A mycobacterium tuberculosis specific drug-loaded dc vaccine membrane nanovesicle and preparation and application thereof

Abstract

The application discloses a drug-loaded DC vaccine film nanovesicle specific to mycobacterium tuberculosis as well as a preparation and application thereof. The drug-loaded DC vaccine film nanovesicle is of a core-shell structure, the inner core is a PLGA nanoparticle loaded with an anti-tuberculosis drug, and the outer shell is a DC vaccine film specific to mycobacterium tuberculosis. The drug-loaded DC vaccine film nanovesicle presents mycobacterium-related antigens on the surface, retains original DC vaccine film surface proteins and molecules, and has the function of activating mycobacterium tuberculosis specific T cells. The drug-loaded DC vaccine film nanovesicle can target lymph nodes, effectively activate initial T cells and play an anti-tuberculosis immunotherapy function on one hand, and the drug-loaded PLGA nanoparticle inner core can gather at a tuberculosis tuberculous lesion site through an EPR effect, thereby playing a role in enhancing targeted tuberculosis nanometer chemotherapy. Through nanometer chemotherapy combined with immunotherapy, the purpose of eliminating mycobacterium tuberculosis is achieved, the treatment cycle of tuberculosis is effectively shortened, and the treatment efficiency of tuberculosis is improved.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and more specifically, relates to a drug-loaded DC vaccine membrane nanovesicle specific to Mycobacterium tuberculosis, its preparation, and its application. This invention relates to the intersection of nanomedicine delivery technology, dendritic cell vaccine technology, and cell membrane biomimetic nanotechnology. Background Technology

[0002] Tuberculosis (TB), caused by Mycobacterium tuberculosis (MTB), is a serious respiratory infectious disease and one of the leading causes of death from single-pathogen infection. Currently, TB treatment primarily relies on long-term (6-12 months) combination chemotherapy with multiple antibiotics; however, this approach has the following serious limitations:

[0003] (1) Low drug delivery efficiency: The effective drug concentration of antibiotics reaching the local tuberculous lesion is low, and the anti-tuberculosis effect is limited;

[0004] (2) Significant toxic side effects: Long-term, high-dose use of the drug has significant toxic side effects on the liver, kidneys and other organs of the patient;

[0005] (3) Poor patient compliance: The treatment cycle is as long as 6 months or more, and patients find it difficult to adhere to the full course of standardized medication, which aggravates the generation and spread of drug-resistant bacteria;

[0006] (4) The problem of drug resistance is serious: In 2019, nearly 500,000 people worldwide were diagnosed with rifampicin-resistant tuberculosis (RR-TB), of which 78% were multidrug-resistant tuberculosis (MDR-TB), and the cure rate of MDR-TB was only about 50%.

[0007] Therefore, in addition to improving the efficiency of antibiotic chemotherapy for tuberculosis and reducing toxic side effects, there is an urgent need to develop non-chemotherapy combination therapies to reduce the use of antibiotics and at the same time improve the treatment efficiency of drug-resistant tuberculosis.

[0008] The occurrence, development, and outcome of tuberculosis are closely related to host immune regulation, and immunotherapy is one of the main directions in the development of novel drugs for tuberculosis. Dendritic cells (DCs) are important cells connecting the innate and adaptive immune responses against tuberculosis, and are among the body's most potent antigen-presenting cells, potentially serving as important targets for anti-tuberculosis immunotherapy. DC vaccines prepared through in vitro antigen induction can bypass the innate immune response step in vivo and directly induce a specific cellular immune response, thereby exerting a specific immunotherapeutic effect.

[0009] However, DC vaccines have the following limitations in clinical application:

[0010] (1) Difficult to preserve: As living cells, DCs cannot be preserved in vitro for a long time, which limits their long-term application and large-scale production;

[0011] (2) Low bioavailability: After DC is injected into the body, the number of cells that home to the lymph nodes is small, resulting in poor bioavailability;

[0012] (3) Single function: Traditional DC vaccines only play an immunotherapy role and cannot simultaneously achieve chemotherapy function.

[0013] In recent years, cell membrane biomimetic nanotechnology has become a research hotspot in the biomedical field. This technology involves coating the surface of various biological cell membranes onto nanoparticles, making the resulting biomimetic nanoparticles appear as part of the organism itself. This allows them to evade the immune system's detection, prolonging their duration of action in the body, while simultaneously inheriting original cell membrane components and performing functions similar to cell membranes. A 2020 study reported the creation of DC vaccine nanovesicles from tumor-bearing dendritic cells (DCs). These nanovesicles retained the original DC vaccine surface proteins and receptors, enabling them to present antigens to T cells and activate tumor-specific adaptive immune responses. Most importantly, these DC vaccine nanovesicles are easy to store and are nanoscale in size, making them easier to penetrate blood vessel walls and target lymph node tissues, thus increasing their bioavailability.

[0014] However, there are currently no reports on the preparation of Mycobacterium tuberculosis-specific DC vaccines into membrane nanovesicles and their combination with nano-drug delivery systems for chemotherapy-immunotherapy of tuberculosis. Therefore, developing a novel drug-loaded DC vaccine membrane nanovesicle, combined with nano-chemotherapy and immunotherapy, is of great significance for improving the treatment efficiency and shortening the treatment cycle of tuberculosis. Summary of the Invention

[0015] In order to overcome the shortcomings and deficiencies of the prior art, the purpose of this invention is to provide a drug-loaded DC vaccine membrane nanovesicle specifically for Mycobacterium tuberculosis.

[0016] Another object of the present invention is to provide a method for preparing the above-mentioned Mycobacterium tuberculosis-specific drug-loaded DC vaccine membrane nanovesicles.

[0017] Another object of the present invention is to provide the application of the above-mentioned Mycobacterium tuberculosis-specific drug-loaded DC vaccine membrane nanovesicles.

[0018] Another object of the present invention is to provide a pharmaceutical composition for treating tuberculosis.

