Injectable polyester polymer / beta-tricalcium phosphate composite microsphere as well as preparation method and application thereof

Abstract

The invention provides injectable polyester polymer / beta-tricalcium phosphate composite microspheres as well as a preparation method and application thereof. The preparation method of the composite microspheres comprises the following steps: (1) providing beta-tricalcium phosphate; (2) mixing a polyester polymer and the beta-tricalcium phosphate according to a mass ratio of (1-20): (0.1-5), and carrying out first homogeneous emulsification treatment to obtain a water-in-oil emulsion; (3) mixing the water-in-oil emulsion with an external water phase solution, and carrying out second homogeneous emulsification treatment to obtain a water-in-oil-in-water emulsion; and (4) curing the water-in-oil-in-water emulsion, washing with water, centrifuging, and freeze-drying to obtain the composite microsphere. The composite microsphere has a porous structure, the particle size is 20-50 microns, fibroblast adhesion and vascular invasion can be promoted, collagen regeneration is synchronously stimulated inside and outside, and the filling and tissue regeneration effects are improved.

Description

Technical Field

[0001] The invention relates to the field of medical materials, and in particular to an injectable polyester polymer / β-tricalcium phosphate composite microsphere and a preparation method and application thereof. Background Art

[0002] Polyester polymers such as polylactic acid (PLA), polyglycolic acid (PGA) and polycaprolactone (PCL) are widely used in the biomedical field due to their good biocompatibility and biodegradability. These materials are often used in surgical sutures, implants, and drug release systems. For example, PLGA, a copolymer of polylactic acid and polyglycolic acid, is often used as a drug carrier due to its adjustable degradation rate and good biocompatibility.

[0003] In addition, polyester polymers can also be combined with other polymers or natural materials to form composite materials to enhance their performance. For example, elastic poly (l-lactide-co-ε-caprolactone) (PLCL) porous microspheres are combined with muscle extracellular matrix (mECM) to form an injectable functional composite material system, which showed good injectability and tissue compatibility in rat subcutaneous injection experiments. This composite material can significantly promote the formation, vascularization and neuralization of new muscle fibers by regulating the behavior of macrophages and myogenic cells in the injured area.

[0004] Polyester polymers can also be chemically modified to improve their hydrophobicity, thereby improving their stability in dermal fillers and comfort during injection. For example, by blending polyamino acid esters with aliphatic polyesters or forming block copolymers, composite micromaterials with good properties can be prepared.

[0005] Polyester polymers have significant advantages in the application of injectable composites, including good biocompatibility, tunable degradation rate, and excellent mechanical properties. These properties make them promising for a wide range of applications in drug delivery systems and tissue engineering.

[0006] Although this material has many advantages, it still faces some challenges in practical applications, such as insufficient mechanical properties. These problems can be solved by further optimizing the preparation process and improving the material formulation. Summary of the invention

[0007] The present invention aims to solve one of the technical problems in the related art to at least a certain extent. The present invention provides a polyester polymer / β-tricalcium phosphate composite microsphere, which can combine the advantages of polyester polymer and β-tricalcium phosphate by compounding polyester polymer and β-tricalcium phosphate, achieve good biocompatibility and degradability, and can be applied to the field of tissue regeneration.

[0008] Specifically, the present invention provides the following technical solutions:

[0009] In a first aspect of the present invention, a method for preparing injectable polyester polymer / β-tricalcium phosphate composite microspheres is provided, comprising:

[0010] (1) Providing β-tricalcium phosphate;

[0011] (2) mixing the polyester polymer and the β-tricalcium phosphate, and performing a first homogenization and emulsification treatment to obtain a water-in-oil emulsion, wherein the mass ratio of the polyester polymer to the β-tricalcium phosphate is: (1-20): (0.1-5);

[0012] (3) mixing the water-in-oil emulsion and the external aqueous phase solution, and performing a second homogenization emulsification treatment to obtain a water-in-oil-in-water emulsion;

[0013] (4) solidifying the water-in-oil-in-water emulsion, washing with water, centrifuging and then freeze-drying to obtain the composite microspheres.

