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Nature Reviews Materials
nature reviews materials https://doi.org/10.1038/s41578-023-00551-3
Review article Check for updates
Extracellular vesicle–matrix
interactions
Koushik Debnath1,2,6, Kevin Las Heras1,2,3,4,6, Ambar Rivera 1,2,5, Stephen Lenzini1,2 & Jae-Won Shin 1,2
Abstract
The extracellular matrix (ECM) harbours various signals to control
cellular functions and the materiality of tissues. Most eorts to
synthetically reconstitute the matrix by biomaterial design have
focused on decoupling cell-secreted and polymer-based cues. Cells
package molecules into nanoscale lipid-membrane-bound extracellular
vesicles (EVs) and secrete them. Thus, EVs inherently interact with
the meshwork of the ECM. In this Review, we discuss various aspects
of EV–matrix interactions. Cells receive feedback from the ECM and
leverage intracellular processes to control the biogenesis of EVs. Once
secreted, various biomolecular and biophysical factors determine
whether EVs are locally incorporated into the matrix or transported
out of the matrix to be taken up by other cells or deposited into
tissues at a distal location. These insights can be utilized to develop
engineered biomaterials in which EV release, retention and production
can be precisely controlled to elicit various biological and therapeutic
outcomes.
Sections
Introduction
Mechanisms of EV biogenesis
in the ECM
Biomolecular interactions
between EVs and the ECM
network
Biophysical EV–ECM network
interactions
Interfacing EVs with
engineered materials
Material-based cell culture
strategies to control EV
secretion from cells
Outlook
1Department of Pharmacology and Regenerative Medicine, University of Illinois at Chicago, Chicago, IL, USA.
2Department of Biomedical Engineering, University of Illinois at Chicago, Chicago, IL, USA. 3NanoBioCel Group,
Laboratory of Pharmaceutics, School of Pharmacy (UPV/EHU), Vitoria-Gasteiz, Spain. 4Bioaraba, NanoBioCel
Research Group, Vitoria-Gasteiz, Spain. 5Department of Chemical Engineering, University of Illinois at Chicago,
Chicago, IL, USA. 6These authors contributed equally: Koushik Debnath, Kevin Las Heras. e-mail: shinjw@uic.edu
Nature Reviews Materials
Review article
this classification is that validating specific cell-secreted EVs based on
biogenesis pathways requires well-controlled investigations, such as
using live cell imaging techniques fused with genetic approaches26.
From a practical point of view, EVs can be classified into large
(>200nm) and small (<200nm) EVs27 and may include various EV
subtypes in addition to apoptotic bodies, ectosomes and exosomes.
Differential centrifugation can be used to separate large EVs (<10,000g)
and small EVs (>100,000g). For instance, exophers are microscale large
EVs that are isolated at ~1,000g and are known to help transport and
eliminate defective mitochondria and protein aggregates
28
. Migras-
omes (>500nm) are large EVs that are produced from long membrane
projections during cell migration on a rigid culture substrate
29,30
. Simi-
larly, filopodia-derived vesicles (>200nm) are formed by scission of
filopodia31. Some of the recently reported small EV subtypes include
arrestin-domain-containing protein 1(ARRDC1)-mediated microvesi-
cles that are formed by budding32 and ECM-bound vesicles, which
are devoid of classical EV markers, tightly bound to the ECM after
decellularization of tissues, and released only after enzyme-mediated
digestion of the ECM13.
Adding to the complexity, recent studies have also shown that
after ultracentrifugation at 100,000g, the pellet contains, in addition
to small EVs, non-vesicular extracellular particles (NVEPs) that do
not contain a lipid bilayer. NVEPs can be separated from small EVs by
high-resolution iodixanol density gradient fractionation, followed
by taking high-density fractions
33
. The supernatant from the first ultra-
centrifugation can be subject to additional overnight ultracentrifuga-
tion at 100,000g to obtain smaller NVEPs (<50nm)
34
, called exomeres,
which were first described by using the asymmetric-flow field-flow
fractionation method
35
. After isolating exomeres, another round of
ultracentrifugation at a higher speed (~360,000g) can be done over-
night on the supernatant to obtain even smaller NVEPs (<30nm), called
supermeres
36
. Some NVEPs were shown to be released via a shared
pathway as exosomes
33
, but the biogenesis pathway of NVEPs remains
relatively unknown compared with that of EVs.
Mechanisms of EV biogenesis in the ECM
EV biogenesis is intricately linked to intracellular transport and secre-
tory pathways and to physicochemical factors in the ECM that regulate
these processes (Fig.2).
Lipid-membrane transport
The unique structural feature of EVs is that they encapsulate various
cargo molecules in the lipid membrane, including proteins, nucleic
acids and various metabolites
37
. Thus, understanding the role of mem-
brane turnover in the context of the ECM will help tounderstand how
EV biogenesis is regulated by the ECM. Lipid rafts are discrete, dynamic
nanoscale domains in the external leaflet of the cell membrane that are
present in a metastable state, but become more stable by undergoing
clustering in response to external signals, including those present in
the ECM
38
. Some lipid raft domains undergo endocytosis
39
, and the
resulting vesicles fuse with early endosomes
40
. Lipid rafts are enriched
with cholesterol and sphingolipids41. Importantly, cholesterol and
ceramide, a simple sphingolipid, have an essential role in the formation
of MVBs: cholesterol recruits the endosomal sorting complex required
for transport (ESCRT) machinery42, and ceramide triggers the negative
curvature of the MVB membrane to form ILVs in an ESCRT-independent
manner
43
. Both cholesterol and ceramide are highly hydrophobic and
intercalate between phospholipid acyl chains of the cell membrane
in a competitive manner
44,45
. Loss of cholesterol not only increases
Introduction
The extracellular matrix (ECM) is a network structure consisting of
various biomolecular and biophysical components essential to cellular
functions and represents the major acellular component of biological
tissues. Tissues are active viscoelastic materials
1
that can change their
properties depending on pathophysiological conditions. The ECM
can determine the rheological properties of tissues both directly as
constituent and indirectly by calibrating how cells generate contractile
forces and tension via mechanotransduction
2,3
, which can influence
the ability of cells to remodel the ECM
4
. Understanding how the ECM is
remodeled and how the materiality of tissue is dynamically controlled
will necessitate biomaterial-based strategies to investigate the interplay
between cell-secreted factors and polymer-based cues.
