Double-targeted cancer cell membrane nanovaccine activated by ginsenoside Rk1 and preparation method and application thereof

Abstract

The present application relates to the technical field of biological medicine engineering, and particularly relates to a ginsenoside Rk1-activated double-targeted cancer cell membrane nano vaccine and a preparation method and application thereof. The present application performs pharmacological regulation on cancer cells through Rk1, significantly remodels the membrane surface antigen spectrum of the cancer cells, enhances the expression of tumor-associated antigens (TAA), and down-regulates the immune inhibitory molecule CD47 and up-regulates the antigen-presenting molecule MHC-I. CpG oligodeoxynucleotides are loaded on a zeolitic imidazolate framework material ZIF-8, and then coated with cancer cell membranes to form a core-shell structure, so as to realize precise and acid-sensitive release. The nano vaccine can significantly improve the uptake and activation efficiency of dendritic cells (DC) on antigens, and enhance the immune effect of CD8+ T cells and the formation of immune memory. Animal experiments show that the nano vaccine has a tumor inhibition rate of about 80% in a 4T1 mouse breast cancer model, and has good immune activation ability and anti-tumor efficiency.

Description

Technical Field

[0001] This invention relates to the field of biomedical engineering technology, specifically to a dual-targeting cancer cell membrane nanovaccine activated by ginsenoside Rk1, its preparation method, and its application. Background Technology

[0002] In recent years, tumor immunotherapy (such as immune checkpoint inhibitors (ICIs) and chimeric antigen receptor T-cell therapy (CAR-T)) has demonstrated significant efficacy in various malignant tumors, particularly achieving breakthroughs in melanoma and lung cancer. However, for solid tumors such as triple-negative breast cancer (TNBC), the overall response rate of ICIs remains below 30%, and the penetration and persistence of cell therapies like CAR-T in solid tumors are limited by tumor microenvironment (TME) suppression. Their common weakness lies in their high dependence on a single antigen target, making it difficult to address the complex and heterogeneous spectrum of tumor antigens. Therefore, developing therapeutic strategies that can activate a broad spectrum of antigens and enhance dendritic cell (DC) uptake and T-cell activation has become an important direction for the development of next-generation tumor vaccines.

[0003] Among numerous antigen delivery carriers, tumor cell membranes (CCMs) are considered ideal materials for tumor vaccines due to their natural ability to carry multiple tumor-associated antigens (TAAs). However, unactivated CCMs still suffer from significant deficiencies in antigen presentation efficiency and immune activation capacity. Traditional methods of inducing membrane antigen expression through chemotherapy drugs (such as DOX) are often limited to single immune-related proteins such as CRTs, resulting in limited activation levels and making it difficult to construct multi-target, potent vaccine systems. In recent years, studies have proposed using natural products to precisely regulate tumor membranes to achieve antigen profile reconstruction and immune pathway activation. Among these, ginsenosides, as natural active triterpenoids, exhibit excellent multi-target immune regulation capabilities and are expected to simultaneously influence antigen-presenting factors (such as MHC-I) and "don't swallow me" signals (such as CD47), providing a novel pathway for constructing multifunctional vaccines. Therefore, exploring the regulation of tumor cell membrane antigen profiles and immune pathways by natural products, and constructing nanovaccine systems with both broad-spectrum antigen presentation and dual-target regulation capabilities, has significant theoretical value and clinical application potential. Summary of the Invention

[0004] The technical problem to be solved by this invention is to provide a dual-target cancer cell membrane nanovaccine activated by ginsenoside Rk1, which addresses the problems of narrow antigen spectrum, weak immunogenicity and poor targeting of existing cancer vaccines, as well as its preparation method and application.

[0005] The present invention specifically adopts the following technical solution:

[0006] In a first aspect, the present invention provides a dual-target cancer cell membrane nanovaccine activated by ginsenoside Rk1, which is a core-shell structured nanovaccine formed by using the tumor cell membrane activated by ginsenoside Rk1 as the shell and an acid-sensitive degradable metal-organic framework material loaded with an immune adjuvant as the shell.

[0007] Furthermore, the tumor cells include breast cancer cells.

[0008] Furthermore, the immune adjuvant includes CpG oligodeoxynucleotides.

[0009] Furthermore, acid-sensitive degradable metal-organic framework materials include the zeolite imidazole ester framework material ZIF-8.

[0010] This invention's nanovaccine utilizes Rk1 to induce in vitro "immunogenic remodeling" of exogenous cancer cell membranes, systematically upregulating the expression of multiple tumor-associated antigens and MHC-I, while simultaneously blocking the "don't eat me" signal CD47, significantly enhancing the recognition and phagocytic efficiency of dendritic cells (DCs). The vaccine core co-encapsulates the TLR9 agonist CpG and the acid-sensitive degradable metal-organic framework material ZIF-8, constructing a core-shell nanostructure that combines high drug loading capacity, blood stability, and targeted drug release from the tumor microenvironment. After in vivo inoculation, this vaccine efficiently drives DC uptake and maturation, significantly expands and infiltrates intratumorally CD8⁺ cytotoxic T cells, induces potent anti-tumor effects and long-term immune memory, achieving complete eradication of tumor cells.

