Gel composite slow-release carrier and preparation method thereof

Abstract

The invention belongs to the technical field of hydrogel, and particularly relates to a gel composite slow-release carrier and a preparation method thereof. The preparation method disclosed by the invention at least comprises the following steps: S1, preparing oldacapib drug-loaded microspheres and S4, preparing a gel composite slow-release carrier, specifically, dissolving oldacapib in an organic solvent, adding a polylactic acid-polyglycolic acid copolymer, and dissolving to obtain a first mixed solution; dissolving polyvinyl alcohol in water to obtain a second mixed solution; dropwise adding the first mixed solution into the second mixed solution while stirring to obtain a third mixed solution; centrifuging the third mixed solution to obtain a first precipitate, cleaning and centrifuging the first precipitate to obtain a second precipitate, and freeze-drying the second precipitate to obtain the oxadeca drug-loaded microspheres; adding a photoinitiator into methacrylated gelatin, and dissolving to obtain a fourth mixed solution; and mixing the fourth mixed solution with the oxadeca drug-loaded microspheres, and gelatinizing under illumination to obtain the gel composite slow-release carrier. The gel composite slow-release carrier disclosed by the invention can continuously release drugs and is beneficial to treatment of periodontitis.

Description

Technical Field

[0001] The present disclosure belongs to the technical field of hydrogels, and particularly relates to a gel composite sustained-release carrier and a preparation method thereof. Background Art

[0002] The incidence of periodontitis is relatively high, and its pathological features are the destruction of the gingiva, periodontal ligament and alveolar bone, resulting in severe bone loss and tooth loosening and exfoliation. Cathepsin K (CTSK) is a key enzyme in the bone resorption process. The expression level of cathepsin K (CTSK) in the periodontal tissue of periodontitis patients is significantly up-regulated, and the concentration and activity in the gingival crevicular fluid are also significantly increased. Inhibiting the expression of cathepsin K (CTSK) has become an effective means to promote the repair of inflammatory alveolar bone.

[0003] Existing periodontitis treatment products often only control the progression of inflammation and cannot inhibit the expression of cathepsin K (CTSK). Odanacatib (ODN) is an inhibitor of cathepsin K (CTSK), but in long-term clinical systemic drug use, the use of odanacatib (ODN) increases the risk of patients suffering from cardiovascular and cerebrovascular diseases, which seriously hinders its further application.

[0004] Therefore, it is necessary to explore a drug delivery system that can safely and efficiently apply odanacatib (ODN) locally to the periodontium and ensure the sustained release of drug components. Summary of the Invention

[0005] The purpose of the present disclosure is to overcome the deficiencies of the prior art and provide a gel composite sustained-release carrier and a preparation method thereof.

[0006] In a first aspect, the present disclosure provides a preparation method of a gel composite sustained-release carrier, and the preparation method at least includes step S1 and step S4; wherein, step S1: preparing odanacatib-loaded microspheres (ODN-MS) specifically includes the following steps: S11: Dissolve odanacatib (ODN) in an organic solvent, and then add poly(lactic-co-glycolic acid) copolymer (PLGA) and dissolve to obtain a first mixed solution; S12: Dissolve polyvinyl alcohol (PVA) in water to obtain a second mixed solution; S13: Drop the first mixed solution into the second mixed solution while stirring to obtain a third mixed solution; S14: Centrifuge the third mixed solution to obtain a first precipitate, wash and centrifuge the first precipitate to obtain a second precipitate, and lyophilize the second precipitate to obtain the odanacatib-loaded microspheres (ODN-MS); Step S4: Preparation of the gel composite sustained-release carrier (ODN-MS-Gel) specifically includes the following steps: S41: Add a photoinitiator to methacrylated gelatin (GelMA) and dissolve it to obtain a fourth mixed solution; S42: Mix the fourth mixed solution with the Odonacatib-loaded microspheres (ODN-MS), and then gelify it under light to obtain the gel composite sustained-release carrier (ODN-MS-Gel).

[0007] In one embodiment, in step S11, the dosage ratio of the odanacatib (ODN), organic solvent, and poly (lactic-co-glycolic acid) copolymer (PLGA) is (0.004~0.005 g): (3~6 ml): (0.035~0.045 g).

[0008] In one embodiment, the detailed method of step S12 is that the polyvinyl alcohol (PVA) is dissolved in pure water by water bath heating at no less than 95°C for 1~2 h, and the dosage ratio of the polyvinyl alcohol (PVA) to pure water is (0.2~0.3 g): (40~60 ml).

[0009] In one embodiment, the detailed method of step S13 is to place the first mixed solution into a syringe, and drop it into the second mixed solution at an injection speed of (0.4~0.6) mL / min, while stirring, and continuously stir at 700~900 rpm for 3~4 h.

[0010] In one embodiment, the detailed method of obtaining the second precipitate in step S14 is to centrifuge the third mixed solution at 3000~4000 rpm for 10~15 min to obtain the first precipitate, then wash the first precipitate with deionized water and centrifuge, and repeat the washing 3 times.

[0011] In one embodiment, the detailed method of freeze-drying in step S14 is to pre-freeze the vacuum freeze dryer at -75~-85°C for 3~4 h, and then place the second precipitate into the vacuum freeze dryer and freeze-dry for 8~12 h.

[0012] In one embodiment, between step S1 and step S4, there is also step S2: Determine that the preparation of the Odonacatib-loaded microspheres (ODN-MS) is successful, specifically including the following steps: S21: Adopt the method of step S1 and remove the odanacatib (ODN) to prepare poly (lactic-co-glycolic acid) copolymer microspheres (MS); S22: Dissolve the odanacatib (ODN), poly (lactic-co-glycolic acid) copolymer microspheres (MS), and odanacatib-loaded microspheres (ODN-MS) in an organic solvent respectively. Observe and record the absorption wavelengths of the odanacatib (ODN), poly (lactic-co-glycolic acid) copolymer microspheres (MS), and odanacatib-loaded microspheres (ODN-MS). Compare the maximum absorption wavelengths to determine the successful preparation of the odanacatib-loaded microspheres (ODN-MS).

[0013] In one embodiment, between step S2 and step S4, there is also step S3: Determine the drug loading and encapsulation efficiency of the odanacatib-loaded microspheres (ODN-MS), which specifically includes the following steps: S31: Fit the standard curve regression equation of the odanacatib (ODN); S32: According to the standard curve regression equation, calculate the drug content of the odanacatib-loaded microspheres (ODN-MS), and then calculate the drug loading and encapsulation efficiency of the odanacatib-loaded microspheres (ODN-MS) according to the formula: Theoretical drug loading (%) = mODN / mtotal × 100%; Actual drug loading (%) = mcalculated ODN / mtotal × 100%; Encapsulation efficiency (%) = actual drug loading / theoretical drug loading × 100%.

[0014] In one embodiment, the specific method of step S41 is to add the photoinitiator to the methacrylated gelatin (GelMA) in the dark and dissolve it in a 40 - 50 °C constant temperature water bath for 20 - 30 min; The photoinitiator is LAP, and the dosage ratio of the methacrylated gelatin (GelMA) to LAP is 1 g:10 ml.

