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Numerous toxicity studies have been conducted to date both in vivo and in vitro on refined and raw Single Walled Carbon Nanotubes (SWCNT). Differences in SWCNT toxicity and biocompatibility have been observed between these studies, and whilst these discrepancies have been attributed to factors such as varying percentages of remnant catalytic particles, differences in dispersion methods etc. the mechanisms underlying these inconsistencies have not been investigated. This study used standard spectroscopic and cellular techniques to elucidate the origins of these inconsistencies and also to estimate the validity of toxicological data evaluated form standard cytotoxic endpoints. Spectroscopic studies were conducted in order to demonstrate and elucidate the interactions of HiPco SWCNT with cell culture medium and its components, prepared both with and without foetal bovine serum. Upon addition of raw SWCNT to the medium a noticeable colour change was observed. UV/Vis absorption spectroscopy revealed a dramatic reduction in the absorption attributable to the phenol red, a pH indicator within the medium, without an associated change in pH. Reductions were also observed in absorbance features attributed to various components of the medium indicating an interaction with the SWCNT. Fluorescence spectroscopy also revealed reductions in emission features associated with the components of the medium giving further support to an interaction. Concentration dependent studies of the fluorescent emission of the various components of the media were modelled to show a differing degree of interaction between the SWCNT and the various components. Finally, notable differences were observed between the behaviour with and without serum. Raman spectroscopy gave no indication of differences between raw SWCNT and those deposited from the medium suspension indicating that no debundling of the SWCNT occurred. The results and their implications for toxicity and in vitro studies are discussed. To investigate if these interactions could induce a secondary toxicity by medium depletion, further spectroscopic and cytotoxicity studies were performed. SWCNT media suspensions were created, centrifuged and filtered (0.2µm cellulose acetate filters) to remove the SWCNT. Spectroscopic analysis was carried out on the filtered samples to verify the removal of the SWCNT from the suspension but also to assess the degree of alteration of the medium due to the aforementioned interactions. Cytotoxicity studies were performed on human alveolar A549 cells with the depleted media. Two cytotoxic endpoints were employed to evaluate cytotoxicity, namely Alamar blue and neutral red. Concentration dependant exposures over different time periods revealed low acute cytotoxicity after 72 and 96 hour exposure to the filtered samples, verifying the proposed notion of a secondary toxicity due to medium depletion. Finally the direct cytotoxicity of single walled carbon nanotubes was evaluated in the A549 human alveolar carcinoma cell line. Cell viability was assessed using the following indicator dyes, Commassie Blue, Alamar Blue , Neutral Red, MTT and WST-1. Exposure of the A549 cells revealed the nanotubes to have low acute toxicity. However considerable variation was found depending on the dye employed. Spectroscopic analysis of the nanotubes’ interactions with the dyes revealed interactions in all cases, resulting in the reduction of the associated absorption/fluorescent emission which is used to evaluate particle toxicity. In addition to being sensitive, simple, safe and cost-effective the ideal test for in vitro cell cytotoxicity must also not interfere with the compound to be tested. The results therefore have comprehensively confirmed that the indicator dyes used in this study were not appropriate for the quantitative toxicity assessment of carbon nanotubes highlighting the pressing need for the development of alternative screening techniques.
Casey, A. (2007). Physiochemical indicators of single walled carbon nanotube toxicity. Doctoral thesis. Technological University Dublin. doi:10.21427/D73P4J