Near-Infrared Light-Responsive Nanomedicine Synergistic Photothermal Chemodynamic Therapy In Application Of Pseudomonas Aeruginosa Infections

Background:Bacterial infection causes great harm to human health,particularly the abuse of antibiotics makes bacteria drug resistant.Therapy targeting of drug-resistant bacteria urgently needs the development of novel antimicrobial strategies.The combination of photothermal therapy（PTT）and chemodynamic therapy（CDT）has been reported to synergistically enhance the antimicrobial effect.Among them,photothermal therapy shows many advantages,such as non-invasive,controllable,broad-spectrum antimicrobial activity,and less susceptible to drug resistance.On this basis,the combination of inorganic nanoparticles with photothermal therapy can enhance its anti-biofilm effect and is less prone to drug resistance,and the nanoparticles can increase the local temperature under near-infrared light irradiation to disrupt the bacterial biofilm and kill the bacteria inside the biofilm.Copper sulfide nanoparticles（CuS NPs）are excellent NIR photothermal conversion materials with the advantages of good biocompatibility,easy synthesis and low toxicity.Chemodynamic therapy is an emerging antimicrobial treatment method that takes advantage of the acidic conditions of the microenvironment of bacterial infection.In the presence of hydrogen peroxide,copper sulfide nanoparticles have peroxidase-like activity,which can effectively catalyze the decomposition of hydrogen peroxide to produce hydroxyl radicals with antimicrobial activity and exert antimicrobial effects.Objective:The aim of this experiment was to rely on the simple synthesis of CuS-BSA NPs with good dispersion,stability and biocompatibility to effectively loading and controlling release of protease and antibiotics under 808 nm NIR laser in combination with chemodynamic and photothermal therapy to enhance its antibacterial effect and minimize bacterial resistance.Methods:Firstly,a novel antibacterial nanoparticle Gen@CuS@BSA/Protease was prepared by encapsulating the antibiotic gentamicin（Gen）within CuS@BSA at high temperature by a one-step synthesis method and further modifying the alkaline protease on the surface.Secondly,Gen@CuS@BSA/Protease was characterized by dynamic light scattering and scanning electron microscopy to analysis the morphology,size and potential of the nanoparticles.Then,the photothermal heating capacity,photothermal stability and photothermal conversion of Gen@CuS@BSA/Protease were evaluated by808 nm NIR laser and UV spectrophotometer.In addition,the peroxidase-like activity and hydroxyl radical generation ability of the nanoparticles were examined by TMB and MB experiments.Finally,the antibacterial and anti-biofilm effects of Gen@CuS@BSA/Protease nanoparticles were further investigated,and the bactericidal and anti-biofilm effects of the nanoparticles were verified by in vitro antibacterial experiments using Pseudomonas aeruginosa as a bacterial infection model.A mouse skin abscess model of P.aeruginosa infection was established to evaluate the antibacterial effect of Gen@CuS@BSA/Protease nanoparticles in vivo.Results:Gen@CuS@BSA/Protease nanoparticles were successfully prepared with a small size of 49.96 nm,uniform size and good stability.After 808 nm NIR light irradiation,25μg/m L of Gen@CuS@BSA/Protease nanoparticles could be rapidly heated up to 50℃ within 10 minutes,and 5 cycles of laser on/off cycle detection showed that the nanoparticles had good photothermal stability and also had a high photothermal conversion efficiency of 37.5%.The results of TMB and MB experiments showed that a characteristic absorption peak at 652 nm can be observed after the addition of hydrogen peroxide to the nanoparticles,and the nanoparticles can catalyze the decomposition of hydrogen peroxide to produce a large number of hydroxyl radicals with concentration dependence and temperature dependence,indicating that Gen@CuS@BSA/Protease nanoparticles possess peroxidase-like activity and can exert chemodynamic therapy by generating hydroxyl radicals to achieve effective antibacterial effects.In vitro antibacterial results showed that Gen@CuS@BSA/Protease nanoparticles had the best antibacterial effect after NIR light irradiation in the presence of hydrogen peroxide,and scanning electron microscopy showed that bacterial cell walls treated with Gen@CuS@BSA/Protease+H₂O₂+NIR became rough and bacterial morphology was disrupted.The crystalline violet and confocal results showed that the Gen@CuS@BSA/Protease+H₂O₂+NIR treatment was able to inhibit the formation of biofilm.The results of in vitro experiments showed that the mice recovered the fastest under the Gen@CuS@BSA/Protease+H₂O₂+NIR treatment and had the smallest scars,indicating that the nanoparticles exerted superior antibacterial ability when chemodynamic therapy was synergized with photothermal therapy.Conclusion:Gen@CuS@BSA/Protease nanomaterials were successfully prepared to synergize with CDT/PTT therapy to enhance the antibacterial effect.