Oral immunization system with cascade response characteristics and preparation method and application thereof

Abstract

The application discloses an oral immune system with a cascade response characteristic as well as a preparation method and application thereof, and belongs to the technical field of biological medicine and nano-preparation. The oral immune system comprises, from inside to outside, a core layer, an intermediate modification layer, a functional unit and a protective shell. The core layer is a live rumen coccus outer membrane vesicle. The intermediate modification layer is a polydopamine coating layer coated on the surface of the core layer. The functional unit is a cerium nanoscale enzyme covalently connected to the surface of the polydopamine coating layer through a thio-ketal bond connecting arm. The protective shell is made of calcium carbonate. The oral immune system integrates oral delivery barrier breakthrough, inflammation microenvironment responsiveness antioxidant and pathogenic bacteria specific immune clearance in the same platform, can break the vicious cycle of flora imbalance, oxidative stress and barrier damage in cooperation, and reduces the interference on beneficial symbiotic flora.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine and nanoparticle technology, and in particular to an oral immune system with cascade response characteristics, its preparation method, and its application. Background Technology

[0002] The pathological features of inflammatory bowel disease (IBD) typically involve the interrelationship and vicious cycle among gut microbiota dysbiosis, oxidative stress microenvironment, and intestinal barrier damage. Current treatments mostly focus on broad-spectrum anti-inflammatory or immunosuppressive therapies, targeting only a single point of action, making it difficult to simultaneously address multiple pathological processes, and are prone to causing systemic side effects.

[0003] Current intervention strategies targeting specific pathogens in dysbiotic gut microbiota mainly rely on antibiotics or probiotics, which cannot achieve precise elimination and may even disrupt the ecological balance of the gut microbiota. Furthermore, oral biologics must overcome gastric acid degradation, intestinal fluid flushing, and the mucus barrier during delivery, resulting in low colonic enrichment efficiency and further limiting the in vivo application of these treatments. Currently, there is a lack of multifunctional synergistic systems capable of simultaneously achieving specific elimination of pathogens, neutralization of reactive oxygen species, and repair of the intestinal barrier. Summary of the Invention

[0004] In view of this, the present invention provides an oral immune system with cascade response characteristics, its preparation method and application, to overcome multiple physiological barriers to oral administration, achieve cascade release in the colon, and synergistically complete pathogen clearance and oxidative stress regulation.

[0005] In a first aspect, the present invention provides an oral immune system with cascading response characteristics, which comprises, from the inside out, a core layer, an intermediate modification layer, a functional unit, and a protective shell. The core layer is composed of active rumenococcal outer membrane vesicles; The intermediate modification layer is a polydopamine coating that covers the surface of the core layer; The functional unit is a cerium oxide nanoenzyme covalently linked to the surface of the polydopamine coating via a thioketal linker arm. The protective shell is made of calcium carbonate.

[0006] Preferably, the particle size of the active rumenococcal outer membrane vesicles is 50~100 nm.

[0007] Secondly, the present invention provides a method for preparing the above-mentioned oral immune system with cascade response characteristics, comprising the following steps: The outer membrane vesicles of active rumenococci are dispersed in a first buffer solution, and dopamine hydrochloride is added to react and form a polydopamine coating on the outer membrane surface of active rumenococci, thus obtaining the first intermediate. The cerium oxide nanozyme was covalently linked to the polydopamine coating surface of the first intermediate via a thioketal linker to obtain the second intermediate. The second intermediate is dispersed in a calcium-containing buffer solution, and carbonate is added to mineralize it in situ to generate calcium carbonate coating the surface of the second intermediate, thus obtaining the oral immune system with cascade response characteristics.

[0008] Preferably, the preparation method of the active rumenococcal outer membrane vesicles is as follows: anaerobic culture of active rumenococci, collection of supernatant followed by microfiltration, ultracentrifugation and sucrose density gradient purification to obtain active rumenococcal outer membrane vesicles.

[0009] Preferably, the protein concentration of active rumenococcal outer membrane vesicles in the first buffer solution is 0.5~2 mg / mL; the ratio of protein mass of active rumenococcal outer membrane vesicles to mass of dopamine hydrochloride is (4~6):1; and the pH of the first buffer solution is 8.0~9.0.

[0010] Preferably, the specific steps for covalently linking cerium oxide nanozymes to the polydopamine coating surface of the first intermediate via thioketal linkage arms are as follows: dissolving the thioketal linkage arms in a solvent and mixing them with a dispersion of activated cerium oxide nanozymes to obtain cerium oxide nanozymes with thioketal linkage arms; then dispersing the first intermediate in a second buffer solution, adding the cerium oxide nanozymes with thioketal linkage arms, and reacting to obtain the second intermediate.

[0011] Furthermore, the mass ratio of the cerium oxide nanozyme connected with the thioketal linker arm to the protein mass of the first intermediate is (0.2~3):1.

[0012] Preferably, the calcium salt is calcium chloride, and the carbonate is selected from at least one of sodium bicarbonate, sodium carbonate, and ammonium carbonate; the molar ratio of calcium salt to carbonate is 1:(0.8~1.2); and the mass ratio of the protein content of the second intermediate to the mass of calcium salt is 1:(1~1.5).

[0013] Thirdly, the present invention provides a pharmaceutical composition comprising the above-described oral immune system with cascade response characteristics or the oral immune system with cascade response characteristics prepared by the above-described preparation method, and pharmaceutically acceptable excipients.

[0014] Fourthly, the present invention provides the use of the above-described oral immune system with cascade response characteristics, or the oral immune system with cascade response characteristics prepared by the above-described preparation method, or the above-described pharmaceutical composition in the preparation of a medicament for treating inflammatory bowel disease.

