Multifunctional hydrogel co-loaded with radix astragali-radix salviae miltiorrhizae components and preparation method of multifunctional hydrogel

Abstract

The invention belongs to the technical field of high polymer materials, and particularly relates to multifunctional hydrogel co-loaded with radix astragali-radix salviae miltiorrhizae components and a preparation method of the multifunctional hydrogel. The preparation method comprises the following steps: dissolving methacrylated silk fibroin and methacrylated astragalus polysaccharide in water containing a photoinitiator to obtain an SFMA-APMA hydrogel solution, then adding tanshinone IIA-loaded nano-micelles, uniformly mixing to obtain an SFMA-APMA-TSIIA-coated HA-PPS hydrogel solution, and irradiating the SFMA-APMA-TSIIA-coated HA-PPS hydrogel solution under ultraviolet light for 2-4 minutes to obtain the multifunctional hydrogel co-loaded with astragalus-salvia miltiorrhiza components. The preparation method comprises the following steps: grafting polypropylene sulfide and hyaluronic acid to form an amphiphilic nano-micelle, loading tanshinone IIA into an inner core of the micelle by utilizing a hydrophobic effect, and loading the nano-micelle into double-network hydrogel formed by cross-linking methylacryloylated astragalus polysaccharide and methylacryloylated silk fibroin. Local medicine storage and targeted slow release of the active ingredients of the traditional Chinese medicine can be realized.

Description

Technical Field

[0001] The invention belongs to the technical field of polymer materials, and particularly relates to a multifunctional hydrogel co-loaded with astragalus and salvia miltiorrhiza components and a preparation method thereof. Background Art

[0002] Hydrogels have good water retention and biocompatibility and have great prospects for use in wound dressings. However, the hydrogel materials currently used in clinical practice are inactive and often need to be loaded with multiple drugs during treatment, which affects the mechanical strength, water retention and other properties of the hydrogels, and the drug release effect is also unsatisfactory.

[0003] Traditional Chinese medicine, with its unique advantages of multiple components, multiple targets, and multiple pathways, exhibits the characteristics of holistic regulation in the treatment of diseases. Among them, Qi deficiency and blood stasis syndrome, as an important syndrome in the TCM differentiation system, is treated with the basic principle of invigorating Qi and activating blood circulation, and Astragalus (a traditional Chinese medicine for invigorating Qi) and Salvia miltiorrhiza (a traditional Chinese medicine for activating blood circulation) are the core drug pair. Astragalus polysaccharide, the main active ingredient of Astragalus, and tanshinone IIA, the main active ingredient of Salvia miltiorrhiza, have anti-inflammatory and antioxidant effects, but due to the influence of their physical and chemical properties, their release effect in hydrogels is not ideal, their bioavailability is low, and their clinical efficacy still needs to be improved. Therefore, there is an urgent need to explore an efficient drug delivery strategy for Astragalus polysaccharide and Tanshinone IIA. Summary of the Invention

[0004] The first object of the present invention is to provide a method for preparing a multifunctional hydrogel loaded with astragalus polysaccharide. The second object of the present invention is to provide a multifunctional hydrogel loaded with astragalus polysaccharide obtained by the preparation method. The third object of the present invention is to provide a method for preparing a multifunctional hydrogel co-loaded with astragalus and salvia miltiorrhiza components. The fourth object of the present invention is to provide a multifunctional hydrogel co-loaded with astragalus and salvia miltiorrhiza components obtained by the preparation method.

[0005] According to a first aspect of the present invention, a method for preparing a multifunctional hydrogel loaded with astragalus polysaccharide is provided, comprising the following steps:

[0006] Dissolving methacrylated silk fibroin and methacrylated astragalus polysaccharide in a first water containing a photoinitiator to obtain an SFMA-APMA hydrogel solution, and then irradiating the SFMA-APMA hydrogel solution under ultraviolet light for 2-4 minutes to obtain;

[0007] The preparation method of methacryloylated astragalus polysaccharide comprises the following steps:

[0008] Astragalus polysaccharide is dissolved in the second water, methacrylic anhydride is added at 45-55°C, and then stirred and reacted at 5-15°C for 12-24 hours. The pH of the reaction system is then adjusted to 9.5-10.5, and the stirring reaction is continued for 12-24 hours. The resulting solution is then dialyzed and subsequently freeze-dried to obtain the product. Thus, the present invention obtains methacrylated astragalus polysaccharide by acylation of the astragalus polysaccharide with the anhydride groups of the methacrylic anhydride and the amino groups in the astragalus polysaccharide molecules.

[0009] After absorbing ultraviolet light energy, the photoinitiator decomposes to produce free radicals. These free radicals trigger a free radical polymerization reaction of the methacryloyl (-C=C-) double bonds in the molecular structure of the methacrylated material, forming covalent crosslinks between the molecular chains of the methacrylated material. As the crosslinking reaction proceeds, the molecular chains of the methacrylated material connect with each other, forming a hydrogel with a three-dimensional network structure. The present invention prepares methacrylated astragalus polysaccharide by methacrylation, and then crosslinks the methacrylated astragalus polysaccharide with methacrylated silk fibroin to form a double-network hydrogel, which can achieve long-term release of the astragalus polysaccharide.

[0010] In some embodiments, the ratio of the mass of astragalus polysaccharide to the volume of the second water is 1:100, and the ratio of the mass of astragalus polysaccharide to the volume of methacrylic anhydride is 1:3.

