Polysaccharide polymer having protein large molecule and preparation method thereofTechnical field
What the present invention relates to is polymkeric substance of a kind of biological technical field and preparation method thereof.A kind of polysaccharide polymer having protein large molecule and preparation method thereof particularly.
Background technology
Bio-compatibility, degradability polymkeric substance have been widely used for disease treatment and other association area.With this class material tempting application prospect that combines with the biotech drug of high speed development in recent years, also run into how to keep the fragile relatively biotech drug of structure low technical bottleneck of stability in this class material simultaneously.Compare with chemical small-molecule drug, protein macromolecule has that specificity is stronger, the selectivity better therapeutic, yet its fragile variable structure becomes in the biomedical material preparation problem to be solved is arranged.The biomedical material that particularly comprises tissue engineering material usually needs to become different shape or coating in the surface of different therapeutic device according to the difference of therapeutic purpose with different prepared.
About the stability of the protein macromolecule of polymeric encapsulate, ideal method is that protein molecular is prepared into solia particle in advance, makes its structure look like to fix.Yet, protein macromolecule is made solia particle and to be not easy when not changing the structure picture, the method for having reported all has various deficiencies.Such as, the method for the mixing solutions of albumen and small molecules carbohydrate or macromolecular polysaccharide being made lyophilized powder is difficult to make the particle of particle diameter less than 10 μ m.The solution spray of albumen and carbohydrate can be run into the high temperature and the high shear force of nozzle when dry.With the solution of albumen and carbohydrate spray into liquid nitrogen then freeze-drying also can run into high shear force, and formed particle is that density is lower than 0.5 vesicular structure, is unfavorable for the protection to protein macromolecule.Protein macromolecule and divalent-metal ion effect are precipitated as the ionic linkage particulate then can cause some proteic irreversible aggrengations.Add inorganic salt or PEG in protein solution, silver protein is separated out when becoming particulate, the former is difficult to remove salt, though and the latter's PEG can remove protein particle and directly contacts polymkeric substance and also can cause irreversible precipitation.
Coat at medical apparatus surface and to add therapeutic scheme in the functional materials film of medicine carrying or the tissue engineering material launch has been arranged.But the biotherapy reagent of coating the film of biologically active or add biologically active in tissue engineering material at medical apparatus surface does not also have product to come out so far, mainly be since biotherapy reagent at easy inactivation of preparation process such as protein, unavoidably to touch organic reagent, high shear force, water-oil interface in preparation process and cause proteinic sex change inactivation, run into local overacidification again at dispose procedure and cause the sex change inactivation, also have a series of problems such as not exclusively release.Although having at present on the surface of cardiac stent the surface that antibody is fixed on support with chemical method catch on the surface of endotheliocyte at support grows; With coat endothelial cell growth factor (ECGF) (VEGF) on its surface and come on the surface of stimulating endothelial cell at support growth to prevent vascular smooth muscle cell curing, but prominent release serious and drug loading too low, also far do not reach the clinical treatment requirement.Also have quiding gene in blood vessel, promote endotheli ocytosis and thrombolysis, the propagation of inhibition smooth muscle cell etc. might reach the purpose and the Klugherz.B.D. of control restenosis, wait the people to study and report controlled release DNA-PLGA cardiac stent.Main gene type has 1. antithrombotic gene, as reorganization t-PA gene; 2. the gene of vaso-active substance is as reorganization NOS gene; 3. the gene of somatomedin and cytokine is as anti-meaning RNA or the DNA of VEGF gene, PDGF; 4. oncogene and antioncogene, as the anti-meaning Ras oncogene etc. of recombinating; 5. Cycle Regulation gene is as Cyclin, CDK and ICE, Bax etc.Gene therapy method commonly used is: therapeutic transgene (comprises; Negative dominant gene treatment, inhibition therapeutic transgene, prodrug gene therapy); Antisense nucleic acid medicament treatment (comprising: antisense oligonucleotide, antisense mRNA etc.).The gene coating support is to adopt by genetic transcription mostly, and as being that carrier carries goal gene to lesion with adenovirus, plasmid DNA etc., target protein matter is synthesized in genetic expression.But because the transfection efficiency and the safety problem of gene, gene therapy now also is in the experimental study stage.
Find [Hennink W.E.and Franssen D. (EN) PROCESS FOR THE PREPARATION OF A CONTROLLED RELEASE SYSTEM (FR) PROCEDEDE PREPARATION D ' UN SYSTEME A LIBERATION RETARDEE by prior art documents.Publication No.:International Application No.:PCT/NL1997/000625]. (Hennink W.E.andFranssen D. method for preparing the controlled release place system, publication number is WO/1998/022093, international application no PCT/NL1997/000625) Hennink W.E. reported that water-water biphasic system prepares polyoses grain.But they have used polysaccharide that can be crosslinked, and a little crosslinked groups be to obtain by chemically modified.So not only can change the character of polysaccharide own, might increase the immunogenicity of polysaccharide, these crosslinked groups also may be crosslinked with protein and polypeptide generation simultaneously, because crosslinked group does not have special selectivity.But also do not see be loaded with proteinic polysaccharide vitreous-report of polymkeric substance.
