Engineering bionic nano dendritic cell vesicle, melanoma vaccine, preparation method and application

Abstract

The invention provides an engineered bionic nano dendritic cell vesicle, a melanoma vaccine, a preparation method and application, and particularly belongs to the technical field of biological medicine. The engineering bionic nano dendritic cell vesicle is loaded with an interferon gene stimulating factor agonist and a holoantigen engineering vesicle which is derived from melanoma cells and is subjected to oxidation treatment. The engineered bionic nano dendritic cell vesicles can be used for preparing melanoma vaccines, are good in safety and strong in stability, and have a good anti-melanoma effect.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to an engineered bionic nano dendritic cell vesicle, a melanoma vaccine, and a preparation method and application thereof. Background Art

[0002] Melanoma vaccines stimulate the body's immune response by displaying specific tumor antigens produced by melanoma, which enables the body to produce anti-tumor immune effects and long-term immune memory, thereby preventing the growth, spread and even recurrence of tumors. How to accurately select effective antigens to stimulate specific immune responses, and how to design and develop new vaccine vectors that can improve vaccine efficacy and safety are the current research focuses of melanoma vaccines. Existing whole-cell vaccines containing all melanoma antigens can theoretically prevent tumors from escaping immune attacks, but their immune effects are not strong enough and need to be improved. Summary of the invention

[0003] The purpose of the present invention is to provide an engineered bionic nano dendritic cell vesicle, a melanoma vaccine, and a preparation method and application thereof. The engineered bionic nano dendritic cell vesicle of the present invention can be used to prepare a melanoma vaccine, has good safety, strong stability, and good immune and anti-melanoma effects.

[0004] The invention provides an engineered bionic nano dendritic cell vesicle, wherein the engineered bionic nano dendritic cell vesicle is loaded with an interferon gene stimulating factor agonist and an oxidized melanoma cell-derived full antigen engineered vesicle.

[0005] Preferably, the particle size of the engineered bionic nano-dendritic cell vesicles is 150-200 nm.

[0006] Preferably, the interferon gene stimulator agonist comprises diABZI.

[0007] Preferably, the method for preparing the oxidized melanoma cell-derived full antigen engineered vesicles comprises the following steps:

[0008] The melanoma cells are subjected to hypochlorous acid oxidation treatment to obtain oxidized melanoma cells;

[0009] incubating the oxidized melanoma cells to obtain incubated melanoma cells;

[0010] The incubated melanoma cells are subjected to membrane filtration by an extrusion method to obtain fully antigen engineered vesicles derived from oxidized melanoma cells.

[0011] Preferably, the concentration of hypochlorous acid used in the oxidation treatment is 300-450 μM.

[0012] The present invention also provides a method for preparing the engineered bionic nano dendritic cell vesicles described in the above technical solution, comprising the following steps:

[0013] The dendritic cells are mixed with the fully antigen-engineered vesicles derived from the oxidized melanoma cells and incubated to obtain the dendritic cells loaded with the fully antigen-engineered vesicles derived from the oxidized melanoma cells;

[0014] Washing the dendritic cells loaded with the oxidized melanoma cell-derived full antigen engineered vesicles, scraping the cells to obtain a cell suspension, centrifuging to obtain a precipitate;

[0015] The precipitate is mixed with an interferon gene stimulator agonist, and is extruded by a gradient extrusion method to obtain engineered bionic nano dendritic cell vesicles.

[0016] Preferably, during the incubation, the protein concentration of the oxidized melanoma cell-derived full antigen engineered vesicles is 5 to 75 μg / mL; the density of the dendritic cells is 5.0×10 5 ~2.0×10 6 Pieces / mL.

[0017] Preferably, the concentration of the interferon gene stimulator agonist is 0.001-2.2 μg / mL.

[0018] The present invention also provides the use of the engineered bionic nano dendritic cell vesicles described in the above technical solution or the engineered bionic nano dendritic cell vesicles prepared by the preparation method described in the above technical solution in the preparation of drugs for preventing and / or treating melanoma.

[0019] The present invention also provides a melanoma vaccine, comprising an active ingredient and a pharmaceutically acceptable excipient, wherein the active ingredient comprises the engineered bionic nano dendritic cell vesicles described in the above technical solution or the engineered bionic nano dendritic cell vesicles prepared by the preparation method described in the above technical solution.

