**5. Cargo loading/sorting**

The cargo that is loaded and carried within EVs include proteins, lipids, nucleic acids, metabolites, and miRNAs. The loading of specific molecules within different types of EVs is dependent on the parent cell and specific loading mechanisms. The enrichment of various types of cargo molecules is also dependent on a particular cell type being subject to pathological conditions, drugs, and various stimuli.

The loading of cargo within EVs may be a means of the cell getting rid of unneeded or excess material. For example, EVs from epithelial cells of the renal tubule are released across the luminal plasma membrane into the filtrate and are subsequently excreted from the body in the form of urine if the EVs are not taken up by recipient cells downstream in the nephron. In fact, a large amount of urinary EVs (uEVs) are those released from renal epithelial cells and bladder cells.

Multiple mechanisms have been proposed for the loading of miRNAs into EVs. One mechanism involves 3′ end uridylation of miRNAs [22]. A second mechanism involves the incorporation of a four nucleotide motif (GGAG) and its interaction with the ribonucleoprotein hnRNPA2B1 [22]. A third mechanism involves the RISC complex associated protein, AGO2 [22]. Another mechanism involves over-expression of nSMase2 [22]. The exosomal protein Annexin A2 was found to play a role in the packaging of miRNAs in EVs [23].

Proteins found to be enriched in EVs include those enriched in lipid rafts, metalloproteinases, tetraspanins, proteins associated with endosomes, heat shock proteins, glycosylphosphatidylinositol (GPI) anchored proteins, and those associated with the exosome biogenesis pathway. Proteomic studies have identified metalloproteinases including ADAMs (a disintegrin and metalloproteinase) with proteolytic activity enriched in EVs [24]. ADAMs are versatile proteins that are involved in cell adhesion, cellular communication, migration, and the regulation of growth factors and cytokines. EVs also contain members of the LAMP family proteins, LAMP-1 [25] and LAMP-2 [26]. These lysosome-associated proteins help maintain the integrity of the lysosomes by forming a glycocalix that protects the membranes from enzymatic degradation. Tetraspanins including CD9, CD63, and CD81 are enriched in exosomes [24]. Heat shock proteins (HSP) including HSP60, HSP70, and HSP90 have been found to be enriched in EVs [24]. Flotillin proteins were found to regulate exosome formation and cargo sorting. Other proteins such as syntenin has been found to contribute to the loading of exosomes with specific cargo [27]. GPI proteins including the complement regulator proteins CD55 and CD50 are enriched in exosomes [28].

Lipids that are enriched in EVs generally share the same features as the cells of origin. Lipids enriched in EVs include cholesterol, ceramides, cholesterol, and phosphatidylserine, which is also enriched in vesicles derived from MVBs. In one study, Glover found 13 ceramides that were significantly reduced in uEVs of hereditary α-tryptasemia (HαT) patients compared to healthy volunteers [29]. In another study, Nouri et al. showed sphingomyelin, phosphatidylethanolamine, and lysophosphatidylcholine were present in greater concentrations in EVs isolated from the conditioned media of human aortic endothelial cells compared to control EVs isolated from the complete growth media of these cells [18].
