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The plastics industry is a major contributor to the world economy, and in 2019 alone, there were some 480 million metric tons of plastic produced worldwide. Although a great deal of attention has been given to measuring and understanding the extent of plastic in the environment and its associated human exposure, much less is known about the human exposure to plastics during its production. The aim of this study is to characterize in detail the particulate matter (PM) emitted from various processes involved in the manufacturing and recycling of plastics. Here we report our findings from an initial campaign carried out at a Danish plastics factory. Online instruments were used to determine the particle number and mass concentrations for particles in the range of 10nm to 10µm, over the course of five hours, at both near- and far-field positions with respect to activities associated with the processing of plastics, including extruding, thermoforming, mixing, crushing, and rolling. For fine particles (

BackgroundAcute phase response (APR) is characterized by a change in concentration of different proteins, including C-reactive protein and serum amyloid A (SAA) that can be linked to both exposure to metal oxide nanomaterials and risk of cardiovascular diseases. In this study, we intratracheally exposed mice to ZnO, CuO, Al2O3, SnO2 and TiO2 and carbon black (Printex 90) nanomaterials with a wide range in phagolysosomal solubility. We subsequently assessed neutrophil numbers, protein and lactate dehydrogenase activity in bronchoalveolar lavage fluid, Saa3 and Saa1 mRNA levels in lung and liver tissue, respectively, and SAA3 and SAA1/2 in plasma. Endpoints were analyzed 1 and 28 days after exposure, including histopathology of lung and liver tissues.ResultsAll nanomaterials induced pulmonary inflammation after 1 day, and exposure to ZnO, CuO, SnO2, TiO2 and Printex 90 increased Saa3 mRNA levels in lungs and Saa1 mRNA levels in liver. Additionally, CuO, SnO2, TiO2 and Printex 90 increased plasma levels of SAA3 and SAA1/2. Acute phase response was predicted by deposited surface area for insoluble metal oxides, 1 and 28 days post-exposure.ConclusionSoluble and insoluble metal oxides induced dose-dependent APR with different time dependency. Neutrophil influx, Saa3 mRNA levels in lung tissue and plasma SAA3 levels correlated across all studied nanomaterials, suggesting that these endpoints can be used as biomarkers of acute phase response and cardiovascular disease risk following exposure to soluble and insoluble particles Background: Acute phase response (APR) is characterized by a change in concentration of different proteins, including C-reactive protein and serum amyloid A (SAA) that can be linked to both exposure to metal oxide nanomaterials and risk of cardiovascular diseases. In this study, we intratracheally exposed mice to ZnO, CuO, Al2O3, SnO2 and TiO2 and carbon black (Printex 90) nanomaterials with a wide range in phagolysosomal solubility. We subsequently assessed neutrophil numbers, protein and lactate dehydrogenase activity in bronchoalveolar lavage fluid, Saa3 and Saa1 mRNA levels in lung and liver tissue, respectively, and SAA3 and SAA1/2 in plasma. Endpoints were analyzed 1 and 28 days after exposure, including histopathology of lung and liver tissues. Results: All nanomaterials induced pulmonary inflammation after 1 day, and exposure to ZnO, CuO, SnO2, TiO2 and Printex 90 increased Saa3 mRNA levels in lungs and Saa1 mRNA levels in liver. Additionally, CuO, SnO2, TiO2 and Printex 90 increased plasma levels of SAA3 and SAA1/2. Acute phase response was predicted by deposited surface area for insoluble metal oxides, 1 and 28 days post-exposure. Conclusion: Soluble and insoluble metal oxides induced dose-dependent APR with different time dependency. Neutrophil influx, Saa3 mRNA levels in lung tissue and plasma SAA3 levels correlated across all studied nanomaterials, suggesting that these endpoints can be used as biomarkers of acute phase response and cardiovascular disease risk following exposure to soluble and insoluble particles.

Materials can be modified for improved functionality. Our aim was to test whether pulmonary toxicity of silica nanomaterials is increased by the introduction of: a) porosity; and b) surface doping with CuO; and whether c) these modifications act synergistically. Mice were exposed by intratracheal instillation and for some doses also oropharyngeal aspiration to: 1) solid silica 100 nm; 2) porous silica 100 nm; 3) porous silica 100 nm with CuO doping; 4) solid silica 300 nm; 5) porous silica 300 nm; 6) solid silica 300 nm with CuO doping; 7) porous silica 300 nm with CuO doping; 8) CuO nanoparticles 9.8 nm; or 9) carbon black Printex 90 as benchmark. Based on a pilot study, dose levels were between 0.5 and 162 µg/mouse (0.2 and 8.1 mg/kg bw). Endpoints included pulmonary inflammation (neutrophil numbers in bronchoalveolar fluid), acute phase response, histopathology, and genotoxicity assessed by the comet assay, micronucleus test, and the gamma-H2AX assay. The porous silica materials induced greater pulmonary inflammation than their solid counterparts. A similar pattern was seen for acute phase response induction and histologic changes. This could be explained by a higher specific surface area per mass unit for the most toxic particles. CuO doping further increased the acute phase response normalized according to the deposited surface area. We identified no consistent evidence of synergism between surface area and CuO doping. In conclusion, porosity and CuO doping each increased the toxicity of silica nanomaterials and there was no indication of synergy when the modifications co-occurred.

