Dendritic cell bionic nano tumor vaccine for targeted therapy of brain glioma as well as preparation method and application of dendritic cell bionic nano tumor vaccine

Abstract

The invention discloses a dendritic cell bionic nano tumor vaccine for targeted therapy of brain glioma as well as a preparation method and application of the dendritic cell bionic nano tumor vaccine. The bionic nano tumor vaccine disclosed by the invention is composed of dendritic cell (DC) membrane protein on an outer layer, polylactic acid-glycolic acid copolymer (PLGA) and a TLR7 / 8 agonist R848 serving as an inner core encapsulation. According to the present invention, the intracerebral delivery efficiency of R848 can be significantly improved, the antitumor activity of T cells at the tumor site can be enhanced, the progress of brain glioma can be inhibited, the systemic toxicity can be reduced, and the safe and efficient new strategy can be provided for the precise immunotherapy of brain glioma.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a dendritic cell biomimetic nano-tumor vaccine for targeted treatment of brain gliomas, and a preparation method and application thereof. Background Art

[0002] Glioblastomas (GBMs), the most common malignant tumor of the central nervous system, are highly invasive and recurrent. The five-year survival rate for glioblastomas (WHO grade 4) is less than 5%, posing significant challenges to clinical treatment. Surgical resection is difficult to achieve due to the blurred tumor margins, while chemoradiotherapy is limited by the dual barriers of the blood-brain barrier (BBB) ​​and the blood-brain tumor barrier (BTB). This results in inefficient brain accumulation of traditional chemotherapy drugs, such as temozolomide, and significant systemic toxicity. Although immune checkpoint inhibitors (such as PD-1 / PD-L1 antibodies) have significant efficacy in some solid tumors, the glioma microenvironment (TME) exhibits typical "cold tumor" characteristics: tumor-associated macrophages (TAMs) account for as high as 30%-40% and secrete a large number of immunosuppressive factors; dendritic cells (DCs) are functionally defective due to blockage of the NF-κB / IRF1 pathway, resulting in insufficient secretion of IL-12 and IFN-γ, which cannot effectively activate CD8+ T cells; at the same time, high expression of PD-L1 induces T cell exhaustion, making it difficult for single immunotherapy to reverse the profound immunosuppression state. This complex immunosuppressive network makes gliomas highly resistant to existing treatments, and there is an urgent need to develop innovative strategies that can simultaneously break through delivery barriers and reshape the immune microenvironment.

[0003] In recent years, biomimetic nano-delivery systems have provided new approaches for the treatment of gliomas. Dendritic cell (DC) membranes have attracted much attention due to their unique biological properties: molecules such as VLA-4 (α4β1 integrin) and LFA-1 (lymphocyte function-associated antigen-1) naturally expressed on their surface can specifically bind to VCAM-1 and ICAM-1 on the surface of brain endothelial cells, mimicking the natural trans-BBB migration behavior of DCs, thereby mediating the active transport of nanocarriers. In addition, the homologous targeting properties of DC membranes can promote the efficient internalization of nanoparticles by DCs residing in the TME, providing the possibility for precise drug delivery to immune cells. However, biomimetic designs that rely solely on DC membranes have significant limitations: first, free DC membranes lack the ability to encapsulate and control the release of drugs, making it difficult to maintain the sustained effect of drugs at the lesion site; second, membrane proteins are prone to denaturation or loss during the extraction and coating process, resulting in attenuation of targeting function; third, existing technologies mostly focus on the single delivery function of DC membranes and fail to deeply integrate them with immune adjuvants and synergistic treatment strategies, making it difficult to achieve multi-dimensional immune regulation.

[0004] In the field of drug carrier materials, the biodegradable polymer PLGA (poly(lactic-co-glycolic acid)) has become an ideal encapsulation material for hydrophobic drugs due to its excellent biocompatibility and sustained-release properties. PLGA can be gradually degraded by hydrolysis, achieving controlled release of drugs. At the same time, its degradation products (lactic acid and glycolic acid) can be metabolized by the body, avoiding long-term toxic accumulation. Studies have shown that the particle size and surface charge of PLGA nanoparticles can regulate their in vivo distribution and cellular uptake efficiency, providing design space for optimizing drug delivery. However, traditional PLGA carriers rely on passive targeting (such as the enhanced permeation and retention effect, EPR effect) and face multiple obstacles in glioma treatment: the tight junctions of the BBB greatly limit the brain penetration of nanoparticles; tumor heterogeneity causes the EPR effect to be unstable in gliomas; and a single PLGA system cannot overcome the immunosuppressive barrier of the TME and needs to be synergistically designed with immune regulatory elements to enhance efficacy.

