Tolerant nano vaccine precursor as well as preparation method and application thereof

Abstract

The invention discloses a tolerant nano vaccine precursor as well as a preparation method and application thereof, the preparation method comprises the following steps: S1, dissolving beta-glucan in dimethyl sulfoxide, adding anhydride under the condition of stirring at the constant temperature of 25-50 DEG C, and reacting for 8-12 hours; s2, adding a carboxyl activating agent-activated carboxyl into the reaction liquid in the step S1, and then adding dopamine hydrochloride to react for 24-48 hours; s3, dialyzing the reaction liquid in the step S2 to obtain colorless transparent liquid I, and performing freeze drying to obtain dopamine modified beta-glucan; s4, respectively preparing a dopamine modified beta-glucan solution and an immune tolerance inducer solution, mixing, and continuously stirring for 12-24 hours; and S5, dialyzing the reaction liquid in the step S4 to obtain colorless transparent liquid II, and freeze-drying to obtain the tolerant nano vaccine precursor. The tolerance nano vaccine precursor prepared by the invention can capture an antigen in situ to form a tolerance nano vaccine, so that immune tolerance to continuously diffused antigens in a body is realized, and the treatment effect is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of drug targeted delivery materials, and particularly relates to a nano vaccine precursor with tolerance and its preparation method and application. Background Art

[0002] Rheumatoid arthritis is one of the most common autoimmune diseases. In recent years, great progress has been made in the treatment field of rheumatoid arthritis. Among them, monoclonal antibody drugs targeting specific cytokines and targeted drugs targeting specific antigens have been widely used in rheumatoid arthritis. In addition, the progress in the field of pharmaceutics enables therapeutic drugs to take effect for a long time and enhances patient compliance.

[0003] However, the treatment of rheumatoid arthritis still faces major challenges. These challenges include: 1. Side effects of monoclonal antibody drugs: Monoclonal antibodies targeting specific inflammatory cytokines play a good role in inhibiting the condition of rheumatoid arthritis, but their systemic inhibitory effect on inflammatory cytokines lacks targeting and easily increases the risk of systemic infection or malignant tumors. 2. Lack of specificity: Currently, the therapeutic drugs used clinically include non-steroidal anti-inflammatory drugs and disease-modifying anti-rheumatic drugs. These drugs can only relieve the condition and cannot treat the cause. They are extremely easy to relapse after stopping the drug and progress. 3. Individualized needs: The pathogenic antigens of rheumatoid arthritis are extensive and complex, and there are differences among different patients. It is extremely difficult to treat the factors causing the disease in oneself.

[0004] Immune tolerance treatment targeting the personalized antigen causing rheumatoid arthritis is an emerging therapy. Currently, antigen-specific immune tolerance strategies mainly induce tolerogenic dendritic cells (tolDCs) through tolerogenic nano vaccines, inhibit the co-stimulatory signals between DCs and T cells, and enhance the interaction of immune privilege molecules, thereby establishing immune tolerance. Currently, nano vaccines mainly induce the generation of tolerogenic dendritic cells (tolDC) by co-delivering antigenic epitope peptides and immune tolerance inducers. TolDC further inhibits activated T cells against antigens, thereby restoring immune tolerance of the body.

[0005] Existing tolerogenic nano vaccines only screen and extract antigenic epitope peptides with relatively high abundance from numerous antigens. However, in the process of rheumatoid arthritis onset, first, due to different pathogenic mechanisms, there are significant differences in autoantigens among different patients; second, the antigenic epitopes are in a state of continuous diffusion and increase, which leads to the fact that the antigenic epitope peptides applied in this way cannot cover all autoantigen lineages, cannot induce multi-antigen immune tolerance, and are difficult to meet the requirements of immune tolerance treatment for rheumatoid arthritis. Summary of the Invention

[0006] Aiming at the above problems, the present invention aims to provide a nano vaccine precursor with tolerance and its preparation method and application.

