Enzyme-loaded calcium-phosphorus modified polyurethane bioactive material and preparation method thereof

Abstract

The application discloses a kind of enzyme-carrying calcium-phosphorus modified polyurethane bioactive materials and preparation method thereof.The application takes polyurethane as basic unit, by introducing the organic calcium / phosphorus small molecule containing hydroxyl, amino, thiol group and the like group in the main chain or side chain of molecular structure, obtains calcium-phosphorus modified bioactive polyurethane carrier, simultaneously, loading and biomineralization related phosphatase, phosphatase and the like, synergic polymer matrix surface organic calcium / phosphorus effect.The obtained material itself can provide nucleation site for biomineralization, secondly can be formed into different carriers by it, and biomineralization related biological enzyme synergic effect, so as to realize the application in the field of regulation mineralization.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological materials, and particularly relates to a calcium-phosphorus modified enzyme-loaded polyurethane bioactive material and a preparation method thereof. BACKGROUND

[0002] Calcium and phosphorus are essential elements for bone mineralization, and play a key role in regulating bone metabolism and mineralization in the steady state of formation and resorption. Both calcium ions (Ca 2+ ) and inorganic phosphate (PO4 3- ) can regulate the proliferation and differentiation of stem cells, bone cells and endothelial cells, which release growth factors through a series of signaling pathways to promote angiogenesis and accelerate bone healing. Due to good hydrophilic-hydrophobic microstructure and excellent chemical designability, polyurethane has been widely used in the field of biological medicine. Organic calcium and phosphorus can be introduced into the polymer matrix through molecular design to provide active sites for high molecular weight polymers with biological activity. In addition to inorganic ions, various biological enzymes with different functions are also involved in mineralization events. In the process of natural bone formation, mineralization is strictly regulated by the enzymes and membrane transport mechanisms of bone cells. A variety of enzyme molecules activate mineralization and participate in bone remodeling. The interaction between the loaded enzyme and the modified molecular group on the calcium-phosphorus modified polyurethane plays an organic synergistic role in promoting mineralization and osteogenesis, which can endow the composite polyurethane with the potential to participate in bone mineralization, and also provides a new idea for the design of enzyme-loaded calcium-phosphorus modified active biomaterials. Polyurethane materials with different physicochemical properties can be formed into scaffold carriers with different morphologies, which can better improve the utilization rate of enzymes when applied in the field of immobilized enzymes, and also achieve the purpose of adjusting the chemical microenvironment of the catalytic region by taking advantage of the designability of polyurethane materials.

[0003] At present, research on calcium-phosphorus modified composite polymer materials has attracted widespread attention. Chinese patent CN114849664A discloses a phosphorous acid modified polyphenylamine material, which is obtained by reacting polyamine with phosphorous acid and formaldehyde to obtain modified polyamine, and then reacting the modified polyamine with tannic acid to obtain the modified polyphenylamine material. The material can be applied in the field of adsorption of metal ions in wastewater. Chinese patent CN114213615A discloses a phosphocholine modified polyurethane material, which grafts glycerophosphocholine on the polyurethane molecular chain to improve the hydrophilicity, mechanical properties and anti-swelling properties of the material. In addition, in the field of immobilized enzymes promoting bone mineralization, Chinese patent CN113831568A discloses a sodium alginate-acrylamide double network hydrogel loaded with alkaline phosphatase, which introduces Ca 2+ , Sr 2+ and Zn 2+The introduction of ions makes the mineralization uniform, and the hydrogel has good mechanical properties and osteogenic induction activity. Chinese Patent CN111892720B discloses a method for inducing silk fibroin solution gelation and promoting mineralization by alkaline phosphatase. Alkaline phosphatase triggers the gelation reaction by catalyzing the phosphate group of small molecule polypeptides, thereby obtaining a hydrogel network loaded with alkaline phosphatase. Chinese Patent CN112625158A discloses a preparation method of enzyme-catalyzed mineralized polyacrylamide hydrogel. Alkaline phosphatase is directly immobilized in the polymer network, thereby enabling mineral deposition inside the gel, and ultimately improving the mechanical properties of the material. Therefore, the prior art is mostly focused on the research of enzyme-loaded polymer materials, and the modification of enzyme carrier functional groups and the role of biological enzymes in jointly regulating mineral deposition are ignored. SUMMARY

[0004] In view of the above prior art, the present application provides a kind of enzyme-loaded calcium-phosphorus modified polyurethane bioactive material and its preparation method, so that the organic calcium-phosphorus modified polyurethane material can not only provide nucleation site for in-situ mineralization, and its processability and surface polar group also provide multiple possibilities for enzyme immobilization method, synergize the effect of biological enzyme, enzyme-loaded calcium-phosphorus modified polyurethane bioactive material has great potential and application prospect in the field of biomineralization.

