CN113599515B - Thermostable nucleic acid vaccine adjuvant and preparation method thereof - Google Patents

Abstract

The invention relates to a thermostable nucleic acid vaccine adjuvant and a preparation method thereof, which belong to the technical field of vaccine adjuvant preparation and comprise the following raw materials in parts by weight: 25.8-31.2 parts of oil phase, 50-60 parts of water phase, 3.5-4.6 parts of modified microsphere and 0.5-1.1 part of inorganic filler, mixing the inorganic filler and the water phase, adding the oil phase and the modified microsphere after ultrasonic dispersion at the frequency of 40-60kHz, and continuing ultrasonic treatment for 20min to obtain the thermostable nucleic acid vaccine adjuvant, wherein after the internal substances adsorb antigen, the thermostable nucleic acid vaccine adjuvant is emulsion, the internal substances have uneven surfaces, the antigen can be adsorbed in gaps of the thermostable nucleic acid vaccine adjuvant, thereby playing a role of protecting the antigen, enabling the antigen to resist thermal injury at a certain temperature, maintaining bioactivity, and in addition, small particle size particles in the emulsion can generate higher stacking efficiency at an oil-water interface to form more uniform particle films, thereby blocking mutual collision between the emulsions and keeping the state of the nucleic acid vaccine adjuvant stable.

Description

Thermostable nucleic acid vaccine adjuvant and preparation method thereof

Technical Field

The invention belongs to the technical field of vaccine adjuvant preparation, and particularly relates to a thermostable nucleic acid vaccine adjuvant and a preparation method thereof.

Background

The nucleic acid vaccine is to introduce the foreign gene encoding some antigen protein into animal cell directly, synthesize antigen protein via the expression system of host cell and induce the host to produce immune response to the antigen protein for the purpose of preventing and treating diseases.

Vaccine adjuvants refer to substances that, either simultaneously with or pre-applied to an antigen, enhance the body's ability to respond to the antigen, or alter the type of immune response, and include inorganic adjuvants (e.g., aluminum hydroxide), organic adjuvants (e.g., lipopolysaccharide, mycobacteria), and synthetic adjuvants such as double-stranded polyinosinic acid, cytidylic acid.

The nucleic acid vaccine is a sensitive product which needs careful management, the protection and transportation of the nucleic acid vaccine at a proper temperature are extremely important, the heat stress can reduce the titer of the vaccine or even deactivate the vaccine, the immune response cannot be activated, and huge potential risks exist.

Disclosure of Invention

The invention aims to provide a thermostable nucleic acid vaccine adjuvant and a preparation method thereof, which are used for solving the technical problems mentioned in the background art.

The aim of the invention can be achieved by the following technical scheme:

a thermostable nucleic acid vaccine adjuvant comprises the following raw materials in parts by weight: 25.8-31.2 parts of oil phase, 50-60 parts of water phase, 3.5-4.6 parts of modified microspheres and 0.5-1.1 parts of inorganic filler;

the thermostable nucleic acid vaccine adjuvant is prepared by the following steps:

mixing inorganic filler and water phase, ultrasonic dispersing at 40-60kHz, adding oil phase and modified microsphere, and continuing ultrasonic treatment for 20min to obtain the heat stable nucleic acid vaccine adjuvant.

Further, the modified microsphere is prepared by the following steps:

step A1, adding polyethylenimine, acetonitrile and triethylamine into a round-bottom flask, shaking uniformly, sequentially adding an acetonitrile solution of hexachlorocyclotriphosphazene and an acetic acid solution of chitosan, sealing a bottle mouth, performing ultrasonic reaction for 8 hours at 40 ℃, performing ultrasonic power for 180W at the frequency of 40Hz, centrifuging for 20-30 minutes at the rotating speed of 1000-1500r/min, washing the precipitate with acetone and absolute ethyl alcohol for 2 times sequentially, and finally drying for 24 hours at the temperature of minus 45 ℃ to obtain composite microspheres, wherein the polyethylenimine, the chitosan and the hexachlorocyclotriphosphazene are used as substrates to prepare the crosslinked polyphosphazene composite microspheres, and the crosslinked polyphosphazene composite microspheres have higher biocompatibility and degradability, and the hexaheterocyclic ring of N, P has higher stability and certain high-temperature resistance;

