BackgroundEngineered nanoparticles are becoming increasingly ubiquitous and their toxicological effects on human health, as well as on the ecosystem, have become a concern. Since initial contact with nanoparticles occurs at the epithelium in the lungs (or skin, or eyes), in vitro cell studies with nanoparticles require dose-controlled systems for delivery of nanoparticles to epithelial cells cultured at the air-liquid interface.ResultsA novel air-liquid interface cell exposure system (ALICE) for nanoparticles in liquids is presented and validated. The ALICE generates a dense cloud of droplets with a vibrating membrane nebulizer and utilizes combined cloud settling and single particle sedimentation for fast (~10 min; entire exposure), repeatable (<12%), low-stress and efficient delivery of nanoparticles, or dissolved substances, to cells cultured at the air-liquid interface. Validation with various types of nanoparticles (Au, ZnO and carbon black nanoparticles) and solutes (such as NaCl) showed that the ALICE provided spatially uniform deposition (<1.6% variability) and had no adverse effect on the viability of a widely used alveolar human epithelial-like cell line (A549). The cell deposited dose can be controlled with a quartz crystal microbalance (QCM) over a dynamic range of at least 0.02-200 μg/cm2. The cell-specific deposition efficiency is currently limited to 0.072 (7.2% for two commercially available 6-er transwell plates), but a deposition efficiency of up to 0.57 (57%) is possible for better cell coverage of the exposure chamber.Dose-response measurements with ZnO nanoparticles (0.3-8.5 μg/cm2) showed significant differences in mRNA expression of pro-inflammatory (IL-8) and oxidative stress (HO-1) markers when comparing submerged and air-liquid interface exposures. Both exposure methods showed no cellular response below 1 μg/cm2 ZnO, which indicates that ZnO nanoparticles are not toxic at occupationally allowed exposure levels.ConclusionThe ALICE is a useful tool for dose-controlled nanoparticle (or solute) exposure of cells at the air-liquid interface. Significant differences between cellular response after ZnO nanoparticle exposure under submerged and air-liquid interface conditions suggest that pharmaceutical and toxicological studies with inhaled (nano-)particles should be performed under the more realistic air-liquid interface, rather than submerged cell conditions.

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In inhalation therapy, drugs are deposited as aerosols onto the air-facing lung epithelium. The currently used in vitro cell assays for drug testing, however, typically dissolve drugs in the medium, completely covering the cells, which represents an unphysiological drug application scenario. Although physiologically realistic in vitro cell culture models of the pulmonary air-blood barrier are available, reliable, easy-to-handle, and efficient technologies for direct aerosol-to-cell delivery are lacking. Here, we introduce the Air-Liquid Interface (ALI) Cell Exposure-Cloud (ALICE-CLOUD) technology, which uses principles of cloud motion for fast and quantitative delivery of aerosolized liquid drugs to pulmonary cells cultured under realistic ALI conditions. Aerosol-to-cell delivery proved to be highly efficient, reproducible, and rapid when using aerosolized fluorescein as surrogate drug. As a proof-of-concept study for the ALICE-CLOUD, we performed functional efficacy studies with the U.S. Food and Drug Administration-approved proteasome inhibitor, Bortezomib, a novel candidate drug for inhalation therapy. Aerosolized Bortezomib had a pronounced anti-inflammatory effect on human epithelial lung cells (A549), as indicated by a significant reduction of (TNFα-induced) IL-8 promoter activation. Importantly, cell-based therapeutic efficacy of aerosolized Bortezomib under ALI conditions was similar to that under dissolved and nonaerosolized submerged conditions, but with faster uptake kinetics. Our data indicate that the ALICE-CLOUD is a reliable tool for aerosolized drug screening with cells cultured under ALI conditions, which combines ease of handling with rapid, efficient, and dosimetrically accurate drug-to-cell delivery. This may pave the way for screening of inhalable drugs under physiologically more relevant and, hence, potentially more predictive conditions than the currently used submerged cell culture systems.

The role of alveolar macrophages in the fate of ultrafine particles in the lung was investigated. Male Wistar-Kyoto rats were exposed to ultrafine gold particles, generated by a spark generator, for 6 h at a concentration of 88 microg/m3 (4 x 10(6)/cm3, 16 nm modal mobility diameter). Up to 7 days, the animals were serially sacrificed, and lavaged cells and lung tissues were examined by transmission electron microscopy. The gold concentration/content in the lung, lavage fluid, and blood was estimated by inductively coupled plasma-mass spectrometry. Gold particles used were spherical and electron dense with diameters of 5-8 nm. The particles were individual or slightly agglomerated. By inductively coupled plasma-mass spectrometry analysis of the lung, 1945 +/- 57 ng (mean +/- SD) and 1512 +/- 184 ng of gold were detected on day 0 and on day 7, respectively, indicating that a large portion of the deposited gold particles was retained in the lung tissue. In the lavage fluid, 573 +/- 67 ng and 96 +/- 29 ng were found on day 0 and day 7, respectively, which means that 29% and 6% of the retained gold particles were lavageable on these days. A low but significant increase of gold (0.03 to 0.06% of lung concentration) was found in the blood. Small vesicles containing gold particles were found in the cytoplasm of alveolar macrophages. In the alveolar septum, the gold particles were enclosed in vesicles observed in the cytoplasm of alveolar type I epithelial cells. These results indicate that inhaled ultrafine gold particles in alveolar macrophages and type I epithelial cells are processed by endocytotic pathways, though the uptake of the gold particles by alveolar macrophages is limited. To a low degree, systemic particle translocation took place.

