A bacterial outer membrane mixed vesicle carrier and its preparation method and application

Abstract

The present invention discloses a bacterial outer membrane mixed vesicle carrier. The mixed vesicle is a mixed membrane structure formed by the fusion of two heterotypic bacterial outer membrane vesicles, one of which is a Gram-positive bacterium and the other is a Gram-negative bacterium. The mixed vesicle carrier can mimic the natural internalization pathway of pathogenic bacteria (G+ / G-) into cells, accurately targeting cells that are susceptible to bacterial invasion. It can also recognize the surface of corresponding homologous bacteria, efficiently enriching drugs at the site of bacterial infection, and effectively improving the treatment of mixed bacterial infections.

Description

Technical Field

[0001] The present invention relates to the technical field of antibacterial drug preparation, in particular to a bacterial outer membrane mixed vesicle carrier and a preparation method and application thereof. Background Art

[0002] Bacterial infectious diseases are contagious and involve a variety of diseases within the host, ranging from minor skin infections to life-threatening deep-seated infections such as sepsis, pneumonia, endocarditis, osteomyelitis, and other complications that may metastasize. When the host is unable to completely eliminate the invading pathogens after the initial infection, persistent infection and intermittent recurrences may occur. At the same time, the six superbugs "ESKAPE" can invade cells and cause latent, recurrent infections. As facultative intracellular bacteria, they can survive within phagocytes and evade the killing effects of antimicrobial drugs and clearance by the host immune system, leading to persistent infection.

[0003] Traditional antimicrobial drugs have poor intracellular permeability, low accumulation capacity, reduced or lost biological activity, making it difficult to achieve effective therapeutic concentrations within the cell. High doses are often used in clinical practice, but this not only leads to drug residue problems, but also accelerates the occurrence of drug resistance, further exacerbating the challenges of treating drug-resistant bacteria. Therefore, the development of new, highly effective, and low-resistance-risk antimicrobial drugs and their treatment strategies are of vital importance for the clinical management of intracellular infections. Precise delivery of antimicrobial drugs to specific sites is an effective option to improve the efficacy of antimicrobial drugs, with the advantages of reducing side effects and reducing the selective pressure of antimicrobial drugs. Therefore, in order to rationally use antimicrobial drugs to combat intracellular bacterial infections, drug-targeted modification has the potential to improve antimicrobial efficacy and reduce selective pressure.

[0004] Bacterial outer membrane vesicles (OMVs) are functional vesicles detached from the bacterial cell surface. They are typically spherical and 10-300 nm in diameter. They possess low immunogenicity, excellent cargo capacity, amenability to modification, and a variety of natural biological properties. OMVs from specific strains exhibit homing properties to specific cell types, which can be enhanced through engineering to develop homologously targeted nanoscale diagnostic and targeted drugs. Furthermore, OMVs from specific bacteria can disrupt the integrity of mucosal and epithelial barriers and promote the transfer of various drugs, facilitating systemic translocation across biological barriers. OMVs exhibit remarkable immunomodulatory effects on the host immune system, including both innate and adaptive immune mechanisms, enabling them to possess dual functions of precise targeting and amelioration of inflammatory responses. Furthermore, OMVs can carry, protect, and deliver a variety of small and large molecules, directly fusing with cell membranes and ultimately entering cells via clathrin-, caveolin-, and lipid raft-mediated pathways, or through phagocytosis and macropinocytosis. OMVs can mimic the internalization pathway of pathogens and target cells susceptible to bacterial invasion (such as phagocytes and dendritic cells). Furthermore, by leveraging the surface recognition properties of homologous OMVs with corresponding bacteria, they can further achieve homologous targeting of bacteria, achieving dual targeting of cells and bacteria, and enabling nanoantimicrobial drugs to target bacteria at different intracellular locations. In summary, bacterial membrane vesicles have the potential to serve as antimicrobial drug carriers to enhance the efficacy of antimicrobial drugs against internalized bacteria, potentially opening up a new avenue for combating multidrug-resistant pathogens.

