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Page 173 Gupta et al. Extracell Vesicles Circ Nucleic Acids 2023;4:170-90 https://dx.doi.org/10.20517/evcna.2023.12

EV CHARACTERIZATION
As aforementioned, EVs are a heterogenous pool of vesicles that contain proteins, a variety of nucleic acid
[4]
species ranging from small RNA to full-length mRNA and DNA, and lipids . Since EVs are smaller than
the wavelength of visible light, reliable detection of EVs is challenging. However, over the years, various
[57]
methodologies have been developed to achieve reliable quantification of EVs .

Levels of any of the biomolecular cargo or the vesicle as a structure can serve as a basis for EV
quantification. Based on this, various means of quantifying EVs have been developed. Technologies which
[58]
[59]
measure hydrodynamic sizes, such as nanoparticle tracking analysis (NTA) and resistive pulse sensing ,
are sensitive and provide a robust means for sizing and determining EV concentrations. However, these
methods fail to distinguish an EV from other non-vesicular particles of similar size. Hence the specificity of
the assay is entirely dependent on the choice of purification method, as certain methods tend to copurify
nonlipoprotein complexes and protein aggregates [22,60,61] .

Recently, flow cytometry-based applications have emerged to quantify EVs at a single vesicle level [62-66] .
However, considering the size of an EV, especially the small EVs, the amount of light scattered fails to
trigger the sensor on conventional flow cytometers. Therefore, a flow cytometry-based application needs to
be coupled with the detection of a fluorescent antibody or a fluorescent lipophilic dye to get a robust
signal [62-66] . Apart from light-scattering-based tools, transmission electron microscopy (TEM) can be used
for the quantification of the EVs, but the size of a vesicle can differ largely due to the process of sample
fixation, which could lead to swelling or shrinking of the EVs .
[67]

EVs can also be quantified by measuring the cargo loaded inside the EVs. In this regard, total protein
amount, total lipid content, and total RNA have been used as a way of determining EV levels [57,68] . Especially
the total protein content is still widely used across the research field . Although these methods are high
[19]
throughput and do not require expensive instrumentation, the propensity of measuring non-vesicular
contaminants in EV preparation is high. Therefore, measuring the levels of EV-associated protein, for
instance, CD63 or CD81, can provide reliable means of quantification but may not reflect the heterogeneity
of the EVs [69-74] .

Overall, despite the technological advancement and availability of a range of highly sensitive methods,
achieving accuracy in EV analytics is still challenging. In addition, the unit particles/mL is an arbitrary unit
as one sample measured on different equipment can yield different values. Reflecting on this, we previously
performed a meta-analysis of 64 pre-clinical studies using EV as a therapeutic intervention and identified
huge variations up to 3 logs in EV doses either based on protein amount or total particles across different
studies . Moreover, this variation in therapeutic dosing could not be resolved even after segregating the
[75]
data either based on the type of disease or purification method used or cell source. This clearly reflects that
every step from purification to characterization in EV-related research leads to its own variation, which
leads to discrepancy in dosing, hence affecting the reproducibility of the work.

TOOLS FOR IMAGING EVS
EV labelling can be achieved in two ways, either by general labelling of EV-associated macromolecules or by
labelling a specific macromolecule in an EV. There is a range of tools available for labelling EVs with a
tracer, both endogenous and exogenous strategies [Figure 1]. This section will discuss a few of the
commonly used EV labelling strategies.