Fusion membrane nano vaccine for promoting up-regulation of co-stimulatory molecule and antigen expression of tumor immune response and preparation method of fusion membrane nano vaccine

Abstract

The invention belongs to the field of nano biomedicine, and particularly discloses a fusion membrane nano vaccine for promoting up-regulated expression of co-stimulatory molecules and antigens of tumor immune response and a preparation method of the fusion membrane nano vaccine. The shell of the vaccine is composed of a fusion membrane prepared from a dendritic cell membrane with up-regulated costimulatory molecule expression and a melanoma tumor cell membrane with up-regulated antigen abundance; and an inner core is formed by metal polyphenol nanoparticles carrying an immune agonist R848. On one hand, the tumor vaccine can target dendritic cells, and antigen cross presentation of the dendritic cells is promoted through up-regulated antigen abundance to promote T cell activation; on the other hand, the tumor vaccine can directly interact with the T cells, the direct activation of the T cells is promoted by up-regulation of the costimulatory molecules, and the activated T cells have a specific killing effect on melanoma tumor cells; the application prospect is wide.

Description

Technical Field

[0001] The present invention belongs to the field of nano-biomedicine and relates to a fusion membrane nano-vaccine that promotes costimulatory molecules and up-regulates antigen expression for promoting tumor immune response and a preparation method thereof. Background Art

[0002] While traditional treatments for malignant tumors, such as surgery, radiotherapy, and chemotherapy, are widely used clinically, they still cannot completely cure tumors. For example, surgery cannot completely eliminate tumor cells, posing the risk of recurrence and metastatic tumor growth. Radiotherapy and chemotherapy also have drawbacks such as drug resistance, side effects, and a high risk of recurrence, leading to poor prognosis. Therefore, developing new tumor treatment strategies to achieve safe and effective tumor treatment is a critical scientific issue that urgently needs to be addressed.

[0003] Tumor vaccines involve screening and extracting tumor-associated antigens from tumor tissue, fusing them with adjuvants, and then injecting them into cancer patients to activate a specific immune response and inhibit tumor growth. Tumor antigens, as a crucial component of tumor vaccines, are essential for activating specific immune responses. Currently, single antigen delivery is difficult to induce effective specific immune responses against mutant tumors due to limited antigen abundance. Therefore, identifying tumor antigens with multiple epitopes has become a key issue in immunotherapy. Tumor cell membranes contain a full repertoire of tumor antigens and exhibit good biocompatibility. Currently, researchers are extracting tumor cell membranes to construct tumor vaccines, which have the potential to safely activate immune responses and inhibit tumor growth. However, due to complex immune escape mechanisms, tumor cells can reduce their immunogenicity by downregulating the expression of surface-associated antigen complexes, promoting immune evasion. Therefore, upregulating the expression of surface antigen complexes on tumor cell membranes and increasing the abundance of immune antigens on tumor cells is key to developing multi-epitope tumor vaccines based on cell membranes.

[0004] Another key scientific challenge in tumor vaccine development is how to effectively activate antigen-presenting cells (APCs) to process and present antigens and activate T cells. Dendritic cells are the immune cells with the strongest antigen-presenting capacity, mediating T cell activation by internalizing and presenting antigens. Mature dendritic cells exhibit decreased phagocytic capacity, while their ability to process and present antigens, home to lymph nodes, and promote T cell activation increases. Mature dendritic cells exhibit various characteristics, primarily manifested by changes in the expression of co-stimulatory molecules and related proteins such as MHC molecules, which are essential for efficient T cell activation. The co-stimulatory molecules expressed by mature dendritic cells provide a secondary signal for T cell activation. In concert with the primary signal provided by the binding of the MHC-I antigen complex to the TCR of the T cell, these activated T cells transform into effector T cells, thereby initiating an anti-tumor immune response. Therefore, it is crucial to induce dendritic cells to upregulate the expression of co-stimulatory molecules and promote their antigen processing and presentation.

[0005] In addition to promoting T cell activation through antigen-presenting cells, current research focuses on the direct activation of T cells by nanomedicines, thereby bypassing the complex antigen presentation process.

[0006] Therefore, on the one hand, the present application utilizes nanovaccines that can be taken up and internalized by dendritic cells, promoting T cell activation by promoting antigen cross-presentation. On the other hand, the nanovaccines can directly interact with T cells and synergistically promote T cell activation, thereby mediating efficient anti-tumor effects. Summary of the Invention

[0007] In response to the problems in the above-mentioned background technology, the present invention aims to provide a fusion membrane nanovaccine that upregulates the expression of costimulatory molecules and antigens that promote tumor immune responses, and a method for preparing the same. The nanovaccine is composed of a fusion membrane derived from tumor cell membranes with enhanced antigen abundance and dendritic cell membranes with upregulated expression of costimulatory molecules, encapsulating a metal polyphenol nanocore loaded with the immune agonist R848. This vaccine can be internalized by dendritic cells through the nanovaccine, promoting T cell activation by promoting antigen cross-presentation. Furthermore, the nanovaccine can directly interact with T cells, synergistically promoting T cell activation, thereby efficiently mediating an anti-tumor immune response.

