Method for increasing yield of extracellular vesicles of lactococcus lactis and application of method

Abstract

The invention discloses a method for increasing the yield of extracellular vesicles of lactococcus lactis and application of the method, and belongs to the technical field of biology. The lysozyme and the ampicillin are used for weakening a lactococcus lactis peptidoglycan layer, so that the yield of the natural bacterial extracellular vesicles of the lactococcus lactis is increased. The technological process is simple, and is suitable for industrial amplification and standardized production; the vesicle yield is high, the amplification effect and the purification effect are good, and the method can be used for large-scale preparation of vesicles. The lactococcus lactis extracellular vesicles have the adjuvant efficacy of stimulating the maturation of dendritic cells and the tropism of targeting solid tumor enrichment, and can be used as an immunopotentiator or a sending carrier of vaccines and nano-drugs.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically to a method for increasing the production of extracellular vesicles of Lactococcus lactis and its application. Background Technology

[0002] Lactococcus lactis is a food-grade bacterium that has been used in food fermentation and preservation for centuries. Its genome is fully sequenced, its genetic background is well-defined, its metabolic pathways are relatively simple, and it has no known virulence genes or plasmid-mediated drug resistance. Furthermore, no harmful metabolites such as hemolysin or biogenic amines have been detected. It is listed as a recognized safe bacterium by the U.S. Food and Drug Administration (FDA), the European Food Safety Authority (EFSA), and the Codex Alimentarius Commission (CAC), deemed safe for animals, consumers, and the environment, and permitted for use in food fermentation and probiotic products. This makes Lactococcus lactis an ideal choice for producing plasmid DNA (pDNA) or recombinant proteins, and it is considered a promising tool for the efficient production of antigens and many other bioactive compounds, widely used in industrial and pharmaceutical applications. In recent years, Lactococcus lactis has been used as a vector for in situ vaccines or immunotherapies due to its safety and ability to express heterologous proteins. However, there are public concerns that direct use of live bacteria may increase the risk of death in immunocompromised and critically ill patients.

[0003] Bacterial membrane vesicles (BMVs) or bacterial extracellular vesicles (BEVs) are tiny vesicle structures secreted by bacterial cells, typically ranging from 20 to 300 nanometers in diameter. BEVs are cup-shaped structures formed by bacteria in response to environmental stress, genetic alterations, antibiotic exposure, etc., and they play important roles in bacterial-to-bacterial, host-to-bacterial, and bacterial physiological processes. Due to their inherent tumor tropism, immunostimulatory properties, and potential for functionalization with therapeutic proteins, they have become promising smart drug delivery systems (SDDS) and universal vaccine platforms. Compared to other drug delivery systems or vaccine platforms, one advantage of BEVs is their ability to load more than one active molecule and maintain these molecules in blood circulation for a longer period, thereby improving bioavailability. Secondly, the rich microbial-associated molecular patterns and nanoscale membrane vesicle structure of BEVs give them high immunogenicity, enabling them to activate innate and adaptive immune responses and recruit more dendritic cells (DCs) and CD8+. +T cells migrate to tumor tissue, improving immune memory in treated mice. Third, the lipid spherical size (50-200 nm) allows BEVs to passively target and deliver to the solid tumor microenvironment through enhanced permeability and retention (EPR), and can also be actively targeted to tumor cells through various modifications. Furthermore, this nanoscale size facilitates entry into lymph nodes and phagocytosis by antigen-presenting cells (APCs), thereby promoting adaptive immune responses. Fourth, unlike live bacteria, BEVs cannot replicate. Genetic engineering techniques can be used to load exogenous proteins into the vesicle cavity or express them on the membrane surface, enabling BEVs to establish controlled immune responses with greater safety. Finally, BEVs are simple to prepare, have low production costs, are easy to quality control, and do not face the ethical barriers associated with the preparation of animal extracellular vesicles. Currently, the clinical application of BEVs mainly faces challenges such as potential biotoxicity and low natural yield. The potential biotoxicity of BEVs is mainly attributed to lipid A on the surface of Gram-negative bacterial vesicles (LPS) or virulence factors such as bacterial adhesins, proteases, and cytotoxins carried by pathogenic bacteria.

[0004] Therefore, using the recognized safe Lactococcus lactis as a source of bacterial extracellular vesicles can eliminate the risk of potential biotoxicity. However, Lactococcus lactis is a Gram-positive bacterium, and its cell wall, composed of a thick peptidoglycan layer, acts as a strong physical barrier, inhibiting the formation and release of extracellular vesicles.

[0005] Therefore, providing a method for increasing the production of extracellular vesicles of Lactococcus lactis and its application is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0006] In view of this, the present invention provides a method for increasing the production of extracellular vesicles of Lactococcus lactis and its application. By weakening the integrity of the Lactococcus lactis cell wall, the production of extracellular vesicles of Lactococcus lactis is significantly increased, providing a safe carrier for drug delivery systems and universal vaccine platforms.

[0007] Bacterial extracellular vesicles (BEVs, all BEVs in the following text refer specifically to Lactococcus lactis BEVs): Non-replicating nanovesicle particles with a phospholipid bilayer naturally secreted by Lactococcus lactis.

[0008] This invention provides a method for increasing the yield of natural BEVs of Lactococcus lactis. The study found that by using ampicillin and lysozyme to influence the cell wall growth of Lactococcus lactis and by selecting the optimal culture temperature and duration, the yield of natural bacterial extracellular vesicles of Lactococcus lactis was increased by more than ten times.

