Apigenin-loaded silk fibroin drug-loaded microspheres and preparation method thereof, broad-spectrum antioxidant hydrogel and preparation method and application thereof

Abstract

The present invention provides a silk fibroin drug-loaded microsphere loaded with apigenin and a preparation method thereof, a broad-spectrum antioxidant hydrogel and a preparation method and application thereof, and relates to the technical field of composite materials. The silk fibroin drug-loaded microsphere loaded with apigenin provided by the present invention has a core-shell structure, the core layer includes silk fibroin and polyvinyl alcohol, and the shell layer includes apigenin. The silk fibroin drug-loaded microsphere loaded with apigenin provided by the present invention can promote angiogenesis and has high biological safety, thereby accelerating the repair of diabetic difficult-to-heal wounds.

Description

Technical Field

[0001] The present invention relates to the technical field of composite materials, and in particular to apigenin-loaded silk fibroin drug-loaded microspheres and a preparation method thereof, a broad-spectrum antioxidant hydrogel and a preparation method and application thereof. Background Art

[0002] Diabetes is one of the most common diseases in the world, which seriously threatens the health of patients. One of its complications, such as diabetic skin ulcers, has become an important cause of disability worldwide, causing great pain to patients and seriously affecting their quality of life. Since the high sugar microenvironment in the diabetic wound microenvironment affects the function of vascular endothelial cells, resulting in vascular damage and microcirculatory dysfunction, which seriously hinders wound repair, the current clinical treatment methods for diabetic wounds are very limited (relying only on surgical debridement, blood sugar control, skin grafting and other treatments), and it is difficult to fundamentally solve the problem of diabetic wounds being difficult to heal.

[0003] Neovascularization in skin tissue is a dynamic process that starts from capillaries and involves the participation of multiple cells, matrices and bioactive factors. The specific process includes: activation of endothelial cells and degradation of surrounding matrix, migration of endothelial cells, formation and proliferation of capillary buds. Neovascularization can promote the formation of granulation tissue, provide oxygen and nutrients to the damaged area, and play an important role in wound repair. However, natural antioxidant enzymes usually involve proteins and RNA molecules, which are susceptible to environmental factors and become inactive or lose their antioxidant function under pathological conditions, causing a large amount of reactive oxygen species (ROS) to accumulate. Excessive ROS causes great damage to endothelial cells and hinders vascular regeneration. Without nutrients and oxygen provided by blood vessels, damaged tissues cannot be repaired, wounds are difficult to heal, and a high disability rate is caused. In short, vascular regeneration is a prerequisite for the repair of diabetic skin damage. It is not only a bridge between skin tissue nutrition and metabolism, but also a provider of skin regeneration microenvironment.

[0004] The ideal scaffold for repairing diabetic skin damage can be matched according to the different conditions of skin damage, build an optimal bionic microenvironment for skin growth, and promote blood vessel and bone tissue regeneration under oxidative stress. Therefore, improving the personalized preparation of scaffolds that can form large skin substitutes with functional vascular systems under oxidative stress is an urgent problem to be solved in the repair of diabetic skin damage. Summary of the invention

[0005] In view of this, the purpose of the present invention is to provide a kind of apigenin-loaded silk fibroin drug-loaded microspheres and preparation method thereof, a broad-spectrum antioxidant hydrogel and preparation method and application thereof. The apigenin-loaded silk fibroin drug-loaded microspheres provided by the present invention can promote angiogenesis and have high biological safety, thereby accelerating the repair of difficult-to-heal diabetic wounds.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The invention provides an apigenin-loaded silk fibroin drug-loaded microsphere, which has a core-shell structure, wherein the shell layer comprises silk fibroin and polyvinyl alcohol, and the core layer comprises apigenin.

[0008] Preferably, the mass ratio of the silk fibroin to apigenin is 1:0.01-0.5.

[0009] The present invention provides a method for preparing apigenin-loaded silk fibroin drug-loaded microspheres according to the above technical solution, comprising the following steps:

[0010] The silk fibroin solution, apigenin solution, polyvinyl alcohol solution and ethanol are mixed and self-assembled to obtain apigenin-silk fibroin nano drug-loaded microspheres.

[0011] Preferably, the ratio of the total mass of the silk fibroin solution and the apigenin solution, the mass of the polyvinyl alcohol solution and the mass of ethanol is 10:0.1-5:1-3.

