Vanadium Oxide Nanozymes With Multiple Enzyme-mimic Activities For Tumor Therapy

Objective:Vanadium oxide(Vnps-x,x=Ⅰ,Ⅱ,Ⅲ,Ⅳ)nanozymes with different V4+/V5+ratios were prepared and characterized.The peroxidase(POD),oxidase(OXD)and catalase(CAT)mimic activities of Vnps-x nanozymes were investigated at different pH(7.4,6.0).The glutathione(GSH)consumption capacity of Vnps-x nanozymes were determined.Meanwhile,the cellular uptake and the inhibitory effect on tumor cell of Vnps-x nanozymes were investigated in vitro.The mechanism of promoting cell death of Vnps-x nanozymes were studied.The tumor inhibition,pharmacokinetics,distribution and safety of Vnps-x nanase were investigated in vivo.Methods:Vnps-x nanozymes were synthesized by REDOX method,and four kinds of Vnps-x were prepared by adjusting the amount of reducing agent and the aging time of nanoparticles.The absorption spectra of Vnps-x between 200-800 nm were measured by ultraviolet method.The morphology of nanozymes were observed by transmission electron microscopy(TEM).The particle size and Zeta potential of Vnps-x were measured by Malvern particle size meter.The elemental composition of Vnps-x were analyzed by energy dispersive X-ray spectroscopy(EDX).The valence of vanadium in Vnps-x were measured by X-ray photoelectron spectrometer(XPS).The XRD pattern of Vnps-x were determined by X-ray diffractometer.The colorimetric method was used to investigate the POD and OXD mimic activities of Vnps-x at different pH.The change of oxygen content in the solution to reflect the CAT mimic activities by Vnps-x.The 5,5 ’-dithiobide(2-nitrobenzoic acid)(DTNB)was the detection substance to investigate the GSH consumption capacity of Vnps-x.The most active nanozymes in Vnps-x were further selected by measuring the steady-state kinetic constants of POD and CAT.The tumor cells were cultured in vitro,Vnps-x was labeled by FITC,and the cellular uptake of nanozymes were investigated.The inhibitory effect of Vnps-x on cells were detected by MTT assay and live/dead cell staining.ROS production in cells induced by Vnps-x were determined by ROS detection kit.The DTNB and[Ru(dpp)3]2+Cl2 were detection substances to investigate the effects of Vnps-x on the GSH and O2 levels in cells.Mitochondrial membrane potential detection kit and apoptosis detection kit were used to detect the effects of Vnps-x on mitochondrial membrane potential and apoptosis rate of tumor cells,respectively.Finally,the best nanozyme was selected according to the above results.The effects of Vnps-x on erythrocyte were investigated by hemolysis test.Subsequently,the tumor-bearing mice model were established.The tumor volume and tumor weight were measured in the process of treatment,and tumor growth inhibition rate of each group was calculated to investigate the tumor inhibitory effect of Vnps-x in vivo.The safety of nanozymes were reflected by the weight changes of mice,the pathological changes of major organs,and the analyzing alanine aminotransferase(ALT),aspartate aminotransferase(AST),urea nitrogen(BUN)and creatinine(CRE)in each group.In addition,the pharmacokinetic curve,parameters and distribution in vivo of the selected Vnps-Ⅲ nanozyme with the best activity were measured within 24 h.Results:The UV spectra showed that the absorption spectra of the four kinds of Vnps-x nanozymes were different,indicating that different nanozymes were synthesized.TEM images showed that Vnps-I,Vnps-Ⅱ and Vnps-Ⅲ were round and spherical,and Vnps-Ⅳ was flake.The Zeta potentials of Vnps-x nanozymes were negative.The elements were C,O,V and Na.In addition,the valence state of vanadium in Vnps-Ⅰand Vnps-Ⅱ was mainly+5,and the XRD results were close to V2O5.The valence state of vanadium in Vnps-Ⅲ and Vnps-Ⅳ was mainly+4,and the XRD results were close to VO2.The POD and OXD mimic activities of the four kinds of Vnps-x nanozymes were in the order of Vnps-Ⅲ>Vnps-Ⅳ>Vnps-Ⅱ>Vnps-Ⅰ,and the simulated activities were enhanced in slightly acidic environment.The order of CAT mimic activities was Vnps-Ⅰ>Vnps-Ⅱ>Vnps-Ⅳ>Vnps-Ⅲ,and the simulated activities were weakened in slightly acidic environment.The order of GSH consumption was:Vnps-Ⅲ>Vnps-Ⅳ>Vnps-Ⅱ>Vnps-Ⅰ.The results of cell experiments in vitro showed that compared with free FITC,the accumulation time of Vnps-x in tumor cells was longer.Among Vnps-x,the Vnps-Ⅲ nanozyme had the strongest inhibitory effect on tumor cells,inducing cells to produce a large number of ROS,effectively reducing the level of intracellular GSH,promoting the decline of mitochondrial membrane potential and apoptosis of tumor cells.The four kinds of Vnps-x did not cause obvious hemolysis.In vivo pharmacodynamic experiment results showed that Vnps-Ⅲ nanozyme in Vnps-x can effectively inhibit tumor growth and have high biosafety.After 24 h injection,there was a special accumulation in tumor tissue,which provided the basis for nanozymes therapy in vivo.Conclusion:In this paper,four kinds of Vnps-x nanozymes with different V4+/V5+were successfully synthesized,and the most active Vnps-Ⅲ nanozyme was screened out.Vnps-Ⅲ had various enzyme activities and GSH consumption ability.It had obvious inhibitory effect on tumor in vivo and in vitro with high safety.This method provided a new idea for the development of vanad-based nanozymes by screening the nanozymes with good activities based on vanadium valence.