CN119055828B

CN119055828B - Apigenin-loaded silk fibroin drug-loaded microspheres and preparation method thereof, broad-spectrum antioxidant hydrogel and preparation method and application thereof

Info

Publication number: CN119055828B
Application number: CN202411545321.XA
Authority: CN
Inventors: 钱智勇; 恩和吉日嘎拉; 李筱贺; 王海燕
Original assignee: Inner Mongolia Medical University
Current assignee: Inner Mongolia Medical University
Priority date: 2024-11-01
Filing date: 2024-11-01
Publication date: 2025-02-28
Anticipated expiration: 2044-11-01
Also published as: CN119055828A

Abstract

The invention provides apigenin-loaded silk fibroin drug-loaded microspheres and a preparation method thereof, broad-spectrum antioxidant hydrogel and a preparation method and application thereof, and relates to the technical field of composite materials. The apigenin-loaded silk fibroin drug-loaded microsphere provided by the invention has a core-shell structure, wherein the core layer comprises silk fibroin and polyvinyl alcohol, and the shell layer comprises apigenin. The apigenin-loaded silk fibroin drug-loaded microsphere provided by the invention can promote vascular regeneration and has high biological safety, so that wound repair difficult to heal due to diabetes is accelerated.

Description

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

Technical Field

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

Background

Diabetes is one of the world-wide high-rise diseases, seriously threatens the health of patients, and one of complications such as diabetic skin ulcer has become an important cause of global disability, brings great pain to patients, and seriously influences the life quality of the patients. Because the high-sugar microenvironment in the diabetes wound surface microenvironment influences the vascular endothelial cell function, the vascular damage, microcirculation dysfunction and the like seriously obstruct wound surface repair, the current method for clinically treating the diabetes wound surface is very limited (only by means of treatment such as surgical debridement, blood sugar control, skin grafting and the like), and the problem that the diabetes wound surface is difficult to heal is difficult to radically solve.

The neovasculature within skin tissue is a dynamic process starting from capillaries involving a variety of cells, substrates and bioactive factors together. The specific process includes activating vascular endothelial cell and degrading peripheral matrix, endothelial cell migration, capillary bud formation and proliferation. The new blood vessel can promote the formation of granulation tissue, provide oxygen and nutrient substances for the damaged area, and play an important role in the wound repair process. However, natural antioxidant enzymes are often related to protein and RNA molecules, which are susceptible to environmental factors, become inactive or lose antioxidant function under pathological conditions, allowing the accumulation of large amounts of reactive oxygen species (reactive oxygen species, ROS). Excessive ROS cause great damage to vascular endothelial cells and inhibit angiogenesis. The wound surface is difficult to heal due to the fact that nutrients and oxygen provided by blood vessels are not available, and the damaged tissue cannot be repaired, so that a high disability rate is caused. In summary, revascularization is a prerequisite for repair of diabetic skin lesions, which is not only a bridge for skin tissue nutrition and metabolism, but also a provider of the skin regeneration microenvironment.

The ideal diabetes skin injury repair bracket can be matched according to different conditions of skin injury, constructs a bionic microenvironment with optimal skin growth, and can promote blood vessels and bone tissue regeneration under oxidative stress. Therefore, the personalized preparation of the large-scale skin substitute capable of having a functionalized vascular system under the oxidative stress state is a difficult problem which needs to be solved urgently for repairing the skin injury of diabetes at present.

Disclosure of Invention

In view of the above, the invention aims to provide apigenin-loaded silk fibroin drug-loaded microspheres, a preparation method thereof, broad-spectrum antioxidant hydrogel, a preparation method thereof and application thereof.

In order to achieve the above object, the present invention provides the following technical solutions:

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

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

The invention provides a preparation method of apigenin-loaded silk fibroin drug-loaded microspheres, which comprises the following steps:

and mixing the silk fibroin solution, the apigenin solution, the polyvinyl alcohol solution and the ethanol, and performing self-assembly to obtain the apigenin-silk fibroin nano drug-loaded microsphere.