[0011] In some embodiments, the photoinitiator is a LAP photoinitiator.

[0012] In some embodiments, based on parts by mass, the amount of methacryloylated silk fibroin is 0.1-0.3 parts, the amount of methacryloylated astragalus polysaccharide is 0.01-0.1 parts, the amount of photoinitiator is 0.001-0.0025 parts, and the amount of the first water is 0.5-2 parts.

[0013] According to a second aspect of the present invention, there is provided a multifunctional hydrogel loaded with astragalus polysaccharide prepared by the above-mentioned preparation method.

[0014] According to a third aspect of the present invention, a method for preparing a multifunctional hydrogel co-loaded with Astragalus and Salvia miltiorrhiza components is provided, comprising the following steps:

[0015] Methacryloylated silk fibroin and methacryloylated astragalus polysaccharide were dissolved in a third water containing a photoinitiator to obtain an SFMA-APMA hydrogel solution, and then the nanomicelles loaded with tanshinone IIA were added and mixed evenly to obtain an SFMA-APMA-TSIIA@HA-PPS hydrogel solution, which was then irradiated under ultraviolet light for 2-4 minutes.

[0016] The preparation method of methacryloylated astragalus polysaccharide comprises the following steps:

[0017] Astragalus polysaccharide is dissolved in the fourth water, methacrylic anhydride is added at 45-55° C., and then stirred and reacted at 5-15° C. for 12-24 hours. Thereafter, the pH of the reaction system is adjusted to 9.5-10.5, and the stirring reaction is continued for 12-24 hours. The resulting solution is then dialyzed and subsequently freeze-dried to obtain the product.

[0018] In some embodiments, the ratio of the mass of astragalus polysaccharide to the volume of the fourth water is 1:100, and the ratio of the mass of astragalus polysaccharide to the volume of methacrylic anhydride is 1:3.

[0019] In some embodiments, the photoinitiator is a LAP photoinitiator.

[0020] In some embodiments, based on parts by mass, the amount of methacryloylated silk fibroin is 0.1-0.3 parts, the amount of methacryloylated astragalus polysaccharide is 0.01-0.1 parts, the amount of photoinitiator is 0.001-0.0025 parts, the amount of the third water is 0.5-2 parts, and the amount of nanomicelles loaded with tanshinone IIA is 0.05-0.15 parts.

[0021] In some embodiments, the nanomicelles loaded with Tanshinone IIA are obtained by grafting polypropylene sulfide and hyaluronic acid to form amphiphilic nanomicelles, and then loading Tanshinone IIA into the core of the micelles by utilizing hydrophobic interaction.

[0022] In some embodiments, the method for preparing nanomicelles loaded with tanshinone IIA comprises the following steps:

[0023] (1) Under ice bath conditions, 30 mL of anhydrous tetrahydrofuran was first added, followed by 100 μL of 3-mercaptopropionic acid, and the mixture was stirred and mixed. 524 μL of 1,8-diazabicyclo[5.4.0]undec-7-ene was then added, and the mixture was stirred for 30 min under a N2 atmosphere. 1.9 mL of propylene sulfide was then added dropwise, and the reaction mixture was stirred at 60°C for 12 h. 5 mL of water was then added to quench the reaction, and the reaction product was precipitated and purified in cold methanol. The solvent was evaporated under reduced pressure to obtain a yellow oily polymer PPS, which was dried for later use.

[0024] (2) Add 158.6 mg of dried polymer PPS to 20 mL of dichloromethane and stir to dissolve. Then add 23 mg of N-hydroxysuccinimide and 48 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and stir for 30 min until dissolved. Then, add 133 μL of ethylenediamine dropwise to the mixture and stir to react for 12 h. Then, add 20 mL of dichloromethane to dilute the reaction solution. Then, wash with water and saturated NaCl solution in sequence, dry with MgSO4, filter, and finally evaporate the solvent under reduced pressure to obtain polymer PPS-NH2, which is dried for later use.

[0025] (3) 100 mg of hyaluronic acid and 10 mL of water were mixed and stirred to dissolve, and then 7 mg of N-hydroxysuccinimide and 14.5 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide were added and the mixture was stirred to dissolve to obtain a mixed solution. Then 40 mg of polymer PPS-NH2 was dissolved in 1 mL of tetrahydrofuran and added dropwise to the above mixed solution. The mixture was stirred and reacted under N2 protection for 24 h. The reaction product was then dialyzed and the solvent was finally removed by freeze-drying to obtain the conjugate HA-PPS.

[0026] (4) The conjugate HA-PPS and tanshinone IIA were dissolved in water, wherein the concentration of the conjugate HA-PPS was 45 mg / mL and the concentration of tanshinone IIA was 0.5 mg / mL, and the mixture was ultrasonically treated in an ice bath for 10 minutes to obtain nanomicelles loaded with tanshinone IIA.

[0027] The tanshinone IIA-loaded nanomicelles of the present invention are amphiphilic polymers whose molecular chains contain both a hydrophilic segment of hyaluronic acid (HA) and a hydrophobic segment of polypropylene sulfide (PPS). When the amphiphilic polymer is dissolved in water, its hydrophobic segments tend to aggregate with each other to reduce contact with water, while the hydrophilic segments tend to come into contact with water. This interaction drives the polymer molecules to self-assemble into micelles in solution. The hydrophobic segments aggregate to form the hydrophobic core of the micelles, while the hydrophilic segments are wrapped around the hydrophilic shell, forming micelles with a "core-shell" structure.

