Preparation Of Cu₂O-Berberine Nanoparticle And Their Synergistic Antibacterial Properties Of Chemodynamic/Photodyna Mic/Photothermal Therapy

Bacterial infections are a serious threat to global public health safety,and antibiotic therapy is considered the main strategy to combat bacterial infections.However,the misuse of antibiotics has led to a dramatic increase in bacterial resistance,so there is an urgent need to develop new and efficient antimicrobial agents with multiple mechanisms of action.Cu₂O nanomaterials not only possess peroxidase-like activity,catalyzing the conversion of H₂O₂to generate·OH,but also exhibit a strong photoresponsiveness in the near-infrared region,exhibiting chemical kinetic antimicrobial activity,which can be used for efficient bactericidal action by synergistic chemical kinetic mechanism through phototherapy,and have wide research prospects in the pharmaceutical field.However,the high toxicity,poor biocompatibility and low stability of Cu₂O seriously limit its further application in the antibacterial field.Therefore,it is important to construct novel,safe and efficient Cu₂O antibacterial nanocomposites.Berberine,a traditional chinese medicine,has been used for more than 2000 years in China,which not only has good antibacterial effect,high biosafety and stability,but also has photosensitivity and can produce photodynamic effect.Based on this,in this thesis,berberine was selected to modify Cu₂O to prepare novel Cu₂O-berberine nanomaterials and to investigate the synergistic antibacterial properties of chemodynamic/photodynamic/photothermal therapy.In the first part of this thesis,Cu₂O-demethylene-berberine（Cu₂O-DMB）nanoparticles with a particle size distribution between 225-375 nm and a positive potential（+5.87 m V）were successfully prepared,which could capture bacteria by electrostatic interaction,and the capture rates of methicillin-resistant Staphylococcus aureus（MRSA）and ampicillin-resistant Escherichia coli（AREC）were 31.1%and22.2%,respectively.Light absorption experiments showed that Cu₂O-DMB had a good near-infrared light response and was able to produce¹O₂under 808 nm light,which showed photodynamic activity and also produced photothermal effect with a conversion efficiency of 31.42%.The enzyme-like activity study showed that Cu₂O-DMB can act as a self-cascade reactor to generate GSSH and H₂O₂by consuming GSH and O₂through glutathione oxidase（GSH-OXD）activity,and then catalyze the conversion of H₂O₂to·OH through peroxidase-like activity（POD）.Therefore,Cu₂O-DMB can catalize the convertion of H₂O₂to·OH with enzyme-like activity and generate ¹O₂with photodynamic activity.The generated·OH causes oxidative damage to the cell membrane of bacteria,which is synergistic with the photothermal effect to sterilize.Antibacterial experiments showed that Cu₂O-DMB could affect bacterial membrane potential and induce protein leakage under 808 nm light,with a minimum inhibitory concentration（MIC）value of 8μg m L^-1,and antibacterial rates of 99.4%and 99.8%for MRSA and AREC,respectively;it could inhibit biofilm formation,with relative biofilm production of 6.86%and 8.55%for MRSA and AREC,respectively.The MIC values did not change significantly within12 incubation cycles.The biocompatibility study showed that Cu₂O-DMB had low toxicity.The hemolysis rate was only 1.31%at a concentration of 8×MIC,and the cell survival rate was over 80%.It could effectively promote wound healing in MRSA-infected mice,and the relative wound area was only 1.56%after 7 days.the H&E staining analysis of wound tissues and organs of mice showed no obvious histopathology and inflammation.The above results indicate that Cu₂O-DMB has a photoresponsive enhanced antimicrobial ability and is an efficient antimicrobial nano-material against drug-resistant bacterial infections by synergistic chemodynamic/photodynamic/photothermal action.Although Cu₂O-DMB has high antibacterial efficiency,it cannot control the release of Cu₂O in the bacterial infection microenvironment,so further optimization of the material is needed.In the second part of this thesis,Cu₂O-dihydrodemethylene-berberine@calcium phosphate（Cu₂O-RDMB@Ca P）nanomaterials with core-shell structure were successfully prepared in a light/pH dual response.The study of metal ion release monitored by inductively coupled plasma spectrometry（ICP）showed that Cu₂O-RDMB@Ca P has higher stability than Cu₂O,Cu₂O-DMB and Cu₂O-RDMB,with a relative decomposition rate of 76.3%for the calcium phosphate shell and 5.2%for the copper ion release after 24 h incubation at pH 4.0,the relative decomposition rate of calcium phosphate shell was only 19.5%and the release of copper ions was 1.2%.Thus Cu₂O-RDMB@Ca P exhibited a pH response that enabled the release of copper ions in the acidic microenvironment of bacterial infection and achieved the antibacterial effect of the material at specific sites.The light absorption experimental study showed that Cu₂O-RDMB@Ca P also has near-infrared light responsiveness and is able to produce¹O₂under 808 nm light,which exhibits photodynamic activity and also produces photothermal effect with a conversion efficiency of 29.81%.Enzyme-like activity studies showed that Cu₂O-RDMB@Ca P could also act as a self-cascade reactor,consuming GSH and O₂to generate H₂O₂,and generating·OH through POD-like activity.Therefore,Cu₂O-RDMB@Ca P has similar chemodynamic/photodynamic/phototherapeutic synergistic antibacterial properties as Cu₂O-DMB,capable of interfering with bacterial membrane potential and causing protein leakage with MIC value of 20μg m L^-1,and antibacterial rates of 98.2%and 98.6%for MRSA and AREC,respectively;effectively inhibited biofilm formation with biofilm production of MRSA and AREC below 5%,with no significant change in MIC value within 12 incubation cycles,and not easily induced resistance in bacteria.Since Cu₂O-RDMB@Ca P has pH responsiveness,the release of copper ions under neutral conditions was less,which reduced the toxicity of the material on normal cells and improved its biocompatibility.When the concentration was 16×MIC,the hemolysis rate was only 1.61%and the cell survival rate was over 90%.Experimental studies in mice showed that Cu₂O-RDMB@Ca P could effectively promote wound healing of MRSA infections,with complete wound healing after 7 days,and H&E staining analysis of wound tissues and organs revealed no obvious lesions or inflammation production.In conclusion,Cu₂O-RDMB@Ca P not only has a photoresponsive enhanced antimicrobial ability to sterilize through chemodynamic/photodynamic/photothermal synergy,but also has a pH-responsive ability to achieve the release of copper ions in the acidic microenvironment of bacterial infection,which improves biosafety and has a good application prospect in the antimicrobial field.The above two parts prepared novel,safe and efficient photo-responsive and photo/pH dual-responsive Cu₂O-berberine nanomaterials,and carried out chemodynamic/photodynamic/photothermal synergistic action mechanism and antibacterial performance studies,which reduced the toxicity of Cu₂O and also broadened the research ideas of berberine in the field of antibacterial materials,providing a theoretical basis for clinical treatment of bacterial infections and solving the problem of bacterial drug resistance.