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

The invention provides a preparation method of broad-spectrum antioxidant hydrogel, which comprises the following steps:

Mixing gelatin solution, sodium alginate solution, apigenin-loaded silk fibroin drug-loaded microspheres and nano silver solution to obtain a mixed solution, wherein the apigenin-loaded silk fibroin drug-loaded microspheres are prepared by the apigenin-loaded silk fibroin drug-loaded microspheres according to the technical scheme or the preparation method of the technical scheme;

And (3) molding the mixed solution to obtain the broad-spectrum antioxidation hydrogel.

Preferably, the mass ratio of the nano silver in the nano silver solution to the apigenin-loaded silk fibroin drug-loaded microsphere is 0.1-1:1.

Preferably, the preparation method of the nano silver solution comprises the steps of mixing silver nitrate, water, isopropanol and glucose, and carrying out reduction reaction to obtain the nano silver solution.

Preferably, the shaping comprises 3D printing.

The invention provides the broad-spectrum antioxidation hydrogel prepared by the preparation method.

The invention provides application of the broad-spectrum antioxidant hydrogel in preparing dressing.

Theinventionprovidesaapigenin-loadedsilkfibroindrug-loadedmicrosphere(SFM-Aforshort),whereinsilkfibroincanbeself-assembledintonanomicrospheres,canbedissolvedinwaterafterbeingoxidizedbyROS,andiswrappedbysilkfibroinself-assemblytechnologytoprepareananodrug-loadedsystem.

The invention provides a preparation method of the apigenin-loaded silk fibroin drug-loaded microsphere. The invention wraps apigenin with silk fibroin to prepare the drug-loaded nano microsphere by a self-assembly technology, has simple operation, mild reaction conditions and low cost, and is suitable for industrial production. The apigenin-loaded fibroin drug-loaded microsphere prepared by the invention can promote vascular regeneration and has high biological safety, thereby accelerating wound repair difficult to heal due to diabetes.

The invention provides a preparation method of broad-spectrum antioxidant hydrogel, which is simple to operate, mild in reaction condition, low in cost and suitable for industrial production. The hydrogel broad-spectrum antioxidation hydrogel prepared by the invention contains a vasogenic active ingredient, eliminates excessive ROS in cells under the condition of not interfering endogenous antioxidation, rebuilds redox steady state, avoids damage to cells, can effectively promote blood vessel regeneration in diabetic skin tissues, accelerates healing of diabetic wounds, has application potential as a wound hydrogel material difficult to heal for diabetes, and provides a new treatment thought and clinical therapy for wound damage repair difficult to heal for diabetes. In addition, the broad-spectrum antioxidant hydrogel prepared by the invention has good degradability and antibacterial property.

Furthermore, the 3D printing technology is used for preparing the broad-spectrum antioxidant hydrogel, so that the shape and the size of the broad-spectrum antioxidant hydrogel can be designed and manufactured with high precision, the structural characteristics can be controlled on a microscopic level, and the prepared broad-spectrum antioxidant hydrogel has application potential as a wound hydrogel material difficult to heal for diabetes, and provides a new treatment idea and clinical therapy for wound injury repair difficult to heal for diabetes.

Drawings

FIG. 1 is a representation of the nano-silver solution prepared in example 1, wherein A is a transmission electron microscope image and B is a nano-silver particle size distribution diagram;

FIG. 2 is a scanning electron microscope image of apigenin-loaded silk fibroin drug-loaded microspheres prepared in example 1;

FIG. 3 is a physical diagram of the broad-spectrum antioxidant hydrogel prepared in example 1;

FIG. 4 is a schematic structural diagram of apigenin-loaded silk fibroin drug-loaded microspheres of the invention.

Detailed Description

The invention provides a apigenin-loaded silk fibroin drug-loaded microsphere, the structural schematic diagram of which is shown in figure 4, and the microsphere has a core-shell structure, wherein a core layer comprises apigenin, and a shell layer comprises silk fibroin and polyvinyl alcohol.

In the invention, the silk fibroin prepared microsphere is oxidized by ROS and becomes a random coiled structure to be dissolved in water, and the invention wraps apigenin by the silk fibroin for treating the wound surface of diabetes, when the drug-loaded microsphere encounters a large amount of ROS, the oxidized microsphere is dissolved to release apigenin, a large amount of ROS on the wound surface is removed, and the apigenin plays a role in promoting vascular regeneration, so that the repair of the wound surface injury of diabetes is accelerated. According to the invention, the ROS content of the microenvironment of the diabetic skin ulcer is regulated through apigenin, so that the vascular regeneration of the damaged part is promoted, and the damage repair of the diabetic refractory ulcer is accelerated.

