Ginsenoside membrane hybrid tumor nanovaccine, pharmaceutical composition, preparation method and application thereof

Abstract

This invention discloses a ginsenoside membrane hybrid tumor nanovaccine, a pharmaceutical composition, its preparation method, and its applications. The ginsenoside membrane hybrid tumor nanovaccine comprises a fusion membrane formed by a macrophage membrane and a lipid membrane; wherein, a tumor antigen peptide-MHC complex is bound to the surface of the macrophage membrane, and the lipid membrane comprises ginsenosides and phospholipids. This ginsenoside membrane hybrid tumor nanovaccine and the pharmaceutical composition containing it simultaneously possess excellent effects such as high antigenicity, broad-spectrum activity, high targeting, high adjuvant potency, and safety, demonstrating superior efficacy in practical applications; furthermore, the entire preparation process is simple to operate and has good reproducibility.

Description

Technical Field

[0001] This invention relates to a ginsenoside membrane hybrid tumor nanovaccine, a pharmaceutical composition, its preparation method, and its application. Background Technology

[0002] Immunotherapy has become a hot topic in cancer treatment in recent years. Among them, cancer vaccines directly deliver tumor antigen information to antigen-presenting cells (APCs), stimulating the body to spontaneously produce tumor cytotoxic T lymphocytes (CTLs), and ultimately inducing an anti-tumor immune response, thereby achieving the prevention and treatment of cancer and bringing hope for improving the efficacy of anti-tumor therapy.

[0003] However, existing cancer vaccines still generally face the following challenges: Clinical studies show that current cancer vaccine response rates are low, with only 5-10% of patients showing a significant response. To date, very few therapeutic cancer vaccines have been approved by the FDA. Sipuleucel-T, used to treat metastatic castration-resistant prostate cancer, has limited efficacy, extending patient survival by only about four months. Furthermore, recent cases of allergic or autoimmune side effects from vaccination have raised public doubts about vaccine safety. The effectiveness and safety issues of cancer vaccines are often due to: (1) The nonspecificity and low immunogenicity of tumor antigens limit the effectiveness of vaccines. Currently, common tumor antigens are tumor cell lysates or single tumor-associated antigen peptides or nucleic acids, which have weak immunogenicity. At the same time, due to the heterogeneity of tumors, the antigens of the same tumor in different patients are often different. However, due to the limitations of antigen identification technology, only the sequences and structures of tumor-associated antigens or tumor-specific antigens of a few tumors have been resolved, which greatly limits the immunogenicity of tumor vaccines.

[0004] (2) Immunoadjuvants struggle to simultaneously meet the requirements of high efficacy and safety: Vaccines typically consist of two parts: antigens and immunoadjuvants. Especially for tumor antigens with low immunogenicity, immunoadjuvants that enhance the immune response are essential for activating T cells. However, current adjuvants generally suffer from the following problems: ① Insufficient immune enhancement and low response rate. For example, aluminum hydroxide can effectively stimulate humoral immune responses, but it has no significant effect on cellular immunity, resulting in insufficient efficacy. ② Excessive immune enhancement, leading to side effects. Studies have shown that most current adjuvants can cause hyperimmunity, resulting in autoimmune / inflammatory syndromes. ③ International adjuvant market monopoly and lack of domestically developed adjuvant technology. Only one type of aluminum adjuvant is available domestically, and most vaccine companies can only obtain adjuvants through cooperation with foreign companies, which is expensive.

[0005] (3) Difficulty in achieving targeted co-delivery of antigen and adjuvant to APC: Currently, commonly used vaccines in clinical practice are often simple mixtures of antigen and adjuvant. Such vaccines cannot achieve targeted co-delivery of APC, thus limiting their effectiveness. Commonly used lipid nanocarriers still have many problems to be solved, mainly including: ① Difficulty in antigen loading, making it difficult to simultaneously load multiple tumor antigens; ② To improve the APC targeting of traditional lipid nanoparticles, active targeting ligands such as mannose or antibodies are often used for surface modification, but this makes the production process of liposomes complex, has poor reproducibility, and increases the difficulty of production transformation.

[0006] Therefore, designing a novel lipid delivery system that can simply and effectively solve the above problems is of great significance for improving the clinical response rate of vaccines. Summary of the Invention

[0007] To address the following technical shortcomings of existing cancer vaccines: (i) narrow antigen sources, weak immunogenicity, high preparation costs, and long cycles; (ii) lack of efficient DCs targeting ability; (iii) insufficient adjuvant potency and safety; and (iv) complex existing targeting strategies, cumbersome synthesis processes, and difficulties in clinical translation, this invention proposes a ginsenoside membrane hybrid tumor nanovaccine, a pharmaceutical composition, its preparation method, and its application. This ginsenoside membrane hybrid tumor nanovaccine, named MAGIN-Vax, possesses excellent effects such as broad antigen spectrum, high targeting, high adjuvant potency, and good safety, demonstrating superior efficacy in practical applications. Furthermore, the entire preparation process is simple to operate and has good reproducibility.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows.

[0009] This invention provides a ginsenoside membrane hybrid tumor nanovaccine, wherein the ginsenoside membrane hybrid tumor nanovaccine comprises a fusion membrane formed by a macrophage membrane and a lipid membrane; wherein: The tumor antigen peptide-MHC complex binds to the surface of the macrophage membrane; The lipid membrane comprises ginsenosides and phospholipids.

[0010] In this invention, the ginsenoside membrane hybrid tumor nanovaccine is named MAGIN-Vax. It mainly enhances the broad spectrum of antigens by hybridizing a biooptimized antigen library (MAtigens)-macrophage membrane with a lipid membrane (GINbranes) based on multifunctional ginsenosides, while ensuring excellent effects such as high targeting, high adjuvant efficacy and safety. In practical applications, it has the advantages of better efficacy. Moreover, the whole process does not require complex chemical modification and can simultaneously load broad-spectrum tumor antigens, DCs targeting units and immune adjuvants.

[0011] in: MAtigens (antigen library): This is obtained by extracting the cell membranes of tumor cells after they have been phagocytosed by macrophages and treated with immunogenic death (ICD) inducers (such as paclitaxel). The membranes are enriched with a variety of tumor antigen peptide-MHC complexes with high MHC (Major Histocompatibility Complex) affinity, processed and presented by macrophages, providing a broad-spectrum, highly immunogenic antigen library covering both membrane and intracellular proteins.

[0012] GINbranes (lipid membranes based on multifunctional ginsenosides): These are stable lipid membranes formed by the self-assembly of ginsenosides and phospholipids. The glycosyl structure of ginsenosides naturally enables them to target the Dectin-1 receptor on the surface of dendritic cells (DCs). Simultaneously, by activating the Dectin-1-NF-κB signaling pathway, ginsenosides effectively promote DC maturation and the secretion of key cytokines such as IL-12 and TNF-α, thus functioning as built-in adjuvants.

[0013] In some embodiments, the tumor antigen peptide-MHC complex originates from the cell membrane of an antigen-presenting cell that recognizes the tumor antigen; the tumor antigen is preferably selected from one or more tumor cells selected from melanoma cells, triple-negative breast cancer cells, pancreatic cancer cells, non-small cell lung cancer cells, prostate cancer cells, and colorectal cancer cells as tumor-specific antigens or tumor-related antigens, for example, tumor-specific antigens or tumor-related antigens of one or more tumor cells selected from melanoma cells, triple-negative breast cancer cells, or pancreatic cancer cells.

[0014] In some embodiments, the macrophage membrane is extracted from macrophages after they engulf tumor cells; the tumor cells are preferably tumor cells that produce an ICD (immunogenic cell death) induction effect.

[0015] The ICD-induced effect can be triggered by an ICD inducer, such as paclitaxel and / or doxorubicin.

[0016] In some embodiments, the macrophage membrane is derived from human macrophages and / or mouse macrophages.

[0017] In some embodiments, the macrophage membrane is derived from macrophages, or from bone marrow hematopoietic stem cells, myeloid progenitor cells, monocytes, premonocytes, monocytes, or macrophages differentiated from them.

[0018] In some preferred embodiments, the macrophages are M0 type macrophages; and / or, the macrophages are THP-1 cells.

[0019] In some embodiments, the ginsenoside is selected from one or more of ginsenoside Rg3, ginsenoside Rb1, ginsenoside Rf, ginsenoside Rd, ginsenoside PPT, ginsenoside Rg1 and ginsenoside Rh1, preferably ginsenoside Rg3.

[0020] In this invention, the phospholipids may be conventional phospholipids in the art, and generally may include natural phospholipids and / or synthetic phospholipids.

[0021] The natural phospholipids preferably include one or more of natural lecithin, soybean lecithin, egg yolk lecithin, and cephalin.

[0022] The synthetic phospholipids preferably include one or more of phosphatidylcholine, phosphatidylglycerol, phosphatidylinositol, phosphatidylethanolamine, phosphatidylserine, and polyethylene glycol-modified phospholipids.

[0023] In some specific embodiments, the phospholipid is egg yolk lecithin and / or soybean lecithin.

[0024] In some embodiments, the mass ratio of membrane proteins in the macrophage membrane to phospholipids in the lipid membrane is 1:(1.25-60), preferably 1:(2-60), for example 1:(10-60), and further for example 1:20. The content of the membrane proteins can be determined by conventional methods in the art, for example using a Micro BCA protein quantification kit.

[0025] In some embodiments, the mass ratio of ginsenosides to phospholipids is 1:(1.5-30), for example, 1:4.

[0026] In this invention, the ginsenoside membrane hybrid tumor nanovaccine can exist in a typical vesicle structure.

[0027] In some embodiments, the hydrated particle size of the vesicles is 90-150 nm.

[0028] In some implementations, the PDI of the vesicle is 0.1-0.2.

[0029] In some embodiments, the vesicle has a zeta potential of -20mV to -30mV.

[0030] The present invention also provides a pharmaceutical composition comprising an immune checkpoint inhibitor and a ginsenoside membrane hybrid tumor nanovaccine as described above.

[0031] In this invention, the immune checkpoint inhibitor refers to a class of targeted therapeutic drugs that activate the body's own immune system to attack tumors by blocking inhibitory signals on immune cells. Research in this invention has found that, in practical applications, combining the aforementioned ginsenoside membrane hybrid tumor nanovaccine with existing immune checkpoint inhibitors can further enhance the immune response rate and complete remission rate of the ginsenoside membrane hybrid tumor nanovaccine, thereby achieving higher therapeutic efficacy.

[0032] In this invention, the immune checkpoint inhibitor can be conventional in the art, such as including PD-1 / PD-L1 inhibitors and / or CTLA-4 inhibitors.

[0033] In some specific embodiments, the PD-1 / PD-L1 inhibitor includes an α-PD-1 antibody.

[0034] In this invention, the dosage of the immune checkpoint inhibitor and the ginsenoside membrane hybrid tumor nanovaccine can be adjusted according to the actual administration method and dosage. Specifically, by intraperitoneal injection, the dosage of the immune checkpoint inhibitor can be 5 μg / kg body weight, and the dosage of the ginsenoside membrane hybrid tumor nanovaccine, based on ginsenosides, can be 2.5 μM / kg body weight.

[0035] In some embodiments, the pharmaceutical composition further includes pharmaceutically acceptable excipients or carriers.

[0036] The present invention also provides the use of the ginsenoside membrane hybrid tumor nanovaccine as described above, or the pharmaceutical composition as described above, in the preparation of immunoprophylactic and / or immunotherapeutic drugs.

[0037] In this invention, the immunoprophylaxis and / or immunotherapy are used for the immunoprophylaxis and / or immunotherapy of tumors, viral infections, or bacterial infections; preferably, the tumors include melanoma, breast cancer such as triple-negative breast cancer, pancreatic cancer, lung cancer such as non-small cell lung cancer, prostate cancer, or colorectal cancer.

[0038] The present invention also provides a method for preparing the ginsenoside membrane hybrid tumor nanovaccine as described above, the method comprising the following steps: S1. Incubate tumor cells that induce ICD effect with macrophages, and extract the cell membrane of the macrophages to obtain the macrophage membrane; S2. Ginsenosides and phospholipids are self-assembled to obtain the lipid membrane; S3. The macrophage membrane and the lipid membrane are fused to obtain the ginsenoside membrane hybrid tumor nanovaccine.

[0039] In step S1, the induction of ICD effect, incubation, and extraction steps can all be performed according to conventional practices in the field.

[0040] In step S2, the self-assembly can be carried out according to conventional methods for preparing liposomes in the art, such as thin-film hydration.

[0041] In step S3, the fusion can be carried out according to conventional methods for preparing hybrid membranes in the art, such as extrusion.

[0042] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0043] The reagents and raw materials used in this invention are all commercially available.

[0044] The positive and progressive effects of this invention are as follows: Compared with existing tumor vaccines, the ginsenoside membrane hybrid tumor nanovaccine of the present invention has the following significant advantages: 1. Antigen advantages: MAtigens provides a "one-stop" antigen solution, which can complete the preparation within 10 days, reducing costs by more than 80%, and has a broad antigen spectrum, avoiding immune escape.

[0045] 2. Adjuvant advantages: Innovative exploration of the adjuvant effect of ginsenosides shows that it is more effective and safer than existing adjuvants QS-21 and LPS.

[0046] 3. Delivery advantages: GINbranes achieves active DCs targeting and LNs enrichment through the interaction of ginsenoside-Dectin-1, solving the spatial delivery problem.

[0047] 4. Functional advantages: The integrated design ensures that the antigen and adjuvant are delivered to the same DCs in a time-space synchronization, efficiently inducing three-signal linkage and activating a strong and long-lasting CTL and memory T cell response.

[0048] 5. Safety and translational advantages: The ingredients are clearly defined, the preparation process is simple and reproducible, no antibody modification is required, the risk of cytokine storm is avoided, and the prospects for clinical translation are broad.

[0049] 6. Outstanding efficacy: In melanoma, triple-negative breast cancer and human pancreatic cancer PDX models, the vaccine demonstrated a 100% immune response rate and an extremely high complete remission rate (up to 100%), with efficacy far exceeding existing vaccine strategies. Attached Figure Description

[0050] Figure 1 This is a schematic diagram illustrating the preparation and action process of ginsenoside membrane hybrid tumor nanovaccines.

[0051] Figure 2 The structure of candidate compounds for ginsenosides is shown.

[0052] Figure 3 Flow cytometry analysis results of the effect of each ginsenoside on DC maturation. n = 3, p < 0.05, p < 0.001, the statistical analysis method is one-way ANOVA with Bonferroni multiple comparisons post-hoc test.

[0053] Figure 4 The results of flow cytometry analysis of the T-cell activation effects of various ginsenosides are as follows: n = 3, p < 0.001, the statistical analysis method is one-way ANOVA with Bonferroni multiple comparisons post-hoc test.

[0054] Figure 5 Quantitative detection of HMGB1 release (A) and ATP secretion (B) in B16-OVA cells after 12 h of incubation with PTX, and observation of CLSM in CRT translocation in B16-OVA cells (C). n = 3, p <0.001, the statistical analysis method is t test, scale bar 50 μm.

[0055] Figure 6 Flow cytometry analysis (A), quantitative statistical plot (B), and CLSM image (C) of phagocytosis of CFSE-labeled B16-OVA cells by CFSE far red-labeled macrophages after PTX treatment. n = 3, p < 0.001, the statistical analysis method was t test, and the scale bar was 5 μm.

[0056] Figure 7 Flow cytometry analysis (A), quantitative statistical plot (B), and CLSM image (C) of macrophage antigen presentation efficiency after PTX treatment and co-culture with tumor cells. n = 3, p <0.001, the statistical analysis method is t test, scale bar is 5 μm.

[0057] Figure 8 Western blot analysis of key functional proteins in BO, Mø, and Mø@BO membrane proteins (A), and semi-quantitative analysis of expression levels of key functional proteins in BO, Mø, and Mø@BO membrane proteins (B). n = 3.

