Yeast micro-capsule packaged modified drug-loaded silicone grease body as well as preparation method and application of yeast micro-capsule packaged modified drug-loaded silicone grease body

Abstract

The invention discloses a yeast microcapsule encapsulated modified drug-loaded silicone grease body as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. The preparation method of the yeast microcapsule encapsulated modified drug-loaded silicone grease comprises the following steps: mixing disulfide bond modified dendritic mesoporous silica, an antibacterial drug, an anti-inflammatory drug and water to prepare drug-loaded dendritic mesoporous silica; the preparation method comprises the following steps: preparing drug-loaded dendritic mesoporous silica, coating the surface of the drug-loaded dendritic mesoporous silica with attenuated salmonella outer membrane vesicles, modifying the surface of the drug-loaded dendritic mesoporous silica with polyethyleneimine and hyaluronic acid, and finally coating with yeast microcapsules to obtain the modified drug-loaded silicone grease encapsulated by the yeast microcapsules. The modified drug-loaded silicone grease can achieve antibacterial, anti-inflammatory and intestinal flora recovery, provides an alternative scheme for traditional antibiotic treatment, and effectively solves the problems that the antibiotic treatment effect on animal salmonella enterica infection is poor and the like.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a modified drug-loaded silicone liposome encapsulated in yeast microcapsules, its preparation method, and its application. Background Technology

[0002] Salmonella is a foodborne, facultative intracellular parasitic bacterium that can cause acute gastroenteritis symptoms such as diarrhea, fever, and abdominal pain. Severe infections can lead to focal infections such as meningitis, endocarditis, and bone and joint infections, as well as sepsis, significantly impacting human morbidity and mortality. Salmonella has evolved complex mechanisms to evade host immunity and antibiotics. The pathogen is induced to internalize into host cells (intestinal epithelial cells and macrophages), forming membrane-bound vacuoles called Salmonella-containing vacuoles (SCVs). Within nutrient-limited SCVs, Salmonella isolates host cell metabolites to support its replication and survival, making intracellular Salmonella difficult to eradicate.

[0003] While some broad-spectrum antibiotics, such as enrofloxacin, have shown good in vitro killing effects against Salmonella, the problem of achieving and maintaining effective concentrations within the host cells where Salmonella resides remains unresolved. Furthermore, high-dose administration can damage surrounding healthy tissues and produce toxic side effects. Additionally, oral administration, as the most convenient and rapid method for treating livestock and poultry diseases, faces the challenge of managing the harsh gastric environment when treating intestinal infections; maintaining the activity of antibacterial drugs is also a pressing issue that needs to be addressed.

[0004] Therefore, developing efficient, precise drug delivery systems that can overcome physiological barriers is crucial. From the perspective of carrier source and design strategy, they can be mainly divided into two categories: synthetic carriers and naturally derived carriers. Synthetic carriers, such as mesoporous silica nanoparticles (MSNs), possess highly controllable amphiphilicity, uniformly tunable particle size and morphology, and modular surface chemical modification capabilities, providing a powerful platform for efficient loading and intracellular delivery of therapeutic drugs. On the other hand, naturally derived carriers exhibit unique biocompatibility and inherent biological functions. For example, bacterial outer membrane vesicles (OMVs), as spherical nanovesicles (20-250 nm) naturally released by Gram-negative bacteria, have a lipid bilayer structure highly similar to that of the parent bacteria and possess the natural ability to fuse with homologous bacterial membranes, offering possibilities for targeted drug delivery, especially against specific pathogens. Furthermore, given the stringent challenges of oral delivery, yeast microcapsules, as a natural carrier derived from yeast cell wall β-glucan, form a porous and rigid structure with a highly cross-linked glucan network. This structure not only effectively loads small molecule drugs or nanoparticles but has also been shown to provide excellent gastric environment protection properties. In the intestinal environment, they are slowly degraded into short-chain fatty acids by specific enzymes to regulate the intestinal flora and achieve controlled drug release, demonstrating great potential as an ideal oral delivery carrier.

[0005] In conclusion, developing a novel drug for treating intestinal Salmonella infection has become one of the most pressing issues to be addressed in this field. Summary of the Invention

[0006] The purpose of this invention is to provide a modified drug-loaded silicone liposome encapsulated with yeast microcapsules, its preparation method, and its application. The modified drug-loaded silicone liposome of this invention can achieve antibacterial, anti-inflammatory, and intestinal flora restoration effects, providing an alternative to traditional antibiotic treatment and effectively solving the problem of poor efficacy of antibiotic treatment for Salmonella infection in animals.

[0007] This invention first provides a method for preparing modified drug-loaded silicone liposomes encapsulated in yeast microcapsules, comprising the following steps: (1) Preparation of disulfide bond modified dendritic mesoporous silica; (2) The disulfide bond modified dendritic mesoporous silica, antibacterial drug, anti-inflammatory drug and water are mixed and stirred to obtain drug-loaded dendritic mesoporous silica; (3) Attenuated Salmonella outer membrane vesicles are wrapped on the surface of the drug-loaded dendritic mesoporous silica to obtain drug-loaded dendritic silica coated with attenuated Salmonella outer membrane vesicles. (4) Modify the surface of the drug-loaded dendritic silica coated with the outer membrane vesicles of the attenuated Salmonella with polyethyleneimine and hyaluronic acid to obtain modified drug-loaded dendritic silica coated with the outer membrane vesicles of attenuated Salmonella. (5) Yeast microcapsules are wrapped around the drug-loaded dendritic silica outer layer of the modified attenuated Salmonella outer membrane vesicles to obtain modified drug-loaded silica liposomes encapsulated with yeast microcapsules.

[0008] In the above preparation method, in step (2), the mass ratio of the sum of the antibacterial drug and the anti-inflammatory drug to the disulfide bond modified dendritic mesoporous silica is 1-2:1; The mass ratio of the antibacterial drug to the anti-inflammatory drug is 1-2:1; The antibacterial drug is enrofloxacin and / or linezolid; The anti-inflammatory drug is cinnamaldehyde and / or paeonol; The concentration of the drug-loaded dendritic mesoporous silica is 1-2 mg / mL, specifically 1 mg / mL.

[0009] The preparation method described above, specifically the preparation method of the disulfide bond-modified dendritic mesoporous silica, includes the following steps: The template agent, organic small molecule amine, pore-forming agent, silicon source and solvent are mixed and reacted to obtain a precipitate of disulfide bond modified dendritic mesoporous silica.