[0014] The preparation method of the composite microspheres provided by the present invention is simple to operate. The prepared composite microspheres combine the advantages of polyester polymers (such as polylactic acid-glycolic acid copolymer PLGA) and tricalcium phosphate (TCP) to achieve good biocompatibility and degradability, and can be widely used in the fields of medical beauty injection filling or tissue engineering. β-tricalcium phosphate, as a natural bone component, has good osteoconductivity and biocompatibility. Adding it to PLGA can reduce the acidic environment generated when PLGA degrades, thereby buffering the stimulation of the degradation products to the surrounding tissues. The porous structure of β-tricalcium phosphate can degrade quickly and release a large amount of Ca 2 + and P3+ ions, promoting the growth of new tissue.

[0015] According to an embodiment of the present invention, the above-mentioned preparation method may further include the following technical features:

[0016] According to an embodiment of the present invention, the β-tricalcium phosphate is prepared by the following method:

[0017] 0.4 mol / L diammonium hydrogen phosphate solution and 0.6 mol / L calcium nitrate tetrahydrate solution are mixed and reacted, wherein the molar ratio of calcium to phosphorus is 1.5;

[0018] During the mixing process, the pH of the mixture is controlled to be 7.3±0.2, centrifuged, and the obtained precipitate is washed and dried, sintered, and ball-milled to obtain the β-tricalcium phosphate.

[0019] According to an embodiment of the present invention, the polyester polymer is selected from at least one of PCL, PLA, PLLA, PLGA, PHA, PLGA-PEG, PLA-PEG, PLLA-PEG, PCL-PEG, PLGA-PEG-PLGA, PLA-PEG-PLGA, PLLA-PEG-PLGA, PCL-PEG-PLGA, PLGA-PEG-PLA, PLA-PEG-PLA, PLLA-PEG-PLA, PCL-PEG-PLA, PLGA-PEG-PLLA, PLA-PEG-PLLA, PLLA-PEG-PLLA, PCL-PEG-PLLA, PLGA-PEG-PCL, PLA-PEG-PCL, PLLA-PEG-PCL and PCL-PEG-PCL.

[0020] According to an embodiment of the present invention, the water-in-oil-in-water emulsion comprises an oil phase, an aqueous phase, and an external aqueous phase, the mass ratio of the oil phase to the aqueous phase is (0.1-10):(0.1-10), and the oil phase comprises a polyester polymer, a surfactant, and an organic solvent;

[0021] The aqueous phase includes a surfactant, a pore-forming agent, β-tricalcium phosphate and purified water;

[0022] The external aqueous phase includes a dispersant and purified water.

[0023] According to an embodiment of the present invention, the organic solvent is one or more of dichloromethane, chloroform, and ethyl acetate.

[0024] According to an embodiment of the present invention, the dispersant is one or more of polyvinyl alcohol (PVA) and polyvinyl butyral (PVB).

[0025] According to an embodiment of the present invention, the surfactant is one or more of sodium dodecyl sulfate (SDS), Tween 80, Tween 20, Span 80, Span 20, and cetyltrimethylammonium bromide (CTAB).

[0026] According to an embodiment of the present invention, the pore-forming agent is at least one of ammonium bicarbonate, sodium bicarbonate, hydrogen peroxide, sodium chloride, and potassium chloride.

[0027] According to an embodiment of the present invention, the first homogenization and emulsification treatment is performed at a speed of 3000-10000 rpm / min, and the time of the first homogenization and emulsification treatment is 2-10 min;

[0028] The second homogenization and emulsification treatment is performed at a rotation speed of 2000-5000 rpm / min, and the time of the second homogenization and emulsification treatment is 2-10 min.

[0029] According to an embodiment of the present invention, step (4) further comprises:

[0030] (4-1) solidifying the water-in-oil-in-water emulsion and volatilizing the organic solvent by vacuuming;

[0031] (4-2) filtering through a 200-600 mesh sieve, washing with water, and centrifuging to obtain a precipitate,

[0032] (4-3) freeze-drying the precipitate to obtain the composite microspheres.

[0033] According to an embodiment of the present invention, the precipitate is frozen at -80°C for 3-4 hours; then the freeze dryer parameters are set to perform freeze drying for 48-72 hours through pre-freezing, sublimation, and analytical drying.

