Preparation method of hydrogel formed by modifying and combining tail end of PluronicF127 with HA and CuS-NDs

Abstract

The invention relates to the technical field of biomedical materials, and particularly discloses a preparation method of hydrogel formed by combining terminal modification of PluronicF127 with HA and CuS-NDs, anhydrous tetrahydrofuran is taken and placed in a container, and then PluronicF127, N, N '-disuccinimido carbonate and 4-dimethylaminopyridine are taken and stirred and mixed together; adding anhydrous dimethyl sulfoxide, stirring and mixing, adding beta-alanine into ultrapure water, adjusting the pH value, and stirring and dissolving; adding the mixture into the mixed solution for stirring reaction; the reaction liquid is placed in a dialysis bag for dialysis, and modified PluronicF127 is obtained; cuCl2. 2H2O and sodium citrate are taken and added into ultrapure water, and after the pH is adjusted, stirring is carried out for dissolution; adding sodium sulfide nonahydrate into ultrapure water, stirring and dissolving; adding the solution, stirring and mixing to obtain a CuS-NDs solution; the CuS-NDs solution is subjected to freeze drying, and freeze-dried CuS-NDs powder is obtained; and mixing and stirring the PluronicCF127 solution, the hyaluronic acid solution and the CuS-NDs solution in an ice-water bath to obtain a hydrogel finished product. The degradable plastic has the characteristics of good mechanical strength and viscosity, and excellent biodegradability, biology and safety.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of biomedical materials, in particular to a preparation method of a hydrogel formed by end modification of Pluronic F127, combination of HA and CuS-NDs. BACKGROUND

[0002] Pluronic F127 is a non-ionic triblock copolymer composed of polyethylene oxide (PEO) and polypropylene oxide (PPO), and the specific structure is PEO-PPO-PEO; the Pluronic F127-based hydrogel has great advantages in the biomedical field due to its unique thermosensitivity, biocompatibility, adjustability and multifunctionality, it can not only realize various functions through simple regulation, but also can further expand the application range through chemical modification, and is a very promising multifunctional material; although the Pluronic F127 copolymer can quickly form a hydrogel at a specific temperature, due to its low molecular weight and low mechanical strength, the hydrogel structure cannot be maintained for a long time under physiological conditions, therefore, the Pluronic F127 concentration used in the hydrogel injected into the body is relatively high, and the hydrogel with a relatively high concentration has potential hazards in the human body. Hyaluronic acid (HA) is a natural polysaccharide, which is alternately connected by N-acetylglucosamine and glucuronic acid through beta-(1→3) and beta-(1→4) glycosidic bonds, and the molecular weight ranges from several thousand to several million daltons, and the different molecular weights significantly affect its functions, it widely exists in human connective tissue, synovial fluid, ocular vitreous body and skin, and has high water retention and biocompatibility.

[0003] Hydrogels are three-dimensional network structures composed of polymer materials that can absorb and retain large amounts of water while maintaining their shape and structure. Although hydrogels have advantages such as high water absorption, biocompatibility, and softness, they have shortcomings in terms of mechanical properties, preparation processes, degradation rates, and biosafety. Many researchers are continuously optimizing the performance of hydrogels through material modification, composite material development, and novel structural design to better meet various application needs. Patent document CN108434091A discloses a self-healing hydrogel that promotes wound healing and tumor treatment, and its preparation method. The self-healing hydrogel is an FCB hydrogel obtained by mixing an 8%-50% (w / w) aqueous solution of a polyether-cationic polymer, a 10%-40% (w / w) aqueous solution of a polyether-p-hydroxybenzaldehyde, and a 2%-10% (w / w) aqueous solution of a dopamine-modified bioactive inorganic material via a Schiff base reaction. F is the polyether-cationic polymer; C is the polyether-p-hydroxybenzaldehyde; and B is the dopamine-modified bioactive inorganic material. It exhibits good biocompatibility, good photothermal properties, and strong antibacterial activity. Under laser irradiation, it can effectively inhibit tumor growth in animals and promote skin repair in mice. However, the hydrogel prepared by this method has insufficient mechanical strength and viscosity, and its biodegradability and safety are also generally poor.

