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

Abstract

人類降鈣素(Human calcitonin, hCT)是由32個胺基酸組成的胜肽荷爾蒙，透過甲狀腺周圍的濾泡旁細胞所分泌。主要的生理功能為維持血液中鈣離子濃度以及骨骼結構之形成，可以用於治療高血鈣症、佩吉特氏病以及骨質疏鬆症等疾病。然而，水溶液中的人類降鈣素具形成類澱粉蛋白纖維之傾向，可能會使其生理功能受到影響並限制其作為胜肽藥物之發展。中孔洞氧化矽奈米粒子有高單位表面積、易官能化修飾、低細胞毒性和高生物相容性等優點，使其在生物醫學相關領域發展相當的廣泛。本實驗室先前研究中，粒徑約1 nm修飾有鄰苯二酚結構的奈米碳點材料成功地抑制人類降鈣素形成類澱粉蛋白纖維，本論文則使用粒徑約45 nm的中孔洞氧化矽奈米粒子亦修飾同樣的鄰苯二酚結構，來探討粒子大小對於抑制人類降鈣素聚集的差異。 另外，有文獻指出人類降鈣素中15-19號序列DFNKF為誘發人類降鈣素聚集的關鍵序列，本實驗想透過五胜肽DFNKF來影響全長的人類降鈣素聚集。同時，由於短胜肽在體內容易發生降解作用，我們亦將五胜肽DFNKF修飾於中孔洞氧化矽奈米粒子(MSN-DFNKF)與人類降鈣素共培養，運用硫磺素-T動力學分析與穿透式電子顯微鏡來觀察纖維數量及纖維形貌的改變。Human calcitonin (hCT) is a 32- residue hormone polypeptide secreted from the C-cells of the thyroid gland. The main physiological function of hCT is to maintain the concentration of calcium ions in the blood and bone structure, so it can be used to treat diseases such as hypercalcemia, Paget's disease, and osteoporosis. However, human calcitonin highly tends to form amyloid fibrils in aqueous solution resulting in loss of physiological function and limiting development of peptide drugs. Mesoporous silica nanoparticles have been widely developed in biomedical related fields, due to high unit surface area, easy functional modification, low cytotoxicity and high biocompatibility. Previously, catechol moiety conjugated carbon dots with diameter of about 1 nm have been shown to successfully inhibit amyloid formation of human calcitonin. In this study, we modified mesoporous silica nanoparticles about 45 nm in diameter with similar catechol moiety (MSN-DOPA) and test whether particle size is an important matter in affecting human calcitonin aggregation.Before, residue 15-19 DFNKF of human calcitonin was found to be the key sequence that induced human calcitonin aggregatioin. In this study, we also prepared pentapeptide DFNKF. Since short peptides are prone to be degraded in the body, we also modified the surface of mesoporous silica nanoparticles with pentapeptide DFNKF (MSN-DFNKF). And then, we investigated the effects of DFNKF and MSN-DFNKF on the aggregation of hCT through Thioflavin-T kinetic assay and transmission electron microscopy.