An inhalable pulmonary targeting hybrid vesicle biomimetic nanomaterial and a preparation method and application thereof

Abstract

The application discloses an inhalable lung-targeting hybrid vesicle biomimetic nanomaterial and a preparation method and application thereof, and belongs to the technical field of biological medicines. 3+ / Sr 2+ The AuSr-GA network obtains the hybrid vesicle biomimetic nanomaterial (AuSr-GA@BMEVs) with synergistic antibacterial, anti-inflammatory and blood vessel repair promoting functions, can systemically cope with drug-resistant bacterial infection, excessive inflammation and tissue damage. The material is suitable for atomization inhalation, can be efficiently delivered to the lung in a targeted manner, has good biological safety, and provides an integrated new strategy with high efficiency and low toxicity for the treatment of pneumonia.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a lung-targeting hybrid vesicle biomimetic nanomaterial that can be atomized and inhaled, its preparation method, and its application. Background Technology

[0002] In recent years, clinical treatment of bacterial pneumonia has primarily relied on traditional antibiotics. However, long-term and irregular use has led to the widespread emergence of methicillin-resistant Staphylococcus aureus (MRSA) and other drug-resistant strains. Simultaneously, bacteria can form biofilms with highly dense extracellular matrix in the lungs, significantly hindering antibiotic penetration and enhancing their resistance to the host immune system, further weakening the efficacy of existing antibiotics. Furthermore, single antibacterial strategies are insufficient to address the excessive inflammatory response triggered by bacterial invasion, specifically the polarization of macrophages towards a pro-inflammatory phenotype (M1) and the release of large amounts of inflammatory factors, causing lung tissue damage and vascular endothelial disruption. Current treatments targeting inflammation regulation and vascular repair remain limited and cannot synergistically address multiple pathological aspects.

[0003] In terms of administration routes, traditional intravenous injection or oral administration lacks targeting to the lungs, leading to systemic drug distribution that can easily cause toxic side effects. Furthermore, higher doses are required to achieve effective therapeutic concentrations, and this increases the risk of drug resistance. While inhaled formulations can achieve local drug delivery and reduce systemic exposure, existing inhalable formulations often have a single function and cannot simultaneously achieve multiple effects such as effective antibacterial activity, immunomodulation, and tissue repair. Therefore, developing a lung-targeted delivery system that integrates highly efficient antibacterial activity, precise immune regulation, and endothelial repair is of significant clinical importance. However, no formulations that integrate these multiple functions and are suitable for nebulized inhalation have been reported in the current technology.

[0004] In summary, existing technologies face the following main challenges: First, there is a lack of synergistic antibacterial and anti-inflammatory strategies that can simultaneously disrupt biofilms, eliminate drug-resistant bacteria, and regulate macrophage polarization; second, conventional drug delivery systems struggle to precisely regulate the inflammatory microenvironment and promote vascular repair within the lungs; and third, most nanocarriers lack sufficient biocompatibility or cannot meet the physical requirements of nebulized inhalation, hindering their clinical application. These issues result in limited efficacy and significant side effects in current pneumonia treatments, necessitating the development of novel, multifunctional, integrated inhalation formulations to overcome these bottlenecks. Summary of the Invention

[0005] The purpose of this invention is to provide a lung-targeting hybrid vesicle biomimetic nanomaterial that can be inhaled via atomization, along with its preparation method and applications, to address the problems existing in the prior art. The AuSr-GA@BMEVs hybrid vesicle biomimetic nanomaterial prepared by this invention, through the synergistic effect of the Au-Sr-GA network and hybrid vesicles, integrates antibacterial, anti-inflammatory, and angiogenesis-promoting effects, systematically addressing drug-resistant bacterial infections, excessive inflammation, and tissue damage. This material is suitable for nebulized inhalation, enabling highly efficient targeted delivery to the lungs while exhibiting good biocompatibility, providing a novel, highly effective, and low-toxicity integrated strategy for pneumonia treatment.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides a lung-targeting hybrid vesicle biomimetic nanomaterial that can be atomized and inhaled, comprising: a hybrid vesicle carrier formed by the hybridization of bacterial exovesicles and milk exovesicles, and an Au-Sr-GA composite nanostructure in situ loaded on the hybrid vesicle carrier; the Au-Sr-GA composite nanostructure exists on the surface of the hybrid vesicle carrier in the form of an Au-Sr-GA coordination network.