The rate of translocation of ingested nanoparticles (NPs) and how the uptake is affected by a food matrix are key aspects of health risk assessment. In this study, female Sprague Dawley rats (N=4/group) received 0, 1.4, or 13 mg of cerium oxide (CeO2 NM-212) NPs/rat/day by gavage or in a chocolate spread snack 5 days/week for 1 or 2 weeks followed by 2 weeks of recovery. A dose and time-dependent uptake in the liver and spleen of 0.1–0.3 and 0.004–0.005 parts per million (ng/mg) of the total administered dose was found, respectively. There was no statistically significant difference in cerium concentration in the liver or spleen after gavage compared to snack dosing. Microscopy revealed indications of necrotic changes in the liver and decreased cellularity in white pulp in the spleen. The snack provided precise administration and a more human-relevant exposure of NPs and could improve animal welfare as alternative to gavage.

Exposure to nanoparticles by various routes results in size-dependent translocation of nanoparticles to the systemic circulation and subsequent accumulation in the liver. The purpose of this study was to determine possible adverse effects in the liver of long-lasting nanoparticle presence in the organ. Mice exposed to a single dose (162 µg/animal equivalent to 9 mg/kg body weight) of TiO2, CeO2 or carbon black nanoparticles by intratracheal instillation or intravenous injection, resulting in relatively low or high liver burdens, were followed for 1, 28 or 180 days. Clinical appearance, feed intake, body and liver weights, hematological indices, and transaminases and alkaline phosphatase activities were unaffected by exposure. Exposure-related foreign material persisted in the liver up to 180 days after intratracheal and intravenous exposure, mainly in sinusoids, near Kupffer cells, or around blood vessels. Increased incidences of histological findings after intratracheal or intravenous exposure included: initially, prominent nuclei of Kupffer cells, the apparent increase in binucleate hepatocytes (TiO2 and carbon black) and inflammatory infiltrations (CeO2), later, cytoplasmic vacuolation, pyknosis and necrosis, especially for CeO2. Thus, neither low nor high nanoparticle burden in the liver affected enzymatic markers of liver injury, but indications of exposure-related necrotic changes, particularly for CeO2 nanoparticles, were noted.

Many chronic diseases manifest in prolonged inflammation and often ignored dysregulated lipid metabolism. When associated with inhalation of nanomaterials, limited information is available on the relevant molecular events and their causal connections. This prevents reliable prediction of outcomes by efficient testing strategies. To unravel how acute nanomaterial exposure leads to chronic conditions, we employed advanced microscopy and omics in vitro and in vivo together with in silico modelling. For selected metal-oxide nanomaterials, we show that lung epithelial cells survive the exposure by excreting internalized nanomaterials and passivating them on the surface, employing elevated lipid synthesis. Macrophages, on the contrary, lose their integrity whilst degrading the passivized bio-nano agglomerates, releasing the nanomaterials, which are taken up again by the epithelial cells. Constant proinflammatory signalling recruits new phagocytes that feed the vicious cycle of events, resulting in a long-lasting response to a single exposure. The proposed mechanism explains the nanomaterial-associated in vivo chronic outcomes and allows its prediction based on in vitro measurements. Similar mechanisms may trigger other chronic diseases affecting millions of lives worldwide.

Background Diesel exhaust is carcinogenic and exposure to diesel particles cause health effects. We investigated the toxicity of diesel exhaust particles designed to have varying physicochemical properties in order to attribute health effects to specific particle characteristics. Particles from three fuel types were compared at 13% engine intake O2 concentration: MK1 ultra low sulfur diesel (DEP13) and the two renewable diesel fuels hydrotreated vegetable oil (HVO13) and rapeseed methyl ester (RME13). Additionally, diesel particles from MK1 ultra low sulfur diesel were generated at 9.7% (DEP9.7) and 17% (DEP17) intake O2 concentration. We evaluated physicochemical properties and histopathological, inflammatory and genotoxic responses on day 1, 28, and 90 after single intratracheal instillation in mice compared to reference diesel particles and carbon black. Results Moderate variations were seen in physical properties for the five particles: primary particle diameter: 15–22 nm, specific surface area: 152–222 m2/g, and count median mobility diameter: 55–103 nm. Larger differences were found in chemical composition: organic carbon/total carbon ratio (0.12–0.60), polycyclic aromatic hydrocarbon content (1–27 μg/mg) and acid-extractable metal content (0.9–16 μg/mg). Intratracheal exposure to all five particles induced similar toxicological responses, with different potency. Lung particle retention was observed in DEP13 and HVO13 exposed mice on day 28 post-exposure, with less retention for the other fuel types. RME exposure induced limited response whereas the remaining particles induced dose-dependent inflammation and acute phase response on day 1. DEP13 induced acute phase response on day 28 and inflammation on day 90. DNA strand break levels were not increased as compared to vehicle, but were increased in lung and liver compared to blank filter extraction control. Neutrophil influx on day 1 correlated best with estimated deposited surface area, but also with elemental carbon, organic carbon and PAHs. DNA strand break levels in lung on day 28 and in liver on day 90 correlated with acellular particle-induced ROS. Conclusions We studied diesel exhaust particles designed to differ in physicochemical properties. Our study highlights specific surface area, elemental carbon content, PAHs and ROS-generating potential as physicochemical predictors of diesel particle toxicity.

