Specialized Pro-Resolving Mediator Loaded Extracellular Vesicles Mitigate Pulmonary Inflammation

Extracellular vesicles (EVs) have emerged as versatile carriers of therapeutic cargo, including nucleic acids, proteins, and small molecules. However, their potential to deliver bioactive lipid mediators remains largely unexplored. Here, we present a novel synthetic biology-based strategy to selectively load EVs with pro-resolving lipid mediators of the Resolvin D and E series by co-expressing the resolvin biosynthetic enzymes cyclooxygenase 2 (COX-2), 5-lipoxygenase (5-LOX), and 15-lipoxygenase (15-LOX) using a custom-designed multigene expression vector. Human embryonic kidney 293T (HEK293T) cells transfected with the multigene expression vector and cultured in the presence of fatty acid free bovine serum albumen (BSA)-complexed docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), and aspirin produced multiple members of the Resolvin D, aspirin-triggered Resolvin D series, and Resolvin E1 and E2, along with their biosynthetic precursors, which were subsequently packaged into EVs (referred to as Resolvin-EVs). Resolvin-EVs attenuated neutrophil adhesion to endothelial cells both under static and flow conditions and preserved endothelial barrier integrity by upregulating VE-cadherin. In macrophages, Resolvin-EVs suppressed nuclear factor kappa B (NF-κB) reporter activity, release of IL6 and TNFα. Effects of Resolvin-EVs on endothelial permeability and macrophage activation were abrogated by pharmacological inhibition of EV uptake using nystatin and cytochalasin D. Furthermore, Resolvin-EVs enhanced efferocytosis in THP-1 derived macrophages compared to Control-EVs. Notably, post-injury administration of Resolvin-EVs attenuated pulmonary inflammation in LPS-treated mice without inducing systemic or pulmonary toxicity. Together, these findings establish a novel, scalable platform for generating Resolvin-loaded EVs and highlight their therapeutic potential for acute lung injury and other chronic inflammatory disorders.