Novel vaccine for preventing and treating colorectal cancer by PLGA (poly (lactic-co-glycolic acid)) loaded whole tumor cell RNA (ribonucleic acid) and APS (astragalus polysaccharide)

Abstract

The invention provides a nano vaccine for colorectal cancer immunotherapy and a preparation method thereof, the nano vaccine takes a biodegradable polylactic acid-glycolic acid copolymer (PLGA) as a carrier, a multiple emulsion solvent evaporation method is adopted, a total RNA antigen of colorectal cancer CT-26 cells and an immunologic adjuvant astragalus polysaccharide (APS) are jointly encapsulated, RNA / APS-coated PLGA nanoparticles are prepared, and the RNA / APS-coated PLGA nanoparticles are used for immunotherapy of colorectal cancer. The particle size of the nano-particles is about 220 nm, the Zeta potential is about-23 mV, the nano-particles have good stability and dispersity, the nano-particles can be efficiently taken by dendritic cells in vivo, the immune response of CD8 + T cells is remarkably enhanced, the proportion of immunosuppressive cells is reduced, and the tumor immune microenvironment is remodeled, so that the growth of colorectal cancer is effectively inhibited. The vaccine has good biological safety, and a novel, safe and effective nano vaccine strategy is provided for immunotherapy of colorectal cancer.

Description

Technical Field

[0001] This invention relates to the field of biomedicine and nanodelivery technology, specifically to a nanovaccine RNA / APS@PLGA that uses polylactic-co-glycolic acid copolymer (PLGA) as a carrier to encapsulate whole colorectal cancer tumor cell RNA and astragalus polysaccharide (APS), for the prevention and immunotherapy of colorectal cancer. Background Technology

[0002] Colorectal cancer (CRC) is the third most common malignant tumor worldwide and the second leading cause of cancer-related deaths. Despite continuous advancements in early screening and comprehensive treatment strategies, approximately 20% of patients have metastases at initial diagnosis, and 40% experience recurrence after radical surgery. The 5-year survival rate for metastatic CRC is less than 20%. Traditional surgery, radiotherapy, chemotherapy, and targeted therapy have limited efficacy in advanced-stage patients and significant toxic side effects. Therefore, developing safe and effective new strategies is an urgent clinical need.

[0003] In recent years, tumor immunotherapy has demonstrated advantages such as durable response and low toxicity by activating the body's immune system to recognize and eliminate tumor cells. Among these, therapeutic tumor vaccines aim to induce tumor antigen-specific T-cell responses, forming immune memory, thereby eliminating residual lesions and preventing recurrence. However, vaccine development still faces the following bottlenecks: 1. Limited selection of tumor antigens: Universal tumor-associated antigens (TAAs) have weak immunogenicity, while personalized neoantigens have long preparation cycles and high costs.

[0004] 2. Poor stability of RNA vaccines: Naked RNA is easily degraded by RNase in vivo, resulting in low cellular uptake efficiency and difficulty in effectively delivering it to antigen-presenting cells (APCs).

[0005] 3. Immunosuppressive microenvironment: The tumor microenvironment (TME) of colorectal cancer is rich in regulatory T cells (Tregs), myeloid-derived suppressor cells (MDSCs), and M2 macrophages, which significantly inhibit vaccine-induced effector T cell function.

[0006] 4. Lack of efficient and safe carriers: Existing liposomes or viral carriers have problems such as insufficient targeting, potential tumorigenicity or inflammatory side effects.

[0007] Therefore, developing a novel vaccine platform that combines "antigen-specific activation" and "tumor microenvironment remodeling" is of great significance for the prevention and treatment of colorectal cancer. Summary of the Invention

[0008] Existing tumor RNA vaccines suffer from drawbacks such as easy RNA degradation, insufficient immunogenicity, and lack of improvement in the immunosuppressive microenvironment, resulting in limited efficacy against colorectal cancer. This invention provides a scalable PLGA nanovaccine that co-encapsulates tumor RNA with the immunoadjuvant astragalus polysaccharide, simultaneously overcoming the aforementioned three drawbacks and thereby enhancing the immunotherapeutic efficacy against colorectal cancer.