[0005] Currently, research primarily focuses on single vesicles as carriers of antimicrobial drugs. However, clinical bacterial infections often involve mixed infections involving multiple bacterial species, such as skin and soft tissue infections, bacterial pneumonia, and otitis media. The limited targeting of single vesicles in these cases prevents them from achieving therapeutic efficacy, significantly hindering their clinical translation. Therefore, developing multifunctional hybrid membrane nanovesicles with excellent adaptability and flexibility has become crucial for expanding their practical applications. Summary of the Invention

[0006] In response to the shortcomings of the existing technology, the purpose of the present invention is to provide a new carrier formed by the fusion of the membranes of heterotypic bacteria (Gram-positive bacteria G+ and Gram-negative bacteria G-). This outer membrane mixed vesicle carrier can simulate the natural internalization pathway of pathogens (G+ / G-) invading cells, accurately target cells that are easily invaded by bacteria, and at the same time recognize the surface of corresponding homologous bacteria, efficiently enriching drugs at the bacterial infection site, effectively improving the treatment of mixed bacterial infection sites.

[0007] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0008] The first aspect of the present invention is to provide a bacterial outer membrane mixed vesicle carrier, wherein the mixed vesicle is a mixed membrane structure formed by the fusion of two heterotypic bacterial outer membrane vesicles, wherein the two heterotypic bacteria are Gram-positive bacteria and Gram-negative bacteria, respectively.

[0009] In an optional embodiment, the Gram-positive bacteria is Staphylococcus aureus, and the Gram-negative bacteria is Escherichia coli.

[0010] The second aspect of the present invention is to provide a method for preparing a bacterial outer membrane mixed vesicle carrier, comprising the following steps:

[0011] Step S1, extracting Gram-positive bacterial outer membrane vesicles and Gram-negative bacterial outer membrane vesicles from two different types of bacterial liquids respectively;

[0012] Step S2: Gram-positive bacterial outer membrane vesicles and Gram-negative bacterial outer membrane vesicles are dispersed in a buffer solution at a concentration ratio of 1:1, and the mixture is placed in an ultrasonic pretreatment. The pretreated mixture is extruded by a membrane extrusion method, and after extrusion, the bacterial outer membrane mixed vesicles are obtained by centrifugation.

[0013] In an optional embodiment, in step S2, the outer membrane vesicles of Gram-positive bacteria and the outer membrane vesicles of Gram-negative bacteria are dispersed into the buffer solution at a concentration ratio of 1: (0.5-1.5).

[0014] In an optional embodiment, in step S1, extracting bacterial outer membrane vesicles from bacterial liquid includes: step S11, centrifuging different types of bacterial liquid respectively, collecting the supernatant, and filtering the supernatant to remove bacteria and bacterial fragments to obtain a sterile supernatant; step S12, ultracentrifuging the sterile supernatant twice, removing the supernatant, and resuspending the obtained precipitate with PBS buffer to obtain bacterial outer membrane vesicles.

[0015] In an optional embodiment, in the step S11, the rotation speed of the centrifugal treatment is 5000-9000g, and the time is 5-10 minutes; in the step S12, the rotation speed of the ultracentrifugal treatment is 100000-200000g, and the time is 2-8 hours.

[0016] In an optional embodiment, the pretreatment is carried out at a temperature of 15 to 30° C., the power of the ultrasonic wave is 150 to 380 W, and the treatment time is 20 to 45 minutes.

[0017] In an optional embodiment, the membrane extrusion method uses an extrusion membrane of 100-400 nm, and the number of extrusions is 50 times; the centrifugation is carried out at a temperature of 3-6°C, a centrifugal speed of 10,000-15,000 rpm, and a centrifugal time of 25-40 min.

[0018] The third aspect of the present invention is to provide an application of a bacterial outer membrane mixed vesicle carrier in the preparation of a drug for treating bacterial infection.

