Bifunctional Nanoenzyme Synergistic Starvation Cell Carcinoma Therapy And Chemodynamic Therapy In Oral Squamous

Objective:The application of nanoenzyme-mediated chemodynamic therapy（CDT）to the treatment of oral squamous cell carcinoma has been extensively studied.However,its therapeutic efficiency is limited by the deficiency of hydrogen peroxide,a substrate for chemodynamic therapy in the tumour microenvironment,which is an important challenge in its translation to the clinic.Glucose oxidase（GOx）can cut off the supply of glucose to cancer cells to inhibit their proliferation,while the generated hydrogen peroxide enhances the efficacy of CDT.Here,we have designed a nanoparticle with dual enzymatic activity by encapsulating glucose oxidase（GOx）in Mn Co MOF,an organometallic framework nanomaterial with POD-like enzymatic activity.The encapsulated GOx can effectively consume glucose at the tumour site,cutting off the tumour energy supply to inhibit tumour growth on the one hand,and generating a large amount of hydrogen peroxide to compensate for the lack of tumour microenvironment on the other hand,and the gluconic acid produced further reduces the p H of the tumour microenvironment.Under acidic conditions,the POD-like enzyme activity of Mn Co MOF can convert hydrogen peroxide into hydroxyl radicals,which can have a killing effect on tumour cells and thus treat oral squamous cell carcinoma.Methods:（1）The nanoparticles GOx@Mn Co MOF with dual enzymatic activity were prep ared and characterised by a one-step method:the morphology and size of GOx@Mn Co MOF nanoparticles were observed by transmission electron microscopy;the hydrated particle size and surface potential of GOx@Mn Co MOF were ass-essed by dynamic light scattering;and the material was characterised using X-ray diffraction mapping and thermogravimetric analysis instruments.（2）Evaluation of enzymatic activity performance of GOx@Mn Co MOF nanoparticles:POD-like enzymatic activity of nanoparticles and in vitro glucose oxidase activity performance studies（including TMB experiments,MB experiments,gluconic acid production,change of solution color,p H changes,etc.）（3）In vitro anti-tumor cell activity studies:killing of Cal-27 cells by GOx@Mn Co MOF,glucose concentration dependence of GOx@Mn Co MOF;dead or alive staining of cells;detection of changes in intracellular reactive oxygen species（ROS）levels.（4）In vivo anti-tumor activity assay:100μL of Cal-27 cell suspension was injected locally in the axilla of mice to establish a subcutaneous oral squamous carcinoma cell model,and the body weight and tumor volume of the mice were measured every 2days after each group of drug treatment;after 18 days of treatment,the mice were executed,and the blood of the mice was taken for biochemical analysis,and the corresponding tumor sites and major organs of the mice were excised and stained with hematoxylin-eosin（H&E）for histological analysis,and the masses were detected by immunofluorescence（IF）.（5）Preliminary safety assessment:serum was collected from all treated groups of mice to assess liver and kidney function;hemolysis of the nanomaterials was measured using fresh blood from the mice.Results:（1）Synthesis and characterization of GOx@Mn Co MOF nanoparticles:GOx@Mn Co MOF nanoparticles with dual enzymatic activity were successfully synthesized by a simple chemical synthesis method,in which GOx was successfully encapsulated into the Mn Co MOF structure.（2）The results of the evaluation of the enzymatic performance of nanoparticles:the results of TMB and MB experiments proved that GOx@Mn Co MOF catalyzed the decomposition of H₂O₂to hydroxyl radicals in a concentration-dependent manner and was time-dependent,and it possessed POD-like enzymatic activity;the gluconic acid generation experiments revealed that in the presence of glucose,GOx solution and GOx@Mn Co MOF showed a characteristic absorption peak at Under acidic conditions,the characteristic absorption peak of GOx@Mn Co MOF was more obvious,and the solution showed a characteristic red-brown complex,and the p H of the solution decreased with the reaction time;after TMB and glucose were added to GOx@Mn Co MOF solution,the typical UV-vis characteristic absorption peak could still be observed at 652 nm The characteristic UV-vis peak at 652 nm was still observed after the addition of TMB and glucose to GOx@Mn Co MOF solution;the characteristic absorption peak at 652 nm was significantly reduced after the addition of MB and glucose to GOx@Mn Co MOF solution.（3）The results of in vitro anti-tumour experiments showed that the toxic effect of GOx@Mn Co MOF on Cal-27 cells was significantly greater than that of Mn Co MOF in a certain concentration range compared to unmodified GOx;no significant specificity was seen in the killing of Cal-27 cells by different concentrations of glucose medium at low concentrations of the material,in that as the concentration of the material increased,the higher the concentration of glucose,the stronger the effect of the killing of cells was presented,showing a dependence on the gradient of glucose concentration.The results of the dead or alive staining illustrated that GOx@Mn Co MOF exhibited a more pronounced killing effect on Cal-27 cells than Mn Co MOF alone,which was in general agreement with the MTT data.the results of the ROS oxygen assay illustrated that almost no green fluorescence was observed in the blank group,a fainter fluorescence could be observed in the Mn Co MOF group,while a more pronounced fluorescence in a glucose concentration gradient-dependent manner.（4）The results of in vivo antitumor assay showed that for the subcutaneous mice in the oral squamous cell carcinoma tumor model,the GOx@Mn Co MOF group showed a more obvious tumor suppressive effect compared with the control group and the Mn Co MOF group;IF assay was performed on the tumor masses of the mice,and CD⁸⁺T cells were activated in the tumor tissue in the GOx@Mn Co MOF experimental group compared with the control group,while The secretion of granzyme B and anti-tumor cytokines TNF-αand INF-γwere also significantly increased in the GOx@Mn Co MOF experimental group compared with the control group.In contrast,the immunosuppressive cytokines IL-10 and TGF-βwere significantly decreased in the experimental group.（5）The results of biosafety studies showed that there was no significant hemolysis of red blood cells after co-culture with GOx@Mn Co MOF,indicating that GOx@Mn Co MOF has good hemocompatibility;there was no significant difference in the biochemical indexes of liver and kidney blood of mice,no significant abnormalities in the H&E staining of important organs,and no significant changes in the daily body weight of mice,proving that GOx@Mn Co MOF has good biosafety.Conclusion:The results of this study show that the novel GOx@Mn Co MOF nanoparticles possess dual enzymatic activities,i.e.POD-like enzymes and glucose oxidase,which improve the anti-tumour effect through synergistic starvation therapy and chemokinetic therapy,providing a new strategy for the clinical treatment of oral cancer.