Although a considerable body of research has been carried on the formation of BEVs, the underlying mechanisms remain incompletely understood. Key periodontal pathogens are mostly Gram-negative obligate and facultative anaerobic bacteria, including Porphyromonas gingivalis (P. gingivalis), Fusobacterium nucleatum (F. nucleatum), Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans), Tannerella forsythia (T. forsythia), and Treponema denticola (T. denticola) (16). The cell walls of Gram-negative bacteria are composed of lipopolysaccharides (LPS) and a thin layer of peptidoglycan, characterized by an outer membrane and an inner membrane separated by the periplasm. The production of Gram-negative bacterial membrane vesicles (MVs) involves mechanisms such as non-lytic vesicle formation (Type B) and explosive cell lysis with subsequent membrane fragment fusion into vesicular structures (Type E) (17). Bacterial outer membrane vesicles (OMVs) represent the primary form of BEVs released by periodontal pathogens. OMVs formation occurs when the expansion of the outer membrane outpaces the peptidoglycan layer, often due to the loss or repositioning of covalent bonds between these two structures (18) ( Figure 1 ). Additionally, peptidoglycan fragments or misfolded proteins may exert expansive pressure on the outer membrane, leading to OMVs formation (19, 20). Specific phospholipids, LPS, and other molecules that accumulate in the bacterial outer membrane can also induce OMVs production by altering membrane curvature (21). For example, OMVs formation in P. gingivalis may be facilitated by the upregulation of certain inner or outer leaflet lipids, linked to the selective incorporation of anionic lipopolysaccharides (A-LPS) and the C-terminal domain (CTD) family of proteins on the bacterial surface (22). Another proposed mechanism involves the VacJ/Yrb ABC phospholipid transporter system, which may regulate OMVs formation (23). OMVs lack DNA and RNA but are enriched in periplasmic proteins and lipids. Conversely, outer-inner MVs contain cytoplasmic components, likely due to the weakening of the bacterial peptidoglycan layer by endolysins (12, 21) ( Figure 1 ). The expression of phage-related endolysins can degrade the peptidoglycan layer, leading to explosive bacterial lysis and the formation of Type E MVs, which include explosive OMVs (EOMVs) and explosive outer-inner MVs (EOIMVs), both containing cytoplasmic contents (24) ( Figure 1 ).

Among periodontal pathogens, Gram-positive bacteria are relatively rare. However, recent oral microbiome studies have highlighted Filifactor alocis (F. alocis), a Gram-positive, obligate anaerobe, as an important periodontal pathogen (25). F. alocis has been frequently detected in chronic periodontitis, aggressive periodontitis and peri-implantitis (21, 26). Gram-positive bacteria, which possess a thick peptidoglycan cell wall, typically release EVs through explosive cell lysis, producing cytoplasmic MVs, a process referred to as “bubbling cell death.” (12, 27) ( Figure 1 ).

Proteins found in BEVs originate from the outer membrane, periplasm, and cytoplasm of the parent bacteria (18). Proteomic analyses have revealed a diverse array of proteins in BEVs, including structural proteins, porins, and transporters involved in various biological processes (28–30). Under specific conditions, certain proteins may be preferentially loaded into EVs. Gingipains, lysine-specific (Kgp) and arginine-specific (RgpA) proteases, are major virulence factors in P. gingivalis OM and OMVs (31, 32). The absence of RgpA reduces OMV secretion (33). Studies comparing P. gingivalis OMVs and outer membrane protein cargo have shown that CTD proteins derived from gingipains are concentrated on OMVs and lipoproteins involved in iron acquisition are also selectively sorted into OMVs (34). Under conditions of hemin excess, some moonlighting cytoplasmic proteins, with adhesive potential, are preferentially loaded onto OMVs, promoting P. gingivalis proliferation and co-aggregation with other bacteria in specific environments (35). Peptidylarginine deiminase (PPAD), detected in P. gingivalis OMVs (36), has been shown to be associated with OMV biogenesis through citrullination activity (37). PPAD also facilitates immune evasion and has been implicated in autoimmune diseases, such as rheumatoid arthritis (38).

OMVs produced by A. actinomycetemcomitans are rich in leukotoxin, which selectively kills host immune cells (39, 40). LtxA can be selectively sorted into large OMVs(>300 nm) due to surface-associated DNA driving (41). Additionally, A. actinomycetemcomitans OMVs can deliver cytolethal distending toxin (CDT) to HeLa cells and human gingival fibroblasts (HGFs), causing the characteristic cytolethal distending effect (42). CDT, a genotoxin, induces DNA damage in mammalian cells, leading to G2 cell cycle arrest, progressive cell enlargement, and/or apoptosis (43, 44). CDT toxicity has been linked to GSK-3-dependent cell cycle arrest in gingival keratinocytes (45).

