International Journal of

Molecular Sciences

Review
Unraveling the Multifaceted Roles of Extracellular Vesicles:
Insights into Biology, Pharmacology, and Pharmaceutical
Applications for Drug Delivery
Ali Al-Jipouri 1 , Àuria Eritja 2 and Milica Bozic 1,2, *

1 Institute for Transfusion Medicine, University Hospital Essen, University of Duisburg-Essen,

D-45147 Essen, Germany; [email protected]
2 Vascular and Renal Translational Research Group, Biomedical Research Institute of Lleida Dr. Pifarré
Foundation (IRBLLEIDA), 25196 Lleida, Spain; [email protected]
* Correspondence: [email protected]

Abstract: Extracellular vesicles (EVs) are nanoparticles released from various cell types that have
emerged as powerful new therapeutic option for a variety of diseases. EVs are involved in the trans-
mission of biological signals between cells and in the regulation of a variety of biological processes,
highlighting them as potential novel targets/platforms for therapeutics intervention and/or delivery.
Therefore, it is necessary to investigate new aspects of EVs’ biogenesis, biodistribution, metabolism,
and excretion as well as safety/compatibility of both unmodified and engineered EVs upon adminis-
tration in different pharmaceutical dosage forms and delivery systems. In this review, we summarize
the current knowledge of essential physiological and pathological roles of EVs in different organs
and organ systems. We provide an overview regarding application of EVs as therapeutic targets,
therapeutics, and drug delivery platforms. We also explore various approaches implemented over
the years to improve the dosage of specific EV products for different administration routes.

Keywords: extracellular vesicles; lipid bilayer vesicles; cell-free therapeutics; pharmacokinetics;

pharmacodynamics; pharmaceutical applications/drug delivery; nano-biocarriers

Citation: Al-Jipouri, A.; Eritja, À.;

Bozic, M. Unraveling the Multifaceted
1. Introduction
Roles of Extracellular Vesicles:
Insights into Biology, Pharmacology, Extracellular vesicles (EVs) are lipid bilayer vesicles released and taken up by diverse
and Pharmaceutical Applications for types of cells, thus playing an important role in different physiological and pathological
Drug Delivery. Int. J. Mol. Sci. 2024, processes. In a physiological context, EVs serve as facilitators of intercellular communica-
25, 485. https://doi.org/10.3390/ tion, whereas in pathological circumstances, they play roles in the onset, aggravation, and
ijms25010485 resilience of various diseases [1]. Owing to their distinctive attributes, EVs hold significant
Academic Editor: Tamás Visnovitz
promise in formulating innovative therapeutic approaches for various diseases.
Comprehension of the regulatory mechanisms of EVs in diverse biological processes
Received: 30 November 2023 and intercellular communication is essential for unlocking their clinical potential and broad-
Revised: 19 December 2023 ening their applications. Various non-clinical trials aim to provide comprehensive data on
Accepted: 25 December 2023 the pharmacodynamics, pharmacokinetics, and toxicity of EV products to support their
Published: 29 December 2023
investigation in future clinical trials, focusing on their efficacy and safety. The therapeutic
trinity concept of EVs encompasses three primary therapeutic applications: (a) EVs as
therapeutic targets; (b) EVs as therapeutics; and (c) EVs as drug delivery platforms.
Copyright: © 2023 by the authors.
The current review outlines important physiological roles of EVs in different organs
Licensee MDPI, Basel, Switzerland. and describes their crucial pathological roles in the development of several diseases. We
This article is an open access article provide an overview of the most recent discoveries regarding the three primary therapeutic
distributed under the terms and uses of EVs: inhibiting their pathological functions for therapeutic targeting, leveraging
conditions of the Creative Commons their natural functions for therapeutic purposes, and employing their in vivo kinetics as a
Attribution (CC BY) license (https:// foundation for drug delivery platforms. Finally, we discuss various strategies that have
creativecommons.org/licenses/by/ been undertaken during recent decades to improve EV-based dosage forms for various
4.0/). routes of administration.

Int. J. Mol. Sci. 2024, 25, 485. https://doi.org/10.3390/ijms25010485 https://www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2024, 25, 485 2 of 51

2. Biology of Extracellular Vesicles

2.1. Biogenesis of Extracellular Vesicles
Extracellular vesicles (EVs) are lipid bilayer vesicles composed of proteins, lipids, and
nucleic acids which are heterogenous in structure and function [2]. EVs are released by cells
from prokaryotes to lower/higher eukaryotes, and plants [3], and play an important role in
mediating physiological and pathological processes [4–7] (Table 1). As per their biogenesis,
EVs are divided into two main types: Exosomes and microvesicles [2,4]. While apoptotic
bodies may also be considered a type of EV, their role in intercellular communication is
less studied and will not be reviewed here. Exosomes (30–150 nm) originate through the
invagination of the limiting membrane of the early endosome, i.e., multivesicular bod-
ies (MVBs) and are released to the extracellular environment upon fusion of MVBs with
the plasma membrane [5]. Microvesicles (MVs) (100–1000 nm) are produced by outward
budding and pinching of the plasma membrane [8,9]. The intracellular membrane is not
involved during the secretion of microvesicles, and thus the membrane composition closely
mirrors that of parent cells, a key difference from exosomes, which are heavily enriched in
phosphatidylserine [4]. Although several other types of EVs released from plasma mem-
branes have been discovered, such as migrasomes, ciliary ectosomes, secreted midbody
remnants, exophers, etc., they have recently been classified into two major categories:
exosomes (originating from the endosomal compartment) and ectosomes (originating from
the plasma membrane) [10]. Recently, several mechanisms have been identified to regulate
the biogenesis of EVs, thereby facilitating the sorting of protein and RNA cargo to generate
EVs with a precise biochemical composition [11,12]. EV contents, size, and membrane
composition are highly heterogeneous, dynamic, and dependent on the cellular source,
state, and environmental conditions.

2.2. Composition of Extracellular Vesicles

The composition of EV subgroups varies greatly depending on their source and iso-
lation or enrichment techniques. Exosomes’ protein topology is the same as that of the
releasing cell plasma membrane due to fusion of MVBs with plasma membrane, whereas
the protein topology of microvesicles is heterogenous due to the direct budding off-plasma
membrane [4]. Because there are no specific markers that differentiate between exosomes
and microvesicles, investigating these two groups individually remains challenging [10].
The content of EV proteins ranges from general EV markers, subdivided into exosome
markers (tetraspanins (CD9, CD63, CD81, and CD82), syntenin-1, TSG101, and Alix) and mi-
crovesicle markers (glycoprotein 1b, actinin-4, heat shock protein (HSP) 90B1, and myosin
light chain) to post-translational protein modifications that specifically reflect vesicle lo-
calization, cellular origin (tissue-specific proteins), and secretion machinery [13,14]. EVs
are highly abundant in cytoskeletal, cytosolic, heat shock, plasma membrane, and vesicle
trafficking proteins, while they are less abundant in intracellular organelle proteins [12].
Furthermore, lipids are major components of EVs and have important roles during EV
biogenesis, release, targeting, and cellular uptake [15]. The lipid composition of EV mem-
brane depends on the type and physio-pathological status of releasing cells and determines
their biological properties. Compared to parental cells, EVs are enriched in sphingolipids
(i.e., sphingomyelin and ceramides) and glycerophospholipids containing saturated fatty
acids [16]. These lipids resemble rafts that are important for increasing EV membrane
rigidity and stability in biological fluids compared to parental cells. Moreover, the phos-
pholipids that make up the EV membrane are also precursors of bioactive molecules (i.e.,
lysophospholipids and eicosanoids) that are able to mediate several processes in target
cells, such as immune signaling and inflammation [16].
Int. J. Mol. Sci. 2024, 25, 485 3 of 51

Table 1. Selected organ-derived physiological “Good” versus pathological “Bad” EVs.

Homeostasis EVs EVs Physiological “Good” Pathological “Bad”

Ref.
State Source Cargo Effect Effect
Diabetic nephropathy
Urinary Tract Nephron collecting duct Balancing overall water and ion (AQP2-AQP5 interaction) and
AQP2
epithelial cell-derived levels in response to blood nephrogenic diabetes insipidus [17]
protein/miRNA
Water-Salt Balance EVs osmolality (AQP2 mutation) result in the
inability to concentrate urine
Gastrointestinal Tract GIT-Microbiota Low (Bacteroidota/Firmicutes)-
High (Bacteroidota/Firmicutes)-
(Bacteroidota—Gram derived OMV/MV ratio reduces
OMVs carrying LPS derived OMV/MV ratio increases
Gut–Brain–Microbiota negative/Firmicutes— GBMAx permeability, producing [18]
cross BBB GBMAx permeability; therefore,
Axis Gram positive)-derived normal child brain development
children are vulnerable to ASDs
(GBMAx) OMV/MV ratio and function
DMD is caused by a mutation in
the dystrophin gene. Therefore,
Upregulation of miR-206 targets
upregulation of miR-206 will
ribosome binding protein 1
further promote collagen
required for collagen synthesis
Musculoskeletal synthesis at the expense of
along with dystrophin, which
System Muscle precursor quiescent satellite cells,
MyomiR miR-206 stimulates asymmetric division of [19]
satellite cell-derived EVs inflammatory cytokine secretion,
satellite cells and will help repair
Myogenesis and disturb
muscle injury and reduce
calcium/mitochondrial
extracellular matrix deposition
homeostasis, contributing to the
ideal for muscle remodeling
replacement of muscles with
fibrous and adipose tissues
STB-EV MICs and pro-apoptotics
maintain semi-allogeneic fetus STB-EVs carrying MICs can
Reproductive Tract NKG2D receptor
immune tolerance by suppressing induce semi-allogeneic fetus
binding MIC-related
immunity at the fetal–maternal rejection, i.e., miscarriage by
Semi-Allogeneic Fetus STB-derived EVs proteins; A, B, and UL16, [20]
interface via downregulating cross-dressing maternal APCs,
Tolerance and Self- pro-apoptotic proteins;
NKG2D NK cells and promoting thereby activating NKG2D NK to
Recognition FASL and TRAIL
Treg cell development through attack fetal cells
HSPEI and their miRNA cargo
Loss-of-function (LOF) mutations
Central Nervous SHH protein stimulates in the ESCRT-III member,
Cerebellar Purkinje
System proliferation of GCPs, a CHMP1A required for vesicular
Cell-derived
SHH protein progenitor cell that generates SHH secretion causes [21]
AXL-RAB18-TMED10
Sonic Hedgehog (SHH) granule neurons, the most microcephaly with
(ART)-EVs
Signaling Pathway abundant neuron in the brain pontocerebellar hypoplasia and
short stature in humans
Human Scott syndrome is a mild
bleeding disorder caused by loss
Platelet EVs mediate the
Cardiovascular System of Ca2+ -dependent scramblase
homeostasis necessary for
Platelet-derived EVs TF-CD142 activity. Upon vascular damage, [22]
embryogenesis, angiogenesis, and
Blood Coagulation the perivascular TF and not the
inflammation
platelet EVs’ TF will initiate the
coagulation process
The participation of EVs in the
MHC-I and -II cross-presentation of exogenous EVs express immunoregulatory
Immune System
versus antigens on MHC-I complexes to molecules: PDL1, CTLA4, FASL,
APCs: DCs, BLs, and immunoregulatory CD8+ T cells has an important and TRAIL, which interact with
Immune Tolerance [20]
MP-derived EVs molecules: PD-L1, role in immunity against viruses cognate ligands and receptors
versus
CTLA4, FASL, and and tumors and in the immune expressed T and NK cells, inhibit
Immune Regulation
TRAIL response upon vaccination and their activity, or induce apoptosis
induction of tolerance
Aquaporin-2 (AQP2); outer membrane vesicles (OMVs)/membrane vesicles (MVs); lipopolysaccharides (LPS);
blood–brain barrier (BBB); autism spectrum disorders (ASDs); Duchenne muscular dystrophy (DMD); Syncy-
tiotrophoblast (STB); natural killer group 2 member D (NKG2D); major histocompatibility complex class-I chain
(MIC); antigen presenting cells (APCs); granule cell precursors (GCPs); endosomal sorting complexes required for
transport (ESCRT); charged multivesicular body protein 1A (CHMP1A); tissue factor (TF); dendritic cells (DCs); B
lymphocytes (BLs); macrophages (MPs); major histocompatibility complexes (MHCs); programmed cell death
ligand 1 (PD-L1); cytotoxic T-lymphocyte-associated antigen 4 (CTLA4).

2.3. Physiological Roles of Extracellular Vesicles

EVs are produced and taken up by all types of cells. Therefore, substances that make
EVs or are enclosed within them represent physiological components of the body. Cells
rely on their secretome, more specifically on EVs, to induce various biological effects
and physiological functions [23,24]. Furthermore, as EVs can sum up a large part of the
parental cell’s biological effects, they are considered potential therapeutic agents. Indeed,
preclinical studies have shown beneficial effects of EVs/secretome from various cell sources
to treat many injuries of the heart, kidney, liver, brain, and skin [25–29]. EVs exert their
basic physiological functions in a pleiotropic manner via (a) protein-/lipid-ligands’ direct
Int. J. Mol. Sci. 2024, 25, 485 4 of 51

cell surface receptor activation, and/or (b) recipient cell plasma membrane fusion and
delivery of effectors (proteins and nucleic acids) [30–33], playing critical roles in stem cell
maintenance [34], tissue repair [35], immunosurveillance [20], and blood coagulation [36].
Body fluid (urine, saliva, synovial, bile, cerebrospinal, bronchoalveolar, nasal, uterine,
amniotic, breast, blood, feces, and seminal)-derived EVs are a mixture of vesicles that
originate from various sources, such as cells in body fluids and/or cells that line extruded
body fluid cavities. Thus, the contents of these EVs act as a source of physiological and
pathological information, which can be transmitted over a long distance. In this section,
we will focus on important physiological roles of EVs in maintaining the homeostasis of
different organs.

