Application of calcium phosphate-loaded bacterial membrane vesicles in preparation of preparation for enhancing prevention effect of bacterial pneumonia
By loading calcium phosphate (CaP) onto bacterial membrane vesicles (BMVs) as an immune adjuvant, a highly efficient drug delivery system was constructed, which solved the problems of insufficient immunogenicity of BMVs and narrow applicability of adjuvants in the prior art, and achieved specific prevention and immune enhancement against Pseudomonas aeruginosa, Klebsiella pneumoniae and Acinetobacter baumannii.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- RUIJIN HOSPITAL AFFILIATED TO SHANGHAI JIAO TONG UNIV SCHOOL OF MEDICINE
- Filing Date
- 2026-02-28
- Publication Date
- 2026-05-15
AI Technical Summary
Existing technologies lack specific preventive measures against Pseudomonas aeruginosa, Klebsiella pneumoniae, and Acinetobacter baumannii. BMVs have insufficient immunogenicity, and existing adjuvants have a narrow range of applications and complex loading processes, resulting in poor efficacy in preventing bacterial pneumonia.
Using calcium phosphate (CaP) as an immune adjuvant, a highly efficient drug delivery system was constructed by loading it onto the surface or interior of bacterial membrane vesicles (BMVs) via ultrasound. Specific BMVs@CaP formulations were then prepared to enhance immunity against different pathogenic bacteria.
It achieves precise prevention of Pseudomonas aeruginosa, Klebsiella pneumoniae and Acinetobacter baumannii, significantly improves the immune response, simplifies the preparation process and improves the stability and safety of the formulation.
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Figure CN122031671A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biomedical technology, and in particular to the application of calcium phosphate-loaded bacterial membrane vesicles in the preparation of formulations that enhance the preventive effect against bacterial pneumonia. Background Technology
[0002] Hospital-acquired pneumonia (HAP) often leads to prolonged hospital stays and poor treatment outcomes. HAP is primarily caused by Gram-negative bacteria, with *Pseudomonas aeruginosa* (PA), *Klebsiella pneumoniae* (KP), and *Acinetobacter baumannii* (AB) being the most common clinical causative agents. These pathogens are not only resistant to multiple antibiotics themselves but also readily acquire new resistance determinants, resulting in a continuous decline in the effectiveness of clinical treatments. Currently, antimicrobial drugs (such as enzyme inhibitor combination preparations and carbapenems) are frequently used to treat refractory bacterial pneumonia. However, their efficacy against extensively or pan-drug-resistant bacteria is poor, and the variety of antimicrobial drugs is limited. Furthermore, many pharmaceutical companies have announced the suspension of antibiotic research projects due to the rapid spread of drug-resistant bacteria. In today's global medical field, antimicrobial resistance has evolved into a serious and urgent emerging threat, with the spread of resistance far outpacing the pace of innovative research and new discoveries. Given the growing problem of drug resistance in PA and the limited effectiveness of traditional antibiotic treatments, it is urgent to develop new prevention and treatment strategies. Preventing drug-resistant bacterial infections is a valuable strategic option.
[0003] Domestic and international studies have reported that bacteria can secrete and release nanoscale spherical vesicles, namely bacterial membrane vesicles (BMVs), which have a structure similar to the bacterial outer membrane and contain a large amount of endogenous substances. Studies have shown that synthesized *E. coli* BMVs and neutrophil hybrid membrane vesicles encapsulating quinolone antibiotics have been validated for their therapeutic and preventative effects against *Klebsiella pneumoniae* infection. The clinical value of BMVs is being continuously explored, for example, in the development of drug carriers, vaccines, or immune adjuvants.
[0004] BMVs retain pathogen-associated pattern molecules and lipopolysaccharides, playing important roles in cell-cell interactions and immune regulation. They pre-activate the body's immune system to defend against bacterial invasion, compensating for the lack of immune regulation function in the use of antibiotics alone. As early as the 1980s, researchers used BMVs derived from group B meningococcus to prevent meningococcal infection, verifying that BMVs produce specific antibodies and have similar protective effects against homologous strains and other strains, exhibiting cross-protective activity. A vaccine marketed as Bexsero was approved in Europe in 2013 and has proven effective in several countries, including New Zealand, Norway, and Cuba. As an emerging nanomaterial with potential immunomodulatory functions, BMVs have become a research hotspot and are expected to play an important role in the prevention of infectious diseases.
