Research On Antibacterial Mechanism Of Gold Nanoclusters And Its Applications

Objective To explore the antibacterial mechanism of p MBA-Au NCs,the efficacy of Au NCs-Cu²⁺complex against drug-resistant bacteria and the preventive and therapeutic ability of Au NCs-Cu²⁺-M⁴⁺coating on implant-related infections.Methods SEM,TEM,AFM,UV absorption spectra,grazing incidence X-ray diffraction and synchrotron radiation grazing incidence wide-angle X-ray scattering and X-ray photoelectron spectroscopy were used to determine the physical and chemical properties of the synthesized materials.Dilution coating method,minimum inhibitory concentration（MIC）,bacterial growth curve and Live/Dead staining were used to explore the antibacterial effect of the materials.TEM,HAADF-STEM,EDS,NPN uptake test and membrane potential detection were used to explore the antibacterial mechanism of the materials.H&E,Geimsa,Masson,CD31 immunohistochemistry and immunofluorescence staining were used to evaluate the histological effect of the material in vivo,and CCK-8,hematological index and H&E staining of important organs were used to evaluate the safety of the material.Results The size of p MBA-Au NCs was between 1-2nm and its antibacterial rate towards ESBL E.coli was much higher than MASA（88 vs 48%,P<0.05）.After protonation,the antibacterial rate towards ESBL E.coli and MASA was increased（88%vs 99%,48%vs 80%,P<0.05）.TEM showed destruction of bacterial cell membrane and vacuole of Escherichia coli cytoplasmic efflux;HAADF-STEM and EDG observed that gold was distributed on the surface and interior of bacteria;NPN staining showed that gold clusters caused the increase of bacterial outer membrane permeability,and Di SC3（5）staining showed that the cytoplasmic membrane potential changed.A low amount of copper ion was introduced into the p MBA system to form Au NCs@Cu by self-assembly.The in vitro antibacterial rate was significantly higher than that of p MBA-Au NCs purely treated（MRSA48%vs 99.5%and ESBL E.coli 88%vs 99.9%,P<0.05）;at the same time,no toxic reaction was found in CCK-8 toxic test on MIC concentration.At9 h after scratch test,the migration rate of Au NCs@Cu treatment group was higher than that of blank group（51.6%vs 21.6%,P<0.05）.In vivo,the wound defect healing rate of Au NCs@Cu group was higher than that of blank group（93%vs 58%,P<0.05）.Immunohistochemistry showed that local inflammation,bacterial residue decreased,collagen thickening and the number of angiogenesis increased,blood routine indexes and H&E staining of important organs were not abnormal.On the basis of Au NCs@Cu,high valence ion(M⁴⁺)was introduced to make the coating more stable.The anti-adhesion rate in vitro was more than 3 orders of magnitude.The animal model of subcutaneous implant infection was established in vivo,and the coating could significantly reduce the bacterial adhesion on the surface of the coating,reduce the local bacterial residue and improve the local inflammation.The vital organs Hype indicates the safety of the body.Conclusion p MBA-Au NCs has excellent antibacterial properties and protonation can destroy the cell membrane by forming hydrogen bond with the hydroxyl structure of bacterial cell membrane.Au NCs@Cu can promote the healing of infectious skin wounds,because copper ions are involved in the process of antibacterial and angiogenesis.The structure of Au NCs-Cu²⁺-M⁴⁺coating is stable and it has a good application prospect for the prevention of implant-related infection.