[0012] The present invention provides a method for preparing a broad-spectrum antioxidant hydrogel, comprising the following steps:

[0013] The gelatin solution, the sodium alginate solution, the apigenin-loaded silk fibroin drug-loaded microspheres and the nanosilver solution are mixed to obtain a mixed solution; the apigenin-loaded silk fibroin drug-loaded microspheres are the apigenin-loaded silk fibroin drug-loaded microspheres described in the above technical solution or the apigenin-loaded silk fibroin drug-loaded microspheres prepared by the preparation method described in the above technical solution;

[0014] The mixed solution is molded to obtain a broad-spectrum antioxidant hydrogel.

[0015] Preferably, the mass ratio of the nanosilver in the nanosilver solution to the apigenin-loaded silk fibroin drug-loaded microspheres is 0.1-1:1.

[0016] Preferably, the method for preparing the nanosilver solution comprises: mixing silver nitrate, water, isopropanol and glucose, and performing a reduction reaction to obtain the nanosilver solution.

[0017] Preferably, the forming comprises 3D printing.

[0018] The present invention provides a broad-spectrum antioxidant hydrogel prepared by the preparation method described in the above technical scheme.

[0019] The present invention provides the use of the broad-spectrum antioxidant hydrogel described in the above technical solution in the preparation of dressings.

[0020] The present invention provides a silk fibroin drug-loaded microsphere loaded with apigenin (abbreviated as SFM-A). Silk fibroin can self-assemble into nano-microspheres, which can be dissolved in water after being oxidized by ROS. The apigenin is wrapped with the silk fibroin self-assembly technology to prepare a nano-drug delivery system, which has a protective effect on apigenin and is also responsive to ROS. After the ROS in the wound microenvironment is cleared, the apigenin exerts its ability to promote angiogenesis and accelerate wound healing.

[0021] The present invention provides a method for preparing the apigenin-loaded silk fibroin drug-loaded microspheres described in the above technical solution. The present invention uses self-assembly technology to wrap apigenin with silk fibroin to prepare drug-loaded nano-microspheres, which is simple to operate, has mild reaction conditions, low cost, and is suitable for industrial production. The apigenin-loaded silk fibroin drug-loaded microspheres prepared by the present invention can promote angiogenesis, have high biosafety, and thus accelerate the repair of diabetic difficult-to-heal wounds.

[0022] The present invention provides a method for preparing a broad-spectrum antioxidant hydrogel. The preparation method provided by the present invention is simple to operate, has mild reaction conditions, is low in cost, and is suitable for industrial production. The broad-spectrum antioxidant hydrogel prepared by the present invention contains vasoactive ingredients, which can remove excess intracellular ROS without interfering with endogenous antioxidant effects, reconstruct redox homeostasis, and avoid cell damage; it can effectively promote angiogenesis in diabetic skin tissue and accelerate the healing of diabetic wounds. It has the potential for application as a hydrogel material for diabetic difficult-to-heal wounds, and provides new treatment ideas and clinical therapies for diabetic difficult-to-heal wound damage repair. In addition, the broad-spectrum antioxidant hydrogel prepared by the present invention has good degradability and antibacterial properties.

[0023] Furthermore, the present invention uses 3D printing technology to prepare broad-spectrum antioxidant hydrogels, which can achieve high-precision design and manufacturing of the shape and size of the broad-spectrum antioxidant hydrogels, thereby being able to control the structural characteristics at the microscopic level. The prepared broad-spectrum antioxidant hydrogel has potential for application as a hydrogel material for diabetic difficult-to-heal wounds, providing new treatment ideas and clinical therapies for the repair of diabetic difficult-to-heal wound injuries. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Characterization diagram of the nanosilver solution prepared in Example 1, wherein A is a transmission electron microscope image and B is a nanosilver particle size distribution diagram;

[0025] Figure 2 This is a scanning electron micrograph of the apigenin-loaded silk fibroin drug-loaded microspheres prepared in Example 1;

[0026] Figure 3 This is a physical picture of the broad-spectrum antioxidant hydrogel prepared in Example 1;

[0027] Figure 4It is a schematic diagram of the structure of the apigenin-silk fibroin drug-loaded microspheres of the present invention. DETAILED DESCRIPTION

[0028] The present invention provides a drug-loaded microsphere loaded with apigenin and silk fibroin, the structural schematic diagram of which is shown in FIG. Figure 4 As shown, it has a core-shell structure, the core layer includes apigenin, and the shell layer includes silk fibroin and polyvinyl alcohol.