[0015] Compared with the prior art, the present invention has achieved the following beneficial effects: This invention utilizes actively active rumen cocci arranged sequentially from the inside out. Ruminococcus gnavus A core-shell oral immune system with cascade response characteristics was constructed, consisting of an outer membrane vesicle core layer, a polydopamine intermediate modification layer, cerium oxide nanozyme functional units covalently linked by thioketal linkage arms, and a calcium carbonate protective shell. This system utilizes the calcium carbonate shell to resist gastric acid degradation. Upon entering the intestine, the shell disintegrates, exposing the polydopamine coating, which enhances interaction with intestinal mucus to prolong colonic retention time. Simultaneously, the thioketal linkage arms break in the highly reactive oxygen species environment at the inflammatory site, releasing cerium oxide nanozymes on demand to scavenge reactive oxygen species. Active rumenococcal outer membrane vesicles act as natural antigen carriers, taken up by dendritic cells in Peyer's patches, activating the mucosal immune system and inducing the production of specific secretory immunoglobulin A in the intestinal mucosa, thus achieving protection against pathogenic bacteria (…). R. gnavus Precise clearance of pathogens. This system integrates oral delivery barrier breakthrough, inflammatory microenvironment-responsive antioxidation, and pathogen-specific immune clearance on the same platform, which can synergistically break the vicious cycle of dysbiosis, oxidative stress, and barrier damage, and reduce interference with beneficial symbiotic flora. Attached Figure Description

[0016] The accompanying drawings, which form part of this specification, are used to provide a further understanding of the invention. The illustrative embodiments and descriptions of the invention are used to explain the invention and do not constitute an undue limitation thereof. Obviously, those skilled in the art can obtain other drawings based on these drawings without any inventive effort.

[0017] Figure 1 These are formulation-related characterizations of Examples 1-3 of the present invention, wherein A is a transmission electron microscope image of Rg-OMV in Example 1; B is a laser confocal microscope image in Example 2; C is a dynamic light scattering particle size distribution map of Examples 1-3; D is a Zeta potential map of Examples 1-3; E is an SDS-PAGE image; F is an X-ray photoelectron spectrum of Example 3; and G is a Fourier transform infrared spectrum of Examples 1-3. Figure 2 This is a characterization of the C-OMV-PDA@Ca formulation in Example 4 of the present invention, wherein A is a scanning electron microscope image; B is a transmission electron microscope image; and C is an energy dispersive spectroscopy (EDS) diagram. Figure 3 This is an evaluation of the in vitro antioxidant capacity in the experimental examples of this invention. Among them, A is a graph of the chemical antioxidant capacity detection results; B is a fluorescence microscopy image of the ROS level in RAW 264.7 cells; and C is a flow cytometry quantitative analysis. Figure 4This invention presents the in vitro mucus penetration and in vivo distribution of the formulations in the experimental examples. Among them, A is a schematic diagram of the artificial mucus penetration experiment; B is a quantitative diagram of the penetration depth of each formulation in artificial mucus after 48 hours; C is an in vivo imaging image of mice; and D is an in vitro fluorescence imaging image of the gastrointestinal tract 8 hours after administration. Figure 5 These are the antibody responses and pathogen clearance effects in the experimental examples of this invention. A shows the change in serum anti-Rg specific IgG antibody titers; B shows the quantitative graph of sIgA antibody content in intestinal mucus; C shows the quantitative graph of sIgA antibody content in feces; and D shows the quantitative graph of sIgA antibody content in feces after challenge. R. gnavus qPCR detection results of relative load; Figure 6 This is an evaluation of the in vivo synergistic therapeutic effect of acute colitis in the experimental examples of the present invention. In this paper, A is a flowchart; B is a graph of relative weight change of mice in each group; C is a representative image of the colon of mice in each group; D is a quantitative graph of colon length of mice in each group; and E is an H&E staining image of distal colon tissue of mice in each group. Detailed Implementation

[0018] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, 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 invention pertains.

[0019] This invention provides an oral immune system with cascading response characteristics, which comprises, from the inside out, a core layer, an intermediate modification layer, a functional unit, and a protective shell. The core layer is composed of active rumenococcal outer membrane vesicles; The intermediate modification layer is a polydopamine coating that covers the surface of the core layer; The functional unit is a cerium oxide nanoenzyme covalently linked to the surface of the polydopamine coating via a thioketal linker arm. The protective shell is made of calcium carbonate.

[0020] In this invention, the oral immune system employs a core-shell structure with sequential encapsulation from the inside out. The core layer contains active rumenococci (…). Ruminococcus gnavus Rg-OMV (Ren-Omniform Vesicles) are naturally derived nanoscale vesicles carrying active rumenococcal-specific antigens on their surface. After oral delivery to the intestine, this core layer can be recognized by the intestinal mucosal immune system, inducing the production of specific secretory immunoglobulin A (sIgA). This allows for the precise elimination of pathogenic active rumenococci through an immune rejection mechanism, avoiding damage to the beneficial symbiotic flora.

[0021] The polydopamine coating coats the surface of the core layer. The catechol groups in the coating molecules can interact with mucins in intestinal mucus, thereby enhancing the adhesion and retention time of the entire immune system in the colonic mucus layer and prolonging the action window of the formulation at the target site. Simultaneously, the abundant active groups on the surface of the polydopamine coating provide reaction sites for the covalent linkage of subsequent functional units.

