Preparation method and application of BMP-2 controllable slow-release flocculent based on self-regulation of lysozyme

Abstract

The invention belongs to the technical field of drug sustained release, and particularly relates to a preparation method and application of a BMP-2 controllable sustained-release flocculent body based on lysozyme self-regulation. The preparation method comprises the following steps: preparing a chitosan solution; sequentially adding a protein stabilizer and glycerol into the chitosan solution obtained in the step A1 to prepare a chitosan composite solution; obtaining a non-specific binding solution; preparing BMP-2 / chitosan solid particles by adopting a freeze drying method; the preparation method comprises the following steps: preparing a PLGA / PEG composite solution to obtain a suspension, and carrying out post-treatment on the suspension to form the BMP-2 controllable slow-release flocculent. The carrier in the BMP-2 controllable slow-release flocculent body prepared by the preparation method can be specifically degraded by lysozyme in a human body, so that the release amount and the release speed of BMP-2 can be automatically adjusted according to a bone healing process, and long-time continuous release of the BMP-2 controllable slow-release flocculent body is realized. The problems that in the prior art, the slow release effect of a hydrophilic material on BMP-2 is poor, sudden release of factors is easily formed, and the slow release time and the slow release dosage are not controllable are solved.

Description

Technical Field

[0001] The invention belongs to the technical field of drug sustained-release, and in particular relates to a preparation method and application of BMP-2 controllable sustained-release floccules based on lysozyme self-regulation. Background Art

[0002] Bone morphogenetic protein-2 (BMP-2) is a potent growth factor that belongs to the transforming growth factor β (TGF-β) family and is primarily used for bone regeneration and repair in orthopedic implants. BMP-2 can induce undifferentiated mesenchymal stem cells to differentiate into osteoblasts, promoting the formation and repair of bone tissue. It has significant effects in fracture healing, spinal fusion, and the treatment of large bone defects. BMP-2 can accelerate the healing process after fractures or bone grafting surgery and reduce the patient's recovery time.

[0003] However, the effect of BMP-2 is very sensitive to dose. Too low a dose may be ineffective, while too high a dose may cause side effects. In some cases, BMP-2 may lead to excessive bone formation or even ectopic bone formation in areas where bone formation is not needed (such as in soft tissue). How to accurately control the dose of BMP-2 to achieve the best therapeutic effect remains a challenge.

[0004] Sustained release means that the drug takes effect relatively slowly in the body, which can keep the fluctuation of drug concentration in the body stable, avoid discomfort symptoms, and have a long duration of action, which can continuously control the condition. In the prior art, in order to achieve sustained release of BMP-2, BMP-2 is combined with collagen sponge or gelatin carrier material and enters the human body. Collagen sponge or gelatin carrier or hydrophilic materials such as sodium alginate or albumin have weak sustained release effect on BMP-2, which is easy to form sudden release of factors, and the sustained release time is short, which is easy to cause excessive BMP-2, resulting in uncontrollable osteogenesis and excessive osteogenesis with irregular new bone structure. Summary of the invention

[0005] The purpose of the present invention is to overcome the defects of the prior art that BMP-2 carried by hydrophilic materials is released quickly, which easily leads to excessive osteogenesis with uncontrollable osteogenesis range and irregular new bone structure, and to provide a preparation method and application of BMP-2 controllable sustained-release flocs based on lysozyme self-regulation with a chitosan-PLGA composite material as a carrier whose degradation is regulated by lysozyme.

[0006] The technical solution adopted by the present invention to solve its technical problem is:

[0007] As a first aspect, a method for preparing a BMP-2 controlled sustained-release floc based on lysozyme self-regulation comprises the following steps:

[0008] A1. Prepare a chitosan solution having a chitosan concentration of 20 mg / mL to 50 mg / mL;

[0009] A2. In step A1, a protein stabilizer and glycerol are sequentially added to the chitosan solution obtained to prepare a chitosan composite solution;

[0010] A3. Add the growth factor BMP-2 to the chitosan composite solution obtained in step A2, and stir ultrasonically until the protein stabilizer and BMP-2 are completely non-specifically bound to obtain a non-specific binding solution, wherein the concentration of BMP-2 in the non-specific binding solution is 0.1 mg / mL to 0.5 mg / mL;

[0011] A4. Prepare BMP-2 / chitosan solid particles with a particle size of 10 μm to 100 μm by freeze drying;

[0012] A5. Prepare a PLGA solution having a PLGA concentration of 45 mg / mL to 55 mg / mL and a polyethylene glycol solution having a polyethylene glycol concentration of 45 mg / mL to 55 mg / mL;

[0013] A6. Mixing the PLGA solution and the polyethylene glycol solution in a ratio of 9:1 to 4:1 to obtain a PLGA / PEG composite solution;

[0014] A7. The BMP-2 / chitosan solid particles obtained in step A4 were mixed with the PLGA / PEG composite solution obtained in step A6 at a ratio of (0.5-1.5) g:100 mL, and ultrasonically stirred to obtain a stable suspension;

[0015] A8. Post-treat the suspension obtained in step A7 to form BMP-2 controllable sustained-release flocs.

