Anti-tumor composite nano vaccine as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological medicines, and discloses an anti-tumor composite nano vaccine, which comprises a dendritic cell source exosome and a tumor cell membrane, the dendritic cell source exosome carries a target pathogen antigen by fusing the tumor cell membrane, more effective antigen delivery and immune activation are realized, the safety is high, and the anti-tumor composite nano vaccine has a good anti-tumor effect. The compound has a certain application prospect in the aspect of preventing and treating malignant tumors such as glioblastoma and the like.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology and relates to an anti-tumor composite nanovaccine and its preparation method and use. Specifically, it relates to an anti-tumor composite vaccine formed by the fusion of dendritic cell exosomes with tumor cell membranes, which is particularly suitable for the prevention and treatment of glioblastoma. Background Art

[0002] Glioblastoma is a common primary brain tumor that grows in an infiltrative manner, exhibiting exceptional proliferation and invasiveness. Glioblastoma (GBM), the most malignant type, accounts for over 50% of cases. Patients with gliomas often lack typical symptoms in their early stages, making it easy for them to miss the optimal treatment window. Furthermore, the high malignancy of gliomas further complicates treatment, leading to suboptimal surgical outcomes and a high recurrence rate in the short term, posing a serious threat to patients' lives.

[0003] Immunotherapy is a biological treatment method that uses the principle of ligand-receptor interaction to stimulate or suppress the immune system. It helps the body fight infectious, immune, and neoplastic diseases by directly killing tumor cells, simulating antibody-dependent cellular phagocytosis, and blocking or activating T cell immune function. Currently, immunotherapy has achieved significant therapeutic effects in diseases such as lymphoma, melanoma, and leukemia. However, the effectiveness of immunotherapy in human glioma is still unsatisfactory.

[0004] Exosomes are membrane vesicles released by cells and present in nearly all bodily fluids. They possess the natural property of transporting molecular cargo (such as DNA, RNA, proteins, and lipids) between different cells. In recent years, their potential as nanocarriers for drug delivery has garnered widespread attention. Using exosomes as vaccine carriers can achieve more effective antigen delivery and immune activation. Dendritic cell-derived exosomes (DC-Exo) offer unique advantages in the field of tumor vaccines, as they retain the immunomodulatory properties of their parent cells. These exosomes not only carry key immunologically active components such as MHC-peptide complexes, co-stimulatory molecules, and cytokines, but also mimic the functions of antigen-presenting cells, enabling efficient T cell activation, subset differentiation, and immune memory formation. Notably, preclinical studies have demonstrated that the route of administration significantly influences the immune efficacy of DC-Exo vaccines: compared with conventional subcutaneous injection, intradermal administration significantly enhances systemic immune responses, a finding validated by quantitative detection of serum proinflammatory cytokines (including IL-12, IFN-γ, and TNF-α). Therefore, DC-Exo-based vaccine design has excellent potential to effectively activate T cells and anti-tumor responses, and is expected to play a role in the prevention and treatment of glioblastoma. Summary of the Invention

[0005] The present invention addresses the technical problems existing in the immunotherapy of glioblastoma and provides an anti-tumor composite nanovaccine. It carries target pathogen antigens through the fusion of dendritic cell-derived exosomes with tumor cell membranes, achieving more effective antigen delivery and immune activation. It has high safety and has certain application prospects in the prevention and treatment of malignant tumors such as glioblastoma.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides an anti-tumor composite nano-vaccine, comprising dendritic cell-derived exosomes and tumor cell membranes, wherein the dendritic cell-derived exosomes carry target pathogen antigens by fusing with tumor cell membranes.

[0008] Furthermore, the tumor cell membrane is a glioblastoma cell membrane.

[0009] In a second aspect, the present invention provides a method for preparing an anti-tumor composite nanovaccine, comprising the following steps: culturing dendritic cells and tumor cells, inducing dendritic cells to secrete dendritic cell-derived exosomes; extracting dendritic cell-derived exosomes; extracting tumor cell membranes; mixing the dendritic cell-derived exosomes with the tumor cell membranes, ultrasonically treating them, and then cyclically extruding them using a polycarbonate membrane to obtain a composite nanovaccine.