2.3.1. Urinary Tract

It has recently been shown that 3% of the total urinary protein content originates
from EVs [37]. Although urinary EVs serve as a reservoir of biomarkers that come from
the kidneys, ureters, urinary bladder, urethra [38], and prostate [39], their secretion and
reuptake are essential in intercellular communication along the nephron and thus preserva-
tion of homeostasis of the urinary tract [40]. To differentiate between kidney-derived EVs
and other infiltrating EVs, CD24 and CD133 may be of interest as kidney-specific urinary
EV markers [41]. In the healthy organism, EVs contribute to the regulation of water–salt
balance, where vasopressin-mediated water channel aquaporin-2 (AQP2), an apical Na+
transporter protein carried by EVs to the collecting duct cells [42], plays an important
role. Thus, EVs control AQP2 trafficking and fusion with the apical plasma membrane,
increasing nephron water permeability and hence water retention within the kidney [17].
Similarly, the direct action of one of EVs’ resident proteins, the angiotensin-converting en-
zyme (ACE) of the renin–angiotensin system (RAS), plays a role in water homeostasis [43].
Additionally, urinary EVs are rich in innate immune effectors (calprotectin and lysozyme
C) that contribute to host defense within the urinary tract [44]. Moreover, urinary EVs
expressing tissue factor (TF) can promote coagulation and hemostasis and thus reduce
blood loss and contribute to host defense by reducing the risk of microorganisms entering
the body through urinary and urethral epithelia [45].

2.3.2. Gastrointestinal Tract

Saliva is the most easily accessible biofluid and is considered as a mirror of general
health. It is currently considered as a biofluid suitable for multilevel assessments [46].
Saliva-derived EVs are biologically active due to their protein and nucleic acid content,
and, upon internalization by oral keratinocytes and macrophages, induce alterations in
gene expression [47,48]. The source of saliva-derived EVs is the epithelial cells lining
the salivary glands, as well as granulocytes found in saliva [49]. Saliva-derived EVs
from healthy subjects have been shown to contain TF and CD26. The former can initiate
blood coagulation (plasma-free clotting) [50], whereas the latter can cleave substance P
and chemokines [51]. The bulk of the human microbiota inhabits the gastrointestinal
tract (GIT), where it modulates diverse aspects such as insulin signaling, behavior, and
allergy [52–55]. Similar to EVs that play a role in the host’s intercellular communication,
microbiota release outer membrane vesicles (OMVs) that play a role in host–microbiota
communication [56]. Diet and lifestyle influence microbiota and thus GIT homeostasis,
which is highly dependent on inter-microbial communication, as well as host–microbial
communication. Compared to eukaryotic EVs, OMVs are slightly smaller, ranging between
20 and 400 nm [57], and yet their physicochemical characteristics are similar. Nevertheless,
the exact physiological role of OMVs is currently unclear. GIT homeostasis depends on
healthy competition of microbiota with each other, and host neutralization of pathogenic
lipopolysaccharide (LPS) components. This balance is necessary to maintain proper energy
levels, lipid homeostasis, inflammatory homeostasis, and an effective GIT–blood barrier
(GBB). EVs from Akkermansia muciniphila (Am), a beneficial bacterium that alleviates gut
Int. J. Mol. Sci. 2024, 25, 485 5 of 51

barrier disruption, have been found in fecal samples of healthy subjects, while in vitro and
in vivo studies have shown their beneficial effect in intestinal barrier integrity [58].

2.3.3. Musculoskeletal System

The musculoskeletal system provides structure for the body; thus, intercellular com-
munication is vital for maintaining bone and muscle homeostasis, as well as for the regen-
eration after injury. EVs derived from skeletal muscle carry myokines, proteins, miRNA,
and mRNA that are essential for the preservation of muscular homeostasis, development,
and myogenesis [59–61]. Skeletal muscle is highly enriched in miRNAs (MyomiRs), such
as miR-1, miR-133, and miR-206 [62]; hence, EVs that carry these miRNAs play a role
in controlling myogenic homeostasis, proliferation, and differentiation, especially after
injury and during exercise [63,64]. Muscle-derived EVs containing miR-16 that is taken
up by the pancreas can modulate beta-cell proliferation and insulin secretion to regulate
metabolism [65]. Furthermore, muscle-derived EVs carrying miR-206 regulate extracellular
matrix collagen expression that facilitates fiber growth during repair [66]. Although the
communication between bone-forming osteoblasts and bone-recycling osteoclasts is well-
documented [67], the role of EVs participating in these processes, as well as in the synovial
fluid production locally, has recently become an area of interest. Namely, primary bone
marrow-derived mast cell EVs carrying mRNA and miRNA were able to drive protein
production in recipient mast cells [68]. Furthermore, osteoblast-derived EVs transported
the receptor activator of nuclear factor kappa-B ligand (RANKL) to osteoclast precursors,
facilitating their formation in vitro [69]. It has been reported that mouse osteoclast-derived
EVs carrying RANKL inhibited secretion of 1,25-dihydroxyvitamin D3, which regulated the
formation of new osteoclasts [70]. The natural aging process is accompanied by bone dete-
rioration, with EVs having a role in this process. Namely, EVs derived from the plasma of
older adults have been shown to inhibit osteogenesis, mostly due to a decrease in Galectin-3
expression, which contributed to age-related loss of capacity for osteogenic differentia-
tion [71]. Conversion of cartilage into a mineralized bone is a process mediated by EVs
derived from calcified tissues carrying ossifying enzymes [72,73]. Thus, these enzymes
mediate the local increment in orthophosphate that drives hydroxyapatite formation at
matrix EV accumulation sites [73]. Therefore, a change in EVs during the aging process
leads to the change in cargo and surface proteins, which may lead to functional changes.

2.3.4. Reproductive Tract

EVs have important roles during all stages of reproduction, starting from sperm and
egg development, through fertilization and implantation, to maternal–fetal communication
later in pregnancy [74]. It has been reported that the fusion of the prostasome (small
vesicles secreted by the prostate) with sperm leads to an increased sperm motility, which
is regulated by intracellular Ca2+ [75]. The process begins with prostasomes transferring
CD38 into sperm and stimulating cyclic ADP-ribose (cADPR) production. In addition,
prostasomes facilitate sperm–oocyte fusion, protect sperm from lysis [76], and have antibac-
terial activity [77]. Of note, luminal fluid EVs from mouse oviduct transported Ca2+ -ATPase
4a (PMCA4) into sperm [78], triggering sperm motility and facilitating sperm–oocyte fusion
required for fertilization [79]. Indeed, during incubation of CD9−/− sperm with CD9−/−
eggs, the fusion was enabled only by the addition of EVs derived from CD9+/+ eggs.
Furthermore, anti-CD9 mAb blocked the binding of sperm with CD9-containing vesicles,
preventing sperm–egg fusion [80].
In addition to preconception, fertilization, and implantation, EVs have also been
shown to play key roles during pregnancy, specifically in early trophoblast development
and maternal–fetal and amniotic fluid signaling. Namely, extravillous trophoblast (EVT)-
EVs express human leukocyte antigen G “HLA-G” and have been shown to be present
in the maternal circulation from the first trimester of pregnancy [81]. Although EVT-
EVs confer maternal tolerance and adaptation to pregnancy, their immunomodulatory
properties ensure uterine tolerance to foreign antigens presented by the developing fetus.
Int. J. Mol. Sci. 2024, 25, 485 6 of 51

In addition, placenta-derived EVs have been shown to reduce cytotoxicity of CD4+, CD8+,
and NK cells through Ig-like receptors and the NKG2D NK cell receptors, respectively [82].
During the late stages of pregnancy, syncytiotrophoblast (STB)-EVs are released directly
into the maternal circulation and can be uniquely recognized by a placental alkaline
phosphatase [83]. STB-EVs have been shown to carry several proteins including endoglin,
plasminogen activator inhibitor, soluble fms-like kinase (sFlt), and endothelial nitric oxide
synthase [84,85], as well as miRNAs [86,87], tRNA [88], and DNA [81,89]. The physiological
functions of peripherally circulating STB-EVs in both in vitro and in vivo investigations
are broad and their molecular contents as well as surface markers are powerful and cannot
be underestimated.

2.3.5. Central Nervous System

The brain is characterized by diverse and dynamic cell populations such as neurons,
astrocytes, microglia, oligodendrocytes, and vascular cells. Since all cells of the central
nervous system (CNS) release EVs, the neurovascular unit requires an efficient process of
intercellular communication. It has been shown that increased neural activity is associated
with increased release of EVs [90], a process vital for the efficient maintenance of synapses.
This EV-driven neuronal activity is especially important during development, when activity-
dependent pruning of synapses forms part of normal brain development [91]. In addition,
intercellular communication also plays a key role in the regional development of the CNS,
e.g., sonic hedgehog (shh) signaling, known to regulate cortical development [92]. Thus,
shh signaling is regulated by differential expression accessory molecules on the surface of
different EV populations. In the adult CNS, there are ongoing and essential interactions
between cells that must be maintained for the CNS to function normally, as microglia
need to remain in a quiescent/observable state, the blood–brain barrier (BBB) must remain
intact, and astrocytes must maintain healthy functionality. The microglia’s quiescent
state stems from the interaction between CX3CL1 (fractalkine “find me” signal) on the
neuron with its receptor CX3CL1 on microglia, thereby reducing proinflammatory cytokine
release (IL-6, IL-1β, and TNFα) and increasing anti-inflammatory cytokine release (IL-10),
with an increased ratio of p-AMPK/AMPK and expression of Nrf2 after germinal matrix
hemorrhage (GMH) [93]. Similarly, in the immune system, endothelial cell-derived EVs bear
fractalkine on their surface to attract CX3CL1+ monocytes, acting as homing signals [94].
The BBB consists of a unit of cells including pericytes, astrocytes, and endothelial cells,
the latter of which are connected via tight junctions preventing the normal migration of
cells and macromolecules that appear in the fenestrated vasculature of the peripheral
circulation [95]. The potential of EVs to cross the BBB to deliver drugs [96], passing from
periphery to brain [97], and from brain to periphery [98], makes them an effective means of
communication across the intact BBB. For example, EVs derived from brain pericytes are
pro-angiogenic and have a role in regulation normal growth and function at the BBB [99].
Although the complexity of the brain requires reductionist approaches, the majority of EV
release mechanisms at the BBB have been implemented in cell culture.

2.3.6. Cardiovascular System (Blood Pressure and Coagulation)

The vascular system maintains blood pressure by generating and releasing vasoactive
chemicals. Enrichment of EVs with angiotensin II type I receptors can alter blood pres-
sure [100], whereas EV-mediated inhibition of endothelial nitric oxide synthase (eNOS)
could play a role in regulating nitric oxide (NO) production [101]. Coagulation is an
important and dynamic process that maintains the integrity of the circulatory system,
where platelet clumping plays the main role. It has been demonstrated that the presence of
activated platelet-derived phospholipid-rich vesicles (EVs) in plasma mediates blood clot-
ting [102]. Some conditions such as exercise, hypoxia, inflammation, and consumption of a
high-fat diet increase the level of circulating platelet EVs [103]. Similar to EVs in general,
platelet EVs can be classified based on their sizes and molecular contents into different sub-
populations [104]. Platelet EVs range in size from large EVs (microparticles) [105], tubular
Int. J. Mol. Sci. 2024, 25, 485 7 of 51

elongated EVs [106], to smaller EVs (70–150 nm) resembling exosomes. Their load includes
proteins from the plasma membrane, cytosol, organelles, adhesion receptors, coagulation
and transcription factors, growth factors, active enzymes, cytokines, and chemokines [105].
Also, the main mediators of interaction with circulatory cells and matrices (fibrin) in-
clude GP IIa/IIIa (CD41/CD61), GP Ib (CD42b), P-selectin (CD62P), and CD40L (CD154),
as well as unique exosomal markers (CD9, CD63, CD81, HSP70, TSG101 [103]). The
content of platelet EVs can be confusing, as factors with opposing functions, e.g., pro-
and anti-coagulant substances, can be detected [107]. In addition, they contain small
metabolites [108], and RNAome comprises microRNAs (miRNAs), YRNAs, and circular-
RNAs (ciRNAs) [31] originating from parent megakaryocytes. Although the lifespan of
platelets is about 10 days, exogenously injected EVs are cleared from the circulation within
10–60 min [103]. The most unique physiological role of platelet EVs is their ability to
promote coagulation and thus participate in hemostasis. Human Scott syndrome abol-
ishes platelet phosphatidylserine (PS) exposure, microvesiculation (i.e., EV formation),
and thrombosis, and results in a mild bleeding disorder [22]. Upon platelet activation,
disruption of the membrane phospholipid bilayer resulted in impaired PS externalization
and decreased procoagulant activity, abolishing fibrin formation at sites of vascular dam-
age [109]. The relationship between membrane phospholipid bilayer scrambling and EV
formation in human Scott syndrome was found to be due to a gene defect encoding the
transmembrane protein 16F (TMEM16F), an occult Ca2+ -activated phospholipid scramblase
(CaPLSase) that passively transports phospholipids down their chemical gradients and
mediates blood coagulation [110]. Physiological hypoxic exercise training increased the
level of pro-coagulant EVs and thus increased thrombin production [111]. Larger EVs
(microparticles) from different cellular sources, rather than exosomes [112], show pro-
coagulant activity in relation to tissue factor (TF+ ) EVs [113], which also act in other body
fluids such as saliva and urine [114].