step A2, placing DMF in a three-neck flask, dropwise adding chlorosulfonic acid into the three-neck flask under ice bath condition, stirring while dropwise adding, keeping the temperature of a reaction system at 0-5 ℃, stirring at room temperature until the reaction system is clear after dropwise adding is finished, obtaining a treating agent, and mixing chlorosulfonic acid and DMF to obtain the treating agent;

and A3, adding the composite microspheres and absolute ethyl alcohol into a three-neck flask, performing ultrasonic dispersion for 30min, dropwise adding a treating agent into the three-neck flask, reacting for 1min under the condition of microwave radiation after the dropwise adding, precipitating for 30min in a refrigerator at 4 ℃ after the reaction is finished, taking out, washing with distilled water, adding a sodium hydroxide solution with the concentration of 2mol/L to adjust the pH value to 7-8, filtering, dialyzing a filter cake in distilled water for 72h, and freeze-drying to constant weight to obtain modified microspheres, and performing substitution reaction on amino groups on the surfaces of the composite microspheres and chlorosulfonic acid to enable the surfaces of the polymer microspheres to be grafted with sulfonic acid groups, thereby improving the water solubility of the composite microspheres.

Further, the dosage ratio of the acetonitrile solution of polyethylenimine, acetonitrile, triethylamine, hexachlorocyclotriphosphazene and the acetic acid solution of chitosan in the step A1 is 0.42-0.48g:80-90mL:4mL:10-15mL:20mL of an acetonitrile solution of hexachlorocyclotriphosphazene from hexachlorocyclotriphosphazene and acetonitrile according to 0.1g:20mL of the mixture, wherein the acetic acid solution of the chitosan is prepared from 0.1g of chitosan and 6-8% acetic acid solution by mass percent: 20mL of the mixture.

Further, in the step A2, the volume ratio of DMF to chlorosulfonic acid is 5:1.

further, in the step A3, the dosage ratio of the composite microsphere, the absolute ethyl alcohol and the treating agent is 50mg:100mL:8.5-9.1mg.

Further, the inorganic filler is prepared by the following steps:

step B1, adding triethylamine, hexadecyl trimethyl ammonium chloride and deionized water into a three-neck flask, performing ultrasonic dispersion for 15min at the frequency of 40-45kHz, heating to 60 ℃, stirring at the rotation speed of 150r/min for reaction for 1h, dropwise adding chlorobenzene into the three-neck flask, stirring for 20min after the dropwise adding, dropwise adding tetraethoxysilane, performing heat preservation and stirring for reaction for 12h, performing centrifugal treatment at the rotation speed of 30000rpm for 10min after the reaction is finished, washing the precipitate with absolute ethyl alcohol for 3-5 times, finally calcining in a muffle furnace at the temperature of 600 ℃ for 8h, and cooling to room temperature to obtain mesoporous silica;

and B2, under the protection of nitrogen, adding mesoporous silica, 2, 3-epoxypropyl trimethyl ammonium chloride and dimethyl sulfoxide into a three-neck flask, controlling the temperature to be 50-60 ℃, stirring and reacting for 10min, then adding potassium hydroxide, heating to 180-185 ℃, stirring at a speed of 120-150r/min, carrying out reflux reaction for 3-5h, centrifuging after the reaction is finished, washing the precipitate with deionized water for 3-5 times, and finally drying at 80 ℃ to constant weight to obtain the inorganic filler.