[0029] In the present invention, the microspheres prepared by silk fibroin are oxidized by ROS to form a random curled structure and dissolved in water. The present invention uses silk fibroin to wrap apigenin for diabetic wound treatment. When the drug-loaded microspheres encounter a large amount of ROS, they are oxidized and dissolved, releasing apigenin, and a large amount of ROS on the wound surface is removed. Apigenin plays a role in promoting angiogenesis, thereby accelerating the repair of diabetic wound damage. The present invention regulates the ROS content of the microenvironment of diabetic skin ulcers through apigenin, promotes angiogenesis at the damaged site, and accelerates the repair of diabetic refractory ulcers.

[0030] In the present invention, the mass ratio of the silk fibroin to apigenin is preferably 1:0.01-0.5, more preferably 1:0.1-0.3.

[0031] In the present invention, the mass ratio of the silk fibroin to the polyvinyl alcohol is preferably 1: 0.1-10, more preferably 1: 4. In the present invention, the molecular weight of the polyvinyl alcohol is preferably 10,000-200,000, more preferably 20,000.

[0032] In the present invention, the diameter of the apigenin-loaded silk fibroin drug-loaded microspheres is preferably 200-500 nm.

[0033] The present invention provides a method for preparing apigenin-silk fibroin drug-loaded microspheres according to the above technical solution, comprising the following steps: mixing a silk fibroin solution, an apigenin solution, a polyvinyl alcohol solution and ethanol, and performing self-assembly to obtain apigenin-silk fibroin nano drug-loaded microspheres.

[0034] In the present invention, the concentration of the silk fibroin solution is preferably 1-100 mg / mL, more preferably 10-60 mg / mL, and further preferably 40 mg / mL. The present invention has no particular limitation on the preparation method of the silk fibroin solution, and any preparation method known to those skilled in the art can be used.

[0035] In a specific embodiment of the present invention, the method for preparing the silk fibroin solution preferably comprises: degumming natural mulberry silk to obtain silk; dissolving the silk in a solvent to obtain a mixed solution; and sequentially performing dialysis treatment and purification treatment on the mixed solution to obtain a silk fibroin solution. In the present invention, the degumming preferably comprises: mixing and boiling natural mulberry silk, sodium carbonate and boiling water. In the present invention, the mass ratio of the natural mulberry silk to sodium carbonate is preferably 1:0.5~1.5, more preferably 1:0.8~1. In the present invention, the boiling time is preferably 20~40min, more preferably 30min; the boiling is preferably carried out under stirring conditions. After the degumming, the present invention preferably further comprises washing the degummed silk with water and then drying to obtain silk. In the present invention, the solvent preferably includes a lithium bromide solution or a calcium chloride ternary solution; the concentration of the lithium bromide solution is preferably 8-12 mol / L, more preferably 9.3 mol / L; the composition of the calcium chloride ternary solution preferably includes calcium chloride, ethanol and water; the molar ratio of calcium chloride, ethanol and water is preferably 1:2:8. In the present invention, the dissolution temperature is preferably 50-70°C, more preferably 60°C; the dissolution time is preferably 3-5h, more preferably 4h. In the present invention, the dialysis treatment preferably includes water dialysis, and the molecular weight cutoff of the dialysis bag used in the dialysis treatment is preferably 3500Da; the dialysis time is preferably 2-4d, more preferably 3d; during the dialysis treatment, the water is preferably replaced every 2-4h (more preferably 3h); the water preferably includes deionized water. In the present invention, the purification preferably includes centrifugal separation, and the supernatant is a protein solution; the rotation speed of the centrifugal separation is preferably 8000~10000r / min, more preferably 9000r / min; the time of the centrifugal separation is preferably 20~40min, more preferably 30min; the temperature of the centrifugal separation is preferably 1~5°C, more preferably 4°C.

[0036] In the present invention, the source of apigenin is preferably Mongolian medicine Echinops cylindrica. The present invention has no special requirements on the method of extracting apigenin from Mongolian medicine Echinops cylindrica, and any extraction method known to those skilled in the art can be used.

