CN113908267A - Vaccine adjuvant and preparation method and application thereof - Google Patents
Vaccine adjuvant and preparation method and application thereof Download PDFInfo
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Images
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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
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
- C08G63/00—Macromolecular compounds obtained by reactions forming a carboxylic ester link in the main chain of the macromolecule
- C08G63/91—Polymers modified by chemical after-treatment
- C08G63/912—Polymers modified by chemical after-treatment derived from hydroxycarboxylic acids
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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
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55511—Organic adjuvants
- A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
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Abstract
The invention provides an immunologic adjuvant which is polylactic acid with modified terminal group; the end group of the end group modified polylactic acid comprises-NH2And/or tertiary amine groups. Compared with the prior art, the amino-modified polylactic acid is used as a vaccine adjuvant, and the vaccine obtained by compounding the composite vaccine antigen is easier to be phagocytized by dendritic cells and start immune reaction, has a better immune effect, and also has higher biocompatibility.
Description
Technical Field
The invention belongs to the technical field of pharmaceutical preparations, and particularly relates to a vaccine adjuvant and a preparation method and application thereof.
Background
Attenuated vaccines, inactivated vaccines, subunit and mRNA vaccines are generally difficult to generate strong immune responses after injection into the body, and the immunogenicity of the vaccines is usually enhanced by combining with immune adjuvants. Adjuvants can be divided into two main categories, traditional adjuvants and novel adjuvants.
Conventional adjuvants generally include aluminum adjuvants, which increase the humoral immune response but are not involved in cellular immunity, and oil emulsion adjuvants; the main component of the oil emulsion adjuvant is oil, which is widely used in animal vaccine producers at present, but the ideal requirements on both activity and safety are difficult to achieve.
The novel adjuvants generally include nano-adjuvants, cytokine adjuvants, immunostimulating complex adjuvants, and other adjuvants, among others. The nano adjuvant is divided into inorganic nano adjuvant and organic nano adjuvant, generally has better biocompatibility, can continuously release antigen, and can carry and transport the antigen to corresponding immune cells to fully exert immune effect. The organic nanometer adjuvant comprises chitosan and its derivatives, polypropylene-ethylene colloid, propolis, etc.; the inorganic nano adjuvant comprises aluminum hydroxide, calcium phosphate and the like. Compared with inorganic nano-adjuvants, the organic nano-adjuvants have better histocompatibility and show larger application potential.
As early as 1995, the FDA (food and Drug administration) in the United states approved polylactic acid and its derivatives for clinical use as a pharmaceutical adjuvant. The polylactic acid is prepared into nano particles, and the nano vaccine prepared by combining the nano particles with the antigen has better application prospect. The preparation of polylactic acid nanoparticles with negative surface charge and integral points and the verification of the immune effect of the positively charged polylactic acid nanoparticles as an immune adjuvant are introduced in the thesis of the university of Beijing chemical industry, Master's academic thesis, that is, the preparation of polylactic acid nanoparticles and the application research of polylactic acid nanoparticles as an immune adjuvant, the polylactic acid nanoparticles with the positive surface charge and integral points can be prepared by a nano-precipitation method, and the obtained polylactic acid nanoparticles with the particle size of 100nm can induce the expression of serum specific antibodies and cytokines at higher levels, show better immune effect, but the immune effect is still to be improved.
Disclosure of Invention
In view of the above, the technical problem to be solved by the present invention is to provide a vaccine adjuvant which is easily phagocytosed by dendritic cells and can initiate immune response, and a preparation method and an application thereof.
The invention provides an immunologic adjuvant which is polylactic acid with modified terminal group; the end group of the end group modified polylactic acid comprises-NH2And/or tertiary amine groups.
Preferably, the terminal group is an alkylamino group containing a thioether bond or a tertiary amine group containing a thioether bond.
Preferably, the end group modified polylactic acid comprises a structure shown in formula (I) or formula (II):
n in the formula (I) and the formula (II) is an integer of 1-5 independently;
m in the formula (I) and the formula (II) is an integer of 1-5 independently;
x in the formula (I) and the formula (II) is an integer of 10-200 independently;
r in the formula (I) and the formula (II) is independently selected from the group consisting of the groups represented by the formulas (1) to (3):
wherein y is an integer of 1 to 6, and z is an integer of 0 to 2;
R1is C1-C10 alkyl;
R2is C1-C5 alkyl.
Preferably, n in the formula (I) and the formula (II) is an integer of 1-3 independently; m in the formula (I) and the formula (II) is an integer of 1-3 independently; x in the formula (I) and the formula (II) is an integer of 20-80 independently; r1Is C2-C5 alkyl; r2Is C1-C2 alkyl.
Preferably, including end groups comprising-NH2The terminal-modified polylactic acid of (a) and the terminal-modified polylactic acid whose terminal contains a group of a tertiary amine; the end group comprises-NH2The mass ratio of the terminal-modified polylactic acid of (a) to the terminal-modified polylactic acid having a terminal containing a tertiary amine group is 1: 10-10: 1.
the invention also provides a preparation method of the immunologic adjuvant, which comprises the following steps:
mixing polylactic acid with an end group containing an acetylene bond with a compound shown as a formula (III) and/or a salt thereof for reaction in a protective atmosphere to obtain an immunologic adjuvant;
n is an integer of 1-5;
r is selected from the group consisting of the groups represented by the formulae (1) to (3):
wherein y is an integer of 1 to 6, and z is an integer of 0 to 2;
R1is C1-C10 alkyl;
R2is C1-C5 alkyl.