Further, the dosage ratio of triethylamine, cetyltrimethylammonium chloride, deionized water, chlorobenzene and ethyl orthosilicate in B1 is 0.18g:2.7-3.3g:36-40mL:2.6-4.1mL:1mL.

Further, the dosage ratio of the mesoporous silica B2 to the 2, 3-epoxypropyl trimethyl ammonium chloride to the dimethyl sulfoxide to the potassium hydroxide is 0.5-0.8g:0.2-0.4g:25.8-26.4mL:0.5g.

Further, the oil phase is one of a biocompatible grease and a mineral oil.

Further, the water phase comprises one or more of purified water, water for injection, glycerol water solution, physiological saline with the mass fraction of 0.9%, phosphate buffer solution, citric acid buffer solution or Tris buffer solution, which are mixed according to any ratio.

The invention has the beneficial effects that:

1) The invention prepares a heat stable nucleic acid vaccine adjuvant by taking an oil phase, a water phase, modified microspheres and inorganic fillers as raw materials, the heat stable nucleic acid vaccine adjuvant is emulsion, after the internal substances adsorb antigens, the internal substances have rugged surfaces, and the antigens can be adsorbed in gaps of the internal substances, so that the heat stable nucleic acid vaccine adjuvant has the effect of protecting the antigens, the antigens can resist heat damage at a certain temperature, the biological activity is kept, in addition, small-particle-size particles in the emulsion can generate higher stacking efficiency at an oil-water interface to form more uniform particle films, so that the mutual collision between the emulsions is blocked, and the state of the nucleic acid vaccine adjuvant is kept stable.

2) According to the invention, polyethyleneimine, chitosan and hexachlorocyclotriphosphazene are used as substrates, so that cross-linked polyphosphazene composite microspheres are prepared, sulfonic acid groups are grafted on the surfaces of the composite microspheres through treatment of a treatment agent, the water solubility of the polymer microspheres is improved, the composite microspheres have higher biocompatibility and degradability, the N, P-containing six-membered heterocycle has higher thermal stability, chitosan has good inherent immunity and specific immunity stimulating capability, can induce mucosal immunity, has high protection effect on antigen, can enhance antigen immunogenicity, can wrap antigens in the antigen, has huge molecular weight and complex space structure, can carry a large amount of positive charges, has strong electrostatic effect to protect vaccines from enzymolysis in the transportation process, has stronger stability when the vaccine is combined with chitosan than the vaccine which is directly exposed, has sustained release characteristic, can change the processing and presenting mode of the antigen in the presenting cells after the absorbed microspheres are ingested into cells, promotes more antigens to realize cross-presenting through MHCI (mobile high-content immune response) paths, and induces high-level cell immunity.

3) The inorganic filler takes mesoporous silica as a carrier, utilizes the ring-opening reaction of hydroxyl groups on the surface of the mesoporous silica and epoxy groups of 2, 3-epoxypropyl trimethyl ammonium chloride under an alkaline condition to obtain the inorganic filler with surface grafted cationic functional groups, and has electropositivity due to the fact that the surface of the inorganic filler has cationic functional groups, can carry out electrostatic adsorption with negatively charged protein antigens and can carry out electrostatic adsorption with the cell surfaces, so that the mesoporous silica has better antigen loading capacity and cell targeting capacity, has the characteristics of high specific surface area and multiple gaps, has multiple attachment sites, can realize stable loading of protein antigens, and further improves the compatibility of the modified mesoporous silica with an oil phase.