[0009] A novel vaccine for the prevention and treatment of colorectal cancer, consisting mainly of the following components, is a polylactic acid-glycolic acid copolymer loaded with whole tumor cell RNA and astragalus polysaccharide: a) Polylactic acid-glycolic acid copolymer nanoparticle carrier; b) Astragalus polysaccharides encapsulated within polylactic acid-glycolic acid copolymer; c) Total RNA of colorectal cancer cells encapsulated within polylactic acid-glycolic acid copolymer.

[0010] The novel vaccine, which uses polylactic acid-glycolic acid copolymer to load whole tumor cell RNA and astragalus polysaccharide, has an average particle size of 200-250 nm, a zeta potential of -20 to -25 mV, an RNA encapsulation efficiency of ≥20%, and an astragalus polysaccharide encapsulation efficiency of ≥55%.

[0011] The monomer molar ratio of polylactic acid-glycolic acid copolymer is 75:25, and the molecular weight is 15-20 kDa; the molecular weight of Astragalus polysaccharide is 50-200 kDa, and the purity is ≥90%; the tumor RNA is derived from the mouse colorectal cancer cell line CT-26, and the concentration is 0.5-2 μg / μL.

[0012] A novel vaccine loaded with whole tumor cell RNA and astragalus polysaccharide on polylactic-co-glycolic acid copolymer is prepared by the following steps: a) Preparation of the internal aqueous phase: Dissolve tumor RNA and Astragalus polysaccharide in RNase-free water; b) Preparation of the oil phase: Dissolve the polylactic acid-glycolic acid copolymer in dichloromethane; c) Preparation of colostrum: Add W1 to O and emulsify by ultrasonication to form colostrum; d) Preparation of double emulsion: Add the colostrum to a polyvinyl alcohol aqueous solution and sonicate again to form a double emulsion; e) Stir to evaporate the organic solvent, centrifuge and wash, freeze dry to obtain nano-vaccine powder, and store at -20℃ for a long time.

[0013] A novel vaccine based on polylactic acid-glycolic acid copolymer loaded with whole tumor cell RNA and astragalus polysaccharide, wherein the polyvinyl alcohol concentration is 2-5% w / v, the stirring speed is 600-1000 rpm, and the evaporation time is 4-6 hours.

[0014] The use of a novel vaccine, consisting of polylactic acid-glycolic acid copolymer loaded with whole tumor cell RNA and astragalus polysaccharide, for the prevention of colorectal cancer, is characterized by: a) Administer a vaccine for prophylactic immunization 21 days prior to tumor inoculation; b) Vaccine immunization is administered subcutaneously every 7 days, for a total of 3 times; c) 100 μL of nano-vaccine is injected each time.

[0015] A novel vaccine, based on polylactic-co-glycolic acid copolymer loaded with whole-cell RNA and astragalus polysaccharide, can induce: a) Increased expression of dendritic cell maturation markers CD80, CD86, and MHC I / II; b) Nanoparticle vaccines significantly enhance tumor-infiltrating CD8 + T cell quantity and function; c) The proportion of M1 macrophages increases and the proportion of immunosuppressive cells such as regulatory T cells, myeloid suppressor cells, and M2 macrophages decreases in the tumor microenvironment; d) Tumor growth inhibition.

[0016] A novel vaccine, in lyophilized powder form, is a polylactic acid-glycolic acid copolymer loaded with whole tumor cell RNA and astragalus polysaccharide.

[0017] Beneficial effects 1. Immune synergy: Physical co-encapsulation of RNA and Astragalus polysaccharide significantly increased T cell activation compared to RNA alone or physical mixing.