2.3.7. Immune System

Cell-to-cell communication is an essential aspect of an efficient immune system ca-
pable of protecting the host from injury, infection, and disease. While soluble factors
such as chemokines and cytokines are known to modulate the immune system, EVs have
been identified as pivotal players in the initiation and resolution of inflammation [1]. EV
signaling plays a key role in the innate immune response to injury or infection [115,116].
EVs derived from neutrophils, monocytes, and macrophages are released upon stimu-
lation by inflammatory and damage mediators and/or pathogen-associated molecular
patterns [117,118]. It has been demonstrated that patients with inflammatory and infectious
diseases have an increased number of circulating EVs derived from immune cells [119,120]
that contribute to the restoration of homeostasis. In addition to antimicrobial effects of
neutrophil-derived EVs [121], they enhance the immunological role of their parent cells by
increasing the expression of IL-6 and ICAM-1 on endothelial cells [122], thereby facilitating
their migration across the endothelial barrier. These EVs are enriched with numerous
chemokines that direct leukocytes to the site of inflammation [115,123]. In contrast, cancer
cell-derived EVs taken up by leukocytes can also trigger a response [124], confirming a
bidirectional communication pathway. Despite the lack of in vivo evidence, investigating
the role of EVs in immune cell activation is currently considered one of the most impor-
tant areas of research in the field of EV biology. Raposo et al., 1996 demonstrated that
Epstein–Barr Virus (EBV)-infected B-cell line released EVs that were able to stimulate
T-cell proliferation and an antigen-specific response [125]. This immune stimulation was
attributed to EVs that served as antigen-presenting vessels, as they were enriched for Major
Histocompatibility Complex (MHC) II and EBV-specific proteins. A similar earlier study
showed that upon activation, dendritic cells (DCs) secrete antigen-presenting EVs, enriched
with MHC complexes and T-cell co-stimulatory molecules that prime a T-cell-specific
cytotoxic response with higher immunogenicity [126]. Subsequent studies showed that
EVs expressed MHC class I and II molecules, and adhesion and co-stimulatory molecules
Int. J. Mol. Sci. 2024, 25, 485 8 of 51

were able directly to stimulate CD8+ and CD4+ T-cells via binding their respective plasma
membrane receptors [127]. Moreover, a comparative study showed that DCs pulsed with
tumor peptides released EVs that could induce a stronger anti-tumor CD4+ T-cell response
than T-cells incubated with the peptides alone, which is evidence for the theory of enhanced
immunity [128].
In terms of homeostatic ability, while some EVs are able to activate the immune system,
other EVs are able to suppress the immune system. For example, natural killer-derived
EVs (NK-EVs) show their cytotoxic activity only on activated, but not resting cells [129].
The suppressive effect of EVs is important during pregnancy, as the pro-inflammatory state
can be harmful. Placental EVs are shed in large quantities during pregnancy [130,131]
and have been associated with TNF-family ligands FasL and TRAIL, leading to apoptosis
in activated lymphocytes [132,133]. In the same context, FasL plus plasma EVs have
been shown to induce apoptosis of CD4+ T-cells [134]. Immunomodulatory EVs play an
important role in the prevention of autoimmunity and chronic inflammation. The term
tolerosomes was coined to demonstrate EV-mediated immune modulation by epithelial
cells [135], which represents the key to the effective development of an allergic response.
The same was demonstrated for EVs isolated from bronchoalveolar lavage fluid (BALF) of
mice immunized against olive pollen allergen [136], and adoptively transferred into naïve
mice. Immunized mice upon exposure to the allergen showed suppression of the immune
response and production of Th2 cytokines. Similarly, responding to ovalbumin-loaded
dendritic cell (DC)-derived EVs from MHC−/− mice stimulated antigen-specific T cells
at the same magnitude as wild type EVs, i.e., MHC-independent immune response [137].
This stimulation of immune tolerance has been exploited therapeutically in diseases such
as post-transplant graft vs. host [138].

2.4. Pathological Roles of Extracellular Vesicles

EVs can be enriched for pathogenic proteins and nucleic acids [139,140]; thus, biofluid
analysis improves our understanding of their pathophysiological roles in different organs.
In this section, we will focus on pathological roles of EVs in the development of several
diseases, and we will explore their potential as biomarkers, as well as tools for potential
therapeutic intervention.

2.4.1. Urinary Tract

Compared with other biological fluids, urine is widely available and relatively easy to
obtain in large quantities, and thus the study of the functional role of EVs in kidney disease
processes is a more accessible area than other areas of EV biology. This accessibility has
made urinary EVs the most studied biomarkers of kidney and urinary tract diseases [141].
The functional unit of the kidney is the nephron, which begins with the glomerulus that
filters the blood, towards the tubules and the collecting duct that regulates the composition
of the urine into the tubular interstitial system. The involvement of EVs in a range of intrin-
sic kidney diseases has been well-established and affects all parts of the nephron, including
glomerular and tubular injury, nephritis, fibrosis, and ion-channels and water transport
disorders [142–145]. Although intra-glomerular communication within the glomerular
vasculature has already been investigated in vasculitis, intra-nephron communication is
challenging to study. In the plasma of patients with vasculitis, leukocyte EVs transferred
B1-kinin receptors to glomerular endothelial cells, and promoted kinin-associated inflam-
mation [143]. This demonstrates the bidirectional nature of EV communication between
the circulatory system and urinary system. In addition, EVs have been shown to contribute
to the outcome of glomerular–tubular inter-communication in disease progression [146].
Indeed, tubular epithelial cell EVs loaded with functional cargoes such as chemokines, TGF-
β1, CCL2 mRNA, and osteopontin transferred these cargoes to interstitial macrophages,
aggravating kidney damage [145]. In kidney diseases such as IgA nephropathy [147] and
diabetic kidney disease [148], increased inflammation leads to cellular oxidative damage.
The spread of inflammation between tubules and renal interstitium may lead to significant
Int. J. Mol. Sci. 2024, 25, 485 9 of 51

kidney damage. Indeed, EVs derived from TGF-β1-treated tubular cells exacerbated kidney
injury and fibrosis [144]. This result was supported by the demonstration that EVs gener-
ated in vivo from animal kidney tissue with ischemia reperfusion injury transferred TGF-β1
into fibroblasts in vitro [149]. Besides communication between vasculature and glomerulus,
as well as tubules and interstitial renal compartments, communication between proximal
and distal tubular cells has also been demonstrated in vitro. For example, proximal tubule
EVs have been shown to reduce sodium channel function in distal cells by transferring
nucleic acid [150,151].

2.4.2. Gastrointestinal Tract

As previously mentioned, microbiota release outer membrane vesicles (OMVs) that
play local and systemic roles in maintaining homeostasis. Similarly, OMVs can also be
released by harmful bacteria, such as the Gram-negative proteobacterium Helicobacter py-
lorus (Hp), whose chronic infection leads to chronic inflammation and the development
of gastric cancer [152,153]. Hp OMVs have been shown to carry virulence genes (CagA
and VacA) of the parent bacteria, which can induce the production of immunomodulatory
cytokines in host macrophages and gastric epithelial cells [154,155]. As seen from in vitro
and in vivo studies, Cy7 labeled Hp OMVs were able to infiltrate the gastric epithelium
and still be detectable even 24 h after injection, causing inflammation and contributing to
the development of gastric cancer [156]. It was later found that the inter-communication be-
tween Hp-infected gastric cancer cells and macrophages involved the internalization of EVs
enriched with phosphorylated active growth factor isoform by macrophages in vitro and
in vivo [157]. The apparent role of growth factor internalization is to educate macrophages
toward a pro-tumorigenic phenotype, including an increase in IL-1β secretion that pro-
motes tumor growth and progression in vivo [158]. These studies reveal the importance of
the role of Hp OMVs in the development of gastric cancer and shed light on the diversity of
OMVs derived from microorganisms. In addition to local effects, the gut also communicates
with other parts of the body, such as the gut–brain–microbiota axis (GBMAx), which is
an important player in psychiatric diseases [159]. During childhood, increased GBMAx
permeability may lead to autism spectrum disorders (ASDs) [160,161], with microbiota
by-products including EVs entering the circulation and interfering with normal devel-
opment [162]. Profiling urinary bacterial OMVs in ASDs, with the aim of prophylaxis
for children with pre- and pro-biotics could definitely be used to combat differences in
microbial diversity [163]. Pathophysiologically, EVs derived from the gut microbiome may
contribute directly or indirectly to a number of mental health disorders.

2.4.3. Musculoskeletal System

Pathologies of the musculoskeletal system are particularly harmful for the body, while
the accompanied pain and lack of movement are significant burdens for the healthcare
system. It requires new intervention strategies in order to understand how EVs partici-
pate in the formation and strengthening of the musculoskeletal system. Various diseases,
especially cancer, chronic infections, and heart failure, often cause Cachexia syndrome, a
complex disease characterized by the loss of skeletal muscle and adipose tissue. About
50–80% of cancer patients suffer from Cachexia syndrome and weight loss with increased
levels of circulating EVs [164]. Tumor EVs contain two markers, HSP70 and HSP90, which
have been shown to effectively induce muscle wasting [165]. Although circulating EV
contents and function in Duchenne muscular dystrophy (DMD) are still under investi-
gation [19], in terms of biomarkers and therapeutics, they may assist in the early diag-
nosis [166]. Recently, it was shown that GW4869 (neutral sphingomyelinase (nSMase)
inhibitor), which reduces EV release in mdx mice (DMD model), was protective against
cardiac stress, which has been attributed to miRNA load [19]. In this sense, KO of the nS-
Mase2/Smpd3 gene in these mice reduced muscle inflammation and improved functional
performance [167]. Diseases of the skeletal system such as osteological tumors, chondro-
cytic disease, and inflammation influence EV populations within the circulation and their
Int. J. Mol. Sci. 2024, 25, 485 10 of 51

downstream effects on cells and organ systems. For example, synovial fibroblasts from
patients with rheumatoid arthritis (RA) produce EVs containing the inflammatory protein
TNF-α and stimulate NFkB production [168]. In contrast, synovial fluid EVs from men and
women with OA showed an ability to reduce cellular metabolic activity [169]. These studies
include the identification of the miRNA content of EVs that have shown enrichment of
targeting of sex-specific signaling pathways and shed light on the importance of combining
clinical data with pre-clinical research.

2.4.4. Reproductive Tract

In contrast to the role of EVs in normal gynecological processes, dysfunctional EV
signaling has been associated with gynecological pathology and diseases occurring during
pregnancy. These include endometriosis [170], polycystic ovary syndrome (PCOS) [171],
pre-eclampsia [172], and gestational diabetes [173]. For example, elevated EV-associated
RNA (DENNDIA gene) in a woman’s urine indicates polycystic ovary syndrome (PCOS) [174].
EV miRNAs involved in estradiol regulation were found to be downregulated; thus,
EVs play a role in the initiation of PCOS [175]. Furthermore, PCOS is associated with a
prothrombotic state, as affected women have been shown to have abundant amounts of
platelet-derived EVs in their plasma, and this contributed to the higher cardiovascular risk
in these patients [176,177]. Worldwide, pre-eclampsia is a leading cause of maternal and
fetal morbidity and mortality [178], showing higher levels of circulating STB-EVs [179].
It has been found that placental EVs of pregnant women with pre-eclampsia can cause
hypertension when injected into non-pregnant mice [180]. Moreover, 12 STB-EV-isolated
miRNAs in peripheral plasma can be used to differentiate normal pregnancies from pre-
eclampsia [181]. Physiologically, normal pregnancy represents a state of relative insulin
resistance, while in some women, insulin resistance becomes pathological, leading to a
disease called gestational diabetes mellitus (GDM). The level of EVs in maternal plasma
is increased in GDM pregnancies compared to normal pregnancies [182,183]. Similarly,
EVs derived from plasma from women with GDM induced glucose tolerance in non-
pregnant mice [182]. These data demonstrate the powerful role that EVs play in maintaining
reproductive health and the development of a successful and healthy pregnancy.

2.4.5. Central Nervous System

Diseases of the CNS can be divided into acute injuries such as stroke and trauma,
and chronic neurodegenerative diseases such as Parkinson’s disease. Acute trauma injury
causes rapid mechanical damage to blood vessels, neurons, and glia, leading to the death
of multiple cell types, as well as the release of damage-associated molecular patterns. In
contrast, neurodegenerative diseases are more limited to single cell types, before becom-
ing more widespread. Therefore, the role that EV signaling plays in these pathologies
depends a great deal on the characteristics of the disease. It has been demonstrated that
traumatic brain injury in mice caused a release of EVs from microglia, while injection of
these EVs into a healthy mouse brain led to neuroinflammation [184]. Intrastriatal injection
of brain-derived EVs has been reported to activate microglia and stimulate the release of
pro-inflammatory mediators; therefore, EVs could act locally to exacerbate central inflam-
mation after initial injury [185]. Of note, it has been demonstrated that after stroke [186] and
TBI [187,188], an increased number of circulating EVs had the potential to induce systemic
immune reaction [186,188], indicating EV-mediated brain–immune system communication.
For example, the multiple sclerosis (MS) relapse group demonstrated an increase in circu-
lating myeloid+ EVs as potential markers of neuroinflammatory attacks [189]. Thus, the
increment in myeloid-derived EVs in an MS patient’s cerebrospinal fluid (CSF) depends on
the patient’s condition (stable vs. acute MS) and is related to the number of neuroinflam-
matory lesions [189]. On the other hand, in chronic CNS diseases such as Parkinson’s and
Alzheimer’s, characterized by a slow degeneration of neurons over a number of years [190],
EVs could propagate the disease. In a mouse model of Alzheimer’s disease [191], as
well as aging [192], the propagation of tau fibrils has been shown to be mediated by EVs.
Int. J. Mol. Sci. 2024, 25, 485 11 of 51

Recent work has demonstrated that familial Alzheimer’s-induced pluripotent stem cell
(IPSC)-derived EVs are able to induce tau pathology in naïve mice [193]. In Parkinson’s
patients, EVs have been proposed as a link for α-synuclein aggregation, as they also have
the potential to be used as diagnostic tools for stratifying patients [194,195].

