CN109364243A

CN109364243A - A kind of antigen thermostabilization lotion and its preparation method and application

Info

Publication number: CN109364243A
Application number: CN201811252290.3A
Authority: CN
Inventors: 马光辉; 吴颉; 吴楠; 苗春宇; 周炜清
Original assignee: Institute of Process Engineering of CAS
Current assignee: Institute of Process Engineering of CAS
Priority date: 2018-10-25
Filing date: 2018-10-25
Publication date: 2019-02-22
Anticipated expiration: 2038-10-25
Also published as: CN109364243B

Abstract

The present invention provides a kind of antigen thermostabilization lotions and its preparation method and application, the antigen thermostabilization lotion includes water phase, oil phase and the inorganic particle for being combined with antigen, the oil is mutually wrapped up by water phase, the inorganic particle dispersion for being combined with antigen in water phase on oil phase interface；The combining form of the antigen and inorganic particle is Electrostatic Absorption；Lotion provided by the invention can significantly improve the thermal stability of organism antigens；Structural water molecule in mineral facies is by tight union around mineralization protein, reduce the hydrogen bond exchange rate of hydrone in protein molecular and external environment, reduce destruction of the warm-up movement process to protein structure, improve vaccine stability in a high temperauture environment, extend the limited period that vaccine saves, can be stored 3 weeks or more at 25 DEG C；In addition, lotion is alternatively arranged as vaccine adjuvant, drug delivery or slow controlled release carrier application, the use of surfactant is avoided, can reduce murder by poisoning and environmental pollution to human body.

Description

A kind of antigen thermostabilization lotion and its preparation method and application

Technical field

The invention belongs to field of biotechnology, it is related to a kind of antigen thermostabilization lotion and its preparation method and application.

Background technique

For a long time, communicable disease is always one of significant threat of human health.Vaccine inoculation is infected as reply One of the effective means of disease, shows extremely important effect in the prevention and control of disease.However, the vaccine of early stage is mostly Attenuation or the whole virus vaccine of inactivation are also deposited although it can excite stronger protection antibody horizontal and cellullar immunologic response Restore and inactivate halfway risk in virulence.Therefore with the development of biotechnology, the cracking epidemic disease with greater security Seedling, subunit vaccine, recombinant protein vaccine, synthetic peptide vaccine, polysaccharide conjugate vaccine etc. in clinic gradually instead of attenuation and Inactivated vaccine.Especially in recent years, the development of Biochemical Engineering helps to realize the extensive expression of antigen and purifying, further pushes away The application of above-mentioned vaccine is moved.These new generation vaccine purity is highs, definite ingredients, safety are good, but exist simultaneously that molecule is small, exempts from Epidemic focus is weak, is used alone and is difficult to induce body to generate effective immune response, needs plus adjuvant to enhance its immune effect.

The main function of addition adjuvant is the immunogenicity of enhancement antigen.Meanwhile part adjuvant can also play adjusting Affinity of antibody, specificity and subtype distribution stimulate cellullar immunologic response, reduce antigen dose, reduce inoculation frequency and mention The effects of response success rate of high immunologic inadequacy person (such as children and old age).

Currently, the most commonly used people of Commercialization application is Alum adjuvant with adjuvant, pass through reservoir of antigen effect and induction The immune-stimulating effects such as inflammation play the effect of enhancing immune response.Alum adjuvant is the assistant for being applied to people by FDA approval earliest Agent.However, there is also application limitations for Alum adjuvant: 1) antigen carried in Alum adjuvant is in the form of soluble antigen It is absorbed by antigen presenting cell (APCs), main lift humoral immune response, and can not inducing cellular immune response.However, right In the intracellular infections disease such as such as virus infection, cellullar immunologic response especially CD8+T cell-mediated cellulotoxic effect is for killing Hurt infected cell, control and removes viral most important in vivo.2) the antigen slow-release time of Alum adjuvant is longer, causes Antigen offers that effect is undesirable, immune response generation time lag.3) occasionally there is serious local reaction in injection site, occurs red Spot, tubercle, granulomatous inflammation etc..Meanwhile it (such as being connect using Non-injective route with the development of vaccine and the expansion of application range Kind vaccine, therapeutic vaccine etc.), Alum adjuvant is gradually unable to satisfy the demand of Vaccine Development, and people is promoted actively to find newly Vaccine adjuvant.

In addition to Alum adjuvant, another kind of is oil emulsion adjuvant using more commercialization adjuvant in clinic, can be effectively It promotes antibody level and extends holding time for antibody.And for the immunologic inadequacies person such as old man, children, using oily cream assistant Agent also can effectively stimulate body to generate higher antibody level.Its Immune-enhancing effect mechanism is the monocyte of induction human body, Neutrophil leucocyte and eosinophil etc. enhance cell to the intake of antigen and offer, simultaneously in injection site rapid aggregation The mature differentiation and the migration to peripheral lymph nodes for promoting Dendritic Cells (DCs).The oil cream assistant of part listing and clinic Agent is as shown in table 1.

Table 1

These oil emulsion adjuvant are usually made of water-oil phase, include at least one surfactant, common surface-active Agent include polyoxyethylene sorbitan esters surfactant (commonly referred to as tween), sorbitan esters (commonly referred to as Sapn), octoxynol 9 (triton x-100 or tert-octylphenoxypolyethoxyethanol) and lecithin etc., especially tween 80 (SPAN 80s), span 85 (anhydrosorbitol trioleate) and triton x-100 are answered With more.Role is mainly stable emulsion to surfactant in the formulation, avoids being demulsified.For this purpose, it needs At least one surfactant is used, and to keep emulsion-stabilizing, content of the surfactant in lotion requires more than cream Change aequum, so as to cause there are free surfactants in water phase or oily phase or both.In addition, CN101365485A is disclosed A kind of immunogenic composition contains cracking influenza antigen and oil-in-water emulsion, wherein the lotion is in its water phase Include free surfactant.But the membrane glycoprotein in the method in antigen is such as contacted with free surfactant for a long time, it can It can cause it that denaturation occurs, must be strictly controlled simultaneously for the additional amount of surfactant, the surface of more amount is added Activating agent may cause haemolysis.

From it is traditional different as the lotion of stabilizer using surfactant, Pickering lotion be one kind by solid Particle replaces the emulsion system of conventional surfactant.Mainly pass through solid particle is adsorbed on grease circle to the stable mechanism of lotion Face forms solid particle single or multi-layer structure, to make emulsion-stabilizing.Compared with lotion of the tradition containing surfactant, It has advantage outstanding: (1) emulsifier greatly reduces, save the cost；(2) small to the toxic side effect of human body；(3) lotion Stability improves, not the influence vulnerable to factors such as temperature, oily phase compositions；(4) environmental pollution is reduced.Therefore its food, medicine, The fields such as cosmetics and material have huge potential using value.

Additionally, it is well known that vaccine has played important function in terms of the prevalence that keeps off infection.Since vaccine is typical Biological products have temperature sensitivity, therefore transporting must be using low temperature environment with preserving process (as freezed).According to statistics, by Improper in storing or transporting, there are about 50% vaccines to be wasted every year.In lacking the development of reliable cold chain infrastructure extensively Country and poverty-stricken area, the permanent cryo-conservation of vaccine are still extremely difficult.The cold chain cost of vaccine occupies most of finance branch Out, annual to exceed 200,000,000 dollars.Especially in developing country, cold chain cost accounts for 80% or more of the total expenditure of national vaccine.Cause This, improves the thermal stability of vaccine, prepares heat-staple vaccine with substantial worth.

The existing method for improving live vaccine stability has: (1) adding stabilizer in liquid or the vaccine of dry powder Such as heavy water, MgCl₂, protein and nonreducing sugar mode increase vaccine stability；Such as Matthew G.Cottingham seminar in vaccine liquid by being added sucrose and trehalose, the mode for then slowly dehydrating vaccine Prepare the high adenovirus of thermal stability and poxvirus thermostabilization dry powder vaccine.(2) in the freeze drying technology of vaccine, using sugared glass Dry, spray drying of glassization etc., to enhance the stability of vaccine.(3) by protein engineering, by increase subunit it Between electrostatic force, the interaction of capsid nucleic acid or introduce the mode of disulfide bond etc. to increase the thermostabilization of vaccine particle Property.The mode of existing preparation thermal-stability vaccine is mainly obtained by preparing the method for dry powder vaccine at present, is generally required Complicated operating procedure is realized with expensive instrument.And many vaccines, it cannot especially lead to the mixed vaccine of adjuvant It crosses and the form of dry powder is made to save.