[0020] The present invention provides an engineered bionic nano dendritic cell vesicle. The engineered bionic nano dendritic cell vesicle of the present invention can prepare a melanoma vaccine, has good safety, can maintain strong stability after repeated freezing and thawing, has a good anti-melanoma effect, and has a maturation-promoting effect on dendritic cells, and effectively presents antigens to lymphocytes, promotes lymphocytes to play an anti-tumor immune role, and in a melanoma B16 tumor-bearing mouse model, by subcutaneous injection of engineered bionic nano dendritic cell vesicles, can cause a strong tumor-specific T cell immune response, reshape the tumor microenvironment, and effectively inhibit tumor growth. The engineered bionic nano dendritic cell vesicle of the present invention is easily recognized by antigen presenting cells (Antigen-presenting cells, APC) and presented to T cells by DC, which can stimulate the promoting immune cells in the tumor immune microenvironment to enhance the immune response. By detecting the expression of surface markers of DCs cells, the engineered bionic nano dendritic cell vesicle is obtained, which is beneficial to promoting the differentiation and maturation of DCs and the effective presentation of antigens in vivo. The melanoma vaccine was tested for stability, and the results showed that the melanoma vaccine maintained good stability at 4°C and -80°C after storage and freeze-thaw processes, with PDI values ​​below 0.1, demonstrating that the particle size distribution was uniform and the particle size could still be maintained. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0022] Figure 1 A schematic diagram of the preparation and use methods and main effects of the engineered bionic nano dendritic cell vesicles provided by the present invention;

[0023] Figure 2 The morphological diagram of melanoma cells provided by the present invention after being treated with hypochlorous acid and then re-incubated for different time periods;

[0024] Figure 3 The results of the concentrations of LDH (A) and ATP (B) in the culture supernatant of melanoma cells treated with hypochlorous acid oxidation provided by the present invention after re-incubation for different time periods (n=3, Mean±SD, ****p<0.0001);

[0025] Figure 4The particle size distribution diagram of the full antigen engineered vesicles derived from oxidized melanoma cells determined by the DLS method provided by the present invention (A) and the morphology diagram of the full antigen engineered vesicles derived from oxidized melanoma cells taken under a transmission electron microscope (scale: 100 nm) (B);

[0026] Figure 5 The particle size distribution diagram of the engineered bionic nano dendritic cell vesicle vaccine determined by the DLS method provided by the present invention (A) and the morphology diagram of the engineered bionic nano dendritic cell vesicle taken under a transmission electron microscope (scale: 100 nm) (B);

[0027] Figure 6 The particle size diagram (A) and polymer dispersibility index PDI (B) change trend result diagram of the engineered bionic nano dendritic cell vesicles provided by the present invention under storage conditions of 4°C and -80°C (n=3, Mean±SD);

[0028] Figure 7 The flow cytometry results of the vaccine provided by the present invention for detecting the proportion of double positive cells of CD86 and CD80, which are the maturation markers of DCs cultured in vitro; A is the expression result of the markers CD11c, CD80 and CD86 of DCs in different treatment groups; B is the expression result of CD11c in DC cells + CD80 + and CD86 + The ratio (%) results are shown in Figure 3 (n=3, Mean±SD, compared with the PBS group, ***p<0.001);

[0029] Figure 8 The graphs are the results of the anti-tumor efficacy evaluation of each treatment group of the tumor treatment model provided by the present invention; wherein A is the weight curve of tumor-bearing mice (n=6); B is the mouse tumor volume (mm 3 )-Time (day) change curve (n=6, Mean±SD, compared with PBS group, ****p<0.0001);

[0030] Fig. 9 The immunohistochemical staining results of tumor tissues of mice in each group provided by the present invention are shown in the figure; wherein, the upper figure is the Ki67 tumor proliferation detection result; the lower figure is the Tunel cell apoptosis detection result (scale: 100 μm);

[0031] Fig.10 This is a diagram of the H&E staining results of each visceral tissue of each group of mice provided by the present invention (scale: 100 μm). DETAILED DESCRIPTION

