CN115804866A - Polyester-based microsphere containing drug-loaded calcium inorganic matter and preparation method and application thereof - Google Patents
Polyester-based microsphere containing drug-loaded calcium inorganic matter and preparation method and application thereof Download PDFInfo
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- CN115804866A CN115804866A CN202211573213.4A CN202211573213A CN115804866A CN 115804866 A CN115804866 A CN 115804866A CN 202211573213 A CN202211573213 A CN 202211573213A CN 115804866 A CN115804866 A CN 115804866A
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- calcium
- polyester
- polydopamine
- tissue regeneration
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- 229940079593 drug Drugs 0.000 title claims abstract description 74
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 title claims abstract description 70
- 239000011575 calcium Substances 0.000 title claims abstract description 70
- 229910052791 calcium Inorganic materials 0.000 title claims abstract description 70
- 229920000728 polyester Polymers 0.000 title claims abstract description 56
- 238000002360 preparation method Methods 0.000 title claims abstract description 13
- 229920001690 polydopamine Polymers 0.000 claims abstract description 73
- 230000017423 tissue regeneration Effects 0.000 claims abstract description 58
- 239000002131 composite material Substances 0.000 claims abstract description 53
- 239000010954 inorganic particle Substances 0.000 claims abstract description 51
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- ZQBZAOZWBKABNC-UHFFFAOYSA-N [P].[Ca] Chemical compound [P].[Ca] ZQBZAOZWBKABNC-UHFFFAOYSA-N 0.000 claims abstract description 23
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- 229910052588 hydroxylapatite Inorganic materials 0.000 claims description 27
- XYJRXVWERLGGKC-UHFFFAOYSA-D pentacalcium;hydroxide;triphosphate Chemical compound [OH-].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O XYJRXVWERLGGKC-UHFFFAOYSA-D 0.000 claims description 27
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- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 claims description 12
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- YIGWVOWKHUSYER-UHFFFAOYSA-F tetracalcium;hydrogen phosphate;diphosphate Chemical compound [Ca+2].[Ca+2].[Ca+2].[Ca+2].OP([O-])([O-])=O.[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O YIGWVOWKHUSYER-UHFFFAOYSA-F 0.000 claims description 8
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- OGSPWJRAVKPPFI-UHFFFAOYSA-N Alendronic Acid Chemical compound NCCCC(O)(P(O)(O)=O)P(O)(O)=O OGSPWJRAVKPPFI-UHFFFAOYSA-N 0.000 claims description 2
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- MPBVHIBUJCELCL-UHFFFAOYSA-N Ibandronate Chemical compound CCCCCN(C)CCC(O)(P(O)(O)=O)P(O)(O)=O MPBVHIBUJCELCL-UHFFFAOYSA-N 0.000 claims description 2
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- GBNXLQPMFAUCOI-UHFFFAOYSA-H tetracalcium;oxygen(2-);diphosphate Chemical compound [O-2].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O GBNXLQPMFAUCOI-UHFFFAOYSA-H 0.000 claims description 2
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Images
Abstract
The invention discloses a polyester-based microsphere containing drug-loaded calcium-containing inorganic substance and a preparation method and application thereof, wherein the polyester-based microsphere combines and uses induced tissue regeneration drugs, calcium-containing inorganic particles, polydopamine, degradable artificially synthesized polyester and mineralized calcium-phosphorus layers, so that the composite microsphere has good effect of inducing the release of the tissue regeneration drugs, has a release period of the induced tissue regeneration drugs of over 35 days, has good biocompatibility and bioactivity, can be used as a scaffold material to support the adhesion and proliferation of cells, and can effectively promote the repair and reconstruction of bone tissues. In addition, the preparation method is simple, the requirement on equipment is not high, the raw materials are industrialized, the sources are easily available, the cost is low, and industrialization is easy to realize.
Description
The technical field is as follows:
the invention relates to the technical field of biomedical materials, in particular to polyester-based microspheres containing drug-loaded calcium-containing inorganic substances, and a preparation method and application thereof.
Background art:
in the treatment of bone tissue diseases, such as tumor chemotherapy, anti-infection and anti-inflammation, systemic administration may result in insufficient content of effective drug at the site of bone tissue "focus", while the distribution of drug in other tissues is liable to cause toxic and side effects. The transhepatic effect of oral administration and the attenuation and loss of the drug during transportation result in very low availability and low blood concentration of the drug. Large amounts of medication are required which in turn increases the hyperactive effects. Therefore, the slow release of the drug is introduced into the bone tissue scaffold, and the bone tissue scaffold material which is loaded and slowly released by combining the drug and the bone tissue scaffold is expected to be developed. Sustained and controlled release Drug Delivery Systems (DDS) are the hot spot of recent domestic and foreign research. A drug sustained-release system taking various bone repair materials as a carrier is a novel drug delivery mode, and after the DDS is implanted into an organism inner skeleton, the drug carried by the carrier can be continuously, stably and efficiently and slowly released, so that the dual purposes of repairing bone defect and drug therapy are achieved. The DDS plays a role in underestimation in the fields of treating osteomyelitis, bone tumor, bone tuberculosis, bone fracture, nonunion, artificial joint replacement and the like.