[0010] Furthermore, the extraction of dendritic exosomes adopts PEG sedimentation method,

[0011] Furthermore, the tumor cell membrane is extracted by liquid nitrogen rapid freezing-mechanical disruption combined with differential centrifugation.

[0012] Furthermore, the dendritic cell-derived exosomes and tumor cell membranes are mixed at a mass ratio of 1:1.2.

[0013] In a second aspect, the present invention provides use of the above-mentioned anti-tumor composite vaccine in the preparation of drugs for preventing and treating brain tumors.

[0014] Furthermore, the brain tumor is glioblastoma.

[0015] It is worth noting that, based on the concept of the present invention, those skilled in the art can fuse dendritic cell-derived exosomes with tumor cell membranes other than glioblastoma cell membranes to prepare corresponding tumor vaccines, thereby achieving the purpose of preventing and treating more tumor diseases.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The anti-tumor composite vaccine of the present invention comprises dendritic cell-derived exosomes and tumor cell membranes. By utilizing the presence of a large number of tumor antigens in the tumor cell membranes, more effective antigen delivery and immune activation are achieved. It is suitable for constructing a variety of tumor vaccines to prevent and treat a variety of tumor diseases and has broad application prospects. Based on dendritic cell-derived exosomes, cell-derived exosomes are naturally occurring cell vesicles with low immunogenicity and toxicity. The composite vaccine components are safe and easy to prepare. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Schematic diagram of the preparation of the DEX / GM composite nanovaccine of the present invention.

[0019] Figure 2 is the particle size of DEX and DEX / GM.

[0020] Figure 3 Western blot results of DEX and DEX / GM.

[0021] Figure 4 These are laser confocal micrographs of DC2.4 cells stably transfected with RFP.

[0022] Figure 5 The co-localization of DEX-RFP / GM-GFP in DC2.4 cells.

[0023] Figure 6 The uptake of DEX-RFP / GM in DC2.4 cells.

[0024] Figure 7 DEX / GM promotes the polarization of M2 macrophages to M1 in vitro.

[0025] Figure 8 DEX / GM promotes DC2.4 cell maturation in vitro.

[0026] Figure 9 The ability of DEX / GM to target lymph nodes in vivo, including: Figure 9 -a is an in vitro picture of each group of lymph nodes; Figure 9 -b is the quantitative results of fluorescence intensity of lymph nodes in each group.

[0027] Figure 10 To verify the optimal number of DEX / GM administration in vivo, including: Figure 10 -a is the experimental design diagram; Figure 10 -b is the flow cytometry of memory T cells in spleen and blood; Figure 10 -c is the quantitative statistical results of memory T cells in spleen and blood; Figure 10 -d is the ELISA analysis of interleukin-10 in blood.

[0028] Figure 11 The results of the DEX / GM immunization in vivo GL261-luc tumor prevention model in mice were as follows: Figure 11 -a is the fluorescent image of the brain of each group of immunized mice after three tumor attacks; Figure 11 -c is the survival curve of each group of immunized mice after multiple tumor attacks; Figure 11 -d~ Figure 11 -h is the quantitative statistical result of in vivo immune cell detection.

[0029] Figure 12 The results of DEX / GM immunization in mice were compared with the CT2A-luc tumor prevention model in vivo, including: Figure 12 -a is the fluorescence quantitative image of the brain of each group of immunized mice after tumor attack; Figure 12 -b is the weight change curve of each group of immunized mice after tumor attack; Figure 12 -c is the survival curve of each group of immunized mice after tumor attack; Figure 12 -d~ Figure 12 -h is the quantitative statistical result of in vivo immune cell detection.

[0030] Figure 13 H&E staining analysis of major organs of immunized mice after CT2A-luc tumor challenge. DETAILED DESCRIPTION

[0031] The following examples are used to illustrate the present invention but are not intended to limit the scope of protection of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. The test methods in the following examples are all conventional methods unless otherwise specified.

