The Role Of Cobalt-doped Ferromagnetic Nanoparticles In The Treatment Of Renal Cell Carcinoma

ObjectiveThe Fe₃O₄ nanoparticles,with intrinsic peroxidase-like activity,has been successfully employed for various diagnostic applications in clinic.However,only a few studies have been reported on the therapeutic applications of the Fe₃O₄nanoparticles partly due to its low affinity to the substrate H₂O₂.Herein,we report a new strategy for improving the peroxidase-like activity and affinity of the Fe₃O₄nanoparticles to H₂O₂ to generate reactive oxygen species?ROS?for renal cell carcinoma?RCC?catalytic therapy.Thus,Fe₃O₄ nanoparticles provide a new way in the treatment of renal cell carcinoma,and at the same time provide experimental and theoretical basis for improving the therapeutic effect of human renal cell carcinoma.Materials and methodsIn this experiment,the A-498 cell line of RCC were cultured in vitro,and controlled with HEK-293 normal cell.we employed the BALB/C nude mice as a tumor model.First of all,The Fe₃O₄ nanoparticles were synthesized according to the solvothermal method with some modifications.The Co@Fe₃O₄ nanoparticles were also synthesized using the same procedure but extra Co?NO₃?₃·6H₂O?0.82g?was added to the reaction system.The morphology and structure of the Fe₃O₄ and Co@Fe₃O₄ nanoparticles were characterized by transmission electron microscopy?TEM?,scanning electron microscopy?SEM?and dynamic light scat-tering?DLS?.The blue precipitate produced by TMB and H₂O₂ under the condition of peroxidase.We can prove that Co@Fe₃O₄ nanoparticles have peroxidase like activity.The kinetic parameters of the Fe₃O₄ and Co@Fe₃O₄ nanoparticles were determined by monitoring the absorbance change at 652 nm using the iMark?Microplate Reader?BioRad,USA?in the time course mode at room temperature.The ESR measurements were carried out using a Bruker electron spin resonance?ESR?spectrometer?A300-10/12,Germany?at ambient temperature.The cytotoxicity of the Fe₃O₄ and Co@Fe₃O₄nanoparticles with the addition of 10 nmol/L H₂O₂ was determined using the CCK-8cell viability assay kit.The fluorescent probe 2',7'-dichlorofluorescin diacetate?H₂DCFDA?was used to measure the intracellular generation of ROS by the Fe₃O₄and Co@Fe₃O₄ nanoparticles.The cellular uptake and distribution of Fe₃O₄ and Co@Fe₃O₄ nanoparticles in human renal tumor cells were investigated by a confocal laser scanning microscope.The apoptosis analysis of the treated tumor cells was conducted by PI and annexin V staining and flow cytometry.Then it was controlled with HEK-293 cells if the Fe₃O₄ and Co@Fe₃O₄ nanoparticles damage the normal cells.Finally,fifteen BALB/C nude mice bearing A-498 tumors were randomly assigned to three groups?n=5 mice per group?.All the mice were intratumorally treated with a single dose of Fe₃O₄ and Co@Fe₃O₄ nanoparticles?3mg/mL,100?L?with 10 nmol/L H₂O₂ when the diameter of the tumors was about 200mm³.For the controls,Normal saline was administered.The tumor size and mice weight were measured every two days.The tumor size was calculated as volume[mm³]=length×width²×1/2.The measured values are presented as mean±SD.ResultsThe EDX spectrum of the Co@Fe₃O₄ nanoparticles indicated that the Fe and Co elements were present in the Co-doped Fe₃O₄ nanoparticles?Co@Fe₃O₄?.Based on the EDX mapping analysis,the content of Fe and Co in the Co@Fe₃O₄ nanoparticles were determined as 33.48%and 16.23%,respectively.In conclusion,herein,the synthesized Co@Fe₃O₄ nanoparticles contained Fe and Co with the ratio of approximately 2:1.To characterize the structure of the Co@Fe₃O₄ nanoparticles,TEM,SEM,DLS and X-ray diffraction?XRD?analysis were performed.The results indicate that the Fe₃O₄ and Co@Fe₃O₄ nanoparticles present a typical spherical morphology.The average size of the Fe₃O₄ nanoparticles was determined to be89.8±7.9 nm by the TEM images,whereas that of the Co@Fe₃O₄ nanoparticles was determined to be 94.6±8.6 nm.Moreover,the Fe₃O₄ and