[0028] The present invention grafts active oxygen-sensitive polypropylene sulfide (PPS) with hyaluronic acid (HA) to form amphiphilic nanomicelles, utilizes hydrophobic interaction to load tanshinone IIA into the micelle core, and then loads the nanomicelles into a double-network hydrogel formed by cross-linking methacryloylated astragalus polysaccharide and methacryloylated silk fibroin. Through in situ administration, the active ingredients of traditional Chinese medicine can avoid the first-pass effect and the limitation of the blood-spinal cord barrier. At the same time, the high level of active oxygen in the spinal cord injury environment is targeted by oxidation of polypropylene sulfide (PPS), destroying the micelle structure and releasing tanshinone IIA, thereby realizing local drug storage and targeted sustained release of the active ingredients of traditional Chinese medicine.

[0029] According to a fourth aspect of the present invention, a multifunctional hydrogel co-loaded with Astragalus and Salvia miltiorrhiza components prepared by the above-mentioned preparation method is provided.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] The test results show that the hydrogel of the present invention has excellent mechanical properties, swelling properties, and biodegradability, and has an excellent sustained-release effect on tanshinone IIA and astragalus polysaccharide. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The characterization results of the micelles prepared by the present invention are shown in Figure 1. (A) is the characterization results of the micelles prepared by the present invention. 1 H NMR spectrum; (B) is the particle size distribution of HA-PPS micelles; (C) is the particle size distribution of TSIIA@HA-PPS micelles; (D) is the transmission electron microscopy image of HA-PPS and TSIIA@HA-PPS micelles.

[0033] Figure 2 The results of characterization of the properties of the hydrogel prepared by the present invention are shown in Figure 1. (A) is the properties of SF and SFMA. 1 H NMR spectra; (B) is AP and APMA 1 H NMR spectrum; (C) is the FT-IR spectrum of SF and SFMA; (D) is the FT-IR spectrum of AP and APMA; (E) is a photograph of the gelation of the hydrogel; (F) is the time scanning curve of the hydrogel; (G) is the frequency scanning curve of the hydrogel; (H) is the stress-strain curve of the hydrogel; (I) is the swelling curve of the hydrogel; (J) is the degradation curve of the hydrogel in PBS buffer and PBS buffer containing lysozyme; (K) is the release curve of TSIIA from the hydrogel in PBS buffer containing H2O2 and PBS buffer, and the release curve of astragalus polysaccharide (APMA) in PBS buffer. DETAILED DESCRIPTION

[0034] The present invention will be further described in detail below with reference to the accompanying drawings. It is worth noting that the following examples are only for better explanation of the present invention and are not intended to limit the scope of protection of the present invention. The undisclosed process steps in the examples are prior art. Unless otherwise specified, the following raw materials are commercially available.

[0035] Some of the reagents and consumables used for material synthesis are shown in Table 1.

[0036] Table 1 Some reagents and consumables used in material synthesis

[0037]

[0038]

[0039] Some of the instruments used for material synthesis and characterization are shown in Table 2.

[0040] Table 2 Some instruments used for material synthesis and characterization

[0041]

[0042]

[0043] Example 1

[0044] The preparation method of the multifunctional hydrogel loaded with astragalus polysaccharide of this embodiment comprises the following steps:

[0045] (1) Synthesis of methacrylylated silk fibroin

[0046] ① Preparation of silk fibroin

[0047] The cocoons were removed of pupae and impurities and chopped into small pieces. A 0.05 mol / L sodium carbonate solution was prepared. 30 g of the chopped cocoons were placed in 15 mL of the 0.05 mol / L sodium carbonate solution and boiled for 30 minutes. The solids were then removed and washed several times with clean water. This step was repeated twice until the boiled water was no longer noticeably yellow, yielding degummed silk fibroin. The degummed silk fibroin was then dried in an oven at 50°C to yield dried silk fibroin (SF).

[0048] ② Preparation of methacrylylated silk fibroin

[0049] 80.72 g of lithium bromide was added to 100 mL of deionized water while stirring continuously, followed by the addition of 0.48 g of sodium hydroxide, followed by the addition of 19.2 g of dried silk fibroin (SF), and then stirred in a 60 ° C water bath for 4 hours until the silk fibroin was completely dissolved, and then 1 mL of 12 mol / L concentrated hydrochloric acid was added to neutralize the sodium hydroxide, and then 12 mL of glycidyl methacrylate was added, and the reaction was stirred at room temperature for 6 hours. The reaction solution was then filtered to remove impurities, and then dialyzed in deionized water with a 1.2-1.4 kDa cellulose dialysis bag to remove salts. After that, the solution was centrifuged at 5000 rpm for 10 minutes, and the supernatant was freeze-dried to obtain methacryloylated silk fibroin (abbreviated as SFMA).

[0050] (2) Synthesis of methacryloylated Astragalus polysaccharide

[0051] 5g of astragalus polysaccharide (AP) was dissolved in 500mL of deionized water. 15mL of methacrylic anhydride (MA) was added at 50°C, and the mixture was stirred at 10±5°C for 24 hours. The pH of the reaction system was then adjusted to 10 with a 2% (w / v) aqueous solution of NaOH, and the mixture was stirred for another 24 hours. The resulting solution was then dialyzed against deionized water (3.5kDa cellulose dialysis tubing) for 5 days and then freeze-dried to obtain methacryloylated astragalus polysaccharide (APMA).