[0018] 2. Safe and biodegradable: PLGA metabolites are human-natured lactic acid / glycolic acid, which do not accumulate; the maximum tolerated dose (mice) is >200 mg / kg, which is more than 20 times the effective dose.

[0019] 3. Process-friendly: The double emulsion method uses an all-aqueous / water-oil system, eliminating the need for high-speed homogenization or microfluidics.

[0020] 4. Universal Expansion: It can be extended to other solid tumors simply by replacing the tumor RNA, meeting the needs of personalized precision treatment. Attached Figure Description

[0021] Figure 1 This is a flowchart illustrating the preparation process of the RNA / APS@PLGA nanovaccine in this invention embodiment; Figure 2 This is a characterization diagram of the RNA / APS@PLGA nanovaccine in an embodiment of the present invention; Figure 3 This is a fluorescence micrograph of the RNA / APS@PLGA nanovaccine being taken up by dendritic cells (DC2.4); Figure 4 These are tumor growth curves and survival curves in a mouse CT-26 model. Figure 5 This is a diagram showing the maturation of dendritic cells induced by nano-vaccines; Figure 6 Nanoparticle vaccines enhance tumor infiltration CD8 + T cell number and function diagram; Figure 7 This is a diagram of the microenvironment in which a nanovaccine reverses tumor immunosuppression. Figure 8 This is a graph showing the safety evaluation results of the RNA / APS@PLGA nanovaccine.

[0022] The present invention will be further described in detail below through specific embodiments.

[0023] Example 1: Preparation of RNA / APS@PLGA nanovaccine 1.1 Total RNA extraction from whole tumor cells (1) Cryopreserved cell thawing: The frozen mouse colon cancer CT-26 (ATCC: CRL-2638, purchased from Wuhan Pusai Life Science Technology Co., Ltd.) cell suspension was removed from liquid nitrogen and thawed rapidly in a 37°C water bath. The thawed cell suspension was then mixed with 4-6 mL of complete culture medium (RPMI-1640 + 10% F) Mix BS (1% P / S) and centrifuge at 1000 RPM for 5 minutes. Discard the supernatant and resuspend the cells in complete culture medium. Then add the cell suspension to a culture flask containing 4-5 mL of complete culture medium and incubate overnight in a cell culture incubator (culture conditions: 95% air + 5% carbon dioxide, temperature: 37℃, humidity 70%-80%).

[0024] (2) Cell passage: When the cell density reaches 80%, passage culture can be performed. Aspirate the original culture medium; Add approximately 2 ml of PBS, gently agitate the culture flask to rinse the cells, and discard the PBS. Add approximately 1 ml of 0.25% trypsin solution (containing EDTA), gently agitate the culture flask to infiltrate all cells. Place the flask in an incubator for digestion. After digestion is complete, collect the cell suspension, centrifuge at 1000 RPM for 5 minutes, and discard the supernatant. Then add fresh culture medium, gently pipette to mix the cells, seed them into new culture flasks at the appropriate ratio, add an appropriate amount of culture medium, and place the flasks in a cell culture incubator for further culture.

[0025] (3) Total RNA extraction: a. Collecting adherent cells: Discard the original culture medium, add 2 mL of 1× PBS to wash, discard the PBS, and repeat twice; 1×10 7Add 1 mL of TRNzol to each cell and lyse at room temperature for 10 minutes. Mix well by pipetting with an RNase-free pipette tip and transfer the solution to a 1.5 mL RNase-free EP tube. b. Chloroform extraction: Add 200 μL of chloroform to each 1 mL of TRNzol (TRNzol:chloroform volume ratio is 5:1). Shake the tube vigorously by hand for 10 seconds, incubate at room temperature for 2-3 minutes to ensure that chloroform and TRNzol are thoroughly mixed; pre-cool the tube in a refrigerated centrifuge beforehand, and centrifuge at 4°C, 12000 RPM (13400 g) for 15 minutes; c. Take the aqueous phase: Carefully aspirate approximately 500 μL of the upper aqueous phase into a new RNase-free EP tube; d. Isopropanol precipitation: Add an equal volume of isopropanol to the aqueous phase, invert to mix, incubate at room temperature for 10 minutes, centrifuge at 12000 RPM (13400 g) for 10 minutes at 4°C. After centrifugation, a small amount of white precipitate can be seen at the bottom of the tube. e. Ethanol washing: Carefully aspirate the supernatant, gently add 1 mL of 75% ethanol to wash, and centrifuge at 10000 RPM for 5 minutes at 4°C. f. Dissolve the precipitate after drying: When the RNA precipitate is slightly moistened and gel-like at room temperature, add 30-100 μL of LDPPC-treated water as needed for the experiment.