[0032] The reagents and consumables used in the following examples are as follows:

[0033] 0.22 μm pore size filter membrane was purchased from Merck Millipore Ltd.; CD63 antibody (67605-1-Ig) and ATP1A1 antibody (14418-1-AP) were purchased from Wuhan Sanying Biotechnology Co., Ltd.; 1640 culture medium, DMEM culture medium, penicillin-streptomycin solution, and non-essential amino acid solution were purchased from Gibco; serum was purchased from Sigma; DC2.4 cells were donated by Professor Yang Weijing of Zhengzhou University and can also be purchased externally; GL261-luc and CT2A-luc cells were donated by Professor Zhang Lei of Shaanxi Normal University and can also be purchased externally; exosome extraction kit was purchased from Zhengzhou Beibei Biotechnology Co., Ltd. (product number: 084001); C57BL / 6 mice were purchased from Beijing Sibeifu Biotechnology Co., Ltd.; cell membrane extraction kit (product number: P0033), puromycin (product number: ST551-10mg ) and polybrene (C0351-1mL) were purchased from Shanghai Biyuntian Biotechnology Co., Ltd.; CD3-PerCP-Cy5.5 (45-0031-82), CD4-FITC (11-0041-82), CD8-PE (12-0081-82), CD11c-FITC (11-0114-81), CD86-APC (17-0862-82), CD8a-PerCP-Cy5.5 (45-0081-82), CD3-FITC (11-0031-82), CD62L-APC (17-0621-82) and CD45-PE-Cyanine7 (25-0451-82) were purchased from Thermo Fisher Scientific; CD80-PE (104707), CD86-Brilliant Violet650 (105036), CD44-PE (103007), CD69-Brilliant Violet421 (104527) and CD45R / B220-APC (103211) were purchased from BioLegend; DAPI staining reagent (ready-to-use) (G1012-100ML), IF488-phalloidin (G1248-100T), and 4% paraformaldehyde (G1101-200ML) were purchased from Wuhan Saiwei Biotechnology Co., Ltd.; BCA quantitative kit (E112-02) was purchased from Nanjing Novozymes Biotechnology Co., Ltd.; IL-10 ELISA kit (JLC2863-97T) was purchased from Shanghai Jingkang Bioengineering Co., Ltd.

[0034] Example 1 Construction of composite nanovaccine using GL261-luc mouse glioblastoma cell membrane

[0035] 1.1 Preparation of DEX / GM composite nanovaccine

[0036] (1) Extraction of dendritic cell-derived exosomes (DEX)

[0037] Exosomes were isolated from DC2.4 cells (mouse bone marrow-derived dendritic cells) using a PEG precipitation method. The specific steps are as follows: ① Preparation of exosome-free culture medium: Normal fetal bovine serum (FBS) was removed by ultrahigh-speed centrifugation (160,000 × g, 4°C, 16 h) to obtain exosome-free serum. High-glucose DMEM medium was supplemented with 1% (v / v) penicillin-streptomycin solution, 1% (v / v) non-essential amino acid solution, and 10% (v / v) exosome-free serum to prepare complete culture medium. The medium was sterilized through a 0.22 μm filter before use. ② Dendritic cell culture and exosome induction: Mouse bone marrow-derived dendritic cells (DC2.4 cells) were seeded in 15 cm culture dishes and supplemented with exosome-free complete culture medium. The cells were cultured under standard culture conditions (37°C, 5% CO2, 95% humidity) for 72 h to promote exosome secretion. ③ Isolation and purification of dendritic cell-derived exosomes: After the culture, the cell supernatant was collected and centrifuged at 4000 × g for 15 min using a Thermo Fisher centrifuge to remove dead cells and cell debris. The supernatant was then filtered through a 0.22 μm pore size PES filter to remove extracellular vesicles with a diameter greater than 200 nm. The filtered supernatant was mixed with 8% (w / v) PEG-8000 solution at a volume ratio of 3:1 and incubated with horizontal shaking at 4°C for 1 hour. The mixture was then centrifuged at 12000 × g for 15 min, and the supernatant was discarded to precipitate the exosomes. Finally, the cells were resuspended in 1× PBS to a final concentration of 4.5 mg / mL (dendritic cell-derived exosomes, DEX) and stored at −80°C until use.

