Surface-Enhanced Raman Scattering to Evaluate Nanomaterial Cytotoxicity on Living Cells

Abstract

The increasing number of reports about false positive or negative results from conventional cytotoxicity assays of nanomaterials (NMs) suggests that more reliable NM toxicity assessment methods should be developed. Here, we report a novel approach for nanotoxicity evaluation based on surface-enhanced Raman spectroscopy (SERS). Three model NMs were tested on two model cell lines and the results were validated by WST-1 cytotoxicity assay and annexin V-FITC/propidium iodide (PI) staining as apoptosis-necrosis assay. The localization of nanoparticles (NPs) in the cells and the cellular conditions upon NP incubation were visualized by transmission electron microscopy (TEM) and enhanced dark-field (EDF) microscopy. SERS revealed a broader view on the consequences of cell-NM interactions compared to the conventional cytotoxicity assays where only one aspect of toxicity can be measured by one assay type. The results suggest that SERS can significantly contribute to the cytotoxicity evaluation bypassing NM or assay component-related complications with less effort.

What do we actually see in intracellular SERS? Investigating nanosensor-induced variation

“…We point out the major spectral differences and discuss their assignment below. ) which implies the destabilisation of protein folding by the breaking of cysteine-cysteine bonds; it is clear following 250 µM AuNP treatment and is in agreement with previous cell stress studies 11,12 . Conversely, the intensity of the C-C ring breathing vibration in Phe (1000 cm -1 ) and that of its acidified form (1030 cm ) in Phe/proteins was also observed with increased AuNP uptake.…”

“…The vibrational mode at 1305 cm -1 relates to CH 2 deformation in lipids, with its decreased intensity inferring possible degradation of phospholipids. This cell stress event has previously been detected by SERS within HSF and A549 cell lines upon stress induced by exposure to ZnO NPs 12 .…”

“…Correlative information provided by DCF-DA ROS assay identified that oxidative stress plays some role in the complex spectral changes observed following excessive NP uptake by cells. Assignment of spectral variations arising from AuNP-induced stress identified SERS peaks which have been previously attributed to oxidative stress following administration of other inorganic NPs, such as protein denaturation and misfolding events, lipid decomposition and DNA fragmentation 11,12 . Thus, overall our study shows that nanosensors themselves can perturb the environment which they measure and therefore care must be taken in the application of NPs, including AuNPs, for intracellular SERS investigations.…”

“…Similarly, organic Raman reporter molecules have been chemisorbed onto Au nanoshells to provide ratiometric, quantitative and reversible measurement of chemically-induced cellular hypoxia 42 . Evaluation of cytotoxicity of nanomaterials (ZnO, TiO 2 NPs and single-walled carbon nanotubes, SWCNTS) towards cells has also been performed using a reporter-free SERS methodology 12 . The study corroborates data obtained by WST-1 cell proliferation and Annexin V-FITC/PI apoptosis and necrosis assay, along with SERS spectra collected using co-incubated AuNPs to provide molecular level insights into the toxic events occurring upon internalisation of the nanomaterials.…”

“…Inconsistencies between the two biological assays, observed in two cell lines verified their unreliable nature induced by NP-dye interactions despite careful washing of samples. More significantly, potential SERS-markers of cell stress as induced by the nanomaterials were identified, with alterations observed in peaks relating to protein structure, ratios of phenylalanine (Phe) peak intensities and redistribution of cellular endoplasmic reticulum and mitochondria 12 .…”