CN113599515A - Thermostable nucleic acid vaccine adjuvant and preparation method thereof - Google Patents
Thermostable nucleic acid vaccine adjuvant and preparation method thereof Download PDFInfo
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- 239000012646 vaccine adjuvant Substances 0.000 title claims abstract description 38
- 108700001237 Nucleic Acid-Based Vaccines Proteins 0.000 title claims abstract description 36
- 229940023146 nucleic acid vaccine Drugs 0.000 title claims abstract description 36
- 238000002360 preparation method Methods 0.000 title abstract description 8
- 239000004005 microsphere Substances 0.000 claims abstract description 49
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- 239000011256 inorganic filler Substances 0.000 claims abstract description 29
- 229910003475 inorganic filler Inorganic materials 0.000 claims abstract description 29
- 238000002156 mixing Methods 0.000 claims abstract description 20
- 239000002994 raw material Substances 0.000 claims abstract description 9
- 238000001132 ultrasonic dispersion Methods 0.000 claims abstract description 9
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- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 45
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims description 45
- 238000003756 stirring Methods 0.000 claims description 38
- 238000006243 chemical reaction Methods 0.000 claims description 34
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 30
- 229920001661 Chitosan Polymers 0.000 claims description 25
- 238000005406 washing Methods 0.000 claims description 22
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- UBIJTWDKTYCPMQ-UHFFFAOYSA-N hexachlorophosphazene Chemical compound ClP1(Cl)=NP(Cl)(Cl)=NP(Cl)(Cl)=N1 UBIJTWDKTYCPMQ-UHFFFAOYSA-N 0.000 claims description 21
- 239000002131 composite material Substances 0.000 claims description 19
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 17
- MVPPADPHJFYWMZ-UHFFFAOYSA-N chlorobenzene Chemical compound ClC1=CC=CC=C1 MVPPADPHJFYWMZ-UHFFFAOYSA-N 0.000 claims description 16
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- 239000000377 silicon dioxide Substances 0.000 claims description 15
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- 239000003795 chemical substances by application Substances 0.000 claims description 14
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- 238000001035 drying Methods 0.000 claims description 11
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- 229920002873 Polyethylenimine Polymers 0.000 claims description 9
- XTHPWXDJESJLNJ-UHFFFAOYSA-N chlorosulfonic acid Substances OS(Cl)(=O)=O XTHPWXDJESJLNJ-UHFFFAOYSA-N 0.000 claims description 9
- BOTDANWDWHJENH-UHFFFAOYSA-N Tetraethyl orthosilicate Chemical compound CCO[Si](OCC)(OCC)OCC BOTDANWDWHJENH-UHFFFAOYSA-N 0.000 claims description 8
- WOWHHFRSBJGXCM-UHFFFAOYSA-M cetyltrimethylammonium chloride Chemical compound [Cl-].CCCCCCCCCCCCCCCC[N+](C)(C)C WOWHHFRSBJGXCM-UHFFFAOYSA-M 0.000 claims description 8
- 239000000203 mixture Substances 0.000 claims description 8
- PUVAFTRIIUSGLK-UHFFFAOYSA-M trimethyl(oxiran-2-ylmethyl)azanium;chloride Chemical compound [Cl-].C[N+](C)(C)CC1CO1 PUVAFTRIIUSGLK-UHFFFAOYSA-M 0.000 claims description 8
- 229910052757 nitrogen Inorganic materials 0.000 claims description 7
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- 102000036639 antigens Human genes 0.000 abstract description 23
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- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 15
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- 239000003921 oil Substances 0.000 description 10
- 230000000052 comparative effect Effects 0.000 description 9
- 239000002671 adjuvant Substances 0.000 description 8
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- 229960005486 vaccine Drugs 0.000 description 8
- 239000012065 filter cake Substances 0.000 description 4
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- 230000028993 immune response Effects 0.000 description 4
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 210000004027 cell Anatomy 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 238000001962 electrophoresis Methods 0.000 description 3
- 230000036039 immunity Effects 0.000 description 3
- 102000039446 nucleic acids Human genes 0.000 description 3
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- 125000003277 amino group Chemical group 0.000 description 1
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- IERHLVCPSMICTF-UHFFFAOYSA-N cytidine monophosphate Natural products O=C1N=C(N)C=CN1C1C(O)C(O)C(COP(O)(O)=O)O1 IERHLVCPSMICTF-UHFFFAOYSA-N 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
