CN112545992A

CN112545992A - Veterinary vaccine nanoemulsion and preparation method thereof

Info

Publication number: CN112545992A
Application number: CN202110050444.6A
Authority: CN
Inventors: 崔海信; 郭亮; 赵翔; 王春鑫; 崔博
Original assignee: Institute of Environment and Sustainable Development in Agriculturem of CAAS
Current assignee: Institute of Environment and Sustainable Development in Agriculturem of CAAS
Priority date: 2021-01-14
Filing date: 2021-01-14
Publication date: 2021-03-26
Anticipated expiration: 2041-01-14
Also published as: CN112545992B

Abstract

The invention discloses a veterinary vaccine nanoemulsion and a preparation method thereof, and the veterinary vaccine nanoemulsion comprises an inner water phase with the droplet average particle size of less than 50nm, an outer water phase with the continuous phase and the dispersion coefficient of less than 0.1, and a middle layer oil phase with the average particle size of less than 100 nm; wherein the weight percentages of the components are as follows: 5-30% of an inner water phase, 20-50% of a middle layer oil phase and 50-85% of an outer water phase; the preparation method of the veterinary vaccine nanoemulsion is simple, and the cost is saved; according to the invention, the antigen and/or immunopotentiator are released layer by layer, so that the stability and safety of the vaccine nanoemulsion are improved; the dispersion of the medicine is reduced through the mutual matching of the inner water phase, the outer water phase and the middle layer oil, and the absorption of the medicine is improved.

Description

Veterinary vaccine nanoemulsion and preparation method thereof

Technical Field

The invention belongs to the technical field of veterinary pharmacy, and particularly relates to a veterinary vaccine nanoemulsion and a preparation method thereof.

Background

The domestic inactivated vaccine for animals is mainly an emulsifiable solution system, namely the inactivated vaccine mainly comprises oil for injection, a surfactant and an antigen solution. At present, most of emulsion vaccines have particle sizes of more than 1 micron, and belong to common milk or coarse milk. The emulsion is a thermodynamically unstable system, is easy to generate layering, flocculation and the like to cause emulsion breaking, and has poor stability. In addition, the emulsion has larger particle size, and the oil phase and the antigen water phase are not fully dispersed, so that the emulsion has larger viscosity, difficult injection and serious side reaction at the injection part. Meanwhile, antigen molecules cannot be fully absorbed by animal bodies, so that the utilization rate of the antigen molecules is low.

In recent years, the rapid development of nanotechnology provides a new scientific methodology for modern agricultural science. Particularly, a drug delivery system is constructed by utilizing nano materials and technologies, and active ingredients are directionally transmitted and released to a target position in a living body, so that the method becomes a research hotspot of novel green veterinary drug products. The nano-emulsion liquid system has wide application prospect in the aspect of upgrading the traditional vaccine dosage form. Compared with common milk and coarse milk, the nano-emulsion is an isotropic transparent liquid and belongs to a thermodynamically stable system. And secondly, the viscosity of the nanoemulsion is low, so that the nanoemulsion is convenient to inject and can reduce the stress response of animals. In addition, the antigen molecules in the nano-emulsion can be fully dispersed in the emulsion by nano-emulsion droplets, so that the antigen molecules have larger specific surface area, the antigen molecules are favorably and fully absorbed by passive objects, and the utilization rate is improved. Antigen molecules in the nano milk can be simultaneously dispersed in the inner core water phase and the outer layer flowing water phase, and after the nano milk is injected into an animal body, the antigen in the outer layer flowing water phase can be quickly absorbed by the body, so that an immune system can be activated in a short time; the antigen of the inner core water phase is slowly and durably released under the protection of the middle layer oil phase, thereby achieving the purpose of slow release. Therefore, the nanoemulsion can combine quick action and long action.

CN105727284 discloses a biphase nano-emulsion adjuvant for a pig and cattle foot-and-mouth disease vaccine, a preparation method and application thereof. The vaccine nanoemulsion prepared by adopting one-step emulsification of the two-phase nanoemulsion adjuvant disclosed by the technology is milk white, the multiple emulsion structure composed of an inner water phase, an outer water phase and a middle-layer oil film phase is not obvious in characteristics, the particle size is not uniform, the ideal targeted transmission, controlled release and isolation characteristics of multiple emulsion nano-drugs cannot be exerted, and quick-acting property and long-acting property cannot be considered.

CN108578688 discloses a preparation method of a multiple nanoemulsion vaccine adjuvant. The raw materials for preparing the vaccine are non-mineral oil, such as peanut oil, squalene or palm oil, and the cost of the vaccine production cannot be reduced due to the generally high price of the raw materials, particularly the peanut oil, so that the economic burden of farmers is increased. The squalene used in the technology has the characteristic of easy oxidation, and the stability of the emulsion finished product is difficult to ensure in the actual production and storage processes. In terms of the preparation method, the shear rate used in the second step of the technology is 8000-10000rpm, which belongs to the range of high energy emulsification, and the initial oil-in-water phase is still likely to be converted into oil-in-water. In addition, the particle size of the nano-emulsion prepared by the method is within 200-500nm, the particle size is relatively coarse, the distribution is not uniform, and the fine emulsion size (< 100 nm) of a nano-drug targeted delivery system is difficult to achieve.

