Bacterial extracellular vesicles in inflammatory diseases: emerging roles and mechanisms.

EVs secreted by innate immune cells and bacteria play pivotal roles in regulating inflammation. Inflammatory cells, such as macrophages, secrete EVs that carry bioactive molecules, including lipids, proteins and miRNAs, to influence immune responses. For instance, macrophage-derived EVs promote osteogenesis and tissue regeneration in periodontitis by modulating M1/M2 macrophage polarization. In contrast, EVs from pro-inflammatory macrophages exacerbate alveolar bone loss by inducing cytokine release and disrupting bone metabolism [ 110 ]. As shown in Figure 2A , bacteroides acidifaciens and its derived extracellular vesicles improve dextran sulfate sodium (DSS)-induced colitis. Figure 2B illustrates how M2 macrophage-derived EVs can contribute to immune reprogramming, reversion of cell states and periodontal protection [ 111 ]. Figure 2C summarizes the results and conclusion of the effects of nonalcoholic steatohepatitis (NASH) fEVs (fecal-derived extracellular vesicles). fEVs contribute to intestinal barrier dysfunction. They carry lipopolysaccharide and act via the non-muscle myosin light-chain kinase (nmMLCK) pathway [ 112 ]. This mediates downstream liver injury. Figure 2D highlights how BEVs interact with host cells via PRRs, triggering inflammatory cytokine production and exacerbating tissue damage [ 111 ]. The dual roles of bacteria-derived EVs acting both as mediators of inflammation and as potential therapeutic agents open up a broad range of applications for EV-based therapies. However, these vesicles also introduce complexities that require careful consideration [ 113 ]. For instance, Hong et al . [ 54 ] observed that the hemoglobin-like cell nucleus of F. nucleatum was enriched in patients with rheumatoid arthritis and was positively correlated with the severity of rheumatoid arthritis. F. nucleatum also exacerbated arthritis in mice with collagen-induced arthritis (CIA). The outer membrane vesicles (OMVs) of F. nucleatum containing the pathogenic determinant FadA migrated to the joints and triggered a local inflammatory response. FadA acted on synovial macrophages, activating the Rab5a GTPase involved in vesicle transport and inflammatory pathways, as well as the key regulatory factor YB-1 of inflammatory mediators. While they may serve as promising vaccine candidates or therapeutic agents to modulate immune responses, their pro-inflammatory properties, particularly in chronic diseases, must be controlled to avoid exacerbating tissue damage [ 114 ]. Understanding the delicate balance between the beneficial and harmful effects of EVs is essential for their effective application in clinical settings [ 115 ], highlighting the need for targeted approaches that can modulate their inflammatory impact without compromising their therapeutic potential. It is worth noting that among various periodontal pathogens, OMVs derived from F. nucleatum play a unique and crucial role in the occurrence and development of periodontitis, and their independent pathogenicity and specific mechanism have been confirmed by the latest research [ 116 , 117 ]. Mechanisms of BEVs in inflammatory diseases: ( A ) the impact of gut microbiota and BEVs on the intestinal barrier. Reprinted with permission [ 110 ]. Copyright 2023, The Author(s). ( B ) Schematic representation of engineered M2 macrophage-derived BEVs for treating inflammatory bone loss in periodontitis through mediating ER stress and immune reprogramming. Reprinted with permission [ 111 ]. Copyright 2023, The Author(s). ( C ) Schematic illustration of the interaction between the intestinal barrier, endothelial cells and stellate cells in the context of gut—liver axis disruption. Reprinted with permission [ 112 ]. Copyright 2023, The Author(s). ( D ) Schematic diagram of engineered M2 macrophage-derived EVs inhibiting ER stress and cell apoptosis for periodontitis treatment. Reprinted with permission [ 111 ]. Copyright 2023, The Author(s). Recent studies show that OMVs from P. gingivalis and Aggregatibacter actinomycetemcomitans activate host cell inflammatory factor release, while M1 macrophage-derived EVs may contribute to periodontitis-associated bone loss. Conversely, M2 macrophage and dendritic cell EVs promote bone regeneration in animal models. Although bacterial EV applications in periodontal therapy are nascent, P. gingivalis OMVs show promise as vaccine candidates [ 118 ]. Periodontitis, a chronic inflammatory disease involving innate and adaptive immunity, is closely tied to immune cell-derived EVs. Modulating these EVs to alter cell behavior and differentiation may effectively combat periodontitis-related bone loss. In experimental periodontitis models, M1 and M2 macrophage EVs inhibited and promoted osteogenesis, respectively [ 16 ]. In vitro , M1 EVs suppressed mesenchymal stem cell (MSC) osteogenic differentiation, while M2 EVs enhanced mineralization. In vivo , preloading M1 EVs with miR-303-5p inhibitors significantly reduced alveolar bone loss [ 111 , 119 ]. The anti-inflammatory roles and mechanisms of bacterial and immune cell-derived EVs present a promising frontier in periodontal therapy. As periodontitis involves complex interactions between innate and adaptive immunity, EVs offer a strategic approach to mitigating inflammation and promoting osteogenesis, particularly those from M2 macrophages and DCs. Experimental evidence confirms that M1 macrophage EVs exacerbate bone loss, whereas M2 EVs enhance mineralization and tissue repair, especially when engineered to suppress pro-inflammatory miRNAs like miR-303-5p [ 119 , 120 ]. Chen et al . [ 121 ] found that M2 EVs promoted bone marrow MSC osteogenesis via multiple miRNAs and IL-10/IL-10R signaling, reducing bone resorption in periodontitis mice. Similarly, Cui et al . [ 111 ] reported melatonin-loaded M2 EVs enhanced osteogenic/cementogenic potential of human periodontal ligament stem cells (hPDLSCs) under inflammation. These EVs also drove M1-to-M2 reprogramming, mitigating inflammation and accelerating healing ( Table 1 ). Classification of anti-inflammatory studies of probiotic-derived BEVs by strength of evidence. DSS colitis mice (oral OMVs pretreatment) ↓Weight loss; improved clinical/histology; TJ changes (↑ZO-1/claudin-14, ↓claudin-2) ↓IL-1α/IL-1β/IL-2/TNF-α; ↑IL-10/TGFβ; ↓NF-κB/iNOS/COX-2; improved DAI/colon length In vitro CHOP siRNA; ER-stress inhibitors; No in vivo KO Mechanism inferred mainly from in vitro perturbations; in vivo lacks genetic validation [ 125 ] DSS acute; chronic colitis in IL-10 mice; Caco-2 assays Attenuated weight loss/colon shortening; restored TJ (ZO-1/claudin-1/occludin); ↓p–NF-κB p65/IκBα Prevented colitis; ↓injury; ↑MUC2; ↓permeability; ↓NO/TNF-α/IL-1β in vitro ; microbiota shifts DSS colitis mice; biodistribution; pseudo-germ-free ↑IL-10; improved survival/weight/DAI; epigenetic reprogramming (↑H3K4me1) TLR2 mediation shown in THP-1; trained-immunity marks; no in vivo KO Polymicrobial sepsis (FIP); macrophage phagocytosis assays ↑Survival; ↑bacterial clearance; ↓serum cytokines; ↓organ injury Correlative in vivo ; partial causal support from in vitro receptor blockade [ 129 ] ↓NF-κB activation; ↓IL-8-dependent) EV surface proteolysis test; partial dependence on SlpB inferred; no animal model In vitro only; effector implication (SlpB) via