A fusion membrane nanovaccine for promoting co-stimulatory molecules and antigen expression up-regulation of tumor immune response and a preparation method thereof

Abstract

The application belongs to the field of nanobiomedicine, and specifically discloses a fusion membrane nanovaccine for promoting the immune response of tumors and up-regulating the expression of costimulatory molecules and antigens and a preparation method thereof. The vaccine shell is composed of a fusion membrane prepared from the membranes of dendritic cells with up-regulated expression of costimulatory molecules and melanoma tumor cells with up-regulated antigen abundance; and the inner core is composed of metal polyphenol nanoparticles loaded with an immune stimulant R848. The tumor vaccine can target dendritic cells, promote the antigen cross-presentation of the dendritic cells by up-regulating the antigen abundance, promote the activation of T cells, directly interact with T cells, promote the direct activation of T cells by up-regulating the costimulatory molecules, and kill melanoma tumor cells specifically, and thus has a wide application prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of nanobiomedicine, and relates to a fusion membrane nanovaccine for promoting the expression of co-stimulatory molecules and antigens and upregulating the immune response to tumors and a preparation method thereof. BACKGROUND

[0002] For the treatment of malignant tumors, traditional treatment methods such as surgery, radiotherapy and chemotherapy have been widely used in clinical practice, but they still cannot completely cure tumors. For example, surgery cannot completely remove tumor cells, and there is a risk of recurrence and metastatic tumor growth; radiotherapy and chemotherapy have defects such as drug resistance, side effects and easy recurrence, resulting in poor prognosis. Therefore, developing new tumor treatment strategies to achieve safe and efficient treatment of tumors is a key scientific problem that needs to be solved at present.

[0003] A tumor vaccine refers to screening and extracting tumor-associated antigens from tumor tissues, fusing the tumor-associated antigens with an adjuvant, and then injecting the tumor vaccine into a tumor patient to activate specific immune responses in the body to achieve the purpose of inhibiting tumor growth. Tumor antigens, as an important component of tumor vaccines, are necessary for activating specific immune responses. At present, single antigen delivery has limited antigen abundance and is difficult to induce effective specific immune responses to mutant tumors. Therefore, finding tumor antigens with multiple epitopes has become a key problem in immunotherapy. Tumor cell membranes have a complete set of tumor antigens on their surface and have good biocompatibility. At present, researchers construct tumor vaccines by extracting tumor cell membranes, which have the potential to safely activate immune responses and inhibit tumor growth. However, due to the complex immune escape mechanisms of tumor cells, tumor cells reduce the immunogenicity of tumor cells by downregulating the expression of membrane surface antigen complex molecules to promote the immune escape of tumor cells. Therefore, upregulating the expression of tumor cell membrane surface antigen complex molecules and increasing the immunogenicity of tumor cells are key to constructing a multi-antigen epitope tumor vaccine based on cell membranes.

[0004] In the development of tumor vaccines, how to effectively activate antigen-presenting cells to process and present antigens and activate T cells is another key scientific problem. Dendritic cells are the most powerful antigen-presenting immune cells, which internalize and process antigens and mediate the activation of T cells. The phagocytic ability of mature dendritic cells decreases, and the antigen processing and presentation ability, homing ability to lymph nodes and T cell activation ability are enhanced. Mature dendritic cells have many characteristics, mainly manifested as changes in the expression levels of related proteins such as costimulatory molecules and MHC molecules, which are essential for the efficient activation of T cells. The costimulatory molecules expressed on mature dendritic cells provide a second signal for the activation of T cells, and under the synergistic action of the first signal provided by the combination of MHC-I antigen complex and TCR of T cells, the activated T cells are converted into effector T cells, which in turn exert an anti-tumor immune response. Therefore, it is also important to induce dendritic cells to up-regulate the expression of costimulatory molecules and promote the processing and presentation of antigens by dendritic cells.

