Preparation Of Glycopeptide Hydrogel Based On Schiff Base Bond And Its Biological Application

As biomimetic biomaterials,hydrogels are non-toxic,harmless,biocompatible and biodegradable.They can be used in tissue engineering,drug delivery and wound hemostasis.Glycopeptide,as a precursor of glycoprotein,has good biological stability and cell compatibility.It is often used as the construction unit of new materials.Glycopeptide hydrogels prepared by traditional chemical methods are complex.The glycopeptide hydrogel prepared by the method has greater cytotoxicity.Based on this,we use carboxymethyl chitosan as the building unit of glycopeptide hydrogel,it is a polysaccharide derivative with good water solubility,which preserves the biological activity of the precursor material chitosan,and its structure is easy to be modified.It is often used to study the modification of polysaccharides.Schiff base reaction is a typical reaction in click chemistry.It has the advantages of simple reaction conditions,high efficiency,by product only water and easy operation.Many experts and scholars have constructed glycosyl hydrogels through Schiff base reaction,and applied them to hemostasis materials,cell culture and drug carriers.In this paper,the oxidation of carboxymethyl chitosan was oxidized by sodium periodate.The dialdehyde carboxymethyl chitosan was successfully prepared.It was reacted with peptide Ac-I₃QGK-NH₂ through Schiff base bond.By varying the concentration of polypeptide Ac-I₃QGK-NH₂,glycopeptide hydrogel with different mechanical strength was prepared.The glycopeptide hydrogel was proved to support osteoblast growth and differentiation.At the same time,it has good hemostatic performance.The main research contents are as follows:（1）Using strong oxidant sodium periodate to oxidize carboxymethyl chitosan,by changing the mass ratio of sodium periodate and carboxymethyl chitosan,three kinds of dialdehyde carboxymethyl chitosan with different oxidation degree were obtained.The successful synthesis of dialdehyde carboxymethyl chitosan was proved by element analyzer,infrared spectroscopy and atomic force microscope.Glycopeptide hydrogel was synthesized by crosslinking of dialdehyde carboxymethyl chitosan with peptide Ac-I₃QGK-NH₂ through Schiff base reaction.The successful synthesis of glycopeptide hydrogel was demonstrated by infrared spectroscopy,and the morphology of glycopeptide hydrogel was observed by atomic force microscopy.（2）Glycopeptide hydrogels were obtained by the reaction of dialdehyde carboxymethyl chitosan and peptide Ac-I₃QGK-NH₂ through Schiff base.The glycopeptide hydrogel was used as the substrate of cell culture to investigate the effect of glycopeptide hydrogel on the biocompatibility and cell differentiation of osteoblast MC-3T3.The results showed that glycopeptide hydrogel could induce osteoblast differentiation.Under low mechanical strength,the cells spread on the surface of glycopeptide hydrogel,but the spreading area was small.At high concentration of mechanical strength,the spreading area of osteoblasts increased,and its degree of differentiation increased with the increase of mechanical strength,which had a certain threshold.When the mechanical strength is about 3 k Pa,the differentiation degree of osteoblasts is the highest.（3）Glycopeptide hydrogels were obtained by the reaction of dialdehyde carboxymethyl chitosan and peptide Ac-I₃QGK-NH₂ through Schiff base.It was proved by experiments that when the concentration of dialdehyde carboxymethyl chitosan was 3 mg/m L,its hemostatic property was better.The concentration of immobilized dialdehyde carboxymethyl chitosan was3 mg/m L.By changing the ratio of carboxymethyl chitosan to polypeptide Ac-I₃QGK-NH₂,glycopeptide hydrogel with different mechanical strength was obtained.When the concentration of Ac-I₃QGK-NH₂ was 4 m M and 8 m M,the time of stopping bleeding was less than 10 s.It is proved by experiments that the hemolysis rate of glycopeptide hydrogel and its raw materials is less than 5%,which accords with the application standard of medical biomaterials.It shows that it has good blood compatibility.Calcein-AM/PI experiments show that it has no cytotoxicity to normal cell NIH-3T3 cells,and glycopeptide hydrogel has good cytocompatibility.Through the rheological characterization of glycopeptide hydrogel and blood hydrogel,and platelet adsorption experiments,glycopeptide hydrogel can prevent blood flow through forming a certain mechanical strength hydrogel,and glycopeptide hydrogel can adsorb and activate platelets,so as to achieve the effect of coagulation.Above all,glycopeptide hydrogel based on Schiff base reaction has good cytocompatibility and can be used to support osteoblast MC-3T3 cell differentiation.It has good blood compatibility and hemostatic properties.It can be used as a good tissue engineering material and hemostasis material.It provides a new idea for preparation of glycopeptide hydrogel through sugar modification and click chemistry reaction in the future.