[0015] In one embodiment, the specific method of step S42 is to mix the fourth mixed solution with the odanacatib-loaded microspheres (ODN-MS) after sterilization and gelate it by irradiating with a light source for 10 - 30 s.

[0016] In one embodiment, after step S4, there is also step S5, which specifically includes the following steps: S51: Observe the poly (lactic-co-glycolic acid) copolymer microspheres (MS), odanacatib-loaded microspheres (ODN-MS), and gel composite sustained-release carrier (ODN-MS-Gel) using a microscope, compare the morphologies, and analyze that the gel composite sustained-release carrier (ODN-MS-Gel) can achieve drug sustained release; S52: Measure the particle sizes of the poly(lactic-co-glycolic acid) copolymer microspheres (MS) and the odanacatib-loaded microspheres (ODN-MS) using a scanning electron microscope, and determine whether the gel composite sustained-release carrier (ODN-MS-Gel) is injectable.

[0017] In one embodiment, after step S5, the method further includes step S6, and the specific method is as follows: S6: Take the odanacatib-loaded microspheres (ODN-MS) and the gel composite sustained-release carrier (ODN-MS-Gel) with the same drug content respectively, add an equal amount of in vitro release medium for sequential culture, and calculate the drug cumulative release rate according to the formula:

[0018] In one embodiment, after step S5, the method further includes step S7: Detect cell compatibility, and the specific steps include: S71: Using the method of step S4, replace the odanacatib-loaded microspheres (ODN-MS) with the poly(lactic-co-glycolic acid) copolymer microspheres (MS) to prepare a microsphere hydrogel (MS-Gel); S72: Prepare the complete medium extracts of the methacrylated gelatin (GelMA), the microsphere hydrogel (MS-Gel), and the gel composite sustained-release carrier (ODN-MS-Gel) respectively, inoculate bone marrow mesenchymal stem cells (BMSCs) for sequential culture, and detect and record the cell number, and compare to determine that the gel composite sustained-release carrier (ODN-MS-Gel) can promote cell proliferation; S73: Prepare the complete medium extracts of the methacrylated gelatin (GelMA), the microsphere hydrogel (MS-Gel), and the gel composite sustained-release carrier (ODN-MS-Gel) respectively, inoculate bone marrow mesenchymal stem cells (BMSCs) for culture, and then stain with a live / dead cell reagent, and compare to determine that the gel composite sustained-release carrier (ODN-MS-Gel) does not affect cell viability.

[0019] In one embodiment, after step S5, the method further includes step S8, and the specific steps include: S81: Using the method of step S4, replace the odanacatib-loaded microspheres (ODN-MS) with the poly(lactic-co-glycolic acid) copolymer microspheres (MS) to prepare a microsphere hydrogel (MS-Gel); S82: After photopolymerizing the methacrylated gelatin (GelMA), the microsphere hydrogel (MS-Gel), and the gel composite sustained-release carrier (ODN-MS-Gel) respectively, inoculate bone marrow mesenchymal stem cells (BMSCs) for culture, and observe the adhesion state using a microscope to determine that the gel composite sustained-release carrier (ODN-MS-Gel) is beneficial to cell adhesion.

[0020] In a second aspect, the present disclosure provides a gel composite sustained-release carrier, which is prepared by using the preparation method of the gel composite sustained-release carrier described in any one of the above embodiments.

[0021] In one embodiment, the application of the gel composite sustained-release carrier in periodontitis treatment products.

[0022] One of the beneficial effects of the present disclosure is that for the gel composite sustained-release carrier prepared in the present disclosure, odanacatib-loaded microspheres (ODN-MS) are first constructed, and then combined with methacrylated gelatin (GelMA) to form a gel composite sustained-release carrier (ODN-MS-Gel). GelMA has good biocompatibility and a hydrophilic surface, which solves the problem of poor retention of the local injection solution of ODN. Moreover, the ODN-MS-Gel of the present disclosure can continuously release drugs, and also has excellent biocompatibility, which can effectively promote cell proliferation, increase cell adhesion and information interaction between cells, and is beneficial to the treatment of chronic periodontitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The drawings incorporated in the specification and constituting a part of the specification illustrate embodiments of the present disclosure and, together with the description, are used to explain the principles of the present disclosure.

[0024] Figure 1 is a flowchart of an embodiment of the present disclosure; Figure 2 is a flowchart of another embodiment of the present disclosure; Figure 3 is a flowchart of still another embodiment of the present disclosure; Figure 4 is a UV-Vis spectrogram of odanacatib, poly (lactic-co-glycolic acid) copolymer microspheres and odanacatib-loaded microspheres in an embodiment of the present disclosure; Figure 5 is a standard curve graph of odanacatib in an embodiment of the present disclosure; Figure 6 is a morphological characterization graph of poly (lactic-co-glycolic acid) copolymer microspheres and odanacatib-loaded microspheres under a light microscope in an embodiment of the present disclosure; Figure 7 is a morphological characterization graph of poly (lactic-co-glycolic acid) copolymer microspheres, odanacatib-loaded microspheres and gel composite sustained-release carrier under an electron microscope in an embodiment of the present disclosure; Figure 8 is an in vitro drug release performance graph of odanacatib-loaded microspheres and gel composite sustained-release carrier in an embodiment of the present disclosure; Figure 9 is a cell proliferation result graph of methacrylated gelatin, microsphere hydrogel and gel composite sustained-release carrier in an embodiment of the present disclosure; Figure 10It is the live / dead staining result diagram of methacrylated gelatin, microsphere hydrogel and gel composite sustained-release carrier according to an embodiment of the present disclosure; Figure 11 It is the cytoskeleton staining diagram of methacrylated gelatin, microsphere hydrogel and gel composite sustained-release carrier according to an embodiment of the present disclosure; Figure 12 It is the scanning electron microscopy diagram of bone marrow mesenchymal stem cells on the surface of methacrylated gelatin, microsphere hydrogel and gel composite sustained-release carrier according to an embodiment of the present disclosure. Detailed implementation manners

[0025] Now, various exemplary embodiments of the present disclosure will be described in detail with reference to the accompanying drawings. It should be noted that: unless otherwise specifically stated, the relative arrangements of components and steps, numerical expressions and values set forth in these embodiments do not limit the scope of the present disclosure.

[0026] The following description of at least one exemplary embodiment is merely illustrative in nature and in no way serves as a limitation to the present disclosure and its application or use.

[0027] Technologies, methods and devices known to those of ordinary skill in the relevant art may not be discussed in detail, but where appropriate, the technologies, methods and devices should be regarded as part of the specification.

[0028] In all the examples shown and discussed herein, any specific value should be construed as merely exemplary and not as a limitation. Therefore, other examples of the exemplary embodiments may have different values.

[0029] It should be noted that: similar reference numerals and letters denote similar items in the following drawings, and thus, once an item is defined in one drawing, it does not need to be further discussed in subsequent drawings.

[0030] In this article, "upper", "lower", "front", "rear", "left", "right", etc. are only used to represent the relative positional relationship between relevant parts, rather than defining the absolute positions of these relevant parts.

[0031] In this article, "first", "second", etc. are only used for distinguishing each other, rather than indicating the degree of importance, order, and the prerequisite for mutual existence, etc.