Detailed Description

The technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Example 1

The modified microsphere is prepared by the following steps:

step A1, adding 0.42g of polyethylenimine, 80mL of acetonitrile and 4mL of triethylamine into a round-bottom flask, shaking uniformly, sequentially adding 10mL of acetonitrile solution of hexachlorocyclotriphosphazene and 20mL of acetic acid solution of chitosan, sealing a bottle mouth, performing ultrasonic reaction for 8h at 40 ℃, performing ultrasonic power of 180W and frequency of 40Hz, centrifuging for 20min at the rotating speed of 1000r/min, washing the precipitate with acetone and absolute ethyl alcohol for 2 times sequentially, and finally drying at-45 ℃ for 24h to obtain composite microspheres, wherein the acetonitrile solution of hexachlorocyclotriphosphazene is prepared by mixing hexachlorocyclotriphosphazene and acetonitrile according to 0.1g:20mL of the mixture, wherein the acetic acid solution of chitosan is prepared from 0.1g of chitosan and 6% acetic acid solution by mass percent: 20mL of the mixture;

step A2, placing 5mL of DMF in a three-neck flask, dropwise adding 1mL of chlorosulfonic acid into the three-neck flask under ice bath condition, stirring while dropwise adding, keeping the temperature of a reaction system at 0 ℃, and stirring at room temperature until the mixture is clarified after the dropwise adding is finished to obtain a treating agent;

and A3, adding 50mg of composite microspheres and 100mL of absolute ethyl alcohol into a three-neck flask, dispersing for 30min by ultrasonic, dripping 8.5mg of treating agent into the three-neck flask, reacting for 1min under the condition of microwave radiation after dripping, precipitating for 30min in a refrigerator at 4 ℃ after reaction, taking out, washing with distilled water, adding 2mol/L sodium hydroxide solution to adjust the pH value to 7, filtering, dialyzing a filter cake in distilled water for 72h, and freeze-drying to constant weight to obtain the modified microspheres.

Example 2

The modified microsphere is prepared by the following steps:

step A1, adding 0.46g of polyethylenimine, 85mL of acetonitrile and 4mL of triethylamine into a round-bottom flask, shaking uniformly, sequentially adding 12mL of acetonitrile solution of hexachlorocyclotriphosphazene and 20mL of acetic acid solution of chitosan, sealing a bottle mouth, performing ultrasonic reaction for 8h at 40 ℃, performing ultrasonic power of 180W and frequency of 40Hz, centrifuging for 25min at the rotating speed of 1200r/min, washing the precipitate with acetone and absolute ethyl alcohol for 2 times sequentially, and finally drying at-45 ℃ for 24h to obtain composite microspheres, wherein the acetonitrile solution of hexachlorocyclotriphosphazene is prepared by mixing hexachlorocyclotriphosphazene and acetonitrile according to 0.1g:20mL of the mixture, wherein the acetic acid solution of chitosan is prepared from 0.1g of chitosan and 7% acetic acid solution by mass percent: 20mL of the mixture;

step A2, placing 5mL of DMF in a three-neck flask, dropwise adding 1mL of chlorosulfonic acid into the three-neck flask under ice bath condition, stirring while dropwise adding, keeping the temperature of a reaction system at 2 ℃, and stirring at room temperature until the mixture is clarified after the dropwise adding is finished to obtain a treating agent;

and A3, adding 50mg of composite microspheres and 100mL of absolute ethyl alcohol into a three-neck flask, dispersing for 30min by ultrasonic, dripping 8.8mg of treating agent into the three-neck flask, reacting for 1min under the condition of microwave radiation after dripping, precipitating for 30min in a refrigerator at 4 ℃ after reaction, taking out, washing with distilled water, adding 2mol/L sodium hydroxide solution to adjust the pH value to 7, filtering, dialyzing a filter cake in distilled water for 72h, and freeze-drying to constant weight to obtain the modified microspheres.

