Hydrogel drug-carrying system for achieving spatiotemporal sequential release and preparation method and application thereof

Abstract

The present invention relates to a hydrogel, and in particular to a hydrogel drug-carrying system and a preparation method and application thereof that realizes spatiotemporal sequential release, including: a first drug, which is icariin; a drug carrier, which encapsulates the first drug to form drug-carrying nanoparticles of a core-shell structure, for realizing long-term release of the first drug; a second drug, which is IL-10; and a hydrogel, in which the drug-carrying nanoparticles are uniformly dispersed in the hydrogel, and the second drug is uniformly dispersed in the hydrogel after electrostatic binding with heparin-modified hyaluronic acid; the drug carrier is PLGA-HA, and the hydrogel is GelMA. Compared with the prior art, the present invention solves the problem that the effective components of the drugs loaded by the hydrogel in the prior art cannot be released and released continuously according to a certain timing. The present scheme realizes the long-term sustained release of icariin and the early release of IL-10 in the hydrogel drug-carrying system, and makes the bone regeneration effect more excellent through orderly and sustained release.

Description

Technical Field

[0001] The invention relates to a hydrogel, and in particular to a hydrogel drug-carrying system for realizing temporal and spatial sequential release, and a preparation method and application thereof. Background Art

[0002] Bone is a strong and dense connective tissue that provides structural support, helps with movement, protects internal organs, and retains and releases minerals. The structure of bone is composed of the inorganic component hydroxyapatite and the organic component collagen. In general, bone tissue has a strong self-repair ability. However, when there is a large bone defect, the intrinsic self-healing ability of the bone is often exceeded, resulting in delayed and impaired bone repair. Severe bone defects are often caused by trauma, infection, and tumors, which is a constant challenge for orthopedic surgeons.

[0003] Hydrogel materials are well-known for their excellent biocompatibility and biosafety, and have recently been widely used in the field of regenerative medicine. Hydrogels have a three-dimensional porous network, similar to the extracellular matrix (ECM) in natural tissues. They can also act as drug carriers, providing drug efficacy and reducing side effects by controlling drug release. Hydrogels are used to encapsulate "drugs" for bone tissue regeneration, such as cells, cytokines, natural products, and exosomes, to form a drug delivery system. For example, CN114246877A discloses a composition containing icariin, a drug, a preparation method thereof, and its use in promoting wound healing, wherein the composition and drug use icariin as the main active ingredient and are loaded on PLGA-PEG-PLGA gel, which can repair cells, accelerate wound healing, promote hair follicle regeneration, and inhibit scar formation, and also has multiple functions such as thermosensitivity, anti-inflammatory, and moisturizing.

[0004] For the creation of hydrogel drug delivery systems for the purpose of bone regeneration, it is crucial to synchronize the timed release of drugs with the dynamic changes of the osteogenic microenvironment. Hydrogel systems for bone regeneration should precisely target and release drugs in response to environmental stimuli. These systems can significantly promote the formation of a bone regeneration microenvironment by promoting angiogenesis, regulating immune responses, and guiding the differentiation of mesenchymal stem cells into osteogenesis. However, the existing technologies all directly load the active components onto the hydrogel without considering aspects such as the release order and sustained effect. It is difficult to match the release order and duration required by the active components, resulting in poor actual effects.

[0005] Although the use of exogenous biologics in biomaterials is a simple and promising approach, an incorrect release pattern may hinder bone regeneration. Therefore, it is urgent to develop a new hydrogel drug delivery system that can achieve spatiotemporal sequential release for bone regeneration. Summary of the invention

[0006] The purpose of the present invention is to solve at least one of the above problems and to provide a hydrogel drug delivery system that realizes spatiotemporal sequential release, as well as a preparation method and application thereof, so as to solve the problem in the prior art that the effective components of the drugs loaded by the hydrogel cannot be released in a certain sequence and released continuously. The present solution realizes the long-term sustained release of icariin and the early release of IL-10 in the hydrogel drug delivery system, and the bone regeneration effect is more excellent through orderly and sustained release.

[0007] The purpose of the present invention is achieved through the following technical solutions:

[0008] The first aspect of the present invention discloses a hydrogel drug delivery system for achieving spatiotemporal sequential release, comprising:

[0009] The first drug, the first drug is icariin;

[0010] A drug carrier, wherein the drug carrier encapsulates the first drug and forms drug-loaded nanoparticles with a core-shell structure through self-assembly, so as to achieve long-term release of the first drug;

[0011] The second drug, the second drug is IL-10;

[0012] as well as,

[0013] The hydrogel, the drug-loaded nanoparticles are uniformly dispersed in the hydrogel, and the second drug is electrostatically bonded to the heparin-modified hyaluronic acid and then uniformly dispersed in the hydrogel;

[0014] The drug carrier is PLGA-HA, and the hydrogel is GelMA.

[0015] Polylactic-co-glycolic acid-hyaluronic acid (PLGA-HA) is used as a drug carrier, in which PLGA, as a hydrophobic core, can encapsulate a variety of hydrophobic drugs, and HA, as a hydrophilic shell, can prolong the retention time of the drug and achieve a long-term release effect.

[0016] GelMA hydrogel is a modified gelatin obtained by the reaction of gelatin and methacrylic anhydride (MA). It has the characteristics of photocrosslinking, excellent biocompatibility, and low immune response.

