80 years of extracellular vesicles: from discovery to clinical translation

Lipid components of EVs

The lipid bilayer of EVs exhibits a composition distinct from the plasma membrane of their parent cells, being notably enriched in cholesterol, sphingomyelin, glycosphingolipids (GSLs), and phosphatidylserine^[106]. Cholesterol contributes to the increased rigidity and structural stability of the EV membrane^[107]. Sphingomyelin and GSLs, including gangliosides, further stabilize membrane structure and participate in cell recognition and signal transduction processes^[108]. These lipids contribute to the formation of microdomains within the EV membrane, which is essential for the docking and fusion processes during EV release and cellular uptake^[109]. Beyond structural lipids, EVs carry bioactive lipids capable of modulating various signaling pathways^[110]. For example, ceramides are directly involved in ESCRT-independent exosome biogenesis^[61], whereas exosomal leukotrienes can influence inflammatory responses of recipient cells. In addition, cancer-derived exosomes often exhibit altered lipid compositions and can transfer bioactive lipids into recipient cells, modulating tumorigenesis and progression^[110,111].

GSLs, a major subclass of glycolipids, are ubiquitous membrane components found in nearly all living organisms and are commonly found on EVs^[112]. Gangliosides - a family of sialic acid-containing GSLs including monosialotetrahexosylganglioside (GM1), its precursor GM2, GM3, and disialodihexosylganglioside (GD3) - are prominently present in exosomes. Several gangliosides such as GM1 on EVs have been shown to regulate pathological processes involved in neurodegenerative disorders and cancers^[113,114].

Protein components of EVs

The EV membrane contains various membrane proteins. Integrins, for example, are present on EV surfaces and play a key role in directing EVs to specific recipient cells^[115]. Major histocompatibility complex (MHC) molecules on the membrane of DC-derived EVs can activate T cells, underscoring the involvement of EVs in immune responses^[116]. Additionally, tetraspanins, such as CD9, CD63, and CD81, are among the most abundant membrane proteins and are commonly used as markers for EV isolation and characterization. These proteins help organize membrane microdomains, facilitating the assembly of protein complexes essential for EV biogenesis and release. Other highly abundant membrane proteins in EVs, such as ESCRT proteins [e.g., Alix and tumor susceptibility gene 101 (TSG101)], have been reported to facilitate the inward budding of endosomal membranes during ILV formation^[3,117].

EVs carry a diverse array of bioactive proteins that influence recipient cell behavior. Cytosolic proteins such as HSP70 and HSP90, which mediate protein folding and stress response, are frequently observed in EVs^[118,119]. Some metabolic enzymes in EVs, such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and pyruvate kinase M2 (PKM2), can regulate metabolic reprogramming and cancer progression. Moreover, EVs can also transfer drug resistance-related proteins, including DNAJB8 [DnaJ Heat Shock Protein Family (Hsp40) Member B8]^[120], TrpC5 (human transient receptor potential canonical 5)^[121], RAB22A (RAB22A, Member RAS Oncogene Family)^[122], and programmed death-ligand 1 (PD-L1)^[123], thereby contributing to therapeutic resistance in recipient cells. EVs from MSCs (MSC-EVs) are notably enriched with proteins that promote tissue repair and regeneration. A comprehensive proteomic analysis by Anderson et al. identified 1,927 proteins in MSC-derived exosomes, including growth factors such as platelet-derived growth factor (PDGF), epidermal growth factor (EGF), and fibroblast growth factor (FGF), and components of the nuclear factor kappa B (NF-κB) signaling pathway^[124].

Nucleic acids in EVs

EVs contain both single-stranded and double-stranded DNA (ssDNA and dsDNA) with various sizes^[125]. However, the mechanisms underlying the sorting of DNA into exosomes are not yet fully understood. This process may involve direct budding of nuclear or mitochondrial fragments into MVBs or the packaging of DNA through interactions with DNA-binding proteins and lipids^[126]. EVs carrying mutant DNA sequences of KRAS (Kirsten rat sarcoma virus) and tumor protein p53 gene (TP53) can drive the malignant transformation of recipient cells^[127]. Furthermore, viral DNA, such as herpes simplex virus and hepatitis B virus (HBV), could be encapsulated into the EVs of infected cells and be delivered to uninfected cells, aiding in the propagation of the infection^[128]. A recent study proposes a new autophagy- and MVB-dependent, but exosome-independent, model for active secretion of extracellular DNA^[129]. Compelling evidence from other studies, however, indicates that DNA is frequently detected in EV preparations and can be functionally significant^[130,131]. This apparent contradiction may be reconciled by considering the heterogeneity of EV populations and diverse biogenesis pathways. For instance, autophagy-dependent secretory mechanisms (a process termed “EV secretion via autophagy”) have been shown to actively package cytoplasmic components, including fragmented nuclear or mitochondrial DNA, into double-membrane vesicles that are released extracellularly^[68]. Similarly, distinct vesicle subtypes such as large oncosomes (shed from cancer cells) or arrestin domain containing 1 (ARRDC1)-mediated microvesicles (ARMMs) are also strongly implicated in the active sequestration and release of genomic and double-stranded DNA^[132]. Consequently, the debate often centers not on the mere presence of DNA in EV preparations, but on its precise localization (intraluminal versus surface-adsorbed) and its biogenetic origin.

A wide variety of RNAs have been identified in EVs. In 2006, Ratajczak et al. revealed for the first time that MVs carried mRNAs that could be absorbed by recipient cells and translated into proteins^[33]. In 2007, Valadi et al. reported that exosomes contain mRNA and miRNAs and can transfer them to recipient cells, thereby regulating their phenotypes^[14]. Since then, an increasing number of RNA species have been observed in EVs. EVs are particularly rich in miRNAs, a type of small non-coding RNA (ncRNA) that regulates gene expression post-transcriptionally. For example, exosomal miR-21 derived from cancer cells promotes tumor progression by targeting multiple target genes in different recipient cells^[133,134]. In addition to miRNAs, EVs also contain other ncRNAs such as lncRNAs, circRNAs and small nucleolar RNAs (snoRNAs)^[42,44,135]. Glioblastoma-derived EVs transport tumor-specific mRNAs such as EGF receptor variant III (EGFRvIII) to promote angiogenesis and serve as diagnostic biomarkers^[136]. Conigliaro et al. revealed that CD90⁺ liver cancer cells regulate endothelial cell (EC) phenotype via exosomal H19^[137]. Moreover, EV RNAs show promise as biomarkers. Li et al. revealed that circRNAs are enriched in EVs and appear to be promising cancer diagnostic biomarkers^[42]. Exosomal miRNAs, snoRNAs, and mRNA are also emerging as potential functional entities and promising candidate biomarkers^[135].

Mechanisms of EV absorption

EVs can be internalized by recipient cells via membrane fusion, a process mediated by several protein families, including SNARE proteins, Rab GTPases, and Sec1/Munc-18-related proteins (SM proteins)^[138]. The primary mechanism for the uptake of EVs by cells is endocytosis, a multifaceted process encompassing macropinocytosis, phagocytosis, clathrin-mediated, caveolin-dependent, lipid raft-dependent, and clathrin/caveolin-independent endocytosis^[139]. During this process, the contents of EVs must be released from the vesicle compartment into the cytoplasm; otherwise, they are transported to lysosomes for degradation. For instance, the cellular uptake of EVs derived from PC12 cells by other cells is regulated by clathrin-mediated endocytosis and macropinocytosis^[140]. The extracellular signal-regulated kinase 1/2 (ERK1/2)-heat shock protein 27 (Hsp27) signaling pathway also mediates EV uptake, which is negatively regulated by Caveolin-1^[141].

Targeting mechanism of EVs depends on the interaction between EV surface proteins, lipids, or other components and receptors or ligands on target cells. This process is influenced by various external factors, enabling the specific targeting of EVs to recipient cells^[142,143]. Protein-mediated targeting mechanisms are complex and diverse. Integrins, tetraspanins, and immune checkpoint molecules are among the most studied examples^[142]. Tetraspanins (such as CD9, CD81, CD151) are highly expressed on the surface of EVs, and promote EV targeting and uptake through interactions with specific proteins on the surface of recipient cells. Immune checkpoint molecule-mediated targeting is a recently emerging area of research. PD-L1 on the surface of EVs binds to programmed death-1 (PD-1) on T cells, inhibiting T cell activity and immune response^[144]. Lipid-mediated targeting is primarily executed by lipid rafts. Lipid rafts form stable microdomains on the exosome membrane, enhancing exosome targeting by interacting with lipid rafts or receptors on the target cell membrane. Additionally, the lipid molecule phosphatidylserine can bind to specific proteins (such as Tim4 and Tim1) on the surface of target cells, mediating exosome targeting^[145]. Furthermore, collagen, fibronectin, and laminin in the extracellular matrix can interact with integrins and other binding proteins on the surface of EVs, regulating their distribution and targeting specificity^[146,147].

The uptake efficiency of EVs is influenced by various factors, including surface proteins and carbohydrates, pH levels, temperature, as well as EV size and concentration. For example, proteins, including integrins, tetraspanins, and MHC molecules, as well as carbohydrates present on the surface of EVs or recipient cells, can affect the binding efficiency of EVs to recipient cells^[148]. Under low pH conditions, EVs exhibit increased rigidity and a higher sphingomyelin-to-ganglioside GM3 content, which facilitates the internalization of EVs into cells^[149]. In contrast, low temperatures can reduce the activity of EV surface proteins, leading to a decrease in EV uptake^[150]. Hypoxic conditions can regulate the generation of EVs and the expression of surface molecules, thereby affecting their targeting and uptake^[151].

