CN1907270A - Method for preparing protein-polysaccharide vitreum slow release microsphere by using low-temperature aqueous-aqueous phase emulsion - Google Patents
Method for preparing protein-polysaccharide vitreum slow release microsphere by using low-temperature aqueous-aqueous phase emulsion Download PDFInfo
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Abstract
The invention discloses a protein-polysaccharide glass slow releasing microball preparing method of low-temperature water phase-water phase emulsion, which comprises the following steps: allocating water-phase-water-phase emulsion; freezing the emulsion; freeze-drying the sample to obtain the protein-polysaccharide glass; dispersing the micro-particle of protein-polysaccharide in the primary emulsion of oil-soluble macromolecular solution; forming water-in-oil in the water-phase; or forming primary emulsion to decompose macromolecular solution; spraying to dry; adding addictive in the gel; fitting for peptide, antibody or grease body agent.
Description
Technical field
What the present invention relates to is a kind of preparation method of biological technical field.Particularly a kind of low temperature aqueous phase-aqueous phase emulsion prepares the method for protein-polysaccharide vitreous sustained-release micro-spheres.
Background technology
Because absorbent properties are poor, the interior half-life of body is short, most pharmaceutical grade protein need pass through frequent drug administration by injection.In order to reduce the frequency of this injection, since the 1970's, the research and development of the slow release formulation of this class medicine have attracted a large amount of concerns of this area scientist, are still the problem of still tackling key problems so far.Yet a large amount of scientific researches drops into and does not obtain expected result.Up to the present, (human growth hormone of unique slow release protein drug dosage form-reorganization listing is just counted out soon, has withdrawn from market the product of a success as yet.Technology impregnability main obstacles of a specified duration is the protein stability problem in the dispose procedure and intravital prominent releasing and not exclusively release in preparation process and body.
In the past few decades, saw various reports in the document about the method that improves the stability of protein in the microcapsule packet procedures.But these methods can only solve one or a part of problem, and can not take into account for other problems, even cause the problem that makes new advances.Also some method only is applicable to specific a kind of protein.The different mutual contradictions that seem of research field that also have some reports also self to be paid close attention to owing to researcher.The explanation of giving an example, for unique like product on the market--controlled-release recombinant human class growth hormone (rhGH), its stability by and zinc form complex and realize.And form complex with zinc is rhGH natural form in vivo.When this zinc complexation preparation method is used for another kind of albumen, as erythropoietin (EPO), accumulative ratio takes place protein molecular can cause to cause immunogenic misgivings up to 40%.For avoiding albumen in the microcapsule packet procedures, to be out of shape inactivation because of removing organic solvent; scientist is made as solid granular with protein molecular and saccharide, inorganic salt or other protective agents in advance; thereby take a kind of solid dispersion at oil phase; (promptly oil wraps solid-oil-in-water, method microcapsule bag protein drug S/O/W) further being dispersed in water.These protein stabilizedization adjuvants cause prominent releasing usually owing to its high-dissolvability produces strong osmotic pressure.Such as, when the high sulfate of dissolubility is used for improving EPO when the element of microcapsule packet procedures is stable, prominent releasing up to 55% of accumulated dose.
[Cleland, J.L., Jones J.S., Stable formulations of recombinant humangrowth hormone and interferon-gama for microencapsulation inbiodegradable microspheres.Pharm.Res., 1996.13:1464-1475.] (Cleland, J.L., Jones J.S., stable recombinant human growth element and an ancient woman's ornament interferon dosage form are used for biodegradable microsphere microencapsulation. study of pharmacy, 1996, the 13rd phase, the 1464-1475 page or leaf); Cleland and Jones have studied in W-O-W (Water-In-Oil-oil-in-water) and S-O-W microencapsulation process various adjuvants to the protective effect of recombinant human growth hormone and interferon, find that mannitol and trehalose can play best effect aspect this preventing that albumen from assembling in the microcapsule process.Sanchez has verified similar excipient for another kind of albumen---and the protective effect of tetanus fermentoid element found that to be identified as in the report of Cleland and Jones and can play the better preserved function to protein in the release stage under the hydrating condition to restoring the glucosan that recombinant human growth hormone and interferon do not play useful effect.As if such experiment has illustrated that monosaccharide can play the better protection effect to albumen in dry run, and macromolecular compound then plays better effect in the release stage.But, the release profiles of the glucosan of Sanchez-PLGA complex microsphere slow release formulation shows that 60% drug loading has taken place by prominent releasing.Perhaps, this prominent release is because oversize the causing of albumen-granules of accessories of direct lyophilization preparation.