[0005] Despite the aforementioned development potential and advantages of BMVs, there are still issues that need to be addressed and explored in depth regarding their immunogenicity, potential risks, and mechanisms of inducing specific immune responses, which limit their application in vaccine development. Insufficient immunogenicity may prevent them from effectively activating the body's immune response, while excessive immunogenicity may trigger an overactive immune response, such as a cytokine storm. The core of preventing bacterial infections lies in enhancing both specific and non-specific immune responses. The synergistic effect of "immunogenic regulation + adjuvant effect" can effectively activate the body's immune system, laying the foundation for infection prevention. To enhance specific immune responses and improve the targeting ability of tissues such as lymph nodes, some researchers have integrated adjuvants with BMVs or optimized membrane structures. Aluminum-containing adjuvants such as alum are widely used in common vaccines such as those for hepatitis B virus and human papillomavirus to confer protective immunity; however, aluminum-based adjuvants lack the ability to induce type I helper T cell activation and produce inflammatory responses at the injection site. Therefore, researchers are continuously developing novel inorganic nanoparticles to overcome the limitations of traditional adjuvants.
[0006] In summary, existing technologies for the prevention of bacterial pneumonia have the following shortcomings: (1) Lack of specific preventive measures against specific pathogens: Existing bacterial pneumonia prevention products (such as broad-spectrum antibiotics and universal vaccines) mostly adopt a "full coverage" design, which makes it difficult to achieve targeted prevention against common pathogens of clinical hospital-acquired infections such as Pseudomonas aeruginosa (PA), Klebsiella pneumoniae (KP), and Acinetobacter baumannii (AB). Routine use of antibiotics can easily lead to problems such as dysbiosis and increased drug resistance, and the preventive effect against specific bacterial infections is limited, which cannot meet the needs of precise prevention and control in clinical practice.
[0007] (2) Insufficient immunogenicity of BMVs and limited preventive effect: Among the existing preventive technologies based on bacterial membrane vesicles, BMVs without immune adjuvant loading have weak immunogenicity and are difficult to effectively stimulate the body to produce a specific immune response, resulting in low protective efficacy in preventing bacterial infectious pneumonia and failing to achieve the ideal preventive effect.
[0008] (3) Lack of broad-spectrum and compatibility in the selection of immune adjuvants: In the existing technology, the adjuvants used to enhance the immunogenicity of BMVs are mostly highly specific and have a narrow range of applications. Some adjuvants may also cause strong immune side effects and are difficult to form good loading compatibility with BMVs from different bacterial sources. They cannot achieve the immune enhancement effect on BMVs from multiple pathogenic bacteria sources, which limits the large-scale application of BMVs preparations.
[0009] (4) The loading process of BMVs and adjuvants is complicated and the stability is poor: The existing binding methods of BMVs and adjuvants mostly adopt complex processes such as chemical cross-linking, which are not only cumbersome and costly, but may also damage the structural integrity of BMVs, resulting in the loss of their antigenicity; at the same time, the complexes formed by chemical cross-linking have poor stability and are prone to dissociation during storage and application, affecting the preventive effect of the formulation. Summary of the Invention
[0010] To address the above technical problems, this invention provides the application of calcium phosphate-loaded bacterial membrane vesicles in the preparation of formulations that enhance the preventive effect against bacterial pneumonia.
[0011] Calcium phosphate (CaP) is a promising next-generation immunoadjuvant. CaP not only possesses good biocompatibility, predictable metabolic pathways, controllable biodegradation, small molecule delivery capabilities, and a simple preparation process, but it also promotes antigen-presenting cell uptake and presentation to lymph nodes, enhancing the immunostimulatory effect. Loading CaP onto the surface or interior of bacterial microvessels (BMVs) combines the advantages of both to construct a highly efficient drug delivery system. This invention prepares and synthesizes CaP-loaded BMVs from different bacterial sources, specifically preventing bacterial infections.