[0017] Interleukin 10 (IL-10), also known as cytokine synthesis inhibitor, is a pleiotropic cytokine that can exert immunosuppressive or immunostimulatory effects in various cell types. IL-10 can inhibit the synthesis and release of inflammatory factors. Both endogenous and exogenous IL-10 can strongly inhibit the synthesis of interleukin-1 (IL-1), IL-6, IL-8, tumor necrosis factor-α (TNF-α), granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) at the transcriptional level, thereby playing an anti-inflammatory role.

[0018] Icariin (ICA) is a Chinese herbal monomer and the main active ingredient of Epimedium. Several studies have shown that icariin can accelerate osteoblast differentiation, promote bone formation, and inhibit osteoclast differentiation and bone resorption. Icariin can activate autophagy, have a significant anti-inflammatory effect on macrophages, restore osteogenic activity of aging BMSCs, and thus alleviate bone loss in osteoporosis.

[0019] HA-Ac first reacts and connects with thiolated heparin to form negatively charged HA-Ac-Heparin, which then combines with positively charged IL-10 through electrostatic interaction and is subsequently dispersed in the hydrogel together with the self-assembled drug-loaded nanoparticles.

[0020] Preferably, the mass ratio of PLGA-HA to icariin in the drug-loaded nanoparticles is 10:330-350; the dosage ratio of the drug-loaded nanoparticles, IL-10 and GelMA is 0.03-0.05 g: 10-15 μL: 0.02-0.03 g.

[0021] The second aspect of the present invention discloses a method for preparing the hydrogel drug delivery system for achieving spatiotemporal sequential release as described above, comprising the following steps:

[0022] S1: Preparation of drug-loaded nanoparticles: preparing a drug carrier loaded with a first drug by a solvent-dialysis method to obtain drug-loaded nanoparticles;

[0023] S2: Preparation of hydrogel drug delivery system: The hydrogel, the second drug, the drug-loaded nanoparticles, the thiolated heparin and the modified hyaluronic acid are stirred and mixed in a solvent to form a hydrogel solution, and the drug-loaded nanoparticles are evenly distributed by ultrasonic stirring. Finally, a photoinitiator is added and photo-crosslinking is performed under light.

[0024] Preferably, step S1 is:

[0025] PLGA-HA and icariin are dissolved in an organic solvent, stirred and mixed, and deionized water is added dropwise. After continuous stirring, the organic solvent is removed by dialysis to obtain drug-loaded nanoparticles.

[0026] Preferably, the mass ratio of PLGA-HA to icariin is 10:330-350; the organic solvent is a mixture of DMSO and DMF, wherein the volume ratio of DMSO to DMF is 3-4:1; and the dialysis time is 12-15h.

[0027] Preferably, the modified hyaluronic acid is HA-Ac (hyaluronic acid-acrylate); and the photoinitiator is LAP.

[0028] Preferably, the dosage ratio of the GelMA, thiolated heparin, IL-10, drug-loaded nanoparticles and HA-Ac is 0.02-0.03g: 0.001-0.002g: 10-15μL: 0.03-0.05g: 0.01-0.02g; the stirring and mixing is magnetic stirring at 37°C for 0.5-1h; the amount of LAP added is 0.05-0.2% of the volume of the hydrogel solution; the wavelength of the light is 390-430nm, and the time is 20-50s.

[0029] Preferably, the modified hyaluronic acid is prepared by the following steps:

[0030] First, hyaluronic acid is activated to obtain HA-ADH (amino-modified hyaluronic acid, prepared with hyaluronic acid and hexahydrazine as raw materials and EDC condensation agent), and then HA-ADH is added to HEPES buffer to react with N-acryloyloxysuccinimide. After dialysis and freeze-drying, sponge-like solid HA-Ac is obtained.

[0031] The third aspect of the present invention discloses the use of the hydrogel drug-carrying system for achieving spatiotemporal sequential release as described above in the preparation of drugs for bone regeneration.

[0032] Preferably, the dosage form of the bone regeneration drug includes an injection.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] In the early stage, a large number of immune cells, including M1 macrophages, are recruited to the bone defect, which produces inflammatory factors, leading to a local inflammatory immune microenvironment. In the inflammatory environment, the osteoblast differentiation effect will be greatly weakened, limiting bone regeneration. IL-10 can inhibit the synthesis and release of inflammatory factors. Both endogenous and exogenous IL-10 can strongly inhibit the synthesis of IL-1, IL-6, IL-8, TNF-α, GM-CSF and G-CSF at the transcriptional level, thereby playing an anti-inflammatory role. As an anti-inflammatory factor, IL-10 has a strong anti-inflammatory effect. It acts on macrophages to polarize them to M2 macrophages, reduce inflammation, and create a local microenvironment that is conducive to regeneration. In the middle and late stages, the release of ICA promotes osteoblast differentiation, produces new bone tissue, and achieves effective repair of bone defect regeneration. ICA can accelerate osteoblast differentiation, promote bone formation, and inhibit osteoclast differentiation and bone resorption. Icariin can activate autophagy, produce significant anti-inflammatory effects on macrophages, restore osteogenic activity of aged BMSCs, and thus alleviate bone loss in osteoporosis. The synergistic effect of the two can form an effective anti-inflammatory and osteogenic effect, which is beneficial to promote the healing of wounds and bone defects.