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- 230000000694 effects Effects 0.000 description 1
- 239000002158 endotoxin Substances 0.000 description 1
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- 125000000524 functional group Chemical group 0.000 description 1
- 239000004519 grease Substances 0.000 description 1
- 230000008642 heat stress Effects 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 210000002865 immune cell Anatomy 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 229920006008 lipopolysaccharide Polymers 0.000 description 1
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- 108090000623 proteins and genes Proteins 0.000 description 1
- 239000008213 purified water Substances 0.000 description 1
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- 230000003685 thermal hair damage Effects 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 239000008215 water for injection Substances 0.000 description 1
Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/04—Immunostimulants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/53—DNA (RNA) vaccination
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55505—Inorganic adjuvants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55511—Organic adjuvants
- A61K2039/55566—Emulsions, e.g. Freund's adjuvant, MF59
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55511—Organic adjuvants
- A61K2039/55583—Polysaccharides
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- Health & Medical Sciences (AREA)
- Immunology (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- General Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- General Health & Medical Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Microbiology (AREA)
- Mycology (AREA)
- Epidemiology (AREA)
- Medicinal Preparation (AREA)
Abstract
The invention relates to a thermostable nucleic acid vaccine adjuvant and a preparation method thereof, belonging to the technical field of vaccine adjuvant preparation and 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 part of inorganic filler, mixing the inorganic filler and the water phase, performing ultrasonic dispersion at the frequency of 40-60kHz, adding the oil phase and the modified microspheres, and continuing ultrasonic treatment for 20min to obtain the heat-stable nucleic acid vaccine adjuvant which is emulsion-shaped, has an uneven surface after antigen is adsorbed by an internal substance, and can adsorb the antigen in gaps of the antigen so as to play a role in protecting the antigen, so that the antigen can resist thermal injury and keep bioactivity at a certain temperature, and in addition, small-particle-size particles in the emulsion can generate higher accumulation efficiency at an oil-water interface to form a more uniform particle film, thereby preventing mutual collision between the emulsions and keeping the state stability of the nucleic acid vaccine adjuvant.
Description
Technical Field
The invention belongs to the technical field of preparation of vaccine adjuvants, and particularly relates to a thermostable nucleic acid vaccine adjuvant and a preparation method thereof.
Background
The nucleic acid vaccine is prepared through introducing exogenous gene (DNA or RNA) encoding certain antigen protein into animal cell, synthesizing antigen protein with the expression system of host cell, and inducing the host to produce immune response to the antigen protein for preventing and treating diseases.
Vaccine adjuvants refer to substances that can be used simultaneously or in advance with an antigen to enhance the immune response of the body to the antigen, or to 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 eye.
The nucleic acid vaccine is a sensitive product which needs to be carefully managed, the protection and transportation of the nucleic acid vaccine are very important when the nucleic acid vaccine is placed at a proper temperature, the titer of the vaccine can be reduced or even the vaccine can be inactivated by heat stress, the immune response cannot be activated, and a huge potential risk exists.
Disclosure of Invention
The present invention aims to provide a thermostable nucleic acid vaccine adjuvant and a method for preparing the same to solve the technical problems mentioned in the background art.
The purpose of the invention can be realized 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 part of inorganic filler;
the thermostable nucleic acid vaccine adjuvant is prepared by the following steps:
mixing inorganic filler and water, performing ultrasonic dispersion at frequency of 40-60kHz, adding oil phase and modified microspheres, and continuing ultrasonic treatment for 20min to obtain the thermostable nucleic acid vaccine adjuvant.
