Pathogen capture driven oral microgel system based on spatial reprogramming, preparation method and application

Abstract

The application belongs to the technical field of biology and biological medicine, and discloses a pathogen capturing driven oral microgel system based on spatial reprogramming, a preparation method and application. The system is composed of a methacrylated hyaluronic acid microgel matrix, salmonella specific nanobodies covalently anchored on the surface and meropenem loaded bacterial membrane vesicles encapsulated in the interior. The nanobodies selectively capture and fix salmonella, the microgel releases the vesicles in response in the intestinal tract, and after the vesicles fuse with the outer membrane of the pathogen, the antibiotic is delivered to the periplasmic space, realizing spatially limited and efficient sterilization. The minimum inhibitory concentration of the system is reduced by 4 times compared with free antibiotics, the pathogen is removed at the same time, the intestinal flora homeostasis is protected, the barrier repair and immune balance are promoted, the problems of flora imbalance and drug resistance caused by non-selective sterilization of traditional antibiotics are solved, and a new paradigm for precise treatment of intestinal bacterial infection is provided.

Description

Technical Field

[0001] This invention belongs to the field of biology and biomedicine, specifically relating to a pathogen capture-driven oral microgel system based on spatial reprogramming, its preparation method, and its application. Background Technology

[0002] salmonella( Salmonella Intestinal infection is a major threat to human health and livestock production worldwide. Salmonella can invade and survive within intestinal epithelial cells, triggering an inflammatory cascade that leads to epithelial damage and barrier dysfunction. Clinical and experimental studies consistently demonstrate that Salmonella infection not only causes acute diarrhea and weight loss but is also often accompanied by intestinal barrier disruption, immune imbalance, and gut microbiota dysbiosis. These pathological processes reinforce each other, increasing the risk of persistent infection, recurrence, and systemic dissemination. Therefore, effective treatment for intestinal Salmonella infection requires not only reducing the pathogen load but also coordinating the restoration of barrier integrity, immune balance, and gut microbiota homeostasis.

[0003] Antibiotics, with their rapid bactericidal activity, are currently the primary means of clinical management of Salmonella infections. However, their inherent limitations are becoming increasingly apparent: broad-spectrum antibiotics inevitably disrupt the symbiotic microbial community while eliminating pathogens, often leading to long-term dysbiosis and delayed mucosal repair; repeated or high-dose use further promotes antibiotic resistance and raises concerns about drug residues and systemic toxicity; more importantly, antibiotic treatment primarily works by directly killing bacteria and is largely ineffective in regulating infection-related inflammatory responses and tissue damage, thus limiting its ability to restore intestinal function and immune homeostasis. Simply eliminating pathogens often fails to translate into satisfactory therapeutic effects.

[0004] In recent years, in order to overcome the aforementioned shortcomings of traditional antibiotics, researchers have explored a variety of novel anti-infective strategies from different perspectives, but each still faces significant technical bottlenecks: Firstly, the application of nanobody therapy in gastrointestinal infections is being explored. Nanobodies (VHHs) have been explored for the treatment of gastrointestinal infections in recent years due to their small size, high stability, ease of engineering, and low-cost production. For example, studies have developed VHHs targeting the adhesion proteins of common bacterial diarrhea pathogens such as Escherichia coli and Salmonella, which exert anti-infective effects by preventing bacterial attachment in the gastrointestinal tract. Professor Wang Hongning's team at Sichuan University developed a novel anti-Salmonella infection strategy using a nanobody (FliC-Nb76) secreted by recombinant Lactococcus lactis targeting the flagellate protein FliC. This nanobody significantly inhibited the motility of Salmonella and prevented its adhesion and invasion of intestinal epithelial cells and macrophages. In animal models, it significantly reduced mortality caused by Salmonella infection and alleviated the inflammatory response (Journal of Nanobiotechnology, 2024, doi: 10.1186 / s12951-024-02904-8). Furthermore, existing technology CN119591700A discloses an anti-Salmonella enteritidis nanobody, kit, and its application. This nanobody contains specific complementarity-determining region sequences, exhibiting strong affinity and high specificity. Other studies have reported the potential of orally administered nanobodies in gastrointestinal-related diseases, including those caused by contaminated food, opportunistic pathogen proliferation due to gut microbiota dysbiosis, and enterotoxin ingestion. However, current nanobody therapies primarily work by neutralizing pathogen virulence factors or blocking their adhesion, lacking active bactericidal capabilities and failing to completely eliminate already colonized pathogens. Simultaneously, orally administered nanobodies are affected by pH and enzymatic degradation in the gastrointestinal tract, resulting in low bioavailability, requiring protection through formulation strategies (such as engineered cell delivery). More critically, existing nanobody strategies have not been effectively integrated with precision bactericidal methods, making it difficult to achieve efficient pathogen clearance and gut microbiota protection while simultaneously capturing pathogens.