[0026] g. RNA quality testing: Take 1 μL of sample, and use NanoDrop to determine 260 / 280 ≥ 1.9 and 260 / 230 ≥ 1.8; use agarose gel electrophoresis to determine the 28S / 18S band brightness ratio ≈ 2.0 and RIN value ≥ 7.0; after 0.22 μm sterile filtration, store at -80 ℃ and use within 4 hours.

[0027] 1.2 Preparation of RNA / APS@PLGA nanovaccine Because the loaded RNA and drug are water-soluble, the RNA / APS@PLGA nanovaccine was prepared using a water-in-oil-in-water (W1 / O / W2) double emulsion solvent evaporation method. (See [link to relevant documentation]). Figure 1 .

[0028] (1) Preparation of the inner aqueous phase (W1): W1 mainly consists of a mixture of 100 μg CT-26 whole tumor cell RNA solution (100 μL) and 10 mg / mL APS aqueous solution (100 μL).

[0029] (2) Preparation of PLGA oil phase (O): Accurately weigh 100 mg PLGA (lactic acid: glycolic acid = 75:25, molecular weight 15000, purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.) and dissolve it in 2 mL of chloroform, vortex until fully dissolved.

[0030] (3) External aqueous phase (W2): Prepare a 5 mg / mL PVA (Mw 9-10 kDa, degree of hydrolysis 85-89%) aqueous solution, and use it after autoclaving at 121℃ for 15 min; before the experiment, add 0.02% (w / v) sodium azide to inhibit microorganisms, or use 0.22 μm filtration for sterilization, and store at 4℃.

[0031] (4) Ultrasonic emulsification: The inner aqueous phase (W1) was added to the PLGA oil phase (O), and ultrasonically disrupted in an ice-water bath for 2 minutes (power 200W, working for 5 seconds, intermittent for 5 seconds) to obtain the primary emulsion (W1 / O). Then, 5 mL of 40 mg / mL PVA aqueous solution (W2) was added to the W1 / O system, and ultrasonically disrupted in an ice-water bath for 5 minutes (power 200W, working for 5 seconds, intermittent for 5 seconds) to obtain the secondary emulsion (W1 / O / W2).

[0032] (5) Evaporation solvent: Add the double emulsion (W1 / O / W2) dropwise to 40 mL of 5 mg / mL PVA aqueous solution, stir at room temperature for 4-5 hours until the trichloromethane is completely evaporated.

[0033] (6) Centrifugation and washing: Centrifuge 12000g at 4℃ for 15 minutes, discard the supernatant, and resuspend and wash 3 times with enzyme-free water.

[0034] (7) Freeze-drying: After pre-freezing the obtained nanoparticle suspension at -80℃, freeze-dry it under vacuum for 24 hours to obtain a white freeze-dried powder, which is stored at -20℃.

[0035] (8) Sterility and endotoxin test: The lyophilized powder was reconstituted with 2 mL of pyrogen-free water, filtered through a 0.22 μm sterile filter, and tested according to the Limulus amebocyte lysate (LAL) reagent method in General Chapter 1143 of Part IV of the 2020 edition of the Chinese Pharmacopoeia. The sterility test met the requirements of General Chapter 1101.