Further, the modified microspheres are prepared by the following steps:
step A1, adding polyethyleneimine, acetonitrile and triethylamine into a round-bottom flask, uniformly shaking, sequentially adding an acetonitrile solution of hexachlorocyclotriphosphazene and an acetic acid solution of chitosan, sealing the flask, carrying out ultrasonic reaction for 8 hours at 40 ℃, carrying out ultrasonic power of 180W and frequency of 40Hz, centrifuging for 20-30 minutes at the condition of the rotation speed of 1000-1500r/min, sequentially washing the precipitate with acetone and absolute ethyl alcohol for 2 times, and finally drying for 24 hours at-45 ℃ to obtain composite microspheres, wherein the polyethyleneimine, chitosan and hexachlorocyclotriphosphazene are used as substrates to prepare the cross-linked polyphosphazene composite microspheres, and the six-membered heterocyclic ring of N, P has high biocompatibility and degradability, high 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 an ice bath condition, stirring while dropwise adding, keeping the temperature of a reaction system at 0-5 ℃, stirring at room temperature until the mixture is clear after dropwise adding is finished to obtain a treating agent, and mixing chlorosulfonic acid and DMF to obtain the treating agent;
step A3, adding the composite microspheres and absolute ethyl alcohol into a three-neck flask, after ultrasonic dispersion for 30min, dropwise adding a treating agent into the three-neck flask, after dropwise adding, reacting for 1min under the condition of microwave radiation, after reaction, precipitating in a refrigerator at 4 ℃ for 30min, 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 filter cakes in distilled water for 72h, freeze-drying to constant weight to obtain modified microspheres, and performing substitution reaction by using amino groups on the surfaces of the composite microspheres and chlorosulfonic acid to graft sulfonic acid groups on the surfaces of the polymer microspheres, so that the water solubility of the composite microspheres is improved.
Further, the dosage ratio of the acetonitrile solution of polyethyleneimine, acetonitrile, triethylamine, hexachlorocyclotriphosphazene and the acetic acid solution of chitosan in the step A1 is 0.42-0.48 g: 80-90 mL: 4mL of: 10-15 mL: 20mL of a solution of hexachlorocyclotriphosphazene in acetonitrile prepared from hexachlorocyclotriphosphazene and acetonitrile in an amount of 0.1 g: 20mL of the chitosan solution is prepared by mixing chitosan and acetic acid solution with the mass fraction of 6-8% according to the weight ratio of 0.1 g: 20mL of the mixture was mixed.
Further, in the step a2, the volume ratio of DMF to chlorosulfonic acid is 5: 1.
further, the dosage ratio of the composite microspheres, the absolute ethyl alcohol and the treating agent in the step A3 is 50 mg: 100mL of: 8.5-9.1 mg.
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 and reacting for 1h under the condition of the rotating speed of 150r/min, dropwise adding chlorobenzene into the three-neck flask, stirring for 20min after dropwise adding, dropwise adding ethyl orthosilicate, performing heat preservation and stirring reaction for 12h, after the reaction is finished, performing centrifugal treatment for 10min at the rotating speed of 30000rpm, washing precipitates for 3-5 times by using absolute ethyl alcohol, finally calcining for 8h in a 600 ℃ muffle furnace, and cooling to room temperature to obtain mesoporous silica;
and step B2, under the protection of nitrogen, adding mesoporous silica, 2, 3-epoxypropyltrimethylammonium chloride and dimethyl sulfoxide into a three-neck flask, controlling the temperature to be 50-60 ℃, stirring for reaction for 10min, then adding potassium hydroxide, heating to 180-185 ℃, stirring at the speed of 120-150r/min, carrying out reflux reaction for 3-5h, centrifuging after the reaction is finished, washing the precipitate for 3-5 times by using deionized water, and finally drying at 80 ℃ to constant weight to obtain the inorganic filler.
Further, the dosage ratio of triethylamine, hexadecyl trimethyl ammonium chloride, deionized water, chlorobenzene and ethyl orthosilicate in the B1 is 0.18 g: 2.7-3.3 g: 36-40 mL: 2.6-4.1 mL: 1 mL.
