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Visualization strategies of extracellular vesicles: Illuminating the invisible
‘dust’ in theranostics
Kaiyue Zhang
*,1
, Jingxuan Hu
1
, Yilan Hu
1
Columbia University, New York City, NY, 10032, USA
ARTICLE INFO
Keywords:
Extracellular vesicles
Visualization labeling methods
Theranostic applications
ABSTRACT
Extracellular vesicles (EVs), lipid bilayer vesicles with diameters of 30-150 nm derived from nearly all cell types,
were once dismissed as “platelet dust” but are now recognized as key mediators of intercellular communication,
playing vital roles in both physiological and pathophysiological processes. However, the clinical applications of
EVs in theranostics remain limited by the challenge of effectively visualizing them at high resolution both in vitro
and in vivo, primarily due to their nanoscale size. To address this limitation, researchers worldwide are devel-
oping innovative methods for labeling and visualizing EVs, aiming to unlock their full potential in optimized
theranostic applications. This review provides a comprehensive overview of current strategies for EV labeling
across various experimental settings and highlights their promising theranostic applications of diverse diseases.
1. Introduction
Extracellular vesicles (EVs), composed of the plasma membrane with
embedded transmembrane proteins and encapsulated contents, play a
critical role in cell-to-cell communication under both physiological and
pathophysiological conditions.
1–3
Based on their size and biogenesis,
EVs are typically categorized into three subtypes: exosomes (30–120
nm), microvesicles (50–1000 nm), and apoptotic bodies (50–2000 nm).
4
Exosomes are generally believed to form and be released from endo-
somes, while microvesicles bud directly from the cell membrane.
Apoptotic bodies, on the other hand, are considered to originate from
the cell membrane during cell apoptosis.
5,6
It is important to note that
there remains some debate over the size distribution and naming con-
ventions of different EV types, so we refer to them collectively as EVs
rather than distinguishing between exosomes and microvesicles here.
All kinds of EVs have been reported to carry a wide range of bioactive
molecules, including proteins, peptides, genetic materials (mRNAs and
microRNAs), and lipids, which can facilitate intercellular communica-
tion by shuttling information in an endocrine or paracrine manner.
1,7
These vesicles transport specic biomolecules-such as proteins, lipids,
mRNAs, microRNAs, and DNAs-to recipient cells, regulating their
physiological and pathological processes (Fig. 1).
8,9
EVs offer advan-
tages such as immune tolerance and stability in the circulatory system
and are widely distributed in body uids, including plasma, urine,
saliva, tears, and cerebrospinal uid.
10
In some cases, the distribution of
EVs varies depending on their sources, for example, tumor-derived EVs
exhibit tumor targeting ability, while the intrinsic properties of EVs can
determine organ-specic targeting and contribute to tumor metastasis
preferences11. Given these features, there is growing interest in the
diagnostic and therapeutic potential of EVs in cancer and other dis-
eases.
12–14
For instance, EVs derived from mesenchymal stem cells
(MSCs) have shown remarkable promise in promoting tissue regenera-
tion and delivering antitumor drugs.
10,15–20
Therefore, it is crucial to
develop a reliable, high-resolution visualization strategy to monitor EVs
in both in vitro and in vivo, to better understand their biodistribution and
pharmacokinetics in current clinical applications. Intense efforts from
global are underway to advance research in EV visualization to maxi-
mize their potential in theranostic applications.
21–25
Here, we will
discuss the current strategies for labeling and visualizing EVs in both in
vitro and in vivo contexts, with a focus on their theranostic applications.
2. EV isolation
Based on current research, EVs are secreted from almost all cell types
and have been isolated from cultured cells, tissues, and a wide range of
bodily uids, including plasma,
26
breast milk,
27
urine,
28
saliva,
29,30
synovial uid,
31
bile,
32
amniotic uid,
33
semen,
34
and ascites uid.
35
Multiple methods have been proved that can be used to isolate EVs. At
* Corresponding author. Columbia University, 3960 Broadway, New York City, NY, 10032, USA.
E-mail address: kz2518@columbia.edu (K. Zhang).
1
There authors contributed equally to this work.
Contents lists available at ScienceDirect
Extracellular Vesicle
journal homepage: www.elsevier.com/locate/vesic
https://doi.org/10.1016/j.vesic.2024.100061
Received 27 October 2024; Received in revised form 2 December 2024; Accepted 4 December 2024
Extracellular Vesicle 4 (2024) 100061
Available online 11 December 2024
2773-0417/© 2024 The Authors. Published by Elsevier Inc. on behalf of American Association of Extracellular Vesicles. This is an open access article under the CC
BY-NC-ND license (
http://creativecommons.org/licenses/by-nc-nd/4.0/ ).
present, there is still debate about the best EV isolation method.
The gold standard for EV isolation is ultracentrifugation. It includes
differential centrifugation and density gradient centrifugation. Differ-
ential centrifugation uses different speeds to gradually precipitate and
remove large cell debris. The nal separated EV pellet can be deter-
mined based on its size.
36
Density gradient centrifugation loads EV
samples into a density gradient medium, usually sucrose. During ultra-
centrifugation, EVs are located in a specic density gradient.
37
The
purity of EVs obtained by this isolation method is relatively high. Other
methods for EV isolation, often used in combination with differential
centrifugation, include: 1) Precipitation. For the precipitation method to
isolate EVs, samples containing EVs are incubated with a buffer (such as
polyethylene glycol, PEG) that changes their solubility or sedimentation
rate. After incubation, high yield EVs can be recovered at low centrifugal
force, usually lower than 4000×g.
38
2) Size exclusion chromatography.
EVs can be separated from other extracellular molecules by exclusion
columns based on their size.
39
3) Immunoafnity isolation.
Immuno-isolation of EVs is achieved through the conjugation of mag-
netic beads with antibodies that can bind to EV proteins.
40
4) Membrane
ltration. Separate EVs through continuous ltration using membranes
of different sizes.
41
The purication efciency is limited by the mem-
brane’s molecular weight cut-off (MWCO). 5) Microuidics techniques.
The recent progress of microuidics platform combined with above
isolation principles (e.g., immune-afnity-based micro-device,
size-based microchip, and multi-approaches integration microuidic
platform) makes integration of EV isolation and further analysis
possible.
24
Lately, more new instruments are being developed for EV isolation.
For example, Luo et al. utilized tangential ow ltration (TFF) systems
to separate the supernatant of NK cells, and ultracentrifugation to obtain
NK cell-derived exosomes (NK-Exo). This represents an efcient and
large-scale method for isolating EVs.
42
An automatic EV isolation system
named EXODUS is another label-free and high-efcient EV isolation
system. Its utilization of negative pressure oscillation and dual-coupled
oscillator membrane vibration enables purication efciency far supe-
rior to other isolation methods.
43
The development of these new in-
struments is ultimately aimed at achieving a streamlined closed
production line to efciently isolate large-scale good manufacturing
practices (GMP)-grade EVs.
For the quality control of isolated EVs, common methods include
using transmission electron microscopy (TEM) and nanoparticle
tracking analysis (NTA) to determine the morphology and size of EVs.