[0007] The technical solution of the present invention is as follows:

[0008] On the one hand, a method for preparing a tolerogenic nano-vaccine precursor is provided, including the following steps:

[0009] S1: Dissolve β-glucan in dimethyl sulfoxide, add an acid anhydride under the condition of constant temperature stirring at 25 - 50 °C, and react for 8 - 12 h;

[0010] S2: Add a carboxyl activator I to activate the carboxyl group to the reaction solution of step S1, and then add dopamine hydrochloride and react for 24 - 48 h;

[0011] S3: Dialyze the reaction solution of step S2 to obtain a colorless and transparent liquid I, and perform lyophilization on the colorless and transparent liquid I to obtain dopamine-modified β-glucan;

[0012] S4: Prepare a dopamine-modified β-glucan solution and an immune tolerance inducer solution respectively, and mix the dopamine-modified β-glucan solution and the immune tolerance inducer solution, and continuously stir for 12 - 24 h;

[0013] S5: Dialyze the reaction solution of step S4 to obtain a colorless and transparent liquid II, and perform lyophilization on the colorless and transparent liquid II to obtain the tolerogenic nano-vaccine precursor.

[0014] Preferably, in step S1, the acid anhydride is succinic anhydride and / or maleic anhydride.

[0015] Preferably, in step S2, the carboxyl activator I is a mixture of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide with a molar ratio of 1:1.

[0016] Preferably, in step S4, the immune tolerance inducer used for preparing the immune tolerance inducer solution is selected from any one of dexamethasone, rapamycin, simvastatin, and curcumin.

[0017] Preferably, in step S1, the dosage of the acid anhydride is 1 - 3 times the mass of the β-glucan; in step S2, the dosage of the dopamine hydrochloride is 1 - 3 times the mass of the β-glucan; in step S4, the dosage of the immune tolerance inducer is 0.1 - 1 times the mass of the dopamine-modified β-glucan.

[0018] Preferably, it further includes the step of encapsulating the tolerogenic nano-vaccine precursor into a hydrogel.

[0019] Preferably, encapsulating the tolerogenic nano-vaccine precursor into a hydrogel specifically includes the following steps:

[0020] S6: Prepare a sodium hyaluronate solution and adjust the pH of the sodium hyaluronate solution to 5 - 6;

[0021] S7: Add a carboxyl activator to the sodium hyaluronate solution to activate the carboxyl group, and then add 3 - aminophenylboronic acid, and stir at room temperature for 24 - 48 h;

[0022] S8: Dialyze the reaction solution of step S7 to obtain a colorless and transparent liquid III, and perform freeze - drying on the colorless and transparent liquid III to obtain phenylboronic acid - modified hyaluronic acid;

[0023] S9: Prepare a tolerogenic nano - vaccine precursor solution and a polyvinyl alcohol solution respectively, and add the phenylboronic acid - modified hyaluronic acid to the tolerogenic nano - vaccine precursor solution to obtain a phenylboronic acid - modified hyaluronic acid solution;

[0024] S10: Mix the phenylboronic acid - modified hyaluronic acid solution and the polyvinyl alcohol solution in equal volumes to obtain a hydrogel encapsulating the tolerogenic nano - vaccine precursor.

[0025] Preferably, in step S7, the dosage of the 3 - aminophenylboronic acid is 1 - 3 times the molar amount of sodium hyaluronate; in step S9, the concentration of the tolerogenic nano - vaccine precursor in the tolerogenic nano - vaccine precursor solution is 6 - 13 mg / mL, the mass - volume concentration of the polyvinyl alcohol solution is 2 - 10%, and the mass - volume concentration of the phenylboronic acid - modified hyaluronic acid solution is 1 - 4%.

[0026] On the other hand, a tolerogenic nano - vaccine precursor prepared by the preparation method of the tolerogenic nano - vaccine precursor described in any one of the above and its application in the treatment of rheumatoid arthritis are also provided.