Direct EV labeling

Agents that can release different signals are loaded onto EVs directly via biological or physical approaches, making this strategy widely used for in vivo EV imaging. Some lipophilic molecules, including the commercial PKH series (PKH26 and PKH67), carbocyanine dyes [DiO (3,3’-dioctadecyloxacarbocyanine perchlorate), DiI (1,1’-dioctadecyl-3,3,3’,3’-tetramethylindocarbocyanine perchlorate), and DiR (1,1’-dioctadecyl-3,3,3’,3’-tetramethylindotricarbocyanine iodide)], and other lipid-conjugated fluorescent probes [e.g., cholesterol- or DSPE (1,2-distearoyl-sn-glycero-3-phosphoethanolamine)-linked dyes], can be attached to or fused with the EV membrane via direct incubation. For example, DSPE-PEG-Cy7 (DSPE-polyethylene glycol with conjugated cyanine 3) was loaded onto exosomes derived from urine samples of prostate cancer patients for tumor monitoring^[182]. Electroporation is another commonly used technique for loading contrast agents, especially for nanoparticles, into EVs^[183]. Although efficient, electroporation can cause unexpected membrane damage and cargo leakage, potentially compromising EV integrity. Alternative internalization strategies, such as membrane fusion or transporter-mediated endocytosis, have also been explored^[184]. For example, glucose-coated gold nanoparticles (GNPs, ~5 nm) were internalized into EVs via glucose transporter type 1 (GLUT-1)^[185]. The GNPs were further applied in the in vivo tracking of MSC-EVs, which displayed distinct migration patterns in different brain pathologies. This specific targeting and accumulation behavior endows MSC-EVs with great potential in diagnosis and evaluation of brain diseases^[186]. Overall, direct labeling is simple, low-cost, and efficient, making it a suitable and reliable approach for various EVs, especially those collected from bodily fluids with unknown cellular origins.

Indirect EV labeling via parent cells

Indirect labeling has been widely demonstrated to be an effective strategy, offering advantages in long-term stability and minimal off-target effects. By modifying or genetically engineering parent cells, signal components or functional groups can be subsequently incorporated into the secreted EVs. A more commonly used indirect labeling method involves co-expressing exosomal marker proteins with fluorescent proteins [such as green fluorescent protein (GFP), enhanced GFP (eGFP), and red fluorescent protein (RFP)] or bioluminescence reporters. CD63, the C1C2 domains of lactadherin, and palmitoylation sequences were frequently selected as targets for EV genetic engineering^[187]. For example, Hikita et al. employed a bioluminescence resonance energy transfer (BRET)-based reporter, Antares2, to monitor the homing behaviors of prostate cancer-derived exosomes, achieving long-term and deep-tissue in vivo imaging of EVs^[188]. However, the process of engineering recombinant proteins is often tedious and time-consuming, which may affect EV biogenesis and cellular recognition processes.

Alternatively, some chemically active groups can be introduced onto EV membrane by metabolic labeling. Phospholipids are regarded as ideal substrates, as they minimize functional interference compared to recombinant fusion proteins. Zhang et al. developed a phospholipid-based biorthogonal labeling for exosomes. An unnatural choline analogue bearing an azide was incubated with parent cells, incorporated into cell membrane, and subsequently transferred to the EV membranes. The azide groups then reacted with aza-dibenzocyclooctyne (DBCO)-conjugated fluorescent dyes, enabling stable imaging of EVs^[189]. A similar strategy has also been applied using azido-sugars, which are metabolically incorporated into exosomal glycans^[190]. This in situ bioorthogonal labeling strategy offers high-yield, operational simplicity, and minimal impact on EV function. In a comparative study evaluating direct and indirect labeling in an acute kidney injury mouse model, indirect imaging demonstrated superior specificity and longer signal retention^[191].

In vivo EV imaging techniques

Reported in vivo tracking techniques for EVs include optical imaging, nuclear imaging, tomographic imaging, and photoacoustic imaging. Optical imaging mainly consists of fluorescent imaging and bioluminescent imaging, which primarily differ in whether an external excitation module is required. Bioluminescent signals generated from the luciferase enzyme-substrate reaction offer higher sensitivity and lower background noise compared to fluorescent imaging. However, the need for substrate injection and the limited half-life of the substrate restrict the broader application of bioluminescent imaging^[192]. Optical imaging is relatively simple and low-cost, but it suffers from limited penetration depth and imaging resolution. Though imaging quality can be improved using long-wavelength light signals, achieving sufficient spatial resolution remains challenging compared to other imaging techniques. Nuclear imaging techniques, such as single-photon emission computed tomography (CT) (SPECT) and positron emission tomography (PET), are often combined with anatomical imaging via tomography to determine EV location. The deep tissue penetration of nuclear imaging enables it to be applicable to any organ. Nuclear agents are typically incorporated into EVs through direct labeling. For instance, ^99mTc- and ¹²⁵I-based EV imaging has been widely reported in preclinical studies^[193].

Magnetic resonance imaging (MRI) and CT are leading clinical imaging techniques and can also be applied to EV tracking with the assistance of metal nanoparticles such as GNPs^[194]. Photoacoustic imaging has emerged recently as a promising tool for EV tracking. Unlike conventional ultrasound imaging, photoacoustic imaging employs contrast agents with optical absorption properties, which can be loaded onto EVs via direct or indirect approaches. This approach combines the spectroscopic specificity of optical imaging with the high spatial resolution of ultrasound imaging^[194].

Taken together, the tissue penetration and homing properties of EVs make them effective tools for in vivo disease monitoring, particularly in inflammation, tumor, and neurological diseases. Notably, different labeling and imaging approaches have their own advantages and limitations, depending on the application scenarios. Optical imaging is highly suitable for localized observation in specific regions, while various tomographic imaging techniques are more suitable for whole-body monitoring.

Physical engineering approaches

Physical engineering approaches are commonly utilized to encapsulate therapeutic agents such as small-molecule drugs, nucleic acids, peptides, and proteins into EVs. These approaches may be categorized into passive loading and active loading. Passive loading typically includes EV-agent co-incubation of EVs with therapeutic agents or donor cells with carrier molecules, whereas active loading involves techniques such as electroporation, sonication, extrusion, freeze-thaw cycling, and other methods. Beyond drug encapsulation, physical engineering approaches are also exploited to functionalize the EV membrane. Strategies such as membrane fusion and membrane coating can enhance the targetability of EVs and reduce their immunogenicity, thereby facilitating their clinical application^[235].

Passive loading

The hydrophobic nature of the EV lipid bilayer allows hydrophobic drugs (e.g., curcumin, paclitaxel, and azithromycin) to passively diffuse into the vesicles along a concentration gradient^[236]. In addition, miRNAs can also be loaded into EVs through passive loading, enabling efficient delivery and targeted therapy^[237].

Donor-carrier cell co-incubation is another passive loading strategy. For instance, apoptotic cells can efficiently encapsulate chemotherapeutic drugs or nanoparticles into EVs^[238]. In addition, autologous malignant cells isolated from malignant pleural effusions can be incubated with methotrexate (MTX) to produce EVs that retain tumor-targeting and immunostimulatory capacities while delivering MTX for synergistic therapy^[239].

Active loading

Electroporation is currently the most widely used active loading method that applies short, high-voltage pulses to transiently permeabilize EV membranes, enabling cargo entry. Many small molecules (e.g., chemotherapeutic drugs, miRNA, siRNA, and luminescent substances)^[183,240] and macromolecules (e.g., DNA^[241] and proteins^[242] can be loaded into EVs via electroporation. However, electroporation may affect EV stability. To address this, microfluidic-assisted electroporation has been developed, which reduces voltage requirements and minimizes membrane damage^[243].

Sonication is another common membrane penetration strategy. Ultrasound-induced mechanical shear disrupts EV membranes, thereby enhancing cargo diffusion. This method is commonly used to load chemotherapeutic agents into EVs, and shows higher loading efficiency than electroporation^[244]. However, several drawbacks, including drug adhesion to EV surfaces and loss of EV structural integrity, limit its widespread application.

Extrusion involves pressing a mixture of EVs and liposomes (Doxil) through polycarbonate membranes (e.g., with pore sizes of 400, 200, or 100 nm) under physical pressure to form hybrid vesicles with more controlled and uniform sizes compared to incubation-based methods^[245]. Although extrusion demonstrates high fusion efficiency, the shear stress generated during the process may disrupt EV membrane integrity^[246].

Freeze-thaw cyclizing is a relatively simple physical process that disrupts membranes through ice crystal formation. This method removes water molecules from the hydrophilic surfaces of lipid bilayer membranes, leading to significant changes in membrane structure and function^[247]. However, this method can induce EV aggregation, leading to increased particle size.

Saponin treatment represents another strategy to alter EV membrane permeability. By selectively removing cholesterol from the lipid bilayer membranes, saponin induces pore formation, thereby facilitating the loading of bioactive molecules such as lipids^[248]. However, the potential cytotoxicity of saponin hinders its widespread applications, necessitating careful control of its concentration and thorough removal of residuals after loading.

Different loading methods result in varying efficiencies for the same cargo. Haney et al. evaluated the loading, storage, and release of peroxidase encapsulated into EVs using several methods, including incubation, ultrasound-induced sonication, extrusion, freeze-thaw cycling, and saponin permeabilization^[249]. Sonication and extrusion showed higher efficiency than simple incubation, whereas freeze-thaw cycling and saponin permeation offered moderate efficiency. The hydrophilicity of the loaded therapeutic agents is a key factor affecting the loading efficiency. Notably, sonication, extrusion, and freeze-thaw cycling tended to increase EV size, whereas co-incubation at room temperature did not significantly alter particle size. Overall, both the passive and active loading strategies described above have their own advantages and limitations. Therefore, the optimal technique should be selected based on the specific cargo and intended application to achieve efficient EV loading.

Chemical engineering approaches

Chemical engineering approaches are commonly exploited to display functional molecules or ligands on the surface of EVs. Based on the functionalization strategy, these approaches can be classified into two categories: covalent modifications, including click chemistry, bioconjugation and aldehyde amine condensation, and non-covalent modifications such as hydrophobic insertion, multivalent electrostatic interaction, and receptor-ligand binding.