Disclosure of Invention

The invention aims to provide a veterinary vaccine nanoemulsion and a preparation method thereof, and aims to solve the problem of how to prepare a clear, transparent and stable vaccine nanoemulsion; how to reduce the production cost of the veterinary vaccine nanoemulsion; how to realize the layer-by-layer release of the drugs through the interaction of the inner water phase, the outer water phase and the middle oil phase, and the preparation problem of the veterinary vaccine nanoemulsion is solved by reducing the dispersion of the drugs and improving the absorption of the drugs.

In order to achieve the purpose, the invention adopts the following technical scheme: a veterinary vaccine nanoemulsion comprises an inner water phase with droplet average particle size less than 50nm, an outer water phase with continuous phase and dispersion coefficient less than 0.1, and an intermediate oil phase with average particle size less than 100 nm. Wherein the weight percentages of the components are as follows: 5-30% of an internal water phase, 20-50% of a middle layer oil phase and 50-85% of an external water phase.

Further, the inner water phase comprises an antigen solution and/or an immunopotentiator, wherein the weight percentage ratio of the antigen solution to the immunopotentiator is that the antigen solution is as follows: immunopotentiator =1: (0.01-0.1).

Further, the middle layer oil phase comprises mineral oil, non-mineral oil and mixed surfactant I; wherein the weight percentage of the mineral oil and the non-mineral oil is mineral oil: non-mineral oil = (1-10): 1; the non-mineral oil is squalane; the weight percentage of the mineral oil, the non-mineral oil and the mixed surfactant I is as follows: mixed surfactant one = 10: (1-10).

Further, the range of the hydrophilic-lipophilic balance value of the first mixed surfactant is 3-8; wherein the first mixed surfactant is propylene glycol monolaurate and polyoxyethylene monolaurate; or span 85 and polyethylene glycol 15-hydroxystearate; or glyceryl monostearate and isopropyl myristate; or a mixture of diethylene glycol monostearate and polyoxyethylene alkyl aryl ether.

Wherein the weight percentage of the propylene glycol monolaurate and the polyoxyethylene monolaurate is that the propylene glycol monolaurate: polyoxyethylene monolaurate = (1-10): (1-10).

The weight percentage of the span 85 and the polyethylene glycol 15-hydroxystearate is that the span 85: polyethylene glycol 15-hydroxystearate = (1-10): (1-10).

The weight percentage of the glyceryl monostearate and the isopropyl myristate is that of the glyceryl monostearate: isopropyl myristate = (1-10): (1-10).

The weight percentage of the diethylene glycol monostearate and the polyoxyethylene alkyl aryl ether is as follows: polyoxyethylene alkylaryl ether = (1-10): (1-10).

Further, the external water phase comprises an antigen solution and/or an immunopotentiator, and a mixed surfactant II; the weight percentage of the antigen solution and/or the immunopotentiator to the mixed surfactant II is as follows: mixed surfactant two =1: (0.1 to 1); the weight percentage of the antigen solution and the immunopotentiator is that the antigen solution is: immunopotentiator =1: (0.01-0.1).

Further, the range of the hydrophilic-lipophilic balance value of the mixed surfactant II is 9-15, wherein the mixed surfactant II is span-60 and polyethylene glycol monopalmitate; or tween 85 and cremophor; or ethylene glycol fatty acid esters and polyoxyethylene monostearate; or a mixture of propylene glycol fatty acid ester and polyoxyethylene monooleate.

Wherein the weight percentage of the span 60 to the polyethylene glycol monopalmitate is span 60: polyethylene glycol monopalmitate = (1-10): (1-10).

The weight percentage of the tween 85 and the polyoxyethylene castor oil is that the tween 85: polyoxyethylene castor oil = (1-10): (1-10).

The weight percentage of the glycol fatty acid ester and the polyoxyethylene monostearate is that the glycol fatty acid ester: polyoxyethylene monostearate = (1-10): (1-10).

The weight percentage of the propylene glycol fatty acid ester and the polyoxyethylene monooleate is that the propylene glycol fatty acid ester: polyoxyethylene monooleate = (1-10): (1-10).

Further, the antigen is at least one of inactivated virus, polypeptide, protein, nucleic acid and virus-like particle; such as inactivated avian influenza virus, inactivated newcastle disease virus, inactivated foot and mouth disease virus, foot and mouth disease virus-like particles, synthetic peptide antigen of foot and mouth disease, inactivated goat pox virus, and the like.

Further, the immunopotentiator is at least one of polysaccharide, active peptide, cytokine, nucleic acid, immunoglobulin or Chinese medicinal extract, such as astragalus polysaccharide, levamisole, grifola polysaccharide, interleukin, etc.

