We have developed structure/toxicity relationships for amorphous silica nanoparticles (NPs) synthesized through low temperature, colloidal (e.g. Stöber silica) or high temperature pyrolysis (e.g. fumed silica) routes. Through combined spectroscopic and physical analyses, we have determined the state of aggregation, hydroxyl concentration, relative proportion of strained and unstrained siloxane rings, and potential to generate hydroxyl radicals for Stöber and fumed silica NPs with comparable primary particle sizes (16-nm in diameter). Based on erythrocyte hemolytic assays and assessment of the viability and ATP levels in epithelial and macrophage cells, we discovered for fumed silica an important toxicity relationship to post-synthesis thermal annealing or environmental exposure, whereas colloidal silicas were essentially non-toxic under identical treatment conditions. Specifically, we find for fumed silica a positive correlation of toxicity with hydroxyl concentration and its potential to generate reactive oxygen species (ROS) and cause red blood cell hemolysis. We propose fumed silica toxicity stems from its intrinsic population of strained three-membered rings (3MRs) along with its chain-like aggregation and hydroxyl content. Hydrogen-bonding and electrostatic interactions of the silanol surfaces of fumed silica aggregates with the extracellular plasma membrane cause membrane perturbations sensed by the Nalp3 inflammasome, whose subsequent activation leads to secretion of the cytokine IL-1β. Hydroxyl radicals generated by the strained 3MRs in fumed silica but largely absent in colloidal silicas may contribute to the inflammasome activation. Formation of colloidal silica into aggregates mimicking those of fumed silica had no effect on cell viability or hemolysis. This study emphasizes that not all amorphous silica is created equal and that the unusual toxicity of fumed silica compared to colloidal silica derives from its framework and surface chemistry along with its fused chain-like morphology established by high temperature synthesis (>1300°C) and rapid thermal quenching.

Functionalized carbon nanotubes (f-CNTs) are being produced in increased volume because of the ease of dispersion and maintenance of the pristine material physicochemical properties when used in composite materials as well as for other commercial applications. However, the potential adverse effects of f-CNTs have not been quantitatively or systematically explored, and in this study we used a library of covalently functionalized multiwall carbon nanotubes (f-MWCNTs), established from the same starting material, to assess the impact of surface charge in a predictive toxicological model that relates the tubes’ pro-inflammatory and pro-fibrogenic effects at cellular level to the development of pulmonary fibrosis. Carboxylated (COOH), polyethylene glycol (PEG), amine (NH2), sidewall amine (sw-NH2) and polyetherimide (PEI) modified MWCNTs were successfully established from raw or as-prepared (AP-) MWCNTs, and comprehensively characterized by TEM, XPS, FTIR and DLS to obtain information about morphology, length, degree of functionalization, hydrodynamic size and surface charge. Cellular screening in BEAS-2B and THP-1 cells showed that, compared to AP-MWCNTs, anionic functionalization (COOH and PEG) decreased the production of pro-fibrogenic cytokines and growth factors (including IL-1β, TGF-β1 and PDGF-AA), while neutral and weak cationic functionalization (NH2 and sw-NH2) showed intermediary effects. In contrast, the strongly cationic PEI-functionalized tubes induced robust biological effects. These differences could be attributed to differences in cellular uptake and NLRP3 inflammasome activation, which depends on the propensity towards lysosomal damage and cathepsin B release in macrophages. Moreover, the in vitro hazard ranking was validated by the pro-fibrogenic potential of the tubes in vivo. Compared to pristine MWCNTs, strong cationic PEIMWCNTs induced significant lung fibrosis, while carboxylation significantly decreased the extent of pulmonary fibrosis. These results demonstrate that surface charge plays an important role in the structure-activity relationships that determine the pro-fibrogenic potential of f-CNTs in the lung.

While it has been shown that high aspect ratio nanomaterials like carbon nanotubes and TiO2 nanowires can induce toxicity by acting as fiber-like substances that damage the lysosome, it is not clear what the critical lengths and aspect ratios are that induce this type of toxicity. To answer this question, we synthesized a series of cerium oxide (CeO2) nanorods and nanowires with precisely controlled lengths and aspect ratios. Both phosphate and chloride ions were shown to play critical roles in obtaining these high aspect ratio nanostructures. High resolution TEM analysis shows that single crystalline CeO2 nanorods/nanowires were formed along the [211] direction by an “oriented attachment” mechanism, followed by Ostwald ripening. The successful creation of a comprehensive CeO2 nanorod/nanowire combinatorial library allows, for the first time, the systematic study of the effect of aspect ratio on lysosomal damage, cytoxicity and IL-1β production by the human myeloid cell line (THP-1). This in vitro toxicity study demonstrated that at lengths ≥200 nm and aspect ratios ≥ 22, CeO2 nanorods induced progressive cytotoxicity and pro-inflammatory effects. The relatively low “critical” length and aspect ratio were associated with small nanorod/nanowire diameters (6–10 nm), which facilitates the formation of stacking bundles due to strong van der Waals and dipole-dipole attractions. Our results suggest that both length and diameter components of aspect ratio should be considered when addressing the cytotoxic effects of long aspect ratio materials.