[0008] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0009] A fusion membrane nanovaccine that upregulates the expression of costimulatory molecules and antigens that promote tumor immune responses. The outer shell is composed of a fusion membrane prepared from dendritic cell membranes with upregulated expression of costimulatory molecules and melanoma tumor cell membranes with upregulated antigen abundance; the inner core is composed of metal polyphenol nanoparticles loaded with the immune agonist R848.

[0010] On the one hand, the vaccine targets dendritic cells and promotes T cell activation through antigen presentation by dendritic cells; on the other hand, the vaccine directly interacts with T cells and promotes T cell activation; the activated T cells have a specific killing effect on melanoma tumor cells.

[0011] The average particle size of the membrane fusion nanovaccine of tumor cells and dendritic cells is 193 nm.

[0012] Furthermore,

[0013] The dendritic cell membrane is derived from DC2.4; the tumor cell membrane is derived from melanoma B16F10-OVA cells.

[0014] Furthermore,

[0015] Dendritic cell membranes with upregulated expression of co-stimulatory molecules were obtained by incubating DC2.4 with R848; tumor cell membranes with upregulated antigen abundance were obtained by incubating B16F10-OVA tumor cells with the mitochondrial fission inhibitor Mdivi-1.

[0016] The present invention also provides a method for preparing the fusion membrane nanovaccine that promotes co-stimulatory molecules and up-regulates antigen expression for promoting tumor immune response, comprising the following steps:

[0017] (1) Induce dendritic cells to upregulate the expression of co-stimulatory molecules and extract cell membrane DCM; induce tumor cells to upregulate antigen abundance and extract cell membrane CM(M), and fuse the two cell membranes by ultrasound to prepare fused cell membrane DCCM(M);

[0018] (2) Tannic acid was mixed with iron ion solution to prepare blank metal polyphenol nanoparticles TF through coordination, and the immune adjuvant R848 was loaded to prepare TF / R848 nanoparticles;

[0019] (3) TF / R848 nanoparticles were mixed with the fusion membrane DCCM(M) solution and the nanovaccine TF / R848@DCCM(M) was prepared by ultrasonication.

[0020] Furthermore,

[0021] The specific method of step (1) is as follows:

[0022] A. Seed DC2.4 cells in a culture dish. After the cells adhere, add R848 diluted in culture medium at a concentration of 5-20 μg / mL and incubate for 24-48 hours. After incubation, harvest the DC2.4 cells and wash with PBS for later use.

[0023] B. Inoculate B16F10-OVA tumor cells into a culture dish. After the cells adhere, add mitochondrial fission inhibitor Mdivi-1 at a concentration of 10-50 μg / mL diluted in culture medium and incubate for 48-72 hours. After incubation, collect the B16F10-OVA tumor cells and wash them with PBS for later use.

[0024] C. Place the cells treated in steps A and B in centrifuge tubes, wash with PBS, and incubate in a hypotonic solution containing 1% PMSF for 10-20 minutes to rupture the cells. Grind the cells on ice and centrifuge the fully ground cell solution at 1800-2700 rpm for at least 10 minutes. Collect the supernatant to separate organelles and unruptured cells. Centrifuge the supernatant at 10,000-12,000 rpm for at least 30 minutes to collect the cell membrane precipitate. Prepare a fused cell membrane by mixing tumor cell membranes and dendritic cell membrane precipitates at a membrane precipitate mass ratio of 1:1-1:4 and sonicate for 3-5 minutes at a power of no more than 125 W.

[0025] The specific method of step (2) is as follows:

[0026] Dissolve ferric chloride hexahydrate in water and adjust its concentration to prepare a 2-8 mg / mL ferric chloride hexahydrate solution. Dissolve tannic acid in methanol to prepare a 10-30 mg / mL tannic acid methanol solution. Add 30-150 μL of ferric chloride hexahydrate solution to 2 mL of 10-50 mM phosphate buffer solution with a pH of 8-10, stir evenly, and then slowly add 10-50 μL of tannic acid methanol solution, stirring at 700-900 rpm for 20-30 min. After the reaction is completed, centrifuge at 8000-10000 rpm for 10-15 min to collect the precipitate, wash and obtain nanoparticles for use. Disperse the obtained nanoparticles in 1 mL of water to obtain a uniformly dispersed nanoparticle suspension. Dissolve the adjuvant R848 in methanol to prepare a solution with a concentration of 5-30 mg / mL. Add 50-100 μL of R848 methanol solution to the suspension and stir at room temperature for 6-12 h.

