CN108324938A - A kind of granular pattern adjuvant and its preparation method and application - Google Patents

Abstract

The present invention provides a kind of granular pattern adjuvants and its preparation method and application, the granular pattern adjuvant includes biocompatibility wall material, biocompatibility grease and all-trans retinoic acid, the kernel that the biocompatibility grease is formed is coated in the shell of biocompatibility wall material formation, the all-trans retinoic acid is coated in the kernel, granular pattern adjuvant provided by the invention, the activation single compared to existing method, the present invention is immunized by the method while activation system of injecting immune and mucosal immunity, realize the dual activation effect for systemic immune and mucosal immunity, antigen and hydrophobicity immune regulator ATRA are loaded jointly for the first time, delivering and release.

Description

A kind of granular pattern adjuvant and its preparation method and application

Technical field

The invention belongs to biotechnology, it is related to a kind of granular pattern adjuvant and its preparation method and application.

Background technology

Mucomembranous immune system, including intestines associated mucosal tissues, nasal membrane tissue, lung mucosal tissue and genital mucosal group It knits, is first of physiologic barrier of human body.Most pathogen, as influenza virus, AIDS virus, hand-foot-and-mouth disease are malicious, deep and remote Door pylori, HPV oncogenic viruses etc. are all to enter human body by mucous membrane.Therefore, mucosal immunity response and systemic immune (body Liquid is immunized and cellular immunity) no less important.

But the activation of mucosal immunity is cannot achieve by multiple proof in such a way that conventional injection is immune.Cause mucous membrane The most direct mode of immune response is exactly to pass through mucosa delivery.But mucous membrane is there are self-cleaning effect, and strong acid, highly basic, height Concentration protein enzyme environment is unfavorable for being stabilized for antigen, and the mucosal immunity depression effect generated under high antigen concentration. Mucosa delivery often cannot achieve efficient antigen delivery, and the mucosal immunity response of initiation and systemic immune are all barely satisfactory. So far, more successful story is exactly sabin's oral vaccine, but since the vaccine uses attenuated live vaccine, meeting Children are caused to generate anus redness, fever, the complication such as diarrhea.

As the rapid development and society of modern biotechnology are for the pay attention to day by day of the adverse reaction caused by vaccine, split Solution vaccine, recombinant subunit vaccine, Anti-idiotype Antibody Vaccine, nucleic acid vaccine and synthetic peptide vaccine etc. obtain Devoting Major Efforts To Developing.This A little vaccine antigen purity are high, and the adverse reaction of vaccine is low, however the immunogenicity of these antigens and immune protective are generally weaker, In order to improve the validity of antigen, it is necessary to add adjuvant in antigen to enhance the ability that it induces immune response.Therefore, it develops A kind of adjuvant by way of injection inoculation, while causing systemic immune and mucosal immunity and is particularly important.

All-trans retinoic acid (all-trans retinoic acid, ATRA) also known as Tretinoin, vitamin A acid etc., chemical name (13E) -3,7- dimethyl -9- (2,6,6- 3-methyl cyclohexanol alkenyl) -2,4,6,8- nona tetraenoic acids, molecular formula C₂₀H₂₈O₂, it is dimension The derivative of raw element A, is the mesostate of vitamin A in vivo.To Cell differentiation inducing activity and immunological regulation play to Close important role.It is widely used in acute promyelocytic leukemia, the treatment of the Hematological Malignancies such as myelodysplastisches, Promote the growth of epithelial cell differentiation, reduce sebum secretion, inhibit inflammation, promotes stem cell differentiation, adjust benchmark animal embryo The fields such as differentiation.

In recent years, be equally proved to can be by activating intracellular RAR and rxr receptor, to induce immunocyte to produce by ATRA Raw mucous membrane homing receptor (CCR9 and α₄β₇) function.Moreover, ATRA inducing mucosals are gone back to the nest, it is thin to may act on panimmunity for effect Born of the same parents, including antigen presenting cell (Dendritic Cells, monocyte and macrophage), T cell, B cell and B cell mucous membrane are anti- The secretion of body IgA.Also, ATRA can also promote these cell height expression RALDH (enzyme for promoting ATRA synthesis), to divide jointly ATRA is secreted, to form positive feedback, more many cells is promoted to go back to the nest to mucous membrane field.

At the same time, ATRA can not only induce the mucous membrane of immunocyte to go back to the nest effect, in swashing for immunity of organism response Aspect living is also equipped with two-sidedness.Under relatively low concentration, ATRA promotes immune cell differentiation, and more obvious be immunized is induced to answer It answers and immunological memory.It is advantageously used for the antibody expression of communicable disease, the fields such as antineoplaston.And in higher concentration Under, ATRA can promote antigen presenting cell and T cell to express FoxP3 genes, to TNF secretion-β and IL-10, promote immune The differentiation of suppressor T lymphocyte (Treg) generates immunosuppressive effect.Can be used for diminishing inflammation response, fight autoimmune Disease and antianaphylactic treatment.

However, the practical application example that ATRA is activated simultaneously in mucosal immunity and systemic immune is rarely reported.U.S. Ah Charles O.Elson of La Bama universities etc. were once reported using ATRA inducing bone marrows source Dendritic Cells (Bone Marrow derived dendritic cells, BMDC) participate in the research that mucosal immunity responds.But this research only demonstrates ATRA can induce BMDC to generate intestinal mucosa homing receptor CCR9 and α₄β₇, and promote the expression of Foxp3, and not by ATRA As a kind of immunostimulation dosage form, lack the research to immune response and characterization.In addition, Hannover medical colleges of Germany Hammershmidt etc. was once reported realizes that the mucous membrane of T cell and B cell is gone back to the nest using ATRA and antigen co-administered.Although This studies the activation for realizing mucosal immunity, including the secretion of IgA and the intestinal mucosa of T cells with antigenic specificity are gone back to the nest.But by It is dissolved in PEG400 and delivers in ATRA, and common loading is not carried out with antigen, cannot achieve long-acting administration, to biology Availability reduces, and causes its systemic immune weaker.Again because administration number of times is frequent, cause it can not extensive use.

In order to realize the method by injection inoculation, while causing the target of systemic immune and mucosal immunity response, assistant Agent dosage form still needs to face following challenge：1) ATRA has photo-labile and thermal instability, is easy to be denaturalized；2) due to ATRA's Poorly water-soluble needs High efficient encapsulation and slow-releasing system to improve its bioavilability；3) ATRA needs and antigen collective effect ability Reach effect；3) activation of intracellular RAR and rxr receptor needs the collaboration of the inflammatory factors such as injection site generation IL-6 and IL-24 Effect；4) the either immune activation of ATRA or immunosupress is required for the embedding amount for ATRA accurately to be controlled. And reported without related preparations in the document or patent being currently known, it is known that particulate adjuvants dosage form without according to not causing simultaneously yet Systemic immune and the target of mucosal immunity response are designed and optimize.

Therefore, a kind of novel adjuvant is developed, by way of injection, realizes while causing systemic immune and mucosal immunity Effect, it appears it is particularly important.

Invention content

The purpose of the present invention is to provide a kind of granular pattern adjuvants and its preparation method and application.

To reach the invention purpose, the present invention uses following technical scheme：

In a first aspect, the present invention provides a kind of granular pattern adjuvant, the granular pattern adjuvant include biocompatibility wall material, Biocompatibility grease and all-trans retinoic acid, the kernel that the biocompatibility grease is formed are coated on biocompatibility wall In the shell that material is formed, the all-trans retinoic acid is coated in the kernel.

Granular pattern adjuvant provided by the invention, the activation single compared to existing method, the present invention pass through injecting immune Activation system is immune simultaneously and mucosal immunity for method, realizes the dual activation effect for systemic immune and mucosal immunity.

The present invention for the first time loads antigen and hydrophobicity immune regulator ATRA jointly, delivers and discharges.Pass through control Its space-time coupling reaches immune cell activated and is migrated to mucous membrane, and generates mucosal immunity response.With traditional medical means phase Than this technology has broken the native physiological barrier of mucosal immunity and systemic immune, realizes produced by injection inoculation for the first time The effect of raw systemic immune and mucosal immunity double response.Prevention suitable for mucous membrane infectious disease and tumour chronic diseases Novel therapeutic means.At the same time, immune activation and immunosuppressive effect can further be adjusted by adjusting the carrying capacity of ATRA.

Granular pattern adjuvant provided by the invention, compared with adjuvant prepared by conventional surfactant, bio-compatible of the present invention Property wall material in the wall material shell that is compounded to form of distinctive high molecular material and liposome assign the splendid storage of system and freeze-drying is steady It is qualitative.