Previous studies with purified ECM proteins have highlighted
the role of polymeric networks in determining rheological properties
essential to tissue integrity, such as strain stiffening5. To date, efforts to
engineer synthetic ECMs to direct cellular functions have focused on
controlling the crosslinking of polymeric networks to tune elasticity6,
viscoelasticity7,8 and plasticity9. However, molecular profiling studies
of decellularized tissues have shown the presence of soluble proteins
tightly bound to fibrous ECM networks10. Cells can secrete soluble pro-
teins directly, but they can also package molecules into nanoscale media-
tors and secrete them, especially in lipid-membrane-bound vesicles,
called extracellular vesicles (EVs). The presence of EVs in the ECM was
documented several decades ago by electron microscopy studies in the
context of vesicle-mediated mineralization
11,12
, but ECM-bound vesicles
were documented in other tissues only recently
13
. Studies with label-free
third-harmonic generation microscopy further showed the enrichment
of EVs in tissue stromal regions, which consist of dense matrix fibres
14,15
.
Vesicles can also be found in blood16 and lymph17, suggesting that some
secreted EVs from cells can transport out of the ECM
18
and end up at a
distal location to be taken up by other cells
19
or deposited into tissues
20
.
Here, we provide a comprehensive review on EV–ECM interactions.
We start by surveying the current knowledge of different cell-secreted
nanoscale mediators. We then elaborate on the role of membrane traf-
ficking in EV biogenesis and its regulatory mechanisms by the ECM as a
key example of how cells leverage biological processes to produce and
secrete nanoscale mediators. We examine biomolecular and biophysi-
cal determinants of EV–ECM polymer interactions and highlight recent
advances in interfacing EVs with engineered hydrogels as biologically
inspired strategies to promote tissue regeneration by controlling trans-
port or retention of EVs. Given the importance of sourcing EVs from
cells, we also review the role of biomaterial design in controlling EV
production from cells. Finally, we explore future areas of investigation
into EVs as essential structural elements of hydrogel-based materials to
better recapitulate mechanisms of health and disease and to develop
a novel class of biologically inspired materials.
Cell-secreted nanoscale mediators
Cell-secreted EVs are traditionally classified into apoptotic bodies,
ectosomes (also called microvesicles or microparticles) and exosomes
on the basis of their distinct biogenesis mechanisms
21
(Fig.1). Apoptotic
bodies are produced during apoptosis of cells by outward budding of the
cell membrane22,23. Ectosomes are also produced by outward budding
of the plasma membrane, but may or may not accompany apoptosis24.
By contrast, exosomes are secreted when early endosomes become
specialized into multivesicular bodies (MVBs) by inward budding of
intraluminal vesicles (ILVs). MVBs then fuse with the plasma membrane
to release ILVs as exosomes that express tetraspanins25. The issue with
Nature Reviews Materials
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membrane fluidity
46
but also promotes membrane–cytoskeleton inter-
actions
47
, thereby stiffening the cell membrane
48
. Thus, endocytosis
of lipid rafts may result in a temporary increase in the cell membrane
tension. However, this increase can be counteracted when MVBs fuse
with the cell membrane to release exosomes, a process that can restore
the membrane pool and decrease the tension49. Similarly, MVB fusion
or exocytosis could potentially serve as a homeostatic mechanism to
counteract the loss of plasma membrane during outward budding
when microvesicles or apoptotic bodies are formed.
Biophysical regulation by the ECM
Because cells pull on and sense the resistive force from the ECM2,3, the
biophysical properties of the ECM can impact membrane trafficking
49,50
and hence EV biogenesis. Caveolae represent a subset of lipid rafts
that contain the protein caveolin51. Caveolae have a role in mecha-
nosensing, because they enable endothelial cells to be responsive to
ECM rigidity52,53 and shear flow54,55, and protect cells from rupture by
undergoing flattening and disassembly in response to acute mechani-
cal stress independently of actin and ATP56. Interestingly, caveolin is
known to be incorporated into MVBs and exosomes, and required for
sorting of some ECM molecules into exosomal cargo, which can then
be transported to distal tissues
20
. Conversely, cells reassemble cave-
olae in an actin-dependent manner in response to stress release
56
; this
also happens in a hydrogel matrix that recapitulates the physiological
stiffness of soft tissue, where cells maintain low membrane tension57.
Consistent with these observations, cells on a soft hydrogel matrix
maintain the nanoscale assembly of short actin filaments, which per-
mits MVBs to readily transport and fuse with the plasma membrane to
release exosomes. By contrast, cells on a stiffer matrix form an extensive
actin network, which serves as a physical barrier for MVB transport
and exosome release26.
