Study On Protein Functionalization And Construction Of Anticoagulant Antibacterial Surface Based On Protein Crosslinking

Blood-contacting medical devices,as exogenous allogeneic devices,the interaction between their materials and blood will cause complex changes in blood components and material surfaces,protein adsorption,platelet activation and adhesion,endogenous,exogenous and common coagulation pathways and complement activation,and ultimately induce abnormal coagulation and thrombosis in the body.In addition,as an important carrier of microbial pathogens,medical devices may cause bacterial colonies to colonize the surface of blood-contacting materials during use,causing more serious local and systemic infections and other adverse events.Thrombosis and infection events restrict the application scenarios and service time of blood-contact medical devices,and have become an important reason for threatening the life safety of patients,reducing medical quality and increasing medical costs.Clinically,anticoagulation and antibiotics are usually given systemically for treatment.However,the side effects caused by systemic administration,such as thrombocytopenia and coagulation dysfunction,increase the risk of surgical bleeding,and the resistance caused by antibiotics.Allergic reactions,toxic and side effects are also restricting its application value.Therefore,the construction of a simple,efficient,non-toxic,sdrug-resistant anticoagulant and antibacterial coating technology is an important research direction to effectively reduce medical device thrombosis and infection events,and has important clinical significance and application prospects.Based on this,this research is committed to developing a blood-contact material coating technology with good blood compatibility and effectively reducing the occurrence of thrombosis and bacterial infection,which not only has clinical application prospects,but also provides anticoagulant and antibacterial surface coatings for medical devices.In this study,a novel anticoagulant antimicrobial protein coating was developed through protein functionalization and protein cross-linking.Firstly,the modified protein molecule(Cu^Ⅱ-DOTA@LAM)was synthesized by grafting macrocyclic polyamine metal chelate(Cu^Ⅱ-DOTA)on the lysozyme（LZM）molecule by carbodiimide chemistry,and then the modified protein molecule(Cu^Ⅱ-DOTA@LAM)was synthesized by oxidizing（sodium persulfate）.A novel protein coating with anticoagulant and antibacterial function was formed by cross-linking and assembling on the surface of the base material silicone rubber.The modified coating catalyzes the endogenous nitric oxide donor RSNO to generate NO through the in situ immobilized Cu ions of macrocyclic polyamines to prevent platelet adhesion and activation,and combined with the bactericidal activity of lysozyme,the coating has anticoagulant and antibacterial functions.A series of material characterization results proved that lysozyme modified molecules DOTA@LAM and Cu^Ⅱ-DOTA@LAM synthesis successfully.The basic properties and surface chemical composition of the coating after oxidative crosslinking were characterized,and the film-forming mechanism was explored.The results showed that the disulfide bond of the protein primary structure of the molecule is oxidized to sulfonic acid group.The sulfonic acid group repels the acidic amino acid in the peptide chain with the same charge,which destroys the formation of hydrogen bond in the chain and leads to the secondary structureα-helix intoβ-sheet and irregular curling.The destruction of the charge balance on the protein surface makes it more inclined to aggregate.Under the action of static electricity,the protein is crosslinked and gradually formed into nano particles,which are deposited under the action of static electricity and gravity to form a coating,and the coating has good binding ability with the material substrate.The results of the in vitro anticoagulation performance evaluation showed that the Cu^Ⅱ-DOTA@LAM coating has the ability to catalyze the release of NO continuously and stably,and platelets have c GMP response behavior to the NO catalytically released,which can effectively inhibit platelet adhesion and activation.The antibacterial experiments show that the Cu^Ⅱ-DOTA@LAM coating has excellent bactericidal properties,which can effectively kill gram-positive and gram-negative bacteria represented by coli and epidermidis.The biocompatibility of medical device materials is very important.We systematically demonstrated the good in vitro biocompatibility of the Cu^Ⅱ-DOTA@LZM coating through various in vitro experimental systems such as hemolysis rate,cytotoxicity,and in vitro coagulation and inflammatory factor detection.However,blood as a complex system,cannot characterize the anticoagulant properties and biocompatibility of materials by controlling a single factor.Therefore,the Cu^Ⅱ-DOTA@LZM coating has a good practical anticoagulant behavior verified by in vivo blood circulation experiments,but after immersion in simulated body fluids for 30 days,the anticoagulant ability of the Cu^Ⅱ-DOTA@LZM coated catheter was reduced under the dynamic blood circulation conditions in vivo.However,the Cu^Ⅱ-DOTA@LZM coating with large area of blood contact will not cause significant adverse effects on blood coagulation,inflammatory response and liver and kidney function indicators in a short period of time under the condition of dynamic elution of blood circulation.The above results show that the Cu^Ⅱ-DOTA@LZM coating has good anticoagulation,antibacterial stability and biocompatibility.The functionalization of proteins through grafting modification and other means to endow them with new functions,and then oxidatively cross-linked by oxidizing agents on the surface of modified substrates to form a multifunctional protein coating has broad application prospects,and also provides a new method for surface functional modification of materials.