Air-liquid Interface Cell Exposure Research Articles

Airborne emissions from combustion of graphene nanoplatelet/epoxy composites and their cytotoxicity on lung cells via air-liquid interface cell exposure in vitro

Graphene nanoplatelet (GNP) as a nanofiller improves the mechanical strength, electrical conductivity, and flame retardancy of the polymers significantly. With an increasing number of GNP-reinforced products, a careful safety assessment is needed to avoid social and economic setbacks. However, no study has addressed the effects of combustion-generated emissions from GNP-reinforced products in the lung, the most sensitive exposure route to airborne particles. Therefore, we studied the influence of GNP as a nanofiller on the emitted particles and polycyclic aromatic hydrocarbons (PAHs), and cytotoxicity of the emissions from the combustion of pure epoxy (EP) and GNP-reinforced epoxy (EP-GNP). GNP was not detected in the airborne emissions. PAHs were found in airborne particles of both emissions from EP and EP-GNP, with some differences in their concentrations. A first hazard assessment was performed on human alveolar epithelial cells exposed to the airborne emissions at air-liquid interface conditions. At 24 h and 96 h after the exposure, similar responses were observed between EP and EP-GNP except an acute transient decrease in mitochondrial activity after exposure to the emissions from EP-GNP. Both emissions from EP and EP-GNP had no acute effects on membrane integrity, cell morphology or expression of anti-oxidative stress markers (HMOX1 and SOD2 genes). Meanwhile, both emissions induced the activation of the aryl hydrocarbon receptor (CYP1A1 gene) and a transient (pro-) inflammatory response (MCP-1), but the effects between EP and EP-GNP were not significantly different.

Comparative anti-inflammatory effect of curcumin at air-liquid interface and submerged conditions using lipopolysaccharide stimulated human lung epithelial A549 cells

BackgroundInhalation of aerosolized drugs is a promising entry route for rapid and non-invasive therapeutics delivery to the lung. Curcumin exhibits potent anti-inflammatory properties, which are effective for use in lung diseases. The anti-inflammatory properties of curcumin have been widely studied in vitro with cells cultured in submerged conditions, however, the effectiveness using air-liquid interface (ALI) exposure is currently unknown. MethodsThe anti-inflammatory effect of curcumin under both ALI and submerged conditions was investigated in the present study. Lipopolysaccharide (LPS) stimulated A549 cells were exposed to curcumin under ALI (10–100 μM) using a dose-controlled air-liquid interface cell exposure (ALICE)-CLOUD system and submerged cell culture conditions (1–20 μM). The expression of pro-inflammatory cytokines (interleukin (IL)-6, IL-8), cell viability and cytotoxicity were studied for each exposure scenario. The cellular uptake behaviour of curcumin was studied with an equipotent cell-based dose (200 pmol/106 cell) at various time points up to 24 h. ResultsThe ALI delivery profile proved to be rapid, efficient and reproducible. For the doses studied, no significant effect on cell viability and cytotoxicity were observed. ALI exposure of curcumin was more effective in reducing pro-inflammatory cytokines expression in lung epithelial cells compared with submerged cell cultures. Furthermore, rapid cellular uptake and higher intracellular doses were achieved by ALI conditions. ConclusionsThe ALICE-CLOUD system combined with lung epithelial cells cultured under ALI conditions offers a reliable and relevant in vitro method for preclinical aerosolized drug screening. Curcumin might be a promising anti-inflammatory candidate drug for inhalation therapy of lung diseases.

