Chinese herbal medicine vesicle-LNP hybrid tumor mRNA vaccine and application thereof

Abstract

The invention relates to the technical field of biological medicine, and provides a Chinese herbal medicine vesicle-LNP hybrid tumor mRNA vaccine and application thereof. The tumor mRNA vaccine disclosed by the invention is obtained by hybridizing Chinese herbal medicine nano-vesicles and lipid nano-particles loaded with tumor antigen mRNA. According to the invention, the characteristic of good biocompatibility of Chinese herbal medicines is utilized, so that the safety risk of the cationic lipid carrier is reduced. By combining the characteristics of Chinese herbal medicines, the nano-vesicles are extracted, so that the LNP is promoted to more effectively target antigen-presenting cells, the uptake level of the antigen-presenting cells is promoted, the phagocytosis of mRNA is enhanced, and the targeting and transcriptional expression level of mRNA can be enhanced. Finally, the Chinese herbal medicine nano vesicle-LNP hybrid tumor mRNA vaccine can strengthen the curative effects of inhibiting tumor progression, tumor recurrence and tumor metastasis after the tumor mRNA vaccine is inoculated, and a path is opened up for the combination of the tumor mRNA vaccine and richer immunotherapy means.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technologies, and particularly relates to a construction strategy of a low-toxic Chinese herbal medicine nanovesicle-LNP hybrid tumor mRNA vaccine and its biomedical applications. Background Art

[0002] With the development of precision medicine, cancer treatment is shifting from "indiscriminate killing" to "individualized targeting". However, drug resistance and recurrence remain major challenges, and there is an urgent need for more efficient and low-toxic new strategies. Immunotherapy is a treatment method that utilizes the human immune system to recognize and kill cancer cells. It does not directly attack cancer cells, but rather activates or enhances the functions of immune cells (such as T cells, NK cells) to more effectively eliminate tumors. Currently, the main types of immunotherapy include: immune checkpoint inhibitors, chimeric antigen receptor T cell therapies, oncolytic viruses, and cancer vaccines, etc. In recent years, the potential for personalized customization of cancer vaccines has made cancer vaccines a research hotspot in immunotherapy.

[0003] Vaccines have evolved from traditional inactivated / attenuated pathogens to subunit vaccines, protein vaccines, viral vector vaccines, whole cell vaccines, DC vaccines (such as Provenge®), and nucleic acid vaccines (DNA / mRNA). Their core goal is to efficiently and safely induce immune memory. In the field of oncology, conventional therapies (surgery, radiotherapy, chemotherapy) are difficult to eliminate metastatic foci or drug-resistant cells, while vaccines are expected to achieve long-term immune surveillance by activating specific T cell responses. However, the heterogeneity of tumor antigens, immune escape mechanisms, and insufficient delivery efficiency have limited the clinical efficacy of most therapeutic cancer vaccines, and there is an urgent need for breakthrough technologies to address the three major challenges of antigen selection, delivery, and immune microenvironment regulation.

[0004] Nucleic acid vaccines have rapidly emerged in recent years, mainly due to factors such as mRNA sequence optimization and the increasing maturity of delivery systems. Among them, mRNA vaccines activate the body's specific immune response by encoding tumor-associated antigens (TAAs) or neoantigens, and have unique advantages in personalized cancer treatment. Compared with traditional vaccines, mRNA does not need to enter the cell nucleus, avoiding the risk of genomic integration, and can quickly respond to mutant antigens through sequence design. In recent years, the success of COVID-19 mRNA vaccines has verified their clinical feasibility, but the immunosuppressive characteristics of the tumor microenvironment (TME) and insufficient delivery efficiency limit their efficacy. Therefore, developing an efficient and low-toxic delivery system is the core challenge for tumor mRNA vaccines.

[0005] mRNA vaccines rely on delivery vectors to introduce the mRNA encoding antigens into the cytoplasm. After translation by ribosomes, the antigens are presented through MHC class I / II molecules, activating CD8 + / CD4 +T cells. However, naked mRNA is easily degraded by serum RNase, and negatively charged mRNA is difficult to penetrate cell membranes. Existing delivery systems (such as LNP) can encapsulate mRNA, but they face problems such as poor targeting and off-target toxicity caused by liver and spleen enrichment. In addition, the immunogenicity of mRNA may trigger an excessive inflammatory response, further restricting its clinical application.

[0006] Currently, mainstream delivery carriers such as lipid nanoparticles (Lipid Nanoparticle, LNP) rely on cationic lipids (such as SM102, DOTAP, etc.) to compress mRNA, but their positive charges lead to binding to serum proteins, accelerated clearance, and cell membrane damage. In addition, although the PEGylation of LNP can extend the circulation time, it may induce anti-PEG antibodies and reduce the effect of repeated dosing. Virus vectors have high transfection efficiency, but there are risks of insertional mutagenesis and pre-existing immunity problems. These defects highlight the urgent need for a new delivery platform.

[0007] Mature cationic lipid carriers include multiple cationic lipid components. Taking SM102 as an example, as the core component of LNP, its effectiveness in mRNA delivery has been proven in COVID-19 vaccines, but its cationic properties may cause cell membrane lysis, complement activation, and liver toxicity. Animal experiments show that high-dose SM102 may induce a cytokine storm, and multiple administrations are required for tumor treatment, further amplifying the safety risks. Therefore, it is urgent to modify the carrier to shield its toxicity while enhancing its efficient delivery ability.

[0008] Currently, the LNP carriers (such as SM102, DOTAP, etc.) relied on by mRNA vaccines have significant defects, including possible toxicity risks, induced inflammatory damage or liver damage, and ultimately the need to limit the dosing doses of cationic lipids and mRNA. At the same time, LNP carriers will accumulate non-specifically. A large amount of LNP remains at the injection site after injection or is captured by the hepatic and splenic reticuloendothelial systems, and the tumor and lymph accumulation rates are less than 5%, severely restricting the efficacy of mRNA. In addition, the LNP-mRNA complex requires low-temperature storage and transportation, increasing the clinical use cost. Summary of the Invention

[0009] To solve the above technical problems, the present invention provides a low-toxicity Chinese herbal medicine nanovesicle-LNP hybrid tumor mRNA vaccine. By utilizing the good biocompatibility of Chinese herbal medicine, the present invention reduces the safety risks of cationic lipid carriers. Combining the characteristics of Chinese herbal medicine, its nanovesicles are extracted to promote the more effective targeting of LNP to antigen-presenting cells, enhance the uptake level of antigen-presenting cells, and strengthen the phagocytosis of mRNA. The maturation of antigen-presenting cells is promoted through Toll-like receptors on the surface of antigen-presenting cells, enhancing the targeting and transcriptional expression levels of mRNA. Ultimately, the tumor mRNA vaccine hybridized with Chinese herbal medicine nanovesicles-LNP can strengthen the efficacy of inhibiting tumor progression, recurrence, and metastasis after the inoculation of tumor mRNA vaccines, opening up a path for its combination with more diverse immunotherapy means.