[0058] Figure 9The particle size distribution of GMø@BO is shown in (A), the average particle size and PDI of GMø@BO and CMø@BO are shown in (B), and the average potential value is shown in (C). n = 3.

[0059] Figure 10 This is a typical TEM image of GMø@BO. The scale bar is 50 nm.

[0060] Figure 11 The fluorescence spectra of C6-labeled Rg3 lipid membranes mixed with different levels of Rhodamine B-labeled Mø@BO are shown in the figure. The proportions in the figure represent the mass ratio of lipid material to Mø@BO protein in the lipid membrane.

[0061] Figure 12 CLSM images of C6-labeled ginsenoside Rg3 lipid membrane and DiD-labeled Mø@BO before and after extrusion, scale bar 5 μm, and fluorescence intensity distribution of each channel along the white dashed line in the figure.

[0062] Figure 13 The 7-day particle size stability of GMø@BO and CMø@BO. n = 3.

[0063] Figure 14 Flow cytometry analysis (A), quantitative statistical plot (B), and CLSM image (C) of 4T1 cells phagocytosed by CFSE-labeled macrophages after PTX treatment with CFSE far red. n = 3, p < 0.001, the statistical analysis method was t test, and the scale bar was 5 μm.

[0064] Figure 15 The particle size distribution of GMø@4T1 is shown in (A), the average particle size and PDI of GMø@4T1 and CMø@4T1 are shown in (B), and the average potential value is shown in (C). n = 3.

[0065] Figure 16 Flow cytometry analysis (A) and CLSM image (B) of CFSE-labeled PDC cells being phagocytosed by CFSE-farred-labeled macrophages after PTX treatment. n = 3, p < 0.001, the statistical analysis method was t test, and the scale bar was 5 μm.

[0066] Figure 17 The particle size distribution of GMø@PDC is shown in Figure (A). The average particle size and PDI of GMø@PDC and CMø@PDC are shown in Figure (B), and the average potential value is shown in Figure (C). n = 3.

[0067] Figure 18 The signal intensity of each liposome on BMDC, n = 3, p < 0.05, p <0.001, the statistical analysis method is Bonferroni multiple comparison post-hoc test one-way ANOVA.

[0068] Figure 19 For each liposome to be coated with DCs, DCs MR- DCs Dectin-1- or DCs TLR4- The strength of the captured signal, n = 3, p <0.001, the statistical analysis method is Bonferroni two-way ANOVA with post-hoc test of multiple comparisons.

[0069] Figure 20 For each liposome in DCs, DCs MR- DCs Dectin-1- or DCs TLR4- CLSM image captured above, scale bar 10 μm.

[0070] Figure 21 This is a 3D diagram of the molecular docking interaction between Dectin-1 and Rg3 (A) or cholesterol (B), with the yellow dashed lines representing hydrogen bonding interactions.

[0071] Figure 22 The presentation levels of OVA antigen epitopes on the surface of DCs after treatment with various liposomes. n = 3, p < 0.05, p <0.001, the statistical analysis method is Bonferroni multiple comparison post-hoc test one-way ANOVA.

[0072] Figure 23 CLSM images showing the distribution of various liposome biomembranes, lipid membranes, and OVA antigen epitopes within DCs, and the signal intensity distribution of each channel at the white dashed line. Scale bar: 10 μm.

[0073] Figure 24 Representative flow cytometry and quantitative statistical plots of mature DCs levels after treatment in each group. n = 3, p <0.05, p<0.001, the statistical analysis method is Bonferroni multiple comparison post-hoc test one-way ANOVA.

[0074] Figure 25 The levels of IL-12, TNF-α, and IL-1β secreted by DCs in each group.

[0075] Figure 26 For each group of processed DCs, DCs MR- DCs Dectin-1- or DCs TLR4- Quantitative statistical chart of maturity level. n =3, p < 0.01, p <0.001, the statistical analysis method is Bonferroni two-way ANOVA with post-hoc test of multiple comparisons.

[0076] Figure 27 To determine the antigen-specific CD8 antigen after co-incubation with DCs treated with different drugs + Representative flow cytometry and quantitative statistical plots of T cell activation levels n = 3, p < 0.05, p <0.01, the statistical analysis method is Bonferroni multiple comparison post-hoc test one-way ANOVA.

[0077] Figure 28 CD8+ after co-incubation with DCs treated with different drugs + T cell proliferation n = 3, p <0.001, the statistical analysis method is Bonferroni multiple comparison post-hoc test one-way ANOVA.

[0078] Figure 29 For each liposome in CD8 + Signal intensity on T lymphocytes, n = 3, p < 0.001, the statistical analysis method was one-way ANOVA with Bonferroni multiple comparisons post-hoc test.

[0079] Figure 30 For each liposome to CD8 + T lymphocyte antigen presentation, n = 3, p < 0.001, the statistical analysis method was one-way ANOVA with Bonferroni multiple comparisons post-hoc test.

[0080] Figure 31 Typical confocal images of liposome uptake and antigen presentation on lymphocytes, and signal intensity distribution of each channel along the white dashed line, scale bar 5 μm.

[0081] Figure 32 For each liposome to CD8 + T lymphocyte activation, n = 3, p < 0.001, the statistical analysis method was one-way ANOVA with Bonferroni multiple comparisons post-hoc test.

[0082] Figure 33 The IFN-γ secretion level of T lymphocytes in each group was measured by ELISA, n = 3. p < 0.001, the statistical analysis method was one-way ANOVA with Bonferroni multiple comparisons post-hoc test.

[0083] Figure 34 This diagram illustrates the direct activation of T cells by GMø@BO as pseudo-APCs.

[0084] Figure 35 To determine the gating strategy of mature DCs and the levels of LNs and mature DCs in the blood of mice after immunization in each group, n = 3. p < 0.05 p < 0.01 and p < 0.001, the statistical analysis method is Bonferroni two-way ANOVA with post-hoc test of multiple comparisons.

[0085] Figure 36 DCs (CD11c green, CD80 red) and CD8+ in LNs after immunization in each group + CLSM full scan image of T (CD8 green) cells, scale bar 500 μm.

[0086] Figure 37 This study investigated antigen-specific T cell gating strategies and the levels of lymphocytes (LNs) and antigen-specific T cells in the blood after immunization in each group. n = 3. p < 0.05 p < 0.01 and p < 0.001, the statistical analysis method is Bonferroni two-way ANOVA with post-hoc test of multiple comparisons.

[0087] Figure 38 For IFN-γ + CD8 + T-cell gate strategy and post-immunization LNs and IFN-γ levels in the blood of each group + CD8 + T cell levels, n = 3, p < 0.05 p < 0.01 and p < 0.001, the statistical analysis method is Bonferroni two-way ANOVA with post-hoc test of multiple comparisons.

[0088] Figure 39 For Grz B + CD8 + T-cell gate strategy and post-immunization LNs and Grz B in the blood of each group + CD8 + T cell levels, n = 3. p < 0.05 p < 0.01 and p < 0.001, the statistical analysis method is Bonferroni two-way ANOVA with post-hoc test of multiple comparisons.

[0089] Figure 40 To study the memory T-cell gating strategy and the post-immunization LNs and blood CD8 memory in each group. + T cell levels, n =3. p < 0.05 p < 0.01 and p < 0.001, the statistical analysis method is one-way ANOVA with Bonferroni multiple comparisons post-hoc test.

[0090] Figure 41 The levels of LNs and serum cytokines in each group after immunization. n = 3. p < 0.001, the statistical analysis method is Bonferroni two-way ANOVA with post-hoc test of multiple comparisons.

[0091] Figure 42 This is a schematic diagram of the experimental protocol for the long-term immune protection efficacy of GMø@BO.

[0092] Figure 43 This is a schematic diagram of the experimental protocol for investigating the tumor prevention effect of GMø@BO.

[0093] Figure 44 Tumor growth curves for each group of tumor-bearing mice.

[0094] Figure 45 The survival time of tumor-bearing mice in each group. n = 6, p < 0.001, the statistical method is log-rank (Mantel–Cox) test.

[0095] Figure 46 Lung images of normal mice and surviving mice from each group 30 days after tail vein injection of B16-OVA cells.

[0096] Figure 47 Tumor growth curves after immunization in each group of mice.

[0097] Figure 48 The melanoma immunity rate after immunization in each group of mice. n = 6, p < 0.05 and p < 0.001, the statistical method is log-rank (Mantel–Cox) test.

[0098] Figure 49 The survival time of mice in each group after immunization. n = 6, p < 0.001, the statistical method is log-rank (Mantel–Cox) test.

[0099] Figure 50 To detect the secretion of IL-4 and IL-10 in lymph nodes and serum of mice after immunization, as well as the levels of IgM and IgA antibodies in serum, using ELISA. n = 3, p < 0.001, the statistical analysis method is Bonferroni two-way ANOVA with post-hoc test of multiple comparisons.

[0100] Figure 51 The curves showing the changes in body weight of mice after immunization in each group are shown. n = 6.

[0101] Figure 52 Images of H&E staining of organs in mice after immunization in each group.

[0102] Figure 53 This is a schematic diagram of the experimental protocol for the long-term immune protection efficacy of GMø@4T1 against TNBC.

[0103] Figure 54 This is a schematic diagram of the experimental protocol for evaluating the pharmacodynamic effect of GMø@4T1 on TNBC prevention.

[0104] Figure 55 Tumor growth curves for TNBC-bearing mice in each group.

[0105] Figure 56 The survival time of TNBC-bearing mice in each group.

[0106] Figure 57 Live BLI imaging of normal mice and surviving mice from each group after tail vein injection of 4T1-Luci cells, and ex vivo lung images 28 days later. The white circles indicate metastatic nodules.

[0107] Figure 58 The number of lung metastatic nodules was calculated for normal mice and surviving mice from each group 28 days after tail vein injection of 4T1-Luci cells. n = 6, p < 0.001, the statistical analysis method is one-way ANOVA with Bonferroni multiple comparisons post-hoc test.

[0108] Figure 59 Tumor growth curves after immunization in each group of mice (A), the percentage of tumor weight to body weight at the end of the experiment (B), and tumor photographs at the end of the experiment (C). n = 6, p <0.01, p < 0.001, the statistical analysis method is one-way ANOVA with Bonferroni multiple comparisons post-hoc test.

[0109] Figure 60 The TNBC exemption rate after immunization in each group of mice. n = 6, p < 0.001, the statistical method is log-rank (Mantel–Cox) test.

[0110] Figure 61 The curves showing the changes in body weight of mice after immunization (A) and the organ coefficients at the experimental endpoint (B) are shown. n = 6.

[0111] Figure 62The dosing regimen is shown in Figure A. The average tumor growth curve is shown in Figure B. The tumor growth curves of mice after immunization are shown in Figure C. The tumor dissection photographs of mice in each group at the end of the experiment are shown in Figure D. n = 4. Detailed Implementation

[0112] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0113] Example

[0114] like Figure 1 The diagram shown illustrates the preparation and action process of the ginsenoside membrane hybrid tumor nanovaccine (MAGIN-Vax) in this embodiment of the invention. Specifically, the preparation method of this ginsenoside membrane hybrid tumor nanovaccine includes the following steps: 1. Screening of ginsenosides

[0115] 1.1 Screening Method

[0116] Based on the differences in glycosidic bond positions, number of sugar moieties, and R / S configurations in the structures of ginsenosides, nine candidate ginsenoside compounds were selected: protopanaxadiol (PPD), Rh2, Rg3, Rd, Rb1, protopanaxatriol (PPT), Rh1, Rf, and Rg1, with structures as shown below. Figure 2 As shown.

[0117] 1.1.1 Analysis of the effect of promoting DCs maturation

[0118] Dendritic cells (DCs) derived from bone marrow of C57BL / 6 mice were extracted. 50 μM of each ginsenoside solution was added to the DC culture medium (a concentrated DMSO solution of 50 mM ginsenoside solution was prepared first, then added to the DC culture medium at a ratio of 1:1000, for example, 2 μL of the concentrated solution was added to 2 mL of DC culture medium). After incubation for 48 h, the cells were washed twice with PBS, blocked with PBS containing 1% BSA for 20 min, and then 0.3 μL of Percp-Cy5.5-CD11c, FITC-CD80, and PE-CD86 antibodies (all purchased from Biolegend) were added. Staining was performed at room temperature for 20 min, followed by washing twice with PBS. Flow cytometry analysis was used to analyze the maturation of DCs in each group after drug administration.

[0119] 1.1.2 Analysis of T cell activation promotion

[0120] Lymphocytes from C57BL / 6 mice were extracted, and 50 μM solutions of each ginsenoside were added. After 48 h, the cells were washed twice with PBS, blocked with PBS containing 1% BSA for 20 min, and then 0.3 μL of Percp-Cy7-CD3 and FITC-CD69 antibodies (both purchased from Biolegend) were added. The cells were stained at room temperature for 20 min, washed twice with PBS, and the ability of different saponins to stimulate T cell activation was compared by flow cytometry.

[0121] 1.2 Screening Results

[0122] 1.2.1 Comparison of the effects of various ginsenosides on promoting DC maturation

[0123] The effects of nine ginsenosides on promoting DC maturation were determined, and the results are as follows: Figure 3 As shown in the figure, PPD without any glycosyl groups did not significantly promote DC maturation; however, the maturation-promoting effect increased with the increase of the number of glycosyl groups at the C-3 position, with Rh2 containing one glycosyl group showing a significantly lower maturation-promoting effect than Rg3 containing two glycosyl groups; however, the increase of the number of glycosyl groups at the C-20 position led to a weakening of the maturation-promoting effect, that is, Rd and Rb1 with glycosyl groups at the C20 position had a significantly lower maturation-promoting ability than ginsenoside Rg3 without glycosyl groups at the C20 position. Therefore, ginsenoside Rg3 has the strongest maturation-promoting effect on DCs. Based on the experimental results and structure-activity relationship analysis, it was found that the number and position of glycosyl groups are crucial to the DC activation effect of ginsenosides. Therefore, this invention hypothesizes that the DC maturation-promoting effect of ginsenosides may be mediated by glycosyl recognition receptors on the surface of DCs.

[0124] 1.2.2 Comparison of the T-cell activation effects of various ginsenosides

[0125] An ideal immune adjuvant should enhance the immunogenicity of the antigen by promoting dendritic cell (DC) maturation, but it should not be immunogenic itself; otherwise, it would induce a non-specific immune response and cause inflammatory side effects. Therefore, an ideal immune adjuvant should not have the ability to activate T cells. Figure 4 It is known that, except for PPD and Rh2, the other ginsenosides did not show any effect in promoting T cell activation and had no immunogenicity, thus they can be used as adjuvants with good safety.

[0126] Based on the combined effects of various saponins on dendritic cells (DCs) and T cells, this invention preliminarily screened ginsenoside Rg3 as the safest and most effective ginsenoside immunoadjuvant for subsequent experiments. It has the strongest effect in promoting DC maturation and can enhance the immunogenicity of antigens. At the same time, it also has strong safety, avoiding direct activation of T cells and preventing the adjuvant itself from triggering non-antigen-specific immune responses in the body, thus exhibiting stronger safety.

[0127] 2. Preparation and Characterization of Ginsenoside Membrane Hybrid Tumor Nanovaccine (MAGIN-Vax)

[0128] 2.1 Preparation and Characterization of GMø@BO (Ginsenoside Membrane Hybrid Tumor Nanovaccine Based on B16-OVA Melanoma Cells)

[0129] 2.1.1 Preparation method: 2.1.1.1 Tumor cell ICD induction and labeling: Mouse melanoma B16-OVA cells were harvested, digested, counted, and then cultured at a density of 1×10⁶ cells per 150 mm culture dish. 7 Density seeding of individual cells.

[0130] Paclitaxel (PTX) was added to Gibico DMEM culture medium (containing 10% (v / v) fetal bovine serum and 1% (v / v) penicillin-streptomycin antibiotics, specifically containing 10,000 units / mL penicillin and 10,000 μg / mL streptomycin) to a final concentration of 10 μg / mL. The mixture was then incubated at 37°C and 5% CO2 for 24 hours to induce immunogenic cell death (ICD).

