CN112704734A - Metallic aluminum nano adjuvant, vaccine composition, preparation method and application thereof - Google Patents

Metallic aluminum nano adjuvant, vaccine composition, preparation method and application thereof Download PDF

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CN112704734A
CN112704734A CN201911020292.4A CN201911020292A CN112704734A CN 112704734 A CN112704734 A CN 112704734A CN 201911020292 A CN201911020292 A CN 201911020292A CN 112704734 A CN112704734 A CN 112704734A
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刘堃
于华
杨成功
孙天盟
王野
朱歌
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Abstract

The invention discloses a metal aluminum nano adjuvant, a vaccine composition, and a preparation method and application thereof. The vaccine adjuvant comprises metal aluminum nanoparticles, and can be used as a candidate adjuvant of preventive vaccines and therapeutic vaccines for various diseases such as infection, autoimmune diseases, tumors and the like. The vaccine adjuvant provided by the invention is combined with antigen for application, so that the humoral immune response and the cellular immune response of the vaccine can be effectively enhanced, and the enhancement effect of the vaccine adjuvant is obviously better than that of a commercial aluminum hydroxide adjuvant.

Description

Metallic aluminum nano adjuvant, vaccine composition, preparation method and application thereof
Technical Field
The invention relates to the field of vaccine preparations, in particular to a metal aluminum nano adjuvant, a vaccine composition, and a preparation method and application thereof.
Background
Vaccines are generally used for preventing infectious diseases such as influenza, for example, a classical diphtheria vaccine and a commonly used influenza vaccine, and in recent years, development of vaccines is proceeding from prevention to treatment. In addition to the treatment of tumors, the development of therapeutic vaccines also includes vaccines for lifestyle-related diseases such as allergy, diabetes and hypertension. Therapeutic vaccines come in various forms, such as short peptide or protein vaccines, RNA or DNA vaccines, cancer cell vaccines, etc., and most fail to demonstrate effective anticancer effects despite years of clinical trials. One of the important reasons for the poor therapeutic vaccine effect is that antigens such as short peptides or proteins have low immunogenicity and cannot effectively cause anti-tumor immune response. Adjuvants are a class of immunostimulants aimed at boosting the immune response to vaccines and are widely used in prophylactic vaccines. By means of the adjuvant, the immune response of antigens such as short peptides or proteins can be effectively improved, and the treatment of diseases such as tumors and the like is realized. How to improve the specificity of the tumor vaccine, regulate and control the immune response type, and increase the safety and effectiveness of the vaccine, and the design of the individual vaccine becomes one of the main research directions at present.
Aluminum salt adjuvants are the most widely used adjuvants in biological products, and include potassium aluminum sulfate, aluminum phosphate, aluminum hydroxide and the like, with aluminum hydroxide being the most commonly used. Since Glenny first used aluminum salt to adsorb diphtheria toxoid had higher antigenicity in 1926, aluminum salt adjuvants have been used in vaccine adjuvants for over 90 years, and were the first adjuvants approved by the U.S. Food and Drug Administration (FDA) for use in human vaccines. The currently available aluminum hydroxide gel adjuvant is a fibrous particle, which exists in loose form after being aggregated, and the particle size is 1-20 microns. Commercial aluminum hydroxide adjuvant is actually Al (OH)3The incomplete dehydration product of (a), fibrous crystalline aluminum metahydroxide, AlO (OH). At present, Alhydrogel produced by Denmark is taken as a recognized standard, and the colloidal particle size is 3.07 mu m.
The mechanism of action of aluminum salt adjuvants is not completely determined at present, and the possible action modes are considered to be mainly according to the existing research results: antigen storage function; enhancing antigen presentation; enhancing a Th2 helper cell (T helper 2cell, Th2) mediated humoral immune response; enhancing the innate immune response of the body; activating complement action, etc. On the other hand, however, the aluminum hydroxide gel adjuvant also has the following disadvantages: the induction of cytotoxic T cell immune response is inefficient, the level of homologous cell-specific antibodies is often increased, and severe local reactions occur at the injection site; it may increase the sensitivity of susceptible individuals and is only suitable as a vaccine antigen adjuvant with strong immunity or in large quantities.
In recent 20 years, with the rapid development of modern biology, novel vaccines such as polypeptide vaccines and recombinant subunit vaccines are emerging continuously, and are equivalent to or more effective than aluminum adjuvants in the aspect of enhancing immune response. Wherein, the nanotechnology provides more new ideas for vaccine research in adjuvant application. Li and the like (J Control Release 2014,173,148) use vaccines containing nano aluminum hydroxide adjuvant and conventional micron aluminum hydroxide adjuvant to respectively immunize mice, and after 40 days, take tissues of injection sites of the mice for observation. The result shows that compared with the conventional aluminum adjuvant, the nano aluminum adjuvant has no obvious inflammatory reaction at the injection site of the mice immunized by the nano aluminum adjuvant, which indicates that the nano aluminum adjuvant has better safety compared with the conventional aluminum adjuvant. The aluminum adjuvant is subjected to nanocrystallization, so that the surface area of the adjuvant can be greatly increased, and the antigen adsorption rate is increased. More surprisingly, Sun et al (Cancer Nanotechnology 2010,1,63) studies found that tumor size of mice immunized with nano-alumina as an adjuvant was significantly reduced compared to conventional aluminum hydroxide adjuvants, which indicates that nano-alumina adjuvants can improve the antitumor effect of Tumor Cell Vaccines (TCV). The study of Jiang et al (adv.Sci.2018,5,1700426) finds that the cross presentation of dendritic cells is effectively improved by using nano AlO (OH) particles and polymer composite as an adjuvant, the response of CD8+ T cells is stimulated, and the anti-tumor effect is good. Li et Al (Nat nanotechnol.2011,18,645) used crystalline alpha-Al2O3When the nano-particles are used as an adjuvant, the dosage of the antigen required by activating T cells is reduced,and simultaneously induces autophagy-mediated cross presentation and effective anti-tumor response.
The preparation method of the metallic aluminum nanoparticles can be divided into a physical method and a chemical method. The physical methods mainly include ball milling, electric explosion, photolithography, vapor phase evaporation, and the like. Generally, although the particle size of the aluminum particles prepared by the electron beam lithography technology is uniform and the purity is good, the electron beam lithography technology is only suitable for a small amount of preparation at present, and has high requirements on instruments and complex equipment. The electric explosion method is that by applying strong current to metal filament or foil, considerable energy is quickly accumulated in the filament or foil by the heating action of resistance, so that the filament or foil is subjected to phase change, accompanied by explosion sound and flash, and simultaneously, an evaporation zone containing metal particles with the particle size of about 100nm is formed, and nano particles are formed after condensation. The electric explosion method has the advantages of high production capacity and high product quality.
Since aluminum compounds are difficult to be reduced by common reducing agents, metallic aluminum is easy to be oxidized, and the environment for synthesizing the metallic aluminum needs to be strictly anhydrous and anaerobic, the chemical synthesis of aluminum nanoparticles is quite challenging. At present, the thermal decomposition method and the catalytic decomposition method are mostly focused. 1998, Haber et al (J.Am. chem. Soc.1998,120,10847) reflux in 1,3, 5-trimethylbenzene using N, N-dimethylethylamine in complex with aluminum hydride as precursor and titanium isopropoxide as catalyst to give nanoscale aluminum with average size of 40-180 nm, wherein the degree of aggregation of the particles depends mainly on the amount of catalyst used. In 2009, Meziani et al (ACS appl. mater. interfaces 2009,1,703) used 1-methylpyrrole and N, N-dimethylethylamine to form complexes with aluminum hydride, respectively, as precursors, and stabilized the particle surface using molecules with carboxylic acid functional groups as ligands, to prepare aluminum nanoparticles with clear boundaries and good dispersibility. In 2015, McClain et al (Nano Lett.2015,15,2751) used oleic acid as a ligand, and adjusted the ratio of tetrahydrofuran and 1, 4-dioxane as a solvent to primarily control the size of aluminum nanocrystals, so as to synthesize 70-220 nm high-purity dispersed aluminum nanoparticles. Due to the lack of proper ligand to stabilize the particle surface, the particle size distribution is not uniform, and the morphology is not easy to control. In 2018, Rugosong et al (J.Am.chem.Soc.2018,146 and 15412) firstly select bis-thioester terminated polystyrene (CDTB-PS) as a ligand to control the nucleation and growth processes of aluminum particles, and synthesize aluminum particle solutions with different colors. The nucleation quantity of the particles is adjusted by changing the dosage of the catalyst, so that the controllable synthesis of the aluminum nanoparticles with the size of 90-250 nanometers and good monodispersity is realized.