Covalent modification

Click chemistry, bioconjugation, and aldehyde-amine condensation are three common modification strategies for covalently linking exogenous functional groups and bioactive molecules to EV membranes. Click chemistry is a copper-catalyzed azide-alkyne cycloaddition process^[250]. Typically, an alkyne group is first attached to EVs via EDC [N-(3-Dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride]-NHS (N-hydroxysuccinimide)-mediated condensation. Subsequently, under copper catalysis, the alkyne moiety reacts with azide-bearing functional molecules to form a stable covalent bond. Smyth et al. utilized the method to prepare Fluor-545-modified EVs without changing the size and function of EVs^[251]. To circumvent potential copper-induced cytotoxicity, copper-free click chemistry has also been developed^[252].

Bioconjugation enables the display of functional macromolecules on EV surfaces. For example, the peptide CP05 can bind specifically to the second extracellular loop of the EV marker CD63^[253]. Gao et al. separately conjugated the muscle-targeting peptide M12 and a Duchenne muscular dystrophy treatment drug PMO to CP05. Incubating EVs with the resulting CP05-M12 and CP05-PMO conjugates yielded dually functionalized EVs with enhanced targeting and therapeutic efficacy^[254]. Theoretically, CP05 can serve as a universal anchor for diverse cargoes, offering broad potential in EV isolation, imaging, and targeted therapy^[255].

Aldehyde-amine condensation reaction offers a direct means to modify EVs with functional nucleic acid aptamers^[256]. Due to the high affinity and specificity of aptamers, aptamer-conjugated EVs represent a promising platform for precision medicine.

Non-covalent modification

Hydrophobic insertion, electrostatic interactions, and receptor-ligand binding are the three most used non-covalent methods for EV surface engineering. EV membranes share a similar amphiphilic lipid bilayer structure with cell membranes, composed mainly of phospholipids, cholesterol, and glycolipids. The synthesized amphiphiles have hydrophilic heads and hydrophobic tails. Therefore, amphiphiles attached to different functional groups and materials can efficiently embed themselves into the cell membranes and EVs through hydrophobic interactions. In hydrophobic insertion, bioactive molecules are typically conjugated to hydrophobic anchors such as DSPE-PEG, palmitic acid, or cholesterol. The strong hydrophobic nature of these anchors facilitates their spontaneous integration into the EV lipid bilayer^[257].

Electrostatic interaction involves the mutual attraction of negative and positive charges. Since most EV surfaces are negatively charged, cationic polymers can readily associate with them through electrostatic interactions. For instance, simple mixing of EVs with cationized pullulan - a polysaccharide targeting hepatocyte asialoglycoprotein receptors - resulted in pullulan-modified EVs that showed enhanced liver accumulation and efficacy in treating liver injury^[258].

Receptor-ligand interaction generally involves modifying EVs with a protein receptor or ligand to enable specific binding to complementary molecules. For instance, Yang et al. used clusters of transferrin, which naturally associate with blood-borne EVs, to couple superparamagnetic nanoparticles for isolation purposes^[259]. Alternatively, non-natural receptor moieties can be engineered onto EVs. Weyant et al. developed a generalized strategy that fuses the biotin-binding proteins with outer membrane scaffolding proteins of bacterial OMVs through genetic engineering^[231].

Biological engineering approaches

Biological engineering is widely used to functionalize EV membranes with fusion proteins or targeting peptides. Owing to the rapid development of genetic engineering and synthetic biology techniques, gene sequences encoding desired proteins or peptides can be designed and fused with selected membrane protein genes. These rationally synthesized gene constructs can be transfected into mammalian, bacterial, or plant cells, leading to the production of EVs that display the functional proteins or peptides on their surface.

Beyond membrane engineering, biological engineering approaches are also utilized to overexpress bioactive proteins, peptides, or miRNAs inside parent cells, thereby enriching the resulting EVs with bioactive cargo^[260]. Furthermore, gene knockout techniques can be applied to eliminate specific genes, generating EVs with reduced toxicity or immunogenicity.

Targeted modification of EVs can be achieved by genetically engineering transmembrane proteins in parent cells. EV transmembrane proteins can be categorized into two categories: nonspecific proteins and receptor membrane proteins. Various nonspecific proteins have been identified, including Lamp2b and CD63, which are naturally enriched on EV membranes from parental cells^[261]. Nonetheless, these conventional scaffolds face limitations in the expression level. To circumvent the expression level limitation, researchers from Codiak BioSciences identified PTGFRN (prostaglandin F2 receptor negative regulator) as the naturally highly abundant surface protein of EVs, enabling its high-density surface display with over 30-fold greater efficiency^[262]. The PTGFRN-based EV bioengineering technique, the engEx^TM platform, facilitates the development of therapeutic candidates such as exoIL-12, the world’s first entry of engineered exosome into Phase I clinical trial^[263]. Besides, clustered regularly interspaced short palindromic repeats-CRISPR associated protein 9 (CRISPR-Cas9)-based knockout systems are available to remove specific effectors such as msbA, lpxL1, or lpxM in Gram-negative strains to produce OMVs with attenuated virulence. Overall, engineering of parental strains can generate EVs with low toxicity and immunogenicity, along with targeting and therapeutic capabilities, thereby improving therapeutic efficiency. Moreover, CRISPR-Cas9-based plant genome editing has been widely reported, offering a promising approach to produce engineered PDEs^[264,265]. However, the limited availability of genetically engineered plants models, plant cell wall barriers, generally low editing efficiencies, and insufficient functional annotation of plant genes make these approaches challenging at present^[266].

EV proteins

EV protein biomarkers primarily fall into two categories: those with aberrant expression levels and those with aberrant posttranslational modification (PTM) patterns, with most studies focusing on the former. Proteomic analyses have identified numerous EV proteins as promising cancer biomarkers across malignancies, including but not limited to prostate^[268], ovarian^[269], breast^[270], bladder^[271] and lung cancers^[272]. Notably, certain protein biomarkers demonstrate enhanced diagnostic or prognostic value when analyzed within EVs compared to their dissociated forms, such as prostate-specific antigen (PSA) for prostate cancer^[273], developmental endothelial locus-1 protein for breast cancer^[274], Eph receptor A2 (EphA2) for pancreatic cancer, and PD-L1 for monitoring responses to anticancer immunotherapy^[123]. Furthermore, specific tumor EV proteins, such as macrophage migration inhibitory factor and integrins (e.g., α6β4 and αvβ5), could be used to predict organ-specific metastasis^[275] [Table 3].

Beyond changes in expression levels, PTMs enhance the structural complexity and functional diversity of proteins via covalent modification by functional groups. Aberrant PTMs are frequently identified in disease-related EVs, such as glycosylation, acylation, and phosphorylation. Glycans attached to proteins with nitrogen or oxygen linkages are classified as N- or O-glycosylation. Glycosylation, which involves the attachment of glycans via nitrogen (N-glycosylation) or oxygen (O-glycosylation) linkages, participates throughout EV biogenesis^[283]. The typical glycoforms include sialic acids (α-2,3 and α-2,6 type), mannose, complex N-glycans, heparan sulfate and polylactosamine. Glycoproteins occupy the major parts of glycoconjugates on EVs, and aberrant glycosylation has been regarded as a disease biomarker. For example, elevated sialic acid levels on EV proteins promote immune inhibition in various cancers via the interaction between sialic acids and receptors (Siglec) on immune cells^[284]. Similarly, glycosylated CD133 ascites-derived exosomes emerge as a potential prognostic biomarker for pancreatic cancer^[285]. Other glycosylation types, such as mucin 1 (MUC1) with aberrant O-glycoforms and terminal fucosylation on haptoglobin, have been reported as potential cancer biomarkers or neoepitopes^[286,287].

Protein acylation is another frequent and essential type of PTMs, including acetylation, crotonylation, lactylation, palmitoylation, myristoylation, succinylation, β-hydroxybutyrylation, and malonylation^[288]. Minic et al. identified upregulated acetylation and specific acetylation patterns in breast cancer-derived sEVs, and revealed that acetylated glycolytic metabolic enzymes in sEVs could be potential biomarkers for the early diagnosis of breast cancer^[289]. Palmitoylation is widely reported in the exosome production including protein sorting and membrane organization^[290]. Mariscal et al. identified palmitoyl-protein ABCC4 (adenosine triphosphate-binding cassette sub-family C member 4), human six-transmembrane epithelial antigen of the prostate (STEAP)1, and STEAP2 in EVs as prostate cancer-specific biomarkers, and inhibition of palmitoylation decreased the sorting of these proteins into EVs^[291].

Phosphorylation is a major regulatory mechanism for protein functions. Advances in phosphoproteomic technologies have enabled extensive profiling of phosphoproteins in EVs. There is evidence that plasma-derived extracellular vesicles carry more than 100 phosphorylated proteins, whose abundances are generally higher in breast cancer patients than in healthy individuals. In addition, Soares Martins et al. identified 150 proteins with altered expression and/or phosphorylation patterns in blood-derived EVs from Alzheimer’s Disease (AD) patients using a high-density phosphoproteome microarray^[292].

EV RNAs

Accumulating evidence indicates that a spectrum of RNA species, including mRNAs, miRNAs, lncRNAs, tsRNAs, and circRNAs, are selectively enriched within EVs. Among these, EV-derived miRNAs have been extensively studied due to their relative abundance and stability, with several EV miRNA-based assays already translated to clinical use. In 2015, Li et al. reported, for the first time, that circRNAs are enriched in exosomes, and serum exosomal circRNAs were able to distinguish cancer patients from healthy controls, highlighting EV circRNAs as promising cancer biomarkers^[42]. They subsequently developed exoRBase database (www.exoRBase.org) that contains more than 160,000 EV long RNAs (termed exLRs), including mRNAs, lncRNAs, and circRNAs, derived from diverse human body fluids^[44-46]. Importantly, about one-third of these EV RNAs reflect their tissue or cell origin, establishing a foundation for EV long RNA biomarker research^[293]. In addition, tsRNAs in EVs also appear as promising biomarkers for various diseases^[294,295].