OMVs derived from T. denticola contain adhesins and serine proteases necessary for adhering to and degrading host cells and mammalian matrix proteins (46). OMVs from T. forsythia harbor several virulence factors, including leucine-rich-repeat family virulence factor BspA, a Toll-like receptors 2 (TLR2) agonist, as well as non-TLR2 agonist virulence factors such as sialidase and GroEL (47).

Lipids are important structural components of BEVs, but their specific composition remains understudied. It has been shown that P. gingivalis can synthesize sphingolipids, which are delivered via OMVs and suppress host immune responses (48, 49). Lipid rafts play a crucial role in OMV-mediated endocytosis by host cells, as seen in both P. gingivalis and A. actinomycetemcomitans OMVs (42, 50).

LPS is the most abundant surface antigen in Gram-negative bacteria and also a critical structural and toxic component of BEVs (51). LPS consists of lipid A, a core oligosaccharide, and an O-antigen polysaccharide chain (52). P. gingivalis expresses two types of LPS: neutral O-LPS and anionic A-LPS, the latter of which is involved in OMV formation (22, 53). In addition, T. forsythia and T. denticola OMVs express low-molecular-weight lipooligosaccharides (54, 55).

BEVs carry genetic material, including DNA, mRNA, sRNA, and other non-coding RNAs from the parent bacteria, which can mediate horizontal gene transfer (HGT) between species (56). HGT is a crucial driver of gene and genome evolution (57). DNA and RNA have been detected in OMVs from P. gingivalis, T. denticola, and T. forsythia, meanwhile, they can activate TLR7, TLR8, and TLR9 receptors (54). Extracellular DNA (eDNA) has also been identified on OMV surfaces, forming an eDNA/OMV network that may aid in nutrient capture for pathogens residing on the surface of polymicrobial biofilms (54).

A. actinomycetemcomitans-derived OMVs contain extracellular RNA (exRNA), which is protected from enzymatic degradation in body fluids by encapsulation within the EVs (58). exRNA is transferred into host cells via OMVs and may be integrated into the host RNA-induced silencing complex, regulating host target transcripts. A. actinomycetemcomitans OMVs and exRNA influence not only local immune responses but may also cross the blood-brain barrier (59). Furthermore, A. actinomycetemcomitans, T. denticola, and P. gingivalis secrete small RNAs of microRNA size (miRNA-size, small RNAs or msRNAs) via OMVs, which are stably transferred to host cells, modulating immune responses and apoptosis (60, 61).

Plaque biofilm formation plays a central role in the onset and progression of periodontitis (64), and bacterial interactions are critical to the formation of subgingival biofilm. Periodontal pathogens can communicate with one another or with other bacteria via BEVs, influencing biofilm formation, bacterial survival, and enhancing their invasive capabilities (65) ( Figure 2 ). A series of studies have shown that OMVs from P. gingivalis, enriched with gingipains or adhesins, significantly promote co-aggregation of various oral pathogens, such as Streptococcus spp., F. nucleatum, Actinomyces naeslundii (A.naeslundii), and Actinomyces viscosus (A. viscosus) (66). Additionally, P. gingivalis OMVs mediate co-aggregation with T. denticola and Lachnoanaerobaculum saburreum (L.saburreum), and enhance the motility of non-motile bacteria, aiding the formation of a multispecies plaque biofilm (67). Moreover, P. gingivalis OMVs enhance T. forsythia adhesion to and invasion of epithelial cells, contributing to its virulence (68). Zhang (69) et al. reported that proteases in P. gingivalis OMVs reduce the expression of adhesion-associated proteins, such as FadA and FomA, on the surface of F. nucleatum, inhibiting its invasion of oral epithelial cells and its auto-aggregation. However, it has no impact on the morphology or proliferation of F. nucleatum. During co-infection with F. nucleatum and P. gingivalis, F. nucleatum paradoxically enhances the invasive capacity of P. gingivalis. By preventing F. nucleatum degradation within cells and maintaining its bioactivity, P. gingivalis promotes deeper infection. Furthermore, periodontal pathogen-derived BEVs have the function of inhibiting and dispersing competitor biofilms. For example, P. gingivalis OMVs appeared to have a negative impact on biofilm formation and the maintenance of Streptococcus gordonii (S. gordonii) in a gingipain-dependent manner, creating a more favorable environment for its own survival (70).

