Preparation Of Cerium Doped Carbon Quantum Dots With High Efficiency In Reactive Oxide Species Scavenging For Biological Antioxidant Reserch

Objective:Reactive oxygen species?ROS?,as a natural product of oxygen metabolism,participate in various normal physiological activities.However,under the influence of disease or some exogenous factors,the balance of free radical metabolism is destroyed,leading to peroxidation damage of cells and biological macromolecules.Therefore,it is an important and urgent problem to control the concentration of ROS in organisms.Keeping them in an appropriate concentration range is also a basic means to reduce a series of oxidative abnormalities.In recent years,cerium oxide has attracted extensive attention of researchers due to its regenerative antioxidant properties,and has been widely used in bioengineering and medical research.Based on the our previous research,a novel Cerium doped carbon quantum dots?Ce-doped CQDs?was synthesized by one-step hydrothermal carbonization using citric acid and Ce?NO₃?₃·6H₂O as precursors and glycine as surface passivators.The as-prepared Ce-doped CQDs retained the low toxicity and good fluorescence properties of traditional carbon quantum dots,and further obtained the ability of scavenging hydroxyl radicals.This study systematically characterized the physical,chemical and optical properties of Ce-doped CQDs,confirmed the hydroxyl radical ability of Ce-doped CQDs,as well as explored the application potential of biological antioxidant in vitro.Methods:1.Ce-doped CQDs were prepared by one-step hydrothermal carbonization using Ce?NO₃?₃·6H₂O and citric acid as precursors and glycine as surface passivator.The physical properties of Ce-doped CQDs were characterized by high resolution transmission electron microscopy?HRTEM?and X-ray diffraction spectra?XRD?;the chemical properties of Ce-doped CQDs were characterized by X-ray photoelectron spectroscopy?XPS?and Fourier transform infrared spectroscopy ?FTIR?;and the optical properties of Ce-doped CQDs were characterized by ultraviolet-visible spectrophotometer and fluorescence spectroscopy.2.Using methyl violet?MV?as color reagent,according to typical Fenton reaction,Fe²⁺can catalyze H₂O₂ to produce hydroxyl radicals.The generated hydroxyl radicals can react with MV,making then colorless or even colorless.Meanwhile,the maximum absorbance value of MV at 582 nm decreases.Direct evidence of the scavenging activity of Ce-doped CQDs on hydroxyl radicals can also be obtained by this simple spectrophotometry,and the scavenging activity of Ce-doped CQDs on hydroxyl radicals can be proved by electron spin resonance spectroscopy.3.The biological safety of Ce-doped CQDs was verified by CCK-8 assay.Laser confocal microscopy was used to observe the phagocytosis of Ce-doped CQDs,as well as its localization and imaging ability in cells.4.The cell model of oxidative stress in vitro was established by H₂O₂ treatment.The antioxidant capacity of Ce-doped CQDs was explored by CCK-8 and fluorescence staining of living and dead cells.Result:1.Through a series of optimized reaction conditions,we finally chose the raw material ratio of citric acid,glycine and Ce?NO₃?₃·6H₂O is 1.68 g:1.50 g:0.8 g,and the reaction volume is 20 mL,as well as the reaction condition is 200? for 4 h.The obtained Ce-doped CQDs had the optimum carbonization effect and fluorescence performance.2.The average size of Ce-doped CQDs is about 2.5 nm,which conforms to the Gauss normal distribution curve.Ce-doped CQDs have good dispersion,spherical shape and mixed phase structure,including both of graphene and graphene oxide characteristic features.Ce-doped CQDs are mainly composed of carbon,nitrogen,oxygen and cerium.The surface of Ce-doped CQDs is rich in carboxyl,hydroxyl and other oxygen-containing groups,which makes it has good water solubility.The proportion of Ce³⁺with high correlation with antioxidation is about 46.03%.Ce-doped CQDs have good photoluminescence properties and good photostability.3.The results of MV colour rendering experiments show that even low concentration of Ce-doped CQDs can quickly restore the color of MV solution and the absorbance at 582 nm,which indicates that Ce-doped CQDs can scavenge hydroxyl radicals quickly and efficiently.At the same time,long-cycle experiments verify that the oxidation-reduction reaction on the surface of Ce-doped CQDs is a continuous process.ESR experiments further confirmed the above results.4.Different concentrations of Ce-doped CQDs were co-incubated with MEF and NIH 3T3 cells,and the cells maintained high activity even at concentrations up to 800?g/mL.Ce-doped CQDs showed no significant toxicity to cells.5.After phagocytosed by cells,Ce-doped CQDs did not significantly influence these cell morphologies.The Ce-doped CQDs were mainly located in the cytoplasm,which emitted bright green fluorescence under blue excitation light.Ce-doped CQDs can rapidly cross cell membrane and enter cytoplasm within 5 minutes,and maintain long-term persistent luminescence.6.Through MEF and NIH 3T3 cells experiments,Ce-doped CQDs were verifided that they could protect cells against oxidative stress damage reduce cell apoptosis and improve cell survival.Conclusion:1.Ce-doped CQDs were successfully prepared by one-step hydrothermal carbonization.Ce-doped CQDs are composed of carbon,nitrogen,oxygen and cerium.The surface of Ce-doped CQDs particles is rich in oxygen-containing groups and nitrogen-containing groups,which make them have good water solubility and fluorescence properties.2.Ce-doped CQDs possess super high specific area,which result in elevating the proportion rate of Ce³⁺/Ce⁴⁺.Furthermore,high proportion of Ce³⁺/Ce⁴⁺enhance the scavenging capacity of hydroxyl radicals.3.Ce-doped CQDs have good biocompatibility and excellent performance in bioimaging.4.Ce-doped CQDs showed excellent biological antioxidant activity in vitro.