CN101816634B - Technology for preparing bone growth factor-carrying microspheres by ultrasonic atomization method - Google Patents
Technology for preparing bone growth factor-carrying microspheres by ultrasonic atomization method Download PDFInfo
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Abstract
The invention relates to a technology for preparing bone growth factor-carrying microspheres by an ultrasonic atomization method, which combines an a (aggregate)/o/w method with an ultrasonic atomization system to simultaneously form drug aggregates and emulsify the drug aggregates into spheres, adopts biodegradable high molecular polylactic acid or polyglycolic acid or lactic acid-glycolic acid copolymer as the initial raw material of the microspheres, embeds bone morphogenetic proteins such as BMP-2, BMP-7, BMP-1 and the like, and achieves the purpose of regulating and controlling the size and the release period of the microspheres mainly by controlling parameters such as the nozzle type, the ultrasonic atomization frequency, the components and the concentration of the raw material and the like in the preparation process. In the process of embedding and releasing the bone growth factor, the factor has a storage-type preservation effect in the microsphere due to the existence of reversible aggregation-dissociation balance, so that the bone growth factor keeps good activity and is completely released, and the release amount of the bone growth factor after being soaked in Phosphate Buffer Solution (PBS) for 45 days in vitro reaches 78.1-92.4%.
Description
Technical field
The invention belongs to medical instruments field, or rather, the present invention relates to a kind of method for preparing of bone growth factor-carrying microspheres.
Technical background
Medicine carrying microballoons is a kind of biomaterial for medical purpose goods that rise gradually along with the development of biomaterial science, clinical medicine and materia medica; Be that a kind of degradable high polymer material that uses is carrier; Parcel or absorption medicine and the spherical or type spheric microgranule processed is suspended in the inertia hydrophilic solution.The injectable microsphere diameter is generally about tens microns, and its substitutive characteristics is that the carrying person as medicine gets into human body, and the position of drug release, speed, mode etc. have alternative and controllability, can bring into play the effectiveness of medicine better.As carrier material, the degradable macromolecule microsphere has its special advantages, such as excellent biological compatibility, hydrophilic and biodegradability.But the release of medicine carrying microballoons Chinese medicine has slow release characteristic, can increase the half-life of medicine, prolongs administration time; Under the situation of Isodose, improved the biological availability of medicine.
The research proof; Bone morphogenetic protein (bone morphogenetic protein; BMP) have another name called: skeletal growth factor; Can increase the marker enzyme of osteoblast differentiation---the isogenic expression of alkali phosphatase and osteocalcin, promote the stratification of new bone formation and bone, in the osteoblast differentiation process, play pivotal role.Drugs approved by FDA in 2002 OP-1 (BMP-7) be used for the damaged clinical treatment of long bone and the collagen carrier microgranule carries the application that recombinant human BMP-2 forwardly merges between lumbar vertebra.Both at home and abroad to BMP, especially BMP-2 and the BMP-7 bone inductive effect in clinical has given very high evaluation at present.Yet it mainly is to be prone to diffusion dilution in vivo that BMP is applied to clinical difficulty, and its bioavailability is extremely low.Therefore, need suitable carriers so that BMP is slowly discharged, and medicine carrying microballoons is a preferable selection, especially size evenly, high carrying drug ratio is arranged, can keep the microsphere of high pharmaceutically active.
In preparation such as emulsion process " w/o/w ", " s (solid)/o/w " bone growth factor-carrying microspheres, the activity of medicine keeps that bad to cause release not exclusively be a common problem.The object of the invention is utilized soft, the low flow velocity that the ultrasonic atomizatio system provides, the advantage of low discharge, the method for preparing of inventing a kind of advanced person's bone growth factor-carrying microspheres.
Summary of the invention:
The present invention provides a kind of method for preparing of bone growth factor-carrying microspheres.
The method of employing of the present invention " a (aggregate)/o/w " method and ultrasonic atomizatio systems incorporate prepares microsphere.
Method for preparing of the present invention may further comprise the steps:
With microsphere raw material and skeletal growth factor wiring solution-forming, produce the microsphere drop through the nebulizer atomizing, volatile dry obtains solid-state-microspherical.
Wherein, the microsphere raw material is selected from: Biodegradable high-molecular polylactic acid (PLA) or polyglycolic acid (PGA) or lactic acid-ethanol copolymer (PLGA), and its molecular weight ranges is 5～100KDa, solvent is selected from: dichloromethane or chloroform.
