Lactic acid bacteria bionic extracellular vesicle as well as preparation method and application thereof

Abstract

The invention discloses a lactic acid bacteria bionic extracellular vesicle as well as a preparation method and application thereof, and belongs to the technical field of medical materials. According to the preparation method of the lactic acid bacteria bionic extracellular vesicles disclosed by the invention, lactic acid bacteria cells are efficiently broken by utilizing a high-pressure homogenization technology, and the lactic acid bacteria bionic extracellular vesicles are separated and purified by adopting differential and ultraspeed centrifugation. The preparation method disclosed by the invention is simple and controllable in process, and large-scale amplification and standardized production are easy to realize; the obtained bionic extracellular vesicles of lactic acid bacteria are higher in yield and uniform in particle size than natural extracellular vesicles, have similar safety to the natural extracellular vesicles of lactic acid bacteria, and can promote immune maturation of dendritic cells and drug delivery carrier activity. The compound can be used as a vaccine adjuvant, an immunopotentiator and a vaccine and nano-drug delivery carrier to be applied to the field of biological medicine.

Description

A biomimetic extracellular vesicle of lactic acid bacteria, its preparation method and application Technical Field

[0001] This invention relates to the field of pharmaceutical materials technology, and more specifically to a biomimetic extracellular vesicle of lactic acid bacteria, its preparation method, and its application. Background Technology

[0002] Bacterial extracellular vesicles (BEVs) are vesicles naturally secreted by bacteria, possessing a phospholipid bilayer structure and a diameter between 50 and 200 nanometers. BEVs carry bioactive substances such as proteins, nucleic acids, and lipids from the parent bacteria and play crucial roles in intercellular communication, antigen presentation, and immune regulation. However, the production of BEVs currently faces significant challenges. First, their natural secretion levels are extremely low, resulting in limited yields and high extraction costs, making it difficult to meet the demands of basic research and clinical applications for large quantities of homogeneous vesicles. Second, existing BEV extraction methods are cumbersome, time-consuming, and difficult to scale up. Furthermore, the biological functions of BEVs, such as immune adjuvant activity or tissue targeting, may suffer from insufficient intensity or uncontrollable limitations, restricting their therapeutic efficacy and application scope.

[0003] To overcome the bottleneck of low BEV yield, several methods have been developed in existing technologies, such as altering the optimal growth conditions of the bacteria or using treatments with glycine, lysozyme, or antibiotics (e.g., ampicillin) to weaken the bacterial cell wall and promote vesicle release. However, these chemical or biological methods may introduce exogenous substances, posing potential residual risks and potentially affecting the natural composition and function of BEVs, while also increasing production costs. Furthermore, the process stability and reproducibility of these methods need improvement, limiting their application in large-scale industrial production.

[0004] Therefore, the technology of preparing bacterial biomimetic extracellular vesicles (BBVs) based on bacterial membranes has attracted attention in recent years. This type of technology breaks down cells through physical means (such as ultrasound and compression), reorganizing the cell membrane into nanoscale vesicle structures. Compared to BEVs, BBVs can retain the membrane components and functions of the parent cell to a greater extent and are expected to be endowed with new properties through engineering. BBVs inherit the core advantages of BEVs and show broad prospects in drug delivery and vaccine development. Similar to BEVs, the clinical application of BBVs also faces challenges such as potential biotoxicity and component heterogeneity. The potential biotoxicity of BBVs, like that of BEVs, is attributed to lipid A of the lipopolysaccharide (LPS) on the surface of Gram-negative bacterial vesicles or virulence factors such as bacterial adhesins, proteases, and cytotoxins carried by pathogenic bacteria.

[0005] Extracellular vesicles of probiotics are biochemically active structures responsible for triggering the biological effects of probiotics. Lactic acid bacteria (LAB), abundant in animals and humans (e.g., the gut), are recognized as probiotics, possessing anti-inflammatory and antibacterial properties and commonly used in food, dietary supplements, and basic research. Increasing evidence suggests that LAB-derived extracellular vesicles have a stronger immunomodulatory capacity than the probiotics themselves. The immunomodulatory effects of LAB extracellular vesicles are related to the ability of different immune cells in the gut (such as intestinal epithelial cells (IECs), monocytes and macrophages, lymphocytes, and dendritic cells (DCs)) to interact. Studies have shown that some LAB extracellular vesicles can induce immune cell proliferation and mimic polarized Th1 immune responses by stimulating the production of the Th1 cytokine IFN-γ, without affecting the production of the Th2 cytokine IL-4 in mouse immune cells. Therefore, LAB-derived BEVs exhibit good biocompatibility and safety, showing great application potential in areas such as vaccine adjuvants and drug delivery vectors. However, as Gram-positive bacteria, natural BEVs of lactic acid bacteria also have limitations, such as low yield, which makes it difficult to meet clinical needs.