Further, the dosage ratio of the mesoporous silica, the 2, 3-epoxypropyltrimethylammonium chloride, the dimethyl sulfoxide and the potassium hydroxide in the B2 is 0.5-0.8 g: 0.2-0.4 g: 25.8-26.4 mL: 0.5 g.
Further, the oil phase is one of biocompatible grease and mineral oil.
Further, the water phase comprises one or more of purified water, water for injection, glycerol aqueous solution, 0.9% by mass of physiological saline, phosphate buffer, citric acid buffer or Tris buffer, and is mixed according to any ratio.
The invention has the beneficial effects that:
1) the invention takes oil phase, water phase, modified microsphere and inorganic filler as raw materials to prepare a thermostable nucleic acid vaccine adjuvant, which is emulsion-shaped, after the antigen is adsorbed by the internal substance, the internal substance has uneven surface, the antigen can be adsorbed in the gap, thus playing the role of protecting the antigen, leading the antigen to resist thermal damage at a certain temperature and keeping the bioactivity, in addition, the small-particle-size particles in the emulsion can generate higher accumulation efficiency at the oil-water interface to form more uniform particle films, thus preventing the mutual collision between the emulsions and keeping the stable state of the nucleic acid vaccine adjuvant.
2) The invention takes polyethyleneimine, chitosan and hexachlorocyclotriphosphazene as substrates to prepare cross-linked polyphosphazene composite microspheres, sulfonic acid groups are grafted on the surfaces of the composite microspheres through treatment of a treating agent, the water solubility of the polymer microspheres is improved, the composite microspheres have higher biocompatibility and degradability, N, P-containing hexahydric heterocycles have higher thermal stability, chitosan has good stimulation capability of inherent immunity and specific immunity, can induce mucosal immunity, has high-efficiency protection against the original, can enhance the immunogenicity of antigens, the antigen can be wrapped in the solution by the huge molecular weight and the complex spatial structure, the solution carries a large amount of positive charges, the powerful electrostatic effect protects the vaccines from being easily hydrolyzed in the transportation process, the vaccine-combined chitosan has stronger stability than the directly naked vaccines, and the antigens after the microspheres are adsorbed have the sustained release characteristic, and after the adsorbed microspheres are taken into cells, the processing and presentation modes of antigens in presenting cells can be changed, more antigens are promoted to realize cross presentation through an MHCI (MHCI) way, and high-level cellular immune response is induced.
3) The inorganic filler is prepared by taking mesoporous silica as a carrier and performing ring-opening reaction on hydroxyl on the surface of the mesoporous silica and epoxy group of 2, 3-epoxypropyltrimethylammonium chloride under an alkaline condition to obtain the inorganic filler with cationic functional groups grafted on the surface.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
The modified microsphere is prepared by the following steps:
step A1, adding 0.42g of polyethyleneimine, 80mL of acetonitrile and 4mL of triethylamine into a round-bottom flask, uniformly shaking, sequentially adding 10mL of acetonitrile solution of hexachlorocyclotriphosphazene and 20mL of acetic acid solution of chitosan, sealing a bottle mouth, carrying out ultrasonic reaction for 8h at 40 ℃, wherein the ultrasonic power is 180W, the frequency is 40Hz, then centrifuging for 20min at the rotation speed of 1000r/min, sequentially washing precipitates with acetone and absolute ethyl alcohol for 2 times, and finally drying at-45 ℃ for 24h to obtain the composite microspheres, wherein the acetonitrile solution of hexachlorocyclotriphosphazene is prepared by mixing hexachlorocyclotriphosphazene and acetonitrile according to the weight ratio of 0.1 g: 20mL of the chitosan solution is prepared by mixing chitosan and acetic acid solution with mass fraction of 6%, wherein the weight ratio of the chitosan solution to the acetic acid solution is 0.1 g: 20mL of the mixture is mixed;