[0032] In this article, "equal", "same", etc. are not strict mathematical and / or geometric limitations, and also include the allowable errors that can be understood by those skilled in the art and are allowed in manufacturing or using, etc.

[0033] In periodontitis, inflammatory factors (such as RANKL) activate osteoclasts, which secrete a large amount of cathepsin K (CTSK), resulting in excessive degradation of alveolar bone.

[0034] In the prior art, periodontitis treatment products can often only control the progression of inflammation, but cannot inhibit the expression of cathepsin K (CTSK). Odanacatib (ODN) is an inhibitor of cathepsin K (CTSK). After the Odanacatib (ODN) solution acts on the periodontal pocket, it can promote the polarization of macrophages into the M2 type, playing a role in regulating the repair of inflammatory alveolar bone defects. However, in long-term systemic clinical use, the use of Odanacatib (ODN) increases the risk of patients suffering from cardiovascular and cerebrovascular diseases, which seriously hinders its further application.

[0035] Therefore, it is necessary to explore a drug delivery system that can safely and efficiently apply Odanacatib (ODN) locally to the periodontium and ensure the sustained release of drug components.

[0036] The present disclosure provides a preparation method of a gel composite sustained-release carrier. The preparation method at least includes step S1 and step S4; wherein, step S1: preparing Odanacatib-loaded microspheres (ODN-MS) specifically includes the following steps: S11: Dissolve Odanacatib (ODN) in an organic solvent, and then add a poly(lactic-co-glycolic acid) copolymer (PLGA) and dissolve it to obtain a first mixed solution; S12: Dissolve polyvinyl alcohol (PVA) in water to obtain a second mixed solution; S13: Drop the first mixed solution into the second mixed solution while stirring to obtain a third mixed solution; S14: Centrifuge the third mixed solution to obtain a first precipitate, wash and centrifuge the first precipitate to obtain a second precipitate, and lyophilize the second precipitate to obtain Odanacatib-loaded microspheres (ODN-MS); Step S4: preparing a gel composite sustained-release carrier (ODN-MS-Gel) specifically includes the following steps: S41: Add a photoinitiator to methacrylated gelatin (GelMA) and dissolve it to obtain a fourth mixed solution; S42: Mix the fourth mixed solution with Odanacatib-loaded microspheres (ODN-MS), and then gelate under light to obtain a gel composite sustained-release carrier (ODN-MS-Gel).

[0037] For the gel composite sustained-release carrier prepared in the present disclosure, Odanacatib-loaded microspheres (ODN-MS) are first constructed, and then it is combined with methacrylated gelatin (GelMA) to form a gel composite sustained-release carrier (ODN-MS-Gel). Methacrylated gelatin (GelMA) has good biocompatibility and a hydrophilic surface, which can solve the problem of poor retention of the Odanacatib (ODN) local injection solution.

[0038] Moreover, the gel composite sustained-release carrier (ODN-MS-Gel) of the present disclosure can continuously release drugs, and also has excellent biocompatibility, which can effectively promote cell proliferation, increase cell adhesion and information interaction between cells, and is beneficial to the treatment of chronic periodontitis.

[0039] The following examples are used to further illustrate the preparation method of the gel composite sustained-release carrier provided by the present disclosure.

[0040] See Figure 1 , in one embodiment, the present disclosure provides a preparation method of a gel composite sustained-release carrier, and the preparation method at least includes step S1 and step S4; wherein, step S1: preparing odanacatib-loaded microspheres (ODN-MS) specifically includes the following steps: S11: Dissolve odanacatib (ODN) in an organic solvent, and then add poly(lactic-co-glycolic acid) copolymer (PLGA) and dissolve it to obtain a first mixed solution; S12: Dissolve polyvinyl alcohol (PVA) in water to obtain a second mixed solution; S13: Drop the first mixed solution into the second mixed solution while stirring to obtain a third mixed solution; S14: Centrifuge the third mixed solution to obtain a first precipitate, wash and centrifuge the first precipitate to obtain a second precipitate, and lyophilize the second precipitate to obtain odanacatib-loaded microspheres (ODN-MS); Step S4: Preparing the gel composite sustained-release carrier (ODN-MS-Gel) specifically includes the following steps: S41: Add a photoinitiator to methacrylated gelatin (GelMA) and dissolve it to obtain a fourth mixed solution; S42: Mix the fourth mixed solution with odanacatib-loaded microspheres (ODN-MS), and then gelate under light to obtain a gel composite sustained-release carrier (ODN-MS-Gel).

[0041] The present disclosure uses the emulsion solvent evaporation method to prepare odanacatib-loaded microspheres (ODN-MS). In step S11, odanacatib (ODN) and poly(lactic-co-glycolic acid) copolymer (PLGA) are dissolved in an organic solvent to prepare a first mixed solution (oil phase). In step S12, polyvinyl alcohol (PVA) is dissolved in water to prepare a second mixed solution (external water phase). Then, the first mixed solution is dropped into the second mixed solution to carry out emulsification, and the organic solvent is fully volatilized to obtain a third mixed solution. Then, post-treatment is carried out. The third mixed solution is poured into a centrifuge tube and centrifuged for 10 min to collect the first precipitate. Deionized water is added to the first precipitate, and ultrasonic cleaning is used, and then centrifuged again. Repeat this three times to obtain a second precipitate. Then, the second precipitate is freeze-dried to obtain odanacatib-loaded microspheres (ODN-MS).

[0042] Further, in step S41, a photoinitiator is added to and dissolved in methacrylated gelatin (GelMA) to obtain a fourth mixed solution. In step S42, the previously prepared odanacatib-loaded microspheres (ODN-MS) are added to the fourth mixed solution, and photo-crosslinking is performed under light irradiation to obtain a gel composite sustained-release carrier (ODN-MS-Gel).

[0043] Among them, the emulsion solvent evaporation method is a technique widely used in the preparation of micron- or nanoscale drug-loaded particles, polymer microspheres, and functional material coatings, especially suitable for drug delivery systems and biomaterial engineering. The solidification and encapsulation of materials are achieved through the formation of an emulsion system and solvent evaporation.

[0044] Compared with traditional antiresorptive drugs (such as bisphosphonates), odanacatib (ODN) only inhibits cathepsin K (CTSK) without affecting osteoclast activity. In theory, it can reduce bone destruction while retaining the ability of bone formation.

[0045] Poly(lactic-co-glycolic acid) (PLGA) is a biodegradable polymer material synthesized by copolymerization of lactic acid (LA) and glycolic acid (GA) monomers. Poly(lactic-co-glycolic acid) (PLGA) has excellent biocompatibility and controllable degradability and is widely used in drug delivery systems, tissue engineering scaffolds, and medical implant materials. Poly(lactic-co-glycolic acid) (PLGA) is gradually hydrolyzed in water into lactic acid and glycolic acid and finally metabolized into CO2 and H2O without toxic residues.

[0046] Polyvinyl alcohol (PVA) is a synthetic, water-soluble polymer that dissolves in water (especially hot water) and is insoluble in most organic solvents.

[0047] Methacrylated gelatin (GelMA) is prepared by reacting gelatin (a collagen hydrolysis product) with methacrylic anhydride (MA), promotes cell adhesion, and can form a hydrogel under ultraviolet / visible light irradiation.