Chemical regulation by the ECM
Chemical factors in the ECM can also impact EV biogenesis by modulat
-
ing membrane trafficking. The ECM is the largest source of free calcium
ions58, which bind to lipid rafts to initiate calcium signalling and have
essential roles in EV biogenesis, including MVB formation and fusion
to the plasma membrane
59,60
. EV release can be enhanced by soluble
extracellular mediators that elevate intracellular calcium, such as
histamine
61,62
. In cancer and tissue injury, some tissues become rigid
Exosomes
Size: <200 nm
Present: LAMP1, syntenin 1
Additional step:
validation of biogenesis
by live imaging
Exomeres
Size: <50 nm
Present: HSP90β, EN01, GANAB
Additional step: asymmetric-low
ield-low fractionation or
167,000g for 16 h
Supermeres
Size: <30 nm
Present: TGFβI, AG02, ACE2, PCSK9
Additional step: 367,000g for 16 h
ultracentrifugation of supernatant
after exomere isolation
NVEPs
Ectosomes
Size: >200 nm
Present: annexin A1,
SLC3A2, BSG
Additional step: validation
of biogenesis by live imaging
Filopodia-derived vesicles
Size: >200 nm
Present: IRS4, RAC1
Absent: PS and CD63
Additional step: validation of
biogenesis by live imaging
Apoptotic bodies
Size: >1,000 nm
Present: caspase 3, PSMC5,
PSMD1, PSMD11, PSME1
Additional step: apoptotic
cell death
2,000g
10,000g
100,000g
360,000g
Large EVs
Large EVs
Small EVs
Migrasomes
Size: >500 nm
Present: α5 integrin, WGA, NDST1, PIGK, CPQ, EOGT
Additional step: ECM-coated culture
system + density gradient ultracentrifugation
Exophers
Size: >3,000 nm
Present: mitochondrial proteins
Additional step: validation of
biogenesis by live imaging
ECM-bound vesicles
Size: 50–400 nm
Absent: CD63, CD9, CD81
Additional step: ECM
enzymatic digestion
ECM
ARMM
Size: 40–200 nm
Present: ARRDC1
Additional step: validation
of biogenesis by live imaging
ARRDC1
Enzymatic digestion
MVB
ILV
Fig. 1 | Cell-secreted nanoscale mediators. Cells secrete a diverse range of
nanoscale mediators with distinct physicochemical properties. In general, these
mediators are classified into lipid-membrane-bound extracellular vesicles (EVs)
and non-vesicular extracellular nanoparticles (NVEPs), which can generally be
separated on the basis of their size by differential ultracentrifugation. Apoptotic
bodies and ectosomes (or microvesicles) are large (>200nm) EVs and produced by
membrane budding. More recently described large EVs are associated with specific
biological processes and include exophers, migrasomes and filopodia-derived
vesicles. Exosomes belong to a subpopulation of small (<200nm) EVs that
originate from intraluminal vesicles in multivesicular bodies (MVBs) and are
released when MVBs fuse with the plasma membrane. In addition to exosomes,
small EVs consist of other subpopulations, including arrestin-domain-containing
protein 1 (ARRDC1)-mediated microvesicles (ARMMs) and extracellular matrix
(ECM)-bound vesicles. NVEPs, including exomeres and supermeres, are generally
smaller (<50nm) than EVs and can be isolated by additional ultracentrifugation
steps. ILV, intraluminal vesicle; PS,phosphatidylserine.
Nature Reviews Materials
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owing to increased ECM crosslinking63, which by itself can impede EV
production
26
. However, in these disease conditions, tissues undergo
hypoxia, which decreases extracellular pH owing to increased anaerobic
metabolism64,65. Hypoxia has been shown to increase membrane traf-
ficking by recruiting short actin filaments
66
, to increase EV number and
to modify EV cargo content to induce pathogenic phenotypes
67–69
. A low
extracellular pH not only enhances the secretion of caveolin-containing
EVs but also makes the EV membrane less fluid owing to increased
incorporation of sphingomyelin, another class of sphingolipids70.
Biomolecular interactions between EVs and the
ECM network
The molecular basis of the interactions between EVs and ECM polymers
can be hypothesized on the basis of the biochemical compositions of
EVs and the ECM and chemical bonds that govern interactions between
the molecules. EVs contain various protein and lipid molecules, some
of which are known to interact with the ECM via covalent or hydrogen
bonds (Fig.3). However, most of these interactions remain to be directly
confirmed in the context of EV–ECM interactions.
Covalent bonds
In principle, covalent bonds can facilitate permanent interactions
between EVs and the ECM. One way that covalent bonding can
occur between EVs and matrix polymers is when proteins on EVs contain
cysteines exposed to the extracellular space, which can form disulfide
bonds with proteins in the ECM network. This interaction can be facili-
tated by an extracellular disulfide catalyst secreted by cells, as exem-
plified by the covalent incorporation of laminin, which is known to be
Lipid-membrane transport
Extracellular
space
Biophysical regulation
Rigid ECM
Actin
Lipid
raft
Clustering
Endocytosis
Endoplasmic
reticulum
Negative
curvature ILVs
Fusion
MVB
Soft ECM
Ca2+
Ca2+
Chemical regulation
Nucleus
Inluences cargo content
and EV production
Early endosome
ESCRT
Loss of cholesterol
Increased
membrane
luidity
Increased
membrane
tension
Stronger
membrane–
cytoskeleton
interactions
Stress
release
Cholesterol
Ceramide
Caveolin
Low pHHypoxia
O2
O2
H+
H+
Histamine
Fig. 2 | Biogenesis mechanisms of EVs in the ECM. Extracellular vesicle (EV)
biogenesis is tightly linked with the lipid-membrane transport process and
physicochemical factors in the extracellular matrix (ECM) that regulate this
process. Lipid rafts serve as precursors of multivesicular bodies (MVBs) by
providing lipids, including cholesterol and ceramide. Cholesterol mediates the
recruitment of the endosomal sorting complexes required for transport (ESCRT),
and ceramide induces negative curvature to form intraluminal vesicles (ILVs).
The loss of membrane during endocytosis of lipid rafts can be counteracted
by the gain of membrane during MVB fusion, thereby balancing membrane
tension. When the ECM is softer, lipid rafts, including caveolae, are more readily
formed because they are not used to counteract mechanical stress. In this case,
lipid rafts can package some ECM molecules, which are shuttled into MVBs and
released via exosomes. In addition, actin cytoskeletons are less dense in cells on
a soft ECM, thereby facilitating MVB fusion and exosome release. The ECM also
offers chemical cues that facilitate EV release, including oxygen tension, pH and
signalling molecules that activate intracellular calcium levels.
Nature Reviews Materials
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present in some EVs71, into the ECM72. Because EVs are enclosed by the
lipid membrane, they can also form covalent bonds with matrix poly-
mers through lipid–protein interactions. ECM-bound vesicles contain
higher levels of oxidized phospholipids than vesicles in fluid
73
. Oxidized
phospholipids that contain carbonyl moieties form Schiff bases by
reacting with a primary amine group of lysine or arginine, whereas
those that contain α,β-unsaturated carbonyl groups form Michael
adducts by reacting with a thiol group of cysteine or basic residues
of histidine
74
. Indeed, oxidized phospholipids were shown to modify
collagen via lipoxidation throughout life and are hence associated with
ageing
75
. Thus, some covalent EV–ECM interactions may be subject to
regulation by the redox state of their environments, which is altered in
various pathological conditions in which EVs have been implicated76,77.