[0012] On the one hand, the present invention provides the application of calcium phosphate-loaded bacterial membrane vesicles in the preparation of formulations that enhance the preventive effect of bacterial pneumonia, wherein the bacterial membrane vesicles are derived from Pseudomonas aeruginosa, Klebsiella pneumoniae or Acinetobacter baumannii. The extraction and purification steps of bacterial membrane vesicles (BMVs) are as follows: Standard strains of Pseudomonas aeruginosa, Klebsiella pneumoniae, and Acinetobacter baumannii were selected and activated in LB liquid medium until the bacteria reached the logarithmic growth phase. After the culture was completed, the bacterial supernatant was collected by centrifugation at 3000 rpm for 10 min at room temperature. The crude BMV extract was purified by gel filtration chromatography. The morphology of BMVs was observed by transmission electron microscopy to ensure that they were typical vesicle structures, and they were frozen and stored at -80℃ for later use.
[0013] Furthermore, the specific procedure for purifying the crude extract of BMVs using a gel filtration chromatography column is as follows: First, the chromatography column is equilibrated with sterile PBS until the baseline is stable. Then, the crude extract is slowly loaded onto the top of the chromatography column. PBS is used as the elution buffer, and the elution flow rate is controlled at 0.5-1.0 mL / min. The elution peaks are monitored using a UV detector. The characteristic elution peak components corresponding to the BMVs are collected.
[0014] Furthermore, the preparation method of calcium phosphate-loaded bacterial membrane vesicles is as follows: bacterial membrane vesicles (BMVs) and calcium phosphate particles are resuspended, mixed evenly in a certain proportion, and then sonicated. After sonication, the system is centrifuged at 4°C and 13,000 rpm for 30 min to remove free BMVs from the supernatant. The precipitate is collected, washed with sterile PBS, and then resuspended in sterile PBS to obtain three specific formulation suspensions: PA-BMVs@CaP, KP-BMVs@CaP, and AB-BMVs@CaP.
[0015] Furthermore, the mass ratio of bacterial membrane vesicles (BMVs) to calcium phosphate particles was 1:1; the ultrasonic treatment conditions were: power 50W, working time 3s, interval time 3s, total ultrasonic time 3min, and the system temperature was maintained at ≤10℃ during the ultrasonic process.
[0016] On the other hand, the present invention also provides an agent that enhances the preventive effect against bacterial pneumonia caused by Pseudomonas aeruginosa, Klebsiella pneumoniae, or Acinetobacter baumannii; the active ingredient of the agent is a bacterial membrane vesicle loaded with calcium phosphate, the bacterial membrane vesicle being derived from Pseudomonas aeruginosa, Klebsiella pneumoniae, or Acinetobacter baumannii.
[0017] Furthermore, in the calcium phosphate-loaded bacterial membrane vesicles, the mass ratio of bacterial membrane vesicles to calcium phosphate particles is 1:1.
[0018] On the other hand, the present invention also provides the application of the above-mentioned preparation in the preparation of vaccines that enhance the preventive effect against bacterial pneumonia.
[0019] On the other hand, the present invention also provides a vaccine that enhances the preventive effect against bacterial pneumonia caused by Pseudomonas aeruginosa, Klebsiella pneumoniae, or Acinetobacter baumannii; the active ingredient of the vaccine is the calcium phosphate-loaded bacterial membrane vesicles as described in claim 1, wherein the bacterial membrane vesicles are derived from Pseudomonas aeruginosa, Klebsiella pneumoniae, or Acinetobacter baumannii; and the mass ratio of the bacterial membrane vesicles to calcium phosphate particles in the calcium phosphate-loaded bacterial membrane vesicles is 1:1.
