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

Abstract

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.

Identification of Tomato Infecting Viruses That Co-Isolate with Nanovesicles Using a Combined Proteomics and Electron-Microscopic Approach

“…Cell-derived submicron particles isolated from plant resources [1][2][3][4][5][6][7] are gaining attention both as complex biomaterials with health-promoting effects [8,9] and as delivery vectors for exogenous substances [10]. Nanometer-sized vesicles (NVs) have been isolated from many different plants, such as ginger [6,7,11], carrots [12], citrus species [4,13], grapes [1,2], tomato [1,14], blueberry [1], coconut [1], broccoli [15], wheat [16], etc., and even dried plant material [5].…”

“…Besides the biological activities, plant-derived NVs are used as carriers for the delivery of exogenous molecules/drug cargo. For instance, grapefruit-juice-derived NVs ameliorated the delivery of exogenous proteins to human cells compared to the same proteins without vesicles [10]. Moreover, the administration of ginger-exosome-like NVs loaded with exogenous therapeutic RNA efficiently inhibited tumor growth in a mice model [10].…”

Stem Cell-Derived Exosomes Potential Therapeutic Roles in Cardiovascular Diseases

“…Exosomes have been classified broadly as natural exosomes ( 13 ) and engineered exosomes ( 85 ) ( Figure 2 ). Naturally occurring exosomes are further classified into animal-derived exosomes ( 16 ) and plant-derived exosomes ( 86 ). As exosomes are synthesized under normal and diseased or tumor conditions, animal exosomes are subdivided further into normal exosomes and tumor exosomes or oncosomes ( 30 , 87 ).…”

In situ and ex situ imaging of plant extracellular vesicles as nanovectors for cross‐domain communication

“…The discrepancies in EV sizes based on dynamic light scattering (DLS) and TEM data resulted from the shrinking effects of drying during TEM preparation; however, cryo‐TEM imaging of EVs from a tobacco leaf apoplast supported the DLS data (Woith & Melzig, 2019). Most grapefruit EVs were round‐shaped and enclosed with a lipid bilayer with an average thickness of ~5 nm in cryo‐TEM imaging (Figure 6a) (Garaeva et al., 2021). They had double or elliptical vesicles (Figure 6b,c).…”

“…In addition, the grapefruits were peeled and manually pressed to collect the juice (Wang et al., 2013). Grapefruit juice was also extracted using a household citrus juicer (Garaeva et al., 2021).…”