EV DNA

In 2014, Thakur revealed, for the first time, the presence of dsDNA in tumor-derived exosomes (exoDNA) that represents the whole genomic DNA and mutational status of parental cancer cells, proposing its translational potential as a circulating cancer biomarker^[125]. Subsequent studies have reported the functions of EV DNA, such as activating immune response through horizontal gene transfer and dsDNA recognition pathways^[296,297]. Interestingly, cells can remove harmful cytoplasmic DNA via exosome secretion to maintain cellular homeostasis^[298]. Wortzel et al. described a unique structural configuration of EV DNA, and revealed that uniquely chromatinized EV DNA can regulate anti-tumor immunity^[299]. These functional insights further support the biomarker potential of EV DNA.

However, a recent publication reported that sEVs are not primary vehicles of active DNA release and proposes an alternative model involving an autophagy- and MVB-dependent, but exosome-independent, mechanism for extracellular DNA secretion^[129]. Given the disparate DNA detection methodologies employed across these studies, the presence and biological relevance of genomic DNA within EVs remains ambiguous. Nonetheless, research on EV DNA has substantially advanced the field^[300].

EV metabolomics

Metabolites, as downstream products of biochemical reaction networks, provide phenotypic information that more directly reflects an individual’s physiological and pathological state than genomic or proteomic data, offering significant potential for assessing disease status. Nishida-Aoki et al. revealed a substantial elevation of exosomal diacylglycerol in highly metastatic triple-negative breast cancer cells compared with those with lower metastatic potential, suggesting its potential as a biomarker for breast cancer progression^[301]. Dorado et al. determined that the lipid composition of plasma EVs could efficiently differentiate breast cancer from healthy control with an accuracy of 93.1%^[302]. Skotland et al. reported that a specific combination of two phosphatidylserine molecules and one lactosylceramide in urinary exosomes could accurately discriminate prostate cancer^[303]. Notably, Chen et al. constructed a biomarker panel based on urinary exosomal metabolic features, which accurately discriminates early gastric cancer patients from healthy controls^[304]. In addition, Krokidis et al. identified plasma EV lipidomic profiling as a powerful biomarker discovery tool in AD and related neurodegenerative conditions^[305]. Collectively, these studies highlight the substantial potential of EV metabolites as circulating biomarkers.

Applications of EVs in cancer therapy

The incorporation of EVs in cancer therapy stands as an emerging and highly promising field. Current approaches to tumor treatment are diverse, including surgical resection, minimally invasive interventional therapy, chemotherapy, radiotherapy, targeted therapy, immunotherapy, and physical therapy^[306]. In clinical settings, patients with early-stage tumors show favorable prognoses after curative surgical excision, whereas the majority of advanced cancer patients require multifaceted, integrated treatment strategies to extend their survival and improve their quality of life^[307]. In this section, we will review the research progress of EVs in cancer therapy.

Applications of EVs in chemotherapy

Chemotherapy holds a cornerstone in cancer therapy. However, toxic side effects and drug resistance significantly limit its clinical effectiveness. Novel liposome-based chemotherapeutic drugs, such as doxorubicin (Dox) liposomes (Doxil), have gained extensive clinical application, enhancing the efficacy and safety of chemotherapy^[308]. Similarly, EVs, as biologically derived lipid nanovesicles, exhibit great potential as effective carriers for chemotherapeutic drug delivery. Analogous to Doxil, Dox-loaded exosomes (Exo-Dox) demonstrated markedly enhanced cellular uptake and anti-tumor efficacy, along with reduced cardiotoxicity^[309]. Li et al. decorated milk exosomes with hyaluronan, a CD44-specific ligand, to specifically deliver Dox into CD44-overexpressing tumor cells^[310]. In addition, they also used this engineered milk exosome system to deliver miR-204-5p, a pan-cancer tumor suppressor, into CD44-expressing cancer cells, efficiently inducing cancer cell death^[311]. Beyond sharing the characteristics of lipid-based vesicles, EVs exhibit a unique capacity to traverse the blood-brain barrier (BBB). Recognizing this potential, Zhu et al. developed dual-targeted, functionalized sEVs modified with Angiopep-2 and Transactivator of Transcription (TAT) peptides to deliver chemotherapeutic agents. This system could traverse the BBB, efficiently inhibit glioma growth and reduce chemotherapy-related toxic side effects^[312].

EVs also show potential application to reverse chemoresistance. For example, miR-21, an oncogenic miRNA, has been shown to foster chemoresistance in multiple cancer types by suppressing targets such as phosphatase and tensin homolog (PTEN), human mutS homolog 2 (hMSH2) and programmed cell death 4 (PDCD4)^[313]. Liang et al. harnessed sEVs to co-deliver miR-21 inhibitors and 5-fluorouracil (5-FU) to tumor cells, overcoming chemoresistance and enhancing treatment effects^[314]. Similarly, cell-derived exosomes or milk exosomes have been used to deliver miR-204-5p, inhibiting drug resistance and tumor growth^[311,315]. These studies underscore the potential of EV-mediated drug delivery in overcoming chemoresistance and enhancing treatment efficacy.

Applications of EVs in targeted therapy and immunotherapy

In recent years, targeted therapy has been extensively applied to cancer patients with specific molecular features. By precisely targeting molecules on tumor cells or immune cells, these therapies can effectively kill cancer cells while minimizing harm to normal cells^[316]. EVs have attracted considerable attention as delivery vehicles or therapeutic agents for both targeted therapy and immunotherapy^[317].

Nucleic acid-based gene therapy is a crucial component of cancer-targeted therapy but faces challenges such as poor membrane permeability and susceptibility to enzymatic degradation. A robust delivery system is essential to protect nucleic acids and ensure their targeted delivery. EVs meet these demands with their excellent loading capacity, high stability, and biocompatibility^[318]. Among emerging technologies, the CRISPR-Cas9 system stands out for its potential in gene-targeted treatments^[319], and EVs serve as promising vectors for this system^[320]. In 2017, Kim et al. reported, for the first time, that tumor-derived EVs serve as a valuable delivery platform of CRISPR-Cas9 for cancer gene editing^[242]. Wan et al. successfully integrated Cas9/single-guide RNA (sgRNA) ribonucleoprotein (Cas9 RNP) into exosomes secreted by hepatic stellate cells (HSCs), creating a precise and tissue-specific gene therapy platform for liver diseases^[321]. Furthermore, the remarkable capability of EVs to encapsulate and deliver nucleic acids has positioned them as crucial tools for advancing gene-targeted tumor therapy, enabling highly effective and precise treatments^[322].

Given that RNA is prone to degradation and has difficulty crossing cell membranes to enter cells, EVs, as natural lipid vesicles, emerge as promising delivery carriers that enhance RNA stability and targeting accuracy^[323]. In numerous preclinical studies, EVs have demonstrated significant potential as delivery tools for nucleic acid drugs, including antisense oligonucleotides (ASOs), siRNA, miRNAs, mRNAs, circRNAs, and lncRNAs^[324]. Codiak Biosciences, a pioneering company in the clinical translation of exosomes, utilized ASO-loaded EVs to knock down STAT6 (signal transducer and activator of transcription 6) in tumor-associated macrophages (TAMs), thereby remodeling TME and inhibiting tumor growth^[325]. Chen et al. developed a dual-targeted milk exosome system to deliver PD-L1 siRNA (siPD-L1) into TAMs, efficiently reprogramming TAMs and TME^[326]. Zhao et al. developed an exosome membrane-coated biomimetic nanoparticle system (CBSA/siS100A4@Exosome) to improve siRNA delivery to the lung pre-metastatic niche of breast cancer, achieving excellent gene knockdown and lung metastases inhibition^[327].

In addition to these small nucleic acids, mRNAs can also be efficiently encapsulated and delivered by EVs. In 2020, Yang et al. developed a method to efficiently encapsulate therapeutic mRNA into exosomes on a large scale. In a PTEN-deficient glioma mouse model, these mRNA-loaded exosomes restored anti-tumor defenses, suppressed tumor growth, and improved survival outcomes^[328]. EV-based nucleic acid delivery enables personalized therapy by selecting optimal nucleic acid types for individual patients, paving the way for precise and effective clinical interventions.

Cancer immunotherapy, a specialized subset of targeted therapy, focuses on reversing immunosuppressive TME. Its central principle is to reinvigorate and enhance the body’s anti-tumor immune response^[329]. EV-based immunotherapy is among the most promising anti-tumor strategies currently available^[330]. In addition to the EV-delivered RNA drugs mentioned above that regulate anti-cancer immune response^[326], engineered EVs from tumor cells or immune cells also show potential as immune mediators. For example, engineered tumor-derived exosomes (TEXs) can serve as an attractive cell-free tumor vaccine platform^[331]. Liang et al. have constructed a biologically self-assembled tumor nanovaccine platform with TEXs as an efficient drug delivery system for tumor antigens^[332]. However, TEXs may contain tumor-promoting molecules, which limits their clinical application.

Apart from TEXs, DC-derived exosomes (DEXs) have also garnered significant attention as tumor vaccines. potentially surpassing DC-based vaccines in evading tumor cell-mediated immune suppression^[332]. A notable example is the “trigger” DEX vaccine (DEXP&A2&N) developed by Zuo et al, which effectively triggers tumor-specific immune responses against hepatocellular carcinoma^[333]. Nevertheless, obtaining sufficient immune cells and their EVs remains challenging. Furthermore, both TEXs and DEXs exhibit limited efficacy in activating anti-tumor immune responses. Consequently, combining these EVs with other treatment modalities is often necessary to achieve optimal therapeutic outcomes^[334].