[0010] In the first aspect of the present invention, there is provided a tumor mRNA vaccine hybridized by Chinese herbal medicine vesicles and LNP, which is obtained by hybridizing Chinese herbal medicine nanovesicles and lipid nanoparticles loaded with tumor antigen mRNA.

[0011] As an alternative, the Chinese herbal medicine includes ginseng; the tumor antigen includes one or a combination of pattern antigens, universal tumor antigens, tumor neoantigens, tumor-associated antigens, or tumor fusion antigens.

[0012] As an alternative, the operation method of the hybridization includes microfluidics, homogenization, extrusion through an extruder, PEG-mediated membrane fusion, calcium ion triggering, charge inversion, biotin-avidin bridging, or low-intensity ultrasound.

[0013] As an alternative, the preparation method of the lipid nanoparticles loaded with tumor antigen mRNA includes: dissolving LNP raw materials containing cationic lipid and co-lipid in alcohol in proportion to form an alcohol phase; dissolving tumor antigen mRNA in an acidic buffer to form an aqueous phase; mixing the alcohol phase and the aqueous phase to obtain a primary emulsion; dialyzing and concentrating the primary emulsion to obtain lipid nanoparticles loaded with tumor antigen mRNA.

[0014] As an alternative, the cationic lipid is SM102, and the co-lipids include DSPC, cholesterol, and DMG-PEG2000; the ratio is SM102:DSPC:cholesterol:DMG-PEG2000 mass ratio = 50:10:38.5:1.5.

[0015] As an alternative, the mixing of the alcohol phase and the aqueous phase includes mixing by microfluidics under the conditions that the molar ratio of nitrogen in SM102 to nucleic acid in mRNA is 6 and the volume ratio of the alcohol phase to the aqueous phase is 3.

[0016] As an alternative, the method for hybridizing the Chinese herbal medicine nanovesicles with lipid nanoparticles loaded with tumor antigen mRNA includes: mixing and assembling the Chinese herbal medicine nanovesicles and the lipid nanoparticles loaded with tumor antigen mRNA by the extrusion method or the microfluidic method according to the ratio of the protein mass of the Chinese herbal medicine nanovesicles to the mass of the tumor antigen mRNA of 40:1 to obtain the tumor mRNA vaccine.

[0017] As an alternative, the cationic lipid includes one or a combination of more than one of SM102, DOTAP, DDAB, DOTMA, Dlin-MC3-DMA, ALC-0315, DOSPA, DOGS, DMG-PEG2000 or DSG-PEG2000.

[0018] As an alternative, the co-lipid includes one or a combination of more than one of cholesterol, DOPE, DSPC, β-sitosterol, natural lecithin, natural plant lipid components or DSPE-PEG2000.

[0019] As an alternative, the administration methods of the tumor mRNA vaccine include nasal instillation, aerosol inhalation, subcutaneous inoculation, intramuscular injection, oral administration, intranodal injection; it also includes injecting the activated dendritic cells into the lymph nodes, veins or subcutaneous tissues after activating the patient's bone marrow-derived dendritic cells with the tumor vaccine in vitro.

[0020] In the second aspect of the present invention, there is provided the use of the tumor mRNA vaccine according to any of the above-mentioned schemes in the preparation of drugs for preventing and treating tumor diseases or inhibiting tumor recurrence or metastasis.

[0021] As an alternative, the tumor includes melanoma, colorectal tumor, breast tumor, lymphoma or bladder tumor.

[0022] In the third aspect of the present invention, there is provided a dendritic cell vaccine, including bone marrow-derived dendritic cells activated by the tumor mRNA vaccine according to any of the above-mentioned schemes.

[0023] In the fourth aspect of the present invention, there is provided a tumor treatment composition, including the tumor mRNA vaccine according to any of the above-mentioned schemes and a tumor treatment drug, and the tumor treatment drug includes a chemotherapy drug, a radiotherapy drug, siRNA, an immune checkpoint blocker, T cells, CAR-T cells or an oncolytic virus.

[0024] The present invention establishes a personalized tumor mRNA vaccine by encapsulating tumor mRNA antigen cationic carrier LNP inside Chinese herbal medicine nanovesicles or chimerizing it with Chinese herbal medicine nanovesicles. It can be prepared on a large scale through hybrid methods such as microfluidics, maintaining the biocompatibility of the personalized vaccine and reducing the safety risks of cationic lipid carriers. At the same time, the Chinese herbal medicine vesicle-LNP hybrid mRNA vaccine can integrate tumor antigens, adjuvants, and delivery carriers, effectively promoting the phagocytosis and translation of mRNA by antigen-presenting cells, promoting the maturation and antigen presentation ability of antigen-presenting cells. Finally, it enhances the generation efficiency of specific T lymphocytes and the anti-tumor immune protection of the vaccine. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is a schematic diagram of the construction process of a low-toxic Chinese herbal medicine vesicle-LNP hybrid tumor mRNA vaccine according to an embodiment of the present invention. Taking the ginseng nanovesicles G-EVLPs of Chinese herbal medicine as an example. The tumor antigen mRNA@SM102 vaccine is prepared by microfluidics. The ginseng nanovesicles G-EVLPs obtained by differential centrifugation are mixed with mRNA and mRNA@SM102 and then extruded to obtain the mRNA@G-EVLPs (denoted as mRNA@G) tumor vaccine and the mRNA@SM102@G-EVLPs (denoted as SM102@G) tumor vaccine.

[0026] Figure 2 It is the dynamic light scattering (DLS) data of the mRNA@SM102 tumor vaccine. The average particle size of the mRNA@SM102 tumor vaccine is about 142.0 nm, the ζ potential is greater than 0, and the distribution is relatively uniform.

[0027] Figure 3 It is the dynamic light scattering (DLS) data of the SM102@G tumor vaccine. The average particle size of the SM102@G tumor vaccine is about 220.0 nm, the ζ potential is less than 0, and the distribution uniformity is insufficient compared with mRNA@SM102.