[0005] Secondly, the development of antimicrobial delivery systems using bacterial outer membrane vesicles (OMVs). Bacterial outer membrane vesicles are naturally secreted lipid bilayer nanostructures (10-250 nm in diameter) by Gram-negative bacteria, possessing the innate ability to deliver biomolecules to specific target cells. In recent years, OMVs have been explored as antimicrobial drug delivery carriers. Existing technology CN115006367A discloses an antibiotic-loaded bacterial outer membrane vesicle, its preparation method, and its application. Specifically, it involves extracting bacterial outer membrane vesicles and encapsulating the antibiotic meropenem for the treatment of common drug-resistant bacteria in clinical practice, such as carbapenem-resistant Acinetobacter baumannii and carbapenem-resistant Pseudomonas aeruginosa. This nanocarrier, by extracting bacterial outer membrane vesicles and encapsulating antibiotics, can improve the efficacy of antimicrobial drugs. Existing technology CN119875903A discloses an antibiotic carrier based on bacterial outer membrane vesicles and its preparation method. Salmonella mutant strain QS0074 (accession number CCTCC M 2023192) is cultured in a medium containing the antibiotic acaricin. After removing viable bacteria, outer membrane vesicles are extracted. The resulting carrier exhibits excellent drug loading capacity and good drug activity stability. Furthermore, previous studies have utilized the homologous membrane fusion properties of OMVs with bacterial outer membranes for drug delivery. However, existing OMV delivery systems mainly rely on single-targeting strategies, lacking the ability to actively capture and spatially concentrate pathogens dispersed in the intestinal lumen. The potential toxicity of OMV surface lipopolysaccharide (LPS) and its susceptibility to immune system clearance have not been fully resolved. More importantly, existing systems are mostly single-stage delivery systems, lacking a multi-stage synergistic architecture of "microgel protection + vesicle release + membrane fusion delivery," making it difficult to achieve the three-stage linkage of gastric acid protection after oral administration, intestinal localization release, and precise delivery near the pathogen. The promoting effects on post-infection tissue repair and microbial reconstruction are also insufficiently considered.

[0006] Thirdly, research progress on oral hydrogel / microgel delivery systems. Hydrogels and microgels, as oral drug delivery carriers, are widely used in the treatment of intestinal diseases due to their excellent biocompatibility, modifiability, and intestinal retention capacity. Yang et al. developed dual-response hydrogel microspheres (HMs) prepared by electrospray irradiation, composed of sodium alginate, hyaluronic acid, and Eudragit S100, achieving a 90% encapsulation efficiency for Salmonella-targeting phage cocktails. In a Salmonella typhimurium-induced colitis model, this formulation reduced the intestinal Salmonella load by nearly 2000-fold and decreased the levels of pro-inflammatory cytokines (TNF-α, IL-6, IL-1β) to 60% of the infected group, achieving efficacy comparable to ciprofloxacin while avoiding antibiotic-associated dysbiosis and diarrhea. Existing technology CN121910656A discloses a ROS-responsive oral microgel-in-situ intestinal bioadhesive patch system, which triggers the in-situ transformation of the microgel into a patch via ROS, forming a natural mucus barrier and simultaneously releasing anti-inflammatory drugs. Another study constructed a PVA-GG-based bilayer heterogeneous microgel delivery system for the treatment of colitis. However, existing hydrogel microsphere systems mainly deliver biological agents such as bacteriophages or probiotics, rather than traditional antibiotic phage therapy, which is limited by its narrow host spectrum and susceptibility to resistance. Probiotic therapy, on the other hand, has limited bactericidal efficiency and slow onset of action. More importantly, existing hydrogels mainly act as passive protective carriers, releasing their contents in response to signals such as pH or enzymes, lacking the ability to actively recognize, capture, and restrict the movement of pathogens. Even when loaded with antibiotics, release is still primarily passive diffusion, failing to deliver antibiotics directionally to the periplasmic space of pathogens, still posing a risk of off-target effects on symbiotic bacteria. Functionally, a complete closed loop of "capture-restriction-targeted killing-tissue repair" has not yet been achieved.

[0007] Fourth, the application limitations of the combined strategy of nanobodies and bacterial membrane vesicles. In recent years, the combined application of nanobodies and bacterial membrane vesicles has gradually attracted attention, but it is mainly concentrated in the field of tumor immunotherapy. For example, some studies have prepared bacterial outer membrane vesicles (OMVs) expressing PD-L1 nanobodies on their surface to explore their blocking effect on the PD-1 / PD-L1 signaling axis. Other studies have used genetic engineering to anchor CD47 nanobodies on the surface of outer membrane vesicles, obtaining engineered nanocarriers OMV-CD47nb-CERX, to enhance the phagocytic capacity of macrophages and induce macrophage polarization. In the field of anti-infection, research at the University of Cincinnati has shown that by orally administering specially engineered bacteria, viral proteins or therapeutic nanobodies can be displayed on their surface and released through outer membrane vesicles, thereby inducing an immune response in animal models. In addition, some studies have used outer membrane vesicles secreted by engineered Escherichia coli Nissle 1917 as natural biocompatible carriers, enabling nanobodies to cross mucosal barriers and extend to sites throughout the body. However, existing strategies primarily modify the surface of nanobodies to target host cells (such as tumor cells and immune cells), rather than the pathogens themselves; combined strategies mainly deliver immunomodulators or chemotherapeutic drugs, without addressing the loading and precise release of antibiotics; and existing systems are mostly for injection, failing to solve the problems of gastric acid protection and intestinal controlled release required for oral delivery. The application of nanobody-bacterial membrane vesicle combined strategies to the field of oral anti-intestinal bacterial infection still presents a significant research gap.