Immunologically engineered EVs, modified with antibodies targeting cancer-promoting molecules such as CD3, human epidermal growth factor receptor 2 (HER2), PD-1, and PD-L1, could be utilized to modulate anti-cancer immune response^[330]. In addition, Chimeric antigen receptor (CAR) T cell-derived EVs also show potential for anti-cancer therapy^[335].

Applications of EVs in physical therapy

In addition to conventional clinical modalities for cancer treatment, innovative therapeutic strategies leveraging physical stimuli such as light, sound, and magnetism are increasingly gaining widespread clinical acceptance. Treatments based on light include photothermal therapy and photodynamic therapy (PDT), among others. PDT has been approved by the FDA for clinical application^[336]. Similarly, most photosensitizers are also sonosensitizers, enabling photoacoustic imaging and sonodynamic therapy (SDT) under ultrasonic stimulation^[337]. A key advantage of SDT is the deeper tissue penetration of ultrasound compared to light, making it suitable for treating tumors located in deeper regions^[337]. Magnetic field-based therapies typically involve magnetic hyperthermia, which relies on appropriate magnetic fields and magnetic materials such as iron oxide nanoparticles, particularly superparamagnetic iron oxide nanoparticles (SPIONs). Similar to sound, magnetic fields overcome penetration limitations, and SPIONs can also enhance MRI^[338].

A long-standing challenge in this field is improving the delivery efficiency of photosensitizers, sonosensitizers, and iron oxide nanoparticles. To address this, Cheng et al. engineered sEVs with a chimeric peptide for dual targeting of the cytoplasmic membrane and nucleus, loading them with photosensitizers to achieve more precise strikes against tumor cells while reducing drug toxicity and side effects^[339]. Liu et al. designed a functionalized smart nano-sonosensitizer by loading sonosensitizers into engineering exosomes. This approach not only enhances the efficiency of drug delivery but also enables ultrasound-controlled drug release, thereby augmenting the anti-tumor efficacy of SDT^[340]. Through innovative EV engineering combined with physical therapy^[341], researchers have significantly bolstered the efficacy of tumor eradication while maintaining rigorous biosafety standards.

Applications of EVs in comprehensive multimodal therapy

Monotherapy often shows limited efficacy in tumor treatment, necessitating combination therapy as a key strategy to improve therapeutic outcomes. Due to their excellent biocompatibility, tumor targeting, long circulation, immune-modulatory properties, and ease of modification, EVs have emerged as versatile platforms for comprehensive multimodal therapy, including phototherapy, chemotherapy, gene therapy, and immunotherapy^[252]. They can co-deliver chemotherapeutic agents, genetic materials, immune modulators, and phototherapeutic compounds, offering a synergistic multimodal approach to combat tumor heterogeneity and resistance.

In a pioneering study, Guo et al. developed a novel therapeutic platform using hollow manganese dioxide (MnO₂) particles loaded with the photosensitizer indocyanine green (ICG) and camouflaged by PD-L1 antibody-modified exosomes^[342]. This platform combines PDT with immunotherapy, simultaneously targeting both the physical and immunological dimensions of tumors. In another study^[343], a cutting-edge nano-polymer (Ir-Fe NP) was developed by synthesizing an Ir(III) photosensitizer with Fe(III) and encapsulating it within melanoma-derived exosomes. This combined photodynamic and chemodynamic approach offers a highly efficient and potent strategy for melanoma treatment. Besides, Cheng et al. developed a hybrid nanovesicle by fusing genetically engineered exosomes with drug-loaded temperature-sensitive Doxil, establishing a promising nanomedicine platform for efficient and safe photothermal-immunotherapy combination therapy^[344].

Chemotherapy can activate anti-tumor immunity by inducing tumor cell death and releasing tumor antigens^[345]. This effect often serves as a precursor to or is synergistically combined with immunotherapy, enhancing the overall treatment effectiveness by enabling the immune system to recognize and eliminate residual tumor cells. For example, a hybrid exosome system was developed by fusing M1-like macrophage-derived exosomes with genetically engineered TEXs carrying CD47^[346]. This complex was loaded with the DNA-damaging agent SN38 and the stimulator of interferon genes (STING)-agonist MnO-₂, resulting in multifunctional hybrid exosomes that exert excellent anti-cancer effects via multiple mechanisms. Moreover, Shan et al. developed a functionalized macrophage-derived exosomal delivery system capable of crossing the BBB, co-delivering the small molecule drug panobinostat and PPM1D (protein phosphatase, magnesium-dependent 1, delta)-siRNA for combined chemo-gene therapy of PPM1D-mutant gliomas^[347]. Other researchers have also developed multifaceted therapeutic platforms that integrate EVs with synthetic components to facilitate tumor-targeted chemical, genetic, and photothermal therapy^[348].

Applications of EVs in CVDs

CVDs represent the leading cause of death worldwide, accounting for nearly one-third of all global deaths^[349]. Despite advances in therapeutic strategies, the prevention and treatment of CVDs remain a major challenge. Emerging evidence indicates that EVs are involved in the pathological progression of CVDs, holding promising potential for monitoring and treating these diseases^[350].

Applications of EVs in atherosclerosis

Atherosclerosis is a major CVD driven by chronic inflammation and lipid deposition within arterial plaques. EVs have been identified in plaques and play a crucial role in the progression of atherosclerosis^[351]. Given that the activation of ECs is a key step in the intimal lesion formation of atherosclerosis, endothelial progenitor cells (EPC)-derived EVs have been shown to regulate the progression of atherosclerosis. Li et al. reported that EPC-EVs transfer miR-199a-3p to inhibit specificity protein 1 (Sp1), suppressing ferroptosis in ECs and delaying atherosclerosis^[352]. Macrophages also play a vital role in atherosclerosis. EVs derived from anti-inflammatory M2 macrophages have been used to reduce inflammation and hematopoiesis in atherosclerotic plaques^[353]. In a recent study, Xie et al. demonstrated that M2 macrophage-derived EVs fused with platelet membranes show enhanced plaque targeting and alleviate atherosclerosis development^[354]. These findings highlight that EVs are promising therapeutic tools for atherosclerosis, warranting further investigation.

Applications of EVs in myocardial ischemia and injury

Myocardial ischemia, injury, and infarction represent distinct types of myocardial tissue damage caused by an imbalance between myocardial blood supply and oxygen demand. Recent studies have reported increased numbers of platelet-, EC-, and leukocyte-derived EVs in myocardial infarction (MI)^[355]. Interestingly, these EVs have been shown to protect the myocardium from ischemia-reperfusion injury^[356]. For example, EVs purified from serum-converted human platelet lysates provide robust protection following cardiac ischemia/reperfusion injury^[357]. Similarly, EVs derived from Krüppel-like Factor 2 (KLF2)-overexpressing ECs attenuate myocardial ischemia/reperfusion injury by shuttling miR-24-3p that inhibits lymphocyte antigen 6C (Ly6C)^high monocyte recruitment^[358]. Apart from these endogenous EVs, EVs derived exogenously from pluripotent stem cells also exert cardioprotective effects. MSC-EVs protect myocardial ischemia-reperfusion injury in mice by transporting miR-182 and thereby modulating macrophage polarization^[359]. Hypoxia-induced MSC-EVs promote ischemic cardiac repair through miR-125b-mediated amelioration of cardiomyocyte apoptosis and prevention of cardiomyocyte death in MI^[360]. Furthermore, sEVs derived from M2 macrophages inhibit pro-inflammatory C-C motif chemokine receptor 2 (CCR2)⁺ macrophage subpopulations to promote cardiac repair in MI^[361]. In summary, EVs hold significant potential for mitigating myocardial ischemia-reperfusion injury by promoting myocardial repair through multiple mechanisms. These multifunctional properties position EVs as promising therapeutic candidates for the treatment of ischemic heart disease.

Applications of EVs in cardiac hypertrophy and heart failure

EVs have been documented to mediate crosstalk between cardiomyocytes and fibroblasts in the context of cardiac hypertrophy and heart failure. Under pathological conditions of these diseases, EVs derived from fibroblasts transfer miR-21 and miR-27a into cardiomyocytes, contributing to cardiac hypertrophy^[362]. Similarly, macrophage-derived EVs transport miR-155 into fibroblasts, exacerbating cardiac fibrosis^[363]. Conversely, stem cell-derived EVs carrying miRNAs such as miR-185-5p and miR-143/145 cluster can reduce the expression of genes associated with cardiac hypertrophy, offering protective effects^[202]. Current research in this area is largely focused on profiling miRNA changes in circulating EVs as potential biomarkers for myocardial hypertrophy and heart failure. Studies exploring the therapeutic application of EVs and their miRNA cargo for treating these conditions are still in the early stages, highlighting a promising avenue for future investigation.

Applications of EVs in metabolic diseases

Applications of EVs in obesity
Obesity is a serious worldwide epidemic characterized by long-term imbalance between caloric intake and energy expenditure. Visceral adipose tissue (VAT) regulates immunometabolic processes and systemic homeostasis through cellular signaling and cytokine release. Given the interplay between lipid metabolism and EVs, and the fact that their imbalance is closely related to obesity, EV-based therapies hold significant potential as a treatment for obesity^[364].

EVs play a dual role in adipocyte differentiation. They can actively promote terminal adipocyte differentiation via the MAPK-ERK signaling pathway. Conversely, Wang et al. found that tumor-derived EVs negatively affected adipose-derived MSCs (AD-MSCs) adipogenesis by activating TGF-β signaling pathway^[365]. Additionally, miR-450a-5p in adipose tissue-derived exosomes (ATDEs) is upregulated during adipogenesis and promotes AD-MSCs adipogenesis by inhibiting WISP2 (WNT1-inducible signaling pathway protein 2)^[366]. Dai et al. further demonstrated that ATDEs induce the expression of adipogenic marker genes in AD-MSCs and enhance the formation of lipid droplets^[367]. Taken together, these findings highlight the crucial role of EVs in adipose differentiation, offering valuable therapeutic strategies for obesity.