Granular pattern adjuvant provided by the invention has the advantage that in terms of systemic immune：(1) as particles used type is helped Agent is adsorbed or is embedded to antigen, then the oil-in-water emulsion, which can play, delays antigen rate of release, and antigen is protected not to be hydrolyzed, Extend antigen residence time in vivo, is conducive to the generation of high-affinity antibody；(2) granular pattern adjuvant can be with activated macrophage And promote the interaction of macrophage and T and β cells, reinforce stimulation to play the role of specificity to lymphocyte；(3) such as Particles used type adjuvant is adsorbed or is embedded to antigen, and antigen can be made to be easy to be swallowed by macrophage；(4) oil-in-water emulsion is also It can cause slight inflammatory reaction in injection site, raise inflammatory cell, stimulate the secretion of inflammatory factor, activate immune response； (5), it can be achieved that the lyase of antigen after certain specific granules, such as chitosan particle with pH sensibility, absorption or embedding antigen Body is escaped, and cellullar immunologic response is enhanced；In terms of mucosal immunity：(6) delivering and sustained release of the present invention to all-trans retinoic acid, promotees Into immunocyte (antigen presenting cell, T cell and B cell) expression mucous membrane homing receptor CCR9 and α₄β₇Expression, induction is immune Cell is migrated to mucosal tissue, causes immune dependent interaction；(7) present invention can express mucous membrane activation with stimulator antigen presenting cell Specific molecular CD103, promotes T cell to Th1 and Th2 type cell differentiations, and activation B cell secretes mucoantibody IgA, induction Generate mucosal immunity memory t cell；(8) the granular pattern adjuvant can induce immunocyte height to express RALDH, promote its secretion More further inducing peripheral immunocytes of ATRA are migrated to mucosal tissue, play the response of its mucosal immunity；(9) it is loading In the case of high concentration ATRA, which can promote the expression of immunocyte FoxP3, with mucous membrane go back to the nest effect synergistic effect, Activation system is immunized and mucosal immunity depression effect.

Preferably, the biocompatibility grease and the mass ratio of biocompatibility wall material are (0.05-4):1, such as can To be 0.05:1、1:1、2:1、3:1 or 4:1.

In the present invention, biocompatibility grease and the mass ratio of biocompatibility wall material are specific, are higher or lower than Above range all can make the grain size of particulate adjuvants and particle diameter distribution obvious deviation occur, and ATRA embedding rates and immune effect are equal There is apparent reduce.

Preferably, the average grain diameter of the granular pattern adjuvant is 10nm-50 μm, such as can be 10nm, 100nm, 500 Nm, 800nm, 1 μm, 2 μm, 5 μm, 8 μm, 9 μm or 10 μm, preferably 50nm-10 μm.

Preferably, the biocompatibility wall material includes biocompatible polymer material and optionally liposome.

Preferably, when biocompatibility wall material includes liposome, the liposome is in biocompatibility wall material Mass percent be less than or equal to 80%, such as can be 0,10%, 20%, 30%, 40%, 50%, 60%, 70% or 80%.

Preferably, mass percent of the liposome in biocompatibility wall material is 5%-30%.

In the present invention, the introducing due to liposome molecule in wall material, macromolecule and liposome molecule compound wall materials, it is simultaneous The advantage for having two kinds of membrane materials, can greatly promote the embedding rate to hydrophobic compound, stablize metabolizable grease, and add more More modification groups is conducive to modification of the later stage for wall material, therefore, mass percent of the liposome in biocompatibility wall material It is best for 5%-30%.Also, biocompatible polymer material can preferably be dissolved in the macromolecule material in organic solvent Material.

Preferably, the biocompatible polymer material include poly- 'alpha '-hydroxy acids and its derivative, polyhydroxybutyrate and Its derivative, polycaprolactone and its derivative, polyorthoester and its derivative, polyanhydride and its derivative or poly- cyanoacrylate In acid esters and its derivative any one or at least two combination.

Preferably, the biocompatible polymer material is poly- 'alpha '-hydroxy acids and its copolymer, and preferably (L- third is handed over autohemagglutination Ester), poly(D,L-lactide) or poly(lactide-co-glycolide), further preferably poly(lactide-co-glycolide).

Preferably, in poly(lactide-co-glycolide), the segment molar ratio of lactide and glycolide is (1-9):(9- 1), such as can be 1:9、1:5、1:3、1:1、2:1、5:1、7:1 or 9:1.

In the present invention, different segment molar ratios can influence the hydrophilic and hydrophobic and degradation speed of material, such as containing The 50 of 50%D, L- lactide and 50% glycolide:The degradation of 50PLGA polymer is very fast, and because lactide component increases, 75:25PLGA degradations are slower.

In the present invention, the above-mentioned polymer that various molecular weight are prepared in already known processes may be used, it can be according to institute The influence factors such as the rate of release and application field that need determine suitable molecular weight.For poly- (L- lactides) and poly- (D, L- third Lactide), such as suitable molecular weight is about 2000-5000 dalton ranks.For PLGA, suitable molecular weight is typically about 10000 to about 200000 dalton, preferably from about 13000 to about 150000 dalton.

Preferably, the liposome includes any one in cationic-liposome, anionic liposome or helper lipids body Kind or at least two combination.

Preferably, the cationic-liposome includes the oily alkenyloxy group propyl ammonium of chlorination trimethyl -2,3- bis-, bromination front three The oily alkenyloxy group propyl ammonium of base -2,3- dioleoyl oxygroups propyl ammonium, chlorination trimethyl -2,3- two, trimethylcetyl base ammonium, Bromoethyl dibasecylammonium bromide, 1,2- dioleoyl -3- succinyl-sn- glycerolcholines ester, lipid poly-l-lysine, 3 β-[in N- (N', N'- dimethyl aminoethyl) amido formacyls or stearic acid any one or at least two combination.

Preferably, the anionic liposome includes phosphatidyl-ethanolamine, phosphatidyl glycerol, phosphatidylinositols, phosphatidyl In serine, phosphatidic acid or phosphatidic acid derivative any one or at least two combination.

Preferably, the helper lipids body includes natural phosphatidyl choline, synthesis lecithin, phosphatidyl choline or phosphatidyl choline In analog any one or at least two combination.

Preferably, the biocompatibility grease include tocopherol and the like, soybean oil, Miglitol, midchain oil, Fish oil, vitamin E, Vitamin E succinate, Vitwas E, safflower oil, corn oil, Seabuckthorn Oil, Linseed oil, peanut Oil, tea oil, sunflower oil, apricot kernel oil, coix seed oil, evening primrose oil, sesame oil, cottonseed oil, castor oil, Tower rape oil, oil Acetoacetic ester, oleic acid, ethyl linoleate, isopropyl laurate, interior isopropyl myristate, coconut oil, ethyl butyrate, ethyl lactate, Trivent OCG, Triglyceride DDD, glycerol tristearate, saturated fatty acid glyceride, tripalmitin, cinnamic acid In glyceride, spermaceti acid Palmitate any one or at least two combination.

In the present invention, the combination of above-mentioned grease can be combined with arbitrary proportion.

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

In the present invention, the squalene is a kind of triterpene compound, and English name is Squalene, molecular structure For the isoprene of 30 light dydrocarbon decahydros, molecular formula is：2,6,10,15,19,23- hexamethyls -2,6,10,14,18,22- 20 Four carbon, six alkene, molecular mass：410.72, animal, plant extract or chemical synthesis can be derived from.Squalene is a kind of metabolizable Oil, because it is the intermediate product of the biosynthesis of cholesterol.This is a kind of all higher organisms, including (skin on mankind Can be found in fat) grease of naturally secret.Lotion (containing surfactant) containing squalene is real in zoopery and clinic It tests and shows excellent immunological enhancement.

The tocol is alpha-tocopherol or derivatives thereof such as alpha-tocofecol succinic acid ester (also referred to as VE succinic acid Ester).Alpha-tocopherol can play enhancing and exempt from the vaccine for gerontal patient (such as age is more than 60 years old or the patient of bigger) The effect of epidemic disease response.Existing tocol includes a variety of tocopherols such as α, β, γ, δ, ε, ζ, preferably alpha-tocopherol, especially DL- α-tocopherol.

In the present invention, since, there are grease, the ATRA embedded is realized interior in preparation process inside particulate adjuvants Portion's inversion of phases, so as to by the ingredient proportion for changing ATRA, wall material and grease, control the embedding rate of ATRA.

Preferably, further include hydrophobic drug in the biocompatibility grease.

In the present invention, hydrophobic drug has strong-hydrophobicity, the characteristics of being not easy to store and discharge, by being helped in granular pattern Coated by hydrophobic drug in agent can further promote to promote the effect that mucous membrane and systemic immune double-bang firecracker are answered.

Preferably, the hydrophobic drug is selected from antitumor broad-spectrum medicinal, immune activation adjuvant substance or antineoplastic target Any one in drug；

Preferably, the antitumor broad-spectrum medicinal include camptothecine, adriamycin class drug, taxanes drug or Along in platinum medicine any one or at least two combination.

Preferably, the immune activation adjuvant substance includes monophosphoryl lipid matter (monphosphoryllipid, MPLA), miaow Quinoline not special (Imiquimod) or poly I:poly C (Poly (I:C)) any one or at least two combination.

