An optimized protocol for plant extracellular vesicle isolation from Ophiopogon japonicus root: a comparative evaluation based on miRNA cargo

Background: Plant extracellular vesicles (PEVs), hold significant therapeutic potential due to their roles in intercellular communication and cross-kingdom regulation, primarily mediated by their miRNA cargo. However, isolating high-purity PEVs from complex plant tissues, such as the tuberous roots of Ophiopogon japonicus , is challenging due to the dense cell wall matrix and high content of contaminants like polysaccharides. Existing isolation methods, including differential centrifugation and density gradient ultracentrifugation, involve trade-offs between yield, purity, and vesicle integrity, necessitating the development of optimized protocols. Results: We developed and systematically optimized an integrated protocol for isolating high-purity EVs from O. japonicus roots. Key optimizations included: 1) refining the differential centrifugation protocol by incorporating a double ultracentrifugation step. 2) implementing a modified density gradient ultracentrifugation approach with a pre-clearing step for superior debris removal; and 3) evaluating enzymatic pre-treatment with cellulase and pectinase to enhance EV release. Comparative analysis demonstrated that the optimized method, particularly utilizing enzymatic pre-processing and double ultracentrifugation, significantly improved EV yield and purity. Small RNA sequencing of the resulting high-purity EVs successfully characterized their functional miRNA cargo profile, validating the efficacy of the isolation strategy. Conclusions: This study establishes a robust and adaptable pipeline for isolating high-quality, functionally intact PEVs from challenging plant root tissues. The optimized protocol effectively addresses the critical methodological challenges of yield and purity, enabling reliable downstream functional characterization and advancing therapeutic investigations of plant-derived vesicles.