[0008] Furthermore, the bacterial exovesicles are derived from Lactobacillus johnsonii.

[0009] Furthermore, the lung-targeting hybrid vesicle biomimetic nanomaterial has a particle size of 130 nm to 135 nm and a zeta potential of -27 mV to -29 mV.

[0010] This invention also provides a method for preparing the lung-targeting hybrid vesicle biomimetic nanomaterial, comprising the following steps:

[0011] S1. Extract bacterial exovesicles and milk exovesicles separately;

[0012] S2. The purified bacterial vesicles and milk vesicles were mixed at a protein concentration of 1:1 and subjected to membrane fusion treatment to obtain hybrid vesicles.

[0013] S3. The hybrid vesicle is combined with Au-containing... 3+ 、Sr 2+ The precursor solution of gallic acid was mixed and reacted in situ under alkaline conditions to generate Au-Sr-GA composite nanostructures on the hybrid vesicles. The lung-targeting hybrid vesicle biomimetic nanomaterials were obtained after purification.

[0014] Furthermore, in step S2, the membrane fusion treatment is performed under the condition of ice bath ultrasonic treatment for 3 minutes.

[0015] Furthermore, after the membrane fusion treatment, it also includes extrusion through a polycarbonate membrane with a pore size of 100 nm.

[0016] Furthermore, in step S3, Au in the precursor solution 3+ 、Sr 2+ The molar ratio of gallic acid to gallic acid is 1:1:2; the pH value of the in-situ reaction is 13, the reaction temperature is 37℃, and the reaction time is 12 hours.

[0017] The present invention also provides the application of the lung-targeting hybrid vesicle biomimetic nanomaterial described above in the preparation of a drug for treating pneumonia.

[0018] Furthermore, the pneumonia in question is bacterial pneumonia.

[0019] Furthermore, the drug is administered via nebulized inhalation.

[0020] The present invention discloses the following technical effects:

[0021] This invention prepares hybrid vesicles (BMEVs) by integrating extracellular vesicles (BEVs) derived from Lactobacillus johnsoni with extracellular vesicles (MEVs) derived from milk, and modifies their surface with Au. 3+ / Sr 2+ -GA network yielded hybrid vesicle biomimetic nanomaterials (AuSr-GA@BMEVs) with synergistic antibacterial, anti-inflammatory, and angiogenesis-promoting effects. Through the unique mechanism of the Au-Sr-GA network, these materials effectively kill drug-resistant bacteria and disrupt biofilms; simultaneously, the hybrid vesicles synergistically regulate macrophage polarization towards an anti-inflammatory phenotype, reducing the level of pro-inflammatory factor IL-6 and increasing the level of anti-inflammatory factor IL-10, significantly inhibiting excessive inflammation; Sr... 2+ The introduction of this technology further promotes vascular endothelial repair. This multi-dimensional synergistic effect overcomes the limitations of single-therapy approaches and systematically addresses the challenges of drug resistance, cytokine storms, and tissue damage in bacterial pneumonia.

[0022] Furthermore, this material exhibits excellent lung targeting and biocompatibility. Its stable nanostructure is well-suited for nebulized inhalation, enabling highly efficient local drug accumulation in the lungs, thus enhancing efficacy while avoiding the toxic side effects of traditional systemic administration. In vivo experiments have demonstrated that nebulized inhalation of this material exhibits significant advantages in pathogen clearance, inflammation regulation, and improved survival rates, providing an innovative solution for the clinical development of safe and effective inhaled pneumonia treatments. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are 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.

[0024] Figure 1 A diagram illustrating the construction and application of hybrid vesicle biomimetic nanomaterials;

[0025] Figure 2 Particle size distribution diagrams for BEVs, MEVs, BMEVs, and AuSr-GA@BMEVs;

[0026] Figure 3 Zeta potential distribution of BEVs, MEVs, BMEVs and AuSr-GA@BMEVs;

[0027] Figure 4 The particle size distribution of AuSr-GA@BMEVs before and after atomization is shown.

