The cellular response of the bronchial epithelium shapes the protein corona of inhaled nanoparticles

Protein adsorption to nanoparticles is a key molecular event that influences their fate, biodistribution and toxicity. In the lung, a mucus layer protects the bronchial epithelium from inhaled pollutants. However, the effect of cell exposure to nanoparticles on the formation of the protein corona in the bronchial mucus is not well understood. This study aimed to uncover how the bronchial epithelial cell response shapes the biomolecular corona on inhaled nanoparticles and whether cell adaptation remodels the nano/bio interface. We reproduced a realistic scenario of lung exposure to silver nanoparticles (AgNPs) <i>in vitro</i> using a 3D human bronchial epithelium model. AgNPs were incubated in the isolated bronchial mucus or directly exposed to Calu-3 cells at the air-liquid interface. The stability of AgNPs in the mucus was characterized by small-angle X-ray scattering, dynamic light scattering, and transmission electron microscopy. The protein corona formed during the exposure of the bronchial epithelium to nanoparticles was analyzed using quantitative mass spectrometry and Reactome pathway analysis as a function of NP concentration and exposure time. Proteomic analysis revealed major differences in the biomolecular corona formed <i>in situ</i> compared to the corona formed in isolated bronchial mucus. Unique proteins expressed in the apical secretome of Calu-3 cells exposed to AgNPs were identified in the protein corona formed <i>in situ</i>. The stress response of the epithelial cells led to a complete reshuffling of the protein corona after initial deposition of AgNPs on mucus. Our results demonstrate that the cellular response of the bronchial epithelium plays a critical role in shaping the protein corona of inhaled particles. The remodeling of the nano/bio interface by cellular secretory mechanisms during exposure calls for a renewed focus on the role of cells and NP-cell interactions in biomolecular corona studies using advances 3D models.