A low-temperature preparation method for nanoparticle vaccines

Abstract

The present invention discloses a low-temperature preparation method of a nanoparticle vaccine. The present invention uses fluorine-substituted functional nucleic acid and antigen peptide as raw materials. Under low-temperature conditions, the two raw materials self-assemble in an aqueous solution through the action of molecular electrostatic attraction and dispersion force to form a nano-vaccine complex. The method for preparing a nano-vaccine of the present invention is simple to prepare, green, mild in reaction conditions and low in cost, and realizes the co-delivery of immune adjuvants and antigen peptides without a carrier, avoids the immunogenicity brought by the carrier, and realizes the assembly of antigens with a high loading rate. The present invention overcomes the limitations of the preparation of traditional nucleic acid nanostructures, avoids the loss of antigens caused by high temperatures, and demonstrates a simple and universal method for realizing the direct self-assembly of any nucleic acid and any antigen peptide, which has important scientific significance for the development and preparation of nano-vaccines with different functions. Therefore, the present invention is expected to provide theoretical and experimental technical support for the preparation of effective tumor nano-vaccines.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and specifically relates to a low-temperature preparation method of a nanoparticle vaccine. Background Art

[0002] Functional nucleic acids are divided into deoxyribonucleic acid and ribonucleic acid. According to the different functions of nucleic acids, they can be divided into siRNA, mRNA and antisense nucleic acids, etc., and are currently widely used in the field of biomedicine. Although functional nucleic acids are widely used, their utilization rate is relatively low, mainly because they are extremely susceptible to nuclease degradation and low transfection efficiency in organisms, which seriously affects the application of functional nucleic acids. Peptides are natural targets for binding to targeted protein targets, especially non-drug targets. Therefore, peptides are immunogenic and are widely used in cancer vaccines. In particular, new antigen peptides are only expressed in tumors but not in normal cells, thus bypassing central thymic tolerance, having high immunogenicity, affinity with major histocompatibility complexes (MHCs), and activating tumor-specific CD4+ and CD8+ T cells to produce immune responses. However, the immunogenicity of new antigens is usually weak, and the cancer vaccine formulations currently explored in clinical practice cannot induce strong tumor antigen-specific CD8+ T cell immune responses, which are crucial for the anti-tumor immunity of tumor vaccines. This is mainly due to inefficient accumulation of tumor vaccines in draining lymph nodes (LNs), insufficient cellular uptake by antigen-presenting cells (APCs), and low levels of antigen cross-presentation.

[0003] Nanoparticle-based cancer vaccines (nanovaccines) are widely used for the co-delivery of antigens and adjuvants, which is attributed to the fact that nanoparticles (<200nm) can not only selectively accumulate in draining lymph nodes to increase the immunogenicity and stability of antigens, but also achieve APC-targeted delivery and enhanced cross-presentation. However, the carrier materials of nanovaccines (such as liposomes, metal organic frameworks, mesoporous silica, polymers) themselves may cause host reactions and produce short-term and long-term toxicity during degradation, metabolism and excretion in the body. In addition, carrier materials can produce unexpected immune responses, such as the common polymer carrier polyethylene glycol (PEG), which has been identified as immunogenic. Injection of PEGylated nanoparticles into mice induces polyethylene glycol (PEG)-specific antibodies, resulting in rapid clearance of PEG-based drug or vaccine delivery systems from the body. In addition, insufficient antigen loading: antigens and adjuvants are mostly encapsulated or coupled to nanoparticles, which leads to insufficient loading of antigens and adjuvants on nanoparticles (less than <20%), which reduces the immunogenicity of nanovaccines in vivo and weakens the immune response, making it difficult to obtain a strong immune response. Furthermore, while protecting the antigens, the nanoframe material also hinders antigen capture and exposure, making it difficult for nanovaccines to mediate effective immune responses by displaying multiple copies like viruses. Therefore, it is still an important technical issue to develop a simple and universal immune adjuvant and antigen direct assembly for co-delivery to reduce the complexity of synthesis and increase the flexibility of synthesis. Further development of a biocompatible, non-toxic and degradable antigen carrier as an immune adjuvant that can effectively present the antigen to the cytoplasm of APCs is still challenging to construct a safe, high-performance, and effectively antigen-loaded nano cancer vaccine.

