A hybrid vesicle loaded with tumor antigen and preparation method and application thereof

Abstract

The application discloses a tumor antigen-loaded hybrid vesicle and a preparation method and application thereof, and belongs to the field of biological medicines. The hybrid vesicle is fused from a bacterial outer membrane vesicle and a red blood cell membrane. The bacterial outer membrane vesicle is an outer membrane vesicle extracted from gram-negative bacteria, and the red blood cell membrane is a mouse red blood cell membrane. The mass ratio of the bacterial outer membrane vesicle to the red blood cell membrane is 1:(1-100). The hybrid vesicle combines the characteristics of the bacterial outer membrane vesicle and the red blood cell membrane, has a stable and uniform structure, can efficiently and rapidly target the spleen and deliver tumor cell antigens, induces the maturation and antigen presentation of antigen-presenting cells, further activates the immune system, and has a remarkable effect on tumor prevention and treatment.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, in particular to a hybrid vesicle loaded with tumor antigen and a preparation method and application thereof. BACKGROUND

[0002] Tumor immunotherapy is a research hotspot in the field of tumor treatment, which can stimulate the body's immune system and activate related immune cells and factors to attack tumor tissue. Nanoparticle-based vaccine, namely nanovaccine, has attracted great attention in the field of tumor immunotherapy. By co-delivering tumor antigens and immunostimulatory adjuvants into antigen-presenting cells (APCs), the maturation and antigen presentation of APCs are induced, and the antigen-specific T cell immune response is further stimulated. Although some progress has been made in the research of tumor vaccine, a fundamental challenge lies in the insufficient ability to induce the full maturation of dendritic cells (DCs), which are the most effective APCs. Based on the efficient recognition and phagocytosis of DCs to bacteria, we speculate that engineered nanovaccines composed of components derived from bacteria will be beneficial to their targeting of DCs. In addition, components derived from bacteria inherit multiple pathogen-associated molecular patterns (PAMPs) from parent bacteria, which are more likely to stimulate effective immune synergy than whole pathogen adjuvants. Spleen is an important lymphoid organ containing various immune factors such as antigen-presenting cells. Therefore, spleen targeting can stimulate effective systemic immune response to attack tumor tissue.

[0003] In view of this, the present application is proposed. SUMMARY

[0004] The purpose of the present application is to provide a hybrid vesicle loaded with tumor antigen and a preparation method and application thereof, so as to solve the problems existing in the prior art. The hybrid vesicle of the present application combines the characteristics of bacterial outer membrane vesicles and red blood cell membranes, and has stable and uniform structure, can efficiently and rapidly target the spleen and promote antigen presentation, further activate the immune system, and has significant effect in the prevention of tumors, etc.

[0005] To achieve the above purpose, the present application provides the following solutions.

[0006] The present application provides a hybrid vesicle loaded with tumor antigen, which is fused from a bacterial outer membrane vesicle and a red blood cell membrane. The bacterial outer membrane vesicle is an outer membrane vesicle extracted from gram-negative bacteria, and the red blood cell membrane is a mouse red blood cell membrane.

[0007] Preferably, the mass ratio of the bacterial outer membrane vesicle to the red blood cell membrane is 1:(1-100).

[0008] Preferably, the mass ratio of the bacterial outer membrane vesicle to the red blood cell membrane is 1:5.

[0009] The application also provides a preparation method of the hybrid vesicle, comprising the steps of mixing the bacterial outer membrane vesicle and the red blood cell membrane, and co-extruding the hybrid vesicle through a filter membrane.

[0010] Preferably, the preparation method comprises the steps of extruding the red blood cell membrane through a filter membrane first, then mixing the bacterial outer membrane vesicle and the red blood cell membrane, ultrasonic treating the mixed system in an ice water bath until the mixed system is slightly transparent, oscillating the mixed system in a water bath, and co-extruding through the filter membrane. The filter membrane is preferably a polycarbonate porous membrane.

