CN102138905B - RhBMP-2-loaded chitosan microspheres, preparation method thereof and application thereof - Google Patents
RhBMP-2-loaded chitosan microspheres, preparation method thereof and application thereof Download PDFInfo
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Abstract
The invention discloses recombinant human bone morphogenetic protein-2 (rhBMP-2)-loaded chitosan microspheres, a preparation method thereof and application thereof. A substrate of the rhBMP-2-loaded chitosan microspheres is chitosan microspheres, and a loaded medicament is rhBMP-2. Chitosan reacts with an ionic cross-linking agent, and a reaction product is processed by solution of sodium hydroxide to prepare the chitosan microspheres; and the rhBMP-2 is loaded by a passive loading mode, so that the rhBMP-2 is adsorbed on the chitosan microspheres, wherein a weight ratio of the rhBMP-2 to thechitosan is (1-30):100. The preparation method is simple and easy, has a mild preparation condition and is favorable for maintaining the bioactivity of the rhBMP-2. The rhBMP-2-loaded chitosan microspheres can control and release the rhBMP-2 for a long term, promote the formation of new bones, and can be used for preparing medicaments for treating bone coloboma, nonunion and delayed union.
Description
Technical field
The present invention relates to a kind of protide bioactie agent carrier and preparation method thereof, relate more specifically to a kind of carrying recombinant human bone morphogenesis protein-2 (rhBMP-2) chitosan microball and preparation method thereof.
Background technology
Along with genetic engineering and development of biology, increasing bioactie agent is used for the treatment of and prevent disease.Bone morphogenesis protein-2 (bone morphogenetic protein-2, BMP-2) be one of transforming growth factor member, have and induce undifferentiated mescenchymal stem cell to the ability of chondroblast and osteoblast directed differentiation and propagation, at (the Reddi that plays an important role aspect repairing bone defect and the promotion knitting, A.H., Bone morphogenetic proteins:from basic science to clinical applications.J Bone Joint Surg Am, 2001,83-A Suppl 1 (Pt 1): S1-6).But pure rhBMP-2 is the same with other cell growth factor, short by the half-life in enzymatic degradation, the body easily, very fast by the dilution metabolism in part and whole body application, bioavailability is very low, so effective performance of rhBMP-2 needs suitable carriers.
Chitosan (chitosan) is the deacetylated product of chitin, is formed by connecting through glycosidic bond by 2-amino-2-deoxidation-β-D-glucoside, is the unique natural alkaline polysaccharide of occurring in nature, also is that minority has one of natural product of positive charge.Many experiments show that chitosan has excellent biological compatibility and biological degradability, and the degraded after product in vivo safely, have no side effect.At present, utilize slow release, the control-release microsphere of medicines such as Preparation of Chitosan chemotherapeutics, albumen and gene to obtain effect preferably.For reaching the lasting release of medicine, adopt cross-linking agent to carry out crosslinked to chitosan microball usually.(Niu such as Feng Qingling, X., et al., Preparation and characterization of chitosan microspheres for controlled release of synthetic oligopeptide derived from BMP-2.J.Microencapsulation, 2009.26 (4): 297-305) adopt emulsifying-ionic cross-linking to prepare load-carrying group bone morphogenesis protein-2 chitosan microball, the mixed solution of albumen and chitosan is splashed in the liquid paraffin that contains surfactant, splash into sodium tripolyphosphate solution after the emulsifying and react the formation microsphere, product adopts bulk petroleum ether, isopropyl alcohol cleans postlyophilization, and cumulative in vitro discharges 7 days and reaches 62.3%.Huang Xin etc. contain the mixed solution adding of rhBMP and chitosan in the octanol solution of surfactant, adopt biological cross-linking agent genipin to carry out crosslinked after the emulsifying, the product microsphere adopts isopropyl alcohol, petroleum ether, water rinsing repeatedly, and the rhBMP-2's chitosan microball release in vitro that makes can reach 30 days.Patent CN101637454 adopts static drop-ionic cross-linking to prepare the human bone morphogenesis protein-2 chitosan microball.Under electrostatic field, the mixed solution of albumen and chitosan is splashed in the coagulation bath that contains sodium tripolyphosphate, form the human bone morphogenesis protein-2 chitosan microball, the external slow release cycle reached more than 4 weeks.But adopt the crosslinked chitosan microsphere inflammatory reaction degree in vivo of cross-linking agent preparations such as glutaraldehyde, genipin and sodium tripolyphosphate obviously to increase than pure chitosan, and the crosslinked microsphere degradation in vivo speed (Mi that reduced, F.-L., et al., In vivo biocompatibility and degradability of a novel injectable-chitosan-based implant.Biomaterials, 2002,23 (1): 181-191).Simultaneously factors such as the interfacial effect in the preparation process, voltage, ultrasonic agitation also easily make albumen lose activity, and the chitosan microball whole process of preparation all will keep the sterile working, and technology is loaded down with trivial details relatively.
