Plasminogen–cyclodextrin aerosol for ARDS: activity retention in simulated oxygen therapy and inflammation-triggered clot lysis

Acute Respiratory Distress Syndrome (ARDS) involves intense pulmonary inflammation, endothelial damage, and fibrin accumulation, often requiring oxygen therapy. Plasminogen (PLG), a fibrinolytic zymogen, has therapeutic potential for resolving fibrin deposits in the lungs but is vulnerable to oxidative degradation during aerosolization. This study presents a clinically feasible inhalable formulation of PLG complexed with hydroxypropyl-β-cyclodextrin (HP-β-CD), intended to preserve enzymatic function during nebulization under oxygen-rich conditions. Using a ready-to-use eye-drop solution (PLG-OMP), the formulation is repurposed for off-label inhalable administration and prepared with minimal handling suitable for hospital pharmacy protocols. Spectroscopic analyses confirmed complexation, and FT-IR demonstrated HP-β-CD’s protective effect against methionine oxidation. Consistently, computer-based investigation at oxidation-relevant sites revealed binding of HP-β-CD close to methionine residues, supporting the spectroscopic evidence of a shielding effect. Aerodynamic evaluation via mesh nebulization showed favorable lung deposition profiles (MMAD ∼ 2.1 μm, FPF ∼ 84 %). Enzymatic activity post-nebulization in oxygen flow remained > 95 % when complexed with HP-β-CD, compared to ∼ 57 % for unprotected PLG. In vitro lysis of human clots was confirmed in both urokinase-triggered and cell-activated degradation models. The latter employed a cell-based model wherein LPS-stimulated macrophages-triggered lysis of human clots through inflammation-induced activation of nebulised PLG. D-dimer quantification verified consistent fibrinolytic performance across both models. These results establish a robust foundation for targeted fibrinolytic therapy in ARDS, combining formulation simplicity with biological relevance to support clinical translation