Molecular Targeting of Ischemic Stroke: The Promise of Naïve and Engineered Extracellular Vesicles

1. Introduction

Stroke is a neurological disorder caused by acute focal injury of the central nervous system (CNS) with a vascular etiology [1]. According to the World Health Organization, stroke is the second leading cause of death and the fourth leading cause of disability worldwide [2,3]. Ischemic stroke (IS), which accounts for approximately 87% of all strokes, has a steadily increasing incidence and is responsible for nearly half of all stroke-related mortality [4]. IS is defined by neurological dysfunction resulting from focal cerebral, spinal, or retinal infarction, most commonly due to cardioembolism or atherosclerosis in the aortic arch or cervical arteries [1,5]. In clinical practice, treatment of IS primarily involves intravenous administration of alteplase, the only tissue-type plasminogen activator (tPA) approved by the USFDA for thrombolysis, mechanical thrombectomy, or a combination of both, as quickly as possible after the onset of ischemic events [5,6,7].

Although advancements in infrastructure and treatment technologies have enabled rapid intervention for IS, more than one-third of patients continue to experience temporary or permanent disabilities, such as motor function impairment or dementia [8,9]. These post-stroke complications are closely correlated with both the severity and the frequency of the stroke [10,11]. The primary mechanism of complications arises from damage to brain parenchymal cells due to nutrient-depleted hypoxia and reperfusion injury [12]. The underlying pathophysiological processes include excitotoxicity, oxidative stress, inflammatory responses, disruption of the blood-brain barrier (BBB), and apoptosis [12,13,14]. Since the 1960s, numerous neuroprotective agents have entered clinical trials, but most candidates failed to demonstrate sufficient efficacy, likely due to a lack of preclinical models that accurately mimic the complex

pathology of stroke, particularly in elderly populations [14,15,16]. Furthermore, clinical trials involving cell therapies aimed at tissue replacement or modulation of inflammation have faced significant challenges, including low therapeutic efficacy and unexpected adverse events [17,18,19,20].

Over the last decade, increasing attention has been directed toward the potential of extracellular vesicles (EVs) as an alternative therapeutic strategy for IS [21]. EVs secreted by nearly all cell types facilitate intercellular communication by delivering their luminal cargos to target cells [22]. Moreover, EVs derived from various cell types have been shown to enhance neurogenesis [23], promote angiogenesis [24], attenuate inflammation [25,26], and reduce oxidative stress [27], all of which are critical for the treatment of IS (Table 1). Additionally, the luminal cargos of EVs from damaged tissues reflect disease progression, allowing the EVs to serve as potential biomarkers in various neurological disorders, including stroke [28,29]. Because they can cross the BBB, and their properties and cargos can be modified relatively easily, EVs have emerged as attractive candidates for therapeutic applications in CNS-related disease [30,31].

In this review, we briefly summarize the general characteristics of EVs, describe the molecular mechanisms underlying the pathophysiology of IS, and discuss the naïve EV cargos that target these mechanisms. We also discuss recent advances in EV engineering that aim to improve the therapeutic efficacy of EVs.

2. General Characteristics of EVs

EVs are non-replicative, membrane-bound particles enclosed by a lipid bilayer that are naturally released by cells [32]. EVs are generally categorized into three main types based on their size and biogenesis: exosomes (40–200 nm), microvesicles (200–1,000 nm), and apoptotic bodies (500–2,000 nm) [33]. Exosomes are formed by inward budding of the endosomal membrane, leading to the generation of intraluminal vesicles. The intraruminal vesicles are released into the extracellular space through the fusion of multivesicular bodies, also known as late endosomes, with the plasma membrane [34]. Microvesicles and apoptotic bodies are produced and released by outward budding of the plasma membrane in normal and apoptotic cells, respectively [35].

EV biogenesis involves several key pathways, one of which requires the endosomal sorting complexes required for transport (ESCRT) protein complex. This complex plays a crucial role in cargo recruitment and sorting during EV formation [36]. EVs can also be produced by ESCRT-independent mechanisms, such as the ceramide-based sphingomyelinase (SMase) pathway or the tetraspanin-dependent pathway [37,38]. Tetraspanins (e.g., CD63, CD81, and CD9), ESCRT-associated proteins (e.g., Alix, TSG101, and Syntenin), and heat shock protein 70 (HSP70) are incorporated into EVs during biogenesis and serve as conventional markers for identifying EVs [32]. EVs are also capable of encapsulating a variety of bioactive molecules, including proteins, lipids, and nucleic acids (double-stranded DNAs, mRNAs, and microRNAs [miRNAs]), which they subsequently deliver to target cells [39]. In the CNS, EVs secreted by stimulated cortical neurons are preferentially taken up by neurons rather than glial cells [40]. Additionally, glioblastoma-derived EVs can modulate the local immune environment by transferring the miRNAs miR-451 and miR-21 to monocytes and macrophages in the brain [41].

