Methods most commonly used for isolation of EVs have recently been comprehensively reviewed and discussed in the terms of their impact on biological function of EVs [34]. However, there is still a long way to develop a unified protocol for the isolation of a particular EV subtype from a particular biological material. One of the promising approaches to isolate EVs from any biological fluid is based upon size-exclusion chromatography that provides high purity of EV isolate [35,36,37].

In multicellular organisms, EVs are a part of physiological communication system. Therefore, they seem to be much more biocompatible than artificially produced vesicles and liposomes. However, EV populations isolated from body fluids by ultracentrifugation are largely heterogeneous. This prompted researchers to develop the procedures of in vitro stimulation of cultured cells to release EVs. However, cell-released EVs from ultracentrifuged supernatants were found to be heterogeneous as well [38]. Thus, EVs’ isolation from both body fluids and cell culture supernatants requires much more precise methods for separation of a particular EV subtype. Those include magnetic bead-based immunoaffinity method [39] and antigen affinity chromatography [40]. By using specific antibodies, both methods should allow the isolation of EVs expressing particular antigen of choice. Otherwise, the use of antigen-coated beads or polymers for chromatography separates EVs that are able to bind the antigen, for example, due to the surface expression of antigen-specific antibody light chains (LCs) [40]. However, the process of EVs’ elution from chromatographic columns or their detaching from the magnetic beads may influence their physical and chemical properties and, consequently, their biological activities. Thus, this aspect requires further investigation. Accordingly, our observations suggest that chromatographically-separated EVs eluted with acidic guanidine from column filled with Sepharose linked with either trinitrophenol (TNP) hapten [40], casein hydrolysate [41], or anti-CD9 monoclonal antibodies [41,42] preserved their biological activity.

One can assume that EV-contained cargo is a main determinant of the therapeutic efficacy of their application. As mention above, EVs may carry a great variety of biologically active molecules, including RNAs, proteins and lipids, which makes them a conveyor of virtually unlimited types of cargos. The cargo can be packed into EVs during their intracellular biogenesis or extracellularly after EVs’ exocytosis. The latter generally happens in in vitro conditions [43], but some results suggest the possibility of loading of freely circulating RNAs into EVs also in vivo [44]. Accordingly, the methods for loading EVs with selected molecules can be classified into two main groups, i.e., those based on modification of parental cells and those adapting physicochemical techniques enabling in vitro loading [45].

Parental cells can be passively loaded with chosen molecules. Along these lines, human gingival mesenchymal stromal cells were shown to uptake the chemotherapeutic drugs during standard cell culture [46]. Furthermore, one of these drugs, namely paclitaxel, was then found in cell-secreted EVs that expressed anti-cancer activity in vitro [47]. Similar activity was observed in the case of paclitaxel-carrying EVs released by mouse mesenchymal stromal cells [48]. In addition, mouse and human tumor cell lines were also shown to release drug-containing EVs after simple culturing in the presence of different chemotherapeutics [49]. In such cases, one can speculate that the drug is passively packaged into EVs during their formation. On the other hand, EV-parental cells can be transfected or transduced by non-viral or viral vectors, respectively, to produce the encoded molecules. These would likely be then actively sorted into EVs during their biogenesis. Accordingly, parental cells transfected with different plasmids were shown to secrete EVs that contained the plasmid-encoded products, including antibody protein and mRNA for enzyme that activates the chemotherapeutic prodrug. As a result, EVs were able to deliver mRNA to the cells of HER2-positive human breast tumor xenografts in a targeted manner due to the surface-expressed anti-HER2 antibody, which inhibited the growth of the xenografts in mice [50]. Interestingly, the later results suggested that EVs may deliver in vitro transcribed enzyme-encoding mRNA, which allows to eliminate the potentially harmful plasmid transfection of EV-parental cells [51]. Another interesting possibility was proposed by Sancho-Albero et al. [31]. The authors reported that hollow gold nanoparticles incubated with EV-parental cells are much more efficiently incorporated into EVs after their PEGylation. Delivery by EVs may greatly improve the accumulation of PEGylated gold nanoparticles in tumors [31]. However, many variables have to be taken into account while using these strategies. Therefore, at present, much more commonly used strategies are based on loading EVs with selected cargo after their isolation [45]. Maintaining the EVs’ integrity, allowing to protect the incorporated cargo from extracellular degradation or inactivation, is one of the features that should be considered while choosing the loading method. 

3.3. Directing EVs towards Desired Target