Multivalent nucleic acid vaccine and preparation method and application thereof

Abstract

The application belongs to the field of biological medicine, and relates to a multivalent nucleic acid vaccine as well as a preparation method and application thereof. The application adopts a modular construction mode of 'arm first and nucleic acid later', that is, first, a DBCO modified nucleic acid chain is coupled with an azidated ursolic acid derivative through click chemistry to form a functional monomer, and then the functional monomer is self-assembled into a spherical nucleic acid structure in an aqueous phase through hydrophilic and hydrophobic interaction. The method can realize accurate regulation of the proportion of different functional nucleic acids (such as mRNA, aptamer, etc.) in the vaccine, and significantly improves the cell uptake efficiency, immune activation capacity and biological safety of the vaccine. The application also relates to the application of the spherical nucleic acid vaccine in freeze-drying preservation, multivalent vaccine construction and targeted delivery, and the spherical nucleic acid vaccine is suitable for rapid response to virus variation and vaccine distribution in resource-limited areas.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and relates to the preparation of nucleic acid vaccines. Background Technology

[0002] The concept of nucleic acid vaccines was proposed at the beginning of this century. Also known as gene vaccines, nucleic acid vaccines involve directly introducing a foreign gene (DNA or RNA) encoding a specific antigen protein into animal somatic cells (e.g., via intramuscular injection, microparticle bombardment, etc.). The host cell's expression system synthesizes the antigen protein, inducing an immune response in the host, thereby preventing and treating diseases. Based on their main components, nucleic acid vaccines are divided into DNA vaccines and RNA vaccines, with RNA vaccines primarily referring to mRNA vaccines. These vaccines have advantages such as long-lasting immune responses, high similarity between protein antigens and natural infections, ease of genetic modification, and the ability to overcome the inhibitory effects of the major histocompatibility complex (MHC), attracting significant attention in the industry. However, researchers subsequently discovered significant safety and delivery system risks associated with nucleic acid vaccines, leading to a decline in vaccine applications in this field. In recent years, research into nucleic acid delivery systems has deepened, resulting in various delivery methods such as biojet injectors, gene guns, microneedle arrays, electroporation, liposomes, virus-like particles, nanoparticles, and cell-penetrating peptides. The number of patent applications related to nucleic acid vaccines has recovered and is now stabilizing.

[0003] Spherical nucleic acids (SNAs), as a novel multivalent nucleic acid delivery vector, have demonstrated significant advantages in gene regulation and targeted therapy due to their unique structural and functional properties, especially in the synergistic regulation of multiple targets or signaling pathways. Traditional nucleic acid delivery systems, such as liposomes or viral vectors, often face problems such as low cellular uptake efficiency, poor intracellular stability, insufficient targeting, and high immunogenicity. SNAs, by anchoring oligonucleotides at high density to the surface of nanoparticles to form a three-dimensional spherical structure, not only significantly enhance nuclease resistance but also greatly improve cellular uptake efficiency through multivalent effects.

[0004] Spherical nucleic acid (SNA) constructs are promising new single-entity gene regulatory materials capable of excellent cell transfection and gene knockdown. However, their hybrid structures, especially multivalent SNAs, have resulted in a complex chemical composition, making it difficult to precisely measure the proportions of different components. This is because their preparation method involves first constructing a spherical core and then modifying the surface with nucleic acids. This method makes it impossible to precisely measure the proportions of different chemical components in the material.

[0005] According to literature, the therapeutic efficacy of multivalent vaccines is directly determined by the different antigens or the combination of antigens and adjuvants. Although constructing spherical nucleic acid structures improves material stability, how to anchor different functional DNA / RNAs (such as dual-antigen vaccine vectors) to the core remains an underdeveloped problem. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention proposes a multivalent nucleic acid vaccine, its preparation method, and its application.

[0007] The technical solution of this invention is implemented as follows:

[0008] A method for preparing a multivalent nucleic acid vaccine, comprising the following steps:

[0009] (1) Synthesis of linkers

[0010] The technical approach is as follows:

[0011]

[0012] Using tetraethylene glycol as a starting material, the hydroxyl groups were activated with TsCl (4-methylbenzenesulfonyl chloride), and then reacted with NaN3 (1.5 equiv.) in DMF solvent under oil bath heating to 90 °C for 6 h to obtain compound S1. S1 reacted with CBr4 (1.3 equiv.) in DCM solvent under the catalysis of PPh3 (1.5 equiv.) for 1 h to obtain compound S2, which serves as a linker for coupling natural small molecules with nucleic acids (Reference: Proc. Natl. Acad. Sci. USA 115 (17), 4340-4344).