The polymeric microspheres function as microreservoirs, storing and protecting certain substances so that they can be released at a desired rate when and where they are needed (multidimensional controlled release). The materials currently used in such microspheres mainly include inorganic materials, natural polymers and synthetic polymers, and are classified into degradable materials and non-degradable materials according to their degradation properties. The polyester is the most studied and widely used biodegradable synthetic polymer material at present, and is degradable artificially synthesized polyester, such as polylactic acid, polyglycolic acid, poly epsilon-caprolactone, poly beta-hydroxybutyric acid, poly beta-hydroxyvaleric acid and their copolymers. However, the degradable artificially synthesized polyester lacks bioactivity and has low mechanical strength, and is difficult to induce tissue regeneration when used alone. Inorganic materials such as calcium phosphate ceramics, calcium carbonate, calcium sulfate and the like are well known for the beneficial effect of bone regeneration, but the materials are difficult to effectively adsorb and load the drugs for inducing tissue regeneration.
The invention content is as follows:
the invention aims to provide polyester-based microspheres containing drug-loaded calcium-containing inorganic matters, and a preparation method and application thereof.
The invention is realized by the following technical scheme:
the polyester-based microsphere containing the drug-loaded calcium-containing inorganic substance sequentially comprises a polydopamine-containing calcium-containing inorganic particle loaded with a tissue regeneration inducing drug, degradable artificially synthesized polyester coating the polydopamine-containing calcium-containing inorganic particle loaded with the tissue regeneration inducing drug and a calcium-phosphorus layer on the surface of the polyester from inside to outside.
The calcium-containing inorganic particles include, but are not limited to, any one of hydroxyapatite, bioglass, octacalcium phosphate, tricalcium phosphate, tetracalcium phosphate, calcium hydrophosphate, amorphous calcium phosphate, biphasic calcium phosphate, calcium carbonate, calcium bicarbonate, and calcium sulfate; the size of the calcium-containing inorganic particles is 0.1-5 um.
The medicine for inducing tissue regeneration comprises, but is not limited to, bone morphogenetic protein, vascular endothelial growth factor, platelet derived factor, fibroblast growth factor, transforming growth factor, insulin-like growth factor, parathyroid hormone, growth hormone, interleukin, alendronate sodium, resveratrol, salmon calcitonin, zoledronate sodium, ibandronate sodium, strontium ranelate, naringin, dexamethasone and vitamin D.
The average particle size of the calcium-containing inorganic particle loaded with the tissue regeneration inducing drug and containing polydopamine is 1-50 um.
The degradable artificially synthesized polyester is selected from any one of polylactic acid, polylactic acid-glycolic acid copolymer, polycaprolactone, poly 3-hydroxyalkanoate, poly (3-hydroxybutyrate), poly 3-hydroxybutyrate-co-3-hydroxyvalerate, polytrimethylene carbonate and polybutylene succinate; preferably, the molecular weight of the degradable polyester is 1.0-10.0 ten thousand daltons.
The preparation method of the polyester-based microsphere containing the medicine-carrying calcium inorganic substance comprises the following steps:
(1) Wrapping calcium-containing inorganic particles with polydopamine, and adsorbing a tissue regeneration inducing drug onto the polydopamine-containing calcium-containing inorganic particles to prepare polydopamine-containing calcium-containing inorganic particles loaded with the tissue regeneration inducing drug;
(2) Calcium-containing inorganic particles containing polydopamine and loaded with a tissue regeneration inducing drug are dispersed into a degradable artificially synthesized polyester solution, then added into a water solution containing a surfactant, and solidified by an emulsion solvent volatilization method to form composite microspheres;
(3) Dispersing the composite microspheres into simulated body fluid with the concentration of 5-10 times, standing, centrifuging and cleaning to obtain the polyester-based microspheres containing the drug-loaded calcium-containing inorganic substance.
The simulated body fluid is an apatite supersaturated solution containing calcium ions and phosphate ions, and the ion composition concentration is as follows:
preferably, step (1) comprises the steps of: dispersing calcium-containing inorganic particles into an aqueous solution of dopamine with the concentration of 0.2-2 mg/ml, stirring for 3-24 h, centrifuging and cleaning to obtain the calcium-containing inorganic particles containing polydopamine, then dispersing the calcium-containing inorganic particles into a solution containing 0.01-0.1 mg/ml of tissue regeneration inducing medicine, centrifuging and freeze-drying to obtain the polydopamine-containing inorganic particles loaded with the tissue regeneration inducing medicine; the mass-volume ratio of the calcium-containing inorganic particles to the dopamine aqueous solution is 1: 20-90 g/ml; the mass-volume ratio of the calcium-containing inorganic particles containing polydopamine to the solution containing the medicine for inducing tissue regeneration is 1:30 to 120g/ml.
