BackgroundSafety assessment of nanoparticles (NPs) requires techniques that are suitable to quantify tissue and cellular uptake of NPs. The most commonly applied techniques for this purpose are based on inductively coupled plasma mass spectrometry (ICP-MS). Here we apply and compare three different ICP-MS methods to investigate the cellular uptake of TiO2 (diameter 7 or 20 nm, respectively) and Ag (diameter 50 or 75 nm, respectively) NPs into differentiated mouse neuroblastoma cells (Neuro-2a cells). Cells were incubated with different amounts of the NPs. Thereafter they were either directly analyzed by laser ablation ICP-MS (LA-ICP-MS) or were lysed and lysates were analyzed by ICP-MS and by single particle ICP-MS (SP-ICP-MS).ResultsAll techniques confirmed that smaller particles were taken up to a higher extent when values were converted in an NP number-based dose metric. In contrast to ICP-MS and LA-ICP-MS, this measure is already directly provided through SP-ICP-MS. Analysis of NP size distribution in cell lysates by SP-ICP-MS indicates the formation of NP agglomerates inside cells. LA-ICP-MS imaging shows that some of the 75 nm Ag NPs seemed to be adsorbed onto the cell membranes and were not penetrating into the cells, while most of the 50 nm Ag NPs were internalized. LA-ICP-MS confirms high cell-to-cell variability for NP uptake.ConclusionsBased on our data we propose to combine different ICP-MS techniques in order to reliably determine the average NP mass and number concentrations, NP sizes and size distribution patterns as well as cell-to-cell variations in NP uptake and intracellular localization.Electronic supplementary materialThe online version of this article (doi:10.1186/s12951-016-0203-z) contains supplementary material, which is available to authorized users.

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The application of appropriate analytical techniques is essential for nanomaterial (NM) characterization.

The biomolecular imaging of cell-nanoparticle (NP) interactions using time-of-flight secondary ion mass spectrometry (ToF-SIMS) represents an evolving tool in nanotoxicology. In this study we present the three dimensional (3D) distribution of nanomaterials within biomolecular agglomerates using ToF-SIMS imaging. This novel approach was used to model the resistance of human alveolar A549 cells against gold (Au) ion toxicity through intra-and extracellular biomineralization. At low Au concentrations (≤1 mM HAuCl 4) 3D-ToF-SIMS imaging reveals a homogenous intracellular distribution of Au-NPs in combination with polydisperse spherical NPs biomineralized in different layers on the cell surface. However, at higher precursor concentrations (≥2 mM) supplemented with biogenic spherical NPs as seeds, cells start to biosynthesize partially embedded long aspect ratio fiber-like Au nanostructures. Most interestingly, A549 cells seem to be able to sense the enhanced Au concentration. They change the chemical composition of the extracellular NP agglomerates from threonine-O-3-phosphate aureate to an arginine-Au(I)-imine. Furthermore they adopt the extracellular mineralization process from spheres to irregular structures to nanoribbons in a dose-dependent manner with increasing Au concentrations. The results achieved regarding size, shape and chemical specificity were cross checked by SEM-EDX and single particle (sp-)ICP-MS. Our findings demonstrate the potential of ToF-SIMS 3D imaging to better understand cell-NP interactions and their impact in nanotoxicology. Au nano-or microparticles are promising candidates for both bioimaging and therapeutic applications 1. Recently, cell based biomineralization has been explored as a novel technique to synthesize versatile 0, 1, and 2 dimensional gold nanostructures under ambient synthesis conditions. For example, cancer cells are known to synthesize NPs de novo 2 from Au ions. However, little is known about how the surrounding chemical environment influences the extracellular biomineralization at the cell membrane interface and what effect this then has on uptake and biocompatibility. Time of Flight-Secondary Ion Mass Spectrometry (ToF-SIMS) is a versatile and noninvasive technique that allows for both label free imaging and analysis of the surface composition. Conventional scanning electron microscope-energy dispersive X-ray (SEM-EDX) imaging or fluorescence labelling of biological cells not only perturbs the cellular state but also limits the analysis to a specific number of fluorescence labeled targets. On the contrary, ToF-SIMS provides a more versatile method for the visualization of multiple chemicals or intracellular metabolites within individual cells 3. The submicron lateral resolution in combination with a high depth resolution for organic samples (down to 10 nm 3) enables the three dimensional (3D) label-free reconstruction of single cells. This makes ToF-SIMS a top emerging and evolving tool for studies relating to the chemical distribution within single cells 4. Here we app...

Inhalation is considered a critical uptake route for NMs, demanding for sound toxicity testing using relevant test systems. This study investigates cytotoxicity and genotoxicity in EpiAirway™ 3D human bronchial models using 16 well-characterized NMs, including surface-functionalized 15 nm SiO (4 variants), 10 nm ZrO (4), and nanosilver (3), ZnO NM-110, TiO NM-105, BaSO NM-220, and two AlOOH NMs. Cytotoxicity was assessed by LDH and ATP assays and genotoxicity by the alkaline comet assay. For 9 NMs, uptake was investigated using inductively coupled plasma-mass spectrometry (ICP-MS). Most NMs were neither cytotoxic nor genotoxic in vitro. ZnO displayed a dose-dependent genotoxicity between 10 and 25 µg/cm. Ag.50.citrate was genotoxic at 50 µg/cm. A marginal but still significant genotoxic response was observed for SiO.unmodified, SiO.phosphate and ZrO.TODS at 50 µg/cm. For all NMs for which uptake in the 3D models could be assessed, the amount taken up was below 5% of the applied mass doses and was furthermore dose dependent. For in vivo comparison, published in vivo genotoxicity data were used and in addition, at the beginning of this study, two NMs were randomly selected for short-term (5-day) rat inhalation studies with subsequent comet and micronucleus assays in lung and bone marrow cells, respectively, i.e., ZrO.acrylate and SiO.amino. Both substances were not genotoxic neither in vivo nor in vitro. EpiAirway™ 3D models appear useful for NM in vitro testing. Using 16 different NMs, this study confirms that genotoxicity is mainly determined by chemical composition of the core material.