perturbation [ 130 ] UF-EVs more abundant/smaller; stronger ↓NF-κB/IL-8 Bacterial mutant (ΔslpB EVs reduced effect); cargo shifts by culture conditions; no animal model In vitro only; producer-factor dependence shown without in vivo testing [ 131 ] In vitro only; pathway modulation without functional genetic tests [ 133 ] Classification of probiotic/commensal-derived BEV studies into evidence categories A–C, noting in vivo anti-inflammatory outcomes and whether causal mechanisms were proven with host knockouts or bacterial mutants. This helps distinguish mechanistic causality from correlative pathway readouts across major BEV reports. Class A: Clear evidence of internal mechanisms (validation of gene manipulation or receptor/host gene necessity); Class B: Preliminary evidence of in vivo relevance (animal studies are effective, and the mechanism is derived from biomarkers or in vitro intervention inferences); Class C: Limited to in vitro observation results. BEVs contribute significantly to antibiotic resistance and systemic inflammation, complicating therapeutic interventions. A recent study by Burt et al . [ 75 ] demonstrated that OMVs from Klebsiella pneumoniae can bind to polymyxins, effectively acting as decoys that shield bacteria from antibiotic action. This protective mechanism is mediated primarily by lipid A, a component of LPS, which is enriched in OMVs. Under antibiotic stress, bacteria increase vesiculation and alter OMV lipid composition, enhancing their capacity to sequester antibiotics and promote bacterial survival [ 75 ]. Periodontal pathogen-derived OMVs are enriched with various virulence factors, such as muropeptides, LPS, fimbriae, gingipains, outer membrane proteins and proteases [ 134 , 135 ]. These OMVs act as representatives of parent bacteria, facilitating communication with other oral bacteria and host cells and adhering to tooth surfaces [ 136 ]. Other species in oral biofilms, such as Tannerella forsythia , also release OMVs associated with biofilm formation. OMVs can accelerate the progression of periodontitis by protecting other microorganisms from complement activity. For instance, Aggregatibacter-derived OMVs serve as decoys, relying on LPS to activate and deplete complement, thereby shielding serum-sensitive bacteria. Furthermore, Pg OMVs induce selective TNF deficiency, impairing the recognition of microbes by macrophages/monocytes. Beyond evading innate immune surveillance, OMVs can also escape attacks from adaptive immune cells. For example, OMVs derived from Aggregatibacter, Pg and Treponema denticola carry sRNAs that inhibit IL-13 and IL-5 release in Jurkat T cells. These findings suggest that periodontal pathogen-derived OMVs promote bacterial survival and aggregation through multiple mechanisms, exacerbating the pathological processes of periodontitis [ 137 ]. OMVs can activate the first line of defense in oral mucosal epithelium through various pathways to resist bacterial infection [ 138 , 139 ]. Pg OMVs are internalized by epithelial and endothelial cells via lipid raft-mediated endocytosis, facilitating the invasion of other pathogens such as T. forsythia [ 140 ]. After invasion, OMVs inhibit the migration and proliferation of oral epithelial cells, leading to dysfunction in periodontal tissue cells. For example, Pg OMVs, upon uptake by human periodontal ligament cells, activate the inflammasome, resulting in apoptosis and pyroptosis [ 141 ]. After breaching the oral epithelial barrier, OMVs enter submucosal tissues and directly interact with the host’s innate or adaptive immune cells. OMVs released by Pg, T. forsythia and T. denticola activate PRRs on macrophages and monocytes, increasing the production of TNF-α, IL-1β and IL-8. Similarly, Aggregatibacter OMVs trigger inflammatory responses in monocytes by activating the NOD1-dependent NF-κB signaling pathway. Additionally, F. nucleatum OMVs promote macrophage polarization toward a pro-inflammatory phenotype [ 116 ]. OMVs may further exacerbate the inflammatory environment by impairing neutrophil chemotaxis and phagocytosis, thereby enhancing their toxicity to gingival fibroblasts and periodontal tissues. The main pathway for F. nucleatum OMVs to enter hPDLSCs is caveolin-dependent endocytosis, which is significantly different from the endocytosis mode of other pathogenic bacteria OMVs [ 142 ]. Internalized OMVs significantly inhibit the migration ability of hPDLSCs, while activating intracellular ROS generation [ 116 , 143 ]. ROS, as a key upstream activator of the NLRP3 inflammasome, further amplifies the inflammatory cascade reaction [ 144 ]. Bacterial OMVs aggravate alveolar bone loss and tissue destruction by delivering toxic substances to susceptible cells in periodontal tissues. For instance, Aggregatibacter OMVs transmit cytotoxic distending toxins, promoting damage to human gingival fibroblasts and leading to the destruction of the junctional epithelium. Recent studies indicate that BEVs from both oral commensal bacteria and periodontal pathogens can trigger osteoclastogenic activity via TLR2 activation [ 145 ]. Moreover, Aggregatibacter OMVs directly induce alveolar bone loss by delivering PG. Similarly, F. nucleatum OMVs accelerate periodontal bone loss in a mouse periodontitis model by increasing osteoclast numbers and the production of inflammatory cytokines such as IL-1β, IL-6 and TNF-α [ 18 ]. In summary, periodontal pathogen-derived OMVs contribute to the formation of an inflammatory microenvironment and bone destruction in periodontal tissues by activating or degrading bioactive substances in host cells, inhibiting cell proliferation, promoting cell death and inducing the release of inflammatory cytokines. Inhalation of organic dust, a significant occupational hazard in concentrated animal feeding operations (CAFOs), is associated with respiratory inflammation and chronic lung diseases. Bioaerosols generated in these environments contain high concentrations of bacteria and their byproducts, including BEVs. Recent studies suggest that BEVs play a critical role in triggering pulmonary inflammation and tissue remodeling, contributing to the pathogenesis of organic dust-related respiratory disorders. Treatment of airway epithelial cells, THP-1 monocytes and macrophages with dust OMVs rapidly induces the expression of IL-8, IL-6, ICAM-1, pro-IL-1β and TNF-α. In airway epithelial cells, the induction of inflammatory mediators is associated with elevated mRNA levels and NF-κB activation [ 146 ]. Polymyxin B fails to inhibit the induction of inflammatory mediators by dust OMVs. Single and repeated exposure to dust OMVs increases levels of KC, IL-6 and TNF-α in mouse lungs, without significantly altering IL-17A levels. The increase in cytokines correlates with enhanced neutrophil infiltration in the lungs. Repeated exposure to dust OMVs results in increased mean linear intercepts and peribronchial collagen deposition in mouse lungs, indicating pulmonary remodeling. Post-treatment, mice exhibit bronchial cellular infiltration and thickened airway epithelium. Due to their nanoscale size, OMVs can reach and accumulate in bronchioles and alveolar regions, causing lung damage and contributing to the development of respiratory diseases. These findings provide new evidence for the presence of BEVs in organic dust and their role in pulmonary inflammation and injury [ 147 ]. In addition to BEVs, host-derived EVs also contribute to rheumatoid arthritis pathogenesis. Irreversible tissue damage in RA, such as cartilage erosion, is attributed to the secretion of matrix-degrading enzymes by synovial fibroblasts, some of which are associated with EVs. Synovial fibroblasts from RA patients secrete vesicles with polymerase activity. Compared to healthy individuals, RA patients exhibit higher levels of platelet-derived microvesicles in their EVs, and platelet-derived microvesicles (PMVs) counts correlate with disease severity. In one study, PMVs and leukocyte-derived microvesicles were detected in the synovial fluid of RA patients. Many studies have reported a decline in PMV counts in the circulation and inflamed joints of RA patients. PMVs carry pro-inflammatory cytokines, and synovial fluid microvesicles can induce coagulation, leading to fibrin deposition and joint inflammation. The abundance of procoagulant microvesicles in RA patients may contribute to cardiovascular comorbidities, as platelet-derived extracellular vesicles (PDEVs) exhibit procoagulant and pro-inflammatory properties, impair endothelial protective functions and induce angiogenesis. Microvesicles isolated from the synovial fluid of RA patients have profound biological effects, including promoting the synthesis of B-cell activating factor and increasing the production of IL-8, CCL5 (RANTES), CCL2 (MCP1), IL-6, ICAM-1 and VEGF by cultured synovial cells. However, the centrifugation methods used in these studies may result in microvesicle samples contaminated with protein aggregates, immunoglobulins and complement proteins. In studies focusing on the effects of leukocyte-derived microvesicles on synovial cells, the risk of protein aggregate contamination in EVs is reduced. Platelet-derived microvesicles can also stimulate synovial cells via IL-1β. Microvesicles from T cells and monocytes activate multiple signaling pathways in synovial fibroblasts, including NF-κB, AP-1, p38 MAPK and JNK. Upon activation, synovial fibroblasts upregulate cyclooxygenase-2 and prostaglandin E synthase, induce the release of matrix metalloproteinases (MMPs), and increase the production of pro-angiogenic chemokines. In summary, microvesicles derived from platelets and leukocytes in the synovial fluid of RA patients play a key role in inflammation and local joint destruction by activating synovial fibroblasts. Additionally, increased EV secretion in RA patients may elevate the risk of cardiovascular disease. From a biomarker perspective, synovial microvesicle profiles are characteristic of RA; compared to patients with osteoarthritis (OA) or juvenile idiopathic arthritis (JIA), RA patients exhibit significantly higher numbers of CD3 - CD8 + T-cell-derived microvesicles [ 148 , 149 ]. The immunomodulatory effect of BEV is much more than the activation of the TLR2/4-NF-κB/MAPK pathway. Different BEVs carry different molecules, and host cells sense them through a variety of pattern recognition receptors, with widely varying effects. For example, some BEVs activate NOD2 via peptidoglycan [ 150 ], some activate endosome TLR9 via DNA [ 151 ] and some are recognized by C-type lectin receptors via surface polysaccharides, which do not cause inflammation but promote immune tolerance [ 152 ]. The content itself is also very particular: the lipopolysaccharide of the probiotic BEV, because of its different structure, only mildly induces IL-10. However, LPS from pathogenic bacteria will continuously activate NF-κB and trigger a strong inflammatory response. The same is true for lipoproteins and nucleic acids, where subtle differences in structure determine whether they are ultimately pro- or anti-inflammatory. For example, the LPS of probiotic bacteria such as B. thetaiotaomicron features a penta-acylated, monophosphorylated lipid A structure that elicits a markedly attenuated inflammatory response compared to pathogenic variants. Subtle variations in fatty acid composition and phosphate placement within the lipid A moiety can produce 100- to 1000-fold differences in LPS potency in a TLR4-dependent manner [ 153 ]. Similarly, the number and length of acyl chains within lipid A directly correlate with the capacity of LPS to activate TNF production by macrophages [ 154 ]. It can be said that BEV acts like an informative package, and the specific morphology of its contents directly determines how the host cell interprets it. The regulation also shows strong cell and tissue specificity. When the same BEV encounters intestinal epithelial cells, macrophages or DCs, the response can differ substantially among cell types, as demonstrated by single-cell RNA sequencing and host-microbe protein-protein interaction network analyses showing that BEV targets differ among dendritic cells, macrophages and monocytes in a cell-type-specific manner [ 155 ]. For intestinal epithelial cells, it mainly helps to strengthen the barrier; macrophages induce anti-inflammatory cytokines such as IL-10 and establish immune tolerance. Cell-specific PRR expression explains much of this difference. Tissue microenvironments add further regulation. Mucus retention, hypoxic clearance and chronic exposure tolerance all contribute. These factors make BEV functions highly flexible [ 155 ]. Under pathological conditions, this fine regulatory network is often disrupted. The anti-inflammatory response of immune cells to BEV is significantly attenuated in inflammatory bowel disease (IBD) patients, while the macrophage response to BEV even changed from anti-inflammatory to pro-inflammatory in people with the NOD2 gene mutation. In the hepatocellular carcinoma (HCC) microenvironment, tumor-associated macrophages fail to respond to certain signaling pathways of BEV and become immunosuppressive. In addition, BEV can leave long-term effects through epigenetic and metabolic reprogramming, such as maintaining high expression of anti-inflammatory factors in some cells for weeks afterwards [ 128 ]. For instance, exposure to P. aeruginosa OMVs has been demonstrated to induce genome-wide loss of DNA methylation at enhancer and DNase hypersensitive site regions in human lung macrophages, indicating that BEVs can directly remodel the host epigenetic landscape [ 156 ]. Furthermore, BEVs transport substantial amounts of tRNA-derived fragments (tRFs) that can form stable secondary structures in the host cytoplasm and post-transcriptionally regulate immune gene expression, providing a mechanistic basis for the sustained anti-inflammatory phenotype [ 157 ]. The pathogenic and probiotic BEVs also have diametrically opposite effects on mitochondrial function, with one activating the inflammasome and the other suppressing excessive inflammation through mitophagy [ 158 ]. Mechanistically, pathogenic BEVs can trigger mitochondrial dysfunction that leads to K + efflux and subsequent assembly of the NLRP3/NLRC4 inflammasome complexes, which activate caspase-1 and caspase-11 to drive pro-inflammatory cytokine release and pyroptosis. In contrast, probiotic BEVs have been shown to enhance mitophagy, the selective autophagic degradation of damaged mitochondria, and negatively regulate NLRP3 inflammasome activation and thereby restrain excessive inflammation [ 159 ]. These interconnected mechanisms form a complex network. This explains how BEVs can both drive and resolve inflammation [ 155 ].