Summary of the invention
Because chitosan has good affinity to albumen, chitosan microball can pass through hydrophobic interaction, electrostatic interaction and interaction of hydrogen bond adhesion protein, mild condition, simple to operate, therefore the object of the present invention is to provide a kind of load rhBMP-2 chitosan microball, rhBMP-2 is adsorbed on the described chitosan microball, adsorption capacity height not only, and rhBMP-2 is difficult for inactivation.
Another object of the present invention is to provide the preparation method of above-mentioned microsphere.
A further object of the present invention is to provide the application of above-mentioned microsphere.
Load rhBMP-2 chitosan microball of the present invention, substrate is chitosan microball, and the medicine of load is rhBMP-2, and described rhBMP-2 is adsorbed on the described chitosan microball, and the weight ratio of rhBMP-2 and chitosan is 1～30: 100.
According to the present invention, the mean diameter of described chitosan microball is 1～300 μ m.
According to the present invention, described chitosan microball is reacted by chitosan and ion crosslinking agent and adopts the sodium hydroxide solution processing to make again.
The preparation method of load rhBMP-2 chitosan microball of the present invention is characterized in that, comprises the steps:
(a), chitosan solution is distributed to the oil phase that contains surfactant and forms w/o type emulsus system;
(b), the ionomer agent solution is joined described w/o type emulsus system reaction 1～12 hour, adding acetone, collection chitosan microball;
(c), after acetone, dehydrated alcohol and water wash successively, chitosan microball is scattered in the water, dripping concentration under slowly stirring is the sodium hydroxide solution of 0.1～5% (w/v), greater than 7, collects chitosan microball until the final pH value of reaction system;
(d1), the postlyophilization that chitosan microball washed with water or in water, dialyse; Or (d2) chitosan microball is soaked 1～60 minute with 0.01～1% (w/w) aqueous sulfuric acid after, postlyophilization again washes with water or dialyses in water;
(e), the chitosan microball after the lyophilizing is scattered in the solution that contains 0.1～10mg/mL rhBMP-2, passive absorption loaded after 0.5～12 hour, and the direct lyophilization of the dispersion of acquisition namely gets described load rhBMP-2 chitosan microball.
According to the present invention, described surfactant is Span-80, Tween-80 or both mixture; The volume ratio of described surfactant and oil phase is 0.5～5: 100.
According to the present invention, described oil phase is the mixed liquor of liquid paraffin, n-octyl alcohol, Oleum Ricini, Oleum Arachidis hypogaeae semen, soybean oil, olive oil, Oleum Helianthi or petroleum ether and liquid paraffin.
According to the present invention, the volume ratio of described chitosan solution and oil phase is 1: 3～10.
According to the present invention, described ion crosslinking agent is sulphuric acid, sodium tripolyphosphate, sodium sulfate or sodium citrate.
According to the present invention, described ion crosslinking agent is sulphuric acid, and described ionomer agent solution is aqueous sulfuric acid, and concentration is 10～30% (w/w).