[0038] (2) Extraction of tumor cell membrane (GM)

[0039] The culture process and conditions of mouse glioblastoma cells (GL261-luc) refer to dendritic cells. After the tumor cell culture medium is collected, 1 mL of PBS is added to the culture dish, the tumor cells are scraped off with a cell scraper, the cell suspension is collected and transferred to a 50 mL centrifuge tube. Centrifuge at 2000 rpm for 5 minutes, discard the supernatant, collect the tumor cell pellet, and then add PBS to the cell pellet and wash it three times. After washing, add cell membrane extraction reagent A (containing 1% protease inhibitor) to the cell pellet and perform a lysis treatment on ice for 15 minutes. Subsequently, the cell pellet is repeatedly frozen and thawed three times with liquid nitrogen, and then centrifuged at a centrifugal force of 700g for 10 minutes to remove the nuclear components, and the supernatant is collected into a new EP tube. Finally, the collected supernatant is centrifuged again at a centrifugal force of 14000×g for 30 minutes. The resulting pellet is the glioblastoma cell membrane (denoted as GM). Quantify it using the BCA method and set aside.

[0040] (3) Preparation of DEX / GM composite nanovaccine

[0041] like Figure 1 The preparation process is shown. The exosome protein content was quantitatively analyzed by BCA protein assay. DC2.4 cell-derived exosomes (DEX, 4.5 mg / mL) and glioblastoma cell membrane fraction (GM, 5.4 mg / mL) were mixed in ice-cold PBS buffer (pH = 7.4) at a protein ratio of 1:1.2. To promote the modification of tumor cell membranes to the exosome surface, the mixed solution of tumor cell membranes and exosomes was sonicated (100W, 10 min, ice bath) to promote the efficient anchoring of GM on the DEX surface, forming a DEX / GM composite nanovaccine (denoted as DEX / GM).

[0042] The particle size distribution of DEX and DEX / GM was determined by dynamic light scattering (DLS) technique. The results showed that the average particle size of DEX / GM was 181.97±1.8nm, which was slightly larger than that of the original DEX (111.17±5.7nm) (see Figure 2 In addition, DEX, DM and DEX / DM were verified by Western blot experiments. The results showed that DEX / GM contained both the CD63 membrane protein unique to DEX and the ATP membrane protein unique to GM. That is, DEX / GM simultaneously expressed the exosome marker CD63 and the cell membrane marker ATP, confirming that membrane fusion was successful (see Figure 3 ).

[0043] 1.2 Characterization of DEX / GM composite nanovaccine

[0044] 1.2.1 Construction of cell lines stably expressing fluorescent proteins

[0045] A cell line stably expressing fluorescent protein was constructed by lentiviral transduction. First, 5×10 5 DC2.4 cells were seeded at a density of 1000 cells / mL in a 6 cm cell culture dish and incubated overnight at 37°C and 5% CO2 under standard culture conditions. The next day, fresh complete culture medium was replaced, 500 μL of lentiviral particles expressing red fluorescent protein (RFP) were added to the cells, and 1.5 μL of polybrene (final concentration 8 μg / mL) was added to improve the efficiency of viral transfection. After incubation for another 6 hours, the medium was replaced with antibiotic-free medium to remove residual viral particles. 48 hours after transfection, the successful expression of RFP in DC2.4 cells was confirmed by fluorescence microscopy (see Figure 4 ).

[0046] The same transfection steps were used for tumor cells (GL261-luc), and 5×105 Cells were seeded in culture dishes of the same specifications. After the cells adhered overnight, 500 μL of lentiviral vector expressing green fluorescent protein (GFP) was added and transduction was performed under the same polybrene conditions. Subsequent culture medium replacement and expression verification steps were consistent with those for DC2.4 cells.