Hydrogen bonds
Hydrogen bonding is ubiquitous in nature and enables the forma-
tion of reversible interactions. One potential way for EVs to interact
with ECM polymers via hydrogen bonds is through heparin-binding
domains, which are rich in basic amino acid residues, such as arginine
and lysine, and are present in a number of ECM molecules, including
fibronectin, vitronectin, collagen and laminin78. Arginine contains the
positively charged guanidinium group, which forms strong hydrogen
bonds with negatively charged phosphate, sulfate and carboxylate
groups79. The same principle applies to lysine, but its interaction with
a negatively charged group is weaker than for arginine because lysine
forms one hydrogen bond, whereas arginine forms a cyclic structure
with a negatively charged group by forming two hydrogen bonds. Thus,
some ECM polymers with heparin-binding domains may interact with
either sulfated molecules on EVs, such as glypican
80
, or phospholipids
on the membrane of vesicles, such as phosphatidylserine, an acidic
phospholipid, which is enriched in matrix-bound vesicles secreted from
cells in cartilage
81
. Conversely, this process can be inhibited when ECM
polymers themselves are phosphorylated by extracellular enzymes
to become more acidic, as occurs in some tissues, such as bones82. In
addition, the EV membrane contains a number of receptors that can
bind to the ECM at places where hydrogen bonding plays important
roles, including αLβ2 integrin(also known asLFA1)83,84, α4β1 integrin85,86
and CD44 (refs. 87,88).
Biophysical EV–ECM network interactions
The ECM consists of a polymer network with meshes that enable the
transport of liquid and solutes. The mesh size ranges from nanometres
to micrometres
89,90
. Whereas small molecules transport freely through
the meshes by diffusion, EVs are often larger and more likely confined
in the nanoporous ECM (r
mesh
/r
EV
≤ 1, where r
mesh
and r
EV
are the mesh
size and the EV radius, respectively) owing to strong steric hinderance
by the polymer. Indeed, the ECM in the interstitium is known to impede
the transport of larger (>100nm) synthetic nanoparticles and their
drainage into the lymphatic system, thereby serving as a barrier for drug
delivery
91
. The presence of matrix remodelling enzymes, such as matrix
metalloproteinases (MMPs)
92
and lysyl oxidases
93
, in EVs suggests that
EVs can potentially modulate the mesh size of the ECM. However, if each
EV relied on its ability to degrade the ECM to transport, the energy cost
of EV transport would be very high. Hence, some EVs may have evolved to
rapidly transport in the nanoporous ECM with minimum energy cost by
leveraging physical interactions with the network. Indeed, the transport
of EVs in the nanoporous ECM does not necessarily require energy, as
long as mechanisms exist to temporarily reduce steric hinderance in
the network, thereby restoring the thermal motion of EVs. The hopping
Covalent bonds
Disulide bond formation
Hydrogen bonds
Hydrogen bond formation
Schi base and Michael addition
Reduction
Oxidation Reduction
Oxidation
S
S
S
SH
SH
S
SH SH
Schi-base
formation
Michael
addition
NH2SH
Schi base
Primary amine: lysine, arginine
Michael adduct
SH: cysteine
Basic residue: histidine
Carbonyl group from
oxidized phospholipids
α,β-Unsaturated carbonyl group
from oxidized phospholipids
Guanidinium group from arginine Ammonium group from lysine
O HN OO
S
NH
HN NH
H H
O O
P
O O
NH
HN NH
H H
O O
S
O O
NH
HN NH
H H
O O
S S
S
S
NH2
H
HO O
P
O O
NH2
H
HO O
NH2
H
HO O
S
O O
Sulfate group from glypican or
phosphate group from phosphatidylserine
Fig. 3 | Biomolecular interactions between EVs and the ECM. Several
biomolecular interactions can determine whether extracellular vesicles (EVs)
bind to or are released from the extracellular matrix (ECM). Disulfide bonds
can form between a cysteine group of an EV membrane protein and that of
an ECM protein and are reversible depending on the redox state of the tissue
environment and the availability of an extracellular enzyme that catalyses this
process. In addition, covalent bonds can form between a lipid molecule of the
EV membrane and an ECM protein as Schiff bases or Michael adducts. EVs can
also interact with the ECM via hydrogen bonds between a negatively charged
heparin sulfate proteoglycan (such as glypican) or a phospholipid (such as
phosphatidylserine) on the EV membrane and a positively charged amino acid
(such as arginine or lysine from heparin-binding domains) in an ECM protein.
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diffusion model offers a physical explanation of this concept, because
it shows that trapped particles larger than the mesh size can escape at
longer timescales by overcoming the free-energy barrier between the
confinement cages94. Supporting this model, synthetic nanoparticles
were observed to exhibit subdiffusive behaviours with infrequent jumps
in mucus
95,96
, which is made of entangled polymers without covalent
crosslinking. In the context of ECM-based polymers, a number of stud-
ies over the past decades showed that the cartilage matrix allows the
transport of molecules larger than its pore size (~6nm)
90
, including
nanoparticles97, a process that is facilitated under mechanical loading
owing to convective flow
98,99
. Convective flow is also known to drive the
transport of nanoparticles with a size of 20–50nm in the interstitial
matrix by lymphatic drainage
91
. However, EVs do not require actomyosin
contractility, convective flow or matrix degradation to transport in the
viscoelastic ECM
18
. Understanding the biophysical basis of EV–ECM
polymer interactions will not only deepen our fundamental understand-
ing of EV transport in the ECM but also inform engineering strategies
to release EVs from or retain EVs in hydrogels (Fig.4).
EV biophysical properties
The rigidity of synthetic nanovesicles impacts their ability to transport
in a confined space by deformation100–103. A broad range of rigidity
has been reported for EVs. The majority of studies use atomic force
microscopy (AFM) to characterize nanoscale vesicle rigidity in terms
of Young’s modulus (E), which is defined by the response of a material
to a force applied along a 1D axis (in Pa or Nm
−2
). Using the Hertz model
of indentation
104
, E for EVs has generally been reported to be in the
megapascal range, variations of which depend on cell types and sub
-
populations. EVs from tissue preparations, including saliva
105
, neuronal
synapses
106
and blood plasma
107
, show E<10MPa, whereas EVs secreted
from cultured mammalian cells
18
and cancer cells
108,109
show E>20MPa.