The content of invention
The objective of the invention is to overcome deficiency of the prior art, a kind of polysaccharide polymer having protein large molecule and preparation method thereof is provided.Make it work as protein drug need be used for interventional therapy the time, how to make this class macromole under different processing condition, remain natural structure picture, the protein macromolecule physical mixed is in the matrix of this class wetting ability polysaccharide particulate simultaneously, avoid the interaction with the polymkeric substance that is used to seal, do not caused proteic gathering or other reaction.
The present invention is achieved by the following technical solutions, polysaccharide polymer having protein large molecule of the present invention contains the reagent of polysaccharide, protein, polymkeric substance, protected protein matter, when preparation concrete protein polysaccharide-polymkeric substance, select different polysaccharide, protein, polymkeric substance, protection reagent according to concrete needs; Polysaccharide is that 0.01%-50%, protein are that 0.01%-50%, polymkeric substance are that the reagent of 20%-80%, protected protein matter is 0%-10%.
Described polysaccharide comprises dextran, starch, Mierocrystalline cellulose and derivative thereof, agarose and water-soluble polymer polysaccharide.
Described protein, its protein macromolecule are distributed in the middle mutually formation vitreous particle of polysaccharide, and albumen-polysaccharide particulate is evenly distributed in the lipophilic polymer matrix.In
Described protein is: erythropoietin (EPO), recombinant methionyl human G-CSF (G-CSF), granulocyte-macrophage colony stimutaing factor (GM-CSF), vaccine, Interferon, rabbit (INF), tethelin (GH), Regular Insulin (Insulin), Urogastron (EGF), fibroblast growth factor (FGF), transforming growth factor (TGF-β), rhIGF-1 (IGF), vascular endothelial growth factor (VEGF), PDGF (PDGF), endothelial cell growth factor (ECGF) (ECGF), nerve growth factor (NGF), bone-derived growth factor (BDGF), bone morphogenetic protein (BMP), tissue polypeptide antigen (TPA), antibody (antibody), blood coagulation factor VIII (VIII) and IX gene and other protein that is used for the treatment of or polypeptide, antisense nucleotide (anti-RNA), microRNA (RNAi), gene (DNA), antibody, vaccine, or liposome.
Described polymkeric substance is for having the functional polymer compound.
Described functional polymer polymkeric substance is: the polyamino acid of polyester, poly-acid anhydrides or skeleton non-peptide bond, comprising: poly(lactic acid), poly(lactic acid)-polyglycolic acid and combination thereof; Comprise that also silicon is as glue, tetrafluoroethylene, polyvinyl chloride, polyethylene, polypropylene, polystyrene, polyethylene terephthalate, polycarbonate, Carmomer, hyaluronic acid, gelatin, collagen protein, poly-glycollide, polycaprolactone, polybutylcyanoacrylate, poly phosphazene, poly phosphate, Fibrinogen, scleroproein, gel comprises: polyethylene glycol-lactic acid, the polyethylene glycol-oxyacetic acid, polyglycolic acid-polyethylene glycol-oxyacetic acid, polylactic acid-polyglycol-poly(lactic acid) temperature-sensitive hydrogel and responsive type material.
Described particulate is finger protein-polysaccharide particulate, and its particle diameter is in 60 nanometers-20 micron.
The reagent of described protected protein matter is: a kind of or arbitrary combination of little sugar, polyol, amino-acid compound, metallic compound.
Described little sugar comprises trehalose, sucrose, lactose.
Described polyol comprises N.F,USP MANNITOL, glycerine.
Described amino-acid compound comprises glycine, arginine.
Described metallic compound comprises zinc, copper, magnesium, iron, calcium.
The present invention may be made in the coating of film, fiber, support, skeleton and device surface; The top coat that also can be used for film, fiber, skeleton and device is when the interventional therapy that is loaded with the human cytokines medicine.