[0004] Therefore, the present invention reports a new strategy to solve these problems. We self-assemble fluorine-substituted functional nucleic acids and antigenic peptides under low temperature conditions; due to the molecular electrostatic attraction and dispersion force, the two can easily form nanomaterials. Fluorine-substituted nucleic acids can destroy the hydrogen bonding between bases, reverse the spatial structure of nucleic acids, increase the active sites of nucleic acids, thereby improving the assembly ability of nucleic acids, and are easier to assemble into nanostructures with antigenic peptides than non-fluorine-substituted ones. In addition, due to the formation of nanoparticles, the resistance of functional nucleic acids and antigenic peptides to protease hydrolysis and the efficiency of cytoplasmic delivery are improved. The hydrophobic and lipophobic nature of fluorine-substituted materials also greatly improves the lysosomal escape ability of nanomaterials, thereby reducing the degradation of functional nucleic acids and antigenic peptides in lysosomes. Due to the size effect of nanomaterials, it can be effectively enriched in draining lymph nodes, and after being taken up by APC cells, it can stimulate DC cell maturation, better stimulate effective antigen-specific CD8+ cytotoxic T lymphocyte (CTL) response and effective immune-mediated tumor therapy. This method of self-assembly of fluorine-substituted functional nucleic acids and antigenic peptides to form nanoparticles has important scientific significance for the development and preparation of nanovaccines with different functions. Summary of the invention

[0005] The purpose of the present invention is to provide a low-temperature preparation method for nanoparticle vaccines, which uses a simple self-assembly method under low-temperature conditions to self-assemble functional nucleic acids and antigen peptides, which can effectively inhibit tumor growth and promote the cytoplasmic delivery of antigen peptides.

[0006] To achieve the above object, the present invention adopts the following technical solution:

[0007] A low-temperature preparation method for a nanoparticle vaccine, characterized in that: a fluorine-substituted functional nucleic acid and an antigen peptide are uniformly mixed in a buffer, first reacted at 37°C for 1h to 24h, then reacted at 4°C for 6h to 72h, and after the reaction is completed, the precipitate is collected by centrifugation and washed to obtain the nanoparticle vaccine.

[0008] Preferably, in the low-temperature preparation method of the above-mentioned nanoparticle vaccine, the fluorine-substituted functional nucleic acid includes a modified fluorine atom nucleic acid derivative, and the nucleic acid in the fluorine-substituted functional nucleic acid is one or more of single-stranded DNA / RNA or double-stranded RNA / DNA; the fluorine in the fluorine-substituted functional nucleic acid is substituted at the 2' position of the five-membered sugar ring in the nucleic acid, or the fluorine atom is modified at any position on the base, and the number of modified fluorine atoms on the base is one or more. The antigen peptide is selected from: (i) protein antigen or fragment thereof; (ii) tumor-associated antigen or tumor neoantigen; (iii) viral antigen; the antigen peptide can specifically be any one of human papillomavirus E6 protein, human papillomavirus E7 protein, mesothelin, OVA antigen peptide (SIINFEKL or ISQAVHAAHAEINEAGR), Obsl1 antigen (REGVELCPGNKYEMRRHGTTHSLVIHD), HBV surface antigen (HBsAg), HBV core antigen (HBcAg), and hemagglutinin antigen (HA). Fluorine-substituted nucleic acids are widely used in nucleic acid aptamers and G-quadruplexes because they can improve the stability of nucleic acids. Fluorine atoms, as the most polar atom, can significantly change the physicochemical properties of nucleic acids. Fluorine-substituted nucleic acids can destroy the hydrogen bonding between bases, reverse the spatial structure of nucleic acids, increase the active sites of nucleic acids, and thus improve the assembly ability of nucleic acids. In the present invention, fluorine-substituted functional nucleic acids (CPG) with immune adjuvant properties are used as raw materials, and further self-assembled with antigen peptides at low temperatures through the effects of molecular electrostatic attraction, molecular stacking and water bridges to form functional nucleic acid nano vaccines, achieving carrier-free delivery of immune adjuvants and antigen peptides, avoiding the immunogenicity brought by the carrier, and achieving high antigen loading rate assembly. The present invention overcomes the limitations of traditional nucleic acid nanostructure preparation, and also avoids the loss of antigens caused by traditional high-temperature preparation of nano vaccines, showing a simple and universal realization of direct assembly of any nucleic acid and antigen peptides, which has important scientific significance for the development and preparation of nano vaccines with different functions.

