CN108403659A - A kind of hard emulsion receives microballoon and its preparation method and application - Google Patents
A kind of hard emulsion receives microballoon and its preparation method and application Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/5005—Wall or coating material
- A61K9/5021—Organic macromolecular compounds
- A61K9/5031—Organic macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, poly(lactide-co-glycolide)
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- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
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- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
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- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/22—Heterocyclic compounds, e.g. ascorbic acid, tocopherol or pyrrolidones
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- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
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- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/44—Oils, fats or waxes according to two or more groups of A61K47/02-A61K47/42; Natural or modified natural oils, fats or waxes, e.g. castor oil, polyethoxylated castor oil, montan wax, lignite, shellac, rosin, beeswax or lanolin
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Abstract
Microballoon and its preparation method and application is received the present invention provides a kind of hard emulsion,It includes biocompatibility wall material that the hard emulsion, which receives microballoon,,Biocompatibility grease and modification group,The kernel that the biocompatibility grease is formed is coated in the shell of biocompatibility wall material formation,The modification group is connected to the case surface,Microballoon is received the present invention provides hard emulsion,Its distinctive macromolecule and liposome compound wall materials shell assign the splendid storage of system and freeze-drying stability,And the system is easy to surface modification and structure regulating,To realize the common loading of multi-products,Targeted delivery and intelligent controlled release,In release,Since the presence of liposome makes small particle hard emulsion coalescence phenomenon occurring close to body temperature region,Reduce specific surface area,The phenomenon of burst release of micron delivery system is received to substantially improve,Realize long-acting slow-release,In cosmetics,Food and field of medicaments have broad application prospects.
Description
Technical field
The invention belongs to biomedicine technical field, it is related to a kind of hard emulsion and receives microballoon and its preparation method and application.
Background technology
Current many drugs (taxol, camptothecine etc.), naturally extract product (color flavones), essence, crosslinking agent and day
Change product core component is often insoluble in water although being proved to have good biological property, either be easy in water or
It is inactivated in alcohol, to restrict its development and application.The main lotion stablized using surfactant carries out indissoluble ingredient at present
Embedding, storage and sustained release.These oily emulsion formulations are usually made of water-oil phase, including at least one surfactant.Commonly
Surfactant includes polyoxyethylene sorbitan esters surfactant (commonly referred to as tween), sorbitan esters
(commonly referred to as sapn), octoxynol 9 (triton x-100 or tert-octylphenoxypolyethoxyethanol) and lecithin etc., it is special
It is not Tween 80 (SPAN 80), span 85 (anhydrosorbitol trioleate) and triton X-
100 application is more.Role is mainly stable emulsion to surfactant in the formulation, avoids demulsification and emulsion droplet coalescence.
To realize this purpose, need using at least one surfactant, and to keep emulsion-stabilizing, surface-active
Content of the agent in lotion requires more than emulsification aequum, and so as to cause among water phase or oil phase or the two, there are free surface activity
Agent.And the often 4 degrees Celsius of storages in liquid form of this preparation, in addition short texture, easy microbiological contamination are denaturalized.It needs to be added a large amount of
Preservative.At the same time, due to lacking rigidity, cause it that can not be lyophilized so that its storage cycle substantially reduces, and increases life
Produce cost.Also, emulsion droplet surface can not be surface modified and structure optimization, limits it and is discharged in environment-responsive, targeting
The application of the numerous areas such as delivering, and intelligent slow-releasing system.
Then, biocompatibility macromolecule receive micron particles appearance.But this particulate carrier, the meeting in forming process
It is separated, active constituent is caused to be deposited in particle surface, or mutually detached together with liquid, lead to embedding efficiency and embedding matter
Amount reduces (embedding rate is only 30% or less), to cause the waste of a large amount of raw material.Also, since drug deposition is in surface,
With the degradation of polymer, it will appear burst release during the release of fat-soluble active substance, phenomena such as rate of release is unstable.
In view of the above problems, it is proposed that receiving micron particles with hard emulsion carries out fat-soluble active substance controllable embedding
With release.Hard emulsion particle is to have a biocompatibility wall material, and inside embeds the particle for receiving micro-meter scale of metabolizable grease.With
Traditional emulsion formulations using surfactant stabilisers are different, the solid emulsion droplet particle degradable grease (coconut oil, castor
Sesame oil, squalene and tocopherol etc.) embedding fat-soluble active ingredient.At the same time, the polymer plating of kernel oil droplets densification
Layer realizes and its rigidity is reinforced and protected.Also, liposome molecule and modification group are introduced, particle environments sound is imparted
Ying Xing.In addition, by Electrostatic Absorption, the modes such as chemical bonding are realized to albumen, the hydrophilic compositions such as polypeptide and nucleic acid it is common
It loads, delivering and release.Compared with traditional preparation, has advantage outstanding:(1) small to the toxic side effect of organism;(2)
Fine and close shell assigns more efficient embedding rate and outstanding sustained release performance;(3) system has environment-responsive;(4) a variety of work
Property molecule load jointly, delivering and controlled release;(5) it can be lyophilized;(6) system stability is strong.However, at present about macromolecule
And grease compounded system is rarely reported, and work few in number also uses the bad glyceride stock of biocompatibility, limitation
, in skin care item, the development in food and drug direction or storage stability are bad for it, also or cannot achieve efficient multigroup
Packing carries and delivering.For example, having been reported that a kind of micro-capsule system of polystyrene embedding hexadecane is used for the embedding of nitro-anisol
With release.Though illustrating preparating mechanism, it is strong that hexadecane used (hexadecane) not only pollutes environment also harmful to human
Health.Meanwhile system does not investigate the stability of storage and freeze-drying, does not have actual application value.In addition there is research using biology
Degradability lactide-co-glycolide (also referred to as Vicryl Rapide or Poly(D,L-lactide-co-glycolide or D,
L- lactide-co-glycolide polymer, English name Poly (lactic-co-glycolic acid), english abbreviation, PLG or
PLGA, lactide are 50 with glycolide ratio:50, viscosity 0.4dl/g) as shell membrane material embedding glycol dicaprate
(propylene glycol dicaprylate), to realize for Hydrophobic small molecules all-trans retinoic acid (all-trans-
Retinoic acid, ATRA) and antiphlogistic antinfan (indomethacin, IDMC) loading and release.Although its preliminary suppression
Make phenomenon of burst release of the nano particle for Hydrophobic small molecules, but slow release effect still unobvious.