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

In the invention, the mass ratio of the silk fibroin to the polyvinyl alcohol is preferably 1:0.1-10, and more preferably 1:4. In the present invention, the molecular weight of the polyvinyl alcohol is preferably 10000 to 200000, more preferably 20000.

In the invention, the diameter of the apigenin-loaded silk fibroin drug-loaded microsphere is preferably 200-500 nm.

The invention provides a preparation method of apigenin-loaded silk fibroin drug-loaded microspheres, which comprises the following steps of mixing a silk fibroin solution, a apigenin solution, a polyvinyl alcohol solution and ethanol, and performing self-assembly to obtain apigenin-silk fibroin drug-loaded microspheres.

In the present invention, the concentration of the silk fibroin solution is preferably 1 to 100mg/mL, more preferably 10 to 60 mg/mL, and even more preferably 40mg/mL. The method for preparing the silk fibroin solution is not particularly limited, and can be prepared by methods well known to those skilled in the art.

In the specific embodiment of the invention, the preparation method of the silk fibroin solution preferably comprises the steps of degumming natural mulberry silk threads to obtain silk, dissolving the silk in a solvent to obtain a mixed solution, and sequentially carrying out dialysis treatment and purification treatment on the mixed solution to obtain the silk fibroin solution. In the present invention, the degumming preferably comprises mixing natural silkworm silk, sodium carbonate and boiling water and boiling. In the invention, the mass ratio of the natural mulberry silk thread to the sodium carbonate is preferably 1:0.5-1.5, and more preferably 1:0.8-1. In the invention, the boiling time is preferably 20-40 min, more preferably 30min, and the boiling is preferably performed under stirring. The degumming method preferably further comprises the step of washing the degummed silk with water and then drying to obtain the silk. In the invention, the solvent preferably comprises 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 comprises calcium chloride, ethanol and water, and the molar ratio of the calcium chloride to the ethanol to the water is preferably 1:2:8. In the invention, the dissolution temperature is preferably 50-70 ℃, more preferably 60 ℃, and the dissolution time is preferably 3-5 h, more preferably 4h. In the invention, the dialysis treatment preferably comprises water dialysis, the molecular weight cut-off of a dialysis bag adopted in the dialysis treatment is preferably 3500Da, the dialysis time is preferably 2-4 d, more preferably 3d, the water is preferably replaced every 2-4 h (more preferably 3 h) in the dialysis treatment process, and the water preferably comprises deionized water. In the invention, the purification preferably comprises centrifugal separation, wherein the supernatant is a protein solution, the rotating speed of the centrifugal separation is preferably 8000-10000 r/min, more preferably 9000r/min, the time of the centrifugal separation is preferably 20-40 min, more preferably 30min, and the temperature of the centrifugal separation is preferably 1-5 ℃ and more preferably 4 ℃.

In the present invention, the source of apigenin is preferably the Mongolian medicine blue-thorn head. The method for extracting apigenin from Mongolian medicine prinsepia utilis royle has no special requirements, and extraction methods well known to those skilled in the art can be adopted.

The preparation method of apigenin preferably comprises the steps of mixing and extracting bluish bone powder (dried) with ethanol water solution to obtain an extracting solution, concentrating the extracting solution to obtain an extract, freeze-drying the extract to obtain 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.

The invention mixes the bluish-thorn head powder (dried) with ethanol water solution for extraction to obtain extract. In the invention, the volume fraction of the ethanol in the ethanol aqueous solution is preferably 30-100%, more preferably 50-75%, and the dosage ratio of the bluish-thorn head powder (dry weight) to the ethanol solution is preferably 1 kg:1-10L, more preferably 1 kg:2-5L. In the invention, the extraction temperature is preferably 25-80 ℃, more preferably 50-60 ℃, the extraction time is preferably 6-20 h, more preferably 12-20 h, and the extraction is preferably carried out under stirring.