[0011] Secondly, the present invention provides a method for preparing a ginsenoside Rk1-activated dual-target cancer cell membrane nanovaccine, comprising the following steps:

[0012] S1. Culture tumor cells to a density of 80%-90%, add ginsenoside Rk1 solution, culture for 18-20 hours, then lyse the cells, discard the cell nucleus precipitate in the lysate, retain the cell membrane components, and then perform particle size reduction and homogenization treatment to obtain tumor cell membranes with uniform particle size and stable morphology.

[0013] S2. Mix the acid-sensitive degradable metal-organic framework material with the immune adjuvant in PBS buffer and incubate under ice bath conditions for 10-12 hours. The resulting precipitate is the acid-sensitive degradable metal-organic framework material loaded with the immune adjuvant.

[0014] S3. The tumor cell membrane is mixed with an acid-sensitive degradable metal-organic framework material loaded with an immune adjuvant in an equal mass ratio, and then coated by ultrasonic treatment to obtain the nanovaccine.

[0015] Furthermore, the concentration of ginsenoside Rk1 solution was 60-100 μg / mL, and the culture time was 18-20 h.

[0016] Furthermore, acid-sensitive degradable metal-organic framework materials and immune adjuvants are incorporated into PBS buffer, with a mass ratio of metal-organic framework materials to immune adjuvants of 25:5~7.

[0017] Thirdly, the present invention provides the application of the aforementioned ginsenoside Rk1-activated dual-target cancer cell membrane nanovaccine in the preparation of antitumor drugs.

[0018] Compared with the prior art, the present invention has the following advantages:

[0019] 1. This invention activates cancer cell membranes through ginsenoside Rk1, systematically upregulates multiple tumor-associated antigens (such as GPA33, EpCAM, ERBB2, etc.) and immune recognition molecules (MHC-I), constructs a broad-spectrum antigen library, and effectively enhances the vaccine's adaptability to tumor heterogeneity and its ability to elicit a strong immune response.

[0020] 2. In the preparation method of the present invention, ginsenoside Rk1 induces downregulation of the immunosuppressive molecule CD47, blocks the "don't swallow me" signaling axis (CD47-SIRPα), and promotes the phagocytosis of vaccine particles by dendritic cells (DCs); at the same time, it upregulates the expression of MHC-I molecules, enhances the activation and infiltration ability of CD8⁺ T cells, and achieves dual targeted regulation of APCs and T cells.

[0021] 3. In the preparation method of this invention, CpG is loaded into the zeolite imidazole ester framework material ZIF-8, and then coated with the Rk1-activated cancer cell membrane to form a core-shell nanovaccine; CpG and Zn are reacted in the acidic tumor microenvironment. 2+ Rapid release of the substance precisely activates DCs, while the outer shell of the cancer cell membrane enhances particle stability and targeting. Attached Figure Description

[0022] Figure 1 This is the structural diagram of ginsenoside Rk1.

[0023] Figure 2 The expression of MHC-I (a) and CD47 (b) in the tumor cell membrane activated by ginsenoside Rk1 obtained in step one of Example 1.

[0024] Figure 3 This is a transmission electron microscope image of the tumor cell membrane activated by ginsenoside Rk1 obtained in step two of Example 1. Figure 4 This is a transmission electron microscope (TEM) image of the ZIF-8 particles obtained in step three of Example 1.

[0025] Figure 5 This is a transmission electron microscope image of the dual-target cancer cell membrane nanovaccine activated by ginsenoside Rk1 in step five of Example 1.

[0026] Figure 6The images show the infrared (a) and XRD (b) test patterns of the ZIF-8 particles in step three of Example 1 and the ZIF-8 loaded with CpG in step four.

[0027] Figure 7 This is a diagram showing the expression of membrane proteins in the ginsenoside Rk1-activated dual-target tumor cell membrane nanovaccine prepared in Example 1.

[0028] Figure 8 The expression diagram of CPG in the ginsenoside Rk1-activated dual-target tumor cell membrane nanovaccine prepared in Example 1 is shown.

[0029] Figure 9 The images show quantitative diagrams (a) and (b) of the dual-target tumor cell membrane nanovaccine activated by ginsenoside Rk1 prepared in Example 1, which are used to activate dendritic cells.

[0030] Figure a shows the activation of dendritic cells by the ginsenoside Rk1-activated dual-target tumor cell membrane nanovaccine prepared in Example 1.