[0048] A photoinitiator is a type of compound that can generate active substances (such as free radicals or cations) under specific wavelength light irradiation, and it initiates the polymerization or crosslinking process through a photochemical reaction.

[0049] In one embodiment, in step S11, the dosage ratio of odanacatib (ODN), organic solvent, and poly(lactic-co-glycolic acid) (PLGA) is (0.004 - 0.005 g) : (3 - 6 ml) : (0.035 - 0.045 g).

[0050] Among them, the organic solvent can be dichloromethane solution. Dichloromethane is a colorless, transparent, and volatile organic solvent with excellent solubility and low boiling point. Optionally, solvents for dissolving Olodanacatl (ODN) can also include various organic solvents such as dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF), ethanol, or acetone, etc., which are not limited in this disclosure.

[0051] Further, in step S11, precisely weigh 0.005 g of Olodanacatl (ODN), pour it into a centrifuge tube containing 5 mL of dichloromethane, ultrasonically dissolve for 1 min, weigh 0.04 g of poly (lactic-co-glycolic acid) copolymer (PLGA) (with a molecular weight range WM: 37000 - 52000) and add it to the above centrifuge tube, ultrasonically dissolve for 1 min to obtain a first mixed solution.

[0052] In one embodiment, the detailed method of step S12 is that polyvinyl alcohol (PVA) is dissolved in pure water by heating in a water bath at no less than 95 °C for 1 - 2 h. The dosage ratio of polyvinyl alcohol (PVA) to pure water is (0.2 - 0.3 g):(40 - 60 ml).

[0053] Optionally, precisely weigh 0.25 g of polyvinyl alcohol (PVA), add it to a beaker containing 50 mL of pure water, heat it in a water bath above 95 °C for 1 h, stir it several times during this period until it is completely dissolved, and cool it at room temperature to obtain a second mixed solution.

[0054] Among them, pure water refers to H2O without impurities. Its purity is extremely high, and usually, salts, organic substances, microorganisms, particulate matters, etc. in the water, even including dissolved gases (such as oxygen, carbon dioxide), are removed through special processes.

[0055] In one embodiment, the detailed method of step S13 is to place the first mixed solution into a syringe, and drop it into the second mixed solution at an injection speed of (0.4 - 0.6) mL / min, while stirring, and continuously stir at 700 - 900 rpm for 3 - 4 h.

[0056] Optionally, place the rotor of a magnetic stirrer into the beaker containing the second mixed solution, stir magnetically at 800 rpm at room temperature, place the first mixed solution into a syringe, and drop it into the second mixed solution being stirred at an injection speed of 0.5 mL / min, and continuously stir for 3 h to fully volatilize dichloromethane to obtain a third mixed solution.

[0057] In one embodiment, the detailed method for obtaining the second precipitate in step S14 is as follows: centrifuge the third mixed solution at 3000 - 4000 rpm for 10 - 15 min to obtain the first precipitate, then wash the first precipitate with deionized water and centrifuge, and repeat the washing 3 times.

[0058] After stirring, pour the third mixed solution into a 50 mL centrifuge tube, centrifuge at 4000 rpm for 10 min, and collect the first precipitate. Add deionized water, ultrasonically wash the first precipitate, and centrifuge again. Repeat this three times to obtain the second precipitate.

[0059] In one embodiment, the detailed method for freeze-drying in step S14 is as follows: pre-freeze the vacuum freeze-dryer at -75~-85°C for 3 - 4 h, then place the second precipitate into the vacuum freeze-dryer and freeze-dry for 8 - 12 h.

[0060] Further, pre-freeze the vacuum freeze-dryer at -80°C for 4 h, then place the second precipitate into the vacuum freeze-dryer and freeze-dry for 12 h to obtain the olodaterol-loaded microspheres (ODN-MS).

[0061] See Figure 2 , in one embodiment, between step S1 and step S4, there is also step S2: determine that the preparation of the olodaterol-loaded microspheres (ODN-MS) is successful, which specifically includes the following steps: S21: Use the method of step S1 and remove olodaterol (ODN) to prepare poly (lactic-co-glycolic acid) microspheres (MS); S22: Dissolve olodaterol (ODN), poly (lactic-co-glycolic acid) microspheres (MS), and olodaterol-loaded microspheres (ODN-MS) in an organic solvent respectively, observe and record the absorption wavelengths of olodaterol (ODN), poly (lactic-co-glycolic acid) microspheres (MS), and olodaterol-loaded microspheres (ODN-MS), compare the maximum absorption wavelengths, and determine that the preparation of the olodaterol-loaded microspheres (ODN-MS) is successful.

[0062] Specifically, through ultraviolet-visible spectroscopy (UV-Vis method), scan the dichloromethane liquid medicine (the ratio of solute to solvent is 1:10 (m / V)) dissolving olodaterol (ODN), poly (lactic-co-glycolic acid) microspheres (MS), and olodaterol-loaded microspheres (ODN-MS) in the full wavelength range of 200 - 800 nm, observe and record the absorption wavelengths of olodaterol (ODN), poly (lactic-co-glycolic acid) microspheres (MS), and olodaterol-loaded microspheres (ODN-MS), and determine the maximum absorption wavelength to detect whether the preparation of the olodaterol-loaded microspheres (ODN-MS) is successful.

[0063] See Figure 4, through full-wavelength scanning within 200 - 800 nm by ultraviolet-visible spectroscopy (UV-Vis method), the poly(lactic-co-glycolic acid) copolymer microspheres (MS) have a main absorption peak between 220 - 240 nm, and olodaterol (ODN) has a main absorption peak between 250 - 280 nm. Further, it is determined that olodaterol (ODN) has a maximum absorbance peak at a wavelength of 264 nm.

[0064] Both olodaterol (ODN) and olodaterol-loaded microspheres (ODN-MS) have an absorption peak between 250 - 280 nm, indicating that the drug olodaterol (ODN) has been loaded onto the poly(lactic-co-glycolic acid) (PLGA) microspheres, and the olodaterol-loaded microspheres (ODN-MS) have been successfully prepared.

[0065] Furthermore, high-performance liquid chromatography can also be used to detect the absorption wavelength, and this disclosure does not limit this. It is sufficient that those skilled in the art can confirm the successful drug loading.

[0066] In one embodiment, between step S2 and step S4, there is also step S3: determining the drug loading amount and encapsulation efficiency of the olodaterol-loaded microspheres (ODN-MS), which specifically includes the following steps: S31: Fitting the standard curve regression equation of olodaterol (ODN); S32: According to the standard curve regression equation, calculate the drug content of the olodaterol-loaded microspheres (ODN-MS), and then according to the formula, calculate the drug loading amount and encapsulation efficiency of the olodaterol-loaded microspheres (ODN-MS): Theoretical drug loading amount (%) = m ODN / m 总1 × 100%; Actual drug loading amount (%) = m 计算ODN / m 总2 × 100%; Encapsulation efficiency (%) = actual drug loading amount / theoretical drug loading amount × 100%.