Once secreted, EVs interact with recipient cells either by being taken up directly or by activating receptors on the plasma membrane [42,43]. Direct uptake of EVs can occur by various mechanisms, including clathrin- or caveolin-mediated endocytosis, phagocytosis, micropinocytosis, lipid raft-mediated endocytosis, and membrane fusion [44]. Specific EV surface proteins, such as lactadherin and CD209, play critical roles in facilitating EV uptake by interacting with corresponding surface proteins on recipient cells [45,46]. EV surface proteins can also prevent uptake by non-target cells; for example, EVs expressing CD47 can evade clearance by monocytes, allowing them to persist and function in an endocrine manner [47].

3. The Molecular Pathophysiology of IS and the Therapeutic Potential of EVs

The complications associated with IS arise from pathophysiological changes within the CNS triggered by restricted nutrient and oxygen supply, resulting in activation of glial cells and irreversible loss of brain parenchymal cells [12]. Omics data analyses have demonstrated that EVs, particularly those derived from mesenchymal stem cells (MSCs), carry a diverse array of anti-inflammatory or anti-apoptotic cargos while simultaneously promoting neurogenesis and angiogenesis for tissue repair [48]. In this section, we explore the molecular mechanisms driving IS pathologies and discuss the therapeutic potential of naïve EV cargos to mitigate these processes.

3.1. Excitotoxicity

Exitotoxicity refers to nerve cell damage or death caused by excessive stimulation by neurotransmitters. IS begins with a reduction in cerebral blood flow, leading to a decrease in ATP levels that disrupts transmembrane ionic gradients (Figure 1A). This disruption triggers anoxic depolarization and excessive release of neurotransmitters into the extracellular space [49]. The released neurotransmitters cannot be sufficiently cleared due to the insufficient ATP supply, resulting in their continued accumulation in the extracellular environment. Glutamate, an excitatory neurotransmitter, induces calcium ion (Ca2+) influx by activating N-methyl-D-aspartate receptors (NMDARs), kainate receptors, and α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptors (AMPARs) [50]. The Ca2+ overload is further exacerbated by Ca2+ release from the endoplasmic reticulum through the mGluR–PLC pathway, calcium-dependent protease (e.g., calpain)-mediated cleavage of the sodium-calcium exchanger (NCX), and activation of other Ca2+-permeable channels such as acid-sensing ion channels (ASICs) and TRPM7 [51,52,53,54]. Excessive Ca2+ accumulation generates free radicals and initiates cell death pathways, ultimately leading to neuronal cell death [55]. Notably, NMDARs containing the GluN2B subunit can directly activate various cell death signals in neurons [55].

The potential use of EVs to treat IS by reducing extracellular glutamate concentrations has not been fully investigated. Recent studies showed that EVs derived from microglia and neurons deliver miR-124 to astrocytes, leading to upregulation of astrocytic glutamate transporter-1 (GLT-1) expression, which enhances the clearance of extracellular glutamate in normal conditions and in glioma [56,57] (Figure 1A). Additionally, EVs originating from astrocytes were found to contain glutamate transporters, suggesting a potential role in scavenging extracellular glutamate [58]. Although investigations of therapeutic EVs remain in the early stages, the results so far suggest that EVs might be useful in treating IS by reducing extracellular glutamate levels.

3.2. Oxidative/Nitrosative Stress

Oxidative stress in IS typically manifests in three distinct phases [59]. Initially, oxygen and glucose deprivation leads to decoupling of the mitochondrial respiratory chain, resulting in the accumulation of reduced intermediates and the generation of reactive oxygen species (ROS) (Figure 1B). The second phase involves the production of hydrogen peroxide (H2O2) as a result of xanthine oxidase (XO) activation. The final phase generally occurs during reoxygenation and is associated with NADPH oxidase (NOX) activation and a subsequent increase in Ca2+ concentrations. Concurrently, nitric oxide (NO) production, driven by the activation of the NMDARs (especially those containing the GluN2B subunit)–PSD95–neuronal nitric oxide synthase (nNOS) complex, further contributes to oxidative stress in the third phase [60]. Oxidative stress can also arise during excitotoxicity, primarily by ROS generation due to NOX and phospholipase A2 (PLA2) activation in response to Ca2+ influx [61,62]. The ROS and reactive nitrogen species (RNS) generated during IS can directly damage DNA, proteins, membranes, and other cellular compounds ultimately leading to cell death [63]. For instance, NO and superoxide anions produced by nNOS and NOX activation combine to form highly reactive peroxynitrite, which induces DNA fragmentation, lipid peroxidation, and disruption of the BBB [64].