[0027] The beneficial effects of the present invention are:

[0028] The present invention can utilize the tolerogenic nano - vaccine precursor to capture antigens and combine with the immune tolerance strategy to achieve continuous immune tolerance to autoantigens, while reducing the extensive immunosuppression and other side effects caused by direct drug administration to the body; in addition, by encapsulating the tolerogenic nano - vaccine precursor into a hydrogel, the tolerogenic nano - vaccine precursor of the present invention can be controllably released and the retention time can be increased. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0030] Figure 1Schematic diagram of the test results of the shape and particle size distribution of the finished products of Example 1 and Comparative Example 1; among them, a is the particle size distribution diagram of the blank nanoparticles in Comparative Example 1, b is the particle size distribution diagram of the tolerogenic nano-vaccine precursor GSDD NPs in Example 1, c is the TEM image of the blank nanoparticles in Comparative Example 1, and d is the TEM image of the tolerogenic nano-vaccine precursor GSDD NPs in Example 1;

[0031] Figure 2 Schematic diagram of the test results of the shape and particle size distribution before and after the formation of the tolerogenic nano-vaccine precursor into a vaccine in Example 1; among them, a is the particle size distribution diagram of the tolerogenic nano-vaccine precursor, b is the particle size distribution diagram of the tolerogenic nano-vaccine, c is the TEM image of the tolerogenic nano-vaccine precursor, and d is the TEM image of the tolerogenic nano-vaccine;

[0032] Figure 3 Schematic diagram of the morphological characterization results of the HPGSDD nanocomposite hydrogel in Example 2; among them, a is the SEM image of the blank hydrogel in Comparative Example 2, and b is the SEM image of the HPGSDD nanocomposite hydrogel in Example 2;

[0033] Figure 4 Schematic diagram of the test results of the gel-forming performance of the HPGSDD nanocomposite hydrogel in Example 2;

[0034] Figure 5 Schematic diagram of the test results of the blood compatibility of the HPGSDD nanocomposite hydrogel in Example 2; among them, a is the photo of the blood compatibility experiment, and b is the hemolysis rate of each group in the blood compatibility experiment;

[0035] Figure 6 Schematic diagram of the test results of the cell compatibility of the HPGSDD nanocomposite hydrogel in Example 2; among them, a is the live / dead staining fluorescence image of chondrocytes and DC2.4 cells cultured in the extract of the nanocomposite hydrogel, and b is the cell survival rate of chondrocytes and DC2.4 cells cultured in the extract of the nanocomposite hydrogel;

[0036] Figure 7 Schematic diagram of the test results of the in vitro immune tolerance induction effect of GSDD NPs in Example 1. Detailed implementation method

[0037] The present invention will be further described below in conjunction with the accompanying drawings and embodiments. It should be noted that, without conflict, the embodiments in the present application and the technical features in the embodiments may be combined with each other. It should be pointed out that unless otherwise specified, all technical and scientific terms used in the present application have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present application belongs. As used in the disclosure of the present invention, words such as "including" or "comprising" and the like are intended to mean that the elements or items appearing before that word cover the elements or items listed after that word and their equivalents, without excluding other elements or items.

[0038] On the one hand, the present invention provides a method for preparing a tolerogenic nano-vaccine precursor, comprising the following steps:

[0039] S1: Dissolve β-glucan in dimethyl sulfoxide, and add an acid anhydride under the condition of constant temperature stirring at 25 - 50 °C, and react for 8 - 12 h.

[0040] In the present invention, the glucan can only be the said β-glucan. On the one hand, it can be used as a material targeting dendritic cells (β-glucan can target the Dectin-1 receptor of dendritic cells). On the other hand, the hydroxyl groups rich in its structure can be structurally modified to achieve more functions of the material. In addition, the solvent for dissolving the β-glucan can only be the said dimethyl sulfoxide. Although other solvents in the prior art such as water, ethanol, etc. can also dissolve β-glucan, they cannot unwind the β-glucan, so that the tolerogenic nano-vaccine precursor described in the present invention cannot be prepared.

[0041] In a specific embodiment, the acid anhydride is succinic anhydride and / or maleic anhydride. It should be noted that in addition to the acid anhydrides preferred in this embodiment, other acid anhydride structures similar to the said succinic anhydride or maleic anhydride in the prior art (having a cyclic acid anhydride structure and capable of combining with hydroxyl groups through ring-opening esterification reaction, such as cyclic acid anhydrides or cyclic dicarboxylic anhydrides) are also applicable to the present invention.

