Biological Activities of Citrus-Derived Extracellular Vesicles on Human Cells: The Role of Preservation

Extracellular vesicles (EVs) have been identified as important mediators for cell-to-cell communication. Citrus-based EVs in particular offer an excellent platform for nutraceutical delivery systems, as their endemic cargo includes micronutrients (e.g., ascorbic acid), which contribute to their antioxidant capacity. Despite being extensively investigated as to their therapeutic and diagnostic potential, their cargo is inherently unstable and thus directly affected by their storage and preservation. In this study, EVs were isolated from citrus fruit using tangential flow filtration and evaluated for their physicochemical characteristics, antioxidant activity and effects on human cells. To assess how their isolation and preservation methods affect these properties, the EVs were tested immediately after isolation (from fresh and freeze-thawed juices) or following freeze-drying. A measurable biological effect of cryoprotection on citrus-derived EVs was evident, whether during or after isolation. This was more pronounced in the cell-based assays, ranging from -4% to +32% in human skin fibroblast proliferation. Nevertheless, the effects on human Cancer cells varied depending on the cell line. Although these results should be considered preliminary observations, subject to further investigation, it is safe to state that any type of preservation is expected to impact the EVs' biological activity.

Akt phosphorylation; antioxidant activity; cell proliferation; freeze-dried EVs; freeze-thawed EVs; fresh EVs; tangential flow filtration.