44
Protein components in EVs can be investigated by western blotting.
45
As
for determining the subtype of EVs, the emergence of nano ow
cytometry offers some new possibilities. The next development trend
will inevitably be a digital and automatic system that integrates
large-scale manufacturing of EVs with a leable quality control system.
3. Conventional labeling strategies for EV visualization
Many label-free EV visualization strategies have been developed to
characterize EVs’ morphology, size, and other physical properties,
including transmission electron microscopy (TEM), cryo-electron mi-
croscopy (cryo-EM), scanning electron microscopy (SEM), nanoparticle
tracking analyzer (NTA), atomic force microscopy (AFM), Raman
spectroscopy, and microuid-based imaging et al. (Table 1). However,
one of the biggest challenges in EV research is investigating how EVs
transfer to recipient cells and where EVs go in the recipient cells in vitro
and in vivo. To visualize these processes, it is important to choose a
suitable method to label EVs for different visualization purposes. As we
Fig. 1. Biogenesis of extracellular vesicles. In addition to apoptotic bodies, EVs are usually categorized into exosomes and microvesicles according to biogenesis and
size. At present, it is typically believed that exosomes are formed and released from endosomes, while microvesicles are generated by budding directly from the cell
membrane. Several proteins are implicated in EV biogenesis and are usually used as EV markers, such as CD9, CD81, CD63, TSG101, Alix et al. Other proteins are may
also present in EVs including adhesive molecules (such as integrins), immunomodulatory proteins (such as PD-1, PD-L1) and more. In general, EVs can contain
different types of cell surface proteins, intracellular proteins, RNA, DNA, amino acids, and metabolites.
K. Zhang et al.
Extracellular Vesicle 4 (2024) 100061
2
know, the EVs have a lipid bilayer containing transmembrane proteins
and carry special cargos including cytosolic proteins, and nucleic acid.
46
Thus, lipids, proteins, and nucleic acids of EVs are all capable of being
labeled directly for tracking EVs in vitro and in vivo (Fig. 2).
3.1. Lipids
Many lipid dyes, such as PKH26/PKH67, DiR/DiD, and MemGlow,
have been widely used for labeling EVs.
60–64
For instance, Hoshino et al.
labeled EVs derived from different tumor cell lines with lipophilic
PKH26 dye (red) to investigate the tumor organotropic metastasis
determined by the tumor EV integrins.
65
Similarly, Grange et al. labeled
the EVs with lipophilic uorescent stain DiD to study the biodistribution
and the renal localization of EVs in acute kidney ischemia mouse
model.
66
These lipophilic uorescent dyes with distinct uorescence
colors, can bind to lipophilic molecules and incorporate into mem-
branes, providing a convenient tool for multicolor imaging of EVs in vitro
and in vivo. Compared to the previous two kinds of lipid dyes, MemGlow
offers superior contrast, brighter imaging, and less tendency to aggre-
gate. It has numerous applications in neuroscience.
67
However, the practical issue of lipid dyes is that their half-life is
much longer than that of EVs. Therefore, when EVs are taken up by cells,
the lipid uorescent dye is absorbed by the cells and redistributed.
68
Thus, lipid dye labeled EVs are only suitable for monitoring bio-
distribution of EVs in a short term. For long-term monitor or pharma-
cokinetic analysis of EV research, using lipid dyes may lead to a false
positive result.
3.2. Proteins
To investigate the stability or pharmacokinetic properties of EVs,
such as blood levels, urine clearance, and tissue retention, various
genetically encoded uorescent or bioluminescent reporters have been
developed for labeling EV proteins. In general, when exogenously
expressing these reporter proteins in the donor cell cytosol, the reporter
proteins can freely shuttle into the lumen of EVs.
69
However, the la-
beling efciency will be extremely limited. To elevate the labeling ef-
ciency, several general methods were developed to label all subtypes of
EVs. For instance, fusing a palmitoylation signal in-frame to the N-ter-
minus of a uorescent protein (eGFP or tdTomato) induces its locali-
zation at the cell membrane and the secreted EVs membrane.
70
By the
uorescent EVs membrane reporters, Lai et al. tracked tumor EV
transference between cells in culture and visualized the EVs in the tumor
process in tumor animal models by intravital microscopy. In addition to
anchoring reporter proteins inside of EV membrane, expression of the
reporter proteins which fused with the C1C2 domain of lactadherin in
Table 1
Label-free EV visualization strategies.
Visualization
methods
EV resources Equipments In
vivo/
in vitro
Target cells or organs Applications Advantages and limitations Refs
TEM Milk TEM In
vitro
RAW264.7,
splenocytes, and
intestinal cells
Observing the particle size of
EVs
Advantages: Low sample size, single
EV analysis, visualization of
membrane structure and intravesicular
structure
Limitations: Complex sample
preparation and high-resolution
limitations
47
Astrocytes TEM In
vitro
Microglial
endosomes
Prove the diameter range of
EVs
48
Urine TEM In
vitro
NA Observe the morphology of EVs
and measure their diameter
49
Cryo-EM Cardiac-resident
progenitor cells
(CPC)
Cryo-EM In
vitro
Cardiomyocytes
(CM)
Verify the correct
transmembrane direction of
receptors in Exo CXCR4
Advantages: Most native EV
morphology, single EV analysis, low
sample size
50
Stem/stromal cells
(SCs)
Cryo-EM In
vitro
Colo-cutaneous
stulas
Prove the high size
polydispersity of EVs
Limitations: Using specialized
equipment, sample preparation is
complex
51
SEM 293T cells SEM In
vitro
Brain neuron cells Study the morphology and size
of exosomes
Advantages: Low sample size, single
EV analysis, and visualization of
surface morphology.
52
Limitations: Risk of agglomeration
and dehydration during sample
preparation
NTA Placenta NTA In
vitro
NA Quickly determine the size and
phenotype of EVs
Advantages: Real time, high-
resolution analysis of individual EVs
53
Human broblasts NTA In
vitro
Mesenchymal
progenitor cells
Analyze the concentration and
size distribution of EVs
Limitations: Unable to provide
information on the composition or
cargo of EVs, unable to distinguish
between individual EVs and
aggregated EVs
54
AFM 143B cells,
osteosarcoma (OS)
cell line
AFM In
vitro
NA Revealing the structure and
nanomechanical properties of
exosomes with high spatial
resolution
Advantages: Single EV analysis, 3D
morphology, the substructure details
of EVs
Limitations: Possible morphological
changes may occur
55
Saliva AFM In
vitro
NA Explain the nanoscale structure
of exosomes under different
forces
56
264.7
macrophages
AFM In
vitro
Mouse brain Observing the morphology of
exoCAT
57
Raman
spectroscopy
Lung cancer cells
and alveolar cells
Raman
spectroscopy
In
vitro
NA Detection of EVs derived from
cancer cells
Advantages: Identify detailed
information and draw spatial
distribution maps, study heterogeneity
and monitor changes in EV cargo
58
Microuid-
based
imaging
Plasma Microuidic
ExosSearch
chip
In
vitro
NA Measurement of EV tumor
markers for cancer diagnosis
Advantages: Low sample size, high
sensitivity, integrated with various
analytical and imaging techniques
59
Limitations: Special equipment
required
K. Zhang et al.
Extracellular Vesicle 4 (2024) 100061
3
the donor cells enables to label EVs via the secreted reporter protein
binding to the phosphatidylserine on the surface of EVs. For example,
Takahashi et al. inserted a Gaussia luciferase (Gluc) into a truncated
lactadherin gene sequence to label B16-BL6 EVs for investigation of
B16-BL6 EV distribution.