[0035] In this scheme, IL-10 and thiolated heparin are electrostatically bonded to achieve early rapid release, and ICA is loaded into PLGA-HA nanoparticles to achieve long-term slow release. Using hydrogel as a carrier, the various components are combined to prepare a new hydrogel drug delivery system that can achieve the effect of timed drug release. The prepared hydrogel has the function of immunomodulation and can promote osteogenic differentiation. It also has multiple properties such as injectability and photocrosslinking. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 The appearance of the composite hydrogel solution (a) and the photocured hydrogel (b) in Example 1;

[0037] Figure 2 is a scanning electron microscope image of the composite hydrogel in Example 1;

[0038] Figure 3 This is a stress-strain test diagram of each component in the composite hydrogel in Example 1;

[0039] Figure 4 The biocompatibility test diagram of the composite hydrogel in Example 1 and Comparative Examples 1-3;

[0040] Figure 5 This is a release test diagram of the composite hydrogel in Example 1;

[0041] Figure 6The test diagrams of the composite hydrogel promoting bone regeneration in Example 1 and Comparative Examples 1-3, wherein: A is a representative microscopic CT image of a skull defect, B and C are quantitative evaluations of osteogenesis parameters (BV represents new bone volume, TV represents total defect volume, and BMD represents new bone density), D is a representative staining image of postoperative regenerated bone tissue (H&E, Masson), E is a representative immunohistochemical staining image of osteogenic markers (OPN, OCN), F is a quantitative analysis image of new bone area, and G and H are quantitative analysis images of OPN and OCN positive areas;

[0042] Figure 7 The test diagrams of the composite hydrogels in Example 1 and Comparative Examples 1-3 for inhibiting inflammation, wherein: A is a representative image of the expression of surface markers CCR7 (red, M1 phenotype) and CD206 (green, M2 phenotype) by immunofluorescence analysis, B is the immunohistochemistry of IL-10 and TNF-α, C and D are the qualitative analysis of CCR7 and CD206, and E and F are the qualitative analysis of IL-10 and TNF-α;

[0043] Figure 8 The preparation method and mechanism of action of the composite drug delivery system of this scheme;

[0044] Fig. 9 The test diagrams of the composite hydrogel regulating macrophage polarization in vitro in Example 1 and Comparative Examples 1-3, wherein: AC is immunofluorescence staining, DG is flow cytometry, and HK is qPCR to measure the expression of inflammation-related genes;

[0045] Fig.10 The test graphs show the effect of the composite hydrogel in Example 1 and Comparative Examples 1-3 on promoting osteogenic differentiation of BMSCs in vitro, wherein: AD is immunofluorescence staining, EH is alkaline phosphatase (ALP) staining and Alizarin red staining (ARS), and IL is qPCR to measure the expression of osteogenic differentiation-related genes. DETAILED DESCRIPTION

[0046] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0047] In the following description, unless otherwise specified, the reagents used are conventional commercially available products, and the methods used are well known in the art.

[0048] like Figure 8 As shown, a hydrogel drug delivery system for achieving spatiotemporal sequential release comprises:

[0049] The first drug, the first drug is icariin;

[0050] A drug carrier, wherein the drug carrier encapsulates the first drug to form drug-loaded nanoparticles with a core-shell structure, and is used to achieve long-term release of the first drug;

[0051] The second drug, the second drug is IL-10;

[0052] as well as,

[0053] The hydrogel, the drug-loaded nanoparticles are uniformly dispersed in the hydrogel, and the second drug is electrostatically bonded with heparin-modified hyaluronic acid (HA-Ac-Heparin) and then uniformly dispersed in the hydrogel;

[0054] The drug carrier is PLGA-HA, and the hydrogel is GelMA.

[0055] The mass ratio of PLGA-HA to icariin in the drug-loaded nanoparticles is 10:330-350; the dosage ratio of the drug-loaded nanoparticles to IL-10 and GelMA is 0.03-0.05 g:10-15 μL:0.02-0.03 g.

[0056] Polylactic-co-glycolic acid-hyaluronic acid (PLGA-HA) is used as a drug carrier, in which PLGA, as a hydrophobic core, can encapsulate a variety of hydrophobic drugs, and HA, as a hydrophilic shell, can prolong the retention time of the drug and achieve a long-term release effect.

[0057] GelMA hydrogel is a modified gelatin obtained by reacting gelatin with methacrylic anhydride (MA), which has the characteristics of photocrosslinking, excellent biocompatibility, low immune response, etc. In the following example, the GelMA hydrogel used was purchased from EFL (Engineering For Life, Suzhou Yongqinquan Intelligent Equipment Co., Ltd.).

[0058] Interleukin 10 (IL-10), also known as cytokine synthesis inhibitor, is a pleiotropic cytokine that can exert immunosuppressive or immunostimulatory effects in various cell types. IL-10 can inhibit the synthesis and release of inflammatory factors. Both endogenous and exogenous IL-10 can strongly inhibit the synthesis of interleukin-1 (IL-1), IL-6, IL-8, tumor necrosis factor-α (TNF-α), granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) at the transcriptional level, thereby playing an anti-inflammatory role.

[0059] Icariin (ICA) is a Chinese herbal monomer and the main active ingredient of Epimedium. Several studies have shown that icariin can accelerate osteoblast differentiation, promote bone formation, and inhibit osteoclast differentiation and bone resorption. Icariin can activate autophagy, have a significant anti-inflammatory effect on macrophages, restore osteogenic activity of aging BMSCs, and thus alleviate bone loss in osteoporosis.

[0060] The method for preparing the hydrogel drug-carrying system for achieving spatiotemporal sequential release as described above comprises the following steps:

[0061] S1: Preparation of drug-loaded nanoparticles: preparing a drug carrier loaded with a first drug by a solvent-dialysis method to obtain drug-loaded nanoparticles;

[0062] S2: Preparation of hydrogel drug delivery system: The hydrogel, the second drug, the drug-loaded nanoparticles, the thiolated heparin and the modified hyaluronic acid are stirred and mixed in a solvent to form a hydrogel solution, and the drug-loaded nanoparticles are evenly distributed by ultrasonic stirring. Finally, a photoinitiator is added and photo-crosslinking is performed under light.