Therefore, on the premise that the effectiveness and safety of the aluminum salt adjuvant are acknowledged, the aluminum adjuvant is modified, so that the immune response of an organism to an antigen is further improved, the efficiency of inducing the cytotoxic T cell immune response is improved, the antigen storage effect of the cytotoxic T cell immune response is enhanced, and the immune stimulation effect is enhanced or improved, so that the adjuvant effect of the cytotoxic T cell immune response is improved, and the technical problem to be solved in the field is urgently needed.
Disclosure of Invention
The invention aims to overcome the defect of low cell immune response induction efficiency of the traditional aluminum salt adjuvant (such as aluminum hydroxide, aluminum phosphate and the like), and provides a metallic aluminum nano adjuvant, a vaccine composition, a preparation method and application thereof. The metallic aluminum nano adjuvant provided by the invention has controllable size, monodisperse particle size (the dispersion coefficient is less than or equal to 0.15) and stable colloid, and can be used as a candidate adjuvant of preventive vaccines and therapeutic vaccines for various diseases such as infection, autoimmune diseases, tumors and the like. The vaccine adjuvant provided by the invention is combined with antigen for application, so that the humoral immune response and the cellular immune response of the vaccine can be effectively enhanced, and the enhancement effect of the vaccine adjuvant is obviously better than that of a commercial aluminum hydroxide adjuvant.
The invention provides a vaccine adjuvant which comprises metal aluminum nanoparticles.
In the invention, the metal aluminum nano particles can be metal aluminum particles with the conventional particle size of nanometer (the particle size is less than or equal to 1000nm) in the field, and the common surface is provided with an amorphous alumina layer with the thickness of 3-5 nm.
In the present invention, the average particle diameter of the metallic aluminum nanoparticles is preferably 10 to 999nm, more preferably 50 to 300 nm; for example 88.85 nm. + -. 8.86nm, 147.14 nm. + -. 11.95nm, 139.76. + -. 42.81nm or 287.82 nm. + -. 24.13 nm.
In the present invention, the metal aluminum nanoparticles are preferably metal aluminum nanoparticles having a coefficient of variation of circumscribed circle diameter of not more than 0.21, for example, metal aluminum nanoparticles having a coefficient of variation of 0.09, a coefficient of variation of 0.08, or a coefficient of variation of 0.10.
In the present invention, it is preferable that the average particle diameter and the dispersion coefficient of the metal aluminum nanoparticles are as follows: 88.85nm plus or minus 8.86nm and a dispersion coefficient of 0.10; 147.14nm +/-11.95 nm, and has a dispersion coefficient of 0.09; alternatively, 287.82 nm. + -. 24.13nm with a coefficient of variation of 0.08.
In the invention, the metal aluminum nanoparticles are preferably size-controllable, monodisperse in particle size (dispersion coefficient is less than or equal to 0.15) and stable in colloid. Generally, the particle size of the metal aluminum nanoparticles can be determined by observing and measuring through an electron microscope, so that the metal aluminum nanoparticles with controllable size, monodisperse particle size and stable colloid can be obtained.
In the present invention, the metal aluminum nanoparticles may be prepared according to a conventional method in the art, such as an electrical explosion method or a ligand modification method.
The electric explosion method can be carried out according to conventional methods in the field, for example, in an inert environment, aluminum wires are gasified by using current to form aluminum vapor, and the aluminum vapor is condensed to obtain the metal aluminum nanoparticles.
The inert environment may be an argon environment.
The current conditions may be: the power supply capacitor is 96 muF, and the electrode discharge voltage is 4.0-4.4 kV.
The aluminum wire can be conventional in the field, such as aluminum wire with the diameter of 0.2mm and the purity of more than or equal to 95 percent.
Wherein the ligand regulation method can be performed according to the conventional method in the field, and preferably, the ligand regulation method comprises the following steps: reacting a ligand solution and a precursor solution in the presence of a titanium catalyst in an atmosphere with water content lower than 10ppm and oxygen content lower than 100 ppm;
the ligand is a polymer with a functional group containing a sulfur atom as a terminal group, and the polymerization degree of the ligand is 10-1000; the structural formula of the precursor is H3Al-X, wherein X is an organic molecule, and the organic molecule contains an atom which can be coordinated with aluminum and has a lone pair of electrons.
In the ligand regulation method, an atmosphere having an extremely low water content and oxygen content can be obtained by a method which is conventional in the chemical field, for example, by a glove box. The water content is preferably less than 1 ppm. The oxygen content is preferably below 50ppm, more preferably below 1 ppm.
In the ligand control method, the titanium catalyst may be an organic titanium catalyst capable of being dissolved in a reaction solvent, which is conventionally used in the art for such a reaction, and is preferably titanium tetraisopropoxide (Ti (i-PrO)4)。
In the ligand regulation method, the ligand is preferably
Figure BDA0002246999020000041
Wherein: r1Is C1-10Alkyl radical, C6-30Aryl or R1aSubstituted C6-30An aryl group; r2Is composed of
Figure BDA0002246999020000042
RaIs C1-10Alkyl or Ra1Substituted C1-10Alkyl radical, Ra1Is C6-30An aryl group; rbIs H or C1-10An alkyl group; rcIs C6-30Aryl radical, Rc1Substituted C6-30Aryl or
Figure BDA0002246999020000043
R1aAnd Rc1Each independently is C1-10Alkyl or halogen.
When R is1Is C1-10When alkyl, said C1-10The alkyl group is preferably C1-6Alkyl, more preferably C1-3Alkyl groups, such as methyl or isopropyl.
When R is1aIs C1-10When alkyl, said C1-10The alkyl group is preferably C1-6Alkyl, more preferably C1-3Alkyl groups, such as methyl or isopropyl.
When R isaIs C1-10Alkyl or Ra1Substituted C1-10When alkyl, said C1-10The alkyl group is preferably C1-6Alkyl, more preferably C1-3Alkyl groups, such as methyl or isopropyl.
When R isbIs C1-10When alkyl, said C1-10The alkyl group is preferably C1-6Alkyl, more preferably C1-3Alkyl groups, such as methyl or isopropyl.
When R is1Is C6-30Aryl or R of1aSubstituted C6-30Aryl is said to C6-30Aryl is preferably C6-10More preferably phenyl.
When R isa1Is C6-30Aryl is said to C6-30Aryl is preferably C6-10More preferably phenyl.
When R iscIs C6-30Aryl or Rc1Substituted C6-30Aryl is said to C6-30Aryl is preferably C6-10More preferably phenyl.
When R is1aAnd/or Rc1In the case of halogen, the halogen is preferably Br or Cl.
Ra1Substituted C1-10Alkyl is preferably phenyl-substituted C1-3Alkyl, more preferably
Figure BDA0002246999020000051
In the ligand regulation method, the degree of polymerization of the ligand is preferably 20 to 1000, more preferably 40 to 240, for example 42.
In the ligand regulation method, the PDI of the ligand is preferably 1 to 2, more preferably 1 to 1.51, for example 1.09.