[0022] Cerium oxide nanozymes are covalently linked to the polydopamine coating surface via thioketal linkage arms. A thioketal bond is a reactive oxygen species (ROS) bond that can be broken. Under high oxidative stress conditions at the site of inflammation (such as the presence of large amounts of hydrogen peroxide and superoxide anions), the thioketal bond breaks, releasing the cerium oxide nanozyme. The cerium oxide nanozyme exhibits activities mimicking superoxide dismutase and catalase, efficiently scavenging superoxide anions, hydroxyl radicals, and hydrogen peroxide, thereby alleviating local oxidative stress damage in the intestine.

[0023] The protective outer shell is made of calcium carbonate. In the acidic environment of the stomach, calcium carbonate can undergo a neutralization reaction with hydrochloric acid and gradually dissolve. This process consumes stomach acid and delays the exposure of the internal core components, thus protecting the core components from rapid destruction by stomach acid. When the formulation enters the intestines, the calcium carbonate outer shell further disintegrates, rapidly releasing the internal functional structures and achieving targeted delivery to the colon.

[0024] Therefore, the oral immune system described in this invention integrates multiple functions such as gastric acid protection, mucus adhesion, ROS response release, precise immune clearance, and antioxidant therapy, forming a cascaded delivery and treatment system.

[0025] In this invention, the particle size of the active rumenococcal outer membrane vesicles is 50-100 nm. Within this particle size range, the outer membrane vesicles can maintain an intact vesicle structure and antigen activity, while also facilitating subsequent coating modification and in vivo delivery. For example, the particle size of the active rumenococcal outer membrane vesicles can be 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, or 100 nm, preferably 60-80 nm, and more preferably 65-75 nm.

[0026] In this invention, the outer membrane vesicles of the active rumenococci carry active rumenococcal-specific antigens on their surface, which are used to induce the production of specific secretory immunoglobulin A. These specific antigens include, but are not limited to, outer membrane proteins, lipopolysaccharides, and capsular polysaccharides, and can be taken up by M cells in Peyre's nodes of the intestine, thereby activating B cells to produce specific sIgA.

[0027] In this invention, the cerium oxide nanozyme is covalently linked to the polydopamine coating surface via a thioketal linker arm. One end of the thioketal linker arm reacts with an amino group on the polydopamine coating surface to form a covalent bond, while the other end reacts with a carboxyl group on the cerium oxide nanozyme surface to form a covalent bond. The thioketal bond is stable under normal physiological conditions and breaks when the ROS concentration exceeds a threshold, thereby enabling on-demand release from the inflamed site.

[0028] In this invention, the overall particle size of the oral immune system is 2-5 μm, more preferably 2-3 μm. This particle size range is beneficial for the adhesion and retention of the formulation on the intestinal mucosa, while avoiding excessively rapid clearance by the intestine.

[0029] The present invention also provides a method for preparing the above-mentioned oral immune system with cascade response characteristics, comprising the following steps: The outer membrane vesicles of active rumenococci are dispersed in a first buffer solution, and dopamine hydrochloride is added to react and form a polydopamine coating on the outer membrane surface of active rumenococci, thus obtaining the first intermediate. The cerium oxide nanozyme was covalently linked to the polydopamine coating surface of the first intermediate via a thioketal linker to obtain the second intermediate. The second intermediate is dispersed in a calcium-containing buffer solution, and carbonate is added to mineralize it in situ to generate calcium carbonate coating the surface of the second intermediate, thus obtaining the oral immune system with cascade response characteristics.

[0030] In one embodiment, the preparation method of the active rumenococcal outer membrane vesicles is as follows: Active rumenococci are anaerobically cultured, and the supernatant is collected and purified by microfiltration, ultracentrifugation, and sucrose density gradient purification to obtain the active rumenococcal outer membrane vesicles. Specifically, active rumenococci are inoculated into an anaerobic culture medium and anaerobically cultured at 37°C until the late logarithmic phase (e.g., OD). 600 When the bacterial concentration reaches 1.0–1.5, collect the bacterial culture and centrifuge at 8,000–12,000 × g for 15–30 minutes at 4°C to remove bacterial cells, collecting the supernatant. Filter the supernatant sequentially through 0.45 μm and 0.22 μm microporous membranes to remove residual bacterial cells and debris. Centrifuge the filtrate at 100,000–200,000 × g for 2–4 hours at 4°C, collecting the precipitate. Resuspend the precipitate in PBS buffer and slowly add it to a 30% sucrose layer, then centrifuge again at 100,000–200,000 × g for 2–3 hours, collecting the milky white band at the interface. Transfer the collected band to a dialysis bag and dialyze with PBS buffer to remove sucrose, yielding purified Rg-OMV.

[0031] In one embodiment, the protein concentration of *Ruminococcus rumeniformis* outer membrane vesicles in the first buffer solution is 0.5–2 mg / mL; the protein concentration is determined by the BCA (Bicinchoninic Acid Assay); the mass ratio of protein in the *Ruminococcus rumeniformis* outer membrane vesicles to dopamine hydrochloride is (4–6):1; and the pH of the first buffer solution is 8.0–9.0. The first buffer solution is preferably Tris-HCl buffer. For example, the protein concentration can be 0.5 mg / mL, 0.8 mg / mL, 1.0 mg / mL, 1.2 mg / mL, 1.5 mg / mL, 1.8 mg / mL, or 2.0 mg / mL, preferably 0.8–1.5 mg / mL, and more preferably 1.0 mg / mL. The mass ratio of protein in the *Ruminococcus rumeniformis* outer membrane vesicles to dopamine hydrochloride is further preferably 5:1. The pH of the first buffer solution can be 8.0, 8.2, 8.5, 8.8, or 9.0, preferably 8.3–8.7, and more preferably 8.5. The reaction conditions are: 25°C, protected from light, for 4–8 hours, for example, 6 hours. After the reaction, unreacted dopamine hydrochloride is removed by ultrafiltration centrifugation (100 kDa molecular weight cutoff) or dialysis to obtain the first intermediate (OMV-PDA).

