The Research And Application Of Tumor Microenvironment-responsive Oxygen Self-supply Nanotheranostics Platform With Bi-modal Imaging Capability

Objective:A multi-functional nanotheranostics platform Mn O₂/Ag₃Sb S₃NPs with high efficiency and good biosafety was prepared.The nanotheranostics platform could enrich the tumor tissue through EPR effect and release Mn²⁺and Ag₃Sb S₃NPs in the tumor site,achieving PDT/PTT/CDT oxygen self-supply combined therapy guided by magnetic resonance imaging and photoacoustic imaging in the biological window of NIR-Ⅱregion.Methods:1.Preparation and characterization of nanotheranostics platformAg NPs and Ag₃Sb S₃NPs were synthesized by typical solvent thermal methods,and then modified with DSPE-PEG-NH₂.The Ag₃Sb S₃-PEG NPs were oxidized with potassium permanganate to form degradable Mn O₂/Ag₃Sb S₃NPs（MA NPs）.The physical and chemical properties of nanoparticles were investigated by TEM、SEM、DLS,UV-vis、EDX、XRD and FT-IR.The morphology changes and ion release of MA NPs at p H 7.4,6.5 and 6.5/GSH were monitored by TEM,UV and ICP-AES.DTNB probe was used to monitor the consumption of GSH by MA NPs.The signal intensity of MA NPs at different concentrations was monitored on magnetic resonance imaging equipment and photoacoustic imaging equipment to evaluate in vitro imaging performance.The ability of MA NPs to catalyze H₂O₂to produce O₂was monitored by an oxygen solute instrument.The ability of MA NPs to produce·OH and ¹O₂was detected by probe MB and DPBF,and double verification was performed by ESR.The photothermal properties of MA NPs were evaluated by thermal imager and the photothermal conversion efficiency was calculated.2.In vitro performance evaluation of the nanotheranostics platformThe potential cytotoxicity of MA NPs was detected by in vitro standard cell counting kit（CCK-8）.Confocal microscopy and flow cytometry were used to detect the uptake capacity of MA NPs in 4T1 cells.DCFH-DA was used as a chemical probe to evaluate ROS production after different treatments at the cell level.The ability of MA NPs to produce O₂at the cellular level was evaluated by[Ru（dpp）₃]Cl₂（RDPP）.AM/PI double staining and flow cytometry were used to detect the apoptosis-inducing effect of MA NPs on cells under different conditions.Mitochondrial membrane potential probe（JC-1 dye）was used to detect the changes of mitochondrial membrane and early apoptosis of MA NPs under different conditions.3.In vivo performance evaluation of nanotheranostics platformMice subcutaneous breast cancer model was established.To assess in vivo imaging performance,MRI and PAI scans of the mouse tumor site were performed before and after drug administration.4T1 tumor bearing mice were randomly divided into different treatments.The weight and tumor volume of the mice were observed 14days after injection,and the mice were euthanized at 14 days.The tumor anatomy was photographed,H&E staining and TUNEL staining,evaluating the therapeutic effect.To evaluate the biosafety of the nanoparticles,blood from mice was subjected for blood biochemical analysis and the main organs were collected for H&E section staining.Results:1.TEM,SEM and DLS results show that MA NPs are uniformly dispersed and the particle size is about 150 nm with honeycomb spherical structure.The element mapping images and EDX analysis also show that the element composition of MA NPs is Sb,S,Ag,Mn and O.FT-IR,UV-vis and X-ray power diffraction demonstrate the successful preparation of MA NPs.MA NPs can be degraded to release Mn²⁺by H⁺and glutathione（GSH）in response to tumor microenvironment,while remaining stable in a neutral environment.Mn²⁺and Ag₃Sb S₃NPs released by degradation have the ability of T1 MRI and PAI in NIR-Ⅱregion respectively,which can be used for bimodal specific imaging of tumors.The Mn²⁺released by degradation can also catalyze H₂O₂to produce·OH and exert CDT performance.The degradation release of Ag₃Sb S₃NPs can produce ¹O₂under the irradiation of 1064 nm laser,and Mn O₂in MA NPs can dehydrolyze and induce oxygen production in tumor microenvironment under the action of H⁺and H₂O₂,relieveing the hypoxic environment of tumor,provide a continuous supply of oxygen for PDT,and improving the efficiency of PDT.Under the irradiation of 1064 nm laser,MA NPs solution showed a concentration-dependent heating trend,and the photothermal conversion efficiency reached 23.15%,showing good potential for NIR-Ⅱregion photothermal treatment.2.CCK-8 method showed that when the concentration of MA NPs reached 200μg m L^-1,the cell survival rate was still more than 80%,indicating that the nanoparticles had good biocompatibility and low toxicity.MA NPs could be taken up by 4T1 cells and reached saturation at the 5 h.MA NPs can produce ROS at the cell level,which can produce ¹O₂at 1064 nm,1 W cm^-2laser irradiation to play the role of PDT,and produce·OH by Fenton-like reaction under the condition of mimicking TME.The combination of both produces more abundant ROS to kill tumor cells at the cellular level,MA NPs can respond to the tumor microenvironment to induce H₂O₂to produce O₂in a concentration-dependent manner.Annexin V-FITC/PI double staining and flow cytometry showed that the combined treatment of PDT/PTT/CDT in MA/GSH/HCO₃^-/H₂O₂/Laser/p H 5.5 group had complementary advantages,and the cells were all apoptotic,implying the superb treatment efficacy.The results of JC-1staining were consistent with those of live/dead cell assay and flow cytometry,indicating that MA NPs could induce cell apoptosis through mitochondrial function injury.3.By monitoring the intensity of MR And PA signals at tumor sites of MA NPs intravenously injected mice,the results showed that MA NPs could gradually enrich at tumor sites through EPR effect,and the signal decreased after the enrichment reached the peak at the 8th hour,so the optimal time point for laser administration was determined to be the 8th hour.By monitoring the mice in different treatment groups,the tumor was almost completely eliminated in the MA+L group after PDT/PTT/CDT combined treatment,showing excellent therapeutic effect.In addition,the blood biochemistry and H&E staining analysis of major organs in each group showed that MA NPs had no obvious toxicity to mice.Conclusion:In conclusion,an intelligent responsive nanotheranostics platform Mn O₂/Ag₃Sb S₃NPs with significant biocompatibility and biodegradability has been successfully synthesized in this project.In the weakly acidic tumor microenvironment,MA NPs is degraded to produce highly toxic hydroxyl radicals through Fenton-like reactions.In addition,MA NPs can regulate TME by catalyzing endogenous hydrogen peroxide to produce O₂and consuming overexpressed GSH,which not only reduces tumor hypoxia and tumor antioxidant capacity,but also improves the efficiency of PDT and CDT.Ag₃Sb S₃NPs dispersed on MA NPs can be used as photosensitizer for NIR-II photodynamic therapy and photothermal therapy to exert anti-tumor role,and achieve a single laser triggered by 1064 nm of NIR-II PTT/PDT effect,achieving significant tumor treatment effect.Moreover,MA NPs has shown great potential in tumor PAI and MRI,greatly improving the detection rate of early malignant tumors.In conclusion,MA NPs provides a promising pathway for the accurate diagnosis of tumors by dual-mode MR/PA imaging and a prospective integrated TME response nanotheranostics platform for oxygen-self-supplied CDT/PTT/PDT combination therapy under 1064 nm laser irradiation.