71
To label specic subtype EVs, researchers usually fuse reporter
proteins to their marker proteins, such as labeling exosomes by CD63
fused eGFP.
72
Using this fusion protein, the author could trace the CD63
positive exosomes by GFP signal in vitro or ex vivo. In addition to CD63,
there are several EV-sorting proteins that can be chosen to label EVs via
a genetic engineering method, including CD81, CD9, CLC1 protein,
TNFR, and so on.
73,74
In some cases, researchers need to label EVs
derived from specic lineage of cells. To solve this problem,
exosome-reporter transgenic mice were generated for in situ
cell-type-specic exosome labeling. Men et al. generated Cre-dependent
exosome reporter (CD63-GFP) mice by inserting a loxP-oxed stop
codon upstream of human CD63 tagged with GFP at its C-terminal.
75
When injected AAV-Cre virus or crossed with a Cre recombinase
expressing strain with a cell type specic promoter (such as CaMKII
promoter, a neural cell marker protein), all exosomes derived from this
kind of cells (neural cells) will be labeled by CD63-GFP. These
Cre-dependent exosome reporter mice provide the possibility to observe
or isolate cell-type specic exosomes in vivo.
While these methods are delicate, dynamic real-time monitoring of
EVs in living animals is still a huge challenge due to the limitation of
uorescence imaging (FI) including restricted penetration depth and
unavoidable background noise. Using luciferase as a reporter protein to
label EVs can solve this problem.
23
Compared to uorescent proteins,
the luciferase family proteins react with their substrate and generate
signature bioluminescence that can be captured by a charge-coupled
device (CCD)-spectrometer system. For example, Takahashi et al.
fused a Gaussia luciferase gene with the C1C2 domain of lactadherin
gene to produce a fusion protein (Gluc-lactadherin) to label exosomes.
71
Gaussia luciferase is from the marine copepod Gaussia princeps, which
emits bioluminescence when coelenterazine is present. Compared with
Firey or Renilla luciferases, Gaussia luciferase has many advantages,
including it is smaller (only 19.9 KDa) and more sensitive. Recently, a
novel bioluminescence platform, NanoLuc luciferase, with enhanced
Fig. 2. Conventional EV labeling methods for visualization. Conventional EV labeling methods could be classied into 3 categories: A. labeling based on EV lipid, B.
labeling based on EV proteins, and C. labeling based on EV nucleic acids.
K. Zhang et al.
Extracellular Vesicle 4 (2024) 100061
4
stability, smallest size (19 kDa), and >150-fold increase in lumines-
cence, was developed for EV imaging.
76,77
All these luciferase genes can
help visualize EV internalization in recipient cells or ex vivo organs, as
well as real-time trace EV distribution in vivo. These protein-based la-
beling methods represent a powerful tool for understanding the molec-
ular mechanisms underlying EV biogenesis, uptake, long-term
distribution, and pharmacokinetic properties.
3.3. Nucleic acids
EVs can also be labeled with nucleic acid using nucleic acid-selective
uorescent cationic dye such as acridine orange.
78,79
Another elegant
approach to directly labeling EVs is tagging EV mRNAs with MS2
binding site sequence and co-expressing a bacteriophage MS2 coat
protein (MCP)-fused GFP in the donor cells. The GFP will bind to EV
mRNAs via the interaction between MCP and MS2 binding site.
70
While
this strategy is promising, studies so far have only demonstrated
EV-mRNA transfer between cultured cells in vitro. To trace EV-RNA
transfer between cells and organs in vivo, some indirect imaging strate-
gies were designed by hiring the Cre-LoxP reporter system.
69,80,81
One of
the Cre-LoxP reporter systems reported by Zomer et al. induces the
conversion of DsRed to eGFP specically in recipient cells that take up
Fig. 3. Novel EV labeling methods for visualization. Some novel methods have been developed, such as methods based on A. metabolic labeling with chemical
reaction, B. hybrid EVs with other particles, and C. other non-optical imaging methods based on electrostatic adsorption or incubation.
K. Zhang et al.
Extracellular Vesicle 4 (2024) 100061
5
EVs derived from donor cells expressing the Cre recombinase.
69
This
ingenious strategy provided direct in vivo evidence of local and systemic
EV-mRNAs exchange between tumor cells. More specically, EV-RNAs
derived from malignant tumor cells could enhance migration and
metastasis of less malignant tumor cells. Similarly, another team used
the Cre-LoxP system and LacZ reporter gene to trace the crosstalk of
EV-mRNAs between the hematopoietic system and various organs in the
inammation condition.
81
In this study, Ridder et al. expressed the Cre
recombinase under a hematopoietic lineage specic promoter in a LacZ
reporter transgenic mice; in other words, Cre mRNAs and proteins only
existed and triggered LacZ expression specically in the hematopoietic
lineage.
81
However, they observed LacZ expression not only in he-
matopoietic cells but also in neurons and other non-hematopoietic cells.
These results indirectly traced the transfer of Cre mRNAs in EVs between
hematopoietic lineage and other tissue cells. As EV research has begun to
progress towards delivering nucleic acid drugs, more labeling methods
for nucleic acid in EVs are urgently to be developed.
4. Novel direction of labeling strategies for EV visualization
Lately, as researchers in the elds of physics and chemistry turn their
attention to EVs, an increased number of novel EV labeling methods
have been developed, such as methods based on physical methods or
chemical reactions (Fig. 3).
82–84
Zhang et al. reported a
phospholipid-based bioorthogonal labeling strategy to endow EVs with
optical probes to trace EVs in vivo.
82
They adopted a metabolic strategy
to incorporate EV phospholipids with an unnatural choline analogu
bearing an azide (N3, a typical bioorthogonal functional group), which
can be conjugated with a uorescent probe using a bioorthogonal re-
action (copper-free click chemistry). Compared with the lipid-based
labeling methods we mentioned above, this strategy has better speci-
city and avoids many deciencies of lipid dyes, including
self-aggregation and non-specic binding.
Furthermore, another direction of EV labeling is transforming
nanoscale EVs, which are difcult to observe, into optically imageable
micrometer scale clusters. Liu et al. reported a pH-mediated EV assem-
bly system through incorporating pH-responsive diacyl lipid-conjugated
polymers (DLP) into the EV membrane. Under acidic conditions, DLP is
highly dispersed, while under neutral conditions, DLP self-assembles
into EV clusters through deprotonation and hydrophobic interactions,
resulting in a size of approximately 1
μ
m, which is larger than the
detection limit of conventional imaging methods such as ow cytometry
or confocal microscope. Therefore, EV imaging can be completed via
conventional methods, overcoming the size limitations of EVs and of-
fering advantages of sensitive and rapid detection.