Within subpopulations, E is lower for larger EVs than for smaller EVs
and NVEPs from cancer cells108. Intriguingly, a study on synthetic
nanovesicles showed that there exists an optimum E ~ 50MPa for which
vesicles show the fastest diffusivity through mucus102. This value is
similar to the value of E of CD63+ EVs from mesenchymal stromal cells
(MSCs, ~100MPa), which were shown to transport in the crosslinked,
viscoelastic ECM18. The Hertz model is widely used because of its sim-
plicity and independence of particle size, but it requires the assumption
that EVs are purely elastic and homogeneous in composition. More
recently, a modified Canham–Helfrich model was used to separately
determine the bending rigidity (κ) of the EV membrane and the osmotic
pressurization of the EV lumen from AFM measurements
110
. The κ is the
energy needed to deform a membrane to a different curvature from its
initial one (in k
b
T, which equals to 4.11×10
−21
J at room temperature)
111
.
Using this model, the κ of EVs from red blood cells was shown to be
~15 k
b
T (ref.
112
), whereas the κ of EVs from breast cancer cell lines was
shown to decrease from ~16 kbT to below 10 kbT with increased malig-
nancy
113
. Systematic studies are still needed to correlate the Young’s
modulus and bending rigidity of EVs from different sources with their
diffusivity in the ECM.
The relationship between nanoparticle rigidity and diffusivity
motivates the important question of what determines the rigidity of
EVs. Synthetic phosphatidylcholine-based nanovesicles exhibit an
E of 2−10MPa
114,115
and a κ of ~14 k
b
T (ref.
110
); the latter was also observed
in microscale unilamellar vesicles
116,117
. The similarity of these values to
those of EVs warrants further examination of the roles of natural lipid
bilayer compositions and lumen fluid properties in determining the
rigidity of EVs. Cholesterol and sphingolipids are the most abundant
lipids in EVs
118–120
and hence have important roles in the structural integ-
rity of EVs. EVs also contain different phospholipids. Early studies with
microscale unilamellar vesicles showed that at a constant temperature,
the presence of cis-double bonds (unsaturated) in the hydrocarbon tails
of phospholipids introduces a structural kink that decreases molecu-
lar packing, thereby increasing membrane fluidity and decreasing
κ (refs.
121,122
). These observations were confirmed by AFM investiga-
tions of synthetic nanovesicles that showed that liposomes with a
liquid-like, disordered membrane have lower κ (ref.
123
). Culturing MSCs
withpolyunsaturated acids increases the content of phospholipids with
unsaturated fatty acyl groups in EVs124, suggesting the possibility
that the bending rigidity of EVs could be tuned ex vivo. By contrast,
ECM-bound vesicles are enriched in phosphatidylglycerol
73
, which
increases the κ of the membrane
125
. Together, lipid-membrane com-
positions could potentially impact the ability of EVs to transport or
remain within the nanoporous ECM by tuning their deformability.
In addition to lipids, the membrane of EV subpopulations con-
sists of different transmembrane proteins21,126. The rigidity of EVs from
red blood cells generally decreases with increased protein-to-lipid
ratios
127
, although this relationship likely depends on how protein
insertion impacts membrane order115,128,129. One important class of
membrane proteins in natural vesicles is channel proteins that medi-
ate membrane transport, because they regulate fluid content and
Lipid-membrane deformability
Water lux
Reversible crosslinking
Aquaporin
Sphingomyelin
Cholesterol
Sphingolipids
Unsaturated
phospholipids
Reversible crosslinker
Fig. 4 | Biophysical mechanisms of EV transport in the extracellular matrix.
Under certain conditions, extracellular vesicles (EVs) can readily transport through
a nanoporous network without relying on polymer degradation or convection.
EVs contain a distinct set of lipids that can make EVs deformable. The ability of EVs
to flux water through aquaporins enables them to deform in the network, thereby
helping them toresist changes in osmotic pressure. In addition to EV deformability,
extracellular matrix crosslinking likely needs to be reversible for EVs to bind to the
crosslinks and to rearrange the network during the transport process.
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properties of the vesicle lumen. To date, a diverse range of ion and
water channel proteins have been identified in EVs
130
. Of these, aqua-
porins are one of the earliest channel proteins discovered in EVs in
urine
131–133
and red blood cells
134
. The amount of aquaporins in EVs
is known to change depending on physiological demands by cells.
For instance, more aquaporin2 is packaged into EVs from the apical
plasma membrane of the renal-collecting ducts when there is an
increased demand to retain water in the body131, whereas red blood
cells secrete EVs with less aquaporin1 under hypertonic conditions
134
.
Interestingly, aquaporin-driven water flux was shown to maintain
stability in plant-derived vesicles under hypertonic conditions
135
,
suggesting its role in resisting mechanical deformation. From a bio-
physical perspective, deformation of EVs would temporarily decrease
the internal volume and hence increase the concentration of sol-
utes in the lumen, thereby creating osmotic pressure and increasing
vesicle rigidity110. Aquaporin1 is essential for EVs to transport in the
nanoporous ECM, and downregulating aquaporin1 rigidifies EVs
18
.
Thus, rapid water flux by aquaporins likely helps toresist changes in
osmotic pressure and rigidification of EVs upon deformation during
the transport process.
ECM biophysical properties
The deformability of EVs alone is likely not sufficient to overcome steric
hinderance by the matrix polymer, because extreme deformation of
EVs would compromise their structures. Successful EV transport also
requires the ability of the ECM polymer to undergo structural reorgani-
zation, which is determined in large part by polymer crosslinking. In
general, a less permanent form of crosslinking, such as electrostatic
and hydrogen bonds, results in a polymeric network that dissipates
energy upon external force, leading to viscoelastic properties136.
Because most tissues are viscoelastic1, it is possible that EV transport
occurs in tissues upon external load. Interestingly, a modelling study
showed that in the absence of external force, a weakly crosslinked ECM
polymer network can still rearrange if nanoparticles or nanovesicles in
the polymer transiently bind to or interfere with the crosslinks of the
polymer, thereby enabling their transport in the ECM137. This concept
remains to be directly tested for EVs in the ECM, but a study supports
this notion, because EVs but not synthetic nanoparticles can trans-
port in ionically crosslinked hydrogels18. This observation raises the
interesting possibility that EVs may be able to transport in viscoelastic
hydrogels by influencing their crosslinks.