[0042] S2: Add a carboxyl activator to activate the carboxyl group to the reaction solution in step S1, and then add dopamine hydrochloride and react for 24 - 48 h.

[0043] In the present invention, by adding the said dopamine hydrochloride, an antigen-capturing group dopamine can be modified on the β-glucan chain. The catechol structure in the dopamine group can form a covalent bond with the thiol or amine in the protein through Michael addition or Schiff base reaction under oxidative conditions, so as to achieve the purpose of capturing antigens, so that the tolerogenic nano-vaccine precursor described in the present invention can capture self-antigens and form a tolerogenic nano-vaccine.

[0044] In a specific embodiment, the first carboxyl activator is a mixture of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide with a molar ratio of 1:1. It should be noted that the role of the first carboxyl activator is to activate the carboxyl group. In addition to the agents used in this embodiment, other carboxyl activators in the prior art that can activate the carboxyl group can also be applied to the present invention, such as DIC, DCC, etc. Optionally, the activation time is 15-30 min.

[0045] S3: Dialyze the reaction solution in step S2 to obtain a colorless and transparent liquid I, and perform freeze-drying on the colorless and transparent liquid I to obtain dopamine-modified β-glucan.

[0046] S4: Prepare a dopamine-modified β-glucan solution and an immune tolerance inducer solution respectively, and mix the dopamine-modified β-glucan solution and the immune tolerance inducer solution, and continuously stir for 12-24 h.

[0047] In the present invention, the triple helix structure of β-glucan can be unwound and rewound, so as to encapsulate drugs into the material and serve as a drug carrier.

[0048] In a specific embodiment, the immune tolerance inducer used to prepare the immune tolerance inducer solution is selected from any one of dexamethasone, rapamycin, simvastatin, and curcumin. It should be noted that the immune tolerance inducer in this embodiment is only a part of the preferred immune tolerance inducers of the present invention, and other immune tolerance inducers in the prior art can also be applied to the present invention. By using different immune tolerance inducers, the present invention can prepare tolerance nano-vaccine precursors for different diseases.

[0049] S5: Dialyze the reaction solution in step S4 to obtain a colorless and transparent liquid II, and perform freeze-drying on the colorless and transparent liquid II to obtain the tolerance nano-vaccine precursor.

[0050] In a specific embodiment, in step S1, the dosage of the anhydride is 1-3 times the mass of the β-glucan; in step S2, the dosage of the dopamine hydrochloride is 1-3 times the mass of the β-glucan; in step S4, the dosage of the immune tolerance inducer is 0.1-1 times the mass of the dopamine-modified β-glucan.

[0051] In a specific embodiment, the method for preparing the tolerance nano-vaccine precursor of the present invention further includes the step of encapsulating the tolerance nano-vaccine precursor into a hydrogel.

[0052] Nanoparticles smaller than 300 nm at the inflammatory site of arthritis can escape from the joint cavity freely through the dilated and hyperplastic lymphatic vessels and capillaries and be rapidly cleared. In the above embodiments, the present invention prolongs the residence time of the tolerogenic nano-vaccine precursor in the joint cavity by introducing a hydrogel carrier with a macroscopic size.

[0053] In a specific embodiment, encapsulating the tolerogenic nano-vaccine precursor into the hydrogel specifically includes the following steps:

[0054] S6: Prepare a sodium hyaluronate solution and adjust the pH of the sodium hyaluronate solution to 5 - 6.

[0055] S7: Add a carboxyl activator to activate the carboxyl group to the sodium hyaluronate solution, and then add 3-aminophenylboronic acid, and stir at room temperature for 24 - 48 h.

[0056] S8: Dialyze the reaction solution in step S7 to obtain a colorless and transparent liquid III, and freeze-dry the colorless and transparent liquid III to obtain phenylboronic acid-modified hyaluronic acid.