[0004] Therefore, existing hydrogels used in biomedicine suffer from insufficient mechanical strength and viscosity, as well as generally poor biodegradability and safety. Summary of the Invention

[0005] In order to solve the above-mentioned technical problems of existing hydrogels used in biomedicine, the present invention provides a method for preparing a hydrogel formed by end modification of Pluronic F127 combined with HA and CuS-NDs, which has the characteristics of good mechanical strength and viscosity, as well as excellent biodegradability, biocompatibility and safety.

[0006] The technical solution of the present invention: A method for preparing a hydrogel formed by end modification of Pluronic F127 combined with HA and CuS-NDs, comprising the following steps: (S01) Taking an appropriate amount of anhydrous tetrahydrofuran and placing it in a container, then taking an appropriate amount of Pluronic F127, N,N′-disuccinimidyl carbonate and 4-dimethylaminopyridine and placing them together in the container and stirring to mix, to obtain mixture I; (S02) Adding an appropriate amount of anhydrous dimethyl sulfoxide to mixture I in step (S01) and stirring to mix, to obtain mixture II; (S03) Taking an appropriate amount of anhydrous dimethyl sulfoxide and placing it in a container together with HA and CuS-NDs, comprising the following steps: (S01) Taking an appropriate amount of anhydrous tetrahydrofuran and placing it in a container, then taking an appropriate amount of anhydrous tetrahydrofuran and placing it in a container and stirring to mix, to obtain mixture II; (S03) Taking an appropriate amount of anhydrous dimethyl sulfoxide and placing it in a container, then taking an appropriate amount of anhydrous tetrahydrofuran ... Add β-alanine to ultrapure water, adjust the pH to 8-9, and stir to dissolve, obtaining a solution; (S04) Add the solution from step (S03) dropwise to mixture II from step (S02) and stir to react, obtaining a reaction solution; (S05) Place the reaction solution from step (S04) in a dialysis bag and dialyze with ultrapure water, take the retentate and freeze-dry to constant weight, obtaining the modified Pluronic F127, denoted as Pluronic CF127; (S06) Take appropriate amounts of CuCl2·2H2O and sodium citrate and add them to ultrapure water. After adjusting the pH to 7-8, stir to dissolve, obtaining solution I; (S07) Take an appropriate amount of sodium sulfide nonahydrate and add it to ultrapure water, stir to dissolve under nitrogen protection, obtaining solution II; (S08) Under nitrogen protection, add solution II from step (S07) dropwise to solution I from step (S06) and stir to mix. After the addition is complete, continue stirring and then heat at a constant temperature to obtain CuS-NDs solution; (S09) After the CuS-NDs solution from step (S08) is cooled with ice water, collected by centrifugation, washed and freeze-dried, to obtain freeze-dried CuS-NDs solution. (S10) Prepare a PBS solution of Pluronic CF127 from step (S05), prepare a PBS solution of hyaluronic acid HA, prepare a PBS solution of hyaluronic acid, prepare a PBS solution of CuS-NDs powder from step (S08), take appropriate amounts of Pluronic CF127 solution, hyaluronic acid solution and CuS-NDs solution, mix and stir in an ice water bath, and then let stand to obtain the hydrogel product, which is denoted as CuS@CF127 / HA.This invention utilizes terminal modification of Pluronic F127 followed by combination with hyaluronic acid and copper sulfide nanodots to form a multifunctional hydrogel. Pluronic F127, as a thermosensitive block copolymer, allows the hydrogel to form at body temperature after injection and undergo a sol-gel transition near body temperature. Through the reaction of N,N′-disuccinimidyl carbonate and β-alanine, the terminal hydroxyl groups of Pluronic F127 are converted to carboxyl groups, enabling them to chemically crosslink with the amino or hydroxyl groups of HA to form a network, thus enhancing the hydrogel's stability. Hyaluronic acid, as a natural polysaccharide, possesses biocompatibility, moisturizing properties, and cell adhesion. The introduction of CuS-NDs may endow the hydrogel with photothermal therapy, imaging, and antibacterial functions. This invention uses N,N′-disuccinimidyl carbonate to activate the terminal hydroxyl groups of Pluronic F127, and then reacts with β-alanine to introduce carboxyl groups, which can enhance the stability of Pluronic F127. The F127 terminal is well carboxylated, and CuS nanoparticles are synthesized by coprecipitation method. The steps are relatively simple. Combining modified polymer, natural polysaccharide and functional nanomaterial can achieve complementary material functions.