In the ligand regulation and control method, the structural formula of the ligand can be shown as a formula (1), wherein Mn is 4.5kg/mol, n is 42, and PDI is 1.09. The preparation method may be a method conventionally used in the art, and preferably styrene, azobisisobutyronitrile and 2-phenyl-2-propylbenzodithio are reacted under anhydrous and anaerobic conditions, and for example, the method can be synthesized by the Journal of Polymer Science Part A, Polymer Chemistry 2001,39,1553, described in the following publications.
Figure BDA0002246999020000052
In the ligand regulation method, in the precursor, X is preferably an organic molecule containing an N atom or an O atom, and more preferably tertiary ammonia (NR)3) Or tetrahydrofuran, e.g.
Figure BDA0002246999020000053
Preferably, the precursor is
Figure BDA0002246999020000054
When the precursor is
Figure BDA0002246999020000055
When the method for producing the same comprises: and (3) dropwise adding 1-methylpyrrolidine into a toluene solution of lithium aluminum hydride and aluminum chloride, and reacting.
The precursor is
Figure BDA0002246999020000061
The preparation process of (a) generally maintains both oxygen and water contents below 1ppm and can be carried out, for example, in a glove box.
The precursor is
Figure BDA0002246999020000062
The mass concentration of the lithium aluminum hydride in the toluene solution of lithium aluminum hydride and aluminum chloride in the preparation process of (1) may be a mass concentration conventional in the art, and is preferably 20 to 1000mg/mL, more preferably 20 to 200mg/mL, for example 83.3 mg/mL. The mass concentration of the aluminum chloride in the toluene solution of lithium aluminum hydride and aluminum chloride can be conventional in the art, and is preferably 10-1000mg/mL, more preferably 10-200mg/mL, such as 91.8 mg/mL.
The precursor is
Figure BDA0002246999020000063
The molar ratio of the lithium aluminum hydride, the aluminum chloride and the 1-methylpyrrolidine in the preparation process of (4) can be the conventional molar ratio in the field, and is preferably (2-4):1 (0.8-1.5), more preferably (2-4):1Preferably 3:1: 1.
The precursor is
Figure BDA0002246999020000064
The parameters and conditions of the reaction in the preparation process of (a) may be those conventional in the art. The temperature of the reaction is generally room temperature, for example 20-30 ℃. The reaction time may be conventional in the art, and is preferably from 2 to 24 hours, for example 12 hours. The stirring speed during the reaction may be conventional in the art, preferably 300-1000rpm, more preferably 800 rpm.
The precursor is
Figure BDA0002246999020000065
In the production process of (3), it is preferable to subject the reaction solution obtained by the reaction to a post-treatment. The operation and conditions of the post-treatment are conventional in the field, and the post-treatment is carried out by general filtration, preferably, a funnel is used for filtering to remove impurities, and the obtained filtrate is filtered by an organic phase filter membrane. The pore size of the organic phase filtration membrane may be conventional in the art, preferably 0.01-1 μm, e.g. 0.22 μm. The organic phase filter membrane can be made of organic materials which are conventionally used as filter membranes in the field. The precursor obtained is generally stored in a glove box refrigerator at a low temperature of-10 ℃.
In the ligand regulation method, the concentration of the precursor in the reaction solution is preferably 15 to 500mM, more preferably 20 to 100mM, for example, 50mM or 80 mM.
When the concentration of the precursor is 15-100mM, the concentration of the titanium catalyst is preferably 0.1-1.5 mM; for example, when the concentration of the precursor is 80mM, the concentration of the titanium catalyst is 0.2 mM.
In the ligand regulation method, the concentration of the ligand solution can be a concentration conventional in the art, and preferably, the molar ratio of the ligand to the precursor is 3 (500- & gt 800), for example, 3: 800.
In the ligand regulation method, the concentration of the titanium catalyst solution can be a concentration conventional in the art, and preferably, the molar ratio of the titanium catalyst to the precursor is 1 (350-550), for example, 1: 400.
In the ligand regulation method, the molar ratio of the ligand, the precursor and the titanium catalyst can be a molar ratio which is conventional in the art, and is preferably 1 (60-520): 0.04-1.6, more preferably 1 (70-500): 0.0.5-1.5), such as 3:800: 2.
In the ligand regulation method, the solvent in the ligand solution and the precursor solution may be a solvent conventional in the art, preferably an aprotic solvent, and more preferably one or more of toluene, tetrahydrofuran, ether solvents, and the like. The aprotic solvent preferably has an oxygen content of less than 10ppm and a water content of less than 10 ppm.
In the ligand regulation method, the reaction is preferably carried out in a glove box.
In the ligand regulation method, the operation and conditions of the reaction may be those conventional in the art. The time of the reaction is preferably 10 minutes to 24 hours, for example 45 minutes, 1.5 hours or 4 hours. The temperature of the reaction is generally from room temperature to the boiling point of the solvent selected in the reaction mixture. For example, when the solvent in the reaction liquid is tetrahydrofuran, the temperature of the reaction is preferably 40 to 60 ℃, more preferably 50 ℃.
In the ligand regulation method, the operation of the reaction is preferably performed by the following steps: and adding the precursor solution and the titanium catalyst solution into the ligand solution in sequence for reaction.
In the ligand regulation method, the reaction is preferably carried out under stirring conditions, wherein the rotation speed of the stirring may be 50 to 3000rpm, preferably 500 rpm.
In the ligand regulation method, the reaction solution obtained by the reaction may be post-treated according to the conventional operations and conditions in the art, for example, after cooling to room temperature, the supernatant is removed by centrifugation, and the reaction solution is washed. The centrifugation conditions may be 8000rpm for 10 minutes. The washing condition can be that the solvent added into the reaction solution is used for shaking, washing and precipitating, and circulating for three times.
The invention also provides a preparation method of the vaccine adjuvant, which comprises the following steps of mixing the metal aluminum nano particles and the solvent A to prepare a vaccine adjuvant suspension.
The solvent a may be a solvent that is conventional in the art and can disperse the metallic aluminum nanoparticles and does not dissolve the metallic aluminum nanoparticles, and is preferably one or more of an alcohol solvent, an ether solvent, a ketone solvent, dimethyl sulfoxide, N-Dimethylformamide (DMF), and tetrahydrofuran, and more preferably N, N-dimethylformamide. The alcohol solvent is preferably methanol and/or ethanol.
In the vaccine adjuvant suspension, the concentration of the metal aluminum nanoparticles is preferably 0.1 to 100mg/mL, more preferably 0.1 to 20mg/mL, even more preferably 0.1 to 5mg/mL, for example, 2 mg/mL.
Wherein the mixing may be by means conventional in the art, such as ultrasonic dispersion. The time for ultrasonic dispersion may be 10-120min, for example 20 min.
In the invention, the vaccine adjuvant can not only induce humoral immunity, but also stimulate cellular immune response.
The invention also provides a vaccine composition, which comprises the vaccine adjuvant and an antigen or DNA encoding the antigen.
In the present invention, the antigen may be a substance that stimulates the body to generate a specific immune response, which is conventional in the art, or may be a small molecule substance (also referred to as a hapten) that alone cannot induce an immune response, but can acquire immunogenicity when it is cross-linked or conjugated with a carrier such as a macromolecular protein or non-antigenic polylysine. The antigen may be one or more of a short peptide, a polypeptide and a protein.
The short peptide can be conventional in the art, and generally refers to a short peptide consisting of 3-9 amino acid residues, such as antigenic peptide OT-1 of ovalbumin OVA, antigenic glycoprotein gp100 of melanoma cells, or apoptosis inhibitor protein survivin/birc 5-1.
The sequence of the antigen peptide OT-1 can be SIINFEKL.
The molecular weight of the antigenic peptide OT-1 may be 963.14 g/mol.