[0052] (3) Preparation of multifunctional hydrogel loaded with astragalus polysaccharide

[0053] 0.2 g of methacryloylated silk fibroin (SFMA) and 0.01 g of methacryloylated astragalus polysaccharide (APMA) were dissolved in 1 mL of deionized water containing 0.25% (w / v, mass volume ratio) of LAP photoinitiator (phenyl (2,4,6-trimethylbenzoyl) lithium phosphate) to obtain an SFMA-APMA hydrogel solution. The SFMA-APMA hydrogel solution was placed in an EP tube and then exposed to ultraviolet light (405 nm) for 3 minutes to obtain the hydrogel. The hydrogel of this example is referred to as a 20% SFMA-1% APMA hydrogel.

[0054] Example 2

[0055] The preparation method of the multifunctional hydrogel loaded with astragalus polysaccharide in this example is basically the same as that in Example 1, except that in step (3), the amount of methacryloylated astragalus polysaccharide (APMA) used is 0.05 g. The hydrogel of Example 2 is designated as 20% SFMA-5% APMA hydrogel.

[0056] Example 3

[0057] The preparation method of the multifunctional hydrogel loaded with astragalus polysaccharide in this example is essentially the same as that in Example 1, with the only difference being that in step (3), the amount of methacryloyl-astragalus polysaccharide (APMA) used is 0.1 g. The hydrogel of Example 3 is designated as 20% SFMA-10% APMA hydrogel.

[0058] Example 4

[0059] The preparation method of the multifunctional hydrogel co-loaded with Astragalus and Salvia miltiorrhiza components of this embodiment comprises the following steps:

[0060] (1) Methacryl-coated silk fibroin (SFMA) was prepared according to the method of Example 1.

[0061] (2) According to the method of Example 1, methacryloylated astragalus polysaccharide (APMA) was prepared.

[0062] (3) Preparation of nanomicelles loaded with tanshinone IIA

[0063] ①Synthetic polymer PPS

[0064] Under an ice bath at 0°C, 30 mL of anhydrous tetrahydrofuran (THF) was added, followed by 100 μL (1.15 mmol) of 3-mercaptopropionic acid (3-MPA). The mixture was mixed by magnetic stirring, followed by 524 μL (3.45 mmol) of 1,8-diazabicyclo[5.4.0]undec-7-ene. The mixture was stirred under a nitrogen atmosphere for 30 min. 1.9 mL (21.15 mmol) of propylene sulfide was then added dropwise, and the reaction mixture was stirred at 60°C overnight (12 h). The reaction was quenched by the addition of 5 mL of water, and the product was purified by precipitation in cold methanol. Finally, the solvent was evaporated under reduced pressure to obtain a yellow oily polymer (abbreviated as PPS), which was dried for later use.

[0065] ②Synthetic polymer PPS-NH2

[0066] To 20 mL of dichloromethane, add 158.6 mg (100 μmol) of dried polymer PPS and dissolve under magnetic stirring. Then, add 23 mg (200 μmol) of N-hydroxysuccinimide (NHS) and 48 mg (250 μmol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI) and stir magnetically at room temperature for 30 minutes until dissolved. Then, add 133 μL (2 mmol) of ethylenediamine dropwise to the mixture and stir at room temperature overnight (12 hours). Then, dilute the reaction solution with 20 mL of dichloromethane, wash with H2O and saturated NaCl solution, dry over MgSO4, filter, and evaporate the solvent under reduced pressure to obtain the polymer (abbreviated as PPS-NH2), which is then dried for later use.

[0067] ③Synthetic conjugate HA-PPS

[0068] Hyaluronic acid sodium salt was dialyzed overnight against a 0.01 mol / L HCl solution and then lyophilized to obtain the acid form of HA. To a 50 mL beaker, 10 mL of H₂O and 100 mg of hyaluronic acid (HA) were added and dissolved under magnetic stirring. Then, 7 mg (60 μmol) of N-hydroxysuccinimide and 14.5 mg (75 μmol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide were added. The mixture was stirred at room temperature for 30 minutes until completely dissolved, yielding a mixed solution. 40 mg of the polymer PPS-NH₂ was dissolved in 1 mL of tetrahydrofuran and added dropwise to the mixed solution. The reaction was stirred at room temperature under nitrogen for 24 hours. The reaction product was then dialyzed against water and methanol in a 1:1 volume ratio three times for a total of one day, and then against distilled water three times for a total of one day. Finally, the solvent was removed by lyophilization to obtain the conjugate (abbreviated as HA-PPS).

[0069] ④ Preparation of nanomicelles loaded with tanshinone IIA

[0070] The HA-PPS conjugate and tanshinone IIA (TSIIA for short) were dissolved in distilled water, wherein the concentration of the HA-PPS conjugate was 45 mg / mL and the concentration of tanshinone IIA was 0.5 mg / mL. The mixture was ultrasonically treated in an ice bath at 0°C for 10 minutes to obtain nanomicelles loaded with tanshinone IIA (TSIIA@HA-PPS for short).

