Abstract
Plant-derived extracellular vesicles (PEVs) have emerged as a promising area of research in biotechnology with enormous potential in drug delivery, skincare, and functional foods. Currently, PEVs are obtained primarily from fresh and dried materials through soaking and extraction; however, little is known about the differences in their contents. Using Portulaca oleracea L. as the research object, this study firstly employed a method that combined differential and ultracentrifugation with membrane filtration to separate and purify exosome-like nanoparticles from dried material (D-PELNs) and fresh material (F-PELNs). Then, multi-omics analysis compared the small-molecule metabolites, lipid profiles, and protein expression patterns. Both D-PELNs and F-PELNs showed typical cup-shaped morphology, with mean particle sizes of 139 nm and 186 nm, and mean zeta potentials of -16.015 ± 0.335 mV and -6.29 ± 0.19 mV, respectively. Both types contained diverse small-molecule metabolites. Among them, terpenoids (e.g., caesaldekarin e) were more abundant in F-PELNs, whereas carboxylic acids and their derivatives (e.g., citric acid) were predominantly found in D-PELNs. Both types had abundant lipids. D-PELNs exhibited greater lipid diversity than F-PELNs, with notable enrichment in phosphatidylcholine (18.48%) and ceramide (17.02%). F-PELNs mainly consisted of functional neutral lipids, such as monoglycerides and triglycerides. Proteins involved in plant morphogenesis and secondary-metabolite biosynthesis were also identified. Proteins from both Portulaca oleracea L.-derived exosome-like nanoparticles (PELNs) were localized to intracellular structures, including the cytoplasm and mitochondria of the cells. D-PELNs had a higher protein content related to carbon metabolism, whereas F-PELNs were more enriched in proteins related to secondary metabolite synthesis. In summary, D-PELNs and F-PELNs were successfully isolated and characterized, and their compositions were analyzed and compared using multi-omics approaches. These findings identify the specific chemical components of PELNs and offer new insights for comparing the compositional differences between exosome-like nanoparticles derived from dried and fresh plant states.