The preparation method of polysaccharide polymer having protein large molecule of the present invention may further comprise the steps:
1. albumen being added polysaccharide soln or albumen is added polysaccharide and polyoxyethylene glycol mixes broad;
2. the polyglycol solution of prepared albumen-polysaccharide soln 6% or 6% above concentration solution and same concentrations is mixed being lower than under the temperature that room temperature is higher than freezing point, or with albumen-polysaccharide 6% or 6% above concentration solution with and the polyglycol solution that contains the polyelectrolyte of 0.2-2% mix in temperature above freezing, or albumen-polysaccharide 6% following concentration solution is mixed in temperature above freezing with the same concentrations polyglycol solution;
3. formed mixing solutions or water-water milkiness liquid cooling freeze-drying is dry;
4. resulting lyophilized powder is added the organic solution of soluble polyethylene glycol washing, remove polyoxyethylene glycol, obtain being loaded with proteic polysaccharide vitreous particle;
5. the solution that resulting albumen-polysaccharide vitreous particle is added lipophilic polymer mixes, and this suspension liquid is shaped to film, fiber, skeleton, coating or is painted on the surface of apparatus, makes after the solvent evaporates stand-by.
Described polyelectrolyte is sodium alginate, the electronegative polyelectrolyte of carboxylic cellulose formiate sodium.
Described polysaccharide soln and identical polyglycol solution be lower than mix under the temperature that room temperature is higher than freezing point or addition polymerization electrolytical, their concentration is 6%-40%; Proteinic concentration is 0.1%-20%.
The solution of described polymkeric substance is one or more functional polymer combination of polymers, and protein also is one or more combination, according to concrete proteinic character and treatment needs, even can add chemical medicine and play the combination therapy purpose.
The present invention can make the top coat of film, fiber, skeleton and other device to be used for interventional therapy or other purposes.The present invention has multiple function, can select different prescriptions to reach the purpose of treatment as required.The support of the coating of the various supports that the preparation method makes, film, skeleton, fiber, tissue engineering material can have protection and slow-releasing and controlled-releasing action to the activity of medicine.
Concrete embodiment
Example 1: the preparation of film control-released agent and release in vitro behavior
One, solution allocation:
1. myohaemoglobin formulations prepared from solutions: take by weighing myohaemoglobin 20.7mg, add choosing dissolving in whirlpool among the ultrapure water 2ml, obtain the about 10mg/ml of myohaemoglobin strength of solution.
2. dextran solution preparation: take by weighing dextran (Mw=64,000-70,000) 1.0g, add ultrapure water to 10.00g, heated and stirred is to dissolving.Obtain dextran solution weight percent about 10%.
3.PEG formulations prepared from solutions: take by weighing dextran (Mw=8,000) 1.0g, add ultrapure water to 10.00g, heated and stirred is to dissolving.Obtain dextran solution weight percent about 10%.
Two, model protein-dextran microparticles preparation:
1. myohaemoglobin dextran microparticles preparation: take by weighing myohaemoglobin solution, dextran solution and PEG solution 0.50g, 0.16g and 1.00g (annotating two parts) respectively, under 0 ℃ condition, employing homogenizer E shelves (22,000rpm) homogenate 3 * 5s.Above-mentioned emulsion is let slip night at-20 ℃, again lyophilize 24h.Dry thing places methylene dichloride 5ml, and 12, the centrifugal 5min of 000rpm inclines the upper strata stillness of night, repeats washed with dichloromethane process three times.The myohaemoglobin dextran microparticles that obtains at last is kept in the 1ml methylene dichloride.
2. blank dextran microparticles preparation: take by weighing dextran solution and PEG solution 0.15g and 1.00g respectively, prepare with method by last noodle producing method.
(annotate: above-mentioned two kinds of particulates are measured its size distribution and size and scanning electron microscopic observation form and big or small as Fig. 1 at PCS, 2, the most of particle diameters of particulate are about 1～2um.)
Three, the preparation of film controlled release preparation:
1:5 film controlled release preparation preparation: take by weighing PLGA (75/253A) 15.1mg, methylene dichloride 1ml, whirlpool choosing dissolving.Add the myohaemoglobin dextran microparticles suspension that 700ul shakes up, the whirlpool choosing makes and is uniformly dispersed.The employing airbrush is sprayed onto above-mentioned solution on the slide glass or on the tinsel, methylene dichloride is removed in the tinsel decompression of preparation.Dry back collection membrane careful from slide glass or on the tinsel, we can be clearly seen that finely dispersed dextran microparticles in the film.
Four, discharging liquid measures:
Discharge the liquid sample and adopt the micro-BCA kit measurement.With myohaemoglobin standard protein preparation standard curve.Myohaemoglobin-dextran film discharges the subduction of liquid absorption value to be proofreaied and correct with the blank film release liquid absorption value of method preparation, brings into to calculate in the above-mentioned typical curve to discharge the liquid myoglobin concentration.Draw the release profiles of myohaemoglobin in different dextran/PLGA ratio coating successively.Its release behavior is good as can be seen, does not have the obviously prominent behavior of releasing.
These films can be directly used in clinical, play result of treatment.Can also be fixed on it on other support, play slow controlled release and therapeutic action.