[0034] The second aspect of the present invention provides a method for preparing injectable polyester polymer / β-tricalcium phosphate composite microspheres, comprising:

[0035] An oil phase consisting of a polyester polymer, a surfactant and an organic solvent and a water phase consisting of β-tricalcium phosphate, a surfactant, a pore-forming agent and purified water are mixed and subjected to a first homogenization treatment to obtain a water-in-oil o / w emulsion;

[0036] Mixing an external aqueous phase consisting of a dispersant and purified water with the water-in-oil (o / w) emulsion, and performing a second homogenization treatment to obtain a water-in-oil-in-water (w / o / w) emulsion;

[0037] The water-in-oil-in-water (w / o / w) emulsion is solidified, the organic solvent is volatilized, and the emulsion is washed with water, centrifuged, and then freeze-dried to obtain polyester polymer / β-tricalcium phosphate composite microspheres;

[0038] Wherein the β-tricalcium phosphate is obtained by the following method:

[0039] 0.4 mol / L diammonium hydrogen phosphate solution and 0.6 mol / L calcium nitrate tetrahydrate solution are mixed and reacted, wherein the molar ratio of calcium to phosphorus is 1.5;

[0040] During the mixing process, the pH of the mixture is controlled to be 7.3±0.2 by adding ammonia water. The mixed product is stirred (for example, it can be stirred for 3 hours) and centrifuged. The obtained precipitate is washed and dried, sintered, and ball-milled to obtain the β-tricalcium phosphate.

[0041] The third aspect of the present invention provides an injectable polyester polymer / β-tricalcium phosphate composite microsphere, comprising a polyester polymer and β-tricalcium phosphate powder doped in the polyester polymer, wherein the mass ratio of the polyester polymer to the tricalcium phosphate powder is (1-20): (0.1-5);

[0042] According to an embodiment of the present invention, the polyester polymer / β-tricalcium phosphate composite microspheres are prepared according to the preparation method described in the first aspect or the second aspect.

[0043] The fourth aspect of the present invention provides the use of the above-mentioned injectable polyester polymer / β-tricalcium phosphate composite microspheres in the preparation of tissue regeneration materials.

[0044] The beneficial effects achieved by the present invention are:

[0045] The composite microspheres provided by the present invention form a porous structure (particle size is, for example, 20-50 μm) during the preparation process, and utilize the difference in degradation rates of β-tricalcium phosphate and polyester polymer to improve the degradation performance of a single material, promote fibroblast adhesion and vascular invasion, and simultaneously stimulate collagen regeneration internally and externally, thereby improving the filling and tissue regeneration effects, and can be applied in the field of tissue regeneration. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 3 is a particle size distribution diagram of the composite microspheres provided according to Example 1 of the present invention.

[0047] Figure 2 1 is a scanning electron microscope image of the composite microspheres provided according to Example 1 of the present invention.

[0048] Figure 3 is a DSC graph of the composite microspheres provided according to Example 1 of the present invention.

[0049] Figure 4 It is a Fourier infrared spectrum of the composite microspheres provided according to Example 1 of the present invention.

[0050] Figure 5 is a thermogravimetric analysis diagram of the composite microspheres provided in Example 1 of the present invention.

[0051] Figure 6 3 is a scanning electron microscope image of the composite microspheres provided according to Example 2 of the present invention. DETAILED DESCRIPTION

[0052] Embodiments of the present invention are described in detail below. Examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present invention, but should not be construed as limiting the present invention.

[0053] The present invention provides a method for preparing injectable polyester polymer / β-tricalcium phosphate composite microspheres, comprising:

[0054] S01. Preparation of β-tricalcium phosphate;

[0055] S02. The polyester polymer and tricalcium phosphate are homogenized and emulsified to obtain a water-in-oil-in-water emulsion;

[0056] S03. freeze-drying the water-in-oil-in-water emulsion to obtain polyester polymer / β-tricalcium phosphate composite microspheres.

[0057] Compared with traditional inorganic hydroxyapatite microspheres, the polyester polymer / β-tricalcium phosphate composite microspheres provided by the present invention form a porous structure during the preparation process, utilize the difference in degradation rates of tricalcium phosphate and polyester polymer to improve the degradation performance of a single material, promote fibroblast adhesion and vascular invasion, stimulate collagen regeneration internally and externally, and improve the filling and tissue regeneration effects.

