Study On The Structure And Hemostatic Performance Of Silk Fibroin Peptide

The effective, fast hemostasis for the partial injury is very important in following situations,such as the emergency treatment of sudden accident in daily life, the trauma hemostasis duringthe surgery for patients in the hospital, and the on-site rescue for injured soldiers in the war.Uncontrolled bleeding has become a threat to the wounded life and the major reason for causingdeath in the accidents, medical surgery and battlefield. Rapid and effective hemostatic materialscan dramatically decrease the mortality of the injured personnel. Therefore, the development ofrapid, safe and effective hemostatic materials has become an important research topic in the fieldof medical materials.Silk fibroin as a natural macromolecular material extracted from silk, with the characteristicssuch as non-toxic, low inflammation, good biocompatibility, biodegradability and so on, iswidely used in drug delivery, artificial skin, artificial ligaments and tissue engineering, etc.Fibroin peptide, as the hydrolyzate of silk fibroin, has been developed and applied in the field ofhousehold chemicals, food processing and bio-medicine, etc. however, the study of silk peptide’sapplication in the hemostatic materials has been rarely reported. Considering the few study abouthemostatic powder and given the hemostatic powder easy to use and store, it is very necessary toexplore the application of fibroin peptide in the hemostatic materials.In this article, we use CaCl2-ethanol-H2O(mole ration of1:2:8), H3PO4and NaOH tohydrolyze degummed silk, to prepare silk fibroin peptides with different molecular weight byspray-drying and freezing-drying, and analyze its structure and performance, apply the silkfibroin peptide to the hemostatic experiment and in vitro cytotoxicity experiment, to study anddiscuss the relationship between the physical and chemical properties of fibroin peptide and itshemostatic performance preliminarily. The main study contents are as follows:1. In the hydrolysis solution of CaCl2-ethanol-H2O(mole ration of1:2:8), with hydrolysistemperature of58℃,68℃,78℃,88℃,98℃and hydrolysis time of1h,3h,9h,24h as differentfactors to hydrolyze the degummed silk, using spray-drying and freezing-drying to prepare silkfibroin peptides. The molecular weight distribution, aggregation structure, surface morphology and amino acid composition of fibroin peptides were characterized by GPC, FTIR, XRD, SEMand amino acid composition analysis. The results showed as follows: the molecular weight ofsilk fibroin peptides decreased with the increase of hydrolysis temperature and hydrolysis time,the aggregation structure was mainly random coil with a small amount of β-sheet, the surfacemorphology was mainly spherical cystic particles with wrinkle surface, also existing a fewspherical particles, the particle size was smaller and more uniform with the decrease ofmolecular weight, the primary amino acid included serine, glycine, alanine and tyrosine, itscomposition and content has no obvious relation with the hydrolysis temperature and hydrolysistime2. In the hydrolysis solution of H3PO4, the orthogonal hydrolysis experiments were carriedout based on the three factors of concentration, hydrolysis temperature and hydrolysis time,fibroin peptide powder was prepared by freezing-drying. The same test methods were applied tothe fibroin peptide. The results showed as follows: the molecular weight decreased significantlywith the distribution of3.8~12.6kDa, the aggregation structure was random coil, exhibitingrandom pieces, the main amino acid contained glycine, alanine, glutamic acid and aspartic acid.3. In the hydrolysis solution of NaOH, the orthogonal hydrolysis experiments were carriedout based on the three factors of concentration, hydrolysis temperature and hydrolysis time,fibroin peptide powder was prepared by freezing-drying. The test methods were the same asdescribed above. The results showed as follows: the molecular weight concentrated in the3.6~18.9kDa, the aggregation structure was random coil, the surface morphology is similar tothat of fibroin peptide prepared by H3PO4hydrolysis, the main amino acid included aspartic acid,glutamic acid, alanine and tyrosine.4. The hemostatic properties and in vitro cytotoxicity of fibroin peptides prepared bydifferent hydrolysis were studied through thromboelastography (TEG), rat liver injury model andin vitro cytotoxicity experiments. The results showed as follows: the fibroin peptide prepared byCaCl2-ethanol-H2O with high molecular weight exhibited the similar hemostatic effect comparedto Arista, however, the fibroin peptide prepared by H3PO4and NaOH with low molecular weightshowed no obvious hemostatic effect. All fibroin peptides prepared by different hydrolysis hadno toxic effects on the growth and proliferation of L929mouse fibroblast cell.