WO2015192603A1 - 一种不含表面活性剂的水包油乳液及其用途 - Google Patents

一种不含表面活性剂的水包油乳液及其用途 Download PDF

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WO2015192603A1
WO2015192603A1 PCT/CN2014/092290 CN2014092290W WO2015192603A1 WO 2015192603 A1 WO2015192603 A1 WO 2015192603A1 CN 2014092290 W CN2014092290 W CN 2014092290W WO 2015192603 A1 WO2015192603 A1 WO 2015192603A1
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oil
antigen
water
emulsion
water emulsion
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PCT/CN2014/092290
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English (en)
French (fr)
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马光辉
吴颉
杨柳青
夏宇飞
齐峰
范清泽
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中国科学院过程工程研究所
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Priority to JP2016573130A priority Critical patent/JP6434995B2/ja
Priority to US14/909,593 priority patent/US20160175432A1/en
Priority to EP14895356.5A priority patent/EP3015114B1/en
Publication of WO2015192603A1 publication Critical patent/WO2015192603A1/zh

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Definitions

  • the present invention relates to a biological product, in particular to an oil-in-water emulsion which can be used in human or other animals. It is particularly important that the oil-in-water emulsion of the present invention contains no surfactant and uses solid particles as an emulsion.
  • Stabilizer which can be used as a vaccine adjuvant or drug delivery or slow release carrier, belongs to the field of biomedical technology.
  • Simultaneous application or synergistic application of adjuvants and antigens can enhance the body's immune response to antigen, or change immunity Type of reaction.
  • Its functions mainly include: collecting immune cells and immune molecules at the site of use, enhancing immune response; enhancing vaccine antigen delivery; enhancing immune exposure; enhancing immunogenicity and immune memory of weak antigens, such as highly purified antigens or recombinant antigens; Vaccination dose and number of vaccinations; promote the immune effect of the vaccine in a population with weak immune response; speed up the immune response and prolong the duration; change the configuration of the antigen; increase the diversity of antibody types, and achieve the crossover of susceptible pathogens Protection (such as influenza virus); changes in the types of body fluid antibodies, IgG subclass and antibody affinity, and stimulate cellular and mucosal immune effects.
  • susceptible pathogens Protection such as influenza virus
  • Aluminum adjuvant is currently the main adjuvant approved for human vaccines. Since 1959, Glenny first applied aluminum salt to adsorb diphtheria toxoid. Up to now, aluminum phosphate and aluminum hydroxide have been widely used in many applications. a vaccine.
  • aluminum adjuvants are mainly humoral immune responses and cannot induce cellular and mucosal immune response effects. In particular, it has been found in recent years that multiple doses of vaccines containing aluminum adjuvants may cause immunosuppression and cumulative poisoning and use them, and aluminum.
  • Adjuvants have poor immune enhancement effects on some vaccine antigens, and occasional severe local reactions at the injection site, including erythema, subcutaneous nodules, contact allergies and granulomatous inflammation, so finding new vaccine adjuvants becomes vaccination Major realities.
  • oil emulsion adjuvants have attracted much attention. Many companies or research institutes have carried out research on oil emulsion adjuvants. Some of the listed and clinical oil emulsion adjuvants are shown in Table 1.
  • oil emulsion adjuvants usually consist of two phases of oil and water and contain at least one surfactant.
  • surfactants include polyoxyethylene sorbitan ester surfactant (commonly known as Tween), sorbitan Alcohol ester (commonly known as Span), octoxynol-9 (Triton X-100 or tert-octylphenoxypolyethoxyethanol) and lecithin, especially Tween 80 (polyoxyethylene loss)
  • Tween 80 polyoxyethylene loss
  • sorbitan monooleate Span 85 (sorbitan trioleate)
  • Triton X-100 Triton X-100.
  • the role of surfactants in the formulation is primarily to stabilize the emulsion and avoid breaking the emulsion.
  • the content of the surfactant in the emulsion needs to exceed the amount required for emulsification, resulting in the presence of a free surface in the aqueous phase or the oil phase or both.
  • Active agent the surfactants used in oil emulsion adjuvants are generally biodegradable (metabolizable) and biocompatible, the use of surfactants may also have some other adverse effects. For example, aliphatic components are often present in oil emulsion adjuvants.
  • MF59 adjuvant comprises squalene, MPL TM monophosphoryl lipid containing fatty acid chains having a plurality of connection to form two deacylated glucosamine backbone of A.
  • aliphatic adjuvants in vaccine compositions may be incompatible with antigens containing surfactant components (CN 101267835A).
  • surfactants 85 and Tween 80 used in MF59 and AS03 although they have been used for a long time in food, cosmetics or as an in vivo injection, are not immune adjuvants themselves and cannot stimulate immune cells. The effect (CN102293743B) will increase the metabolic burden of the body.
  • membrane glycoproteins in the antigen may cause denaturation (CN 101365485A).
  • the amount of surfactant added must be strictly controlled, and the addition of a large amount of surfactant may cause hemolysis.
  • Pickering emulsions are emulsion systems that replace traditional surfactants with solid particles.
  • the stabilizing mechanism of the emulsion is mainly to adsorb the solid particles at the oil-water interface to form a single layer or a multi-layer structure of the solid particles, thereby stabilizing the emulsion.
  • it has outstanding advantages: (1) low toxicity to human body; (2) reducing environmental pollution; (3) strong emulsion stability, and even preparation of high internal phase emulsion.
  • the Pickering emulsions reported in the literature are generally prepared using solid particles or oil phases which are not biocompatible, and thus their application in the field of biomedicine is limited.
  • the literature proposes the preparation of water using polystyrene colloidal particles.
  • the oil phase used is a mixture of octanol and ethyl acetate, which cannot be used in pharmaceutical systems, and polystyrene is not biodegradable.
  • Literature Catherine P.
  • the oil phase examined in this document includes dodecane, polydimethylsiloxane, toluene, isopropyl myristate, in which PLGA nanoparticles stabilize the oil phase.
  • one of the objects of the present invention is to provide an oil-in-water emulsion containing no surfactant. It can be used as a vaccine adjuvant.
  • a surfactant-free oil-in-water emulsion comprising a metabolizable oil phase, an aqueous phase, and biocompatible oil-water amphiphilic solid particles dispersed in an aqueous phase, wherein
  • the oil phase comprises squalene or/and tocol
  • the aqueous phase is any one or a combination of at least two of purified water, water for injection, aqueous glycerin solution, buffered saline solution or clinically available infusion.
  • the average particle size of the particles is on the order of nanometers to micrometers.
  • the solid particles have oil-water amphiphilicity and can be adsorbed onto the liquid-liquid interface between the aqueous phase and the oil phase to stabilize the emulsion droplets, and the average particle diameter of the solid particles. On the nanometer to micron level.
  • the nanoparticle-sized solid particles can function as an immunoadjuvant, and the mechanism of action of the adjuvant can be mainly attributed to the following aspects: 1) the nanoparticle can specifically activate the antigen presenting cell, increasing The amount of uptake; 2) embedding, adsorbing or coupling antigen with nanoparticle, sustainable release of antigen, prolonging cell absorption and antigen expression time; 3) partial nanoparticle (such as positively charged chitosan nano Particles, can achieve lysosomal escape of antigen through proton pump effect, achieve antigen cross-presentation, can promote cellular immune response; 4) some nano-particles can also recruit inflammatory cells, thereby enhancing antigen and antigen presenting cells The role between.
  • the use of solid particles instead of surfactants to prepare a surfactant-free oil-in-water emulsion as described above not only avoids the negative effects of surfactants on vaccine formulations, but also can be immunized by solid particles and oil-in-water emulsions. Synergistically, a more comprehensive, significant, and lasting immune protection effect is obtained.
  • the oil-in-water emulsion does not contain a surfactant, which avoids the effect of the surfactant on the antigen, the product has good safety and stability, and can be used for different vaccination routes of the vaccine.
  • solid particles as an emulsion stabilizer to prepare an oil-in-water emulsion
  • the solid particles themselves can also act as an immunoadjuvant.
  • the immune enhancement and regulation of the two can be synergistically utilized, the amount of antigen can be reduced, the antibody level can be increased, and the diversity of antibody types can be increased, resulting in a wider range of different types. Antigen antibody.
  • the oil phase is preferably a mixture of one or both of squalene and tocopherol.
  • the squalene is a triterpenoid compound whose English name is Squalene, whose molecular structure is isocyanate of thirty carbons and fifty hydrogens, and has the molecular formula: 2, 6, 10, 15, 19, 23-six. Methyl-2,6,10,14,18,22-tetracosahexaene, CAS: 111-02-4, molecular mass: 410.72, may be derived from animal, plant extraction or chemical synthesis. Squalene is a metabolisable oil because it is an intermediate in the biosynthesis of cholesterol (Merck Index, 10th edition, registration number 8619). This is a natural organism that is naturally secreted by all higher organisms, including humans (which can be found in sebum). Emulsions containing squalene (including surfactants) exhibit excellent immunopotentiating effects in animal experiments and clinical trials.
  • the tocol is alpha-tocopherol or a derivative thereof such as alpha-tocopherol succinate (also known as vitamin E succinate).
  • Alpha-tocopherol acts to enhance the immune response in vaccines against elderly patients, such as patients older than 60 years of age or older.
  • the tocols present include various tocopherols such as ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , and the like, preferably ⁇ -tocopherol, especially DL- ⁇ -tocopherol.
  • the oil phase is immiscible with water and may also include other metabolisable oils.
  • the oil phase of the oil-in-water emulsion of the present invention is a metabolisable oil.
  • metabolized oil is meant to be well known in the art.
  • Metabolizable can be defined as “capable of being transformed by metabolism” (Dorland's Medical Dictionary Interpretation, W.B. Sanders, 25th Edition (1974)).
  • An exemplary metabolisable oil can be any vegetable oil, fish oil, animal oil or synthetic oil that is non-toxic to the receptor and can be converted by metabolism, including but not limited to soybean oil, miglitol (Miglyol 812), medium chain oil , fish oil, vitamin E, Vitamin E succinate, vitamin E acetate, safflower oil, corn oil, sea buckthorn oil, linseed oil, peanut oil, tea oil, sunflower oil, almond oil, coix seed oil, evening primrose oil, sesame oil, cottonseed oil, Castor oil, canola oil, ethyl oleate, oleic acid, ethyl linoleate, isopropyl laurate, isopropyl myristate, ethyl butyrate, ethyl lactate, caprylic triglyceride or Any one or a combination of at least two of citric acid triglycerides. Nuts, seeds and grains are a common source of
  • the aqueous phase of the oil-in-water emulsion in the present invention is preferably any one of water for injection, phosphate buffer, citrate buffer or Tris buffer or a combination of at least two.
  • the combination is, for example, a combination of water for injection and phosphate buffer, a combination of citrate buffer and Tris buffer, a combination of water for injection, phosphate buffer and citrate buffer, Tris buffer, water for injection, phosphate buffer. Combination of liquid, citrate buffer and Tris buffer.
  • the pH of the phosphate buffer, citrate buffer or Tris buffer is independently 5.0 to 8.1, such as 5.2, 5.4, 5.6, 5.8, 6, 6.2, 6.4, 6.6, 6.8, 7, 7.2. 7.4, 7.6, 7.8 or 8, preferably 6.0 to 8.0.
  • the aqueous phase of the oil-in-water emulsion of the present invention may contain a monovalent or multivalent antigen, including but not limited to human antigen, non-human animal antigen, plant antigen, bacterial antigen, fungal antigen, viral antigen, parasite Any one or combination of at least two of an antigen or a tumor antigen.
  • a monovalent or multivalent antigen including but not limited to human antigen, non-human animal antigen, plant antigen, bacterial antigen, fungal antigen, viral antigen, parasite Any one or combination of at least two of an antigen or a tumor antigen.
  • the combination for example, a combination of a human antigen and a non-human animal antigen, a mixture of a plant antigen and a bacterial antigen, a combination of a fungal antigen, a viral antigen, and a parasite antigen, a tumor antigen, a human antigen, a non-human animal antigen, a plant antigen, a bacterial antigen Combination with a fungal antigen, a combination of a viral antigen, a parasite antigen, a tumor antigen, a human antigen, a non-human animal antigen, and a plant antigen, a combination of a bacterial antigen, a fungal antigen, a viral antigen, a parasite antigen, and a tumor antigen.
  • the antigen may be derived from, but not limited to, chicken embryo culture, cell culture, purified from a carrier's body fluid, organs or tissues, recombinant gene expression or chemical synthesis, preferably the antigen includes, but is not limited to, an attenuated vaccine, an inactivated vaccine. Any one or a combination of at least two of a split vaccine, a subunit vaccine, a polysaccharide conjugate vaccine, a recombinant vaccine, or a DNA vaccine.
  • the combination for example, a combination of an attenuated vaccine and an inactivated vaccine, a combination of a split vaccine and a subunit vaccine, a combination of a polysaccharide-conjugated vaccine, a recombinant vaccine, a combination of a DNA vaccine and an attenuated vaccine, a combination of an inactivated vaccine and a split vaccine, A combination of a subunit vaccine, a polysaccharide conjugate vaccine, a recombinant vaccine, and a DNA vaccine.
  • the antigen may be a viral antigen or antigenic preparation comprising at least three influenza seasonal (pandemic) strains, and optionally comprising at least one influenza associated with a pandemic outbreak or having a potential associated with a pandemic outbreak.
  • a viral antigen or antigenic preparation of a virus strain, wherein the influenza virus strain associated with a pandemic outbreak or having a potential associated with a pandemic outbreak is selected from the group consisting of human influenza viruses A, B, and C, including H1N1, H2N2, and H3N2.
  • the aqueous phase in the present invention further includes a pharmaceutically acceptable auxiliary substance such as a pH adjuster or/and a buffer, etc., preferably from sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, human serum albumin, Any one or a combination of at least two of an essential amino acid, a non-essential amino acid, L-arginine hydrochloride, sucrose, anhydrous D-trehalose, mannitol, mannose, starch or gelatin.
  • a pharmaceutically acceptable auxiliary substance such as a pH adjuster or/and a buffer, etc., preferably from sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, human serum albumin, Any one or a combination of at least two of an essential amino acid, a non-essential amino acid, L-arginine hydrochloride, sucrose, anhydrous D-trehalose, mannitol, mannose, starch or gelatin.
  • the combination such as a combination of sodium acetate and sodium lactate, a combination of sodium chloride and potassium chloride, a combination of calcium chloride, human serum albumin and essential amino acids, a non-essential amino acid, L-arginine hydrochloride, sucrose and Combination of anhydrous D-trehalose, combination of mannitol, mannose, starch and gelatin, combination of sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride and human serum albumin, essential amino acids, non-essential Amino acid, L-arginine hydrochloride, sucrose, anhydrous D-trehalose, A combination of mannitol, mannose, starch and gelatin.
  • the oil-in-water emulsion of the present invention contains at least one solid particle.
  • the solid particles in the oil-in-water emulsion of the present invention are biocompatible, and are selected from any one or a mixture of at least two of an aluminum salt, a calcium salt, a polysaccharide, a polysaccharide derivative or a high molecular polymer.
  • the aluminum salt is aluminum hydroxide or/and aluminum phosphate.
  • the calcium salt is calcium phosphate or/and calcium carbonate.
  • the polysaccharide is selected from the group consisting of chitosan, alginic acid, gelatin, starch, dextran, konjac glucomannan, heparin, pectin polysaccharide, hyaluronic acid, chondroitin sulfate, chitosan salt, seaweed Any one or a combination of at least two of an acid salt, a gelatin salt, a glucose salt, a konjac glucomannan salt, a heparin salt, a pectin-like polysaccharide salt, a hyaluronate salt or a chondroitin sulfate salt, preferably chitosan Any one or a combination of at least two of alginate, gelatin, starch or dextran.
  • the polysaccharide derivative is a polysaccharide derivative obtained by subjecting a polysaccharide to quaternization, carboxymethylation, hydroxylation, alkylation, acylation, sulfonation, nitration or halogenation, etc., preferably, preferably Any one or a combination of at least two of chitosan, alginic acid, gelatin, starch or dextran for quaternization, carboxymethylation, hydroxylation, alkylation, acylation, sulfonation, nitration or A polysaccharide derivative obtained after a derivatization reaction such as halogenation.
  • a suitable molecular weight is determined depending on factors such as the size of the antigen used, the desired release rate, and the like.
  • a suitable molecular weight is from about 50,000 to 900,000 Daltons, preferably from 100,000 to 800,000 Daltons.
  • the high molecular polymer comprises poly( ⁇ -hydroxy acid), polyhydroxybutyric acid, polycaprolactone, polyorthoester, polyanhydride or polycyanoacrylate and copolymer thereof, the copolymerization
  • the comonomer of the material is preferably any one or a combination of at least two of poly( ⁇ -hydroxy acid), polyhydroxybutyric acid, polycaprolactone, polyorthoester, polyanhydride or polycyanoacrylate.
  • the combination is, for example, a combination of poly( ⁇ -hydroxy acid) and polyhydroxybutyric acid, a combination of polycaprolactone, polyorthoester and polyanhydride, polycyanoacrylate, poly( ⁇ -hydroxy acid), poly Combination of hydroxybutyric acid, polycaprolactone and polyorthoester, combination of polyanhydride, polycyanoacrylate and poly( ⁇ -hydroxy acid), polyhydroxybutyric acid, polycaprolactone, polyortho A combination of an ester, a polyanhydride, and a polycyanoacrylate.