Current nothing in the prior art is based on vaccine combination Pickering lotion formation vaccine adjuvant after biomimetic mineralization Research, it is known that Pickering emulsion system be not also designed and optimize according to the demand of vaccine preparation thermal stability.Cause This, develops a kind of Pickering emulsion adjuvant based on biomimetic mineralization, while realizing the thermal stability of vaccine adjuvant and effect is immunized Fruit, it appears particularly important.

Summary of the invention

In view of the deficiencies of the prior art, the purpose of the present invention is to provide a kind of antigen thermostabilization lotions and preparation method thereof And application realizes the antigen thermal stability for improving organism to develop a kind of antigen thermostabilization lotion based on biomimetic mineralization, from And the products such as vaccine adjuvant is made to reach good stability and immune effect.

In order to achieve that object of the invention, the invention adopts the following technical scheme:

In a first aspect, the antigen thermostabilization lotion includes water phase, oil the present invention provides a kind of antigen thermostabilization lotion Phase and the inorganic particle for being combined with antigen, the oil are mutually wrapped up by water phase, and the inorganic particle dispersion for being combined with antigen is in water Mutually and on oily phase interface；The combining form of the antigen and inorganic particle is Electrostatic Absorption.

Antigen thermostabilization lotion provided by the invention, distinctive mineralising strategy can significantly improve organism, especially Bacterial antigens, fungal antigen, viral antigen thermal stability.Structural water molecule in mineral facies is by tight union in mineralising egg Around white, to form a kind of stable hydration status, protein molecular is reduced with the hydrogen bond of hydrone in external environment and exchanges speed Rate reduces destruction of the warm-up movement process to protein structure, is not influencing the basic biology of vaccine itself and do not losing its immunogene Property under conditions of, improve vaccine stability in a high temperauture environment, vaccine and its similar heat under dry powder can be assigned Stability reduces requirement of the vaccine to cold chain transportation, reduces vaccine transport and saves cost, extends the limited period that vaccine saves, It can store 3 weeks or more, or be stored at 37 DEG C 1 week or more at 25 DEG C, antigen active is still able to maintain 80% or more, and lotion Without surfactant, influence of the surfactant to antigen is avoided, so that product has good safety and stability, And it can be used for the different vaccination approach of vaccine.

Antigen thermostabilization lotion provided by the invention has better antigen heat steady compared to existing Pickering lotion It is qualitative.

In the present invention, on the emulsion droplet oil-water interfaces that inorganic particle dispersion is formed in lotion.

In the present invention, antigen can be protected from the external world by receiving the oil-in-water emulsion that is prepared of inorganic particle of particle diameter Heat inactivation caused by hot environment, mechanism of action can be mainly due to the following aspects: (1) being combined with the inorganic particulate of antigen Antigen thermostabilization lotion prepared by grain is made except particle crystal structure itself can provide the protection that a fixed gap is played to antigen Except, its interaction of the adjacent particle of lotion surface accumulation further fights original shape into frame protection.By particle and resist Gap between former charge adsorption and particle, the adsorption capacity of enhancement antigen make antigen rather than are scattered in outer aqueous phase, into And enhance stability of the antigen under thermal environment；(2) antigenic surface, which introduces inorganic particle shell, can reduce and completely cut off inside Directly contacting between antigen and extraneous water environment, reduces the hydrogen bond exchange rate of hydrone in protein molecular and external environment, The destruction that warm-up movement process conceives albumen is reduced, the extraneous warm-up movement rate that internally antigen transmits is thereby reduced；(3) nothing Machine particle shell can be used as a kind of physical barriers and antigen coat protein hindered to expand and some apparent structure changes； (4) inorganic particle shell can be by forming Electrostatic Absorption between polar amino acid on ion and antigen coat protein, and then enhances Stability of the antigen protein structure under thermal environment, also can be enhanced the stabilization combined between antigen coat protein difference subunit Property.

In the present invention, antigen thermostabilization lotion includes at least one inorganic particle, the Zeta potential one of inorganic particle As be+40mV~+50mV.

Preferably, the inorganic particle includes aluminium hydroxide, aluminum phosphate, aluminum sulfate, calcium carbonate, calcium phosphate, calcium oxalate, four In Fe 3 O, ferrous sulfate, ferric phosphate or silica any one or at least two combination.

Preferably, the inorganic particle be aluminium hydroxide, aluminum phosphate, calcium phosphate or calcium carbonate in any one or at least Two kinds of combination.

Wherein, aluminium hydroxide is γ-AlOOH crystal structure, and 2 θ of diffraction maximum position (020) is 13.68 °~14.86 °, brilliant Interplanar distance d (020) is 0.612~0.644nm, and grain size L (020) is 9.4~14.6nm.

In the present invention, referred to as " aluminium hydroxide " be usually aluminum oxyhydroxide salt, aluminum oxyhydroxide is with molecular formula AlO (OH) it indicates, with other aluminium compounds, such as aluminium hydroxide Al (OH)₃Difference be infrared (IR) spectrum, especially exist 1070cm^-1Place is there are absorption band and in 3090-3100cm^-1There are strong acromions at place.The pI value of aluminum hydroxide adjuvant is normally about 11, i.e., adjuvant itself has positive surface charge at physiological ph.When pH value is 7.4, the adsorption capacity of aluminium hydroxide is at every milligram Al³⁺Between absorbable 1.8-2.6mg protein.Since there are certain differences between the different production batch of business aluminium hydroxide Different, at present using the Alhydrogel of Denmark's production as recognised standard, colloidal solid size is 3.07 μm.Referred to as " aluminum phosphate " Usually Adju-Phos also often contain a small amount of sulfate radical (i.e. aluminium hydroxyphosphate sulfate).These assistants can be obtained by precipitating Agent.Aluminum phosphate is usually granular, and representative diameter of these particles after adsorbing any antigen is 0.5-20 μm (for example, about 5-10μm).When pH is 7.4, the adsorption capacity of aluminum phosphate is in every mg Al³⁺It absorbs between 0.7-1.5mg protein.

When inorganic particle is selected from various calcium salts, preferably calcium phosphate is as inorganic particle of the invention.It has been reported that phosphorus The various adjuvant forms of sour calcium, the present invention can use any adjuvant form.Adjuvant can form the needle-shaped of size about 10nm × 150nm The piece of the irregular shape of particle and diameter about 20-30nm.There are also documents to disclose particle calcium phosphate (" CAP "), wherein institute The diameter for stating particle is 300-4000nm (nano particle), and shape is spherical shape, smooth surface.This hair can be achieved in above-mentioned calcium phosphate It is bright.

Preferably, the shape of the inorganic particle be spherical, rodlike, sheet, fusiform, plate-like, cube, polyhedron or Any one in amorphous.

Preferably, the pattern of the inorganic particle be rough surface, porous surface, internal multi-chamber, it is hollow or simple eye in Any one or at least two combination.

In the present invention, inorganic particle can be the particle of different crystallinity, and those skilled in the art can be according to used Grease phase and antigenic property screening optimized to obtain meeting the oil-in-water emulsion of application demand by limited technique.

In the present invention, surface-active based on biomimetic mineralization and is free of using inorganic particle substitution surfactant preparation The emulsion oil-in-water of agent, not only can negative effect to avoid surfactant to vaccine preparation, but also pass through inorganic particle Epidemic disease can be improved under conditions of not influencing the basic biology of vaccine itself and not losing its immunogenicity with the combination of vaccine The thermal stability of seedling assigns vaccine and its similar thermal stability under dry powder, obtains more fully, significantly, persistently Immune protective effect.Surfactant is free of in lotion, avoids influence of the surfactant to antigen, and product has good Safety and stability, and can be used for the different vaccination approach of vaccine.

Preferably, the surface modification of the inorganic particle has modification group.

Preferably, the modification group for the modification group with amino or has sulfonic modification group.