CN105311631A discloses a kind of CaP-PLA/PLGA and receives microballoon and the preparation method and application thereof:(1) by PLA or
PLGA is dissolved in organic solvent, forms oil phase O;(2) contain the aqueous solution of 0.1M~1M calcium ions as inner aqueous phase W1, by oil phase
O and W1 mixing prepares colostric fluid W1/O by ultrasound or homogeneous method;(3) aqueous solution of polyethylene dissolving alcohol (PVA) is as outer water
Phase W2, colostric fluid is added in W2, and mechanical agitation forms pre- double emulsion W1/O/W2;(4) pre- double emulsion W1/O/W2 is shifted
It is emulsified in storage tank to fast film, crosses film repeatedly under appropriate gas pressure, obtain the double emulsion W1/O/W2 of uniform particle diameter;(5)
The solution of the phosphate anion containing 0.1M~1M, mixing are added into double emulsion W1/O/W2 systems, centrifugal filtration removes solution;It goes
Ion water washing is dried to obtain and receives microballoon, but the method will not receive microballoon functionalization, limit it and further apply.
Do not embed the system research of the hard emulsion nano-particle of grease in existing method about functionalization at present, it is known that
Macromolecule and grease compounded system also not according to cosmetics, the demand of food and drug is designed and optimizes again.
Invention content
In view of the deficiencies of the prior art, the purpose of the present invention is to provide a kind of hard emulsion receive microballoon and preparation method thereof and
Using.
To reach the invention purpose, the present invention uses following technical scheme:
In a first aspect, receive microballoon the present invention provides a kind of hard emulsion, it includes biocompatibility that the hard emulsion, which receives microballoon,
Wall material, biocompatibility grease and modification group, the kernel that the biocompatibility grease is formed are coated on biocompatibility
In the shell that wall material is formed, the modification group is connected to the case surface.
Hard emulsion provided by the invention receives microballoon (as shown in Figure 1), is solid entrapping liquid system, outside the nanosphere of formation
Shell is hard macromolecule wall material, and the biocompatibility grease that kernel is liquid, the lotion stablized with conventional surfactant
It compares, the distinctive macromolecule of the present invention and liposome compound wall materials shell assign the splendid storage of system and freeze-drying stability, with
This simultaneously, which is easy to surface modification and structure regulating, to realize the common loading of multi-products, targeted delivery and intelligence
Type controlled release, release when, due to the presence of liposome make small particle hard emulsion close to body temperature region (skin surface,
It is oral cavity and alimentary canal, subcutaneous or intracellular) there is coalescence phenomenon, reduce specific surface area, receives micron to substantially improve and pass
The phenomenon of burst release of system is sent, realizes long-acting slow-release, in cosmetics, food and field of medicaments have broad application prospects.
Further, the hard emulsion in the present invention receives the distinctive wall material shell of microballoon, and many advantages are provided for system:Greatly
Its storage stability and application field are improved greatly;Double octadecyl bromination ammoniums, 3 β-[N- (N', N'- bis- are added in shell membrane material
Methyl amine ethyl)] amido formacyl, cholesterol, polyetherimide, chitosan etc. may be implemented for big point of hydrophily biology
Son is (such as:Albumen, calculates drug or hydrophilic compounds at polypeptide) common loading;Double octadecyl brominations are added in membrane material
The substances such as ammonium, 3 β-[N- (N', N'- dimethyl aminoethyl)] amido formacyl, cholesterol, lecithin, phosphatide and its derivative
Afterwards, small particle receives micron hard emulsion close to body temperature region (skin surface, oral cavity and alimentary canal, subcutaneous or intracellular) meeting
It is heated coalescence phenomenon occur, reduce specific surface area, realizes long-acting slow-release;Shell provides excellent modification for hard emulsion nano-particle
Site so that system is easy to surface modification and structure regulating, to the targeted delivery for realizing product and intelligent controlled release.
In addition, for the lotion stablized compared to surfactant, hard emulsion provided by the invention receives microballoon with more preferable
Rigidity, can be lyophilized, extend its storage time, it is cost-effective.
In the present invention, hard emulsion receives microballoon and includes:Embedding biocompatibility grease to have biocompatibility wall material hard
Lotion receives microballoon;The polymer hard emulsion of embedding hydrophobic drug and biocompatibility grease receives microballoon;Embed hydrophobicity chemical combination
The hard emulsion of object and biocompatibility grease and the microballoon of receiving for being modified with group;The polymer for embedding biocompatibility grease is firmly newborn
Liquid and the microballoon of receiving for being modified with group;It embeds the polymer hard emulsion of biocompatibility grease and is modified with group while being loaded with
Hydrophilic compounds receive microballoon.
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 is less than or equal to 80%.
In the present invention, the biocompatibility wall material has biocompatibility, and preferably wall material is by biocompatibility high score
Sub- material and liposome mix.Due to introducing of the liposome molecule in wall material, macromolecule and liposome molecule composite
Material has both the advantage of two kinds of membrane materials, can greatly promote the embedding rate to hydrophobic compound, stablizes metabolizable grease, and
More modification groups are added, modification of the later stage for wall material, biocompatible polymer material and liposome molecule material are conducive to
Material is with (1-9):1 is more preferably weight ratio, also, biocompatibility macromolecule polymer can be preferably dissolved in organic solvent
High molecular 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 group.
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), answer
For abbreviation (PLG or PLGA).
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、1:4、1:1、2:3、5:8、6:1 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, mass percent of the biocompatibility grease in hard emulsion receives microballoon is 5%-80%, such as
Can be 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70% or 80%.
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;
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-
Alpha-tocopherol.
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.
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 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.
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.
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, further include hydrophilic biological macromolecule in the biocompatibility grease.
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), the agonist of pattern recognition receptors
Alum, MPL, saponin(e (such as QS-21, Quil-A, iscoMs, the iscomatrix of specific bindingTM);Liposome and liposome are matched
Product includes AS01, AS02 etc.;The microparticle of synthesis, the microcarrier of synthesis can be neisseria gonorrhoeaes
(N.gonorrheae), the outer film bubble (OMV) derived from bacterium of chlamydia trachomatis and other bacteriums;Polysaccharide can be chitosan;
Special sex modification or the peptide of preparation can be muramyl dipeptides;Pathogen-associated molecular pattern is selectable pathogen-associated molecular mould
Formula (PAMPS);Aminoalkyl glucosaminide 4- phosphates can be RC529;Protein can be bacterial toxoid or toxin
Segment.
