Protein aerosol deposition to air-liquid interface (ALI)

Purpose Pulmonary delivery of proteins offers a promising noninvasive alternative to injections, driving interest in pulmonary delivery systems for protein therapeutics. New approach methods (NAMs) are increasingly applied in inhalation toxicology. Here, we employed an NAM-based in vitro air–liquid interface (ALI) exposure system to evaluate protein aerosol deposition. Methods A protein suspension was aerosolized using an atomizer and delivered to a 6-transwell ALI system for 2 hours at 12 or 30 mL/min flow rates. Results Aerosol exposures were stable within ± 20%. The generated aerosols had a median diameter of 128 nm, with number and mass concentrations of 6.59 × 10 ⁵ particles/cm ³ and 17.13 mg/m ³ , respectively. The average deposition was 4.40 µg (12 mL/min) and 3.73 µg (30 mL/min), with a higher efficiency at 12 mL/min. Modeling indicated diffusion as the dominant deposition mechanism, consistent with particle size and theoretical collection diameters. In vitro deposition efficiencies (19.7% and 13.9%) were comparable to pulmonary deposition predicted by the MPPD model (12.8%). Conclusions Our study identified key dose metrics (particle size, concentration, deposition stability) for protein aerosol testing and established diffusion as the primary deposition pathway in an ALI system. Comparable efficiencies with in silico predictions support the use of this in vitro approach for evaluating pulmonary delivery and safety of protein therapeutics.