A dose-controlled system for air-liquid interface cell exposure and application to zinc oxide nanoparticles

<p>Abstract</p> <p>Background</p> <p>Engineered 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), <it>in vitro </it>cell studies with nanoparticles require dose-controlled systems for delivery of nanoparticles to epithelial cells cultured at the air-liquid interface.</p> <p>Results</p> <p>A 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.</p> <p>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/cm²ZnO, which indicates that ZnO nanoparticles are not toxic at occupationally allowed exposure levels.</p> <p>Conclusion</p> <p>The 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.</p

Observations of Particulates within the North Atlantic Flight Corridor: POLINAT 2, September-October 1997

This paper discusses particulate concentration and size distribution data gathered using the University of Missouri-Rolla Mobile Aerosol Sampling System (UMR-MASS), and used to investigate the southern extent of the eastern end of the North Atlantic Flight Corridor (NAFC) during project Pollution From Aircraft Emissions in the North Atlantic Flight Corridor/Subsonic Assessment (SASS) Ozone and Nitrogen Oxide Experiment (POLINAT 2/SONEX) from September 19 to October 23, 1997. The analysis presented in this paper focuses on the corridor effect, or enhancement of pollutants by jet aircraft combustion events. To investigate the phenomena, both vertical and horizontal profiles of the corridor, and regions immediately adjacent to the corridor, were performed. The profiles showed a time-dependent enhancement of particulates within the corridor, and a nonvolatile (with respect to thermal volatilization at 300° C) aerosol enhancement at corridor altitudes by a factor of 3.6. The southern extent of the North Atlantic Flight Corridor was established from a four flight average of the particulate data and yielded a boundary near 42.5° N during the study period. A size distribution analysis of the nonvolatile particulates revealed an enhancement in the \u3c40 nm particulates for size distributions recorded within the flight corridor

An inter-laboratory effort to harmonize the cell-delivered in vitro dose of aerosolized materials

Air-liquid interface (ALI) lung cell models cultured on permeable transwell inserts are increasingly used for respiratory hazard assessment requiring controlled aerosolization and deposition of any material on ALI cells. The approach presented herein aimed to assess the transwell insert-delivered dose of aerosolized materials using the VITROCELL® Cloud12 system, a commercially available aerosol-cell exposure system. An inter-laboratory comparison study was conducted with seven European partners having different levels of experience with the VITROCELL® Cloud12. A standard operating procedure (SOP) was developed and applied by all partners for aerosolized delivery of materials, i.e., a water-soluble molecular substance (fluorescence-spiked salt) and two poorly soluble particles, crystalline silica quartz (DQ12) and titanium dioxide nanoparticles (TiO2 NM-105). The material dose delivered to transwell inserts was quantified with spectrofluorometry (fluorescein) and with the quartz crystal microbalance (QCM) integrated in the VITROCELL® Cloud12 system. The shape and agglomeration state of the deposited particles were confirmed with transmission electron microscopy (TEM). Inter-laboratory comparison of the device-specific performance was conducted in two steps, first for molecular substances (fluorescein-spiked salt), and then for particles. Device- and/or handling-specific differences in aerosol deposition of VITROCELL® Cloud12 systems were characterized in terms of the so-called deposition factor (DF), which allows for prediction of the transwell insert-deposited particle dose from the particle concentration in the aerosolized suspension. Albeit DF varied between the different labs from 0.39 to 0.87 (mean (coefficient of variation (CV)): 0.64 (28%)), the QCM of each VITROCELL® Cloud 12 system accurately measured the respective transwell insert-deposited dose. Aerosolized delivery of DQ12 and TiO2 NM-105 particles showed good linearity (R2 > 0.95) between particle concentration of the aerosolized suspension and QCM-determined insert-delivered particle dose. The VITROCELL® Cloud 12 performance for DQ12 particles was identical to that for fluorescein-spiked salt, i.e., the ratio of measured and salt-predicted dose was 1.0 (29%). On the other hand, a ca. 2-fold reduced dose was observed for TiO2 NM-105 (0.54 (41%)), which was likely due to partial retention of TiO2 NM-105 agglomerates in the vibrating mesh nebulizer of the VITROCELL® Cloud12. This inter-laboratory comparison demonstrates that the QCM integrated in the VITROCELL® Cloud 12 is a reliable tool for dosimetry, which accounts for potential variations of the transwell insert-delivered dose due to device-, handling- and/or material-specific effects. With the detailed protocol presented herein, all seven partner laboratories were able to demonstrate dose-controlled aerosolization of material suspensions using the VITROCELL® Cloud12 exposure system at dose levels relevant for observing in vitro hazard responses. This is an important step towards regulatory approved implementation of ALI lung cell cultures for in vitro hazard assessment of aerosolized materials