2.4.6. Cardiovascular System (Blood Pressure and Coagulation)

EVs have been shown to participate in the pathogenesis of different cardiovascular
diseases (CVDs) [196–198]. Direct contact between blood vessel endothelial cells and
peripheral blood allows endothelial cell (EC)-EVs to be rapidly released and delivered to
distant organs, thus influencing the onset of different CVDs. Although EVs may also have
downstream pathogenic consequences, they have been shown to have a hemostatic blood
pressure modulating effect. In rats, circulating EVs have been shown to be able to suppress
vasodilation [199,200] and thus play an important role in hypertension. Namely, it has
been shown that hypertensive patients show an increase in EC-EVs in the plasma [201]
and urine [202]. Thus, blocking the generation and release of EVs was found to reduce
blood pressure in hypertensive rats [203]. Furthermore, an increase in circulatory EC-EV
levels has been found in patients with endothelial dysfunction [204], obstructive sleep
apnea [205], obesity [206,207], renal failure [208], coronary artery disease [209], myocardial
infarction [210–212], β-thalassemia [213], and stroke [214]. Nevertheless, it is not clear if
the increase in their number contributes to these conditions.
Although the coagulation process is a homeostatic response to injury, inappropriate
platelet activation can lead to pathological thrombosis and atherosclerosis [215]. It has been
estimated that platelet EVs account for 25% of the procoagulant/anticoagulant activity
in blood [216] and exhibit 50–100-fold higher procoagulant activity than the surface of
activated platelets [217]. Thus, blocking the generation and release of platelet EVs has been
found to lead to an increase in abnormal bleeding [218]. Fibrin fibers have been shown to
contain EV-positive CD61, which plays a role in platelet aggregation [219]. Platelet EVs
provided pro-hemostasis support during uncontrolled bleeding by modulating the kinetics
of fibrin formation, clot structure, as well as fibrinolysis, thus preventing hemorrhagic
shock [220].
Studies on the role of platelet EVs related to inflammation have been reported to
increase their numbers in response to infection with viruses and parasites [221]. Platelet
EVs orchestrate the immune response by modulating the performance of other immune
cells [222]. Certainly, platelet EVs generated during inflammation [223] and acute liver
injury and infiltrating into the bone marrow were essential for the regulation of megakary-
ocyte formation [224]. The results were corroborated in human bone marrow biopsies [223].
This demonstrates that platelet EV proxies mediate their pathological functions, thus
hindering the identification of their roles in vivo. Implications for platelet EVs in autoim-
munity [225], e.g., in RA, have shown that platelet EVs contain IL-1α and IL-1β in synovial
fluid. These EVs are triggered by collagen receptor GPVI and promoted synovial cell
activation and production of IL-6 and IL-8 (CINC-1) [226]. It has also been reported in RA
that peripheral blood-derived regulatory T-cells transduced with platelet EVs ceased to
differentiate into IL-17 and IFN-γ producing proinflammatory cells [227,228]. The role of
platelet EVs in inflammation highlights the importance of studying them in a system of
multiple cell types.

2.4.7. Immune System

Autoimmune or inflammatory diseases are involved in creating a pro-inflammatory
environment that is associated with an increment in circulating EVs. Furthermore, based on
the knowledge on the involvement of EVs in intercellular communication between cells of
the immune system, inappropriate EV-mediated activation can contribute to pathological
conditions. Thus, platelet EVs in the synovial fluid of RA patients is likely responsible for
triggering an inflammatory reaction within the affected joints [229]. Similarly, in patients with
inflammatory bowel disease, granulocyte-derived EVs enriched for metalloproteinases and
Int. J. Mol. Sci. 2024, 25, 485 12 of 51

pro-inflammatory cytokines have been shown to increase intestinal permeability [230]. Other
studies have linked EV-mediated inflammation to CVDs [206,231–234], MS [189,235–237], and
more. Autoimmune diseases can stem from EV-presenting self-antigens that auto-activate
lymphocytes and trigger immune cells with antigen specificity for EVs’ own parent cells.
Circulating EVs in systemic lupus erythematosus (SLE) that are enriched for antigenic
DNA may act as an autoantigen to accelerate disease progression [238]. Similarly, the
interaction between EVs and autoreactive T- and B-cells has been shown to trigger pancre-
atic inflammation and lead to the development of diabetes in non-obese diabetic (NOD)
mice [239–241]. In contrast to activation of the immune system, EV-mediated suppression
can be devastating. Currently, there is a large body of evidence showing the participation
of EVs in the development of the metastatic niche by suppressing the circulating immune
response to migrate tumor cells [242,243]. An active inflammatory response to cellular
growth is a key mechanism to reduce tumor burden, which is currently being exploited, as
EVs play an important role in this process. In vitro and in vivo studies showed that tumor-
derived EVs expressing FasL and TRAIL activated regulatory T-cells and myeloid-derived
suppressor cells (MDSCs), which prevented CD8+ T-cell from targeting the tumor [244].
FasL+ EVs were found in the sera of oral squamous cell carcinoma patients, and their level
correlated with tumor burden and nodal involvement [245]. In addition, EVs suppress
the immune system by reducing cytotoxic NK cells circulating in the lung and spleen,
allowing metastatic niches to form in those organs. It has been demonstrated that neu-
trophil mobilization is required for tumor growth; thus, targeting EV release by GTPase
RAB27A/B blockade in mice decreased primary mammary carcinoma tumor growth and
its dissemination into the lung [246].

3. Pharmacology of Extracellular Vesicles

Pharmacology branches include pharmacodynamics (PD), which studies the action of
the drug on the organism (primary intended effects and secondary unintended effects) [247],
and pharmacokinetics (PK), which studies the effect of the organism on the drug related
to absorption, distribution, metabolism, and elimination (ADME) [248]. The objective of
non-clinical trials is to provide in vitro, ex vivo, and in vivo data on the PD, PK, and toxicity
profile of the given EV product for the chosen route of administration in order to support
its investigation in a clinical trial in terms of efficacy and safety [249]. Principally, the PD
and PK studies are performed earlier in the development phase, while toxicity tests are
performed later. EVs generated from non-pretreatment and genetically modified cells that
do not contain any transgenic product belong to the category of biomedical products. In
contrast, EVs generated from genetically modified cells containing a transgenic product
are considered gene therapy products (GTPs), a subclass of advanced therapy medicinal
products (ATMPs) [250]. The European Medicine Agency (EMA) supports the classification
of ATMPs through the committee for advanced therapy (CAT) with the recommendation
that recombinant RNA-containing EVs be considered as GTPs [251,252], as the effects are
directly related to these molecules. In contrast, recombinant peptides or protein-containing
EVs are products of biotechnology. The following sections will address pharmacological
and pharmaceutical aspects of EVs that stem from their composition and PK-PD model
similarities to liposomes (LPs) [253] (Figure 1), where both are lipid bilayer vesicles (LBVs).
 ports the classification of ATMPs through the committee for advanced therapy (CAT) with
the recommendation that recombinant RNA-containing EVs be considered as GTPs
[251,252], as the effects are directly related to these molecules. In contrast, recombinant
peptides or protein-containing EVs are products of biotechnology. The following sections
Int. J. Mol. Sci. 2024, 25, 485
will address pharmacological and pharmaceutical aspects of EVs that stem from their
13 of 51
composition and PK-PD model similarities to liposomes (LPs) [253] (Figure 1), where both
are lipid bilayer vesicles (LBVs).

Figure 1. Schematic of pharmacokinetics–pharmacodynamics (PK-PD) modeling of the lipid bilayer

Figure 1. Schematic of pharmacokinetics–pharmacodynamics (PK-PD) modeling of the lipid bilayer
vesicles (LBVs): liposome (LP), extracellular vesicle (EV), and hybrid liposoxome (LO) drug delivery
vesicles
system (LBVs): liposome
development. (A)(LP),
PK-PDextracellular vesicle (EV),
modeling connects anddose
the drug hybrid liposoxome (LO) drug
(encapsulated/engineered delivery
vesi-
system development. (A) PK-PD modeling connects the drug dose (encapsulated/engineered
cles) to the physiological response, related to the drug delivery system properties and the physio- vesicles)
logical factors. A series of events describes the flow from administration, drug exposure
to the physiological response, related to the drug delivery system properties and the physiological (plasma
and target
factors. site),ofreceptor
A series binding and
events describes theactivation,
flow from transduction to effect,
administration, drugand effect on(plasma
exposure physiological
and target
site), receptor binding and activation, transduction to effect, and effect on physiological response.
(B) Physiologically based PK (PBPK) modeling for nanodrugs (encapsulated/engineered vesicles)
consists of LBVs and released small molecules. LBVs are linked via small orange arrows to drug
release. LBV tissue distribution is convection-driven (unidirectional blue arrows) except in the tumor,
where passive diffusion is the main distribution mechanism due of the high interstitial pressure.
In contrast, the release of small molecules is tissue-specific (i.e., phagocytosis, low tumor pH, etc.),
whereas drug distribution is bidirectional (blue arrows). Enhanced accumulation of LBVs in the
lung, spleen, and liver is associated with the leaky vascular structures and sequestration of the
mononuclear phagocytic system (MPS) (small thicker orange arrows) with the exception of the higher
tumor accumulation of LBVs, attributable to the enhanced permeation and retention (EPR) effect. The
model also includes the lymphatic system to recycle LBVs (blue dashed arrows) from the interstitial
space (created with https://app.biorender.com/ (accessed on 25 December 2023)).
Int. J. Mol. Sci. 2024, 25, 485 14 of 51

3.1. Pharmacodynamics of Extracellular Vesicles

EVs are involved in diverse biological processes, such as cell motility [254–256], differ-
entiation [257–259], proliferation [260,261], apoptosis [262,263], reprogramming [264–266],
and immunity [267,268], through regulation of intercellular communication. Understand-
ing the regulatory mechanisms of EVs in these biological processes and in intercellular
communication is crucial for their clinical potential [269,270] and applications [271–279].
This section will focus on therapeutic applications of EVs as therapeutic targets by blocking
their pathological roles, as well as their use as therapeutics by employing their physiological
roles. The use of EVs as drug delivery platforms by utilizing their in vivo kinetics will be
discussed under Section 4. Pharmaceutical Applications of Extracellular Vesicles.

3.1.1. Therapeutic Targeting of Extracellular Vesicles

Physiologically, EVs are mediators of intercellular communication, while patholog-
ically, they are involved in the initiation, exacerbation, and resistance in various dis-
eases [280]. This leads to a large proportion of unsuccessful treatment regimens, specifically
in cancer, due to the role of EVs in conferring resistance to cancer cells, via immune evasion
and metastasis, as well as to chemotherapy [281,282]. These unfavorable pathological roles
of EVs can be intercepted by targeting the three main parts of EVs machinery: biogenesis
cargo sorting, release, and uptake [283]. For instance, to abrogate the suppressive effect
of multiple myeloma (MM) cell-derived EVs on the cytotoxic activity of natural killer
(NC) cells, two long-chain omega-3 polyunsaturated fatty acids, eicosapentaenoic acid
(EPA) and docosahexaenoic acid (DHA), should be used at the biogenesis level [284]. Thus,
pre-treatment of MM cell lines with either EPA or DHA could largely reverse EVs’ natural
killer-suppressing effects, hindering their biogenesis [284]. Similarly, the pro-angiogenic
effects of breast cancer cell-derived EVs can be nullified using DHA, which alters the cargo
sorting of EVs in favor of anti-tumor effects [285,286]. To disrupt the release of EVs from
parent cells, efforts have been made using bioinformatics methods to hypothetically screen
a large number of approved drugs to identify those with potential EV release-inhibiting
effects [287–289]. For example, the natural antibiotic Manumycin A has been shown to
inhibit prostate cancer cell EV secretion by blocking the Ras/Raf/ERK1/2 and hnRNP H1
pathways in vitro [278,290]. Targeting cancer cell-derived EV-mediated shedding of NK
cell inhibitory ligands, MICA and MICB, prevented these cells from evading the immune
system in mice [291,292]. Likewise, inhibition of lung tumor cells’ kras-derived EVs could
reverse the induced immunosuppression and chemoresistance [293]. In the same sense,
targeting the biogenesis of OMVs from prokaryotic cells (e.g., extrinsic pathogens) has the
same physiological and pathological significance as targeting host cell biogenesis of EVs.
These results suggest that the above-described targeting methods may be of interest in
developing new therapies for some bacterial diseases.

3.1.2. Extracellular Vesicles as Therapeutics

Different cell types, such as mesenchymal stromal cells (MSCs), specific tumor cells
(TCs), and immune professional antigen presenting cells (APCs), such as dendritic cells
(DCs), B lymphocytes (BLs), and macrophages (MPs), produce EVs that can be used as drugs
without any specific manipulation of their contents and/or associated molecules [294,295].
Also, further education of EV-producing parent cells via genetic engineering or pharmaco-
logical treatments could add more potential over their untreated/engineered counterparts,
because produced EVs can carry a cargo of interest that aligns with therapeutic goals.

Mesenchymal Stromal Cell-Derived Extracellular Vesicles (MSC-EVs)

Five decades ago, MSCs were first recognized for their pluripotent potential, and recent
findings suggest their regenerative and modulatory properties [296]. MSCs are the source
of a myriad of active molecules [297]. However, concerns about the safety of cell-based
therapies still challenge the applicability of MSCs for use in humans [298]. Two mechanisms
by which MSCs can exert their beneficial effects are direct cell-to-cell contact with their
Int. J. Mol. Sci. 2024, 25, 485 15 of 51

target cells, and by release of soluble factors (including EVs) as a means of modulating their
targets remotely [299–301]. The anti-apoptotic, pro-angiogenic, anti-inflammatory, prolif-
erative, and trophic nature of MSC-EVs provide potent intrinsic regenerative properties
that have been demonstrated in numerous organs [302–306]. Thus, mouse bone marrow
endothelial progenitor cell-derived EVs improved the hemodynamic status of murine mod-
els of myocardial infarction (MI), showing significant pro-angiogenic effects [307]. Bone
marrow, adipose tissue, and umbilical cord MSC-EVs inhibit cardiomyocyte apoptosis and
promote angiogenesis, thus improving cardiac function and protecting myocardium [308].
The mechanism by which natural umbilical cord MSC-EVs alleviate liver injury after is-
chemia/reperfusion is due to the binding of miR-20a and two upregulated genes, Beclin-1
and FAS 3′ UTRs, thereby inhibiting apoptosis [309]. Injecting MSC-EVs locally into a
murine model of retinal detachment (RD) significantly reduces levels of inflammatory
cytokines TNF-α and IL-1β, Atg5 cleavage, and apoptosis of photoreceptor cells, thereby
preserving the normal structure of the retina [310]. The existence of miRNAs targeting
TLR4/NF-kB within MSC-EVs suppresses inflammation associated with peripheral neu-
ropathy in a murine diabetic model by decreasing the expression of inflammatory cytokines
and regulating the ratio of M1 and M2 macrophages, thereby improving neurovascular
architecture [311].
In contrast to the aforementioned natural pharmacodynamics of MSC-EVs, engineer-
ing EV-producing MSCs leverages them towards producing stronger specialized EVs. The
methods that can be employed range from stimulating EV-producing cells with, e.g., drugs,
cytokines, growth factors, altering cell culture conditions (e.g., hypoxic vs. normoxic, 3D
vs. 2D culture), and genetic engineering with genetic constructs (e.g., plasmids) [312]. The
goals of these methods may be to increase the amount(s) of a specific molecule(s) in the EVs,
alter the sorting of small RNAs, or even add/delete a specific gene in the final structure
of the released EVs. Natural regenerative effects of human adipose tissue derived-MSC
secretome in the lungs have been revealed to include proteins and lipids essential for main-
taining protease/anti-protease homeostasis and anti-microbial activity. In vitro stimulation
of MSCs with dexamethasone and IL-1β along with starvation leads to an increment in
Alpha-1 antitrypsin (AAT), the major elastase-inhibitory enzyme in the lung [313]. Al-
though MSC-EVs carrying miR-20a partially alleviated liver IR-induced injuries in rats,
boosting these EVs with mimics of this miRNA resulted in the complete alleviation of the
injury [314]. Similarly, MSC-EVs transfected with a miR-20b-3p mimic reduced calcium
oxalate accumulation in rat kidneys, with downregulation of oxalate-induced autophagy
and inflammation as responsible therapeutic effects [315].

