Nicotine Drives Acute Epithelial Injury in Region-Specific Human Airway Models at the Air-Liquid Interface after E-Cigarette Aerosol Exposure

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