Skeletal growth factor is selected from bone morphogenetic proteins such as BMP-2, BMP-7 or BMP-1, and its solvent adopts PBS (PBS).
Concrete method for preparing:
(1). the preparation of solution: preparation microsphere material solution with skeletal growth factor solution;
(2). reinforced: as to add the ultrasonic atomizatio generator to microsphere material solution (organic facies) and skeletal growth factor solution (water) with constant flow velocity respectively through syringe pump;
(3). atomizing: produce little drop with constant atomizing frequency of vibration;
(4). the volatilization of solvent: collect little drop that (3) are produced with surfactant, and continue mechanical agitation, make the volatilization of dichloromethane or chloroform;
(5). washing and lyophilization.
The concentration of the material solution that obtains after step (1) preparation is between 2～15% (w/v), and the concentration of skeletal growth factor solution is between 5～25ug/ml.
The flow velocity of material solution and factor solution is respectively 0.2～0.4mL/ minute, 1～3mL/ minute in the step (2), and the atomizing frequency of vibration is 25～60kHz;
Surfactant is low-molecular-weight polyvinyl alcohol (PVA) in the step (3), its outside the concentration range of water be 0.1～1% (w/v).
Mixing time is 3～4 hours in the step (4).
The most preferred method for preparing of the present invention in an embodiment.
The sustained-release micro-spheres particle diameter of the present invention's preparation is even, and the mean diameter size is 15.5～31.4 microns, can change preparation parameter (mainly being burner design and ultrasonic atomizatio frequency) as required and control.
In the sustained-release micro-spheres of the present invention's preparation, the release of its skeletal growth factor has the adjustable slow release cycle, and the burst size that external phosphate buffer (PBS) soaked after 45 days reaches 78.1～92.4%.
The method for preparing of microsphere is simple among the present invention, adopts " a (aggregate)/o/w " method to combine the ultrasonic atomizatio system, and the formation of medicine aggregation and emulsifying balling-up are carried out simultaneously.The factor is in by the process of embedding and release, owing to there is reversible " assembling-dissociate " balance, the factor has storage formula preservation effect therein; Make it keep good activity, discharge comparatively fully, the polymeric microspheres stabilize property for preparing of the present invention is good in addition; Safe, long shelf-life, slow release controlling cycle; With low cost, be fit to large-scale production.
The specific embodiment
To combine the more detailed explanation the present invention of embodiment below, but not play restriction.
The preparation process of PLA (5KDa) microsphere of embodiment 1, year BMP-2 is following:
(1). the preparation of solution: the PBS solution of dichloromethane solution and BMP-2 5ug/ml of the PLA of preparation 2%.
(2). reinforced: as to pass through syringe pump to PLA solution of (1) preparation and BMP-2 solution respectively with the flow velocity adding ultrasonic atomizatio generator of 0.2mL/ minute, 1mL/ minute.
(3). atomizing: the atomizing frequency of vibration with 60kHz produces little drop.
(4). the volatilization of solvent: the PVA aqueous solution with 0.1% is collected little drop that (3) are produced, and continues mechanical agitation 4 hours, makes the dichloromethane volatilization.
(5). washing and lyophilization.
The PLA microsphere that carries BMP-2 of this example preparation has uniform particle diameter, and the mean diameter size is 15.5 microns, and the burst size that external PBS soaks BMP-2 after 45 days reaches 92.4%.
The preparation process of PLGA (20KDa) microsphere of embodiment 2, year BMP-1 is following:
(1). the preparation of solution: dichloromethane solution and the 10ug/ml solution of the PLGA of preparation 15%,
(2) the solution of PLGA and the BMP-1 of (1) preparation through syringe pump respectively with the flow velocity adding ultrasonic atomizatio generator of 0.1mL/ minute, 1mL/ minute,
(3). atomizing: the atomizing frequency of vibration with 40kHz produces little drop,
(4). the volatilization of solvent: the PVA aqueous solution with 0.5% is collected little drop that (3) are produced, and continues mechanical agitation 3 hours, makes the dichloromethane volatilization,
(5). washing and lyophilization.
The PLGA microsphere average grain diameter size of carrying BMP-1 of this example preparation is 26.8 microns, and the burst size that external PBS soaks BMP-1 after 45 days reaches 85%.
The preparation process of PLGA (50KDa) microsphere of embodiment 3, year BMP-7 is following:
(1). the preparation of solution: the PBS solution of the dichloromethane solution of the PLGA of preparation 5% and the BMP-7 of 10ug/ml.