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

[0006] Therefore, in one aspect of the present application, the nanovaccine can be taken up and internalized by dendritic cells, and the activation of T cells is promoted by promoting antigen cross-presentation. On the other hand, the nanovaccine can directly interact with T cells, synergistically promoting the activation of T cells, thereby mediating efficient anti-tumor effects. SUMMARY

[0007] To solve the problems in the above background art, the purpose of the present application is to provide a fusion membrane nanovaccine for promoting tumor immune response and up-regulating expression of costimulatory molecules and antigens and a preparation method thereof. The nanovaccine is a fusion membrane obtained from tumor cell membranes with enhanced antigen abundance and dendritic cell membranes with up-regulated expression of costimulatory molecules, and is composed of a metal polyphenol nanocore loaded with an immune stimulant R848. The vaccine can be taken up and internalized by dendritic cells on one hand, and the activation of T cells is promoted by promoting antigen cross-presentation. On the other hand, the nanovaccine can directly interact with T cells, synergistically promoting the activation of T cells, thereby efficiently mediating anti-tumor immune response.

[0008] To achieve the purpose of the present application, the following technical solutions are adopted:

[0009] A fusion membrane nanovaccine for promoting tumor immune response and up-regulating expression of costimulatory molecules and antigens, the outer shell is composed of a fusion membrane prepared from dendritic cell membranes with up-regulated expression of costimulatory molecules and melanoma tumor cell membranes with up-regulated antigen abundance; the inner core is composed of metal polyphenol nanoparticles loaded with an immune stimulant R848;

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

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

[0012] Further,

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

[0014] Further,

[0015] The dendritic cell membrane with up-regulated co-stimulatory molecule expression is obtained after incubation of DC2.4 with R848; and the tumor cell membrane with up-regulated antigen abundance is obtained after incubation of B16F10-OVA tumor cells with mitochondrial fission inhibitor Mdivi-1.

[0016] The application further provides a preparation method of the fusion membrane nanovaccine with up-regulated co-stimulatory molecule and antigen expression for promoting tumor immune response, comprising the following steps:

[0017] (1) inducing dendritic cells to up-regulate the expression of co-stimulatory molecules, and extracting cell membrane DCM; inducing tumor cells with up-regulated antigen abundance, and extracting cell membrane CM(M), and fusing the two kinds of cell membranes to prepare fusion cell membrane DCCM(M) by ultrasonic treatment;

[0018] (2) mixing tannic acid and a solution of iron ions to prepare blank metal polyphenol nanoparticles TF through coordination, and loading immune adjuvant R848 to prepare TF / R848 nanoparticles;

[0019] (3) mixing the TF / R848 nanoparticles and the fusion membrane DCCM(M) solution, and preparing nanovaccine TF / R848@DCCM(M) by ultrasonic treatment.

[0020] Further,

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

[0022] A. inoculating DC2.4 into a culture dish, adding R848 diluted in culture medium with a concentration of 5-20 μg / mL after the cells adhere to the dish, and incubating for 24-48 h; after the incubation, collecting the DC2.4 cells, and washing with PBS for standby;

[0023] B. B16F10-OVA tumor cells are inoculated in a culture dish, after the cells adhere, mitochondrial fission inhibitor Mdivi-1 with a concentration of 10-50 μg / mL diluted in culture medium is added for incubation for 48-72 h; after the incubation is completed, the B16F10-OVA tumor cells are collected and washed with PBS for standby;

[0024] C. The cells treated in steps A and B are respectively placed in centrifuge tubes, after being washed with PBS by centrifugation, low osmotic solution containing 1% PMSF is added for incubation for 10-20 min to make the cells swell and break, then the cells are ground on ice, the cell liquid after sufficient grinding is centrifuged in a centrifuge at 1800-2700 rpm for at least 10 min, and the supernatant is collected to separate organelles and unbroken cells; the supernatant is centrifuged at 10000-12000 rpm for at least 30 min to collect cell membrane precipitates; the tumor cell membrane precipitates and the dendritic cell membrane precipitates are mixed at a mass ratio of 1:1-1:4, and are ultrasonicated for 3-5 min at a power of not more than 125 W to prepare fused cell membranes.