[0011] Preferably, the ultrasonic treating in the ice water bath is performed at 100-200 W for 20-30 min, and the oscillating in the water bath is performed at 35-40 ℃ for 0.5-2 h.

[0012] The application also provides a hybrid vesicle nanoparticle loaded with tumor antigens, comprising the hybrid vesicle and the tumor antigens wrapped by the hybrid vesicle.

[0013] Preferably, the hybrid vesicle is in a 1:1 ratio with the tumor antigens, and the tumor antigens are derived from mouse T lymphoma cells.

[0014] The application also provides a preparation method of the hybrid vesicle nanoparticle, comprising the steps of ultrasonic breaking the tumor antigens, ultrasonic mixing the tumor antigens with the hybrid vesicle, and extruding through a filter membrane to obtain the hybrid vesicle nanoparticle.

[0015] The application also provides application of the hybrid vesicle or the hybrid vesicle nanoparticle in preparation of a drug for preventing and / or treating melanoma, wherein the prevention and / or treatment of melanoma comprises inhibiting growth and metastasis of tumors.

[0016] The application discloses the following technical effects:

[0017] 1) The application can fuse the bacterial outer membrane vesicle and the red blood cell membrane well, so as to form an engineered hybrid vesicle, which has the characteristics of targeting the spleen of damaged red blood cell membranes and the adjuvant characteristics of bacterial outer membrane vesicles.

[0018] 2) The hybrid vesicle construction method in the application is simple, efficient, easy to operate, and the formed structure is stable and uniform. The vesicle can deliver tumor antigens to the target site, effectively avoiding premature separation in the biological environment.

[0019] 3) Based on the good characteristics of the hybrid vesicle, tumor endogenous antigens derived from tumor cells are loaded as training clues, and different T cell libraries are created by amplifying the processing and presentation of tumor-specific antigens. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 The diagram shows the particle size, potential distribution, and stability distribution of the bacterial outer membrane vesicles, erythrocyte membranes, hybrid vesicles, and nanoparticles of this invention.

[0022] Figure 2 The results of morphological observation of bacterial outer membrane vesicles (OMVs), erythrocyte membrane RBCs, and their hybrid vesicles (ROs) by transmission electron microscopy.

[0023] Figure 3 SDS-PAGE protein electrophoresis images of bacterial outer membrane vesicles, erythrocyte membranes, tumor antigens, and nanoparticles in this invention;

[0024] Figure 4 These are laser confocal fluorescence images of bacterial outer membrane vesicles after extrusion and fusion with erythrocyte membranes and images of physical mixing only, as described in this invention.

[0025] Figure 5 The stimulation effect of different ratios of hybrid membranes on BMDCs cells in this invention is shown by the average fluorescence intensity and positive rate of CD80 and CD86 molecules on the surface of BMDCs; and the antigen presentation effect on the model antigen OVA.

[0026] Figure 6 The results of immunoassay of each component in mice in this invention;

[0027] Figure 7 This is a schematic diagram of a mouse melanoma prevention model experiment, with the groups being PBS, TA, TA-R, TA-O, Mix group (TA+RBCs+OMVs) and TA@RO group, respectively.

[0028] Figure 8 This is a comparative image of the final anatomical structure of tumors in mice under different drug treatments during a mouse melanoma prevention model experiment.

[0029] Figure 9 The growth curves of the whole body and tumors in each group of mice under different drug treatments in a mouse melanoma prevention model experiment;

[0030] Figure 10 This image shows the tumor weight of mice in different treatment groups during a mouse melanoma prevention model experiment.

[0031] Figure 11 The schematic diagram of mouse lung metastasis model experiment, the groups are PBS, TA-R, TA-O and TA@R-O groups respectively;

[0032] Figure 12 The mouse lung dissection diagram under different drug treatments in the mouse lung metastasis model experiment. DETAILED DESCRIPTION

[0033] A number of illustrative embodiments of the present application are described in detail below, which should not be considered limiting of the application, but rather as being exemplary. Specific aspects, features and embodiments of the application are described in this detailed description.

