In vitro monitoring of time and dose dependent cytotoxicity of aminated nanoparticles using Raman spectroscopy

Abstract

Investigation of possible adverse health effects of nanomaterials, in a rapid multi-parametric fashion, has become increasingly important, due to their increased production and potential uses in a wide range of application areas, from cosmetics to pharmaceutics. Although conventional in-vitro cytotoxicological techniques provide valuable information about the particle toxicity, the importance of gaining high content information in a single assay with the analysis of multiple parameters in a non-invasive and label-free way is still one of the biggest challenges in nanotoxicology. As a vibrational spectroscopic technique, the power of Raman spectroscopy for the analysis of cells, tissues and also nanoparticle localization within cells has been shown previously. In this study, the ability of Raman spectroscopy to fingerprint the dose and time dependent cellular responses and effect of cytotoxic events on biochemical constituents of the cells is monitored. A549 human lung carcinoma cells and aminated polystyrene nanoparticles (PS-NH 2 ) are used as a model cell line and nanoparticle, respectively. Following the determination of cellular responses in the presence of toxic PS-NH 2 by using conventional cellular assays, Alamar Blue (AB) and (3-(4,5-Dimethylthiazol-2-yl)-2,5diphenyltetrazoliumbromid (MTT), and calculation of EC 50 values for both assays, Raman spectroscopy was employed at response related doses and time points. Multiple point spectra from the cytoplasm, nucleus and nucleolus of 20 cells were acquired using Raman spectroscopy for each exposure dose and timepoint. Unsupervised principle components analysis (PCA) was applied to the Raman data sets for the comparison of exposed and unexposed cells as well as different exposure doses and times. The study shows the ability of Raman spectroscopy to provide information about cellular responses at different particle concentrations and exposure times with the aid of multivariate analysis. In the chosen range of concentrations, the most significant changes were observed in the cytoplasm for both time dependent and dose dependent cases due to the route of endocytosis The Raman spectral markers for lipidosis, ROS formation and oxidative stress related biochemical damage are determined and correlated with exposure dose and time, and the responses are correlated with conventional cytotoxicity assays.

In vitro comparative cytotoxicity study of aminated polystyrene, zinc oxide and silver nanoparticles on a cervical cancer cell line

“…In the case of AmPs NPs, it has previously been reported that ROS is first generated around cell membrane and in the cytosol region of lysosomes, where they are trafficked from endosomes to lysosomes, which subsequently impacts on the mitochondria (Lunov et al 2011, Efeoglu et al 2016) and the previously observed MTT cytotoxicity data would support this (Table 2). For ZnO NPs, the increase in ROS formation can attribute to the extracellular dissolution of ZnO NPs due to the acidic microenvironment of cancer cells as was observed previously from the AB cytotoxicity data (Figure 2(b)).…”

“…Table 2 displays the cytotoxicity data, in the form of IC 50 values, obtained for the AB, NR, and MTT viability assays for all three NPs. These assays showed a doseand time-dependent decrease in cell viability from 24 to 72 h. This effect was mirrored in the calculated IC 50 values for all three NPs, indicative of a complex cascade of events triggered by different mechanisms, giving rise to subsequent oxidative stress, and apoptosis induced cell death (Maher et al 2014, Efeoglu et al 2016.…”

“…It is widely accepted that AmPs NPs induce toxicity which is initiated by the formation of ROS following the endocytosis of NPs (Lunov et al 2011, Efeoglu et al 2016. Furthermore, due to induced oxidative stress, inflammatory factors are release and apoptosis is triggered (Lunov et al 2011, Efeoglu et al 2016. But it has not yet been confirmed that AmPs NPs induced cytotoxicity can be caspase-mediated, triggering the apoptotic pathway due to their cationic properties.…”

“…Early endosomes carry the NPs to the lysosomes across the cell membrane (Treuel et al 2013) and they are further engulfed by the lysosomes and carried to the endoplasmic reticulum (Shapero et al 2011, Mu et al 2012, Efeoglu et al 2016. It is widely accepted that AmPs NPs induce toxicity which is initiated by the formation of ROS following the endocytosis of NPs (Lunov et al 2011, Efeoglu et al 2016. Furthermore, due to induced oxidative stress, inflammatory factors are release and apoptosis is triggered (Lunov et al 2011, Efeoglu et al 2016.…”

“…In most studies, endocytosis has been described as the primary mechanism for AmPs NPs uptake into the cells. Early endosomes carry the NPs to the lysosomes across the cell membrane (Treuel et al 2013) and they are further engulfed by the lysosomes and carried to the endoplasmic reticulum (Shapero et al 2011, Mu et al 2012, Efeoglu et al 2016. It is widely accepted that AmPs NPs induce toxicity which is initiated by the formation of ROS following the endocytosis of NPs (Lunov et al 2011, Efeoglu et al 2016.…”