A composite drug-loaded hydrogel and a preparation method and application thereof

Abstract

The scheme discloses the field of biomedical materials and bone tissue engineering technology, and discloses a composite drug-loaded hydrogel and a preparation method and application thereof.The composite drug-loaded hydrogel comprises a self-assembled peptide hydrogel base, hollow mesoporous dopamine nanoparticles and simvastatin, the simvastatin is loaded in the hollow mesoporous structure of the hollow mesoporous dopamine nanoparticles to form SIM@H-MPDA drug-loaded nanoparticles, and the drug-loaded nanoparticles are stably dispersed in the three-dimensional network structure of RADA16-I.The preparation method of the composite drug-loaded hydrogel is also disclosed, and the preparation method comprises H-MPDA preparation, SIM@H-MPDA drug-loaded nanoparticle preparation and composite hydrogel assembly.The composite drug-loaded hydrogel has good injectability and in-situ gelation, can realize smooth and slow release of simvastatin, significantly promotes osteogenic differentiation and new bone formation, effectively repairs non-infectious bone defects, and has no adverse effects on main organs of the body.

Description

Technical Field

[0001] This invention relates to the fields of biomedical materials and bone tissue engineering technology, and particularly to a composite drug-loaded hydrogel, its preparation method, and its application. Background Technology

[0002] Bone defects are a common clinical challenge in orthopedics, oral and maxillofacial surgery, and trauma repair. They are caused by a variety of factors, including severe trauma, tumor resection, congenital malformations, and degenerative diseases. The repair outcome directly affects the patient's limb function and quality of life. Small bone defects can heal on their own, but large defects, those with limited blood supply, or those accompanied by chronic inflammation are more likely to have their natural repair process inhibited, leading to delayed bone healing, nonunion, or even permanent functional impairment.

[0003] Currently, the "gold standard" for clinical bone defect repair is autologous or allogeneic bone transplantation, but this method has many limitations: autologous bone transplantation has a limited source of donors and can cause secondary damage to the donor site; allogeneic bone transplantation carries the risk of immune rejection, along with potential risks of infection and disease transmission. In recent years, bone tissue engineering technology has provided new solutions for bone defect repair. Ideal bone tissue engineering repair materials need to simultaneously possess good biocompatibility, injectability, shapeability, cell adhesion and proliferation support capabilities, sustained release of osteogenic drugs, and certain mechanical support properties.

[0004] Simvastatin (SIM), a widely used statin lipid-lowering drug in clinical practice, has been found in recent years to have significant osteogenic induction activity, promoting osteogenic differentiation and upregulating the expression of bone morphogenetic protein 2 (BMP2). It has been attempted for use in the repair of periodontal bone defects. However, simvastatin has poor water solubility and low oral bioavailability, making it difficult for conventional formulations to achieve targeted delivery and continuous administration to bone defect sites.

[0005] RADA16-I, a self-assembled peptide hydrogel, is a biomimetic nanofiber material that can mimic the extracellular matrix microenvironment, providing a scaffold for cell adhesion and proliferation. It also exhibits good biocompatibility and injectability. However, its mechanical properties are weak, and it lacks self-drug sustained-release capability, making it difficult to achieve long-term delivery of osteogenic drugs. Hollow mesoporous dopamine nanoparticles (H-MPDA) possess high specific surface area, good biocompatibility, and a good ability to load small molecule drugs, making them excellent hydrophobic drug carriers. They can effectively load simvastatin and achieve initial sustained release; however, H-MPDA particles are prone to aggregation, resulting in poor stability when directly applied to bone defects, and they cannot form a sustained osteogenic induction microenvironment.

[0006] Combining the drug-loaded sustained-release properties of H-MPDA with the scaffold support and in-situ gelation properties of RADA16-I hydrogel to construct a composite drug-loaded hydrogel system is an effective way to solve key problems in bone defect repair, such as low local drug delivery efficiency, unsustainable osteogenic induction, and poor material stability. Currently, there are no reports on the application of combining H-MPDA and RADA16-I to construct a simvastatin-loaded composite drug-loaded hydrogel for bone defect repair. Summary of the Invention

[0007] The present invention aims to provide a composite drug-loaded hydrogel, its preparation method and application, and to address the shortcomings of existing bone defect repair materials by providing a composite drug-loaded hydrogel that can achieve efficient loading and continuous, stable sustained release of simvastatin.

[0008] This invention provides a composite drug-loaded hydrogel comprising a hydrogel matrix formed by a self-assembled peptide RADA16-I and hollow mesoporous dopamine nanoparticles (H-MPDA) loaded with an osteogenic inducing drug. The H-MPDA nanoparticles are surface-modified mesoporous dopamine nanoparticles. The osteogenic inducing drug is simvastatin (SIM) loaded into the hollow mesoporous structure of the hollow mesoporous dopamine nanoparticles. The hollow mesoporous dopamine nanoparticles form a stable and dispersed composite hydrogel drug-loaded system within the three-dimensional network structure of the RADA16-I hydrogel. This composite hydrogel drug-loaded system enables the continuous release of simvastatin at the bone defect site.

[0009] Furthermore, the concentration of the self-assembled peptide RADA16-I in the hydrogel is 5-7 mg / mL; the final concentration of simvastatin in the composite drug-loaded hydrogel is 10 mg / mL. -7 mol / L; the hollow mesoporous dopamine nanoparticles are spherical with a particle size of 50-300 nm, and possess both hollow and mesoporous structures.