Preferably, the cell factor include granulocyte macrophage colony stimulating factor, interferon, interleukins,
In 3 ligand of tyrosine kinase or tumor necrosis factor-alpha any one 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, it further includes medical additive that the hard emulsion, which receives microballoon,.
Preferably, mass fraction of the medical additive in hard emulsion receives microballoon is 0-20%, such as can be 0,
1%, 5%, 10%, 12%, 15%, 18% or 20%.
Preferably, the medical additive includes any one in diluent, stabilizer or preservative or at least two
The combination of kind.
In the present invention, hard emulsion microballoon 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 adjusting agent or and and buffer, preferably sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, human serum albumin,
Essential amino acid, nonessential amino acid, L-arginine hydrochloride, sucrose, anhydrous D- trehaloses, mannitol, mannose, starch or
In gelatin any one or at least two combination.It is wherein typical but non-limiting to be combined as:Sodium acetate and sodium lactate
Combination;The combination of sodium chloride and potassium chloride;The combination of calcium chloride, human serum albumin and essential amino acid;Non-essential amino
The combination of acid, L-arginine hydrochloride, sucrose and anhydrous D- trehaloses;The combination of mannitol, mannose, starch and gelatin, second
Sour sodium, sodium lactate, sodium chloride, potassium chloride, calcium chloride and the combination of human serum albumin;Essential amino acid, nonessential amino acid,
The combination of L-arginine hydrochloride, sucrose, anhydrous D- trehaloses, mannitol, mannose, starch and gelatin.
Preferably, the hard emulsion receive microballoon average grain diameter be 10nm-50 μm, such as can be 10nm, 100nm,
400nm, 700nm, 1 μm, 10 μm, 20 μm, 30 μm, 40 μm or 50 μm, preferably 50nm-20 μm.
In the present invention, the hard emulsion particle in preferred scope has a better particle diameter distribution, storage stability, and
Better bioactivity, average grain diameter are higher or lower than above range, can all make the stability of particulate adjuvants, embedding rate and exempt from
There is the trend being substantially reduced in epidemic disease effect.
Second aspect, receives the preparation method of microballoon the present invention provides hard emulsion as described in relation to the first aspect, the method
Include the following steps:Biocompatibility wall material is dissolved in include biocompatibility grease solvent in, by disperseing, solid
Change, the isolated hard emulsion receives microballoon after the modification of modification group.
Preparation method provided by the invention prepares oil phase first, and target is embedded biocompatibility grease and biocompatibility wall
The substances such as material and the indissoluble object that can be added according to purpose demand as described above and modification group are dissolved in oil phase solvent.
Preferably, the solvent is oil phase solvent and the mixed solvent that aqueous phase solvent forms.
Preferably, the mass ratio of the oil phase solvent and aqueous phase solvent is 1:2-499, such as can be 1:2、1:10、1:
100、1:200、1:300、1:400 or 1:499.
Preferably, the oil phase solvent includes good solvent and poor solvent, volume of the good solvent in oil phase solvent
Score is 40%-100%, such as can be 40%, 50%, 60%, 70%, 80%, 90% or 100%, the poor solvent
Volume fraction in oil phase solvent is 0-60%, such as can be 0,10%, 20%, 30%, 40%, 50% or 60%.
In the present invention, no poor solvent can also form nanosphere, and there may also be lifes when poor solvent volume is 0
Object compatibility grease.
Preferably, the volume ratio of the good solvent and poor solvent is 9:1 or 8:2.
In the present invention, mixed solvent is conducive to grease and is separated in inner emulsion with wall material.It is good molten by adjusting
The rate of the proportioning of agent and poor solvent, phase separation can be regulated and controled, and the interior of microballoon is received to which optimization is formed by hard emulsion
Portion's structure achievees the purpose that promote embedding rate, but if poor solvent may excessively make active ingredient can not in system
It is dissolved in oil phase, causes system unstable.
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, the aqueous phase solvent includes emulsifier and/or salting liquid.
Preferably, the mass fraction of the emulsifier and/or salting liquid in aqueous phase solvent is 0-40%, such as can be
0,10%, 20%, 25%, 30%, 35% or 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 amine 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 mode of the dispersion includes that ultrasonic, micro-fluidic, homogeneous, ultrasound, syringe pair push away emulsification, spraying, micro-
Jet stream, microchannel, film emulsification, stirring, oscillation, reverse or it is hand in any one.
In the present invention, as long as the mode of dispersion can be equal by the parameters such as power, time, number arrival target particle sizes
Can, used dispersing mode will not cause to significantly affect to biocompatibility wall material and biocompatibility grease, can be according to institute
Suitable dispersing mode and concrete operations parameter are selected with water phase and solid particle property and the experimental facilities condition of itself.
Hybrid mode can preferably can obtain in micro-fluidic, microchannel or film emulsification etc. in disperseing according to different requirements,
Uniform particle size is distributed the mode of lotion, preferably microjet, syringe pair can also push away emulsification, stirring or oscillation etc. convenient for scale
The hybrid mode of preparation.It should be clear that different dispersing modes, preparing power, time, number etc. and necessarily affecting gained
Particle size, dispersibility and the stability etc. of grain, will also influence final embedding efficiency, release, targeted delivery and other effects.Cause
This, it is suitable to be determined according to the influence factors such as grease phase used and solid particle property, the emulsion droplet size range of required preparation
Hybrid mode and concrete operations parameter.
Preferably, hard emulsions of the Span less than 1.0 receives microballoon and has higher embedding rate and stability, and has more preferable
Sustained release performance.
Preferably, the cured method include solvent evaporation method, physical crosslinking method, chemical crosslink technique, oxidation-reduction method,
High-temperature calcination adjusts pH methods, adjusts any one in salinity method, extraction or the precipitation method.
In the work that macromolecule receives microballoon embedding hydrophobic compound, since hydrophobic molecule is (oily in phase separation
Mutually and water phase) tend to interface deposit so that hydrophobic compound solidification when with liquid mutually detach together, cause embedding rate with
The reduction of bioavilability.And the present invention, using internal phase transfer principle (as shown in Figure 2), is introduced by a step emulsion method
The grease insoluble with wall material is as third phase.By adjusting the ratio of solvent, inside phase separation (grease and the oil of the membrane material containing shell
Phase) it is separated (oil phase and outer aqueous phase of the membrane material containing shell) prior to outside, cause hydrophobic molecule to be deposited in internal fat drips interface.