[0063] in:

[0064] Step S1 is:

[0065] PLGA-HA and icariin are dissolved in an organic solvent, stirred and mixed, and deionized water is added dropwise. After continuous stirring, the organic solvent is removed by dialysis to obtain drug-loaded nanoparticles.

[0066] The mass ratio of PLGA-HA to icariin is 10:330-350; the organic solvent is a mixture of DMSO and DMF, wherein the volume ratio of DMSO to DMF is 3-4:1; and the dialysis time is 12-15h.

[0067] In step S2:

[0068] The modified hyaluronic acid is HA-Ac; and the photoinitiator is LAP.

[0069] The dosage ratio of GelMA, thiolated heparin, IL-10, drug-loaded nanoparticles and HA-Ac is 0.02-0.03g: 0.001-0.002g: 10-15μL: 0.03-0.05g: 0.01-0.02g; the stirring and mixing is magnetic stirring at 37°C for 0.5-1h; the amount of LAP added is 0.05-0.2% of the volume of the hydrogel solution; the wavelength of the light is 390-430nm, and the time is 20-50s.

[0070] The modified hyaluronic acid is prepared by the following steps:

[0071] First, hyaluronic acid is activated to obtain HA-ADH, and then HA-ADH is added to HEPES buffer to react with N-acryloyloxysuccinimide. After dialysis and freeze-drying, sponge-like solid HA-Ac is obtained.

[0072] HA-Ac first reacts and connects with thiolated heparin to form negatively charged HA-Ac-Heparin, which then combines with positively charged IL-10 through electrostatic interaction and is subsequently dispersed in the hydrogel together with the self-assembled drug-loaded nanoparticles.

[0073] A use of the hydrogel drug-carrying system that realizes spatiotemporal sequential release as described above in the preparation of drugs for bone regeneration.

[0074] Wherein, the dosage form of the bone regeneration drug includes injection.

[0075] Example 1

[0076] Preparation of PLGA-HA nanoparticles: PLGA-HA nanoparticles were prepared by solvent-dialysis method. The specific method is as follows: 10 mg PLGA-HA and 338 mg ICA were dissolved in DMSO / DMF (v / v=3:1) at the same time, and then deionized water was slowly added to the organic phase (DMSO / DMF containing drugs and polymers) under stirring. After stirring for 15 minutes, the mixture was transferred to a dialysis bag and dialyzed for 12 hours to remove the organic solvent to obtain drug-loaded nanoparticles (NPs).

[0077] Preparation of composite hydrogel (hydrogel drug-carrying system):

[0078] Modified hyaluronic acid (HA-Ac): The synthesis process is divided into two steps: first, HA is activated to amino-modified hyaluronic acid (HA-ADH), and then reacted with N-acryloyloxysuccinimide in HEPES buffer, followed by dialysis and freeze-drying to produce sponge-like solid HA-Ac.

[0079] Specifically: First, HA (1 mmol) and NHS (2 mmol) were fully dissolved in 80 mL of deionized water with stirring at room temperature. Then, EDCI (3 mmol) in solid form was added to the mixture solution and reacted for 2 hours to activate the carboxyl group of HA. It was then added to 50 mL of ADH (10 mmol) deionized solution, and the pH of the mixed solution was maintained at 4.75. After 24 hours, the solution was transferred to a dialysis tube (8000–14000 MWCO) and dialyzed with a NaCl solution (concentration gradient from 100 mM to 0) for 72 hours. The purified intermediate HA-ADH was lyophilized and stored at -20 °C. Next, HA-ADH (1 mmol) was reacted with NHS-Ac (5 mmol) in HEPES buffer (10 mM HEPES, 150 mM NaCl, 10 mM EDTA, pH = 7.2) at room temperature for 12 h, and then dialyzed for 72 h under the same conditions. The solution was then lyophilized to obtain HA-Ac as a spongy solid.

[0080] Thiolated heparin (Heparin-SH): Heparin is mixed with EDC, HOBT and cysteamine, dialyzed after sufficient reaction, and then DTT is added to break the disulfide bond, dialyzed again, and freeze-dried. Finally, cysteamine is connected to the carboxyl group in the heparin structure to obtain thiolated heparin (Heparin-SH).

[0081] Preparation of thiolated heparin, specifically:

[0082] 1. Take 0.2g heparin (Mw=15000) and dissolve it in 20mL ultrapure water to make a solution with a concentration of 10mg / mL. Weigh the mass of heparin to calculate the moles of carboxyl groups (1mol heparin contains about 25mol carboxyl groups). Calculate the required mass of EDC, HOBT and cysteamine according to Table 1, add EDC, HOBT and cysteamine in sequence while stirring, and mix thoroughly.

[0083] 2. Mix well using a magnetic stirrer at room temperature for 5 hours.

[0084] 3. Place the reaction solution in a dialysis bag with a molecular weight of 3500 and dialyze it in double distilled water for 12 hours to completely remove the unreacted molecules in the synthesis.

[0085] 4. Place the solution in a beaker, accurately weigh 0.5142 g of DTT and add it to the solution and mix thoroughly. Adjust the pH to 7.5 and use a magnetic stirrer to mix and react at room temperature for 3-5 hours.

[0086] 5. Take 29.25g of sodium chloride and 1.4612g of EDTA and dissolve them in 5L of double distilled water (to prepare a solution containing 0.1M NaCl and 1mM EDTA), and adjust the pH to 3.5 to prepare a dialysate.