[0027] The specific method of step (3) is as follows:

[0028] Take an appropriate amount of TF / R848 and ultrasonically disperse it evenly; mix the fusion membrane DCCM(M) and TF / R848 nanoparticles with a solid mass ratio of 1:1-1:2 evenly, and ultrasonicate in a 4°C cold water bath for 5-10 min with a power not exceeding 125 W to complete the fusion membrane coating.

[0029] Beneficial effects of the present invention

[0030] 1. The present invention has developed a fusion membrane nanovaccine composed of metal polyphenol nanoparticles loaded with the immune agonist R848 as a core, encapsulated by tumor cell membranes with enhanced antigen abundance and dendritic cell membranes with upregulated co-stimulatory molecule expression, for use in tumor immunotherapy. This tumor vaccine can directly activate naive T cells into specific anti-tumor effector cells. It can also function as a vaccine by promoting antigen processing and cross-presentation by dendritic cells through homologous targeting, thereby activating specific anti-tumor immunity.

[0031] 2. The nanovaccine provided by the present invention has dendritic cell membrane components, which can promote the uptake of dendritic cells through homologous targeting and has a targeting effect.

[0032] 3. The nano vaccines involved in the present invention have low toxic side effects on normal tissue cells, mouse epithelial fibroblasts and dendritic cells, and have good biocompatibility. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is a flow cytometric analysis of upregulated dendritic cell costimulatory molecules.

[0034] Figure 2 This is a flow cytometric analysis of the induced upregulation of tumor cell antigen abundance.

[0035] Figure 3 It is the ultraviolet absorption spectrum of tannic acid TA, immune adjuvant R848, metal polyphenol nanoparticles TF and TF / R848 prepared by the present invention.

[0036] Figure 4 It is the fluorescence emission spectra of TF / R848 prepared by the present invention and free adjuvant R848.

[0037] Figure 5 These are the fluorescence emission images of DiO-labeled tumor cell membrane CM(M), DiI-labeled dendritic cell membrane DCM, and fusion membrane DCCM(M).

[0038] Figure 6 The particle size distribution diagram and transmission electron micrograph of TF / R848@DCCM(M) prepared by the present invention are shown;

[0039] in Figure 6 A is the particle size distribution diagram of TF / R848@DCCM(M) prepared in the present invention; Figure 6 B is a transmission electron microscopy image of TF / R848@DCCM(M) prepared in the present invention.

[0040] Figure 7 This is the fluorescence intensity graph of dendritic cells uptake of TF / NR@CM(M), TF / NR@DCM, and TF / NR@DCCM(M) detected by high-content imaging;

[0041] NR is the fluorescent dye Nile Red.

[0042] Figure 8 The cytotoxicity of metal polyphenol nanoparticles TF, TF / R848, and nanovaccine TF / R848@DCCM(M) on dendritic cells and normal tissue cells mouse epithelial-like fibroblast L929 was studied;

[0043] in Figure 8 A is the study of the cytotoxicity of metal polyphenol nanoparticles TF, TF / R848, and nanovaccine TF / R848@DCCM(M) on dendritic cells; Figure 8 B is the cytotoxicity study of metal polyphenol nanoparticles TF, TF / R848 and nanovaccine TF / R848@DCCM(M) on normal tissue cells L929.

[0044] Figure 9 This is a study on the antigen presentation of dendritic cells after incubation of TF / R848, TF / R848@DCCM and the antigen abundance-inducing upregulation vaccine TF / R848@DCCM(M) with dendritic cells.

[0045] Figure 10 This is a study on the promotion of T cell proliferation by TF / R848, TF / R848@DCCM and antigen abundance-induced upregulation vaccine TF / R848@DCCM(M).

[0046] Figure 11 This is a study on the direct activation of T cells by TF / R848, TF / R848@DCCM and the antigen abundance-induced upregulation vaccine TF / R848@DCCM(M).

[0047] Figure 12 This is a study on the indirect activation of T cells by TF / R848, TF / R848@DCCM and the antigen abundance-induced upregulation vaccine TF / R848@DCCM(M).

[0048] Figure 13 This study studies the killing effects of T cells activated by TF / R848, TF / R848@DCCM and antigen abundance-induced upregulation vaccine TF / R848@DCCM(M) on melanoma tumor cells B16F10-OVA.

[0049] Figure 14 This study studies the killing effects of activated T cells on tumor cells melanoma B16F10-OVA, breast cancer cells 4T1 and mouse epithelial fibroblasts L929.

[0050] Figure 9-13TF / R848@DCCM is a nanovaccine prepared from fused cell membranes in which antigen abundance is not induced to increase; TF / R848@DCCM(M) is a nanovaccine prepared from fused cell membranes in which antigen abundance is induced to increase. DETAILED DESCRIPTION

[0051] In order to better illustrate the present invention, the present invention will be further described below with reference to the embodiments.