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

[0026] Iron chloride hexahydrate is dissolved in water and its concentration is adjusted to prepare an iron chloride hexahydrate solution with a concentration of 2-8 mg / mL; tannic acid is dissolved in methanol to prepare a tannic acid methanol solution with a concentration of 10-30 mg / mL; 30-150 μL of the iron chloride hexahydrate solution is added to 2 mL of 10-50 mM phosphate buffer solution with a pH of 8-10, after being stirred uniformly, 10-50 μL of the tannic acid methanol solution is slowly added, and stirring is performed at 700-900 rpm for 20-30 min; after the reaction is completed, the precipitates are collected by centrifugation at 8000-10000 rpm for 10-15 min, and the nanoparticles are obtained after being washed; the obtained nanoparticles are dispersed in 1 mL of water to obtain a uniformly dispersed nanoparticle suspension; R848 is dissolved in methanol to prepare a solution with a concentration of 5-30 mg / mL, and 50-100 μL of the R848 methanol solution is added to the suspension, and stirring is performed at room temperature for 6-12 h.

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

[0028] An appropriate amount of TF / R848 is ultrasonicated to disperse uniformly; the fused membrane DCCM(M) and the TF / R848 nanoparticles are mixed uniformly at a solid mass ratio of 1:1-1:2, and ultrasonication is performed at 4°C in a cold water bath for 5-10 min at a power of not more than 125 W to complete the coating of the fused membrane.

[0029] Advantages of the present application

[0030] 1. The application prepares a fusion membrane nano-vaccine with a metal polyphenol nanoparticle loaded with an immune agonist R848 as the inner core, and an antigen abundance enhanced tumor cell membrane and a dendritic cell membrane with up-regulated co-stimulatory molecule expression as the outer package, which is used for the immunotherapy of tumors. The tumor vaccine can directly activate naive T cells into specific anti-tumor effector cells, and can also act as a vaccine to promote antigen processing and cross-presentation of dendritic cells through homologous targeting, and activate specific anti-tumor immunity.

[0031] 2. The nano-vaccine provided by the application has dendritic cell membrane components, can promote the uptake of dendritic cells through homologous targeting, and has targeting effect.

[0032] 3. The nano-vaccine involved in the application has low toxic and side effects on normal tissue cells, mouse epithelioid fibroblasts and dendritic cells, and has good biocompatibility. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1 is a flow cytometry analysis diagram of up-regulating dendritic cell co-stimulatory molecules.

[0034] Figure 2 is a flow cytometry analysis diagram of tumor cell antigen abundance induced up-regulation.

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

[0036] Figure 4 is the fluorescence emission spectrum diagram of TF / R848 prepared by the application and free adjuvant R848.

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

[0038] Figure 6 is the particle size distribution diagram and transmission electron microscope diagram of TF / R848@DCCM(M) prepared by the application;

[0039] wherein Figure 6 A is the particle size distribution diagram of TF / R848@DCCM(M) prepared by the application; Figure 6 B is the transmission electron microscope diagram of TF / R848@DCCM(M) prepared by the application.

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

[0041] wherein NR is the fluorescent dye Nile Red.

[0042] Figure 8 Cytotoxicity study of metal polyphenol nanoparticles TF, TF / R848 and nanovaccine TF / R848@DCCM(M) on dendritic cells and normal tissue cells mouse epithelioid fibroblasts L929;

[0043] wherein Figure 8 Cytotoxicity study 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 TF / R848, TF / R848@DCCM and antigen abundance induced up-regulation vaccine TF / R848@DCCM(M) on dendritic cells after incubation with dendritic cells antigen presentation study of dendritic cells.

[0045] Figure 10 TF / R848, TF / R848@DCCM and antigen abundance induced up-regulation vaccine TF / R848@DCCM(M) on T cell proliferation-promoting study.

[0046] Figure 11 TF / R848, TF / R848@DCCM and antigen abundance induced up-regulation vaccine TF / R848@DCCM(M) on T cell direct activation study.

[0047] Figure 12 TF / R848, TF / R848@DCCM and antigen abundance induced up-regulation vaccine TF / R848@DCCM(M) on T cell indirect activation study.

[0048] Figure 13 TF / R848, TF / R848@DCCM and antigen abundance induced up-regulation vaccine TF / R848@DCCM(M) activated T cells on melanoma tumor cells B16F10-OVA killing effect study.