[0009] Preferably, in the above-mentioned low-temperature preparation method of nanoparticle vaccine, the pH of the buffer solution is 6-8.

[0010] Preferably, the low-temperature preparation method of the above-mentioned nanoparticle vaccine specifically comprises the following steps:

[0011] (1) Preparation of fluorine-substituted functional nucleic acid stock solution: dissolve the fluorine-substituted functional nucleic acid in sterile water, and pipette evenly at room temperature to obtain a fluorine-substituted functional nucleic acid stock solution with a concentration of 50 μM to 200 μM;

[0012] (2) Preparation of antigen peptide stock solution: dissolving the antigen peptide in a solvent and mixing evenly at room temperature to obtain an antigen peptide stock solution with a concentration of 1 mM to 10 mM;

[0013] (3) Preparation of nanoparticle vaccine: The fluorine-substituted functional nucleic acid stock solution and the antigen peptide stock solution are uniformly mixed in a buffer solution, and the resulting mixture is first reacted at 37° C. for 1 h to 24 h, and then reacted at 4° C. for 6 h to 72 h. After the reaction is completed, the precipitate is collected and washed at 5000 to 10000 rpm for 1 min to 60 min to obtain the nanoparticle vaccine;

[0014] Wherein, in step (2), the solvent is one or more of water, dimethyl sulfoxide, dimethylformamide, acetic acid, acetonitrile, methanol, propanol, and isopropanol.

[0015] Wherein, in step (3), the molar ratio of the fluorine-substituted functional nucleic acid to the antigen peptide in the mixed solution is 1:1 to 1:100.

[0016] The resulting nanoparticle vaccine was prepared using the low temperature preparation method described above.

[0017] The use of the above-mentioned nanoparticle vaccine in the preparation of tumor immunotherapy drugs.

[0018] The beneficial effects of the present invention are:

[0019] The temperature of traditional self-assembly preparation reaches 90℃~100℃, and the reaction time is 0.1h~1.5h. Under this condition, the antigen is prone to inactivation (Food.Funct.2021,12,10083-10096;J.Funct.Foods.2015,18,28-34;J.Agric.Food.Chem.2005,53,4962-4967), and as the reaction time increases, the antigen loss becomes more serious, or even completely lost, which causes the binding ability of antigen to IgG and IgM to drop sharply, greatly reducing the immune efficiency of nano vaccines. A simple and universal method for preparing nano vaccines at low temperature provided by the present invention can realize the self-assembly of antigen peptides under low temperature conditions (reacting at 37℃ for 1h~24h, and then reacting at 4℃ for 6h~72h) to form nanoparticle vaccines, 37℃ is the most active temperature of antigen, and it is most suitable for preparing nano vaccines. In addition, nano vaccines can be grown to a suitable nano size under 4℃ conditions. The nanoparticle vaccine prepared by the present invention can realize the carrier-free co-delivery of immune adjuvants and antigen peptides, avoid the immunogenicity brought by the carrier, and achieve high antigen loading. At the same time, under low temperature conditions, the loss of antigens caused by the traditional high-temperature preparation of nano vaccines can be avoided. The present invention demonstrates a simple and universal method for realizing the direct self-assembly of any nucleic acid and any antigen peptide, which has important scientific significance for the development and preparation of nano vaccines with different functions. The method is simple, the raw materials are easily available, no carrier is added, and it has wide versatility. The resulting nanostructure has high stability and exceeds the antigen loading efficiency. The ultra-high new antigen density increases the uptake of new antigen nano vaccines by antigen presenting cells (APCs) and presents antigens, thereby enhancing the body's innate immune response and driving subsequent adaptive immune responses. As a general method, the present invention provides a reference for the design of minimalist combination vaccines in future clinical environments.

