Preparation method of a BMP-2 sustained-release material and its application in bone defect repair

Abstract

The present invention discloses a preparation method of a BMP-2 sustained-release material and an application thereof in bone defect repair, belonging to the technical field of drug sustained-release. The present invention uses boric acid, urea and PMMA microspheres to prepare a boron nitride porous material. After the boron nitride porous material is modified by treating it with an O₂ / Ar mixed gas, the modified boron nitride porous material is soaked in an SBF solution and a BMP-2 solution. The prepared BMP-2 sustained-release material serves as a sustained-release material, has a slow self-degradation rate, can achieve the slow release of BMP-2 in the body, and has high cellular ALP activity.

Description

Technical Field

[0001] The present invention belongs to the technical field of drug sustained-release technology, and in particular relates to a preparation method of a BMP-2 sustained-release material and an application thereof in bone defect repair. Background Art

[0002] Four common bone transplant methods are available: autologous bone transplantation, allogeneic bone transplantation, xenogeneic bone transplantation, and bone replacement material transplantation. Autologous bone transplantation, typically obtained from the patient, offers the best results, but extracting bone tissue from the patient presents secondary trauma and limited sources. Furthermore, allogeneic and xenogeneic bone transplantation present the risk of immune rejection.

[0003] Delivering the drug bone morphogenetic protein-2 (BMP-2) via a sustained-release carrier material is currently the best treatment option for replacing autologous bone grafts. BMP-2 is a growth factor with a pronounced osteoblastic effect. However, BMP-2's clinical efficacy has been suboptimal. Under physiological conditions, BMP-2 rapidly degrades and inactivates, resulting in a shortened half-life. Low doses of BMP-2 have suboptimal osteogenesis. Furthermore, allergic doses of BMP-2 can cause a range of side effects, including ectopic bone formation, abnormal inflammatory responses, nerve damage, and cancer. Summary of the Invention

[0004] In order to overcome the problems existing in the prior art, the present invention proposes a preparation method of a BMP-2 sustained-release material and its application in bone defect repair.

[0005] To achieve the above object, the present invention is implemented through the following technical solutions:

[0006] The first aspect of the present invention provides a method for preparing a BMP-2 sustained-release material, comprising the following steps:

[0007] S1, dissolving boric acid and urea in deionized water at a molar ratio of 1:(3-4), adding polyethylene glycol, and stirring until completely dissolved to obtain a mixed solution;

[0008] S2, adding PMMA microspheres with a particle size of 300-500 μm to the mixed solution of step S1, assisting the immersion at -0.1 MPa for 2-3 hours, and then freeze-drying to remove moisture to obtain a composite;

[0009] S3, the composite is kept at 300°C in an air atmosphere for 2 hours. After the holding period, it is transferred to an ammonia atmosphere, heated at 5°C / min to 1000°C and held for 4 hours, and then sintered at 1400°C in nitrogen for 2 hours to form a porous boron nitride material;

[0010] S4, after cleaning the boron nitride porous material, place it in a microwave plasma system cavity and evacuate it to 0.5-0.8 Pa, then introduce an O2 / Ar mixed gas at a flow rate of 15-25 sccm, stabilize the cavity pressure to 40-60 Pa, and then pre-treat it with a plasma excitation power of 50 W for 2-3 minutes, then increase it to 100 W for 10-12 minutes, turn off the plasma, maintain the gas flow for 5-7 minutes, and slowly cool it to room temperature to obtain the modified boron nitride porous material;

[0011] S5, soaking the modified boron nitride porous material in 5×SBF for 24 h; then transferring it to 1×SBF and soaking it for 7 d, during which: the pH was controlled at 7.2-7.6 and the SBF was changed every 48 h;

[0012] S6, washing the soaked boron nitride porous material with deionized water three times, vacuum drying at 37°C for 24 hours, and then transferring it to a 10 mg / ml BMP-2 solution and soaking it for 2 hours to obtain a BMP-2 sustained-release material.

[0013] Furthermore, in step S1, the amount of polyethylene glycol added is 5-8 wt % of the mixed solution of boric acid and urea.

[0014] Furthermore, in the S4 step, the volume ratio of O2 to Ar is 1:(1-4).

[0015] Furthermore, in step S4, the boron nitride porous material is cleaned by ultrasonic cleaning with acetone, ethanol, and deionized water for 15 minutes each to remove surface organic matter and dust, and then dried at 80°C for later use.

[0016] The second aspect of the present invention provides a sustained-release material prepared according to the method described in the first aspect.

[0017] The third aspect of the present invention provides the use of the sustained-release material prepared according to the method described in the first aspect in the preparation of a drug delivery vehicle for bone defect repair.