[0019] In an alternative embodiment, the bacterial infection is a mixed bacterial infection.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] The present invention constructs a new model of heterotypic bacterial (G+ / G-) membrane fusion, which can not only simulate the natural internalization pathway of pathogens invading cells and accurately target cells that are easily invaded by bacteria, but also has a highly specific recognition ability. It can recognize the surface of corresponding homologous bacteria, efficiently enrich the drug at the bacterial infection site, and achieve a dual and precise attack on cells and bacteria, effectively achieving a dual and efficient killing effect on intracellular and extracellular bacteria, and improving the effect of treating mixed bacterial infections. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a transmission electron micrograph of the mixed vesicle HOMV of Example 1 of the present invention.

[0023] Figure 2 This is a diagram showing the particle size of the mixed vesicle HOMV in Example 1 of the present invention.

[0024] Figure 3 This is a confocal visualization image of the mixed vesicle HOMV in Example 1 of the present invention.

[0025] Figure 4 This is an SDS-PAGE image of Staphylococcus aureus T144 OMV, Escherichia coli B2 OMV and mixed vesicle HOMV in Example 1 of the present invention.

[0026] Figure 5 This is a laser confocal visualization image of the mixed vesicle HOMV homologously targeting T144 and B2 in Example 1 of the present invention.

[0027] Figure 6 This is the flow cytometry data of the mixed vesicle HOMV homologous targeting T144 and B2 in Example 1 of the present invention.

[0028] Figure 7 Figure 3 shows the clearance effect of drug carbon dots (CD) and mixed vesicles CD@HOMV carrying drug carbon dots (CD) on intracellular bacteria in HaCaT cells.

[0029] Figure 8 The plate count results of the clearance of intracellular bacteria in HaCaT cells by drug carbon dots (CD) and mixed vesicles CD@HOMV carrying drug carbon dots (CD).

[0030] Figure 9 This is the cytotoxicity test result of the mixed vesicle HOMV in Example 1 of the present invention. DETAILED DESCRIPTION

[0031] In order to help those skilled in the art better understand the present invention, the following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of this application.

[0032] Example 1

[0033] 1. Preparation of Bacterial Outer Membrane Mixed Vesicles (HOMVs)

[0034] (1) Escherichia coli E. coli Preparation of B2 outer membrane vesicles

[0035] Escherichia coli E. coli Streak B2 on LB agar, then place in an incubator and culture overnight at 37°C; pick a single colony and inoculate it into a sterile test tube containing 3 mL of LB medium, set the shaker at 37°C, 220 rpm, and culture for 14-16 hours; transfer the bacterial liquid in the test tube to a conical flask containing liquid LB at a ratio of 1:100 and culture overnight until the bacteria reach the logarithmic growth phase (OD600≈1.2). The cultured bacterial liquid was transferred to a 50 mL centrifuge tube and centrifuged at 6000 g for 20 min at 4°C using an ST8R high-speed refrigerated centrifuge. The supernatant was collected after centrifugation; the supernatant was filtered through a 0.45 μm pore size cellulose acetate filter; a 100 kDa ultrafiltration tube was used for centrifugation at 6000 g for 5 min, and the liquid in the inner tube was collected to a total volume of approximately 25 mL. The collected liquid was filtered through a 0.45 μm pore size cellulose acetate filter; then, an ultraspeed refrigerated centrifuge was used for centrifugation at 150,000 g for 4 h at 4°C. The supernatant was removed, and the precipitate was resuspended in PBS and centrifuged again at 150,000 g for 2 h. The supernatant was discarded, and the OMVs obtained after resuspending the precipitate in PBS were frozen and stored at -80°C.