[0067] Among them, m ODN is the mass of olodaterol (ODN); m 总1 is the total sample amount; m 计算ODN is the mass of olodaterol (ODN) in the olodaterol-loaded microspheres (ODN-MS); m 总2 is the total mass of the olodaterol-loaded microspheres (ODN-MS).

[0068] Specifically, in step S31, standard dichloromethane solutions of odanacatib (ODN) with concentrations of 2, 4, 6, 8, 10, and 12 μg / mL are prepared, with dichloromethane solution as the control. Selecting the maximum absorption wavelength of 264 nm of the drug determined above, the absorbance A is measured. With the odanacatib (ODN) concentration C as the abscissa and the absorbance A as the ordinate, a linear fitting is performed to determine the regression equation of the standard curve.

[0069] Set the wavelength of the UV–Vis to 264 nm, detect the absorbance of odanacatib (ODN) at different concentrations, and the standard curve obtained is as Figure 5 shown. The concentration of odanacatib (ODN) (2 - 12 μg / mL) has a linear relationship with the absorbance line, and the regression equation obtained by fitting is: A = 0.07121*C - 0.01071, R 2 = 0.99919.

[0070] In step S32, accurately weigh 1 mg of the odanacatib-loaded microspheres (ODN-MS) sample into a 15 mL centrifuge tube, add 10 mL of dichloromethane, and calculate the drug content in the odanacatib-loaded microspheres (ODN-MS) according to the maximum absorption wavelength of the drug at 264 nm and the regression equation. Each group is detected 3 times, and the average value is taken.

[0071] Furthermore, through the calculation formula of the odanacatib (ODN) standard curve of the present disclosure, the drug loading of the odanacatib-loaded microspheres (ODN-MS) is calculated to be (9.1 ± 0.18)%, and the encapsulation efficiency is (81 ± 1.34)%.

[0072] In one embodiment, the specific method of step S41 is to add a photoinitiator to methacrylated gelatin (GelMA) in the dark and dissolve it in a constant temperature water bath at 40 - 50 °C for 20 - 30 min; the photoinitiator is LAP, and the dosage ratio of methacrylated gelatin (GelMA) to LAP is 1 g:10 ml.

[0073] Optionally, accurately weigh 1 g of dry methacrylated gelatin (GelMA) into a 15 mL centrifuge tube, add 10 mL of the photoinitiator LAP in the dark. Dissolve it in a constant temperature water bath at 40 - 50 °C for 20 - 30 min, and shake it several times during this period to obtain the fourth mixed solution.

[0074] Among them, LAP is a highly efficient and low-toxic photoinitiator, and its core characteristic lies in its visible light initiation ability, significantly reducing the damage of ultraviolet light to cells. Compared with ultraviolet light photoinitiators (such as Irgacure2959), LAP combined with visible light (405 nm blue light) can reduce DNA damage and cell apoptosis.

[0075] See Figure 1, in one embodiment, the specific method of step S42 is to mix the fourth mixed solution with the oteracil-loaded microspheres (ODN-MS) after sterilization, and gelate it by irradiating with a light source for 10 - 30 s.

[0076] Further, sterilize the fourth mixed solution with a 0.22 μm sterile filter, and use ultraviolet sterilization for the oteracil-loaded microspheres (ODN-MS). After sufficient mixing, irradiate the fourth mixed solution with 405 nm blue light for 10 - 30 s to gelate it, and finally obtain the gel composite sustained-release carrier (ODN-MS-Gel).

[0077] See Figure 3 , in one embodiment, after step S4, step S5 is further included, which specifically includes the following steps: S51: Observe the poly(lactic-co-glycolic acid) microspheres (MS), oteracil-loaded microspheres (ODN-MS), and the gel composite sustained-release carrier (ODN-MS-Gel) using a microscope, compare their morphologies, and analyze that the gel composite sustained-release carrier (ODN-MS-Gel) can achieve drug sustained release; S52: Measure the particle sizes of the poly(lactic-co-glycolic acid) microspheres (MS) and oteracil-loaded microspheres (ODN-MS) using a scanning electron microscope, and judge that the gel composite sustained-release carrier (ODN-MS-Gel) is injectable.

[0078] Specifically, in step S51, the present disclosure uses an optical microscope to place the centrifuged and washed poly(lactic-co-glycolic acid) microspheres (MS) and oteracil-loaded microspheres (ODN-MS) in a beaker containing an appropriate amount of deionized water, and observe whether they are dispersed and whether their sizes are uniform under the optical microscope.

[0079] See Figure 6 , the poly(lactic-co-glycolic acid) microspheres (MS) and oteracil-loaded microspheres (ODN-MS) prepared by the emulsion solvent evaporation method are both uniformly distributed and have uniform sizes.

[0080] In step S52, use a scanning electron microscope to observe. Fix the freeze-dried poly(lactic-co-glycolic acid) microspheres (MS), oteracil-loaded microspheres (ODN-MS), and the gel composite sustained-release carrier (ODN-MS-Gel) on a flat plate with conductive glue, sputter gold under vacuum conditions, observe the material morphology using a scanning electron microscope, and simultaneously randomly select multiple microspheres in different regions under the electron microscope for particle size measurement.

[0081] See Figure 7Both the poly(lactic-co-glycolic acid) copolymer microspheres (MS) and the odanacatib-loaded microspheres (ODN-MS) are spherical. However, the surface of the poly(lactic-co-glycolic acid) copolymer microspheres (MS) is smooth without obvious pores, while the surface of the odanacatib-loaded microspheres (ODN-MS) presents a fine porous structure. Analyzing the porous spherical structure of the odanacatib-loaded microspheres (ODN-MS) is not only beneficial for encapsulating high-dose drugs but also enables the sustained release of drugs. After wrapping the odanacatib-loaded microspheres (ODN-MS) with methacrylated gelatin (GelMA), it was observed by scanning electron microscopy that the hydrogel has a porous structure, and the odanacatib-loaded microspheres (ODN-MS) are distributed in the porous structure of the hydrogel. It is speculated that the porous structure of the hydrogel is conducive to loading more microspheres and the uniform release of drugs. In addition, the particle size ranges of both the poly(lactic-co-glycolic acid) copolymer microspheres (MS) and the odanacatib-loaded microspheres (ODN-MS) are between 40–120 μm, indicating that the odanacatib-loaded microspheres (ODN-MS) have injectability after being mixed with the hydrogel. Among them, the poly(lactic-co-glycolic acid) copolymer microspheres (MS) are used as a control group for observation.

[0082] Spraying gold under vacuum is a physical vapor deposition technique commonly used for manufacturing thin films or coatings, mainly to provide a conductive layer for electron microscopy observation.

[0083] In one embodiment, after step S5, step S6 is further included, and the specific method is as follows: S6: Take the odanacatib-loaded microspheres (ODN-MS) and the gel composite sustained-release carrier (ODN-MS-Gel) with the same drug content respectively, and add an equal amount of in vitro release medium for sequential culture. Calculate the drug cumulative release rate according to the formula:

[0084] In the formula, V0 is the total volume of the release medium; C t is the drug concentration in the release medium at the last sampling time point; V is the volume of each sampling; C i is the drug mass concentration in the release medium at the i-th time; m is the total mass of the drug in the drug preparation.