In the present invention, the surface of inorganic particle can modify a variety of groups, and preferably modification has amino or sulfonic Group.

Inorganic particle in lotion of the present invention has grease amphipathic, is stably dispersed in oil-water two-phase interfaces, plays steady Determine the effect of lotion.For different grease systems, the solid particle with different hydrophilic and hydrophobics may be selected with stable emulsion, Hydrophilic or hydrophobic modification, coating or graft modification etc. can be carried out to the surface of inorganic particle, with obtain suitable hydrophilic and hydrophobic (or (particle wettability generally uses oil-water-solid contact angle θ ow table to particle wettability Show)).The surface of inorganic particle is adsorbable, coupling targeting substance, fluorescent marker, isotopic label, environmental response substance, The functional mass such as cell factor, antibody or immunomodulator.Wherein environmental response substance is selected from sensitive, thermo-responsive with pH or raw The group of active substances sensitivity etc..Inorganic particle can save in aqueous solution or in buffer solution in the present invention, can also do It is stand-by after dry.

Preferably, the oil mutually includes biocompatibility grease and/or mineral oil.

In the present invention, hydrophobic drug can be also embedded in oily phase, such as anti-tumor drug or immune activation adjuvant substance.It dredges Aqueous pharmaceutical has strong-hydrophobicity, is not easy the characteristics of storing and discharging, can be into one by the coated by hydrophobic drug in oily phase Step promotes the effect of lifting system immune response.Immune activation adjuvant substance may include monophosphoryl lipid matter Times of (monphosphoryllipid, MPLA), imiquimod (Imiquimod) or poly I:poly C (Poly (I:C)) It anticipates a kind of or at least two combinations.Antineoplastic target drug include everolimus, according to Shandong for Buddhist nun, Imatinib, Wei Mode Ji, Wei Luofeini, tesirolimus, Sony De Ji, Sorafenib, Sutent, match it is vertical for Buddhist nun, Rui Gefeini, Trimetinib, General sodium cuts down for Buddhist nun, bortezomib, pazopanib, Pa Boxini, pabishta, nilotinib, romidepsin, pleasure for Buddhist nun, La Pa For Buddhist nun, gram azoles for Buddhist nun, Carfilzomib, card it is rich for Buddhist nun, card than for Buddhist nun, Gefitinib, Vorinostat, Vande Thani, Tarceva, Di Nuosaimai, Dasatinib, dabrafenib, bosutinib, Baily department he, difficult to understand this replace Buddhist nun, olaparib, Ai Le to replace Buddhist nun, A Xi For in Buddhist nun, Afatinib or VEGF Trap any one or at least two combination.

Preferably, the biocompatibility grease includes squalene, soybean oil, Miglitol, midchain oil, fish oil, dimension life Plain E, Vitamin E succinate, Vitwas E, safflower oil, corn oil, Seabuckthorn Oil, Linseed oil, peanut oil, tea oil, Sunflower oil, apricot kernel oil, tocopherol, coix seed oil, evening primrose oil, sesame oil, cottonseed oil, castor oil, castor oil analog, low mustard Sauerkraut seed oil, ethyl oleate, oleic acid, ethyl linoleate, isopropyl laurate, interior isopropyl myristate, ethyl butyrate, lactic acid second In ester, Trivent OCG or Triglyceride DDD any one or at least two combination.

Preferably, the biocompatibility grease is any in squalene, tocopherol, castor oil or castor oil analog It is a kind of or at least two combination, preferably squalene.

In the present invention, mutually it is preferable to use squalenes for oil.Squalene is a kind of triterpene compound, and English name is Squalene, molecular structure are the isoprene of 30 light dydrocarbon decahydros, molecular formula are as follows: 2,6,10,15,19,23- hexamethyl -2, 6,10,14,18,22- tetracosa carbon, six alkene, CAS:111-02-4, molecular mass: 410.72, animal, plant extract can be derived from Or chemical synthesis.Squalene is a kind of metabolizable oil, because it is intermediate product (the Merck rope of the biosynthesis of cholesterol Draw, the 10th version, registration number 8619).This is a kind of all higher organisms, including (can be found in sebum) on mankind certainly The grease so secreted.Lotion (containing surfactant) containing squalene shows excellent in zoopery and clinical trial Immunological enhancement.

Preferably, the water phase includes that purified water, water for injection, glycerine water solution, 0.9% physiological saline, phosphate are slow In fliud flushing, citrate buffer solution or Tris buffer any one or at least two combination.

Preferably, the water phase be phosphate buffer, citrate buffer solution or Tris buffer in any one or The combination of person at least two.

Preferably, the pH value of the water phase is 5.5-8.5, for example, can be 5.5,5.6,5.8,6.0,6.2,6.4,6.5, 6.6,6.8,7.0,7.2,7.4,7.5,7.6,7.8,8.0 or 8.5 etc., preferably 6.0-8.0.

Preferably, the antigen is anti-selected from human antigen, non-human animal antigen, plant antigen, bacterial antigens, fungi It is former, in viral antigen any one or at least two combination, wherein typical but non-limiting combination includes: that the mankind are anti- Former and non-human animal antigen combination；The mixture of plant antigen and bacterial antigens；Fungal antigen, viral antigen and helminth The combination of antigen；Tumour antigen, human antigen, non-human animal antigen, plant antigen, bacterial antigens and fungal antigen group It closes；The combination of viral antigen, parasite antigen, tumour antigen, human antigen, non-human animal antigen and plant antigen；Bacterium Antigen, fungal antigen, viral antigen, parasite antigen and combination of tumour antigen etc..

In the present invention, the antigen in lotion can be monovalent antigen either polyvalent antigen；Antigen can come from but not Be limited to chick embryo culture, cell culture, carrier's body fluid, in organ or tissue obtained by purifies and separates, recombinant gene expression or chemistry Synthesis gained, preferred antigens include but is not limited to attenuated vaccine, inactivated vaccine, split vaccine, subunit vaccine, polysaccharide combination epidemic disease In seedling, recombinant vaccine or DNA vaccination etc. any one or at least two combination, such as attenuated vaccine and inactivated vaccine Combination, the combination of split vaccine and subunit vaccine, the combination of polysaccharide conjugate vaccine, recombinant vaccine, DNA vaccination and attenuated vaccine Combination, the combination of inactivated vaccine and split vaccine, the group of subunit vaccine, polysaccharide conjugate vaccine, recombinant vaccine and DNA vaccination It closes.

Preferably, the oil is mutually (0.05-3) with the mass ratio of inorganic particle: 1, such as can be 0.05:1,1:1,2: 1 or 3:1.

Preferably, the average grain diameter of the inorganic particle be 30nm-3 μm, such as can be 30nm, 50nm, 100nm, 500nm, 950nm, 1 μm, 1.5 μm, 2.1 μm, 2.4 μm, 2.8 μm or 3 μm etc..

Preferably, the average grain diameter of the inorganic particle is 50nm-3 μm.

Preferably, the average grain diameter of the inorganic particle and antigen ratio be (10-30): 1, for example, can be 10:1,11:1, 12:1、13:1、14:1、15:1、16:1、17:1、18:1、19:1、20:1、21:1、22:1、23:1、24:1、25:1、26:1、 27:1,28:1,29:1 or 30:1 etc..

In the present invention, when inorganic particle particle size and antigen are close, gap can not provide between particle for antigen Frame protection effect；And when inorganic particle partial size is greater than 30 times of antigen partial size or more, Antigen adsorption fails shape in particle surface At wrapping layer, cannot play a protective role to antigen.Therefore, the average grain diameter of inorganic particle and antigen is preferably controlled in above-mentioned model When enclosing interior, effect is more preferable.

The average grain diameter of inorganic particle and antigen in the present invention is the inorganic particulate measured respectively by particle size analyzer than range Grain partial size and antigen partial size later comment antigen thermal stability in the lower preparation for carrying out lotion of the ratio between different-grain diameter in vitro Valence so that it is determined that proportional region.

Preferably, the inorganic particle and the contact angle of oily phase are 45 ° -90 °, for example, can be 45 °, 48 °, 50 °, 55 °, 60 °, 65 °, 70 °, 75 °, 80 °, 85 ° or 90 ° etc..

