Composition for preventing and treating oral disease or condition, preparation method therefor, and use thereof

Abstract

Provided are a composition for preventing and treating an oral disease or condition, a preparation method therefor, and the use thereof. The composition comprises extracellular vesicles derived from oral pathogenic bacteria. The extracellular vesicles are nanoscale vesicles rich in immune activation and defensive factors against pathogenic bacterial infection. Upon administration via the oral mucosa into a body, the vesicles are passively aimed at lymph nodes associated with the oral mucosa to induce the expression of a mucosal immunity-specific antibody, thereby enhancing immune defense against pathogenic bacteria. The extracellular vesicles of oral pathogenic bacteria are coated with a calcium phosphate shell, and the shell is soluble only under acidic conditions and degrades only after the extracellular vesicles are aimed at the lymph nodes and are phagocytosed into lysosomes of dendritic cells, thereby avoiding inflammatory reactions in an administration area. By using a sublingual disintegrating tablet as the form of administration, the bioavailability of a vaccine can be enhanced while achieving the local administration at the mucosa and activating mucosal immunity. Moreover, the composition is convenient to administer, which is conducive to the diagnosis and treatment of oral bacterial infectious diseases.

Description

Compositions for the prevention and treatment of oral diseases or conditions, their preparation methods and uses Technical Field

[0001] This disclosure relates to the prevention and treatment of oral bacterial infectious diseases or conditions, and more specifically to compositions for the prevention and treatment of oral bacterial infectious diseases or conditions, methods of their preparation, and uses. Background Technology

[0002] Oral pathogens can cause a variety of diseases, ranging from skin and soft tissue infections to invasive systemic infections. For example, chronic periodontitis is a chronic infectious disease that leads to the slow loss of periodontal tissues such as the periodontal ligament and alveolar bone, eventually resulting in loose teeth and tooth loss. It is one of the leading oral diseases causing tooth loss in my country. As a long-term, persistent source of infection, periodontitis can affect overall health; patients with periodontitis have a significantly increased risk of cardiovascular disease, myocardial infarction, diabetes, rheumatoid arthritis, lupus, and Alzheimer's disease. Staphylococcus aureus, or Staphylococcus aureus for short, can cause serious oral infections when the oral mucosa is damaged (such as ulcers, periodontal disease, tooth extraction wounds, or oral surgical wounds) or when the body's immunity is weakened.

[0003] The initiating factor for bacterial infections such as periodontitis is the invasion of local plaque microorganisms and their toxin products, causing an inflammatory response in the local tissues, which in turn leads to damage and destruction of periodontal tissues. Currently, the main clinical treatments for periodontitis are basic treatments aimed at removing local plaque, such as supragingival scaling and subgingival curettage. However, due to the dysfunction of host immune cells caused by the local inflammatory microenvironment, although basic periodontal treatment can control plaque and restore the tissue's healthy color, shape, and texture, if there is a lack of immune defense against reinfection by pathogenic bacteria, periodontitis carries the risk of repeated relapses and even difficulty in tissue repair. Therefore, there is an urgent need to develop new clinical strategies and formulations for preventing the onset and recurrence of periodontitis.

[0004] The information in the background section is intended only to illustrate the general context of this disclosure and should not be construed as an admission or implication in any way that such information constitutes prior art known to those skilled in the art. Summary of the Invention

[0005] Through large-scale clinical sample collection and correlation analysis, the applicant discovered that a lack of pathogen-specific sIgA antibodies in saliva is a key risk factor for poor treatment prognosis and high recurrence rate in patients with periodontitis, and that oromandibular lymphoid tissues, represented by the submandibular lymph nodes, are key sites for sIgA antibody production and expression. Based on this, the applicant further discovered that targeting and activating B cell-related functions in the submandibular lymph nodes and upregulating the level of pathogen-specific sIgA antibodies in saliva can effectively prevent and treat periodontitis. Specifically, this disclosure includes the following:

[0006] A first aspect of this disclosure provides a composition for preventing and treating oral bacterial infectious diseases or conditions, comprising bacterial exovesicles derived from oral pathogens such as periodontal pathogens.

[0007] In some embodiments, the composition according to this disclosure for preventing oral bacterial infectious diseases or conditions includes, wherein the pathogenic bacteria include conditionally pathogenic and / or absolutely pathogenic bacteria, including at least one of Porphyromonas gingivalis, Actinobacillus actinomycetii, Forsythorbium, Fusobacterium nucleatum, Prevotella intermedius, and Staphylococcus aureus.

[0008] In some embodiments, the composition according to this disclosure for preventing and treating oral bacterial infectious diseases or conditions, wherein the oral disease is an oral bacterial infectious inflammatory disease, such as periodontitis or ulcers.

[0009] In some embodiments, the composition for preventing or treating oral bacterial infections or conditions according to this disclosure further comprises a calcium salt, preferably, the calcium salt coating the surface of the outer vesicle to form a shell.

[0010] In some embodiments, the composition for preventing or treating oral bacterial infections or conditions according to this disclosure is a vaccine, such as a periodontitis vaccine or an ulcer vaccine.

[0011] In some embodiments, the composition for preventing or treating oral bacterial infections or conditions according to this disclosure is a sublingual disintegrating tablet, oral lozenge, oral spray, or hydrogel formulation.

[0012] A second aspect of this disclosure provides a method for preparing a composition for preventing or treating oral bacterial infectious diseases or conditions, comprising the steps of providing exovesicles derived from oral pathogens and coating the surface of the exovesicles with calcium salts to obtain calcium-coated exovesicles.

[0013] In some embodiments, the method of preparing a composition for preventing or treating oral bacterial infections or conditions according to the present disclosure further includes the step of forming a formulation, preferably a tablet, using calcium-coated exovesicles.

[0014] A third aspect of this disclosure provides the use of external vesicles derived from oral pathogens in the preparation of medicaments for increasing oral levels of specific IgG and / or sIgA antibodies against the source bacteria and for increasing serum levels of specific IgG and / or IgA antibodies against the source bacteria.

