A Toll-like receptor 7 agonist nanoparticle and its preparation method and application

Abstract

The present invention discloses a Toll-like receptor 7 agonist nanoparticle, which is first prepared into lipoic acid nanoparticles using lipoic acid and amine heterocyclic compounds as raw materials, and then reacts with a TLR7 agonist having a structure as shown in Formula I to obtain TLR7 agonist-lipoic acid nanoparticles. The TLR7 agonist-lipoic acid nanoparticles of the present invention are easier to pass through the lymphatic vessel terminals and selectively enter the drainage lymph nodes, thereby avoiding the TLR7 agonist activating systemic inflammatory factors and inducing adverse reactions to the greatest extent; the nanoparticles deliver the TLR7 agonist to the lymph nodes in a targeted manner, which can mobilize antigen-presenting cells such as DCs and macrophages, induce stronger humoral immunity and cellular immunity, and exert an immune enhancement effect on vaccine antigens. The present invention also discloses the use of Toll-like receptor 7 agonist nanoparticles as veterinary vaccine adjuvants.

Description

Technical Field

[0001] The present invention relates to the field of chemical pharmacy or vaccine adjuvant or veterinary vaccine adjuvant, and relates to a Toll-like receptor 7 agonist nanoparticle and a preparation method and application thereof. Background Art

[0002] The immune protection efficacy of vaccines is the core indicator of product quality. Using specific immune enhancers to activate signal pathways and enhance the body's immune response to antigens is a flexible and efficient way to significantly improve the immune efficacy of vaccines. With the cross-integration and innovative development of immunology and nanomaterials, new immune-enhancing molecular structures are constantly emerging.

[0003] Toll-like receptors (TLRs) on immune cells are important targets for mediating immune responses. They can specifically recognize the structure of foreign pathogens and enhance immune responses. Among them, TLR7 is one of the important immune targets in the TLR family and is highly expressed in the endosomes of B cells and pDCs. The natural ligand of TLR7 is a viral ssRNA rich in uracil and guanine nucleosides. After the two are combined, they can stimulate the secretion of a large amount of type I interferon and inflammatory cytokines through the IRF7 and NF-κB signaling pathways, inducing a strong humoral immune response. However, as a nucleic acid molecule, ssRNA is not stable enough in the body. Mimicking the characteristics of its key groups, artificially synthesized TLR7 agonists are mainly small molecule compounds of imidazoquinoline and its derivative structures. Among them, R837 and R848 developed by 3M Company in the United States have been approved by the FDA for topical preparations for the treatment of condyloma caused by human papillomavirus (HPV). However, although artificially synthesized small molecule TLR7 agonists have strong immune enhancement effects, no products have been approved for vaccine manufacturing. The main reasons are (1) safety issues: small molecule TLR7 agonists quickly enter the blood after injection, inducing the secretion of systemic inflammatory cytokines and causing serious adverse reactions. (2) solubility issues: most existing small molecule TLR7 agonists have poor water solubility and are difficult to be incorporated into conventional vaccine formulations. Summary of the invention

[0004] In order to solve the above problems existing in the prior art, the object of the present invention is to provide a Toll-like receptor 7 (TLR7) agonist nanoparticle.

[0005] The objective of the present invention is achieved through the following technical solutions:

[0006] A Toll-like receptor 7 agonist nanoparticle, which uses lipoic acid and an amine heterocyclic compound as raw materials to first prepare lipoic acid nanoparticles (denoted as LP), and then reacts the lipoic acid nanoparticles with a TLR7 agonist to prepare TLR7 agonist-lipoic acid nanoparticles (denoted as T7-LP); wherein the structure of the TLR7 agonist is shown in Formula I:

[0007]

[0008] The particle size of the TLR7 agonist nanoparticles is 20 to 50 nm.

[0009] Another object of the present invention is to provide a method for preparing Toll-like receptor 7 agonist nanoparticles, comprising:

[0010] Step (1), preparing lipoic acid nanoparticles: dissolving lipoic acid and an amine heterocyclic compound in DMSO, wherein the lipoic acid and the amine heterocyclic compound are paired one by one to form an amphiphilic composition through the attraction of positive and negative charges; slowly dropping the solution containing the amphiphilic composition into water under rapid stirring to form nanomicelles; adjusting the pH of the solution containing the nanomicelles to 7.0-8.0, cross-linking under ultraviolet light to obtain core-cross-linked lipoic acid-amine heterocyclic nanoparticles; adjusting the pH to 10.0-11.0, dissociating the amine heterocyclic compound from the surface of the lipoic acid nanoparticles, removing the amine heterocyclic compound through dichloromethane extraction, and then dialyzing and freeze-drying to obtain lipoic acid nanoparticles;

[0011] Step (2), preparing TLR7 agonist-lipoic acid nanoparticles: redissolving the lipoic acid nanoparticles in a lower alcohol aqueous solution, adjusting the pH to 7.0-9.0, adding 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) in sequence to activate the carboxylic acid on the surface of the lipoic acid nanoparticles, then adding a lower alcohol aqueous solution of the TLR7 agonist, adjusting the pH of the reaction system to 6.5-8.0, stirring the reaction, dialyzing, and freeze-drying to obtain TLR7 agonist-lipoic acid nanoparticles.

[0012] In step (1), the amine heterocyclic compound is piperazine, piperidine or morpholine.

[0013] The molar ratio of thioctic acid to the amine heterocyclic compound is 2:1 to 1:4; considering the yield, the molar ratio of thioctic acid to the amine heterocyclic compound is preferably 1:1 to 1:4.

[0014] The volume ratio of DMSO to water is 1:1 to 1:10.

[0015] When the amphiphilic composition is added dropwise to water, the stirring speed of the water is 200 to 1400 rpm; considering the yield, the stirring speed of the water is preferably 700 to 1400 rpm; after the dropwise addition is completed, the stirring time is 5 min to 2 h.

[0016] The cross-linking conditions are: wavelength 365nm, cross-linking time 10min-4h, distance from light source to liquid surface of reaction system 50-200mm. Under ultraviolet light, disulfide bonds of lipoic acid are broken and recombined to form a core cross-linking structure.

[0017] The dichloromethane extraction is as follows: add 2 times the volume of dichloromethane to the obtained solution, extract for 3 times in total, and discard the dichloromethane layer.

[0018] The dialysis is as follows: the water layer obtained by dichloromethane extraction is placed in a dialysis bag with a molecular weight cutoff (MWCO) of 1000, and dialyzed in deionized water for 24 hours.