[0005] As a potent immune adjuvant, TLR7 / 8 agonist R848 can activate TLR signaling pathways in DCs, promote the expression of co-stimulatory molecules (CD80 / CD86) and IL-12 secretion, and drive CD8 + T cell activation and proliferation. Preclinical studies have shown that R848 can significantly enhance the antigen presentation efficiency of tumor vaccines, but its clinical application faces severe challenges: the strong hydrophobicity of R848 makes it difficult to penetrate the BBB, and only a small amount of the drug can reach the brain lesions after systemic administration; free R848 lacks targeting and is easily cleared by the monocyte-macrophage system in the circulation, and excessive activation of peripheral immune cells may trigger systemic inflammatory responses (such as fever and cytokine release syndrome); in addition, DCs in the glioma TME are sparsely distributed and functionally inhibited, further reducing the therapeutic efficacy of R848. Existing technologies attempt to encapsulate R848 in liposomes or polymer micelles to improve solubility, but these carriers still rely on passive targeting and cannot break through the BBB and accurately deliver to DCs in the TME, resulting in insufficient effective concentrations of the drug in the target cells.

[0006] To address the dual bottlenecks of delivery and immune regulation, an innovative strategy integrating biomimetic targeting, intelligent controlled release, and immune synergy is urgently needed. Therefore, the development of a biomimetic nanodrug system combining dendritic cell (DC) membranes, the TLR7 / 8 agonist R848, and PLGA has become the key to breaking through the bottleneck of glioma treatment through the synergistic effects of DC membrane-mediated brain-targeted delivery, PLGA-controlled intracellular release of R848, and immune activation of the DC-T cell axis. Summary of the Invention

[0007] This invention addresses the problem of insufficient efficacy in treating gliomas due to the difficulty of drugs penetrating the blood-brain barrier (BBB) ​​and the immunosuppressive microenvironment. It provides a dendritic cell (DC) biomimetic nano-tumor vaccine for the targeted treatment of gliomas, as well as its preparation method and application. This vaccine, through the synergistic effects of biomimetic delivery, intelligent controlled release, and immune activation, breaks through the BBB delivery barrier, reverses the immunosuppression of the tumor microenvironment (TME), and significantly enhances anti-tumor efficacy.

[0008] The technical solution adopted by the present invention to solve the technical problem is:

[0009] In the first aspect, the present invention provides a dendritic cell biomimetic nano-tumor vaccine DCM@PLGA / R848 for the targeted treatment of brain glioma. The tumor vaccine is mainly composed of dendritic cell (DC) membrane-coated poly(lactic acid-co-glycolic acid) copolymer (PLGA) nanoparticles, and the core of the PLGA nanoparticles encapsulates the TLR7 / 8 agonist Resiquimod (R848); the DC membrane surface retains VLA-4 and LFA-1 molecules, which are used to penetrate the blood-brain barrier and target dendritic cells in the tumor microenvironment.

[0010] The biomimetic nano-tumor vaccine of the present invention has excellent biocompatibility and brain targeting ability. It can actively recognize brain endothelial cell VCAM-1 / ICAM-1 through VLA-4 and LFA-1 molecules on the surface of DC membrane, efficiently penetrate the blood-brain barrier (BBB), and target DCs enriched in the microenvironment of brain glioma. The PLGA core releases R848 through hydrolysis, activates the TLR7 / 8 signaling pathway in DCs, promotes the secretion of IL-12 and IFN-γ, and drives CD8 + T cell maturation and infiltration, reversing the immunosuppressive microenvironment. Experiments have shown that the biomimetic nanovaccine of the present invention significantly improves the efficiency of R848 delivery into the brain, enhances the anti-tumor activity of T cells at the tumor site, inhibits glioma progression, and reduces systemic toxicity.

[0011] In a specific embodiment, the nanoparticles have an average particle size of 100.18±0.774 nm and a surface charge of -28.1±0.378 mV.