Preferably, the step (2) comprises the steps of: calcium-containing inorganic particles loaded with a tissue regeneration inducing drug and containing polydopamine are dispersed in a degradable artificially synthesized polyester solution; slowly dripping the solution into 300-800ml of 1.5-40 mg/ml surfactant aqueous solution, continuously stirring at 250-800 rpm for 12-24 h, and separating out the composite microspheres at the bottom of the container to prepare composite microspheres; the mass ratio of the calcium-containing inorganic particles containing polydopamine and loaded with the induced tissue regeneration medicine to the degradable artificially synthesized polyester is 0.02-0.35: 1; the surfactant includes, but is not limited to, polyvinyl alcohol, gelatin, methyl cellulose, carboxymethyl cellulose.
Preferably, the standing time in the step (3) is 12-48 h, and the mass-to-volume ratio of the composite microspheres to the simulated body fluid is 1:20 to 160g/ml.
The invention also protects the application of the polyester-based microsphere containing the drug-loaded calcium-containing inorganic substance, and the polyester-based microsphere is used as a support material to support the adhesion and proliferation of cells and promote the repair and reconstruction of bone tissues under bacterial infection.
The invention has the following beneficial effects:
1) The method comprises the steps of firstly wrapping calcium-containing inorganic particles with polydopamine, adsorbing induced tissue regeneration drugs onto the calcium-containing inorganic particles with the polydopamine, then dispersing the drugs into a degradable artificially synthesized polyester network, solidifying the particles into microspheres by an emulsion solvent volatilization method, and forming a calcium-phosphorus layer on the surfaces of the microspheres.
2) In the application process of the polyester-based microsphere containing the drug-loaded calcium-containing inorganic substance, polydopamine can 1) effectively fix and induce tissue regeneration drugs; 2) The release rate of the induced tissue regeneration medicine is regulated and controlled together with the degradable artificially synthesized polyester, the calcium-containing inorganic particles and the calcium-phosphorus layer, and the release period of the induced tissue regeneration medicine can reach more than 35 days; the calcium-phosphorus layer 1) on the surface of the microsphere can promote the adhesion and proliferation of cells on the surface of the microsphere; 2) Calcium ions and other medicines capable of synergistically inducing tissue regeneration and synergistically releasing calcium inorganic particles to play a role in promoting tissue regeneration and repair
In a word, the invention combines and uses the tissue regeneration inducing medicine, the calcium-containing inorganic particles, the polydopamine, the degradable artificially synthesized polyester and the mineralized calcium-phosphorus layer, so that the composite microsphere has good tissue regeneration inducing medicine release effect, the tissue regeneration inducing medicine release period can reach more than 35 days, and the composite microsphere has good biocompatibility and bioactivity, can be used as a bracket material to support the adhesion and proliferation of cells, and can effectively promote the repair and reconstruction of bone tissues. In addition, the preparation method is simple, the requirement on equipment is not high, the raw materials are industrialized, the sources are easily available, the cost is low, and industrialization is easy to realize.
Description of the drawings:
FIG. 1 is a graph showing the drug release performance of the composite microspheres prepared in examples 1 to 5 and comparative examples 2 to 6 in vitro induced tissue regeneration as a scaffold material;
FIG. 2 shows osteogenic differentiation performance of preosteoblasts induced in vitro by using the composite microspheres prepared in examples 1 to 5 and comparative examples 1 to 6 as scaffold materials.
The specific implementation mode is as follows:
the following is a further description of the invention and is not intended to be limiting.
Example 1:
dispersing 200mg of bioglass in 36ml of dopamine aqueous solution with the concentration of 2mg/ml, stirring for 3 hours, centrifuging, and cleaning to obtain polydopamine-containing bioglass; 100mg of polydopamine-containing bioglass is dispersed in 12ml of solution containing 0.01mg/ml bone morphogenetic protein 7, and the polydopamine-containing bioglass is obtained after centrifugation and freeze-drying. Dispersing 20mg of bioglass carrying polydopamine in 10ml of poly 3-hydroxybutyrate-co-3-hydroxyvalerate (molecular weight: 10 ten thousand daltons) solution to obtain polydopamine-carrying bioglass/poly 3-hydroxybutyrate-co-3-hydroxyvalerate solution blending solution; and preparing 800ml of 1.5mg/ml gelatin aqueous solution by using deionized water, then slowly dropwise adding the blending solution into the gelatin aqueous solution, continuously stirring at 400rpm for 18 hours, and separating out the composite microspheres at the bottom of the container to obtain the composite microspheres. Dispersing 1g of composite microspheres in 20ml of 10-time concentration simulated body fluid, standing for 12 hours, centrifuging, and cleaning to obtain the microspheres with the calcium-phosphorus layer.