[0010] Furthermore, the simvastatin is loaded into hollow mesoporous dopamine nanoparticles through one or more of the following methods: physical adsorption, pore encapsulation, and hydrogen bonding; the in vitro drug release curve of the composite drug-loaded hydrogel exhibits sustained-release characteristics with no obvious burst release stage, and it is injectable and can gel in situ under physiological ionic conditions.

[0011] This composite drug-loaded hydrogel has no obvious explosive drug release phase, and its in vitro release curve shows a smooth, sustained-release characteristic. At the same time, it has good injectability and can rapidly gel in situ under physiological ionic conditions such as PBS and cell culture medium to fill irregularly shaped bone defect areas and provide scaffold support for bone tissue regeneration.

[0012] A method for preparing a composite drug-loaded hydrogel includes the following steps: Step 1: In a water-alcohol mixed solvent reaction system containing a template agent, a dopamine self-polymerization reaction is initiated under alkaline buffer conditions to prepare hollow mesoporous dopamine nanoparticles; Step 2: Remove the template agent from the hollow mesoporous dopamine nanoparticles, and after washing and drying, obtain purified hollow mesoporous dopamine nanoparticles. Step 3: Simvastatin is dissolved in an organic solvent and mixed with the purified hollow mesoporous dopamine nanoparticles to load simvastatin. After separation, washing and drying, hollow mesoporous dopamine nanoparticles loaded with simvastatin (SIM@H-MPDA drug-loaded nanoparticles) are obtained. Step 4: Disperse the SIM@H-MPDA drug-loaded nanoparticles in a solution of the self-assembled peptide RADA16-I, adjust the system to physiological ionic conditions, and induce RADA16-I to self-assemble into a three-dimensional network structure to obtain the composite drug-loaded hydrogel.

[0013] Furthermore, in step one, the template agent comprises 360 mg Pluronic F-127 and 420 μL 1,3,5-trimethylbenzene; in the water-alcohol mixed solvent, the volume ratio of deionized water to anhydrous ethanol is 65:60; the alkaline buffer condition is provided by a Tris-HCl buffer system, which consists of 90 mg Tris-HCl dissolved in 10 mL of deionized water.

[0014] Preferably, the dopamine self-polymerization reaction is carried out under aerobic, light-protected, and room temperature conditions for 24 hours, and the amount of dopamine added is 60 mg.

[0015] Furthermore, in step two, an anhydrous ethanol / acetone mixed solution with a volume ratio of 2:1 is used as the washing solvent, and the template agent is removed by a combination of ultrasonic vibration and centrifugation.

[0016] Preferably, the centrifugation conditions are 4℃, 12000rpm / min, and 15min; the drying method is freeze drying, and the drying time is 24h.

[0017] Furthermore, in step three, the mass ratio of the hollow mesoporous dopamine nanoparticles to simvastatin is 2:1.

[0018] Preferably, the mixing and contact conditions are room temperature, protection from light, and stirring for 24 hours; the separation method is centrifugation at 12000 rpm / min for 15 minutes; the washing solvent is a mixed solution of ultrapure water and anhydrous ethanol; and the drying method is vacuum freeze drying.

[0019] Furthermore, in step four, the solvent for the self-assembled peptide RADA16-I solution is deionized water containing 10% sucrose; after the drug-loaded nanoparticles are mixed with the RADA16-I solution, they are first stirred at room temperature and 550 rpm for 24 h to form a homogeneous suspension system, and then the ionic strength and pH value are adjusted to physiological conditions by adding PBS buffer or complete cell culture medium to induce self-assembly.

[0020] Application of a composite drug-loaded hydrogel in the preparation of medical materials for bone defect repair.

[0021] Furthermore, the bone defects are non-infectious bone defects, including skull defects, alveolar bone defects, and long bone defects.

[0022] Furthermore, the medical material is applied to the bone defect site through local implantation or injection, and gels in situ under physiological conditions to fill the defect area.

[0023] The working principle and beneficial effects of this scheme are as follows: This scheme disperses hollow mesoporous dopamine nanoparticles loaded with osteogenic induction drugs in a self-assembled peptide hydrogel to construct a composite drug-loaded hydrogel system, thereby achieving local sustained release of drugs and osteogenic promotion at bone defect sites.

[0024] 1. Achieving efficient drug loading and dual sustained release: The hollow mesoporous structure of H-MPDA provides a large number of loading sites for simvastatin, achieving efficient loading of hydrophobic simvastatin through physical adsorption, pore encapsulation, and hydrogen bonding; The three-dimensional network structure of RADA16-I hydrogel stably embeds SIM@H-MPDA drug-loaded nanoparticles, forming a dual sustained-release system of "nanoparticle-hydrogel", effectively inhibiting the burst release of drugs and achieving continuous and stable release of simvastatin at bone defect sites, thus prolonging osteogenic induction time.

