1989-1992.
57. Lu, Y.-C., et al., Augmented cellular uptake of nanoparticles using tea catechins: effect of surface modification on nanoparticle–cell interaction. Nanoscale, 2014. 6(17): p. 10297-10306.
58. Zeta-potential & Particle size Analyzer ELSZ-2000. Available from: https://www.otsukael.jp/product/detail/productid/92.
59. Biancalana, M. and S. Koide, Molecular mechanism of Thioflavin-T binding to amyloid fibrils. Biochimica et Biophysica Acta (BBA)-Proteins and Proteomics, 2010. 1804(7): p. 1405-1412.
60. Parthemore, J., L. Deftos, and D. Bronzert, The regulation of calcitonin in normal human plasma as assessed by immunoprecipitation and immunoextraction. The Journal of clinical investigation, 1975. 56(4): p. 835-841.