EVs play a crucial role in regulating lipid metabolism and hold promise for obesity treatment. Zhao et al. reported that M2 macrophages treated with EVs derived from adipose-derived stem cells (ADSCs) express high levels of tyrosine hydroxylase, which in turn promotes ADSC proliferation and lactate production, thereby facilitating white adipose tissue (WAT) beiging and improving metabolic homeostasis^[368]. Conversely, exosomal miR-155 from adipose tissue macrophage (ATM) is upregulated in high-fat diet mice and inhibits WAT beiging and thermogenesis, partly by targeting peroxisome proliferator-activated receptors (PPAR)γ^[369]. Other obesity-related EV miRNAs, such as miR-122, miR-192 and miR-27a-3p, may serve as therapeutic targets for obesity^[364]. In another study, melatonin treatment increased exosomal α-ketoglutarate levels and exosome release from adipose tissues, promoting anti-inflammatory ATM polarization^[370]. TEXs also modulate lipid metabolism. For example, pancreatic cancer-derived exosomes carrying adrenomedullin trigger lipolysis, with similar effects observed with cancer-associated fibroblast-derived EVs^[371,372]. Collectively, these findings underscore the pivotal role of EVs in lipid metabolism and their potential as therapeutic tools for obesity.

Applications of EVs in liver regeneration

The liver, a highly regenerative organ, relies on hepatocyte proliferation to maintain physiological homeostasis. Despite this capacity, end-stage liver disease remains a leading cause of death, with transplantation being the only curative option - limited by donor shortages. As an alternative, EVs have emerged as promising modulators of liver regeneration due to their cargo of growth factors, cytokines, and miRNAs that enhance hepatocyte proliferation and survival^[373,374]. During liver injury, EVs mediate intercellular communication among hepatocytes, sinusoidal ECs, and immune cells to coordinate regenerative responses^[375]. For instance, hepatocyte-derived exosomal miR-146a-5p induces HSC inactivation by promoting mesenchymal transition^[376]. Conversely, mannan-binding lectin-associated serine protease-1 (MASP1) and the lncRNA cytoskeleton regulator RNA (CYTOR) in hepatocyte-derived EVs can activate HSCs and promote liver fibrosis^[377,378]. Notably, HSC autophagy can suppress fibrosis by limiting fibrogenic EV release^[379]. Exogenous EVs, particularly from stem cells, can deliver pro-regenerative and anti-inflammatory factors that enhance liver repair while avoiding risks such as tumorigenicity and immune rejection associated with cell therapy^[380].

Owing to their size and composition, EVs and other nanoparticles preferentially accumulate in the liver after intravenous injection. However, the primary liver cell type responsible for EV uptake remains debated. Hepatocytes, Kupffer cells, and ECs have all been shown to internalize EVs and contribute to regenerative signaling^[381,382]. Notably, lipid nanoparticle (LNP)-mRNA delivery studies indicate hepatocytes as the primary target, followed by endothelial and Kupffer cells^[383], highlighting the importance of EV-cell interactions in liver repair.

Applications of EVs in diabetes

Accumulating evidence suggests that EVs, particularly exosomes, can serve as reliable, non-invasive biomarkers for early diabetes detection and disease progression monitoring^[384,385]. Beyond diagnostic applications, EVs also play active roles in modulating metabolic pathways. Some miRNAs, proteins, and lipids encapsulated within EVs can influence key signaling cascades involved in glucose homeostasis, enhancing pancreatic β-cell functions and insulin sensitivity in peripheral tissues^[386]. MSC-EVs have shown therapeutic promise for their capabilities in attenuating systemic inflammation, reducing oxidative stress, and promoting regeneration of pancreatic islets^[275,387]. These regenerative and immunomodulatory effects make MSC-EVs attractive candidates for cell-free therapeutic strategies in both type 1 and type 2 diabetes. Taken together, EVs represent a versatile platform for next-generation diabetes therapeutics.

Applications of EVs in aging and NDs

EVs have emerged as key players in aging and rejuvenation^[388,389]. Studies show that EVs from young plasma or cultured cells can rejuvenate aged tissues by enhancing mitochondrial function, reducing oxidative stress, and reversing cellular senescence^[390,391]. MSC-EVs are particularly promising for combating aging-associated diseases by mitigating oxidative damage, promoting tissue repair, and extending health span in progeroid mice^[392,393]. Conversely, senescent cells release significantly more EVs, which transmit paracrine senescence to neighboring cells, a process partially mediated by sEV IFITM3 (interferon-induced transmembrane protein 3)^[394]. This functional duality underscores the importance of source selection in EV-based therapy. Future research should focus on optimizing therapeutic EVs from young or engineered cells while mitigating the adverse effects of senescence-associated EVs. Overall, EVs represent a compelling avenue for combating age-related decline and extending healthy lifespan.

NDs, characterized by progressive loss of neurons due to the accumulation of abnormal proteins or peptides in the central and peripheral nervous systems, account for millions of global deaths and disabilities annually^[395]. Despite ongoing research, effective treatments remain limited, largely due to the BBB that restricts most therapeutic agents from entering the brain. EVs offer a promising solution due to their ability to cross the BBB and represent an ideal carrier for therapeutic molecules, enabling the delivery of repair and regulatory factors from stem cells to damaged neural tissue^[396].

EVs in AD

There are two main pathological mechanisms for AD: (1) Excessive cleavage of amyloid precursor protein (APP) by β-secretase rather than α-secretase, resulting in the accumulation of polymerized amyloid-β in the brain; and (2) Hyperphosphorylation of Tau proteins, forming neurofibrillary tangles that lead to neuronal dysfunction and cell death^[397]. As EVs have been identified as carriers of pathological molecules during disease progression, pharmacological modulation of the release of EVs containing pathogenic cargos represents a common approach for ND therapy development^[398]. For example, neuron-derived exosomes aid in forming non-toxic amyloid β (Aβ) protofibrils and enhance Aβ uptake and degradation by microglia. Their secretion is regulated by enzymes such as nSMase2 (neutral sphingomyelinase 2) and sphingomyelin synthase (SMS)2, positioning them as potential therapeutic targets for AD^[399].

Several studies demonstrate the therapeutic potential of engineered EVs in AD models. Liu et al. found that bone marrow MSCs (BMSCs)-derived exosomes alleviate cognitive decline in a constructed AD mouse model by improving brain-derived neurotrophic factor (BDNF)-related neuropathology^[400]. Similarly, engineered MSC-EVs with high SHP2 (Src-homology 2 domain-containing phosphatase 2) expression significantly promote neuronal mitophagy, thereby reducing mitochondria damage-induced apoptosis and NLRP3 (NOD-, LRR- and pyrin domain-containing protein 3) inflammasome activation^[401]. In addition, AD-MSC exosomes loaded with miR-122 show promising therapeutic potential in AD^[402]. Yu et al. constructed engineered exosomes modified with rabies virus glycoprotein (RVG) peptide (RVG-EXO) and loaded with a neprilysin variant to target α7-nAChR in the brain’s hippocampus. RVG-EXO administration reduced pro-inflammatory markers (IL-1α, TNF-α, NF-ĸB) and elevated anti-inflammatory IL-10 in the brain, providing a novel therapeutic strategy for AD^[403].

EVs in Parkinson’s disease

The pathological features of Parkinson’s disease (PD) include the progressive degeneration of dopaminergic neurons in the substantia nigra, resulting in reduced dopamine levels and motor symptoms such as tremor and rigidity^[404]. EVs not only participate in PD development and progression but also show potential as biomarkers and therapeutic tools^[405]. In 2011, Alvarez-Erviti first demonstrated the therapeutic potential of exosomes for brain delivery by engineering immature DEXs expressing the fusion protein of Lamp2b-RVG. The endogenous engineered EVs were utilized to deliver siRNA into the brain to knock down β‑secretase 1 (BACE1, a therapeutic target in AD).

Liu et al. developed a core-shell hybrid nanosystem (REXO-C/ANP/S) coated with RVG-modified exosomes derived from immature DCs to deliver siRNA targeting α-synuclein (siSNCA), which can efficiently eliminate α-synuclein aggregates and reduce cytotoxicity in PD neurons^[406]. Additionally, preventing dopaminergic neuron loss in the substantia nigra represents a promising therapeutic strategy for PD. Researchers employed umbilical cord blood mononuclear cell-derived sEVs to deliver miR-124-3p. These miR-124-3p-loaded sEVs successfully protected dopaminergic neurons from the 6-hydroxydopamine toxicity in a PD mouse model^[407].

Encapsulating therapeutic agents with low biocompatibility into EVs has emerged as a major research strategy for PD treatment. For instance, blood-derived exosomes loaded with dopamine significantly enhanced striatal and nigral dopamine distribution while showing increased therapeutic efficacy and reduced systemic toxicity compared to free dopamine in PD models^[408]. Similarly, Luo et al. developed a milk exosome-based delivery system that efficiently delivers epicatechin gallate into SH-SY5Y cells and exhibits enhanced neuroprotective effects^[409]. Stem cell-derived EVs also show therapeutic potential. EVs derived from human NSCs exhibit significant neuroprotective effects in PD models by reducing reactive oxygen species (ROS) and pro-inflammatory factors^[410].

Applications of EVs in trauma medicine

EVs in regeneration of the musculoskeletal system
The musculoskeletal system, essential for movement and postural maintenance, is vulnerable to various injuries and degenerative conditions^[411]. Although surgical options exist, outcomes are often suboptimal, highlighting the need for innovative regenerative strategies. EVs have gained prominence in regenerative medicine, demonstrating their potential to facilitate the healing of wounds, bones, cartilage, menisci, spinal cord, rotator cuffs, tendons, and muscle fibers. For example, Sahu et al. revealed that EVs derived from young animals rejuvenate aged skeletal muscle regeneration^[412]. MSC-EVs appear to be a promising novel therapy for musculoskeletal regeneration. In addition, hybridized nanovesicles based on stem cell-derived exosomes and hydrogels have shown enhanced effects in musculoskeletal regeneration^[413]. A recent review summarized the therapeutic potential of EVs in musculoskeletal regeneration^[414].