[0036] (II) Preparation of Staphylococcus aureus MRSA T144 Outer Membrane Vesicles

[0037] Streak Staphylococcus aureus MRSA T144 on LB agar, then incubate at 37°C overnight in an incubator. Pick a single colony and inoculate it into a sterile test tube containing 3 mL of LB medium. Set the shaker at 37°C, 220 rpm, and incubate for 14-16 hours. The bacterial liquid in the test tube was transferred to a conical flask containing liquid LB at a ratio of 1:100 and incubated overnight until the bacteria reached the logarithmic growth phase (OD600≈1.2). The cultured bacterial liquid was transferred to a 50 mL centrifuge tube and centrifuged at 6000 g for 20 min at 4°C using an ST8R high-speed refrigerated centrifuge. The supernatant was collected after centrifugation; the supernatant was filtered through a 0.45 μm pore size cellulose acetate filter; a 100 kDa ultrafiltration tube was used for centrifugation at 6000 g for 5 min, and the liquid in the inner tube was collected to a total volume of approximately 25 mL. The collected liquid was filtered through a 0.45 μm pore size cellulose acetate filter; then, an ultraspeed refrigerated centrifuge was used for centrifugation at 150,000 g for 4 h at 4°C. The supernatant was removed, and the precipitate was resuspended in PBS and centrifuged again at 150,000 g for 2 h. The supernatant was discarded, and the OMVs obtained after resuspending the precipitate in PBS were frozen and stored at -80°C.

[0038] (III) Mixing of bacterial outer membrane vesicles

[0039] Take the Escherichia coli prepared above E. coli B2 outer membrane vesicles and Staphylococcus aureus MRSAT144 outer membrane vesicles were dispersed in PBS at a concentration ratio of 1:1 (500 μg / mL). The resulting mixed solution was then placed in an ultrasonic bath at 20°C for 30 minutes and physically extruded 50 times through a polycarbonate porous membrane (400 nm) using a micro-extruder. The mixture was then centrifuged at 4°C and 12,000 rpm for 30 minutes to obtain bacterial outer membrane mixed vesicles (HOMVs).

[0040] 2. Characterization of Bacterial Outer Membrane Hybrid Vesicles (HOMVs)

[0041] The basic characteristics of HOMV were determined by transmission electron microscopy (TEM), Leica TCS SP8 confocal microscopy (CLSM, DEU), and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The prepared HOMV were dispersed in PBS and the particle size distribution was measured using a Zetasizer Nano ZS. Figure 1 、 Figure 2 、 Figure 3 as well as Figure 4 shown.

[0042] The prepared HOMV was dropped onto a copper mesh, and excess water was absorbed by filter paper. After drying at room temperature, the nanoparticles were observed for morphology using a transmission electron microscope, and the particle size was measured by a laser particle size analyzer after dilution to an appropriate concentration. Figure 1 and Figure 2 As shown, from Figure 1 It can be seen that HOMV has a monodisperse structure and a uniform spherical structure. Figure 2 It can be seen that the particle size of HOMV is about 35 nm, which preliminarily indicates that the HOMV nanostructure is successfully synthesized.

[0043] To further confirm that HOMV is formed by the complete fusion of two single vesicles, MRSA T144 OMV and E. coli B2 OMV in HOMV were stained with DiO (green) and Dil (red), respectively, and the components were observed by laser confocal microscopy (CLSM) co-localization technology. Figure 3 shown.

[0044] from Figure 3 As can be seen in the figure, perfect overlap of DiO-labeled T144 OMV (green) and Dil-labeled B2 OMV (red) was observed in the HOMV, which turned yellow, indicating that the HOMV was formed by the fusion of two single vesicles (T144 OMV and B2 OMV).

[0045] Meanwhile, HOMV (10 μL, 10 mg / mL) was mixed with loading buffer (5 μL), vortexed at 100°C, electrophoresed at 120 V, stained with Coomassie Brilliant Blue, and washed three times. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) confirmed the results. Figure 4 As shown, the characteristic proteins of T144 OMV and B2 OMV were retained in HOMV, indicating that HOMV was successfully synthesized.