The polypeptide may be a polypeptide conventional in the art, and generally refers to a compound formed by dehydration condensation of a 10-100 amino acid molecule, such as telomerase activity catalytic unit TERT, or "T cell recognition melanoma antigen MART-1, MOG35-55, PADRE, Trp2 or survivin/birc 5-2".
The telomerase activity catalytic unit TERT can have a sequence of DLQPYMGQFLKHLQDSDASALRNSVVI.
The telomerase activity catalytic unit TERT can have a molecular weight of 3046.70 g/mol.
The protein can be a protein conventional in the art, and generally refers to a substance with a certain spatial structure formed by a polypeptide chain formed by amino acids in a dehydration condensation mode through a convoluted fold, such as ovalbumin OVA.
The molecular weight of the OVA may be 298.4 g/mol.
In the present invention, the mass ratio of the metal aluminum nanoparticle to the antigen is preferably 2 (0.1-10), such as 2 (1-3), and further such as 2:3 or 1: 1.
When the antigen is a short peptide and/or a polypeptide, the mass ratio of the metal aluminum nanoparticle to the antigen is preferably 1: 1.
When the antigen is a protein, the mass ratio of the metal aluminum nanoparticle to the antigen is preferably 2: 3.
The invention also provides a preparation method of the vaccine composition, which comprises the following steps: and mixing the vaccine adjuvant with a solution containing the antigen, and carrying out incubation reaction.
In the present invention, the solution containing the antigen may be prepared by a method conventional in the art, for example, by mixing the antigen with the solvent B.
Wherein, the solvent B can be a solvent which is conventional in the art and can be used for dissolving the antigen, and is preferably water, PBS buffer solution, DMF or an alcohol solvent, and is more preferably PBS buffer solution or DMF.
In the present invention, the concentration of the antigen in the solution containing the antigen is preferably 0.01 to 10mg/mL, more preferably 0.1 to 5mg/mL, for example, 2.0mg/mL or 3.0 mg/mL.
When the vaccine adjuvant is mixed with the antigen-containing solution in the form of the vaccine adjuvant suspension, it is preferable that the solvent a and the solvent B are compatible.
Wherein, preferably, when the antigen is a short peptide and/or a polypeptide, the solvent A and the solvent B are the same; for example, the solvent A is DMF and the solvent B is DMF.
Wherein, preferably, when the antigen is a protein, the solvent a and the solvent B are different; for example, the solvent A is DMF, and the solvent B is PBS buffer.
Preferably, the solution containing the antigen is added into the vaccine adjuvant suspension, and after ultrasonic dispersion, a cultivation reaction is performed. This mixing sequence enables more uniform antigen coating.
In the present invention, the operation and conditions of the incubation reaction may be those conventional in the art, such as ultrasonic dispersion followed by incubation. The time for ultrasonic dispersion may be 5-120min, for example 10-120min, preferably 30 min.
In the present invention, the incubation reaction may be carried out in a shaker.
In the present invention, it is preferred that the incubation reaction is carried out at 80-800rpm, preferably 240-320rpm, for example 320 rpm.
In the present invention, the incubation reaction time may be 1-24h, for example 12 h.
In the present invention, the temperature of the incubation reaction is generally room temperature, for example, 20 to 30 ℃.
In the present invention, after the incubation reaction, the vaccine composition may be centrifuged to remove the supernatant, as is conventional in the art.
Wherein the operation and conditions of the centrifugation may be those conventional in the art. The rotation speed of the centrifugation is preferably 3000-15000rpm, more preferably 5000-15000rpm, for example 6000rpm or 12000 rpm. The time of the centrifugation is preferably 5-20min, for example 10 min.
The invention also provides a freeze-dried vaccine which comprises the metal aluminum nano particle and the antigen.
Preferably, the freeze-dried vaccine is prepared by mixing the vaccine composition with water and freeze-drying.
The invention also provides application of the metal aluminum nanoparticles as a vaccine adjuvant in a vaccine.
Wherein the metallic aluminum nanoparticles are as described above.
On the basis of the common knowledge in the field, the above preferred conditions can be combined randomly to obtain the preferred embodiments of the invention.
The reagents and starting materials used in the present invention are commercially available.
The positive progress effects of the invention are as follows:
(1) the invention provides a vaccine adjuvant prepared by taking metal aluminum nanoparticles as a raw material, and the vaccine adjuvant and an antigen are combined for application, so that the humoral immune response and the cellular immune response of a vaccine can be effectively enhanced, and the enhancement effect of the vaccine adjuvant is obviously better than that of a commercial aluminum hydroxide adjuvant.
(2) The metal aluminum nanometer adjuvant has good stability and safety, and can be used as a candidate adjuvant of preventive vaccines and therapeutic vaccines for various diseases such as infection, autoimmune diseases, tumors and the like.
Drawings
FIG. 1 shows the precursor H of example 23Nuclear magnetic resonance hydrogen spectrum (deuterated benzene) of Al (1-MP).
FIG. 2 shows the precursor H of example 23Nuclear magnetic resonance aluminum spectrum (deuterated benzene) of Al (1-MP).
FIG. 3 shows the NMR spectrum (deuterated chloroform) of ligand (2-phenyl-2-propylbenzodithio) terminated polystyrene (CDTB-PS) in example 2.
FIG. 4 is a permeation gel chromatogram (GPC) of ligand (2-phenyl-2-propylbenzodithio) terminated polystyrene (CDTB-PS) of example 2.
FIG. 5 shows the Th2 antibody subtype Ig G1 titer after AlNPs-OVA induction in example 1.
FIG. 6 shows the Th1 antibody subtype Ig G2a titer after AlNPs-OVA induction in example 1.
FIG. 7 is the percentage of the number of antigen-specific T cells in draining lymph node cells after AlNPs-OVA induction in effect example 2.
FIG. 8 is a graph showing the percentage of the number of antigen-specific T cells in splenocytes after AlNPs-OVA induction in effect example 2.
FIG. 9 is a graph showing the effect of Al-OVA on tumor growth in example 3.
FIG. 10 is a graph showing the effect of Al-OVA on tumor growth in example 4.
FIG. 11 is a graph showing the effect of Al-OT-1 on the prevention of tumor growth in example 5.
FIG. 12 is a graph showing the effect of Al-Tert on tumor growth in example 6.
Detailed Description
The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.
In the following examples and comparative examples:
ovalbumin OVA was purchased from sigma-aldrich and has a molecular weight of 298.4 g/mol;
short peptides or polypeptides were purchased from gill biochemical (shanghai) ltd, for the following specific information:
polypeptide Tert (DI-27): the sequence is DLQPYMGQFLKHLQDSDASALRNSVVI, the molecular weight is 3046.70 g/mol;
short peptide OT-1: the sequence is SIINFEKL, and the molecular weight is 963.14 g/mol.
EXAMPLE 1 preparation of metallic aluminum nanoparticles by electric explosion method
Fixing an aluminum wire with the diameter of 0.2mm and the purity of more than or equal to 95 percent between high-voltage electrodes in an argon environment, continuously supplying power by a mechanical device, wherein the power supply capacity is 96 mu F, the electrode discharge voltage is 4.0-4.4kV, heating the aluminum wire under high-density current and enabling the aluminum wire to radially expand, vaporizing the aluminum wire to form aluminum vapor, then condensing to form aluminum particles with the average particle size of 139.76 +/-42.81 nm, and collecting in a collector filled with argon. (see the literature: Passivation Process for Superfine Aluminum Powders infected by electric amplification of Wires. appl. Sun. Sci.2003,211, 57-67.)