[0020] The advantages and positive effects of using the calcium phosphate-loaded bacterial membrane vesicles described in this invention in the preparation of formulations that enhance the preventive effect against bacterial pneumonia are as follows: 1. Highly specific and targeted prevention: This invention prepares BMVs@CaP preparations derived from three common pathogenic bacteria: Pseudomonas aeruginosa (PA), Klebsiella pneumoniae (KP), and Acinetobacter baumannii (AB). These preparations can precisely stimulate the body to produce a specific immune response against the corresponding pathogens, effectively preventing pneumonia caused by specific bacteria. This avoids problems such as dysbiosis caused by broad-spectrum antibiotics and meets the needs of precise clinical prevention and control.
[0021] 2. Significantly enhanced immune response and improved preventive efficacy: This invention loads calcium phosphate particles (CaP) into BMVs using an ultrasonic method. CaP can act as a highly effective immune adjuvant to significantly enhance the immunogenicity of BMVs, effectively stimulate humoral and cellular immune responses, and greatly improve the preventive and protective efficacy of the formulation, thus solving the problem of insufficient immunogenicity of existing BMVs without adjuvant loading.
[0022] 3. Calcium phosphate particles have broad compatibility and broad-spectrum adjuvant properties: The calcium phosphate particles in this invention can achieve efficient loading with BMVs from different bacterial sources (PA, KP, AB), and have significant immune enhancement effects on various BMVs. This breaks through the limitation of the narrow applicability of existing adjuvants and provides a universal immune enhancement solution for the development of preventive agents for various bacterial infectious pneumonias.
[0023] 4. Simple and efficient loading process, excellent formulation stability: This invention uses an ultrasonic method to load calcium phosphate particles with BMVs. The operation is simple and the cost is low. Moreover, the ultrasonic process is gentle and will not damage the structural integrity of BMVs, thus preserving their complete antigenicity. At the same time, the complex formed by CaP and BMVs has good stability and is not easy to dissociate during storage and application, ensuring the stability of the preventive effect of the formulation.
[0024] 5. High biocompatibility and few side effects: The calcium phosphate particles in this invention have excellent biocompatibility and no obvious immunotoxicity. Furthermore, the BMVs are derived from the target pathogens and have good biocompatibility. The two are combined through physical loading without introducing harmful components such as chemical cross-linking agents, which greatly reduces the immune side effects of the formulation and improves the safety of clinical application.
[0025] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description
[0026] Figure 1 This is a flowchart illustrating the preparation process of PA, KP, or AB-BMVs@CaP in the embodiments of the present invention; Figure 2 For the characterization and identification of BMVs@CaP in the embodiments of the present invention, A is the particle size distribution of PA-BMVs@CaP identified by NTA method, B is the particle size distribution of KP-BMVs@CaP identified by NTA method, C is the particle size distribution of KP-BMVs@CaP identified by NTA method, and D is the protein electrophoresis bands of BMVs and BMVs@CaP obtained by Coomassie brilliant blue staining. Figure 3To illustrate the preventive effect of PA-BMVs@CaP on PA bacterial pneumonia in this embodiment of the invention, A is a statistical chart of lung bacterial load, B is the concentration of lung pro-inflammatory factor TNFα detected by ELISA, and C is the concentration of peripheral blood pro-inflammatory factor TNFα detected by ELISA. Figure 4 To illustrate the preventive effect of KP-BMVs@CaP on KP bacterial pneumonia in this embodiment of the invention, A is a statistical graph of lung bacterial load, B is the concentration of pro-inflammatory factor TNFα in peripheral blood of mice with KP lung infection detected by ELISA, and C is the concentration of pro-inflammatory factor TNFα in lungs of mice with KP lung infection detected by ELISA. Figure 5 To illustrate the preventive effect of AB-BMVs@CaP on AB bacterial pneumonia in this embodiment of the invention, A is a statistical graph of lung bacterial load, B is the concentration of TNFα pro-inflammatory factor in peripheral blood of AB lung-infected mice detected by ELISA, and C is the concentration of TNFα pro-inflammatory factor in lungs of AB lung-infected mice detected by ELISA. Figure 6 In this embodiment of the invention, PA-BMVs@CaP has no preventive effect on KP and AB bacterial pneumonia. In this embodiment, A is the colony count in the lungs of mice with KP lung infection, B is the level of pro-inflammatory factor IL-1β in the lungs of mice with KP lung infection detected by ELISA, C is the colony count in the lungs of mice with AB lung infection, and D is the level of pro-inflammatory factor IL-1β in the lungs of mice with AB lung infection detected by ELISA. Detailed Implementation
[0027] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.
