Cathelicidin LL37-loaded extracellular vesicles from Edwardsiella piscicida promote antibacterial and wound-healing activity

Bacterial extracellular vesicles (BEVs) have emerged as a promising therapeutic platform, offering the potential to be engineered for targeted molecular delivery. In this study, the human cathelicidin antimicrobial peptide (LL37) was encapsulated into extracellular vesicles derived from the fish pathogen Edwardsiella piscicida (EpEVs) through co-incubation. LL37-encapsulated EpEVs (EpEVs-LL37) were isolated by ultracentrifugation and characterized with a mean particle size of 73.6 ± 1.4 nm and a zeta potential of -11.27 ± 0.49 mV. Morphology of EpEVs-LL37 was confirmed as spherical-shaped particles. Enzymatic stability evaluation results revealed a proportionate increase in mean size and zeta potential with the pepsin concentration (0.4-2 mg/mL) at pH 2.0. EpEVs-LL37 exhibited lower toxicity than free LL37 in both Raw 264.7 cells and zebrafish larvae, while maintaining a similar level of cellular internalization as naïve EpEVs. Notably, EpEVs-LL37 demonstrated enhanced antibacterial activity against E. piscicida compared to free LL37, as evidenced by time-kill kinetics and bacterial viability assays. Mechanistically, EpEVs-LL37 damaged the bacterial cell membrane and induced higher levels of reactive oxygen species (ROS), along with bacterial membrane permeability, as observed through confocal microscopy. Additionally, EpEVs-LL37 upregulated the expression of immunomodulatory genes (Tlr2, Il1β, Il10) in both Raw 264.7 cells and zebrafish larvae. Furthermore, treatment with EpEVs-LL37 (10 µg/mL) significantly enhanced wound-healing in vitro and in vivo, reducing the cellular wound area to 11.01 ± 0.81% and increasing larval fin regeneration to 1.64 ± 0.07 mm², both outperforming free LL37. Collectively, these findings highlight EpEVs-LL37 as a promising therapeutic and drug-delivery platform for the treatment of E. piscicida-associated infections.