[0087] 6. The pH of the solution after the reaction in step 4 is adjusted to 3.5 to obtain an acidified solution.

[0088] 7. The acidified solution was placed in a dialysis bag with a molecular weight of 3500 again, and dialyzed in the dialysate in step 5 for 24 hours.

[0089] 8. Subsequently, the desired solution was placed in a solution containing 1 mM EDTA, pH = 3.5 and continued to be dialyzed for about 36 hours.

[0090] 9. After dialysis is completed, take out the solution and place it in a beaker, adjust the pH to 5-6, and then place it in a -20℃ refrigerator overnight.

[0091] 10. Take out the solidified liquid and freeze-dry it in a freeze dryer.

[0092] Table 1 The mass ratio of heparin (carboxyl) to EDC, HOBT and cysteamine

[0093]

[0094] HA-Ac (0.015 g), GelMA (0.025 g), Heparin-SH (0.0015 g), drug-loaded nanoparticles (0.04 g) and IL-10 (12 μL) were added to the LAP solvent. The mixture was magnetically stirred in a water bath at 37 degrees Celsius for 1 hour. The stirred hydrogel solution was then ultrasonically stirred to uniformly distribute the drug-loaded nanoparticles in the hydrogel solution. Finally, 0.1% v / v LAP was added for photocrosslinking under 405 nm wavelength for 30 seconds to obtain a cured composite hydrogel IL-10 / ICA@gel.

[0095] Appearance of composite hydrogel:

[0096] After the components of the composite hydrogel are mixed in a certain proportion, a mixed solution can be obtained. After the solution is irradiated with ultraviolet light for 30 seconds, if the gel does not flow when the test tube is tilted and can be maintained, the transition from liquid to gel state has occurred. Figure 1 As shown, it is confirmed that the composite hydrogel has the property of photo-crosslinking.

[0097] Microstructure of composite hydrogel:

[0098] The dried composite hydrogel (whether containing drug-loaded nanoparticles or not) was fixed on the platform with conductive glue. Then, gold was sputter-coated on it and observed using a scanning electron microscope, as shown in Figure 2. Figure 2 The results show that the composite hydrogel has a loose and porous structure, which is conducive to cell adhesion and proliferation.

[0099] Characterization of composite hydrogels:

[0100] The composite hydrogel samples were prepared into strips (10 cm × 1 cm × 0.2 cm), and the stress-strain tests of the components in the composite hydrogel were performed at a speed of 20 mm / min at 25°C using a universal testing machine to test its tensile properties. At the same time, the stress-strain tests of the HA-Ac prepared above, the purchased GelMA, and the HA-Ac / GelMA formed by mixing HA-Ac and GelMA in a mass ratio of 1:1 were performed under the same conditions as a comparative reference. Figure 3 As shown, the composite hydrogel has good mechanical properties.

[0101] Comparative Example 1

[0102] This comparative example is gel, that is, no active pharmaceutical ingredient is added, and only hydrogel is contained.

[0103] Preparation of hydrogel:

[0104] GelMA (0.025 g) was added to LAP solvent. The mixture was magnetically stirred in a water bath at 37 degrees Celsius for 1 hour. The stirred hydrogel solution was then ultrasonically stirred. Finally, 0.1% v / v LAP was added for photocrosslinking under 405 nm wavelength for 30 seconds to obtain a cured hydrogel gel.

[0105] Comparative Example 2

[0106] This comparative example is IL-10@gel, that is, only IL-10 is added without icariin.

[0107] Preparation of composite hydrogel:

[0108] Modified hyaluronic acid (HA-Ac): The synthesis process is divided into two steps: first, HA is activated to amino-modified hyaluronic acid (HA-ADH), and then reacted with N-acryloyloxysuccinimide in HEPES buffer, followed by dialysis and freeze-drying to produce sponge-like solid HA-Ac.

[0109] Thiolated heparin (Heparin-SH): Heparin is mixed with EDC, HOBT and cysteamine, dialyzed after sufficient reaction, and then DTT is added to break the disulfide bond, dialyzed again, and freeze-dried. Finally, cysteamine is connected to the carboxyl group in the heparin structure to obtain thiolated heparin (Heparin-SH).

[0110] The specific preparation methods of HA-Ac and Heparin-SH are the same as those described in Example 1.

[0111] HA-Ac (0.015 g), GelMA (0.025 g), Heparin-SH (0.0015 g), and IL-10 (12 μL) were added to the LAP solvent. The mixture was magnetically stirred in a water bath at 37 degrees Celsius for 1 hour. The stirred hydrogel solution was then ultrasonically stirred. Finally, 0.1% v / v LAP was added for photocrosslinking under 405 nm wavelength for 30 seconds to obtain a cured composite hydrogel IL-10@gel.

[0112] Comparative Example 3

[0113] This comparative example is ICA@gel, that is, only icariin is added without IL-10.

[0114] Preparation of PLGA-HA nanoparticles: PLGA-HA nanoparticles were prepared by solvent-dialysis method. The specific method is as follows: 10 mg PLGA-HA and 338 mg ICA were dissolved in DMSO / DMF (v / v=3:1) at the same time, and then deionized water was slowly added to the organic phase (DMSO / DMF containing drugs and polymers) under stirring. After stirring for 15 minutes, the mixture was transferred to a dialysis bag and dialyzed for 12 hours to remove the organic solvent to obtain drug-loaded nanoparticles (NPs).

