Reliable and predictive in vitro assays for hazard assessments of manufactured nanomaterials (MNMs) are still limited. Specifically, exposure systems which more realistically recapitulate the ...physiological conditions in the lung are needed to predict pulmonary toxicity. To this end, air-liquid interface (ALI) systems have been developed in recent years which might be better suited than conventional submerged exposure assays. However, there is still a need for rigorous side-by-side comparisons of the results obtained with the two different exposure methods considering numerous parameters, such as different MNMs, cell culture models and read outs. In this study, human A549 lung epithelial cells and differentiated THP-1 macrophages were exposed under submerged conditions to two abundant types of MNMs i.e., ceria and titania nanoparticles (NPs). Membrane integrity, metabolic activity as well as pro-inflammatory responses were recorded. For comparison, A549 monocultures were also exposed at the ALI to the same MNMs. In the case of titania NPs, genotoxicity was also investigated. In general, cells were more sensitive at the ALI compared to under classical submerged conditions. Whereas ceria NPs triggered only moderate effects, titania NPs clearly initiated cytotoxicity, pro-inflammatory gene expression and genotoxicity. Interestingly, low doses of NPs deposited at the ALI were sufficient to drive adverse outcomes, as also documented in rodent experiments. Therefore, further development of ALI systems seems promising to refine, reduce or even replace acute pulmonary toxicity studies in animals.
Investigations on adverse biological effects of nanoparticles (NPs) in the lung by in vitro studies are usually performed under submerged conditions where NPs are suspended in cell culture media. ...However, the behaviour of nanoparticles such as agglomeration and sedimentation in such complex suspensions is difficult to control and hence the deposited cellular dose often remains unknown. Moreover, the cellular responses to NPs under submerged culture conditions might differ from those observed at physiological settings at the air-liquid interface.
In order to avoid problems because of an altered behaviour of the nanoparticles in cell culture medium and to mimic a more realistic situation relevant for inhalation, human A549 lung epithelial cells were exposed to aerosols at the air-liquid interphase (ALI) by using the ALI deposition apparatus (ALIDA). The application of an electrostatic field allowed for particle deposition efficiencies that were higher by a factor of more than 20 compared to the unmodified VITROCELL deposition system. We studied two different amorphous silica nanoparticles (particles produced by flame synthesis and particles produced in suspension by the Stöber method). Aerosols with well-defined particle sizes and concentrations were generated by using a commercial electrospray generator or an atomizer. Only the electrospray method allowed for the generation of an aerosol containing monodisperse NPs. However, the deposited mass and surface dose of the particles was too low to induce cellular responses. Therefore, we generated the aerosol with an atomizer which supplied agglomerates and thus allowed a particle deposition with a three orders of magnitude higher mass and of surface doses on lung cells that induced significant biological effects. The deposited dose was estimated and independently validated by measurements using either transmission electron microscopy or, in case of labelled NPs, by fluorescence analyses. Surprisingly, cells exposed at the ALI were less sensitive to silica NPs as evidenced by reduced cytotoxicity and inflammatory responses.
Amorphous silica NPs induced qualitatively similar cellular responses under submerged conditions and at the ALI. However, submerged exposure to NPs triggers stronger effects at much lower cellular doses. Hence, more studies are warranted to decipher whether cells at the ALI are in general less vulnerable to NPs or specific NPs show different activities dependent on the exposure method.
In vitro toxicity testing of airborne particles usually takes place in multi-well plates, where the cells are exposed to a suspension of particles in cell culture medium. Due to the artefacts caused ...by particle collection and preparation of suspensions, the air–liquid interface (ALI) exposure is challenging this conventional exposure technique to become the method of choice. The ALI technique allows for direct sampling of an aerosol and exposure of cell cultures to airborne particles. At the same time, it reflects the physiological conditions in the lung to a greater extent. So far, the available ALI systems have mostly been laboratory set-ups of the single components. Here, we present a mobile and complete system providing all process technology required for cell exposure experiments at dynamic aerosol sources. The system is controlled by a human machine interface (HMI) with standard routines for experiments and internal testing to assure reproducibility. It also provides documentation of the exposure experiment regarding process parameters and measured doses. The performance of this system is evaluated using fluorescein-sodium dosimetry, which is also used to determine the factor of dose enhancement by optional electrostatic deposition. The application of the system is shown for two different technical aerosol sources: wood smoke particles emitted by a household log wood stove and emissions from a ship diesel engine. After exposure of lung cells, cytotoxicity and gene regulation on a genome-wide scale were analysed.
•We present a fully automated air–liquid-interface exposure system.•System characterisation shows a high reproducibility.•Wood combustion aerosol causes cytotoxicity in human lung epithelial cells.•Gene regulation caused by ship diesel emissions in human lung epithelial cells.