[0057] S9: Prepare a tolerogenic nano-vaccine precursor solution and a polyvinyl alcohol solution respectively, and add the phenylboronic acid-modified hyaluronic acid to the tolerogenic nano-vaccine precursor solution to obtain a phenylboronic acid-modified hyaluronic acid solution.

[0058] In a specific embodiment, in step S7, the dosage of the 3-aminophenylboronic acid is 1 - 3 times the molar amount of sodium hyaluronate; in step S9, the concentration of the tolerogenic nano-vaccine precursor in the tolerogenic nano-vaccine precursor solution is 6 - 13 mg / mL, the mass-volume concentration of the polyvinyl alcohol solution is 2 - 10%, and the mass-volume concentration of the phenylboronic acid-modified hyaluronic acid solution is 1 - 4%.

[0059] S10: Mix the phenylboronic acid-modified hyaluronic acid solution and the polyvinyl alcohol solution in equal volume to obtain a hydrogel encapsulating the tolerogenic nano-vaccine precursor.

[0060] In the above embodiments, phenylboronic acid-modified hyaluronic acid and polyvinyl alcohol are used to construct a hydrogel network through dynamic borate ester bonds to encapsulate the tolerogenic nano-vaccine precursor into the hydrogel. It can reduce the injection frequency of the tolerogenic nano-vaccine precursor, increase the residence time of the tolerogenic nano-vaccine precursor, and achieve the controlled release of the tolerogenic nano-vaccine precursor, and capture antigens to construct specific tolerogenic nano-vaccines.

[0061] On the other hand, the present invention also provides a tolerogenic nano-vaccine precursor prepared by the preparation method of the tolerogenic nano-vaccine precursor described in any one of the above, and its application in the treatment of rheumatoid arthritis.

[0062] Example 1

[0063] A nano-vaccine precursor with tolerance is prepared by the following steps:

[0064] (1) Dissolve 0.25 mmol of β-glucan in dimethyl sulfoxide, and add 0.25 mmol of anhydride (succinic anhydride) under the condition of constant temperature stirring at 50 °C, and react for 8 h;

[0065] (2) Cool the reaction solution of step (1) to 25 °C, then add carboxyl activator I (0.375 mmol of EDC and 0.375 mmol of NHS) to the reaction solution of step (1) to activate the carboxyl group for 30 min, and then add 0.375 mmol of dopamine hydrochloride and react for 48 h;

[0066] (3) Transfer the reaction solution of step (2) into a dialysis bag, dialyze with deionized water for 72 h to obtain a colorless and transparent liquid I, and freeze-dry the colorless and transparent liquid I to obtain dopamine-modified β-glucan (GSD); measure the absorbance of the dopamine-modified β-glucan at 278 nm by an ultraviolet spectrophotometer, and determine that the dopamine grafting amount is 0.41%;

[0067] (4) Take 20 mg of the dopamine-modified β-glucan and dissolve it in 10 mL of dimethyl sulfoxide, stir at 30 °C for 6 h to obtain a dopamine-modified β-glucan solution; take 2 mg of an immune tolerance inducer (dexamethasone) and dissolve it in 2 mL of dimethyl sulfoxide to obtain an immune tolerance inducer solution; mix the dopamine-modified β-glucan solution and the immune tolerance inducer solution, and continuously stir for 12 h;

[0068] (5) Transfer the reaction solution of step (4) into an MCWO 3500Da dialysis bag, dialyze with deionized water for 72 h to obtain a colorless and transparent liquid II, and freeze-dry the colorless and transparent liquid II to obtain a nano-vaccine precursor with tolerance (GSDDNPs); measure the absorption value of the GSDD NPs at 240 nm by liquid chromatography analysis, and determine that the dexamethasone loading amount is 9.1%.

