胸腺五肽超分子水凝胶的制备及其免疫调节作用研究

Preparation of thymopentin supramolecular hydrogel and its application in immunomodulatory

目的制备胸腺五肽(RKDVY)超分子水凝胶,对其纳米形貌及力学性能进行表征,并研究其体外增强细胞摄取及免疫调节的性能。方法通过固相合成制备含有胸腺五肽的自组装多肽(NapG^DF^DF^DYGRKDVY),纯化目的多肽后使用高效液相色谱和液质联用色谱分别对其分子质量和纯度进行分析鉴定。通过加热-冷却的方法制备胸腺五肽超分子水凝胶,并分别利用透射电镜和流变仪对其纳米形貌及其力学性能进行表征分析。FITC标记相应多肽后,通过荧光显微镜观察RAW 264.7细胞对RKDVY及NapG^DF^DF^DYGRKDVY纳米纤维的细胞摄取行为。通过酶联免疫吸附试验测试RKDVY和NapG^DF^DF^DYGRKDVY刺激RAW 264.7细胞产生肿瘤坏死因子-α(TNF-α)的能力。结果合成的自组装RKDVY能够在加热-冷却下形成宏观可见的超分子水凝胶,该水凝胶由交联的长纤维构成并且具有良好的力学延展性。相较于游离RKDVY,NapG^DF^DF^DYGRKDVY能够更多地被RAW 264.7细胞摄取并刺激其分泌出较高含量的TNF-α。结论通过合理结构修饰后,形成纳米药物的RKDVY的细胞摄取性能和免疫调节活性显著增强,本研究能够为改善RKDVY在临床应用中的疗效提供新的方法和指导。

Objective To prepare a dlymopentin-contained supramolecular hydrogel, and characterize its micromoqohology and mechanical property, and further investigate its effects on the cellular uptake and the immunomodulatolT peerformance in vitro. Methods The self-assembling peptide containing thymopentin was prepared by solid phase synthesis method and identified using LC-MS after being purified by high performance liquid chromatography （HPLC）. Supramolecular hydrogel was prepared through a heating-cooling process, and its micromoqohology and mechanical property were characterized using transmission electron microscopy （TEM） and rheology. The cellular uptake efficiencies of free thymopentin and thymopentin nanofibers were observed by inverted fluorescence microscope after FITC-labeling. The abilities of fiee thymopentin and thymopentin nanofibers to stimulate RAW 264.7 cells to secrete tumor necrosis factor （TNF- o0 were studied by enzyme-linked immunosorbent assay （ELISA）. Results A macroscopic visible supramolecular hydrogel was obtained by a heating-cooling process, composing with long cross-linked nanofibers and possessing good ductility of mechanics. Compared with free thymopentin, thymopentin nanofibers showed much more enhanced cellular uptake, and better immunomodulation property as stimulating the RAW 264.7 cells to produce much higher concentration of TNF-o~ irk vitro. Conclusion After rational structural modification, the cellular uptake and immunomodulatory activity of thymopentin, which formed nanomedicine, were significantly enhanced. This study can provide new methods and guidance for improving the therapeutic effect of thymopentin in clinical application.