Oxidative stress in IS can be mitigated by overexpression and nuclear translocation of the nuclear factor-erythroid 2-related factor 2 (Nrf2) transcription factor. In the nucleus, Nrf2 binds to the antioxidant response element (ARE), promoting the expression of antioxidant enzymes such as heme oxygenase (HO-1), glutathione peroxidase (GPx), catalase (CAT), and superoxide dismutase (SOD) [65,66]. Recent studies showed that neural stem cell (NSC)-derived EVs can stimulate Nrf2 translocation and increase the expression of SOD1, CAT, and GPx-1, thereby reducing intracellular ROS levels in neuronal hypoxia/reperfusion models [67] (Figure 1B). Additionally, EVs from mini-pig adipose-derived MSCs (AD-MSCs) were shown to decrease the expression of iNOS, NOX-1, and NOX-2 and reduce oxidized proteins in a rat model of acute IS [68]. Furthermore, human AD-MSC-derived EVs were found to lower ROS levels and restore mitochondrial respiratory chain function in H2O2-treated endothelial cells by delivering miR-146a-5p [69]. Recent studies demonstrated that EVs from young human donors are enriched with GST or NAMPT and can ameliorate age-related tissue damage by enhancing antioxidant capacity [70,71]. These findings highlight the potential of stem cell-derived EVs in IS therapy by restoring antioxidant balance and counteracting oxidative stress.

3.3. Inflammation and Ischemia/Reperfusion (I/R) Injury

As IS progresses, excitotoxicity and excessive ROS/RNS generation occurs from the center of the infarction (ischemic core). This process escalates with reperfusion, leading to brain cell death and the release of various danger-associated molecular patterns (DAMPs) such as ATP, heat shock protein (HSP), and high mobility group box 1 (HMGB1) [72]. Microglia and astrocytes are subsequently activated by DAMPs, oxidative stress, and other inflammatory signals. Along with other brain cells such as neurons and endothelial cells, the activated microglia and astrocytes release pro-inflammatory cytokines (e.g., TNF-α, IL-1β, and IL-6), chemokines (e.g., MCP-1 and MIP-1a), matrix metalloproteinases (MMPs), ROS, and NO, which collectively induce inflammation (Figure 1C) [72,73,74]. These released molecules stimulate the expression of cell adhesion molecules (e.g., ICAM-1 and E/P-selectin) in endothelial cells, initiating infiltration of peripheral immune cells, including leukocytes, monocytes, and lymphocytes [75]. Neutrophils, the first leukocytes to infiltrate, release large amounts of pro-inflammatory mediators and neutrophil extracellular traps, which further exacerbate brain injury by intensifying inflammation, releasing ROS/NO, and disrupting the BBB [76]. For instance, MMP-9 contributes to BBB disruption by degrading endothelial cell tight junction proteins (e.g., claudin-5) and cerebrovascular basal lamina proteins (e.g., collagen-4), resulting in detrimental effects including brain edema and hemorrhagic transformation [77]. The complement cascade also plays a role in this process; for example, C3a, an anaphylatoxin, and the C1 protein complex exacerbate I/R injury by promoting leukocyte infiltration and activating endothelial cells [78,79].

The inflammatory reactions in IS are mediated by various signaling pathways. Members of the mitogen-activated protein kinase (MAPK) family, including extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal kinase (JNK), and p38, as well as the NF-κB subunits p65/RelA and p50, are activated in response to external signals such as DAMPs, ROS, and inflammatory cytokines, leading to upregulation of pro-inflammatory mediators that exacerbate IS pathology [80,81,82,83,84] (Figure 1C). Additionally, Toll-like receptors (TLRs), which serve as upstream regulators of MAPKs and NF-κB, induce inflammation by responding to DAMPs [85].