[0046] Example 2

[0047] Escherichia coli in this example E. coli The preparation method of B2 outer membrane vesicles and Staphylococcus aureus MRSA T144 outer membrane vesicles and the mixing method of bacterial outer membrane vesicles are the same as those in Example 1, except that the bacterial outer membrane vesicles are mixed with Escherichia coli during the mixing process. E. coli The concentration ratio of B2 outer membrane vesicles and Staphylococcus aureus MRSA T144 outer membrane vesicles, in this example Escherichia coli E. coli The concentration ratio of B2 outer membrane vesicles to Staphylococcus aureus MRSA T144 outer membrane vesicles was 1:0.5.

[0048] Example 3

[0049] Escherichia coli in this example E. coli The preparation method of B2 outer membrane vesicles and Staphylococcus aureus MRSA T144 outer membrane vesicles and the mixing method of bacterial outer membrane vesicles are the same as those in Example 1, except that the bacterial outer membrane vesicles are mixed with Escherichia coli during the mixing process. E. coli The concentration ratio of B2 outer membrane vesicles and Staphylococcus aureus MRSA T144 outer membrane vesicles, in this example Escherichia coli E. coli The concentration ratio of B2 outer membrane vesicles to Staphylococcus aureus MRSA T144 outer membrane vesicles was 1:1.5.

[0050] Example 4

[0051] Escherichia coli in this example E. coli The preparation method of B2 outer membrane vesicles and Staphylococcus aureus MRSA T144 outer membrane vesicles and the mixing method of bacterial outer membrane vesicles are the same as those in Example 1, except that the bacterial outer membrane vesicles are mixed during the mixing process. The specific mixing process is as follows:

[0052] Take the Escherichia coli prepared above E. coli B2 outer membrane vesicles and Staphylococcus aureus MRSAT144 outer membrane vesicles were dispersed in PBS at a concentration ratio of 1:1 (500 μg / mL). The resulting mixed solution was then placed in an ultrasonic bath at 30°C for 20 minutes and physically extruded 50 times through a polycarbonate porous membrane (200 nm) using a micro-extruder. The mixture was then centrifuged at 3°C ​​and 15,000 rpm for 25 minutes to obtain bacterial outer membrane mixed vesicles (HOMVs).

[0053] Example 5

[0054] Escherichia coli in this example E. coli The preparation method of B2 outer membrane vesicles and Staphylococcus aureus MRSA T144 outer membrane vesicles and the mixing method of bacterial outer membrane vesicles are the same as those in Example 1, except that the bacterial outer membrane vesicles are mixed during the mixing process. The specific mixing process is as follows:

[0055] Take the Escherichia coli prepared above E. coli B2 outer membrane vesicles and Staphylococcus aureus MRSAT144 outer membrane vesicles were dispersed in PBS at a concentration ratio of 1:1 (500 μg / mL), and the resulting mixed solution was placed in an ultrasonic bath at 15°C for 45 minutes. The mixture was then physically extruded 50 times through a polycarbonate porous membrane (300 nm) using a micro-extruder. The mixture was then centrifuged at 6°C and 10,000 rpm for 40 minutes to obtain bacterial outer membrane mixed vesicles (HOMVs).

[0056] Test Case

[0057] 1. Verification of Source Targeting Capabilities

[0058] To investigate whether the drug-encapsulated hybrid HOMV vesicles described in Example 1 could be internalized by homologous bacteria, flow cytometry was used to analyze bacterial uptake of both free drug and the hybrid vesicle-encapsulated drug system. Carbon dots (CDs) were used as fluorescent markers and loaded into the hybrid HOMV vesicles. Drug internalization was visualized using confocal laser scanning microscopy (CLSM). The specific steps were as follows:

[0059] Carbon dots (CD) were loaded as fluorescent markers in the mixed vesicles HOMV, and CD with a final concentration of 10 μg / mL and the corresponding concentration of CD@HOMV were mixed with two bacteria (≈10 8 CFU mL -1 ) and incubated at 37°C for 3 h. The cells were washed three times with PBS to remove excess fluorescent marker CD, and the bacteria suspended in PBS were monitored using a flow cytometer (BD FACSAria™). Simultaneously, a 10 µL sample suspension was placed on a glass slide, covered with a coverslip, and imaged on a Zeiss LSM 880 NLO. The results are shown in Figure 2. Figure 5 、 Figure 6 as well as Figure 7 shown.