In the S-O-W process, the size of the protein body of pre-preparation is very important.[Takahiro.Morita, Yuji Horikiri, Hiroshi Yamahara, Takehiko Suzuki, and Hiroyuki Yoshino.Formation and Isolation of Spherical fine protein Microparticles throughLyophilization of protein-poly (ethylene glycol) Aqueous Mixture.Pharm.Res., 17:1376-1373 (2000)], (Takahiro.Morita, Yuji Horikiri, HiroshiYamahara, Takehiko Suzuki, and Hiroyuki Yoshino. prepares the ball-type protein microsphere by freezing albumen and Polyethylene Glycol water mixed solution, study of pharmacy, 2000, the 17th phase, the 1376-1373 page or leaf); Morita find when the particulate diameter of solid protein matter when 5 microns increase to 20 microns, not only the rate of release at initial stage is double, its microencapsulation rate drops to 20% from 80%.Cleland has discussed at S-O-W and has crossed the method that the Cheng Qian dwindles protein microbeads volume size.Further pulverize the protein lyophilized powder and can not obtain diameter, grind the protein change that heat causes and the method that these powder grind to form littler size is hampered by less than 10 microns microgranule.Though the atomizing seasoning can obtain expecting the protein microbeads of size, but the heat of shear force therebetween and generation is understood owing to causing property of the intervention change that the gas liquid interface has been arranged.Spray drying method perhaps can provide enough little protein body, but high shear force and the interfacial tension between liquid-to-air near the nozzle can make albuminous degeneration.What more be far from good is, must use surfactant in spray drying or the atomizing lyophilization, and surfactant has and facilitated protein to interact with solvent in next step preparation program.Maa etc. have reported the gathering that in advance method of recombinant human growth hormone and zinc complexation was prevented protein component before spray drying.But zinc complexation meeting causes growth hormone albuminous degeneration in addition.The method of usefulness PEG precipitating proteins such as Motita has prepared the albumen ultramicro powder.But these microgranules still directly are under the effect of organic solvent in the microcapsule packet procedures.Because protein is not subjected to the protein of any protection and direct contact of PLGA can cause incomplete release for the adsorption on the surface, inside of macromolecular compound substrate.For fear of hydrophilic-hydrophobic separating surface, prepare the macromolecular compound microgranule with the hydrophilic diphasic system.Yet, must lean on covalency or the synchronous crosslinked action of ion to come cured granulate at emulsifying stage, thereby protein is exposed in the active reaction thing.
The exploitation of the slow release formulation of the protein macromolecule medicine of fragile structure needs a technical scheme that can comprehensively solve above-mentioned all problems and simple and easy to do (such as not relying on the liquid nitrogen preparation).Find by prior art documents, find [CHEN, Li by prior art documents; ZHU, Hua; JIN, Tuo; THE PREPARATION METHOD OF A STABLE POLYMER AQUEOUS PHASE-AQUEOUS PHASEEMULSION AND ITS USE, Publication Number International:WO/2002/000778, Application No.:PCT/CN2001/001033] (Chen Li; Zhu Hua; Jin Tuo; The preparation of stable polymer aqueous phase-aqueous phase emulsion and application thereof, publication number is, WO/2002/000778, international application no is PCT/CN2001/001033), this patent has disclosed a kind of novel stable emulsion (polymer aqueous phase-aqueous phase emulsion) and preparation method thereof and in pharmacy and Application in Biotechnology.The fundamental difference of this emulsion and conventional emulsions is that its decentralized photo and continuous phase are aqueous solution.Promptly produce diffuse double layer, thereby developed the material system of a uniqueness: stable do not need continuous stirring or (to decentralized photo) to solidify macromolecule water-water Emulsion immediately by polyelectrolyte being introduced two systems of high molecular weight hydrophilic (aqueous two-phase system).This system is used for the preparation of protein macromolecule medicament slow-release microsphere dosage form, has obtained ideal results.Yet some protein molecular may interact with polyelectrolyte, causes gathering.