[0036] Example 2: Characterization of Nanovaccines The nanovaccine prepared in Example 1 was characterized as follows: The particle size, zeta potential, and PDI (polydispersity index) of the nanoparticles were determined using a laser diffraction particle size analyzer. See [link to relevant documentation]. Figure 2 AC analysis showed that the vaccine particle size was 228.8±4.6nm, PDI was 0.20±0.03, and surface potential was -23.2±1.5mV, which met the design requirements. This indicates that the vaccine particle size was uniform, the system was stable, and it conformed to the microstructure characteristics of nano-vaccines.

[0037] Morphological observation: Weigh 2 mg of lyophilized RNA / APS@PLGA nanoparticles into an EP tube and resuspend in 500 μL of deionized water to achieve the appropriate concentration. Place a sample onto a copper grid specifically designed for electron microscopy. After the sample dries, add 2% (w / v) phosphotungstic acid (PTA) staining solution for 15 min. Then, gently blot away excess PTCA solution with filter paper along the edge of the copper grid. Allow the sample to dry completely before observing its morphology under a transmission electron microscope (TEM). See also... Figure 2 D. The results showed that the RNA / APS@PLGA nanoparticles were smooth, near-spherical particles, consistent with the ultrastructural characteristics of nanovaccines.

[0038] Example 3: Evaluation of the in vitro cellular uptake effect of nano-vaccines To verify whether RNA / APS@PLGA nanoparticles could be taken up by mouse dendritic cell DC2.4 cells (ATCC: CRL-2887), DC2.4 cells were co-incubated with fluorescently labeled RNA / APS@PLGA nanovaccine for 24 hours (cell nuclei were labeled with blue DAPI, and nanoparticles were labeled with green FITC). See also Figure 3 Fluorescence microscopy showed that the FITC fluorescence signal intensity of the RNA / APS@PLGA nanovaccine group was significantly higher than that of the negative control group, and a large number of green fluorescence signals were clearly visible in the cells, proving that the RNA / APS@PLGA nanovaccine could be phagocytosed by mouse dendritic cells DC2.4.

[0039] Example 4: Evaluation of the in vivo tumor prevention effect of nano-vaccines 4.1 Laboratory Animals Ten SPF-grade 4–5-week-old male BALB / c mice were housed at the Experimental Animal Center of Ningxia Medical University. The experimental protocol was approved by the university's Animal Ethics Committee (approval number IACUC-NYLAC-2023-161), and all procedures complied with the "Regulations on the Management of Laboratory Animals".

[0040] 4.2 Vaccination and Grouping Mice were randomly divided into 2 groups (n=5): ① PBS group (blank control); ② RNA / APS@PLGA group (prevention group).

[0041] See Figure 4 Timeline A: On days -21, -14, and -7, 100 μL of PBS or RNA / APS@PLGA nanovaccine (containing 20 μg RNA and 10 μg APS) were subcutaneously injected to complete three basic immunizations.

[0042] 4.3 Establishment of a subcutaneous model of colon cancer On day 7 after the last immunization, mice were subcutaneously injected with 100 μL (5 × 10⁶) of CT-26 cell suspension under the right axilla. 5 (One per animal). Shave the animal and disinfect with 75% ethanol before vaccination.

[0043] 4.4 Observation and Measurement Results Starting from day 3 post-inoculation, mouse weight, behavior, and injection site reactions were monitored every other day. Once the tumor was palpable, the major axis (a) and minor axis (b) were measured every two days using digital calipers, and the values ​​were calculated using the formula V = a × b. 2 / 2 Calculate the volume. See also Figure 4 Compared with the PBS-treated group, mice treated with RNA / APS@PLGA nanovaccine showed reduced subcutaneous tumor volume and significantly decreased tumor weight after resection.

