| In recent years,emerging infectious diseases caused by pathogens have seriously endangered human health.Bacterial infection has become one of the diseases with high incidence rate and mortality in the world.However,the misuse or abuse of traditional antibiotics leads to continuous drug resistance of bacteria.Therefore,it is urgent to explore and develop new and effective methods to treat bacterial infection.Zeolite imidazole framework-8(ZIF-8),as a metal-organic framework(MOF)material,has the advantages of stable physical and chemical properties,adjustable performance,and good biological safety.ZIF-8 is often used as a protective carrier for functional antibacterial substances,which enhances the drug delivery efficiency and maximizes the antibacterial effect.Nanozyme can effectively kill bacteria by simulating the catalytic activity of enzyme.However,nanozyme is often combined with other ways owing to low catalytic efficiency,which achieves synergistic and efficient antibacterial.In this paper,MOF and nanozyme were respectively used as the main body to construct two kinds of composite nano-antibacterial materials for the collaborative treatment of bacterial infection.The research content of this paper is mainly divided into the following two parts:Design Ⅰ: ZIF-8 was used as mineralized material to encapsulate glucose oxidase(GOx)and horseradish peroxidase(HRP),and to load an antisense oligonucleotide(fts Z ASO)by electrostatic adsorption,which was used to construct a biomineralized nanoplatform(GOx&HRP@ZIF-8/ASO).(1)Scanning electron microscopy(SEM),transmission electron microscopy(TEM),fourier transform infrared spectroscopy(FT-IR),ultraviolet visible spectroscopy(UV-vis),Zeta potential and other characterization methods were used to confirm that the particle morphology was spherical,the particle size was about 410 nm,and the encapsulation efficiency and loading rate reached～63.62%(GOx),～91.46%(HRP)and more than 80%(fts Z ASO)respectively.The ability of hydroxyl radical(·OH)generation and sustained release in GOx&HRP@ZIF-8/ASO system was successfully verified by active oxygen experiment.(2)The growth inhibition curve,MIC value and bacteriostatic rate of GOx&HRP@ZIF-8/ASO system verified that it had effective antibacterial activity in vitro against Escherichia coli(E.coli),Staphylococcus aureus(S.aureus)and methicillin-resistant Staphylococcus aureus(MRSA),among which the inhibition effect on MRSA was the best,with MIC value of only 16 μg/m L.Through the bacterial live/dead double staining test,bacterial content leakage test,anti-biofilm activity test and bacterial morphology observation,the highly effective antibacterial effect of GOx&HRP@ZIF-8/ASO nanoparticles in vitro and the ability to destroy biofilm were confirmed,among which the biofilm removal efficiency was up to88.2%.At the same time,it was confirmed that its synergistic antibacterial activity mainly came from metal ions,reactive oxygen species and antisense nucleic acids,and its antibacterial mechanism of destroying bacterial cell wall was revealed.(3)The cytotoxicity assay and hemolysis assay showed that GOx&HRP@ZIF-8/ASO nanoparticles had good biological safety in vitro.The research of antiinfection treatment in vivo confirmed that GOx&HRP@ZIF-8/ASO nanoparticles have excellent MRSA killing effect,wound repair ability and good biological safety in vivo.Design Ⅱ: Copper sulfide(Cu S)was used as the basic carrier,Au nanozyme and Pt nanozyme were deposited on the surface of Cu S in situ,and the nucleic acid aptamer(Aptamer)was co-loaded to construct a composite nano-antibacterial material with near-infrared light response(Cu S@Pt-Au/Apt).(1)Through a series of characterization methods such as SEM,TEM,EDS mapping and Zeta potential,Cu S@Pt-Au/Apt nanomaterial was proved that the particle morphology was spherical,and the particle size was about 180 nm.The nearinfrared light irradiation experiment proved that Cu S@Pt-Au/Apt had excellent photothermal properties.The activity of oxidase-peroxidase(OXD-POD)in Cu S@PtAu/Apt system was successfully verified by the detection of active oxygen species.(2)The growth curve assay and bacteriostatic rate assay verified that Cu S@PtAu/Apt system had efficient and sustained bacteriostatic effect on E.coli and S.aureus,and the MIC value is only 128 μg/m L.In vitro experiments,such as bacterial live/dead double staining test,bacterial content leakage test,bacterial morphology observation and glutathione consumption test,had confirmed that Cu S@Pt-Au/Apt nanoparticles had the antibacterial effect and the ability to destroy bacterial membrane of highly effective synergetic chemodynamic therapy(CDT)and photothermal therapy(PTT).(3)The cytotoxicity assay and hemolysis assay verified that Cu S@Pt-Au/Apt nanoparticles had good biological safety in vitro.The acute toxicity test in mice confirmed that Cu S@Pt-Au/Apt nanoparticles had no obvious toxicity in vivo.The fluorescence imaging experiment,thermal imaging experiment and in vivo wound infection treatment experiment of diabetes mice verified that Cu S@Pt-Au/Apt nanoparticles had excellent antibacterial infection treatment in vivo,wound repair ability and biological safety in vivo.To sum up,this paper has successfully constructed two synergetic antibacterial systems,GOx&HRP@ZIF-8/ASO system cooperates with metal ions,chemodynamic therapy and antisense therapy,Cu S@Pt-Au/Apt system combines with targeting,chemodynamic therapy and photothermal therapy,which both show high antibacterial activity in vitro,excellent wound healing ability in vivo and good biological safety. |