[0028] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.
[0029] Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this invention. Experimental methods in the following embodiments that do not specify specific conditions are generally determined according to national standards. Experimental instruments, equipment, and reagents in the following embodiments that do not specify their sources are all commercially available materials.
[0030] Unless otherwise defined or stated, all technical and scientific terms used in this invention have the same meaning as those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of this invention.
[0031] Pseudomonas aeruginosa PAO1, Klebsiella pneumoniae ATCC 700603, and Acinetobacter baumannii ATCC 19606 were obtained from the Shanghai Key Laboratory for Emergency Prevention and Treatment of Respiratory Infectious Diseases, Ruijin Hospital, affiliated with Shanghai Jiao Tong University. The public can contact us for more information.
[0032] Example 1: Extraction and purification of bacterial membrane vesicles (BMVs) from specific pathogenic bacteria Standard strains of *Pseudomonas aeruginosa* (PA, e.g., PAO1), *Klebsiella pneumoniae* (KP, e.g., ATCC700603), and *Acinetobacter baumannii* (AB, e.g., ATCC 19606) were selected and activated using LB liquid medium. The culture conditions were 37℃ with constant temperature shaking at 220 rpm for 12–18 h, until the bacteria reached the logarithmic growth phase, ensuring good bacterial activity and freedom from contamination. After culture, the bacterial supernatant was collected by centrifugation at 3000 rpm for 10 min at room temperature. The crude BMV extract was purified using gel filtration chromatography. First, the column was equilibrated with sterile PBS until baseline stability was achieved. Then, the crude extract was slowly loaded onto the top of the column, using PBS as the elution buffer at a flow rate of 0.5–1.0 mL / min. The elution peaks were monitored using a UV detector; characteristic elution peak components corresponding to the BMVs were collected. The morphology of the BMVs was observed using transmission electron microscopy (TEM) to ensure typical vesicle structures without obvious impurities. Store frozen at -80℃ for later use.
[0033] Example 2: Preparation of calcium phosphate particles (1) Preparation of aqueous solution: Mix 1.8 mg / mL NaH2PO4 and 2.13 mg / mL Na2HPO4 to prepare 0.03 M phosphate buffer, then add 4 mM sodium silicate to dissolve it as solution 1; take another 6.50 mL 0.05 M CaCl2 solution as solution 2.
[0034] (2) Preparation of oil phase dispersion: Solution 1 and Solution 2 were added dropwise to a mixture of 40 mL ethylene glycol monobutyl ether and 100 mL cyclohexane, and stirred for 5 min to disperse them evenly.
[0035] (3) Reaction and modification: Mix the two oil phase dispersions thoroughly, add 2.25 mL of 1 mM sodium citrate dropwise while stirring continuously, and continue stirring for 10 min to promote the reaction.
[0036] (4) Separation and purification: Centrifuge the reaction solution at 11000×g for 15 min, collect the precipitate, wash it twice with ethanol and twice with PBS, and then resuspend it in PBS to obtain CaP nanoparticles.
