Nanozyme Single-atom Fe Combat Multidrug-resistant Bacterial Infections

Objective:The abuse of antibiotics leads to the emergence of many drug-resistant strains,which seriously affects human’s life and health.Nanozymes have been considered as new antibacterial agents because of its wide antibacterial spectrum,low toxicity and low possibility of inducing multidrug resistance.In this study,we aimed to synthesize nanozymes iron single-atom catalysts（Fe-SACs）and investigated their catalytic activities and photothermal properties.The surface of Fe-SACs were later coated with CCS-PBA and their antibacterial activities against Methicillin-resistant Staphylococcus aureus（MRSA）were explored in vitro and in vivo.Methods:Fe-ZIF8 was firstly prepared by one-pot synthesis method and then Si Ox was coated on the surface of Fe-ZIF8.Single-atom dispersed Fe-SACs were obtained after calcinating and etching.The morphology,particle size and composition of Fe-SACs were characterized by transmission electron microscopy（TEM）,fourier transform infrared spectroscopy（FTIR）,high-angle annular dark-field scanning transmission electron microscopy（HAADF-STEM）,X-ray diffraction（XRD）and X-ray photoelectron spectroscopy（XPS）.The valence and coordination environments of Fe-SACs were obtained from X-ray absorption near edge structures（XANES）spectrum and extended X-ray absorption of fine structures（EXAFS）spectrum.In the study of catalytic and photothermal performance of Fe-SACs,the O₂production capacity of H₂O₂ catalyzed by Fe-SACs was studied by dissolved oxygen instrument.3,3’,5,5’-tetramethylbenzidine（TMB）,dihydroethidium（DHE）,o-phenylenediamine（OPD）and methylene blue（MB）were used as indicators to study the production of reactive oxygen species（ROS）via the enzyme-like activities of Fe-SACs.The photothermal conversion efficiency（PCE）,heating capacity and photothermal stability of Fe-SACs were investigated using near-infrared（NIR）laser irradiation at 808 nm as light source.In order to enhance the water-solubility of Fe-SACs and improve the antibacterial performance,CCS-PBA was selected to coated on the surface of Fe-SACs.FTIR,NMR（nuclear magnetic resonance）and Zeta potential analyzer were used to verify whether the modification was successful.The antibacterial properties of Fe-SACs@CCS-PBA were investigated against two strains of gram-negative bacteria and two strains of gram-positive bacteria.Considering the current situation of bacterial drug resistance,MRSA was further taken as the object to investigate the catalytic antibacterial,photothermal antibacterial and synergistic antibacterial properties of Fe-SACs@CCS-PBA.Their antibacterial mechanism was revealed as well.The antibacterial activity of Fe-SACs@CCS-PBA in vivo was studied against MRSA infected mouse models.The body weight and wound area of each group were recorded during treatment for comprehensive evaluating the wound healing effect and biosafety of Fe-SACs@CCS-PBA in vivo.Results:The porous Fe-SACs with particle size of～100 nm were successfully synthesized.Fe-SACs was composed of C,N,O and Fe,with a specific surface area of844.86 m²g^-1 and an average pore size of 3.808 nm.The content of Fe was 0.3054 wt%.Fe-SACs contained pyridinic nitrogen,pyrrolic nitrogen,graphitic nitrogen and oxidized nitrogen,among which the content of pyridine nitrogen was the highest,providing abundant ligand sites with Fe.XANES and EXAFS spectra showed that the valence of Fe in Fe-SACs was between 0～+3 and the coordination structure was Fe-N₄.Fe-SACs not only had excellent enzyme-like activity,but also showed excellent photothermal properties.Fe-SACs could catalyze H₂O₂ to produce O₂,exhibiting excellent catalase-like activity.Fe-SACs could catalyze O₂ to produce O₂^·-and change the color and fluorescence intensity of TMB and DHE indicators,indicating the outstanding oxidase-like activity.Fe-SACs could also catalyze H₂O₂ to produce·OH and significantly change the color of the indicators（TMB,OPD and MB）,revealing the excellent peroxidase-like activity.Fe-SACs exerted superb photothermal performance as well.With the NIR irradiation,the temperature climbed rapidly to 85℃,and the PCE was determined to be 25.60%.The results of FTIR,NMR and Zeta potential confirmed that Fe-SACs@CCS-PBA were successfully prepared.Fe-SACs@CCS-PBA showed anti-bactericidal effects on both gram-negative bacteria（Escherichia coli and Multidrug-resistant Acinetobacter baumannii）and gram-positive bacteria（Staphylococcus aureus and MRSA）.Taking consideration of the current situation of bacterial drug resistance,MRSA was taken as the object to further explore the antibacterial mechanism.It was concluded that the Fe-SACs@CCS-PBA caused damage to bacterial cell wall or membrane,resulting in the leakage of proteins and ds DNA,and effective eliminated biofilm,thus exhibiting excellent anti-MRSA activities.Systemical in vivo experiments indicated that the synergistic catalytic therapy and photothermal therapy of Fe-SACs@CCS-PBA significantly accelerated the wound healing process with the wound area decreased to 22.11%at the 8^th day,which was significantly higher than that of the control group,indicating that Fe-SACs@CCS-PBA had excellent antibacterial activity in vivo.Furthermore,Fe-SACs@CCS-PBA did not cause damage to liver and kidney of the mice,revealing the good biosafety of Fe-SACs@CCS-PBA.Conclusion:Fe-SACs were successfully prepared and exhibited outstanding enzyme-like activities,which could catalyze H₂O₂ and O₂ to produce ROS.Moreover,Fe-SACs exerted excellent photothermal properties,which accelerated the catalytic process while elevating the systemic temperature.In vitro and in vivo studies demostrated that the synergistic catalytic therapy and photothermal therapy of Fe-SACs@CCS-PBA could effectively kill MRSA bacteria and accelerate wound healing of MRSA bacteria-infected mice without causing damage to liver and kidney function.The present work indicated that nanozymes single-atom catalysts were of great potential in overcoming infectious diseases caused by drug-resistant bacteria.