[0131] Discard the drug-containing culture medium and gently wash the cells twice with PBS. Add PBS solution containing 5 μM CFSE fluorescent dye (Ex / Em = 492 / 517 nm) and incubate at 37°C in the dark for 20 minutes to fluorescently label the cells. Then wash three times with complete culture medium (Gibco DMEM with 10% (v / v) fetal bovine serum and 1% (v / v) penicillin-streptomycin antibiotics, specifically containing 10,000 units / mL penicillin and 10,000 μg / mL streptomycin) to remove free dye.

[0132] 2.1.1.2 Macrophage preparation and phagocytosis: Bone marrow-derived macrophages (BMDMs) were extracted from C57BL / 6 mice, counted, and then stored at 2 × 10⁶ cells per dish. 7 Density seeding of individual cells.

[0133] Macrophages were labeled using CFSE far red fluorescent dye (Ex / Em = 648 / 668 nm), in the same manner as tumor cells.

[0134] Labeled macrophages were starved in Gibico DMEM serum-free medium for 12 hours to enhance their phagocytic capacity.

[0135] Discard the serum-free culture medium containing macrophages and add B16-OVA cells treated with PTX and labeled with CFSE. Culture macrophages and tumor cells at a ratio of 2:1 in DMEM complete medium. This co-culture system should be incubated at 37°C for 48 hours to allow phagocytosis to occur.

[0136] 2.1.1.3 Sorting and Membrane Extraction (Preparation with MAtigens - Mø@BO): After digestion and collection of co-cultured cells, a double-positive cell population exhibiting both CFSE farred (macrophages) and CFSE (phagocytosed tumor cells) fluorescence signals was collected using flow cytometry (FACS).

[0137] The sorted cells were resuspended and washed with PBS and then processed using a membrane protein extraction kit (Beyotime), or the cell membrane was extracted using the following physical method: the cells were resuspended in PBS containing 10 μg / mL cytochalasin B and treated at 4°C for 2 hours; then vortexed vigorously for 2 minutes, centrifuged at 1,000 rpm for 10 minutes to remove the cell nucleus and unbroken cells, and the supernatant was centrifuged at 4,500 rpm for 15 minutes. The resulting precipitate was the crude cell membrane.

[0138] The crude membrane fraction was resuspended in a solution containing 0.25 mM EDTA and washed twice, and finally resuspended in an appropriate amount of PBS.

[0139] Membrane protein concentrations were determined using the Micro BCA Protein Quantification Kit, and the aliquots were stored at -80°C. This product is known as MAtigens, denoted as Mø@BO.

[0140] 2.1.1.4 Assembly of Nanovaccine (GMø@BO): Accurately weigh 4 mg of egg yolk lecithin (PL-100M) and 1 mg of ginsenoside Rg3, and dissolve them together in 2 mL of a chloroform:ethanol (1:1, v / v) mixed solvent.

[0141] Organic solvents were removed using a rotary evaporator in a 45°C water bath, forming a uniform lipid film on the bottle wall, denoted as GINbrane.

[0142] Take 150 μL of Mø@BO suspension equivalent to 2 mg of membrane protein and add it to 2 mL of PBS. Use this hydration solution to mix the above lipid membrane (i.e., hydration treatment) and sonicate it in an ice-water bath at 4°C for 3 minutes to obtain colostrum.

[0143] By extruding the colostrum through polycarbonate membranes (with pore sizes of 0.2 μm and 0.1 μm, respectively) 11 times each, a GMø@BO nanovaccine suspension with uniform particle size can be obtained.

[0144] 2.1.2 Characterization: 2.1.2.1 PTX-induced immunogenic cell death (ICD) in tumor cells: Existing studies have confirmed that PTX can induce ICD in tumor cells, and the biomarkers of ICD are CRT translocation and the release of HMGB1 and ATP. During ICD, endoplasmic reticulum homeostasis is disrupted, and CRT translocates from the endoplasmic reticulum to the cell membrane surface. Cells exposed to CRT release "eat me" signals to APCs (mainly macrophages), promoting macrophage recognition and phagocytosis of tumor cells. Macrophages process and present tumor antigens, triggering related immune responses. HMGB1 released during ICD can bind to macrophage Toll-like receptors, promoting macrophage activation and expression of co-stimulatory molecules CD80 / CD86 on the cell surface. Tumor cells produce ATP after ICD and release "find me" signals to macrophages.

[0145] To evaluate the ICD effect level, this invention measured the expression of CRT, HMGB1, and ATP release on the surface of B16-OVA cells after co-incubation with PTX using confocal imaging, enzyme-linked immunosorbent assay (ELISA), and ATP bioluminescence techniques, respectively. The results are as follows: Figure 5 As shown.

[0146] 2.1.2.2 Macrophage Phagocytosis: CFSE-labeled B16-OVA cells and CFSE-farred-labeled macrophages were co-incubated for 12 h. Flow cytometry and confocal imaging were used to investigate the phagocytic and processing effects of macrophages on B16-OVA cells. Figure 6 It was found that macrophages exhibited a significantly higher phagocytic rate for tumor cells induced by ICD than for those not induced by ICD. After ICD induction, nearly 80% of tumor cells were phagocytosed by macrophages, while the phagocytic rate in the PBS group (without ICD induction) was only 10.6%. Furthermore, confocal laser microscopy (CLSM) images showed a significant increase in the number of phagocytic vesicles in the macrophage cytoplasm after co-incubation with ICD-induced B16-OVA cells, and these vesicles primarily contained B16-OVA cell debris displaying CFSE signals. All these results indicate that ICD induction significantly enhances the phagocytic capacity of macrophages for tumor cells.

[0147] 2.1.2.3 Macrophage Antigen Presentation: CRT-mediated phagocytosis facilitates antigen processing and presentation by macrophages. This invention uses SIINFEKL–H2kb staining and evaluates the expression level of the MHCⅠ-OVA antigenic epitope (SIINFEKL–H2kb) on the surface of macrophages after phagocytosis of tumor cells using flow cytometry and confocal imaging. This is to assess the efficiency of macrophage antigen processing and presentation to tumor cells after ICD induction. The results are as follows: Figure 7 As shown in the figure, compared with the PBS group, after tumor cells produced the ICD effect, the proportion of SIINFEKL–H2kb+ macrophages co-cultured with them increased significantly (62.45 ± 2.93%), and CLSM imaging results showed that after phagocytizing tumor B16-OVA cells, the SIINFEKL–H2kb signal on the macrophage surface was significantly enhanced, indicating that as macrophages phagocytose tumor cells, the macrophage surface can present related tumor antigen epitopes.

[0148] 2.1.2.4 Macrophage membrane extraction and characterization of key proteins after phagocytosis of B16-OVA cells: Western blotting results showed that antigen-presenting related proteins on Mø@BO, such as co-stimulatory molecules CD80 / CD86, MHCⅠ, and the level of B16-OVA-specific antigen OVA, were upregulated compared to Mø. Figure 8 Furthermore, the levels of cell migration-related proteins CCR7 and ICAM-1 on Mø@BO were significantly increased compared to other groups, indicating a stronger ability to migrate to lymph nodes (LNs). This suggests that tumor cells undergoing ICD can stimulate macrophage activation and phagocytosis. Subsequently, macrophages that phagocytose tumor cells present multiple related tumor antigen epitopes on their membranes and express co-stimulatory molecules and migration molecules.

[0149] 2.1.2.5 Particle Size and Distribution: Using dynamic light scattering (DLS) analysis, the hydrated particle size of GMø@BO was determined to be 99.8 ± 3.5 nm, and the polydispersity index (PDI) was 0.121 ± 0.015, indicating uniform particle size and narrow distribution. The results are as follows: Figure 9 As shown in A and B.

[0150] 2.1.2.6 Zeta potential: The Zeta potential value is -28.4 ± 1.7 mV, indicating good system stability, as shown in the results. Figure 9 As shown in C.

[0151] 2.1.2.7 Morphology: After negative staining with phosphotungstic acid, transmission electron microscopy (TEM) revealed ( Figure 10 GMø@BO exhibits a typical spherical or near-spherical vesicle structure with clear lipid bilayer boundaries, and its particle size is consistent with the DLS results.

[0152] 2.1.2.8 Membrane fusion validation: Validation was performed using the Förster resonance energy transfer (FRET) assay. GINbrane was labeled with the donor dye Courmarin-6 (C6), and Mø@BO was labeled with the acceptor dye Rhodamine B (RhB). Figure 11 As shown, with the addition of Mø@BO (the mass ratio of lipid material to Mø@BO protein in the lipid membrane gradually changed from 2:0 to 2:1.6), after GINbrane and Mø@BO hybridized, the fluorescence intensity at 534 nm (C6 emission peak) under 460 nm excitation light significantly decreased, while a significant enhancement occurred at 583 nm (RhB emission peak), indicating that the two membranes successfully fused and the distance between dye molecules entered the effective range of FRET (<10 nm). Figure 12 As shown, under confocal microscopy (CLSM), the fluorescence signals of DiD-labeled GINbrane (red) and C6-labeled Mø@BO (green) also showed a high degree of overlap (appearing yellow), further confirming the successful hybridization of the two.

[0153] 2.1.2.9 Stability Study: This invention also investigated the particle size stability of GMø@BO and cholesterol membrane hybrid liposome nanovaccine CMø@BO (prepared by replacing ginsenoside Rg3 with cholesterol as described above, followed by assembly of the nanovaccine) at 4°C. The results are as follows: Figure 13 As shown, CMø@BO liposomes began to aggregate and their particle size increased from day 5. Unlike CMø@BO, GMø@BO showed significantly lower particle size changes over 7 days, demonstrating superior particle size stability compared to CMø@BO.

[0154] 2.2 Preparation and Characterization of GMø@4T1 (Ginsenoside Membrane Hybrid Tumor Nanovaccine Based on 4T1 Triple-Negative Breast Cancer Cells)

[0155] 2.2.1 Preparation method: 2.2.1.1 Tumor cell treatment: Mouse triple-negative breast cancer 4T1 cells were obtained and ICD induction and labeling were performed using step 2.1.1.1 in the preparation of GMø@BO.

[0156] 2.2.1.2 Macrophage preparation and phagocytosis: The procedure is the same as step 2.1.1.2 in the preparation of GMø@BO. BMDMs derived from BALB / c mice were used.

[0157] 2.2.1.3 Sorting and Membrane Extraction (MAtigens - Mø@4T1 Preparation): The procedure is the same as step 2.1.1.3 in the GMø@BO preparation. Macrophage membranes that have engulfed 4T1 cells were sorted and extracted by flow cytometry, and the product was denoted as Mø@4T1.

[0158] 2.2.1.4 Assembly of the nanovaccine (GMø@4T1): The procedure is the same as step 2.1.1.4 in the preparation of GMø@BO. Replace Mø@BO with Mø@4T1 of equal membrane protein content.

[0159] 2.2.2 Characterization: 2.2.2.1 Macrophage Phagocytosis: CFSE-labeled 4T1 cells and CFSE-farred-labeled macrophages were co-incubated for 12 h. Flow cytometry and confocal imaging were used to investigate the phagocytic and processing effects of macrophages on 4T1 cells. Figure 14 It was found that macrophages exhibited a significantly higher phagocytic rate for tumor cells that had undergone ICD than for those that had not. Following ICD induction, nearly 70% of 4T1 tumor cells were phagocytosed by macrophages. Furthermore, confocal laser microscopy (CLSM) images showed a significant increase in the number of phagocytic vesicles within the macrophage cytoplasm after co-incubation with ICD-treated 4T1 cells, and these vesicles primarily contained 4T1 cell debris displaying CFSE signals. All these results indicate that ICD induction significantly enhances the phagocytic capacity of macrophages for tumor cells.

[0160] 2.2.2.2 Particle Size and Distribution: DLS analysis showed that the hydrated particle size of GMø@4T1 was 101.5 ± 4.1 nm, and the PDI was 0.130 ± 0.022. The results are as follows: Figure 15 As shown in A and B.

[0161] 2.2.2.3 Zeta potential: The Zeta potential value is -26.9 ± 2.2 mV, indicating good system stability, as shown in the results. Figure 15 As shown in C.

[0162] 2.3 Preparation and Characterization of GMø@PDC (Ginsenoside Membrane Hybrid Tumor Nanovaccine Based on Human Pancreatic Cancer Cells)

[0163] 2.3.1 Preparation method: 2.3.1.1 Isolation and processing of patient-derived tumor cells (PDC): With informed consent from the patient and approval from the ethics committee, a fresh surgical sample of pancreatic ductal adenocarcinoma (PDAC) was obtained. Under aseptic conditions on ice, the capsule and necrotic tissue were removed, and the tumor tissue was minced to approximately 1 mm. 3Small pieces. Add 2 mM EDTA solution, gently shake, centrifuge, and discard the supernatant. Add type II collagenase solution and digest on a 37°C shaker for about 1 hour, pipetting every 15 minutes until most of the tissue has dissolved. Filter the digestion solution using a 40 μm cell sieve, collect the filtrate, centrifuge to obtain the cell pellet, resuspend in complete culture medium, and culture. Change half the medium every 3 days. The adherent cells are primary pancreatic cancer cells (PDC).

[0164] Take the cultured PDC, and follow the same steps as step 2.1.1.1 in the previous GMø@BO preparation.

[0165] 2.3.1.2 Preparation and phagocytosis of human macrophages: Human monocyte cell line THP-1 was cultured and stimulated for 12 hours with medium containing 100 ng / mL PMA and LPS to differentiate into M0 macrophages. Human macrophages were then labeled with CFSE far red dye.

[0166] The subsequent starvation treatment and co-incubation with labeled PDC are the same as step 2.1.1.2 in the preparation of GMø@BO.

[0167] 2.3.1.3 Sorting and Membrane Extraction (Preparation with MAtigens - Mø@PDC): The procedure is the same as step 2.1.1.3 in the preparation of GMø@BO. Human macrophage membranes that have engulfed human PDCs are sorted and extracted by flow cytometry, and the product is denoted as Mø@PDC.

[0168] 2.3.1.4 Assembly of Nanovaccine (GMø@PDC): The procedure is the same as step 2.1.1.4 in the preparation of GMø@BO. Replace Mø@BO with Mø@PDC of equal membrane protein content.

[0169] 2.3.2 Characterization: 2.3.2.1 Macrophage Phagocytosis: CFSE-labeled PDC cells and CFSE-farred-labeled macrophages were co-incubated for 12 h. Flow cytometry and confocal imaging were used to investigate the phagocytic and processing effects of macrophages on 4T1 cells. Figure 16It was found that macrophages exhibited a significantly higher phagocytic rate for tumor cells that had undergone ICD than for those that had not. Following ICD induction, nearly 80% of PDC tumor cells were phagocytosed by macrophages. Furthermore, confocal laser microscopy (CLSM) images showed a significant increase in the number of phagocytic vesicles within the macrophage cytoplasm after co-incubation with ICD-treated PDCs, and these vesicles primarily contained PDC cell debris displaying CFSE signals. All these results indicate that ICD induction significantly enhances the phagocytic capacity of macrophages for tumor cells.

[0170] 2.3.2.2 Particle Size and Distribution: DLS analysis showed that the hydrated particle size of GMø@PDC was 103.2 ± 3.8 nm, and the PDI was 0.118 ± 0.019. The results are as follows: Figure 17 As shown in A and B.

[0171] 2.3.2.3 Zeta potential: The Zeta potential value is -27.5 ± 1.9 mV, as shown in the figure. Figure 17 As shown in C.

[0172] Furthermore, the present invention summarizes the corresponding information of the three ginsenoside membrane hybrid tumor nanovaccines mentioned above in Table 1 below.

[0173] Table 1. Physicochemical properties of MAGIN-Vax

[0174] 3. Study on the lipid membrane-mediated targeting effects of ginsenosides on dendritic cells (DCs) and lymph nodes.

