# Plant Exosomal Vesicles: Perspective Information Nanocarriers in Biomedicine
## 6 Isolation and Purification of Plant Nanovesicles
The polyethylene glycol (PEG) method is based on changing the solubility or dispersibility of nanovesicles using water-exclusive polymers, such as PEG, in the range of a molecular weight of more than 8000 [71]. This displacement agent can create a network-like structure, trapping nanovesicles before their precipitation. By adjusting the concentration of PEG from 8%, 10%, 12% to 15%, the nanoparticles of 365 nm, 304 nm, 256 nm, and 252 nm are able to be obtained, respectively, while the ultracentrifugation method results in the formation of nanoparticles of 403 nm [77]. The highest yield of nanovesicles from ginger using PEG with the different molecular weights (4000, 6000, and 8000) was observed with the use of PEG4000, with its residual concentration at the end of preparation remaining acceptable for consumption [78]. In general, this method results in high yield but low-quality isolation due to its non-specific mechanism. However, combining the PEG-based isolation with other isolation methods is an effective strategy to avoid the disadvantages associated with using PEG alone. There are many benefits of such a strategy providing the processing of multiple samples simultaneously: it is easier, faster, and is relatively low cost, without damaging the nanovesicles. However, lengthy sample preparation and purification procedures are needed, while the proper selective isolation mechanism is absent. These factors inevitably compromise the purity of the isolated nanovesicles, thereby impairing subsequent analysis [79]. Size exclusion chromatography (SEC) is based on the size separation of biomolecules. Sample components with a small hydrodynamic radius may pass through the pores, resulting in a late elution. The components with a larger hydrodynamic radius, including nanovesicles, cannot enter the pores, thereby eluting by the first washing solution volume [71]. For example, in one study, the nanovesicles were isolated from two types of cabbage using three methods, including SEC, and it was concluded that the nanovesicles isolated by SEC were the most homogeneous and purified from protein impurities, as well as retaining their biological activity [80]. This method of isolation can be used as an additional method for the purification of nanovesicles obtained by ultracentrifugation. Thus, the nanovesicles obtained from _Cucunis sativus_ by differential ultracentrifugation was further successfully purified by SEC to remove the protein impurities [81]. The SEC isolation method has a great potential to produce highly-purified nanovesicles whose integrity and biological activity are largely preserved, probably because SEC relies on the use of gravity rather than centrifugal force reaching high values. Additionally, there are plenty of pre-packed commercial columns, specially designed for the isolation of exosomes from various biofluids. Nevertheless, its long runtime limits the SEC application in any large-scale production [79]. The method of immunoprecipitation or immunoaffinity consists of coating magnetic beads with the antibodies that target the proteins present on the surface of exosomes [71]. This method is based on the surface biomarkers that are uniquely expressed in nanovesicles, allowing the isolation of specific nanovesicles. Despite its high potential, the immunoaffinity method has not been extensively studied for the isolation of plant nanovesicles, probably due to the lack of extensive knowledge of the surface composition and antibody-antigen interactions that could be used to isolate them. However, in a study on the isolation of the nanovesicles from _Arabidopsis thaliana_, this method was used in conjunction with ultracentrifugation and, as a result, the nanovesicles were separated from the TET8 proteins mix by adding the appropriate antibodies, then passed through electrophoresis and isolated from the gel [82]. Although it provides high purity isolation, the overall yield of nanovesicles is reduced as only those recognized by the antibodies are captured.