85
In addition to labeling EV itself, the emergence of hybrid EV provides
new possibilities to label EVs by labeling the hybrid ligand. For example,
EV-liposome hybrid particles, which combine the advantages of EVs and
liposomes, can be labeled via both EV and liposome components. In
detail, liposomes can be labeled by their lipid composition and their
cargos. Fluorescent dyes can be conjugated to lipids, nucleic acids, or
small molecule drugs according to the liposome formulations.
86,87
This
strategy also helps to characterize the fusion efciency of EV-liposome
hybrid. According to research by Zhang et al., they analyzed these
particles using the uorescence resonance energy transfer (FRET)
method to conrm membrane fusion by labeling EVs with DiI and li-
posomes with DiO.
88
Another example is the hybridization of EVs with
pH-sensitive liposomes to deliver doxorubicin (Dox), which used the
cargo Dox as a uorescent probe as well as a chemotherapeutic drug for
investigation of their endosomal escape capacity.
89
Recent breakthroughs have also been made in labeling EVs in vivo.
90
Excepted for the use of transgenic mice to label EVs from lineage specic
cells, one study has reported that EVs derived from foam cells can be
labeled in vivo.
83
To achieve this, Ji et al. designed a foam cell-targeting
uorescent nanoprobe that can be specically degraded inside the cells
to produce a metabolite: triuoromethyl-bearing boron-dipyrromethene
uorophore (termed B-CF3). This metabolite B-CF3 was then enriched
into all membrane structures including EVs of the recipient foam cells
via hydrophobic interaction. This strategy overcame the limitation of
endogenous EV labeling methods in transgenic mice and provided a
practice platform to label specic EVs by an exogenous approach.
83
In addition to the uorescent dyes and various reporter proteins
mentioned above, more and more new luminescent materials, such as
quantum dots and aggregation-induced emission agents (AIEgens), have
been developed for labeling EVs.
84,91,92
Lin et al. developed a novel type
of AIEgens and camouaged it with engineered EV-derived mem-
branes.
92
Since AIEgens are highly luminescent and stable luminophores
capable of generating reactive oxygen species (ROS) in their aggregated
state, they serve as ideal uorescent materials for image-guided tumor
cell therapy and diagnosis. By incorporating them into EVs, in vivo
photodynamic and photothermal therapy of EVs targeting lung cancer
cells can be achieved. The development of these new materials makes it
possible to integrate EV imaging and therapeutic functions in a single
system.
The uorescence and bioluminescence labeling methods described
above are very effective for investigating the behavior of EVs in pre-
clinical research. However, optical imaging still has some intrinsic
limitations that prevent it to be transformed into clinical use. Thus, lots
of researchers devote themselves to developing new approaches to trace
EVs using advanced modern medical technologies. Case in point, la-
beling EVs with radioisotope is suitable for tracing EVs in vivo via nu-
clear imaging.
26
Do Won Hwang et al. used
99m
Tc-
hexamethylpropyleneamineoxime (HMPAO) to label macrophage
derived EVs for dynamic monitoring of EVs in living mice by
SPECT/CT.
27
Once the uncharged and highly lipophilic
99m
Tc-HMPAO
enters the EVs, it will be trapped inside them because the endogenous
glutathione in EVs can convert lipophilic
99m
Tc-HMPAO to a hydrophilic
form. By using SPECT/CT, the radioisotope labeled EVs can be virtual-
ized with great clarity and resolution in mice after injection. Apart from
this, Hu et al. labeled EVs with superparamagnetic iron oxide nano-
particles (SPIONs) via electroporation.
28
After labeling EVs with
SPIONs, melanoma EVs were found distributed in lymph nodes in a
C57BL/6 mouse model by a standard MRI approach. Like the above two
methods, labeling EVs with modern medical imaging contrast agents can
help understand the natural behavior of EVs in vivo at a super high
resolution and inspire clinical transformation.
5. Applications of EV visualization in theranostics
EV labeling and visualization strategies have seen signicant ad-
vancements in recent years, enabling researchers to better track and
study EV behavior in biological systems. A growing number of imaging
technologies are now available for EV visualization, including uores-
cence imaging (FI), bioluminescence imaging (BLI), computerized to-
mography (CT) scan, magnetic resonance imaging (MRI), radionuclide
imaging, and photoacoustic imaging (PAI). These imaging modalities,
along with the development of specialized imaging probes and labeling
techniques, have greatly expanded the potential for EV applications in
clinical diagnostics and therapeutics (Table 2). By enabling early
screening and precise tracking of EVs in vivo, these technologies pave the
way for improved understanding and utilization of EVs in disease
diagnosis and treatment (Fig. 4).
5.1. Cancer theranostics
In cancer theranostics, EV visualization integrates early diagnosis,
targeted drug delivery, and real-time treatment monitoring into a uni-
ed framework, emerging as a groundbreaking tool in the realm of early
cancer diagnosis. Early detection is a critical component of cancer
diagnosis and treatment. EVs derived from circulating tumor cells carry
specic biomarkers, enhancing the sensitivity of tumor identication.
113
Researchers can detect the unique biomolecular signatures carried by
K. Zhang et al.
Extracellular Vesicle 4 (2024) 100061
6
Table 2
Visualization methods of labeled EVs.