Calibration of Gas Flow Meters using Choked Flow and an Evacuated Vessel

The measurement of gas flow rates is of great importance in a wide range of modern technologies. This paper introduces a simple, yet accurate technique for in-house calibration of gas FMs (mass and volumetric) even under harsh environmental conditions such as encountered during field measurement campaigns. The method requires only readily available, low cost components: a vessel of known volume, an air pump, a pressure sensor and a metal plate orifice or a needle valve to act as a CO. The unique property of choked flow in the CO is used here for flow calibration. In the method presented here a vessel is evacuated to below the critical pressure ( \u3c 0.53 of upstream pressure) and then allowed to refill with ambient air (or some other process gas) under so-called choked flow conditions through the CO. The method presented here leverages that the flow rate upstream of the CO is not only constant but readily determined from (a) the known V VESS, (b) the measured time rate of change of the absolute pressure in the vessel and (c) the ideal gas law. This calculated flow rate can be used for calibration of FMs. The accuracy of the method depends only on the accuracy of the pressure measurement, the timer and the value of the V VESS. The flow rate computed in this way is found to be in excellent agreement (typically 1% difference) with the flow rate measured by a soap film FM (Gilibrator). As expected from theory this method is found to work for all kinds of CFRs (here: various types of metal plate orifices and needle valves were tested), gas types (here: air, Argon, and CO2) and upstream pressures (here: between 650 hPa and 1400 hPa). The accuracy of this technique (∼1%) is as good as that of standard volume displacement methods (e.g. soap film FMs) (typically 1% difference), the standard of laboratory-based flow calibrators, but less expensive and more suitable for harsh environments

Quantitative detection of drug dose and spatial distribution in the lung revealed by Cryoslicing Imaging

AbstractAdministration of drugs via inhalation is an attractive route for pulmonary and systemic drug delivery. The therapeutic outcome of inhalation therapy depends not only on the dose of the lung-delivered drug, but also on its bioactivity and regional distribution. Fluorescence imaging has the potential to monitor these aspects already during preclinical development of inhaled drugs, but quantitative methods of analysis are lacking. In this proof-of-concept study, we demonstrate that Cryoslicing Imaging allows for 3D quantitative fluorescence imaging on ex vivo murine lungs. Known amounts of fluorescent substance (nanoparticles or fluorophore–drug conjugate) were instilled in the lungs of mice. The excised lungs were measured by Cryoslicing Imaging. Herein, white light and fluorescence images are obtained from the face of a gradually sliced frozen organ block. A quantitative representation of the fluorescence intensity throughout the lung was inferred from the images by accounting for instrument noise, tissue autofluorescence and out-of-plane fluorescence. Importantly, the out-of-plane fluorescence correction is based on the experimentally determined effective light attenuation coefficient of frozen murine lung tissue (10.0±0.6cm−1 at 716nm). The linear correlation between pulmonary total fluorescence intensity and pulmonary fluorophore dose indicates the validity of this method and allows direct fluorophore dose assessment. The pulmonary dose of a fluorescence-labeled drug (FcγR-Alexa750) could be assessed with an estimated accuracy of 9% and the limit of detection in ng regime. Hence, Cryoslicing Imaging can be used for quantitative assessment of dose and 3D distribution of fluorescence-labeled drugs or drug carriers in the lungs of mice