The application of load rhBMP-2 chitosan microball provided by the invention is for the preparation of the medicine for the treatment of bone injury.
The present invention adopts first emulsifying ionomer; and then partly removing cross-linked structure prepares chitosan microball; adopt gentle absorption strategy preparation to carry the chitosan microball of bone morphogenesis protein-2; adsorption capacity height to somatomedin; operation is simple, and preparation process is difficult for microbiological contamination, not only is conducive in the preparation process activity of somatomedin and keeps; and the microsphere supported somatomedin of protecting improves bioavailability in its body not by the enzymatic degradation in the body.Vitro drug release has the prominent feature of releasing with the later stage slow release in early stage, and zoopery shows that load rhBMP-2 chitosan microball of the present invention can promote new bone formation better.
Description of drawings
Fig. 1 is infrared spectrogram, and a is the chitosan raw material, and b is the first ionomer chitosan microball of naoh treatment again.
Fig. 2 is XRD figure, and a is the chitosan raw material, and b is the first ionomer chitosan microball of naoh treatment again.
Fig. 3 is the chitosan microball stereoscan photograph.
Fig. 4 is load rhBMP-2 chitosan microball elution profiles in PBS (pH=7.4) buffer.
Fig. 5 is hematoxylin-Yihong colored graph of load rhBMP-2 chitosan microball dystopy skeletonization, * 20.
Fig. 6 is load rhBMP-2 chitosan microball dystopy osteogenic activity.
The specific embodiment
Below in conjunction with specific embodiment, the present invention will be further described.Should be understood that following examples are only for explanation the present invention but not for limiting the scope of the invention.
Universal method
(1) slow release of load rhBMP-2 chitosan microball
The load rhBMP-2 chitosan microball of 20mg is put into the 10mL plastic test tube, add PBS (pH=7.4) solution 5mL, at 37 ℃ of isothermal vibrations, (be respectively 1,2,3 in different time points, 5,7,10,14,21,28,35 and 42 days) carry out centrifugalize, collect supernatant in the test tube, replenish new PBS (pH=7.4) solution 5mL simultaneously.Get supernatant 1mL solution, add Coomassie brilliant blue G250 developer 5mL, mixing is measured absorbance with ultraviolet spectrophotometer behind the placement 2min in the 595nm place, be blank not add protein solution.Then by rhBMP-2 concentration in the rhBMP-2 standard curve Equation for Calculating supernatant.Each sample parallel assay three times calculates rhBMP-2 accumulative total and discharges percentage ratio and draw release profiles.
Be that 30KDa, deacetylation are 90% chitosan with molecular weight, be dissolved in 2% (v/v) acetic acid solution, be mixed with 4 (w/v) % chitosan solution, (Span-80 and Tween-80 volume ratio are 2: 1, and the volume ratio of surfactant and oil phase is 1: 100 slowly to join the surfactant that contains of 5 times of volumes after the deaeration.) the oil phase liquid paraffin body in, stir 3h, form the W/O emulsion, add isopyknic 20% (w/w) sulfuric acid solution and carry out crosslinkedly, continue to stir after crosslinked 4 hours, add the acetone dehydration, standing demix is with acetone, the washing precipitation respectively of dehydrated alcohol distilled water.Then precipitation is scattered in the water, dripping concentration under magnetic agitation is the sodium hydroxide solution of 4% (w/v), and greater than 7, reuse acetone, dehydrated alcohol and water fully wash until the final pH value of reaction system, namely get chitosan microball after the lyophilization.Adopt infrared spectrum and the chitosan microball of XRD and chitosan raw material to analyze, the result shows that chitosan microball has identical chemical constitution with the chitosan raw material, does not introduce other group respectively as shown in Figure 1 and Figure 2; And chitosan microball is after NaOH handles, and its degree of crystallinity descends, and mainly exists with amorphous state.Adopt the scanning electron microscopic observation chitosan microball, as shown in Figure 3, chitosan microball outward appearance rounding, mean diameter is 94 ± 53 μ m.
