Targeting NETosis in the Lung: Respiratory Delivery of Neutrophil-Specific NEBP-Liposomes for Enhanced Therapy of Acute Lung Injury.

The activation, adhesion, and migration of neutrophils are one of the main reasons for the progression of acute lung injury (ALI) and a new therapeutic target for ALI. Although respiratory administration for ALI has the characteristics of targeted drug delivery, some issues, such as the drug's short residence time in the lungs, rapid clearance rate, and lack of specificity for neutrophils, make it difficult to maintain an effective drug concentration. Here, we designed and evaluated a neutrophil-targeting peptide NEBP-modified andrographolide liposome (N-Lip@A) that can be administered via the respiratory tract to inhibit the formation of neutrophil extracellular traps, aiming to provide a rapid, efficient, and precise intervention strategy for ALI. N-Lip@A liposomes were synthesized by the traditional thin-film hydration method. Cell immunofluorescence and scanning electron microscopy observations indicated that N-Lip@A could reduce the formation of neutrophil extracellular traps (NETs). Quantitative analysis using the PicoGreen assay showed that the release of double-stranded DNA (dsDNA) in the N-Lip@A group was reduced to 57.9% compared with the untreated PMA group, and its effect was superior to that of the nontargeted liposome formulation (Lip@A). SYTOX staining, CCK8 assay, and Calcein-AM/PI staining results demonstrated that N-Lip@A could reduce the death of PMA-stimulated neutrophils. Besides, compared with the untreated group, the supernatant from neutrophils pretreated with N-Lip@A increased the survival rate of alveolar epithelial MLE-12 cells. In a lipopolysaccharide-induced murine model of ALI, compared with the nontargeted Lip@A group or the untreated group, the pathological lung injury score decreased to 52.6% and 31.6%, respectively. Significant disease remission, including reduced pulmonary edema, restored integrity of the blood-air barrier, and improved alveolar architecture were also demonstrated through EB staining analysis, lung wet-to-dry weight ratio determination, and HE staining analysis. Collectively, N-Lip@A enables precise delivery of AGL to activated neutrophils via NEBP-mediated targeting, effectively inhibiting NETs formation and mitigating ALI-associated pathological damage. This strategy provides an expandable targeted drug delivery platform for the treatment of various acute or chronic pulmonary inflammatory diseases driven mainly by neutrophils.