[0007] Preferably, the container used in step (S01) is a 100ml glass bottle. Preferably, the amount of anhydrous tetrahydrofuran used in step (S01) is 30ml. Preferably, the specifications of the anhydrous tetrahydrofuran used in step (S01) are: CAS: 109-99-9, purity ≥99.9%.

[0008] Preferably, the amount of Pluronic F127 used in step (S01) is 12.6 g. Preferably, the specification of Pluronic F127 in step (S01) is MW=12600Da. Preferably, the amount of N,N′-disuccinimidyl carbonate used in step (S01) is 1.025 g.

[0009] Preferably, the N,N′-disuccinimidyl carbonate in step (S01) has the following specifications: CAS: 74124-79-1, purity ≥98%.

[0010] Preferably, the amount of 4-dimethylaminopyridine used in step (S01) is 0.489 g. Preferably, the 4-dimethylaminopyridine in step (S01) has the following specifications: CAS: 1122-58-3, purity ≥99%. Preferably, the stirring and mixing in step (S01) is magnetic stirring co-mixing. Preferably, the magnetic stirring speed is 400 rpm to 600 rpm. More preferably, the magnetic stirring speed is 500 rpm.

[0011] Preferably, the mixing temperature in step (S01) is 24°C to 26°C. More preferably, the mixing temperature in step (S02) is 25°C. Preferably, the mixing time in step (S01) is 10 min to 20 min. More preferably, the mixing time in step (S01) is 15 min. Preferably, in step (S01), the container is pre-dried and the operation is carried out under nitrogen or argon protection. Preferably, in step (S01), based on the terminal hydroxyl group of Pluronic F127, the molar ratio of Pluronic F127, N,N′-disuccinimidyl carbonate, and 4-dimethylaminopyridine is 1:1.5 to 2.5:0.15 to 0.25. More preferably, the molar ratio of Pluronic F127, N,N′-disuccinimidyl carbonate, and 4-dimethylaminopyridine is 1:2:0.2. Preferably, in step (S01), the volume ratio of 4-dimethylaminopyridine to anhydrous tetrahydrofuran is 1:5 to 7. More preferably, the volume ratio of 4-dimethylaminopyridine to anhydrous tetrahydrofuran is 1:6. Preferably, in step (S02), the amount of anhydrous dimethyl sulfoxide added is 5 ml. Preferably, in step (S02), the anhydrous dimethyl sulfoxide has the specification CAS: 67-68-5 and a purity ≥99.8%. Preferably, the stirring and mixing in step (S02) is magnetic stirring. Preferably, the magnetic stirring speed is 400 rpm to 600 rpm. More preferably, the magnetic stirring speed is 500 rpm. Preferably, the stirring and mixing temperature in step (S02) is 24°C to 26°C. More preferably, the stirring and mixing temperature in step (S02) is 25°C. Preferably, the stirring and mixing time in step (S02) is 12 h to 36 h. More preferably, the mixing time in step (S02) is 18h to 30h. More preferably, the mixing time in step (S02) is 24h. Preferably, the amount of β-alanine used in step (S03) is 0.3564g. Preferably, the β-alanine in step (S03) has a CAS number of 107-95-9 and a purity of ≥99%. Preferably, the amount of ultrapure water used in step (S03) is 5ml to 15ml. More preferably, the amount of ultrapure water used in step (S03) is 10ml. Preferably, in step (S03), the pH is adjusted to 8-9 using a 1M NaOH solution. More preferably, the pH is adjusted to 8.5 using a 1M NaOH solution. Preferably, the dissolution process in step (S03) is carried out in a vortex mixer. Preferably, the dissolving solution in step (S04) is added slowly dropwise. Preferably, the