Summary of the invention
The objective of the invention is to overcome deficiency of the prior art, provide a kind of low temperature aqueous phase-aqueous phase emulsion to prepare the method for protein-polysaccharide vitreous sustained-release microparticle.Make it prepare the method for protein-polysaccharide vitreous particle without organic solvent and surfactant, the structure and the activity of protein, polypeptide, DNA, RNA, anti-RNA, vaccine, antibody, liposome and virus etc. can be preserved effectively, and the requirement of clinical practice slow release can be reached effectively.
The present invention is achieved by the following technical solutions, and a kind of low temperature aqueous phase-aqueous phase emulsion prepares the method for protein-polysaccharide vitreous sustained-release micro-spheres.The present invention includes following steps:
1. will wait and polysaccharide wiring solution-forming soluble in water, albumen is added polysaccharide solution (as decentralized photo) and is prepared into aqueous phase-aqueous phase emulsion with the immiscible water soluble polymer of polysaccharide solution [Polyethylene Glycol (PEG), poly(ethylene oxide) (PEO) or polypyrrole alkane ketone (PVP), ficoll (Ficoll), polyethylene-ethylene acetate copolymer (PEV) or polyvinyl alcohol (PVA) or their combination are hereinafter referred to as continuous phase] solution being lower than under the temperature that room temperature is higher than freezing point;
2. completing steps aqueous phase-aqueous phase emulsion 1. is freezing;
3. with completing steps sample lyophilizing 2.;
4. completing steps sample 3. being removed PEG, PEO or PVP continuous phase with solvent wash obtains protein-polysaccharide vitreous.
5. form colostrum (S/O) by albumen-polysaccharide vitreous particle being dispersed in the molten macromolecular solution of oil, redispersion forms the i.e. method of oil bag solid-oil-in-water (S/O/W) of emulsion at aqueous phase, perhaps form colostrum (S/O) by polysaccharide vitreous particle being dispersed in the molten degradable macromolecule solution of oil, redispersion forms the i.e. method of oil bag solid-oil bag oil (S/O/O) of emulsion in another oil phase, perhaps, perhaps add in the various gels by the spray drying method for preparation microsphere.
Described albumen-polysaccharide vitreous is characterized by protein 0.01%-50%, polysaccharide is 0.01%-50%, protective agent 0%-10%, and Polyethylene Glycol, poly(ethylene oxide) or polypyrrole alkane ketone are 20%-80%.
So-called low temperature aqueous phase-aqueous phase emulsion method is meant at-7 ℃-10 ℃, with 0 ℃-4 ℃ is good, proteoglycan solution and Polyethylene Glycol, poly(ethylene oxide) (PEO) or polypyrrole alkane ketone (PVP) solution mixed forming emulsion, freeze overnight then is again in vacuum lyophilization.Sample after the vacuum lyophilization also obtains the albumen-polysaccharide vitreous particle of 0.5-5 micron equally with organic solvent flush away PEG continuous phase.
Described albumen-polysaccharide vitreous contains buffer substance, salt and micromolecule saccharide material, and its concentration is below the 0-10% of percentage by weight of polysaccharide; Being good less than 1-5%.
Described buffer substance is Mg (OH)
2, ZnCO
3, and MgCO
3, salt is Zn (AC)
2, ZnCl
2, CaSO
4, MgSO
4, the micromolecule saccharide is trehalose, sucrose, glucose, sorbitol, mannitol and their combination.
Described macromolecule refers to the polyamino acid of polyester, poly-anhydride or skeleton non-peptide bond, comprising: polylactic acid, polylactic acid-polyglycolic acid and combination thereof; Comprise that also silicon is as glue, politef, polrvinyl chloride, polyethylene, polypropylene, polystyrene, polyethylene terephthalate, Merlon, Carmomer, hyaluronic acid, gelatin, collagen protein, poly-Acetic acid, hydroxy-, bimol. cyclic ester, polycaprolactone, polybutylcyanoacrylate, poly phosphazene, poly phosphate, Fibrinogen, fibrin and their combination, gel comprises: polyethylene glycol-lactic acid, the polyethylene glycol-hydroxyacetic acid, polyglycolic acid-polyethylene glycol-hydroxyacetic acid, polylactic acid-polyglycol-polylactic acid temperature-sensitive hydrogel, quick type gel of sugar and acid-sensitive type gel, their combination.