[0008] Fifth, the functional expansion of hyaluronic acid-based hydrogel materials: Hyaluronic acid (HA) is widely used as a carrier material for oral drug delivery due to its excellent biocompatibility, mucosal adhesion, and modifiability. Existing technology CN120754023A discloses a hyaluronic acid-based nanogel with intestinal targeting and inflammatory responsiveness, its preparation method, and applications. Another study encapsulated thiolized hyaluronic acid-curcumin self-assembled nanoparticles in sodium alginate hydrogel microspheres, constructing a pH, GSH-responsive, and macrophage-targeting drug delivery system for the precise treatment of ulcerative colitis. Existing technology CN120899768A discloses a probiotic-polyphenol nanoparticle colon-targeting co-delivery system, which combines polyphenol nanoparticles with hyaluronic acid through non-covalent interactions, giving the hydrogel both colon-targeting and mucosal adhesion properties. Studies have shown that methacryloyl hyaluronic acid (HAMA) can form hydrogels through photocrosslinking, which can be used to encapsulate and deliver therapeutic substances. However, existing hyaluronic acid-based hydrogels mainly serve as single-function protective carriers, with contents limited to probiotics or single drugs. They have not yet achieved multi-component functional integration of "targeting elements (nanobodies) + bactericidal elements (drug-loaded vesicles)". Existing HA hydrogels are mostly monolithic gels or large-sized microspheres, making it difficult to achieve monodispersity and precise control of particle size, which affects batch consistency in oral delivery. More importantly, there is a lack of spatially confined delivery design for antibiotics, and antibiotics are still mainly released by passive diffusion, making it impossible to achieve precise control over the release location and target of antibiotics.

[0009] In summary, although researchers have made progress in single-technology dimensions such as nanobody targeting (e.g., FliC-Nb76 nanobody), bacterial membrane vesicle delivery, and hydrogel carriers (e.g., dual-response hydrogel microspheres), existing technologies still suffer from the following common defects: functional fragmentation: targeted capture, bactericidal delivery, and tissue repair functions are dispersed in different systems, failing to form a synergistic closed loop; lack of spatial constraint: antibacterial effects are still mainly based on non-selective diffusion, making it impossible to confine bactericidal activity to the microenvironment near the pathogen, thus hindering the protection of the microbiome; insufficient oral integration: lacking a multi-level delivery architecture that can simultaneously satisfy gastric acid protection, intestinal responsive release, and precise delivery near the pathogen.

[0010] Therefore, there is an urgent need in the field for a novel strategy that can synergistically integrate pathogen selective capture, spatially confined sterilization, and intestinal homeostasis protection into a single oral delivery platform. Summary of the Invention

[0011] To address the technical problems of existing oral antibiotic treatments for Salmonella infections, such as non-selective bactericidal action, disruption of gut microbiota balance, easy induction of drug resistance, and difficulty in simultaneously promoting tissue repair, the primary objective of this invention is to provide a pathogen-capture-driven oral microgel system based on spatial reprogramming. The core of this system is the selective capture and concentration of dispersed Salmonella bacteria using surface-anchored specific nanobodies, confining them spatially to the microgel surface. This triggers the release of antibiotic-loaded vesicles, which are then directly delivered to the periplasmic space adjacent to the pathogen through outer membrane fusion. This avoids the diffusion-dependent dilution of free antibiotics and minimizes off-target bacterial disturbance, thereby achieving efficient pathogen clearance and synergistic restoration of host gut homeostasis. This system functionally decouples pathogen confinement, antibacterial delivery, and post-infection repair at different spatial scales, establishing a new paradigm for precision treatment of intestinal bacterial infections.

[0012] To achieve the above objectives, the present invention adopts the following technical solution: A pathogen capture-driven oral microgel system based on spatial reprogramming comprises a hyaluronic acid-based microgel matrix, pathogen-specific nanobodies covalently anchored to the surface of the hyaluronic acid-based microgel matrix, and antibiotic-loaded bacterial membrane vesicles encapsulated within the hyaluronic acid-based microgel matrix. The pathogen-specific nanobody is Salmonella-specific nanobody Nb76; The antibiotic is a carbapenem antibiotic; The bacterial membrane vesicles are derived from probiotics or attenuated bacteria; The pathogen-specific nanobody captures and confines the pathogen on the surface of the microgel. After the bacterial membrane vesicles are released in response to intestinal fluid, they fuse with the outer membrane of the pathogen and deliver antibiotics into the periplasmic space of the pathogen, thus completing targeted delivery.

[0013] Preferably, the hyaluronic acid-based microgel matrix is ​​prepared by methacrylamide hyaluronic acid through microfluidic droplet generation combined with ultraviolet light crosslinking; The molecular weight of the methacrylamide hyaluronic acid is 50-150 kDa; The intensity of the ultraviolet crosslinking light is 5~40 mW / cm². 2 Crosslinking time: 10-120 seconds; The microgel exhibits a swelling rate of 200-500% in simulated intestinal fluid at pH 6.8. The microgel releases bacterial membrane vesicles in the intestinal environment within 2 to 8 hours.