Recent findings suggest that EVs secreted by various cell types may target inflammatory pathways and offer therapeutic benefits in IS. For example, MSC-derived EVs reduced inflammation in a rodent model of transient middle cerebral artery occlusion (tMCAO), potentially by reversing CysLT2R-ERK1/2–mediated M1 polarization of microglia or by inhibiting immune cell infiltration from the blood [86,87]. Furthermore, astrocyte-derived EVs carrying miR-34c alleviated I/R injury in in vitro models of oxygen-glucose deprivation/reperfusion and in vivo tMCAO models by downregulating TLR7, NF-κB, and MAPK pathways [88] (Figure 1C). Anti-inflammatory effects can also be achieved by activating regulatory T cells that secrete anti-inflammatory cytokines such as IL-10 and TGF-β. Embryonic stem cells also release EVs containing TGF-β, Smad2, and Smad4 and were shown to reduce peripheral immune cell infiltration and neuroinflammation by promoting regulatory T cell expansion [89].

It is important to note that certain molecules and cells that promote inflammation and I/R injury might paradoxically play beneficial roles in IS under specific conditions. For example, ERK1/2 activated by brain-derived neurotrophic factor (BDNF) was shown to inhibit apoptosis by reducing caspase-3 activity in hypoxic-ischemic brain injury [90]. Additionally, MMP-9 and proliferating microglia were shown to contribute to neurovascular remodeling during the later stages of cerebral ischemia [91,92]. Therefore, the therapeutic application of EVs harboring immunomodulatory agents in IS requires careful consideration of the target signaling molecules and the time window for treatments.

3.4. Ischemic Brain Cell Death

Various molecular mechanisms contribute to ischemic brain cell death (Figure 2) [93]. One major pathway involves either activation of the Ca2+-dependent protease calpain [94,95] or induction of the mitochondrial permeability transition due to Ca2+ overload and oxidative stress [96,97]. These processes activate the intrinsic apoptosis pathway by causing the release of cytochrome C or apoptosis inducing factor (AIF) from mitochondria. Additionally, signaling molecules such as death-associated protein kinase 1 (DAPK1), JNKs, p38, and Notch, which are activated during IS, can induce p53 activation [98]. Once activated, p53 promotes the transcription of proapoptotic genes such as PUMA and NOXA, which can directly interact with Bcl-xL to permeabilize the outer mitochondrial membrane, ultimately triggering mitochondrial apoptosis [99,100,101].

Extracellular factors released during inflammatory responses, including TNF-α [102], Fas ligand (FasL) [103,104], and TNF-related apoptosis-inducing ligand (TRAIL) [104,105], can induce caspase-8 activation through ligand-receptor interactions, initiating both the extrinsic and the mitochondrial apoptosis pathways [106,107] (Figure 2). However, caspase activity may also be reduced under IS conditions [108,109]. In such cases, external signals, particularly TNF-α, prompt formation of the necrosome complex, which consists of receptor-interacting protein kinase (RIPK) 1, RIPK3, and mixed lineage kinase domain-like pseudokinase (MLKL), and induces necroptosis [109,110,111].

Under the acidic conditions of the ischemic brain, the intracellular free iron level increases as a result of ferritinophagy and iron dissociation from transferrin [112,113]. The accumulation of free iron, combined with ROS, enhances lipoxygenase-mediated lipid peroxidation, resulting in the formation of lipid hydroperoxides that trigger ferroptosis [114]. Additionally, excessive intracellular Ca2+ activates cytosolic PLA2, promoting the production of arachidonic acid (AA) [115] (Figure 2). The AA is then esterified into phosphatidylethanolamines by acyl-CoA synthetase long-chain family member 4 (ACSL4), further contributing to ferroptosis [116]. Conversely, GPx-4 utilizes glutathione (GSH) as a substrate to reduce lipid hydroperoxides, thereby inhibiting an iron-dependent cell death ferroptosis [117]. Excessive extracellular glutamate in IS inhibits the cystine/glutamate antiporter (System Xc-), impairing the uptake of cystine, an essential precursor of GSH, thereby reducing GPx-4 activity and increasing susceptibility to ferroptosis [118] (Figure 2). Moreover, DNA damage caused by excitotoxicity and ROS/RNS activates poly(ADP-ribose) polymerase 1 (PARP1), initiating a series of events including cytoplasmic translo-cation of poly(ADP-ribose), AIF release from mitochondria, AIF/macrophage migration inhibitory factor (MIF) translocation, and DNA cleavage, ultimately resulting in the induction of a cell death pathway known as parthanatos [119] (Figure 2). Cell death can also be mediated by the inflammasome, a multi-protein complex composed of a single type of sensor protein, such as NLRP or AIM2, along with the adaptor ASC and pro-caspase-1. This complex activates caspase-1 and eventually induces pyroptosis, a type of inflammatory cell death [120,121]. Alternatively, exposure of phosphatidylserine (PS) on the outer leaflet of the plasma membrane and expression of proteins that mediate PS recognition (e.g., MerTK and MFG-e8) can trigger phagoptosis, the cell death induced by phagocytosis [122].