Preferably, the antineoplastic target drug includes everolimus, replaces Buddhist nun, Imatinib, vismodegib, dimension sieve according to Shandong Non- Buddhist nun, tesirolimus, Sony De Ji, Sorafenib, Sutent, match are stood to be replaced for Buddhist nun, Rui Gefeini, Trimetinib, general sodium Buddhist nun, bortezomib, pazopanib, Pa Boxini, pabishta, nilotinib, romidepsin, it is happy cut down for Buddhist nun, Lapatinib, gram Azoles is won for Buddhist nun, card for Buddhist nun, Carfilzomib, card than for Buddhist nun, Gefitinib, Vorinostat, Vande Thani, Tarceva, Di Nuosai Wheat, Dasatinib, dabrafenib, bosutinib, Baily department he, it is difficult to understand this for Buddhist nun, olaparib, Ai Le for Buddhist nun, Axitinib, Ah Method in Buddhist nun or VEGF Trap any one or at least two combination.

Preferably, the granular pattern adjuvant further includes modification group, and the modification group is connected to biocompatibility wall material The case surface of formation, the modification group are provided by the donor of modification group.

In the present invention, the modification group is connected by modes such as hydrophilic modification, hydrophobic modification, coating or graft modifications In wall material surface.

In the present invention, wall material is further modified by modification group, mucous membrane and systemic immune double-bang firecracker can also be promoted The effect answered.

Preferably, the donor of the modification group includes hydrophilic biological macromolecule, coupling targeting substance, fluorescent marker Object, isotopic label, environmental response substance, immunomodulator, pattern recognition receptors, immunopotentiator, cell factor become Change the factor in any one or at least two combination.

Preferably, the hydrophilic biological macromolecule includes protein matter, polypeptides matter, small peptide and amino acid derived Object or nucleic acid material any one or at least two combination.

Preferably, the protein matter include antigen (including be not limited to chick embryo culture, cell culture, carrier's body fluid, In organ or tissue obtained by purifies and separates, obtained by recombinant gene expression or chemical synthesis), medical albumen (including be not limited to day So extraction product and recombinant protein combination) antibody substance in any one or at least two combination.

Preferably, the antibody substance include thunder not Lu Dankang, the trastuzumab of resistance to former times, receive military monoclonal antibody, pyridine aldoxime methyliodide (PAM) monoclonal antibody, song Trastuzumab, Victibix, Cetuximab, difficult to understand, Ao Binyou trastuzumabs, this appropriate former times monoclonal antibody, Rituximab, Ibritumomab tiuxetan, tositumomab, pyridine aldoxime methyliodide (PAM) monoclonal antibody, Avastin, Herceptin, up to thunder wood monoclonal antibody, angstrom sieve trastuzumab, Beaune spits the combination of any one or at least two in monoclonal antibody or ground Nu Tuxi monoclonal antibodies.

Preferably, the environmental response substance is selected from group sensitive, thermo-responsive or sensitive bioactive substance pH Substance.

Preferably, the environmental response substance includes poly-N-isopropyl acrylamide, ferrocene, graphene, maleimide In amine any one or at least two combination.

Preferably, the pattern recognition receptors include the stimulant of Toll-like receptor, the stimulant of RIG-1 receptors, NOD samples Stimulant, mineral salt, saponin(e, liposome, liposome formulation product, the microparticle of synthesis, the microcarrier of synthesis, the trachoma clothing of receptor Substance, the outer film bubble of bacterium, polysaccharide, the peptide of special sex modification, the peptide of preparation, protein, pathogen-associated molecular pattern or amino In alkyl glucosaminide 4- phosphates any one or at least two combination.

In the present invention, the stimulant of RIG-1 and NOD samples receptor can be the oligonucleotides or double-stranded RNA of the motif containing CpG The series containing the palindrome oligonucleotides or containing poly- (dG) sequence oligonucleotides；Mineral salt can be alum, with enterobacteria (such as Escherichia coli, salmonella minnesota, Salmonella typhimurtum or shigella flexneri) monophosphoryl lipid matter (monphosphoryllipid, MPLA) combine alum or respectively with(AS04), above-mentioned bacterium MPLA specificity knots Alum, MPL, saponin(e (such as QS-21, Quil-A, iscoMs, the iscomatrix of conjunction^TM)；Liposome and liposome formulation product include AS01, AS02 etc.；The microparticle of synthesis, the microcarrier of synthesis can be neisseria gonorrhoeae (N.gonorrheae), trachoma clothing The outer film bubble (OMV) derived from bacterium of substance and other bacteriums；Polysaccharide can be chitosan；Special sex modification or the peptide of preparation can To be muramyl dipeptide；Pathogen-associated molecular pattern is selectable pathogen-associated molecular pattern (PAMPS)；Aminoalkyl amino Glucoside 4- phosphates can be RC529；Protein can be bacterial toxoid or toxin Fragment.

Preferably, the cell factor include granulocyte macrophage colony stimulating factor (GM-CSF), it is interferon, white thin Any one in born of the same parents' interleukin, 3 ligand of tyrosine kinase (FIt3L) or tumor necrosis factor-alpha (TNF-α) or at least two Combination.

In the present invention, interferon can be interferon-' alpha ' (IFN-α), interferon-beta (IFN-β), interferon-γ (IFN- γ) etc.；Interleukins can be interleukin-1 alpha (IL-1 α), interleukin-1 ' beta ' (IL-1 β), interleukin 2 (IL-2), interleukin 4 (IL-4), interleukin 7 (IL-7), interleukin 12 (IL-12), interleukins- 15 (IL-15), interleukin-18 (IL-18) etc..

Preferably, the granular pattern adjuvant further includes antigen.

Preferably, the antigen is embedded in granular pattern adjuvant or is adsorbed on the surface of granular pattern adjuvant.

In the present invention, antigen can load antigen by way of embedding, Electrostatic Absorption or chemical coupling, to real Common loading, delivering and the release of existing ATRA and antigen.

Second aspect, the present invention provides a kind of preparation method of granular pattern adjuvant as described in relation to the first aspect, the sides Method includes the following steps：

(1) biocompatibility wall material, biocompatibility grease and all-trans retinoic acid are dissolved in oil phase solvent, obtain oil Phase mixture, while aqueous phase solvent is prepared, obtain aqueous mixture；

(2) oil mixture object mixed with water is mixed in proportion, the particle is obtained by dispersion, solidification, washing Type adjuvant.

Preferably, oil phase solvent described in step (1) includes good solvent and poor solvent.

Preferably, the volume ratio of the good solvent and poor solvent is (1-9):1, such as can be 1:1、2:1、3:1、4: 1、5:1、 6:1、7:1、8:1 or 9:1.

Preferably, the volume ratio of the good solvent and poor solvent is 9:1 or 8:2.

Preferably, the good solvent includes isopentane, pentane, hexane, hexamethylene, isooctane, trifluoroacetic acid, trimethyl Pentane, pentamethylene, heptane, trichloro ethylene, carbon tetrachloride, chlorotrifluoroethane, propyl ether, toluene, paraxylene, chlorine isobutyl Alcohol, diethyl ether, tetrahydrofuran, petroleum ether, dichloromethane, chloroform, isopropanol, dimethyl sulfoxide (DMSO), acetone, ethyl acetate or In acetonitrile any one or at least two combination.

Preferably, the poor solvent includes appointing in methanol, ethyl alcohol, propyl alcohol, ethylene glycol, formamide, trifluoroacetic acid, water It anticipates a kind of or at least two combinations.

Preferably, aqueous phase solvent described in step (1) is emulsifier aqueous solution and/or salting liquid.

Preferably, the mass fraction of the emulsifier and/or salting liquid in aqueous phase solvent is 0-40%.

Preferably, the emulsifier includes PVAC polyvinylalcohol, polyetherimide PEI, polylactic acid-methoxy polyethylene glycol PLA-PEG, polyoxyethylene sorbitan esters surfactant (tween), sorbitan esters (sapn), Octoxinol- 9 (triton x-100 or tert-octylphenoxypolyethoxyethanol), enuatrol, poloxamer, lecithin, double dimethyls In base ammonium bromide or cetyl trimethylammonium bromide any one or at least two combination.

Preferably, the salting liquid includes that phosphate buffer, sodium citrate buffer solution, sodium chloride solution, potassium chloride are molten In liquid, potassium nitrate solution, metabisulfite solution or Tris-HCl buffer solutions any one or at least two combination.

Preferably, the volume ratio of oil mixture object mixed with water described in step (2) is 1:2-499, such as can be with It is 1:2、 1:10、1:20、1:100、1:220、1:350、1:400、1:450 or 1:499.

In the present invention, preparation method is a step emulsion method, prepares oil phase first, oil phase is comprising mass volume ratio The biocompatibility wall material of 0.01%-40% (g/mL), mass volume ratio are the biocompatibility of 0.01%-40% (g/mL) Grease, mass volume ratio are the all-trans retinoic acid of 0%-50% (g/mL), and mass volume ratio is the dress of 0%-50% (g/mL) Loading matter or modification group.Oil phase solvent includes good solvent and poor solvent.