[0010] In the above preparation method, the template agent is at least one selected from hexadecyltrimethylammonium chloride, hexadecyltrimethylammonium bromide, dodecyltrimethylammonium chloride, dodecyltrimethylammonium bromide, octadecyltrimethylammonium chloride, and octadecyltrimethylammonium bromide; The organic small molecule amine is at least one of triethanolamine, urea and ethanolamine; The pore-forming agent is bis-(γ-triethoxysilylpropyl)tetrasulfide and / or bis-(γ-triethoxysilylpropyl)disulfide; The silicon source is tetraethyl orthosilicate; The solvent is water; The mass ratio of the template agent to the small organic molecule amine is 8-9:1; The template agent is 0.4-0.6g per 1mL of silicon source. The volume ratio of the silicon source to the pore-forming agent is 1:1-1.5; The reaction temperature is 80-100℃; The reaction time is 3-5 hours; The reaction was carried out under stirring conditions; Specifically, the stirring speed is 200-400 rpm.

[0011] Specifically, the method for preparing the disulfide bond-modified dendritic mesoporous silica includes the following steps: S1. Dissolve the template agent in a solvent to obtain a template agent solution; dissolve the small organic molecule amine in a solvent, and then mix it with the template agent solution; S2. Mix the pore-forming agent and the silicon source, and then add it dropwise to the mixture in step S1. React, centrifuge, and wash the resulting precipitate with water and anhydrous ethanol in sequence, and dry to obtain a crude dendritic mesoporous silica precursor. S3. The crude dendritic mesoporous silica precursor is mixed with anhydrous ethanol, concentrated hydrochloric acid is added, and the mixture is sonicated and then extracted under heating conditions with stirring. After centrifugation, the precipitate is washed with a mixture of anhydrous ethanol and water and dried to obtain disulfide bond modified dendritic mesoporous silica.

[0012] In the above preparation method, in step S1, the concentration of the template agent in the template agent solution is 2.5-3 g / 100 mL; In the step of dissolving the organic small molecule amine in the solvent, the mass-to-volume ratio of the organic small molecule amine to the solvent is 0.3-0.4 g: 7.5 mL; In steps S2 and S3, the drying is performed at room temperature; In step S3, the mass-to-volume ratio of the crude dendritic mesoporous silica precursor to anhydrous ethanol is 50-60 mg: 100 mL. In the step of mixing the crude dendritic mesoporous silica precursor with anhydrous ethanol and adding concentrated hydrochloric acid, the volume ratio of anhydrous ethanol to concentrated hydrochloric acid is 10-20:1. In step S3, the power of the ultrasound is 80-100W; the duration of the ultrasound is 1.5-2.5h; and the temperature of the ultrasound is room temperature. The stirring extraction temperature is 60-80℃, the time is 5-8h, and the stirring speed is 500-800rpm; In step S3, the volume ratio of anhydrous ethanol to water in the mixture of anhydrous ethanol and water is 1:1.

[0013] In the above preparation method, step (3) specifically includes the following steps: c. Centrifuge the attenuated Salmonella ATCC202165 bacterial culture, collect the supernatant and filter; then centrifuge the filtrate using a 100-200 kDa ultrafiltration tube, collect the liquid and filter; centrifuge the obtained filtrate, resuspend the obtained precipitate in PBS solution, centrifuge, and the obtained precipitate is bacterial outer membrane vesicles; mix the bacterial outer membrane vesicles with physiological saline to obtain a bacterial outer membrane vesicle suspension; d. Mix the drug-loaded dendritic silica and the bacterial outer membrane vesicle suspension, and sonicate to coat the bacterial outer membrane vesicles onto the drug-loaded dendritic silica particles, thereby obtaining drug-loaded dendritic silica coated with attenuated Salmonella outer membrane vesicles.

[0014] In the above preparation method, the OD of the attenuated Salmonella ATCC202165 bacterial suspension... 600 It is 0.8-1; Step c is as follows: The attenuated Salmonella ATCC202165 bacterial suspension is centrifuged at 6,000-8,000g for 20-30 min at 4°C. The supernatant is collected and filtered through a 0.45 μm pore size cellulose acetate filter. The filtrate is then centrifuged at 6,000-8,000g for 5-6 min using a 100 kDa ultrafiltration tube. The liquid in the inner tube is collected to a total volume of 20-30 mL. The collected liquid is filtered through a 0.22 μm pore size cellulose acetate filter. The resulting filtrate is centrifuged at 150,000-170,000g for 2-4 h at 4°C. The resulting precipitate is resuspended in PBS solution and centrifuged at 150,000-170,000g for 1-2 h. The resulting precipitate is bacterial outer membrane vesicles. The bacterial outer membrane vesicles are mixed with physiological saline to obtain a bacterial outer membrane vesicle suspension. In step d, the volume ratio of the drug-loaded dendritic silica to the bacterial outer membrane vesicle suspension is 1-2:1; specifically, it can be 2:1. The concentration of the drug-loaded dendritic silica is 1-2 mg / mL; specifically, it can be 1 mg / mL. The concentration of the bacterial outer membrane vesicle suspension is 0.3-0.6 mg / mL, specifically 0.384-0.512 mg / mL; more specifically, 0.384 mg / mL. The ultrasound is performed using a probe; the ultrasound power is 40-60W; and the ultrasound time is 3-6 minutes.

[0015] In the above preparation method, step (4) specifically includes the following steps: e. Drop the drug-loaded dendritic silica coated with the outer membrane vesicles of the attenuated Salmonella into a polyethyleneimine solution and stir to obtain PEI-OMV@DMSN; f. Mix the PEI-OMV@DMSN and hyaluronic acid solution and react to obtain the drug-loaded dendritic silica coated with the modified attenuated Salmonella outer membrane vesicles.

[0016] In the above preparation method, in step e, the concentration of the polyethyleneimine solution is 2-5 mg / mL; The volume ratio of the drug-loaded dendritic silica and polyethyleneimine solution coated with the outer membrane vesicles of the attenuated Salmonella is 3-6:1. The stirring time is 20-40 minutes; In step f, the volume ratio of PEI-OMV@DMSN to hyaluronic acid solution is 7-10:1; The concentration of the hyaluronic acid solution is 10-12 mg / mL; The reaction temperature is 22℃-25℃; the reaction time is 0.5-1.5h.

[0017] Step (5) specifically includes the following steps: g. Disperse yeast powder in 1-1.2M NaOH solution, stir continuously at 80-85℃ for 0.5-1.5h, centrifuge at 2000-3000rpm for 10-15min, and collect the insoluble substance containing yeast cell walls; then suspend the insoluble substance in 1L water, adjust the pH to 4-5 with HCl solution, and incubate at 50-70℃ for 1-1.5h; collect the insoluble residue again by centrifugation, and then wash with water, isopropanol and acetone in sequence; freeze-dry the obtained slurry to obtain yeast microcapsules; h. Mix the yeast microcapsules with water and incubate them; then add the drug-loaded dendritic silica solution coated with the modified attenuated Salmonella outer membrane vesicles, incubate, and centrifuge; resuspend the solid in water, and then freeze-dry and hydrate the suspension to obtain modified drug-loaded silica liposomes encapsulated in yeast microcapsules.

