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

In the field of nanotoxicology, the detection and size characterization of nanoparticles (NPs) in biological tissues become increasingly important. To gain information on both particle size and particle distribution in histological sections, laser ablation and single particle inductively coupled plasma-mass spectrometry (LA-spICP-MS) was used in combination with a liquid calibration of dissolved metal standards via a pneumatic nebulizer. In the first step, the particle size distribution of Ag NPs embedded in matrix-matched gelatine standards introduced via LA was compared with that of Ag NPs in a suspension and nebulizer-based ICP-MS. The data show that the particles remained intact by the ablation process as confirmed by transmission electron microscopy. Moreover, the optimized method was applied to CeO2 NPs that are highly relevant for (eco-)toxicological research but, unlike Ag NPs, are multi-shaped and have a broad particle size distribution. Upon analyzing the particle size distribution of CeO2 NPs in cryosections of rat spleen, CeO2 NPs were found to remain unchanged in size over 3 h, 3 d, and 3 weeks post-intratracheal instillation, with the fraction of smaller particles reaching the spleen first. Overall, LA-spICP-MS combined with a calibration based on dissolved metal standards is a powerful tool to simultaneously localize and size NPs in histological sections in the absence of particle standards.

Surface Treatment With Hydrophobic Coating Reagents (Organosilanes) Strongly Reduces the Bioactivity of Synthetic Amorphous Silica in vitro.

Synthetic amorphous silica (SAS) is industrially relevant material whose bioactivity in vitro is strongly diminished, for example, by protein binding to the particle surface. Here, we investigated the in vitro bioactivity of fourteen SAS (pyrogenic, precipitated, or colloidal), nine of which were surface-treated with organosilanes, using alveolar macrophages as a highly sensitive test system. Dispersion of the hydrophobic SAS required pre-wetting with ethanol and extensive ultrasonic treatment in the presence of 0.05% BSA (Protocol 1). Hydrophilic SAS was suspended by moderate ultrasonic treatment (Protocol 2) and also by Protocol 1. The suspensions were administered to NR8383 alveolar macrophages under serum-free conditions for 16 h, and the release of LDH, GLU, H2O2, and TNFα was measured in cell culture supernatants. While seven surface-treated hydrophobic SAS exhibited virtually no bioactivity, two materials (AEROSIL® R 504 and AEROSIL® R 816) had minimal effects on NR8383 cells. In contrast, non-treated SAS elicited considerable increases in LDH, GLU, and TNFα, while the release of H2O2 was low except for CAB-O-SIL® S17D Fumed Silica. Dispersing hydrophilic SAS with Protocol 1 gradually reduced the bioactivity but did not abolish it. The results show that hydrophobic coating reagents, which bind covalently to the SAS surface, abrogate the bioactivity of SAS even under serum-free in vitro conditions. The results may have implications for the hazard assessment of hydrophobic surface-treated SAS in the lung.

Evaluating nanobiomaterial-induced DNA strand breaks using the alkaline comet assay.

Due to their unique chemical and physical properties, nanobiomaterials (NBMs) are extensively studied for applications in medicine and drug delivery. Despite these exciting properties, their small sizes also make them susceptible to toxicity. Whilst nanomaterial immunotoxicity and cytotoxicity are studied in great depth, there is still limited data on their potential genotoxicity or ability to cause DNA damage. In the past years, new medical device regulations, which came into place in 2020, were developed, which require the assessment of long-term NBM exposure; therefore, in recent years, increased attention is being paid to genotoxicity screening of these materials. In this article, and through an interlaboratory comparison (ILC) study conducted within the Horizon 2020 REFINE project, we assess five different NBM formulations, each with different uses, namely, a bio-persistent gold nanoparticle (AuNP), an IR-780 dye-loaded liposome which is used in deep tissue imaging (LipImage™815), an unloaded PACA polymeric nanoparticle used as a drug delivery system (PACA), and two loaded PACA NBMs, i.e. the cabazitaxel drug-loaded PACA (CBZ-PACA) and the NR668 dye-loaded PACA (NR668 PACA) for their potential to cause DNA strand breaks using the alkaline comet assay and discuss the current state of genotoxicity testing for nanomaterials. We have found through our interlaboratory comparison that the alkaline comet assay can be suitably applied to the pre-clinical assessment of NBMs, as a reproducible and repeatable methodology for assessing NBM-induced DNA damage. Workflow for assessing the applicability of the alkaline comet assay to determine nanobiomaterial (NBM)-induced DNA strand breaks, through an interlaboratory comparison study (ILC).

