Iron oxide nanoparticle agglomeration influences dose rates and modulates oxidative stress-mediated dose–response profiles in vitro

Abstract

Spontaneous agglomeration of engineered nanoparticles (ENPs) is a common problem in cell culture media which can confound interpretation of in vitro nanotoxicity studies. The authors created stable agglomerates of iron oxide nanoparticles (IONPs) in conventional culture medium, which varied in hydrodynamic size (276 nm–1.5 μm) but were composed of identical primary particles with similar surface potentials and protein coatings. Studies using C10 lung epithelial cells show that the dose rate effects of agglomeration can be substantial, varying by over an order of magnitude difference in cellular dose in some cases. Quantification by magnetic particle detection showed that small agglomerates of carboxylated IONPs induced greater cytotoxicity and redox-regulated gene expression when compared with large agglomerates on an equivalent total cellular IONP mass dose basis, whereas agglomerates of amine-modified IONPs failed to induce cytotoxicity or redox-regulated gene expression despite delivery of similar cellular doses. Dosimetry modelling and experimental measurements reveal that on a delivered surface area basis, large and small agglomerates of carboxylated IONPs have similar inherent potency for the generation of ROS, induction of stress-related genes and eventual cytotoxicity. The results suggest that reactive moieties on the agglomerate surface are more efficient in catalysing cellular ROS production than molecules buried within the agglomerate core. Because of the dynamic, size and density-dependent nature of ENP delivery to cells in vitro, the biological consequences of agglomeration are not discernible from static measures of exposure concentration (μg/ml) alone, highlighting the central importance of integrated physical characterisation and quantitative dosimetry for in vitro studies. The combined experimental and computational approach provides a quantitative framework for evaluating relationships between the biocompatibility of nanoparticles and their physical and chemical characteristics.

Keywords::

• nanotoxicology
• agglomeration
• iron oxide nanoparticles
• oxidative stress
• gene expression
• dosimetry modelling

Acknowledgements

Support for this research was provided by Battelle Memorial Institute under a cooperative research and development agreement (#PNNL 2119) and by the National Institutes of Health grant U19 ES019544. Pacific Northwest National Laboratory is operated by Battelle for the US Department of Energy under contract ACO6 76RLO 1830. The authors also thank Dr. Libor Kovarik for technical support with TEM images conducted at the Environmental Molecular Sciences Laboratory, a DOE-funded National User Facility at PNNL.