[0018] In the above preparation method, in step h, the mass ratio of the yeast microcapsules to the volume ratio of water is 1-1.2 mg: 1 mL; The yeast microcapsules were incubated with water at a temperature of 37-42°C for 0.5-1 hour. The mass ratio of the yeast microcapsules and the drug-loaded dendritic silica coated with the modified attenuated Salmonella outer membrane vesicles is 2:1.5-2, specifically 2:1.536-1.920. The concentration of the drug-loaded dendritic silica solution coated with the modified attenuated Salmonella outer membrane vesicles is 0.2-0.4 mg / mL, specifically 0.256-0.384 mg / mL; The incubation time after adding the drug-loaded dendritic silica solution coated with the modified attenuated Salmonella outer membrane vesicles is 0.5-1.5 h; The freeze-drying time for the freeze-drying-hydration process is 12-30 hours.

[0019] In the above preparation method, the room temperature is known to those skilled in the art and is generally 15-35°C; specifically, it can be 25°C.

[0020] The present invention further provides modified drug-loaded silicone liposomes encapsulated in yeast microcapsules prepared by the above preparation method.

[0021] Finally, the present invention also provides the application of the modified drug-loaded silicone liposomes encapsulated in yeast microcapsules in the preparation of drugs for treating bacterial infectious diseases; Specifically, the bacterial infection is at least one of intestinal infection, lung infection, and breast infection; The bacteria in question is Salmonella.

[0022] Preferably, the intestinal bacterial infection is intestinal inflammation caused by Salmonella infection.

[0023] The concentration ratio of OMV@DMSN to PEI solution, electrostatic adsorption time, and reaction time provided by this invention are all appropriate parameters set after determining the particle size distribution, encapsulation efficiency, and drug loading rate using a Malvern particle size analyzer and liquid chromatography-mass spectrometry (LC-MS / MS).

[0024] Compared with the prior art, the present invention has the following beneficial effects: (1) The modified drug-loaded silicone liposomes for treating Salmonella infection of the present invention have a uniform particle size, very stable structure and properties, and naturally target macrophages and inflamed intestinal epithelial cells, thereby enhancing the host cells’ ability to clear intracellular bacteria.

[0025] (2) The yeast microcapsule encapsulation technology of the present invention effectively solves the problem of gastric environment interference faced by oral administration and achieves the purpose of targeted drug release in the intestine; at the same time, yeast microcapsules have good safety and are widely available and inexpensive.

[0026] (3) Using modified drug-loaded silicone liposomes encapsulated with yeast microcapsules to treat intestinal Salmonella infection solves the problems of poor efficacy of antibiotics in clearing intracellular bacteria and the destruction of intestinal flora by long-term use of broad-spectrum antibiotics. It has high safety and few toxic side effects, and is a very promising drug delivery platform for treating intracellular bacterial infections, providing an efficient and safe new paradigm for the treatment of intestinal Salmonella infection. Attached Figure Description

[0027] Figure 1 Transmission electron microscopy (TEM) image of DMSN prepared in Example 1 in GSH and PBS solutions.

[0028] Figure 2 Transmission electron microscopy image of DMSN-E / C prepared in Example 2.

[0029] Figure 3 The particle size distribution diagram is shown for DMSN-E / C prepared in Example 2.

[0030] Figure 4 Zeta potential map of DMSN-E / C prepared in Example 2.

[0031] Figure 5 The image shows a transmission electron microscope (TEM) image of the O@DE / C prepared in Example 3.

[0032] Figure 6 The particle size distribution diagram of O@DE / C prepared in Example 3 is shown.

[0033] Figure 7 The Zeta potential diagram of O@DE / C prepared in Example 3.

[0034] Figure 8 Transmission electron microscopy (TEM) image of H / PO@DE / C prepared in Example 4.

[0035] Figure 9 The permeable particle size distribution of H / PO@DE / C prepared in Example 4 is shown.

[0036] Figure 10 Zeta potential diagram of H / PO@DE / C prepared in Example 4.

[0037] Figure 11 Transmission electron microscopy (TEM) image of YP@H / PO@DE / C prepared in Example 5.

[0038] Figure 12The time-killing kinetics curve of O@DE / C prepared in Example 3.

[0039] Figure 13 The figure shows the statistical results of intracellular fluorescence intensity of Raw264.7 macrophages and Caco-2 intestinal epithelial cells after treatment in each group of the endocytosis experiment.

[0040] Figure 14 The bacterial count of intracellular Salmonella was performed on a plate after treating macrophages with Raw264.7 cells in each treatment group for the intracellular bacterial clearance assay.

[0041] Figure 15 Laser confocal microscopy images of intracellular Salmonella in macrophages after treatment with Raw264.7 cells in each treatment group for the intracellular bacterial clearance assay.

[0042] Figure 16 The bacterial count of intracellular Salmonella after Caco-2 treatment of intestinal epithelial cells in each treatment group was performed as part of the intracellular bacteria clearance assay.

[0043] Figure 17 Laser confocal microscopy images of intracellular Salmonella after Caco-2 treatment of intestinal epithelial cells in each treatment group of the intracellular bacteria clearance test.

[0044] Figure 18 Drug release curves of modified drug-loaded silicone liposomes encapsulated in yeast microcapsules in simulated gastric and cecal fluids.

[0045] Figure 19 A statistical graph showing the bacterial count in the cecal segment of mice after treatment with an acute intestinal infection model in each treatment group.

[0046] Figure 20 HE-stained sections of cecal tissue from mice in each treatment group after developing an acute intestinal infection model.

[0047] Figure 21 Abundance analysis of genus-level gut microbiota in the cecum of mice after treatment with an acute intestinal infection model in each treatment group. Detailed Implementation

[0048] The present invention will be further described in detail below with reference to specific embodiments. The embodiments given are only for illustrating the present invention and are not intended to limit the scope of the present invention.

[0049] Unless otherwise specified, the experimental methods described in the following examples are conventional methods.

[0050] Unless otherwise specified, the quantitative experiments in the following examples are all repeated three times, and the results are averaged.