Example 2:
dispersing 450mg of biphasic calcium phosphate into 36ml of dopamine aqueous solution with the concentration of 1.5mg/ml, stirring for 9 hours, centrifuging and cleaning to obtain the biphasic calcium phosphate containing the polydopamine; 100mg of polydopamine-containing biphasic calcium phosphate is dispersed in 3ml of solution containing 0.06mg/ml transforming growth factor, and the polydopamine-containing biphasic calcium phosphate is obtained after centrifugation and freeze-drying. Dispersing 200mg of double-phase calcium phosphate carrying polydopamine in 10ml of polycaprolactone (molecular weight: 4 kilodalton) solution to obtain a double-phase calcium phosphate/polycaprolactone solution blending solution carrying polydopamine; and then, preparing 400ml of a 8mg/ml methyl cellulose aqueous solution by using deionized water, slowly dropwise adding the blended solution into the methyl cellulose aqueous solution, continuously stirring at 250rpm for 15 hours, and separating out the composite microspheres at the bottom of the container to obtain the composite microspheres. Dispersing 1g of composite microspheres in 80ml of simulated body fluid with 6 times concentration, standing for 20h, centrifuging, and cleaning to obtain the microspheres with the calcium-phosphorus layer.
Example 3:
dispersing 200mg of hydroxyapatite in 36ml of dopamine aqueous solution with the concentration of 0.5mg/ml, stirring for 12 hours, centrifuging, and cleaning to obtain the dopamine-containing hydroxyapatite; dispersing 100mg of poly-dopamine-containing hydroxyapatite in 8ml of solution containing 0.05mg/ml of bone morphogenetic protein 2, centrifuging and freeze-drying to obtain the drug-loaded poly-dopamine-containing hydroxyapatite. Dispersing 100mg of hydroxyapatite carrying polydopamine in 10ml of polylactic acid-glycolic acid copolymer (molecular weight: 3 ten thousand daltons) solution to obtain a hydroxyapatite/polylactic acid-glycolic acid copolymer solution blend carrying polydopamine; then, 300ml of 10mg/ml polyvinyl alcohol 1799 aqueous solution is prepared by using deionized water, then the blended solution is slowly dripped into the polyvinyl alcohol 1799 aqueous solution, and after the mixture is continuously stirred at 300rpm for 12 hours, the composite microspheres at the bottom of the container are separated out, so that the composite microspheres are prepared. Dispersing 1g of composite microspheres in 50ml of simulated body fluid with 5 times concentration, standing for 24h, centrifuging, and cleaning to obtain the microspheres with the calcium-phosphorus layer.
Example 4:
dispersing 300mg of calcium carbonate into 36ml of dopamine aqueous solution with the concentration of 0.2mg/ml, stirring for 24 hours, centrifuging, and cleaning to obtain calcium carbonate containing polydopamine; 100mg of calcium carbonate containing polydopamine is dispersed in 12ml of solution containing 0.04mg/ml of fibroblast growth factor, and the calcium carbonate carrying the polydopamine is obtained by centrifugation and freeze-drying. Dispersing 150mg of calcium carbonate carrying polydopamine in 10ml of polylactic acid (molecular weight: 5 kilodalton) solution to obtain calcium carbonate/polylactic acid solution blending solution carrying polydopamine; then, 600ml of 20mg/ml gelatin aqueous solution is prepared by using deionized water, then the blended solution is slowly dripped into the gelatin aqueous solution, the mixture is continuously stirred at 600rpm for 16 hours, and then the composite microspheres at the bottom of the container are separated out, so that the composite microspheres are prepared. Dispersing 1g of composite microspheres in 120ml of simulated body fluid with 5 times concentration, standing for 16h, centrifuging, and cleaning to obtain the microspheres with the calcium-phosphorus layer.