[0014] Preferably, the molecular weight of the methacrylamide hyaluronic acid is 100 kDa; The intensity of the ultraviolet crosslinking light irradiation is 20 mW / cm².2 Crosslinking time: 40 s; The microgel exhibited a swelling rate of 300% in simulated intestinal fluid at pH 6.8. The microgel releases bacterial membrane vesicles within 4 hours in the intestinal environment.

[0015] Preferably, the pathogen-specific nanobody is covalently linked to the microgel surface via maleimide-NHS coupling chemical; The anchoring density of the pathogen-specific nanobodies on the microgel surface is 1~10 μg / mg microgel; The bacterial membrane vesicles were derived from Escherichia coli Nissle 1917; The antibiotic in question is meropenem; The loading amount of meropenem in bacterial membrane vesicles is 50~500 μg / mg vesicle protein; The bacterial membrane vesicles have a particle size of 50~300 nm; The minimum inhibitory concentration (MIC) of the microgel system, based on meropenem equivalents, is 10-50 ng / mL.

[0016] Preferably, the anchoring density of the pathogen-specific nanobody on the microgel surface is 5 μg / mg microgel; The loading amount of meropenem in bacterial membrane vesicles is 200 μg / mg vesicle protein; The bacterial membrane vesicles have a particle size of 150 nm; The minimum inhibitory concentration (MIC) of the microgel system, based on meropenem equivalents, is 25 ng / mL.

[0017] A method for preparing a pathogen capture-driven oral microgel system based on spatial reprogramming, comprising the following steps: S1. Preparation of antibiotic-loaded bacterial membrane vesicles: Bacterial membrane vesicles were cultured to the logarithmic growth phase, and the bacterial cells were collected. They were treated with a buffer containing 0.5-5 mM EDTA to soften the bacterial outer membrane. Then, they were homogenized under high pressure at 800-1500 bar for 2-5 cycles. The membrane fragments were then co-assembled with antibiotics into vesicles using an extrusion process. Finally, the free drug was removed by ultracentrifugation at 100,000-400,000 × g to obtain purified antibiotic-loaded bacterial membrane vesicles. S2. Preparation of microgels encapsulating vesicles: The antibiotic-loaded bacterial vesicles obtained in S1 were mixed with a methacrylamide hyaluronic acid precursor solution and a photoinitiator. Monodisperse droplets were generated using a microfluidic device with an oil / water phase flow ratio of 5:1 to 20:1. The mixture was then subjected to a wavelength of 365 nm and an intensity of 5 to 20 mW / cm².2 In-situ cross-linking was performed by irradiating with ultraviolet light for 10-60 seconds to obtain HAMA@BM intermediate microgel; S3. Covalent anchoring of pathogen-specific nanobodies: The microgels obtained from S2 were activated with maleimide-NHS to introduce maleimide groups. After washing, they were incubated with pathogen-specific nanobodies at 2-8°C for 4-24 hours to form a targeted microgel system.

[0018] Preferably, in step S1, the bacterial outer membrane is softened by treatment with a buffer containing 1 mM EDTA, followed by three cycles of high-pressure homogenization at 1200 bar; finally, free drugs are removed by ultracentrifugation at 300,000 × g. In S2, a microfluidic device with an oil / water flow rate ratio of 12:1 is used; the wavelength is 365 nm and the intensity is 10 mW / cm. 2 In-situ crosslinking was performed by irradiating with ultraviolet light for 30 seconds; In step S3, after washing, the nanobody is incubated with pathogen-specific nanobody at 4°C for 12-16 hours. The precursor solution of the methacryloyl hyaluronic acid has a mass concentration of 1~8 mg / mL; The photoinitiator is lithium phenyl-2,4,6-trimethylbenzoyl phosphite, with a concentration of 1~5 mg / mL; The bacterial membrane vesicles were loaded into the microgel at a rate of 10–50 μg / mg microgel.

[0019] Preferably, the precursor solution of the methacrylamide hyaluronic acid has a mass concentration of 3.5 mg / mL; The concentration of the photoinitiator is 3 mg / mL; The bacterial membrane vesicles were loaded into the microgel at a rate of 20 μg / mg microgel.

[0020] Application of a pathogen capture-driven oral microgel system based on spatial reprogramming in the preparation of oral drugs against enteric bacterial infections, wherein the enteric bacterial infection is enteritis caused by Salmonella; The drug is used to prevent / treat enteritis caused by Salmonella infection.

[0021] Preferably, the microgel system is used in any of the following situations: 1) Reduce the pathogen load in the gut and system; 2) Relieves weight loss, abnormal body temperature, and shortened colon caused by bacterial enteritis; 3) Promotes the repair of the intestinal epithelial barrier and the reconstruction of the mucus layer; 4) It inhibits the expression of pro-inflammatory factor TNF-α and increases the level of anti-inflammatory factor TGF-β; 5) Promotes macrophage polarization from M1 to M2 type; 6) Maintain the α-diversity, β-diversity, and core taxonomic composition of the gut microbiota; 7) It protects gut microbiota homeostasis while eliminating intestinal pathogens; 8) Promotes intestinal barrier repair and regulates the immune microenvironment.