[0052] Example 1: Preparation and characterization of nanovaccine TF / R848@DCCM(M)

[0053] (1) DC2.4 is divided into 5×10 5 The cells were seeded at a density of 10 μg / mL at 400 μL / well in a 6-well plate. After the cells adhered to the wall, blank culture medium and 10 μg / mL R848 diluted in culture medium were added and incubated for 24 h. After incubation, DC2.4 cells were collected and washed twice with PBS. DC2.4 cells in different wells were stained with anti-CD11c-FITC, anti-CD80-PE and anti-CD86-APC flow cytometry antibodies at 4°C for 30 min. After staining, they were washed once with PBS by centrifugation and resuspended in 400 μL PBS. The maturation ratio of DC2.4 was analyzed by flow cytometry. The results are shown in Figure 2. Figure 1 As shown in the figure, compared with the control group without drug addition, the experimental group incubated with drug addition significantly promoted the expression of costimulatory molecules CD80 and CD86. Therefore, dendritic cells with upregulation of costimulatory molecules were successfully induced.

[0054] (2) Melanoma B16F10-OVA tumor cells carrying the model antigen were cultured at a rate of 5×10 5 The cells were seeded at a density of 10 μg / mL / well in a 6-well plate. After the cells adhered to the wall, blank culture medium and mitochondrial fission inhibitor Mdivi-1 diluted in culture medium at concentrations of 10 μg / mL, 30 μg / mL, and 50 μg / mL were added and incubated for 72 h. After incubation, B16F10-OVA tumor cells were collected and washed twice with PBS. Cells in different wells were stained with anti-H-2Kb-SIINFEKL-APC flow cytometry antibody at 4°C for 30 minutes. After staining, they were washed once with PBS by centrifugation and resuspended in 400 μL PBS. The upregulation of MHC-antigen complexes was analyzed by flow cytometry. The results are shown in the figure. Figure 2 As shown in the figure, compared with the untreated control group, the expression of MHC-antigen complexes increased gradually with increasing drug concentration, and the induction effect was the best at 50 μg / mL. Therefore, pretreatment can effectively increase the antigen abundance of tumor cells.

[0055] (3) Dissolve ferric chloride hexahydrate in water and adjust its concentration to prepare a 2.7 mg / mL ferric chloride hexahydrate solution. Dissolve tannic acid in methanol to prepare a 17 mg / mL tannic acid methanol solution. Add 150 μL of ferric chloride hexahydrate solution to 2 mL of 10 mM phosphate buffer solution with a pH of 8, stir evenly, and then slowly add 50 μL of tannic acid methanol solution. Stir at 700 rpm for 30 min at room temperature. After the reaction is completed, centrifuge at 10,000 rpm to collect the precipitate, wash it twice and set aside. Disperse the nanoparticles obtained above in 1 mL of water, and add the evenly dispersed nanoparticle suspension to a penicillin bottle. Dissolve the adjuvant R848 in methanol to prepare a solution with a concentration of 5 mg / mL. Add 50 μL of R848 methanol solution to the TF suspension respectively, and stir at room temperature for 12 h. Weigh a certain amount of tannic acid, R848, and metal polyphenol nanoparticles TF. TF / R848 was dispersed in water. 700 μL of each was placed in an analytical dish for spectral scanning. The scanning wavelength range was 250-400 nm. The results were as follows: Figure 3 As shown in the figure, it was found that TF / R848 had characteristic absorption peaks of drug R848 at 307 and 322 nm, indicating that TF / R848 was successfully prepared.

[0056] (4) Weigh a certain amount of TF / R848 nanoparticles and R848 powder, dissolve the R848 powder and dilute it to a concentration corresponding to that of the TF / R848 nanoparticles. Add the two solutions to the analysis dish, excite at an excitation wavelength of 322 nm, and observe their emission spectra in the range of 335–400 nm. The results are as follows: Figure 4 As shown in the figure, the fluorescence emission spectrum of TF / R848 shows fluorescence quenching at 335-400 nm, which is equivalent to the free R848 solution. This may be due to the interaction between R848 and the carrier, which inhibits the energy release of the excited state, indicating the successful preparation of TF / R848 nanoparticles.

[0057] (5) The cells treated in steps (1) and (2) were placed in centrifuge tubes, washed by centrifugation with PBS, and then incubated with a hypotonic solution containing 1% PMSF for 15 min to rupture the cells. The cells were then ground on ice. The fully ground cell liquid was centrifuged at 2000 rpm for 10 min, and the supernatant was collected to separate organelles and unbroken cells. The supernatant was centrifuged at 12000 rpm for 30 min to collect the cell membrane precipitate. The cell membrane DCM of dendritic cells with upregulated expression of costimulatory molecules and the cell membrane CM (M) of tumor cells with upregulated antigen abundance were obtained respectively.