[0020] The present invention prepares the nanoparticle vaccine under low temperature conditions without causing the antigen of the antigen peptide to be lost.

[0021] The nanoparticle vaccine provided by the present invention is non-toxic and harmless and does not involve any organic solvent.

[0022] The method for preparing the nanoparticle vaccine of the present invention is simple, green, mild and low-cost.

[0023] The nanoparticle vaccine provided by the present invention has high stability, can circulate well in the body, and is enriched in the lymph node area.

[0024] The preparation strategy of the present invention can be applied to the design of minimalist combination vaccines in future clinical settings. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1Flow chart for the preparation of nanoparticle vaccines.

[0026] Figure 2 Characterization diagram of nanoparticle vaccine. Among them, (A) is the transmission electron microscopy image and particle size distribution diagram of FCPG / Obsl1 nanoparticle vaccine; (B) is the transmission electron microscopy image and particle size distribution diagram of FCPG / Obsl1-FITC nanoparticle vaccine; (C) is the ultraviolet absorption diagram of nanoparticle vaccine; (D) is the potential change diagram of FCPG / Obsl1 nanoparticle vaccine. The transmission electron microscope scale is 200nm.

[0027] Figure 3 The performance test results of FCPG / Obsl1 nanoparticle vaccine are shown in Figure 1. (A) and (B) show the stimulation and maturation of mouse bone marrow derived dendritic cells (BMDC) by nanoparticle vaccine; (C) shows the cytotoxicity of nanoparticle vaccine to BMDC; (D) shows the confocal imaging of BMDC cell uptake by nanoparticle vaccine.

[0028] Figure 4 The figure shows the situation of FCPG / Obsl1 nanoparticle vaccine preventing tumor growth. Among them, (A) is the operation flow chart of preventing growth; (B) is the schematic diagram of tumor growth; (C) is the mouse survival rate chart.

[0029] Figure 5 The figure shows the situation of FCPG / Obsl1 nanoparticle vaccine inhibiting tumor growth. Among them, (A) is the operation flow chart of inhibiting growth; (B) is the schematic diagram of tumor growth; (C) is the mouse survival rate chart. DETAILED DESCRIPTION

[0030] In order to make the contents of the present invention easier to understand, the technical solution of the present invention is further described below in conjunction with specific implementation methods, but the present invention is not limited thereto.

[0031] Example 1

[0032] The nanoparticle vaccine was assembled by using fluorine-substituted functional nucleic acid FCPG (sequence: 5'-(fA)TCCATGACGTTCCTGACGTT-3') and antigen peptide Obsl1 (sequence: N-terminal-REGVELCPGNKYEMRRHGTTHSLVIHD-C-terminal) Figure 1 ), and its specific preparation steps are as follows:

[0033] (1) Dissolve FCPG in sterile water and pipette at room temperature to evenly disperse the nucleic acid to form a 100 μM FCPG stock solution, which is then stored at -20°C.

[0034] (2) Dispersing the antigen peptide Obsl1 in secondary water at room temperature to obtain a 2 mM Obsl1 stock solution, which was then stored at -20°C;

[0035] (3) Thoroughly mix 60 μL of 100 μM FCPG stock solution, 10 μL of 2 mM Obsl1 stock solution, 12 μL of 10×PBS buffer (pH=7.2-7.4) and 38 μL of secondary water, then react the mixture at 37°C for 2 h and then at 4°C overnight. After the reaction, centrifuge at 8500 rpm for 5 min, collect the precipitate and wash twice with 1×PBS buffer (pH=7.2-7.4) to obtain FCPG / Obsl1 nanoparticle vaccine.

