Construction And Evaluation Of Antibacterial Surface Based On Immobilized Lysozyme

Ventilator-associated pneumonia(VAP)is hospital-acquired pneumonia in patients 48hours after mechanical ventilation via endotracheal tube(ETT)or tracheostomy tube.VAP has a high incidence in intensive care units,leading to serious complications,and is an important factor threatening the lives of critically ill patients.Therefore,the development of a mechanical ventilation circuit with bacteriostatic ability is of great significance for the prevention of VAP.In this study,based on the antibacterial properties of lysozyme,it was applied to the construction of bacteriostatic functional surfaces to inhibit the formation of biofilms on ventilator pipeline materials.The specific research idea is based on 316 L stainless steel and polyethylene,which are commonly used pipeline materials for ventilators.The study proposes and establishes stainless steel surface,which is enriched first and then photocrosslinked to couple lysozyme and a protein immobilization strategy on polyethylene surface with plasma activation first and lysozyme adsorption later.Two strategies have significant antibacterial effects and can inhibit biofilm formation.The specific conclusions are as follows:(1)Lysozyme was successfully coupled to the tyrosine functional interface by a two-step immobilization strategy of enrichment-photocrosslinking.The optimal reaction conditions were as follows:p H 6.5;lysozyme:APS:Ru catalyst=40:20:1;photocrosslinking reaction for 5 min.Contact angle analysis,surface zeta potential,FITC-labeled lysozyme fluorescence and elemental analysis further proved that the construction of functional surface was in line with expectations.The antibacterial rate of the functionalized stainless steel surface against S.aureus can reach 99.2%.It was proved that the functional surface has significant antibacterial effect by living and dead bacteria staining and electron microscope observation on the number of bacteria on the surface of the material.The persistence of the antibacterial activity of the functional interface was verified using E.coli.The results show that after 50 days at room temperature,it still has a bacteriostatic rate of more than 70%.(2)By treating the polyethylene surface with oxygen plasma,the activation of carboxyl groups on the surface of the material was successfully achieved,and the lysozyme could be effectively adsorbed by electrostatic action.When the plasma treatment time was 180 s,the highest bacteriostatic rate against E.coli was 96.9%.Contact angle analysis,infrared spectroscopy analysis,fluorescence of FITC-labeled lysozyme,surface topography analysis,and elemental analysis further demonstrated that the functional surface construction was as expected.The density of lysozyme modification on the surface of functionalized polyethylene was determined up to 0.32 nmol/cm~2by a highly sensitive colorimetric assay with sulfo-SDTB.Studies have shown that the system has long-lasting antibacterial activity and can delay biofilm colonization at an early stage,and has a significant inhibitory effect on biofilm formation.The technical strategies established in this study based on photocrosslinking and plasma treatment are easy to operate,simple and fast,and can quickly modify the ventilator pipeline components with lysozyme,which is expected to improve its antibacterial effect during use.It has potential application value in VAP prevention and treatment.