[0037] In the present invention, the preparation method of apigenin preferably comprises: mixing Echinops blue powder (dried) with ethanol aqueous solution to extract and obtain an extract; concentrating the extract to obtain an extract; freeze-drying the extract to obtain a freeze-dried powder; re-dissolving the freeze-dried powder, and separating the obtained freeze-dried powder solution by high performance liquid chromatography-tandem mass spectrometry to obtain apigenin.

[0038] The present invention mixes the blue echinops powder (dried) with an ethanol aqueous solution for extraction to obtain an extract. In the present invention, the volume fraction of ethanol in the ethanol aqueous solution is preferably 30-100%, more preferably 50-75%; the amount ratio of the blue echinops powder (dry weight) to the ethanol solution is preferably 1kg:1-10L, more preferably 1kg:2-5L. In the present invention, the extraction temperature is preferably 25-80°C, more preferably 50-60°C; the extraction time is preferably 6-20 h, more preferably 12-20 h; the extraction is preferably carried out under stirring conditions.

[0039] After the extraction is completed, the present invention preferably further comprises performing solid-liquid separation on the obtained extraction system to obtain an extract. The present invention has no particular limitation on the solid-liquid separation, and a solid-liquid separation method well known to those skilled in the art can be used, such as filtration; the purpose of the solid-liquid separation is to remove residues.

[0040] After obtaining the extract, the present invention concentrates the extract to obtain an extract. In the present invention, the concentration preferably includes rotary evaporation; the rotary evaporation temperature is preferably 25-80°C, more preferably 60-80°C; the rotary evaporation time is preferably 2-10 hours, more preferably 4-5 hours.

[0041] After obtaining the extract, the present invention freeze-dries the extract to obtain freeze-dried powder. The present invention has no special limitation on the freeze-drying, as long as the solvent in the extract can be completely removed.

[0042] After obtaining the freeze-dried powder, the present invention re-dissolves the freeze-dried powder, and separates the obtained freeze-dried powder solution by high performance liquid chromatography-tandem mass spectrometry to obtain apigenin. In the present invention, the concentration of the freeze-dried powder solution is preferably 20-100 mg / mL, more preferably 50-80 mg / mL; the solvent in the freeze-dried powder solution preferably includes a lower alcohol aqueous solution, and the volume fraction of the lower alcohol in the lower alcohol aqueous solution is preferably 20-100%, more preferably 30-90%, and further preferably 40-50%; the lower alcohol preferably includes methanol and / or ethanol. In the present invention, the conditions for high performance liquid chromatography-tandem mass spectrometry separation preferably include: high performance liquid chromatography-tandem mass spectrometry separation conditions: using Vanquish UHPLC (Thermo Scientific, Waltham, MA) ultra-high performance liquid chromatography system combined with ACQUITY UPLC HSS T3 (2.1 mm × 100 mm, 1.8 µm) chromatographic column for separation, column temperature is 35°C; flow rate is 0.3 mL / min; mobile phase: mobile phase A is 0.1 v / v% formic acid aqueous solution, mobile phase B is 0.1 v / v% formic acid-acetonitrile; elution mode is gradient elution, and the gradient elution program is shown in Table 1. The Q-Exactive HFX mass spectrometer was used to collect the primary and secondary spectra of the samples. The Q-Exactive HFX mass spectrometer sample was connected to the UHPLC system. The electrospray ionization (ESI) positive and negative ion modes were used for mass spectrometry acquisition. The spray voltage was 3800V (ESI+) / 3500V (ESI-), the sheath gas pressure was 45arb, the auxiliary gas pressure was 20arb, the ion transfer tube temperature was 320℃, and the nebulization temperature was 350℃. The detection mode was full scan / data-dependent secondary scan (Full-MS / dd-MS2) mode. The primary and secondary resolutions were 60000 and 15000, respectively. The top 10 MS1 ions obtained MS / MS spectra and collision energies (CEs). The step normalization energy levels were 20, 40, and 60; the primary mass-to-charge ratio scanning range was 90~1300. 2 μL of EC-sp solution was accurately aspirated and analyzed by LC-MS injection. The EC-sp sample was injected 5 times.