  • the high molecular polymer is poly( ⁇ -hydroxy acid) and a copolymer thereof, preferably self-polymerized (L-lactide), poly(D,L-lactide) or poly(lactide- Co-glycolide, the most preferred polymer is poly(lactide-co-glycolide) called "PLG” or "PLGA” (also known as glycolide lactide copolymer or poly A lactic acid-glycolic acid copolymer or a poly(D,L-lactide-co-glycolide) polymer having the English name Poly(lactic-co-glycolic acid), abbreviated as PLG or PLGA).
  • the molar ratio of lactide to glycolide is from 10:90 to 90:10.
  • Different chain molar ratios affect the hydrophilicity and degradation rate of the material.
  • the 50:50 PLGA polymer containing 50% D, L-lactide and 50% glycolide degrades faster, because of the lactide component. Increased, 75:25 PLGA degradation is slower.
  • An exemplary surfactant-free oil-in-water emulsion comprising a metabolizable oil phase, an aqueous phase, and a biocompatible oil-water amphiphilic solid particle dispersed in the aqueous phase, wherein
  • the oil phase is squalene
  • the aqueous phase is any one or a combination of at least two of purified water, water for injection, aqueous glycerin solution, buffered saline solution or clinically available infusion, the solid particles being PLGA. Its average particle size is on the order of nanometers to micrometers.
  • polymers of various molecular weights can be obtained by a known process, and the appropriate molecular weight can be determined depending on factors such as the size of the antigen used, the desired release rate, and the like.
  • a suitable molecular weight is on the order of about 2000-5000 Daltons.
  • Suitable molecular weights for PLGA are typically from about 10,000 to about 200,000 Daltons, preferably from about 13,000 to about 150,000 Daltons.
  • the solid particles are selected from the group consisting of aluminum hydroxide, aluminum phosphate, calcium phosphate, calcium carbonate, chitosan, alginate, polylactic acid, polylactic acid-glycolic acid copolymer or polyethylene glycol-lactic acid copolymer. Any one or a mixture of at least two, further preferably from aluminum hydroxide, aluminum phosphate, calcium phosphate, polylactic acid, polylactic acid-glycolic acid copolymer or polyethylene Any one or a mixture of at least two of the alcohol-lactic acid copolymers is most preferably a polylactic acid-glycolic acid copolymer.
  • aluminum hydroxide is a hydroxyaluminum salt which is typically at least partially crystalline.
  • the aluminum oxyhydroxide is represented by the formula AlO(OH), which differs from other aluminum compounds, such as aluminum hydroxide Al(OH) 3 by infrared (IR) spectroscopy, especially at 1070 cm -1 and at 3090-3100 cm. There is a strong shoulder at -1 .
  • Aluminum hydroxide is in a typical fiber form, and the hydrate of the aluminum hydroxide adjuvant is usually about 11, that is, the adjuvant itself has a positive surface charge at physiological pH. At pH 7.4, the adsorption capacity of aluminum hydroxide is between 1.8 and 2.6 mg protein per mg Al 3+ .
  • the colloidal particle size is 3.07 ⁇ m.
  • aluminum phosphate is aluminum hydroxyphosphate, which also often contains a small amount of sulfate (i.e., aluminum hydroxyphosphate). These adjuvants can be obtained by precipitation.
  • Aluminum phosphate is usually granular. Typical diameters of these particles after adsorption of any antigen are from 0.5 to 20 [mu]m (e.g., from about 5 to 10 [mu]m). At pH 7.4, the adsorption capacity of aluminum phosphate is between 0.7 and 1.5 mg protein per mg Al 3+ .
  • calcium phosphate is preferred as the solid particles of the present invention.
  • Various adjuvant forms of calcium phosphate have been reported, and any of these forms can be used in the present invention.
  • the adjuvant may form acicular particles having a size of about 10 nm x 150 nm and irregularly shaped sheets having a diameter of about 20-30 nm.
  • particulate calcium phosphate (“CAP"), wherein the particles have a diameter of 300-4000 nm (nanoparticles), are spherical in shape, and have a smooth surface.
  • CAP particulate calcium phosphate
  • the above calcium phosphate can achieve the present invention.
  • the shape of the solid particles in the present invention may be spherical, rod-shaped, spindle-shaped, disc-shaped, cubic, peanut-shaped or amorphous, and the solid particles may have a smooth surface, a porous surface, and an internal multi-chamber.
  • a variety of topographies, hollow or monocular, and those skilled in the art can optimize the screening according to the oil-water phase and antigen properties used by a limited process to obtain an oil-in-water emulsion that meets the application requirements.
  • the solid particles in the oil-in-water emulsion of the invention have oil-water amphiphilic properties and can be stably dispersed in the oil-water two-phase interface to stabilize the emulsion.
  • solid particles with different hydrophilic and hydrophobic properties may be selected to stabilize the emulsion, and the surface of the solid particles may be subjected to hydrophilic or hydrophobic modification, coating or graft modification to obtain suitable hydrophobicity ( Or particle wettability (usually expressed by oil-water-solid contact angle ⁇ ow )).
  • the surface or interior of the solid particles may also adsorb, couple or embed functional substances such as targeting substances, fluorescent labels, isotopic labels, environmental responsive substances, cytokines, antibodies or immunomodulators.
  • the environmentally responsive substance is selected from the group consisting of pH sensitive, heat sensitive or sensitive to biologically active substances.
  • the surface or the inside of the solid particles can also adsorb, couple or embed an antigen, and as an antigen delivery system, the stability of the antigen can be improved, the antigen-uptake of the antigen-promoting cells can be promoted, and the immune response can be enhanced.
  • the solid particles can be prepared in a variety of ways.
  • the poly(lactide-co-glycolide) solid particles can be prepared by various methods such as solvent evaporation, solvent extraction, and precipitation.
  • chitosan solid particles it can be prepared by single emulsion method (forming water-in-oil emulsion) combined with chemical crosslinking method (such as cross-linking with glutaraldehyde dissolved in oil phase), or by spray drying or precipitation method. .
  • the solid particles may be stored in an aqueous solution or a buffer solution, or may be lyophilized for use.
  • the solid particles have an average particle diameter of between 1 nm and 10 ⁇ m, such as 5 nm, 10 nm, 50 nm, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1 ⁇ m, 2 ⁇ m, 3 ⁇ m, 4 ⁇ m. 5 ⁇ m, 6 ⁇ m, 7 ⁇ m, 8 ⁇ m or 9 ⁇ m, preferably between 10 nm and 5 ⁇ m.
  • the solid particle size distribution coefficient span value is less than 1.0.
  • the solid particles have a mass concentration in the aqueous phase of from 0.1 to 20% by weight, such as 0.5% by weight, 1% by weight, 2% by weight, 3% by weight, 4% by weight, 5% by weight, 6% by weight, 7% by weight, 8% by weight, and 9% by weight. , 10wt%, 11wt%, 12wt%, 13 wt%, 14 wt%, 15 wt%, 16 wt%, 17 wt%, 18 wt% or 19 wt%, preferably 0.5 to 10 wt%, further preferably 1 to 8 wt%.
  • the mass concentration of the solid particles in the aqueous phase is the mass of the solid particles divided by the mass of the solid particles and the aqueous phase.
  • the oil-water two-phase volume ratio of the oil-in-water emulsion of the present invention is 1:100 to 9:1, for example, 1:90, 1:80, 1:70, 1:60, 1:50, 1: 40, 1:30, 1:20, 1:10, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1 or 8:1, preferably 1: 50 to 1:2.
  • the average particle diameter of the emulsion droplets in the oil-in-water emulsion is between 50 nm and 300 ⁇ m, for example, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 800 nm, 1 ⁇ m, 10 ⁇ m, 30 ⁇ m, 50 ⁇ m, 80 ⁇ m, 110 ⁇ m, 140 ⁇ m, 170 ⁇ m, 200 ⁇ m, 230 ⁇ m, 260 ⁇ m or 290 ⁇ m, preferably between 100 nm and 100 ⁇ m.
  • the oil-in-water emulsion of the present invention may further comprise a pharmaceutical additive comprising, for example, any one or a combination of at least two of a diluent, a stabilizer or a preservative.
  • the oil-in-water emulsion of the present invention may further include the following adjuvants, but are not limited to: pattern recognition receptors (for example, Toll-like receptors, RIG-1 and NOD-like receptors (NLR) stimulants, such as CpG-containing groups) Oligonucleotides or double-stranded RNA or oligonucleotides containing palindromic sequences or oligonucleotides containing poly(dG) sequences, mineral salts (eg alum, and intestinal bacteria (eg E.
  • pattern recognition receptors for example, Toll-like receptors, RIG-1 and NOD-like receptors (NLR) stimulants, such as CpG-containing groups
  • mineral salts eg alum
  • intestinal bacteria eg E.
  • MPL Monophosphoryllipid
  • AS04 Monophosphoryllipid
  • MPLA Monophosphoryllipid
  • MPL Monophosphoryllipid
  • AS04 Monophosphoryllipid
  • MPL Monophosphoryllipid
  • MPL Monophosphoryllipid
  • AS04 Monophosphoryllipid
  • MPL Monophosphoryllipid
  • MPL Monophosphoryllipid
  • AS04 Monophosphoryllipid
  • MPL Monophosphoryllipid
  • MPL Monophosphoryllipid
  • AS04 Monophosphoryllipid
  • MPL Monophosphoryllipid
  • AS04 Shigella flexneri
  • MPLA specifically binds alum
  • MPL a saponin
  • QS-21 e.g., QS-21, Quil-A, iscoMs, iscomatrix TM
  • liposomes and liposome formulations e.g., AS01
  • Synthetic or specially prepared microparticles and microcarriers such as N.
  • gonorrheae Chlamydia trachomatis and other bacterial bacterial outer membranes (OMV)
  • polysaccharides such as chitosan
  • specific sexually modified or prepared peptides such as muramyl dipeptide
  • aminoalkyl glucosaminyl 4-phosphates such as RC529
  • proteins such as bacterial toxoids or toxin fragments
  • PAMPS pathogen-associated molecular patterns
  • SIP small molecule immune enhancers
  • cytokines and chemokines cytokines and chemokines.
  • Cytokines include, but are not limited to, granulocyte macrophage colony-stimulating factor (GM-CSF), interferons (such as interferon- ⁇ (IFN- ⁇ ), interferon- ⁇ (IFN- ⁇ ), interferon- ⁇ (IFN) - ⁇ , etc.), interleukin (such as interleukin-1 ⁇ (IL-1 ⁇ ), interleukin-1 ⁇ (IL-1 ⁇ ), interleukin-2 (IL-2), interleukin-4 (IL) -4), IL-7, IL-12, IL-15, IL-18, Fetus Hepatic tyrosine kinase 3 ligand (FIt3L) or tumor necrosis factor- ⁇ (TNF- ⁇ ).
  • GM-CSF granulocyte macrophage colony-stimulating factor
  • interferons such as interferon- ⁇ (IFN- ⁇ ), interferon- ⁇ (IFN- ⁇ ), interferon- ⁇ (IFN) - ⁇ , etc.
  • interleukin
  • oil-in-water emulsions of the present invention can be prepared by a variety of methods. Specifically, the oil-in-water emulsion of the present invention can be produced by the following method, but is not limited by the production method described below.
  • the oil-in-water emulsion of the present invention can be prepared by first dispersing solid particles in an aqueous phase and then mixing the oil phase with an aqueous phase.
  • the manner in which the solid particles are dispersed may be selected by various methods such as shaking, stirring, ultrasonic dispersion, etc., to achieve good dispersion of the solid particles in the aqueous phase. As long as the particles can be well dispersed in the aqueous phase, the dispersion method does not significantly affect the properties of the oil-in-water emulsion.
  • the proper dispersion can be selected according to the nature of the water phase and solid particles and the conditions of the experimental equipment. And specific operating parameters.
  • the mixing of the oil phase and the aqueous phase can be selected by Microfluidization, Homogenization, Ultrasound, Two-Syringe emulsification, Spray, Micro Jet, Microchannel emulsification, Membrane Emulsification ( Membrane emulsification), stirring, shaking, inverting or hand mixing.
  • the mixing mode may preferably be a microfluidic, microchannel or membrane emulsification, etc., or a uniform particle size distribution emulsion may be obtained, or a microjet, a syringe double push emulsification, homogenization, stirring or shaking may be preferred to facilitate scale.
  • the mixing method of preparation may be preferably be a microfluidic, microchannel or membrane emulsification, etc., or a uniform particle size distribution emulsion may be obtained, or a microjet, a syringe double push emulsification, homogenization, stirring or shaking may be preferred to facilitate scale.
  • the solid particles, the oil phase and the water phase in the oil-in-water emulsion of the present invention may be separately packaged separately, and may be temporarily mixed according to the aforementioned preparation method before application, or two or three of them may be mixed in advance according to the aforementioned preparation method.
  • the method for sterilizing the oil-in-water emulsion in the present invention may be wet heat sterilization or filter sterilization.
  • the particle size of the particles used is less than 220 nm, it is preferred to adopt a method of filtration sterilization.
  • the oil-in-water emulsion may be packaged separately from the antigen, temporarily mixed prior to immunization or inoculated to the same in a short interval (usually within 1 hour, including 1 hour).
  • the part may also be pre-mixed and packaged according to the aforementioned preparation method, and may be directly applied at the time of immunization.
  • Another object of the present invention is to provide a surfactant-free immunogenic composition
  • a surfactant-free immunogenic composition comprising: (1) an antigen or antigen composition, and (2) an adjuvant composition, the adjuvant composition consisting of The oil-in-water emulsion composition as described above.
  • Another object of the present invention is to provide the use of the above oil-in-water emulsion as a vaccine adjuvant, drug delivery or controlled release carrier.
  • the method of immunization or administration includes intravenous injection, intraspinal injection, intramuscular injection, subcutaneous injection, intradermal injection, respiratory injection or inhalation, intraperitoneal injection, nasal administration, ocular administration, Oral administration, rectal administration, vaginal administration, topical administration or transdermal administration.
  • the oil-in-water emulsion can be used as a vaccine adjuvant for humans, livestock, poultry and aquatic products.
  • the present invention has the following beneficial effects:
  • the present invention combines solid particles with an oil emulsion preparation (oil-in-water emulsion) for the first time to prepare an oil-in-water emulsion which does not require a surfactant, and is applied to the development field of vaccine adjuvants.
  • the addition of solid particles not only improves the biocompatibility of the preparation, but also avoids the adverse effects of the surfactant on the human body, animals or vaccines, and can stimulate the immune cells more effectively and stimulate the immune regulation function; at the same time, the solid particles
  • the nature is easy to control, and it can be surface modified or coated, or solid particles with different properties (such as composition, morphology, structure, particle size, etc.) can be selected to exert different immune enhancement mechanisms, and the antigen is embedded and adsorbed. Or coupled, as a carrier for the antigen, the ability to control the release of the antigen to regulate the immune response, can also be applied to a variety of immunization methods.
  • the main immunopotentiating mechanisms of the oil-in-water emulsions of the present invention include:
  • the oil-in-water emulsion can delay the release rate of the antigen, protect the antigen from hydrolysis, prolong the retention time of the antigen in the body, and facilitate the production of high affinity antibodies; 2)
  • the particles can activate macrophages and promote the interaction of macrophages with T and ⁇ cells, thereby specifically enhancing the stimulation of lymphocytes; (3) if the particles used adsorb or embed the antigen, it may increase The surface area of the antigen makes the antigen easily phagocytosed by macrophages;
  • the oil-in-water emulsion can also cause a slight inflammatory reaction at the injection site, recruit inflammatory cells, stimulate the secretion of inflammatory factors, and activate the immune response; (5)
  • Special particles, such as pH-sensitive chitosan particles can achieve lysosomal escape of the antigen and enhance cellular immune response after adsorption or embedding of the antigen.
  • the oil-in-water emulsion of the present invention can also be used for drug delivery or slow release carrier by dispersing a fat-soluble drug, a fluorescent label or other biologically active substance in an oil phase, or by using a drug, a fluorescent label or the like.
  • the biologically active substance is embedded or adsorbed on the surface of the particle to achieve controlled release of the drug.
  • the oil-in-water emulsion containing the particles can also be used for drugs, fluorescent labels or other biologically active substances by coupling or embedding a targeting substance (such as Fe3O4, folic acid, mannose, etc.) on or in the surface of the particles. Targeted delivery.
  • oil-in-water emulsion disclosed in the present invention can be used both as an immunological adjuvant for vaccines and as a delivery or slow release carrier for drugs or other biologically active substances.
  • Figure 1 is a schematic diagram of a water-in-water Pickering emulsion
  • Figure 2 is a particle size distribution diagram of the PLGA particles prepared in Example 1;
  • Figure 3 is a scanning electron micrograph of the PLGA particles prepared in Example 1;
  • Figure 4 is a photomicrograph of a Pickering emulsion prepared in Example 1 (magnification 20 times);
  • Figure 5 is a photograph of the Pickering emulsion prepared in Example 1 before and after centrifugation (1 before centrifugation, 2 after centrifugation);
  • Figure 6 is a particle size distribution diagram of the aluminum hydroxide particles prepared in Example 2.
  • Figure 7 is a photomicrograph of a Pickering emulsion prepared in Example 2 (magnification 20 times);
  • Figure 8 is a photograph of a Pickering emulsion prepared in Example 4.
  • micron-sized particles or emulsion droplets The particle size distribution of micron-sized particles or emulsion droplets is determined by laser particle size analyzer. The specific measurement steps are as follows: 5 mg micron-sized particles are added to 50 mL of deionized water, sonicated for 5 min to uniformly disperse, or 50 mL of emulsion is taken to suspend the particles. The liquid or emulsion was added to the sample cell and assayed using a laser particle sizer (Ma1vern Instruments, United Kingdom Coulter Co., USA).