Co@Fe₃O₄ nanoparticles exhibited the average size of 90.31±0.62 nm and 95.82±3.57 nm in solution,respectively.Moreover,each characteristic diffraction peak of the Co@Fe₃O₄nanoparticles was similar to that of the Fe₃O₄ nanoparticles;this indicated that Co-doping of the Fe₃O₄ nanoparticles did not affect the phase pattern of Fe₃O₄.To directly compare the peroxidase-like activity of the Fe₃O₄ and Co@Fe₃O₄nanoparticles,we performed typical catalytic experiments using the peroxidase substrate 3,3',5,5'-tetramethylbenzidine?TMB?and H₂O₂ as previously reported.The results showed that both the Fe₃O₄ and Co@Fe₃O₄ nanoparticles catalyzed the oxidation of TMB with H₂O₂ to produce blue color products with absorption at 652 nm.Moreover,the results demonstrated that the Co@Fe₃O₄nanoparticles exhibited a significant improvement in the peroxidase-like activity as compared to the Fe₃O₄ nanoparticles;this indicated that a significant improvement in the nanoparticles activity was achieved by Co doping of the Fe₃O₄ nanoparticles.To obtain the apparent kinetic parameters of the Co@Fe₃O₄ nanoparticles,the Michaelis–Menten experiments were performed.The Km value to H₂O₂ for the Co@Fe₃O₄ nanoparticles was much lower than that for the Fe₃O₄ and Co₃O₄nanoparticles;this indicated that there was a significant improvement in the affinity of the nanoparticles towards substrates after Co doping.More importantly,the Km value to H₂O₂ for Co@Fe₃O₄ was nearly 100-fold and 500-fold lower than that of the HRP enzyme and the Fe₃O₄ nanoparticles,respectively;this demonstrated that the Co@Fe₃O₄ nanoparticles exhibited much higher affinity to H₂O₂ than HRP and the other nanoparticles.The Vmax values to H₂O₂ for the Co@Fe₃O₄ nanoparticles were also significantly improved.The intracellular ROS levels in the tumor cells were detected by employing 2',7'-dichlorofluorescein diacetate?H₂DCFDA?,a typical ROS fluorescent dye.The tumor cells treated with only 10 nmol/L H₂O₂ exhibited no significant ROS signal.After incubation with the Fe₃O₄ nanoparticles and 10 nmol/L H₂O₂,the green fluorescence intensity increased.In contrast,the tumor cells treated with the Co@Fe₃O₄ nanoparticles and 10 nmol/L H₂O₂ presented strong green fluorescence intensity,indicating that the Co@Fe₃O₄ nanoparticles catalyzed the decomposition of H₂O₂ to generate a ROS burst to cause cell apoptosis.The tumor cells treated with the Co@Fe₃O₄ nanoparticles and 10 nmol/L H₂O₂ exhibited a significant apoptosis pattern.When the tumor cells were stimulated with the nanoparticles at same concentration,the apoptosis induced by the Co@Fe₃O₄ nanoparticles in the tumor cells was much higher than that of the Fe₃O₄ nanoparticles.Compared with the the control experiment,Co@Fe₃O₄ nanoparticles had little damage and caused slight apoptosis to normal renal cells.To further evaluate the antitumor activity of the Co@Fe₃O₄ nanoparticles in vivo,we employed human renal cancer cell A-498 xenograft in nude mice as tumor model.The Co@Fe₃O₄ nanoparticles exhibited excellent in vivo renal tumor catalytic therapy activity,whereas the Fe₃O₄ nanoparticles only partially inhibited the renal tumor growth due to their relative low peroxidase activity and low binding affinity to H₂O₂.Conclusion1.Co-doping in Fe₃O₄ nanoparticles did not affect the phase pattern of Fe₃O₄.2.The peroxidase-like activity of the Co-doped Fe₃O₄ nanoparticles is dependented pH value.3.Transition metal doping has been demonstrated to be an effective and easy way to improve the peroxidase-like activity of Fe₃O₄ nanoparticles.4.The antitumor activity of Co@Fe₃O₄ nanoparticles are attributed to the catalytic generation of ROS by decomposition of hydrogen peroxide,resulting in the oxidative stress of tumor cells.5.Co@Fe₃O₄ nanoparticles are less harmful to normal renal cells.6.In our work importantly,the Co@Fe₃O₄ nanoparticles exhibited excellent antitumor activities both in vitro and in vivo for renal cell carcinoma catalytic therapy.