Extracellular vesicles (EVs) have gained considerable attention for their roles in various biological processes, particularly their mechanisms in inflammatory diseases [ 1–3 ]. Inflammation represents a defensive reaction of the organism against noxious stimuli. Such stimuli encompass pathogens, damaged cells or irritants. Inflammation assumes a vital physiological function in the maintenance of internal homeostasis. Additionally, it triggers tissue repair [ 4 ]. However, when inflammation becomes chronic or dysregulated, it can lead to tissue damage and a series of pathological changes. Bacterial extracellular vesicles (BEVs) participate in this process by delivering pathogen-associated molecular patterns (PAMPs), which bind to pattern recognition receptors (PRRs) on various cell types, including macrophages, dendritic cells (DCs), epithelial cells and even endothelial cells [ 5 ]. Receptor activation promotes the secretion of pro-inflammatory cytokines and chemokines. This incites local inflammation. Unchecked, it may become systemic [ 6–8 ]. Recent advances have revealed that BEVs are intimately involved in the pathogenesis of many inflammatory diseases. These range from chronic respiratory ailments to gastrointestinal disorders. They also include systemic conditions such as sepsis and neuroinflammatory disorders [ 9–13 ]. BEVs are nanoscale membrane-bound vesicles released by bacteria, with diameters ranging from 20 to 400 nm [ 14 ]. BEVs carry proteins, lipids, nucleic acids and metabolites. This cargo transfers to recipient cells without direct bacterial contact [ 5 ]. The cargo of BEVs exhibits remarkable diversity, which reflects the physiological state of the bacterial producer. These cargo molecules encompass classical virulence determinants, such as toxins and adhesins, as well as molecules that regulate host immune function, including nucleic acids and small regulatory RNAs [ 15 , 16 ]. Upon interacting with host cells, these EVs initiate immune recognition through surface binding to receptors. These receptors encompass TLR2, TLR4, TLR7 and TLR8. Additionally, they activate intracellular pathways related to nucleotide-binding oligomerization domain (NOD)-like receptors [ 8 , 17 ]. This activates downstream cascades, including NF-κB and MAPK pathways. Ultimately, this activation leads to the secretion of pro-inflammatory mediators. These mediators include interleukin (IL)-1β, IL-6 and tumor necrosis factor-α (TNF-α). They also include chemokines that coordinate recruitment of additional immune effector cells [ 10 ]. This intercellular communication allows BEVs to modulate host immunity. In turn, they influence inflammatory disease progression. Studies have shown that BEVs exhibit dual regulatory roles in inflammatory diseases [ 18 ]. Pathogenic BEVs often carry virulence factors that induce pro-inflammatory responses. This leads to tissue damage and disease progression. In contrast, BEVs from commensal or probiotic bacteria may exert anti-inflammatory effects. These effects help restore immune homeostasis and alleviate disease symptoms [ 19–23 ]. This review summarizes recent advances in EV research. We focus on mechanisms relevant to inflammatory diseases. We will delve into the biological significance of inflammation, the stages of the inflammatory cascade, as well as the origin, structural features and molecular composition of BEVs. This article will analyze the dual regulatory mechanisms of BEVs in inflammation. We will explore their potential therapeutic applications in inflammatory diseases. We will also assess the challenges and prospects for clinical translation. Better understanding of BEV-host interactions could lead to new anti-inflammatory strategies. Clinical outcomes may improve as a result.

BEVs are nanoscale membrane-bound structures released by both Gram‑negative and Gram‑positive bacteria through outer membrane budding or cell lysis and include subtypes such as OMVs, CMVs and OIMVs [ 102 , 220 ]. These vesicles transport a range of biomolecules like proteins, lipids and nucleic acids. They are vital for bacterial survival, immune modulation and material exchange [ 223 ]. BEVs play dual roles in inflammation. Pathogenic BEVs carry LPS and other virulence factors. They activate inflammatory pathways and induce cytokine release. This exacerbates tissue damage in periodontitis and rheumatoid arthritis [ 220 ]; conversely, BEVs from symbiotic or probiotic bacteria exert anti-inflammatory effects by promoting M2 macrophage polarization, enhancing tight junction protein expression and supporting microbiota balance [ 224 ]. For instance, BEVs from Akkermansia muciniphila alleviate colitis, while probiotic‑derived BEVs inhibit bone resorption [ 128 ]. The nanocarrier properties of BEVs make them highly promising for applications in vaccine development, targeted drug delivery and the treatment of inflammatory diseases [ 225 ]. OMV-based vaccines, such as the meningococcal vaccine, demonstrate strong immunogenic potential. Additionally, engineered BEVs show high efficacy and low toxicity in cancer and infection treatments [ 226 ]. Despite these advantages, several challenges hinder their clinical translation, including product heterogeneity, the need for optimized targeting specificity and biocompatibility, large-scale production and long-term safety data. Three actionable research priorities emerge from the current state of the field. (i) Integrated manufacturing and Quality-by-Design (QbD) frameworks: To overcome BEV heterogeneity and enable scalable clinical production, closed-loop dynamic bioreactor systems must be paired with inline microfluidic separation and real-time process analytical technologies (PAT) to shift production from static flask-based cultures to GMP-compliant pipelines. The QbD approach, entailing systematic identification of critical quality attributes (CQAs), critical process parameters (CPPs) and in-process analytical methods, has been successfully applied to the purification of bacterial outer membrane vesicle-based vaccines (GMMA platform) and provides a regulatory-endorsed framework for BEV manufacturing development [ 227 ]. (ii) Target-specific delivery and conditional activation: Next-generation BEV engineering should move beyond bulk cargo loading toward conditional activation strategies such as protease-activatable surface ligands or pH-responsive linkers that confine therapeutic action to disease sites, thereby enhancing bioavailability and precisely controlling immunogenicity. (iii) Clinical validation focused on unique BEV properties: Clinical trials should prioritize therapeutic endpoints where BEVs offer functional advantages over synthetic nanocarriers, such as mucosal vaccination for respiratory pathogens or the restoration of homeostatic immune tone in chronic inflammatory disorders, validating both safety and unique efficacy in well-defined patient cohorts. As these three axes converge, BEV-based therapeutics may transition from proof-of-concept studies into a distinct class of biologics, where the informational complexity of the bacterial vesicle is no longer a manufacturing obstacle but a therapeutic asset.

