Targeted delivery of peptidoglycan hydrolases (PGH) via engineered extracellular vesicles (EVs) to clear intracellular bacterial infections.

Bacterial infectious diseases remain a major global health threat due to increasing antibiotic resistance. Further complicating effective treatment, some pathogens such as Staphylococcus aureus persist within host cells. Peptidoglycan hydrolases (PGHs), including bacteriophage-derived endolysins, represent a promising class of novel antimicrobials due to their rapid bacteriolytic activity and low risk of resistance emergence. However, their clinical application is limited by an unfavourable PK profile and inefficient delivery into infected host cells. Here, we engineered extracellular vesicles (EVs) for the targeted, intracellular delivery of GH15, a novel endolysin against S. aureus. GH15-loaded EVs, functionalized with antibodies targeting αvβ3 integrin on bacterially infected cells, efficiently delivered GH15 into S. aureus-positive endothelial cells and macrophages in vitro and eliminated intracellular bacteria. In addition, GH15-loaded EVs promoted the clearance of intracellular bacteria in macrophages of infected zebrafish larvae in vivo. Our findings establish EVs as effective vehicles for intracellular delivery of antimicrobial enzymes and highlight their potential for the treatment of intracellular bacterial infections.