Described protein is: erythropoietin (EPO), recombinant human granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimutaing factor (GM-CSF), vaccine, interferon (INF), growth hormone (GH), insulin (Insulin), epidermal growth factor (EGF), fibroblast growth factor (FGF), transforming growth factor (TGF-β), insulin like growth factor (IGF), vascular endothelial cell growth somatomedin (VEGF), PDGF (PDGF), endothelial cell growth factor (ECGF) (ECGF), nerve growth factor (NGF), bone-derived growth factor (BDGF), bone morphogenetic protein(BMP) (BMP), tissue polypeptide antigen (TPA), antibody (antibody), blood coagulation factor VIII (VIII) and IX genetic factor and the protein or polypeptide and their combination that are used for the treatment of.
Reagent beyond the described albumen is: antisense nucleotide (anti-RNA), microRNA (RNAi), gene (DNA), antibody, vaccine, liposome or their combination.
Described microsphere is characterized by protein 0.01%-50%, polysaccharide is that 0.01%-50%, protective agent 0%-10%, macromolecule are 20%-80%, and its particle diameter is 1 micron-120 microns.
Described temperature sensitive type gel is characterized by the albumen that is stated from albumen-polysaccharide vitreous particle or the polysaccharide solution and optionally is distributed in polysaccharide and is on the thermodynamics more stable status in mutually.In addition, the temperature sensitive type gel ties up in intensification phase transformation (from liquid to the glue) process prominent the releasing that syneresis causes is taken place, and is distributed in the albumen of polysaccharide in mutually and can avoids to a great extent then that this class is prominent to be released.Described polysaccharide vitreous or contain proteic polysaccharide solution and can be introduced directly into the temperature sensitive type gel that is in liquid condition under the low temperature or their combination without emulsifying is freeze dried.
Described protein-polysaccharide vitreous or can be solution state or nanoparticle state without the freeze dried albumen that contains in the proteic polysaccharide solution of emulsifying.
Described polysaccharide vitreous except protected protein in the preparation process; also have by the prepared polysaccharide vitreous particle of the method among the present invention and under body temperature and hydrating condition, to keep protein active for a long time, and improve the effect of albumen release dynamics.
Also can be earlier in the described low temperature aqueous phase-aqueous phase emulsion method adding polyvalent metal ion in protein solution makes it to become microgranule to be prepared into albumen-polysaccharide vitreous particle by lyophilization and flush away continuous phase again in mutually adding polysaccharide to the protein molecular complexation.
The invention provides a kind of easy and effective scheme, the medium-term and long-term technical barrier that exists of R﹠D process of protein macromolecule slow release formulation is better solved in the lump.
The specific embodiment
The primary condition of implementing: the protein-polysaccharide vitreous preparation and the preparation of microsphere or year protein-polysaccharide vitreous responsive type preparing gel.
Embodiment 1: the preparation of microsphere
1. preparation albumen-polysaccharide vitreous
Prepare 10% Polyethylene Glycol and 10% polysaccharide solution respectively with ultra-pure water, accurately taking by weighing 10 gram PEG and 10 gram polysaccharide water is placed on respectively in 100 milliliters the beaker and adds water 90 grams, then two beakers are placed on the magnetic agitation 30min of heating, the polysaccharide for the treatment of PEG and 10% all dissolving to take off cooling stand-by.Accurately taking by weighing 800 milligrams of albumen with electronic balance is dissolved in 7.2 ml waters stand-by.
1. under 0 ℃ of-4 ℃ of condition, above-mentioned polysaccharide, albumen and PEG aqueous solution are respectively 1: 1: 5,1: 1: 10,1: 1: 20,1: 1: 40 at the abundant mixing of cillin bottle vortex 30s-60s according to volume ratio, form aqueous phase-aqueous phase emulsion; Or to take by weighing mass ratio according to polysaccharide and PEG be to be dissolved in water at cillin bottle elder generation mixing in 1: 1: 5,1: 1: 10,1: 1: 20,1: 1: 40 again, is to add at 1: 1 according to polysaccharide and proteic weight ratio again, makes it to form aqueous phase-aqueous phase emulsion.
2. pre-freeze albumen, PEG and polysaccharide aqueous phase-aqueous phase emulsion
To prepare albumen, PEG and polysaccharide and form aqueous phase-aqueous phase emulsion, some are not assigned to the protein of polysaccharide phase, are redistributed in the polysaccharide decentralized photo freeze overnight in temperature-fall period.