step A2, placing 5mL of DMF in a three-neck flask, dropwise adding 1mL of chlorosulfonic acid into the three-neck flask under the ice bath condition, stirring while dropwise adding, keeping the temperature of a reaction system at 0 ℃, and stirring at room temperature until the solution is clear after dropwise adding is finished to obtain a treating agent;
step A3, adding 50mg of composite microspheres and 100mL of absolute ethyl alcohol into a three-neck flask, performing ultrasonic dispersion for 30min, then dropwise adding 8.5mg of treating agent into the three-neck flask, reacting for 1min under the condition of microwave radiation after dropwise adding, precipitating for 30min in a refrigerator at 4 ℃ after reaction, taking out, washing with distilled water, adding a 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 polyethyleneimine, 85mL of acetonitrile and 4mL of triethylamine into a round-bottom flask, uniformly shaking, sequentially adding 12mL of acetonitrile solution of hexachlorocyclotriphosphazene and 20mL of acetic acid solution of chitosan, sealing a bottle mouth, carrying out ultrasonic reaction for 8h at 40 ℃, wherein the ultrasonic power is 180W, the frequency is 40Hz, then centrifuging for 25min at the rotation speed of 1200r/min, sequentially washing precipitates with acetone and absolute ethyl alcohol for 2 times, and finally drying at-45 ℃ for 24h to obtain the composite microspheres, wherein the acetonitrile solution of hexachlorocyclotriphosphazene is prepared by mixing hexachlorocyclotriphosphazene and acetonitrile according to the weight ratio of 0.1 g: 20mL of the chitosan solution is prepared by mixing chitosan and an acetic acid solution with the mass fraction of 7% according to the weight ratio of 0.1 g: 20mL of the mixture is mixed;
step A2, placing 5mL of DMF in a three-neck flask, dropwise adding 1mL of chlorosulfonic acid into the three-neck flask under the ice bath condition, stirring while dropwise adding, keeping the temperature of a reaction system at 2 ℃, and stirring at room temperature until the solution is clear after dropwise adding is finished to obtain a treating agent;
step A3, adding 50mg of composite microspheres and 100mL of absolute ethyl alcohol into a three-neck flask, ultrasonically dispersing for 30min, then dropwise adding 8.8mg of treating agent into the three-neck flask, reacting for 1min under the condition of microwave radiation after dropwise adding, precipitating for 30min in a refrigerator at 4 ℃ after reaction, taking out, washing with distilled water, adding a 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 polyethyleneimine, 90mL of acetonitrile and 4mL of triethylamine into a round-bottom flask, uniformly shaking, sequentially adding 15mL of acetonitrile solution of hexachlorocyclotriphosphazene and 20mL of acetic acid solution of chitosan, sealing a bottle opening, carrying out ultrasonic reaction for 8h at 40 ℃, wherein the ultrasonic power is 180W, the frequency is 40Hz, then centrifuging for 30min at the rotation speed of 1500r/min, sequentially washing precipitates with acetone and absolute ethyl alcohol for 2 times, and finally drying at-45 ℃ for 24h to obtain the composite microspheres, wherein the acetonitrile solution of hexachlorocyclotriphosphazene is prepared by mixing hexachlorocyclotriphosphazene and acetonitrile according to the weight ratio of 0.1 g: 20mL of the chitosan solution is prepared by mixing chitosan and 8% acetic acid solution by mass percent according to the ratio of 0.1 g: 20mL of the mixture is mixed;
step A2, placing 5mL of DMF in a three-neck flask, dropwise adding 1mL of chlorosulfonic acid into the three-neck flask under the ice bath condition, stirring while dropwise adding, keeping the temperature of a reaction system at 5 ℃, and stirring at room temperature until the solution is clear after dropwise adding is finished to obtain a treating agent;