[0036] Example 2

[0037] The nanoparticle vaccine was assembled by using fluorine-substituted functional nucleic acid FCPG (sequence: 5'-(fA)TCCATGACGTTCCTGACGTT-3') and antigen peptide Obsl1-FITC (sequence: N-terminal-FITC-REGVELCPGNKYEMRRHGTTHSLVIHD-C-terminal), and the specific preparation steps are as follows:

[0038] (1) Dissolve FCPG in sterile water and pipette at room temperature to evenly disperse the nucleic acid to form a 100 μM FCPG stock solution, which is then stored at -20°C.

[0039] (2) Disperse the antigen peptide Obsl1-FITC in secondary water at room temperature to obtain a 2 mM Obsl1-FITC stock solution, which is then stored at -20°C;

[0040] (3) Thoroughly mix 60 μL of 100 μM FCPG stock solution, 10 μL of 2 mM Obsl1-FITC stock solution, 12 μL of 10×PBS buffer (pH=7.2-7.4) and 38 μL of secondary water, then react the mixture at 37°C for 2 h and then at 4°C overnight. After the reaction, centrifuge at 8500 rpm for 5 min, collect the precipitate and wash twice with 1×PBS buffer (pH=7.2-7.4) to obtain FCPG / Obsl1-FITC nanoparticle vaccine.

[0041] Characterization of Nanoparticle Vaccines

[0042] Figure 2Characterization diagrams of the nanoparticle vaccine prepared in Examples 1 to 2; wherein, (A) is a transmission electron microscopy image and a particle size distribution diagram of the FCPG / Obsl1 nanoparticle vaccine; (B) is a transmission electron microscopy image and a particle size distribution diagram of the FCPG / Obsl1-FITC nanoparticle vaccine; (C) is a UV absorption diagram of the nanoparticle vaccine; and (D) is a potential change diagram of the FCPG / Obsl1 nanoparticle vaccine. As can be seen from the figure, the nanoparticle vaccine is uniform in size under a transmission electron microscope; the diameter of the nanoparticles is measured by dynamic light scattering to obtain an average hydrated particle size of 81.42 nm for the FCPG / Obsl1 nanoparticle vaccine, and a surface charge of -5 mV; the average hydrated particle size of the FCPG / Obsl1-FITC nanoparticle vaccine is 93.01 nm.

[0043] Performance test

[0044] 1. Extraction of mouse bone marrow derived dendritic cells (BMDC): C57BL / 6 mice were killed by cervical dislocation, and the femur and tibia were taken under sterile conditions and immersed in RPMI-1640 culture medium. The RPMI-1640 culture medium was drawn with a 1 ml syringe, and the bone cavity was pierced from one end of the bone shaft, and the bone was flushed into a sterile culture dish. Each bone was repeated 4 to 6 times, and the bone marrow cell suspension in the culture dish was collected and centrifuged at 1500 rpm for 10 minutes: then the supernatant was discarded, and 5 ml of sterile Tris-NH4C1 solution was added to suspend the cells and dissolve the red blood cells. After standing at room temperature for 2 minutes to dissolve the red blood cells, the mixture was centrifuged again at 1500 rpm for 5 minutes, and the supernatant was discarded. Wash once with RPMI-1640 culture medium; centrifuge at 1500rpm for 5min; suspend the cells with complete culture medium (RPMI-1640; 10% FBS and containing penicillin 100U / mL and streptomycin 100μg / mL), divide into 6-well culture plates, add complete culture medium to each well to 4ml, then add rmGM-CSF (Novoprotein; CK02) to a final concentration of 20ng / ml, IL-4 (Novoprotein; CK74) to a final concentration of 10ng / ml. Place the cell culture plate in a 37℃, 5% CO2 incubator and culture for 48 hours; take out the cells and blow gently, aspirate the suspended cells together with the culture medium, and only retain the attached cells. Add fresh complete culture medium and the same concentration of rmGM-CSF, and continue to culture until the 4th day. Replace half of the medium and replenish rmGM-CSF; try to retain the suspended cells. The culture was continued until the 6th day, and all the suspended cells were collected by gently blowing with a pipette, which were the enriched mouse bone marrow derived dendritic cells (BMDC).