[0025] 2. Excellent Comprehensive Material Performance: The composite drug-loaded hydrogel combines the drug delivery capabilities of H-MPDA with the biomimetic scaffold properties of RADA16-I hydrogel. It exhibits excellent biocompatibility and provides a suitable microenvironment for osteoblast adhesion, proliferation, and differentiation. Simultaneously, the material possesses excellent injectability and in-situ gelling properties, adapting to irregularly shaped bone defects and improving the fit between the material and the defect site. It eliminates the need for complex shaping operations, making it suitable for minimally invasive repair. Furthermore, it continuously releases osteogenic induction drugs, creating a stable osteogenic stimulation environment locally and promoting new bone formation.

[0026] 3. Significant osteogenic induction activity: Simvastatin, released by the composite drug-loaded hydrogel, can effectively upregulate the expression of osteogenic-related genes (RUNX2) and proteins (BMP2, ALP), significantly increase the activity of alkaline phosphatase in osteoblasts, and promote the formation of mineralized nodules. In vivo experiments have confirmed that this material can significantly promote new bone formation in rabbit skull defect areas, increase bone volume fraction (BV / TV) and bone mineral density (BMD), and the bone defect repair effect is significantly better than that of simple RADA16-I hydrogel.

[0027] 4. High biocompatibility: In vitro cell experiments show that the composite drug-loaded hydrogel has no significant cytotoxicity to osteoblast precursor cells and can support cell survival, adhesion and proliferation; in vivo animal experiments confirm that the material does not have adverse effects on the tissue structure and function of major organs such as the heart, liver, spleen, lungs and kidneys, and serum liver and kidney function indicators are normal, and there is no obvious inflammatory reaction when applied locally, indicating high safety for clinical application.

[0028] 5. Simple and controllable preparation method: The preparation method of this invention uses the soft template method to prepare H-MPDA. Drug loading and hydrogel composite are achieved through simple physical mixing. The entire preparation process does not require complicated instruments and equipment. The reaction conditions are mild (room temperature and normal pressure). The process parameters of each step are clear and controllable, and it is easy to scale up production. Attached Figure Description

[0029] Figure 1 A schematic diagram of the design of S-HM / RA16 composite hydrogel for bone defect repair; Figure 2 A schematic diagram of the preparation of SIM@H-MPDANPs; Figure 3 (A) TEM image of H-MPDA nanoparticles, (B) Particle size distribution of H-MPDA, (C) Zeta potential of H-MPDANPs and SIM@H-MPDANPs in water (n=3), (D) Infrared spectra of Simvastatin, H-MPDANPs and SIM@H-MPDANPs. Figure 4 To characterize the gelling properties, injectability, and microstructure of the RADA16-I composite hydrogel, (A) the in vitro gelling properties of RADA16-I alone, (B) the in vitro gelling properties of the S-HM / RA16 composite hydrogel, (C) the injectability test of the S-HM / RA16 composite hydrogel, (D) the scanning electron microscopy (SEM) observation results of the RADA16-I group hydrogel, and (E) the scanning electron microscopy (SEM) observation results of the S-HM / RA16 composite hydrogel. Figure 5 The in vitro release curves of SIM@H-MPDANPs and their composite RADA16-I hydrogel are shown. Figure 6 To assess the cell compatibility of H-MPDANPs, SIM@H-MPDANPs and composite hydrogels, (A) cell viability detection of MC3T3-E1 cells by H-MPDANPs, (B) cell viability detection of MC3T3-E1 cells by SIM@H-MPDANPs, (C) cell viability detection of MC3T3-E1 cells by each composite hydrogel, (D) observation of live / dead cell fluorescence staining, (E) and (F) detection of adhesion of MC3T3-E1 cells to the surface of each group of hydrogels; Figure 7 To illustrate the promoting effect of S-HM / RA16 composite hydrogel on osteogenic differentiation of MC3T3-E1 cells, (A) ALP staining results of MC3T3-E1 cells, (B) ARS mineralized nodules staining results of MC3T3-E1 cells, (C) semi-quantitative analysis of ALP staining, and (D) semi-quantitative analysis of ARS staining. Figure 8 To assess the in vivo bone repair effect of S-HM / RA16 composite hydrogel in a rabbit skull defect model, (A) establishment of a rabbit skull critical size defect model, and (B) gross observation of tissue samples taken from each group at 4 and 8 weeks post-surgery. Figure 8 Micro-CT analysis of skull defect area in CF rabbits: (C) Representative three-dimensional and two-dimensional reconstructed images of skull defect area at 4 and 8 weeks postoperatively, (D) Micro-CT cross-sectional images of local skull defect area in each group, (E) and (F) Quantitative statistical results of bone volume fraction (BV / TV) and bone mineral density (BMD) of new bone in each group. Figure 9 To assess the biocompatibility of the composite hydrogel in vivo, (A) results of liver and kidney function indicators in each group of animals at 8 weeks post-surgery, and (B) HE staining of major organs (heart, liver, spleen, lung, and kidney) at 4 and 8 weeks post-surgery. Detailed Implementation

[0030] The following detailed explanation illustrates the specific implementation methods: Example 1 A composite drug-loaded hydrogel, Preparation of SIM@H-MPDA drug-loaded nanoparticles 1. Preparation of H-MPDA nanoparticles (1) Take 65 mL of deionized water and 60 mL of anhydrous ethanol and place them in a three-necked flask. Stir magnetically to mix them evenly. Then add 360 mg of Pluronic F-127 and 420 μL of 1,3,5-trimethylbenzene (TMB) in sequence. Continue stirring magnetically for 30 min to obtain a homogeneous template solution. (2) Weigh 90 mg of tris(hydroxymethyl)aminomethane hydrochloride Tris-HCl, dissolve it in 10 mL of ultrapure water, slowly add the buffer solution dropwise to the template solution, and stir for 10 min; (3) Then weigh 60 mg of dopamine hydrochloride and add it to the above reaction system. Stir until completely dissolved. Wrap the three-necked flask with tin foil and stir magnetically for 24 h under oxygen and room temperature conditions to carry out the dopamine self-polymerization reaction.