[0016] Specifically, the preparation process of the chitosan solution in step A1 includes the following contents:

[0017] Weigh chitosan with a molecular weight of 100 kDa to 200 kDa; the deacetylation degree of the chitosan is 85% to 95%;

[0018] Dissolving chitosan in 1.5% to 3% acetic acid solution to obtain an initial solution;

[0019] Using a buffer solution to adjust the pH value of the initial solution to 5.5-6 to obtain a chitosan solution;

[0020] Specifically, the buffer solution includes at least one of a phosphate buffer, an acetate buffer, a sodium acetate buffer and a bicarbonate buffer.

[0021] Specifically, the preparation of the chitosan composite solution in step A2 comprises the following steps:

[0022] A201. Add a protein stabilizer to the chitosan solution, stir and fuse to form a chitosan-protein mixed solution, wherein the protein concentration in the chitosan-protein mixed solution is 0.1% to 1% (w / v);

[0023] A202. Add glycerol to the chitosan-protein mixed solution, and stir evenly at room temperature to form a chitosan composite solution, wherein the volume ratio of glycerol in the chitosan composite solution is 5% to 20% (v / v).

[0024] Specifically, in step A4, the preparation of the BMP-2 / chitosan solid particles includes the following:

[0025] freeze-drying the non-specific binding solution obtained in step A3 to obtain a solid material;

[0026] The solid material is ball-milled with zirconium oxide ball milling beads, and the BMP-2 / chitosan solid particles are obtained by controlling the ball milling parameters.

[0027] Specifically, the preparation process of the PLGA solution in step A5 is as follows:

[0028] Obtaining polylactic acid-glycolic acid copolymer (PLGA), wherein the molar ratio of lactic acid to glycolic acid is 20:80 to 50:50; and the molecular weight of the polylactic acid-glycolic acid copolymer is 10 kDa to 30 kDa;

[0029] The poly(lactic acid-glycolic acid) copolymer is dissolved in dichloromethane or chloroform to obtain a PLGA solution with a concentration of 45 mg / mL to 55 mg / mL.

[0030] Specifically, the preparation process of the polyethylene glycol solution is as follows: obtain polyethylene glycol with a molecular weight of 200Da to 6000Da, dissolve it in dichloromethane or chloroform, and stir at room temperature to obtain a polyethylene glycol solution with a concentration of 45mg / mL to 55mg / mL.

[0031] Specifically, the post-processing of the suspension obtained in step A7 comprises the following steps:

[0032] A801. The suspension obtained in step A7 was dripped into deionized water at a rate of 5 mL / min to 50 mL / min while stirring, and the stirring speed was controlled at 100 rpm to 200 rpm to obtain a liquid having a white flocculent precipitate;

[0033] A802. Obtain flocculent precipitate by filtration and wash the flocculent precipitate with deionized water;

[0034] A803. The flocculent precipitate washed in step A802 is dried by freeze drying to obtain dry BMP-2 controlled sustained-release floccules.

[0035] As a second aspect, a BMP-2 controllable sustained-release floc is prepared by the above-mentioned method for preparing BMP-2 controllable sustained-release floc based on lysozyme self-regulation.

[0036] As a third aspect, the use of the BMP-2 controlled sustained-release floccules as described above is characterized by comprising the following contents:

[0037] Pressing the BMP-2 controlled sustained-release floccules into the same shape according to the shape of the bone defect site;

[0038] Fill in the corresponding bone defect.

[0039] The preparation method and application of the BMP-2 controllable sustained-release flocs based on lysozyme self-regulation of the present invention have the following beneficial effects:

[0040] The present invention mixes BMP-2 / chitosan solid particles prepared in a special way with a PLGA / PEG composite solution to obtain a suspension in which chitosan / growth factor BMP-2 composite particles, PLGA and polyethylene glycol are uniformly distributed, and post-processes the suspension to form a BMP-2 controllable sustained-release floc. The carrier in the BMP-2 controllable sustained-release floc prepared by the preparation method can be specifically degraded by lysozyme in the human body, and the lysozyme concentration at the bone defect site is affected by the severity of the bone defect site, that is, the larger the wound surface at the bone defect site, the greater the lysozyme concentration. By utilizing that the degradation of chitosan in the BMP-2 controllable sustained-release floc is regulated by lysozyme, the release amount and release speed of BMP-2 can be automatically adjusted according to the progress of bone healing (that is, the concentration of lysozyme), so as to achieve long-term continuous release of the BMP-2 controllable sustained-release floc instead of instantaneous large-scale release, thereby enhancing the safety and effect of treatment. The invention solves the problems in the prior art that the sustained release effect of BMP-2 using hydrophilic materials is not strong, sudden release of factors is easily formed, and the sustained release time and sustained release dosage are uncontrollable.

