Enzyme-assisted extraction of plant-derived extracellular vesicles: recent advances, methodological considerations,...

Plant-derived extracellular vesicles (PDEVs) have attracted increasing attention as bioactive nanostructures with emerging applications in medicine, functional foods, and cosmetics. However, unlike mammalian extracellular vesicle systems, the recovery of PDEVs is fundamentally constrained by plant-specific structural features, particularly the rigid cell wall and the limited accessibility of vesicles within plant tissues. In plant systems, extracellular vesicles are thought to be retained, at least in part, within the apoplastic space between the plasma membrane and the cell wall, which restricts the direct application of conventional animal cell-based isolation workflows. Conventional extraction approaches based on mechanical disruption enable bulk recovery of vesicle-containing material but are frequently associated with intracellular contamination, cell wall-derived debris, increased extract viscosity, and poor process reproducibility. To address these limitations, recent studies have explored enzyme-based extraction strategies that partially relax the plant cell wall to facilitate the selective release of apoplastic vesicles while reducing nonspecific contamination. In this mini-review, we summarize recent advances in enzyme-based extraction strategies for PDEVs, with particular emphasis on their structural rationale, methodological advantages over mechanical disruption, and associated limitations. We further discuss key challenges in quantitative evaluation, purity assessment, and reporting standardization, highlighting the need for methodologically rigorous frameworks to enable reproducible isolation and reliable interpretation of PDEV-associated biological activities.