[0031] (4) After the reaction is complete, transfer the reaction solution to a centrifuge tube and centrifuge at 4°C and 12,000 rpm / min for 15 min. Discard the supernatant and collect the black precipitate. Add anhydrous ethanol / acetone mixed solution (volume ratio 2:1) to the precipitate, sonicate for 30 min, and centrifuge again at 4°C and 12,000 rpm / min for 15 min. Discard the supernatant. Repeat the above washing steps 3 times to remove the template agent. (5) The washed precipitate was resuspended in ultrapure water, centrifuged and washed twice to remove organic solvents, and finally the precipitate was freeze-dried for 24 hours to obtain purified H-MPDA nanoparticles with a particle size of 50-300 nm. The nanoparticles were sealed and stored in a refrigerator at 4°C for later use.

[0032] 2. Preparation of SIM@H-MPDA drug-loaded nanoparticles, such as... Figure 2 As shown Weigh appropriate amounts of simvastatin and H-MPDA nanoparticles separately and prepare a 2 mg / mL solution with anhydrous ethanol. Place equal volumes of the two solutions in centrifuge tubes at a H-MPDA to simvastatin mass ratio of 2:1, and stir magnetically for 24 h at room temperature in the dark. After stirring, centrifuge at 12000 rpm / min for 15 min, discard the supernatant, and collect the black precipitate. Wash the precipitate three times with a mixture of ultrapure water and anhydrous ethanol (volume ratio 1:1), centrifuging at 12000 rpm / min for 15 min each time to remove unloaded free simvastatin. Dry the resulting precipitate in a vacuum freeze dryer for 24 h to obtain SIM@H-MPDA drug-loaded nanoparticles, which are then sealed and stored at 4℃ for later use.

[0033] Structural characterization of the obtained H-MPDA and SIM@H-MPDA: Transmission electron microscopy (TEM) observation showed that, as Figure 3 (A) H-MPDA is a regular sphere with a distinct internal cavity structure and a mesoporous outer shell, exhibiting good dispersibility, such as... Figure 3 (B); Zeta potential detection shows, as Figure 3 (C) The potential of the nanoparticles loaded with simvastatin changed significantly, further confirming successful drug loading; Fourier transform infrared spectroscopy (FT-IR) showed that, as Figure 3(D) The simultaneous appearance of characteristic absorption peaks of simvastatin and H-MPDA in SIM@H-MPDA confirms the successful loading of simvastatin.

[0034] Example 2: Preparation and physicochemical characterization of S-HM / RA16 composite drug-loaded hydrogel 1. Preparation of S-HM / RA16 composite drug-loaded hydrogel Take 10 mg / tube of RADA16-I lyophilized powder, add 2 mL of deionized water containing 10% sucrose, and sonicate for 30 min until completely dissolved to prepare a 5 mg / mL RADA16-I solution. Store at 4°C for later use. Take 1 mL of the above RADA16-I solution and add a SIM@H-MPDA drug-loaded nanoparticle suspension that has been pre-dispersed evenly in ultrapure water (to bring the final concentration of simvastatin to 10). -7 The mixture was magnetically stirred at 550 rpm for 24 h at room temperature to form a homogeneous suspension. 0.2 mL of PBS buffer (pH=7.4) was added to the suspension, and the mixture was gently mixed and allowed to stand at room temperature for 5 min to induce RADA16-I self-assembly, forming a transparent and elastic S-HM / RA16 composite drug-loaded hydrogel.

[0035] Meanwhile, a simple RADA16-I hydrogel was prepared as a control group. The preparation method was the same as above, except that SIM@H-MPDA drug-loaded nanoparticles were not added.

[0036] 2. Physicochemical property characterization, such as Figure 4 Scale bar: 100μm (1) Gel forming properties 300 μL of RADA16-I blank hydrogel suspension and S-HM / RA16 composite hydrogel suspension were added to culture dishes containing 400 μL of complete cell culture medium, respectively, and incubated at room temperature to observe the gelation process. The results showed that both suspensions completed in-situ gelation within 5 minutes, forming stable hydrogel blocks without dissolution, indicating that the addition of SIM@H-MPDA does not affect the self-assembly gelation performance of RADA16-I. Figure 4 (A) and Figure 4 (B).