Interfacing EVs with engineered materials
After systemic injection in vivo in solution form, EVs are dispersed
and cleared by the liver with a half-life of hours or less
138
. Analogous
to controlled drug delivery
139
, material-based strategies, often based
on engineered hydrogels, can be used to control either the release or
the retention of EVs in a specific tissue. Implantation
140,141
, injection of
bulk hydrogels
142
, in situ gelation
143–150
and microgels
151
have been used
to deliver hydrogels containing EVs to the host. The majority of these
strategies used EVs from MSCs as a means to restore damaged tissues,
because they are known to contain cargo molecules with potential
immunomodulatory and regenerative effects152,153.
Controlled release of EVs to the host
Diffusion. The ability to gradually release EVs from hydrogels will help
tocontrol the rate at which EVs become available to occupy tissue
over time to achieve therapeutic effects. The first important step to
achieve this goal is to crosslink hydrogels from polymer solutions in
the presence of EVs, so that EVs can gradually diffuse from hydrogels
over time (Fig.5). EV transport is generally more sensitive to crosslink-
ing than small molecule transport owing to the large particle–mesh
size ratios. Thus, the choice of crosslinking strategy determines both
the kinetics and the maximum amount of EV release by diffusion.
A delayed release of EVs was obtained from alginate hydrogels with
high molecular weight
142
. The release might have been facilitated by
the use of CaCl2 as an ionic crosslinking agent, which results in a rapid
but non-uniform gelation
154
. Viscoelastic hydrogels from purified algi-
nate can release a substantial fraction of EVs at an optimum elasticity
when crosslinked with CaSO
4
, which offers a slower, more uniform
gelation. Diffusion is partially facilitated by the ability of the EV to
control deformation via water flux in confined spaces
18
. In addition
to partial or reversible crosslinking of hydrogels, temperature-sensitive
crosslinking of hydrogels can be effective in achieving controlled EV
release, while being a useful strategy to obtain injectable materials.
A recent study loaded EVs in chitosan with glycerol-2-phosphate, which
undergoes ionic crosslinking after injection at 37°C, with an optimum
porosity controlled by polymer concentration. EVs were gradually
released and promoted corneal regeneration145. Another study used
methylcellulose-based hydrogels with xylitol and polyethylene glycol
(PEG), which undergo gelation at 37°C via hydrogen bonds, to control
the release of EVs, which is accelerated at lower temperatures. This
system can potentially be useful in some disease conditions, such as
critical limb ischaemia, in which the temperature of the damaged tissue
is known to decrease owing to reduced blood flow146.
Erosion. To ensure that EVs are as completely released as possible from
hydrogels in a localized manner, several studies have used strategies to
induce the erosion of the polymer backbone. These strategies can be
categorized on the basis of the degradation mechanisms (Fig.5). The
simplest strategy is to engineer polymer networks so that they undergo
hydrolytic degradation over time to gradually release EVs148,151,155. For
example, cleavage of the ester bonds present in poly (lactic acid)-based
3D-engineered scaffolds results in sustained release of EVs from human
gingival MSCs, which can treat bone defects
155
. Similarly, clickable
PEG-based hydrogels, in which cleavage of the ester bonds in PEG-thiol
derivatives leads to gradual swelling and sustained release of encapsu
-
lated EVs from MSCs over 4 weeks, were used to treat an animal model
of chronic liver failure
148
. In addition, aldehyde-containing oxidized
sodium alginate hydrogels with a low degree of oxidation were used to
achieve prolonged release of dermal papilla-derived EVs over a period
of 7 days, resulting in improved hair growth151.
In many cases, it is desirable to erode the polymer backbone
in response to specific conditions in the host tissue. In a number of
diseases, such as cancer and diabetic wounds, tissue environments
become acidic, presenting opportunities to release EVs in a pH-sensitive
manner. For example, EVs were encapsulated in a hydrogel formed by
Schiff-base reaction between the aldehyde group of oxidized hya-
luronic acid and the primary amine group of a polypeptide, such as
ε-poly--lysine. Because Schiff bases hydrolyse under weak acidic
conditions, this hydrogel system enables EV release in response to
low pH, which was shown to be effective in treating an animal model
of chronic diabetic wounds147.
Enzyme-based degradation mechanisms can also be used to erode
the polymer backbone and release EVs. In particular, naturally derived
hydrogels or synthetic hydrogels with peptide-based crosslink-
ers can be used to encapsulate EVs, so that they can be released
when various cells in the host tissue secrete MMPs. For instance,
Nature Reviews Materials
Review article
gelatin-methacrylate hydrogels are degraded by both collagenases
and gelatinases156 and were used to encapsulate and locally release
EVs for the treatment of myocardial infarction
149
and for cartilage
regeneration140. In addition, MMP2-cleavable self-assembling peptides
were used to form hydrogels and deliver EVs in the context of renal
ischaemia–reperfusion injury150.
Light
pH
Hydrolysis
Enzyme
Lys‐Leu‐Asp‐Leu‐Pro‐Val‐Gly‐Leu‐
Ile‐Gly‐Lys‐Leu‐Asp‐Leu
25–32 ºC 37 ºC
Methyl cellulose
PEG
Xylitol
+
+
Tetra-PEG-thiol + tetra-PEG-maleimidePolylactic acid Oxidized sodium alginate
Thermosensitive crosslinking
Bifunctional thiol–acrylate photocleavable linker Thiolated hyaluronic acid
+
+
Primary amine from
polypeptide (poly-L-lysine)
Schi-base bond formation
Gelatin methacrylateMMP-responsive
self-assembling peptide
pH change
<pH 5.5
UV light
Aldehyde group from
oxidized hyaluronic acid
Enzymatic
cleavage
Ionic crosslinking
Partial or viscoelastic
O
O
SN
O
O
O
NH
HO
O
O
H
O
O
nm
OO
OH
HO
HO
NH
OO
HO
HO OH
OH
NH
O
SH
O
O
S
O
O
O
ONO2
3
O
O
3
O
O
HO
OH
OH
OH
OH
O
O
O
HO
n
OH
OO
OH
OO
HO
O
OH
OO
O
O
OO
O
O
O
O
O
O
O
OH
HO
mn
OO
O
O
O
HO
HO
NH
O
HOOC
O
Hn
H
N
O
NH2
n
H
N
O
NO
O
N
HNH
N
H
O
O
H
N
N
O
OH
O
O
O
H
N
NH
N
HO
H2N
O O
DiusionErosion
Chitosan
Glycerol-2-phosphate
+
O
NH2
HO HO
OH
O
NH2
OHO
OH
n
Na
HO
HO
P
O
OO
OH
Na
Nature Reviews Materials
Review article
The light-sensitive degradation of hydrogels addresses a need
for non-contact-based strategies to externally trigger EV release inde-
pendently of host tissue conditions. The ortho-nitrobenzyl-based
photocleavable linker, which contains both thiol and acrylate groups,
was used for this purpose to promote wound healing
143
. The linker
molecules were first attached to EVs via disulfide bonds and then mixed
with cysteine-conjugated hyaluronic acid to induce gelation via thiol-
acrylate Michael addition. The amount of released EVs was shown to
be proportional to the duration of UV light irradiation, suggesting the
utility of this approach for on-demand EV release.
