**3.6 Pulmonary barrier**

Located in the lungs, the blood-air barrier (BAB) possesses characteristics that enable it to guard against pathogenic invasion. For instance, the lung mucosa is a rich source of immune cells [106, 159], and lung epithelial cells can sense a wide range of bacteria and viruses *via* a broad array of membrane-bound, endosomal and cytosolic pattern-recognition receptors (PRR) [106, 160]. In response to the presence of pathogens, the BAB regulates paracellular flow, cell-to-cell communication, synthesis of mucus and the composition of periciliary fluid [160], which complements the removal of foreign substances *via* ciliary action [106, 161]. While the passage of EVs across the BAB is still largely unexplored [106], exosomes derived from bronchoalveolar lavage fluid (BALF) have been discovered to possess a similar protein profile to mesenchymal derived dendritic cells, given that they carry CD54, CD63, CD86 and in particular, MHC classes I and II [162, 163], implying their involvement in triggering

an immune response against pathogenic invasion in the BAB. In light of this, BALF exosomes might potentially be used as diagnostic biomarkers for pathogenic detection, in addition to engineering them as personalised medicine for effective drug delivery across the BAB.

### **3.7 Renal barrier**

The nephron's ability to efficiently filter out waste materials from the blood into the urine is attributed to the high pressure in the glomerulus due to high blood flow, as well as the presence of the glomerular filtration barrier consisting of three layers—fenestrated endothelium, glomerular basement membrane and glomerular epithelium [66, 164–166]. Despite the tiny pores (2.5–2.8 nm) of the glomerular basement membrane [166, 167] which are smaller than the smallest EVs (30 nm [16–18]), and the presence of filter proteins lining the slits in the glomerular epithelium [165, 166, 168], the urine is surprisingly a rich source of EVs from both renal and non-renal sources. While the majority of EVs found in urine originate from the kidney, urinary bladder, testis, prostate, epididymis and seminal vesicle [169–172], studies have also identified EVs from outside the urinary tract, such as those carrying biomarkers of acute myocardial infarction [173]. EVs injected into the bloodstream of rodents in one study were found to accumulate in the kidneys, and eventually the urine, without undergoing degradation, as indicated by their ability to be internalized by HEK293 cells after being retrieved from the urine and introduced to the cells [166]. The presence of EVs in urine might imply that EVs can squeeze through the tiny pores of the glomerular filtration barrier due to their fluid membranes, or undergo mechanisms like transcytosis to reach the glomerular filtrate. It is also logical to deduce that EVs can cross the glomerular filtration barrier better in pathological conditions like diabetic nephropathy when the glomerular filtration barrier becomes more porous due to injury [166].