step A3, adding 50mg of composite microspheres and 100mL of absolute ethyl alcohol into a three-neck flask, ultrasonically dispersing for 30min, then dropwise adding 9.1mg of treating agent into the three-neck flask, reacting for 1min under the condition of microwave radiation after dropwise adding, precipitating for 30min in a refrigerator at 4 ℃ after reaction, taking out, washing with distilled water, adding a 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 hexadecyltrimethylammonium chloride and 36mL of deionized water into a three-neck flask, ultrasonically dispersing for 15min at the frequency of 40kHz, heating to 60 ℃, stirring and reacting for 1h at the rotation speed of 150r/min, dropwise adding 2.6mL of chlorobenzene into the three-neck flask, stirring for 20min after dropwise adding, dropwise adding 1mL of tetraethoxysilane, stirring and reacting for 12h while keeping the temperature, after the reaction is finished, centrifugally treating for 10min at the rotation speed of 30000rpm, washing precipitates for 3 times with absolute ethyl alcohol, finally calcining for 8h in a 600 ℃ muffle furnace, and cooling to room temperature to obtain mesoporous silicon dioxide;
and step B2, under the protection of nitrogen, adding 0.5g of mesoporous silica, 0.2g of 2, 3-epoxypropyltrimethylammonium chloride and 25.8mL of dimethyl sulfoxide into a three-neck flask, controlling the temperature to be 50 ℃, stirring for reaction 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 for 3 times by deionized water, and finally drying at the temperature of 80 ℃ to constant weight 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 hexadecyltrimethylammonium chloride and 38mL of deionized water into a three-neck flask, ultrasonically dispersing 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 dropwise adding, dropwise adding 1mL of tetraethoxysilane, stirring and reacting for 12h while keeping the temperature, after the reaction is finished, centrifugally treating for 10min at the rotating speed of 30000rpm, washing precipitates for 4 times by using absolute ethyl alcohol, finally calcining for 8h in a 600 ℃ muffle furnace, and cooling to room temperature to obtain mesoporous silicon dioxide;
and step B2, under the protection of nitrogen, adding 0.7g of mesoporous silica, 0.3g of 2, 3-epoxypropyltrimethylammonium chloride and 26.1mL of dimethyl sulfoxide into a three-neck flask, controlling the temperature to be 55 ℃, stirring for reaction 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 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 hexadecyltrimethylammonium chloride and 40mL of deionized water into a three-neck flask, ultrasonically dispersing for 15min at the frequency of 45kHz, heating to 60 ℃, stirring and reacting for 1h at the rotation speed of 150r/min, dropwise adding 4.1mL of chlorobenzene into the three-neck flask, stirring for 20min after dropwise adding, dropwise adding 1mL of tetraethoxysilane, stirring and reacting for 12h while keeping the temperature, after the reaction is finished, centrifugally treating for 10min at the rotation speed of 30000rpm, washing precipitates for 5 times with absolute ethyl alcohol, finally calcining for 8h in a 600 ℃ muffle furnace, and cooling to room temperature to obtain mesoporous silicon dioxide;
and step B2, under the protection of nitrogen, adding 0.8g of mesoporous silica, 0.4g of 2, 3-epoxypropyltrimethylammonium chloride and 26.4mL of dimethyl sulfoxide into a three-neck flask, controlling the temperature to be 60 ℃, stirring for reaction for 10min, then adding 0.5g of potassium hydroxide, heating to 185 ℃, stirring at the speed of 150r/min, carrying out reflux reaction for 5h, centrifuging after the reaction is finished, washing the precipitate for 5 times by using deionized water, and finally drying at 80 ℃ to constant weight to obtain the inorganic filler.
Comparative example 1
The modified microsphere is prepared by the following steps:
step A1, adding 0.42g of polyethyleneimine, 80mL of acetonitrile and 4mL of triethylamine into a round-bottom flask, uniformly shaking, sequentially adding 10mL of acetonitrile solution of hexachlorocyclotriphosphazene and 20mL of acetic acid solution of chitosan, sealing a bottle mouth, carrying out ultrasonic reaction for 8h at 40 ℃, wherein the ultrasonic power is 180W, the frequency is 40Hz, then centrifuging for 20min at the rotation speed of 1000r/min, sequentially washing precipitates with acetone and absolute ethyl alcohol for 2 times, 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 the weight ratio of 0.1 g: 20mL of the chitosan solution is prepared by mixing chitosan and acetic acid solution with the mass fraction of 6-8% according to the weight ratio of 0.1 g: 20mL of the mixture was mixed.
