**3. The EV journey—overcoming biological barriers**

To reach their target sites, EVs need to overcome various biological barriers (**Figure 2**). Complementing these barriers are blood vessels (capillaries in particular). EVs can enter and extravasate from these vessels *via* diffusion, due to their lipidic nature which enables them to pass through the highly-lipidic capillary endothelium and their small size that enables them to pass or squeeze through fenestrations in the capillary wall [84].

#### **Figure 2.**

*Biological barriers encountered by extracellular vesicles (EVs). (A) Neurological barriers include the blood-brain barrier (BBB), blood-labyrinth barrier (BLaB) and blood-retinal barrier (BRB). (B) The pulmonary barrier, or blood-air barrier (BAB), guards against the invasion of pathogens in the lungs via its immune cell-rich lung mucosa, lung epithelial cells and ciliary action. (C) Immunological barriers eliminate pathogens and perceived foreign substances from the body via the mononuclear phagocyte system and the adaptive immune system. (D) The placental barrier consists of an inner blood-vessel-rich layer with the syncytiotrophoblast facing the bloodstream and an outer layer of trophoblasts. (E) The lymphatic barrier, or blood-lymph barrier (BLyB), is regulated by various mechanisms including extravasation, overcoming the interstitium, diffusion, and passage through the mucosal barrier. The collagen reticular network (RN) also hinders soluble substances from passing through. (F) The renal barrier, or glomerular filtration barrier, composes of the fenestrated endothelium, glomerular basement membrane and glomerular epithelium, and this hinders the passage of large molecules across the barrier. (G) Gastrointestinal barriers are associated with digestive enzyme degradation, harsh stomach acidic conditions and the small intestinal barrier. (H) Cellular barriers of the target cell include the plasma membrane, endosomal membrane and lysosomal membrane. EVs internalised by cells via endocytosis are packaged into endosomes which may risk fusing with lysosomes to undergo degradation (created with BioRender.com).*

#### **3.1 Gastrointestinal barriers**

EVs administered orally need to overcome digestive enzymatic degradation, harsh stomach acidic conditions and the small intestinal barrier before entering the bloodstream for systemic absorption. As milk and plant-derived EVs are delivered into the body naturally *via* oral consumption, they might provide key insights into how EVs can be used and/or engineered for oral administration. *In vivo* evidence in rodents showed that unmodified bovine milk-derived EVs naturally containing immune-active proteins were able to cross the intestinal barrier *via* endocytosis to treat inflammatory bowel disease (IBD) [85], and were distributed significantly in the bloodstream 24 h post-oral consumption [86]. EVs can pass through the intestinal barrier *via* intestinal epithelial cell (IEC) mediated transendocytosis, a process that requires surface glycoproteins on both EVs and target cells, based on *in vitro* findings of skimmed bovine milk-derived EVs being internalised by human colon carcinoma Caco-2 cells and rodent small intestinal IEC-6 cells [87, 88]. Paracellular translocation is another possible mechanism by which EVs may cross the intestinal epithelium,

#### *Extracellular Vesicles and Their Interplay with Biological Membranes DOI: http://dx.doi.org/10.5772/intechopen.101297*

through tight junctions between adjacent epithelial cells [76, 87, 89]. Although *in vivo* evidence is lacking, it is possible that EVs might cross the intestinal epithelium paracellularly to a greater extent in pathological conditions like IBD as the tight junctions would be disrupted [90], making the intestinal epithelium more penetrable.

Milk-derived EVs have been shown to withstand acidic and enzymatic conditions [87, 91]. However, their ability to do so might be dependent on the milk source, as EVs from processed milk would have undergone more damage than those from unprocessed milk and hence possess less integrity [87, 92–97]. Although bovine milkderived EV surface proteins CD9 and CD81 were found to be partially degraded by acidification at pH 4.6 in one study [98], these findings did not demonstrate whether these EVs can survive stomach acidic conditions, which are usually characterised by a much lower pH. Moreover, the study was focused on evaluating the effectiveness of acidification in ultracentrifugation to isolate EVs. Thus, these conditions would have differed vastly from true gastrointestinal conditions. Although the underlying mechanism is unclear, the ability of both processed and unprocessed milk-derived EVs to withstand harsh conditions might be correlated with milk calcium content [87]. This could be due to the adhering of milk calcium to the surface of EVs, which might strengthen their membrane integrity against acidic and enzymatic degradation. Another hypothesis is that calcium might influence milk-derived EV biogenesis pathways in alveoli cells to increase the expression of certain proteins or transporters in secreted EVs that enable them to withstand gastrointestinal conditions.