[0037] (2) Injectability The prepared S-HM / RA16 composite drug-loaded hydrogel suspension was loaded into a 1mL syringe and slowly injected, observing its flowability. The results showed that the suspension passed smoothly through the syringe needle and rapidly gelled in the culture medium after injection, indicating that the composite drug-loaded hydrogel has good injectability and is suitable for minimally invasive injection repair. Figure 4 (C) (3) Microstructure RADA16-I blank hydrogel and S-HM / RA16 composite hydrogel were fixed with 5% glutaraldehyde solution for 12 h, followed by gradient ethanol dehydration and critical point drying with carbon dioxide. The microstructure was then observed using scanning electron microscopy (SEM). Figure 4 (D) and Figure 4 (E). The results showed that both hydrogels exhibited a continuous porous three-dimensional network structure with uniform pore size; uniformly dispersed nanoparticles were visible in the network structure of the S-HM / RA16 composite hydrogel, with no obvious aggregation, indicating that SIM@H-MPDA and RADA16-I hydrogels have good structural compatibility and can be stably dispersed in the hydrogel matrix.

[0038] The above results demonstrate that SIM@H-MPDA nanospheres can be stably dispersed within the three-dimensional network structure formed by RADA16 hydrogel without affecting its self-assembly and gelation process, indicating good structural compatibility between the two. The resulting composite hydrogel exhibits a continuous porous structure, which is beneficial for material exchange and cell growth-related processes. The functional groups on the surface of polydopamine can interact with peptide molecules, thereby improving the structural stability of the system. This composite system can achieve in-situ gelation under physiological conditions and exhibits good injectability, making it suitable for minimally invasive filling and repair of bone defects, such as... Figure 1 .

[0039] Example 3: In vitro drug release characteristics of S-HM / RA16 composite drug-loaded hydrogel The in vitro drug release characteristics of the composite drug-loaded hydrogel were studied using the dialysis method. Three groups were set up: free simvastatin solution, SIM@H-MPDA drug-loaded nanoparticles, and S-HM / RA16 composite hydrogel, with three parallel samples in each group.

[0040] 1. Dialysis bag pretreatment: Take a dialysis bag with a molecular weight cutoff of 3500 Da, cut it into 10 cm long segments, rinse it repeatedly with ultrapure water, and soak it in ultrapure water for later use; 2. Sample preparation: Weigh out 400 μL of free simvastatin solution containing 1 mg simvastatin, SIM@H-MPDA drug-loaded nanoparticles, and S-HM / RA16 composite hydrogel, and disperse them in 2 mL of PBS buffer (pH=7.4) containing 1% SDS. Transfer the solutions to the pretreated dialysis bags and seal both ends of the dialysis bags. 3. In vitro release: Place the dialysis bag in a centrifuge tube containing 20 mL of the above release medium and shake it at 100 r / min in a constant temperature shaker at 37°C; at predetermined time points (0.5 h, 2 h, 6 h, 12 h, 24 h, 2 d, 3 d, 4 d, 5 d, 6 d, 7 d), take out 3 mL of release medium each time, and add an equal volume of fresh release medium at the same time; 4. Drug concentration determination: After filtering the released medium through a 0.45μm microporous membrane, the absorbance was measured at the characteristic wavelength of simvastatin using a UV-Vis spectrophotometer. The drug concentration was calculated based on the pre-established standard curve, and the cumulative release rate was calculated.

[0041]

[0042] Where Q is the cumulative drug release rate, V0 is the total volume of the release medium (mL), Ci is the drug concentration of the sample taken at time point i, Cn is the drug concentration of the sample taken at time point n, Vi is the sampling volume (mL), and m is the total mass of the drug.

[0043] Results of the in vitro release model are as follows Figure 5 As shown, (n=3) The results showed that the free simvastatin solution group had a cumulative release rate of over 90% within 24 hours, exhibiting a significant burst release; the drug release rate of the SIM@H-MPDA drug-loaded nanoparticle group was significantly reduced, with a cumulative release rate of approximately 70% over 7 days; the drug release process of the S-HM / RA16 composite hydrogel group was more gradual, without a significant burst release phase, with a cumulative release rate of approximately 60% over 7 days, and the release curve showed a continuous upward trend. This indicates that the "nanoparticle-hydrogel" dual sustained-release system formed by the composite hydrogel can effectively delay the release of simvastatin and achieve long-acting drug delivery.

[0044] Example 4: In vitro biosafety evaluation of S-HM / RA16 composite drug-loaded hydrogel Mouse osteogenic progenitor cells MC3T3-E1Subclone14 were selected as experimental cells. The in vitro biosafety of the composite hydrogel was evaluated by cell viability assay (CCK-8 assay), live / dead cell fluorescence staining, and cell adhesion assay. A blank control group (no material), a RADA16-I blank hydrogel group, and an S-HM / RA16 composite hydrogel group were set up, with 3 replicates in each group.

[0045] 1. Cell Culture Frozen MC3T3-E1 cells were removed from the -80℃ freezer, rapidly thawed in a 37℃ water bath, transferred to α-MEM complete medium containing 10% fetal bovine serum, centrifuged at 1200 rpm for 3 min, the supernatant was discarded, the cells were resuspended and seeded into culture flasks, and cultured in a 37℃, 5% CO2 cell culture incubator. The medium was changed every 2-3 days, and the cells were passaged when the confluence reached 80%.

[0046] 2. Preparation of material extract RADA16-I blank hydrogel and S-HM / RA16 composite hydrogel were added to 12-well plates respectively. After gelation induced by PBS, 10 times the volume of complete culture medium was added, and the plates were soaked at 37°C for 24 hours. The supernatant was collected to obtain the material extract, which was then filtered through a 0.22 μm filter membrane for sterilization before use.