[0034] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. Additionally, for a range of values of a parameter, unless otherwise stated, each intervening value of the parameter is also specifically included within the scope of the present application. The intervening values of the parameter are combined with a specific value of the parameter in range to each encompass every combination of the specific value and the intervening values. These intervening values are specifically included within the scope of the present application.

[0035] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although preferred methods and materials are described, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application. All documents mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the documents are cited. In case of conflict, the content of the present specification will control.

[0036] Various modifications and changes can be made to the specific embodiments of the present application described herein without departing from the scope or spirit of the application. Other embodiments of the application will be apparent to those of ordinary skill in the art from the description and examples presented herein. The description and examples are illustrative of the application and are not intended to limit the scope of the application.

[0037] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having", "contains", "containing", or variations thereof, are intended to be open-ended terms that mean including, but not limited to.

[0038] Example 1 Preparation method of hybrid vesicle R-O

[0039] 1. Extraction of bacterial outer membrane vesicles OMVs

[0040] The activated MG1655 E. coli was inoculated into 1 L of LB medium and cultured at 37°C and 220 rpm. When the OD 600 was 1, the shaking was stopped, the bacterial solution was centrifuged at 8000 g and 4°C for 10 min, and the supernatant was collected. Then, the bacterial solution was vacuum filtered through a 0.45 μm acetic anhydride membrane to further remove the bacterial bodies. The filtrate was centrifuged at 2500 g for 10 min using a 100 kd, 15 mL ultrafiltration tube, and the concentrated solution was collected. The concentrated solution was filtered through a 0.45 μm microporous filter and then ultracentrifuged at 150000 g and 4°C for 3 h. The supernatant was discarded, the precipitate was resuspended in sterile 1 mL PBS buffer, quantified by the BCA method, and stored at -80°C, thereby obtaining bacterial outer membrane vesicles OMVs.

[0041] 2. Extraction of red blood cell membranes RBCs

[0042] The mouse was subjected to orbital blood collection in an anticoagulant tube. The blood was centrifuged at 800 g and 4°C for 10 min, and the supernatant was discarded. The red cell precipitate was resuspended in 2 mL of PBS buffer and centrifuged again at 800 g and 4°C for 5 min, and the supernatant was discarded. This step was repeated until the supernatant was clear and transparent. The precipitate was resuspended in 1 / 4 PBS buffer and placed on ice for 40 min. The precipitate was centrifuged at 9000 rpm and 4°C for 10 min. 1.5 mL of the supernatant was discarded, and 1.5 mL of PBS buffer was added. The precipitate was centrifuged again at 9000 rpm and 4°C for 10 min, and the supernatant was discarded. The precipitate was resuspended in 1 / 4 PBS buffer, placed on ice for 10 min, and centrifuged at 9000 rpm and 4°C for 10 min. The supernatant was discarded, and this step was repeated until the supernatant was clear and transparent. The precipitate was washed twice with PBS solution and placed at 37°C for 1 h and at 4°C for storage.

[0043] 3. Preparation of hybrid vesicles R-O

[0044] The red cell body precipitate was suspended in pre-cooled isotonic saline and passed through polycarbonate porous membranes with pore sizes of 800 nm, 400 nm, and 200 nm in sequence, thereby obtaining red blood cell membrane vesicles RBCs. OMVs and RBCs were mixed at a desired ratio [mass:volume ratio 1 : (1-100)], and the system was subjected to ultrasonic treatment at 100 W in an ice water bath for 30 min until the system was slightly transparent. The system was placed in a 37°C water bath and shaken for 1 h. Then, the system was extruded 21 times at 200 nm, thereby obtaining R-O hybrid membranes.