3. completing steps sample 2. is in the vacuum freeze drier lyophilizing;
4. completing steps sample is 3. removed PEG continuous phase for three times with washed with dichloromethane and is obtained to carry the proteoglycan vitreous body.
The albumen that obtains-polysaccharide vitreous particle diameter obtains the smooth rounding of these vitreous body mostly at 300nm-5 μ m, and particle size distribution is even.The protection that proteinic structure is obtained has been avoided at dosage form preparation process inactivation.
2. be loaded with protein-polysaccharide vitreous microsphere preparation
Take by weighing PVA and the NaCl of 1g and 5g, add ultra-pure water 94g, heat simultaneously, stir,, stop heating, continue slowly to stir to the PVA complete swelling, treat that solution is clear and does not have bubble after, stop to stir, it is stand-by to be put in 4 ℃ of refrigerators.This solution concentration is 1%PVA (W/W) and 5%NaCl (W/W).
Accurately take by weighing polylactic acid-polyglycolic acid (PLGA) 200mg, the 980mg that adds methylene chloride, vortex oscillation is dissolved in the dichloromethane PLGA, gets the dichloromethane solution of PLGA, and concentration is 20% (W/W), and matching while using is in case the dichloromethane volatilization.
Take by weighing protein-polysaccharide vitreous particle 10mg, be dispersed in the dichloromethane solution of PLGA of 0.5g, the concentration of PLGA is 20% (W/W), magnetic agitation 15 minutes, rotating speed 1800rpm, fully stirring makes protein-polysaccharide vitreous particle be dispersed in the PLGA solution, the gained colostrum is scattered in the solution that contains 1%PVA and 10% sodium chloride once more, rotating speed homogenate with 2200rpm becomes emulsion (S/O/W), in one minute, transfer in 4 ℃ 10% sodium-chloride water solution, stir and collect aged protein microsphere after 3～4 hours, with ultra-pure water washing three times, spend the night ice phase pre-freeze, vacuum lyophilization again, making particle diameter is the microsphere of 50 μ m-120 μ m.
The microsphere of preparation can be used for subcutaneous injection, plays slow release and therapeutical effect.
Embodiment 2: preparation controlled release and slow release PLGA microsphere
One, preparation albumen-polysaccharide vitreous according to example 1 method
Two, be loaded with albumen-polysaccharide vitreous microsphere preparation
Take by weighing polylactic acid-polyglycolic acid (PLGA) 100mg, make it fully to be dissolved under the effect of eddy mixer in the acetonitrile of 1ml, a step granule that takes by weighing 10mg then adds in the solution of acetonitrile, and fully disperses under magnetic agitation.The surfactant span80 that outside a certain amount of, adds 200ml Oleum Gossypii semen in the oil-phase solution, abundant mixing under motor stirrer stirs with 200rpm.Dropwise add in the outer oil-phase solution in the stirring being dispersed with particulate acetonitrile solution of a step, stir and solidified 5 hours, sucking filtration, the microsphere particle that obtains is given a baby a bath on the third day after its birth inferior with the 50ml petroleum ether, drying under reduced pressure 10 hours, collect microsphere, the particle diameter of observing microsphere at existing micro mirror is the microsphere of 50 μ m～120 μ m, meets hypodermic requirement.Can also controlled condition make 1 μ m-10 μ m and be used for bacterin preparation.
The microsphere of preparation can be used for subcutaneous injection, plays slow release and therapeutical effect.
Embodiment 3: be loaded with albumen-polysaccharide vitreous temperature sensitive type preparing gel
One, preparation is carried the proteoglycan vitreous body according to example 1 method
Two, being loaded with albumen-polysaccharide vitreous temperature-sensitive liquid-glue system is equipped with
Take by weighing albumen-polysaccharide vitreous particle 10mg, in the aqueous solution of 4 ℃ of PLGA-PEG-PLGA that are dispersed in 0.5g, the concentration of PLGA-PEG-PLGA is 20% (W/W), magnetic agitation 15 minutes, rotating speed 1800rpm, fully stirring makes albumen-polysaccharide vitreous particle be dispersed in the PLGA-PEG-PLGA solution, and the suspension that gained is dispersed in PLGA-PEG-PLGA is heated to 37 ℃ of curing, does release in vitro.Clinical practice is then now with the current.