[0047] 3. Cell viability assay, such as Figure 6 (A) Figure 6 (B) Figure 6 (C) MC3T3-E1 cells were loaded at 1×10 4 Cells were seeded at a density of 100 cells / well in 96-well plates and cultured for 24 hours until adherence. The original culture medium was discarded, and 100 μL of blank culture medium, RADA16-I extract, and S-HM / RA16 extract were added to each well. After culturing for 24 hours, 48 ​​hours, and 72 hours, 10 μL of LCK-8 reagent was added to each well and incubated for 1.5 hours. The absorbance (OD450) was measured at 450 nm using a microplate reader, and the cell viability was calculated.

[0048] The results showed that after 24h, 48h, and 72h of culture, the cell survival rate of each material group was above 90%, with no significant difference compared with the blank control group (P>0.05), indicating that the composite drug-loaded hydrogel had no obvious in vitro cytotoxicity and could support normal cell survival.

[0049] 4. Live / dead cell fluorescent staining, such as Figure 6 (D) Add 300 μL of LRADA16-I blank hydrogel and S-HM / RA16 composite hydrogel to a 12-well plate, gel with PBS, and soak in complete culture medium for 24 h; then add 1×10⁻⁶ μL of PBS to the wells of the plate. 5 MC3T3-E1 cells were seeded at a density of cells / well and cultured for 48 h. After washing twice with PBS, Calcein AM / PI staining solution was added and incubated at 37 °C for 40 min. After washing three times with PBS, live / dead cells were observed using a fluorescence microscope.

[0050] The results showed that the cells in each material group were mainly live cells (green) with very few dead cells (red), and there was no significant difference from the blank control group, further confirming that the composite drug-loaded hydrogel has good in vitro biocompatibility.

[0051] 5. Cell adhesion assay, such as Figure 6 (E) and Figure 6 (F), Scale bar: 100 μm. Data are expressed as mean ± SD (n=3), compared with the control group. p<0.05, p<0.01, p<0.001, p<0.0001. Data are expressed as mean ± SD (n=3). Compared with the control group, p<0.05, p<0.01, p<0.001, p<0.0001.

[0052] 300 μL of pre-prepared RADA16-I blank hydrogel and S-HM / RA16 composite hydrogel were placed in 24-well plates, and after gelation, they were soaked in complete culture medium for 24 h; then, 1×10⁻⁶ μL of the medium was added. 5 MC3T3-E1 cells were seeded at a density of cells / well, and the number of adherent cells was observed and counted using an inverted optical microscope at 10 min, 30 min, and 90 min after seeding.

[0053] The results showed that the number of cells adhering in the S-HM / RA16 composite hydrogel group was not significantly different from that in the RADA16-I blank hydrogel group, and both increased with time. This indicates that the composite drug-loaded hydrogel can provide a good scaffold surface for cell adhesion and does not affect the early adhesion of cells.

[0054] Example 5: Evaluation of the in vitro osteogenic induction activity of S-HM / RA16 composite drug-loaded hydrogel The in vitro osteogenic induction activity of the composite hydrogel was evaluated by alkaline phosphatase (ALP) staining and alizarin red (ARS) mineralized nodule staining. A blank control group, a RADA16-I blank hydrogel group, and an S-HM / RA16 composite hydrogel group were set up, with 3 replicates in each group.

[0055] 1. Cell seeding and osteogenic induction Coat 12-well plates (with Transwell chambers, 8 μm pore size) with 0.1% gelatin solution, incubate at 37°C for 2 h, discard the coating solution, and dry for later use; MC3T3-E1 cells are then cultured at 5 × 10⁻⁶ cells / well. 4 The cells were seeded at a density of cells / well in coated 12-well plates and cultured for 24 h until the cells adhered. 200 μL of each hydrogel sample was placed in the upper chamber of the Transwell plate, and the culture medium was replaced with osteogenic induction medium (containing 10 mmol / L sodium β-glycerophosphate, 50 μg / mL ascorbic acid, and 100 nmol / L dexamethasone). The culture medium was changed every 48-72 h.

[0056] 2. Alkaline phosphatase (ALP) staining, such as... Figure 7 (A) After 7 days of osteogenic induction culture, the culture medium was discarded, and the cells were fixed with 4% paraformaldehyde for 30 min, rinsed 3 times with PBS, added with the working solution of the BCIP / NBT alkaline phosphatase staining kit, incubated at room temperature for 30 min, rinsed with distilled water to stop staining, observed and photographed with an optical microscope, and semi-quantitative analysis was performed using image analysis software.

[0057] The results showed that the ALP-positive area in the cells of the S-HM / RA16 composite hydrogel group was significantly larger than that in the blank control group and the RADA16-I blank hydrogel group. Semi-quantitative analysis showed that its ALP activity was significantly higher than that of the two control groups (P<0.01). Figure 7 (C) indicates that the composite drug-loaded hydrogel can significantly increase the ALP activity of osteoblasts and promote early osteogenic differentiation.

