Lung Lining Fluid Research Articles

A Comparative Pharmacokinetics Study of Orally and Intranasally Administered 8-Nitro-1,3-benzothiazin-4-one (BTZ043) Amorphous Drug Nanoparticles.

BTZ043 is an 8-nitro-1,3-benzothiazin-4-one with potency against multidrug-resistant Mycobacterium tuberculosis. Low solubility and hepatic metabolism are linked to poor oral bioavailability. Amorphous drug nanoparticles (ADN) were formulated to improve the bioavailability. Comparative pharmacokinetics of BTZ043 ADN following intranasal (2.5 mg kg-1) and oral administration (25 mg kg-1) in Balb/c mice was investigated using oral BTZ043 drug suspensions (neat; 25 mg kg-1) as a standard-of-care reference. Plasma exposure following oral ADN administration was 8-fold higher than for oral neat BTZ043. Intranasal ADN increased plasma exposure 18-fold compared to oral neat BTZ043 after dose normalization. BTZ043 was detectable in lung lining fluid following ADN administration, but not after oral neat BTZ043 dosing. BTZ043 was cleared faster from the lung and plasma following intranasal administration with a shorter time above the minimum inhibitory concentration (MIC) compared to oral ADN. Since time > MIC is reported to drive activity, oral ADN may represent a promising delivery strategy for BTZ043.

Poly(malic acid)-budesonide nanoconjugates embedded in microparticles for lung administration.

To improve the therapeutic activity of inhaled glucocorticoids and reduce potential side effects, we designed a formulation combining the advantages of nanoparticles, which have an enhanced uptake by alveolar cells, allow targeted delivery and sustained drug release, as well as limited drug systemic passage, with those of microparticles, which display good alveolar deposition. Herein, a polymer-drug conjugate, poly(malic acid)-budesonide (PMAB), was first synthesized with either 11, 20, 33, or 43mol% budesonide (drug:polymer from 1:8 to 3:4), the drug creating hydrophobic domains. The obtained conjugates self-assemble into nanoconjugates in water, yielding excellent drug loading of up to 73 wt%, with 80-100nm diameters. In vitro assays showed that budesonide could be steadily released from the nanoconjugates, and the anti-inflammatory activity was preserved, as evidenced by reduced cytokine production in LPS-activated RAW 264.7 macrophages. Nanoconjugates were then embedded into microparticles through spray-drying with L-leucine, forming nano-embedded microparticles (NEMs). NEMs were produced with an aerodynamic diameter close to 1µm and a density below 0.1 g.cm-3, indicative of a high alveolar deposition. NEMs spray-dried with the less hydrophobic nanoconjugates, PMAB 1:4, were readily dissolved in simulated lung fluid and were chosen for in vivo experiments to study pharmacokinetics in healthy rats. As it was released in vivo from NEMs, sustained distribution of budesonide was obtained for 48 h in lung tissue, cells, and lining fluid. With high loading rates, modulable release kinetics, and low cytotoxicity, these nanoconjugates delivered by NEMs are promising for the more efficient treatment of pulmonary inflammatory diseases.

Drug solubility in biorelevant media in the context of an inhalation-based biopharmaceutics classification system (iBCS)

An inhalation-based Biopharmaceutics Classification System for pulmonary drugs (iBCS) holds the perspective to allow for scientifically sound prediction of differences in the in vivo performance of orally inhaled drug products (OIDPs).A set of nine drug substances were selected, that are administered via both the oral and pulmonary routes. Their solubility was determined in media representative for the oral (Fasted State Simulated Intestinal Fluid (FaSSIF)) and pulmonary (Alveofact medium and Simulated Lung Fluid (SLF)) routes of administration to confirm the need for a novel approach for inhaled drugs. The complexity of these media was then stepwise reduced with the purpose of understanding the contribution of their components to the solubilizing capacity of the media. A second reason for varying the complexity was to identify a medium that would allow robust but accurate dissolution testing. Hence, Hank’s balanced salt solution (HBSS) as a medium used in many in vitro biological tests, non-buffered saline solution, and water were included.For some drug substances (salbutamol sulfate, tobramycin, isoniazid, and tiotropium bromide), no significant differences were observed between the solubility in the media used. For other drugs, however, we observed either just small (rifampicin, budesonide, salmeterol) or unexpectedly large differences (beclomethasone dipropionate).Based on the minimum theoretical solubility required for their common pulmonary dose in 10 ml of lung lining fluid, drug solubility was classified as either high or low. Two high solubility and two low solubility compounds were then selected for refined solubility testing in pulmonary relevant media by varying their content of phospholipids, surfactant proteins and other proteins.The solubility of drug substances in simulated lung lining fluids was found to be dependent on the physicochemical properties of the drug substance and the composition of the media. While a pulmonary dissolution medium that would fit all drugs could not be established, our approach may provide guidance for finding the most suitable dissolution medium for a given drug substance and better designing in vitro tests for predicting the in vivo performance of inhalable drug products.