stirring in step (S04) is magnetic stirring.Preferably, the stirring reaction temperature in step (S04) is 24℃~26℃. More preferably, the stirring reaction temperature in step (S04) is 25℃. Preferably, the stirring reaction time in step (S04) is 36h~60h. More preferably, the stirring reaction time in step (S04) is 42h~54h. More preferably, the stirring reaction time in step (S04) is 48h. Preferably, in step (S04), the reaction endpoint is monitored by TLC. Preferably, in step (S05), the molecular weight cutoff of the dialysis bag is 950Da~1050Da. More preferably, the molecular weight cutoff of the dialysis bag in step (S05) is 1000Da. Preferably, in step (S05), the ultrapure water is replaced every 6h~10h until the conductivity of the dialysate is <5μS / cm. More preferably, in step (S05), the ultrapure water is replaced every 8 hours until the conductivity of the dialysate is <5 μS / cm. Preferably, the dialyzing time with ultrapure water in step (S05) is 3 to 7 days. More preferably, the dialyzing time with ultrapure water in step (S05) is 4 to 6 days. More preferably, the dialyzing time with ultrapure water in step (S05) is 5 days. Preferably, the lyophilization in step (S05) is performed in a lyophilizer. Preferably, the lyophilization time in step (S05) is 2 to 4 days. More preferably, the lyophilization time in step (S05) is 3 days. Preferably, after lyophilization in step (S05), the lyophilized product is sealed and stored at 24°C to 26°C. More preferably, after lyophilization in step (S05), the lyophilized product is sealed and stored at 25°C. Preferably, an appropriate amount of the lyophilized product from step (S05) is used to perform an NMR 1H2O spectrum, and the synthesis is analyzed using MestReNova software. If successful, it is stored for later use; if unsuccessful, steps (S01) to (S04) are repeated. Preferably, 10 mg of the lyophilized product from step (S05) is dissolved in D2O to perform an NMR 1H2O spectrum, and the synthesis is analyzed using MestReNova software. If a characteristic peak of -CH2CH2COOH appears at δ 2.5 ppm to 3.0 ppm, the synthesis is considered successful, and the product is stored in a sealed, light-protected desiccator. If unsuccessful, steps (S01) to (S04) are repeated. Preferably, the amount of CuCl2·2H2O used in step (S06) is 0.252 mmol. Preferably, the CuCl2·2H2O in step (S06) has the following specifications: CAS: 10125-13-0, purity ≥99%. Preferably, the amount of sodium citrate used in step (S06) is 0.4 mmol. Preferably, the sodium citrate used in step (S06) has the specification CAS: 68-04-2 and a purity ≥99%. Preferably, the amount of ultrapure water used in step (S06) is 20 ml.Preferably, the stirring temperature in step (S06) is 24℃~26℃. More preferably, the stirring temperature in step (S06) is 25℃. Preferably, the stirring time in step (S06) is 20min~40min. More preferably, the stirring time in step (S06) is 25min~35min. More preferably, the stirring time in step (S06) is 30min. Preferably, in step (S06), the pH is adjusted to 7~8 using 0.1M NaOH solution. More preferably, the pH is adjusted to 7.5 using 0.1M NaOH solution. Preferably, the amount of sodium sulfide nonahydrate used in step (S07) is 0.258mmol. Preferably, the sodium sulfide nonahydrate in step (S07) has the specification CAS: 1313-84-4 and a