Preferably, mass concentration of the inorganic particle in water phase is 1wt.%-6wt.%, such as be can be 1wt.%, 1.5wt.%, 2wt.%, 2.5wt.%, 3wt.%, 3.5wt.%, 4wt.%, 4.5wt.%, 5wt.%, 5.5wt.% or 6wt.% etc..

In the present invention, mass concentration of the inorganic particle in water phase is that the quality of inorganic particle removes inorganic body particle and water The ratio of phase quality sum.If inorganic particle excessive concentration is unfavorable for the formation of lotion；If concentration is too low, it is not achieved bionical The effect of mineralising.

Preferably, the oil is mutually 1:100-9:1 with the volume ratio of water phase, such as can be 1:100,1:50,1:32,1: 21,1:10,1:1,2:1,3:1,5:1,8:1 or 9:1 etc..

Preferably, it is described oil mutually and water phase volume ratio be 1:(4-50), such as can be 1:4,1:6,1:10,1:14, 1:18,1:20,1:25,1:30,1:35,1:40,1:45 or 1:50 etc..

Preferably, emulsion droplet average grain diameter formed in the antigen thermostabilization lotion is 800nm-3 μm, such as be can be 800nm, 900nm, 1 μm, 1.5 μm, 2 μm, 2.5 μm or 3 μm etc..

Preferably, the partial size polydispersity coefficient PDI value of the emulsion droplet is less than 0.6.

Second aspect, the present invention provides a kind of preparation method of antigen thermostabilization lotion as described in relation to the first aspect, institutes Stating preparation method includes: that antigen is added in the water phase containing inorganic particle, is incubated for after suspension, is then added to oil It obtains in water phase, is mixed to get the antigen thermostabilization lotion.

Antigen thermostabilization lotion can be used first by inorganic particle dispersion in water phase in the present invention, then by oily phase and water phase It is mixed with.It is more that the dispersing mode of inorganic particle can choose oscillation, stirring, ultrasound (Ultrasonic dispersion) etc. Kind mode, to realize fine dispersion of the inorganic particle in water phase.As long as being able to achieve fine dispersion of the particle in water phase, adopted Dispersing mode will not cause to significantly affect to the property of oil-in-water emulsion, can according to water phase used and solid particle property and The experimental facilities condition of itself selects suitable dispersing mode and concrete operations parameter.The mixing of oily phase and water phase can choose micro- Flow control (Microfluidization), homogeneous (Homogenization), ultrasound, syringe are double to push away emulsification (Two-syringe Emulsification), spraying, microjet, microchannel (Microchannel emulsification), film emulsification The various ways such as (Membrane emulsification), stirring, oscillation, reversing or hand mixing.According to different requirements, Hybrid mode can be in such a way that preferred micro-fluidic, microchannel or film emulsification etc. can obtain uniform particle size distribution lotion, can also be with It is preferred that microjet, syringe are double to push away the hybrid mode that emulsification, homogeneous, stirring or oscillation etc. are convenient for large-scale preparation.It should define , different greases mix mode, prepare power, time, number etc. necessarily affect emulsion droplet in gained lotion partial size it is big Small, dispersed and stability etc. also will affect final immune or administering effect.It therefore, should be according to grease phase used and nothing The influence factors such as machine particle properties, the emulsion droplet size range of required preparation determine suitable hybrid mode and concrete operations parameter.

Preferably, the amount of the inorganic particle of every 2-55 μ g/mL antigen binding is 50-1000 μ g/mL.

Preferably, the temperature of the incubation be 25 DEG C -45 DEG C, such as can be 25 DEG C, 28 DEG C, 30 DEG C, 32 DEG C, 35 DEG C, 40 DEG C, 42 DEG C, 43 DEG C or 45 DEG C etc..

Preferably, the time of the incubation be 0.5h-3h, such as can be 0.5h, 1h, 1.2h, 1.5h, 1.7h, 1.8h, 2h, 2.3h, 2.5h, 2.7h, 2.9h or 3h etc..

The third aspect, the present invention provides a kind of antigen thermostabilization lotions as described in relation to the first aspect to prepare vaccine assistant Agent, the drug with inhibitive ability of immunity, fights answering in the gentle controlled release carrier of therapeutic agent of chronic disease at drug delivery preparation With.

In the present invention, chronic disease includes human nipple virus infection, Infected With Polioviruses In Vitro, coxsackie virus sense Dye, norwalk virus infection, AIDS, anthrax, malaria, hepatitis B, hepatitis A, hepatitis C, cervical carcinoma or lung cancer In any one.

In the present invention, antigen thermostabilization lotion by immunity inoculation progress drug administration by injection in use, can generally be given The mode of medicine may include a variety of, for example, can be subcutaneous injection, intracutaneous injection, the administration of vertebra chamber, intramuscular injection, in lymph node Injection, intratumor injection, intraperitoneal injection, intravenous injection or foot injection in any one.

Compared with the existing technology, the invention has the following advantages:

(1) antigen thermostabilization lotion provided by the invention for the first time ties biomimetic mineralization and oily emulsion formulation (oil-in-water emulsion) The oil-in-water emulsion that preparation is not necessarily to surfactant is closed, applied to the development field of vaccine adjuvant, and passes through inorganic particle It is added, the biocompatibility of preparation not only can be improved, avoid surfactant to human body, animal or the bad shadow of vaccine bring It rings, and can more efficiently stimulate immunocyte, excite immunoloregulation function.

(2) inorganic particle property used in antigen thermostabilization lotion provided by the invention, can be to it convenient for regulation It is surface modified or coating, or the inorganic particle of selection heterogeneity (such as pattern, structure, partial size, crystallinity), plays Different Immune-enhancing effect mechanism adjusts immune response using the ability of its antigen control release as the transport carrier of antigen, It can also be applied to panimmunity vaccination ways, have wide range of applications.

(3) antigen thermostabilization lotion provided by the invention, distinctive mineralising strategy can significantly improve organism, especially It is the thermal stability such as bacterial antigens, fungal antigen, viral antigen, polypeptide, there is universality.It is substantially raw not influencing vaccine itself Object and under conditions of not losing its immunogenicity, improves vaccine stability in a high temperauture environment, reduces vaccine and transport to cold chain Defeated requirement reduces vaccine transport and saves cost, extends the limited period that vaccine saves, can store at 25 DEG C 3 weeks or more, and Antigen active is still able to maintain 80% or more.It largely solves developing country and poverty-stricken area shortage is reliable cold extensively The problem of chain infrastructure, the cold chain that vaccine is greatly saved is spent, conducive to being widely popularized.

Detailed description of the invention

The grain size distribution of the aluminum hydroxide particles prepared in Fig. 1 embodiment of the present invention 1.

The stereoscan photograph (scale 100nm) of the aluminum hydroxide particles prepared in Fig. 2 embodiment of the present invention 1.

The grain size distribution based on biomimetic mineralization lotion prepared in Fig. 3 embodiment of the present invention 1.

The optical microscope (100 times of amplification) based on biomimetic mineralization lotion prepared in Fig. 4 embodiment of the present invention 1.

The horizontal checkout knot of 11 moderate stimulation of Fig. 5 embodiment of the present invention generation mouse boosting cell secrete cytokines IFN-γ Fruit.

Specific embodiment

The technical scheme of the invention is further explained by means of specific implementation.Those skilled in the art should be bright , the described embodiments are merely helpful in understanding the present invention, should not be regarded as a specific limitation of the invention.

