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Analytical and Bioanalytical Chemistry, February 2017, Volume 409, Issue 5, pp 1333–1346.


Raman microspectroscopy is a non-invasive, in vitro analytical tool which is being increasingly explored for its potential in clinical applications and monitoring the uptake, mechanism of action and cellular interaction at a molecular level of chemotherapeutic drugs, ultimately as a potential label-free preclinical screening and companion diagnostic tool. In this study, doxorubicin (DOX), a “gold standard” chemotherapeutic drug, is employed as a model in the in vitro lung cancer cell line A549 in order to demonstrate the potential of Raman microspectroscopy to screen and identify spectroscopic markers of its trafficking and mechanism of action. Confocal laser scanning microscopy (CLSM) was used in parallel to illustrate the uptake and subcellular localisation, and cytotoxicity assays were employed to establish the toxicity profiles for early and late exposure times of A549 to DOX. Multivariate statistical analysis, consisting of principal components analysis (PCA), partial least squares regression (PLSR) and independent component analysis (ICA), was used to elucidate the spectroscopic signatures associated with DOX uptake and subcellular interaction. Raman spectroscopic profiling illustrates both drug kinetics and its pharmacodynamics in the cell and associated biochemical changes, demonstrating that DOX is mainly localised in the nuclear area, saturating the nucleolus first, within ~6 h of exposure, before the surrounding nuclear areas after ~12 h, and only accumulates in the cytoplasm after 48 h. PLSR over varying time intervals enables identification of DOX–DNA binding at early stages of exposure (0–12 h), while regression over longer time periods (24–72 h) reveals spectroscopic signatures associated with the metabolic cellular response.



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