[0058] Tumor cell membranes (CM(M)) were stained with DiO (excitation / emission = 484 / 501), while dendritic cell membranes (DCM) were stained with DiI (excitation / emission = 549 / 565). Equal amounts of the two stained membranes were mixed and sonicated for 5 minutes to prepare fused membranes (DCCM(M). Tumor cell membranes (CM(M), dendritic cell membranes (DCM), and fused membranes (DCCM(M)) were measured using a fluorescence spectrophotometer with excitation at 484 nm and emission between 495 and 620 nm. The results are shown in Figure 2. Figure 5 As shown in the figure, compared with the unfused CM(M) and DCM, the fluorescence intensity of the fused membrane DCCM(M) at 501 nm was weakened, while the fluorescence intensity at 565 nm was enhanced, and the fluorescence resonance shift phenomenon occurred, which indicated the successful fusion of the two cell membranes.

[0059] (6) Take an appropriate amount of TF / R848 and ultrasonicate for 1 minute to disperse it evenly. Mix the two cell membranes with a membrane mass ratio of 1:1 and ultrasonicate for 5 minutes (125W) to complete the cell membrane fusion. Mix the fusion membrane DCCM(M) with a solid mass ratio of 1:1 and TF / R848 nanoparticles evenly. Ultrasonicate in a cold water bath for 5 minutes to complete the coating of the fusion membrane. Collect the precipitate by centrifugation. The particle size distribution of the nanomedicine was detected by dynamic light scattering and the morphological characteristics were observed by transmission electron microscopy. The results are as follows: Figure 6 As shown in the figure, the average particle size of the nanovaccine is 193 nm. TEM observation shows that the prepared nanovaccine has a clear core-membrane structure, and its particle size is basically consistent with the DLS results, further indicating that the fusion membrane-coated nanovaccine TF / R848@DCCM(M) was successfully prepared.

[0060] Example 2: Cellular uptake of nanovaccines

[0061] High-content imaging was used to investigate the uptake of nanoformulations by dendritic cells. The model drug Nile red (NR) was loaded onto metal polyphenol nanoparticles to characterize the cellular uptake. 4 Each well was inoculated in a 96-well plate. After 24 h of culture, the supernatant was discarded and the tumor cell membrane-encapsulated nanodrug TF / NR@CM(M), dendritic cell membrane-encapsulated nanodrug TF / NR@DCM and fusion membrane-encapsulated nanodrug TF / NR@DCCM(M) diluted in culture medium were added and incubated for 4 h. After the incubation, the supernatant was discarded and the cells were washed twice with culture medium. Then, the nuclear staining solution Hoechst33342 diluted in culture medium was added and incubated at room temperature for 10 min. After the incubation, the cells were washed once with culture medium. After the incubation, 100 μL of blank culture medium was added to each well for imaging. The results are shown in Figure 2. Figure 7As shown, dendritic cells showed low uptake of tumor cell membrane-coated nanoparticles TF / NR@CM(M), resulting in weak fluorescence intensity. In contrast, nanoparticles TF / NR@DCCM(M) and TF / NR@DCM coated with dendritic cell membrane components showed stronger fluorescence intensity, indicating increased uptake of these nanoparticles by dendritic cells. This suggests that the nanovaccine prepared by the present invention has a certain degree of homologous targeting.

[0062] Example 3: Cytotoxicity of Nanovaccines

[0063] Dendritic cells were inoculated with mouse epithelial fibroblast L929 cells at a rate of 1×10 4 Cells were seeded in 96-well plates and cultured in an incubator for 24 hours. The supernatant was discarded and different concentrations of TF, TF / R848, and TF / R848@DCCM(M) (5 μg / mL, 10 μg / mL, 20 μg / mL, 30 μg / mL, and 40 μg / mL) diluted in culture medium were added. Each group had 5 replicate wells, with 100 μL per well. A blank experimental group (containing only culture medium) and a control group (cells + blank culture medium) were also set up. After incubation for 24 hours, the supernatant was discarded and culture medium containing CCK8 was added for 1 hour. The absorbance at 450 nm was measured using a microplate reader. The results are shown in Figure 2. Figure 8 As shown. After incubation of dendritic cells with different concentrations of TF, TF / R848, and TF / R848@DCCM(M) nanovaccines for 24 hours, their cytotoxic effects on dendritic cells were negligible, and the cell survival rates were all above 80%. Similarly, after incubation of L929 cells with different concentrations of TF, TF / R848, and TF / R848@DCCM(M) nanovaccines for 24 hours, their cytotoxic effects on L929 cells were minimal, and the cell survival rates were all above 75%, indicating that the constructed nanovaccines have good biocompatibility.

