# Origin and Composition of Exosomes as Crucial Factors in Designing Drug Delivery Systems
## 8 Loading Drugs to Exosomes  
While designing DDS, one of the most important features is the method of drug loading. It should be chosen carefully based on the drug and the exosomes properties. The biogenesis of exosomes makes them promising drug carriers due to their biocompatibility, stability, and preferred tumor targeting [123]. They are built similarly to double-membared liposomes and the same methods can be used for drug loading. Exosomes can be loaded with numerous drugs, macromolecules, and other substances either by passive or active methods (Figure 3). Passive methods are simple and based on drug incubation with exosomes or with cells with further isolation of released exosomes containing drugs (Figure 3I,II). This method depends on the difference in the drug concentration gradient; thus, during the incubation with exosomes, the substance itself will migrate to the vesicle. This process can be intensified by applying an additional force, such as shaking or stirring [9,124]. In a similar matter, the incubation of cells with a drug will result in the release of exosomes loaded with the drug. The downsides of this method are low loading efficiency and low-output drug concentration (about 1%) [124]. Incubation of drugs with exosomes or other DDS vehicles is a very common and simple method, especially popular with hydrophobic drugs [122,125]. Higher loading efficiency can be achieved with active loading methods, including sonication (\(\sim\)28%), electroporation (\(\sim\)5%), extrusion, freeze-thaw, pH gradient (1.7%), and conjugation methods [124]. Both sonication and electroporation will result in a temporary disruption of the exosome membrane, mediated by sonic waves or electric pulses, respectively (Figure 3 III,IV). Afterward, exosomes will accumulate the drug from the solutionthrough small membrane pores induced by mentioned agents. These methods do not seem to alter exosome membrane properties but cause an increase in exosomes size [124, 126]. The extrusion method is also based on an exosome membrane disruption by a syringe-based lipid extruder (Figure 3V) [127]. In one experiment, the freeze-thaw loading method was used with unknown efficacy but observable therapeutic effects (Figure 3 VI) [128]. In addition, a method based on a pH-driven drug loading was presented but needs extended research due to the very low drug loading efficiency [129]. Another approach is to conjugate drugs to exosomes via antibodies or click-chemistry. The antibodies will bind non-covalently to the exosomes surface via their affinity to structural elements of the exosomes (Figure 3 VII). The chemical synthesis will result in covalently bound drugs to exosomes via, e.g., PEG or membrane proteins, and the further release dependent on exosomes degradation. These methods can also serve to target exosomes to desired cells by the introduction of specific receptors to the membrane [7]. Mentioned methods allow for the incorporation of drugs into the inside of exosomes or the phospholipid bilayer depending on their hydro- and lipophilic properties. Hydrophilic substances will preferably locate in the lumen of exosomes, whilst substances soluble in lipids will accumulate at the membrane of exosomes (Figure 2). Some studies report that hydrophilic substances are easily loaded into exosomes, while lipophilic substances are loaded less efficiently. Generally, the substances are released from exosomes in about 50% during 24 h in vitro [122]. An extensive evaluation is needed for the assessment of the influence of mentioned loading methods on the structure and properties of exosomes. To date, aggregation and fusion were confirmed for electroporation [130]. Thus far, exosomes were used to deliver a variety of substances including chemotherapeutics (paclitaxel, doxorubicin, and taxol), RNA, and DNA (Table 4). Exosome-based designs of vaccines against bacterial (toxicDissosis and salmonellosis), viral (AIDS and hepatitis B), cancer (lung, pancreatic, colon, brain, and breast), and other diseases were also proposed [131].