[0031] Figure b shows the activation of CTLL-2 cytotoxic T cells in the ginsenoside Rk1-activated dual-target tumor cell membrane nanovaccine prepared in Example 1.

[0032] Figure 10 The figure shows the inhibition of tumor growth by the dual-target tumor cell membrane nanovaccine activated by ginsenoside Rk1 prepared for the example. Detailed Implementation

[0033] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0034] The structural formula of ginsenoside Rk1 in the following examples is as follows: Figure 1 As shown, the CAS number is 494753-69-4, purchased from the Institute of Biology and Medicine, Northwest University; Ginsenoside Rk1 is a rare dammarane-type triterpenoid saponin with a ginsenoside diol-type core structure, two glucose residues linked at the C-3 position, side chains containing C20–C21 and C24–C25 double bonds, and hydroxyl groups at the C-3 and C-12 positions.

[0035] The sequence of CpG oligodeoxynucleotides: 5′-TCCATGACGTTCCTGACGTT-3′, purchased from Shanghai Sangon Biotech Co., Ltd.; 4T1 cells purchased from Shanghai Gaining Biotechnology Co., Ltd.; PE-Cyanine5-CD80, FITC-CD86, and APC-CD69 purchased from BioLegend; zinc nitrate hexahydrate (Zn(NO3)2·6H2O), CAS No. 10196-18-6, purchased from Maclean's Reagent Company; dimethylimidazole, CAS No. 693-98-1, purchased from Maclean's Reagent Company.

[0036] Example 1

[0037] This embodiment provides a method for preparing a dual-targeting cancer cell membrane nanovaccine activated by ginsenoside Rk1, comprising the following steps:

[0038] Step 1: Transfer 4T1 breast cancer cells to 10 cm diameter culture dishes and culture until the density reaches 80%-90%. Add a 100 μg / mL solution of ginsenoside Rk1 and continue culturing for 18 hours. Collect the cells by centrifugation at 1000 rpm and wash 2-3 times with PBS. Figure 2 The results showed that ginsenoside Rk1 activated the expression of CD47 and MHC-I in the membrane of breast cancer cells.

[0039] Step 2: Resuspend the cells collected in Step 1 in a hypotonic lysis buffer containing the following components: 20 mM Tris hydrochloric acid buffer (pH 7.4), 10 mM potassium chloride, 2 mM magnesium chloride, and an EDTA-free protease inhibitor mixture. This results in 20 mL of Tris hydrochloric acid buffer containing 14.91 mg of potassium chloride and 3.81 mg of magnesium chloride. Under ice bath conditions, sonicate the cell suspension 25 times at 10% amplitude (1 second shaking, 5 second interval) to fully lyse the cells while maintaining membrane integrity. Centrifuge the lysate at 5000 × g for 10 minutes at 4°C, discard the nuclear pellet, and collect the supernatant. Then centrifuge the supernatant at 16000 × g for 30 minutes and collect the pellet, which is the cell membrane component. The obtained membrane precipitate was resuspended in 5 mL of PBS, and polycarbonate membranes with pore sizes of 400 nm and 200 nm were sequentially mechanically extruded through a small nanoextruder (extrusion at least 17 times per pore size), ultimately forming tumor cell membrane nanovesicles with uniform particle size and stable morphology (denoted as G-CCM). Figure 3 The transmission electron microscopy image shown reveals that tumor cell membrane nanovesicles have a clearly visible hollow cell membrane structure.

[0040] Step 3: At room temperature, dissolve 9.7 g of 2-methylimidazole in 20 mL of pure water, and then dissolve 0.5 g of zinc nitrate in 30 mL of pure water. Slowly add the zinc nitrate solution to the 2-methylimidazole solution and stir at 500 rpm for 8 hours to form a white emulsion. Centrifuge the obtained liquid at 10,000 rpm for 10 minutes to collect the precipitate. Dissolve the precipitate completely with ethanol by sonication, then centrifuge at 10,000 rpm for 10 minutes to collect the precipitate. Wash the sample twice with ethanol and collect the precipitate. Finally, dry the sample overnight in a vacuum oven at 40°C. After drying, collect the solid, which is the ZIF-8 zeolite imidazole ester framework material. Figure 4 The transmission electron microscope image shown indicates that the ZIF-8 particles are uniformly distributed, structurally intact, and irregularly distributed.

[0041] Step 4: Add the ZIF-8 obtained in Step 3 to PBS to form a 0.5 mg / mL solution. Mix ZIF-8 and CpG at a ratio of 25:7, and incubate overnight on ice at 800 rpm. Centrifuge the resulting liquid at 10,000 rpm for 10 minutes and collect the precipitate, which is the CpG-loaded ZIF-8 (denoted as ZIF-CPG or ZC).

