Potential of Plant Exosome Vesicles from Grapefruit (Citrus × paradisi) and Tomato (Solanum lycopersicum) Juices as Functional Ingredients and Targeted Drug Delivery Vehicles

Abstract

Plant-derived extracellular vesicles (PEVs) have gained attention as promising bioactive nutraceutical molecules; their presence in common fruit juices has increased their significance because human interaction is inevitable. The goal of this study was to evaluate the potential of PEVs derived from grapefruit and tomato juices as functional ingredients, antioxidant compounds, and delivery vehicles. PEVs were isolated using differential ultracentrifugation and were found to be similar in size and morphology to mammalian exosomes. The yield of grapefruit exosome-like vesicles (GEVs) was higher than that of tomato exosome-like vesicles (TEVs), despite the latter having larger vesicle sizes. Furthermore, the antioxidant activity of GEVs and TEVs was found to be low in comparison to their juice sources, indicating a limited contribution of PEVs to the juice. GEVs showed a higher efficiency in being loaded with the heat shock protein 70 (HSP70) than TEVs, as well as a higher efficiency than TEV and PEV-free HSP70 in delivering HSP70 to glioma cells. Overall, our results revealed that GEVs present a higher potential as functional ingredients present in juice and that they exert the potential to deliver functional molecules to human cells. Although PEVs showed low antioxidant activity, their role in oxidative response in cells should be further addressed.

Keywords: antioxidant activity; drug delivery; fruit juices; grapefruit exosomes; plant exosomes; tomato exosomes.

Figure 1

Characterization and particle size of grapefruit exosome vesicles (GEVs) and tomato exosome vesicles (TEVs). Nanoparticle tracking analysis (NTA) of (A) concentration as well as (B) hydrodynamic size and size mode of GEVs and TEVs. GEVs showed a higher hydrodynamic size and mode distribution than TEVs (p < 0.05 with unpaired t-student test, n = 3). Representative Dynamic light scattering (DLS) vesicle size distributions of GEVs and TEVs determined by (C) Photocor Compact-Z and (D) Brookhaven Mod 90 Plus systems. (E) Resume of size distribution of GEVs and TEVs by NTA, DLS, and atomic force microscopy (AFM). Overall, TEVs show a higher size distribution than GEVs (p < 0.05 with unpaired t-student test, n = 3). (F) Z-potential of GEVs and TEVs. TEVs show a minor Z-potential than GEVs by Photocor Compact-Z system (p < 0.01 with unpaired t-student test, n = 3).

Figure 2

Atomic force microscopy (AFM) and field emission scanning electron microscope (FESEM) micrographs of grapefruit exosome vesicles (GEVs) and tomato exosome vesicles (TEVs). AFM topography scan (left) and cross section (right) of (A) GEVs and (B) TEVs. On the right of topography scans is the pseudo color ruler indicating the particles’ height (nm). FESEM micrographs of (C) GEVs and (D) TEVs. A representative group of vesicles has been magnified.

Figure 3

Antioxidant activity by (A,C) DPPH and (B,D) ABTS radicals assays of (A,B) tomato exosome vesicles (TEVs) and (C,D) grapefruit exosome vesicles (GEVs). Overall, pure GEVs and TEVs showed a lower antioxidant activity than tomato (TJ) and grapefruit juices (GJ) (*** p < 0.001, ** p < 0.01, * p < 0.05, ns p > 0.05; One-way ANOVA with Tukey’s post hoc test, n = 3). Results are expressed as mg of Trolox equivalent (TE) per mL of sample ± standard deviation (SD). SD < 0.01 were not plotted.

Figure 4

Grapefruit exosome-like vesicles (GEVs) and tomato exosome-like vesicles (TEVs) mediated the delivery efficiency of exogenous HSP-70 proteins to human cells. (A) Fluorescence of Alexa Fluor 647 (AF647) labeled-HSP70 in the initial mixture and loaded samples of GEVs and TEVs, as well as washing filtrates (F1 and F10). After loading, excess free proteins were washed out, as shown from F1 to F10. Similar to TEVs, GEVs showed a loading efficiency of 1.10% when compared to the initial amount added to the vesicle suspension (sonicated mixture) (*** p < 0.001; One-way ANOVA with Dunnett’s post hoc test, n = 3). (B) Fluorescence intensity of the uptake of GEVs and TEVs loaded with HSP70-AF647 by glioma (Gl-Tr) cells. (C) Delivery efficiency of protein to recipient cells by GEVs or TEVs analyzed by flow cytometry. Fluorescence signal accumulation into Gl-Tr cells was higher by loaded GEVs than the control (untreated cells) as well as 2% of free protein and TEVs (*** p < 0.001, ** p < 0.01, * p < 0.05; One-way ANOVA with Dunnett’s post hoc test, n = 3). (D) Cytotoxicity of grapefruit or tomato vesicles loaded with recombinant HSP70 for Gl-Tr glioma cells. The cytotoxic effects have been studied by the AlamarBlue cell viability assay after 48 h of incubation. No cytotoxic effect was determined when treatments were compared to untreated cells (Control) (p > 0.05; One-way ANOVA with Dunnett’s post hoc test). (E) Cell index in real-time after treatment of Gl-Tr glioma cells with HSP70, GEVs, or GEVs loaded with HSP70. Control cells were incubated with culture medium only. The average of two replicates is shown for each condition. Error bars represent standard deviations.