EVs in wound healing

Wound healing is a complex clinical challenge involving various injuries, including infectious, diabetic, and burn wounds. This intricate process requires a coordinated interplay of cellular elements, growth factors, extracellular matrix, nerve fibers, and blood vessels. MSCs are key players in skin regeneration and repair, influencing all phases of wound healing from inflammation to tissue reconstruction^[415]. MSC-EVs replicate the biological effects of their parent MSCs and have demonstrated potential applications in wound repair^[416]. For example, MSC-EVs can promote M2 macrophage polarization and enhance cutaneous wound healing^[417]. Additionally, EVs from M2 macrophages can induce the transition from M1 to M2 phenotype, accelerating wound healing by enhancing angiogenesis, re-epithelialization, and collagen deposition^[418]. In addition, engineered MSC-EVs or MSC-EV-based hybrid vesicles have been constructed to enhance the effects of MSC-EVs in wound healing^[419]. Furthermore, EVs from other stem cell types, such as ADSCs, have also shown potential in wound healing^[420].

EVs in bone regeneration

Bone defects pose significant clinical challenges. BMSCs play a pivotal role in bone regeneration, and BMSC-derived EVs produce therapeutic effects comparable to those of BMSCs in stimulating bone formation^[421]. EVs enhance osteogenesis by stimulating the differentiation of MSCs into osteoblasts and promoting the deposition of extracellular matrix. On the one hand, EVs are commonly used to modify scaffolds for the repair of bone defects. For example, Youseflee et al. developed a hydroxyapatite scaffold loaded with EVs derived from endometrial MSCs, exhibiting satisfactory osteogenic and angiogenic characteristics^[422]. Wang et al. developed an acellular fish scale scaffold enriched with BMSC-EVs for treating calvarial defects, demonstrating that the scaffold significantly promotes bone regeneration by enhancing the osteogenic differentiation of BMSCs^[423]. BMSC-EV-loaded hydrogels have also been used to repair calvarial defects, exhibiting excellent thermosensitive properties and further enhancing bone regeneration. Additionally, stem cell-derived EVs or bacterial EVs can be employed to deliver therapeutic agents, such as growth factors, directly targeting injury sites to enhance bone healing^[424,425].

In addition to bone defects and fractures, EVs have been studied for the treatment of femoral head necrosis. Chen et al. developed an extracellular matrix-mimicking hydrogel composed of methacryloylated type I collagen-loaded EVs, which promotes bone repair in glucocorticoid-induced osteonecrosis of the femoral head by enhancing macrophage M2 polarization, osteogenesis, and angiogenesis to^[426]. Overall, EVs represent a promising therapeutic tool for bone defect repair, fracture repair, and femoral osteonecrosis by promoting osteogenic differentiation of BMSCs and stimulating neovascularization.

EVs in cartilage regeneration

Cartilage is a connective tissue composed of chondrocytes that generate a collagen-rich extracellular matrix abundant with proteoglycans and elastin. However, it has a limited capacity for self-repair due to its poor vascularization, sparse cellular content, and scarcity of progenitor cells. Cartilage defect is a common clinical challenge with limited treatment options. EVs derived from MSCs or ADSCs have garnered attention as a viable therapeutic for post-traumatic cartilage injury and osteoarthritis of the knee^[427]. However, clinical translation has been hindered by challenges such as inconsistent dosing and rapid in vivo clearance. To address these limitations, scaffolds or hydrogels loaded with EVs have been recently investigated.

Li et al. designed a biomimetic double-network hydrogel scaffold via 3D printing using tissue-specific decellularized extracellular matrix and human adipose MSC-EVs. This scaffold significantly enhanced simultaneous regeneration of cartilage and subchondral bone in a rat model^[428]. A recent systematic review summarized the effect of EVs-loaded scaffolds on post-traumatic cartilage injury and knee osteoarthritis^[429]. Collectively, EVs represent a promising future treatment option for cartilage defects and osteoarthritis.

EVs in meniscus injury repair

Meniscal injuries, commonly caused by sports activities or age-related degeneration, often lead to impaired knee function and increase the risk of osteoarthritis. Current surgical treatments yield limited success, especially in the avascular zones of the meniscus. EVs, particularly MSC-EVs, have shown great potential in promoting meniscal regeneration through enhancing meniscal cell migration and ECM synthesis, as well as inhibiting inflammatory responses^[430]. In addition to MSC-EVs, exosomes from skeletal stem cells also offer a promising therapeutic strategy for meniscal injury repair^[431]. Recent research has increasingly focused on combining EVs with bioengineered scaffolds to enhance meniscus regeneration.

EVs in spinal cord injury repair

Spinal cord injuries (SCI) are one of the most debilitating musculoskeletal injuries, often leading to permanent loss of motor and sensory functions. The CNS has limited capacity for self-repair. EVs can effectively cross the BBB, making them ideal candidates for drug delivery. Stem cell-derived EVs have emerged as a novel therapeutic approach for SCI due to their neuroregenerative properties and ability to modulate the post-injury microenvironment^[432]. Specifically, EVs derived from NSCs and MSCs carry neuroprotective molecules, including miRNAs, growth factors, and anti-apoptotic proteins that can promote axonal growth and prevent neuronal cell death^[433,434]. In addition, autologous plasma-derived EVs have been utilized for targeted delivery of growth-facilitating peptides to promote recovery after SCI^[435]. These findings highlight the dual potential of EVs as both direct therapeutic agents and targeted delivery systems in SCI treatment.

In summary, EVs represent a versatile therapeutic tool for trauma medicine. Their integration into biomaterials such as hydrogels and scaffolds further enhances their regenerative potential, offering new avenues for treating complex musculoskeletal injuries.

Applications of EVs in infectious diseases

Infectious diseases, caused directly or indirectly by microorganisms, pose a significant threat to global public health. Chemotherapy remains the most effective option for the clinical management of infected patients. However, the emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains has introduced challenges, including complex drug regimens and prolonged treatment durations^[436]. To address these issues, EV-based nanotechnologies have been designed and developed for the diagnosis and treatment of infectious diseases^[437].

EVs in viral infection

EVs are closely linked to viral infection and body immunity. Many EV-based strategies have been designed to resist viral infection, including HIV^[438], severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)^[439,440] and HBV^[441]. For example, Li et al. reported biomimetic macrophage EV-like nanoparticles coated with an aggregation-induced emission (AIE) molecule (TBD@M NPs) for virus transmission blockage and targeted photothermal therapy in monkeypox^[442]. This pioneering study demonstrates the potential of nanovesicles in monkeypox theranostics and may pave the way for the development of promising therapeutic strategies for monkeypox management in clinical trials. Engineered EVs modified with the receptor-specific ligands have been applied to deliver therapeutic drugs to infected tissues or organs. Cas9/guide RNA (gRNA)-loaded EVs decorated with vesicular stomatitis virus-glycoprotein (VSV-G) significantly enhanced the endosomal escape of Cas9 protein and increased its gene-editing activity in recipient cells, effectively reducing viral antigens and HBV covalently closed circular DNA (cccDNA) levels in the HBV-replicating and infected cells^[441]. In addition, the innovative approach of incorporating virus-encoded envelope proteins into EVs carrying genetic material or therapeutic biomolecules confers exceptional binding or entry specificity, thereby enhancing their delivery efficiency.

EVs are assessed as promising cell-free vaccines for the prevention and control of infectious diseases^[443]. In 2004, Aline and coworkers reported the effectiveness of EVs derived from Toxoplasma gondii antigen-pulsed DC as an innovative cell-free vaccine against toxoplasmosis^[444]. In addition to their capacity to foster protective immunity against Toxoplasma infection, these EVs possess the immunoprophylactic potential against various viral infections. In all, these findings indicated that the EV-based nanomedicine offers a promising strategy for the prevention and blockade of viral infection^[445].

EVs in bacterial infection

Tuberculosis (TB), caused by Mycobacterium tuberculosis (M. tuberculosis), is a serious disease characterized by symptoms such as blood-tinged mucus, fever, and potentially fatal complications. The emergence of MDR and XDR strains has further exacerbated the challenges in TB treatment^[446]. To address this issue, Li et al. developed a lesion-pathogen dual-targeting strategy for TB treatment, which involved coating mycobacterium-activated macrophage membrane vesicles onto polymeric cores encapsulated with an AIE photothermal agent^[447]. These engineered vesicles-based nanoparticles can simultaneously target tuberculous granulomas and internalized M. tuberculosis. In a TB mouse model, intravenous administration of these nanoparticles effectively eradicated the bacteria, alleviated pathological damage, and reduced excessive inflammation in the lungs, demonstrating superior therapeutic efficacy compared with conventional first-line antibiotics. This membrane vesicle-based dual-targeted photothermal modality represents a promising strategy for TB management.

Infectious wounds are often complicated by persistent MDR bacterial infection and sustained inflammatory responses, which delay the wound-healing process. In addition, excess ROS at the wound site can further impede tissue repair^[448]. An intelligent dual-layered hydrogel loaded with stem cell-derived vesicles was designed to regulate wound repair^[449]. Specifically, the inner hydrogel layer responds to bacterial hyaluronidase by releasing ADSC-EVs functionalized with an AIE photosensitizer, which generate ROS upon light irradiation to inhibit bacterial growth. The outer hydrogel layer continuously scavenges excess ROS levels in the wound site, thereby promoting tissue regeneration through the action of ADSC-EVs. This study presents a smart ROS-modulating treatment platform that provides a multifunctional wound dressing for comprehensive repair of infected wounds.