[0015] This disclosure discovers that oral bacterial vesicles are nanoscale vesicles rich in immune-activating and defense factors against periodontitis. After administration via the oral mucosa, these vesicles passively target the oral mucosa-associated lymph nodes, inducing mucosal immune-specific antibody expression, thereby enhancing immune defense against pathogenic bacteria. In a preferred embodiment, the vesicles are bacterial vesicles such as *Porphyromonas gingivalis*, *Actinomyces actinomycetes*, *Focusae*, *Fusobacterium nucleatum*, *Prevotella intermedius*, and *Staphylococcus aureus*. In a preferred embodiment, the vesicles of this disclosure are coated with a calcium phosphate shell, which is only soluble in acidic conditions. This allows the engineered vesicles to target the lymph nodes and be phagocytosed into dendritic cell lysosomes before degradation, avoiding inflammatory reactions at the administration site. In a preferred embodiment, the composition of this disclosure is prepared as a sublingual disintegrating tablet. This dosage form allows for local administration to activate mucosal immunity while improving vaccine bioavailability. The convenient dosage form also facilitates the promotion and popularization of periodontitis vaccines in chairside clinics or primary communities. Attached Figure Description

[0016] Figure 1. Application scenarios and mechanism of action of the sublingual disintegrating tablet periodontitis vaccine based on Porphyromonas gingivalis external vesicles (OMV@CaP ODT) for the prevention of periodontitis.

[0017] Figure 2. Electron micrograph of Porphyromonas gingivalis external vesicles (OMV).

[0018] Figure 3. Protein composition of Porphyromonas gingivalis external vesicles (OMV).

[0019] Figure 4 shows the average particle size analysis results of OMV.

[0020] Figure 5. Average particle size analysis results of OMV and OMV@CaP.

[0021] Figure 6. Transmission electron microscopy (TEM) images and thermogravimetric (TGA) results. (A) TEM images of OMV and OMV@CaP; (B) TGA results of OMV@CaP.

[0022] Figure 7. Activation effect of OMV and OMV@CaP on mouse dendritic cells.

[0023] Figure 8. Disintegration of OMV@CaP ODT periodontitis vaccine under the tongue of mice.

[0024] Figure 9. Disintegration of OMV@CaP ODT periodontitis vaccine under the tongue of cynomolgus monkeys.

[0025] Figure 10. Lymph node targeting of OMV@CaP ODT periodontitis vaccine after intraoral administration in mice. (A) Small animal imaging results show that the fluorescently labeled OMV@CaP ODT periodontitis vaccine significantly accumulated in the submandibular lymph nodes of mice 12 hours after intraoral administration; (B) Small animal imaging observation of the submandibular lymph nodes of mice at different administration time points confirmed that both OMV@CaP and OMV@CaP ODT can effectively deliver vesicles to the submandibular lymph nodes of mice after administration, and OMV@CaP ODT has higher bioavailability.

[0026] Figure 11. Lymph node targeting of OMV@CaP ODT periodontitis vaccine after intraoral administration to cynomolgus monkeys. Photoacoustic results showed that the fluorescently labeled OMV@CaP ODT periodontitis vaccine was significantly enriched in the submandibular lymph nodes of cynomolgus monkeys 6 hours after intraoral administration.

[0027] Figure 12 shows that after administration of OMV@CaP and OMV@CaP ODT, the submandibular lymph nodes of mice were significantly enlarged and the number of B cell follicles increased.

[0028] Figure 13. Activation of submandibular lymph nodes in cynomolgus monkeys after administration of OMV@CaP ODT periodontitis vaccine. (A) Ultrasound observation of submandibular lymph nodes in cynomolgus monkeys at different time points after administration showed enlargement of submandibular lymph nodes in the OMV@CaP ODT administration group, proving that the drug activated the immune response within the lymph nodes; (B) Gross observation showed that the submandibular lymph nodes in cynomolgus monkeys significantly enlarged after administration of OMV@CaP ODT periodontitis vaccine, consistent with the ultrasound results; (C) Histological staining results confirmed the enlargement of submandibular lymph nodes in cynomolgus monkeys after administration, consistent with the ultrasound results and gross observation.

[0029] Figure 14. The promoting effects of OMV@CaP and OMV@CaP ODT on the levels of antigen-specific antibodies in mouse saliva and serum. (A) OMV@CaP and OMV@CaP ODT upregulated the level of antigen-specific sIgA antibody in mouse saliva; (B) OMV@CaP and OMV@CaP ODT upregulated the level of antigen-specific IgG antibody in mouse saliva; (C) OMV@CaP and OMV@CaP ODT upregulated the level of antigen-specific IgG antibody in mouse serum; (D) OMV@CaP and OMV@CaP ODT upregulated the level of antigen-specific IgA antibody in mouse serum; (E) The promoting effect of OMV@CaP and OMV@CaP ODT on the level of antigen-specific sIgA antibody in mouse saliva is long-lasting, and the effect of OMV@CaP ODT is better than that of OMV@CaP.

[0030] Figure 15. The promoting effects of OMV@CaP and OMV@CaP ODT on the levels of antigen-specific antibodies in the saliva and serum of cynomolgus monkeys. (A) OMV@CaP and OMV@CaP ODT upregulated the level of antigen-specific sIgA antibody in the saliva of cynomolgus monkeys; (B) OMV@CaP and OMV@CaP ODT upregulated the level of antigen-specific IgG antibody in the saliva of cynomolgus monkeys; (C) OMV@CaP and OMV@CaP ODT upregulated the level of antigen-specific IgG antibody in the serum of cynomolgus monkeys; (D) OMV@CaP and OMV@CaP ODT upregulated the level of antigen-specific IgA antibody in the serum of cynomolgus monkeys; (E) The promoting effect of OMV@CaP and OMV@CaP ODT on the level of antigen-specific sIgA antibody in the saliva of cynomolgus monkeys was long-lasting, and the effect of OMV@CaP ODT was better than that of OMV@CaP.