[0041] At the same time, the composite degradable polymer material (i.e. PLGA) can adapt to various shapes of bone defects at the initial stage of implantation after adjusting its plasticity, provide necessary structural support, and is not easily lost to soft tissues, thereby improving the utilization rate of BMP-2. The slow degradation characteristics of PLGA further extend the degradation time of the overall material to further control the sustained release of BMP-2 in PLGA. In addition, the addition of protein stabilizers and the non-specific binding of growth factors added later can help maintain the structural stability of BMP-2 and prevent protein denaturation and inactivation during processing and storage, so as to achieve the effectiveness and sustainability of BMP-2 in bone regeneration and repair. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments.

[0043] Figure 1 It is a flow chart of the steps of the method for preparing BMP-2 controlled sustained-release floccules according to an embodiment of the present invention.

[0044] Figure 2 It is a flowchart of post-processing in step A8 of an embodiment of the present invention.

[0045] Figure 3 It is a comparative curve diagram of BMP-2 release between Example 1 of the present invention and Comparative Examples 1, 2 and 3.

[0046] Figure 4 It is a comparative curve diagram of the BMP-2 release of Example 2 of the present invention and Comparative Examples 4, 5 and 6. DETAILED DESCRIPTION

[0047] The present invention will now be described in further detail with reference to the accompanying drawings. These drawings are simplified schematic diagrams, which only illustrate the basic structure of the present invention in a schematic manner, and therefore only show the components related to the present invention.

[0048] like Figure 1 The method for preparing a BMP-2 controlled slow-release floc based on lysozyme self-regulation of the present invention comprises the following steps:

[0049] A1. Prepare a chitosan solution having a chitosan concentration of 20 mg / mL to 50 mg / mL;

[0050] A2. In step A1, a protein stabilizer and glycerol are sequentially added to the chitosan solution obtained to prepare a chitosan composite solution;

[0051] A3. Add the growth factor BMP-2 to the chitosan composite solution obtained in step A2, and stir ultrasonically until the protein stabilizer and BMP-2 are completely non-specifically bound to obtain a non-specific binding solution, wherein the concentration of BMP-2 in the non-specific binding solution is 0.1 mg / mL to 0.5 mg / mL;

[0052] A4. Prepare BMP-2 / chitosan solid particles with a particle size of 10 μm to 100 μm by freeze drying;

[0053] A5. Prepare a PLGA solution having a PLGA concentration of 45 mg / mL to 55 mg / mL and a polyethylene glycol solution having a polyethylene glycol concentration of 45 mg / mL to 55 mg / mL;

[0054] A6. Mixing the PLGA solution and the polyethylene glycol solution in a ratio of 9:1 to 4:1 to obtain a PLGA / PEG composite solution;

[0055] A7. The BMP-2 / chitosan solid particles obtained in step A4 were mixed with the PLGA / PEG composite solution obtained in step A6 at a ratio of (0.5-1.5) g:100 mL, and ultrasonically stirred to obtain a stable suspension;

[0056] A8. Post-treat the suspension obtained in step A7 to form BMP-2 controllable sustained-release flocs.

[0057] The above preparation steps are specifically described as follows. The preparation process of the chitosan solution in step A1 includes the following: weighing chitosan with a molecular weight of 100kDa to 200kDa; the deacetylation content in the chitosan is 85% to 95%; dissolving the chitosan in a 1.5% to 3% acetic acid solution to obtain an initial solution; using a buffer solution to adjust the pH value of the initial solution to 5.5 to 6 to obtain a chitosan solution. It should be explained that the pH value of the chitosan solution in this embodiment is adjusted to 5.5 to 6 to ensure that the chitosan solution is in an acidic dissolution environment, and at the same time, the pH value of the solution needs to be within a range that can maintain the stability of the structure of the growth factor BMP-2.

[0058] It should be understood that the buffer solution in the present invention includes at least one of phosphate buffer, acetate buffer, sodium acetate buffer and bicarbonate buffer. When used specifically, it can be selected according to the specific usage scenario or the amount of substance retained. There is no absolute limitation on the specific usage type of the buffer solution here.

[0059] The preparation of the chitosan composite solution described in step A2 comprises the following steps:

[0060] A201. Add a protein stabilizer to the chitosan solution, stir and fuse to form a chitosan-protein mixed solution, wherein the protein concentration in the chitosan-protein mixed solution is 0.1% to 1% (w / v);

[0061] A202. Add glycerol to the chitosan-protein mixed solution, and stir evenly at room temperature to form a chitosan composite solution, wherein the volume ratio of glycerol in the chitosan composite solution is 5% to 20%.