[0005] In order to achieve the above purpose, the technical scheme adopted by the present application is to provide an enzyme-loaded calcium-phosphorus modified polyurethane bioactive material, which comprises a polyurethane carrier and a biological enzyme loaded on the polyurethane carrier by at least one of physical adsorption method, covalent binding method, embedding method and crosslinking method; the molecular chain of the polyurethane carrier is connected with an organic calcium / phosphorus small molecule containing hydroxyl, amino and / or thiol group by grafting, block or crosslinking method; the molecular chain of the polyurethane carrier includes hard segment and soft segment, wherein the hard segment monomer is aliphatic diisocyanate, and the soft segment monomer is a polyol containing terminal hydroxyl group; the biological enzyme is an enzyme involved in the human mineralization process and promoting angiogenesis.

[0006] On the basis of the above technical scheme, the present application can also be improved as follows.

[0007] Further, the organic calcium / phosphorus small molecule is at least one of glycerophosphate calcium, glycerophosphoric acid, dibutyryl cyclic phosphoadenosine calcium, fosfomycin calcium, 3-phosphorylbenzoic acid, calcium citrate and glycerophosphoric acid sodium.

[0008] Further, the hard segment monomer is isophorone diisocyanate or hexamethylene diisocyanate; the soft segment monomer is polytetrahydrofuran ether diol with a molecular weight of 1000-4000 or poly-caprolactone diol with a molecular weight of 1000-4000.

[0009] Further, the biological enzyme is at least one of tissue non-specific alkaline phosphatase, phosphoaminoethanol / phosphocholine phosphatase and extracellular nucleotide pyrophosphatase / phosphodiesterase 1.

[0010] The application further provides a preparation method of the enzyme-loaded calcium-phosphorus modified polyurethane bioactive material, comprising the following steps:

[0011] S1: mixing hard segment monomers and soft segment monomers at a molar ratio of 1-4:1, adding a catalyst accounting for 0.05-1% of the total mass of the hard segment monomers and the soft segment monomers, and reacting at 65-75°C for 90-150 min; the catalyst is an organic metal catalyst or an amine catalyst;

[0012] S2: adding an organic calcium / phosphorus small molecule to the system after the reaction of S1, and continuing to heat and react for 4-9 h to obtain a polyurethane matrix; the molar ratio of the added organic calcium / phosphorus small molecule to the soft segment monomer is 1:1-5;

[0013] S3: processing the polyurethane matrix obtained in S2 to obtain a polyurethane carrier;

[0014] S4: loading a biological enzyme on the polyurethane carrier by at least one of a physical adsorption method, a covalent binding method, an embedding method and a cross-linking method.

[0015] Further, the catalyst is dibutyltin dilaurate, stannous octoate or triethanolamine.

[0016] Further, the polyurethane carrier is a polyurethane fiber membrane, which is prepared by the following steps:

[0017] S1: dissolving the calcium-phosphorus modified polyurethane in an organic solvent at a solid-liquid ratio of 1g:20-30mL to obtain an electrospinning solution; the organic solvent is at least one of trifluoroethanol, chloroform, dichloromethane, hexafluoroisopropanol, tetrahydrofuran, acetone, N,N-dimethylformamide and N,N-dimethylacetamide;

[0018] S2: preparing a polyurethane fiber membrane from the electrospinning solution by electrospinning, and the electrospinning conditions are as follows:

[0019] the negative pressure on the receiving plate side is 0-5kV, and the positive pressure on the electrospinning solution side is 5-12kV,

[0020] the flow rate of the electrospinning solution is 0.25-3mL / h,

[0021] the distance between the electrospinning solution jetting structure and the receiving plate is 10-20cm,

[0022] the environmental temperature is 10-40°C,

[0023] the environmental relative humidity is 20-70%.