[0013] (2) Azide modification of ursolic acid

[0014] The technical approach is as follows:

[0015]

[0016] S2 and UA (1-3 equiv.) were reacted in DMF solvent under the catalysis of K2CO3 (2-6 equiv.) for 24 h to obtain an azide-modified ursolic acid monomer, compound S3.

[0017] (3) Synthesis of nucleic acid-ursolic acid conjugates

[0018] The technical approach is as follows:

[0019]

[0020] Synthesis of UA-RNA: DBCO-modified miR-122 nucleic acid and azide-modified ursolic acid in DMSO solution (1 equiv., 20-500 μM) were reacted in a H2O / DMSO mixture at room temperature for 24-48 hours. After the reaction, lyophilization yielded the first nucleic acid UA-RNA monomer, S4. The sequence of miR-122 was synthesized using the sense primer Sense and the antisense primer Antisense.

[0021] The sequence of the positive primer Sense is shown in SEQ ID No. 1: 5'-DBCO-UGGAGUGUGACAAUGGUGUGUUUGU-3';

[0022] The sequence of the antisense primer Antisense is shown in SEQ ID No. 2: 5'-AAACACCAUUGUCACACUCCAUU-3';

[0023] (4) Preparation of SNA nucleic acid vaccine

[0024] ①SNA nucleic acid vaccine: UA was mixed thoroughly with S4 in 20 μL of DMSO solution at different ratios (1, 2, 3, 4, 5 equiv.). The mixture was then rapidly added to 1000 μL of ddH2O under sonication. After sonication for 10 min, the mixture was placed in a 4℃ refrigerator for 2 h. The mixture was then centrifuged at 13000 r for 30 min, and the supernatant was discarded, leaving the precipitate to obtain five different ratios of SNA nucleic acid vaccine.

[0025] ②Ce6 fluorescently labeled SNA nucleic acid vaccine: UA was thoroughly mixed with Sora and S4 in 20 μL of DMSO solution at different ratios (5 equiv.), and the fluorescent probe Ce6 (0.15 equiv) was added simultaneously. The mixture was rapidly added to 1000 μL of ddH2O under sonication for 10 min, and then incubated at 4℃ for 2 h. Due to the presence of the Ce6 fluorescent probe, the reaction process must be conducted in the dark. The mixture was centrifuged at 13000 r for 30 min, the supernatant was discarded, and the precipitate was retained to obtain the Ce6 fluorescently labeled SNA nucleic acid vaccine.

[0026] Secondly, this application also seeks protection for the nucleic acid vaccine prepared by the above method. Its key advantages lie in the improved stability and delivery efficiency. Nucleic acid monomers can be stored at -20°C for a certain period of time in a lyophilized state, which lays a theoretical foundation and practical accumulation for the development of lyophilized dosage forms.

[0027] Thirdly, this application provides the application of the aforementioned nucleic acid vaccine in the preparation of freeze-dried formulations of vaccines with stable properties and enhanced antigen delivery efficiency. For example, it applies to the preparation of nucleic acid vaccines such as virus-like particles (antigen hybridization-spherical nucleic acid).

[0028] The present invention has the following beneficial effects:

[0029] 1. This invention employs a "arm-first, core-later" preparation strategy. First, functional nucleic acids are coupled with a natural small molecule (ursolic acid) possessing self-assembly properties. Then, spherical nucleic acid vaccines are prepared using the self-assembly of ursolic acid. The greatest advantage of this synthesis method lies in fully utilizing the "programmability" of nucleic acid vaccines. By pre-preparing multiple nucleic acid-ursolic acid monomers, and then through a simple and mild self-assembly process in an aqueous phase (controlling the feeding ratio of different monomers), more precise multivalent vaccine materials can be easily obtained.

