- 學位論文
人髮角蛋白的萃取及其止血和抗菌材料上之應用
Extraction of Human Hair Keratin Proteins and Their Hemostatic and Antibacterial Applications
摘要
人髮角蛋白因其高生物相容性、非免疫原性與生物降解性，而被廣泛應用於生物材料，包括藥物遞送、組織工程、傷口癒合和促進細胞生長及分化。我們使用兩步驟還原萃取法從人髮中萃取出角蛋白相關蛋白(KAPs)和角蛋白並詳細分析，而分離出的KAPs和角蛋白均可利用它們本身不同的理化性質以促進生醫應用。傷口不受控的出血與感染被認為是創傷後造成生命威脅的兩個主要原因。然而，開發具有高止血能力及有效抗菌功效的安全傷口敷料仍然是一個嚴峻的挑戰。在先前的研究中，已發現角蛋白本身具有促進血小板活化和纖維蛋白聚合的能力。第一部分，我們探討KAPs材料作為止血劑的應用，因為之前的研究沒有報導其止血性能。KAPs奈米粒子通過去溶劑化法合成，這些粒子在顯微外觀下呈現球形型態且平均流體動力學直徑約為170 nm。從體外止血實驗結果表示，使用濃度為50 mg/mL KAPs奈米粒子與新鮮的血液混合處理後，所需的凝血時間可從原先16分鐘減少到4分鐘內。溶血試驗的數據證明KAPs奈米粒子具有低溶血百分比。綜合上述結果表示KAPs奈米粒子在未來臨床止血應用上的實踐有很高的機會。第二部分，我們構思了一種具有良好生物降解性、生物相容性、止血能力和抗菌功能的新型止血材料，以解決普通止血材料的缺點。通過冷凍凝膠法製備了亞甲基藍摻雜的角蛋白/藻酸鹽複合支架，此複合支架具有超過1600％的液體吸收率、互相連通性的孔洞結構及良好的生物相容性及生物降解性。體外藥物釋放實驗指出複合支架能夠透過強大的液體吸收性能確保初期高爆發釋放，從而在傷口癒合的初始階段防止感染。通過體外抗菌光動力測試獲得的結果指出，亞甲基藍摻雜的複合支架可以觸發抗菌光動力作用來防止菌落快速生長。
並列摘要
Human hair keratin had been widely used in biomaterials including drug delivery, tissue engineering, wound healing and promotion of cell growth and differentiation, because of their high biocompatibility, non-immunogenicity, inherent biodegradability. We used the two-step reductive extraction method to analyze the keratin and keratin-associated proteins (KAPs) extracted from human hair in detail. The separation of keratin and KAPs could take advantage of their different physicochemical properties and facilitate biomedical applications. These results provided new ideas for the extraction of keratin and KAPs from human hair and had an impact on its use as a biomaterial. Uncontrollable hemorrhage and infection had been considered two of the major reasons leading to life threats caused by traumas. However, the development of safe materials with high hemostatic and effective antibacterial effectiveness remained a strait challenge. The keratin-based biomaterials had been reported to exhibit the functions of enhancing platelet binding and activation as well as facilitating fibrinogen polymerization. First part, we investigated the application of KAPs material as a hemostatic agent, because previous studies had not reported its hemostatic performance. The KAPs nanoparticles were synthesized by the desolvation method. The prepared KAPs nanoparticles demonstrated a spherical morphology and had an average hydrodynamic diameter of about 170 nm. In vitro hemostasis experiments had showed that the clotting time decreased from 16 min to 4 min after treatment with KAPs nanoparticles at a concentration of 50 mg/mL. The results from hemolysis tests showed that KAPs nanoparticles had a low hemolysis percentage. The above results indicated that KAPs nanoparticles had a high chance in clinical hemostasis application practice. The second part, we conceived a novel hemostatic material with good biodegradability, biocompatibility, hemostatic ability and antibacterial function to solve the shortcomings of common hemostatic materials. The methylene blue loaded keratin/alginate composite scaffolds were prepared by freeze-gelation method. The composite scaffolds exhibited over 1600% liquid absorption, well interconnected pores, good biocompatibility and biodegradability. The drug-loaded scaffolds could achieve high burst release through the absorption of wound exudate in the initial stage of wound healing to prevent infection. The results obtained by the antimicrobial photoinactivation assay in vitro suggested that the antimicrobial photodynamic effect could be triggered, thereby preventing colonies fast growth.