EVs are a large family of membrane-enclosed extracellular nanoparticles secreted by cells into their surrounding environment. They are typically classified based on physical features such as size and density, biochemical composition, or cell of origin; however, the distinctness of subtypes can be somewhat ambiguous due to overlapping characteristics (13, 14). The International Society for Extracellular Vesicles (MISEV2018) has launched guidelines to facilitate a more uniform classification of EVs, requiring thorough multi-parametric characterization to prevent mislabeling (15). The MISEV 2023, given new nomenclature recommendations, separation protocols, EV release/uptake assays, and in vivo protocols. These guidelines strongly emphasize strict reporting of EV purity (including non-vesicular contaminants), detection instrument limits, dose–response and time-course functional assays, and the use of appropriate non-EV biological controls (16). Despite these efforts, many reviewed studies still fall short of full compliance, highlighting the need for transparent reporting. EVs, including exosomes, microvesicles, and apoptotic bodies, are recognized into three prominent subtypes, each with different functions in health and disease (17).

EVs are produced through distinct but sometimes overlapping cellular pathways, which can contribute to the structural and functional diversity. Their formation involves specialized molecular machinery that governs their size, cargo composition, and eventual secretion into the extracellular environment. Among the different types of EVs, exosomes are the most well-characterized in terms of EV subpopulation, characterized by their consistent size and morphology. They are derived from the endosomal system, beginning with the inward budding of the endosomal membrane, which leads to the formation of intraluminal vesicles (ILVs) within multivesicular bodies (MVBs) (42). This budding process is primarily driven by the endosomal sorting complex required for transport (ESCRT), a group of conserved protein complexes that selectively sort and package cargoes, such as ubiquitinated proteins and nucleic acids, into newly developed ILV. Besides ESCRT-dependent mechanisms, other factors, such as ceramide, lipids, and tetraspanin-enriched microdomains, also contribute to ILV formation, highlighting the flexibility in exosome biogenesis. Once MVBs are fully formed, they can either fuse with lysosomes for content degradation or be moved toward the plasma membrane with the help of Rab GTPases (e.g., Rab27a/b) and SNARE proteins, ultimately releasing ILVs into the extracellular space as exosomes (43–45).

In sharp contrast, microvesicles (MVs), which are produced by outward budding of the plasma membrane, are driven by cytoskeletal rearrangements and mechanical signals with Ca²⁺ dependent downstream communication (46). High intracellular calcium levels then activate enzymes (e.g., flippases and scramblases) that change the distribution of phosphatidylserine from the inside to the outer leaflet, a characteristic feature of MVs surfaces (46). The simultaneous contraction of actin-myosin filaments, operating via RhoA/ROCK pathways, generates the force needed for membrane protrusion and fission. Unlike exosomes, MVs contain cytoplasmic components nonspecifically like the mother cell into the offspring; this often reflects the cell’s actual state, including metabolites, cytosolic proteins, and even organelles (47). The size and component heterogeneity reflect the diverse construction of these vesicles, which can be increased under stress or disease conditions, such as cancer or inflammation.

Apoptotic bodies are the most considerable EV fraction, formed exclusively during cell apoptosis. During apoptosis, caspase activation leads to the widespread denaturation and breakdown of the cell’s cytoskeleton, ultimately resulting in the cell’s demise through blebbing and fragmentation. Apoptotic bodies contain blebs with nuclear remnants, organelles, and degraded genomic DNA, distinguishing them from all other EVs (34, 35). Their release is a final event, often acting as an “eat me” signal for phagocytes. However, recent findings suggest that apoptotic bodies may also carry signaling function as they sometimes contain intact, functional molecules such as growth factors, regulatory RNAs, that can influence neighboring cells (48, 49).

Emerging EV subtypes have unique biogenesis mechanisms that go beyond these classical pathways. For example, migrasomes are generated when vesicles and cytoskeleton elements are pinched off at retraction fibers in migrating cells as they move forward. Oncosomes released by metastatic cancer cells originate from abnormal membrane shedding, a feature of oncogenic metabolic reprogramming, often containing cytoplasmic material that enables their extensive dissemination (50, 51). Smaller nanoparticles (exomeres; without a lipid bilayer) also challenge traditional EV concepts, as exomeres arise from non-vesicular secretory routes, although their biogenesis is not well understood (52).