Be 1: 10 according to rhBMP-2 and chitosan microball weight ratio, chitosan microball be scattered in the aqueous solution that contains rhBMP-2 that passive absorption loaded after 6 hours, and the direct lyophilization of suspension is obtained load rhBMP-2 chitosan microball.As shown in Figure 4, in PBS solution (pH=7.4), rhBMP-2 slow release in early stage is very fast, and cumulative release 65% in 7 days, slowly discharges subsequently to reach more than 4 weeks.
The Kunming mouse of 2w of will growing earlier carries out the pentobarbital sodium intraperitoneal injection of anesthesia by 3% of body weight, lie on the back and be fixed on the operation plate, the hair sterilization is shaved in the outside behind the right thigh, cut the skin incision of about 1cm, passivity is separated thigh flesh bag, after implanting the chitosan microball (rhBMP-2 that wherein contains 100 micrograms) of the load rhBMP-2 that as above prepares, sew up sarolemma layer and skin.Put into after the operation and raise box, carry out normal clean grade standard forage feed.4 weeks were put to death mice in the operation back, wherein 1 mice is taken out embedded material and surrounding soft tissue thereof rapidly and immerses in 10% the formalin solution fixing, section, hematoxylin-Yihong dyeing is observed the skeletonization situation and is carried out histological observation, as shown in Figure 5, find visible osseous tissue in the striated muscle tissue, comprise bone trabecula and bone marrow.Other 4 mices are dissected its right lower limb and take out new long bone, and as shown in Figure 6, the average fresh weight of new bone is 300mg; New bone is placed in the high temperature furnace 600 ℃ of calcining 6h down, behind the furnace cooling, takes out and claim that its ash average weight is 39mg, confirm that prepared carrying recombinant human bone morphogenesis protein-2 chitosan microball has good induced osteogenesis activity.
Be that 50.5KDa, deacetylation are 90% chitosan with molecular weight, be dissolved in 3% (v/v) acetic acid solution, after being mixed with 3% chitosan solution deaeration, slowly join in the oil phase liquid paraffin body that contains surfactant Span-80 (volume ratio of surfactant and oil phase is 1: 100) of 7 times of volumes, stir 4h, form the W/O emulsion, 10% (w/w) sulfuric acid solution that adds 2 times of volumes carries out crosslinked, continue to stir after crosslinked 6 hours, add the acetone dehydration, standing demix is with acetone, the washing precipitation respectively of dehydrated alcohol distilled water.Then precipitation is scattered in the water, dripping concentration under magnetic agitation is the sodium hydroxide solution of 2% (w/v), until the final pH value of reaction system greater than 7; After again chitosan microball being soaked 1 minute with 1% (w/w) aqueous sulfuric acid, reuse acetone, dehydrated alcohol and water fully wash, and namely get chitosan microball after the lyophilization.The chitosan microball mean diameter is 62 ± 25 μ m.It is 1: 20 according to rhBMP-2/chitosan microball weight ratio, chitosan microball is scattered in the solution that contains the rhBMP-2, passive absorption loaded after 8 hours, the direct lyophilization of suspension is obtained carrying recombinant human bone morphogenesis protein-2 chitosan microball, rhBMP-2's slow release in early stage is very fast in PBS solution (pH=7.4), and cumulative release 55% in 7 days; Slowly discharge subsequently and reach more than 4 weeks.
Sample is implanted into dystopy osteogenic activity experimental test through as above mice flesh bag and shows, the sample that contains the microsphere of 100 microgram rhBMP-2 after 4 weeks forms fresh bone in the flesh bag average weight is 312mg; The ash average weight is 46mg, confirms that prepared carrying recombinant human bone morphogenesis protein-2 chitosan microball has good induced osteogenesis activity.