[0085] In step S6, to address the problem of poor water solubility of odanacatib (ODN), referring to the relevant content in the Pharmacopoeia of the People's Republic of China: PBS solution (phosphate buffer solution) can be selected as the in vitro release medium for conventional sustained-release and controlled-release preparations. For poorly soluble drugs, a small amount of surfactant can be added to the release medium. According to the pre-experiment results, 0.5% SDS-PBS solution is finally selected as the in vitro release medium.

[0086] Take the odanacat-loaded microspheres (ODN-MS) and the gel composite sustained-release carrier (ODN-MS-Gel) with the same drug content and place them in a 15 mL centrifuge tube. After adding 10 mL of 0.5% SDS-PBS solution, place it in a constant temperature vertical shaking incubator at a rotation speed of 100 rpm and a temperature of 37°C. At 4 h, 8 h, 1 d, 2 d, 3 d, 5 d, 7 d, 10 d, 14 d, 21 d, 28 d, 35 d, 42 d, 48 d, 55 d, and 62 d, take out 3 mL of the release solution in the test tube for absorbance measurement, and at the same time supplement 3 mL of 0.5% SDS-PBS solution at the same temperature in the centrifuge tube. Measure the absorbance at the maximum absorption wavelength of the drug by ultraviolet-visible spectroscopy (UV-Vis method) to determine the drug content in the medium and calculate the cumulative drug release rate.

[0087] Based on the odanacat (ODN) standard curve, further characterize the in vitro drug release performance of the odanacat-loaded microspheres (ODN-MS) and the gel composite sustained-release carrier (ODN-MS-Gel).

[0088] The release curve is as Figure 8 shown: Compared with the odanacat-loaded microspheres (ODN-MS), the in vitro drug release curve of the gel composite sustained-release carrier (ODN-MS-Gel) shows a trend of slowly rising to a stable state. The release rate of the gel composite sustained-release carrier (ODN-MS-Gel) within 4 h is only 0.45%, which is significantly lower than that of the odanacat-loaded microspheres (ODN-MS) group (3.04%, P < 0.05). On the 62nd day, the in vitro drug release rates of the odanacat-loaded microspheres (ODN-MS) and the gel composite sustained-release carrier (ODN-MS-Gel) are 82.7% and 65.96% respectively, indicating that both the odanacat-loaded microspheres (ODN-MS) and the gel composite sustained-release carrier (ODN-MS-Gel) can achieve a sustained-release effect, while the gel composite sustained-release carrier (ODN-MS-Gel) system is more sluggish and has the potential for continuous sustained release.

[0089] See Figure 3 , in one embodiment, after step S5, it further includes step S7: detecting cell compatibility, and the specific steps include: S71: Using the method of step S4, replace the odanacat-loaded microspheres (ODN-MS) with poly (lactic-co-glycolic acid) microspheres (MS) to prepare microsphere hydrogel (MS-Gel); S72: Respectively prepare complete medium extracts of methacrylated gelatin (GelMA), microsphere hydrogel (MS-Gel), and gel composite sustained-release carrier (ODN-MS-Gel), inoculate bone marrow mesenchymal stem cells (BMSCs) for sequential culture, and detect and record the cell number, and compare to determine that the gel composite sustained-release carrier (ODN-MS-Gel) can promote cell proliferation; S73: Prepare the complete medium extracts of methacrylated gelatin (GelMA), microsphere hydrogel (MS-Gel), and gel composite sustained-release carrier (ODN-MS-Gel) respectively, inoculate bone marrow mesenchymal stem cells (BMSCs) for culture, and then stain with a live / dead cell reagent to determine by comparison that the gel composite sustained-release carrier (ODN-MS-Gel) does not affect cell viability.

[0090] Specifically, in step S72, through a cell proliferation experiment: Prepare the complete medium extracts of methacrylated gelatin (GelMA), microsphere hydrogel (MS-Gel), and gel composite sustained-release carriers (ODN-MS-Gel) with different concentrations (such as 250, 500 μg / mL), and the soaking time is 24 h. Inoculate bone marrow mesenchymal stem cells (BMSCs) in a 96-well plate, set 3 replicates at each time point, and inoculate 2×10 3 cells per well. Replace the sterilized material extract after inoculation for 24 h, and use the complete medium for the control group. Change the medium every 2 days. Discard the medium at 1 d, 3 d, and 5 d respectively, wash with PBS, add 10 μL of CCK-8 solution, incubate at 37 °C for 1 h, and measure the absorbance value at 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0091] WST-8 in the CCK-8 reagent can be reduced by dehydrogenases in living cells to generate a highly water-soluble orange-yellow formazan product under the action of the electron carrier 1-Methoxy PMS. The formation amount of this formazan is proportional to the number of living cells. Therefore, the number of living cells or cell activity can be indirectly reflected by measuring the absorbance at a wavelength of 450 nm.

[0092] An ELISA reader, also known as a microplate reader, is a laboratory device mainly used to detect the optical properties of samples placed in a microplate. The ELISA reader can measure various types of spectral data, including absorbance, fluorescence intensity, luminescence, etc.

[0093] The detection results by the CCK-8 method are as Figure 9As shown, after bone marrow mesenchymal stem cells (BMSCs) were co-cultured in the leaching solutions of different concentrations of gel composite sustained-release carriers (ODN-MS-Gel) for 1 day, 3 days, and 5 days: The bone marrow mesenchymal stem cells (BMSCs) in the groups of methacrylated gelatin (GelMA), microsphere hydrogel (MS-Gel), and gel composite sustained-release carrier (ODN-MS-Gel) all proliferated normally, indicating that the gel composite sustained-release carrier (ODN-MS-Gel) is non-toxic. There was no significant statistical difference in absorbance among the control group, the methacrylated gelatin (GelMA) group, and the microsphere hydrogel (MS-Gel) group; while on the 3rd day, the absorbance of the gel composite sustained-release carrier (ODN-MS-Gel) groups at 250 μg / mL and 500 μg / mL was significantly higher than that of the control group (P<0.05). Therefore, the gel composite sustained-release carrier (ODN-MS-Gel) can promote cell proliferation.

[0094] In step S73, through a cell viability experiment, among which, the blank control group used complete medium, and the remaining groups were added with the material leaching solutions of methacrylated gelatin (GelMA), microsphere hydrogel (MS-Gel), and gel composite sustained-release carrier (ODN-MS-Gel). Bone marrow mesenchymal stem cells (BMSCs) were inoculated in a 24-well plate, with 1×10 5 cells inoculated in each well, and 3 replicate wells were set in each group. After culturing for 3 days, staining was performed according to the instruction manual of the live / dead staining kit, and observation and photographing records were made through an inverted fluorescence microscope.

[0095] Calcein AM and PI are two commonly used fluorescent dyes, which can label live cells and dead cells respectively. By using these two dyes in combination and merging the fluorescence images of the two through image processing software, the distribution of live cells and dead cells in the sample can be observed simultaneously.

[0096] As Figure 10 shown, taking the gel composite sustained-release carrier (ODN-MS-Gel) with a concentration of 250 μg / mL as an example, its effect on cell viability was detected. Live cells showed green fluorescence, and dead cells showed red fluorescence. Although there were a few dead cells in each group, the vast majority of cells had good viability.