The clinical translation of BEVs as therapeutic agents faces several significant challenges, primarily due to their heterogeneity, cargo loading and delivery, bioavailability, scalability and ethical considerations [ 194 ]. BEVs are highly diverse in size, cargo, membrane composition and biological function, which complicates their isolation, characterization and standardization [ 195 ]. This variability impedes reproducibility and accurate data interpretation, hindering the development of reliable therapies [ 195 , 196 ]. Additionally, efficiently loading therapeutic cargos into BEVs and ensuring controlled release at target sites is challenging [ 197 ]. Innovative delivery methods such as 3D bioprinting show promise, but targeted delivery remains an issue as EVs can be taken up by various cell types [ 198 ]. Bioavailability presents another obstacle. BEVs are rapidly cleared by the immune system after administration. Enzymatic degradation further limits their stability and effectiveness [ 199 ]. Scalable production methods are also needed to meet clinical demands, as current culture systems are limited to two-dimensional static flasks [ 200 ]. Advances in 3D cell culture and dynamic bioreactor systems may address these scalability issues. Ethical concerns related to sourcing biological materials from donors and ensuring proper consent further complicate clinical applications, especially given the lack of a universally accepted international directive for EV use in clinical studies [ 201 ]. Many BEV activities are hybrid: the membrane can determine uptake route and endosomal escape, enabling cargo delivery, while cargo can tune the magnitude or direction of PRR-driven responses. In our opinion, when the mechanisms of loading effect and carrier effect are not yet fully understood, the key issue in drug design is not the cargo or the membrane, but rather, what is the dominant controllable knob under a certain therapeutic endpoint? Practical discriminators include biochemical protection assays to localize active principles to lumen vs surface [ 202 ]; host receptor perturbation to test membrane-PAMP dependence [ 160 , 203 ]; and composition engineering to shift signaling without changing cargo identity [ 204 ]. Reviews caution that purification can leave contaminants and that genetic deletions may confound fitness or vesiculation, underscoring the need for orthogonal controls and potency assays tied to mechanism [ 202 ]. As a regenerative biomaterial, the clinical translation challenges of BEVs are more complex than those of common mammalian extracellular vesicles. Three core challenges are reflected: the endotoxin problem, the drift of immunogenicity caused by engineering, and the regulatory dilemma caused by cross-species sources. The LPS carried by BEVs is not only the functional basis of BEV but also the source of its toxicity. This homology of function and toxicity property poses a dilemma for drug development. Conventional methods poorly remove membrane-integrated LPS. Strong detergents destroy vesicle structure. Polymyxin affinity chromatography removes less than 20%. Ultrafiltration and size exclusion are largely ineffective. More troublesome is that the structure of LPS from different strains is different, and improper processing will either sacrifice the activity or cause instability from batch to batch. Therefore, it is necessary to redefine the standard of endotoxin in quality control and combine the limbocyte lysate method with the TLR4 reporter gene detection at the cellular level to establish the limit standard of ‘functional endotoxin unit’. Functional readouts attributed to BEVs can be confounded by ‘pseudo-effects’ from endotoxin that is free in solution or co-purified with vesicles, other co-isolated soluble bacterial products or viable bacteria carried over into the vesicle preparation. Because OMVs intrinsically carry potent pathogen-associated molecular patterns, particularly LPS, many host responses can be driven by TLR4 signaling unless explicitly controlled [ 205 , 206 ]. The controls below are widely recommended to distinguish vesicle-intrinsic activities from these artifacts ( Table 4 ). Key control experiments to distinguish true vesicular functions from artifacts. Use chromogenic or gel-clot LAL kits on vesicle preparations, comparing against a buffer blank; often involves dilution to fit a standard curve [ 208 ] Relies on engineered cell lines which may lack the full physiological complexity or alternative receptors of primary immune cells; free LPS vs. OMV-bound LPS can behave differently [ 205 , 207 ] Polymyxin B binding efficiency can be reduced by specific LPS structural modifications; it may not neutralize all diverse LPS chemotypes completely [ 207 ] Preparation is sterile; effects are due to the vesicles or co-isolated nonliving components [ 208 , 209 ] Immunogenicity drift caused by engineering modification is another potential hazard. Whether it is drug delivery, surface modification or construction of eukaryotic/prokaryotic hybrid vesicles, it may change the original immune recognition characteristics of BEV or even induce unexpected immune responses. In terms of raw materials, cultivation conditions and plasmid stability of engineered strains can affect the yield and purity of BEV. At the product level, a multiparameter quality control system should be established for particle size, density, charge and drug loading efficiency. The stability assessment should take into account the mechanical stress caused by storage, in vivo environment and even atomization inhalation. Nonclinical safety evaluation also faces the issue of species selection, and humanized TLR4 mouse models or nonhuman primates may need to be considered. A pragmatic preclinical safety pipeline for BEV therapeutics could thus proceed in three sequential stages. First, GLP-compliant repeated-dose toxicology studies should be combined with multiplex cytokine profiling to map the systemic inflammatory landscape, as has been recommended for EV-based therapeutics in recent preclinical evaluation frameworks [ 210 ]. Second, given the central role of LPS-TLR4 interactions in BEV-mediated immunotoxicity, confirmatory immunotoxicity testing in humanized TLR4/MD-2 mice—which express human TLR4 and MD-2 from BAC transgenes on a mouse TLR4/MD-2 double-knockout background and enable species-specific TLR4 response discrimination—should be performed on the most promising candidates [ 211 ]. Third, for BEV formulations intended for systemic or mucosal administration, radiolabeled biodistribution studies are essential; recent work demonstrated that E. coli OMVs labeled with 64 Cu can be quantitatively tracked in mice using PET/MRI imaging, with the highest organ uptake observed in the liver and spleen at 3, 6 and 12 h post-administration [ 212 ]. The breakthrough lies in multidimensional collaboration: establishing a new endotoxin release standard with function as the end point, developing a prediction platform for the immunogenicity of engineered BEV, promoting the dialogue between regulators and the academic community to form chemistry, manufacturing, and controls (CMC) guidelines for BEV products, and exploring a feasible path for registration of combined products. These problems require systematic solutions. BEV-based therapies can then transition from laboratory studies to clinical practice [ 213 ] ( Table 5 ). Bacterial EV subtypes, cargo and expected host immune responses. Surface-exposed LPS; outer-membrane proteins; periplasmic components PG fragments packaged/transported into host cells; outer membrane scaffold that enables entry NOD1/NOD2 → RIP2 → NF-κB after OMV-mediated PG delivery; responses can be TLR-independent in vivo in some settings NF-κB-driven epithelial inflammatory programs; innate and adaptive immune activation dependent on NOD1 [ 214 , 215 ] TLR5 → NF-κB/MAPK; NLRC4/caspase-1 activation when flagellin reaches cytosol Pro-inflammatory cytokines; pyroptosis signatures when cytosolic sensing occurs [ 160 ] Double bilayer; cytoplasmic material including DNA and ATP; inner-membrane and cytosolic proteins Increased likelihood of endosomal TLR9 and cytosolic