[0045] Example 2: Mouse bone marrow-derived dendritic cell (BMDC) stimulation experiment

[0046] Four-week-old male C57BL / 6 mice were purchased. Surgical instruments were autoclaved. The mice were euthanized and completely immersed in 75% alcohol for 20 minutes. The entire leg was removed and immersed in 75% alcohol. The tendons, femur, and tibia joints and excess muscles were removed. The tibia and femur were immersed in PBS. The ends of the femur or tibia were cut off. The bone marrow was blown out into a 50mL centrifuge tube using a 1mL syringe containing sterile PBS (containing antibiotics). The tube was rinsed repeatedly until clean. The mixture was mixed by pipetting and filtered through a 40μm filter. The tube was centrifuged at 800g at 4℃ for 8 minutes. 1× erythrocyte lysis buffer (1mL 10× erythrocyte lysis buffer + 9mL pure water) was prepared. The supernatant was discarded, 10mL of erythrocyte lysis buffer was added, and the mixture was mixed by pipetting and incubated at 37℃ for 10 minutes. The tube was centrifuged at 800g at 4℃ for 8 minutes. The supernatant was discarded. The tube was resuspended in PBS and centrifuged at 800g. Centrifuge at 4℃ for 8 min; prepare DCs medium (50 mL): add 10 μL LGM-CSF (10 ng / mL) and 10 μL IL-4 (20 ng / mL) to the medium containing 1% antibiotics and 10% FBS1640; discard the supernatant, resuspend the DCs in the medium, count the cells, and repeat at 5 × 10⁻⁶. 5 Seeds were seeded per well in 24-well plates, 1 mL per well, and incubated at 37°C with 5% CO2. After 48 hours of incubation, the medium was changed, and 800 μL of culture medium was added to 900 μL of culture medium, and incubated at 37°C with 5% CO2 (Day 2). After 48 hours of incubation, the medium was changed again, and 500 μL of culture medium was added to 1 mL of culture medium (Day 4). After 24 hours of incubation, the medium was changed again, and 1 mL of culture medium was added to 500 μL of culture medium (Day 5). After 24 hours of incubation, the medium was changed again, and 500 μL of culture medium was added to 500 μL of culture medium. (Day 6); Different groups (PBS, OMVs and RBCs ratios of 1:0, 1:5, 1:25, 1:50, 0:1, 1:100) were co-incubated with BMDCs for 48 h; the supernatant was aspirated, and cells were collected by blowing off the supernatant with 1 mL of PBS; the cells were centrifuged at 800 g at 4 °C for 8 min, and washed once with 0.5-1 mL of flow cytometry buffer; antibody staining solution was prepared: 1 μL each of PerCP-CD11c, FITC-CD80, APC-CD86, and PE-H-2Kb bound to SIINFEKL were added to 100 μL of PBS; the supernatant was discarded, 100 μL of antibody was added, and the mixture was incubated at 4 °C for 30 min in the dark; 1 mL of PBS was added, and the mixture was mixed, and centrifuged at 800 g at 4 °C for 8 min; the supernatant was discarded, and 1 mL of antibody was added. Add PBS, mix well, centrifuge at 800g at 4℃ for 8 min; discard the supernatant, add 200μL PBS, mix well, transfer to a flow cytometer, and analyze.

[0047] The results are as follows Figure 5 As shown, when the ratio of OMVs to RBCs is 1:5, heterozygous vesicles can significantly accelerate the maturation of DCs in vivo, while also exhibiting a high antigen cross-presentation capacity.

[0048] Extraction of tumor antigen TA

[0049] Mouse T lymphoma cells (EG.7-OVA) were collected, resuspended in culture medium to 10 7 mL; transferred to a cryopreservation tube, frozen in liquid nitrogen and thawed in a 37°C water bath, repeated 3-5 times to fully lyse the cells; centrifuged at 12000g at 4°C for 10 min, and the supernatant was collected to remove organelles, cell membranes and other substances; the supernatant was quantified by BCA and stored at -20°C. The tumor antigen was obtained.