[0019] In step (2), the lower alcohol aqueous solution is obtained by mixing lower alcohol and water in a volume ratio of 0.05:1 to 1:1. Preferably, the lower alcohol aqueous solution is obtained by mixing lower alcohol and water in a volume ratio of 0.05:1 to 0.4:1. The lower alcohol aqueous solution is used to dissolve lipoic acid nanoparticles and TLR7 agonists.

[0020] The lower alcohol is selected from methanol and ethanol.

[0021] The molar ratio of the lipoic acid nanoparticles (calculated as lipoic acid) to 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 1:1-1:5.

[0022] The molar ratio of the lipoic acid nanoparticles (calculated as lipoic acid) to N-hydroxysuccinimide is 1:1 to 1:5.

[0023] Preferably, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is added, stirred at room temperature for 10 min to 4 h, and then N-hydroxysuccinimide is added, and stirring is continued at room temperature for 10 min to 4 h to fully activate the carboxylic acid on the surface of the lipoic acid nanoparticles.

[0024] The molar ratio of the lipoic acid nanoparticles (calculated as lipoic acid) to the TLR7 agonist is 1:0.5 to 1:4.

[0025] The reaction time of the lipoic acid nanoparticles and the TLR7 agonist is 4 to 48 hours. The present invention has no special requirements on the reaction temperature of the lipoic acid nanoparticles and the TLR7 agonist, and is generally carried out at room temperature (25° C.).

[0026] The dialysis is performed by using a dialysis bag with a molecular weight cutoff of 1000 and dialyzing in deionized water for 24 hours.

[0027] The Toll-like receptor 7 agonist nanoparticles of the present invention have the synergistic advantages of Toll-like 7 receptor activation and nanometer size. On the one hand, nanoparticles tend to enter the lymphatic system in the body and are enriched in lymph nodes, and there are abundant antigen-presenting cells such as DCs and macrophages in the lymph nodes. The TLR7 agonist is efficiently delivered here by nanoparticles, activating the Toll-like 7 receptor signaling pathway in antigen-presenting cells, which can induce a stronger immune response. On the other hand, nanoparticles of this size are difficult to enter the blood, which can minimize the excessive secretion of inflammatory factors in the blood circulation after the TLR7 signaling pathway is activated, which induces systemic inflammatory response. In the immunization experiments of mice and chickens, compared with small molecule TLR7 agonists, TLR7 agonist-lipoic acid nanoparticles significantly increased the antibody level after vaccine immunization, and had no inhibitory effect regardless of the dose. In the evaluation of the activation level of DCs recruited at the injection site in the early stage of immunization, the surface MHCⅡ, CD80, CD86, and CD40 of DCs recruited at the injection site of mice were highly expressed when immunized with granulated TLR7 agonists as adjuvants, and the peak was reached 3 days after immunization. The activation level and activation time of DCs are better than those of the same dose of TLR7 agonists, which will prompt DCs to present antigens to B cells and T cells faster and more, stimulating subsequent strong acquired immune responses. This shows that compared with TLR7 agonists, the Toll-like receptor 7 agonist nanoparticles of the present invention have an immune enhancement effect on vaccine antigens.

[0028] Another object of the present invention is to provide the use of the Toll-like receptor 7 agonist nanoparticles as an adjuvant for veterinary vaccines.

[0029] Another object of the present invention is to provide a veterinary vaccine, which comprises adding Toll-like receptor 7 agonist nanoparticles to a PBS solution of pH 7.4, stirring at room temperature to uniformly disperse the nanoparticles in the PBS solution, making the solution clear, filtering and sterilizing, and then mixing with an inactivated antigen solution to serve as a vaccine aqueous phase; mixing the vaccine aqueous phase with sterile Montanide ISA206 (oil phase) at a volume ratio of 1:1, and emulsifying to obtain a water-in-oil-in-water (W / O / W) formulation of the vaccine;

[0030] Alternatively, the Toll-like receptor 7 agonist nanoparticles are added to a PBS solution of pH 7.4, stirred at room temperature to uniformly disperse the nanoparticles in the PBS solution until the surface is clear, filtered for sterilization, and then mixed with an inactivated antigen solution and sterile Tween 80 to serve as the vaccine aqueous phase; the vaccine aqueous phase is mixed with sterile Marcol 52 pharmaceutical-grade white oil containing 4% Span 80 at a volume ratio of 1:2, and emulsified to obtain a water-in-oil (W / O) dosage form vaccine.

[0031] The stirring speed is 700-1400 rpm, and the stirring time is 5 min-12 h.

[0032] The antigens of the vaccine include but are not limited to inactivated antigens of porcine O-type foot-and-mouth disease virus, inactivated antigens of porcine pseudorabies virus, inactivated antigens of porcine epidemic diarrhea virus, and inactivated antigens of avian influenza H9N2 virus.

[0033] In the vaccine, the concentration of Toll-like receptor 7 agonist nanoparticles is 0.01 to 10 mg / mL.

[0034] Beneficial effects of the present invention:

[0035] Traditional amphiphilic compounds can form nanomicelles by self-assembly, but the instability of the micelle structure restricts its application. The present invention first uses the reversible attraction of positive and negative charges to form an amphiphilic composition of lipoic acid-heterocyclic amine, and then self-assembles into nanomicelles in water, and then irradiates the nanomicelle core with ultraviolet light to break and reorganize the disulfide bonds, forming a core cross-linked structure. The chemical bond connection of the cross-linked structure is significantly higher than the hydrophilic and hydrophobic forces of the conventional micelle structure, and the structural stability is greatly improved. Since the pKa of lipoic acid is 5.4 and the pKa of heterocyclic amine is 8.0-10.0, the present invention controls the solution pH at pH 7.0-8.0 during the reaction process of converting nanomicelles into nano cross-linked structures, which is conducive to the full ionization of the carboxyl group of lipoic acid and the secondary amine of heterocyclic amine, ensuring that the amphiphilic composition formed by the attraction of positive and negative charges is stable, thereby ensuring the stability of the self-assembled nanostructure. After the cross-linking reaction is completed, the solution pH is adjusted to 10.0-11.0 to convert the heterocyclic amine from an ionic state to a molecular state, which is then dissociated and removed from the surface of the nanoparticles, exposing the carboxyl groups on the surface of the nanoparticles for the next coupling step.