[0062] The addition of the protein stabilizer in this example and the non-specific binding of the growth factor added later help maintain the structural stability of BMP-2 and prevent the protein from denaturing and inactivating during processing and storage.

[0063] In step A4, the preparation of the BMP-2 / chitosan solid particles includes the following: freeze-drying the non-specific binding solution obtained in step A3 to obtain a solid material; ball-milling the solid material with zirconium oxide ball milling beads, and controlling the parameters of the milling beads to obtain BMP-2 / chitosan solid particles. The freeze-drying method generally includes rapid freezing, placing the frozen solution in a vacuum drying chamber, reducing the pressure in the vacuum drying chamber, removing moisture, performing a secondary drying treatment on the solution after the moisture is removed, and performing a secondary moisture removal treatment to obtain the final solid material. The parameters in the specific freeze-drying process are set according to the specific use and are not described in detail here.

[0064] The preparation process of the PLGA solution described in step A5 is as follows: obtain polylactic acid-glycolic acid copolymer (PLGA), wherein the molar ratio of lactic acid and glycolic acid is 20: 80 ~ 50: 50; the molecular weight of the polylactic acid-glycolic acid copolymer is 10kDa ~ 30kDa; the polylactic acid-glycolic acid copolymer is dissolved in dichloromethane or chloroform to obtain a PLGA solution with a concentration of 45mg / mL ~ 55mg / mL. The molecular weights of lactic acid and glycolic acid in the present embodiment are both 10kDa ~ 30kDa, the use of low molecular weight and the addition ratio of lactic acid and glycolic acid effectively increase the plasticity of the final BMP-2 controlled sustained-release floccules, and prepare a polyethylene glycol solution at the same time, and mix the polyethylene glycol solution with the PLGA solution in a ratio of 9: 1 ~ 4: 1 to obtain a PLGA / PEG composite solution, and the increase of polyethylene glycol in the PLGA / PEG composite solution further increases the plasticity. The PLGA / PEG composite solution of this embodiment is designed to adapt to bone defects of various properties in the early stage of implantation after adjusting the plasticity of the final BMP-2 controlled sustained-release floccules, provide necessary structural support, and is not easily lost into the soft tissue. In addition, the BMP-2 controlled sustained-release floccules prepared using the PLGA / PEG composite solution of this embodiment have a slow degradation characteristic, which prolongs the degradation time of the overall material.

[0065] The preparation process of the polyethylene glycol solution is as follows: obtain polyethylene glycol with a molecular weight of 200Da to 6000Da, dissolve it in dichloromethane, and stir at room temperature to obtain a polyethylene glycol solution with a polyethylene glycol concentration of 45mg / mL to 55mg / mL. The BMP-2 / chitosan solid particles obtained in step A4 are mixed with the PLGA / PEG composite solution obtained in step A6 at a ratio of (0.5 to 1.5) g: 100mL. In order to obtain a stable suspension, an ultrasonic stirrer is used here for 10 minutes to 30 minutes to ensure that the particles are evenly distributed in the solution.

[0066] See also Figure 2 As shown, the post-treatment of the suspension obtained in step A7 in this embodiment includes the following steps:

[0067] A801. The suspension obtained in step A7 was dripped into deionized water at a rate of 5 mL / min to 50 mL / min while stirring, and the stirring speed was controlled at 100 rpm to 200 rpm to obtain a liquid having a white flocculent precipitate;

[0068] A802. Obtain flocculent precipitate by filtration and wash the flocculent precipitate with deionized water;

[0069] A803. The flocculent precipitate washed in step A802 is dried by freeze drying to obtain dry BMP-2 controlled sustained-release floccules.

[0070] In this embodiment, BMP-2 / chitosan solid particles prepared in a special manner and a PLGA / PEG composite solution are mixed to obtain a suspension in which chitosan, growth factor BMP-2, PLGA and polyethylene glycol are uniformly distributed. The suspension is post-processed to form a BMP-2 controllable sustained-release floc. The carrier in the BMP-2 controllable sustained-release floc prepared by the preparation method can be specifically degraded by lysozyme in the human body, and the lysozyme concentration in the bone defect site is affected by the severity of the bone defect site, that is, the larger the wound surface of the bone defect site, the greater the lysozyme concentration. The degradation of chitosan in the BMP-2 controllable sustained-release floc is regulated by lysozyme, so that the release amount and release rate of BMP-2 can be adjusted according to the progress of bone healing (that is, the concentration of lysozyme), so as to achieve long-term continuous release of the BMP-2 controllable sustained-release floc instead of instantaneous large-scale release, thereby enhancing the safety and effect of treatment. The invention solves the problems in the prior art that the sustained release effect of BMP-2 using hydrophilic materials is not strong, sudden release of factors is easily formed, and the sustained release time and sustained release dosage are uncontrollable.