[0050] Preparation of nanoparticles TA@R-O

[0051] The tumor antigen in Example 3 was ultrasonically broken, mixed with hybrid vesicles at a mass-volume ratio of 1:1 under 100-150W ultrasonic conditions, and passed through a 200nm polycarbonate porous membrane to obtain nanoparticles.

[0052] Characterization of Example 4

[0053] The OMVs, RBCs, hybrid vesicles R-O extracted in Example 1 and TA@R-O prepared in Example 4 were characterized, and the experimental steps were as follows:

[0054] 1. Particle size and potential

[0055] The particle size and potential distribution of the above four kinds of particles were measured by Malvern particle size analyzer, and the results are shown in Figure 1 , wherein the particle size of the bacterial outer membrane vesicle is 111.2nm, and the potential is -4.25mV; the particle size of the red blood cell membrane after being extruded through a 200nm polycarbonate porous membrane is 184.9nm, and the potential is -9.12mV; the particle size of the hybrid vesicle is 200.9nm, and the potential is -9.27mV; the particle size of the nanoparticle is 214.1nm, and the potential is -10.5mV.

[0056] 2. Electron microscopy

[0057] 3-5μL of each of the above four component solutions was dropped on an amorphous carbon film copper grid carrier, dried for 1min, then 4μL of 2% uranyl acetate dye was dropped on the copper grid, and immediately dried. OMVs, RBCs, R-O and TA@R-O were characterized by FEI Tecnai Spirit transmission electron microscope, and the results are shown in Figure 2 , which all showed typical vesicle morphology similar to OMVs, and TA@R-O formed a more complex morphology.

[0058] 3. SDS-PAGE protein analysis

[0059] First, the protein of each component was quantified by BCA method; 20 mL 12% separation gel was prepared [6.6 mL H2O, 8.0 mL 30% acrylamide, 5.0 mL 1.5 M Tris-HCl (pH 8.80), 0.2 mL 10% SDS, 0.2 mL 10% ammonium persulfate, 0.008 mL tetramethyl ethylenediamine (TEMED)], mixed quickly, poured into the plate, sealed, and allowed to solidify for 30 min; 4 mL concentrated gel was prepared [2.7 mL H2O, 0.67 mL 30% acrylamide, 0.5 mL 1.0 M Tris (pH 6.8), 0.04 mL 10% SDS, 0.04 mL 10% ammonium persulfate, 0.004 mL TEMED], the water seal was discarded, the concentrated gel was added, and a comb was immediately inserted, and allowed to solidify for 30 min; 500 mL 1x electrophoresis buffer was added to the electrophoresis tank, and the sample marker and each group of samples were added in turn, and electrophoresis was performed at 80 V and 200 mA for 30 min, and then at 120 V until the buffer reached the bottom of the gel; the gel was removed, 100 mL coomassie brilliant blue staining solution was added, and staining was performed for 30 min, and the decolorizing solution was washed until the solution was clear.

[0060] The results are shown in Figure 3 The specific components of the red blood cell membrane and the bacterial outer membrane vesicle are shown.

[0061] 4. Laser confocal imaging

[0062] 500 μL 1% polylysine was added to the confocal dish, and the dish was placed in a cell incubator for 4 h to overnight, the excess liquid was removed, and PBS was washed 3 times for standby; FITC-labeled LPS antibody was added to the OMVs, mixed evenly, and stained at 37°C for 1.5 h; 300 kd ultrafiltration tube was used, centrifugation was performed at 5000 g for 10 min, and the free antibody was removed; RBCs were labeled with DiI dye, and staining was performed at 37°C for 1 h; 150 μL of each of the two components after staining was mixed, and a manual liposome extruder was used to extrude through a 200 nm polycarbonate porous membrane for 21 times as hybrid vesicles; the physical mixing Mix group was mixed evenly by taking 100 μL of each of the above FITC-OMVs solution and RBCs solution; the prepared RBCs, OMVs, Mix, and R-O were added to the coated confocal dish, and the dish was placed in a 37°C incubator for 1 h, and the excess liquid was removed; PBS was added gently, and the fluorescence distribution was observed by laser scanning confocal microscopy.

