Ginsenoside Rk1 activated double-targeting cancer cell membrane nano vaccine as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biomedical engineering, in particular to a ginsenoside Rk1 activated double-targeting cancer cell membrane nano vaccine as well as a preparation method and application thereof. Pharmacological regulation is performed on cancer cells through Rk1, the membrane surface antigen spectrum of the cancer cells is remarkably remodeled, the expression of tumor associated antigen (TAA) is enhanced, an immunosuppressive molecule CD47 is down-regulated, and an antigen presenting molecule MHC-I is up-regulated. CpG oligodeoxynucleotide is loaded on a zeolite imidazate framework material ZIF-8, and then the zeolite imidazate framework material ZIF-8 is coated with a cancer cell membrane to form a core-shell structure, so that accurate and acid-sensitive responsive release is realized. The nano vaccine can significantly improve the antigen uptake and activation efficiency of dendritic cells (DC), and enhance the immune effect and immune memory formation of CD8T cells. Animal experiments show that the nano vaccine shows about 80% of tumor inhibition rate in a 4T1 mouse breast cancer model, and has good immune activation ability and anti-tumor efficacy.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine engineering, and in particular to a ginsenoside Rk1-activated double-targeted cancer cell membrane nano vaccine and a preparation method and application thereof. BACKGROUND

[0002] In recent years, tumor immunotherapy (such as immune checkpoint inhibitors ICIs, chimeric antigen receptor T cell therapy CAR-T, etc.) has shown significant efficacy in various malignancies, especially in melanoma and lung cancer, and has made breakthrough progress. However, for solid tumors such as triple-negative breast cancer (TNBC), the overall response rate of ICIs is still less than 30%, and the penetration and persistence of cell therapies such as CAR-T in solid tumors are limited by tumor microenvironment (TME) inhibition, etc. The common shortcoming is that it is highly dependent on a single antigen target, and it is difficult to cope with complex and heterogeneous tumor antigen spectrum. Therefore, developing a treatment strategy that can activate a broad spectrum of antigens, enhance dendritic cell (DC) uptake and T cell activation ability, has become an important direction for the development of the next generation of tumor vaccines.

[0003] Among the many antigen delivery carriers, tumor cell membrane (CCM) is considered an ideal material for tumor vaccines because it naturally carries a variety of tumor-associated antigens (TAA). However, unactivated CCM still has significant deficiencies in antigen presentation efficiency and immune activation ability. Traditional methods of inducing membrane antigen expression using chemotherapy drugs (such as DOX) are often limited to a single immune-related protein such as CRT, with limited activation, making it difficult to construct a multi-target, strong immune vaccine system. In recent years, some studies have proposed using natural products to precisely regulate tumor membranes to achieve the goal of antigen spectrum reconstruction and immune pathway activation. Among them, ginsenosides, as natural active triterpenoids, exhibit good multi-target immune regulation ability, and are expected to simultaneously affect antigen presentation factors (such as MHC-I) and "eat me" signals (such as CD47), providing a new path for constructing multifunctional vaccines. Therefore, exploring the regulation of tumor cell membrane antigen spectrum and immune pathways by natural products to construct a nano vaccine system with broad antigen presentation and double-targeted regulation ability has important theoretical value and clinical application potential. SUMMARY

[0004] The technical problem to be solved by the present application is to provide a ginsenoside Rk1-activated double-targeted cancer cell membrane nano vaccine and a preparation method and application thereof, which solves the problems of narrow antigen spectrum, weak immunogenicity and poor targeting of existing cancer vaccines.

[0005] The present application specifically adopts the following technical solutions: In a first aspect, the present application provides a ginsenoside Rk1-activated double-targeted cancer cell membrane nanovaccine, which is a nanovaccine with a core-shell structure, taking tumor cell membranes activated by ginsenoside Rk1 as the shell and acid-sensitive degradable metal-organic framework materials loaded with immune adjuvants as the shell.

[0006] Further, the tumor cells include breast cancer cells.

[0007] Further, the immune adjuvant includes CpG oligodeoxynucleotides.