[0028] Figure 4 It is the total protein SDS-PAGE images of ginseng nanovesicles (G-EVLPs / GdNPs), mRNA, mRNA@SM102, mRNA@G, and SM102@G. mRNA@G and SM102@G still have the total protein band characteristics of G-EVLPs after extrusion.

[0029] Figure 5These are transmission electron microscope (TEM) images of mRNA@SM102, G-EVLPs, and SM102@G. mRNA@SM102 is characterized by a solid spherical structure. After G-EVLPs encapsulate mRNA@SM102, SM102@G still has the characteristics of a bilayer lipid membrane, and the particle size is approximately 200 nm.

[0030] Figure 6 This shows the expression of CD80 and CD86 proteins on the surface of mature BMDCs analyzed by flow cytometry after co-incubation of mRNA@SM102, G-EVLPs, SM102@G, and bone marrow-derived dendritic cells (BMDCs). SM102@G can significantly promote the maturation of BMDCs.

[0031] Figure 7 This shows the uptake of mature BMDCs analyzed by flow cytometry (ICG + ) after co-incubation of fluorescently labeled mRNA@SM102, G-EVLPs, SM102@G, and bone marrow-derived dendritic cells (BMDCs). SM102@G can significantly promote the uptake level of mRNA@SM102.

[0032] Figure 8 These are the tumor volumes, mouse weights, complete response rates (CR, no recurrence of tumors in mice), and lymph node pictures of the tumor recurrence model and long-term anti-tumor recurrence model after subcutaneous vaccination with the melanoma B16F10-OVA vaccine. The figure shows that the SM102@G tumor vaccine can effectively inhibit the recurrence of B16F10-OVA melanoma and significantly prolong the survival period of mice. In long-term anti-tumor protection, the recurrence rate can still be lower than 20% 60 days after vaccination. At the same time, after vaccination with the SM102@G vaccine, the lymph nodes of mice are significantly enlarged compared to the mRNA@SM102 group, indicating a significant enhancement of the vaccine effect.

[0033] Figure 9 These are the levels of pro-inflammatory cytokines in the sera of mice after vaccination with the SM102@G tumor vaccine, including IFN-γ, IL-1β, IL-6, IL-12p70, and TNF-α. The figure shows that the SM102@G tumor vaccine can effectively stimulate the secretion level of anti-tumor cytokines.

[0034] Figure 10 These are the tumor volumes, mouse weights, mouse survival rates, and tumor weights of the tumor vaccine treatment model after subcutaneous vaccination with melanoma B16F10-OVA. This treatment model is combined with the treatment of the immune checkpoint blocker PD-1 antibody. The SM102@G tumor vaccine combined with PD-1 can effectively inhibit the progression of B16F10-OVA melanoma and significantly prolong the survival period of mice.

[0035] Figure 11 Tumor growth curve and partial remission rate (PR, tumor size in mice decreased and was less than 150 mm after treatment) of the tumor vaccine treatment model after subcutaneous inoculation with melanoma B16F10-OVA 3 ). In this treatment model, immune checkpoint blocker PD-1 antibody was used for treatment. The SM102@G tumor vaccine combined with PD-1 could effectively inhibit the progression of B16F10-OVA melanoma and increase the partial remission rate of tumors in treated mice.

[0036] Figure 12 Mouse tumor photos of the tumor vaccine treatment model after subcutaneous inoculation with melanoma B16F10-OVA. In this treatment model, immune checkpoint blocker PD-1 antibody was used for treatment. The SM102@G tumor vaccine combined with PD-1 could effectively inhibit the growth of B16F10-OVA melanoma, and some tumors almost completely disappeared.

[0037] Figure 13 Immunohistochemical staining (PD-L1) pictures of mouse tumor sections of the tumor vaccine treatment model after subcutaneous inoculation with melanoma B16F10-OVA. In this treatment model, immune checkpoint blocker PD-1 antibody was used for treatment. G-EVLPs could reduce the expression of PD-L1 in tumors, and SM102 would further enhance the expression of PD-L1 in tumors. The SM102@G tumor vaccine combined with PD-1 could effectively inhibit the increase in PD-L1 expression caused by SM102, laying a foundation for the combination of PD-1 immunotherapy.

[0038] Figure 14 Immunofluorescence staining (CD8+ T lymphocytes) pictures of mouse tumors of the tumor vaccine treatment model after subcutaneous inoculation with melanoma B16F10-OVA. In this treatment model, immune checkpoint blocker PD-1 antibody was used for treatment. The SM102@G tumor vaccine combined with PD-1 could effectively increase the infiltration level of CD8+ T lymphocytes in B16F10-OVA melanoma tumors, enhancing the effect of the combined treatment.

[0039] Figure 15 Mouse inguinal lymph node mass of the tumor vaccine treatment model after subcutaneous inoculation with melanoma B16F10-OVA. In this treatment model, immune checkpoint blocker PD-1 antibody was used for treatment. The lymph nodes of mice in the SM102@G tumor vaccine group were significantly larger than those in the mRNA@SM102 group, indicating a significant enhancement of the vaccine effect.

[0040] Figure 16This is a photo of the inguinal lymph nodes of mice in a tumor vaccine treatment model after subcutaneous inoculation with melanoma B16F10-OVA. This treatment model is combined with immunotherapy using the checkpoint inhibitor anti-PD-1 antibody. The lymph nodes of mice in the SM102@G tumor vaccine group were significantly enlarged compared to those in the mRNA@SM102 group, indicating a significant enhancement of the vaccine effect.

[0041] Figure 17 This shows the levels of pro-inflammatory cytokines in the serum of mice after inoculation with the SM102@G tumor vaccine in a subcutaneous B16F10-OVA treatment model, including IFN-γ, IL-1β, IL-6, IL-12p70, and TNF-α. The figure shows that the SM102@G tumor vaccine can effectively stimulate the secretion level of anti-tumor cytokines.

[0042] Figure 18 This shows the data of serum biochemical indexes of mice after inoculation with the SM102@G tumor vaccine, including liver and kidney indexes such as AST, ALT, BUN, CREA, ALP, and γ-GT. The figure shows that the SM102@G tumor vaccine has high safety.

[0043] Figure 19 This shows the blood cell data of mice after inoculation with the SM102@G tumor vaccine, including indexes such as the number of white blood cells, lymphocytes, red blood cells, platelets, blood protein level, and percentage of monocytes. The figure shows that the SM102@G tumor vaccine has high safety. Detailed implementation mode

[0044] The technical solution of the present invention will be described in detail below in conjunction with the accompanying drawings and specific implementation modes.