DNA sensors; also delivers other MAMPs that can co-stimulate TLR2/TLR4 Highly heterogeneous; envelope fragments reassemble; can enclose released DNA and cytoplasmic material; higher nucleic-acid-positive fraction during lysis Enriched nucleic acid cargo shifts sensing toward TLR9, TLR8, AIM2 inflammasome; cytosolic access can also enable caspase-4/5/11 → pyroptosis and downstream NLRP3; DNA can also drive cGAS-STING-IRF3 → IFN-β Increased propensity for type I IFN and inflammasome-linked outcomes, relative to strictly budding OMVs; response breadth expected to be larger due to cytoplasmic cargo [ 160 , 217 , 218 ] DNA, RNA, PG; lipoproteins TLR2, TLR7/8/9, NOD2; MV cargo can also be consistent with NLRP3 engagement via lipoprotein-related pathways Designed antigen display; retain intrinsic PAMPs unless detoxified Can induce DC maturation and MyD88-dependent programming for cross-presentation; also inherently engage PRRs such as TLR2/4/5 and intracellular NOD/TLR9 depending on cargo To enhance the reproducibility and scientific rigor of BEV research, a minimum set of quality control standards should be applied across isolation, characterization, functional studies and data reporting. During sample preparation and purification, strain cultivation conditions, including origin, medium type, culture time distinguishing between log phase and stationary phase, pH and temperature, must be recorded to avoid vesicle release artifacts induced by nonphysiological stress. For vesicle separation, differential centrifugation, such as 10 000× g filtration followed by 100 000× g ultracentrifugation for at least 2 h or size exclusion chromatography, is recommended, while reliance on filtration alone should be avoided. Contamination controls are mandatory: a negative control consisting of sterile culture medium processed in parallel must be included, and bacterial lysis contamination should be verified by qPCR targeting genomic DNA or by Western blot for intracellular proteins such as GroEL or EF-Tu [ 221 ]. Basic characterization of BEVs requires three essential items. Morphology should be confirmed by transmission electron microscopy to reveal typical cup-shaped or spherical vesicles with a lipid bilayer. Particle size distribution, determined by nanoparticle tracking analysis or dynamic light scattering, should show a main peak between 20 and 200 nm, with a polydispersity index below 0.3 considered optimal [ 221 ]. Marker detection must include at least two BEV‑enriched components, such as OmpA, LPS or small RNAs, as positive markers, while the absence of intracellular protein contamination such as GroEL or ribosomal proteins should be verified as negative markers [ 201 ]. For sample analysis and credibility management, component identification by proteomics requires filtering out common contaminants like keratin and bovine serum albumin, and nucleic acid analysis should remove ribosomal RNA while verifying the specific packaging of small RNAs [ 222 ]. Reliability of findings should be labeled accordingly: high reliability for results verified by multiple independent experiments, specifically at least three, with no detection in negative controls; medium confidence for two-step verification where function is supported but with some fluctuations; and low confidence or suspected contamination for detections that occur only once or are also present in controls, which require annotation and cautious interpretation. Finally, data reporting and transparency demand full disclosure of methodological details, including centrifugation parameters, filter membrane pore size, storage conditions and number of freeze-thaw cycles, and raw data from sources such as mass spectrometry, nanoparticle tracking analysis or RNA-seq should be deposited in public databases such as Mass Spectrometry (ProteomeXchange) or the Sequence Read Archive.

BEVs have emerged as critical mediators of intercellular communication between the microbiota and the host, offering new opportunities for diagnosis and treatment of inflammatory diseases. In recent years, significant advances have been made in understanding the molecular composition, biogenesis, immunomodulatory functions and therapeutic applications of BEVs [ 160 , 161 ]. BEVs exhibit dual roles in inflammatory diseases, acting either as anti-inflammatory mediators or pro-inflammatory triggers, depending on their origin, cargo, modification methods, diameters and mechanisms of action [ 128 ]. Researchers have demonstrated that BEVs derived from commensal and probiotic bacteria can enhance intestinal barrier integrity, stimulate regulatory immune responses and reduce pro-inflammatory cytokine production. Conversely, BEVs from pathogenic bacteria may exacerbate inflammation by engaging PRRs and activating inflammasome pathways [ 162 ]. Their unique properties, such as biocompatibility, natural targeting ability and immunomodulatory effects, make them promising candidates for diverse applications in disease treatment. To clarify the distinct roles of BEV subtypes, their primary mechanisms and inflammatory outcomes are summarized in Table 2 . Subtype-specific mechanisms of BEVs in inflammatory regulation. Lipopolysaccharide (LPS), gingipains, FadA The therapeutic potential of BEVs extends far beyond metabolic regulation, with OMVs emerging as particularly versatile nanocarriers that demonstrate breakthrough capabilities across multiple therapeutic domains. Engineered OMV systems exhibit remarkable dual functionality, positioning them as next-generation multimodal nanoplatforms capable of addressing two critical healthcare challenges: cancer therapy and antimicrobial resistance. In targeted oncology therapeutics ( Figure 3A ), DOX-loaded OMVs derived from attenuated K. pneumoniae demonstrate rapid cellular uptake in NSCLC A549 cells, potent tumoricidal activity ( in vitro and in vivo ) and innate immunostimulatory properties through tumor-associated macrophage (TAM) recruitment, enabling synergistic chemoimmunotherapeutic effects [ 164 ]. Simultaneously, in precision anti-infective delivery, as shown in Figure 3B , antibiotic-encapsulated OMVs achieve localized intestinal drug retention (>36 h) with minimal systemic exposure, significantly enhancing pathogen clearance while potentially circumventing antibiotic resistance development [ 168 ]. This dual therapeutic paradigm establishes OMVs as a transformative platform for next-generation nanomedicine, bridging critical gaps in both oncological and anti-infective therapies. Figure 3C showcases a pH-responsive (6.8) combinatorial nanotherapy system that orchestrates sequential release of paclitaxel (PTX) and Redd1-siRNA, demonstrating remarkable multifactorial efficacy in triple-negative breast cancer—achieving 68% tumor suppression through PTX-mediated M2 macrophage glycolytic activation and subsequent TAM repolarization, while simultaneously remodeling the immune microenvironment and inhibiting metastasis [ 166 ]. Moreover, parallel innovations in genetic engineering have produced scalable tRNA-pre-miRNA OMV platforms, as illustrated in Figure 3D . These platforms leverage a unique tRNA scaffold mechanism to achieve 82% CXCR4 oncogene silencing through efficient eukaryotic processing of pre-miR-126, delivering potent antiproliferative effects in breast cancer models [ 167 ]. These advances move beyond passive drug carriers. New systems offer stimulus-responsive release, genetic modulation and microenvironment engineering. OMVs are now versatile platforms for next-generation therapeutics. BEVs in cancer therapy and inflammation: ( A ) overview of anti-NSCLC effect triggered by DOX-OMV. Reprinted with permission [ 164 ]. Copyright 2020, The Author(s). ( B ) Schematic diagram of the design of novel nanoantibiotics using an OMV-based drug efflux mechanism. Reprinted with permission [ 165 ]. Copyright 2019 Elsevier B.V. All rights reserved. ( C ) The impact of BEVs in tumor growth, metastasis and confinement. Reprinted with permission [ 166 ]. Copyright 2021, The Author(s). ( D ) Schematic illustration of the bacterial OMV over-expressed pre-miRNA as a delivery vehicle for 4T1 breast cancer therapy. Reprinted with permission [ 167 ]. Copyright 2022, The Author(s). IBD is one of the most extensively studied conditions with respect to BEV applications. BEVs isolated from gut commensal bacteria, particularly from B. thetaiotaomicron and Bacteroides fragilis , have demonstrated promising anti-inflammatory effects in several murine models of colitis. Administration of these BEVs has been associated with reductions in colonic inflammation, improved epithelial barrier function and modulation of cytokine profiles toward an anti-inflammatory state, marked by increased IL-10 secretion and suppression of TNF-α and IL-1β [ 155 , 169–171 ]. In addition, BEVs have been shown to condition bone marrow-derived monocytes to produce a higher IL-10/TNF-α ratio upon LPS challenge, suggesting long-term modulation of innate immune responses that may protect against recurrent inflammatory insults [ 128 ]. Recent studies have further explored the use of BEVs as delivery vehicles for therapeutic agents in IBD. For example, BEVs derived from E. coli Nissle 1917 have been engineered to encapsulate anti-inflammatory agents and demonstrate improved targeting to inflamed colonic tissues, leading to attenuation of experimentally induced colitis [ 161 ]. The incorporation of nanotechnology has enabled the development of ‘nanoprobiotics’ in which BEVs are encapsulated with nanoparticles to enhance their stability, bioavailability and targeted delivery to inflamed regions of the gut [ 169 ]. Such approaches have shown increased efficacy in reducing oxidative stress and inflammatory cytokine production, suggesting that BEVs may provide a minimally invasive alternative to conventional IBD therapies [ 172 ]. For instance, a very recent study by Chen et al . [ 173 ] demonstrated that Lactobacillus plantarum EVs restore the intestinal barrier, drive macrophage polarization toward M2 and markedly increase IL‑10 and TGF‑β while reducing pro‑inflammatory cytokines in DSS colitis, with clear improvement in disease scores and histology. In periodontitis, BEVs derived from periodontal pathogens and commensal species have been implicated in modulating local immune responses. While pathogenic BEVs from bacteria such as P. gingivalis are known to promote inflammatory cascades leading to tissue damage [ 174 ], beneficial BEVs from commensal species have the potential to counteract these effects by modulating dendritic cell responses and regulating cytokine production [ 172 , 175 ]. These findings raise the possibility of using engineered BEVs as therapeutic agents for periodontal regeneration and in reducing the inflammatory burden associated with chronic periodontitis [ 175 , 176 ]. In periodontitis treatment, BEVs from P. gingivalis (Pg-EVs) exhibit vaccine potential. Pg-EVs are structurally and functionally stable. They resist proteinase K degradation. They deliver bacterial components to the host immune system. Intranasal immunization with OMVs combined with the TLR3 agonist Poly(I: C) significantly reduced P. gingivalis colonization while elevating IgG (including IgG1 and IgG2a) and s-IgA levels in saliva and serum. Pg-EV safety was confirmed via intracerebral injection, with further studies indicating their strong immunogenicity and antigenicity, likely attributable to LPS and A-LPS-modified proteins in OMVs [ 177 ]. Thus, Pg-EVs or OMVs from other periodontal pathogens demonstrate potential as periodontitis therapeutic vaccines, offering bacterial clearance, safety, low cost, high efficacy and ease of transport. The ability to simultaneously deliver antigens and modulate immune responses positions BEVs as truly versatile platforms capable of addressing diverse medical challenges through a unified nanotechnology approach. Beyond gastrointestinal and periodontal inflammation, BEVs have also been explored in the context of metabolic and systemic inflammatory diseases. BEVs from Bacteroides species have been shown to enhance gut barrier integrity and modulate systemic metabolic responses, thereby ameliorating conditions such as nonalcoholic fatty liver disease (NAFLD) and type 2 diabetes mellitus (T2DM) [ 176 ]. These vesicles, often rich in enzymes and bioactive lipids, contribute to improved lipid metabolism and reduced systemic inflammation, which are critical for maintaining metabolic homeostasis in chronic inflammatory states [ 176 , 178 ]. Neuroinflammatory conditions, including Alzheimer’s disease (AD) and Parkinson’s disease (PD), represent another frontier for BEV application. BEVs are capable of crossing the blood–brain barrier (BBB) and modulating neuronal and glial functions. Research has demonstrated that BEVs derived from both pathogenic and probiotic sources can influence neuroinflammatory processes: while BEVs from pathogenic bacteria may activate microglia and astrocytes to produce pro-inflammatory mediators [ 179 ], BEVs from probiotic bacteria such as Lactobacillus have been associated with neuroprotective effects through the promotion of M2 macrophage phenotypes and increased production of brain-derived neurotrophic factor (BDNF) [ 179 ]. These dual roles illustrate the potential for employing BEVs in designing novel nanotherapeutic approaches aimed at reducing neuroinflammation and protecting neuronal integrity [ 179 ]. BEVs have also been leveraged for their diagnostic potential in inflammatory diseases. Recent pilot studies have demonstrated that BEVs circulating in blood or present in other body fluids can serve as biomarkers indicative of underlying inflammatory states. For instance, plasma-derived BEVs have been profiled in patients with IBD, revealing distinct differences in microbial diversity and EV cargo when compared to healthy individuals. Such findings suggest that BEV profiling may aid in the early diagnosis of inflammatory diseases and in monitoring treatment responses [ 129 , 180 ]. Endometriosis, a chronic inflammatory condition affecting reproductive tissues, has also been linked to altered BEV profiles. Recent studies have shown that BEVs in endometriosis patients carry elevated levels of pro-inflammatory cytokines that may contribute to disease progression and pain. Moreover, alterations in the cargo composition of BEVs from both host and bacterial origins have been implicated in promoting aberrant tissue remodeling and angiogenesis within endometrial lesions, suggesting that BEVs could serve as potential biomarkers and therapeutic targets in endometriosis [ 181 ]. Another promising application area involves the use of BEVs to modulate the gut-brain axis. As key mediators of interkingdom communication, BEVs derived from gut microbiota influence neural signaling, stress responses and cognitive functions. Preclinical evidence indicates that BEVs can ameliorate depressive and anxiety-like behaviors in animal models by modulating neuroinflammatory pathways and influencing central cytokine production [ 179 ]. These findings open new avenues for leveraging BEVs as adjunctive therapies in neuropsychiatric disorders with an inflammatory component. In summary, BEVs represent a novel and versatile platform for the modulation of inflammatory responses, offering opportunities not only to alleviate chronic inflammation but also to restore tissue homeostasis and protect against disease progression. Ongoing research continues to reveal new insights into their molecular mechanisms and therapeutic applications, paving the way for future clinical trials and eventual integration into the standard of care for inflammatory diseases [ 80 ]. OMVs exhibit a dual role in inflammatory processes, serving as both mediators of pathological inflammation and platforms for innovative therapies. While native OMVs can induce severe inflammatory responses through the release of pro-inflammatory cytokines (IL-6, IL-8, IL-1β, TNF-α) that cause vascular dysfunction and tissue damage [ 6 ], recent advances in bioengineering have transformed these vesicles into powerful therapeutic tools. Inflammation plays a critical role in tumor initiation and progression, making it a key therapeutic target. BEVs, owing to their strong pro-inflammatory properties, can activate host immunity and enhance immune cell infiltration into tumors, showing