In addition, the invention also provides a preparation method of the veterinary vaccine nanoemulsion, which comprises the following specific steps.

s1, preparing an internal water phase; dispersing an immunopotentiator in an antigen solution, magnetically stirring at 600-1000 rpm for 5min, uniformly mixing, and adjusting the pH value to 6.8-7.4;

s2, preparing a middle layer oil phase; the weight percentage of the mineral oil, the non-mineral oil and the mixed surfactant I is as follows: mixed surfactant one = 10: (1-10), dissolving the first mixed surfactant in mineral oil and non-mineral oil; uniformly mixing the mixture by magnetic stirring at 600-1000 rpm for 5min to prepare an intermediate oil phase;

s3, quickly pouring the intermediate layer oil phase of s2 into the inner water phase of s1, emulsifying for 5min at the temperature of 25 ℃ and the shearing speed of 5000-8000 rpm, and standing for 10-20 min to obtain water-in-oil primary emulsion;

s4, preparing an external water phase; the weight percentage of the mixed surfactant II to the rest antigen solution in the s1 is that the mixed surfactant II: adding a second mixed surfactant into the rest antigen solution in s1, and magnetically stirring and uniformly mixing at 600-1000 rpm to obtain an outer water phase, wherein the rest antigen solution in s1 is =1 (1-10);

s5, pouring the external water phase and the water-in-oil colostrum into a constant-temperature pipeline type ultrahigh-speed homogenizing emulsifying machine, wherein the mass ratio of the external water phase to the water-in-oil colostrum is = (2-5): 1, emulsifying at the temperature of 25 ℃, under the pressure of 0.2-0.5 MPa and at the shearing speed of 500-1000 rpm, and then standing for 10-20 min; the prepared veterinary vaccine nanoemulsion has the mass percentage of antigen not more than 80%.

Further, the preparation of the internal and external aqueous phases in steps s1 and s4 may be performed by adding the immunopotentiator and/or antigen to physiological saline or a culture medium solution, and then concentrating or diluting according to the concentration requirement of the target vaccine to obtain internal and external aqueous phases with corresponding concentrations.

Further, the storage temperature range of the veterinary vaccine nanoemulsion prepared in s5 is 4-12 ℃, the normal-temperature storage adaptive region and season period of the vaccine is prolonged, the refrigeration transportation cost is reduced, and the quality guarantee period of the product is prolonged.

Compared with the prior art, the invention has the following beneficial effects:

1. the invention relates to a veterinary vaccine nanoemulsion and a preparation method thereof, wherein the veterinary vaccine nanoemulsion is a clear and transparent liquid with low viscosity and good fluidity; the three-layer structure of oil-in-water and water-in-oil is formed, so that the effective components of antigen and immunopotentiator are gradually released, the loss in a pharmacokinetic phase and a pharmacodynamic phase is reduced, and the drug absorption is improved by reducing the dispersion of the drug; the use level of the effective components is reduced, and simultaneously, a better absorption effect is achieved.

2. The invention relates to a veterinary vaccine nanoemulsion and a preparation method thereof.A middle layer oil phase is a mixture of mineral oil and non-mineral oil, and compared with the traditional paraffin oil or mineral oil with single component, the veterinary vaccine nanoemulsion ensures that a medicament which is easy to hydrolyze is protected by the oil phase, has a slow release effect and is gradually absorbed by an organism, and gradually absorbs to improve the absorption and metabolism efficiency of an animal organism. In addition, the method does not increase excessive production cost.

3. The invention relates to a veterinary vaccine nanoemulsion and a preparation method thereof, the veterinary vaccine nanoemulsion comprises an inner water phase, an outer water phase and an intermediate oil phase which are mixed to form a transparent multiple emulsion system, wherein the average particle size of microdroplets of the inner water phase is less than 50nm, the average particle size of an oil film phase is less than 100nm, the outer water phase is a continuous phase, and the rapid-acting and long-acting function regulation of the vaccine immune effect can be realized through the multiple emulsion structure and the accurate control of the content and the proportion of antigens and immune enhancing substances in the inner water phase and the outer water phase.

4. The veterinary vaccine nanoemulsion has a certain broad spectrum and is applicable to common antigens of poultry, pigs, cattle and sheep and the like.

5. The veterinary vaccine nanoemulsion has the effects of high immune activity, quick response, long lasting period, low residue and toxic and side effects, stable product performance and excellent cost performance, and is favorable for remarkably improving the production efficiency and the product quality safety of animals.

6. The preparation method is simple, the veterinary vaccine nanoemulsion can be rapidly produced, and the veterinary vaccine nanoemulsion can be stably stored at 4-12 ℃, so that the purposes of expanding the adaptive area and season limit of vaccine storage at normal temperature, reducing the production and refrigerated transportation cost and improving the production efficiency are achieved while the vaccine effect is not influenced.

Additional features and advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by the practice of the invention. The primary objects and other advantages of the invention may be realized and attained by the instrumentalities particularly pointed out in the specification.

Reference numerals:

FIG. 1: photo of the foot-and-mouth disease inactivated vaccine nanoemulsion.

FIG. 2: and (3) a frozen transmission electron microscope photo of the foot-and-mouth disease inactivated vaccine nanoemulsion.

FIG. 3: the particle size distribution map of the foot-and-mouth disease inactivated vaccine nanoemulsion.

FIG. 4-1: photograph of the foot-and-mouth disease inactivated vaccine nanoemulsion stored at 4 ℃.

FIG. 4-2: photograph of the inactivated foot-and-mouth disease vaccine nanoemulsion of fig. 4-1 stored for one year at 4 ℃.

FIG. 5-1: photo of foot-and-mouth disease inactivated vaccine nanoemulsion stored at 10 ℃.

FIG. 5-2: photograph of the inactivated foot-and-mouth disease vaccine nanoemulsion of fig. 5-1 stored for one year at 10 ℃.