Preferably, the polylactic acid with the end group containing the acetylene bond is prepared according to the following method:
in a protective atmosphere, mixing lactide, alkynol and a catalyst in an organic solvent, and heating for reaction to obtain polylactic acid with an end group containing an alkyne bond;
the mol ratio of the polylactic acid with the end group containing the acetylene bond to the compound shown in the formula (III) and/or the salt thereof is 1: (5-15); the mixed reaction is carried out in an organic solvent; the molar concentration of the polylactic acid with the acetylene bond at the end group in the mixed reaction system is 0.03-0.1 mol/L;
the mixed reaction is initiated by ultraviolet irradiation in the presence of a photoinitiator; the photoinitiator is selected from one or more of a photoinitiator 2959, a photoinitiator 651 and a diphenylmethyl ether photoinitiator; the mole number of the photoinitiator is 30-80% of that of the polylactic acid of which the end group contains an acetylene bond; the mixed reaction is irradiated by ultraviolet light for 30-90 min and then reacts for 8-30 h;
after mixing and reaction, settling by using a glacial alcohol solvent, centrifuging and drying to obtain the immunologic adjuvant; the volume of the glacial alcohol solvent is 6-10 times of that of the mixed reaction system.
The invention also provides a vaccine comprising one or more of the above immunoadjuvants.
The invention also provides a preparation method of the vaccine, which comprises the following steps:
mixing the immune adjuvant with water, and ultrasonically stirring to obtain a nano micelle solution;
and mixing the nano micelle solution with a vaccine antigen to obtain the vaccine.
Preferably, the mass ratio of the immunological adjuvant to the vaccine antigen is 10: (1-5).
The invention also provides the application of the end group modified polylactic acid as an immunologic adjuvant; the end group of the end group modified polylactic acid comprises-NH2And/or tertiary amine groups.
The invention provides an immunologic adjuvant which is polylactic acid with modified terminal group; the end group of the end group modified polylactic acid comprises-NH2And/or tertiary amine groups. Compared with the prior art, the amino-modified polylactic acid is used as a vaccine adjuvant, and the vaccine obtained by compounding the composite vaccine antigen is easier to be phagocytized by dendritic cells and start immune reaction, has a better immune effect, and also has higher biocompatibility.
The invention also provides a composite immunologic adjuvant, wherein the immunologic adjuvant is terminated by-NH2The composite immunologic adjuvant has better antigen loading capacity and particle size distribution.
Drawings
In FIG. 1, A is PLLA-NH obtained in example 1 of the present invention2And PDLA-NH obtained in example 22The nuclear magnetic resonance hydrogen spectrum of (a); b is PLLA-NH obtained in example 1 of the present invention2And PDLA-NH obtained in example 22An infrared spectrum of (1);
FIG. 2A is a graph showing the distribution of the particle size distribution of the vaccine obtained in example 1 of the present invention; b is the particle size distribution diagram of the vaccine obtained in example 2 of the invention;
FIG. 3A is a bar graph of the vaccine obtained in example 1 and example 2 of the present invention activating cells to produce interleukin-6 secretion; b is a bar graph of the vaccine obtained in the embodiment 1 and the embodiment 2 of the invention for activating cells to produce and secrete interleukin-12;
FIG. 4 is a graph showing the activation of the lymph node after vaccine derived from mice in example 1 and example 2;
FIG. 5 is a graph showing the effect of different vaccines on tumor prevention, wherein A is a graph showing the change in tumor volume of B16 melanoma-bearing mice treated with the vaccines obtained in examples 1 and 2; and B is the survival rate curve of B16 melanoma-bearing mice treated by the vaccine obtained in example 1 and example 2 of the invention.
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.
The invention provides an immunologic adjuvant which is polylactic acid with modified terminal group; the end group of the end group modified polylactic acid comprises-NH2And/or tertiary amine groups.
In the present invention, the terminal group is preferably a C2-C10 group containing-NH2More preferably from C2 to C8, containing-NH2More preferably from C2 to C6, containing-NH2Most preferably C2-C4-NH containing groups2A group of (1).
Or the terminal group is preferably a tertiary amine-containing group of C2-C20, more preferably a tertiary amine-containing group of C4-C16, and still more preferably a tertiary amine-containing group of C4-C10.
In the present invention, the terminal-modified polylactic acid is preferably terminal-modified L-polylactic acid and/or terminal-modified D-polylactic acid.
In the present invention, it is further preferred that the terminal group of the terminal-modified polylactic acid is an alkylamino group containing a thioether bond, preferably an alkylamino group containing a thioether bond of C2 to C10, more preferably an alkylamino group containing a thioether bond of C2 to C8, still more preferably an alkylamino group containing a thioether bond of C2 to C6, and most preferably an alkylamino group containing a thioether bond of C2 to C4.
Or more preferably, the end group of the end group-modified polylactic acid is a tertiary amine group containing a thioether bond, preferably a tertiary amine group containing a thioether bond of C2 to C20, more preferably a tertiary amine group containing a thioether bond of C4 to C16, and still more preferably a tertiary amine group containing a thioether bond of C4 to C10.