[0008] Further, the acid-sensitive degradable metal-organic framework material includes zeolitic imidazolate framework material ZIF-8. The nanovaccine of the present application performs in vitro "immunogenic remodeling" on exogenous cancer cell membranes by Rk1, systematically up-regulates a variety of tumor-related antigens and MHC-I expression, and blocks the "don't eat me" signal CD47, significantly enhancing the recognition and phagocytosis efficiency of dendritic cells (DCs). The vaccine core co-encapsulates TLR9 agonist CpG and acid-sensitive degradable metal-organic framework material ZIF-8, constructing a core-shell nanostructure, which has high drug loading capacity, blood stability and tumor microenvironment targeted drug release characteristics. After in vivo inoculation, the vaccine can efficiently drive DC uptake and maturation, significantly expand and infiltrate CD8+ cytotoxic T cells in the tumor, induce strong anti-tumor effects and long-term immune memory, and achieve complete removal of tumor cells.

[0009] In a second aspect, the present application provides a preparation method of the ginsenoside Rk1-activated double-targeted cancer cell membrane nanovaccine, which includes the following steps: S1, culturing tumor cells to a density of 80%-90%, adding a ginsenoside Rk1 solution, culturing for 18-20 h, then lysing the cells, discarding the cell nucleus precipitate in the lysate, retaining the cell membrane components, then performing particle size nanofication and uniformization treatment to obtain tumor cell membranes with uniform particle size and stable morphology; S2, mixing acid-sensitive degradable metal-organic framework materials and immune adjuvants in PBS buffer, and incubating under ice bath conditions for 10-12 h, and the obtained precipitate is the acid-sensitive degradable metal-organic framework material loaded with immune adjuvants; S3, mixing the tumor cell membranes and the acid-sensitive degradable metal-organic framework material loaded with immune adjuvants in equal mass ratio, and performing ultrasonic treatment for coating to obtain the nanovaccine.

[0010] Further, the concentration of the ginsenoside Rk1 solution is 60-100 μg / mL, and the culturing time is 18-20 h.

[0011] Further, the acid-sensitive degradable metal-organic framework material and the immune adjuvant are mixed into PBS buffer, and the mass ratio of the metal-organic framework material to the immune adjuvant is 25:5-7.

[0012] 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.

[0013] Compared with the prior art, the present invention has the following advantages: 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.

[0014] 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.

[0015] 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

[0016] Figure 1 This is the structural diagram of ginsenoside Rk1.

[0017] 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.

[0018] 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.

[0019] 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.

[0020] Figure 6 The 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.

[0021] 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.

[0022] Figure 8 The expression diagram of CPG in the ginsenoside Rk1-activated dual-target tumor cell membrane nanovaccine prepared in Example 1 is shown.

[0023] Figure 9 The images show quantitative diagrams (a) and (b) of the ginsenoside Rk1-activated dual-target tumor cell membrane nanovaccine prepared in Example 1, which are used to activate dendritic cells.

[0024] Figure a shows the activation of dendritic cells by the ginsenoside Rk1-activated dual-target tumor cell membrane nanovaccine prepared in Example 1.

[0025] 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.

[0026] 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

[0027] 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.

[0028] 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.

[0029] The sequence of the CpG oligodeoxynucleotide is 5'-TCCATGACGTTCCTGACGTT-3', purchased from Shanghai Sangon Biological Engineering Co., Ltd.; 4T1 cells were purchased from Shanghai Gaining Biological Technology Co., Ltd.; PE-Cyanine5-CD80, FITC-CD86, and APC-CD69 were purchased from BioLegend; zinc nitrate hexahydrate (Zn(NO3)2·6H2O) with CAS number 10196-18-6 was purchased from Macklin Reagent Co., Ltd.; dimethylimidazole with CAS number 693-98-1 was purchased from Macklin Reagent Co., Ltd.

[0030] Example 1 The present embodiment provides a preparation method of a ginsenoside Rk1-activated double-targeted cancer cell membrane nanovaccine, comprising the following steps: Step one, 4T1 breast cancer cells were cultured in a 10 cm diameter culture dish, and the density reached 80%-90% after culture. Then, a ginsenoside Rk1 solution with a concentration of 100 μg / mL was added, and the culture was continued for 18 hours. The cells were collected by centrifugation at 1000 rpm, and finally washed with PBS for 2-3 times. Figure 2 The results showed that ginsenoside Rk1 activated the expression of CD47 and MHC-I in the membrane of breast cancer cells.