Influence of wood species on toxicity of log-wood stove combustion aerosols: a parallel animal and air-liquid interface cell exposure study on spruce and pine smoke

BackgroundWood combustion emissions have been studied previously either by in vitro or in vivo models using collected particles, yet most studies have neglected gaseous compounds. Furthermore, a more accurate and holistic view of the toxicity of aerosols can be gained with parallel in vitro and in vivo studies using direct exposure methods. Moreover, modern exposure techniques such as air-liquid interface (ALI) exposures enable better assessment of the toxicity of the applied aerosols than, for example, the previous state-of-the-art submerged cell exposure techniques.MethodsWe used three different ALI exposure systems in parallel to study the toxicological effects of spruce and pine combustion emissions in human alveolar epithelial (A549) and murine macrophage (RAW264.7) cell lines. A whole-body mouse inhalation system was also used to expose C57BL/6 J mice to aerosol emissions. Moreover, gaseous and particulate fractions were studied separately in one of the cell exposure systems. After exposure, the cells and animals were measured for various parameters of cytotoxicity, inflammation, genotoxicity, transcriptome and proteome.ResultsWe found that diluted (1:15) exposure pine combustion emissions (PM1 mass 7.7 ± 6.5 mg m− 3, 41 mg MJ− 1) contained, on average, more PM and polycyclic aromatic hydrocarbons (PAHs) than spruce (PM1 mass 4.3 ± 5.1 mg m− 3, 26 mg MJ− 1) emissions, which instead showed a higher concentration of inorganic metals in the emission aerosol. Both A549 cells and mice exposed to these emissions showed low levels of inflammation but significantly increased genotoxicity. Gaseous emission compounds produced similar genotoxicity and a higher inflammatory response than the corresponding complete combustion emission in A549 cells. Systems biology approaches supported the findings, but we detected differing responses between in vivo and in vitro experiments.ConclusionsComprehensive in vitro and in vivo exposure studies with emission characterization and systems biology approaches revealed further information on the effects of combustion aerosol toxicity than could be achieved with either method alone. Interestingly, in vitro and in vivo exposures showed the opposite order of the highest DNA damage. In vitro measurements also indicated that the gaseous fraction of emission aerosols may be more important in causing adverse toxicological effects. Combustion aerosols of different wood species result in mild but aerosol specific in vitro and in vivo effects.

Exposure of Lung Epithelial Cells to Photochemically Aged Secondary Organic Aerosol Shows Increased Toxic Effects

Adverse health effects due to exposure to particulate matter (PM) are among the most important global environmental health risks. However, the effects of exposure to secondary organic aerosols (SOA), a major component of the global aerosol, are largely unknown. Here we exposed lung epithelial cells (A549) to fresh and aged SOA particles and investigated the effect of SOA atmospheric aging on cell viability and gene expression. Naphthalene- and α-pinene-derived SOA were formed in an oxidation flow reactor that simulates atmospheric SOA formation and aging dominated by OH radical oxidation under NOx-free conditions. The SOA mass and chemical composition were characterized on-line using a scanning mobility particle sizer and aerosol mass spectrometer. Fresh and aged SOA were directed to an air–liquid interface cell exposure system. Aged naphthalene- and α-pinene-derived SOA were somewhat more toxic than fresh SOA. Aged naphthalene SOA contained peroxide levels that were higher than those of fresh SOA. The le...

Acute effects of multi-walled carbon nanotubes on primary bronchial epithelial cells from COPD patients

The risks of occupational exposure during handling of multi-walled carbon nanotubes (MWCNTs) have received limited attention to date, in particular for potentially susceptible individuals with highly prevalent chronic obstructive pulmonary disease (COPD). In this in vitro study, we simulated acute inhalation of MWCNTs employing an air–liquid interface cell exposure (ALICE) system: primary human bronchial epithelial cells from COPD patients and healthy donors (controls), cultured at the air–liquid interface (ALI) were exposed to MWCNTs. To study acute health effects on the respiratory epithelium, two different concentrations (0.16; 0.34 µg/cm2) of MWCNTs were aerosolized onto cell cultures followed by analysis after 24 h. Following MWCNT exposure, epithelial integrity and differentiation remained intact. Electron microscopy analyses identified MWCNTs both extra- and intracellular within vesicles of mucus producing cells. In both COPD and healthy control cultures, MWCNTs neither caused increased release of lactate dehydrogenase (LDH), nor alterations in inflammatory responses, as measured by RNA expression and protein secretion of the cytokines IL-6, IL-8, CXCL10, IL-1β and TGF-β and oxidative stress markers HMOX-1 and SOD-2. No short-term alteration of epithelial cell function, as determined by ciliary beating frequency (CBF), occurred in any of the conditions tested. In conclusion, the present study provided a reliable and realistic in vitro acute-exposure model of the respiratory tract, responsive to positive controls such as Dörentruper Quartz (DQ12) and asbestos. Acute exposure to MWCNTs did not affect epithelial integrity, nor induce increased cell death, apoptosis or inflammatory changes.