[0032] In one embodiment, the specific steps for covalently linking cerium oxide nanozymes to the polydopamine coating surface of a first intermediate via a thioketene linker are as follows: The thioketene linker is dissolved in a solvent and mixed with a dispersion of activated cerium oxide nanozymes to obtain cerium oxide nanozymes (TK-CeO2) linked with the thioketene linker; then, the first intermediate is dispersed in a second buffer solution, and the cerium oxide nanozymes linked with the thioketene linker are added, followed by a reaction to obtain a second intermediate. The thioketene linker (TK linker) contains a thioketene bond (-SC(CH3)2-S-) and active groups at both ends (preferably carboxyl and amino groups). In one or more embodiments of the present invention, the thioketene linker is preferably COOH-PEG-TK-NH2. The solvent can be selected from dimethyl sulfoxide, dimethylformamide, or anhydrous ethanol. The activation process uses EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and NHS (N-hydroxysuccinimide) to activate the carboxyl groups on the surface of cerium oxide nanozymes.

[0033] In this invention, the mass ratio of the cerium oxide nanozyme connected with the thioketal linker arm to the protein mass of the first intermediate is (0.2~3):1, more preferably (1~3):1, further preferably (1.5~2.5):1, and most preferably 2:1.

[0034] In one or more embodiments of the present invention, COOH-CeO2 is dispersed in MES buffer (pH 5.5-6.0), and EDC and NHS are added for activation at room temperature for 1-3 hours. After ultrafiltration purification, activated CeO2-NHS ester is obtained. COOH-PEG-TK-NH2 is added, and the molar ratio of TK linker to carboxyl groups on the CeO2 surface is controlled at (4-6):1. The reaction is carried out at room temperature for 3-5 hours, and CeO2-PEG-TK-COOH is obtained after ultrafiltration purification. CeO2-PEG-TK-COOH is then added to EDC and NHS for reactivation for 1-3 hours. The first intermediate (OMV-PDA) is dispersed in a second buffer. The second buffer is preferably PBS buffer (pH 7.2-7.4). The two are mixed and reacted overnight at 4°C in the dark. After ultrafiltration centrifugation (100 kDa) and washing three times, the second intermediate (C-OMV-PDA) is obtained.

[0035] In one embodiment, the calcium salt is calcium chloride, and the carbonate is selected from at least one of sodium bicarbonate, sodium carbonate, and ammonium carbonate, more preferably sodium bicarbonate. The molar ratio of calcium salt to carbonate is 1:(0.8~1.2), more preferably 1:1; the mass ratio of protein content of the second intermediate to calcium salt is 1:(1~1.5), more preferably 1:(1.2~1.3), and even more preferably 1:1.25.

[0036] In this invention, the second intermediate (C-OMV-PDA) is dispersed in a calcium-containing buffer solution, preferably HEPES buffer (pH 7.2-7.6) or Tris-HCl buffer (pH 7.4). The concentration of the second intermediate can be 0.5 mg / mL, 0.8 mg / mL, 1.0 mg / mL, 1.2 mg / mL, 1.5 mg / mL, or 2.0 mg / mL, preferably 0.8-1.2 mg / mL, and more preferably 0.8 mg / mL.

[0037] The in-situ mineralization steps are as follows: Under stirring conditions, the carbonate solution is slowly added dropwise to a buffer solution containing the second intermediate and calcium salt, and the reaction is carried out at room temperature for 2-10 minutes. After the reaction is complete, the precipitate is collected by centrifugation (3,000-5,000×g, 5-10 minutes), washed 2-3 times with deionized water, and then freeze-dried or stored at low temperature to obtain an oral immune system with cascade response characteristics (C-OMV-PDA@Ca).

[0038] The present invention also provides a pharmaceutical composition comprising the above-described oral immune system with cascade response characteristics or the oral immune system with cascade response characteristics prepared by the above-described preparation method, and pharmaceutically acceptable excipients.

[0039] Pharmaceutically acceptable excipients include, but are not limited to, fillers, binders, disintegrants, lubricants, flow aids, flavoring agents, and preservatives. For example, fillers may be selected from one or more of lactose, starch, microcrystalline cellulose, and mannitol; binders may be selected from one or more of hydroxypropyl methylcellulose, polyvinylpyrrolidone, and starch paste; disintegrants may be selected from one or more of croscarmellose sodium, carboxymethyl starch sodium, and croscarmellose; and lubricants may be selected from one or more of magnesium stearate, stearic acid, and talc.

[0040] In this invention, the pharmaceutical composition is an oral dosage form. For example, the oral dosage form may be a capsule, tablet, granule, powder, or oral suspension. The oral immune system is mixed with pharmaceutically acceptable excipients and then formulated into the above dosage form using conventional formulation techniques. For capsules, the oral immune system can be mixed with fillers and disintegrants and then filled into gelatin capsules or hydroxypropyl methylcellulose capsules; for tablets, binders and lubricants can be further added, and the tablets can be compressed using a tableting machine; for granules, wet granulation or dry granulation can be used followed by packaging.

[0041] The present invention also provides the use of the above-described oral immune system with cascade response characteristics, or the oral immune system with cascade response characteristics prepared by the above-described preparation method, or the above-described pharmaceutical composition in the preparation of a medicament for treating inflammatory bowel disease.