Method provided by the invention avoids hydrophobic compound and is detached together in nano-particle outside deposition or with water phase, to significantly
The efficiency for promoting embedding and loading.Also, the method is easy to operate, and the production cost in actual production and sterilizing is greatly saved
Cost, conducive to being widely popularized.
The third aspect, the present invention provides a kind of hard emulsion as described in relation to the first aspect receive microballoon prepare vaccine adjuvant or
Application in drug delivery vehicle.
Hard emulsion provided by the invention receive microballoon can make hydrophobic perfume, crosslinking agent, drug, immune activation substance with
And other compositions will realize more efficient embedding, storage protection and sustained release.
Compared with the existing technology, the invention has the advantages that:
(1) hard emulsion provided by the invention receives microballoon, and compared with the lotion that conventional surfactant is stablized, the present invention is peculiar
Macromolecule and liposome compound wall materials shell assign the splendid storage of system and freeze-drying stability, at the same time, the system is easy
It is being released in surface modification and structure regulating to realize the common loading of multi-products, targeted delivery and intelligent controlled release
When putting, since the presence of liposome makes small particle hard emulsion close to body temperature region (skin surface, oral cavity and alimentary canal, skin
It is lower or intracellular) there is coalescence phenomenon, reduce specific surface area, to substantially improve receive micron delivery system burst release it is existing
As realizing long-acting slow-release, in cosmetics, food and field of medicaments have broad application prospects.
(2) invention is by a step emulsion method, using internal phase transfer principle, introduces the grease insoluble with wall material as the
Three-phase, by adjusting the ratio of solvent, inside phase separation (grease and the oil phase of the membrane material containing shell) (contains shell prior to outside phase separation
The oil phase and outer aqueous phase of tunic material), cause hydrophobic molecule to be deposited in internal fat drips interface;Method provided by the invention avoids
Hydrophobic 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.
Description of the drawings
Fig. 1 is that the hard emulsion of the present invention receives the structural schematic diagram of microballoon.
Fig. 2 be the present invention preparation method in phase transfer principle schematic diagram inside a step emulsion process.
Fig. 3 is that the oil polymers hard emulsion prepared in the embodiment of the present invention 1 receives the grain size distribution of microballoon.
Fig. 4 is that the oil polymers hard emulsion prepared in the embodiment of the present invention 1 receives the scanning electron microscope (SEM) photograph of microballoon.
Fig. 5 is that the polymer hard emulsion for the embedding biocompatibility grease that modification group is modified in the embodiment of the present invention 3 is received
The grain size distribution of microballoon.
Fig. 6 is that the polymer hard emulsion for the embedding biocompatibility grease that modification group is modified in the embodiment of the present invention 3 is received
The scanning electron microscope (SEM) photograph of microballoon.
Fig. 7 A are that oil polymers hard emulsion receives microballoon lyophilized preceding scanning electron microscope (SEM) photograph in the embodiment of the present invention 7.
Fig. 7 B be in the embodiment of the present invention 7 oil polymers hard emulsion receive it is microballoon lyophilized after scanning electron microscope (SEM) photograph.
Fig. 8 be in the embodiment of the present invention 8 oil polymers hard emulsion receive microballoon the protection of the compound that is embedded is stored it is steady
Qualitative fluorescence collection of illustrative plates.
Fig. 9 is that oil polymers hard emulsion receives the elution profiles figure that microballoon changes over time in the embodiment of the present invention 9.
Figure 10 is that oil polymers hard emulsion receives distribution map characterization Evaluation on Its Targeting Performance figure in microsphere in the embodiment of the present invention 12.
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.
The raw material used in the embodiment of the present invention is as shown in table 1 below:
Table 1
The instrument used in the embodiment of the present invention is as shown in table 2 below:
Table 2
Grease embedding rate and the assay method of carrying capacity are as follows in particle in following embodiment:
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=(inhale
Antigen or drug concentration in supernatant after attached pro-antigen or drug concentration-absorption)/absorption 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).
Embodiment 1
The PLGA polymer hard emulsions that the present embodiment is prepared by the following method embedding biocompatibility grease receive microballoon
PLGA (the LA of 0.30g are accurately weighed using electronic balance:GA is 50:50, molecular weight is 110,000 dalton),
0.30g squalenes are dissolved in the ethyl alcohol of 1mL and the mixed solution of dichloromethane, and solution is poured into rapidly to 20mL aqueous solutions and (is contained
The alcoholysis degree of the PVA of 1wt.%, PVA are 99%, viscosity 5.0mPas), dispersion (50W, 2min, the interval under ultrasound condition
Time 4s).It is stirred overnight (magnetic agitation, rotating speed 500rpm) at 25 DEG C, at normal temperatures, utilizes solvent evaporation method, removing body
Ethyl alcohol in system and dichloromethane solvent cure hard emulsion nano-particle.After the completion of solidification, 20min is centrifuged under 20000g, is abandoned
Supernatant is removed, 10mL deionized waters are added into precipitation, ultrasonic disperse centrifuges 5min under 20000g, and after discarding supernatant, precipitation is frozen
The dry PLGA polymer hard emulsions for obtaining preparing embedding biocompatibility grease receive microballoon, are placed in 4 DEG C of preservations in refrigerator.
To be prepared PLGA polymer hard emulsions receive microballoon carry out particle size distribution measuring:
The particle diameter distribution of micron particles or emulsion droplet is measured using laser particle analyzer, and specific determination step is:By 5mg
Micron particles are added in the deionized water of 50mL, and ultrasonic 5min keeps its evenly dispersed, or take 50mL lotions, and particle is suspended
Liquid or lotion are added in sample cell, using laser particle analyzer (Malvern Instruments, United Kingdom
Coulter Co., USA) it is measured.
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:Span=
(d90-d10)/d50, value is smaller to show that grain diameter is more uniform, wherein d10, d50And d90Respectively particle cumulative volume is
10%, 50%, grain size when 90%.
The morphology observation of particle uses scanning electron microscopic observation:1mg particles are weighed, are added in 10mL deionized waters, ultrasound
5min 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.
Aluminium foil is affixed on conducting resinl on sample stage, under vacuum metal spraying (suitable metal spraying condition is chosen according to properties of samples)
Afterwards, it is observed with scanning electron microscope.