[0024] Further, the polyurethane carrier is a polyurethane porous scaffold, which is prepared by the following steps:

[0025] S1: dissolving the calcium-phosphorus modified polyurethane in an organic solvent to obtain an electro-spraying solution, the organic solvent being at least one of trifluoroethanol, chloroform, dichloromethane, hexafluoroisopropanol, tetrahydrofuran, acetone, N,N-dimethylformamide and N,N-dimethylacetamide, the ratio of the polyurethane to the organic solvent being 1 g:30-50 mL;

[0026] S2: preparing the polyurethane microspheres by electro-spraying the electro-spraying solution, the electro-spraying conditions being:

[0027] the negative pressure on the receiving plate side being 0-5 kV, and the positive pressure on the electro-spraying solution side being 5-10 kV,

[0028] the flow rate of the electro-spraying solution being 0.5-5 mL / h,

[0029] the distance between the syringe needle and the receiving plate being 10-20 cm,

[0030] the ambient temperature being 10-40℃,

[0031] the relative humidity being 20-70%; or, the polyurethane microspheres are prepared by the following steps:

[0032] S1: dissolving the calcium-phosphorus modified polyurethane in an organic solvent to obtain an oil phase, the organic solvent being at least one of dichloromethane, chloroform, toluene and ethyl acetate; dissolving a surfactant in water to obtain a solution with a concentration of 0.2-3 wt%, the surfactant being at least one of a polyoxyethylene fatty alcohol ether series, an alkyl glycoside compound, a polyoxyethylene fatty amine, a polyethylene glycol fatty acid ester and a polyvinyl alcohol;

[0033] S2: mixing the oil phase and the water phase at a volume ratio of 1:3-10, stirring at a speed of 1000-2000 rpm for 2-6 h to obtain a polyurethane microsphere emulsion, and then centrifuging, washing and drying to obtain the polyurethane microspheres.

[0034] Further, the polyurethane carrier is a polyurethane porous scaffold, which is prepared by the following steps:

[0035] S1: dissolving the calcium-phosphorus modified polyurethane in an organic solvent to obtain a printing solution, the organic solvent being at least one of acetone, ethanol, chloroform and dichloromethane;

[0036] S2: preparing the polyurethane porous scaffold by 3D printing the printing solution, the 3D printing conditions being:

[0037] The printing platform temperature is 25-40℃;

[0038] The air pressure is 0.1-0.6 MPa;

[0039] The printing speed is 1-5 mm / min;

[0040] The layer height is 0.1-0.15 mm;

[0041] The filament spacing is 400-1000 mm.

[0042] Further, the physical adsorption method is to immerse the polyurethane carrier in a biological enzyme solution with a concentration of 1-5 mg / mL for 10-48 h.

[0043] The present application has the following beneficial effects: the present application takes polyurethane as a basic unit, introduces organic calcium / phosphorus small molecules containing hydroxyl, amino, sulfydryl and the like groups into the main chain or side chain of the molecular structure, obtains calcium / phosphorus modified bioactive polyurethane material, and simultaneously loads phosphatase, dephosphorylase, oxidoreductase and the like related to biological mineralization, and cooperates with the surface organic calcium / phosphorus of the polymer matrix. The obtained material itself can provide nucleation sites for biological mineralization, and secondly, by forming the material into different carriers, the mineralization-related biological enzymes are cooperated to accelerate or regulate mineral generation, so as to achieve the purpose of bone repair. BRIEF DESCRIPTION OF DRAWINGS

[0044] Figure 1 The calcium / phosphorus element distribution diagram and the morphology diagram of the surface of the calcium / phosphorus modified polyurethane prepared in Example 10 are shown in the following figures:

[0045] Figure 2 The surface morphology diagram of the enzyme-loaded biomaterial prepared in Example 10 is shown in the following figure:

[0046] Figures 3-4 The surface morphology diagram and the XRD diagram of the surface layer apatite crystal formed by in-vitro mineralization of the enzyme-loaded biomaterial are shown in the following figures. DETAILED DESCRIPTION

[0047] The specific embodiments of the present application will be described in detail below in combination with examples.

