Unlike traditional submerged cell cultures, nebulized drug delivery to air-liquid interface (ALI)-cultured lung cells provides a clinically relevant route for in vitro testing of inhalable drugs. Current nebulizer systems deliver non-physiologically large drug volumes. Here, we introduce the Cloud MAX technology, characterize its accuracy of dosimetry, and apply it to toxicity, efficacy, and biokinetics or permeability testing of a locked nucleic acid-modified antisense oligonucleotide (ASO). On average the Cloud MAX delivers 44% of 3–20 μL aerosolized liquid to ALI-cultured cells, the coefficient of variation (CV) of repeated drug dosing is <11%-rel. and as little as 1 μL of drug/insert (=0.25 μL/cm2) can be delivered to ALI-cultured cells. As proof of concept for dose-controlled in vitro drug testing, we demonstrated target engagement of a LIM and Calponin Homology domain 1 (LIMCH1)-specific ASO in primary lung fibroblasts from Idiopathic Pulmonary Fibrosis (IPF) patients “shielded” by an A549 alveolar epithelial layer, mimicking inhalation therapy. LIMCH1-ASO was effective in LIMCH1-silencing even at the lowest dose (0.64 nmol/cm2). Nebulization did neither impair LIMCH1-ASO efficacy nor induce cytotoxicity for the highest tested dose (3.21 nmol/cm2.) The permeability (biokinetics) of nebulized ASO through the A549 cell barrier was 14-fold faster (“burst-like”) than for larger volume (pipetted) ASO application. Computational modelling revealed that this well-known “burst-like” pharmaco−/biokinetics profile is due to small-volume drug application, not due to nebulizer-induced changes on barrier tightness (apparent permeability Papp). Thus, the Cloud MAX is a substance-efficient platform for in vitro pharmacokinetics/−dynamics testing of inhalable drugs for targeted pulmonary therapy.

Abstract Safety assessment of engineered and incidental nanomaterials (NM) is a costly and time-consuming process, which is currently mainly relying on in vivo (animal) testing. A mechanism-aware prediction of the in vivo hazard profile from readily available material properties and in chimico assays could help avoid animal experiments and save time and resources during product design. We present two machine-learning models for the prediction of the acute lung inflammation quantified via neutrophil influx into mouse lungs at 1 d and 28 d after intratracheal NM instillation: one using advanced experimental descriptors such as the BET surface area and ROS production rate, and the other using purely in silico-derived extrinsic properties of the NMs such as interaction energies with key biomolecules (amino acids and lipids). Using the first model, we show that in vivo respiratory toxicity for a diverse set of 50 NMs, including carbon nanotubes, carbonaceous and metal oxide particles, asbestos, and graphene, can be confidently predicted using only 5 NM descriptors derived from the surface area, shape, and oxidative potency measurements. The analysis of experimental and computational data enables us to categorize NMs into toxicity classes based on their inflammogenic potential and advanced descriptors, thereby relating them to their potential mode of action. The second model, also successfully predicting in vivo outcomes, provides further insights into the toxicity mechanisms by revealing the correlation of inflammogenicity with NM-lipid interactions. The models can be used to group NMs based on in-chimico and/or in-silico characterisation and to predict inflammogenicity for novel materials outside the training set.

Pulmonary vascular disease (PVD) is a major contributor to morbidity in preterm infants as it is associated with a significant risk to develop pulmonary hypertension, especially in infants diagnosed with prematurity-associated lung disease (PLD), also known as bronchopulmonary dysplasia (BPD). However, the earliest events of vascular injury triggered by postnatal mechanical and oxygen-related stress remain poorly understood, largely due to the limitations of existing in vitro models. We therefore developed a biomimetic, miniaturized pulmonary in vitro perfusion (PIPE) system that integrates pathophysiologically relevant shear stress with controlled oxygen exposure for the exposure of a human coculture of pulmonary microvascular endothelial cells and pulmonary artery smooth muscle cells. Advancing the system to a triple coculture, circulating THP-1 monocytes capture early endothelial-smooth muscle-immune cell interactions. Shear stress alone induced early proliferative and extracellular matrix-related responses in endothelial cells and resulted in enhanced monocyte recruitment without disrupting barrier integrity. When combined with oxygen exposure, the model revealed a dose-dependent injury pattern: moderate hyperoxia [fraction of inspired oxygen ([Formula: see text] = 0.40)] had minimal acute effects, whereas severe hyperoxia ([Formula: see text] = 0.85) impaired endothelial barrier function, increased reactive oxygen species (ROS) production, promoted monocyte transmigration, activated apoptosis (caspase 3), and elevated soluble collagen synthesis. This dynamic in vitro system reveals early drivers in vascular injury and recapitulates key characteristics of PVD, thereby introducing a translational platform for the dissection of disease mechanisms and evaluation of therapeutic strategies targeting vascular injury in the developing lung.NEW & NOTEWORTHY We present a biomimetic, miniaturized in vitro model that replicates key features of neonatal pulmonary vascular injury. By integrating physiologically relevant cues, this platform enables early detection of injury-associated molecular markers and mechanistic interrogation of disease onset. This scalable model offers a powerful tool for studying neonatal lung vascular pathology and for accelerating the discovery of early diagnostic and therapeutic strategies.