[0006] Therefore, developing a biomimetic extracellular vesicle of lactic acid bacteria with a simple process, high yield, easy standardization and scale-up production, and the ability to enhance or retain its immune activity, as well as its preparation method and application, has important theoretical significance and practical application value. Summary of the Invention

[0007] In view of this, the present invention provides a biomimetic extracellular vesicle of lactic acid bacteria, its preparation method, and its application. The present invention utilizes high-pressure homogenization technology to efficiently disrupt lactic acid bacteria cells, and employs differential and ultracentrifugation to separate and purify the biomimetic extracellular vesicle of lactic acid bacteria. This provides a safe adjuvant and immune activator for vaccine development, and a safe carrier for vaccine platforms and drug delivery systems.

[0008] Lactic acid bacterial extracellular vesicles (BEVs, all BEVs in the following text refer specifically to lactic acid bacterial BEVs): Non-replicating vesicle particles with a phospholipid bilayer naturally secreted by lactic acid bacteria.

[0009] Lactic acid bacterial biomimetic extracellular vesicles (BBVs, hereinafter, all BBVs refer specifically to lactic acid bacteria BBVs): These are non-replicable vesicle particles with a phospholipid bilayer, artificially prepared using lactic acid bacteria cell membranes as the basic raw material through physical, chemical, or bioengineering techniques.

[0010] This invention provides a method for preparing biomimetic extracellular vesicles of lactic acid bacteria. The optimal high-pressure homogenization conditions for disrupting *Lactococcus lactis* cells were studied and screened. The biomimetic extracellular vesicles of *Lactococcus lactis* were then purified by differential and ultracentrifugation, resulting in a 260-fold increase in yield compared to natural extracellular vesicles. The use of high-pressure homogenization technology to prepare biomimetic extracellular vesicles of lactic acid bacteria overcomes the bottlenecks of low yield and large batch-to-batch variation of natural bacterial extracellular vesicles, significantly improving the efficiency of bacterial vesicle acquisition and reducing production costs.

[0011] To achieve the above objectives, the present invention adopts the following technical solution:

[0012] A method for preparing biomimetic extracellular vesicles of lactic acid bacteria includes the following steps: 1) Cultivating lactic acid bacteria to the late exponential growth stage under aseptic conditions to obtain bacterial suspension; 2) Collecting the bacterial suspension, centrifuging the bacterial suspension and collecting the precipitate, washing the bacterial precipitate twice with phosphate buffered saline (PBS) at pH 7.4, and diluting the bacterial cells with PBS to an OD600 value of 0.5-1.5 to obtain bacterial suspension 1; 3) Filtering bacterial suspension 1 through an 80-mesh sieve to obtain bacterial suspension 2, and then homogenizing bacterial suspension 2 using a high-pressure homogenizer to obtain a homogenized liquid; 4) Collecting the homogenized liquid, centrifuging the homogenized liquid at 2000g for 30min to remove impurities, collecting supernatant 1, and then centrifuging it at 20000g for 60min to remove impurities, collecting supernatant 2; 5) Calcium chloride solution for supernatant 2. 6) The concentrated liquid after ultrafiltration is centrifuged at 150000g for 120min to obtain biomimetic extracellular vesicles of lactic acid bacteria.

[0013] Furthermore, the lactic acid bacteria include *Bifidobacterium*, *Lactobacillus*, *Lactococcus*, *C. casei*, *Lactobacillus*, *Streptococcus*, *Pediococcus*, *Leuconostoc*, and *Lactobacillus mucinus*. Specifically, it is *Lactococcus lactis*.

[0014] Further, in step 2), the centrifugation is performed at 5000-10000g for 20-60 minutes.

[0015] Furthermore, the high-pressure homogenization conditions in step 3) are 60-120 MPa, and the number of cycles is 5-30.

[0016] Further, in step 5), the ultrafiltration concentration is 100-5000g and centrifuged for 10-100min.

[0017] Furthermore, the method described yields biomimetic extracellular vesicles of lactic acid bacteria.

[0018] Furthermore, the application of the described lactic acid bacteria biomimetic extracellular vesicles in the preparation of vaccine adjuvants, immune activators, and vaccine delivery carriers.

[0019] Furthermore, the application of the described lactic acid bacteria biomimetic extracellular vesicles in the preparation of drug delivery carriers.