[0048] Example 1

[0049] An enzyme-loaded calcium / phosphorus modified polyurethane bioactive material is prepared by the following steps:

[0050] (1) Preparation of calcium / phosphorus modified polyurethane

[0051] Into a 100 mL three-necked flask filled with nitrogen, 7.5 g of polytetramethylene ether glycol (PTMEG) (molecular weight 2000, 3.75 mmol), 1.8 g of isophorone diisocyanate (IPDI) (molecular weight 222.28, 8.1 mmol), and 0.0125 g of dibutyltin dilaurate were added. After reaction at 70 °C for 2 h, 0.4 g of calcium citrate (molecular weight 498.433, 0.8 mmol) was added, and the reaction was continued for 9 h to obtain a calcium-phosphorus modified polyurethane.

[0052] (2) Preparation of a polyurethane electrospun fiber membrane

[0053] After 0.25 g of the calcium-phosphorus modified polyurethane was dissolved in 5 mL of hexafluoroisopropanol to obtain a polyurethane electrospinning solution, the polyurethane electrospinning solution was loaded into a syringe at room temperature of 25 °C and in an environment with a relative humidity of 40%. After the receiving device was connected to a positive high voltage of 1 kV, the receiving distance was controlled to be 15 cm, a positive high voltage of 8 kV was applied, the liquid flow rate was adjusted to be 1.05 mL / h, and a 22-gauge flat needle was used. After about 8 h, a polyurethane fiber membrane was obtained.

[0054] (3) Preparation of an enzyme-loaded biomaterial

[0055] After the extracellular nucleotide pyrophosphatase / phosphodiesterase 1 was dissolved in ultrapure water to obtain an enzyme solution with a concentration of 0.1 mg / mL, the polyurethane fiber membrane obtained in step (2) was soaked in the enzyme solution and placed at 37 °C for 24 h of adsorption to obtain an enzyme-loaded biomaterial.

[0056] Example 2

[0057] An enzyme-loaded calcium-phosphorus modified polyurethane bioactive material was prepared by the following steps:

[0058] (1) Preparation of a calcium-phosphorus modified polyurethane

[0059] Into a 100 mL three-necked flask filled with nitrogen, 7.5 g of polytetramethylene ether glycol (PTMEG) (molecular weight 2000, 3.75 mmol), 1.9 g of isophorone diisocyanate (IPDI) (molecular weight 222.28, 8.54 mmol), and 0.0125 g of triethanolamine were added. After reaction at 70 °C for 2 h, 0.6 g of glycerophosphoric acid (molecular weight 172.08, 3.48 mmol) was added, and the reaction was continued for 8 h to obtain a calcium-phosphorus modified polyurethane.

[0060] (2) Preparation of a polyurethane electrospun fiber membrane

[0061] 0.25 g of the calcium-phosphorus modified polyurethane was dissolved in 5 mL of trifluoroethanol to obtain a polyurethane electrospinning solution; the polyurethane electrospinning solution was loaded into a syringe in an environment of 25 °C and 40% relative humidity, and a negative high voltage of 1 kV was applied to the receiving device; the receiving distance was controlled to be 12 cm, a positive high voltage of 8 kV was applied, the liquid flow rate was adjusted to be 1.05 mL / h, and a 22-gauge flat needle was used; and polyurethane fiber membranes were obtained after about 12 h.

[0062] (3) Preparation of enzyme-loaded biomaterials

[0063] Phosphoaminoethanol / phosphocholine phosphatase was dissolved in ultrapure water to prepare an enzyme solution with a concentration of 0.2 mg / mL; the polyurethane fiber membranes obtained in step (2) were soaked in the enzyme solution and allowed to stand for 24 h at 37 °C to adsorb the enzyme, thereby obtaining enzyme-loaded biomaterials.

[0064] Example 3

[0065] An enzyme-loaded calcium-phosphorus modified polyurethane bioactive material was prepared by the following steps:

[0066] (1) Preparation of calcium-phosphorus modified polyurethane

[0067] 7.5 g of polytetramethylene ether glycol (PTMEG) (molecular weight 2000, 3.75 mmol), 1.8 g of isophorone diisocyanate (IPDI) (molecular weight 222.28, 8.1 mmol), and 0.0125 g of dibutyltin dilaurate were added to a 100 mL three-necked flask filled with nitrogen, and the mixture was reacted at 70 °C for 2 h; then 0.3 g of phosphomycin calcium (molecular weight 176.12, 1.7 mmol) was added, and the reaction was continued for 8 h to obtain calcium-phosphorus modified polyurethane.

