Toxicological assessment of nanomaterials: the role of in vitro Raman microspectroscopic analysis

Abstract

The acceleration of nanomaterials research has brought about increased demands for rapid analysis of their bioactivity, in a multi-parametric fashion, to minimize the gap between potential applications and knowledge of their toxicological properties. The potential of Raman microspectroscopy for the analysis of biological systems with the aid of multivariate analysis techniques has been demonstrated. In this study, an overview of recent efforts towards establishing a 'label-free high content nanotoxicological assessment technique' using Raman microspectroscopy is presented. The current state of the art for cellular toxicity assessment and the potential of Raman microspectroscopy are discussed, and the spectral markers of the cellular toxic responses upon exposure to nanoparticles, changes on the identified spectral markers upon exposure to different nanoparticles, cell death mechanisms, and the effects of nanoparticles on different cell lines are summarized. Moreover, 3D toxicity plots of spectral markers, as a function of time and dose, are introduced as new methodology for toxicological analysis based on the intrinsic properties of the biomolecular changes, such as cytoplasmic RNA aberrations, protein and lipid damage associated with the toxic response. The 3D evolution of the spectral markers are correlated with the results obtained by commonly used cytotoxicity assays, and significant similarities are observed between band intensity and percentage viability obtained by the Alamar Blue assay, as an example. Therefore, the developed 3D plots can be used to identify toxicological properties of a nanomaterial and can potentially be used to predict toxicity, which can provide rapid advances in nanomedicine. Graphical Abstract Spectral markers of cytotoxicity as a function of time and dose.

Genotoxicity of Nanomaterials: Advanced In Vitro Models and High Throughput Methods for Human Hazard Assessment—A Review

“…The literature search resulted in 59 papers for “genotoxicity & 3D in vitro model”, 73 papers for “genotoxicity & advanced in vitro model”, 134 papers for “genotoxicity & high throughput”, eight papers for “genotoxicity & high throughput & nanomaterials” [ 15 , 16 , 17 , 18 , 19 , 20 , 21 ] and six papers for “genotoxicity & high throughput & nanoparticles” (4 are the same as for “genotoxicity & high throughput & nanomaterials”) [ 16 , 17 , 18 , 19 , 21 , 22 ], seven papers for “genotoxicity & organ on chip“ [ 23 , 24 , 25 , 26 , 27 , 28 , 29 ], seven papers for “genotoxicity & 3D models & nanoparticles” [ 30 , 31 , 32 , 33 , 34 , 35 , 36 ], 11 papers for “genotoxicity & 3D models & nanomaterials” [ 30 , 31 , 33 , 36 , 37 , 38 , 39 , 40 , 41 ] for the period of 20 years (2000–2020), whereby the year 2020 was considered only from January to August. ( Table 1 ).…”

“…If genotoxicity is measured after, e.g., three and 24 h exposure, as is common for the comet assay, then cytotoxicity should also be measured after three and 24 h’ exposure. The alamarBlue™ assay is a convenient and reliable cytotoxicity assay to be used in combination with the comet assay, which has been tested and found to be applicable with several different NMs [ 6 , 26 , 36 ].…”

Biochemical Changes in Human Cells Exposed to Low Concentrations of Gold Nanoparticles Detected by Raman Microspectroscopy

“…Recently, Efeoglu et al [17,18,19] showed the ability of Raman spectroscopy to provide information about cellular responses at different concentrations of aminated polystyrene (PS-NH 2 ) nanoparticles and exposure times with the aid of multivariate analysis. In particular, they identified specific spectral markers reflecting changes in nucleic acid, protein, and lipid components for different cell lines exposed to such NPs at sub-lethal and lethal doses.…”

Raman spectroscopy detects biochemical changes due to different cell culture environments in live cells in vitro

“…(Fig. 3), have previously been observed in studies of oxidative stress in nanoparticle exposed cell cultures, and may be related to noncoding RNA formation as a result of reactive oxygen species generation [22]. This band has also been seen to be prominent in the study of the subcellular interactions of the chemotherapeutic agent, doxorubicin, particularly in the cell nucleus and nucleolus [40].…”

“…Due to these advantages, Raman spectroscopy offers a better possibility to study cells in an aqueous environment and thus keep cells alive during the experiments under normal physiological conditions [20]. The technique has thus been widely used for the analysis of cell-drug and cell nanoparticle interaction at a sub-cellular level [21,22]. Currently, in bio-spectroscopy, one of the most widely used substrates is CaF2 windows, as the material permits the transmission of visible, near and mid IR light with low losses and UV grade CaF2 does not have any characteristic Raman bands in the cellular spectral fingerprint region (400-1800 cm -1 ) which allows the analysis of biological samples [23].…”