[0045] 2. Stimulation and maturation of BMDCs: 1×10 cells / well were seeded in each well of a 12-well plate. 6 BMDCs were then added with antigen peptide Obsl1 (final concentration 11 μg / ml), CPG+Obsl1 (physical mixture of CPG and Obsl1, final concentration [Obsl1] = 11 μg / ml) or FCPG / Obsl1 nanoparticle vaccine (final concentration [Obsl1] = 11 μg / ml) and incubated for 24 hours before collecting BMDCs. BMDCs were resuspended in flow cytometry buffer (PBS containing 1% FBS), incubated with anti-mouse CD16 / 32 antibody (BioLegend, Cat. No.: 101302) at 4°C for 15 minutes, and stained with anti-CD11c-APC (BioLegend, Cat. No.: 117310), anti-CD86-PE (BioLegend, Cat. No.: 105007) and anti-CD80-FITC (BioLegend, Cat. No.: 104705), and then analyzed by flow cytometry.

[0046] 3. Cytotoxicity experiment of nanoparticle vaccine on BMDC. BMDCs were inoculated in 96-well plates, with 1×10 4 BMDCs cells were added with FCPG / Obsl1 nanoparticle vaccine (final concentration [Obsl1] = 0, 1, 5, 10, 20, 40 μg / ml, respectively) and cultured in a humidified environment at 37°C and 5% CO2 for 24 h. Cell viability was detected using Enhanced Cell Counting Kit-8 (Biyuntian Company, Cat. No.: C0043), and the absorbance at 450 nm was measured on an SH-1000Lab microplate reader.

[0047] 4. Confocal imaging of BMDC cell uptake of nanoparticle vaccines. 4 BMDCs were inoculated in 20 mm glass-bottomed culture dishes and then incubated for 6 h with antigen peptide Obsl1-FITC (final concentration 11 μg / ml), CPG+Obsl1 (physical mixture of CPG and Obsl1, final concentration [Obsl1] = 11 μg / ml) or FCPG / Obsl1-FITC nanoparticle vaccine (final concentration [Obsl1-TITC] = 11 μg / ml). Cell nuclei were stained with Hoechst 33342 at a dilution of 1:300. After washing with PBS, the cells were observed in a laser scanning confocal microscope system.

[0048] 5. Establishment of a mouse tumor-bearing model for FCPG / Obsl1 nanoparticle vaccine to prevent tumor growth: We randomly divided female C57BL / 6 mice into four groups, with 5 mice in each group. 180 μl of 1×PBS (pH=7.2-7.4), antigen peptide Obsl1 (final concentration 167 μg / ml), CPG+Obsl1 (physical mixture of CPG and Obsl1, final concentration [Obsl1]=167 μg / ml) or FCPG / Obsl1 nanoparticle vaccine (final concentration [Obsl1]=167 μg / ml) were injected into the tail base of the mice 21, 14, and 7 days before tumor cell inoculation. Then, on day 0, each mouse was subcutaneously inoculated with 3×10 5 The melanoma cells B16F10 were then inoculated with a vernier caliper to record the tumor growth trend and the survival of the mice was observed at the same time.

[0049] 6. Establishment of a mouse tumor-bearing model in which FCPG / Obsl1 nanoparticle vaccine inhibits tumor growth: We randomly divided female C57BL / 6 mice into four groups, with 5 mice in each group. On day 0, each mouse was subcutaneously inoculated with 3×10 5 melanoma cells; then, on days 3, 10, and 17, the mice were injected with 180 μl of 1×PBS (pH=7.2-7.4), antigen peptide Obsl1 (final concentration 167 μg / ml), CPG+Obsl1 (physical mixture of CPG and Obsl1, final concentration [Obsl1]=167 μg / ml), or FCPG / Obsl1 nanoparticle vaccine (final concentration [Obsl1]=167 μg / ml) at the tail base. The tumor growth trend was then recorded with a vernier caliper, and the survival of the mice was observed.

