In-house Fabrication of Nanoplastics of Tunable Composition and Application: Assessment of Bioelectric Changes in Primary Rat Lung Alveolar Epithelial Cell Monolayers Exposed to Nanoplastics

Plastic pollution presents a looming danger to the environment and virtually all life on planet Earth. Especially pernicious are nanoplastics (NPs), which are plastic fragments with dimensions ≤1 μm. Conventional detection methods are ineffective for NPs, while their high specific surface area renders them efficient carriers of toxic substances; additionally, they may even be inherently toxic. Although NP waste chiefly arises from environmental weathering of larger plastic fragments, most published studies employed manufactured pristine NPs of uniform size and shape. Furthermore, almost all NP effects were studied using polystyrene (PS) as a convenient model material, despite PS accounting for <6% of all plastic pollution. There is thus an urgent need to expand investigations of environmental NP pollution and effects on biota. The present work provides a comprehensive roadmap for studying the effects of "real-world" NP pollution on living systems, using, for example, lung alveolar epithelial cells on which such NPs deposit by breathing ambient air. Herein, we describe detailed in-house methods to fabricate various NPs that are weathered with UV light and O₃ gas exposure to more closely mimic real environmental NPs. We also illustrate a simple and straightforward bioelectrical method for assessing passive and active ion transport properties of primary rat lung alveolar epithelial cell monolayers as a model for the distal mammalian lung exposed to one of the generated NPs. This protocol allows researchers to rapidly and more accurately assess the biological impact of various simulated environmental NPs on a vulnerable air-blood barrier in the lung. Key features • Many simulated weathered environmental NPs can be produced at high concentrations (up to 120 mg/mL) and yields (up to 12 mg/g bulk plastic). • Any plastic waste can be "nano-sized" with this protocol and then studied for impacts on active and passive ion transport properties of cell monolayer models. • Methods described herein are very relevant for studying environmental pollution effects, since NPs are found in many different shapes, sizes, and compositions. • NP weathering and generation methods do not require any expensive or specialty lab instruments.