Example 5:
dispersing 100mg of octacalcium phosphate into 36ml of dopamine aqueous solution with the concentration of 0.8mg/ml, stirring for 15, centrifuging, and cleaning to obtain octacalcium phosphate containing polydopamine; 100mg of octacalcium phosphate containing polydopamine is dispersed in 5ml of solution containing 0.1mg/ml of parathyroid hormone, and the octacalcium phosphate carrying the polydopamine is obtained by centrifugation and freeze-drying. Dispersing 350mg of octacalcium phosphate carrying polydopamine in 10ml of polylactic acid-glycolic acid copolymer (molecular weight: 1 ten thousand daltons) solution to obtain octacalcium phosphate/polylactic acid-glycolic acid copolymer solution blending solution carrying polydopamine; then, 500ml of 40mg/ml polyvinyl alcohol 1799 aqueous solution is prepared by deionized water, then the blended solution is slowly dripped into the polyvinyl alcohol 1799 aqueous solution, and after stirring is continuously carried out for 24 hours at 800rpm, the composite microspheres at the bottom of the container are separated out, thus obtaining the composite microspheres. Dispersing 1g of the composite microspheres in 160ml of simulated body fluid with 8 times of concentration, standing for 8h, centrifuging, and cleaning to obtain the microspheres with the calcium-phosphorus layer.
Comparative example 1:
the comparative example provides a preparation method of polyester-based microspheres containing drug-loaded calcium inorganic substance, which is substantially the same as that of example 3, except that: does not contain a drug for inducing tissue regeneration, and comprises the following steps:
dispersing 200mg of hydroxyapatite in 36ml of dopamine aqueous solution with the concentration of 0.5mg/ml, stirring for 12 hours, centrifuging, and cleaning to obtain the poly-dopamine-containing hydroxyapatite. Dispersing 100mg of poly-dopamine-containing hydroxyapatite in 10ml of poly (lactic acid-co-glycolic acid) (molecular weight: 3 kilodaltons) solution to obtain poly-dopamine-containing hydroxyapatite/poly (lactic acid-co-glycolic acid) solution blend; then, 300ml of 10mg/ml polyvinyl alcohol 1799 aqueous solution is prepared by using deionized water, then the blended solution is slowly dripped into the polyvinyl alcohol 1799 aqueous solution, and after the mixture is continuously stirred at 300rpm for 12 hours, the composite microspheres at the bottom of the container are separated out, so that the composite microspheres are prepared. Dispersing 1g of composite microspheres in 50ml of simulated body fluid with 5 times concentration, standing for 24h, centrifuging, and cleaning to obtain the microspheres with the calcium-phosphorus layer.
Comparative example 2:
the comparative example provides a preparation method of polyester-based microspheres containing drug-loaded calcium inorganic substance, which is substantially the same as that of example 3, except that: does not contain calcium-containing inorganic particles, comprising the steps of:
dispersing 0.36mg of bone morphogenetic protein 2 into 10ml of polycaprolactone (molecular weight: 4 ten thousand daltons) solution to obtain a polycaprolactone solution blending solution carrying the bone morphogenetic protein 2; and then, preparing 400ml of a 8mg/ml methyl cellulose aqueous solution by using deionized water, slowly dropwise adding the blended solution into the methyl cellulose aqueous solution, continuously stirring at 250rpm for 15 hours, and separating out the composite microspheres at the bottom of the container to obtain the composite microspheres. Dispersing 1g of composite microspheres in 80ml of simulated body fluid with 6 times concentration, standing for 20h, centrifuging, and cleaning to obtain the microspheres with the calcium-phosphorus layer.
Comparative example 3:
the comparative example provides a preparation method of polyester-based microspheres containing drug-loaded calcium inorganic substance, which is substantially the same as that of example 3, except that: does not contain polydopamine, comprising the following steps:
dispersing 100mg of hydroxyapatite in 8ml of solution containing 0.05mg/ml of bone morphogenetic protein 2, centrifuging and freeze-drying to obtain the drug-loaded poly-dopamine-containing hydroxyapatite. Dispersing 100mg of drug-loaded hydroxyapatite in 10ml of polylactic acid-glycolic acid copolymer (molecular weight: 3 kilodaltons) solution to obtain drug-loaded hydroxyapatite/polylactic acid-glycolic acid copolymer solution blend; then, 300ml of 10mg/ml polyvinyl alcohol 1799 aqueous solution is prepared by using deionized water, then the blended solution is slowly dripped into the polyvinyl alcohol 1799 aqueous solution, and after the mixture is continuously stirred at 300rpm for 12 hours, the composite microspheres at the bottom of the container are separated out, so that the composite microspheres are prepared. Dispersing 1g of composite microspheres in 50ml of simulated body fluid with 5 times concentration, standing for 24h, centrifuging, and cleaning to obtain the microspheres with the calcium-phosphorus layer.