Although controversial findings exist, excessive autophagy can exacerbate IS by promoting neuron death [123]. In IS, inhibition of the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) pathway [124] and activation of ERK1/2 [125] both trigger autophagy-related cell death by inhibiting mammalian target of rapamycin complex 1 (mTORC1) activation (Figure 2). Additionally, activation of AMP-activated protein kinase (AMPK), either due to a high AMP/ATP ratio [126,127] or by calmodulin-dependent protein kinase kinase β (CaMKKβ) [128], causes autophagy-induced cell death by inhibiting mTORC1. Hypoxia-inducible factor 1 (HIF-1) is also activated in response to hypoxia, leading to upregulation of p53 and Bcl-2/adenovirus E1B 19-kDa-interacting protein 3 (BNIP3) [129,130], which contributes to excessive autophagy by promoting the release of beclin-1 from the Bcl-2/beclin-1 complex [131] or by inhibiting mTORC1 activation through its binding to Ras homolog enriched in brain (Rheb) [132] (Figure 2). In addition, p53 enhances expression of the damage-regulated autophagy modulator (DRAM) [133], potentially leading to excessive autophagy. Furthermore, the forkhead box O (FOXO) family proteins, which are regulated by Sirt1, AKT, and signal inducer and activator of transcription 3 (STAT3), can worsen IS outcomes by increasing the expression of autophagy-related proteins such as ATG7 [134,135,136,137,138]. This dysregulated autophagy can further exacerbate neuronal impairments in IS.

Recent studies suggest that the various forms of cell death observed in IS can be mitigated by EVs. MSC-derived EVs were shown to alleviate both extrinsic and intrinsic apoptosis [139,140]. Specifically, miR-134 delivered by exosomes inhibited apoptotic cell death of oligodendrocytes by targeting caspase-8 [140] (Figure 2). In addition, circBBS2 and miR-760-3p enriched in MSC-EVs targeted miR-494 and glutathione-specific gamma-glutamylcyclotransferase 1, respectively, thereby inhibiting ferroptosis by increasing System Xc- activity and boosting GSH levels [141,142] (Figure 2). Furthermore, EVs derived from bone marrow MSCs were shown to inhibit pyroptosis by reducing the expression of NLRP3, ASC, gasdermin D, and mature IL-1β [143]. Neuronal EVs containing miR-98 were also reported to inhibit phagoptosis by reducing the expression of platelet-activating factor receptor (PAFR) [144]. Moreover, EVs from AD-MSCs and induced pluripotent stem cell-derived MSCs were shown to inhibit autophagy-associated cell death by modulating p53–BNIP3 signaling and STAT3 expression, respectively [24,145] (Figure 2). Although direct studies in IS models are limited, inhibition of RIPK1/3 and PARP1 expression by MSC-derived EVs in other injury models suggests that these EVs hold potential for inhibiting necroptosis and parthanatos [146,147,148].

4. EV Engineering Methods

Advances in understanding the molecular mechanisms underlying IS pathophysiology have led to the development of strategies to target these mechanisms using bioactive molecules. The low permeability of the BBB poses a significant obstacle to non-invasive drug delivery for IS treatment; however, EVs exhibit a range of beneficial effects in IS-related pathologies and can cross the BBB, maintain circulation stability, and protect internal cargos with their lipid bilayer. These advantages, along with the therapeutic efficacy of EVs in IS, can be enhanced by modifications that enable EVs to precisely regulate signaling pathways in specific target cells (Table 2) [149].

Generally, EV engineering methods can be categorized depending on whether modifications are made before or after EV isolation (Figure 3). Pre-isolation modifications involve various pretreatments or gene transfection of EV-producing cells, whereas post-isolation modifications involve passive methods, such as co-incubation with desired target molecules, as well as active methods that use physicochemical stimulation to enable loading of exogenous factors [31].