(2). reinforced: as to pass through syringe pump to the solution of PLGA solution of (1) preparation and BMP-7 respectively with the flow velocity adding ultrasonic atomizatio generator of 0.4mL/ minute, 3mL/ minute.
(3). atomizing: the atomizing frequency of vibration with 25kHz produces little drop.
(4). the volatilization of solvent: the PVA aqueous solution with 0.5% is collected little drop that (3) are produced, and continues mechanical agitation 4 hours, makes the dichloromethane volatilization.
(5). washing and lyophilization.
The mean diameter size of the PLGA microsphere that carries BMP-7 of this example preparation is 31.4 microns, and the burst size that external PBS soaks BMP-2 after 45 days reaches 89.2%.
The preparation process of PGA (10KDa) microsphere of embodiment 4, year BMP-2 is following:
(1). the preparation of solution: the PBS solution of dichloromethane solution and BMP-2 20ug/ml of the PGA of preparation 10%.
(2). reinforced: as to pass through syringe pump to the solution of PGA solution of (1) preparation and BMP-2 respectively with the flow velocity adding ultrasonic atomizatio generator of 0.4mL/ minute, 3mL/ minute.
(3). atomizing: the atomizing frequency of vibration with 40kHz produces little drop.
(4). the volatilization of solvent: the PVA aqueous solution with 1% is collected little drop that (3) are produced, and continues mechanical agitation 3 hours, makes the dichloromethane volatilization.
(5). washing and lyophilization.
The sustained-release micro-spheres of this example preparation has uniform particle diameter, and the mean diameter size is 23.7 microns, and the burst size that external PBS soaks BMP-2 after 45 days reaches 80.3%.
The preparation process of PLA (100KDa) microsphere of embodiment 5, year BMP-2 is following:
(1). the preparation of solution: the PBS solution of chloroform soln and BMP-2 25ug/ml of the PLA of preparation 10%.
(2). reinforced: as to pass through syringe pump to the solution of PLA solution of (1) preparation and BMP-2 respectively with the flow velocity adding ultrasonic atomizatio generator of 0.3mL/ minute, 2mL/ minute.
(3). atomizing: the atomizing frequency of vibration with 60kHz produces little drop.
(4). the volatilization of solvent: the PVA aqueous solution with 0.1% is collected little drop that (3) are produced, and continues mechanical agitation 4 hours, makes the chloroform volatilization.
(5). washing and lyophilization.
PLA (100KDa) microsphere that carries BMP-2 of this example preparation has uniform particle diameter, and its mean diameter size is 28.4 microns, and the burst size that external PBS soaks BMP-2 after 45 days reaches 78.1%.
Claims (6)
1. the method for preparing of a bone growth factor-carrying microspheres, step is following:
(1). the preparation of solution: preparation microsphere material solution and skeletal growth factor solution;
(2). reinforced: as to add the ultrasonic atomizatio generator to microsphere material solution and skeletal growth factor solution with constant flow velocity respectively through syringe pump;
(3). atomizing: produce little drop with constant atomizing frequency of vibration;
(4). the volatilization of solvent: collect little drop that (3) are produced with surfactant, and continue mechanical agitation;
(5). washing and lyophilization;
Wherein, the microsphere raw material is selected from: polylactic acid, polyglycolic acid or lactic acid-ethanol copolymer, its molecular weight ranges are 5～100KDa, and microsphere raw material solvent is selected from: dichloromethane or chloroform,
Wherein, skeletal growth factor is selected from BMP-2, BMP-7 or BMP-1, and solvent is a PBS,
Wherein, the concentration of the material solution that obtains after step (1) preparation is between 2～15% (w/v), and the concentration of skeletal growth factor solution is between 5～25 μ g/ml;
Wherein, the flow velocity of material solution and factor solution is respectively 0.2～0.4mL/ minute, 1～3mL/ minute in the step (2), and the atomizing frequency of vibration is 25～60kHz;
Wherein, surfactant is low-molecular-weight polyvinyl alcohol in the step (4), its outside the concentration range of water be 0.1～1% (w/v);
Wherein, mixing time is 3-4 hour in the step (4).