  • the particle size distribution of nano-sized particles or emulsion droplets is determined by Zeta potential and particle size analyzer.
  • the specific measurement steps are as follows: 1 mg of nano-sized particles are added to 10 mL of deionized water, sonicated for 5 min to uniformly disperse, or 2 mL of emulsion is taken.
  • the particle suspension or emulsion was added to the sample cell and placed in a Zeta Potential Analyzer (Zeta Potential Analyzer, Brookhaven Instruments Corporation) for measurement.
  • the uniformity of the particles or emulsion droplets is represented by the particle size distribution coefficient (Span) value.
  • Span is calculated as follows, the smaller the value, the more uniform the particle size.
  • d 10 , d 50 and d 90 are particle diameters when the cumulative volume of the particles is 10%, 50%, and 90%, respectively.
  • the morphology of the particles was observed by scanning electron microscopy: 1 mg of the particles were weighed, added to 10 mL of deionized water, and uniformly dispersed by ultrasonication for 5 min. Pipette 1 mL of the suspension, drop it on the aluminum foil, spread it evenly on the aluminum foil, and dry it naturally. The aluminum foil was adhered to the sample stage with a conductive paste, and gold was sprayed under vacuum (according to the nature of the sample to select a suitable gold-spraying condition), and then observed with a scanning electron microscope.
  • the morphology of the emulsion droplets was observed by optical microscopy: a small amount of oil-in-water emulsion was taken and dropped on a glass slide and observed under an optical microscope.
  • the stability of the emulsion was determined by centrifugation: 5 mL of oil-in-water emulsion was taken, added to a 15 mL centrifuge tube, and centrifuged for 10 min under a centrifugal force of 2000 g to observe the stratification.
  • the microspheres can be degraded by adding NaOH solution or acetonitrile; for chitosan-like micro--
  • the ball can be degraded by adding dilute hydrochloric acid.
  • the degradation solution is neutralized with NaOH or hydrochloric acid to have a pH of 7, and then made up to 2 mL.
  • the antigen or drug content is determined using a BCA kit or micro-BCA kit or other suitable assay.
  • the antigen or drug embedding rate is calculated according to the following formula:
  • Embedding rate (the amount of antigen or drug in the measured particles / the amount of antigen or drug added during actual preparation) ⁇ 100%
  • the loading of the antigen or drug on the particles is calculated according to the following formula:
  • Loading (the amount of antigen or drug in the measured particle / the mass of the measured particle)
  • the antigen or drug content is determined using a BCA or micro-BCA kit or other suitable assay.
  • the antigen or drug adsorption rate is calculated by the following formula:
  • Adsorption rate (anti-adsorption antigen or drug concentration - antigen or drug concentration in supernatant after adsorption) / antigen or drug concentration before adsorption ⁇ 100%
  • the loading of the antigen or drug on the particles is calculated according to the following formula:
  • Load (measured antigen or drug amount on the particle / mass of the measured particle)
  • mice used in the experiment were provided by Vitalliwa.
  • the immunization procedure was basically as follows: mice were randomly divided into groups, and each group was subjected to experiments using 6 or more mice, and the mice were grouped and immunized according to the specific description of the examples. Before inoculation, 200 ⁇ L of blood was taken and immediately centrifuged at 12,000 rpm for 5 min to separate serum, and the IgG antibody level was measured, and the IgG antibody level at this time was used as an initial value, and then the mouse was immunized. After immunization, blood was taken from the eye or the tip of the mouse at a time, and 200 ⁇ L of blood was taken each time, and the IgG antibody level was measured.
  • mice Two weeks later, the mice were given a second immunization, and the mice were sacrificed at 35 days, and blood was taken to measure the IgG antibody level (for influenza vaccine, blood coagulation titer (HI) was also measured).
  • Mouse spleen cells were cultured, and the secretion of IL-4 and IFN- ⁇ cytokines in the supernatant of mouse spleen cell culture medium was detected by enzyme-linked immunosorbent assay (ELISA).
  • ELISA enzyme-linked immunosorbent assay
  • Example 1 Preparation of oil-in-water emulsion using poly-PLGA particles
  • aqueous solution containing 1 wt.% of PVA (melanol degree: 99%, viscosity: 5.0 mPa.s), magnetic stirring, rotation speed of 500 rpm), stirring at 25 ° C overnight, centrifugation at 20000 g for 20 min, discarding
  • 5 mL of deionized water was added, ultrasonically dispersed, centrifuged at 20000 g for 5 min, and the supernatant was discarded, and the precipitate was freeze-dried to obtain PLGA pellets, which were stored in a refrigerator at 4 ° C.
  • the prepared PLGA particles had an average particle diameter of 226 nm and a Span of 0.535. Scanning electron microscopy showed that the prepared particles had a smooth surface and a spherical shape.
  • the particle size distribution of the particles is shown in Figure 2, and the morphology is shown in Figure 3.
  • Oil-in-water emulsion preparation oil-in-water emulsion preparation:
  • PLGA particles 0.50 g were accurately weighed by an electronic balance, added to 40 mL of deionized water, and uniformly dispersed by ultrasonic for 1 min to obtain an aqueous phase suspension in which particles were dispersed.
  • the aqueous phase has a pH of 6.5.
  • 1 mL of squalene was pipetted into the above aqueous suspension, and an oil-in-water emulsion was prepared by homogenization (15,000 rpm, 3 min). The droplets are well dispersed and the spheres are regular.
  • the average droplet size of the emulsion droplets was 18.9 ⁇ m and the Span was 0.895.
  • the stability of the emulsion was measured by centrifugation.
  • the appearance of the prepared oil-in-water emulsion was the same as that of the non-centrifugal emulsion, and the oil-free phase of the upper layer was precipitated.
  • the photomicrograph of the emulsion is shown in Figure 4, and the photograph of the emulsion before and after centrifugation is shown in Figure 5.
  • the oil-in-water emulsion can also be prepared by using other PLA-type particles, and the preparation steps are similar to those in the first embodiment.
  • the specific process parameters and results are shown in Table 1.
  • Example 2 Preparation of an oil-in-water emulsion using aluminum hydroxide particles
  • Triton X-100, n-butanol and cyclohexane were mixed in a volume ratio of 1:0.5:20 and magnetically stirred (800 rpm, 5 min) to obtain an oil phase.
  • a 1 mol/L AlCl 3 solution (2 mL) was added dropwise to the oil phase (20 mL) at a magnetic stirring (500 rpm) at a rate of 2 mL/min using a syringe propelling pump to obtain a reverse phase microemulsion of aluminum chloride.
  • a syringe propelling pump was used to add ammonia water to the above-mentioned reverse microemulsion at a dropping rate of 0.5 mL/min, and the pH of the reaction system was maintained above 10.0.
  • Oil-in-water emulsion preparation oil-in-water emulsion preparation:
  • Example 3 Preparation of an oil-in-water emulsion using calcium phosphate particles
  • Oil-in-water emulsion preparation oil-in-water emulsion preparation:
  • Example 4 Preparation of an oil-in-water emulsion using a chitosan-coated alginic acid granule
  • petroleum ether (boiling range of 60-90 ° C) and liquid paraffin are mixed at a volume ratio of 2:1, and an emulsifier Span 80 having a mass fraction of 4 wt.% is added to the mixed organic phase, and the above mixture is used as an oil phase.
  • the aqueous phase was an aqueous solution of sodium alginate (1.0 wt%).
  • the fine emulsion was mixed with the uniform emulsion obtained above, and then stirred in a 37 ° C water bath for 5 hours (250 rpm) to solidify the emulsion to obtain colloidal particles, which were respectively washed three times with petroleum ether, ethanol and water to obtain alginic acid particles.
  • the chitosan coating step of the alginic acid particles comprises first dispersing 1 g of the alginic acid particles into a 0.7 wt% chitosan acetic acid solution (20 mL, the chitosan molecular weight is 800,000 Daltons, and the degree of deacetylation is 90%) After stirring for 1 h (200 rpm), the particles were washed with acetic acid buffer solution (pH 4 and pH 5.5) and deionized water to obtain a chitosan-coated alginic acid. Particles.
  • the particles may also be plated multiple times by dispersing the coated colloidal particles (1 g) into a 0.5 wt% aqueous solution of sodium alginate (20 mL), stirring for 1 h (200 rpm), and washing once with deionized water. The particles were again dispersed in a 0.7 wt% chitosan acetic acid solution (20 mL) and stirred for 1 h (200 rpm). The particles were then washed with an acetic acid buffer solution (pH 4 and pH 5.5) and deionized water to obtain a double-coated chitosan-alginic acid granule.
  • an acetic acid buffer solution pH 4 and pH 5.5
  • alginic acid particles having multiple coatings of chitosan can be obtained.
  • the alginic acid particles were three times coated, the average particle diameter of the particles was 457 nm, and the Span of the particles was 0.839.
  • the prepared particles have a rough surface and a spherical shape.
  • Oil-in-water emulsion preparation oil-in-water emulsion preparation:
  • Example 5 Preparation of an oil-in-water emulsion using monomethoxypolyethylene glycol-lactic acid copolymer (PELA) porous particles
  • PELA monomethoxypolyethylene glycol-lactic acid copolymer
  • PELA mPEG:PLA molar ratio of 1:19, average molecular weight of 40 kDa
  • 7.5 mL of acetone 100 mg was dissolved in 7.5 mL of acetone, and 7.5 mL of absolute ethanol solution was added, and the above solution was dropped (1 drop/s) with rapid stirring (750 rpm).
  • deionized water 90 mL, containing 10 g / L SDS
  • stirring was continued for 24 h (750 rpm). After centrifugation 5 times with deionized water, the precipitate was suspended in 10 mL of deionized water and stored as a suspension of PELA particles.
  • the structured PELA particles have an average particle diameter of 78.55 nm and a Span of 0.331, and the particles are spherical structures having a superficial surface.
  • Oil-in-water emulsion preparation oil-in-water emulsion preparation:
  • PELA particles were accurately weighed by an electronic balance, added to 10 mL of water for injection, and dispersed uniformly for 1 min to obtain an aqueous suspension in which particles were dispersed.
  • the aqueous phase has a pH of 7.0.
  • the oil phase is 2mL ⁇ -tocopherol, using a cis-cone microfluidic emulsification method (the cone port diameter ranges from 20-40 ⁇ m, the oil phase flow rate ranges from 500-600 ⁇ L/h, and the aqueous phase flow rate ranges from 1000-1200 ⁇ L/h).
  • the average droplet size of the emulsion droplets was 70 ⁇ m and the Span was 0.096.
  • the appearance of the prepared oil-in-water emulsion was the same as that of the non-centrifugal emulsion, and the oil-free phase of the upper layer was precipitated.
  • Example 6 Preparation of an oil-in-water emulsion using peanut-like calcium carbonate particles
  • Peanut-like calcium carbonate particles were prepared by liquid phase direct mixing precipitation method:
  • the calcium acetate and trisodium citrate were weighed and dissolved in 200 mL of distilled water (the mass concentration of trisodium citrate was 10 wt.% and 30 wt.%, respectively), and 10 wt.% of sodium carbonate aqueous solution (50 mL) was added to the solution.
  • the mixture was stirred under stirring (300 rpm) for 3 hours, filtered, and the precipitate was washed three times with distilled water and absolute ethanol, and then dried at 70 to obtain peanut-like calcium carbonate particles.
  • the length of the particles was 7.2 ⁇ m, the ratio of the length to the minor axis was 2:1, and the morphology of the prepared particles was peanut-like.
  • Oil-in-water emulsion preparation oil-in-water emulsion preparation:
  • Example 7 Preparation of an oil-in-water emulsion using polylactic acid (PLA) particles embedded with antigen
  • PLA 200 mg was dissolved in 4.0 mL of ethyl acetate, 0.4 mL of 5% (w/v) HBsAg was added, and initial emulsification was carried out in an ice water bath using an ultrasonic cell disrupter (power 12%, time 15 s) Then, the initial emulsion was poured into 200 mL of an aqueous solution (external aqueous phase) containing 1.0 wt.% of PVA, and pre-emulsified (300 rpm, 50 s) with magnetic stirring.
  • an aqueous solution external aqueous phase
  • pre-emulsified 300 rpm, 50 s
  • the pre-emulsion was poured into a storage tank of a rapid membrane embedding apparatus, and the pre-emulsion was pressed through a SPG membrane (film pore size of 5.2 ⁇ m) with a pressure of 300 KPa of nitrogen to obtain a double emulsion.
  • the double emulsion was poured into 800 mL of a 0.9 wt.% NaCl aqueous solution (curing solution) and magnetically stirred at 500 rpm for 10 min to cure the microspheres.
  • the solidified microspheres were washed three times with deionized water (4000 r/min, 5 min) and finally freeze-dried to obtain a finished product.
  • the particles had an average particle diameter of 2.32 ⁇ m and a Span of 0.496, and the particles were spherical structures having a smooth surface.
  • the embedding rate of the antigen was 90%, and the antigen-loading amount of the particles was 0.09 mg antigen/g microspheres.
  • Oil-in-water emulsion preparation oil-in-water emulsion preparation:
  • Example 8 Preparation of an oil-in-water emulsion using chitosan particles
  • aqueous phase Preparation of aqueous phase: accurately weigh a certain amount of chitosan (molecular weight 50,000 Daltons, deacetylation degree is 80%) dissolved in 9mL acetic acid solution (0.1mol / L), fully dissolved under magnetic stirring Chitosan acetic acid solution; another amount of sodium glycerophosphate was dissolved in 1 mL of deionized water. After the chitosan acetic acid solution and the sodium glycerophosphate solution were respectively incubated at 4 ° C for 10 min, the sodium glycerophosphate solution was slowly added dropwise to the chitosan acetic acid solution, and the mixture was uniformly stirred by magnetic stirring (300 rpm, 10 min).
  • This solution was centrifuged at 20,000 rpm to remove insoluble impurities, and the supernatant was retained as an aqueous phase for use.
  • the concentration of chitosan in the aqueous phase was 3.5 wt.%, and the concentration of sodium glycerophosphate in the aqueous phase was 10.0 wt.%.
  • Oil phase preparation Add oil-soluble emulsifier PO-500 to a mixture of 60 mL of liquid paraffin and petroleum ether (petroleum boiling point of 60-90 ° C) (volume ratio of 5:7), PO-500 in oil phase The concentration in the solution was 4 wt.%, stirred until completely dissolved, and kept at 4 ° C for 10 min as an oil phase.
  • Emulsion preparation 2 mL of the aqueous phase was mixed with 50 mL of the oil phase at 4 ° C and emulsified with a homogenizer at 6000 rpm for 1 min to form a pre-emulsion.
  • the obtained pre-emulsion was quickly poured into a pre-emulsion reservoir of a rapid membrane embedding apparatus, and rapidly passed through a SPG microporous membrane (membrane pore diameter of 2.8 ⁇ m) under a nitrogen pressure of 5.0 MPa to obtain a uniform particle size W/.
  • the obtained emulsion was again passed through the SPG microporous membrane as a pre-emulsion under a nitrogen pressure of 5.0 MPa, and repeated emulsification five times to finally obtain a W/O type emulsion having a uniform particle size; the emulsification process took about 10 minutes, and the emulsification was completed. Thereafter, the W/O type emulsion was placed in a water bath at 35 ° C and solidified by mechanical stirring (200 rpm) for 1 h. After completion of the curing reaction, the mixture was centrifuged at 10,000 rpm, and washed successively with petroleum ether, ethanol and deionized water to obtain chitosan particles. The average diameter of the particles is At 870 nm, the Span value is 0.487, and the particles are loosely porous globular structures.
  • Oil-in-water emulsion preparation oil-in-water emulsion preparation:
  • chitosan particles were accurately weighed by an electronic balance, added to a 20 mL PBS buffer solution, and uniformly dispersed by ultrasonic for 1 min to obtain an aqueous phase suspension in which particles were dispersed.
  • the aqueous phase has a pH of 8.1.
  • 2 mL of ⁇ -tocopherol was pipetted into the above aqueous suspension, and an oil-in-water emulsion was prepared by vortexing (10 min). The droplets are well dispersed and the spheres are regular.
  • the average droplet size of the emulsion droplets was 18.10 ⁇ m and the Span was 0.935.
  • the appearance of the prepared oil-in-water emulsion was the same as that of the non-centrifugal emulsion, and the oil-free phase of the upper layer was precipitated.
  • chitosan materials can also be used to prepare the granules and the accompanying oil-in-water emulsion, and the preparation steps are similar to those of the embodiment 8, except that the chitosan with different molecular weight and deacetylation degree is used, and the oil-water phase composition and preparation process are changed.
  • the specific process parameters and results are shown in Table 2. Under the same degree of deacetylation, the smaller the molecular weight, the lower the viscosity, and the smaller the particle size of the particles prepared by the same membrane emulsification conditions, the smaller the particle size of the prepared emulsion. Under the same molecular weight conditions, the higher the degree of deacetylation, the lower the viscosity, and the smaller the particle size of the particles prepared by the same membrane emulsification conditions, the smaller the particle size of the prepared emulsion.
  • Example 9 Preparation of oil-in-water emulsion using chitosan particles adsorbed with antigen
  • the preparation method of the particles is the same as that of Example 8, except that the chitosan particles adsorb the H5N1 avian influenza split vaccine:
  • H5N1 avian influenza split vaccine (HA concentration 150 ⁇ g / mL) in PBS buffer, shake at 4 ° C (120 rpm, 24 h), centrifuge at 10,000 rpm, use The ionized water was washed three times to obtain chitosan particles adsorbed with the H5N1 avian influenza split vaccine.
