Optimization of the filtration and centrifugation steps for the isolation of exosome-like nanoparticles (ELNs) from grapes

Exosome-like nanoparticles (ELNs) are a class of nanosized extracellular vesicles from plants that can deliver drugs, siRNA, DNA, and proteins to various types of cells. Purification of ELNs from plants are tricky when compared to that from body fluids or cell culture medium because the initial suspension of the former contains high amounts of sugar, fiber, fat, etc. In this study, we have optimized the different steps for the isolation of ELNs from grapes. After preparing the juice, the bulk density was measured by Brix analysis and followed the same bulk density whenever doing the isolation. Differential centrifugation at various speeds and times and membrane filtration using different pore-size membranes were also optimized before the final step of ELN separation by ultracentrifugation. The size, zeta potential, and PDI value of the isolated ELNs were measured using a Malvern Zeta Sizer. SEM analysis showed that the grape ELN has an average size of 140.8 +/- 10.75 nm. The results indicated that filtration and centrifugation at different levels are essential before the ultracentrifugation for isolating homogenous grape ELNs with desired size, zeta potential and PDI value. From the optimized method, we have obtained 4.8 +/- 0.509 mg protein equivalents of ELNs from 100 g of grapes.