[0009] To achieve the above objectives, the present invention adopts the following technical solution:

[0010] A method for increasing the production of extracellular vesicles of Lactococcus lactis, comprising the following steps:

[0011] 1) After the lactococcus lactis is revived and activated, it is inoculated into M17 liquid medium containing 5% glucose after being centrifuged by ultracentrifugation, and 250-4000 μg / ml lysozyme is added at the same time. It is then placed in a 30℃ incubator for static culture.

[0012] 2) When the OD600 value reaches 0.8-0.1, add 125-2000 μg / ml ampicillin and continue to incubate at 37℃ for 12-72 h.

[0013] 3) Collect the bacterial culture, centrifuge the bacterial culture and collect the supernatant. Filter the supernatant through a 0.2-0.5μm filter to remove bacteria;

[0014] 4) The filtrate is concentrated by ultrafiltration;

[0015] 5) The precipitate obtained by ultrafiltration concentration followed by ultracentrifugation is bacterial extracellular vesicles.

[0016] Preferably, the lysozyme in step 1) is administered at doses of 250 μg / ml, 500 μg / ml, 1000 μg / ml, 2000 μg / ml, and 4000 μg / ml; more preferably, the optimal dose is 1000-2000 μg / ml, specifically including 1000 μg / ml and 2000 μg / ml.

[0017] Preferably, the ampicillin (AMP) in step 2) is administered at doses of 125 μg / ml, 250 μg / ml, 500 μg / ml, 1000 μg / ml, and 2000 μg / ml; more preferably, the optimal dose is 125-250 μg / ml, specifically including 125 μg / ml and 250 μg / ml.

[0018] Preferably, the culture time in step 2) specifically includes 12h, 24h, 36h, 48h, 60h, and 72h; more preferably, the optimal culture time is 60-72h, specifically including 60h and 72h.

[0019] Furthermore, the ultracentrifugation in step 1) is centrifugation at 150,000 rpm for 2 hours.

[0020] Furthermore, in step 3), the centrifugation is performed at 100-10000g for 10-60 minutes.

[0021] Further, the supernatant from step 3) is filtered and sterilized using a 0.22 μm filter.

[0022] Further, in step 4), the ultrafiltration concentration is achieved by centrifuging at 100-5000g for 10-100min; the ultrafiltration tube is 100kDa.

[0023] Furthermore, in step 5), the ultracentrifugation is performed at 10,000-150,000g for 60-600 minutes.

[0024] Furthermore, the method described prepares extracellular vesicles of Lactococcus lactis.

[0025] Furthermore, the application of the aforementioned Lactococcus lactis extracellular vesicles in the preparation of vaccine vectors.

[0026] Furthermore, the application of the aforementioned Lactococcus lactis extracellular vesicles in the preparation of tumor tissue-targeted delivery carriers for antitumor drugs.

[0027] Furthermore, the application of the aforementioned Lactococcus lactis extracellular vesicles in the preparation of immune enhancers.

[0028] As can be seen from the above technical solution, compared with the prior art, this invention discloses a method for increasing the yield of extracellular vesicles (BEVs) of *Lactococcus lactis* and its application. By using lysozyme and ampicillin to weaken the *Lactococcus lactis* peptidoglycan layer, increasing the growth temperature, and prolonging the growth time of *Lactococcus lactis*, the yield of natural *Lactococcus lactis* BEVs is increased by approximately 13 times. During the preparation process, ultrafiltration removes residual lysozyme and ampicillin, avoiding residual irritants and their adverse effects on membrane vesicle application. Simultaneously, the process is simple, suitable for industrial scale-up and standardized production; the vesicle yield is high, with good scale-up and purification effects, and can be used for large-scale vesicle preparation. *Lactococcus lactis* BEVs exhibit a tendency to stimulate dendritic cell maturation and target solid tumor enrichment, and can be directly used as carriers for immune enhancers, vaccines, and nanomedicine delivery. Attached Figure Description

[0029] 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.

[0030] Figure 1 Flowchart for the preparation and purification of BEVs;

[0031] Figure 2 Yield of BEVs per 100 ml of culture medium before and after optimization (Std: standard operation, Opt: optimized operation);

[0032] Figure 3 Characterization of BEVs; Left: Transmission electron microscope image of BEVs (scale bar 20 nm); Right: Particle size range and dispersion coefficient of BEVs under dynamic light scattering;

[0033] Figure 4 Characterization of BEVs promoting DC maturation: Specific surface molecules were significantly higher than those in immature DCs;

[0034] Figure 5 Characteristic 2 of BEVs promoting DC maturation: The secretion of pro-inflammatory cytokines was significantly higher than that of immature DCs;

[0035] Figure 6 Characteristic 3 for promoting DC maturation with BEVs: Migration ability is significantly higher than immature DCs; Left: Number of migrating DCs after BEV treatment; Right: CD11c of DCs after BEV treatment. + CCR7 + The expression status was as follows: Compared with the untreated group, **P<0.01, ***P<0.001; compared with each treatment group, ###P<0.001.

[0036] Figure 7 Characterization of BEVs promoting DC maturation: 4. Phagocytic capacity was significantly lower than that of immature DCs; Left: Effect of BEVs treatment on DC phagocytic capacity; Right: Flow cytometry histogram of BEVs treatment on DC phagocytic capacity; Compared with the untreated control group, *P<0.05, **P<0.01, ***P<0.001.