Visualization
methods
EV resources Imaging probes Equipments In
vivo/
in
vitro
Target cells or
organs
Applications Advantages and
limitations
Refs
FI Human placental
and umbilical
derived
mesenchymal stem
cells (hP-MSCs and
hUC-MSCs)
CM-DiI Fluorescence
microscope
In
vitro
Chemotherapy-
damaged cells
Verify that EVs are
internalized by target
cells
Advantages: High
sensitivity, low cost, high
signal contrast, and easy
detection
Limitations: Limited
penetration depth
93
Human placenta-
derived
mesenchymal stem
cells (hP-MSCs)
Cy5.5 Fluorescence
microscope
In
vitro
Kidneys Detection of organ
distribution of EV and
PBP-EV in renal IRI mice
94
Urine Ce6 Fluorescence
microscope
In
vitro
Human gastric
carcinoma cells
(MGC-803)
Detecting the absorption
capacity of target cells for
EVs
95
Colon tumor GP-ACM Fluorescence
microscope
In
vivo
NA Real time monitoring of
CD26 activity in EVs
secreted by tumor cells
96
Murine lung cancer
(LL/2) cells, murine
colon cancer (MC-
38) cells, human
lung cancer (A549)
cells, and healthy
human liver biopsy
samples
DiD Fluorescence
microscope
In
vivo
+in
vitro
NA In vivo monitoring the
biodistribution of EVs, in
vitro characterizing the
targeting properties of
EVs
97
BLI Mesenchymal stem
cells (MSCs)
Gaussia
luciferase
IVIS Lumina
imaging system
(Xenogen
Corporation)
In
vivo
+in
vitro
Kidney Detecting biological
distribution of EVs and
indicating EV stability in
vivo
Advantages:
High sensitivity, can
quantify the intensity of
bioluminescence signals
to estimate the number of
EVs
Limitations:
Low spatial resolution,
limited organizational
penetration
98
Human cancer cell
lines (human colon
cancer cell lines
HCT116, HT29 cells
and human lung
cancer cell lines
A431, A549 cells)
Nano luciferase IVIS® Lumina III
imaging system
In
vivo
+in
vitro
NA Detecting long-term
biodistribution analysis
77
HEK-293T, Huh7,
B16F10 cells
Firey
luciferase,
Super Rluc8,
Thermoluc,
Nanoluc
IVIS® Spectrum
imaging system
In
vivo
NA Monitoring the
biodistribution of EVs
99
CT Mesenchymal stem
cells (MSCs)
Glucose-coated
gold
nanoparticle
(GNP)
Micro-CT scanner In
vivo
Brain Imaging and tracking of
EV in vivo
Advantages:
High resolution imaging,
real-time imaging
capability, can be
combined with other
imaging technologies
such as PET-CT
Limitations:
Radiation dose has low
resolution for soft tissues
and is limited to static
imaging
100
Mesenchymal stem
cells (MSCs)
Glucose-coated
gold
nanoparticle
(GNP)
Micro-CT scanner In
vivo
Liver, spleen, and
kidney
Visualize the in vivo
distribution of EVs
101
Mesenchymal stem
cells (MSCs)
Glucose-coated
gold
nanoparticle
(GNP)
Micro-CT scanner In
vivo
Brain Visualize the distribution
of EVs in the brain
through imaging
100
MRI Melanoma SPION5 iron Varian small
animal scanner
In
vivo
+in
vitro
Lymph nodes In vitro detection and in
vivo monitoring of the
biological distribution of
exosomes and exosomes
in lymph nodes
Advantages:
High resolution, good
biosafety, and high image
depth
Limitations:
Low sensitivity, long
scanning time, stability of
contrast agent labeling
still needs to be improved
28
Hepatic cancer cells
(HCCs)
GOD-
ESIONs@EVs
(GE@EVs)
Clinic MR scanner In
vivo
+in
vitro
HCC cells Study the targeting
characteristics and in vivo
and in vitro biological
distribution of EVs
102
Human bone
marrow
mesenchymal stem
cells
Molday ION 7T scanner In
vitro
NA Real-time imaging of EVs 103
Radionuclide
imaging
Goat milk
99m
Tc (IV) The SPECT (single
photon emission
computed
tomography)
In
vivo
NA In vivo tracking of EVs and
evaluating their
pharmacokinetic
characteristics
Advantages:
More suitable for
evaluating the in vivo
distribution of EVs, with
high stability, innite
tissue penetration, high
104
(continued on next page)
K. Zhang et al.
Extracellular Vesicle 4 (2024) 100061
7
tumor cell-derived EVs using sophisticated imaging techniques, such as
super-resolution uorescent imaging or microuidic-based imaging.
This provides a noninvasive strategy for diagnosing cancer at early
stages.
113–116
For instance, Villa et al. demonstrated that blood-derived
EVs from colorectal cancer (CRC) patients exhibit a stronger
tumor-targeting ability compared to those from healthy individuals.
They incubated EVs with indocyanine green (ICG) for labeling and used
uorescence microscopy to observe the EV distribution. In a
patient-derived xenograft (PDX) mouse model, these EVs recognized the
homologous tumor, suggesting a novel strategy for cancer patient
diagnosis.
117
Furthermore, EVs can also enhance the accuracy of cancer
diagnosis. Guo et al. (2018) utilized microuidic technology to detect
biomarkers such as DNA, miRNA, and ncRNA in body uid-derived EVs.
These biomarkers were used to assess the tumor microenvironment and
identify the type and stage of tumors. For instance, the validation of 8
miRNAs was conducted, proposing their potential use in diagnosing the
stage of ovarian cancer. They also envisioned the integration of
emerging articial intelligence with microuidic systems in the future,
which may enhance pre-diagnostic capabilities regarding cancer.
118
EV-based cancer screening has also made signicant progress. Several
companies have captured and detected EVs from blood to aid potential
prostate cancer patients in deciding whether to undergo a biopsy. They
employed Fe
3
O
4
@SiO
2
@TiO
2
particles to adsorb exosomes and detected
the presence of tumor-derived EVs using a uorescence-responsive
PSMA aptamer sensor. This method has been successfully applied in
clinical settings, highlighting its accuracy and speed advantages in
enhancing clinical diagnosis efciency.
119
Therefore, this early diag-
nostic capability of EV visualization and detection not only facilitates
timely intervention but also lays the foundation for precise and
personalized treatment tailored to the molecular prole of the detected
cancer.
115,120
Besides early diagnosis, EV visualization is also invaluable in moni-
toring treatment response in modern therapeutics. Researchers can
assess treatment-induced changes by real-time visualization and anal-
ysis of EVs after treatment.
120
For example, Takahashi et al. designed
EVs derived from B16-BL6 mouse melanoma cells containing Gaussia
luciferase protein by inserting Gaussia luciferase fragment into the C1C2
domain of EV specic protein MFGE8.
71
After injection into mice,
bioluminescence imaging revealed that these EVs initially accumulated
in the liver and subsequently spread to the lungs, enabling real-time
tracking of EVs in vivo and facilitating pharmacokinetic studies on
EVs.
71
Similarly, Shi et al. used positron emission tomography (PET) for
non-invasive in vivo monitoring of copper-64 (^64Cu) and polyethylene
glycol (PEG)-modied exosomes. The addition of PEG enabled the suc-
cessful accumulation of EVs around tumor cells and reduced hepatic
clearance. PET images demonstrated high-quality imaging and quanti-
tatively measured EVs accumulation in tumors, providing an effective
method for accurately assessing the in vivo biological distribution of
Table 2 (continued )
Visualization
methods
EV resources Imaging probes Equipments In
vivo/
in
vitro
Target cells or
organs
Applications Advantages and
limitations
Refs
sensitivity, and easy
clinical translation
Limitations:
High cost, radioactive
exposure
4T1 cells, HEK-293
cells
111
in-oxine MicroSPECT/CT
4R
In
vivo
M2 macrophages Detect the targeting
ability and in vivo
distribution of EVs
105
RAW264.7
99m
Tc-HMPAO SPECT/CT
scanner
In
vivo
NA Evaluate the targeting
and pharmacokinetic
characteristics of EVs in
mice
27
Tumor cells,
myeloid-derived
suppressor cells, and
endothelial
progenitor cells
111
I SPECT/CT
scanner
In
vivo
NA Evaluate the biological
distribution of different
EVs in vivo
106
Murine mammary
carcinoma 4T1 cells
64
Cu or
68
Ga PET In
vivo
NA Monitoring the biological
distribution of
extracellular vesicles in
vivo, as well as the
metastasis pathways of
EVs through lymph and
bloodstream
107
Pancreatic cancer
sEVs (PANC1)
[
89
Zr] Zr
(oxinate)
4
PET In
vivo
NA In vivo tracking of EVs and
detection of the biological
distribution of REVS
108
Moues liver
proliferative cells
124INaI PET In
vivo
NA Evaluate the distribution
of surface glycosylation
complexes modied EVs
109
PAI Tumor cells Au nanostars Vevo LAZR
photoacoustic
imaging system
In
vivo
Mouse tumor
tissue
Detect the PA imaging
performance of TDSP-
Exos in vivo
Advantages:
High resolution
visualization of the
structure and function in
biological tissues, with
high sensitivity and
spatial resolution,
achieving high contrast
imaging at the molecular
level
Limitations:
Image depth limit
110
Red blood cells Exosome-like
GQDzyme/
ABTS
nanoparticle
Not indicated In
vivo
Nasopharyngeal
cancer (NPC)
tumor tissue
Targeted accumulation of
EVs detected in vivo
111
HEK-293T cells SBC-EV(ICG/
PTX)
