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Page 181 Gupta et al. Extracell Vesicles Circ Nucleic Acids 2023;4:170-90 https://dx.doi.org/10.20517/evcna.2023.12

life of EVs.

PEG
The most commonly used strategy in pharmaceutics to enhance drug pharmacokinetic properties is the
conjugation of PEG to prevent hydrophilic and electrostatic interaction of serum proteins with the delivery
vector . This strategy has been used in various clinically approved delivery vectors, such as LNPs and
[176]
[177]
liposomes . Similarly, EVs have also been exogenously engineered with PEG, which in turn enhanced the
plasma half-life, decreased hepatic uptake, and improved extrahepatic delivery .
[178]
Although only a few strategies have been explored in augmenting EVs plasma half-life, it is evident that for
enhancing tissue targeting properties of EVs, preventing phagocytic clearance is advantageous.

Factors governing EVs in vivo biodistribution
Route of administration
Route of administration can play a critical role in the in vivo biodistribution of EVs, as different injection
routes will present a different kind of barrier for EVs to achieve tissue-specific delivery. Even within
systemic routes, substantial differences exist between the three most commonly used methods; intravenous
(IV), intraperitoneal (IP), and subcutaneous (SC) administration. For instance, drugs administered through
SC routes into hypodermis will face adipose tissue and a fibrovascular network of blood and lymphatic
vessels [179,180] . Drugs smaller than 16 KDa tend to diffuse through blood capillaries, while larger molecular
weight drugs such as antibodies or LNPs adopt an indirect route first by diffusion through the cells and
[179]
ECM, followed by uptake into the lymphatic system to enter the systemic circulation . Since lymphatic
vessels lack basement membrane, the permeability is much higher as compared to continuous endothelium,
and hence allows diffusion of large drug carriers. However, this model is entirely dependent on diffusion
through the extracellular matrix, and larger molecules may be rather inefficient in diffusing through the
ECM. In addition, lymphocyte-mediated clearance in the lymphatic system will limit the release of cargo
[180]
into the systemic circulation . A similar trend is also observed with EVs, where we and others have seen
very small levels of EVs entering systemic circulation . Similar barriers also need to be surpassed upon IP
[80]
injection. The peritoneal cavity consists of a mesothelial cell monolayer attached to multiple layers of
connective tissues with embedded vascular and lymphatic system networks . In general, two different
[181]
mechanisms have been proposed for drug uptake from the peritoneal cavity. Similar to SC administration,
the nanoparticles enter the systemic circulation through lymphatic drainage. Importantly, IP administration
of EVs shows a delay in entering systemic circulation , and this could be due to the lymph flow rate, which
[80]
is 400-700 times slower than the blood flow rate in humans .
[182]
For local administration routes such as intramuscular, intratumoral, intracerebral, intranasal, or
intracerebroventricular, the majority of the EV dose is localized in the injected tissue interstitium, and only
a small fraction enters systemic circulation [76,80,87,183,184] . This is primarily due to the above-discussed tight
endothelial barriers present in these tissues, which regulate the exchange of materials. In addition to the
previously discussed strategies to manipulate distribution and pharmacokinetics, several other strategies
have been developed for specific tissue targeting, as discussed below.

Targeting strategies
The appreciation of EVs as potential therapeutic agents owes not only to their ability to serve as a protective
natural delivery vector but also to the numerous reports demonstrating that EVs are targetable. Tissue
targeting of functional EVs was successfully demonstrated more than ten years ago, using an approach
[93]
where the cell source was engineered to produce EVs displaying targeting moieties . In this pioneering