The Fate of Inhaled Nanoparticles: Detection and Measurement by Enhanced Dark-field Microscopy

Mercer, Robert R.; Scabilloni, James F.; Wang, Liying; Battelli, Lori; Antonini, James M.; Roberts, Jenny R.; Qian, Yong; Sisler, Jennifer D.; Castranova, Vincent; Porter, Dale W.; Hubbs, Ann F.

doi:10.1177/0192623317732321

Cited by 22 publications

(17 citation statements)

References 50 publications

(96 reference statements)

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“…Because of the significant scattering of light by nanoparticles, the enhanced dark-field microscope is able to detect nanoparticles in tissues and sections which could not otherwise be detected by a standard light microscope. The theory and application of this microscopy method to detect numerous types of nanoparticles (particles with dimensions less than 100 nm) in a variety of nanoparticle studies are described in detailed elsewhere [100].…”

Section: Methodsmentioning

confidence: 99%

Mitsui-7, heat-treated, and nitrogen-doped multi-walled carbon nanotubes elicit genotoxicity in human lung epithelial cells

et al. 2019

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Background The unique physicochemical properties of multi-walled carbon nanotubes (MWCNT) have led to many industrial applications. Due to their low density and small size, MWCNT are easily aerosolized in the workplace making respiratory exposures likely in workers. The International Agency for Research on Cancer designated the pristine Mitsui-7 MWCNT (MWCNT-7) as a Group 2B carcinogen, but there was insufficient data to classify all other MWCNT. Previously, MWCNT exposed to high temperature (MWCNT-HT) or synthesized with nitrogen (MWCNT-ND) have been found to elicit attenuated toxicity; however, their genotoxic and carcinogenic potential are not known. Our aim was to measure the genotoxicity of MWCNT-7 compared to these two physicochemically-altered MWCNTs in human lung epithelial cells (BEAS-2B & SAEC). Results Dose-dependent partitioning of individual nanotubes in the cell nuclei was observed for each MWCNT material and was greatest for MWCNT-7. Exposure to each MWCNT led to significantly increased mitotic aberrations with multi- and monopolar spindle morphologies and fragmented centrosomes. Quantitative analysis of the spindle pole demonstrated significantly increased centrosome fragmentation from 0.024–2.4 μg/mL of each MWCNT. Significant aneuploidy was measured in a dose-response from each MWCNT-7, HT, and ND; the highest dose of 24 μg/mL produced 67, 61, and 55%, respectively. Chromosome analysis demonstrated significantly increased centromere fragmentation and translocations from each MWCNT at each dose. Following 24 h of exposure to MWCNT-7, ND and/or HT in BEAS-2B a significant arrest in the G1/S phase in the cell cycle occurred, whereas the MWCNT-ND also induced a G2 arrest. Primary SAEC exposed for 24 h to each MWCNT elicited a significantly greater arrest in the G1 and G2 phases. However, SAEC arrested in the G1/S phase after 72 h of exposure. Lastly, a significant increase in clonal growth was observed one month after exposure to 0.024 μg/mL MWCNT-HT & ND. Conclusions Although MWCNT-HT & ND cause a lower incidence of genotoxicity, all three MWCNTs cause the same type of mitotic and chromosomal disruptions. Chromosomal fragmentation and translocations have not been observed with other nanomaterials. Because in vitro genotoxicity is correlated with in vivo genotoxic response, these studies in primary human lung cells may predict the genotoxic potency in exposed human populations.

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Section: Methodsmentioning

confidence: 99%

Mitsui-7, heat-treated, and nitrogen-doped multi-walled carbon nanotubes elicit genotoxicity in human lung epithelial cells

et al. 2019

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“…Enhanced dark-field microscopy (DFM) detects NP down to a size of 15 nm [ 28 ] and was, therefore, used as a sensitive tool to visualize the Fe-Si-NP in tissue sections prior to ToF-SIMS analysis. Overall, the ToF-SIMS 54 Fe + signal and the DFM signal showed a high degree of similarity ( Figure 5 A,B).…”

Section: Resultsmentioning

confidence: 99%

Distribution of Paramagnetic Fe2O3/SiO2–Core/Shell Nanoparticles in the Rat Lung Studied by Time-of-Flight Secondary Ion Mass Spectrometry: No Indication for Rapid Lipid Adsorption