Example 2 preparation of Metal-aluminum nanoparticles by ligand Regulation
(1) Precursor H3Synthesis and characterization of Al (1-MP)
In a glove box (oxygen and water content below 1ppm), 3.748g of lithium aluminum hydride and 4.132g of aluminum chloride were added to a flask containing 45mL of anhydrous toluene. Under vigorous stirring (stirring speed 800rpm), 11.65mL of 1-methylpyrrolidine (1-methylpyrrolidine) was added dropwise. Wherein the molar ratio of the lithium aluminum hydride to the aluminum chloride to the 1-methylpyrrolidine is 3:1: 1. After overnight reaction at room temperature, the reaction solution was filtered through a funnel to remove solid impurities. For further purification, the obtained filtrate is filtered again by an organic phase filter membrane with the pore diameter of 0.22 mu m, and the obtained filtrate is H3Al (1-MP) (structural formula is
Figure BDA0002246999020000111
Yield 90%) and stored cryogenically in a refrigerator in a glove box at-10 ℃. The concentration can be calibrated by nuclear magnetic resonance hydrogen spectrum.
FIG. 1 shows the precursor H of example 23Nuclear magnetic resonance hydrogen spectrum (deuterated benzene) of Al (1-MP).1H NMR(C6D6):δ4.13(s,br,3H,H3Al),2.38(s,4H,N(CH2CH2)2),2.02(s,3H,NCH3),1.37(m,4H,N(CH2CH2)2)；27Al NMR(C6D6):δ140.87(s,br)。
FIG. 2 shows the precursor H of example 23Nuclear magnetic resonance aluminum spectrum (deuterated benzene) of Al (1-MP).27Al NMR(C6D6):δ140.87(s,br)。
(2) Synthesis and characterization of ligand (2-phenyl-2-propylbenzodithio) terminated polystyrene (CDTB-PS)
90.90g of styrene, 0.0576g of azobisisobutyronitrile and 0.4107g of 2-phenyl-2-propylbenzodithiol (CDTB) were introduced into a Schelenk flask. After three times of liquid nitrogen freezing, vacuumizing and thawing treatment, the mixed solution is stirred and reacts in an oil bath at 60 ℃ for 12 hours, and then the reaction solution is cooled to room temperature. Most of the unreacted styrene was then removed by rotary evaporator. Finally, the process is carried out in a batch,the reaction solution was precipitated and washed with methanol and centrifuged by ultrasound three times to remove the reactant. And (3) placing the product in a vacuum oven at 120 ℃ for 1 day, and placing the final solid product in a glove box refrigerator for low-temperature storage. Molecular weight of the resulting product (M)n) About 4.5kg/mol, and a dispersibility index (PDI) of 1.09. The product was 7.64g, 8.5% yield. The structure is as follows:
Figure BDA0002246999020000121
(n is 42).
FIG. 3 shows the NMR spectrum (deuterated chloroform) of ligand (2-phenyl-2-propylbenzodithio) terminated polystyrene (CDTB-PS) in example 2.1H NMR：CDTB-PS(Mn＝4.5kg/mol)(CDCl3):δ7.85(br,-S-CS-C6H5),6.37-7.31(br,5H,Ph),1.85(br,1H,CHCH2),1.37(br,2H,CHCH2)。
FIG. 4 is a permeation gel chromatogram (GPC) of ligand (2-phenyl-2-propylbenzodithio) terminated polystyrene (CDTB-PS) of example 2.
(3) Synthesis and characterization of aluminum nanoparticles
Preparing a solution:
preparing an anhydrous THF solution with the CDTB-PS concentration of 20mM, roughly adding a little excessive CDTB-PS according to the molecular weight addition, and quantitatively calibrating the accurate concentration by ultraviolet.
Preparation H3The preparation method of the anhydrous THF solution with the Al (1-MP) concentration of 1M comprises the following steps: according to nuclear magnetism of liquid1The relation of the integral area of the specific peak of H can determine the prepared H3Concentration of Al (1-MP) solution (usually greater than 1M), calculated by adding a specific volume of THF, 1M H concentration is obtained3Al (1-MP) anhydrous THF solution.
Preparing Ti (i-PrO)4An anhydrous THF solution having a concentration of 100mM, Ti (i-PrO) was taken out by balance4A specified volume of THF was added.
In a glove box (oxygen and water content below 1ppm) to 4.425mL of anhydrous Tetrahydrofuran (THF) was added 75 μ L of 20mM CDTB-PS in anhydrous THF, heated and stabilized to 50 ℃.Under vigorous stirring (500rpm), 400. mu.L of 1M H was added3Al (1-MP) in anhydrous THF and 100. mu.L of 10mM Ti (i-PrO)4Anhydrous THF solution. Wherein, the ligand, the precursor and Ti (i-PrO)4In a molar ratio of 3:800: 2; after completion of the charging, H was added to the whole reaction solution (solvent: 5.0mL)3The concentration of the Al (1-MP) precursor was 80mM, and the concentration of the titanium catalyst was 0.2 mM. Reacting at 50 ℃ under vigorous stirring (the rotating speed is 500rpm), cooling the reaction liquid to room temperature within 45 minutes, 1.5 hours and 4 hours respectively, centrifuging the reaction liquid at 8000rpm for 10 minutes, removing supernatant, adding equivalent anhydrous THF, shaking, washing and precipitating, and circulating for three times to obtain particles with the size of 88.85nm +/-8.86 nm and the dispersion coefficient of 0.10 respectively; 147.14nm +/-11.95 nm, and has a dispersion coefficient of 0.09; 287.82nm +/-24.13 nm and a dispersion coefficient of 0.08.
Example 3
Mixing the electric explosion metal aluminum nano particles with OVA to prepare the vaccine AlNPs-OVA. The method comprises the following specific steps:
(1) the electrically exploding metal aluminum nanoparticles prepared in example 1 were dissolved in DMF (DMF) at a concentration of 2.0 mg/mL) in a solution of 0.1-5.0mg/mL, and ultrasonically dispersed for 20 minutes.
(2) Preparing 0.1-5.0mg/mL egg white albumin OVA PBS solution, wherein the concentration of the egg white albumin OVA in the implementation is 3.0 mg/mL.
(3) Mixing the PBS solution of ovalbumin OVA and the DMF solution of nano aluminum particles according to the volume of 1:1, ultrasonically dispersing for half an hour, transferring to a shaking table, and shaking at 320rpm at room temperature for 12 hours. After stopping shaking, the mixture was centrifuged at 12000rpm for 10 minutes, and the supernatant was removed to freeze-dry the sample, and the remaining small amount of the solvent was removed.
Example 4
287.82nm metal aluminum nano particles are mixed with OVA to prepare 287.82nm Al-OVA vaccine. The method comprises the following specific steps:
the same procedure as in example 3 was repeated except for using 287.82 nm. + -. 24.13nm aluminum particles having a dispersion coefficient of 0.08 obtained in example 2.
Example 5
147.14nm metal aluminum nano particles are mixed with OVA to prepare 147.14nm Al-OVA vaccine. The method comprises the following specific steps:
the same procedure as in example 3 was repeated except for using 147.14 nm. + -. 11.95nm aluminum particles having a dispersion coefficient of 0.09 prepared in example 2.
Example 6
Mixing 88.85nm metal aluminum nano particles with OVA to prepare 88.85nm Al-OVA vaccine. The method comprises the following specific steps:
the same procedure as in example 3 was repeated except for using 88.85 nm. + -. 8.86nm of the aluminum particles prepared in example 2 and having a dispersion coefficient of 0.10.
Example 7
147.14nm metal aluminum nano particles are mixed with short peptide OT-1 to prepare the vaccine 147.14nm Al-OT-1. The method comprises the following specific steps:
(1) the 147.14 nm. + -. 11.95nm metal aluminum nanoparticles with a dispersion coefficient of 0.09 prepared in example 2 were mixed to 0.1-5.0mg/mL (DMF was used as solvent), the concentration of the metal aluminum nanoparticles in this example was 2.0mg/mL, and the mixture was ultrasonically dispersed for 20 minutes.