[0030] 2. The purpose of this invention is to develop a modular development strategy for nucleic acid vaccines. By conjugating synthetic nucleic acids with natural small molecules and utilizing the self-assembly properties of these small molecules to prepare modular antigen designs such as virus-like particles, flexible assembly and rapid iteration of vaccines can be achieved, adapting to viral mutations. This method enables flexible assembly and rapid iteration of vaccine components, effectively adapting to rapid viral mutations. From the practical perspective of vaccine development and application, especially for "nucleic acid vaccines," its key advantages lie in improved stability and delivery efficiency. Nucleic acid monomers can be stored at -20°C for a certain period in a freeze-dried state, laying a theoretical foundation and practical experience for the development of freeze-dried dosage forms. By combining freeze-drying technology with nanocarrier technology, not only is the stability of the vaccine at room temperature significantly enhanced, but antigen delivery efficiency is also significantly improved. This technological combination is particularly suitable for vaccine distribution in resource-limited areas, solving the problems of cold chain dependence and transportation. Furthermore, freeze-drying-reconstitution experiments demonstrate that this spherical nucleic acid vaccine retains a relatively uniform morphology after reconstitution, making it suitable for vaccine distribution in resource-limited areas. In the precise targeted design of peptide vaccines, organic chemistry and synthetic biology techniques facilitate the accurate screening of key viral antigenic epitopes, enabling the design of synthetic peptide vaccines with high immunogenicity and low side effects, thereby improving vaccine specificity and safety. In the biomedical field, the innovation of this vaccine technology is mainly reflected in two aspects: precise design and flexible construction. ① Modular construction methods are more conducive to the precise targeted design of peptide vaccines, facilitating the convenient design and synthesis of vaccines with high immunogenicity and low side effects. This construction strategy significantly improves vaccine specificity and safety. ② Modular development of nucleic acid vaccines: A modular strategy is adopted to develop nucleic acid vaccines. Core technologies include conjugating synthetic nucleic acids with natural small molecules and utilizing the self-assembly properties of small molecules to construct modular antigen structures such as virus-like particles (antigen hybridization-spherical nucleic acid).

[0031] 3. The main advantages of this invention are: ① From a biomedical perspective: In terms of precise targeted design of peptide vaccines, the use of organic chemistry and synthetic biology methods facilitates the precise screening of key viral antigenic epitopes, enabling the design of synthetic peptide vaccines with high immunogenicity and low side effects, thereby improving the specificity and safety of the vaccine. ② A modular development strategy for nucleic acid vaccines is constructed, utilizing the conjugation of synthetic nucleic acids with natural small molecules, and leveraging the self-assembly properties of small molecules to prepare virus-like particles (see...). Figure 10 Modular antigen design, such as [missing information], enables flexible assembly and rapid iteration of vaccines, adapting to viral mutations. ③ From the perspective of antiviral vaccine development and application, monomeric nucleic acids can be stored long-term at -20℃ under lyophilized conditions, which is beneficial for the development of lyophilized formulations. Combining lyophilization technology with nanocarrier technology improves vaccine stability at room temperature and enhances antigen delivery efficiency (see [missing information]). Figure 9 This is applicable to vaccine distribution in resource-limited areas. Attached Figure Description

[0032] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0033] Figure 1 NMR characterization of the linker.

[0034] Figure 2 NMR characterization of azide monomers.

[0035] Figure 3 Mass spectrometry characterization of azide monomers.

[0036] Figure 4 HPLC characterization of two ursolic acid-nucleic acid pairs.

[0037] Figure 5 SEM characterization of ursolic acid-nucleic acid assembly into SNA.

[0038] Figure 6 Gel electrophoresis for SNA.

[0039] Figure 7 The content of two monomers in SNA was determined.

[0040] Figure 8 For the toxicity test of SNA.

[0041] Figure 9 Cellular uptake experiment for SNA.

[0042] Figure 10TEM and mapping elemental analysis characterized the SNA, revealing virus-like particles. Detailed Implementation

[0043] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0044] Unless otherwise specified, the experimental methods used in the following experimental examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0045] Example 1

[0046] The preparation method of the spherical nucleic acid vaccine in this embodiment includes the following steps:

[0047] (1) Using tetraethylene glycol as a raw material, the hydroxyl groups were activated with TsCl (4-methylbenzenesulfonyl chloride), and then reacted with NaN3 (1.5 equiv.) in DMF solvent and heated to 90°C in an oil bath for 6 h to obtain compound S1. S1 reacted with CBr4 (1.3 equiv.) in DCM solvent under the catalysis of PPh3 (1.5 equiv.) for 1 h to obtain compound S2 as a linker for coupling natural small molecules with nucleic acids (Reference Proc. Natl. Acad. Sci. USA 115 (17), 4340-4344).