It is raw materials used as shown in table 2 below in following embodiment of the present invention:

Table 2

Title	Specification	Manufacturer
			Aluminium isopropoxide	Purity >=98%	Aladdin reagent (Shanghai) Co., Ltd.
Squalene	Purity >=98%	Sigma Aldrich
			Castor oil	Purity >=98%	Sigma Aldrich
Trishydroxymethylaminomethane (Tris)	It analyzes pure	Sigma Aldrich
			Citric acid	It analyzes pure	Sinopharm Chemical Reagent Co., Ltd.
Sodium chloride	It analyzes pure	Sinopharm Chemical Reagent Co., Ltd.
			PEG6000	It analyzes pure	Sinopharm Chemical Reagent Co., Ltd.
Disodium hydrogen phosphate	It analyzes pure	Sinopharm Chemical Reagent Co., Ltd.
			Magnesium chloride	It analyzes pure	Sinopharm Chemical Reagent Co., Ltd.
Tween 80	It analyzes pure	Sinopharm Chemical Reagent Co., Ltd.
			Span 85	It analyzes pure	Sinopharm Chemical Reagent Co., Ltd.
Calcium acetate	It analyzes pure	Sinopharm Chemical Reagent Co., Ltd.
			Trisodium citrate	It analyzes pure	Sinopharm Chemical Reagent Co., Ltd.
Sodium carbonate	It analyzes pure	Sinopharm Chemical Reagent Co., Ltd.
			Anhydrous calcium chloride	It analyzes pure	Sinopharm Chemical Reagent Co., Ltd.
Dehydrated alcohol	It analyzes pure	Sinopharm Chemical Reagent Co., Ltd.
			Magnesium dichloride hexahydrate	It analyzes pure	Sinopharm Chemical Reagent Co., Ltd.
OVA	Purity >=98%	Sigma Aldrich
			Catalase	Purity >=98%	Hui Lindun company of the U.S.

Instrument is as shown in table 3 below:

Table 3

Title	Specification	Producer
			Electronic balance	TB-214	Beijing Sai Duolisi instrument system Co., Ltd
Centrifuge	Avanti-J-E	Beckman-Coulter
			Supersonic wave cleaning machine	25-12	NingBo XinZhi Biology Science Co., Ltd
Magnetic stirring apparatus	Color Squid	IKA
			Scanning electron microscope	JSM-6700F	JEOL
Refrigerator	MDF-382E(N)	SANYO
			Optical microscopy	Olympus BX51	Olympus
Homogenizer	T18Basic	IKA
			Liquid-transfering gun	200μL、1mL、5mL、10mL	Eppendorf
Dynamic light scattering particle size instrument particle size analyzer	Zetasizer Nano ZS	Malvern
			Ultrasonic cell disruption instrument	S-450D	Branson Inc.
Vortex oscillator	Vortex-Genie 2	Scientific Industries,Inc.
			Flow cytometer	Arial III	BD
Microplate reader	Infinite M200	Tecan

The present invention about particle property representation with the following method:

The particle size distribution measuring of particle:

The particle diameter distribution of nano-scale particle or emulsion droplet is measured using dynamic light scattering particle size instrument, specific determination step Are as follows: by 1mg nano-scale particle, it is added in 10mL deionized water, ultrasonic 5min keeps its evenly dispersed, or takes 1mL lotion, by particle Suspension or lotion are added in sample cell, are put into dynamic light scattering particle size instrument and are measured.

The homogeneity of particle or emulsion droplet is indicated by partial size polydispersity coefficient (PDI) value.PDI value is smaller to show that grain diameter is got over It is uniform.

The morphology observation of particle uses scanning electron microscopic observation: 1mg particle weighed, is added in 10mL deionized water, ultrasound 5min keeps its evenly dispersed.1mL suspension is drawn, is dripped on aluminium foil, spreads it uniformly on aluminium foil out, naturally dry. Aluminium foil is affixed on sample stage with conducting resinl, under vacuum conditions metal spraying (suitable metal spraying condition is chosen according to properties of samples) Afterwards, it is observed with scanning electron microscope.

The morphology observation of emulsion droplet uses optical microscopy: drawing a small amount of oil-in-water emulsion, drips on glass slide, be placed in optics Microscopically observation.

Emulsion intercalation method is detected using centrifugal process: take 5mL oil-in-water emulsion, be added in 15mL centrifuge tube, 2000g from After being centrifuged 10min under mental power function, delamination is observed.

Zoopery measurement:

C57BL/6 mouse used by testing is provided by dimension tonneau company of China.Immune step is substantially as follows are as follows: first by mouse Random grouping, every group is tested using 6 or more mouse, and according to the embodiment illustrate is grouped mouse and exempts from Epidemic disease inoculation.Before inoculation, 200 μ L of blood is first taken, is centrifuged 10min at 10000rpm immediately, isolate serum, measures IgG antibody Then level is immunized mouse using IgG antibody level at this time as initial value.After immune, periodically from mouse eye circumference or Tail point takes blood, takes 200 μ L of blood every time, and measures IgG antibody level.Secondary immunity is carried out after two weeks, and 35 days execution mouse take Blood, measurement IgG antibody are horizontal.Mouse boosting cell is taken to be cultivated, it is thin with enzyme-linked immunosorbent assay (ELISA) detection mice spleen The secretion situation of IFN-γ cell factor in born of the same parents' culture solution supernatant.

Embodiment 1

The present embodiment prepares antigen thermostabilization lotion using aluminum hydroxide particles biomimetic mineralization

(1) aluminum hydroxide particles are prepared using hydrolysis of alkoxide-hydro-thermal method: weighs a certain amount of deionized water with graduated cylinder and is placed in In 100mL three-necked flask, 85 DEG C are heated to, aluminium isopropoxide powder is added later.Aluminium isopropoxide and deionized water are according to 1:100's Molar ratio mixing, is stirred to react 2h, cooled to room temperature.Above-mentioned reaction solution is put into the stainless of polytetrafluoroethyllining lining In steel high-pressure bottle, hydro-thermal process at a certain temperature.After hydro-thermal reaction, material natural cooling is centrifugated out Solid product is washed with deionized 3 times, aluminum hydroxide particles is obtained after vacuum drying.Aluminum hydroxide particles are characterized.

Particle diameter distribution is as shown in Figure 1, can show that the average grain diameter of particle is 50nm, the PDI value of particle is 0.149；Scanning Electron microscope is as shown in Figure 2.

(2) biomimetic mineralization of protein:

1.00g aluminum hydroxide particles are accurately weighed using electronic balance, are added in 20mL citric acid solution, ultrasound 2min makes it be uniformly dispersed, and obtains the suspension liquid of aqueous phase for being dispersed with particle.Wherein aqueous pH values are 7.5.The suspension is added Into OVA solution (1mg/mL), after being sufficiently mixed, 37 DEG C of incubation 2h obtain the protein solution of aluminium hydroxide mineralising.

(3) prepared by oil-in-water emulsion:

It draws 2mL squalene with liquid-transfering gun to be added in the good suspension liquid of aqueous phase of above-mentioned incubation, using ultrasonication (power 25%, 2min) emulsification method, prepare oil-in-water emulsion.

For obtained emulsion particle diameter distribution map as shown in figure 3, the average grain diameter of emulsion droplet is 1.6 μm, PDI value is 0.268；Optics Lotion figure under microscope is as shown in Figure 4, it can be seen that emulsion droplet favorable dispersibility, it is spherical regular.Prepared oil-in-water emulsion Appearance is precipitated with lotion indifference, the oil-free phase in upper layer is not centrifuged.

Embodiment 2

The present embodiment prepares antigen thermostabilization lotion using calcium phosphate granules biomimetic mineralization

(1) hollow calcium phosphate granules are prepared using template method:

By 3.0g Tween 80, the Tris-HCl (pH8.0) and 1.5mL of 0.25g PEG 6000,3.0mL 0.5mol/L are gone After ionized water is mixed evenly, ultrasonic 20min forms nonionic surfactant vesicle, instills 3.55mL CaCl₂ (0.175mol/L) solution adds 3.55mL Na after stirring 0.5h₂HPO₄Sodium phosphate can be obtained in (0.175mol/L) solution 0.83mL MgCl is added in the suspension of grain₂Solution (0.075mmol/L) stablizes newly formed calcium phosphate granules, continues to stir Centrifuge washing after 2h obtains hollow calcium phosphate granules after vacuum drying.The average grain diameter of particle is 210nm, shell thickness 30- 40nm, the PDI value of particle are 0.289.Prepared particle is hollow sphere.

(2) biomimetic mineralization of protein:

The hollow calcium phosphate granules of 1.00g are accurately weighed using electronic balance, are added in 20mL citric acid solution, are surpassed Sound 2min makes it be uniformly dispersed, and obtains the suspension liquid of aqueous phase for being dispersed with particle.Wherein aqueous pH values are 7.5.The suspension is added Enter into OVA solution (1mg/mL), after being sufficiently mixed, 37 DEG C of incubation 2h obtain hollow calcium phosphate mineralized protein solution.