Q-switched pump
laser, optical
parametric
oscillator laser, 5
MHz US
transducer
In
vivo
+in
vitro
Mouse tumor
tissue
Evaluate the PA
characteristics of ICG
encapsulated EVs in vitro,
monitor the biological
distribution of EV based
imaging agents in vivo,
and achieve pH
responsive PA imaging
guided chemoacoustic
dynamic combination
therapy
112
K. Zhang et al.
Extracellular Vesicle 4 (2024) 100061
8
EVs.
121
Furthermore, Wu et al. developed a nano-catalyst GOD--
ESIONs@EVs (GE@EVs) for targeted therapy of advanced hepatocellu-
lar carcinoma (HCC). GE@EVs served as nano-scale contrast agents,
allowing real-time monitoring of EVs’ tumor targeting through magnetic
resonance imaging (MRI), proposing a feasible strategy for precise EV
visualization.
102
Moreover, combining EV visualization with other detection tech-
nologies, such as nanoow cytometry or high-throughput sequencing,
allows researchers to monitor alterations in the EV prole, including
changes in protein expression or nucleic acid content. This provides a
dynamic and comprehensive overview of treatment response, enabling
real-time adjustments.
116
For example, Takahashi et al. employed Taq-
Man MicroRNA assay to measure plasma exosomes in patients with
pancreatic ductal adenocarcinoma (PDAC). They found that miR-451a
in EVs is correlated with tumor size and stage, and patients with
higher levels of miR-451a have lower survival rates. The EV miR-451a
can effectively detect the potential risk of postoperative recurrence in
PDAC patients.
122
Furthermore, integrated nanouidic chips can be
utilized to detect miRNAs from breast cancer-derived EVs
123
as well as
miR-25-3p and miR-92a-3p produced by adipose tissue sarcoma cells.
124
This serves as an effective strategy for monitoring tumor recurrence and
can be used for postoperative adjuvant therapy. EV visualization in
cancer theranostics enhances the precision of personalized medicine and
contributes to our understanding of the molecular landscape of
treatment response.
Furthermore, EV visualization plays a critical role in the develop-
ment of targeted drug delivery strategies for cancer therapeutics.
Beneting from their natural ability to carry and transfer biomolecules,
EVs can be used as effective carriers to deliver therapeutic agents
directly to tumor cells while minimizing off-target effects.
116,125
Re-
searchers engineer EVs to load specic drugs, proteins, and RNA mole-
cules to maximize therapeutic efcacy. For instance, Kim et al.
engineered dendritic cell-derived EVs expressing human leukocyte an-
tigen (HLA)-A2 and co-stimulatory molecules. These EVs effectively
stimulate antigen-specic CD8
+
T cell immune responses, enhancing
anti-tumor immunity.
126
Additionally, Nie et al. utilized engineered EVs
to deliver miRNA-126, inhibiting lung cancer cell growth.
127
By inte-
grating imaging reporter genes into EV cargo, the release kinetics of
therapeutic drugs can be effectively tracked, providing deep insights
into therapeutic efcacy.
64,128
For example, Gong et al. validated the
effective delivery of EVs to target tumor cells by monitoring
Cy5-Cho-miRNA, which emits a purple uorescence, using confocal
laser microscopy. Imaging techniques further validate the accuracy of
targeted drug delivery by visualizing interactions between EVs and
tumor cells.
129
For example, Peng et al. developed magnetic particle
imaging (MPI) as an emerging imaging modality. MPI offers high reso-
lution and sensitivity, making it suitable for studying the relationship
between tumor cells and EVs. Efforts are currently underway to develop
Fig. 4. Summary of EV visualization strategies and their applications in theranostics.
K. Zhang et al.
Extracellular Vesicle 4 (2024) 100061
9
human MPI scanners and other devices, showing promising clinical
potential.
130
In conclusion, the multiple roles of EV visualization in early
diagnosis, treatment response monitoring, and targeted drug delivery
underscore its signicance in advancing precise, personalized, and
effective cancer theranostics.
5.2. Neurodegenerative diseases
In neurodegenerative disease research, EV visualization has emerged
as a powerful tool for identifying biomarkers and therapeutic in-
terventions, offering valuable insights into accurate diagnosis and
addressing neurodegenerative diseases.
131–133
As EVs are derived from
cells and contain proteins, nucleic acids, and lipids reecting the phys-
iological and pathological states of donor cells, they serve as potential
biomarkers for neurodegenerative diseases like Alzheimer’s disease and
Parkinson’s disease.
132,134–137
For instance, neuronal EVs can be infer-
red through L1 cell adhesion molecule (L1CAM, CD171),
138
microglial
EVs through CD11b or transmembrane protein 119 (TMEM119),
139
and
oligodendrocyte EVs through myelin oligodendrocyte glycoprotein
(MOG).
140
Biomarker specic EV visualization enables scientists to
identify specic EVs associated with disease progression. Using multiple
visualization and analysis techniques, researchers study EV composi-
tions isolated from cerebrospinal uid and blood to identify early in-
dicators of neurodegenerative disorders, which are critical for timely
diagnosis and intervention.
135,141
For example, Xylaki et al. revealed a
signicant increase in
α
-synuclein (aSyn) and leucine-rich repeat kinase
2 (LRRK2) in neuronal EVs from Parkinson’s disease (PD) patients,
proposing these biomarkers for early diagnosis.
142
Similarly, visualiza-
tion techniques like MRI, CT, and PET have been used to detect
EV-carried AD-related proteins Aβ1-42 and miR-193b as biomarkers for
diagnosing Alzheimer’s disease (AD).
141
In advancing therapeutic interventions, EV visualization plays an
irreplaceable role. EVs have gained attention as potential carriers for
targeted drug delivery to the central nervous system due to their ability
to cross the blood-brain barrier.
57,143
By loading therapeutic cargos and
imaging probes into EVs, researchers can simultaneously track their
biodistribution and optimize drug efcacy in the central nervous system
using imaging techniques, thereby increasing treatment precision.
144,145
This imaging-assisted targeted therapy minimizes off-target effects and
improves the overall effectiveness of therapeutic interventions. For
example, Perets et al. (2019) developed an in vivo neuroimaging method
for tracking EV migration using X-ray computed tomography (CT) and
mesenchymal stem cell-derived EVs modied with gold nanoparticles.