[0037] Figure 8 Characterization of BEVs-Promoted DC Maturation (Part 5): Mature DCs promote T cell proliferation; DCs treated with BEVs inhibit CD4+. + T (left) and CD8 + Effect on T (right) cells; compared with untreated, *P<0.05, ***P<0.001; compared with each treatment group, ##P<0.01, ###P<0.001

[0038] Figure 9 Encapsulation efficiency and drug loading rate of BEVs and DOX at different mass ratios; Left: Encapsulation efficiency of BEV-DOX; Right: Drug loading rate of BEV-DOX;

[0039] Figure 10 Physical characterization of BEV-DOX and drug release at different pH values; A: TEM image of BEV-DOX (scale bar 200 nm); B: DLS analysis image of BEV-DOX; C: Drug release of BEV-DOX at different pH values.

[0040] Figure 11 Effects of BEV, the chemotherapy drug doxorubicin (DOX), and BEV carrying doxorubicin (BEV-DOX) on the viability of B16 tumor cells; A: Confocal cell viability assay results under Control, BEV, DOX, and BEV-DOX treatments; B: Flow cytometry apoptosis rate in different treatment groups; Compared with the untreated group, ***P<0.001; Comparison between different treatment groups, ###P<0.001;

[0041] Figure 12 Pharmacokinetic diagrams of DOX and BEV-DOX; A: DOX fluorescence in blood at different time points; B: Statistical graphs of average fluorescence of DOX and BEV-DOX at different time points; Parallel comparisons, ***P<0.001; Conclusion: BEV significantly prolongs the retention time of DOX-carrying cells in serum;

[0042] Figure 13 In vivo distribution experiments of DiR and DiR-BEV: A: Fluorescence expression at different time points detected using the FOBI small animal imaging system; B: Statistical graph of average fluorescence intensity at tumor sites at different time points; C: Fluorescence map of organ tissues in mice after 24 hours; D: Statistical graph of average fluorescence intensity of different organs in mice after 24 hours; Parallel comparison of DiR and DiR-BEV, ***P<0.001; Conclusion: BEV has the ability to target and enrich tumor tissues.

[0043] Figure 14 The following is an in vivo antitumor experiment of BEV-DOX: A: Schematic diagram of the in vivo antitumor experiment; B: Tumor volume change in mice during the experiment; C: Body weight change in mice; D: Schematic diagram of mouse tumor tissue at 18 days; E: Tumor weight in mice; Compared with the untreated group, *P<0.05, **P<0.01, ***P<0.001; Compared with different treatment groups, ##P<0.01, ###P<0.001;

[0044] Figure 15 Serum biochemical markers of BEV-DOX were detected (n=5); A: Liver and kidney damage markers under different treatments; B: Cardiotoxicity markers under different treatments; C: Serum IL-4 level detection; D: Serum IFN-γ level detection; Compared with the untreated group, *P<0.05, **P<0.01, ***P<0.001; Compared with different treatment groups, #P<0.05, ##P<0.01, ###P<0.001; The results showed that BEV could attenuate the damage of DOX to the liver, kidneys and heart of mice, and could induce humoral and cellular immune responses in vivo. Detailed Implementation

[0045] 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.

[0046] Example 1: A method to increase the yield of extracellular vesicles of *Lactococcus lactis* by weakening the cell wall, increasing the growth temperature, and prolonging the growth time.

[0047] A method for separating and purifying bacterial extracellular vesicles by weakening the peptidoglycan layer of *Lactococcus lactis*. Figure 1 The process includes the following steps:

[0048] 1) Resuscitate Lactococcus lactis (L. lactis subsp. cremoris MG1363) at -80℃ and streak onto Elliker agar plates (Shanghai Kanglang Biotechnology Co., Ltd.), then incubate at 30℃ for 36 hours;

[0049] 2) Single colonies were picked from Elliker agar plates and inoculated into 10 mL of M17 liquid medium (GM17, Shanghai Kanglang Biotechnology Co., Ltd.) containing 5% glucose (all M17 liquid medium used in this study were centrifuged at 150,000 rpm for 2 h before sterilization to remove possible vesicle contamination), and incubated at 30°C for 24 h.

[0050] 3) After culturing for 24 hours, inoculate the bacterial culture into 1L of GM17 medium, and add 2000μg / mL lysozyme (Sangon Biotech (Shanghai) Co., Ltd.: A610308, Potency≥20000U / mg). Incubate at 37℃ until OD reaches 100000U / mg. 600 Value = 1.0.

[0051] 4) When OD 600 When the value reaches 1.0, add 250 μg / mL ampicillin and continue to incubate at 37°C.

[0052] 5) After adding ampicillin (Sangon Biotech (Shanghai) Co., Ltd.: A610028), continue culturing for 48 hours. Collect the bacterial culture, centrifuge at 5000g for 30 minutes, and collect the supernatant to remove most bacteria and impurities. This step needs to be repeated 3 times until there is no precipitate in the supernatant after centrifugation. This step is crucial for the subsequent extraction of membrane vesicles, ensuring the minimization of impurities in the extract.

[0053] 6) Pre-treat the supernatant after centrifugation through a 0.22 μm filter to remove larger particles. Add the filtered liquid to an ultrafiltration tube (100 kDa membrane) and concentrate by ultrafiltration at 5000 g for 15 minutes. This step helps concentrate membrane vesicles and reduce other unwanted components.

[0054] 7) Filter the concentrated liquid again through a 0.22 μm filter and transfer the filtrate to an ultracentrifuge tube. Centrifuge at 150,000 g for 2 hours. After centrifugation, collect the membrane vesicle precipitate into the centrifuge tube, wash with 1 mL of PBS buffer, and resuspend.

[0055] 8) Centrifuge again at 150,000g for 2 hours to ensure the purity of the membrane vesicles. Finally, resuspend the precipitate in PBS, filter through a 0.22μm filter, quantify using a BCA protein concentration assay kit (Beyotime: P0012), and store at -80℃ to ensure long-term preservation of the membrane vesicles.