[0063] The results are shown in Figure 4 Green is the FITC antibody fluorescence on OMVs, red is the DiI fluorescence of RBCs, and compared with the Mix group, the two fluorescence channels show red and green alternately, and the two fluorescence channels in the R-O group are mixed as gold, indicating that OMVs and RBCs are successfully fused.

[0064] In summary, bacterial outer membrane vesicles (OMVs) and erythrocyte membrane RBCs were successfully fused into hybrid vesicles (ROs) using a manual liposome extruder.

[0065] Example 5: Mouse Immunization Effect Test

[0066] The groups were PBS, TA, TA-R, TA-O, Mix (TA+RBCs+OMVs), and TA@RO, with dosages of 10 μg / mouse for OMVs, 50 μg / mouse for RBCs, and 120 μg / mouse for TA@RO, respectively. C57 mice were administered the drugs via tail vein injection on days 0, 2, and 4. Blood was collected from mice at the end of day 6, allowed to stand at room temperature for 2 hours, centrifuged, and the serum was collected and stored at -80°C for later use. Mice were euthanized, and the spleen was dissected, photographed, and weighed. The spleen was ground and used for flow cytometry analysis. The heart, liver, spleen, lungs, and kidneys of mice were fixed in 4% tissue fixative for subsequent HE staining.

[0067] The results are as follows Figure 6 As shown, the TA@RO group significantly accelerated the maturation of DCs in vivo and exhibited high antigen cross-presentation capacity; compared with other groups, TA@RO significantly reduced the activity of CD4+ in the spleen. + T cells and CD8 + T cells have good expansion and activation effects and effectively stimulate the secretion of IFN-γ.

[0068] Example 6: Mouse melanoma prevention model experiment

[0069] Experimental protocol as follows Figure 7 As shown, the groups were PBS, TA, TA-R, TA-O, Mix (TA+RBCs+OMVs), and TA@RO. The nano-vaccine was administered via tail vein injection on Day 9, Day 6, and Day 3, and B16-OVA cells were subcutaneously inoculated on Day 0 at a dose of 1×10⁻⁶. 6 Cell / mouse; mouse body weight and tumor size were measured every two days until the tumor size in the PBS group reached 1000 mm. 3 At this point, the mouse was dissected and the tumor was removed.

[0070] The results are as follows Figure 8-10 As shown, the results indicate that the TA@RO group significantly inhibited tumor development, with two tumor-free cells among them. The remaining groups showed a certain preventive effect compared to the PBS group.

[0071] Example 7: Mouse Lung Metastasis Model Experiment

[0072] Experimental protocol as follows Figure 11As shown, the groups are PBS, TA-R, TA-O and TA@R-O groups. The mice were injected with the nano-vaccine through tail vein at Day-4, Day 3, Day 6 and Day 9, and injected with B16-OVA-luc cells through tail vein at Day 0, and dissected at Day 28. The results are shown in the following table. Figure 12 As shown, the results show that the TA@R-O group can significantly inhibit tumor metastasis, and the lung has only a small amount of nodules, followed by the TA-R group.

[0073] In summary, the present application provides a hybrid vesicle of bacteria and red blood cell sources, which can load tumor antigens, and the hybrid vesicle comprises a mixed membrane structure of a bacterial outer membrane vesicle and a red blood cell membrane, wherein the bacteria are gram-negative bacteria, and the red blood cell membrane is derived from mouse red blood cells. The bacterial outer membrane vesicle contains multiple pathogen-associated molecular patterns (PAMPs) derived from parent bacteria, which can interact with pattern recognition receptors on dendritic cells, thereby triggering effective immune synergy. The spleen is the largest lymphatic organ in the human body, which has the functions of storing blood, producing blood, removing senescent red blood cells and responding to immune responses. When blood is transported to the spleen, newly born red blood cells can continue to stay in the plasma and pass through the spleen by cell membrane deformation, while senescent or damaged red blood cells are intercepted in the spleen and further phagocytosed and removed. Therefore, it is suggested that damaged red blood cells have the ability to target the spleen. On the basis of the hybrid vesicle, tumor antigens are loaded, i.e. the antigens are wrapped in the hybrid vesicle, and tumor endogenous antigens derived from tumor cells serve as training clues to create different T cell libraries by amplifying the processing and presentation of tumor-specific antigens. In addition, the tumor antigens wrapped in the hybrid vesicle avoid premature separation in the biological environment.