[0050] Figure 3The performance test results of the FCPG / Obsl1 nanoparticle vaccine are shown in Figure 1. (A) and (B) show the stimulation of BMDC maturation by the nanoparticle vaccine. Mature BMDC cells highly express CD11c+ / CD86+ and CD80+. The expression of CD11c+ / CD86+ and CD80+ in the nanoparticle vaccine group was significantly higher than that in other groups. This shows that the prepared nanoparticle vaccine can effectively stimulate BMDC cell maturation. (C) is a graph showing the cytotoxicity of the nanoparticle vaccine to BMDC. Even in the case of the FCPG / Obsl1 nanoparticle vaccine (final concentration [Obsl1] = 4 After treatment with 50 μg / ml of 5% MgCl2, the survival rate of BMDC cells was still above 93% on average, indicating that the prepared nanoparticle vaccine had low toxicity; (D) is a confocal imaging diagram of the cell uptake of nanoparticle vaccine by BMDC. The BMDC cell uptake rate of nanoparticle vaccine was significantly higher than that of other groups, indicating that the prepared nanoparticle vaccine was well internalized in the cytoplasm; in addition, in the co-localization with lysosomes, it was found that the co-localization rate of nanoparticle vaccine and lysosomes was low, indicating that the nanovaccine escaped from the lysosomes, which made the antigen peptide and FCPG in the nanoparticle vaccine less likely to be degraded in the lysosomes and better able to play the function of antigen presentation.

[0051] Figure 4 Figure 2 is a diagram of the prevention of tumor growth by FCPG / Obsl1 nanoparticle vaccine; (A) is a flowchart of the prevention of growth, where PBS, antigen peptide Obsl1, CPG+Obsl1 or nanoparticle vaccine were injected into the tail base of mice on days 0, 7 and 14, and then melanoma cells B16F10 were subcutaneously inoculated in each mouse on day 21; (B) is a schematic diagram of tumor growth, where we can see that in the nanoparticle vaccine group, the nanoparticle vaccine activated the immune response when the vaccine was first administered, and when the tumor cells were inoculated, the immune system was able to immediately recognize and kill the tumor cells. cells, thereby delaying tumor growth. In addition, FCPG, as a stimulating adjuvant for antigen presenting cells, can activate antigen presenting cells (APCs) to enhance the presentation ability of antigens, further synergistically enhancing the body's immune response. Therefore, the nanoparticle vaccine exhibits stronger anti-tumor ability. (C) is a graph of mouse survival rate. It can be clearly seen that the survival rate of mice in the nanoparticle vaccine group has increased, and compared with the survival rates of other groups, the PBS group, the Obsl1 group, and the CPG+Obsl1 group, it was found that there were significant differences between them, indicating that the prepared nanoparticle vaccine has the effect of preventing tumor growth.

[0052] Figure 5Figure 1 is a diagram showing the effect of the FCPG / Obsl1 nanoparticle vaccine prepared in the example on inhibiting tumor growth; wherein, (A) is a flow chart showing the operation of inhibiting growth, in which each mouse was subcutaneously inoculated with melanoma cells on day 0; then on days 3, 10, and 17, the tail bases of the mice were injected with PBS, Obsl1, CPG+Obsl1, or nanoparticle vaccine; (B) is a schematic diagram showing tumor growth, in which we can see that the tumor growth rate in the nanoparticle vaccine group was significantly slowed down, indicating that the nanoparticle vaccine activated the immune system of the mice and improved the anti-tumor ability of the mice; (C) is a graph showing the survival rate of the mice, in which we can also see that the survival rate of the nanoparticle vaccine group was significantly improved and was significantly different from that of the other groups, indicating that the prepared nanoparticle vaccine has a good effect in inhibiting tumor growth.