[0048] (2) Compound S2 and UA (2.8 equiv.) were reacted in DMF solvent under the catalysis of K2CO3 (5.8 equiv.) for 24 h to obtain the azide-modified ursolic acid monomer, compound S3.

[0049] (3) Synthesis of nucleic acid-ursolic acid conjugates

[0050] Synthesis of UA-RNA: miR-122 with nucleic acid sequences Sense 5'-DBCO-UGGAGUGUGACAAUGGUGUUUGU-3' and Antisense 5'-AAACACCAUUGUCACACUCCAUU-3' was reacted with an azide-modified ursolic acid solution in DMSO (1 equiv., 20-500 μM) at room temperature in a H2O / DMSO mixture for 24-48 hours. The first nucleic acid monomer UA-RNA (S4) was obtained by lyophilization after the reaction.

[0051] (4) Preparation of SNA nucleic acid vaccine

[0052] ①SNA Nucleic Acid Vaccine: Compound UA (1, 2, 3, 4, 5 equiv.) was thoroughly mixed with Sora and S4 in 20 μL of DMSO solution. The mixture was then rapidly added to 1000 μL of ddH2O under sonication for 10 min, followed by sonication at 4°C for 2 h. The mixture was then centrifuged at 13000 r for 30 min, and the supernatant was discarded, leaving the precipitate to obtain SNA nucleic acid vaccines with five different adjuvant ratios.

[0053] ②Ce6 fluorescently labeled SNA nucleic acid vaccine: UA (5 equiv.) and S4 were thoroughly mixed in 20 μL of DMSO solution. Simultaneously, the fluorescent probe Ce6 (0.15 equiv.) was added rapidly to 1000 μL of ddH2O under sonication. After sonication for 10 min, the mixture was placed in a 4℃ refrigerator for 2 h. Due to the presence of the Ce6 fluorescent probe, the reaction process must be conducted in the dark. The mixture was centrifuged at 13000 r for 30 min, the supernatant was discarded, and the precipitate was retained to obtain the Ce6 fluorescently labeled SNA nucleic acid vaccine.

[0054] Example 2

[0055] The preparation method of the spherical nucleic acid vaccine in this embodiment includes the following steps:

[0056] (1) Using tetraethylene glycol as a raw material, the hydroxyl groups were activated with TsCl (4-methylbenzenesulfonyl chloride), and then reacted with NaN3 (1.5 equiv.) in DMF solvent and heated to 90°C in an oil bath for 6 h to obtain compound S1. S1 reacted with CBr4 (1.3 equiv.) in DCM solvent under the catalysis of PPh3 (1.5 equiv.) for 1 h to obtain compound S2 as a linker for coupling natural small molecules with nucleic acids (Reference Proc. Natl. Acad. Sci. USA 115 (17), 4340-4344).

[0057] (2) Compound S2 reacted with UA (1 equiv.) in DMF solvent under the catalysis of K2CO3 (2 equiv.) for 24 h to obtain the azide-modified ursolic acid monomer, compound S3.

[0058] (3) Synthesis of nucleic acid-ursolic acid conjugates

[0059] Synthesis of UA-RNA: miR-122 with nucleic acid sequences Sense 5'-DBCO-UGGAGUGUGACAAUGGUGUUUGU-3' and Antisense 5'-AAACACCAUUGUCACACUCCAUU-3' was reacted with an azide-modified ursolic acid solution in DMSO (1 equiv., 20-500 μM) at room temperature in a H2O / DMSO mixture for 24-48 hours. The first nucleic acid monomer UA-RNA (S4) was obtained by lyophilization after the reaction.

[0060] (4) Preparation of SNA nucleic acid vaccine

[0061] ①SNA Nucleic Acid Vaccine: Compound UA (5 equiv.) was thoroughly mixed with Sora and S4 in 20 μL of DMSO solution. This mixture was then rapidly added to 1000 μL of ddH2O under sonication for 10 min, followed by incubation at 4°C for 2 h. After centrifugation at 13000 r for 30 min, the supernatant was discarded, and the precipitate was retained to obtain SNA nucleic acid vaccines with five different adjuvant ratios.