Although this crosstalk indicates other complexities of EV biology, although exosomes and MVs are constantly released, this might result from transcriptional stress, hypoxia, or disease influencing cargo selection and secretion rates (53). Apoptotic bodies are closely linked to cell death. Although their utility overlaps with that of EVs, this complicates their classification. Live-cell imaging and CRISPR-based screens are now beginning to dissect these pathways, identifying conserved molecular regulators that connect distinct biogenesis mechanisms, such as TSG101 or ARF6 (53, 54). Nonetheless, the question of cargo sorting remains open. Additionally, membrane lipids are vital, and it is unclear how EV subtypes represent distinct populations versus a smear of vesiculation events.

EVs are proteins involved in vesicle formation, trafficking, and uptake within vesicles. Exosomes are identified by tetraspanins (CD9, CD63, CD81) that are organized into functional microdomains on the EVs surface and are essential for cargo selection, recognition, and recipient cell interactions. Common exosomal proteins include ESCRT components (Alix and TSG101), Heat shock proteins (Hsp70 and Hsp90), and fusion membrane regulators (Rab GTPases and annexin (22, 73). These proteins determined both the identity of exosomes and their release/uptake. While microvesicles typically contain a diverse array of plasma membrane proteins, such as integrins, selectins, and flotillins, similar to those directly budding from the cell surface. They can also carry cell-specific receptors, such as the epidermal growth factor receptor (EGFR), in exosomes derived from cancer cells. These may include MITF expression and are believed to induce signaling pathways in recipient cells (74). It is hard to mistake apoptotic bodies for other EVs due to their content of cell debris, including histones and nuclear proteins, as well as the confirmed release of activated caspase from fragmented cells. Besides these common markers, EVs contain cell-type-specific proteins that reflect the characteristics of their parental cells. For example, neuronal EVs are enriched with synaptophysin, a synaptic protein, and EVs from the immune system display major histocompatibility complex (MHC) molecules involved in antigen processing (75, 76). Tumor-derived EVs are especially rich in oncoproteins (eg, mutant KRAS, MET) and proteases (MMP2/9), which contribute to invasive and metastatic properties. Notably, the proteomic landscape of EVs offers excellent potential as yet-to-be-undetermined biomarkers of diseases, including cancer, neurodegenerative disorders, and cardiovascular injuries ( Table 1 ).

The lipid bilayers of EVs are not just static structural components, but also serve as the programming machinery that regulates vesicle robustness, bioactivity, targeting, and functionality. The exosomal membrane is extremely rich in cholesterol, sphingomyelin, and ceramide, which are relatively complex and less degradable (77). Ceramide plays a key role in exosome biogenesis by facilitating the budding of intraluminal vesicles into multivesicular bodies and interacting with other proteins that promote exocytosis. The lipid profile of microvesicles is less consistent than that of EVs, because it mainly reflects the apolar phospholipid asymmetry across the parental plasma membrane (78). Notably, platelet-derived microvesicles contain a high amount of phosphatidylserine due to its externalization, which acts as a phagosomal recognition marker and as a procoagulant biomarker. They also have elevated levels of glycosphingolipids and gangliosides to influence EV interaction with target cells (79). Variations in the lipid content of EVs impact their native functions and have implications for therapeutic interventions, such as lipid modification on EVs for drug delivery ( Table 1 ) (80).