[0053] In summary, the present invention provides a low-temperature preparation method for nanoparticle vaccines. The present invention uses fluorine-substituted functional nucleic acids and antigen peptides as raw materials under low temperature conditions. The two raw materials self-assemble to form a nano-vaccine complex in a phosphate buffer through molecular electrostatic attraction, dispersion force and hydrophobic interaction. The present invention has developed a simple, safe, green and mild method. The new antigen nano-vaccine FCPG / Obsl1 with a small amount of non-toxic and degradable immune adjuvant as a carrier is synthesized by directly self-assembling the immune adjuvant FCPG and the new antigen peptide Obsl1, thereby avoiding the high-temperature inactivation of the antigen peptide. The synthesized FCPG / Obsl1 new antigen nano-vaccine has low biological toxicity, good biocompatibility and a new antigen loading rate of 85.27%. The ultra-high new antigen density increases the uptake of the new antigen nano-vaccine by antigen presenting cells (APC). This nano-vaccine realizes the co-delivery of new antigens and adjuvants, and is one of the simplest nano-vaccines to date. It can target draining lymph nodes, enhance the uptake of antigens by APCs, and present antigens, enhance innate immune responses and drive subsequent adaptive immune responses. In murine B16F10 melanoma, significant tumor suppression was achieved by potent nanovaccine-induced cytotoxic T lymphocytes. This strategy may be applicable to minimalist combinatorial vaccine design in future clinical settings.

[0054] The above description is only a preferred embodiment of the present invention. All equivalent changes and substitutions made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims

1. A low temperature preparation method for nanoparticle vaccines, characterized in that: The fluorine-substituted functional nucleic acid and the antigen peptide are mixed evenly in a buffer solution, reacted at 37°C for 1h to 24h, and then reacted at 4°C for 6h to 72h. After the reaction, the precipitate is collected by centrifugation and washed to obtain the nanoparticle vaccine. The method comprises the following steps: (1) Preparation of fluorine-substituted functional nucleic acid stock solution: dissolve the fluorine-substituted functional nucleic acid in sterile water and pipette evenly at room temperature to obtain a fluorine-substituted functional nucleic acid stock solution with a concentration of 50 μM to 200 μM; (2) Preparation of antigen peptide stock solution: Disperse the antigen peptide in secondary water and disperse it evenly to obtain an antigen peptide stock solution with a concentration of 1 mM to 20 mM; (3) Preparation of nanoparticle vaccine: The fluorine-substituted functional nucleic acid stock solution is mixed evenly with the antigen peptide stock solution, buffer solution and secondary water. The resulting mixture is first reacted at 37°C for 1 h to 24 h, and then reacted at 4°C for 6 h to 72 h. After the reaction is completed, the mixture is centrifuged at 5000 to 10000 rpm for 1 min to 60 min, and the precipitate is collected and washed to obtain the nanoparticle vaccine. The pH of the buffer solution is 6 to 8; The antigen peptide is Obsl1, and its amino acid sequence is REGVELCPGNKYEMRRHGTTHSLVIHD.

2. The low-temperature preparation method of a nanoparticle vaccine according to claim 1, characterized in that: The fluorine-substituted functional nucleic acid includes a nucleic acid derivative modified with a fluorine atom, and the nucleic acid in the fluorine-substituted functional nucleic acid is selected from single-stranded DNA, single-stranded RNA, double-stranded RNA, and double-stranded DNA.

3. The low temperature preparation method of a nanoparticle vaccine according to claim 1, characterized in that: In the fluorine-substituted functional nucleic acid, the fluorine is substituted at the 2' position of the five-membered sugar ring in the nucleic acid, or the fluorine atom is modified at any position on the base, and the number of modified fluorine atoms on the base is one or more.

4. The low temperature preparation method of a nanoparticle vaccine according to claim 1, characterized in that: In step (3), the molar ratio of the fluorine-substituted functional nucleic acid to the antigen peptide in the mixed solution is 1:1 to 1:

100.

5. The nanoparticle vaccine prepared by the low-temperature preparation method as claimed in claim 1.

6. Use of the nanoparticle vaccine according to claim 5 in the preparation of tumor immunotherapy drugs.

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