Extensive production and use of nanomaterials (NMs), such as titanium dioxide (TiO
), raises concern regarding their potential adverse effects to humans. While considerable efforts have been made to ...assess the safety of TiO
NMs using in vitro and in vivo studies, results obtained to date are unreliable, possibly due to the dynamic agglomeration behavior of TiO
NMs. Moreover, agglomerates are of prime importance in occupational exposure scenarios, but their toxicological relevance remains poorly understood. Therefore, the aim of this study was to investigate the potential pulmonary effects induced by TiO
agglomerates of different sizes at the air-liquid interface (ALI), which is more realistic in terms of inhalation exposure, and compare it to results previously obtained under submerged conditions. A nano-TiO
(17 nm) and a non-nano TiO
(117 nm) was selected for this study. Stable stock dispersions of small agglomerates and their respective larger counterparts of each TiO
particles were prepared, and human bronchial epithelial (HBE) cells were exposed to different doses of aerosolized TiO
agglomerates at the ALI. At the end of 4h exposure, cytotoxicity, glutathione depletion, and DNA damage were evaluated. Our results indicate that dose deposition and the toxic potential in HBE cells are influenced by agglomeration and exposure via the ALI induces different cellular responses than in submerged systems. We conclude that the agglomeration state is crucial in the assessment of pulmonary effects of NMs.
Several in vitro studies have suggested that silica nanoparticles (NPs) might induce adverse effects in gut cells. Here, we used the human colon cancer epithelial cell line HCT116 to study the ...potential cytotoxic effects of ingested silica NPs in the presence or absence of serum. Furthermore, we evaluated different physico-chemical parameters important for the assessment of nanoparticle safety, including primary particle size (12, 70, 200, and 500 nm) and surface modification (-NH
and -COOH). Silica NPs triggered cytotoxicity, as evidenced by reduced metabolism and enhanced membrane leakage. Automated microscopy revealed that the silica NPs promoted apoptosis and necrosis proportional to the administered specific surface area dose. Cytotoxicity of silica NPs was suppressed by increasing amount of serum and surface modification. Furthermore, inhibition of caspases partially prevented silica NP-induced cytotoxicity. In order to investigate the role of specific cell death pathways in more detail, we used isogenic derivatives of HCT116 cells which lack the pro-apoptotic proteins p53 or BAX. In contrast to the anticancer drug cisplatin, silica NPs induced cell death independent of the p53-BAX axis. In conclusion, silica NPs initiated cell death in colon cancer cells dependent on the specific surface area and presence of serum. Further studies in vivo are warranted to address potential cytotoxic actions in the gut epithelium. The unintended toxicity of silica NPs as observed here could also be beneficial. As loss of p53 in colon cancer cells contributes to resistance against anticancer drugs, and thus to reoccurrence of colon cancer, targeted delivery of silica NPs could be envisioned to also deplete p53 deficient tumor cells.
Manufactured nanomaterials (MNMs) selected from a library of over 120 different MNMs with varied compositions, sizes, and surface coatings were tested by four different laboratories for toxicity by ...high-throughput/-content (HT/C) techniques. The selected particles comprise 14 MNMs composed of CeO
2
, Ag, TiO
2
, ZnO and SiO
2
with different coatings and surface characteristics at varying concentrations. The MNMs were tested in different mammalian cell lines at concentrations between 0.5 and 250 µg/mL to link physical–chemical properties to multiple adverse effects. The cell lines are derived from relevant organs such as liver, lung, colon and the immune system. Endpoints such as viable cell count, cell membrane permeability, apoptotic cell death, mitochondrial membrane potential, lysosomal acidification and steatosis have been studied. Soluble MNMs, Ag and ZnO, were toxic in all cell types. TiO
2
and SiO
2
MNMs also triggered toxicity in some, but not all, cell types and the cell type-specific effects were influenced by the specific coating and surface modification. CeO
2
MNMs were nearly ineffective in our test systems. Differentiated liver cells appear to be most sensitive to MNMs, Whereas most of the investigated MNMs showed no acute toxicity, it became clear that some show adverse effects dependent on the assay and cell line. Hence, it is advised that future nanosafety studies utilise a multi-parametric approach such as HT/C screening to avoid missing signs of toxicity. Furthermore, some of the cell type-specific effects should be followed up in more detail and might also provide an incentive to address potential adverse effects in vivo in the relevant organ.
