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Current methods for the evaluation of cellular interactions with nanoparticles are non-specific, relatively slow and invasive to the cell. Raman spectroscopy is a non invasive technique whose potential in the biosciences has already been demonstrated and has been used in the investigation of cell interactions with various external agents. The main focus of this study is to employ Raman spectroscopy to investigate the interaction of A549 human lung cells with single walled carbon nanotubes. · Carbon nanotubes have attracted considerable interest not only for their outstanding physical and electronic properties, promising a potentially vast number of applications, but also for their potential toxicological risks as nanoparticles. Determination of their toxic response using classical cyto-toxicological assays has proven problematic due to their interaction with colorimetric assay methods. In this work, in vitro samples were prepared similar to samples used for clonogenic assays and were examined using Raman spectroscopy. The acquired spectra of the treated and untreated cells were analysed for spectral changes in the fingerprint region to try to establish correlations between changes to the cellular spectra and the results of the clonogenic assay. Peak area ratio analysis suggests a dose dependent response which correlates to previous toxicological studies. Multivariate techniques such as principal component analysis were employed to further classify cellular response as a function of dose and to examine differences between spectra as a function of carbon nanotube concentration . It is thus shown that the exposed and unexposed cells can be well differentiated on the basis of Raman spectral features. The project is basically divided into four sections, each of them assesses one aspect of the toxic effects of SWCNTs and the investigation of those by Raman spectroscopy. The responses of cells in the presence of nanoparticles, the effect of oxidant production and medium depletion on the Raman spectral response and toxicology of the cell are examined independently. In all cases the changes to the spectral response will be correlated with literature data from classical assays. These spectral data build knowledge on the effects of the individual response mechanisms, on the overall spectral content of the cell in the presence of SWCNTs, and allow the interpretation of subsequent measurements of spectral content after exposure to a wider range of concentrations of SWCNTs. As the development of Raman spectroscopy, for the analysis of cellular toxicity, is a key feature of this work, the development of applicable processing methods is a crucial part. Multivariate analysis (e.g. independent component analysis (ICA) partial least squares regression PLS regression and principal component analysis PCA) are employed to analyse these aspects differentially and to classify and model the cellular responses. Finally a comprehensive demonstration of the ability of Raman spectroscopy to characterise the cellular response of A549 cells to the presence of Carbon nanotubes is made, demonstrating it to be a realistic alternative to classical cytotoxicological techniques though highlighting the increased demands to experimental designs while employing nano particles.
Knief, P.: Interactions of Carbon Nanotubes with Human Lung Epithelial Cells In Vitro, Assessed by Raman Spectroscopy. Doctoral Thesis. Dublin Institute of Technology, 2010.