Abstract Introduction Understanding the dynamic processes of nanoparticle (NP) deposition and subsequent biokinetics in the lung plays a pivotal role in inhalation nanotoxicology and nanomedicine. Here we introduce LungVis1.0, an artificial intelligence (AI) powered imaging ecosystem that enables precise, single-cell resolution mapping of NP distribution and NP-cell interaction in non-dissected lungs. Method NPs (600 nm, melamine) were delivered to murine lungs using four different bulk-liquid and aerosol-based delivery modalities. Whole lung samples were collected at 0 h, 2 h, 1 d, and 14 d post-administration. Tissue clearing and light sheet fluorescence microscopy processed with active/deep learning AI algorithms allowed localization of NPs in precisely segmented, non-dissected airway trees. Complementary data from intravital microscopy, perfused lung models, and flow cytometry is available. Results LungVis1.0 revealed substantial differences in bronchial and acinar NP distribution patterns depending on NP delivery route at macroscopical and microscopical levels. Briefly, bulk-liquid delivery results in patchy NP distribution with elevated bronchial-to-acinar dose ratio (B/A > 0.2), whereas aerosol inhalation achieves globally uniform, more alveolar NP deposition with local dose hot-spots in the proximal acinar region. Moreover, lung tissue-resident macrophages (TRMs) exhibited dynamic behaviour, actively patrolling and redistributing NPs within alveoli, challenging the traditional view of TRMs as static cells. Discussion/Conclusions LungVis1.0 provides a comprehensive framework for studying the delivery, biokinetics and cell interaction of NPs in the lung fostering progress in inhalation nanotoxicology/-medicine. The findings underscore the advantages of aerosol delivery for achieving uniform pulmonary NP distribution and the presence of high NP doses in the acinar region, potentially bridging the “sensitivity gap” between in vitro and in vivo models in nanotoxicology.

Lung resident immune cells are essential for initiating defenses against inhaled air pollutants, including nanoparticles (NPs), which contribute to pulmonary disease progression. Here, lung intravital microscopy was used to examine the pulmonary innate immune responses in mice, during acute aerosol exposure to carbon NPs, a common environmental pollutant, or fluorescent quantum dot NPs. We found that inhaled NPs triggered rapid, neutrophil recruitment, localized to alveolar NP deposition hotspots, orchestrated by alveolar macrophages (AMs) through both their motility and phagocytic activity. AM motility inhibition in the alveoli via intercellular adhesion molecule-1/LFA-1 blockade reduced neutrophil recruitment, as did impaired AM phagocytosis through C5a receptor 1/Fc-γ receptor I inhibition or by stealth NP surfaces. In addition, cellular degranulation inhibition indicated the importance of spatially focused cytokine release in neutrophil recruitment. Collectively, our study elucidates AM-epithelial interactions as a critical key event for NPs triggered neutrophilia, with AM motility and phagocytosis driving recruitment and site-specific immune responses in the alveolar microenvironment.

