The Therapeutic Effect And Mechanism Of Light Triggered Nanoprobe On Hypoxic Triple Negative Breast Cancer

Objective Triple Negative Breast Cancer（TNBC）is the most lethal subtype in female Breast Cancer for its high heterogeneity,aggressiveness and lack of therapeutic targets.Photodynamics Therapy（PDT）has gradually become a new treatment method for TNBC due to its advantages of non-invasive,non-invasive,maximum preservation of breast organs and low cost.However,the oxygen dependence of PDT in the treatment process is limited by the hypoxic microenvironment of tumor tissue,which greatly weakens the anti-tumor effect.Therefore,in this study,a light tirggered nano diagnostic and therapeutic nanoprobe integrating targeting,fenton reaction of self oxygen production and magnetic resonance imaging（MRI）/fluorescence dual-mode imaging was prepared,and its excellent performance was verified,morever,the diagnosis,PDT therapeutic effect and mechanism of the nanoprobe was explored on hypoxic TNBC from the cellular and animal levels.Methods 1.The carboxylated Fe₃O₄was linked to the aminoated mesoporous silica（MSN）by aminocarboxylated reaction,and then the light triggered nanoprobe MMSN-c RGD@Ce6 was synthesized by the surface chelation targeting arginine-glycine-aspartic acid（Ary-Gly-Asp,c RGD）and internal adsorption of photosensitizing agent chlorin e6（Ce6）.Using Scanning electron Microscope（SEM）,Dynamic light scatterer（DLS）,N₂ adsorption-desorption experiment,and Energy dispersive spectroscopy（EDS）,Elemental Mapping,Vibrating sample-Magnetometer（VSM）,small animal fluorescence imager and MRI to analyze and test the morphology,particle size,surface potential,Fe loading,Ce6 loading rate,stability,fluorescence/MR dual-mode imaging performance and fenton reaction self-generated oxygen performance in vitro.2.The targeting effect of c RGD cyclic peptide was verified by detecting the expression of integrinαVβ3 on the surface of human TNBC cells MBA-MD-231and the phagocytosis of the nanoprobes.CCK 8 assay,Reactive oxygen species（ROS）probe and Calcein AM/PI living and dead cell staining were used to detect the ROS producing efficiency of light tirggeder nanoprobe MMSN-c RGD@Ce6.The ameliorative effect of light triggered nanoprobe MMSN-c RGD@Ce6 on hypoxia was detected by detection of hypoxia probe and the expression of hypoxia protein HIF-1α.The cytotoxicity mechanism of the light triggered nanoprobe MMSN-c RGD@Ce6 in vitro was detected by optical microscopy,bioscanning electron microscopy,immunofluorescence and Western blot.3.The biosafety of the nanoprobes in vivo was verified by liver and kidney function analysis and HE staining of major organs in mice.The fluorescence imaging effect of targeted nanoprobe MMSN-c RGD@Ce6 was compared with that of non-targeted nanoprobe MMSN@Ce6.The MR imaging effect of the targeted nanoprobe MMSN-c RGD@Ce6 was compared with that of the non-targeted nanoprobe MMSN@Ce6.4.Establish the transplantable tumormodel of hypoxic human TNBC MBA-MD-231 cells in nude mice.The antitumor effect of light triggered nanoprobe MMSN-c RGD@Ce6 on hypoxic human breast cancer model was detected.Using HE staining,TUNEL staining and immunohistochemistry staining to detect the effects of light triggered nanoprobe on tumor cell necrosis,apoptosis,pyroptosis,proliferation,hypoxia and angiogenesis.Result 1.In this part,a highly effective light triggered nanoprobe therapeutic agent MMSN-c RGD@Ce6 integrat targeting,imaging and oxygen production was successfully constructed.Various characterization tests showed that the nanoprobe MMSN-c RGD@Ce6 had uniform size,good dispersion,high drug loading rate,high stability,and characteristics of targeted MR/fluorescence dual-mode imaging and self-oxygen generation,which laid a foundation for the further application of nanoparticle MMSN-c RGD@Ce6 in vitro and vivo.2.This part proves that the expression of integrinαVβ3 is higher in breast cancer cells than that in normal breast epithelial cells.The phagocytosis of human breast cancer MBA-MD-231 cells of targeted nanoprobes MMSN-c RGD@Ce6was higher than that to non-targeted nanoprobes MMSN@Ce6.The Co Cl₂-induced chemohypoxic model of human TNBC MBA-MD-231 cell was successfully constructed.The nanoprobe MMSN-c RGD@Ce6 inhibited cell proliferation and increased ROS production under the irradiation of 660nm Laser.Light triggered nanoprobes promoted the antitumor effect of PDT in vitro by alleviating hypoxia and inducing pyroptosis and apoptosis of human TNBC MBA-MD-231 cells.3.The biosafety of the nanoprobe MMSN-c RGD@Ce6 in vivo was confirmed by liver and kidney function analysis and HE staining of major organs in mice.Compared with non-targeted nanoprobe,targeted nanoprobe MMSN-c RGD@Ce6 have more enrichment amount,longer enrichment time,and better fluorescence/MR dual-mode imaging effect in tumor,which achieves targeted dual-mode imaging of TNBC from the level of molecular imaging.4.The model of breast cancer TNBC transplantation in hypoxic nude mice was successfully constructed.The light triggered nanoprobe MMSN-c RGD@Ce6has obvious antitumor effect in vivo.The antitumor effect of PDT was enhanced by nanoprobe MMSN-c RGD@Ce6 through targeting,alleviating tumor hypoxia,inducing pyroptosis/apoptosis/necrosis,inhibit tumor angiogenesis in vivo.Conclusion In this study,we synthesized a highly effective light triggered nano therapeutic agent MMSN-c RGD@Ce6 integrat targeting,imaging and oxygen production.Various characterization tests,in vitro and in vivo experiments verified that the probe greatly enhanced the anti-tumor effect of PDT through the mechanism of targeting,alleviating tumor hypoxia and inducing pyroptosis/apoptosis of tumor cells.