[0042] The inflammatory bowel disease includes ulcerative colitis and Crohn's disease. The treatment includes at least one of the following effects: reducing the disease activity index (DAI), reducing colonic shortening, reducing the levels of pro-inflammatory factors (such as TNF-α, IL-6, IL-1β) in colonic tissue, clearing accumulated reactive oxygen species, upregulating the expression of tight junction proteins (such as ZO-1, Occludin), and repairing the intestinal mucus barrier (increasing the number of goblet cells).

[0043] The technical solution of the present invention will be further described below with reference to specific embodiments. The present invention does not impose any special restrictions on the source of reagents used in the following embodiments; commercially available products well known to those skilled in the art can be used.

[0044] Example 1 This embodiment provides the preparation of active rumenococcal outer membrane vesicles (Rg-OMV). (1) Active rumen cocci ( Ruminococcus gnavus ATCC 29149 strain was inoculated into anaerobic medium and strictly anaerobic cultured at 37°C until the late logarithmic growth phase (OD2). 600 =1.2).

[0045] (2) Collect the bacterial culture, centrifuge at 10,000×g for 20 minutes at 4℃ to remove the bacterial cells, and collect the supernatant. Filter the supernatant through 0.45 μm and 0.22 μm microporous membranes in sequence to remove residual bacterial cells and debris.

[0046] (3) The filtrate was centrifuged at 150,000×g for 3 hours at 4℃, and the precipitate was collected, which was the crude OMV extract.

[0047] (4) Resuspend the crude extract in PBS buffer, carefully add it to the 30% sucrose pad, and centrifuge again at 150,000×g and 4℃ for 2 hours. Collect the milky white OMV band at the interface between the sucrose pad and the sample solution.

[0048] (5) The collected OMV was resuspended in PBS buffer, transferred to a dialysis bag (molecular weight cutoff 100 kDa), and dialyzed overnight at 4°C to remove sucrose, thus obtaining a high-purity Rg-OMV preparation.

[0049] like Figure 1 As shown in A, the obtained Rg-OMV, observed by transmission electron microscopy, exhibits a typical bilayer membrane vesicle structure with a particle size of 50–100 nm. Figure 1 As shown in C and D, the average particle size of the obtained Rg-OMV is 68.5 nm and the Zeta potential is -28.3 mV, as determined by dynamic light scattering (DLS) testing.

[0050] Example 2 This embodiment provides the preparation of a first intermediate, namely, active rumen cocci outer membrane vesicles (OMV-PDA) modified with a polydopamine coating.

[0051] (1) The Rg-OMV prepared in Example 1 was dispersed in Tris-HCl buffer (pH 8.5) and the protein concentration of Rg-OMV was controlled to be 1 mg / mL.

[0052] (2) Add dopamine hydrochloride and control the ratio of protein content of Rg-OMV to mass of dopamine hydrochloride to 5:1. React at 25°C in the dark for 6 hours to allow dopamine to oxidize and self-polymerize on the surface of OMV to form a polydopamine coating.

[0053] (3) After the reaction is complete, use an ultrafiltration centrifuge tube (molecular weight cutoff 100 kDa) to centrifuge at 4,000×g for 15 minutes to remove unreacted dopamine monomers and oligomers. Wash with PBS buffer 3 times to obtain the first intermediate OMV-PDA preparation.

[0054] Figure 1 Laser confocal microscopy in B showed that the DID-labeled Rg-OMV and the FITC-labeled PDA were highly co-localized, confirming successful PDA encapsulation. Figure 1 As shown in C and D, the average particle size of the obtained OMV-PDA is 135.8 nm and the Zeta potential is -24.7 mV, as determined by dynamic light scattering (DLS) testing.

[0055] Example 3 This embodiment provides the preparation of the second intermediate, namely the first intermediate of the cerium oxide nanozyme coupling (C-OMV-PDA).

[0056] (1) Activation of carboxyl groups on CeO2 surface: COOH-CeO2 was dispersed in MES buffer (0.1 M, pH 5.5-6.0), and EDC and NHS (molar ratio COOH:EDC:NHS = 1:100:200) were added. The mixture was activated at 4°C for 30 minutes, and then the reaction was continued at room temperature for 2 hours. After the reaction was completed, unreacted EDC / NHS was removed by ultrafiltration centrifugation (10 kDa). The mixture was washed three times with PBS buffer (pH 7.4) to obtain activated CeO2-NHS ester, which was then resuspended in PBS buffer for later use.

[0057] (2) Linking the TK linker: COOH-PEG-TK-NH2 (thioketal linker arm) was dissolved in anhydrous dimethyl sulfoxide to prepare a 10 mM TK linker stock solution. The CeO2-NHS ester dispersion activated in step (2) was mixed with the TK linker stock solution, and the molar ratio of TK linker to carboxyl groups on the CeO2 surface was controlled at 5:1. The reaction was carried out at room temperature for 4 hours. After the reaction was completed, unreacted TK linker was removed by ultrafiltration centrifugation (10 kDa), and washed three times with PBS to obtain CeO2-PEG-TK-COOH.

[0058] (3) Disperse the OMV-PDA prepared in Example 2 in PBS buffer (pH 7.4). Take CeO2-PEG-TK-COOH prepared in step (2), add EDC and NHS again (molar ratio COOH:EDC:NHS = 1:100:200) to activate the terminal carboxyl group. After activating at room temperature for 2 hours, add OMV-PDA solution and control the protein mass ratio of CeO2 to OMV-PDA to be 2:1. React at 4°C overnight (12 hours).

[0059] (4) After the reaction is complete, remove unbound TK-CeO2 by ultrafiltration centrifugation (100 kDa) and wash three times with PBS buffer to obtain the second intermediate C-OMV-PDA preparation.