[0031] Step two, the cells collected in step one were resuspended in a hypotonic lysis buffer containing the following components: 20 mM Tris-HCl buffer (pH 7.4), 10 mM potassium chloride, 2 mM magnesium chloride, and a proteinase inhibitor cocktail without EDTA, i.e., 20 mL Tris-HCl buffer containing 14.91 mg potassium chloride and 3.81 mg magnesium chloride. Under ice bath conditions, the cell suspension was treated with 10% amplitude ultrasonic waves for a total of 25 times (1 second of oscillation, 5 seconds of interval) to fully lyse the cells while maintaining the integrity of the membrane structure. The lysate was centrifuged at 5000 x g for 10 minutes at 4°C, and the cell nucleus precipitate was discarded. The supernatant was collected. The supernatant was then centrifuged at 16000 x g for 30 minutes, and the precipitate was collected, which was the cell membrane component. The obtained membrane precipitate was resuspended in 5 mL PBS, and then mechanically extruded through a small nanometer extruder with 400 nm and 200 nm pore size polycarbonate membranes in sequence (at least 17 extrusions per pore size), finally forming tumor cell membrane nanovesicles with uniform particle size and stable morphology (denoted as G-CCM). As shown in the transmission electron microscopy image, Figure 3 The transmission electron microscopy image shows that the tumor cell membrane nanovesicles have a clear hollow cell membrane structure.

[0032] Step three, 9.7 g of 2-methylimidazole was dissolved in 20 mL of pure water, and 0.5 g of zinc nitrate was dissolved in 30 mL of pure water. The zinc nitrate solution was slowly added to the 2-methylimidazole solution, and stirred at 500 rpm for 8 hours to form a white emulsion. The obtained liquid was centrifuged at 10,000 rpm for 10 minutes to collect the precipitate, which was completely dissolved in ethanol by ultrasonic, and then centrifuged at 10,000 rpm for 10 minutes to collect the precipitate. The precipitate was repeatedly washed with ethanol for 2 times, and then collected. Finally, the sample was dried in a vacuum drying oven at 40°C overnight. After drying, the solid was collected, which was ZIF-8 particles. As shown in the transmission electron microscopy (TEM) image in FIG. 1, Figure 4 As shown in the transmission electron microscopy (TEM) image in FIG. 1,

[0033] Step four, the ZIF-8 obtained in step three was added to PBS to form a solution of 0.5 mg / mL, and mixed according to the ratio of ZIF-8:CpG of 25:7. The rotation speed was 800 rpm, and the incubation was performed on ice overnight. The obtained liquid was centrifuged at 10,000 rpm for 10 minutes to collect the precipitate, which was CpG-loaded ZIF-8 (denoted as ZIF-CPG or ZC).

[0034] Step five, the tumor cell membrane activated by ginsenoside Rk1 obtained in step two was coated on the CpG-loaded ZIF-8 obtained in step four to form a core-shell structure of CpG-loaded ZIF-8 coated ginsenoside-activated cancer cell membrane nanovaccine (ZC@G-CCM). The coating method was ultrasonic method. Two kinds of materials were mixed in the form of 1:1 by mass, and a 10W ultrasonic treatment instrument was used in a cycle mode of working for 2 minutes and intermittent for 2 minutes for 4 cycles. Finally, the liquid was collected, and centrifuged at 10,000 rpm for 10 minutes to collect the precipitate. As shown in the transmission electron microscopy (TEM) image in FIG. 3, Figure 5 As shown in the transmission electron microscopy (TEM) image in FIG. 3,

[0035] Figure 6 FIG. 4a shows the infrared spectra of ZIF-8 particles and CpG-loaded ZIF-8. In the low wave number region, the characteristic absorption peak of Zn–N bond is clearly visible, which indicates that the coordination structure between zinc and imidazole ligand in ZIF-8 remains stable and does not change significantly after loading CpG. In the medium wave number region, the characteristic vibration peak of C–N bond is also retained, which is a typical feature of imidazole ring structure. These results show that even after modification by CpG, the core framework structure of ZIF-8 remains intact, and its chemical nature is not destroyed. ZIF-CPG successfully retains the structural features of ZIF-8.