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

Due to the increasing use and production of CeO2 nanoparticles (NPs), the likelihood of exposure especially via the air rapidly grows. However, the uptake of CeO2 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 CeO2 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 low-resolution 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.

Subcellular detection of PEBCA particles in macrophages: combining darkfield microscopy, confocal Raman microscopy, and ToF-SIMS analysis.

The detection of biomedical organic nanocarriers in cells and tissues is still an experimental challenge. Here we developed an imaging strategy for the label-free detection of poly (ethylbutyl cyanoacrylate) (PEBCA) particles. Experiments were carried out with phagocytic NR8383 macrophages exposed to non-toxic and non-activating concentrations of fluorescent (PEBCA NR668 and PEBCA NR668/IR), non-fluorescent (PEBCA), and cabazitaxel-loaded PEBCA particles (PEBCA CBZ). Exposure to PEBCA NR668 revealed an inhomogeneous particle uptake similar to what was obtained with the free modified Nile Red dye (NR668). In order to successfully identify the PEBCA-loaded cells under label-free conditions, we developed an imaging strategy based on enhanced darkfield microscopy (DFM), followed by confocal Raman microscopy (CRM) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). Nitrile groups of the PEBCA matrix and PEBCA ions were used as suitable analytes for CRM and ToF-SIMS, respectively. Masses found with ToF-SIMS were further confirmed by Orbitrap-SIMS. The combined approach allowed to image small (< 1 µm) PEBCA-containing phagolysosomes, which were identified as PEBCA-containing compartments in NR8383 cells by electron microscopy. The combination of DFM, CRM, and ToF-SIMS is a promising strategy for the label-free detection of PEBCA particles.

Classes of organic pigments meet tentative PSLT criteria and lack toxicity in short-term inhalation studies.

Here, we present a non-animal testing battery to identify PSLT (poorly soluble, low toxicity) substances based on their solubility in phagolysosomal lung fluid simulant, surface reactivity and effects on alveolar macrophages in vitro. This is exemplified by eleven organic pigments belonging to five chemical classes that cover a significant share of the European market. Three of the pigments were tested as both, nanoform and non-nanoform. The results obtained in this integrated non-animal testing battery qualified two pigments as non PSLT, one pigment as poorly soluble and eight pigments as poorly soluble and low toxicity in vitro. The low toxic potency of the eight PSLT and the one poorly soluble pigment was corroborated by short-term inhalation studies with rats. These pigments did not elicit apparent toxic effects at 10 mg/m3 (systemic and in the respiratory tract). One of the pigments, Diarylide Pigment Yellow 83 transparent, however, caused minimal infiltration of neutrophils; hence its low toxicity is ambiguous and needs further verification or falsification. The present test battery provides an opportunity to identify PSLT-properties of test substances to prioritise particles for further development. Thus, it can help to reduce animal testing and steer product development towards safe applications.

Spatially and size-resolved analysis of gold nanoparticles in rat spleen after intratracheal instillation by laser ablation-inductively coupled plasma-mass spectrometry.