[0097] Through the above experiments, it was determined that the gel composite sustained-release carrier (ODN-MS-Gel) has excellent cell compatibility and has the potential for clinical application.

[0098] In one embodiment, after step S5, step S8 is further included, and the specific steps include: S81: Using the method of step S4, replace the odanacatib-loaded microspheres (ODN-MS) with poly (lactic-co-glycolic acid) microspheres (MS) to prepare microsphere hydrogel (MS-Gel). S82: Respectively, after photocuring methacrylated gelatin (GelMA), microsphere hydrogel (MS-Gel) and gel composite sustained-release carrier (ODN-MS-Gel), inoculate bone marrow mesenchymal stem cells (BMSCs) for culture, and use a microscope to observe the adhesion state to determine that the gel composite sustained-release carrier (ODN-MS-Gel) is conducive to cell adhesion.

[0099] In step S82, methacrylated gelatin (GelMA), microsphere hydrogel (MS-Gel) and gel composite sustained-release carrier (ODN-MS-Gel) are respectively spread on a 24-well plate. After photocuring, inoculate bone marrow mesenchymal stem cells (BMSCs) at a density of 1×10 5 cells per well. After culturing for 1 day, take them out, fix them with 4% paraformaldehyde at room temperature, permeabilize them with 0.4% Triton X-100 PBS solution at room temperature, then add phalloidin and DAPI staining solution respectively, incubate in the dark at room temperature, and wash with PBS 5 minutes each time after each step is completed, wash 3 times, and observe and photograph under an inverted fluorescence microscope.

[0100] The results are as Figure 11 shown. Use phalloidin to label the cytoskeleton of bone marrow mesenchymal stem cells (BMSCs) to detect the effect of the gel composite sustained-release carrier (ODN-MS-Gel) on cell adhesion ability. The cytoskeleton structure of the gel composite sustained-release carrier (ODN-MS-Gel) group is clear, showing good adhesion ability. It is worth noting that the cells in the gel composite sustained-release carrier (ODN-MS-Gel) group have more cell pseudopodia formation, increasing cell adhesion and information interaction between cells, which is conducive to cell recruitment at the wound to promote tissue damage repair.

[0101] Furthermore, use a scanning electron microscope to observe: spread methacrylated gelatin (GelMA), microsphere hydrogel (MS-Gel) and gel composite sustained-release carrier (ODN-MS-Gel) on a 24-well plate. After photocuring, inoculate bone marrow mesenchymal stem cells (BMSCs) at a density of 1×10 5 cells per well. After culturing for 1 day, take them out, fix them with 4% paraformaldehyde at room temperature, and perform gradient dehydration with ethanol at concentrations of 50%, 70%, 80%, 90% and 100% for 20 minutes each time after cell fixation. After dehydration, add an appropriate amount of hexamethyldisilane and volatilize and dry it in a fume hood. Fix the dried material on a flat plate with conductive glue, sputter gold under vacuum conditions, and observe cell adhesion under a scanning electron microscope.

[0102] The results are as follows Figure 12 As shown, bone marrow mesenchymal stem cells (BMSCs) are spindle-shaped and distributed on the surfaces of methacrylated gelatin (GelMA), microsphere hydrogel (MS-Gel), and gel composite sustained-release carrier (ODN-MS-Gel), indicating good cell adhesion ability. However, compared with the microsphere hydrogel (MS-Gel) group, more cells can be observed recruiting around the gel composite sustained-release carrier (ODN-MS-Gel) and adhering to the microsphere surface in the gel composite sustained-release carrier (ODN-MS-Gel) group. This is consistent with the results we observed in Figure 11 which is that the surface of the porous spherical structure of odanacatib-loaded microspheres (ODN-MS) is rough, facilitating cell adhesion. At the same time, the analysis may also be related to the release of odanacatib (ODN).

[0103] In addition, steps S6, S7, and S8 are used to verify the performance of the gel composite sustained-release carrier (ODN-MS-Gel) prepared by the present disclosure through a comparison test, and the order can be unrestricted.

[0104] In one embodiment, the present disclosure provides a gel composite sustained-release carrier prepared by using the preparation method of the gel composite sustained-release carrier in any of the above embodiments.

[0105] The gel composite sustained-release carrier prepared by the present disclosure solves the problem of poor retention of the local injection liquid of odanacatib (ODN), can continuously release the drug, and is beneficial to the treatment of chronic periodontitis.

[0106] In one embodiment, the application of the gel composite sustained-release carrier of the present disclosure in periodontitis treatment products.

[0107] In summary, the present disclosure proposes a novel local drug delivery system, the gel composite sustained-release carrier (ODN-MS-Gel), which solves the problems of poor retention of the local injection liquid of odanacatib (ODN) and the adverse reactions caused by the rapid diffusion of odanacatib (ODN). The results of the drug release curve show that the gel composite sustained-release carrier (ODN-MS-Gel) releases the drug more continuously than the odanacatib-loaded microspheres (ODN-MS), which is beneficial to the treatment of chronic periodontitis. In addition, cell experiments show that the gel composite sustained-release carrier (ODN-MS-Gel) has excellent biocompatibility, can effectively promote cell proliferation, increase cell adhesion and information interaction between cells, and provides a new strategy for the treatment of periodontitis.

[0108] The embodiments of the present disclosure have been described above. The above description is exemplary, not exhaustive, and is not limited to the disclosed embodiments. Many modifications and variations are obvious to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments. The choice of terms used herein is intended to best explain the principles of the embodiments, practical applications, or improvements to technologies in the market, or to enable other ordinary skill in the art to understand the embodiments disclosed herein. The scope of the present disclosure is defined by the appended claims.

Claims

1. A preparation method of a gel composite sustained-release carrier, characterized in that, The preparation method at least includes step S1 and step S4; wherein, step S1: preparing the odanacatib-loaded microspheres (ODN-MS) specifically includes the following steps: S11: Dissolve odanacatib (ODN) in an organic solvent, then add poly (lactic-co-glycolic acid) copolymer (PLGA) and dissolve to obtain a first mixed solution; S12: Dissolve polyvinyl alcohol (PVA) in water to obtain a second mixed solution; S13: Drop the first mixed solution into the second mixed solution while stirring to obtain a third mixed solution; S14: Centrifuge the third mixed solution to obtain a first precipitate, wash and centrifuge the first precipitate to obtain a second precipitate, and lyophilize the second precipitate to obtain the odanacatib-loaded microspheres (ODN-MS); Step S4: Preparing the gel composite sustained-release carrier (ODN-MS-Gel) specifically includes the following steps: S41: Add a photoinitiator to methacrylated gelatin (GelMA) and dissolve to obtain a fourth mixed solution; S42: Mix the fourth mixed solution with the odanacatib-loaded microspheres (ODN-MS), and then gel under light irradiation to obtain the gel composite sustained-release carrier (ODN-MS-Gel).

2. The preparation method of the gel composite sustained-release carrier according to claim 1, wherein In step S11, the dosage ratio of the odanacatib (ODN), organic solvent and poly (lactic-co-glycolic acid) copolymer (PLGA) is (0.004 - 0.005 g): (3 - 6 ml): (0.035 - 0.045 g).