Embodiment 4: the microsphere that is loaded with albumen-polysaccharide vitreous gel
One, preparation is carried the proteoglycan vitreous body according to example 1 method
Two, be loaded with the microsphere of albumen-polysaccharide vitreous hydrogel
The macromolecular material that we have also synthesized two block mPEG (5K)-PLGA, three block PLGA-PEG (6K)-PLGA, PEG-DTC, PEG-ε-CL ourselves has prepared the hydrogel particle that has loaded GL-PP.We find that the time that discharges shortens and rate of release is accelerated.
Concrete preparation method is as follows:
The preparation of 1 solution
(1) PVA, NaCl aqueous solution
Take by weighing a certain proportion of PVA and NaCl, add ultra-pure water, heat simultaneously, stir,, stop heating, continue slowly to stir to the PVA complete swelling, treat that solution is clear and does not have bubble after, stop to stir, it is stand-by to be put in 4 ℃ of refrigerators.This solution concentration is 1%PVA (W/W) and 5%NaCl (W/W).
(2) dichloromethane solution of PLGA
Respectively weighing m PEG (5K)-PLGA, three block PLGA-PEG (6K)-PLGA, PEG-DTC, PEG-ε-CL are an amount of, it is an amount of to add methylene chloride, vortex oscillation, make them be dissolved in the dichloromethane respectively, get the dichloromethane solution of mPEG (5K)-PLGA, three block PLGA-PEG (6K)-PLGA, PEG-DTC, PEG-ε-CL, concentration is 20% (W/W), and matching while using is in case the dichloromethane volatilization.
The preparation of 2 microspheres
Take by weighing albumen-polysaccharide vitreous particle 10mg, be dispersed in the above-mentioned dichloromethane solution of 0.5g, mPEG (5K)-PLGA, three block PLGA-PEG (6K)-PLGA, PEG-DTC, the concentration of PEG-ε-CL is 20% (W/W), magnetic agitation 15 minutes, rotating speed 1800rpm, fully stirring makes albumen-polysaccharide vitreous particle be dispersed in mPEG (5K)-PLGA, three block PLGA-PEG (6K)-PLGA, PEG-DTC, in PEG-ε-CL solution, the gained colostrum is scattered in the solution that contains 1%PVA and 10% sodium chloride once more, rotating speed homogenate with 2200rpm becomes emulsion (S/O/W), in one minute, transfer in 4 ℃ 10% sodium-chloride water solution, stir and collect aged protein microsphere after 3～4 hours, with ultra-pure water washing three times, spend the night ice phase pre-freeze, vacuum lyophilization again, making particle diameter is the microsphere of 50 μ m～120 μ m.
Embodiment 5: the preparation of microsphere
One, preparation is carried the proteoglycan vitreous body according to example 1 method
Two, be loaded with albumen-polysaccharide vitreous microsphere preparation
It is an amount of accurately to take by weighing PLGA, and it is an amount of to add methylene chloride, and vortex oscillation is dissolved in the dichloromethane PLGA, gets the dichloromethane solution of PLGA, and concentration is 20% (W/W), and matching while using is in case the dichloromethane volatilization.Take by weighing albumen-polysaccharide vitreous particle 10mg, be dispersed in the dichloromethane solution of PLGA of 0.5g, the concentration of PLGA is 20% (W/W), magnetic agitation 15 minutes, rotating speed 1800rpm, fully stirring makes albumen-polysaccharide vitreous particle be dispersed in the PLGA solution, then by the spraying instrument, it is sprayed onto in liquid nitrogen and the ethanol, again by temperature programming sclerosis microsphere.The microspherulite diameter of preparation is the microsphere of 50 μ m～120 μ m.