After the extraction is completed, the invention preferably further comprises solid-liquid separation of the obtained extraction system to obtain an extraction solution. The solid-liquid separation is not particularly limited, and may be performed by a solid-liquid separation method known to those skilled in the art, specifically, filtration, and the purpose of the solid-liquid separation is to remove the residue.

After the extracting solution is obtained, the extracting solution is concentrated to obtain extractum. In the invention, the concentration preferably comprises rotary steaming, the temperature of the rotary steaming is preferably 25-80 ℃, more preferably 60-80 ℃, and the time of the rotary steaming is preferably 2-10 h, more preferably 4-5 h.

After the extract is obtained, the extract is freeze-dried to obtain freeze-dried powder. The present invention is not particularly limited to the above-mentioned lyophilization, and the solvent in the extract may be completely removed.

After the freeze-dried powder is obtained, the freeze-dried powder is redissolved, and the obtained freeze-dried powder solution is subjected to high performance liquid chromatography-tandem mass spectrometry to obtain apigenin. In the 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 comprises a lower alcohol aqueous solution, the volume fraction of lower alcohol in the lower alcohol aqueous solution is preferably 20-100%, more preferably 30-90%, further preferably 40-50%, and the lower alcohol preferably comprises methanol and/or ethanol. In the invention, the conditions for high performance liquid chromatography-tandem mass spectrometry preferably comprise high performance liquid chromatography-tandem mass spectrometry separation conditions, wherein a Vanquish UHPLC (Thermo Scientific, waltham, mass.) ultra-high performance liquid chromatography system is combined with a ACQUITY UPLC HSS T3 (2.1 mm multiplied by 100 mm, 1.8 mu m) chromatographic column for separation, the column temperature is 35 ℃, the flow rate is 0.3 mL/min, the mobile phase is 0.1v/v% formic acid aqueous solution, the mobile phase B is 0.1v/v% formic acid-acetonitrile, the elution mode is gradient elution, and the gradient elution program is shown in Table 1. The method comprises the steps of collecting a primary spectrum and a secondary spectrum of a sample by adopting a Q-Exactive HFX mass spectrometer, combining the sample of the Q-Exactive HFX mass spectrometer with a UHPLC system, respectively collecting mass spectra by adopting an electrospray ionization (ESI) positive ion mode and an electrospray ionization negative ion mode, wherein the spray voltage is 3800V (ESI+)/3500V (ESI-), the sheath gas pressure is 45arb, the auxiliary gas pressure is 20arb, the temperature of an ion transmission tube is 320 ℃, the atomization temperature is 350 ℃, the detection mode is a Full-scanning/data-dependent secondary scanning (Full-MS/dd-MS 2) mode, the primary resolution and the secondary resolution are 60000 and 15000, and the top 10 MS1 ion obtains an MS/MS spectrum and Collision Energy (CEs). The step normalization energy levels 20, 40 and 60 are adopted, and the scanning range of the primary mass-to-charge ratio is 90-1300. 2 mu L of EC-sp solution is precisely sucked, LC-MS sample injection analysis is carried out, and EC-sp samples are repeatedly injected for 5 times.

TABLE 1 gradient elution procedure

In the invention, the concentration of the apigenin solution is preferably 10 mu mol/L-10 mmol/L, more preferably 1-5 mmol/L, and even more preferably 2-3 mmol/L. In the present invention, the solvent of the apigenin solution is preferably absolute ethanol.

In the invention, apigenin has anti-inflammatory and antioxidant effects, can accelerate the rapid repair of wound parts, and can effectively promote the regeneration of blood vessels.

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 5wt%, more preferably 1 to 2 wt%.

In the present invention, the mixing of the silk fibroin solution, the apigenin solution, the polyvinyl alcohol solution and the ethanol preferably comprises mixing the silk fibroin solution and the apigenin solution, and adding the polyvinyl alcohol solution and the ethanol. In the invention, 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 the ethanol is preferably 10:0.1-5:1-3, more preferably 10:0.15-1:1-2, and even more preferably 10:0.2:1.

In the present invention, the self-assembly preferably includes sequentially freezing and thawing. In the invention, the freezing temperature is preferably-60-25 ℃, more preferably-20 ℃, and the freezing time is preferably 12-72 hours, more preferably 36-48 hours.