Inhalation therapy represents a promising strategy for the delivery of biopharmaceuticals for the local treatment of respiratory diseases. Purified polyclonal serum immunoglobulin G (IgG), also known as IVIg products, exhibit a solid reactivity against common viral and bacterial antigens. However, IVIg are usually delivered intravenously or subcutaneously, thus, not at the sites where most infections originate. Accordingly, a respiratory mucosal delivery of IVIg may have the potential to prevent infections at the mucosal barrier. To evaluate the feasibility and efficacy of inhalable IVIg for the prevention and therapy of respiratory infections, this study examined nebulization and its impact on protein quality, as well as potential effects on in vitro cytotoxicity and immunogenicity. IVIg were formulated with either 0, 200, or 400 μg/mL of polysorbate 80 (PS80). Formulation with polysorbate 80 resulted in less IgG aggregation during nebulization and thereby reduced in vitro immunogenicity. Further, the transepithelial transport was analyzed using two different airway epithelial models, with no effects observed due to either nebulization or formulation. Finally, the efficacy of formulated aerosolized IVIg against Streptococcus pneumoniae TIGR4 model bacteria was assessed. The results demonstrated a dose-dependent binding of relevant S. pneumoniae antigens and efficient dose-dependent opsonophagocytosis of S. pneumoniae. In conclusion, this study indicated the promising potential of inhaled polyclonal IVIg as an effective therapy against respiratory infections.

Rationale: The airway epithelium serves as the first protective barrier against cigarette smoke (CS), which is a major cause of chronic lung disease (CLD). While transient and persistent transcriptomic changes in the bronchial epithelium as a result of CS exposure have been described, proteomic studies are lacking in this context. Objective: To uncover CS-induced effects on the human airway proteome and identify the cellular source of transient and persistent changes. Methods: Label-free tandem mass spectrometry-based proteomics data of bronchoalveolar lavage fluid (BALF) from a CLD patient cohort mainly composed of interstitial lung disease (ILD) patients (n=124; 16 idiopathic pulmonary fibrosis (IPF), 79 non-IPF ILD, 29 non-ILD1) was analyzed in respect of patients’ smoking history (ex-, active- and never-smokers). Results were compared to the proteome of chronically CS-exposed primary human bronchial epithelial cells (phBECs) relative to unexposed controls. Cellular sources of persistent protein changes were explored using immunofluorescent stainings of human lung tissue and whole lung single cell RNA-Seq analysis. Results: Smoking was associated with transient (up in active but lost in ex-smokers) and persistent (up in active and sustained in ex-smokers) protein changes in the BALF samples. We observed an overlap between CS-induced protein changes in phBECs and transient changes in BALF. Proteins associated with the persistent response primarily localized to macrophages as well as to basal and ciliated cells, but not to secretory cells, in the bronchial epithelium. Conclusion: CS triggers both transient and persistent protein changes in BALF of CLD patients. While the transient expression changes likely originate from the bronchial epithelium, persistent changes stem from both bronchial epithelial cells and macrophages. The results warrant further investigation into the relevance of these changes for onset and progression of CS-associated CLD. 1 Mayr CH, et al. Integrative analysis of cell state changes in lung fibrosis with peripheral protein biomarkers. EMBO Mol Med 13: e12871, 2021.

Multi-component nanomaterials (MCNM) and High Aspect Ratio Nanomaterials (HARN) are advanced materials that present innovation potential but also challenge the innovation by Safe and Sustainable by Design (SSbD) principles. In 2021 to 2025, three EU-funded sister projects developed and implemented SSbD concepts in digital tools that are applicable to MCNM and HARNs despite their respective unique properties. The projects jointly established a tiered suite of tools that serves both industrial, SME (small and medium enterprise) and regulatory stakeholders. The tools for innovators are tiered by StageGate phases (projects SUNSHINE and HARMLESS) or organized in transversal topics (project DIAGONAL). Also the tools for regulatory preparedness comprise a range from simple to elaborate approaches. Key achievements include the alignment of tools for innovators and for regulators via a high overlap of the questions asked, and the systematic tiering of targeted input that is required. The suite of tools thus supports the OECD's Safe and Sustainable Innovation Approach (SSIA). With specific value to SME innovators, tools were developed that lower the previously perceived hurdles to implementation of SSbD concepts in product development. The suite of tools is demonstrated on three MCNM or HARN case studies provided by partners from industry and SMEs, specifically anti-stick coatings for baking trays (SiC@TiO2), automotive catalysts (doped oxide perovskites) and flexible electronics (Ag nanowires). However, the cases also shed a light on remaining challenges in the SSbD concept that are not solved by tools alone, most notably the uncertainty of decision-support at early stages and the complexity of data gathering at later stages, which also implies a need of increasing data exchange among value chain actors while allowing actors to generate and protect intellectual property.