[0028] Figure 5 The diagram shows the zeta potential distribution of AuSr-GA@BMEVs before and after atomization;

[0029] Figure 6 Image showing cell viability of BEAS-2B cells after treatment with different concentrations of AuSr-GA@BMEVs;

[0030] Figure 7 Image showing cell viability of RAW264.7 cells after treatment with different concentrations of AuSr-GA@BMEVs;

[0031] Figure 8 The survival results (A) and survival statistics (B) of MRSA after treatment with different concentrations of AuSr-GA@BMEVs;

[0032] Figure 9 A statistical graph showing the damage rate of MRSA biofilm after treatment with different concentrations of AuSr-GA@BMEVs;

[0033] Figure 10 A statistical chart showing the percentage of M2 macrophages in different treatment groups;

[0034] Figure 11 Statistical graph of IL-10 levels in the supernatant of BMDMs in different treatment groups;

[0035] Figure 12 Statistical graph of VEGF levels in HUVECs supernatant in different treatment groups;

[0036] Figure 13 A statistical graph showing the viable bacterial count in the lungs after different treatment groups;

[0037] Figure 14 A statistical graph showing the survival rate of mice during different treatment groups;

[0038] Figure 15 A statistical graph showing the concentration of IL-6 in the BALF of mice after different treatment groups;

[0039] Figure 16 A statistical graph showing the concentration of IL-10 in the BALF of mice after different treatment groups. Detailed Implementation

[0040] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0041] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0042] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0043] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0044] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0045] This invention prepares hybrid vesicles (BMEVs) by integrating extracellular vesicles (BEVs) derived from Lactobacillus johnsoni with extracellular vesicles (MEVs) derived from milk, and modifies their surface with Au. 3+ / Sr 2+ -GA network yields hybrid vesicle biomimetic nanomaterials (AuSr-GA@BMEVs) with synergistic antibacterial, anti-inflammatory, and pro-angiogenic repair properties. These nanomaterials remain stable during atomization, allowing for inhalation drug delivery via microneedling devices, and enabling targeted delivery by depositing nanoparticles in the lungs. Figure 1 ).

[0046] Example 1: Preparation of hybrid vesicle biomimetic nanomaterials

[0047] 1. Preparation of bacterial extravesicles (BEVs)

[0048] Take 10 mL of Lactobacillus johnsoni bacterial culture in the logarithmic growth phase, centrifuge at 8,500 g for 30 min, and collect the supernatant. Centrifuge again at 20,000 g for 45 min, and filter the supernatant through a 0.22 μm microporous membrane. Centrifuge the filtered solution at 120,000 g for 2 h. Discard the supernatant, resuspend the precipitate in PBS buffer, and repeat the ultracentrifugation twice at 120,000 g to obtain purified BEVs. Finally, resuspend the purified BEV precipitate in 1 mL PBS, determine its protein concentration using a BCA protein quantification kit, and store at -80℃ for later use.

[0049] 2. Preparation of Milk Extravesicles (MEVs)

[0050] Centrifuge 50 mL of fresh milk at 10,000 g for 30 min, carefully removing the upper fat layer and precipitate. Collect the whey fraction in the middle layer and centrifuge again at 10,000 g for 30 min to thoroughly remove residual fat particles and casein microclusters. Collect the supernatant and centrifuge at 160,000 g for 90 min. Discard the supernatant, resuspend the precipitate in PBS buffer, and repeat the ultracentrifugation twice at 160,000 g to obtain purified MEVs. Finally, resuspend the purified MEV precipitate in 1 mL PBS, determine the protein concentration, and store at -80℃ for later use.