Veith¹,

Vennemann²,

Breitenstein³

et al. 2018

Nanomaterials

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Amorphous silica nanoparticles comprise a class of widely used industrial nanomaterials, which may elicit acute inflammation in the lung. These materials have a large specific surface to which components of the pulmonary micro-milieu can bind. To conduct appropriate binding studies, paramagnetic Fe2O3/SiO2 core/shell nanoparticles (Fe-Si-NP) may be used as an easy-to-isolate silica surrogate, if several prerequisites are fulfilled. To this end, we investigated the distribution of Fe, Si, protein and phosphatidylcholine (PC) by Time-of-Flight secondary ion mass spectrometry (ToF-SIMS) in cryo-sections from the rat lungs to which Fe-Si-NP had been administered for 30 min. Regions-of-interest were identified and analyzed with incident light and enhanced dark-field microscopy (DFM). Fe-Si-NP particles (primary particle size by electron microscopy: 10–20 nm; aggregate size by tracking analysis: 190 ± 20 nm) and agglomerates thereof were mainly attached to alveolar walls and only marginally internalized by cells such as alveolar macrophages. The localization of Fe-Si-NP by DFM was confirmed by ToF-SIMS signals from both, Fe and Si ions. With respect to an optimized signal-to-noise ratio, Fe+, Si+, CH4N+ and the PC head group (C5H15NO4P+) were the most versatile ions to detect iron, silica, protein, and PC, respectively. Largely congruent Fe+ and Si+ signals demonstrated that the silica coating of Fe-Si-NP remained stable under the conditions of the lung. PC, as a major lipid of the pulmonary surfactant, was colocalized with the protein signal alongside alveolar septa, but was not detected on Fe-Si-NP, suggesting that silica nanoparticles do not adsorb lipids of the lung surfactant under native conditions. The study shows that ToF-SIMS is a valuable technique with adequate spatial resolution to analyze nanoparticles together with organic molecules in the lung. The paramagnetic Fe-Si-NP appear well suited to study the binding of proteins to silica nanomaterials in the lung.

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“…Acetone is a standard reagent in most research and commercial laboratories. Other glass microscopy slides and coverslips could potentially be used instead of the cleanroom-cleaned slides and coverslips (Roth et al, 2015b; Mercer et al, 2017) used here only if they have been shown to be free of contaminants, like dust or other particulates, that may interfere with analysis. In future work, various cleaning and decontamination methods will be assessed for their ability to appropriately remove particulates from microscopy slides.…”

Section: Discussionmentioning

confidence: 99%

Sample preparation method for visualization of nanoparticulate captured on mixed cellulose ester filter media by enhanced darkfield microscopy and hyperspectral imaging

Neu‐Baker

Eastlake

Brenner

2019

Microscopy Res & Technique

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A significant hurdle in conducting effective occupational health and safety hazard analysis and risk assessment for the nanotechnology workforce is the lack of a rapid method for the direct visualization and analysis of filter media used to sample nanomaterials from work environments that represent potential worker exposure. The current best-known methods include transmission electron microscopy (TEM), often coupled with energy dispersive x-ray spectroscopy (EDS) for elemental identification. TEM-EDS is considerably time-, cost- and resource-intensive, which may prevent timely health and safety recommendations and corrective actions. A rapid screening method is currently being explored using enhanced darkfield microscopy with hyperspectral imaging (EDFM-HSI). For this approach to be effective, rapid and easy sample preparation that is amenable to the analytical technique is needed. Here, we compare the sample preparation steps for mixed cellulose ester (MCE) filter media specified in NIOSH Method 7400 – Asbestos and Other Fibers by Phase Contrast Microscopy (PCM) – against a new method. NIOSH Method 7400 was chosen as a starting point since it is an established technique for preparing transparent MCE filters for optical microscopy. Limitations in this method led to the development and comparison of a new sample preparation method. The new method, which involves saturation of the filter media with acetone, was faster, easier, and rendered filters more transparent, resulting in improved visualization and analysis of nanomaterials captured on MCE filter media via EDFM-HSI.

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The Fate of Inhaled Nanoparticles: Detection and Measurement by Enhanced Dark-field Microscopy

Cited by 22 publications

References 50 publications

Mitsui-7, heat-treated, and nitrogen-doped multi-walled carbon nanotubes elicit genotoxicity in human lung epithelial cells

Mitsui-7, heat-treated, and nitrogen-doped multi-walled carbon nanotubes elicit genotoxicity in human lung epithelial cells

Distribution of Paramagnetic Fe2O3/SiO2–Core/Shell Nanoparticles in the Rat Lung Studied by Time-of-Flight Secondary Ion Mass Spectrometry: No Indication for Rapid Lipid Adsorption

Sample preparation method for visualization of nanoparticulate captured on mixed cellulose ester filter media by enhanced darkfield microscopy and hyperspectral imaging

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