[0068] (2) Preparation of polyurethane electrospun fiber membranes

[0069] 0.17 g of the calcium-phosphorus modified polyurethane was dissolved in 5 mL of hexafluoroisopropanol to obtain a polyurethane electrospinning solution; the polyurethane electrospinning solution was loaded into a syringe in an environment of 25 °C and 40% relative humidity, and a negative high voltage of 1 kV was applied to the receiving device; the receiving distance was controlled to be 15 cm, a positive high voltage of 8 kV was applied, the liquid flow rate was adjusted to be 0.7 mL / h, and a 22-gauge flat needle was used; and polyurethane fiber membranes were obtained after about 8 h.

[0070] (3) Preparation of enzyme-loaded biomaterials

[0071] Phosphoaminoethanol / phosphocholine phosphatase was dissolved in ultrapure water to prepare an enzyme solution with a concentration of 0.5 mg / mL; the polyurethane fiber membranes obtained in step (2) were soaked in the enzyme solution and allowed to stand for 24 h at 37 °C to adsorb the enzyme, thereby obtaining enzyme-loaded biomaterials.

[0072] Example 4

[0073] An enzyme-loaded calcium-phosphorus modified polyurethane bioactive material is prepared by the following steps:

[0074] (1) Preparation of calcium-phosphorus modified polyurethane

[0075] 4 g of polytetramethylene ether glycol (PTMEG) (molecular weight 1000, 4 mmol), 0.89 g of isophorone diisocyanate (IPDI) (molecular weight 222.28, 4 mmol), and 2.445 mg of triethanolamine were added to a 100 mL three-necked flask filled with nitrogen, and after reaction at 65°C for 1.5 h, 0.7 g of phosphomycin calcium (molecular weight 176.12, 4 mmol) was added, and the reaction was continued for 8 h to obtain a calcium-phosphorus modified polyurethane.

[0076] (2) Preparation of polyurethane electrospun fiber membrane

[0077] 0.1 g of the calcium-phosphorus modified polyurethane was dissolved in 3 mL of acetone to obtain a polyurethane electrospinning solution; the polyurethane electrospinning solution was loaded into a syringe in an environment of 10°C and 20% relative humidity, and after the receiving device was connected to a negative high voltage of 0 kV, the receiving distance was controlled to be 10 cm, a positive high voltage of 5 kV was applied, the liquid flow rate was adjusted to be 3 mL / h, and a 22-gauge flat needle was used, and about 8 h obtained a polyurethane fiber membrane.

[0078] (3) Preparation of enzyme-loaded biomaterial

[0079] Phosphoaminoethanol / phosphocholine phosphatase was dissolved in ultrapure water to prepare an enzyme solution of 0.1 mg / mL, and the polyurethane fiber membrane obtained in step (2) was soaked in the enzyme solution, and after standing at 37°C for 24 h, an enzyme-loaded biomaterial was obtained.

[0080] Example 5

[0081] An enzyme-loaded calcium-phosphorus modified polyurethane bioactive material is prepared by the following steps:

[0082] (1) Preparation of calcium-phosphorus modified polyurethane

[0083] 4 g of polytetramethylene ether glycol (PTMEG) (molecular weight 4000, 1 mmol), 0.89 g of isophorone diisocyanate (IPDI) (molecular weight 222.28, 4 mmol), and 0.0489 g of stannous octoate were added to a 100 mL three-necked flask filled with nitrogen, and after reaction at 75°C for 2.5 h, 0.9 g of phosphomycin calcium (molecular weight 176.12, 5 mmol) was added, and the reaction was continued for 9 h to obtain a calcium-phosphorus modified polyurethane.

[0084] (2) Preparation of polyurethane electrospun fiber membrane

[0085] 0.1 g of the calcium-phosphorus modified polyurethane was dissolved in 2 mL of acetone to obtain a polyurethane electrospinning solution; the polyurethane electrospinning solution was loaded into a syringe in an environment of 40 °C and 70% relative humidity, and a negative high voltage of 5 kV was applied to the receiving device; the receiving distance was controlled to be 20 cm, a positive high voltage of 12 kV was applied, the liquid flow rate was adjusted to be 0.25 mL / h, and a 22-gauge flat needle was used; and after about 8 h, a polyurethane fiber membrane was obtained.