[0043] Table 1 Gradient elution program

[0044]

[0045] In the present invention, the concentration of the apigenin solution is preferably 10 μmol / L to 10 mmol / L, more preferably 1 to 5 mmol / L, and further preferably 2 to 3 mmol / L. In the present invention, the solvent of the apigenin solution is preferably anhydrous ethanol.

[0046] In the present invention, apigenin has anti-inflammatory and antioxidant effects, can accelerate the rapid repair of the wound site; and can also effectively promote blood vessel regeneration.

[0047] In the present invention, the solvent in the polyvinyl alcohol solution is preferably water, and the concentration of the polyvinyl alcohol solution is preferably 0.1 to 5 wt %, more preferably 1 to 2 wt %.

[0048] In the present invention, the mixing of the silk fibroin solution, apigenin solution, polyvinyl alcohol solution and ethanol preferably comprises: mixing the silk fibroin solution and apigenin solution, and adding polyvinyl alcohol solution and ethanol. In the present invention, the ratio of the total mass of the silk fibroin solution and apigenin solution, the mass of the polyvinyl alcohol solution and the mass of ethanol is preferably 10:0.1~5:1~3, more preferably 10:0.15~1:1~2, and further preferably 10:0.2:1.

[0049] In the present invention, the self-assembly preferably includes freezing and thawing in sequence. In the present invention, the freezing temperature is preferably -60 to 25°C, more preferably -20°C; the freezing time is preferably 12 to 72 hours, more preferably 36 to 48 hours.

[0050] After the thawing is completed, the present invention preferably further comprises centrifuging the obtained thawing system, washing the obtained solid components and then drying them to obtain apigenin-silk fibroin nanoparticles. In the present invention, the speed of the centrifugation is preferably 8000-18000 r / min, more preferably 10000-12000 r / min; the time of the centrifugation is preferably 10-120 min, more preferably 30-100 min. In the present invention, the washing preferably comprises washing with deionized water 2-3 times. In the present invention, the drying preferably comprises freeze-drying, and the freeze-drying temperature is preferably -60-25°C, more preferably -20°C; the freeze-drying time is preferably 48-72 h.

[0051] The present invention provides a method for preparing a broad-spectrum antioxidant hydrogel, comprising the following steps:

[0052] The gelatin solution, the sodium alginate solution, the apigenin-loaded silk fibroin drug-loaded microspheres and the nanosilver solution are mixed to obtain a mixed solution; the apigenin-loaded silk fibroin drug-loaded microspheres are the apigenin-loaded silk fibroin drug-loaded microspheres described in the above technical solution or the apigenin-loaded silk fibroin drug-loaded microspheres prepared by the preparation method described in the above technical solution;

[0053] The mixed solution is molded to obtain a broad-spectrum antioxidant hydrogel.

[0054] The invention mixes gelatin solution, sodium alginate solution, apigenin-loaded silk fibroin drug-loaded microspheres and nano silver solution to obtain a mixed solution.

[0055] In the present invention, the concentration of the gelatin solution is preferably 0.1-20 wt %, more preferably 1-10 wt %. In the present invention, the gelatin solution is preferably heated to dissolve before use; the heating temperature is preferably 70-90° C., more preferably 80° C.

[0056] In the present invention, the concentration of the sodium alginate solution is preferably 0.1-5 wt%, more preferably 2-4 wt%. In the present invention, the volume ratio of the gelatin solution to the sodium alginate solution is preferably 1:0.1-5, more preferably 1:1-3. In the present invention, the ratio of the mass of the apigenin silk fibroin drug-loaded microspheres to the total volume of the gelatin solution and the sodium alginate solution is preferably 1-1000 mg:10 mL, more preferably 200-500 mg:10 mL.

[0057] In the present invention, the mass ratio of the nanosilver in the nanosilver solution to the apigenin silk fibroin drug-loaded microspheres is preferably 0.1-1:1, more preferably 0.2:1. In the present invention, the particle size of the nanosilver is preferably 30-151 nm. In the present invention, the concentration of the nanosilver solution is preferably 1-50 mg / mL, more preferably 2-10 mg / mL.

[0058] In the present invention, the method for preparing the nano silver solution preferably comprises: mixing silver nitrate, water, isopropanol and glucose, and performing a reduction reaction to obtain the nano silver solution.