[0020] As can be seen from the above technical solution, compared with the prior art, this invention discloses a lactic acid bacteria biomimetic extracellular vesicle, its preparation method, and its application. High-pressure homogenization technology is used to break lactic acid bacteria cells, and the optimal pressure, number of cycles, and bacterial concentration are selected. The prepared lactic acid bacteria biomimetic extracellular vesicles yield 260 times that of natural extracellular vesicles. During the preparation process, centrifugation removes macromolecular residual membrane impurities, and ultrafiltration removes residual small molecule impurities (DNA, protein), avoiding adverse effects of residual impurities on the application of biomimetic extracellular vesicles. Simultaneously, the process is simple, suitable for industrial scale-up and standardized production; the vesicle yield is high, and the scale-up and purification effects are good, making it suitable for the large-scale preparation of nanoscale vesicles. Lactic acid bacteria BBVs exhibit a tendency to stimulate DC maturation, macrophage M1 polarization, and targeted enrichment of solid tumors, and can be directly used as carriers for immune enhancers, vaccines, and nanomedicines. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0022] Figure 1 shows the yield of biomimetic extracellular vesicles (BBVs) and natural extracellular vesicles (BEVs) per gram of wet Lactococcus lactis.

[0023] Figure 2 shows the characteristics of Lactococcus lactis BBVs and BEVs; A: Transmission electron micrograph of BBVs (scale bar 200 nm); B: Transmission electron micrograph of BEVs (scale bar 200 nm); C: Particle size range and dispersion coefficient of BBVs under dynamic light scattering; D: Particle size range and dispersion coefficient of BEVs under dynamic light scattering.

[0024] Figure 3 is a schematic diagram of the safety evaluation of *Lactococcus lactis* BBVs. A: Hemolytic activity test results; B: Endotoxin content test results; C: Animal cell viability MTT assay results; D: Flow cytometry scatter plot of animal cell apoptosis level detection; E: Animal cell apoptosis level detection plot. DDP-30: 30 µg / ml cisplatin.

[0025] Figure 4 shows the detection of significant upregulation of surface molecules CD40 (A), CD86 (B), MHC-II (C) and secreted cytokines IL-1β (D), IL-6 (E), and TNF-α (F) by Lactococcus lactis BBVs and BEVs.

[0026] Figure 5 shows the detection of the expression of different OVA antigens carried by Lactococcus lactis on the surface of DCs and the expression of Th1 antigen peptide complexes. A: Loaded with OVA protein antigen; B: Loaded with OVA DNA antigen; C: Loaded with complex membrane antigen fused with B16-OVA containing OVA.

[0027] Figure 6 shows the evaluation of the ability of Lactococcus lactis BBVs as a drug carrier; where A: encapsulation efficiency and drug loading rate of Lactococcus lactis BBVs carrying DOX at different mass ratios; B: release rate of DOX carried by Lactococcus lactis BBVs in different pH environments.

[0028] Figure 7 shows the in vitro apoptosis and activity analysis of mouse breast cancer 4T1 cells by Lactococcus lactis BBVs encapsulated with DOX (BBV-DOX). In the figure, A: scatter plot of apoptosis by flow cytometry; B: bar chart of apoptosis; C: cell activity results by MTT assay.

[0029] Figure 8 shows the in vivo anti-B16-OVA melanoma results of Lactococcus lactis BBVs encapsulated with DOX (BBV-DOX). A: Tumor growth trend; B: Tumor weight in mice at the end of the experiment; C: Mouse body weight change trend during the experiment; D: Serum alanine aminotransferase (ALT) activity detection; E: Serum aspartate aminotransferase (AST) activity detection; F: Serum blood urea nitrogen (BUN) content; G: Serum creatinine (CRE) content; H: Serum creatine kinase (CK) content; I: Serum lactate dehydrogenase (LDH) content.

[0030] Figure 9 shows HE-stained sections of the heart, liver, and kidney of mice after intravenous treatment with DOX-encapsulated Lactococcus lactis BBVs (BBV-DOX). Detailed Implementation

[0031] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0032] Example 1 Lactococcus lactis subsp. Cremoris (MG1363) BBV has the ability to stimulate DC maturation and cross-present protein antigens. 1.1 Preparation and physical characterization of Lactococcus lactis BBVs A method for preparing biomimetic extracellular vesicles of Lactococcus lactis includes the following steps: 1) Culturing Lactococcus lactis strain MG1363 under aseptic conditions to the late exponential growth stage (approximately 20 h) to obtain bacterial suspension; 2) Collecting the bacterial suspension, centrifuging the bacterial suspension at 8000 g for 30 min, collecting the precipitate, washing the bacterial precipitate twice with PBS at pH=7.4, and diluting the bacterial cells with PBS to an OD600 value of approximately 1.0 to obtain bacterial suspension 1; 3) culturing bacterial suspension 1 at 80°C... After filtration through a mesh screen, bacterial suspension 2 was obtained. Bacterial suspension 2 was then homogenized using a high-pressure homogenizer to obtain a lysate. The homogenization conditions were 100 MPa and 20 cycles. 4) The lysate was collected and centrifuged at 2000 g for 30 min to remove impurities. The supernatant 1 was collected and then centrifuged at 20000 g for 60 min to remove impurities again. The supernatant 2 was collected. 5) Supernatant 2 was concentrated by ultrafiltration at 5000 g for 30 min using a 100 kDa ultrafiltration flask. 6) The concentrated liquid after ultrafiltration was centrifuged at 150000 g for 120 min to obtain BBVs precipitate. The BBVs precipitate was collected into a centrifuge tube and washed and resuspended with 1 mL of PBS buffer. It was then centrifuged again at 150000 g for 120 min to ensure the purity of the biomimetic extracellular vesicles of lactic acid bacteria. Finally, the precipitate was resuspended in PBS, quantified using a BCA protein concentration assay kit (Beyotime: P0012), and stored at -80℃ to ensure long-term preservation of the biomimetic extracellular vesicles of lactic acid bacteria.