[0042] Step 5: The tumor cell membrane activated by ginsenoside Rk1 obtained in Step 2 is coated with CpG-loaded ZIF-8 obtained in Step 4 to form a core-shell structure of CpG-loaded ZIF-8-encapsulated ginsenoside-activated cancer cell membrane nanovaccine (ZC@G-CCM). The coating method is ultrasound. The two are mixed in a 1:1 mass ratio and a 10W ultrasound therapy device is used in a 2-minute on / 2-minute off cycle for a total of 4 cycles. Finally, the liquid is collected, centrifuged at 10,000 rpm for 10 minutes, and the precipitate is collected as shown in the figure. Figure 5 The transmission electron microscope image shown indicates that ZC@G-CCM has a clearly visible, complete core-shell structure.

[0043] Figure 6 Figure a shows the infrared spectra of ZIF-8 particles and their CpG-loaded counterparts. In the low wavenumber region, the characteristic absorption peaks of the Zn–N bond are clearly visible, indicating that the coordination structure between zinc and the imidazole ligand in ZIF-8 remains stable and has not changed significantly due to CpG loading. In the mid-wavenumber region, the characteristic vibrational peaks of the C–N bond are also preserved, which is typical of the imidazole ring structure. These results demonstrate that even after CpG modification, the core framework structure of ZIF-8 remains intact, and its chemical nature is not destroyed; ZIF-CPG successfully preserves the structural characteristics of ZIF-8.

[0044] Figure 6Figure b presents the X-ray diffraction (XRD) patterns of ZIF-8 particles and their CpG-loaded forms. The diffraction peaks of the 011, 002, 112, 013, and 222 crystal planes are clearly marked. These peaks are typical characteristics of the ZIF-8 crystal structure, reflecting its highly ordered crystal structure. After CpG loading, these characteristic peaks remain and their positions are essentially unchanged, indicating that ZIF-CPG not only retains the basic structural framework of ZIF-8 but also maintains the integrity of its crystal structure. This further confirms that the CpG loading process did not damage the crystal structure of ZIF-8, and that ZIF-CPG is structurally highly consistent with ZIF-8, providing a solid structural foundation for subsequent application research. Figure 7 The expression of membrane proteins in a ginsenoside Rk1-activated dual-target tumor cell membrane nanovaccine was depicted. In the electrophoresis images, neither ZIF-8 nor ZC showed obvious protein bands, indicating that these two materials themselves do not contain protein components. In contrast, G-CCM (ginsenoside Rk1-activated tumor cell membrane) exhibited a unique protein distribution pattern, with multiple characteristic protein bands clearly visible in its electrophoresis image, reflecting the rich membrane protein composition of G-CCM.

[0045] More importantly, the electrophoresis results of ZC@G-CCM (a composite of ZC and G-CCM) show a protein band combination highly consistent with that of G-CCM, while retaining the characteristics of ZC. This result clearly demonstrates that the ZC@G-CCM composite successfully integrates the components of both ZC and G-CCM, preserving not only the integrity of the unique membrane proteins in G-CCM but also ensuring the structural properties of ZC. This successful integration provides strong experimental evidence for constructing tumor cell membrane nanovaccines with dual targeting functions, proving the effectiveness and feasibility of this composite material in nanovaccine design.

[0046] Figure 8 The expression of CpG in a dual-target tumor cell membrane nanovaccine activated by ginsenoside Rk1 was demonstrated. In the electrophoresis image, ZIF-8 did not show a DNA band, indicating that it does not contain DNA. In contrast, CpG, ZC, and ZC@G-CCM all showed clear DNA bands.

[0047] This result clearly confirms the successful loading of CpG. Specifically, the DNA bands of CpG itself are clearly identifiable, indicating that it contains a specific DNA sequence. DNA bands consistent with CpG were also observed in ZC and ZC@G-CCM, which not only demonstrates the successful loading of CpG onto ZC but also further indicates that the CpG DNA bands remain stable in the ZC@G-CCM composite material, suggesting that CpG was not lost or degraded during the composite material preparation process. This discovery provides crucial experimental evidence for constructing dual-targeting tumor cell membrane nanovaccines with immune-activating functions, demonstrating the stability and functionality of CpG in nanovaccine systems.

[0048] Example 2

[0049] This embodiment provides a method for preparing a dual-targeting cancer cell membrane nanovaccine activated by ginsenoside Rk1, comprising the following steps:

[0050] Step 1: Transfer 4T1 breast cancer cells to T75 culture dishes and culture until the density reaches 80%-90%. Add ginsenoside Rk1 solution at a concentration of 80 μg / mL and continue culturing for 20 hours. Collect cells by centrifugation at 1000 rpm and wash 2-3 times with PBS.