This technique enables real-time monitoring of EVs and has demon-
strated targeted effects in various neurodegenerative diseases such as
stroke, autism, Parkinson’s, and Alzheimer’s.
146
Moreover, EV visualization can also monitor treatment responses,
providing noninvasive real-time feedback on therapy impact within the
brain. For instance, Shi et al. monitored the severity of Parkinson’s
disease (PD) by detecting levels of radiolabeled EV
α
-synuclein in blood
samples.
147
Like their application in cancer theranostics, EV visualiza-
tion in neurodegenerative diseases offers dual benets in biomarker
discovery and therapeutic interventions, enhancing our understanding
of these diseases and improving clinical outcomes.
5.3. Tissue injuries
Like in the above applications, EVs can also be considered as bio-
markers of tissue injury. EV-based noninvasive visualization is a trans-
formative technique for diagnosing tissue injury.
148
After injury, cells at
the injury site release EVs with a specic biomolecular prole reecting
the type and severity of the injury.
149
These EVs can traverse biological
barriers and protect their cargo under harsh conditions, enhancing their
accessibility for sampling.
12,150,151
This makes EVs particularly suitable
for minimally invasive diagnostic procedures, such as liquid biopsies,
where the analysis of circulating EVs in bodily uids can provide
valuable information about the status of distant tissues.
152–154
By
analyzing individual EVs and their cargo using visualizing modalities
and other downstream methods, researchers can obtain information on
potential tissue injuries, enabling timely diagnosis.
153
This noninvasive
approach holds promise for personalized medicine, allowing for tailored
regenerative strategies based on the molecular signatures detected
through EV visualization. For example, Ji et al. designed a foam
cell-targeting uorescent nanoprobe that can be specically degraded
inside the cells to produce a metabolite: triuoromethyl-bearing bor-
on-dipyrromethene uorophore (termed B-CF3). This metabolite B-CF3
was then enriched into all membrane structures including EVs of the
recipient foam cells via hydrophobic interaction. This strategy overcame
the limitation of endogenous EV labeling methods in transgenic mice
and provided a practice platform to label specic EVs by an exogenous
approach.
83
Another key application of EV visualization in tissue injury is
monitoring EV-based therapies. EVs possess unique homing features,
targeting the cell type from which they are released, and can be engi-
neered with desired ligands to target specic sites with overexpressed
cell surface biomarkers.
155–161
Imaging techniques allow researchers to
track the biodistribution of EVs, ensuring therapeutic payloads reach the
intended site of action. Additionally, visualization of EV treatments can
indicate the location and severity of tissue injury in vivo, integrating
diagnosis and treatment into a single process.
98
Zhang, K. et al. reported
a Cy5 and P-selectin binding peptide modied EVs, which can target
bind to ischemia induced P-selectin expression on renal endothelial
cells, could indicate the kidney injury degree and achieve the target
treatment at the same time.
98
The combination of EV-based treatment
and imaging technologies holds great promise for developing precise
and personalized therapeutic strategies for tissue regeneration.
Currently, ongoing research includes real-time tracking of EVs in liver
regeneration, as reported by Cao, H. et al. They utilized
aggregation-induced emission luminogenic DPA-SCP to label EVs
derived from human placental MSCs and applied them for the treatment
of acute liver injury in mice. DPA-SCP features high brightness and
biocompatibility, exhibiting superior labeling efciency and tracking
capability compared to traditional commercial membrane dyes, without
affecting the therapeutic efcacy of EVs on liver tissue. The use of
DPA-SCP enables long-term and safe tracking of EVs in liver regenera-
tion through real-time in vivo imaging.
84
Cardiovascular disease (CVD)
remains a leading cause of morbidity and mortality worldwide, and EVs
showed effectively therapeutic functions in promoting cardiac repair
after myocardial infarction.
162
Wang et al. designed EVs containing the
ischemic myocardium-targeting peptide CSTSMLKAC (IMTP). Flow
cytometry and uorescence imaging showed these EVs effectively
accumulated in the ischemic heart region, signicantly reducing
myocardial cell apoptosis, decreasing brosis, and increasing angio-
genesis.
163
In another study, EVs derived from cardio sphere-derived
cells (CDCs) fused with the myocardial cell-specic peptide (CMP)
also achieved myocardial cell targeting, reduced apoptosis, and
improved cardiac injury through real-time uorescence imaging.
164
In
conclusion, EV visualization plays a crucial role in diagnosing and
real-time EV tracing in tissue injury theranostic, offering a promising
approach for personalized and regenerative medicine.
5.4. Metabolic diseases
EVs are also emerging as effective tools for early screening, accurate
diagnosis, and treatment of metabolic diseases and their associated
complications. Diabetes mellitus (DM) is a major metabolic disorder
characterized by elevated blood glucose levels due to pancreatic β-cell
damage and insulin resistance. Its complications can cause severe
damage to the retina, central nervous system, kidneys, and other organs.
Early screening and diagnosis of DM patients are crucial, and timely
intervention can alleviate the symptoms of diabetes. Since the comple-
tion of the Human Genome Project, various microRNAs (miRNAs) have
K. Zhang et al.
Extracellular Vesicle 4 (2024) 100061
10
been found to be closely associated with DM and its complications.
Therefore, cell-secreted EVs miRNAs can serve as DM biomarkers for
detection.
165
EVs miRNAs are easily extracted, highly specic, and can
be detected by qRT-PCR and in situ hybridization.
166
Recent studies
have shown distinct differences in EVs miRNAs in the serum and urine of
healthy individuals compared to diabetes patients. For example, sig-
nicant differences in miR-1 and miR-133a in serum-derived EVs can
distinguish between diabetes and non-diabetes.
167
MiR-30a, miR-342,
and miR-133b in urine isolated EVs can also serve as markers for
screening diabetes patients.
168
EVs are also being used to treat DM, offering new strategies and hope
for a diabetes cure. In a recent study, EVs were combined with super-
paramagnetic iron oxide nanoparticles (SPIONs) and loaded with the
insulin-promoting peptide BAY55-9837. Confocal microscopy and IVIS
images revealed that they could be magnetically targeted to pancreatic
islets, signicantly increasing insulin secretion and lowering blood sugar
levels.
169
In another study, fusogenic EVs induced by the viral fusogenic
vesicular stomatitis virus (VSV) successfully transferred glucose trans-
porter 4 (GLUT4-GFP) to the recipient cell membrane, and the locali-
zation of the protein was monitored using confocal microscopy. This
EV-mediated delivery of GLUT4 has high clinical value and can ach-
ieve higher glucose uptake.
170
5.5. Bone and cartilage diseases
EV visualization also shows application value in some other diseases,
such as bone diseases.
171
In bone diseases, EVs secreted by mesenchymal
stem cells (MSCs) are important therapeutic tools in bone regenerative
medicine. These MSC-derived EVs possess the ability to promote
angiogenesis, alleviate inammation, and regulate cell proliferation and
differentiation.