[0059] In the present invention, the usage ratio of the silver nitrate to isopropanol is preferably 1:0.01-0.5, more preferably 1:0.1-0.2; the mass ratio of the silver nitrate to glucose is preferably 1:0.1-10, more preferably 1:1-2. In the present invention, the glucose is preferably in powder form.

[0060] In the present invention, the mixing of silver nitrate, water, isopropanol and glucose preferably includes: mixing silver nitrate and part of the water to obtain a silver nitrate solution; mixing glucose and the remaining water to obtain a glucose solution; heating the silver nitrate solution to the temperature of the reduction reaction, and sequentially adding isopropanol and glucose solution. In the present invention, the concentration of the silver nitrate solution is preferably 0.1 to 50 mg / mL, more preferably 10 to 30 mg / mL. In the present invention, the concentration of the glucose solution is preferably 10 to 200 mg / mL, more preferably 50 to 100 mg / mL.

[0061] In the present invention, the temperature of the reduction reaction is preferably 25-100° C., more preferably 50-80° C.; the time of the reduction reaction is preferably 30-300 min, more preferably 60-100 min.

[0062] In the present invention, after the reduction reaction, the obtained reaction system is preferably centrifuged to remove the aggregated nanosilver particles to obtain a nanosilver solution. In the present invention, the centrifugal speed is preferably 1000-5000 G, more preferably 2000-4000 G; the centrifugal time is preferably 1-30 min, more preferably 5-20 min.

[0063] Diabetic wounds are difficult to heal, and the sugar content in the wound microenvironment is relatively high, which can easily lead to infection. The present invention introduces broad-spectrum antibacterial nanosilver into the hydrogel dressing, making it antioxidant and anti-inflammatory while also having broad-spectrum antibacterial effects.

[0064] After obtaining the mixed solution, the present invention shapes the mixed solution to obtain a broad-spectrum antioxidant hydrogel. In the present invention, the molding preferably includes 3D printing. In the present invention, the conditions of the 3D printing preferably include: the barrel temperature is 85~145°C, the platform temperature is -10~0°C, the extrusion pressure is 0.3~0.8 MPa, the extrusion speed is 0.1~0.3 mm / s, the wire is advanced by 30~60 ms, the wire is closed in advance by 1~10 ms, and the broken wire is raised by 10~20 mm; the printing parameters preferably include: the printing model is cylindrical, linear filling is adopted, the filling spacing is 0.1~5 mm, the number of rotations is 80~100 times, and the rotation angle is 90°; the number of printed hydrogel layers is 10~20 layers, and the total thickness is 8~20 mm.

[0065] 3D printed scaffolds can be customized according to the different shapes and depths of skin injuries. The present invention utilizes the advantages of Mongolian medicine to combine the effective active ingredients in the Mongolian medicine blue spinulosa that promote angiogenesis with the gelatin-sodium alginate scaffold to prepare a skin repair scaffold containing angioactive ingredients. The implant can stimulate the body to produce and recruit growth factors required for bone repair and angiogenesis, reduce the negative impact of ROS on cells, and promote the rapid repair of difficult-to-heal wounds.

[0066] The present invention provides a broad-spectrum antioxidant hydrogel prepared by the preparation method described in the above technical scheme.

[0067] The present invention provides the use of the broad-spectrum antioxidant hydrogel described in the above technical solution in the preparation of a dressing, wherein the dressing preferably includes a diabetic skin ulcer dressing.

[0068] The technical solutions in the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0069] Example 1

[0070] Preparation of apigenin-loaded silk fibroin drug-loaded microspheres:

[0071] 1 kg of dried blue echinops powder was mixed with 2 L of 75% ethanol aqueous solution, heated to 60°C for mechanical stirring, extracted for 12 h, filtered, and the residue was removed. The obtained extract was rotary evaporated at 80°C for 4 h to evaporate the ethanol to obtain a drug extract, which was frozen at -20°C for 12 h and freeze-dried in a freeze dryer to obtain a freeze-dried powder. The freeze-dried powder was dissolved in a 40v / v% methanol aqueous solution, vortexed for 30 s, centrifuged at 16000 g and 4°C for 15 min, and the supernatant was a freeze-dried powder solution. The freeze-dried powder solution was separated by high performance liquid chromatography-tandem mass spectrometry to obtain apigenin. The solid-liquid ratio of the freeze-dried powder to the methanol aqueous solution was 20 mg: 0.3 mL.