Despite broad application of magnetic nanoparticles in biomedicine and electronics, only a few in vivo studies on biocompatibility are available. In this study, toxicity of magnetic metal oxide nanoparticles on the respiratory system was examined in vivo by single intratracheal instillation in mice. Bronchoalveolar lavage fluid (BALF) samples were collected for proteome analyses by LC-MS/MS, testing Fe3O4 nanoparticles doped with increasing amounts of cobalt (Fe3O4, CoFe2O4 with an iron to cobalt ratio 5:1, 3:1, 1:3, Co3O4) at two doses (54 μg, 162 μg per animal) and two time points (day 1 and 3 days postinstillation). In discovery phase, in-depth proteome profiling of a few representative samples allowed for comprehensive pathway analyses. Clustering of the 681 differentially expressed proteins (FDR n = 166) were measured label-free as single-injections after a short gradient (21 min) LC separation using the Evosep One system, validating the findings from the discovery and defining protein signatures which enable discrimination of lung inflammation. We demonstrate a proteomics-based toxicity screening with high sample throughput easily transferrable to other nanoparticle types. Data are available via ProteomeXchange with identifier PXD016148.

Increased use of nanomaterials in industry, medicine, and consumer products has raised concerns over their toxicity. To ensure safe use of nanomaterials, understanding their biological effects at the molecular level is crucial. In particular, the regulatory mechanisms responsible for the cascade of genes activated by nanomaterial exposure are not well-characterized. To this end, we profiled the genome-wide usage of gene transcription start sites and linked active enhancer regions in lungs of C57BL/6 mice 24 h after intratracheal instillation of a single dose of the multiwalled carbon nanotube (MWCNT) Mitsui-7. Our results revealed a massive gene regulatory response, where expression of key inflammatory genes (e.g., Csf3, Il24, and Fgf23) was increased100-fold 24 h after Mitsui-7 exposure. Many of the Mitsui-7-responsive transcription start sites were alternative transcription start sites for known genes, and the number of alternative transcription start sites used in a given gene was correlated with overall Mitsui-7 response. Strikingly, genes that were up-regulated after Mitsui-7 exposure only through their main annotated transcription start site were linked to inflammatory and defense responses, while genes up-regulated only through alternative transcription start sites were functionally heterogeneous and not inflammation-associated. Furthermore, we identified almost 12 000 active enhancers, many of which were Mitsui-7-responsive, and we identified similarly responding putative target genes. Overall, our study provides the location and activity of Mitsui-7-induced enhancers and transcription start sites, providing a useful resource for targeted experiments elucidating the biological effects of nanomaterials and the identification of biomarkers for early detection of MWCNT-induced inflammation.

Nanomaterial (NM) characteristics may affect the pulmonary toxicity and inflammatory response, including specific surface area, size, shape, crystal phase or other surface characteristics. Grouping of TiO

Background Little is known about the exposure levels and adverse health effects of occupational exposure to airplane emissions. Diesel exhaust particles are classified as carcinogenic to humans and jet engines produce potentially similar soot particles. Here, we evaluated the potential occupational exposure risk by analyzing particles from a non-commercial airfield and from the apron of a commercial airport. Toxicity of the collected particles was evaluated alongside NIST standard reference diesel exhaust particles (NIST2975) in terms of acute phase response, pulmonary inflammation, and genotoxicity after single intratracheal instillation in mice. Results Particle exposure levels were up to 1 mg/m3 at the non-commercial airfield. Particulate matter from the non-commercial airfield air consisted of primary and aggregated soot particles, whereas commercial airport sampling resulted in a more heterogeneous mixture of organic compounds including salt, pollen and soot, reflecting the complex occupational exposure at an apron. The particle contents of polycyclic aromatic hydrocarbons and metals were similar to the content in NIST2975. Mice were exposed to doses 6, 18 and 54 μg alongside carbon black (Printex 90) and NIST2975 and euthanized after 1, 28 or 90 days. Dose-dependent increases in total number of cells, neutrophils, and eosinophils in bronchoalveolar lavage fluid were observed on day 1 post-exposure for all particles. Lymphocytes were increased for all four particle types on 28 days post-exposure as well as for neutrophil influx for jet engine particles and carbon black nanoparticles. Increased Saa3 mRNA levels in lung tissue and increased SAA3 protein levels in plasma were observed on day 1 post-exposure. Increased levels of DNA strand breaks in bronchoalveolar lavage cells and liver tissue were observed for both particles, at single dose levels across doses and time points. Conclusions Pulmonary exposure of mice to particles collected at two airports induced acute phase response, inflammation, and genotoxicity similar to standard diesel exhaust particles and carbon black nanoparticles, suggesting similar physicochemical properties and toxicity of jet engine particles and diesel exhaust particles. Given this resemblance as well as the dose-response relationship between diesel exhaust exposure and lung cancer, occupational exposure to jet engine emissions at the two airports should be minimized. Electronic supplementary material The online version of this article (10.1186/s12989-019-0305-5) contains supplementary material, which is available to authorized users.