[0051] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0052] The reagents and strains used in the following examples are from the following sources: CTAC (hexadecyltrimethylammonium chloride) was purchased from Adamas-beta, model number 01008865-14644B; TEA (triethanolamine) was purchased from Sigma, model number V900257-500ML; TEOS (tetraethyl orthosilicate) was purchased from Beijing Bailingwei Technology Co., Ltd., model number 308971; BTES (bis(r-triethoxysilylpropyl)tetrasulfide) was purchased from Shanghai Yuanye Biotechnology Co., Ltd., model number S31121- 25g; Cinnamaldehyde, purchased from Shanghai Maclean Biochemical Technology Co., Ltd., purity ≥95% (GC), model C80500; Enrofloxacin, purchased from Shanghai Maclean Biochemical Technology Co., Ltd., model E809130; Glutathione (GSH) purchased from Sigma-Aldrich, USA; High-activity instant dry yeast powder purchased from LESAFFRE; Salmonella standard strain ATCC14028 and attenuated Salmonella typhimurium serotype ATCC202165 purchased from the American Center for Type Culture Collection.

[0053] Example 1: Preparation of disulfide bond modified dendritic mesoporous silica (DMSN) The specific preparation method is as follows: (1) Weigh 2.5g CTAC into a flask and add 100mL of deionized water; weigh 0.3g TEA into a 5mL centrifuge tube, then rinse the centrifuge tube with 7.5mL of deionized water and add it to the above CTAC solution. (2) Mix 5.376 mL TEOS (note that TEOS should not be shaken, as it is prone to self-polymerization) and 6.018 mL BTES. Slowly invert the TEOS and BTES (orange-yellow) mixture to make it evenly mixed. Then slowly add the above mixture dropwise to the flask of the system in step (1) to make the above orange-yellow mixture evenly dispersed. Stir the system at 95°C and 300 rpm for 4 hours. (3) After step (2), the liquid was aliquoted and centrifuged at 25°C and 14,000 rpm to obtain an orange-red precipitate. A certain volume (7-10 mL) of ultrapure water was used to resuspend the precipitate, and the precipitate was sonicated and blown around for 2 min. Then an equal volume of anhydrous ethanol was added to continue resuspension, and the precipitate was sonicated for 2 min and centrifuged at 14,000 rpm for 10 min. The above steps were repeated 3 times. The precipitate was left at room temperature for 12 h until it was dry, which is the crude dendritic mesoporous silica precursor (MONs).

[0054] (4) Weigh 50 mg of the above MONs into a 250 mL round bottom flask, then add 100 mL of anhydrous ethanol, add 5 mL of concentrated hydrochloric acid (mass fraction 37%) into a fume hood, and sonicate in a water bath at room temperature and 100 W for 2 h. After sonication, place an A30 rotor into the flask and place it in a 60 °C water bath. Install an extraction device at the opening of the flask and stir at 600 rpm for 6 h.

[0055] (5) Centrifuge at 12000 rpm for 10 min and discard the supernatant. After washing the precipitate with a mixture of anhydrous ethanol and ultrapure water in a volume ratio of 1:1, sonicate it to disperse it, and centrifuge at 12000 rpm for 10 min. (6) Repeat step (5) centrifugation-washing operation 3 times. Centrifuge at 12000rpm for 10min, discard the supernatant, and dry the precipitate at room temperature to form powder, which is disulfide bond modified dendritic mesoporous silica (DMSN).

[0056] The above steps employed an organic-inorganic self-assembly method, doping the template solution with tetrasulfide to successfully synthesize a silica framework (DMSN) bridged by disulfide bonds. Transmission electron microscopy (TEM) was used to observe the DMSN framework and evaluate its structural stability at different time points. Results are as follows: Figure 1 As shown, the DMSN backbone is structurally stable in PBS solution. After incubation in GSH solution for 24 h, the DMSN backbone gradually dissolves; after incubation for 48 h, the DMSN backbone completely disintegrates, indicating that the DMSN backbone has significant responsive degradation characteristics in the GSH environment, enabling the gradual release of the drug.

[0057] Figure 1 The experimental method is as follows: DMSN is placed in PBS solution or GSH solution (10mM) and incubated at room temperature.

[0058] Example 2: Preparation of drug-loaded dendritic mesoporous silica (DMSN-E / C) The specific preparation method is as follows: Steps (1)-(6) are the same as in Example 1.

[0059] (7) Prepare 1 mL of 2 mg / mL DMSN solution and 1 mL of 2 mg / mL enrofloxacin solution using ultrapure water, mix them to obtain 2 mL of mixed solution, add 1 μL of 1 g / mL cinnamaldehyde (the mass ratio of DMSN, enrofloxacin and cinnamaldehyde is 2:2:1), stir at 600 rpm overnight to obtain drug-loaded dendritic mesoporous silica (DMSN-E / C).

[0060] The prepared DMSN-E / C was characterized by transmission electron microscopy, such as... Figure 2As shown, DMSN-E / C exhibits uniform particle size and dendritic structure, demonstrating good dispersibility and stable properties. The particle size and potential of the prepared DMSN-E / C were characterized, as shown... Figure 3 and Figure 4 As shown, its hydrated particle size is 37.84±4.98nm and its Zeta potential is -14.3±4.04mV.

[0061] Example 3: Preparation of drug-loaded dendritic silica (O@DE / C) coated with outer membrane vesicles of attenuated Salmonella. The difference from Example 2 is that a layer of bacterial outer membrane vesicles is coated on the surface of DMSN-E / C to prevent drug leakage. The specific preparation method is as follows: Steps (1)-(7) are the same as in Example 2.

[0062] (8) Pick a single colony of attenuated Salmonella ATCC202165 and inoculate it into sterile LB medium. Incubate on a shaker at 37°C and 220 rpm for 10-12 h. Transfer the bacterial solution from the test tube into an Erlenmeyer flask containing liquid LB at a ratio of 1:100 and incubate overnight for 14 h until the bacterial growth reaches the logarithmic growth phase (OD). 600 ≈0.8); (9) Transfer the cultured bacterial solution into a 50 mL centrifuge tube and centrifuge at 4 °C and 6,000 g for 20 min using an ST8R high-speed refrigerated centrifuge. Collect the supernatant. Filter the supernatant through a 0.45 μm pore size cellulose acetate filter (purchased from Millipore, USA). Centrifuge at 6,000 g for 5-6 min using a 100 kDa ultrafiltration tube (purchased from Millipore, USA). Collect the liquid in the inner tube to a total volume of about 20-30 mL. Filter the collected liquid through a 0.22 μm pore size cellulose acetate filter (purchased from Millipore, USA). (10) Collect the filtrate and centrifuge at 150,000g for 4 h at 4 °C. Resuspend the precipitate in 2 mL PBS and centrifuge again at 150,000g for 2 h. Carefully discard the supernatant. The precipitate is the bacterial outer membrane vesicle (OMV). After resuspending in physiological saline, use the BCA kit to determine its concentration, which is 1 mg / mL. (11) Take 2 mL of DMSN-E / C (concentration of 1 mg / mL) obtained in step (7) and mix it with 1 mL of OMV suspension (concentration of 0.384 mg / mL, obtained by dilution with physiological saline) obtained in step (10) under vortex. Then, use a probe to sonicate (50 W, 3 min) to coat OMV onto nanoparticles to obtain drug-loaded dendritic silica (O@DE / C, concentration of 0.512 mg / mL, calculated based on the actual loading amount of Enr) coated with attenuated Salmonella outer membrane vesicles.