Use
The therapeutant of protein, liposome, virion equistability difference can be avoided factor affecting such as organic solvent, concentrated salt, extreme pH value, cross-linking agent, strong shearing force, high temperature and high interfacial tension equally, be encapsulated in the decentralized photo vitreous body microdroplet by two alternate distribution, after further handling by lyophilization or other drying meanss, form the particle size distribution homogeneous, vitreous body microsphere closely, (can be used for protein drug sucks or slow release is carried).Employed adjuvant all is that human body can be injected in the preparation process.The protein-polysaccharide vitreous of preparation can be directly used in inhalant dosage form, the biological reagent such as the medicines such as protein and polypeptide of protection fragile structure; Microsphere and be loaded with protein-polysaccharide vitreous temperature-sensitive hydrogel and can be directly used in clinical subcutaneous injection administration or implant subcutaneous the proteinic effect of slow controlled release.Albumen-polysaccharide microgranule is further used for protein drug coating bracket, protein drug sustained-release matrix.Polysaccharide mutually not only can be in the preparation process protected protein, can also be used to improving its release dynamics.For the microsphere of given albumen loading, the consumption of polysaccharide be one easily factor can be used to the rate of release and the release mode of modulin molecule.Increase the consumption of polysaccharide, then rate of release improves, and not exclusively the problem that discharges improves, but the prominent probability of releasing increases.The consumption that reduces polysaccharide then plays reverse effect.The consumption of carefully selecting polysaccharide can be able to reach in conjunction with the molecular weight of degradable macromolecule and hydrophilic-hydrophobic simultaneously both having controlled prominent releasing, and avoided the purpose that not exclusively discharges again.
Claims (8)
1, a kind of low temperature aqueous phase-aqueous phase emulsion prepares the method for protein-polysaccharide vitreous sustained-release micro-spheres, it is characterized in that, may further comprise the steps:
1. will wait and polysaccharide wiring solution-forming soluble in water, with albumen add polysaccharide solution as decentralized photo and with the immiscible water soluble polymer of polysaccharide solution as being prepared into aqueous phase-aqueous phase emulsion being lower than under the temperature that room temperature is higher than freezing point for continuous phase solution;
2. completing steps aqueous phase-aqueous phase emulsion 1. is freezing;
3. with completing steps sample lyophilizing 2.;
4. completing steps sample 3. being removed Polyethylene Glycol, poly(ethylene oxide), polypyrrole alkane ketone, ficoll, polyethylene-ethylene acetate copolymer, polyvinyl alcohol or their combination continuous phase with solvent wash obtains protein-polysaccharide vitreous;
5. form colostrum by albumen-polysaccharide vitreous particle being dispersed in the molten macromolecular solution of oil, redispersion forms the i.e. oil bag solid-oil-in-water method of emulsion at aqueous phase, perhaps form colostrum by polysaccharide vitreous particle being dispersed in the molten degradable macromolecule solution of oil, redispersion forms the i.e. method of oil bag solid-oil bag oil of emulsion in another oil phase, perhaps, perhaps add in the various gels by the spray drying method for preparation microsphere.
2. low temperature aqueous phase-aqueous phase emulsion according to claim 1 prepares the method for protein-polysaccharide vitreous sustained-release micro-spheres, it is characterized in that, described low temperature aqueous phase-aqueous phase emulsion method is meant at-7 ℃-10 ℃, proteoglycan solution and Polyethylene Glycol, poly(ethylene oxide) or polypyrrole alkane ketone solution are mixed the formation emulsion, freeze overnight then, in vacuum lyophilization, the sample after the vacuum lyophilization also obtains albumen-polysaccharide vitreous particle of 0.5 micron-20 microns equally with organic solvent flush away PEG continuous phase again.
3, the method for preparing protein-polysaccharide vitreous sustained-release micro-spheres according to claim 1 or 2 described low temperature aqueous phase-aqueous phase emulsions, it is characterized in that described polysaccharide is glucosan, starch, cellulose and derivant thereof, agarose and water soluble polymer polysaccharide or their combination.
4, the method for preparing protein-polysaccharide vitreous sustained-release micro-spheres according to claim 1 or 2 described low temperature aqueous phase-aqueous phase emulsions, it is characterized in that, described protein is: described protein is: erythropoietin, recombinant human granulocyte colony stimulating factor, granulocyte-macrophage colony stimutaing factor, vaccine, interferon, growth hormone, insulin, epidermal growth factor, fibroblast growth factor, transforming growth factor, insulin like growth factor, the vascular endothelial cell growth somatomedin, PDGF, endothelial cell growth factor (ECGF), nerve growth factor, bone-derived growth factor, bone morphogenetic protein(BMP), tissue polypeptide antigen, antibody, blood coagulation factor VIII and IX genetic factor and the protein or the polypeptide that are used for the treatment of, or their combination; Protein is that 0.01%-50%, polysaccharide are 0.01%-50%, and protective agent 0%-10%, Polyethylene Glycol are 20%-80%; Treatment reagent is antisense nucleotide, microRNA, gene, antibody, vaccine, liposome or their combination;
5, the method for preparing protein-polysaccharide vitreous sustained-release micro-spheres according to claim 1 or 2 described low temperature aqueous phase-aqueous phase emulsions; it is characterized in that; described protective agent is trehalose, mannitol, sucrose, lactose, glycerol or their combination, and metallic compound comprises the ionic compound of zinc, copper, magnesium, ferrum, calcium or their combination.
