**3.8 Cellular barriers**

EVs that eventually reach the target cell has to overcome the plasma membrane, escape the endosome and evade lysosomal degradation to release their cargo into the cytosol (**Figure 3**).

The plasma membrane is an intricate structure consisting of various domains formed *via* different mechanisms. Some of these mechanisms include the formation of plasma membrane protein fences to reduce lateral diffusion in the plasma membrane, the arrangement of plasma membrane proteins into a scaffold that interacts with certain plasma membrane lipids, and protein-lipid interaction to form lipid rafts [174]. These domains represent the first barrier that a freshly-secreted EV needs to cross and determine the way the target cell internalizes EVs. Environmental factors also influence the interaction of EVs with the plasma membrane. For instance, the uptake of EVs *via* fusion with the cell membrane is observed to occur at a higher rate under acidic conditions [175], while endocytosis is shown to be hindered by neutral pH or high cholesterol levels [14, 176].

EVs can be internalised by target cells *via* endocytosis, be it caveolae-dependent endocytosis, flotillin-dependent endocytosis, ARF6-dependent endocytosis or other forms of endocytosis [105]. Clathrin-mediated endocytosis (CME), or "receptor-mediated endocytosis", plays an especially prominent role in the uptake of small EVs [105],

#### **Figure 3.**

*Internalisation, lysosomal degradation and lysosomal escape mechanisms of extracellular vesicles (EVs) in target cells. Upon reaching the target cell, EVs may be internalised by the cell via endocytosis or direct fusion with the plasma membrane. EVs that are internalised via endocytosis are packaged into endosomes, which may fuse with lysosomes to degrade the EVs. To escape lysosomal degradation, endocytosed EVs may undergo retrograde trafficking to the trans-Golgi network, endocytic recycling to be secreted out of the cell, or another mechanism altogether. Endocytosed EVs that do not undergo lysosomal degradation fuse with the endosomal membrane via the mediation of soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) proteins to release their cargo into the cytosol. EVs that fuse directly with the plasma membrane to release their cargo into the cytosol evade endosomal and lysosomal activity completely (created with BioRender.com).*

as supported by recent studies on the uptake of human epidermoid carcinoma EVs [177] and rat pheochromocytoma EVs [178] by human cervical carcinoma (HeLa) cells and rat bone marrow-derived mesenchymal stromal cells respectively. During CME, a temporary membrane scaffold forms as a result of membrane binding of Bin/amphiphysin/Rvs (BAR) domain-containing proteins which recruit clathrin. Clathrin then binds to the cytoplasmic tails of membrane proteins with the help of adaptor proteins, resulting in a clathrin-coated pit that internalises the EV [105]. EVs internalised *via* endocytosis are packaged into endosomes. These EVs then proceed to release their cargo into the cytoplasm by fusion of their membranes with the endosomal membrane, a process mediated by soluble *N*-ethylmaleimide sensitive factor attachment protein receptor (SNARE) proteins which join the cytosolic sides of the EV and endosomal membranes together [3].

In general, the CME of EVs is initiated through the mediation of lectins, tetraspanins, cell adhesion proteins and other receptor-ligand interactions [18, 179]. For instance, exosomes from macrophages possess C-type lectin, which interacts with the C-type lectin receptor found on dendritic and brain endothelial cells [179, 180]. Galectin-5 on red blood cell (RBC) EVs enables them to be internalised by macrophages [45, 179]. Integrins on tumour EVs have been associated with the uptake of these EVs by lung fibroblasts and liver macrophages [42, 179]. Exosomes and target cells can exploit the interaction between intercellular adhesion molecule 1 (ICAM-1) and lymphocyte function-associated antigen 1 (LFA-1) in exosomal uptake [179–181]. Heparan sulphate proteoglycans on target cells bind to EV fibronectin to facilitate uptake of EVs [54, 179, 182]. The high levels of outward-facing PS on the surface of exosomes also enable the recognition and

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

uptake of these exosomes by antigen-presenting cells *via* T-cell immunoglobulin and mucin domain (TIM) receptors located on the antigen-presenting cells' surface [63, 179, 183].

EVs internalised *via* endocytosis might risk being degraded by lysosomes in the cytosol [3]. The fusion of the membrane of endosomes containing the endocytosed EV with the lysosomal membrane is mediated by SNARE proteins and involves the active transport of vesicles along the cytoskeleton [18]. EV size may play a role in determining the fate of EVs upon uptake *via* endocytosis, as EVs larger than 100 nm may require macropinocytosis for their uptake, which tends towards lysosomal degradation more than other internalisation mechanisms accessible to smaller EVs [177, 184, 185]. Endocytosed EVs might escape lysosomes *via* pathways similar to those of viruses, like the CD81 positive lysosome-associated membrane protein 1 (Lamp-1) negative route in dendritic cells which resembles that of HIV-1 uptake [186]. A study showed that HEK293 exosomes internalised by human fibroblastic, hepatic and renal cells were transported to the endoplasmic reticulum where they released their cargo, a pathway that might be a potential escape route from lysosomal degradation [187]. EVs may also evade lysosomal degradation *via* endocytic recycling out of the cell [188] or retrograde trafficking from the endosomal pathway to the trans-Golgi network [189].

EVs have also been reported to deliver their cargo into target cells *via* direct membrane fusion with the cell membrane, with EV surface proteins syncytin-1 and syncytin-2 seemingly playing a significant role in this [190–192]. Originally found on the plasma membranes of placental trophoblast cells [190, 191], gamete cells [190, 193] and various cancerous and non-cancerous cells known to fuse directly with other cells [190, 194–197], these proteins have also been detected on EVs secreted from these cells [190, 192]. In light of this, incorporating these surface proteins into EVs to increase their uptake *via* direct membrane fusion might be a possible way to evade endocytosis and lysosomal degradation completely.