[0063] The prepared O@DE / C was characterized by transmission electron microscopy, such as... Figure 5 As shown, O@DE / C exhibits uniform particle size and a spherical structure, demonstrating good dispersibility and stable properties. The prepared O@DE / C was characterized for particle size and potential, as shown in the figure. Figure 6 and Figure 7 As shown, its hydrated particle size is 43.82±6.93nm and its Zeta potential is -14.5±3.25mV.

[0064] Example 4: Preparation of drug-loaded dendritic silica (H / PO@DE / C) coated with modified attenuated Salmonella outer membrane vesicles The difference from Example 3 lies in the adjustment of the surface charge of the bacterial outer membrane vesicles, which is sequentially modified with polyethyleneimine (PEI) and hyaluronic acid (HA). The specific preparation method is as follows: Steps (1)-(11) are the same as in Example 3.

[0065] 3 mL of the prepared O@DE / C was added dropwise to PEI solution (Mw25000, 500 μL, 5 mg / mL, solvent: ultrapure water), and stirred for 40 min to complete electrostatic adsorption, yielding PEI-OMV@DMSN; then 3.5 mL of PEI-OMV@DMSN was added to HA solution (Mw8000, 500 μL, 10 mg / mL, solvent: ultrapure water), and stirred at 25 °C for 1 h. After centrifugation at 6000 × g for 15 min, the resulting precipitate was the drug-loaded dendritic silica (H / PO@DE / C) coated with the outer membrane vesicles of modified attenuated Salmonella.

[0066] The prepared H / PO@DE / C was characterized by transmission electron microscopy, such as... Figure 8 As shown; by Figure 8 As can be seen, H / PO@DE / C possesses uniform particle size and a spherical structure, exhibiting good dispersibility and stable properties. The prepared H / PO@DE / C was characterized by its particle size and potential, as shown below. Figure 9 and Figure 10 As shown, its hydrated particle size is 52.4±2.67nm and its Zeta potential is 22.3±5.84mV.

[0067] Example 5: Preparation of modified drug-loaded silicone liposomes (YP@H / PO@DE / C) encapsulated with yeast microcapsules The difference from Example 4 lies in the extraction of a Saccharomyces cerevisiae cell wall for encapsulating the drug-loaded dendritic silica coated with the modified attenuated Salmonella outer membrane vesicles described in Example 4, overcoming the difficulty of oral administration. The preparation method is as follows: Steps (1) to (12) are the same as in Example 4.

[0068] (13) Disperse 100g of yeast powder in 1L of 1M NaOH solution, stir continuously at 80℃ for 1h, centrifuge at 2000rpm for 10min, and collect the insoluble material containing yeast cell walls. Then suspend the insoluble material in 1L of water, adjust the pH to 4-5 with HCl solution, and incubate at 55℃ for 1h. Then collect the insoluble residue again by centrifugation, wash once with 1L of deionized water, wash four times with 200mL of isopropanol, wash twice with 200mL of acetone, and wash three times with 500mL of ultrapure water. Place the obtained slurry in a plastic tray, freeze immediately with liquid nitrogen, and then freeze-dry to obtain the powder, which is the yeast microcapsule.

[0069] (14) The 2 mg yeast microcapsules prepared above were mixed with 2 mL of deionized water and incubated at 37 °C for 30 min. Then, 4 mL of H / PO@DE / C solution (dissolved in ultrapure water) with a concentration of 0.384 mg / mL was added dropwise. After stirring at 200 rpm and incubating for 1 h, the mixture was centrifuged at 4000 rpm for 10 min to obtain a precipitate. The precipitate was resuspended in 6 mL of deionized water, and the suspension was subjected to a freeze-drying-hydration cycle. The above resuspension-freeze-hydration process was repeated twice to finally obtain 6 mL of modified drug-loaded silicone liposomes (YP@H / PO@DE / C, where the actual encapsulation concentration of Enr was 0.256 mg / mL) encapsulated in yeast microcapsules.

[0070] The modified drug-loaded silicone liposomes encapsulated in yeast microcapsules were characterized by transmission electron microscopy, such as... Figure 11 As shown, the modified drug-loaded silicone liposomes encapsulated in yeast microcapsules have an average particle size of 4-5 μm. They are porous on the surface and hollow inside, with the modified drug-loaded silicone liposomes encapsulated within the cavity.

[0071] Example 6 1. The morphology of different dendritic mesoporous silica obtained in Examples 2, 3 and 4 was characterized and statistically analyzed. The encapsulation efficiency and drug loading rate of the antibacterial drug enrofloxacin and the anti-inflammatory drug cinnamaldehyde were measured by liquid chromatography-mass spectrometry (LC-MS / MS). The specific data are shown in Table 1.

[0072] Table 1 Drug loading rate and encapsulation efficiency

[0073] According to the data in Table 1, the DMSN, O@DE / C, and H / PO@DE / C prepared in Examples 2, 3, and 4 all possess uniform particle size and porous structure. Compared with Example 2, Example 3 shows a significant improvement in encapsulation efficiency and drug loading rate, indicating that OMV encapsulation can effectively reduce drug leakage. Compared with Example 3, Example 4 shows a certain degree of improvement in encapsulation efficiency and drug loading rate, suggesting that the modification with polyethyleneimine (PEI) and hyaluronic acid (HA) may alter the charge and chemical properties of the drug-loaded silicone liposome surface, resulting in a tighter bond between drug molecules and the silicone liposome, thereby improving encapsulation efficiency and drug loading rate. In summary, the modified OMV silicone liposome is a drug loading platform with uniform particle size and stable properties, exhibiting good loading efficiency for both hydrophilic (Enr) and hydrophobic (CA) drugs.

[0074] 2. The bactericidal kinetics of the drug-loaded dendritic silica (O@DE / C) coated with the outer membrane vesicles of attenuated Salmonella prepared in Example 3 against the Salmonella standard strain ATCC14028 were tested.