Although EVs are involved in horizontal gene transfer, they contain a wide variety of nucleic acids, including messenger RNA (mRNA), microRNA (miRNA), long non-coding RNA (lncRNA), and even mitochondrial DNA (mtDNA). Exosomal miRNAs, which are strongly linked to various aspects of gene expression in recipient cells, such as miR-21 and miR-155, play a roles in cancer, immune modulation, and tissue repair (81). These miRNAs are typically protected from degradation via ribonucleoprotein complexes, ensuring their functional delivery. Microvesicles and Annexin V apoptotic bodies can carry larger amounts of the genetic material; they are capable of transporting chromosomal DNA fragments that may be involved in oncogenic transformation or autoimmune responses (35, 82). For example, tumor EVs contain oncogenic DNA sequences (e.g., amplified EGFRvIII) that can integrate into the genome of the recipient cell, resulting in malignant transformation. Increasing evidence shows that EVs can carry nucleic acids, such as reporter genes, which may be helpful in liquid biopsies for early cancer detection (e.g., Data in Table 1 ).

Recent research suggests that EVs may also play a role in systemic metabolic functioning by releasing metabolites, such as glucose, lactic acid, and amino acids, which can affect the metabolic state of recipient cells. In the stromal cells, tumor-derived EVs often carry signaling molecules associated with the Warburg effect, such as elevated levels of metabolites like lactate that promote glycolysis and support tumor growth. EVs also contain signaling molecules such as prostaglandins and cytokines, which can modulate inflammation ( Table 1 ) (83, 84).

HIV-infected cells release EVs that carry the accessory protein Nef, which triggers some of the disease-causing processes: It induces CD4⁺ T cell apoptosis by binding to CXCR4 and inhibiting CD4 and MHC-I surface expression, disrupting adaptive immunity (88). It mediates TNFα release by sequestering ADAM17/TACE, leading to persistent inflammation and reactivation of latent HIV reservoirs (89). Additionally, EVs also carry trans-activation response (TAR) RNA, which, upon delivery to target cells, downregulates pro-apoptotic proteins Bim and Cdk9, promoting cell survival, and increases susceptibility to HIV infection (90). Viral RNA transfer via EVs also activates innate receptors (e.g., TLR8), further promoting chronic inflammation through cytokines like IL-6 and TNFβ (91). EVs containing host-derived pro-inflammatory mediators, including miRNAs miR−155, miR−223, and miR−29a, as well as proteins HIF−1α, have been linked to endothelial activation, immune cell dysfunction, and neuroinflammatory cascades, contributing to HIV-related comorbidities (92).

EVs have the potential to serve as HIV biomarkers and therapeutic agents. Elevated platelet- and endothelial-derived EVs correlate with immune activation and vascular dysfunction in HIV-infected patients. EVs containing Nef protein and TAR RNA have been found in patient plasma even after the administration of combination antiretroviral therapy (cART), making them potential markers for residual viral activity detection (93). Moreover, EVs are also antiviral, some APOBEC3G and cyclic GMP−AMP (cGAMP), which interfere with viral replication and trigger antiviral signaling in target cells (85). This seemingly contradictory role, facilitating HIV transmission on one hand and blocking it on the other, indicates a multifunctional role of EVs that could be harnessed for therapeutic purposes ( Table 3 ).

New studies have confirmed that HBV-infected hepatocytes secrete EVs-CD63⁺/CD81⁺ exosomes containing HBV DNA and intact virions. They demonstrated that these HBV DNA-loaded exosomes infect naive hepatocytes via a ceramide-dependent, ESCRT-independent pathway; these exosomes are resistant to neutralizing antibodies (100). They extended their findings to patient sera and showed that exosomes infect NK cells and inhibit their function (101). Additionally, they demonstrated that exosomal virions carry large surface antigens (LHBs) and enter target cells through NTCP-dependent entry, establishing an alternative entry pathway of HBV. HBV-derived EVs radically reorganize innate immunity. They demonstrated that exosomes from infected hepatocytes increase PD-L1 expression in monocytes and macrophages, inhibiting T-cell activation through PD−1 signaling (101). At the same time, pathogen-induced exosomes both stimulate NK cell activity through MyD88/TICAM-1/MAVS pathways and inhibit IL−12 through microRNAs (e.g., miR−21, miR−29), reducing innate antiviral immunity (71). Proteomics also identified complement factor, lipopolysaccharide-binding protein, and extracellular matrix protein enriched within plasma EVs, linking them to disease progression and potential as biomarkers in chronic hepatitis ( Table 3 ) (53).