After the thawing is finished, the invention preferably further comprises the steps of centrifugally separating the thawing system, washing and drying the solid component to obtain the apigenin-silk fibroin nano drug-loaded microsphere. In the invention, the rotation speed of the centrifugation is preferably 8000-18000 r/min, more preferably 10000-12000 r/min, and the time of the centrifugation is preferably 10-120 min, more preferably 30-100 min. In the present invention, the washing preferably includes washing with deionized water 2 to 3 times. In the invention, the drying preferably comprises freeze-drying, wherein the temperature of the freeze-drying is preferably-60-25 ℃, more preferably-20 ℃, and the time of the freeze-drying is preferably 48-72 h.

The invention mixes gelatin solution, sodium alginate solution, apigenin-loaded silk fibroin drug-loaded microsphere and nano silver solution to obtain mixed solution.

In the present invention, the concentration of the gelatin solution is preferably 0.1 to 20 wt%, more preferably 1 to 10 wt%. In the invention, the gelatin solution is preferably heated to be dissolved before use, and the heating temperature is preferably 70-90 ℃, more preferably 80 ℃.

In the invention, the concentration of the sodium alginate solution is preferably 0.1-5 wt%, more preferably 2-4 wt%. In the invention, the volume ratio of the gelatin solution to the sodium alginate solution is preferably 1:0.1-5, and more preferably 1:1-3. In the invention, the ratio of the mass of the apigenin-loaded silk fibroin drug-loaded microsphere 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.

In the invention, the mass ratio of the nano silver in the nano silver solution to the apigenin-loaded silk fibroin drug-loaded microsphere is preferably 0.1-1:1, and more preferably 0.2:1. In the invention, the particle size of the nano silver is preferably 30-151 nm. In the invention, the concentration of the nano silver solution is preferably 1-50 mg/mL, more preferably 2-10 mg/mL.

In the invention, the preparation method of the nano silver solution preferably comprises the steps of mixing silver nitrate, water, isopropanol and glucose, and carrying out reduction reaction to obtain the nano silver solution.

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

In the invention, the mixing of the silver nitrate, the water, the isopropanol and the glucose preferably comprises the steps of mixing the silver nitrate with part of the water to obtain a silver nitrate solution, mixing the glucose with the rest of the water to obtain a glucose solution, heating the silver nitrate solution to the temperature of a reduction reaction, and sequentially adding the isopropanol and the 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.

In the invention, the temperature of the reduction reaction is preferably 25-100 ℃, more preferably 50-80 ℃, and the time of the reduction reaction is preferably 30-300 min, more preferably 60-100 min.

After the reduction reaction, the obtained reaction system is centrifuged to remove the aggregated nano silver particles, so as to obtain the nano silver solution. In the invention, the rotation speed of the centrifugation is preferably 1000-5000G, more preferably 2000-4000G, and the centrifugation time is preferably 1-30 min, more preferably 5-20 min.

The wound surface of diabetes is difficult to heal, the content of sugar in the microenvironment of the wound surface is high, and the infection is easy to cause.

After the mixed solution is obtained, the mixed solution is molded to obtain the broad-spectrum antioxidation hydrogel. In the present invention, the molding preferably includes 3D printing. In the invention, the 3D printing conditions preferably comprise a charging barrel temperature of 85-145 ℃, a platform temperature of-10-0 ℃, an extrusion pressure of 0.3-0.8 MPa, an extrusion speed of 0.1-0.3 mm/s, a wire discharge advance of 30-60 ms, a wire closing advance of 1-10 ms, a wire breakage elevation of 10-20 mm, printing parameters preferably comprise a cylindrical printing model, linear filling, a filling interval of 0.1-5 mm, a rotation time of 80-100 times, a rotation angle of 90 degrees, a number of layers of printing hydrogel of 10-20 layers and a total thickness of 8-20 mm.

The 3D printing bracket can be customized in an individualized way according to different shapes and different depths of skin injury, and the invention combines the effective active components for promoting angiogenesis in Mongolian medicine blue thorn head with the gelatin-sodium alginate bracket to prepare the skin repair bracket containing the active components for promoting the angiogenesis, so that the implant can stimulate the organism to generate and recruit growth factors required by bone repair and angiogenesis, reduce the negative influence of ROS on cells and promote the rapid repair of wound surfaces difficult to heal.

