Application Of Gox-loaded Fe₃O₄ Nanoparticles In Tumor Diagnosis And Treatment

Objective:Cancer is a major disease to endanger human health seriously.Development of effective diagnostic and therapeutic approaches has always been a hot topic in research.Glucose oxidase（GOx）is able to catalyze glucose in living organisms,producing large amounts of hydrogen peroxide（H₂O₂）to kill tumor cells.Thus GOx can be used as an effective molecule for tumor starvation therapy.In this study,a superparamagnetic iron oxide nanoparticles（SPIONs）with a hollow porous structure（Fe₃O₄ NPs）was synthesized,which can be used as a magnetic resonance imaging（MRI）contrast agents,and has good biocompatibility and photothermal properties.The multifunctional nanoparticles（Fe₃O₄-GOx NPs）were constructed by loading GOx in Fe₃O₄ NPs.We investigated the T2 imaging capabilities of Fe₃O₄-GOx NPs,as magnetic resonance negative contrast agents.The ability of Fe₃O₄-GOx NPs to synergistically treat tumors in vitro and in vivo through starvation/photothermal therapy was also studied.Methods:Superparamagnetic Fe₃O₄ NPs with hollow porous structure were synthesized by hydrothermal reaction and characterized by transmission electron microscopy（TEM）and measurements for hydration particle size and Zeta potential.The photothermal properties of Fe₃O₄ NPs were measured by infrared thermal image instrument.GOx was loaded in Fe₃O₄NPs to construct the multifunctional Fe₃O₄-GOx NPs.The production capacity of total reactive oxygen species（ROS）of Fe₃O₄-GOx NPs and the production capacity of H₂O₂ were evaluated.The ability of Fe₃O₄-Gox NPs as T2 contrast agent was evaluated by 7.0 T animal magnetic resonance imaging system.Murine breast cancer 4T1 cells were incubated with Fe₃O₄ NPs and Fe₃O₄-GOX NPs to evaluate the in vitro biocompatibility of nanomaterials.The intracellular ROS level after Fe₃O₄-Gox NPs treatment was evaluated by DCFH-DA probe.Western Blot was used to explore the cell damage mechanism induced by the generated ROS after Fe₃O₄-Gox NPs treatment.4T1 cells were injected subcutaneously into BALB/c female to establish a tumor model.After tail vein injection of Fe₃O₄-Gox NPs,T2MRI in a mouse tumor model was performed to evaluate the ability of Fe₃O₄-Gox NPs to act as a T2-negative contrast agent.The tumor treatment effect and biosecurity of Fe₃O₄-GOx NPs were evaluated by observing changes in tumor volume and mice body weight,as well as pathological analysis of tumors and major organs.Results:TEM images showed that the particle size of the prepared Fe₃O₄ NPs was about 200nm and the shell thickness was about 50 nm.The hydration particle size was 346.5±3.38nm.Zeta potential test results showed that Fe₃O₄ NPs had a negative charge on the surface.The temperature rise curve of Fe₃O₄ NPs aqueous solution by near-infrared（NIR）laser irradiation showed that Fe₃O₄ NPs had excellent photothermal conversion capability,and the photothermal performance was time-dependent,concentration-dependent and power density-dependent.In vitro DCF fluorescence intensity test showed that Fe₃O₄-GOx NPs could catalyze glucose to produce ROS,and the production capacity was concentration-dependent and time-dependent.By quantitative detection of H₂O₂,it was proved that Fe₃O₄-GOx NPs could catalyze glucose to produce H₂O₂.In vitro MRI results confirmed that Fe₃O₄-GOx NPs can be used as a T2-negative contrast agent.Fe₃O₄ NPs had no significant cell cytotoxicity in the concentration range of 0-100μg/m L,indicating good biocompatibility.Fe₃O₄-GOx NPs exhibited significant cell killing capacity in the concentration range of 25-100μg/m L.When combined with NIR laser irradiation,Fe₃O₄-GOx NPs significantly reduced the survival rate of tumor cells.Western Blot results showed that Fe₃O₄-GOx NPs could significantly increase the expression of heme oxygenase-1（HO-1）protein in 4T1 cells.Animal experiments showed that Fe₃O₄-GOx NPs had a significant negative contrast effect in the tumor area after 6 h of in vivo injection,and it could further improve the effect of photothermal therapy through starvation therapy,significantly inhibit tumor growth,and the synergistic therapy has good biosecurity.Conclusion:This paper successfully synthesized Fe₃O₄ NPs with hollow porous structures,which could be used as carriers to load GOx,thereby constructing multifunctional nanoparticles Fe₃O₄-GOx NPs.The nanoparticles are biocompatible and can be used as T2 contrast agent for diagnostic imaging of tumors.Fe₃O₄-GOx NPs can catalyze glucose to produce high concentrations of H₂O₂,playing a role in starvation therapy.Moreover,under the NIR laser irradiation,Fe₃O₄-GOx NPs have good photothermal properties.Therefore,Fe₃O₄-GOx NPs can kill tumors through the synergistic effect of starvation therapy and photothermal therapy,and their effect is more significant than that of monotherapy.The multifunctional nanoparticles that integrate MRI and starvation-photothermal combined therapy provide a new treatment strategy for tumor treatment.