[0044] 4.5 Endpoint Processing On day 21 post-inoculation, the patient was euthanized by CO2 asphyxiation. The tumor was completely removed, and after the surface blood was blotted dry with filter paper, it was photographed, weighed, and the maximum values ​​of a and b were measured to calculate the final volume. At the same time, draining lymph nodes and organs of the heart, liver, spleen, lungs, and kidneys were collected for subsequent immunological and safety analysis.

[0045] Example 5: Study on the immune mechanism of nano-vaccines 5.1 Tissue Sampling and Lymphocyte Isolation After the mice were euthanized at the endpoint, tumor tissue was aseptically obtained. The tumor tissue was minced and digested with collagenase IV (1 mg / mL) at 37 °C for 30 min. The tissue was then passed through a steel mesh and centrifuged with tumor-infiltrating lymphocyte separation medium to obtain tumor-infiltrating lymphocytes (TILs). The suspension was washed twice with PBS and counted with trypan blue. The viable cells were >90%.

[0046] 5.2 Flow Cytometry Antibody Labeling and Detection 5.2.1 DC Maturity Marker and Detection Take 1×10 6 For each cell / tube, add the following surface antibody combination: CD11c-APC, CD80-PE, CD86-APC-Cy7, MHC I (H-2Kb)-BV421, MHC II (IA / IE)-BV510, incubate at 4°C for 30 minutes, wash twice with PBS, and immediately collect data using a CytoFLEX flow cytometer and analyze with FlowJo v10.

[0047] 5.2.2 T cell activation markers and detection Take 1×10 6For each cell / tube, add the following surface antibody combination: CD3ε-FITC, CD4-FITC, CD8-PE, incubate at 4°C for 30 minutes, wash twice with PBS, and immediately collect data using a CytoFLEX flow cytometer and analyze with FlowJo v10.

[0048] Intracellular factor staining: Following the instructions of the eBioscience fixation / permeabilization kit, TNF-α-PE and IFN-γ-PerCP-Cy5.5 were added sequentially, incubated at 4 °C for 35 min, washed, and resuspended in 300 μL PBS. Immediately, samples were collected using a CytoFLEX flow cytometer and analyzed using FlowJo v10.

[0049] 5.2.3 Tumor Microenvironment Markers and Detection Take 1×10 6 For each cell / tube, add the following surface antibody combination: CD4-FITC, FOXP3-eFluor™450, PE-CD11b, APC-GR-1, CD86-eFluor™450, F4 / 80-APC-eFluor™780, CD206-APC, incubate at 4°C for 30 minutes, wash twice with PBS, and immediately collect cells using a CytoFLEX flow cytometer and analyze with FlowJo v10.

[0050] 5.3 Results 5.3.1 DC Maturity: See Figure 5 AD, vaccine group CD11c + In cells, the proportion of double positivity for CD80, CD86, MHC I, and MHC II was significantly higher than that in the PBS group.

[0051] 5.3.2 Tumor infiltration CD8 + T cell activation: See also Figure 6 AC, compared with the PBS group, CD8 in tumor tissue of the vaccine group + T cells, CD8 + IFN-γ + and CD8 + TNF-α + The significantly increased proportion of T cells indicates that the vaccine can significantly activate the body's cellular immune response and promote tumor-infiltrating CD8 cells. + T cells are activated, thereby exerting an anti-tumor effect.

[0052] 5.3.3 Nanoparticle vaccines reverse the tumor immunosuppressive microenvironment: See Figure 7In the tumor microenvironment of the AD vaccine group, compared with the PBS group, the ratio of Treg, MDSC, and M2 macrophages was significantly reduced, while the ratio of M1 macrophages was significantly increased, indicating that the immunosuppressive state of the tumor microenvironment was significantly reversed after vaccine prevention.