[0036] The present invention further realizes the coupling of TLR7 agonist and lipoic acid nanoparticles by coupling agent EDC / NHS. However, lipoic acid nanoparticles are uniformly dispersed rather than dissolved in aqueous solution, so the coupling reaction is a heterogeneous reaction. The present invention promotes lipoic acid nanoparticles to transfer appropriately to the dissolved state during the reaction by adding a small amount of methanol in the reaction system, and then by adjusting the pH of the reaction system to neutral-weak alkaline (pH7.0~9.0), the ionization level of the carboxyl group is improved, and then the activation efficiency of the carboxyl group is improved. At the same time, the TLR7 agonist of the present invention has a certain solubility in weakly acidic aqueous solution, and is insoluble in the neutral-weakly alkaline range, which is inconsistent with the pH required for the activation of the carboxyl group on the surface of lipoic acid nanoparticles. Therefore, the present invention adds a small amount of methanol in the solvent system, promotes the moderate dissolution of the TLR7 agonist in the neutral-weakly alkaline range (pH 6.5~8.0), and improves the coupling efficiency of the TLR7 agonist on the surface of lipoic acid nanoparticles.

[0037] The TLR7 agonist-lipoic acid nanoparticles of the present invention have the following advantages: (1) Size targeting of nanoparticles. The particle size of the TLR7 agonist-lipoic acid nanoparticles of the present invention is 20 to 50 nm. Nanoparticles of this size are easier to pass through the lymphatic terminal and selectively enter the draining lymph nodes, but are not easy to pass through blood vessels and enter the blood circulation. Therefore, the activation of systemic inflammatory factors and the induction of adverse reactions by the TLR7 agonist can be avoided to the greatest extent. (2) The enrichment effect of the TLR7 agonist in the lymph nodes. The nanoparticles deliver the TLR7 agonist to the lymph nodes in a targeted manner, which can mobilize the abundant antigen presenting cells such as DCs and macrophages there, and induce stronger humoral immunity and cellular immunity. (3) The compatibility of the dosage form of the nanoparticles. Compared with the TLR7 agonist with extremely poor water solubility, the TLR7 agonist-lipoic acid nanoparticles are evenly dispersed in water, and are uniform and stable for at least 18 months, meeting the shelf life requirements of the vaccine adjuvant in the vaccine product. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 The present invention is a preparation route of lipoic acid nanoparticles (LP).

[0039] Figure 2 This is the nuclear magnetic resonance hydrogen spectrum of TLR7 agonist-lipoic acid nanoparticles (T7-LP).

[0040] Figure 3 This is the UV wavelength-absorbance relationship diagram of TLR7 agonist (T7).

[0041] Figure 4 This is the relationship diagram between ultraviolet wavelength and absorbance of lipoic acid nanoparticles (LP).

[0042] Figure 5 The concentration-absorbance standard curve of TLR7 agonist (T7) at a wavelength of 270 nm.

[0043] Figure 6 The concentration-absorbance standard curve of lipoic acid nanoparticles (LP) at a wavelength of 270 nm.

[0044] Figure 7 TEM images of lipoic acid nanoparticles (LP) and TLR7 agonist-lipoic acid nanoparticles (T7-LP); wherein, Figure 7 A is LP, Figure 7 B is T7-LP.

[0045] Figure 8 The antibody levels of mice 28 days after subcutaneous immunization with TLR7 agonist-lipoic acid nanoparticles (T7-LP) as an immune adjuvant combined with O-type foot-and-mouth disease inactivated vaccine (FMDV); wherein the tested T7-LP was prepared according to Example 6.

[0046] Fig. 9 After mice were immunized with T7-LP and T7-adjuvanted FMDV vaccines in the quadriceps of the hind leg, DCs (CD11b + , CD11c + ) activation status; wherein, the tested T7-LP was prepared according to Example 6.

[0047] Fig.10 The antibody level of chickens 21 days after intramuscular immunization with TLR7 agonist-lipoic acid nanoparticles (T7-LP) as an immune adjuvant combined with avian influenza H9N2 vaccine; wherein the tested T7-LP was prepared according to Example 6. DETAILED DESCRIPTION

[0048] The structure of the TLR7 agonist (T7) used in the examples is shown in Formula I:

[0049]

[0050] Chemical name: 9-(4-(aminomethyl)benzyl)-8-methoxy-2-(2-methoxyethoxy)-9H-purin-6-amine.

[0051] Example 1

[0052] like Figure 1 As shown, lipoic acid (1.0 mmol) and piperazine (2.0 mmol) were dissolved in DMSO (0.5 mL), and lipoic acid and piperazine were paired one by one through the attraction of positive and negative charges to form an amphiphilic composition; the solution containing the amphiphilic composition was slowly added to water (4 mL) stirred at a high speed (1400 rpm), and after the addition was completed, stirring was continued for 30 minutes to form nano micelles; the pH of the aqueous solution containing the nano micelles was adjusted to 7.5 with 1 M hydrochloric acid, and cross-linked under ultraviolet light (wavelength: 365 nm, cross-linking time: 4h, distance from light source to liquid surface of reaction system: 200mm), to obtain core-crosslinked lipoic acid-piperazine nanoparticles; then adjust pH to 10.5 with 1M NaOH solution, piperazine dissociates from the surface of lipoic acid nanoparticles, add 2 times volume of dichloromethane to the solution obtained above, extract 3 times in total, discard the dichloromethane layer; then put the solution extracted with dichloromethane into dialysis bag (MWCO: 1000), dialyze in deionized water for 24h, freeze-dry, to obtain lipoic acid nanoparticles (LP), with a yield of 92.39%.

[0053] Example 2

[0054] Lipoic acid (1.0 mmol) and morpholine (1.0 mmol) were dissolved in DMSO (0.5 mL) to form an amphiphilic composition; the solution containing the amphiphilic composition was slowly added to water (2 mL) stirred at a high speed (1000 rpm), and after the addition was completed, the stirring was continued for 1.5 h to form nanomicelles; the pH of the aqueous solution containing the nanomicelles was adjusted to 7.0 with 1 M hydrochloric acid, and cross-linked under ultraviolet light (wavelength: 365 nm, light cross-linking time: 20 mi) was performed. n, the distance from the light source to the liquid surface of the reaction system: 50mm), to obtain core-crosslinked lipoic acid-amine heterocyclic nanoparticles; then adjust the pH to 10.0 with a 1M NaOH solution, dissociate morpholine from the surface of the lipoic acid nanoparticles, add 2 times the volume of dichloromethane to the above-obtained solution, extract 3 times in total, discard the dichloromethane layer; then put it into a dialysis bag (MWCO: 1000), dialyze in deionized water for 24h, and freeze-dry to obtain lipoic acid nanoparticles (LP), with a yield of 87.66%.

