Exosome-like Nanoparticles, High in Trans-δ-Viniferin Derivatives, Produced from Grape Cell Cultures: Preparation, Characterization, and Anticancer Properties

Abstract

Background: Recent interest in plant-derived exosome-like nanoparticles (ENs) has surged due to their therapeutic potential, which includes antioxidant, anti-inflammatory, and anticancer activities. These properties are attributed to their cargo of bioactive metabolites and other endogenous molecules. However, the properties of ENs isolated from plant cell cultures remain less explored. Methods: In this investigation, grape callus-derived ENs (GCENs) were isolated using differential ultracentrifugation techniques. Structural analysis through electron microscopy, nanoparticle tracking analysis, and western blotting confirmed that GCENs qualify as exosome-like nanovesicles. Results: These GCENs contained significant amounts of microRNAs and proteins characteristic of plant-derived ENs, as well as trans-δ-viniferin, a notable stilbenoid known for its health-promoting properties. Functional assays revealed that the GCENs reduced the viability of the triple-negative breast cancer cell line MDA-MB-231 in a dose-dependent manner. Moreover, the GCENs exhibited negligible effects on the viability of normal human embryonic kidney (HEK) 293 cells, indicating selective cytotoxicity. Notably, treatment with these GCENs led to cell cycle arrest in the G1 phase and triggered apoptosis in the MDA-MB-231 cell line. Conclusions: Overall, this study underscores the potential of grape callus-derived nanovectors as natural carriers of stilbenoids and proposes their application as a novel and effective approach in the management of cancer.

Keywords: Vitis vinifera; callus culture; cancer; extracellular vesicles; salicylic acid.

Figure 1

Scanning electron microscopy images and nanoparticle tracking analysis of GCENs (A) and GENs (B). The presence of HSP70 and TET8 proteins in ENs are shown in a western blot (C).

Figure 2

Expression levels of VvTET8 and VvPEN1 genes in grape berries and callus cultures (A). Biomass accumulation (B), and GCEN concentration (C) in grape calluses grown under control conditions and with 200 µM of salicylic acid (SA). Nanoparticle tracking analysis of GCENs isolated from control (D) and SA-treated calluses (E). Data shown are mean ± SE; n = 3. Different letters in the heatmap and asterisks denote significant differences at p < 0.05 (*) with Fisher’s LSD.

Figure 3

Expression levels of exosomal miRNA in GCENs and GENs. Data shown are mean ± SE; n = 3. Asterisks denote significant differences at p < 0.05 (*) with Student’s t-test.

Figure 4

HPLC–UV (320 nm) profiling of stilbene derivatives from crude GCEN extracts, with peak numbers corresponding to the compounds listed in Table 3 and Supplementary Table S3. A schematic representation of the structure of trans-δ-viniferin (compound 6) is also included.

Figure 5

Confocal fluorescence imaging of GCENs labeled with PKH26, showing internalization in MDA-MB-231 cells after 4 and 24 h of incubation (A). The merged images display three channels: blue—DAPI stain for nuclei (excitation at 405 nm), red—PKH26 stain for exosomes (excitation at 543 nm), and transmitted light detection for a common view. Merged images from two channels show only the DAPI stain (blue) and PKH26 stain (red) for clarity. Light microscopy imaging of MDA-MB-231 cells illustrating cell morphology at different concentrations of GCENs (B), and the cytotoxic effect of GCENs on MDA-MB-231 and HEK-293 cells assessed by the MTT assay (C). Scale bar: 20 µm.

Figure 6

Analysis of cell death induced by GCENs in MDA-MB-231 cells. Flow cytometric analysis of apoptotic cell death using the fluorescent dye Annexin V-AF 488 (A,B) and a Caspase 3/7 activation assay (C,D) were performed after 24 h. Samples without EN treatment served as controls in each case.

Figure 7

Flow cytometric analysis of MDA-MB-231 cell cycle arrest induced by GCEN treatment after 24 h (A), and quantitative results of cell cycle distribution (B). A sample without EN treatment served as the control. Green denotes the G1 phase, yellow denotes the S phase, and blue denotes the G2/M phase.