Isolation, extraction, and physicochemical characterization of exosome-like nanovesicles from ginseng adventitious roots in tissue culture

This study aimed to establish a method for isolating exosome-like nanovesicles from ginseng adventitious roots and their culture supernatant, to investigate the effects of methyl jasmonate (MeJA) induction on vesicle yield and composition, and to provide experimental evidence for the scalable production of ginseng-derived exosome-like nanovesicles. Exosome-like nanovesicles were isolated from ginseng adventitious roots and culture supernatant using low-speed centrifugation combined with stirred-cell ultrafiltration. Physicochemical characterization was performed using transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), Zeta potential analysis, and Q-ToF LC-MS lipidomics. Ginseng adventitious roots were treated with 200 M MeJA to evaluate changes in vesicle particle concentration, total protein, total saponins, and miRNA expression before and after induction. Paraffin section fluorescence staining was used to observe the distribution of vesicle-related signals in adventitious root tissues. Exosome-like nanovesicles with typical bilayer membrane structures were isolated from both ginseng adventitious roots and culture supernatant.The particle size ranged from 77.333 to 171.733 nm, and the particle concentration ranged from 2.1E+8 to 7.6E+10 /mL. The vesicles contained lipid components including glycerides, phospholipids, and sphingolipids. Induction with 200 M MeJA increased vesicle particle concentration and total protein content, significantly upregulated the total saponin level in vesicles derived from adventitious roots, and altered the miRNA expression pattern of the vesicles. Tissue section fluorescence observation showed overlap between vacuolar markers and membrane vesicle dye signals. Low-speed centrifugation combined with ultrafiltration can effectively prepare ginseng-derived exosome-like nanovesicles. MeJA induction can regulate vesicle yield and active components, providing foundational data for the scalable process development of such vesicles.