REPOSITORY > RESULTS

Abstract

The potential toxicity of nanoparticles in our environment and consumer products is currently determined by costly and timely animal-based testing, which limits the rate of nanoparticle testing, causing a desperate need for alternative testing strategies. A promising alternative – mechanism-based prediction – employs a set of high-throughput cell-based tests that target the key events connecting nanoparticle exposure to the adverse outcomes. However, this approach requires understanding of the molecular mechanisms of nanoparticle toxicity, which is sadly still lacking.
To alleviate this knowledge gap and devise targeted in vitro tests for mechanism-based toxicity prediction, we discerned the early molecular events caused by exposure of alveolar cells to TiO2 nanotubes – a model nanoparticle that interacts strongly with biomolecules and causes long-lasting lung inflammation in mice. We investigated the relations between these events in epithelial and macrophage cell lines by live-cell fluorescence microscopy – including 3D- and time-acquisition, lifetime imaging and super-resolution STED microscopy – and tracked the nanoparticles by fluorescent labelling and reflectance microscopy. We further confirmed our findings by transcriptomics and in vivo experiments performed using the same nanotubes.
Importantly, we discovered nanoquarantine: a novel cell-defense mechanism in which lung epithelial cells excrete and quarantine nanoparticles on their surface, forming large bio-nano composites termed cauliflowers and effectively lowering the nanoparticle dose. We also observed it with other nanoparticles (e.g. crystalline silica DQ12, TiO2 rutile NM-105, but not TiO2 anatase NM-101 or carbonaceous nanoparticles) and other cell lines (neurons, but not alveolar macrophages). The other key events in chronic inflammation are immune cell death (re-exposing other cells to the nanoparticles) and pro-inflammatory signaling (causing the influx of fresh immune cells). The combination of these processes leads to continuous cycling of nanoparticles between the alveolar cells, causing continuous pro-inflammatory signaling, observed as chronic inflammation.
Further, we described the cell response by a theoretical model which can mimic the inflammatory outcomes by modifying only the three nanoparticle-specific rates of the key events. To determine them, we devised three simple and quick in vitro assays, the results of which can be combined with the theoretical model to predict the inflammatory potential of nanoparticles.
As shown in this dissertation, microscopy-based real-time tracking of cellular events coupled with complementary approaches improves our understanding of nanotoxicity mechanisms, providing the much-needed boost for mechanism-based prediction of nanosafety, a time- and cost-efficient animal-free alternative for toxicity testing.