Abstract
The scientific interest in cannabis plants is continuously growing, with heightened interest in properties of plant-derived extracellular vesicles. This manuscript focuses on isolating and characterizing vesicles originating from cannabis plants. Establishing the most appropriate and efficient isolation procedure for plant vesicles remains a challenge due to vast differences in the physio-structural characteristics of different plant cultivars within the same species, and different species within the same genera. In this study, we employed a crude but standard isolation procedure for the extraction of apoplastic wash fluid, which is known to contain plant-derived extracellular vesicles. This method includes a detailed stepwise process of plant-derived extracellular vesicles extraction from two (2) cultivars of cannabis plants, namely: Citrus and BaOx. Approximately, 150 leaves were collected from each plant strain. In order to collect plant-derived extracellular vesicles pellets, apoplastic wash fluid was extracted from the plants via negative pressure permeabilization and infiltration followed by high-speed differential ultracentrifugation. Apoplastic wash fluid fractions were collected for Citrus and BaOx fractions, P100 and P40. Particle tracking analysis of plant-derived extracellular vesicles revealed particle sizes ranging from 60 to 160 nanometers. Both cultivar fractions yielded high levels of plant-derived extracellular vesicles, and contained enriched plant-derived extracellular vesicles RNA levels. Our results suggest that the cannabis apoplastic wash fluid fractions (P100 and P40) yielded plant-derived extracellular vesicles. In total, the results provide a guide for the selection and optimization of cannabis-derived extracellular vesicles. Subsequently, these cannabis plant-derived vesicles can be used for further biomedical uses.