[0115] Preparation of composite hydrogel:

[0116] GelMA (0.025 g) and drug-loaded nanoparticles (0.04 g) were added to the LAP solvent. The mixture was magnetically stirred in a water bath at 37 degrees Celsius for 1 hour. The stirred hydrogel solution was then ultrasonically stirred to uniformly distribute the drug-loaded nanoparticles in the hydrogel solution. Finally, 0.1% v / v LAP was added for photocrosslinking under 405 nm wavelength for 30 seconds to obtain a cured composite hydrogel ICA@gel.

[0117] Biocompatibility test of composite hydrogel:

[0118] After the composite hydrogel was co-cultured with BMSCs, a cell counting kit-8 (CCK-8) test was performed on the 1st, 4th, and 7th days to evaluate cell viability. BMSCs were cultured normally, and when the cell growth reached 80% confluence, the cells were digested and passaged. When the third generation was observed, the cell growth was stable and reached 80% confluence, and the cells were digested and counted. 5000 cells per well were inoculated in a 96-well culture plate (100 μL / well), and the 96-well plate was placed in a 37°C, 5% CO2 incubator for 24 hours until the cells adhered to the wall and grew and then the original culture medium was discarded. After rinsing with PBS solution 3 times, 100 μL of hydrogel (the hydrogel / composite hydrogel of Example 1 and Comparative Examples 1-3 were added respectively), and a negative control group (α-MEM culture medium containing 10% fetal bovine serum by volume) was set up at the same time, and 3 wells were set up in each group. For CCK-8 assay, the hydrogel was rinsed with PBS and incubated in a medium containing 10% CCK-8 reagent at 37 °C for 2 h, and then the absorbance was measured at 450 nm using a spectrophotometer. Figure 4 ,According to the experimental results, the composite hydrogels in each group have good ,biocompatibility and no cytotoxicity.

[0119] Release rate detection of composite hydrogel:

[0120] The composite hydrogel was placed in a PBS buffer solution with pH = 7.2 and placed on a constant temperature oscillator at 37°C. At the designated time points, the released supernatant was collected and replaced with fresh PBS. The IL-10 concentration was measured using a commercial ELISA kit according to the manufacturer's instructions. The absorbance value of the solution at 268 nm was measured using a UV-visible instrument to calculate the concentration of ICA. The cumulative release rate was calculated and the release curve was plotted. Figure 5 .

[0121] In vivo study of composite hydrogels:

[0122] 1. To investigate the effect of composite hydrogel in promoting bone regeneration.

[0123] Establishment of rat skull defect model: After anesthesia, shaving and disinfection, 6-8 week old SD rats were subjected to longitudinal incision along the skull to separate the skin, muscle and periosteum. The full-thickness bone tissue was removed with a dental implant, and two full-thickness bone defects with a diameter of 5 mm were created on both sides of the skull. Then, the composite hydrogel was filled in the defect and the skin was sutured. The experimental group was implanted with gel (Comparative Example 1), IL-10@gel (Comparative Example 2), ICA@gel (Comparative Example 3) and IL-10 / ICA@gel (Example 1), and the control group (con) was not implanted with any gel. Eight weeks after the operation, the skull was sampled, and the skull tissue was placed in 4% paraformaldehyde fixative for preservation and Micro-CT photography was performed. After routine dehydration, decalcification, paraffin embedding, sectioning, HE staining, Masson staining, observation under a microscope and photography were performed, as shown in the figure. Figure 6 shown.

[0124] Figure 6 The skull defect repair status and quantitative analysis of rats in each group. It can be seen from the pictures of each group that IL-10 / ICA@gel can significantly promote bone regeneration compared with other groups. BV / TV and BMD were calculated. Eight weeks after surgery, the newly generated bone tissue of IL-10 / ICA@gel was more than that of other groups, almost completely covering the wound. The results of HE and Masson staining also confirmed this. Therefore, the ICA released by the composite hydrogel produces more new bone tissue during the bone defect repair process and promotes bone regeneration.

[0125] 2. Investigate the inhibitory effect of composite hydrogel on early excessive inflammation of wounds

[0126] Immunofluorescence staining was used to detect the expression of pro-inflammatory M1 macrophages CCR7 and anti-inflammatory M2 macrophages CD206 in local tissues on the seventh day to evaluate the level of wound inflammation in different groups of hydrogel treatment. Immunohistochemistry was used to detect the markers of M1 macrophages TNF-α and M2 macrophages IL-10 to evaluate the early regulation of wound inflammation. Figure 7 shown.

[0127] Figure 7The figure shows the effect of composite hydrogel in inhibiting local excessive inflammation. From the representative fluorescent images in the figure, it can be seen that the control group has more CCR7 positive cells and fewer CD206 positive cells, while the IL-10 / ICA@gel group has fewer CCR7 positive cells and more CD206 positive cells, which indicates that the IL-10 released by the composite hydrogel in the IL-10 / ICA@gel group produced higher levels of M2 macrophages with anti-inflammatory effects locally, while inhibiting M1 macrophages. Immunohistochemistry further confirmed that there were more M2 macrophages and fewer M1 macrophages in the injured area. IL-10 / ICA@gel can significantly reduce the expression of TNF-α and increase the expression of IL-10 on the seventh day of injury.

[0128] Therefore, the results showed that the IL-10 released by the composite hydrogel could significantly inhibit M1 macrophages and promote the phenotype of M2 macrophages, showing a good anti-inflammatory effect.

[0129] In vitro study of composite hydrogels:

[0130] 1. Investigate the role of composite hydrogels in regulating macrophage polarization in vitro.