[0045] The present invention provides a tumor mRNA vaccine hybridized by Chinese herbal medicine vesicles and LNP. This tumor mRNA vaccine is obtained by hybridizing Chinese herbal medicine nanovesicles with lipid nanoparticles loaded with tumor antigen mRNA (denoted as mRNA LNP).

[0046] For the Chinese herbal medicine nanovesicles, the Chinese herbal medicine nanovesicles are nanovesicles or exosomes extracted from fresh Chinese herbal medicine plants. The types of Chinese herbal medicine include ginseng, etc. The extraction methods of Chinese herbal medicine nanovesicles include differential centrifugation, dialysis, ultracentrifugation, density gradient centrifugation, ultrafiltration, magnetic bead immunoisolation, size exclusion, and cationic polymer precipitation, etc.

[0047] Exosomes or nanovesicles derived from Chinese herbal medicines or plants have natural biocompatibility, low immunogenicity, target organ specificity, and the ability to cross biological barriers. These exosomes or nanovesicles have a phospholipid bilayer structure that can effectively encapsulate mRNA or cationic lipid carriers, and surface proteins can mediate natural targeting. Research has shown that some exosomes or nanovesicles derived from Chinese herbal medicines or plants can evade immune surveillance, prolong the in vivo circulation time, and can be further optimized for targeting ability through genetic engineering or chemical modification. Compared with synthetic carriers, the characteristics of exosomes or nanovesicles derived from Chinese herbal medicines or plants are more in line with clinical safety requirements.

[0048] For tumor antigen mRNA, including model antigen mRNA, universal tumor antigen mRNA, neoantigen mRNA, tumor-associated antigen mRNA, tumor fusion antigen mRNA, etc.

[0049] For the method of loading tumor mRNA with LNP, including: dissolving the LNP raw materials containing cationic lipids and co-lipids in alcohol in proportion to form an alcohol phase; dissolving tumor antigen mRNA in an acidic buffer to form an aqueous phase; mixing the alcohol phase and the aqueous phase to obtain a primary emulsion; dialyzing and concentrating the primary emulsion to obtain lipid nanoparticles loaded with tumor antigen mRNA. LNP is a composite system composed of multiple components. The LNP raw materials mentioned above are the respective components of the LNP composite delivery system, including ionizable cationic lipids (such as SM102) and co-lipids (such as DSPC, cholesterol, DMG-PEG2000). As an alternative LNP formulation, the cationic lipid can also be one or a combination of more than one of SM102, DOTAP, DDAB, DOTMA, Dlin-MC3-DMA, ALC-0315, DOSPA, DOGS, DMG-PEG2000, or DSG-PEG2000. The co-lipid can also be one or a combination of more than one of cholesterol, DOPE, DSPC, β-sitosterol, natural lecithin, natural plant lipid components, or DSPE-PEG2000.

[0050] In a preferred embodiment, the components of the LNP composite system used are SM102, DSPC, cholesterol, and DMG-PEG2000. After multiple experiments and optimizations, the mass ratios of the above components are 50:10:38.5:1.5 in sequence. Dissolve the above components in an alcohol such as ethanol to form an alcohol phase. Dissolve the tumor antigen mRNA to be loaded in an acidic buffer such as sodium citrate buffer at pH 4.0 to form an aqueous phase. Then mix the alcohol phase and the aqueous phase to form a crude suspension, i.e., the primary emulsion. More specifically, for the mixing ratio of the alcohol phase and the aqueous phase, after multiple experiments and optimizations, it is preferably mixed under the conditions of a nitrogen-phosphorus ratio of 6 and an alcohol phase / aqueous phase volume ratio of 3. When mixing specifically, the microfluidic method can be used, i.e., prepared on a microfluidic device to obtain the primary emulsion (i.e., the suspension). Then dialyze the primary emulsion for a certain time, such as 2 h, and then concentrate it to the required concentration through an ultrafiltration tube to prepare lipid nanoparticles loaded with mRNA (mRNA LNP). Through further optimization of the above various ratios and other parameter values, the LNP can preferably load the tumor antigen mRNA and achieve good results when hybridized with Chinese herbal medicine nanovesicles subsequently.

[0051] The hybridization of Chinese herbal medicine nanovesicles and mRNA LNP refers to combining Chinese herbal medicine nanovesicles with lipid nanoparticles LNP loaded with tumor antigen mRNA to form a composite structure in the form of fusion, encapsulation, or chimerism.

[0052] More specific hybridization operation methods also include methods such as the microfluidic method, the homogenization method, the extrusion method using an extruder, the PEG-mediated membrane fusion method, the calcium ion triggering method, the charge reversal method, the biotin-avidin bridging method, or the low-intensity ultrasound method. Among them, the microfluidic method refers to using a microfluidic chip (such as the laminar flow focusing method) to control the precise mixing of LNP and vesicles, and forming a uniform encapsulation structure (the particle size can be controlled within 80 - 150 nm) by adjusting the flow rate ratio (aqueous phase:oil phase) and the channel size. The homogenization method refers to mixing LNP and plant vesicles, adding the above mixed liquid to a homogenizer, and circulating it repeatedly 20 - 25 times under a pressure of 200 - 800 Pa. Use a condensation device to ensure that the temperature of the mixed liquid is 4 - 8 °C to obtain it. The extrusion method using an extruder refers to mixing LNP and plant vesicles and then extruding them through a polycarbonate membrane (such as a pore size of 100 - 200 nm) multiple times, and using the shear force to reorganize the vesicle membrane and encapsulate LNP. The PEG-mediated membrane fusion method refers to modifying short-chain PEG (such as DSPE-PEG2000) on the surfaces of LNP and vesicles respectively, and promoting membrane fusion through the dehydration effect of PEG. The calcium ion triggering method refers to using Ca 2+Neutralize the negative charges of the vesicles and the phospholipids of the LNP membrane, inducing temporary membrane fusion, and subsequently remove free calcium ions with EDTA. The charge reversal method involves mixing LNP (positively charged) with negatively charged plant vesicles (such as soybean vesicles), forming a loose complex through electrostatic adsorption, and then promoting the internalization of LNP by the vesicles through mild sonication or pH adjustment (such as pH 5.0). The biotin-avidin bridging method refers to modifying biotin and streptavidin on the surfaces of LNP and vesicles respectively, and achieving directional encapsulation by utilizing the high-affinity binding. The encapsulation efficiency of this method is high (>90%). The low-intensity sonication method means that after mixing LNP and plant vesicles, the above mixed liquid is placed under an ultrasonic probe for sonication. Under ice bath conditions, sonicate for 5 min with an ultrasonic intensity of 125 - 250 W, stopping for 2 s every 3 s of sonication, and then it is obtained. For the specific protocols and implementation effects of hybridization, for example, in one embodiment of the present invention (as Figure 1 shown, it is the specific construction method of the tumor mRNA vaccine of this protocol), hybridize the mRNA encapsulated by LNP (i.e., lipid nanoparticles loaded with tumor antigen mRNA) with plant-derived nanovesicles to form a structure similar to a "shell-core" structure. The inner layer of the hybrid particles, SM102, is used to compress the tumor antigen mRNA, and the outer layer utilizes Chinese herbal medicine vesicles to provide a biocompatibility barrier and a targeting barrier. This design uses Chinese herbal medicine vesicles to neutralize the surface charge of LNP, reducing non-specific adsorption; its natural membrane proteins and natural sterol compounds can promote enrichment at the tumor site, while the pH-responsive phospholipids of LNP can trigger the release of mRNA in the acidic environment of the lysosomes of antigen-presenting cells (APCs). This structure combines the high efficiency of synthetic materials and the safety of natural carriers, breaking through the bottleneck of the existing technology.