[0062] ②Ce6 fluorescently labeled SNA nucleic acid vaccine: UA (5 equiv.) and S4 were thoroughly mixed in 20 μL of DMSO solution. Simultaneously, the fluorescent probe Ce6 (0.15 equiv.) was added rapidly to 1000 μL of ddH2O under sonication. After sonication for 10 min, the mixture was placed in a 4℃ refrigerator for 2 h. Due to the presence of the Ce6 fluorescent probe, the reaction process must be conducted in the dark. The mixture was centrifuged at 13000 r for 30 min, the supernatant was discarded, and the precipitate was retained to obtain the Ce6 fluorescently labeled SNA nucleic acid vaccine.

[0063] Example 3

[0064] The preparation method of the spherical nucleic acid vaccine in this embodiment includes the following steps:

[0065] (1) Using tetraethylene glycol as a raw material, the hydroxyl groups were activated with TsCl (4-methylbenzenesulfonyl chloride), and then reacted with NaN3 (1.5 equiv.) in DMF solvent and heated to 90°C in an oil bath for 6 h to obtain compound S1. S1 reacted with CBr4 (1.3 equiv.) in DCM solvent under the catalysis of PPh3 (1.5 equiv.) for 1 h to obtain compound S2 as a linker for coupling natural small molecules with nucleic acids (Reference Proc. Natl. Acad. Sci. USA 115 (17), 4340-4344).

[0066] (2) Compound S2 reacted with UA (3 equiv.) in DMF solvent under the catalysis of K2CO3 (6 equiv.) for 24 h to obtain the azide-modified ursolic acid monomer, compound S3.

[0067] (3) Synthesis of nucleic acid-ursolic acid conjugates

[0068] Synthesis of UA-RNA: miR-122 with nucleic acid sequences Sense 5'-DBCO-UGGAGUGUGACAAUGGUGUUUGU-3' and Antisense 5'-AAACACCAUUGUCACACUCCAUU-3' was reacted with an azide-modified ursolic acid solution in DMSO (1 equiv., 20-500 μM) at room temperature in a H2O / DMSO mixture for 24-48 hours. The first nucleic acid monomer UA-RNA (S4) was obtained by lyophilization after the reaction.

[0069] (4) Preparation of SNA nucleic acid vaccine

[0070] ①SNA Nucleic Acid Vaccine: Compound UA (5 equiv.) was thoroughly mixed with Sora and S4 in 20 μL of DMSO solution. This mixture was then rapidly added to 1000 μL of ddH2O under sonication for 10 min, followed by incubation at 4°C for 2 h. After centrifugation at 13000 r for 30 min, the supernatant was discarded, and the precipitate was retained to obtain SNA nucleic acid vaccines with five different adjuvant ratios.

[0071] ②Ce6 fluorescently labeled SNA nucleic acid vaccine: UA (5 equiv.) and S4 were thoroughly mixed in 20 μL of DMSO solution. Simultaneously, the fluorescent probe Ce6 (0.15 equiv.) was added rapidly to 1000 μL of ddH2O under sonication. After sonication for 10 min, the mixture was placed in a 4℃ refrigerator for 2 h. Due to the presence of the Ce6 fluorescent probe, the reaction process must be conducted in the dark. The mixture was centrifuged at 13000 r for 30 min, the supernatant was discarded, and the precipitate was retained to obtain the Ce6 fluorescently labeled SNA nucleic acid vaccine.

[0072] Implementation Results Example

[0073] The linkers prepared in Example 1 were characterized by NMR, and the results are as follows: Figure 1 As shown, after comparing with the literature (J. Am. Chem. Soc. 2021, 143, 47, 19844–19855), it can be seen that the hydrogen on the methylene group next to the azide, the hydrogen on the methylene group bonded to the bromine atom, and other hydrogens are all in the correct positions. The carbon spectrum on the right is the same. It can be seen that the structure of the material is the expected molecular structure.