Strategies to increase EV retention within hydrogels
Previous studies suggest that EVs deposited on a cell culture surface
facilitate cell migration
157–159
, raising the possibility that EVs can be used
as haptotactic cues to recruit cells in the vicinity of hydrogels via jux-
tacrine interactions. In addition, when EVs are entrapped in hydrogels,
soluble factors from EVs can be released in a controlled manner160. Some
of these factors are chemotactic signals
161,162
, which can recruit cells
from a distance. Thus, increasing the retention of EVs in hydrogels offers
opportunities to recruit, program and deploy host cells in a localized
manner. Indeed, physical entrapment of EVs in nanoporous hydrogels
was shown to increase EV retention in vivo after delivery163–165.
Hydrogels can be engineered to increase the retention of EVs by
leveraging non-selective or selective molecular interactions (Fig.6).
The advantage of using non-selective interactions is that they can be
generalized to different types of EVs regardless of their subpopulations
or sources. Because the EV membrane is often negatively charged166,167,
positively charged materials can be used to increase the retention of EVs
via electrostatic interactions, promoting regeneration168 and immuno-
modulation
169
. EVs can also be grafted to materials more permanently
by covalent bonds. One study used a photoinduced imine crosslinking
hydrogel to graft EVs upon gelation and showed sustained EV retention
over 2 weeks
164
. More recently, a copper-free click chemistry strategy was
described, in which EVs were collected from cells that were metabolically
labelled with azide-containing amino acids and encapsulated in collagen
hydrogels that were modified with dibenzocyclooctyne to conjugate
EVs. This strategy resulted in increased recruitment of macrophages and
vascular growth in hydrogels
170
. By contrast, selective molecular interac-
tions are desirable if the goal is to elicit specific biological responses by
immobilizing a subset of EVs. This has been achieved by grafting peptide
sequences that bind to specific integrins present on the EV membrane
to promote EV retention and tissue regeneration. Examples include the
Arg-Gly-Asp (RGD) peptide
171,172
that binds to α5β1 and αVβ3 integrins
173
to promote kidney and bone repair and a laminin-derived peptide
174
that
binds to α3β1 integrin175 to treat spinal cord injury.
Material-based cell culture strategies to control
EV secretion from cells
Most studies on the controlled release and retention of EVs via engi-
neered materials collect EVs from cells on 2D tissue culture plas-
tic and interface them with materials. However, as we discussed,
physicochemical factors related to materials used in cell culture can
impact the quantity and the properties of EVs, which may subsequently
influence their downstream applications. Thus, it is important to under-
stand how materials impact EV production by cells. These insights
can be helpful not only to improve the production of EVs that will be
interfaced with materials but also to inspire material-based strategies
for sustained EV release via cells. Advances in biomaterial design and
biomanufacturing strategies have led to tunable engineered systems
that recapitulate physical, chemical and structural properties of native
tissues. These systems have been leveraged to uncover new insights on
cellular functions that cannot be readily studied under standard tissue
culture conditions
153,176
. Recent studies have used these advances to
control and improve EV production.
Fig. 5 | Biomaterial strategies to control EV release. Extracellular vesicle (EV)
release can be controlled by either diffusion or erosion-based mechanisms. EVs
can diffuse out of partially crosslinked or viscoelastic hydrogels. Thermosensitive
crosslinking can be used to tune EV diffusion from hydrogels as a function of
temperature. For a more complete local release of EVs, erosion of a hydrogel
network can be achieved either spontaneously through hydrolytic degradation
or conditionally in response to external stimuli. The external stimuli that result
in EV release by erosion of a hydrogel network can be classified into those that
depend on host tissue conditions, such as pH and the presence of enzymes, and
those that enable on-demand release, such as light. Specific examples are shown
for each category. MMP, matrix metalloproteinase; PEG, polyethylene glycol.
Electrostatic interactions Imine bonding
Sequence-specific interactionsClick chemistry
Peptide
Integrins
o-Nitrobenzyl
DBCO
Azide
+ Charged domains
O
O
ON
N
N
NNN
O
O
O
ON
N
O
O
ON
N
N
NNN
O
N
NNN
O
Fig. 6 | Biomaterial strategies to promote EV retention. Introducing molecular
interactions between extracellular vesicles (EVs) and a polymer network helps
toretain EVs within biomaterials to recruit and locally program cells. These
interactions can be general, such as electrostatic interactions, imine bonding and
click chemistry (for example, with dibenzocyclooctyne (DBCO)–azide covalent
bonds) of metabolically labelled EVs, to accommodate different types of EV
subpopulations. Conversely, introducing a molecular sequence to a polymer
network, such as an adhesion peptide that binds to integrins, enables the capture
of a defined EV subpopulation to elicit a specific biological response.
Nature Reviews Materials
Review article
One important advance was the development of bioreactor sys-
tems in which cells can be cultured and a medium can be perfused so
that EVs can be collected over time. Hollow-fibre bioreactor systems
(such as Fibercell) have emerged as one of the major methods to scale
up the production of EVs, because hollow fibres offer a high surface area
to attach a large number of cells (over 10
9
) per setup, while enabling the
circulation of the medium for nutrient exchange
177–180
. In addition to
concentrating EVs in a small medium volume, the system also produces
more proteins associated with small EVs per protein preparation com-
pared with plastic culture. This suggests that a hollow-fibre geometry
and mass transfer have potential to increase small EV secretion or to
decrease EV reuptake. It is also possible to customize a bioreactor sys-
tem by replacing hollow fibres with a 3D-printed scaffold from a com-
mercial stereolithography instrument, which was shown to increase EV
production from endothelial cells181. These studies used rigid materials
to attach cells; using a hydrogel-based cell culture surface or a scaffold
with physiological biophysical properties26 will likely further increase
the yield of EVs from bioreactor systems.