[0055] Example 3

[0056] Lipoic acid (1.0 mmol) and piperazine (2.0 mmol) were dissolved in DMSO (0.5 mL), and lipoic acid and piperazine were paired one by one through the attraction of positive and negative charges to form an amphiphilic composition; the solution containing the amphiphilic composition was slowly added into water (4 mL) stirred at a speed of 200 rpm, and after the addition was completed, the stirring was continued for 30 minutes to form nanomicelles; the pH of the aqueous solution containing the nanomicelles was adjusted to 7.5 with 1 M hydrochloric acid, and cross-linked under ultraviolet light (wavelength: 365 nm, cross-linking time: 4h, distance from light source to liquid surface of reaction system: 200mm), to obtain core-crosslinked lipoic acid-piperazine nanoparticles; then adjust pH to 10.5 with 1M NaOH solution, piperazine dissociates from the surface of lipoic acid nanoparticles, add 2 times volume of dichloromethane to the above solution, extract 3 times in total, discard the dichloromethane layer; then put into dialysis bag (MWCO: 1000), dialyze in deionized water for 24h, freeze-dry, to obtain lipoic acid nanoparticles (LP), with a yield of 67.45%.

[0057] Example 4

[0058] Lipoic acid (1.0 mmol) and piperazine (0.5 mmol) were dissolved in DMSO (0.5 mL), and lipoic acid and piperazine were paired one by one through the attraction of positive and negative charges to form an amphiphilic composition; the solution containing the amphiphilic composition was slowly added into water (4 mL) with high-speed stirring (1400 rpm), and after the addition was completed, the stirring was continued for 30 minutes to form nanomicelles; the pH of the aqueous solution containing the nanomicelles was adjusted to 7.5 with 1 M hydrochloric acid, and cross-linked under ultraviolet light (wavelength: 365 nm); m, cross-linking time: 4h, distance from light source to liquid surface of reaction system: 200mm), to obtain core-crosslinked lipoic acid-piperazine nanoparticles; then adjust the pH to 10.5 with 1M NaOH solution, piperazine dissociates from the surface of lipoic acid nanoparticles, add 2 times volume of dichloromethane to the above solution, extract 3 times in total, discard the dichloromethane layer; then put into dialysis bag (MWCO: 1000), dialyze in deionized water for 24h, freeze-dry, to obtain lipoic acid nanoparticles (LP), with a yield of 79.26%.

[0059] Comparative Example 1

[0060] The method comprises the following steps: dissolving lipoic acid (1.0 mmol) and piperazine (0.5 mmol) in DMSO (0.5 mL) to form an amphiphilic composition; slowly adding the solution containing the amphiphilic composition into water (4 mL) stirred at a high speed (1400 rpm), and continuing stirring for 30 min to form nano micelles; adjusting the pH of the aqueous solution containing the nano micelles to 10.0 with a 1 M NaOH solution, dissociating piperazine from the surface of lipoic acid nanoparticles, adding 2 times the volume of dichloromethane to the solution obtained above, extracting for 3 times, and discarding the dichloromethane layer; and then placing the solution into a dialysis bag (MWCO: 1000), dialyzing in deionized water for 24 h to obtain lipoic acid nano micelles with a yield of 42.95%.

[0061] Comparative Example 2

[0062] Lipoic acid (1.0 mmol) and piperazine (2.0 mmol) were dissolved in DMSO (0.5 mL). Lipoic acid and piperazine were paired one by one through the attraction of positive and negative charges to form an amphiphilic composition. The solution containing the amphiphilic composition was slowly added into water (4 mL) with high-speed stirring (1400 rpm). After the addition was completed, the stirring was continued for 30 minutes to form nanomicelles. At this time, the pH of the solution was about 9.0. Cross-linking was carried out under ultraviolet light (wavelength: 365 nm, cross-linking time: 200 ℃). 4h, the distance from the light source to the liquid surface of the reaction system: 200mm), to obtain core-crosslinked lipoic acid-piperazine nanoparticles; then adjust the pH to 10.5 with a 1M NaOH solution, piperazine dissociates from the surface of the lipoic acid nanoparticles, add 2 times the volume of dichloromethane to the above solution, extract 3 times in total, discard the dichloromethane layer; then put it into a dialysis bag (MWCO: 1000), dialyze in deionized water for 24h, and lyophilize to obtain lipoic acid nanoparticles (LP) with a yield of 32.02%.

[0063] Example 5

[0064] Utilize instrument Zetasizer Nano ZS (Malvern, UK) to measure the particle diameter and polydispersity index (PDI) of the thioctic acid nanoparticles prepared by embodiment 1-embodiment 2 and the thioctic acid nano micelle prepared by comparative example 1. As table 1, the sample is mixed with the water dispersion solution of different concentrations (in thioctic acid). When the sample of comparative example 1 is diluted to 0.001M, the particle diameter is reduced to single digit, indicating that the concentration is lower than the critical colloid concentration (CMC) of thioctic acid nano micelle, and now micelle collapses. And under the same concentration, the thioctic acid nanoparticles particle diameter that embodiment 1 and 2 prepare still remains stable, indicating that the particle kernel is completely cross-linked, and its nanostructure is not affected by CMC.

[0065] Table 1. Particle size and PDI of lipoic acid nanoparticles and nanomicelles at different concentrations

[0066]

[0067] The samples were stored at 4°C, and appropriate amounts were taken out at the time of preparation (0 month), 1 month, 3 months, 6 months, 12 months, and 18 months, respectively, and prepared into a 0.1M aqueous dispersion solution, and its particle size and PDI were measured to evaluate its long-term storage stability. The results are shown in Table 2. Within 18 months, the particle size of the lipoic acid nanoparticles prepared in Examples 1 and 2 was stable and uniform. The lipoic acid nanomicelles prepared in Comparative Example 1 showed a significant increase in particle size and a deterioration in dispersibility within 1 month. At 6 months, obvious precipitation was visible in the solution, and the PDI detection was 1 (reaching the upper limit of detection). It is shown that the stability of the lipoic acid nanoparticles prepared by ultraviolet light cross-linking of the present invention is significantly better than that of the nanomicelles that have not been cross-linked.