The average grain diameter that PLGA particles are obtained by above-mentioned test is 257nm, Span 0.335.
It is scanned Electronic Speculum observation, surface is smooth, spherical regular.The grain size distribution of particle is as shown in Figure 3, it can be seen that
Average grain diameter is 10nm-50 μm;Pattern is as shown in Figure 4, it can be seen that grain diameter is more uniform.
The present embodiment, which also can be used other PLA family macromolecules and prepare the hard emulsion of embedding biocompatibility grease, receives microballoon,
Its preparation process is similar to Example 1, design parameter and the results are shown in Table 3.
Table 3
Embodiment 2
The hard emulsion that the present embodiment prepares embedding hydrophobic drug by following steps receives microballoon
2mg all-trans retinoic acid is accurately weighed using electronic balance and is dissolved in 0.30g squalenes, weighs 0.30g PLA, and
Be dissolved in the ethyl alcohol of 1mL and the mixed solution of dichloromethane, solution is poured into rapidly 20mL aqueous solutions (PVA containing 1wt.%,
The alcoholysis degree of PVA is 99%, viscosity 5.0mPas), disperse (50W, 2min, interval time 4s) under ultrasound condition.25℃
Under be stirred overnight (magnetic agitation, rotating speed 500rpm), at normal temperatures, using solvent evaporation method, ethyl alcohol in removal system and
Dichloromethane solvent cures hard emulsion nano-particle.After the completion of solidification, 20min is centrifuged under 20000g, is discarded supernatant, Xiang Chen
10mL deionized waters are added in shallow lake, ultrasonic disperse centrifuges 5min under 20000g, after discarding supernatant, centrifuges, and be lyophilized,
It obtains hard emulsion and receives microballoon.
The present embodiment can also receive microballoon embedding various hydrophobic drug using hard emulsion, and obtained microballoon of receiving is provided with height
The embedding rate of effect, preparation process is similar to Example 2, specific process parameter and the results are shown in Table 4.
Table 4
Embodiment 3
The polymer that the present embodiment prepares the embedding biocompatibility grease of modification group modification by following steps is firmly newborn
Liquid receives microballoon
Electronic balance is used accurately to weigh the PLGA (molecular weight is 150,000 dalton) of 1.0g, 0.50g squalenes, 0.2g bromines
Change trimethyl -2,3- dioleoyl oxygroup propyl ammonium, is dissolved in the ethyl alcohol and dichloromethane (1 of 10mL:It 9), will be molten in mixed solution
Liquid pours into rapidly 200mL aqueous solutions, and (PVA containing 0.5wt.%, the wherein alcoholysis degree of PVA are 99%, viscosity 3.0mPa
S), disperse (40W, 2min, interval time 4s) under ultrasound condition.It is stirred overnight that (magnetic agitation, rotating speed are at 25 DEG C
500rpm), the organic solvent in solvent is removed at room temperature, to cure hard emulsion particle.20min is centrifuged under 5000g, is discarded
Supernatant, 5mL deionized waters are added into precipitation, and ultrasonic disperse centrifuges 5min under 5000g, after discarding supernatant, precipitation is lyophilized
The PLGA polymer hard emulsions for being modified with the embedding grease of cationic-liposome group receive microballoon, are placed in 4 DEG C of preservations in refrigerator.
Surface by particle prepared by scanning electron microscopic observation is smooth, spherical regular.The grain size distribution of particle such as Fig. 5 institutes
Show, average grain diameter 2689nm, Span 0.405;Pattern is as shown in Figure 6, it can be seen that pattern becomes larger, and shape is uniform.
Other modification groups (liposome, targeted molecular, environment-responsive substance etc.) also can be used to prepare in the present embodiment
The polymer hard emulsion for being modified with the embedding grease of group receives microballoon, and preparation process is similar to Example 3, specific process parameter
And the results are shown in Table 5 (aglucon modification rate=unit mass receive microballoon in ligand content (mg)/receive microspheres quality * 100%).
Table 5
Embodiment 4
The oil polymers with modification group that the present embodiment prepares embedding hydrophobic drug by following steps are firmly newborn
Liquid receives microballoon
Electronic balance is used accurately to weigh the PLGA (molecular weight is 430,000 dalton) of 2.0g, 1.0g squalenes, 0.3g bromines
Change dimethyl dioctadecyl ammonium and 10mg all-trans retinoic acid, is dissolved in the ethyl alcohol, acetone and dichloromethane (1/1/9) of 30mL
In mixed solution, solution is poured into rapidly to 400mL aqueous solutions, and ((alcoholysis degree 99%, viscosity are the PVA containing 1wt.%
5.0mPas), disperse (100W, 3min, interval time 4s) under ultrasound condition.Be stirred overnight at 25 DEG C (magnetic agitation, turn
Speed is 500rpm), to remove organic solvent, realize the solidification of system.20min is centrifuged under 15000g, is discarded supernatant, into precipitation
10mL deionized waters are added, ultrasonic disperse centrifuges 20min under 15000g, after discarding supernatant, will precipitation freeze-drying, be placed in 4 in refrigerator
DEG C preserve.The average grain diameter of prepared particle is 302nm, Span 0.501, embedding rate 85.2%, modification rate 9.2%.
The present embodiment can also be received by the oil polymers hard emulsion with modification group microballoon embedding various hydrophobic medicine
Object is selected from antitumor broad-spectrum medicinal, and immune activation adjuvant substance, antineoplastic target drug, fluorescence substance etc., receiving for obtaining be micro-
Ball is provided with efficient embedding rate.
Embodiment 5
The oil polymers hard emulsion that the present embodiment prepares embedding hydrophilic compounds by following steps receives microballoon
Model proteins OVA 10mg are accurately weighed using electronic balance, are dissolved in 1mL deionized waters, are weighed 0.2g's
PLGA (molecular weight is 430,000 dalton), 0.1g squalenes, 0.04g bromoethyl dibasecylammonium bromides and 10mg total trans dimensions
Formic acid is dissolved in the acetone and dichloromethane (1 of 30mL:9) in mixed solution.Aqueous solution containing OVA is poured into rapidly this to mix
It closes in solution and forms colostrum, magnetic agitation 2min (500rpm).Colostrum is poured into rapidly to 50mL aqueous solutions again (containing 1wt.%'s
PVA, wherein alcoholysis degree are 99%, viscosity 5.0mPas), disperse (200W, 2min, interval time 4s) under ultrasound condition.