[0058] S01. Preparation of β-tricalcium phosphate

[0059] The β-tricalcium phosphate mentioned can be prepared by the following method:

[0060] A mixed product is obtained by mixing a 0.4 mol / L diammonium hydrogen phosphate solution and a 0.6 mol / L calcium nitrate tetrahydrate solution, wherein the molar ratio of calcium to phosphorus is 1.5;

[0061] During the mixing process, the pH of the solution is controlled to be 7.3±0.2 (for example, the pH of the solution can be controlled by gradually adding ammonia water). After the dropwise addition is completed, the solution is stirred for a certain period of time (for example, it can be stirred for 2-4 hours), centrifuged, and the precipitate is washed and dried, sintered, and ball-milled to obtain the β-tricalcium phosphate.

[0062] According to a specific embodiment, the steps for preparing β-tricalcium phosphate are as follows:

[0063] It is prepared by mixing 0.4 mol / L diammonium hydrogen phosphate solution and 0.6 mol / L calcium nitrate tetrahydrate solution, wherein the molar ratio of calcium to phosphorus is 1.5. Ammonia water is used to adjust the pH to 7.3±0.2, and after the addition is completed, the mixture is stirred for 2-3 hours, centrifuged to discard the supernatant, washed with purified water for 3-5 times, dried for 48 hours, sintered at 1000°C for 3 hours, cooled to room temperature, and ball-milled to obtain β-tricalcium phosphate powder.

[0064] S02. The polyester polymer and β-tricalcium phosphate are homogenized and emulsified to obtain a water-in-oil-in-water emulsion.

[0065] The polyester polymer and β-tricalcium phosphate are homogenized and emulsified, and in the formed emulsion, β-tricalcium phosphate is doped in the polyester polymer, and the mass ratio of the β-tricalcium phosphate to the polyester polymer is (0.1-5):(1-20). According to a specific embodiment, the composite microspheres include a polyester polymer and a tricalcium phosphate powder doped in the polyester polymer, and the mass ratio of the tricalcium phosphate powder to the polyester polymer is (0.1-5):(1-20) (for example, (0.1-2):(1-20)).

[0066] The polyester polymer is selected from PCL (poly-ε-caprolactone), PLA (polylactic acid), PLLA (levorotatory polylactic acid), PLGA (polylactic acid-co-glycolic acid), PHA (polyhydroxyalkanoate), PLGA-PEG (polylactic acid-co-glycolic acid-polyethylene glycol block copolymer), PLA-PEG (polylactic acid-polyethylene glycol block copolymer), PLLA-PEG (levorotatory polylactic acid-polyethylene glycol block copolymer), PCL-PEG (poly-ε-caprolactone-polyethylene glycol block copolymer), PLGA-PEG-PLGA (polylactic acid-co-glycolic acid-polyethylene glycol block copolymer-polylactic acid-hydroxy acetic acid copolymer block copolymer), PLA-PEG-PLGA (polylactic acid-polyethylene glycol block copolymer-polylactic acid-glycolic acid copolymer block copolymer), PLLA-PEG-PLGA (L-polylactic acid-polyethylene glycol block copolymer-polylactic acid-glycolic acid copolymer block copolymer), PCL-PEG-PLGA (poly-ε-caprolactone-polyethylene glycol block copolymer-polylactic acid-glycolic acid copolymer block copolymer), PLGA-PEG-PLA (polylactic acid-glycolic acid copolymer-polyethylene glycol block copolymer-polylactic acid block copolymer), PLA-PEG-PLA (polylactic acid-polyethylene glycol block copolymer Block copolymer-polylactic acid block copolymer), PLLA-PEG-PLA (levorotatory polylactic acid-polyethylene glycol block copolymer-polylactic acid block copolymer), PCL-PEG-PLA (poly-ε-caprolactone-polyethylene glycol block copolymer-polylactic acid block copolymer), PLGA-PEG-PLLA (polylactic acid-co-glycolic acid copolymer-polyethylene glycol block copolymer-levorotatory polylactic acid block copolymer), PLA-PEG-PLLA (polylactic acid-polyethylene glycol block copolymer-levorotatory polylactic acid block copolymer), PLLA-PEG-PLLA (levorotatory polylactic acid-polyethylene glycol block copolymer-levorotatory polylactic acid block copolymer) ), PCL-PEG-PLLA (poly-ε-caprolactone-polyethylene glycol block copolymer-levorotatory polylactic acid block copolymer), PLGA-PEG-PCL (polylactic acid-glycolic acid copolymer-polyethylene glycol block copolymer-poly-ε-caprolactone block copolymer), PLA-PEG-PCL (polylactic acid-polyethylene glycol block copolymer-poly-ε-caprolactone block copolymer), PLLA-PEG-PCL (levorotatory polylactic acid-polyethylene glycol block copolymer-poly-ε-caprolactone block copolymer) and PCL-PEG-PCL (poly-ε-caprolactone-polyethylene glycol block copolymer-poly-ε-caprolactone block copolymer).