In the present invention, it is further preferred that the end group-modified polylactic acid comprises a structure represented by formula (I) or formula (II):
n in the formula (I) and the formula (II) is independently an integer of 1-5, preferably independently an integer of 1-4, more preferably independently an integer of 1-3, even more preferably independently an integer of 1-2, and most preferably 1;
m in the formula (I) and the formula (II) is independently an integer of 1-5, preferably independently an integer of 1-4, more preferably independently an integer of 1-3, even more preferably independently an integer of 1-2, and most preferably 1;
x in the formula (I) and the formula (II) is independently an integer of 10-200, preferably independently an integer of 20-150, more preferably independently an integer of 20-120, more preferably independently an integer of 20-100, more preferably independently an integer of 20-80, more preferably independently an integer of 20-50, and most preferably independently an integer of 25-30; in the embodiments provided by the present invention, x is specifically 28;
r in the formula (I) and the formula (II) is independently selected from the group consisting of the groups represented by the formulas (1) to (3):
wherein y is an integer of 1 to 6, and z is an integer of 0 to 2; y represents the number of carbon atoms on the heterocycle, namely when y is 1, the heterocycle is a ternary heterocycle, when y is 2, the heterocycle is a quaternary heterocycle, and the like; z represents the number of substituents on the heterocycle.
R1Is C1-C10 alkyl, preferably C2-C8 alkyl, and more preferably C2-C5 alkyl;
R2is an alkyl group having 1 to 5 carbon atoms, preferably an alkyl group having 1 to 3 carbon atoms, and more preferably an alkyl group having 1 to 2 carbon atoms.
Further preferably, said R is selected from the group consisting of:
the immunological adjuvant provided by the invention is further preferably a terminal group containing-NH2A mixture of the terminal-modified polylactic acid of (a) and a terminal-modified polylactic acid whose terminal contains a group of a tertiary amine; the end group comprises-NH2The mass ratio of the terminal-modified polylactic acid of (a) to the terminal-modified polylactic acid whose terminal contains a group of a tertiary amine is preferably 1: 10-10: 1, more preferably 1: 5-5: 1, more preferably 1: 2-2: 1, most preferably 1: 1. the composite immunologic adjuvant has better antigen loading capacity and particle size distribution.
The amino-modified polylactic acid is used as a vaccine adjuvant, and the vaccine obtained by compounding the compound vaccine antigen is easier to be phagocytized by dendritic cells and start immune reaction, so that the amino-modified polylactic acid vaccine has a better immune effect and higher biocompatibility.
The invention also provides a preparation method of the immunological adjuvant, which comprises the following steps:
mixing polylactic acid with an end group containing an acetylene bond with a compound shown as a formula (III) and/or a salt thereof for reaction in a protective atmosphere to obtain an immunologic adjuvant;
n is an integer of 1-5, preferably an integer of 1-4, more preferably an integer of 1-3, and most preferably 1 or 2; the salt of the compound represented by the formula (III) is preferably a hydrochloride thereof.
R is a group represented by the formula (1) to (3):
wherein y is an integer of 1 to 6, and z is an integer of 0 to 2;
R1is C1-C10 alkyl;
R2is C1-C5 alkyl.
In the present invention, the sources of all raw materials are not particularly limited, and they may be commercially available.
In the present invention, the polylactic acid having an acetylene bond at the terminal is preferably prepared by the following method: in a protective atmosphere, mixing lactide, alkynol and a catalyst in an organic solvent, and heating for reaction to obtain the polylactic acid with the end group containing an alkyne bond.
Mixing lactide, alkynol and a catalyst in an organic solvent in a protective atmosphere; the protective atmosphere is not particularly limited as long as it is known to those skilled in the art, and nitrogen is preferred in the present invention; the lactide is preferably recrystallized lactide, more preferably L-lactide and/or D-lactide; the alkynol is preferably C3-C10, more preferably C3-C8, still more preferably C3-C6, still more preferably C3-C5, and most preferably C3-C4; the mole number of the alkynol is preferably 5 to 10 percent, more preferably 6 to 8 percent and even more preferably 7 percent of the mole number of the lactide; the catalyst is a catalyst well known to those skilled in the art, and is not particularly limited, and in the present invention, a tin catalyst is preferred, and stannous octoate is more preferred; the mole number of the catalyst is preferably 0.01 to 0.1 percent of the mole number of the lactide, more preferably 0.03 to 0.08 percent, still more preferably 0.04 to 0.06 percent, and most preferably 0.045 percent; the organic solvent is not particularly limited as long as it is well known to those skilled in the art, and toluene is preferable in the present invention; since the reaction is preferably carried out under anhydrous conditions, in the present invention, it is preferred that the lactide is dried by vacuum pumping and then mixed with the alkynol and the catalyst in the organic solvent; the vacuumizing time is preferably 20-50 min, and more preferably 30-40 min; the alkynol and the catalyst are preferably dissolved in a solvent and added in the form of an alkynol solution and a catalyst solution; the solvent may be any organic solvent known to those skilled in the art, and is not particularly limited, and in the present invention, toluene is preferred; the concentration of the alkynol in the alkynol solution is preferably 0.05-0.5 g/g, more preferably 0.1-0.3 g/g, still more preferably 0.1-0.2 g/g, and most preferably 0.14 g/g; the concentration of the catalyst in the catalyst solution is preferably 0.005-0.02 g/g, more preferably 0.005-0.01 g/g, still more preferably 0.006-0.008 g/g, and most preferably 0.0064 g/g; the concentration of lactide in the mixed system is preferably 0.5-1 mol/L, more preferably 0.6-0.8 mol/L, and still more preferably 0.7 mol/L.