(2) DMF solution with the concentration of 0.1-5.0mg/mL OT-1 is prepared, and the concentration of OT-1 in the present embodiment is 2.0 mg/mL.
(3) Mixing the OT-1 solution and the metal aluminum nanoparticle solution according to the volume ratio of 1:1, ultrasonically dispersing for half an hour, transferring to a shaking table, and shaking at 320rpm at room temperature for 12 hours. After stopping shaking, centrifugation was carried out at 6000rpm for 10 minutes, the supernatant was removed and the sample was lyophilized to remove the remaining small amount of solvent.
Example 8
147.14nm metal aluminum nano-particles are mixed with polypeptide Tert (DI-27) to prepare the vaccine 147.14nm Al-Tert.
The operation and conditions of this example were the same as those of example 7 except that the following conditions were used:
DMF solution with the concentration of 2.0mg/mL Tert (DI-27) is prepared and mixed with the metal aluminum nano particle solution.
Comparative example 1
Mixing aluminum hydroxide nanoparticles with OVA to prepare vaccine (nano Al (OH)3-OVA)。
The preparation method of the aluminum hydroxide nano-particles comprises the following steps: 5ml of 3.6mg/ml aluminum chloride hexahydrate AlCl is added into a 20ml white bottle in sequence3·6H2O, 5ml of 0.04M NaOH solution, followed by adjustment of the reaction solution pH to 7 with 0.01M NaOH. After stirring at room temperature for 20 minutes, the supernatant was removed by centrifugation at 8000 rpm. After washing with ultrapure water for 2 times, the mixture was drained, and DMF was added to the mixture to prepare a solution having a concentration of 2.0 mg/ml. The mean particle size was determined by TEM to be 150.35. + -. 32.84 nm.
Vaccine nano Al (OH)3The preparation method of OVA is as follows: the vaccine was prepared by mixing the aluminium hydroxide nanoparticles with OVA, and the rest of the conditions were the same as in example 3.
Comparative example 2
Preparation of vaccine (Al (OH) Using commercial aluminum hydroxide gel adjuvant in combination with OVA3-OVA)。
Specifically, the method comprises the following steps: vaccines were prepared by mixing OVA with aluminium hydroxide gel adjuvant (available from Thermo Scientific, particle size 4-10 μm) and the rest of the conditions were the same as in example 3.
Effect example 1 humoral immune response
The lyophilized vaccines prepared in example 3, comparative example 1 and comparative example 2 were tested for their activity of stimulating humoral immune response.
The experimental conditions are as follows:
1. animal immunization:
female C57BL/6 mice at 7 weeks were randomly divided into 5 groups of 3 mice each. OVA is taken as antigen, and the groups are respectively 2.5mg/ml OVA group and 10mg/ml Al (OH)3OVA group, 10mg/ml nano Al (OH)3Mice were given subcutaneous injections of 100. mu.L each at day 0 and day 7 in the right inguinal region on OVA group, 10mg/ml AlNPs-OVA group and control PBS group, and serum was collected from the eye at day 14 for Ig G1 and Ig G2a tests.
2. Mouse serum preparation:
1) eye picking and blood sampling: grabbing double ears and skin behind the neck of a mouse by a left thumb and a forefinger, and fixing the tail of the mouse by a little finger; pressing the left forelimb of the mouse on the sternum and the heart with the middle finger, pressing the ring finger on the abdomen, twirling the thumb, and pressing the eye skin on the blood side with the middle finger to make the eyeball congestion and protrusion; thirdly, taking the eyeballs by using elbow tweezers; twisting the thumb and the index finger according to the needs to make the blood vertically flow into the centrifuge tube from the orbit at different speeds; lightly pressing the heart part of the mouse with the middle finger of the left hand to accelerate the blood pumping speed of the heart; when the blood has run out, the mice are sacrificed by dislocation.
2) Separating serum: placing blood in a centrifuge tube at room temperature for 2 hours; ② 3 hours in a refrigerator at 4 ℃; ③ centrifuging at 4000rpm for 10 minutes after the blood coagulation blood clot shrinks; fourthly, the supernatant is taken out and stored in a clean EP tube at the temperature of minus 80 ℃ for standby.
ELISA detection of specific antibody subtypes:
the detection is carried out according to the instructions of the Mouse Anti-Ovalbumin IgG2a ELISA Kit,96tests and the Quantitative Kit produced by Alpha Diagnostic International and the Mouse Anti-Ovalbumin IgG1 ELISA Kit,96tests and the Quantitative Kit.
The specific experimental results can be seen in table 1, fig. 5 and fig. 6. Wherein the antibody titer value is an OD 450nm value measured by an enzyme-labeling instrument (EXL-800; Bio-Tek, Winooski, VT, USA).
TABLE 1
Figure BDA0002246999020000151
Note: p is less than 0.05, P is less than 0.01, and the AlNPs-OVA group has significant difference with other four groups.
As can be seen from table 1, fig. 5 and fig. 6:
the ability of AlNPs-OVA as vaccine to induce the generation of Th2 antibody subtype Ig G1 and Th1 antibody subtype Ig G2a is obviously higher than that of other four groups.
Effect example 2 cellular immune response
The lyophilized vaccines prepared in example 3, comparative example 1 and comparative example 2 were taken and tested for their activity of stimulating cellular immune responses.
The experimental conditions are as follows:
1. animal immunization:
female C57BL/6 mice at 7 weeks were randomly divided into 5 groups of 2 mice each. OVA is taken as antigen, and the groups are respectively 2.5mg/ml OVA group and 10mg/ml Al (OH)3Group OVA、10mg/ml nano Al(OH)3The OVA group, the 10mg/ml AlNPs-OVA group and the control group PBS group are respectively given to mice on the 0 th day and the 7 th day for subcutaneous injection immunization of the right inguinal region, each time of 100 mu L, and the mice are killed by cervical vertebra removal on the 14 th day, and spleen and drainage lymph node are taken for flow detection.
2. Mouse splenocyte preparation
1) Removing cervical vertebrae to kill mice, taking spleen to a 60mm dish containing 5mL PBS, grinding and dispersing cells, filtering the cells into a 15mL centrifuge tube through a 200-mesh filter screen, centrifuging the cells for 5min at 1200rpm and 4 ℃, and removing supernatant;
2) adding 1mL of erythrocyte lysate, standing at 4 ℃ for 15min, shaking every 5min, adding 10mL of PBS for termination, centrifuging at 1200rpm at 4 ℃ for 5min, discarding the supernatant, resuspending spleen cells by 1mL of FACS, and counting.
3. Mouse draining lymph node preparation
The mice were sacrificed by cervical dislocation, the right inguinal lymph node was taken to a 60mm dish containing 5mL PBS, dispersed cells were ground, filtered through a 200 mesh screen into a 15mL centrifuge tube, centrifuged at 1200rpm at 4 ℃ for 5min, the supernatant was discarded, and 1mL FACS (FACS means a PBS solution containing 0.1% bovine serum albumin) was counted after resuspension of lymphocytes.
4. Flow detection of antigen-specific T lymphocytes
Take 1X 106Spleen/lymph node cells were subjected to flow tube administration of CD45-Pacific Blue, CD3-PE-Dazzle-594, CD4-BV421, CD8-PE-Cy5, CD19-BV650, CD11c-APC-Cy7, CD11b-BV711, Anti-SIINFEKL-H-2kb-PE flow antibody staining (all antibodies were purchased from BD Co., USA (Becton, Dickinson and Company)), FACS staining at 4 ℃ for 30min in the dark, 3ml (FACS refers to a 0.1% bovine serum albumin in PBS solution), centrifugation at 1200rpm and 4 ℃ for 5min, supernatant was discarded, cells were vortexed, and assayed by Cytek Aurora flow analyzer.