We studied if the pulmonary and systemic toxicity of nanofibrillated celluloses can be reduced by carboxylation. Nanofibrillated celluloses administered at 6 or 18 mu g to mice by intratracheal instillation were: 1) FINE NFC, 2-20 mu m in length, 2-15 nm in width, 2) AS (-COOH), carboxylated, 0.5-10 mu m in length, 4-10 nm in width, containing the biocide BIM MC4901 and 3) BIOCID FINE NFC: as (1) but containing BIM MC4901. FINE NFC administration increased neutrophil influx in BAL and induced SAA3 in plasma. AS (-COOH) produced lower neutrophil influx and systemic SAA3 levels than FINE NFC. Results obtained with BIOCID FINE NFC suggested that BIM MC4901 biocide did not explain the lowered response. Increased DNA damage levels were observed across materials, doses and time points. In conclusion, carboxylation of nanofibrillated cellulose was associated with reduced pulmonary and systemic toxicity, suggesting involvement of OH groups in the inflammatory and acute phase responses.

Nanomaterial (NM) characteristics may affect the pulmonary toxicity and inflammatory response, including specific surface area, size, shape, crystal phase or other surface characteristics. Grouping of TiO2 in hazard assessment might be challenging because of variation in physicochemical properties. We exposed C57BL/6 J mice to a single dose of four anatase TiO2 NMs with various sizes and shapes by intratracheal instillation and assessed the pulmonary toxicity 1, 3, 28, 90 or 180 days post-exposure. The quartz DQ12 was included as benchmark particle. Pulmonary responses were evaluated by histopathology, electron microscopy, bronchoalveolar lavage (BAL) fluid cell composition and acute phase response. Genotoxicity was evaluated by DNA strand break levels in BAL cells, lung and liver in the comet assay. Multiple regression analyses were applied to identify specific TiO2 NMs properties important for the pulmonary inflammation and acute phase response. The TiO2 NMs induced similar inflammatory responses when surface area was used as dose metrics, although inflammatory and acute phase response was greatest and more persistent for the TiO2 tube. Similar histopathological changes were observed for the TiO2 tube and DQ12 including pulmonary alveolar proteinosis indicating profound effects related to the tube shape. Comparison with previously published data on rutile TiO2 NMs indicated that rutile TiO2 NMs were more inflammogenic in terms of neutrophil influx than anatase TiO2 NMs when normalized to total deposited surface area. Overall, the results suggest that specific surface area, crystal phase and shape of TiO2 NMs are important predictors for the observed pulmonary effects of TiO2 NMs.

Background With ever-increasing exposure to engineered nanomaterials (NMs), there is an urgent need to evaluate the probability of consequential adverse effects. The potential for NM translocation to distal organs is a realistic prospect, with the liver being one of the most important target organs. Traditional in vitro or ex vivo hepatic toxicology models are often limiting (i.e. short life-span, reduced metabolic activity, lacking important cell populations, etc.). In this study, we scrutinize a 3D human liver microtissue (MT) model (composed of primary hepatocytes and non-parenchymal cells). This unique experiment benefits from long-term (3 weeks) repeated very low exposure concentrations, as well as incorporation of recovery periods (up to 2 weeks), in an attempt to account for the liver’s recovery capacity in vivo. As a means of assessing the toxicological potential of NMs, cell cytotoxicity (cell membrane integrity and aspartate aminotransferase (AST) activity), pro/anti-inflammatory response and hepatic function were investigated. Results The data showed that 2 weeks of cell culture might be close to limits before subtle ageing effects start to overshadow low sub-lethal NM-induced cellular responses in this test system (adenylate kinase (AK) cytotoxicity assay). We showed that in vitro AST measurement are not suitable in a nanotoxicological context. Moreover, the cytokine analysis (IL6, IL8, IL10 and TNF-α) proved useful in highlighting recovery periods as being sufficient for allowing a reduction in the pro-inflammatory response. Next, low soluble NM-treated MT showed a concentration-dependent penetration of materials deep into the tissue. Conclusion In this study the advantages and pitfalls of the multi-cellular primary liver MT are discussed. Furthermore, we explore a number of important considerations for allowing more meaningful in vitro vs. in vivo comparisons in the field of hepatic nanotoxicology.

