# Plant Extracellular Vesicles: Investigating Their Utilization as Beneficial Nutrients in Diet
## 3 Conventional Methods for P-EV Isolation and Characterization
Size exclusion chromatography is based on particle size for separation [43]. Larger vesicles are eluted earlier, as they cannot enter the pores of the column, while vesicles with a smaller hydrodynamic radius are retained, resulting in late elution [13]. Although this approach has great potential to preserve the integrity and biological activities of vesicles, as it relies on passive gravity flow, the long run time limits its application in large-scale production [44]. Immunoprecipitation consists of magnetic beads coated with antibodies that target vesicle surface markers, such as the tetraspanins CD63 and CD81, allowing for the isolation and identification of specific extracellular vesicles [25]. Despite the high-purity isolation, the immunoaffinity method is not widely used, probably because protein markers for the plant vesicle surface are not yet well known [45]. In addition, the integrity of vesicles may be disrupted if the antibodies cannot be easily removed after precipitation [25]. Polymer-based precipitation methods have been successfully used to isolate P-EVs [43]. Highly hydrophilic polymers, such as polyethylene glycol (PEG), are used as crowding reagents to form a net-like structure that traps vesicles, allowing them to precipitate at low-speed centrifugation by reducing their solubility in solution [13]. In this procedure, PEG with different molecular weights (6000-20,000 Da) [46] is added to the samples in an appropriate amount (5-12%) to recover between 60-90% of EVs, with a yield comparable to the differential centrifugation method [42]. Therefore, this precipitation method provides a scalable and cost-effective alternative for the purification of P-EVs. Polymers with higher molecular weight are more efficient, but they may increase the viscosity of fluids, exacerbating the purification process [46]. Moreover, due to similar solubility, other biological contaminants are co-precipitated, thereby compromising the purity of the isolated vesicles [47]. In plants, EVs were first detected in carrot cell cultures in 1967, using transmission electron microscopy (TEM) [34]. Since then, various physical and biochemical approaches have been developed for vesicle characterization in terms of size, morphology, and zeta potential to determine the type and stability of the particles [11]. TEM is one of the most common imaging techniques for observing nanoscale samples because its resolution, of about 0.5 nm, is smaller than that of exosomes, providing detailed structural information about EVs [13]  
A good alternative to TEM is cryo-electron microscopy, in which samples are vitrified and cryo-immobilized at extremely low temperatures (\(-175\)\({}^{\circ}\)C) by cooling with liquid ethane, preserving the native hydrated status of samples and thus avoiding artifacts usually caused by conventional TEM [48]. In combination with immunogold labeling, cryo-TEM has been successfully used to distinguish EVs containing specific proteins and to track their uptake by recipient cells [25]. Scanning electron microscopy (SEM) scans the surface of samples by using a focused beam of high-energy electrons to provide information about the three-dimensional structure of the vesicle surface [49]. However, as the sample needs to be dried for imaging, the natural morphology may change, typically resulting in the formation of cup-shaped structures. In addition, during the preparation, the sample is coated with a thin layer of conductive material, which may affect the surface structure of EVs [49]. Recently, a novel technique in the form of nanoparticle tracking analysis (NTA) has been developed to analyze a population of polydisperse nanoparticles in solution, ranging from 10 nm up to 2 \(\upmu\)m in size [50]. During NTA, the sample is irradiated with a laser beam, and based on the light scattered by individual particles and relative Brownian motion in the solution, a camera tracks the path of each particle to determine the average concentration and size [13].