[0067] The obtained water-in-oil-in-water emulsion consists of an oil phase, an aqueous phase and an external aqueous phase, and the mass ratio of the oil phase to the aqueous phase is (0.1-10):(0.1-10); the oil phase includes a polyester polymer, a surfactant and an organic solvent, the aqueous phase includes a surfactant, a pore-forming agent, β-tricalcium phosphate and purified water, and the external aqueous phase includes a dispersant and purified water.

[0068] The organic solvent mentioned is one or more of dichloromethane, chloroform and ethyl acetate. According to a preferred embodiment, the organic solvent is ethyl acetate.

[0069] The dispersant mentioned is one or more of polyvinyl alcohol (PVA) and polyvinyl butyral (PVB).

[0070] The surfactant mentioned is one or more of sodium dodecyl sulfate (SDS), Tween 80, Tween 20, Span 80, Span 20, and cetyltrimethylammonium bromide (CTAB).

[0071] The pore-forming agent mentioned is at least one of ammonium bicarbonate, sodium bicarbonate, hydrogen peroxide, sodium chloride and potassium chloride. According to a preferred embodiment, the pore-forming agent mentioned is ammonium bicarbonate.

[0072] In step S02, the polyester polymer and the β-tricalcium phosphate are mixed and subjected to a first homogenization and emulsification treatment, wherein the first homogenization and emulsification time is 2-10 minutes and the speed of the homogenizer is 3000-10000 rpm / min. The first homogenization and emulsification obtains an oil-in-water emulsion (o / w) emulsion.

[0073] Then the water-in-oil emulsion and the external aqueous phase solution are mixed and subjected to a second homogenization emulsification treatment to obtain a water-in-oil-in-water emulsion (w / o / w). According to a specific embodiment, the second homogenization emulsification treatment is carried out at a rotation speed of 2000-5000 rpm / min, and the time of the second homogenization emulsification treatment is 2-10 minutes.

[0074] After the above two homogenization treatments, the emulsion is pumped by a vacuum pump for 1-3 hours. Then it is passed through a 200-600 mesh screen. The polyester polymer / tricalcium phosphate solution after screening is washed and centrifuged several times, the centrifugal speed is 3000-5000rpm / min, and the centrifugal time is 5-20min.

[0075] S03. Freeze-drying the water-in-oil-in-water emulsion

[0076] The freeze-drying process conditions in step S03 are: placing the precipitate after centrifugation in a container and freezing it in a -80°C refrigerator for 4-8 hours; then directly placing it in a freeze dryer that has been cooled and vacuumed in advance, and freeze-drying it for 48-72 hours.

[0077] The freeze dryer should be turned on 1.5 hours in advance to cool the plate layer before loading. Sample loading should start when the plate layer drops below -55℃. The loading speed should be fast to ensure that the loading is completed within 3 minutes.

[0078] Freeze-drying process curve:

[0079] Prefreeze at -55°C for 0 min, 30 min;

[0080] Sublimation 5℃ 60min, 2000min;

[0081] Desorption 15℃ 0 min, 240 min

[0082] After freeze-drying, directly pack the surface dish and add desiccant to prevent moisture.

[0083] The prepared composite microspheres can be used for fat reduction, subcutaneous filling, and bioactive composite materials, etc. These materials have broad application prospects in the fields of medicine and bioengineering, such as tissue engineering and drug delivery.

[0084] The technical scheme of the present invention is described below by specific examples. It should be noted that these examples are only used to facilitate the understanding of those skilled in the art and should not be regarded as limiting the scope of protection of the present invention. Unless otherwise specified, the reagents used in the examples can be purchased.

[0085] Example 1

[0086] Example 1 Composite microspheres were prepared by the following method, comprising:

[0087] (1) Add 0.5 mL of 5% span 20 and 0.5 g of PLGA (poly(lactic-co-glycolic acid)) into 20 mL of ethyl acetate and stir to dissolve (until the solution is clear and transparent).