Tumor Cell-Derived Extracellular Vesicles (TC-EVs)

TC-EVs are rich in immunogenic tumor antigens [316], which, when taken up by DCs,
address antigen cross-presentation by complexing with major histocompatibility complex
type 1 (MHC I) [317] to both T-helper (Th) and cytotoxic T lymphocytes (CTL), thereby cre-
ating a potent anti-tumor response [318–320]. There are two main groups of tumor antigens,
namely tumor-specific antigens (TSAs) and tumor-associated antigens (TAAs). The former
represents the unique antigens (neoantigens) [321] resulting from mutagenic events, while
the latter is present on nonmalignant cells with changes in their expression [322,323]. The
difficulties of TSAs to be identified make TAAs the most promising antitumor therapeutics
and vaccines. Examples of TAAs are carcinoembryonic antigen (CEA), the transmembrane
glycoprotein Mucin 1 (MUC1), and melanoma-associated antigen (MAGE), each of which
ranges from overexpression to aberrant expression in specific tumor types. However, their
use is limited due to their limited number and poor immunogenicity [324,325]. Further-
more, the reason for designing a single vaccine against several types of tumors is a myth
due to the inability of shared/overlapping antigens to be cross-presented efficiently by
APCs. Interestingly, TC-EVs were shown to contain shared tumor antigens that were effi-
ciently cross-presented by DCs and lead to cancer rejection in mice [326]. The reason for this
finding lies in the presence of a separate group of proteins critical for a robust anti-tumor
Int. J. Mol. Sci. 2024, 25, 485 16 of 51

response, the original tumor antigens, and the overexpression of specific molecules and
receptors that are essential for antigen sampling by APCs [327]. In this sense, the use
of TC-EVs as natural samples of tumor antigens may be a viable option for developing
effective antigen-based immunotherapies. Although the components of TC-EVs are diverse,
one of the many strategies is to employ TC-EVs in ex vivo maturation and induction of
DCs to induce robust CTL responses rather than additional activation stimuli in DCs, as
well as to combine different oncolytic peptides to increase coverage of designed thera-
pies/vaccines [324]. Like MSCs, tumor cells can be primed to produce EVs with enhanced
therapeutic activities that, when presented to the host directly, elicit effects of interest, such
as targeting the delivery of specific molecules. The complex composition of TC-EVs could
be a major drawback of this strategy, which could lead to undesirable effects. This can
be solved by exploiting the surface properties of TC-EVs without undesirable effects to
produce biomimetics in which the drug-carrying core is coated with the outer covering of
the TC-EVs [328,329]. One study showed HepG2 and SKBR3 TC-EVs transfected with a
therapeutic anti-miR-21 to activate synthetic gold iron oxide nanoparticles and harness their
potential in tumor targeting [330]. Thus, the targeting behavior of TC-EVs depends on the
type of cancer cell from which they were isolated. The larger amounts of TC-EVs produced
by tumor cells compared to their non-malignant counterparts [331] and their targeting
behaviors based on their parent cells show that TC-EVs may be unusual candidates for
serving as novel targeting therapies [332].

Immune Cell-Derived Extracellular Vesicles (IC-EVs)

IC-EVs include DC-EVs (dexosomes) [126,333], BL-EVs, and MP-EVs, which carry
MHC I and II, required for antigen presentation, as well as necessary co-stimulatory
molecules [20,125]. It has been shown that adhesion molecules that direct EVs towards
effector cells such as CD11b, CD9, and lactadherin are also released upon selective enrich-
ment by these immune cells [333,334]. MP-EVs are part of the immune system, and they are
not trapped or eliminated by the phagocytic system; therefore, they are superior to other
microparticles used for drug delivery [335]. It has been shown that DC-EVs pulsing with
tumor peptides can be used as an effective non-cellular vaccine to prime CTLs in murine
tumors [126]. The DC-EVs used in this study efficiently presented tumor antigens loaded
to effector cells, which finally eradicated tumors in an animal model of P815-mastocytoma
and TS/A-mammary carcinoma [336]. Although this study and several similar studies
demonstrated the potential of cell-free DC-EV vaccines, drawbacks, such as difficulties in
long-term storage and challenges with targeted delivery, limit their applicability [337]. In
addition to the antigen presenting potential of MP-EVs, they express adhesion molecules,
e.g., LFA-1, where overexpression of complementary adhesion molecules, e.g., ICAM-1,
facilitates EV–target interaction. As inflammation is an inseparable component of many
conditions, this natural feature of MP-EVs can be used to deliver targeted drugs to inflamed
sites [338]. This knowledge supports further exploration of the physiological properties of
IC-EVs as well as the study of less-studied NK cell-derived EVs (NK-EVs), which could
open new windows for designing novel therapeutics.

Human Microbiome-Derived Extracellular Vesicles (HMB-EVs)

Bacteria-derived EVs, on the basis of their source and biogenesis, can be classified into
membrane vesicles (MVs), originating from the inner membranes of Gram-positive bacteria,
and OMVs, released by Gram-negative bacteria [339,340]. The roles of HMB-EVs in host
cell homeostasis range from nutrient sources to horizontal gene transfer, and even nucleic
acid delivery [341]. Lactobacillus acidophilus-derived MVs contain bacteriocins capable of
eliminating opportunistic pathogens in vitro [342]. Similarities between prokaryotic EVs
and their paternal microbes regarding the interaction of pathogen-associated molecular
patterns (PAMPs) with their corresponding pattern recognition receptors (PRRs) combined
with natural adjuvant properties could lead to the same antimicrobial immune response as
that of the pathogen itself [343,344]. EVs derived from Staphylococcus aureus as a vaccine
Int. J. Mol. Sci. 2024, 25, 485 17 of 51

candidate trigger immune responses via the toll-like receptor (TLR) pathway in Staphylococ-
cus aureus-induced pneumonia in mice [344]. Although microbial EV lipopolysaccharide
(LPS) mediate immunomodulation, further study of the therapeutic opportunities of other
immunomodulatory components and their potential risks is needed.

Breast Milk-Derived Extracellular Vesicles (BM-EVs)

BM-EVs are characterized by their large numbers, high diversity [345], and enrich-
ment of immune-related miRNAs capable of conferring immunomodulatory effects to
the infant [346]. Recently, various studies have shown that BM-EVs have direct pharma-
cological effects, including significant anti-inflammatory, tolerogenic, and anti-apoptotic
effects. Porcine BM-EVs promoted cell proliferation and reduced cell apoptosis by de-
creasing deoxynivalenol-induced injury via upregulation of miRNAs in the p53 pathway
in vitro [347], as well as preventing LPS-induced injury via downregulation of inflam-
matory cytokines [348]. Human BM-EVs had a potency to protect against necrotizing
enterocolitis by increasing cellular proliferation and decreasing apoptosis in vitro [349].
Interestingly, bovine BM-EVs attenuated colitis by upregulating the inflammatory protein
A20 in the NF-κβ pathway and downregulating colitis-associated miRNAs in vitro [350].
Furthermore, bovine BM-EVs protected against cisplatin-induced toxicity in vitro by in-
creasing macrophage proliferation and expression of β-catenin, p21, and p53 [351]. Addi-
tionally, BM-EVs diminished arthritis via improving cartilage pathology and bone mar-
row inflammation [352]. The same EVs were proven effective against breast cancer via
promoting apoptosis and reducing oxidative stress and inflammation markers in vitro
and in vivo [353]. Regardless of the source, BM-EVs in general can have potent anti-
inflammatory, immunomodulatory, and anti-apoptotic effects, which can be used to treat
various inflammatory disorders.

3.2. Pharmacokinetics of Extracellular Vesicles

The concept that EVs are conveyors of information and functionality enhances their
application as drug delivery platforms; thus, it is essential to understand the pharma-
cokinetics of EVs. This section addresses issues concerning machinery involved in the
biogenesis of intrinsic EVs (release and uptake), as well as the biokinetics of extrinsic EVs,
including absorption, distribution, metabolism, and excretion (ADME).

3.2.1. Pharmacokinetics of Intrinsic Extracellular Vesicles

Endocytosis of molecular cargo and early endosome formation is the first step in EV
biogenesis [354,355]. Cargo sorting within the early endosome involves three pathways.
Cargo that needs to be recycled will be placed in the peripheral tubular domains of en-
dosomes that will separate to integrate into the Golgi network or the plasma membrane.
These cargoes fused to the plasma membrane are either conveyed directly into pinched
microvesicles or incorporated into released exosomes. Cargo not destined for recycling
will concentrate in the central endosomal region and maturate to form late endosomes.
These late endosomes either fuse into lysosomes and then degrade or fuse into the plasma
membrane and are subsequently released as exosomes [354]. In contrast to changes in
subcellular localization, the maturation of endosomes is accompanied by changes in their
membrane. Changes in membrane composition allow downstream mobility and sorting,
such as ceramides, instead of sphingomyelin, and Rab11, essential for trafficking in late
endosomes, instead of Rab5 in early endosomes [356,357]. Endosomal vesicular maturation
of certain membranous regions initiates engulfment and budding away from the cytoplasm
to generate ILVs that enclose the cargo into late multivesicular endosomes, i.e., MVBs [3].
MVBs that have fused into lysosomes lead to the degradation of ILV cargo, whereas MVBs
that have fused into plasma membrane lead to the secretion of ILVs into the extracellular
space as exosomes.
Int. J. Mol. Sci. 2024, 25, 485 18 of 51

Extracellular Vesicle Release

After MVBs are delivered to the plasma membrane, they undergo docking and fusion
with the cell membrane via key players such as v-SNAREs (on vesicles), t-SNAREs (on
target membranes), Rab GTPases, tethers, and additional proteins [358]. Complexes of one
v-SNARE molecule and three t-SNAREs molecules occur between the fused membranes.
Rabs include Rab27a, Rab27b, and Rab35, which recruit tethers for the binding of SNARE
proteins (i.e., v-SNAREs and t-SNAREs) and vesicular (i.e., EVs) docking at the cell mem-
brane [358–360]. SNARE proteins, such as VAMPs (v-SNAREs), syntaxins (t-SNAREs),
and SNAPs (t-SNAREs), play a role in facilitating the fusion and hence secretion of EVs.
For example, VAMP7 localized to late endosomes forms VAMP7-syntaxin1-SNAP25 and
VAMP7-syntaxin3-SNAP23 complexes, promoting fusion [361]. In an Alzheimer’s dis-
ease model, tau-bearing vesicles fuse to the cellular membrane of neurons through late
endosomal v-SNARE VAMP8 participation [362]. Hepatitis C virus-carrying MVBs fuse
to the cellular membrane via syntaxin4 of infected cells and hence spread infection via
released EVs [363,364]. In models of Parkinson’s disease, the correlation between increased
α-syn concentration and decreased interaction between syntaxin4 and VAMP2 results in
decreased EV secretion [365]. In prostate cancer cells, knockdown of t-SNARE (syntaxin 6)
significantly decreased EV production and thus reduced drug resistance conferred by this
secretion [366]. Although most SNARE proteins are cell type-specific, it is clear that VAMP7
and SNAP23 are ubiquitous hubs of the membrane fusion process [361]. Post-translational
modifications of SNARE proteins, such as decreased SNAP23 O-GlcNAcylation, enhances
its interaction with syntaxin4 and VAMP8 leads to increased secretion of EVs, whereas a
similar effect is produced by phosphorylation of SNAP23 and H1 receptor activation in
Hela cells [367,368]. In addition, a study in cancer cells showed that PKM2 involved in the
phosphorylation of SNAP23 at Ser95 upregulates the secretion of EVs [369]. In contrast to
phosphorylation, RNAs have been employed in the regulation of SNARE and the secretion
of EVs. For example, in models of non-small cell lung cancer, miRNAs (134, and 135b)
inhibit the SNARE protein YKT6 and reduce the secretion of EVs [334]. Similarly, in pancre-
atic cancer cells, long non-coding RNA (lncRNA) PVT-1 and HOTAIR regulates YKT6 and
VAMP3 [370] and SNAP23 with VAMP3 colocalization [371], respectively, thereby playing
a role in the fusion of MVBs with the plasma membrane.

Extracellular Vesicle Uptake

Generally, fusion of MVB with the cell membrane is followed by secretion of ILVs into
the extracellular space as EVs (i.e., exosomes). Mechanisms and players of EV targeting
are still unknown, and the question of how many EVs will be delivered randomly rather
than specifically remains unanswered [372]. There are three pathways through which EVs
interact with target cells: (a) direct interaction between cell membrane receptors and EV
surface proteins; (b) cell–EV membrane fusion; and (c) endocytosis [359,373–376]. More-
over, to date, EV surface molecules such tetraspanins, immunoglobulins, proteoglycans,
and lectin receptors are implicated in EV target cell binding through unknown mecha-
nisms [359,377,378]. The most therapeutically important EV ligands that have receptors on
cancer cell surfaces are PD-L1, TNF, FasL, and TRAIL, and they are considered potential
anti-cancer targets. The most efficient pathway for intracellular delivery of EV cargo is
through EV–cell membrane direct fusion. Indeed, such a mechanism does not always occur,
as evidence indicates that the dominant mechanism for uptake of EVs by the cell is endo-
cytosis, where intact EVs are engulfed, bound by the plasma membrane, and eventually
joined to endosomes [372]. This would exacerbate the dilemma of intracellular delivery,
given the needs of EV cargo to escape from endosomes into the cytoplasm, i.e., “endosomal
escape”, avoiding lysosomal degradation, cellular recycling, or exile into the extracellular
space [379–381]. There are a few proposed mechanisms based on pH-dependent perme-
ability for endosomal escape of cargo into the cytoplasm, where it can carry out its specific
function [381,382]. One of the major hurdles in utilizing EVs for clinical application is
the understanding of the mechanisms of EV cargo release into the cytoplasm and the
Int. J. Mol. Sci. 2024, 25, 485 19 of 51

poor predictability of the process across different cell types [381,383,384]. Similarly, much
remains to be understood about the transmission of EVs in the blood and the crossing of
the endothelial layers of blood vessels. However, recent studies on the passage of EVs
across the blood–brain barrier (BBB) have indicated that transcytosis is the most widely
accepted mechanism for transporting EVs across the endothelium [385]. Breast-cancer
derived EVs are taken up by endothelial cells via clathrin-mediated endocytosis, sorted by
Rab11 for exocytosis at the basolateral membrane, and finally secreted from the cell through
interactions between EVs v-SNARE and VAMP-3 and membrane-associated t-SNAREs
SNAP23 and syntaxin 4 [386]. Although it is not clear whether endothelial cells are involved
in the internalization or attachment of EVs, surface heparan sulfate proteoglycans have
been shown to be involved in cellular endocytosis of EVs [387]. Another mechanism that
facilitates this is EVs’ adsorptive transcytosis through interactions between positive and
negative molecular charges [388].