[0071] It should be understood that the BMP-2 controlled sustained-release flocs prepared by the above preparation method should also be within the scope of protection of the present invention. The application of the BMP-2 controlled sustained-release flocs prepared by the above preparation method includes the following contents: the BMP-2 controlled sustained-release flocs are pressed into the same shape according to the shape of the bone defect, and filled in the corresponding bone defect.

[0072] The BMP-2 controlled sustained-release flocs prepared by the preparation method of this embodiment have good plasticity, are easily pressed into bone defect fillers of various desired shapes, are easy to use, and can be adaptively adjusted in shape according to the patient's bone defect site, ensuring accuracy of use and complete coverage.

[0073] Embodiment 1

[0074] The first embodiment of the present invention adopts the slow-release method of the present invention to obtain a BMP-2 controlled slow-release flocculent, which is specifically as follows: chitosan with a deacetylation degree of 85% and a molecular weight of 100 kDa is dissolved in a 2% acetic acid solution at a concentration of 20 mg / mL, and stirred evenly until completely dissolved to obtain a chitosan initial solution. The chitosan initial solution is adjusted to a pH value of 5.5 by an acetic acid buffer. A protein stabilizer (albumin Bovine Serum Albumin, BSA) is added to the chitosan solution, and the concentration is controlled at 0.1% (w / v), and then glycerol (Glycerol) is added at a volume ratio of 5% to prepare a chitosan composite solution, and the growth factor BMP-2 is added to the chitosan composite solution, and ultrasonic stirring is performed until the protein stabilizer and BMP-2 are completely non-specifically bound to obtain a non-specific binding solution, wherein the concentration of BMP-2 in the non-specific binding solution is 0.1 mg / mL. The non-specific binding solution was dried using a freeze-drying method to obtain a solid material, and the obtained material was ball-milled with zirconium oxide ball milling beads, and the ball milling parameters were controlled to obtain particles with a particle size of 10 microns. Polylactic acid-glycolic acid copolymer, wherein the molar ratio of lactic acid to glycolic acid was 20:80, was obtained, dissolved in dichloromethane, and stirred until completely dissolved to obtain a PLGA solution with a concentration of 50 mg / mL. Polyethylene glycol (PEG) with a molecular weight of 200 Da was dissolved in dichloromethane and stirred until completely dissolved to obtain a polyethylene glycol solution with a concentration of 50 mg / mL.

[0075] PLGA solution and polyethylene glycol solution are mixed in a ratio of 9:1, stirred until uniformly mixed, and a PLGA / PEG composite solution is obtained. The BMP-2 / chitosan particles prepared above are mixed with the prepared PLGA / PEG composite solution in a ratio of 1g:100mL, and an ultrasonic agitator is used to process for 10 minutes to form a stable suspension. The above suspension is then dripped into deionized water at a speed of 20mL / minute, stirred simultaneously, and the stirring speed is controlled at 100rpm to obtain a white flocculent precipitate, and the flocculent precipitate is obtained by filtering, washed with deionized water, and finally freeze-dried to obtain a dry BMP-2 controlled sustained-release flocculent.

[0076] Embodiment 2

[0077] In the present embodiment 1, the controlled slow-release floccules of BMP-2 are obtained by the slow-release method of the present invention, which is as follows: chitosan with a deacetylation degree of 90% and a molecular weight of 200 kDa is dissolved in a 2.5% acetic acid solution at a concentration of 50 mg / mL, and stirred evenly until completely dissolved to obtain a chitosan initial solution. The chitosan initial solution is adjusted to a pH value of 6 by an acetic acid buffer. A protein stabilizer (albumin Bovine Serum Albumin, BSA) is added to the chitosan solution at a concentration controlled at 1% (w / v), and then glycerol (Glycerol) is added at a volume ratio of 20% to prepare a chitosan composite solution, and the growth factor BMP-2 is added to the chitosan composite solution, and ultrasonic stirring is performed until the protein stabilizer and BMP-2 are completely non-specifically bound to obtain a non-specific binding solution, wherein the concentration of BMP-2 in the non-specific binding solution is 0.5 mg / mL. The non-specific binding solution was dried using a freeze-drying method to obtain a solid material, and the obtained material was ball-milled with zirconium oxide ball milling beads, and the ball milling parameters were controlled to obtain particles with a particle size of 50 microns. Polylactic acid-glycolic acid copolymer, wherein the molar ratio of lactic acid to glycolic acid was 50:50, was obtained, dissolved in dichloromethane, and stirred until completely dissolved to obtain a PLGA solution with a concentration of 45 mg / mL. Polyethylene glycol (PEG) with a molecular weight of 200 Da was dissolved in dichloromethane, and stirred until completely dissolved to obtain a polyethylene glycol solution with a concentration of 45 mg / mL.