Example 2: the preparation of bracket coating model control-released agent and release in vitro behavior
One, model protein-dextran microparticles preparation is according to example 1 preparation
Two, model protein (myohaemoglobin) coating preparation:
1:5 coating preparation: take by weighing PLGA (50/502A) 15.1mg, methylene dichloride 1ml, whirlpool choosing dissolving.Add the myohaemoglobin dextran microparticles suspension that 700ul shakes up, the whirlpool choosing makes and is uniformly dispersed.Adopt airbrush that above-mentioned solution is sprayed onto 316L stainless steel metal sheet, methylene dichloride is removed in the tinsel decompression of preparation.
1:10 coating preparation: take by weighing PLGA (50/502A) 29.9mg, be equipped with coating with legal system by top method.
1:15 coating preparation: take by weighing PLGA (50/502A) 45.6mg, be equipped with coating with legal system by top method.
Blank coating preparation: take by weighing respectively with above-mentioned three kinds of coatings that PLGA (50/502A) is an amount of, methylene dichloride 1ml, whirlpool choosing dissolving.Adopt airbrush that above-mentioned solution is sprayed onto 316L stainless steel metal sheet, methylene dichloride is removed in the tinsel decompression of preparation.
Three, coating release test: discharge liquid according to example 1 and measure; Its release profiles such as Fig. 6:
The coating of these medicine equipments can be directly used in clinical, plays result of treatment.
Example 3: the preparation of cardiac stent coating control-released agent and release in vitro behavior
One, model protein-dextran microparticles preparation is according to example 1 preparation;
Two, the cardiac stent coating is according to example 1 preparation
1:5 coating preparation: take by weighing PLGA (75/253A) 15.1mg, methylene dichloride 1ml, whirlpool choosing dissolving.Add the myohaemoglobin dextran microparticles suspension that 700ul shakes up, the whirlpool choosing makes and is uniformly dispersed.Adopt airbrush that above-mentioned solution is sprayed onto on the cardiac stent surface, methylene dichloride is removed in the cardiac stent decompression of preparation.The surface of cardiac stent can be clearly seen that by scanning electron microscope its smooth surface is smooth, does not have burr, and bracket coating is fit to clinical application.
Three, cardiac stent coating release test: discharge liquid according to example 1 and measure; Its release in vitro curve its release behavior as can be seen is good, does not have the obviously prominent behavior of releasing.
The coating of these medicine equipments can be directly used in clinical, plays result of treatment, and this support not only has supporting role, also has slow-releasing and controlled-releasing action and result of treatment.
Example 4: the preparation of framework controlled release agent and release in vitro behavior
Model protein-dextran microparticles preparation is according to example 1 preparation;
Two, the preparation of framework controlled release agent
The preparation of 1:5 framework controlled release agent: take by weighing PLGA (75/253A) 300.1mg, methylene dichloride 5ml, whirlpool choosing dissolving.Add in the 60mg myohaemoglobin dextran microparticles appeal PLGA solution, the whirlpool is outstanding to be uniformly dispersed it.Adopt airbrush that above-mentioned solution is sprayed onto or is poured onto in the mould device, dry up with nitrogen on one side then, continue solution again and be sprayed onto or be poured onto in the mould device, and then continue to dry up, till satisfying the demand.Clearly see and have microparticulate inside.
Three, the release test of framework controlled release agent: discharge liquid according to example 1 and measure; By optimizing preparation prescription, can be clearly seen that the obviously not prominent release profiles of releasing.
This skeleton not only has supporting role, also has slow controlled release treatment effect.
Example 5: the inventive method is to the provide protection of β-half sugared lactoside zymoprotein
In order to verify that the present invention has the better protecting effect to biologically active substance; design following experiment: dextran microparticles is carried dextran microparticles to the sugared lactoside enzyme of β-half (have quaternary structure, molecular weight is the enzyme of 500KD) and is tested the provide protection of the protein macromolecule of structure mutability under the organic solution existence condition.Earlier protein is dissolved in dextran solution with the sugared lactoside enzyme of 0.67mg/ml β-half,, then it is added emulsification in the example 1 described PEG solution according to 1: 20 ratio.After the lyophilize, several all over removing PEG with the methylene dichloride repetitive scrubbing.In the following step, the proteinic dextran microparticles that carries that is reclaimed is dissolved in damping fluid again, add o-nitrophenyl-β-D-galactopyranoside (ONPG), test is to the hydrolyzation catalysis activity of substrate-ONPG.As shown in Figure 13, the katalysis of enzyme has just descended less than 10% having experienced from example 1 to example after 2 the operation described processes (comprising emulsification, lyophilize and washed with dichloromethane).This activity of 10% descends and has also comprised the protein-active that loses owing in Partial Protein that is dispensed on the PEG phase in emulsion process and the freezing dry process.That is to say, with the contacting of the used organic solution-methylene dichloride of micro-capsule packet procedures in, dextran microparticles has been preserved protease activities effectively.