光谱学与光谱分析, 2018, 38 (9): 2770, 网络出版: 2018-10-02   

三维拉曼成像技术用于纳米金刚石与细胞相互作用过程的研究

Visualization of the Interaction between NDs and Cells with 3D Raman Imaging
作者单位
1 中国科学院理化技术研究所, 北京 100190
2 中国科学院大学, 北京 100049
3 北京大学药学院, 北京 100191
摘要
纳米金刚石(NDs), 作为一种具备良好生物兼容性、 化学稳定性、 药物负载能力和众多不可比拟优越性能的材料, 其在生物医学领域的应用被广泛关注, 尤其是在生物成像和抗癌药物传输领域。 首先对不同尺寸纳米金刚石的拉曼性能进行评价, 确定了100 nm高温高压合成的NDs更适宜作为拉曼生物探针。 之后, 为了生物领域的应用, 这些NDs表面的杂质经过羧基化方式处理获得均一表面性能, 并采用扫描电镜、 红外、 拉曼和粒径分析手段对该过程进行验证。 然后, NDs作为拉曼探针被用于快速定位HeLa细胞内NDs的分布, 验证了HepG2细胞对NDs内吞过程的时间依赖性。 此外, 借助非侵入性的三维(3D)共聚焦拉曼成像技术, 可视化观察了四种不同细胞(HeLa, HepG2, C6和MDCK)对NDs内吞量和滞留量的差异。 其中, MDCK这种正常细胞内部极少发现NDs, 而其他三种癌细胞中有大量NDs信号, 显示出不同种类细胞对于NDs的吞入和滞留量的明显差异。 实验结果表明, 纳米金刚石拉曼生物探针不仅可以用于生物成像, 更为癌症的定位和诊断提供可能性。
Abstract
Nanodiamonds, with good bio-compatibility, chemical stability, drug accommodatingability, fluorescence signals and many other superior properties, have attracted growing attention for anti-cancer drug delivery and bio-imaging applications in recent decades. In this work, the intrinsic Raman signal (1 332 cm-1) of nanodiamonds (NDs) with different sizes synthesized by high temperature high pressure (HTHP) and explosion methods were investigated and compared, in order to select suitable NDs as Raman probes for bio-application, indicating that HTHP synthesized NDs with the size of 100 nm were preferable. NDs were surface homogenized to remove the impurity for further bio-application through acid carboxylation, which was verified with Scanning Electron Microscopy (SEM), Frourier Transform Infared Spectroscopy (FTIR), Raman Spectroscopy and size analysis. And the cytotoxicity of NDs to different cell lines was confirmed with Cell Counting Kit-8 (CCK-8) assay, assuring the good bio-compatibility of NDs and their potential application in bio-system. Besides, based on the sharp Raman peak of NDs at 1 332 cm-1 and the representative Raman vibration band of cells in 2 800~3 000 cm-1 range, NDs were used as Raman bio-probes for the fast localization of NDs in HeLa cells, and the non-invasive dual-color two-dimensional (2D) confocal Raman imaging technique with 532 nm laser was successfully realized. Furthermore, the uptake process of NDs into HepG2 cells was visualized with Raman mapping, revealing the time-dependent behavior of this internalization process. Besides, the endocytosis and localization of NDs as Raman probes by different cells, including HeLa, HepG2, C6 and MDCK cells, were further investigated using the advanced three-dimensional (3D) confocal Raman imaging microscopy, which could render detailed information about the interaction of NDs and cells. And it was observed that the endocytosis of NDs was influenced by cell lines, not only for cancer and non-cancer cells, but also for different cancer cell lines, providing further potential applications for bio-imagingand cancer diagnosis. These results were encouraging and sufficient for following bio-medical studies.

李丹丹, 陈鑫, 王宏, 付杨, 余愿, 只金芳. 三维拉曼成像技术用于纳米金刚石与细胞相互作用过程的研究[J]. 光谱学与光谱分析, 2018, 38(9): 2770. LI Dan-dan, CHEN Xin, WANG Hong, FU Yang, YU Yuan, ZHI Jin-fang. Visualization of the Interaction between NDs and Cells with 3D Raman Imaging[J]. Spectroscopy and Spectral Analysis, 2018, 38(9): 2770.

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