FIG. 6-1: section of the mouse liver after inoculation of the foot-and-mouth disease inactivated vaccine nanoemulsion.

FIG. 6-2: section of mouse liver without inactivated foot and mouth disease vaccine nanoemulsion.

FIGS. 6-3: section of mouse spleen after inoculation of foot-and-mouth disease inactivated vaccine nanoemulsion.

FIGS. 6 to 4: section of mouse spleen without inactivated foot-and-mouth disease vaccine nanoemulsion.

FIGS. 6 to 5: section of mouse lung after inoculation of foot-and-mouth disease inactivated vaccine nanoemulsion.

FIGS. 6 to 6: section of mouse lung without inactivated foot and mouth disease vaccine nanoemulsion.

FIGS. 6 to 7: section image of mouse kidney after inoculation of foot-and-mouth disease inactivated vaccine nanoemulsion.

FIGS. 6 to 8: section image of mouse kidney without inactivated foot and mouth disease vaccine nanoemulsion.

FIGS. 6 to 9: and (3) a section of the muscle of the mouse after the foot-and-mouth disease inactivated vaccine nanoemulsion is inoculated.

FIGS. 6 to 10: section of mouse muscle without inactivated foot and mouth disease vaccine nanoemulsion.

FIG. 7: and (3) an antibody detection result graph of mice of different groups inoculated with the foot-and-mouth disease inactivated vaccine nanoemulsion.

Detailed Description

The present invention will be described in detail with reference to specific examples. The following examples will assist those skilled in the art in further understanding the invention, but are not intended to limit the invention in any way. It should be noted that variations and modifications can be made by persons skilled in the art without departing from the spirit of the invention. All falling within the scope of the present invention.

Firstly, preparing avian influenza H7N9 inactivated vaccine nanoemulsion sample

Example 1: preparation of avian influenza H7N9 inactivated vaccine nanoemulsion 1:

s1, preparing an internal water phase; dispersing levamisole (mass fraction is 1%) in an antigen solution, magnetically stirring at 800rpm for 5min, uniformly mixing, adjusting pH value to 6.9 to obtain an antigen dispersion liquid, and taking 5g as an internal water phase;

s2, preparing a middle layer oil phase; uniformly stirring and mixing propylene glycol monolaurate and polyoxyethylene monolaurate according to the ratio of 3:1 to prepare a first mixed surfactant; and then uniformly mixing the mineral oil, squalane and the mixed surfactant I according to the proportion of 5:1:3, and magnetically stirring at 800rpm for 5min to prepare the middle-layer oil phase.

s3, quickly pouring 25g of the oil phase in the middle layer of 2 into 5g of the inner water phase of 1, emulsifying for 5min at the temperature of 25 ℃ and the shearing speed of 8000rpm, and standing for 10-20 min to obtain the water-in-oil primary emulsion.

s4, preparing an external water phase; taking 7g of span 60, 7g of polyethylene glycol monopalmitate and 56g of antigen dispersion liquid in s1 (the proportion of the three is 1:1: 8), magnetically stirring at 700rpm for 5min, and uniformly mixing to obtain an external water phase.

s5, pouring 70g of the external water phase of 4 and 30g of the 3 water-in-oil primary emulsion (the ratio of the two is 2.33: 1) into a constant-temperature pipeline type ultrahigh-speed homogenizing emulsifying machine, and emulsifying for 5min under the conditions that the temperature is kept at 25 ℃, the pressure is 0.2MPa and the shearing speed is 800rpm to prepare the avian influenza H7N9 inactivated vaccine nanoemulsion 1, wherein the mass percentage of the antigen dispersion liquid is 61%.

Example 2: preparation of avian influenza H7N9 inactivated vaccine nanoemulsion 2:

s1, preparing an internal water phase; dispersing levamisole (mass fraction is 1%) in an antigen solution, magnetically stirring at 800rpm for 5min, uniformly mixing, adjusting pH value to 6.9 to obtain an antigen dispersion liquid, and taking 10g as an internal water phase;

s2, preparing a middle layer oil phase; uniformly stirring and mixing span 85 and polyethylene glycol 15-hydroxystearate according to the proportion of 2.5:1 to prepare a first mixed surfactant, and magnetically stirring and uniformly mixing mineral oil, squalane and the first mixed surfactant at the proportion of 4:1:2 and 720rpm for 5min to prepare an intermediate oil phase;

s3, quickly pouring 20g of the middle layer oil phase of 2 into 10g of the inner water phase of 1, and emulsifying for 5min at the temperature of 25 ℃ and the shearing speed of 8000rpm to obtain water-in-oil primary emulsion;

s4, preparing an external water phase; taking 7g of span 60, 14g of polyethylene glycol monopalmitate and 49g of antigen dispersion liquid in s1 (the proportion of the three is 1:2: 7), and magnetically stirring at 700rpm for 5min to mix uniformly to obtain an external water phase.

s5, pouring 70g of the external water phase of 4 and 30g of the 3 water-in-oil primary emulsion (the ratio of the two is 2.33: 1) into a constant-temperature pipeline type ultrahigh-speed homogenizing emulsifying machine, and emulsifying for 5min under the conditions that the temperature is kept at 25 ℃, the pressure is 0.2MPa and the shearing speed is 800rpm to prepare the avian influenza H7N9 inactivated vaccine nanoemulsion 2, wherein the mass percentage of the antigen dispersion liquid is 59%.

