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Gupta et al. Extracell Vesicles Circ Nucleic Acids 2023;4:170-90 https://dx.doi.org/10.20517/evcna.2023.12 Page 180

[147]
of enveloped viruses . Similar to viruses, several energy-dependent pathways such as micropinocytosis,
clathrin-dependent, caveolin-dependent or lipid raft-mediated endocytosis processes have been reported to
be involved in the uptake of EVs into recipient cells [148-150] . Due to the existence of a heterogenous EV
population and a variable surface protein composition, the involvement of multiple uptake pathways is
expected. The presence of lectins, tetraspanins, integrins and proteoglycans on the EVs surface have shown
to be the major driver of EV uptake through energy-dependent endocytosis [151-155] . In contrast, evidence of
direct fusion with the cell membrane is limited .
[156]

Importantly, evidence of how the luminal cargo of EVs escapes the endocytic compartment is still largely
lacking, with the possibility of either a full fusion between EV and endosomal membrane or complete cargo
degradation in lysosomes. This is a critical aspect on which contradictory research exists. Some recent
studies have shown that the EVs lack the ability to induce endosomal escape and viral fusogenic proteins are
needed for efficient intracellular cargo delivery [157-159] . On the contrary, some studies have shown that EVs
have an inherent ability to escape from endosomes [160-163] . However, there are various factors that could be
driving this discrepancy. The difference in cell source could be a potential player as some early
developmental cells express ERV ENV genes such as syncytin-1 and syncytin-2, which could potentially
induce endosomal escape [164-167] . Furthermore, recent evidence suggests that a small percentage of ILVs that
are secreted as exosomes tend to retrogradely fuse with the MVB membrane during biogenesis, indicating a
[168]
possibility that a small proportion of exosomes may be able to induce membrane fusion . Importantly, the
majority of the studies have addressed biodistribution of the EVs in a specific tissue which may not reflect
the bioavailability of the cargo. In addition, with the existence of so many biological barriers, it can be
speculated that different cell sources may have similar biodistribution profiles but different bioavailability
profiles in different tissues. Therefore, much of the focus should now be on addressing the bioactivity of
EVs on a cellular level.

Strategies to enhance the in vivo pharmacokinetics of EVs
To circumvent the issue of clearance by MPS and enhance extra hepatic delivery of EVs, various
engineering strategies, both endogenous and exogenous, have been employed on the surface of EVs.

CD47-Don’t eat me signal
CD47 is a cell surface protein expressed on various normal and malignant tissues. Cancer cells utilize CD47
to prevent phagocytosis by interacting with SIRP alpha present on macrophages and thereby evading the
innate immune system. In the past decade, various delivery vectors, such as lentiviruses, have been
[169]
engineered with CD47 to prevent phagocytosis and liver clearance . Similar strategies have been explored
in the EV space as well, where EVs engineered with CD47 prevented the uptake of EVs in circulating
monocytes in vivo and extended EV half-life by 3-fold . Similar observations were made in other studies
[170]
where the expression of CD47 prevented macrophage clearance [171,172] . Furthermore, the expression of other
[173]
CD47-like molecules, such as CD55 and CD59, on EVs surface prevented complement activation in vitro .
Albumin
Albumin is the most abundant plasma protein and has a serum half-life of 3 weeks in humans. Albumin
interaction with neonatal Fc receptors allows for pH-dependent recycling . This recycling property of
[174]
albumin has attracted a lot of interest in the drug delivery field for enhancing the plasma half-life of a range
of biotherapeutics either by directly fusing albumin or introducing albumin binding domains on the surface
of the vector for interacting with endogenous albumin . Similarly, we recently developed a novel platform
[174]
for enhancing EV plasma half-life by introducing albumin-binding peptides on the extracellular loop of
CD63 . This EV engineering step allowed for enhanced extrahepatic delivery and enhanced plasma half-
[175]