[0031] Figure 16. Protective effects of OMV@CaP and OMV@CaP ODT against periodontitis in mice in vivo. Histological staining results showed that OMV@CaP and OMV@CaP ODT effectively inhibited alveolar bone resorption and destruction caused by pathogenic bacterial infection, and maintained a protective effect against periodontitis with long-term reinfection.

[0032] Figure 17. In vivo protective effect of OMV@CaP ODT against periodontitis in cynomolgus monkeys. (A) Intraoral examination results showed that after modeling, the gingival redness, bleeding on probing, and probing depth of the affected teeth in cynomolgus monkeys were significantly improved. Various clinical manifestations of the affected teeth in the OMV@CaP ODT group were significantly improved. (B) CBCT results showed that OMV@CaP ODT could effectively inhibit alveolar bone resorption and destruction caused by pathogenic bacterial infection, and it still had a protective effect against periodontitis that was reinfected in the future. (CD) Histological staining results showed that OMV@CaP ODT effectively improved irregular proliferation and inflammatory cell infiltration caused by local inflammatory stimulation in periodontal tissues. (E) Intraoral examination results showed that after modeling, the probing depth (PD), bleeding index (BI), and positive rate of bleeding sites (BOP) of the affected teeth in cynomolgus monkeys were significantly increased. Various disease indicators of the affected teeth in the OMV@CaP ODT group were significantly improved, and it still had a protective effect against periodontitis that was reinfected in the future.

[0033] Figure 18. The immune-activating effect of OMV@CaP ODT on clinical volunteers. (A) OMV@CaP ODT disintegrated under the tongue of volunteers within 1 minute; (B) After immunization with OMV@CaP ODT, the submandibular lymph nodes of all 10 volunteers were transiently enlarged, indicating local immune response activation; (C) OMV@CaP ODT immunization significantly increased the level of antigen-specific sIgA antibody in the saliva of all subjects.

[0034] Figure 19 Transmission electron micrograph of Staphylococcus aureus external vesicles (MV).

[0035] Figure 20 Transmission electron micrograph of mineralized Staphylococcus aureus exovesicles (MV@CaP).

[0036] Figure 21. Lymph node targeting of Staphylococcus aureus exovesical sublingual disintegrating tablet vaccine (MV@CaP ODT) after intraoral administration in mice. (A) Gross view of the sublingual disintegrating tablet vaccine (scale bar: 2 mm); (B) Schematic diagram of intraoral disintegration of the sublingual disintegrating tablet; (C) Small animal imaging observation showing that MV@CaP ODT effectively accumulates in the submandibular lymph nodes of mice 24 hours after administration.

[0037] Figure 22. The promoting effect of MV@CaP ODT (ODT) on the level of Staphylococcus aureus-specific antibodies in mouse saliva. (A) MV@CaP ODT upregulated the level of Staphylococcus aureus-specific sIgA antibodies in mouse saliva, and this promoting effect was long-lasting; (B) The in vitro killing ability of MV@CaP ODT-immunized mouse saliva against Staphylococcus aureus was improved; (C) The resistance of mice immunized with MV@CaP ODT to oral Staphylococcus aureus infection was enhanced.

[0038] Figure 23. The promoting effect of MV@CaP ODT (ODT) on the level of Staphylococcus aureus-specific antibodies in cynomolgus monkey saliva. (A) MV@CaP ODT upregulated the level of Staphylococcus aureus-specific sIgA antibodies in cynomolgus monkey saliva, and this promoting effect was long-lasting; (B) The in vitro killing ability of cynomolgus monkey saliva immunized with MV@CaP ODT was improved.

[0039] Figure 24. The promoting effect of MV@CaP ODT (ODT for short) on the level of Staphylococcus aureus-specific antibodies in mouse saliva and serum.

[0040] Figure 25. The promoting effect of MV@CaP ODT (ODT for short) on the level of Staphylococcus aureus-specific antibodies in the saliva and serum of cynomolgus monkeys.

[0041] Figure 26. The therapeutic effect of MV@CaP ODT (ODT) on delayed healing of oral ulcers caused by Staphylococcus aureus infection in mice. (A) Quantitative fluorescence analysis of Luciferella showed that MV@CaP ODT effectively suppressed the abundance of Staphylococcus aureus infection in the local area after oral ulceration in mice; (B) Compared with the control group, MV@CaP ODT improved the ulcer healing rate and effectively rescued the delayed healing of oral ulcers caused by Staphylococcus aureus infection.

[0042] Figure 27. The promoting effect of MV@CaP ODT (ODT) on the level of Staphylococcus aureus-specific antibodies in the saliva of clinical subjects. (A) MV@CaP ODT upregulated the level of Staphylococcus aureus-specific sIgA antibodies in the saliva of subjects; (B) The in vitro killing ability of Staphylococcus aureus in the saliva of subjects immunized with MV@CaP ODT was improved. Detailed Implementation

[0043] Various exemplary embodiments of this disclosure are now described in detail. This detailed description should not be considered as a limitation of this disclosure, but rather as a more detailed description of certain aspects, features, and implementations of this disclosure.

[0044] It should be understood that the terminology used in this disclosure is for describing particular embodiments only and is not intended to limit the disclosure. Furthermore, for numerical ranges in this disclosure, it should be understood that an upper and lower limit of the range and each intermediate value between them are specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this disclosure. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0045] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. While this disclosure describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may be used in implementation or testing of this disclosure. All references in this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0046] In this article, the term "oral bacterial infectious disease" refers to diseases caused by oral flora imbalance or pathogenic bacterial infection, preferably inflammatory diseases, examples of which include, but are not limited to, postoperative oral infections, refractory periapical diseases, gingivitis, periodontitis, ulcers, etc.