[0012] In a specific embodiment, the molar ratio of lactic acid to glycolic acid in the PLGA is 30-70:30:70, preferably 45-55:45-55, more preferably 50:50, the drug loading of R848 is 5.15±1%, and the encapsulation efficiency is 37.3±5%.

[0013] In a specific embodiment, the mass ratio of the DC membrane to the PLGA nanoparticles is 1:20-40, preferably 1:30.

[0014] In a specific embodiment, the DC membrane is extracted from DC2.4 cells by repeated freezing and thawing, retains VLA-4 and LFA-1 molecules on the surface, and actively penetrates the BBB and targets DC in the TME through VCAM-1 / ICAM-1 mediation.

[0015] In a specific embodiment, the extraction of the DC membrane specifically comprises the following steps:

[0016] (1) DC2.4 cells were digested and separated from the culture dish using trypsin, and then resuspended in pre-chilled PBS. The cells were subjected to three freeze-thaw cycles: freezing at -80 ± 5 °C for 30 ± 3 minutes and thawing at 37 ± 3 °C for 30 ± 3 minutes, repeated three times.

[0017] (2) Collect the frozen-thawed cell suspension and sonicate in an ice bath for 3 to 8 times at a power of 100 W, with each sonication lasting 1 to 3 seconds, preferably 2 seconds, and an interval of 3 to 8 seconds, preferably 5 seconds;

[0018] (3) Centrifuge at 4°C at a slow speed, such as 500-1000g, preferably 600g, discard the pellet (nuclei), and collect the supernatant;

[0019] (4) Centrifuge rapidly at 4°C, such as 40,000-60,000g, preferably 50,000g, and discard the supernatant. The precipitate is the DC membrane.

[0020] In a specific embodiment, the extracted DC membranes are stored in PBS solution at 4°C.

[0021] The present invention utilizes DC membrane for the first time to achieve dual-stage delivery of BBB penetration and DC-specific targeting; the nano-tumor vaccine DCM@PLGA / R848 controlled-release system synergizes with the immune regulatory function of the DC membrane, and a single system realizes the entire "delivery-release-activation" chain; and the biocompatible materials (DC membrane, PLGA) significantly reduce systemic toxicity.

[0022] In a specific embodiment, the vaccine further contains other pharmaceutically acceptable excipients.

[0023] In a second aspect, the present invention provides a method for preparing any of the above-mentioned dendritic cell biomimetic nano-tumor vaccines, comprising the following steps:

[0024] (1) PLGA was dissolved in a mixed solution of ethanol and acetonitrile, and R848 was added. Poly(lactic-co-glycolic acid) nanoparticles with the core encapsulated with the TLR7 / 8 agonist R848 were prepared by nanoprecipitation. The organic solvent was then removed by rotary evaporation and ultrafiltration to prepare drug-loaded PLGA nanoparticles.

[0025] (2) Coating the DC membrane and the PLGA nanoparticles prepared in step (1) by ultrasonic fusion:

[0026] (A1) DC membrane was sonicated in an ice bath (power 100 W, 2 seconds each time, repeated 3 times) to form a nanoscale membrane; (A2) PLGA nanoparticles were mixed with DC membrane and sonicated in an ice bath (power 100 W, 1 second each time, repeated 3 times);

[0027] (A3) The mixed solution was extruded through a polycarbonate membrane and the precipitate was collected by centrifugation to obtain DCM@PLGA / R848.

[0028] In a specific embodiment, in step (1), the mass ratio of polylactic acid-glycolic acid copolymer to R848 is 10-50:4, preferably 20-35:4.

[0029] In a specific embodiment, in step (1), the volume ratio of ethanol to acetonitrile is 2:1-5, preferably 2:1-3, 2:3-5, and more preferably 2:1, 2:2, 2:3, 2:4, 2:5.

[0030] In a specific embodiment, the aqueous phase of the nanoprecipitation method in step (1) is a 0.5% Tween 80 solution.

[0031] More preferably, the specific preparation method of step (1) is as follows:

[0032] (B1) PLGA and R848 were dissolved in an ethanol-acetonitrile mixed solvent according to the mass ratio to form an organic phase;

[0033] (B2) adding 0.5% Tween 80 aqueous solution dropwise to the organic phase in step (B1), stirring magnetically at room temperature for 2-5 hours, and then subjecting to reduced pressure rotary evaporation at 45°C for 20-50 minutes;

[0034] (B3) The resulting solution was purified by 10 kDa ultrafiltration tube and centrifuged, and the filtrate was dispersed in ultrapure water to obtain PLGA / R848 nanoparticles.