[0069] Example 2

[0070] Different from Example 1, this example further includes the following steps:

[0071] (6) Prepare a sodium hyaluronate solution, and adjust the pH of the sodium hyaluronate solution to 6 with 0.1 M hydrochloric acid aqueous solution;

[0072] (7) Add a carboxyl activator bis(4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl-chloramine salt) to the sodium hyaluronate solution to activate the carboxyl group, and then add 3-aminophenylboronic acid in a molar amount three times that of sodium hyaluronate, and stir at room temperature for 24 h;

[0073] (8) Transfer the reaction solution of step (7) to a dialysis bag and dialyze for 5 days to obtain a colorless and transparent liquid III, and freeze-dry the colorless and transparent liquid III to obtain phenylboronic acid-modified hyaluronic acid;

[0074] (9) Weigh 30 mg of the phenylboronic acid-modified hyaluronic acid and add it to 1 mL of a solution of GSDD NPs containing 12.5 μg of an immune tolerance inducer dexamethasone to obtain a phenylboronic acid-modified hyaluronic acid solution containing 3% phenylboronic acid-modified hyaluronic acid; Prepare a polyvinyl alcohol solution containing 6% polyvinyl alcohol;

[0075] (10) Mix the phenylboronic acid-modified hyaluronic acid solution and the polyvinyl alcohol solution in equal volumes to obtain a hydrogel (HPGSDD nanocomposite hydrogel) encapsulating the tolerance nano-vaccine precursor.

[0076] Comparative Example 1

[0077] Different from Example 1, the dosage of the immune tolerance inducer in step (4) of this comparative example is 0 mg to obtain blank nanoparticles.

[0078] Comparative Example 2

[0079] Different from Example 2, the dosage of GSDD NPs in step (9) of this comparative example is 0 to obtain a blank hydrogel.

[0080] Test Example

[0081] The shape and particle size distribution of the tolerance nano-vaccine precursor of Example 1 were determined by transmission electron microscopy (TEM), dynamic light scattering (DLS), and Zeta potential. The results are as Figure 1 shown. As can be seen from Figure 1 a and b, the particle size of the blank nanoparticles in Comparative Example 1 is 183.3 ± 12.48 nm, and the potential is -19.2 ± 1.72; the average particle size of the tolerance nano-vaccine precursor of the present invention is 200.2 ± 3.5 nm, and the potential is 23.1 ± 1.16. As can be seen from Figure 1 c and d in, the tolerance nano-vaccine precursor of the present invention is a spherical structure.

[0082] The shape and particle size distribution of the tolerance nano-vaccine precursor of Example 1 after capturing the antigen were determined by transmission electron microscopy (TEM), dynamic light scattering (DLS), and Zeta potential. The results are as Figure 2 shown. As can be seen fromFigure 2 As can be seen from a and b, the particle size of the nano-vaccine formed after the nano-vaccine precursor of Example 1 captures the antigen is 255.1 ± 30.72 nm, and the potential is -8.18 ± 0.82. From Figure 2 As can be seen from c and d, the nano-vaccine formed after the nano-vaccine precursor of Example 1 captures the antigen is also in a spherical structure.

[0083] The morphology of the HPGSDD nano-composite hydrogel of Example 2 was observed by scanning electron microscopy, and the results are as Figure 3 shown. From Figure 3 a, it can be seen that the HPGSDD nano-composite hydrogel presents a loose and porous morphology; from Figure 3 b, it can be seen that the nano-vaccine precursor described in the present invention adheres to the hydrogel.

[0084] The gel-forming property of the HPGSDD nano-composite hydrogel of Example 2 was tested by a rotational rheometer. Specifically: the hydrogel was prepared into a sheet with a thickness of 2 mm and a radius of 1 cm, and the change trends of G' and G'' of the HPGSDD nano-composite hydrogel with time at 25 °C were detected by a rotational rheometer. Among them, G' is the storage modulus during the gel-forming process, which reflects the elastic properties of the material, and G'' is the loss modulus during the gel-forming process. When G' > G'', the material is in a gel state. The test results are as Figure 4 shown. From Figure 4 it can be seen that the HPGSDD nano-composite hydrogel described in the present invention is in a gel state (G' > G'') at 25 °C, and the gel strength is stable.