[0033] As shown in Figure 1, the method of this invention can yield approximately 7727.18 μg of BBVs per gram of wet bacterial cells; this is 260 times the yield of 29.48 μg of BEVs per gram of wet bacterial cells in Lactococcus lactis culture medium that has been statically cultured at 30°C for 48 hours.

[0034] A small amount of BBVs were taken and subjected to dynamic light scattering (DLS) and transmission electron microscopy (TEM) to observe the particle size and vesicle characteristics. DLS and TEM showed that BBVs have similar lipid bilayer vesicle characteristics and particle size to BEVs (see Figure 2).

[0035] 1.2 Safety evaluation of Lactococcus lactis BBVs 1.2.1 Hemolysis test (1) Whole blood was obtained from healthy C57 female mice through the ocular sinus vein, collected in anticoagulant tubes containing EDTA, shaken evenly, and centrifuged at 3000 rpm for 20 min to collect blood cells.

[0036] (2) Prepare the test solution. Take 1 ml of ultrapure water, PBS solution and BBVs (1, 10, 50 and 100 μg / ml) into a 1.5 ml conical centrifuge tube. Add 20 μl of blood cells to each tube and incubate all centrifuge tubes at 37°C for 4 h.

[0037] (3) After incubation, all centrifuge tubes were centrifuged at 3000 rpm for 20 min. Then, all samples were gently removed, placed on the same horizontal line, and their hemolysis phenomenon was photographed.

[0038] (4) After taking the photos, aspirate 100 μl of supernatant from each centrifuge tube into the microplate. Use a microplate reader to measure the absorbance of each well at OD542 and calculate the hemolysis rate. The formula for calculating the hemolysis rate is as follows: Hemolysis rate = (OD542 + 0.0542) / ... 样本 -OD PBS ) / (OD 超纯水 -OD PBS ) x 100%.

[0039] 1.2.2 Endotoxin Content Detection: The endotoxin content in BBV was detected using the endotoxin detection kit (Limulus Amebocyte Lysate (LIL) colorimetric method) (product number: C0273S) from Shanghai Beyotime Biotechnology Co., Ltd. Specific procedures were performed according to the kit instructions. The negative control was endotoxin-free ultrapure water (Endotoxin-free H2O, EF H2O) provided in the kit.

[0040] 1.2.3 BBV activity assay in animal cells BBV activity assay in animal cells was performed using the MTT Cell Proliferation and Cytotoxicity Assay Kit (product code: M1020) from Beijing Solarbio Science & Technology Co., Ltd.

[0041] (1) After the cells were passaged to the third generation, they were digested, centrifuged and collected, and counted. 4T1 cells (5000 cells / well) were seeded into 96-well plates. 100 μl of cell suspension was added to each well of the 96-well plate. The wells around the perimeter of the plate were filled with sterile PBS. After gently shaking, the plates were incubated in a 37°C incubator.

[0042] (2) After 1 day of culture, examine the cell status and density under a microscope. Add 100 μL of 30 μg / ml cisplatin (DDP), DMEM cell culture medium (Con), and 1, 10, 50 or 100 μg / ml BBVs to each well. Set up 5 replicates for each gradient. After gently shaking, incubate in a 37 ℃ incubator.

[0043] (3) After 8 h of drug treatment, the well plate was removed and the cell state was observed under a microscope. After centrifuging the well plate at 1200 rpm for 7 min, the liquid in the plate was gently shaken off, and 100 μl of MTT solution (0.5 mg / mL) was added to each well under dark conditions and incubated in a cell culture incubator for 4 h.

[0044] (4) After incubation, remove the plate, centrifuge at 1200 rpm for 7 min and then remove the liquid from the plate. Add 150 μL DMSO to each well, place on a shaker and shake for 15 min. After the crystals are fully dissolved, use an ELISA reader to measure the absorbance of each well at OD490.

[0045] (5) Calculate cell viability and half-maximal inhibitory concentration. The cell viability calculation formula is as follows: Cell viability (%) = (OD value of experimental group - OD value of blank group) / (OD value of control group - OD value of blank group) x 100%.

