| Objective:Malignant tumors have become an important disease threatening human’s health.Traditional cancer therapies cannot meet people’s needs due to many side effects.In recent years,sonodynamic therapy(SDT)as an emerging form of non-invasive cancer treatment has attracted widespread attention,it has high tissue penetrability and can activate the sonosensitizers accumulated in deep tumors by ultrasound,which produces reactive oxygen species(ROS)to effectively kill tumor cells.This research both synthesizes and characterizes a multifunctional biomimetic drug-loaded nanocarrier that can respond to sonodynamic therapy,then verifies the killing effect on tumor cells on the cellular level and evaluates its’biosafety,which may provide a theoretical basis for further research on collaborative anti-tumor therapy in vivo.Methods:The drug-loaded nanoparticles(apatinib/Ce6@ZIF-8,a CZ)based on the metal organic framework ZIF-8(Zeolitic imidazolate framework-8)as the platform were synthesized by in-situ encapsulation.The surface of the nanocarrier was modified with the purified cell membrane to construct a biomimetic drug-loaded nanoparticle(apatinib/Ce6@ZIF-8@Membrane,a CZM),which was further characterized and tested,and SDS-PAGE gel electrophoresis was performed at the same time to verify the membrane protein on the surface.The CCK-8(Cell Counting Kit-8)method was used to test the toxicity of different concentrations of drug-loaded nanocarriers in mouse breast cancer cell line(4T1).The uptake of nanoparticles by cells under different conditions was analyzed by flow cytometry and confocal laser microscope.SOSG(Singlet Oxygen Sensor Green)singlet oxygen fluorescent probe,ESR(electron spin resonance spectroscopy)and DCFH-DA fluorescent probe method were carried out to verify the production of sonodynamic-mediated ROS.Subsequently,CCK-8,live cell dead cell(Calcein/PI)staining,and flow cytometry apoptosis analysis were performed to verify the anti-tumor effect of the nanomaterial under the activation of ultrasound in vitro.Finally,the in vitro hemolysis test,blood routine and biochemical examinations,and HE staining of major organs were further analyzed for biological safety of the biomimetic nanocarriers.Results:We successfully synthesized the drug-loaded nanoparticles(a CZ)with an average particle size of 169.83nm,and the distribution range was mainly from 140 to 190nm.When surface of nanomaterial was modified with cell membrane(a CZM),the average size will increase to about 210.26nm,and the distribution range was concentrated in 190-255nm.The stability test of a CZM for 7 days found that it is stable and dispersible in the solution,the particle size fluctuates around 205-230nm,although the polydispersity index(PDI)fluctuated slightly in the previous few days,it was stable below 0.3 to the end.SDS-PAGE gel electrophoresis experiment results show that a CZM has a band similar to that of pure cell membrane protein.CCK-8 show that there is no obvious toxicity to cells in the low concentration range,and the relative cell survival rate can reach more than 95%,the cell survival rate is still higher than85%at a concentration of 20μg/ml.The results of confocal laser confocal microscopy and flow cytometry analysis consistently showed that the intracellular fluorescence intensity of the nanocarrier groups carrying the sonosensitizer Ce6(Chlorin e6)in the p H 5.5 environment was higher than that in p H 7.4,the a CZM group with cell membrane modification has the strongest fluorescence and the largest amount of internalization.The extracellular SOSG and ESR method and the intracellular DCFH-DA fluorescent probe method both showed that the a CZM+SDT group produced the most ROS,followed by the a CZM group,and the single SDT group was the weakest.The CCK-8 was performed to detect the killing effect of the biomimetic drug-loaded nanoparticles under SDT-mediated,the results show that when the ultrasonic intensity is fixed at 0.5 W/cm~2,the relative survival rate of the cells in the medium concentration group was(10μg/ml)(5.54±1.26%)and the high concentration group(20μg/ml)(2.14±1.63%)was significantly lower than the low concentration group(5μg/ml)(53.40±4.25%),(P<0.0001),and within a certain range,the cell killing effect is depending on concentration and intensity.Flow cytometry results also showed that SDT-mediated nanomaterial has a strong ability to promote apoptosis.The apoptosis rate of the a CZM in ultrasound group(44.95±3.03%)was significantly higher than that of the non-ultrasound(17.00±2.26%)and a CZ+SDT group(24.85±3.08%)(P<0.0001),the live and dead cells staining(Calcein/PI)also supports this trend.Finally,in vitro 4 and 24 hours hemolysis test showed that the hemolysis rate of the highest concentration group was still less than 1%,and the blood routine,biochemistry and HE staining results of major organs in mice undergoing nano-treatments showed no obvious functional abnormalities and tissue damage in a short term.Conclusion:We successfully constructed and characterized a multifunctional biomimetic drug-loaded nano-system(a CZM)based on the ZIF-8 platform,which has high stability under physiological p H condition and can target tumors through its’surface cell membrane modification,and also the ability to disintegrate and release drugs in the acidic tumor environment.It can produce a higher level of ROS under low-intensity ultrasound,and then powerfully kill tumor cells.Within a certain nano-concentration range(<20μg/ml),as the concentration increases,the SDT-mediated anticancer effect is of significance,showing a concentration-based effect;when at the same concentration,especially at a low concentration(5μg/ml),the combined cell killing effect show a positive correlation with intensity of SDT.In vivo and in vitro experiments have confirmed that the biomimetic drug-loaded nanocarrier(a CZM)has good biosafety and compatibility characteristics for major tissues and organs of the body in the short term. |
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