# Plant Exosomal Vesicles: Perspective Information Nanocarriers in Biomedicine
## 6 Isolation and Purification of Plant Nanovesicles  
Various parts of plants are used to isolate the nanovesicles, mainly the fruits, roots, and apoplastic fluid from their leaves. Currently, five approaches are applicable for plant nanovesicle isolation, namely differential and gradient density ultracentrifugation, precipitation using highly hydrophilic polymers (e.g., protamine, dextran, polyethylene glycol), size exclusion chromatography, and immunoprecipitation (Figure 2), with preference of the methods based on ultracentrifugation [70]. The methods based on ultracentrifugation are preferred due to resulting in a larger amount of the material [42]. Differential ultracentrifugation includes series of centrifugation cycles with different centrifugal forces and durations, providing differences in the densities and sizes of nanovesicles and other cellular components, including organelles, microvesicles, and apoplastic bodies [71]. Typically, the biological fluids are successively centrifuged at 5000\(\times\), 10,000\(\times\), and 20,000\(\times\)\(g\), followed by the ultracentrifugation step. For ultracentrifugation, the speed in the range of 100,000\(\times\)\(g\) to 120,000\(\times\)\(g\) is sufficient to pellet nanovesicles [72]. However, for the study on extracellular vesicles from the apoplastic fluids of _A. thaliana_ leaves, recovery efficiency and quality were maximally improved by the ultracentrifugation at 40,000\(\times\)\(g\) that allowed them not to use the speed of 100,000\(\times\)\(g\)[35]. Later, the research of [PERSON] et al. demonstrated the opposite results for the same model plant [72]. In another study, the nanovesicles were isolated from _Nicotiana tabacum_ L., _Vinca minor_ L., and _Viscum album_ L. by differential ultracentrifugation at 50,000\(\times\)\(g\) (the optimal speed for the nanovesicles sedimentation), followed by agarose gel electrophoresis purification to increase their purity [73]. Additionally, the method was improved by adding a thin pad (Optiprep(tm)) to the bottom of the centrifuge tube that prevented the damage of nanovesicles [74]. This means that the issue of the most suitable ultracentrifugation conditions is still open and they should obviously be optimized for each plant species and type of the biofluids as the starting material. Moreover, the differential ultracentrifugation method should be used in combination with the additional purification methods, since the disadvantage of this method is the contamination of the resulting vesicles with protein molecules similar in density and size to the nanovesicles [42]. Density gradient ultracentrifugation is based on the separation of nanovesicles depending on their size, mass, and density in a centrifuge tube with a pre-designed medium with a gradually decreasing gradient. The sample is subjected to a long cycle of ultracentrifugation, whereby the solutes of the sample, including the nanovesicles, move as separate zones through the medium with a density gradient to the bottom, each with its own specific sedimentation rate [71]. The samples of nanovesicles isolated by this method are much more homogeneous and of less foreign biomolecules. When both methods of  
Figure 2: Schematic flow diagram of the isolation and purification of plant nanovesicles. ultracentrifugation are applied together, the yield of the purified nanovesicles is much higher. Thus, the ginger nanovesicles were isolated firstly by differential centrifugation and then purified in a sucrose gradient [75]. The nanovesicles from the plant _Asparagus cochinchinensis_ were also obtained by a combination of differential and gradient ultracentrifugation methods [76]. The disadvantage of density gradient ultracentrifugation is the comparatively lower yield and contamination of the isolated nanovesicles, but their quality is higher than that of isolation by differential ultracentrifugation.