Multi-walled carbon nanotubes (MWCNT) are widely used nanomaterials that cause pulmonary toxicity upon inhalation. The physicochemical properties of MWCNT vary greatly, which makes general safety evaluation challenging to conduct. Identification of the toxicity-inducing physicochemical properties of MWCNT is therefore of great importance. We have evaluated histological changes in lung tissue 1 year after a single intratracheal instillation of 11 well-characterized MWCNT in female C57BL/6N BomTac mice. Genotoxicity in liver and spleen was evaluated by the comet assay. The dose of 54 μg MWCNT corresponds to three times the estimated dose accumulated during a work life at a NIOSH recommended exposure limit (0.001 mg/m3). Short and thin MWCNT were observed as agglomerates in lung tissue 1 year after exposure, whereas thicker and longer MWCNT were detected as single fibres, suggesting biopersistence of both types of MWCNT. The thin and entangled MWCNT induced varying degree of pulmonary inflammation, in terms of lymphocytic aggregates, granulomas and macrophage infiltration, whereas two thick and straight MWCNT did not. By multiple regression analysis, larger diameter and higher content of iron predicted less histopathological changes, whereas higher cobalt content significantly predicted more histopathological changes. No MWCNT-related fibrosis or tumours in the lungs or pleura was found. One thin and entangled MWCNT induced increased levels of DNA strand breaks in liver; however, no physicochemical properties could be related to genotoxicity. This study reveals physicochemical-dependent difference in MWCNT-induced long-term, pulmonary histopathological changes. Identification of diameter size and cobalt content as important for MWCNT toxicity provides clues for designing MWCNT, which cause reduced human health effects following pulmonary exposure.

Background Little is known about the mechanism underlying the genotoxicity observed in the liver following pulmonary exposure to carbon black (CB) nanoparticles (NPs). The genotoxicity could be caused by the presence of translocated particles or by circulating inflammatory mediators released during pulmonary inflammation and acute-phase response. To address this, we evaluated induction of pulmonary inflammation, pulmonary and hepatic acute-phase response and genotoxicity following exposure to titanium dioxide (TiO2), cerium oxide (CeO2) or CB NPs. Female C57BL/6 mice were exposed by intratracheal instillation, intravenous injection or oral gavage to a single dose of 162 μg NPs/mouse and terminated 1, 28 or 180 days post-exposure alongside vehicle control. Results Liver DNA damage assessed by the Comet Assay was observed after intravenous injection and intratracheal instillation of CB NPs but not after exposure to TiO2 or CeO2. Intratracheal exposure to NPs resulted in pulmonary inflammation in terms of increased neutrophils influx for all NPs 1 and 28 days post-exposure. Persistent pulmonary acute phase response was detected for all NPs at all three time points while only a transient induction of hepatic acute phase response was observed. All 3 materials were detected in the liver by enhanced darkfield microscopy up to 180 days post-exposure. In contrast to TiO2 and CeO2 NPs, CB NPs generated ROS in an acellular assay. Conclusions Our results suggest that the observed hepatic DNA damage following intravenous and intratracheal dosing with CB NPs was caused by the presence of translocated, ROS-generating, particles detected in the liver rather than by the secondary effects of pulmonary inflammation or hepatic acute phase response. Electronic supplementary material The online version of this article (10.1186/s12989-017-0238-9) contains supplementary material, which is available to authorized users.

Vertical arrays of nanostructures (NSs) are emerging as promising platforms for probing and manipulating live mammalian cells. The broad range of applications requires different types of interfaces, but cell settling on NS arrays is not yet fully controlled and understood. Cells are both seen to deform completely into NS arrays and to stay suspended like tiny fakirs, which have hitherto been explained with differences in NS spacing or density. Here, a better understanding of this phenomenon is provided by using a model that takes into account the extreme membrane deformation needed for a cell to settle into a NS array. It is shown that, in addition to the NS density, cell settling depends strongly on the dimensions of the single NS, and that the settling can be predicted for a given NS array geometry. The predictive power of the model is confirmed by experiments and good agreement with cases from the literature. Furthermore, the influence of cell-related parameters is evaluated theoretically and a generic method of tuning cell settling through surface coating is demonstrated experimentally. These findings allow a more rational design of NS arrays for the numerous exciting biological applications where the mode of cell settling is crucial.

