BackgroundMost in vitro studies investigating nanomaterial pulmonary toxicity poorly correlate to in vivo inhalation studies. Alveolar macrophages (AMs) play an outstanding role during inhalation exposure since they effectively clear the alveoli from particles. This study addresses the applicability of an in vitro alveolar macrophage assay to distinguish biologically active from passive nanomaterials.MethodsRat NR8383 alveolar macrophages were exposed to 18 inorganic nanomaterials, covering AlOOH, BaSO4, CeO2, Fe2O3, TiO2, ZrO2, and ZnO NMs, amorphous SiO2 and graphite nanoplatelets, and two nanosized organic pigments. ZrO2 and amorphous SiO2 were tested without and with surface functionalization. Non-nanosized quartz DQ12 and corundum were used as positive and negative controls, respectively. The test materials were incubated with the cells in protein-free culture medium. Lactate dehydrogenase, glucuronidase, and tumour necrosis factor alpha were assessed after 16 h. In parallel, H2O2 was assessed after 1.5 h. Using the no-observed-adverse-effect concentrations (NOAECs) from available rat short-term inhalation studies (STIS), the test materials were categorized as active (NOAEC < 10 mg/m3) or passive.ResultsIn vitro data reflected the STIS categorization if a particle surface area-based threshold of <6000 mm2/mL was used to determine the biological relevance of the lowest observed significant in vitro effects. Significant effects that were recorded above this threshold were assessed as resulting from test material-unspecific cellular ‘overload’. Test materials were assessed as active if ≥2 of the 4 in vitro parameters undercut this threshold. They were assessed as passive if 0 or 1 parameter was altered. An overall assay accuracy of 95 % was achieved.ConclusionsThe in vitro NR8383 alveolar macrophage assay allows distinguishing active from passive nanomaterials. Thereby, it allows determining whether in vivo short-term inhalation testing is necessary for hazard assessment. Results may also be used to group nanomaterials by biological activity. Further work should aim at validating the assay.Electronic supplementary materialThe online version of this article (doi:10.1186/s12951-016-0164-2) contains supplementary material, which is available to authorized users.

One of the key challenges in the field of nanoparticle (NP) analysis is in producing reliable and reproducible characterisation data for nanomaterials. This study looks at the reproducibility using a relatively new, but rapidly adopted, technique, Nanoparticle Tracking Analysis (NTA) on a range of particle sizes and materials in several different media. It describes the protocol development and presents both the data and analysis of results obtained from 12 laboratories, mostly based in Europe, who are primarily QualityNano members. QualityNano is an EU FP7 funded Research Infrastructure that integrates 28 European analytical and experimental facilities in nanotechnology, medicine and natural sciences with the goal of developing and implementing best practice and quality in all aspects of nanosafety assessment. This study looks at both the development of the protocol and how this leads to highly reproducible results amongst participants. In this study, the parameter being measured is the modal particle size.

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Lung lining fluid is the first biological barrier nanoparticles (NPs) encounter during inhalation. As previous inhalation studies revealed considerable differences between surface functionalized NPs with respect to deposition and toxicity, our aim was to investigate the influence of lipid and/or protein binding on these processes. Thus, we analyzed a set of surface functionalized NPs including different SiO2 and ZrO2 in pure phospholipids, CuroSurf(TM) and purified native porcine pulmonary surfactant (nS). Lipid binding was surprisingly low for pure phospholipids and only few NPs attracted a minimal lipid corona. Additional presence of hydrophobic surfactant protein (SP) B in CuroSurf(TM) promoted lipid binding to NPs functionalized with Amino or PEG residues. The presence of the hydrophilic SP A in nS facilitated lipid binding to all NPs. In line with this the degree of lipid and protein affinities for different surface functionalized SiO2 NPs in nS followed the same order (SiO2 Phosphate ∼ unmodified SiO2 < SiO2 PEG < SiO2 Amino NPs). Agglomeration and biomolecule interaction of NPs in nS was mainly influenced by surface charge and hydrophobicity. Toxicological differences as observed in short-term inhalation studies (STIS) were mainly influenced by the core composition and/or surface reactivity of NPs. However, agglomeration in lipid media and lipid/protein affinity appeared to play a modulatory role on short-term inhalation toxicity. For instance, lipophilic NPs like ZrO2, which are interacting with nS to a higher extent, exhibited a far higher lung burden than their hydrophilic counterparts, which deserves further attention to predict or model effects of respirable NPs.

BackgroundOxidative stress, a commonly used paradigm to explain nanoparticle (NP)-induced toxicity, results from an imbalance between reactive oxygen species (ROS) generation and detoxification. As one consequence, protein carbonyl levels may become enhanced. Thus, the qualitative and quantitative description of protein carbonylation may be used to characterize how biological systems respond to oxidative stress induced by NPs.MethodsWe investigated a representative panel of 24 NPs including functionalized amorphous silica (6), zirconium dioxide (4), silver (4), titanium dioxide (3), zinc oxide (2), multiwalled carbon nanotubes (3), barium sulfate and boehmite. Surface reactivities of all NPs were studied in a cell-free system by electron spin resonance (ESR). NRK-52E cells were treated with all NPs, analyzed for viability (WST-1 assay) and intracellular ROS production (DCFDA assay). Carbonylated proteins were assessed by 1D and/or 2D immunoblotting and identified by matrix assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF/TOF). In parallel, tissue homogenates from rat lungs intratracheally instilled with silver NPs were studied.ResultsEleven NPs induced elevated levels of carbonylated proteins. This was in good agreement with the surface reactivity of the NPs as obtained by ESR and the reduction in cell viability as assessed by WST-1 assay. By contrast, results obtained by DCFDA assay were deviating. Each NP induced an individual pattern of protein carbonyls on 2D immunoblots. Affected proteins comprised cytoskeletal components, proteins being involved in stress response, or cytoplasmic enzymes of central metabolic pathways such as glycolysis and gluconeogenesis. Furthermore, induction of carbonyls upon silver NP treatment was also verified in rat lung tissue homogenates.ConclusionsAnalysis of protein carbonylation is a versatile and sensitive method to describe NP-induced oxidative stress and, therefore, can be used to identify NPs of concern. Furthermore, detailed information about compromised proteins may aid in classifying NPs according to their mode of action.Electronic supplementary materialThe online version of this article (doi:10.1186/s12989-015-0108-2) contains supplementary material, which is available to authorized users.

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.

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, β-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.

One of the greatest challenges in the manufacturing and development of nanotechnologies is the requirement for robust, reliable, and accurate characterization data. Presented here are the results of an interlaboratory comparison (ILC) brought about through multiple rounds of engagement with NanoSight Malvern and ten pan-European research facilities. Following refinement of the nanoparticle tracking analysis (NTA) technique, the size and concentration characterization of nanoparticles in liquid suspension was proven to be robust and reproducible for multiple sample types in monomodal, binary, or multimodal mixtures. The limits of measurement were shown to exceed the 30–600 nm range (with all system models), with percentage coefficients of variation (% CV) being calculated as sub 5% for monodisperse samples. Particle size distributions were also improved through the incorporation of the finite track length adjustment (FTLA) algorithm, which most noticeably acts to improve the resolution of multimodal sample mixtures. The addition of a software correction to account for variations between instruments also dramatically increased the accuracy and reproducibility of concentration measurements. When combined, the advances brought about during the interlaboratory comparisons allow for the simultaneous determination of accurate and precise nanoparticle sizing and concentration data in one measurement.