The results of the specific experiments are shown in tables 2-3, FIG. 7 and FIG. 8. Wherein, the percentage of the antigen-specific T cells is the percentage of the number of the cells obtained by analyzing the data by using Flowjo software after the Cytek Aurora flow analyzer detects the percentage of the antigen-specific T cells.
TABLE 2 mouse spleen cell and draining lymph node cell counts
Group of Draining lymph node cell/1X 106Cells Spleen cells/1X 106Cells
PBS 1.85±0.28 74.00±9.19
OVA 2.76±0.83 69.5±10.61
Al(OH)3-OVA 8.61±2.25 151.75±34.29
nano Al(OH)3-OVA 1.70±0.78 133.5±45.25
AlNPs-OVA 13.65±2.65 109.75±8.84
As shown in Table 2, the ability of AlNPs-OVA as a vaccine to induce generation of spleen cells and draining lymph node cells was significantly higher than that of the other four groups.
TABLE 3 percentage of antigen-specific T cells in draining lymph node cells and spleen cells of mice
Figure BDA0002246999020000171
Note: p is less than 0.05, P is less than 0.01, and the AlNPs-OVA group has significant difference with other four groups.
As can be seen from table 3, fig. 7 and fig. 8, the ability of AlNPs-OVA as a vaccine to induce the generation of antigen-specific T lymphocytes was significantly higher than that of the other four groups.
Effect example 3
The lyophilized vaccines prepared in examples 4 and 5 were tested for their ability to stimulate an immune response.
The experimental conditions are as follows:
female C57BL/6 mice, 8 weeks, purchased from Wittisley, Beijing;
the samples were randomly divided into 5 groups of 4 each, namely PBS group, OVA protein group (160. mu.g OVA/OVA), metallic aluminum nanoparticle group (1 mg/OVA, wherein 1mg metallic aluminum nanoparticles consist of 0.5mg 287.82nm Al and 0.5mg147.14nm Al, 287.82nm Al-OVA group (1mg Al/160. mu.g OVA/OVA), 147.14nm Al-OVA group (1mg Al/160. mu.g OVA/OVA).
Mice were immunized by intradermal injection into the left dorsal side on days 0 and 7, at 100. mu.l/mouse/time.
Day 14 mice were inoculated subcutaneously on the right back of the mice at 5X 105B16F10 cells/mouse.
Tumor measurements were taken every 2 days starting on day 21, tumor volume ═ length × width/2.
The specific experimental results can be seen in table 4 and fig. 9.
TABLE 4
Group of Days after tumor inoculation/d Tumor volume/mm3
PBS 26 974.15±351.57
OVA(Free-OVA) 26 886.91±431.86
Metal aluminium nanoparticles (Free-Al) 26 551.36±481.76
287.82nm Al-OVA 26 44.71±18.16*
147.14nm Al-OVA 26 21.63±15.58*
Note: p is less than 0.05, the 147.14nm Al-OVA group is significantly different from the PBS group and the Free-Al group, and the 287.82nm Al-OVA group is significantly different from the PBS group and the Free-Al group.
As can be seen from table 4 and fig. 9, the aluminum metal nano protein vaccine can significantly slow down the growth of tumor in mice, and the prevention effect is superior to other three groups.
Effect example 4
The lyophilized vaccines prepared in example 5 and comparative example 2 were taken and tested for their ability to stimulate an immune response.
The experimental conditions are as follows:
female C57BL/6 mice, 8 weeks, purchased from Wittisley, Beijing;
right dorsal subcutaneous inoculation 1X 10 on day 06B16F10 cells/mouse.
Randomly divided into 4 groups of 2 each, PBS group, OVA protein group (100. mu.g OVA/protein), commercial aluminum hydroxide gel group (1.332mg Al (OH)3100. mu.g OVA/mouse), 147.14nm Al-OVA group (0.23mg Al/100. mu.g OVA/mouse).
Mice were injected subcutaneously in the right groin every 3 days, 6 times every 3 days, 100 μ l/mouse/time, starting on day 7.
Tumor measurements were taken every 2 days starting on day 7, tumor volume ═ length × width/2.
The specific experimental results can be seen in table 5 and fig. 10.
TABLE 5
Group of Days after tumor inoculation/d Tumor volume/mm3
PBS 16 727.47±132.65
OVA(Free-OVA) 16 467.39±88.51
Al(OH)3-OVA 16 144.50±31.94**
147.14nm Al-OVA 16 37.75±16.91**
Note: p < 0.01, 147.14nm Al-OVA group and PBS group, Free-OVA group have significant difference, Al (OH)3There was a significant difference between the OVA group and the PBS group.
As shown in Table 5 and FIG. 10, the aluminum metal nano protein vaccine can significantly slow down the growth of mouse tumor, and the therapeutic effect is better than other three groups.
Effect example 5
The lyophilized vaccines prepared in example 7 and comparative example 2 were tested for their ability to stimulate an immune response.
The experimental conditions are as follows:
female C57BL/6 mice, 8 weeks, purchased from Wittisley, Beijing;
randomly divided into 5 groups of 3, PBS group, 147.14nm Al group (0.8 mg/piece), OT1 short peptide group (50 μ g OT 1/piece), and commercial aluminum hydroxide gel group (0.8mg Al (OH)350 μ g OT 1/mouse), 147.14nm Al-OVA group (0.8mg Al/50 μ g OVA/mouse).
Mice were immunized subcutaneously in the right groin on days 0 and 7 at 100. mu.l/mouse.
Day 14 mice were inoculated subcutaneously on the right back of the mice at 1X 106B16F10 cells/mouse.
Tumor measurements were taken every 2 days starting on day 25, tumor volume ═ length × width/2.
The specific experimental results can be seen in table 6 and fig. 11.
TABLE 6
Figure BDA0002246999020000191
Figure BDA0002246999020000201
Note: p < 0.05, the significant difference exists between the Al-OT1 group with 147.14nm < 0.001 and Al-OT1 group with PBS group and Free-Al group, and Al (OH)3There was a significant difference between the-OT 1 group and the PBS group, and a significant difference between the OT1(Free-OT1) group and the PBS group.
As shown in Table 6 and FIG. 11, the aluminum nano-short peptide vaccine can delay the growth of the tumor in mice, and the tumor volume is reduced compared with other four groups.
Effect example 6
The lyophilized vaccine prepared in example 8 was tested for its ability to stimulate an immune response.
The experimental conditions are as follows:
female C57BL/6 mice, 8 weeks, purchased from Wittisley, Beijing;
the samples were randomly divided into 4 groups of 3, 3 each, PBS group, 147.14nm Al group (2.5mg Al/one), Tert (DI-27) polypeptide group (250. mu.g Tert (DI-27)/one), 147.14nm Al-Tert (DI-27) group (2.5mg Al/250. mu.g Tert (DI-27)/one).
Mice were immunized subcutaneously in the right groin on days 0 and 7 at 100. mu.l/mouse.
Day 14 mice were inoculated subcutaneously on the right back of the mice at 5X 105B16F10 cells/mouse.
Tumor measurements were taken every 2 days starting on day 21, tumor volume ═ length × width/2.
The specific experimental results can be seen in table 7 and fig. 12.
TABLE 7
Group of Days after tumor inoculation/d Tumor volume/mm3
PBS 21 748.43±159.59
Metal aluminium nanoparticles (Free-Al) 21 722.58±97.72
Tert(Free-Tert) 21 604.92±167.18
147.14nm Al-Tert 21 134.49±38.63**
Note: p < 0.01, significant differences were found between the 147.14nm Al-tart group and the PBS, Free-Al, and tart (Free-tart) groups.
As can be seen from Table 7 and FIG. 12, the aluminum metal nano-polypeptide vaccine can significantly delay the growth of mouse tumors, and the prevention effect is superior to the other three groups.