Another emerging approach consists in collecting EVs from cell
spheroids formed in microwells or on non-adhesive materials182. In
one study, spheroids from gastric cancer cells were formed in an aga-
rose microwell array and shown to increase the number of EVs per
cell, with a decreased average EV size. Spheroid-derived EVs show an
increased level of microRNAs, which subsequently downregulate pro-
teins involved in the ADP-ribosylation factor 6 pathway to inhibit large
EV production, while promoting small EV production
183
. Consistently,
another study showed that MSC spheroids formed by a hanging-drop
method or on an anti-adhesive, a poly(2-hydroxyethyl methacrylate)-
coated surface, increase the EV number per cell compared with 2D
cultures180. In a therapeutic context, cell spheroids were formed from
lung biopsy tissues on an anti-adhesive surface, followed by cell expan-
sion and collection of EVs, which were shown to be effective in treating
preclinical models of fibrotic lung injury184. Overall, these studies
suggest the utility of forming spheroids in promoting EV production.
Given the diffusion limit of spheroids for nutrient exchange, the size
of spheroids needs to be kept below 100µm to avoid the necrotic
core185. Combining this strategy with a bioreactor system or using
vascularization strategies will enable the use of larger spheroids with
high viability to increase the yield of EVs. From a mechanistic per-
spective, micropatterning-based strategies to decouple cell–cell con-
tact and cell–material interactions
186
will help todissect the relative
contributions of these interactions to EV production.
In principle, encapsulation in engineered materials provides cells
with physiologically relevant cues in 3D microenvironments, which could
be optimal for EV production compared with standard culture condi-
tions. One study showed that the amount of EV proteins secreted per
cell increases when the medium is collected from MSCs in a 3D collagen
gel compared with cells on a 2D plastic culture, and that EVs from MSCs
in a 3D collagen gel with a pore size of 1–3µm (ref. 187) show efficacy in an
animal model of traumatic brain injury188. Another study showed that
encapsulating HeLa cells in a peptide nanofibre-based hydrogel with
a pore size of ~500nm increases cell spheroid formation compared
with a 2D plastic culture, resulting in a more gradual release of EVs with a
unimodal size distribution and a similar microRNA expression profile as
that of plasma of patients with cervical cancer
189
. More studies are needed
to understand how 3D environments improve EV production, because
these observations can be attributed to several factors arising from dif-
ferences in the presentation of both physical and biochemical cues by
3D hydrogels compared with 2D plastic cultures. Unlike in 2D cultures,
where EVs are directly secreted into a liquid medium, in 3D environments,
EVs can interact with a polymeric network, a factor that needs to be taken
into consideration when evaluating EV production.
Outlook
Understanding EVs in the context of the ECM can inspire various strate-
gies to interface them with engineered hydrogels as a means to improve
their therapeutic efficacy by locally controlling their release or reten-
tion. Making advances in this field requires the convergence of mul-
tiple fields, including cell and matrix biology, chemistry, membrane
biophysics, biomaterial design and nanotechnology.
The presence of EVs in the ECM is reminiscent of synthetic nano-
composite hydrogels
190
, materials with distinct properties arising from
the inclusion of nanoparticles
190
, which were developed to achieve
advanced material properties, such as rapid self-healing
191
and tough-
ness
192
. Polymer physics teaches us that nanoparticles or nanovesicles
can crosslink a polymer chain if they bind to the polymer with strong
affinity and multivalency, provided that they are small enough to be
bridged by the network193. This principle suggests the possibility that
some cell-secreted nanoscale mediators may serve as primary or sec-
ondary crosslinkers of the ECM polymers and hence influence ECM
structure and ultimately function. Large EVs will likely offer greater
multivalency, but small EVs may be better suited to be bridged by the
network. Exomeres are smaller and more rigid than EVs35, suggesting
the possibility that NVEPs may remain in nanoporous hydrogels after
encapsulation and contribute to mechanical rigidity.
A simple negative feedback loop can be envisioned in which cells
initially secrete more EVs when the ECM is softer26, but when some EVs
are deposited into the ECM20, they stiffen the network by crosslinking
and limit the ability of cells to further produce EVs in a physiological
condition. Testing this possibility will necessitate the development of
materials with properties that can be dynamically tuned by incorpo-
ration of EVs from material-interfacing cells. This is also important in
modelling diseases, such as cancer194 and fibrosis195, in which the ECM
stiffens and EVs play important roles in disease progression196,197. Under-
standing the interplay between cell-secreted EVs and the ECM and its
impact on cellular functions will help toadvance our understanding of
pathological processes that accompany substantial structural changes
in tissue microenvironments.
It has become clear that cells secrete both EVs and NVEPs and that
they have distinct properties
33–36
. Because this insight has emerged very
recently, it is likely that most studies to date interfaced both EVs and
NVEPs with biomaterials simultaneously. Future efforts will benefit from
the implementation of fractionation strategies to separate or deplete
EVs and NVEPs, such as immunoaffinity-based approaches
198
, before
interfacing with biomaterials. In addition, biogenesis mechanisms and
biomolecular compositions are beginning to be better understood
for different types of EVs and NVEPs, offering opportunities to design
biomaterials that can release or retain specific subpopulations171,172,174.
The field is still young and rapidly redefined, but it is clear that combin-
ing cell-secreted nanoscale mediators with biomaterial design offers
a novel platform to advance materials science, biology and medicine.
Published online: xx xx xxxx
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Acknowledgements
This work was supported by NIH Grants R01-GM141147 (J.-W.S.),R01-HL141255(J.-W.S.)
and T32-HL007829 (A.R.), National Science Foundation CAREER Grant 2143857 (J.-W.S.) and
Hebrew University of Jerusalem and University of Illinois Joint Research and Innovation
Seed Grant(J.-W.S.). The authors acknowledge S. Badylak and G. Hussey (University of
Pittsburgh)for initial discussion of the topic.
Author contributions
All the authors researched data for the article and wrote the article. All the authors
contributed substantially to discussion of the content and reviewed the manuscript before
submission.
Competing interests
The authors declare no competing interests.
Additional information
Peer review information Nature Reviews Materials thanks Mei He and the other, anonymous,
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