[0019] The objective of this invention is achieved through the following technical solution:

[0020] In a first aspect, the present invention provides a drug-loaded DC vaccine membrane nanovesicle specifically for Mycobacterium tuberculosis, wherein the nanovesicle has a core-shell structure and comprises:

[0021] Core: PLGA nanoparticles loaded with anti-tuberculosis drugs;

[0022] The outer shell is derived from cell membrane vesicles of a Mycobacterium tuberculosis-specific DC vaccine;

[0023] The drug-loaded DC vaccine membrane nanovesicles exhibit mycobacterial-associated antigens on their surface, retaining the original DC vaccine membrane surface proteins and molecules, and possessing the function of activating Mycobacterium tuberculosis-specific T cells.

[0024] Preferably, the Mycobacterium tuberculosis-specific DC vaccine is obtained by inducing DC cells with inactivated Mycobacterium tuberculosis at an MOI of 1 to 10 (preferably for 2 days).

[0025] Preferably, the cell membrane vesicles are obtained by cryolysis using a hypotonic cell lysis buffer containing protease inhibitors, followed by mechanical disruption and homogenization, and then differential centrifugation.

[0026] More preferably, the differential centrifugation is performed by first centrifuging at 2–6°C and 3000–3500 g for 4–6 min and collecting the supernatant; then centrifuging at 2–6°C and 18000–22000 g for 20–30 min and collecting the supernatant; and then centrifuging at 2–6°C and 90000–110000 g for 30–40 min. The white precipitate obtained is then resuspended and stabilized with PBS to obtain the cell membrane vesicle solution.

[0027] More preferably, the differential centrifugation is performed by first centrifuging at 3200 g for 5 min at 4°C and collecting the supernatant; then centrifuging at 20000 g for 25 min at 4°C and collecting the supernatant; and then centrifuging at 100000 g for 35 min at 4°C. The white precipitate collected is then resuspended and stabilized with PBS to obtain the cell membrane vesicle solution.

[0028] Preferably, the mass ratio of the anti-tuberculosis drug to PLGA is (2.5-15):15.

[0029] Preferably, the anti-tuberculosis drug is selected from at least one of rifampin and isoniazid.

[0030] Preferably, the mass ratio of the cell membrane vesicles to the PLGA nanoparticles loaded with anti-tuberculosis drugs is 1:2 to 5.

[0031] Preferably, the hydrated particle size of the drug-loaded DC vaccine membrane nanovesicles is 250–350 nm, and the surface potential is negative.

[0032] Preferably, the surface of the DC vaccine membrane expresses CD11c and CD80 molecules.

[0033] Secondly, the present invention provides a method for preparing the Mycobacterium tuberculosis-specific drug-loaded DC vaccine membrane nanovesicles, comprising the following steps:

[0034] S1. DC vaccine induction: DC cells are co-incubated with inactivated Mycobacterium tuberculosis to induce mature Mycobacterium tuberculosis-specific DC vaccine.

[0035] S2. Cell membrane extraction: Collect DC vaccines and extract cell membranes through hypotonic lysis, homogenization, and differential centrifugation to prepare Mycobacterium tuberculosis-specific DC vaccine membrane nanovesicles;

[0036] S3. Preparation of drug-loaded PLGA nanoparticles: PLGA nanoparticles loaded with anti-tuberculosis drugs were prepared by water-emulsion method.

[0037] S4. Fusion of membrane vesicles and nanoparticles: DC vaccine membrane nanovesicles are mixed with PLGA nanoparticles loaded with anti-tuberculosis drugs, ultrasonically treated, and then incubated. After centrifugation and washing, drug-loaded DC vaccine membrane nanovesicles are obtained.

[0038] Preferably, step S1 specifically involves: collecting mouse bone marrow cells, inducing the bone marrow cells with 20 ng / mL GM-CSF + 10 ng / mL IL-4 for 6 days to obtain immature DC cells; then adding inactivated Mycobacterium tuberculosis and inducing for 2 days with MOI=1-10; and using flow cytometry to detect the expression of CD11c, CD86, and MHCII molecules to confirm the maturity of DCs, thereby obtaining a mature Mycobacterium tuberculosis-specific DC vaccine.

[0039] Preferably, the inactivation method includes chemical inactivation or thermal inactivation.

[0040] Preferably, step S2 specifically includes:

[0041] S21. Collect the DC vaccine after immunization with inactivated Mycobacterium tuberculosis, centrifuge and wash 3 times, resuspend the cell pellet in hypotonic cell lysis buffer containing protease inhibitors, and lyse it at -20℃.

[0042] S22. The lysed cells are mechanically disrupted and homogenized.

[0043] Centrifuge at 2-6℃, 3000-3500 g for 4-6 min to remove large fragments and collect the supernatant;

[0044] Centrifuge at 2-6℃, 18000-22000 g for 20-30 min, discard the precipitate, and collect the supernatant;

[0045] S25. The supernatant is further centrifuged at 2-6°C and 90,000-110,000 g for 30-40 min. The supernatant is discarded, and the white precipitate, i.e., the cell membrane, is collected. The cell membrane is then resuspended in PBS to obtain the Mycobacterium tuberculosis-specific DC vaccine membrane nanovesicle solution.

[0046] Preferably, in step S21, the protease inhibitor includes benzyl sulfonyl fluoride (PMSF); further, the concentration of benzyl sulfonyl fluoride is 1-2 mmol / L PMSF.

[0047] Preferably, in step S23, the centrifugation conditions are 4°C, 3200 g for 5 min;

[0048] Preferably, in step S24, the centrifugation conditions are 4°C, 20000 g for 25 min;

[0049] Preferably, in step S25, the centrifugation conditions are 4°C, 100,000 g for 35 min.

[0050] Preferably, step S3 specifically includes:

[0051] S31. Dissolve PLGA in an organic solvent, dissolve the anti-tuberculosis drug in an organic solvent, and then mix thoroughly to obtain an organic solution of PLGA containing the anti-tuberculosis drug.

[0052] S32. Rapidly inject the PLGA organic solution containing anti-tuberculosis drugs into the aqueous phase, with the volume ratio of organic solution to aqueous phase being 1:4 to 10.

[0053] S33. After ultrasonic emulsification, stirring was performed to evaporate the organic solvent, resulting in an aqueous solution of drug-loaded PLGA nanoparticles.

