Pharmaceutical Sciences Research Center, Shiraz University of Medical Sciences, Shiraz, Iran.
Department of Pharmaceutical Biotechnology, School of Pharmacy, Shiraz University of Medical Sciences, Shiraz PO Box 71468-64685, Iran.
Sci Rep. 2017 Feb 10;7:42258. doi: 10.1038/srep42258.
Proteins have short half-life because of enzymatic cleavage. Here, a new protein nanocarrier made of graphene oxide (GO) + Chitosan (CS) is proposed to successfully prevent proteolysis in protein and simultaneously retain its activity. Bovine serum albumin (BSA) and collagenase were loaded on GO and GO-CS to explore the stability and activity of proteins. SEM, AFM, TEM, DSC, UV-Vis, FT-IR, RBS, Raman, SDS-PAGE and zymography were utilized as characterization techniques. The protecting role of GO and GO-CS against enzymatic cleavage was probed by protease digestion analysis on BSA, where the protease solution was introduced to GO-BSA and GO-CS-BSA at 37 °C for 0.5-1-3-6 hours. Characterizations showed the successful synthesis of few layers of GO and the coverage by CS. According to gelatin zymographic analysis, the loaded collagenase on GO and GO-CS lysed the gelatin and created non-staining bands which confirmed the activity of loaded collagenase. SDS-PAGE analysis revealed no significant change in the intact protein in the GO-BSA and GO-CS-BSA solution after 30-minute and 1-hour exposure to protease; however, free BSA was completely digested after 1 hour. After 6 hours, intact proteins were detected in GO-BSA and GO-CS-BSA solutions, while no intact protein was detected in the free BSA solution.
蛋白质由于酶切而半衰期较短。在这里，提出了一种由氧化石墨烯（GO）+壳聚糖（CS）制成的新型蛋白质纳米载体，以成功防止蛋白质的蛋白水解，并同时保持其活性。牛血清白蛋白（BSA）和胶原酶被加载到 GO 和 GO-CS 上，以探索蛋白质的稳定性和活性。SEM、AFM、TEM、DSC、UV-Vis、FT-IR、RBS、拉曼、SDS-PAGE 和胶凝电泳被用作表征技术。通过蛋白酶消化分析研究了 GO 和 GO-CS 对 BSA 酶切的保护作用，其中在 37°C 下将蛋白酶溶液引入 GO-BSA 和 GO-CS-BSA 中 0.5-1-3-6 小时。特性表明成功合成了几层 GO 并被 CS 覆盖。根据明胶胶凝电泳分析，负载在 GO 和 GO-CS 上的胶原酶裂解了明胶并产生无染色条带，这证实了负载胶原酶的活性。SDS-PAGE 分析显示，在暴露于蛋白酶 30 分钟和 1 小时后，GO-BSA 和 GO-CS-BSA 溶液中的完整蛋白没有明显变化；然而，游离 BSA 在 1 小时后完全被消化。6 小时后，在 GO-BSA 和 GO-CS-BSA 溶液中检测到完整的蛋白质，而在游离 BSA 溶液中未检测到完整的蛋白质。