3.2.2. Pharmacokinetics of Extrinsic Extracellular Vesicles

Compared to many synthetic drug delivery systems, the exceptional EVs that are well-
tolerated in vivo as mediators of intercellular communication are beginning to highlight
their usefulness as effective drug delivery platforms for a range of therapeutic macro-
molecules. These advances and their applications can be made possible by technological
advances in the labeling and understanding of the in vivo pharmacokinetics of exogenously
administered EVs. EVs have the unique attributes of stability in circulation, biocompatibil-
ity, immune tolerance, and the ability to cross all biological barriers, entering all organs,
including the central nervous system [389,390]. Although therapeutic EV research has
evolved from in vitro studies to pre-clinical models to clinical trials [389], successful clinical
translation has various obstacles. This section will focus on labeling and engineering EVs
as tools to study their in vivo kinetics and potential for drug delivery and targeting.

Extracellular Vesicle Labelling

In general, labelling of EVs can be performed in two ways, either by general la-
belling of EV-associated macromolecules or by labelling of an EV-associated specific
macromolecule [391]. The bond established between the lipophilic functional groups
of the fluorophore-conjugated dye and the EV lipid bilayer will be non-covalent. Various
lipophilic tracer dyes, such as PKH67 and DiR/DiL/DiD, cover a wide range of emission
wavelengths for better in vivo penetration through tissues [392–394]. Although these dyes
are capable of rapid and efficient labeling of EVs without altering the EV-producing cells,
they tend to aggregate into micelles similar in size to EVs and also potentially label non-EV
particles [395]. In addition, the non-covalent bond promotes a high risk of transferring the
EV-bound dye to the plasma membrane, as well as altering the properties of EVs, thereby
affecting the biodistribution of EVs in vivo [394,396]. In contrast to the non-covalent an-
choring of lipids, the fluorophore NHS ester covalently binds to the surface protein amine
groups of EVs [397,398]. These covalent conjugations can alter the surface proteome of EVs,
thereby affecting their interactions with other proteins. Furthermore, these dyes can label
non-vesicular proteins, thus making them inaccurate. Nevertheless, dyes/tracers that are
currently being produced are very stable, with a half-life of a few days to weeks [399,400].
Apart from fluorescent dyes, EVs can be labelled with various radiotracers, such as 99m Tc-
HMPAO [401], 125 I-IBB [402], and 111 Indium-oxine [403]. Superparamagnetic iron oxide
nanoparticle (SPION)-loaded EVs can be employed for biodistribution studies utilizing
magnetic resonance imaging (MRI) [396,404]. Despite the high sensitivity in vivo of radiola-
beling and MRI, the high infrastructure cost makes it difficult to implement in basic science
research. In contrast to exogenous EV labelling, genetically engineered cells can generate
fluorescent or bioluminescent protein-labeled EVs [396,398,402,405–407]. To label a specific
population, genetically engineered producer cells express a reporter protein fused to the
EV sorting domain to allow loading of the reporter protein during EV biogenesis. Thus, the
CD63 and eGFP fusion protein can promote eGFP sorting in 30–40% of EVs; therefore, each
Int. J. Mol. Sci. 2024, 25, 485 20 of 51

carries 30–60 eGFP molecules on average [408]. This approach can similarly be exploited to
label other EV sorting domains, such as CD9, CD81, syntenin, and Gag [408,409]. However,
not all EV protein domains, such as ALIX, SIMPLE, and syndecan, can be engineered and
characterized because the efficiency is relatively low [408]. Generally, genetic engineering
approaches provide an effective way to tag a specific group of EVs, either with fluores-
cent proteins, e.g., GFP, RFP, etc., or bioluminescent proteins, e.g., Gaussia-, Firefly-, and
Nano-luciferase. Disadvantages of these approaches include failure to label all EVs and
the requirement for genetic engineering of the produced cells, which is challenging for
some cell sources. Furthermore, overexpression of specific EV-sorting proteins may change
EV biogenesis and/or proteome, thereby changing the biodistribution of EVs. Because
there is no perfect EV reporter or labeling method, and because each method has a certain
degree of advantages and disadvantages, the choice of labeling method should be based on
indication and feasibility.

Extracellular Vesicle Engineering

To neutralize the problem of EV clearance and to promote additional hepatic delivery
of EVs, endogenous and exogenous EV surface engineering strategies have been utilized.
CD47 surface protein, a potent “do not eat me” signal, has been shown to be expressed
in many normal and tumor cells. Namely, CD47-expressing TC-EVs can inhibit phagocy-
tosis by interacting with SIRPα on macrophages, by which tumors can evade the innate
immune system. Previously, there have been several delivery vectors, such as lentiviruses
engineered with CD47 to inhibit phagocytosis and liver clearance [410]. Similarly, EVs
engineered with CD47 inhibit their uptake by monocytes and prolong their half-life in
circulation [411]. Similar observations have been made in other studies that have utilized
CD47 overexpression [412,413], as well as expression of CD47-resembling molecules such
as CD55 and CD59 on the surface of EVs [330]. The relatively long in vivo half-life of
3 weeks of albumin, the most abundant human plasma protein, has brought much interest
for its use in drug delivery for a range of biotherapeutics, either by direct incorporation or
via a binding domain on the surface of the carrier [414]. Similarly, insertion of albumin-
binding peptides into the extracellular loop of CD63 prolongs the circulation half-life of
EVs [415]. The goals behind this EV engineering are to improve extrahepatic delivery and
to extend the plasma half-life of EVs. A very common strategy in pharmaceutics to im-
prove therapeutic pharmacokinetic properties is the hydrophilic coupling of polyethylene
glycols (PEGs, PEGylation) to prevent the electrostatic interaction between plasma proteins
and the delivery carrier [416]. Lipid nanoparticles (LNPs) and LPs are the most clinically
validated delivery systems utilizing this strategy [417]. Similarly, the use of this strategy in
EV research will result to an extension of the plasma half-life, a decrease in hepatic uptake,
and an increase in extrahepatic delivery [418].

4. Pharmaceutical Applications of Extracellular Vesicles

In contrast to LPs that have been used clinically as well-established nanocarriers for
drugs in the treatment of various diseases [419], the development of EVs [420] as well as
hybrid liposoxomes (LOs) [421] is still in the pre-clinical stage. One of the hurdles with EV-
based therapies is designing them as suitable dosage forms for specific applications. With
the wide applications of EVs in the management of various diseases, delivery strategies
can be critical to achieve the optimal therapeutic effect. Although intravenous (IV) injection
is the most common method of administering EVs, other drug delivery routes may be
considered, including oral, nasal, or pulmonal, depending on the application of EVs
(Figure 2). This section will closely look at several decades of designing LP formulation
dosage forms for various routes of administration, establishing dosing regimens, and
in vitro–in vivo translational studies, as well as pharmacokinetic evaluations that may help
pave the way for the formulation of better EV-based dosage forms.
Int. Int.
J. Mol. Sci.Sci.
J. Mol. 2024, 25,25,
2024, 485x FOR PEER REVIEW 22 of 21
51 of 51

Figure 2. Challenges of administration of lipid bilayer vesicles (LBVs: LPs, EVs, and LOs) in vivo.
Figure 2. Challenges of administration of lipid bilayer vesicles (LBVs: LPs, EVs, and LOs) in vivo.
The formulation of LBVs ranges from free suspensions to bulk composites and 3D scaffolds within
The formulation
functional of LBVsThe
biomaterials. ranges
routefrom free suspensions
of administration to bulk the
determines composites and 3D scaffolds
most appropriate LBV formu-within
functional biomaterials.
lation to use Theintended
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administration
By adjustingdetermines the most
the composition and appropriate
administration LBV
of for-
LBVs, it is
mulation topossible to facilitate
use to achieve the their delivery
intended across
effect. By normal physiological
adjusting barriers.
the composition andEpithelial and
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ofendothelial
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facilitate theirinternalize LBVs by
delivery across different
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physiological (createdEpithelial
barriers. with
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and endothelial cells lining these barriers internalize LBVs by different mechanisms (created with
https://app.biorender.com/ (accessed on 25 December 2023)).
4.1. Applications of Lipid Bilayer Vesicles for Drug Delivery
4.1. Applications
Compared of toLipid Bilayer
several Vesicles systems,
NP delivery for Drug lipid
Delivery
bilayer vesicles (LBVs) are versatile
platforms for drug
Compared packaging
to several NPand delivery.
delivery Based on
systems, their
lipid origin,vesicles
bilayer they are(LBVs)
classified
areinto
versa-
synthetically originated LPs, biologically originated EVs, as well as hybrid LOs
tile platforms for drug packaging and delivery. Based on their origin, they are classified originat-
ing synthetically
into from the fusion of LPs and
originated EVsbiologically
LPs, [253]. LPs are self-assembled
originated EVs, assynthetic
well as NPs thatLOs
hybrid pro-orig-
vide a prominent platform consisting of fatty acids and lipids centered
inating from the fusion of LPs and EVs [253]. LPs are self-assembled synthetic NPs in a spherical bi-that
layer membrane surrounding an aqueous chamber [422] in which both hydrophilic
provide a prominent platform consisting of fatty acids and lipids centered in a spherical and
hydrophobic
bilayer molecules
membrane can be encapsulated.
surrounding LPs improve
an aqueous chamber [422] the pharmacokinetics
in which of the and
both hydrophilic
incorporated molecules by increasing their circulation time and overcoming
hydrophobic molecules can be encapsulated. LPs improve the pharmacokinetics barriers, suchof the
incorporated molecules by increasing their circulation time and overcoming barriers, such
as the BBB [423]. LPs were classified based on structure into unilamellar, multilamellar,
and multivesicular constructs. The LP preparation method determines the structure and
Int. J. Mol. Sci. 2024, 25, 485 22 of 51

size of the LPs produced, which in turn determines the encapsulation capacity and the
drug release [424]. The membrane fluidity of LPs associated with their composition has
also been the basis for their classification into either non-flexible (non-deformable, clas-
sical, and conventional) or flexible (deformable and elastic) LPs. The charge of the LPs’
phospholipid head groups depends on the characteristics of the surrounding environment,
including pH, temperature, and ionic strength. Thus, LPs’ ζ-potential is a key parameter
affecting the stability of liposomal dispersions and plays a role in the interaction between
LPs and the biological environment [425,426]. Studying these interactions is crucial for
predicting the biological fate of LPs, including corona formation and adsorption onto the
cell membrane [427].
In the context of drug delivery, all advantages of LPs reported in the literature also
apply to EVs. EVs have revealed great potential for integrating many small molecules [428],
proteins [429], nucleic acids [430], and theranostics [431] to be loaded and transported
via EVs for therapeutic and diagnostic applications. Moreover, EVs in a hybrid plat-
form [432,433] incorporate other nanovesicles [434,435] that provide them with superior
biomimetic or drug loading purposes. In contrast, diverse combinations of LPs with chemi-
cal and biological entities improve their physicochemical properties and stability, which in
turn enable controlled drug release and optimize their interactions with the biological envi-
ronment [436]. These combination approaches include modification with polymers [437],
peptides and proteins [438], and nucleic acids [439], as well as coating with [440] or en-
capsulating other [441] nano-entities and hybridizing with the cell membrane [442] or
EVs [443]. Apart from these approaches, intact EVs are more complex due to their biolog-
ical origin, and thus meet the complexity requirements of the optimal biological level of
nanomedicine. This ideality is due to the hundreds of different types of lipids, proteins, and
carbohydrates as well as internal cargoes and surface-bound molecules [253]. In addition,
further design of EVs can be accomplished using EV parent cells engineering [444–446].
Although very simple LP systems can be produced on a large scale, EVs may offer the
possibility to design more complex membrane nanovesicles. EVs outperform LPs through
their remarkable similarity to the cell membrane and are thus more biocompatible and
safer [447]. Employing patient-derived EVs makes it a very promising tool in the context
of personalized medicine [448]. From a pharmacokinetic point of view, EVs, compared
to LPs, have superb circulation time [393,449,450] in crossing biological barriers and ex-
ert physiological, pathological, and therapeutic effects [388,451]. In order to bridge the
bench-to-market gap in the clinical translation of both LBV (LP and EV) drug delivery
products, a range of hurdles must be overcome. These barriers include (1) fully disclosing
the physicochemical properties of the interaction of the lipid bilayer with the biological
environment [452], (2) employing smart strategies to control drug release and concentration
at the site of action [453], (3) advanced production techniques with the highest levels of
particle homogeneity, drug content uniformity, and batch reproducibility, scalability, and
sterility [454], (4) preserving storage stability through innovative formulations [427], and
(5) ensuring clinical trial success by fitting in silico, in vitro, and in vivo models to provide
the highest simulation of the PK-PD of the human body in in vivo studies [253,455]. The
following parts will deal with LP-based dosage forms for various routes of administration
and how they may pave the way for better EV-based dosage forms.