  • the adsorption rate of the antigen was 60%, and the antigen load on the particles was 900 ⁇ g HA/g particles.
  • Oil-in-water emulsion preparation oil-in-water emulsion preparation:
  • chitosan particles adsorbed with antigen were accurately weighed by an electronic balance, added to a 20 mL PBS buffer solution, and uniformly dispersed for 1 min to obtain an aqueous phase suspension in which particles were dispersed.
  • the aqueous phase has a pH of 8.1.
  • 2 mL of ⁇ -tocopherol was pipetted into the above aqueous suspension, and an oil-in-water emulsion was prepared by vortexing (10 min). The droplets are well dispersed and the spheres are regular.
  • the average droplet size of the emulsion droplets was 19.70 ⁇ m and the Span was 0.941.
  • the appearance of the prepared oil-in-water emulsion was the same as that of the non-centrifugal emulsion, and the oil-free phase of the upper layer was precipitated.
  • Example 1 and Example 2 were administered to the rabbit ear, respectively, once a day for 3 consecutive days. Result: home There was no obvious change in the rabbit ear vein administration site. The histopathological section microscopic examination showed that the blood vessel at 1 cm from the injection site was continuous and intact at 5 cm, and no hyperplasia or swelling was observed. No inflammatory cell infiltration and necrosis were observed in the perivascular tissue. There is no thrombosis in the lumen. This product showed no obvious stimulating effect on rabbit ear veins.
  • Example 1 and Example 2 were mixed with a 2% red blood cell suspension by a conventional in vitro test method (visual observation), and no hemolysis and red blood cell aggregation were observed within 3 hours.
  • Example 1 and Example 2 were respectively administered to rabbit quadriceps injections, and 1 mL was administered per side. After 48 hours, the administration site was visually observed and histopathological examination was performed. The results showed that this product is not irritating to rabbit quadriceps.
  • Example 11 Effect of mixing of aqueous and oil phases in oil-in-water emulsion on antibody titer production
  • PLGA particles were prepared in accordance with the procedure of Example 1.
  • Oil-in-water emulsion preparation oil-in-water emulsion preparation:
  • PLGA particles were accurately weighed by an electronic balance, added to a 40 mL citric acid buffer solution, and uniformly dispersed by ultrasonication for 5 minutes to obtain an aqueous phase suspension in which particles were dispersed.
  • the aqueous phase has a pH of 6.0.
  • 2 mL of squalene was pipetted into the above aqueous suspension, and the mixture was equally divided into two portions, which were respectively subjected to rapid membrane emulsification (film pore size of 40.2 ⁇ m, membrane pressure of 500 KPa, and membrane passage three times).
  • the oil-in-water emulsion was prepared by emulsification by mechanical stirring (500 rpm, 5 min).
  • the emulsion droplets of the prepared emulsion have good dispersibility and spherical regularity.
  • the average particle size of the emulsion droplets in the emulsion prepared by rapid membrane emulsification was 32.20 ⁇ m, and the Span was 0.372; the average particle diameter of the emulsion droplets in the emulsion prepared by mechanical stirring was 45.32 ⁇ m, and the Span was 0.839.
  • the appearance of the prepared oil-in-water emulsion was the same as that of the non-centrifugal emulsion, and the oil-free phase of the upper layer was precipitated.
  • the oil-in-water emulsion prepared above was mixed with H5N1 avian influenza split vaccine (with a hemagglutinin (HA) content of 4.5 ⁇ g/100 ⁇ L) (the emulsion to vaccine mixed volume ratio was 1:1, ultrasound (10 W, 3 min). (mixed), another split vaccine with the same hemagglutinin content was used as a control.
  • H5N1 avian influenza split vaccine with a hemagglutinin (HA) content of 4.5 ⁇ g/100 ⁇ L
  • Example 12 Effect of mixing of oil-in-water emulsion with antigen on antibody titer production
  • the oil-in-water emulsion prepared according to the method of Example 11 was mixed with H5N1 avian influenza inactivated whole virus vaccine and split vaccine (hemagglutinin (HA) content of 4.5 ⁇ g/100 ⁇ L, respectively) (mixed volume of emulsion and vaccine)
  • the ratio was 1:1, using different mixing methods, including shaking (5 min), ultrasound (10 W, 3 min), homogenization (10000 rpm, 1 min), two weeks after the injection of Balb/c mice into the left hind leg muscles. After the second immunization, the mice were sacrificed on the 35th day.
  • the oil-in-water emulsion could exert a good adjuvant effect, and the experimental animals produced high levels of IgG and HI titers, and the single antigen injection group. There is a significant difference in which the homogeneously mixed oil-in-water emulsion injection group has higher antibody levels.
  • Antigen type Adjuvant type Mixed mode Serum IgG level HI level Inactivated whole virus vaccine Example 1 oscillation 320000 720 Inactivated whole virus vaccine Example 1 Ultrasound 510000 2580 Inactivated whole virus vaccine Example 1 Homogenization 600000 3600 Inactivated whole virus vaccine no no 80000 64 Lysis vaccine Example 1 oscillation 300000 640 Lysis vaccine Example 1 Ultrasound 480000 2560 Lysis vaccine Example 1 Homogenization 580000 3200 Lysis vaccine no no 62000 32
  • Example 13 Effect of oil-in-water emulsion and antigen inoculation method on adjuvant
  • the oil-in-water emulsion prepared according to the method of Example 11 was separately mixed with a hepatitis B surface antigen recombinant vaccine (recombinant Hansenula vaccine, antigen concentration of 1.9 mg/mL) (the emulsion to vaccine mixed volume ratio was 1:1,
  • the above vaccine composition was injected intramuscularly, subcutaneously and intraperitoneally into Balb/c mice by homogenization (10000 rpm, 1 min). Two weeks later, the mice were subjected to secondary immunization, and the mice were sacrificed at 35 days. The results are shown in Table 5.
  • the intramuscular group showed higher levels of humoral antibody (IgG) and cytokine secretion (IL-4 and IFN- ⁇ ).
  • Antigen type Adjuvant type Inoculation method Serum IgG level IL-4 IFN- ⁇ Hepatitis B vaccine
  • Example 1 Intramuscular injection 256000 32 11240 Hepatitis B vaccine
  • Example 1 Subcutaneous injection 184000 28 10020 Hepatitis B vaccine
  • Example 1 Intraperitoneal injection 102400 18 8000 Hepatitis B vaccine no Intramuscular injection 20480 5 2000 Hepatitis B vaccine no Subcutaneous injection 14560 4 1540 Hepatitis B vaccine no Intraperitoneal injection 10240 2 1000
  • Example 14 Comparison of adjuvant effects of oil-in-water emulsion with aluminum adjuvant and MF59 adjuvant
  • the oil-in-water emulsion, aluminum hydroxide adjuvant and MF59 in Example 11 were mixed with the H1N1 influenza A inactivated whole virus vaccine at a ratio of 1:1 by volume to prepare a vaccine adjuvant composition, and blood coagulation in the composition.
  • the concentration of the hormone was 15 ⁇ g/mL, 0.5 mL per dose.
  • Another inactivated whole virus vaccine with a hemagglutinin concentration of 15 ⁇ g/mL was used as a control.
  • the Balb/c mice were intramuscularly immunized with an equal volume of the above three vaccine adjuvant compositions and a control vaccine, and the mice were sacrificed two weeks later, and the mice were sacrificed at 35 days. The results of the immunization are shown in Table 6.
  • the enhancement effect of oil-in-water emulsion on humoral immunity is comparable to that of MF59, higher than that of aluminum hydroxide adjuvant, all of which are higher than the vaccine without adjuvant; in terms of cellular immunity, the oil-in-water emulsion group is superior to MF59 and hydroxide.
  • the aluminum adjuvant group mainly because the particles in the oil-in-water emulsion promote the phagocytosis of the antigen by the antigen-presenting cells, activate the lymphocytes, stimulate their differentiation and secrete cytokines.
  • Example 15 Effect of the amount of oil-in-water emulsion added on the immune effect