[0049] Figure 14 Activated T cells on tumor cells melanoma B16F10-OVA, breast cancer cells 4T1 and mouse epithelioid fibroblasts L929 killing effect study.

[0050] Figures 9-13TF / R848@DCCM is a nano-vaccine prepared from fusion cell membrane with antigen abundance uninduced up-regulation; and TF / R848@DCCM(M) is a nano-vaccine prepared from fusion cell membrane with antigen abundance induced up-regulation. DETAILED DESCRIPTION

[0051] In order to better illustrate the present application, the present application is further described below in combination with examples.

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

[0053] (1) DC2.4 was inoculated in a 6-well plate at a density of 5×10 5 / well, and after the cells adhered, blank medium, 10 μg / mL R848 diluted in medium was added respectively for incubation for 24 h. After the incubation, the DC2.4 cells were collected and washed with PBS twice. The DC2.4 cells in different wells were respectively dyed with anti-CD11c-FITC, anti-CD80-PE and anti-CD86-APC flow cytometry antibodies at 4°C for 30 min, and after the dyeing, the cells were centrifuged and washed with PBS once, resuspended with 400 μL PBS, and the maturation proportion of the DC2.4 was analyzed by flow cytometry. The results are shown in Figure 1 , compared with the control group without drug, the experimental group with drug incubation can significantly promote the expression of costimulatory molecules CD80 and CD86. Therefore, the dendritic cells with up-regulated costimulatory molecules are successfully induced.

[0054] (2) B16F10-OVA tumor cells with model antigen were inoculated in a 6-well plate at a density of 5×10 5 / well, and after the cells adhered, blank medium, Mdivi-1 with concentrations of 10 μg / mL, 30 μg / mL and 50 μg / mL diluted in medium was added respectively for incubation for 72 h. After the incubation, the B16F10-OVA tumor cells were collected and washed with PBS twice. The cells in different wells were respectively dyed with anti-H-2Kb-SIINFEKL-APC flow cytometry antibodies at 4°C for 30 min, and after the dyeing, the cells were centrifuged and washed with PBS once, resuspended with 400 μL PBS, and the up-regulation of MHC-antigen complex was analyzed by flow cytometry. The results are shown in Figure 2 , compared with the control group without drug, with the increase of the drug concentration, the expression of MHC-antigen complex was up-regulated in a gradient manner, and the induction effect of 50 μg / mL was the best. Therefore, the antigen abundance of tumor cells can be effectively increased by pretreatment.

[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 the ferric chloride hexahydrate solution to 2 mL of 10 mM phosphate buffer solution with pH 8, stir uniformly, slowly add 50 μL of the tannic acid methanol solution, and stir at 700 rpm at room temperature for 30 min. After the reaction, collect the precipitate by centrifugation at 10,000 rpm, and wash twice for standby. Disperse the nanoparticles obtained above in 1 mL of water, and add the uniformly dispersed nanoparticle suspension to a Schlenk flask. Dissolve the adjuvant R848 in methanol to prepare a 5 mg / mL solution, and add 50 μL of the R848 methanol solution to the TF suspension, and stir at room temperature for 12 h to obtain the TF / R848. Weigh a certain amount of tannic acid, R848, and metal polyphenol nanoparticle TF, and disperse TF / R848 in water. Take 700 μL of each and place in an analysis dish for spectral scanning in the wavelength range of 250-400 nm. The results are shown in FIG. 6, and it is found that TF / R848 has characteristic absorption peaks of the drug R848 at 307 and 322 nm, indicating the successful preparation of TF / R848. Figure 3

[0056] (4) Weigh a certain amount of TF / R848 nanoparticles and R848 powder, dissolve and dilute the R848 powder to the corresponding concentration of the TF / R848 nanoparticles. Add the two solutions to an analysis dish, excite at an excitation wavelength of 322 nm, and observe the emission spectrum in the range of 335-400 nm. The results are shown in FIG. 7, and it is found that the fluorescence emission spectrum of TF / R848 has 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. Figure 4