Biokinetics of Aerosolized Liposomal Ciclosporin A in Human Lung Cells In Vitro Using an Air-Liquid Cell Interface Exposure System.

Inhalation of aerosolized drugs is a promising route for noninvasive targeted drug delivery to the lung. Nanocarrier systems such as liposomes have been explored for inhalation therapy opening new avenues, including stabilization of nonsoluble drugs (e.g., Ciclosporin A [CsA]) and controlled release. The biokinetic behavior of the immunosuppressive drug CsA encapsulated in liposomes (L-CsA) at the lung epithelial barrier was studied in vitro. Human lung epithelial cells (alveolar A549 and bronchial 16HBE14o- epithelial cells) were exposed to aerosolized L-CsA at the air-liquid interface (ALI) using a dose-controlled air-liquid interface cell exposure (ALICE) system and the temporal profile of the L-CsA dose in the apical, basal, and cell compartment was monitored up to 24 hours. Aerosolization of different volumes of L-CsA solution with the ALICE resulted in dose-controlled, spatially uniform, and reproducible L-CsA delivery. Cell viability at 24 hours postexposure was not impaired and immunofluorescence staining revealed the typical epithelial cell morphology in control as well as in L-CsA-exposed cells. The (pro-)inflammatory interleukin-8 levels were not elevated under any condition. The biokinetic analysis revealed that both cell types formed a tight, but imperfect, barrier for L-CsA resulting in initially high transbarrier L-CsA transport rates, which ceased after about 4 hours. Although substantial transbarrier L-CsA transport was observed for both cell types, respectively, a 150-fold higher L-CsA concentration was established in the apical and cell compared to the basal compartment. Most importantly, for pulmonary drug targeting, a high cellular L-CsA dose level (20%-25% of the delivered dose) was obtained rapidly (<1 hour) and maintained for at least 24 hours. The ALICE system combined with lung epithelial cells cultured at the ALI offers a reliable and relevant in vitro platform technology to study the effects of inhalable substances such as L-CsA under biomimetic conditions.

Human Asthmatic Bronchial Cells Are More Susceptible to Subchronic Repeated Exposures of Aerosolized Carbon Nanotubes At Occupationally Relevant Doses Than Healthy Cells.

Although acute pulmonary toxicity of carbon nanotubes (CNTs) has been extensively investigated, the knowledge of potential health effects following chronic occupational exposure is currently limited and based only upon in vivo approaches. Our aim was to realistically mimic subchronic inhalation of multiwalled CNTs (MWCNTs) in vitro, using the air-liquid interface cell exposure (ALICE) system for aerosol exposures on reconstituted human bronchial tissue from healthy and asthmatic donors. The reliability and sensitivity of the system were validated using crystalline quartz (DQ12), which elicited an increased (pro-)inflammatory response, as reported in vivo. At the administrated MWCNT doses relevant to human occupational lifetime exposure (10 μg/cm2 for 5 weeks of repeated exposures/5 days per week) elevated cilia beating frequency (in both epithelial cultures), and mucociliary clearance (in asthmatic cells only) occurred, whereas no cytotoxic reactions or morphological changes were observed. However, chronic MWCNT exposure did induce an evident (pro-)inflammatory and oxidative stress response in both healthy and asthmatic cells. The latter revealed stronger and more durable long-term effects compared to healthy cells, indicating that individuals with asthma may be more susceptible to adverse effects from chronic MWCNT exposure. Our results highlight the power of occupationally relevant subchronic exposures on human in vitro models in nanosafety hazard assessment.