[0051] Step 2: Resuspend the cells collected in Step 1 in a hypotonic lysis buffer containing the following components: 20 mM Tris hydrochloric acid buffer (pH 7.4), 10 mM potassium chloride, 2 mM magnesium chloride, and an EDTA-free protease inhibitor mixture (i.e., 50 mL Tris hydrochloric acid buffer containing 37.28 mg potassium chloride and 9.52 mg magnesium chloride). Under ice bath conditions, sonicate the cell suspension 25 times at 20% amplitude (1 second shaking, 5 second interval) to fully lyse the cells while maintaining membrane integrity. Centrifuge the lysis buffer at 5000 × g for 10 minutes at 4°C, discard the nuclear pellet, and collect the supernatant. Then centrifuge the supernatant at 16000 × g for 30 minutes and collect the pellet, which is the cell membrane component. The obtained membrane precipitate was resuspended in 5 mL PBS, and polycarbonate membranes with pore sizes of 800 nm, 400 nm and 200 nm were mechanically extruded sequentially through a small nano-extruder (extrusion at least 17 times per pore size) to finally form tumor cell membrane nanovesicles with uniform particle size and stable morphology.

[0052] Step 3: At room temperature, dissolve 29.1 g of 2-methylimidazole solution in 60 mL of pure water, and then dissolve 1.5 g of zinc nitrate in 90 mL of pure water. Slowly add the zinc nitrate solution to the 2-methylimidazole solution and stir at 500 rpm for 8 hours to form a white emulsion. Centrifuge the obtained liquid at 10,000 rpm for 10 minutes to collect the precipitate. Dissolve the precipitate completely with ethanol by sonication, then centrifuge at 10,000 rpm for 10 minutes to collect the precipitate. Wash the sample twice with ethanol and collect the precipitate. Finally, dry the sample overnight in a vacuum drying oven at 40°C. After drying, collect the solid, which is the ZIF-8 zeolite imidazole ester framework material.

[0053] Step 4: Add the ZIF-8 obtained in Step 3 to PBS to form a 0.5 mg / mL solution. Mix ZIF-8 and CpG at a ratio of 25:7, and incubate overnight on ice at 800 rpm. Centrifuge the resulting liquid at 10,000 rpm for 10 minutes and collect the precipitate, which is the CpG-loaded ZIF-8.

[0054] Step 5: The tumor cell membrane activated by ginsenoside Rk1 obtained in Step 2 is coated with CpG-loaded ZIF-8 obtained in Step 4 to form a core-shell structured CpG-loaded ZIF-8-encapsulated ginsenoside-activated cancer cell membrane nanovaccine (ZC@G-CCM). The coating method is ultrasound. The two are mixed in a 1:1 mass ratio and a 10W ultrasound therapy device is used in a 2-minute on / 2-minute off cycle for a total of 4 cycles. Finally, the liquid is collected and centrifuged at 10,000 rpm for 10 minutes to collect the precipitate.

[0055] Example 3

[0056] This embodiment provides a method for preparing a dual-targeting cancer cell membrane nanovaccine activated by ginsenoside Rk1, comprising the following steps:

[0057] Step 1: Transfer 4T1 breast cancer cells to T25 culture dishes and culture until the density reaches 80%-90%. Add ginsenoside Rk1 solution at a concentration of 60 μg / mL and continue culturing for 24 hours. Collect cells by centrifugation at 1000 rpm and wash 2-3 times with PBS.

[0058] Step 2: Resuspend the cells collected in Step 1 in a hypotonic lysis buffer containing the following components: 20 mM Tris hydrochloric acid buffer (pH 7.4), 10 mM potassium chloride, 2 mM magnesium chloride, and an EDTA-free protease inhibitor mixture (30 mL Tris hydrochloric acid buffer containing 22.37 mg potassium chloride and 5.71 mg magnesium chloride). Under ice bath conditions, sonicate the cell suspension 25 times at 30% amplitude (1 second shaking, 5 second interval) to fully lyse the cells while maintaining membrane integrity. Centrifuge the lysis buffer at 5000 × g for 10 minutes at 4°C, discard the nuclear pellet, and collect the supernatant. Centrifuge the supernatant again at 16000 × g for 30 minutes, and collect the pellet, which is the cell membrane component. The obtained membrane precipitate was resuspended in 5 mL PBS, and polycarbonate membranes with pore sizes of 800 nm, 400 nm and 200 nm were mechanically extruded sequentially through a small nano-extruder (extrusion at least 17 times per pore size) to finally form tumor cell membrane nanovesicles with uniform particle size and stable morphology.

