Mechanisms of nebulized anti-FAP-engineered exosomes encapsulating pirfenidone for targeted therapy of pulmonary fibrosis

Pulmonary fibrosis (PF) is a progressive and often fatal interstitial lung disease with limited therapeutic options. Although pirfenidone (PFD) is an approved antifibrotic agent, its clinical efficacy is constrained by poor lesion-specific delivery and systemic adverse effects associated with conventional administration. In this study, we developed anti-FAP-engineered exosomes encapsulating PFD for nebulization-based targeted therapy of PF. Exosomes derived from 293T cells were functionalized with anti-FAP using copper-free click chemistry and loaded with PFD. They were then characterized by transmission electron microscopy, nanoparticle tracking analysis, Western blot, and high-performance liquid chromatography. A bleomycin-induced mouse model of PF was established to compare tail vein injection with nebulization delivery. In vivo fluorescence imaging, confocal microscopy, histological staining, western blot, single-cell RNA sequencing, and cell-based functional assays were performed to evaluate therapeutic efficacy and underlying mechanisms. Nebulization significantly enhanced pulmonary accumulation and retention of the engineered exosomes compared with systemic administration. This resulted in improved lung histopathology, reduced collagen deposition, and stronger suppression of the TGF-β1/p-Smad3 signaling axis. Additionally, nebulization remodeled the fibrotic microenvironment by attenuating fibroblast and myofibroblast activation while promoting polarization toward a repair-associated state. Proteomic analysis and tissue immunofluorescence further identified AXL as a candidate regulatory molecule associated with these effects. Functional studies in MLE12 cells and polarized BMDMs demonstrated that modulation of AXL influenced pro-fibrotic cellular behaviors, TGF-β1/p-Smad3 signaling, and oxidative stress. Overall, these findings demonstrated that nebulized anti-FAP-engineered exosomes represented a promising targeted delivery platform for PF and highlighted AXL-associated signaling as a potential mechanistic node and therapeutic target in microenvironment-focused antifibrotic intervention.