Preparation Of Metal-organic Framework Nanozyme And Their Antibacterial Properties

ObjectiveThe use of antibiotics has effectively reduced the risk of bacterial infections,but the problem of bacterial resistance caused by long-term antibiotic use poses a great risk to human health.In recent years,antibiotic-free therapies such as photothermal therapy(PTT),photodynamic therapy(PDT),and chemodynamic therapy(CDT)have been developed and are widely used in the field of antimicrobial therapy.However,single antimicrobial modalities and complex trauma environments often limit their practical application.Therefore,the preparation of multimodal synergistic metal-organic framework nanozyme for use against multidrug-resistant bacteria is essential.In this paper,Au nanoclusters(Au NCs)modified zirconium-based porphyrin metal-organic frameworks(PCN-224)were prepared by an in situ reduction method,and the photothermal properties of Au NCs@PCN,the photocatalytic and nanozyme activities were investigated.Under Near-Infrared(NIR)laser irradiation,Au NCs@PCN is expected to produce large amounts of reactive oxygen species(ROS)in combination with high local temperature to rapidly eliminate bacterial infections and thus shorten the healing process of diabetic wounds under the synergistic effect of various antimicrobial therapies.MethodsUnder heating conditions,benzoic acid was used as a capping agent,and Zr4+ was interlinked to form Zr6 clusters with a D3 d symmetric structure.The Zr6 clusters were then coordinated with porphyrins to obtain PCN-224 with a stable three-dimensional framework structure.The particle size of PCN-224 and Au NCs@PCN was analyzed by dynamic laser scattering(DLS).The successful preparation of Au NCs@PCN was characterized by observation of color changes,scanning electron microscopy(SEM),transmission electron microscopy(TEM),ultraviolet-visible absorption spectroscopy(UV-Vis),and Fourier transform infrared spectroscopy(FTIR).The distribution of O,N,Zr and Au elements in Au NCs@PCN was examined by energy spectrometry(EDS).The temperature variation of Au NCs@PCN was recorded by thermal imager under NIR laser irradiation as a means to evaluate its photothermal properties and photothermal stability.3,3’,5,5’-tetramethylbenzidine(TMB)was used to detect the generation of hydroxyl radicals catalyzed by the nanoenzyme activity of Au NCs@PCN,as well as the high level of nanoenzyme activity exhibited by Au NCs@PCN with or without NIR laser irradiation.9,10-anthracenyl-bis(methylene)dipropionic acid(ABDA)was used as a probe to detect the production of singlet oxygen under NIR laser irradiation and the real-time changes with increasing irradiation time.Staphylococcus aureus(ATCC25923),methicillin-resistant Staphylococcus aureus(ATCC43300),Escherichia coli(ATCC25922)and ampicillin-resistant Escherichia coli(ATCC35218)were used to evaluate the antimicrobial efficacy of Au NCs@PCN by Oxford cup method and plate diffusion method.The levels of ROS in bacteria after different treatments were detected using a 2’,7’-dichlorodihydrofluorescein diacetate(DCFH-DA)fluorescent probe.The inhibition of Au NCs@PCN against MRSA biofilm model was detected by crystalline violet staining.Protein leakage triggered by the disruption of biofilm structure was detected by using a protein quantification kit(BCA).The cytotoxicity of Au NCs@PCN on HUVECs and HACATs at different concentration gradients was assessed by MTT method.The ability of Au NCs@PCN to promote angiogenesis and cell migration was assessed by matrix gel method and scratch assay.The expression of related proteins in HUVECs and HACATs promoted by Au NCs@PCN was probed using laser confocal scanning microscopy and protein blotting.Au NCs@PCN combined with NIR laser was used to treat a diabetic rat trauma infection model as a way to evaluate the in vivo antimicrobial effect.The protein pathway mechanism of Au NCs@PCN induced diabetic wound repair at the molecular level was investigated by protein blotting assay,and the in vivo biosafety of Au NCs@PCN was evaluated by HE staining.ResultsThe DLS test