4.1. Pre-Isolation Modification: Pretreatment and Gene Transfection

Various pretreatments of EV-producing cells can enhance the efficacy of EVs for IS treatment by altering the composition of the EV cargo. In vitro treatments with cytokines [150,151], metallic compounds [152], magnetic nanoparticles [153], drugs [154], and therapeutic biomolecules [155] under normal or hypoxic culture conditions [156] have been shown to modulate the activity or polarization state of EV-producing cells, including NSCs, MSCs, and macrophages (Figure 3A). These methods either facilitate the loading of extrinsic therapeutic agents into EVs or upregulate the intrinsic levels of neurotrophic, angiogenic, and anti-inflammatory cytokines or cell survival-related miRNAs within the EVs. These modifications potentiate the ability of EVs to inhibit ischemic brain damage while promoting tissue regeneration and neurological recovery.

Enrichment of TGF-β, miR-124, and miR-133a in EVs, achieved by hypoxic conditioning or inflammatory cytokine treatment of EV-producing cells, has been shown to reduce neural cell death in tMCAO animal models [150,151,154]. In addition, systemic administration of EVs derived from magnetic nanoparticle-treated MSCs improved the efficiency of EV delivery to IS lesions in a rat MCAO model [153]. Furthermore, exosomes from melatonin-treated rat plasma inhibited microglial and neuronal pyroptosis, partially by downregulating the TLR4/NF-κB pathway [157]. Additionally, mice that underwent moderate exercise before MCAO surgery exhibited increased levels of miR-126 in EVs obtained from circulating endothelial progenitor cells, which enhanced neurogenesis and angiogenesis by promoting BDNF secretion and PI3K/AKT signaling activation [158]. Exercise also elevated miR-484 levels in skeletal muscle-derived EVs, which targeted ACSL4 and thereby inhibited neuronal ferroptosis in a rat model of I/R injury [159].

Gene transfection of EV-producing cells is another commonly used method to modify the contents of EVs. Overexpression to increase the cytosolic levels of therapeutic proteins or RNAs in EV-producing cells can lead to enrichment of these molecules within EVs [160,161,162,163] (Figure 3A). Alternatively, specific proteins can be selectively loaded into EVs by creating EV-related fusion proteins [164], using the protein loading platform known as the exosomes for protein loading via optically reversible protein-protein interaction(EXPLORs) [165], or employing VSV-G and split GFP complementation [166]. In addition, specific RNAs can be loaded into EVs by incorporating specific motif sequences (e.g. hnRNPA2B1 binding motif [167] and Zip code-like sequence [168]) in the RNAs or by using the Targeted and Modular EV Loading (TAMEL) platform [169].

4.2. Post-Isolation Modification: Passive and Active Methods

One of the simplest methods for engineering EVs is to incubate isolated EVs with molecules that enhance the therapeutic potential of the EVs. Molecules conjugated to hydrophobic lipid derivatives, such as cholesterol and phospholipid-polyethylene glycol, can integrate into the EV membrane during simple incubations [170,171] (Figure 3B). Non-hydrophobic molecules that have an affinity for EV surface molecules can also be attached to the periphery of EVs by incubation. Additionally, physical stimulation allows incorporation of non-hydrophobic molecules in the luminal space of EVs by creating micropores in the EV membrane. Sonication or electroporation of a mixture of EVs and therapeutic molecules is the most common method for actively loading desired cargos into EVs via micropores. Repeated freeze-thaw cycles provide another physical way to actively load molecules into EVs. Cargos can also be loaded into EVs by extrusion, dialysis, or permeabilization with surfactants (e.g., saponin, Triton X-100, or Tween-80) [172,173,174,175]. Recent studies demonstrated that saponin-mediated cargo loading achieved an 11-fold higher efficiency for loading hydrophilic porphyrins compared with passive loading [174]. Furthermore, EVs that were actively loaded with catalase using saponin achieved higher neuron survival rates and more effective ROS removal compared with passively loaded EVs [173].

The surface of EVs can be chemically modified by carbodiimide coupling or copper-catalyzed azide-alkyne cycloaddition (click chemistry). Compounds with carbodiimide functional groups, such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, act as crosslinkers by catalyzing linkages between carboxyl and amino groups. Using this chemical method, EV surfaces can be conjugated with antibodies that bind to specific molecules highly expressed in target cells or tissues under pathological conditions (Figure 3B). Additionally, carbodiimide chemistry was used to introduce alkyne groups onto EV surface proteins, which were then conjugated with azide-fluor 545 by click chemistry for fluorescent labeling of the EVs [176].