[0071] (4) Preparation of multifunctional hydrogels co-loaded with Astragalus and Salvia miltiorrhiza components

[0072] 0.2 g of methacryloylated silk fibroin (SFMA) and 0.1 g of methacryloylated astragalus polysaccharide (APMA) were dissolved in 1 mL of deionized water containing 0.1% (w / v, mass volume ratio) of LAP photoinitiator (phenyl (2,4,6-trimethylbenzoyl) lithium phosphate) to obtain SFMA-APMA hydrogel solution. Then, 0.1 g of tanshinone IIA-loaded nanomicelles (TSIIA@HA-PPS) were added and mixed uniformly to obtain SFMA-APMA-TSIIA@HA-PPS hydrogel solution. The solution was then irradiated with ultraviolet light (405 nm, 30 W / cm 2 ) was irradiated for 180 s to obtain a multifunctional hydrogel co-loaded with Astragalus and Salvia miltiorrhiza components (abbreviated as 20% SFMA-10% APMA-10% TSIIA@HA-PPS).

[0073] Next, the prepared materials were characterized and their performance tested.

[0074] 1. Characterization and performance testing

[0075] 1. NMR characterization

[0076] (1) Samples: SF, SFMA; AP, APMA; HA, PPS, HA-PPS

[0077] (2) Test: Weigh 3-5 mg of sample and dissolve it in an appropriate amount of deuterated reagent (heavy water D2O). Then put it into a clean NMR tube and perform NMR structure determination at room temperature using an NMR spectrometer. Use MestReNova software for spectral analysis.

[0078] 2. Infrared characterization

[0079] (1) Sample: SF, SFMA; AP, APMA

[0080] (2) Test: First, take 3-5 mg of sample and an appropriate amount of dry potassium bromide powder (mass ratio is about 5%) in an agate mortar and grind them thoroughly to make them evenly mixed. Then, take an appropriate amount of the ground sample powder and press it into a tablet (vacuum pressure 20 mmHg, tableting for 5 minutes) to obtain a sample slice. Set the scanning range to 4000-400 cm -1 , detected by Fourier transform infrared spectrometer.

[0081] 3. Transmission electron microscopy and dynamic light scattering analysis

[0082] (1) Sample: HA-PPS, TSIIA@HA-PPS

[0083] (2) Testing: HA-PPS and TSIIA@HA-PPS were dispersed in an appropriate amount of deionized water and ultrasonicated for 30 seconds to obtain a dispersion. The dispersion was dropped onto a copper mesh and the morphology was photographed after it was completely dried. The microscopic morphology of HA-PPS and TSIIA@HA-PPS was observed by selecting the observation range using a transmission electron microscope (TEM). In addition, the HA-PPS and TSIIA@HA-PPS dispersions were placed in a potentiometer and their hydrated particle size was measured using a nano-laser particle size analyzer (DLS). The test was repeated at least three times.

[0084] 4. Hydrogel material gelation test

[0085] SFMA-APMA hydrogel solutions with different mass fractions were prepared according to the methods of Examples 1-3, and the SFMA-APMA hydrogel solutions were placed in EP tubes. Then, the SFMA-APMA hydrogel solutions were exposed to ultraviolet light (405 nm) for 3 minutes, and the formation of hydrogels was observed.

[0086] 5. Hydrogel rheological properties test

[0087] (1) Samples: 20% SFMA-1% APMA hydrogel of Example 1, 20% SFMA-5% APMA hydrogel of Example 2, and 20% SFMA-10% APMA hydrogel of Example 3.

[0088] (2) Test: Use a 24-well plate, add 400 μL of hydrogel solution to each well, and crosslink under UV light for 3 minutes to form a gel. Rheological measurements are performed using a stainless steel parallel plate rotor with a diameter of 25 mm. G' characterizes the elastic modulus of the sample, and G" characterizes the viscous modulus of the sample. Dynamic strain scanning is performed from 0.1 to 10 rad / s at room temperature to determine the linear viscoelastic range of the hydrogel, and the storage modulus (G') and loss modulus (G") change curves are recorded.

[0089] 6. Hydrogel compression performance test

[0090] (1) Samples: 20% SFMA-1% APMA hydrogel of Example 1, 20% SFMA-5% APMA hydrogel of Example 2, and 20% SFMA-10% APMA hydrogel of Example 3.

[0091] (2) Testing: Use a 48-well plate, add 600 μL of hydrogel solution to each well, 5 wells per group, and crosslink under UV light for 3 minutes to form a gel. Place the hydrogel on a sample stage, and then use a universal testing machine to press the hydrogel sample at a constant rate of 0.05 mm / s until it breaks.

[0092] 7. In vitro swelling test of hydrogel

[0093] (1) Samples: 20% SFMA-1% APMA hydrogel of Example 1, 20% SFMA-5% APMA hydrogel of Example 2, and 20% SFMA-10% APMA hydrogel of Example 3.

