Preparation Of Metal-organic Framework Nanoparticles Based On MnO₂ And Its Synergistic Effect In HIFU Treatment

BackgroundHigh Intensity Focused Ultrasound（HIFU）tumor treatment system is a new method for image-guided targeted ablation of local tumor tissues^[1].In recent years,the development of nanoparticles in targeted functionalization,stimulation response,in vivo imaging,combined chemotherapy and other aspects has provided a new idea for the combined therapy of HIFU^[2].However,the traditional HIFU synergist has the problems of large volume,poor stability and low drug loading^[3,4].To solve these problems,In this study,the metal organic framework nanoparticles(GOD-MnO₂-Fe³⁺-DOX NPs)was prepared by using Glucose Oxidase（GOD）,manganese dioxide（MnO₂）,ferric ion(Fe³⁺)and doxorubicin hydrochloride（DOX·HCl）.The nanoparticles can be dissociated under the stimulation of the weak acidic microenvironment in tumor,the released MnO₂can produce O₂to enhance HIFU treatment,the released GOD and DOX can treat the residual tumor cells after HIFU ablation together.ObjectivePreparation of GOD-MnO₂-Fe³⁺-DOX metallic organic nanoparticles,and explore the synergistic effect of HIFU in the treatment of tumor.Methods1.Manganese dioxide（MnO₂）nanoparticles were prepared and characterized by REDOX reaction.GOD-MnO₂-Fe³⁺-DOX nanoparticles were self-assembly by physical adsorption of glucose oxidase（GOD）,MnO₂,ferric ion(Fe³⁺)and doxorubicin（DOX）.2.The uptake and distribution of GOD-MnO₂-Fe³⁺-DOX NPs in cells and the killing effect of GOD-MnO₂-Fe³⁺-DOX NPs on 4T1 mouse breast cancer cells were investigated by flow cytometry,laser confocal and CCK-8 cytotoxicity assay.3.Free DOX and GOD-MnO₂-Fe³⁺-DOX NPs were injected into tail vein,blood samples were collected at different time points.After treatment,drug concentration in plasma was detected to evaluate pharmacokinetics.After the red blood cells were extracted and co-incubated with different concentrations of GOD-MnO₂-Fe³⁺-DOX NPs,the hemolysis phenomenon was observed and the hemolysis rate was calculated.4.A tumor-bearing mouse model of 4T1 breast cancer cells was established for tumor inhibition experiment.DOX and GOD-MnO₂-Fe³⁺-DOX NPs were injected into the tail vein.After 24 h,the mice were sacrificed and the tumor tissue was prepared for frozen sections.5.A tumor-bearing mouse model of 4T1 breast cancer cells was established.When the tumor volume reached 80-100 mm³,the mice were randomly divided into control group,DOX group,GOD-MnO₂-Fe³⁺-DOX NPs group for treatment.After treatments,the mice were sacrificed,tumors and main organs of heart,liver,spleen,lung and kidney were collected.The toxic and side effects of nanoparticles on each organ and the killing ability of tumors after circulating in vivo were detected by paraffin section of histomathology.6.A tumor-bearing mouse model of 4T1 breast cancer cells was established.When the tumor volume reached 300-500 mm³,the mice were randomly divided into blank control group,HIFU group and GOD-MnO₂-Fe³⁺-DOX NPs+HIFU group for treatment.The sound power of HIFU treatment was 90 W,and the total treatment time was 3 s.The changes of tumor gray values before and after HIFU irradiation were observed before and after HIFU treatment,and the volume of tumor coagulant necrosis was calculated.Tumor volume,body weight and survival were measured after treatment regularly.Results1.Preparation of GOD-MnO₂-Fe³⁺-DOX NPs with particle size of131.23±0.84 nm and surface potential of 21.87±1.72 m V.The drug loading rate was about 40.18%,In the neutral environment with pH=7.4,the cumulative drug release rate within 48h was only 23.13%,while in the weak acid condition with pH=5,the cumulative drug release rate within 4 h was 57.37%,and the cumulative drug release rate within 48h was about87.85%.2.The results of cell cytotoxicity in vitro showed that GOD-MnO₂-Fe³⁺-DOX NPs had higher toxicity to cancer cells than DOX alone.Confocal laser scanning images and flow cytometry results showed that GOD-MnO₂-Fe³⁺-DOX NPs could be effectively taken up and released by tumor cells after co-incubation with 4T1 mouse breast cancer cells,respectively from qualitative and quantitative points of view,and GOD-MnO₂-Fe³⁺-DOX NPs had a higher killing effect on tumor cells than DOX alone.3.The results of pharmacokinetics and in vivo safety experiments showed that GOD-MnO₂-Fe³⁺-DOX NPs have good biocompatibility and longer circulation time.Confocal laser scanning showed that the DOX fluorescence of the GOD-MnO₂-Fe³⁺-DOX NPs group remained in the tumor cells,while the DOX alone group had no obvious fluorescence.4.The results of animal experiments in vivo showed that free DOX and GOD-MnO₂-Fe³⁺-DOX NPs both had different degrees of inhibitory effects on tumor growth.However,the body weight and survival time of mice in the free drug group were significantly shorter,no significant difference was showed in NPs and the control group.5.The results of GOD-MnO₂-Fe³⁺-DOX NPs combined with HIFU showed that the gray-scale enhancement（25.5±4.5）of GOD-MnO₂-Fe³⁺-DOX NPs group was significantly higher than that of HIFU irradiation group（18.7±3.9）,the coagulant necrosis volume of GOD-MnO₂-Fe³⁺-DOX NPs group（105.80±1.21）was significantly higher than that of HIFU irradiation group（38.02±0.34）,and the energy efficiency factor of GOD-MnO₂-Fe³⁺-DOX NPs group（t=1.79）was significantly lower than normal saline group（t=4.97）,and the differences were statistically significant.Tumor inhibition results within two weeks after treatment showed that GOD-MnO₂-Fe³⁺-DOX NPs could effectively inhibit the further growth and metastasis of tumors.ConclusionsThe metal-organic nanoparticles(GOD-MnO₂-Fe³⁺-DOX NPs)prepared in this study were enriched to the tumor site by EPR effect to change the acoustic environment,and disintegrated under the influence of tumor microenvironment to produce O₂,thus enhancing the ablation effect of HIFU on tumor tissue of tumor mice,and releasing the anticancer drug DOX to treat the residual tumor tissue.