BEVs from periodontal pathogens have significant immunomodulatory effects on host cells including both triggering the activation of the immune system and contributing to immune evasion ( Figure 3 ).

In addition to their immunomodulatory effects, periodontal pathogen-derived BEVs can also exert the ability to destroy periodontal tissues through various mechanisms. The process of periodontal destruction by BEVs includes direct damage and invasion of the epithelial barrier, inhibition of angiogenesis, and the creation of an immunological microenvironment that induces bone resorption ( Figure 4 ).

Oral biofluids, such as saliva and gingival crevicular fluid (GCF), are rich of biomolecules from both host cells and resident microorganisms, and are commonly used to identify diagnostic markers for periodontitis (95). EVs found in these fluids are emerging as potential biomarkers for periodontal diseases (96–102). While most current research focuses on human-derived components, much less is known about BEVs. Han (102) et al. utilized LPS to label bacterial OMVs in saliva and found a significant increase in the amount of LPS+ OMVs in the saliva of patients with periodontitis compared to healthy individuals. Quantitative PCR analysis of genomic DNA from saliva small EVs (sEVs) indicated a marked increase in four periodontal pathogens (T. denticola, E. corrodens, P. gingivalis, and F. nucleatum) in the periodontitis group, with P. gingivalis and T. denticola being the most sensitive. These findings suggest that LPS+ OMVs, along with OMVs from P. gingivalis and T. denticola, could serve as potential diagnostic biomarkers for periodontitis. However, this study had a small sample size, and pure OMVs from specific periodontal pathogens were not isolated, warranting further investigation.

Other studies have explored the expression of virulence factors in OMVs as a diagnostic tool. For example, monoclonal antibodies have been developed to recognize the conserved P. gingivalis virulence factor RgpA-Kgp complex, forming the basis for a saliva-based diagnostic kit to detect P. gingivalis and its OMVs (103). However, the challenge remains in distinguishing BEVs from human-derived EVs in saliva or GCF due to the lack of specific markers (104). Therefore, separating BEVs for clinical diagnosis remains complex and difficult.

BEVs are highly stable under various temperature and treatment conditions, and do not possess the ability to self-replicate, thus providing strong biosafety profiles (105). Due to their nanoscale size, BEVs are preferentially taken up by dendritic cells (DCs) (106). They also contain numerous immunogenic surface- and membrane-associated components of their parent bacterium, which can induce host immune responses (107, 108). Consequently, BEVs hold great promise as immunogenic biological agents that activate the immune system to combat bacterial infections. Moreover, BEVs can be bioengineered to express target antigens with reduced toxicity (109), making them promising candidates for vaccine development (110, 111).

For example, OMVs derived from P. gingivalis maintain the immunodominant epitopes of the bacterium, while animal studies by Nakao et al. (112) have demonstrated that intranasal administration of P. gingivalis OMVs in mice induced the dose-dependent production of salivary IgA, as well as serum IgG and IgA. Previous studies have estabilished that Poly(I:C), a TLR3 agonist, significantly increased antibody production and enhanced the clearance of P. gingivalis (112, 113). Moreover, the study confirmed the safety of low-dose intranasal immunization for adjacent organs and the central nervous system (113). The strong immunogenicity of P. gingivalis OMVs mainly originates from LPS and A-LPS-modified proteins, such as gingipains in the OMVs, while immune reactivity was significantly reduced after serum absorption of LPS (114).

Despite the promising progress, BEVs are still far from being used in clinical settings for the prevention of periodontal diseases. This is primarily due to the high toxicity of BEVs, as well as difficulties in isolating and characterizing them. Therefore, standard methods are urgently needed to isolate and characterize BEVs.

Traditional antibiotic administration delivers drugs systemically via the bloodstream, allowing them to target pathogenic bacteria located in different parts of the body. However, this approach often lacks specificity, requiring higher doses and increased frequency to reach therapeutic concentrations at the infection site, which can lead to side effects and accelerate the development of antibiotic resistance (115). In recent years, the use of nanoparticle-based drug delivery systems has been extensively studied. These systems can enhance drug solubility, modulate drug release, target specific sites, and simultaneously deliver multiple therapeutic agents (116). Compared to synthetic nanoparticle carriers, naturally derived BEVs offer several advantages, including small particle size, stable cargo-carrying capacity, and high biocompatibility (117). BEVs possess immunogenic antigens on their surface, which can elicit robust immune responses against invading pathogens (107).