[0094] (2) Test: The initial weight of the hydrogel is recorded as M0. Then the hydrogel is completely immersed in PBS buffer for a certain period of time, the hydrogel is taken out, and the excess water is wiped off with weighing paper. The weight after swelling at different times is recorded as M t , the swelling ratio is calculated as follows:

[0095] Swelling ratio (%) = M t / M0×100%

[0096] 8. In vitro degradation test of hydrogel

[0097] (1) Sample: 20% SFMA-10% APMA hydrogel of Example 3

[0098] (2) Test method: The initial weight of the hydrogel sample after freeze-drying is recorded as W0. Then the hydrogel is completely immersed in PBS buffer or PBS buffer containing 1000U / mL lysozyme, and placed in a 37°C constant temperature shaker. The PBS buffer and lysozyme are replaced every 3 days. The hydrogel is taken out at different time points, the excess degradation solution is discarded, the sample is freeze-dried, and the weight of the hydrogel is recorded as W. t The degradation rate is calculated as follows:

[0099] Degradation rate (%) = (W0-W t ) / W0×100%

[0100] 9. Drug release performance test

[0101] (1) Tanshinone IIA response release

[0102] Sample: 20% SFMA-10% APMA-10% TSIIA@HA-PPS of Example 4

[0103] Methods: Hydrogel samples were immersed in 2 mL of PBS buffer or PBS buffer containing 1 mmol / L H₂O₂ (referred to as the extract). At specific time points (days 1, 3, 7, 14, and 21), 2 mL of the extract was removed and simultaneously added with 2 mL of PBS buffer or PBS buffer containing 1 mmol / L H₂O₂, maintaining the extract volume constant. Tanshinone IIA concentrations in the extracts removed at different time points were measured, and the release rate was calculated according to the following formula:

[0104] Release rate (%) = total release amount / total load amount × 100%

[0105] (2) Release of Astragalus Polysaccharide

[0106] Sample: 20% SFMA-10% APMA-10% TSIIA@HA-PPS of Example 4

[0107] Methods: Hydrogel samples were immersed in 2 mL of PBS buffer (referred to as the extract). At specific time points (days 1, 3, 7, 14, and 21), 2 mL of the extract was removed and 2 mL of PBS buffer was added simultaneously, maintaining the extract volume constant. The concentration of APS in the extracts removed at different time points was measured, and the release rate was calculated according to the following formula:

[0108] Release rate (%) = total release amount / total load amount × 100%

[0109] 2. Characterization and Performance Test Results

[0110] 1. Characterization results of micelle properties

[0111] The characterization results of micelle properties are as follows Figure 1 As shown in the figure, (A) is HA, PPS and HA-PPS 1 H NMR spectrum; (B) is the particle size distribution of HA-PPS micelles; (C) is the particle size distribution of TSIIA@HA-PPS micelles; (D) is the transmission electron microscopy image of HA-PPS and TSIIA@HA-PPS micelles.

[0112] from Figure 1 As shown in Figure A, the HA-PPS conjugate shows the presence of methyl groups in HA and PPS near δ = 2.0 ppm and δ = 1.0 ppm, respectively, indicating the successful synthesis of HA-PPS. HA-PPS micelles were formed based on the amphiphilic self-assembly present when HA and PPS bind. The hydrophobic drug TSIIA was loaded at the center of the micelles to produce TSIIA@HA-PPS micelles. The morphology and size of the HA-PPS and TSIIA@HA-PPS micelles were characterized by DLS and TEM. Figure 1 B and Figure 1 C shows the particle size distribution histograms of HA-PPS and TSIIA@HA-PPS micelles. The average particle sizes of HA-PPS and TSIIA@HA-PPS micelles are 137.243±1.01853 nm (PDI=0.092) and 142±2.00245 nm (PDI=0.02), respectively. The average particle size increases slightly after loading with TSIIA. Figure 1 D are the morphological images of HA-PPS and TSIIA@HA-PPS micelles, respectively, confirming that HA-PPS was successfully loaded with TSIIA.

[0113] 2. Characterization results of hydrogel properties

[0114] The hydrogel characterization results are as follows Figure 2 As shown in the figure, (A) is SF and SFMA 1 H NMR spectra; (B) is AP and APMA 1 H NMR spectrum; (C) is the FT-IR spectrum of SF and SFMA; (D) is the FT-IR spectrum of AP and APMA; (E) is a photograph of the gelation of the hydrogel; (F) is the time scanning curve of the hydrogel; (G) is the frequency scanning curve of the hydrogel; (H) is the stress-strain curve of the hydrogel; (I) is the swelling curve of the hydrogel; (J) is the degradation curve of the hydrogel in PBS buffer and PBS buffer containing lysozyme; (K) is the release curve of TSIIA from the hydrogel in PBS buffer containing H2O2 and PBS buffer, and the release curve of astragalus polysaccharide (APMA) in PBS buffer.

[0115] Figure 2A is the H NMR spectrum of SF and SFMA. The chemical shifts of the methacryloyl group in SFMA are 5.5 and 6.0 ppm, respectively, indicating that SF is successfully methylated. AP is a natural polysaccharide with many hydroxyl groups in its molecular chain, which provides more sites for the reaction between AP and MA, enabling it to be photocrosslinked. 1 H NMR confirmed that MA was successfully grafted onto AP (5.2 and 5.5 ppm), with the signal peaks corresponding to methacrylate ( Figure 2 B), demonstrating that AP is acidified by methacrylic acid.

[0116] The FT-IR spectrum of SFMA is at 1166 cm -1 There is a secondary amine stretching vibration peak at ( Figure 2 C). When SF reacts with glycidyl methacrylate, one hydrogen atom on the primary amine is replaced to form a secondary amine, further demonstrating the successful synthesis of SFMA. Figure 2 D is the FTIR spectrum of AP and APMA, showing that AP was successfully methylated. -1 The C=O peak intensity increases at 1409cm -1 The C=C peak at 935cm -1 The out-of-plane bending vibration of =CH further confirmed the successful synthesis of APMA.