The invention provides application of the broad-spectrum antioxidant hydrogel in preparing a dressing, wherein the dressing preferably comprises a dressing for treating diabetic skin ulcer.

The technical solutions of the present invention will be clearly and completely described in the following in connection with the embodiments of the present invention. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Example 1

Preparing apigenin silk fibroin loaded drug-loaded microspheres:

Mixing 1 kg dry blue thorn head powder with 75% ethanol water solution 2L, heating to 60deg.C, mechanically stirring, extracting 12h, filtering, removing residue, steaming the obtained extractive solution at 80deg.C for 4 hr, volatilizing ethanol to obtain medicinal extract, freezing at-20deg.C 12h, and lyophilizing with a lyophilizing machine to obtain lyophilized powder. Dissolving the freeze-dried powder in 40v/v% methanol water solution, carrying out vortex for 30 s, centrifuging for 15 min at the temperature of 16000 g and 4 ℃, obtaining a supernatant as a freeze-dried powder solution, and carrying out high performance liquid chromatography-tandem mass spectrometry separation on the freeze-dried powder solution to obtain apigenin. Wherein, freeze-dried powder and methanol aqueous solution the solid-to-liquid ratio of (C) was 20mg:0.3mL.

The conditions of high performance liquid chromatography-tandem mass spectrometry include that a Vanquish UHPLC (Thermo Scientific, waltham, MA) ultra-high performance liquid chromatography system is combined with a ACQUITY UPLC HSS T (2.1 mm multiplied by 100mm, 1.8 μm) chromatographic column for separation, the column temperature is 35 ℃, the flow rate is 0.3 mL/min, the mobile phase is 0.1v/v% formic acid aqueous solution of mobile phase A, 0.1v/v% formic acid-acetonitrile of mobile phase B, the elution mode is gradient elution, and the gradient elution program is shown in Table 1. The method comprises the steps of collecting a primary spectrum and a secondary spectrum of a sample by adopting a Q-Exactive HFX mass spectrometer, combining the sample of the Q-Exactive HFX mass spectrometer with a UHPLC system, respectively collecting mass spectra by adopting an electrospray ionization (ESI) positive ion mode and an electrospray ionization negative ion mode, wherein the spray voltage is 3800V (ESI+)/3500V (ESI-), the sheath gas pressure is 45arb, the auxiliary gas pressure is 20arb, the temperature of an ion transmission tube is 320 ℃, the atomization temperature is 350 ℃, the detection mode is a Full-scanning/data-dependent secondary scanning (Full-MS/dd-MS 2) mode, the primary resolution and the secondary resolution are 60000 and 15000, and the top 10 MS1 ion obtains an MS/MS spectrum and Collision Energy (CEs). The step normalization energy levels 20, 40 and 60 are adopted, and the scanning range of the primary mass-to-charge ratio is 90-1300. 2 mu L of EC-sp solution is precisely sucked, LC-MS sample injection analysis is carried out, and EC-sp samples are repeatedly injected for 5 times.

Adding 2.24 g anhydrous sodium carbonate and natural silkworm silk thread 5g into a water bath kettle, heating to 100 ℃, adding 4.24 g anhydrous sodium carbonate and natural silkworm silk thread, boiling 30min to remove sericin in the natural silkworm silk thread, stirring in the middle of the heating process, repeatedly washing the removed silk with deionized water, putting into a baking oven at 60 ℃ to dry to obtain silk, adding 9.3 mol/L lithium bromide solution into the silk according to the ratio of the silk to lithium bromide solution of 27 g to 100mL, sealing preservative film and puncturing holes, continuously baking at 60 ℃ to 4h, filling the obtained silk solution into a dialysis bag (dialysis bag with molecular weight cutoff of 3500 Da), sealing two ends, putting into deionized water for dialysis of 3d, replacing deionized water every 3 h in the dialysis process, filling the dialyzed silk protein solution into a centrifuge tube of 50mL, centrifuging under the conditions that the centrifugal speed is 9000 r/min and the temperature is 30min, discarding the lower layer of sediment, collecting the upper layer sediment, putting the silk protein solution into a refrigerator, and detecting the concentration of the silk protein solution of which is 40 ℃ to obtain the total concentration of the silk protein solution, and storing the total concentration of the silk protein solution in a BCA box of 40.

