Characterization of cannabis strain-plant-derived extracellular vesicles as potential biomarkers

The scientific interest in cannabis plants' beneficial properties has recently sparked certain interest in the possible functional characterization of plant-derived extracellular vesicles (PDEVs). Establishing the most appropriate and efficient isolation procedure for PDEVs remains a challenge due to vast differences in the physio-structural characteristics of different plants within the same genera and species. In this study, we employed a crude but standard isolation procedure for the extraction of apoplastic wash fluid (AWF) which is known to contain the PDEVs. This method includes a detailed stepwise process of PDEV extraction from five (5) cultivars of cannabis plants, namely: Citrus (C), Henola (HA), Bialobrezenski (BZ), Southern-Sunset (SS), and Cat-Daddy (CAD). Approximately, 150 leaves were collected from each plant strain. In order to collect PDEV pellets, apoplastic wash fluid (AWF) was extracted from plants via negative pressure permeabilization and infiltration followed by high-speed differential ultracentrifugation. Particle tracking analysis of PDEVs revealed particle size distribution in the range of 20 to 200 nm from all plant strains, while PDEV total protein concentration from HA was higher than that of SS. Although HA-PDEVs' total protein was higher than SS-PDEVs, SS-PDEVs' RNA yield was higher than that of HA-PDEVs. Our result suggests that the cannabis plant strains contain EVs, and PDEV concentration from the cannabis plant could be age or strain dependent. Overall, the results provide a guide for the selection and optimization of PDEV isolation methods for future studies.