[0058] 3. Alizarin Red (ARS) staining of mineralized nodules, such as... Figure 7 (B) After 21 days of osteogenic induction culture, the culture medium was discarded, and the cells were washed three times with PBS and fixed with 4% paraformaldehyde for 30 min. 1% Alizarin Red S solution (pH=4.2) was added, and staining was performed at room temperature in the dark for 30 min. The cells were repeatedly rinsed with distilled water until the background was colorless. The formation of mineralized nodules was observed using an optical microscope, and semi-quantitative analysis was performed using image analysis software. Figure 7 (D), (a is the control group, b is the RADA16-I group, c is the S-HM / RA16 composite hydrogel group) Scale bar: 200 μm. Data are expressed as mean ± SD (n=3), compared with the control group. p<0.01.

[0059] The results showed that the number and size of mineralized nodules in the S-HM / RA16 composite hydrogel group were significantly better than those in the blank control group and the RADA16-I blank hydrogel group. Semi-quantitative analysis showed that the area of ​​mineralized nodules in the S-HM / RA16 composite hydrogel group was significantly higher than that in the two control groups (P<0.01), indicating that the composite drug-loaded hydrogel can significantly promote osteoblast mineralization and accelerate bone matrix formation.

[0060] Example 6: Evaluation of the in vivo bone defect repair effect and biosafety of S-HM / RA16 composite drug-loaded hydrogel Establish a non-infectious skull bone defect model in New Zealand white rabbits, such as... Figure 8(A), Scale bar: 5mm, to evaluate the in vivo bone defect repair effect and biosafety of composite hydrogel, set up blank control group (no material), RADA16-I group, S-HM / RA16 low dose group (simvastatin 0.2mg / defect), S-HM / RA16 high dose group (simvastatin 0.5mg / defect), 4 bone defect sites in each group.

[0061] 1. Establishment of experimental animals and models Ten 3-month-old male New Zealand white rabbits, weighing 2.5-3.0 kg, were selected and acclimatized for one week. They were anesthetized by intravenous injection of 20% urethane (5 mL / kg) in the ear. The heads were shaved and disinfected with povidone-iodine. The skin was cut along the midline of the skull, the periosteum was separated, and the skull was exposed. A standardized circular bone defect model was prepared at a symmetrical position on the parietal bone using a 5 mm diameter trephine. The model was continuously rinsed with sterile saline to avoid overheating of the bone tissue.

[0062] 2. Material implantation and postoperative management Each experimental material was implanted into the bone defect site, and the skin was sutured layer by layer. Gentamicin sulfate (80,000 U / rabbit) was injected intramuscularly for 3 consecutive days after the operation to prevent infection. The rabbits were fed normally, and 5 rabbits were sacrificed at 4 weeks and 8 weeks after the operation for material testing.

[0063] 3. General observation, such as Figure 8 (B) Rabbits were euthanized at 4 and 8 weeks post-surgery, and skull specimens were extracted, repeatedly rinsed with PBS, and the repair of bone defects was observed. Results showed that at 4 weeks post-surgery, no significant new bone formation was observed in the blank control group and the RADA16-I group, and the defect boundaries were clear. In the low- and high-dose S-HM / RA16 groups, a small amount of new bone formation was observed in the defect sites, but the defect boundaries were blurred. At 8 weeks post-surgery, significant bone defects remained in the blank control group and the RADA16-I group. In the low-dose S-HM / RA16 group, the defect sites were mostly filled with new bone, and in the high-dose group, the defect sites were almost completely filled with new bone, with the defect boundaries essentially disappearing, indicating the best repair effect.

[0064] 4. Micro-CT scanning and quantitative analysis, such as Figure 8 (C~F) The skull specimen was fixed with 4% paraformaldehyde and subjected to Micro-CT scanning to perform three-dimensional reconstruction of the bone defect area, such as... Figure 8 (C) and Figure 8 (D), and calculate bone volume fraction (BV / TV) and bone mineral density (BMD), such as Figure 8 (E) and Figure 8(F), a, control group; b, RADA16-I group; c, SIM@H-MPDA / RADA16-I low-dose group; d, SIM@H-MPDA / RADA16-I high-dose group. Scale bar: 2 mm. Data are expressed as mean ± SD (n=4), compared with the control group. p<0.05, p<0.001. The results showed that at 4 and 8 weeks postoperatively, the BV / TV and BMD of the low- and high-dose S-HM / RA16 groups were significantly higher than those of the blank control group and the RADA16-I group (P<0.05), and the high-dose group was significantly higher than the low-dose group (P<0.05). At 8 weeks postoperatively, the BV / TV and BMD of all experimental groups were higher than those at 4 weeks postoperatively, indicating that the composite drug-loaded hydrogel can significantly promote new bone formation in vivo, and the repair effect increases with time, showing a certain dose-dependent effect.

[0065] 5. In vivo biosafety evaluation (1) Serum biochemical index detection Eight weeks post-surgery, peripheral blood was collected from the ear vein of rabbits. Serum was separated by centrifugation, and liver function indicators (ALT, AST) and kidney function indicators (BUN, CREA) were detected using a fully automated biochemical analyzer. Figure 9 (A), data are expressed as mean ± SD (n=5). The results showed that there were no significant differences in serum liver and kidney function indicators between the material groups and the blank control group (P>0.05), and all were within the normal physiological range, indicating that the in vivo application of the composite drug-loaded hydrogel did not have adverse effects on the liver and kidney function of rabbits.