In a dual approach, laser ablation-inductively coupled plasma-mass spectrometry was applied to investigate spleen samples of rats after intratracheal instillation of polyvinylpyrrolidone-coated gold nanoparticles. First, spatially resolved imaging analysis was deployed to investigate gold translocation from the lungs to the spleen and to investigate the distribution pattern of gold in the spleen parenchyma itself. Using the same instrumental setup, laser ablation-inductively coupled plasma-mass spectrometry in single particle mode was applied to determine the species of translocated gold. Single particle analysis allows the determination of particle size distributions and therefore to distinguish between ionic species, intact nanoparticles, and agglomerates. A translocation of instilled gold from the lungs to the spleen was demonstrated for gold nanoparticles of 30 and 50 nm diameter. Furthermore single particle analysis revealed the translocation of intact gold nanoparticles in a non-agglomerated state.

Serum Lowers Bioactivity and Uptake of Synthetic Amorphous Silica by Alveolar Macrophages in a Particle Specific Manner.

Various cell types are compromised by synthetic amorphous silica (SAS) if they are exposed to SAS under protein-free conditions in vitro. Addition of serum protein can mitigate most SAS effects, but it is not clear whether this is solely caused by protein corona formation and/or altered particle uptake. Because sensitive and reliable mass spectrometric measurements of SiO2 NP are cumbersome, quantitative uptake studies of SAS at the cellular level are largely missing. In this study, we combined the comparison of SAS effects on alveolar macrophages in the presence and absence of foetal calf serum with mass spectrometric measurement of 28Si in alkaline cell lysates. Effects on the release of lactate dehydrogenase, glucuronidase, TNFα and H2O2 of precipitated (SIPERNAT® 50, SIPERNAT® 160) and fumed SAS (AEROSIL® OX50, AEROSIL® 380 F) were lowered close to control level by foetal calf serum (FCS) added to the medium. Using a quantitative high resolution ICP-MS measurement combined with electron microscopy, we found that FCS reduced the uptake of particle mass by 9.9% (SIPERNAT® 50) up to 83.8% (AEROSIL® OX50). Additionally, larger particle agglomerates were less frequent in cells in the presence of FCS. Plotting values for lactate dehydrogenase (LDH), glucuronidase (GLU) or tumour necrosis factor alpha (TNFα) against the mean cellular dose showed the reduction of bioactivity with a particle sedimentation bias. As a whole, the mitigating effects of FCS on precipitated and fumed SAS on alveolar macrophages are caused by a reduction of bioactivity and by a lowered internalization, and both effects occur in a particle specific manner. The method to quantify nanosized SiO2 in cells is a valuable tool for future in vitro studies.

Aerogels are not regulated as nanomaterials, but can be assessed by tiered testing and grouping strategies for nanomaterials.

Aerogels contribute to an increasing number of novel applications due to many unique properties, such as high porosity and low density. They outperform most other insulation materials, and some are also useful as carriers in food or pharma applications. Aerogels are not nanomaterials by the REACH definition but retain properties of nanoscale structures. Here we applied a testing strategy in three tiers. In Tier 1, we examined a panel of 19 aerogels (functionalized chitosan, alginate, pyrolyzed carbon, silicate, cellulose, polyurethane) for their biosolubility, and oxidative potential. Biosolubility was very limited except for some alginate and silicate aerogels. Oxidative potential, as by the ferric reduction ability of human serum (FRAS), was very low except for one chitosan and pyrolyzed carbon, both of which were <10% of the positive control Mn2O3. Five aerogels were further subjected to the Tier 2 alveolar macrophage assay, which revealed no in vitro cytotoxicity, except for silicate and polyurethane that induced increases in tumor necrosis factor α. Insufficiently similar aerogels were excluded from a candidate group, and a worst case identified. In the Tier 3 in vivo instillation, polyurethane (0.3 to 2.4 mg) elicited dose-dependent but reversible enzyme changes in lung lavage fluid on day 3, but no significant inflammatory effects. Overall, the results show a very low inherent toxicity of aerogels and support a categorization based on similarities in Tier 1 and Tier 2. This exemplifies how nanosafety concepts and methods developed on particles can be applied to specific concerns on advanced materials that contain or release nanostructures.

Revealing Silver Nanoparticle Uptake by Macrophages Using SR-µXRF and LA-ICP-MS.