Example 3

The modified microsphere is prepared by the following steps:

step A1, adding 0.48g of polyethylenimine, 90mL of acetonitrile and 4mL of triethylamine into a round-bottom flask, shaking uniformly, sequentially adding 15mL of acetonitrile solution of hexachlorocyclotriphosphazene and 20mL of acetic acid solution of chitosan, sealing a bottle mouth, performing ultrasonic reaction for 8 hours at 40 ℃, performing ultrasonic power of 180W and frequency of 40Hz, centrifuging for 30 minutes at the rotating speed of 1500r/min, washing the precipitate with acetone and absolute ethyl alcohol for 2 times sequentially, and finally drying at-45 ℃ for 24 hours to obtain composite microspheres, wherein the acetonitrile solution of hexachlorocyclotriphosphazene is prepared by mixing hexachlorocyclotriphosphazene and acetonitrile according to 0.1g:20mL of the mixture, wherein the acetic acid solution of chitosan is prepared from 0.1g of chitosan and 8% acetic acid solution by mass percent: 20mL of the mixture;

step A2, placing 5mL of DMF in a three-neck flask, dropwise adding 1mL of chlorosulfonic acid into the three-neck flask under ice bath condition, stirring while dropwise adding, keeping the temperature of a reaction system at 5 ℃, and stirring at room temperature until the mixture is clarified after the dropwise adding is finished to obtain a treating agent;

and A3, adding 50mg of composite microspheres and 100mL of absolute ethyl alcohol into a three-neck flask, dispersing for 30min by ultrasonic, dripping 9.1mg of treating agent into the three-neck flask, reacting for 1min under the condition of microwave radiation after dripping, precipitating for 30min in a refrigerator at 4 ℃ after reaction, taking out, washing with distilled water, adding 2mol/L sodium hydroxide solution to adjust the pH value to 8, filtering, dialyzing a filter cake in distilled water for 72h, and freeze-drying to constant weight to obtain the modified microspheres.

Example 4

The inorganic filler is prepared by the following steps:

step B1, adding 0.18g of triethylamine, 2.7g of hexadecyl trimethyl ammonium chloride and 36mL of deionized water into a three-neck flask, performing ultrasonic dispersion for 15min at the frequency of 40kHz, heating to 60 ℃, stirring and reacting for 1h at the rotating speed of 150r/min, dropwise adding 2.6mL of chlorobenzene into the three-neck flask, stirring for 20min after the dropwise adding is finished, dropwise adding 1mL of tetraethoxysilane, performing heat-preserving and stirring for 12h, performing centrifugal treatment at the rotating speed of 30000rpm for 10min after the reaction is finished, washing the precipitate with absolute ethyl alcohol for 3 times, calcining for 8h in a muffle furnace at the temperature of 600 ℃, and cooling to room temperature to obtain mesoporous silica;

and B2, under the protection of nitrogen, adding 0.5g of mesoporous silica, 0.2g of 2, 3-epoxypropyl trimethyl ammonium chloride and 25.8mL of dimethyl sulfoxide into a three-neck flask, controlling the temperature to be 50 ℃, stirring and reacting for 10min, then adding 0.5g of potassium hydroxide, heating to 180 ℃, stirring at the speed of 120r/min, carrying out reflux reaction for 3h, centrifuging after the reaction is finished, washing the precipitate with deionized water for 3 times, and finally drying to constant weight at the temperature of 80 ℃ to obtain the inorganic filler.

Example 5

The inorganic filler is prepared by the following steps:

step B1, adding 0.18g of triethylamine, 2.9g of hexadecyl trimethyl ammonium chloride and 38mL of deionized water into a three-neck flask, performing ultrasonic dispersion for 15min at the frequency of 42kHz, heating to 60 ℃, stirring and reacting for 1h at the rotating speed of 150r/min, dropwise adding 3.8mL of chlorobenzene into the three-neck flask, stirring for 20min after the dropwise adding is finished, dropwise adding 1mL of tetraethoxysilane, performing heat-preserving and stirring for 12h, performing centrifugal treatment at the rotating speed of 30000rpm after the reaction is finished, washing the precipitate with absolute ethyl alcohol for 4 times, calcining for 8h in a muffle furnace at the temperature of 600 ℃, and cooling to room temperature to obtain mesoporous silica;

and B2, under the protection of nitrogen, adding 0.7g of mesoporous silica, 0.3g of 2, 3-epoxypropyl trimethyl ammonium chloride and 26.1mL of dimethyl sulfoxide into a three-neck flask, controlling the temperature to be 55 ℃, stirring and reacting for 10min, then adding 0.5g of potassium hydroxide, heating to 182 ℃, stirring at the speed of 130r/min, carrying out reflux reaction for 4h, centrifuging after the reaction is finished, washing the precipitate with deionized water for 4 times, and finally drying at the temperature of 80 ℃ to constant weight to obtain the inorganic filler.

