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.

中文翻译：

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由活化的中性粒细胞和丰富的杀菌蛋白组成的工程纳米囊泡具有分子清创能力并促进感染性伤口愈合


背景 细菌感染对皮肤伤口构成相当大的威胁，尤其是在难以治疗的糖尿病伤口的情况下。全身性抗生素通常难以渗透到深层伤口组织，局部应用抗生素可能导致致敏，因此需要开发有效治疗深层伤口组织中细菌的新方法。中性粒细胞是血液中的主要免疫细胞，在激活后通过脱颗粒迅速释放出大量分子，具有直接消除病原体的能力。本研究旨在开发高产的新型中性粒细胞工程纳米囊泡 （NVs），并探索其杀菌特性和促进感染性伤口愈合的应用。方法 从外周血中分离中性粒细胞，并通过佛波肉豆蔻酸酯 （PMA） 刺激在体外激活。通过依次挤出活化的嗜中性粒细胞，然后超速离心制备工程化 NVs，并在形态、大小分布和蛋白质含量方面与嗜中性粒细胞衍生的外泌体进行比较。采用展板技术、LIVE/DEAD 背光细菌测定和细菌形态观察评价 NVs 的体外杀菌效果。使用伤口收缩面积测量、组织病理学检查、炎症因子评估和免疫化学染色评估 NVs 在体内的治疗效果。结果 体外 PMA 刺激的活化中性粒细胞迅速释放大量杀菌蛋白。NV 在形态和粒径方面与外泌体相似，但它们表现出显着更高的杀菌蛋白富集度。 在体外，NVs 显示出显着的杀菌作用，可能是由溶菌酶等杀菌蛋白的富集介导的。这些 NVs 显着加速伤口愈合，导致全层感染性皮肤缺损模型中细菌载量显着降低、炎症因子下调和胶原蛋白沉积增强。结论 我们开发了源自活化中性粒细胞的工程化 NVs，作为一种针对深部组织中细菌的新型清创方法，最终促进感染性伤口愈合。