[0060] like Figure 1 As shown in C and D, dynamic light scattering measurements revealed that the C-OMV-PDA particle size is 148.7 nm and the Zeta potential is -18.2 mV. Figure 1 As shown in F, X-ray photoelectron spectroscopy detected Ce 3d characteristic peaks, Ce 3+ / Ce 4+ The ratio is consistent with that of free CeO2NPs. For example... Figure 1 As shown in G, the Fourier transform infrared spectrum is displayed at 650 cm⁻¹. -1 The presence of a TK bond characteristic peak confirms successful coupling.

[0061] Example 4 This embodiment provides the preparation of an oral immune system (C-OMV-PDA@Ca) with cascade response characteristics.

[0062] (1) The C-OMV-PDA prepared in Example 3 was dispersed in HEPES buffer (pH 7.4) containing CaCl2, and the concentration of C-OMV-PDA was adjusted to an appropriate value (800 μg / mL based on OMV protein). The mass ratio of OMV protein to CaCl2 was controlled to be 1:1.25.

[0063] (2) Under magnetic stirring, slowly add an equal volume of NaHCO3 solution of equal concentration (the molar ratio of CaCl2 to NaHCO3 is 1:1).

[0064] (3) After the addition is complete, continue stirring for 3 minutes to allow calcium carbonate to mineralize in situ on the carrier surface and form a core-shell structure. Collect the precipitate by centrifugation, wash it three times with deionized water, and freeze-dry it to obtain the C-OMV-PDA@Ca preparation.

[0065] like Figure 2 As shown in Figure A, scanning electron microscopy revealed that C-OMV-PDA@Ca exhibits a regular spherical structure with a smooth surface and a particle size of approximately 2–3 μm. Figure 2 As shown in B, a clear core-shell structure is visible under a transmission electron microscope, with a dense outer layer of CaCO3. Figure 2 As shown in C, energy dispersive spectroscopy analysis reveals that Ca, N, Ce, and O elements are uniformly distributed on the surface of the microspheres.

[0066] Comparative Example 1 This comparative example provides a formulation of unmodified PDA and cerium oxide nanozyme, namely OMV@Ca.

[0067] The specific preparation method is similar to that of Example 4, except that in this comparative example, Rg-OMV from Example 1 is used instead of C-OMV-PDA to finally obtain OMV@Ca.

[0068] Comparative Example 2 This comparative example provides a formulation of unmodified cerium oxide nanozyme, namely OMV-PDA@Ca.

[0069] The specific preparation method is similar to that of Example 4, except that in this comparative example, OMV-PDA from Example 2 is used instead of C-OMV-PDA to finally obtain OMV-PDA@Ca.

[0070] Test case All experimental data are expressed as mean ± standard deviation (Mean ± SD). One-way ANOVA was used for comparisons between groups, followed by Tukey's multiple comparison test. P < 0.05 was considered statistically significant, and P < 0.01 was considered highly significant.

[0071] 1. Antioxidant capacity test (1) Chemical antioxidant capacity test C-OMV-PDA formulations with different mass ratios of CeO2:OMV were prepared according to Example 3. Following the instructions of the commercially available kit, their effects on superoxide anion (O2) were measured. - The scavenging capacity of the formulation for hydroxyl radicals (•OH) and hydrogen peroxide (H2O2) was evaluated. Free CeO2NPs were used as a control. The free radical scavenging rate of the formulation at each concentration was calculated, and concentration-scavenging rate curves were plotted.

[0072] The results are as follows Figure 3 As shown in Figures A, B, C, and D, the carbon dioxide (C-OMV-PDA) exhibit different scavenging abilities against the three types of free radicals at different ratios. At a mass ratio of 2:1, C-OMV-PDA demonstrates the optimal scavenging efficiency for ROS.

[0073] (2) Intracellular oxidative stress model testing RAW 264.7 cells were seeded in 6-well plates and cultured overnight to allow them to adhere. The experimental groups were as follows: blank control group, model group (H2O2 stimulation), OMV group (Example 1), CeO2 group (free cerium oxide nanozyme), OMV-PDA group (Example 2), and C-OMV-PDA group (Example 3). Except for the blank control group, each group was stimulated with H2O2 (400 μM) for 4 hours, followed by co-incubation with the corresponding formulation (50 μg / mL). Intracellular ROS levels were detected using the DCFH-DA probe method. After drug administration, the culture medium was aspirated, and DCFH-DA working solution was added to a final concentration of 10 μM. The cells were incubated at 37°C in the dark for 30 minutes. Cells were washed three times with serum-free culture medium to remove probes that had not entered the cells. Fluorescence intensity was detected using a fluorescence microplate reader (excitation wavelength 488 nm, emission wavelength 525 nm), and images were simultaneously taken using a fluorescence microscope.

[0074] like Figure 3As shown in Figure D, fluorescence microscopy revealed strong green fluorescence in the model group, indicating that H2O2 stimulation induced a large amount of ROS production; the green fluorescence in the C-OMV-PDA group was significantly weakened, approaching the level of the blank control group. Figure 3 As shown in E, the flow cytometry results are consistent.

[0075] 2. In vitro mucus permeability test An artificial mucus model was constructed using 5% porcine gastric mucin, and the mucus layer was placed in a flow... Tube In this study, Cy5.5-labeled OMV (Example 1), OMV-PDA (Example 2), and C-OMV-PDA (Example 3) were added above the mucus layer, and the penetration depth of each formulation in the mucus layer was detected using in vivo imaging at 0, 24, and 48 hours.