[0051] 3. Preparation of hybrid vesicles (BMEVs)

[0052] The prepared BEVs and MEVs solutions were mixed at a protein concentration ratio of 1:1 according to the protein concentration determined by the BCA method. 50 mL of the mixture was then sonicated in an ice bath for 3 min. The mixture was extruded through a 100 nm polycarbonate membrane using a micro-extruder to promote membrane fusion. The extruded product was ultracentrifuged at 100,000 g for 2 h, and the precipitate was resuspended in PBS buffer to obtain a BMEVs hybrid vesicle dispersion.

[0053] 4. Preparation of Nanomaterial Loading and Biomimetic Coating (AuSr-GA@BMEVs)

[0054] (1) Preparation of precursor solution: Add 2 mL of 30 mM gallic acid (GA) solution, 3 mL of 30 mM chloroauric acid (HAuCl4) solution, and 2 mL of 60 mM strontium chloride (SrCl2) solution to a beaker in sequence, stir at room temperature for 30 min to obtain precursor solution.

[0055] (2) Vesicle mixing: Add 5 mL of BMEVs solution of 20 mg / mL (based on total vesicle protein) to the above precursor solution and continue stirring for 30 min to allow BMEVs to fully contact and adsorb the precursor ions.

[0056] (3) In-situ mineralization pre-assembly: Transfer the beaker to a 37°C water bath, add sodium hydroxide to adjust the pH of the solution to 13, and continue stirring for 12 h to allow the gold-strontium-gallic acid nanostructure to be generated and assembled in situ on the surface and inside of BMEVs.

[0057] (4) Purification: The obtained mixture was placed in a dialysis bag (MwCO: 3500 Da) and dialyzed for 3 days. The purified solution was freeze-dried to obtain the hybrid vesicle biomimetic nanomaterial AuSr-GA@BMEVs.

[0058] Example 2 Characterization and property determination of hybrid vesicle biomimetic nanomaterials

[0059] 1. Determination of particle size and potential of nanoparticles

[0060] The BEVs, MEVs, BMEVs, and AuSr-GA@BMEVs nanoparticle dispersions prepared in Example 1 were diluted with sterile phosphate-buffered saline (PBS, pH 7.4) and filtered through a 0.22 μm microporous membrane to remove any dust or large particle aggregates. The particle size distribution and zeta potential of BEVs, MEVs, BMEVs, and AuSr-GA@BMEVs were determined using a dynamic light scattering particle size analyzer.

[0061] Particle size distribution results are as follows Figure 2 As shown, the zeta potential measurement results are as follows: Figure 3 As shown. By Figure 2 and Figure 3 The average particle sizes of BEVs and MEVs are 110.4 nm and 118.5 nm, respectively. The hybrid membrane vesicles BMEVs formed by membrane fusion have a particle size of 105.2 nm, while the particle size of AuSr-GA@BMEVs increases to 132.3 nm, indicating that the metal-phenolic network structure was successfully loaded onto the vesicle surface. The average Zeta potentials of BEVs, MEVs, and BMEVs are -18.2 mV, -17.5 mV, and -18.3 mV, respectively, while the Zeta potential of AuSr-GA@BMEVs decreases to -28.2 mV. This is because the GA loaded on the vesicle surface carries a negative charge, which further enhances the electrostatic repulsion of nanoparticles in solution, which is beneficial for long-term storage stability and preventing non-specific protein adsorption in vivo.

[0062] 2. Evaluation of the atomization stability of nanoparticles

[0063] The AuSr-GA@BMEVs solution was nebulized using a vibrating screen nebulizer. The nebulized aerosol was then rapidly condensed on ice and collected in centrifuge tubes for reliquefaction. Changes in particle size and zeta potential of the AuSr-GA@BMEVs before and after nebulization were analyzed. Results are as follows: Figure 4 and Figure 5 As shown.

[0064] Depend on Figure 4 and Figure 5 It can be seen that AuSr-GA@BMEVs maintained a stable particle size and zeta potential after nebulization, which meets the requirements for nebulized inhalation drug delivery.

[0065] Example 3: Biosafety Evaluation of Hybrid Vesicle Biomimetic Nanomaterials

[0066] Lung epithelial cells (BEAS-2B) and monocytes / macrophages (RAW264.7) were cultured at 5 × 10⁶ cells per well. 3 Cells were seeded at a density of 100 μL into each well of a 96-well cell culture plate. The plate was pre-cultured in an incubator for 24 hours to allow for full cell adhesion and entry into the logarithmic growth phase. After good cell adhesion, the old culture medium was discarded from each well. Experimental groups, negative control groups, and blank control groups were set up.