[0175] 3.1 Research Methods

[0176] 3.1.1 DCs uptake experiment

[0177] BMDCs were extracted from C57BL / 6 mice and processed at 2 × 10⁻⁶. 5 Cells were seeded at a density of 10 cells / well into 12-well plates and co-incubated for 4 h (C6 500 ng / mL) with C6-labeled C-Lp (liposomes made from cholesterol and phospholipids), Rg3-Lp (liposomes made from ginsenoside Rg3 and phospholipids), CMø / BO (membrane hybrid liposomes synthesized from cholesterol lipid membranes, macrophage membranes, and BO tumor cell membranes), CMø@BO, or GMø@BO. After washing three times with PBS, the cells were co-incubated at room temperature for 20 min with PE-CD8 and Percp-cy5.5CD11c antibodies to label CD8. + T cells were then washed three times with PBS, and the signal intensity of each liposome on the DCs was determined by flow cytometry.

[0178] 3.1.2 Knockdown experiment of DC surface pattern recognition receptor expression

[0179] BMDC with 2 × 10 5 Cells were seeded at a density of 10 cells / well in 12-well plates. After 12 h, siRNA and siRNA-mate of mannose receptor (MR, 5'-CCGUGUUGAACCUCUUAAATT-3' [SEQ ID NO: 1]), Dectin-2 receptor (5'-GAGGUCUACAACCAAAUCUTT-3' [SEQ ID NO: 2]), Toll-like receptor-4 (TLR4, 5'-GGACAGCUUAUAACCUUAATT-3' [SEQ ID NO: 3]), or negative control (NC, 5'-UUCUCCGAACGUGUCACGUTT-3' [SEQ ID NO: 4]) were mixed with each other in Gibico DMEM serum-free medium and incubated at room temperature for 20 min to form siRNA-siRNA-mate complexes. After replacing the medium with fresh medium, the siRNA-siRNA-mate complexes were added and incubated for 72 hours. After h, DCs with knocked-down expression of pattern recognition receptors MR, Dectin-1, or TLR4 can be obtained (DCs MR- DCs Dectin-1- or DCs TLR4- ).

[0180] 3.1.3 Investigation of DCs Targeting Mechanism

[0181] 3.1.3.1 DCs uptake experiment after pattern recognition receptor expression knockdown

[0182] DCs, DCs MR- DCs Dectin-1- or DCs TLR4- With 2 × 10 5 Cells were seeded at a density of 500 ng / mL into 12-well plates and co-incubated with C6-labeled Rg3-Lp or GMø@BO for 4 h (C6 500 ng / mL). After washing three times with PBS, the cells were co-incubated with Percp-cy5.5 CD11c antibody at room temperature for 20 min to label the DCs. After washing three times with PBS, the signal intensity of liposomes on the DCs was determined by flow cytometry.

[0183] DCs or DCs respectively Dectin-1- With 2 × 10 5Cells were seeded at a density of 500 ng / mL into 12-well plates and co-incubated for 4 h with C6-labeled Rg3-Lp or GMø@BO (C6 500 ng / mL). After washing the cells three times with PBS, the cells were fixed with 4% paraformaldehyde, blocked with 2% BSA at room temperature for 30 min, incubated overnight at 4°C with Dectin-1 antibody (1 / 200 dilution), washed three times with PBS, incubated with secondary antibody Cy3-goat anti-rabbit IgG H&L at room temperature for 2 h, washed three times with PBS, and finally stained with DAPI for 10 min, washed three times with PBS, and observed using confocal microscopy.

[0184] 3.1.3.2 Dectin-1-Rg3 Molecular Docking Experiment

[0185] The lowest-energy 3D conformations of Rg3 and Chol were constructed using Chem3D. The structures of Rg3, Cholesterol (Chol), and Dectin-1 (PDB ID 2CL8) were imported into Schrödinger Maestro 11.8. After running the Ligprep program, 32 possible conformations of Rg3 and Chol were generated from the library. Energy minimization of Rg3 and Chol was performed, including hydrogen addition, ionization, and the allocation of appropriate bond lengths, bond angles, torsion angles, correct chirality, stereochemistry, and ring conformation 3D coordinates. The 3D structure data of Dectin-1 was downloaded from the RCSB protein database, opened in Schrödinger Maestro 11.8, and the protein structure was optimized before docking. The optimal docking conformation of the compound was determined based on the required docking energy, docking score, and hydrogen bond interactions. 3D models of the corresponding protein-ligand complexes were generated using PyMol.

[0186] 3.1.4 Research on DCs Replacement Phenomenon

[0187] DCs are divided into 2 × 10 5 Cells were seeded at a density of 500 ng / mL into 12-well plates and co-incubated with each C6-labeled liposome for 4 h (C6 500 ng / mL). After washing three times with PBS, the plates were blocked with 1% BSA for 20 min and then co-incubated with Percp-cy5.5-CD11c and APC-SIINFEKL–H2kb antibodies at room temperature for 20 min. After washing three times with PBS, the signal intensity of each liposome and surface antigen epitope on the DCs was determined by flow cytometry.

[0188] DCs are divided into 2 × 10 5Cells were seeded at a density of 10 cells / well in 12-well plates. Lipid membranes and cell membranes were labeled with C6 and DiD, respectively, following the same procedure as step 2.1.2.8 in the GMø@BO preparation. Each labeled liposome was co-incubated with DCs for 4 h (C6 500 ng / mL). After washing three times with PBS, the cells were blocked with 1% BSA for 20 min, then co-incubated with PE-SIINFEKL–H2kb antibody at room temperature for 20 min. After washing three times with PBS, the cells were co-incubated with 2 mg / mL Hoechst 33258 for 15 min, washed three times with PBS, and then observed using confocal microscopy.

[0189] 3.1.5 In vivo distribution study of GMø@BO

[0190] C57BL / 6 mice were injected with DiR-encapsulated C-Lp, Rg3-Lp, CMø / BO, CMø@BO, or GMø@BO at the base of their tails. Near-infrared fluorescence imaging of the mice was performed using an IVIS imaging system at 2, 4, 8, 12, and 24 h post-injection. 24 h later, mice in the GMø@BO group were subjected to 3D fluorescence imaging under a small animal in vivo three-dimensional multimodal imaging system. Subsequently, the mice were sacrificed, and draining lymph nodes, heart, liver, spleen, lungs, and kidneys were collected for in vitro fluorescence imaging. The lymph nodes were fixed in 4% paraformaldehyde, embedded, and frozen sectioned. The obtained lymph node sections were stained overnight at 4°C with mouse anti-CD11c antibody and rabbit anti-Dectin-1 (both purchased from Abcam), washed three times with PBS, and then incubated at 37°C for 2 h with Alexa Fluor 488-labeled goat anti-rabbit IgG (H+L) and Cy3-labeled goat anti-mouse IgG (H+L) (both purchased from Yisheng Biotechnology), washed three times with PBS, and stained with DAPI for 10 min. Another serially obtained lymph node section with similar morphology was stained overnight at 4°C with rabbit anti-CD8 antibody (Abcam), washed three times with PBS, incubated at 37°C for 2 h with Alexa Fluor 488-labeled goat anti-rabbit IgG (H+L) (Yisheng Biotechnology), washed three times with PBS, stained with DAPI for 10 min, washed three times with PBS, mounted with glycerol-gelatin mounting solution, and observed via confocal microscopy.

[0191] 3.1.6 Study on the distribution of GMø@BO cells in lymph nodes

[0192] C57BL / 6 mice were injected with DiD-loaded C-Lp, Rg3-Lp, CMø / BO, CMø@BO, or GMø@BO at the base of their tails. Twenty-four hours after injection, the mice were sacrificed, and inguinal lymph nodes were collected and ground. The resulting cell suspension was passed through a 200-mesh sieve. The obtained lymph node cells were blocked with 1% BSA for 20 min, followed by staining with PE-Cy7, PE-CD8, and APC-CD11c at room temperature for 20 min. After washing three times with PBS, flow cytometry analysis was performed.

[0193] 3.2 Experimental Results

[0194] 3.2.1 GMø@BO achieves DC targeting through Rg3-Dectin-1 interaction

[0195] like Figure 18 As shown, compared with C-Lp, DCs uptake of Rg3-Lp increased by nearly 50%, indicating that Rg3-Lp has a DC-targeting effect. Therefore, compared with CMø@BO, GMø@BO, which is hybridized with an Rg3 lipid membrane, has significantly stronger DC uptake. Meanwhile, compared with CMø / BO, CMø@BO uptake on DCs is slightly increased, possibly due to the expression of more antigenic epitopes on GMø@BO. To elucidate the targeting mechanism of Rg3-Lp on DCs, this invention knocked down the expression levels of typical pattern recognition receptors on DCs, such as the mannose receptor (MR), Toll-like receptor-4 (TLR4), and Dectin-1, and observed the changes in DC uptake of Rg3-Lp after the reduction of related receptor expression. The results are as follows: Figure 19 As shown.

[0196] Notably, decreased Dectin-1 expression significantly reduced the uptake of Rg3-Lp or GMø@BO by DCs. However, the uptake of C-Lp- or CMø@BO by DCs did not change significantly before and after the decrease in Dectin-1 expression. Furthermore, knockdown of TLR4 and MR expression in DCs did not significantly alter the uptake of Rg3-Lp and GMø@BO. CLSM plots further confirmed this trend; observation of CLSM images revealed strong co-localization between the C6-labeled Rg3 lipid membrane and the Dectin-1 signal on DCs. Figure 20 This suggests that the lipid membrane of ginsenoside Rg3 likely targets DCs through the DCs surface pattern recognition receptor Dectin-1.

[0197] Further molecular docking experiments were used to investigate the interaction between Rg3 or cholesterol and Dectin-1. The molecular docking scores of Rg3 and cholesterol with Dectin-1 were -9.3 and -4.7, respectively, indicating that Rg3 has a stronger affinity for Dectin-1. 3D imaging was performed using Pymol, and the interaction between Rg3 or cholesterol and Dectin-1 was analyzed. The results are as follows: Figure 21 As shown.

[0198] The glycosyl moiety of Rg3 can form hydrogen bonds with the amino acid residues GLU232, ALA152, GLU194, SER148, and LYS242 of Dectin-1. However, due to the lack of glycosyl groups, cholesterol cannot form hydrogen bonds with Dectin-1. Combined with the evidence in "1.2.1 Comparison of the effects of various ginsenosides on DC maturation," which demonstrates that Rg3 can expose its glycosyl group on the lipid membrane surface, this suggests that GMø@BO, which possesses a hybrid Rg3 lipid membrane, may be able to target DCs through the specific interaction of Rg3 with the highly expressed Dectin-1 on DCs.

[0199] 3.2.2 DCs replacement phenomenon of GMø@BO surface antigen epitopes

[0200] Studies have shown that dendritic cells (DCs) can directly transfer pMHC complexes from the donor cell membrane to the host DC membrane, a phenomenon known as "transferring." Therefore, this invention uses flow cytometry to detect the expression level of the OVA epitope SIINFEKL–H2kb on the surface of DCs after co-incubation with GMø@BO to verify whether GMø@BO can transfer the epitope from Mø@BO to the DC surface. The results are as follows: Figure 22 As shown.

[0201] After co-incubation with CMø@BO, the level of OVA antigenic epitopes on the surface of DCs was significantly higher than that in the CMø / BO group. This is because the macrophage membrane extracted after phagocytosis and treatment of tumor cells, Mø@BO, expresses more OVA antigenic epitopes on its surface compared to the mixture of tumor cell membrane and macrophage membrane alone, Mø / BO. Furthermore, the level of antigenic epitopes on the surface of DCs in the GMø@BO group was still significantly higher than that in the CMø@BO group. This is mainly because, as demonstrated above, GMø@BO, hybridized with Rg3 lipid membrane, can contact DCs more effectively, thus transferring more Mø@BO antigenic epitopes to the DC surface. Subsequently, cell membranes and lipid membranes were stained separately, and CLSM imaging was performed to observe the distribution of various components of GMø@BO on DCs after contact. The results are as follows: Figure 23 As shown.

[0202] In GMø@BO, DiD-labeled Mø@BO was completely transferred to the surface of dendritic cells (DCs), while the C6-labeled Rg3 lipid membrane portion was dispersed in the DC cytoplasm. Furthermore, the GMø@BO group exhibited the strongest antigenic epitope signal (SIINFEKL-H2kb) on the DC surface and strong co-localization with the DiD-labeled biomembrane signal, indicating successful translocation of the antigenic epitope from Mø@BO to the DC surface. The hybridization of the Rg3 lipid membrane facilitated contact and translocation between Mø@BO and DCs, resulting in a stronger antigenic epitope signal on the DC surface in the GMø@BO group compared to the CMø@BO group. This unconventional antigen presentation process bypasses complex antigen processing, which is beneficial for subsequent T cell activation responses.

[0203] 4. Study on the adjuvant effect mediated by ginsenoside lipid membrane

[0204] 4.1 Research Methods

[0205] 4.1.1 Determination of DCs Maturity Level

[0206] DCs are divided into 2 × 10 5 Cells were seeded at a density of 1000 cells / well into 12-well plates and co-incubated for 48 h with C-Lp, Rg3-Lp, CMø / BO, CMø@BO, or GMø@BO (Rg3 or 50 μM cholesterol). After centrifugation at 1000 g for 15 min, the supernatant was collected. The levels of IL-12, TNF-α, and IL-1β in the cell supernatant of each group were detected according to the instructions of the IL-12, TNF-α, and IL-1β ELISA kits (purchased from Lianchuan Biotechnology). Simultaneously, cells were collected, washed three times with PBS, blocked with 1% BSA for 20 min, and co-incubated with FITC-CD80 and PE-CD86 antibodies at room temperature for 20 min to label mature DCs. Cells were washed three times with PBS and then analyzed by flow cytometry.

[0207] 4.1.2 Research on the mechanism of promoting DC maturation

[0208] DCs, DCs Dectin-1- or DCs TLR4- With 2 × 10 5 Cells were seeded at a density of 10 cells / well into 12-well plates and co-incubated for 48 h with C-Lp, Rg3-Lp, CMø@BO, or GMø@BO (Rg3 or 50 μM cholesterol). After washing three times with PBS, the cells were blocked with 1% BSA for 20 min, followed by co-incubation at room temperature with FITC-CD80 and PE-CD86 antibodies for 20 min to label mature DCs. The cells were washed three times with PBS, and the maturity level of the DCs was determined by flow cytometry.

[0209] 4.1.2 Investigation into the effect of DCs on promoting T cell activation

[0210] Extract BMDC, in 2 × 10 5 Cells were seeded at a density of 10 cells / well into 12-well plates and co-incubated with C-Lp, Rg3-Lp, CMø / BO, CMø@BO, or GMø@BO (Rg3 or 50 μM cholesterol) for 48 h. Mouse lymphocytes were extracted and stained with CFSE. The DCs treated in each group were co-incubated with CFSE-labeled lymphocytes for 48 h (DCs: lymphocytes 10:1). After three PBS cycles, the cells were blocked with 1% BSA for 20 min and then co-incubated with PE-CD8, APC-CD69, and PE-Cy7-SIINFEKL-H2Kb antibodies at room temperature for 20 min. The cells were washed three times with PBS. Flow cytometry analysis was performed to analyze the proliferation and activation of T cells after co-incubation with DCs in each group.

[0211] 4.2 Research Results

[0212] 4.2.1 GMø@BO promotes DC maturation through Rg3-Dectin-1 interaction

[0213] Rg3 can be used as a vaccine adjuvant to promote DC maturation. Further investigation of the DC maturation-promoting effect of GMø@BO was conducted using flow cytometry, and the results are as follows... Figure 24 As shown in the figure. The results showed that Rg3-Lp and GMø@BO exhibited stronger effects on promoting DC maturation than CMø@BO, and the co-stimulatory molecules CD80 / CD86 on DCs were significantly upregulated. Moreover, the effect of Rg3 on promoting DC maturation was not significantly different from that of the positive control (typical adjuvant lipopolysaccharide LPS, purchased from InvivoGen).