[0059] Step 3: At room temperature, dissolve 29.1 g of 2-methylimidazole solution in 60 mL of pure water, and then dissolve 1.5 g of zinc nitrate in 90 mL of pure water. Slowly add the zinc nitrate solution to the 2-methylimidazole solution and stir at 500 rpm for 10 hours to form a white emulsion. Centrifuge the obtained liquid at 10,000 rpm for 10 minutes to collect the precipitate. Dissolve the precipitate completely with ethanol by sonication, then centrifuge at 10,000 rpm for 10 minutes to collect the precipitate. Wash the sample twice with ethanol and collect the precipitate. Finally, dry the sample overnight in a vacuum drying oven at 40°C. After drying, collect the solid, which is the ZIF-8 particle.

[0060] Step 4: Add the ZIF-8 obtained in Step 3 to PBS to form a 0.5 mg / mL solution. Mix ZIF-8 and CpG at a ratio of 25:6, and incubate overnight on ice at 1000 rpm. Centrifuge the resulting liquid at 10000 rpm for 10 minutes and collect the precipitate, which is the CpG-loaded ZIF-8.

[0061] Step 5: The tumor cell membrane activated by ginsenoside Rk1 obtained in Step 2 is coated with CpG-loaded ZIF-8 obtained in Step 4 to form a core-shell structured CpG-loaded ZIF-8-encapsulated ginsenoside-activated cancer cell membrane nanovaccine (ZC@G-CCM). The coating method is ultrasound. The two are mixed in a 1:1 mass ratio and a 10W ultrasound therapy device is used in a 2-minute on / 2-minute off cycle for a total of 4 cycles. Finally, the liquid is collected and centrifuged at 10,000 rpm for 10 minutes to collect the precipitate.

[0062] Example 4

[0063] This embodiment provides a method for preparing a dual-targeting cancer cell membrane nanovaccine activated by ginsenoside Rk1, comprising the following steps:

[0064] Step 1: Transfer 4T1 breast cancer cells to T25 culture dishes and culture until the density reaches 80%-90%. Add ginsenoside Rk1 solution at a concentration of 60 μg / mL and continue culturing for 24 hours. Collect cells by centrifugation at 1000 rpm and wash 2-3 times with PBS.

[0065] Step 2: Resuspend the cells collected in Step 1 in a hypotonic lysis buffer containing the following components: 20 mM Tris hydrochloric acid buffer (pH 7.4), 10 mM potassium chloride, 2 mM magnesium chloride, and an EDTA-free protease inhibitor mixture (40 mL Tris hydrochloric acid buffer containing 29.82 mg potassium chloride and 7.62 mg magnesium chloride). Under ice bath conditions, sonicate the cell suspension 25 times at 30% amplitude (1 second shaking, 5 second interval) to fully lyse the cells while maintaining membrane integrity. Centrifuge the lysis buffer at 5000 × g for 10 minutes at 4°C, discard the nuclear pellet, and collect the supernatant. Centrifuge the supernatant again at 16000 × g for 30 minutes, and collect the pellet, which is the cell membrane component. The obtained membrane precipitate was resuspended in 5 mL PBS, and polycarbonate membranes with pore sizes of 1000 nm, 800 nm, 400 nm and 200 nm were mechanically extruded sequentially through a small nano extruder (extrusion at least 17 times per pore size) to finally form tumor cell membrane nanovesicles with uniform particle size and stable morphology.

[0066] Step 3: At room temperature, dissolve 29.1 g of 2-methylimidazole solution in 60 mL of pure water, and then dissolve 1.5 g of zinc nitrate in 90 mL of pure water. Slowly add the zinc nitrate solution to the 2-methylimidazole solution and stir at 500 rpm for 10 hours to form a white emulsion. Centrifuge the obtained liquid at 10,000 rpm for 10 minutes to collect the precipitate. Dissolve the precipitate completely with ethanol by sonication, then centrifuge at 10,000 rpm for 10 minutes to collect the precipitate. Wash the sample twice with ethanol and collect the precipitate. Finally, dry the sample overnight in a vacuum drying oven at 40°C. After drying, collect the solid, which is the ZIF-8 zeolite imidazole ester framework material.

[0067] Step 4: Add the ZIF-8 obtained in Step 3 to PBS to form a 0.5 mg / mL solution. Mix ZIF-8 and CpG at a ratio of 25:5, and incubate overnight on ice at 1000 rpm. Centrifuge the resulting liquid at 10000 rpm for 10 minutes and collect the precipitate, which is the CpG-loaded ZIF-8.

[0068] Step 5: The tumor cell membrane activated by ginsenoside Rk1 obtained in Step 2 is coated with CpG-loaded ZIF-8 obtained in Step 4 to form a core-shell structured CpG-loaded ZIF-8-encapsulated ginsenoside-activated cancer cell membrane nanovaccine (ZC@G-CCM). The coating method is ultrasound. The two are mixed in a 1:2 mass ratio and a 10W ultrasound therapy device is used in a 2-minute on / 2-minute off cycle for a total of 4 cycles. Finally, the liquid is collected and centrifuged at 10,000 rpm for 10 minutes to collect the precipitate.