Arrays of nanowires (NWs) are currently being established as vehicles for molecule delivery and electrical- and fluorescence-based platforms in the development of biosensors. It is conceivable that NW-based biosensors can be optimized through increased understanding of how the nanotopography influences the interfaced biological material. Using state-of-the-art homogenous NW arrays allow for a systematic investigation of how the broad range of NW densities used by the community influences cells. Here it is demonstrated that indium arsenide NW arrays provide a cell-promoting surface, which affects both cell division and focal adhesion up-regulation. Furthermore, a systematic variation in NW spacing affects both the detailed cell morphology and adhesion properties, where the latter can be predicted based on changes in free-energy states using the proposed theoretical model. As the NW density influences cellular parameters, such as cell size and adhesion tightness, it will be important to take NW density into consideration in the continued development of NW-based platforms for cellular applications, such as molecule delivery and electrical measurements.

Lung deposition of multi-walled carbon nanotubes (MWCNT) induces pulmonary toxicity. Commercial MWCNT vary greatly in physicochemical properties and consequently in biological effects. To identify determinants of MWCNT-induced toxicity, we analyzed the effects of pulmonary exposure to 10 commercial MWCNT (supplied in three groups of different dimensions, with one pristine and two/three surface modified in each group). We characterized morphology, chemical composition, surface area and functionalization levels. MWCNT were deposited in lungs of female C57BL/6J mice by intratracheal instillation of 0, 6, 18 or 54 μg/mouse. Pulmonary inflammation (neutrophil influx in bronchoalveolar lavage (BAL)) and genotoxicity were determined on day 1, 28 or 92. Histopathology of the lungs was performed on day 28 and 92. All MWCNT induced similar histological changes. Lymphocytic aggregates were detected for all MWCNT on day 28 and 92. Using adjusted, multiple regression analyses, inflammation and genotoxicity were related to dose, time and physicochemical properties. The specific surface area (BET) was identified as a positive predictor of pulmonary inflammation on all post-exposure days. In addition, length significantly predicted pulmonary inflammation, whereas surface oxidation (–OH and –COOH) was predictor of lowered inflammation on day 28. BET surface area, and therefore diameter, significantly predicted genotoxicity in BAL fluid cells and lung tissue such that lower BET surface area or correspondingly larger diameter was associated with increased genotoxicity. This study provides information on possible toxicity-driving physicochemical properties of MWCNT. The results may contribute to safe-by-design manufacturing of MWCNT, thereby minimizing adverse effects.

The human malaria parasite Plasmodium falciparum detoxifies the heme byproduct of hemoglobin digestion in infected red blood cells by sequestration into submicron-sized hemozoin crystals. The crystal is composed of heme units interlinked to form cyclic dimers via reciprocal Fe─O (propionate) bonds. Templated hemozoin nucleation was envisaged to explain a classic observation by electron microscopy of a cluster of aligned hemozoin crystals within the parasite digestive vacuole. This dovetails with evidence that acylglycerol lipids are involved in hemozoin nucleation in vivo, and nucleation of β-hematin, the synthetic analogue of hemozoin, was consistently induced at an acylglycerol–water interface via their {100} crystal faces. In order to ascertain the nature of hemozoin nucleation in vivo, we probed the mutual orientations of hemozoin crystals in situ within RBCs using synchrotron-based X-ray nanoprobe Fe fluorescence and diffraction. The X-ray patterns indicated the presence of hemozoin clusters, each comprising several crystals aligned along their needle c axes and exposing {100} side faces to an approximately cylindrical surface, suggestive of nucleation via a common lipid layer. This experimental finding, and the associated nucleation model, are difficult to reconcile with recent reports of hemozoin formation within lipid droplets in the digestive vacuole. The diffraction results are verified by a study of the nucleation process using emerging tools of three-dimensional cellular microscopy, described in the companion paper.

Recent findings show that cerium oxide (CeO2) nanoparticles may undergo in vivo-induced size transformation with the formation of smaller particles that could result in a higher translocation following pulmonary exposure compared to virtually insoluble particles, like titanium dioxide (TiO2). Therefore, we compared liver deposition of CeO2 and TiO2 nanoparticles of similar primary sizes 1, 28 or 180 days after intratracheal instillation of 162 μg of NPs in female C57BL/6 mice. Mice exposed to 162 μg CeO2 or TiO2 nanoparticles by intravenous injection or oral gavage were included as reference groups to assess the amount of NPs that reach the liver bypassing the lungs and the translocation of NPs from the gastrointestinal tract to the liver, respectively. Pulmonary deposited CeO2 nanoparticles were detected in the liver 28 and 180 days post-exposure and TiO2 nanoparticles 180 days post-exposure as determined by darkfield imaging and by the quantification of Ce and Ti mass concentration by inductively coupled plasma-mass spectrometry (ICP-MS). Ce and Ti concentrations increased over time and 180 days post-exposure the translocation to the liver was 2.87 ± 3.37% and 1.24 ± 1.98% of the initial pulmonary dose, respectively. Single particle ICP-MS showed that the size of CeO2 nanoparticles in both lung and liver tissue decreased over time. No nanoparticles were detected in the liver following oral gavage. Our results suggest that pulmonary deposited CeO2 and TiO2 nanoparticles translocate to the liver with similar calculated translocation rates despite their different chemical composition and shape. The observed particle size distributions of CeO2 nanoparticles indicate in vivo processing over time both in lung and liver. The fact that no particles were detected in the liver following oral exposure showed that direct translocation of nanoparticles from lung to the systemic circulation was the most important route of translocation for pulmonary deposited particles.