[0022] Compared with the prior art, the present invention has at least the following technical effects: This invention provides a pathogen capture-driven oral microgel system based on spatial reprogramming. The system comprises a methacryloyl hyaluronic acid (HAMA) microgel matrix, Salmonella-specific nanobodies (Nb76) covalently anchored to the microgel surface, and meropenem-loaded bacterial membrane vesicles (BMs) encapsulated within the microgel. The nanobodies selectively recognize, capture, and immobilize Salmonella, restricting its motility. The microgel responsively swells in the intestinal environment and sequentially releases vesicles. After the vesicles fuse with the pathogen's outer membrane, they directly deliver antibiotics into the periplasmic space, achieving highly efficient bactericidal action within a confined space. This system achieves a minimum inhibitory concentration (MIC) as low as 25 ng / mL (based on meropenem equivalents), four times lower than free antibiotics, rapidly reducing bacterial numbers, effectively inhibiting biofilm formation, and delaying bacterial regeneration after withdrawal.

[0023] The core of this system is the selective capture and concentration of dispersed Salmonella bacteria using surface-anchored specific nanobodies, confining them to the surface of a microgel. This triggers the release of antibiotic-loaded vesicles, which are then delivered directly to the periplasmic space of adjacent pathogens via outer membrane fusion. This avoids the diffusion-dependent dilution of free antibiotics and minimizes off-target bacterial disturbances, thereby achieving efficient pathogen clearance and synergistic restoration of host gut homeostasis. It breaks the self-limiting cycle of "bactericidal-dysbiosis-repair" and provides a universal design principle for precision anti-infective therapy.

[0024] In vivo cell experiments showed that the system significantly reduced the intestinal pathogen load, achieved high cell survival rates, and did not cause significant cell death or skeletal disorder. At the same time, it protected intestinal flora homeostasis, promoted inflammation resolution, epithelial barrier repair, and immune balance. It solved the problems of flora imbalance and drug resistance caused by the non-selective bactericidal effect of traditional antibiotics, and provided a new paradigm for precision treatment of intestinal bacterial infections. Attached Figure Description

[0025] Figure 1 A schematic diagram illustrating the construction and physicochemical characterization of BM; Figure 2 A schematic diagram illustrating the construction and physicochemical characterization of HAMA-Nb@BM; Figure 3 This is a schematic diagram illustrating the targeted capture, motility restriction, and efficient killing of Salmonella typhimurium by HAMA-Nb@BM. Figure 4 This is a schematic diagram of how HAMA-Nb@BM synergistically inhibits bacterial invasion and reprograms the host defense transcriptional network in an in vitro epithelial infection model. Figure 5 A schematic diagram illustrating the in vivo biocompatibility and immune safety of HAMA-Nb@BM; Figure 6 This is a schematic diagram illustrating the in vivo anti-Salmonella performance evaluation of HAMA Nb@BM. Detailed Implementation

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the present invention will be briefly introduced below in conjunction with the accompanying drawings and descriptions of the embodiments or the prior art. Obviously, the following description of the structure of the accompanying drawings is only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. It should be noted that the description of these embodiments is for the purpose of helping to understand the present invention, but does not constitute a limitation of the present invention.

[0027] Example 1: Materials and Reagents Reagents: Hyaluronic acid (HA, molecular weight ~100 kDa), methacrylic anhydride (MA), NHS-maleimide (NHS-Mal) and related coupling reagents, meropenem trihydrate (MEM), EDTA, paraffin oil, photoinitiator, and other analytical grade reagents were purchased from MedChemExpress (USA). Sterile phosphate buffer (PBS, 1×, pH 7.4) was used throughout the process.

[0028] Biological raw materials: Salmonella Enteritidis ATCC13076-specific nanobody Nb76 was derived from previous work; its construction, expression, and purification have been reported and validated. Escherichia coli Nissle 1917 was used to prepare bacterial membrane vesicles (BMVs). Salmonella Typhimurium was used for in vitro and in vivo infection studies; some experiments employed stable expression of mCherry (Amp... + The strains were Lactobacillus rhamnosus ATCC 39595 and Enterococcus faecalis ATCC29212 as commensal control strains.

[0029] Cell lines: Human colonic epithelial cells Caco-2, mouse macrophages RAW264.7, and mouse fibroblasts L929 were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin in a humidified incubator at 37°C with 5% CO2. Mycoplasma contamination was detected periodically.

[0030] Laboratory animals: Four-week-old female SPF BALB / c mice were purchased from Chengdu Dashuo Laboratory Animal Co., Ltd., and were housed under specific pathogen-free (SPF) conditions. The mice were acclimatized for 7 days before modeling. All animal procedures were approved by the Institutional Animal Management and Use Committee of Sichuan University (Approval No. SCU251009001).

[0031] Example 2: Preparation and purification of meropenem-loaded bacterial membrane vesicles Escherichia coli Nissle 1917 was inoculated into LB medium and cultured at 37°C on a shaker (200 rpm) until the mid-log phase (OD2). 600 = 0.7). Bacterial cells were collected by centrifugation at 5000g for 10 min, washed twice with PBS, and resuspended in buffer containing 1 mM EDTA. The cells were incubated at room temperature for 1 h to gently disrupt the outer membrane. Subsequently, high-pressure homogenization (1200 bar, 3 cycles) was performed to obtain membrane fragments. Unbroken cells and large debris were removed by centrifugation at 5000×g for 10 min. The supernatant was then sequentially centrifuged, followed by ultracentrifugation at 300,000×g for 2 h to collect the BMVs precipitate. The precipitate was resuspended in PBS to obtain empty BMVs.