[0037] Example 3: Preparation of bacterial membrane vesicles (BMVs@CaP) loaded with calcium phosphate particles In sterile centrifuge tubes, the prepared PA-BMVs, KP-BMVs, and AB-BMVs suspensions were added to calcium phosphate particle suspensions at a BMVs to calcium phosphate particle mass ratio of 1:1. The total volume was then increased to 4 mL with sterile PBS. The mixture was gently inverted to ensure homogeneity. The centrifuge tubes were placed in an sonicator and subjected to low-frequency sonication for loading. The sonication conditions were: 50 W power, 3 s working time, 3 s interval, and a total sonication time of 3 min. The system temperature was maintained ≤10℃ during sonication to avoid damaging the BMVs structure and antigenicity. During sonication, CaP entered the BMVs, forming BMVs@CaP complexes. After ultrasonic treatment, the system was centrifuged at 4℃ and 13000 rpm for 30 min to remove free CaP from the supernatant; the precipitate was collected, washed twice with sterile PBS, and then resuspended in sterile PBS to obtain three specific formulation suspensions: PA-BMVs@CaP, KP-BMVs@CaP, and AB-BMVs@CaP. Figure 1 The particle size distribution of the product was observed using nano-tracer technology (NTA). Figure 2 It can be seen that the particle size of BMVs@CaP from the aforementioned three pathogens is maintained in the nanometer range, which is beneficial for subsequent in vivo applications. Figure 2 (AC); simultaneously, SDS-PAGE electrophoresis was used to verify the protein integrity of BMVs. The protein profile stained with Coomassie Brilliant Blue showed that the process of CaP encapsulation into BMVs did not disrupt the protein composition of the latter. Figure 2 (D).
[0038] Example 4: Application of BMVs@CaP formulation for specific prevention of bacterial pneumonia The three BMVs@CaP formulations prepared in this invention were used to prevent pneumonia caused by corresponding pathogenic bacteria. The preventive effects of the formulations were verified in a mouse model. The specific methods are as follows: Subcutaneous injection was administered to the dorsal skin of C57 / BL6 mice (6 weeks old, male); the single dose was 5 μg of PA-BMVs, CaP, and PA-BMVs@CaP, dissolved in sterile PBS to 100 μL; booster immunizations were performed one week apart, for a total of two immunizations; one week after the treatment, the mice were administered nasally (1×10⁻⁶ g of PA-BMVs@CaP). 6 A bacterial pneumonia mouse model was constructed using one PA (part of a PCR agent). One day after acute infection, mice were sacrificed, and lung colony counts, lung supernatant analysis, and serum inflammatory factor detection were performed. Figure 3 It can be seen that PA-BMVs@CaP pretreatment reduced the bacterial load in the lungs of mice and the level of TNFα pro-inflammatory factor, indicating that PA-BMVs@CaP has a preventive effect on PA-induced lung infection. Figure 3Using the same method, mice were immunized with BMVs@CaP derived from KP or AB to construct KP or AB (1×10⁻⁶). 6 We will develop a KP or AB lung infection model and evaluate its preventive effect.
[0039] like Figure 4 and Figure 5 As shown, KP- or AB-BMVs@CaP can reduce the pulmonary bacterial load and TNFα level induced by KP or AB, respectively, indicating that CaP modification can enhance the preventive effect of KP-BMVs and AB-BMVs against pulmonary infections caused by the corresponding pathogens. However, due to... Figure 6 As shown, PA-BMVs@CaP pretreatment did not reduce the bacterial load of KP and AB in the lungs, nor did it reduce the concentration of pro-inflammatory factors in the lungs, indicating that PA-BMVs@CaP has no significant preventive effect against KP or AB lung infections. Figure 6 This suggests the specificity and targeted preventive effect of BMVs@CaP formulations.
[0040] Therefore, the present invention utilizes the above-mentioned calcium phosphate-loaded bacterial membrane vesicles in the preparation of formulations that enhance the preventive effect of bacterial pneumonia. Calcium phosphate is used as a broad-spectrum immune adjuvant, and its combined immune enhancement system with bacterial membrane vesicles is highly efficient and versatile, with strong targeting specificity, and can accurately prevent pneumonia caused by corresponding pathogenic bacteria, significantly improving the preventive efficacy of the formulation. At the same time, the preparation process is simple and mild, and has high biosafety.
[0041] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.