[0047] In this article, the term "condition" refers to the symptoms and signs caused by oral bacterial infections. This article primarily focuses on gingival redness and swelling, pain, abscesses, which may be accompanied by bleeding, alveolar bone resorption, tooth loosening, wound pain, purulent discharge, hyperplasia, ulceration, recurrent periapical abscesses accompanied by discomfort when biting, and bad breath.

[0048] In this text, the term "prevention and treatment" refers to therapeutic treatments and preventive or preventative measures aimed at preventing or mitigating (reducing) undesirable physiological changes or disorders, such as the onset, progression, or recurrence of inflammatory diseases. Beneficial or desired clinical outcomes include, but are not limited to, symptom relief, reduction of disease severity, decrease in susceptibility to disease, stabilization of the disease state (i.e., no worsening), delay or slowing of disease progression, improvement or mitigation of the disease state, and ablation. Those requiring treatment include individuals who already have the condition or disorder, those susceptible to the condition or disorder, or those who require prevention of the condition or disorder.

[0049] [Composition]

[0050] A first aspect of this disclosure provides a composition for preventing or treating oral bacterial infectious diseases or conditions, sometimes referred to herein as a "vaccine," comprising exovesicles derived from oral pathogens.

[0051] In this disclosure, pathogenic bacteria are microorganisms present in the oral cavity that can cause, but are not limited to, periodontitis or other infectious diseases. Examples include, but are not limited to, *Porphyromonas gingivalis*, *Actinomyces actinomycete*, *Fusobacterium nucleatum*, *Prevotella intermedius*, and *Staphylococcus aureus*. In some embodiments, the periodontitis pathogen of this disclosure is *Porphyromonas gingivalis*. In other embodiments, the periodontitis pathogen of this disclosure is *Staphylococcus aureus*. In this disclosure, the oral pathogenic bacteria used to produce external vesicles can be one of the above-mentioned bacteria or a combination of two or more. The bacteria disclosed in this disclosure can be natural bacteria or engineered bacteria obtained through genetic engineering.

[0052] In this disclosure, external vesicles refer to bacterial outer membrane vesicles (OMVs) secreted by bacteria, which have a vesicle-like structure with a lipid bilayer. Preferably, the external vesicles are naturally secreted bacterial nanoparticles, more preferably nanostructures secreted by the outer membrane of Gram-negative bacteria, with an average diameter generally 1-1000 nm, preferably 5-800 nm, more preferably 10-700 nm, further preferably 15-600 nm, and most preferably 20-250 nm, such as 20 nm, 50 nm, 80 nm, 100 nm, 120 nm, 150 nm, 180 nm, 200 nm, 250 nm, 300 nm, 350 nm, 400 nm, 450 nm, 500 nm, 550 nm, etc. In some embodiments, the particle size of the external vesicles of this disclosure is, for example, between 20-250 nm, thereby allowing them to passively accumulate in lymph nodes and activate antigen-specific immune responses. OMVs contain various bacterial-derived components, such as enzymes, virulence factors, and bacterial-specific antigens, which can effectively promote the recognition and activation of immune cells, giving them a unique advantage in the development of adjuvants and vaccines. The form of the exovesicles in the composition is not limited; they can be in dry powder, solution, or suspension form, etc.

[0053] In some embodiments, the exovesicles of this disclosure include a shell on their outer surface, said shell being, for example, bound to or encapsulated on the outer surface of the exovesicle by mineralization. Exemplarily, the shell comprises a calcium salt. Examples of calcium salts are not limited, but are generally slightly soluble salts; preferably, the calcium salt is soluble in acid but slightly soluble or insoluble in water, thereby ensuring that the exovesicles are only degraded after targeting lymph nodes and being phagocytosed into the lysosomes of dendritic cells, avoiding inflammatory responses at the administration site. Examples of such calcium salts include, but are not limited to, calcium phosphate and calcium carbonate.

[0054] In some embodiments, the compositions disclosed herein are vaccines used to treat or prevent oral infectious diseases or conditions, particularly periodontal disease, including improving or reducing the severity of inflammation, swelling, and improving or reducing bone resorption, bone loss, etc. caused by periodontal disease.

[0055] The compositions disclosed herein can be in any suitable dosage form, such as injections, suspensions, emulsions, tablets, etc. The compositions disclosed herein can be administered into the body in known ways, such as by intramuscular injection to the tissue of interest, or alternatively by intravenous, percutaneous, intranasal, oral, mucosal, or other delivery methods. Such administration can be performed via a single dose or multiple doses. Those skilled in the art will understand that the actual dose to be administered herein can vary considerably depending on a variety of factors, such as the target cells, biological type or tissue thereof, the general condition of the subject to be treated, the route of administration, the manner of administration, etc.

[0056] In this disclosure, the compositions further include pharmaceutically acceptable carriers, which are well known in the art and can be determined by those skilled in the art to meet clinical standards. Pharmaceutically acceptable carriers include diluents and excipients, a pharmaceutically acceptable material, composition, or carrier, such as liquid or solid fillers, diluents, excipients, solvents, or encapsulating materials, and such pharmaceutically acceptable materials, compositions, or carriers participate in the delivery or transport of a drug from one organ or site of the body to another organ or site of the body. Each carrier must be "acceptable," meaning it is compatible with other components of the formulation and does not harm the patient. Some examples of pharmaceutically acceptable carriers include: sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives and analogs such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; tragacanth gum powder; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; polyols such as glycerol, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl lauryl ester; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginate; pyrogen-free water; isotonic saline; Ringer's solution; ethanol; phosphate buffer; and other non-toxic and compatible substances used in pharmaceutical preparations. Wetting agents, emulsifiers and lubricants, such as sodium dodecyl sulfonate, magnesium stearate, and polyoxyethylene-polypropylene copolymers, as well as colorants, release agents, coating agents, sweeteners, flavorings and fragrances, preservatives and antioxidants may also be present in the composition.

