Raman micro spectroscopy for in vitro drug screening: subcellular localisation and interactions of doxorubicin

Abstract

Raman spectroscopy is used for the localization and tracking of chemotherapeutic drug, doxorubicin, in the intracellular environment of lung cancer cell line. Results show the potential of the technique to monitor the mechanisms of action and response on a molecular level, with subcellular resolution.Please check this proof carefully. Our staff will not read it in detail after you have returned it.Translation errors between word-processor files and typesetting systems can occur so the whole proof needs to be read. Please pay particular attention to: tabulated material; equations; numerical data; figures and graphics; and references. If you have not already indicated the corresponding author(s) please mark their name(s) with an asterisk. Please e-mail a list of corrections or the PDF with electronic notes attached -do not change the text within the PDF file or send a revised manuscript. Corrections at this stage should be minor and not involve extensive changes. All corrections must be sent at the same time.Please bear in mind that minor layout improvements, e.g. in line breaking, table widths and graphic placement, are routinely applied to the final version.We will publish articles on the web as soon as possible after receiving your corrections; no late corrections will be made. Please ensure that all queries are answered when returning your proof corrections so that publication of your article is not delayed. Vibrational spectroscopy, including Raman spectroscopy, has been widely used over the last few years to explore potential biomedical applications. Indeed, Raman 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 study paper, Raman 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 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 an...

Spectroscopic studies of anthracyclines: Structural characterization and in vitro tracking

“…The impact on cells of DOX exposure has been assessed in various in vitro tests [14,17,[48][49][50]. Spectroscopic methods have also been employed to study the interactions in vitro [25,[51][52][53]. The development of a new therapeutic agent always involves a preclinical screening stage.…”

“…Anthracyclines, especially DNR and DOX have been widely investigated using various spectroscopic methods, e.g. Raman spectroscopy [24,25], Resonance Raman (RR) [26][27][28], Surface Enhanced Raman Spectroscopy (SERS) [29][30][31][32], UV-Vis absorption [26,33] and FT-IR absorption spectroscopy [24,34,35]. The visible absorption spectra of DOX and DNR are, however, almost identical, having a maximum of absorption near to 480-490 nm.…”

Cellular discrimination using in vitro Raman micro spectroscopy: the role of the nucleolus

“…The normal and cancerous cell lines are now largely discriminated by PC1 and almost the same discriminating features can be found in the loadings of PC1 for the nucleolar and nuclear regions, while additional peaks in the loading of PC1 for the cytoplasm of the normal versus cancerous cells are observed at 760 and 820 (tryptophan ring) and 1250 (Amide III), related to normal cells and ones at 717 (CN + (CH 3 ) 3 stretching), 1400 (CH deformation) and 1578 cm −1 ( protein) 1661 cm −1 (lipids CvC stretching) related to cancerous cell lines. 4,17,30,47 Thus using PCA allows a separation between normal and cancerous cell lines. Notably, however, no differentiation of the cancerous cell lines is evident.…”

“…16,17 Thus, the potential applications extend beyond disease diagnostics to the label free in vitro screening of cytological processes, such as drug or nanoparticle uptake and mechanisms of interaction, and toxicology. 16,[18][19][20] There has been a wide range of studies to date demonstrating the potential of Raman micro spectroscopy to map live and fixed cells with subcellular resolution, [21][22][23][24][25] profile the distribution of anticancer agents [26][27][28][29][30] and nanoparticles in cells 16,31,32 and monitor subcellular processes 33 and toxicological responses. [34][35][36][37] Fundamental to the development of applications of Raman micro spectroscopy for disease diagnostics as well as analysis of cytological processes is an understanding of the variability of the spectral signatures across the subcellular environment, their potential for differentiation of cell phenotype or diseased state, and their sensitivity to external perturbation, such as viral infection, radiation damage, or chemical stress due to, for example, toxic or chemotherapeutic agents.…”

Spectral pre and post processing for infrared and Raman spectroscopy of biological tissues and cells

“…IR absorption and Raman spectroscopy are very distinct physical processes [2], and therefore the spectral distortions can be of very different physical origin. Although many biomedical applications of the techniques have been targeted towards disease diagnostics, for which high specificity and sensitivity classification algorithms are desirable, more recent applications have been targeted at, for example, disease aetiology [3,4], radiation dosimetry [5], drug screening [6][7][8][9][10] and nanotoxicology [11][12][13], for which a range of other data mining and analysis protocols have been explored [14]. It is important, however, that these protocols are well validated, in order to progress the field with confidence.…”