[0054] S34. After centrifugation and washing, the PLGA nanoparticles were resuspended in PBS to obtain a solution loaded with anti-tuberculosis drugs.

[0055] Preferably, in step S31, the organic solvent includes dichloromethane, etc.

[0056] Preferably, in step S32, the aqueous phase is an aqueous phase containing polyvinyl alcohol; further, the concentration of polyvinyl alcohol in the aqueous phase is 0.5-2% (w / v) (preferably 1%).

[0057] Preferably, in step S33, the ultrasonic emulsification conditions are 80-100 W ultrasound for 5-10 min; more preferably, 100 W ultrasound for 5 min.

[0058] Preferably, in step S34, the centrifugation speed is 6000-8000 g; more preferably 7000 g.

[0059] Preferably, step S4 specifically involves: mixing DC vaccine membrane nanovesicles with PLGA nanoparticles loaded with anti-tuberculosis drugs at a mass ratio of 1:2 to 5, sonicating in a 100 W water bath for 3 to 5 minutes, letting stand overnight at 4°C, and centrifuging and washing 3 times to obtain the final product.

[0060] Thirdly, the present invention provides a pharmaceutical composition comprising the drug-loaded DC vaccine membrane nanovesicles described above;

[0061] Furthermore, the pharmaceutical composition also includes a pharmaceutically acceptable carrier.

[0062] Fourthly, the present invention provides the application of the drug-loaded DC vaccine membrane nanovesicles or pharmaceutical compositions described herein, which may be one or more combinations of the following applications:

[0063] (a) Its use in the preparation of drugs that kill Mycobacterium tuberculosis;

[0064] (b) Its use in the preparation of medicines for treating tuberculosis;

[0065] (c) Application in the preparation of drugs that kill Mycobacterium marinum;

[0066] (d) Application in the preparation of drugs for treating diseases related to Mycobacterium marinum.

[0067] Preferably, the drug exerts its anti-tuberculosis effect through nanochemotherapy combined with immunotherapy.

[0068] Preferably, the drug is capable of activating CD4. + T cells promote T cell proliferation and release anti-tuberculosis-related cytokines.

[0069] The present invention has the following advantages and effects compared with the prior art:

[0070] (1) Dual treatment mechanism: This invention innovatively combines nanochemotherapy with immunotherapy. On the one hand, the drug-loaded PLGA nanonuclei accumulate at the tuberculous nodule lesion site through the EPR effect, enhancing the targeted effect of nanochemotherapy for tuberculosis; on the other hand, the DC vaccine membrane vesicles can target lymph nodes, effectively activating naïve T cells and exerting anti-tuberculosis immunotherapy function. Through the combination of nanochemotherapy and immunotherapy, the goal of clearing Mycobacterium tuberculosis is achieved, effectively shortening the treatment cycle of tuberculosis and improving the treatment efficiency of tuberculosis.

[0071] (2) Improve drug delivery efficiency: PLGA nanocarriers can significantly improve the efficiency of anti-tuberculosis drugs entering macrophages. Experiments have shown that the amount of drug-loaded nanoparticles entering the cell is much higher than that of free drugs, thereby enhancing the intracellular bactericidal effect.

[0072] (3) Specific immune activation: The drug-loaded DC vaccine membrane nanovesicles of the present invention retain the surface antigens and functional molecules of the original DC vaccine, and can specifically activate CD4. + T cells promote T cell proliferation and release anti-tuberculosis-related cytokines such as TNF-α and IFN-γ, thereby exerting a specific immune bactericidal effect.

[0073] (4) Improvement of the limitations of DC vaccines: Compared with traditional DC cell vaccines, the membrane nanovesicles of the present invention have the following advantages: ① They are easy to store for a long time without the need for live cell preservation conditions; ② Their nanoscale size is conducive to crossing blood vessel walls, targeting lymph nodes, and improving bioavailability; ③ They can be mass-produced and their quality is controllable.

[0074] (5) Enhanced therapeutic effect: Experiments have shown that the drug-loaded DC vaccine membrane nanovesicles of the present invention have a stronger effect on killing intracellular mycobacteria than drug-loaded PLGA nanoparticles alone, and have added chemotherapy function compared with simple DC vaccine membrane vesicles, achieving a synergistic therapeutic effect of 1+1>2.

[0075] (6) Good biocompatibility: PLGA is an FDA-approved biodegradable material. The DC cell membrane is derived from autologous cells, has good biocompatibility, and has no obvious immunogenicity or toxic side effects. Attached Figure Description

[0076] Figure 1 The images show the preparation and characterization of rifampicin-loaded PLGA nanoparticles (RIF-PLGA NPs); where: A: Transmission electron microscopy (TEM) image of RIF-PLGA NPs with a drug dosage of 15 mg (Scale bar: 100 nm); B: Hydration particle size and potential characterization of PLGA NPs and RIF-PLGA NPs with a drug dosage of 15 mg; C: UV-Vis characteristic spectrum characterization of RIF-PLGA NPs with a drug dosage of 15 mg; D: Encapsulation efficiency of RIF in RIF-PLGA NPs with different drug dosages.

[0077] Figure 2 The diagram shows the cytotoxicity and mycobactericidal activity of RIF-PLGA NPs; where: A: Cell survival rate of macrophages after 24 h of RIF treatment; B: Cell survival rate of macrophages after 24 h of RIF-PLGA NPs treatment; C: Quantitative analysis of CFU in macrophages infected with mycobacteria by RIF and RIF-PLGA NPs treatment; D: Quantitative fluorescence analysis of macrophages infected with fluorescent mycobacteria by RIF and RIF-PLGA NPs treatment.

[0078] Figure 3The graph shows the efficiency of RIF entry into cells; where: A: quantitative fluorescence values ​​of RIF-CY5 at different time points in free or loaded RIF-PLGA NPs; B: representative fluorescence graph of RIF and RIF-PLGA NPs labeled with CY5 after co-incubation with macrophages for 4 h.