(3) prepared by oil-in-water emulsion:

With liquid-transfering gun draw 2mL squalene be added in above-mentioned suspension liquid of aqueous phase, using magnetic agitation (500rpm, 10min) the method emulsified, prepares oil-in-water emulsion.Emulsion droplet favorable dispersibility, it is spherical regular.The average grain diameter of emulsion droplet is 3.2 μ M, PDI value are 0.296.The appearance of prepared oil-in-water emulsion is precipitated with lotion indifference, the oil-free phase in upper layer is not centrifuged.

Embodiment 3

The present embodiment prepares antigen thermostabilization lotion using the calcium carbonate granule biomimetic mineralization of peanut shape

(1) using the calcium carbonate granule of the direct mixed precipitation method preparation peanut shape of liquid phase:

Calcium acetate is weighed with electronic balance and trisodium citrate is dissolved in 200mL distilled water the (quality of trisodium citrate Concentration is respectively 10wt.% and 30wt.%), the aqueous sodium carbonate (50mL) of 10wt.%, stirring are added into the solution 3h is reacted under (300rpm), is filtered, and after washing precipitating respectively three times with distilled water and dehydrated alcohol, is dried, is obtained at 70 DEG C The calcium carbonate granule of peanut shape.The length of particle is 7.2 μm, axial ratio 2:1, and the pattern of prepared particle is peanut shape.

(2) biomimetic mineralization of protein:

3.1g calcium carbonate granule is accurately weighed using electronic balance, is added in 40mL phosphate buffer solution, ultrasound 2min makes it be uniformly dispersed, and obtains the suspension liquid of aqueous phase for being dispersed with particle.Wherein aqueous pH values are 7.5.The suspension is added Into OVA solution (1mg/mL), after being sufficiently mixed, 37 DEG C of incubation 2h obtain the protein solution of calcium carbonate mineralising.

(3) prepared by oil-in-water emulsion:

2mL squalene is drawn with liquid-transfering gun to be added in above-mentioned suspension liquid of aqueous phase, and (Shirasu is emulsified using fast film Porous Glass (SPG) micropore membrane aperture be 50.2 μm, cross film pressure be 200KPa, cross film three times) method, prepare water packet Fat liquor.Emulsion droplet favorable dispersibility, it is spherical regular.The average grain diameter of emulsion droplet is 45.10 μm, and PDI value is 0.726.Prepared water After the centrifugation of packet fat liquor, the oily phase of 0.5cm is precipitated in upper layer.

Embodiment 4

The present embodiment prepares traditional antigen thermostabilization lotion using aluminum hydroxide particles

The aluminum hydroxide particles prepared in a certain amount of embodiment 1 are accurately weighed using electronic balance, are added to 20mL lemon In acid buffering solution, ultrasonic 2min makes it be uniformly dispersed, and the Tween 80 that mass fraction is 5wt.% is added, and wherein aqueous pH values are 7.5, said mixture is as water phase.The Span85 that mass fraction is 5wt.% is added into 2mL squalene and is used as oily phase, it will be oily It is added into water phase, the method emulsified using ultrasonication (power 25%, 2min) prepares oil-in-water emulsion.Emulsion droplet is put down Equal partial size is 1.6 μm, and PDI value is 0.238.The appearance of prepared oil-in-water emulsion and it is not centrifuged lotion indifference, upper layer is oil-free Mutually it is precipitated.

Embodiment 5

The present embodiment is free of the antigen thermostabilization lotion of surfactant using aluminum hydroxide particles preparation

The aluminum hydroxide particles prepared in a certain amount of embodiment 1 are accurately weighed using electronic balance, are added to 20mL lemon In acid buffering solution, ultrasonic 2min makes it be uniformly dispersed, and obtains the suspension liquid of aqueous phase for being dispersed with particle.Wherein aqueous pH values are 7.5.It draws 2mL squalene with liquid-transfering gun later to be added in the good suspension liquid of aqueous phase of above-mentioned incubation, using ultrasonication (power 25%, 2min) emulsification method, prepare oil-in-water emulsion.The average grain diameter of emulsion droplet is 1.5 μm, and PDI value is 0.279.It is prepared The appearance of oil-in-water emulsion is precipitated with lotion indifference, the oil-free phase in upper layer is not centrifuged.

Embodiment 6

The present embodiment tests stability of emulsion using different mineralization methods

1, mineralising method in situ prepares lotion

Hydrogen peroxide enzyme powder is dissolved in the Tris-HCl of pH8.0, the solution that concentration is 0.2mg/mL is configured to.Later By enzyme solutions and 5mmol/L CaCl₂With 5mmol/L MgCl₂After common incubation 30min, it is slowly added to 3mmol/L Na₂HPO₄ Normal-temperature reaction 4h, the protein solution of final obtained amorphous calcium phosphate mineralising.2mL squalene is drawn with liquid-transfering gun later to be added to In the good suspension liquid of aqueous phase of above-mentioned incubation, the method emulsified using ultrasonication (power 25%, 2min) prepares oil-in-water cream Liquid.Emulsion droplet favorable dispersibility, it is spherical regular.After prepared oil-in-water emulsion centrifugation, the oily phase of 2cm or so is precipitated in upper layer.This is Since protein is not completely encapsulated in the inside of inorganic particle, and then it is difficult to adjust hydrophilic and hydrophobic, therefore be unable to get stable Pickering lotion.

2, ex situ mineralising method prepares lotion

By 12.5mmol/L CaCl₂It is added in beaker, is placed on magnetic stirring apparatus, in whipping process, be slowly added dropwise same The 12.5mmol/L Na of volume₂HPO₄Aqueous solution.15.6mmol/L sodium citrate aqueous solution is slowly added dropwise again, stirs 12h. 15min is centrifuged under 15000g centrifugal force.Calcium phosphate is obtained after vacuum drying.1.00g calcium phosphate is accurately weighed using electronic balance Particle is added in 20mL citric acid solution, and ultrasonic 2min makes it be uniformly dispersed, and obtains the aqueous-phase suspending for being dispersed with particle Liquid.Wherein aqueous pH values are 7.5.The suspension is added in Catalase solution (0.2mg/mL), after being sufficiently mixed, 37 DEG C be incubated for 2h, obtain calcium phosphate mineralized protein solution.It is good that above-mentioned incubation is added to liquid-transfering gun absorption 2mL squalene later In suspension liquid of aqueous phase, the method emulsified using ultrasonication (power 25%, 2min) prepares oil-in-water emulsion.Emulsion droplet dispersibility Well, spherical regular.The average grain diameter of emulsion droplet is 2.1 μm, and PDI value is 0.219.Prepared oil-in-water emulsion appearance with not from Heart lotion indifference, the oil-free phase in upper layer are precipitated.This is because inorganic particle forms wrapping layer outside protein, for protein While protection is provided, stable Pickering lotion can be prepared by adjusting the hydrophilic and hydrophobic of inorganic particle.

Embodiment 7

The present embodiment measures the influence of inorganic particle and antigen binding ratio to stability of emulsion

The aluminum hydroxide particles prepared in a certain amount of embodiment 1 are accurately weighed using electronic balance, are added to 20mL lemon In acid buffering solution, ultrasonic 2min makes it be uniformly dispersed, and obtains the suspension liquid of aqueous phase for being dispersed with particle.Wherein aqueous pH values are 7.5.The suspension is added to (1 × 10 in EV71 vaccine liquid⁸PFU/mL), after being sufficiently mixed, 37 DEG C of incubation 2h obtain hydrogen-oxygen Change the vaccine liquid of aluminium ore.2mL squalene is drawn with liquid-transfering gun later to be added in the good suspension liquid of aqueous phase of above-mentioned incubation, is used The method of ultrasonication (power 25%, 2min) emulsification, prepares oil-in-water emulsion.Inorganic particle and antigen binding ratio are to lotion Stability is as shown in table 4.