[0032] The present invention provides an engineered bionic nano dendritic cell vesicle, wherein the engineered bionic nano dendritic cell vesicle is loaded with an interferon gene stimulating factor agonist and an oxidized melanoma cell-derived full antigen engineered vesicle. The interior of the engineered bionic nano dendritic cell vesicle of the present invention encapsulates the interferon gene stimulating factor (STING) agonist, and the external vesicle (which may be referred to as an engineered nano vesicle) is derived from a dendritic cell (DC) loaded with a full antigen engineered vesicle derived from an oxidized melanoma cell, that is, the surface of the engineered bionic nano dendritic cell vesicle is loaded with an oxidized melanoma cell-derived full antigen engineered vesicle. In the present invention, the particle size of the engineered bionic nano dendritic cell vesicle is preferably 150 to 200 nm. Nanovesicles within this range can be better engulfed and processed by DC cells in vivo. In the present invention, the interferon gene stimulating factor agonist preferably includes diABZI. The present invention uses STING agonists as vaccine adjuvants, and STING-dependent type I interferon signal transduction can induce tumor antigen cross-presentation, thereby achieving the cytotoxic T cell response required for effective anti-tumor immunity. Interferon gene stimulator (STING) agonists can be used as attractive adjuvants to induce type I interferon (IFN-I) responses and inflammatory cytokines, and can induce the activation of type I interferon and proinflammatory cytokines in vitro and in vivo, enhance the antigen presentation of DC in vivo, and further activate the immune killing effect of T cells, resulting in a sustained anti-tumor immune effect. The present invention does not specifically limit the source of the interferon gene stimulator agonist, and conventional commercial products can be used. In the present invention, the commercial information of the diABZI is preferably diABZI STING agonist (Compound 3) (brand; Selleck, catalog number: S8796, purity: 99.86%). After structural characterization and stability testing, the particle size of the engineered bionic nanodendritic cell vesicles of the present invention is normally distributed and has good storage stability.

[0033] In the present invention, the method for preparing the oxidized melanoma cell-derived full antigen engineered vesicles comprises the following steps:

[0034] The melanoma cells are subjected to hypochlorous acid oxidation treatment to obtain oxidized melanoma cells;

[0035] incubating the oxidized melanoma cells to obtain incubated melanoma cells;

[0036] The incubated melanoma cells are subjected to membrane filtration by an extrusion method to obtain fully antigen engineered vesicles derived from oxidized melanoma cells.

[0037] The present invention performs hypochlorous acid oxidation treatment on melanoma cells to obtain oxidized melanoma cells. In the present invention, the melanoma cells preferably include B16 cells. The present invention uses hypochlorous acid to oxidize melanoma cells to cause cell apoptosis. After cell apoptosis, a large number of damage pattern molecules and immune cytokines can be produced and a large number of tumor antigens can be released, which can trigger tumor-specific dendritic cell activation and provide key activation signals for the immune system, thereby enhancing the anti-tumor immune response. In the present invention, the density of melanoma cells during the oxidation treatment is preferably 1.0×10 6 ~3.0×10 6 / mL, more preferably 2.0×10 6 / mL. In the present invention, the concentration of hypochlorous acid used in the oxidation treatment is preferably 300-450 μM, more preferably 350 μM. In the present invention, hypochlorous acid is preferably dissolved in PBS buffer. The degree of oxidative damage to melanoma cells treated in this concentration range is better. If the concentration is lower than this range, the cells cannot apoptosis within the specified time. If the concentration is higher than this range, the content of inflammatory factors produced after cell apoptosis no longer increases. The results of the detection of melanoma cell lines treated with oxidation showed that the cell morphology changed and vacuoles appeared inside, indicating that the cells were damaged and immunogenic death, and a large amount of LDH and ATP were produced outside the cells.

[0038] After obtaining the oxidized melanoma cells, the present invention incubates the oxidized melanoma cells to obtain incubated melanoma cells. In the present invention, the incubation temperature is preferably 36 to 38° C., more preferably 37° C. In the present invention, the incubation time is preferably 1 to 4 hours, more preferably 4 hours.

[0039] After obtaining the incubated melanoma cells, the present invention uses an extrusion method to filter the incubated melanoma cells to obtain oxidized melanoma cells derived from full antigen engineered vesicles. In the present invention, before the membrane filtration, the density of B16 cells is preferably adjusted to 3.0×10 6 ~5.0×10 6 / mL. In the present invention, the membrane filtration is preferably repeated extrusion on polycarbonate membranes with different pore sizes. In the present invention, the polycarbonate membranes with different pore sizes are preferably 3.0μm→0.45μm→0.22μm in sequence. In the present invention, the number of repeated extrusion is preferably 3 times.

