Result 4 – PS-Eu affects surfactant secretion/production and boosts metabolic activity in Sub and ALI samples, while changing cell morphology (TEM) in ALI (increased authophagy and protein aggregation, reduced Mitochondria/ER contacts and lamellar bodies). Sub samples ultrastructure is still under evaluation. No effect on barrier integrity, PS-Eu translocation or cytotoxicity was found. Result 2 – PS-Eu affect acidic organelle activity (NRU) in a dose-dependent manner and are uptaken by A549 grown under standard submerged conditions (culture method A). 600nm * N LD * PS-Eu 50 µg/ml PS-Eu 100 µg/ml 200nm Control LB * PS-Eu 25 µg/ml 200nm 600nm Particle uptake (TEM) Impact of Polystyrene Nanoparticles on Lung Epithelial Barrier Formation and Functionality Sara Michelini, Neža Repar, Ema Kurešepi, Katarina Šimunović, Francesco Barbero, Martin Schicht, Katharina Jüngert, Victor Ganin, Ivana Fenoglio, Friedrich Paulsen, Andreja Erman, Barbara Šetina Batić, Ines Mandič-Mulec, Anita Jemec Kokalj, Damjana Drobne. Background: Micro- and nanoplastics (MNPs) are one of the most widespread pollutants of our time. They can be manufactured in specific sizes for a specific purpose or be released into the environment through the degradation of plastic products. Humans are generally exposed to MNPs via two main routes: food ingestion or inhalation of air. In the latter case, it has already been reported that airborne particles can enter and accumulate in the lower respiratory tract, leading to occupational diseases. This study aims to assess the impact of 274 nm europium-doped polystyrene (PS-Eu) particles on the integrity and functionality of the lung barrier, especially in terms of surfactant production. The presented results were produced both on A549 cells grown in “standard” submerged (Sub) conditions as well as by performing the assays in Air-liquid interface conditions (ALI) using transwells which resemble more closely the real-life settings. Result 3 – Model characterization. Epithelial barrier forms 10 days post seeding. After 17 days ALI-grown A549 produce more surfactants, and have more microvilli than Sub cells. hello@plasticsfate.eu www.plasticsfate.eu plasticsfate Linkedin.com/groups/12519441 PlasticsFatE has received funding from the European Union’s Horizon 2020 Research and Innovation programme, under the Grant Agreement number 965367 Conclusion and outlook: PS-Eu has been found able to increase acidic organelle activity and, in cells grown on transwells, also surfactant production and metabolic activity. Additionally in ALI, which is a more physiological model, TEM analysis revealed that PS-Eu caused a dose-dependent reduction of both LB and mitochondria-ER contacts (structures known to be critically involved in surfactant production/secretion), possibly by inducing autophagy upon uptake. Many LB were in fact found in autophagic vacuoles. Taken together these results may indicate that PS-Eu, upon uptake, are able to affect LB trafficking, vesicle acidification and mitochondria/ER activity causing a dose-dependent reduction of intracellular surfactant production which possibly will affect, at later timepoints, the extracellular surfactant pool, which is up to now not drastically affected with the exception of SP-D and SP-D. Sub samples ultrastructural analysis might shed more light on the mechanisms underlying the shown effects. Acknowledgements: Method images were created with BioRender.com. Figure 2: Impact of different concentrations of PS-Eu on A549 cells in terms of A) cytotoxicity (LDH assay), B) metabolic activity (Resazurin assay), C) protein content (CBB assay) and D) acidic organelles activity (Neutral Red Uptake assay). Particle uptake was confirmed via TEM E). LB, lamellar body; N, nucleus; LD, lipid droplet; asterisk, PS-Eu. Figure 3: Model characterization. A) OT permeability and TEER of A549 cells grown in ALI or submerged conditions on transwells (culture method B). B) Confocal microscopy showing OT barrier for