Example 6

The inorganic filler is prepared by the following steps:

step B1, adding 0.18g of triethylamine, 3.3g of hexadecyl trimethyl ammonium chloride and 40mL of deionized water into a three-neck flask, performing ultrasonic dispersion for 15min at the frequency of 45kHz, heating to 60 ℃, stirring and reacting for 1h at the rotating speed of 150r/min, dropwise adding 4.1mL of chlorobenzene into the three-neck flask, stirring for 20min after the dropwise adding is finished, dropwise adding 1mL of tetraethoxysilane, performing heat-preserving and stirring for 12h, performing centrifugal treatment at the rotating speed of 30000rpm after the reaction is finished, washing the precipitate with absolute ethyl alcohol for 5 times, calcining for 8h in a muffle furnace at the temperature of 600 ℃, and cooling to room temperature to obtain mesoporous silica;

and B2, under the protection of nitrogen, adding 0.8g of mesoporous silica, 0.4g of 2, 3-epoxypropyl trimethyl ammonium chloride and 26.4mL of dimethyl sulfoxide into a three-neck flask, controlling the temperature to be 60 ℃, stirring and reacting for 10min, then adding 0.5g of potassium hydroxide, heating to 185 ℃, stirring at 150r/min, refluxing and reacting for 5h, centrifuging after the reaction is finished, washing the precipitate with deionized water for 5 times, and finally drying to constant weight at 80 ℃ to obtain the inorganic filler.

Comparative example 1

The modified microsphere is prepared by the following steps:

step A1, adding 0.42g of polyethylenimine, 80mL of acetonitrile and 4mL of triethylamine into a round-bottom flask, shaking uniformly, sequentially adding 10mL of acetonitrile solution of hexachlorocyclotriphosphazene and 20mL of acetic acid solution of chitosan, sealing a bottle mouth, performing ultrasonic reaction for 8h at 40 ℃, performing ultrasonic power of 180W and frequency of 40Hz, centrifuging for 20min at the rotating speed of 1000r/min, washing the precipitate with acetone and absolute ethyl alcohol for 2 times sequentially, and finally drying at-45 ℃ for 24h to obtain modified microspheres, wherein the acetonitrile solution of hexachlorocyclotriphosphazene is prepared by mixing hexachlorocyclotriphosphazene and acetonitrile according to 0.1g:20mL of the mixture, wherein the acetic acid solution of the chitosan is prepared from 0.1g of chitosan and 6-8% acetic acid solution by mass percent: 20mL of the mixture.

Comparative example 2

The inorganic filler is prepared by the following steps:

and B1, adding 0.18g of triethylamine, 2.7g of hexadecyl trimethyl ammonium chloride and 36mL of deionized water into a three-neck flask, performing ultrasonic dispersion for 15min at the frequency of 40kHz, heating to 60 ℃, stirring at the rotating speed of 150r/min for reaction for 1h, dropwise adding 2.6mL of chlorobenzene into the three-neck flask, stirring for 20min after the dropwise adding is finished, dropwise adding 1mL of tetraethoxysilane, performing heat preservation and stirring for reaction for 12h, performing centrifugal treatment at the rotating speed of 30000rpm after the reaction is finished, washing the precipitate with absolute ethyl alcohol for 3 times, calcining for 8h in a muffle furnace at the temperature of 600 ℃, and cooling to room temperature to obtain the inorganic filler.