Lung transplantation (LTx) offers life-saving therapy for end-stage lung disease, yet primary graft dysfunction (PGD) and bronchiolitis obliterans syndrome remain major complications, which limit long-term outcomes. The risk of PGD increases with prolonged storage and reperfusion injury. This study investigated the protective effects of cystatin C (CysC), a cysteine protease inhibitor, against PGD, through neutralization of cathepsin B activity. LTx recipients exhibited reduced levels of CysC and greater lung damage reflected by increased γ-H2AX and ACSL4 expression, correlating with poorer outcome. Using an orthotopic LTx model, we engineered CysC-Alb, an albumin-fused, cell-permeable CysC derivative for enhanced lung preservation. Adding CysC-Alb to preservation solutions during cold storage and ex vivo lung perfusion improved oxygenation, reduced DNA damage, and minimized cell death, particularly in alveolar type 2 cells. In murine grafts, CysC-Alb decreased γ-H2AX and ACSL4 expression, markers of DNA damage and ferroptosis, respectively. These findings highlight CysC-Alb as an effective additive to mitigate early pulmonary dysfunction after LTx and improve lung graft viability and transplantation outcomes.

To facilitate Safe-and-Sustainable-by-Design (SSbD), the HARMLESS project developed a practical SSbD approach and SSbD Decision Support System (DSS) for advanced materials. The HARMLESS SSbD approach and SSbD-DSS are in alignment with the EU-recommended SSbD framework and methods. The HARMLESS SSbD approach recommends New Approach Methodologies (NAMs) tailored to advanced materials and the innovation stage of the material under development. The approach follows a flexible stage-gate model with three innovation stages: 1) Ideation & Business Case Phase, 2) Lab Phase, and 3) Pilot Phase. The HARMLESS SSbD-DSS guides designers through a workflow starting with the Advanced Material Earliest Assessment (AMEA) tool. AMEA contains three questions enabling categorization and subsequently advice on design principles. A second tool, named Warning flags, Design Advice, Screening Priorities (WASP), consists of 12 questions to identify early warning flags and provide design and assessment advice. A third tool, named Alternative SSbD Design Inspector (ASDI), provides guidance on descriptors to measure in the Lab Phase for informed decision making on the most optimal SSbD version. By adapting the data and resource requirements to each innovation stage, the SSbD-DSS facilitates practical implementation of SSbD, supporting the user to find a transparent balance between safety, sustainability, and performance.

An integrated framework is introduced and applied to assess the health impact of airborne pollution with greater physiological relevance, moving beyond conventional exposure metrics. Measured particle number size distribution data was integrated with a regional respiratory tract deposition fractions to estimate total and alveolar deposited particle surface area concentrations. Land use regression modeling, combined with randomized commuting patterns, enabled the evaluation of city-specific alveolar surface area deposition doses, providing new insight into localized average exposure and its implications for public health. The results showed that although the mean street-level air pollution in Lithuania is higher than in other European cities, the urban background levels are on the same level. We found that the total respiratory deposited surface area concentration is up to 18-fold higher due to coarse particles, which also determines alveolar deposited particle surface area dose. Our findings advocate for using integrated pollution assessments and region-specific policies rather than broad diesel vehicle-targeted bans. The proposed methodology is expected to enhance traditional exposure assessments by switching to lung deposited surface area, which can be further refined by incorporating daytime activity patterns, socio-economic status, and personal health conditions.

There is an ongoing demand to develop options to reduce hazard testing of substances and materials on a case-by-case basis. Grouping approaches offer a way to share or re-use safety-related information between similar substances, providing insights that can inform the Safe and Sustainable by-Design (SSbD)2 of new materials. Here, an existing grouping hypothesis template for single-component nanomaterials (NMs)3 is expanded to facilitate systematic consideration of grouping for multicomponent nanomaterials (MCNMs)4 relevant to SSbD. Modifications to the template include additional information on a) the complexity of physical and chemical composition; b) the emerging properties driving the MCNM functionality; c) the potential for MCNM components to transform with different rates, leading to complex exposure scenarios; d) prioritisation and simplification of grouping decisions related to material properties (what they are), fate/toxicokinetics (where they go) and the hazard mechanisms (what they do). Existing information and data are used to formulate a matrix of sub-hypotheses that individually relate one (or more) indicators of 'what they are' to a single indicator of either 'where they go' or 'what they do'. The resultant sub-hypotheses are easier to assess than the all-encompassing over-arching hypothesis required for regulatory application of grouping. The estimated level of impact of each indicator is used to prioritise the sub-hypothesis assessment. Accepting or rejecting each prioritised sub-hypothesis is facilitated by the application of tiered testing strategies promoting the use of relevant existing data, new approach methodologies and machine learning-based models. A case study of SiO2@ZnO MCNM is provided to demonstrate the template's usefulness in an SSbD context.