purity ≥98%. Preferably, the amount of ultrapure water used in step (S07) is 20mL. Preferably, solution II in step (S07) is an aqueous solution of sodium sulfide nonahydrate. Preferably, the concentration of the sodium sulfide nonahydrate aqueous solution is 0.0129 mol / L. Preferably, the stirring in step (S08) is magnetic stirring. Preferably, the stirring speed in step (S08) is 450 rpm to 550 rpm. More preferably, the stirring speed in step (S08) is 500 rpm. Preferably, the stirring time in step (S08) is 10 min to 20 min. More preferably, the stirring time in step (S08) is 15 min. Preferably, the continued stirring time in step (S08) is 8 min to 12 min. More preferably, the continued stirring time in step (S08) is 10 min. Preferably, the isothermal heating in step (S08) is an oil bath. Preferably, the isothermal heating temperature in step (S08) is 80°C to 95°C. More preferably, the isothermal heating temperature in step (S08) is 85℃~90℃. Preferably, the isothermal heating time in step (S08) is 0.5h~1.5h. More preferably, the isothermal heating time in step (S08) is 1h. Preferably, the isothermal heating in step (S08) continues until the solution color changes from brown to dark green. Preferably, the ice water cooling temperature in step (S09) is 3℃~5℃. More preferably, the ice water cooling temperature in step (S09) is 4℃. Preferably, the centrifugation speed for centrifugation collection in step (S09) is 11000rpm~13000rpm, and the centrifugation time is 20min~40min. More preferably, the centrifugation speed for centrifugation collection in step (S09) is 11500rpm~12500rpm, and the centrifugation time is 25min~35min.More preferably, in step (S09), the centrifugation speed for collection is 12000 rpm, and the centrifugation time is 30 min. Preferably, the washing in step (S09) involves ultrasonically redispersing the collected precipitate with an ethanol-water solution containing 1 mM sodium citrate, followed by centrifugation. Preferably, the volume ratio of ethanol to water is 1:1. Preferably, the washing is performed two to four times. More preferably, the washing is performed three times. Preferably, the lyophilization is carried out in a vacuum freeze dryer. Preferably, in step (S10), the concentration of the Pluronic CF127 solution is 20% w / v, the concentration of the hyaluronic acid solution is 2% w / v, and the concentration of the CuS-NDs solution is 0.5 mg / mL. Preferably, the volume of the Pluronic CF127 solution used is 3 mL, the volume of the hyaluronic acid solution used is 1.5 mL, and the volume of the CuS-NDs solution used is 2.37 mL. Preferably, the stirring time is 20 min to 40 min. More preferably, the stirring time is 25 min to 35 min. Even more preferably, the stirring time is 30 min. Preferably, the mixing container in step (S10) is a 5 ml glass bottle. Preferably, the mixing in step (S10) is performed on a vortex mixer. Preferably, the solution being mixed in step (S10) is sealed and shaken. Preferably, the shaking is performed on a shaker. Preferably, the shaking temperature is 3°C to 5°C. More preferably, the shaking temperature is 4°C. Preferably, the shaking time is overnight. Preferably, the settling temperature is 3°C to 5°C, and the settling time is 1 h to 3 h. More preferably, the settling temperature is 4°C, and the settling time is 2 h.

