Advancing Vibrational Spectroscopy for Cellular and Sub Cellular Analysis: Raman Spectroscopy as a Novel In Vitro Nanotoxicological Assessment Protocol

This work is designed to establish a ‘High content Nanotoxicological Screening method’ using in vitro Raman microspectroscopy. The undeniable increase of nanotechnology based products has brought challenges in terms of determining their toxicological properties. Considering the total time and cost of screening nanomaterials by conventional methods, the need for a rapid, label-free technique which will provide a wide range of information on multiple parameters is unquestionable. This study investigated the applicability of Raman microspectroscopy as a High Content Screening technique to clarify cell-nanoparticle interaction by determining the localisation of the nanoparticles and consequent effects in these localised areas in terms of cyto- and geno- toxicity. For this purpose, in the first part of the study, the potential of Raman spectroscopy has been explored to monitor sequential trafficking of nanoparticles in cellular organelles and to determine the differing spectral signatures of the organelles. Human lung carcinoma cells (A549) were exposed to non-toxic carboxylate-modified and fluorescently-labelled polystyrene nanoparticles for 4, 12 and 24 hours and Raman spectral maps were acquired from the subcellular regions to determine their localisation. With the aid of multivariate analysis techniques, the study demonstrated the applicability of Raman microspectroscopy to provide information regarding localisation and to determine the local environment based on differing signatures of intracellular compartments such as endosomes, lysosomes and endoplasmic reticulum, in a completely label free manner. Aminated polystyrene nanoparticles (PS-NH2) and polyamidoamine (PAMAM) dendrimers are known to show acute toxicity and in order to observe this toxicity and corresponding responses, time and dose dependant Raman spectroscopic markers of cellular toxic events were systematically monitored upon nanoparticle exposure to A549 cells. Alamar Blue (AB) and 3-(4,5-Dimethylthiazol-2-yl)-2,5diphenyltetrazoliumbromid (MTT) assays were employed to determine the mean effective concentration, EC50 and Raman spectroscopy was used to acquire multiple point spectra from the cytoplasm, nucleus and nucleolus. The most significant changes were observed in the cytoplasm for both time and dose dependent cases. The Raman spectral markers for lipidosis and oxidative stress were determined as a function of dose and time, and the responses were correlated with conventional cytotoxicity assays. With the aid of multivariate analysis techniques, the study showed the ability of Raman spectroscopy to provide information about cellular responses at different particle concentrations and exposure times. Following this, the potential of Raman microspectroscopy was analysed by comparing spectral marker evolution in non-cancerous cells (immortalized human bronchial epithelium) with cancerous cells (A549 and human lung epidermoid). Spectral markers of oxidative stress, cytoplasmic RNA aberrations and liposomal rupture were identified and cell-line dependent variations in these spectral markers were observed, and were correlated with cellular assays and imaging techniques. The findings from the comparison of spectral markers, especially in the low wavenumber region, have shown the applicability of Raman spectroscopy to identify different cell death pathways in cancerous and non-cancerous cell lines. Different cell death mechanisms were also identified based on common and/or differing spectral markers of cyto- and geno- toxicity upon PS-NH2 and PAMAM exposure. The results were correlated with flow cytometry and cytotoxicity assays. The study further demonstrated the potential of Raman microspectroscopy to iii differentiate apoptotic and necrotic cell death mechanisms, as a function of time (from 4 to 72 h) and applied dose (sub-lethal/lethal). Finally, in order to establish a toxicological assessment protocol based on Raman microspectroscopy, 3D graphs of biomarker intensities are plotted as a function of time and dose and also intensities are correlated with % viability values. The established 3D models can be used to predict nanotoxicity, which can also be applied to nanomedicine