Enrichment and Characterization Protocol for Plant-Derived Nanovesicles from the Aquatic Plant Wolffia globosa

This article presents a rigorous and reproducible protocol for the enrichment of plant-derived nanovesicles (PDNVs) from the aquatic plant Wolffia globosa using differential ultracentrifugation combined with sucrose density gradient enrichment. The protocol is specifically optimized to address the technical challenges associated with PDNV enrichment from extremely small aquatic plants that require whole-plant homogenization, a process that inherently increases contamination from intracellular organelles, soluble proteins, and chlorophyll. The workflow integrates gentle homogenization, sequential low- and medium-speed centrifugation, sterile filtration, and an initial ultracentrifugation step to obtain crude PDNVs, followed by density-based separation using a discontinuous sucrose gradient (8%-30% w/v). This combined size- and density-based strategy enables effective enrichment of PDNVs while substantially reducing non-vesicular contaminants. Quality control and characterization are performed in accordance with MISEV2023 recommendations using nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). Gradient-enriched PDNVs exhibit a predominantly nanoscale, homogeneous particle population with a mean diameter of approximately 105.3 nm and a particle concentration on the order of 10¹⁰ particles/ml. TEM analysis confirms well-preserved membrane-bound vesicles with characteristic round-to-cup-shaped morphology and minimal background contamination, demonstrating a clear improvement over crude PDNV preparations. Quantitative normalization of PDNV yield to plant biomass and assessment of particle-to-protein ratios provide standardized metrics for evaluating preparation quality and reproducibility across independent preparations. Overall, this protocol provides a robust, scalable approach for obtaining enriched PDNVs prepared from W.globosa using commonly available laboratory equipment. The resulting PDNV preparations are suitable for downstream physicochemical characterization, molecular profiling, and functional studies, supporting their application in plant PDNV biology and emerging biomedical research. © 2026 Wiley Periodicals LLC. Basic Protocol 1: Enrichment of plant-derived nanovesicles (PDNVs) from the aquatic plant W. globosa by differential ultracentrifugation and sucrose density gradient.