Nanotherapeutic System With Effective Microwave Sensitization And Pyroptosis Programming Enable Synergistic Microwave-Immunotherapy In Bladder Cancer

Background: bladder cancer is one of the ten most popular cancers in the world.The prognosis of patients with advanced bladder cancer is still poor despite the combination of neoadjuvant chemotherapy and surgery.At present,studies have confirmed that cancer immunotherapy including immune checkpoint blocking therapy can achieve good survival benefits for patients with advanced bladder cancer,but only a few patients are sensitive to immune checkpoint inhibitors.In recent years,it has been found that pyroptosis can release tumor antigen,induce anti-tumor immunity and improve the efficacy of immunosuppressive agents at immune checkpoints,but it is difficult to trigger pyroptosis in bladder tumors.Therefore,it is of great significance to explore new and effective treatment methods for bladder cancer and develop combined treatment strategies to improve the efficacy of immunotherapy for improving the prognosis of patients.Aim: This study intends to develop an effective combination therapy strategy and design a multifunctional nanoparticles Mn-ZrMOF@DAC and will be based on Mn-ZrMOF@DAC the application of microwave therapy of nanoparticles in the treatment of bladder cancer can not only play a significant anti-tumor effect,but also activate anti-tumor immunity by inducing pyroptosis,enhance the efficacy of immunotherapy for bladder cancer,and provide new ideas for the treatment of bladder cancer.Methods: in this study,Mn-ZrMOF nanoparticles were synthesized by one pot hydrothermal method and loaded with dicitabine(DAC)to construct Mn-ZrMOF@DAC nanoparticles.Firstly,the unique morphology,element distribution,hydrated particle size and ZETA potential of the nanoparticle particles were detected by scanning electron microscope,transmission electron microscope,nanometer size and ZETA potential analyzer;Determination by UV absorption spectroscopy Mn-ZrMOF@DAC Drug loading and drug release curve of nanoparticles;The in vitro temperature rise experiment and TMB color experiment verified that the nanoparticles can enhance the microwave thermal effect and catalyze hydrogen peroxide to produce hydroxyl radical(· OH).Then,through CCK-8 and live cell staining experiments,a preliminary exploration was conducted based on Mn-ZrMOF@DAC the effect of microwave therapy of nanoparticles on the cell viability of mouse bladder cancer cell MB49;Through reactive oxygen species detection experiments,protein immunoblotting experiments,LDH release experiments,ATP detection experiments,and immunofluorescence experiments,we explored the application of Mn-ZrMOF@DAC microwave therapy of nanoparticles induced pyroptosis of mouse bladder cancer cell MB49 and related mechanisms.Finally,by constructing an immunocompetent C57 mouse bladder cancer model Mn-ZrMOF@DAC In vivo anti-tumor effect of microwave therapy of nanoparticles;Exploring the detection of serum cytokines through flow cytometry and ELISA based on Mn-ZrMOF@DAC the effect of microwave therapy of nanoparticles on the infiltration of immune cells at tumor sites and on inflammatory factors in serum;Exploring the combination therapy of constructing a bilateral tumor bearing mouse model and applying anti PD-1 immune checkpoint inhibitors based on Mn-ZrMOF@DAC microwave therapy of nanoparticles can activate anti-tumor immunity and enhance the efficacy of immunotherapy;By detecting the liver and kidney functions of mice and evaluating important organs with HE staining Mn-ZrMOF@DAC biocompatibility of nanoparticles.Result: this study successfully synthesized Mn-ZrMOF@DAC nanoparticles have a unique porous cube shape.The average hydrated particle size is 192.3nm,the dispersion coefficient(PDI)is 0.301,and the Zeta potential is-14.6m V.Mn-ZrMOF@DAC nanoparticles can enhance the microwave thermal effect and catalyze hydrogen peroxide to generate hydroxyl radical(· OH)under microwave radiation.Mn-ZrMOF@DAC nanoparticles can release DAC better under acidic conditions.In vitro cell experiments,the results showed that,Mn-ZrMOF@DAC nanoparticles can directly kill mouse bladder cancer cell MB49 through microwave heating effect and production of reactive oxygen species;Mn-ZrMOF@DAC DAC released by nanoparticles can up regulate the expression of GSDME,a key protein in pyroptosis;Based on Mn-ZrMOF@DAC After microwave irradiation of nanoparticles,the production of reactive oxygen species in MB49 cells increased,the activated caspase-3 protein increased,the NGSDME protein with perforation activity increased,the release of LDH and ATP increased,and the release of HMGB1 increased.Moreover,under the light microscope,the special pyroptosis morphology with bubble like swelling was observed.These results all indicate that Mn-ZrMOF@DAC microwave therapy of nanoparticles can induce MB49 cells to scorch.In vivo animal experiments,the results showed that,Mn-ZrMOF@DAC nanoparticles have good biocompatibility.After the microwave treatment of nanoparticles,the tumor of mice was significantly ablated and its growth was significantly inhibited.Flow cytometry results showed that the number of mature DC cells at the tumor site was increased,and the infiltration of T lymphocytes was increased.ELISA showed that the level of inflammatory factors in serum was increased,indicating the activation of anti-tumor immunity.More importantly,based on Mn-ZrMOF@DAC microwave therapy of nanoparticles significantly enhanced the efficacy of anti PD-1immunocheckpoint inhibitor.Conclusion: this study has developed an effective combination therapy strategy that will be based on Mn-ZrMOF@DAC microwave therapy of nanoparticles applied to the treatment of bladder cancer can not only play a significant anti-tumor effect,but also enhance the efficacy of immunotherapy for bladder cancer by inducing pyroptosis to activate anti-tumor immunity.