Antitumor Study Of Mannose-modified Iron-based Nanozyme Stimulated By Endoplasmic Reticulum Stress-induced Pyroptosis

Objective:The incidence of oral cancer ranks sixth among malignant tumors in the world.The complex anatomical structure and important physiological functions of cavity squamous cell carcinoma determine that the purpose of treatment is not only to improve the life cycle of patients,but also to protect the important functions of the oral cavity.Treatment for oral squamous cell carcinoma mainly includes early surgery or radiotherapy,while advanced treatment requires chemotherapy combined with surgery and radiotherapy.Overall,the 5-year survival rate of patients with oral squamous cell carcinoma remains only 40%-50%.Therefore,the development of novel therapeutic strategies has important clinical significance for prolonging the survival period of patients and improving the prognosis of patients.The rise of immunotherapy has brought new hope to most patients.Tumor immunotherapy aims to activate the human immune system and kill cancer cells by relying on autoimmune functions.However,strict screening of indications and contraindications is required before administration of immunotherapy,and only a few patients are eligible for immunotherapy.Therefore,to overcome the limitations of current immunotherapy strategies and break through the bottleneck of immunotherapy,it is an urgent problem to be solved in the field of immunotherapy to develop new therapeutic strategies for reducing toxicity and enhancing efficacy.As a new member in the frontier strategy of immunotherapy,pyroptosis provides a new solution for the development of new immunotherapy methods.Pyroptosis is a newly discovered method of programmed cell death.The Gasdermins protein family is the main executive molecule of pyroptosis.In addition,pyroptosis mediates the recruitment of CD8+T cells and other immune cells to increase the infiltration of local tumors,further expanding the inflammatory response,activate the body’s anti-tumor immunity.Therefore,induction of pyroptosis in tumors can induce high-efficiency anti-tumor immune activity in the body,thereby inhibiting tumor growth.Therefore,inducing pyroptosis in tumor cells is a new way to treat malignant tumors.Some current studies have shown that chemotherapy drugs such as decitabine,cisplatin and doxorubicin can inhibit tumor growth by causing tumor cells to undergo pyroptosis.However,due to the lack of targeting of chemotherapeutic drugs and the accumulation in normal tissues of the body,the damage to normal tissues and the side effects of multidrug resistance seriously affects the therapeutic effect of tumors and the quality of life of patients.Exploring immunomodulatory drugs with reduced side effects and enhanced targeting is a new idea for tumor immunotherapy.Iron-based nanozymes are a new generation of artificial mimic enzymes that not only have the physicochemical properties of nanomaterials,but also contain enzymatic catalytic functions.They show significant advantages in the field of tumor immunotherapy because they not only overcome the limitations brought by traditional chemotherapy and radiotherapy methods,but also avoid the additional harm and toxic side effects of radiotherapy and chemotherapy.However,the therapeutic effects of iron-based nanozymes reported so far are still limited by technical problems such as low transmembrane efficiency and poor targeting.Therefore,the development of new iron-based nanozymes with good transmembrane transport ability and certain targeting is a key strategy to solve the above problems.Methods:1.Nano-Fe₃O₄was prepared by solvothermal method,and Fe₃O₄@NH₂-MIL-100（FNM）was prepared by repeatedly coating Fe₃O₄as the core surface with amino-modified iron-based metal-organic framework.2.To improve the endocytosis efficiency of nanoparticles,mannose was continuously modified on the surface of FNM to finally make the iron-based nanozyme Mannose@Fe₃O₄@NH₂-MIL-100（M-FNM）.3.The morphology and size of M-FNM were characterized by high-resolution transmission electron microscopy and dynamic light scattering;the mannose modification was characterized by infrared spectroscopy.4.Observing the effect of mannose modification on cellular uptake efficiency by laser confocal,using mannose receptor inhibitors to discover the important role of MR in transmembrane transport.5.Preliminarily explore the effect of M-FNM on cell viability by CCK-8 experiment.6.To construct 3D organoids and in vitro culture models of oral squamous cell carcinoma cell line Cal-27 to explore the cell-killing mode of M-FNM nanozymes.7.To observe the changes of the morphological markers of pyroptosis by M-FNM nanozyme by laser confocal microscope.8.To detect the expression of the caspase-1 p10,ASC and N-GSDMD protein by western blotting.Therefore,it is suggested that the mode of cell death by M-FNM is pyroptosis.In addition,9.The related protein molecular markers of ER Stress and PERK signaling pathway were detected by western blotting.10.To explore the targeting effect and inhibitory effect of M-FNM nanozyme on tumor and the activation effect of anti-tumor immune response in tumor-bearing mice.11.To explore the tumor targeting ability of M-FNM by establishing Cal-27 tumor implantation model subcutaneously in nude mice.12.To explore the effect of M-FNM nanozyme on the infiltration of immune cells in local tumor by histological fluorescence staining.13.The release of LDH and IL-1βfrom cells was detected by enzyme-linked immunosorbent assay and LDH kit.14.Statistical analysis:All in vitro experiments were repeated 3 times,and in vivo animal experiments were repeated at least 2 times,and the experimental results were expressed as mean±standard deviation.The two groups of data that conformed to the normal distribution were statistically analyzed by Student’s t test.P<0.05 indicates statistical difference.The research results:1.Nano Fe₃O₄was prepared by solvothermal method,and Fe₃O₄@NH₂-MIL-100（FNM）was prepared by wrapping Fe₃O₄as the core surface with amino-modified iron-based metal-organic framework.2.The morphology and size of M-FNM were characterized by high-resolution transmission electron microscopy and dynamic light scattering.The M-FNM nanozyme has a typical core-shell structure,and the size of the M-FNM nanozyme is about 115 nm;3.Infrared spectroscopy results indicate successful modification of mannose.4.Observing the effect of mannose modification on cellular uptake efficiency by laser confocal,using the mannose receptor inhibitor to discover the important role of MR in transmembrane transport.5.It was initially found by CCK-8 experiment that M-FNM had obvious inhibitory effect on cell viability.6.M-FNM nanozyme promotes the morphological markers of pyroptosis,which are water-absorbing swelling,and promotes the release of LDH and IL-1β;in addition,it also up-regulates the molecular markers of pyroptosis,Caspase-1 p10,ASC and N-GSDMD expression at the protein level.7.By detecting the protein molecular markers of ER Stress and PERK signaling pathway,we found that M-FNM nanozyme induced ER Stress in cells and activated PERK signaling pathway;under the action of ER Stress inhibitor 4-PBA,pyroptosis was rescued,and the expression of molecular markers of pyroptosis was significantly inhibited.8.By establishing a Cal-27 tumor implantation model subcutaneously in nude mice,it was found that M-FNM was more localized in the tumor than FNM;Nanozyme treatment significantly inhibited tumor growth.9.Histological fluorescence staining results showed that M-FNM nanozyme changed the infiltration of immune cells in the local tumor,promoted the infiltration of antigen-presenting cells（DCs）,and mediated the infiltration of DCs.CD8+T and CD4+T infiltration leading to anti-tumor immunity increased and Treg cells with immunosuppressive effects were significantly decreased.10.ELISA analysis showed that M-FNM increased the level of inflammatory factors in the plasma of mice after administration.Conclusion:We have successfully prepared a mannose-modified iron-based nanozyme M-FNM that is easy to be phagocytosed by cells;M-FNM nanozyme promotes the activation of cellular ER Stress and PERK signaling pathway,and then activates the caspase-1 cascade to cleave activation of N-GSDMD eventually leads to the occurrence of pyroptosis;M-FNM nanozyme has a certain active targeting ability and regulates the immune system to inhibit tumor growth.