Abstract: BACKGROUND: Nanocell vesicles possess functions such as re-epithelialization, antioxidation, anti-inflammation, and regulation of extracellular matrix remodeling. Meanwhile, apoptotic bodies have the immunomodulatory effects. Therefore, the combination of the two to form nanofusion vesicles can synergistically promote the healing of diabetic skin wounds. 
OBJECTIVE: To elucidate the impact of nanofusion vesicles on skin wound healing in a diabetic murine model.
METHODS: (1) Material preparation and characterization: The primary bone marrow mesenchymal stem cells of C57BL/6J neonatal mice and the neutrophil apoptotic bodies of C57BL/6J mice were isolated and extracted. The nanofusion vesicles were prepared by micro-extrusion mechanism. (2) In vitro experiment: MTT assay was used to detect the proliferative effect of different concentrations of nanofusion vesicles on NIH-3T3 cells and human umbilical vein endothelial cells. Reactive oxygen species fluorescence probe was used to detect the antioxidant effect of nano-fusion vesicles on NIH-3T3 cells treated with hydrogen peroxide (H2O2). The inhibitory effect of nanofusion vesicles on RAW 264.7 macrophage inflammation induced by lipopolyside was detected by real-time quantitative RT-qPCR. (3) In vivo experiment: 36 male C57BL/6J mice were employed to develop a murine model of diabetes mellitus. Following the successful induction of diabetes, two circular full-thickness wounds, each with a diameter of 6 mm, were created on either side of the diabetic mice’s spine using a skin punch. The mice were divided into three groups by random number table method. The control group was injected with 0.1 mL of phosphate buffer solution. The nanovesicle group was injected with 0.1 mL nanovesicles (25 μg/mL). The nanofusion vesicle group was injected with 0.1 mL nanofusion (25 μg/mL) vesicles. After treatment for three consecutive days, the wound healing and histomorphological changes were observed.
RESULTS AND CONCLUSION: (1) In vitro experiment: nanofusion vesicles, when administered at concentrations ranging from 0 to 100 μg/mL, exhibited no toxic effects and promoted the proliferation of NIH-3T3 and HUVEC cell lines. Notably, a concentration of 25 μg/mL nanofusion vesicle significantly enhanced the proliferation of NIH-3T3 cells. Furthermore, the survival rate of human umbilical vein endothelial cells was observed to increase in correlation with escalating concentrations of nanofusion vesicles. Nanofusion vesicles had a good antioxidant effect. In comparison to the H2O2 group, the fluorescence signal indicative of reactive oxygen species was progressively diminished in both the nanovesicle group and the nanofusion vesicle group. Furthermore, nanofusion vesicles possessed anti-inflammatory capabilities, effectively mitigating the inflammatory response in macrophages triggered by lipopolysaccharide stimulation. (2) In vivo experiment: Hematoxylin-eosin and Masson's trichrome staining revealed that in comparison to the control group, both the nanovesicle group and the nanofusion vesicle group exhibited a significant increase in granulation tissue formation and collagen fiber deposition within the wounds by day 6. Notably, the nanofusion vesicle group displayed the most pronounced effects. On day 12, the wound of nanofusion vesicle group was significantly reduced, and the healing rate was significantly faster than that of other groups (P < 0.01), and the effect of promoting wound healing was the most significant. Our findings demonstrated that nanofusion vesicles exhibited superior pro-cell proliferative, antioxidant, and anti-inflammatory properties, thereby exerting a beneficial effect on the promotion of skin wound healing in diabetic mouse models.

Key words: bone marrow mesenchymal stem cell, neutrophil, apoptotic body, nanofusion vesicle, anti-oxidation, anti-inflammatory, diabetes, skin wound