HCV also hijacks EVs for infectivity and immune evasion. eHAV-like EVs from HCV consist of stable viral RNA-AGO2-HSP90-miR-122 complexes, shielding the RNA from neutralizing antibodies and facilitating target-cell entry (68). EVs also transfer glycoproteins E1/E2 and immunomodulatory miRNAs (e.g., miR−19a, miR−192), triggering fibrosis and inhibiting immune responses through RUNXOR-mediated induction of ARG1, iNOS, STAT3, and ROS-induced T-cell apoptosis. In contrast, EVs secreted by non-parenchymal liver cells (e.g., activated macrophages or LSECs) bearing type I/III interferons or antiviral miRNAs may suppress HCV replication, which suggests opposite roles based on their origin (102). EVs are emerging as promising therapeutic delivery platforms. In HBV, Tenofovir-treated patient serum EVs and macrophage EVs exert antiviral activity, inhibiting HBsAg, HBeAg, HBV DNA, and cccDNA in hepatoma cells—processes mediated by exosomal lncRNA HOTTIP (103). Furthermore, HBsAg-bearing exosomes or adjuvant-modified exosomes elicited robust CTL responses, enabling antigen cross-priming (103). In HCV, EVs carrying let-7f, miR-145, miR-199a, and miR-221delivered from MSCs strongly inhibit viral replication, while strategies targeting EVs release pathway (e.g., ESCRT, ceramide) also reduced HCV infection in vitro (100).

EVs resemble enveloped viruses in size, composition, and biogenesis which makes conventional separation methods challenging (104). This biophysical mimicry has been exploited for therapy: ACE2-positive EVs (ACE2-EVs), either endogenous or genetically modified, act as decoys by binding the SARS-CoV-2 spike protein and blocking viral attachment to host cells (105). It has been demonstrated that TMPRSS2-enhanced ACE2-extracellular vesicles (ACE2-EVs) neutralize spike-pseudotyped lentiviruses with significant efficacy, which is more potent than the soluble form. Moreover, circulating ACE2-EVs from COVID-19 patients neutralized wild-type and variant strains (α, β, δ) in vitro, and protected hACE2-transgenic mice against lung injury (106). Several studies have designed ACE2-expressing EVs as antiviral treatments, showing that intranasal administration of engineered EVs−ACE2 profoundly blocked SARS−CoV−2 pseudovirus infection in mice (107). Nanodecoys, as reported in a study that included ACE2 and cytokine−receptor EVs, not only neutralized the virus but also bound inflammatory cytokines such as IL−6 and GM−CSF, thereby reducing pulmonary injury in pneumonia mouse models (108). In another study it is shown that ACE2−EVs are 500–1000 times more potent than spike−EVs for certain strains, although the potency of EV decoys may vary with modified variants such as Omicron (109). ACE2−EVs exhibit significantly higher blocking activity compared to soluble recombinant ACE2—approximately 135−fold higher potency in spike−binding assays, and provide 60-80-fold higher protection in cell culture and animal models (110). Besides their antiviral decoy function, EVs are also used as vaccine vectors. EVs expressing the SARS-CoV-2 spike protein have been employed in preclinical studies, eliciting robust humoral and cellular immune responses in mice, even without the use of adjuvants. EV-mediated antigen diversion improves antigen presentation and neutralizing antibody responses, offering a safer alternative to viral vectors ( Table 3 ) (111).

This makes EVs very promising as diagnostic tools for identifying viral infections. Their ability to encapsulate and transport viral proteins, RNA, and intact viral particles makes EVs an easily accessible source of viral biomarkers. For example, in HBV and HCV-infected patients, extracted EVs carry viral RNA that can be identified using technologies such as PCR (9). Similarly, EVs isolated from HIV and influenza patients contain viral materials that reflect the infection condition. Therefore, they can be very reliable for noninvasive diagnosis (123). Another advantage of EVs is that they can be easily separated from readily accessible biological fluids, such as blood, saliva, and urine, thereby further enhancing their diagnostic utility. Traditional diagnostic technique often relies on biopsy, which is invasive and risky. In contrast, EV-based diagnostics have the potential to be less invasive and more effective in monitoring viral infections. For example, circulating EVs present in the plasma of patients with COVID-19 contain viral RNA and are therefore potentially used as targets for the early detection of SARS-CoV-2 infection (98, 124).