[0036] Figure 6The b presents the X-ray diffraction (XRD) patterns of ZIF-8 particles and ZIF-CPG after loading CpG. The diffraction peaks of 011, 002, 112, 013, 222 and other crystal planes are clearly marked in the figure, which are the typical characteristics of the crystal structure of ZIF-8, reflecting its highly ordered crystal structure. After loading CpG, these characteristic peaks still exist and the position is basically unchanged, which shows 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 loading process of CpG does not destroy the crystal structure of ZIF-8, and ZIF-CPG is highly consistent with ZIF-8 in structure, which provides a solid structural foundation for subsequent application research. Figure 7 The expression of membrane proteins in the ginsenoside Rk1-activated dual-targeted tumor cell membrane nanovaccine is depicted. In the electrophoretogram, neither ZIF-8 nor ZC shows obvious protein bands, indicating that these two materials themselves do not contain protein components. In contrast, G-CCM (ginsenoside Rk1-activated tumor cell membrane) presents a unique protein distribution pattern, with multiple characteristic protein bands clearly visible in the electrophoretogram, which reflects the rich composition of membrane proteins in G-CCM.

[0037] More importantly, in the electrophoretogram of ZC@G-CCM (ZC and G-CCM composite material), the combination of protein bands is highly consistent with G-CCM, while retaining the characteristics of ZC. This result clearly shows that ZC@G-CCM composite material successfully integrates the components of ZC and G-CCM, not only retaining 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 the construction of a dual-targeted tumor cell membrane nanovaccine, demonstrating the effectiveness and feasibility of the composite material in nanovaccine design.

[0038] Figure 8 The expression of CpG in the ginsenoside Rk1-activated dual-targeted tumor cell membrane nanovaccine is shown. In the electrophoretogram, ZIF-8 does not show DNA bands, indicating that it does not contain DNA components. In contrast, CpG, ZC and ZC@G-CCM all present clear DNA bands.

[0039] This result clearly confirmed the successful loading of CpG. Specifically, the DNA band of CpG itself was clearly discernible, indicating that it contained a specific DNA sequence. The presence of a DNA band consistent with CpG in ZC and ZC@G-CCM not only indicates that CpG can be successfully loaded onto ZC, but further indicates that in the ZC@G-CCM composite material, the DNA band of CpG remains stable, indicating that CpG has not been lost or degraded during the preparation of the composite material. This finding provides key experimental evidence for the construction of a double-targeted tumor cell membrane nanovaccine with immune activation function, proving the stability and functionality of CpG in the nanovaccine system.

[0040] Example 2 The present embodiment provides a preparation method of a ginsenoside Rk1-activated double-targeted cancer cell membrane nanovaccine, comprising the following steps: Step one, 4T1 breast cancer cells are cultured in a T75 culture dish until the density reaches 80%-90%, and then a ginsenoside Rk1 solution with a concentration of 80 μg / mL is added and the culture is continued for 20 hours. The cells are collected by centrifugation at 1000 rpm, and finally washed with PBS for 2-3 times.

[0041] Step two, the cells collected in step one are resuspended in a hypotonic lysis buffer containing the following components: 20 mM Tris-HCl buffer (pH 7.4), 10 mM potassium chloride, 2 mM magnesium chloride, and a proteinase inhibitor cocktail without EDTA. The cell suspension is treated with 20% amplitude ultrasound for a total of 25 times (1 second of oscillation, 5 seconds of interval) under ice bath conditions, to fully lyse the cells but maintain the integrity of the membrane structure. The lysate is centrifuged at 5000 x g for 10 minutes at 4°C, and the cell nucleus precipitate is discarded, and the supernatant is collected. The supernatant is then centrifuged at 16000 x g for 30 minutes, and the precipitate is collected, which is the cell membrane component. The obtained membrane precipitate is resuspended in 5 mL of PBS, and is mechanically extruded through a small nanometer extruder with 800 nm, 400 nm, and 200 nm pore size polycarbonate membranes in sequence (at least 17 extrusions per pore size), to finally form tumor cell membrane nanovesicles with uniform particle size and stable morphology.