Oral ulcers are refractory superficial lesions of the oral mucosa that are highly susceptible to inflammatory storms and secondary infections. Ge et al. constructed a silk fibroin microneedle patch incorporating exosomes derived from LPS-prestimulated BMSCs and zeolitic imidazolate framework-8, localized at the tip and base of the microneedles, respectively^[450]. Upon coating on the oral superficial lesions, the patch enables sustained release of EVs and Zn²⁺ ions, synergistically enhancing tissue healing and exerting potent antimicrobial effects, thereby accelerating ulcer healing. In another study, EVs derived from Apis mellifera honey demonstrated significant antibacterial activity against oral streptococci, revealing a novel source of EVs enriched with antibacterial peptides^[451].

EVs in fungal infection

Fungal pathogens can cause many diseases and clinical symptoms by invading the human tissues, triggering inflammatory reactions, inducing tissue damage, and leading to organ dysfunction^[452]. Recent advances have highlighted the “double-edged sword” role of fungal EVs in mediating intercellular communication and pathogen-host interactions^[453]. On the one hand, fungal EVs contribute to infection progression and transmission, facilitate fungal-host cell interactions, and deliver virulence factors^[454]. On the other hand, they can exert protective effects by regulating fungal growth and stimulating adaptive immune responses in the host^[455].

Fungal EVs have demonstrated considerable potential for diverse applications. For instance, they can be used to construct drug delivery systems. Moreover, fungal EVs are known to induce protective immune responses against fungal infections, highlighting their promising role in clinical theranotics and disease prevention^[456,457]. Notably, the surface of fungal EVs contains some known protective antigens, such as those from Mp88 and Gox (galactose oxidase-like) families, providing a strong theoretical support for early prevention and treatment of fungal-related diseases^[458]. Overall, advances in EV-based nanotechnology are expected to enhance the treatment and prevention of infections by viruses, bacteria, fungi, and other pathogens.

EVs have demonstrated established therapeutic roles in three main areas: as biocompatible drug delivery vectors that improve the bioavailability of therapeutics while mitigating off-target toxicity; as immunomodulators via MSC- or DC-derived EVs that regulate immune cell activity; and as promoters of tissue repair and regeneration through the transfer of bioactive molecules that promote proliferation, extracellular matrix deposition, and neovascularization. Their emerging potential, supported by preclinical studies but still awaiting clinical translation, includes delivering therapeutics across biological barriers for NDs, acting synergistically with photothermal or sonodynamic therapies and immunomodulatory agents, and utilizing microbial EVs as vaccines or drug carriers. However, these applications remain tentative due to unclear mechanisms, limited reproducibility, and a lack of direct clinical evidence.

Notably, although EVs are often thought to act through miRNA delivery, there is ongoing debate about whether the miRNA content in single EVs is sufficient to induce functional changes in recipient cells. This uncertainty underscores key limitations in our current understanding of EV cargo functionality and supports the rationale for engineering strategies - such as overexpression or selective loading of specific miRNAs or other bioactive molecules - to enhance signal intensity and therapeutic reliability.

In different disease contexts, EVs show distinct advantages: tumor-targeting capability in oncology, inherent BBB penetration for neurodegenerative conditions, cardiac repair and anti-inflammatory functions in cardiovascular disease, and the dual targeting-immune activation features of microbial EVs in infections. Nevertheless, they are concomitantly constrained by inherent limitations, such as the potential of tumor-derived EVs to facilitate immune escape, limited brain-targeting efficiency, rapid systemic clearance, and technical challenges in standardizing microbial EV isolation. Broader translational hurdles include insufficient specificity and controllability, difficulties in dose standardization due to EV heterogeneity, and the risk of overgeneralizing preclinical data to clinical settings.

EVs have demonstrated definitive therapeutic value in drug delivery, immunomodulation, and tissue repair, with promising emerging potential in barrier-crossing delivery and combination therapies. In contrast, their actual clinical effectiveness requires systematic validation through well-designed preclinical and clinical studies. Future research should prioritize optimizing EV specificity and controllability, establishing standardized production and quality control systems, and conducting large-scale clinical trials to fully realize their therapeutic potential.

EVs for disease diagnosis

Early disease screening, especially for malignant tumors, can effectively improve the 5-year survival rate of patients and even offer a chance for radical cure. Liquid biopsy has a wide range of applications across various diseases. Over the past decade, liquid biopsy techniques, primarily based on circulating tumor cells (CTC) and circulating tumor DNA (ctDNA), have become essential tools for the precise diagnosis of tumors^[480]. Although EVs (exosomes) hold great potential as a basic component of a liquid biopsy system, their clinical applications still lag behind those of CTC and ctDNA^[481].

EV-based biomarkers have been rapidly applied in clinical practice. For example, the first prostate cancer diagnostic test using exosomal RNA has already helped over 50,000 patients in clinical decision-making and has been included in the National Comprehensive Cancer Network Guidelines for Early Detection of Prostate Cancer^[482]. As emphasized previously, the intrinsic properties of EVs confer unique advantages for early disease screening, disease classification, and prognosis assessment.

Future clinical translation of EVs as medical tools will focus on enhancing diagnostic and therapeutic strategies. Researchers aim to capitalize on the early diagnostic capabilities of EVs, by further exploring the diagnostic information encoded in their specific biomolecular cargo. This will require the development of ultra-sensitive and highly specific detection technologies, enabling not only early disease detection but also precise molecular stratification of pathologies at initial stages, thereby advancing personalized medicine.

For EV-based early disease screening, the preliminary basic research is crucial. Key priorities includes but not limits to: (1) accurate capture of disease-related EVs, such as those derived from tumor cells or other lesion cells; (2) comprehensive understanding of disease mechanisms to allow accurate classification and subtyping; (3) development of signal amplification strategies for EV cargo, given the limited quantity of EVs in bodily fluids during early disease stages, to enable more efficient in vitro analysis.

EVs for disease treatment

EVs also hold significant translational potential for disease treatment. Among them, MSC-EVs are the most studied, exerting immunomodulatory, anti-inflammatory, anti-fibrotic, and antioxidant effects, as well as promoting angiogenesis, owing to their unique protein and nucleic acid components^[387,483]. These vesicles have demonstrated considerable clinical value in various human diseases, including neurological, cardiovascular, respiratory, and kidney diseases, as well as metabolic disorders, dermatological conditions, immune regulation, and medical aesthetics^[484-486]. In particular, the skincare and medical aesthetics industries have been at the forefront of integrating MSC-EVs into commercial products such as creams, essences, masks, and other similar formulations^[487]. Although MSC-EVs are derived from stem cells and share similar contents to stem cells, they offer improved safety and stability compared to whole stem cells^[48,488,489]. Therefore, MSC-EVs are expected to advance more rapidly toward clinical translation and application in the near future.

Beyond MSC-EVs, EVs derived from other sources also show high clinical translation potential. For instance, EVs derived from cardiac spheroid-derived cells hold promise for treating Duchenne muscular dystrophy^[490], while milk-derived EVs offer broader application prospects due to their superior safety and cost-effectiveness compared with EVs from other sources. Additionally, EVs from DCs have been shown to enhance the body’s ability to clear tumors or infections^[491]. Despite their promise, these EV-based strategies still require further research and development to effectively advance toward clinical application.

Despite the numerous advantages of EVs as drug delivery vehicles, two major challenges must be overcome before they can be successfully translated into clinical use. First, the large-scale production of EVs remains challenging. Second, although multiple methods exist for drug loading and engineering of EVs, current loading efficiencies are generally low and inconsistent. Compared with natural EVs, engineered EVs show enhanced physicochemical properties and improved therapeutic efficiency. Nevertheless, as discussed previously, clinical translation of EV-based therapies remains hindered by technical challenges, requiring further development before widespread clinical adoption.

LNP-mediated mRNA delivery represents one of the most significant recent advances in drug delivery. Similarly, EVs also offer distinct advantages in delivering small-molecule drugs and nucleic acid therapeutics, particularly mRNA. A direct comparison with EVs highlights a complementary landscape of advantages and challenges for therapeutic applications.

LNPs, although efficacious, can induce acute inflammatory responses and dose-dependent toxicities (e.g., complement activation, hepatic effects)^[492]. Their cationic or ionizable lipids may cause longer-term accumulation concerns. In contrast, EVs, as natural biological entities, generally exhibit superior biocompatibility and lower inherent toxicity. Their immunogenicity can be tunable; naive EVs may be low-immunogenic or even immuno-tolerant, while engineered EVs can be designed to either avoid immune clearance or specifically modulate immune responses - a dual capability LNPs lack^[493].

LNP targeting is primarily passive (e.g., hepatic accumulation via ApoE adsorption) or reliant on the incorporation of complex targeting ligands, which can affect formulation stability. EVs possess intrinsic homing capabilities due to their surface adhesion proteins and glycans^[493,494]. Furthermore, their natural composition facilitates membrane engineering for enhanced active targeting, enabling more efficient and specific tissue delivery with reduced off-target effects compared to current LNP technologies.

Currently, the major advantage of LNPs lies in their fully synthetic nature, which allows for scalable, GMP-compliant manufacturing with well-defined chemistry, manufacturing, and controls (CMC), resulting in lower and predictable cost of goods sold (COGS). In contrast, EV production faces significant biomanufacturing challenges, including low yields from cell cultures, complex purification requirements, and inherent batch-to-batch variability^[495-497]. These factors currently result in high COGS, a major hurdle for clinical translation.

In December 2025, regulatory authorities in China classified certain products containing exosomal membrane components, such as exosome-containing hyaluronic acid dressings, as medical devices, thereby offering a reference regulatory route for specific types of EV-based products. In the same year, a Chinese study^[498] reported that nebulized administration of human MSC-derived exosomes rapidly improved key pathological and functional parameters in patients with pulmonary fibrosis, further supporting the feasibility and translational potential of inhaled EV-based therapies for fibrotic lung diseases.