A rapid capillary-channeled polymer (C-CP) fiber spin-down tip approach for the isolation of plant-derived extracellular vesicles (PDEVs) from 20 common fruit and vegetable sources

Abstract

In the emerging field of phyto-nanotechnology, 30-200 nm plant-derived extracellular vesicles (PDEVs) are now known to contain active biomolecules that mediate cell-to-cell communication processes in a manner very similar to exosomes in mammalian cells. The ability to deliver cargo across cellular membranes suggests that botanical systems could be used in the mass production of therapeutic vectors to transport exogenous molecules into human cells. The fundamental biochemical characteristics of PDEVs remain poorly understood due to the lack of efficient methods to isolate and characterize these nanovesicles. Described here is a rapid PDEV isolation method using a hydrophobic interaction chromatography (HIC)-based extraction performed on a capillary-channeled polymer (C-CP) fiber spin-down tip. The C-CP solid-phase extraction method is performed using a standard table-top centrifuge, enabling the isolation and concentration of PDEVs (>1 × 10¹⁰ particles from 100 μL of sample). PDEVs of 189 nm average diameter were obtained from 20 common fruit and vegetable stocks. The size, integrity, and purity of the recovered PDEVs were assessed using transmission electron microscopy (TEM), multi-angle light scattering (MALS), absorbance quantification, a protein purity assay, and an enzyme-linked immunosorbent assay (ELISA) to the PEN1 PDEV surface marker protein. The HIC C-CP tip isolation method allows for concentrated PDEV recoveries (up to 2 × 10¹¹ EVs) on reasonable time scales (<15 min) and low cost (<$1), with the purity and integrity fit for fundamental research and downstream applications.

Keywords: Capillary-channeled polymer (C-CP); Exosomes; Isolation; Plant materials; Plant-derived extracellular vesicles (PDEVs); Solid-phase extraction (SPE).