Strawberry-Derived Exosome-Like Nanoparticles Prevent Oxidative Stress in Human Mesenchymal Stromal Cells

Abstract

Plant-derived exosome-like nanovesicles (EPDENs) have recently been isolated and evaluated as potential bioactive nutraceutical biomolecules. It has been hypothesized that EPDENs may exert their activity on mammalian cells through their specific cargo. In this study, we isolated and purified EPDENs from the strawberry juice of Fragaria x ananassa (cv. Romina), a new cultivar characterized by a high content of anthocyanins, folic acid, flavonols, and vitamin C and an elevated antioxidant capacity. Fragaria-derived EPDENs were purified by a series of centrifugation and filtration steps. EPDENs showed size and morphology similar to mammalian extracellular nanovesicles. The internalization of Fragaria-derived EPDENs by human mesenchymal stromal cells (MSCs) did not negatively affect their viability, and the pretreatment of MSCs with Fragaria-derived EPDENs prevented oxidative stress in a dose-dependent manner. This is possibly due to the presence of vitamin C inside the nanovesicle membrane. The analysis of EPDEN cargo also revealed the presence of small RNAs and miRNAs. These findings suggest that Fragaria-derived EPDENs may be considered nanoshuttles contained in food, with potential health-promoting activity.

Keywords: Fragaria x ananassa; ascorbic acid; edible plant-derived exosome-like nanoparticles (EPDENs); extracellular vesicles (EVs); mesenchymal stromal cells; miRNA; oxidative stress.

Figure 1

Isolation and characterization of Fragaria-derived exosome-like nanovesicles (EPDENs). (a) Schematic representation of the method used to isolate and purify EPDENs from Fragaria x ananassa. Transmission electron microscopy analysis of nanovesicles isolated from (b) Fragaria x ananassa, (c) Citrus limon L., and (d) adipose-derived mesenchymal stem cells (ADMSCs) (scale bar: 100 nm).

Figure 2

Effects of Fragaria-derived EPDENs on ADMSCs. (a) Uptake of Fragaria-derived EPDENs by ADMSCs. The uptake of the fluorescently labeled EPDENs (red) was evident in ADMSCs after 24 h of incubation. No stain was revealed in the untreated cells (CTR). Actin filaments were stained with a FITC-conjugated phalloidin (green). Scale bar: 10 µm. (b) Effect of Fragaria-derived EPDENs on ADMSC viability. Cells were treated with the indicated concentration of EPDENs for 48 h and 120 h. Data are reported as the cell number (mean ± SD, * p < 0.05).

Figure 3

Effect of Fragaria-derived EPDENs on oxidative stress in human ADMSCs. (a) Dose-dependent effect of H₂O₂ on cell viability (n = 3) (*** p < 0.001 vs. CTR); (b) Dose-dependent effect of Fragaria-derived EPDENs (µg/mL) on H₂O₂ (400 µM)-induced cytotoxicity (n = 4) (*** p < 0.001).

Figure 4

Effect of Fragaria-derived EPDENs on ROS production in human ADMSC. (A) Dose-dependent effect of H₂O₂ on ROS production (* p < 0.05 vs. CTR and ** p < 0.01 vs. CTR); (B) Effect of Fragaria-derived EPDENs (0.5 µg/mL) on H₂O₂ (600 µM)-induced ROS production (* p < 0.05 vs. H₂O₂).

Figure 5

Size distribution of small RNAs of (a) Fragaria-derived EPDENs with respect to (b) Fragaria whole juice. GC frequency of sequences of (c) Fragaria-derived EPDENs with respect to (d) Fragaria whole juice.