[0040] Compared with the existing methods, the preparation method of the present invention is more convenient and has lower cost, and the nanoscale full-antigen engineered vesicles can also be better loaded and phagocytosed by dendritic cells in vitro.

[0041] The present invention also provides a method for preparing the engineered bionic nano dendritic cell vesicles described in the above technical solution, comprising the following steps:

[0042] The dendritic cells are mixed with the fully antigen-engineered vesicles derived from the oxidized melanoma cells and incubated to obtain the dendritic cells loaded with the fully antigen-engineered vesicles derived from the oxidized melanoma cells;

[0043] Washing the dendritic cells loaded with the oxidized melanoma cell-derived full antigen engineered vesicles, scraping the cells to obtain a cell suspension, centrifuging to obtain a precipitate;

[0044] The precipitate is mixed with an interferon gene stimulator agonist, and is extruded by a gradient extrusion method to obtain engineered bionic nano dendritic cell vesicles.

[0045] The present invention uses dendritic cells loaded with full-antigen engineered vesicles derived from oxidized melanoma cells as materials to prepare engineered nanovesicles, and uses STING agonists as adjuvants to encapsulate the engineered nanovesicles, which can induce the activation of type I interferon and proinflammatory cytokines in vivo and in vitro, greatly improving the ability of engineered bionic nano-dendritic cell vesicles to present loaded antigen vesicles to dendritic cells in vivo, migrate to lymph nodes, and activate anti-tumor specific immune responses.

[0046] The present invention mixes dendritic cells with full antigen engineered vesicles derived from oxidized melanoma cells for incubation to obtain dendritic cells loaded with full antigen engineered vesicles derived from oxidized melanoma cells. That is, the full antigen engineered vesicles derived from oxidized melanoma cells are loaded on engineered biomimetic nano dendritic cell vesicles by co-incubation. In the present invention, the dendritic cells preferably include DC2.4 cells. DC2.4 cells are immortalized mouse dendritic cells produced by transducing bone marrow isolates of C57BL / 6 mice with retroviral vectors expressing mouse granulocyte-macrophage CSF (GM-CSF) and MYC and RAF oncogenes. DC2.4 cells exhibit the cell morphology of dendritic cells and have the expression of specific markers, as well as the ability to phagocytose and present exogenous antigens on MHC class and class II molecules. In the present invention, the culture temperature of all cells is preferably 37°C. In the present invention, during the incubation process, the protein concentration of the oxidized melanoma cell-derived full antigen engineered vesicles is preferably 5 to 75 μg / mL; the density of the dendritic cells is 5.0×10 5 ~2.0×10 6 / mL, preferably 1.0×10 6 In the present invention, the density of dendritic cells during extrusion is preferably 2.0×10 6 ~5.0×10 6 / mL, more preferably 3×10 6 ~4.5×10 6 The protein concentration detection method used in the present invention is the BCA protein concentration detection method.

[0047] After obtaining the dendritic cells loaded with the full antigen engineered vesicles derived from oxidized melanoma cells, the present invention preferably uses PBS buffer to wash the dendritic cells loaded with the full antigen engineered vesicles derived from oxidized melanoma cells. After washing, the present invention preferably scrapes the cells to obtain a cell suspension, and then centrifuges the cell suspension to obtain a precipitate.

[0048] After obtaining the precipitate, the present invention resuspends the precipitate with PBS and then mixes it with an interferon gene stimulating factor agonist, and uses a gradient extrusion method to perform extrusion treatment to obtain engineered bionic nano dendritic cell vesicles. In the present invention, the concentration of the interferon gene stimulating factor agonist is preferably 0.001-2.2 μg / mL, more preferably 0.55 μg / mL. In the present invention, the extrusion treatment preferably includes repeated extrusion, passing through 3.0 μm, 0.45 μm and 0.22 μm polycarbonate membranes in sequence. In the present invention, the number of repeated extrusions is preferably 1 to 10 times. In the present invention, the engineered bionic nano dendritic cell vesicles are preferably stored in a PBS buffer containing trehalose. In the present invention, the mass percentage of trehalose in the PBS buffer is preferably 1 to 5%, more preferably 2%.

[0049] The present invention also provides the use of the engineered bionic nano dendritic cell vesicles described in the above technical solution or the engineered bionic nano dendritic cell vesicles prepared by the preparation method described in the above technical solution in the preparation of drugs for preventing and / or treating melanoma. Figure 1 The figure is a schematic diagram of the preparation, use and main effects of the engineered bionic nano dendritic cell vesicles of the present invention.