Example 3: preparation of avian influenza H7N9 inactivated vaccine nanoemulsion 3:

s1, preparing an internal water phase; dispersing levamisole (mass fraction is 1%) in an antigen solution, magnetically stirring at 800rpm for 5min, uniformly mixing, adjusting pH value to 6.9 to obtain an antigen dispersion liquid, and taking 15g as an internal water phase;

s2, preparing a middle layer oil phase; uniformly stirring and mixing glyceryl monostearate and isopropyl myristate according to the ratio of 4:1 to prepare a first mixed surfactant, and uniformly stirring and mixing mineral oil, squalane and the first mixed surfactant according to the ratio of 5:2:2 to prepare an intermediate oil phase;

s3, quickly pouring 15gs of the middle layer oil phase of 2 into 15gs of the inner water phase of 1, and emulsifying for 5min at the temperature of 25 ℃ and the shearing speed of 8000rpm to obtain water-in-oil primary emulsion;

s4, preparing an external water phase; taking 7g of span 60, 21g of polyethylene glycol monopalmitate and 42g of antigen dispersion liquid in s1 (the proportion of the three is 1:3: 6), and magnetically stirring at 700rpm for 5min to mix uniformly to obtain an external water phase.

s5, pouring 70g of an external water phase of 4 and 30g of water-in-oil colostrum of 3 (the proportion of the two is 2.33: 1) into a constant-temperature pipeline type ultra-high-speed homogenizing emulsifying machine, emulsifying for 5min at 25 ℃, 0.2MPa and 800rpm to prepare the avian influenza H7N9 inactivated vaccine nanoemulsion 3, wherein the mass percentage of the antigen dispersion is 57%.

Comparative example 1: preparation of avian influenza H7N9 inactivated vaccine ordinary emulsion:

selecting commercially available water-in-oil adjuvant (MONTANI DETM ISA 71VG), and emulsifying 70% of the MONTANI DETM ISA 71VG adjuvant and 30% of H7N9 antigen solution at the rotation speed of 300-500rpm for 15-25min at 15-25 ℃. The avian influenza H7N9 inactivated vaccine common emulsion is prepared, wherein the mass percentage of the antigen solution is 30%.

Second, the physical and chemical property detection of the avian influenza H7N9 inactivated vaccine nanoemulsion sample

The appearances, particle size ranges, viscosities and stabilities of 3 avian influenza H7N9 inactivated vaccine nano emulsions 1-3 prepared in the examples and common emulsions are characterized and compared with comparative example 1.

The appearance adopts a visual observation method to judge the transparency degree of the emulsion.

The particle size range was measured using a Malvern particle sizer, three times for each sample, and the mean value was taken.

The viscosity test is carried out by sucking 1mL of the emulsion with a 1mL glass pipette (lower mouth having an inner diameter of 1.2mm and upper mouth having an inner diameter of 2.7mm) at room temperature of 25 ℃, allowing the emulsion to flow out vertically and naturally, recording the time required for 0.4mL of the emulsion to flow out, measuring the time for three times, and averaging the values.

And the centrifugal stability test is to centrifuge a proper amount of emulsion for 30min at 6000rpm/min of a centrifuge and observe whether the emulsion is layered and demulsified.

The storage stability test is to take 5 bottles of each emulsion, place the bottles at 4 ℃ and 10 ℃ for one year, and observe whether the emulsions are layered or demulsified.

The results of comparing the appearance, particle size range, viscosity, stability and other physicochemical properties of the avian influenza H7N9 inactivated vaccine nano-emulsions 1-3 prepared in 3 examples with those of a common emulsion are shown in Table 1.

Table 1: the physicochemical properties of the veterinary vaccine nanoemulsions of examples 1 to 3 and comparative example 1 were compared.

Thirdly, safety evaluation of avian influenza H7N9 inactivated vaccine nanoemulsion sample

The safety test is to inject 10 SPF (specific pathogen free) chickens with 7 days old into 1mL of emulsion, observe whether the test animals have side reaction, feed intake change, morbidity and mortality, influence on egg laying and the like, and make relevant records. The result is characterized in that all the animals in the grouping test have no side effect, no death and normal laying rate.

The immunopotency test used 50 SPF chickens 7 days old, divided into 5 groups of 10. The emulsions prepared in examples 1 to 3 and comparative example 1 were injected intramuscularly into each chicken, wherein, in order to ensure that the amount of antigen injected into each chicken was consistent, the experiment employed 0.49mL per chicken in example 1 group, 0.51 mL per chicken in example 2 group, 0.53 mL per chicken in example 3 group and 1mL per chicken in comparative example group. Control group each chicken was injected with 1mL of physiological saline. Sera were taken at 7, 14, 21 and 28 days post immunization and tested for immunopotency as shown in table 2.

Table 2: examples 1-3 and comparative example 1 a comparison of the immunological activities of different emulsions was prepared.