[0068] Table 2. Particle size and PDI of lipoic acid nanoparticles and nanomicelles at different storage time points

[0069]

[0070]

[0071] Example 6

[0072] 1.87 mmol of the lipoic acid nanoparticles prepared in Example 1 (calculated as lipoic acid) were redissolved in 5 mL of methanol aqueous solution (the volume ratio of methanol to water was 0.05:1), and the pH was adjusted to 7.5 with a 1 M NaOH solution; 3.74 mmol of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) was added, and the mixture was stirred at room temperature for 20 min, and then 3.74 mmol of N-hydroxysuccinimide (NHS) was added, and the mixture was stirred for 2 h at room temperature. To fully activate the carboxylic acid on the surface of lipoic acid nanoparticles, add 2mL of TLR7 agonist (T7) methanol aqueous solution (TLR7 agonist 1.12mmol, volume ratio of methanol to water is 0.05:1), adjust the pH to 6.8 with 1M hydrochloric acid, continue stirring at room temperature for 24h, put into a dialysis bag (MWCO: 1000), dialyze in deionized water for 24h, and lyophilize to obtain TLR7 agonist-lipoic acid nanoparticles (T7-LP) with a yield of 84.23%.

[0073] Example 7

[0074] 1.87 mmol of the lipoic acid nanoparticles prepared in Example 1 (calculated as lipoic acid) were redissolved in 5 mL of methanol aqueous solution (the volume ratio of methanol to water was 0.4:1), and the pH was adjusted to 8.5 with a 1 M NaOH solution; 7.48 mmol of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride was added, and the mixture was stirred at room temperature for 2 h, then 7.48 mmol of N-hydroxysuccinimide was added, and the mixture was stirred for 4 h at room temperature, 4 mL of a methanol aqueous solution of a TLR7 agonist was added (TLR7 agonist 3.74 mmol, the volume ratio of methanol to water was 0.4:1), the reaction pH was adjusted to 7.5 with 1 M hydrochloric acid, and the mixture was stirred for 48 h at room temperature, and the mixture was placed in a dialysis bag (MWCO: 1000), dialyzed in deionized water for 24 h, and lyophilized to obtain TLR7 agonist-lipoic acid nanoparticles (T7-LP) with a yield of 78.42%.

[0075] Comparative Example 3

[0076] 1.87 mmol of the lipoic acid nanoparticles prepared in Example 1 (calculated as lipoic acid) were redissolved in 5 mL of water, and the pH was adjusted to 7.5 with a 1 M NaOH solution; 3.74 mmol of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride was added, and the mixture was stirred at room temperature for 20 min, then 3.74 mmol of N-hydroxysuccinimide was added, and the mixture was stirred for 2 h at room temperature; 2 mL of an aqueous solution of a TLR7 agonist (1.12 mmol) was added, the pH was adjusted to 6.8 with 1 M hydrochloric acid, the mixture was stirred for 24 h at room temperature, and the mixture was placed in a dialysis bag (MWCO: 1000), dialyzed in deionized water for 24 h, and freeze-dried to obtain TLR7 agonist-lipoic acid nanoparticles with a yield of 45.17%.

[0077] Comparative Example 4

[0078] 1.87 mmol of the lipoic acid nanoparticles prepared in Example 1 (calculated as lipoic acid) were redissolved in 5 mL of methanol aqueous solution (the volume ratio of methanol to water was 0.02:1), and the pH was adjusted to 7.5 with a 1 M NaOH solution; 3.74 mmol of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride was added, and the mixture was stirred at room temperature for 20 min, then 3.74 mmol of N-hydroxysuccinimide was added, and the mixture was stirred for 2 h at room temperature, 2 mL of TLR7 agonist methanol aqueous solution (TLR7 agonist 1.12 mmol, the volume ratio of methanol to water was 0.02:1) was added, the pH was adjusted to 6.8 with 1 M hydrochloric acid, and the mixture was stirred for 24 h at room temperature, and the mixture was placed in a dialysis bag (MWCO: 1000), dialyzed in deionized water for 24 h, and lyophilized to obtain TLR7 agonist-lipoic acid nanoparticles (T7-LP) with a yield of 51.63%.

[0079] Comparative Example 5

[0080] 1.87 mmol of the lipoic acid nanoparticles prepared in Example 1 (calculated as lipoic acid) were redissolved in 5 mL of methanol aqueous solution (the volume ratio of methanol to water was 0.05:1), and the pH of the solution was about 5.3; 3.74 mmol of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride was added, and the mixture was stirred at room temperature for 20 min, then 3.74 mmol of N-hydroxysuccinimide was added, and the mixture was stirred at room temperature for 2 h, 2 mL of a methanol aqueous solution of a TLR7 agonist (T7) was added (1.12 mmol of the TLR7 agonist, and the volume ratio of methanol to water was 0.05:1), and the mixture was stirred at room temperature for 24 h, and the mixture was placed in a dialysis bag (MWCO: 1000), dialyzed in deionized water for 24 h, and freeze-dried to obtain TLR7 agonist-lipoic acid nanoparticles (T7-LP) with a yield of 57.25%.

[0081] Example 8

[0082] The structure of TLR7 agonist-lipoic acid nanoparticles (T7-LP) can be 1 H NMR spectrum characterization. Figure 2 As shown, compared with lipoic acid nanoparticles (LP, Example 1), the hydrogen spectrum of T7-LP (Example 6) added methylene peaks at positions 16 and 23, methyl peaks at positions 14 and 26, amino peak at position 15, and peaks on benzene rings at positions 18, 19, 21, and 22 on the molecular structure of the TLR7 agonist, proving that the TLR7 agonist was successfully coupled to the surface of the lipoic acid nanoparticles.