[0064] Example 4: Antigen presentation analysis after incubation of nanovaccine with dendritic cells

[0065] Dendritic cells were cultured at a rate of 5 × 10 5 The cells were seeded at a density of 100 μg / well in a 6-well plate. After 24 h of culture, the supernatant was removed and TF / R848, TF / R848@DCCM, and the antigen abundance-enhancing vaccine TF / R848@DCCM(M) diluted in culture medium were added. Cells incubated with blank culture medium served as the control group. After 36 h of incubation, the dendritic cells were collected, washed once with PBS, and stained with flow cytometry antibodies CD11c-FITC and H-2kb-SIINFEKL-APC at 4°C for 30 min. The cells were washed once with PBS, and 400 μL of pre-chilled PBS was added to each tube for flow cytometry analysis. The results are shown in Figure 2. Figure 9 As shown in the figure. After 24 hours of incubation with dendritic cells, the TF / R848, TF / R848@DCCM (fusion membranes formed by the fusion of tumor cell membranes without upregulated antigens and dendritic cell membranes with upregulated co-stimulatory molecules), and TF / R848@DCCM(M) nanovaccines showed significantly greater antigen presentation than the other groups. Therefore, nanovaccines with upregulated antigen abundance constructed through pretreatment can effectively promote cross-presentation by dendritic cells, laying the foundation for further T cell activation.

[0066] Example 5: Study on the Proliferation of T Lymphocytes by Nanovaccines

[0067] Dissolve CFSE in DMSO and adjust its concentration to 10 mM as a stock solution. Take an appropriate amount of the stock solution and add it to serum-free culture medium to prepare a working solution with a concentration of 5 μM. Pipette 1 mL of the working solution and add it to the extracted mouse T lymphocytes. Incubate at room temperature for 10 minutes. After the incubation, wash twice with PBS to remove the free dye. After centrifugation, collect and disperse in 1640 complete culture medium containing IL-2 (10 ng / mL) and adjust the concentration to the appropriate level. T lymphocytes are plated at 1×10 per well. 6 Cells were seeded at a density of 100 μg / well in a 6-well plate, and then TF / R848, TF / R848@DCCM, and TF / R848@DCCM(M) were added, respectively. Cells incubated with blank medium served as the control group and incubated for 72 h. After the incubation, T lymphocytes were collected and washed with PBS. The proliferation of T lymphocytes was detected by flow cytometry (FITC channel). The results are shown in Figure 2. Figure 10 As shown. Compared with the TF / R848 experimental group without fusion membrane coating, the fusion membrane-coated nanoparticle group can significantly promote the proliferation of T lymphocytes. The proliferation promotion rate of the TF / R848 experimental group without fusion membrane coating was 9.86%, while that of the fusion membrane-coated experimental group reached 12.2% and 18.1%, respectively. Moreover, compared with the TF / R848@DCCM group in which antigen abundance was not induced to increase, the proliferation promotion effect of the TF / R848@DCCM(M) group in which antigen abundance was induced to increase was more obvious. Therefore, the constructed fusion membrane-coated nanovaccine directly promotes the proliferation of T lymphocytes by upregulating the expression of MHC-antigen complexes and directly interacting with T lymphocytes.

[0068] Example 6: Study on direct activation of T lymphocytes by nanovaccines

[0069] Mouse T lymphocytes were extracted and dispersed in 1640 complete medium containing IL-2. T lymphocytes were cultured at a concentration of 1×10 6The cells were seeded at a density of 100 μg / well in a 6-well plate. TF / R848, TF / R848@DCCM, and TF / R848@DCCM(M) were then added, respectively. T lymphocytes incubated with blank culture medium served as a control group and incubated for 48 h. After incubation, T lymphocytes were collected and washed with PBS. Flow cytometry staining with antibodies CD3-PE, CD8-APC, and CD69-FITC was performed at 4°C for 30 min. The cells were washed once with PBS, and 400 μL of pre-chilled PBS was added to each tube for flow cytometry analysis. The results are shown in Table 1. Figure 11 The TF / R848 group and the Control group without fusion membrane had no obvious direct activation effect on T lymphocytes. + The average proportion of T cells was 3.3% and 3.6% respectively. This indicates that the nanoparticles without fusion membrane coating lack the first and second signals for activating T lymphocytes and do not have the ability to directly activate T lymphocytes. Compared with the TF / R848 group without fusion membrane coating, the TF / R848@DCCM and TF / R848@DCCM(M) coated with fusion membrane can significantly activate T lymphocytes, CD69 + The average proportions of T cells were 8.9% and 18%, respectively. This suggests that the fusion membrane-coated nanoparticles, composed of tumor cell membranes and dendritic cell membranes, contain MHC-antigen complexes and co-stimulatory molecules on the nanoparticle surface. These fusion membrane-coated nanoparticles can directly interact with and activate T lymphocytes. Furthermore, compared with the TF / R848@DCCM group, the direct activation of T lymphocytes by TF / R848@DCCM(M) was more pronounced, suggesting that inhibiting mitochondrial fission in tumor cells can increase the expression of MHC-antigen complexes. Fusion membrane-coated nanovaccines with high expression of MHC-antigen complexes can increase direct antigen presentation and directly promote efficient activation of T lymphocytes.

