Pulsed Focused Ultrasound-Mediated Mechanopriming Enhances the Therapeutic Efficacy of Mesenchymal Stromal Cell-Derived Extracellular Vesicles in Acute Respiratory Distress Syndrome

Extracellular vesicles (EVs) derived from mesenchymal stem or stromal cells (MSCs) have shown therapeutic potential for acute respiratory distress syndrome (ARDS), yet reproducible and scalable methods to enhance EV yield and therapeutic potency remain limited. Here, we describe a biophysical priming strategy using pulsed focused ultrasound (pFUS) to apply controlled mechanical stimulation to umbilical cord (UC)-derived MSCs. A single, 1-min exposure generated primed EVs (pUC-EVs) with a 1.9-fold increase in concentration and a therapeutically enhanced molecular cargo, without changes in vesicle size distribution. We demonstrate that this process is mechanistically dependent on calcium influx, as calcium chelation with EGTA markedly reduced pFUS‑induced enhancement of EV secretion. Transcriptomic profiling of UC-MSCs indicated that pFUS activates calcium-dependent mechanotransduction pathways associated with endosomal sorting complexes required for transport (ESCRT) machinery, leading to coordinated upregulation of ESCRT components, vesicle trafficking regulators, while proteomics analysis of UC-EVs revealed enrichment of mitochondrial proteins that support oxidative phosphorylation and ATP generation. Functionally, pUC-EVs exhibit enhanced uptake by macrophages and alveolar epithelial cells, where they increase ATP levels, improve mitochondrial respiration, and promote a reparative, anti-inflammatory macrophage phenotype in vitro. In a lipopolysaccharide-induced juvenile ARDS model, intratracheal delivery of pUC-EVs localized treatment to the injured lung and significantly improved epithelial-endothelial barrier integrity, as shown by reduced vascular leakage, bronchoalveolar lavage protein content, inflammatory mediators, and neutrophil infiltration, alongside improved histological preservation compared with unprimed UC-EVs. A comprehensive in vivo safety assessment demonstrated that pUC-EVs exhibited significantly reduced serum IgG binding compared to UC-EVs, maintained normal coagulation parameter, and preserved organ function (liver, kidney, spleen) with no adverse histopathological changes, supporting a favorable biocompatibility profile. Together, these findings identify mechanopriming by pFUS as a mechanistically defined, well‑tolerated, and scalable approach to enhance EV biogenesis and therapeutic function, supporting the potential application of EVs as a therapy for ARDS and related inflammatory lung diseases.