[0053] Based on the tumor mRNA vaccines of the present invention constructed according to the above various protocols, the dosage forms include liquid preparations, etc., and the final vaccination methods include but are not limited to: nasal instillation, aerosol inhalation, subcutaneous injection, intramuscular injection, oral administration, intranodal injection, injection into lymph nodes after in vitro activation of bone marrow-derived dendritic cells of patients, injection into veins after in vitro activation of bone marrow-derived dendritic cells of patients, injection subcutaneously after in vitro activation of bone marrow-derived dendritic cells of patients, etc. Correspondingly, the present invention also provides a dendritic cell vaccine, which is the bone marrow-derived dendritic cells activated by the tumor mRNA vaccine of the present invention.

[0054] Thus, the tumor mRNA vaccine of the present invention can also be used as the main component or combined component of a tumor treatment drug in tumor treatment; more specifically, the present invention also provides the application of the tumor mRNA vaccine in the preparation of drugs for preventing and treating tumor diseases or inhibiting tumor recurrence or metastasis. In some protocols, the tumors include melanoma, colorectal tumors, breast tumors, lymphoma, or bladder tumors, etc.

[0055] In addition, the Chinese herbal medicine vesicle-LNP hybrid tumor mRNA vaccine of the present invention can also be used in combination in conventional and immunotherapy, and the conventional and immunotherapy includes chemotherapy, radiotherapy, siRNA therapy, PD-1 / PD-L1 immune checkpoint blockade therapy, adoptive T cell transfer therapy, CAR-T therapy, oncolytic virus therapy, etc., or CRISPR technology or small molecule drugs, etc.

[0056] That is, the present invention also provides a tumor treatment composition, which includes the above-mentioned tumor mRNA vaccine of the present invention and a tumor treatment drug, and the tumor treatment drug includes a chemotherapeutic drug, a radiotherapy drug, an siRNA drug, an immune checkpoint inhibitor, a T cell, a CAR-T cell or an oncolytic virus, etc.

[0057] Furthermore, the tumor mRNA vaccine solution of the present invention can also be combined with gene-editable oncolytic virus therapy to establish a universal Chinese herbal medicine vesicle-LNP hybrid tumor mRNA vaccine, reducing the antigen screening step.

[0058] In addition, as a vaccine, the present invention also provides a storage method thereof, including storing in a liquid form at 4°C, preparing a freeze-dried powder injection for storage, storing by combining with a gel matrix, storing by adding a preservative, or storing by adding stabilizers such as a co-emulsifier, etc.

[0059] During the process, the methods for verifying the activity of the prepared tumor mRNA vaccine include but are not limited to: in vitro verification of the uptake and activation of bone marrow-derived dendritic cells, in vivo verification of lymph node accumulation, and in vivo verification of the activation of lymph node dendritic cells, etc.

[0060] Taking ginseng nano-vesicles (G-EVLPs) as an example of the Chinese herbal medicine vesicles and SM102 as an example of the cationic lipid of LNP, in one embodiment, the establishment of the low-toxicity Chinese herbal medicine vesicle-LNP hybrid tumor mRNA vaccine (SM102@G) mainly includes three steps: The first step is to obtain Chinese herbal medicine nano-vesicles by density gradient centrifugation as an integrated platform for adjuvant and carrier for tumor antigen delivery.

[0061] The second step is to use microfluidic technology to establish cationic lipid particles LNP loaded with tumor antigen mRNA, and establish mRNA LNP loaded with tumor antigen (mRNA@SM102).

[0062] The third step is to hybridize the above-mentioned Chinese herbal medicine nano-vesicles and mRNA LNP to establish a Chinese herbal medicine nano-vesicle-LNP hybrid tumor mRNA vaccine (SM102@G), which can deliver tumor antigen mRNA, promote the translation of mRNA by antigen-presenting cells, and enhance the maturation of dendritic cells and the expression of MHC-I-antigen peptide complexes.

[0063] Compared with the prior art, the present invention has obvious innovation. Compared with a single carrier, this composite system has three-fold innovation: (1) Chinese herbal medicine nanovesicles reduce the systemic toxicity, non-specific adsorption and targeting of SM102, reducing the safety risk of SM102; (2) Through the surface protein and glycolipid characteristics of the Chinese herbal medicine nanovesicles themselves, the purpose of active targeting is achieved, enhancing the delivery efficiency of SM102 in lymph nodes; (3) The characteristic of the Chinese herbal medicine nanovesicles themselves to synergistically activate immune responses can enhance the antigen peptide presentation level of SM102 to dendritic cells. Experiments have confirmed that this carrier significantly enhances the antigen presentation ability of antigen-presenting cells in a mouse model, and significantly reverses the expression levels such as PD-L1, improving whole blood indexes and serum biochemical indexes.

[0064] In the following more specific Examples 1-10, the model antigen OVA mRNA was used as the research object to verify the universality of the above platform. The cationic lipid and Chinese herbal medicine nanovesicles were verified using SM102 and ginseng nanovesicles respectively.

[0065] Specifically, the prepared ginseng nanovesicle-LNP hybrid tumor mRNA vaccine (SM102@G) was verified for its effectiveness at the cellular level and animal level respectively. Based on the established tumor recurrence, tumor progression, orthotopic tumor and tumor metastasis models, the therapeutic effect of the tumor vaccine SM102@G was verified. Tumor models from different sources were established to repeatedly verify the efficacy and conduct safety evaluations on it.