[0076] The results are as follows Figure 4 As shown in Figure A, after 24 hours, the penetration depth was 0.8 ± 0.2 cm for the OMV group, 1.2 ± 0.2 cm for the OMV-PDA group, and 1.3 ± 0.2 cm for the C-OMV-PDA group; Figure 4 As shown in Figure B, at 48 hours, the penetration depth of the OMV group was 1.5 ± 0.2 cm, the OMV-PDA group was 2.2 ± 0.2 cm, and the C-OMV-PDA group was 2.3 ± 0.2 cm. The penetration depth of the PDA-modified groups (OMV-PDA and C-OMV-PDA) was significantly higher than that of the unmodified group (P < 0.01), confirming that the PDA coating enhances the diffusion ability of the formulation in mucus.

[0077] 3. Distribution in mice and evaluation of colon targeting Cy7 fluorescent dye was used to label OMV@Ca (Comparative Example 1), OMV-PDA@Ca (Comparative Example 2), C-OMV-PDA (Example 3), and C-OMV-PDA@Ca (Example 4), respectively. The specific groups were as follows: OMV@Ca group (Comparative Example 1), OMV-PDA@Ca group (Comparative Example 2), C-OMV-PDA group (Example 3), and C-OMV-PDA@Ca group (Example 4).

[0078] Each formulation was administered to mice (n=3) via gavage, and the dosage for each mouse was normalized to Cy7 fluorescence intensity. Fluorescence imaging was performed using a small animal in vivo imaging system at 0, 2, 4, 6, 8, 12, and 24 hours after administration to observe the distribution of fluorescence signals in vivo.

[0079] like Figure 4As shown in Figure C, 2 hours after administration, the fluorescence signals in all groups were mainly concentrated in the stomach and small intestine; at 4 hours, the fluorescence signals in the OMV-PDA@Ca group and the C-OMV-PDA@Ca group began to migrate towards the colonic region; from 6 to 8 hours, the fluorescence signals in the above two groups were significantly enriched in the colon, and the signal intensity was significantly higher than that in the OMV@Ca group and the C-OMV-PDA group. Figure 4 As shown in D, 8-hour ex vivo imaging showed that the colonic fluorescence signal of the OMV-PDA@Ca group and the C-OMV-PDA@Ca group was significantly higher than that of the other two groups, confirming that the CaCO3 shell protection and PDA coating adhesion synergistically achieved good colonic targeted delivery, and that the coupling of CeO2 did not affect the delivery efficiency.

[0080] 4. Antibody response and pathogen clearance effect test (1) Immunization protocol and sample collection Female BALB / c mice aged 6-8 weeks were randomly divided into four groups: a blank control group, OMV@Ca (Comparative Example 1), OMV-PDA@Ca (Comparative Example 2), C-OMV-PDA (Example 3), and C-OMV-PDA@Ca (Example 4), with 8 mice in each group. Mice in each group were administered the corresponding formulation (5 μg / mouse / time, based on Rg-OMV protein content) via gavage on days 0, 2, 4, and 6. The blank control group received an equal volume of PBS. Serum samples were collected from mice on days 7, 14, and 21 after the first immunization, and intestinal mucus and fecal samples were collected on day 21.

[0081] (2) Antibody detection The titer of anti-Rg specific IgG antibodies in serum was detected by indirect ELISA: Rg-OMV was coated onto an ELISA plate, serially diluted mouse serum was added, incubated, HRP-labeled secondary antibody was added, and OD was read after color development. 450 The antibody titer was calculated. The sIgA antibody content in intestinal mucus and feces was quantitatively analyzed using an ELISA kit, following the instructions of the kit manufacturer.

[0082] like Figure 5As shown in Figure A, specific IgG antibodies were detectable in all groups of mice at all time points. On day 7, specific IgG antibodies were detectable in the C-OMV-PDA@Ca group, significantly higher than in other groups; the OMV-PDA@Ca group was next; the OMV@Ca and C-OMV-PDA groups had lower titers, with no significant difference between the two groups. On day 14, antibody titers increased in all groups, with the C-OMV-PDA@Ca group still having the highest titer, followed by the OMV-PDA@Ca group, while the OMV@Ca and C-OMV-PDA groups had lower titers. On day 21, the C-OMV-PDA@Ca group reached its peak antibody titer, significantly higher than the OMV-PDA@Ca group, while the OMV@Ca and C-OMV-PDA groups remained at lower levels. Figure 5 As shown in Figure BC, the intestinal mucus sIgA level in the OMV-PDA@Ca group was significantly higher than that in the OMV@Ca group and the C-OMV-PDA group. The fecal sIgA detection results were consistent with those in the intestinal mucus, confirming that the oral delivery system effectively induced a local mucosal immune response in the intestine.

[0083] (3) Pathogenic bacteria challenge experiment On day 21 after the last immunization (the peak antibody period), mice in each group were subjected to... R. gnavus Live bacteria challenge experiment: Each mouse was administered 1×10⁻⁶ bacteria via gavage. 8 CFU R. gnavus Live bacterial suspension. Fecal samples were collected on day 3 post-challenge, and fecal genomic DNA was extracted. Specific primers were used to detect the presence of genomic DNA in the feces via real-time quantitative PCR (qPCR). R. gnavus The relative load. Using the 16S rRNA gene as an internal reference, through 2 -ΔΔCt Method to calculate each group R. gnavus The fold change in expression relative to the blank control group.

[0084] like Figure 5 As shown in D, on day 3 post-challenge, the feces of group C-OMV-PDA@Ca were positive. R. gnavus The relative viral load was significantly lower than that in the model group and other formulation groups (P<0.01), indicating that the specific sIgA antibody effectively mediated the clearance of pathogens. The OMV@Ca group and the C-OMV-PDA group also showed some clearance effects, but were significantly weaker than those in the C-OMV-PDA@Ca group.