[0047] To remove untransfected cells, DC2.4-RFP and GM-GFP cells were selected in medium containing 2 μg / mL puromycin dihydrochloride for 72 hours, with the medium replaced every 24 hours to maintain selection pressure. Cells that survived the selection and stably expressed fluorescence were expanded in complete medium for three passages and subsequently used in subsequent experiments. From these validated cells, DC2.4-RFP exosomes (denoted as DEX-RFP) and GL261-GFP cell membranes (denoted as GM-GFP) were collected for subsequent colocalization and cellular uptake experiments.

[0048] 1.2.2 Cell co-localization ability of DEX / GM composite nanovaccine in vitro

[0049] In the colocalization experiment, the specific steps are as follows: First, non-fluorescently labeled DC2.4 cells were plated at 1×10 6 DC2.4 cells were seeded at a density of 500 μg / mL in 12-well culture plates and incubated at 37°C, 5% CO₂ for 12 hours to ensure adequate cell attachment and growth. Subsequently, DC2.4 cells were co-cultured with DEX-RFP / GM-GFP nanoparticles for 4 hours at a DEX concentration of 52.5 μg / mL and a GM concentration of 63 μg / mL.

[0050] After co-culture, the cells were fixed with 4% paraformaldehyde solution for 10 minutes to maintain cell morphology and enhance the stability of the fluorescence signal. After fixation, the cells were thoroughly washed three times with PBS buffer to remove residual paraformaldehyde. To visualize the nuclear structure, 4',6-diamidino-2-phenylindole (DAPI) dye was added to the fixed and washed cells and stained for 10 minutes. DAPI can specifically bind to DNA and emit blue fluorescence under ultraviolet light excitation, thereby clearly marking the outline of the cell nucleus. After staining, the cells were washed again with PBS buffer three times to remove unbound DAPI dye.

[0051] Finally, confocal laser scanning microscopy (CLSM) was used to observe and analyze the treated cells, focusing on the co-localization of DEX-RFP / GM-GFP in DC2.4 cells. Figure 5It can be clearly seen that the red light emitted by DEX-RFP is highly consistent with the green light emitted by GM-GFP, which strongly proves that DEX and GM have a good co-localization situation.

[0052] 1.2.3 Cellular Uptake Ability of DEX / GM Composite Nanovaccine in Vitro

[0053] At the beginning of the experiment, 1×10 6 Non-fluorescently labeled DC2.4 cells were seeded into 12-well culture plates and incubated for 12 hours at 37°C in a 5% CO2 environment. Subsequently, DC2.4 cells were co-cultured with DEX-RFP or DEX-RFP / GM nanoparticles for 4 hours.

[0054] After the co-culture, the cells were immediately fixed with 4% paraformaldehyde solution for 10 minutes, and then washed three times with PBS buffer to completely remove the residual fixative. Next, the cytoskeleton was stained with phalloidin dye for 30 minutes. Phalloidin can specifically bind to actin fibers and produce a strong fluorescent signal, thereby clearly presenting the morphological structure of the cytoskeleton. After the staining is completed, the cells were thoroughly washed again with PBS buffer for 3 times to remove the unbound dye. Afterwards, the cell nucleus was stained with DAPI dye for 10 minutes, and after the staining was completed, it was also washed three times with PBS buffer.

[0055] Finally, the uptake of DEX-RFP and DEX-RFP / GM in DC2.4 cells was observed and analyzed using confocal laser scanning microscopy (CLSM). Figure 6 It can be clearly seen that DEX-RFP and DEX-RFP / GM have similar fluorescence intensities, which fully proves that the packaging of GM has no effect on the fluorescence of DEX.