Influence of aerosol acidity and organic ligands on transition metal solubility and oxidative potential of fine particulate matter in urban environments

The adverse health effects of air pollution around the world have been associated with the inhalation of fine particulate matter (PM2.5). Such outcomes are thought to be related to the induction of oxidative stress due to the excess formation of reactive oxygen species (ROS) in the respiratory and cardiovascular systems. The ability of airborne chemicals to deplete antioxidants and to form ROS is known as oxidative potential (OP). Here we studied the influence of aerosol acidity and organic ligands on the solubility of transition metals, in particular iron (Fe) and copper (Cu), and on the OP of PM2.5 from Canadian National Air Pollution Surveillance urban sites in Toronto, Vancouver, and Hamilton. Using chemical assays and model simulations of the lung redox chemistry, we quantified ROS formation in the lung lining fluid, targeting superoxide anion (O2•-), hydrogen peroxide (H2O2), and hydroxyl radical (•OH), as well as the PM2.5 redox potential (RP). Experimental •OH formation (OPOH) showed high correlations with RP and model-predicted ROS metrics. Both aerosol acidity and oxalate content enhanced the solubility of transition metals, with oxalate showing a stronger association. While experimental OP metrics were primarily associated with species of primary origin such as elemental carbon, Fe, and Cu, model-predicted ROS were associated with secondary processes including proton- and ligand-mediated dissolution of Fe. Model simulations showed that water-soluble Cu was the main contributor to O2•- formation, while water-soluble Fe dominated the formation of highly reactive •OH radical, particularly at study sites with highly acidic aerosol and elevated levels of oxalate. This study underscores the importance of reducing transition metal emissions in urban environments to improve population health.

Exposure to phagolysosomal simulated fluid altered the cytotoxicity of PET micro(nano)plastics to human lung epithelial cells

The occurrence of micro(nano)plastics into various environmental and biological settings influences their physicochemical and toxic behavior. Simulated body fluids are appropriate media for understanding the degradation, stability, and interaction with other substances of any material in the human body. When the particles enter the human body via inhalation, which is one of the avenues for micro(nano)plastics, they first come into contact with the lung lining fluid under neutral conditions and then are phagocytosed under acidic conditions to be removed. Therefore, it is important to examine the physicochemical transformation and toxicity characteristics after interaction with phagolysosomal simulant fluid (PSF). Here, we focused on exploring how the physicochemical differences (e.g. surface chemistry, elemental distribution, and surface charge) of micro(nano)plastics under pH 4.5 phagolysosome conditions impact cytotoxicity and the oxidative characteristics of lung epithelia cells. The cytotoxicity of lung epithelia cells to those treated with PSF and non-treated micro(nano)plastics was tested by various viability indicators including cell counting kit-8 (CCK-8), MTT, and LDH. Furthermore, the cytotoxicity background was examined through the oxidative processes (e.g. reactive oxygen species, antioxidant, superoxide dismutase (SOD), catalase, and reduced glutathione). The results showed that all tested surface physicochemical characteristics were significantly influenced by the phagolysosome conditions. The staged responses were observed with the treatment duration, and significant changes were calculated in carbonyl, carbon-nitrogen, and sulfonyl groups. Moreover, the negativity of the zeta potentials declined between exposure of 2–40 h and then increased at 80 h compared to control owing to the chemical functional groups and elemental distribution of the plastic particles. The tested viability indicators showed that the micro(nano)plastics treated with PSF were cytotoxic to the lung epithelia cells compared to non-treated micro(nano)plastics, and SOD was the dominant enzyme triggering cytotoxicity due to the particle degradation and instability.