great potential in cancer immunotherapy. Ban et al . [ 182 ] developed a microbial nanocomplex by encapsulating oncolytic viruses (OVs) into calcium phosphate (CaP)-coated, pyranose oxidase (P 2 O)-engineered OMVs, thereby enhancing autophagy-mediated antitumor immunity ( Figure 4A ). The CaP shell protects OVs from immune clearance and improves tumor accumulation. Inside tumor cells, P2O generates ROS from glucose, promoting autophagy and viral replication, ultimately inducing cancer cell death. This strategy leverages OMV-mediated tumor microenvironment (TME) modulation to boost oncolytic efficacy. BEVs have also emerged as promising immunoadjuvants in tumor vaccine development. Zhang et al . [ 183 ] constructed HMVs by fusing cytomegalovirus-derived membranes expressing viral antigens with bacterial OMVs ( Figure 4B ). These HMVs, carrying native spike proteins and PAMPs, enhanced DC uptake and antigen presentation, markedly boosting B-cell activation, T-cell proliferation, DC maturation and antigen-specific antibody production in mice. BEVs in tumor therapy and immunization: ( A ) the biomineralized microbial nanocomposite engineered from OVs for autophagy-cascade-augmented immunotherapy. Reprinted with permission [ 182 ]. Copyright 2023, Springer Nature BV. ( B ) The development of virus-mimetic hybrid nanovesicles (HMVs) for vaccination, where spike protein-displaying SMVs and OMVs activate DCs and induce antibody production and T cell proliferation. Reprinted with permission [ 183 ]. Copyright 2022, The Author(s). ( C ) Preparation and therapeutic strategy of mU@OMVs for in situ cancer vaccination. Reprinted with permission [ 184 ]. Copyright 2023, The Author(s). ( D ) The preparation and the immune-protective mechanism of HMVs. Reprinted with permission [ 185 ]. Copyright 2024, The Author(s). Recent studies have combined BEVs with other therapeutic modalities to enhance antitumor responses. Zhuang et al . [ 184 ] developed a multifunctional OMV-based platform integrating MerTK inhibition and antigen delivery ( Figure 4C ). The MerTK inhibitor UNC2025 was encapsulated in maleimide-modified OMVs (mU@OMVs), which were engulfed by TAMs upon peritumoral injection. This blocked MerTK phosphorylation, induced secondary necrosis and enabled OMVs to capture tumor-associated antigens (TAAs) via thioether bonds. The co-delivery of TAAs and OMVs to lymph nodes stimulated potent CD8 + T cell responses, effectively suppressing tumor growth, metastasis and recurrence. This synergistic immune activation strategy has also been applied to infectious disease prevention. Jiang et al . [ 185 ] engineered HMVs by fusing K. pneumoniae OMVs with alveolar macrophage membranes for pulmonary immunization ( Figure 4D ). Administered via intratracheal instillation to mimic inhaled vaccines, HMVs rapidly recruited lung immune cells, induced pathogen-specific responses and established local mucosal immunity. In acute pneumonia mouse models (strains 43816 and 729088), HMV immunization significantly reduced bacterial load, improved survival and alleviated inflammation. These OMV-based strategies demonstrate the vast potential of engineered BEVs in immunotherapy, vaccine development and targeted delivery. However, challenges remain. Gram-negative bacterial vesicles contain virulence factors and can trigger systemic inflammatory response syndrome (SIRS), characterized by leukopenia, thrombocytopenia and elevated IL-6 and TNF-α levels. Studies show that blocking IL-6 or TNF-α improves survival in septic patients. Using EV-based screening, salbutamol, a β2-adrenergic agonist and nortriptyline, a tricyclic antidepressant were identified as effective inhibitors of IL-6 and TNF-α release in EV-stimulated macrophages. In sublethal SIRS models, salbutamol and nortriptyline significantly reduced inflammatory cytokine levels in a dose-dependent manner, and their combination synergistically suppressed IL-6 and TNF-α secretion. BEV inhibitors offer a promising strategy for treating bacterial infections and systemic inflammatory response syndrome (SIRS). Various chemical and natural compounds have shown inhibitory activity against BEV formation, release, host cell uptake and inflammatory responses [ 11 ]. These inhibitors not only function independently but can also be used alongside antibiotics to enhance efficacy and reduce resistance. Targeting BEVs is particularly crucial due to their multifaceted role in infection [ 10 , 87 , 186 ]. Gut microbes utilize BEVs to impair host defenses by neutralizing antimicrobial peptides (AMPs), promoting biofilm formation and facilitating toxin delivery [ 187 ]. For instance, V. cholerae OMVs transport cholera toxin (CT) into intestinal epithelial cells via GM1 receptor-mediated uptake, elevating intracellular cAMP [ 188 ]. Similarly, Listeria monocytogenes uses cytolysin-bearing CMVs to exert cytotoxic effects. Nonetheless, BEVs can also enhance host defense. OMVs deliver LPS to macrophages, activating caspase-11-dependent responses that promote bacterial clearance. Pretreatment with E. coli C25-derived OMVs has been shown to reduce bacterial internalization by inducing protective pro-inflammatory responses in epithelial cells. BEVs exhibit dual roles in inflammatory diseases. Pathogenic BEVs exacerbate inflammation via TLR/NF-κB pathways, contributing to diseases such as periodontitis and rheumatoid arthritis. In contrast, BEVs from commensals or probiotics can attenuate inflammation by promoting M2 macrophage polarization, enhancing epithelial barrier function and maintaining microbiota balance. As natural nanocarriers, BEVs hold promise in vaccine development like meningococcal vaccines and drug delivery. However, clinical translation faces challenges, including heterogeneity, targeting efficiency, large-scale production and long-term safety. Future research should focus on optimizing BEV isolation and engineering, exploring synergistic functions of BEV subtypes and validating therapeutic efficacy in clinical trials to advance their application in precision medicine ( Table 3 ). Application of BEVs in inflammatory diseases. Biomineral shells mask surface antigens of OMVs. Overexpressed Pyranose Oxidase (P 2 O) catalyzes the accumulation of excess H 2 O 2 within tumor cells. High oxidative stress triggers cellular autophagy. Genetic engineering Endogenous modification Genetic engineering Exogenous modification Prevent MerTK phosphorylation in tumor-associated macrophages (TAMs). Captures released antigens via the formation of universal thioether bonds. Utilize the intrinsic features of OMVs for lymph node targeting. Rapid mobilization of immune cells. Promotion of pathogen-specific immunity. Establishment of local mucosal immunity. Genetic engineering Exogenous modification Induces substantial tumor growth inhibition. Prime the immune system. Enhances the overall antitumor efficacy of Doxorubicin (DOX). Genetic engineering Exogenous modification Bacteria excrete vesicles via high efflux pump expression coupled with OMV secretion. Exerts passive and active bactericidal effects. Genetic engineering Exogenous modification Endogenous modification Dual regulation of the tumor microenvironment. Inhibition of tumor progression Suppression of tumor metastasis. Genetic engineering Exogenous modification Endogenous modification Utilization of a ‘tRNA scaffold’ camouflage. Significant inhibition of CXCR4 expression in breast cancer tissues. Significant restraint of tumor proliferation. Intracellular Delivery. Activation of proteasome-dependent antigen presentation pathways. Generation of subsequent robust CD8+ T cell responses. Genetic engineering Exogenous modification Endogenous modification Induction of photothermal-photodynamic responses. Activation of TRAIL-induced apoptosis. Genetic engineering Exogenous modification Endogenous modification Genetic engineering Exogenous modification Endogenous modification Activation of the BMP/SMAD signaling pathway. Enhances osteogenic differentiation Genetic engineering Exogenous modification Endogenous modification VEGF-Mediated Endothelial Activation Lactate-Induced Immunomodulation Suppresses adipogenic differentiation