[0018] Through the above technical solution, the present invention can achieve at least the following beneficial effects:

[0019] The present invention uses boric acid, urea and PMMA microspheres to prepare a boron nitride porous material. After the boron nitride porous material is modified by treating it with an O2 / Ar mixed gas, the modified boron nitride porous material is soaked in an SBF solution and a BMP-2 solution. The prepared BMP-2 sustained-release material serves as a sustained-release material. The material has a slow self-degradation rate and can achieve slow release of BMP-2 in the body, and has high cell ALP activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a scanning electron micrograph of the sustained-release material obtained in Example 1 of the present invention;

[0021] Figure 2 This is a graph showing the results of a drug release experiment using a sustained-release material according to the present invention;

[0022] Figure 3 This is a graph showing the results of a degradation experiment of the sustained-release material of the present invention;

[0023] Figure 4 It is a diagram of the alkaline phosphatase (ALP) detection results in the present invention. DETAILED DESCRIPTION

[0024] Unless otherwise stated, all materials and reagents used in the present invention are commercially available.

[0025] The PMMA microspheres in the present invention are polymethyl methacrylate microspheres, which are commercially available products;

[0026] The SBF in the present invention is a simulated body fluid and is a commercially available product.

[0027] Example 1: A method for preparing a BMP-2 sustained-release material, comprising the following steps:

[0028] S1, dissolving boric acid and urea in deionized water at a molar ratio of 1:3.5, adding 7 wt % of polyethylene glycol, and stirring until completely dissolved to obtain a mixed solution;

[0029] S2, adding PMMA microspheres with a particle size of 400 μm to the mixed solution of step S1, assisting the immersion at -0.1 MPa for 2.5 h, and then freeze-drying to remove moisture to obtain a composite;

[0030] S3, the composite is kept at 300°C in an air atmosphere for 2 hours. After the holding period, it is transferred to an ammonia atmosphere, heated at 5°C / min to 1000°C and held for 4 hours, and then sintered at 1400°C in nitrogen for 2 hours to form a porous boron nitride material;

[0031] S4, ultrasonically cleaning the boron nitride porous material with acetone, ethanol, and deionized water for 15 minutes each to remove surface organic matter and dust, drying at 80°C, placing it in a microwave plasma system cavity and evacuating it to 0.6Pa, then introducing an O2 / Ar mixed gas at a flow rate of 20sccm, stabilizing the cavity pressure to 50Pa, and then pre-treating it with a plasma power of 50W for 2.5min, then increasing it to 100W for 11min, turning off the plasma, maintaining the gas flow for 6min, and slowly cooling it to room temperature to obtain the modified boron nitride porous material; wherein the volume ratio of O2 to Ar is 1:3;

[0032] S5, soaking the modified boron nitride porous material in 5×SBF for 24 h; then transferring it to 1×SBF and soaking it for 7 d, during which: the pH was controlled at 7.2-7.6 and the SBF was changed every 48 h;

[0033] S6, washing the soaked boron nitride porous material with deionized water three times, vacuum drying at 37°C for 24 hours, and then transferring it to a 10 mg / ml BMP-2 solution and soaking it for 2 hours to obtain a BMP-2 sustained-release material.

[0034] Example 2: A method for preparing a BMP-2 sustained-release material, comprising the following steps:

[0035] S1, dissolving boric acid and urea in deionized water at a molar ratio of 1:3, adding 5 wt % polyethylene glycol, and stirring until completely dissolved to obtain a mixed solution;

[0036] S2, adding PMMA microspheres with a particle size of 300 μm to the mixed solution of step S1, assisting immersion at -0.1 MPa for 2 h, and then freeze-drying to remove moisture to obtain a composite;

[0037] S3, the composite is kept at 300°C in an air atmosphere for 2 hours. After the holding period, it is transferred to an ammonia atmosphere, heated at 5°C / min to 1000°C and held for 4 hours, and then sintered at 1400°C in nitrogen for 2 hours to form a porous boron nitride material;

[0038] S4, ultrasonically cleaning the boron nitride porous material with acetone, ethanol, and deionized water for 15 minutes each to remove surface organic matter and dust, drying at 80°C, placing it in a microwave plasma system cavity and evacuating it to 0.5Pa, then introducing an O2 / Ar mixed gas at a flow rate of 15sccm, stabilizing the cavity pressure to 40Pa, and then pre-treating it with a plasma power of 50W for 2min, then increasing it to 100W for 10min, turning off the plasma, maintaining the gas flow for 5min, and slowly cooling it to room temperature to obtain a modified boron nitride porous material; wherein the volume ratio of O2 to Ar is 1:1;

[0039] S5, soaking the modified boron nitride porous material in 5×SBF for 24 h; then transferring it to 1×SBF and soaking it for 7 d, during which: the pH was controlled at 7.2-7.6 and the SBF was changed every 48 h;

[0040] S6, washing the soaked boron nitride porous material with deionized water three times, vacuum drying at 37°C for 24 hours, and then transferring it to a 10 mg / ml BMP-2 solution and soaking it for 2 hours to obtain a BMP-2 sustained-release material.