  • the oil-in-water emulsion prepared according to the method of Example 11 and the EV71 hand-foot-inactivated virus vaccine are mixed in a ratio of 2:1, 1:1, 1:2, 1:4, etc. to form a vaccine adjuvant composition, wherein The antigen concentration was 0.05 mg/mL, 0.2 mL per dose.
  • Balb/c mice were intramuscularly inoculated with the above vaccine adjuvant composition, and secondary immunization was performed two weeks later. The results are shown in Table 7. The results indicate that increasing the adjuvant dose in the vaccine adjuvant composition can enhance the level of immune response produced by quantitative antigen production, and the dose of adjuvant is positively correlated with the intensity of adjuvant action.
  • Antigen type Adjuvant type Adjuvant to antigen mixing ratio Serum IgG level EV71 hand, foot and mouth inactivated virus vaccine
  • Example 16 Effect of vaccines with different antigen concentrations on immune response
  • the oil-in-water emulsion prepared according to the method of Example 11 and the H5N1 influenza inactivated whole virus vaccine constitute a vaccine adjuvant composition having a hemagglutinin concentration of 37.5 ⁇ g/mL, 75 ⁇ g/mL, and 150 ⁇ g/mL, each dose It is 0.1 mL.
  • Balb/c mice were immunized intramuscularly with two vaccine adjuvant compositions and two adjuvant-free vaccines of the same hemagglutinin concentration. Two weeks later, the mice were immunized twice, and the mice were sacrificed at 35 days. The results are shown in Table 8. .
  • Example 17 Adjuvant effect of oil-in-water emulsion for nasal mucosal immunization
  • the oil-in-water emulsion prepared according to the method of Example 11 and the H7N9 influenza split vaccine constituted a vaccine adjuvant composition having a hemagglutinin concentration of 150 ⁇ g/mL, each of which was 0.03 mL.
  • the Balb/c mice were immunized intranasally with the above-mentioned vaccine adjuvant composition and the adjuvant-free vaccine of the same hemagglutinin concentration. Two weeks later, the mice were immunized twice, and the mice were sacrificed at 35 days. The immune results are shown in Table 9.
  • Example 18 Partial enhancement mechanism of oil-in-water emulsion as an immunoadjuvant (local inflammation)
  • chicken egg albumin (OVA) was used as an antigen and three emulsion vaccines.
  • the adjuvant is mixed to form an adjuvant vaccine composition.
  • the above adjuvant vaccine composition was immunized by intramuscular injection of the right hind leg of Balb/c mice, and the mice were sacrificed at different times. The injection site was sectioned and the sections were stained by H&E and TUNEL. An inflammatory response caused by an emulsion-type vaccine adjuvant in the body. Local tissue section observation showed that the three emulsion-type vaccine adjuvants could simultaneously cause certain inflammatory reactions and recruit inflammatory cells, but the degree of inflammation was mild. After seven days, the inflammatory reaction subsided and had good biocompatibility.
  • Example 19 Partial enhancement mechanism of oil-in-water emulsion as an immunoadjuvant (promoting cell phagocytosis)
  • the mouse macrophage cell line RAW264.7 was used as an antigen-presenting cell, and an adjuvant vaccine composition was prepared by mixing OVA as an antigen with an oil-in-water emulsion.
  • In vitro cell experiments were performed to investigate the effect of the oil-in-water emulsion (prepared according to Example 11) on the amount of antigen-presenting cells phagocytic antigen. The results showed that the adjuvant group significantly increased the amount of phagocytic antigen of RAW264.7 cells.
  • Example 20 Oil-in-water emulsion as a drug release carrier
  • An oil-in-water emulsion was prepared as in Example 4, except that paclitaxel having a mass concentration of 0.5 wt.% was added to the oil phase.
  • Example 21 Oil-in-water emulsion as a drug release carrier
  • An oil-in-water emulsion was prepared as in Example 5, except that the PELA particles were embedded with insulin at a mass concentration of 1 wt.%.

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Abstract

提供一种不含表面活性剂的水包油乳液，所述乳液包括可代谢的油相、水相以及分散在水相中的具有生物相容性的油水两亲性的固体颗粒，其中，油相包括角鲨烯或/和母育酚，所述水相为纯化水、注射用水、甘油水溶液、缓冲盐水溶液或临床上可输液的任意一种或至少两种的组合，所述固体颗粒的平均粒径在纳米到微米级别。该乳液可作为疫苗佐剂、药物递送或缓控释载体应用，避免了表面活性剂的使用，降低对人体的毒害和环境污染。

Description

一种不含表面活性剂的水包油乳液及其用途 技术领域
本发明涉及一种生物制品，特别是涉及一种可以用于人或其他动物体内的水包油乳液，特别重要的是本发明所述水包油乳液不含表面活性剂，采用固体颗粒作为乳液稳定剂，可作为疫苗佐剂或药物递送或缓控释载体，属于生物医药技术领域。
背景技术
随着现代生物技术的迅速发展和社会对于减毒疫苗所导致的不良反应的日益重视，裂解疫苗、重组亚单位疫苗、抗独特型抗体疫苗、核酸疫苗及合成肽疫苗等得到开发。这些疫苗抗原纯度高，相对分子量小，疫苗的不良反应低，然而这些抗原的免疫原性和免疫保护性普遍较弱，为了提高抗原的有效性，必须在抗原中添加佐剂以增强它们诱导免疫反应的能力。
佐剂与抗原的同时应用或协同应用(在较短的间隔时间内(通常为1小时或更短时间)先后接种到同一或邻近部位)，能增强机体针对抗原的免疫应答能力，或改变免疫反应类型。其功能主要包括：在使用部位募集免疫细胞和免疫分子，增强免疫应答；增强疫苗抗原传递；增进免疫接触；增强弱抗原的免疫原性和免疫记忆，如高度纯化的抗原或重组抗原；减少抗原接种剂量和接种次数；促进疫苗在免疫应答能力弱的群体中的免疫效果；加快免疫应答的速度和延长持续时间；改变抗原的构型；增加抗体类型的多样性，实现对易变异病原体的交叉防护(如流感病毒)；改变体液抗体的种类、IgG亚类和抗体的亲和性，以及激发细胞免疫和黏膜免疫效应等。
铝佐剂是目前普遍被批准用于人类疫苗的主要佐剂，自1926年Glenny首先应用铝盐吸附白喉类毒素开始，至今已有磷酸铝和氢氧化铝两种铝盐佐剂广泛应用于多种疫苗。但是铝佐剂以体液免疫应答为主，不能诱导细胞免疫和黏膜免疫应答效应，特别是近几年发现多次接种含铝佐剂的疫苗可能出现免疫抑制和累积中毒而使其使用，并且铝佐剂对一些疫苗抗原的免疫增强效果不佳，注射部位偶有严重的局部反应，包括出现红斑、皮下结节、接触性过敏和肉芽肿性炎症，因此寻找新的疫苗佐剂成为疫苗学的重大现实问题。
近年来油乳佐剂备受关注，多家企业或科研机构开展了油乳佐剂方面的研究，其中部分上市及临床的油乳佐剂如表1所示。
表1 部分上市及临床的油乳佐剂
Figure PCTCN2014092290-appb-000001
Figure PCTCN2014092290-appb-000002
这些油乳佐剂通常由油水两相组成，包含至少一种表面活性剂，常用的表面活性剂包括聚氧乙烯失水山梨糖醇酯表面活性剂(通常称为吐温)、失水山梨糖醇酯(通常称为司盘)、辛苯聚醇-9(曲通X-100或叔辛基苯氧基聚乙氧基乙醇)以及卵磷脂等，特别是吐温80(聚氧乙烯失水山梨糖醇单油酸酯)、司盘85(失水山梨糖醇三油酸酯)和曲通X-100的应用较多。表面活性剂在制剂中所起的作用主要是稳定乳液，避免破乳。为实现这一目的，需要使用至少一种表面活性剂，并且为保持乳液稳定，表面活性剂在乳液中的含量需超过乳化所需量，从而 导致水相或油相或二者中存在游离表面活性剂。而且，虽然油乳佐剂中所使用的表面活性剂通常是生物可降解(可代谢)和生物相容的，但表面活性剂的使用还可能带来一些其他的不利影响。例如，油乳佐剂中往往存在脂肪族组分。如，MF59佐剂包含角鲨烯，MPLTM包含带有多个脂肪酸链连接于二葡糖胺主链的脱酰基化形式的单磷酰基脂质A。有研究发现疫苗组合物中的脂肪族佐剂可能与含有表面活性剂组分的抗原不相容(CN 101267835A)。同时，MF59和AS03中使用的表面活性剂司盘85和吐温80，虽然已被长久使用于食品、化妆品或作为体内注射使用，但其本身并不是免疫佐剂，无法起到刺激免疫细胞的作用(CN102293743B)，反而会增加机体的代谢负担。此外，抗原中的膜糖蛋白如长期与游离表面活性剂接触，可能导致其发生变性作用(CN 101365485A)。同时对于表面活性剂的加入量必须严格控制，加入较多量的表面活性剂，可能导致溶血现象。
与传统的采用表面活性剂作为稳定剂的乳液不同，Pickering乳液是一种由固体颗粒代替传统表面活性剂的乳液体系。乳液的稳定机理主要是通过固体颗粒吸附在油水界面，形成固体颗粒单层或多层结构，从而使乳液稳定。与传统含有表面活性剂的乳液比较，它具有突出的优势：(1)对人体的毒副作用小；(2)降低环境污染；(3)乳液稳定性强，甚至可以制备高内相的乳液。然而，目前文献中所报道的Pickering乳液普遍采用不具有生物相容性的固体颗粒或油相制备，因此其在生物医药领域的应用受到限制。例如，文献(Zhuo Ao，Zhi Yang，Jianfang Wang，Guangzhao Zhang，To Ngai，Emulsion-Templated Liquid Core-Polymer Shell Microcapsule Formation，Langmuir，2009，25：2572-2574)提出了采用聚苯乙烯胶体颗粒制备水包油型(o/w)Pickering乳液，并在乳滴表面沉淀一层聚(丙交酯-共-乙交酯)(也可称作乙交酯丙交酯共聚物或聚乳酸-羟基乙酸共聚物或聚(D，L-丙交酯-共-乙交酯)聚合物，其英文名称为Poly(1actic-co-glycolic acid)，英文缩写为PLG或PLGA)壳层，形成微囊结构。所用的油相为辛醇和乙酸乙酯的混合液，不能用于药物体系，并且聚苯乙烯也不能生物降解。文献(Catherine P.Whitby，Li Hui Lim，Nasrin Ghouchi Eskandar，Spomenka Simovic，Clive A.Prestidge，Poly(lactic-co-glycolic acid)as a particulate emulsifier，Journal of Colloid and Interface Science，2012，375：142-147)报道，直接采用生物降解性和生物相容性良好的高分子材料PLGA(丙交酯与乙交酯的聚合比例为50∶50，分子量40-75kDa)纳米颗粒制备Pickering乳液。该文献中考察的油相包括十二烷(dodecane)、聚二甲硅氧烷(polydimethylsiloxane)、甲苯(toluene)、十四酸异丙酯(isopropyl myristate)，其中PLGA纳米颗粒可以稳定油相为十二烷和聚二甲硅氧烷的水包油型(o/w)乳液，但上述油相并不能作为药用辅料应用，因此也无法作为疫苗佐剂应用。文献(Zengjiang Wei，Chaoyang Wang，Hao Liu，Shengwen Zou，Zhen Tong，Facile fabrication of biocompatible PLGA drug-carrying microspheres by O/W pickering emulsions，Colloids and Surfaces B：Biointerfaces，2012，9l：97-105)采用二氧化硅纳米粒作为固体颗粒，含有PLGA的二氯甲烷作为油相，制备了二氧化硅纳米粒覆层的PLGA微球，但该体系中的二氧化硅和二氯甲烷在临床应用上都受到限制，该Pickering乳液无法直接在生物医药领域应用。并且，目前已知的文献或专利中均没有关于作为疫苗佐剂的Pickering乳液的研究，已知的Pickering乳液体系也没有根据疫苗制剂的需求来进行设计、优化。
发明内容
针对已有技术的问题，本发明的目的之一在于提供一种不含表面活性剂的水包油乳液， 其可以作为疫苗佐剂。
为了达到上述目的，本发明采用了如下技术方案：
一种不含表面活性剂的水包油乳液，所述乳液包括可代谢的油相、水相以及分散在水相中的具有生物相容性的油水两亲性的固体颗粒，其中，所述油相包括角鲨烯或/和母育酚，所述水相为纯化水、注射用水、甘油水溶液、缓冲盐水溶液或临床上可用输液中的任意一种或者至少两种的组合，所述固体颗粒的平均粒径在纳米到微米级别。
在本发明中，固体颗粒具有油水两亲性，可被吸附到所述水相和所述油相之间的液-液界面之上，起到稳定乳滴的作用，固体颗粒的平均粒径在纳米到微米级别。
在本发明中，纳微粒径的固体颗粒可发挥免疫佐剂的作用，其佐剂作用机制可主要归因于以下几个方面：1)纳微颗粒能够特异性激活抗原提呈细胞，增加其摄取量；2)用纳微颗粒包埋、吸附或偶联抗原，可持续释放抗原，延长细胞吸收和抗原表达时间；3)部分纳微颗粒(如带有正电荷的壳聚糖纳微颗粒)，可以通过质子泵效应等实现抗原的溶酶体逃逸，实现抗原的交叉提呈，可促进机体细胞免疫反应；4)部分纳微颗粒还可募集炎症细胞，从而增强抗原与抗原呈递细胞之间的作用。
因此，采用固体颗粒替代表面活性剂制备如上所述的不含表面活性剂的水包油乳液，不仅可以避免表面活性剂对疫苗制剂的负面影响，而且可以通过固体颗粒和水包油乳液的免疫协同作用，获得更为全面、显著、持久的免疫保护效果。该水包油乳液中不含表面活性剂，避免了表面活性剂对抗原的影响，产品具有良好的安全性和稳定性，并可用于疫苗的不同接种途径。
