The Role And Mechanism Of Zinc-based Metal-organic Frameworks In Infectious Wound Healing And Breast Cancer Therapy

Objective:Two novel zinc-based metal-organic frameworks were designed according to the different roles of zinc,and their specific mechanisms of action in wound healing of drug-resistant bacterial infection and breast cancer treatment were studied respectively.Methods:1.Preparation of zinc-regulated Fe-MOFs（ZFMs）and its application in infectious wound healing1.1 Preparation and characterization of ZFMs:ZFMs was synthesized by solvothermal method;Scanning electron microscope（SEM）,transmission electron microscope（TEM）,Zeta potential,hydrated particle size（DLS）,inductively coupled plasma mass spectrometry（ICP-MS）,energy spectrometer（EDS）,X-ray photoelectron spectroscopy（XPS）,X-ray diffraction（XRD）and fourier transform infrared absorption spectroscopy（FTIR）were characterized ZFMs;3,3’,5,5’-tetramethylbenzidine（TMB）method,electron spin resonance（ESR）method were used to detect the enzyme activity of ZFMs.1.2 Antibacterial and cell experiments of ZFMs:The antibacterial effect of ZFMs was studied by flat plate bacterial cloning experiment and SYTO/PI staining of live/dead bacteria;The morphological changes of ZFMs after treatment of drug-resistant E.coli were photographed by TEM;ROS production by bacteria was studied with DCFH-DA probe;BCA kit and agarose gel electrophoresis were used to study bacterial protein leakage and DNA degradation;CCK-8 cytotoxicity test and scratch test were used to study the effect of ZFMs on fibroblast activity;Expression of angiogenesis-related protein VEGFαwas detected by western blot;Tubule formation assay was used to detect the formation of blood vessel-like cells.1.3 Construction of rat wound infection model:A circular wound model with a diameter of 1 cm was established on the back of the rat,and then extended-spectrumβ-lactamase-producing Escherichia coli was dripped into the wound;The experiment was divided into 6 groups:PBS,H₂O₂,FMs,FMs+H₂O₂,ZFMs,ZFMs+H₂O₂.The change of wound area in mice was measured;Pathological observation on mouse wounds was conducted by H&E staining and immunohistochemistry;Biotoxicity evaluation was conducted by biochemical analysis and H&E staining.2.Preparation of ultra-small ZIF-8 nanoparticles functionalized with C-phycocyanin（ZIF-8@C-PC）and its anti-tumor mechanism2.1 Preparation and characterization of ZIF-8@C-PC:ZIF-8 and ZIF-8@C-PC were synthesized by self-assembly method;ZIF-8 and ZIF-8@C-PC were characterized by SEM,Zeta potential,DLS,UV-Vis spectrophotometer,XRD,FTIR,and thermogravimetric analysis.2.2 Cell experiment of ZIF-8@C-PC:the effect of ZIF-8 and ZIF-8@C-PC on the activity of breast cancer MCF-7 cells was detected by MTT cytotoxicity assay;plate cell cloning assay was used to study cell colony formation;the expression of autophagy-related protein LC3 was studied by western blot;ROS and apoptosis were detected by flow cytometry;Cellular uptake was studied by confocal microscopy..Results:1.ZFMs promoted bacteria-infected wound healingThe morphology of ZFMs was hexagonal or spindle-shaped,and the-COOH skeleton in its structure was increased in physiological environments.ZFMs can turn TMB blue in the presence of H₂O₂,and the generation of·OH was detected by TA and ESR methods,showing peroxidase（POD）-like activity.In vitro antibacterial experiment,the inactivation efficiency of ZFMs against 1×10⁶ CFU/m L drug-resistant Escherichia coli was greater than98%,and it could increase the ROS in the bacteria,cause bacterial protein leakage,and DNA degradation.The viability of fibroblasts didn’t decrease with ZFMs.In scratch experiments,scratch width narrowed in ZFMs-treated group.ZFMs can upregulate VEGFαand promote angiogenesis.In vivo experiments showed that ZFMs was able to heal wounds within 10 days with negligible functional impact on vital organs.2.ZIF-8@C-PC inhibited breast cancer growthThe hydrated particle size of ZIF-8 nanoparticles was 40～50 nm,and that of ZIF-8@C-PC was 200～300 nm.The killing ability of ZIF-8 and ZIF-8@CPC on tumor cells was dose-dependent,and the cell viability decreased as the concentration increased.ZIF-8and ZIF-8@C-PC can promote the increase of intracellular ROS and increase the expression of LC3-II.Cell viability after ZIF-8 and ZIF-8@C-PC treatment was reversed by autophagy inhibitors.Compared with ZIF-8 and C-PC alone,ZIF-8@C-PC is more easily taken up by tumor cells,and the autophagic flux is also increased,which increased the apoptosis rate of tumor cells.Conclusions:1.ZFMs maintained long-term stability under physiological conditions,and have superior POD-like activity,which can increase ROS in bacteria and destroy bacterial cell membranes,proteins,and DNA.In addition,ZFMs have good biocompatibility,which can promote fibroblast proliferation and up-regulate the expression of VEGFα,accelerate angiogenesis,and achieve rapid wound healing in a rat wound infection model.2.ZIF-8@C-PC can induce cellular oxidative stress and induce tumor cell apoptosis by stimulating pro-death autophagy.The natural protein C-PC camouflages ZIF-8,making ZIF-8@C-PC easier to be taken up by tumor cells,so that it can kill tumor cells at low concentration（50μg/m L）.Compared with ZIF-8 and C-PC alone,ZIF-8@C-PC had a more efficient anticancer effect.