Assessing toxicity of amorphous nanoplastics in airway- and lung epithelial cells using air-liquid interface models

Inhalation is one of the main exposure routes to nanoplastics. Knowledge of the toxicological impact of nanoplastics on the airway- and lung epithelium is limited and almost exclusively based on submerged in vitro models using spherical polystyrene (PS) particles. Mono-cultures and advanced (co-)cultures of human bronchial- and alveolar epithelial cells, all air-liquid interface (ALI) cultures, were exposed to nanoplastics and reference nanoparticles. Alveolar models included A549 mono-cultures and A549 cells co-cultured with endothelial cells (Ea.hy926) and macrophage-like cells (differentiated THP-1). Bronchial models included BEAS-2B cells and differentiated primary bronchial epithelial cells (PBEC). Cultures were exposed to PS, copper(II) oxide (CuO) or titanium dioxide (TiO 2 ) nanoparticles (50 nm). Additionally, BEAS-2B cells were exposed to well-characterised, amorphous polyvinyl chloride (PVC), polypropylene (PP), or polyamide (PA) nanoplastics. Cytotoxicity and inflammation (IL-8 secretion and IL-8 transcript levels) were assessed after 24 h of exposure. Cell viability remained unaffected by all exposures in all models. Unlike PS and TiO 2 , CuO exposure dose-dependently induced IL-8 protein secretion and mRNA levels. Although the extent of IL-8 secretion differed between models, the relative response to CuO was similar in both mono-cultures and advanced (co-)cultures. None of the environmentally relevant nanoplastics (PVC, PA or PP) impacted inflammation or cell viability in BEAS-2B ALI cultures. Although CuO induced inflammation, PS failed to elicit an inflammatory response in any of our models. For the first time, we show that PVC, PA and PP do not induce cell death or inflammation in a BEAS-2B ALI model. • Mono-cultures and advanced (co-) cultures responded similar to nanoparticles. • None of the tested nanoplastics induced cytotoxicity. • Polystyrene did not induce inflammation in air-liquid interface cultures. • Environmentally relevant nanoplastics did not impact inflammation in bronchial cells.