Orange-Derived Extracellular Vesicles: Characterization and Therapeutic Applications in Normal and Diabetic Wound Healing in In Vivo Models

Extracellular vesicles (EVs) of human origin show promise for regenerative medicine and wound healing. However, they have limitations regarding scalability, variability, safety, and costs. Plant-derived EVs may represent a valid alternative. This study investigated the regenerative potential of EVs extracted from orange juice (oEVs). oEVs obtained by standard ultracentrifugation were compared with oEVs purified by tangential flow filtration (TFF), a scalable technique suitable for large-scale and regulatory-compliant manufacturing. Comparisons included size, morphology, pH, Zeta potential, protein and RNA content, Raman spectroscopy, and proteomic, metabolomic, and RNA sequencing. The regenerative potential of oEVs was tested in vitro, with cell migration, endothelial tube formation, and proliferation assays performed. Antioxidant ability was tested on endothelial cells stressed by hyperglycemia or pro-inflammatory cytokine cocktails. Next, oEVs were formulated with different hydrogels and tested at different doses on skin ulcers on healthy and diabetic mice. TFF oEVs showed the same physio-chemical characteristics and a comparable molecular content as those isolated by ultracentrifugation, confirming the path to scalability. In vitro oEVs promoted cell migration, formation of capillary-like structures, cell proliferation, and strong antioxidant activity. Moreover, oEVs effectively accelerated in vivo wound closure in healthy and diabetic mice. Thus, oEVs may provide a useful and cost-effective ingredient for improved and effective wound treatment strategies.