[0214] Mature dendritic cells (DCs) often secrete cytokines that promote T cell proliferation and differentiation, such as IL-12, TNF-α, and IL-1β. Therefore, this invention used an ELISA kit to detect the levels of IL-12, TNF-α, and IL-1β in the culture medium of DCs from different groups. The results are as follows: Figure 25 As shown.

[0215] Consistent with DC maturation levels, the levels of cytokines IL-12, TNF-α, and IL-1β secreted by mature DCs in the Rg3-Lp and GMø@BO groups were significantly increased. This indicates that the Rg3 lipid membrane can act as a vaccine adjuvant to promote DC maturation, and GMø@BO, which incorporates the Rg3 lipid membrane, can achieve co-delivery of antigen and adjuvant. As mentioned above, Rg3-Lp can interact with Dectin-1, a typical pattern recognition receptor on the surface of DCs. Interaction with Dectin-1 activates the downstream ITAM region molecule, spleen tyrosine kinase Syk, inducing the expression of co-stimulatory molecules CD80 / CD86, thereby promoting DC maturation. To explore whether the DC-promoting effect of the Rg3 lipid membrane is mediated by Dectin-1, this invention used flow cytometry to determine the expression of co-stimulatory molecules in DCs co-incubated with Rg3-Lp or GMø@BO before and after Dectin-1 expression knockdown. The results are as follows: Figure 26 As shown.

[0216] Knockdown of Dectin-1 expression on the surface of dendritic cells (DCs) significantly reduced the maturation-promoting effects of Rg3-Lp and GMø@BO compared to their efficacy on normal DCs. However, knockdown of TLR4 expression did not significantly reduce the maturation-promoting effects of Rg3-Lp and GMø@BO compared to their efficacy on normal DCs. This suggests that the Rg3 lipid membrane can activate downstream signaling pathways of Dectin-1 through interaction with it, thus inducing DC maturation and potentially functioning as a vaccine adjuvant.

[0217] 4.2.2 GMø@BO indirectly activates tumor-killing CTLs

[0218] The results above indicate that the Rg3 lipid membrane can act as an immune adjuvant, promoting DC maturation (signal 2) and the secretion of related cytokines that promote T cell activation and proliferation (signal 3). GMø@BO, a hybrid of the Rg3 lipid membrane and Mø@BO (signal 1), can induce DCs to simultaneously provide three signals for T cell activation to T cells, thereby maximizing T cell activation and proliferation. Therefore, this invention co-incubates GMø@BO-pretreated DCs with lymphocytes and detects the activation and proliferation effects of GMø@BO-pretreated DCs on T lymphocytes. The results are as follows: Figure 27 and Figure 28 As shown.

[0219] GMø@BO pretreated dendritic cells (DCs), when co-cultured with T lymphocytes, can provide antigen signals (signal 1) to T lymphocytes, resulting in pMHC signaling on the T cell surface. This, along with co-stimulatory molecules on the surface of mature DCs and secreted cytokines, promotes T cell activation. Therefore, as... Figure 27 As shown, the antigen-specific CD8 of the GMø@BO group+ The activation level of T cells was 1.7 times that of the CMø@BO group, indicating the important role of immune adjuvants in promoting antigen-specific T cell activation.

[0220] As mentioned above, GMø@BO pretreated DCs exhibit higher maturation levels and secrete high levels of cytokines such as IL-12, TNF-α, and IL-1β. These cytokines promote T cell proliferation and differentiation, providing cytokine signals to T lymphocytes (signal 3). CFSE-labeled T lymphocytes were co-cultured with pretreated DCs from each group to detect the pro-T lymphocyte proliferation effect of pretreated DCs. CFSE can non-specifically covalently bind to amino acids on intracellular proteins. During cell division, these CFSEs are distributed to daughter cells, and their fluorescence weakens. The intensity of the CFSE fluorescence signal can be used to evaluate lymphocyte proliferation. Figure 28 As shown, the CFSE signal intensity was lowest in the GMø@BO group, indicating that DCs pretreated with GMø@BO had the strongest efficacy in promoting T cell proliferation.

[0221] 5. Study on the direct activation effect of ginsenoside membrane hybrid tumor nanovaccine on T cells

[0222] 5.1 Experimental Materials and Instruments

[0223] The IFN-γ ELISA kit was purchased from Shanghai Lianke Biotechnology Co., Ltd.; the Brefeldin A solution (1000X) was purchased from eBioscience.

[0224] C57BL / 6 mice (female, 6-8 weeks old) were purchased from Shanghai Lingchang Biotechnology Co., Ltd. and housed at the Experimental Animal Center of the School of Pharmacy, Fudan University.

[0225] The other reagents and instruments used are the same as those described above.

[0226] 5.2 Experimental Methods

[0227] 5.2.1 Detection of GMø@BO interaction with T cells

[0228] Primary lymphocytes were isolated from C57BL / 6 mice and cultured at 2 × 10⁻⁶ cells per cell line. 5 Cells were seeded at a density of 500 ng / mL into 12-well plates and co-incubated for 4 h with C6-labeled C-Lp, Rg3-Lp, CMø / BO, CMø@BO, or GMø@BO (C6 500 ng / mL). After washing three times with PBS, the plates were blocked with 1% BSA at room temperature for 20 min, and then co-incubated with PE-CD8 antibody at room temperature for 20 min to label CD8. +T cells were washed three times with PBS and CD8 count was determined by flow cytometry. + Signal intensity of each liposome on T lymphocytes.

[0229] 5.2.2 Detection of T cell surface antigen epitope levels

[0230] Lymphocytes at 2 × 10 5 Cells were seeded at a density of 500 ng / mL into 12-well plates and co-incubated for 4 h with C6-labeled C-Lp, Rg3-Lp, CMø / BO, CMø@BO, or GMø@BO (C6 500 ng / mL). After washing three times with PBS and blocking with 1% BSA for 20 min, the following procedures were performed: Flow cytometry: CD8 was labeled by incubation at room temperature for 20 min with PE-CD8 and APC-SIINFEKL–H2kb antibodies. + T cells and MHCⅠ-OVA antigen complex. Washed three times with PBS and analyzed by flow cytometry.

[0231] Confocal microscopy: MHC-OVA antigen complex was labeled by incubating with APC-SIINFEKL–H2kb antibody at room temperature for 20 min. Then, the cells were co-incubated with 2 mg / mL Hoechst 33258 for 15 min to stain the cell nuclei. After washing three times with PBS, the expression of the preparation and MHC-OVA antigen complex on lymphocytes in each group was observed using a rotating confocal microscope.

[0232] 5.2.3 Measurement of T cell activation level

[0233] Lymphocytes at 2 × 10 5 Cells were seeded at a density of 100 cells / well into 12-well plates and co-incubated for 8 h with C-Lp, Rg3-Lp, CMø / BO, CMø@BO, or GMø@BO (Rg3 or 50 μM cholesterol). The medium was then replaced with fresh medium containing 20 μM OVA, and incubation continued for 12 h. The culture medium was then aspirated, centrifuged at 1000 g for 15 min, and the supernatant was collected. The IFN-γ levels in the cell supernatant of each group were detected using an IFN-γ ELISA kit according to the manufacturer's instructions. Simultaneously, cells were collected, washed three times with PBS, blocked with 1% BSA for 20 min, and co-incubated with PE-CD8 and APC-CD69 antibodies at room temperature for 20 min to label CD8. + T cells and CD69, a marker of early T cell activation, were then washed three times with PBS and analyzed by flow cytometry.

[0234] 5.3 Experimental Results

[0235] 5.3.1 GMø@BO can be used with CD8 + T lymphocyte interaction

[0236] GMø@BO and CD8 were analyzed by flow cytometry. + The interaction between T lymphocytes was characterized, and the results were as follows: Figure 29 As shown.

[0237] Compared to macrophage membranes or tumor cell membranes, macrophage membranes that have engulfed tumor cells present more antigenic epitopes. These epitopes can interact with the TCR on T cells; therefore, compared to CMø / BO, the signal values ​​of GMø@BO and CMø@BO on T cells are significantly increased. This suggests that GMø@BO and CMø@BO can interact with CD8+ via the pMHC antigenic epitopes on the surface of Mø@BO. + T cell-mediated TCR interaction, on CD8 + T lymphocytes have a stronger targeting effect.

[0238] 5.3.2 GMø@BO vs CD8 + T lymphocytes have antigen-presenting function

[0239] Using the MHC-OVA antigen peptide complex H2Kb (MHC I fragment)-SIINFEKL (OVA fragment) as a typical antigenic epitope, flow cytometry analysis was performed on the antigen presentation on the surface of T cells in each group to further verify the interaction between the pMHC antigenic epitope on the Mø@BO surface and the TCR. The results are as follows: Figure 30 As shown.

[0240] After co-incubation with CMø@BO or GMø@BO, CD8 + The level of the antigenic epitope SIINFEKL–H2kb on the surface of T lymphocytes was nearly 7 times that of other groups. Furthermore, CLSM plots showed that CMø@BO or GMø@BO exhibited stronger formulation signal intensity (green) on lymphocytes than other groups, and the lymphocyte surface also showed stronger antigenic epitope signal (red), with good co-localization of the formulation signal and antigenic epitope signal. Figure 31 This indicates that CMø@BO or GMø@BO can act as pseudo-APCs, interacting with T cells and presenting their surface MHC-antigen peptide complexes to T cells.

[0241] 5.3.3 GMø@BO can directly activate CD8 + T lymphocytes

[0242] CTL activation requires the combined action of pMHC-TCR signaling (signal 1) and co-stimulatory signaling (signal 2). The above experiments show that the antigenic epitopes on the surface of Mø@BO can provide signal 1 to T cells. Simultaneously, the Western blot results in "2.1.2.4 Macrophage membrane extraction and characterization of key proteins after phagocytosis of B16-OVA cells" also demonstrate that the surface of Mø@BO highly expresses the co-stimulatory molecules CD80 / CD86, which can provide signal 2 to T cells. Therefore, GMø@BO or CMø@BO has the potential to promote T cell activation. Thus, this invention uses CD69, an early activation marker on the T cell surface, and IFN-γ secreted by activated T cells as indicators to verify the level of T cell activation promoted by GMø@BO or CMø@BO. The results are as follows: Figure 32 and Figure 33 As shown.

[0243] After co-incubating T cells with each liposome, they were co-incubated with the antigenic peptide OVA for 12 h, and then CD8 in each group was analyzed by flow cytometry. + The expression level of CD69 on the surface of T cells can be observed by examining CD8 expression levels after treatment with CMø@BO or GMø@BO. + T cells exposed to antigens interact with CD8 cells treated with CMø / BO. + Compared to T cells, the expression level of CD69 on the cell surface was significantly increased, indicating that CMø@BO or GMø@BO can promote CD8 expression. + T cell activation.

[0244] Activated T cells secrete large amounts of IFN-γ. ELISA analysis of IFN-γ secretion levels in T cells from different groups revealed that T cells sensitized with CMø@BO and GMø@BO secreted 2.53 times more IFN-γ after exposure to the OVA antigen than the CMø@BO group. This indicates that GMø@BO or CMø@BO can provide activation signals to T cells via antigenic epitopes and co-stimulatory molecules on GMø@BO, enhancing T cell activation and IFN-γ secretion upon antigen exposure.

[0245] summary

[0246] This section demonstrates the role of GMø@BO as a pseudo-APC directly sensitizing T cells. Firstly, flow cytometry and confocal imaging revealed that GMø@BO interacts more extensively with T cells, presenting pMHC on GMø@BO to CD8. + T lymphocytes provide activation signals to T cells (1) Figure 34 Under the combined action of co-stimulatory molecules on Mø@BO, T cells stimulated by GMø@BO and exposed to tumor antigens undergo T cell activation and secrete high levels of IFN-γ. Therefore, GMø@BO can directly act on T cells, inducing a direct T cell-specific activation response.

[0247] 6. In vivo immune response analysis of ginsenoside membrane hybrid tumor nanovaccine

[0248] 6.1 Experimental Methods

[0249] 6.1.1 Peripheral blood immune cell analysis

[0250] C57BL / 6 mice were randomly divided into 7 groups of 3 mice each. Each group was inoculated at the base of the tail with C-Lp, Rg3-Lp, LPS, CMø / BO, CMø@BO, CMø@BO+LPS (50 μL of 10 mg / mL LPS solution added to 500 μL of CMø@BO solution), or GMø@BO (10 mg / kg cholesterol, Rg3, or 1 mg LPS per mouse), for a total of three inoculations, once a week. Seven days after the last inoculation, blood was collected by enucleation, and erythrocyte lysis buffer was added for 5 min. Cells were then centrifuged at 400 g for 5 min, collected, and incubated for 12 minutes in fresh medium (Gibico DMEM with 10% (v / v) fetal bovine serum and 1% (v / v) penicillin-streptomycin antibiotics, specifically containing 10,000 units / mL penicillin and 10,000 μg / mL streptomycin) containing 20 μM OVA. h, washed three times with PBS, and stained for cell surface proteins and nucleoproteins using the following fluorescent labels: ① BV510-CD45, PerCP-Cy5.5-CD11c, FITC-CD80, PE-CD86 antibodies; ② BV510-CD45, PE-Cy7-CD3, PE-CD8, APC-IFN-γ, PerCP-Cy5.5-CD44, FITC-CD127, PE-Cy7-SIINFEKL-H2Kb antibodies; ③ BV510-CD45, PE-Cy7-CD3, PE-CD8, APC-IFN-γ, PerCP-Cy5.5-Granzyme B (Grz B) antibodies (all purchased from Biolegend). After staining, washed three times with PBS and detected by flow cytometry.

[0251] 6.1.2 Analysis of lymph node immune cells

[0252] After enucleation of the above-mentioned mice, lymph nodes were collected, washed twice with PBS, and then ground and filtered to obtain single-cell suspensions. These suspensions were centrifuged at 400 g and 4℃ for 5 min, the supernatant was discarded, and the cell pellet was collected. The pellets were incubated in fresh medium containing 20 μM OVA for 12 h, washed three times with PBS, and then stained with the lymph node single-cell suspensions according to the same staining protocol as in "6.6.1 Peripheral Blood Immune Cell Analysis". Simultaneously, the collected lymph nodes from each group were paraffin-embedded, serially sectioned to ensure that the cross-sectional morphology of the lymph nodes was basically consistent, and dewaxed with xylene to repair antigens. The sections were blocked with 10% normal rabbit serum for 30 min, and mature DCs (CD11c, CD80) and CD8+ were scanned according to the procedure. + T-cell (CD8) staining was observed, photographed, and analyzed under a fluorescence microscope.

[0253] 6.1.3 Cytokine Level Analysis

[0254] C57BL / 6 mice were randomly divided into 7 groups of 3 mice each. Each group was inoculated at the base of its tail with C-Lp, Rg3-Lp, LPS, CMø / BO, CMø@BO, CMø@BO+LPS, or GMø@BO (10 mg / kg cholesterol, Rg3, or 1 mg LPS per mouse), for a total of three inoculations, administered weekly. Seven days after the last inoculation, blood was collected from all mice by enucleation, placed in EP tubes, incubated at room temperature for 1 h, and centrifuged at 3000 rpm for 10 min to obtain mouse serum. Lymph nodes were collected after enucleation, and homogenates were added to pre-chilled PBS (5 mL PBS per 1 g sample). The homogenate was centrifuged at 5000 g for 5 min, and the supernatant was collected. The levels of IL-12, TNF-α, and IL-1β in the serum and lymph nodes of each group were measured according to the ELISA kit instructions.