We report the straightforward, time‐efficient synthesis of radical core–shell nanoparticles (NPs) by polymerization‐induced self‐assembly. A nitroxide‐containing hydrophilic macromolecular precursor ...was prepared by ring‐opening metathesis copolymerization of norbornenyl derivatives of TEMPO and oligoethylene glycol and was chain‐extended in situ with norbornene in ethanolic solution, leading to simultaneous amphiphilic block copolymer formation and self‐assembly. Without any intermediate purification from the monomers to the block copolymers, radical NPs with tunable diameters ranging from 10 to 110 nm are obtained within minutes at room temperature. The high activity of the radical NPs as chemoselective and homogeneous, yet readily recyclable catalysts is demonstrated through oxidation of a variety of alcohols and recovery by simple centrifugation. Furthermore, the NPs show biocompatibility and antioxidant activity in vitro.
Particularly radical: Radical core–shell nanoparticles can be synthesized by ring‐opening metathesis polymerization‐induced self‐assembly (ROMPISA). The radical nanoparticles exhibited high chemoselectivity, catalytic, and recycling capacity in oxidation reactions, and biocompatibility and antioxidant activity in vitro.
Abstract Although the technological and economic benefits of engineered nanomaterials are obvious, concerns have been raised about adverse effects if such material is inhaled, ingested, applied to ...the skin or even released into the environment. Here we studied the cytotoxic effects of the most abundant nanomaterial, silica nanoparticles (SiO2 -NPs), in murine RAW264.7 macrophages. SiO2 -NPs dose-dependently induce membrane leakage and cell death without obvious involvement of reactive oxygen species. Interestingly, at low concentrations SiO2 -NPs trigger autophagy, evidenced by morphological and biochemical hallmarks such as autophagolysosomes or increased levels of LC3-II, which serves to protect cells from cytotoxicity. Hence SiO2 -NPs initiate an adaptive stress response which dependent on dose serve to balance survival and death and ultimately dictates the cellular fate.
Engineered amorphous silica nanoparticles (SiO
2
-NPs) are widely used in dyes, varnishes, plastics and glue, as well as in pharmaceuticals, cosmetics and food. Novel composite SiO
2
-NPs are ...promising multifunctional devices and combine labels for subsequent tracking and are functionalized e.g. to specifically target cells to deliver their cargo. However, biological and potential toxic effects of SiO
2
-NPs are insufficiently understood. The aim of this study was to determine the uptake and fate of SiO
2
-NPs in mammalian cells. Also, silica submicron particles (SiO
2
-SMPs) were included in the studies in order to identify effects, which are only observed for nano-sized SiO
2
particles. Fluorescently labelled SiO
2
-NPs (nominal size 70 nm) and SiO
2
-SMPs (nominal size 200 and 500 nm) were used to examine cytotoxicity, cellular uptake and localization in human cervical carcinoma cells (HeLa). Particle uptake and intracellular localization in mitochondria, endosomes, lysosomes and nuclei were studied by wide field and confocal laser scanning fluorescence microscopy. Physicochemical characterization of SiO
2
-NPs by transmission electron microscopy and dynamic light scattering revealed a spherical morphology and a monodisperse size distribution. In the presence of serum, all SiO
2
particles are non-toxic. However, in the absence of serum SiO
2
-NPs but not SiO
2
-SMPs are highly toxic. SiO
2
particles, irrespective of size, were detected in the cytosol and accumulated in endosomal compartments of HeLa cells. No accumulation of SiO
2
particles in nuclei or mitochondria of HeLa cells could be observed. In contrast to SiO
2
-SMPs, SiO
2
-NPs are preferentially localized in lysosomes.
In this article, we review and analyze different modes of exposure to ultrafine particles in order to assess particle-induced inflammatory responses and the underlying mechanisms in vitro and in ...vivo. Based on results from monocytic cells cultured under submerged conditions, we discuss (1) the impact of particle properties such as surface area and oxidative potential on lipid metabolism as a highly sensitive regulatory pathway and (2) the interference of diesel exhaust particles with toll-like receptor-mediated inflammatory responses. Furthermore, new developments of air-liquid interface exposure used as an alternative approach to simulate cell particle interactions are presented. In addition to the in vitro approaches, animal exposure studies are described that apply selected mouse models to elucidate potential allergic and inflammatory pulmonary responses and mast-cell-related mechanisms after particle exposure. Long-term inhalation of ultrafine particles might lead to irreversible changes in lung structure and function. Clinical studies addressing the characteristics of inflammatory airway cells are a promising approach to understand underlying pathophysiological mechanisms in chronic obstructive pulmonary disease. Finally, a potential outcome of human particle exposure is chronic cough in children. Here, discrimination between asthmatic and nonasthmatic cough by means of immunological parameters appears to be an important step toward improving diagnosis and therapy.
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