Engineered nanovesicles from activated neutrophils with enriched bactericidal proteins have molecular debridement ability and promote infectious wound healing.

ABSTRACT

Background: Bacterial infections pose a considerable threat to skin wounds, particularly in the case of challenging-to-treat diabetic wounds. Systemic antibiotics often struggle to penetrate deep wound tissues and topically applied antibiotics may lead to sensitization, necessitating the development of novel approaches for effectively treating germs in deep wound tissues. Neutrophils, the predominant immune cells in the bloodstream, rapidly release an abundance of molecules via degranulation upon activation, which possess the ability to directly eliminate pathogens. This study was designed to develop novel neutrophil cell engineered nanovesicles (NVs) with high production and explore their bactericidal properties and application in promoting infectious wound healing. Methods: Neutrophils were isolated from peripheral blood and activated in vitro via phorbol myristate acetate (PMA) stimulation. Engineered NVs were prepared by sequentially extruding activated neutrophils followed by ultracentrifugation and were compared with neutrophil-derived exosomes in terms of morphology, size distribution and protein contents. The bactericidal effect of NVs in vitro was evaluated using the spread plate technique, LIVE/DEAD backlight bacteria assay and observation of bacterial morphology. The therapeutic effects of NVs in vivo were evaluated using wound contraction area measurements, histopathological examinations, assessments of inflammatory factors and immunochemical staining. Results: Activated neutrophils stimulated with PMA in vitro promptly release a substantial amount of bactericidal proteins. NVs are similar to exosomes in terms of morphology and particle size, but they exhibit a significantly higher enrichment of bactericidal proteins. In vitro, NVs demonstrated a significant bactericidal effect, presumably mediated by the enrichment of bactericidal proteins such as lysozyme. These NVs significantly accelerated wound healing, leading to a marked reduction in bacterial load, downregulation of inflammatory factors and enhanced collagen deposition in a full-thickness infectious skin defect model. Conclusions: We developed engineered NVs derived from activated neutrophils to serve as a novel debridement method targeting bacteria in deep tissues, ultimately promoting infectious wound healing.