It is stirred overnight at 25 DEG C (magnetic agitation, rotating speed 500rpm), centrifuges 20min under 10000g, discard supernatant, be added into precipitation
10mL deionized waters, ultrasonic disperse centrifuge 20min under 10000g, after discarding supernatant, will precipitation freeze-drying, be placed in 4 DEG C of guarantors in refrigerator
It deposits.
The average grain diameter for preparing particle is 451nm, and Span 0.567, ATRA embedding rates are 85.2%, and albumen embedding rate is
61.7%, modification rate 9.2%.
Embodiment 6
The oil polymers hard emulsion that the present embodiment is prepared by the following method absorption hydrophilic compounds receives microballoon
Oil polymers hard emulsion is prepared using method in embodiment 3-5 and receives microballoon, and is lyophilized and obtains product.Weigh 2mg
Microballoon and the OVA albumen obtained received is with 2000:1,1000:1,500:1,200:1,100:1,50:1 and 10:1 ratio mixing,
After 4 DEG C of vertical suspensions overnight, its adsorption rate is tested, result is summarized as follows shown in table 6:
Table 6
The results show that hard emulsion nano-particle has high OVA load factors.The present embodiment also can be used the method poly- by grease
It closes object hard emulsion and receives microballoon and adsorb a variety of hydrophilic compounds, wherein hydrophilic compounds are selected from protein matter, polypeptide
Matter, small peptide and amino acid derivativges, nucleic acid material any one or at least two combination, obtained microballoon of receiving has
Standby efficient adsorption rate.
Embodiment 7
It measures oil polymers hard emulsion and receives the freeze-drying stability of microballoon
Oil polymers hard emulsion is prepared using method in embodiment 1-6 and receives microballoon, and is lyophilized, before freeze-drying and will be frozen
Microballoon sample preparation is received after dry, its form of scanning electron microscopic observation is used in combination in metal spraying.As a result as shown in figures 7 a and 7b, (scheme with before freeze-drying
Shown in 7A) it compares, it receives the grain size of microballoon (shown in Fig. 7 B) after freeze drying and significant change does not occur in structure, it was demonstrated that the oil of preparation
Lipopolymer hard emulsion receives microballoon and has good freeze-drying stability, is suitable for content grease or the long Kubo of hydrophobic molecule
It deposits.
Such as can be amino acid, L-arginine to be further ensured that receive microballoon is added freeze drying protectant in freeze-drying process
Hydrochloride, sucrose, anhydrous D- trehaloses, mannitol, mannose, starch or gelatin any one or at least two combination.
Embodiment 8
Test oil polymers hard emulsion receive microballoon for target embedded object long-term storage
It is accurate to weigh 20mg ATRA (thermal instability, photo-labile hydrophobic compound), and use embodiment 2,4,5
With 6 in method, be embedded in oil polymers hard emulsion and receive microballoon, the ATRA that is embedded with after 2.0g is lyophilized receives microballoon and phase
With aimed quality ATRA powder be separately stored in 25 DEG C store 14 days.By the ATRA before storage, after storage receive microballoon and
ATRA is dissolved with 10 microlitres of chloroforms, and carries out full spectral scan using Fluorescence Spectrometer (Hitachi, F-4600), as a result such as Fig. 8
It is shown, it is stored in and receives the fluorescence spectrum of ATRA (green) and the ATRA (red) not stored of microballoon and apparent fluorescence do not occur
Deviate, and there is apparent offset in unprotected ATRA (blue), shows that long term storage so that the configuration of ATRA changes, and
It is stored in hard emulsion particle and greatly improves its stability, it was demonstrated that oil polymers hard emulsion receives microballoon can be thin with effective protection
Aqueous small molecule improves its storage stability.
Embodiment 9
Test oil polymers hard emulsion receives the slow release effect of microballoon
Using preparation method in embodiment 1-6, receives microballoon using oil polymers hard emulsion and embed Imatinib, freeze-drying.
The microballoon of receiving that 100mg is embedded with Imatinib is accurately weighed using electronic balance, is dissolved in 0.5mL dialysis tubings, is placed in
25mL PBS carry out sustained release experiment in 37 degree.1mL outer aqueous phases are drawn for every eight hours, and add 1mL PBS.Sustained release experiment terminates
Afterwards, Imatinib is detected using high performance liquid chromatography (HPLC), obtains elution profiles.As shown in figure 9, oil polymers
Hard emulsion receive microballoon be effectively relieved high molecular polymer microballoon burst release the phenomenon that, realize the long-acting slow-release to drug target.
Embodiment 10
The comparison of macromolecule wall material hard emulsion nano-particle and the compound wall material hard emulsion nano-particle of macromolecule/liposome
The macromolecule wall material hard emulsion nano-particle and high score for being embedded with Imatinib are prepared using method in embodiment 1-5
Son/liposome compound wall materials nano-particle, and be lyophilized and obtain product.It weighs two kinds of 10mg and receives microballoon, it is molten using chromatographic grade acetonitrile
Solution, measures its embedding rate for Imatinib using high performance liquid chromatography, is summarized as follows table 7.In addition, as described in Example 9,
It weighs made from 100mg two kinds and receives microballoon, be dissolved in 0.5mL dialysis tubings, be placed in 25mL PBS, be sustained in 37 DEG C
Experiment.1mL outer aqueous phases are drawn for every eight hours, and add 1mL PBS.After sustained release experiment terminates, utilize high performance liquid chromatography (HPLC)
Imatinib is detected, obtains elution profiles, it is as a result close with Fig. 9.Use half release rate for detection slow release effect
(Imatinib of 50% embedding is released required time, R for evaluation50), R50Its result is summarized as follows shown in table 7.
The results show that compound wall material has higher embedding rate and more preferably slow release effect.