[0057] In some embodiments, the compositions disclosed herein are solid dosage forms for oral administration, including tablets, pills, powders, granules, or capsules. In these solid dosage forms, the active ingredient is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with: (a) fillers or compatibilizers, such as starch, lactose, sucrose, glucose, mannitol, and silica; (b) binders, such as hydroxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and gum arabic; (c) humectants, such as glycerin; (d) disintegrants, such as agar, calcium carbonate, potato starch or cassava starch, alginate, certain complex silicates, and sodium carbonate; (e) slowing agents, such as paraffin; (f) absorption accelerators, such as quaternary ammonium compounds; (g) wetting agents, such as cetyl alcohol and glyceryl monostearate; (h) adsorbents, such as kaolin; and (i) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium dodecyl sulfate, or mixtures thereof. Buffers may also be included in capsules, tablets, and pills.

[0058] In some embodiments, the formulations disclosed herein are substances suitable for rapid disintegration into fine particles in the oral mucosa region, thereby enabling the functional ingredients to dissolve and be absorbed rapidly to exert their effects, such as sublingual disintegrating tablets, oral lozenges, oral sprays, hydrogel formulations, etc. Their components include at least one of the following: dry starch, sodium carboxymethyl starch, low-substituted hydroxypropyl methylcellulose, croscarmellose, croscarmellose sodium, micronized silica gel, microcrystalline cellulose, and effervescent disintegrants.

[0059] In some embodiments, the formulation of this disclosure is a sublingual disintegrating tablet comprising microcrystalline cellulose, croscarmellose sodium, starch, and micronized silica. Preferably, based on weight, the amount of microcrystalline cellulose is generally 50-80%, more preferably 60-70%, and more preferably 60-65%. The amount of starch is generally 10-40%, preferably 10-30%, and more preferably 10-20%. The amount of croscarmellose sodium is generally 10-40%, preferably 10-30%, and more preferably 15-25%. The amount of micronized silica is generally 1-5%, preferably 1-4%, and even more preferably 1-3%. In this disclosure, the protective agent prepared by comprising exovesicles coated with a calcium phosphate shell and sublingual disintegrating tablet excipients is in tablet form, which can be stably stored and transported.

[0060] In some embodiments, the composition of this disclosure disintegrates under the tongue after being taken orally, releasing calcium phosphate-coated engineered Porphyromonas gingivalis exovesicles which target the oral mucosa-associated lymphoid tissue (OMA) into the oral cavity. After being phagocytosed by dendritic cells, the OMA presents the antigenic components of the pathogen and activates T cells and B cells, promoting the expression of high-affinity, specific, long-acting IgG and sIgA antibodies in saliva, and inhibiting the colonization and infection of the pathogen, thereby achieving an effective, efficient, and long-lasting preventive effect against periodontitis.

[0061] [Preparation Method]

[0062] A second aspect of this disclosure provides a method for preparing a composition for preventing or treating oral bacterial infectious diseases or conditions, comprising the steps of providing exovesicles derived from oral pathogens and coating the surface of the exovesicles with calcium salts to obtain calcium-coated exovesicles.

[0063] In this disclosure, the external vesicles derived from oral pathogenic bacteria can be obtained by directly purchasing existing external vesicles or by preparing them using known methods. The known methods for preparing external vesicles are not particularly limited, and any method can be used. Exemplarily, they are obtained by culturing bacteria such as *Porphyromonas gingivalis* into a culture medium, removing bacterial precipitate by centrifugation, and then ultracentrifuging. Further preferably, a step of filtration after bacterial removal to remove residual bacteria is performed.

[0064] In some embodiments of this disclosure, the calcium salt used to coat the surface of the calcium-coated exovesicle is generally a slightly soluble or insoluble salt, preferably an acid-soluble salt. The step of coating the exovesicle surface with a calcium salt to obtain calcium-coated exovesicles is not limited, but preferably includes a step of contacting and reacting a soluble calcium salt solution with the exovesicle, for example, mineralizing it, thereby making the calcium salt on the exovesicle surface a slightly soluble or insoluble salt.

[0065] In this disclosure, the mineralization step includes first adding OMVs to DMEM medium and equilibrating overnight at a low temperature (e.g., below 10°C, preferably 4°C). Then, CaCl2 (preferably 0.1-5M, more preferably 0.5-2M) is added to the reaction system to initiate the mineralization reaction, with a reaction time preferably of 1-5 hours and a reaction temperature generally of 20-45°C. An exemplary reaction, for example, involves adding OMVs containing 1 mg of protein to DMEM medium and equilibrating overnight at 4°C. Then, 10 μL of 1M CaCl2 is added to the reaction system to initiate the mineralization reaction, with a reaction time preferably of 1-5 hours. The reaction temperature is generally of 20-45°C, preferably 25-38°C, such as 30°C, 32°C, 34°C, 36°C, 37°C, etc. Under these conditions, the vesicles react with a solution of a soluble calcium salt, such as calcium chloride. Calcium ions bind to the negatively charged vesicles on their surface, and then calcium phosphate crystallizes to form nuclei, thereby forming vesicles coated with a calcium phosphate shell. Furthermore, preferably, the preparation method of this disclosure further includes a step of centrifugation to collect the precipitate.

[0066] In some embodiments, the preparation method of this disclosure further includes the step of freeze-drying the calcium salt-coated exovesicles.

[0067] [use]

[0068] A third aspect of this disclosure provides the use of external vesicles derived from oral pathogens in the preparation of medicaments for increasing oral IgG and / or sIgA antibody levels or for increasing serum IgG and / or IgA antibody levels.

[0069] This disclosure reveals that the outer vesicles of pathogenic bacteria such as Porphyromonas gingivalis and Staphylococcus aureus contain a large number of periodontitis-related immune activation and defense factors in their nanoscale vesicles. Through formulation, these vesicles can effectively target the mucosa-associated lymphoid tissue to activate the body's mucosal immunity, thereby generating a large number of specific IgG and sIgA antibodies in the oral cavity.

