Unlocking the stability and storage conditions of plant-derived nanovesicles through metabolomic and lipidomic profiling

Extracellular vesicles (EVs) are nanoscale lipid bilayer carriers that mediate intercellular communication through the transport of bioactive cargo. Plant-derived nanovesicles (PDNVs) have emerged as sustainable platforms for therapeutic, nutraceutical, and biotechnological applications; however, their physicochemical stability under storage and handling conditions remains insufficiently characterized. Here, we present an integrated dual-omics framework combining untargeted metabolomics and lipidomics with ontology-based analyses to systematically evaluate PDNV stability. Vesicles isolated in a single batch were subjected to controlled variations in temperature (+ 25 °C, + 4 °C, -20 °C, -80 °C), storage duration (1 week-3 months), buffer composition (acidic, neutral, basic), lyophilization, and freeze-thaw (F/T) cycling (1x, 3x, 5x), followed by GC-MS and LC-MS analyses. Multivariate (PLS-DA) and univariate analyses revealed that − 80 °C best preserved native metabolite and lipid signatures, whereas + 25 °C induced pronounced remodeling, including ceramide and sugar acid accumulation and depletion of phosphatidylcholine, hexosylceramides, and N-acylethanolamines. Storage at -20 °C maintained short-term stability (~ 1 week) but showed progressive molecular drift over time. Buffer composition exerted modest yet reproducible effects, with PBS maintaining near-native lipid profiles. Lyophilization caused immediate lipid reorganization that remained largely stable during storage, while F/T cycling emerged as the most disruptive stressor. Notably, although multiple conditions altered molecular composition, most changes remained within 10%, underscoring the relative robustness of PDNVs and supporting − 80 °C storage and short-term lyophilization as preferred preservation strategies.