[0067] Experimental group: Replace the old medium with complete medium containing different concentrations of AuSr-GA@BMEVs. Set final concentration gradients including 25 µg / mL, 50 µg / mL, 100 µg / mL, 250 µg / mL, and 500 µg / mL. Each concentration had at least 5 replicates.

[0068] Negative control group: replaced with an equal volume of fresh complete culture medium that does not contain nanomaterials.

[0069] Blank control group: Only an equal volume of complete culture medium was added, without cells.

[0070] Each group was incubated for 24 hours, and the in vitro biosafety of the hybrid vesicle biomimetic nanomaterials was evaluated using the Cell Counting Kit-8 (CCK-8) assay. Results are as follows: Figure 6 and Figure 7 As shown.

[0071] Depend on Figure 6 and Figure 7 It was found that within the concentration range of ≤500 µg / mL, AuSr-GA@BMEVs had no significant effect on the viability of lung epithelial cells and macrophages, demonstrating that the material has good biocompatibility.

[0072] Example 4: In vitro bioactivity evaluation of hybrid vesicle biomimetic nanomaterials

[0073] 1. Evaluation of the antibacterial activity of nanoparticles

[0074] Methicillin-resistant Staphylococcus aureus (MRSA, ATCC 43300) was cultured in a shaker at 37°C and 220 rpm until the mid-log phase (OD). 600 =0.5). After washing with PBS, AuSr-GA@BMEVs at different concentrations (0 µg / mL, 25 µg / mL, 50 µg / mL, 100 µg / mL, and 250 µg / mL) were added and incubated for 8 h. The resulting serially diluted solutions were then plated on LB agar plates and incubated at 37°C for 24 h. The number of colonies grown on each plate was counted, and the MRSA survival rate was calculated. Results are as follows: Figure 8 As shown.

[0075] 1 mL of MRSA suspension was inoculated into 24-well plates and incubated statically at 37°C for 6 h to form a mature biofilm. Unattached bacteria and culture medium were gently washed away with PBS. Fresh culture medium containing different concentrations (0 µg / mL, 25 µg / mL, 50 µg / mL, 100 µg / mL, and 250 µg / mL) of AuSr-GA@BMEVs was added, and the plates were incubated at 37°C for another 12 h. After gentle washing with PBS, the plates were stained with crystal violet, and the biofilm disruption rate was calculated. Results are shown below. Figure 9 As shown.

[0076] Depend on Figure 8 and Figure 9It is known that, at a non-cytotoxic dose, AuSr-GA@BMEVs can significantly inhibit the growth of MRSA and significantly disrupt the biofilm formed by MRSA.

[0077] 2. Evaluation of the anti-inflammatory activity of nanoparticles (induction of macrophage M2 polarization)

[0078] Bone marrow-derived macrophages (BMDMs) were obtained by induced differentiation of bone marrow mesenchymal stem cells (BMSCs) from the femoral and tibial mesenchymal cavities of BALB / c mice. After 6 days of culture in DMEM medium containing macrophage colony-stimulating factor M-CSF (20 ng / mL), BMDMs were collected and seeded into 12-well plates and cultured for 24 h. 100 µg / mL AuSr-GA@BMEVs were added to the culture medium, and control groups were established with blank culture medium and single vesicle nanoparticles (AuSr-GA@BEVs and AuSr-GA@MEVs). Cells were cultured for another 48 h. Cells were collected, stained with CD206 antibody (a marker on the surface of M2 macrophages), and data were acquired using flow cytometry to analyze the percentage of M2 macrophages. Results are as follows: Figure 10 As shown.

[0079] Simultaneously, after culture, the cell supernatant was collected, cell debris was removed by centrifugation, and the clarified supernatant was transferred to a new centrifuge tube. IL-10 cytokine levels were detected using a commercially available ELISA kit. Results are as follows: Figure 11 As shown.