[0078] PLGA solution and polyethylene glycol solution are mixed in a ratio of 4: 1, stirred until uniformly mixed, and PLGA / PEG composite solution is obtained. The BMP-2 / chitosan particles prepared above are mixed with the PLGA / PEG composite solution prepared in a ratio of 1.5g: 100mL, and an ultrasonic agitator is used to process 30 minutes to form a stable suspension. Then the above suspension is dripped into deionized water at a speed of 50mL / min, and stirred simultaneously, and the stirring speed is controlled at 200rpm to obtain a white flocculent precipitate, and the flocculent precipitate is obtained by filtering, and washed with deionized water, and finally freeze-drying method obtains dry BMP-2 controlled sustained-release flocculent.

[0079] The first simulated body fluid was prepared, and the pH of the simulated body fluid was adjusted to pH=7.4 using a phosphate buffer solution. 10 μg / mL lysozyme, 50 μg / mL lysozyme, and 100 μg / mL lysozyme were added to the first simulated body fluid, respectively. The degradation rate of the BMP-2 controlled sustained-release floccules of Example 1 was recorded, as shown in Table 1 below:

[0080]

[0081] 10 μg / mL lysozyme, 50 μg / mL lysozyme, and 100 μg / mL lysozyme were added to the first simulated body fluid, respectively, and the degradation rate of the BMP-2 controlled sustained-release floccules of Example 2 was recorded, as shown in Table 2 below:

[0082]

[0083] The simulated body fluids with different lysozyme concentrations in Table 1 and Table 2 regulate the degradation rate of the BMP-2 controlled sustained-release flocs of Examples 1 and 2. The higher the lysozyme concentration, the faster the degradation rate of the BMP-2 controlled sustained-release flocs. When the wound surface of the human bone defect is severe, more lysozyme is gathered here, and the degradation of the BMP-2 controlled sustained-release flocs is controlled to be relatively accelerated, and the BMP-2 in the BMP-2 controlled sustained-release flocs is quickly released. When the wound surface of the human bone defect is not severe or has slowly healed, less lysozyme is gathered here, and a low lysozyme concentration slows down the degradation rate of the BMP-2 controlled sustained-release flocs, and the BMP-2 in the BMP-2 controlled sustained-release flocs is slowly released, effectively avoiding the occurrence of excessive BMP-2, resulting in uncontrollable osteogenesis and excessive osteogenesis with irregular new bone structure.

[0084] Comparative Example 1 is a first BMP-2 sustained-release carrier prepared by the prior art using collagen sponge as a carrier material.

[0085] Comparative Example 2 is a second BMP-2 sustained-release carrier prepared by the prior art using collagen sponge as a carrier material, wherein the ratio of raw materials for preparing the first BMP-2 sustained-release carrier and the second BMP-2 sustained-release carrier is different.

[0086] Comparative Example 3 is a BMP-2 sustained-release carrier prepared by the prior art using gelatin as a carrier material.

[0087] The second simulated body fluid was prepared, the pH of the simulated body fluid was adjusted to pH=7.37 using phosphate buffer, and an appropriate amount of lysozyme was added to simulate the in vivo environment. The BMP-2 controlled slow-release flocs obtained in Example 1 and the BMP-2 slow-release carriers purchased in Comparative Examples 1, 2 and 3 were placed in the simulated body fluid environment prepared above and sample solutions were collected regularly. The BMP-2 release data were collected and recorded at different time periods as shown in Table 3 below.

[0088] Table 3:

[0089]

[0090] The carriers of Example 1, Comparative Example 1, Comparative Example 2 and Comparative Example 3 were simultaneously placed into the above-mentioned simulated in vivo environment on the morning of February 11, 2023, and data were collected every evening. From Table 3 and Figure 3 It can be seen that the release rate of BMP-2 in the BMP-2 controlled sustained-release flocs loaded with chitosan-PLGA composite materials obtained in Example 1 of the present invention is slow, and is sustained-released at a certain ratio close to time. The BMP-2 controlled sustained-release flocs prepared by the preparation method of the present invention load BMP-2, and the degradation rate of the BMP-2 controlled sustained-release flocs is regulated by the change in the body's own lysozyme concentration, thereby achieving self-regulation of the BMP-2 release rate, avoiding side effects caused by excessive release of BMP-2, such as ectopic bone formation or excessive bone formation, and ensuring the accuracy of treatment.