Comparative example 4:
this comparative example provides a method for preparing polyester-based composite microspheres, which is substantially the same as example 3, except that: does not contain a calcium-phosphorus layer, comprising the steps of:
dispersing 200mg of hydroxyapatite in 36ml of dopamine aqueous solution with the concentration of 0.5mg/ml, stirring for 12 hours, centrifuging, and cleaning to obtain the dopamine-containing hydroxyapatite; dispersing 100mg of poly-dopamine-containing hydroxyapatite in 8ml of solution containing 0.05mg/ml of bone morphogenetic protein 2, centrifuging and freeze-drying to obtain the drug-loaded poly-dopamine-containing hydroxyapatite. Dispersing 100mg of hydroxyapatite carrying polydopamine in 10ml of polylactic acid-glycolic acid copolymer (molecular weight: 3 ten thousand daltons) solution to obtain a hydroxyapatite/polylactic acid-glycolic acid copolymer solution blend carrying polydopamine; then, 300ml of 10mg/ml polyvinyl alcohol 1799 aqueous solution is prepared by using deionized water, then the blended solution is slowly dripped into the polyvinyl alcohol 1799 aqueous solution, and after the mixture is continuously stirred at 300rpm for 12 hours, the composite microspheres at the bottom of the container are separated out, so that the composite microspheres are prepared.
Comparative example 5:
this comparative example provides a method for preparing polyester-based composite microspheres, which is substantially the same as example 3, except that: does not contain degradable polyester, and comprises the following steps:
dispersing 200mg of hydroxyapatite in 36ml of dopamine aqueous solution with the concentration of 0.5mg/ml, stirring for 12 hours, centrifuging, and cleaning to obtain the dopamine-containing hydroxyapatite; 100mg of poly-dopamine-containing hydroxyapatite is dispersed in 8ml of solution containing 0.05mg/ml of bone morphogenetic protein 2, and the drug-loaded poly-dopamine-containing hydroxyapatite is obtained through centrifugation and freeze-drying. Dispersing 1g of poly-dopamine-containing hydroxyapatite in 50ml of 5-fold simulated body fluid, standing for 24 hours, centrifuging, and cleaning to obtain the microsphere with the calcium-phosphorus layer.
Comparative example 6:
the comparative example provides a preparation method of polyester-based composite microspheres, which is substantially the same as that of example 3, except that: contains no Ca-P layer, polydopamine, and calcium-containing inorganic particles.
Test example 1:
the microspheres prepared in examples 1 to 5 and comparative examples 1 to 6 were subjected to the following property evaluations, and the results are shown in table 1.
1. In vitro cytotoxicity assessment
The prepared bracket is taken and evaluated and scored according to the requirements of GB/T16886.5. The results of the experiment are as follows:
TABLE 1 in vitro cytotoxicity scores of composite microspheres prepared in examples and comparative examples
2. In vitro induced tissue regeneration drug release performance detection
This test example tested the performance of the composite microspheres prepared in the examples and comparative examples. The specific method and the result are as follows:
the composite microspheres prepared in examples 1 to 5 and comparative examples 1 to 6 were evaluated for solute release in vitro, and the results are shown in fig. 1.
Environment: the temperature is 37 ℃, the stirring speed is 60rpm, and the temperature control device is a constant temperature shaking table;
liquid slow-release medium: PBS (pH =7.4 phosphate buffer);
the proportion is as follows: 50mg of composite microspheres: 20mLPBS;
and (3) measuring the release rate of the small-molecule water-soluble drug: periodically collecting PBS test solution, supplementing equivalent PBS, and measuring the content of the medicine for inducing tissue regeneration by High Performance Liquid Chromatography (HPLC);
substituting the absorbance of the solute at a certain time point into the standard curve to obtain the actual amount of the drug for inducing tissue regeneration at the time point; and dividing the actual amount by the total amount of the loaded tissue regeneration inducing drug in the composite microspheres to obtain the cumulative release amount of the tissue regeneration inducing drug at the time point.
3. In vitro induction preosteoblast osteogenic differentiation performance detection
After radiation sterilization, the composite microspheres are soaked in a DMEM basal medium according to the concentration of 10mg/mL, and then are put into a shaking table at 37 ℃ and extracted for 24 hours at 120 rpm. After completion of the leaching, the microspheres and the medium were centrifuged at 1000rpm and the supernatant was collected. And (3) diluting the collected leaching liquor by 2 times by using corresponding DMEM culture media respectively, and finally adding 10% fetal calf serum to obtain a complete culture medium.
MC3T3-E1 cells were plated at 1X 10 per well 5 The density of each seed was inoculated on a 24-well plate, and after adherent culture for 24 hours, the complete medium was replaced respectively, and the culture was carried out in an incubator at 37 ℃ and under 5% carbon dioxide atmosphere. The culture medium is changed every 2-3d, after 7 days of culture, the osteogenic differentiation performance of MC3T3-E1 cells is detected by secreted alkaline phosphatase, and the determination is carried out by using a pNPP method, and the specific steps are as follows: after the cells were washed with PBS solution, they were immersed in PBS solution containing 0.1M glycine, 1mM magnesium chloride and 0.05% Triton X-100. After the cells are dissolved, the dissolving solution is uniformly mixed with the p-nitrophenyl phosphate disodium salt, and the mixed solution is placed at 37 ℃ for 30min. Subsequently, the mixture was dropped into a 96-well plate, and the absorbance of each well at a wavelength of 405nm was measured with a microplate reader. The actual alkaline phosphatase content in the cells on each scaffold was calculated according to the formula.
