Construction Of Manganese Dioxide Nanoparticles And Experimental Study On Photochemotherapy Synergistic Anti-tumor

The rapid proliferation of tumor cells and abnormal vascular structure and function lead to the tumor in a low pH and hypoxia microenvironment.Glycolysis is a feature that distinguishes tumor cells from normal cells.Lonidamine(LND)is an indole small molecule inhibitor of hexokinase(HK),which inhibits the generation of glucose-6-phosphate from glucose and blocks the metabolic pathway mainly derived from glucose.It has shown potential application value in anti-tumor research.However,LND has poor water solubility and low bioavailability,so it is difficult to deliver to the lesion site effectively.As a nanomaterial with redox properties,manganese dioxide nanosheets can interact with excessive hydrogen peroxide in tumors to generate Mn2+ and release oxygen to improve the effect of oxygen-dependent photodynamic therapy(PDT).In addition,combining the synthesized TPP-LND with mitochondrial targeting and high-loading capacity MnO2 nanosheets can enhance the enrichment efficiency in tumors,realize the controlled drug release,and help to improve the anti-tumor effect.In this study,the response of the tumor microenvironment of manganese dioxide nanosheets as a carrier,co-loaded Chlorin e6(Ce6)and glycolysis inhibitor LND,to build a photo-chemotherapy combined therapy system based on MnO2-Ce6@TPP-LND(MCTL)targeted inhibition of energy metabolism,and carried out a study on the synergistic enhancement of anti-breast cancer efficacy and mechanism between tumor mitochondrial damage induced by PDT and blocked glycolysis pathway.This paper mainly analyzed the effect and mechanism of combined therapy from the perspectives of the synthesis and identification of MCTL,light-induced 4T1 cell response and glycolysis inhibition,so as to provide ideas and research basis for intervention of tumor cell glucose metabolism.Research contents and resultsPart ? Preparation and characterization of MnO2-Ce6@TPP-LNDMnO2 nanosheets were prepared by redox method,MnO2 nanosheets loaded Ce6 via ?-? stacking to obtain MnO2-Ce6 complex(MC),TPP-LND was obtained by amide reaction between LND and TPP-NH2,MC and TPP-LND were used to construct the composite MnO2-Ce6@TPP-LND(MCTL)through physical interaction.The particle size,morphology,spectrum and other physical and chemical properties of MnO2 nanosheets,MC and MCTL were identified by DLS,transmission electron microscope and fluorescence spectrophotometer,etc.Under laser irradiation,singlet oxygen production rate of different particles and Ce6 release under different pHs were detected.The result shows that the MnO2 nanosheets,MC and MCTL were relatively uniform in size and stable in properties.MCTL successfully carried Ce6 and TPP-LND,Ce6 has certain fluorescence quenching in MnO2 nanosheets.Under acidic conditions,MCTL combined with PDT treatment can increase singlet oxygen yield and enhance PDT effect.After laser irradiation of MCTL,Ce6 can be released quickly in simulated tumor acid environment.Part ? The anti-breast cancer effect of PDT mediated by MCTL in vitroTo evaluate the effects of MnO2 nanosheets,MC,and MCTL on the survival rate of mouse breast cancer 4T1 and human breast cancer MDA-MB-231 cells,analyzed the enrichment of free Ce6 and MCTL in 4T1 cells and explored the cytotoxic effect of MCTL combined with PDT;DCFH-DA and Rho 123 were used to detect ROS production and mitochondrial membrane potential changes under different treatments;Seahorse energy detector,ATP kit and glucose content kit were used to detect the effect of combined treatment on glycolysis.The results shows that:(1)MnO2 nanosheets and MC had no obvious toxicity to the two kinds of cells.TPP-LND improved the solubility of LND,and MCTL showed certain cytotoxicity,the fluorescence signal of MCTL uptake by 4T1 cells was stronger than that of free Ce6,and the fluorescence intensity reached a higher level after 12 h.(2)MCTL combined with PDT alleviated hypoxia and significantly improved the anti-cancer effect and produced a large amount of ROS,which triggered the decrease of mitochondrial membrane potential and induced 4T1 cell apoptosis.(3)PDT mediated MCTL inhibited the glycolysis pathway,reduced the level of ATP production,and resulted in impaired mitochondrial function.Part ? Effect of PDT combined with MCTL against breast cancer in vivo4T1 mouse xenograft tumor model was established to explore the enrichment pattern of MCTL in main organs and tumor sites,and the antitumor effect of MCTL combined with PDT treatment was evaluated from tumor volume,paraffin section,H?E staining etc.The results are as follows:(1)The concentration of MCTL reached the maximum at 3 h after i.v.injection,and this time point was selected as the time for subsequent photodynamic therapy.(2)MCTL combined with PDT induced tumor tissue damage and apoptosis,effectively inhibited tumor growth and prolonged the survival time of mice.(3)MCTL combined PDT significantly inhibited the metastasis of lung nodules in mice,down-regulated the expression of HK2 in tumor tissues,and effectively inhibited glycolysis pathway.(4)There was no obvious damage to the main organs during the combined treatment,and the safety was good.ConclusionsIn this paper,we successfully prepared MCTL,a nano-medicine therapy platform responsive to tumor microenvironment,which has good responsiveness both in vivo and in vitro.After entering tumor cells through endocytosis,MCTL can degrade and release the loaded drug,alleviate tumor hypoxia,inhibit glycolysis metabolic pathway,and enhance the therapeutic effect of combined photochemotherapy.The combination of drug delivery and targeted therapy can provide experimental basis for expanding the application of photochemotherapy in clinical tumor treatment.