results showed that the average particle sizes of PCN-224 and Au NCs@PCN nanoparticles were 183 nm and 196 nm,respectively.During the preparation of Au NCs@PCN,it can be clearly observed that the solution changed from purple to purple-red.The SEM and TEM images clearly showed that PCN-224 was a homogeneous ellipsoidal nanoparticle,and Au NCs@PCN also showed a clear ellipsoidal morphology with a large number of Au NCs of 2-5 nm scattered on the surface.UV-Vis and FTIR test results also verified the successful preparation of Au NCs@PCN.The results of thermal imaging experiments showed that the introduction of Au NCs made Au NCs@PCN exhibit good photothermal properties and photothermal stability.the results of TMB and ABDA experiments showed that the introduction of Au NCs imparted Au NCs@PCN with stronger reactive oxygen generation ability.In vitro antibacterial experiments showed that Au NCs@PCN combined with NIR laser achieved 95.3% and 90.6% inhibition of MRSA and Ampr E.coli,respectively.The results of intrabacterial reactive oxygen species detection showed that a large amount of green fluorescence could be observed in the Au NCs@PCN group under NIR laser irradiation,which indicated that the photocatalytic and nanoenzyme catalytic activities of Au NCs@PCN induced a large amount of reactive oxygen species production.Crystalline violet staining experiments showed a near-transparent mauve color in the near-infrared laser-irradiated Au NCs@PCN group,exhibiting a good biofilm inhibition effect.The protein leakage assay experiment showed the highest absorbance in the Au NCs@PCN combined with NIR laser treatment group,which further verified the damage of bacterial membrane structure.MTT assay results showed that HUVECs and HACATs co-cultured with Au NCs@PCN at a concentration of 200 μg/m L for 24 h did not produce significant cytotoxicity to the cells,and the cell survival rate remained at 90% around.The results of cell scratch assay showed that the scratch area of Au NCs@PCN group was significantly reduced at 12 h and 24 h,and the cell migration rate reached 55.6% and 82.3%,respectively,compared with the other two groups.The results of angiogenic assay showed that HUVECs in the Au NCs@PCN group formed a tight and continuous reticular vascular tissue,indicating that AuNCs@PCN could stimulate the angiogenic potential of HUVECs.Cellular immunofluorescence images showed that the expression levels of both CD31 in HUVECs and b FGF in HACATs cells were significantly increased under Au NCs@PCN induction,which was consistent with the protein expression trend demonstrated by protein blotting experiments.In vivo antibacterial experiment results showed that the treatment temperature of Au NCs@PCN combined with near infrared laser irradiation group could rise to 55.4°C,and the wound healing rate reached 97.3% after 21 days of treatment.Western blotting showed that Au NCs@PCN combined with NIR laser irradiation significantly upregulated the expression of p-PI3 K,p-AKT,p-CREB and SR-B1,which accelerated wound healing by inducing cell proliferation and migration based on the stimulation of PI3K/AKT and CREB pathways.Biosafety assays showed no inflammatory response or accumulation in heart,liver,spleen,lung and kidney,and Au NCs@PCN was compatible,excreted and degraded in vivo.ConclusionsIn this study,Au NCs@PCN with photocatalytic and nanoenzyme activities were successfully prepared by in situ growth of gold nanoclusters on PCN-224 through Na BH4 reduction for efficient sterilization and promotion of diabetic wound healing at relatively low photothermal temperature(<57°C).Under NIR laser irradiation,Au NCs@PCN could rapidly warm up and generate large amounts of reactive oxygen species for sterilization,thus improving the microenvironment of diabetic wounds.Cytotoxicity experiments and organ toxicity experiments showed that Au NCs@PCN exhibited good biocompatibility.Based on these studies,the Au NCs@PCN antimicrobial nanoplatform with photothermal and nanozyme activities may provide new ideas for clinical treatment of diabetic infected wounds.