An enzyme-based system for extraction of small extracellular vesicles from plants

Abstract

Plant-derived nanovesicles (NVs) and extracellular vesicles (EVs) are the next generation of nanocarrier platforms for biotherapeutics and drug delivery. EVs exist not only in the extracellular space, but also within the cell wall. Due to the limitations of existing isolation methods, the EVs extraction efficiency is low, and a large amount of plant material is wasted, which is of concern for rare and expensive medicinal plants. We proposed and validated a novel method for isolation of plant EVs by enzyme degradation of the plant cell wall to release the EVs. The released EVs can easily be collected. The new method was used for extraction of EVs from the roots of Morinda officinalis (MOEVs). For comparison, nanoparticles from the roots (MONVs) were extracted using the grinding method. The new method yielded a greater amount of MOEVs, and the vesicles had a smaller diameter compared to MONVs. Both MOEVs and MONVs were readily absorbed by endothelial cells without cytotoxic effect and promoted the expression of miR-155. The promotion of miR-155 by MOEVs was dose-dependent. More importantly, we found that MOEVs and MONVs were enriched toward bone tissue. These results support our hypothesis that EVs in plants could be efficiently extracted by enzymatic cell wall digestion and confirm the potential of MOEVs as therapeutic agents and drug carriers.

Consensus statement on research and application of Chinese herbal medicine derived extracellular vesicles-like particles (2023 edition)

“…Some researchers have compared the wall-breaking and osmotic centrifugation methods, and found that Arabidopsis EVs obtained by vacuum osmosis method had smaller particle size, lower potential, smaller density and higher lipid film thickness ( Liu et al, 2020 ). Zhao et al suggested employing cellulase and pectinase enzymes to facilitate the digestion and disruption of cell walls to obtain the EVs derived from Morindae Officinalis Radix (Bajitian in Chinese) ( Zhao et al, 2023 ).…”

“…The Bicinchoninic Acid Assay (BCA) is utilized to determine the concentration of protein in CHM-EVLP, while NTA or nFCM techniques are employed to measure particles concentration, which is subsequently converted into the number of particles per milligram of vesicles protein (particles/mg) ( Zhao et al, 2023 ).…”

“…Following incubation and lysis with Triton X-100, the NTA or nFCM techniques are employed to quantify the proportion of membranous particles present in CHM-EVLP. The measurement serves as an indirect indicator of the sample's purity to a certain degree ( Zhao et al, 2023 ).…”

Plant‐derived nanovesicles offer a promising avenue for anti‐aging interventions

“…Simultaneously, this combination approach is applicable for isolating PDNVs with high purity and stability from the model plant Arabidopsis . Additionally, several experts have suggested a novel pretreatment technique for isolating NVs from plants (Zhao et al 2023b). The plant cell walls were broken down using specific digestive enzymes that targeted the primary components of the wall.…”