[0032] For drug loading, meropenem (final concentration 1000 μg / mL) was added during the recombination stage of the homogenized membrane to co-assemble into vesicles. Repeated ultracentrifugation was used to remove free drug, yielding meropenem-loaded BMVs (BMs). The vesicle formulation was normalized to protein content before use.

[0033] Example 3: Physicochemical characterization of bacterial membrane vesicles (BMVs) Vesicle morphology was observed using transmission electron microscopy (TEM): Samples were fixed with 2% paraformaldehyde, adsorbed onto a Formvar-carbon-coated copper grid, negatively stained with 1% uranium acetate, and imaged using a JEOL JEM-2100F electron microscope (200 kV). Nanoparticle size distribution and concentration were determined using a ZetaView system (Particle Metrix) with NTA software v3.1. The measurement temperature was 20℃, and three 30-second videos were recorded for each sample, with detection parameters manually set (camera gain=4; detection threshold=17).

[0034] The results showed that the vesicle size ranged from 50 to 300 nm, with an average size of about 150 nm.

[0035] Example 4: Construction of HAMA-based microgels encapsulating BMVs HAMA was synthesized using a known method: HA was reacted with methacrylic anhydride under alkaline conditions (pH 8.0), followed by lyophilization by dialyzing. Amino-functionalized HAMA (HAMA-NH2) was obtained via EDC / NHS-mediated diamine coupling. The structure was confirmed by ¹H NMR and ATR-FTIR.

[0036] Microgel preparation: A HAMA precursor solution (50 mg / mL) containing the photoinitiator LAP (3 mg / mL) was mixed with BMVs (20 μg / mg microgel) normalized to protein content. Monodisperse droplets were generated using a flow-focusing microfluidic device (oil-to-water ratio 12:1), and in-situ crosslinked by UV light (365 nm, 10 mW / cm², 30 seconds) to obtain HAMA@BM microgels. The particles were repeatedly washed to remove unreacted components and resuspended in sterile PBS.

[0037] Example 5: Nanobody Conjugation and Preparation of HAMA-Nb@BM HAMA@BM microgel: Nb76 was chemically modified with NHS-maleimide. The microgel was first reacted with NHS-Mal to introduce maleimide groups. After thorough washing, it was incubated with Nb76 (5 μg / mg microgel) at 4°C with gentle shaking for 12 h. Excess nanobodies were washed away to obtain HAMA-Nb@BM. The vesicle content and nanobodies density of each batch were normalized before use.

[0038] Example 6: Extraction and Characterization of BMVs like Figure 1 The diagram shows the construction and physicochemical characterization of BM. A) Schematic diagram of the BM preparation process; B) Photograph of the BM precipitate after ultracentrifugation; C) Electron microscopy showing naturally secreted extracellular vesicles (indicated by red arrows); D) TEM showing increased electron density within vesicles after drug loading (scale bar 200 nm); E) NTA analysis of vesicle size distribution (50–300 nm). Results combined Figure 1 The results showed that BMVs were successfully isolated from the probiotic (Escherichia coli 1917).

[0039] Example 7: Microgel characterization and gastrointestinal responsiveness The morphology and size of the microgels were observed using an optical microscope.

[0040] Simulated gastrointestinal conditions: HAMA-Nb@BM was placed in simulated gastric fluid (SGF, pH 1.2) or simulated intestinal fluid (SIF, pH 6.8) and incubated at 37°C. Particle swelling and structural integrity were monitored at specific time points.

[0041] like Figure 2The diagram shows the construction and physicochemical characterization of HAMA-Nb@BM. A) Schematic diagram of microfluidic preparation of microgels; B) Image of monodisperse microgels under an optical microscope; C) Schematic diagram of structural response in simulated gastrointestinal fluid; D) Microgel morphology under different digestion conditions.

[0042] The results, as shown in Figure D, indicate that the microgels maintained their structural integrity within 2 hours in SGF, and gradually swelled, disintegrated, and released vesicles within 4–8 hours in SIF. Vesicle release in SIF was quantified using NTA.

[0043] Example 8: In vitro targeting assay like Figure 3 The diagram shows the targeted capture, motility restriction, and efficient killing of Salmonella typhimurium by HAMA-Nb@BM. A) Fluorescent images of vesicles binding to different bacteria; B) Inhibition of colony expansion in semi-solid agar; C) MIC determination (25 ng / mL); D) Bacterial nucleic acid staining and membrane damage confocal image.

[0044] 7.1 Targeted binding: Salmonella expressing mCherry was incubated with HAMA@BM or HAMA-Nb@BM at 37°C for 2 hours and then confocal imaging was performed.

[0045] The results, as shown in Figure A, indicate that HAMA-Nb@BM has selective binding ability to Salmonella, but significantly less binding to Lactobacillus rhamnosus and Enterococcus faecalis.

[0046] 7.2 Bacterial motility was assessed using the semi-solid agar method and real-time microscopic tracking.

[0047] The results, as shown in Figure B, indicate that HAMA-Nb@BM significantly inhibited the diffusion of Salmonella in semi-solid agar.