In the present invention, dispersing mode can select oscillation, stirring, ultrasonic (Ultrasonic dispersion) etc. more Kind mode, as long as can be by power, the time, the parameters such as number arrival target particle sizes, used dispersing mode will not be right Biocompatibility wall material and biocompatibility grease cause to significantly affect, can according to water phase used and solid particle property and itself Experimental facilities condition select suitable dispersing mode and concrete operations parameter.The mixing of oil phase and water phase can select micro-fluidic (Microfluidization), homogeneous (Homogenization), ultrasound, syringe are double pushes away emulsification (Two-syringe Emulsification), spraying, microjet, microchannel (Microchannel emulsification), film emulsification The various ways such as (Membrane emulsification), stirring, oscillation, reversing or hand mixing.

In the present invention, hybrid mode can preferably micro-fluidic, microchannel or film emulsification etc. can be with according to different requirements, The mode of uniform particle size distribution lotion is obtained, preferably microjet, syringe pair can also push away emulsification, homogeneous, stirring or oscillation etc. The hybrid mode being convenient for large-scale preparation.It should be clear that different dispersing modes, it is inevitable to prepare power, time, number etc. Particle size, dispersibility and the stability etc. for influencing gained particle, will also influence final embedding efficiency, discharge, and targeting is passed Give and other effects.It therefore, should be according to the influence factors such as grease phase used and solid particle property, the emulsion droplet size range of required preparation Determine suitable hybrid mode and concrete operations parameter.

Preferably, granular pattern adjuvants of the Span less than 1.0 has higher embedding rate and stability, and has preferably Sustained release performance.

In the present invention, according to different requirements, curing mode can with preferred solvent volatility process (including room temperature volatilize, very Empty negative-pressure volatilizing, low temperature volatilization and the modes such as vaporization at high temperature), physical crosslinking or chemical crosslinking, redox, high-temperature calcination, Adjust the various ways such as pH and salinity, extraction, precipitation.

In the present invention, granular pattern adjuvant can be packed by the form of liquid suspension or in a manner of dried frozen aquatic products respectively Storage.Further include medical aid matter in the present invention to ensure fluid present invention suspension and freeze-drying stability and storage stability, Such as pH conditioning agents or and buffer, preferably be selected from the clear egg of sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, human serum In vain, essential amino acid, nonessential amino acid, L-arginine hydrochloride, sucrose, anhydrous D- trehaloses, mannitol, mannose, shallow lake In powder or gelatin any one or at least two combination.It is wherein typical but non-limiting to be combined as：Sodium acetate and breast The combination of sour sodium；The combination of sodium chloride and potassium chloride；The combination of calcium chloride, human serum albumin and essential amino acid；It is nonessential The combination of amino acid, L-arginine hydrochloride, sucrose and anhydrous D- trehaloses；The group of mannitol, mannose, starch and gelatin It closes；Sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride and the combination of human serum albumin；Essential amino acid, nonessential ammonia The combination of base acid, L-arginine hydrochloride, sucrose, anhydrous D- trehaloses, mannitol, mannose, starch and gelatin.

Irradiation sterilization, moist heat sterilization or filtration sterilization may be used in the sterilization method of heretofore described granular pattern adjuvant, When particles used grain size is less than 220nm, it is preferred to use the mode of filtration sterilization.

In the present invention, due to hydrophobic molecule, (oil phase and water phase) is tended to deposit at interface in phase separation, makes It obtains hydrophobic compound mutually to detach together with liquid in solidification, causes the reduction of embedding rate and bioavilability.And the present invention is logical A step emulsion method is crossed, using internal phase transfer principle (as shown in Figure 8), introduces the grease insoluble with wall material as third phase. By adjusting the ratio of solvent, inside phase separation (grease and the oil phase of the membrane material containing shell) is prior to outside phase separation (film containing shell The oil phase and outer aqueous phase of material), cause hydrophobic molecule to be deposited in internal fat drips interface.Method provided by the invention avoids hydrophobic Property compound detaches together in nano-particle outside deposition or with water phase, to greatly promote the efficiency of embedding and loading.Also, The method is easy to operate, the production cost in actual production and sterilizing cost is greatly saved, conducive to being widely popularized.

Preferably, the administering mode of the granular pattern adjuvant is drug administration by injection.

Preferably, the drug administration by injection includes injection, intratumor injection, abdominal cavity note in hypodermic injection, intramuscular injection, lymph node Penetrate, be injected intravenously or foot injection in any one.

In the present invention, granular pattern adjuvant can be caused by way of injection T cell, B cell, eosinophil, The generations such as basophilic granulocyte, neutrophil leucocyte and antigen presenting cell (macrophage, Dendritic Cells and monocyte) are viscous Film homing receptor.

The third aspect, the present invention provides a kind of granular pattern adjuvants as described in relation to the first aspect to prepare vaccine adjuvant, exempt from Application in drug, the drug of confrontation mucosal tissue relevant disease or the medicine of anti-chronic disease that epidemic disease inhibits.

Preferably, the mucosal tissue relevant disease and chronic disease include human nipple virus infection, poliovirus Infection, coxsackie virus infection, enterovirus infection, norwalk virus infection, AIDS, anthrax, malaria, hepatitis B, Any one in hepatitis A, hepatitis C, colon cancer, colorectal cancer, cervical carcinoma, carcinoma of small intestine or lung cancer.

Preferably, when granular pattern adjuvant is used to prepare immunosuppressive drug, such as preventative vaccine, therapeutic vaccine Charging ratio Deng, all-trans retinoic acid is 2%-25%, for example, can be 2%, 5%, 8%, 10%, 12%, 13%, 15%, 20% or 25%.

Preferably, when granular pattern adjuvant is used to prepare the medicine of vaccine adjuvant or anti-chronic disease, such as itself exempt from Epidemic disease disease, antianaphylactic treatment etc., the charging ratio of all-trans retinoic acid are 0.01%-2%, such as can be 0.01%, 0.1%, 0.2%, 0.5%, 0.6%, 0.8%, 1%, 1.3%, 1.5%, 1.8% or 2%.

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

(1) granular pattern adjuvant provided by the invention, the activation single compared to existing method, the present invention pass through injecting immune Method activation system is immune simultaneously and mucosal immunity, realize and the dual activation of systemic immune and mucosal immunity imitated It answers.

(2) present invention for the first time loads antigen and hydrophobicity immune regulator ATRA jointly, delivers and discharges.Pass through control Its space-time coupling is made, reaches immune cell activated and is migrated to mucous membrane, and generates mucosal immunity response.With traditional medical means phase Than this technology has broken the native physiological barrier of mucosal immunity and systemic immune, realizes produced by injection inoculation for the first time The effect of raw systemic immune and mucosal immunity double response.Prevention suitable for mucous membrane infectious disease and tumour chronic diseases Novel therapeutic means.At the same time, immune activation and immunosuppressive effect can further be adjusted by adjusting the carrying capacity of ATRA.

(3) granular pattern adjuvant provided by the invention, compared with adjuvant prepared by conventional surfactant, biofacies of the present invention The wall material shell that distinctive high molecular material and liposome are compounded to form in capacitive wall material assigns system splendid storage and freeze-drying Stability.

(4) method provided by the invention avoids hydrophobic compound and divides together in nano-particle outside deposition or with water phase From to greatly promote the efficiency of embedding and loading.Also, the method is easy to operate, and the life in actual production is greatly saved Cost and sterilizing cost are produced, conducive to being widely popularized.

Description of the drawings

Fig. 1 is the scanning electron microscope (SEM) photograph (scale 500nm) of granular pattern adjuvant prepared by the embodiment of the present invention 1.

Fig. 2 is the survey of granular pattern adjuvant splenocyte secretion interferon-γ (IFN-γ) level prepared by the embodiment of the present invention 2 Attempt.

Fig. 3 is EV71 specific killing T cells in granular pattern adjuvant intestinal mucosa lymph node prepared by the embodiment of the present invention 2 Ratio chart.

Fig. 4 is the survey of granular pattern adjuvant splenocyte secretion interferon-γ (IFN-γ) level prepared by the embodiment of the present invention 3 Attempt.

Fig. 5 is HIV specific killing T cells in granular pattern adjuvant intestinal mucosa lymph node prepared by the embodiment of the present invention 3 Ratio chart.

Fig. 6 is the treatment of colon cancer vaccine effect that granular pattern adjuvant is directed to CEA short peptide antigens in the embodiment of the present invention 4 Survivorship curve figure.

Fig. 7 is the ratio chart of immune-suppressing T cell in granular pattern adjuvant intestinal mucosa lymph node in the embodiment of the present invention 5.

Fig. 8 be the present invention preparation method in phase transfer principle schematic diagram inside a step emulsion process.

Specific implementation mode

The technical solution further illustrated the present invention below by specific implementation mode.Those skilled in the art should be bright , the embodiment, which is only to aid in, understands the present invention, should not be regarded as a specific limitation of the invention.