[0070] Example 7: Study on indirect activation of T lymphocytes by nanovaccines

[0071] Dendritic cells were cultured at a rate of 1×10 5 The cells were seeded at a density of 100 μg / well in a 6-well plate and cultured for 24 h until the cells adhered to the wall. The supernatant was removed and TF / R848, TF / R848@DCCM and TF / R848@DCCM(M) diluted in culture medium were added. The cells incubated with blank culture medium were used as the control group and incubated for 24 h. After the incubation, 10 T lymphocytes were added to each well. 6After incubation, the cells were collected, washed once with PBS, and stained with flow cytometry antibodies CD3-PE, CD8-APC, and CD69-FITC at 4°C for 30 minutes. The cells were washed once with PBS, and 400 μL of pre-cooled PBS was added to each tube for flow cytometry. The results were as follows: Figure 12 As shown. CD69 in the control group + The average proportion of T cells was 32.8%, which may be due to the low expression of co-stimulatory molecules in dendritic cells, which can promote the maturation of T lymphocytes. + The average proportion of T cells reached 60.2%, which may be due to the adjuvant nanoparticles promoting the maturation of dendritic cells. Dendritic cells promote T lymphocyte maturation by upregulating the expression of costimulatory molecules and secreting proinflammatory factors. In addition, compared with the adjuvant nanoparticle group without fusion membrane coating, the experimental group coated with fusion membrane significantly promoted T lymphocyte maturation, with the fusion membrane-coated TF / R848@DCCM group reaching 70.2% and the TF / R848@DCCM(M) group reaching 75.8%. This may be because the fusion membrane coating promoted the uptake of the nanovaccine and antigen presentation by dendritic cells. BMDC promoted T lymphocyte activation by upregulating the expression of costimulatory molecules and MHC-antigen complexes on the membrane surface. Moreover, compared with the TF / R848@DCCM group, the fusion membrane group with increased antigen abundance TF / R848@DCCM(M) significantly promoted T lymphocyte activation. Therefore, the constructed tumor vaccine with increased antigen abundance and enhanced immune stimulation has excellent immune activation ability and can efficiently promote the activation of T lymphocytes through antigen cross-presentation.

[0072] Example 8: Study on the killing effect of T lymphocytes activated by nano-vaccines on melanoma tumor cells B16F10-OVA.

[0073] Dendritic cells were cultured at a density of 1×10 5 Each well was seeded in a 6-well plate. After the cells adhered to the wall, the supernatant was removed and TF / R848, TF / R848@DCCM and TF / R848@DCCM(M) diluted in culture medium were added and incubated for 24 h. 6 After incubation, activated T cells were collected and added to a 96-well plate pre-seeded with tumor cells at a ratio of 20:1. The cells were cultured for 24 hours and the cell viability of the different groups was measured using an LDH kit. Figure 13As shown. Compared with the control group, TF / R848 can promote the activation of T cells by promoting the activation of BMDCs. The activated T cells have an enhanced killing effect on tumor cells and increase the release of lactate dehydrogenase by tumor cells. In addition, compared with TF / R848, the fusion membrane-coated nanoparticle group can promote the activation of T cells. Among them, the fusion membrane group TF / R848@DCCM(M) with upregulated antigen abundance can significantly promote the activation of T cells. The activated T cells have a more obvious killing effect on tumor cells and release the highest content of lactate dehydrogenase. Therefore, the constructed fusion membrane-coated nanovaccine with upregulated antigen abundance can significantly promote the activation of T cells and increase the killing effect on tumor cells.

[0074] Example 9: Study on the specific killing effect of T lymphocytes activated by nano-vaccines on tumor cells.

[0075] Melanoma tumor cells B16F10-OVA, breast cancer cells 4T1 and mouse epithelial fibroblast L929 were cultured at a concentration of 10 4 / well in a 96-well plate. TF / R848@DCCM(M) pretreated dendritic cells were incubated with T cells for 48 h. Activated T cells were collected and added to 96-well plates at a ratio of 20:1 for co-culture for 24 h. The cell viability of different groups was measured using an LDH kit. The results are shown in Figure 2. Figure 14 As shown in the results, T cells have little effect on the viability of normal tissue cells and secrete lower levels of lactate dehydrogenase. Compared with 4T1 tumor cells, activated T cells have the strongest killing effect on B16F10-OVA tumor cells, significantly reducing their cell viability. Therefore, the tumor vaccine constructed using B16F10-OVA tumor cell membranes, which has increased antigen abundance and enhanced immune stimulation, has a specific killing effect on B16F10-OVA tumor cells.

[0076] The results of Examples 4 to 9 above demonstrate the activation effect of the present invention on immune cells and the killing effect on tumor cells.

[0077] The above embodiments are preferred implementations of the present invention and are only used to illustrate the technical solutions of the present invention rather than to limit the present invention. It should be understood that there are many alternative solutions within the scope of the principles of the present invention, which do not affect the essential content of the present invention.