[0060] from Figure 5 As can be seen in the figure, CD autofluorescence (green) coated with HOMV, compared with CD alone, HOMV-coated CD was efficiently internalized into the interior of both bacteria.

[0061] from Figure 6 As can be seen from the figure, HOMV specifically enhanced the internalization of T144 and B2 into CD, and the mean fluorescence intensity was significantly shifted to the right.

[0062] These phenomena are primarily attributed to the dual homologous targeting capabilities conferred by the system through the encapsulation of extracellular vesicles from two different strains. Studies have demonstrated that the emergence of this novel HOMV form facilitates the fusion of intrinsic biological characteristics and overlapping functional properties of Gram-positive and Gram-negative strains. This hybrid approach significantly enhances internalization and increases the sensitivity of target bacteria to therapeutic agents, thereby achieving broad-spectrum antimicrobial efficacy.

[0063] 2. Verification of intracellular bacterial clearance

[0064] For mixed bacterial infection of skin and soft tissue, human epidermal keratinocytes HaCaT were used as model cell line, Staphylococcus aureus MRSA T144 and Escherichia coli E. coli B2 is a model strain for constructing an intracellular bacteria model to explore whether the mixed vesicle HOMV in Example 1 can carry drugs into the cell through a bionic pathway and kill intracellular bacteria.

[0065] Intracellular bacteria model: MRSA T144 labeled with green fluorescent protein and E. coli B2 was adjusted to a 0.5 McFarland turbidity standard and incubated with HaCaT cells at 37°C for 2 hours to construct an intracellular bacterial model. Bacteria that did not invade the cells were eliminated with 100µg / mL gentamicin. Subsequently, the cells were treated with drug-containing carbon dots (CDs) and HOMVs carrying drug-containing carbon dots (CDs) for 4 hours. HACAT cells were lysed with 0.1% Triton X100, and the number of intracellular bacteria was counted using the plate count method. The results are shown in Figure 2. Figure 7 As shown in the figure, the clearance rates of bacteria T144 and B2 in HaCaT cells by CD@HOMV were reduced by 4.8 and 3.5 orders of magnitude, respectively, while those by CD were only reduced by 2.2 and 2.1 orders of magnitude.

[0066] To further observe the eradication effect of intracellular bacteria, a GFP-carrying plasmid was transferred into the two bacteria for fluorescence tracking. HaCaT cells were plated as a monolayer on a confocal plate and infected and killed with the above-mentioned intracellular bacteria. After removing the supernatant, the cells were fixed, permeabilized, and labeled with Actired 555 ReadyProbes Reagent and DAPI. Images were acquired using a Leica TCS SP8 confocal microscope. The results are shown in Figure 2. Figure 8 As shown, Figure 8 It was shown that although the bacterial count (green fluorescence) in the CD treatment group was reduced, the antibacterial effect was significantly lower than that in the CD@HOMV treatment group, indicating that CD@HOMV has excellent intracellular bacterial clearance ability.

[0067] In summary, it can be proved that HOMV can target and enter the cell by imitating the internalization pathway of homologous bacteria, thereby effectively killing intracellular bacteria.

[0068] 3. Cytotoxicity Verification

[0069] The toxicity of HOMV in Example 1 to the model cell line human epidermal keratinocytes HaCaT was evaluated by CCK8 assay. A series of system solutions were prepared using sterile PBS. RAW 264.7 cells were seeded in a 96-well plate at a cell density of 5×10 4After culturing for 12 hours at 37°C in a 5% CO2 incubator, 100 μL of a series of HOMV concentrations (final concentrations of 0.2, 0.4, 0.8, and 1 mg / mL) were added. A control group (10% FBS DMEM medium + cells) and a blank group (10% FBS DMEM medium) were also set up. All groups were placed in a 37°C, 5% CO2 incubator and incubated for another 24 hours. After that, 100 μL of 10% CCK8 culture medium was added to each well and incubated for 1 hour. The corresponding absorbance was measured at 450 nm using a microplate reader. Cell viability can be calculated using the following formula:

[0070]

[0071] Where As is the absorbance of the experimental group, Ab is the absorbance of the blank group, and Ac is the absorbance of the control group.

