石墨烯量子点与体外巨噬细胞生物相容性的研究  

Study on Biocompatibility of Graphene Quantum Dots With Macrophages in vitro

作　　者：刘琦 许海燕 苏雨轩 周开红[1] 李常艳[1] LIU Qi;XU Hai-Yan;SU Yu-Xuan;ZHOU Kai-Hong;LI Chang-Yan(College of Chemistry and Chemical Engineering,Inner Mongolia University,Huhhot 010021,China)

机构地区：[1]内蒙古大学化学化工学院,呼和浩特010021

出　　处：《生物化学与生物物理进展》2024年第11期2971-2982,共12页Progress In Biochemistry and Biophysics

基　　金：国家自然科学基金(22067015);内蒙古自治区研究生精品课程建设项目(JP20231008)资助。

摘　　要：目的探讨石墨烯量子点(GQDs)的对体外RAW264.7巨噬细胞存活率、细胞凋亡及炎症因子表达的影响和细胞成像能力,为GQDs在生物医学领域的安全应用提供理论基础。方法采用改性Hummer’s法制备了氧化石墨烯。利用H2O2和W_(18)O_(49)在水热条件下产生的羟基自由基,采用自上而下的方法将氧化石墨烯切割成GQDs。利用X射线粉末衍射、X光电子能谱、透射电镜、原子力显微镜、扫描电镜和傅里叶红外变换等技术对GQDs微观结构进行详细分析。通过CCK-8、流式细胞、激光共聚焦方法和实时荧光定量PCR(RT-qPCR),评价GQDs对巨噬细胞的生物兼容性和对炎症因子表达的影响。通过CCK-8、流式细胞术和RT-qPCR,评价GQDs对巨噬细胞的生物兼容性和炎症因子的影响。激光共聚焦显示GQDs在巨噬细胞中成像情况。结果水热条件下,以W_(18)O_(49)为催化剂可将氧化石墨烯切割为3~5 nm蓝光GQDs,产率为43%,荧光寿命(τ)=1.67 ns。三重态碳烯自由基的Zigzag型位点和缺陷态导致GQDs表现出激发波长的依赖性,其最佳激发和发射波长分别为330 nm和400 nm。GQDs表面丰富的含氧官能团和其亲水性使其具有良好的水溶性。CCK-8和死活染色表明,GQDs具有较高的生物兼容性。RT-qPCR分析结果显示,GQDs对体外RAW264.7细胞有生长抑制作用,可刺激RAW264.7细胞提高TNF-α的表达,使细胞膜破裂并产生IL-1β炎症因子诱导细胞凋亡。激光共聚焦结果显示,蓝光GQDs具有一定的体外细胞成像能力。结论石墨烯量子点的水溶性、低毒、荧光特性和炎症因子的诱导效应,为其在免疫标记和细胞成像领域的应用提供了广阔的前景。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 W_(18)O_(49) 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 W_(18)O_(49) 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

关 键 词：W18O49 石墨烯量子点 RAW264.7巨噬细胞 生物相容性 炎症因子 

分 类 号：Q23[生物学—细胞生物学]