| Objective Glucose oxidase(GOx)modified magnetic nanoparticles(MNPs)(GMNPs)were prepared,and the inhibitory activity of GMNPs against Enterococcus faecalis and Candida albicans in persistently infected root canals was investigated to evaluate their ability to scavenge biofilms of both bacteria,and to assess their cytocompatibility and haemocompatibility.Methods In the present study,we combined GOx and MNPs to form an enzyme-inorganic nanomaterial.The positively charged MNPs were first prepared by the classical hydrothermal method and bound to the MNPs by cross-linking with glutaraldehyde and electrostatic attraction under the influence of the p H of the buffer to form GMNPs.The composition and morphological structure of the MNPs and GMNPs were analyzed using Fourier Transform Infrared Spectroscopy(FT-IR)and Transmission electron microscope(TEM).Nanoparticle size potentiometry and vibrating sample magnetometer were used to analyze the Zeta potential and magnetic intensity of MNPs and GMNPs,respectively.The inhibitory activity of GMNPs against Enterococcus faecalis and Candida albicans was assayed by agar plate diffusion method and growth curve assay.Intracellular reactive oxygen species(ROS)levels of Enterococcus faecalis and Candida albicans were measured using a reactive oxygen species assay kit.The scavenging potential of Enterococcus faecalis and Candida albicans biofilms was further investigated using Confocal laser scanning microscopy(CLSM).The biocompatibility of MNPs and GMNPs was also evaluated by CCK-8 assay,cell adhesion assay and hemolysis assay.Statistical analysis was carried out using Graph Pad PRISM 8.0 and all data were analyzed using a t-test to determine differences between groups.Results TEM images showed that the MNPs and GMNPs were uniformly circular and,that there was a thin GOx film outside the GMNPs at high magnification.The particle size of the GMNPs(100.70 ± 17.33 nm)was larger than that of the MNPs(87.90 ± 13.90nm).the FT-IR of the GMNPs contained all the characteristic peaks of the MNPs and GOx,indicating that the MNPs were successfully modified by GOx.This indicates that the MNPs were successfully modified by GOx.The results of the inhibition and growth curve experiments showed that GMNPs showed better inhibition against Enterococcus faecalis and Candida albicans at the same concentrations of MNPs and GMNPs(P<0.05),and the inhibition activity of GMNPs was concentration-dependent,GMNPs showed a sustained bactericidal effect within 24 h.The ROS release level assay detected a significant increase in intracellular fluorescence intensity of both Enterococcus faecalis and Candida albicans after 12 h of MNPs treatment compared to the blank control group,with the GMNPs group showing significantly higher fluorescence intensity than the MNPs group(P < 0.001).CLSM observations from biofilm experiments revealed that GMNPs in the presence of a magnetic field caused a significant decrease in the biomass and mean thickness of Enterococcus faecalis and Candida albicans biofilms and an increase in the surface area to volume ratio.The CCK-8 assay yielded a cell survival rate of over 80% for all samples after 36 h of cell culture;the cell adhesion assay showed no significant difference between the groups by counting cell density,cell spreading area and cell elongation;the haemolysis rate of different concentrations of MNPs and GMNPs was less than 2%.Conclusion In this study,MNPs and GMNPs were successfully prepared.both MNPs and GMNPs showed significant inhibitory effects on Enterococcus faecalis and Candida albicans and had significant scavenging ability on their biofilms,with GMNPs being more effective.GMNPs have good biocompatibility and therefore MNPs carrying GOx have promising applications for killing biofilms. |
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