[0131] Immunofluorescence staining of osteogenic differentiation markers: Raw264.7 cells were cultured with high-glucose DMEM medium containing 100 ng / mL lipopolysaccharide (LPS) for 24 h to generate an inflammatory microenvironment. Then, each group of composite hydrogels (gel, IL-10@gel, ICA@gel, and IL-10 / ICA@gel) were co-cultured with Raw264.7 cells and cultured for another 24 h. The treated Raw264.7 cells were fixed with 4% paraformaldehyde solution for 30 min, permeabilized with 0.5% Triton, and blocked with 10% BSA. Immunopolyclonal anti-CD206 was co-incubated overnight, followed by goat anti-rabbit IgG secondary antibody for 1 h. Then, the cell nuclei were stained with DAPI and observed and photographed by confocal microscopy. Similarly, immunopolyclonal CCR7 antibody was used for immunofluorescence staining. The fluorescence intensity was semi-quantitatively analyzed using Image J software.

[0132] Flow cytometry: Raw264.7 cells were cultured with high-glucose DMEM medium containing 100 ng / mL lipopolysaccharide (LPS) for 24 hours to produce an inflammatory microenvironment. Then, each group of composite hydrogels (gel, IL-10@gel, ICA@gel, and IL-10 / ICA@gel) were co-cultured with Raw264.7 cells and cultured for another 24 hours. The treated Raw264.7 cells were fixed with 4% paraformaldehyde solution for 30 minutes and blocked with Fc receptor binding blocking agent for 10 minutes. Then, the cells were incubated with the primary antibody for 30 minutes. The samples were loaded into the flow cytometer for analysis.

[0133] qPCR determination of inflammation-related gene expression: Raw264.7 cells were cultured with high-glucose DMEM medium containing 100 ng / mL lipopolysaccharide (LPS) for 24 hours to produce an inflammatory microenvironment. Then, each group of composite hydrogels (gel, IL-10@gel, ICA@gel, and IL-10 / ICA@gel) were co-cultured with Raw264.7 cells and cultured for another 24 hours. RNA was extracted from each group of cells using an RNA extraction kit from the treated Raw264.7 cells, and the gene expression of IL-1β, iNOS, IL-1ra, and IL-4 was analyzed by RT-qPCR. The results were normalized to the expression of the gene GAPDH.

[0134] The test results are as follows Fig. 9 shown.

[0135] Fig. 9 Figures AC in the figure are immunofluorescence staining to verify the polarization characteristics of macrophages after gel, IL-10@gel, ICA@gel and IL-10 / ICA@gel were co-cultured with Raw264.7 cells for 1 day in vitro. The results show that the fluorescence staining images of CD206 (M2 macrophage marker) and CCR7 (M1 macrophage marker) and quantitative analysis show that the green fluorescence intensity of the IL-10@gel and IL-10 / ICA@gel groups is higher, indicating that the inhibitory effect on inflammation is more significant, and it can promote the M2 polarization of Raw264.7 cells in vitro and inhibit M1 polarization, and IL-10 / ICA@gel has the best effect.

[0136] Fig. 9 Figure DG in the figure is the flow cytometry verification of the macrophage polarization characteristics of gel, IL-10@gel, ICA@gel and IL-10 / ICA@gel after co-culture with Raw264.7 cells for 1 day. The results show that IL-10 / ICA@gel has more CD206 (M2 macrophage marker) positive cells and fewer CD86 (M1 macrophage marker) positive cells. Compared with gel treatment, IL-10@gel, ICA@gel and IL-10 / ICA@gel can promote the M2 polarization of Raw264.7 cells in vitro and inhibit M1 polarization, and IL-10 / ICA@gel has the best effect.

[0137] Fig. 9 The HK figure in the figure shows the expression of inflammation-related genes determined by qPCR, indicating that IL-10 / ICA@gel can promote the expression of anti-inflammatory factors and inhibit the expression of pro-inflammatory factors. It proves that IL-10 / ICA@gel can effectively inhibit inflammation and regulate the polarization of macrophages.

[0138] 2. Investigate the effect of composite hydrogel in promoting osteogenic differentiation of BMSCs in vitro.

[0139] Immunofluorescence staining of osteogenic differentiation markers: Each group of hydrogels was co-cultured with BMSCs for 7 days. The treated BMSCs were fixed with 4% paraformaldehyde solution for 30 minutes, permeabilized with 0.5% Triton and blocked with 10% BSA. They were co-incubated with polyclonal anti-OPN overnight, then incubated with goat anti-rabbit IgG secondary antibody for 1 hour, and incubated with phalloidin for 1 hour. Then, the cell nuclei were stained with DAPI and observed and photographed by laser confocal microscopy. Similarly, polyclonal RUNX2 antibody was used for immunofluorescence staining. The fluorescence intensity was semi-quantitatively analyzed using Image J software.

[0140] Alkaline phosphatase (ALP) staining: Using a 24-well plate, each group of hydrogels was co-cultured with BMSCs, and the culture medium was changed every 3 days. After 7 days of culture, BMSCs were washed with PBS and fixed with 4% paraformaldehyde at room temperature for 10 minutes. After that, the induced differentiated cells were stained according to the instructions of the BCIP / NBT alkaline phosphatase colorimetric kit. Microscope was used for photography and observation. Image J software was used to semi-quantitatively analyze the fluorescence intensity.

[0141] Alizarin red staining (ARS): Using a 24-well plate, each group of hydrogels was co-cultured with BMSCs, and the culture medium was changed every 3 days. After 7 days of culture, BMSCs were washed with PBS and fixed with 4% paraformaldehyde for 10 minutes at room temperature. The cells were stained with Alizarin red staining kit according to the instructions, and photographed and observed under a microscope. The fluorescence intensity was semi-quantitatively analyzed using Image J software.