[0085] To determine the blood compatibility of the HPGSDD nano-composite hydrogel of Example 2, a hemolysis study was carried out. Take a 2% (v / v) concentration of blood cell solution, incubate 0.5 mL of nano-composite hydrogel leachate with different concentrations (2 mg, 1.5 mg, 1 mg, 0.5 mg, and 0.1 mg) with 0.5 mL of red blood cell suspension for 2 hours (37 °C), and centrifuge at 1500 rpm for 10 minutes. Measure the absorbance of the diluted supernatant at 540 nm in an enzyme-linked immunosorbent assay (ELISA) reader. The test results are as Figure 5 shown, and from Figure 5 it can be seen that the highest hemolysis rate of the HPGSDD nano-composite hydrogel described in the present invention does not exceed 2%, so it does not affect its in vivo use.

[0086] The cell compatibility of the HPGSDD nano-composite hydrogel of Example 2 was verified by cell live / dead fluorescence staining and the CCK-8 method, and the results are as Figure 6 shown. From Figure 6It can be seen that both rat chondrocytes and DC2.4 cells incubated with nano-composite hydrogel extracts at different concentrations were stained with uniform green fluorescence by AM, and almost no red fluorescence was observed, indicating good cell viability. At the same time, CCK-8 detection showed that the viability of chondrocytes and DC cells at different nano-material concentrations was higher than 90%, which met the experimental and treatment requirements.

[0087] To prove that the GSDD NPs in Example 1 can effectively induce immune tolerance in dendritic cells (DCs), the maturity of DC cells after immune tolerance was investigated. As a professional antigen-presenting cell, DC plays a crucial role in activating a series of acquired immune responses. The maturation of DC (dendritic cell) is closely related to immune stimulation. During the maturation process of DC cells, a large number of CD80 / 86 co-stimulatory signals will be expressed. These molecules are responsible for providing activation signals to immune cells after presenting antigens, thereby triggering an immune response. Therefore, we detected the expression of CD80 and CD86 in bone marrow-derived dendritic cells (BMDCs) treated with different sample groups after lipopolysaccharide (LPS) stimulation, and the results are as Figure 7 shown. As Figure 7 can be seen, the maturity of the control group (without adding any preparations, used to detect the state of normal dendritic cells) was 24.18%, the maturity of the LPs stimulation group (added LPS, used to detect the state of dendritic cells under inflammatory stimulation) was 79.95%, the dexamethasone group (simply added dexamethasone, used to detect the state of dendritic cells protected by dexamethasone alone) was 14.64%, the blank nanoparticle group (GSD NPs in Comparative Example 1) was 73.47%, and the nano-vaccine precursor group (tolerant nano-vaccine precursor GSDDNPs in Example 1) was 27.53%. The nano-vaccine group 1 (tolerant nano-vaccine precursor captures antigen to form tolerant nano-vaccine GSDD@Antigen NPs). It can be seen from this that the tolerant nano-vaccine precursor of the present invention showed a good down-regulation effect on DC cells, a decrease of 52.42% compared with the LPS group, and the tolerant nano-vaccine precursor of the present invention performed even better after capturing the antigen, a decrease of 59% compared with the LPS group. This proves that the antigen and the tolerance inducer can effectively induce tolerance and maintain a protective effect under the stimulation of LPS.

[0088] It should be noted that the above embodiments are only partial embodiments of the present invention. The tolerant nano-vaccine precursors prepared by changing the types, dosages, or temperature and time parameters of the various agents in the preparation method of the present invention have similar properties.

[0089] In summary, the present invention can in-situ capture antigens to form tolerogenic nano-vaccines, thereby achieving immune tolerance to continuously diffusing antigens in the body, and further improving the treatment effect. Compared with the prior art, the present invention has made significant progress.