Table 4

Embodiment 8

The antigen thermostabilization lotion safety evaluatio that the present embodiment is prepared based on biomimetic mineralization

1. vascular stimulation tests

By embodiment 1 and embodiment 2 respectively at family's rabbit ear vein drug administration by injection, once a day, successive administration 3 days.As a result: Family's rabbit ear vein medicine-feeding part visually observes no significant change；Tissue pathological slice microscope inspection is shown away from injection site 1cm Blood vessel endothelium is continuous, complete at blood vessel, 5cm, has no hyperplasia, swelling；Tissues surrounding vascular has no inflammatory cell infiltration and necrosis； Without thrombosis in lumen.Display this product has no that obvious stimulation acts on to family's rabbit ear vein blood vessel.

2. haemolysis and agglutination test

Using routine in vitro test tube method (observation method of naked eye), embodiment 1 and embodiment 2 are suspended with 2% red blood cell respectively Liquid mixing, had no haemolysis and red blood cell condensation phenomenon in 3 hours.

3. muscle irritation is tested

Embodiment 1 and embodiment 2 are respectively used to rabbit quadriceps muscle of thigh drug administration by injection, l mL is administered in every side.After 48 hours It takes medicine-feeding part to visually observe and makees histopathologic examination.The result shows that this product is nonirritant to rabbit quadriceps muscle of thigh.

Embodiment 9

The present embodiment determines the influence of inorganic particle and antigen particle size ratio to albumen vitro stability in lotion

Lotion is prepared according to the method in embodiment 7 using the aluminum hydroxide particles of different-grain diameter size, later to lotion Middle albumen vitro stability is investigated, and the results are shown in Table 5.When the ratio between inorganic particle and antigen particle size are 1:1, epidemic disease Seedling deactivation rate is higher, and as the ratio between inorganic particle and antigen particle size increase, vaccine deactivation rate is reduced；But when partial size is big It is the ratio between small be 40:1 when, vaccine again easy in inactivation.The above results show when inorganic particle particle size and antigen it is close when, particle it Between gap can not be provided for antigen frame protection effect；And when inorganic particle partial size is greater than 30 times of antigen partial size or more, antigen It is adsorbed in particle surface, fails to form wrapping layer, therefore cannot play a protective role to antigen.

Table 5

Inorganic particle and antigen particle size ratio	Vaccine deactivation rate (log10PFUd^-1)
		1:1	0.77
10:1	0.015
		20:1	0.022
30:1	0.029
		40:1	0.85

Embodiment 10

Evaluation of Thermal Stability outside proteosome in the antigen thermostabilization lotion that the present embodiment is prepared based on biomimetic mineralization

(1) calcium phosphate

External Evaluation of Thermal Stability is carried out using thermostabilization lotion prepared by ex situ mineralising method in embodiment 6.

External Evaluation of Thermal Stability:

By the lotion prepared based on biomimetic mineralization and traditional lotion (first preparing lotion, then adsorption antigen) respectively in water-bath It is incubated for 30min under the different temperatures of case, passes through enzymatic activity test proteins residual activity.The experimental results showed that when temperature is lower than 45 DEG C when, non-mineralising catalase activity is relatively stable, and as the temperature rises, it is left that activity only slightly reduces by 10% It is right.When temperature is higher than 45 DEG C, protein starts to become thermally labile and gradually loss of biological activity.And when temperature reaches 65 DEG C When, non-mineralization protein only maintains 10% or so of original activity, and the activity of mineralization protein is then remained well, greatly Generally maintain 70% of original activity or so.Even if being incubated for 1h at 65 DEG C, the activity of mineralization protein is still also retained nearly 60%.This illustrates that calcium phosphate mineralized shell has apparent high temperature protection effect to protein, can greatly improve protein Thermal stability.

(2) aluminium hydroxide

The biomimetic mineralization of virus:

The aluminum hydroxide particles prepared in embodiment 1 are accurately weighed using electronic balance, are added to 20mL lemon acid buffering In solution, ultrasonic 2min makes it be uniformly dispersed, and obtains the suspension liquid of aqueous phase for being dispersed with particle.Wherein aqueous pH values are 7.5.It should Suspension is added to (1 × 10 in EV71 vaccine liquid⁸PFU/mL), the content of aluminium element is 420 μ g, and after being sufficiently mixed, 37 DEG C are incubated 2h is educated, the vaccine liquid of aluminium hydroxide mineralising is obtained.

Oil-in-water emulsion preparation:

It draws 2mL squalene with liquid-transfering gun to be added in the good suspension liquid of aqueous phase of above-mentioned incubation, using ultrasonication (power 25%, 2min) emulsification method, prepare oil-in-water emulsion.Emulsion droplet favorable dispersibility, it is spherical regular.The average grain diameter of emulsion droplet is 1.8 μm, PDI value is 0.245.The appearance of prepared oil-in-water emulsion is precipitated with lotion indifference, the oil-free phase in upper layer is not centrifuged.

External Evaluation of Thermal Stability:

By the lotion being prepared in embodiment 4,5 and EV71 vaccine liquid (1 × 10⁸PFU/mL sagging at 4 DEG C after) mixing Straight suspension 2h.The lotion of the above-mentioned lotion mixed and biomimetic mineralization preparation is stored 12 days, 42 DEG C at 25 DEG C respectively later Store 12h.At 25 DEG C after storage in 12 days, the emulsion vaccines deactivation rate that embodiment 4 is prepared is 2log₁₀PFU·d^-1, the emulsion vaccines deactivation rate that embodiment 5 is prepared is 0.15log₁₀PFU·d^-1, lotion after mineralising Vaccine deactivation rate is 0.015log₁₀PFU·d^-1.At 42 DEG C after 12h is stored, lotion epidemic disease that embodiment 4 is prepared Seedling deactivation rate is 2.5log₁₀PFU·d^-1, the emulsion vaccines deactivation rate that embodiment 5 is prepared is 0.19log₁₀PFU· d^-1, the emulsion vaccines deactivation rate after mineralising is 0.040log₁₀PFU·d^-1.And virus only carry out biomimetic mineralization (such as Disclosed in CN107041952A) after, it is respectively 0.037log in the vaccine deactivation rate of 25 DEG C and 42 DEG C₁₀PFU·d^-1 And 0.066log₁₀PFU·d^-1.The above results show emulsion vaccines that embodiment 4 is prepared since antigen is distributed in water mostly Xiang Zhong, and not on the oil-water interfaces of emulsion droplet, so inorganic particle does not play the protective effect to antigen.Furthermore biomimetic mineralization The thermal stability of emulsion adjuvant vaccine improves 2 times than only carrying out the vaccine of biomimetic mineralization.This illustrates biomimetic mineralization emulsion adjuvant The stability of vaccine can be significantly improved.

Embodiment 11

Evaluation of Thermal Stability in proteosome in the antigen thermostabilization lotion that the present embodiment is prepared based on biomimetic mineralization

Mouse experiment in vivo:

By the emulsion adjuvant prepared in embodiment 1 and with the emulsion adjuvant (after adsorption antigen) in embodiment 5 in room temperature 25 It is handled 14 days in DEG C environment, and mouse is immunized to detect the ability of the vaccine-induced immune response after heat treatment with them.Experiment The result shows that treated that traditional emulsion adjuvant inducing mouse generates IgG antibody and the ability of neutralizing antibody compares respectively for room temperature Traditional emulsion adjuvant before processing has dropped 3~4 times and 6 times or so.Likewise, the traditional emulsion adjuvant thorn after heat treatment Swash the horizontal also than having dropped one times or more before processing of splenocyte secretion gamma interferon.But the emulsion adjuvant through biomimetic mineralization Handle at room temperature after a week still can effectively inducing mouse humoral and cellular immune response.Biomimetic mineralization lotion assistant after heat treatment The serum IgG antibody levels of agent induction are similar to before processing, and serum neutralizing antibody level is than decline (decline degree faint before handling In 25%-30%).

IFN-γ secretion cellular level characterizes bionical mine in the mouse of further ELISPOT measuring Immune-enhancing effect Change the ability that emulsion adjuvant induces T cell response.Experimental result as shown in figure 5, the emulsion adjuvant after biomimetic mineralization can induce it is small Mouse generates the IFN-γ secretion cellular level for being significantly higher than pure antigen group and traditional emulsion adjuvant group mouse, this illustrates bionical mine Changing emulsion adjuvant can more effectively induce body to generate cellular immunity.