[0072] HPLC-MS / MS separation conditions: Vanquish UHPLC (Thermo Scientific, Waltham, MA) ultra-high performance liquid chromatography system combined with ACQUITY UPLC HSS T3 (2.1 mm × 100 mm, 1.8 µm) column was used for separation. The column temperature was 35 °C. The flow rate was 0.3 mL / min. The mobile phases were: mobile phase A was 0.1 v / v% formic acid in water, and mobile phase B was 0.1 v / v% formic acid-acetonitrile. The elution mode was gradient elution. The gradient elution program is shown in Table 1. The Q-Exactive HFX mass spectrometer was used to collect the primary and secondary spectra of the samples. The Q-Exactive HFX mass spectrometer sample was connected to the UHPLC system. The electrospray ionization (ESI) positive and negative ion modes were used for mass spectrometry acquisition. The spray voltage was 3800V (ESI+) / 3500V (ESI-), the sheath gas pressure was 45arb, the auxiliary gas pressure was 20arb, the ion transfer tube temperature was 320℃, and the nebulization temperature was 350℃. The detection mode was full scan / data-dependent secondary scan (Full-MS / dd-MS2) mode. The primary and secondary resolutions were 60000 and 15000, respectively. The top 10 MS1 ions obtained MS / MS spectra and collision energies (CEs). The step normalization energy levels were 20, 40, and 60; the primary mass-to-charge ratio scanning range was 90~1300. 2 μL of EC-sp solution was accurately aspirated and analyzed by LC-MS injection. The EC-sp sample was injected 5 times.

[0073] 2 L of deionized water was added to a water bath, heated to 100 °C, and then 4.24 g of anhydrous sodium carbonate and 5 g of natural mulberry silk were added. The mixture was boiled for 30 min to remove the sericin in the natural silk thread, and stirred at intervals during the heating process. The de-sericin-free silk was repeatedly rinsed with deionized water and dried in an oven at 60 °C to obtain silk. According to the ratio of silk to lithium bromide solution of 27 g:100 mL, 9.3 mol / L lithium bromide solution was added to the silk, and the silk was sealed with plastic wrap and pierced with holes. The silk was dried at 60 °C for 4 h. The obtained silk solution was placed in a dialysis bag (with a molecular weight cutoff of 3500 Da), sealed at both ends, and dialyzed in deionized water for 3 d. The deionized water was replaced every 3 h during the dialysis process. The dialyzed silk fibroin solution was divided into 50 mL centrifuge tubes and centrifuged at a speed of 9000 r / min, a time of 30 min, and a temperature of 4 The mixture was centrifuged at ℃, the lower precipitate was discarded, the supernatant was collected to obtain a silk fibroin solution, and the solution was stored in a 4℃ refrigerator. The concentration of the silk fibroin solution was detected by a BCA total protein kit, and the concentration of the silk fibroin solution was 40 mg / mL.

[0074] Polyvinyl alcohol (PVA) with a molecular weight of 20,000 was placed in deionized water, heated in a water bath at 95° C., and stirred until completely dissolved to obtain a 2 wt % polyvinyl alcohol aqueous solution.

[0075] The self-assembly method was used to prepare silk fibroin drug-loaded microspheres loaded with apigenin. A 40 mg / mL silk fibroin solution and a 100 μM apigenin ethanol solution were mixed in a mass ratio of 10:1 to obtain a silk fibroin-apigenin mixed solution, and a 2 wt% PVA aqueous solution and anhydrous ethanol were added and mixed. The mixture was frozen at -20 °C for 48 h, thawed, centrifuged (12000 r / min, 30 min), the supernatant was discarded, the precipitate was collected, washed with deionized water 3 times, and freeze-dried at -20 °C to obtain silk fibroin drug-loaded microspheres loaded with apigenin. The mass ratio of silk fibroin-apigenin mixed solution, PVA aqueous solution and anhydrous ethanol was 10:2:1.

[0076] Preparation of nanosilver solution:

[0077] Weigh 50 mg of silver nitrate and dissolve it in 10 mL of deionized water, and stir it magnetically for 30 minutes; heat it to 80°C, add 1 mL of isopropanol, and continue stirring for 10 minutes to obtain a mixed solution of silver nitrate and isopropanol; dissolve 100 mg of glucose powder in 1 mL of deionized water, add it to the mixed solution of silver nitrate and isopropanol after it is fully dissolved, continue stirring for 60 minutes, and centrifuge it at 2000G for 5 minutes to remove the aggregated nanosilver particles to obtain a nanosilver solution.