After mixing, heating for reaction; the reaction temperature is preferably 100-140 ℃, and more preferably 110-120 ℃; the reaction time is preferably 15-30 h, more preferably 20-30 h, and still more preferably 20-24 h.
After the reaction is finished, preferably pouring the reaction solution into an ice alcohol solvent for sedimentation, centrifugation and drying to obtain a crude product; the alcohol solvent is preferably ethanol; the volume of the alcohol solvent of the ice is preferably 6-10 times, more preferably 7-9 times, and even more preferably 8 times of the volume of the reaction solution; the crude product is preferably dissolved by a small amount of organic solvent, and then is settled, centrifuged and dried by using an ice alcohol solvent to obtain the polylactic acid with the end group containing the acetylene bond; the organic solvent is preferably dichloromethane; the alcohol solvent is preferably an ethanol solvent; the alcohol solvent of the ice is preferably 6-10 times, more preferably 7-9 times and even more preferably 8 times of the volume of the organic solvent.
Mixing and reacting polylactic acid with an end group containing an acetylene bond with a compound shown as a formula (III) and/or a salt thereof in a protective atmosphere; the protective atmosphere is not particularly limited as long as it is known to those skilled in the art, and nitrogen is preferred in the present invention; the molar ratio of the polylactic acid with the end group containing the acetylene bond to the compound represented by the formula (III) and/or the salt thereof is preferably 1: (5-15), more preferably 1: (8-12), and more preferably 1: 10; the mixing reaction is preferably carried out in an organic solvent; the organic solvent is not particularly limited as long as it is well known to those skilled in the art, but N, N-Dimethylformamide (DMF) is preferable in the present invention; the mol concentration of the polylactic acid with the acetylene bond at the end group in the mixed reaction system is preferably 0.03-0.1 mol/L, more preferably 0.05-0.08 mol/L, and still more preferably 0.06-0.07 mol/L; the mixing reaction is preferably initiated by ultraviolet irradiation in the presence of a photoinitiator; the photoinitiator is preferably one or more of a photoinitiator 2959, a photoinitiator 651 and a diphenylmethyl ether photoinitiator; the mole number of the photoinitiator is preferably 30 to 80 percent, more preferably 40 to 70 percent, and still more preferably 50 to 60 percent of the mole number of the polylactic acid of which the end group contains the acetylene bond; the mixing reaction is preferably carried out for 30-90 min through ultraviolet irradiation and then is carried out for 8-30 h; the wavelength of the ultraviolet light is preferably 365 nm; the time for the ultraviolet irradiation is preferably 40-80 min, more preferably 50-70 min, and further preferably 60 min; the reaction time is preferably 10-25 h, and more preferably 10-15 h.
After the mixing reaction is finished, preferably using a glacial alcohol solvent for settling, centrifuging and drying; the glacial alcohol solvent is preferably glacial ethanol; the volume of the glacial alcohol solvent is preferably 6-10 times, more preferably 7-9 times and even more preferably 8 times of that of the mixed reaction system; drying the obtained product, preferably precipitating and centrifuging the product by using an organic solvent again and a glacial alcohol solvent, and drying to obtain the immune adjuvant; the organic solvent is preferably dichloromethane and/or DMF; the glacial alcohol solvent is preferably 6-10 times, more preferably 7-9 times and even more preferably 8 times of the volume of the organic solvent.
The invention also provides the application of the end group modified polylactic acid as an immunologic adjuvant; the end group of the end group-modified polylactic acid is a group containing an amino group and/or a tertiary amine.
The end group modified polylactic acid is the same as that described above and is not described in detail herein.
The invention also provides a vaccine comprising the immunological adjuvant. The content of the surface amino group of the vaccine is preferably 0-99%, more preferably 10-90%, and even more preferably 40-90% of the amino group content of the immunologic adjuvant.
The invention also provides a preparation method of the vaccine, which comprises the following steps: mixing one or more of the immune adjuvants with water, and ultrasonically stirring to obtain a nano micelle solution; and mixing the nano micelle solution with a vaccine antigen to obtain the vaccine.
Mixing an immunologic adjuvant with water, and obtaining a nano micelle solution in an ultrasonic-assisted stirring manner; the water is preferably ultrapure water; the ratio of the immunological adjuvant to water is preferably (0.5-5) mg:1mL, more preferably (0.5-4) mg:1mL, still more preferably (1-3) mg:1mL, and most preferably 2mg:1 mL; the ultrasonic power of the ultrasonic stirring is preferably 200-800 watts, more preferably 300-600 watts, further preferably 400-500 watts, and most preferably 450 watts; the stirring time is preferably 0.5-3 h, more preferably 0.5-2 h, and still more preferably 0.5-1 h; after ultrasonic stirring, preferably continuously stirring to obtain a nano micelle solution; the rotation speed of the continuous stirring is preferably 300-1000 rpm, more preferably 400-800 rpm, still more preferably 500-700 rpm, and most preferably 600 rpm.