Repeated exposure to carbon nanotube-based aerosols does not affect the functional properties of a 3D human epithelial airway model

Carbon nanotubes (CNTs) represent one of the most promising engineered nanomaterials, with possible applications in advanced engineering and biomedical technologies. During their production, human exposure to CNTs may occur via inhalation. Therefore, the aim of this study was to mimic inhalation of multi-walled CNTs (MWCNTs) in vitro as realistically as possible, by producing MWCNTs aerosols via an Air–Liquid Interface Cell Exposure System (ALICE), combined with a 3D epithelial airway barrier model cultivated at the air–liquid interface (ALI). To address the consequences of an extended exposure period, repeated exposures of MWCNTs (total deposition 1.15 μg/cm2) were applied to the co-culture system, either over one day (one day repeated exposure) or three days (three day repeated exposure scenario). Although in both repeated exposure scenarios MWCNTs were found to interact with the different cell types, they did not induce any cytotoxicity or alterations in cell morphology, nor did they elucidate any significant increase in pro-inflammatory markers compared to control cultures. Similar results were also observed following single MWCNTs exposures at deposited concentrations of 0.14, 0.20 and 0.39 µg/cm2. Cells exposed repeatedly to MWCNTs for three days, however did show a decrease in reduced glutathione levels, although not significant (p > 0.05). In conclusion, we have presented a realistic in vitro alternative to mimic occupational exposure of MWCNTs and by applying this approach it was shown that repeated MWCNT exposures to lung cell cultures at the ALI elicit a limited biological impact over a three day period.

An in vitro testing strategy towards mimicking the inhalation of high aspect ratio nanoparticles.

BackgroundThe challenge remains to reliably mimic human exposure to high aspect ratio nanoparticles (HARN) via inhalation. Sophisticated, multi-cellular in vitro models are a particular advantageous solution to this issue, especially when considering the need to provide realistic and efficient alternatives to invasive animal experimentation for HARN hazard assessment. By incorporating a systematic test-bed of material characterisation techniques, a specific air-liquid cell exposure system with real-time monitoring of the cell-delivered HARN dose in addition to key biochemical endpoints, here we demonstrate a successful approach towards investigation of the hazard of HARN aerosols in vitro.MethodsCellulose nanocrystals (CNCs) derived from cotton and tunicates, with differing aspect ratios (~9 and ~80), were employed as model HARN samples. Specifically, well-dispersed and characterised CNC suspensions were aerosolised using an “Air Liquid Interface Cell Exposure System” (ALICE) at realistic, cell-delivered concentrations ranging from 0.14 to 1.57 μg/cm2. The biological impact (cytotoxicity, oxidative stress levels and pro-inflammatory effects) of each HARN sample was then assessed using a 3D multi-cellular in vitro model of the human epithelial airway barrier at the air liquid interface (ALI) 24 hours post-exposure. Additionally, the testing strategy was validated using both crystalline quartz (DQ12) as a positive particulate control in the ALICE system and long fibre amosite asbestos (LFA) to confirm the susceptibility of the in vitro model to a fibrous insult.ResultsA rapid (≤4 min), controlled nebulisation of CNC suspensions enabled a dose-controlled and spatially homogeneous CNC deposition onto cells cultured under ALI conditions. Real-time monitoring of the cell-delivered CNC dose with a quartz crystal microbalance was accomplished. Independent of CNC aspect ratio, no significant cytotoxicity (p > 0.05), induction of oxidative stress, or (pro)-inflammatory responses were observed up to the highest concentration of 1.57 μg/cm2. Both DQ12 and LFA elicited a significant (p < 0.05) pro-inflammatory response at sub-lethal concentrations in vitro.ConclusionIn summary, whilst the present study highlights the benign nature of CNCs, it is the advanced technological and mechanistic approach presented that allows for a state of the art testing strategy to realistically and efficiently determine the in vitro hazard concerning inhalation exposure of HARN.Electronic supplementary materialThe online version of this article (doi:10.1186/s12989-014-0040-x) contains supplementary material, which is available to authorized users.