[0050] The present invention also provides a melanoma vaccine, comprising an active ingredient and a pharmaceutically acceptable excipient, wherein the active ingredient comprises the engineered bionic nano dendritic cell vesicles described in the above technical solution or the engineered bionic nano dendritic cell vesicles prepared by the preparation method described in the above technical solution. In the present invention, in the melanoma vaccine, the protein concentration of the engineered bionic nano dendritic cell vesicles is preferably 200-300 μg / mL, more preferably 250 μg / mL. In the present invention, the pharmaceutically acceptable excipients preferably include trehalose, more preferably a PBS buffer containing trehalose. In the present invention, trehalose is added to the vaccine as a cryoprotectant. In the present invention, the mass percentage of trehalose in the PBS buffer is preferably 1-5%, more preferably 2%. Trehalose solution can protect the immune effect of the engineered bionic nano dendritic cell vesicle vaccine. In the present invention, the preparation method of the melanoma vaccine is preferably to mix the engineered bionic nano dendritic cell vesicles with a PBS solution containing trehalose. In the present invention, the storage method of the engineered nanovesicles is preferably refrigerated at 4°C (without trehalose) or frozen at -80°C (with trehalose added). It can be stored for about 1 month at 4°C and can be stored for at least one month at -80°C. Whole-cell vaccines contain all tumor antigens, are not restricted by antigens, and can induce immune effector cells to target most of the antigens expressed by tumors, thereby avoiding immune escape caused by the loss of certain antigens during tumor development to a certain extent. Since DC cells can migrate to lymph nodes through the CCR7 signaling pathway and activate specific T cell responses. Then the melanoma vaccine with dendritic cells as carriers directly presents tumor antigens to DC in the body by utilizing the natural chemotaxis of DC cells to lymph nodes. STING agonists enhance DC antigen presentation ability, activate lymphocytes in the body, and stimulate specific immune responses to fight tumors. Compared with the complicated production and purification process and rare yield of exosomes, cell vesicles can be squeezed through the gradient pore size of polycarbonate membranes in vitro, and micro-nano-level vaccine vesicles can also be better engulfed and processed by DC.

[0051] In the present invention, the melanoma vaccine is preferably administered by injection. In the present invention, the injection preferably includes 5 immunizations, and the interval between two adjacent immunizations is preferably 3 days. In the present invention, the immunization dose of the tumor vaccine is preferably 25 to 75 μg / mouse, more preferably 25 μg / mouse, and the mice are C57BL / 6J mice, each weighing 15 to 20 g.

[0052] In the present invention, after the therapeutic melanoma vaccine prepared by engineered bionic nano dendritic cell vesicles was used to treat the melanoma mouse model, the tumor volume was smaller than that of the blank control and the nano vesicles without adjuvant, and the survival period of the mouse model was longer. TUNEL method for detecting cell apoptosis. Ki-67 is a nuclear proliferation antigen of cells, which reflects the proliferation of cells. In the embodiments of the present invention, the results of TUNEL staining were consistent with the results of tumor proliferation antigen Ki67, indicating that the melanoma vaccine prepared by engineered bionic nano dendritic cell vesicles effectively inhibits tumor growth and promotes apoptosis of tumor tissue.

[0053] To further illustrate the present invention, an engineered bionic nano-dendritic cell vesicle, a melanoma vaccine, a preparation method and an application provided by the present invention are described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0054] Example 1

[0055] 1. Hypochlorous acid-induced apoptosis of melanoma cells and its evaluation

[0056] (1) Adjust the density of melanoma cells (B16 cells) to 2.0×10 6 / mL, take 10mL of cell suspension and transfer it to the cell culture dish for later use;

[0057] (2) The cells in the culture dish of step (1) were digested and centrifuged, resuspended in PBS buffer containing 350 μM hypochlorous acid, and cultured for 0.5 h. The cell morphology was observed and photographed under a light microscope. Fresh culture medium (RPMI-1640 + 10% FBS + 1% penicillin-streptomycin-amphotericin B triple antibody) was replaced and incubated at 37° C. for 1 h, 2 h, and 4 h. The cell morphology was observed and photographed under a light microscope.