In the context of the EU Chemicals Strategy for Sustainability toward a Toxic-free Environment, and in the aim to facilitate Safe-and-Sustainable-by-Design (SSbD) development of new materials and products, the EU-funded project HARMLESS built a Decision Support System (DSS) to support innovators in SSbD assessments of products at early design stages. The HARMLESS DSS is tailored to enable Advanced Materials (AdMa) screening, using a combination of New Approach Methodologies and guided by Integrated Approaches to Testing and Assessment. Three online tools for SSbD screenings are developed and tested on case study materials, namely: Advanced Materials Earliest Assessment; Warning flags, design Advice, Screening Priorities; and Alternative SSbD Inspector. The following case studies are assessed: oxide perovskites for automotive catalysts, imogolites for agricultural solutions, aerogel mats for facade insulation, and colloidal silica for paint formulations. The case study materials are provided by both large and small enterprises, they represent diverse industry sectors and different stages in the value chain. Results show the DSS ability to guide innovators in developing SSbD AdMa, by facilitating the assessment of most SSbD dimensions and visualizing the results in a comprehensive, yet detailed way, enabling their own balancing of SSbD benefits and trade-offs.

Advanced Materials (AdMa) play a crucial role for numerous strategies that address global challenges. They are being developed fast, making it increasingly challenging for regulation to keep pace with innovation. Existing frameworks, which are either not designed for AdMa or lack adequate filtering to identify AdMa of high concern, do not (yet) effectively support regulatory preparedness. The HARMLESS Early Warning System (EWS), in contrast, is a practically applicable tool for screening plenty of materials in a reasonable time. It is organized in two tiers, each underpinned by a specific methodology and facilitated by a dedicated online tool. The initial Tier 0 categorizes the materials using the Advanced Materials Earliest Assessment (AMEA) tool. Tier 1 first screens materials asking only 15 questions and is ideal for data-poor materials at early innovation stages. These questions cover issues related to human/ environmental exposure and hazard, sustainability and applicability of existing regulations. In a more elaborated version, experimental testing based on New Approach Methodologies (NAMs) is suggested. As outcome, the user is provided with 1) material-related concerns, 2) prioritization of AdMa and 3) recommendations for (regulatory) follow-up actions. Data from two industrial case studies is presented to demonstrate the applicability of the HARMLESS EWS.

Understanding how nanomaterial properties drive acute lung inflammation is critical for the development of safer materials, but for low solubility carbon-based nanomaterials (CBNs) the initiation of the inflammatory response is still poorly understood. Leveraging single-cell RNA sequencing of mouse lungs, 12 h after intratracheally instillation with different CBN spherical carbon nanoparticles (CNP), tangled double-walled (DWCNT), and rigid multiwalled carbon nanotubes (MWCNT) and lipopolysaccharide (LPS) as positive control, we identified 41 cell states and delineated material-specific molecular initiation events at single-cell resolution. CBN doses were chosen to cause equal levels of moderate inflammation, assessed by airspace neutrophilia, and exposure-triggered cellular activation was tested for in vitro reproducibility. To advance future development of cell-based assays, we developed a webtool, ToxAtlas, mapping CBN-specific gene responses of interest. Despite chemical similarity, CBN elicited distinct inflammatory cytokine and cell responses via different modes of action. CNP triggered neutrophilia through alveolar epithelial activation and Cxcl1 and Csf2 expression but without apparent cell damage or macrophage activation. In contrast, CNT induced epithelial and macrophage damage, with alarmin release (IL-1α, IL-33) dominating the MWCNT response. DWCNT caused alveolar epithelial injury, and pro-inflammatory macrophage and fibroblast-derived monocyte attractant (Ccl2, Ccl7) activation. Our initiating cell circuits identify epithelial as well as early fibroblast activation, especially from alveolar type 2 cell-adjacent lipofibroblasts, as central to orchestrating the initiation of CBN-induced inflammation. These findings support the role of mesenchymal cells in early pulmonary defense, eventually priming chronic inflammation, a known cause of MWCNT exposure.

Nanotechnology-based platforms are being explored for the delivery of therapeutics directly to the lungs through inhalation. However, to ensure translational relevance between preclinical animal models and human applications, it is essential to accurately quantify and report the lung-deposited dose, rather than relying solely on the nominally administered dose.