Fruit and vegetable-derived EVs have been shown to withstand gastrointestinal conditions and eventually be internalised by rodent intestinal tissue *in vivo*, though their passage across the intestinal barrier into the bloodstream cannot be concluded in some studies [77, 99–102]. Grape EVs derived *via* cold-pressing have been discovered to enter rodent IECs *via* macropinocytosis [100], while a previous analysis of grapefruit EVs derived *via* homogenization revealed their internalisation by intestinal macrophages *via* macropinocytosis and clathrin-mediated endocytosis [101]. Watermelon EVs were also observed to be taken up by human IECs in an *in vitro* experiment *via* clathrin-mediated endocytosis, causing the cells to multiply rapidly and their basal secretome to change [103]. Ginger EVs were found to accumulate in rodent liver tissue 12 h post-oral consumption, implying that the EVs were able to withstand gastrointestinal conditions and cross the intestinal barrier into the bloodstream while remaining intact [104]. Though unconfirmed, the uptake of plant-derived EVs *via* clathrinmediated endocytosis and macropinocytosis probably indicates that these EVs possess receptor tyrosine kinases, G protein-coupled receptors (GPCRs) and transferrin receptors [105], while passage across the intestinal barriers into the bloodstream might imply that these plant-derived EVs undergo transendocytosis like milk-derived EVs, a mechanism which requires EVs to possess surface glycoproteins [87, 88]. The ability of milk and plant-derived EVs to withstand and overcome gastrointestinal conditions and barriers makes them highly suitable as DDSs *via* the oral route as a non-invasive alternative to intravenous DDSs.

#### **3.2 Placental barrier**

The placenta supports foetal growth and development while secreting female hormones [106–111]. The placental barrier (PB) is suggested to be selectively penetrable, given that drugs administered to pregnant women can either cause adverse side effects in both the mother and the fetus or not penetrate the PB at all. It consists of an inner blood-vessel-rich layer with the syncytiotrophoblast facing the bloodstream

and an outer layer of trophoblasts [106, 112–114]. Occurring in large amounts during pregnancy [115, 116], placental exosomes exert their functions during foetal growth and development, being involved in processes like angiogenesis regulation and cell migration [106, 116–126]. This implies that they can overcome the PB, though the underlying mechanism is unclear. Placental exosomes have also been tested as diagnostic biomarkers for foetal development [106, 115] and gestational diabetes [106, 127].

Although placental EVs may be used to pass through the PB, the use of non-placental EVs to deliver drugs across the PB is a potential area for exploration. The placenta can respond to signals from immune cells and exert an inflammatory response during infection. An *in vitro* study revealed that THP-1 monocyte-derived exosomes were internalised by human placental trophoblast cells *via* clathrin-mediated endocytosis, exerting a pro-inflammatory effect that caused the cells to release cytokines [128]. Provided that this mechanism can be proven *in vivo*, packaging drugs in EVs derived from immune cells might be one way to deliver drugs across the PB. Another possible method to deliver drug-containing EVs across the PB might be *via* administering EVs that target IECs instead of placental cells, as IECs can communicate with the placenta [103]. IECs that internalise watermelon EVs can secrete watermelon EV contents *via* the formation of intestinal exosomes, which are shown to be taken up by placental cells *via* clathrin-mediated endocytosis [103]. This concept, however, is deduced from a few *in vitro* studies and has yet to be proven in a single *in vivo* experiment. Nevertheless, being able to deploy non-placenta-derived EVs to treat placental pathological conditions like chorioamnionitis may offer some flexibility in EV engineering, as researchers would not need to adhere strictly to using placental EVs.