4.1.1. Oral Delivery

Oral administration of LPs is hampered by their instability and difficulties in bypass-
ing bio-membranes, as their initial application with insulin delivery [456] was neither
reproducible nor predictable [457]. Attempts have been made to improve their oral deliv-
ery [458–463] by adding polymers or ligands to modify their lipid compositions, which can
enhance their stability and permeation. In parallel, naturally produced EVs have recently
gained much research interest as a platform for miRNA and drug delivery. The speculation
that lipids support the intestinal transmission of miRNAs [464] has opened up another re-
search direction, namely studies on EV-based transmission and function after oral delivery
Int. J. Mol. Sci. 2024, 25, 485 23 of 51

both locally and systemically. The fact that most water and nutrient absorption takes place
in the gut could also be true for orally delivered EVs [465]. The digestion stability of bovine
milk EVs containing miRNAs was evaluated in vitro by subsequent incubation in three
solutions simulating oral, gastric, and intestinal phases of digestion, respectively. It has
been shown that about 50% of all miRNAs survive the oral and gastric phases of digestion.
Moreover, in in vivo oral administration in mice, EVs were detected in various distant
tissues [466]. These results provide indirect evidence of the digestive stability of EVs, al-
lowing them to reach the intestine after oral administration. Furthermore, after absorption
through the intestine [467], therapeutic EVs can exert predictable effects at distant sites. The
present observations suggest the involvement of the “neonatal” Fc receptor in the uptake
of intact EVs [468] and the role of integrins in both tissue trafficking [469] and subsequent
EV uptake by cells [470]. Attempts to evaluate orally administered fluorochrome-labeled
EV bioavailability and tissue biodistribution in mice after oral gavage have demonstrated
the presence of vesicles in the intestine, liver, spleen, kidney, lung, heart, and brain [471].
However, these in vivo attempts have failed to estimate the precise efficacy of EVs’ passage
through the GIT, [472] because it relied on the detection of EV-containing miRNAs rather
than the vesicles themselves [473]. Oral drug administration is the preferred route for clini-
cians and patients. EVs’ superiority to LPs in oral delivery is due to features including fast
internalization, low immunogenicity even at repeated doses, physiological stability, and
feasibility of modification of internal and surface components, which generates specific and
controlled release of internalized or loaded therapeutic molecules. In general, nanocarriers
(including LPs and EVs) are a colloidal delivery system for drugs with a particle size of
less than 500 nm [474]. Different types of nanocarriers cross the intestinal epithelium using
different mechanisms [475]. One such mechanism is paracellular transport, which covers
the diffusion of particles between 0.5 and 20 nm across the intestinal epithelial barrier and
is therefore impractical due to the limited physical dimensions between cells [476,477].
Conversely, disruption of the intestinal barrier, either due to inflammatory diseases or treat-
ments that reduce the tightness of the epithelial barrier, allows the passage of larger EVs
over 200 nm [478]. The other mechanism is transcellular transport, mainly via endocytosis
by epithelial cells and phagocytosis by M cells, where the former accounts for 90–95% and
the latter 1% of the total cells of the GIT [475]. Wu et al., 2022 found insulin-loaded bovine
milk EVs showing efficient internalization through multiple active endocytic pathways into
the epithelium [479]. The authors, as well as Betker et al., 2019, suggested that since milk
is a nutrient, milk EV uptake is mediated by peptides, amino acids, glucose transporters,
and the neonatal Fc receptor (FcRn) [468,479]. In contrast to several studies demonstrating
a rapid clearance rate of circulating exogenous EVs after IV injection (~2–30 min) medi-
ated by the reticuloendothelial system (RES), mainly macrophages [480], Munagala et al.,
2016 found that bovine milk EVs remained circulating for at least 24 h after oral admin-
istration in nude mice [481]. The same group tested milk EVs for oral paclitaxel (PTX)
administration in a lung tumor xenograft model, demonstrating that orally administered
PTX-EVs significantly inhibited tumor growth compared to the same dose administering
PTX intraperitoneally. These PTX-EVs showed significantly less systemic and immuno-
logic toxicities compared to IV PTX [482]. Soo Kim et al., 2016 showed that murine RAW
264.7 macrophage-derived EVs loaded with PTX are more than 50-fold cytotoxic to drug-
resistant MDCKMDR1 (Pgp+ ) cells in vitro [483].
To understand the true clinical potential of oral administration of EVs, the question
that remains to be answered is why EVs absorbed from the GIT have a longer circulating
half-life than observed in systemically injected EVs. Bardonnet et al., 2006 suggested that
NP size is necessary for gastric retention, as particles < 7 mm are evacuated efficiently [484].
Thus, the size range of EVs of 50–200 nm [5] is unlikely to have any biological effect in
the stomach due to poor gastric retention. However, modifying EVs with mucoadhesion
strategies using polymers or phospholipids in their surface membrane could give them
time to induce the desired GIT biological changes [475] as well as drug delivery. In
accordance with bovine milk EVs, the addition of casein has been shown to enhance the
Int. J. Mol. Sci. 2024, 25, 485 24 of 51

uptake of EVs derived from human cardiosphere stromal cells. Modification of EVs with
casein also presented an increased biological effect compared to unmodified EVs in cardiac
dysfunction [485]. Munagala et al., 2016 showed that the addition of folic acid to the surface
of bovine milk EVs loaded with withaferin A resulted in a reduction in tumor size in a
murine model of lung cancer. This response was attributed to folic acid, which either
enhanced the stability of EVs in the GIT or targeted cancer cells after systemic circulation
was reached [481]. These data indirectly support bovine milk EVs as nanocarriers for
oral drug delivery. Similar to PEGylated LPs, Warren et al., 2021 modified the surface
of milk EVs with PEG, thereby decreasing hydrophobic interactions with mucin lining
the intestinal lumen, increasing uptake by epithelial cells, and delivering siRNA loaded
in vitro [486]. Although oral delivery of EVs offers various physiological and practical
advantages compared to other routes, there is still a need for further investigation into their
safety, stability, pharmacokinetics, and biodistribution features before they can be widely
used as drug vehicles or nutritional supplements.

4.1.2. Dermal Delivery

Exogenous molecules can cross the skin by transcellular permeation, paracellular
transport, and absorption via skin appendages, including hair follicles and glands [487].
LPs and EVs have tremendous potential to deliver active pharmaceutical ingredients (APIs)
to skin structures [488,489]. Some studies have shown that intact LPs permeate the stratum
corneum (SC), the outermost layer of the epidermis, after topical application [490]. The
composition of LPs [491], size, ζ-potential, and membrane fluidity and elasticity play an
important role in the rate and depth of skin penetration. A higher proportion of studies re-
ported hydrogel formulations of LPs compared to other semisolid dosage forms, including
ointments and creams for topical applications. Ex vivo models of human skin have been
used to study the time-dependent penetration of stem cell-derived EVs through the SC and
their internalization by dermal fibroblasts [492]. Zhang et al., 2021 reported topical appli-
cation of an aqueous dispersion cream (oil-in-water emulsion) of MSC-EVs on explanted
human skin cultures, resulting in less than 1% of the particles penetrating beyond the
SC [493]. Furthermore, in vivo experiments in rat models involved very limited infiltration
of MSC-EVs into the SC when administered topically [494]. However, OMVs of the skin
pathogens Staphylococcus aureus (S. aureus) [495] and Malassezia furfur [496] may penetrate
deeper layers of the skin, especially when the SC is removed or damaged. Furthermore,
engineering Escherichia coli (E. coli) OMVs with integrin-targeting peptides, RGP, resulted
in excellent infiltration across epidermal barriers, mainly via the skin appendages and
intracellular pathway, resulting in OMVs being widely present in the dermis [497,498].
Among many advanced formulation-based strategies, hydrogels stand out for their ver-
satility and attractive properties as suitable dermatological dosage forms of LPs and EVs.
Hydrogel-forming polysaccharides such as chitosan, alginate, and hyaluronic acid are a
class of hydrogel biomaterials that are widely used in the food and pharmaceutical industry
due to their abundance in nature, biodegradability, and biocompatibility.

4.1.3. Parenteral Delivery

The development of new drug molecules for the alleviation and treatment of various
diseases is an ongoing and continuous process. However, at present, most of the developed
new chemical entities have poor aqueous solubility and many undesirable physicochemical
properties such as short half-life, extensive degradation, high protein binding, first-pass
metabolism, and poor intestinal permeability [499]. Novel formulations are being devel-
oped for parenteral applications, which has improved PD-PK behavior of the drug with
lower dosing frequency and minimal adverse effects [500]. Many APIs, especially small
molecules, are not suitable for encapsulation in LP formulations intended for parenteral
administration due to their inherent permeability and lipophilicity (partition coefficient).
An API with a high permeability leads to premature leakage of vesicles, while too low a
permeability means an inability to cross biological barriers. Solutions to such a problem
Int. J. Mol. Sci. 2024, 25, 485 25 of 51

include modifying lipid composition of LPs, adding a permeation enhancer, or modifying

the chemical structure of the API as a prodrug. Balouch et al., 2023 reported that with
modification of the chemical structure of the four parental drugs (abiraterone, cytarabine, 5-
fluorouracil, and paliperidone), both permeability and lipophilicity could be systematically
converted to the desired LP formulability window [501]. In contrast, EVs, as novel drug de-
livery platforms with amphiphilic loading capacity, offer several advantages that overcome
many limitations imposed by conventional and advanced LP parenteral nanoformulations.
Among these, the factor that plays a role in the usefulness of EVs for systemic adminis-
tration is the surface protein CD47, which limits the uptake of EVs by macrophages, thus
prolonging the circulatory half-life of exogenously administered EVs. The same benefit has
been reported for synthetic NPs such as LPs decorated with CD47-derived peptides [502].
Selection of a pharmaceutical nanocarrier requires the fulfillment of two basic principles:
protecting the contained drugs from inactivation in the in vivo environment and releasing
the contained drugs without inducing an immune response to the nanocarriers. In this
sense, the employment of EVs in drug delivery is superior to existing nanocarriers like
LP- and polymer-based nanocarriers. This superiority is based on several features [389].
EVs arise naturally from normal cells, where their inclusions can transfer and alter the
function of recipient cells. Compared with LPs, EVs can effectively attract nucleic acids, i.e.,
hydrophilic, and greatly improve the packaging efficiency. The ability of surface molecules
of EVs to evade interaction with opsonin, antibodies, and coagulation factors helps avoid
immune responses in vivo. Compared with LP- and polymer-based nanocarriers, EVs
have higher stability in body fluids such as blood. Finally, EVs derived from special cells
including MSCs or immature DCs as well as EVs derived through native or engineered
molecules present on the surface can have a targeting effect by selectively binding to recipi-
ent cells [389]. Although cargo-loaded EVs are futuristic multifunctional nanotherapeutics,
the combination of LPs and EVs (i.e., hybrid liposoxomes (LOs)) promotes drug delivery
systems, as each system contributes to improved stability, drug loading capacity, and drug
release controllability [503]. Among the various avenues of parenteral administration (IV,
intramuscular, intradermal, subcutaneous, intraperitoneal, intra-articular, intrathecal, intra-
tumoral, etc.), to date, IV administration of EVs as therapeutic agents or carriers prevails
in treatment strategies for various diseases [504]. However, there will be a future trend to
integrate EVs with smart technologies to achieve real-time detection and control of drug
release as well as personalized drug therapy and precision medicine.

4.1.4. Pulmonal Delivery

Inhalation therapy offers an attractive and noninvasive method of drug delivery for
local and systemic treatments. By directly inhaling drugs, pulmonary bioavailability can
be improved, while subsequent adverse effects can be reduced [505,506]. However, drug
formulation and aerosol deposition are critical obstacles that hinder therapeutic efficacy.
Nanomaterials provide a solution by altering the drug’s size, solubility and surface chem-
istry to become compatible with the pulmonary microenvironment [506–508]. Developing
uniform, loadable NPs for a range of pulmonary therapeutic applications and determin-
ing their distribution characteristics upon inhalation would clarify cellular targeting and
optimize drug dosage. mRNA-loaded lipid NPs have demonstrated therapeutic efficacy
in eliciting systemic immunity against severe acute respiratory syndrome coronavirus 2
(SARS-CoV-2) infection as a vaccine for intramuscular (IM) injection [509–512], opening the
application of mRNA-based therapeutics for the treatment of other lung diseases across
different inhalation devices, such as nebulizers [513,514]. However, extensive formulation
of lipid NPs is needed to improve mRNA translation and pulmonary bioavailability for
inhaled delivery. Therefore, EVs of natural origin provide bio-alternatives to synthetic
lipid NPs that are naturally optimized for mRNA encapsulation and cellular delivery [515].
Recently, Wang et al., 2022 successfully designed an inhalable vaccine using recombinant
SARS-CoV-2 receptor-binding domain (RBD) conjugated to lung-derived EVs (lung EVs)
to elicit local lung immunity against SARS-CoV-2 infection. In addition, its stability at
Int. J. Mol. Sci. 2024, 25, 485 26 of 51

room temperature for three months outperforms the requirement of mRNA-loaded lipid
NP vaccines for cold chain transportation [516]. The use of lung EVs as nanovesicles for
inhaled drug delivery may increase drug retention and efficacy by more efficiently avoiding
immune clearance and targeting pneumocytes. Besides drug delivery, lung EVs themselves
have demonstrated therapeutic benefits. In a rodent model of idiopathic pulmonary fibro-
sis, lung EVs better restore lung function and reduce the severity of fibrosis compared to
their MSC-EV counterpart [517]. Furthermore, inhaled EV therapeutics are superior and
outperform LPs, as they are naturally optimized to distribute and retain mRNA and protein
cargo components to the lung after inhaled delivery [518].
Targeting EVs to a specific organ presents the challenge of rapid clearance after sys-
temic administration, mainly via the liver. A study to minimize liver clearance was per-
formed by Cober et al., 2023, which is based on the ability of porous microgels to engraft
and increase the survival of transplanted cells. They encapsulated EVs and showed that
lung targeting was improved, thanks to EVs’ size-based retention within the pulmonary
microcirculation [519]. The existence of lung EVs in human airway mucus and their less
obstructed movement facilitates crosstalk between lung-resident parenchymal cells and/or
immune cells. This concession was used by Kwak et al., 2023, who demonstrated that
Adeno-associated virus serotype 6 (AAV6) associated with EVs and secreted from vector-
producing HEK-293 cells was a safe and effective platform for inhaled gene delivery. In
contrast, standard preparations of AAV6 alone as well as physical mixtures of individu-
ally prepared EVs and AAV6 failed to mediate EV-AAV6 interaction or to improve gene
transfer efficacy [520]. EVs, as cell-free therapeutics naturally loaded with various bioactive
molecules, offer several advantages for clinical respiratory applications. First, small-sized
EVs facilitate their inhalation and deposit within the small bronchioles and alveoli. Second,
the lipid bilayer structure of EVs grants them stability in tissues and body fluids. Third,
EVs show lower levels of immunogenicity and toxicity compared to cell therapies.