[0070] Example 1

[0071] I. Preparation

[0072] This embodiment is an exemplary example of tablet preparation using Porphyromonas gingivalis exovesicles, specifically including:

[0073] 1. Isolation and acquisition of Porphyromonas gingivalis exovesicles (OMV)

[0074] *Porphyromonas gingivalis* was cultured in a special medium (3.7% brain heart extract powder, 1% heme chloride, 0.1% vitamin K) for 48 h. The bacterial precipitate was removed by centrifugation, and residual bacteria in the supernatant were removed using a 0.22 μm filter. The collected supernatant was centrifuged at 110,000 g for 4 h. The precipitate was collected and resuspended in PBS to obtain *Porphyromonas gingivalis* exovesicles, which can be stored for short periods at 4 °C or long periods at -20 °C.

[0075] 2. Preparation of *Porphyromonas gingivalis* exovesicles coated with calcium phosphate shells (OMV@CaP)

[0076] In the preferred embodiment, 1 mg of protein OMV is first added to DMEM medium and equilibrated overnight at 4°C. Then, 10 μL of 1M CaCl2 is added to the reaction system to initiate the mineralization reaction. After the reaction, the precipitate is collected by centrifugation at 14000g for 15 min and washed twice with ultrapure water to obtain *Porphyromonas gingivalis* exovesicles coated with a calcium phosphate shell (OMV@CaP). These can be stored for short-term preservation at 4°C or freeze-dried and then stored long-term at -80°C.

[0077] 3. Preparation of periodontitis vaccine based on engineered Porphyromonas gingivalis exovesicles in sublingual disintegrating tablets

[0078] In a preferred embodiment, after preparing *Porphyromonas gingivalis* exovesicles coated with a calcium phosphate shell (OMV@CaP), 10 μg (for tablets with a 2 mm diameter) or 100 μg (for tablets with a 6 mm diameter) of OMV@CaP is lyophilized and then thoroughly mixed with tablet excipients (based on weight: 62% microcrystalline cellulose, 20% croscarmellose sodium, 16% starch, and 2% micronized silica gel). The mixture is then further compressed using a 2 mm or 6 mm die tableting machine to obtain the corresponding sublingual disintegrating tablets (OMV@CaP ODT). These tablets can be stored for short periods at 4°C or for long periods at -80°C.

[0079] The following experiments used OMV solution, OMV@CaP solution, and OMV@CaP ODT tablets as subjects.

[0080] II. Results

[0081] 1. Isolation and characterization of Porphyromonas gingivalis exovesicles

[0082] In this embodiment, *Porphyromonas gingivalis* outer vesicles (OMVs) were isolated and collected using ultracentrifugation. The morphology of the OMVs was observed using transmission electron microscopy, as shown in Figure 2. Next, the protein composition of *Porphyromonas gingivalis* and its OMVs was analyzed using a proteomics chip, as shown in Figure 3. This confirmed that the OMVs contain various pathogenic antigens and outer membrane components of *Porphyromonas gingivalis*. The average particle size analysis of the OMVs, as shown in Figure 4, confirmed that the average particle size of the OMVs is approximately 50.75 nm.

[0083] 2. Preparation of periodontitis vaccine using sublingual disintegrating tablets of Porphyromonas gingivalis exovesicles and its activation effect on immune cells.

[0084] Following the method described in Part 1, *Porphyromonas gingivalis* exovesicles coated with a calcium phosphate shell (OMV@CaP) were prepared. As shown in Figure 5, the average particle size of the calcium phosphate-coated OMV@CaP was approximately 105.7 nm. Transmission electron microscopy (TEM) images, as shown in Figure 6, show that the outer shell is coated with calcium phosphate, and the overall particle size is larger than that of OMV. Mouse dendritic cells were isolated and cultured in vitro, and treated with 1 μg / mL LPS, OMV, and OMV@CaP for 48 hours. Flow cytometry was used to detect dendritic cell activation. The results, as shown in Figure 7, show that OMV and OMV@CaP significantly upregulated the expression of antigen-presenting markers MHC-I, MHC-II, CD40, CD80, and CD86 in dendritic cells, with OMV@CaP showing a more significant upregulating effect than OMV.

[0085] Following the methods described in Part I, sublingually disintegrating periodontitis vaccine tablets for mice and cynomolgus monkeys were prepared, and the intraoral disintegration of the sublingually disintegrating periodontitis vaccine tablets (OMV@CaP ODT) was observed. As shown in Figures 8 (mice) and 9 (cynomolgus monkeys), OMV@CaP ODT disintegrated rapidly within 1 minute after being placed in the mouth. In vivo tracing techniques, including photoacoustic and small animal imaging, confirmed that OMV@CaP could target the submandibular lymph nodes of mice and cynomolgus monkeys after administration, as shown in Figures 10 (mice) and 11 (cynomolgus monkeys). Gross observation and histological staining of the submandibular lymph nodes in mice and cynomolgus monkeys after administration also confirmed that the OMV@CaP ODT periodontitis vaccine activated a local immune response, resulting in increased lymph node volume, as shown in Figures 12 and 13.

[0086] 3. The promoting effect of OMV@CaP and OMV@CaP ODT on the level of antigen-specific antibodies in saliva and serum.

[0087] After drug administration, saliva and serum from mice and cynomolgus monkeys in each group were continuously collected to detect the titer levels of anti-Porphyromonas gingivalis-specific IgG and sIgA antibodies, as shown in Figure 14 (mice) and Figure 15 (cynomolgus monkeys). It can be seen that OMV@CaP and OMV@CaP ODT significantly promoted the expression of anti-Porphyromonas gingivalis-specific IgG and sIgA antibodies in the samples, and their effects were long-term.

[0088] 4. Protective effects of OMV@CaP and OMV@CaP ODT on periodontitis in mice in vivo.