As can be seen from the results of in vitro cytotoxicity evaluation (Table 1) of the examples and comparative examples, the composite microspheres prepared by the method of the present invention were all non-cytotoxic. As can be seen from the results of the in vitro tissue regeneration drug release performance test (FIG. 1 and Table 2), examples 1 to 5 all had long-lasting tissue regeneration drug release performance, and example 3 and comparative examples 2 to 6 were all based on composite microspheres loaded with tissue regeneration drug. In the process of preparing the composite microspheres in the embodiment 3, firstly, polydopamine is used for wrapping calcium-containing inorganic particles, then induced tissue regeneration drugs are adsorbed on the polydopamine-containing calcium-containing inorganic particles, then the drug-loaded polydopamine-containing calcium-containing inorganic particles are dispersed in a degradable artificially synthesized polyester network, and then the drug-loaded polydopamine-containing calcium-containing inorganic particles are solidified into microspheres by an emulsion solvent volatilization method, and a calcium-phosphorus layer is formed on the surfaces of the microspheres; comparative example 2 contains no calcium-containing inorganic particles and has less influence on the release of the drug for inducing tissue regeneration; in the comparative example 3, dopamine is not used for adsorbing and loading the induced tissue regeneration medicine, and the induced tissue regeneration medicine in the microspheres is released in 21 days, so that the long-acting sustained and controlled release effect cannot be achieved; comparative example 4 no calcium-phosphorus layer was formed on the surface of the microspheres, which resulted in a slightly faster release rate of the drug inducing tissue regeneration; comparative example 5 contains no degradable polyester, and the induced tissue regeneration drug is released in 21d, so that the long-acting sustained and controlled release effect cannot be achieved; comparative example 6 does not contain a calcium-phosphorus layer, polydopamine, calcium-containing inorganic particles, and the tissue regeneration inducing drug is released within 35 days, and the effect of long-acting sustained and controlled release cannot be achieved. Using the microspheres of example 3, the cumulative release on day 21 was 49.95%, i.e., the drug residue was 50.05%. While the cumulative release on day 21 using comparative examples 5 and 6 was 100% and 83.16%, respectively, i.e., the drug residue was 0% and 16.84%, respectively. The sum of the drug residue rates of comparative examples 5 and 6 is far lower than that of example 3, which shows that the polydopamine, the degradable artificially synthesized polyester, the calcium-containing inorganic particles and the calcium-phosphorus layer have synergistic effect, and the combination of the four components can achieve good slow release effect.
As can be seen from the osteogenic differentiation performance of preosteoblasts induced in vitro (FIG. 2), examples 1 to 5 all had a good effect of inducing cells to secrete alkaline phosphatase. Compared with example 3, comparative example 1, which is not loaded with the tissue regeneration-inducing drug, has significantly reduced osteoblast osteogenic differentiation performance before induction, and thus has the lowest concentration of alkaline phosphatase secreted from the cells; comparative example 2, in which calcium-containing inorganic particles were not used, osteoblasts had poor osteogenic differentiation properties before induction, and thus the concentration of alkaline phosphatase secreted from the cells was low; comparative example 3 in which polydopamine was not used, the amount of the loaded tissue regeneration-inducing drug was small, and thus osteoblasts were relatively weakly induced to differentiate into osteogenesis; comparative example 4, which does not contain a calcium-phosphorus layer, shows poor osteogenic differentiation of osteoblasts before induction, and thus the concentration of alkaline phosphatase secreted from the cells is also low; comparative example 5, which does not contain a degradable polyester, shows weak osteoblast osteogenic differentiation properties before induction, and thus shows low cell-secreted alkaline phosphatase concentration; comparative example 6 contains no calcium-phosphorus layer, polydopamine, calcium-containing inorganic particles, and the group of cells secretes alkaline phosphatase between example 3 and comparative example 5.
TABLE 2 cumulative drug release rates for the examples and comparative examples
Claims (10)
1. The polyester-based microsphere containing the medicine-carrying calcium-containing inorganic substance is characterized by sequentially comprising a calcium-containing inorganic particle containing polydopamine and used for loading the induced tissue regeneration medicine, degradable artificially synthesized polyester coating the calcium-containing inorganic particle containing polydopamine and used for loading the induced tissue regeneration medicine and a calcium-phosphorus layer on the surface of the polyester from inside to outside.
