分子表面修飾的氧化鐵奈米粒子用於穩定人類降鈣素

Abstract

類澱粉蛋白纖維的形成在類澱粉蛋白變性中是重要的關鍵。此外，一些多肽類賀爾蒙，如人類降鈣素 (human calcitonin, hCT) ，因具有調節血鈣水平的作用而聞名，但由於hCT具高度傾向形成類澱粉蛋白纖維，因此作為藥物的潛力有限。奈米材料在各個科學領域具有許多優勢，通常具有獨特的物理及化學性質，如體積小、表面積大和與其他物質反應時活性極高。最近，研究指出氧化鐵奈米粒子經過適當表面修飾能夠抑制類澱粉蛋白纖維的形成。在這項研究中，我們發現通過不同方法合成的 Fe3O4@Chl/Fe (Fe-C) 、Fe3O4@Chl/Cu (Fe-CCu) 和 Fe3O4@hematoxylin (Fe-SU) 會影響hCT的成核和聚集過程。為了獲取雙重的驗證，我們使用兩種放光區域不同的苯並噻唑螢光探針來監測 hCT 類澱粉蛋白纖維的形成，以避免氧化鐵奈米粒子自身光學特性潛在的干擾，並使用電子顯微鏡觀察樣品終點樣貌。額外使用動態光散射粒徑分析儀輔助監測hCT纖維的形成 。由凝膠電泳來佐證出終點樣品的單體含量，和使用4,4'-二苯胺基-1,1'-聯二萘-5,5'-二磺酸與尼羅紅觀測共培育後的纖維含量。我們證實Fe-C為效果最佳的奈米粒子用作抑制hCT類澱粉蛋白纖維的產生，可使hCT更加穩定，經共培養後依然保留最多蛋白質單體，得以與細胞膜上的受體結合，增加其生物活性。此外，我們發現Fe-C以及Fe-CCu能夠降解預先形成的hCT纖維。我們的目標是希望在 hCT 製劑中添加一種新穎材料，以增加 hCT 作為藥物活性成分的機會。

Accumulation of amyloid fibrils plays an important role in the development of amyloidosis. Besides, some hormone peptide such as human calcitonin (hCT) known for its effect in regulating blood calcium levels has limited pharmaceutical potential due to a high tendency to form amyloid.Nanotechnology offers many advantages in various fields of science, they usually have unique physicochemical properties such as small size, large surface area, and high reactivity. Recently, research has emphasized that appropriate surface modifications of nanoparticles can inhibit amyloid fibril formation. In this study, we found that Fe3O4@Chl/Fe (Fe-C), Fe3O4@Chl/Cu (Fe-CCu), and Fe3O4@hematoxylin (Fe-SU) synthesized via different approaches influence the nucleation and aggregation process of peptides. To obtain unbiased data, we used two different benzothiazole-based fluorescent probes to monitor hCT amyloid formation to avoid potential interference from the optical properties of nanoparticles. The appearance of hCT samples from the ThT endpoint was observed by transmission electron microscopy. Besides, the time course size distribution of hCT and nanoparticles mixture was monitored by dynamic light scattering. The monomer content from the ThT end product was checked by gel electrophoresis, and the fiber content of the ThT end product was measured using bis-ANS and nile red assay. The intracellular activated cAMP level was measured to confirm that Fe-C can inhibit hCT amyloid formation and major content with monomer type has larger remained. In addition, we found that Fe-C and Fe-CCu can dissociate pre-formed hCT fibrils. Our ultimate goal is to apply new material in hCT formulation to increase the opportunity of hCT to serve as active pharmaceutical ingredients in the medicine.