[0086] (3) Preparation of enzyme-loaded biomaterials

[0087] Phosphoaminoethanol / phosphocholine phosphatase was dissolved in ultrapure water to prepare an enzyme solution with a concentration of 0.3 mg / mL; the polyurethane fiber membrane obtained in step (2) was soaked in the enzyme solution and allowed to stand for 24 h at 37 °C to adsorb the enzyme, thereby obtaining enzyme-loaded biomaterials.

[0088] Example 6

[0089] An enzyme-loaded calcium-phosphorus modified polyurethane bioactive material was prepared by the following steps:

[0090] (1) Preparation of calcium-phosphorus modified polyurethane

[0091] 7.5 g of polycaprolactone diol (molecular weight 2000, 3.75 mmol), 1.8 g of isophorone diisocyanate (IPDI) (molecular weight 222.28, 8.1 mmol), and 0.0125 g of dibutyltin dilaurate were added to a 100 mL three-necked flask filled with nitrogen, and the mixture was reacted at 70 °C for 2 h; then 0.3 g of phosphomycin calcium (molecular weight 176.12, 1.7 mmol) was added, and the reaction was continued for 8 h to obtain calcium-phosphorus modified polyurethane.

[0092] (2) Preparation of polyurethane electrospun fiber membrane

[0093] 0.17 g of the calcium-phosphorus modified polyurethane was dissolved in 5 mL of hexafluoroisopropanol to obtain a polyurethane electrospinning solution; the polyurethane electrospinning solution was loaded into a syringe in an environment of 25 °C and 40% relative humidity, and a negative high voltage of 1 kV was applied to the receiving device; the receiving distance was controlled to be 15 cm, a positive high voltage of 8 kV was applied, the liquid flow rate was adjusted to be 0.7 mL / h, and a 22-gauge flat needle was used; and after about 8 h, a polyurethane fiber membrane was obtained.

[0094] (3) Preparation of enzyme-loaded biomaterials

[0095] Phosphoaminoethanol / phosphocholine phosphatase was dissolved in ultrapure water to prepare an enzyme solution with a concentration of 0.5 mg / mL; the polyurethane fiber membrane obtained in step (2) was soaked in the enzyme solution and allowed to stand for 24 h at 37 °C to adsorb the enzyme, thereby obtaining enzyme-loaded biomaterials.

[0096] Example 9

[0097] An enzyme-loaded calcium-phosphorus modified polyurethane bioactive material is prepared by the following steps:

[0098] (1) Preparation of calcium-phosphorus modified polyurethane

[0099] 7.4 g of polytetramethylene ether glycol (PTMEG) (molecular weight 2000, 3.7 mmol), 1.7 g of isophorone diisocyanate (IPDI) (molecular weight 222.28, 7.65 mmol), and 0.0125 g of dibutyltin dilaurate were added to a 100 mL three-necked flask filled with nitrogen, and after reaction at 70°C for 2 h, 0.8 g of glycerol sodium phosphate (molecular weight 216.037, 3.7 mmol) was added, and the reaction was continued for 8 h to obtain a calcium-phosphorus modified polyurethane.

[0100] (2) Preparation of polyurethane microspheres

[0101] 0.4 g of the calcium-phosphorus modified polyurethane was dissolved in 10 mL of dichloromethane, and the obtained solution was used as an oil phase, and 50 mL of deionized water containing 2% polyvinyl alcohol was used as an aqueous phase; the oil phase was quickly poured into the aqueous phase under stirring at 1000 rpm, and the stirring was continued for 3 h, and the obtained emulsion was centrifuged, washed, and freeze-dried to obtain polyurethane microspheres.

[0102] (3) Preparation of enzyme-loaded biomaterials

[0103] Alkaline phosphatase was dissolved in ultrapure water to prepare an enzyme solution of 1 mg / mL, and the polyurethane electrosprayed microspheres obtained in step (2) were soaked in the enzyme solution, and adsorption was carried out at 37°C for 24 h to obtain enzyme-loaded biomaterials.

[0104] Example 10

[0105] An enzyme-loaded calcium-phosphorus modified polyurethane bioactive material is prepared by the following steps:

[0106] (1) Preparation of calcium-phosphorus modified polyurethane

[0107] 7.4 g of polytetramethylene ether glycol (PTMEG) (molecular weight 2000, 3.7 mmol), 1.7 g of isophorone diisocyanate (IPDI) (molecular weight 222.28, 7.65 mmol), and 0.0125 g of dibutyltin dilaurate were added to a 100 mL three-necked flask filled with nitrogen, and after reaction at 70°C for 2 h, 0.8 g of glycerol calcium phosphate (molecular weight 212.152, 3.77 mmol) was added, and the reaction was continued for 8 h to obtain a calcium-phosphorus modified polyurethane.