[0080] Depend on Figure 10 and Figure 11 It was found that, at a non-cytotoxic dose, AuSr-GA@BMEVs could significantly induce macrophages to differentiate into the M2 type and significantly upregulate the level of the anti-inflammatory cytokine IL-10 secreted by macrophages. Moreover, the effect was significantly better than that of using a single vesicle, indicating that AuSr-GA@BMEVs hybrid vesicles can synergistically reprogram the inflammatory microenvironment and exert an anti-inflammatory effect.

[0081] 3. Evaluation of the angiogenesis-promoting activity of nanoparticles

[0082] Human umbilical vein endothelial cells (HUVECs) were seeded into culture flasks and cultured for 12 h. An inflammatory environment was simulated by adding 0.5 mM H2O2 and 20 μg / mL LPS. 100 µg / mL AuSr-GA@BMEVs were then added, and blank culture medium (without Sr) was also provided. 2+ The group containing Au-GA@BMEVs nanoparticles served as a control and was cultured for another 6 hours. Cell supernatant was collected, cell debris was removed by centrifugation, and the clarified supernatant was transferred to a new centrifuge tube. VEGF levels were detected using a commercially available ELISA kit.

[0083] Depend on Figure 12 It can be seen that, at a non-cytotoxic dose, AuSr-GA@BMEVs can significantly promote VEGF secretion by HUVECs, and the effect is significantly better than that of Sr-free products. 2+ Nanoparticles indicate that Sr 2+ Key role of AuSr-GA@BMEVs in promoting angiogenesis repair.

[0084] Example 5: In vivo bioactivity evaluation of hybrid vesicle biomimetic nanomaterials

[0085] 1. Animal model establishment and grouping for drug administration

[0086] (1) Experimental animals: Healthy female BALB / c mice aged 6-8 weeks and weighing 18-22 g were selected and acclimatized in an SPF-grade animal room for one week.

[0087] (2) Establishment of the pneumonia model: MRSA (ATCC 43300) bacterial culture in the logarithmic growth phase was centrifuged, resuspended in sterile PBS, and the bacterial concentration was adjusted to 1.5 × 10⁻⁶. 9 CFU / mL. Mice were anesthetized by isoflurane inhalation. Using a microsyringe and a dedicated endotracheal intubation device, 50 µL of the above bacterial suspension was slowly injected into each mouse via the trachea, equivalent to an infection of 7.5 × 10⁻⁶ CFU / mL per mouse. 8 CFU MRSA was used to establish an acute bacterial pneumonia model.

[0088] (3) Experimental grouping and administration: The mice that were successfully modeled were randomly divided into three groups of 10 each.

[0089] Model control group (PBS group): nebulized inhalation of sterile phosphate-buffered saline (PBS).

[0090] Positive drug control group (Cip group): Ciprofloxacin injection was administered via tail vein at a dose of 80 mg / kg.

[0091] Experimental treatment group (AuSr-GA@BMEVs group): nebulized inhalation of AuSr-GA@BMEVs nanomaterial suspension (prepared with PBS), at a dose of 40 mg / kg (based on the total solid mass of the nanomaterials).

[0092] (4) Administration regimen: Administer the appropriate treatment once at 12 h, 24 h, and 36 h post-infection (i.e., every 12 hours). For nebulized inhalation, use a small animal-specific nebulizer. Place the mouse in a sealed nebulizer chamber and nebulize until the medication is completely depleted. For intravenous administration, follow standard procedures. Observe and record the survival status, activity level, and weight changes of the mice daily.

[0093] 2. Evaluation Indicators and Detection Methods for Treatment Efficacy

[0094] Twelve hours after the last administration, five mice in each group were euthanized and dissected, and lung tissue was extracted and homogenized in 1 mL of sterile PBS. Serial dilutions were prepared with PBS and spread on plates. After overnight incubation, the number of bacterial colonies was counted, and the results were expressed as colony-forming units (CFU / g) per gram of lung tissue.

[0095] The levels of IL-6 and IL-10 in bronchoalveolar lavage fluid (BALF) were measured using ELISA to investigate changes in the alveolar immune microenvironment.