[0079] Figure 4 The diagram shows the induction and characterization of the DC vaccine; where: A: Schematic diagram of the experimental principle of DC vaccine induction and characterization; B: Flow cytometry diagram of DC vaccine expression of CD11c, CD80 and MHC II; Mm 1 represents chemically inactivated Mycobacterium marineum; Mm 2 represents heat-inactivated Mycobacterium marineum; ISO represents the untreated group (i.e. blank control).

[0080] Figure 5 The diagram shows the immune function verification of the DC vaccine; where: A: flow cytometry of CD69 expression in T cells after DC vaccine treatment; B: quantitative analysis of intracellular mycobacterial bactericidal activity of T cells after DC vaccine treatment, and CTL represents the blank control (Control).

[0081] Figure 6 Figure 1 shows the preparation and characterization of drug-loaded DC vaccine membrane nanovesicles (RIF@DNPs); where: A: Schematic diagram of RIF@DNPs preparation principle; B: Transmission electron microscopy (TEM) image of DNPs (Scale bar: 100 nm); C: TEM image of RIF@DNPs (Scale bar: 100 nm); D: Particle size potential characterization of DNPs and RIF@DNPs; E: UV-Vis characteristic spectrum of RIF@DNPs.

[0082] Figure 7 The image shows the protein characterization of RIF@DNPs. A: SDS-PAGE protein electrophoresis analysis of unactivated DC lysates, activated DC vaccine lysates, RIF-PLGA NPs, DNPs, and RIF@DNPs; B: Western blot analysis of the expression of important surface membrane protein receptors in unactivated DC lysates, activated DC vaccine lysates, RIF-PLGA NPs, DNPs, and RIF@DNPs.

[0083] Figure 8 The diagram shows the direct bactericidal effect of RIF@DNPs; where: A: CFU statistics of residual bacteria after treatment of intracellular mycobacteria with DNPs, RIF-PLGA NPs, and RIF@DNPs; B: CFU statistics of residual bacteria after treatment of intracellular mycobacteria with ordinary DC membrane nanovesicles (unactived DNPs) and DNPs.

[0084] Figure 9The diagram shows the bactericidal effect of PLGA@DNPs; where: the CFU count of residual bacteria after treatment with ordinary T cells, DNPs, and T cells pretreated with PLGA@DNPs are respectively.

[0085] Figure 10 DNPs promote CD4 + T cell activation diagram; where: A: PLGA NPs, DNPs, and PLGA@DNPs interact with CD4+. + and CD8 + Flow cytometry plot of CD69 expression after T cell administration; B: Effects of PLGA NPs, DNPs, and PLGA@DNPs on CD4. + Quantitative analysis of CD69 expression after T cell administration; C: Effects of PLGA NPs, DNPs, and PLGA@DNPs on CD8. + Quantitative analysis of CD69 expression after T cell transplantation.

[0086] Figure 11 Diagram showing the effect of DNPs on T cell proliferation; where: A: PLGA NPs, DNPs, and PLGA@DNPs on CD4. + Flow cytometry plot of cell proliferation after T cells; B: CD4 + Quantitative analysis of T cell proliferation; C: Effects of PLGA NPs, DNPs, and PLGA@DNPs on CD8. + Flow cytometry plot of cell proliferation after T cells; D: CD8 + Quantitative analysis of the number of proliferating T cells.

[0087] Figure 12 The diagram shows how DNPs promote the release of cytokines from T cells; where: A: Changes in TNF-α concentration in the supernatant of T cells after treatment with PLGA NPs, DNPs, and PLGA@DNPs; B: Changes in IFN-γ concentration; C: Changes in IL-2 concentration. Detailed Implementation

[0088] The present invention will now be described in detail with reference to specific embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention. These all fall within the scope of protection of the present invention.

[0089] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by those skilled in the art. The technical terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the scope of the invention.

[0090] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be purchased from the market or prepared by existing methods.

[0091] It should be noted that *Mycobacterium marineum* is an excellent model for studying *Mycobacterium tuberculosis*, sharing over 95% genetic similarity with it. This instruction manual uses *Mycobacterium marineum* as a substitute bacterium for *Mycobacterium tuberculosis* in experiments, and the experimental conclusions are also applicable to *Mycobacterium tuberculosis*.

[0092] Example 1: Preparation and characterization of rifampicin-loaded PLGA nanoparticles (RIF-PLGA NPs)

[0093] 1. Materials and Reagents

[0094] PLGA (polylactic acid-glycolic acid copolymer, 50:50, molecular weight 15000), rifampicin (RIF), polyvinyl alcohol (PVA, molecular weight 30000-70000), dichloromethane, ultrapure water (ddH2O).

[0095] 2. Experimental Methods

[0096] (1) Clean the glassware in advance using an ultrasonic cleaner and let it dry.

[0097] (2) Weigh 15 mg of PLGA and add it to 2 mL of dichloromethane. Mix thoroughly to obtain a PLGA solution.

[0098] (3) Weigh 2.5, 5, 10 and 15 mg of RIF respectively, and dissolve them completely in 500 μL of dichloromethane to obtain RIF solutions;

[0099] (4) Mix the RIF solution and PLGA solution thoroughly by sonication for 3 min to obtain a mixed solution of RIF and PLGA;

[0100] (5) Add the RIF and PLGA mixed solution to 10 mL of 1% (w / v) PVA in ddH2O and use a probe to sonicate (100 W, 5 min) to fully emulsify it;

[0101] (6) Take the emulsified mixture and stir it overnight in a fume hood to allow the organic solvent to evaporate completely;

[0102] (7) Centrifuge the final drug-loaded aqueous solution at 7000 g, wash twice with ddH2O, and resuspend in PBS to obtain RIF-PLGA NPs aqueous solution, and store at 4℃.

[0103] An aqueous solution of unencapsulated PLGA nanoparticles (PLGA NPs) was prepared according to this method.

[0104] 3. Characterization methods

[0105] (1) Encapsulation rate determination: The RIF content was determined by high performance liquid chromatography and the encapsulation rate was calculated.