[0035] In a specific embodiment, after ultrasonic fusion in step (2), a liposome extruder is used to sequentially extrude through polycarbonate membranes with pore sizes of 800 nm, 400 nm, and 200 nm for 10 to 20 times each.

[0036] Preferably, in said (A3), the centrifugation condition is: centrifugation at 20,000 g for 30 minutes.

[0037] In a third aspect, the present invention also protects the use of the dendritic cell biomimetic nano-tumor vaccine described in any of the above items in the preparation of a product for treating brain glioma.

[0038] In a fourth aspect, the present invention also protects the use of the dendritic cell biomimetic nano-tumor vaccine described in any of the above items in the preparation of a product for inhibiting the proliferation and / or production of brain glioma cells.

[0039] Preferably, the brain glioma is glioblastoma.

[0040] Preferably, the product is a medicine;

[0041] Preferably, the drug further contains other pharmaceutically acceptable carriers.

[0042] Beneficial effects

[0043] Compared with the existing technology, the nano-tumor vaccine DCM@PLGA / R848 of the present invention has the following advantages: (1) Highly efficient brain targeting: DC membrane-mediated active delivery breaks through the BBB restriction and improves the drug accumulation rate in the tumor site.

[0044] (2) Local-systemic immune activation: R848 release drives DC maturation, which in turn drives cold tumors to heat through the three-level immune network, synergistically enhancing local-lymph node-systemic anti-tumor responses, CD4 + T cells dynamically balance helper / killer functions, simulating systemic regulation of therapeutic tumor vaccines.

[0045] (3) Scalability: It can be combined with immune checkpoint inhibitors or chemotherapy drugs to achieve multimodal synergistic treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 This is a transmission electron microscopy image of the dendritic cell biomimetic nano-tumor vaccine DCM@PLGA / R848 of the present invention;

[0047] Figure 2 Laser particle size analyzer is used to measure the particle size of nanoparticles;

[0048] Figure 3 This is the sodium dodecyl sulfate-polyacrylamide gel electrophoresis image of DCM@PLGA / R848 in Example 2;

[0049] Figure 4 Western blot of DCM@PLGA / R848;

[0050] Figure 5 This is the blood-brain barrier migration diagram of DCM@PLGA / R848;

[0051] Figure 6 This is a graph showing the expression of the blood-brain barrier tight junction protein ZO-1 in vitro;

[0052] Figure 7 In vivo fluorescence distribution diagram (A) and semi-quantitative diagram (B, C) of DCM@PLGA / R848 in treating brain glioma;

[0053] Figure 8This is the tumor fluorescence change and survival curve of DCM@PLGA / R848 in the treatment of brain glioma;

[0054] Figure 9 Figure 4: Activation of DC cells in the brain during the treatment of glioma with DCM@PLGA / R848 (AC)

[0055] and T cell population diagram (DF);

[0056] Figure 10 Figure 4 shows DC cell activation (AC) and T cell population (DF) in cervical lymph node of brain glioma treated with DCM@PLGA / R848.

[0057] Figure 11 Figures 2 and 3 show the activation of splenic DC cells (AC) and T cell clustering (DF) in the treatment of brain glioma with DCM@PLGA / R848. DETAILED DESCRIPTION

[0058] The present invention is further described below with reference to specific examples and accompanying drawings. The specific examples are carried out under the preferred conditions of the present invention. The methods described are conventional methods unless otherwise specified, and the raw materials described can be obtained from public commercial channels unless otherwise specified.

[0059] Example 1 Preparation and Characterization of DCM@PLGA / R848

[0060] This example discloses the preparation method and structural characterization of DCM@PLGA / R848. The specific implementation steps are as follows:

[0061] 1. Preparation of PLGA / R848 Nanoparticles

[0062] Step 1: PLGA and R848 were dissolved in an ethanol-acetonitrile mixed solvent (volume ratio 2:3) at a mass ratio of 25:4 to form an organic phase;

[0063] Step 2: Add 0.5% Tween 80 aqueous solution dropwise to the organic phase, stir magnetically at room temperature for 4 h, and then evaporate under reduced pressure at 45 °C for 30 min;

[0064] Step 3: The obtained solution was purified by 10 kDa ultrafiltration tube, centrifuged at 5000 rpm for 15 min × 3 times, and the filtrate was dispersed in ultrapure water to obtain PLGA / R848 nanoparticles.

