Towards the identification of structural determinants of toxicity of amorphous silica nanoparticles and carbon nanotubes: an in vitro study

Nanomaterials (NM) contain particles, in an unbound state or as an aggregate or as an agglomerate, which, for a percentage of 50% or more, have one or more external dimensions in the range 1-100 nm. The great development of nanotechnology has produced an increasing quantity of nanomaterials of different types in several productive sectors (food, chemicals, pharmaceuticals). For this reason, several studies are aimed at characterizing the physical and chemical properties of nanomaterials and the determination of their effects on human health and the environment. Multi walled carbon nanotubes (MWCNT) and amorphous silica nanoparticles (ASNP) are examples of nanomaterials widely used in many industrial fields. The overall aim of this thesis is the elucidation of the potential hazards of MWCNT and ASNP, evaluating their interaction with relevant cell models. Attention is given to the assessment of potential toxic effects on cells of innate immunity and to the identification of structural determinants of toxicity. Since inhalation is the major way of interaction with nanomaterials, we decided to study the biological effects of MWCNT and ASNP on two cell lines (MH-S and RAW264.7), as representative models of macrophages, which are the first to contact the inhaled particles, and on airway epithelial cells (Calu-3), which represents one of the first body barriers encountered by nanomaterials dispersed in the environment. The first part of the thesis is focused on the identification of structural determinants of toxicity, in vitro, of four preparations of multi-walled carbon nanotubes with different length, morphology (rigid, needle-like or flexible, tangle-like shape), and level of metal contaminants. We have assessed the biological effects of the four MWCNT preparations (NM400, NM401, NM402 and MWCNT-SA) on macrophages and airway epithelial cells, in order to identify the determinants of toxicity, thus far incompletely elucidated. To study the biological effects of MWCNT on macrophage cell lines we analyzed different endpoints, such as cell viability, phagocytic activity and pro-inflammatory M1 macrophage activation. We found that the main determinants of toxicity for macrophages are the length and the needle-like shape, which hinder, or even prevent, phagocytosis. Indeed, the greater toxicity of NM401 and MWCNT-SA, as demonstrated by the decrease in cell viability and the alteration of functional activity, are ascribable to their greater length and to their morphological features. On the contrary, reduced length and tangle-like shape (NM400 and NM402) promote M1 macrophage activation. Since these materials can be engulfed by macrophages, these results suggest that phagocytosis is a main step for the M1 macrophage activation by nanomaterials, endowed with low acute toxicity. Given the high tendency of MWCNT to aggregate and the presence of aggregates in the airway walls of exposed animals, as reported in several in vivo studies, we have investigated if MWCNT produced a barrier impairment. The behavior of epithelial cells was studied both at the monolayer (cell population) and at the single-cell level. At a cell-population level, Trans-Epithelial Electrical Resistance (TEER) was used as a synthetic indicator of barrier competence, caspase activity was assessed with standard biochemical assays, and cell viability was investigated with both standard biochemical techniques and an high throughput (HTP) technique, based on automated epifluorescence microscopy; at single-cell level, cell responses to MWCNT were investigated with confocal microscopy, by evaluating cell death (calcein/propidium iodide), proliferation (Ki-67), inflammation triggering (NF-B) and apoptosis (caspase activity). We found that the main determinant of toxicity for epithelial cells depends on the actual shape in which MWCNT get in contact with the cells and, in particular, if they form aggregates. The second part of the thesis is focused on the identification of structural determinants of toxicity of two preparations of amorphous silica nanoparticles (ASNP, a material usually considered endowed with modest toxicity). This study has evaluated the capability of ASNP, of comparable size and specific surface area, but produced through different synthetic procedures (colloidal NM200 vs pyrogenic NM203), to induce macrophage activation in MH-S and RAW264.7 cell lines. To study the biological effects of ASNP we analyzed different endpoints, such as cell viability, oxidative stress (ROS formation and the induction of Hmox-1), the induction of the inducible nitric oxide synthase Nos2, the production of NO and the secretion of cytokines like TNF-α, IL-6 and IL-1β. Helium Ion microscopy (HIM) and confocal microscopy were adopted for imaging the interaction between ASNP and the cell surface. The results demonstrate that pyrogenic ASNP are more potentially inflammogenic than colloidal ASNP. Moreover, an additional mechanism of toxicity is proposed, consisting in the greater capability of pyrogenic ASNP to bind biologically active compounds, such as LPS, enhancing their effects. Thus we found that the preparation route procedure may constitute a main determinant of toxicity of ASNP, likely because of the different surface chemistry established by high-temperature synthesis. In conclusion, this thesis highlights that determinants of toxicity of nanomaterials are strongly dependent on several parameters. The identification of these determinants, which appear essential for a “safety-by-design” approach, will therefore require an in-depth characterization of the toxicological properties of each type of nanomaterial.