3. The preparation method of the gel composite sustained-release carrier according to claim 2, wherein, The detailed method of step S12 is that the polyvinyl alcohol (PVA) is dissolved in pure water by water bath heating at not less than 95 °C for 1 - 2 h, and the dosage ratio of the polyvinyl alcohol (PVA) and pure water is (0.2 - 0.3 g): (40 - 60 ml).

4. The preparation method of the gel composite sustained-release carrier according to claim 3, characterized in that, The detailed method of step S13 is to place the first mixed solution into a syringe, and drop it into the second mixed solution at an injection rate of (0.4 - 0.6) mL / min while stirring, and continuously stir at 700 - 900 rpm for 3 - 4 h.

5. The preparation method of the gel composite sustained-release carrier according to claim 4, characterized in that, The detailed method of obtaining the second precipitate in step S14 is to centrifuge the third mixed solution at 3000 - 4000 rpm for 10 - 15 min to obtain the first precipitate, then wash the first precipitate with deionized water and centrifuge, and repeat the washing 3 times.

6. The preparation method of the gel composite sustained-release carrier according to claim 5, wherein The detailed method of lyophilization in step S14 is to pre-freeze the vacuum freeze dryer at -75 to -85 °C for 3 - 4 h, and then place the second precipitate into the vacuum freeze dryer and freeze-dry for 8 - 12 h.

7. The preparation method of the gel composite sustained-release carrier according to claim 1, wherein, Between step S1 and step S4, there is also step S2: determining that the preparation of the odanacatib-loaded microspheres (ODN-MS) is successful, specifically including the following steps: S21: Adopt the method of step S1 and remove the odanacatib (ODN) to prepare poly (lactic-co-glycolic acid) copolymer microspheres (MS); S22: Dissolve the odanacatib (ODN), poly (lactic - co - glycolic acid) copolymer microspheres (MS), and odanacatib - loaded drug microspheres (ODN - MS) in an organic solvent respectively. Observe and record the absorption wavelengths of the odanacatib (ODN), poly (lactic - co - glycolic acid) copolymer microspheres (MS), and odanacatib - loaded drug microspheres (ODN - MS). Compare the maximum absorption wavelengths to determine the successful preparation of the odanacatib - loaded drug microspheres (ODN - MS).

8. The preparation method of the gel composite sustained-release carrier according to claim 7, wherein, Between step S2 and step S4, there is also step S3: Determine the drug - loading amount and encapsulation efficiency of the odanacatib - loaded drug microspheres (ODN - MS), which specifically includes the following steps: S31: Fit the standard curve regression equation of the odanacatib (ODN). S32: According to the standard curve regression equation, calculate the drug content of the odanacatib - loaded drug microspheres (ODN - MS), and then calculate the drug - loading amount and encapsulation efficiency of the odanacatib - loaded drug microspheres (ODN - MS) according to the formula: Theoretical drug loading amount (%) = m ODN / m 总 × 100%; Actual drug loading amount (%) = m 计算ODN / m 总 × 100%; Encapsulation efficiency (%) = actual drug - loading amount / theoretical drug - loading amount × 100%.

9. The preparation method of the gel composite sustained-release carrier according to claim 1, wherein The specific method of step S41 is to add the photo - initiator to the methacrylated gelatin (GelMA) in the dark and dissolve it in a 40 - 50 °C constant temperature water bath for 20 - 30 min. The photo - initiator is LAP, and the dosage ratio of the methacrylated gelatin (GelMA) to LAP is 1 g: 10 ml.

10. The preparation method of the gel composite sustained-release carrier according to claim 9, wherein, The specific method of step S42 is to mix the fourth mixed solution with the odanacatib - loaded drug microspheres (ODN - MS) after sterilization and gel them by irradiating with a light source for 10 - 30 s.

11. The preparation method of the gel composite sustained-release carrier according to claim 8, wherein, After step S4, there is also step S5, which specifically includes the following steps: S51: Observe the poly (lactic - co - glycolic acid) copolymer microspheres (MS), odanacatib - loaded drug microspheres (ODN - MS), and gel composite sustained - release carrier (ODN - MS - Gel) using a microscope, and compare their morphologies to analyze that the gel composite sustained - release carrier (ODN - MS - Gel) can achieve drug sustained - release. S52: Measure the particle sizes of the poly (lactic - co - glycolic acid) copolymer microspheres (MS) and odanacatib - loaded drug microspheres (ODN - MS) using a scanning electron microscope to determine whether the gel composite sustained - release carrier (ODN - MS - Gel) is injectable.

12. The preparation method of the gel composite sustained-release carrier according to claim 11, wherein After step S5, there is also step S6, and the specific method is: S6: Take the odanacatib - loaded drug microspheres (ODN - MS) and the gel composite sustained - release carrier (ODN - MS - Gel) with the same drug content respectively, and add an equal amount of in vitro release medium for sequential culture. Calculate the drug cumulative release rate according to the formula: 。 13. The preparation method of the gel composite sustained-release carrier according to claim 12, wherein, After step S5, there is also step S7: Detect the cell compatibility, and the specific steps include: S71: Using the method of step S4, replace the odanacatib - loaded drug microspheres (ODN - MS) with the poly (lactic - co - glycolic acid) copolymer microspheres (MS) to prepare microsphere hydrogels (MS - Gel). S72: Prepare the complete medium extracts of the methacrylated gelatin (GelMA), microsphere hydrogel (MS-Gel), and gel composite sustained-release carrier (ODN-MS-Gel) respectively, inoculate bone marrow mesenchymal stem cells (BMSCs) for sequential culture, detect and record the cell number, and compare to determine that the gel composite sustained-release carrier (ODN-MS-Gel) can promote cell proliferation; S73: Prepare the complete medium extracts of the methacrylated gelatin (GelMA), microsphere hydrogel (MS-Gel), and gel composite sustained-release carrier (ODN-MS-Gel) respectively, inoculate bone marrow mesenchymal stem cells (BMSCs) for culture, then stain with a live / dead cell reagent, and compare to determine that the gel composite sustained-release carrier (ODN-MS-Gel) does not affect cell viability.

14. The preparation method of the gel composite sustained-release carrier according to claim 13, wherein, After the step S5, the following step S8 is further included, and the specific steps are as follows: S81: Using the method of step S4, replace the odanacatib-loaded microspheres (ODN-MS) with the poly (lactic-co-glycolic acid) microspheres (MS) to prepare the microsphere hydrogel (MS-Gel); S82: After photopolymerizing the methacrylated gelatin (GelMA), microsphere hydrogel (MS-Gel), and gel composite sustained-release carrier (ODN-MS-Gel) respectively, inoculate bone marrow mesenchymal stem cells (BMSCs) for culture, and use a microscope to observe the adhesion state to determine that the gel composite sustained-release carrier (ODN-MS-Gel) is beneficial to cell adhesion.

15. A gel composite sustained-release carrier, characterized in that, Prepared by using the preparation method of the gel composite sustained-release carrier according to any one of claims 1-14.

16. The gel composite sustained-release carrier according to claim 15, characterized in that, The application of the gel composite sustained-release carrier in periodontitis treatment products.

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