Protease-Loaded CuS Nanoparticles With Synergistic Photothermal/Dynamic Therapy Against F. Nucleatum-Induced Periodontitis

Objective:To prepare CuS nanoparticles(CuS NPs)modified by Alcalase 2.4 L FG(CuS@A NPs),and to observe the antibacterial activity against on F.nucleatum and its related biofilms in vitro,the cytocompatibility and blood compatibility,and the therapeutic effect on periodontitis in rats.Methods:Alcalase 2.4L FG was immobilized onto CuS NPs covalently by amide bond through carbodiimide as a coupling agent.The carboxyl acid group in CuS NPs was activated by EDC to generate an O-acylisourea intermediate which further reacted with the amino group in Alcalase 2.4L FG to form CuS@A NPs;CuS@A NPs were detected chemical composition,material morphology,surface positive and negative charges,particle size,and the photothermal and photodynamic efficiency using Fourier transform infrared spectrometer(FTIR),Energy dispersive spectrometer(EDS),Transmission electron microscope(TEM),Nano-NIR real-time living imaging system and other instruments.The antibacterial effect of CuS@A NPs on free F.nucleatum and F.nucleatum on cementum was studied by plate count method(CFU)and SEM,the ability of antimicrobial biofilm was detected by standard crystal violet staining and live/dead fluorescent staining and then observed by CLSM.Then,the cytocompatibility and blood compatibility of CuS@A NPs were detected by cell proliferation and hemolysis assay.Finally,the therapeutic effect of CuS@A NPs on periodontitis was evaluated by an animal model.Statistical analysis was performed using Graph Pad Prism 8.0.2.One-way analysis of variance(ANOVA)with Tukey’s test was used to analyze differences between groups.Results:The FTIR spectra of CuS and CuS@A NPs were evaluated and showed a characteristic peak signal of the–CO–NH–bond.Compared with CuS NPs,the EDS spectra of CuS@A NPs showed the presence of P elements and increased the proportion of O elements.Through TEM,the CuS NPs are relatively uniform and dispersed in an ellipsoidal shape with an average diameter of around 14 nm,and a very thin layer on CuS@A NPs was observed.After treatment of CuS NPs under NIR laser irradiation for different periods,all samples showed a shift in absorbance,suggesting the generation of ROS.CuS@A NPs efficiently converted NIR laser into heat and the NIR-light-to-heat conversion ability was not compromised after three cycles of heating and cooling process.CuS@A NPs(250μg/m L)with relatively low laser intensity(1.5 W/cm~2)could achieve100%antibacterial effects on F.nucleatum and could completely kill F.nucleatum on cementum surface.After staining via standard crystal violet,the untreated biofilm(blank)displayed deep purple color,but the treatment of nanoparticles no matter with or without NIR all expressed a shallowness of purple.Live/dead fluorescent staining showed the thickness and area size of the biofilm treated by CuS and CuS@A NPs decreased significantly,while the ratio of dead to alive bacteria increased.The standard CCK-8assay showed the cell viability was higher than 80%after 36 h incubation with CuS@A NPs at various concentrations ranging from 31.3 to 250μg/m L.The hemolysis rate of CuS@A NPs was much lower than the recommended safety value(2%)of biological materials.The CuS@A NPs effectively reduced gingival inflammation and inhibit alveolar bone resorption in animal models of periodontitis.Conclusions:A novel nanoparticle CuS@A NPs was successfully prepared by modifying Alcalase 2.4L FG onto CuS nanoparticles.The CuS@A NPs possessed superior F.nucleatum-related antibacterial/biofilm activities which are attributed to the lytic ability of Alcalase 2.4L FG as well as the photodynamic and photothermal effect of CuS NPs synergistically.Moreover,the CuS@A NPs possessed excellent biocompatibility as tested in vitro.The CuS@A NPs have an obvious therapeutic effect on periodontitis in rats.Thus,the designed CuS@A NPs offered a novel strategy to eradicate bacteria and relate biofilm,displaying promising potential for the therapy of periodontitis.