[0056] 1.2.4 DEX / GM composite nanovaccine promotes M2 macrophage polarization and dendritic cell maturation in vitro

[0057] Mouse mononuclear macrophage leukemia cells (RAW264.7) were cultured at 1.2×10 5 The cells were seeded in a 6-well plate at a density of 10 cells and cultured for 12 minutes. Subsequently, the cells were stimulated with the polarization factor IL-4 (20 ng / mL) for 24 minutes. After incubation, the cells were washed three times with PBS to completely remove IL-4, and supplemented with 1 mL of fresh culture medium containing DEX / GM (DEX concentration: 200 μg / mL; GM concentration: 240 μg / mL). After an additional 24 hours of incubation, the cells were collected and stained with anti-CD86-APC and anti-CD206-PE antibodies. Finally, the stained cells were analyzed using a flow cytometer (BD FACSCalibur). Figure 7 As shown, DEX / GM effectively promoted the polarization of M2 macrophages to M1 macrophages.

[0058] DC2.4 cells were plated at 1×10 5 The cells were seeded at a density of 100 cells / mL in a 12-well plate and cultured for 12 hours. Then, the cells were treated with lipopolysaccharide LPS (1.2 μg / mL) or DEX / GM (DEX concentration: 200 μg / mL; GM concentration: 240 μg / mL) for 24 hours. After incubation, the cells were collected and stained with anti-CD86-BV650 and anti-CD80-PE antibodies. Finally, the stained cells were analyzed using a flow cytometer (BD FACSCalibur). Figure 8 As shown, DEX / GM significantly promoted the maturation of DC2.4 cells.

[0059] 1.2.5 Lymph node targeting ability of DEX / GM composite nanovaccine in vivo

[0060] The Cy5 dye solution and the DEX solution were mixed and first sonicated at 100W for 10 minutes. The mixture was then incubated in a 37°C oven for 1 hour to promote the binding of the dye to the DEX. After incubation, the mixture was washed with PBS buffer and centrifuged at 140,000 × g for 20 minutes in an ultracentrifuge to remove unbound free Cy5 dye.

[0061] Cy5-labeled DEX (denoted as DEX-Cy5) or a DEX-Cy5 / GM complex (DEX concentration: 11.25 mg / kg, GM concentration: 13.5 mg / kg) was injected intradermally into mice bearing GL261-luc tumors to evaluate their lymph node targeting ability. Four hours after injection, tumor-bearing mice were euthanized, and lymph nodes were isolated. Fluorescence imaging of the lymph nodes was performed using an IVIS Lumina III fluorescence imaging system (Caliper, MA, USA), and the lymph node targeting efficiency of DEX-Cy5 and DEX-Cy5 / GM was assessed by quantitative analysis of fluorescence intensity.

[0062] like Figure 9 -a The isolated lymph node image shows that DEX-Cy5 and DEX-Cy5 / GM have similar Cy5 enrichment abilities. Figure 9 The fluorescence semi-quantitative analysis of lymph nodes (b) also showed the same experimental results as above, indicating that DEX / GM has lymph node targeting ability and the introduction of GM has no effect on the lymph node targeting ability of DEX.

[0063] 1. Verification of the optimal administration frequency of 3DEX / GM composite nanovaccine in vivo

[0064] C57BL / 6 mice were randomly divided into 6 groups and received 5, 4, 3, 2, 1 administrations or PBS control group (0 administrations), with 7 days between each administration. Seven days after the last administration, mice were challenged with orthotopic GL261-luc cells. Blood and spleen samples were collected from mice 7 days after the tumor attack ( Figure 10 -a).

[0065] The collected mouse blood was centrifuged at 800×g for 10 minutes, the plasma was discarded, the lower layer of cells was lysed with red blood cell lysis buffer, and then washed once with PBS. Afterwards, anti-CD3-FITC, anti-CD8-Cy5.5, anti-CD62-APC and anti-CD44-PE antibodies were added for staining according to the dosage recommended in the instructions. The spleen was gently separated into a single cell suspension through a 150-mesh sieve, and then the cells were collected by centrifugation at 1500rpm for 3 minutes, and the cells were stained with the same antibody and dose. Finally, the memory T cells in the blood and spleen were quantitatively analyzed by flow cytometry (BD FACSCalibur), and the data were processed using FlowJo software (version 10.6.2). From Figure 10 -b~ Figure 10 -d shows that mice that were immunized three times produced more memory T cells in the spleen and blood.