[0041] Example 3: A method for preparing a BMP-2 sustained-release material, comprising the following steps:

[0042] S1, dissolving boric acid and urea in deionized water at a molar ratio of 1:4, adding 8 wt % polyethylene glycol, and stirring until completely dissolved to obtain a mixed solution;

[0043] S2, adding PMMA microspheres with a particle size of 500 μm to the mixed solution of step S1, assisting the immersion at -0.1 MPa for 3 h, and then freeze-drying to remove moisture to obtain a composite;

[0044] S3, the composite is kept at 300°C in an air atmosphere for 2 hours. After the holding period, it is transferred to an ammonia atmosphere, heated at 5°C / min to 1000°C and held for 4 hours, and then sintered at 1400°C in nitrogen for 2 hours to form a porous boron nitride material;

[0045] S4, ultrasonically cleaning the boron nitride porous material with acetone, ethanol, and deionized water for 15 minutes each to remove surface organic matter and dust, drying at 80°C, placing it in a microwave plasma system cavity and evacuating it to 0.8Pa, then introducing an O2 / Ar mixed gas at a flow rate of 25sccm, stabilizing the cavity pressure to 60Pa, and then pre-treating it with a plasma power of 50W for 3min, then increasing it to 100W for 12min, turning off the plasma, maintaining the gas flow for 7min, and slowly cooling it to room temperature to obtain the modified boron nitride porous material; wherein, the volume ratio of O2 to Ar is 1:4;

[0046] S5, soaking the modified boron nitride porous material in 5×SBF for 24 h; then transferring it to 1×SBF and soaking it for 7 d, during which: the pH was controlled at 7.2-7.6 and the SBF was changed every 48 h;

[0047] S6, washing the soaked boron nitride porous material with deionized water three times, vacuum drying at 37°C for 24 hours, and then transferring it to a 10 mg / ml BMP-2 solution and soaking it for 2 hours to obtain a BMP-2 sustained-release material.

[0048] Comparative Example 1:

[0049] The difference between Comparative Example 1 and Example 1 is that in step S4, the porous boron nitride material is ultrasonically cleaned with acetone, ethanol, and deionized water for 15 minutes each to remove surface organic matter and dust, and then dried at 80°C before steps S5 and S6 are performed.

[0050] Comparative Example 2:

[0051] The difference between Comparative Example 2 and Example 1 is that the operation of "immersing the modified porous boron nitride material in 5×SBF for 24 hours" is omitted in step S5.

[0052] Experimental example:

[0053] 1. Drug release experiment of sustained-release materials

[0054] Take the BMP-2 sustained-release materials prepared in Examples 1 to 3 and Comparative Examples 1 and 2, weigh 50 mg each, soak them in 4 mL of PBS solution (pH = 7.4) (with a centrifuge tube as a container), and then place the centrifuge tube in a thermostat and oscillator at 37°C for incubation. Take out 100 L of supernatant at the selected time, and add an equal volume of PBS solution to the centrifuge tube. Store the supernatant in a refrigerator at -80°C for further use. Finally, calculate the total BMP-2 content in the supernatant according to the steps of the human bone morphogenetic protein-2 (rhBMP-2) ELISA kit. The results are shown in the attached figure. Figure 2 Three parallel samples were collected for each group.

[0055] from Figure 2 It can be seen that the drug release of the sustained-release materials of Examples 1 to 3 is not much different from that of Comparative Examples 1 and 2.

[0056] 2. Degradation experiment of sustained-release materials

[0057] The degradation test of the sustained-release material was conducted by first weighing the total mass of the composite material W0, immersing the sustained-release material in 10ml phosphate buffered saline (PBS), and incubating the solution at 37°C on an oscillator at 50rpm for 30 days. The sustained-release material was then collected every two days and the remaining sustained-release material was air-dried at 25°C for 6h. The weighed mass was W1. The results of the sustained-release material degradation test are shown in the attached figure. Figure 3 Retention rate (%) = (W1 / W0) * 100%, and three parallel experiments were performed for each sample.

[0058] From the attached Figure 3 It can be seen that the 30d retention rates of the sustained-release materials of Examples 1 to 3 are higher than those of Comparative Examples 1 and 2, indicating that their degradation rates are slow.

[0059] 3. Alkaline phosphatase (ALP) test

[0060] Alkaline phosphatase (ALP) is a standard characteristic of stem cell differentiation into osteoblasts and can also be used to reflect the activity of osteoblasts and the functional status of osteoblasts.