[0074] The azide monomer prepared in Example 1 was characterized by NMR, and the results are as follows: Figure 2 As shown, the left image is 1 In the ¹H NMR spectrum, all peaks except the water peak in the solvent can be attributed to the structure, such as the tetraethylene glycol peak at 3.8-3.5 ppm, the characteristic shift peak of the hydrogen on the C adjacent to the carboxyl group of the ester bond after the reaction of the linker with the carboxyl group of the UA monomer at position b in the left figure, and the hydrogen on the olefin bond of UA also conforms to the predicted value; the right figure is... 13 C NMR, where the ursolic acid moiety retains the pentacyclic triterpenoid skeleton, with the carboxyl group (–COOH) replaced by an ester group (–COO–); the linker moiety connected to the ester bond is a triethylene glycol (tetraethylene glycol) chain, terminated by an azide group (–N3). σ 170-175 ppm represents the high-field shift of the original carboxyl carbon of ursolic acid (~180 ppm) after esterification, a key reaction marker; 100-160 ppm represents the shift peaks of the alkene, quaternary, and oxygen-containing carbons in the ursolic acid skeleton; 60-80 ppm represents the carbons on the -O-CH2CH2O- linker, with multiple peaks appearing due to the chemical shifts of multiple methylene carbons connected to oxygen atoms within this range; ~50-55 ppm represents the carbons on the methylene group (-CH2-N3) connected to the azide; 10-50 ppm represents the aliphatic carbons (CH3, CH2, CH) in the ursolic acid skeleton, including multiple methyl and methylene signals, hence the multiple peaks.

[0075] Further mass spectrometric characterization of the azide monomers yielded the following results: Figure 3 As shown, since this molecule is a completely new compound (although literature reports have shown that some have an additional ethoxy unit in the linker), we still need to characterize it using high-resolution mass spectrometry. The chemical formula of the azide monomer is known to be C1. 38 H 63 The theoretical molecular weight of N3O6 with sodium ions is 680.4609, and the molecular weight of the synthesized material was measured to be 680.4597. The measured value is within the allowable error range, so it can be determined that the material is the target molecule. The theoretical molecular weight with potassium ions is 696.4354, and the experimental measurement was 696.4331, yielding the same conclusion.

[0076] HPLC characterization of the three ursolic acid-nucleic acid preparations prepared in Example 1 yielded the following results: Figure 4 As shown, the high-performance liquid chromatography (HPLC) spectra of the three nucleic acid-modified ursolic acid conjugate monomers are shown below, with the peak positions of the three nucleic acid monomers being 6.29, 6.51, and 6.98, respectively.

[0077] SEM characterization of the ursolic acid-nucleic acid assembly into SNA prepared in Example 1 yielded the following results: Figure 5As shown, this application presents scanning electron microscopy (SEM) characterizations of five SNAs with different nucleic acid ratios prepared in two separate periods (winter and summer). SEM characterization reveals that materials containing more UA are smaller in size. This may be due to the increased assembly difficulty caused by the three-dimensional conformation of miR-122, leading to a smaller assembly size. The morphology and trends of the SNA vaccines prepared in June and October show high reproducibility. The third row, "lyophilized-reconstituted," represents the experimental data from the second preparation of five SNAs, which were lyophilized, stored at -20°C for 7 days, and then reconstituted at room temperature. The reconstituted materials were then ultrasonically dispersed and observed using SEM. The data shows that the reconstituted SEM characterization is similar in morphology to the newly prepared materials. Since the properties of nanomaterials are closely related to their morphology, it is inferred that the materials possess a certain degree of stability. Currently, 20 mg of each of the five molecularly spherical nucleic acid vaccines have been obtained (samples already available).

[0078] Electrophoresis was performed on the materials in a 1% agarose gel. From left to right, the samples are DBCO-miRNA-122, SNA, and the supernatant obtained after centrifugation following assembly. Results are as follows: Figure 6 As shown, by Figure 6 It can be seen that the molecular weight (MW) increased significantly after UA-RNA assembly, and the migration of RNA in the agarose gel was inhibited, indicating that ursolic acid and nucleic acid were successfully assembled.

[0079] SNAs prepared from UA / UA-RNA with a starting material ratio ranging from 1 / 1 to 5 / 1 were quantified by HPLC peak area analysis to determine the proportion of nucleic acids in assembled SNAs with different ratios of nucleic acid monomers. The results are as follows: Figure 7 As shown in the table on the left, by controlling the ratio of the two nucleic acid monomers, the proportions of different nucleic acids can be precisely controlled. The ideal control ratio range is 1 / 1 to 5 / 1 (UA / UA-RNA), which can control the ratio of the two monomers (UA and miR-122) to change linearly from 0.5 to 4.

