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Page 448 Shami-shah et al. Extracell Vesicles Circ Nucleic Acids 2023;4:447-60 https://dx.doi.org/10.20517/evcna.2023.14

INTRODUCTION
Extracellular vesicles (EVs) are nanoscopic, membrane-bound, phospholipid-rich vesicles that are secreted
[1-3]
by both prokaryotic and eukaryotic cells into the extracellular space . EVs play a crucial role in
intercellular communication because they can transport a wide variety of biomolecules such as proteins,
[2-4]
lipids, nucleic acids, and metabolites between cells in a protected lipid shell . Historically, EVs were
regarded as cellular debris only responsible for clearing biomolecular byproducts . Recently, there has been
[5]
a tremendous rise in interest towards EVs because they have been established as a highly promising source
of circulating biomarkers .
[2,4]

EVs encompass several different types of vesicles, including exosomes, microvesicles, and apoptotic bodies,
[2]
which differ in size and origin . Exosomes are the smallest type of EVs (usually ranging from 30-100 nm)
and are formed by endosomal cell sorting pathways in carefully orchestrated packing, budding, and fusion
processes regulated by the ESCRT complex. In this pathway, intraluminal vesicles are formed by the
invagination of multivesicular bodies. Various cargo molecules, such as proteins, nucleic acids, and
metabolites, are packed inside these intraluminal vesicles, and they are eventually released as exosomes into
[6,7]
the extracellular space through exocytosis . Microvesicles are larger than exosomes (ranging from
100-1,000 nm), and are formed directly when a piece of the plasma membrane buds off into the extracellular
space forming a vesicle . The outward budding process involves the physical bending of the phospholipid
[6]
membrane, which is tightly regulated by a series of calcium-dependent enzymatic steps involving
[6]
scramblases, flippases, and aminophospholipid translocases . Apoptotic bodies are another type of EVs
(ranging from 1-5 µm), and are formed during apoptosis when the plasma membrane of dying cells
becomes blebbed and forms vesicles . The EV biogenesis process is discussed in greater detail in other
[2,6]
reviews [2,8,9] .

EVs are involved in a wide range of pathophysiological processes, including immune responses ,
[10]
a
inflammation , cancer , neuropsychiatric , n d neurodegenerative d i s e a s e s . F o r e x a m p l e ,
[12]
[13]
[11]
[10]
microvesicles and exosomes released by immune cells can elicit an immune response by presenting antigens
to other immune cells and initiating immune infiltration . Moreover, exosomes have been shown to play a
[10]
role in the spread of cancer cells, as they can transport oncogenic proteins and RNA to other cells, leading
to tissue invasion and metastasis [11,14] . Additionally, there is increasing evidence suggesting that cancer-
derived EVs carry differentially expressed molecular signatures than EVs from healthy tissues, suggesting
the applicability of EVs for diagnostics [1,4,11] .
With the growing understanding that EVs comprise a new class of biomarkers, EVs are being increasingly
investigated as diagnostic and therapeutic tools. EVs can be isolated from biological fluids such as blood,
cerebrospinal fluid, urine, saliva, and breast milk, and their biomolecular contents may be analyzed to
provide information about the disease progression of patients . Additionally, EVs can be engineered to
[2]
deliver therapeutic agents to specific cells or tissues, making them a potential delivery vehicle for drugs .
[15]
Other excellent reviews focus more thoroughly on the utility of EVs as sources of biomarkers in
diagnostics [16,17] and as delivery vehicles in therapeutic [15,16,18] applications.

To be effectively utilized for biomedical applications, high-quality EV isolation techniques are required. The
heterogeneity of EV size and their relatively low abundance compared to other contaminants in biological
fluids make high-quality EV isolation a difficult challenge [4,19] . Many techniques have been developed for the
isolation and characterization of EVs, including reduced solubility approaches, ultracentrifugation, density
[20]
gradient separation, size-exclusion chromatography, microfluidics, and magnetic bead-based methods .
While these techniques have limitations, they have aided in advancing the field of EV research. This review