[0106] Encapsulation efficiency = (Drug content in nanoparticles / Total drug dosage) × 100%;

[0107] (2) Transmission electron microscopy (TEM) characterization: 10 μL of RIF-PLGA NPs aqueous solution was suspended on a 300-mesh copper grid and allowed to settle for 5 minutes. Then, 10 μL of uranium acetate was suspended and stained for 20 seconds. Excess uranium acetate was removed with the tip of filter paper and the mixture was dried at room temperature before being tested on the TEM.

[0108] (3) Particle size and potential characterization: The aqueous solution of the analyte was placed in a disposable plastic cuvette and a potentiometer, and the hydrated particle size and surface potential were detected in a zetasizer.

[0109] (4) Ultraviolet absorption spectroscopy measurement: The test solution was tested using the same solvent as a blank control and detected by an ultraviolet-visible spectrophotometer with a range of 200-800 nm.

[0110] 4. Experimental Results

[0111] (1) Encapsulation ratio: such as Figure 1 As shown in D, the encapsulation efficiency of RIF was highest at a dosage of 2.5 mg, followed by 15 mg. Considering the low drug loading of 2.5 mg RIF, 15 mg RIF was subsequently selected as the dosage for synthesizing RIF-loaded PLGA nanoparticles.

[0112] (2) Morphological characteristics: such as Figure 1 As shown in A, the TEM image shows that the RIF-PLGA NPs are nearly spherical with a diameter of about 100 nm.

[0113] (3) Particle size and potential: such as Figure 1 As shown in B, the hydrated particle size of the uncoated PLGA nanoparticles is about 180 nm, while the particle size of the coated nanoparticles increases to about 185 nm. The potential is negative, around -37 mV, both before and after coating.

[0114] (4) Ultraviolet spectrum: such as Figure 1 As shown in Figure C, RIF-PLGA NPs exhibit characteristic absorption peaks of RIF at around 310 nm and 490 nm, indicating successful drug loading.

[0115] Conclusion: Stable RIF-loaded PLGA nanoparticles were successfully constructed.

[0116] Example 2: Cytotoxicity and mycobactericidal activity of RIF-PLGA NPs

[0117] 1. Materials and Reagents

[0118] Mouse mononuclear macrophage cell line RAW 264.7, GFP-labeled Mycobacterium marinum, CCK-8 kit, 7H9 medium, 7H10 agar plates.

[0119] 2. Experimental Methods

[0120] (1) Cytotoxicity test:

[0121] ① RAW 264.7 cells were cultured at a concentration of 1×10⁻⁶. 4 Cells were seeded per well in a 96-well plate;

[0122] ② Different concentrations of RIF and RIF-PLGA NPs were added (final concentrations of 0, 10, 20, 40, 80, and 160 μg / mL, respectively); the treatment group without drugs was used as the control group.

[0123] ③ After co-incubation for 24 h, CCK-8 reagent was added, and the absorbance at 450 nm was measured to calculate the cell viability.

[0124] (2) Intracellular bactericidal experiment:

[0125] ① RAW 264.7 cells were cultured at a rate of 2 × 10⁻⁶. 5 Cells were seeded per well in a 24-well plate;

[0126] ② Macrophages RAW 264.7 were infected with GFP-labeled Mycobacterium marineum at MOI=10 and incubated for 4 h;

[0127] ③ Wash three times with PBS to remove extracellular bacteria;

[0128] ④ Add free RIF or RIF-PLGA NPs to a final concentration of 5 μg / mL;

[0129] ⑤ Continue incubation for another 36 hours:

[0130] ⑥ Observe the intracellular GFP fluorescence intensity using a fluorescence microscope;

[0131] ⑦ Lyse cells, serially dilute them, spread them on 7H10 plates, incubate at 37°C for 14 days, and count CFU.

[0132] (3) Cell uptake experiment:

[0133] ① Label the RIF with CY5 fluorescence;

[0134] ② RAW 264.7 cells were co-incubated with CY5-labeled free RIF or RIF-PLGA NPs;

[0135] ③ Cells were collected at 1, 2, 4, 6, and 12 h, and the fluorescence intensity was detected by flow cytometry;

[0136] ④ After co-incubation for 4 hours, observe under a fluorescence microscope.

[0137] 3. Experimental Results

[0138] (1) Cytotoxicity: such as Figure 2 A and Figure 2 As shown in B, RIF and RIF-PLGA NPs alone showed no significant toxicity to macrophages at concentrations below 40 μg / mL. In subsequent cell experiments, the concentration of RIF-PLGA NPs used was 5 μg / mL, far below the cytotoxic concentration.

[0139] (2) Bactericidal effect: such as Figure 2 As shown in CD:

[0140] ① The intracellular green fluorescence of infected cells treated with free RIF and RIF-PLGA NPs was significantly reduced;

[0141] ②CFU counting showed that free RIF had a significant bactericidal effect on intracellular mycobacteria;

[0142] ③ Compared with free RIF, RIF-PLGA NPs at the same concentration have a stronger effect on killing intracellular mycobacteria (*p ≤ 0.05).

[0143] (3) Cellular uptake: such as Figure 3 As shown:

[0144] ①After incubation for 4 hours, the intracellular drug concentration of free RIFs reached its peak.

[0145] ②RIF-PLGA NPs reached their peak at 6 h, and the amount of drug entering the cell at each time point was much higher than that of free RIF;

[0146] ③The fluorescence spectrum at 4 h also showed that RIF-PLGA NPs could promote macrophages to phagocytose more RIFs.

[0147] Conclusion: RIF loaded with PLGA NPs also has bactericidal activity, and has better intracellular bactericidal function compared with free RIF. This is mainly attributed to the fact that the PLGA nanocarrier promotes drug entry into the cell.

[0148] Example 3: Induction and Functional Verification of DC Vaccine

[0149] 1. Materials and Reagents

[0150] 6-8 week old SPF-grade BALB / c female mice, GM-CSF, IL-4 cytokines, inactivated Mycobacterium marineum, anti-CD11c, CD80, MHC II, CD69 flow cytometry antibodies, lymphocyte separation medium, CD3 + T-cell sorting kit.

[0151] Among them, Mycobacterium marineum (Mm) is disclosed in the literature "CN115252778A, A biomimetic vesicle complex based on preactivated macrophage membrane and its preparation method and application".