[0088] (2) Add 0.2 g of β-TCP (β-tricalcium phosphate) powder and 0.5 mL of 10% Tween 20 to 5 mL of 5% ammonium bicarbonate and stir to dissolve.

[0089] (3) The solution in the above step (1) is placed under a homogenizer. After the speed stabilizes at 7500 rpm / min, the liquid in step (2) is added thereto. The timing is stopped after 2 minutes to obtain an o / w emulsion.

[0090] (4) Place 200 mL of 1% polyvinyl alcohol (PVA) aqueous solution under a homogenizer. After the speed stabilizes at 3200 rpm / min, add the o / w emulsion in step (3) thereto. Stop after 5 minutes to obtain a w / o / w emulsion.

[0091] (5) Set up the three-necked flask in advance and start stirring at 400 rpm / min. Continue stirring until the ethyl acetate is completely evaporated.

[0092] (6) The product obtained in step (5) was sieved and centrifuged at 5000 rpm / min for 10 min to obtain a precipitate, which was then resuspended in water, washed and centrifuged 5 times. After centrifugation, the microsphere product was freeze-dried to obtain the microsphere product.

[0093] Example 2

[0094] Example 2 Composite microspheres were prepared by the following method:

[0095] (1) Add 1 mL of 3% span80 and 1 g of PLGA into 40 mL of ethyl acetate and stir to dissolve (until the solution is clear and transparent).

[0096] (2) Add 0.5 g of β-TCP powder and 10 mL of 5% Tween 80 into 10 mL of 3% sodium chloride and stir to dissolve.

[0097] (3) The solution in the above step (1) is placed under a homogenizer. After the speed stabilizes at 7500 rpm / min, the liquid in step (2) is added thereto. The timing is stopped after 2 minutes to obtain an o / w emulsion.

[0098] (4) Place 400 mL of 2% polyvinyl alcohol (PVA) aqueous solution under a homogenizer. After the speed stabilizes at 3000 rpm / min, add the o / w emulsion in step (3) thereto. Stop after 5 minutes to obtain a w / o / w emulsion.

[0099] (5) Set up the three-necked flask in advance and start stirring at 400 rpm / min. Continue stirring until the ethyl acetate is completely evaporated.

[0100] (6) The product obtained in step (5) was sieved and centrifuged at 5000 rpm / min for 10 min to obtain a precipitate, which was then resuspended in water, washed and centrifuged 5 times. After centrifugation, the microsphere product was freeze-dried to obtain the microsphere product.

[0101] Example 3

[0102] Example 3 Composite microspheres were prepared by the following method:

[0103] (1) Add 1 mL of 5% span 20 and 1 g of PLLA into 30 mL of dichloromethane and stir to dissolve (until the solution is clear and transparent).

[0104] (2) Add 0.5 g of β-TCP powder and 2 mL of 10% Tween 20 into 10 mL of 5% ammonium bicarbonate and stir to dissolve.

[0105] (3) The solution in the above (1) was placed in a homogenizer. After the speed stabilized at 7500 rpm / min, the liquid in step (2) was added thereto. The timing was stopped after 2 minutes to obtain an o / w emulsion.

[0106] (4) Place 400 mL of 2% polyvinyl alcohol (PVA) aqueous solution under a homogenizer. After the speed stabilizes at 3200 rpm / min, add the o / w emulsion in (3) thereto. Stop the process after 5 minutes to obtain a w / o / w emulsion.

[0107] (5) Set up the three-necked flask in advance and start stirring at 400 rpm / min. Continue stirring until the dichloromethane is completely evaporated.

[0108] (6) The product obtained in step (5) was sieved and centrifuged at 5000 rpm / min for 10 min to obtain a precipitate, which was then resuspended in water, washed and centrifuged 5 times. After centrifugation, the microsphere product was freeze-dried to obtain the microsphere product.

[0109] Example 4

[0110] Example 4 Composite microspheres were prepared by the following method:

[0111] (1) Add 1 mL of 1% span 80 and 1 g of PLLA (poly-L-lactic acid) into 40 mL of dichloromethane and stir to dissolve (until the solution is clear and transparent).