[0078] Preparation of broad-spectrum antioxidant hydrogel:

[0079] 5 mL of 10 wt% gelatin solution was heated to 80°C. After fully dissolved, 5 mL of 2 wt% sodium alginate solution was added under mechanical stirring conditions, and stirring was continued at 80°C for 30 min; 200 mg of the apigenin silk fibroin drug-loaded microspheres were added, and stirring was continued for 60 min; 200 μL of the nanosilver solution was added, and stirring was continued at 80°C for 60 min to obtain a mixed solution; the bubbles in the mixed solution were eliminated, and the mixed solution was loaded into the 3D printer barrel (the barrel was preheated to 80°C in advance), and a 300 μm print nozzle was selected to complete the barrel loading; it was installed on the 3D bioprinter mechanical arm, the barrel temperature was set to 85°C, the platform temperature was set to 0°C, the extrusion pressure was 0.6 MPa, the extrusion speed was 0.1 mm / s, the wire was advanced 50 ms, the wire was closed 1 ms in advance, and the broken wire was raised 15 mm; the printing parameters were set as follows: the printing model was cylindrical, linear filling was used, the filling spacing was 1 mm, the number of rotations was 88 times, and the rotation angle was 90°. The number of printed hydrogel layers is 10 and the thickness is 8 mm.

[0080] Test Case

[0081] The characterization diagram of the nano silver solution prepared in Example 1 is as follows: Figure 1 As shown, A is a transmission electron microscope image, B is a nanosilver particle size distribution diagram, Figure 1 It can be seen that the particle size of nanosilver is between 30 and 151 nm.

[0082] The scanning electron microscopy image of the apigenin silk fibroin drug-loaded microspheres prepared in Example 1 is as follows: Figure 2 As shown, it can be seen that the apigenin-loaded silk fibroin drug-loaded microspheres form a topological structure, and the apigenin-loaded silk fibroin drug-loaded microspheres are spherical with a diameter between 200 and 500 nm.

[0083] The actual picture of the broad-spectrum antioxidant hydrogel prepared in Example 1 is as follows Figure 3 As shown by Figure 3 It can be seen that the hydrogel is loaded with apigenin silk fibroin drug-loaded microspheres.

[0084] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing an antioxidant hydrogel, comprising the following steps: The gelatin solution, the sodium alginate solution, the apigenin-loaded silk fibroin drug-loaded microspheres and the nanosilver solution are mixed to obtain a mixed solution; The apigenin-loaded silk fibroin drug-loaded microspheres have a core-shell structure, the shell layer includes silk fibroin and polyvinyl alcohol, and the core layer includes apigenin; the mass ratio of the silk fibroin to apigenin is 1:0.01-0.5; the mass ratio of the silk fibroin to polyvinyl alcohol is preferably 1:0.1-10; the molecular weight of the polyvinyl alcohol is 10000-20000; The mass ratio of the nanosilver in the nanosilver solution to the apigenin silk fibroin drug-loaded microspheres is 0.1-1:1; The mixed solution is molded to obtain a broad-spectrum antioxidant hydrogel.

2. The preparation method according to claim 1, characterized in that: The method for preparing the apigenin-loaded silk fibroin drug-loaded microspheres comprises the following steps: The silk fibroin solution, apigenin solution, polyvinyl alcohol solution and ethanol are mixed and self-assembled to obtain apigenin-silk fibroin nano drug-loaded microspheres; The ratio of the total mass of the silk fibroin solution and the apigenin solution, the mass of the polyvinyl alcohol solution and the mass of ethanol is 10:0.1-5:1-3.

3. The preparation method according to claim 1, characterized in that: The preparation method of the nano silver solution comprises: mixing silver nitrate, water, isopropanol and glucose, and performing a reduction reaction to obtain the nano silver solution.

4. The preparation method according to claim 1, characterized in that: The molding includes 3D printing.

5. The antioxidant hydrogel prepared by the preparation method according to any one of claims 1 to 4.

6. Use of the antioxidant hydrogel according to claim 5 in preparing a dressing.

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