[0048] 7.3 Minimum inhibitory concentration and bacterial biofilm staining As shown in Figure C, the minimum inhibitory concentration (MIC) of HAMA-Nb@BM against Salmonella enteritidis is 25 ng / mL, which is 4 times lower than that of free MEM.

[0049] As shown in Figure D, HAMA-Nb@BM treatment caused bacterial biofilm disruption, while the bactericidal effect of low-dose free MEM was relatively limited.

[0050] Example 9: Cell Culture Infection Model and Transcriptome Analysis Caco-2 cells were infected with Salmonella (MOI=10) and treated with PBS, free meropenem, blank microgel, and HAMA-Nb@BM, respectively. Cell integrity was assessed by phalloidin / DAPI staining and CFU counting.

[0051] like Figure 4 The diagram shown illustrates the synergistic inhibition of bacterial invasion and reprogramming of the host defense transcriptional network by HAMA-Nb@BM in an in vitro epithelial infection model. A) Experimental procedure; B) Caco-2 cell viability; C) Cytoskeleton confocal image; D) Intracellular bacterial load.

[0052] Results combined Figure 4 Overall, the HAMA-Nb@BM treatment group showed intact cytoskeleton and significantly reduced intracellular bacterial load.

[0053] Example 10: Biosafety Evaluation BALB / c mice were orally administered HAMA Nb@BM, and major organs (heart, liver, spleen, lung, and kidney) were extracted 72 hours later. The organs were photographed and recorded in their gross form, and then sectioned using HE after paraffin embedding.

[0054] Figure 5 This diagram illustrates the in vivo biocompatibility and immune safety of HAMA Nb@BM. (AC) Mouse hematology, organ morphology, and tissue sections.

[0055] Combination Figure 5 The results show that HAMA-Nb@BM has good biocompatibility and does not have significant toxic effects on animal organs.

[0056] Example 11: Animal infection model and in vivo evaluation BALB / c mice were orally administered Salmonella (1×10⁻⁶). 8 (CFU / animal). Patients were randomly assigned to one of the following groups: PBS control group, free meropenem group, blank drug-loaded vesicle group, or HAMA-Nb@BM treatment group. They received oral PBS, free meropenem (MEM), BM, or HAMA-Nb@BM once daily for 3 consecutive days at a dose of 6 mg / animal / day. Body weight, temperature, and clinical scores were monitored to assess disease progression. Tissue bacterial load was determined by CFU counting. Histological analysis was performed in a blinded manner.

[0057] Figure 6 This is a schematic diagram illustrating the in vivo anti-Salmonella performance evaluation of HAMA Nb@BM. AB) Mouse gavage administration protocol and gross tube; C) Daily changes in body weight; D) Intestinal Salmonella load.

[0058] Results combined Figure 6 The results showed that, compared with the PBS group, mice in the HAMA-Nb@BM treatment group recovered body weight significantly faster, their body temperature returned to normal, and their colon length was significantly longer than that of the untreated group. Colon and cecal CFU counts showed that the pathogen load in the HAMA-Nb@BM group was reduced by 3-4 orders of magnitude.

[0059] Example 12: Statistical Analysis Data are expressed as mean ± sd. Statistical analysis was performed using GraphPad Prism v9.1. Two-tailed Student's t-tests were used for comparisons between two groups; one-way ANOVA and appropriate post-hoc tests were used for comparisons among multiple groups. Survival data were analyzed using Kaplan-Meier curves and log-rank tests. A p-value < 0.05 was considered statistically significant. No data were excluded from the analysis.

[0060] Finally, it should be noted that the above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. 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. A pathogen capture-driven oral microgel system based on spatial reprogramming, characterized in that, The product comprises a hyaluronic acid-based microgel matrix, pathogen-specific nanobodies covalently anchored to the surface of the hyaluronic acid-based microgel matrix, and antibiotic-loaded bacterial membrane vesicles encapsulated within the hyaluronic acid-based microgel matrix. The pathogen-specific nanobody is Salmonella-specific nanobody Nb76; The antibiotic is a carbapenem antibiotic; The bacterial membrane vesicles are derived from probiotics or attenuated bacteria; The pathogen-specific nanobody captures and confines the pathogen on the surface of the microgel. After the bacterial membrane vesicles are released in response to intestinal fluid, they fuse with the outer membrane of the pathogen and deliver antibiotics into the periplasmic space of the pathogen, thus completing targeted delivery.

2. The oral microgel system according to claim 1, characterized in that, The hyaluronic acid-based microgel matrix was prepared by microfluidic droplet generation combined with ultraviolet light crosslinking of methacryloyl hyaluronic acid. The molecular weight of the methacrylamide hyaluronic acid is 50-150 kDa; The intensity of the ultraviolet crosslinking light is 5~40 mW / cm². 2 Crosslinking time: 10-120 seconds; The microgel exhibits a swelling rate of 200-500% in simulated intestinal fluid at pH 6.

8. The microgel releases bacterial membrane vesicles in the intestinal environment within 2 to 8 hours.

3. The oral microgel system according to claim 2, characterized in that, The molecular weight of the methacrylamide hyaluronic acid is 100 kDa; The intensity of the ultraviolet crosslinking light irradiation is 20 mW / cm². 2 Crosslinking time: 40 s; The microgel exhibited a swelling rate of 300% in simulated intestinal fluid at pH 6.