[0056] Results: The method of this invention (Opt) yielded BEVs with a 13-fold increase in yield compared to Lactococcus lactis cultured at 30℃ for 60 h (Std) (see...). Figure 2 Transmission electron microscopy revealed typical vesicle characteristics and particle size (see...). Figure 3 ).

[0057] Example 2: Evaluation of the efficacy of BEVs as vaccine carriers and adjuvants

[0058] 2.1 Granulocyte-macrophage colony-stimulating factor (GM-CSF)-induced culture of mouse bone marrow cell-derived dendritic cells (GM-DC, hereinafter referred to as DC)

[0059] (1) Remove the tibia and femur of 6-8 week old C57BL / 6 mice, remove the muscle tissue, soak them in medical disinfectant alcohol for 2 minutes, and then wash them 3 times with PBS solution.

[0060] (2) Use RPMI-1640 complete medium containing double antibiotics (Seville Bio: G4531PS-500ML) and 10% inactivated fetal bovine serum (Gibco original, Beyotime: C0230) to flush out bone marrow cells with a syringe and blow them evenly with a syringe to disperse the cells in the bone marrow.

[0061] (3) Collect bone marrow cells, centrifuge at 1200 rpm for 7 min, discard the supernatant; resuspend the cells in complete culture medium containing 20 ng / mL GM-CSF (Beyotime: P6006), and concentrate to a final concentration of 1×10⁻⁶. 6Cells were transferred at a concentration of 1 cell / mL to 60 mm cell culture dishes and cultured in 6 mL culture solutions at 37 °C.

[0062] (4) On day 2, replace half of the RPMI-1640 complete medium containing 10% serum. On day 3, replace the entire medium. On days 5 and 6, replace half of the RPMI-1640 complete medium containing 10% serum each time. On day 7, collect the Fixed Viability Dyee Fluor. TM 780 negative (eBioscience) TM :65-0865-14), anti-CD11c-PE (Elabscience: E-AB-F0991D) positive cells are GM-CSF-induced DCs from bone marrow.

[0063] 2.2 Flow cytometry detection of surface molecules on DCs treated with BEVs

[0064] DCs induced and cultured to day 7 were collected and diluted with cryopreserved BEVs to concentrations of 1 μg / mL, 5 μg / mL, and 10 μg / mL, respectively, and then used to treat DCs (1×10⁻⁶) 6 DCs (1×10⁻⁶ cells / mL) treated with 40 ng / mL LPS (lipopolysaccharide, Beyotime: S1732) 6 DCs treated with PBS (number of cells / mL) served as the positive control group, while DCs treated with PBS served as the negative control group. Cells were collected after 24 hours and incubated with the appropriate flow cytometry antibody, as follows:

[0065] (1) Centrifuge each group of DC solutions at 1200 rpm for 7 min, collect the supernatant for subsequent cytokine detection, add 1 mL of PBS to each DC precipitate and wash, then centrifuge at 1200 rpm for 7 min. Repeat the washing step 3 times.

[0066] (2) Add the following Elabscience DC surface molecular flow cytometry antibodies to the cell pellet: anti-CD40-FITC (E-AB-F1028C), anti-CD86-APC (E-AB-F0994E), anti-CD80-APC (E-AB-F0992E), anti-MHC I-FITC (AN00429C), and anti-MHC II-APC (E-AB-F0990E), and incubate at room temperature in the dark.

[0067] (3) After 25 min, add 1 mL of PBS, mix well, centrifuge at 1200 rpm for 7 min, and wash away the unbound antibody.

[0068] (4) The cell pellet was resuspended in 200 μL PBS and filtered through a 200-mesh copper grid into a flow cytometer.

[0069] (5) Finally, flow cytometer (BD FACSCalibur) was used to collect molecular information on the surface of DCs, and the information was analyzed using FlowJo software.

[0070] Results: Compared with the negative control group treated with PBS, the positive control LPS group significantly increased the expression levels of all biomarkers, validating the effectiveness of the experimental system. In different concentrations of BEVs, both 5 μg / ml and 10 μg / ml of BEVs significantly promoted the expression of CD40, CD80, CD86, MHC-I, and MHC-II (p<0.001), indicating that BEVs at concentrations above 5 μg / ml significantly promoted DC maturation in a dose-dependent manner, with no significant difference between the two concentrations (see [link to relevant documentation]). Figure 4 ).

[0071] 2.3 ELISA detection of DC pro-inflammatory factor secretion after BEVs treatment

[0072] Cytokine levels were determined according to the instructions of the Elabscience 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), Mouse IL-6 (Interleukin 6) ELISA Kit (E-EL-M0044), and Mouse IL-10 (Interleukin 10) ELISA Kit (E-EL-M0046). Unless otherwise specified, all reagents used were from the respective assay kits.

[0073] (1) Each test reagent should be placed at room temperature for 30 minutes.

[0074] (2) Dilute the sample to be tested to an appropriate concentration using the sample diluent in the kit.

[0075] (3) Sample addition: Add 100 μL of the corresponding cytokine standard dilution solution provided in different kits and the DCs (1×10⁻⁶) collected in section 2.2 treated with different concentrations of BEVs, 40 ng / mL LPS, and PBS to each well of the ELISA plate. 6 After the culture medium supernatant was collected (samples / mL), each sample was repeated in 3 replicates and incubated at 37°C for 90 min.

[0076] (4) Biotinylated antibody incubation: Discard the liquid in the ELISA plate, add 100 μL of biotinylated antibody diluted 100 times to each well, and incubate at 37°C for 60 min.