Be that 50KDa, deacetylation are 95% chitosan with molecular weight, be dissolved in 2% (v/v) acetic acid solution, after being mixed with 3% chitosan solution deaeration, what slowly join 3 times of volumes contains surfactant Tween-80 (volume ratio of surfactant and oil phase is 2: 100).The oil phase liquid paraffin body in, stir 2h, form the W/O emulsion, add isopyknic 30% (w/w) sulfuric acid solution and carry out crosslinkedly, continue to stir after crosslinked 1 hour, add the acetone dehydration, standing demix is with acetone, the washing precipitation respectively of dehydrated alcohol distilled water.Then precipitation is scattered in the water, dripping concentration under magnetic agitation is the sodium hydroxide solution of 4% (w/v), until the final pH value of reaction system greater than 7; Reuse acetone, dehydrated alcohol and water fully wash, and namely get chitosan microball after the lyophilization.The chitosan microball mean diameter is 27 ± 10 μ m.It is 1: 8 according to rhBMP-2/chitosan microball weight ratio, chitosan microball is scattered in the solution that contains the rhBMP-2, passive absorption loaded after 4 hours, the direct lyophilization of suspension is obtained carrying recombinant human bone morphogenesis protein-2 chitosan microball, 7 days cumulative release 68% of rhBMP-2 in PBS solution (pH=7.4).Sample is implanted into dystopy osteogenic activity experimental test through as above mice flesh bag and shows, the sample that contains the microsphere of 100 microgram rhBMP-2 after 4 weeks forms fresh bone in the flesh bag average weight is 330mg; The ash average weight is 45mg, confirms that prepared carrying recombinant human bone morphogenesis protein-2 chitosan microball has good induced osteogenesis activity.
Be that 30KDa, deacetylation are 98% chitosan with molecular weight, be dissolved in 1.5% (v/v) acetic acid solution, after being mixed with 3% chitosan solution deaeration, what slowly join 10 times of volumes contains surfactant (Span-80 and Tween-80 volume ratio are 1: 1, and the volume ratio of surfactant and oil phase is 5: 100).The oil phase liquid paraffin body in, stir 2h, form the W/O emulsion, 10% (w/w) sulfuric acid solution that adds 1.5 times of volumes carries out crosslinked, continues to stir after crosslinked 6 hours, adds the acetone dehydration, standing demix is with acetone, the washing precipitation respectively of dehydrated alcohol distilled water.Then precipitation is scattered in the water, dripping concentration under magnetic agitation is the sodium hydroxide solution of 0.1% (w/v), until the final pH value of reaction system greater than 7; After again chitosan microball being soaked 60 minutes with 0.01% (w/w) aqueous sulfuric acid, reuse acetone, dehydrated alcohol and water fully wash, and namely get chitosan microball after the lyophilization.The chitosan microball mean diameter is 5 ± 4 μ m.It is 30: 100 according to rhBMP-2/chitosan microball weight ratio, chitosan microball is scattered in the solution that contains the rhBMP-2, passive absorption loaded after 0.5 hour, the direct lyophilization of suspension is obtained carrying recombinant human bone morphogenesis protein-2 chitosan microball, 7 days cumulative release 73% of rhBMP-2 in PBS solution (pH=7.4).Sample is implanted into dystopy osteogenic activity experimental test through as above mice flesh bag and shows, the sample that contains the microsphere of 100 microgram rhBMP-2 after 4 weeks forms fresh bone in the flesh bag average weight is 350mg; The ash average weight is 48mg, confirms that prepared carrying recombinant human bone morphogenesis protein-2 chitosan microball has good induced osteogenesis activity.