Embodiment 12

Water phase is with the hybrid mode of oily phase to generation in the antigen thermostabilization lotion that the present embodiment is prepared based on biomimetic mineralization The influence of antibody titer

Aluminum hydroxide particles are prepared according to method in embodiment 1.

The biomimetic mineralization of protein:

Oil-in-water emulsion preparation:

2mL squalene is drawn with liquid-transfering gun to be added in the good suspension liquid of aqueous phase of above-mentioned incubation, and above-mentioned mixed liquor is averaged It is divided into two parts, the method that ultrasonication (power 25%, 2min) and mechanical stirring (500rpm, 5min) is respectively adopted carries out cream Change, prepares oil-in-water emulsion.The emulsion droplet Monodispersed of prepared lotion is good, spherical regular.Wherein cream prepared by ultrasonication The average grain diameter of emulsion droplet is 1.6 μm in liquid, and Span value is 0.296；The average grain diameter of emulsion droplet is in lotion prepared by mechanical stirring 2.7 μm, Span 0.339.The appearance of prepared oil-in-water emulsion is precipitated with lotion indifference, the oil-free phase in upper layer is not centrifuged.

Take simple antigen group as control.After the injection of C57BL/6 mouse left back leg muscle, secondary exempt from is carried out after two weeks Epidemic disease, 35 days execution mouse, the results are shown in Table 6, and compared with mechanical stirring group, ultrasonication group can play good adjuvant effect It answers, generates high-caliber IgG, HI titre in experimental animal body, that there is conspicuousnesses between single antigen injection group is poor for adjuvant group It is different.

Table 6

Antigen type	Adjuvant type	Lotion preparation method	Serological IgG level	HI is horizontal
					OVA	Embodiment 1	Ultrasonication	480000	2560
OVA	Embodiment 1	Mechanical stirring	320000	640
					OVA	Nothing	Nothing	62000	32

Embodiment 13

The influence that the present embodiment acts on adjuvant based on antigen thermostabilization lotion vaccination ways prepared by biomimetic mineralization

C57BL/6 mouse will be carried out respectively according to oil-in-water emulsion made from the method in embodiment 1 muscle, it is subcutaneous and Intraperitoneal injection, carries out secondary immunity, 35 days execution mouse, the results are shown in Table 7, and intramuscular injection group shows higher after two weeks Humoral antibody level (IgG) and levels of cytokine secretion (IL-4 and IFN-γ).

Table 7

Antigen type	Adjuvant type	Vaccination ways	Serological IgG level	IL-4	IFN-γ
						OVA	Embodiment 1	Intramuscular injection	256000	32	629
OVA	Embodiment 1	Subcutaneous injection	184000	28	565
						OVA	Embodiment 1	Intraperitoneal injection	102400	18	463
OVA	Nothing	Intramuscular injection	20480	5	31
						OVA	Nothing	Subcutaneous injection	14560	4	36
OVA	Nothing	Intraperitoneal injection	10240	2	24

The Applicant declares that the present invention is explained by the above embodiments antigen thermostabilization lotion and its preparation side of the invention Method and application, but the invention is not limited to above-mentioned processing steps, that is, do not mean that the present invention must rely on above-mentioned processing step It could implement.It should be clear to those skilled in the art, any improvement in the present invention, to raw material selected by the present invention Equivalence replacement and addition, the selection of concrete mode of auxiliary element etc., all fall within protection scope of the present invention and the open scope Within.

Claims

1. a kind of antigen thermostabilization lotion, which is characterized in that the antigen thermostabilization lotion includes water phase, oil mutually and is combined with anti- Former inorganic particle, the oil are mutually wrapped up by water phase, and the inorganic particle dispersion for being combined with antigen is mutually demarcated in water phase with oily On face；The combining form of the antigen and inorganic particle is Electrostatic Absorption.

2. antigen thermostabilization lotion according to claim 1, which is characterized in that the inorganic particle include aluminium hydroxide, Appointing in aluminum phosphate, aluminum sulfate, calcium carbonate, calcium phosphate, calcium oxalate, ferroso-ferric oxide, ferrous sulfate, ferric phosphate or silica It anticipates a kind of or at least two combinations；Preferably in aluminium hydroxide, aluminum phosphate, calcium phosphate or calcium carbonate any one or at least Two kinds of combination；

Preferably, the shape of the inorganic particle be spherical, rodlike, sheet, fusiform, plate-like, cube, polyhedron or without calmly Any one in shape；

Preferably, the pattern of the inorganic particle be rough surface, porous surface, internal multi-chamber, it is hollow or simple eye in appoint It anticipates a kind of or at least two combinations；

Preferably, the surface modification of the inorganic particle has modification group；

3. antigen thermostabilization lotion according to claim 1 or 2, which is characterized in that the oil mutually includes biocompatibility Grease and/or mineral oil；

Preferably, the biocompatibility grease include squalene, soybean oil, Miglitol, midchain oil, fish oil, vitamin E, Vitamin E succinate, Vitwas E, safflower oil, corn oil, Seabuckthorn Oil, Linseed oil, peanut oil, tea oil, sunflower Seed oil, apricot kernel oil, tocopherol, coix seed oil, evening primrose oil, sesame oil, cottonseed oil, castor oil, castor oil analog, low erucic acid dish Seed oil, ethyl oleate, oleic acid, ethyl linoleate, isopropyl laurate, interior isopropyl myristate, ethyl butyrate, ethyl lactate, In Trivent OCG or Triglyceride DDD any one or at least two combination；

Preferably, the biocompatibility grease is any one in squalene, tocopherol, castor oil or castor oil analog Or at least two combination, preferably squalene.

4. antigen thermostabilization lotion according to any one of claim 1-3, which is characterized in that the water phase includes purifying In water, water for injection, glycerine water solution, 0.9% physiological saline, phosphate buffer, citrate buffer solution or Tris buffer Any one or at least two combination；

Preferably, the water phase be phosphate buffer, citrate buffer solution or Tris buffer in any one or extremely Few two kinds of combination；

Preferably, the pH value of the water phase is 5.5-8.5, preferably 6.0-8.0.

5. antigen thermostabilization lotion described in any one of -4 according to claim 1, which is characterized in that the antigen is selected from the mankind Antigen, non-human animal antigen, plant antigen, bacterial antigens, fungal antigen, viral antigen, parasite antigen or tumour antigen In any one or at least two combination.

6. antigen thermostabilization lotion according to any one of claims 1-5, which is characterized in that the oil phase and inorganic particulate The mass ratio of grain is (0.05-3): 1.

7. antigen thermostabilization lotion according to claim 1 to 6, which is characterized in that the inorganic particle is put down Equal partial size is 30nm-3 μm；

Preferably, the average grain diameter of the inorganic particle is 50nm-3 μm；

Preferably, the average grain diameter of the inorganic particle and antigen ratio is (10-30): 1；

Preferably, the inorganic particle and the contact angle of oily phase are 45 ° -90 °；

Preferably, mass concentration of the inorganic particle in water phase is 1wt.%-6wt.%；

Preferably, the oil is mutually 1:100-9:1 with the volume ratio of water phase；

Preferably, the oil is mutually 1:(4-50 with the volume ratio of water phase)；

Preferably, emulsion droplet average grain diameter formed in the antigen thermostabilization lotion is 800nm-3 μm；

8. the preparation method of antigen thermostabilization lotion described in any one of -7 according to claim 1, which is characterized in that the system Preparation Method includes: that antigen is added in the water phase containing inorganic particle, is incubated for after suspension, then is added to obtain by oil In water phase, it is mixed to get the antigen thermostabilization lotion.

9. according to the method described in claim 8, it is characterized in that, the amount of the inorganic particle of every 2-55 μ g/mL antigen binding is 50-1000μg/mL；

Preferably, the temperature of the incubation is 25 DEG C -45 DEG C；

Preferably, the time of the incubation is 0.5h-3h.

10. antigen thermostabilization lotion described in any one of -7 is preparing vaccine adjuvant, drug delivery system according to claim 1 Agent, the drug with inhibitive ability of immunity, fight chronic disease therapeutic agent ease up controlled release carrier in application.