[0046] The control group consisted of untreated cell solutions grown in culture medium; the blank group consisted of pure culture medium solutions containing no cells.

[0047] 1.2.4 Detection of BBV apoptosis activity in animal cells The apoptosis activity of BBV in animal cells was detected using the Annexin V-FITC / PI apoptosis detection kit (product code: 40302ES60) from Yisheng Biotechnology (Shanghai) Co., Ltd.

[0048] (1) Cell plating: Cells passaged to the third generation were digested, centrifuged, collected, and counted at 5 × 10⁻⁶ cells per cell line. 5 At a cell density of 4 T1 cells per well, three replicates were set up for each treatment group. After gently shaking, the cells were incubated in a 37 ℃ incubator.

[0049] (2) Drug treatment: Discard the culture medium from the cell well plate after 1 day of culture, and add 30 μg / ml cisplatin (DDP), DMEM cell culture medium, 1, 10, 50 or 100 μg / ml BBVs respectively, and culture for 5 h.

[0050] (3) After the culture is completed, the culture medium is aspirated and washed 3 times with PBS; trypsin without EDTA is added to digest the cells and collect the cells. Centrifuge at 1200 rpm for 7 min, discard the supernatant, and wash the collected cells 3 times with PBS.

[0051] (4) Resuspend the cells in 1× Binding Buffer, mix Annexin V and PI to prepare (PI: Annexin=2:1), and incubate at room temperature in the dark for 15-20 min.

[0052] (5) Add 300 µl of 1× Binding Buffer to each group, mix well, place on ice, and pass through a copper mesh into a clean flow cytometer.

[0053] (6) Flow cytometry detection.

[0054] Conclusions: As shown in Figure 3A (hemolysis assay), BBVs concentrations below 100 μg / ml did not cause significant lysis of blood cells in mouse venous anticoagulated blood. Figure 3B (endotoxin content assay) showed that the endotoxin content in a 1 μg / ml BBV solution was below 0.5 EU / μg. Figure 3C (cell proliferation activity assay) showed that BBVs concentrations below 100 μg / ml did not induce significant 4T1 cell death. Figures 3D-E (apoptosis assay) showed that BBVs concentrations below 100 μg / ml did not induce significant apoptosis in 4T1 cells. These results indicate that the endotoxin content in the BBV solution is low; BBVs concentrations below 100 μg / ml neither cause hemolysis nor affect animal cell activity through apoptosis, demonstrating high safety.

[0055] 1.3 The immunostimulatory activity of BBVs was evaluated using 1×10⁻⁶ BBVs. 6Mouse bone marrow-derived dendritic cells (GM-DCs, hereinafter referred to as DCs) induced by granulocyte-macrophage colony-stimulating factor (GM-CSF) were co-incubated with different concentrations of BBVs (0.1 μg / mL, 0.5 μg / mL, and 1.0 μg / mL) for 24 h. The levels of DC surface molecules before and after treatment were detected by flow cytometry using Elabscience's DC surface molecular molecules: anti-CD40-FITC (E-AB-F1028C), anti-CD86-APC (E-AB-F0994E), and anti-MHC II-APC (E-AB-F0990E). The levels were also measured using Elabscience's cytokine assay kits: Mouse IL-1β (Interleukin 1 Beta) ELISA Kit (E-EL-M0037), Mouse TNF-α (Tumor Necrosis Factor Alpha) ELISA Kit (E-EL-M3063), and Mouse IL-6 (Interleukin 1 Beta) ELISA Kit. 6) The ELISA Kit (E-EL-M0044) was used to detect changes in the concentration of inflammatory cytokines secreted by dendritic cells (DCs) in the culture medium, evaluating the effect of *Lactococcus lactis* BBVs on DC maturation. Figure 4A-C shows that the flow cytometry results indicated that, compared with the negative control group treated with PBS, the positive control group (LPS) significantly increased the expression levels of all biomarkers, validating the effectiveness of the experimental system. In different concentrations of BBVs and BEVs, 0.1 µg / ml, 0.5 µg / ml, and 1.0 µg / ml BBVs and 0.5 µg / ml and 1.0 µg / ml BEVs significantly promoted the upregulation of CD40, CD86, and MHC-II molecules (p<0.001). Furthermore, at concentrations of 0.1 µg / ml and 0.5 µg / ml, BBVs significantly promoted the expression of CD40, CD86, and MHC-II compared to BEVs (p<0.001). Figure 4D-F shows the ELISA results: Compared with the PBS control group, BEVs at different concentrations (0.5 µg / ml and 1 µg / ml) significantly promoted the release of IL-6, IL-1β, and TNF-α (p<0.001), indicating that BEVs at concentrations above 0.5 µg / ml significantly promoted DC maturation in a dose-dependent manner. BBVs at concentrations above 0.1 µg / ml also significantly promoted DC maturation in a dose-dependent manner, and at the same dose, their activation of DCs was significantly higher than that of BEVs. This indicates that both BBVs and BEVs can significantly promote DC maturation in a dose-dependent manner and function as DC immune activators, making them suitable for use as adjuvants in vaccines.