此外，采用固体颗粒作为乳液稳定剂制备水包油型乳液，除可以发挥水包油乳液的佐剂作用外，固体颗粒本身也能起到免疫佐剂的作用。通过将固体颗粒与水包油乳液结合，可协同发挥二者的免疫增强及调节作用，降低抗原用量，提高抗体水平，同时还可增加抗体类型的多样性，产生范围更为广泛的针对不同类型抗原的抗体。
油相优选角鲨烯和生育酚中的一种或两种的混合物。所述角鲨烯是一种三萜类化合物，其英文名称是Squalene，分子结构为三十碳五十氢的异戊二烯，分子式为：2，6，10，15，19，23-六甲基-2，6，10，14，18，22-二十四碳六烯，CAS：111-02-4，分子质量：410.72，可来源于动物、植物提取或化学合成。角鲨烯是一种可代谢的油，因为它是胆固醇的生物合成的中间产物(Merck索引，第10版本，登记号8619)。这是一种所有高等生物，包括在人类身上(皮脂中可以找到)自然分泌的油脂。含有角鲨烯的乳液(含表面活性剂)在动物实验和临床实验上表现出优异的免疫增强作用。
所述母育酚是α-生育酚或其衍生物如α-生育酚琥珀酸酯(也称作维生素E琥珀酸酯)。α-生育酚在针对老年患者(如年龄大于60岁或更大的患者)的疫苗中，可以起到增强免疫应答的作用。存在的母育酚包括α、β、γ、δ、ε、ζ等多种生育酚，优选α-生育酚，尤其是DL-α-生育酚。优选地，所述油相与水相互不相溶，还可以包括其他可代谢的油。
为了使所述水包油乳液适合于疫苗或药物制剂，本发明中所述水包油乳液的油相为可代谢的油。术语“代谢的油”意思已为本领域所熟知。“可代谢的”可被定义为“能够通过代谢作用转变”(Dorland的医学词典解释，W.B.Sanders公司，第25版本(1974))。
示例性的可代谢的油可以是任何对受体无毒并可通过代谢作用转化的植物油、鱼油、动物油或合成油，包括但不限于大豆油、米格列醇(Miglyol 812)、中链油、鱼油、维生素E、 维生素E琥珀酸酯、维生素E醋酸酯、红花油、玉米油、沙棘油、亚麻子油、花生油、茶油、葵花籽油、杏仁油、薏仁油、月见草油、芝麻油、棉籽油、蓖麻油、低芥酸菜子油、油酸乙酯、油酸、亚油酸乙酯、月桂酸异丙酯、内豆蔻酸异丙酯、丁酸乙酯、乳酸乙酯、辛酸甘油三酯或癸酸甘油三酯中的任意一种或者至少两种的组合。坚果、种子和谷物是常见的植物油来源。
本发明中所述水包油乳液的水相优选为注射用水、磷酸盐缓冲液、柠檬酸缓冲液或Tris缓冲液中的任意一种或者至少两种的组合。所述组合例如注射用水和磷酸盐缓冲液的组合，柠檬酸缓冲液和Tris缓冲液的组合，注射用水、磷酸盐缓冲液和柠檬酸缓冲液的组合，Tris缓冲液、注射用水、磷酸盐缓冲液、柠檬酸缓冲液和Tris缓冲液的组合。
优选地，所述磷酸盐缓冲液、柠檬酸缓冲液或Tris缓冲液的pH值独立地为5.0～8.1，例如5.2、5.4、5.6、5.8、6、6.2、6.4、6.6、6.8、7、7.2、7.4、7.6、7.8或8，优选为6.0～8.0。
本发明中所述水包油乳液的水相中可含有单价或多价抗原，所述抗原包括但不限于人类抗原、非人类动物抗原、植物抗原、细菌抗原、真菌抗原、病毒抗原、寄生虫抗原或肿瘤抗原中的任意一种或者至少两种的组合。所述组合例如人类抗原和非人类动物抗原的组合，植物抗原和细菌抗原的混合物，真菌抗原、病毒抗原和寄生虫抗原的组合，肿瘤抗原、人类抗原、非人类动物抗原、植物抗原、细菌抗原和真菌抗原的组合，病毒抗原、寄生虫抗原、肿瘤抗原、人类抗原、非人类动物抗原和植物抗原的组合，细菌抗原、真菌抗原、病毒抗原、寄生虫抗原和肿瘤抗原的组合。
所述抗原可以来自但不限于鸡胚培养、细胞培养、携带者体液、器官或组织中纯化分离所得、重组基因表达或化学合成所得，优选所述抗原包括但不限于减毒疫苗、灭活疫苗、裂解疫苗、亚单位疫苗、多糖结合疫苗、重组疫苗或DNA疫苗等中的任意一种或者至少两种的组合。所述组合例如减毒疫苗和灭活疫苗的组合，裂解疫苗和亚单位疫苗的组合，多糖结合疫苗、重组疫苗的组合，DNA疫苗和减毒疫苗的组合，灭活疫苗和裂解疫苗的组合，亚单位疫苗、多糖结合疫苗、重组疫苗和DNA疫苗的组合。
所述抗原可为包含来自至少三种流感季节性(大流行间)株的病毒抗原或抗原性制剂，并且任选包含至少一种与大流行爆发相关或具有与大流行爆发相关的潜能的流感病毒株的病毒抗原或抗原性制剂，其中，与大流行爆发相关或具有与大流行爆发相关的潜能的流感病毒株选自：人类流感病毒中A、B、C型，包含H1N1，H2N2，H3N2，H5N1，H7N7，H1N2，H9N2，H7N3，H10N7；猪型流感病毒H1N1，H1N2，H3N1，H3N2；狗或马型流感病毒H7N7，H3N8；或禽流感病毒H5N1，H7N2，H1N7，H7N3，H13N6，H5N9，H11N6，H3N8，H9N2，H5N2，H4N8，H10N7，H2N2，H8N4，H14N5，H6N5和H12N5中的一种或一种以上的流感病毒组合。
本发明中所述水相中还包括药用辅助物质，如pH调节剂或/和缓冲剂等，优选自乙酸钠、乳酸钠、氯化钠、氯化钾、氯化钙、人血清清蛋白、必需氨基酸、非必需氨基酸、L-精氨酸盐酸盐、蔗糖、无水D-海藻糖、甘露醇、甘露糖、淀粉或明胶中的任意一种或者至少两种的组合。所述组合例如乙酸钠和乳酸钠的组合，氯化钠和氯化钾的组合，氯化钙、人血清清蛋白和必需氨基酸的组合，非必需氨基酸、L-精氨酸盐酸盐、蔗糖和无水D-海藻糖的组合，甘露醇、甘露糖、淀粉和明胶的组合，乙酸钠、乳酸钠、氯化钠、氯化钾、氯化钙和人血清清蛋白的组合，必需氨基酸、非必需氨基酸、L-精氨酸盐酸盐、蔗糖、无水D-海藻糖、 甘露醇、甘露糖、淀粉和明胶的组合。
本发明中所述水包油乳液中含有至少一种固体颗粒。本发明中所述水包油乳液中的固体颗粒具有生物相容性，选自铝盐、钙盐、多糖、多糖衍生物或高分子聚合物中的任意一种或者至少两种的混合物。
优选地，所述铝盐为氢氧化铝或/和磷酸铝。优选地，所述钙盐为磷酸钙或/和碳酸钙。
优选地，所述多糖选自壳聚糖、海藻酸、明胶、淀粉、葡聚糖、魔芋葡甘聚糖、肝素、果胶类多糖、透明质酸、硫酸软骨素、壳聚糖盐、海藻酸盐、明胶盐、葡萄糖盐、魔芋葡甘聚糖盐、肝素盐、果胶类多糖盐、透明质酸盐或硫酸软骨素盐中的任意一种或者至少两种的组合，优选壳聚糖、海藻酸盐、明胶、淀粉或葡聚糖中的任意一种或者至少两种的组合。
优选地，所述多糖衍生物为对多糖进行季铵化、羧甲基化、羟基化、烷基化、酰基化、磺化、硝化或卤化等衍生反应后所得的多糖衍生物，优选为对壳聚糖、海藻酸、明胶、淀粉或葡聚糖中的任意一种或者至少两种的组合进行季铵化、羧甲基化、羟基化、烷基化、酰基化、磺化、硝化或卤化等衍生反应后所得的多糖衍生物。
在本发明中，根据所用抗原的大小、所需的释放速率等影响因素确定合适分子量。对于壳聚糖，例如合适的分子量是约5万-90万道尔顿，优选为10万-80万道尔顿。
优选地，所述高分子聚合物包括聚(α-羟酸)、聚羟基丁酸、聚己酸内酯、聚原酸酯、聚酐或聚氰基丙烯酸酯及其共聚物，所述共聚物的共聚单体优选聚(α-羟酸)、聚羟基丁酸、聚己酸内酯、聚原酸酯、聚酐或聚氰基丙烯酸酯中的任意一种或者至少两种的组合。所述组合例如聚(α-羟酸)和聚羟基丁酸的组合，聚己酸内酯、聚原酸酯和聚酐的组合，聚氰基丙烯酸酯、聚(α-羟酸)、聚羟基丁酸、聚己酸内酯和聚原酸酯的组合，聚酐、聚氰基丙烯酸酯和聚(α-羟酸)的组合，聚羟基丁酸、聚己酸内酯、聚原酸酯、聚酐和聚氰基丙烯酸酯的组合。
优选地，所述高分子聚合物是聚(α-羟酸)及其共聚物，优选自聚(L-丙交酯)、聚(D，L-丙交酯)或聚(丙交酯-共-乙交酯)，最优选的聚合物是称为“PLG”或“PLGA”的聚(丙交酯-共-乙交酯)(也可称作乙交酯丙交酯共聚物或聚乳酸-羟基乙酸共聚物或聚(D，L-丙交酯-共-乙交酯)聚合物，其英文名称为Poly(lactic-co-glycolic acid)，英文缩写为PLG或PLGA)。
优选地，在聚(丙交酯-共-乙交酯)中，丙交酯与乙交酯的链段摩尔比为10∶90-90∶10。不同的链段摩尔比会影响材料的亲疏水性以及降解速度，例如含有50％D，L-丙交酯和50％乙交酯的50∶50PLGA聚合物的降解较快，而因为丙交酯分量增加，75∶25PLGA降解较慢。
示例性的一种不含表面活性剂的水包油乳液，所述乳液包括可代谢的油相、水相以及分散在水相中的具有生物相容性的油水两亲性的固体颗粒，其中，所述油相为角鲨烯，所述水相为纯化水、注射用水、甘油水溶液、缓冲盐水溶液或临床上可用输液中的任意一种或者至少两种的组合，所述固体颗粒为PLGA，其平均粒径在纳米到微米级别。
采用已知工艺可获得各种分子量的这些聚合物，可以根据所用抗原的大小、所需的释放速率等影响因素确定合适分子量。对于聚(L-丙交酯)和聚(D，L-丙交酯)，例如合适的分子量是约2000-5000道尔顿级别的。对于PLGA，合适的分子量通常是约10000-约200000道尔顿，优选约13000-约150000道尔顿。
优选地，所述固体颗粒选自氢氧化铝、磷酸铝、磷酸钙、碳酸钙、壳聚糖、海藻酸盐、聚乳酸、聚乳酸-羟基乙酸共聚物或聚乙二醇-乳酸共聚物中的任意一种或者至少两种的混合物，进一步优选自氢氧化铝、磷酸铝、磷酸钙、聚乳酸、聚乳酸-羟基乙酸共聚物或聚乙二 醇-乳酸共聚物中的任意一种或者至少两种的混合物，最优选聚乳酸-羟基乙酸共聚物。
称为″氢氧化铝″的一般是羟基氧化铝盐，其通常至少部分结晶。羟基氧化铝以分子式AlO(OH)表示，其与其它铝化合物，例如氢氧化铝Al(OH)3的区别在于红外(IR)光谱，特别是在1070cm-1处存在吸收带和在3090-3100cm-1处存在强烈的肩峰。氢氧化铝呈典型的纤维形态，氢氧化铝佐剂的pI通常约11，即在生理pH下佐剂本身具有表面正电荷。pH7.4时，氢氧化铝的吸附能力在每mg Al3+1.8-2.6mg蛋白质之间。由于商业氢氧化铝的不同生产批次之间存在一定的差异，目前以丹麦生产的
Figure PCTCN2014092290-appb-000003
为公认标准，其胶体颗粒大小为3.07μm。称为“磷酸铝”的一般是羟基磷酸铝，其也常含有少量硫酸根(即，羟基磷酸硫酸铝)。可通过沉淀获得这些佐剂。磷酸铝通常是颗粒状的。这些颗粒在吸附任何抗原后的典型直径是0.5-20μm(例如，约5-10μm)。pH 7.4时，磷酸铝的吸附能力在每mg Al3+0.7-1.5mg蛋白质之间。
在各种钙盐中，首选磷酸钙作为本发明的固体颗粒。现已报道了磷酸钙的各种佐剂形式，本发明可用任何这些形式。佐剂可以形成尺寸约10nm×150nm的针状颗粒以及直径约20-30nm的不规则形状的片。还有文献披露了颗粒磷酸钙(“CAP”)，其中所述颗粒的直径为300-4000nm(纳米颗粒)，形状为球形，表面平滑。上述磷酸钙均可实现本发明。本发明中所述固体颗粒的形状可以是球形、棒状、纺锤状、盘状、立方体、花生形状或无定形等多种形状，固体颗粒的形貌可以为表面光滑、表面多孔、内部多腔室、中空或单眼等多种形貌，本领域技术人员可以根据所用的油水相及抗原性质通过有限的工艺进行优化筛选以得到满足应用需求的水包油乳液。
本发明中所述水包油乳液中的固体颗粒具有油水两亲性，可稳定分散于油水两相界面，起到稳定乳液的作用。针对不同的油水体系，可选择具有不同亲疏水性的固体颗粒以稳定乳液，也可对固体颗粒的表面可以进行亲水或疏水修饰、覆层或接枝改性等，以获得适宜的亲疏水性(或颗粒可润湿性(particle wettability，一般用oil-water-solid contact angleθow表示))。
优选地，所述固体颗粒的表面或内部还可吸附、偶联或包埋靶向物质、荧光标记物、同位素标记物、环境响应物质、细胞因子、抗体或免疫调节剂等功能物质。所述环境响应物质选自带有pH敏感、热敏感或生物活性物质敏感等的基团。
所述固体颗粒的表面或内部还可吸附、偶联或包埋抗原，作为抗原的输送系统，可提高抗原稳定性，促进抗原提呈细胞对抗原的摄取，增强免疫应答。
所述固体颗粒可采用多种方法制备。例如，对于聚(丙交酯-共-乙交酯)固体颗粒，可以采用溶剂蒸发法、溶剂萃取法、沉淀法等多种方法制备。对于壳聚糖固体颗粒，可以采用单乳法(形成油包水乳液)结合化学交联法(如采用溶解在油相中的戊二醛交联)制备，也可以采用喷雾干燥或沉淀法制备。
制备后，可将固体微粒保存在水溶液或缓冲溶液中，也可冻干待用。
优选地，所述固体颗粒的平均粒径在1nm～10μm之间，例如5nm、10nm、50nm、100nm、200nm、300nm、400nm、500nm、600nm、700nm、800nm、900nm、1μm、2μm、3μm、4μm、5μm、6μm、7μm、8μm或9μm，优选10nm～5μm之间。
优选地，所述固体颗粒粒径分布系数span值低于1.0。
优选地，固体颗粒在水相中的质量浓度在0.1～20wt％，例如为0.5wt％、1wt％、2wt％、3wt％、4wt％、5wt％、6wt％、7wt％、8wt％、9wt％、10wt％、11wt％、12wt％、 13wt％、14wt％、15wt％、16wt％、17wt％、18wt％或19wt％，优选为0.5～10wt％，进一步优选为1～8wt％。所述固体颗粒在水相中的质量浓度为固体颗粒的质量除以固体颗粒和水相质量和的比值。
优选地，本发明中所述水包油乳液的油水两相体积比为1∶100～9∶1，例如1∶90、1∶80、1∶70、1∶60、1∶50、1∶40、1∶30、1∶20、1∶10、1∶1、2∶1、3∶1、4∶1、5∶1、6∶1、7∶1或8∶1，优选为1∶50～1∶2。
优选地，所述水包油乳液中乳滴的平均粒径在50nm～300μm之间，例如100nm、200nm、300nm、400nm、500nm、600nm、800nm、1μm、10μm、30μm、50μm、80μm、110μm、140μm、170μm、200μm、230μm、260μm或290μm，优选在100nm～100μm之间。
本发明中所述水包油乳液中还可以包含药用添加剂，所述药用添加剂包括例如稀释剂、稳定剂或防腐剂中的任意一种或者至少两种的组合。
本发明中所述水包油乳液中还可以包括以下佐剂但不限于：模式识别受体(例如Toll样受体、RIG-1和NOD样受体(NLR)的刺激剂，如含CpG基序的寡核苷酸或双链RNA或含回文系列的寡核苷酸或含聚(dG)序列的寡核苷酸)、矿物盐(例如明矾，与肠细菌(例如大肠杆菌、明尼苏达沙门菌、鼠伤寒沙门菌、或弗氏志贺菌)的单磷酰脂质(monphosphoryllipid，MPL)A结合的明矾或分别与
Figure PCTCN2014092290-appb-000004
(AS04)、以上提到的细菌MPLA特异性结合的明矾)、MPL、皂苷(如QS-21、Quil-A，iscoMs，iscomatrixTM)、脂质体和脂质体配制品(如AS01)、合成的或特别制备的微颗粒和微载体(如淋病奈瑟球菌(N.gonorrheae)、沙眼衣原体和其他细菌的源于细菌的外膜泡(OMV))、多糖(如壳聚糖)、特异性修饰或制备的肽(例如胞壁酰二肽)、氨基烷基氨基葡糖苷4-磷酸酯(如RC529)、或蛋白质(如细菌类毒素或毒素片段)、可选择的病原相关分子模式(PAMPS)、小分子免疫增强剂(SMIP)、细胞因子和趋化因子。细胞因子包括但不限于粒细胞巨噬细胞集落刺激因子(GM-CSF)、干扰素(如干扰素-α(IFN-α)、干扰素-β(IFN-β)、干扰素-γ(IFN-γ等)、白细胞介素(如白细胞介素-1α(IL-1α)、白细胞介素-1β(IL-1β)、白细胞介素-2(IL-2)、白细胞介素-4(IL-4)、白细胞介素-7(IL-7)、白细胞介素-12(IL-12)、白细胞介素-15(IL-15)、白细胞介素-18(IL-18))、胎儿肝酪氨酸激酶3配体(FIt3L)或肿瘤坏死因子-α(TNF-α)等。
本发明中所述水包油乳液可采用多种方法制备。具体地，本发明中所述水包油乳液可采用下述方法制备，但不受下述制备方法的限制。
本发明中所述水包油乳液可采用先将固体颗粒分散在水相中，然后将油相和水相混合制备。固体颗粒的分散方式可以选择振荡、搅拌、超声(Ultrasonic dispersion)等多种方式，以实现固体颗粒在水相中的良好分散。只要能实现颗粒在水相中的良好分散，所采用的分散方式不会对水包油乳液的性质造成明显影响，可根据所用水相及固体颗粒性质及自身的实验设备条件选择合适的分散方式及具体操作参数。油相和水相的混合可以选择微流控(Microfluidization)、均质(Homogenization)、超声、注射器双推乳化(Two-syringe emulsification)、喷雾、微射流、微通道(Microchannel emulsification)、膜乳化(Membrane emulsification)、搅拌、振荡、倒转或手摇混合等多种方式。根据不同的需求，混合方式可以优选微流控、微通道或膜乳化等可以获得均一粒径分布乳液的方式，也可以优选微射流、注射器双推乳化、均质、搅拌或振荡等便于规模化制备的混合方式。应该明确的是，不同的油水相混合方式必然影响所得乳液中乳滴的粒径大小、乳液稳定性等，也将影响到最终的免 疫或给药效果。如本发明中的一个实施例所提到的与机械搅拌相比较，采用膜乳化方式可以得到粒径分布更为均一、乳滴更小的乳液，可以获得更高的抗体水平。因此，应根据所用油水相及固体颗粒性质、所需制备的乳滴粒径范围等影响因素确定合适的混合方式及具体操作参数。
本发明中所述水包油乳液中的固体颗粒、油相、水相可以分别独立包装，在应用前根据前述制备方法临时混合，也可以根据前述制备方法事先混合其中的两种或三种。
本发明中所述水包油乳液的灭菌方式可以采用湿热灭菌或过滤除菌，当所用颗粒的粒径小于220nm时，优选采用过滤除菌的方式。
当水包油乳液中含有抗原时，所述水包油乳液可以和抗原独立包装，在免疫接种前临时混合或在较短的时间间隔内(通常为1小时以内，包含1小时)接种到同一部位，也可预先根据前述制备方法先混合后包装，在免疫接种时可以直接应用。
本发明的另一目的在于提供一种不含表面活性剂的免疫原性组合物，其包含：(1)抗原或抗原组合物，和(2)佐剂组合物，所述佐剂组合物由如上所述的水包油乳液组成。
本发明的另一目的在于提供上述水包油乳液作为疫苗佐剂、药物递送或缓控释载体的用途。在本发明中，免疫接种或给药方式包括静脉注射、脊椎腔注射、肌内注射、皮下注射、皮内注射、经呼吸道喷入或吸入、腹腔注射、经鼻给药、经眼给药、经口给药、直肠给药、阴道给药、局部给药或透皮给药。所述水包油乳液作为疫苗佐剂可用于人类、畜类、禽类及水产类。
与已有技术相比，本发明具有如下有益效果：
本发明首次将固体颗粒与油乳制剂(水包油乳液)结合制备不需表面活性剂的水包油乳液，应用于疫苗佐剂的开发领域。固体颗粒的加入，不仅可以提高制剂的生物相容性，避免表面活性剂对人体、动物或疫苗带来的不良影响，并且能够更加有效地刺激免疫细胞，激发免疫调节功能；同时，固体颗粒的性质便于调控，可以对其进行表面修饰或覆层，或者选择不同性质(如组成、形貌、结构、粒径等)的固体颗粒，发挥不同的免疫增强机制，以及对抗原进行包埋、吸附或偶联，作为抗原的运送载体，利用其抗原控制释放的能力来调节免疫应答，还可以应用于多种免疫接种方式。
本发明中水包油乳液的主要免疫增强机制包括：
