Faculty of Physics and Applied Computer Science, AGH University of Krakow, Mickiewicza 30, 30-059 Krakow, Poland.
Institute of Zoology and Biomedical Research, Jagiellonian University, Gronostajowa 9, 30-387 Krakow, Poland.
Spectrochim Acta A Mol Biomol Spectrosc. 2024 Dec 15;323:124888. doi: 10.1016/j.saa.2024.124888. Epub 2024 Jul 26.
An important issue in the context of both potenial toxicity of iron oxide nanoparticles (IONP) and their medical applications is tracking of the internalization process of these nanomaterials into living cells, as well as their localization and fate within them. The typical methods used for this purpose are transmission electron microscopy, confocal fluorescence microscopy as well as light-scattering techniques including dark-field microscopy and flow cytometry. All the techniques mentioned have their advantages and disadvantages. Among the problems it is necessary to mention complicated sample preparation, difficult interpretation of experimental data requiring qualified and experienced personnel, different behavior of fluorescently labeled IONP comparing to those label-free or finally the lack of possibility of chemical composition characteristics of nanomaterials. The purpose of the present investigation was the assessment of the usefulness of Raman microscopy for the tracking of the internalization of IONP into cells, as well as the optimization of this process. Moreover, the study focused on identification of the potential differences in the cellular fate of superparamagnetic nanoparticles having magnetite and maghemite core. The Raman spectra of U87MG cells which internalized IONP presented additional bands which position depended on the used laser wavelength. They occurred at the wavenumber range 1700-2400 cm for laser 488 nm and below the wavenumber of 800 cm in case of laser 532 nm. The intensity of the mentioned Raman bands was higher for the green laser (532 nm) and their position, was independent and not characteristic on the primary core material of IONP (magnetite, maghemite). The obtained results showed that Raman microscopy is an excellent, non-destructive and objective technique that allows monitoring the process of internalization of IONP into cells and visualizing such nanoparticles and/or their metabolism products within them at low exposure levels. What is more, the process of tracking IONP using the technique may be further improved by using appropriate wavelength and power of the laser source.
在氧化铁纳米粒子（IONP）的潜在毒性及其医学应用的背景下，一个重要问题是跟踪这些纳米材料进入活细胞的内化过程，以及它们在细胞内的定位和命运。为此目的通常使用的方法是透射电子显微镜、共聚焦荧光显微镜以及包括暗场显微镜和流式细胞术在内的光散射技术。所有提到的技术都有其优点和缺点。在需要提及的问题中，包括复杂的样品制备、需要合格和经验丰富的人员进行实验数据解释、荧光标记的 IONP 与未标记的或最终缺乏纳米材料化学组成特征的 IONP 行为不同等问题。本研究的目的是评估拉曼显微镜在跟踪 IONP 内化到细胞中的有用性，以及优化该过程。此外，该研究侧重于鉴定具有磁铁矿和磁赤铁矿核心的超顺磁性纳米粒子的细胞命运的潜在差异。内化了 IONP 的 U87MG 细胞的拉曼光谱呈现出与所使用的激光波长有关的额外谱带。当激光波长为 488nm 时，这些谱带出现在 1700-2400cm-1 的波数范围内，而当激光波长为 532nm 时，这些谱带出现在 800cm-1 以下的波数范围内。这些拉曼谱带的强度对于绿光激光（532nm）更高，其位置与 IONP 的主要核心材料（磁铁矿、磁赤铁矿）无关且不具有特征性。所得结果表明，拉曼显微镜是一种极好的、非破坏性的和客观的技术，它允许在低暴露水平下监测 IONP 内化到细胞中的过程，并可视化这些纳米粒子和/或它们在细胞内的代谢产物。更重要的是，通过使用适当的激光源波长和功率，可以进一步改进使用该技术跟踪 IONP 的过程。