Therefore, compared with the comparative example 1 in the prior art, the nano emulsion for the veterinary vaccine provided by the invention has the advantages of clear and transparent appearance, small particle size which is below 100nm, stability after being stored at 4 ℃ and 10 ℃ for 1 year, and no layering phenomenon. In the comparative example, one bottle was delaminated at each of month 4 and month 8 under the storage condition at 4 ℃. One bottle was stratified at month 2, month 3, month 5 and month 9 under 10 ℃ storage conditions. From the results of animal experiments, compared with comparative example 1, the veterinary vaccine nanoemulsion provided by the invention stimulates the animals to produce higher antibodies at day 7 (especially example 3), and the serum antibody titer is higher than that of comparative example 1 after 28 days of immunization. The results show that the veterinary vaccine nanoemulsion provided by the invention has good stability at 4 ℃ and 10 ℃, can provide faster immune response after being used for immunizing animals, and has better comprehensive antibody level than the nano vaccine emulsion prepared by the prior art.

Fourthly, preparing the nano emulsion sample of the inactivated foot-and-mouth disease vaccine

Example 4. preparation of inactivated foot-and-mouth disease vaccine nanoemulsion:

s1, preparing an internal water phase; dispersing ganoderan (1% by mass) in antigen solution, magnetically stirring at 800rpm for 5min, mixing, adjusting pH to 7.0 to obtain antigen dispersion, and taking 10g as internal water phase; (ii) a

s2, preparing a middle layer oil phase; uniformly stirring and mixing glyceryl monostearate and isopropyl myristate in a ratio of 1:1 to obtain a first mixed surfactant, and uniformly stirring and mixing mineral oil, squalane and the first mixed surfactant in a ratio of 5:2:2 to obtain an intermediate oil phase;

s4, preparing an external water phase; mixing 15g propylene glycol fatty acid ester, 30g polyoxyethylene monooleate and 75g s1 antigen dispersion solution (ratio of 1.5:3: 7.5) at 700rpm under magnetic stirring for 5min to obtain external water phase.

s5, pouring 120g of external water phase 4 and 30g of water-in-oil primary emulsion 3 (the ratio of the two is 4: 1) into a constant-temperature pipeline type ultrahigh-speed homogenizing emulsifying machine, and emulsifying for 5min under the conditions that the temperature is kept at 25 ℃, the pressure is 0.4MPa and the shearing speed is 600rpm to prepare the foot-and-mouth disease inactivated vaccine nanoemulsion, wherein the mass percentage of the antigen solution is 56.7%.

Physicochemical property of nano emulsion of foot-and-mouth disease inactivated vaccine

As shown in figure 1, the whole appearance of the foot-and-mouth disease inactivated vaccine nanoemulsion is clear and transparent, and the content of the periphery of a bottle for storing the emulsion is background and is not related to the invention.

As shown in figure 2, the frozen transmission electron microscope photograph of the nano-emulsion of the inactivated foot-and-mouth disease vaccine shows that the nano-emulsion of the inactivated foot-and-mouth disease vaccine has uniform particle size distribution and the structures of the oil film of the middle layer and the water phase of the inner layer are clear and visible. Wherein the average particle diameter of the middle layer oil film is 80 nm, and the average particle diameter of the inner layer water phase is 50 nm.

As shown in figure 3, a Malvern particle sizer is used to test the particle size distribution emulsion graph of the nano-emulsion of the inactivated foot-and-mouth disease vaccine, the average hydrated particle size is 70nm, and the data is basically consistent with the data obtained by a frozen transmission electron microscope.

The average viscosity of the nano emulsion of the foot-and-mouth disease inactivated vaccine measured by a capillary method is 5.7 s.0.4 ml^-1Meet the requirement of vaccine viscosity (<8s·0.4ml^-1）。

As shown in fig. 4-1, 4-2, 5-1 and 5-2, the photographs of the inactivated foot-and-mouth disease vaccine nanoemulsion at 4 ℃ and 10 ℃ during and after one year of storage can be seen, and no delamination or emulsion breaking occurs, indicating that the stability is good; the red horizontal lines and black letters in fig. 4-1, 4-2 and 5-1, 5-2 are included in the photographic background and are not relevant to the present invention.

And evaluating the safety of the foot-and-mouth disease inactivated vaccine nanoemulsion:

12 BALB/C mice weighing around 20g were divided into 2 groups of 8 mice each. The first group of the animals are injected with 0.2 mL of foot-and-mouth disease inactivated vaccine nanoemulsion through the muscle, and the second group of the animals are injected with 0.2 mL of normal saline (blank group), and no death or obvious adverse local or systemic reaction is caused after two weeks of observation. The tissue sections of the main organs are shown in figures 6-1 to 6-10, and the results show that the foot-and-mouth disease inactivated vaccine nanoemulsion has good safety and no obvious toxic or side effect.

And evaluating the immune effect of the foot-and-mouth disease inactivated vaccine nanoemulsion:

24 BALB/C mice weighing around 20g were divided into 3 groups of 8 mice each. In order to ensure that the amount of antigen injected into each chicken is consistent, 0.18 mL of inactivated vaccine nanoemulsion of foot and mouth disease is injected subcutaneously into the first group, 0.2 mL of commercial inactivated vaccine of foot and mouth disease (provided by Zhongnong W ü te, Lanzhou, wherein the mass percentage of the antigen solution is 50%) is injected intramuscularly into the second group, 0.2 mL of physiological saline (blank group) is injected intramuscularly into the third group, venous blood is collected respectively 7 days, 14 days, 21 days and 28 days after immunization, and the serum is subjected to liquid phase blocking ELISA test to determine the comprehensive antibody level (figure 7). The result shows that compared with the commercial foot-and-mouth disease inactivated vaccine, the speed of generating the antibody by the foot-and-mouth disease inactivated vaccine nanoemulsion is high, and the content of the antibody generated by the foot-and-mouth disease inactivated vaccine nanoemulsion is far higher than that of the commercial group after the vaccine is inoculated for 28 days.