[0083] The coupling efficiency of TLR7 agonist in TLR7 agonist-lipoic acid nanoparticles was determined by ultraviolet spectrophotometry. The ultraviolet wavelength-absorbance relationship diagrams of TLR7 agonist (T7) and lipoic acid nanoparticles (LP, prepared in Example 1) are shown as follows: Figure 3 and 4 As shown in Figure 2, T7 has a distinct characteristic peak at 230 nm, while the absorbance of LP is close to the background at this wavelength. Therefore, 230 nm was selected as the measurement wavelength, and the concentration-absorbance relationship diagrams of TLR7 agonist (T7) and lipoic acid nanoparticles (LP) were established, and the standard curves were obtained by regression (see Figure 2, respectively). Figure 5 and Figure 6 ). TLR7 agonist-lipoic acid nanoparticles (Example 6) were diluted with methanol to a suitable concentration, and the absorbance value A at 230 nm was measured. Assuming that the absorbance value of the TLR7 agonist in the nanoparticles is X, the absorbance value of the lipoic acid nanoparticles is AX, and the concentrations of the TLR7 agonist and lipoic acid in the nanoparticles are substituted into the respective standard curves, and the mass fraction of the TLR7 agonist in the TLR7 agonist-lipoic acid nanoparticles, i.e., the coupling rate, was further calculated. According to the detection and calculation, in Example 6, Example 7, Comparative Example 3, Comparative Example 4, and Comparative Example 5, the coupling rates of the TLR7 agonist in T7-LP were 45.8%, 67.2%, 5.2%, 7.1%, and 6.7%, respectively. It shows that the reaction solvent and pH will affect the yield of TLR7 agonist-lipoic acid nanoparticles and the coupling rate of TLR7 agonist. Adding an appropriate amount of methanol and adjusting the solution pH during carboxyl activation and TLR7 agonist coupling can significantly improve the yield of TLR7 agonist-lipoic acid nanoparticles and the coupling rate of TLR7 agonist.

[0084] Example 9

[0085] The particle size and polydispersity index (PDI) of the TLR7 agonist-lipoic acid nanoparticles (T7-LP) prepared in Examples 6 and 7 were measured using a Zetasizer Nano ZS (Malvern, UK). The sample storage condition was 4°C, and appropriate amounts were taken out at the time of preparation (0 month), 1 month, 3 months, 6 months, 12 months, and 18 months, and the particle size and PDI were measured to evaluate the long-term storage stability. The results are shown in Table 3. Within 18 months, the particle size of the nanoparticles prepared in Examples 6 and 7 was stable and uniform.

[0086] The particle size and morphology of lipoic acid nanoparticles (LP, Example 1) and TLR7 agonist-lipoic acid nanoparticles (T7-LP, Example 6) were characterized by transmission electron microscopy (TEM, Hitachi, Japan). The results showed that the lipoic acid nanoparticles ( Figure 7 A) has a particle size of about 20 nm, TLR7 agonist-lipoic acid nanoparticles ( Figure 7 B) has a particle size between 20-50nm, which is basically spherical and has good uniformity. It is worth noting that the particle size shown by TEM is smaller than that measured by Zetasizer. This is because the Zetasizer measures the hydrated particle size, while TEM shows the bulk particle size of the particles.

[0087] Table 3. Particle size and PDI of T7-LP at different storage time points

[0088]

[0089] Example 10

[0090] The veterinary vaccine containing TLR7 agonist-lipoic acid nanoparticles (T7-LP) is prepared by the following method: adding different masses of TLR7 agonist-lipoic acid nanoparticles (Example 6) to a PBS solution of pH 7.4, stirring at a stirring speed of 1400 rpm for 30 minutes at room temperature, sterilizing through a filter membrane with a pore size of 0.22 μm, and then uniformly mixing with inactivated swine O-type foot-and-mouth disease virus (FMDV) antigen (Inner Mongolia Jinyu Group) to obtain a vaccine aqueous phase; then the above-mentioned aqueous phase is mixed with a sterile MontanideISA206 adjuvant (French SEPPIC Company, oil phase) at a volume ratio of 1:1, and emulsified to obtain a water-in-oil-in-water (W / O / W) dosage form vaccine. Among them, the final concentration of FMDV antigen in the vaccine is 10 μg / mL, and the final concentration of T7-LP in the vaccine is 0.1 mg / mL, 0.5 mg / mL, and 2.5 mg / mL, respectively.

[0091] The veterinary vaccine containing TLR7 agonist (T7) is prepared by the following method: adding different masses of T7 to DMSO, then adding to a PBS solution of pH 7.4, stirring at room temperature at a stirring speed of 1400 rpm for 30 minutes, sterilizing through a filter membrane with a pore size of 0.22 μm, and then uniformly mixing with inactivated swine type O foot-and-mouth disease virus (FMDV) antigen (Inner Mongolia Jinyu Group) to obtain a vaccine aqueous phase; then mixing the above aqueous phase with a sterile Montanide ISA206 adjuvant (SEPPIC, France, oil phase) at a volume ratio of 1:1, and emulsifying to obtain a water-in-oil-in-water (W / O / W) dosage form vaccine. Among them, the final concentration of DMSO in the vaccine is 0.5% (v / v), the final concentration of FMDV antigen in the vaccine is 10 μg / mL, and the final concentration of T7 in the vaccine is 0.1 mg / mL, 0.5 mg / mL, and 2.5 mg / mL, respectively.

[0092] The method of vaccine immunization and antibody detection is as follows: 6-week-old ICR female mice were randomly divided into groups according to Table 4, with 8 mice in each group, and the volume of subcutaneous immunization vaccine was 100 μL / mouse. 28 days after immunization, mouse serum was collected and separated, and the antibody titer of each group after vaccine immunization was detected using O-type foot-and-mouth disease liquid phase blocking ELISA kit (Lanzhou Veterinary Research Institute).

[0093] The results are as follows Figure 8 As shown in the figure, low and medium doses of TLR7 agonists as vaccine adjuvants can increase the vaccine antibody titer by more than 1, but the antibody level mediated by high doses of TLR7 agonists is lower than that of the vaccine control. It is speculated that high concentrations of T7 induce a large amount of inflammatory factors in the body, causing systemic toxic side effects, which manifest as immunosuppression. In contrast, nano-particle T7-LP can significantly increase the antibody level (≥2 titers) when used in combination with vaccines, regardless of high, medium or low doses, showing excellent immune enhancement effects.

[0094] Table 4. Grouping of mice immunized with FMDV vaccine

[0095]

[0096]

[0097] Embodiment 11

[0098] The ability of TLR7 agonist-lipoic acid nanoparticles (T7-LP) as vaccine adjuvants to enhance immune responses can also be evaluated by the activation level of DCs recruited to the injection site in the early stage of immunization.

[0099] According to Example 10, FMDV vaccines containing T7-LP (medium dose, i.e., the final concentration of T7-LP in the vaccine is 0.5 mg / mL) and FMDV vaccines containing T7 (medium dose, i.e., the final concentration of T7 in the vaccine is 0.5 mg / mL) were prepared respectively.