Table 7
Embodiment 11
Oil polymers hard emulsion receives microballoon temperature sensitivity after test modification
Electronic balance is used accurately to weigh PLA-PEG (molecular weight is 230,000 dalton), the blueberry flavor 10mg of 0.5g,
0.5g squalenes, 0.2g bromination trimethyl -2,3- dioleoyl oxygroup propyl ammoniums are dissolved in the ethyl alcohol and dichloromethane (2/8) of 10mL
Mixed solution in, solution is poured into rapidly to 50mL aqueous solutions, and ((alcoholysis degree 99%, viscosity are the PVA containing 0.5wt.%
3.0mPas), disperse (200W, 2min, interval time 4s) under ultrasound condition.Be stirred overnight at 25 DEG C (magnetic agitation, turn
Speed is 500rpm), 20min is centrifuged under 20000g, is discarded supernatant, and is added 5mL deionized waters into precipitation, ultrasonic disperse,
Centrifuge 5min under 20000g, after discarding supernatant, by precipitation be lyophilized temperature sensitivity oil polymers hard emulsion receives microballoon, put down
Equal grain size is 312nm, Span 0.581, is placed in 4 DEG C of preservations.
The microballoon of receiving for being embedded with blueberry flavor that 4 DEG C are placed is taken out, is smeared in subjects skin, and rub 5min, by
Examination person smells strong blueberry flavor taste in 1min, it was demonstrated that a degree of solution occurs under shell temperature for the microballoon of receiving of formation
It is poly-, cause it to release the squalene oil droplet of embedding blueberry flavor so that active constituent environment-responsive discharges.
Embodiment 12
Oil polymers hard emulsion receives microballoon targeting after test modification
Prepare hard emulsion according to method in embodiment 3-6 and receive microballoon, accurately weighed using electronic balance 2.0g PLGA (point
Son amount is 130,000 dalton), 0.5mg Cy7 (fluorescent marker), 1.0g squalenes, 0.2g bromination trimethyl -2,3- dioleoyls
Oxygroup propyl ammonium is dissolved in the ethyl alcohol of 10mL and the mixed solution of dichloromethane (2/8), solution is poured into rapidly to 50mL aqueous solutions
(PVA (alcoholysis degree 99%, viscosity 3.0mPas) containing 0.5wt.%, disperse under ultrasound condition (200W, 2min,
Interval time 4s).It is stirred overnight at 25 DEG C (magnetic agitation, rotating speed 500rpm), centrifuges 20min under 20000g, discard supernatant,
5mL deionized waters are added into precipitation, ultrasonic disperse centrifuges 5min under 20000g, after discarding supernatant, 5mL is added in precipitation and is contained
The RGD small peptides solution (targeting group) for having 0.5wt.% amido modified, room temperature reaction overnight, centrifuges 20min under 20000g, detach
Product, be lyophilized the oil polymers hard emulsion of targeting base group modification receives microballoon, average grain diameter 97.2nm, Span are
0.281, it is placed in 4 degrees Celsius of preservations.
Above-mentioned nano-particle is redissolved, the injection of 1mg/mL is configured to using PBS.Select 6-8 week old females Balb/c
Mouse carries out tail vein injection.After injection 6 hours, using small animal imaging instrument (In Vivo Image
System.Kodak) the internal fluorescence distribution for receiving microballoon of observation injection Cy7 labels, as shown in Figure 10, in addition to part liver assembles
Except, most grease polymer hard emulsion receives microballoon and is all targeted to tumor locus, it was demonstrated that efficient tumor-targeting.
Embodiment 13
Oil polymers hard emulsion receives microballoon as vaccine adjuvant evaluation test after modification
Oil polymers hard emulsion is prepared using method in embodiment 1-6 and receives microballoon, 2.0g is accurately weighed with electronic balance
PLGA (molecular weight is 130,000 dalton), 2.0g squalenes, 0.4g bromoethyl dibasecylammonium bromides are dissolved in the ethyl alcohol of 10mL
With the mixed solution of dichloromethane (1/9), solution is poured into rapidly to the (PVA (alcoholysis containing 1.0wt.% of 100mL aqueous solutions
Degree is 99%, viscosity 5.0mPas), disperse (200W, 3min, interval time 5s) under ultrasound condition.It is stirred at 25 DEG C
Night (magnetic agitation, rotating speed 500rpm) centrifuges 20min under 15000g, discards supernatant, and 5mL deionized waters are added into precipitation,
Ultrasonic disperse centrifuges 5min under 15000g, after discarding supernatant, will precipitation freeze-drying, be stored in 4 DEG C.
By oil polymers hard emulsion obtained above receive microballoon redissolve be made 1mg/mL solution, it is entirely sick with H1N1 respectively
Malicious vaccine (a concentration of 20 μ g/100 μ L of holoprotein) (serial number 1), (hemagglutinin (HA) content is H5N1 bird flus split vaccine
4.5 μ g/100 μ L) (serial number 3), hand-foot-and-mouth disease poison (EV71) recombinant vaccine (a concentration of 10 μ g/100 μ L of holoprotein) (serial number 5),
Human nipple virus (HPV) subunit vaccine (serial number 7), melanoma personalization small peptide (MUC1, serial number 9) and anti-AIDS
(mixed volume ratio of microballoon and vaccine received is 1 for DNA vaccination (serial number 11) and mixing:1, mixed at room temperature 1 hour), separately take and equally contain
The vaccine of amount is as a contrast (serial number 2,4,6,8,10,12).After being injected to the left leg muscle of Balb/c mouse, carry out after two weeks secondary
It is immune, blood is taken in 28 days eye sockets, detaches serum, and indirect type ELISA inspections are carried out to antigen-specific antibodies IgG levels in serum
It surveys, obtains antibody titer.It puts to death mouse within 35 days, detaches each mouse boosting cell, by splenocyte in 37 degrees Celsius, 5% carbon dioxide
It is cultivated 24 hours under gnotobasis, centrifuges splenocyte, take supernatant, IFN-γ secretion level is examined using ELISA kit
It surveys.
The results are shown in Table 8, and compared with simple antigen group, oil polymers hard emulsion receives microballoon, and to be obviously improved H1N1 complete
Viral vaccine, H5N1 bird flu split vaccines, hand-foot-and-mouth disease poison (EV71) recombinant vaccine, human nipple virus (HPV) subunit epidemic disease
The antigen-specific antibodies level and IFN-γ secretion of seedling and anti-AIDS DNA vaccination are horizontal, and that there is conspicuousnesses is poor
It is different.The humoral immunity and cellullar immunologic response of antigen can be obviously improved simultaneously by demonstrating oil polymers hard emulsion and receiving microballoon,
It is had broad application prospects in vaccine adjuvant field.