[0066] In addition, the mouse blood was centrifuged at 800×g for 10 min to obtain serum, and the level of anti-inflammatory cytokine interleukin-10 (IL-10) in the serum was analyzed using an enzyme-linked immunosorbent assay (ELISA) kit. The entire operation was carried out strictly in accordance with the ELISA kit instructions. Figure 10 -e As can be seen, mice that received three immunization injections produced fewer anti-inflammatory cytokines in their blood.

[0067] 1.4 Effect of DEX / GM composite nanovaccine on in vivo tumor prevention model

[0068] Female C57BL / 6 mice (6-8 weeks old, n=10 / group) received intradermal injections of PBS, DEX (11.25 mg / kg), or DEX / GM (DEX 11.25 mg / kg; GM 13.5 mg / kg) once a week for 3 consecutive weeks. Seven days after the last immunization, 1×10 5An orthotopic glioblastoma model was established with GL261-luc cells (inoculated on Day 1). Tumor bioluminescence signals were monitored every 48 hours using an in vivo imaging system (IVIS Lumina III, PerkinElmer), and mouse survival was recorded. Three mice per group were sacrificed on Day 7, and blood, lymph nodes, and spleens were collected for immune cell phenotyping analysis. Three mice per group were sacrificed on Day 20, and spleens were isolated to assess memory T cell subsets.

[0069] like Figure 11 As shown in Figure 2a, after the initial tumor challenge, DEX monotherapy demonstrated a moderate tumor growth inhibitory effect compared to the PBS control group, while incorporation of tumor cell membranes into DEX / GM significantly enhanced the anti-tumor efficacy. Notably, 60% of DEX-treated mice (6 / 10) and 100% of DEX / GM-treated mice (10 / 10) exhibited undetectable tumor bioluminescence signals during the monitoring period. DEX / GM achieved complete tumor eradication by day 42.

[0070] To further evaluate the long-term immune memory effect, PBS control mice and DEX / GM-immunized mice of the same age were given 1.5×10 5 The second in situ challenge of GL261-luc cells and 3.0 × 10 5 The third attack of GL261-luc cells. The results showed that the tumors of all mice in the PBS group progressed rapidly, with significant weight loss within 20 days and a mortality rate of 100% ( Figure 11 -b, Figure 11 -c). In contrast, DEX / GM-immunized mice remained 100% tumor-free for 91 days. 5 During the third attack of tumor cells, the DEX / GM immunized mice still did not develop tumors and their body weight remained stable ( Figure 11 -b), while the median survival time of mice in the initial PBS control group was only 13 days ( Figure 11 Notably, DEX / GM immunization demonstrated durable protection, with all vaccinated mice showing no tumor recurrence 150 days after the final challenge ( Figure 11 -c).

[0071] Flow cytometry analysis showed that the CD4 + / CD8 + The proportion of T cells increased significantly, and CD62L + and CD44 + The proportion of cell subpopulations was significantly better than that of the PBS control group ( Figure 11 -d- Figure 11 -h).

[0072] The above results indicate that DEX / GM vaccine achieves significant anti-tumor effects by synergistically activating cytotoxic T lymphocytes (CTLs) and inducing long-term immune memory.

[0073] Taken together, these results collectively demonstrate that DEX / GM vaccines can not only effectively activate anti-tumor immune responses but also induce the formation of long-term immune memory.

[0074] Example 2 Construction of composite nanovaccine using CT2A-luc mouse glioblastoma cell membrane

[0075] In view of the good tumor prevention effect of the DEX / GM vaccine in the GL261-luc mouse brain glioblastoma model demonstrated in Example 1, this example further explores its potential to inhibit tumor growth in the CT2A-luc glioblastoma model with phosphatase and tensin homolog (PTEN) gene deletion. To this end, an in situ CT2A-luc GBM prevention model was constructed to verify this hypothesis. In this example, DC2.4 cell-derived exosomes (DEX) and mouse glioblastoma cell (CT2A) cell membranes were used to construct a DEX / GM composite nanovaccine. The steps of exosome and cell membrane extraction and composite vaccine construction are the same as in Example 1 and will not be repeated here.

