Delivery of Functional Exogenous Proteins by Plant Vesicles to Human Cells in Vitro

Background: Plant-derived extracellular vesicles increasingly gain attention as promising carriers of exogenous bioactive molecules to the human cells. Due to their various edible sources they are distinctly biocompatible, biodegradable and easily available in significant amounts. Methods: In present work, extracellular vesicles from grapefruit juice were isolated by differential centrifugation and characterized in terms of size, quantity, and morphology by nanoparticle tracking analysis, dynamic light scattering, atomic force microscopy, and cryo-electron microscopy (Cryo-EM). In Cryo-EM experiments we have visualized grapefruit vesicles with average size of 41 ± 13 nm, confirmed their round-shaped morphology and estimated the thickness of their lipid bilayer as 5.3 ± 0.8 nm. Results: Using in vitro cell culture models, we have shown that grapefruit-derived extracellular vesicles (GF-EVs) are highly efficient carriers for the delivery of the exogenous Alexa flour 647 labelled BSA and HSP70 proteins into human colon cancer HCT-116 and DLD1 cells. Both proteins when loaded to plant vesicles were captured by human intestinal cells much more efficiently compare to their free state. Additionally, the functional activity of human recombinant HSP70 delivered by GF-EVs in the tissue culture cells has been confirmed. Conclusions: The results clearly indicate the high potential of native plant vesicles for the safe delivery of therapeutic proteins into human cells. Here, we reported the first demonstration of effective loading of natural plant-derived extracellular nanovesicles with exogenous proteins and their successful delivery into human cells.