[0100] Six-week-old ICR female mice were randomly divided into groups, with 15 mice in each group. Each mouse was injected with 50 μL of the vaccine into the quadriceps muscle of the hind leg. Three mice were killed in each group at 0.5, 1, 3, 5, and 7 days after immunization, and the muscles at the injection site were taken, cut into small pieces, placed in HBSS with 0.05% type II collagenase, and digested with continuous stirring at 37°C for 30 minutes. After centrifugation (speed 400g) for 5 minutes, the cell suspension was resuspended in DMEM culture medium and filtered through a 70μm nylon mesh. DCs were labeled with fluorescently labeled antibodies anti-CD11b PE-Cy7 and anti-CD11c FITC in turn, and finally anti-MHCⅡAPC, anti-CD80 APC, anti-CD86 APC, and anti-CD40 APC were used to label DCs surface molecules for flow cytometric analysis. DCs recruited at the injection site (CD11b + , CD11c + ), the degree of activation can be analyzed and compared by the expression levels of MHCⅡ, CD80, CD86, and CD40 on the surface of DCs (mean fluorescence intensity of markers).

[0101] After vaccination, the recruitment of DCs at the injection site is the first step in the body's immune response, and the degree of DC activation determines its ability to capture, process, and present antigens. It is well known that the activation of Toll-like 7 receptors can significantly increase the activation level of DCs. Fig. 9 As shown in the figure, after immunization with the granulated TLR7 agonist T7-LP as an adjuvant, the surface MHCⅡ, CD80, CD86, and CD40 of DCs recruited at the injection site of mice were highly expressed, and the peak was reached 3 days after immunization. The activation level and activation time of DCs are better than those of T7 at the same dose, which will prompt DCs to present antigens to B cells and T cells faster and more, stimulating subsequent strong acquired immune responses.

[0102] Example 12

[0103] The veterinary vaccine containing TLR7 agonist-lipoic acid nanoparticles (T7-LP) is prepared by the following method: adding different masses of TLR7 agonist-lipoic acid nanoparticles (Example 6) to a PBS solution of pH 7.4, stirring at a stirring speed of 1400 rpm for 30 minutes at room temperature, sterilizing through a filter membrane with a pore size of 0.22 μm, and then uniformly mixing with inactivated avian influenza H9N2 antigen (Nanjing Tianbang Company) and sterile Tween 80 to obtain a vaccine aqueous phase (Tween 80 accounts for 4% v / v of the aqueous phase); then mixing the above aqueous phase with sterilized Marcol 52 pharmaceutical grade white oil containing 4% v / v Span 80 (French Esso Company, oil phase) at a volume ratio of 1:2, and emulsifying to obtain a vaccine in a water-in-oil (W / O) dosage form. The final content of H9N2 antigen in the vaccine is 10 8 Chicken embryo half infectious dose / mL (10 8 EID 50 / mL), and the final concentrations of T7-LP in the vaccine were 0.05 mg / mL, 0.25 mg / mL, and 1.25 mg / mL, respectively.

[0104] The veterinary vaccine containing TLR7 agonist (T7) is prepared by the following method: adding different masses of T7 to DMSO, then adding to a PBS solution of pH 7.4, stirring at room temperature at a stirring speed of 1400rpm for 30 minutes, sterilizing through a filter membrane with a pore size of 0.22μm, and then uniformly mixing with inactivated avian influenza H9N2 antigen (Nanjing Tianbang Company) and sterile Tween 80 to obtain a vaccine aqueous phase (Tween 80 accounts for 4% of the aqueous phase); then mixing the above aqueous phase with sterilized Marcol 52 pharmaceutical grade white oil containing 4% Span 80 (French Esso Company, oil phase) at a volume ratio of 1:2, and emulsifying to obtain a water-in-oil (W / O) dosage form vaccine. Among them, the final concentration of DMSO in the vaccine is 0.25% (v / v), and the final content of H9N2 antigen in the vaccine is 10 8 Chicken embryo half infectious dose / mL (10 8 EID 50 / mL), and the final concentrations of T7-LP in the vaccine were 0.05 mg / mL, 0.25 mg / mL, and 1.25 mg / mL, respectively.

[0105] The method of vaccine immunization and antibody detection is as follows: 10-week-old chickens are randomly divided into groups according to Table 5, with 10 chickens in each group, and the volume of intramuscular immunization vaccine is 400 μL / bird. 21 days after immunization, chicken serum is collected and separated, and the serum antibody titer is detected by microhemagglutination inhibition test (HI) using H9 avian influenza detection antigen.

[0106] The results are as follows Fig.10As shown in the figure, nano-particle T7-LP combined with avian influenza antigens in immunization of animals still exerted a significant immune enhancement effect, and the medium dose was the best, showing potential application prospects. However, T7 has a strong immunosuppressive effect at high doses, and the antibody levels at medium and low doses are not as good as T7-LP.

[0107] Table 5. Grouping of chickens immunized with avian influenza H9N2 vaccine

[0108] Vaccine grouping Vaccine composition Adjuvant and concentration 1 H9N2 vaccine none 2 H9N2 vaccine + TLR7 agonist (low dose) T7 (0.05 mg / mL) 3 H9N2 vaccine + TLR7 agonist (medium dose) T7 (0.25 mg / mL) 4 H9N2 vaccine + TLR7 agonist (high dose) T7 (1.25 mg / mL) 5 H9N2 vaccine + TLR7 agonist nanoparticles (low dose) T7-LP (0.05 mg / mL) 6 H9N2 vaccine + TLR7 agonist nanoparticles (medium dose) T7-LP (0.25 mg / mL) 7 H9N2 vaccine + TLR7 agonist nanoparticles (high dose) T7-LP (1.25 mg / mL)

Claims

1. A Toll-like receptor 7 agonist nanoparticle, characterized in that: The method uses lipoic acid and an amine heterocyclic compound as raw materials to first prepare lipoic acid nanoparticles, and then reacts the lipoic acid nanoparticles with a TLR7 agonist to prepare TLR7 agonist-lipoic acid nanoparticles; wherein the amine heterocyclic compound is piperazine, piperidine or morpholine; and the structure of the TLR7 agonist is shown in Formula I: The Toll-like receptor 7 agonist nanoparticles are prepared by the following method: Step (1), preparing thioctic acid nanoparticles: dissolving thioctic acid and an amine heterocyclic compound in DMSO, pairing thioctic acid and the amine heterocyclic compound one by one to form an amphiphilic composition; dropping the solution containing the amphiphilic composition into water under rapid stirring to form nanomicelles; adjusting the pH of the solution containing the nanomicelles to 7.0-8.0, cross-linking under ultraviolet light to obtain core-cross-linked thioctic acid-amine heterocyclic nanoparticles; adjusting the pH to 10.0-11.0, dissociating the amine heterocyclic compound from the surface of the thioctic acid nanoparticles, extracting with dichloromethane to remove the amine heterocyclic compound, and then dialyzing and freeze-drying to obtain thioctic acid nanoparticles; Step (2), preparing TLR7 agonist-lipoic acid nanoparticles: redissolving the lipoic acid nanoparticles in a lower alcohol aqueous solution, adjusting the pH to 7.0-9.0, adding 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide in sequence to activate the carboxylic acid on the surface of the lipoic acid nanoparticles, then adding a lower alcohol aqueous solution of the TLR7 agonist, adjusting the pH of the reaction system to 6.5-8.0, stirring the reaction, dialyzing, and freeze-drying to obtain the TLR7 agonist-lipoic acid nanoparticles; the lower alcohol is selected from methanol and ethanol.

