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Organisationseinheit der BAM
Development and market introduction of new nanomaterials trigger the need for an adequate risk assessment of such products alongside suitable risk communication measures. Current application of classical and new nanomaterials is analyzed in context of regulatory requirements and standardization for chemicals, food and consumer products. The challenges of nanomaterial characterization as the main bottleneck of risk assessment and regulation are presented. In some areas, e.g., quantification of nanomaterials within complex matrices, the establishment and adaptation of analytical techniques such as laser ablation inductively coupled plasma mass spectrometry and others are potentially suited to meet the requirements. As an example, we here provide an approach for the reliable characterization of human exposure to nanomaterials resulting from food packaging. Furthermore, results of nanomaterial toxicity and ecotoxicity testing are discussed, with concluding key criteria such as solubility and fiber rigidity as important parameters to be considered in material development and regulation. Although an analysis of the public opinion has revealed a distinguished rating depending on the particular field of application, a rather positive perception of nanotechnology could be ascertained for the German public in general. An improvement of material characterization in both toxicological testing as well as end-product control was concluded as being the main obstacle to ensure not only safe use of materials, but also wide acceptance of this and any novel technology in the general public.
The impact of nanoparticles, NPs, at the single cell level has become a major field of toxicological research and different analytical methodologies are being investigated to obtain biological and toxicological information to better understand the mechanisms of cell–NP interactions. Here, inductively coupled plasma mass spectrometry in the single cell measurement mode (SC-ICP-MS) is proposed to study the uptake of silver NPs, AgNPs, with a diameter of 50 nm by human THP-1 monocytes in a proof-ofprinciple experiment. The main operating parameters of SC-ICP-MS have been optimized and applied for subsequent quantitative analysis of AgNPs to determine the number of particles in individual cells using AgNP suspensions for calibration. THP-1 cells were incubated with AgNP suspensions with concentrations of 0.1 and 1 µg/mL for 4 and 24 hours. The results reveal that the AgNP uptake by THP-1 monocytes is minimal at the lower dose of 0.1 µg/mL (roughly 1 AgNP per cell was determined), whereas a large cell-to-cell variance dependent on the exposure time is observed for a 10 times higher concentration (roughly 7 AgNPs per cell). The method was further applied to monitor the AgNP uptake by THP-1 cells differentiated macrophages incubated at the same AgNP concentration levels and exposure times demonstrating a much higher AgNP uptake (roughly from 9 to 45 AgNPs per cell) that was dependent on exposure concentration and remained constant over time. The results have been compared and validated by sample digestion followed by ICP-MS analysis as well as with other alternative promising techniques providing single cell analysis.
A simple metal staining procedure for identification and visualization of single cells by LA-ICP-MS
(2017)
High lateral resolution of metal detection in single cells by use of laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) demands powerful staining methods. In this work different staining procedures for the single cell analysis with LA-ICP-MS were optimized. An iridium intercalator was utilized to stain the cell nuclei whereas the whole cell was stained by the use of maleimido-mono-amide-DOTA (mDOTA) complexing lanthanide(III) ions. The content of the artificially introduced metals per cell was quantified using a matrix matched calibration approach based on cellulose membranes onto which standards were spotted by a microarray spotter. Absolute metal stain amounts in the range of 2.34 to 9.81 femtomole per cell were determined. The metal staining procedures allow direct identification and visualization of single cells and their cell compartments by element microscopy without the use of bright field images of the sample.
Quantification of nanoparticle (NP) uptake in cells or tissues is very important for safety assessment. Often, electron microscopy based approaches are used for this purpose, which allow imaging at very high resolution. However, precise quantification of NP numbers in cells and tissues remains challenging. The aim of this study was to present a novel approach, that combines precise quantification of NPs in individual cells together with high resolution imaging of their intracellular distribution based on focused ion beam/ scanning electron microscopy (FIB/SEM) slice and view approaches.
Safety 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).
In the last decades, significant efforts have been made to investigate possible cytotoxic effects of metallic nanoparticles (NPs). Methodologies enabling precise information regarding uptake and intracellular distribution of NPs at the single cell level remain to be established. Mass cytometry (MC) has been developed for high-dimensional single cell analyses and is a promising tool to quantify NP−cell interactions. Here, we aim to establish a new MC-based quantification procedure to receive absolute numbers of NPs per single cell by using a calibration that considers the specific transmission efficiency (TE) of suspended NPs. The current MC-quantification strategy accept TE values of complementary metal solutions. In this study, we demonstrate the different transmission behavior of 50 nm silver NPs (AgNP) and silver nitrate solution. We have used identical AgNPs for calibration as for in vitro-differentiated macrophages (THP-1 cell line) in a time- and dose-dependent manner. Our quantification relies on silver intensities measuring AgNPs in the same detection mode as the cells. Results were comparable with the TE quantification strategy using AgNPs but differed when using ionic silver. Furthermore, intact and digested cell aliquots were measured to investigate the impact of MC sample processing on the amount of AgNPs/cell. Taken together, we have provided a MC-specific calibration procedure to precisely calculate absolute numbers of NPs per single cell. Combined with its unique feature of multiplexing up to 50 parameters, MC provides much more information on the single cell level than single cell-inductively coupled plasma mass spectrometry (SC-ICP-MS) and, therefore, offers new opportunities in nanotoxicology.