[0065] 2. Extraction of Dendritic Cell Membranes (DCM)

[0066] Step 1: DC2.4 cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (37°C, 5% CO2) and passaged at a 1:3 ratio when the density reached 80%.

[0067] Step 2: After trypsinization, resuspend DC2.4 cells in pre-chilled PBS and lyse the cells by freeze-thaw cycles (-80°C / 37°C × 3 times) and ice-bath probe sonication (power 100 W, 3 times × 2 seconds);

[0068] Step 3: The lysate was purified by gradient centrifugation (600 g × 10 min to remove debris, 50,000 g × 30 min to collect the DCM precipitate).

[0069] 3. Assembly of DCM@PLGA / R848

[0070] Step 1: Treat DCM into nano-scale membrane by ultrasonic treatment in ice bath (power 100W, 2 seconds each time, repeated 3 times)

[0071] Step 2: PLGA / R848 and DCM were mixed at a mass ratio of 1:30 and sonicated three times in an ice-water bath (power 100 W, 1 s each time);

[0072] Step 3: The mixture was extruded through 800 nm, 400 nm, and 200 nm polycarbonate membranes 20 times to obtain DCM-coated nanoparticles (see Figure 1 Schematic diagram of core-shell structure);

[0073] Step 4: Laser particle size analyzer measurement display ( Figure 2 ), the average particle size of DCM@PLGA / R848 is 100 nm.

[0074] Example 2

[0075] This example verifies the membrane protein integration effect of DCM@PLGA / R848 by SDS-PAGE. The specific implementation method is as follows:

[0076] Sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) was used to analyze the protein expression profiles of membrane proteins, DCM@PLGA / R848 prepared in Example 1, and naked PLGA / R848. After quantifying the protein concentration of each group using the BCA method, each group was diluted to the same concentration using RIPA lysis buffer, mixed evenly with 5X loading buffer, heated at 100°C for 10 minutes, and the sample was added to the loading wells of a 4-20% Bis-Tris protein gel. The sample volume per well was 20 μL, containing 40 μg of protein. After electrophoresis, the gel was stained with Coomassie brilliant blue staining solution. The protein expression band diagram showed that DCM@PLGA / R848 had an expression profile similar to that of dendritic cell membrane proteins, indicating that dendritic cell membrane proteins were successfully integrated into the biomimetic nanoformulation. The results are shown in Figure 3 .

[0077] Example 3 Western blotting experiment of DCM@PLGA / R848

[0078] In this example, the key membrane protein integration effect of DCM@PLGA / R848 was verified by Western blotting, and the specific implementation is as follows:

[0079] Each sample was lysed with RIPA solution and centrifuged at 12,000 g for 20 minutes. The supernatant was collected and protein concentration was quantified. After SDS-PAGE separation, the proteins were transferred to a 0.22 μm pore size PVDF membrane. The PVDF membrane was blocked with 5% nonfat dry milk in TBST and incubated with shaking at room temperature for 1 hour. The membrane was then incubated with primary antibodies against VLA-4 (1:1000) and LFA-1 (1:1000) at 4°C overnight with shaking. Finally, the membrane was incubated with an HRP-conjugated IgG secondary antibody (1:5000) at room temperature for 1 hour. The strips were then immersed in ECL solution and exposed on a Tanon 4600 series fully automated chemiluminescence image analysis system.

[0080] The results are as follows Figure 4 , indicating that the key membrane proteins VLA-4 and LFA-1 of dendritic cells can be successfully transferred to the surface of biomimetic nanoparticles.