Additionally, BEVs are involved in signal transduction, facilitating inter-bacterial communication and exchange, and can easily fuse with bacterial membranes (118), delivering bioactive molecules to their parent bacteria and surrounding microbes. Through genetic engineering of parent bacteria, BEVs can be modified with targeting ligands, enhancing drug accumulation at desired sites (119). BEVs themselves can also be surface-modified to improve cellular or site-specific targeting (120). Accordingly, BEVs have been explored as drug delivery vehicles to enhance bacterial uptake of loaded antibiotics. Previous evidence suggests that BEVs are more potent in delivering autolysins and peptidoglycan hydrolases, resulting in higher bacterial killing efficiency compared to gentamicin (121). Another study developed a novel antibiotic delivery system using OMVs isolated from E. coli as a shell and rifampicin-loaded mesoporous silica nanoparticles (MSNs) as the core. In contrast to conventional antibiotics, BEVs could enhance antibiotic uptake and achieve superior antibacterial effects (122).

Currently, the mechanisms of BEV-mediated delivery are not fully understood, and there is a need to improve purification processes and production yields, as well as to standardize the techniques and analyses (123). While the application of BEV-based drug delivery systems in clinical practice remains distant, BEVs hold great promise as novel antibiotic delivery vehicles or potential antimicrobial agents to effectively kill or inhibit periodontal pathogens.

Periodontopathic bacteria-derived EVs contain various toxic factors that contribute significantly to periodontal tissue destruction, as discussed previously. Inhibiting the release of these EVs from periodontal pathogens could represent a therapeutic approach to managing periodontitis. Peptidylarginine deiminases (PADs), a group of calcium-activated enzymes, serve as toxic components in P. gingivalis EVs. PADs convert arginine residues into citrulline residues, which lead to the citrullination of host proteins such as fibrinogen and α-enolase. This modification is crucial for various physiological processes, including the biogenesis of OMVs and the initiation of pathological inflammation (124). The use of PAD inhibitors, such as GSK 199, BB-Cl-amidine, Cl-amidine, and AMF30a, has been shown to effectively reduce BEVs production (125, 126). Additionally, cannabidiol (CBD) has been reported to inhibit the release of BEVs from Gram-negative bacteria, potentially reducing antibiotic resistance (127).

Given BEVs’ ability to mediate immune-inflammatory responses and promote periodontitis progression and tissue damage, finding agents to reduce the release of pro-inflammatory factors induced by BEVs opens new therapeutic avenues. Hop bract polyphenol (HBP), for example, has been shown to inhibit the expression of cyclooxygenase (COX)-2, IL-6, IL-8, and matrix metalloproteinases (MMP)-1 and -3 in human gingival epithelial (HGE) cells challenged with P. gingivalis EVs in a dose-dependent manner. This makes HBP a promising inhibitor of the cell inflammatory response induced by P. gingivalis EVs. Key active components of HBP, such as 2-[(2-methylpropanoyl)-phloroglucinol]1-O-β-D-glucopyranoside (MPPG) and kaempferol 3-O-β-glucopyranoside (astragalin), have been identified as effective in mediating these anti-inflammatory effects (128). Curcumin also shows notable efficacy, significantly inhibiting P. gingivalis OMV-stimulated gene expression and protein production of IL-6, IL-1β, and TNF-α in HGE cells, in a dose-dependent manner. Additionally, curcumin attenuates the cytotoxic effects of OMVs on cell migration and reduces OMV adhesion, cell entry, and apoptosis, also in a dose-dependent manner (129).

Probiotic therapy aimed at neutralizing the toxic effects of periodontal pathogen-derived EVs may represent an emerging area of research. Microbial dysbiosis is critical in the progression of periodontitis, typically involving a reduction in probiotic bacteria and/or an increase in periodontal pathogens (130). Lactobacillus reuteri (L. reuteri) is one of the most extensively studied probiotics and shows potential as an adjunctive treatment for chronic periodontitis (131). L. reuteri has been found to downregulate key virulence factors of periodontal pathogens, interfere with interspecies communication among pathogens, inhibit pathogenic adhesion and invasion, and reduce virulence (132). Probiotic-derived EVs also possess anti-infective properties (133). Notwithstanding current evidence on the efficacy of probiotic-derived EVs in periodontal inflammation control is limited, this approach does indicate a promising direction for future research.