Mimicking exposures to acute and lifetime concentrations of inhaled silver nanoparticles by two different in vitro approaches.

In the emerging market of nano-sized products, silver nanoparticles (Ag NPs) are widely used due to their antimicrobial properties. Human interaction with Ag NPs can occur through the lung, skin, gastrointestinal tract, and bloodstream. However, the inhalation of Ag NP aerosols is a primary concern. To study the possible effects of inhaled Ag NPs, an in vitro triple cell co-culture model of the human alveolar/airway barrier (A549 epithelial cells, human peripheral blood monocyte derived dendritic and macrophage cells) together with an air–liquid interface cell exposure (ALICE) system was used in order to reflect a real-life exposure scenario. Cells were exposed at the air–liquid interface (ALI) to 0.03, 0.3, and 3 µg Ag/cm2 of Ag NPs (diameter 100 nm; coated with polyvinylpyrrolidone: PVP). Ag NPs were found to be highly aggregated within ALI exposed cells with no impairment of cell morphology. Furthermore, a significant increase in release of cytotoxic (LDH), oxidative stress (SOD-1, HMOX-1) or pro-inflammatory markers (TNF-α, IL-8) was absent. As a comparison, cells were exposed to Ag NPs in submerged conditions to 10, 20, and 30 µg Ag/mL. The deposited dose per surface area was estimated by using a dosimetry model (ISDD) to directly compare submerged vs ALI exposure concentrations after 4 and 24 h. Unlike ALI exposures, the two highest concentrations under submerged conditions promoted a cytotoxic and pro-inflammatory response after 24 h. Interestingly, when cell cultures were co-incubated with lipopolysaccharide (LPS), no synergistic inflammatory effects were observed. By using two different exposure scenarios it has been shown that the ALI as well as the suspension conditions for the lower concentrations after 4 h, reflecting real-life concentrations of an acute 24 h exposure, did not induce any adverse effects in a complex 3D model mimicking the human alveolar/airway barrier. However, the highest concentrations used in the ALI setup, as well as all concentrations under submerged conditions after 24 h, reflecting more of a chronic lifetime exposure concentration, showed cytotoxic as well as pro-inflammatory effects. In conclusion, more studies need to address long-term and chronic Ag NP exposure effects.

Risk assessment of released cellulose nanocrystals – mimicking inhalatory exposure

Cellulose nanocrystals (CNCs) exhibit advantageous chemical and mechanical properties that render them attractive for a wide range of applications. During the life-cycle of CNC containing materials the nanocrystals could be released and become airborne, posing a potential inhalatory exposure risk towards humans. Absent reliable and dose-controlled models that mimic this exposure in situ is a central issue in gaining an insight into the CNC-lung interaction. Here, an Air Liquid Interface Cell Exposure system (ALICE), previously designed for studies of spherical nanoparticles, was used for the first time to establish a realistic physiological exposure test method for inhaled fiber shaped nano-objects; in this case, CNCs isolated from cotton. Applying a microscopy based approach the spatially homogenous deposition of CNCs was demonstrated as a prerequisite of the functioning of the ALICE. Furthermore, reliability and controllability of the system to nebulise high aspect ratio nanomaterials (HARN, e.g. CNCs) was shown. This opens the potential to thoroughly investigate the inhalatory risk of CNCs in vitro using a realistic exposure system.

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

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.