[0091] It can be seen from the data of Comparative Example 2 that on 2023.02.13, the release rate of BMP-2 reached 86% in a short time. It can be seen that the BMP-2 sustained-release carrier using collagen sponge as the carrier material is prone to instantaneous release, resulting in excessive release of BMP-2 and causing harm to patients.

[0092] To further verify the stability of the data, multiple groups of experiments lasting up to 42 days were conducted on Example 2 and Comparative Examples 4, 5 and 6. Comparative Examples 4, 5 and 6 corresponded to the BMP-2 sustained-release carriers prepared by the above-mentioned Comparative Examples 1, 2 and 3.

[0093] The third simulated body fluid was prepared, and the pH of the simulated body fluid was adjusted to pH=7.3 using bicarbonate buffer, and an appropriate amount of lysozyme was added to simulate the in vivo environment. The BMP-2 controlled slow-release flocs obtained in Example 2 and the BMP-2 slow-release carriers purchased in Comparative Examples 4, 5, and 6 were subjected to regular solution sample collection tests in the simulated body fluid environment prepared above, and the BMP-2 release data collection records for different time periods are shown in Table 4 below.

[0094] Table 4:

[0095]

[0096] The carriers of Example 2, Comparative Example 4, Comparative Example 5 and Comparative Example 6 were placed in a second simulated in vivo environment at the same time, and data were collected every night. From Table 4 and Figure 4 It can be seen that the BMP-2 carriers of Comparative Examples 1, 2 and 3 in the prior art are almost completely released on the 14th day, while the release rate of BMP-2 in the BMP-2 controlled sustained-release floccules with composite materials such as chitosan, PLGA / PEG, etc. obtained by Example 2 of the present invention is about 50% on the 14th day. The release rate of BMP-2 in the BMP-2 controlled sustained-release floccules of Example 2 of the present invention is significantly slower, and there is still BMP-2 that has not been released until the 42nd day. Figure 4 As shown, the release rate of BMP-2 is close to a certain proportion of time for sustained release, which further proves that the BMP-2 controlled sustained release flocs prepared by the method for preparing BMP-2 controlled sustained release flocs of the present invention can be regulated by lysozyme in the human body to the chitosan-PLGA multi-composite material, thereby controlling and slowing down the release dose of BMP-2.

[0097] The present invention utilizes the characteristics of "enzyme responsive materials" to prepare BMP-2 controllable sustained-release flocs that can be specifically degraded by lysozyme. In bone defect repair, the inner layer of the BMP-2 controllable sustained-release flocs is decomposed by lysozyme in the body during the bone tissue healing period, releasing the growth factor BMP-2, thereby controlling the release rate of BMP-2. The human body fluid is used to self-regulate the BMP-2 release rate of the BMP-2 controlled sustained-release flocs according to the severity of the wound surface, and the lysozyme content in the body fluid near the wound surface is different, so as to achieve a controllable bone tissue regeneration effect. At the same time, the composite degradable polymer material, that is, the PLGA / PEG composite material in this embodiment, provides the necessary structural support for the bone defect site at the initial stage of implantation, and has a slow degradation characteristic that prolongs the degradation time of the overall material.

[0098] According to the above-mentioned Example 1 and Example 2 and multiple groups of experimental data, it can be clearly seen that the BMP-2 controllable sustained-release flocs prepared by the preparation method of the present invention have a long release period, and can control the release rate according to the concentration of lysozyme in human body fluids, realize the controlled release of BMP-2, can automatically adjust the release of BMP-2 according to the progress of bone healing, realize long-term sustained release, rather than instantaneous large-scale release, and enhance the safety and effect of treatment. The BMP-2 controllable sustained-release flocs prepared by the preparation method of this embodiment can control the release dose of BMP-2, avoid side effects caused by its excessive dosage, such as ectopic bone formation or excessive bone formation, and ensure the accuracy of treatment.

[0099] It should be understood that the specific embodiments described above are only used to explain the present invention, and are not used to limit the present invention. Obvious changes or modifications derived from the spirit of the present invention are still within the protection scope of the present invention.

Claims

1. A method for preparing BMP-2 controlled slow-release flocs based on lysozyme self-regulation, characterized in that: The following steps are involved: A1. Prepare a chitosan solution having a chitosan concentration of 20 mg / mL to 50 mg / mL; A2. In step A1, a protein stabilizer and glycerol are sequentially added to the chitosan solution obtained to prepare a chitosan composite solution; A3. Add the growth factor BMP-2 to the chitosan composite solution obtained in step A2, and stir ultrasonically until the protein stabilizer and BMP-2 are completely non-specifically bound to obtain a non-specific binding solution, wherein the concentration of BMP-2 in the non-specific binding solution is 0.1 mg / mL to 0.5 mg / mL; A4. Prepare BMP-2 / chitosan solid particles with a particle size of 10 μm to 100 μm by freeze drying; A5. Prepare a PLGA solution having a PLGA concentration of 45 mg / mL to 55 mg / mL and a polyethylene glycol solution having a polyethylene glycol concentration of 45 mg / mL to 55 mg / mL; A6. Mixing the PLGA solution and the polyethylene glycol solution in a ratio of 9:1 to 4:1 to obtain a PLGA / PEG composite solution; A7. The BMP-2 / chitosan solid particles obtained in step A4 were mixed with the PLGA / PEG composite solution obtained in step A6 at a ratio of (0.5-1.5) g:100 mL, and ultrasonically stirred to obtain a stable suspension; A8. Post-treat the suspension obtained in step A7 to form BMP-2 controllable sustained-release flocs.

