LA-ICP-MS and Immunohistochemical Staining with Lanthanide-Labeled Antibodies to Study the Uptake of CeO₂ Nanoparticles by Macrophages in Tissue Sections

Abstract

Due to the increasing use and production of CeO 2 nanoparticles (NPs), the likelihood of exposure especially via the air rapidly grows. However, the uptake of CeO 2 NPs via the lung and the resulting distribution into various cell types of remote organs are not well understood because classical analytical methods provide limited spatial information. In this study, laser ablation− inductively coupled plasma−mass spectrometry (LA-ICP-MS) was combined with immunohistochemical (IHC) staining with lanthanide-labeled antibodies to investigate the distribution of intratracheally instilled CeO 2 NPs from the rat lung to lymph nodes, spleen, and liver after 3 h, 3 days, and 21 days. We selected regions of interest after fast imaging using LA-ICP-MS in lowresolution mode and conducted high-resolution LA-ICP-MS in combination with IHC for cellular localization. The lanthanide labeling, which was largely congruent with conventional fluorescent labeling, allowed us to calculate the association rates of Ce to specific cell types. Major portions of Ce were found to be associated with phagocytic cells in the lung, lymph nodes, spleen, and liver. In the lung, almost 94% of the Ce was co-localized with CD68-positive alveolar macrophages after 21 days. Ce was also detected in the lymph nodes outside macrophages 3 h post instillation but shifted to macrophage-associated locations. In the liver, Ce accumulations associated with Kupffer cells (CD163-positive) were found. Ce-containing populations of metallophilic and marginal zone macrophages (both CD169-positive) as well as red pulp macrophages (CD68-positive) were identified as major targets in the spleen. Overall, high-resolution LA-ICP-MS analysis in combination with IHC staining with lanthanide-labeled antibodies is a suitable tool to quantify and localize Ce associated with specific cell types and to estimate their particle burden under in vivo conditions.

Calibration Strategy to Size and Localize Multi-Shaped Nanoparticles in Tissue Sections Using LA–spICP–MS

“…To confirm the suitability of applied ion cloud integration parameters, the mean particle size should not vary among different concentrations. In an earlier study, LA–ICP–MS results revealed that particle concentration maxima are expected to be up to 1 μg·g –1 CeO 2 NPs in spleen tissue sections . Therefore, gelatine standards spiked with concentrations of CeO 2 NM212 up to 5 μg·g –1 were prepared to cover the expected particle concentration range and locally higher concentration maxima.…”

“…In an earlier study, LA−ICP−MS results revealed that particle concentration maxima are expected to be up to 1 μg•g −1 CeO 2 NPs in spleen tissue sections. 26 Therefore, gelatine standards spiked with concentrations of CeO 2 NM212 up to 5 μg•g −1 were prepared to cover the expected particle concentration range and locally higher concentration maxima. For concentrations above 0.5 μ• g −1 , the deviation of the mean particle size was less than 2 nm along with similar particle size distributions (Supporting Information, Figure S5).…”

“…Nevertheless, the differentiation between the two pulps based on the microscopic image was difficult. Previous studies utilized the iron distribution in the spleen to distinguish between the two pulps , to allocate inorganic NPs directly to one of the pulps. Quadrupole-based ICP–MS restricts the analysis as only one isotope can be examined at any given time, so the iron distribution cannot be determined simultaneously with the NP distribution.…”

“…CeO 2 NM212-loaded spleen tissue was taken from an earlier study on female Wistar rats (ethically approved by LANUV, Dortmund, Germany, accession no. 84 02.04.2022.A157) . Animals had received 0.6 mg of CeO 2 NM212 NPs per lung of a well-dispersed aqueous CeO 2 suspension via intratracheal instillation (for details, see Seiffert et al) and were sacrificed 3 h, 3 d, and 3 weeks post-particle administration.…”

“…84 02.04.2022.A157). 26 Animals had received 0.6 mg of CeO 2 NM212 NPs per lung of a well-dispersed aqueous CeO 2 suspension via intratracheal instillation (for details, see Seiffert et al 26 ) and were sacrificed 3 h, 3 d, and 3 weeks post-particle administration. Spleen blocks were taken from representative animals embedded in cryomatrix, and 7 μm thick cryo-sections were prepared which were mounted on glass slides for LA−spICP−MS analysis.…”