[0042] Step three, dissolve 29.1 g of 2-methylimidazole solution in 60 mL of pure water, and dissolve 1.5 g of zinc nitrate in 90 mL of pure water, slowly add the zinc nitrate solution to the 2-methylimidazole solution, stir at 500 rpm for 8 hours, and form a white emulsion liquid. The obtained liquid is centrifuged at 10000 rpm for 10 minutes to collect the precipitate, the precipitate is completely dissolved with ethanol by ultrasonic, then centrifuged at 10000 rpm for 10 minutes to collect the precipitate, the precipitate is repeatedly washed with ethanol for 2 times, the precipitate is collected, and finally the sample is dried in a vacuum drying oven at 40℃ overnight, and the solid is collected after drying, which is the zeolitic imidazolate framework material ZIF-8 particles.

[0043] Step four, add the ZIF-8 obtained in step three into PBS to form a solution of 0.5 mg / mL, mix according to the ratio of ZIF-8:CpG of 25:7, rotate at 800 rpm, and incubate on ice overnight. The obtained liquid is centrifuged at 10000 rpm for 10 minutes to collect the precipitate, which is the CpG-loaded ZIF-8.

[0044] Step five, coat the tumor cell membrane activated by ginsenoside Rk1 obtained in step two on the CpG-loaded ZIF-8 obtained in step four to form a core-shell structure of CpG-loaded ZIF-8 coated ginsenoside-activated cancer cell membrane nano vaccine (ZC@G-CCM). The coating method is ultrasonic method, mix the two in the form of 1:1 by mass, use a 10W ultrasonic treatment instrument, and work in a cycle mode of 2 minutes / intermittent 2 minutes for 4 cycles. Finally, collect the liquid, centrifuge at 10000 rpm for 10 minutes to collect the precipitate.

[0045] Example 3 The present embodiment provides a preparation method of a ginsenoside Rk1-activated double-targeted cancer cell membrane nano vaccine, comprising the following steps: Step one, culture 4T1 breast cancer cells in a T25 culture dish, and culture until the density reaches 80%-90%, then add a ginsenoside Rk1 solution with a concentration of 60 μg / mL, and continue to culture for 24 hours. Centrifuge the cells at 1000 rpm, and finally wash with PBS for 2-3 times.

[0046] Step two, resuspend the cells collected in step one in hypotonic lysis buffer containing the following components: 20 mM Tris hydrochloride buffer (pH 7.4), 10 mM potassium chloride, 2 mM magnesium chloride, and a protease inhibitor cocktail without EDTA. The lysis buffer is prepared by adding 22.37 mg of potassium chloride and 5.71 mg of magnesium chloride to 30 mL of Tris hydrochloride buffer. The cell suspension is treated with 30% amplitude ultrasound for a total of 25 times (1 second of oscillation, 5 seconds of interval) in an ice bath to fully lyse the cells while maintaining the integrity of the membrane structure. The lysate is centrifuged at 5000 x g for 10 minutes at 4°C, and the cell nucleus precipitate is discarded. The supernatant is collected and centrifuged at 16000 x g for 30 minutes. The precipitate is collected, which is the membrane component of the cells. The obtained membrane precipitate is resuspended in 5 mL of PBS and mechanically extruded through a small nano-extruder with 800 nm, 400 nm, and 200 nm pore size polycarbonate membranes in sequence (at least 17 times per pore size). Finally, tumor cell membrane nanovesicles with uniform particle size and stable morphology are formed.

[0047] Step three, at room temperature, dissolve 29.1 g of 2-methylimidazole 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, stir at 500 rpm for 10 hours, and form a white emulsion liquid. Centrifuge the obtained liquid at 10000 rpm for 10 minutes to collect the precipitate. Dissolve the precipitate with ethanol by ultrasonic, then centrifuge at 10000 rpm for 10 minutes to collect the precipitate. Repeat the washing with ethanol for 2 times, collect the precipitate, and finally dry the sample in a vacuum drying oven at 40°C overnight. After drying, collect the solid, which is ZIF-8 particles.