Mixing the nanomicelle solution (i.e., vaccine adjuvant) with a vaccine antigen; the mass ratio of the immune adjuvant to the vaccine antigen is preferably 10: (0.1 to 10), more preferably 10: (0.5 to 8), and more preferably 10: (1-5); in the embodiment provided by the invention, the mass ratio of the immune adjuvant to the vaccine antigen is specifically 10: 1. 10: 2. 10:5 or 10: 10; the vaccine antigen is preferably mixed with the nanomicelle solution in the form of an aqueous solution thereof; the concentration of the vaccine antigen aqueous solution is preferably 0.1-1 mg/ml, more preferably 0.1-0.8 mg/ml, still more preferably 0.3-0.6 mg/ml, and most preferably 0.4-0.5 mg/ml; the volume ratio of the nano-micelle solution to the vaccine antigen aqueous solution is preferably 1: (0.5 to 2), more preferably 1: (0.5 to 1.5), and preferably 1: (0.8 to 1.2), most preferably 1: 1; the mixing temperature is preferably 2-25 ℃, more preferably 3-20 ℃, and further preferably 4-20 ℃; the mixing method is preferably stirring; the rotation speed of the stirring is preferably 300-1000 rpm, more preferably 400-800 rpm, still more preferably 500-700 rpm, and most preferably 600 rpm; the mixing time is preferably 10-60 min, more preferably 10-45 min, and further preferably 15-30 min, so that the nano micelle adsorbs the vaccine antigen.
Mixing, and preferably centrifuging to obtain vaccine; the centrifugal rotating speed is preferably 10000-30000 g, more preferably 12000-25000 g, still more preferably 14000-20000 g, and most preferably 16000-18000 g; the time for centrifugation is preferably 20-40 min, more preferably 25-35 min, and still more preferably 30 min.
In order to further illustrate the present invention, the following examples are provided to describe a vaccine adjuvant, its preparation method and application in detail.
The reagents used in the following examples are all commercially available.
Example 1
1.1 baking the ampoule with proper size in advance, and pumping nitrogen for three times to ensure that the ampoule is anhydrous and anaerobic. 5.0g of previously recrystallized L-lactide was weighed into the above-mentioned dry ampoule, connected to a double calandria and dried again after evacuation for 30 min. The mixture was introduced into a glove box under negative pressure, and 1.01g of an initiator propargyl alcohol solution (c: 0.14g/g) and 1.01g of a catalyst stannous octoate solution (c: 0.0064g/g) were weighed into an ampoule. The ampoule was taken out, the stopper was tightly wound with iron wire, and 50ml of anhydrous toluene was injected from the branch of the latex tube with a dry syringe. The ampoule was stirred in an oil bath for 24 hours at 110 ℃. After the reaction, the reaction solution was slowly poured into 8 times the volume of the reaction solution of glacial ethanol for settling. Then centrifuged, the supernatant decanted and the product dried in a desiccator under vacuum. After drying, the mixture was dissolved in a small amount of methylene chloride, and the resulting solution was centrifuged using 8 times the volume of iced ethanol and dried. Pure product 3.8g was obtained in 76% yield.
1.2 the polymer 3.8g, 0.0019mol was weighed into a clean 100ml quartz flask, and 30ml DMF was added and dissolved with stirring. After complete dissolution, 2.159g of cysteamine hydrochloride, 0.019mol, and @ 29590.213 g of photoinitiator, 0.00095mol are added. After completely dissolvingNitrogen was passed through for 20 minutes. Then 365nm ultraviolet irradiation is carried out for 1h, and the reaction is carried out overnight. The next day, 8 times the volume of ice absolute ethanol was used for settling, centrifugation, and drying. Finally, the product is dissolved again, settled, centrifuged and dried to obtain the pure product PLLA-NH22.5g, 66% yield.
Nuclear magnetic resonance for PLLA-NH product obtained in 1.22The nuclear magnetic resonance hydrogen spectrum obtained by detection is shown as A in figure 1.
Fourier infrared spectroscopy is used for the product PLLA-NH obtained in 1.22The infrared spectrum obtained by detection is shown as B in figure 1.
1.3 weighing 4.0mg of the product, adding the product into 2ml of ultrapure water, carrying out ultrasonic-assisted stirring for preparation, wherein the ultrasonic power is 450 watts, carrying out ultrasonic treatment for 1 hour, and finally stirring at 600rpm for 15 minutes to obtain the nano PLLA vaccine adjuvant for subsequent use.
1.4 the resulting micellar solution was mixed with an equal volume of Ovalbumin (OVA) solution (0.4mg/ml) of the model antigen at 600rpm at 4 ℃ for 15 minutes to allow the adjuvant to adsorb OVA, giving a vaccine as PLLA-OVA.