[0142] qPCR determination of osteogenic differentiation-related gene expression: After 7 days of co-culture, RNA was extracted from each group of BMSCs using an RNA extraction kit, and the gene expression of OPN, OCN, COL-1, and RUNX2 was analyzed by RT-qPCR. The results were normalized to the expression of the gene GAPDH.

[0143] The test results are as follows Fig.10 shown.

[0144] Fig.10 Figures AD in the figure are immunofluorescence staining to verify the osteogenic properties of gel, IL-10@gel, ICA@gel and IL-10 / ICA@gel after 7 days of in vitro co-culture. The results show that the fluorescence intensity of the ICA@gel and IL-10 / ICA@gel groups is higher, indicating that the osteogenic effect is more significant and can promote the osteogenic differentiation of BMSCs in vitro, and IL-10 / ICA@gel has the best effect.

[0145] Fig.10 Figure EH in the figure uses alkaline phosphatase (ALP) staining and Alizarin red staining (ARS) to verify the osteogenic properties of gel, IL-10@gel, ICA@gel and IL-10 / ICA@gel after 14 days of in vitro co-culture. The results show that IL-10 / ICA@gel has a darker blue color and more red nodules. Compared with gel treatment, IL-10@gel, ICA@gel and IL-10 / ICA@gel can promote the osteogenic differentiation of BMSCs in vitro, and IL-10 / ICA@gel has the best effect.

[0146] Fig.10 The IL-10 in the figure is the expression of osteogenic differentiation-related genes determined by qPCR, indicating that IL-10 and ICA in IL-10 / ICA@gel can synergistically enhance the differentiation of BMSCs into osteoblasts and stimulate the expression of osteogenic biomarkers. It proves that IL-10 / ICA@gel can effectively promote the osteogenic differentiation of BMSCs.

[0147] The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. A hydrogel drug delivery system for achieving spatiotemporal sequential release, characterized in that: include: The first drug, the first drug is icariin; A drug carrier, wherein the drug carrier is PLGA-HA, and the drug carrier encapsulates the first drug to form drug-loaded nanoparticles of a core-shell structure, so as to achieve long-term release of the first drug; The second drug, the second drug is IL-10; as well as, Hydrogel, the hydrogel is GelMA; The drug-loaded nanoparticles are uniformly dispersed in the hydrogel, and the second drug is electrostatically bonded to the heparin-modified hyaluronic acid and then uniformly dispersed in the hydrogel; The mass ratio of PLGA-HA to icariin in the drug-loaded nanoparticles is 10:338; The heparin-modified hyaluronic acid is formed by the reaction of modified hyaluronic acid HA-Ac and thiolated heparin; The HA-Ac is prepared by the following steps: First, hyaluronic acid is activated to obtain amino-modified hyaluronic acid HA-ADH, and then HA-ADH is added to HEPES buffer to react with N-acryloyloxysuccinimide, and then dialyzed and freeze-dried to obtain sponge-like solid HA-Ac; The thiolated heparin is prepared by the following steps: Heparin is mixed with EDC, HOBT and cysteamine, dialyzed after sufficient reaction, and then DTT is added to break the disulfide bond, dialyzed again, and freeze-dried to obtain thiolated heparin; The dosage ratio of GelMA, thiolated heparin, IL-10, drug-loaded nanoparticles and HA-Ac was 0.025 g: 0.0015 g: 12 μL: 0.04 g: 0.015 g.

2. A method for preparing a hydrogel drug delivery system for achieving spatiotemporal sequential release as claimed in claim 1, characterized in that: The steps include: S1: Preparation of drug-loaded nanoparticles: preparing a drug carrier loaded with a first drug by a solvent-dialysis method to obtain drug-loaded nanoparticles; S2: Preparation of hydrogel drug delivery system: The hydrogel, the second drug, the drug-loaded nanoparticles, the thiolated heparin and the modified hyaluronic acid are stirred and mixed in a solvent to form a hydrogel solution, and the drug-loaded nanoparticles are evenly distributed by ultrasonic stirring. Finally, a photoinitiator is added and photo-crosslinking is performed under light.

3. The method for preparing a hydrogel drug delivery system for achieving spatiotemporal sequential release according to claim 2, characterized in that: Step S1 is: PLGA-HA and icariin are dissolved in an organic solvent, stirred and mixed, and deionized water is added dropwise. After continuous stirring, the organic solvent is removed by dialysis to obtain drug-loaded nanoparticles.

4. The method for preparing a hydrogel drug delivery system for achieving spatiotemporal sequential release according to claim 3, characterized in that: The organic solvent is a mixture of DMSO and DMF, wherein the volume ratio of DMSO to DMF is 3-4:1; and the dialysis time is 12-15 hours.

5. The method for preparing a hydrogel drug delivery system for achieving spatiotemporal sequential release according to claim 2, characterized in that: The modified hyaluronic acid is HA-Ac; and the photoinitiator is LAP.

6. The method for preparing a hydrogel drug delivery system for achieving spatiotemporal sequential release according to claim 5, characterized in that: The stirring and mixing is magnetic stirring at 37° C. for 0.5-1 h; the amount of LAP added is 0.05-0.2% of the volume of the hydrogel solution; the wavelength of the light irradiation is 390-430 nm, and the time is 20-50 s.

7. Use of the hydrogel drug delivery system realizing spatiotemporal sequential release as claimed in claim 1 in the preparation of drugs for bone defects.

8. The use of a hydrogel drug delivery system for achieving spatiotemporal sequential release according to claim 7, characterized in that: The dosage form of the bone defect medicine includes injection.

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