[0077] (5) Washing: Shake off the liquid in the ELISA plate and pat dry. Add 350 μL of washing solution, soak and shake for 1 min, discard the liquid, pat dry the ELISA plate, and repeat the washing 3 times.

[0078] (6) Incubation with enzyme-conjugated working solution: Add 100 μL of enzyme-conjugated working solution to each well and incubate at 37°C for 30 min.

[0079] (7) Washing: Shake off the liquid in the ELISA plate and pat dry. Wash 5 times according to the washing steps in (6).

[0080] (8) Color development: Add 90 μL of color development solution to each well and react at room temperature for 5-15 min. Terminate when gradient color development occurs.

[0081] (9) Termination: Add 50 μL of stop solution to each well to terminate the reaction, shake well, and measure OD using a microplate reader. 450 Absorbance value.

[0082] (10) Calculation of cytokine levels: The OD of the sample was calculated. 450 Substitute the absorbance value into the standard curve prepared by the cytokine standard dilution solution in the kit, and multiply the resulting value by the dilution factor to obtain the cytokine content.

[0083] Results: Compared with the PBS control group, the positive control LPS group significantly increased cytokine secretion, validating the effectiveness of the experimental system. In different concentrations of BEVs treatment groups, both 5 μg / ml and 10 μg / ml concentrations of BEVs significantly promoted the release of IL-6, IL-1β, and TNF-α (p<0.001), while having less effect on IL-10 secretion; indicating that BEVs at concentrations above 5 μg / ml can significantly promote DC maturation in a dose-dependent manner (see...). Figure 5 ).

[0084] 2.4 BEVs stimulated an increase in the migration ability of dendritic cells.

[0085] Transwell BD Matrigel (FN) was used to detect the in vitro migration of dendritic cells (DCs).

[0086] (1) DCs were treated with 1 μg / mL and 10 μg / mL BEVs for 24 h, respectively. The positive control group was treated with 40 ng / mL LPS, and the negative control group was treated with PBS.

[0087] (2) 2×10 5DCs (cells resuspended in 0.5 mL of RPMI-1640 complete medium) were added to the upper chamber of the transwell, and 0.5 mL of complete RPMI-1640 medium containing 100 ng / mL CCR19 was added to the lower chamber of the transwell.

[0088] (3) After incubation for 3 hours, cells were collected from the lower chamber and subjected to anti-CD11c-PE (Elabscience: E-AB-F0991D) and anti-CCR7-APC (eBioscience). TM (17-1971-81) After treatment, the cells were counted by flow cytometry.

[0089] Results: Compared with the control group, the positive control LPS group significantly increased DC migration and CCR7 expression, validating the effectiveness of the experimental system. In different BEV treatment groups, both 1 μg / ml and 10 μg / ml BEVs significantly promoted DC migration (p<0.001) and CD11C expression. + CCR7 + The expression of [the substance] indicates that BEVs at concentrations above 1 μg / ml can significantly promote DC maturation in a dose-dependent manner (see [reference]). Figure 6 ).

[0090] 2.5 BEVs stimulated a decrease in DC antigen phagocytosis.

[0091] The effect of membrane vesicles on DC maturation was evaluated based on the characteristic that DCs have reduced phagocytic capacity after maturation.

[0092] (1) After all DC cells were blown off on day 7 of induction culture, they were centrifuged, resuspended, and counted. Then, they were seeded into 24-well plates, with 1×10⁶ cells per well. 6 Cells were then treated with 1 μg / mL and 10 μg / mL BEVs, respectively (1 × 10⁻⁶ cells). 6 The positive control group was treated with LPS (40 ng / mL), and the negative control group was treated with PBS. All samples were incubated at 37°C for 24 hours.

[0093] (2) Phagocytosis and detection. After DC treatment for 24 h, 50 μg / mL FITC-Dextran was added and cultured for another 1 h.

[0094] (3) Collect cells, wash them three times with PBS, and then pass them through a 200-mesh copper grid into a flow cytometer tube for flow cytometry detection of FITC fluorescence.

[0095] Results: Compared with the control group, the positive control LPS group significantly reduced DC phagocytosis.

[0096] The efficacy of FITC-Dextran validated the effectiveness of the experimental system. In different BEV treatment groups, 10 μg / ml of BEV significantly promoted a decrease in DC phagocytic capacity (p<0.05); indicating that BEV concentrations above 10 μg / ml can significantly promote DC maturation (see...). Figure 7 ).

[0097] 2.6 The immune-enhancing effect of BEVs—Mature DCs can stimulate T cell proliferation

[0098] Mixed lymphatic reaction (MLR)

[0099] (1) DCs collected on day 7 were treated with 1 μg / mL and 10 μg / mL BEVs for 24 h. The positive control group was LPS (40 ng / mL) and the negative control group was treated with PBS.

[0100] (2) The spleen of BALB / c mice was isolated and ground into a cell suspension on a 70μm sterile copper grid. The spleen cells were collected, treated with red blood cell lysis buffer for 1 min, terminated with 3 mL PBS, and centrifuged at 1200 rpm for 7 min to obtain spleen cells.

[0101] (3) Resuspend spleen cells in PBS, stain with 1 μM CFSE for 10 min, add cold PBS to stop staining, centrifuge at 1200 rpm for 7 min, wash twice with PBS, and resuspend in complete culture medium.

[0102] (4) Splenic cells (1×10⁻⁶) 6 The ratio of 1000 cells to DC was 5:1, and the cells were co-cultured for 72 hours. The culture volume was 1 mL.

