| PART I PREPARATION AND CHARACTERIZATION OF NANOMATERIALSObjective To prepare the metal-organic framework PCN-224,which was loaded with MTH1 inhibitor TH588,and then modified with polydopamine on the surface of metal-organic framework to detect and characterize the basic physical and chemical properties and cytotoxicity of nanoparticles.Methods The particle size distribution and Zeta potential distribution of nanoparticles were detected by Malvern laser particle size and potentiometer.The microstructure and morphology of the nanoparticles were detected by transmission electron microscopy and scanning electron microscopy,and the elemental distribution in the nanoparticles was analyzed by energy dispersive spectroscopy.The specific surface area and pore size of nanoparticles were measured by automatic specific surface area pore-able analyzer.The UV-vis absorption spectra of nanoparticles were measured by UV-vis spectrophotometer.1,3-diphenylisophenylfuran(DPBF)probe was used to detect the singlet oxygen production efficiency of nanoparticles.CCK8 method was used to detect the cytotoxicity of PCN-224@PDA nanoparticles.Confocal laser microscopy,flow cytometry and transmission electron microscopy were used to evaluate the phagocytosis and uptake of nanoparticles by breast cancer cells.Results Firstly,the metal-organic framework PCN-224 was prepared.Under electron microscope,it was spherical with regular morphology,specific surface area of 394.25 m2/g,average pore size of 4.54 nm,particle size of(105 ± 16.86)nm,potential of(21.69 ± 2.85)m V.Further detection shows that PCN-224 can significantly produce singlet oxygen under lowintensity focused ultrasound irradiation,and the singlet oxygen generation ability is dependent on ultrasonic irradiation time.After the metal-organic framework was modified with polydopamine,CCK-8 method showed that PCN-224@PDA nanoparticles had less toxicity to cells,and the cell survival rate was still more than 85% when the concentration of nanoparticles was up to 200 μg/m L.The nanoparticle PCN-224@PDA@TH588 was characterized by electron microscope,which showed regular spherical shape,uniform size distribution and good dispersion.Energy spectrum analysis showed that the nanoparticle was mainly composed of four chemical elements C,N,O and Zr,with an average particle size of(118 ± 18.75)nm.The potential was(-30.35 ± 0.46)m V.The maximum absorption peaks of PCN-224@PDA and PCN-224@PDA@TH588 are around 413 nm.Confocal laser observation,flow cytometry quantitative analysis and TEM showed that breast cancer cells could effectively phagocytosis and uptake of nanoparticles after 4 hours of co-incubation with them.Conclusion Multifunctional nanoparticles PCN-224@PDA@TH588 containing MTH1 inhibitor TH588 with regular morphology,uniform particle size,good dispersity and good biocompatibility were successfully prepared.The nanoparticles can be used as a sound sensitizer and a good drug carrier.It can realize the targeted delivery of insoluble small molecules or toxic chemotherapy drugs to the tumor region through EPR effect,laying a foundation for the next in vivo and in vitro research.PART II STUDY ON PHOTOACOUSTIC IMAGING,BIOSAFETY AND BIODISTRIBUTION OF NANOMATERIALSObjective To investigate the photoacoustic imaging ability,biodistribution and biosafety of PCN-224@PDA@TH588 nanoparticles in vivo and in vitro.Methods PCN-224@PDA@TH588 nanoparticles with different concentrations were scanned by small animal photoacoustic imager in vitro,and their photoacoustic images and signal values were collected and measured.Blood routine indexes,blood biochemical indexes and pathological sections of major organs of healthy mice were analyzed at different time points(1,7 and 14 days)after tail vein injection of nanoparticles,and the biosafety of nanoparticles in vivo was systematically detected.The photoacoustic signals in the tumor region of mice were observed before and after the injection of nanoparticles,and the signal intensity was measured.The model of orthotopic transplanted tumor of breast cancer in mice was established,and the biological distribution was evaluated by injecting DIR dye labeled nanoparticles into mice.Results In vitro photoacoustic imaging results showed that the photoacoustic signal of nanoparticles increased linearly with the increase of concentration.The results of photoacoustic imaging in vivo showed that the photoacoustic signal of the tumor site in mice increased gradually with time, and reached the peak at 24 h after the injection of nanoparticles,then gradually decreased.Compared with the control group,there were no significant abnormalities in blood routine,liver and kidney function and H&E staining sections of main organs of mice after injection of nanoparticles,and the differences were not statistically significant.In vivo imaging results of small animals showed that the fluorescence intensity in tumor region reached the maximum 24 h after injection,and the nanoparticles mainly accumulated in liver and spleen 48 h after injection.Conclusion The prepared PCN-224@PDA@TH588 nanomaterial has good photoacoustic imaging effect both in vitro and in vivo,and has potential as a photoacoustic imaging contrast agent.PCN-224@PDA @TH588 has good biocompatibility and safety in vivo,which lays a foundation for the later experimental application of therapy.PART III TO EVALUATE THE EFFICACY AND RELATED MECHANISM OF TH588 LOADED NANOMATERIALS COMBINED WITH ULTRASOUND IN THE TREATMENT OF BREAST CANCERObjective To evaluate the therapeutic effect of PCN-224@PDA @Th588 nanoparticles combined with low intensity focused ultrasound on mouse breast cancer cells and the synergistic mechanism.Methods 4T1 cells were divided into the following groups: Control group,LIFU group,PCN-224@PDA@TH588 group,PCN-224@PDA + LIFU group,PCN-224@PDA@TH588 + LIFU group,CCK-8 assay,live/dead cell staining and flow cytometry were used to detect the effects of nanoparticles treated by different groups on the activity of breast cancer cells at the cellular level and the related mechanisms.A mouse mammary gland orthotopic tumor model was established,and the tumor-bearing mice were randomly divided into the following groups: Control group(PBS),LIFU group,PCN-224@PDA@TH588 group,PCN-224@PDA + LIFU group,PCN-224@PDA@TH588 + LIFU group.To evaluate the effect of low intensity focused ultrasound combined with PCN-224@PDA@TH588 nanoparticles on mouse breast cancer in vivo.Results Compared with other treatment groups,PCN-224@PDA@TH588 nanoparticles combined with ultrasonic irradiation showed stronger cell killing effect,and the difference was statistically significant(P<0.05);The study found that breast cancer 4T1 cells produced a large amount of reactive oxygen species under the irradiation of ultrasound,and with the gradual release of nano-particle loading agent TH588,the activity of MTH1 was inhibited,the content of 8-oxo-d G was increased,and the ROS defense system of cancer cells was destroyed,and the killing effect of intracellular reactive oxygen species was significantly improved.It has the effect of synergistic sonodynamic therapy.In the 4T1 tumor-bearing mice,PCN-224@PDA@TH588 nanoparticles combined with low-intensity focused ultrasound irradiation can significantly inhibit the growth of tumor,and at the end of treatment,tumor volume and tumor weight in mice were statistically significant compared with other groups(P< 0.05).Conclusion The results showed that the anti-tumor effect of PCN-224@PDA@TH588 nanoparticles combined with ultrasound irradiation is significantly better than TH588 alone and sonodynamic therapy alone both in vitro and in vivo.The excellent cell killing efficiency is attributed to the reduction of MTH1 expression induced by TH588,the MTH1 inhibitor released by nanoparticles,which prevents the intracellular self-repair process and leads to the destruction of cancer cells’ ROS defense system,thus significantly enhancing the oxidative damage caused by SDT,thus synergistically killing tumor cells.This provides a new method and strategy for the treatment of tumors. |
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