Anti-tumor Research Of Mesothelin Targeted Nanoparticles MTM@Lox@HFe-TA Combined With Tirapazamine

Background:The main methods for tumors treatments in clinic,include surgery,chemotherapy,radiotherapy,immunotherapy and combination therapy.However,chemotherapy and radiotherapy are not targeted and have toxic side effects.Therefore,there is an urgent need for a new strategy with tumor targeting and low side effects.Nanomedicine can be enriched in tumor with low toxicity,nano-materials can be precisely targeted and delivered by coating with targeting cell membranes to treat malignancy.Methods:Mesothelin antibody was expressed on 293T cell membrane,and mesothelin targeted membrane（MTM）was prepared.Its targeting property was verified by Co-IP and live animal imaging.experiments.We synthesized HFe-TA and load lactate oxidase（Lox）on it to obtain Lox@HFe-TA.Lox@HFe-TA was coated with MTM to prepare MTM@Lox@HFe-TA nanoparticles.In vitro,we have tested the anti-tumor effect of MTM@Lox@HFe-TA combined with TPZ through live&dead cell staining,flow cytometry,Edu staining and other experiments,and investigated its anti-tumor mechanism.In vivo experiments,we validated the anti-tumor effect of MTM@Lox@HFe-TA combined with Tirapazamine（TPZ）through the mouse subcutaneous tumor model and lung metastasis,we also tested the biological safety of these combine therapy.Results:（1）Material preparation:First,the cell membrane that can target mesothelin was prepared.Then we synthesized HFe-TA nanomaterials.The material contains Fe²⁺and has Fenton effect.It can react with H₂O₂to generate a large number of free radicals,and the generated Fe³⁺can react with TA to generate Fe²⁺.After that,Lox was loaded on HFe-TA and related verification expriments were performed.MTM@Lox@HFe-TA nanoparticles were synthesized by coating Lox@HFe-TA with MTM.We observed the morphological structure and Zeta potential of MTM@Lox@HFe-TA nanoparticles,it was found that the cell membrane can smoothly encapsulated Lox@HFe-TA.MTM@Lox@HFe-TA has the ability to decompose lactate,consume oxygen,and generate hydrogen peroxide.Finally,cell experiment and live animal imaging were performed to prove that MTM@Lox@HFe-TA has tumor cell targeting property.（2）In vitro experiment:MTT experiment,live&dead cell staining and flow cytometry were used to detect the anti-tumor effect of MTM@Lox@HFe-TA combined with TPZ on tumors.Ed U experiment proved that MTM@Lox@HFe-TA combined with TPZ can inhibit cell proliferation.At the same time,the combined application of TPZ can reverse the upregulation of Hif-1αcaused by Lox,reverse the epithelial-mesenchymal transition,and inhibit the migration of 4T1 cells.We also studied the anti-tumor mechanism of MTM@Lox@HFe-TA combined with TPZ.It was found that the main mechanism is the decomposition of lactic acid by Lox,which causes hypoxia and leads to the production of hydrogen peroxide.HFe-TA causes Fenton reaction and reacts with hydrogen peroxide to produce reactive oxygen species（ROS）.At the same time,TPZ was further activated under hypoxia.TPZ can reverse the upregulation of Hif-1αcaused by Lox,which is mainly through phosphorylation of e IF2α,negatively regulates Hif-1α,and ultimately inhibits tumor migration.（3）In vivo experiment:Balb/c mice were used to construct the subcutaneous tumor model.After tumor formation,the experiment group was injected with MTM@Lox@HFe-TA and intraperitoneal injection of TPZ（every 4 days）.During this period,we recorded the body weight and tumor size of the mice every two days.The results found that:MTM@Lox@HFe-TA+TPZ has a tumor inhibition rate of 85%and a significant anti-tumor effect.Through the lung metastasis model,the effect of MTM@Lox@HFe-TA+TPZ on tumor metastasis was tested,and the results showed that the lung metastasis of the MTM@Lox@HFe-TA+TPZ group was significantly reduced.We collected tumor tissues and organs,detected H&E staining and immunohistochemistry.The results showed that:MTM@Lox@HFe-TA+TPZ inhibited tumor proliferation,lead to tumor apoptosis;HE staining of heart,liver,spleen,lung,and kidney sections showed there is no damage to the main organs.We checked the biochemical indicators of the blood supernatant,the results showed that:MTM@Lox@HFe-TA+TPZ has no effect on organ function,ALT,AST,CREA levels were normal,it is biological safety.Conclusion:MTM@Lox@HFe-TA combined with TPZ produced ROS through the Fenton reaction of Fe²⁺and the decomposition of lactate by Lox,created hypoxic environment to activate TPZ,which can significantly kill tumor cells and inhibit proliferation;TPZ can phosphorylate e IF2α,and negatively regulate Hif-1α,inhibit tumor metastasis.MTM@Lox@HFe-TA combined with TPZ has good biological safety.