Quantitative Evaluation of Protein Nanoparticle Aerosol Deposition in an Air–Liquid Interface (ALI) System and Comparison with In Silico Lung Deposition Modeling

Purpose This study quantitatively evaluated the deposition of protein nanoparticle aerosols in an in vitro air–liquid interface (ALI) exposure system and compared experimental results with in silico lung deposition predictions using the Multiple-Path Particle Dosimetry (MPPD) model. Methods A whey protein suspension was aerosolized at 3 L/min and delivered to a custom-built flow-through ALI system containing six transwells. Aerosols were sampled at impinger flow rates of 12 or 30 mL/min for 2 h using a low-flow pump. Particle size distribution and concentration were continuously monitored using a scanning mobility particle sizer. Deposited protein mass on apical and basolateral compartments was quantified by UV–Vis spectrophotometry. Deposition efficiency was calculated and compared with MPPD model predictions. Results Aerosol exposures remained stable within ± 20% of the mean over 2 h. Generated protein aerosols had median diameters of 128 and 98 nm at downstream impinger pump flow rates of 12 and 30 mL/min, with mass concentrations of 17.1 and 10.1 mg/m 3 , respectively. Mean deposited mass was 4.40 µg (CV 19.7%) at 12 mL/min and 3.73 µg (CV 18.6%) at 30 mL/min of impinger flow rate. Deposition efficiency at 30 mL/min (13.9%) closely matched the MPPD-predicted pulmonary deposition fraction (12.8%). Modeling analysis identified diffusion as the dominant deposition mechanism under the tested conditions. Conclusions Protein aerosol deposition in the ALI system was stable, diffusion-driven, and quantitatively consistent with in silico predictions of pulmonary deposition. These findings demonstrate that this non-animal ALI approach provides a reproducible and physiologically relevant framework for evaluating aerosol dosimetry in inhalation toxicology and pulmonary drug delivery research.