To better study the impact of nanoparticles on both in vitro and in vivo models, tissue distribution and cellular doses need to be described more closely. Here silver nanoparticles were visualized in alveolar macrophages by means of synchrotron radiation micro X-ray fluorescence spectroscopy (SR-µXRF) with high spatial resolution of 3 × 3 µm2. For the spatial allocation of silver signals to cells and tissue structures, additional elemental labeling was carried out by staining with eosin, which binds to protein and can be detected as bromine signal with SR-µXRF. The method was compatible with immunostaining of macrophage antigens. We found that the silver distribution obtained with SR-µXRF was largely congruent with distribution maps from a subsequent laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) of the same tissue sites. The study shows a predominant, though not exclusive uptake of silver into alveolar macrophages in the rat lung, which can be modeled by a similar uptake in cultured alveolar macrophages. Advantages and limitations of the different strategies for measuring nanoparticle uptake at the single cell level are discussed.

Lung Toxicity Analysis of Nano-Sized Kaolin and Bentonite: Missing Indications for a Common Grouping.

Kaolin and bentonite (nanoclay NM-600) are nanostructured aluminosilicates that share a similar chemical composition, platelet-like morphology, and high binding capacity for biomolecules. To investigate if these material-based criteria allow for a common grouping, we prepared particle suspensions of kaolin and bentonite with a similar hydrodynamic diameter and administered them to NR8383 alveolar macrophages in vitro and also to a rat lung using quartz DQ12 as a reference material. Bentonite was far more bioactive in vitro, indicated by a lower threshold for the release of enzymes, tumor necrosis factor α, and H2O2. In addition, in the lung, the early effects of bentonite exceeded those of kaolin and even those of quartz, due to strongly increased numbers of inflammatory cells, and elevated concentrations of total protein and fibronectin within the bronchoalveolar lavage fluid. The pro-inflammatory effects of bentonite decreased over time, although assemblies of particle-laden alveolar macrophages (CD68 positive), numerous type-2 epithelial cells (immunopositive for pro-surfactant protein C), and hypertrophic lung epithelia persisted until day 21. At this point in time, kaolin-treated lungs were completely recovered, whereas quartz DQ12 had induced a progressive inflammation. We conclude that bentonite is far more bioactive than equally sized kaolin. This argues against a common grouping of aluminosilicates, previously suggested for different kaolin qualities.

Effects of Ultrasonic Dispersion Energy on the Preparation of Amorphous SiO2 Nanomaterials for In Vitro Toxicity Testing.

Synthetic amorphous silica (SAS) constitute a large group of industrial nanomaterials (NM). Based on their different production processes, SAS can be distinguished as precipitated, fumed, gel and colloidal. The biological activity of SAS, e.g., cytotoxicity or inflammatory potential in the lungs is low but has been shown to depend on the particle size, at least for colloidal silica. Therefore, the preparation of suspensions from highly aggregated or agglomerated SAS powder materials is critical. Here we analyzed the influence of ultrasonic dispersion energy on the biologic activity of SAS using NR8383 alveolar macrophage (AM) assay. Fully characterized SAS (7 precipitated, 3 fumed, 3 gel, and 1 colloidal) were dispersed in H2O by stirring and filtering through a 5 µm filter. Aqueous suspensions were sonicated with low or high ultrasonic dispersion (USD) energy of 18 or 270 kJ/mL, respectively. A dose range of 11.25⁻90 µg/mL was administered to the AM under protein-free conditions to detect particle-cell interactions without the attenuating effect of proteins that typically occur in vivo. The release of lactate dehydrogenase (LDH), glucuronidase (GLU), and tumor necrosis factor α (TNF) were measured after 16 h. Hydrogen peroxide (H2O2) production was assayed after 90 min. The overall pattern of the in vitro response to SAS (12/14) was clearly dose-dependent, except for two SAS which showed very low bioactivity. High USD energy gradually decreased the particle size of precipitated, fumed, and gel SAS whereas the low adverse effect concentrations (LOECs) remained unchanged. Nevertheless, the comparison of dose-response curves revealed slight, but uniform shifts in EC50 values (LDH, and partially GLU) for precipitated SAS (6/7), gel SAS (2/3), and fumed SAS (3/3). Release of TNF changed inconsistently with higher ultrasonic dispersion (USD) energy whereas the induction of H2O2 was diminished in all cases. Electron microscopy and energy dispersive X-ray analysis showed an uptake of SAS into endosomes, lysosomes, endoplasmic reticulum, and different types of phagosomes. The possible effects of different uptake routes are discussed. The study shows that the effect of increased USD energy on the in vitro bioactivity of SAS is surprisingly small. As the in vitro response of AM to different SAS is highly uniform, the production process per se is of minor relevance for toxicity.