The significance of their role as diagnostic agents, EVs, is vital for determining the course and prognosis of viral infections. Generally, the content of the majority of EVs reflects the progression of disease and may be associated with the host immune response or the level of viral spread. For example, in HIV infection, the number of EVs containing viral proteins and host molecules, such as pro-inflammatory cytokines, correlates with the viral load and the degree of immune activation, predicting disease progression (125). In chronic viral infections like HCV, a specific set of miRNAs is trapped inside EVs, including miR-122, which is involved in liver disease progression and fibrosis (68). Its monitoring provides prognostic value, which potentially leads to clinical decisions regarding antiviral therapy or even transplantation at advanced stages. Additionally, research on EVs carrying viral RNA and proteins in emerging viral infections, such as Zika and Ebola, shows that they carry transport viral RNAs and proteins, which have been used to evaluate disease-level patient prognosis (11, 123).

EVs, particularly exosomes, offer an exciting delivery system for antiviral drugs because they naturally carry bioactive molecules from one cell to another. EVs enable the direct delivery of therapeutics to infected tissues. They are biocompatible, allowing them to cross biological barriers such as the blood-brain barrier, making them ideal candidates for targeted drug delivery systems (126, 127). EVs can be engineered to transport antiviral agents, including small-molecule drugs, nucleic acids like siRNA and mRNA, and proteins that suppress viral replication or enhance host immune responses (11). For example, exosomes filled with antiviral siRNAs have been studied as a therapeutic strategy to silence key viral genes in HCV infection (128). Loading EVs with therapeutic siRNAs reduces off-target effects and lowers the toxicity of the treatment compared to systemic administration of the drug. Further, new antiviral therapies are being developed with EVs, including the delivery of CRISPR Cas systems aimed at correcting the viral genome in infected cells ( Table 6 ) (137, 138).

They have also demonstrated significant potential in developing vaccines and immunotherapies for viral infections. Exosomes derived from either infected or antigen-presenting cells can carry viral antigens, making them valuable tools for stimulating the immune response (139). These EVs trigger both innate and adaptive immune pathways, which are essential for an effective vaccine response. For example, exosomes containing viral proteins can provoke a potent immune response against HIV. Various studies have shown that exosomes pre-loaded with HIV antigens can be stimulate T-cell responses with broad specificity, offering a new approach for vaccine development (69). This method is also being utilized in the development of vaccines against emerging viruses, such as SARS-CoV-2. Such vaccines may include viral spike proteins or other epitopes to trigger the production of neutralizing antibodies (140). In tumor immunotherapy, EVs have already proved their concept by presenting tumor antigens. Similarly, in viral infections, these principles are applied in the use of EV-based vaccines, which now show promise for the inducing long-term immunity (115).

EVs can be engineered for targeted therapies. By modifying the surface proteins of EVs, researchers can design them to target specific virus-infected cells or tissues. This specificity enhances therapeutic outcomes by minimizing the risks of side effects on healthy tissues and targeting antiviral agents where they are needed (139). For example, exosomes can be designed to deliver surface ligands that bind specifically to receptors on infected cells, such as CD4 receptors in HIV-infected cells (139, 141). In this case, targeted delivery increases the effectiveness of the therapeutic agents by delivering their payload directly to the infection site, enhancing the antiviral response.

Additionally, EVs can be engineered to carry immune-modulating molecules, which can boost host immunity against the virus and further expand their therapeutic potential. Another promising area involves developing EVs as platforms for combination therapies. For example, EVs can be loaded with multiple therapeutic agents, such as a combination of antiviral drugs and immune modulators, leading to more effective and adaptable treatments for both chronic and acute viral infections. In case of HBV infections, EVs can deliver siRNAs along with nucleoside analogs to simultaneously inhibit viral replication through a dual blockade of the viral life cycle ( Table 6 ) (95).