Interactions between CuO NPs and PS: The release of copper ions and oxidative damage

Copper oxide nanoparticles (CuO NPs) can adversely affect lung health possibly by inducing oxidative damage through the release of copper ions. However, the migration and transformation processes of CuO NPs in lung lining fluid is still unclear, and there are still conflicting reports of redox reactions involving copper ions. To address this, we examined the release of copper ions from CuO NPs in simulated lung fluid supplemented with pulmonary surfactant (PS), and further analyzed the mechanisms of PS-CuO NPs interactions and the health hazards. The results showed that the phospholipid of PS was adsorbed on the particle surface, which not only induced aggregation of the particles but also provided a reaction environment for the interaction of PS with CuO NPs. PS was able to promote the release of ions from CuO NPs, of which the protein was a key component. Lipid peroxidation, protein destabilization, and disruption of the interfacial chemistry also occurred in the PS-CuO NPs interactions, during which copper ions were present only as divalent cations. Meanwhile, the contribution of the particle surface cannot be neglected in the oxidative damage to the lung caused by CuO NPs. Through reacting with biomolecules, CuO NPs accomplished ion release and induced oxidative damage associated with PS. This research was the first to reveal the mechanism of CuO NPs releasing copper ions and inducing lipid oxidative damage in the presence of PS, which provides a new idea of transition metal-induced health risk in human body.

Role of PPARγ in dyslipidemia and altered pulmonary functioning in mice following ozone exposure.

Exposure to ozone causes decrements in pulmonary function, a response associated with alterations in lung lipids. Pulmonary lipid homeostasis is dependent on the activity of peroxisome proliferator activated receptor gamma (PPARγ), a nuclear receptor that regulates lipid uptake and catabolism by alveolar macrophages (AMs). Herein, we assessed the role of PPARγ in ozone-induced dyslipidemia and aberrant lung function in mice. Exposure of mice to ozone (0.8 ppm, 3 h) resulted in a significant reduction in lung hysteresivity at 72 h post exposure; this correlated with increases in levels of total phospholipids, specifically cholesteryl esters, ceramides, phosphatidylcholines, phosphorylethanolamines, sphingomyelins, and di- and triacylglycerols in lung lining fluid. This was accompanied by a reduction in relative surfactant protein-B (SP-B) content, consistent with surfactant dysfunction. Administration of the PPARγ agonist, rosiglitazone (5 mg/kg/day, i.p.) reduced total lung lipids, increased relative amounts of SP-B, and normalized pulmonary function in ozone-exposed mice. This was associated with increases in lung macrophage expression of CD36, a scavenger receptor important in lipid uptake and a transcriptional target of PPARγ. These findings highlight the role of alveolar lipids as regulators of surfactant activity and pulmonary function following ozone exposure and suggest that targeting lipid uptake by lung macrophages may be an efficacious approach for treating altered respiratory mechanics.

140 A Framework for Grouping Inhaled Multi-Component Nanomaterials to Streamline Hazard Assessment

Abstract The GRACIOUS Framework (www.h2020gracious.eu) supports the grouping of nanomaterials to streamline hazard testing of nanomaterials. The GRACIOUS Framework includes a template to generate grouping hypotheses based upon use and life cycle stage (to inform exposure route), physicochemical properties (what they are), fate or toxicokinetics (where they go) and hazard (what they do). The gathering of evidence to test these hypotheses was supported by tailored Integrated Approaches to Testing and Assessment (IATAs). Subsequent NMBP-16 projects have developed the hypothesis template further for multicomponent nanomaterials. Modifications include additional information requirements to accommodate the complexity of composition (what they are) and enhanced properties, with an aim to incorporate the relationship of these to the mechanism of hazard. Furthermore, the template considers potential for the components to dissociate, disintegrate or dissolve, with different kinetics, leading to a complex exposure scenario both in lung lining fluid and inside cells. This knowledge informs the hazard assessment (whether hazard is driven by the intact multicomponent nanomaterial or various components) and whether interactions between the components may lead to antagonism, synergism or potentiation of response. Hazard assessment outcome can then inform Safe-by-Design modifications. The template has been adapted to formulate clear questions needed to test all aspects of the hypothesis, with these questions being used to formulate the IATA. Case studies of different multicomponent nanomaterials are being used to inform the design process, and demonstrate its usefulness. Funding acknowledgement European Commission Horizon 2020 for GRACIOUS, SUNSHINE, HARMLESS and DIAGONAL, Grant Agreement No. 760840, 952924, 953183 953152 respectively.

