二硫键交联的巯基化壳聚糖水凝胶对蛋白质还原响应的控制释放

Synthesis of Thiolated Chitosan Hydrogel for Redox-responsive Controlled Release of Protein

摘要:

合成了接枝N-乙酰-L-半胱氨酸（NAC）的巯基化壳聚糖（CS-NAC），通过1HNMR和FTIR表征了CS-NAC的结构，通过Ellman法测定其巯基含量为275.8 μmol/g。于CS-NAC的磷酸盐缓冲溶液（PBS）中氧化交联巯基形成二硫键制备了CS-NAC水凝胶。结果表明：当CS-NAC的质量分数为1.5%~3.0%时可以形成稳定的凝胶结构，当CS-NAC质量分数从1.5%增加至3.0%时，水凝胶溶胀比从45.5降低至31.2，网孔尺寸从340nm减小至242nm。该二硫键交联的水凝胶可发生还原响应的降解，在含10mmol/L二硫苏糖醇（DTT）的PBS溶液中400min内质量降解80%，在含50mmol/L L-半胱氨酸（L-Cys）的PBS溶液中180min可被完全降解。原位装载牛血清蛋白（BSA）的CS-NAC水凝胶可实现对BSA还原响应的控制释放，在含10mmol/L DTT的PBS溶液中BSA累积释放量可高达80%，表现出典型的Fickian扩散控制释放行为。

Abstract:

The thiolated chitosan (CS-NAC) was synthesized by grafting N-acetyl-L-cysteine (NAC) onto chitosan chain and the structure was characterized by 1HNMR and FTIR. The content of thiol group in CS-NAC was determined to be 275.8μmol/g by Ellman’s method. The disulfide bond crosslinked CS-NAC hydrogel was prepared by oxidation coupling of thiol with oxygen. The stable hydrogel was obtained in the CS-NAC concentration range of 1.5wt% to 3.0wt%. The swelling ratio of CS-NAC hydrogel decreased from 45.5 to 31.2 and the mesh size (ξ) diminished from 340nm to 242nm with increasing the concentration of CS-NAC from 1.5wt% to 3.0wt%. The redox-responsive degradation of CS-NCA hydrogel was observed. About 80% hydrogel was degraded in 10mmol/L dithiothreitol (DTT) solution within 400min, and complete degradation was observed in 50mmol/L L-cysteine (L-Cys) solution. Bovine serum albumin (BSA) was used as model protein and encapsulated into the CS-NAC hydrogel during the oxidation crosslinking reaction. Redox-responsive release of BSA in vitro from CS-NAC hydrogel was obtained. More than 80% of encapsulated BSA was released from the hydrogel in 10mmol/L DTT solution within 30 h, while only 58% of BSA was released from the hydrogels in PBS. The release kinetics of BSA in 10mmol/L DTT solution is closed to first order and the encapsulated protein was released via a typical Fickian diffusion-controlled form from the CS-NAC hydrogel.