[0096] We continuously observed and recorded the survival status of the remaining 5 mice in each group from the time of infection to day 7, and plotted survival curves.

[0097] 3. Results and Conclusions

[0098] (1) Antibacterial effect: such as Figure 13 As shown, compared with the PBS model control group, the bacterial load (CFU / g) in the lung tissue of mice in the AuSr-GA@BMEVs experimental treatment group was significantly reduced, and its clearance effect was comparable to that of the intravenous ciprofloxacin (Cip) positive control group, indicating that nebulized inhalation of AuSr-GA@BMEVs can effectively clear pulmonary MRSA infection.

[0099] (2) Inflammation regulation: such as Figure 15 and Figure 16 As shown, AuSr-GA@BMEVs treatment significantly reduced the level of the pro-inflammatory factor IL-6 in BALF while significantly increasing the level of the anti-inflammatory factor IL-10, effectively reversing the excessive inflammatory state in the pneumonia model. While the Cip group could clear bacteria, its regulatory effect on the inflammatory microenvironment was not significant.

[0100] (3) Survival benefits: such as Figure 14 As shown, the survival rate of mice in the PBS model control group decreased sharply after infection. AuSr-GA@BMEVs treatment significantly improved the survival rate of infected mice, and its protective effect was superior to that of the positive control group.

[0101] This embodiment demonstrates that the atomizable inhalable hybrid vesicle biomimetic nanomaterial AuSr-GA@BMEVs can effectively treat bacterial pneumonia by clearing lung bacteria and reversing the inflammatory microenvironment.

[0102] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A lung-targeted hybrid vesicle biomimetic nanomaterial for nebulized inhalation, characterized in that, Comprising: A hybrid vesicle carrier formed by hybridizing bacterial extracellular vesicles and milk extracellular vesicles, and an Au-Sr-GA composite nanostructure in-situ loaded on the hybrid vesicle carrier; the Au-Sr-GA composite nanostructure exists in the form of an Au-Sr-GA coordination network on the surface of the hybrid vesicle carrier; The bacterial extracellular vesicles are derived from Lactobacillus johnsonii.

2. The lung-targeting hybrid vesicle biomimetic nanomaterial according to claim 1, wherein The particle size of the lung-targeting hybrid vesicle biomimetic nanomaterial is 130 nm to 135 nm, and the Zeta potential is -27 mV to -29 mV.

3. A method for preparing the lung-targeted hybrid vesicle biomimetic nanomaterial according to claim 1 or 2, characterized in that, Including the following steps: S1. Extract bacterial extracellular vesicles and milk extracellular vesicles respectively; S2. Mix the purified bacterial extracellular vesicles and milk extracellular vesicles at a protein concentration ratio of 1:1, and perform membrane fusion treatment to obtain hybrid vesicles; S3. Mix the hybrid vesicles with a precursor solution containing Au 3+ , Sr 2+ and gallic acid, and conduct an in-situ reaction under alkaline conditions to form an Au-Sr-GA composite nanostructure on the hybrid vesicles, and the lung-targeted hybrid vesicle biomimetic nanomaterial is obtained through purification.

4. The preparation method according to claim 3, characterized in that, In step S2, the conditions for the membrane fusion treatment are ice bath ultrasonic treatment for 3 min.

5. The preparation method according to claim 4, wherein After the membrane fusion treatment, it further includes extrusion through a polycarbonate membrane with a pore size of 100 nm.

6. The preparation method according to claim 3, wherein In step S3, the molar ratio of Au 3+ , Sr 2+ and gallic acid in the precursor solution is 1:1:2; the pH value of the in-situ reaction is 13, the reaction temperature is 37 °C, and the reaction time is 12 hours.

7. Use of the lung-targeting hybrid vesicle biomimetic nanomaterial according to claim 1 or 2 in the preparation of a drug for treating pneumonia.

8. The application according to claim 7, wherein The pneumonia is bacterial pneumonia.

9. The application according to claim 7, characterized in that, The administration method of the drug is aerosol inhalation.

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