Micro contact printing (microCP) has been established as a simple technique for high-resolution protein patterning for micro- and nanoarrays. However, as biochemical assays based on immobilized protein arrays progress from immunoassays to more delicate functional assays, the demand for methods of miniaturized, gentle, and oriented immobilization, which are applicable to many different target proteins, becomes larger. In this study, we present a novel microCP templated assembly approach, based on a recombinant SNAP-FLAG-HIS 10 (SFH) immobilization vehicle, which exploits the recently developed SNAP-tag protein. The SNAP-tag is derived from the human DNA repair protein hAGT, which covalently transfers the alkyl group of benzyl guanine (BG) substrates onto itself. We have designed a model SFH cassette carrying three tags (SNAP-tag, FLAG-tag, and HIS-tag), each of which can be used for fluorescence labeling or surface immobilization. When patterns of streptavidin modified with BG-biotin (streptavidin-BG) are stamped onto a surface, the SFH can subsequently assemble on the ligand pattern from solution, functioning as a general immobilization vehicle for high-resolution patterning of any protein expressed in the SFH cassette, in a gentle and oriented manner. Alternatively, the SFH can be site-selectively biotinylated using BG-biotin and, subsequently, assemble on stamped streptavidin. We exploit several ways to biotinylate the SFH protein via the SNAP-tag, promoting its templated assembly on micropatterns of streptavidin in four complementary formats. Quantitative analysis of the obtained patterns, revealed by immunostaining, indicates that all four approaches resulted in proper SFH immobilization and antibody recognition, demonstrating the versatility of the SFH cassette and the potential for high resolution patterning applications. Also, our data confirm that streptavidin can be stamped directly on surfaces, without loss of activity. While three strategies resulted in similar patterning efficiencies, one particular approach--namely templated assembly of SFH directly on streptavidin-BG patterns--resulted in an order of magnitude increase in patterning efficiency.

We investigated toxicity of 2-3 layered >1 μm sized graphene oxide (GO) and reduced graphene oxide (rGO) in mice following single intratracheal exposure with respect to pulmonary inflammation, acute phase response (biomarker for risk of cardiovascular disease) and genotoxicity. In addition, we assessed exposure levels of particulate matter emitted during production of graphene in a clean room and in a normal industrial environment using chemical vapour deposition. Toxicity was evaluated at day 1, 3, 28 and 90 days (18, 54 and 162 μg/mouse), except for GO exposed mice at day 28 and 90 where only the lowest dose was evaluated. GO induced a strong acute inflammatory response together with a pulmonary (Serum-Amyloid A, Saa3) and hepatic (Saa1) acute phase response. rGO induced less acute, but a constant and prolonged inflammation up to day 90. Lung histopathology showed particle agglomerates at day 90 without signs of fibrosis. In addition, DNA damage in BAL cells was observed across time points and doses for both GO and rGO. In conclusion, pulmonary exposure to GO and rGO induced inflammation, acute phase response and genotoxicity but no fibrosis.

Protein microarrays are valuable tools for protein assays. Reducing spot sizes from micro- to nano-scale facilitates miniaturization of platforms and consequently decreased material consumption, but faces inherent challenges in the reduction of fluorescent signals and compatibility with complex solutions. Here we show that vertical arrays of nanowires (NWs) can overcome several bottlenecks of using nanoarrays for extraction and analysis of proteins. The high aspect ratio of the NWs results in a large surface area available for protein immobilization and renders passivation of the surface between the NWs unnecessary. Fluorescence detection of proteins allows quantitative measurements and spatial resolution, enabling us to track individual NWs through several analytical steps, thereby allowing multiplexed detection of different proteins immobilized on different regions of the NW array. We use NW arrays for on-chip extraction, detection and functional analysis of proteins on a nano-scale platform that holds great promise for performing protein analysis on minute amounts of material. The demonstration made here on highly ordered arrays of indium arsenide (InAs) NWs is generic and can be extended to many high aspect ratio nanostructures.

New developments in X-ray instrumentation and analysis have facilitated the development and improvement of various scanning X-ray microscopy techniques. In this contribution, we offer an overview of recent scanning hard X-ray microscopy measurements performed at the Swiss Light Source. We discuss scanning transmission X-ray microscopy in its transmission, phase contrast, and dark-field imaging modalities. We demonstrate how small-angle X-ray scattering analysis techniques can be used to yield additional information. If the illumination is coherent, coherent diffraction imaging techniques can be brought to bear. We discuss how, from scanning microscopy measurements, detailed measurements of the X-ray scattering distributions can be used to extract high-resolution images. These microscopy techniques with their respective imaging power can easily be combined to multimodal X-ray microscopy.