[0075] The time-killing kinetics experiment was conducted using the method reported in (Yingxian Chen and Xiaowei Ma*. “A Precision-Guided 'Trojan Horse' for Tissue-Cell-Bacteria Targeted Therapy of Salmonella Intestinal Infections.” ACS Nano 19 21 (2025): 20012-20028.). Using PBS without any added antibacterial substances as the control group, free enrofloxacin (Enr concentration of 0.125 μg / mL), free cinnamaldehyde (CA concentration of 0.125 μg / mL), free dual-drug formulations (Enr / CA concentration of 0.125 μg / mL), and drug-loaded silicone liposomes (O@DE / C, diluted 4096 times with ultrapure water to achieve an actual Enr encapsulation concentration of 0.125 μg / mL) prepared according to the method described in Example 3 were co-incubated with Salmonella standard strain ATCC14028 at 37°C in a shaker. At 0, 1, 2, 4, 8, 12, and 24 hours, 100 µL of bacterial suspension was plate-coated for colony counting. The resulting bactericidal kinetic curves are shown in [Figure 1]. Figure 12 .

[0076] like Figure 12 As shown, the O@DE / C prepared in Example 3 still maintained good antibacterial efficacy after 24 hours, showing better bactericidal activity compared to the free dual-drug group. This may be due to the bacterial targeting properties of the drug-loaded OMV liposome itself.

[0077] 3. To detect the endocytosis effect of RAW264.7 macrophages / Caco-2 intestinal epithelial cells on drug-loaded silicone liposomes and modified drug-loaded silicone liposomes.

[0078] The endocytosis assay was performed using the method reported in the literature (Zhao Q, Liu J, Liu S, et al. Multipronged Micelles-Hydrogel for Targeted and Prolonged Drug Delivery in Chronic Wound Infections[J]. ACS Applied Materials & Interfaces, 2022, 14(41): 46224-46238.). RAW264.7 / Caco-2 cells were used as the experimental cell line. PBS without any drugs was used as the control group (NC). Luciferin coumarin 6 (C6) was used as the small molecule drug group. Coumarin 6-loaded silicone liposomes (OD@C6) were used as the nanoparticle group (prepared according to Example 3, except that "DMSN, enrofloxacin, and cinnamaldehyde were mixed in a 2:2:1 mass ratio" was replaced with "DMSN and C6 were mixed in a 1:1 mass ratio"). Coumarin 6-loaded modified silicone liposomes (H / POD@C6) (prepared according to Example 4, except that "DMSN, enrofloxacin, and cinnamaldehyde were mixed in a 2:2:1 mass ratio" was replaced with "DMSN and C6 were mixed in a 1:1 mass ratio") were used as the nanoparticle group with added modifying molecules. Different groups of drugs were co-incubated with macrophages and intestinal epithelial cells, respectively. The cells were then washed with PBS to remove fluorescent drugs that failed to enter the cells. The average fluorescence intensity of the cells was detected by flow cytometry to assess the endocytosis of macrophages.

[0079] Statistical results are as follows Figure 13As shown, compared to the PBS group and the small molecule drug group, the use of C6-loaded silicone liposomes (OD@C6) and modified silicone liposomes (H / POD@C6) as nanoparticles significantly increased the concentration of C6 in macrophages / intestinal epithelial cells. This is because small molecule drugs can only enter the cell via passive transport, resulting in a lower intracellular drug accumulation concentration, manifested as a lower fluorescence intensity, highlighting the advantage of nanoparticles as carriers. Furthermore, in contrast, since OMV does not have the function of targeting macrophages / intestinal epithelial cells, while hyaluronic acid (HA) can actively target cells through the CD44 receptor, the modified silicone liposomes (H / POD@C6) exhibited higher fluorescence intensity than silicone liposomes (OD@C6), indicating higher intracellular drug accumulation. This suggests that modified silicone liposomes (H / POD@C6) can enhance the endocytosis of small molecule drugs by host cells and are superior to ordinary silicone liposomes (OD@C6). This process improves drug bioavailability while reducing drug accumulation in non-target tissues, reducing drug toxicity and improving biosafety.

[0080] 4. Detect the clearance effect of H / PO@DE / C on intracellular Salmonella parasitic in macrophages / intestinal epithelial cells. The intracellular bacterial clearance experiment was conducted using the method reported in the literature (Junhua H, yingxian C, et al. A Comparative Analysis of the Antibacterial Spectrum of Ultrasmall Manganese Ferrite Nanozymes with Varied Surface Modifications[J].ACS Applied Materials & Interfaces, 2024,16(12):14385-14404.). Mouse macrophages / intestinal epithelial cells invaded by the Salmonella fluorescent strain GFP14028 were used as experimental cell lines. PBS without any drugs served as the control group. Mouse macrophages / intestinal epithelial cells were treated with free enrofloxacin (Enr concentration 0.125 μg / mL), free cinnamaldehyde (CA concentration 0.125 μg / mL), free dual-drug regimen (Enr / CA concentrations both 0.125 μg / mL), and H / PO@DE / C prepared according to the method described in Example 4 (diluted 3072 times with ultrapure water to achieve an actual Enr encapsulation concentration of 0.125 μg / mL), for 4 hours. After cell disruption, bacterial cells were collected and plate-coated for counting to determine the clearance effect of different treatment groups on intracellular Salmonella.

[0081] Furthermore, the effect of H / PO@DE / C on scavenging intracellular bacteria was analyzed using confocal fluorescence microscopy. Data on surviving fluorescent bacterial strains within the cells were observed and statistically analyzed under an inverted fluorescence microscope.

[0082] Statistical results are as follows Figures 14-17 As shown, the clearance effect of each treatment group on intracellular bacteria, from strongest to weakest, is H / PO@DE / C > Enr / CA > Enr > CA > PBS. Compared with other treatment groups, after H / PO@DE / C treatment, HA naturally targets macrophages / inflammatory epithelial cells that highly express the CD44 receptor, enabling them to efficiently transport their internal Enr and CA into the cells, effectively clearing intracellular Salmonella and treating intracellular bacterial infections.

[0083] 5. Detect the drug release curves of yeast microencapsulated drug-loaded modified silicone liposomes (YP@H / PO@DE / C) under different body fluid simulation environments.

[0084] The drug release behavior of YP@H / PO@DE / C and O@DE / C was investigated using the dialysis bag diffusion method. For both samples, simulated gastric fluid (SGF) or simulated cecal fluid (SCF) at 37°C was used as the release medium.

[0085] Using SGF as the release medium: A certain amount of YP@H / PO@DE / C and O@DE / C, along with free Enr, were dispersed in 2 mL of release medium. This medium was placed in a cellulose acetate dialysis bag (molecular weight cutoff = 1000 Da, allowing only free drug molecules Enr to pass through the dialysis bag), and the dialysis bag was immersed in 100 mL of release medium. The release system was placed in a stirrer at 100 rpm. At predetermined time points of 0, 2, 4, 6, 8, 10, 12, and 24 h, 4 mL of release medium was taken from outside the dialysis bag each time, and an equal volume of medium solution was added. Subsequently, the concentration of Enr in the release medium was analyzed by liquid chromatography-mass spectrometry.