4.3. EV Engineering for IS Treatment

The therapeutic potential of EVs in IS can be enhanced by modifying the luminal or surface cargo molecules in EVs before or after their isolation using several approaches (Figure 3C). Recent studies demonstrated that increasing the levels of therapeutic proteins, mRNAs, miRNAs, and circRNAs within EVs can enhance the therapeutic efficacy of the EVs for treating IS. For example, induction of BDNF overexpression in MSCs by lentiviral transfection led to BDNF enrichment in MSC-derived EVs, which reduced apoptosis and inflammation and promoted behavioral recovery and neural repair by activating BDNF–tropomyosin receptor kinase B (TrkB) signaling [160] (Figure 3C). Furthermore, EVs derived from MSCs induced to overexpress the miR-17-92 cluster by electroporation and lentiviral infection were shown to increase neuronal plasticity, promote neurogenesis and oligodendrogenesis, and improve behavioral outcomes. This effect was achieved by remodeling the cortico-spinal tract and enhancing neuronal innervation via PTEN downregulation and PI3K/AKT/mTOR signaling activation [161,162]. In another study, EVs derived from circSCMH1-overexpressing HEK293T cells enhanced neuronal plasticity, reduced glial activation, and decreased peripheral immune cell infiltration in a mouse model of IS. These therapeutic benefits were mediated by direct binding between circSCMH1 and methyl CpG binding protein 2 (MeCP2), which inhibited the nuclear localization of MeCP2 [163]. Notably, circSCMH1-containing EVs also facilitated functional recovery in a nonhuman primate model of stroke [163].

Pre-incubation of MSC-derived EVs with cholesterol-conjugated miR-210 promoted vascular endothelial growth factor (VEGF) expression and angiogenesis after administration into a mouse MCAO model [170]. Additionally, bone marrow stromal cell-derived EVs that were loaded with the hydrophobic anti-inflammatory agent curcumin caused significant reduction of pro-inflammatory cytokine secretion and apoptosis after intravenous administration in a mouse model of IS [177]. Strong free radical scavengers quercetin and baicalin, which were incorporated into EVs by creating micropores using sonication, enhanced the innate potential of the EVs to inhibit ROS generation through the Nrf2/heme oxygenase pathway [172,178]. Additionally, EVs loaded with miR-124 or HMGB1 siRNA by electroporation enhanced neurogenesis and reduced TNF-α expression and apoptosis, respectively [179,180]. Furthermore, EVs that encapsulated the antioxidant drug edaravone by sonication improved the safety and bioavailability of the drug, thereby intensifying its neuroprotective effect [181]. Mouse embryonic stem cell-derived EVs enriched with curcumin via two or three rapid freeze-thaw cycles enhanced abilities to reduce inflammation, glial activation, and loss of vascular integrity in a mouse model of IS [182].

The expression of specific peptides on the EV surface can enhance the targeted uptake of EVs by ischemic brain lesions. For instance, the ischemic brain can be targeted by fusing the Lamp2b EV membrane protein with the rabies viral glycoprotein (RVG) peptide, which specifically binds to acetylcholine receptors [163] (Figure 3C). Lamp2b can also be fused with a peptide (RBP) that binds to the receptor for advanced glycation end products (RAGE), which is highly expressed in hypoxic cells of the ischemic brain, providing the additional benefit of potentially alleviating DAMP-induced inflammation by directly blocking RAGE [183]. Additionally, a fusion protein consisting of the PS-binding domain (C1C2) of MFG-e8 and the Arg-Gly-Asp (RGD)-4C peptide (ACDCRGDCFC) was attached to EVs by incubation, facilitating targeted delivery of the EVs to ischemic brain [184]. During the incubation, the C1C2-RGD fusion protein bound to PS, which is abundant on the EV surface. After systemic injection, the interaction between the RGD peptide on EVs and integrin αvβ3, which is highly expressed on reactive cerebral vascular endothelial cells in brain ischemia, enhanced the EV targeting efficiency. Sustained EV delivery was also achieved by directly mixing embryonic NSC-derived EVs with a glucose/ROS dual-responsive hydrogel [185]. Transplantation of this mixture into the cortex of infarcted brain hemispheres enhanced angiogenesis and improved neurobehavioral recovery. Carbodiimide coupling can be used to conjugate EV surface proteins with an antibody targeting growth-associated protein-43 (GAP43), which shows increased neuronal expression in a rat model of IS [172] (Figure 3C). This modification effectively enhanced targeted delivery of the EVs to ischemic brain tissues. Furthermore, EVs treated with dibenzocyclooctyne-sulfo-N-hydroxysuccinimidyl ester (DBCO-sulfo-NHS) can be reacted with the azide-containing cyclo(Arg-Gly-Asp-D-Tyr-Lys) peptide [c(RGDyK)], which has a high affinity for integrin αvβ3 [177]. This copper-free click chemistry method covalently attached c(RGDyK) to the EV surface, thereby facilitating targeted delivery of EVs to the lesion area in the ischemic brain.