[0056] 1.4 Evaluation of the antigen delivery vector function of BBVs GM-CSF-induced dendritic cells (DCs) were collected and treated with OVA protein (Solepro, catalog number: A8041), a mixture of 60 ng / ml LPS (lipopolysaccharide, Beyotime, catalog number: S1732) and OVA protein, BBV-OVA solution, and 8 μg / ml BBVs and PBS solution, respectively (1×10⁻⁶ DCs). 6 Cells were loaded with OVA at a concentration of 2 μg / mL in each experimental group. Cells were collected after 24 hours and treated with OVA. 257-264 The cross-presentation effect of BBVs on protein antigens using a mouse fluorescently labeled APC antibody (Invitrogen, catalog number: 17-5743-82) with the epitope peptide (SIINFEKL) was evaluated by flow cytometry. Figure 5A shows that loading DCs with OVA protein carried by BBVs significantly increased the content of the MHC-I-SIINFEKL complex on the surface of DCs compared with loading DCs with OVA alone.

[0057] Evaluation of BBVs as a DNA vaccine delivery vector: A DNA vesicle vaccine (BBV-DNA) was prepared by cyclically extruding 1 μg of pcDNA3-OVA plasmid (the CDs sequence of GenBank: V00383.1 inserted between EcoR1 and XhoI in pcDNA3) and BBVs at a mass ratio of 1:50 using a liposome extruder (Genizer, USA) with a 200 nm filter membrane 20 times. 40 ng LPS and 10 μg OVA were also included. 257-264 Peptide Mixture Solution (LPS+OVA) P A mixture of 50 μg BBVs, 1 μg pcDNA3-OVA plasmid, and 2 μl Lipofectamine® 3000 (Ivitrogen, catalog number: L3000001) (lip-DNA) was added to 1 × 10⁻⁶ ppm of BBVs. 6 Cells were collected after co-incubation with DCs at a density of 1 cell / mL for 24 hours and then treated with OVA. 257-264 The cross-presentation effect of BBVs on protein antigens was evaluated by flow cytometry using fluorescently labeled APC antibodies against epitope peptides (SIINFEKL) in mice.

[0058] Evaluation of BBVs as cell membrane vaccine delivery vectors: 40 ng LPS and 10 μg OVA were administered separately. 257-264 Peptide Mixture Solution (LPS+OVA) P40 μg of B16-OVA tumor cells were sonicated in an ice-water bath for 5 minutes (2 seconds on / off, 60 W power), then centrifuged at 1000g for 10 min at 4°C to obtain cancer cell membrane vesicles (CMV) in the supernatant. An equal mass of BBVs was then cyclically extruded 20 times through a liposome extruder using a 200 nm filter membrane to prepare a fusion membrane vesicle vaccine (CBV). 20 μg of BBVs and 20 μg of CMV were then mixed with 1×10⁻⁶ ions of BBVs. 6 Cells were collected after co-incubation with DCs at a density of 1 cell / mL for 24 hours and then treated with OVA. 257-264 The cross-presentation effect of BBVs on protein antigens was evaluated by flow cytometry using fluorescently labeled APC antibodies against epitope peptides (SIINFEKL) in mice.

[0059] DC loading results showed that BBVs carrying DNA antigen (BBV-DNA) (Figure 5B) significantly increased the expression of MHC-I and OVA cell immune antigen peptides on the DC surface. 257-264 The efficacy of the content; although there was no significant difference in antigen presentation levels between fusion membrane vesicle vaccines (CBVs) and cancer cell membrane vesicles, the combined effect of the aforementioned BBVs on dendritic cell (DC) maturation activity can prevent the risk of immune tolerance caused by unadjuvanted antigen loading on DCs alone (Figure 5C). These results indicate that BBVs have the potential to promote cellular immunity after loading protein, DNA, and membrane antigens onto DCs.