2. A method for preparing the Toll-like receptor 7 agonist nanoparticles according to claim 1, characterized in that: include: Step (1), preparing thioctic acid nanoparticles: dissolving thioctic acid and an amine heterocyclic compound in DMSO, pairing thioctic acid and the amine heterocyclic compound one by one to form an amphiphilic composition; dropping the solution containing the amphiphilic composition into water under rapid stirring to form nanomicelles; adjusting the pH of the solution containing the nanomicelles to 7.0-8.0, cross-linking under ultraviolet light to obtain core-cross-linked thioctic acid-amine heterocyclic nanoparticles; adjusting the pH to 10.0-11.0, dissociating the amine heterocyclic compound from the surface of the thioctic acid nanoparticles, extracting with dichloromethane to remove the amine heterocyclic compound, and then dialyzing and freeze-drying to obtain thioctic acid nanoparticles; Step (2), preparing TLR7 agonist-lipoic acid nanoparticles: redissolving the lipoic acid nanoparticles in a lower alcohol aqueous solution, adjusting the pH to 7.0-9.0, adding 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide in sequence to activate the carboxylic acid on the surface of the lipoic acid nanoparticles, then adding a lower alcohol aqueous solution of the TLR7 agonist, adjusting the pH of the reaction system to 6.5-8.0, stirring the reaction, dialyzing, and freeze-drying to obtain the TLR7 agonist-lipoic acid nanoparticles; the lower alcohol is selected from methanol and ethanol.

3. The method for preparing Toll-like receptor 7 agonist nanoparticles according to claim 2, characterized in that: In step (1), the amine heterocyclic compound is piperazine, piperidine or morpholine; and the molar ratio of thioctic acid to the amine heterocyclic compound is 2:1 to 1:

4.

4. The method for preparing Toll-like receptor 7 agonist nanoparticles according to claim 3, characterized in that: In step (1), the molar ratio of thioctic acid to the amine heterocyclic compound is 1:1 to 1:

4.

5. The method for preparing Toll-like receptor 7 agonist nanoparticles according to claim 2, characterized in that: In step (1), the volume ratio of DMSO to water is 1:1 to 1:10; when the amphiphilic composition is added dropwise to water, the stirring speed of the water is 200 to 1400 rpm.

6. The method for preparing Toll-like receptor 7 agonist nanoparticles according to claim 5, characterized in that: In step (1), when the amphiphilic composition is added dropwise into water, the stirring speed of the water is 700 to 1400 rpm.

7. The method for preparing Toll-like receptor 7 agonist nanoparticles according to claim 2, characterized in that: In step (1), the cross-linking conditions are: wavelength 365nm, cross-linking time 10min to 4h, and the distance from the light source to the liquid surface of the reaction system is 50 to 200mm.

8. The method for preparing Toll-like receptor 7 agonist nanoparticles according to claim 2, characterized in that: In step (1), the dichloromethane extraction to remove the amine heterocyclic compound is as follows: adding 2 times the volume of dichloromethane to the obtained solution, extracting for 3 times, and discarding the dichloromethane layer; the dialysis is as follows: placing the water layer obtained by dichloromethane extraction into a dialysis bag with a molecular weight cutoff of 1000, and dialyzing in deionized water for 24 hours; In step (2), the dialysis is performed by using a dialysis bag with a molecular weight cut-off of 1000 and dialyzing in deionized water for 24 hours.

9. The method for preparing Toll-like receptor 7 agonist nanoparticles according to claim 2, characterized in that: In step (2), the lower alcohol aqueous solution is obtained by mixing lower alcohol and water in a volume ratio of 0.05:1 to 1:

1.

10. The method for preparing Toll-like receptor 7 agonist nanoparticles according to claim 9, characterized in that: In step (2), the lower alcohol aqueous solution is obtained by mixing lower alcohol and water in a volume ratio of 0.05:1 to 0.4:

1.

11. The method for preparing Toll-like receptor 7 agonist nanoparticles according to claim 2, characterized in that: In step (2), the amount of the lipoic acid nanoparticles is calculated based on lipoic acid, and the molar ratio of the lipoic acid nanoparticles to 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 1:1 to 1:5; the molar ratio of the lipoic acid nanoparticles to N-hydroxysuccinimide is 1:1 to 1:5; and the molar ratio of the lipoic acid nanoparticles to the TLR7 agonist is 1:0.5 to 1:

4.

12. Use of the Toll-like receptor 7 agonist nanoparticles according to claim 1 as an adjuvant for veterinary vaccines.

13. A veterinary vaccine, characterized in that: The method comprises adding the Toll-like receptor 7 agonist nanoparticles described in claim 1 to a PBS solution of pH 7.4, stirring at room temperature, filtering and sterilizing, and then mixing with an inactivated antigen solution to obtain a vaccine aqueous phase; mixing the vaccine aqueous phase with sterile Montanide ISA206 at a volume ratio of 1:1, and emulsifying to obtain a water-in-oil-in-water dosage form vaccine; Or it is adding the Toll-like receptor 7 agonist nanoparticles described in claim 1 to a PBS solution of pH 7.4, stirring at room temperature, filtering and sterilizing, and then mixing with an inactivated antigen solution and sterile Tween 80 to serve as a vaccine aqueous phase; the vaccine aqueous phase is mixed with sterile Marcol 52 pharmaceutical grade white oil containing 4% Span 80 at a volume ratio of 1:2, and emulsified to obtain a water-in-oil dosage form vaccine; Among them, the antigens of the veterinary vaccine are inactivated antigens of swine O-type foot-and-mouth disease virus, inactivated antigens of swine pseudorabies virus, inactivated antigens of porcine epidemic diarrhea virus, and inactivated antigens of avian influenza H9N2 virus.

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