[0058] The results showed that the cell morphology after oxidation treatment changed significantly. The cell morphology gradually changed from spindle to round. The cells ruptured and vacuoles appeared inside, indicating that the organelles and other structures in the cells changed, and obvious cell damage and apoptosis occurred, confirming that the cells were damaged ( Figure 2 ).

[0059] (3) Take the supernatant after the culture in step (2), and use the lactate dehydrogenase cytotoxicity assay kit (LDHCytotoxicity Assay Kit) (Biyuntian) and the adenosine triphosphate (ATP) assay kit (Biyuntian) respectively, according to the operating steps in the instructions, to determine the content of LDH and ATP in the culture medium.

[0060] The results showed that after the oxidatively treated B16 cells were re-incubated at 37°C, the extracellular production of LDH and ATP ( Figure 3 ) to confirm that cells underwent apoptosis.

[0061] 2. Preparation and evaluation of fully antigen-engineered vesicles derived from oxidative melanoma cells

[0062] (1) Melanoma cells (B16 cells) were digested, centrifuged, and resuspended in PBS buffer containing 350 μM hypochlorous acid. The B16 cell density was adjusted to 2.0×10 6 / mL, after culturing for 0.5h, replace with fresh culture medium (RPMI-1640+10% FBS+1% penicillin-streptomycin-amphotericin B triple antibody) and incubate at 37°C for 4h to obtain a suspension of B16 cells in apoptotic state after oxidation treatment;

[0063] (2) Adjust the density of oxidized B16 cells to 3.0×10 6 ~5.0×10 6 / mL, resuspended in PBS, and the cell suspension was repeatedly extruded three times through a polycarbonate membrane with different pore sizes (3.0μm→0.45μm→0.22μm) using a high-precision, high-pressure airtight sterile filter extruder (brand: PhD Technology LLC, model: NanoAble-150) to obtain fully antigen-engineered vesicles derived from oxidized melanoma cells.

[0064] Transmission electron microscopy images of the prepared oxidized melanoma cell-derived full antigen engineered vesicles are shown in Figure 2. Figure 4 As shown. Figure 4 It can be concluded that the morphology of the prepared vesicles is vacuolar membrane-like, with a particle size of about 178.6nm, and the particle size is normally distributed. The fully antigen-engineered vesicles derived from oxidized melanoma cells are called oM-NV. The vaccine enhances the ability of DC to capture tumor antigens in vitro by loading nano-engineered extracellular vesicles oM-NV, which can activate DC, load DC with antigens, and then present them to DC in vivo, thereby activating lymphocytes, killing tumors, releasing more tumor antigens, and enhancing the immunotherapy effect of tumor vaccines.

[0065] Example 2

[0066] 1. Preparation of engineered bionic nano-melanoma vaccine

[0067] (1) Dendritic cells (DC2.4 cells) were placed in a culture dish and cultured with culture medium (RPMI-1640 + 10% FBS + 1% penicillin-streptomycin-amphotericin B triple antibody), and oM-NV with a protein concentration of 50 μg / mL was added to adjust the DC2.4 cells to 1.0×10 6 / mL, and the final culture system is 10mL, incubated to obtain DC2.4 cells loaded with oM-NV; during the incubation process, the protein concentration of the oxidized melanoma cell-derived full antigen engineering vesicles is preferably 5.00-75.00 μg / mL; the density of dendritic cells is 1.0×10 6 Pieces / mL.

[0068] (2) DC2.4 cells loaded with oM-NV are called DCVac. The DCVac cells are washed with PBS buffer, scraped to obtain a cell suspension, centrifuged to obtain a precipitate, and resuspended with PBS to obtain a DCVac cell suspension;

[0069] (3) Adjust the cell density in the DCVac cell suspension to 3.5×10 6 ~4.5×10 6 / mL; add diABZI solution (DMSO as solvent) to the cell suspension to make the diABZI concentration 550ng / mL, and control the volume of diABZI solution in 5-15μL.

[0070] (4) The cell suspension containing diABZI was extruded three times through polycarbonate membranes with different pore sizes (3.0 μm→0.45 μm→0.22 μm) using a high-precision, high-pressure airtight extruder to obtain engineered bionic nano-dendritic cell vesicles.