[0108] (2) Preparation of polyurethane microspheres

[0109] Dissolve 0.3 g of calcium-phosphorus modified polyurethane in 10 mL of dichloromethane, and the obtained solution is used as an oil phase, and 50 mL of deionized water containing 1% polyvinyl alcohol is used as an aqueous phase; under a stirring speed of 1200 rpm, the oil phase is quickly poured into the aqueous phase, and stirring is continued for 5 h; the obtained emulsion is centrifuged, washed, and freeze-dried to obtain polyurethane microspheres.

[0110] (3) Preparation of enzyme-loaded biomaterials

[0111] Dissolve 2 mg of alkaline phosphatase in 1 mL of ultrapure water to obtain an enzyme solution; then dissolve 5 mg of polyvinyl alcohol in 1 mL of the enzyme solution to prepare an enzyme solution containing 0.5% (W / V) polyvinyl alcohol as an internal aqueous phase. Dissolve 0.18 g of the polyurethane microspheres prepared in step (2) in 19 mL of dichloromethane as an oil phase, and use 50 mL of deionized water containing 2% polyvinyl alcohol as an external aqueous phase. The internal aqueous phase is quickly poured into the oil phase, and ultrasonic emulsification is performed for 3 min (ultrasonic power 100 W, ultrasonic 5 s, and stop 1 s) to obtain a W / O emulsion. Under a stirring speed of 1000 rpm, the W / O emulsion is quickly poured into the external aqueous phase, and stirring is continued for 5 h; the obtained emulsion is centrifuged, washed, and freeze-dried to obtain polyurethane microspheres embedded with enzymes, i.e., enzyme-loaded biomaterials.

[0112] Result analysis

[0113] The performance of the materials obtained by using the technical solutions in the present application is analyzed by taking the enzyme-loaded biomaterials prepared in Example 10 as an example.

[0114] The present application modifies polyurethane by using organic calcium / phosphorus small molecules, and the successful introduction of calcium and phosphorus elements into polyurethane is proved by electron spectroscopy and surface morphology analysis (). Figure 1 The successful loading of ALP is observed by scanning electron microscopy (). Figure 2 The scanning electron microscopy results show that crystals with a structure similar to apatite are formed on the enzyme-loaded calcium-phosphorus modified polyurethane matrix. Figure 3 The X-ray diffraction proves that the crystals are apatite crystals. Figure 4

[0115] Although the specific embodiments of the present application are described in detail in combination with the examples, it should not be understood as a limitation on the protection scope of the present patent. Various modifications and variations made by those skilled in the art within the scope described in the claims are still within the protection scope of the present patent.​

Claims

1. A method for preparing an enzyme-loaded calcium-phosphorus modified polyurethane bioactive material, characterized in that, Includes the following steps: S1: Mix the hard segment monomer and the soft segment monomer at a molar ratio of 1 to 4:1, then add a catalyst accounting for 0.05 to 1% of the total mass of the hard segment monomer and the soft segment monomer, and react at 65 to 75 °C for 90 to 150 min; the catalyst is an organometallic catalyst or an amine catalyst; the hard segment monomer is isophorone diisocyanate or hexamethylene diisocyanate; the soft segment monomer is polytetrahydrofuran ether diol with a molecular weight of 1000 to 4000 or polycaprolactone diol with a molecular weight of 1000 to 4000. S2: Add organic calcium / phosphorus small molecules to the system after reaction S1, and continue the reaction at a constant temperature for 4-9 hours to obtain a polyurethane matrix; the molar ratio of the added organic calcium / phosphorus small molecules to the soft segment monomer is 1:1-5; the organic calcium / phosphorus small molecules are at least one of calcium glycerophosphate, glycerophosphate, dibutyryl cyclic adenosine monophosphate calcium, fosfomycin calcium, 3-phosphorylbenzoic acid, calcium citrate and sodium glycerophosphate. S3: The polyurethane matrix obtained in S2 is processed and shaped to obtain a polyurethane carrier; S4: Load the polyurethane carrier obtained in S3 with a biological enzyme by at least one of the following methods: physical adsorption, covalent bonding, encapsulation, and cross-linking; the biological enzyme is an enzyme that participates in the human mineralization process and promotes angiogenesis; the biological enzyme is at least one of tissue nonspecific alkaline phosphatase, phosphatamidoethanol / phosphocholine phosphatase, and extracellular nucleotide pyrophosphatase / phosphodiesterase 1.