[0060] Example 2 Evaluation of the efficacy of BBVs as a chemotherapy drug delivery carrier (1) Detection of drug loading rate and release level of BBV-DOX DOX (Shanghai Yuanye Biotechnology Co., Ltd.: S17092) and BBVs were gently mixed in PBS at different mass ratios and then incubated overnight at 37°C. BBV-DOX was concentrated using a 100 kDa ultrafiltration tube (Solepro, Cat: YA1854) to remove free DOX. After dissolving the BBV-DOX sample in PBS containing 1% Triton X-100 for 10 minutes, the absorbance was measured at 484 nm wavelength at 37°C to determine the amount of encapsulated DOX. As shown in Figure 6A: In terms of drug loading rate, the mass ratio of BBV to DOX reached a maximum of approximately 20% at 1:2. The in vitro release of BBV-DOX was evaluated by dialysis. Five mL of 0.3 mg / mL BEV-DOX solution was placed in a sealed MWCO 1000 Da dialysis bag and then in PBS solution at pH 6.5 (simulating the tumor microenvironment) or pH 7.4 (simulating the blood environment), and shaken at 37 °C for 72 h. Dialysate was collected at 0, 3, 6, 9, 12, 24, and 48 h, and each sample was analyzed using a microplate reader. Figure 6B shows that BBV-DOX was released faster in an acidic environment than in a neutral blood environment, indicating that BBVs have the potential to release drugs in a slightly acidic tumor microenvironment.

[0061] (2) Effect of BBV-DOX on tumor cell viability in vitro In order to evaluate the effects of BBVs and BBV-DOX on cell viability, apoptosis and activity analysis of mouse breast cancer 4T1 cells were performed by Annexin V-FITC / PI staining and MTT assay, respectively.

[0062] Apoptosis level detection (Yisheng Biotechnology, product code: 40302ES60): (1) Cell plating: After passage to the third generation, cells were digested, centrifuged and collected, and counted at 5×10⁻⁶. 5 At a cell density of 4 T1 cells per well, three replicates were set up for each treatment group. After gently shaking, the cells were incubated in a 37 ℃ incubator.

[0063] (2) Drug treatment: Filter the BBVs or BBV-DOX stock solution through a sterile filter and dilute it to 5 μg / ml with DMEM medium. Prepare fresh before use. Discard the medium from cell plates cultured for 1 day, add 5 μg / mL of the diluted drug solution, and incubate for 5 h.

[0064] (3) After the culture is completed, the culture medium is aspirated and washed 3 times with PBS; trypsin without EDTA is added to digest the cells and collect the cells. Centrifuge at 1200 rpm for 7 min, discard the supernatant, and wash the collected cells 3 times with PBS.

[0065] (4) Resuspend the cells in 1× Binding Buffer, mix Annexin V and PI to prepare (PI: Annexin=2:1), and incubate at room temperature in the dark for 15-20 min.

[0066] (5) Add 300 μl of 1× Binding Buffer to each group, mix well, place on ice, and pass through a copper mesh into a clean flow cytometer.

[0067] (6) Flow cytometry detection.

[0068] MTT cell viability assay (Solepro, product code: M1020): (1) After the cells were passaged to the third generation, they were digested, centrifuged and collected, and counted. 4T1 cells (5000 cells / well) were seeded into a 96-well plate. 100 μl of cell suspension was added to each well of the 96-well plate. The wells around the edges of the plate were filled with sterile PBS. After gently shaking, the plate was placed in a 37 ℃ incubator for culture.

[0069] (2) After 1 day of culture, examine the cell state and density under a microscope. Pass the stock solution of the drug through a sterile filter and dilute it with culture medium to the required concentration. After discarding the culture medium, add 100 μL of the diluted drug solution of different concentrations to each well, with 5 replicates for each gradient. After gently shaking, incubate in a 37 ℃ incubator.

[0070] (3) After 8 h of drug treatment, the well plate was removed and the cell state was observed under a microscope. After centrifuging the well plate at 1200 rpm for 7 min, the liquid in the plate was gently shaken off, and 100 μl of MTT solution (0.5 mg / mL) was added to each well under dark conditions and incubated in a cell culture incubator for 4 h.

[0071] (4) After incubation, remove the plate, centrifuge at 1200 rpm for 7 min and then remove the liquid from the plate. Add 150 μL DMSO to each well, place on a shaker and shake for 15 min. After the crystals are fully dissolved, use an ELISA reader to measure the absorbance of each well at OD490.

[0072] (5) Calculate cell viability and half-maximal inhibitory concentration. The cell viability calculation formula is as follows: Cell viability (%) = (OD value of experimental group - OD value of blank group) / (OD value of control group - OD value of blank group) x 100%.

[0073] The control group consisted of untreated cell solutions grown in culture medium; the blank group consisted of pure culture medium solutions containing no cells.

[0074] Results: Apoptosis assays (Figures 7A-B) showed that BBV-DOX containing 3 or 5 μg / ml DOX induced significantly higher apoptosis rates in 4T1 cells (39.8% / 55.1%) than DOX at the same concentration (35.8% / 49.7%, p<0.05 / p<0.001). Figure 7C showed that DOX containing 1.0 μg / ml or higher significantly affected cell viability, but BBV-carried DOX had a more significant effect on inducing 4T1 cell death than free DOX. This confirms that BBV-DOX exhibits a significant advantage in promoting apoptosis compared to DOX alone.

