Farhane Z, Bonnier F, Casey A, Byrne H J
FOCAS Research Institute, Dublin Institute of Technology, Kevin Street, Dublin 8, Ireland.
Analyst. 2015 Jun 21;140(12):4212-23. doi: 10.1039/c5an00256g. Epub 2015 Apr 28.
Vibrational spectroscopy, including Raman micro spectroscopy, has been widely used over the last few years to explore potential biomedical applications. Indeed, Raman micro spectroscopy has been demonstrated to be a powerful non-invasive tool in cancer diagnosis and monitoring. In confocal microscopic mode, the technique is also a molecularly specific analytical tool with optical resolution which has potential applications in subcellular analysis of biochemical processes, and therefore as an in vitro screening tool of the efficacy and mode of action of, for example, chemotherapeutic agents. In order to demonstrate and explore the potential in this field, established, model chemotherapeutic agents can be valuable. In this study paper, Raman micro spectroscopy coupled with confocal microscopy were used for the localization and tracking of the commercially available drug, doxorubicin (DOX), in the intracellular environment of the lung cancer cell line, A549. Cytotoxicity assays were employed to establish clinically relevant drug doses for 24 h exposure, and Confocal Laser Scanning Fluorescence Microscopy was conducted in parallel with Raman micro spectroscopy profiling to confirm the drug internalisation and localisation. Multivariate statistical analysis, consisting of PCA (principal components analysis) was used to highlight doxorubicin interaction with cancer cells and spectral variations due to its effects before and after DOX spectral features subtraction from nuclear and nucleolar spectra, were compared to non-exposed control spectra. Results show that Raman micro spectroscopy is not only able to detect doxorubicin inside cells and profile its specific subcellular localisation, but, it is also capable of elucidating the local biomolecular changes elicited by the drug, differentiating the responses in different sub cellular regions. Further analysis clearly demonstrates the early apoptotic effect in the nuclear regions and the initial responses of cells to this death process, demonstrating the potential of the technique to monitor the mechanisms of action and response on a molecular level, with subcellular resolution.
在过去几年中，振动光谱学，包括拉曼显微光谱学，已被广泛用于探索潜在的生物医学应用。事实上，拉曼显微光谱学已被证明是癌症诊断和监测中的一种强大的非侵入性工具。在共聚焦显微镜模式下，该技术还是一种具有光学分辨率的分子特异性分析工具，在生化过程的亚细胞分析中具有潜在应用，因此可作为例如化疗药物疗效和作用方式的体外筛选工具。为了证明和探索该领域的潜力，已有的模型化疗药物可能很有价值。在本研究论文中，拉曼显微光谱学与共聚焦显微镜相结合，用于在肺癌细胞系A549的细胞内环境中定位和追踪市售药物阿霉素（DOX）。采用细胞毒性试验来确定24小时暴露的临床相关药物剂量，并与拉曼显微光谱分析并行进行共聚焦激光扫描荧光显微镜检查，以确认药物的内化和定位。由主成分分析（PCA）组成的多变量统计分析用于突出阿霉素与癌细胞的相互作用以及由于其作用导致的光谱变化，在从细胞核和核仁光谱中减去DOX光谱特征前后，将光谱变化与未暴露的对照光谱进行比较。结果表明，拉曼显微光谱学不仅能够检测细胞内的阿霉素并描绘其特定的亚细胞定位，而且还能够阐明药物引起的局部生物分子变化，区分不同亚细胞区域的反应。进一步分析清楚地证明了核区域的早期凋亡效应以及细胞对这种死亡过程的初始反应，证明了该技术在分子水平上以亚细胞分辨率监测作用机制和反应的潜力。