Ultrafine carbon, metal, and metal oxide particles were generated with a commercially available spark generator designed for the production of carbon particles. Aerosols with number concentrations up to 10 7 cm −3 were produced at flow rates up to 150 lpm. Lognormal size distributions with modal diameters in the range of 18-150 nm and geometric standard deviations of about 1.5 were obtained. The chemical composition, size, number concentration, morphology, and surface area of the particles were varied, and the generation of particles with fixed characteristics could be maintained over many hours. The particle characteristics, however, could not be varied independently. for cadmium oxide, 120 m 2 g −1 for iridium, and 300 m 2 g −1 for ferric oxide), and carbon with a large BET surface area (750 m 2 g −1 ). Iridium, on the other hand, has a huge volume-related BET surface area (2800 m 2 cm −3 ). It was not possible to generate ultrafine carbon particles without contaminations with the generator. However, these contaminations could be decreased in this study from 25% to 6% by replacing organic components of the generator by pure inorganic components.

High levels of particulate matter in ambient air are associated with increased respiratory and cardiovascular health problems. It has been hypothesised that it is the ultrafine particle fraction (diameter ,100 nm) that is largely responsible for these effects. To evaluate the associated mechanisms on a molecular level, the current authors applied an expression profiling approach.Healthy mice were exposed to either ultrafine carbon particles (UFCPs; mass concentration 380 mg?m -3 ) or filtered air for 4 and 24 h. Histology of the lungs did not indicate any pathomorphological changes after inhalation. Examination of the bronchoalveolar lavage fluid revealed a small increase in polymorphonuclear cell number (ranging 0.6-1%) after UFCP inhalation, compared with clean air controls, suggesting a minor inflammatory response. However, DNA microarray profile analysis revealed a clearly biphasic response to particle exposure. After 4 h of inhalation, mainly heat shock proteins were induced, whereas after 24 h, different immunomodulatory proteins (osteopontin, galectin-3 and lipocalin-2) were upregulated in alveolar macrophages and septal cells.In conclusion, these data indicate that inhalation of ultrafine carbon particles triggers a biphasic pro-inflammatory process in the lung, involving the activation of macrophages and the upregulation of immunomodulatory proteins.

Based on epidemiologic observations, the issue of adverse health effects of inhaled ultrafine particles (UFP) is currently under intensive discussion. We therefore examined cardiovascular effects of UFP in a controlled animal exposure on young, healthy WKY rats. Short-term exposure (24 h) to carbon UFPs (38 nm, 180 microg m (-3)), generated by spark discharging, induced a mild but consistent increase in heart rate (18 bpm, 4.8%), which was associated with a significant decrease in heart-rate variability during particle inhalation. The timing and the transient character of these responses point to a particle induced alteration of cardiac autonomic balance, mediated by a pulmonary receptor activation. After 24 h of inhalation exposure, bronchoalveolar lavage revealed significant but low-grade pulmonary inflammation (clean air 1.9% vs. UFPs 6.9% polymorphonuclear cells) and on histopathology sporadic accumulation of particle-laden macrophages was found in the alveolar region. There was no evidence of an inflammation-mediated increase in blood coagulability, as UFP inhalation did not induce any significant changes in plasma fibrinogen or factor VIIa levels and there were no prothrombotic changes in the lung or the heart at both the protein and mRNA level. Histological analysis revealed no signs of cardiac inflammation or cardiomyopathy. This study therefore provides toxicological evidence for UFP-associated pulmonary and cardiac effects in healthy rats. Our findings suggest that the observed changes are mediated by an altered sympatho-vagal balance in response to UFP inhalation, but do not support the concept of an inflammation-mediated prothrombotic state by UFP.

Female Fischer 344 rats were exposed to ultrafine cadmium oxide particles, generated by spark discharging, for 6 h at a concentration of 70 microg Cd/m(3) (1 x 10(6)/cm(3)) (40 nm modal diameter). Lung morphology and quantification of Cd content/concentration by inductively coupled plasma (ICP)-mass spectrometry were performed on days 0, 1, 4, and 7 after exposure. Cd content in the lung on day 0 was 0.53 +/- 0.12 microg/lung, corresponding to 19% of the estimated total inhaled cumulative dose, and the amount remained constant throughout the study. In the liver no significant increase of Cd content was found up to 4 days. A slight but statistically significant increase was observed in the liver on day 7. We found neither exposure-related morphological changes of lungs nor inflammatory responses in lavaged cells. Another group of rats were exposed to a higher concentration of ultrafine CdO particles (550 microg Cd/m(3) for 6 h, 51 nm modal diameter). The rats were sacrificed immediately and 1 day after exposure. The lavage study performed on day 0 showed an increase in the percentage of neutrophils. Multifocal alveolar inflammation was seen histologically on day 0 and day 1. Although the Cd content in the lung was comparable between day 0 and day 1 (3.9 microg/lung), significant elevation of Cd levels in the liver and kidneys was observed on both days. Two of 4 rats examined on day 0 showed elevation of blood cadmium, indicating systemic translocation of a fraction of deposited Cd from the lung in this group. These results and comparison with reported data using fine CdO particles indicate that inhalation of ultrafine CdO particles results in efficient deposition in the rat lung. With regard to the deposition dose, adverse health effects of ultrafine CdO and fine CdO appear to be comparable. Apparent systemic translocation of Cd took place only in animals exposed to a high concentration that induced lung injury.