(1)如所用颗粒对抗原进行吸附或包埋，则该水包油乳液可起到延缓抗原释放速度，保护抗原不被水解，延长抗原在体内滞留时间，有利于高亲和力抗体的产生；(2)颗粒可以活化巨噬细胞并促进巨噬细胞与T和β细胞的相互作用，从而对淋巴细胞有特异性加强刺激的作用；(3)如所用颗粒对抗原进行吸附或包埋，可增加抗原的表面积，使抗原易于被巨噬细胞吞噬；(4)水包油乳液还可在注射部位引起轻微的炎症反应，募集炎症细胞，刺激炎症因子的分泌，激活免疫反应；(5)某些特殊颗粒，如具有pH敏感性的壳聚糖颗粒，吸附或包埋抗原后，可实现抗原的溶酶体逃逸，增强细胞免疫应答。
此外，本发明中水包油乳液还可用于药物递送或缓控释载体，通过将脂溶性的药物、荧光标记物或其他生物活性物质分散在油相中，或将药物、荧光标记物或其他生物活性物质包埋或吸附在颗粒表面，实现药物的缓控释放。通过在颗粒表面或内部偶联或包埋靶向物质(如Fe3O4、叶酸、甘露糖等)，还可将包含有该种颗粒的水包油乳液用于药物、荧光标记物或其他生物活性物质的靶向递送。
因此，本发明中公开的水包油乳液既可作为疫苗的免疫佐剂，又可作为药物或其他生物活性物质的递送或缓控释载体。
附图说明
图1水包油型Pickering乳液示意图；
图2实施例1中制备PLGA颗粒的粒径分布图；
图3实施例1中制备PLGA颗粒的扫描电镜照片；
图4实施例1中制备Pickering乳液的光镜照片(放大20倍)；
图5实施例1中制备Pickering乳液在离心前后的照片(1为离心前，2为离心后)；
图6实施例2中制备氢氧化铝颗粒的粒径分布图；
图7实施例2中制备Pickering乳液的光镜照片(放大20倍)；
图8实施例4中制备Pickering乳液的照片。
具体实施方式
下面结合附图并通过具体实施方式来进一步说明本发明的技术方案。
本发明可以通过以下实施例实现，但不受以下实施例中原料、仪器、比例、组成、方法、步骤的限制。应该理解，这里讨论的实施例和实施方案只是为了说明，对熟悉该领域的人可以提出各种改进和变化，这些改进和变化将包括在本申请的精神实质和范围以及所附的权利要求范围内。
原料列表：
Figure PCTCN2014092290-appb-000005
Figure PCTCN2014092290-appb-000006
Figure PCTCN2014092290-appb-000007
仪器
Figure PCTCN2014092290-appb-000008
Figure PCTCN2014092290-appb-000009
性质表征：
颗粒或乳滴的粒径分布测定：
微米级颗粒或乳滴的粒径分布采用激光粒度仪进行测定，具体测定步骤为：将5mg微米级颗粒加入到50mL的去离子水中，超声5min使其均匀分散，或取50mL乳液，将颗粒悬浮液或乳液加入到样品池中，采用激光粒度仪(Ma1vern Instruments，United Kingdom Coulter Co.，USA)进行测定。
纳米级颗粒或乳滴的粒径分布采用Zeta电位及粒度分析仪进行测定，具体测定步骤为：将1mg纳米级颗粒，加入10mL去离子水中，超声5min使其均匀分散，或取2mL乳液，将颗粒悬浮液或乳液加入到样品池中，放入Zeta电位仪(Zeta Potential Analyzer，Brookhaven Instruments Corporation)中进行测定。
颗粒或乳滴的均一性由粒径分布系数(Span)值表示。Span计算公式如下，其值越小表明颗粒粒径越均一。
Span＝(d90-d10)/d50       (1)
式(1)中，d10，d50和d90分别为颗粒累计体积为10％，50％，90％时的粒径。
颗粒的形貌观察采用扫描电镜观察：称量1mg颗粒，加入10mL去离子水中，超声5min使其均匀分散。吸取1mL悬浮液，将其滴在铝箔上，使其在铝箔上均匀摊开，自然晾干。将铝箔用导电胶贴于样品台上，在真空条件下喷金(根据样品性质选取合适的喷金条件)后，用扫描电子显微镜进行观察。
乳滴的形貌观察采用光学显微镜：吸取少量水包油乳液，滴于载玻片上，置于光学显微镜下观察。
乳液的稳定性采用离心法检测：取5mL水包油乳液，加入15mL离心管中，在2000g离心力作用下离心10min后，观察分层情况。
颗粒中抗原或药物包埋率及载量的测定：
准确称量10mg载药颗粒冻干粉，采用适当的方法使微球完全降解(例如，对于聚乳酸类微球，可采用加入NaOH溶液或乙腈的方法使微球降解；对于壳聚糖类微球，可采用加入稀盐酸的方法使微球降解)。待颗粒完全降解后，用NaOH或盐酸中和降解液，使其pH＝7，再定容至2mL。抗原或药物含量采用BCA试剂盒或micro-BCA试剂盒或其他适宜的检测方法测定。抗原或药物包埋率按以下公式计算：
包埋率＝(实测颗粒中抗原或药物量/实际制备时抗原或药物加入量)×100％
抗原或药物在颗粒上的载量按以下公式计算：
载量＝(实测颗粒中抗原或药物量/所测颗粒的质量)
颗粒上吸附抗原或药物的吸附率及载量的测定：
取出吸附抗原或药物后的颗粒悬浮液，离心取上清(根据颗粒的大小和密度选择合适的离心条件)，测量上清中抗原或药物浓度，从而间接计算出吸附到颗粒表面的抗原或药物的量。抗原或药物含量采用BCA或micro-BCA试剂盒或其他适宜的检测方法测定。抗原或药物吸附率按以下公式计算：
吸附率＝(吸附前抗原或药物浓度-吸附后上清中抗原或药物浓度)/吸附前抗原或药物浓度×100％
抗原或药物在颗粒上的载量按以下公式计算：
载量＝(实测颗粒上抗原或药物量/所测颗粒的质量)
动物实验测定：
实验所采用的Balb/c小鼠由维通利华公司提供。免疫步骤基本如下为：先将小鼠随机分组，每组采用6只以上的小鼠进行实验，根据实施例的具体说明对小鼠进行分组和免疫接种。在接种前，先取血200μL，立即在12000rpm下离心5min，分离出血清，测定IgG抗体水平，以此时的IgG抗体水平作为初始值，然后对小鼠进行免疫。免疫后，定时从小鼠眼周或尾尖取血，每次取血200μL，并测定IgG抗体水平。两周后进行二次免疫，35天处死小鼠，取血，测定IgG抗体水平(对于流感疫苗，还要测定血凝滴度(HI))。取小鼠脾细胞进行培养，用酶联免疫吸附试验(ELISA)检测小鼠脾细胞培养液上清中的的IL-4和IFN-γ细胞因子的分泌情况。
实施例1：采用聚PLGA颗粒制备水包油乳液
采用纳米沉淀法制备PLGA颗粒：
采用电子天平准确称取0.30g的PLGA(LA∶GA为50∶50，分子量为11万道尔顿)，溶于1mL的丙酮中，将该溶液用9号针头以每秒1滴的速度滴加到20mL水溶液(含有1wt.％的PVA(醇解度为99％，粘度为5.0mPa.s)，磁力搅拌，转速为500rpm)中，25℃下搅拌过夜，20000g下离心20min，弃去上清，向沉淀中加入5mL去离子水，超声分散，20000g下离心5min，弃去上清后，将沉淀冻干得到PLGA颗粒，置于冰箱中4℃保存。所制备PLGA颗粒的平均粒径为226nm，Span为0.535。扫描电镜观察所制备颗粒的表面光滑，球形规整。颗粒的粒径分布图如图2所示，形貌如图3所示。
水包油乳液制备：
采用电子天平准确称取0.50g PLGA颗粒，加入到40mL去离子水中，超声1min使其分散均匀，得到分散有颗粒的水相悬浮液。其中水相pH值为6.5。用移液枪吸取1mL角鲨烯加入到上述水相悬浮液中，采用均质(15000rpm，3min)乳化的方法，制备水包油乳液。乳滴分散性良好，球形规整。乳滴的平均粒径为18.9μm，Span为0.895。乳液的稳定性采用离心法检测，所制备水包油乳液的外观与未离心乳液无差异，上层无油相析出。乳液的光镜照片如图4所示，乳液在离心前后的照片如图5所示。
还可采用其他PLA类颗粒制备水包油乳液，其制备步骤与实施例1类似，具体工艺参数及结果如表1所示。
表1
Figure PCTCN2014092290-appb-000010
Figure PCTCN2014092290-appb-000011
Figure PCTCN2014092290-appb-000012
实施例2：采用氢氧化铝颗粒制备水包油乳液
采用微乳液法制备氢氧化铝颗粒：
将Triton X-100、正丁醇与环己烷按照1∶0.5∶20的体积比混合并磁力搅拌(800rpm，5min)混合得到油相。采用注射器推进泵以2mL/min的速率向磁力搅拌(500rpm)下的油相 (20mL)中滴加1mol/L的AlCl3溶液(2mL)，得到氯化铝的反相微乳液。采用注射器推进泵将氨水以0.5mL/min的滴加速率滴加到上述反相微乳液中，保持反应体系的pH值高于10.0，反应2h后，加入10mL丙酮破乳，离心(15000g，5min)后弃去上清液，用无水乙醇反复洗涤3次，去离子水洗涤1次，最后经冻干得到氢氧化铝颗粒，置于冰箱中4℃保存。颗粒的平均粒径为120.5nm，Span为0.486。所制备颗粒的形貌接近球体。颗粒的粒径分布图如图6所示。
水包油乳液制备：
采用电子天平准确称取0.72g氢氧化铝颗粒，加入到40mL磷酸盐缓冲溶液中，超声1min使其分散均匀，得到分散有颗粒的水相悬浮液。其中水相pH值为7.4。用移液枪吸取2mL角鲨烯加入到上述水相悬浮液中，采用均质(20000rpm，5min)乳化的方法，制备水包油乳液。乳滴分散性良好，球形规整。乳滴的平均粒径为7.4μm，Span为0.696。所制备水包油乳液的外观与未离心乳液无差异，上层无油相析出。乳液的光镜照片如图7所示。
实施例3：采用磷酸钙颗粒制备水包油乳液
采用模板法制备中空磷酸钙颗粒：
将3.0g Tween 80，0.25g PEG 6000，3.0mL 0.5mol/L的Tris-HCl(pH8.0)和1.5mL去离子水混合搅拌均匀后，超声20min(10W)形成非离子表面活性剂囊泡，滴入3.55mLCaCl2(0.175mol/L)溶液，搅拌0.5h后，再加入3.55mL Na2HPO4(0.175mol/L)溶液可得到磷酸钠颗粒的悬浮液，加入0.83mL MgCl2溶液(0.075mmol/L)来稳定刚形成的磷酸钙颗粒，继续搅拌2h后离心洗涤，真空干燥后得到中空磷酸钙颗粒。颗粒的平均粒径为210nm，壳层厚度为30-40nm，颗粒的Span为0.349。所制备颗粒为中空球体。
水包油乳液制备：
采用电子天平准确称取1.00g中空磷酸钙颗粒，加入到20mL柠檬酸缓冲溶液中，超声2min使其分散均匀，得到分散有颗粒的水相悬浮液。其中水相pH值为6.0。用移液枪吸取2mL角鲨烯加入到上述水相悬浮液中，采用磁力搅拌(500rpm，10min)乳化的方法，制备水包油乳液。乳滴分散性良好，球形规整。乳滴的平均粒径为10.2μm，Span为0.696。所制备水包油乳液的外观与未离心乳液无差异，上层无油相析出。
实施例4：采用壳聚糖覆层的海藻酸颗粒制备水包油乳液
采用快速膜乳化技术制备壳聚糖覆层的海藻酸颗粒：
首先将石油醚(沸程为60-90℃)和液体石蜡以体积比为2∶1混合，并向混合有机相中加入质量分数为4wt.％的乳化剂Span 80，上述混合物作为油相，水相为海藻酸钠水溶液(1.0wt％)。将2mL水相与60mL油相在均质(3600rpm，1min)的作用下乳化形成预乳液，接着将预乳液加入到快速膜乳化储罐中，在氮气压力下(1MPa)，预乳液通过SPG膜(膜孔径为1.4μm)，循环5次，就得到了较为均一的乳液。CaCl2溶液(5mol/L，12mL)作为固化剂，在超声(120W，1min)的作用下分散到油相(24mL)中形成细乳。将该细乳与上面得到的均一的乳液混合后在37℃水浴中搅拌5h(250rpm)使乳液固化得到胶体颗粒，用石油醚、乙醇、水各分别洗涤三次，就得到了海藻酸颗粒。
海藻酸颗粒的壳聚糖覆层步骤包括：首先将1g海藻酸颗粒分散到0.7wt％的壳聚糖乙酸溶液(20mL，壳聚糖分子量为80万道尔顿，脱乙酰度为90％)中，搅拌1h(200rpm)后，将颗粒用乙酸缓冲溶液(pH 4和pH 5.5)以及去离子水洗涤干净，获得壳聚糖覆层的海藻酸 颗粒。还可对颗粒进行多次镀层，步骤为：将已覆层的胶体颗粒(1g)分散到0.5wt％的海藻酸钠水溶液(20mL)中，搅拌1h(200rpm)，用去离子水洗涤一次后，再将颗粒分散到0.7wt％的壳聚糖乙酸溶液(20mL)中，并搅拌1h(200rpm)。然后将颗粒用乙酸缓冲溶液(pH 4和pH 5.5)和去离子水洗涤后，就得到了双层覆层的壳聚糖-海藻酸颗粒。按上述步骤重复多次，可获得壳聚糖多次覆层的海藻酸颗粒。本发明中对海藻酸颗粒进行三次覆层，颗粒的平均粒径为457nm，颗粒的Span为0.839。所制备颗粒表面粗糙，球形规整。
水包油乳液制备：
采用电子天平准确称取1.22g壳聚糖覆层海藻酸颗粒，加入到10mL磷酸盐缓冲溶液中，超声1min使其分散均匀，得到分散有颗粒的水相悬浮液。其中水相pH值为8.0。用移液枪吸取2mL角鲨烯加入到上述水相悬浮液中，采用漩涡振荡(10min)乳化的方法，制备水包油乳液。乳滴分散性良好，球形规整。乳滴的平均粒径为10.2μm，Span为0.741。所制备水包油乳液离心后，上层析出1cm左右的油相。乳液的照片如图8所示。
实施例5：采用单甲氧基聚乙二醇-乳酸共聚物(PELA)多孔颗粒制备水包油乳液
采用相分离-溶剂去除法制备PELA多孔颗粒：
将100mg PELA(mPEG∶PLA摩尔比为1∶19，平均分子量为40kDa)溶于7.5mL丙酮中，加入7.5mL无水乙醇溶液，将上述溶液滴入(1滴/s)快速搅拌(750rpm)下的去离子水(90mL，含10g/L SDS)中，滴加完毕后，继续搅拌24h(750rpm)。用去离子水离心洗涤5次后，将沉淀悬浮在10mL去离子水中保存，为PELA颗粒悬浮液。
向3mL去离子水中加入200μL二氯甲烷，置于超声波细胞破碎机(400W，60s)中混合均匀作为溶胀剂。取上述2mL PELA颗粒悬浮液加入到溶剂剂中，维持磁力搅拌(250rpm)溶胀30min，静置1h后快速放入液氮中使其迅速冷冻，保存在-20℃中使有机溶剂挥发得到具有多孔结构的PELA颗粒，颗粒的平均粒径为78.55nm，Span为0.331，颗粒为表面多孔的球形结构。
水包油乳液制备：
采用电子天平准确称取0.351g PELA颗粒，加入到10mL注射用水中，超声1min使其分散均匀，得到分散有颗粒的水相悬浮液。其中水相pH值为7.0。油相为2mLα-生育酚，采用顺锥微流控乳化的方法(锥形端口直径范围为20-40μm，油相流速范围为500-600μL/h，水相流速范围为1000-1200μL/h)，制备水包油乳液。乳滴分散性良好，球形规整。乳滴的平均粒径为70μm，Span为0.096。所制备水包油乳液的外观与未离心乳液无差异，上层无油相析出。
实施例6：采用花生状的碳酸钙颗粒制备水包油乳液
采用液相直接混合沉淀法制备花生状的碳酸钙颗粒：
用电子天平称取醋酸钙和柠檬酸三钠溶解在200mL蒸馏水中(柠檬酸三钠的质量浓度分别为10wt.％和30wt.％)，向该溶液中加入10wt.％的碳酸钠水溶液(50mL)，搅拌(300rpm)下反应3h，过滤，用蒸馏水及无水乙醇分别洗涤沉淀三次后，在70下烘干，得到花生状的碳酸钙颗粒。颗粒的长度为7.2μm，长短轴比为2∶1，所制备颗粒的形貌为花生状。
水包油乳液制备：
采用电子天平准确称取3.15g碳酸钙颗粒，加入到40mL磷酸盐缓冲溶液中，超声1min使其分散均匀，得到分散有颗粒的水相悬浮液。其中水相pH值为7.4。用移液枪吸取2mL 角鲨烯加入到上述水相悬浮液中，采用快速膜乳化(Shirasu Porous Glass(SPG)微孔膜孔径为50.2μm，过膜压力为200KPa，过膜三次)的方法，制备水包油乳液。乳滴分散性良好，球形规整。乳滴的平均粒径为45.10μm，Span为0.726。所制备水包油乳液离心后，上层析出0.5cm的油相。
实施例7：采用包埋有抗原的聚乳酸(PLA)颗粒制备水包油乳液
采用快速膜乳化结合复乳溶剂去除法制备包埋有乙肝抗原(HBsAg)的PLA颗粒：
将200mg的PLA溶于4.0mL的乙酸乙酯中，加入0.4mL的5％(w/v)的HBsAg，用超声波细胞破碎仪在冰水浴条件下进行初乳化(功率12％，时间为15s)，然后将初乳液倒入200mL的含1.0wt.％PVA的水溶液(外水相)中以磁力搅拌进行预复乳化(300rpm，50s)。预复乳化后将预复乳液倒入快速膜乳化装置的储存罐里，以压力为300KPa的氮气把预复乳液压过SPG膜(膜孔径为5.2μm)得到复乳液。将复乳液倒入800mL 0.9wt.％NaCl水溶液(固化溶液)中500rpm磁力搅拌10min，以固化微球。固化后的微球用去离子水离心(4000r/min，5min)洗涤3次，最后冷冻干燥制成成品。颗粒的平均粒径为2.32μm，Span为0.496，颗粒为表面光滑的球形结构。抗原的包埋率为90％，颗粒的载抗原量为0.09mg抗原/g微球。
水包油乳液制备：
采用电子天平准确称取7.22g PLA颗粒，加入到40mL可注射用水中，漩涡振荡5min使其分散均匀，得到分散有颗粒的水相悬浮液。其中水相pH值为7.0。用移液枪吸取2mLα-生育酚加入到上述水相悬浮液中，采用微射流乳化(12000psi)的方法，制备水包油乳液。乳滴分散性良好，球形规整。乳滴的平均粒径为42.39μm，Span为0.742。所制备水包油乳液的外观与未离心乳液无差异，上层无油相析出。
实施例8：采用壳聚糖颗粒制备水包油乳液
采用快速膜乳化结合温度固化法制备壳聚糖颗粒：
水相配制：准确称取一定量的壳聚糖(分子量为5万道尔顿，脱乙酰度为80％)溶于9mL醋酸溶液(0.1mol/L)中，磁力搅拌下使其充分溶解得到壳聚糖醋酸溶液；另将一定量的甘油磷酸钠溶解于1mL去离子水中。将壳聚糖醋酸溶液及甘油磷酸钠溶液分别在4℃下保温10min后，将甘油磷酸钠溶液缓慢滴加至壳聚糖醋酸溶液中，磁力搅拌(300rpm，10min)将其混合均匀。将此溶液在20000rpm下离心除去不溶性杂质，保留上清液作为水相备用。壳聚糖在水相中的浓度为3.5wt.％，甘油磷酸钠在水相中的浓度为10.0wt.％。
油相制备：将油溶性乳化剂PO-500加入到60mL的液体石蜡和石油醚(石油醚沸程为60-90℃)的混合物(体积比为5∶7)中，PO-500在油相中的浓度为4wt.％，搅拌至完全溶解，在4℃下保温10min作为油相。
乳液制备：在4℃下，将2mL的水相与50mL的油相混合并用均质乳化器在6000rpm下乳化1min，形成预乳液。将所得预乳液迅速倒入快速膜乳化装置的预乳液储存器中，在5.0MPa的氮气压力下，使其快速通过SPG微孔膜(膜孔径为2.8μm)，得到粒径比较均一的W/O型乳液，将所得乳液作为预乳液在5.0MPa的氮气压力下再次通过SPG微孔膜，反复乳化五次，最终得到粒径均一的W/O型乳液；乳化过程耗时约10min，乳化完毕后，将W/O型乳液放入35℃的水浴中，机械搅拌(200rpm)下固化1h。固化反应结束后，在10000rpm下离心，依次用石油醚、乙醇和去离子水洗涤，得到壳聚糖颗粒。颗粒的平均直径为 870nm，Span值为0.487，颗粒为表面疏松多孔的球状结构。
水包油乳液制备：