[0012] Preferably, the container used in step (S01) is a 100L metering tank. Preferably, in step (S01), the container is pre-dried and the operation is carried out under nitrogen or argon protection. Preferably, the amount of anhydrous tetrahydrofuran used in step (S01) is 30L.

[0013] Preferably, the amount of Pluronic F127 used in step (S01) is 12.6 kg. Preferably, the amount of N,N′-disuccinimidyl carbonate used in step (S01) is 1.025 kg. Preferably, the amount of 4-dimethylaminopyridine used in step (S01) is 0.489 kg. Preferably, the mixing in step (S01) is mechanical stirring. Preferably, the mechanical stirring speed is 200 rpm to 300 rpm. More preferably, the mechanical stirring speed is 250 rpm. Preferably, the mixing time in step (S01) is 10 min to 20 min. More preferably, the mixing time in step (S01) is 15 min. Preferably, the amount of anhydrous dimethyl sulfoxide added in step (S02) is 5 L. Preferably, the mixing in step (S02) is mechanical stirring. Preferably, the stirring and mixing temperature in step (S02) is 24℃~26℃. More preferably, the stirring and mixing temperature in step (S02) is 25℃. Preferably, the mechanical stirring speed is 200rpm~300rpm. More preferably, the mechanical stirring speed is 250rpm. Preferably, the stirring and mixing time in step (S02) is 12h~36h. More preferably, the stirring and mixing time in step (S02) is 18h~30h. More preferably, the stirring and mixing time in step (S02) is 24h. Preferably, in step (S02), the stirring and mixing continues until the solid is completely dissolved and the reaction solution is homogeneous and clear. Preferably, TLC monitoring shows that N,N′-disuccinimidyl carbonate is completely consumed, yielding a clear mixture II. Preferably, the amount of β-alanine used in step (S03) is 0.3564kg. Preferably, the amount of ultrapure water used in step (S03) is 50 ml to 100 ml. More preferably, the amount of ultrapure water used in step (S03) is 60 ml to 90 ml. More preferably, the amount of ultrapure water used in step (S03) is 70 ml to 80 ml. Preferably, the dissolution process in step (S03) is carried out in a vortex mixer. Preferably, the stirring in step (S04) is mechanical stirring. Preferably, the amount of CuCl2·2H2O used in step (S06) is 0.252 mol. Preferably, the amount of sodium citrate used in step (S06) is 0.4 mol. Preferably, the amount of ultrapure water used in step (S06) is 20 L. Preferably, the amount of sodium sulfide nonahydrate used in step (S07) is 0.252 mol. Preferably, the amount of ultrapure water used in step (S07) is 20L. Preferably, the stirring in step (S08) is mechanical stirring.

[0014] Preferably, in step (S10), the amount of Pluronic CF127 solution used is 3L, the amount of hyaluronic acid solution used is 1.5L, and the amount of CuS-NDs solution used is 2.37L. Preferably, the mixing container in step (S10) is a 10L stainless steel mechanically stirred tank with a cooling jacket. Preferably, the mixing in step (S10) is performed on a vortex mixer.

[0015] Preferably, the biocompatibility of the hydrogel product is tested, including the following steps: (A01) Take an appropriate amount of rat blood, centrifuge it, remove the supernatant, and obtain a collection; (A02) In step (A01), add an appropriate amount of PBS solution to the collection for resuspending, centrifuge again to remove the supernatant, repeat 2 to 3 times, and take the final resuspended solution; (A03) Divide the final resuspended solution from step (A02) into groups, and place each group of resuspended solution in the wells of a plate; take an appropriate amount of the hydrogel product, immerse it in physiological saline, extract it, and filter it to obtain the extract; divide the wells of the plate into three groups, and add an appropriate amount of extract, PBS solution, and 1% Triton solution to the wells of the three groups, respectively. After X-100 solution, place in an incubator for static culture; (A04) Use a pipette to take appropriate amounts of culture medium from different wells in step (A03) into PE tubes for centrifugation, and take an appropriate amount of supernatant to read the absorbance OD value at 545nm using an ELISA reader; (A05) Calculate the hemolysis rate of the hydrogel product extract based on the different absorbance values ​​in step (A04). If the hemolysis rate is less than 5%, the hydrogel product is considered to have no potential hemolysis.