It is raw materials used as shown in table 1 below in following embodiment of the present invention：

Table 1

Instrument is as shown in table 2 below：

Table 2

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

The particle size distribution measuring of particle：

The particle diameter distribution of nano-scale particle or emulsion droplet is measured using Zeta potential and Particle Size Analyzer, specific to measure step Suddenly it is：1mg nano-scale particles are added in 10mL deionized waters, ultrasonic 5min keeps its evenly dispersed, or takes 2mL lotions, general Grain suspension or lotion be added in sample cell, be put into zeta potential instrument (Zeta Potential Analyzer, Brookhaven Instruments Corporation) in be measured.

The homogeneity of particle or emulsion droplet is indicated by particle diameter distribution coefficient (Span) value.Span calculation formula are as follows, Span= (d₉₀- d₁₀)/d₅₀, wherein d₁₀, d₅₀And d₉₀Respectively particle cumulative volume is 10%, 50%, grain size when 90%, Span values It is smaller to show that grain diameter is more uniform.

The morphology observation of particle uses scanning electron microscopic observation：1mg particles are weighed, are added in 10mL deionized waters, ultrasound 5 Min keeps its evenly dispersed.1mL suspension is drawn, is dropped on aluminium foil, it is made uniformly to be spread out on aluminium foil, naturally dry.It will Aluminium foil is affixed on conducting resinl on sample stage, under vacuum after metal spraying (choosing suitable metal spraying condition according to properties of samples), It is observed with scanning electron microscope.

The measurement of grease embedding rate and carrying capacity in particle：

Quickly detection embedding grease straightforward procedure：It takes and carries medicine particle freeze-dried powder, being placed under high temperature makes microballoon degrade, if occurring Oil droplet, expression embed successfully

Accurate detecting method：Precise 10mg carries medicine particle freeze-dried powder, keeps microballoon degradable using method appropriate (for example, for polylactic acid microsphere, it can be used and chromatographic grade acetonitrile or acetone is added；For chitosan class microballoon, it can be used and add Enter chromatographic grade dimethyl sulfoxide, wait while dissolving the solvent of shell membrane material and grease or mixed solvent makes microballoon degrade).It waits for After grain is degradable, using high performance liquid chromatography (HPLC) or High Performance Liquid Chromatography-Mass Spectrometry (HPLC-MS) or gas phase color Spectrum or other suitable detection methods measure.

The measurement of antigen or drug embedding rate and carrying capacity in particle：

Precise 10mg carries medicine particle freeze-dried powder, keeps microballoon degradable (for example, for poly- breast using method appropriate Acids microballoon, the method that NaOH solution or acetonitrile is added, which can be used, makes microballoon degrade；For chitosan class microballoon, addition can be used The method of dilute hydrochloric acid makes microballoon degrade).After particle is degradable, in NaOH or hydrochloric acid and degradation solution, make its pH=7, then It is settled to 2mL.Hydrophilic compounds content uses BCA kits or micro-BCA kits or ELISA kit or other to fit Suitable detection method measures.Hydrophobic compound uses high performance liquid chromatography (HPLC) or High Performance Liquid Chromatography-Mass Spectrometry (HPLC-MS) or other suitable detection methods measure.Antigen or drug embedding rate calculate as follows：Embedding rate=(real Survey antigen or drug addition when in particle prepared by antigen or medication amount/reality) × 100%

The carrying capacity of antigen or drug on particle calculates as follows：Carrying capacity=(antigen or medication amount in actual measurement particle/ The quality of surveyed particle)

The measurement of the adsorption rate and carrying capacity of adsorption antigen or drug on particle：

The particle suspension liquid after adsorption antigen or drug is taken out, centrifuging and taking supernatant (is selected according to the size of particle and density Suitable centrifugal condition), measure supernatant in antigen or drug concentration, to calculate indirectly the antigen for being adsorbed onto particle surface or The amount of drug.Hydrophilic compounds content uses BCA kits or micro-BCA kits or ELISA kit or other to fit Suitable detection method measures.Hydrophobic compound uses high performance liquid chromatography (HPLC) or High Performance Liquid Chromatography-Mass Spectrometry (HPLC-MS) or other suitable detection methods measure.

Antigen or Drug absorbability rate calculate as follows：Adsorption rate=(on after absorption pro-antigen or drug concentration-absorption Antigen or drug concentration in clear)/adsorb pro-antigen or drug concentration × 100%

The carrying capacity of antigen or drug on particle calculates as follows：Carrying capacity=(antigen or medication amount on actual measurement particle/ The quality of surveyed particle)

Zoopery measures：

C57BL/6 mouse used by experiment are provided by company of dimension tonneau China.Immune step the following is substantially：First by mouse Random grouping, every group is tested using 6 or more mouse, and according to the embodiment illustrate is grouped mouse and exempts from Epidemic disease is inoculated with.Before inoculation, 200 μ L of blood are first taken, centrifuge 5min at 12000rpm immediately, isolate serum, measures IgG antibody water It is flat, using IgG antibody level at this time as initial value, then mouse is immunized.After immune, periodically from mouse eye circumference or tail Point takes blood, takes 200 μ L of blood every time, and measures IgG antibody level.Secondary immunity is carried out after two weeks, is put to death mouse within 35 days, is taken blood, It is horizontal to measure IgG antibody.It takes mouse boosting cell to be cultivated, mouse boosting cell is detected with enzyme-linked immunosorbent assay (ELISA) The secretion situation of IL-4 and IFN-γ cell factor in culture solution supernatant.

For tumor experiment, C57BL/6 mouse (8/group) inoculated tumour cell first, later after 1 week, after 2 weeks and 3 Vaccine inoculation after week.Day detects the volume of a tumour every three days.The calculation formula of gross tumor volume is：Long × wide × wide/2. Show loving care in humanity, when gross tumor volume is more than 1500mm³When, put to death mouse.

Embodiment 1

The present embodiment is prepared by the following method the biocompatibility grease and biocompatibility macromolecule for being embedded with ATRA Material cladding type particulate adjuvants

Electronic balance is used accurately to weigh the PLGA (molecular weight is 130,000 dalton) of 2.0g, 1.0g squalenes, 0.3g bromines Change dimethyl dioctadecyl ammonium and 1mg all-trans retinoic acid, is dissolved in the ethyl alcohol, acetone and dichloromethane (1 of 30mL:1:9) In mixed solution, solution is poured into rapidly to 250mL aqueous solutions, and ((alcoholysis degree 99%, viscosity are the PVA containing 1wt.% 5.0mPas), disperse (100W, 2min, interval time 4s) under ultrasound condition.Be stirred overnight at 25 DEG C (magnetic agitation, turn Speed is 500rpm), carry out the organic solvent in removing system, realizes consolidation effect.10min is centrifuged under 25000g, is discarded supernatant, to 10mL deionized waters are added in precipitation, ultrasonic disperse centrifuges 10min under 25000g, after discarding supernatant, will precipitation freeze-drying, be placed in 4 DEG C of preservations in refrigerator.

Obtained particulate adjuvants 20mg is mixed 4 hours with the model antigen of 10mg/mL at 4 DEG C, adsorption antigen is obtained Particulate adjuvants.Antigen adsorption rate is characterized by the above method, the adsorption rate for obtaining OVA is 81.5 ± 10.1%.

It is as shown in Figure 1 that its particle diameter distribution is observed using scanning electron microscope.The average grain diameter of prepared particle is 150nm ± 4.7 nm, Span 0.501, Zeta potential are that 38.2 ± 5.8, ATRA embedding rates are 85.2 ± 8.2%.

In the method for the present embodiment, other modification groups also can be used, wall material and grease are embedded with ATRA's to prepare The immune particulate adjuvants with mucosal immunity of activation system simultaneously, for loading different antigen, preparation process and embodiment 1 It is similar, specific process parameter and the results are shown in Table 3：

Table 3

Embodiment 2

The present embodiment is prepared by the following method causes intestinal mucosa and systemic immune pair as EV71 recombinant vaccine adjuvants The particulate adjuvants for being embedded with ATRA of response：

Electronic balance is used accurately to weigh the PLGA (molecular weight is 130,000 dalton) of 1.0g, 1.0g squalenes, 0.2g bromines Change dimethyl dioctadecyl ammonium and 0.1mg all-trans retinoic acid, is dissolved in the ethyl alcohol and dichloromethane (1 of 30mL:9) mixing is molten In liquid, solution is poured into rapidly to 200mL aqueous solutions, and ((alcoholysis degree 99%, viscosity are 8.0 mPa to the PVA containing 2wt.% S), fast film emulsification (membrane aperture is 1.4 μm, and it is 3MPa to cross film pressure, crosses film 5 times).It is stirred overnight that (magnetic force stirs at 25 DEG C Mix, rotating speed 500rpm), cure hard emulsion particle using room temperature solvent evaporation method.5min is centrifuged under 15000g, is discarded supernatant, 10mL deionized waters are added into precipitation, ultrasonic disperse centrifuges 5min under 15000g, after discarding supernatant, will precipitation freeze-drying, be placed in 4 DEG C of preservations in refrigerator.