Claims

1. A fusion membrane nanovaccine that promotes the expression of co-stimulatory molecules and antigens that promote tumor immune response, characterized in that: The shell is composed of a fusion membrane prepared from dendritic cell membranes with upregulated co-stimulatory molecule expression and melanoma tumor cell membranes with upregulated antigen abundance; the core is composed of metal polyphenol nanoparticles loaded with the immune agonist R848; On the one hand, the vaccine targets dendritic cells, promoting antigen cross-presentation by dendritic cells and activating T cells by upregulating the abundance of antigens. On the other hand, the vaccine directly interacts with T cells, upregulating co-stimulatory molecules and promoting T cell activation. The activated T cells have a specific killing effect on melanoma tumor cells. The dendritic cell membrane is derived from DC2.4; the tumor cell membrane is derived from melanoma B16F10-OVA cells; Dendritic cell membranes with upregulated expression of co-stimulatory molecules were obtained by incubating DC2.4 with R848; tumor cell membranes with upregulated antigen abundance were obtained by incubating B16F10-OVA tumor cells with the mitochondrial fission inhibitor Mdivi-1.

2. The method for preparing the fusion membrane nanovaccine with upregulated expression of co-stimulatory molecules and antigens that promote tumor immune response according to claim 1, characterized in that: The following steps are involved: (1) Induce dendritic cells to upregulate the expression of costimulatory molecules and extract cell membrane DCM; induce tumor cells to upregulate antigen abundance and extract cell membrane CM(M), and fuse the two cell membranes by ultrasound to prepare fused cell membrane DCCM(M); (2) Tannic acid was mixed with iron ion solution to prepare blank metal polyphenol nanoparticles TF through coordination, and the immune adjuvant R848 was loaded to prepare TF / R848 nanoparticles; (3) TF / R848 nanoparticles were mixed with the fusion membrane DCCM(M) solution and the nanovaccine TF / R848@DCCM(M) was prepared by ultrasonication.

3. The preparation method according to claim 2, characterized in that The specific method of step (1) is as follows: A. Seed DC2.4 cells in a culture dish. After the cells adhere, add R848 diluted in culture medium at a concentration of 5-20 μg / mL and incubate for 24-48 hours. After incubation, harvest the DC2.4 cells and wash with PBS for later use. B. Inoculate B16F10-OVA tumor cells into a culture dish. After the cells adhere, add mitochondrial fission inhibitor Mdivi-1 at a concentration of 10-50 μg / mL diluted in culture medium and incubate for 48-72 hours. After incubation, collect the B16F10-OVA tumor cells and wash them with PBS for later use. C. Place the cells treated in steps A and B in centrifuge tubes, wash with PBS by centrifugation, and incubate in a hypotonic solution containing 1% PMSF for 10-20 minutes to rupture the cells. Grind the cells on ice and centrifuge the fully ground cell solution at 1800-2700 rpm for at least 10 minutes. Collect the supernatant to separate organelles and unruptured cells. Centrifuge the supernatant at 10,000-12,000 rpm for at least 30 minutes to collect the cell membrane precipitate. Prepare a fused cell membrane by mixing tumor cell membranes and dendritic cell membranes at a mass ratio of 1:1-1:4 and sonicating at a power of no more than 125 W for 3-5 minutes.

4. The preparation method according to claim 2, characterized in that The specific method of step (2) is as follows: Ferric chloride hexahydrate was dissolved in water and its concentration was adjusted to prepare a 2-8 mg / mL ferric chloride hexahydrate solution. Tannic acid was dissolved in methanol to prepare a 10-30 mg / mL tannic acid methanol solution. 30-150 μL of the ferric chloride hexahydrate solution was added to 2 mL of a 10-50 mM phosphate buffer solution with a pH of 8-10, stirred evenly, and then 10-50 μL of the tannic acid methanol solution was slowly added. The mixture was stirred at 700-900 rpm for 20-30 min. After the reaction was completed, the precipitate was collected by centrifugation at 8000-10000 rpm for 10-15 min, and the nanoparticles were obtained after washing for use. The obtained nanoparticles were dispersed in 1 mL of water to obtain a uniformly dispersed nanoparticle suspension. The adjuvant R848 was dissolved in methanol to prepare a solution with a concentration of 5-30 mg / mL. 50-100 μL of the R848 methanol solution was added to the suspension and stirred at room temperature for 6-12 h.

5. The preparation method according to claim 2, characterized in that The specific method of step (3) is as follows: Take an appropriate amount of TF / R848 and ultrasonically disperse it evenly; mix the fusion membrane DCCM(M) and TF / R848 nanoparticles with a solid mass ratio of 1:1-1:2 evenly, and ultrasonicate in a 4°C cold water bath for 5-10 min with a power not exceeding 125 W to complete the fusion membrane coating.

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