2. The polyester-based microspheres according to claim 1, wherein the calcium-containing inorganic particles are selected from any one of hydroxyapatite, bioglass, octacalcium phosphate, tricalcium phosphate, tetracalcium phosphate, calcium hydrogen phosphate, amorphous calcium phosphate, biphasic calcium phosphate, calcium carbonate, calcium bicarbonate, calcium sulfate; the size of the calcium-containing inorganic particles is 0.1-5 um.
3. The polyester-based microspheres according to claim 1, wherein the tissue regeneration-inducing drug is selected from any one of bone morphogenetic protein, vascular endothelial growth factor, platelet-derived factor, fibroblast growth factor, transforming growth factor, insulin-like growth factor, parathyroid hormone, growth hormone, interleukin, alendronate sodium, resveratrol, salmon calcitonin, zoledronate sodium, ibandronate sodium, strontium ranelate, naringin, dexamethasone, vitamin D.
4. The polyester-based microspheres according to claim 1, wherein the average particle size of the calcium-containing inorganic particles containing polydopamine loaded with a drug inducing tissue regeneration is 1 to 50um.
5. The polyester-based microspheres of claim 1, wherein the degradable synthetic polyester is selected from any one of polylactic acid, polylactic acid-glycolic acid copolymer, polycaprolactone, poly 3-hydroxyalkanoate, poly (3-hydroxybutyrate), poly 3-hydroxybutyrate-co-3-hydroxyvalerate, polytrimethylene carbonate, polybutylene succinate; the molecular weight of the degradable polyester is 1.0-10.0 ten thousand daltons.
6. The method for preparing the polyester-based microspheres containing drug-loaded calcium inorganic substance according to claim 1, is characterized by comprising the following steps:
(1) Wrapping calcium-containing inorganic particles with polydopamine, and adsorbing a tissue regeneration inducing drug onto the polydopamine-containing calcium-containing inorganic particles to prepare polydopamine-containing calcium-containing inorganic particles loaded with the tissue regeneration inducing drug;
(2) Calcium-containing inorganic particles containing polydopamine and loaded with a tissue regeneration inducing drug are dispersed into a degradable artificially synthesized polyester solution, then added into a water solution containing a surfactant, and solidified by an emulsion solvent volatilization method to form composite microspheres;
(3) Dispersing the composite microspheres into simulated body fluid with the concentration of 5-10 times, standing, centrifuging and cleaning to obtain the polyester-based microspheres containing the drug-loaded calcium-containing inorganic substance.
7. The method for preparing polyester-based microspheres loaded with drugs containing calcium inorganic substance according to claim 6, wherein the step (1) comprises the following steps: dispersing calcium-containing inorganic particles into an aqueous solution of dopamine with the concentration of 0.2-2 mg/ml, stirring for 3-24 h, centrifuging and cleaning to obtain the calcium-containing inorganic particles containing polydopamine, then dispersing the calcium-containing inorganic particles into a solution containing 0.01-0.1 mg/ml of induced tissue regeneration medicine, centrifuging and freeze-drying to obtain the calcium-containing inorganic particles containing polydopamine and loaded with the induced tissue regeneration medicine; the mass-volume ratio of the calcium-containing inorganic particles to the dopamine aqueous solution is 1: 20-90 g/ml; the mass-volume ratio of the calcium-containing inorganic particles containing polydopamine to the solution containing the medicine for inducing tissue regeneration is 1:30 to 120g/ml.
8. The method for preparing polyester-based microspheres loaded with drugs containing calcium inorganic substance according to claim 6, wherein the step (2) comprises the following steps: calcium-containing inorganic particles loaded with a tissue regeneration inducing drug and containing polydopamine are dispersed in a degradable artificially synthesized polyester solution; slowly dripping the solution into 300-800ml of 1.5-40 mg/ml of surfactant aqueous solution, continuously stirring at 250-800 rpm for 12-24 h, and separating out the composite microspheres at the bottom of the container to obtain composite microspheres; the mass ratio of the calcium-containing inorganic particles containing polydopamine and loaded with the induced tissue regeneration medicine to the degradable artificially synthesized polyester is 0.02-0.35: 1; the surfactant is selected from any one of polyvinyl alcohol, gelatin, methyl cellulose and carboxymethyl cellulose.
9. The preparation method of the polyester-based microspheres containing drug-loaded calcium inorganic substance according to claim 6, wherein the standing time in the step (3) is 12-48 h, and the mass-to-volume ratio of the composite microspheres to the simulated body fluid is 1:20 to 160g/ml.
10. The use of the polyester-based microspheres loaded with drugs containing calcium minerals as claimed in claim 1, wherein the polyester-based microspheres are used as a scaffold material to support the adhesion and proliferation of cells and promote the repair and reconstruction of bone tissue under bacterial infection.
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