4.1.5. Local Delivery

Local EV administration is beneficial for delivering EVs to well-defined lesions, thereby
limiting systemic circulation [521]. This type of EV application ranges from topical ad-
ministration to more complex radiological, ultrasound-, or endoscopy-guided routes. For
instance, EVs embedded in hydrogels facilitate their delivery and retention at the site of
action while providing a combined mechanical effect [522]. Although orally administered
EVs were distributed to the liver, lung, spleen, ovary, colon, kidney, pancreas, and finally
the brain four days after administration, IV-administered EVs accumulate mostly in the
liver [481]. The ability of EVs to cross the BBB bidirectionally [385,523] makes EVs attractive
as nano-biocarriers for drug delivery to the brain. Betzer et al., 2017 found that intranasal
administration of the EVs resulted in a significant degree of enrichment of EVs in the
brain [524]. Similarly, Han et al., 2022 devised an inhalation nebulizer for EVs and found
that they are almost exclusively enriched in the lungs and not in other non-target organs,
within 7 days [525]. Local application of EVs either by injection or direct coverage of the
trauma site reduces their clearance by circulation and enrichment in non-target organs.
However, due to the complexity of the trauma environment, EVs easily degraded and
become inactive [526]. In order to evade the premature clearing and maintain the desired
therapeutic effect over time, biodegradable, sparse, and porous hydrogels can be employed
to carry EVs [527,528]. Wang et al., 2022 evaluated the bio-removal rates of EVs applied
directly to local wounds versus those loaded with hydrogel. It was found that the former
was almost completely removed within four days, whereas the latter was uniformly re-
tained on the fourth day [529]. Similarly, Kwak et al., 2022 found that PEG-based hydrogels
loaded with EVs for wound application barely reached the liver or kidneys and mainly
acted on the skin [530]. To achieve a more localized and targeted delivery of EVs, hydrogels
can be injected locally into the target organ or prepared as microneedle patches for topical
application [528]. In contrast, the challenges of delivering hydrophobic chemotherapies
require the development of a drug delivery system that targets tumor sites. Thus, EVs
Int. J. Mol. Sci. 2024, 25, 485 27 of 51

loaded with anticancer drugs can improve their solubility and reduce toxicity, while the
use of ligands grafted onto the surface of engineered EVs can improve their targeting and
efficacy [531]. Similar to chemotherapies’ poor pharmacokinetics, the susceptibility of
current mRNA therapeutics (Pfizer–BioNTech’s mRNA-BNT162b2 and Moderna’s mRNA-
1273 COVID-19 vaccines) to degradation [532] increases the need for an effective delivery
system. Although lipid NPs could efficiently deliver mRNA intracellularly, a portion of the
internalized mRNA continued to function through EV secretion, containing more molecules
with similar biological functions. Thus, EVs can be considered a functional expansion of
lipid NPs [533] and are best used to protect mRNAs as their loading vehicle.

4.2. Applications of Hydrogel Platforms for Lipid Bilayer Vesicle Delivery

Hydrogels are three-dimensional reticulation structures based on cross-linked hy-
drophilic polymers with excellent ability to absorb and retain water and biological flu-
ids [534]. They can be classified based on several characteristics, including source (nat-
ural vs. synthetic), chemistry (polysaccharide, peptide/protein, miscellaneous; homo-
vs. co-polymer), charge (neutral, cationic, anionic), cross-linking mechanism (physical
vs. chemical), and biodegradation. Due to their biological tissue similarity, drug and NP
loading capacity, and sustained release property, hydrogels have been extensively used in
drug delivery and tissue engineering [535].
Approaches for loading LBV (prepared LPs, extracted EVs, or LOs)-based hydrogel
platforms include the following: (i) breathing method—beyond removing excess water from
the swollen hydrogel with a solvent, the exposed voids will be occupied by LBVs to obtain
LBV-loaded hydrogel platforms [536]; (ii) mix and crosslink—by directly mixing LBVs with
the hydrogel precursor solution followed by the addition of a crosslinking agent or by
a physical crosslinking method [537]; and (iii) in situ gel formation—by mixing LBVs and
polymers and injecting them with a crosslinking agent into the target site using a double-
lumen syringe [538]. Biocompatibility and structural porosity allow hydrogels to act as
carriers, prolonging the retention time of LBVs at the site of action and slowing their
release [539,540]. Adjusting the swelling rate, surface charge, and degradation rate are
all methods to tune the porosity of hydrogels, thereby tuning the loading and release of
LBVs [541,542]. In contrast, the LBV-related properties (particle size and lipid composition)
dictate the membrane stiffness of LBVs as well as the interaction with the hydrogel matrix,
which directly affects the release kinetics of LBVs from the hydrogel platform [543,544]. The
diffusion phenomena mainly describe the release pattern of LBVs’ payload from hydrogels,
which is directly controlled by the mesh size, swelling deformation, and degradation of the
polymeric network [545]. In addition, internal factors such as temperature [546], pH and
ionic strength [547], specific enzymes [548], and oxidative state [549], as well as external
factors such as electromagnetic waves [550], ultrasound waves [551], electric current [552],
and magnetic field [553], can be introduced at the research level to trigger the release of
LBVs’ payload from hydrogels. These factors that affect the release of LBVs from hydrogel
platforms also affect the release of cargo from those LBVs, thereby measuring the ratio
of the amount of drug released in the form of intact LBVs to the total amount of drug
released, i.e., free drug and drug incorporated into released LBVs separately [544]. Thus,
more controllable drug delivery can be obtained through sustained and multi-step-release
LBV–hydrogel composites. In this section, we will review some of the innovative platforms
with a focus on LBV-based hydrogels.

4.2.1. Wound Dressings

Hydrogels are an ideal alternative for skin and wound dressing, because of their
ability to eliminate infection, absorb trauma exudate, maintain water balance and gas
exchange, and enclose, protect, and deliver bioactive molecules [554]. Trauma dressings
are used to protect damaged tissue from environmental contaminants and infections.
Dressings effectively support the healing process by creating an ideal hermetic wound
environment [555] characterized by its porous structure, viscoelasticity, and water content.
Int. J. Mol. Sci. 2024, 25, 485 28 of 51

Zhao et al., 2020 [556] incorporated human umbilical vein endothelial cell-derived EVs
(HUVEC-EVs) into well-designed gelatin methacryloyl (GelMA) hydrogels, and completely
dressed skin wounds with them. They demonstrated in vivo and in vitro that GelMA
hydrogel dressings not only helped repair injured tissue, but also achieved prolonged
release of loaded HUVEC-EVs.

4.2.2. Microneedle Patches

In order to overcome the limitations of delivering conventional hydrogels to deep
tissues across the skin barrier, a method involving the use of microneedles (MNs) has
been implemented [557]. Common materials used in MN preparations include gelatin,
polylactic acid–hydroxy acetic acid co-polymer (PLGA), polyvinyl alcohol (PVA), and
chitosan, which have been used to deliver LBVs [558,559]. The soluble shell and core
structural properties of MNs facilitate deep and sustained delivery of the bioactive payload,
which synergistically promotes wound healing [558]. Yuan et al., 2022 prepared an MN
patch comprising methacrylate gelatin/polyethylene glycol diacrylate (GelMA/PEGDA)
hydrogel. Subsequently, the preloaded MN molds are subjected to optical or chemical
crosslinking, followed by freeze-drying to obtain hydrogel MN-encapsulated HUVEC-EVs
and tazarotene. After in vitro application, the active ingredients are released around the
wound site; they promote collagen deposition, epithelial regeneration, and angiogene-
sis [559]. In contrast, Ma et al., 2022 designed a novel core–shell hyaluronic acid (HA) MN
patch with ferrum-MSC-derived artificial nanovesicles (Fe-MSC-NVs) and polydopamine
NPs (PDA NPs) encapsulated in the needle tips. The Fe-MSC-NVs loaded with cytokines
are encapsulated in the inner HA core, whereas PDA NPs are encapsulated in the outer
methacrylated HA (HAMA) shell of the MN tips [558]. Hierarchically, these procedures
involve encapsulating LBVs into the shell/core of the needle tip and freeze-drying to
complete the construction of MN composites.

4.2.3. Injectable Applications

Injectable hydrogels can be applied not only to superficial wounds, but also to deeper
tissues and organs [560]. The application of direct injection of hydrogels loaded with active
ingredients, such as drugs, growth factors, and cells, into the damaged area allows for effec-
tive repair while reducing the need for tedious surgical procedures and hence the burden on
patients. Thus, local injection of LBV-loaded hydrogels resulted in sustained local release
of LBVs, which promoted repair and regeneration of injured tissues [560]. These results
required hydrogels with shear thinning rheology (sol state) before injection and in situ
gelation rheology (gel state) after injection via physiologically induced crosslinking [561].
Cao et al., 2021 [562] injected a hydrogel loaded with human urine stem cell-derived
EVs (USC-EVs) intrathecally, which promoted angiogenesis and repair of spinal cord
injury (SCI).

4.2.4. Bioink-3D Bioprinting

Bioink-3D bioprinting is a method for creating hierarchically complex and customiz-
able geometric shapes using computer-aided design software. Due to the excellent rheo-
logical properties of hydrogels, they can be used as bioinks in 3D printers for bioprinting
scaffolds with tactile structure, porosity, and mechanical properties that can effectively load
LBVs [563]. Born et al., 2022 [564] demonstrated that a 3D GelMA hydrogel loaded with
MSC-EVs maintained their biological activity beyond 3D printing and photo-crosslinking.
They also showed that the burst release of EVs could be reduced by optimizing the
crosslinker concentration, while the porosity of the hydrogel and meshwork could be
changed by altering the GelMA synthesis and crosslinking parameters, which in turn
significantly affected the release of EVs.
Int. J. Mol. Sci. 2024, 25, 485 29 of 51

5. Conclusions
EVs play crucial roles in various biological processes and diverse cellular activities
by mediating intercellular communications. Conversely, in pathological conditions, they
contribute to the initiation, worsening, and resilience in various diseases. Understanding
EVs’ regulatory mechanisms and function in different biological processes is deemed crucial
for unlocking their clinical potential and applications. This, indeed, involves developing
new therapeutic strategies or interventions based on regulatory mechanisms of EVs.
EVs derived from MSCs, specific tumor cells, dendritic cells, B lymphocytes, and
macrophages hold significant therapeutic potential, without the necessity for specific
manipulation, and they elicit anti-inflammatory, anti-apoptotic, pro-angiogenic, and prolif-
erative effects. Additionally, engineering EV-producing cells will enhance their therapeutic
potential, and this can be achieved through genetic engineering, changing cell culture con-
ditions, and stimulating cells with factors such as drugs and cytokines. Specific examples,
such as the regenerative effects of MSCs in the lungs and breast milk-derived EVs, with
anti-inflammatory and immunomodulatory properties, highlight the diverse applications
of EVs in treating various disorders. The modulation of EV content, including proteins,
small RNAs, and lipids, emerges as a key strategy for tailoring therapeutic effects, showing
promise for future developments in regenerative medicine and disease treatment.
Besides their use as therapeutics, EVs hold a great potential as therapeutic nano-
biocarriers for drug delivery. Namely, EVs can encapsulate drugs or be decorated with
specific ligands for targeted delivery. The pharmacokinetics of intrinsic and extrinsic
EVs make them good candidates for drug delivery platforms owing to their in vivo toler-
ance and ability to cross biological barriers. Thus, different EV labeling and engineering
strategies were developed to understand the in vivo pharmacokinetics of exogenously
administered EVs. Labeling EVs can be performed with fluorescent dyes, radiotracers, or
employing genetically engineered producer cells that express a reporter protein fused to
the EV sorting. However, evident flaws of labeling using covalent conjugations include
the alteration of the surface proteome of EVs, which affects their interactions with other
proteins. Furthermore, these dyes can label non-vesicular proteins, which makes them im-
precise. Additionally, EV engineering strategies, including the use of surface proteins like
CD47, albumin, and polyethylene glycols (PEGs) to enhance circulation half-life, prevent
clearance, and improve extrahepatic delivery, could also enhance the potential of EVs for
drug delivery and targeting. Genetic engineering approaches also have drawbacks, such
as the inability to label all EVs, as well as problems related with genetic engineering of
producing cells. Additionally, the overexpression of specific EV-sorting proteins has the
potential to alter EV biogenesis and/or proteome, impacting the biodistribution of EVs.
Pharmaceutical application of EVs in drug delivery is currently in the pre-clinical stage.
EVs present challenges in designing suitable dosage forms for specific applications. De-
spite challenges, EVs demonstrate great potential in integrating small molecules, proteins,
nucleic acids, and theranostics for therapeutic and diagnostic purposes. Being biologi-
cally derived, EVs offer complexity requirements ideal for nanomedicine. Importantly,
patient-derived EVs hold promise for personalized medicine due to their biocompatibility
and safety. From a pharmacokinetic point of view, EVs exhibit superior circulation time
compared to LPs, enabling them to cross biological barriers effectively. Overcoming ob-
stacles in clinical translation involves disclosing physicochemical properties, controlling
drug release, employing advanced production techniques, ensuring storage stability, and
fitting models for successful clinical trials. The exploration of LP-based dosage forms for
various administration routes may pave the way for improved EV-based dosage forms in
the future.

Author Contributions: Conceptualization, A.A.-J. and M.B.; literature review, A.A.-J., À.E. and M.B.;
writing—original draft preparation, A.A.-J., À.E. and M.B.; writing—review and editing, A.A.-J., À.E.
and M.B.; scientific illustrations, figures and tables, A.A.-J.; supervision and project administration,
M.B. All authors have read and agreed to the published version of the manuscript.
Int. J. Mol. Sci. 2024, 25, 485 30 of 51

Funding: M.B. was supported by the Miguel Servet contract from the Instituto de Salud Carlos III
(ISCIII) (CP19/00027), ESF “Investing in your future”, and MV22/00017 grant.
Data Availability Statement: Not applicable.
Conflicts of Interest: The authors declare no conflicts of interest.

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