Polyvinyl alcohol (PVA) with molecular weight of 20000 is placed in deionized water, and stirred under the condition of heating in a water bath at 95 ℃ until the PVA is completely dissolved, so as to obtain a 2 wt% polyvinyl alcohol aqueous solution.

The preparation of the apigenin-loaded silk fibroin drug-loaded microsphere is carried out by adopting a self-assembly method, silk fibroin solution with the concentration of 40 mg/mL and apigenin ethanol solution with the concentration of 100 mu M are mixed according to the mass ratio of 10:1 to obtain a silk fibroin-apigenin mixed solution, PVA aqueous solution with the concentration of 2 wt% and absolute ethanol are added to be mixed, the mixture is frozen for 48 and h at the temperature of minus 20 ℃, the mixture is thawed and centrifuged (12000 r/min and 30 min), the supernatant is discarded, the precipitate is collected and washed for 3 times by deionized water, and the mixture is freeze-dried at the temperature of minus 20 ℃ to obtain the apigenin-loaded silk fibroin drug-loaded microsphere. Wherein the mass ratio of the silk fibroin-apigenin mixed solution, the PVA aqueous solution and the absolute ethyl alcohol is 10:2:1.

Preparing a nano silver solution:

Weighing 50mg of silver nitrate, dissolving in 10mL of deionized water, magnetically stirring for 30min, heating to 80 ℃, adding 1mL of isopropanol, continuously stirring for 10min to obtain a mixed solution of silver nitrate and isopropanol, dissolving 100mg of glucose powder in 1mL of deionized water, fully dissolving, adding the dissolved glucose powder into the mixed solution of silver nitrate and isopropanol, continuously stirring for 60min, centrifuging for 5min in a centrifuge 2000G, and removing aggregated nano silver particles to obtain a nano silver solution.

Preparing broad-spectrum antioxidation hydrogel:

Heating 5mL wt% gelatin solution to 80 ℃, fully dissolving, adding 5mL sodium alginate solution with 2wt% gelatin solution under mechanical stirring conditions, continuously stirring for 30min under 80 ℃, adding 200mg apigenin-loaded silk fibroin drug-loaded microspheres, continuously stirring for 60min, adding 200 mu L nano silver solution, continuously stirring for 60min under 80 ℃ to obtain mixed solution, eliminating bubbles in the mixed solution, loading the mixed solution into a 3D printer cylinder (the cylinder is preheated at 80 ℃ in advance), selecting a 300 mu m printing spray head, completing the cylinder loading, mounting the mixed solution onto a three-dimensional bio-printer mechanical arm, setting the temperature of the cylinder to 85 ℃, setting the platform temperature to 0 ℃ and the extrusion pressure to 0.6MPa, and the extrusion speed to 0.1mm/s, and starting silk feeding for 50ms in advance, closing for 1ms and lifting the silk breakage for 15mm, wherein the printing parameters are set as follows, the printing model is cylindrical, linear filling is adopted, the filling interval is 1mm, the rotation time is 88 times, and the rotation angle is 90 ℃. The number of layers of the printed hydrogel was 10 and the thickness was 8 mm.

Test case

The characterization diagram of the nano silver solution prepared in the embodiment 1 is shown in fig. 1, wherein a is a transmission electron microscope diagram, B is a nano silver particle size distribution diagram, and as can be seen from fig. 1, the nano silver particle size is 30-151 nm.

The scanning electron microscope image of the apigenin-loaded silk fibroin drug-loaded microsphere prepared in the embodiment 1 is shown in fig. 2, and it can be seen that the apigenin-loaded silk fibroin drug-loaded microsphere forms a topological structure, and the apigenin-loaded silk fibroin drug-loaded microsphere is spherical and has a diameter of 200-500 nm.

A physical diagram of the broad-spectrum antioxidant hydrogel prepared in the example 1 is shown in fig. 3, and as can be seen from fig. 3, the hydrogel is provided with apigenin silk fibroin loaded drug-carrying microspheres.

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope 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 the apigenin is 1:0.01-0.5; the mass ratio of the silk fibroin to the 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 preparation method of 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 is 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 is characterized in that the molding comprises 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.