EVs Biodistribution and Antifibrotic Impact in Aged Lung Fibrosis Model

Pulmonary fibrosis (PF) is a progressive, life-threatening disease marked by excessive scarring of lung tissue. Recently, extracellular vesicles (EVs) have emerged as a promising antifibrotic therapy due to their regenerative and anti-inflammatory properties. However, the success of EV-based therapies depends on the route of administration, which can significantly influence their biodistribution and therapeutic effects. Furthermore, in PF, aging is a significant risk factor for the disease and, until today, EV treatment efficacy has not been studied in aged tissues. Specifically, we studied EVs derived from human umbilical cord mesenchymal stem cells and compared the biodistribution of these vesicles delivered via three routes: intravenous (IV), intrapleural (IP), and intratracheal (IT). A protocol was developed to set EV staining and concentration, minimizing animal use while maximizing the accuracy of results. To evaluate therapeutic effects, we conducted three experimental setups: (i) to assess their ability to reverse established fibrosis; (ii) to evaluate their effect on fibrosis progression; and (iii) to study early inflammation and macrophage polarization. Lung fibrosis and inflammation were assessed by analyzing fibrotic markers, inflammatory cytokines, collagen deposition, and bronchoalveolar lavage (BAL) fluid cell analysis, providing insights into EVs therapeutic potential in aged, fibrotic lung tissue. In the biodistribution study, IV administration was identified as the most effective route, successfully delivering EVs to both normal and fibrotic lung tissues. In the therapeutic study, antifibrotic effects were observed only when EVs were administered prophylactically, before the establishment of fibrosis. Under this protocol, IV-administered EVs reduced fibrotic mRNA biomarkers, collagen deposition, inflammatory cell infiltration, and macrophage polarization in BAL, as well as altering cytokine. Our findings emphasize the critical importance of selecting the appropriate route of administration for EV-based therapies. Notably, our work with an aging model reveals that EV treatments primarily exhibit prophylactic effects, with a marked reduction in their regenerative potential compared to previous studies conducted in younger models.