[0086] Using SCF as the release medium: YP@H / PO@DE / C, 0.5% β-glucanase + YP@H / PO@DE / C, O@DE / C, and free Enr were dispersed in 2 mL of release medium and placed in a cellulose acetate dialysis bag (molecular weight cutoff = 1000 Da, allowing only free drug molecules Enr to pass through the dialysis bag). The dialysis bag was then immersed in 100 mL of release medium. The release system was placed in a stirrer at 100 rpm. At predetermined time points of 0, 2, 4, 6, 8, 10, 12, and 24 h, 4 mL of release medium was taken from outside the dialysis bag each time, and an equal volume of medium solution was added. Subsequently, the concentration of Enr in the release medium was analyzed by high performance liquid chromatography-mass spectrometry.

[0087] The results are as follows Figure 18As shown, in simulated gastric fluid, the drug-loaded modified silicone liposomes (YP@H / PO@DE / C) encapsulated with yeast microcapsules released drugs at a slower rate than unencapsulated silicone liposomes (O@DE / C), verifying the gastric environmental protection effect of yeast microcapsules and preventing premature drug release. In simulated cecal fluid and β-glucanase environment, the yeast microcapsules were degraded, and the drug release rate of YP@H / PO@DE / C increased, suggesting that the drug-loaded modified silicone liposomes (YP@H / PO@DE / C) encapsulated with yeast microcapsules can effectively release drugs into target tissues as a drug delivery system.

[0088] 6. Evaluate the therapeutic effect of yeast microencapsulated drug-loaded modified silicone liposomes (YP@H / PO@DE / C) in a mouse model of acute intestinal infection induced by Salmonella.

[0089] The testing method was adopted for animal experiments as reported in the literature (A Precision-Guided "Trojan Horse" for Tissue-Cell-Bacteria Targeted Therapy of Salmonella Intestinal Infections.[J].ACS Nano,2025, 19: 0.).

[0090] C57BL / 6 mice, weighing approximately 18-22 g, were pretreated with streptomycin one week after acclimatization, i.e., by gavage administration of 100 μL of 200 mg / mL streptomycin (dissolved in sterile water). 24 hours later (during which time they drank sterile water), they were then gavage with 10 μL of streptomycin... 7 The dosage of CFUs was used for Salmonella 14028; PBS without any added antimicrobial substances was used as the control group G1, and the following groups were treated with free enrofloxacin (G2), free dual-drug regimen (G3), pure carrier (disulfide bond modified dendritic mesoporous silica prepared in Example 1, G4), modified drug-loaded silicone liposomes encapsulated with yeast microcapsules prepared in Example 5 (G5), and untreated group (G6); the dosage of each group was based on the concentration of antibiotic Enr, and the dosage was 10 mg / kg bw.

[0091] Three days after treatment, the mice were sacrificed for tissue sampling. The contents of the cecum and colon were collected for Salmonella counting and 16S rDNA sequencing. A portion of the intestinal tissue (washed with sterile PBS to remove contents) was fixed in 4% paraformaldehyde for tissue section staining.

[0092] Statistical results of bacteria in the cecum and colon are as follows: Figure 19 As shown, the clearance effect of each treatment group on intestinal bacteria from strong to weak is as follows: modified drug-loaded silicone liposomes encapsulated with yeast microcapsules (G5) > free dual-drug group (G3) > free single-drug group enrofloxacin (G2) > pure carrier group (G4) > PBS group (G1).

[0093] The cecal tissue section results for each treatment group are as follows: Figure 20 As shown, the intestines of the PBS treatment group (G1) exhibited coagulative necrosis, epithelial shedding, and acellular structures, while the other treatment groups showed varying degrees of goblet cell loss and unclear epithelial structures. However, the yeast microencapsulated drug-loaded silicone liposome treatment group (G5) showed significant recovery of intestinal crypts and intestinal mucosa, while maintaining a largely intact cecal tissue structure.

[0094] Figure 21 The results showed that after treatment with yeast microencapsulated drug-loaded silicone liposomes (G5), the relative abundance of harmful bacteria (Salmonella and Shigella coli) in the intestinal flora of mice decreased to 9.07% and 8.25%, respectively, which were the lowest among all treatment groups; the sum of the relative abundance of common probiotics (Lactobacillus, Bifidobacterium, Ekkermansia, and Trichophyton) was 36.46%, which was the highest among all treatment groups, suggesting that yeast microencapsulated drug-loaded silicone liposomes (G5) can play a good role in restoring the intestinal flora.

[0095] In summary, this invention prepared three components (dendritic mesoporous silica, attenuated Salmonella outer membrane vesicles, and yeast microcapsules) for oral drug-loaded modification liposomes that can be encapsulated in yeast microcapsules using a specific method and completed their assembly. The prepared modified drug-loaded liposomes significantly enhanced the targeting effect on macrophages and intestinal epithelial cells, and had a high intracellular bacterial clearance efficiency. The modified drug-loaded liposomes encapsulated in yeast microcapsules showed good biocompatibility and demonstrated good therapeutic effects in a mouse model of acute intestinal infection.

Claims

1. A method for preparing a modified drug-loaded silicone liposome encapsulated in yeast microcapsules, comprising the following steps: (1) Preparation of disulfide bond modified dendritic mesoporous silica; (2) The disulfide bond modified dendritic mesoporous silica, antibacterial drug, anti-inflammatory drug and water are mixed and stirred to obtain drug-loaded dendritic mesoporous silica; (3) Attenuated Salmonella outer membrane vesicles are wrapped on the surface of the drug-loaded dendritic mesoporous silica to obtain drug-loaded dendritic silica coated with attenuated Salmonella outer membrane vesicles. (4) Modify the surface of the drug-loaded dendritic silica coated with the outer membrane vesicles of the attenuated Salmonella with polyethyleneimine and hyaluronic acid to obtain modified drug-loaded dendritic silica coated with the outer membrane vesicles of attenuated Salmonella. (5) Yeast microcapsules are wrapped around the drug-loaded dendritic silica outer layer of the modified attenuated Salmonella outer membrane vesicles to obtain modified drug-loaded silica liposomes encapsulated with yeast microcapsules.

2. The preparation method according to claim 1, characterized in that: In step (2), the mass ratio of the sum of the antibacterial drug and the anti-inflammatory drug to the mass of the disulfide bond modified dendritic mesoporous silica is 1-2:1; The mass ratio of the antibacterial drug to the anti-inflammatory drug is 1-2:1; The antibacterial drug is enrofloxacin and / or linezolid; The anti-inflammatory drug is cinnamaldehyde and / or paeonol.