[0048] Step four, add the ZIF-8 obtained in step three to PBS to form a 0.5 mg / mL solution, and mix according to the ratio of ZIF-8:CpG of 25:6. Incubate overnight on ice at a speed of 1000 rpm. Centrifuge the obtained liquid at 10000 rpm for 10 minutes to collect the precipitate, which is CpG-loaded ZIF-8.

[0049] Step five, coat the human ginsenoside Rk1-activated tumor cell membrane obtained in step two on the CpG-loaded ZIF-8 obtained in step four to form a core-shell structure of CpG-loaded ZIF-8 coated human ginsenoside-activated cancer cell membrane nanovaccine (ZC@G-CCM). The coating method is ultrasonic method. Mix the two according to the mass ratio of 1:1, use a 10W ultrasonic treatment instrument, and work in a cycle mode of 2 minutes / intermittent 2 minutes for 4 cycles. Finally, collect the liquid and centrifuge at 10000 rpm for 10 minutes to collect the precipitate.

[0050] Example 4 The embodiment provides a preparation method of a ginsenoside Rk1-activated double-targeted cancer cell membrane nano vaccine, and comprises the following steps: Step one, 4T1 breast cancer cells are cultured in a T25 culture dish, and the culture is continued until the density reaches 80%-90%, and then a ginsenoside Rk1 solution with a concentration of 60 μg / mL is added, and the culture is continued for 24 hours. The cells are collected by centrifugation at a speed of 1000 revolutions per minute, and finally washed with PBS for 2-3 times.

[0051] Step two, the cells collected in step one are resuspended in a hypotonic lysis buffer containing the following components: 20 mM Tris hydrochloride buffer (pH 7.4), 10 mM potassium chloride, 2 mM magnesium chloride, and a proteinase inhibitor cocktail without EDTA, that is, 40 mL of the Tris hydrochloride buffer contains 29.82 mg of potassium chloride and 7.62 mg of magnesium chloride. The cell suspension is treated with ultrasonic waves with an amplitude of 30% for a total of 25 times (1 second of oscillation, 5 seconds of interval) under ice bath conditions to fully lyse the cells while maintaining the integrity of the membrane structure. The lysate is centrifuged at 5000xg for 10 minutes at 4°C, the cell nucleus precipitate is discarded, and the supernatant is collected. The supernatant is then centrifuged at 16000xg for 30 minutes, and the precipitate is collected, which is the cell membrane component. The obtained membrane precipitate is resuspended in 5 mL of PBS, and is mechanically extruded through a small nano extruder in sequence through polycarbonate membranes with pore sizes of 1000 nm, 800 nm, 400 nm and 200 nm (at least 17 extrusions per pore size), and finally forms tumor cell membrane nano vesicles with uniform particle size and stable morphology.

[0052] Step three, 29.1 g of a 2-methylimidazole solution is dissolved in 60 mL of pure water, and 1.5 g of zinc nitrate is dissolved in 90 mL of pure water. The zinc nitrate solution is slowly added to the 2-methylimidazole solution, and stirred at 500 revolutions per minute for 10 hours to form a white emulsion liquid. The obtained liquid is centrifuged at 10000 revolutions per minute for 10 minutes to collect the precipitate, which is completely dissolved by ultrasonic treatment with ethanol. Then, the precipitate is collected by centrifugation at 10000 revolutions per minute for 10 minutes, and is repeatedly washed with ethanol for 2 times. Finally, the sample is dried in a vacuum drying oven at 40°C overnight, and the solid is collected after drying, which is the zeolite imidazolate framework material ZIF-8 particles.

[0053] Step four, the ZIF-8 obtained in step three is added to PBS to form a solution with a concentration of 0.5 mg / mL, and is mixed according to the ratio of ZIF-8:CpG of 25:5, and is incubated on ice at a speed of 1000 revolutions per minute overnight. The obtained liquid is centrifuged at 10000 revolutions per minute for 10 minutes to 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 1:2 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.