172
The role of EVs in regulating bone cell regeneration
has been conrmed through EV visualization. For instance, Qin et al.
constructed an EV delivery system to investigate its effect on bone for-
mation in Sprague-Dawley (SD) rats with cranial defects. Observation of
PKH67-labeled EVs under confocal microscopy conrmed that EVs
entered osteoblasts via endocytosis and promoted their differentiation.
Micro-CT analysis in in vivo experiments also showed a signicant in-
crease in newly formed bone quantity due to EV administration. These
novel EV labeling and visualizing methods contribute to identifying the
specic roles of EVs in bone cells.
173
Osteoporosis is a chronic disease of the skeletal system, primarily
characterized by reduced bone density due to insufcient osteoblast
formation from bone marrow stromal cells (BMSCs), leading to
increased bone fragility and susceptibility to damage. EVs offer prom-
ising approaches for treating osteoporosis. It has been reported that EVs
derived from bone marrow stromal cells (STExos) can promote osteo-
blast formation in vitro. Moreover, when specic antibodies targeting
BMSCs are fused to the surface of STExos, analysis using a uorescence
tomography imaging system has revealed their ability to effectively
target BMSCs in the skeleton, promote bone regeneration, and signi-
cantly enhance bone mass.
174
In a recent study, the application of EV visualization helps scientists
elucidate the role of bone-derived EVs in aging. Shen et al. isolated and
characterized EVs from bone tissues of different age groups by TEM and
NTA, revealing that their properties changed with age. Furthermore, the
study demonstrated that EVs from aging bone tissues can induce age-
related skeletal diseases and impact the aging of other systems and tis-
sues in the body through the circulatory system, such as triggering age-
related cardiovascular diseases, metabolic diseases, and neurodegener-
ative diseases. Therefore, they proposed using aging EVs as biomarkers
for age-related diseases and introducing EVs from young bone cells as a
treatment method for these conditions. However, more research is
needed to clarify the association between EVs and human aging.
175
Due to the limited regenerative capacity of cartilage, injuries often
lead to osteoarthritis (OA), a degenerative joint disease. EVs derived
from mesenchymal stem cells (MSCs) or chondrocytes have been shown
to induce chondrocyte proliferation and differentiation in vitro. EV
visualization could be employed to study the pathogenesis of OA. Re-
sults from luciferase activity assays indicate that lncRNA-KLF3-AS1
within EVs can inhibit miR-206, which in turn suppresses the expres-
sion of GIT1 protein. GIT1 is known to induce chondrocyte apoptosis.
Therefore, EVs can guide chondrocyte differentiation and promote
proliferation by inhibiting miR-206.
176
Additionally, OA is closely
associated with cartilage degradation, primarily mediated by the protein
Wnt5A. According to research reports, MSC-EVs containing miR-92a-3p
can effectively inhibit cartilage degradation in mice. Luciferase assays
revealed that this inhibition is mainly due to miR-92a-3p within the EVs
directly targeting Wnt5A and suppressing its expression, demonstrating
the potential of EVs as a cell-free therapy for OA.
177
5.6. Inammation
EVs can also be utilized for alleviating inammation. EVs derived
from M2 macrophages contain various anti-inammatory cytokines,
making them suitable for treating inammatory diseases.
178,179
Several
targeted inammation therapy strategies using EVs derived from M2
macrophages have been reported, and EV visualization has important
applications in all of these. In one recent study, the anti-inammatory
drug curcumin was incorporated into EVs derived from macrophages
to create a novel drug delivery vehicle. This EV-based drug delivery
system, which is stable and maintains high concentrations in the
bloodstream, holds the potential for treating numerous inammatory
conditions. Moreover, in studies based on Odyssey infrared uorescence
imaging technology, the uorescence signals of EVs are primarily
observed in the liver, kidney, and spleen tissues, highlighting the tar-
geting capabilities associated with EVs.
129
In another study, Tang et al. utilized microvesicles (MVs) derived
from macrophages to targeted deliver dexamethasone (DEX) to inamed
kidneys. DEX, a synthetic glucocorticoid, is an effective drug for treating
renal diseases. However, its side effects such as hypertension, hyper-
glycemia, and obesity limit its dosage. Conrmed by confocal uores-
cence microscopy, the MV-DEX drug delivery system successfully
reduced systemic adverse reactions of glucocorticoids by targeting the
kidneys. This increased the drug utilization of dexamethasone and
effectively suppressed renal inammation. This represents a safe drug
delivery strategy with promising medical applications.
180
In summary,
EVs demonstrate outstanding imaging potential and therapeutic ef-
cacy, offering novel methods and strategies for curing a multitude of
complex inammatory diseases.
6. Outlook
The eld of EV visualization is set to progress in multiple directions,
emphasizing enhanced insights into cell-to-cell communication, early
disease diagnosis, and targeted therapeutic applications. One major
focus will likely be the development of innovative imaging contrast
agents, EV labeling methodologies with high resolution and specicity,
and new instruments of EV visualization both in vivo and in vitro.
Technologies like super-resolution microscopy and advanced contrast
agents are expected to allow researchers to observe single EVs at the
nanoscale, offering new levels of detail into their formation, cargo
composition, and interactions with target cells.
23
The use of articial
intelligence (AI) and machine learning (ML) algorithms is also antici-
pated to become a key part of EV visualization, enabling the analysis of
large datasets and the detection of complex patterns that may not be
immediately apparent to human observers.
In addition, the pursuit of multiplexed EV visualization techniques
will likely take a central role in upcoming research, as these methods
allow for the simultaneous detection of various EV-derived signals-such
as size, concentration, and specic molecular contents like proteins and
nucleic acids-yielding a detailed molecular prole. Such information is
invaluable for accurate diagnostics, especially in complex diseases like
K. Zhang et al.
Extracellular Vesicle 4 (2024) 100061
11
cancer and neurodegenerative conditions. Recent advancements, for
example, include a single-EV analysis platform using a microuidic
chamber and immunouorescence for cancer-related EV analysis,
enabling robust measurements of individual EV biomarkers.
181,182
Similarly, dual imaging approaches combining uorescence and biolu-
minescence have shown promise for tracking EV biodistribution and
therapeutic contents in vivo.
70,183
These advanced multiplexed imaging
techniques will likely be instrumental in rening EV-based therapies to
improve targeting, distribution, and therapeutic outcomes.
In conclusion, the evolution of EV visualization appears to be driven
by the integration of innovative technologies such as high-resolution
microscopy, AI, multiplexed imaging approaches, and noninvasive in
vivo tracking. Together, these advancements are expected to reshape our
understanding of EV biology, while paving the way for diagnostic and
therapeutic innovations. Looking forward, interdisciplinary collabora-
tions among biologists, imaging specialists, and engineers will be
essential to unlocking the full potential of EV visualization in healthcare
and regenerative medicine.
CRediT authorship contribution statement
Kaiyue Zhang: Writing – review & editing, Writing – original draft,
Project administration, Methodology, Investigation, Conceptualization.
Jingxuan Hu: Writing – original draft, Investigation, Data curation.
Yilan Hu: Visualization, Investigation.
Declaration of competing interest
None.
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