采用电子天平准确称取1.00g壳聚糖颗粒，加入到20mL PBS缓冲溶液中，超声1min使其分散均匀，得到分散有颗粒的水相悬浮液。其中水相pH值为8.1。用移液枪吸取2mLα-生育酚加入到上述水相悬浮液中，采用漩涡振荡(10min)的方法，制备水包油乳液。乳滴分散性良好，球形规整。乳滴的平均粒径为18.10μm，Span为0.935。所制备水包油乳液的外观与未离心乳液无差异，上层无油相析出。
还可采用其他壳聚糖材料制备颗粒以及随之的水包油乳液，其制备步骤与实施例8类似，区别在于采用不同分子量及脱乙酰度的壳聚糖及改变油水相组成和制备工艺，具体工艺参数及结果如表2所示。在同样的脱乙酰度条件下，分子量越小，其粘度越低，用同样的膜乳化条件制备的颗粒粒径越小，所制备乳液的粒径也越小。在同样的分子量条件下，脱乙酰度越高，其粘度越低，用同样的膜乳化条件制备的颗粒粒径越小，所制备乳液的粒径也越小。
表2
Figure PCTCN2014092290-appb-000013
Figure PCTCN2014092290-appb-000014
实施例9：采用吸附有抗原的壳聚糖颗粒制备水包油乳液
颗粒的制备方法同实施例8，区别在于壳聚糖颗粒吸附H5N1禽流感裂解疫苗：
准确称取1g制备得到的壳聚糖颗粒，加入10mL含有H5N1禽流感裂解疫苗(HA浓度为150μg/mL)的PBS缓冲液中，4℃下振荡(120rpm，24h)，10000rpm下离心，用去离子水洗涤三次，得到吸附有H5N1禽流感裂解疫苗的壳聚糖颗粒。抗原的吸附率为60％，颗粒上的抗原载量为900μgHA/g颗粒。
水包油乳液制备：
采用电子天平准确称取1.00g吸附有抗原的壳聚糖颗粒，加入到20mLPBS缓冲溶液中，超声1min使其分散均匀，得到分散有颗粒的水相悬浮液。其中水相pH值为8.1。用移液枪吸取2mLα-生育酚加入到上述水相悬浮液中，采用漩涡振荡(10min)的方法，制备水包油乳液。乳滴分散性良好，球形规整。乳滴的平均粒径为19.70μm，Span为0.941。所制备水包油乳液的外观与未离心乳液无差异，上层无油相析出。
实施例10：水包油乳液的安全性评价
1.血管刺激性试验
将实施例1和实施例2分别于家兔耳静脉注射给药，每日一次，连续给药3天。结果：家 兔耳静脉给药部位肉眼观察无明显变化；组织病理切片显微镜检显示距注射部位1cm处血管、5cm处血管内皮连续、完整，未见增生、肿胀；血管周围组织未见炎性细胞浸润及坏死；管腔内无血栓形成。显示本品对家兔耳静脉血管未见明显刺激作用。
2.溶血与凝聚试验
采用常规体外试管法(肉眼观察法)，将实施例1和实施例2分别与2％红细胞混悬液混合，3小时内未见溶血与红细胞凝聚现象。
3.肌肉刺激性试验
将实施例1和实施例2分别用于家兔股四头肌注射给药，每侧给药1mL。48小时后取给药部位肉眼观察并作病理组织学检查。结果表明，本品对家兔股四头肌无刺激性。
实施例11：水包油乳液中水相与油相的混合方式对产生抗体滴度的影响
按照实施例1中方法制备PLGA颗粒。
水包油乳液制备：
采用电子天平准确称取2g PLGA颗粒，加入到40mL柠檬酸缓冲溶液中，超声5min使其分散均匀，得到分散有颗粒的水相悬浮液。其中水相pH值为6.0。用移液枪吸取2mL角鲨烯加入到上述水相悬浮液中，将上述混合液平均分为两份，分别采用快速膜乳化(膜孔径为40.2μm，过膜压力为500KPa，过膜三次)和机械搅拌(500rpm，5min)的方法进行乳化，制备水包油乳液。所制备乳液的乳滴均分散性良好，球形规整。其中快速膜乳化所制备乳液中乳滴的平均粒径为32.20μm，Span为0.372；机械搅拌所制备乳液中乳滴的平均粒径为45.32μm，Span为0.839。所制备水包油乳液的外观与未离心乳液无差异，上层无油相析出。
将上述制得的水包油乳液分别与H5N1禽流感裂解疫苗(血凝素(HA)含量均为4.5μg/100μL)混合(乳液与疫苗的混合体积比为1∶1，超声(10W，3min)混合)，另取同样血凝素含量的裂解疫苗作为对照。对Balb/c小鼠左后腿肌肉注射后，两周后进行二次免疫，35天处死小鼠，结果如表3所示，与机械搅拌组相比，快速膜乳化组能发挥良好的佐剂效应，实验动物体内产生高水平的IgG、HI滴度，佐剂组均与单抗原注射组间存在着显著性差异。
表3
抗原种类 佐剂种类 乳液制备方式 血清IgG水平 HI水平
裂解疫苗 实施例1 快速膜乳化 480000 2560
裂解疫苗 实施例1 机械搅拌 320000 640
裂解疫苗 无 无 62000 32
实施例12：水包油乳液与抗原的混合方式对产生抗体滴度的影响
将按照实施例11中方法制得的水包油乳液分别与H5N1禽流感灭活全病毒疫苗和裂解疫苗(血凝素(HA)含量均为4.5μg/100μL)混合(乳液与疫苗的混合体积比为1∶1，采用不同的混合方式，包括振荡(5min)、超声(10W，3min)、均质(10000rpm，1min))，对Balb/c小鼠左后腿肌肉注射后，两周后进行二次免疫，35天处死小鼠，结果如表4所示，水包油乳液均能发挥良好的佐剂效应，实验动物体内产生高水平的IgG、HI滴度，与单抗原注射组间存在着显著性差异，其中均质法混合的水包油乳液注射组具有更高的抗体水平。
表4
抗原种类 佐剂种类 混合方式 血清IgG水平 HI水平
灭活全病毒疫苗 实施例1 振荡 320000 720
灭活全病毒疫苗 实施例1 超声 510000 2580
灭活全病毒疫苗 实施例1 均质 600000 3600
灭活全病毒疫苗 无 无 80000 64
裂解疫苗 实施例1 振荡 300000 640
裂解疫苗 实施例1 超声 480000 2560
裂解疫苗 实施例1 均质 580000 3200
裂解疫苗 无 无 62000 32
实施例13：水包油乳液与抗原接种方式对佐剂作用的影响
将按照实施例11中方法制得的水包油乳液分别与乙肝表面抗原重组疫苗(重组汉逊酵母疫苗，抗原浓度为1.9mg/mL)混合(乳液与疫苗的混合体积比为1∶1，采用均质(10000rpm，1min)混合)，将上述疫苗组合物对Balb/c小鼠分别进行肌肉、皮下和腹腔注射，两周后进行二次免疫，35天处死小鼠，结果如表5所示，肌肉注射组表现出较高的体液抗体水平(IgG)及细胞因子分泌水平(IL-4及IFN-γ)。
表5
抗原种类 佐剂种类 接种方式 血清IgG水平 IL-4 IFN-γ
乙肝疫苗 实施例1 肌肉注射 256000 32 11240
乙肝疫苗 实施例1 皮下注射 184000 28 10020
乙肝疫苗 实施例1 腹腔注射 102400 18 8000
乙肝疫苗 无 肌肉注射 20480 5 2000
乙肝疫苗 无 皮下注射 14560 4 1540
乙肝疫苗 无 腹腔注射 10240 2 1000
实施例14：水包油乳液与铝佐剂、MF59佐剂的佐剂作用比较
将实施例11中水包油乳液、氢氧化铝佐剂及MF59分别与H1N1甲型流感灭活全病毒疫苗按照体积比1∶1的比例混合制成疫苗佐剂组合物，组合物中血凝素浓度为15μg/mL，每剂0.5mL。另取血凝素浓度15μg/mL的灭活全病毒疫苗作为对照。分别将等体积上述三种疫苗佐剂组合物及对照疫苗对Balb/c小鼠进行肌肉注射免疫接种，两周后进行二次免疫，35天处死小鼠，免疫结果如表6所示。结果表明，本发明的水包油乳液可以显著提高机体对流感灭活全病毒疫苗的体液免疫应答水平及细胞免疫水平。水包油乳液在体液免疫方面的增强效果与MF59相当，高于氢氧化铝佐剂，三者均高于不加佐剂的疫苗；在细胞免疫方面，水包油乳液组优于MF59及氢氧化铝佐剂组，这主要是由于水包油乳液中的颗粒可促进抗原被抗原提呈细胞吞噬，并激活淋巴细胞，刺激其分化并分泌细胞因子。
表6
Figure PCTCN2014092290-appb-000015
实施例15：水包油乳液加入量对免疫效果的影响
将按照实施例11中方法制得的水包油乳液与EV71手足口灭活病毒疫苗以2∶1、1∶1、1∶2、1∶4等不同比例混合组成疫苗佐剂组合物，其中抗原浓度均为0.05mg/mL，每剂0.2mL。以上述疫苗佐剂组合物对Balb/c小鼠进行肌肉注射接种，两周后进行二次免疫。结果见表7。结果表明，提高疫苗佐剂组合物中佐剂量，可以增强定量抗原产生的免疫应答水平，佐剂剂量与佐剂作用强度正相关。
表7
抗原种类 佐剂种类 佐剂与抗原混合比例 血清IgG水平
EV71手足口灭活病毒疫苗 实施例1 2∶1 204800
EV71手足口灭活病毒疫苗 实施例1 1∶1 145000
EV71手足口灭活病毒疫苗 实施例1 1∶2 81400
EV71手足口灭活病毒疫苗 实施例1 1∶4 51200
EV71手足口灭活病毒疫苗 无 无 10240
实施例16：不同抗原浓度的疫苗对免疫效果的影响
将按照实施例11中方法制得的水包油乳液与H5N1流感灭活全病毒疫苗组成血凝素浓度为37.5μg/mL、75μg/mL、150μg/mL的疫苗佐剂组合物，每剂均为0.1mL。以上述两种疫苗佐剂组合物及同样血凝素浓度的两种不含佐剂疫苗经肌肉接种免疫Balb/c小鼠，两周后二次免疫，35天处死小鼠，结果见表8。结果表明，本发明的水包油乳液对H5N1流感灭活全病毒疫苗具有良好的佐剂作用，与相同血凝素浓度的不含佐剂疫苗相比，佐剂疫苗能够明显提高小鼠体内免疫应答强度。
表8
Figure PCTCN2014092290-appb-000016
Figure PCTCN2014092290-appb-000017
实施例17：水包油乳液用于鼻腔粘膜免疫接种的佐剂效果
将按照实施例11中方法制得的水包油乳液与H7N9流感裂解疫苗组成血凝素浓度为150μg/mL的疫苗佐剂组合物，每剂均为0.03mL。以上述疫苗佐剂组合物及同样血凝素浓度的不含佐剂疫苗经鼻腔滴鼻接种免疫Balb/c小鼠，两周后二次免疫，35天处死小鼠，免疫结果见表9。结果表明，本发明的水包油乳液对H7N9流感裂解疫苗具有良好的佐剂作用，与相同血凝素浓度的不含佐剂疫苗相比，佐剂疫苗能够明显提高小鼠血清及粘膜处的免疫应答强度。
表9
Figure PCTCN2014092290-appb-000018
实施例18：水包油乳液作为免疫佐剂的部分增强机理(局部致炎作用)
为观察MF59、AS03与本发明的水包油乳液(按照实施例11制备)这三种不同乳剂型疫苗佐剂所引发的免疫反应，将鸡卵白蛋白(OVA)作为抗原与三种乳剂型疫苗佐剂混合制成佐剂疫苗组合物。将上述佐剂疫苗组合物经Balb/c小鼠右后腿肌肉注射接种免疫，不同时间处死小鼠，将小鼠注射部位组织切片，以H&E、TUNEL两种方法对切片组织进行染色，观察三种乳剂型疫苗佐剂在体内引起的炎症反应。局部组织切片观察显示，三种乳剂型疫苗佐剂均能同时造成一定的炎症反应，募集炎症细胞，但炎症程度较轻，七天后炎症反应消退，具有良好的生物相容性。
实施例19：水包油乳液作为免疫佐剂的部分增强机理(促进细胞吞噬作用)
以小鼠的巨噬细胞株RAW264.7作为抗原递呈细胞，以OVA作为抗原与水包油乳液混合制成佐剂疫苗组合物。进行体外细胞实验，考察水包油乳液(按照实施例11制备)对抗原递呈细胞吞噬抗原量的影响，结果表明佐剂组可以显著增加RAW264.7细胞吞噬抗原的量。
实施例20：水包油乳液作为药物释放载体
按照实施例4制备水包油乳液，区别在于油相中加入质量浓度为0.5wt.％的紫杉醇。
实施例21：水包油乳液作为药物释放载体
按照实施例5制备水包油乳液，区别在于PELA颗粒中包埋有质量浓度为1wt.％的胰岛素。
申请人声明，本发明通过上述实施例来说明本发明的详细方法，但本发明并不局限于上述详细方法，即不意味着本发明必须依赖上述详细方法才能实施。所属技术领域的技术人员应该明了，对本发明的任何改进，对本发明产品各原料的等效替换及辅助成分的添加、具体方式的选择等，均落在本发明的保护范围和公开范围之内。

Claims (10)

  1. 一种不含表面活性剂的水包油乳液，其特征在于，所述乳液包括可代谢的油相、水相以及分散在水相中的具有生物相容性的油水两亲性的固体颗粒，其中，所述油相包括角鲨烯或/和母育酚，所述水相为纯化水、注射用水、甘油水溶液、缓冲盐水溶液或临床上可用输液中的任意一种或者至少两种的组合，所述固体颗粒的平均粒径在纳米到微米级别。
  2. 如权利要求1所述的水包油乳液，其特征在于，所述母育酚是α-生育酚或其衍生物；
    优选地，所述油相还包括任何对受体无毒并可通过代谢作用转化的植物油、鱼油、动物油或合成油，优选自大豆油、米格列醇、中链油、鱼油、维生素E、维生素E琥珀酸酯、维生素E醋酸酯、红花油、玉米油、沙棘油、亚麻子油、花生油、茶油、葵花籽油、杏仁油、薏仁油、月见草油、芝麻油、棉籽油、蓖麻油、低芥酸菜子油、油酸乙酯、油酸、亚油酸乙酯、月桂酸异丙酯、内豆蔻酸异丙酯、丁酸乙酯、乳酸乙酯、辛酸甘油三酯或癸酸甘油三酯中的任意一种或者至少两种的组合。
  3. 如权利要求1或2所述的水包油乳液，其特征在于，所述水相为注射用水、磷酸盐缓冲液、柠檬酸缓冲液或Tris缓冲液中的任意-种或者至少两种的组合；
    优选地，所述磷酸盐缓冲液、柠檬酸缓冲液或Tris缓冲液的pH值独立地为5.0～8.1，优选为6.0～8.0。
  4. 如权利要求1-3之一所述的水包油乳液，其特征在于，所述水相中含有单价或多价抗原，所述抗原选自人类抗原、非人类动物抗原、植物抗原、细菌抗原、真菌抗原、病毒抗原、寄生虫抗原或肿瘤抗原中的任意一种或者至少两种的组合；
    优选地，所述抗原来自鸡胚培养、细胞培养、携带者体液、器官或组织中纯化分离所得、重组基因表达或化学合成所得，优选所述抗原选自减毒疫苗、灭活疫苗、裂解疫苗、亚单位疫苗、多糖结合疫苗、重组疫苗或DNA疫苗等中的任意一种或者至少两种的组合；
    优选地，所述抗原为包含来自至少三种流感季节性株的病毒抗原或抗原性制剂，并且任选包含至少一种与大流行爆发相关或具有与大流行爆发相关的潜能的流感病毒株的病毒抗原或抗原性制剂，其中，与大流行爆发相关或具有与大流行爆发相关的潜能的流感病毒株选自：
    人类流感病毒中A、B、C型，包含H1N1，H2N2，H3N2，H5N1，H7N7，H1N2，H9N2，H7N3，H10N7；
    猪型流感病毒H1N1，H1N2，H3N1，H3N2；
    狗或马型流感病毒H7N7，H3N8；
    或禽流感病毒H5N1，H7N2，H1N7，H7N3，H13N6，H5N9，H11N6，H3N8，H9N2，H5N2，H4N8，H10N7，H2N2，H8N4，H14N5，H6N5和H12N5中的一种或一种以上的流感病毒组合；
    优选地，所述水相中还包括药用辅助物质，优选pH调节剂或/和缓冲剂，进一步优选自乙酸钠、乳酸钠、氯化钠、氯化钾、氯化钙、人血清清蛋白、必需氨基酸、非必需氨基酸、L-精氨酸盐酸盐、蔗糖、无水D-海藻糖、甘露醇、甘露糖、淀粉或明胶中的任意一种或者至少两种的组合。
  5. 如权利要求1-4之一所述的水包油乳液，其特征在于，所述固体颗粒选自铝盐、钙盐、多糖、多糖衍生物或高分子聚合物中的任意一种或者至少两种的混合物；
    优选地，所述铝盐为氢氧化铝或/和磷酸铝；
    优选地，所述钙盐为磷酸钙或/和碳酸钙；
    优选地，所述多糖选自壳聚糖、海藻酸、明胶、淀粉、葡聚糖、魔芋葡甘聚糖、肝素、果胶类多糖、透明质酸、硫酸软骨素、壳聚糖盐、海藻酸盐、明胶盐、葡萄糖盐、魔芋葡甘聚糖盐、肝素盐、果胶类多糖盐、透明质酸盐或硫酸软骨素盐中的任意一种或者至少两种的组合，优选壳聚糖、海藻酸盐、明胶、淀粉或葡聚糖中的任意一种或者至少两种的组合；
    优选地，所述多糖衍生物为对多糖进行季铵化、羧甲基化、羟基化、烷基化、酰基化、磺化、硝化或卤化等衍生反应后所得的多糖衍生物，优选为对壳聚糖、海藻酸、明胶、淀粉或葡聚糖中的任意一种或者至少两种的组合进行季铵化、羧甲基化、羟基化、烷基化、酰基化、磺化、硝化或卤化等衍生反应后所得的多糖衍生物；
    优选地，所述高分子聚合物包括聚(α-羟酸)、聚羟基丁酸、聚己酸内酯、聚原酸酯、聚酐或聚氰基丙烯酸酯及其共聚物，所述共聚物的共聚单体优选聚(α-羟酸)、聚羟基丁酸、聚己酸内酯、聚原酸酯、聚酐或聚氰基丙烯酸酯中的任意一种或者至少两种的组合。
    优选地，所述高分子聚合物是聚(α-羟酸)及其共聚物，优选自聚(L-丙交酯)、聚(D，L-丙交酯)或聚(丙交酯-共-乙交酯)，最优选聚(丙交酯-共-乙交酯)；
    优选地，在聚(丙交酯-共-乙交酯)中，丙交酯与乙交酯的链段摩尔比为10∶90-90∶10；
    优选地，所述固体颗粒选自氢氧化铝、磷酸铝、磷酸钙、碳酸钙、壳聚糖、海藻酸盐、聚乳酸、聚乳酸-羟基乙酸共聚物或聚乙二醇-乳酸共聚物中的任意一种或者至少两种的混合物，进一步优选自氢氧化铝、磷酸铝、磷酸钙、聚乳酸、聚乳酸-羟基乙酸共聚物或聚乙二醇-乳酸共聚物中的任意一种或者至少两种的混合物，最优选聚乳酸-羟基乙酸共聚物。
  6. 如权利要求1-5之一所述的水包油乳液，其特征在于，对固体颗粒的表面进行亲水修饰、疏水修饰、覆层或接枝改性；
    优选地，所述固体颗粒的表面或内部吸附、偶联或包埋靶向物质、荧光标记物、同位素标记物、环境响应物质、细胞因子、抗体或免疫调节剂；
    优选地，所述环境响应物质选自带有pH敏感、热敏感或生物活性物质敏感的基团；
    优选地，所述固体颗粒的表面或内部吸附、偶联或包埋抗原；
    优选地，所述固体颗粒的平均粒径在1nm～10μm之间，优选10nm～5μm之间；
    优选地，所述固体颗粒粒径分布系数span值低于1.0；
    优选地，固体颗粒在水相中的质量浓度在0.1～20wt％，优选为0.5～10wt％，进一步优选为1～8wt％。
  7. 如权利要求1-6之一所述的水包油乳液，其特征在于，所述乳液包括可代谢的油相、水相以及分散在水相中的具有生物相容性的油水两亲性的固体颗粒，其中，所述油相为角鲨烯，所述水相为纯化水、注射用水、甘油水溶液、缓冲盐水溶液或临床上可用输液中的任意一种或者至少两种的组合，所述固体颗粒为PLGA，其平均粒径在纳米到微米级别；
    优选地，所述水包油乳液的油水两相体积比为1∶100～9∶1，优选为1∶50～1∶2；
    优选地，所述水包油乳液中乳滴的平均粒径在50nm～300μm之间，优选在100nm～100μm之间；
    优选地，所述水包油乳液中还包含药用添加剂，所述药用添加剂包括稀释剂、稳定剂或防腐剂中的任意一种或者至少两种的组合。
  8. 一种不含表面活性剂的免疫原性组合物，其特征在于，其包含：(1)抗原或抗原组合物，和(2)佐剂组合物，所述佐剂组合物由如权利要求1-7之一所述的水包油乳液组成。
  9. 一种如权利要求1-7之一所述的水包油乳液作为疫苗佐剂、药物递送或缓控释载体的用途。
  10. 如权利要求9所述的用途，其特征在于，免疫接种或给药方式包括静脉注射、脊椎腔注射、肌内注射、皮下注射、皮内注射、经呼吸道喷入或吸入、腹腔注射、经鼻给药、经眼给药、经口给药、直肠给药、阴道给药、局部给药或透皮给药。
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