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 10