[0117] like Figure 2 As shown in Figure E, SFMA-APMA hydrogel solutions with different mass fractions can form stable hydrogels by UV irradiation in the presence of LAP photoinitiator. Rheological characterization of the SFMA-APMA hydrogels with different mass fractions was then performed to determine the hardness of the hydrogels. Figure 2 F. Figure 2 G is the change of elastic modulus (G′) and viscous modulus (G″) of hydrogel with time and frequency, respectively. It can be seen from the figure that the three groups of hydrogels, 20% SFMA-1% APMA, 20% SFMA-5% APMA and 20% SFMA-10% APMA, all show a state of G′>G″ under the change of time and frequency, indicating that the hydrogel can stably exist in the form of gel under appropriate conditions. The elastic modulus of the hydrogel with the addition of 5% APMA is higher than that of the hydrogel with the addition of 1% APMA. This is because the mass fraction of APMA increases, resulting in an increase in cross-linking density, which leads to an increase in elastic modulus. The elastic modulus of the hydrogel with the addition of 10% APMA decreases instead. This is because if the polysaccharide concentration is too high, it may lead to competition for interaction, affecting the aggregation state of silk fibroin, thereby reducing the elastic modulus.

[0118] Figure 2A plot of the compressive stress versus compressive strain relationship for three hydrogel groups (H: 20% SFMA-1% APMA, 20% SFMA-5% APMA, and 20% SFMA-10% APMA) tested in a universal mechanical tester shows that the Young's modulus of the hydrogels first increases, then decreases, and then increases again with increasing compressive strain. Furthermore, increasing APMA mass fraction causes the strain to first increase and then decrease. When the APMA mass fraction is 1%, the hydrogel ruptures at a strain of approximately 59.3%, with a Young's modulus of 201.9 kPa. When the APMA mass fraction increases to 5%, the hydrogel ruptures at a strain of 62.6%, achieving its highest compressive strength with a Young's modulus of 338.5 kPa. When the APMA mass fraction reaches 10%, the hydrogel ruptures at a compression of approximately 57.9%, with a Young's modulus of approximately 53.7 kPa.

[0119] The appropriate swelling ability of the hydrogel can release the stored drugs into the surrounding environment. Figure 2 Figure 1 shows the swelling performance test results of three groups of hydrogels: 20% SFMA-1% APMA, 20% SFMA-5% APMA, and 20% SFMA-10% APMA in PBS solution. As can be seen from the figure, the swelling rate of the 20% SFMA-5% APMA hydrogel is the lowest, followed by the 20% SFMA-1% APMA hydrogel, and the 20% SFMA-10% APMA hydrogel has the highest swelling rate. The swelling rate of the 20% SFMA-5% APMA hydrogel is 25.1±13.1%, and the swelling rate of the 20% SFMA-10% APMA hydrogel is as high as 78.7±30.8%. Figure 2 The degradation curve of the 20% SFMA-10% APMA hydrogel shows good biodegradability, reaching a degradation rate of 80.67% within 21 days in the presence of lysozyme. The degradation rate of the hydrogel in PBS buffer without lysozyme was significantly slower, due to the presence of only hydrolysis.

[0120] In vitro response release curve of Tanshinone IIA ( Figure 2 K) showed that the TSIIA release rate of 20% SFMA-10% APMA-10% TSIIA@HA-PPS in PBS reached 38.44%, while the TSIIA release rate of 20% SFMA-10% APMA-10% TSIIA@HA-PPS in PBS (H₂O₂) reached 76.64%. This indicates that the addition of H₂O₂ accelerated the release rate of the hydrogel. The presence of reactive oxygen species increased the rate and extent of TSIIA release, achieving ROS-responsive release. Furthermore, the release of APS from 20% SFMA-10% APMA-10% TSIIA@HA-PPS reached equilibrium on day 10, with a release rate of 72.24%.

[0121] The above descriptions are only some specific embodiments of the present invention. For those skilled in the art, several modifications and improvements can be made without departing from the creative concept of the present invention, and these all fall within the scope of protection of the present invention.

Claims

1. A method for preparing a multifunctional hydrogel loaded with astragalus polysaccharide, characterized in that: The following steps are involved: Dissolving methacrylated silk fibroin and methacrylated astragalus polysaccharide in a first water containing a photoinitiator to obtain an SFMA-APMA hydrogel solution, and then irradiating the SFMA-APMA hydrogel solution under ultraviolet light for 2-4 minutes to obtain; The preparation method of the methacryloylated astragalus polysaccharide comprises the following steps: Astragalus polysaccharide is dissolved in the second water, methacrylic anhydride is added at 45-55° C., and then stirred and reacted at 5-15° C. for 12-24 hours. Thereafter, the pH of the reaction system is adjusted to 9.5-10.5, and the stirring reaction is continued for 12-24 hours. The resulting solution is then dialyzed and subsequently freeze-dried to obtain the product.

2. The method for preparing the multifunctional hydrogel loaded with astragalus polysaccharide according to claim 1, characterized in that: The ratio of the mass of the astragalus polysaccharide to the volume of the second water is 1:100, and the ratio of the mass of the astragalus polysaccharide to the volume of methacrylic anhydride is 1:

3.