[0103] (5) Cells were collected and treated with anti-CD3-PE / Cyanine 5.5 (Elabscience: E-AB-F1013I), anti-CD4-PE (Elabscience: E-AB-F1097D), and anti-CD8-APC (Elabscience: E-AB-F1104E), respectively. CD4+ was then analyzed by flow cytometry. + T and CD8 + T cell proliferation.

[0104] Results: Compared with the control group, the positive control LPS group significantly promoted T cell proliferation, validating the effectiveness of the experimental system. Among different concentrations of BEV treatment groups, 10 μg / ml of BEV significantly promoted CD4+ proliferation. + T and CD8 +T cell proliferation (p<0.001); indicating that BEV at concentrations above 10 μg / ml can significantly promote DC maturation (see...). Figure 8 ).

[0105] Example 3: Evaluation of the efficacy of BEVs as tumor tissue-targeted delivery carriers for chemotherapy drugs

[0106] 3.1 Preparation and drug loading rate of BEV-DOX

[0107] (1) DOX (Shanghai Yuanye Biotechnology Co., Ltd.: S17092) and BEVs were gently mixed in PBS at different mass ratios and then incubated overnight at 37°C.

[0108] (2) BEV-DOX was concentrated and free DOX was removed using a 100 kDa ultrafiltration membrane.

[0109] (3) Then continue to wash BEV-DOX several times with PBS to remove free DOX.

[0110] (4) After dissolving the BEV-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 DOX encapsulated.

[0111] (5) The method for calculating DOX encapsulation efficiency is the percentage of encapsulated DOX in BEVs relative to the initial DOX amount.

[0112] (6) The DOX loading efficiency is calculated as [mass of original DOX - mass of free DOX) / mass of BEV + mass of original DOX - mass of free DOX] × 100%.

[0113] Results: As the mass ratio of BEVs to DOX decreased from 10:1 to 1:10, the encapsulation efficiency showed a significant decreasing trend. At higher mass ratios (e.g., 10:1), the encapsulation efficiency approached 75%, indicating that a larger amount of BEVs could effectively encapsulate doxorubicin. However, as the mass ratio of BEVs gradually decreased, the encapsulation efficiency decreased significantly, especially at 1:10, where the encapsulation efficiency was almost zero. Regarding drug loading, the loading capacity gradually increased with the increase of the mass ratio of BEVs to DOX, reaching a maximum of approximately 16% at 1:2. Therefore, a preparation method with a mass ratio of 1:2 was ultimately selected (see...). Figure 9 ).

[0114] 3.2 Drug Release of BEV-DOX

[0115] The in vitro release of BEV-DOX was assessed by dialysis.

[0116] (1) Place 5 mL of 0.3 mg / mL BEV-DOX solution into a sealed MWCO 1000 Da dialysis bag and place it in a PBS solution at pH 6.5 or pH 7.4 to simulate the tumor and blood system environment. Shake the dialysis bag containing BEV-DOX at 37°C for 72 h.

[0117] (2) Dialysate was collected at 0, 0.5, 1, 2, 6, 12, 24, 48 and 72 h respectively, and each sample was analyzed by an enzyme-linked immunosorbent assay (ELISA) reader.

[0118] Results: Transmission microscopy images of DOX-loaded BEVs showed that the vesicles maintained a typical spherical structure with uniform size distribution and no obvious morphological changes or structural damage, indicating that the DOX loading process did not negatively affect the integrity of the BEVs. Particle size distribution analysis showed that the average particle size of the BEVs was 184.6 ± 2.055 nm, and the PDI value was 0.163, indicating that the particle size distribution of the BEVs was uniform and within the size range of passive accumulation of nanomaterials in tumor tissue. Drug release experiments showed that BEV-DOX was released faster in an acidic environment than in a neutral blood environment, indicating that BEV-loaded drugs have the potential to release drugs in the slightly acidic tumor microenvironment (see...). Figure 10 ).

[0119] 3.3 Effects of BEV-DOX on tumor cell viability in vitro

[0120] (1) After digesting and centrifuging the cells that have been passaged to the third generation, count them at a ratio of 1×10⁻⁶. 5 B16-OVA cells were evenly seeded into confocal dishes at a cell density of cells / dish, gently shaken, and then incubated at 37°C.

[0121] (2) Pass the BEV-DOX stock solution through a sterile filter and dilute it to the working concentration with DMEM medium. Prepare fresh each time (5 μg / mL). Remove the culture medium from the dish after 1 day of culture, add 1 mL of the diluted drug solution, shake gently, and continue to culture for 5 hours.

[0122] (3) After the culture is completed, take out the small dish, remove the culture medium and add PBS to wash the cells 3 times to remove the residual drug; add 1 mL of 4% paraformaldehyde fixative to the dish and fix for 15 min, discard the fixative, and then wash the cells 3 times with PBS.

[0123] (4) Add 1 mL of Calcein / PI detection working solution to cover the cells, and incubate at 37°C in the dark for 30 min. After incubation, discard the working solution and wash three times with PBS. Add 200 μL of PBS to prevent the cells from drying out.

[0124] (5) Laser confocal microscopy detection.

[0125] Results: BEVs did not affect tumor cell viability, DOX induced significant cell death, and the same concentration of BEV-DOX induced a large number of tumor cell deaths (see [link to study]). Figure 11 A).

[0126] 3.4 Detection of apoptosis levels induced by BEVs

[0127] (1) Cell plating: Cells passaged to the third generation were digested, centrifuged, collected, and counted at 5 × 10⁻⁶ cells per cell line. 5 B16-OVA cells were seeded into 12-well plates at a cell density of cells / well, with three replicates for each treatment group. After gentle shaking, the cells were incubated at 37°C.