[0081] Example 4 Investigation of the blood-brain barrier migration ability and pattern of DCM@PLGA / R848

[0082] This example verifies the blood-brain barrier penetration mechanism of DCM@PLGA / R848 through an in vitro Transwell model and tight junction protein analysis. The specific implementation method is as follows:

[0083] The ability of DCM@PLGA / R848 to penetrate bEnd.3 cell monolayers in vitro was evaluated using a Transwell assay. 4 bEnd.3 cells were carefully seeded into the upper chamber of a 24-well Transwell plate with a pore size of 1 μm. ERS-2 voltage resistance meter measures transmembrane resistance (TEER), and the TEER value is higher than 200Ω.cm 2The monolayer of cells was used as a BBB experimental model. The cells were cultured for a whole week at the same time. The culture medium was changed every two days. Subsequently, 200 μL of DMEM culture medium containing coumarin 6-labeled nanoparticles was added to the upper chamber of the Transwell system, and 500 μL of fresh DMEM culture medium was added to the lower chamber. At different time points, 100 μL aliquots were collected from the lower chamber, and their fluorescence intensity was analyzed using a fluorescence microplate reader. In order to verify the cell penetration method, after one week of culture of the above cells, a series of different preparations were applied to the bEnd.3 cell monolayer in groups for 24 hours. After this incubation period, the wells were carefully washed three times with PBS, and 0.5 mL of 10 kDa FITC-dextran solution with a concentration of 1 mg / mL was added to the upper chamber. After another hour, the fluorescence intensity of FITC-dextran in the lower chamber was measured using a fluorescence microplate reader. The results are shown in Figure 2. Figure 5 As shown, DCM@PLGA / R848 can well penetrate the bEnd.3 cell monolayer ( Figure 5 A), and the fluorescence of FITC-dextran in the lower chamber was detected to increase ( Figure 5 B), indicating that DCM@PLGA / R848 can promote the enlargement of intercellular spaces across the cell monolayer.

[0084] Next, we characterized the tight junction protein ZO-1 in vitro. bEnd.3 cells were cultured at 5×10 4 Cells were seeded at a density of 100 cells / well on 12-well plates. After 24 hours of standard culture, cells were treated for 1 hour as follows: ① PBS control group; ② PLGA group; ③ DCM@PLGA group; ④ DCM@PLGA combined with LFA-4 and VLA-4 antibodies. After treatment, cells were rinsed three times with PBS and incubated overnight at 4°C with rabbit ZO-1 primary antibody (1:200). Following incubation, cells were washed three times with PBS and incubated with fluorescently labeled goat anti-rabbit secondary antibody (1:500) for 30 minutes in the dark. Nuclear staining with DAPI was performed for 5 minutes. ZO-1 protein expression was observed using a fluorescence microscope.

[0085] The results are as follows Figure 6 As shown in the data, DCM@PLGA / R848 treatment can downregulate the expression of ZO-1 in bEnd.3 cells, which suggests that DCM@PLGA / R848 may affect the tight junctions of endothelial cells; after blocking the VLA-4 and LFA-1 proteins on the surface of DCM@PLGA / R848, the fluorescence intensity of ZO-1 expression was upregulated, indicating that VLA-4 and LFA-1 proteins played an important role in the effect of DCM@PLGA / R848 on ZO-1 expression. DCM@PLGA / R848 can reduce the integrity of tight junctions and regulate the permeability of the blood-brain barrier, thereby promoting the formation of cell gaps, allowing it to safely cross the blood-brain barrier.

[0086] Example 5 In vivo distribution and targeting investigation of DCM@PLGA / R848

[0087] This example verifies the brain tumor targeting of DCM@PLGA / R848 by fluorescence imaging. The specific implementation method is as follows:

[0088] C57B6 / J healthy male mice were randomly divided into three groups: targeted drug delivery system group (DCM@PLGA / R848), non-targeted drug delivery system group (PLGA / R848), and sham operation group (Sham). After the glioma mouse model was established, each group was injected with the corresponding group of vectors labeled with Cy7 fluorescence through the tail vein. At 4h and 8h after administration, the distribution of each group of preparations in the brain of tumor-bearing mice was observed using a small animal in vivo imaging system (InVivo Imaging System, IVIS). Figure 7 A. Semi-quantitative analysis was performed by Region-Of-Interest (ROI) analysis. Figure 7 As shown in Figures B and 7C, at different time points, the fluorescence signal of the DCM@PLGA / R848 group was significantly increased compared with the PLGA / R848 group and the Sham group. Furthermore, after mouse autopsy, it was observed that DCM@PLGA / R848 was specifically enriched in the glioma area, indicating that coating the dendritic cell membrane on the nanoparticle surface enables DCM@PLGA / R848 to be targeted and enriched in the diseased brain area.