[0055] 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.

[0056] Verification Example 1: Verification of the effect of ZC@G-CCM on activating dendritic cells I. Experimental Methods 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).

[0057] 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.

[0058] 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.

[0059] II. Test Results Double positivity for CD80 and CD86 indicates that BMDCs are activated; therefore, flow cytometry was used to detect the expression of both markers. Figure 9The experimental results show that ZC@G-CCM significantly improves the maturation rate of dendritic cells (DC), and performs best in all treatment groups, indicating that it has a strong effect on antigen presentation and immune initiation. CCM-NV is second, indicating that it has certain immune activation ability; while ZIF-CPG, ZIF-8 and CPG perform relatively medium, indicating that the immune stimulation effect of single use or simple combination is limited. The PBS group as a negative control has the lowest maturation rate, effectively verifying the reliability of the experiment.

[0060] Verification Example 2: Verification of the activation effect of ZC@G-CCM on CTLL-2 cytotoxic T cells I. Test method Step one, CTLL-2 cells were inoculated in a 6-well plate at a density of 1 × 10 5 Each well was added with ZC@G-CCM tumor vaccine to make the final concentration reach 10 μg / mL, and incubated at 37℃ in a 5% CO2 incubator for 24 hours.

[0061] Step two, after incubation, the cells were collected, resuspended with flow cytometry staining buffer, and added with APC-labeled anti-mouse CD69 antibody, and incubated at 4℃ in the dark for 30 minutes. Then the cells were washed with PBS for 2 times to remove the free antibody.

[0062] Step three: the stained cells were detected by flow cytometry, and the expression level of CD69 was recorded.

[0063] II. Test results CD69 is an early activation marker of T cells, and its expression level can reflect the initial activation state of the immune system. Therefore, flow cytometry was used to detect the expression of CD69 in each treatment group to evaluate the immune activation effect. As shown in Figure 9 The experimental results shown in b show that ZC@G-CCM significantly improves the expression level of CD69, and performs best in all treatment groups, indicating that it has a strong effect on inducing T cell early activation, further supporting its potential in immune initiation. CCM-NV is second, indicating that it itself has certain immune activation ability and can effectively promote T cell response. ZIF-CPG, ZIF-8 and CPG perform relatively medium, indicating that their single use or simple combination has limited effect on activating T cells. The PBS group as a negative control has the lowest CD69 expression, verifying the reliability and specificity of the experimental system.

[0064] Verification Example 3: Verification of the anti-tumor effect of ZC@G-CCM I. Test method Step one, select 4~6 weeks old female BALB / c mice, adaptive feeding for 1 week before the experiment. The mice were randomly divided into 6 groups, 5 in each group, and inoculated with the following vaccine preparations: saline (PBS) control group; zeolite imidazole framework material (ZIF-8) group; CpG oligodeoxynucleotide group; ZIF-8 / CpG complex (ZIF-CPG) group; cancer cell membrane nanovaccine (CCM-NV) group; ginsenoside Rk1 activated vaccine (ZC@G-CCM) group. Each group of mice was subcutaneously injected 3 times (7 days apart) before tumor cell inoculation, with a dose of 50 μL, which contained 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.

[0065] Step two, after the last injection of vaccine for 7 days, each mouse was subcutaneously injected with 100 μL PBS suspension containing 2 × 10 6 4T1 breast cancer cells, to establish a subcutaneous tumor model. Subsequently, the same dose of vaccine or control treatment was given on the 9th, 12th and 15th day.

[0066] Step three, from the inoculation day, the body weight and tumor volume of each group of mice were recorded regularly. The tumor volume was calculated by the following formula:

[0067] Tumor volume (mm³) = length × (width²) / 2.