Phosphonate coating of SiO2 nanoparticles abrogates inflammatory effects and local changes of the lipid composition in the rat lung: a complementary bioimaging study.

BACKGROUND: The well-known inflammatory and fibrogenic changes of the lung upon crystalline silica are accompanied by early changes of the phospholipid composition (PLC) as detected in broncho-alveolar lavage fluid (BALF). Amorphous silica nanoparticles (NPs) evoke transient lung inflammation, but their effect on PLC is unknown. Here, we compared effects of unmodified and phosphonated amorphous silica NP and describe, for the first time, local changes of the PLC with innovative bioimaging tools. METHODS: Unmodified (SiO2-n), 3-(trihydroxysilyl) propyl methylphosphonate coated SiO2-n (SiO2-p) as well as a fluorescent surrogate of SiO2-n (SiO2-FITC) nanoparticles were used in this study. In vitro toxicity was tested with NR8383 alveolar macrophages. Rats were intratracheally instilled with SiO2-n, SiO2-p, or SiO2-FITC, and effects on lungs were analyzed after 3 days. BALF from the right lung was analyzed for inflammatory markers. Cryo-sections of the left lung were subjected to fluorescence microscopy and PLC analyses by matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MS), Fourier transform infrared microspectroscopy (FT-IR), and tandem mass spectrometry (MS/MS) experiments. RESULTS: Compared to SiO2-p, SiO2-n NPs were more cytotoxic to macrophages in vitro and more inflammatory in the rat lung, as reflected by increased concentration of neutrophils and protein in BALF. Fluorescence microscopy revealed a typical patchy distribution of SiO2-FITC located within the lung parenchyma and alveolar macrophages. Superimposable to this particle distribution, SiO2-FITC elicited local increases of phosphatidylglycerol (PG) and phosphatidylinositol (PI), whereas phoshatidylserine (PS) and signals from triacylgyceride (TAG) were decreased in the same areas. No such changes were found in lungs treated with SiO2-p or particle-free instillation fluid. CONCLUSIONS: Phosphonate coating mitigates effects of silica NP in the lung and abolishes their locally induced changes in PLC pattern. Bioimaging methods based on MALDI-MS may become a useful tool to investigate the mode of action of NPs in tissues.

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.

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.

In Vitro and In Vivo Short-Term Pulmonary Toxicity of Differently Sized Colloidal Amorphous SiO2.

In vitro prediction of inflammatory lung effects of well-dispersed nanomaterials is challenging. Here, the in vitro effects of four colloidal amorphous SiO2 nanomaterials that differed only by their primary particle size (9, 15, 30, and 55 nm) were analyzed using the rat NR8383 alveolar macrophage (AM) assay. Data were compared to effects of single doses of 15 nm and 55 nm SiO2 intratracheally instilled in rat lungs. In vitro, all four elicited the release of concentration-dependent lactate dehydrogenase, ß-glucuronidase, and tumor necrosis factor alpha, and the two smaller materials also released H2O2. All effects were size-dependent. Since the colloidal SiO2 remained well-dispersed in serum-free in vitro conditions, effective particle concentrations reaching the cells were estimated using different models. Evaluating the effective concentration-based in vitro effects using the Decision-making framework for the grouping and testing of nanomaterials, all four nanomaterials were assigned as "active." This assignment and the size dependency of effects were consistent with the outcomes of intratracheal instillation studies and available short-term rat inhalation data for 15 nm SiO2. The study confirms the applicability of the NR8383 AM assay to assessing colloidal SiO2 but underlines the need to estimate and consider the effective concentration of such well-dispersed test materials.