[0089] Example 6 In vivo efficacy evaluation of DCM@PLGA / R848

[0090] This example verifies the in vivo anti-glioma effect of DCM@PLGA / R848 by bioluminescent in vivo imaging and survival analysis, which is specifically implemented as follows:

[0091] Quantitative assessment of tumor growth:

[0092] Seven days after intracranial inoculation of GL261-Luc cells, in vivo imaging showed bioluminescent signals in the brains of mice in all experimental groups, confirming the successful establishment of an orthotopic glioma model. By day 14, the bioluminescent signal in the control group continued to increase, while the signal intensity in the DCM@PLGA / R848 group decreased by 86.05% after five doses (day 21) compared to day 14 (PLGA / R848 group: decreased by 57.61%; control group: signal continued to increase, Figure 8 AB), indicating that the preparation significantly inhibited tumor progression.

[0093] Survival prolongation effect:

[0094] Survival curve analysis ( Figure 8C) shows that the average survival of mice in the DCM@PLGA / R848-treated group reached 81.6 days, significantly longer than the control group (39.4 days), the PLGA group (39.3 days), and the PLGA / R848 group (48.0 days). Three mice survived for up to 120 days, with no obvious signs of tumor recurrence observed.

[0095] Verification of immune memory effect:

[0096] Mice that survived for 120 days were subjected to a second intracranial rechallenge experiment with GL261-Luc cells ( Figure 8 D) The results showed that compared with the primary vaccination control group, the survival time of mice in the DCM@PLGA / R848 treatment group exceeded 60 days, indicating that it can induce long-term anti-tumor immune memory.

[0097] Example 7: In vivo immune activation of DCM@PLGA / R848

[0098] This example demonstrates that DCM@PLGA / R848 significantly improves the glioma immune microenvironment through a three-level immune synergistic mechanism:

[0099] Local immune activation: In brain tumor tissue, the preparation significantly increased the proportion of mature DCs to 16.07% (5.37% in the control group, an increase of 3.0 times, such as Figure 9 AB), while MHC II + The DC ratio reached 39.97% (23.37% in the control group, an increase of 1.7 times, Figure 9 C), promote CD8 + T cells (33.10% vs 6.93% in the control group) and CD4 + T cell infiltration (33.60% vs 6.22%) Figure 9 DF), effectively reversing the “cold tumor” immunosuppressive state;

[0100] Regional immune cascade: In the cervical lymph nodes, the proportion of mature DCs increased to 25.10% (9.63% in the control group, Figure 10 AB), MHC II + DC reached 38.17% (control group 6.67%, Figure 10 C), drive CD8 + T (32.10% vs 25.00%) and CD4 + T (34.70% vs 17.83%) synergistically amplified ( Figure 10 DF), CD4 + / CD8 + The ratio increased from 0.71 to 1.08, indicating effective activation of antigen presentation and adaptive immune response;

[0101] Systemic immune regulation: The proportion of mature DCs in the spleen increased to 22.03% (11.48% in the control group, Figure 11 AB), CD8 + The T ratio increased significantly to 52.87% (39.80% in the control group, Figure 11 DE), and CD4 + The proportion of T decreased to 42.17% (55.37% in the control group, Figure 11 F), indicating CD4 + T cells migrate to tumors / lymph nodes to enhance local helper function, while the spleen enhances CD8 + T-directed cytotoxicity establishes systemic immune surveillance.

[0102] The above results show that this embodiment achieves multi-level anti-tumor immune synergy by simulating the vaccine mechanism through local DC-T axis activation, regional lymphoid immune cascade and systemic effector T cell polarization, proving that it has significant clinical application prospects.

[0103] The protection content of the present invention is not limited to the above embodiments. Without departing from the spirit and scope of the inventive concept, changes and advantages that can be thought of by those skilled in the art are included in the present invention and are protected by the appended claims.

Claims

1. A dendritic cell biomimetic nano-tumor vaccine for targeted treatment of brain glioma, characterized in that: The tumor vaccine is mainly composed of dendritic cell membrane-coated polylactic acid-glycolic acid copolymer nanoparticles, the core of the polylactic acid-glycolic acid copolymer nanoparticles encapsulates TLR7 / 8 agonist R848; the surface of the dendritic cell membrane retains VLA-4 and LFA-1 molecules.

2. The dendritic cell biomimetic nano-tumor vaccine according to claim 1, characterized in that: The average particle size of the nanoparticles is 100.18±0.774 nm, and the surface charge is -28.1±0.378 mV.