[0066] Explanation of the abbreviations that may be involved in the following specific examples: Control group (Control), a completely blank group without any experiments; SM102 vector group of the model antigen OVA mRNA (OVA mRNA@SM102, mRNA@SM102), only using the cationic lipid SM102 to encapsulate the model antigen OVA mRNA for stimulation, that is, only tumor model antigen OVA mRNA stimulation and vector stimulation; Ginseng nanovesicle group (G-EVLPs), only using ginseng nanovesicles for stimulation, that is, only the role of adjuvant, without containing tumor model antigen; Ginseng nanovesicle-LNP hybrid group (SM102@G), using microfluidics and other methods to encapsulate G-EVLPs outside SM102 to form a "shell-core" structure, that is, the tumor model antigen OVA mRNA and immune adjuvant are delivered simultaneously. Among them, combined immunotherapy with the checkpoint inhibitor PD-1 was carried out for synergy.

[0067] Example 1: Isolation and extraction of ginseng nanovesicles, and the acquisition process is as follows: ① Obtaining crude fresh ginseng extract: Take fresh ginseng (genuine medicinal material from September to October), wash it with double-distilled water, add it to a blender to extract the crude extract juice of fresh ginseng, and centrifuge it at 200 rpm for 15 min using a high-speed centrifuge; centrifuge it at 2000 rpm for 30 min; then centrifuge it at 10000 rpm for 60 min, and repeat this step 3 - 5 times.

[0068] ② Extracting fresh ginseng nanovesicles: Collect the supernatant after the above centrifugation, centrifuge it at 100000 rpm for 60 min using an ultra-high-speed centrifuge, collect the precipitate, add 1×PBS solution, and gently mix it with an injector. Use a BCA protein quantification kit to determine the concentration of fresh ginseng nanovesicles. Store the obtained fresh ginseng nanovesicles at -80°C for long-term preservation.

[0069] Example 2: Establishment of fresh ginseng nanovesicle-LNP hybrid tumor mRNA vaccine (as Figure 1 ), and the specific process is as follows: ① Preparation of mRNA@SM102: Dissolve SM102, DSPC, cholesterol, and DMG-PEG2000 in ethanol at a ratio of 50:10:38.5:1.5 to form an alcohol phase. Dissolve mRNA in sodium citrate buffer at pH 4.0 to form an aqueous phase. Prepare it on a microfluidic device under the conditions of a nitrogen-to-phosphorus ratio (the molar ratio of nitrogen in SM102 to nucleic acid in mRNA) of 6 and an alcohol-phase / aqueous-phase volume ratio of 3 to obtain the primary emulsion. Dialyze the primary emulsion for 2 h and concentrate it to the required concentration through an ultrafiltration tube.

[0070] ② Preparation of fresh ginseng nanovesicle-LNP hybrid tumor mRNA vaccine: Mix the above G-EVLPs and mRNA@SM102 according to the concentration (G-EVLPs protein amount: mRNA mass = 40:1 ratio), and prepare SM102@G by extrusion using an extruder / microfluidic device preparation method.

[0071] Example 3: Characterization of fresh ginseng nanovesicle-LNP hybrid tumor mRNA vaccine, and the specific process is as follows: ① Particle size distribution characterization: Collect the above mRNA@SM102 and SM102@G solutions, and use a dynamic light scattering instrument and a nanoparticle size analyzer to characterize the particle size distribution of the above two solutions ( Figure 2 and Figure 3 ).

[0072] ② Component characteristic characterization: Use SDS-PAGE, proteomics, and non-target lipidomics techniques to characterize the obtained G-EVLPs and SM102@G solutions ( Figure 4 ).

[0073] ③ Particle morphology characterization: Collect the above G-EVLPs, mRNA@SM102, and SM102@G solutions, and use a transmission electron microscope to characterize the particle morphology of the above three solutions ( Figure 5 ).

[0074] As shown in the figure, the average particle size of SM102@G is about 220.0 nm, and the ζ potential is less than 0 mV. The protein electrophoresis strip of SM102@G has obvious characteristics, and the SDS-PAGE band situation is similar to that of ginseng nanovesicles G-EVLPs. G-EVLPs contain various lipid, protein, nucleic acid, and organic acid components and have a lipid bilayer structure, which is a significant exosome characteristic.

[0075] Example 4: Effects of fresh ginseng nanovesicle-LNP hybrid tumor mRNA vaccine on the uptake and maturation of dendritic cells in vitro. The specific process is as follows: ① Activation of bone marrow-derived dendritic cells (BMDCs) by SM102@G: Extract, induce, and culture BMDCs, and co-incubate the above G-EVLPs, mRNA@SM102, and SM102@G with BMDCs for 24 hours (the administration concentration of mRNA is 1 μg / mL, and the administration concentration of G-EVLPs is 10 μg / mL). Use a flow cytometer to analyze the levels of BMDC activation markers CD80 and CD86 ( Figure 6 ).

[0076] ② Uptake of SM102@G by BMDCs: Extract, induce, and culture BMDCs. Encapsulate ICG inside SM102, and use DiD to label G-EVLPs. Co-incubate the above fluorescently labeled G-EVLPs, mRNA@SM102, and SM102@G with BMDCs for 24 hours (the administration concentration of mRNA is 1 μg / mL, and the administration concentration of G-EVLPs is 10 μg / mL). Use flow cytometry to analyze the uptake of G-EVLPs and mRNA@SM102 by BMDCs ( Figure 7 ).

[0077] ③ Specific anti-tumor immune response of SM102@G: Extract BMDCs, co-incubate SM102@G with BMDCs, and isolate T lymphocytes from the spleen to the above BMDC cells. Use flow cytometry to analyze CD45 + CD3 + CD8 +The proportion of T cells; The cells in the supernatant were collected and added to a 24-well plate coated with tumor cells (B16F10-OVA). The LDH content in the supernatant (a marker of tumor cell death) was measured using a kit, and the release level of IFN-γ in the supernatant was measured using an ELISA kit, which was the activation level of specific anti-tumor immune response.

[0078] As Figure 6 、 Figure 7 shown, SM102@G can significantly enhance the maturation level of BMDCs, and the expression levels of surface CD80 and CD86 molecular markers increase significantly. At the same time, SM102@G hybridized with G-EVLPs can significantly improve the phagocytosis level of BMDCs for SM102 (and its internal mRNA).