[0255] 6.2 Results and Discussion

[0256] 6.2.1 GMø@BO promotes the maturation of DCs in vivo

[0257] As shown in "5. Study on the Direct Activation Effect of Ginsenoside Membrane Hybrid Tumor Nanovaccine on T Cells", GMø@BO can directly activate T cells as a pseudo-APC, and indirectly activate T cells by targeting DCs through the Rg3 lipid membrane to promote DC maturation and antigen presentation. To verify the level of immune response induced by GMø@BO in vivo under the synergistic effect of these two mechanisms, this invention used flow cytometry to analyze the changes in the types and proportions of DCs and T cells in lymph nodes and peripheral blood of mice after immunization. First, the DC maturation level in vivo after immunization was quantitatively analyzed in each group, and the results are as follows: Figure 35 As shown.

[0258] Compared to C-Lp, Rg3-Lp significantly increased the proportion of mature dendritic cells (DCs) in vivo, exhibiting adjuvant activity. The proportion of mature DCs in lymph nodes and blood was 1.8-fold and 2.0-fold higher in the GMø@BO group than in the CMø@BO group, indicating the self-adjuvant properties of GMø@BO after incorporating the Rg3 lipid membrane. Notably, the level of mature DCs in the GMø@BO group was significantly higher than that in the CMø@BO+LPS group, possibly attributed to GMø@BO enabling targeted co-delivery of the antigen Mø@BO and adjuvant Rg3 in lymph nodes. To further observe the distribution of mature DCs within lymph nodes (LNs), CD11c, CD80 double staining, and CD8 single staining were performed on collected mouse lymph nodes from each group. The confocal imaging results are shown below. Figure 36 As shown.

[0259] In the figure, the yellow signal represents the co-localization of the CD11c green signal and the CD80 red signal, indicating that the yellow signal represents mature dendritic cells (DCs). The significant increase in yellow signal in LNs after GMø@BO immunization indicates that GMø@BO has a significant effect on promoting DC maturation. Furthermore, the distribution of mature DCs in LNs in the figure shows that mature DCs immunized with GMø@BO can migrate to T cell regions for antigen presentation, subsequently promoting CD80 maturation. + T cell proliferation was observed. Consequently, the lymph nodes of the GMø@BO group showed the strongest CD8+ T cell (green) signal.

[0260] 6.2.2 GMø@BO increases the level of antigen-specific T cells in vivo.

[0261] As described above, mature dendritic cells (DCs) migrate to the T cell region of lymph nodes to present antigens, displaying their surface pMHC markers to T cells. Therefore, T cell surfaces exhibit antigen-specific pMHC signals. To explore the levels of antigen-specific T cells in each group after immunization, this invention uses MHC I-OVA as a typical antigenic epitope and analyzes CD8+ in each group. + Flow cytometry was used to detect pMHC signals on the surface of T cells, and the results are as follows: Figure 37 As shown.

[0262] The results showed that the CMø@BO group had antigen-specific CD8 + The T cell level was significantly higher in the Mø@BO group than in the CMø / BO group. This may be because Mø@BO presents more pMHC on its surface, and CMø@BO can act as a pseudo-APC to directly bind to T cells and activate them into antigen-specific CD8. + T cells. Compared to CMø@BO, the GMø@BO group showed antigen-specific CD8+. +The proportion of T cells still increased significantly, which may be due to the lymph node targeting of the Rg3 lipid membrane. GMø@BO, which is hybridized with the Rg3 lipid membrane, can reach more lymph nodes and thus present more pMHC directly or indirectly to T cells.

[0263] 6.2.3 GMø@BO increases the level of cytotoxic T cells in vivo.

[0264] Under the combined action of pMHC-TCR and CD80 / CD86-CD28 signals, antigen-specific T cells are activated. When activated T cells re-examine the antigen, they secrete IFN-γ and granzyme B (Grz B), exerting a tumor-killing effect. Therefore, this invention further investigates the effects of IFN-γ on various groups. + CD8 + T cells and Grz B + CD8 + T cell levels were detected by flow cytometry, and the results were as follows: Figure 38 and 39 As shown.

[0265] Rg3-Lp itself does not induce T cell activation, but only promotes DC maturation, indicating that Rg3-Lp as a vaccine adjuvant is non-immunogenic and has strong safety. Conversely, LPS induces mild T cell activation and has some immunogenicity, posing a safety risk as an adjuvant. Although there was no significant difference in the proportion of mature DCs between the CMø@BO and CMø / BO groups (…),… Figure 39 ), but IFN-γ in the CMø@BO group + CD8 + T cells and Grz B + CD8 + T cell levels were significantly higher in the CMø / BO group than in the CMø / BO group. Figure 38 , Figure 39 This indicates that even without the assistance of mature DCs, CMø@BO, with its antigen self-presentation properties, can still act as a pseudo-APC to directly present antigens to T cells, and the synergistic effect of co-stimulatory molecules on Mø@BO activates T cells to some extent. Notably, compared to CMø@BO, the GMø@BO group showed increased activation of IFN-γ. + CD8 + T cells and Grz B + CD8 + The proportion of T cells was significantly increased because GMø@BO, in addition to directly activating T cells as a pseudo-APC, can also target DCs and promote DC maturation. Mature DCs can transmit pMHC signals and co-stimulatory signals to T cells and secrete corresponding cytokines, further enhancing the activation level of T cells.

[0266] 6.2.4 GMø@BO increases the level of memory T cells in vivo.

[0267] To further explore the long-term immune protection potential of GMø@BO, this invention performed flow cytometry analysis on the levels of memory T cells in peripheral blood and lymphocytes (LNs). The results are as follows: Figure 40 As shown.

[0268] Cytokine signaling contributes to the generation and maintenance of memory T cells. For example, cytokines IL-12 and IL-1β are crucial for the induction and proliferation of memory T cells, and these cytokines are primarily derived from mature dendritic cells (DCs). However, pseudo-APCs relying solely on Mø@BO cannot secrete the relevant cytokines that promote memory T cell differentiation like living APCs. Therefore, memory CD8+ cells in the GMø@BO group... + The level of T cells was 2.38 times that of the CMø@BO group, indicating that Rg3 lipid membrane hybridization can enhance immune memory and that GMø@BO has the potential for long-term immune protection.

[0269] 6.2.5 GMø@BO promotes the secretion of Th1 cytokines

[0270] Subsequently, the levels of IL-12, IL-1β, and TNF-α in the lymph nodes and serum of mice after immunization were measured using ELISA. The results are as follows: Figure 41 As shown. Compared with C-Lp, Rg3-Lp significantly promoted the secretion of IL-12, IL-1β, and TNF-α. This is mainly because Rg3-Lp can act as an adjuvant to promote DC maturation. Mature DCs secrete Th1-type cytokines such as IL-12, IL-1β, and TNF-α, participating in cellular immune responses and promoting T cell differentiation and proliferation. From the previous... Figure 35 It was found that GMø@BO exhibited the strongest effect in promoting DC maturation. Therefore, the GMø@BO group had the highest levels of IL-12, IL-1β, and TNF-α cytokines in LNs and serum, which could effectively induce T cell activation and proliferation.

[0271] 7. Tumor prevention and treatment effects of ginsenoside membrane hybrid tumor nanovaccines

[0272] 7.1 The role of GMø@BO in the prevention and treatment of melanoma

[0273] 7.1.1 Experimental Methods

[0274] 7.1.1.1 Study on the therapeutic effect of GMø@BO on melanoma

[0275] C57BL / 6 mice were randomly divided into 9 groups of 6 mice each, and each group was subcutaneously injected with 5×10⁻⁶ mice. 5B16-OVA cells were inoculated into the lower right back of mice, and tumor growth was observed every two days. On day 10, mice were inoculated at the base of the tail with PBS, Rg3-Lp, LPS, PD-1 antibody (PD-1 antibody, α-PD-1), CMø / BO, CMø@BO, CMø@BO + LPS, GMø@BO, or GMø@BO + α-PD-1 (GMø@BO was injected intramuscularly into the tail first, followed by 10.3 μg α-PD-1 intraperitoneally) (10 mg / kg cholesterol or Rg3 or 1 mg LPS / mouse). Immunization was repeated every week for a total of three immunizations. The length and width of the tumor were recorded every two days, the tumor volume was calculated, and the survival time of mice in each group was recorded.

[0276]

[0277] 7.1.1.2 Study on the long-term immune protective efficacy of GMø@BO against melanoma

[0278] Tumor rechallenge experiments were conducted on tumor-exempt mice that were still alive at the endpoint of the efficacy study of "GMø@BO in the treatment of melanoma". Figure 42 ), that is, 5×10 mmol / L was injected into the tail vein on the 60th day after the initial modeling. 5 A melanoma lung metastasis model was constructed using B16-OVA cells for tumor re-challenge. Mice were euthanized by cervical dislocation after 30 days, and their lungs were removed for photographic observation of metastasis.

[0279] 7.1.1.3 Study on the preventive effect of GMø@BO on melanoma

[0280] C57BL / 6 mice were randomly divided into 8 groups of 6 mice each. Each group was subcutaneously injected with PBS, Rg3-Lp, LPS, CMø / BO, CMø@BO, CMø@BO + LPS, GMø@BO, or GMø@BO + CD8 at the base of the tail. + T-cell neutralizing antibody (CD8 antibody, α-CD8) (10 mg / kg cholesterol or Rg3 or 1 mg LPS / animal). Immunize weekly for a total of 3 weeks, then administer 5×10⁵ T-cell neutralizing antibodies 7 days later. 5 B16-OVA cells were subcutaneously injected into the lower right back of mice. Tumor growth was observed every two days, and the length and width of the tumor were measured. The survival time of mice in each group was recorded. Figure 43 ).

[0281] 7.1.1.4 Security Assessment of GMø@BO

[0282] 7.1.1.4.1 Detection of in vivo cytokine and antibody secretion

[0283] C57BL / 6 mice were randomly divided into 7 groups of 3 mice each. Each group was inoculated at the base of its tail with C-Lp, Rg3-Lp, LPS, CMø / BO, CMø@BO, CMø@BO + LPS, or GMø@BO (10 mg / kg cholesterol or Rg3 or 1 mg LPS per mouse), for a total of three inoculations, administered weekly. On day 7 after the last inoculation, blood was collected from all mice by enucleation, placed in EP tubes, incubated at room temperature for one hour, and centrifuged at 3000 rpm for 10 min to obtain mouse serum. Lymph nodes were collected after enucleation, and homogenized with pre-chilled PBS (5 mL PBS for 1 g sample). The homogenate was centrifuged at 5000 g for 5 min, and the supernatant was collected. The levels of IL-4 and IL-10 in the serum and lymph nodes of each group were detected according to the ELISA kit instructions. Simultaneously, the levels of IgM and IgA antibodies in the serum were detected using IgM and IgA ELISA kits.

[0284] 7.1.1.4.2 Examination of weight changes

[0285] In the efficacy experiment of “6.1.1.2 Study on the long-term immune protection efficacy of GMø@BO against melanoma”, the mice in each group were weighed every two days, and the weight changes of the mice after inoculation with each group of preparations were recorded.

[0286] 7.1.1.4.3 Organ coefficients and H&E staining analysis

[0287] At the endpoint of the efficacy experiment "6.1.1.3 Investigation of the Tumor Prevention Effect of GMø@BO", the heart, liver, spleen, lungs, and kidneys were dissected, weighed, fixed with 4% paraformaldehyde solution, dehydrated in a gradient of ethanol, embedded in paraffin, and sectioned. The paraffin sections of the organs were removed and stained with H&E according to the procedure, observed, photographed, and pathologically analyzed under a fluorescence microscope.

[0288] 7.1.2 Results and Discussion

[0289] 7.1.2.1 GMø@BO has therapeutic effects on melanoma.

[0290] B16-OVA tumor-bearing mice were randomly divided into 9 groups of 6 mice each. Each group was injected with PBS, Rg3-Lp, LPS, α-PD-1, CMø / BO, CMø@BO, CMø@BO + LPS, GMø@BO, or GMø@BO + α-PD-1 at the base of the tail, respectively. Immunization was performed weekly for three consecutive weeks. Tumor growth curves were plotted, and survival time was recorded. Results are as follows: Figure 44 and Figure 45 As shown.

[0291] Throughout the efficacy observation period, the tumor volume of mice in the PBS, LPS, and Rg3-Lp groups increased rapidly, with a median survival of only about 30 days. In contrast, the tumor growth rate of mice in the α-PD-1 and CMø / BO groups decreased, and the median survival increased from 30 days to 37 days. The tumor growth rate of mice in the CMø@BO group was significantly slower compared to the α-PD-1 and CMø / BO groups, and the median survival also increased from 37 days to 43 days (Table 2), indicating that the direct T-cell activation mediated by Mø@BO can enhance the therapeutic effect on tumors.

[0292] When CMø@BO was mixed with the adjuvant LPS, the tumors of two mice completely regressed, indicating that adjuvant-mediated indirect T-cell activation significantly enhanced the immunoprotective effect of Mø@BO. Furthermore, hybridizing Mø@BO with an Rg3 lipid membrane further improved the tumor therapeutic efficacy, resulting in complete tumor regression in four mice with an immunoprotective efficacy of approximately 70%. The tumor growth rate in the remaining two mice was also significantly slower compared to the CMø@BO + LPS group. This is because the Rg3 lipid membrane not only acts as a vaccine adjuvant to mediate indirect T-cell activation, but its hybridization with Mø@BO also mediates lymph node targeting, achieving efficient co-delivery of antigen and adjuvant, maximizing the synergistic effect of direct and indirect T-cell activation. The combination of tumor vaccines and immune checkpoint inhibitors is also a commonly used immunotherapy approach in clinical practice. Notably, when GMø@BO was combined with α-PD-1, the tumor protective efficacy reached 100%, with complete tumor regression in all mice within the group. This is because the primary tumor has already created a tumor immunosuppressive microenvironment. Adding PD-1 antibodies to improve the primary tumor microenvironment can further enhance the tumor treatment efficacy of GMø@BO.

[0293] Table 2. Median survival time of tumor-bearing mice in each group

[0294] 7.1.2.2 GMø@BO has a long-term immune protective effect against melanoma.

[0295] This section further investigates the long-term immunoprotective efficacy of GMø@BO. Mice surviving at the efficacy endpoint of "GMø@BO's therapeutic effect on melanoma" were administered B16-OVA cells via tail vein injection. The long-term anti-tumor immunoprotective effect was evaluated by examining the efficacy of GMø@BO against tumor re-challenge. The results are as follows: Figure 46 As shown.

[0296] The black areas in the lungs of the image represent melanoma metastases that formed in the lungs after tail vein injection of B16-OVA cells. The results show that in unimmunized normal mice (NC group), tail vein injection of B16-OVA cells resulted in rapid colonization and metastasis of the cells in the lungs, with the metastatic lesions rapidly expanding throughout the entire lung. In contrast, two mice in the CMø@BO + LPS group whose tumors had regressed after treatment, although the degree of metastasis was significantly reduced compared to the NC group after B16-OVA cell rechallenge, still showed numerous black metastatic nodules in the lungs. Conversely, surviving mice in the GMø@BO group and the GMø@BO + αPD-1 group showed almost no metastatic nodules in the lungs after B16-OVA rechallenge, indicating that GMø@BO can induce strong immune memory. This is due to the potent memory T cell induction ability of GMø@BO demonstrated by the experimental results in "5.2.4 GMø@BO enhances the level of memory T cells in vivo". Following immunization with GMø@BO, the body produces a large number of memory T cells, which exert a long-lasting immune protective effect when tumor cells re-invade. Therefore, GMø@BO, as a therapeutic vaccine for melanoma, possesses long-term immune protective efficacy.

[0297] 7.1.2.3 GMø@BO has a melanoma prevention effect.

[0298] The melanoma prevention efficacy of GMø@BO was subsequently evaluated. Three immunizations were administered, followed by subcutaneous injection of B16-OVA cells on day 7 post-immunization. The melanoma prevention effect of each liposome was assessed by plotting tumor growth curves and calculating tumor immunity rates in mice. Results are as follows: Figure 47 and Figure 48 As shown.

