Study On The Effect And Mechanism Of CuCo₂S₄ Nanozymes Against Bacterial Infection

Background:Bacterial infection is a global health problem that seriously threatens human life.Bacterial infections are usually treated with antibiotics,but the abuse of antibiotics has caused problems with bacterial resistance,resulting in poor treatment and increased infection-related mortality.Moreover,bacteria tend to form bacterial biofilms on the surface of living or non-living bodies,making it difficult for antibacterial drugs to penetrate and fully sterilize,while incompletely removed bacteria and biofilms can cause persistent infections that are difficult to cure.Therefore,it is essential to develop antibacterial materials that have the role of antibiotics while preventing bacteria from developing resistanceThe discovery of nanomaterials with the function of mimic enzymes,that is,the discovery of nanozymes,provides a new way of thinking and methods for preventing bacterial infections.Nano-enzymes not only have the advantages of high stability,low cost,and large-scale production,but also have rich catalytic activity that mimics natural enzymes Among them,nano-enzymes that can promote the generation of reactive oxygen species have attracted much attention,and have broad application prospects in biomedical fields such as antibacterial,tumor therapy,and immunodetection.Based on the above research background,we have developed a new type of copper-cobalt-sulfur(CuCo2S4)nanozymes.At the same time,we compared the bimetallic nanozymes CuCo2S4 with CuS and CoS single-metal nanozymes.The structure,its mimic enzyme activity and catalytic antibacterial effect were evaluated in detail.Investigate the ability of CuCo2S4 nanozymes to catalyze the generation of reactive oxygen species at low concentrations of H2O2,evaluated the inhibition of the growth of various bacteria and bacterial biofilms after synergy between them,and explored their antibacterial mechanism.Finally,the mouse model of burn infections was established and the therapeutic effect against bacterial infection was evaluated.Purpose:In this paper,the construction of Dextran-modified CuCo2S4 nanozymes was synthesized by hydrothermal method.In order to better illustrate the effect of nanozymes constructed from bimetals on the activity of peroxidase,we used the same method to synthesize CuS and CoS single-metal nanozymes,and systematically studied the morphology and particle size,as well as the peroxidase mimic enzyme activity and other properties of CuCo2S4.Subsequently,the in vitro antibacterial experiment and the treatment of scald infection wounds verified the catalytic treatment effect of CuCo2S4 nanozymes,and provided a new idea and method for fighting bacterial infections.Methods:Chapter 1 Preparation and Characterization of CuCo2S4 nanozymesIn this chapter,first,CuCo2S4 nanozymes was synthesized by hydrothermal method.The morphology and size were then characterized by transmission electron microscopy,X-ray diffractometer and high-resolution transmission electron microscopy were used to analyze its crystal structure,and its elemental composition was analyzed by infrared spectroscopy,energy spectrum analysis and X-ray photoelectron spectroscopy.At the same time,we prepared CuS and CoS nanoparticles under the same conditions as a reference,observed their morphology by scanning electron microscope,and analyzed their crystal structure and elemental composition by X-ray diffractometer and X-ray photoelectron spectroscopy.Then,the peroxidase mimic enzyme activities of CuCo2S4,CuS and CoS nanozymes were tested,and their peroxidase mimic enzyme activities were compared by analyzing the parameters of the enzymatic reaction kinetics.Finally,the ability of CuCo2S4 nanozymes to promote the generation of reactive oxygen species was studied by methylene blue fading and electron paramagnetic resonance spectroscopy.Chapter 2 Antibacterial and anti-biofilm effects of CuCo2S4 nanosuspensions and evaluation of therapeutic effects on burn infectionsBased on the results of detailed analysis and comparison of the peroxidase activity of CuCo2S4,CuS and CoS nanozymes,in this chapter,we prepared CuCo2S4 nanosuspensions,and then we investigated the antibacterial properties and the bacterial infections treatment effect of CuCo2S4 nanosuspensions.Firstly,Gram-negative bacteria Escherichia coli(E.coli)and Pseudomonas aeruginosa(P.aeruginosa),Gram-positive bacteria Staphylococcus aureus(S.aureus)and Methicillin-resistant Staphylococcus aureus(MRSA)were used as the object of the bacteriostatic experiment,antibacterial property of CuCo2S4 nanosuspension was tested the in different pH environments.Secondly,we compared and analyzed the inhibitory effects of CuCo2S4,CuS and CoS nanosuspensions catalysts with H2O2 on E.coli,S.aureus and drug-resistant strain MRSA.Then the morphology,internal structure and redox level of bacteria were further analyzed and observed,and the mechanism of CuCo2S4 nanozymes catalyzed antibacterial was explored.Finally,the inhibitory effect of CuCo2S4 nanosuspensions on MRSA biofilm was investigated,and an animal model of burn infection of MRSA on mice was established.CuCo2S4 nanosuspensions and low concentration H2O2 were coated on the wound for treatment.Moreover,healing and pathological analysis of burn wounds was observed after the treatment.Results:Chapter 1:The CuCo2S4 nanozymes prepared in this chapter had a uniform size,and a particle size of about 30nm with good crystallinity.Through energy spectrum analysis,it was found that CuCo2S4 nanoparticles mainly contained elements of Cu,Co,and S,as well as C and O elements derived from dextran.The results of infrared spectroscopy proved that there was dextran on the surface of CuCo2S4 nanoparticles.XPS analysis results showed that copper and cobalt in CuCo2S4 nanoparticles coexist in a mixed valence state,which was conducive to accelerating the redox reaction of the enzyme-catalyzed process.At the same time,the CuS and CoS nanozymes prepared by the same method in this chapter are nanoparticles with a size of about 40 nm and 80 nm sheets,respectively.XRD results showed that the crystallinity of CuS and CoS nanoparticles was good.Peroxidase activity analysis results showed that the peroxidase activity of CuCo2S4 nanozymes was better than that of CuS and CoS nanozymes,indicating that the composition of copper and cobalt bimetals was beneficial to the improvement of enzyme activity.At the same time,our experimental results showed that the peroxidase activity of CuCo2S4 nanozymes in neutral pH is much higher than that in acidic environment.Finally,in the study of CuCo2S4 nanozymes promoting free radical generation,we found that CuCo2S4 nanozymes catalyzing the hydroxyl radical generated by hydrogen peroxide under neutral conditions was much stronger than in an acidic environment.Chapter 2:This chapter studied the bactericidal effect of the nanosuspensions prepared by CuCo2S4 nanozymes.First of all,CuCo2S4 nanosuspensions exhibited optimal growth inhibition effect on Gram-negative bacteria(E.coli and P.aeruginosa)and Gram-positive bacteria as well as drug-resistant strains(MRSA)in a neutral pH environment.In neutral pH environment,compared with CuS and CoS nanozymes,CuCo2S4 nanozymes showed a more significant catalytic sterilization effect,which was related to its higher efficient of peroxidase mimic enzyme activity.Subsequently,in the study of the antibacterial mechanism of CuCo2S4 nanozymes,we found that due to the increase of ROS levels in bacteria,lipid peroxidation and DNA degradation occurred,cell wall/cell membrane deformation and rupture,which eventually led to the death of bacteria.Finally,our results showed that CuCo2S4 nanozymes can significantly inhibit the growth of MRS A biofilms under neutral conditions.In the study of treating scald infections in mice,the experimental results showed that CuCo2S4 nanosuspensions could effectively promoting the healing of bacterial burn infection wounds.