3. The method for preparing the multifunctional hydrogel loaded with astragalus polysaccharide according to claim 1 or 2, characterized in that: The photoinitiator is a LAP photoinitiator; In parts by mass, the amount of methacryloylated silk fibroin is 0.1-0.3 parts, the amount of methacryloylated astragalus polysaccharide is 0.01-0.1 parts, the amount of photoinitiator is 0.001-0.0025 parts, and the amount of the first water is 0.5-2 parts.

4. The multifunctional hydrogel loaded with astragalus polysaccharide prepared by the preparation method according to any one of claims 1 to 3.

5. A method for preparing a multifunctional hydrogel co-loaded with Astragalus and Salvia miltiorrhiza components, characterized in that: The following steps are involved: Methacryloylated silk fibroin and methacryloylated astragalus polysaccharide were dissolved in a third water containing a photoinitiator to obtain an SFMA-APMA hydrogel solution, and then the nanomicelles loaded with tanshinone IIA were added and mixed evenly to obtain an SFMA-APMA-TSIIA@HA-PPS hydrogel solution, which was then irradiated under ultraviolet light for 2-4 minutes. The preparation method of the methacryloylated astragalus polysaccharide comprises the following steps: Astragalus polysaccharide is dissolved in the fourth water, methacrylic anhydride is added at 45-55° C., and then stirred and reacted at 5-15° C. for 12-24 hours. Thereafter, the pH of the reaction system is adjusted to 9.5-10.5, and the stirring reaction is continued for 12-24 hours. The resulting solution is then dialyzed and subsequently freeze-dried to obtain the product.

6. The method for preparing the multifunctional hydrogel co-loaded with Astragalus and Salvia miltiorrhiza components according to claim 5, characterized in that: The ratio of the mass of the astragalus polysaccharide to the volume of the fourth water is 1:100, and the ratio of the mass of the astragalus polysaccharide to the volume of methacrylic anhydride is 1:

3.

7. The method for preparing the multifunctional hydrogel co-loaded with Astragalus and Salvia miltiorrhiza components according to claim 5 or 6, characterized in that: The photoinitiator is a LAP photoinitiator; In parts by mass, the amount of methacryloylated silk fibroin is 0.1-0.3 parts, the amount of methacryloylated astragalus polysaccharide is 0.01-0.1 parts, the amount of photoinitiator is 0.001-0.0025 parts, the amount of the third water is 0.5-2 parts, and the amount of nanomicelles loaded with tanshinone IIA is 0.05-0.15 parts.

8. The method for preparing the multifunctional hydrogel co-loaded with Astragalus and Salvia miltiorrhiza components according to claim 5 or 6, characterized in that: The nano micelles loaded with tanshinone IIA are obtained by grafting polypropylene sulfide and hyaluronic acid to form amphiphilic nano micelles, and then loading tanshinone IIA into the core of the micelles by utilizing hydrophobic interaction.

9. The method for preparing the multifunctional hydrogel co-loaded with Astragalus and Salvia miltiorrhiza components according to claim 8, characterized in that: The preparation method of the nanomicelles loaded with tanshinone IIA comprises the following steps: (1) Under ice bath conditions, 30 mL of anhydrous tetrahydrofuran was first added, followed by 100 μL of 3-mercaptopropionic acid, and the mixture was stirred and mixed. 524 μL of 1,8-diazabicyclo[5.4.0]undec-7-ene was then added, and the mixture was stirred for 30 min under a N2 atmosphere. 1.9 mL of propylene sulfide was then added dropwise, and the reaction mixture was stirred at 60°C for 12 h. 5 mL of water was then added to quench the reaction, and the reaction product was precipitated and purified in cold methanol. The solvent was evaporated under reduced pressure to obtain a yellow oily polymer PPS, which was dried for later use. (2) Add 158.6 mg of dried polymer PPS to 20 mL of dichloromethane and stir to dissolve. Then add 23 mg of N-hydroxysuccinimide and 48 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and stir for 30 min until dissolved. Then, add 133 μL of ethylenediamine dropwise to the mixture and stir to react for 12 h. Then, add 20 mL of dichloromethane to dilute the reaction solution. Then, wash with water and saturated NaCl solution in sequence, dry with MgSO4, filter, and finally evaporate the solvent under reduced pressure to obtain polymer PPS-NH2, which is dried for later use. (3) 100 mg of hyaluronic acid and 10 mL of water were mixed and stirred to dissolve, and then 7 mg of N-hydroxysuccinimide and 14.5 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide were added and the mixture was stirred to dissolve to obtain a mixed solution. Then 40 mg of polymer PPS-NH2 was dissolved in 1 mL of tetrahydrofuran and added dropwise to the above mixed solution. The mixture was stirred and reacted under N2 protection for 24 h. The reaction product was then dialyzed and the solvent was finally removed by freeze-drying to obtain the conjugate HA-PPS. (4) The conjugate HA-PPS and tanshinone IIA were dissolved in water, wherein the concentration of the conjugate HA-PPS was 45 mg / mL and the concentration of tanshinone IIA was 0.5 mg / mL, and the mixture was ultrasonically treated in an ice bath for 10 minutes to obtain nanomicelles loaded with tanshinone IIA.

10. The multifunctional hydrogel co-loaded with Astragalus and Salvia miltiorrhiza components prepared by the preparation method according to any one of claims 5 to 9.

Images