[0299] like Figure 47 As shown, in addition to its excellent efficacy in treating tumors, GMø@BO can also prevent the occurrence of tumors; the melanoma exemption rate was 100% in mice vaccinated with GMø@BO. Figure 48 It was found that in mice immunized with PBS, LPS, and Rg3, melanoma developed in all mice approximately 10 days after B16-OVA inoculation. Tumors developed in the CMø@BO group approximately 30 days later. However, when CMø@BO was used in combination with LPS, three mice in the group remained tumor-free, achieving a tumor immunity rate of 50%. This demonstrates the important role of adjuvant-mediated indirect T-cell activation in tumor prevention and treatment. Consequently, GMø@BO, incorporating the Rg3 lipid membrane with self-adjuvant and lymph node-targeting effects, exhibited the strongest tumor prevention effect, with no tumors developing in any mice during the treatment period, effectively resisting tumor development and achieving a 100% tumor immunity rate.

[0300] It is worth noting that injecting CD8 neutralizing antibodies depletes CD8 in experimental mice.+ After T cell administration, the immunoprotective effect of GMø@BO essentially disappeared, showing no significant difference compared to the PBS group. Figure 48 This indicates that the antitumor immunoprotective efficacy of GMø@BO mainly depends on CD8. + T cell activation, such as... Figure 48 As shown in Table 3, the prophylactic efficacy of CMø@BO against melanoma was significantly reduced in the absence of the Rg3 lipid membrane, demonstrating the crucial role of the Rg3 lipid membrane as an adjuvant in promoting DC maturation and indirect T cell activation. Although the tumor-preventive effect of CMø@BO was weakened compared to GMø@BO, its efficacy was still superior to CMø / BO or CD8. + The tumor-resistance effect of GMø@BO after T cell depletion may be attributed to the direct T cell activation properties of GMø@BO. Therefore, the superior tumor-preventive efficacy of GMø@BO stems from its activation of potent CD8+. + The immune protective efficacy of T cells is conferred by Mø@BO, which has antigen self-presentation function, and Rg3 lipid membrane, which has self-adjuvant and lymph node targeting efficacy.

[0301] Table 3. Median tumor-free period in each group of tumor-bearing mice

[0302] To further characterize the tumor prevention effects of each group, the survival time of mice after tumor inoculation was statistically analyzed, and the results are as follows: Figure 49 As shown in Table 4.

[0303] Mice in the PBS, LPS, and Rg3-Lp groups all developed tumors around day 10, and the tumors grew rapidly, with a median survival of approximately 30 days. While the CMø@BO group had a tumor clearance rate of 0%, it significantly slowed tumor growth; all mice in this group developed tumors around day 30, extending the median survival to 55 days. This may be because CMø@BO directly activates T cells, delaying tumor development. Mice in the GMø@BO group did not develop tumors throughout the entire treatment period, achieving a 100% tumor clearance rate, and therefore no mice died during treatment. CD8+ in mice... + Even after T cell depletion, GMø@BO immunization failed to prevent tumor development and progression, indicating that the anti-tumor protective efficacy of GMø@BO is mediated by activated CD8+. + T cell-mediated. Therefore, the potent anti-tumor immune response of GMø@BO is the result of the combined effects of direct T cell activation by Mø@BO and indirect T cell activation mediated by the Rg3 lipid membrane.

[0304] Table 4. Median survival time of mice after immunization in each group

[0305] 7.1.2.4GMø@BO has good security.

[0306] 7.1.2.4.1 GMø@BO has a lower risk of inducing inflammation and autoimmune diseases.

[0307] Most vaccines, while inducing cellular immunity, also promote the production of large amounts of Th2 inflammatory factors, such as IL-4 and IL-10, and may induce elevated levels of IgM and IgA, leading to allergic reactions and autoimmune diseases, thus posing a risk to vaccine safety. Therefore, this invention uses ELISA to detect the levels of Th2 inflammatory factors IL-4 and IL-10, as well as antibodies IgM and IgA, to examine the safety of GMø@BO.

[0308] The results showed that no increase in the levels of related inflammatory factors and autoimmune-related antibodies was observed in mice after multiple immunizations with GMø@BO. The levels of IL-4, IL-10, IgM, and IgA in the GMø@BO group were not significantly increased compared to the PBS group. However, in the serum of the CMø@BO + LPS group, the levels of Th2-type inflammation-related cytokines (IL-4 and IL-10) and IgM and IgA antibodies were significantly increased compared to the GMø@BO group. Figure 50 This indicates that LPS, as an adjuvant, carries the potential risk of inducing excessive inflammatory responses and autoimmunity, a finding consistent with previous studies. Therefore, Rg3-Lp, as the prepared GMø@BO, exhibits a higher safety profile compared to LPS.

[0309] 7.1.2.4.2 GMø@BO did not induce weight loss

[0310] This invention monitors the body weight of tumor-bearing mice in each group during treatment, and the results are as follows: Figure 51 As shown in the figure, the body weight of mice did not decrease after immunization with any of the formulations, and their body weight remained stable during the treatment period. This indicates that GMø@BO does not induce weight loss and has a high safety profile.

[0311] 7.1.2.4.3 GMø@BO has organ safety features.

[0312] The H&E staining results of the heart, liver, spleen, lungs, and kidneys of tumor-bearing mice after immunization at the experimental endpoint are as follows: Figure 52 As shown, no obvious organ damage or pathological abnormalities were observed after immunization in any group, demonstrating high organ safety for GMø@BO, which lays the foundation for its clinical application.

[0313] 7.2 Study on the preventive and therapeutic effects of ginsenoside membrane hybrid tumor nanovaccine on triple-negative breast cancer

[0314] 7.2.1 Experimental Methods

[0315] 7.2.1.1 Study on the therapeutic effect of GMø@4T1 on triple-negative breast cancer

[0316] Will contain 5×10 5 A suspension of 4T1 cells was injected into the right lower quadrant mammary fat pad of Balb / c mice to establish a TNBC orthotopic tumor model. Tumor growth was observed every two days. On day 10, TNBC-bearing mice were randomly divided into 9 groups of 6 mice each. Each group was inoculated at the base of its tail with PBS, Rg3-Lp, LPS, α-PD-1, CMø / 4T1, CMø@4T1, CMø@4T1 + LPS, GMø@4T1, or GMø@4T1 + α-PD-1 (10 mg / kg cholesterol or Rg3 or 1 mg LPS / mouse), respectively. Immunization was performed every week for a total of 3 times. Tumor length and width were recorded every two days, tumor volume was calculated, and the survival time of mice in each group was also recorded.

[0317]

[0318] 7.2.1.2 Study on the long-term immune protective efficacy of GMø@4T1 against triple-negative breast cancer

[0319] Tumor rechallenge experiments were conducted on tumor-exempt mice that were still alive at the endpoint of the efficacy study of "GMø@4T1 in the treatment of triple-negative breast cancer" in this section. Figure 53 ), that is, 5×10 mmol / L was injected into the tail vein on the 72nd day after the initial modeling. 5 A TNBC lung metastasis model was constructed using 4T1-Luci cells for tumor re-challenge. Starting from day 7 after injection, in vivo BLI imaging was performed every 5 days to observe the metastasis status. On day 100 after the initial modeling, the mice were sacrificed by cervical dislocation, and the lungs were removed for in vitro photography to count the number of metastatic nodules.

[0320] 7.2.1.3 Study on the preventive effect of GMø@4T1 on triple-negative breast cancer

[0321] Balb / c mice were randomly divided into 8 groups of 6 mice each. Each group was subcutaneously injected with PBS, Rg3-Lp, LPS, CMø / 4T1, CMø@4T1, CMø@4T1 + LPS, GMø@4T1, or GMø@4T1 + α-CD8 (10 mg / kg cholesterol or Rg3 / mouse or 1 mg LPS / mouse). Immunization was performed weekly for a total of 3 times. Seven days after the last immunization, mice containing 5 × 10⁻⁶ α-CD8 molecules were immunized. 5A suspension of 4T1 cells was injected into the right lower quadrant mammary fat pad of mice. Tumor growth was observed every two days, and the length and width of the tumor were measured. On day 28 after tumor inoculation, the mice were sacrificed by cervical dislocation, and the tumors were removed, photographed, and weighed. Figure 54 ).

[0322] 7.2.1.4 Safety Evaluation

[0323] In the pharmacodynamic experiment of "Study on the preventive effect of GMø@4T1 on triple-negative breast cancer", mice in each group were weighed every two days to record the weight changes after inoculation with each formulation. At the end of the pharmacodynamic experiment, the heart, liver, spleen, lungs, and kidneys were dissected, weighed, and organ coefficients were calculated.

[0324] 7.2.2 Results and Discussion

[0325] 7.2.2.1 GMø@4T1 has therapeutic effects on TNBC.

[0326] Given the revealed mechanisms of Rg3 membrane hybrid liposome antigen self-presentation and self-adjuvant action, the in vivo immunoprotective efficacy of GMø@4T1 against TNBC was further evaluated to investigate the universality of this Rg3 membrane hybrid liposome tumor nanovaccine strategy. TNBC-bearing mice were randomly divided into 9 groups of 6 mice each. They were immunized at the base of their tails with PBS, Rg3-Lp, LPS, α-PD-1, CMø / 4T1, CMø@4T1, CMø@4T1 + LPS, GMø@4T1, or GMø@4T1 + α-PD-1, respectively. Immunization was performed weekly for three consecutive weeks. Tumor growth curves were plotted, and survival time was recorded. The results are as follows: Figure 55 and Figure 56 As shown.

[0327] Throughout the efficacy observation period, α-PD-1 and CMø / 4T1 slightly slowed tumor growth in mice, but the effect was not significant, mainly due to the immunosuppressive properties of TNBC. The TNBC tumor growth rate in the CMø@4T1 group was slower compared to the α-PD-1 and CMø / 4T1 groups, and the median survival was extended from 42 days to 49.5 days (Table 5), indicating that Mø@4T1 may self-present TNBC antigens and has the potential to directly activate T cells, thereby improving the efficacy of anti-TNBC immunotherapy.

[0328] Hybridizing Mø@4T1 with the Rg3 lipid membrane further enhanced the therapeutic efficacy of TNBC tumors, achieving complete tumor regression in four mice with an immunoprotective efficacy of approximately 70%, superior to the CMø@4T1 + LPS group. This is because the Rg3 lipid membrane not only acts as a vaccine adjuvant mediating indirect T cell activation, but also mediates targeted co-delivery of antigens and adjuvants after hybridization with Mø@4T1. Notably, while the immunoprotective efficacy of GMø@4T1 increased from 70% to 83% when combined with α-PD-1, it was still lower than the 100% melanoma immunoprotective efficacy of GMø@BO + α-PD-1. This may be because TNBC, as a "cold tumor," exhibits significantly stronger immunosuppression than melanoma, and its responsiveness to α-PD-1 is less pronounced than that of melanoma.

[0329] Table 5. Median survival of TNBC-bearing mice in each group

[0330] 7.2.2.2 GMø@4T1 has long-term anti-TNBC immune protection.

[0331] The experimental results of “GMø@BO enhances the level of memory T cells in vivo” indicate that Rg3 membrane hybrid liposomes can induce the production of memory T cells and induce long-term immune memory. Therefore, this section studies the long-term anti-TNBC immunoprotective efficacy of GMø@4T1. Mice that were still alive at the efficacy endpoint of “GMø@4T1 has a TNBC therapeutic effect” were injected with 4T1-Luci via the tail vein. The efficacy of GMø@4T1 against 4T1-Luci rechallenge was examined by BLI imaging, and its long-term anti-TNBC immunoprotective effect was evaluated. The results are as follows. Figure 57 As shown.

[0332] Bioluminescent signals indicate TNBC metastatic lesions formed after tail vein injection of 4T1-Luci cells. The results showed that unimmunized normal mice (NC group) rapidly metastasized to the lungs after tail vein injection of 4T1-Luci, and all mice died on day 27 post-injection. Figure 57 In contrast, surviving mice in the GMø@4T1 and GMø@4T1 + αPD-1 groups showed weak tumor BLI signal and a significantly lower number of lung nodules after 4T1-Luci rechallenge. Figure 58 This indicates that, similar to GMø@BO, GMø@4T1 can induce immune memory and has long-term immune protection against TNBC.

[0333] 7.2.2.3 GMø@4T1 has a preventive effect against TNBC.

[0334] The TNBC prevention efficacy of GMø@4T1 was subsequently evaluated. Mice underwent three immunizations, and on day 7 post-immunization, 4T1 cells were injected into the right lower quadrant mammary fat pad. Tumor growth curves were plotted, and tumor weight, morphology, and tumor immunity rates at the experimental endpoint were recorded to assess the TNBC prevention effect of each liposome as a tumor vaccine. Results are as follows: Figure 59 and 60 As shown.

[0335] like Figure 60 As shown, four mice vaccinated with GMø@4T1 did not develop TNBC at the end of the experiment, with a tumor exemption rate of 70%. Even when two mice developed TNBC, their growth process was significantly slower compared to other groups, indicating that GMø@4T1 can effectively prevent the occurrence and development of TNBC.

[0336] It is worth noting that injecting CD8 neutralizing antibodies depletes CD8 in experimental mice. + After T cell administration, the immunoprotective effect of GMø@4T1 essentially disappeared, showing no significant difference compared to the PBS group. Figure 59 , Figure 60 This indicates that the antitumor immunoprotective efficacy of GMø@4T1 is primarily dependent on CD8. + T cell activation, such as... Figure 59 As shown in Table 6, the prophylactic efficacy of CMø@4T1 against melanoma was significantly reduced in the absence of the Rg3 lipid membrane, demonstrating the crucial role of the Rg3 lipid membrane as an adjuvant. The tumor prophylactic effect of CMø@4T1 was superior to that of CMø / 4T1 or CD8. + The presence of GMø@4T1 after T cell depletion suggests that GMø@4T1 itself may possess the property of direct T cell activation. Therefore, the remarkable tumor-preventive efficacy of GMø@4T1 stems from its activation of potent CD8+. + The immune protective efficacy of T cells is jointly conferred by the lipid membranes of Mø@4T1 and Rg3, and its mechanism of action is consistent with that of GMø@BO.

[0337] Table 6. Median tumor-free period in each group of tumor-bearing mice

[0338] 7.2.2.4 GMø@4T1 has good safety features.

[0339] The body weight of tumor-bearing mice in each group was monitored during treatment, and their organ coefficients were measured at the experimental endpoint. The results are as follows: Figure 61As shown, the body weight of mice did not decrease after immunization with any of the formulations, and the body weight of mice remained stable during the treatment period. Furthermore, the organ coefficients of tumor-bearing mice in each immunized group were not significantly different from those in the PBS group at the experimental endpoint. GMø@4T1 demonstrated high biocompatibility, laying the foundation for its clinical application.

[0340] 7.3 Therapeutic effect of ginsenoside membrane hybrid tumor nanovaccine on human pancreatic cancer PDX model

[0341] 7.3.1 Experimental Methods

[0342] 7.3.1.1 Establishment of a humanized mouse model of pancreatic cancer patient-derived xenograft (PDX)

[0343] After one week of environmental acclimatization, each NCG mouse was randomly assigned to a group. Freshly resected pancreatic tumor samples from patients were cut into 3–4 mm pieces. 3 Small pieces were subcutaneously implanted on both sides of the back of immunodeficient NCG mice. When the tumor volume grew to approximately 1500 mm... 3 At that time, continuous passaging was performed to establish a stable fourth-generation (P4) patient-derived xenograft model (PDX). Subsequently, approximately 6 × 10⁶ cells were injected via the tail vein. 5 Human peripheral blood mononuclear cells (PBMCs) were introduced into mice carrying PDX. CD45 levels in peripheral blood and various tissues were detected by flow cytometry. + The proportion of white blood cells is used to monitor the remodeling of the human immune system. Circulating human CD45 + Mice with a cell proportion >25% were defined as humanized mice (Hu-NCG) and included in subsequent treatment evaluation after immune reconstitution was confirmed.