Nanomaterial Characterization in Complex Media-Guidance and Application.

A broad range of inorganic nanoparticles (NPs) and their dissolved ions possess a possible toxicological risk for human health and the environment. Reliable and robust measurements of dissolution effects may be influenced by the sample matrix, which challenges the analytical method of choice. In this study, CuO NPs were investigated in several dissolution experiments. Two analytical techniques (dynamic light scattering (DLS) and inductively-coupled plasma mass spectrometry (ICP-MS)) were used to characterize NPs (size distribution curves) time-dependently in different complex matrices (e.g., artificial lung lining fluids and cell culture media). The advantages and challenges of each analytical approach are evaluated and discussed. Additionally, a direct-injection single particle (DI sp)ICP-MS technique for assessing the size distribution curve of the dissolved particles was developed and evaluated. The DI technique provides a sensitive response even at low concentrations without any dilution of the complex sample matrix. These experiments were further enhanced with an automated data evaluation procedure to objectively distinguish between ionic and NP events. With this approach, a fast and reproducible determination of inorganic NPs and ionic backgrounds can be achieved. This study can serve as guidance when choosing the optimal analytical method for NP characterization and for the determination of the origin of an adverse effect in NP toxicity.

Interaction of TiO2 nanoparticles with lung fluid proteins and the resulting macrophage inflammatory response.

Inhalation is a major exposure route to nanoparticles. Following inhalation, nanoparticles first interact with the lung lining fluid, a complex mixture of proteins, lipids, and mucins. We measure the concentration and composition of lung fluid proteins adsorbed on the surface of titanium dioxide (TiO2) nanoparticles. Using proteomics, we find that lung fluid results in a unique protein corona on the surface of the TiO2 nanoparticles. We then measure the expression of three cytokines (interleukin 6 (IL-6), tumor necrosis factor-alpha (TNF-α), and macrophage inflammatory protein 2 (MIP-2)) associated with lung inflammation. We find that the corona formed from lung fluid leads to elevated expression of these cytokines in comparison to bare TiO2 nanoparticles or coronas formed from serum or albumin. These experiments show that understanding the concentration and composition of the protein corona is essential for understanding the pulmonary response associated with human exposure to nanoparticles.

Is the oxidative potential of components of fine particulate matter surface-mediated?

Redox-active substances in fine particulate matter (PM) contribute to inhalation health risks through their potential to generate reactive oxygen species in epithelial lung lining fluid (ELF). The ELF’s air–liquid interface (ALI) can play an important role in the phase transfer and multi-phase reactions of redox-active PM constituents. We investigated the influence of interfacial processes and properties by scrubbing of coated nano-particles with simulated ELF in a nebulizing mist chamber. Weakly water-soluble redox-active organics abundant in ambient fine PM were reproducibly loaded into ELF via ALI mixing. The resulting oxidative potential (OP) of selected quinones and other PAH derivatives were found to exceed the OP resulting from bulk mixing of the same amounts of redox-active substances and ELF. Our results indicate that the OP of PM components depends not only on the PM substance properties but also on the ELF interface properties and uptake mechanisms. OP measurements based on bulk mixing of phases may not represent the effective OP in the human lung.

Inter-comparison of oxidative potential metrics for airborne particles identifies differences between acellular chemical assays

Oxidative potential (OP) has been identified as an important factor underlying the health effects of airborne particulate matter (PM). OP denotes the ability of PM to deplete antioxidants and to form reactive oxygen species (ROS) in the lung. OP can be quantified using a variety of chemical assays and analytical techniques. Despite the widespread use of various OP metrics, there is no consensus or comprehensive inter-laboratory assessment on how these assays compare. In this work for the first time, we compared 11 OP indicators from acellular assays using standard reference material of urban PM. The OP indicators included ascorbic acid, glutathione, glutathione disulfide, cysteine, cystine, dithiothreitol, H2O2, •OH, O2•-, and empirical and theoretical redox potential of simulated lung lining fluid (SLF). The indicators showed first-order kinetics at low PM concentration (25 μg mL−1), whereas the kinetics were non-linear at higher PM concentrations. The indicators demonstrated mainly linear dose-response relationships at PM concentrations 25–100 μg mL−1, following similar trends with water-soluble transition metals, but they were not always proportional to PM concentrations, and demonstrated substantial differences in their sensitivities to PM. The results indicate the importance of using reduced reaction time for reliable OP quantification due to non-linearity in assay responses at high PM concentrations. This work shows that the choice of molecular probes and measurement techniques must be carefully considered when planning studies on OP of ambient air and should importantly include the association of OP metrics with health outcomes.

