Preparation Of GOx Functionalized Double Rare Earth Doped CDs For Nanocatalytic/Starving-Like Synergistic Therapy

Traditional chemotherapy,radiotherapy and surgical treatment of tumors have some problems,such as insufficiency,low specificity and serious side effects,which lead to unsatisfactory therapeutic result.In recent years,considerable progress has been made in tumor treatment methods such as photodynamic therapy（PDT）,photothermal therapy（PTT）,starvation therapy and nanocatalysis therapy.These new methods have the advantages of high curative effect,good selectivity and little side effects.For example,starvation therapy uses glucose oxidase（GOx）to catalyze glucose oxidation to gluconic acid and toxic hydrogen peroxide（H₂O₂）.It has attracted increasing attention because of the biocompatibility and degradation of GOx itself,especially the high catalytic efficiency towardβ-D-glucose.More interestingly,the generated H₂O₂has highly efficient oxidative and photolytic properties,and make it easy to be combined with other therapeutic agents for combination therapy,which has considerable application prospects in tumor treatment.In this paper,double rare earth doped CDs（Ce-Gd@CDs）were prepared by a simple one-step hydrothermal method using citric acid（CA）as carbon source and Ethylene Glycol（EDA）as surface passivation agent.Transmission electron microscopy（TEM）results showed that the as-prepared CDs had the diameter of 2.3±0.6 nm and exhibited good monodispersity.GOx was covalently conjugated with the amino group of Ce-Gd@CDs through the amide bond to obtain a functionalized nanoenzyme Ce-Gd@CDs-GOx with the diameter of 25.5 nm.The surface functional groups and elemental composition of Ce-Gd@CDs-GOx were analyzed by Fourier transform infrared（FT-IR）spectroscopy and X-ray photoelectron（XPS）spectroscopy to determine the successful doping of Ce and Gd and the conjugation of Gox.In the fluorescence spectra,these nanozymes exhisited a maximum emission at 450 nm under the exhibited wavelength of 350 nm,demonstrating the excellent fluorescence properties of Ce-Gd@CDs-GOx.Furthermore,these nanozymes also had a high relaxation ralaxivity of r₁(10.97 m M^-1s^-1),indicating the excellent T₁-weighted MR imaging（MRI）performance.The regulation extent of Ce-Gd@CDs-GOx nanoenzymes towards TME was explored by in vitro experiments.The oxidation effect of the nanoenzymes on glucose was investigated,and the color of the Ce-Gd@CDs-GOx solution changed from light yellow to bright yellow and the H₂O₂production increases with glucose concentration,revealing the efficient catalytic performance of the as-prepared nanoenzymes on glucose.The oxygen production efficiency of the nanoenzymes was evaluated by dissolved oxygen assay.The generation of O₂amount increased to 33 mg L^-1upon the Ce-Gd@CDs-GOx concentration of 100μg m L^-1within 5 min incubation,illustrating that the nanoenzymes could rapidly decompose H₂O₂to produce O₂and relieve hypoxia.The simultaneously producing·OH and consuming GSH by Ce-Gd@CDs-GOx under different conditions were analyzed by ESR spectroscopy.In the presence of glucose and GSH,the H₂O₂generated by GOx-catalyzed glucose decomposition promoted the Ce³⁺-Fenton reaction,and the efficiency of·OH generation by nanoenzymes was significantly improved.At the same time,the reducing capacity of GSH converted Ce⁴⁺into Ce³⁺,and thus accelerated the oxidization of GSH to oxidized glutathione（GSSH）,indicating that the nanoenzyme could consume GSH and increase the production of·OH,thus obtaining a higher catalytic therapeutic effect.The cellular uptake,cell toxicity and the killing effect of nanoenzymes to tumor cells were investigated by cellular experiments.The fluorescence intensity of 4T1 cells treated by Ce-Gd@CDs-GOx increased up to 47.3 after 8 h,confirming that the nanoenzymes could be efficiently uptaken by 4T1 cells.Furthermore,most of the 4T1 cells treated with Ce-Gd@CDs-GOx nanozymes were dead and the survival rate was only 23.3%in the CCK-8 and Calcein-AM/PI staining experiments,demonstrating the excellent effect of intracellular synergistic therapy.Moreover,the mechanism of apoptosis was investigated using DCFH-DA probe,which revealed that 4T1 cells incubated with Ce-Gd@CDs-GOx and GOx emitted strong fluorescence,elucidating that Ce-Gd@CDs-GOx nanoenzymes could effectively promote intracellular ROS production to inhibited the proliferation of tumor cells.In summary,this study offered a new strategy to design an“all-in-one”nanoplatform combining MR/FL imaging and nanocatalytic/starvation-like synergistic therapy,which could become a promising application as a novel nanoplatform for future clinical cancer diagnosis and treatment.