[0344] 7.3.1.2 Study on in vivo immunotherapy and tumor immunotherapy of GMø@PDC in pancreatic cancer

[0345] Hu-NCG mice carrying pancreatic cancer PDX received subcutaneous injections at the base of their tails once a week of the following formulations: PBS, tumor lysate (2 mg protein / mouse), CMø@PDC, and GMø@PDC (Rg3 or cholesterol 10 mg / kg, α-PD-1 10.3 μg / mouse). CMø@PDC combined with QS-21 (a natural saponin compound derived from the bark of the soapberry tree) (5 μM / mouse) served as a positive control. Tumor growth and survival were monitored every 4 days. On day 24 post-immunization, blood and tumor tissue were collected from the mice, weighed, and photographed for tumor recording.

[0346] 7.3.2 Research Results

[0347] To further verify the tumor-killing activity of the ginsenoside membrane hybrid nanovaccine under human immune conditions and to shorten the gap between preclinical and clinical trials, this invention evaluated the therapeutic effect of GMø@PDC in humanized mice carrying patient-derived pancreatic cancer xenografts (PDX). Figure 62 The results showed that GMø@PDC achieved significant tumor reduction in all tested mice (100%) on day 21, far superior to the tumor lysate group and the CMø@PDC+QS-21 group (QS21 mice were administered 6.5 μg / kg (calculated based on the clinical human shingles vaccine dose), and were first diluted with enzyme-free water to a 5 mg / mL concentrate, with 0.312 μL of concentrate added to every 1.2 ml of formulation). At the experimental endpoint, the tumor volume in the tumor lysate group and the CMø@PDC+QS-21 group was 13 times and 7 times that of the GMø@PDC group, respectively. More importantly, in the GMø@PDC group, approximately 25% of the pancreatic cancer PDX mice achieved complete tumor clearance at the experimental endpoint.

[0348] Summarize

[0349] This invention, based on the tumor immunomodulatory effects of ginsenosides, further explores their potential as vaccine adjuvants. An ideal adjuvant can enhance the immunogenicity of antigens by promoting dendritic (DC) maturation, but it should not possess immunogenicity itself; otherwise, it would induce non-specific immune responses and produce inflammatory side effects. Therefore, based on the differences in glycosidic bond position, number of glycosyl groups, and R / S configuration of ginsenosides, nine candidate ginsenoside compounds were selected, and their effects on promoting DC maturation and T cell activation were investigated. The results showed that Rg3 was the safest and most effective adjuvant among the nine ginsenosides. It not only had the strongest effect on promoting DC maturation but also did not directly activate T cells, exhibiting strong safety. Therefore, Rg3 was selected as a vaccine adjuvant for subsequent research.

[0350] Tumor antigens are the most important components of tumor vaccines. Due to the heterogeneity of tumors and the limitations of current sequencing technology, the identification and analysis of tumor antigens remain incomplete, posing challenges to antigen design for tumor vaccines. This also results in a severe shortage of antibodies that can detect tumor antigen epitopes, making it difficult to analyze vaccine immune mechanisms (antigen presentation efficiency, antigen-specific immune response level detection, etc.). This invention employs a top-down biomimetic technique, extracting macrophage membranes that self-present various antigens after phagocytizing and processing tumor cells as the antigen source for tumor vaccines, thus avoiding the difficulties of tumor antigen identification. Currently, the most common commercially available tumor antigen epitope antibody is the model antigen OVA-MHCⅠ antigen epitope, namely the SIINFEKL–H2kb antibody. Therefore, to further investigate the presentation efficiency of tumor antigens by macrophages after phagocytizing tumor cells, the commonly available commercially available melanoma B16-OVA cells expressing OVA were used as a model.

[0351] First, low-dose PTX was used to induce ICD effects in B16-OVA, promoting CRT translocation, HMGB1 release, and ATP secretion. This released "eat me" and "find me" signals to macrophages, promoting macrophage activation and phagocytosis of B16-OVA. Mø@BO, the macrophage membrane after phagocytosis of B16-OVA cells, was extracted. SDS-PAGE, proteomic analysis, and Western blotting revealed that Mø@BO presents multiple tumor antigen epitopes pMHC, including SIINFEKL–H2kb. Simultaneously, the expression of co-stimulatory molecules CD80 / CD86 and migration molecules was upregulated on Mø@BO, indicating its potential as a pseudo-APC to directly activate T cells. GMø@BO was synthesized by hybridizing the Mø@BO biomembrane with an Rg3 lipid membrane using a membrane hydration-extrusion method, achieving efficient co-delivery of tumor antigens and adjuvants. Fret and confocal co-localization experiments confirmed the successful fusion of the Rg3 lipid membrane and Mø@BO. Physicochemical characterization results show that GMø@BO is a spherical vesicle structure with a uniform and stable particle size of approximately 100 nm, and has stronger particle size stability than CMø@BO during storage.

[0352] pMHC-TCR signaling and co-stimulatory signals are both indispensable for T cell activation. Since the Mø@BO sample presented multiple pMHCs and its co-stimulatory molecules were upregulated, Mø@BO has the potential to directly activate T cells as a pseudo-APC. Flow cytometry and confocal microscopy observations demonstrated that CMø@BO and GMø@BO can interact with T cells, presenting pMHCs from Mø@BO to CD8 cells. + T lymphocytes provide activation signals to T cells, which, under the combined action of co-stimulatory molecules, directly promote T cell activation.

[0353] The cytokines secreted by mature dendritic cells (DCs) are known as the third signal for T cell activation, crucial for promoting T cell differentiation, proliferation, and expanding the immune cascade. Tumor antigens cannot effectively induce DC maturation, requiring adjuvants. Flow cytometry, CLSM, and DC surface pattern recognition receptor (DCR) knockdown experiments have demonstrated that GMø@BO can target DCs by interacting with the overexpressed DCR on DCs via its hybrid Rg3 lipid membrane. DCs can then transfer more pMHC from GMø@BO to their surface, providing a signal to T lymphocytes. Simultaneously, Rg3 interaction with DCR triggers subsequent pathway activation, inducing DC maturation and expression of co-stimulatory molecules, providing a signal. ELISA experiments have shown that mature DCs secrete high levels of Th1 cytokines IL-12, TNF-α, and IL-1β, providing a signal. Therefore, GMø@BO indirectly provides the three signals for T cell activation by acting on DCs, maximizing the activation and proliferation of antigen-specific T cells.

[0354] Because Mø@BO in GMø@BO can directly interact with T cells and its hybrid Rg3 lipid membrane has DC-targeting properties, GMø@BO showed the strongest lymph node aggregation in small animal in vivo imaging experiments, while showing no significant accumulation in other organs. Flow cytometry, immunofluorescence staining, and CLSM imaging demonstrated that GMø@BO, which combines Rg3 lipid membrane and Mø@BO, is mainly distributed in lymph nodes among DCs and T cells, exhibiting the strongest lymph node targeting efficacy.

[0355] LNs are the germinal centers of the immune response, and GMø@BO, upon reaching the lymph nodes, can induce a series of immune responses. Using the cytokine adjuvant LPS as a positive control, flow cytometry analysis of LNs and peripheral blood in mice immunized with C-Lp, Rg3-Lp, LPS, CMø / BO, CMø@BO, CMø@BO + LPS, or GMø@BO was performed. Cytokine ELISA assays and immunohistochemical staining of LN sections were also conducted to qualitatively and quantitatively examine the level of the in vivo immune response induced by GMø@BO. The results showed that GMø@BO maximally promotes the maturation of dendritic cells (DCs). Mature DCs, while providing pMHC and co-stimulatory signals to T cells, secrete cytokines IL-12, IL-1β, and TNF-α, thereby promoting T cell differentiation, activation, and proliferation, maximizing the immune response.

[0356] The in vitro and in vivo efficacy of GMø@BO against melanoma was then evaluated. In vitro IFN-γ ELISPOT, B16-OVA cell apoptosis, and LDH assays showed that GMø@BO-immunized mouse lymphocytes exposed to B16-OVA cell-associated antigen secreted large amounts of IFN-γ, promoting melanoma cell apoptosis and exhibiting potent tumor-killing activity. In vivo melanoma treatment, rechallenge, and prevention experiments showed that, as a therapeutic vaccine, the combination of GMø@BO and α-PD-1 resulted in a 100% melanoma regression rate, with therapeutic efficacy far exceeding that of GMø@BO + LPS. Furthermore, mice immunized with GMø@BO exhibited long-term anti-melanoma immunoprotective efficacy, resisting B16-OVA cell rechallenge. As a prophylactic vaccine, GMø@BO also prevented the occurrence of melanoma; almost no tumors formed in mice immunized with GMø@BO after B16-OVA cell injection. Therefore, the tumor nanovaccine constructed by hybridizing the antigen-presenting Mø@BO biomembrane with the Rg3 lipid membrane into the same system exhibits potent melanoma prevention and treatment effects. In vivo safety experiments demonstrate that GMø@BO is far safer than LPS-type vaccines; it does not induce the secretion of Th2-type inflammatory factors IL-4 and IL-10, nor does it cause weight loss or organ toxicity, demonstrating strong safety.

[0357] The above experiments, using GMø@BO as a typical example, successfully revealed the antigen self-presentation and self-adjuvant mechanism of Rg3 membrane hybrid liposomes. However, melanoma is a highly immunoreactive tumor. To further verify the effectiveness of this ginsenoside membrane hybrid tumor nanovaccine strategy against cold tumors, represented by TNBC, a macrophage membrane hybrid liposome Mø@4T1-Rg3 lipid membrane (GMø@4T1) was constructed following the same preparation procedure as GMø@BO, and its TNBC prevention and treatment efficacy was investigated. TNBC treatment and prevention experiments showed that GMø@4T1 achieved a TNBC protection efficiency of 70%, far exceeding that of CMø@4T1 + LPS. Furthermore, mice immunized with GMø@4T1 exhibited long-term anti-TNBC immunoprotective efficacy. Therefore, this ginsenoside membrane hybrid tumor nanovaccine strategy is not only suitable for highly immunoreactive melanoma but can also effectively prevent and treat "cold tumors" such as TNBC, demonstrating broad application prospects.

[0358] In summary, this invention develops a novel tumor nanovaccine with both antigen self-presentation and self-adjuvant efficacy. The antigen-self-presenting macrophage membrane, obtained using a biomimetic strategy, can directly activate T cells. Simultaneously, the Rg3 lipid membrane can target lymph nodes, promote DC maturation, and indirectly activate T cells and amplify the immune cascade after hybridization with the antigen-self-presenting macrophage membrane. Through the synergistic effect of both, the ginsenoside membrane-hybrid tumor nanovaccine exhibits excellent and long-lasting tumor prevention and treatment effects. Given the ability to manipulate the phagocytosis of various tumor cells or pathogens by macrophages in vitro, this study provides a universal and effective strategy for the prevention and treatment of different tumors or other diseases, avoiding the difficulties of individual antigen isolation and identification, as well as the safety issues associated with traditional adjuvants and live cell vaccines.

Claims

1. A ginsenoside membrane hybrid tumor nanovaccine, characterized in that, The ginsenoside membrane hybrid tumor nanovaccine comprises a fusion membrane formed by macrophage membranes and lipid membranes; wherein: The tumor antigen peptide-MHC complex binds to the surface of the macrophage membrane; The lipid membrane comprises ginsenosides and phospholipids.

2. The ginsenoside membrane hybrid tumor nanovaccine as described in claim 1, characterized in that, The ginsenoside membrane hybrid tumor nanovaccine meets one or more of the following conditions (1)-(4): (1) The tumor antigen peptide-MHC complex is derived from the cell membrane of the antigen-presenting cell after recognizing the tumor antigen; the tumor antigen is preferably selected from one or more tumor cell types, including melanoma cells, triple-negative breast cancer cells, pancreatic cancer cells, non-small cell lung cancer cells, prostate cancer cells, and colorectal cancer cells, as tumor-specific antigens or tumor-associated antigens. (2) The macrophage membrane is obtained by extracting macrophages after they have phagocytosed tumor cells; Preferably, the tumor cells are tumor cells that produce an ICD-induced effect; (3) The macrophage membrane is derived from human macrophages and / or mouse macrophages; and, (4) The macrophage membrane is derived from macrophages, or bone marrow hematopoietic stem cells, myeloid progenitor cells, monocytes, premonocytes, monocytes or macrophages differentiated from them; the macrophages are preferably M0 type macrophages; and / or, the macrophages are preferably THP-1 cells.

3. The ginsenoside membrane hybrid tumor nanovaccine as described in claim 1 or 2, characterized in that, The ginsenoside membrane hybrid tumor nanovaccine meets the following conditions (1) and / or (2): (1) The ginsenosides are selected from one or more of ginsenoside Rg3, ginsenoside Rb1, ginsenoside Rf, ginsenoside Rd, ginsenoside PPT, ginsenoside Rg1, and ginsenoside Rh1, preferably ginsenoside Rg3; and, (2) The phospholipids include natural phospholipids and / or synthetic phospholipids; The natural phospholipids preferably include one or more of natural lecithin, soybean lecithin, egg yolk lecithin, and cephalin; The synthetic phospholipids preferably include one or more of phosphatidylcholine, phosphatidylglycerol, phosphatidylinositol, phosphatidylethanolamine, phosphatidylserine, and polyethylene glycol-modified phospholipids. Preferably, the phospholipid is egg yolk lecithin and / or soybean lecithin.

4. The ginsenoside membrane hybrid tumor nanovaccine as described in any one of claims 1-3, characterized in that, The ginsenoside membrane hybrid tumor nanovaccine meets the following conditions (1) and / or (2): (1) The mass ratio of membrane proteins in the macrophage membrane to phospholipids in the lipid membrane is 1:(1.25-60), preferably 1:(2-60), for example 1:(10-60), and further for example 1:20; and, (2) The mass ratio of ginsenosides to phospholipids is 1:(1.5-30), for example, 1:

4.

5. The ginsenoside membrane hybrid tumor nanovaccine according to any one of claims 1-4, characterized in that, In the ginsenoside membrane hybrid tumor nanovaccine, the fusion membrane exists in a vesicle structure; preferably, the ginsenoside membrane hybrid tumor nanovaccine satisfies one or more of the following conditions (1)-(3): (1) The hydrated particle size of the vesicle is 90-150 nm; (2) The PDI of the vesicles is 0.1-0.2; and, (3) The zeta potential of the vesicle is -20mV to -30mV.

6. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises an immune checkpoint inhibitor and a ginsenoside membrane hybrid tumor nanovaccine as described in any one of claims 1-5.

7. The pharmaceutical composition according to claim 6, characterized in that, The immune checkpoint inhibitors include PD-1 / PD-L1 inhibitors and / or CTLA-4 inhibitors; wherein, the PD-1 / PD-L1 inhibitors preferably include α-PD-1 antibodies; And / or, the pharmaceutical composition may further include pharmaceutically acceptable excipients or carriers.

8. The use of a ginsenoside membrane hybrid tumor nanovaccine as described in any one of claims 1-5, or a pharmaceutical composition as described in claim 6 or 7, in the preparation of a medicament for immunoprophylaxis and / or immunotherapy.

9. The application as described in claim 8, characterized in that, The immunoprophylaxis and / or immunotherapy are for the treatment of tumors, viral infections, or bacterial infections. Preferably, the tumor includes melanoma, breast cancer such as triple-negative breast cancer, pancreatic cancer, lung cancer such as non-small cell lung cancer, prostate cancer, or colorectal cancer.

10. A method for preparing a ginsenoside membrane hybrid tumor nanovaccine as described in any one of claims 1-5, characterized in that, The method includes the following steps: S1. Incubate tumor cells that induce ICD effect with macrophages, and extract the cell membrane of the macrophages to obtain the macrophage membrane; S2. Ginsenosides and phospholipids are self-assembled to obtain the lipid membrane; S3. The macrophage membrane and the lipid membrane are fused to obtain the ginsenoside membrane hybrid tumor nanovaccine.

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