Delivery of functional exogenous proteins by plant-derived vesicles to human cells in vitro.

Plant-derived extracellular vesicles (EVs) gain more and more attention as promising carriers of exogenous bioactive molecules to the human cells. Derived from various edible sources, these EVs are remarkably biocompatible, biodegradable and highly abundant from plants. In this work, EVs from grapefruit juice were isolated by differential centrifugation followed by characterization of their 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 visualized grapefruit EVs with the 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. Further, using cell culture models, we have successfully demonstrated that native grapefruit-derived extracellular vesicles (GF-EVs) are highly efficient carriers for the delivery of the exogenous Alexa Fluor 647 labeled bovine serum albumin (BSA) and heat shock protein 70 (HSP70) into both human peripheral blood mononuclear cells and colon cancer cells. Interestingly, loading to plant EVs significantly ameliorated the uptake of exogenous proteins by human cells compared to the same proteins without EVs. Most importantly, we have confirmed the functional activity of human recombinant HSP70 in the colon cancer cell culture upon delivery by GF-EVs. Analysis of the biodistribution of GF-EVs loaded with 125I-labeled BSA in mice demonstrated a significant uptake of the grapefruit-derived extracellular vesicles by the majority of organs. The results of our study indicate that native plant EVs might be safe and effective carriers of exogenous proteins into human cells.

Cryo-electron microscopy of extracellular vesicles from cerebrospinal fluid.

Extracellular vesicles (EVs) are membrane-enclosed vesicles which play important role for cell communication and physiology. EVs are found in many human biological fluids, including blood, breast milk, urine, cerebrospinal fluid (CSF), ejaculate, saliva etc. These nano-sized vesicles contain proteins, mRNAs, microRNAs, non-coding RNAs and lipids that are derived from producing cells. EVs deliver complex sets of biological information to recipient cells thereby modulating their behaviors by their molecular cargo. In this way EVs are involved in the pathological development and progression of many human disorders, including neurodegenerative diseases. In this study EVs purified by ultracentrifugation from CSF of patients with Parkinson's disease (PD) and individuals of the comparison group were characterized using nanoparticle tracking analysis, flow cytometry and cryo-electron microscopy. Vesicular size and the presence of exosomal marker CD9 on the surface provided evidence that most of the EVs were exosome-like vesicles. Cryo-electron microscopy allowed us to visualize a large spectrum of extracellular vesicles of various size and morphology with lipid bilayers and vesicular internal structures. Thus, we described the diversity and new characteristics of the vesicles from CSF suggesting that subpopulations of EVs with different and specific functions may exist.