3. The preparation method according to claim 1, characterized in that: The method for preparing the disulfide bond-modified dendritic mesoporous silica includes the following steps: The template agent, organic small molecule amine, pore-forming agent, silicon source and solvent are mixed and reacted to obtain a precipitate of disulfide bond modified dendritic mesoporous silica.

4. The preparation method according to claim 3, characterized in that: The template agent is at least one selected from hexadecyltrimethylammonium chloride, hexadecyltrimethylammonium bromide, dodecyltrimethylammonium chloride, dodecyltrimethylammonium bromide, octadecyltrimethylammonium chloride, and octadecyltrimethylammonium bromide; The organic small molecule amine is at least one of triethanolamine, urea and ethanolamine; The pore-forming agent is bis-(γ-triethoxysilylpropyl)tetrasulfide and / or bis-(γ-triethoxysilylpropyl)disulfide; The silicon source is tetraethyl orthosilicate; The solvent is water; The mass ratio of the template agent to the small organic molecule amine is 8-9:1; The template agent is 0.4-0.6g per 1mL of silicon source. The volume ratio of the silicon source to the pore-forming agent is 1:1-1.5; The reaction temperature is 80-100℃; The reaction time is 3-5 hours; The reaction was carried out under stirring.

5. The preparation method according to any one of claims 1-4, characterized in that: Step (3) specifically includes the following steps: c. Centrifuge the attenuated Salmonella ATCC202165 bacterial culture, collect the supernatant and filter; then centrifuge the filtrate using a 100-200 kDa ultrafiltration tube, collect the liquid and filter; centrifuge the obtained filtrate, resuspend the obtained precipitate in PBS solution, centrifuge, and the obtained precipitate is bacterial outer membrane vesicles; mix the bacterial outer membrane vesicles with physiological saline to obtain a bacterial outer membrane vesicle suspension; d. Mix the drug-loaded dendritic silica and the bacterial outer membrane vesicle suspension, and sonicate to coat the bacterial outer membrane vesicles onto the drug-loaded dendritic silica particles, thereby obtaining drug-loaded dendritic silica coated with attenuated Salmonella outer membrane vesicles.

6. The preparation method according to claim 5, characterized in that: The OD of the attenuated Salmonella ATCC202165 bacterial suspension 600 It is 0.8-1; Step c is as follows: The attenuated Salmonella ATCC202165 bacterial suspension is centrifuged at 6,000-8,000g for 20-30 min at 4°C. The supernatant is collected and filtered through a 0.45 μm pore size cellulose acetate filter. The filtrate is then centrifuged at 6,000-8,000g for 5-6 min using a 100 kDa ultrafiltration tube. The liquid in the inner tube is collected to a total volume of 20-30 mL. The collected liquid is filtered through a 0.22 μm pore size cellulose acetate filter. The resulting filtrate is centrifuged at 150,000-170,000g for 2-4 h at 4°C. The resulting precipitate is resuspended in PBS solution and centrifuged at 150,000-170,000g for 1-2 h. The resulting precipitate is bacterial outer membrane vesicles. The bacterial outer membrane vesicles are mixed with physiological saline to obtain a bacterial outer membrane vesicle suspension. In step d, the volume ratio of the drug-loaded dendritic silica to the bacterial outer membrane vesicle suspension is 1-2:1; The concentration of the drug-loaded dendritic silica is 1-2 mg / mL; The concentration of the bacterial outer membrane vesicle suspension is 0.3-0.6 mg / mL; The ultrasound is performed using a probe; the ultrasound power is 40-60W; and the ultrasound time is 3-6 minutes.

7. The preparation method according to any one of claims 1-6, characterized in that: Step (4) specifically includes the following steps: e. Drop the drug-loaded dendritic silica coated with the outer membrane vesicles of the attenuated Salmonella into a polyethyleneimine solution and stir to obtain PEI-OMV@DMSN; f. Mix the PEI-OMV@DMSN and hyaluronic acid solution and react to obtain the drug-loaded dendritic silica coated with the modified attenuated Salmonella outer membrane vesicles; Step (5) specifically includes the following steps: g. Disperse yeast powder in 1-1.2M NaOH solution, stir continuously at 80-85℃ for 0.5-1.5h, centrifuge at 2000-3000rpm for 10-15min, and collect the insoluble substance containing yeast cell walls; then suspend the insoluble substance in 1L water, adjust the pH to 4-5 with HCl solution, and incubate at 50-70℃ for 1-1.5h; collect the insoluble residue again by centrifugation, and then wash with water, isopropanol and acetone in sequence; freeze-dry the obtained slurry to obtain yeast microcapsules; h. Mix the yeast microcapsules with water and incubate them; then add the drug-loaded dendritic silica solution coated with the modified attenuated Salmonella outer membrane vesicles, incubate, and centrifuge; resuspend the solid in water, and then freeze-dry and hydrate the suspension to obtain modified drug-loaded silica liposomes encapsulated in yeast microcapsules.

8. The preparation method according to claim 7, characterized in that: In step e, the concentration of the polyethyleneimine solution is 2-5 mg / mL; The volume ratio of the drug-loaded dendritic silica and polyethyleneimine solution coated with the outer membrane vesicles of the attenuated Salmonella is 3-6:

1. The stirring time is 20-40 minutes; In step f, the volume ratio of PEI-OMV@DMSN to hyaluronic acid solution is 7-10:1; The concentration of the hyaluronic acid solution is 10-12 mg / mL; The reaction temperature is 22℃-25℃; the reaction time is 0.5-1.5h; In step h, the mass ratio of the yeast microcapsules to the volume ratio of water is 1-1.2 mg: 1 mL; The yeast microcapsules were incubated with water at a temperature of 37-42°C for 0.5-1 hour. The mass ratio of the yeast microcapsules and the drug-loaded dendritic silica coated with the modified attenuated Salmonella outer membrane vesicles is 2:1.5-2. The concentration of the drug-loaded dendritic silica solution coated with the modified attenuated Salmonella outer membrane vesicles is 0.2-0.4 mg / mL; The incubation time after adding the drug-loaded dendritic silica solution coated with the modified attenuated Salmonella outer membrane vesicles is 0.5-1.5 h.

9. The modified drug-loaded silicone liposome encapsulated with yeast microcapsules prepared by the preparation method of any one of claims 1-8.

10. The use of the modified drug-loaded silicone liposome encapsulated with yeast microcapsules as described in claim 9 in the preparation of a medicament for treating bacterial infectious diseases; Specifically, the bacterial infection is at least one of intestinal infection, lung infection, and breast infection; The bacteria in question is Salmonella.

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