5. Discussion

EVs have emerged as important messengers of intercellular crosstalk and gained attention for their crucial roles in diverse physiological and pathological processes across many organs. Numerous studies have explored the application of EVs for the treatment of various diseases. EV-based therapies offer several advantages over traditional cell therapies by addressing issues such as low cell viability, thromboembolism following intravenous injection, and vascular occlusion due to cell entrapment [186]. EVs also provide benefits of low toxicity, high stability in circulation, scalable production and storage, ability to cross the BBB, and enhanced targeting efficiency [186]. Furthermore, recent findings have demonstrated that administration of EVs in IS can achieve therapeutic outcomes comparable to those of direct cell injections [187]. Engineered EVs in particular have shown improved efficiency in targeting ischemic brain lesions, resulting in reduced inflammation and cell death and enhanced neurogenesis and angiogenesis compared with naïve EVs, leading to safer and more effective functional recovery.

Another promising application of EVs is the rapid diagnosis of IS. Patients with IS often experience slow disease progression due to high collateral blood flow capacity and overall cerebral ischemic tolerance [188]. Alteplase, the only drug approved by the USFDA for IS treatment, is a recombinant tPA that is most effective when administered early, ideally within 4¬–5 hours of IS onset, highlighting the importance of rapid diagnosis [6]. EVs derived from blood or cerebrospinal fluid of patients with IS or animal models show altered levels of non-coding RNAs and proteins, offering potential diagnostic markers [189]. For example, circulating EVs from patients with IS and tMCAO rat models exhibit increased levels of miR-20b-5p and miR-93-5p [190]. Inflammatory proteins such as C-reactive protein (CRP) are also elevated in EVs derived from the serum of patients with acute IS [191]. Furthermore, 67 miRNAs in blood-circulating EVs were found to differ significantly between the ischemic and hemorrhagic subtypes of stroke [192]. This suggests that EVs may help diagnose disease progression and distinguish between major stroke subtypes, enabling more rapid and effective treatments.

The limited blood flow in ischemic regions can be restored by tPA-induced clot lysis; however, tPA may also promote neutrophil degranulation and MMP-9 release, potentially increasing the risk of hemorrhagic transformation [193]. Therefore, several clinical studies are currently investigating EVs as promising alternative treatments for IS (NCT06138210, NCT03384433, and NCT05326724) [194]. A phase 1 clinical trial (NCT06138210) conducted by Xuanwu Hospital is assessing the safety and preliminary efficacy of intravenous administration of exosomes derived from human induced pluripotent stem cells (GD-iExo-003) in patients with acute IS. Another phase 1/2 trial (NCT03384433) by Isfahan University of Medical Sciences is investigating the therapeutic effects of allogenic MSC-derived exosomes transfected with miR-124 in patients with acute IS. A pilot study involving five participants with IS who received allogenic placental MSC-derived exosomes showed no adverse effects [195].

The therapeutic application of EVs is still in early stages, and several challenges must be addressed before EVs can be used as therapeutic agents for IS [186,196]. One critical challenge is the reproducible production and quality control of EVs. The characteristics and purity of EVs can vary depending on their source, the environment of the EV-producing cells, and the techniques used for EV isolation and storage. Therefore, it is essential to establish precise and strictly monitored conditions for EV harvesting, along with the use of appropriate markers and analytical methods for quality control. Additionally, a detailed understanding of the in vivo biological activity of administered EVs is needed. Because of the diversity of EV cargos, EV application can affect not only the ischemic brain environment but also the overall physiological conditions of the body. Furthermore, different modes of action may be required depending on whether IS is in the acute or chronic phase. Therefore, therapeutic parameters including administration routes, biodistribution profiles, comprehensive biological functions of EV cargos, optimal treatment timing and dosages, and cytotoxicity profiles must be thoroughly studied to advance the application of EVs as therapeutic agents for IS.