[0074] The hybrid vesicle of the present application combines the characteristics of both OMVs and RBCs, and is hybridized. The OMVs trigger effective immune synergy, and the damaged RBCs target the spleen, induce the maturation and antigen presentation of APCs, and have significant effects on the prevention of tumors after loading tumor endogenous antigens. In addition, the hybrid vesicle has a stable and uniform structure, and the particle size is 200nm-230nm. The proteins on the hybrid vesicle are analyzed by SDS-PAGE and Western Blot methods, which are derived from the bacterial outer membrane vesicle and the red blood cell membrane. The immune regulation of different proportions of hybrid vesicles in DCs cells, when the fusion ratio is 1:5, the hybrid vesicle can significantly promote the maturation of DCs, and has the highest antigen presentation efficiency; by co-incubating different proportions of hybrid vesicles and DCs, the hybrid vesicle can significantly up-regulate the co-stimulatory molecules CD86 and CD86 of DCs, and effectively present the model antigen OVA. The physiological characteristics of the fusion vesicle of the present application have good spleen targeting efficiency and antigen delivery capacity.

[0075] The above described embodiments are only to illustrate the preferred modes of the present application, and are not intended to limit the scope of the present application. Any modification and improvement made by those skilled in the art to the technical solutions of the present application without departing from the design spirit of the present application shall fall within the protection scope of the present application as defined by the claims.

Claims

1. A hybrid vesicular nanoparticle loaded with a tumor antigen, characterized in that, The hybrid vesicle and a tumor antigen wrapped by the hybrid vesicle; The hybrid vesicle is fused from a bacterial outer membrane vesicle and a red blood cell membrane, the bacterial outer membrane vesicle is an outer membrane vesicle extracted from a gram-negative bacterium, and the red blood cell membrane is a mouse red blood cell membrane; The mass ratio of the bacterial outer membrane vesicle to the red blood cell membrane is 1:

5.

2. The hybrid vesicular nanoparticle of claim 1, wherein, The hybrid vesicle is 1:1 with the tumor antigen, and the tumor antigen is derived from a mouse T lymphoma cell.

3. A method for preparing the hybrid vesicle as set forth in claim 1, characterized by, The method comprises the steps of mixing a bacterial outer membrane vesicle and a red blood cell membrane and co-extruding the hybrid vesicle through a filter membrane.

4. The production method according to claim 3, wherein The preparation method comprises the following steps: first, extruding the red blood cell membrane through a filter membrane, then mixing the bacterial outer membrane vesicle with the red blood cell membrane, ultrasonic treating the mixed system to be micro-transparent in an ice water bath, then oscillating the mixed system in a water bath, and finally co-extruding through the filter membrane.

5. The production method according to claim 4, wherein The ice water bath ultrasonic treatment is performed at 100-200 W for 20-30 min, and the oscillation treatment in the water bath is performed at 35-40 ℃ for 0.5-2 h.

6. A method of producing the hybrid vesicular nanoparticle of claim 1 or 2, wherein the method comprises, The method comprises the following steps: The tumor antigen is ultrasonically broken, then ultrasonically mixed with the hybrid vesicle, and then extruded through a filter membrane to obtain the hybrid vesicle nanoparticles.

7. Use of the hybrid vesicular nanoparticle of claim 1 or 2 for the preparation of a medicament for the prevention and / or treatment of melanoma. Preventing and / or treating melanoma comprises inhibiting the growth and metastasis of a tumor.

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