Electronic cigarette aerosols are complex mixtures whose respiratory toxicity depends on formulation chemistry and regional deposition. This study investigated how nicotine presence, nicotine protonation state, and propylene glycol/vegetable glycerin (PG/VG) ratio shape acute epithelial injury under controlled air–liquid interface (ALI) exposure. Well-defined e-liquid formulations differing in PG/VG ratio (70/30 or 30/70, w/w), nicotine concentration (0 or 20 mg/mL), and nicotine form (freebase or salicylic-acid salt) were aerosolized using a fourth-generation pod-based device and delivered at the ALI to human epithelial models representing the pharynx (Detroit 562), bronchi (Calu-3), and distal-airway (NCI-H441, alveolar type II–like). Cells were exposed to low and high aerosol doses corresponding to 2 and 20 puffs, resulting in deposited surface doses of approximately 33 and 330 µg/cm², respectively, as quantified gravimetrically. Cytotoxicity, epithelial barrier integrity, and inflammatory signaling were assessed 24 h post-exposure. Nicotine emerged as the primary driver of acute epithelial injury. Toxicity was modulated by formulation chemistry: freebase nicotine elicited more pronounced barrier-disruptive responses than the corresponding nicotine salt, while nicotine salts induced comparatively modest inflammatory signaling. PG-rich formulations amplified toxicity, particularly in distal airway - like cells. In contrast, pharyngeal epithelial cells remained largely unresponsive under identical conditions. Pro-inflammatory responses, assessed by interleukin-8 release, were limited and observed at high deposited doses. Overall, these findings demonstrate that acute e-cigarette aerosol toxicity under ALI conditions is nicotine-driven and influenced by nicotine speciation, solvent composition, and airway region, highlighting the importance of region-specific ALI models and surface-based dosimetry for toxicological assessment of inhaled products. • Standardized ALI exposure (2 and 20 puffs) yielded gravimetrically validated doses of ≈33 and 330 µg/cm² • Nicotine was the primary driver of membrane damage and barrier dysfunction • Nicotine-containing aerosols increased LDH release in Calu-3 and NCI-H441, while Detroit 562 remained largely unresponsive • Freebase nicotine induced stronger barrier disruption (TEER↓) than salicylate salt under identical PG/VG conditions • PG-rich (70/30) formulations amplified cytotoxicity compared with VG-rich (30/70) matrices • Pro-inflammatory responses (IL-8) were modest, dose-dependent, and most pronounced in distal airway–like cells
Köse et al. (Sun,) studied this question.