[0128] (2) Drug treatment: The BEV-DOX stock solution was filtered through a sterile filter and diluted to 5 μg / mL with DMEM medium. It was prepared fresh for each use. After 1 day of cell culture, the medium was discarded from the well plate, and 5 μg / mL of the diluted drug solution was added. The plate was then incubated for 5 hours.

[0129] (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 the cells are collected. The cells are centrifuged at 1200 rpm for 7 min, the supernatant is discarded, and the collected cells are washed 3 times with PBS.

[0130] (4) Resuspend the cells in 1× Binding Buffer, mix Annexin V and PI (PI:Annexin = 2:1), and incubate at room temperature in the dark for 15-20 min.

[0131] (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.

[0132] (6) Flow cytometry detection.

[0133] Results: The apoptosis rate of tumor cells induced by BEV-DOX (56.1%) was significantly higher than that of the BEV group (0.21%) and the DOX group (20.1%), and the difference was statistically significant (P<0.01) (see...). Figure 11 B).

[0134] 3.5 In vivo pharmacokinetics of intravenously administered BEV-DOX

[0135] (1) Six mice were randomly divided into two groups: the DOX group and the BEV-DOX group.

[0136] (2) 5 mg / kg of free DOX and DOX encapsulated in BEV were intravenously injected into tumor-bearing mice (average weight 16-18 g). The plasma fluorescence value of DOX content was used to evaluate pharmacokinetic parameters.

[0137] (3) 150 μL of blood samples were collected at 0, 0.5, 1, 2, 4, 6, 8 and 12 h after administration and placed into EP tubes. The DOX content in plasma at different time points was observed using a small animal imaging instrument.

[0138] Results: The fluorescence intensity of DOX decreased rapidly over time, indicating a short half-life and rapid metabolism and clearance in vivo, which may result in a short duration of efficacy and limit its efficiency in delivering drugs to lesions. In contrast, the fluorescence intensity of BEV-DOX decreased relatively slowly, indicating a longer retention time in vivo and a slower metabolic process. This suggests that BEVs can increase the retention time of DOX in vivo (see...). Figure 12 ).

[0139] 3.6 Evaluation of in vivo distribution of BEVs

[0140] (1) Female C57BL / 6J mice aged 4-6 weeks were purchased from the Experimental Animal Center of Xinjiang Medical University. They were acclimatized for 7 days, during which time the mice were observed for any abnormalities in their appearance and behavior. Healthy mice were selected for subsequent modeling.

[0141] (2) Adjust the number of B16-OVA tumor cells to 1×10⁻⁶ using pre-cooled sterile PBS. 6 100 μL of B16-OVA cell suspension was injected subcutaneously into the right hind limb of each C57 mouse at a rate of 1 cell / mL.

[0142] (3) When a significant mass can be palpated under the skin of the inoculated area after mice are inoculated with tumor cells, the tumor inoculation is considered successful.

[0143] (4) When the mouse tumor volume is approximately 100 mm² 3 At that time, mice were injected with a single dose of 5 mg / kg DiR-labeled BEV and the same dose of free DiR via the tail vein.

[0144] (5) At 0, 0.5, 2, 4, 6, 12 and 24 h, mice were randomly selected to obtain tumors and major organs. DiR distribution and tumor targeting were analyzed by small animal in vivo imaging (FOBI imaging system, CELLGENTEK). The fluorescence intensity of DiR in each organ was calculated by NEO Image software to perform biodistribution analysis.

[0145] Results: At 0 h post-administration, both drugs showed very low fluorescence intensity in mice. Over time, DiR-BEV began to accumulate significantly at the tumor site, especially between 12 and 24 h, where the fluorescence intensity of DiR-BEV in the tumor region was significantly higher than that of free DiR at the same time point. Free DiR was more widely distributed, accumulating particularly in tissues such as the liver, lungs, and spleen, but its fluorescence intensity at the tumor site was relatively weak, indicating a lack of tumor targeting specificity. In contrast, DiR-BEV was more abundant at the tumor site and less distributed in other tissues. This suggests that BEV has better tumor targeting ability. Figure 13 ).

[0146] 3.7 Evaluation of the antitumor effect of BEV-DOX

[0147] (1) Female C57BL / 6J mice aged 4-6 weeks were purchased from the Experimental Animal Center of Xinjiang Medical University. They were acclimatized for 7 days, during which time the mice were observed for any abnormalities in their appearance and behavior. Healthy mice were selected for subsequent modeling.

[0148] (2) Adjust the number of B16-OVA tumor cells to 1×10⁻⁶ using pre-cooled sterile PBS. 6 100 μL of B16-OVA cell suspension was injected subcutaneously into the right hind limb of each C57 mouse at a rate of 1 cell / mL.

[0149] (3) In mice inoculated with tumor cells, tumor inoculation was considered successful when a palpable mass was felt under the skin at the inoculation site. The experimental mice were randomly divided into 5 groups of 6 mice each. The tumor-bearing mice were administered different doses and treatment frequencies according to experimental requirements. The doses were as follows: the PBS control group, DOX treatment group, BEVs treatment group, and BEV-DOX treatment group were administered via tail vein injection (iv), while one group received BEV-DOX via intraperitoneal injection (ip). The DOX dose was 2 mg / kg, and the BEV-DOX dose was maintained at the same level as the DOX dose. The BEV content in the BEV-DOX was the same as that in the BEV-DOX. The mice were treated every two days for a total of 4 treatments.

[0150] (4) After treatment begins, the long diameter (A) and short diameter (B) of the mouse tumor are measured with calipers on the same day and every 48 hours and recorded. These measurements are used to calculate the tumor volume. Mouse weight is also measured. Treatment is stopped when the long diameter of the tumor is ≥15mm, and the mice are euthanized. Tumor growth curves and mouse weight change curves are plotted. The tumor volume calculation formula is as follows: Vmm 3 =AB 2 / 2(mm 3 ).