[0071] Transmission electron microscope image of the engineered bionic nano-melanoma vaccine prepared Figure 5 As shown. Figure 5 It can be concluded that the morphology of the prepared vesicles is vacuolar membrane, the particle size is about 166.8nm, and the particle size is normally distributed. The prepared engineered bionic nano-melanoma vaccine is called diA-MADCVac, the vesicles without oM-NV loading and diABZI added are called DCNV, and the vaccine without diABZI added is called MADCVac.

[0072] 2. Storage stability test of engineered bionic nano-melanoma vaccine

[0073] To investigate the storage stability of the vaccine, the prepared diA-MADCVac was dispersed in PBS (pH 7.4), and after adding 2wt.% trehalose as a cryoprotectant, it was divided into 3mL centrifuge tubes and stored in refrigerators at 4°C and -80°C for 26 days (the vaccine stored at 4°C was not added with cryoprotectant). The particle size and PDI of the vaccine were monitored by a potentiometric particle size analyzer at 0, 3, 6, 9, 12, 19 and 26 days to investigate its stability under storage conditions of 4°C and -80°C ( Figure 6 ), under the storage conditions of 4℃ and -80℃, the particle size and PDI of the vesicles in each group did not change much after different storage days, and the PDI was all below 0.1, indicating that the vesicles in each group remained evenly distributed after 26 days, and the storage method had no destructive effect on the vesicles.

[0074] Example 3

[0075] Effect of engineered biomimetic nano-melanoma vaccine on maturation of mouse bone marrow-derived dendritic cells (BMDC)

[0076] In order to detect the effect of the vaccine on the maturation of mouse bone marrow-derived dendritic cells (BMDC), flow cytometry was used to detect the expression of CD80 and CD86 in BMDC after vaccine stimulation. BMDC cells were extracted from mouse bone marrow and the cells were divided into 2×10 6 / well inoculated in a 6-well plate, BMDC-specific complete medium (RPMI-1640 + 10% FBS + 1% penicillin-streptomycin-amphotericin B triple antibody + 20ng / mL GM-CSF + 10ng / mL IL-4) was used to culture the cells, the culture system was 2ml / well, and half of the medium was replaced every three days. About the seventh day, 80-90% of the cells were observed to differentiate into BMDC under an optical microscope, and the supernatant was discarded and replaced with fresh BMDC-specific complete medium. Different vaccine groups and negative and positive control group drugs (PBS, 1μg / mL LPS, oM-NV, DCNV, MADCVac, diA-MADCVac, the concentration of the vesicles administered protein was 25μg / mL) were added to the cells of each group, and the LPS used was lipopolysaccharide, which can be conventionally purchased on the market, so that the culture system of each well is 2mL. After 24 hours of co-incubation, use a cell scraper to gently scrape the DCs in the well plate, use a low-temperature centrifuge to centrifuge at 4°C, discard the supernatant, resuspend the cells in 100μL PBS, add 1μL of CD 86, CD 80, and CD11c flow cytometry antibodies, mix them thoroughly by vortexing, and co-incubate for 30 minutes at 4°C in the dark. After the incubation, use a low-temperature centrifuge to centrifuge at 4°C, discard the supernatant, resuspend in pre-cooled PBS, and transfer the cell suspension into a flow tube so that the volume of each tube of cell suspension is 700μL. The expression of mature BMDCs surface markers CD11c, CD80, and CD86 is detected by flow cytometry (FCM) to evaluate the degree of activation of DCs by different vaccine groups to simulate the in vivo vaccine activation effect on DC. The results of the diA-DCVac group showed that the proportion of CD80+CD86+DC cells was the highest. The results show that the diA-DCVac group has the best effect in activating DC maturation in vitro, and the effect is the best ( Figure 7 ).

[0077] Example 4

[0078] Evaluation of the anti-tumor efficacy of engineered bionic nano-melanoma vaccine in in vivo treatment of melanoma mice

[0079] (1) Healthy 5-week-old C57BL / 6 female mice were selected and a mouse melanoma model was established by subcutaneously injecting B16 cells into the right back of the mice. 1.0×10 6 B16 cells were recorded as D0. The mice were randomly divided into PBS, oM-NV, DCNV, MADCVac, and diA-MADCVac groups, with 6 mice in each group. The vaccine was injected on the 3rd, 6th, 9th, 12th, and 15th days. The protein concentration of the vesicles in the injected vaccine was 25 μg / mouse. The weight of each mouse was 14-19 g. The mice were killed by cervical dislocation on the 21st day.

