Study on Biocompatibility of Graphene Quantum Dots With Macrophages in vitro
Objective GQDs has become a superstar among zero-dimensional carbon-based materials.As one of the most abundant and important biological elements,its unique optical properties,high dispersion and biocompatibility have attracted extensive attention from scientists.This paper aims to investigate the effect of GQDs on cell viability,apoptosis and inflammatory factor expression in RAW264.7 macrophages and evaluate cell imaging capability of GQDs in vitro,which could provide theoretical basis for the safe application of GQDs in biomedical field.Methods Graphene oxide was prepared by modified Hummer's method.H2O2 and W18O49 interacted with each other under hydrothermal conditions to produce hydroxyl radicals,which can cut graphene oxide into GQDs using a top-down approach.The microstructure of GQDs was analyzed in detail by X-ray powder diffraction,X-ray photoelectron spectroscopy,transmission electron microscopy,atomic force microscopy,scanning electron microscopy and Fourier infrared transform.The biocompatibility of GQDs on macrophage was evaluated by CCK-8 and dead/alive staining.Flow cytometry results showed the apoptosis of RAW264.7 macrophages induced by GQDs.mRNA expression of inflammatory factors was evaluated by RT-qPCR.Cell imaging was exhibited by laser scanning confocal.Results Hydroxyl radicals are produced by H2O2 and W18O49 under hydrothermal conditions,which contribute to cut graphene oxide into 3-5 nm GQDs in one step.The quantum yield of this method is 43％.Fluorescence lifetime of these blue GQDs is 1.67 ns.The Zigzag-type site and defect state of the triplet carbene radical lead to the excitation wavelength dependence of GQDs,and the optimal excitation and emission wavelengths are 330 nm and 400 nm,respectively.The boundary effect and amphiphilicity of quantum dots make GQDs possess abundant functional groups,vacancy defects and high dispersion,which results in GQDs exhibits good water solubility.RAW264.7 macrophages are incubated with different concentration in DEME medium for 24 h,48 h and 72 h to evaluate cell.The survival rate of RAW264.7 cells is signifiicantly dependent on the concentration and time of GQDs.CCK-8 and dead/alive staining show that GQDs have high biocompatibility.The effect of 200 mg/L GQDs on apoptosis of RAW264.7 cells is revealed by the scatter plot of bivariate flow cytometry.Under the stimulation of LPS+INF-γ,the expression of TNF-α was increased in RAW264.7 cells,which co-acted with other cytokines to participate in the immune response of RAW264.7 cells in vitro,and mediated the production of IL-1β inflammatory factor in RAW264.7 cells,thereby inducing apoptosis of RAW264.7 cells.The results of RT-qPCR showed that GQDs can inhibit the growth of RAW264.7 cells in vitro,and stimulate them to increase TNF-α expression in RAW264.7 cells,which make cell membrane rupture and produce IL-1β inflammatory factors to induce cell apoptosis.The high biocompatibility of GQDs is attributed to the rich oxygen-containing functional groups(—COOH,—OH,and C ＝ O)on the surface of GQDs,which makes its surface negatively charged and easy to be swallowed into the cell interior when interacting with the cell membrane with low affinity.Transmission electron microscopy(TEM)observed that the GQDs were swallowed into the cells.Furthermore,laser confocal results displayed that blue GQDs has certain ability of cell imaging in vitro.Conclusion The water solubility,low toxicity,fluorescence properties and the induction effect of inflammatory factors of GQDs provide broad prospects for their application in the field of immunotherapy and cell imaging in the future.
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