[0075] (3) Effect of intravenous BBV-DOX on in vivo antitumor efficacy: To evaluate the effects of BBVs and DOX-carrying agents on tumor activity in vivo, a subcutaneous transplantation tumor model was established using B16 cells to study the inhibitory effects of BBV, DOX, and BBV-DOX delivered by the system on tumor cell growth. When the volume of the subcutaneous transplanted tumor reached approximately 100 mm... 3 In this study, tumor-bearing mice were randomly divided into four treatment groups: Control group (PBS), BBV group (10 mg / kg), DOX group (2 mg / kg), and BBV-DOX group (BBV : DOX = 10 mg / kg : 2 mg / kg). Mice were treated every three days for a total of four treatments. Figures 8A-B show that the BBV-DOX group exhibited a significant anti-tumor effect in mice. Changes in tumor volume and weight indicate that the BBV-DOX group effectively inhibited tumor growth. During the 16-day experiment, compared with the untreated control, both the DOX group and the BBV-DOX group significantly slowed tumor growth and had significantly lighter tumor weights. Compared with the effect of DOX alone, the BBV carrier significantly improved the therapeutic effect of DOX. Figure 8C shows that in the experiment, none of the experimental groups had a significant effect on the weight of mice at the existing doses, indicating that BBV has good biocompatibility and did not cause serious side effects during drug delivery, maintaining low toxicity. Figure 8D-I shows that, compared with the increase in liver alanine aminotransferase (ALT, D), aspartate aminotransferase (AST, E) and kidney creatinine (CRE, G) levels caused by DOX, BBVs did not produce significant changes in liver and kidney biochemical indicators, and could reduce the effect of DOX on the increase of liver and kidney biochemical indicators to a certain extent.

[0076] (4) Effects of BBV-DOX on the heart, liver, and kidneys after intravenous injection: Histopathological results (Figure 9) were similar to serum biochemical results. In the BBV and Con groups, only mild inflammatory cell infiltration was observed in the heart, liver, and kidney tissues. However, DOX caused severe inflammatory damage and fibrosis in the heart, liver, and kidney tissues, as well as persistent damage and necrosis of epithelial cells in some renal tubules. BBV-DOX treatment could alleviate the inflammatory damage and fibrosis caused by DOX in the heart, liver, and kidney tissues.

[0077] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing biomimetic extracellular vesicles of lactic acid bacteria, characterized in that, Includes the following steps: 1) Under aseptic conditions, lactic acid bacteria were cultured to the late exponential growth stage to obtain bacterial suspension; 2) The bacterial suspension was collected, centrifuged and the precipitate was collected. The precipitate was washed twice with phosphate buffer solution at pH 7.4, and the bacterial cells were diluted with PBS to an OD600 value of 0.5-1.5 to obtain bacterial suspension 1; 3) Bacterial suspension 1 was filtered through an 80-mesh sieve to obtain bacterial suspension 2. Bacterial suspension 2 was then homogenized using a high-pressure homogenizer to obtain the homogenized liquid; 4) Collect the lysate. After centrifuging the lysate at 2000g for 30 minutes, collect the supernatant 1. Then, after centrifuging at 20000g for 60 minutes, collect the supernatant 2. 5) Concentrate the supernatant 2 by ultrafiltration in a 100kDa 250ml flat-bottom centrifuge bottle. 6) The concentrated liquid after ultrafiltration is centrifuged at 150,000g for 120 minutes to obtain biomimetic extracellular vesicles of lactic acid bacteria.

2. The method for preparing biomimetic extracellular vesicles of lactic acid bacteria according to claim 1, characterized in that, The lactic acid bacteria mentioned are Lactococcus lactis.

3. The method for preparing biomimetic extracellular vesicles of lactic acid bacteria according to claim 1, characterized in that, Step 2) The centrifugation is performed at 5000-10000g for 20-60 minutes.

4. The method for preparing biomimetic extracellular vesicles of lactic acid bacteria according to claim 1, characterized in that, Step 3) The high-pressure homogenization conditions are 60-120 MPa, and the number of cycles is 5-30.

5. The method for preparing biomimetic extracellular vesicles of lactic acid bacteria according to claim 1, characterized in that, Step 5) The ultrafiltration concentration is 100-5000g centrifuged for 10-100min.

6. The biomimetic extracellular vesicles of lactic acid bacteria prepared by the method according to any one of claims 1-5.

7. The use of the lactic acid bacteria biomimetic extracellular vesicles as described in claim 6 in the preparation of vaccine adjuvants, immune activators, and vaccine delivery carriers.

8. The application of the lactic acid bacteria biomimetic extracellular vesicles according to claim 6 in the preparation of drug delivery carriers.

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