[0080] To assess the cytotoxicity of SNA: An MTT assay was designed using the HepG2 cell model to verify the cytotoxicity of the material. Cells were seeded in 96-well plates (4 × 10⁶ cells per well). 3 After culturing for 24 hours, three different ratios of SNA (1 / 1, 3 / 1, 5 / 1) were treated with different concentrations (calculated as UA) for 24 hours. The SNA assemblies also exhibited some cytotoxicity against HepG2 tumor cells, primarily due to exposure to the UA assemblies (according to literature, the UA assemblies themselves have antitumor activity, Int J Biol Sci. 2024, 20(11): 4190-4208.). The MTT assay results are as follows... Figure 8As shown: from left to right, the SNAs are 1 / 1, 3 / 1, and 5 / 1. The data shows that the higher the UA content of the SNA, the greater its nuclear toxicity and the more sensitive it is to mass dependence.

[0081] Cellular uptake assay of SNA: The confocal observation results of Ce6-labeled SNA are shown below. Hepa1-6 cells were used, and the incubation times were 0, 1, 3, and 6 hours. Results are as follows... Figure 9 As shown, by Figure 9 It can be seen that SNA cellular uptake is time-dependent.

[0082] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a multivalent nucleic acid vaccine, characterized in that, Includes the following steps: (1) Using triethylene glycol as raw material, the hydroxyl group was activated with TsCl, and then the reaction was carried out in DMF solution of NaN3 by oil bath heating to obtain compound S1. Compound S1 reacted with CBr4 in DCM solvent under the catalysis of PPh3 to obtain compound S2. (2) Compound S2 and ursolic acid were reacted completely in DMF under the catalysis of K2CO3 to obtain the azide-modified ursolic acid monomer, namely compound S3; (3) React nucleic acid with DMSO solution of compound S3 in a mixed solvent at room temperature, and freeze-dry the reaction solution to obtain nucleic acid monomer; when the nucleic acid sequence is miR-22 as shown in SEQ ID No.1 of the positive primer Sense and SEQ ID No.2 of the antisense primer Antisense and the 5' end is modified with -DBCO-, nucleic acid monomer UA-RNA is obtained. (4) Mix UA with nucleic acid monomer UA-RNA, add it to ddH2O under sonication, sonicate thoroughly to allow all components to be fully dispersed in water, then let it stand to allow it to fully self-assemble, centrifuge and discard the supernatant. The resulting precipitate is the multivalent nucleic acid vaccine.

2. The method for preparing a multivalent nucleic acid vaccine according to claim 1, characterized in that: The structural formula of compound S1 in step (1) is: The structural formula of compound S2 is: .

3. The method for preparing a multivalent nucleic acid vaccine according to claim 2, characterized in that: In step (1), the oil bath heating reaction is carried out at a temperature of 90°C for 6 hours; the molar ratio of triethylene glycol, NaN3, CBr4 and PPh3 is 1:1.5:1.3:1.

5.

4. The method for preparing a multivalent nucleic acid vaccine according to claim 1, characterized in that: In step (2), the molar ratio of compound S2, ursolic acid and K2CO3 is 1:1-3:2-6.

5. The method for preparing a multivalent nucleic acid vaccine according to claim 4, characterized in that: The structural formula of compound S3 is: .

6. The method for preparing a multivalent nucleic acid vaccine according to claim 1, characterized in that: In step (3), the molar ratio of nucleic acid to compound S3 is 1:1; the concentration of compound S3 is 20-500 μM.

7. The method for preparing a multivalent nucleic acid vaccine according to claim 1, characterized in that: In step (4), the molar ratio of UA to the nucleic acid monomer UA-RNA is 1-5:

1.

8. A multivalent nucleic acid vaccine prepared using the method described in any one of claims 1-7.

9. The multivalent nucleic acid vaccine according to claim 8, characterized in that: The multivalent nucleic acid vaccine is a freeze-dried vaccine.

10. The use of the multivalent nucleic acid vaccine according to claim 8 or 9 in the preparation of a freeze-dried formulation vaccine with stable properties and enhanced antigen delivery efficiency.

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