[0068] II. Experimental results Tumor volume is an important indicator for evaluating the effect of anti-tumor treatment, so this experiment compares the treatment effects of different groups by measuring the changes in tumor volume regularly. For example, Figure 10The experimental results show that ZC@G-CCM significantly inhibits tumor growth, the tumor volume grows most slowly in the whole observation period, and shows the best anti-tumor effect, which indicates that it has a strong effect in tumor immunotherapy. CCM-NV is second, and its tumor inhibition ability is significantly better than that of most control groups, which shows that it has certain treatment potential. ZIF-8, ZC and CPG perform moderately, and the tumor volume grows faster, which indicates that their therapeutic effect is limited when used alone or simply compounded. The PBS group is used as a negative control. Compared with the PBS group, the tumor inhibition rates (based on tumor volume) of each group are as follows: ZIF-8: 16.05%, ZC: 17.44%, CPG: 34.41%, CCM-NV: 52.44%, ZC@G-CCM: 83.53%, which further quantifies the anti-tumor effect of each treatment group and highlights the significant advantage of ZC@G-CCM.

[0069] It should be noted that when the present application claims involving numerical ranges, it should be understood that each numerical range of two endpoints and any number between the two endpoints can be selected. In order to prevent repetition, the present application describes the preferred embodiments.

[0070] Although the preferred embodiments of the present application have been described, those skilled in the art can make further changes and modifications to these embodiments once they know the basic inventive concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications falling within the scope of the present application.

Claims

1. A ginsenoside Rk1-activated dual-targeted cancer cell membrane nanovaccine, characterized in that, A nano-vaccine with a core-shell structure, in which a tumor cell membrane activated by ginsenoside Rk1 is used as the shell and an acid-sensitive degradable metal-organic framework material loaded with an immune adjuvant is used as the core.

2. The ginsenoside Rk1-activated dual-targeted cancer cell membrane nanovaccine according to claim 1, characterized in that, The tumor cells include breast cancer cells.

3. The ginsenoside Rk1-activated dual-targeted cancer cell membrane nanovaccine according to claim 1, characterized in that, The immune adjuvant includes CpG oligodeoxynucleotides.

4. The ginsenoside Rk1-activated dual-targeted cancer cell membrane nanovaccine according to claim 1, characterized in that, The acid-sensitive degradable metal-organic framework material includes zeolitic imidazolate framework-8 (ZIF-8).

5. The method for preparing a ginsenoside Rk1-activated dual-targeting cancer cell membrane nanovaccine according to any one of claims 1 to 4, characterized in that, The method comprises the following steps: S1. Culturing tumor cells to a density of 80-90%, adding a ginsenoside Rk1 solution, culturing for 18-20 hours, then lysing the cells, discarding the cell nucleus precipitate in the lysate, retaining the cell membrane components, and then performing particle size nanofication and uniformization treatment to obtain tumor cell membranes with uniform particle sizes and stable morphologies; S2. Mixing an acid-sensitive degradable metal-organic framework material and an immune adjuvant in a PBS buffer, incubating under ice bath conditions for 10-12 hours, and obtaining the precipitate as the acid-sensitive degradable metal-organic framework material loaded with the immune adjuvant; S3. Mixing the tumor cell membranes and the acid-sensitive degradable metal-organic framework material loaded with the immune adjuvant in an equal mass ratio, performing ultrasonic treatment for coating, and obtaining the nano-vaccine.

6. The method according to claim 5, wherein the preparation method of the ginsenoside Rk1-activated dual-targeted cancer cell membrane nano-vaccine is characterized in that, The concentration of the ginsenoside Rk1 solution is 60-100 μg / mL, and the culturing is performed for 18-20 hours.

7. The method according to claim 5, wherein the preparation method of the ginsenoside Rk1-activated dual-targeted cancer cell membrane nano-vaccine is characterized in that, The mass ratio of the acid-sensitive degradable metal-organic framework material to the immune adjuvant is 25:5-7.

8. Use of a ginsenoside Rk1-activated double-targeted cancer cell membrane nano-vaccine according to any one of claims 1-4 in the preparation of an antitumor drug.

9. The use of the ginsenoside Rk1-activated dual-targeted cancer cell membrane nanovaccine of claim 8 in the preparation of an antitumor drug, characterized in that, The tumor includes breast cancer. 10.The use of the ginsenoside Rk1-activated double-targeted cancer cell membrane nano-vaccine of claim 9 in the preparation of an antitumor drug. The drug further comprises a pharmaceutically acceptable excipient.

Images