[0112] (2) Add 0.4 g of β-TCP powder and 1 mL of 10% Tween 80 into 10 mL of 3% ammonium bicarbonate and stir to dissolve.

[0113] (3) The solution in the above (1) was placed in a homogenizer. After the speed stabilized at 8000 rpm / min, the liquid in step (2) was added thereto. The timing was stopped after 2 minutes to obtain an o / w emulsion.

[0114] (4) Place 400 mL of 1% polyvinyl alcohol (PVA) aqueous solution under a homogenizer. After the speed stabilizes at 3000 rpm / min, add the o / w emulsion in (3) and stop after 5 minutes to obtain a w / o / w emulsion.

[0115] (5) Set up the three-necked flask in advance and start stirring at 400 rpm / min. Continue stirring until the dichloromethane is completely evaporated.

[0116] (6) The product obtained in step (5) was sieved and centrifuged at 5000 rpm / min for 10 min to obtain a precipitate, which was then resuspended in water, washed and centrifuged 5 times. After centrifugation, the microsphere product was freeze-dried to obtain the microsphere product.

[0117] Taking Example 1 as an example, the prepared composite microspheres were characterized as follows.

[0118] Figure 1 FIG. 1 is a particle size distribution diagram of the composite microspheres provided in Example 1 of the present invention. Figure 1 It can be seen that the D10 of the prepared composite microspheres is 14.30, D50 is 38.61, and D90 is 62.80. D10 means that in the particle size distribution, particles less than or equal to this particle size account for 10% of the total sample; D50 means that in the particle size distribution, particles less than or equal to this particle size account for 50% of the total sample. D90 means that in the particle size distribution, particles less than or equal to this particle size account for 90% of the total sample. It can be seen that the average particle size of the prepared composite microspheres is about 30 to 40 microns.

[0119] Figure 2 1 is a scanning electron microscope image of the composite microspheres provided according to Example 1 of the present invention. Combining the scanning electron microscope results, it can be seen that the surface of the composite microspheres is porous.

[0120] Figure 3 is a DSC graph of the composite microspheres provided according to Example 1 of the present invention.

[0121] Figure 4 It is a Fourier infrared spectrum of the composite microspheres provided according to Example 1 of the present invention.

[0122] Figure 5 is a thermogravimetric analysis diagram of the composite microspheres provided in Example 1 of the present invention.

[0123] The composite microspheres prepared in Example 2 were characterized, and the SEM scanning electron microscopy results were as follows: Figure 6 As shown. Combined Figure 6 The results given show that the pore-forming effect of sodium chloride is not as good as that of ammonium bicarbonate.

[0124] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific implementation", or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic expressions of the above terms do not necessarily refer to the same embodiment or example.

[0125] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A method for preparing injectable polyester polymer / β-tricalcium phosphate composite microspheres, characterized in that: include: (1) Providing β-tricalcium phosphate; (2) mixing the polyester polymer and the β-tricalcium phosphate, and performing a first homogenization and emulsification treatment to obtain a water-in-oil emulsion, wherein the mass ratio of the polyester polymer to the β-tricalcium phosphate is: (1-20): (0.1-5); (3) mixing the water-in-oil emulsion and the external aqueous phase solution, and performing a second homogenization emulsification treatment to obtain a water-in-oil-in-water emulsion; (4) solidifying the water-in-oil-in-water emulsion, washing with water, centrifuging and then freeze-drying to obtain the composite microspheres.

2. The preparation method according to claim 1, characterized in that: The β-tricalcium phosphate is prepared by the following method: 0.4 mol / L diammonium hydrogen phosphate solution and 0.6 mol / L calcium nitrate tetrahydrate solution are mixed and reacted, wherein the molar ratio of calcium to phosphorus is 1.5; During the mixing process, the pH of the mixture is controlled to be 7.3±0.2, centrifuged, and the obtained precipitate is washed and dried, sintered, and ball-milled to obtain the β-tricalcium phosphate.

3. The preparation method according to claim 1, characterized in that: The polyester polymer is selected from PCL, PLA, PLLA, PLGA, PHA, PLGA-PEG, PLA-PEG, PLLA-PEG, PCL-PEG, PLGA-PEG-PLGA, PLA-PEG-PLGA, PLLA-PEG-PLGA, PCL-PEG-PLGA, PLGA-PEG-PLA, PLA-PEG -At least one of PLA, PLLA-PEG-PLA, PCL-PEG-PLA, PLGA-PEG-PLLA, PLA-PEG-PLLA, PLLA-PEG-PLLA, PCL-PEG-PLLA, PLGA-PEG-PCL, PLA-PEG-PCL, PLLA-PEG-PCL and PCL-PEG-PCL.