Comparative example 2
The inorganic filler is prepared by the following steps:
and step B1, adding 0.18g of triethylamine, 2.7g of hexadecyl trimethyl ammonium chloride and 36mL of deionized water into a three-neck flask, ultrasonically dispersing 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 dropwise adding, dropwise adding 1mL of tetraethoxysilane, stirring and reacting for 12h while keeping the temperature, after the reaction is finished, centrifugally treating for 10min at the rotating speed of 30000rpm, washing precipitates for 3 times by using absolute ethyl alcohol, finally calcining for 8h in a 600 ℃ muffle furnace, 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 a water 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, performing ultrasonic dispersion at the frequency of 40kHz, adding an oil phase and modified microspheres, and continuing ultrasonic treatment for 20min to obtain the thermostable 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 a water 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, performing ultrasonic dispersion at the frequency of 50kHz, adding an oil phase and modified microspheres, and continuing ultrasonic treatment for 20min to obtain the thermostable 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 a water 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, performing ultrasonic dispersion at the frequency of 60kHz, adding an oil phase and modified microspheres, and continuing ultrasonic treatment for 20min to obtain the thermostable 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 preparation process were unchanged.
Comparative example 4
The inorganic filler of example 8 was replaced with the inorganic filler of comparative example 2, and the remaining raw materials and preparation process were unchanged.
The vaccine adjuvants of examples 7-9 and comparative examples 3-4 were subjected to performance tests, the test criteria being as follows:
compatibility of adjuvant with antigen:
3nM of DNA was complexed with 9nM of the adjuvant: standing at room temperature for 30min, performing agarose gel electrophoresis experiment to examine the ability of the composite nucleic acid, wherein the electrophoresis experiment condition is 110V, the electrophoresis time is 30min, and observing under an ultraviolet lamp of 254nm after the electrophoresis is finished.
The observation result shows that the liposome nucleic acid vaccine adjuvants prepared in examples 7-9 have 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 the adjuvant:
the vaccine adjuvants of examples 7-9 and comparative examples 3-4 were left in a water bath at 50 ℃ for 2 hours, and the emulsion state, whether or not they were layered, and even whether or not there was any precipitate generated, were observed;
complex stability of adjuvant with antigen:
the vaccine adjuvants of examples 7-9 and comparative examples 3-4 were added to EV71 vaccine solution (1X 10)8pfu/mL), repeatedly mixing, incubating at 37 ℃ for 2h with power of 25%, performing ultrasonic treatment for 2min, observing oil-free chromatography, standing for 5d, and observing oil-free layer precipitation, wherein the test results are shown in the following table:
as can be seen from the above table, the adjuvants of examples 7-9 have good binding ability with the vaccine, and can ensure the stable state of the vaccine during transportation, improve the heat resistance, and ensure that the active components of the vaccine do not lose efficacy.
In the description herein, references to the description of "one embodiment," "an example," "a specific example" or the like are intended to 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 invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. 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 illustrative and explanatory only and is not intended to be exhaustive or to limit the invention to the precise embodiments described, and various modifications, additions, and substitutions may be made by those skilled in the art without departing from the scope of the invention or exceeding the scope of the claims.
Claims (9)
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 part of inorganic filler;
the modified microsphere is prepared by the following steps:
step A1, mixing polyethyleneimine, acetonitrile and triethylamine, adding an acetonitrile solution of hexachlorocyclotriphosphazene and an acetic acid solution of chitosan, sealing, carrying out 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 while stirring in ice bath, keeping the temperature of the reaction system at 0-5 ℃, and stirring at room temperature until the mixture is clear after dropwise adding to obtain a treating agent;
and A3, ultrasonically dispersing the composite microspheres and absolute ethyl alcohol, dropwise adding a treating agent, performing 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 the modified microspheres.
2. The thermostable nucleic acid vaccine adjuvant according to claim 1, wherein the acetonitrile solution of hexachlorocyclotriphosphazene in step A1 is prepared from hexachlorocyclotriphosphazene and acetonitrile in an amount of 0.1 g: 20mL of the chitosan is mixed, and the acetic acid solution of the chitosan is prepared by mixing chitosan and acetic acid solution with the mass fraction of 6-8% according to the weight ratio of 0.1 g: 20mL of the mixture was mixed.
