自组装多肽凝胶的构建及其在肝实质创伤性出血中的止血应用研究

Preparation of self-assembling peptide-based hydrogels and its application in traumatic hemorrhage of hepatic parenchymal

目的制备FLIVIGSII多肽(FI肽),探究其物理化学性质及其体外凝血和体内止血效应。方法将FI肽与水混合,制备样品,直接观察FI肽水凝胶的状态,通过扫描电子显微镜(scanning electron microscope,SEM)、透射电子显微镜(transmission electron microscope,TEM)观察其微观纤维结构,并通过动态光散射(dynamic light scattering,DLS)分析其纤维尺寸。将FI肽与3.8%柠檬酸钠抗凝处理后的健康人血液混合,直接观察FI肽的凝血效应,并通过SEM观察其血凝块的微观结构。溶血实验和CCK-8细胞毒性实验用于验证其生物相容性。最后,构建大鼠肝脏实质穿孔出血模型,将15只体质量为150 g的6~8周龄雌性SD大鼠按随机数字表法分为3组:对照组、FI肽组和纤维蛋白胶组,通过各组治疗后观察分析FI肽对肝脏实质创伤性出血的止血效应及其预后,并探讨其止血机理。结果成功制备FI肽。FI肽与血液接触后快速成胶,使血液凝固形成血凝块。SEM结果显示FI肽与水混合后自组装形成纤维网状水凝胶。TEM结果验证FI肽在水中成胶后形成直径为(13.70±2.31)nm的纳米纤维,DLS结果验证其在水中形成多分散多尺寸(148.2~208.0 nm或575.0~807.0 nm)的纳米纤维。FI肽与血液混合后形成的纤维网状水凝胶包裹住红细胞,从而形成物理性止血屏障以在数秒内实现凝血。同时,FI肽水凝胶对正常肝细胞(L-O2)无细胞毒性,且不会造成红细胞溶血。在SD大鼠体内肝脏止血实验中,FI肽与血液接触时,迅速形成纳米纤维水凝胶,从而形成物理性止血屏障以在数秒内实现体内止血(止血时间<5 s)。结论FI肽具有快速、高效的止血效应,在肝脏实质创伤性出血的止血治疗中具有较好的临床应用前景。

Objective To prepare FLIVIGSII peptide(FI peptide)and investigate its physicochemical properties and hemostatic effect in vivo and in vitro.Methods The self-assembling peptide-based hydrogels were prepared by the FI peptide mixed with water.After gross observation for the hydrogel state of the FI peptide,scanning electron microscopy(SEM)and transmission electron microscopy(TEM)were used for its microstructure,and dynamic light scattering(DLS)was performed for its size.The hemostatic effect of FI peptide after being mixed with blood samples treated with 3.8%sodium citrate was observed,and the microstructure of the blood clot was observed with SEM.CCK-8 assay and hemolysis assay were performed to verify its biocompatibility.After a rat model of hepatic parenchymal perforation and hemorrhage was established,15 female SD rats(6~8 weeks old,weighing 150 g)were randomly divided into control group,FI peptide group and fibrin sealant group.The hemostatic effect of FI peptide and prognosis was observed and analyzed after treatment in each group,and the hemostatic mechanism was also investigated.Results FI peptides were successfully prepared,and it could rapidly self-assemble into a nanofiber network hydrogel in water,and further cause formation of blood clots.SEM showed that FI peptides self-assembled to form fibrous hydrogels after mixing with water.TEM results verified that the FI peptide formed into nanofibers in a diameter of 13.70±2.31 nm after gelatinization in water,and DLS results verified that the FI peptide formed polydisperse and multi-size nanofibers in water(in a range of 148.2~208.0 nm or 575.0~807.0 nm).The fibrous hydrogel formed by the FI peptide mixed with the blood could envelop the red blood cells,thus form a physical hemostatic barrier to achieve blood clotting in seconds.FI peptide hydrogel had no cytotoxicity to normal hepatocytes(L-O2 cells)and did not cause hemolysis of red blood cells.In in vivo experiment,FI peptide quickly formed nanofiber hydrogel when in contact with blood,thus formed physical hemostasis barrier to achieve hemostasis within a few seconds(hemostasis time<5 s).Conclusion The FI peptide exhibits a rapid and efficient hemostatic effect,indicating a promising clinical application in the hemostasia of hepatic parenchymal traumatic hemorrhage.