Deducing in Vivo Toxicity of Combustion-Derived Nanoparticles from a Cell-Free Oxidative Potency Assay and Metabolic Activation of Organic Compounds

BACKGROUND: The inhalation of combustion-derived nanoparticles (CDNPs) is believed to cause an oxidative stress response, which in turn may lead to pulmonary or even systemic inflammation. OBJECTIVE AND METHODS: In this study we assessed whether the in vivo inflammatory response-which is generally referred to as particle toxicity-of mice to CDNPs can be predicted in vitro by a cell-free ascorbate test for the surface reactivity or, more precisely, oxidative potency (Ox(Pot),) of particles. RESULTS: For six types of CDNPs with widely varying particle diameter (10-50 nm), organic content (OC; 1-20%), and specific Brunauer, Emmett, and Teller (BET) surface area (43-800 m(2)/g), Ox(Pot) correlated strongly with the in vivo inflammatory response (pulmonary polymorphonuclear neutrophil influx 24 hr after intratracheal particle instillation). However, for CDNPs with high organic content, Ox(Pot) could not explain the observed inflammatory response, possibly due to shielding of the Ox(Pot) of the carbon core of CDNPs by an organic coating. On the other hand, a pathway-specific gene expression screen indicated that, for particles rich in polycyclic aromatic hydrocarbon (PAHs), cytochrome P450 1A1 (CYP1A1) enzyme-mediated biotransformation of bioavailable organics may generate oxidative stress and thus enhance the in vivo inflammatory response. CONCLUSION: The compensatory nature of both effects (shielding of carbon core and biotransformation of PAHs) results in a good correlation between inflammatory response and BET surface area for all CDNPs. Hence, the in vivo inflammatory response can either be predicted by BET surface area or by a simple quantitative model, based on in vitro Ox(Pot) and Cyp1a1 induction

Effects of ultrafine particles on the allergic inflammation in the lung of asthmatics : results of a double-blinded randomized cross-over clinical pilot study

Background: Epidemiological and experimental studies suggest that exposure to ultrafine particles (UFP) might aggravate the allergic inflammation of the lung in asthmatics. Methods: We exposed 12 allergic asthmatics in two subgroups in a double-blinded randomized cross-over design, first to freshly generated ultrafine carbon particles (64 μg/m3; 6.1 ± 0.4 × 105 particles/cm3 for 2 h) and then to filtered air or vice versa with a 28-day recovery period in-between. Eighteen hours after each exposure, grass pollen was instilled into a lung lobe via bronchoscopy. Another 24 hours later, inflammatory cells were collected by means of bronchoalveolar lavage (BAL). (Trial registration: NCT00527462) Results: For the entire study group, inhalation of UFP by itself had no significant effect on the allergen induced inflammatory response measured with total cell count as compared to exposure with filtered air (p = 0.188). However, the subgroup of subjects, which inhaled UFP during the first exposure, exhibited a significant increase in total BAL cells (p = 0.021), eosinophils (p = 0.031) and monocytes (p = 0.013) after filtered air exposure and subsequent allergen challenge 28 days later. Additionally, the potential of BAL cells to generate oxidant radicals was significantly elevated at that time point. The subgroup that was exposed first to filtered air and 28 days later to UFP did not reveal differences between sessions. Conclusions: Our data demonstrate that pre-allergen exposure to UFP had no acute effect on the allergic inflammation. However, the subgroup analysis lead to the speculation that inhaled UFP particles might have a long-term effect on the inflammatory course in asthmatic patients. This should be reconfirmed in further studies with an appropriate study design and sufficient number of subjects

Effects of physicochemical properties of TiO2 nanomaterials for pulmonary inflammation, acute phase response and alveolar proteinosis in intratracheally exposed mice

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.Peer reviewe