To determine the Encapsulation Efficiency (EE) and Loading Capacity (LC) of OVA micelles, OVA-loaded PLLA-OVA was ultracentrifuged at 16000g for 30min and the amount of unbound OVA in the supernatant was determined using the Bio-Rad protein assay kit (BioRad, CA, USA). The calculation formula for EE and LC is as follows: EE ═ (total protein-unbound protein)/total protein 100%; LC ═ (total protein-unbound protein)/total dry weight of the nano vaccine 100%. EE was measured to be 99.9% and LC 19.9%.
The particle size of the vaccine obtained in 1.4 was analyzed, and the particle size distribution diagram thereof was shown as a in fig. 2. As can be seen from FIG. 2, the vaccine had a uniform particle size and a hydrodynamic diameter of 87.3. + -. 21.1 nm.
The vaccine obtained in 1.4 was co-cultured with mouse bone marrow-derived dendritic cells (BMDCs), and the activation of DC cells by the vaccine was verified. Specifically, after co-culturing physiological saline, OVA antigen, PLLA-OVA and PDLA-OVA with BMDC cells at 37 ℃ (5% carbon dioxide concentration) for 12h, the culture supernatant is tested, wherein the PLA concentration of the vaccine is 10 mug/ml. Finally, the column diagram of the vaccine activated cell for producing and secreting interleukin-6 is shown as A in figure 3; the bar graph showing the secretion of interleukin-12 by activated cells is shown in B of FIG. 3.
The nano vaccine with uniform particle size can be formed by electrostatic compounding with antigen protein through an ultrasonic-assisted stirring method, can be compounded with different amounts of antigen protein, and the optimal vaccine can be obtained by adjusting the particle size and surface charge of the vaccine by changing the mass ratio of the adjuvant to the antigen, specifically, the optimal vaccine can be obtained by adjusting the concentration of the antigen protein and finally obtaining the relationship between the proportion of the adjuvant to the antigen protein and the particle size as shown in table 1. As shown in Table 1, the ratio of the adjuvant to the antigen protein is 10: 0-10: 10, the optimal ratio is 10: 1-10: 5, and the vaccine with the proper particle size can be assembled by 10: 1-10: 5.
After injecting the vaccine obtained in 1.4 into mice (injection dose is 20 mug/mouse, with PLA-NH adjuvant)2Counting), lymph node analysis is carried out after three days, and a lymph node activation condition graph of the mice immunized by different vaccines is obtained, as shown in figure 4.
The effect of the vaccine obtained in example 1 on tumor prevention was examined, and the graph showing the effect on tumor prevention by activation thereof was obtained as shown in FIG. 5; wherein A is a tumor volume change chart of a B16 melanoma-bearing mouse after treatment, and B is a survival rate curve of a B16 melanoma-bearing mouse. Wherein Alum + OVA is commercial aluminum hydroxide adjuvant (Imject)TMAlum Adjuvant; thermo Fisher Scientific, Waltham, MA, USA) and model antigen complex (1: 1 antigen to aluminum hydroxide mass ratio). The mice are B16 mouse melanin models, after three times of immunization at days-21, -14 and-7, tumor cells are inoculated on the backs of the mice at day 0 to establish a model (the inoculation dose is 20 mu g/mouse, and PLA-NH adjuvant is added2Meter).
Example 2
2.1 baking the ampoule with proper size in advance, and pumping nitrogen for three times to ensure that the ampoule is anhydrous and anaerobic. 5.0g of previously recrystallized D-lactide was weighed into the above-mentioned dry ampoule, connected to a double calandria and dried again by evacuation for 30 min. The mixture was introduced into a glove box under negative pressure, and 1.01g of an initiator propargyl alcohol solution (c: 0.14g/g) and 1.01g of a catalyst stannous octoate solution (c: 0.0064g/g) were weighed into an ampoule. The ampoule was taken out, the stopper was tightly wound with iron wire, and 50ml of anhydrous toluene was injected from the branch of the latex tube with a dry syringe. The ampoule was stirred in an oil bath for 24 hours at 110 ℃. After the reaction, the reaction solution was slowly poured into 8 times the volume of the reaction solution of glacial ethanol for settling. Then centrifuged, the supernatant decanted and the product dried in a desiccator under vacuum. After drying, the mixture was dissolved in a small amount of methylene chloride, and the resulting solution was centrifuged using 8 times the volume of iced ethanol and dried. Pure product 3.8g was obtained in 76% yield.
2.2 3.8g of the above polymer (0.0019 mol) was weighed into a clean 100ml quartz flask, and 30ml of DMF was added thereto and dissolved with stirring. After complete dissolution, 2.159g of cysteamine hydrochloride, 0.019mol, and @ 29590.213 g of photoinitiator, 0.00095mol are added. After complete dissolution, nitrogen was introduced for 20 min. Then 365nm ultraviolet irradiation is carried out for 1h, and the reaction is carried out overnight. The next day, 8 times the volume of ice absolute ethanol was used for settling, centrifugation, and drying. Finally, the product is dissolved again, settled, centrifuged and dried to obtain the pure product PDLA-NH22.5g, yield 87%.
Nuclear magnetic resonance for PDLA-NH product obtained in 2.22The nuclear magnetic resonance hydrogen spectrum obtained by detection is shown as A in figure 1.
Fourier infrared spectroscopy is utilized to measure the product PDLA-NH obtained in 2.22The infrared spectrum obtained by detection is shown as B in figure 1.