[0151] (5) Two days after the last administration, the mice were euthanized after blood was collected, and the tumors and major organs were removed for physiological and biochemical analysis. Blood samples were also collected for biochemical testing.

[0152] Results: Changes in tumor volume and weight showed that, compared with the PBS negative control, the DOX, BEV, and BEV-DOX groups significantly slowed tumor growth and had significantly lighter tumor weights. Compared with the tumor-suppressing effect of DOX alone, the BEV carrier significantly improved the therapeutic effect of DOX. The experimental results also showed that BEV's own immune cell-activating activity gave it the ability to inhibit tumor cell growth. The fact that there were no significant changes in mouse body weight at the current dosage in each experimental group also demonstrated that each treatment group maintained low toxicity while improving tumor treatment efficacy. A comparison of tumor volume and weight between the intraperitoneal injection and tail vein injection groups of BEV-DOX showed no significant difference, indicating that the BEV-DOX combination had superior anti-tumor effects in both treatment routes. Figure 14 ).

[0153] 3.8 Serum biochemical factor level detection

[0154] (1) Place the EP tube containing mouse blood in a 37°C constant temperature incubator for 2 hours, then incubate overnight at 4°C, centrifuge at 3000 rpm for 20 minutes at 4°C, and transfer the supernatant to a new EP tube to obtain the serum of the tumor model mouse.

[0155] (2) The levels of cytokines IFN-γ (Elabscience: E-MSEL-M0048) and IL-4 (Elabscience: E-MSEL-M0008) in the supernatant were detected at 450 nm using an ELISA kit and a microplate reader (BioTek, Santa Clara, CA, USA) according to the manufacturer’s instructions.

[0156] (3) According to the manufacturer's instructions, various cardiac, hepatic and renal injury indicators in the supernatant were detected by microplate method using the creatine kinase (CK) kit (Solarbio: BC1145), lactate dehydrogenase (LDH) kit (Solarbio: BC0685), blood urea nitrogen (BUN) test kit (Solarbio: BC1535), alanine aminotransferase (ALT / GPT) test kit (Solarbio: BC1555), aspartate aminotransferase (AST / GOT) test kit (Solarbio: BC1565) and creatinine (CRE) assay kit (Solarbio: BC4910).

[0157] Results: Serum biochemical tests showed that the levels of CRE, BUN, ALT, AST, LDH, and CK in the BEV-DOX-treated group of mice were not significantly elevated, indicating that BEV-DOX, like the organ indices, did not induce significant organ damage. However, the DOX-only treatment group showed elevated levels of CRE, ALT, AST, LDH, and CK, suggesting that DOX at this dose has certain toxic effects on the liver, kidneys, and heart. BEV may have the effect of reducing the organ toxicity of DOX, and both intravenous and intraperitoneal administration can reduce the risk of organ toxicity of DOX (see...). Figure 15 (AB). There was no significant difference in serum IL-4 levels among the groups, while all experimental groups of DOX significantly increased serum IFN-γ levels (see AB). Figure 15 CD), which may be related to DOX inducing immunogenic cell death in tumor cells.

[0158] 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 increasing the production of extracellular vesicles of Lactococcus lactis, characterized in that, The specific steps are as follows: 1) After the lactococcus lactis is revived and activated, it is inoculated into M17 liquid medium containing 5% glucose after being centrifuged by ultracentrifugation, and 250-4000 μg / ml lysozyme is added at the same time. It is then placed in a 30℃ incubator for static culture. 2) When the OD600 value reaches 0.8-0.1, add 125-2000 μg / ml ampicillin and continue to incubate at 37℃ for 12-72 h. 3) Collect the bacterial culture, centrifuge the bacterial culture and collect the supernatant. Filter the supernatant through a 0.2-0.5μm filter to remove bacteria; 4) The filtrate is concentrated by ultrafiltration; 5) The precipitate obtained by ultrafiltration concentration followed by ultracentrifugation is bacterial extracellular vesicles.

2. The method for increasing the production of extracellular vesicles of Lactococcus lactis according to claim 1, characterized in that, Step 1) The ultracentrifugation is performed at 150,000 rpm for 2 hours.

3. The method for increasing the production of extracellular vesicles of Lactococcus lactis according to claim 1, characterized in that, Step 3) The centrifugation is performed at 100-10000g for 10-60 minutes.

4. The method for increasing the production of extracellular vesicles of Lactococcus lactis according to claim 1, characterized in that, The supernatant from step 3) was sterilized by filtration through a 0.22 μm filter.

5. The method for increasing the production of extracellular vesicles of Lactococcus lactis according to claim 1, characterized in that, Step 4) The ultrafiltration concentration is 100-5000g centrifuged for 10-100min; the ultrafiltration tube is 100kDa.

6. The method for increasing the production of extracellular vesicles of Lactococcus lactis according to claim 1, characterized in that, Step 5) The ultracentrifugation is performed at 10,000-150,000g for 60-600 minutes.

7. Extracellular vesicles of *Lactococcus lactis* prepared by the method according to any one of claims 1-6.

8. The use of the Lactococcus lactis extracellular vesicles according to claim 7 in the preparation of vaccine vectors.

9. The use of the Lactococcus lactis extracellular vesicles according to claim 7 in the preparation of tumor tissue-targeted delivery carriers for antitumor drugs.

10. The use of the Lactococcus lactis extracellular vesicles according to claim 7 in the preparation of an immune enhancer.

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