Boosting lung accumulation of gallium with inhalable nano-embedded microparticles for the treatment of bacterial pneumonia

The potential of intra-venous gallium nitrate (GaN) administration against Pseudomonas aeruginosa pneumonia was recently demonstrated in mice and in cystic fibrosis (CF) patients. Likewise, the added value of direct lung delivery of Ga(III) has been shown in rats. Therefore, the design of a drug delivery system specifically engineered for Ga(III) inhalation is imperative to improve its accumulation in lungs. To this purpose, Ga(III) was efficiently encapsulated into hyaluronic acid/chitosan nanoparticles (Ga_HA/CS NPs), whose features were tuned to facilitate access to the target by overcoming mucus and biofilm surrounding bacteria. Then, to improve in vivo lung deposition, Ga_HA/CS NPs were engineered into mannitol-based NEM (Ga_Man NEM). The powders showed optimal in vitro aerosol performance, and sustained release kinetics in lung lining fluids. Moreover, good tolerability and antimicrobial properties were shown in vitro. Intratracheal insufflation of Ga_Man NEM in rats resulted in a significant improvement of Ga(III) persistence in the lungs coupled to a lower Ga(III) concentration in plasma and urine, compared to GaN solution. Noteworthy, the developed formulation significantly modifies the unfavorable Ga(III) kinetic increasing the Ga(III) to the lung and preventing Ga(III) accumulation in the kidney, key responsible for adverse effects, conclusively demonstrating the benefit of Ga_Man NEM to exploit the therapeutic effect of Ga(III) via inhalation route.

Regulation of macrophage activation by S-Nitrosothiols following ozone-induced lung injury

Acute exposure to ozone causes oxidative stress, characterized by increases in nitric oxide (NO) and other reactive nitrogen species in the lung. NO has been shown to modify thiols generating S-nitrosothiols (SNOs); this results in altered protein function. In macrophages this can lead to changes in inflammatory activity which impact the resolution of inflammation. As SNO formation is dependent on the redox state of both the NO donor and the recipient thiol, the local microenvironment plays a key role in its regulation. This dictates not only the chemical feasibility of SNO formation but also mechanisms by which they may form. In these studies, we compared the ability of the SNO donors, ethyl nitrite (ENO), which targets both hydrophobic and hydrophilic thiols, SNO-propanamide (SNOPPM) which targets hydrophobic thiols, and S-nitroso-N-acetylcysteine. (SNAC) which targets hydrophilic thiols. to modify macrophage activation following ozone exposure. Mice were treated with air or ozone (0.8 ppm, 3 h) followed 1 h later by intranasal administration of ENO, SNOPPM or SNAC (1–500 μM) or appropriate controls. Mice were euthanized 48 h later. Each of the SNO donors reduced ozone-induced inflammation and modified the phenotype of macrophages both within the lung lining fluid and the tissue. ENO and SNOPPM were more effective than SNAC. These findings suggest that the hydrophobic SNO thiol pool targeted by SNOPPM and ENO plays a major role in regulating macrophage phenotype following ozone induced injury.