[0089] A mouse periodontitis model was established using suture ligation and bacterial smear. The protective effects of prophylactic administration of OMV@CaP and OMV@CaP ODT on periodontitis in mice were investigated in vivo. The results are shown in Figure 16. The results indicate that for primary periodontitis, both OMV@CaP and OMV@CaP ODT effectively inhibited alveolar bone resorption and destruction caused by pathogenic bacteria. OMV@CaP ODT was more effective than OMV@CaP, and there was no significant difference compared to the clinically commonly used antibiotic minocycline hydrochloride. However, because OMV@CaP and OMV@CaP ODT activated the long-term expression of the key sIgA antibody in saliva, they still had a protective effect against long-term reinfection of periodontitis. However, the antibiotic group showed no advantage in protecting against periodontitis recurrence, exhibiting severe alveolar bone resorption upon reinfection with pathogenic bacteria.

[0090] 5. The protective effect of OMV@CaP ODT against periodontitis in cynomolgus monkeys in vivo.

[0091] A periodontitis model in cynomolgus monkeys was established using a combination of suture ligation and bacterial smear. The protective effect of OMV@CaP ODT against periodontitis was investigated, and the results are shown in Figure 17. The results indicate that, similar to the mouse model, OMV@CaP ODT not only effectively inhibited alveolar bone resorption and destruction caused by primary infection with pathogenic bacteria, but also demonstrated a long-term protective effect against periodontitis-causing bacteria. This protective effect persisted even in a recurrent periodontitis model with subsequent reinfection.

[0092] 6. The antibody-boosting effect of OMV@CaP ODT on the saliva of healthy subjects in vivo.

[0093] Ten healthy subjects were recruited to investigate the effect of OMV@CaP ODT on enhancing human saliva antibodies. The results are shown in Figure 18. The results indicate that OMV@CaP ODT can effectively activate the immune response of the submandibular lymph nodes. All subjects showed a significant increase in sIgA antibodies specific to periodontitis pathogens in their saliva, and it also showed a significant in vitro antibacterial effect against the pathogens.

[0094] Example 2

[0095] I. Preparation

[0096] This embodiment is an exemplary example of preparing a tablet using Staphylococcus aureus exovesicles, specifically including:

[0097] 1. Isolation and acquisition of Staphylococcus aureus exovesicles (MV)

[0098] Staphylococcus aureus was cultured in standard Staphylococcus aureus medium for 48 hours. The bacterial precipitate was removed by centrifugation, and residual bacteria in the supernatant were removed using a 0.22 μm filter. The collected supernatant was centrifuged at 110,000 g for 4 hours. The precipitate was collected and resuspended in PBS to obtain Staphylococcus aureus exovesicles, which can be stored for short periods at 4°C or long periods at -20°C.

[0099] 2. Preparation of Staphylococcus aureus exovesicles coated with calcium phosphate shells (MV@CaP)

[0100] In the preferred embodiment, 1 mg of protein MV is first added to DMEM medium and equilibrated overnight at 4°C. Then, 10 μL of 1M CaCl2 is added to the reaction system to initiate the mineralization reaction. After the reaction, the precipitate is collected by centrifugation at 14000g for 15 min and washed twice with ultrapure water to obtain Staphylococcus aureus exovesicles coated with a calcium phosphate shell (MV@CaP). These can be stored for short-term preservation at 4°C or freeze-dried and then stored long-term at -80°C.

[0101] 3. Preparation of periodontitis vaccine based on engineered Staphylococcus aureus exovesicles sublingual disintegrating tablets

[0102] In a preferred embodiment, after preparing Staphylococcus aureus exovesicles coated with a calcium phosphate shell (MV@CaP), 10 μg (for tablets with a 2 mm diameter) or 100 μg (for tablets with a 6 mm diameter) of MV@CaP is lyophilized and then thoroughly mixed with tablet excipients (based on weight: 62% microcrystalline cellulose, 20% croscarmellose sodium, 16% starch, and 2% micronized silica gel). The mixture is then further compressed using a 2 mm or 6 mm tableting machine to obtain the corresponding sublingual disintegrating tablets (MV@CaP ODT). These tablets can be stored for short periods at 4°C or for long periods at -80°C.

[0103] II. Results

[0104] 1. Isolation and characterization of Staphylococcus aureus exovesicles

[0105] In this embodiment, Staphylococcus aureus exovesicles (MVs) were isolated and collected by ultracentrifugation. The morphology of the MVs was observed using transmission electron microscopy. The results are shown in Figure 19. The average particle size of the MVs is approximately 80.14 nm (the scale bar in the figure is 200 nm).

[0106] 2. Preparation of sublingual disintegrating tablets of Staphylococcus aureus exovesicles for periodontitis vaccine and their effect on promoting the levels of antigen-specific antibodies in saliva and serum.

[0107] Following the method described in Part 1, Staphylococcus aureus exovesicles (MV@CaP) coated with a calcium phosphate shell were prepared. The average particle size of the calcium phosphate-coated MV@CaP was approximately 140.6 nm. The transmission electron microscopy (TEM) image is shown in Figure 20, which shows that the outer shell is coated with a calcium phosphate shell, and the overall particle size is larger than MV (scale bar in the figure is 200 nm).

[0108] Following the methods described in Part 1, sublingual disintegrating tablets of Staphylococcus aureus vaccine were prepared for mice, and the intraoral disintegration of the sublingual disintegrating tablets was observed. As shown in Figure 21B, the sublingual disintegrating tablets disintegrated rapidly within 1 minute after being placed in the mouth. Small animal imaging tracing technology was used to confirm that the mineralized vesicles could target the submandibular lymph nodes of mice after administration, as shown in Figure 21C.

[0109] After administration, saliva and serum were collected from mice and cynomolgus monkeys in each group, and the titer levels of anti-Staphylococcus aureus specific IgG and IgA antibodies were detected. As shown in Figures 22-25, it can be seen that the sublingual disintegrating Staphylococcus aureus vaccine significantly promoted the expression of anti-Staphylococcus aureus specific IgG and IgA antibodies in saliva and serum samples.

[0110] 3. The in vivo preventive and therapeutic effects of MV@CaP ODT on ulcers.