2.3 weighing 4.0mg of the product, adding the product into 2ml of ultrapure water, carrying out ultrasonic-assisted stirring for preparation, wherein the ultrasonic power is 450 watts, carrying out ultrasonic treatment for 1 hour, and finally stirring at 600rpm for 15 minutes to obtain the nano PDLA vaccine adjuvant for subsequent use.
2.4 the micelle solution was mixed with an equal volume of Ovalbumin (OVA) solution (0.4mg/ml) of the model antigen at 4 ℃ for 15 minutes at 600rpm to allow the adjuvant to adsorb the OVA antigen, giving a vaccine, which was designated PDLA-OVA.
To determine the Encapsulation Efficiency (EE) and Loading Capacity (LC) of OVA micelles, OVA-loaded PLLA/OVA was ultracentrifuged at 16000g for 30min and the amount of unbound OVA in the supernatant was determined using the Bio-Rad protein assay kit (BioRad, CA, USA). The calculation formula for EE and LC is as follows: EE ═ (total protein-unbound protein)/total protein 100%; LC ═ (total protein-unbound protein)/total dry weight of the nano vaccine 100%. EE was measured to be 99.9% and LC 19.9%.
The particle size distribution of the vaccine obtained in 2.4 was analyzed and shown as B in fig. 2. As can be seen from FIG. 2, the vaccine had a uniform particle size and a hydrodynamic diameter of 68.5. + -. 7.1 nm.
The vaccine obtained in 2.4 was co-cultured with mouse bone marrow-derived dendritic cells (BMDCs), and the activation of DC cells by the vaccine was verified. Specifically, after co-culturing physiological saline, OVA antigen, PLLA-OVA and PDLA-OVA with BMDC cells at 37 ℃ (5% carbon dioxide concentration) for 12h, the culture supernatant is tested, wherein the PLA concentration of the vaccine is 10 mug/ml. Finally, the column diagram of the vaccine activated cell for producing and secreting interleukin-6 is shown as A in figure 3; the bar graph showing the secretion of interleukin-12 by activated cells is shown in B of FIG. 3. As can be seen from FIG. 3, the vaccine prepared by the present invention can effectively activate dendritic cells, has a lower activation capacity of PDLA-OVA, and can stimulate DCs to produce more cytokines.
The nano vaccine with uniform particle size can be formed by electrostatic compounding with antigen protein through an ultrasonic-assisted stirring method, can be compounded with different amounts of antigen protein, and the optimal vaccine can be obtained by adjusting the particle size and surface charge of the vaccine by changing the mass ratio of the adjuvant to the antigen, specifically, the optimal vaccine can be obtained by adjusting the concentration of the antigen protein and finally obtaining the relationship between the proportion of the adjuvant to the antigen protein and the particle size as shown in table 1. As shown in Table 1, the ratio of the adjuvant to the antigen protein is 10: 0-10: 10, the optimal ratio is 10: 1-10: 5, and the vaccine with the proper particle size can be assembled by 10: 1-10: 5.
After the vaccine obtained in 2.4 was injected into mice (injection dose 20. mu.g/mouse, with PLA-NH as adjuvant)2Counting), lymph node analysis is carried out after three days, and lymph node stimulation is carried out after the mice are immunized by different vaccinesThe situation is shown in figure 4. As can be seen from FIG. 4, the residual amino groups of PDLA-OVA are about 70% of those in the best immune effect, and have better immunostimulation ability.
The tumor prevention effect of the vaccine obtained in example 2 was examined, and the graph of the tumor prevention effect of the vaccine obtained in example 2 is shown in fig. 5; wherein A is a tumor volume change chart of a B16 melanoma-bearing mouse after treatment, and B is a survival rate curve of a B16 melanoma-bearing mouse. Wherein Alum + OVA is commercial aluminum hydroxide adjuvant (Imject)TMAlumAdjuvant; thermo Fisher Scientific, Waltham, MA, USA) and model antigen complex (1: 1 antigen to aluminum hydroxide mass ratio). The mice are B16 mouse melanin models, after three times of immunization at days-21, -14 and-7, tumor cells are inoculated on the backs of the mice at day 0 to establish a model (the inoculation dose is 20 mu g/mouse, and PLA-NH adjuvant is added2Meter). As can be seen from FIG. 5, PDLA-OVA was effective in preventing tumorigenesis and in prolonging survival of mice.
Example 3
3.1 terminating the group by-NH2The polylactic acid and the polylactic acid with the end group of tertiary amino are mixed according to the mass ratio of 1:1 to prepare a composite polylactic acid vaccine adjuvant, and the composite polylactic acid vaccine is prepared by compounding the polylactic acid and the polylactic acid with OVA according to the methods of the embodiments 1 and 2, specifically, the polylactic acid vaccine adjuvant and the OVA are compounded according to the mass ratio of 5:5:2, and the antigen loading capacity and the average particle size of the polylactic acid vaccine adjuvant are shown in a table 2(PLLA) and a table 3 (PDLA).