[0057] (5) Place the cells treated in steps (1) and (2) in centrifuge tubes, wash with PBS by centrifugation, and then incubate in a hypotonic solution containing 1% PMSF for 15 min to make the cells swell and break, and then grind on ice. Grind the cell liquid thoroughly, centrifuge at 2000 rpm for 10 min in a centrifuge, and collect the supernatant to separate organelles and unbroken cells. Centrifuge the supernatant at 12,000 rpm for 30 min to collect the cell membrane precipitate. Thus, we obtain the cell membrane DCM of dendritic cells with up-regulated expression of costimulatory molecules, and the cell membrane CM(M) of tumor cells with up-regulated antigen abundance.

[0058] ​​DiO (excitation / emission = 484 / 501) staining solution was used to stain the tumor cell membrane CM(M), and DiI (excitation / emission = 549 / 565) staining solution was used to stain the dendritic cell membrane DCM. The same mass of the two kinds of stained cell membranes was mixed and ultrasonicated for 5 min to prepare the fusion membrane DCCM(M). The tumor cell membrane CM(M), the dendritic cell membrane DCM, and the fusion membrane DCCM(M) were respectively detected by a fluorescence spectrophotometer at 484 nm excitation and 495-620 nm emission. The results are shown in Figure 5 FIG. 2, compared with the un-fused CM(M) and DCM, the fluorescence intensity of the fusion membrane DCCM(M) at 501 nm is weakened, while the fluorescence intensity at 565 nm is enhanced, and the fluorescence resonance transfer phenomenon occurs, which indicates the successful fusion of the two kinds of cell membranes.

[0059] (6) An appropriate amount of TF / R848 was ultrasonicated for 1 min to make it uniformly dispersed, and two kinds of cell membranes with a mass ratio of 1:1 were mixed and ultrasonicated for 5 min (125 W) to complete the fusion of the cell membranes. The fusion membrane DCCM(M) with a solid mass ratio of 1:1 was mixed with the TF / R848 nanoparticles uniformly, and the coating of the fusion membrane was completed by ultrasonication in a cold water bath for 5 min. The precipitate was collected by centrifugation. Dynamic light scattering was used to detect the particle size distribution of the nanodrug, and transmission electron microscopy was used to observe the morphological characteristics. The results are shown in Figure 6 FIG. 3, the average particle size of the nanovaccine is 193 nm. It is observed by TEM that the prepared nanovaccine has a clear core-membrane structure, and the particle size is basically consistent with the DLS result, which further indicates that the fusion membrane-coated nanovaccine TF / R848@DCCM(M) is successfully prepared.

[0060] Example 2: Cell uptake of nanovaccine

[0061] The cell uptake of the nanovaccine was investigated by high-content imaging. The model drug Nile Red (NR) was loaded on the metal polyphenol nanoparticles to characterize the cell uptake. The dendritic cells were seeded in a 96-well plate at a density of 1×10 4 / well, and cultured for 24 h. The supernatant was discarded, and the tumor cell membrane-coated nanodrug TF / NR@CM(M), the dendritic cell membrane-coated nanodrug TF / NR@DCM, and the fusion membrane-coated nanodrug TF / NR@DCCM(M) diluted in the culture medium were added and incubated for 4 h. After incubation, the supernatant was discarded, and the cells were washed twice with the culture medium. Then, the nuclear staining solution Hoechst 33342 diluted in the culture medium was added and incubated at room temperature for 10 min. After incubation, the cells were washed once with the culture medium, and 100 μL of blank culture medium was added to each well for imaging. The results are shown in Figure 7As shown, the dendritic cells have less uptake of the tumor cell membrane-coated nanoparticles TF / NR@CM(M), and the fluorescence intensity of the cells is weak. In contrast, the fluorescence intensity of the nanoparticles TF / NR@DCCM(M) and TF / NR@DCM with dendritic cell membrane components is strong, and the dendritic cells have increased uptake of the nanoparticles with dendritic cell membrane components. This shows that the prepared nanovaccine has certain homologous targeting effect.