[0076] like Figure 12 As shown in Figure 2-a, the tumors in the PBS group of mice proliferated rapidly. Although DEX alone had a partial delay effect on tumor growth in the early stages, the inhibitory effect of the DEX / GM vaccine was significantly better than that of DEX alone. It is worth noting that the body weight of mice in the PBS and DEX groups decreased significantly, while the body weight of mice in the DEX / GM treatment group did not change significantly ( Figure 12 -b). In addition, the survival of mice immunized with DEX / GM was as long as 40 days, compared with 23 days in the PBS group and 29 days in the DEX group ( Figure 12 -c).

[0077] To comprehensively evaluate the immune response induced by the vaccine, flow cytometry was used to systematically analyze the immune cell phenotypes of lymph nodes, blood, and spleen samples. Experimental data showed that the DEX and DEX / GM immune groups showed significant improvements in several key immunological indicators: CD80 in lymph nodes + / CD86 + The proportion of mature cells increased significantly ( Figure 12 -d); CD3 + / CD8 + T cells (cytotoxic T cells) and CD3 + / CD4 +The proportion of T cells (helper T cells) increased significantly ( Figure 12 -e~12-f); effector memory T cells in the spleen (CD3 + CD8 + CD62L - CD44 + , T EM ) and central memory T cells (CD3 + CD8 + CD62L + CD44 + , T CM ) population significantly expanded ( Figure 12 More importantly, compared with the DEX group, the DEX / GM group showed a more significant immune-enhancing effect due to the addition of cell membrane components, which fully demonstrates that cell membrane components have a synergistic effect on vaccine efficacy.

[0078] CD8 + In-depth analysis of T cell activation markers showed that CD69 + The proportion of activated T cells was significantly higher than that of other groups ( Figure 12 -h), which strongly proves that it has stronger T cell activation ability. In addition, the results of bone marrow microenvironment analysis showed that the DEX / GM group significantly promoted the proliferation of B cells ( Figure 12 -i).

[0079] Toxicity evaluation of major organs (heart, liver, spleen, lung and kidney) was performed by H&E staining, and the results showed that the DEX / GM vaccine had good biocompatibility ( Figure 13 ).

[0080] The embodiments described above are only preferred embodiments of the present invention and are only used to explain the present invention, not to limit the scope of implementation of the present invention. For those skilled in the art, it is of course possible to easily make other implementation methods by replacing or changing the technical content disclosed in this specification. Therefore, all changes and improvements made on the principles of the present invention should be included in the scope of the patent application of the present invention.

Claims

1. An anti-tumor composite nano vaccine, characterized in that: It includes dendritic cell-derived exosomes and tumor cell membranes. The dendritic cell-derived exosomes carry target pathogen antigens by fusing with the tumor cell membranes.

2. The anti-tumor composite nano vaccine according to claim 1, characterized in that The tumor cell membrane is a glioblastoma cell membrane.

3. The method for preparing the anti-tumor composite nanovaccine according to any one of claims 1 to 2, characterized in that: The following steps are involved: Dendritic cells and tumor cells are cultured to induce the dendritic cells to secrete dendritic cell-derived exosomes; the dendritic cell-derived exosomes are extracted; the tumor cell membranes are extracted; the dendritic cell-derived exosomes are mixed with the tumor cell membranes, and ultrasonically treated to obtain a composite nanovaccine.

4. The preparation method according to claim 3, characterized in that The extraction of dendritic exosomes adopts the PEG sedimentation method.

5. The preparation method according to claim 3, characterized in that The tumor cell membrane is extracted by using liquid nitrogen rapid freezing-mechanical disruption combined with differential centrifugation.

6. The preparation method according to claim 3, characterized in that The dendritic cell-derived exosomes and tumor cell membranes are mixed at a mass ratio of 1:1.

2.

7. Use of the anti-tumor composite vaccine according to any one of claims 1 to 2 in the preparation of drugs for preventing and treating brain tumors.

8. The use according to claim 7, characterized in that The brain tumor is glioblastoma.

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