How Aging and Oxidative Stress Influence the Cytopathic and Inflammatory Effects of SARS-CoV-2 Infection: The Role of Cellular Glutathione and Cysteine Metabolism

SARS-CoV-2 infection can cause a severe respiratory distress syndrome with inflammatory and thrombotic complications, the severity of which increases with patients’ age and presence of comorbidity. The reasons for an age-dependent increase in the risk of severe COVID-19 could be many. These include defects in the homeostatic processes that control the cellular redox and its pivotal role in sustaining the immuno-inflammatory response to the host and the protection against oxidative stress and tissue degeneration. Pathogens may take advantage of such age-dependent abnormalities. Alterations of the thiol redox balance in the lung tissue and lining fluids may influence the risk of infection, and the host capability to respond to pathogens and to avoid severe complications. SARS-CoV-2, likewise other viruses, such as HIV, influenza, and HSV, benefits in its replication cycle of pro-oxidant conditions that the same viral infection seems to induce in the host cell with mechanisms that remain poorly understood. We recently demonstrated that the pro-oxidant effects of SARS-CoV-2 infection are associated with changes in the cellular metabolism and transmembrane fluxes of Cys and GSH. These appear to be the consequence of an increased use of Cys in viral protein synthesis and to ER stress pathway activation that interfere with transcription factors, as Nrf2 and NFkB, important to coordinate the metabolism of GSH with other aspects of the stress response and with the pro-inflammatory effects of this virus in the host cell. This narrative review article describes these cellular and molecular aspects of SARS-CoV-2 infection, and the role that antivirals and cytoprotective agents such as N-acetyl cysteine may have to limit the cytopathic effects of this virus and to recover tissue homeostasis after infection.

Probability of Target Attainment of Tobramycin Treatment in Acute and Chronic Pseudomonas aeruginosa Lung Infection Based on Preclinical Population Pharmacokinetic Modeling.

Biofilms and infectious process may alter free antimicrobial concentrations at the site of infection. Tobramycin (TOB), an aminoglycoside used to treat lung infections caused by Pseudomonas aeruginosa, binds to alginate present in biofilm extracellular matrix increasing its minimum inhibitory concentration (MIC). This work aimed to investigate the impact of biofilm-forming P. aeruginosa infection on TOB lung and epithelial lining fluid (ELF) penetration, using microdialysis, and to develop a population pharmacokinetic (popPK) model to evaluate the probability of therapeutic target attainment of current dosing regimens employed in fibrocystic and non-fibrocystic patients. The popPK model developed has three compartments including the lung. The ELF concentrations were described by a penetration factor derived from the lung compartment. Infection was a covariate in lung volume (V3) and only chronic infection was a covariate in central volume (V1) and total clearance (CL). Simulations of the recommended treatments for acute and chronic infection achieved >90% probability of target attainment (PTA) in the lung with 4.5 mg/kg q24h and 11 mg/kg q24h, respectively, for the most prevalent P. aeruginosa MIC (0.5 mg/mL). The popPK model was successfully applied to evaluate the PTA of current TOB dosing regimens used in the clinic, indicating the need to investigate alternative posology.

Inhalation toxicity of copper compounds: Results of 14-day range finding study for copper sulphate pentahydrate and dicopper oxide and 28-day subacute inhalation exposure of dicopper oxide in rats

Inhalation exposure to copper may occur during a range of occupational activities and the purpose of this study was to characterise the toxicological response to repeated inhalation of two copper compounds, representative of copper substances in large-scale production/use. Crl:CD(SD) rats were repeatedly exposed to aerosols of dicopper oxide (Cu2O) or copper sulphate pentahydrate (CuSO4.5 H2O) for 14-days as part of a range finding study at normalised copper doses of 0.18, 0.71, 1.78 and 8.9 mg/m3 Cu. Within a 28-days main study (Cu2O only), animals were repeatedly exposed to 0.2, 0.4, 0.8 and 2.0 mg/m3 Cu2O following OECD TG 412. The main study also consisted of satellite groups exposed for 1-, 2- or 3- weeks as well as a 13-week post-exposure recovery period group. Repeated exposure for 14-days to both copper compounds, normalised for copper content, led to an acute influx of polymorphonuclear leukocytes (neutrophils) and macrophages whilst only CuSO4.5 H2O exposure resulted in epithelial hyperplasia. This differential response may reflect the highly dissolvable nature of CuSO4.5 H2O in lung lining fluid leading to a release of copper ions at the epithelial surface whilst Cu2O is relatively indissolvable at neutral pH. In the 28-day study with Cu2O, an increase in cellularity was also evident in both histological and BALF samples and was dose-related with minimal to mild (neutrophilic) inflammation observed > 0.4 mg/m3 in the lung tissue sections and significant increases from 0.2 mg/m3 in BALF. There were no minimal haematological findings, no clinical findings and systemic organs were unaffected by inhalation exposure to dicopper oxide. The lung cellular response was limited to alveolar histiocytosis and neutrophil influx with no evidence of epithelial hyperplasia or fibrosis and all lung biomarkers returned to control levels within the post-exposure recovery period. Interestingly, the satellite groups showed that this acute cellular response followed a biphasic rather than monotonic pattern with a peak in lung biomarkers between weeks 1–3 and reduction thereafter. This reduction in lung biomarkers occurred during continued exposure and may indicate an adaptive response to copper exposure. Overall, these results show that repeated exposure to copper compounds results in an acute cellular response with no associated pathology and which fully resolved after the cessation of exposure. Therefore, the cellular response is evidence of a controlled and adaptive response associated with the removal of Cu2O from the alveolar surface.