[0152] Inactivated marine mycobacteria include heat-inactivated marine mycobacteria and chemically inactivated marine mycobacteria;

[0153] Among them, the preparation of heat-inactivated Mycobacterium marineis: the prepared Mycobacterium marineis was boiled at 90℃ for 30 min to inactivate it; 100 μL of the inactivated Mycobacterium marineis solution was evenly coated on the surface of LB solid medium plate and cultured for 2 days. No colonies were produced, which proved that the inactivation was successful.

[0154] Preparation of chemically inactivated Mycobacterium marineis: The prepared Mycobacterium marineis was centrifuged, resuspended in 4% paraformaldehyde, fixed at room temperature for 1 h, washed twice with PBS at 3000 rpm, and finally resuspended in PBS. 100 μL of the resuspension was evenly coated on the surface of an LB agar plate. After culturing for 2 days, no colonies were produced, indicating successful inactivation.

[0155] 2. Experimental Methods

[0156] (1) DC vaccine induction:

[0157] ① Collect bone marrow cells from the femoral cavity of mice;

[0158] ②Induce bone marrow cells for 6 days using 20 ng / mL GM-CSF + 10 ng / mL IL-4;

[0159] ③ Add inactivated Mycobacterium marineum (MOI=1) for 2 days to induce induction;

[0160] ④ Flow cytometry analysis of CD11c expression confirms DC induction efficiency;

[0161] ⑤ Flow cytometry was used to detect molecules such as CD80 and MHC II to determine the maturity of DCs.

[0162] (2) T cell activation experiment:

[0163] ①Isolate lymphocytes from mouse spleen and sort CD3 cells. + T cells;

[0164] ② Incubate the DC vaccine and T cells at a ratio of 1:10 for 24 hours;

[0165] ③ Flow cytometry was used to detect CD69 expression on the surface of T cells.

[0166] (3) Immunosteric assay:

[0167] ① Incubate T cells or T cells activated by DC vaccine with RAW 264.7 macrophages infected with Mm for 36 h;

[0168] ② Lyse macrophages, serially dilute them and spread them on 7H10 plates, and count CFU.

[0169] 3. Experimental Results

[0170] (1) Characterization of DC vaccines: such as Figure 4 As shown:

[0171] ① Bone marrow cells induced by GM-CSF and IL-4 showed high expression of CD11c on their cell surface, indicating a high DC induction rate;

[0172] ② Both thermal inactivation and chemical inactivation methods can significantly increase the expression of CD80 and MHC II, two mature biomarkers, on the DC surface; thermal inactivation method will be selected uniformly in the future.

[0173] ③ This indicates that both inactivated antigens can be recognized and presented by DCs, and the mycobacterial-specific DC vaccine has been successfully prepared.

[0174] (2) T cell activation: such as Figure 5 As shown in Figure A, the expression of CD69 on the surface of T cells after incubation with DC vaccine is significantly increased, indicating that DC vaccine can significantly activate T cells.

[0175] (3) Immune bactericidal effect: such as Figure 5 As shown in B, T cells activated by the DC vaccine are more effective at clearing intracellular mycobacteria than untreated T cells.

[0176] Conclusion: The prepared DC vaccine can activate T cells and promote the clearance of intracellular mycobacteria by T cells, suggesting that mycobacteria-specific DC vaccines have an immunotherapeutic effect in tuberculosis.

[0177] Example 4: Preparation and characterization of drug-loaded DC vaccine membrane nanovesicles (RIF@DNPs)

[0178] 1. Materials and Reagents

[0179] Hypotonic cell lysis buffer (1 mmol / L NaHCO3, 0.2 mmol / L EDTA), protease inhibitor (phenylmethylsulfonyl fluoride, PMSF), Dunns homogenizer, BCA protein quantification kit, SDS-PAGE gel preparation kit, anti-CD11c, CD80 primary antibodies and corresponding secondary antibodies.

[0180] 2. Experimental Methods

[0181] (1) Extraction of DC vaccine membrane:

[0182] ① Collect DCs immunized with heat-inactivated Mycobacterium marineum and centrifuge and wash 3 times;

[0183] ② The cell pellet was resuspended in hypotonic cell lysis buffer containing protease inhibitor (1 mmol / L PMSF) and lysed overnight at -20°C;

[0184] ③Place the cell resuspension into a Durns homogenizer and homogenize the cells up and down 20 times on ice;

[0185] ④ Centrifuge at 3200 g for 5 min at 4℃ to remove large fragments and collect the supernatant;

[0186] ⑤ Centrifuge at 20000 g for 25 min at 4℃, discard the precipitate, and collect the supernatant;

[0187] ⑥ The supernatant was further centrifuged at 4°C and 100,000 g for 35 min, and the white precipitate (cell membrane) was collected, which is the DC vaccine membrane nanovesicles (DNPs).

[0188] ⑦ Resuspend in 500 μL 1×PBS, quantify membrane proteins using the BCA method, and store at -80℃ for later use.

[0189] (2) Synthesis of RIF@DNPs:

[0190] ① Take 0.5 mg of DC vaccine membrane nanovesicles (DNPs) and 1 mg of RIF-loaded PLGA nanoparticles (i.e., RIF-PLGANPs), mix and stir evenly.

[0191] ②The mixture was ultrasonicated in a 100 W water bath for 3 min, left to stand overnight at 4℃, and then centrifuged and washed 3 times to obtain biomimetic DC vaccine membrane nanoparticles, denoted as RIF@DNPs.

[0192] (3) Characterization methods:

[0193] ①TEM, particle size potential, and ultraviolet spectroscopy characterization are the same as in Example 1;

[0194] ② Protein characterization: Cell lysis buffer, DC vaccine membrane nanovesicles, and RIF@DNPs were lysed on ice for 30 min using protein lysis buffer, and then the proteins were quantified and analyzed by SDS-PAGE and Western blot, respectively.