[0062] Example 3: Cytotoxicity of the nanovaccine

[0063] The dendritic cells and mouse epithelioid fibroblasts L929 were inoculated at a density of 1×10 4 After 24 h of culture in the incubator, the upper culture medium 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 the culture medium were added to each well at 100 μL, and a blank experiment group (containing only the culture medium) and a control group (cells + blank culture medium) were set. After 24 h of incubation, the upper liquid was discarded, and the culture medium containing CCK8 was added for 1 h of incubation. The absorbance at 450 nm was detected by an enzyme marker, and the results are shown in Figure 8 The cytotoxicity of different concentrations of TF, TF / R848, and TF / R848@DCCM(M) nanovaccine to dendritic cells after 24 h of incubation can be ignored, and the cell survival rate is above 80%. Similarly, the cytotoxicity of different concentrations of TF, TF / R848, and TF / R848@DCCM(M) nanovaccine to L929 cells after 24 h of incubation is less, and the cell survival rate is above 75%, which shows that the constructed nanovaccine has good biocompatibility.

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

[0065] The dendritic cells were inoculated in a 6-well plate at a density of 5×10 5 After 24 h of culture, the supernatant was removed, and the culture medium-diluted TF / R848, TF / R848@DCCM, and antigen abundance up-regulation vaccine TF / R848@DCCM(M) were added, and the cells added with blank culture medium were used as a control group. After 36 h of incubation, the incubated dendritic cells were collected, washed once with PBS, and stained with flow antibodies CD11c-FITC and H-2kb-SIINFEKL-APC at 4°C for 30 min. PBS was added at 400 μL per tube for washing once, and flow detection was performed. The results are shown inFigure 9 As shown in FIG. 6, after incubation of TF / R848, TF / R848@DCCM (fusion membrane is fused from tumor cell membrane without up-regulated antigen and dendritic cell membrane with up-regulated costimulatory molecules) and TF / R848@DCCM(M) nanovaccine with dendritic cells for 24 h, the antigen abundance up-regulation group TF / R848@DCCM(M) has more significant antigen presentation effect than other groups. Therefore, the nanovaccine with up-regulated antigen abundance constructed by pretreatment can effectively promote the cross-presentation of dendritic cells, and lay a foundation for further mediation of T cell activation.

[0066] Example 5: Study on proliferation promotion of T lymphocytes by nanovaccine

[0067] CFSE was dissolved in DMSO to adjust its concentration to 10 mM as a stock solution. An appropriate amount of stock solution was added to serum-free medium to configure a working solution with a concentration of 5 μM. 1 mL of working solution was added to the extracted mouse T lymphocytes, incubated at room temperature for 10 min, washed twice with PBS after incubation to remove free dye, centrifuged and dispersed in 1640 complete medium containing IL-2 (10 ng / mL) to adjust the appropriate concentration. T lymphocytes were inoculated in 6-well plates at a density of 1×10 6 cells / well, and then TF / R848, TF / R848@DCCM and TF / R848@DCCM(M) were added, respectively. Cells added with blank medium were used as a control group, and incubated for 72 h. After incubation, T lymphocytes were collected and washed with PBS, and the proliferation of T lymphocytes was detected by flow cytometry (FITC channel), and the results are shown in FIG. 5. Figure 10 Compared with the TF / R848 experimental group without fusion membrane coating, the nanovaccine group with fusion membrane coating can significantly promote the proliferation of T lymphocytes. The proliferation promotion rate of the TF / R848 experimental group without fusion membrane coating is 9.86%, while the experimental groups with fusion membrane coating reach 12.2% and 18.1%, respectively. Compared with the TF / R848@DCCM group without up-regulation of antigen abundance, the TF / R848@DCCM(M) group with up-regulation of antigen abundance has more obvious proliferation promotion effect. Therefore, the nanovaccine with fusion membrane coating constructed by up-regulating the expression of MHC-antigen complex can directly interact with T lymphocytes, and directly promote the proliferation of T lymphocytes.

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

[0069] Mouse T lymphocytes were extracted and dispersed in 1640 complete medium containing IL-2. T lymphocytes were inoculated in 6-well plates at a density 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 tumor cell mitochondrial fission 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 T lymphocyte activation.

[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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