The average grain diameter of prepared particle is 1230nm ± 25.9nm, and Span 0.315, Zeta potential is 27.6 ± 3.8, ATRA embedding rates are 81.3 ± 7.5%.

By the hand-foot-and-mouth disease (enteron aisle of obtained particulate adjuvants (Particulate Adjuvant, PA) 20mg and 10mg/mL Virus) antigen EV71 mixes 3 hours at 4 DEG C, obtain the particulate adjuvants of adsorption antigen.By the above method to antigen adsorption rate It is characterized, the adsorption rate for obtaining EV71 is 90.3 ± 8.7%.

Granular pattern adjuvant is subjected to systemic immune and mucosal immunity activation characterization：

C57BL/6 mouse (n=6) are grouped at random, are inoculated with primary each experimental group vaccine adjuvant (referring to table 4) every two weeks, It is inoculated with altogether twice.Blood, 200 μ L are taken to mouse orbit weekly, and measure IgG antibody level.It puts to death mouse within 35 days, takes blood, measure IgG antibody is horizontal.It takes mouse boosting cell to be cultivated, mouse boosting cell culture is detected with enzyme-linked immunosorbent assay (ELISA) The secretion situation of IFN-γ cell factor in liquid supernatant, the results are shown in Figure 2.Take mouse gut associated lymphatic knot (mLNs), EV71 pentamers, CD8, CD3 cell (EV71-specific lethal to wherein EV71 specific cells are utilized Cytotxic T cell) it is dyed, and it is detected using flow cytometer, the results are shown in Figure 3.

Table 4

In terms of systemic immune, as shown in table 4 and Fig. 2, embeds ATRA and the particulate adjuvants for adsorbing EV71 have more preferably Activation serum IgG (humoral immunity) and splenocyte in interferon-γ (IFN-γ, cellular immunity) it is horizontal.In mucosal immunity Aspect embeds ATRA and the particulate adjuvants for adsorbing EV71 has preferably activation intestinal mucosa IgA (body fluid as shown in table 4 and Fig. 3 It is immune) and intestinal mucosa in antigen specific killer cells (EV71-specific cytotxic T cell, cellular immunity) It is horizontal.And free ATRA and activation immune response not yet in effect.The time and space usage of ATRA is imitated importantly, lacking the present invention The particulate adjuvants absorption EV71 for not embedding ATRA of release property is answered to mix (ATRA+PA-EV71) with free ATRA also without effective Activate immunity of organism response.It demonstrates the mechanism of the present invention and strategy has to become and by injecting immune mode while activating body The potentiality of systematicness and mucosal immunity response.

Embodiment 3

The present embodiment is prepared by the following method causes genital mucosal and systemic immune pair as HIV nucleic acid vaccine adjuvants Response is embedded with the particulate adjuvants of ATRA：

Electronic balance is used accurately to weigh the PLGA (molecular weight is 130,000 dalton) of 2.0g, 1.0g ethyl linoleates/rice Lattice row alcohol (1:1), 0.2g bromoethyl dibasecylammonium bromides and 0.1mg all-trans retinoic acid, are dissolved in the ethyl alcohol of 20mL, acetone With dichloromethane (1:2:7) in mixed solution, solution is poured into rapidly to the (PVA (alcoholysis containing 2wt.% of 100mL aqueous solutions Degree is 99%, viscosity 5.0mPas), high-pressure homogeneous homogeneous (10000rpm, 5.0MPa, 5min).It is stirred overnight at 25 DEG C (magnetic agitation, rotating speed 500rpm), to cured granulate.10min is centrifuged under 15000g, is discarded supernatant, is added into precipitation 10mL deionized waters, ultrasonic disperse centrifuge 10min under 15000g, after discarding supernatant, will precipitation freeze-drying, be placed in 4 DEG C of guarantors in refrigerator It deposits.

The average grain diameter of prepared particle is 810.7 ± 15.4nm, and Span 0.705, Zeta potential is 35.1 ± 4.7, ATRA embedding rates are 85.1 ± 8.4%.

By the AIDS virus DNA epidemic diseases of obtained particulate adjuvants (Particulate Adjuvant, PA) 20mg and 10mg/mL Seedling mixes 2 hours at 4 DEG C, obtains the particulate adjuvants of adsorption antigen.Antigen adsorption rate is characterized by the above method, is obtained The adsorption rate of HIV is 87.9 ± 7.5%.

Particulate adjuvant is subjected to systemic immune and mucosal immunity activation characterization：

C57BL/6 mouse (n=6) are grouped at random, are inoculated with primary each experimental group vaccine adjuvant (referring to table 5) every two weeks, It is inoculated with altogether twice.Blood, 200 μ L are taken to mouse orbit weekly, and measure IgG antibody level.It puts to death mouse within 35 days, takes blood, measure IgG antibody is horizontal.It takes mouse boosting cell to be cultivated, mouse boosting cell culture is detected with enzyme-linked immunosorbent assay (ELISA) The secretion situation of IFN-γ cell factor in liquid supernatant, the results are shown in Figure 4.Take mouse gut associated lymphatic knot (mLNs), HIV pentamers, CD8, CD3 cell (HIV-specific lethal to wherein HIV specific cells are utilized Cytotxic T cell) it is dyed, and it is detected using flow cytometer, the results are shown in Figure 5.

Table 5

In terms of systemic immune, as shown in table 5 and Fig. 4, embeds ATRA and adsorb the particulate adjuvants (ATRA-PA- of HIV HIV) has interferon-γ (IFN-γ, cellular immunity) water in preferably activation serum IgG (humoral immunity) and splenocyte It is flat.In terms of mucosal immunity, as shown in table 5 and Fig. 5, embeds ATRA and the particulate adjuvants for adsorbing HIV have preferably activation intestines Antigen specific killer cells in mucous membrane IgA (humoral immunity) and intestinal mucosa (HIV-specific cytotxic T cell, Cellular immunity) it is horizontal.

Embodiment 4

The present embodiment is prepared by the following method the therapeutic vaccine embedding as anti-intestinal mucosa related neoplasms (colon cancer) There are the particulate adjuvants of ATRA：

Described in the preparation method is the same as that of Example 1, being prepared for embedding ATRA using a step emulsion method, (0.001%, quality is divided Number) granular pattern adjuvant (Particulate Adjuvant, PA), and adjuvant CPG is added with oil phase in preparation process.

The average grain diameter of prepared particle is 150.7 ± 5.1nm, and Span 0.354, Zeta potential is 32.7 ± 6.5, The charging ratio that the embedding rate that ATRA embedding rates are 81.2 ± 4.5%, CPG is 78.5 ± 6.2%, CPG is 0.009%.

By the carcinomebryonic antigen for being usually used in treatment of colon cancer of obtained particulate adjuvants 20mg and 20mg/mL (Corcinoembryonic Antigen, CEA) is mixed 4 hours at 4 DEG C, obtains the particulate adjuvants of adsorption antigen.By above-mentioned Method characterizes antigen adsorption rate, and the adsorption rate for obtaining HIV is 91.4 ± 4.1%.

The particulate adjuvants of preparation carry out anticancer effect evaluation：

C57BL/6 mouse (n=8) are grouped at random, and colon cancer primary tumor cells are inoculated with to experimental animal colon portion (105/) is inoculated with three times every each group adjuvant of inoculation in 7 days altogether after two weeks, including：PBS negative control groups (PBS), not It embeds ATRA and adsorbs the particulate adjuvants (PA-CEA) of CEA, embed ATRA and adsorb the particulate adjuvants (ATRA-PA- of CEA CEA), the particulate adjuvants absorption HIV for not embedding ATRA is mixed (ATRA+PA-CEA) with free ATRA.Day detection one every three days The volume of secondary tumour.The calculation formula of gross tumor volume is：Long × wide × wide/2.Show loving care in humanity, when gross tumor volume is more than When 1500mm3, mouse is put to death.The results are shown in Figure 6.

As shown in fig. 6, the particulate adjuvants of embedding ATRA and absorption CEA significantly inhibit effect for the development of colon cancer, And substantially prolong its life span.And there are 2 mouse to be successfully treated.And other control groups are all unable to reach close effect Fruit.The results show that the present invention has confrontation mucous membrane related neoplasms, the ability of body therapeutic immunization effect is activated, having becomes Fight the great potential of the therapeutic vaccine of mucous membrane related neoplasms.