[0069] The ginsenoside Rk1-activated dual-target cancer cell membrane nanovaccines prepared in Examples 1-4 have basically the same performance. The vaccine prepared in Example 1 is used as an example to demonstrate the effectiveness of the vaccine through verification experiments.

[0070] Verification Example 1: Verification of the effect of ZC@G-CCM on activating dendritic cells

[0071] I. Experimental Methods

[0072] Step 1: Female BALB / c mice were sacrificed, and the femur and tibia were aseptically separated. The bone marrow cavity was washed with PBS to collect bone marrow cells. The cell concentration was adjusted to 1 × 10⁻⁶. 6 Bone marrow cells were seeded at a rate of 1 mL / well in 6-well plates. They were cultured in complete RPMI-1640 medium supplemented with granulocyte-macrophage colony-stimulating factor (GM-CSF, 30 ng / mL) and interleukin-4 (IL-4, 20 ng / mL) for 6 days at 37°C in a 5% CO2 incubator to induce differentiation of bone marrow cells into dendritic cells (BMDCs).

[0073] Step 2: On day 6 of differentiation, the induced BMDCs were recovered and re-seeded into new 6-well plates. The test drug was added to a final concentration of 10 μg / mL, and the plates were incubated at 37°C for another 24 hours.

[0074] Cells were collected after incubation and surface labeled using flow cytometry: FITC-labeled anti-mouse CD86 antibody and PE-Cyanine5-labeled anti-mouse CD80 antibody. Antibody staining was performed and incubated at 4°C in the dark for 30 minutes. Cells were then washed twice with PBS. Finally, the expression levels of CD80 and CD86 molecules on the surface of dendritic cells were detected by flow cytometry.

[0075] II. Test Results

[0076] Double positivity for CD80 and CD86 indicates that BMDCs are activated; therefore, flow cytometry was used to detect the expression of both markers. Figure 9 The experimental results showed that ZC@G-CCM significantly improved the maturation rate of dendritic cells (DCs), exhibiting the best performance among all treatment groups, indicating its potent effect in antigen presentation and immune initiation. CCM-NV was the second best, suggesting it possesses some immune activation capacity. ZIF-CPG, ZIF-8, and CPG showed relatively moderate performance, indicating that their effects as a single treatment or simple combination of these treatments are limited. The PBS group, serving as a negative control, had the lowest maturation rate, effectively validating the reliability of the experiment.

[0077] Verification Example 2: Verification of the activation effect of ZC@G-CCM on CTLL-2 cytotoxic T cells

[0078] I. Experimental Methods

[0079] Step 1: CTLL-2 cells were introduced at a density of 1 × 10⁻⁶. 5 ZC@G-CCM tumor vaccine was inoculated into 6-well plates at a concentration of 2 mL per well. The final concentration of ZC@G-CCM tumor vaccine was added to each well to achieve a final concentration of 10 μg / mL, and the plates were incubated at 37°C in a 5% CO2 incubator for 24 hours.

[0080] Step 2: After incubation, collect the cells, resuspend them in flow cytometry staining buffer, add APC-labeled anti-mouse CD69 antibody, and incubate at 4°C in the dark for 30 minutes. Then wash the cells twice with PBS to remove free antibodies.

[0081] Step 3: Use flow cytometry to analyze the stained cells and record the expression level of CD69.

[0082] II. Test Results

[0083] CD69 is a marker of early T cell activation, and its expression level can reflect the initial activation state of the immune system. Therefore, this experiment used flow cytometry to detect the expression of CD69 in each treatment group to evaluate the immune activation effect. Figure 9The experimental results shown in Figure b indicate that ZC@G-CCM significantly increased CD69 expression levels, exhibiting the best performance among all treatment groups, demonstrating its potent effect in inducing early T cell activation and further supporting its potential in immune initiation. CCM-NV followed, indicating that it possesses certain immune activation capabilities and can effectively promote T cell responses. ZIF-CPG, ZIF-8, and CPG showed relatively moderate performance, suggesting that their individual use or simple combination has limited effect in activating T cells. The PBS group, serving as a negative control, showed the lowest CD69 expression, validating the reliability and specificity of the experimental system.

[0084] Verification Example 3: Verification of the antitumor effect of ZC@G-CCM

[0085] I. Experimental Methods

[0086] Step 1: Select 4-6 week old female BALB / c mice and acclimatize them for 1 week before the experiment. Randomly divide the mice into 6 groups of 5 mice each, and administer the following vaccine formulations to each group: saline (PBS) control group; zeolite imidazole ester backbone material (ZIF-8) group; CpG oligodeoxynucleotide group; ZIF-8 / CpG complex (ZIF-CPG) group; cancer cell membrane nanovaccine (CCM-NV) group; and ginsenoside Rk1 activated vaccine (ZC@G-CCM) group. Before tumor cell inoculation, mice in each group were subcutaneously injected three times consecutively (7 days apart), with each injection dose being 50 μL, containing the following substances: PBS group: 50 μL PBS; ZIF-8 group: 40 μg ZIF-8; CpG group: 10 μg CpG; ZIF-CPG group: 50 μg ZIF-8 / CpG; CCM-NV group: 50 μg unactivated cancer cell membrane vaccine; G-CCM-DTCV group: 50 μg ginsenoside Rk1 activated cancer cell membrane nanovaccine.