3. The thermostable nucleic acid vaccine adjuvant according to claim 1, wherein the volume ratio of DMF and chlorosulfonic acid in step A2 is 5: 1.
4. the thermostable nucleic acid vaccine adjuvant according to claim 1, wherein the dosage ratio of the composite microspheres, absolute ethyl alcohol and treating agent in step A3 is 50 mg: 100mL of: 8.5-9.1 mg.
5. The thermostable nucleic acid vaccine adjuvant according to claim 1, wherein said inorganic filler is prepared by the following steps:
under the protection of nitrogen, mixing mesoporous silica, 2, 3-epoxypropyl trimethyl ammonium chloride and dimethyl sulfoxide, stirring and reacting at 50-60 ℃ for 10min, adding potassium hydroxide, heating to 180-185 ℃, stirring and refluxing for reacting for 3-5h, centrifuging, washing and drying to obtain the inorganic filler.
6. The thermostable nucleic acid vaccine adjuvant according to claim 5, wherein the mesoporous silica is prepared by the following steps:
ultrasonically dispersing triethylamine, hexadecyl trimethyl ammonium chloride and deionized water, heating to 60 ℃, stirring for reacting for 1h, dropwise adding chlorobenzene, stirring for 20min after dropwise adding, dropwise adding ethyl orthosilicate, keeping the temperature, stirring for reacting for 12h, centrifuging, washing, calcining, and cooling to obtain the mesoporous silica.
7. The thermostable nucleic acid vaccine adjuvant according to claim 5, wherein the ratio of the amount of mesoporous silica, 2, 3-epoxypropyltrimethylammonium chloride, dimethyl sulfoxide and potassium hydroxide is 0.5-0.8 g: 0.2-0.4 g: 25.8-26.4 mL: 0.5 g.
8. The thermostable nucleic acid vaccine adjuvant according to claim 6, wherein the dosage ratio of triethylamine, cetyltrimethylammonium chloride, deionized water, chlorobenzene and ethyl orthosilicate is 0.18 g: 2.7-3.3 g: 36-40 mL: 2.6-4.1 mL: 1 mL.
9. The method for preparing a thermostable nucleic acid vaccine adjuvant according to claim 1, which comprises the following steps:
mixing inorganic filler and water, performing ultrasonic dispersion at frequency of 40-60kHz, adding oil phase and modified microspheres, and continuing ultrasonic treatment for 20min to obtain the thermostable nucleic acid vaccine adjuvant.
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| US5855895A (en) * | 1995-06-07 | 1999-01-05 | Virus Research Institute | Polyphosphazene polyelectrolyte immunoadjuvants |
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| CN102573802A (en) * | 2009-08-12 | 2012-07-11 | 希格默伊德药业有限公司 | Immunomodulatory compositions comprising a polymer matrix and an oil phase |
| EP2711022A2 (en) * | 2011-05-19 | 2014-03-26 | Universidade de Santiago de Compostela | Polymer microspheres as adjuvants in the production of vaccines against scuticociliate fish parasites |
| CN109364243A (en) * | 2018-10-25 | 2019-02-22 | 中国科学院过程工程研究所 | A kind of antigen thermostabilization lotion and its preparation method and application |
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| US5855895A (en) * | 1995-06-07 | 1999-01-05 | Virus Research Institute | Polyphosphazene polyelectrolyte immunoadjuvants |
| CN102573802A (en) * | 2009-08-12 | 2012-07-11 | 希格默伊德药业有限公司 | Immunomodulatory compositions comprising a polymer matrix and an oil phase |
| CN101721377A (en) * | 2010-01-18 | 2010-06-09 | 上海交通大学 | Method for preparing microspheres with solid-in-oil-in-hydrophilic oil-in-ethanol |
| EP2711022A2 (en) * | 2011-05-19 | 2014-03-26 | Universidade de Santiago de Compostela | Polymer microspheres as adjuvants in the production of vaccines against scuticociliate fish parasites |
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