Embodiment 5

The present embodiment is prepared by the following method to be embedded with as the treatment adjuvant of confrontation intestinal mucosa acute allergic reaction The particulate adjuvants of ATRA：

Different from embodiment 1-4, the present embodiment improves the loading of ATRA to cause intestinal mucosal immune inhibition response Rate increases ATRA in preparation process and feeds intake rate (500 times).PLA-PEG (the molecules of 1.0g are accurately weighed using electronic balance Amount is 130,000 dalton), 1.0g squalenes and 50mg all-trans retinoic acid are dissolved in the ethyl alcohol and dichloromethane (1/9) of 30mL In mixed solution, solution is poured into rapidly to 200mL aqueous solutions, and ((alcoholysis degree 99%, viscosity are the PVA containing 2wt.% 8.0mPas), disperse (200W, 2min, interval time 4s) under ultrasound condition.Be stirred overnight at 25 DEG C (magnetic agitation, turn Speed is 500rpm), cure hard emulsion particle using solvent evaporation method.10min is centrifuged under 25000g, is discarded supernatant, into precipitation 10mL deionized waters are added, ultrasonic disperse centrifuges 10min under 25000g, after discarding supernatant, will precipitation freeze-drying, be placed in 4 in refrigerator DEG C preserve.

The average grain diameter of prepared particle is 105nm ± 4.7, and Span 0.427, Zeta potential is -27.2 ± 8.8, ATRA embedding rates are 83.2 ± 5.4%.

Particulate adjuvants are subjected to intestinal mucosal immune inhibition response evaluation：

C57BL/6 mouse (n=6) are grouped at random, every the primary each experimental group vaccine adjuvant (referring to table 6) of inoculation in 1 day, altogether Inoculation is secondary three times.Mouse intestinal mucosa IgE antibody level is detected using elisa technique after 7 days, extracts intestines associated lymphatic knot (mLNs) interleukin-10 of its medium size lymphocyte (IL-10) and immune-suppressing T cell (Treg) level are evaluated, is tied Fruit is summarized as shown in table 6 and Fig. 7.

Table 6

As shown in table 6, mucous membrane IgE levels can effectively be reduced after promoting the dosage of ATRA and promote IL-10 concentration, but It is that the time and space usage for being delivered and being discharged for ATRA due to particulate adjuvants of the present invention optimizes, ATRA-PA (H) causes brighter Aobviously for the inhibition situation of anaphylaxis emergency reaction.Importantly, Treg (such as Fig. 7 in gut associated lymphatic tissue It is shown), it is also activated by ATRA-PA (H) conspicuousness in the present invention, it was demonstrated that this research has as novel antiallergy and itself exempts from The great potential of the therapeutic vaccine of epidemic disease disease.

Embodiment 6

The particulate adjuvants for embedding ATRA are subjected to safety evaluatio

1. vascular stimulation tests

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

2. haemolysis and agglutination test

Using routine in vitro test tube method (observation method of naked eye), embodiment 1 and embodiment 5 are suspended with 2% red blood cell respectively Liquid mixes, and haemolysis and red blood cell condensation phenomenon are had no in 3 hours.

3. muscle irritation is tested

Embodiment 1 and embodiment 5 are respectively used to rabbit quadriceps muscle of thigh drug administration by injection, the administration l mL per side.After 48 hours It takes medicine-feeding part to visually observe and makees histopathologic examination.The result shows that this product is nonirritant to rabbit quadriceps muscle of thigh.

Comparative example 1

It is identical in the method for embodiment 1 using different biocompatibility greases and biocompatibility wall material mass ratio (embedding rate of ATRA is different, and also respective change makes it obtain to inventory for the particulate adjuvants of ATRA charging ratios preparation embedding ATRA Same charging ratio).As shown in table 7, when biocompatibility grease and biocompatibility wall material mass ratio are less than 0.05:(table when 1 Middle mass ratio is 1:100), obtained particulate adjuvants particle diameter distribution occurs that difference is smaller, but the embedding rate of ATRA, muscle are noted IgA antibody secretion level is decreased obviously in IgG titres and intestinal mucosa in serum after penetrating 28 days.And work as biocompatibility grease and life Object compatibility wall material mass ratio is higher than 4:(mass ratio is 5 in table when 1:1) particulate adjuvants of homogeneous grain diameter, can not be formed, and Gained particulate adjuvants grain size is larger, IgA antibody in IgG titres and intestinal mucosa in serum after ATRA embedding rates, intramuscular injection 28 days More significantly reducing all occurs in secretion level.This comparative example proves, preferred biocompatibility grease and biocompatibility wall The mass ratio of material is (0.05-4):1, it is higher or lower than this range, the grain size of particulate adjuvants, particle diameter distribution can all occurs Obvious deviation, and significantly reducing occur in ATRA embedding rates and immune effect.

Table 7

Comparative example 2

Using identical membrane material, identical biocompatibility grease and biocompatibility wall material mass ratio, identical ATRA dresses Load rate prepares grain size with different ultrasonic powers：5nm, 30nm, 1 μm and 60 μm of particulate adjuvants (as shown in table 8).It is right Than different-grain diameter for particulate adjuvants in zeta current potentials (measurement of storage stability), ATRA embedding rates, serum after injection 28 days IgG antibody titre, and inject the influence of 28 days hindgut mucosal IgA antibodies titres.When particulate adjuvants grain size is less than 10 nm-50 μ When m (second row grain size is 5nm in table 8), obtained particulate adjuvants zeta current potentials are relatively low, illustrate that it is easier to assemble, and IgG titres, intestinal mucosa IgA titres are all substantially reduced in its ATRA embedding rates, serum.And when particulate adjuvants grain size is higher than excellent When 10nm-50 μm of the particle size range of choosing (last column grain size is 60 μm in table 8), under obtained particulate adjuvants zeta current potentials Drop, it was demonstrated that its storage stability is bad.At the same time, IgG titres, intestinal mucosa IgA titres have in ATRA embedding rates, serum It is apparent to reduce phenomenon.This comparative example illustrates that preferred particle size range is 10nm-50 μm, is higher or lower than this range, all The zeta current potentials (stability) of particulate adjuvants, ATRA embedding rates and immune effect can be made the trend being substantially reduced occur.

Table 8

Applicant states, the present invention by above-described embodiment come illustrate the present invention granular pattern adjuvant and preparation method thereof and Using, but the invention is not limited in above-mentioned processing steps, that is, do not mean that the present invention has to rely on above-mentioned processing step ability Implement.Person of ordinary skill in the field it will be clearly understood that any improvement in the present invention, to raw material selected by the present invention etc. Effect is replaced and the addition of auxiliary element, the selection etc. of concrete mode, all falls within protection scope of the present invention and the open scope.

Claims (10)

1. a kind of granular pattern adjuvant, which is characterized in that the granular pattern adjuvant includes biocompatibility wall material, biocompatibility oil Fat and all-trans retinoic acid, the kernel that the biocompatibility grease is formed are coated on the shell of biocompatibility wall material formation Interior, the all-trans retinoic acid is coated in the kernel.

2. granular pattern adjuvant according to claim 1, which is characterized in that the biocompatibility grease and biocompatibility The mass ratio of wall material is (0.05-4):1；

Preferably, the average grain diameter of the granular pattern adjuvant is 10nm-50 μm, preferably 50nm-10 μm.

3. granular pattern adjuvant according to claim 1 or 2, which is characterized in that the biocompatibility wall material includes biology Compatible polymeric material and optionally liposome；

Preferably, when biocompatibility wall material includes liposome, quality of the liposome in biocompatibility wall material Percentage is less than or equal to 80%；

Preferably, mass percent of the liposome in biocompatibility wall material is 5%-30%；

Preferably, the biocompatible polymer material includes poly- 'alpha '-hydroxy acids and its derivative, polyhydroxybutyrate and its spreads out Biology, polycaprolactone and its derivative, polyorthoester and its derivative, polyanhydride and its derivative or polybutylcyanoacrylate And its in derivative any one or at least two combination；

Preferably, the biocompatible polymer material be poly- 'alpha '-hydroxy acids and its copolymer, preferably autohemagglutination (L- lactides), Poly(D,L-lactide) or poly(lactide-co-glycolide), further preferably poly(lactide-co-glycolide)；

Preferably, in poly(lactide-co-glycolide), the segment molar ratio of lactide and glycolide is (1-9):(9-1)；

Preferably, the liposome include in cationic-liposome, anionic liposome or helper lipids body any one or At least two combination；

Preferably, the cationic-liposome includes the oily alkenyloxy group propyl ammonium of chlorination trimethyl -2,3- bis-, bromination trimethyl -2, The oily alkenyloxy group propyl ammonium of 3- dioleoyl oxygroups propyl ammonium, chlorination trimethyl -2,3- two, trimethylcetyl base ammonium, bromination Dimethyl dioctadecyl ammonium, 1,2- dioleoyl -3- succinyl-sn- glycerolcholines ester, lipid poly-l-lysine, 3 β-[N- In (N', N'- dimethyl aminoethyl) amido formacyl or stearic acid any one or at least two combination；

Preferably, the anionic liposome includes phosphatidyl-ethanolamine, phosphatidyl glycerol, phosphatidylinositols, phosphatidyl silk ammonia In acid, phosphatidic acid or phosphatidic acid derivative any one or at least two combination；

Preferably, the helper lipids body includes that natural phosphatidyl choline, synthesis lecithin, phosphatidyl choline or phosphatidyl choline are similar In object any one or at least two combination.