[0195] 3. Experimental Results

[0196] (1) Morphological characteristics: such as Figure 6 As shown:

[0197] ① Transmission electron microscopy revealed that the DC vaccine membrane nanovesicles (DNPs) were nearly concave disc-shaped with a diameter of approximately 100-200 nm. Figure 6 (B in the middle)

[0198] ② After encapsulating RIF-PLGA NPs, RIF@DNPs exhibit a typical core-shell circular structure under electron microscopy. Figure 6 (C in the middle).

[0199] (2) Particle size and potential: such as Figure 6 As shown in D:

[0200] ①The hydrated particle size of individual DNPs is approximately 225 nm;

[0201] ②After adding RIF-PLGA NPs, the hydrated particle size increased to nearly 300 nm;

[0202] ③ The membrane potential displayed by DNPs is negative. After encapsulating RIF-PLGA NPs, the negative value of the RIF@DNPs potential increases slightly.

[0203] (3) Ultraviolet spectrum: such as Figure 6 As shown in E, RIF@DNPs have the same characteristic absorption peaks as RIF around 310 nm and 490 nm, indicating that RIF@DNPs have successfully encapsulated RIF-PLGA NPs.

[0204] (4) Protein characterization: such as Figure 7 As shown:

[0205] ① SDS-PAGE showed that PLGA nanoparticles loaded with RIF alone (i.e., RIF-PLGA NPs) did not express proteins because they did not cover the cell membrane. The protein bands of RIF@DNPs were basically the same as those of DNPs. Figure 7 (A in the middle)

[0206] ②Western blot showed that CD11c was expressed in DC lysate (unactive DC), DC vaccine lysate (active DC), DNPs, and RIF@DNPs;

[0207] ③CD80 is mainly expressed in DC vaccine lysates, DNPs, and RIF@DNPs, with less expression in ordinary unactivated DCs. Figure 7 (B in the middle).

[0208] Conclusion: RIF@DNPs derived from DC vaccine membranes have similar protein profiles to DC vaccine membranes and inherit DC vaccine-related functional receptors, suggesting that the constructed RIF@DNPs may also have DC vaccine-related immune activation functions.

[0209] Example 5: The Mycobacterial Scavenging Effect of RIF@DNPs

[0210] 1. Materials and Methods

[0211] The experimental materials and methods were the same as in Examples 2 and 3.

[0212] 2. Experimental Results

[0213] (1) Direct bactericidal effect: such as Figure 8 As shown:

[0214] ① RIF-PLGA NPs coated with DC vaccine nanovesicles (i.e., RIF@DNPs) have stronger antibacterial activity than uncoated RIF-PLGA NPs. Figure 8 (A in the middle)

[0215] ② Individual DNPs also have antibacterial effects;

[0216] ③ Only DNPs have a bactericidal effect on intracellular Mycobacterium tumefaciens in macrophages; the bactericidal effect of ordinary DC membrane nanovesicles (unactivated DNPs) is not obvious. Figure 8 (B in the middle).

[0217] (2) Immune bactericidal effect: such as Figure 9 As shown:

[0218] ① T cells activated by DNPs and PLGA@DNPs have a significantly stronger bactericidal effect than unactivated T cells;

[0219] ② The results of DNPs alone and PLGA@DNPs in promoting the bactericidal effect of T cells were similar, indicating that the encapsulation of PLGA NPs had little effect on the function of DNPs.

[0220] Conclusion: Compared with RIF-PLGA NPs, RIF@DNPs have enhanced intracellular bactericidal activity while retaining immune bactericidal function, suggesting that they may have better dual effects of nanochemotherapy and immunotherapy.

[0221] Example 6: Study on the immune activation mechanism of DNPs

[0222] 1. Materials and Methods

[0223] The CFSE cell proliferation assay kit, the TNF-α, IFN-γ, and IL-2 ELISA kit, and other materials are the same as in Example 3.

[0224] 2. Experimental Methods

[0225] (1) T cell activation detection:

[0226] ①Sorting CD3 + T cells were co-incubated with DNPs and PLGA@DNPs for 24 h, respectively.

[0227] ② Flow cytometry detection of CD4 + and CD8 + CD69 expression on the surface of T cells.

[0228] (2) T cell proliferation detection:

[0229] ①CD3 + T cells were stained with CFSE.

[0230] ② Co-incubate with DNPs and PLGA@DNPs for 72 h;

[0231] ③ Flow cytometry detection of CD4 + and CD8 + T cell proliferation.

[0232] (3) Cytokine detection:

[0233] ① T cells were co-incubated with DNPs and PLGA@DNPs for 48 h;

[0234] ② Centrifuge and collect the supernatant, then use ELISA to detect the concentrations of TNF-α, IFN-γ, and IL-2.

[0235] 3. Experimental Results

[0236] (1) CD4 T cell activation: such as Figure 10 As shown:

[0237] ① Both DNPs and PLGA@DNPs significantly increased CD4. + CD69 expression on the surface of T cells;

[0238] ②For CD8 + The role of T cells is not obvious;

[0239] ③ Explanation: DNPs and PLGA@DNPs primarily activate CD4. + T cells.

[0240] (2) T cell proliferation: such as Figure 11 As shown:

[0241] ① T cells showed significant proliferation after treatment with DNPs and PLAG@DNPs;

[0242] ② Mainly for CD4 + T cells have a significant proliferative effect on CD8. + T cells have a weak proliferative effect;

[0243] ③ Individual PLGA NPs for CD4 + T cells and CD8 + T cells had no significant effect.

[0244] (3) Cytokine release: such as Figure 12 As shown:

[0245] ① The concentration of TNF-α in the supernatant of T cells incubated with DNPs and PLGA@DNPs was significantly higher than that in the control group;

[0246] ②IFN-γ concentration increased slightly;

[0247] ③The IL-2 concentration did not change significantly.

[0248] Conclusion: DNPs and PLGA@DNPs mainly act on CD4. + T cells, promoting CD4 + T cell activation and proliferation, along with increased release of anti-tuberculosis-related cytokines (especially TNF-α), are the main physiological mechanisms by which they exert their immune-killing function.