Example 7

A thermostable nucleic acid vaccine adjuvant comprises the following raw materials in parts by weight: 25.8 parts of an oil phase, 50 parts of an aqueous phase, 3.5 parts of the modified microspheres of example 1 and 0.5 part of the inorganic filler of example 3;

the thermostable nucleic acid vaccine adjuvant is prepared by the following steps:

mixing inorganic filler and water phase, ultrasonic dispersing at 40kHz, adding oil phase and modified microsphere, and continuing ultrasonic treatment for 20min to obtain the heat stable nucleic acid vaccine adjuvant.

Example 8

A thermostable nucleic acid vaccine adjuvant comprises the following raw materials in parts by weight: 28.6 parts of an oil phase, 55 parts of an aqueous phase, 4.1 parts of the modified microspheres of example 2 and 0.8 part of the inorganic filler of example 4;

the thermostable nucleic acid vaccine adjuvant is prepared by the following steps:

mixing inorganic filler and water phase, ultrasonic dispersing at frequency of 50kHz, adding oil phase and modified microsphere, and continuing ultrasonic treatment for 20min to obtain the heat stable nucleic acid vaccine adjuvant.

Example 9

A thermostable nucleic acid vaccine adjuvant comprises the following raw materials in parts by weight: 31.2 parts of an oil phase, 60 parts of an aqueous phase, 4.6 parts of the modified microspheres of example 3 and 1.1 parts of the inorganic filler of example 6;

the thermostable nucleic acid vaccine adjuvant is prepared by the following steps:

mixing inorganic filler and water phase, ultrasonic dispersing at 60kHz, adding oil phase and modified microsphere, and continuing ultrasonic treatment for 20min to obtain the heat stable nucleic acid vaccine adjuvant.

Comparative example 3

The modified microspheres in example 7 were replaced with the modified microspheres in comparative example 1, and the remaining raw materials and the preparation process were unchanged.

Comparative example 4

The inorganic filler in example 8 was replaced with the inorganic filler in comparative example 2, and the remaining raw materials and the preparation process were unchanged.

The vaccine adjuvants of examples 7-9 and comparative examples 3-4 were tested for performance by the following criteria:

adjuvant to antigen compatibility:

compounding 3nM DNA with 9nM of the above adjuvant under the following conditions: standing at room temperature for 30min, and then performing agarose gel electrophoresis experiment to examine the capability of the compound nucleic acid, wherein the electrophoresis experiment condition is that the voltage is 110V, the electrophoresis time is 30min, and after electrophoresis is finished, observing under an ultraviolet lamp of 254 nm.

The observation results are that the liposome nucleic acid vaccine adjuvants prepared in examples 7-9 all have very strong nucleic acid complexing ability, can load a large amount of nucleic acid antigens, and increase the effective antigen amount entering immune cells.

Thermal stability of adjuvants:

placing the vaccine adjuvants of examples 7-9 and comparative examples 3-4 in a water bath at 50deg.C for 2h, observing the emulsion state, and whether layering or not, even whether precipitation occurs;

stability of adjuvant to antigen complex:

the vaccine adjuvants of examples 7-9 and comparative examples 3-4 were added to EV71 vaccine solution (1X 10) ⁸ pfu/mL), after repeated mixing, incubation at 37 ℃ for 2 hours, power 25%, ultrasonic treatment for 2 minutes, observation of oil-free chromatography, standing for 5 days, observation of oil layer precipitation, test results are shown in the following table:

from the above table, it can be seen that the adjuvant of examples 7 to 9 has a good binding ability with the vaccine, and can ensure that the vaccine is stable in state during transportation, improve the heat resistance, and ensure that the active components of the vaccine will not fail.

In the description of the present specification, the descriptions of the terms "one embodiment," "example," "specific example," and the like, mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms do not necessarily refer to the same embodiments or examples. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

The foregoing is merely illustrative and explanatory of the invention, as various modifications and additions may be made to the particular embodiments described, or in a similar manner, by those skilled in the art, without departing from the scope of the invention or exceeding the scope of the invention as defined in the claims.