[0066] (2) HE staining of major organs Rabbits were euthanized at 4 and 8 weeks post-surgery. Major organs such as the heart, liver, spleen, lungs, and kidneys were harvested, fixed in 4% paraformaldehyde, dehydrated with graded ethanol, embedded in paraffin, sectioned (4-5 μm), stained with hematoxylin and eosin (HE), and their tissue structures were observed under a light microscope. Figure 9 (B), Scale bar: 50 μm. The results showed that the major organ tissue structures of each material group were intact, without pathological changes such as edema, necrosis, or inflammatory cell infiltration, and there was no significant difference from the blank control group, indicating that the composite drug-loaded hydrogel has good in vivo biocompatibility.

[0067] The S-HM / RA16 composite drug-loaded hydrogel of this invention has good biocompatibility, injectability, in-situ gelation and drug sustained-release properties. It can effectively promote osteogenic differentiation and in vivo new bone formation. Moreover, the preparation method is simple and controllable, the raw materials are readily available, and it can be mass-produced. This composite hydrogel can be used as a medical material for bone defect repair. It can be applied to the repair of non-infectious bone defects (skull, alveolar bone, long bones, etc.) through minimally invasive injection or local implantation. It has broad clinical application prospects and industrial promotion value.

[0068] The above descriptions are merely embodiments of the present invention, and common knowledge regarding specific structures and characteristics is not elaborated upon here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the structure of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. A composite drug-loaded hydrogel, characterized in that: The composite hydrogel drug delivery system comprises a hydrogel matrix formed by the self-assembled peptide RADA16-I and hollow mesoporous dopamine nanoparticles loaded with an osteogenic inducing drug, wherein the osteogenic inducing drug is simvastatin and is loaded into the hollow mesoporous structure of the hollow mesoporous dopamine nanoparticles, and the hollow mesoporous dopamine nanoparticles form a stable and dispersed composite hydrogel drug delivery system in the three-dimensional network structure of the RADA16-I hydrogel.

2. The composite drug-loaded hydrogel according to claim 1, characterized in that: The concentration of the self-assembled peptide RADA16-I in the hydrogel is 5-7 mg / mL; the final concentration of simvastatin in the composite drug-loaded hydrogel is 10 mg / mL. -7 mol / L; the hollow mesoporous dopamine nanoparticles are spherical with a particle size of 50-300 nm, and possess both hollow and mesoporous structures.

3. The composite drug-loaded hydrogel according to claim 2, characterized in that: Simvastatin is loaded into hollow mesoporous dopamine nanoparticles through one or more of the following mechanisms: physical adsorption, pore encapsulation, and hydrogen bonding.

4. A method for preparing a composite drug-loaded hydrogel according to any one of claims 1 to 3, characterized in that: The process includes the following steps: Step 1: In a water-alcohol mixed solvent reaction system containing a template agent, dopamine self-polymerization reaction is initiated under alkaline buffer conditions to prepare hollow mesoporous dopamine nanoparticles; Step 2: Remove the template agent from the hollow mesoporous dopamine nanoparticles, and after washing and drying, obtain purified hollow mesoporous dopamine nanoparticles. Step 3: Simvastatin is dissolved in an organic solvent and mixed with the purified hollow mesoporous dopamine nanoparticles to load simvastatin. After separation, washing and drying, SIM@H-MPDA drug-loaded nanoparticles are obtained. Step 4: Disperse the SIM@H-MPDA drug-loaded nanoparticles in a solution of the self-assembled peptide RADA16-I, adjust the system to physiological ionic conditions, and induce RADA16-I to self-assemble into a three-dimensional network structure to obtain the composite drug-loaded hydrogel.

5. The composite drug-loaded hydrogel according to claim 4, its preparation method and application, characterized in that: In step one, the template agent includes Pluronic F-127 and 1,3,5-trimethylbenzene; in the water-alcohol mixed solvent, the volume ratio of deionized water to anhydrous ethanol is 65:60; the alkaline buffer condition is provided by a Tris-HCl buffer system, which is Tris-HCl dissolved in deionized water.

6. The composite drug-loaded hydrogel according to claim 5, its preparation method and application, characterized in that: In step two, an anhydrous ethanol / acetone mixed solution with a volume ratio of 2:1 is used as the washing solvent, and the template agent is removed by a combination of ultrasonic vibration and centrifugation.

7. The composite drug-loaded hydrogel according to claim 6, its preparation method and application, characterized in that: In step three, the mass ratio of the hollow mesoporous dopamine nanoparticles to simvastatin is 2:

1.

8. The composite drug-loaded hydrogel according to claim 7, its preparation method and application, characterized in that: In step four, the solvent for the self-assembled peptide RADA16-I solution is deionized water containing 10% sucrose. After the drug-loaded nanoparticles are mixed with the RADA16-I solution, they are first stirred at room temperature and 550 rpm for 24 hours to form a homogeneous suspension system. Then, the ionic strength and pH value are adjusted to physiological conditions by adding PBS buffer or complete cell culture medium to induce self-assembly.

9. The application of a composite drug-loaded hydrogel according to any one of claims 1 to 3, 5 to 8 in the preparation of medical materials for bone defect repair.

10. The application of the composite drug-loaded hydrogel according to claim 9, characterized in that: The bone defect is a non-infectious bone defect, including skull defects, alveolar bone defects, and long bone defects; the medical material is applied to the bone defect site through local implantation or injection, and gels in situ under physiological conditions to fill the defect area.

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