2. The method for preparing a BMP-2 controlled sustained-release floc based on lysozyme self-regulation according to claim 1, characterized in that: The preparation process of the chitosan solution in step A1 includes the following contents: Weigh chitosan with a molecular weight of 100 kDa to 200 kDa; the deacetylation degree of the chitosan is 85% to 95%; Dissolving chitosan in 1.5% to 3% acetic acid solution to obtain an initial solution; The pH value of the initial solution is adjusted to 5.5-6 using a buffer solution to obtain a chitosan solution.

3. The method for preparing a BMP-2 controlled sustained-release floc based on lysozyme self-regulation according to claim 2, characterized in that: The buffer solution includes at least one of a phosphate buffer, an acetate buffer, a sodium acetate buffer and a bicarbonate buffer.

4. The method for preparing a BMP-2 controlled sustained-release floc based on lysozyme self-regulation according to claim 1, characterized in that: The preparation of the chitosan composite solution described in step A2 comprises the following steps: A201. Add a protein stabilizer to the chitosan solution, stir and fuse to form a chitosan-protein mixed solution, wherein the protein concentration in the chitosan-protein mixed solution is 0.1% to 1% (w / v); A202. Add glycerol to the chitosan-protein mixed solution, and stir evenly at room temperature to form a chitosan composite solution, wherein the volume ratio of glycerol in the chitosan composite solution is 5% to 20% (v / v).

5. The method for preparing a BMP-2 controlled sustained-release floc based on lysozyme self-regulation according to claim 1, characterized in that: In step A4, the preparation of the BMP-2 / chitosan solid particles includes the following steps: freeze-drying the non-specific binding solution obtained in step A3 to obtain a solid material; The solid material is ball-milled with zirconium oxide ball milling beads, and the BMP-2 / chitosan solid particles are obtained by controlling the ball milling parameters.

6. The method for preparing a BMP-2 controlled sustained-release floc based on lysozyme self-regulation according to claim 1, characterized in that: The preparation process of the PLGA solution in step A5 is as follows: Obtaining a polylactic acid-glycolic acid copolymer, wherein the molar ratio of lactic acid to glycolic acid is 20:80 to 50:50; and the molecular weight of the polylactic acid-glycolic acid copolymer is 10 kDa to 30 kDa; The poly(lactic acid-glycolic acid) copolymer is dissolved in dichloromethane or chloroform to obtain a PLGA solution with a concentration of 45 mg / mL to 55 mg / mL.

7. The method for preparing a BMP-2 controlled sustained-release floc based on lysozyme self-regulation according to claim 1, characterized in that: The preparation process of the polyethylene glycol solution is as follows: polyethylene glycol with a molecular weight of 200Da to 6000Da is obtained, dissolved in dichloromethane or chloroform, and stirred at room temperature to obtain a polyethylene glycol solution with a polyethylene glycol concentration of 45mg / mL to 55mg / mL.

8. The method for preparing a BMP-2 controlled sustained-release floc based on lysozyme self-regulation according to claim 1, characterized in that: The post-treatment of the suspension obtained in step A7 comprises the following steps: A801. The suspension obtained in step A7 was dripped into deionized water at a rate of 5 mL / min to 50 mL / min while stirring, and the stirring speed was controlled at 100 rpm to 200 rpm to obtain a liquid having a white flocculent precipitate; A802. Obtain flocculent precipitate by filtration and wash the flocculent precipitate with deionized water; A803. The flocculent precipitate washed in step A802 is dried by freeze drying to obtain dry BMP-2 controlled sustained-release floccules.

9. A BMP-2 controlled sustained-release flocculent, characterized in that: The flocculent is prepared by the method for preparing BMP-2 controllable sustained-release flocs based on lysozyme self-regulation as described in any one of claims 1 to 8.

10. The use of the BMP-2 controlled sustained-release flocculent as described in claim 9, characterized in that: Includes the following: Pressing the BMP-2 controlled sustained-release floccules into the same shape according to the shape of the bone defect site; Fill in the corresponding bone defect.

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