[0085] 5. Evaluation of the efficacy of synergistic in vivo treatment for acute colitis (1) Animal model and dosing regimen Female BALB / c mice aged 6-8 weeks were randomly divided into the following groups, with 5 mice in each group: blank control group, model group, OMV@Ca group (comparative example 1), OMV-PDA@Ca group (comparative example 2), C-OMV-PDA group (example 3), C-OMV-PDA@Ca group (example 4) and 5-ASA group (positive control, 5-aminosalicylic acid, 100 mg / kg).

[0086] like Figure 6 As shown in A, except for the blank control group, the other groups were allowed free access to 3% DSS solution for 7 days to induce acute colitis, while simultaneously being administered the corresponding preparation (10 μg / vial, calculated as OMV protein) by gavage for 4 days, once every other day. The 5-ASA group was administered 5-aminosalicylic acid (100 mg / kg) by gavage.

[0087] (2) Weight change and colon length measurement The body weight of mice in each group was recorded daily, and the relative body weight percentage was calculated (with the body weight on day 0 as 100%). At the end of the experiment, the mice were sacrificed, and the colonic tissue from the cecum to the anus was separated, its length was measured, and images were taken.

[0088] like Figure 6 As shown in B, the C-OMV-PDA@Ca group mice experienced the smallest decrease in body weight, significantly better than the model group (P<0.01) and other formulation groups. Figure 6 As shown in C and D, the colon of the C-OMV-PDA@Ca group was significantly longer than that of the model group (P<0.01), and close to the level of the blank control group.

[0089] (3) Histopathological evaluation Distal colon tissue was collected, fixed in 4% paraformaldehyde, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E). Histopathological changes were observed under a light microscope, and a pathological score (0-4 points) was assigned. Figure 6 As shown in Figure E, H&E staining revealed that the colonic mucosa structure in the model group was severely damaged, crypts disappeared, and a large number of inflammatory cells infiltrated; the intestinal tissue structure in the C-OMV-PDA@Ca group was relatively intact, with only mild inflammatory infiltration, which was significantly lower than that in the model group (P<0.01) and other preparation groups.

[0090] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. An oral immune system with cascade response characteristics, characterized in that, From the inside out, it consists of a core layer, an intermediate decorative layer, functional units, and a protective outer shell; The core layer is composed of active rumenococcal outer membrane vesicles; The intermediate modification layer is a polydopamine coating that covers the surface of the core layer; The functional unit is a cerium oxide nanoenzyme covalently linked to the surface of the polydopamine coating via a thioketal linker arm. The protective shell is made of calcium carbonate.

2. The oral immune system with cascade response characteristics as described in claim 1, characterized in that, The outer membrane vesicles of the active rumenococci have a particle size of 50-100 nm.

3. The method for preparing an oral immune system with cascade response characteristics as described in any one of claims 1 to 2, characterized in that, Includes the following steps: The outer membrane vesicles of active rumenococci are dispersed in a first buffer solution, and dopamine hydrochloride is added to react and form a polydopamine coating on the outer membrane surface of active rumenococci, thus obtaining the first intermediate. The cerium oxide nanozyme was covalently linked to the polydopamine coating surface of the first intermediate via a thioketal linker to obtain the second intermediate. The second intermediate is dispersed in a calcium-containing buffer solution, and carbonate is added to mineralize it in situ to generate calcium carbonate coating the surface of the second intermediate, thus obtaining the oral immune system with cascade response characteristics.

4. The preparation method according to claim 3, characterized in that, The preparation method of the active rumenococcal outer membrane vesicles is as follows: anaerobic culture of active rumenococci, collection of supernatant followed by microfiltration, ultracentrifugation and sucrose density gradient purification to obtain active rumenococcal outer membrane vesicles.

5. The preparation method according to claim 3, characterized in that, The protein concentration of active rumenococcal outer membrane vesicles in the first buffer solution was 0.5–2 mg / mL; the protein content of active rumenococcal outer membrane vesicles to the mass ratio of dopamine hydrochloride was (4–6):1; and the pH of the first buffer solution was 8.0–9.

0.

6. The preparation method according to claim 3, characterized in that, The specific steps for covalently linking cerium oxide nanozymes to the polydopamine coating surface of the first intermediate via thioketal linkage arms are as follows: dissolve the thioketal linkage arms in a solvent and mix them with the dispersion of activated cerium oxide nanozymes to obtain cerium oxide nanozymes with thioketal linkage arms; then disperse the first intermediate in a second buffer solution, add the cerium oxide nanozymes with thioketal linkage arms, and obtain the second intermediate after the reaction.

7. The preparation method according to claim 6, characterized in that, The mass ratio of the cerium oxide nanozyme with the thioketal linker arm to the protein mass of the first intermediate is (0.2~3):

1.

8. The preparation method according to claim 3, characterized in that, The calcium salt is calcium chloride, and the carbonate is selected from at least one of sodium bicarbonate, sodium carbonate, and ammonium carbonate; the molar ratio of calcium salt to carbonate is 1:(0.8~1.2); the mass ratio of protein in the second intermediate to the mass of calcium salt is 1:(1~1.5).

9. A pharmaceutical composition, characterized in that, The oral immune system with cascade response characteristics as described in claim 1 or 2, or the oral immune system with cascade response characteristics prepared by the preparation method described in any one of claims 3 to 8, and pharmaceutically acceptable excipients.

10. The use of the oral immune system with cascade response characteristics as described in claim 1 or 2, or the oral immune system with cascade response characteristics prepared by the preparation method according to any one of claims 3 to 8, or the pharmaceutical composition according to claim 9, in the preparation of a medicament for treating inflammatory bowel disease.

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