2. method for preparing according to claim 1 is characterized in that, step is:
(1). the preparation of solution: the PBS solution of dichloromethane solution and the BMP-2 5 μ g/ml of the PLA of preparation 2%;
(2). reinforced: as to pass through syringe pump to the solution of PLA solution of (1) preparation and BMP-2 respectively with the flow velocity adding ultrasonic atomizatio generator of 0.2mL/ minute, 1mL/ minute;
(3). atomizing: the atomizing frequency of vibration with 60kHz produces little drop;
(4). the volatilization of solvent: the PVA aqueous solution with 0.1% is collected little drop that (3) are produced, and continues mechanical agitation 4 hours, makes the dichloromethane volatilization;
(5). washing and lyophilization.
3. method for preparing according to claim 1 is characterized in that, step is:
(1). the preparation of solution: the PBS solution of the dichloromethane solution of the PLGA of preparation 5% and the BMP-7 of 10 μ g/ml;
(2). reinforced: as to pass through syringe pump to the solution of PLGA solution of (1) preparation and BMP-7 respectively with the flow velocity adding ultrasonic atomizatio generator of 0.4mL/ minute, 3mL/ minute;
(3). atomizing: the atomizing frequency of vibration with 25kHz produces little drop;
(4). the volatilization of solvent: the PVA aqueous solution with 0.5% is collected little drop that (3) are produced, and continues mechanical agitation 4 hours, makes the dichloromethane volatilization;
(5). washing and lyophilization.
4. method for preparing according to claim 1 is characterized in that, step is:
(1). the preparation of solution: the PBS solution of the dichloromethane solution of the PGA of preparation 10% and the BMP-2 of 20 μ g/ml;
(2). reinforced: as to pass through syringe pump to the solution of PGA solution of (1) preparation and BMP-2 respectively with the flow velocity adding ultrasonic atomizatio generator of 0.4mL/ minute, 3mL/ minute;
(3). atomizing: the atomizing frequency of vibration with 40kHz produces little drop;
(4). the volatilization of solvent: the PVA aqueous solution with 1% is collected little drop that (3) are produced, and continues mechanical agitation 3 hours, makes the dichloromethane volatilization;
(5). washing and lyophilization.
5. method for preparing according to claim 1 is characterized in that, step is:
(1). the preparation of solution: the PBS solution of chloroform soln and the BMP-2 25 μ g/ml of the PLA of preparation 10%;
(2). reinforced: as to pass through syringe pump to the solution of PLA solution of (1) preparation and BMP-2 respectively with the flow velocity adding ultrasonic atomizatio generator of 0.3mL/ minute, 2mL/ minute;
(3). atomizing: the atomizing frequency of vibration with 60kHz produces little drop;
(4). the volatilization of solvent: the PVA aqueous solution with 0.1% is collected little drop that (3) are produced, and continues mechanical agitation 4 hours, makes the chloroform volatilization;
(5). washing and lyophilization.
6. method for preparing according to claim 1, the microsphere average grain diameter size that obtains is 15.5～31.4 microns.
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| CN110403736A (en) * | 2019-08-09 | 2019-11-05 | 常州市第二人民医院 | A kind of super porous titanium alloy of 3D printing facilitates bone surface method of modifying |
| CN113456893B (en) * | 2021-07-26 | 2022-04-26 | 温州医科大学附属眼视光医院 | A kind of preparation method of blue-stained amniotic basement membrane coated with fibrinogen |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1163780A (en) * | 1997-04-17 | 1997-11-05 | 中国人民解放军第四军医大学全军创伤骨科研究所 | Osteogenesis stimulin injection and its preparing process |
| CN1443571A (en) * | 2003-04-21 | 2003-09-24 | 李亚非 | Bone growth factor injection and its preparation method |
| CN101152564A (en) * | 2007-09-18 | 2008-04-02 | 江苏省人民医院 | Preparation method of composite biotinylated chitosan film and its application in periodontal tissue regeneration |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1163780A (en) * | 1997-04-17 | 1997-11-05 | 中国人民解放军第四军医大学全军创伤骨科研究所 | Osteogenesis stimulin injection and its preparing process |
| CN1443571A (en) * | 2003-04-21 | 2003-09-24 | 李亚非 | Bone growth factor injection and its preparation method |
| CN101152564A (en) * | 2007-09-18 | 2008-04-02 | 江苏省人民医院 | Preparation method of composite biotinylated chitosan film and its application in periodontal tissue regeneration |
Non-Patent Citations (1)
| Title |
|---|
| Fa-ming Chen,et al.Release of bioactive BMP from dextran-derived microspheres:A novel delivery concept.《International Journal of Pharmaceutics》.2005,第307卷(第1期),第24-32页. * |
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