8. The microgel releases bacterial membrane vesicles within 4 hours in the intestinal environment.

4. The oral microgel system according to claim 1, characterized in that, The pathogen-specific nanobodies are covalently linked to the surface of the microgel via maleimide-NHS coupling chemical; The anchoring density of the pathogen-specific nanobodies on the microgel surface is 1~10 μg / mg microgel; The bacterial membrane vesicles were derived from Escherichia coli Nissle 1917; The antibiotic in question is meropenem; The loading amount of meropenem in bacterial membrane vesicles is 50~500 μg / mg vesicle protein; The bacterial membrane vesicles have a particle size of 50~300 nm; The minimum inhibitory concentration (MIC) of the microgel system, based on meropenem equivalents, is 10-50 ng / mL.

5. The oral microgel system according to claim 4, characterized in that, The pathogen-specific nanobody is anchored at a density of 5 μg / mg microgel on the microgel surface. The loading amount of meropenem in bacterial membrane vesicles is 200 μg / mg vesicle protein; The bacterial membrane vesicles have a particle size of 150 nm; The minimum inhibitory concentration (MIC) of the microgel system, based on meropenem equivalents, is 25 ng / mL.

6. A method for preparing a pathogen capture-driven oral microgel system based on spatial reprogramming as described in any one of claims 1 to 5, characterized in that, Includes the following steps: S1. Preparation of antibiotic-loaded bacterial membrane vesicles: Bacterial membrane vesicles were cultured to the logarithmic growth phase, and the bacterial cells were collected. They were treated with a buffer containing 0.5-5 mM EDTA to soften the bacterial outer membrane. Then, they were homogenized under high pressure at 800-1500 bar for 2-5 cycles. The membrane fragments were then co-assembled with antibiotics into vesicles using an extrusion process. Finally, the free drug was removed by ultracentrifugation at 100,000-400,000 × g to obtain purified antibiotic-loaded bacterial membrane vesicles. S2. Preparation of microgels encapsulating vesicles: The antibiotic-loaded bacterial vesicles obtained in S1 were mixed with a methacrylamide hyaluronic acid precursor solution and a photoinitiator. Monodisperse droplets were generated using a microfluidic device with an oil / water flow ratio of 5:1 to 20:

1. The mixture was then subjected to a wavelength of 365 nm and an intensity of 5 to 20 mW / cm². 2 In-situ cross-linking was performed by irradiating with ultraviolet light for 10-60 seconds to obtain HAMA@BM intermediate microgel; S3. Covalent anchoring of pathogen-specific nanobodies: The microgels obtained from S2 were activated with maleimide-NHS to introduce maleimide groups. After washing, they were incubated with pathogen-specific nanobodies at 2-8°C for 4-24 hours to form a targeted microgel system.

7. The preparation method according to claim 6, characterized in that, In step S1, the bacterial outer membrane is softened by treatment with a buffer containing 1 mM EDTA, followed by three cycles of high-pressure homogenization at 1200 bar; finally, free drug is removed by ultracentrifugation at 300,000 × g. In S2, a microfluidic device with an oil / water flow rate ratio of 12:1 is used; the wavelength is 365 nm and the intensity is 10 mW / cm. 2 In-situ crosslinking was performed by irradiating with ultraviolet light for 30 seconds; In step S3, after washing, the nanobody is incubated with pathogen-specific nanobody at 4°C for 12-16 hours. The precursor solution of the methacryloyl hyaluronic acid has a mass concentration of 1~8 mg / mL; The photoinitiator is lithium phenyl-2,4,6-trimethylbenzoyl phosphite, with a concentration of 1~5 mg / mL; The bacterial membrane vesicles were loaded into the microgel at a rate of 10–50 μg / mg microgel.

8. The preparation method according to claim 7, characterized in that, The precursor solution of the methacrylamide hyaluronic acid has a mass concentration of 3.5 mg / mL; The concentration of the photoinitiator is 3 mg / mL; The bacterial membrane vesicles were loaded into the microgel at a rate of 20 μg / mg microgel.

9. The application of the pathogen capture-driven oral microgel system based on spatial reprogramming as described in any one of claims 1 to 5 in the preparation of oral medicaments against intestinal bacterial infections, characterized in that, The intestinal bacterial infection was enteritis caused by Salmonella; The drug is used to prevent / treat enteritis caused by Salmonella infection.

10. The application according to claim 9, characterized in that, The microgel system is used in any of the following situations: 1) Reduce the pathogen load in the gut and system; 2) Relieves weight loss, abnormal body temperature, and shortened colon caused by bacterial enteritis; 3) Promotes the repair of the intestinal epithelial barrier and the reconstruction of the mucus layer; 4) It inhibits the expression of pro-inflammatory factor TNF-α and increases the level of anti-inflammatory factor TGF-β; 5) Promotes macrophage polarization from M1 to M2 type; 6) Maintain the α-diversity, β-diversity, and core taxonomic composition of the gut microbiota; 7) It protects gut microbiota homeostasis while eliminating intestinal pathogens; 8) Promotes intestinal barrier repair and regulates the immune microenvironment.

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