Claims
1. Use of calcium phosphate-loaded bacterial membrane vesicles for the preparation of a formulation for the prevention of bacterial pneumonia with enhanced efficacy, characterized in that: Bacterial membrane vesicles originate from Pseudomonas aeruginosa, Klebsiella pneumoniae, or Acinetobacter baumannii. The extraction and purification steps of bacterial membrane vesicles (BMVs) are as follows: Standard strains of Pseudomonas aeruginosa, Klebsiella pneumoniae, and Acinetobacter baumannii were selected and activated in LB liquid medium until the bacteria reached the logarithmic growth phase. After the culture was completed, the bacterial supernatant was collected by centrifugation at 3000 rpm for 10 min at room temperature. The crude BMV extract was purified by gel filtration chromatography. The morphology of BMVs was observed by transmission electron microscopy to ensure that they were typical vesicle structures, and they were frozen and stored at -80℃ for later use.
2. The application of the calcium phosphate-loaded bacterial membrane vesicles according to claim 1 in the preparation of formulations that enhance the preventive effect against bacterial pneumonia, characterized in that, The specific procedure for purifying the crude extract of BMVs using a gel filtration chromatography column is as follows: First, the chromatography column is equilibrated with sterile PBS until the baseline is stable. Then, the crude extract is slowly loaded onto the top of the chromatography column. PBS is used as the elution buffer, and the elution flow rate is controlled at 0.5-1.0 mL / min. The elution peaks are monitored using a UV detector. The characteristic elution peak components corresponding to the BMVs are collected.
3. The application of the calcium phosphate-loaded bacterial membrane vesicles according to claim 1 in the preparation of formulations that enhance the preventive effect against bacterial pneumonia, characterized in that, The preparation method of calcium phosphate-loaded bacterial membrane vesicles is as follows: bacterial membrane vesicles (BMVs) and calcium phosphate particles are resuspended, mixed evenly in proportion, and then sonicated. After sonication, the system is centrifuged at 4℃ and 13000rpm for 30min to remove free CaP from the supernatant. The precipitate was collected, washed with sterile PBS, and then resuspended in sterile PBS to obtain three specific formulation suspensions: PA-BMVs@CaP, KP-BMVs@CaP, and AB-BMVs@CaP.
4. The application of the calcium phosphate-loaded bacterial membrane vesicles according to claim 3 in the preparation of formulations that enhance the preventive effect against bacterial pneumonia, characterized in that: The mass ratio of bacterial membrane vesicles (BMVs) to calcium phosphate particles was 1:1; the ultrasonic treatment conditions were: power 50W, working time 3s, interval time 3s, total ultrasonic time 3min, and the system temperature was maintained ≤10℃ during the ultrasonic process.
5. A preparation for enhancing the preventive effect against bacterial pneumonia, characterized in that: The bacterial pneumonia is caused by Pseudomonas aeruginosa, Klebsiella pneumoniae, or Acinetobacter baumannii; the active ingredient of the preparation is the calcium phosphate-loaded bacterial membrane vesicles as described in claim 1, wherein the bacterial membrane vesicles are derived from Pseudomonas aeruginosa, Klebsiella pneumoniae, or Acinetobacter baumannii.
6. The formulation for enhancing the preventive effect against bacterial pneumonia according to claim 5, characterized in that: In the bacterial membrane vesicles loaded with calcium phosphate, the mass ratio of bacterial membrane vesicles to calcium phosphate particles is 1:
1.
7. The use of the formulation according to claim 5 or 6 in the preparation of a vaccine that enhances the preventive effect against bacterial pneumonia.
8. A vaccine that enhances the preventive effect against bacterial pneumonia, characterized in that: The bacterial pneumonia is caused by Pseudomonas aeruginosa, Klebsiella pneumoniae, or Acinetobacter baumannii; the active ingredient of the vaccine is the calcium phosphate-loaded bacterial membrane vesicles as described in claim 1, wherein the bacterial membrane vesicles are derived from Pseudomonas aeruginosa, Klebsiella pneumoniae, or Acinetobacter baumannii; and the mass ratio of the bacterial membrane vesicles to calcium phosphate particles in the calcium phosphate-loaded bacterial membrane vesicles is 1:1.