TABLE 1 relationship between mass ratio of vaccine adjuvant to antigen and particle size
TABLE 2 average particle size and antigen-carrying efficiency of PLLA composite vaccines
| End group R structure | 2-1 | 2-2 | 2-3 | 2-4 | 3-1 | 3-2 | 3-3 | 3-4 | 3-5 | 3-6 | 3-7 |
| Particle size (nm) | 77.3 | 75.5 | 62.8 | 73.1 | 65.0 | 66.4 | 65.5 | 63.7 | 65.4 | 70.2 | 66.2 |
| EF(％) | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 |
TABLE 3 average particle size and antigen-carrying efficiency of PDLA composite vaccines
| End group R structure | 2-1 | 2-2 | 2-3 | 2-4 | 3-1 | 3-2 | 3-3 | 3-4 | 3-5 | 3-6 | 3-7 |
| Particle size (nm) | 79.2 | 79.1 | 64.0 | 76.4 | 68.2 | 67.2 | 65.2 | 66.7 | 67.5 | 75.9 | 69.1 |
| EF(％) | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 |
Claims (10)
1. An immunological adjuvant, characterized in that the immunological adjuvant is a terminal group modified polylactic acid; the end group of the end group modified polylactic acid comprises-NH2And/or tertiary amine groups.
2. The immunoadjuvant of claim 1, wherein the terminal group is an alkylamino group containing a thioether bond or a tertiary amine containing a thioether bond.
3. The immunoadjuvant of claim 1, wherein the end-group-modified polylactic acid comprises a structure of formula (I) or formula (II):
n in the formula (I) and the formula (II) is an integer of 1-5 independently;
m in the formula (I) and the formula (II) is an integer of 1-5 independently;
x in the formula (I) and the formula (II) is an integer of 10-200 independently;
r in the formula (I) and the formula (II) is independently selected from the group consisting of the groups represented by the formulas (1) to (3):
wherein y is an integer of 1 to 6, and z is an integer of 0 to 2;
R1is C1-C10 alkyl;
R2is C1-C5 alkyl.
4. The immunoadjuvant of claim 3, wherein n in formula (I) and formula (II) is independently an integer of 1 to 3; m in the formula (I) and the formula (II) is an integer of 1-3 independently; x in the formula (I) and the formula (II) is an integer of 20-80 independently; r1Is C2-C5 alkyl; r2Is C1-C2 alkyl.
5. The immunoadjuvant of claim 1, comprising a terminal group comprising-NH2The terminal-modified polylactic acid of (a) and the terminal-modified polylactic acid whose terminal contains a group of a tertiary amine; the end group comprises-NH2End group modification ofThe mass ratio of the sexual polylactic acid to the end group modified polylactic acid of which the end group contains a tertiary amine group is 1: 10-10: 1.
6. the preparation method of the immunological adjuvant is characterized by comprising the following steps:
mixing polylactic acid with an end group containing an acetylene bond with a compound shown as a formula (III) and/or a salt thereof for reaction in a protective atmosphere to obtain an immunologic adjuvant;
n is an integer of 1-5;
r is selected from the group consisting of the groups represented by the formulae (1) to (3):
wherein y is an integer of 1 to 6, and z is an integer of 0 to 2;
R1is C1-C10 alkyl;
R2is C1-C5 alkyl.
7. The method according to claim 6, wherein the polylactic acid having an acetylene bond at the end is prepared by the following method:
in a protective atmosphere, mixing lactide, alkynol and a catalyst in an organic solvent, and heating for reaction to obtain polylactic acid with an end group containing an alkyne bond;
the mol ratio of the polylactic acid with the end group containing the acetylene bond to the compound shown in the formula (III) and/or the salt thereof is 1: (5-15); the mixed reaction is carried out in an organic solvent; the molar concentration of the polylactic acid with the acetylene bond at the end group in the mixed reaction system is 0.03-0.1 mol/L;
the mixed reaction is initiated by ultraviolet irradiation in the presence of a photoinitiator; the photoinitiator is selected from one or more of a photoinitiator 2959, a photoinitiator 651 and a diphenylmethyl ether photoinitiator; the mole number of the photoinitiator is 30-80% of that of the polylactic acid of which the end group contains an acetylene bond; the mixed reaction is irradiated by ultraviolet light for 30-90 min and then reacts for 8-30 h;
after mixing and reaction, settling by using a glacial alcohol solvent, centrifuging and drying to obtain the immunologic adjuvant; the volume of the glacial alcohol solvent is 6-10 times of that of the mixed reaction system.
8. A vaccine comprising one or more of the immunoadjuvant of any one of claims 1 to 5 or the immunoadjuvant prepared by the preparation method of any one of claims 6 to 7.
9. A method of preparing a vaccine, comprising the steps of:
mixing one or more of the immunologic adjuvant according to any one of claims 1 to 4 or the immunologic adjuvant prepared by the preparation method according to any one of claims 5 to 6 with water, and ultrasonically stirring to obtain a nano micelle solution;
and mixing the nano micelle solution with a vaccine antigen to obtain the vaccine.
10. The method according to claim 9, wherein the mass ratio of the immunoadjuvant to the vaccine antigen is 10: (1-5).
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| WO2023098456A1 (en) * | 2021-11-30 | 2023-06-08 | 中国科学院长春应用化学研究所 | Vaccine adjuvant, and preparation method therefor and use thereof |
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| WO2023098456A1 (en) * | 2021-11-30 | 2023-06-08 | 中国科学院长春应用化学研究所 | Vaccine adjuvant, and preparation method therefor and use thereof |
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