The endeavor of exploiting arrays of vertical one-dimensional (1D) nanostructures (NSs) for cellular applications has recently been experiencing a pronounced surge of activity. The interest is rooted in the intrinsic properties of high-aspect-ratio NSs. With a height comparable to a mammalian cell, and a diameter 100-1000 times smaller, NSs should intuitively reach far into a cell and, due to their small diameter, do so without compromising cell health. Single NSs would thus be expedient for measuring and modifying cell response. Further organization of these structures into arrays can provide up-scaled and detailed spatiotemporal information on cell activity, an achievement that would entail a massive leap forward in disease understanding and drug discovery. Numerous proofs-of-principle published recently have expanded the large toolbox that is currently being established in this rapidly advancing field of research. Encouragingly, despite the diversity of NS platforms and experimental conditions used thus far, general trends and conclusions from combining cells with NSs are beginning to crystallize. This review covers the broad spectrum of NS materials and dimensions used; the observed cellular responses with specific focus on adhesion, morphology, viability, proliferation, and migration; compares the different approaches used in the field to provide NSs with the often crucial cytosolic access; covers the progress toward biological applications; and finally, envisions the future of this technology. By maintaining the impressive rate and quality of recent progress, it is conceivable that the use of vertical 1D NSs may soon be established as a superior choice over other current techniques, with all the further benefits that may entail.

The perspectives offered by vertical arrays of nanowires for biosensing applications in living cells depend on the access of individual nanowires to the cell interior. Recent results on electrical access and molecular delivery suggest that direct access is not always obtained. Here, we present a generic approach to directly visualize the membrane conformation of living cells interfaced with nanowire arrays, with single nanowire resolution. The method combines confocal z-stack imaging with an optimized cell membrane labelling strategy which was applied to HEK293 cells interfaced with 2-11 μm long and 3-7 μm spaced nanowires with various surface coatings (bare, aminosilane-coated or polyethyleneimine-coated indium arsenide). We demonstrate that, for all commonly used nanowire lengths, spacings and surface coatings, nanowires generally remain enclosed in a membrane compartment, and are thereby not in direct contact with the cell interior.

Nanowires (NWs) are attracting more and more interest due to their potential cellular applications, such as delivery of compounds or sensing platforms. Arrays of vertical indium-arsenide (InAs) NWs are interfaced with human embryonic kidney cells and rat embryonic dorsal root ganglion neurons. A selection of critical cell functions and pathways are shown not to be impaired, including cell adhesion, membrane integrity, intracellular enzyme activity, DNA uptake, cytosolic and membrane protein expression, and the neuronal maturation pathway. The results demonstrate the low invasiveness of InAs NW arrays, which, combined with the unique physical properties of InAs, open up their potential for cellular investigations.

Multi-walled carbon nanotubes (MWCNTs), which vary in length, diameter, functionalization and specific surface area, are used in diverse industrial processes. Since these nanomaterials have a high aspect ratio and are biopersistant in the lung, there is a need for a rapid identification of their potential health hazard. We assessed in Sprague-Dawley rats the pulmonary toxicity of two pristine MWCNTs (the “long and thick” NM-401 and the “short and thin” NM-403) following either intratracheal instillation or 4-week inhalation in order to gain insights into the predictability and intercomparability of the two methods. The deposited doses following inhalation were lower than the instilled doses. Both types of carbon nanotube induced pulmonary neutrophil influx using both exposure methods. This influx correlated with deposited surface area across MWCNT types and means of exposure at two different time points, 1–3 days and 28–30 days post-exposure. Increased levels of DNA damage were observed across doses and time points for both exposure methods, but no dose-response relationship was observed. Intratracheal instillation of NM-401 induced fibrosis at the highest dose while lower lung deposited doses obtained by inhalation did not induce such lung pathology. No fibrosis was observed following NM-403 exposure. When the deposited dose was taken into account, sub-acute inhalation and a single instillation of NM-401 and NM-403 produced very similar inflammation and DNA damage responses. Our data suggest that the dose-dependent inflammatory responses observed after intratracheal instillation and inhalation of MWCNTs are similar and were predicted by the deposited surface area.

Nanowires (NWs) are attracting more and more interest due to their potential cellular applications, such as delivery of compounds or sensing platforms. Arrays of vertical indium-arsenide (InAs) NWs are interfaced with human embryonic kidney cells and rat embryonic dorsal root ganglion neurons. A selection of critical cell functions and pathways are shown not to be impaired, including cell adhesion, membrane integrity, intracellular enzyme activity, DNA uptake, cytosolic and membrane protein expression, and the neuronal maturation pathway. The results demonstrate the low invasiveness of InAs NW arrays, which, combined with the unique physical properties of InAs, open up their potential for cellular investigations.