Preparation Of Blue-head Fungus Polysaccharide Sulfate And Its Study On Surface Modification Of Medical Materials

In recent years,with the changes in people’s living habits and diet structure,cardiovascular diseases such as coronary heart disease and coronary atherosclerosis have become important factors affecting human health.Metal vascular stent implantation is currently the main method for the treatment of cardiovascular system diseases,but high-pressure expansion when the metal stent is released will cause vascular intimal damage,induce vascular intima and smooth muscle cell proliferation,and complement activation will lead to thrombosis and coagulation.The vascular restenosis induced by complications of flexion mismatch has affected the mid-and long-term efficacy of intra-arterial stent implantation.Biodegradable Endovascular Stents（BES）is a kind of stent made of high-molecular polymer.This kind of stent can be degraded in the blood vessels of animals and humans within a certain period of time.The products are non-toxic to the body and local blood vessels.Which be hydrolyzed in the body and discharged in the form of carbon dioxide.The stent can be restored to normal when absorbed contractility is conducive to the remodeling of blood vessels;multiple interventions can also be performed at the same lesion without the problems caused by overlapping stent.Polyhydroxyalkanoates（PHA）can be used to prepare BES,but PHA is a strongly hydrophobic material.Its surface hydrophobicity will affect the compatibility between the material and the blood,and it may easily cause adverse reactions such as inflammation and thrombus in the stent.Therefore,its surface should be modified to improve its biocompatibility.In this study,polysaccharides were extracted from the fruit bodies of Russula virescens,and sulfated to prepare Russula virescens polysaccharide sulfates.Then,layer-by-layer assembly was used to construct SRVP/CS and DA-g-SRVP/CS multilayer film was modified on its surface to prepare PHA biomaterials with good biocompatibility and biological function.The main researches are as follows:First,the polysaccharides were extracted from the fruit body of Russula virescens using the hot water extract and alcohol precipitation method.Single factor experiments,the Box-Behnken central composite design and response surface analysis of Russula virescens polysaccharide extraction conditions were optimized,the optimal extraction process parameters of Russula virescens polysaccharide:solid-liquid ratio 1:20,extraction temperature 75°C,extraction time 90 min,extraction number 2 times.The response surface analysis showed that the optimal extraction rate was 9.91%,while the verified extraction rate was 10.08%,which verified the feasibility of the model.Then the neutral Russula virescens polysaccharide（RVP）was obtained by purification.The RVP was sulfated using sulfur trioxide-pyridine complex method under different reaction conditions.Five sulfated RVP derivatives(SRVP_1-5、SRVP_1-10、SRVP_1-15、SRVP_1-20 and SRVP₁–₂₅)with different degrees of substitution（DS）ranging from 0.34 to0.73 were prepared.The preliminary results of chemical composition determination show that natural RVP is soluble in water and SRVPs is more soluble in water.Several structural features of RVP and SRVPs including chemical structure,monosaccharide composition,molecular weight and chain conformation were investigated.The spectra results indicated that the sulfate groups were successfully introduced on RVP.The molecular weights of SRVP decreased with the increasing DS.Both RVP and SRVPs were mainly composed of mannose,glucose and galactose,with different molar ratios.The triple-helical conformation was broken down into a random coil when the DS exceeded 0.34.In vitro activity test results indicated that the SRVPs showed better antioxidant,anticoagulant,antibacterial and antitumor activities compared with RVP.Among the five sulfated derivatives,SRVP_1–25exhibited the strongest ABTS and hydroxyl radical scavenging activities and anticoagulant activity,while SRVP_1–20 showed the best antibacterial activity and anti-tumor activity.These results demonstrated that the SRVPscould bedevelopedasone of potential antioxidant,anticoagulant,antibacterial and anti-tumor agents for industrial and biomedical use.SRVP_1-25 and chitosan（CS）were deposited alternately on PHA surface by LBL technique to construct SRVP/CS multilayer film.X-ray photoelectron spectroscopy（XPS）,scanning electron microscope（SEM）,water contact Angle tester（WCA）and ultraviolet visible spectrophotometer（uv-vis）were used to characterize the surface chemical composition,morphology,hydrophilic/hydrophobic properties and film growth pattern of multilayer films.XPS results showed that new S and N feature elements appeared on SRVP/CS multilayer films,and the content of S and N elements in multilayer films increased with the progress of layer self-assembly.SEM results showed that when SRVP and CS were deposited alternately on the surface of PHA film,a membranous coating appeared on the surface of PHA film,and the size of cracks gradually decreased with the increase of the number of layers of self-loading.The water contact Angle test showed that with the increase of the number of assembly layers,WCA showed zigzag alternations,and the WCA of SRVP in the outermost layer was lower than that of CS,and the hydrophilicity of the modified multilayer film was improved.SRVP/CS multilayer films are uniformly and continuously growing on the PHA film surface.Through the study of the biological activity of different sample evaluation and get the following conclusion:the blood compatibility test results show that the SRVP/CS modified multilayer membrane can effectively reduce the BSA adsorption amount,decrease the rate of platelet adhesion and activation effect,effectively prolong APTT,TT and PT clotting time,inhibit blood coagulation activation and hemolysis rate below 5%,shows that material conform to the requirements of the hemolysis test of biological materials;The modified PHA film showed excellent inhibitory effect on e.coli and s.aureus,and its inhibition rate was nearly 100%.Da-g-SRVP was obtained by dopamine（DA）treatment of SRVP_1-25,and DA-g-SRVP was fixed on PHA surface by LBL technology to obtain DA-g-SRVP/CS multilayer membrane,which improved the stability of multilayer membrane in buffer solution compared with SRVP/CS.With gentamicin（GS）as the model drug,SRVP_1-25 and CS were alternately deposited on PHA film.The GS drug loading was tracked by UV-Vis,and the maximum drug load of multilayer film was 1124.84 g/cm².The drug release behavior of multi-layer film under different conditions was studied.The drug release curve was fitted by zero-order kinetics,first-order kinetics,Higuchi equation and Riger-Peppars model.The drug-loaded multilayer film has antibacterial effect on escherichia coli and staphylococcus aureus,which is expected to be used as antibacterial material.