4. granular pattern adjuvant according to any one of claim 1-3, which is characterized in that the biocompatibility grease packet Include tocopherol and the like, soybean oil, Miglitol, midchain oil, fish oil, vitamin E, Vitamin E succinate, vitamin E acetates, safflower oil, corn oil, Seabuckthorn Oil, Linseed oil, peanut oil, tea oil, sunflower oil, apricot kernel oil, coix seed oil, the moon are shown in Careless oil, sesame oil, cottonseed oil, castor oil, Tower rape oil, ethyl oleate, oleic acid, ethyl linoleate, isopropyl laurate, Interior isopropyl myristate, coconut oil, ethyl butyrate, ethyl lactate, Trivent OCG, Triglyceride DDD, glycerine three are stearic In acid esters, saturated fatty acid glyceride, tripalmitin, Chinese cassia tree acid glyceride, spermaceti acid Palmitate any one or The combination of person at least two；

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

5. the granular pattern adjuvant according to any one of claim 1-4, which is characterized in that in the biocompatibility grease It further include hydrophobic drug；

Preferably, the hydrophobic drug is selected from antitumor broad-spectrum medicinal, immune activation adjuvant substance or antineoplastic target drug In any one；

Preferably, the antitumor broad-spectrum medicinal includes camptothecine, adriamycin class drug, taxanes drug or cis-platinum In class drug any one or at least two combination；

Preferably, the immune activation adjuvant substance includes the arbitrary of monophosphoryl lipid matter, imiquimod or poly I:poly C It is a kind of or at least two combination；

Preferably, the antineoplastic target drug includes everolimus, replaces Buddhist nun, Imatinib, vismodegib, dimension Rofe according to Shandong Buddhist nun, tesirolimus, Sony De Ji, Sorafenib, Sutent, match it is vertical for Buddhist nun, Rui Gefeini, Trimetinib, general sodium for Buddhist nun, Bortezomib, pazopanib, Pa Boxini, pabishta, nilotinib, romidepsin, pleasure are cut down for Buddhist nun, Lapatinib, gram azoles For Buddhist nun, Carfilzomib, card it is rich for Buddhist nun, card than for Buddhist nun, Gefitinib, Vorinostat, Vande Thani, Tarceva, Di Nuosaimai, Dasatinib, dabrafenib, bosutinib, Baily department he, difficult to understand this replace Buddhist nun, Axitinib, A Fa for Buddhist nun, olaparib, Ai Le For in Buddhist nun or VEGF Trap any one or at least two combination.

6. granular pattern adjuvant according to any one of claims 1-5, which is characterized in that the granular pattern adjuvant further includes Modification group, the modification group are connected to the case surface of biocompatibility wall material formation, and the modification group is by modification base The donor of group provides；

Preferably, the donor of the modification group include hydrophilic biological macromolecule, it is coupling targeting substance, fluorescent marker, same The plain marker in position, environmental response substance, immunomodulator, pattern recognition receptors, immunopotentiator, cell factor or chemotactic factor (CF) In any one or at least two combination；

Preferably, the hydrophilic biological macromolecule include protein matter, polypeptides matter, small peptide and amino acid derivativges or Nucleic acid material any one or at least two combination；

Preferably, the protein matter includes any one in antigen, medical albumen or antibody substance or at least two The combination of kind；

Preferably, the antibody substance include thunder not Lu Dankang, the trastuzumab of resistance to former times, receive military monoclonal antibody, pyridine aldoxime methyliodide (PAM) monoclonal antibody, toltrazuril Monoclonal antibody, Cetuximab, difficult to understand, Ao Binyou trastuzumabs, this appropriate former times monoclonal antibody, Rituximab, replaces her at Victibix Not monoclonal antibody, tositumomab, pyridine aldoxime methyliodide (PAM) monoclonal antibody, Avastin, Herceptin, up to thunder wood monoclonal antibody, angstrom sieve trastuzumab, Beaune Spit in monoclonal antibody or ground Nu Tuxi monoclonal antibodies any one or at least two combination；

Preferably, the environmental response substance is selected from sensitive, thermo-responsive or group sensitive bioactive substance the object with pH Matter；

Preferably, the environmental response substance includes in poly-N-isopropyl acrylamide, ferrocene, graphene, maleimide Any one or at least two combination；

Preferably, the pattern recognition receptors include the stimulant of Toll-like receptor, the stimulant of RIG-1 receptors, NOD sample receptors Stimulant, mineral salt, saponin(e, liposome, liposome formulation product, the microparticle of synthesis, the microcarrier of synthesis, trachoma clothing it is former Body, the outer film bubble of bacterium, polysaccharide, the peptide of special sex modification, the peptide of preparation, protein, pathogen-associated molecular pattern or amino alkane In base glucosaminide 4- phosphates any one or at least two combination；

Preferably, the cell factor includes granulocyte macrophage colony stimulating factor, interferon, interleukins, junket ammonia In 3 ligand of acid kinase or tumor necrosis factor-alpha any one or at least two combination.

7. the granular pattern adjuvant according to any one of claim 1-6, which is characterized in that the granular pattern adjuvant further includes Antigen；

Preferably, the antigen is embedded in granular pattern adjuvant or is adsorbed on the surface of granular pattern adjuvant.

8. the preparation method of the granular pattern adjuvant according to any one of claim 1-7, which is characterized in that the method packet Include following steps：

(1) biocompatibility wall material, biocompatibility grease and all-trans retinoic acid are dissolved in oil phase solvent, it is mixed obtains oil phase Object is closed, while preparing aqueous phase solvent, obtains aqueous mixture；

(2) oil mixture object mixed with water is mixed in proportion, obtaining the granular pattern by dispersion, solidification, washing helps Agent；

Preferably, oil phase solvent described in step (1) includes good solvent and poor solvent；

Preferably, the volume ratio of the good solvent and poor solvent is (1-9):1；

Preferably, the volume ratio of the good solvent and poor solvent is 9:1 or 8:2；

Preferably, the good solvent includes isopentane, pentane, hexane, hexamethylene, isooctane, trifluoroacetic acid, trimethyl penta Alkane, pentamethylene, heptane, trichloro ethylene, carbon tetrachloride, chlorotrifluoroethane, propyl ether, toluene, paraxylene, chlorine isobutyl Alcohol, diethyl ether, tetrahydrofuran, petroleum ether, dichloromethane, chloroform, isopropanol, dimethyl sulfoxide (DMSO), acetone, ethyl acetate or In acetonitrile any one or at least two combination；

Preferably, the poor solvent includes any one in methanol, ethyl alcohol, propyl alcohol, ethylene glycol, formamide, trifluoroacetic acid, water Kind or at least two combination；

Preferably, aqueous phase solvent described in step (1) is emulsifier aqueous solution and/or salting liquid；

Preferably, the mass fraction of the emulsifier and/or salting liquid in aqueous phase solvent is 0-40%；

Preferably, the emulsifier includes polyvinyl alcohol, polyetherimide, polylactic acid-methoxy polyethylene glycol, polyoxyethylene mistake Water sorbitan ester surfactants, sorbitan esters, octoxynol 9, enuatrol, poloxamer, lecithin, lemon In sour sodium, double lauryl dimethyl amine bromides or cetyl trimethylammonium bromide any one or at least two group It closes；

Preferably, the salting liquid includes phosphate buffer, sodium citrate buffer solution, sodium chloride solution, Klorvess Liquid, nitre In sour potassium solution, metabisulfite solution or Tris-HCl buffer solutions any one or at least two combination；

Preferably, the volume ratio of oil mixture object mixed with water described in step (2) is 1:2-499.

9. the granular pattern adjuvant according to any one of claim 1-7, which is characterized in that the administration of the granular pattern adjuvant Mode is drug administration by injection；

Preferably, the drug administration by injection include hypodermic injection, intramuscular injection, injection in lymph node, intratumor injection, intraperitoneal injection, Intravenous injection or foot injection in any one.

10. granular pattern adjuvant according to any one of claim 1-7 prepare vaccine adjuvant, immunosuppressive drug, Fight the application in the drug of mucosal tissue relevant disease or the medicine of anti-chronic disease；

Preferably, the mucosal tissue relevant disease and chronic disease include human nipple virus infection, Infected With Polioviruses In Vitro, Coxsackie virus infection, enterovirus infection, norwalk virus infection, AIDS, anthrax, malaria, hepatitis B, A type liver Any one in inflammation, hepatitis C, colon cancer, colorectal cancer, cervical carcinoma, carcinoma of small intestine or lung cancer；

Preferably, when granular pattern adjuvant is used to prepare immunosuppressive drug, the charging ratio of all-trans retinoic acid is 2%- 25%；

Preferably, when granular pattern adjuvant is used to prepare the medicine of vaccine adjuvant or anti-chronic disease, all-trans retinoic acid Charging ratio is 0.01%-2%.