[0072] The toxicity of the drug to the cells was evaluated based on the obtained cell viability (CV%). Each group was run six times in parallel and the average value was taken. Figure 9 As shown, the results showed that HOMV had no significant effect on cell viability. When its concentration reached 1 mg / mL, the cell survival rate remained above 90%, which fully demonstrated that HOMV has good cell compatibility.

[0073] The applicant declares that the present invention is illustrated by the above-described embodiments, but the present invention is not limited to the above-described embodiments. This does not mean that the present invention must rely on the above-described embodiments in order to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent replacements for raw materials in the present invention, additions of auxiliary ingredients, and selection of specific methods, etc., fall within the scope of protection and disclosure of the present invention.

[0074] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the scope of protection of the present invention.

[0075] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any appropriate manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. A bacterial outer membrane mixed vesicle carrier, characterized in that: The mixed vesicles are a mixed membrane structure obtained by pre-treating a mixture of Gram-positive bacterial outer membrane vesicles and Gram-negative bacterial outer membrane vesicles and then extruding them through a membrane extrusion method; Wherein, in the mixed solution, the concentration ratio of Gram-positive bacterial outer membrane vesicles to Gram-negative bacterial outer membrane vesicles is 1:(0.5-1.5); The Gram-positive bacteria is Staphylococcus aureus, and the Gram-negative bacteria is Escherichia coli.

2. The method for preparing the bacterial outer membrane mixed vesicle carrier according to claim 1, characterized in that: The following steps are involved: Step S1, extracting Gram-positive bacterial outer membrane vesicles and Gram-negative bacterial outer membrane vesicles from two different types of bacterial liquids respectively; Step S2: dispersing the outer membrane vesicles of Gram-positive bacteria and the outer membrane vesicles of Gram-negative bacteria into a buffer solution, and then placing the mixed solution in ultrasound for pretreatment. The pretreated mixed solution is extruded by a membrane extrusion method, and then centrifuged to obtain bacterial outer membrane mixed vesicles.

3. The method for preparing the bacterial outer membrane mixed vesicle carrier according to claim 2, characterized in that: In step S1, extracting bacterial outer membrane vesicles from bacterial liquid includes: step S11, centrifuging different types of bacterial liquid respectively, collecting the supernatant, filtering the supernatant to remove bacteria and bacterial fragments, and obtaining a sterile supernatant; step S12, subjecting the sterile supernatant to two ultracentrifugations, removing the supernatant, and resuspending the obtained precipitate in PBS buffer to obtain bacterial outer membrane vesicles.

4. The method for preparing the bacterial outer membrane mixed vesicle carrier according to claim 3, characterized in that: In the step S11, the rotation speed of the centrifugal treatment is 5000-9000 g, and the time is 5-10 min; in the step S12, the rotation speed of the ultracentrifugal treatment is 100000-200000 g, and the time is 2-8 h.

5. The method for preparing the bacterial outer membrane mixed vesicle carrier according to claim 2, characterized in that: In the step S2, the pretreatment is performed at a temperature of 15 to 30°C, the power of the ultrasonic wave is 150 to 380 W, and the treatment time is 20 to 45 minutes.

6. The method for preparing the bacterial outer membrane mixed vesicle carrier according to claim 2, characterized in that: The membrane extrusion method uses an extrusion membrane of 100-400 nm, and the number of extrusions is 50 times; the centrifugation is carried out at a temperature of 3-6° C., a centrifugal speed of 10,000-15,000 rpm, and a centrifugal time of 25-40 minutes.

7. Use of the bacterial outer membrane mixed vesicle carrier according to claim 1 in preparing a drug for treating mixed bacterial infection.

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