3. The dendritic cell biomimetic nano-tumor vaccine according to claim 1, characterized in that: The molar ratio of lactic acid to glycolic acid in the polylactic acid-glycolic acid copolymer is 30-70:30-70, preferably 50:

50. Preferably, the drug loading of R848 is 5.15±1%, and the encapsulation efficiency is 37.3±5%.

4. The dendritic cell biomimetic nano-tumor vaccine according to claim 1, characterized in that: The mass ratio of the dendritic cell membrane to the poly(lactic-co-glycolic acid) nanoparticles is 1:20-40, preferably 1:

30.

5. The dendritic cell biomimetic nano-tumor vaccine according to claim 1, characterized in that: The dendritic cell membranes were extracted from DC2.4 cells by repeated freeze-thaw method; Specifically, the extraction of dendritic cell membranes comprises the following steps: (1) Resuspend DC2.4 cells in pre-chilled PBS and perform three freeze-thaw cycles: freeze at -80 ± 5°C for 30 ± 3 minutes, thaw at 37 ± 3°C for 30 ± 3 minutes, and repeat three times; (2) Collect the frozen-thawed cell suspension and sonicate in an ice bath for 3–8 times at 100 W power, 1–3 seconds each time, with an interval of 3–8 seconds; (3) Centrifuge at 4°C, discard the precipitate, and collect the supernatant; (4) Centrifuge at 4°C, discard the supernatant, and the precipitate is the dendritic cell membrane.

6. The dendritic cell biomimetic nano-tumor vaccine according to claim 5, characterized in that: The dendritic cell membrane obtained in step (4) was stored in a PBS solution at 4°C.

7. The method for preparing the dendritic cell biomimetic nano-tumor vaccine according to any one of claims 1 to 6, comprising the following steps: (1) Poly(lactic acid-co-glycolic acid) copolymer was dissolved in a mixed solution of ethanol and acetonitrile, and the TLR7 / 8 agonist R848 was added to prepare poly(lactic acid-co-glycolic acid) copolymer nanoparticles with the core encapsulated with the TLR7 / 8 agonist R848 by a nanoprecipitation method. The organic solvent was then removed by rotary evaporation and ultrafiltration to prepare drug-loaded poly(lactic acid-co-glycolic acid) copolymer nanoparticles; (2) coating the dendritic cell membrane with the poly(lactic acid-co-glycolic acid) nanoparticles prepared in step (1) by ultrasonic fusion, as follows: (A1) Dendritic cell membranes were sonicated in an ice bath to form nanoscale membranes; (A2) mixing the polylactic acid-co-glycolic acid nanoparticles prepared in step (1) with the dendritic cell membrane and sonicating in an ice bath; (A3) extruding the mixture through a polycarbonate membrane and collecting the precipitate by rapid centrifugation to obtain the dendritic cell biomimetic nano-tumor vaccine DCM@PLGA / R848.

8. The method according to claim 7, characterized in that In step (1), the mass ratio of polylactic acid-glycolic acid copolymer to R848 is 10-50:4, preferably 20-35:4; Preferably, the volume ratio of ethanol to acetonitrile is 2:1 to 5, preferably 2:3; Preferably, the aqueous phase of the nanoprecipitation method in step (1) is a 0.5% Tween 80 solution.

9. The method according to claim 7, characterized in that In step (2), after ultrasonic fusion, the liposomes are extruded through 800 nm, 400 nm, and 200 nm pore size polycarbonate membranes 10 to 20 times each in sequence using a liposome extruder; Preferably, in said (A1), the ultrasonic power is 100W, 2 seconds each time, repeated 3 times; Preferably, in said (A2), the ultrasonic power is 100W, 1 second each time, repeated 3 times; Preferably, in said (A3), the centrifugation condition is: centrifugation at 20,000 g for 30 minutes.

10. Use of the dendritic cell biomimetic nano-tumor vaccine according to any one of claims 1 to 6 in the following (B1) or (B2): (B1) Application in the preparation of products for treating brain glioma; (B2) Use in the preparation of products for inhibiting the proliferation and / or production of brain glioma cells; Preferably, the brain glioma is glioblastoma; Preferably, the product is a medicine; Preferably, the drug further contains other pharmaceutically acceptable carriers.

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