Emerging investigator series: deposited particles and human lung lining fluid are dynamic, chemically-complex reservoirs leading to thirdhand smoke emissions and exposure

Tobacco combustion products in deposited particles and bodily reservoirs enable persistent thirdhand smoke contamination and transport.

Systematic Analysis of Composition, Interfacial Performance and Effects of Pulmonary Surfactant Preparations on Cellular Uptake and Cytotoxicity of Aerosolized Nanomaterials

The interplay of particles with pulmonary surfactant, a lipid‐protein material pivotal for lung function, is hypothesized as a key factor that has not been routinely considered in the current in vitro models when determining the fate of inhaled nanomaterials. To explain its influence on cellular uptake and protective effects, nanoparticles are studied on two models of alveolar cells, in the absence or presence of pulmonary surfactant. Composition and interfacial performance of native human and porcine surfactants, a commercially available bovine surfactant (Alveofact), and an artificial lung lining fluid are characterized using shotgun lipidomics and biophysical approaches (i.e., Langmuir surface balances and captive bubble surfactometry). Plain and aminofunctionalized silica nanoparticles and a novel antimycobacterial nanoformulated benzothiazinone (BTZ043) are selected as examples of neutral, positively charged and therapeutically relevant nanoparticles, respectively. They are deposited onto monocultures of human alveolar epithelial and phagocytic cell lines in the presence or absence of the surfactant preparations, modeling the alveolar milieu. Only surfactant preparations with high interfacial activity and distinctive composition mitigated the toxicity of aerosolized particles, along with a tendency of aerosolized particles to aggregate. Key requirements of surfactant preparations needed when studying interactions of nanomaterials with the pulmonary air‐blood barrier in vitro are identified.

An Integrated Approach to Testing and Assessment to Support Grouping and Read-Across of Nanomaterials After Inhalation Exposure.

Introduction: Here, we describe the generation of hypotheses for grouping nanoforms (NFs) after inhalation exposure and the tailored Integrated Approaches to Testing and Assessment (IATA) with which each specific hypothesis can be tested. This is part of a state-of-the-art framework to support the hypothesis-driven grouping and read-across of NFs, as developed by the EU-funded Horizon 2020 project GRACIOUS.Development of Grouping Hypotheses and IATA: Respirable NFs, depending on their physicochemical properties, may dissolve either in lung lining fluid or in acidic lysosomal fluid after uptake by cells. Alternatively, NFs may also persist in particulate form. Dissolution in the lung is, therefore, a decisive factor for the toxicokinetics of NFs. This has led to the development of four hypotheses, broadly grouping NFs as instantaneous, quickly, gradually, and very slowly dissolving NFs. For instantaneously dissolving NFs, hazard information can be derived by read-across from the ions. For quickly dissolving particles, as accumulation of particles is not expected, ion toxicity will drive the toxic profile. However, the particle aspect influences the location of the ion release. For gradually dissolving and very slowly dissolving NFs, particle-driven toxicity is of concern. These NFs may be grouped by their reactivity and inflammation potency. The hypotheses are substantiated by a tailored IATA, which describes the minimum information and laboratory assessments of NFs under investigation required to justify grouping.Conclusion: The GRACIOUS hypotheses and tailored IATA for respiratory toxicity of inhaled NFs can be used to support decision making regarding Safe(r)-by-Design product development or adoption of precautionary measures to mitigate potential risks. It can also be used to support read-across of adverse effects such as pulmonary inflammation and subsequent downstream effects such as lung fibrosis and lung tumor formation after long-term exposure.