Table 8
Embodiment 14
Safety evaluatio is tested:
1. vascular stimulation tests
The embodiment 1-4 hard emulsions prepared are received microballoon continuously to give once a day respectively at family's rabbit ear vein drug administration by injection
Medicine 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 note
It penetrates that blood vessel at the 1cm of position, blood vessel endothelium is continuous, complete at 5cm, has no hyperplasia, swelling;Tissues surrounding vascular has no inflammatory cell
Infiltration and necrosis;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), the embodiment 1-4 hard emulsions prepared are received into microballoon respectively with 2%
Red blood cell suspension mixes, and haemolysis and red blood cell condensation phenomenon are had no in 3 hours.
3. muscle irritation is tested
The embodiment 1-4 hard emulsions prepared are received into microballoon and are respectively used to rabbit quadriceps muscle of thigh drug administration by injection, l is administered per side
mL.It takes medicine-feeding part to visually observe after 48 hours and makees histopathologic examination.The result shows that this product to rabbit quadriceps muscle of thigh without
Irritation.
Comparative example 1
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:8nm, 20nm, 0.8 μm and 55 μm of particulate adjuvants are (such as 9 institute of table
Show).Different-grain diameter is compared for particulate adjuvants in zeta current potentials (measurement of storage stability)/ATRA embedding rates/injection 28 days
The influence of 28 days hindgut mucosal IgA antibodies titres of serum IgG antibody titre and injection afterwards.When particulate adjuvants grain size is less than
At 10nm-50 μm (second row grain size is 8nm in table 9), obtained particulate adjuvants zeta current potentials are relatively low, and it is poly- to illustrate that it is easier
Collection, and IgG titres, intestinal mucosa IgA titres are all substantially reduced in its ATRA embedding rates, serum.And work as particulate adjuvants grain
(last column grain size is 55 μm in table 9), obtained particulate adjuvants zeta when diameter is higher than 10nm-50 μm of preferred particle size range
Current potential declines, it was demonstrated that its storage stability is bad.At the same time, IgG titres, intestinal mucosa IgA drops in ATRA embedding rates, serum
Degree has apparent reduction phenomenon.This comparative example illustrates that preferred particle size range is 10nm-50 μm, is higher or lower than this
Range can all make what being substantially reduced occurred in zeta current potentials (stability), ATRA embedding rates and the immune effect of particulate adjuvants to become
Gesture.
Table 9
Applicant states that the present invention illustrates that the hard emulsion of the present invention receives microballoon and preparation method thereof by above-described embodiment
And application, 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
It can implement.Person of ordinary skill in the field is it will be clearly understood that any improvement in the present invention, to raw material selected by the present invention
The addition of equivalence replacement and auxiliary element, selection of concrete mode etc., all fall within protection scope of the present invention and the open scope it
It is interior.
Claims (10)
1. a kind of hard emulsion receives microballoon, which is characterized in that it includes biocompatibility wall material, bio-compatible that the hard emulsion, which receives microballoon,
Property grease and modification group, the kernel that the biocompatibility grease is formed is coated on the shell of biocompatibility wall material formation
Interior, the modification group is connected to the case surface.
2. hard emulsion according to claim 1 receives microballoon, which is characterized in that the biocompatibility wall material includes biofacies
Capacitive high molecular 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%.
3. hard emulsion according to claim 1 or 2 receives microballoon, which is characterized in that the biocompatible polymer material
Including poly- 'alpha '-hydroxy acids and its derivative, polyhydroxybutyrate and its derivative, polycaprolactone and its derivative, polyorthoester and
In its derivative, polyanhydride and its derivative or polybutylcyanoacrylate and its derivative any one or at least two group
It closes;
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. hard emulsion according to any one of claim 1-3 receives microballoon, which is characterized in that the biocompatibility grease
Mass percent in hard emulsion receives microballoon is 5%-80%;
Preferably, the biocompatibility grease includes 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;
Preferably, the biocompatibility grease is any one in squalene, tocopherol, castor oil or castor oil analog
Or at least two combination.
5. the hard emulsion according to any one of claim 1-4 receives microballoon, which is characterized in that the biocompatibility grease
In 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. hard emulsion according to any one of claims 1-5 receives microballoon, which is characterized in that the donor of the modification group
Including hydrophilic biological macromolecule, coupling targeting substance, fluorescent marker, isotopic label, environmental response substance, immune tune
Save in agent, pattern recognition receptors, immunopotentiator, cell factor or chemotactic factor (CF) 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, further include hydrophilic biological macromolecule in the biocompatibility grease;
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 hard emulsion according to any one of claim 1-6 receives microballoon, which is characterized in that the hard emulsion receives microballoon also
Including medical additive;
Preferably, mass fraction of the medical additive in hard emulsion receives microballoon is 0-20%;
Preferably, the medical additive includes any one in diluent, stabilizer or preservative or at least two
Combination.
8. the hard emulsion according to any one of claim 1-7 receives microballoon, which is characterized in that the hard emulsion receives microballoon
Average grain diameter is 10nm-50 μm, preferably 50nm-20 μm.
9. the hard emulsion according to any one of claim 1-8 receives the preparation method of microballoon, which is characterized in that the method
Include the following steps:Biocompatibility wall material is dissolved in include biocompatibility grease solvent in, by disperseing, solid
Change, the isolated hard emulsion receives microballoon after the modification of modification group;
Preferably, the solvent is oil phase solvent and the mixed solvent that aqueous phase solvent forms;
Preferably, the mass ratio of the oil phase solvent and aqueous phase solvent is 1:2-499;
Preferably, the oil phase solvent includes good solvent and poor solvent, volume fraction of the good solvent in oil phase solvent
For 40%-100%, volume fraction of the poor solvent in oil phase solvent is 0-60%;
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, the aqueous phase solvent includes emulsifier 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, double ten
In dialkyl dimethyl amine bromide or cetyl trimethylammonium bromide any one or at least two combination;
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 mode of the dispersion includes that ultrasonic, micro-fluidic, homogeneous, ultrasound, syringe pair push away emulsification, spraying, micro- penetrate
Stream, microchannel, film emulsification, stirring, oscillation, reverse or it is hand in any one;
Preferably, the cured method includes solvent evaporation method, physical crosslinking method, chemical crosslink technique, oxidation-reduction method, high temperature
Calcination method adjusts pH methods, adjusts any one in salinity method, extraction or the precipitation method.
10. the hard emulsion according to any one of claim 1-8 receives microballoon and is preparing vaccine adjuvant or drug delivery vehicle
In application.
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