Be that 90KDa, deacetylation are 95% chitosan with molecular weight, be dissolved in 3% (v/v) acetic acid solution, after being mixed with 3% chitosan solution deaeration, (volume ratio of surfactant and oil phase is 0.5: 100 slowly to join the surfactant Span-80 that contains of 3 times of volumes.) the oil phase liquid paraffin body and petroleum ether in, stir 4h, form the W/O emulsion, adding equal-volume 20% (w/w) sulfuric acid solution carries out crosslinked, continues to stir after crosslinked 12 hours, adds the acetone dehydration, standing demix is with acetone, the washing precipitation respectively of dehydrated alcohol distilled water.Then precipitation is scattered in the water, dripping concentration under magnetic agitation is the sodium hydroxide solution of 5% (w/v), until the final pH value of reaction system greater than 7; Reuse acetone, dehydrated alcohol and water fully wash, and namely get chitosan microball after the lyophilization.The chitosan microball mean diameter is 265 ± 35 μ m.It is 1: 100 according to rhBMP-2/chitosan microball weight ratio, chitosan microball is scattered in the solution that contains the rhBMP-2, passive absorption loaded after 12 hours, the direct lyophilization of suspension is obtained carrying recombinant human bone morphogenesis protein-2 chitosan microball, rhBMP-2's slow release in early stage is very fast in PBS solution (pH=7.4), and cumulative release 52% in 7 days; Slowly discharge subsequently and reach more than 4 weeks.
Sample is implanted into dystopy osteogenic activity experimental test through as above mice flesh bag and shows, the sample that contains the microsphere of 100 microgram rhBMP-2 after 4 weeks forms fresh bone in the flesh bag average weight is 302mg; The ash average weight is 35mg, confirms that prepared carrying recombinant human bone morphogenesis protein-2 chitosan microball has good induced osteogenesis activity.
The performance of the microsphere of table 1 embodiment 6-12 preparation
Embodiment 6-12
Repeat the preparation process of embodiment 1, difference only is: use the corresponding conditions in the following condition alternative embodiment 1 respectively.
In embodiment 6, adopt Oleum Ricini to replace liquid paraffin.
In embodiment 7, adopt the mixed liquor (volume ratio of petroleum ether and liquid paraffin is 1: 1) of petroleum ether and liquid paraffin to replace liquid paraffin.
In embodiment 8, adopt n-octyl alcohol to replace liquid paraffin.
In embodiment 9, adopt soybean oil to replace liquid paraffin.
In embodiment 10, ion crosslinking agent is the tripolyphosphate sodium water solution of 2% (w/w).
In embodiment 11, ion crosslinking agent is the aqueous sodium persulfate solution of 1% (w/w).
In embodiment 12, ion crosslinking agent is the sodium citrate of 3% (w/w).
As a result, prepared chitosan microball equally, after tested, these microsphere average grain diameters are 15～250 μ m, may be used to adsorb load rhBMP-2, and the slow release cycle reached more than 4 weeks, concrete, the concrete mean diameter of each embodiment, slow release cycle, to survive the active testing result as shown in table 1.
The present invention adopts the emulsifying ionomer earlier, and then partly removing cross-linked structure prepares chitosan microball, and prepared microsphere pattern rounding, good dispersion, size and crosslinking degree all are easy to control.Adopt gentle absorption strategy preparation to carry the chitosan microball of bone morphogenesis protein-2; adsorption capacity height to somatomedin; operation is simple; preparation process is difficult for microbiological contamination; not only being conducive in the preparation process activity of somatomedin keeps; and the microsphere supported somatomedin of protecting improves bioavailability in its body not by the enzymatic degradation in the body.Carrying recombinant human bone morphogenesis protein-2 chitosan microball can discharge the rhBMP-2 in long period (6 week) control, vitro drug release has the prominent feature of releasing with the later stage slow release in early stage, can promote new bone formation better, can be used for that the bone that a variety of causes causes is damaged, the filling reparation of bone does not connect, bone delay in healing, aspects such as union of fracture promoter.Can directly be filled in the damaged place of bone, also can be scattered in the method that adopts injection in the injection, can also the compound use together of other medicines carrier.
Claims (8)
1. load rhBMP-2 chitosan microball, substrate is chitosan microball, the medicine of load is rhBMP-2, it is characterized in that, described rhBMP-2 is adsorbed on the described chitosan microball, and the weight ratio of rhBMP-2 and chitosan is 1～30: 100, and the mean diameter of described chitosan microball is 1～300 μ m, and described chitosan microball is reacted by chitosan and ion crosslinking agent and adopts the sodium hydroxide solution processing to make again.