[0249] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A drug-loaded DC vaccine membrane nanovesicle specifically for Mycobacterium tuberculosis, characterized in that, The nanovesicles have a core-shell structure and include: a core: PLGA nanoparticles loaded with anti-tuberculosis drugs; The outer shell is derived from cell membrane vesicles of a Mycobacterium tuberculosis-specific DC vaccine; The drug-loaded DC vaccine membrane nanovesicles exhibit mycobacterial-associated antigens on their surface, retaining the original DC vaccine membrane surface proteins and molecules, and possessing the function of activating Mycobacterium tuberculosis-specific T cells.

2. The drug-loaded DC vaccine membrane nanovesicle according to claim 1, characterized in that: The anti-tuberculosis drug is selected from at least one of rifampin and isoniazid; And / or, the mass ratio of the anti-tuberculosis drug to PLGA is (2.5–15):15; And / or, the mass ratio of the cell membrane vesicles to the PLGA nanoparticles loaded with anti-tuberculosis drugs is 1:2 to 5; And / or, the Mycobacterium tuberculosis-specific DC vaccine is obtained by inducing DC cells with inactivated Mycobacterium tuberculosis at an MOI of 1 to 10; And / or, the cell membrane vesicles are obtained by cryolysis using a hypotonic cell lysis buffer containing protease inhibitors, followed by mechanical disruption and homogenization, and then differential centrifugation; And / or, the hydrated particle size of the drug-loaded DC vaccine membrane nanovesicles is 250–350 nm, and the surface potential is negative.

3. The drug-loaded DC vaccine membrane nanovesicle according to claim 2, characterized in that: The differential centrifugation process involves first centrifuging at 3000–3500 g for 4–6 min at 2–6 °C and collecting the supernatant; then centrifuging at 18000–22000 g for 20–30 min at 2–6 °C and collecting the supernatant; and finally centrifuging at 90000–110000 g for 30–40 min at 2–6 °C. The resulting white precipitate is then resuspended in PBS and stabilized to obtain the cell membrane vesicle solution. Furthermore, the differential centrifugation involves first centrifuging at 3200 g for 5 min at 4°C and collecting the supernatant; then centrifuging at 20000 g for 25 min at 4°C and collecting the supernatant; and finally centrifuging at 100000 g for 35 min at 4°C. The white precipitate collected is then resuspended and stabilized with PBS to obtain the cell membrane vesicle solution.

4. The method for preparing Mycobacterium tuberculosis-specific drug-loaded DC vaccine membrane nanovesicles according to any one of claims 1 to 3, characterized in that, Includes the following steps: S1. DC vaccine induction: DC cells are co-incubated with inactivated Mycobacterium tuberculosis to induce mature Mycobacterium tuberculosis-specific DC vaccine. S2. Cell membrane extraction: Collect DC vaccines and extract cell membranes through hypotonic lysis, homogenization, and differential centrifugation to prepare Mycobacterium tuberculosis-specific DC vaccine membrane nanovesicles; S3. Preparation of drug-loaded PLGA nanoparticles: PLGA nanoparticles loaded with anti-tuberculosis drugs were prepared by water-emulsion method. S4. Fusion of membrane vesicles and nanoparticles: DC vaccine membrane nanovesicles are mixed with PLGA nanoparticles loaded with anti-tuberculosis drugs, ultrasonically treated, and then incubated. After centrifugation and washing, drug-loaded DC vaccine membrane nanovesicles are obtained.

5. The preparation method according to claim 4, characterized in that: In step S1, the inactivation method includes chemical inactivation or thermal inactivation; And / or, step S3 specifically includes: S31. Dissolve PLGA in an organic solvent, dissolve the anti-tuberculosis drug in an organic solvent, and then mix thoroughly to obtain an organic solution of PLGA containing the anti-tuberculosis drug. S32. Rapidly inject the PLGA organic solution containing anti-tuberculosis drugs into the aqueous phase, with the volume ratio of organic solution to aqueous phase being 1:4 to 10. S33. After ultrasonic emulsification, stirring was performed to evaporate the organic solvent, resulting in an aqueous solution of drug-loaded PLGA nanoparticles. S34. After centrifugation and washing, the PLGA nanoparticles loaded with anti-tuberculosis drugs were resuspended in PBS to obtain a solution of PLGA nanoparticles loaded with anti-tuberculosis drugs. And / or, step S4 specifically involves: mixing DC vaccine membrane nanovesicles with PLGA nanoparticles loaded with anti-tuberculosis drugs at a mass ratio of 1:2 to 5, sonicating in a 100 W water bath for 3 to 5 minutes, letting stand overnight at 4°C, and washing by centrifugation 3 times to obtain the final product.

6. The preparation method according to claim 5, characterized in that: In step S31, the organic solvent includes dichloromethane; And / or, in step S32, the aqueous phase is an aqueous phase containing polyvinyl alcohol; And / or, in step S33, the conditions for ultrasonic emulsification are 80-100 W ultrasound for 5-10 min; And / or, in step S34, the centrifugation speed is 6000-8000 g.

7. The preparation method according to claim 6, characterized in that: In step S32, the concentration of polyvinyl alcohol in the aqueous phase is 0.5–2% w / v; And / or, in step S33, the conditions for ultrasonic emulsification are 100 W ultrasound for 5 min; And / or, in step S34, the centrifugation speed is 7000 g.

8. A pharmaceutical composition, characterized in that, It includes drug-loaded DC vaccine membrane nanovesicles as described in any one of claims 1 to 3.

9. The application of the drug-loaded DC vaccine membrane nanovesicles according to any one of claims 1 to 3 or the pharmaceutical composition according to claim 8, characterized in that, For one or more of the following applications: (a) Its use in the preparation of drugs that kill Mycobacterium tuberculosis; (b) Its use in the preparation of medicines for treating tuberculosis; (c) Application in the preparation of drugs that kill Mycobacterium marinum; (d) Application in the preparation of drugs for treating diseases related to Mycobacterium marinum.

10. The application according to claim 9, characterized in that: The drug exerts its anti-tuberculosis effect through nanochemotherapy combined with immunotherapy; And / or, the drug is able to activate CD4 + T cells promote T cell proliferation and release anti-tuberculosis-related cytokines.

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