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Page 171 Gupta et al. Extracell Vesicles Circ Nucleic Acids 2023;4:170-90 https://dx.doi.org/10.20517/evcna.2023.12

[4]
active molecules, including various RNAs, proteins and bioactive lipids derived from their producer cell .
The intercellular transfer of these functional cargo renders EVs an essential player in physiology. As nature’s
very own nanoparticles, EVs intrinsically benefit from stability in circulation, biocompatibility, immune
tolerance and the ability to cross biological barriers to reach hard-to-target organs such as the brain and
muscles . These unique properties of EVs have inspired many to use them as a next-generation drug
[5,6]
delivery tool. Therapeutic EV research has seen tremendous development in the past decade, from in vitro
studies towards pre-clinical models to clinical trials . Even so, the road towards successful clinical
[5]
translation has various obstacles, primarily due to the knowledge gap in EV biology. One of the primary
challenges is to achieve reliable detection of exogenous EVs in vivo and to understand their in vivo
pharmacokinetic properties. This is particularly challenging given the fact that the extracellular space is rich
in various nanoparticulate species ranging from apoptotic bodies to exosomes, lipoprotein complexes, and
ribonucleoprotein complexes, etc. . Therefore, tools for characterizing their spatiotemporal properties have
[7]
been developed to dissect their biological input in pathophysiology, development, and biotherapeutic
delivery . By harnessing the power of these tools, various studies have shed light on EV in vivo
[7]
biodistribution and underpinned various factors governing in vivo biodistribution. This review discusses the
current state of the art for the technologies used in purifying, characterizing and labelling EVs, followed by
general trends in vivo biodistribution of EVs, and discusses various biological barriers EVs experience in
vivo before entering specific tissue. Furthermore, the review discusses the major challenges that need to be
addressed to achieve targeted delivery of EVs.

PURIFICATION OF EVS
Purification of EVs is a significant challenge as the extracellular environments in both biofluids and cell
culture supernatants are rich in protein aggregates, lipoprotein complexes, non-EV bound RNA, cell debris
and a heterogeneous pool of EVs [8-11] . Each of these components can have a physiological effect on the
recipient cell . Therefore, it is critical to have a robust EV purification method that enriches EVs to dissect
[12]
their biological, therapeutic, and diagnostic roles. Different purification methods may need to be combined
based on the desired EV application to achieve a highly pure EV preparation [13-15] . For example, EV
purification from plasma is challenging as it is rich in albumin, lipoproteins and protein aggregates, and
these contaminants overlap with EVs with regard to various physiochemical parameters [16-18] . Hence, the
purification of EVs is of great importance in all areas of EV research. Since the first use of high-speed
centrifugation for purifying EVs, the purification methods have evolved rapidly with the help of expanding
knowledge about EVs and can now be isolated based on various physicochemical properties such as density,
size, charge, or affinity to specific biomolecules on the surface of EVs .
[18]

Ultracentrifugation
[19]
Centrifugation-based EV purification is still the most widely used method across the EV field . The
traditional process involves a series of low-speed spins to clear cell culture supernatants or biological fluids
from cells, apoptotic cell debris, and other large microscopic particles. The precleared supernatant is further
processed using multiple high-speed centrifugations ranging from 10,000 g to 200,000 g to recover large
vesicles to small vesicles, including exomeres [20,21] . Since the separation of EVs is based on size and density,
protein aggregates tend to copurify with EVs . For enhanced EV purity, density gradient centrifugations
[22]
with 30%-60% sucrose cushion or iodixanol can be employed to remove vesicle-free proteins or protein-
RNA aggregates [23,24] . Although ultracentrifugation (UC) on a density gradient yields pure EV populations,
the method itself is variable as the yields are dependent on user handling and experience. Furthermore,
particle disruption, aggregation, and lack of scalability are still significant limitations of the
ultracentrifugation-based purification methods [25,26] .