4. The preparation method according to claim 1, characterized in that: The water-in-oil-in-water emulsion comprises an oil phase, an aqueous phase, and an external aqueous phase, and the mass ratio of the oil phase to the aqueous phase is (0.1-10): (0.1-10); The oil phase includes a polyester polymer, a surfactant and an organic solvent; The aqueous phase includes a surfactant, a pore-forming agent, β-tricalcium phosphate and purified water; The external aqueous phase includes a dispersant and purified water.

5. The preparation method according to claim 4, characterized in that: The organic solvent is one or more of dichloromethane, chloroform, and ethyl acetate; Optionally, the dispersant is one or more of polyvinyl alcohol (PVA) and polyvinyl butyral (PVB); Optionally, the surfactant is one or more of sodium dodecyl sulfate (SDS), Tween 80, Tween 20, Span 80, Span 20, and cetyltrimethylammonium bromide (CTAB); Optionally, the pore-forming agent is at least one of ammonium bicarbonate, sodium bicarbonate, hydrogen peroxide, sodium chloride, and potassium chloride.

6. The preparation method according to claim 1, characterized in that: The first homogenization and emulsification treatment is performed at a speed of 3000-10000 rpm / min, and the time of the first homogenization and emulsification treatment is 2-10 min; The second homogenization and emulsification treatment is performed at a rotation speed of 2000-5000 rpm / min, and the time of the second homogenization and emulsification treatment is 2-10 min.

7. The preparation method according to claim 1, characterized in that: Step (4) further comprises: (4-1) solidifying the water-in-oil-in-water emulsion and volatilizing the organic solvent by vacuuming; (4-2) filtering through a 200-600 mesh sieve, washing with water, and centrifuging to obtain a precipitate, (4-3) freeze-drying the precipitate to obtain the composite microspheres; Optionally, the precipitate is frozen at -80°C for 3-4 hours; then the freeze dryer parameters are set to perform freeze drying for 48-72 hours through pre-freezing, sublimation, and analytical drying to obtain the composite microspheres.

8. A method for preparing injectable polyester polymer / β-tricalcium phosphate composite microspheres, characterized in that: include: An oil phase consisting of a polyester polymer, a surfactant and an organic solvent and a water phase consisting of β-tricalcium phosphate, a surfactant, a pore-forming agent and purified water are mixed and subjected to a first homogenization treatment to obtain a water-in-oil o / w emulsion; Mixing an external aqueous phase consisting of a dispersant and purified water with the water-in-oil (o / w) emulsion, and performing a second homogenization treatment to obtain a water-in-oil-in-water (w / o / w) emulsion; The water-in-oil-in-water (w / o / w) emulsion is solidified, the organic solvent is volatilized, and the emulsion is washed with water, centrifuged, and then freeze-dried to obtain polyester polymer / β-tricalcium phosphate composite microspheres; Wherein the β-tricalcium phosphate is obtained by the following method: 0.4 mol / L diammonium hydrogen phosphate solution and 0.6 mol / L calcium nitrate tetrahydrate solution were mixed and reacted, wherein the molar ratio of calcium to phosphorus was 1.5; During the mixing process, the pH of the mixture is controlled to be 7.3±0.2 by adding ammonia water dropwise. The mixed product is stirred and centrifuged, and the obtained precipitate is washed and dried, sintered, and ball-milled to obtain the β-tricalcium phosphate.

9. An injectable polyester polymer / β-tricalcium phosphate composite microsphere, characterized in that: It comprises a polyester polymer and β-tricalcium phosphate powder doped in the polyester polymer, wherein the mass ratio of the polyester polymer to the tricalcium phosphate powder is (1-20): (0.1-5); Optionally, the polyester polymer / β-tricalcium phosphate composite microspheres are prepared according to the preparation method according to any one of claims 1 to 8.

10. Use of the injectable polyester polymer / β-tricalcium phosphate composite microspheres according to claim 9 in the preparation of tissue regeneration materials.

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