Development of surface-enhanced Raman scattering-based nanospectroscopic methods for toxicity determination of nanomaterials

The intensive research on nanomaterials (NMs) in the last decades resulted in broad applications in many industries ranging from energy deposition to medicine, cosmetics, food, textile, military or communication. The tremendous utilization of these newly discovered materials, however, also raised concerns about their possible side effects. On the other hand, the efforts to determine NM toxicity faced problems in terms of obtaining accurate results; originating from the physicochemical properties of NMs. Most of the conventional cytotoxicity assays have been reported to give false results due to interaction with assay components or absorbance artefacts. Therefore, in vitro nanotoxicity evaluation field is in need of alternative approaches. In an attempt to propose a new perspective, in the present work, surface-enhanced Raman scattering (SERS) was utilized on four cell lines; lung adenocarcinoma (A549), human and mouse fibroblasts (HSF, L929), and human umbilical vein endothelial cell line (HUVEC), to test in vitro nanotoxicity of a panel of NMs; zinc oxide and titanium dioxide nanoparticles, single- and multi-walled carbon nanotubes, and quantum dots. Before that, SERS substrate localization within the cells as well as the spectral contributors were evaluated. Then, SERS spectra obtained from NM-exposed cells were carefully investigated for possible toxicity markers and seven of them were proposed to be in good correlation with the conventional cytotoxicity assays; apoptosis/necrosis and WST-1 cell proliferation assays, as well as the transmission electron microscopy and enhanced-dark field microscopy images. Altogether the results were in agreement with the nanotoxicity studies in the literature. The seven intensity ratios gave information about the rates of collagen, fibronectin, cholesterol depletion, lipid stability, tyrosine phosphorylation, phenylalanine to tyrosine conversion and protein C-S to S-S bond conversion, all of which can also be utilized for various further cellular SERS studies.