Plant hairy roots for the production of extracellular vesicles with antitumor bioactivity

Abstract

Plant extracellular vesicles (EVs) concentrate and deliver different types of bioactive molecules in human cells and are excellent candidates for a next-generation drug delivery system. However, the lack of standard protocols for plant EV production and the natural variations of their biomolecular cargo pose serious limitation to their use as therapeutics. To overcome these issues, we set up a versatile and standardized procedure to purify plant EVs from hairy root (HR) cultures, a versatile biotechnological system, already successfully employed as source of bioactive molecules with pharmaceutical and nutraceutical relevance. Herewith, we report that HR of Salvia dominica represent an excellent platform for the production of plant EVs. In particular, EVs derived from S. dominica HRs are small round-shaped vesicles carrying typical EV-associated proteins such as cytoskeletal components, chaperon proteins and integral membrane proteins including the tetraspanin TET-7. Interestingly, the HR-derived EVs showed selective and strong pro-apoptotic activity in pancreatic and mammary cancer cells. These results reveal that plant hairy roots may be considered a new promising tool in plant biotechnology for the production of extracellular vesicles for human health.

Fig. 1. Experimental design for the production and characterization of S. dominica hairy root derived EVs.

a Plantlets of Salvia dominica grown in vitro. b Sterile leaf sections infected by A. rhizogenes. c Hairy roots (HRs) development from infected leaves. d Subcultures of excised hairy roots in solid hormone free medium. e HR in the hormone-free liquid MS medium. f Stable integration of the rolB gene in HRs assessed by polymerase chain reaction (PCR) in three independent transformation events. Amplicon length: 451 base pairs (bp) and 142 bp for rolB and actin genes, respectively. NTC, no template control. g Workflow planned for the purification and characterization steps of extracellular vesicles released by S. dominica HRs.

Fig. 2. Biophysical characterization of HR-derived EVs.

a EVs size distribution curve obtained by dynamic light scattering (DLS). b Nanoparticle Tracking Analysis (NTA) measurements show the distribution of the EV size with the major peak at 151 nm. c TEM image of isolated vesicles exhibiting round-shaped morphology. d, e Close-up view images of individual HR-derived EVs. f Protein pattern of HR-derived EVs and EV depleted-medium (EDM) visualized by SDS-PAGE and silver staining. Silver staining of EDM did not show detectable proteins. Scale bars: 500 nm in c, 50 nm in d, 50 nm in e.

Fig. 3. Uptake and biological effects of HR-derived EVs in human control and cancer cell lines.

a–d Confocal analysis of HaCaT and MIA PaCa-2 cells treated with Bodipy-stained EVs (Fluo-EVs) (1.2 × 10⁸ particles/mL) for 24 h. Cells have been stained for annexin A1 protein in red. Nuclei were stained with DAPI. Magnification 63×/1.4 numerical aperture. Scale bar = 100 μm. e, f MTT colorimetric assay on HaCaT and MIA PaCa-2 cells after 24 h and 48 h of EV treatments, respectively. Absorbance relative to controls was used to determine the percentage of cells treated with varying concentrations of gemcitabine and EVs. g, h Analyses of apoptotic cells by cytofluorimetric assay in gemcitabine- and EVs-treated MIA PaCa-2 cells upon 24 h and 48 h exposure. The values reported in the graphs are the mean ± SD from at least 3 independent experiments performed in technical triplicates. The asterisks denote significant differences between treatments and untreated controls (*p < 0.05; **p < 0.01; ***p < 0.001) according to Student’s t-test. i Western blot analyses of protein extracts from cells treated for 24 h with different concentrations of gemcitabine and EVs. Levels of cleaved proteins involved in the apoptosis (Procaspase 3 and PARP-1) were evaluated. β−actin was used to check equal loading of protein extracts.