[0087] Step 2: Seven days after the last vaccination, each mouse was subcutaneously injected with 100 μL of PBS suspension containing 2 × 10⁻⁶ ppm. 6 A subcutaneous tumor model was established using 4T1 breast cancer cells. Subsequently, the same dose of vaccine or control was administered on days 9, 12, and 15, respectively.

[0088] Step 3: Starting from the date of inoculation, regularly record the body weight and tumor volume of mice in each group. Tumor volume is calculated using the following formula:

[0089] Tumor volume (mm³) = length × (width²) / 2.

[0090] II. Test Results

[0091] Tumor volume is an important indicator for evaluating the efficacy of anti-tumor treatment; therefore, this experiment compared the treatment effects of different treatment groups by periodically measuring changes in tumor volume. Figure 10 The experimental results showed that ZC@G-CCM significantly inhibited tumor growth, exhibiting the slowest tumor volume growth throughout the observation period and demonstrating the best anti-tumor effect, suggesting its potent role in tumor immunotherapy. CCM-NV followed, with its tumor inhibition ability significantly superior to most control groups, indicating its potential therapeutic efficacy. ZIF-8, ZC, and CPG showed moderate performance, with faster tumor volume growth, suggesting limited therapeutic effects when used alone or in simple combinations. The PBS group served as a negative control. Compared with the PBS group, the tumor inhibition rates (based on tumor volume) of each group were: ZIF-8: 16.05%, ZC: 17.44%, CPG: 34.41%, CCM-NV: 52.44%, and ZC@G-CCM: 83.53%, further quantifying the anti-tumor effects of each treatment group and highlighting the significant advantages of ZC@G-CCM.

[0092] It should be noted that when numerical ranges are mentioned in the claims of this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, the present invention describes preferred embodiments.

[0093] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

Claims

1. A dual-targeting cancer cell membrane nanovaccine activated by ginsenoside Rk1, characterized in that, The nanovaccine is a core-shell structured nanovaccine formed by using tumor cell membranes activated by co-culturing with ginsenoside Rk1 as the shell and an acid-sensitive degradable metal-organic framework material loaded with an immune adjuvant as the core. The tumor cells are breast cancer cells, the immune adjuvant is CpG oligodeoxynucleoside, and the acid-sensitive degradable metal-organic framework material is zeolite imidazole ester framework material ZIF-8.

2. The method for preparing a ginsenoside Rk1-activated dual-targeting cancer cell membrane nanovaccine according to claim 1, characterized in that, Includes the following steps: S1. Culture tumor cells to a density of 80%-90%, add ginsenoside Rk1 solution, culture for 18-20 hours, then lyse the cells, discard the cell nucleus precipitate in the lysate, retain the cell membrane components, and then perform particle size reduction and homogenization treatment to obtain tumor cell membranes with uniform particle size and stable morphology. S2. Mix the acid-sensitive degradable metal-organic framework material with the immune adjuvant in PBS buffer and incubate under ice bath conditions for 10-12 hours. The resulting precipitate is the acid-sensitive degradable metal-organic framework material loaded with the immune adjuvant. S3. The tumor cell membrane is mixed with an acid-sensitive degradable metal-organic framework material loaded with an immune adjuvant in an equal mass ratio, and then coated by ultrasonic treatment to obtain the nanovaccine.

3. The method for preparing a ginsenoside Rk1-activated dual-targeting cancer cell membrane nanovaccine according to claim 2, characterized in that, The concentration of ginsenoside Rk1 solution was 60-100 μg / mL, and the culture time was 18-20 h.

4. The method for preparing a ginsenoside Rk1-activated dual-targeting cancer cell membrane nanovaccine according to claim 3, characterized in that, The mass ratio of acid-sensitive degradable metal-organic framework material to immune adjuvant is 25:5~7.

5. The application of the ginsenoside Rk1-activated dual-targeting cancer cell membrane nanovaccine of claim 1 in the preparation of antitumor drugs, characterized in that, The tumor is breast cancer.

6. The application of the ginsenoside Rk1-activated dual-targeting cancer cell membrane nanovaccine according to claim 5 in the preparation of antitumor drugs, characterized in that, The drug also includes pharmaceutically acceptable excipients.

Images