[0061] Alkaline phosphatase (ALP) content is determined using an alkaline phosphatase assay kit. The basic principle is that disodium p-nitrophenyl phosphate reacts with alkaline phosphatase to produce pale yellow p-nitrophenol. P-nitrophenol exhibits a specific absorption peak at 405 nm. The absorption intensity at 405 nm corresponds to the alkaline phosphatase content in the sample.

[0062] The prepared sustained-release material was placed in a 48-well plate after sterilization, and 2×10 5Cells were cultured in fresh adipose-derived mesenchymal stem cell basal culture medium (BCM) every two days. On days 3 and 15 of culture, the BCM was aspirated and the sustained-release material was repeatedly washed with PBS to remove residual BCM. Following the kit instructions, 600 μL of lysis buffer was added to each well (ensuring that the sample was completely submerged in the lysis buffer). After lysis on ice for 1 hour, ultra-vigorous shaking was performed to ensure complete cell disruption, followed by cold centrifugation at 1000 rpm for 5 minutes. For the assay of holoenzyme activity, 20 μL of sample was added to 200 μL of disodium p-nitrophenylphosphate solution and incubated at room temperature for 15 minutes. 100 μL of 0.1 M sodium hydroxide solution was then added and mixed. The absorbance at 405 nm (OD405) was read using a multifunctional microplate reader. The holoenzyme activity of the sample was calculated. Detection of total protein content: Take 20 μL of the same sample and add 160 μL of protein test solution respectively. Incubate at room temperature for 15 minutes. Use a multifunctional microplate reader to read the absorbance at 450 nm (OD450). The total protein content of the sample can be calculated.

[0063] The alkaline phosphatase (ALP) content in the sample can be calculated using the formula: alkaline phosphatase activity = (holoenzyme activity / total protein content).

[0064] The results are as attached Figure 4 shown.

[0065] from Figure 4 It can be seen that the cell ALP activity of the sustained-release materials of Examples 1 to 3 was significantly higher on the 15th day than on the third day, and the cell ALP activity of the sustained-release materials was significantly higher than that of Comparative Example 1 and Comparative Example 3.

[0066] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and are not limiting. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made in form and details without departing from the scope defined by the claims of the present invention.

Claims

1. A method for preparing a BMP-2 sustained-release material, characterized in that: The following steps are included: S1, dissolving boric acid and urea in deionized water at a molar ratio of 1:(3-4), adding polyethylene glycol, and stirring until completely dissolved to obtain a mixed solution; S2, adding PMMA microspheres with a particle size of 300-500 μm to the mixed solution of step S1, assisting the immersion at -0.1 MPa for 2-3 hours, and then freeze-drying to remove moisture to obtain a composite; S3, the composite is kept at 300°C in an air atmosphere for 2 hours. After the holding period, it is transferred to an ammonia atmosphere, heated at 5°C / min to 1000°C and held for 4 hours, and then sintered at 1400°C in nitrogen for 2 hours to form a porous boron nitride material; S4, after cleaning the boron nitride porous material, place it in a microwave plasma system cavity and evacuate it to 0.5-0.8 Pa, then introduce an O2 / Ar mixed gas at a flow rate of 15-25 sccm, stabilize the cavity pressure to 40-60 Pa, and then pre-treat it with a plasma excitation power of 50 W for 2-3 minutes, then increase it to 100 W for 10-12 minutes, turn off the plasma, maintain the gas flow for 5-7 minutes, and slowly cool it to room temperature to obtain the modified boron nitride porous material; S5, soaking the modified boron nitride porous material in 5×SBF for 24 h; then transferring it to 1×SBF and soaking it for 7 d, during which: the pH was controlled at 7.2-7.6 and the SBF was changed every 48 h; S6, washing the soaked boron nitride porous material with deionized water three times, vacuum drying at 37°C for 24 hours, and then transferring it to a 10 mg / ml BMP-2 solution and soaking it for 2 hours to obtain a BMP-2 sustained-release material.

2. The method for preparing a BMP-2 sustained-release material according to claim 1, characterized in that: In step S1, the amount of polyethylene glycol added is 5-8 wt % of the mixed solution of boric acid and urea.

3. The method for preparing a BMP-2 sustained-release material according to claim 1, characterized in that: The volume ratio of O2 to Ar in the S4 step is 1:(1-4).

4. The method for preparing a BMP-2 sustained-release material according to claim 1, characterized in that: In step S4, the boron nitride porous material is cleaned by ultrasonic cleaning with acetone, ethanol, and deionized water for 15 minutes each to remove surface organic matter and dust, and then dried at 80° C. for later use.

5. The sustained-release material obtained by the method according to any one of claims 1 to 4.

6. Use of the sustained-release material prepared by the method according to any one of claims 1 to 4 in the preparation of a drug delivery vehicle for bone defect repair.

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