[0079] Example 5: Evaluation of the activation of specific anti-tumor immune response in vivo of fresh ginseng nanovesicle-LNP hybrid tumor mRNA vaccine. Taking the model antigen OVA mRNA as an example, the specific measurement process is as follows: ① Lymph node targeting of SM102@G vaccine: The G-EVLPs were labeled with the fluorescent molecule DiD, and ICG was encapsulated in mRNA@SM102 to prepare the fluorescently labeled SM102@G vaccine. The above-prepared mRNA vaccine was subcutaneously inoculated, and the targeting situation was analyzed by small animal imaging, and the fluorescence intensity of each tissue organ (especially lymph nodes) was counted. Single cells of the lymph nodes were isolated, and the phagocytosis efficiency of CD11c + cells for the above was analyzed by flow cytometry, which was the lymph node targeting efficiency of SM102@G.

[0080] ② Formation of immune memory by SM102@G vaccine: The SM102@G vaccine was subcutaneously injected, and then the spleen after vaccine inoculation was taken out and a single cell suspension was prepared. Flow cytometry was used to analyze CD45 + CD3 + CD8 + CD44 high CD62L low The proportion of memory T cells, which was the formation intensity of immune memory.

[0081] The results showed that SM102@G can significantly improve the targeting effect of mRNA on lymph nodes and DC cells in lymph nodes, and enhance the generation of central memory T cells in the spleen.

[0082] Example 6: Pharmacodynamic evaluation of anti-tumor recurrence in vivo of fresh ginseng nanovesicle-LNP hybrid tumor mRNA vaccine. Taking the model antigen OVA mRNA as an example, the specific measurement process is as follows: ① Establishment of melanoma recurrence model: 3×10 melanoma B16F10-OVA cells were subcutaneously inoculated under the axilla of mice5 mice, 95% of the tumors were surgically resected 15 days after inoculation. The above tumor vaccines (G-EVLPs, mRNA@SM102, and SM102@G) were subcutaneously inoculated 1 day, 4 days, and 8 days after tumor resection, and the tumor volume, tumor weight, body weight, survival rate, serum cytokine levels, and complete response rate were detected ( Figure 8 ).

[0083] ② Evaluation of long-term immune protection: The above mice underwent complete tumor resection on day 24, and 3×10 5 B16F10-OVA melanoma cells were re-subcutaneously inoculated on day 30, and the tumor volume, tumor weight, body weight, survival rate, serum cytokine levels, and complete response rate were detected ( Figure 8 ). The mice were euthanized on day 40, and the levels of inflammatory cytokines in the mouse serum were measured.

[0084] The results are as Figure 8 shown. The tumor volume data show that the SM102@G vaccine can significantly inhibit the recurrence of B16F10-OVA tumors and form long-term anti-tumor protection. At the same time, the complete response rate (complete inhibition rate) of B16F10-OVA tumors after SM102@G vaccine inoculation can be increased to 80%, effectively prolonging the survival period of mice, and the survival rate is about 100%. There was no significant change in body weight before and after vaccination and tumor inoculation, indicating that SM102@G has high biocompatibility. As Figure 9 shown, the SM102@G vaccine can significantly increase the levels of pro-inflammatory cytokines in the serum after tumor inoculation.

[0085] Example 7: Pharmacodynamic study of the anti-tumor acute metastasis effect of fresh ginseng nanovesicle-LNP hybrid tumor mRNA vaccine in vivo. Taking the model antigen OVA mRNA as an example, the specific determination process is as follows: Establishment of melanoma B16F10-OVA tumor acute metastasis model: Vaccines (G-EVLPs, mRNA@SM102, and SM102@G) were subcutaneously inoculated on days 1, 4, and 8. Five days later, tumor cells (B16F10-OVA, 1×10 5 ) were injected via the tail vein to simulate the acute metastasis of tumors during tumor resection, and the body weight, number of tumor lung metastases, and survival rate of mice were monitored after acute metastasis modeling.

[0086] Example 8: Pharmacodynamic study of the anti-tumor effect of fresh ginseng nanovesicle-LNP hybrid tumor mRNA vaccine in vivo. Taking the model antigen OVA mRNA as an example, the specific determination process is as follows: Establishment of melanoma progression model: First, 3×10 5mice were subcutaneously inoculated with the vaccine on days 1, 4, and 8 after tumor inoculation, and immune checkpoint blocker PD-1 was administered at intervals. Subsequently, the body weight, tumor volume, tumor weight, lymph node mass, survival rate, intratumoral PD-L1 expression, and intratumoral CD8 + T cell infiltration were continuously detected.

[0087] The results were as Figure 9 - 16 shown. Tumor data showed that SM102@G vaccine + PD-1 could significantly inhibit the progression of B16F10-OVA tumors and form long-term anti-tumor protective ability. At the same time, the partial response rate of B16F10-OVA tumors (tumor regression and final volume less than 100 mm 3 ) after vaccination with SM102@G vaccine + PD-1 could be increased to 100%, effectively prolonging the survival period of mice. The survival rate of mice in the SM102@G vaccine + PD-1 group was about 100%. Before and after vaccination and tumor inoculation, the body weight of mice did not change significantly, indicating that SM102@G combined with PD-1 had high biocompatibility, but could enhance the vaccine activity of SM012 in lymph nodes, increasing the volume and mass of lymph nodes. SM102@G could significantly enhance the infiltration level of intratumoral CD8+ T cells. More importantly, G-EVLPs could significantly reverse the high expression of intratumoral PD-L1 caused by mRNA@SM102 injection, alleviate the immunosuppressive microenvironment, and enhance the effect of immunotherapy. As Figure 17 shown, SM102@G vaccine + PD-1 could significantly increase the levels of pro-inflammatory cytokines in serum after tumor inoculation Example 9: Pharmacodynamic study of the combination of fresh ginseng nanovesicle-LNP hybrid tumor mRNA vaccine and oncolytic virus. Taking the model antigen OVA mRNA as an example, the specific determination process is as follows: ① Pharmacodynamic evaluation of tumor cells mixed in proportion: The B16F10 and B16F10-OVA cell lines were mixed evenly at ratios of 10:0, 9:1, 7:3, 5:5, and 0:10. 3×10 5 of the above-mentioned evenly mixed melanoma B16F10 cells were subcutaneously administered. The SM102@G vaccine was subcutaneously inoculated on days 1, 4, and 8 after tumor inoculation, and immune checkpoint blocker PD-1 was administered at intervals. Subsequently, the body weight, tumor volume, and survival rate of mice were continuously detected.

[0088] ② Oncolytic virus pharmacodynamics and OVA editing activity: An oncolytic virus strain with OVA editing activity was established. 3×10 5 of the above-mentioned evenly mixed melanoma B16F10 cells were subcutaneously administered. The above oncolytic virus was intratumorally inoculated on days 10 and 20 after tumor inoculation. The body weight, tumor volume, survival rate of mice, and positive expression rate of OVA in B16F10 cells were continuously detected.