In addition, healthy pigs weighing about 40kg were selected for challenge tests. The results show that: the foot-and-mouth disease inactivated vaccine nanoemulsion is the same as a commercially available group, and can prevent and control foot-and-mouth disease in percentage.

Preparation method of other veterinary vaccine nanoemulsion

In addition, the method for preparing the veterinary vaccine nanoemulsion can also be used for preparing the following vaccine nanoemulsions. But are not limited to, the following vaccine nanoemulsions.

Example 5: preparing the nano emulsion of the inactivated vaccine against the newcastle disease:

s1, preparing an internal water phase; preparing an internal water phase; dispersing polyporus polysaccharide (mass fraction 1%) in antigen solution, magnetically stirring at 800rpm for 5min, mixing, adjusting pH to 7.1 to obtain antigen dispersion, and taking 10g as internal water phase;

s2, preparing a middle layer oil phase; uniformly stirring and mixing diethylene glycol monostearate and polyoxyethylene alkyl aryl ether in a ratio of 2:1 to prepare a first mixed surfactant, and uniformly stirring and mixing mineral white oil, squalane and the first mixed surfactant in a ratio of 4:1:2 to prepare an intermediate oil phase;

s3, quickly pouring 20g of the middle layer oil phase of 2 into 10g of the inner water phase of 1, emulsifying for 5min at the temperature of 25 ℃ and the shearing speed of 5000 rpm, and standing for 10-20 min to obtain water-in-oil primary emulsion;

s4, preparing an external water phase; uniformly mixing 8.75g of propylene glycol fatty acid ester, 17.5g of polyoxyethylene monooleate and 43.75g of antigen dispersion liquid in 43.75g s1 (the ratio of the three is 1:2: 5) under magnetic stirring at 700rpm for 5min to obtain an external water phase for preparing an external water phase;

s5, pouring 70g of the external water phase 4 and 30g of the 3 water-in-oil primary emulsion (the ratio of the two is 2.33: 1) into a constant-temperature pipeline type ultrahigh-speed homogenizing emulsifying machine, emulsifying for 5min under the conditions that the temperature is kept at 25 ℃, the pressure is 0.4MPa and the shearing speed is 600rpm, and standing for 10-20 min to prepare the newcastle disease inactivated antigen solution, wherein the mass percentage of the antigen solution is 53.75%.

Example 6 preparation of inactivated vaccine nanoemulsion for porcine diarrhea:

s1, preparing an internal water phase; dispersing lentinan (mass fraction 1%) in antigen solution, magnetically stirring at 800rpm for 5min, mixing, adjusting pH to 7.3 to obtain antigen dispersion, and taking 12g as internal water phase;

s2, preparing a middle layer oil phase; uniformly mixing span 85 and polyethylene glycol 15-hydroxystearate for 5min at a ratio of 2:1 to obtain a first mixed surfactant, and uniformly mixing mineral white oil, squalane and the first mixed surfactant at a ratio of 6:1:2 to obtain an intermediate oil phase.

s3, quickly pouring 18g of the intermediate oil phase of 2 into 12g of the inner water phase of 1, and emulsifying for 5min at the temperature of 25 ℃ and the shearing speed of 7000 rpm to obtain water-in-oil primary emulsion;

s4, preparing an external water phase; taking 24g of tween 85, 6g of polyoxyethylene castor oil and an antigen dispersion liquid in 60g s1 (the ratio of the three is 4:1: 10), magnetically stirring at 700rpm for 5min, and uniformly mixing to obtain an external water phase for preparing an external water phase;

s5, pouring 90g of external water phase 4 and 30g of water-in-oil primary emulsion 3 (the ratio of the two is 3: 1) into a constant-temperature pipeline type ultrahigh-speed homogenizing emulsifying machine, emulsifying for 5min under the conditions that the temperature is kept at 25 ℃, the pressure is 0.4MPa and the shearing speed is 900rpm, and standing for 10-20 min to obtain the inactivated vaccine nanoemulsion for porcine diarrhea, wherein the mass percentage of the antigen solution is 60%.