2. the preparation method of the described load rhBMP-2 of claim 1 chitosan microball is characterized in that, comprises the steps:
(a), chitosan solution is distributed to the oil phase that contains surfactant and forms w/o type emulsus system;
(b), the ionomer agent solution is joined described w/o type emulsus system reaction 1～12 hour, adding acetone, collection chitosan microball;
(c), after acetone, dehydrated alcohol and water wash successively, chitosan microball is scattered in the water, dripping concentration under slowly stirring is the sodium hydroxide solution of 0.1～5% (w/v), greater than 7, collects chitosan microball until the final pH value of reaction system;
(d1), the postlyophilization that chitosan microball washed with water or in water, dialyse; Or (d2) chitosan microball is soaked 1～60 minute with 0.01～1% (w/w) aqueous sulfuric acid after, postlyophilization again washes with water or dialyses in water;
(e), the chitosan microball after the lyophilizing is scattered in the solution that contains 0.1～10mg/mL rhBMP-2, passive absorption loaded after 0.5～12 hour, and the direct lyophilization of the dispersion of acquisition namely gets described load rhBMP-2 chitosan microball.
3. preparation method according to claim 2 is characterized in that, described surfactant is Span-80, Tween-80 or both mixture; The volume ratio of described surfactant and oil phase is 0.5～5: 100.
4. preparation method according to claim 2 is characterized in that, described oil phase is the mixed liquor of liquid paraffin, n-octyl alcohol, Oleum Ricini, Oleum Arachidis hypogaeae semen, soybean oil, olive oil, Oleum Helianthi or petroleum ether and liquid paraffin.
5. preparation method according to claim 2 is characterized in that, the volume ratio of described chitosan solution and oil phase is 1: 3～10.
6. preparation method according to claim 2 is characterized in that, described ion crosslinking agent is sulphuric acid, sodium tripolyphosphate, sodium sulfate or sodium citrate.
7. preparation method according to claim 6 is characterized in that, described ion crosslinking agent is sulphuric acid, and described ionomer agent solution is aqueous sulfuric acid, and concentration is 10～30% (w/w).
8. the application of the described load rhBMP-2 of claim 1 chitosan microball in the medicine of preparation treatment bone injury.
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| CN102430129B (en) * | 2011-12-17 | 2013-06-26 | 浙江大学 | Bone morphogenetic protein-2/basic fibroblast growth factor (BMP-2/bFGF) double-gene chitosan nano-microcapsules and application thereof |
| CN102755669B (en) * | 2012-07-16 | 2014-04-30 | 姚琦 | Preparation method and application of fibrin glue composite recombinant human bone morphogenetic protein-2 (rhBMP-2) microsphere |
| CN103536563A (en) * | 2013-10-15 | 2014-01-29 | 海南卫康制药(潜山)有限公司 | Levorotatory-pantoprazole sodium composition freeze-dried powder for injection |
| CN104353121A (en) * | 2014-11-24 | 2015-02-18 | 吴志宏 | BMP microsphere loaded 3D printing porous metal stent and preparation method thereof |
| CN104353122B (en) * | 2014-11-24 | 2017-04-12 | 吴志宏 | 3D printed porous metal with bionic three-dimensional (3D) micro-scaffold and preparation method of 3D printed porous metal |
| CN109700783A (en) * | 2019-03-04 | 2019-05-03 | 江西派尼生物药业有限公司 | A kind of preparation method of chitosan coating tilmicosin microspheres |
| CN111849957B (en) * | 2020-07-31 | 2023-03-28 | 中国农业大学 | PH-responsive aminopeptidase-loaded microsphere and preparation method and application thereof |
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| Title |
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| 黄鑫.rh-BMP-2壳聚糖微球的制备及体外检测.《中国矫形外科杂志》.2009,第17卷(第15期),全文. * |
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