6. low temperature aqueous phase-aqueous phase emulsion according to claim 1 prepares the method for protein-polysaccharide vitreous sustained-release micro-spheres, it is characterized in that the albumen that described microsphere supported, polypeptide, DNA, RNA, anti-RNA, antibody or liposome have slow release, controlled-release function;
7. freezing being separated according to claim 1 prepares the method for slow released microsphere of protein-polysaccharide vitreous particle; it is characterized in that; described microsphere is characterized by protein 0.01%-50%, polysaccharide is that 0.01%-50%, protective agent 0%-10%, macromolecule are 20%-80%, and its particle diameter is 1 micron-120 microns.
8. low temperature aqueous phase-aqueous phase emulsion according to claim 1 prepares the method for protein-polysaccharide vitreous sustained-release micro-spheres, it is characterized in that, described macromolecule is the polyamino acid of polyester, poly-anhydride or skeleton non-peptide bond, comprising the polyamino acid that comprises polyester, poly-anhydride or skeleton non-peptide bond, comprising: polylactic acid, polylactic acid-polyglycolic acid and combination thereof; Comprise that also silicon is as glue, politef, polrvinyl chloride, polyethylene, polypropylene, polystyrene, polyethylene terephthalate, Merlon, Carmomer, hyaluronic acid, gelatin, collagen protein, poly-Acetic acid, hydroxy-, bimol. cyclic ester, polycaprolactone, polybutylcyanoacrylate, poly phosphazene, poly phosphate, Fibrinogen, fibrin and their combination, gel comprises: polyethylene glycol-lactic acid, the polyethylene glycol-hydroxyacetic acid, polyglycolic acid-polyethylene glycol-hydroxyacetic acid, polylactic acid-polyglycol-polylactic acid temperature-sensitive hydrogel and responsive type gel and their combination.
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| WO2008141496A1 (en) * | 2007-05-23 | 2008-11-27 | Tuo Jin | Sustained-release system for epo and gm-csf |
| CN102144977A (en) * | 2011-04-14 | 2011-08-10 | 上海交通大学 | Preparation method of growth hormone nanoparticles with biological activity |
| CN102178654A (en) * | 2011-05-04 | 2011-09-14 | 深圳赛保尔生物药业有限公司 | Sustained-release microspheres and preparation method thereof |
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| WO2008141496A1 (en) * | 2007-05-23 | 2008-11-27 | Tuo Jin | Sustained-release system for epo and gm-csf |
| CN102144977A (en) * | 2011-04-14 | 2011-08-10 | 上海交通大学 | Preparation method of growth hormone nanoparticles with biological activity |
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| CN104208661B (en) * | 2013-09-29 | 2016-04-06 | 郑州后羿制药有限公司 | A kind of pig interleukin-16 injection long-acting slow-release microball preparation and preparation method thereof |
| CN106511315A (en) * | 2016-12-27 | 2017-03-22 | 苏州科技大学 | Degradable polymer core-shell microsphere loaded with protein drugs |
| CN107158752A (en) * | 2017-05-25 | 2017-09-15 | 中原工学院 | A kind of responsive to temperature type keratin base inhales the preparation method for releasing light wood material |
| CN107158752B (en) * | 2017-05-25 | 2019-04-12 | 中原工学院 | The preparation method of light wood material is released in a kind of responsive to temperature type keratin base suction |
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| CN110237046A (en) * | 2019-03-29 | 2019-09-17 | 福格森（武汉）生物科技股份有限公司 | A kind of preparation method of L-5- methyl tetrahydrofolate micro-capsule |
| CN110237046B (en) * | 2019-03-29 | 2021-07-30 | 福格森（武汉）生物科技股份有限公司 | Preparation method of L-5-methyltetrahydrofolic acid microcapsules |
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