**3.3 Immunological barriers**

The body is heavily guarded by immune cells responsible for eliminating pathogens and perceived foreign substances. As such, nanoparticles injected into the bloodstream risk being removed by phagocytes of the mononuclear phagocyte system (including those in the liver and spleen), or the adaptive immune system *via* antibody production [8]. Thus, in conventional non-EV drug therapy, immune cells can potentially hinder the therapeutic effects of nanoparticles by decreasing their systemic circulation half-life [10]. EVs, on the other hand, can evade removal by immune cells naturally. CD47 is a prominent component found on EVs that binds to signal regulatory protein alpha (SIRPα) on dendritic cells and macrophages, which inhibits phagocytosis *via* a "don't eat me" signal [129–131]. Other EV components found on both cancer and non-cancer cell-derived EVs like CD24, CD31 and PD-L1 have been associated with exerting a similar "don't eat me" signal, with PD-L1 also inhibiting T-cell activation [130, 132–134].

A recent study on an *in vivo* rodent tumour model seems to suggest that it may be possible for EVs to be phagocytosed by Kupffer cells in the liver and eliminated *via* biliary excretion, given that the fluorescent markers tagged to the U937 human myeloid leukaemia EVs used in the study were found to accumulate in the liver and eventually detected in the faeces [10]. However, these fluorescent markers were also predominantly detected in CT26 mouse colon adenocarcinoma cells targeted by the EVs, probably because the EVs might have already undergone disintegration in the cells, and the florescent marker component might have been excreted *via* exocytosis before being transported to the liver *via* systemic circulation. In other words, the accumulation of dyes associated with EVs in the liver is not synonymous with a

definite uptake of EVs by Kupffer cells. Nevertheless, EVs may still be removed by immune cells, as shown in another study where melanoma, myoblast, fibroblast, aortic endothelial and macrophage-like cell exosomes from rodents were eliminated by rodent liver macrophages *in vivo*, most likely due to the presence of PS on EVs which is recognised by macrophages [135]. As to whether these EVs possess high amounts of CD47, CD24, CD31 or PD-L1, the study did not include such findings.

### **3.4 Neurological barriers**

The blood-brain barrier (BBB) is characterized by an innermost layer of endothelial cells (which prevents blood and extracellular fluid from mixing), pericytes surrounding the endothelial cells and astrocyte end-feet acting as a sheath in the outermost layer. Though the movement of substances across the BBB is tightly regulated [136], different EVs have been observed to cross the BBB. One study demonstrated the ability of exosomes to carry miR-193b-3p across the BBB to exert an anti-inflammatory effect on rodent brain cells with subarachnoid haemorrhage [137], although the mechanism of crossing was unclear. Other studies involving the BBB in zebrafish showcased the ability of various human breast cancer cell EVs and brain endothelial cell EVs to cross the BBB *via* clathrin-mediated endocytosis [7, 138] and macropinocytosis [138], a notable surface protein that enabled clathrin-mediated endocytosis being CD63 [7]. Another study conducted on rodent BBB showed that human and rodent EVs derived from both cancer and non-cancer cells were able to cross the BBB *via* adsorptive-mediated transcytosis, which correlated with the presence of CD46 on the surface of EVs [11].

Modifications have also been made to EVs to enhance their ability to cross the BBB. In one experiment, after overexpressing the rabies virus glycoprotein (RVG) peptide on their surface, dendritic exosomes became significantly localized in rodents' brain cells [139]. Mouse L929 fibroblastic cell exosomes loaded with methotrexate and functionalized with LDL peptide in another experiment showed enhanced BBB exosome extravasation in rodents [140]. When miR-210-loaded mesenchymal stromal cell-derived exosomes were coupled with c(RGDyK) peptide in another experiment, they displayed enhanced targeting of rodent ischaemic brain cells, indicating greater angiogenesis and improving animal survival significantly [141]. Another experiment showed that RGE-Exo EVs demonstrated greater accumulation and duration of accumulation in murine glioma tumour cells than free exosomes [142].

Apart from surface components, the size of EVs might also be a crucial factor in determining whether EVs can cross the BBB, as deduced from another study where intranasal administration of exosomes to rodent microglial cells *via* the extra-neuronal pathway showed rapid translocation of exosomes to target cells, in contrast to larger microparticles of at least 500 nm in diameter which did not reach these target cells [143]. However, surface components of EVs might be a more vital factor than the size of EVs in enabling passage across the BBB, as proven by how larger brain endothelial EVs can penetrate the BBB better than smaller EVs of the same cell source, due to the higher levels of CD63 in the larger EVs [7, 106].

The blood-labyrinth barrier (BLaB) and blood-retinal barrier (BRB) are two other neurological barriers pertaining to the ear and eye, respectively. The BLaB consists of five layers, namely, the blood-endolymph barrier, blood-perilymph barrier, cerebrospinal-fluid-perilymph barrier, middle-ear-labyrinth barrier and endolymphperilymph barrier [106, 144]. The BRB consists of the retinal vascular endothelium and the retinal pigment epithelium (RPE) [106]. These two barriers share similarities

with each other and the BBB, though the number of EV studies on these two barriers is smaller than that involving passage across the BBB [106]. Nevertheless, the utilization of EVs as potential drug carriers targeting the ear and eye with negligible side effects is worth further research, especially when current drug treatments have resulted in adverse side effects [106]. EVs from RPE cells are involved in the progression of age-related macular degeneration *via* regulating the production of pigment granule and lipid balance in RPE cells [106, 145]. They also promote vascular leakage *via* miR-105 which interferes with the tight cellular junctions of barriers [106, 146]. It is hoped that these seemingly destructive EV mechanisms can be manipulated to enable drug delivery across the BLaB and BRB, by modifying these EVs in a way that does not harm the barriers yet still permits their passage across them.