Detection of ZrO2 Nanoparticles in Lung Tissue Sections by Time-of-Flight Secondary Ion Mass Spectrometry and Ion Beam Microscopy.

The increasing use of nanoparticles (NP) in commercial products requires elaborated techniques to detect NP in the tissue of exposed organisms. However, due to the low amount of material, the detection and exact localization of NP within tissue sections is demanding. In this respect, Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS) and Ion Beam Microscopy (IBM) are promising techniques, because they both offer sub-micron lateral resolutions along with high sensitivities. Here, we compare the performance of the non-material-consumptive IBM and material-consumptive ToF-SIMS for the detection of ZrO2 NP (primary size 9-10 nm) in rat lung tissue. Unfixed or methanol-fixed air-dried cryo-sections were subjected to IBM using proton beam scanning or to three-dimensional ToF-SIMS (3D ToF-SIMS) using either oxygen or argon gas cluster ion beams for complete sample sputtering. Some sample sites were analyzed first by IBM and subsequently by 3D ToF-SIMS, to compare results from exactly the same site. Both techniques revealed that ZrO2 NP particles occurred mostly agglomerated in phagocytic cells with only small quantities being associated to the lung epithelium, with Zr, S, and P colocalized within the same biological structures. However, while IBM provided quantitative information on element distribution, 3D ToF-SIMS delivered a higher lateral resolution and a lower limit of detection under these conditions. We, therefore, conclude that 3D ToF-SIMS, although not yet a quantitative technique, is a highly valuable tool for the detection of NP in biological tissue.

Silver Nanoparticles in the Lung: Toxic Effects and Focal Accumulation of Silver in Remote Organs.

The distribution of silver (Ag) into remote organs secondary to the application of Ag nanoparticles (Ag-NP) to the lung is still incompletely understood and was investigated in the rat with imaging methods. Dose-finding experiments were carried out with 50 nm- or 200 nm-sized polyvinyl pyrrolidine (PVP)-coated Ag-NP using alveolar macrophages in vitro and female rats, which received Ag-NP via intratracheal instillation. In the main study, we administered 37.5-300 µg per rat lung of the more toxic Ag50-PVP and assessed the broncho-alveolar lavage fluid (BALF) for inflammatory cells, total protein and fibronectin after three and 21 days. In parallel, lung tissue was analysed for DNA double-strand breaks and altered cell proliferation. While 75-150 µg Ag50-PVP per rat lung caused a reversible inflammation, 300 µg led to DNA damage, accelerated cell proliferation and progressively increasing numbers of neutrophilic granulocytes. Ag accumulation was significant in homogenates of liver and other peripheral organs upon lung dose of ≥75 µg. Quantitative laser-ablation inductively-coupled plasma mass spectrometry (LA-ICP-MS) combined with enhanced dark field microscopy and autometallography revealed focal accumulations of Ag and/or Ag-NP in sections of peripheral organs: mediastinal lymph nodes contained Ag-NP especially in peripheral macrophages and Ag in argyrophilic fibres. In the kidney, Ag had accumulated within proximal tubuli, while renal filter structures contained no Ag. Discrete localizations were also observed in immune cells of liver and spleen. Overall, the study shows that concentrations of Ag-NP, which elicit a transient inflammation in the rat lung, lead to focal accumulations of Ag in peripheral organs, and this might pose a risk to particular cell populations in remote sites.

Differential Effects of Surface-Functionalized Zirconium Oxide Nanoparticles on Alveolar Macrophages, Rat Lung, and a Mouse Allergy Model.