[0053] Example 6: Safety Evaluation of Nanoparticle Vaccines 6.1 Histopathology: See Figure 8 A. After euthanasia, mice were dissected and their major organs, including the heart, liver, spleen, lungs, and kidneys, were removed. These organs were fixed in 4% paraformaldehyde, dehydrated, embedded in paraffin, sectioned (4 μm), stained with H&E, and then their morphology was observed under a microscope. The results showed that the major organ tissues of the vaccine-treated mice had intact structures, normal cell morphology, and uniform staining, with no obvious pathological changes such as inflammation or necrosis, showing no significant difference compared to the PBS control group.

[0054] 6.2 Serum biochemistry: See Figure 8 B. Blood was collected from the eyeballs of mice, and the serum was separated and tested using a fully automated biochemical analyzer. The results showed that the serum ALT, AST, BUN, and CREA levels of the vaccine-prevented mice were all within the normal reference range, and there was no significant difference compared with the PBS control group (P>0.05), indicating that the vaccine had no obvious liver and kidney toxicity and had high biosafety.

[0055] The embodiments of this solution have been described in detail above. However, this solution is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of this patent.

Claims

1. A novel vaccine for the prevention and treatment of colorectal cancer, comprising polylactic acid-glycolic acid copolymer loaded with whole tumor cell RNA and astragalus polysaccharide, characterized in that, The vaccine consists of the following components: a) Polylactic acid-glycolic acid copolymer nanoparticle carrier; b) Astragalus polysaccharides encapsulated within polylactic acid-glycolic acid copolymer; c) Total RNA of colorectal cancer cells encapsulated within polylactic acid-glycolic acid copolymer.

2. The novel vaccine as described in claim 1, characterized in that: The novel vaccine has an average particle size of 200-250 nm, a zeta potential of -20 to -25 mV, an RNA encapsulation rate of ≥20%, and an astragalus polysaccharide encapsulation rate of ≥55%.

3. The novel vaccine as described in claim 1, characterized in that: The monomer molar ratio of polylactic acid-glycolic acid copolymer is 75:25, and the molecular weight is 15-20 kDa; the molecular weight of Astragalus polysaccharide is 50-200 kDa, and the purity is ≥90%; the tumor RNA is derived from the mouse colorectal cancer cell line CT-26, and the concentration is 0.5-2 μg / μL.

4. The novel vaccine according to claim 1, wherein the preparation method comprises the following steps: a) Preparation of the internal aqueous phase: Dissolve tumor RNA and Astragalus polysaccharide in RNase-free water; b) Preparation of the oil phase: Dissolve the polylactic acid-glycolic acid copolymer in dichloromethane; c) Preparation of colostrum: Add W1 to O and emulsify by ultrasonication to form colostrum; d) Preparation of double emulsion: Add the colostrum to a polyvinyl alcohol aqueous solution and sonicate again to form a double emulsion; e) Stir to evaporate the organic solvent, centrifuge and wash, freeze dry to obtain nano-vaccine powder, and store at -20℃ for a long time.

5. The novel vaccine according to claim 4, wherein the polyvinyl alcohol concentration is 2-5% w / v, the stirring speed is 600-1000 rpm, and the evaporation time is 4-6 hours.

6. The novel vaccine according to claim 1, used for the prevention of colorectal cancer, is characterized in that: a) Administer a vaccine for prophylactic immunization 21 days prior to tumor inoculation; b) Vaccine immunization is administered subcutaneously every 7 days, for a total of 3 times; c) 100 μL of nano-vaccine is injected each time.

7. The novel vaccine according to claim 6, wherein the nanovaccine can induce: a) Increased expression of dendritic cell maturation markers CD80, CD86, and MHC I / II; b) Nanovaccines significantly enhance the number and function of tumor-infiltrating CD8 cells; + T c) The proportion of M1 macrophages increases and the proportion of immunosuppressive cells such as regulatory T cells, myeloid suppressor cells, and M2 macrophages decreases in the tumor microenvironment; d) Tumor growth inhibition.

8. The novel vaccine according to any one of claims 1-7, characterized in that, The vaccine is in the form of freeze-dried powder.

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