[0111] Modeling of tongue mucosal ulcers in mice: C57BL / 6J mice immunized with PBS or ODT were anesthetized. A 2mm diameter filter paper saturated with 50% acetic acid was precisely applied to the posterior 1 / 3 of the tongue dorsum mucosa. After 60 seconds of incubation, the paper was removed, and the area was immediately wiped with a saline-soaked cotton swab. This intervention was repeated for three consecutive days, with daily observation of the ulcer area and calculation of the ulcer healing rate. On day 7 after modeling, animals were sacrificed, and tongue tissue was collected for subsequent histological evaluation.

[0112] The evaluation system includes: 1) Evaluation of ulcer healing rate: ulcer area was measured on days 1, 3 and 7 of ulcer modeling, and the healing rate was calculated; 2) Quantitative assessment of Staphylococcus aureus infection abundance at the ulcer site using PCR; 3) Collection of saliva from mice in each group and in vitro assessment of the in vitro killing ability of saliva against Staphylococcus aureus.

[0113] The efficacy of MV@CaP ODT (ODT) on delayed healing of oral ulcers caused by Staphylococcus aureus infection in mice is shown in Figure 26. Quantitative fluorescence analysis of *Lucernea* bacteria indicated that MV@CaP ODT effectively suppressed the abundance of *S. aureus* infection in the local area after oral ulceration in mice (Figure 26, A). Furthermore, compared with the control group, MV@CaP ODT increased the ulcer healing rate, effectively rescuing the delayed healing of oral ulcers caused by *S. aureus* infection (Figure 26, B).

[0114] 4. The promoting effect of MV@CaP ODT on the level of Staphylococcus aureus-specific antibodies in the saliva of clinical subjects.

[0115] The promoting effect of MV@CaP ODT (ODT) on the level of Staphylococcus aureus-specific antibodies in clinical patients is shown in Figure 26. The results showed that MV@CaP ODT upregulated the level of Staphylococcus aureus-specific sIgA antibodies in the saliva of the subjects (Figure 27, A). Furthermore, the in vitro killing ability of Staphylococcus aureus in the saliva of subjects immunized with MV@CaP ODT was enhanced (Figure 27, B).

[0116] Although this disclosure has been described with reference to exemplary embodiments, it should be understood that this disclosure is not limited to the exemplary embodiments disclosed. Various adjustments or changes may be made to the exemplary embodiments of this disclosure without departing from the scope or spirit of this disclosure. The scope of the claims should be interpreted in the broadest possible sense to cover all modifications and equivalent structures and functions.

Claims

1. A composition for the control of a bacterial infectious disease or condition of the oral cavity, characterized in that, This includes external vesicles derived from oral pathogens, such as periodontal pathogens, and optional pharmaceutically acceptable carriers.

2. The composition for controlling oral bacterial infectious diseases or conditions according to claim 1, characterized in that, The pathogenic bacteria include conditionally pathogenic bacteria and / or absolutely pathogenic bacteria, including at least one of Porphyromonas gingivalis, Actinobacillus actinomycetii, Forsythorbium, Fusobacterium nucleatum, Prevotella intermedius, and Staphylococcus aureus.

3. The composition for controlling oral bacterial infectious diseases or conditions according to claim 1, characterized in that, The oral diseases mentioned are inflammatory diseases caused by oral bacterial infections, such as periodontitis or ulcers.

4. The composition for controlling oral bacterial infectious diseases or conditions according to claim 1, characterized in that, It further includes calcium salts, preferably, the surface of the outer vesicle is bound with calcium salts, preferably, the calcium salts are coated on the surface of the outer vesicle to form a shell, and the calcium salts on the surface of the outer vesicles are slightly soluble or insoluble salts, preferably acid-soluble salts.

5. The composition for controlling oral bacterial infectious diseases or conditions according to claim 1, wherein The composition is a vaccine, such as a periodontitis vaccine or an ulcer vaccine.

6. The composition for controlling oral bacterial infectious diseases or conditions according to claim 1, wherein The composition is in the form of a sublingual disintegrating tablet, oral lozenge, oral spray, or hydrogel.

7. A method of making a composition for the prevention or treatment of a bacterial infectious disease or condition in the oral cavity, characterized in that, This includes providing external vesicles derived from oral pathogens and contacting calcium salts with the external vesicles so that the surface of the external vesicles is bound with calcium salts, preferably so that the calcium salts coat the surface of the external vesicles to obtain calcium-coated external vesicles.

8. The method of claim 7, wherein, This includes providing an external vesicle derived from oral pathogens and reacting a soluble calcium salt with the external vesicle, such as by mineralization, so that the surface of the external vesicle is bound with the calcium salt. Preferably, the calcium salt is coated on the surface of the external vesicle to obtain a calcium-coated external vesicle. The calcium salt on the surface of the external vesicle is a slightly soluble salt or an insoluble salt, preferably an acid-soluble salt.

9. The method of claim 7, wherein, The method further includes the step of preparing a formulation, preferably a tablet, using calcium-coated exovesicles.

10. Use of external vesicles derived from oral pathogens in the preparation of medicaments for increasing oral levels of specific IgG and / or sIgA antibodies against the source bacteria and for increasing serum levels of specific IgG and / or IgA antibodies against the source bacteria.

11. A method of preventing or treating a bacterial infectious disease or condition in the oral cavity, comprising administering to the oral cavity of a subject in need thereof an effective amount of a composition comprising a compound of Formula (I) or a pharmaceutically acceptable salt thereof. This includes administering an effective dose of an external vesicle derived from oral pathogens or a preparation containing such a pathogen to subjects in need.

12. A mineralized extracellular vesicle, characterized in that, It includes external vesicles derived from oral pathogens and calcium salts bound to the surface of the external vesicles. Preferably, the calcium salts coat the surface of the external vesicles to form a shell. The calcium salts on the surface of the external vesicles are slightly soluble or insoluble salts, preferably acid-soluble salts.

Images