2. The preparation method according to claim 1, characterized in that: The catalyst is dibutyltin dilaurate, stannous octoate, or triethanolamine.

3. The preparation method according to claim 1, characterized in that, The polyurethane carrier is a polyurethane fiber membrane, which is prepared through the following steps: S1: Dissolve calcium-phosphorus modified polyurethane in an organic solvent at a material-to-liquid ratio of 1 g: 20-30 mL to obtain an electrospinning solution; the organic solvent is at least one of trifluoroethanol, chloroform, dichloromethane, hexafluoroisopropanol, tetrahydrofuran, acetone, N,N-dimethylformamide and N,N-dimethylacetamide. S2: Polyurethane fiber membranes are produced from the electrospinning solution by electrospinning. The electrospinning conditions are as follows: The negative pressure on the receiving plate side is 0~5 kV, and the positive pressure on the electrospinning solution side is 5~12 kV. The flow rate of the electrospinning solution is 0.25~3 mL / h. The distance between the electrospinning solution spinneret and the receiving plate is 10~20 cm. The ambient temperature is 10~40℃. The relative humidity of the environment is 20-70%.

4. The preparation method according to claim 1, characterized in that, The polyurethane carrier is polyurethane microspheres, which are prepared through the following steps: S1: Dissolve calcium-phosphorus modified polyurethane in an organic solvent at a material-to-liquid ratio of 1 g: 30~50 mL to obtain an electrospray liquid. The organic solvent is at least one of trifluoroethanol, chloroform, dichloromethane, hexafluoroisopropanol, tetrahydrofuran, acetone, N,N-dimethylformamide, and N,N-dimethylacetamide. S2: Polyurethane microspheres are fabricated from the electrospray liquid using electrostatic spraying. The electrostatic spraying conditions are as follows: The negative voltage on the receiving plate side is 0~5 kV, and the positive voltage on the electrospinning solution side is 5~10 kV. The flow rate of the electro-sprayed solution is 0.5~5 mL / h. The distance between the syringe needle and the receiving plate is 10-20 cm. The ambient temperature is 10~40℃. Relative humidity is 20-70%; Alternatively, the polyurethane microspheres may be prepared through the following steps: S1: Dissolve calcium-phosphorus modified polyurethane in an organic solvent at a feed-to-liquid ratio of 1 g: 25-35 mL to obtain an oil phase. The organic solvent is at least one of dichloromethane, chloroform, toluene, and ethyl acetate. Dissolve a surfactant in water to prepare a solution with a concentration of 0.2-3 wt% to obtain an aqueous phase. The surfactant is at least one of polyoxyethylene fatty alcohol ethers, alkyl glycosides, polyoxyethylene fatty amines, polyethylene glycol fatty acid esters, and polyvinyl alcohol. S2: Mix the oil phase and water phase at a volume ratio of 1:3~10, stir at a rate of 1000~2000 rpm for 2~6 h to obtain a polyurethane microsphere emulsion, then centrifuge, wash and dry to obtain polyurethane microspheres.

5. The preparation method according to claim 1, characterized in that, The polyurethane carrier is a porous polyurethane scaffold, which is prepared through the following steps: S1: Dissolve calcium-phosphorus modified polyurethane in an organic solvent at a material-to-liquid ratio of 1 g:10 mL to obtain printing liquid, wherein the organic solvent is at least one of acetone, ethanol, chloroform, and dichloromethane; S2: The printing fluid is used to create a polyurethane porous scaffold using 3D printing. The 3D printing conditions are as follows: The printing platform temperature is 25~40℃; The air pressure is 0.1~0.6 MPa; Printing speed is 1~5 mm / min; Floor height: 0.1~0.15 mm; The wire spacing is 400~1000 mm.

6. The preparation method according to claim 1, characterized in that, The physical adsorption method involves immersing the polyurethane carrier in a biological enzyme solution with a concentration of 1-5 mg / mL for 10-48 h.

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