Claims (10)

1. A vaccine adjuvant characterised in that it comprises metallic aluminium nanoparticles.
2. The vaccine adjuvant according to claim 1, wherein the metallic aluminum nanoparticles have an average particle size of 10-999nm, preferably 50-300nm, such as 88.85nm ± 8.86nm, 147.14nm ± 11.95nm, 139.76 ± 42.81nm or 287.82nm ± 24.13 nm;
and/or the metal aluminum nano particles are metal aluminum nano particles with the diameter of the circumscribed circle and the dispersion coefficient less than or equal to 0.21.
3. The vaccine adjuvant according to claim 1 or 2, wherein the metallic aluminum nanoparticles are prepared by an electrical explosion method or a ligand regulation method, wherein:
preferably, the electrical explosion method comprises the steps of: in an inert environment, gasifying the aluminum wire by using current to form aluminum steam, and condensing to obtain metal aluminum nano particles;
preferably, the ligand modulation method comprises the following steps: reacting a ligand solution and a precursor solution in the presence of a titanium catalyst in an atmosphere with water content lower than 10ppm and oxygen content lower than 100 ppm; the ligand is a polymer with a functional group containing a sulfur atom as a terminal group, and the polymerization degree of the ligand is 10-1000; the structural formula of the precursor is H3Al-X, wherein X is an organic molecule, and the organic molecule contains an atom which can be coordinated with aluminum and has a lone pair of electrons.
4. The vaccine adjuvant of claim 3 wherein in the ligand modulation method, the titanium catalyst is titanium tetraisopropoxide;
and/or, in the ligand regulation method, the ligand is
Figure FDA0002246999010000011
Wherein: r1Is C1-10Alkyl radical, C6-30Aryl or R1aSubstituted C6-30An aryl group; r2Is composed of
Figure FDA0002246999010000012
RaIs C1-10Alkyl or Ra1Substituted C1-10Alkyl radical, Ra1Is C6-30An aryl group; rbIs H or C1-10An alkyl group; rcIs C6-30Aryl radical, Rc1Substituted C6-30Aryl or
Figure FDA0002246999010000013
R1aAnd Rc1Each independently is C1-10Alkyl or halogen;
and/or, in the ligand regulation method, the polymerization degree of the ligand is 20-1000, preferably 40-240;
and/or, in the ligand regulation method, the PDI of the ligand is 1-2, preferably 1-1.51;
or in the ligand regulation and control method, the structural formula of the ligand is shown as a formula (1), wherein Mn is 4.5kg/mol, n is 42, and PDI is 1.09;
Figure FDA0002246999010000021
and/or, in the ligand regulation method, in the precursor, X is an organic molecule containing N atom or O atom, preferably tertiary ammonia (NR)3) Or tetrahydrofuran;
and/or, in the ligand regulation method, the concentration of the precursor in the reaction solution is 15-500mM, preferably 20-100 mM;
and/or in the ligand regulation method, the molar ratio of the ligand to the precursor is 3 (500- & lt800- & gt), and preferably 3: 800;
and/or in the ligand regulation method, the molar ratio of the titanium catalyst to the precursor is 1 (350-550), preferably 1: 400;
and/or in the ligand regulation method, the molar ratio of the ligand, the precursor and the titanium catalyst is 1 (60-520) to (0.04-1.6), preferably 1 (70-500) to (0.0.5-1.5);
and/or in the ligand regulation method, the solvent in the ligand solution and the precursor solution is an aprotic solvent, preferably one or more of toluene, tetrahydrofuran, ether solvents and the like.
5. A method for preparing the vaccine adjuvant according to any one of claims 1 to 4, comprising the steps of mixing the metallic aluminum nanoparticles with a solvent A to prepare a vaccine adjuvant suspension; wherein
The solvent A is preferably one or more of an alcohol solvent, an ether solvent, a ketone solvent, dimethyl sulfoxide, N-dimethylformamide and tetrahydrofuran, and is more preferably N, N-dimethylformamide; the alcohol solvent is preferably methanol and/or ethanol;
in the vaccine adjuvant suspension, the concentration of the metal aluminum nanoparticles is preferably 0.1-100mg/mL, more preferably 0.1-20mg/mL, and further preferably 0.1-5mg/mL, for example 2 mg/mL;
the mixing mode is preferably ultrasonic dispersion, and the time of ultrasonic dispersion is preferably 10-120 min.
6. A vaccine composition comprising the vaccine adjuvant of any one of claims 1-4, and an antigen or DNA encoding the antigen; wherein:
the antigen is preferably one or more of a short peptide, a polypeptide and a protein.
7. The vaccine composition according to claim 6, wherein the short peptide is an antigenic peptide OT-1 of ovalbumin OVA, an antigenic glycoprotein gp100 of melanoma cells, or an apoptosis inhibitor protein survivin/birc 5-1; the sequence of the antigen peptide OT-1 is preferably SIINFEKL; the molecular weight of the antigen peptide OT-1 is preferably 963.14 g/mol;
and/or, the polypeptide is telomerase activity catalytic unit TERT, or 'T cell recognition melanoma antigen MART-1, MOG35-55, PADRE, Trp2 or survivin/birc 5-2'; the sequence of TERT is preferably DLQPYMGQFLKHLQDSDASALRNSVVI; the molecular weight of the TERT is preferably 3046.70 g/mol;
and/or, the protein is ovalbumin OVA; the molecular weight of the OVA is preferably 298.4 g/mol;
and/or the mass ratio of the metal aluminum nano particles to the antigen is 2 (0.1-10), preferably 2 (1-3), more preferably 2:3 or 1: 1; when the antigen is a short peptide and/or a polypeptide, the mass ratio of the metal aluminum nanoparticle to the antigen is preferably 1: 1; when the antigen is a protein, the mass ratio of the metal aluminum nanoparticle to the antigen is preferably 2: 3.
8. A process for the preparation of the vaccine composition according to claim 6 or 7, comprising the steps of: mixing the vaccine adjuvant and the solution containing the antigen, and carrying out cultivation reaction; wherein:
preferably, the solution containing the antigen is prepared by the following method: mixing the antigen with a solvent B; the solvent B is preferably water, PBS buffer solution, DMF or an alcohol solvent, and more preferably PBS buffer solution or DMF;
the concentration of the antigen in the solution containing the antigen is preferably 0.01-10mg/mL, more preferably 0.1-5mg/mL, such as 2.0mg/mL or 3.0 mg/mL;
when the vaccine adjuvant is mixed with the antigen-containing solution in the form of a vaccine adjuvant suspension according to claim 5, preferably, the solvent A and the solvent B are compatible; preferably, when the antigen is a short peptide and/or polypeptide, the solvent a and the solvent B are the same, for example: the solvent A is DMF, and the solvent B is DMF; preferably, when the antigen is a protein, the solvent a and the solvent B are different, for example: the solvent A is DMF, and the solvent B is PBS buffer solution;
preferably, the incubation reaction is carried out as follows: firstly, carrying out ultrasonic dispersion and then culturing; the time of the ultrasonic dispersion is preferably 5-120min, such as 10-120min, more preferably 30 min;
preferably, the incubation reaction is carried out at 80-800 rpm;
preferably, the incubation reaction is for a period of 1 to 24 hours, for example 12 hours;
preferably, after the incubation reaction, the vaccine composition is centrifuged to remove the supernatant.
9. A lyophilized vaccine comprising a metallic aluminum nanoparticle and an antigen, wherein the metallic aluminum nanoparticle is as defined in any one of claims 1 to 4, and the antigen is as defined in claim 6 or 7;
wherein, preferably, the freeze-dried vaccine is prepared by mixing the vaccine composition of claim 8 and water and freeze-drying.
10. Use of metallic aluminium nanoparticles as defined in any one of claims 1 to 4 as vaccine adjuvants in vaccines.
CN201911020292.4A 2019-10-25 2019-10-25 Metallic aluminum nano adjuvant, vaccine composition, preparation method and application thereof Pending CN112704734A (en)

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