Nanoparticles (NPs) may affect the lung via their chemical composition on the surface. Here, we compared the bioactivity of zirconium oxide (ZrO2) NPs coated with either aminopropilsilane (APTS), tetraoxidecanoic acid (TODS), polyethyleneglycol (PGA), or acrylic acid (Acryl). Supernatants from NPs-treated cultured alveolar macrophages (NR8383) tested for lactate dehydrogenase, glucuronidase, tumor necrosis factor α, and H2O2 formation revealed dose-dependent effects, with only gradual differences among particles whose gravitational settling and cellular uptake were similar. We selected TODS- and Acryl-coated NPs for intratracheal administration into the rat lung. Darkfield and hyperspectral microscopy combined with immunocytochemistry showed that both NPs qualities accumulate mainly within the alveolar macrophage compartment, although minute amounts also occurred in neutrophilic granulocytes. Dose-dependent signs of inflammation were found in the broncho-alveolar lavage fluid on day 3 but no longer on day 21 post-application of ≥1.2 mg per lung; again only minor differences occurred between TODS- and Acryl-coated NPs. In contrast, the response of allergic mice was overall higher compared to control mice and dependent on the surface modification. Increases in eosinophils, lymphocytes and macrophages were highest following ZrO2-PGA administration, followed by ZrO2-Acryl, ZrO2-TODS, and ZrO2-APTS. We conclude that surface functionalization of ZrO2 NPs has minor effects on the inflammatory lung response of rats and mice, but is most relevant for an allergic mouse model. Allergic individuals may therefore be more susceptible to exposure to NPs with specific surface modifications.

Pro-Inflammatory versus Immunomodulatory Effects of Silver Nanoparticles in the Lung: The Critical Role of Dose, Size and Surface Modification.

The growing use of silver nanoparticles (Ag-NPs) in consumer products raises concerns about their toxicological potential. The purpose of the study was to investigate the size- and coating-dependent pulmonary toxicity of Ag-NPs in vitro and in vivo, using an ovalbumin (OVA)-mouse allergy model. Supernatants from (5.6-45 µg/mL) Ag50-PVP, Ag200-PVP or Ag50-citrate-treated NR8383 alveolar macrophages were tested for lactate dehydrogenase and glucuronidase activity, tumor necrosis factor (TNF)-α release and reactive oxygen species (ROS) production. For the in vivo study, NPs were intratracheally instilled in non-sensitized (NS) and OVA-sensitized (S) mice (1-50 µg/mouse) prior to OVA-challenge and bronchoalveolar lavage fluid (BALF) inflammatory infiltrate was evaluated five days after challenge. In vitro results showed a dose-dependent cytotoxicity of Ag-NPs, which was highest for Ag50-polyvinilpyrrolidone (PVP), followed by Ag50-citrate, and lowest for Ag200-PVP. In vivo 10-50 µg Ag50-PVP triggered a dose-dependent pulmonary inflammatory milieu in NS and S mice, which was significantly higher in S mice and was dampened upon instillation of Ag200-PVP. Surprisingly, instillation of 1 µg Ag50-PVP significantly reduced OVA-induced inflammatory infiltrate in S mice and had no adverse effect in NS mice. Ag50-citrate showed similar beneficial effects at low concentrations and attenuated pro-inflammatory effects at high concentrations. The lung microbiome was altered by NPs instillation dependent on coating and/or mouse batch, showing the most pronounced effects upon instillation of 50 µg Ag50-citrate, which caused an increased abundance of operational taxonomic units assigned to Actinobacteria, Bacteroidetes, Firmicutes and Proteobacteria. However, no correlation with the biphasic effect of low and high Ag-NPs dose was found. Altogether, both in vitro and in vivo data on the pulmonary effects of Ag-NPs suggest the critical role of the size, dose and surface functionalization of Ag-NPs, especially in susceptible allergic individuals. From the perspective of occupational health, care should be taken by the production of Ag-NPs-containing consumer products.