Example 7 preparation of goatpox inactivated vaccine nanoemulsion:

s1, preparing an internal water phase; dispersing astragalus polysaccharide (mass fraction is 1%) in an antigen solution, magnetically stirring at 800rpm for 5min, mixing uniformly, adjusting the pH value to 6.8 to obtain an antigen dispersion, and taking 12g as an internal water phase;

s2, preparing a middle layer oil phase; uniformly stirring and mixing propylene glycol monolaurate and polyoxyethylene monolaurate according to the ratio of 2:1 to prepare a first mixed surfactant, and uniformly stirring and mixing mineral oil, squalane and the first mixed surfactant according to the ratio of 6:2:3 to prepare an intermediate oil phase;

s3, quickly pouring 18g of the middle layer oil phase of 2 into 12g of the inner water phase of 1, and emulsifying for 5min at the temperature of 25 ℃ and the shearing speed of 6000rpm to obtain water-in-oil primary emulsion;

s4, preparing an external water phase; taking 7.5g of span 60, 22.5g of polyethylene glycol monopalmitate and an antigen dispersion liquid in 60g s1 (the ratio of the three is 1:3: 8), magnetically stirring at 700rpm for 5min, and uniformly mixing to obtain an external water phase and prepare an external water phase;

s5, pouring 90g of external water phase 4 and 30g of water-in-oil primary emulsion 3 (the ratio of the two is 3: 1) into a constant-temperature pipeline type ultrahigh-speed homogenizing emulsifying machine, emulsifying for 5min under the conditions that the temperature is kept at 25 ℃, the pressure is 0.5MPa and the shearing speed is 700rpm, and standing for 10-20 min to prepare the goat pox inactivated vaccine nanoemulsion, wherein the mass percentage of the antigen solution is 60%.

In summary, the present invention is only a preferred embodiment, and not intended to limit the scope of the invention, and all equivalent changes and modifications in the shape, structure, characteristics and spirit of the present invention described in the claims should be included in the scope of the present invention.

Claims

1. A veterinary vaccine nanoemulsion is characterized by comprising an inner water phase with droplet average particle size less than 50nm, an outer water phase with continuous phase and dispersion coefficient less than 0.1, and an intermediate oil phase with average particle size less than 100 nm;

wherein the weight percentages of the components are as follows: 5-30% of an internal water phase, 20-50% of a middle layer oil phase and 50-85% of an external water phase.

2. The veterinary vaccine nanoemulsion of claim 1, wherein the internal aqueous phase comprises an antigen solution and/or an immunopotentiator, wherein the weight percentage of immunopotentiator is antigen solution: immunopotentiator =1: (0.01-0.1).

3. The veterinary vaccine nanoemulsion of claim 1, wherein said middle oil phase comprises a mineral oil, a non-mineral oil and a first mixed surfactant; wherein the weight percentage of the mineral oil and the non-mineral oil is mineral oil: non-mineral oil = (1-10): 1; the non-mineral oil is squalane; the weight percentage of the mineral oil, the non-mineral oil and the mixed surfactant I is as follows: mixed surfactant one = 10: (1-10).

4. The veterinary vaccine nanoemulsion of claim 1, wherein the range of the hydrophilic-lipophilic balance value of the first mixed surfactant is between 3 and 8; wherein the first mixed surfactant is propylene glycol monolaurate and polyoxyethylene monolaurate; or span 85 and polyethylene glycol 15-hydroxystearate; or glyceryl monostearate and isopropyl myristate; or a mixture of diethylene glycol monostearate and polyoxyethylene alkyl aryl ether;

wherein the weight percentage of the propylene glycol monolaurate and the polyoxyethylene monolaurate is that the propylene glycol monolaurate: polyoxyethylene monolaurate = (1-10): (1-10);

the weight percentage of the span 85 and the polyethylene glycol 15-hydroxystearate is that the span 85: polyethylene glycol 15-hydroxystearate = (1-10): (1-10);

the weight percentage of the glyceryl monostearate and the isopropyl myristate is that of the glyceryl monostearate: isopropyl myristate = (1-10): (1-10);

5. The veterinary vaccine nanoemulsion of claim 1, wherein said external aqueous phase comprises an antigen solution and/or an immunopotentiator, and a second mixed surfactant; the weight percentage of the antigen solution and/or the immunopotentiator to the mixed surfactant II is as follows: mixed surfactant two = 10: (1-10); the weight percentage of the antigen solution and the immunopotentiator is that the antigen solution is: immunopotentiator =1: (0.01-0.1).

6. The veterinary vaccine nanoemulsion according to claim 1, wherein the hydrophilic-lipophilic balance value of the second mixed surfactant is in the range of 9-15, wherein the second mixed surfactant is span 60 and polyethylene glycol monopalmitate; or tween 85 and cremophor; or ethylene glycol fatty acid esters and polyoxyethylene monostearate; or a mixture of propylene glycol fatty acid ester and polyoxyethylene monooleate;

wherein the weight percentage of the span 60 to the polyethylene glycol monopalmitate is span 60: polyethylene glycol monopalmitate = (1-10): (1-10);

the weight percentage of the tween 85 and the polyoxyethylene castor oil is that the tween 85: polyoxyethylene castor oil = (1-10): (1-10);

the weight percentage of the glycol fatty acid ester and the polyoxyethylene monostearate is that the glycol fatty acid ester: polyoxyethylene monostearate = (1-10): (1-10);

7. The veterinary vaccine nanoemulsion according to claim 2 or 5, wherein said antigen is at least one of inactivated virus, polypeptide, protein, nucleic acid, virus-like particle.

8. The veterinary vaccine nanoemulsion of claim 2 or 5, wherein the immunopotentiator is at least one of polysaccharides, active peptides, cytokines, nucleic acids, immunoglobulins or herbal extracts.

9. A method for preparing a veterinary vaccine nanoemulsion according to any of claims 1-6, characterized by the following steps:

10. The method of claim 9, wherein the temperature range of the nano emulsion for veterinary vaccine prepared in s5 is 4-12 ℃.