[0080] (2) The mice were weighed and the long and short diameters of the tumors were measured every 3 days to calculate the tumor volume. Figure 8 Figure 2 shows the weight change curve of mice under each group of vaccine treatment and the tumor volume-time change curve of mice. The tumors of mice in the diA-MADCVac group were smaller than those in other groups, indicating that diA-MADCVac has a good anti-tumor effect on mouse melanoma.

[0081] (3) Tumor tissues of mice in each group were collected for Tunel and Ki67 immunohistochemical staining. Fig. 9 The images were taken under a microscope and the TUNEL method was used to detect cell apoptosis. Ki-67 is a nuclear proliferation antigen of cells, which reflects the proliferation of cells. The results of immunohistochemical TUNEL staining of tumor tissue in the embodiment of the present invention are basically consistent with the results of tumor proliferation antigen Ki67, which further illustrates that the vaccine has a good anti-tumor effect on mouse melanoma.

[0082] Example 5

[0083] Safety evaluation of engineered bionic nano-melanoma vaccine in treating melanoma in mice

[0084] The hearts, livers, spleens, lungs, and kidneys of mice in each group were sliced ​​for H&E staining. Fig.10 The pictures were taken under a microscope. The cell structures of the heart, liver, spleen, lung, and kidney of mice in each group were not damaged, indicating that the vaccine did not cause damage to the heart, liver, spleen, lung, and kidney of mice in each group during the treatment of melanoma mice, and the vaccine has good safety.

[0085] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. An engineered bionic nano dendritic cell vesicle, characterized in that: The engineered bionic nano dendritic cell vesicles are loaded with interferon gene stimulating factor agonist and oxidized melanoma cell-derived full antigen engineered vesicles.

2. The engineered biomimetic nano dendritic cell vesicle according to claim 1, characterized in that: The particle size of the engineered bionic nano dendritic cell vesicle is 150-200 nm.

3. The engineered biomimetic nano dendritic cell vesicle according to claim 1, characterized in that: The interferon gene stimulator agonist includes diABZI.

4. The engineered biomimetic nano dendritic cell vesicle according to claim 1, characterized in that: The method for preparing the oxidized melanoma cell-derived full antigen engineered vesicles comprises the following steps: The melanoma cells are subjected to hypochlorous acid oxidation treatment to obtain oxidized melanoma cells; incubating the oxidized melanoma cells to obtain incubated melanoma cells; The incubated melanoma cells are subjected to membrane filtration by an extrusion method to obtain fully antigen engineered vesicles derived from oxidized melanoma cells.

5. The engineered biomimetic nano dendritic cell vesicle according to claim 4, characterized in that: The concentration of hypochlorous acid used in the oxidation treatment is 300-450 μM.

6. The method for preparing the engineered biomimetic nano dendritic cell vesicles according to any one of claims 1 to 5, comprising the following steps: The dendritic cells are mixed with the fully antigen-engineered vesicles derived from the oxidized melanoma cells and incubated to obtain the dendritic cells loaded with the fully antigen-engineered vesicles derived from the oxidized melanoma cells; Washing the dendritic cells loaded with the oxidized melanoma cell-derived full antigen engineered vesicles, scraping the cells to obtain a cell suspension, centrifuging to obtain a precipitate; The precipitate is mixed with an interferon gene stimulator agonist, and is extruded by a gradient extrusion method to obtain engineered bionic nano dendritic cell vesicles.

7. The preparation method according to claim 6, characterized in that: During the incubation process, the protein concentration of the oxidized melanoma cell-derived full antigen engineered vesicles was 5-75 μg / mL; the density of the dendritic cells was 5.0×10 5 ~2.0×10 6 Pieces / mL.

8. The preparation method according to claim 6, characterized in that: The concentration of the interferon gene stimulator agonist is 0.001-2.2 μg / mL.

9. Use of the engineered bionic nano dendritic cell vesicles according to any one of claims 1 to 5 or the engineered bionic nano dendritic cell vesicles prepared by the preparation method according to any one of claims 6 to 8 in the preparation of drugs for preventing and / or treating melanoma.

10. A melanoma vaccine comprising an active ingredient and a pharmaceutically acceptable excipient, characterized in that: The active ingredient comprises the engineered bionic nano dendritic cell vesicles according to any one of claims 1 to 5 or the engineered bionic nano dendritic cell vesicles prepared by the preparation method according to any one of claims 6 to 8.

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