[0090] As described above, these are only representative embodiments of the present invention and do not impose any formal limitations on the present invention. Any person skilled in the relevant art can, without departing from the technical solution of the present invention, make some modifications or improvements based on the disclosed technical content, and such embodiments are equivalent embodiments of the present invention. However, any simple modifications, equivalent changes, and improvements made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A method for preparing a tolerable nano vaccine precursor, characterized in that: The following steps are involved: S1: dissolve β-glucan in dimethyl sulfoxide, add acid anhydride under constant temperature stirring at 25-50°C, and react for 8-12h; S2: adding a carboxyl activator to the reaction solution of step S1 to activate the carboxyl group, and then adding dopamine hydrochloride to react for 24-48 hours; S3: dialyzing the reaction solution of step S2 to obtain a colorless transparent liquid 1, and freeze-drying the colorless transparent liquid 1 to obtain dopamine-modified β-glucan; S4: preparing a dopamine-modified β-glucan solution and an immune tolerance inducer solution respectively, and mixing the dopamine-modified β-glucan solution and the immune tolerance inducer solution, and continuously stirring for 12-24 hours; S5: dialyzing the reaction solution of step S4 to obtain a colorless transparent liquid 2, and freeze-drying the colorless transparent liquid 2 to obtain the tolerant nano vaccine precursor.

2. The method for preparing a tolerant nano vaccine precursor according to claim 1, characterized in that: In step S1, the acid anhydride is succinic anhydride and / or maleic anhydride.

3. The method for preparing a tolerant nano vaccine precursor according to claim 1, characterized in that: In step S2, the carboxyl activating agent 1 is a mixture of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide in a molar ratio of 1:

1.

4. The method for preparing a tolerant nano vaccine precursor according to claim 1, characterized in that: In step S4, the immune tolerance inducing agent used to prepare the immune tolerance inducing agent solution is selected from any one of dexamethasone, rapamycin, simvastatin, and curcumin.

5. The method for preparing a tolerant nano vaccine precursor according to claim 1, characterized in that: In step S1, the amount of the acid anhydride is 1-3 times the mass of the β-glucan; in step S2, the amount of dopamine hydrochloride is 1-3 times the mass of the β-glucan; in step S4, the amount of the immune tolerance inducer is 0.1-1 times the mass of the dopamine-modified β-glucan.

6. The method for preparing a tolerable nano vaccine precursor according to any one of claims 1 to 5, characterized in that: The method also includes the step of encapsulating the tolerized nano vaccine precursor into a hydrogel.

7. The method for preparing a tolerable nano vaccine precursor according to claim 6, characterized in that: Encapsulating the tolerized nano vaccine precursor into the hydrogel specifically comprises the following steps: S6: preparing a sodium hyaluronate solution, and adjusting the pH of the sodium hyaluronate solution to 5-6; S7: adding a carboxyl activator to the sodium hyaluronate solution to activate the carboxyl group, then adding 3-aminophenylboronic acid, and stirring at room temperature for 24-48 hours; S8: dialyzing the reaction solution of step S7 to obtain a colorless transparent liquid three, and freeze-drying the colorless transparent liquid three to obtain phenylboronic acid-modified hyaluronic acid; S9: preparing a tolerant nano vaccine precursor solution and a polyvinyl alcohol solution respectively, and adding the phenylboronic acid-modified hyaluronic acid to the tolerant nano vaccine precursor solution to obtain a phenylboronic acid-modified hyaluronic acid solution; S10: mixing the phenylboronic acid-modified hyaluronic acid solution and the polyvinyl alcohol solution in equal volumes to obtain a hydrogel encapsulating the tolerized nano-vaccine precursor.

8. The method for preparing a tolerable nano vaccine precursor according to claim 6, characterized in that: In step S7, the amount of 3-aminophenylboronic acid used is 1-3 times the molar amount of sodium hyaluronate; in step S9, the concentration of the tolerant nanovaccine precursor in the tolerant nanovaccine precursor solution is 6-13 mg / mL, the mass volume concentration of the polyvinyl alcohol solution is 2-10%, and the mass volume concentration of the phenylboronic acid modified hyaluronic acid solution is 1-4%.

9. A tolerable nano vaccine precursor, characterized in that: The tolerable nano vaccine precursor is prepared by the method for preparing the tolerable nano vaccine precursor according to any one of claims 1 to 8.

10. Use of the tolerized nano vaccine precursor according to claim 9 in the treatment of rheumatoid arthritis.

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