Claims (4)

1. The thermostable nucleic acid vaccine adjuvant is characterized by comprising the following raw materials in parts by weight: 25.8-31.2 parts of oil phase, 50-60 parts of water phase, 3.5-4.6 parts of modified microspheres and 0.5-1.1 parts of inorganic filler;

the modified microsphere is prepared by the following steps:

step A1, mixing polyethylenimine, acetonitrile and triethylamine, adding an acetonitrile solution of hexachlorocyclotriphosphazene and an acetic acid solution of chitosan, sealing, performing ultrasonic reaction for 8 hours at 40 ℃, centrifuging, washing and drying to obtain composite microspheres;

step A2, placing DMF in a three-neck flask, dropwise adding chlorosulfonic acid under ice bath, stirring while dropwise adding, keeping the temperature of a reaction system at 0-5 ℃, and stirring at room temperature until the reaction system is clear after dropwise adding is finished to obtain a treating agent;

step A3, after ultrasonic dispersion of the composite microsphere and absolute ethyl alcohol, dropwise adding a treating agent, carrying out microwave radiation reaction for 1min, precipitating in a refrigerator at 4 ℃ for 30min, taking out, washing, adding a sodium hydroxide solution to adjust the pH value to 7-8, dialyzing, and freeze-drying to obtain modified microspheres;

the acetonitrile solution of hexachlorocyclotriphosphazene in step A1 was prepared from hexachlorocyclotriphosphazene and acetonitrile according to 0.1g:20mL of the chitosan solution is mixed, and the chitosan solution is prepared from 0.1g of chitosan and 6-8% of acetic acid solution by mass percent: 20mL of the mixture;

the volume ratio of DMF to chlorosulfonic acid in step A2 is 5:1, a step of;

in the step A3, the dosage ratio of the composite microsphere, the absolute ethyl alcohol and the treating agent is 50mg:100mL:8.5-9.1mg;

the inorganic filler is prepared by the following steps:

mixing mesoporous silica, 2, 3-epoxypropyl trimethyl ammonium chloride and dimethyl sulfoxide under the protection of nitrogen, stirring and reacting for 10min at 50-60 ℃, adding potassium hydroxide, heating to 180-185 ℃, stirring and refluxing for reacting for 3-5h, centrifuging, washing and drying to obtain inorganic filler;

the mesoporous silica is prepared by the following steps:

and (3) ultrasonically dispersing triethylamine, hexadecyl trimethyl ammonium chloride and deionized water, heating to 60 ℃, stirring and reacting for 1h, dropwise adding chlorobenzene, stirring for 20min after the dropwise adding is finished, dropwise adding tetraethoxysilane, stirring and reacting for 12h under heat preservation, centrifuging, washing, calcining, and cooling to obtain mesoporous silica.

2. The thermostable nucleic acid vaccine adjuvant according to claim 1, wherein the ratio of the amount of mesoporous silica, 2, 3-epoxypropyltrimethylammonium chloride, dimethyl sulfoxide and potassium hydroxide is 0.5-0.8g:0.2-0.4g:25.8-26.4mL:0.5g.

3. The thermostable nucleic acid vaccine adjuvant according to claim 1, wherein the dosage ratio of triethylamine, cetyltrimethylammonium chloride, deionized water, chlorobenzene and ethyl orthosilicate is 0.18g:2.7-3.3g:36-40mL:2.6-4.1mL:1mL.

4. The method for preparing a thermostable nucleic acid vaccine adjuvant according to claim 1, characterized by the steps of:

mixing inorganic filler and water phase, ultrasonic dispersing at 40-60kHz, adding oil phase and modified microsphere, and continuing ultrasonic treatment for 20min to obtain the heat stable nucleic acid vaccine adjuvant.