[0089] ③ Efficacy of combined treatment with SM102@G and oncolytic virus: Subcutaneously administer 3×10 5 of the above - mixed melanoma B16F10 cells. Intratumorally inoculate the oncolytic virus 5 days and 10 days after tumor inoculation. Subcutaneously inoculate the SM102@G vaccine 3 days, 8 days, and 15 days after tumor inoculation. Intermittently administer the immune checkpoint blocker PD - 1, and then continuously detect the body weight, tumor volume, and survival rate of the mice.

[0090] The results show that SM102@G can significantly inhibit the volume of mixed tumors in mice; the oncolytic virus can, while inhibiting the tumor volume, promote the tumor to express heterologous antigens not encoded by the tumor itself; the combination of SM102 and the oncolytic virus can be used as a tumor - universal vaccine strategy to inhibit or reverse tumor growth.

[0091] Example 10: In - vivo biocompatibility study of fresh ginseng nanovesicle - LNP hybrid tumor mRNA vaccine. Taking the model antigen OVA mRNA as an example, the specific determination process is as follows: ① Pathological detection of main organs after administration of SM102@G vaccine: Take the main organs such as the heart, liver, spleen, lungs, kidneys, and brain of the mice inoculated with the above vaccine, and perform pathological (H&E) staining.

[0092] ② Detection of blood and main serum biochemical indexes after administration of SM102@G vaccine: Take the blood of the mice inoculated with the above vaccine. For one part, directly measure the blood cell indexes; for the other part, coagulate at room temperature for 30 min, centrifuge at 2500 rpm for 5 min, and then detect the biochemical indexes (AST, ALT, BUN, CREA, ALP, and γ - GT) after obtaining the serum.

[0093] The results are as Figure 18 and Figure 19 shown. After administration of the SM102@G vaccine, there are no obvious damage changes in the morphology of the main tissues and organs, indicating good biocompatibility. The results are as Figure 18 and Figure 19 shown. The detection results of serum biochemical indexes such as AST, ALT, BUN, CREA, ALP, and γ - GT after administration of the SM102@G vaccine show no significant changes, indicating the good biocompatibility and safety of the SM102@G vaccine.

Claims

1. A tumor mRNA vaccine hybridized with Chinese herbal vesicles and LNP, characterized in that: It is obtained by hybridizing Chinese herbal medicine nanovesicles with lipid nanoparticles loaded with tumor antigen mRNA.

2. The tumor mRNA vaccine according to claim 1, characterized in that The Chinese herbal medicine includes ginseng; the tumor antigen includes a combination of one or more of pattern antigens, universal tumor antigens, new tumor antigens, tumor-associated antigens or tumor fusion antigens.

3. The tumor mRNA vaccine according to claim 1, characterized in that The hybridization operation method includes microfluidics, homogenization, extruder extrusion, PEG-mediated membrane fusion, calcium ion triggering, charge reversal, biotin-avidin bridging or low-intensity ultrasound.

4. The tumor mRNA vaccine according to claim 1, characterized in that The method for preparing lipid nanoparticles loaded with tumor antigen mRNA comprises: dissolving LNP raw materials containing cationic lipids and auxiliary lipids in alcohols in proportion to form an alcohol phase; dissolving the tumor antigen mRNA in an acidic buffer to form an aqueous phase; mixing the alcohol phase with the aqueous phase to obtain colostrum; dialyzing and concentrating the colostrum to obtain lipid nanoparticles loaded with tumor antigen mRNA.

5. The tumor mRNA vaccine according to claim 4, characterized in that The cationic lipid is SM102, and the auxiliary lipid includes DSPC, cholesterol and DMG-PEG2000; the mass ratio of SM102: DSPC: cholesterol: DMG-PEG2000 is 50:10:38.5:1.

5.

6. The tumor mRNA vaccine according to claim 5, characterized in that The mixing of the alcohol phase and the water phase includes mixing by microfluidics under the conditions that the molar ratio of nitrogen in SM102 to nucleic acid in tumor antigen mRNA is 6 and the volume ratio of the alcohol phase to the water phase is 3.

7. The tumor mRNA vaccine according to claim 6, characterized in that The method for hybridizing the Chinese herbal medicine nanovesicles and lipid nanoparticles loaded with tumor antigen mRNA includes: mixing and assembling the Chinese herbal medicine nanovesicles and lipid nanoparticles loaded with tumor antigen mRNA in a ratio of Chinese herbal medicine nanovesicle protein mass: tumor antigen mRNA mass = 40:1, using an extruder extrusion method or a microfluidics method to obtain the tumor mRNA vaccine.

8. The tumor mRNA vaccine according to claim 4, characterized in that The cationic lipid includes one or more combinations of SM102, DOTAP, DDAB, DOTMA, Dlin-MC3-DMA, ALC-0315, DOSPA, DOGS, DMG-PEG2000 or DSG-PEG2000.

9. The tumor mRNA vaccine according to claim 4, characterized in that The auxiliary lipid includes one or more combinations of cholesterol, DOPE, DSPC, β-sitosterol, natural lecithin, plant natural lipid components or DSPE-PEG2000.

10. The tumor mRNA vaccine according to any one of claims 1 to 9, characterized in that: The administration methods of the tumor mRNA vaccine include nasal drops, aerosol inhalation, subcutaneous inoculation, intramuscular injection, oral administration, and lymph node injection; and also include activating the patient's bone marrow-derived dendritic cells with the tumor vaccine in vitro and then injecting the activated dendritic cells into the lymph nodes, intravenously, or subcutaneously.

11. Use of the tumor mRNA vaccine according to any one of claims 1 to 9 in the preparation of drugs for preventing or treating tumor diseases or for inhibiting tumor recurrence or metastasis.

12. The use according to claim 11, characterized in that: The tumor includes a melanoma, a colorectal tumor, a breast tumor, a lymphocytic tumor, or a bladder tumor.

13. A dendritic cell vaccine, characterized in that: It includes bone marrow-derived dendritic cells activated by the tumor mRNA vaccine described in any one of claims 1-9.

14. A tumor treatment composition, characterized in that: It includes the tumor mRNA vaccine and tumor therapeutic drug described in any one of claims 1-9, and the tumor therapeutic drug includes chemotherapy drugs, radiotherapy drugs, siRNA, immune checkpoint blockers, T cells, CAR-T cells or oncolytic viruses.

Images