[0016] Preferably, the amount of rat blood used in step (A01) is 0.8 ml to 1.2 ml. More preferably, the amount of rat blood used in step (A01) is 1 ml. Preferably, the centrifugation temperature in step (A01) is 23°C to 27°C. More preferably, the centrifugation temperature in step (A01) is 25°C. Preferably, the centrifugation speed in step (A01) is 3000 rpm to 4000 rpm. More preferably, the centrifugation speed in step (A01) is 3500 rpm. Preferably, the centrifugation time in step (A01) is 4 min to 6 min. More preferably, the centrifugation time in step (A01) is 5 min. Preferably, the amount of PBS solution added in step (A02) is 8 ml to 12 ml. More preferably, the amount of PBS solution added in step (A02) is 10 ml. Preferably, the final resuspension volume in step (A02) is 8 ml to 12 ml. More preferably, the volume of the resuspension in step (A02) is 10 ml. Preferably, the plate in step (A03) is a 24-well plate. Preferably, the amount of resuspension in each well in step (A03) is 0.4 ml to 1 ml. More preferably, the amount of resuspension in each well in step (A03) is 0.5 ml to 0.8 ml. Preferably, the hydrogel product in step (A03) is extracted in physiological saline at a ratio of 0.1 g / mL. Preferably, the extraction temperature in step (A03) is 35°C to 40°C. More preferably, the extraction temperature in step (A03) is 37°C to 38°C. Preferably, the extraction time in step (A03) is 18 h to 30 h. More preferably, the extraction time in step (A03) is 21 h to 27 h. More preferably, the extraction time in step (A03) is 24 h. Preferably, in step (A03), the amounts of the extract, PBS solution, and 1% Triton X-100 solution added are all 0.4 ml to 1 ml. More preferably, the amounts of the extract, PBS solution, and 1% Triton X-100 solution added in step (A03) are all 0.5 ml to 0.8 ml. Preferably, the incubation temperature in step (A03) is 35°C to 39°C. More preferably, the incubation temperature in step (A03) is 37°C. Preferably, the incubation time in step (A03) is 2 h to 4 h. More preferably, the incubation time in step (A03) is 3 h. Preferably, the volume of liquid drawn by the pipette in step (A04) is 0.4 ml to 0.6 ml. More preferably, the volume of liquid drawn by the pipette in step (A04) is 0.5 ml. Preferably, the centrifugation temperature in step (A04) is 23°C to 27°C. More preferably, the centrifugation temperature in step (A04) is 25°C.Preferably, the centrifugation speed in step (A04) is 1500 rpm to 2500 rpm. More preferably, the centrifugation speed in step (A04) is 2000 rpm. Preferably, the centrifugation time in step (A04) is 4 min to 6 min. More preferably, the centrifugation time in step (A04) is 5 min. Preferably, the amount of supernatant used in step (A04) is 90 μL to 110 μL; or the amount of supernatant used in step (A04) is 100 μL.

[0017] Preferably, the hemolysis rate in step (A05) is calculated as follows: Hemolysis rate (%) = [(OD sample - OD negative) / (OD positive - OD negative)] × 100%.

[0018] Preferably, the human umbilical vein endothelial cell staining experiment using the hydrogel product includes the following steps: (B01) Human umbilical vein endothelial cells are collected, digested, and made into a cell suspension, and the cells are counted; (B02) The cells from step (B01) are evenly distributed into the wells of a plate, and an appropriate amount of complete culture medium is added to each well before being placed in a CO2 incubator for adherent culture; (B03) An appropriate amount of the hydrogel product is immersed in physiological saline, extracted, centrifuged, and the supernatant is collected and filtered through a filter membrane to obtain the extract; The wells of the plate in step (B02) are divided into two groups, and transwell chambers are placed on the corresponding wells of the two groups of wells. After adding appropriate amounts of extract and fresh complete culture medium to the upper chamber of the transwell, continue co-culturing in a CO2 incubator; (B04) After the co-culturing in step (B03) is completed, remove the transwell chamber, aspirate the culture medium in the lower chamber, wash the cells in the lower chamber, add an appropriate amount of staining working solution to each well of the lower chamber, and continue to culture in a CO2 incubator in the dark; (B05) After the culture in step (B04) is completed, aspirate the staining working solution and wash the cells, then add an appropriate amount of PBS solution to each well, and observe under a fluorescence imaging microscope. Use Calcein-AM and propidium iodide for imaging, record and analyze cell morphology and the ratio of live to dead cells.

[0019] Preferably, the human umbilical vein endothelial cells in step (B01) are human umbilical vein endothelial cells of the fifth generation or less in good growth condition.

[0020] Preferably, the well plate in step (B02) is a 24-well plate. Preferably, in step (B02), the adherent cell culture reaches a cell count of 2.5 × 10⁻⁶ cells per well. 4 ~3.5×10 4 More preferably, in step (B02), the adherent cell culture reaches a cell count of 3 × 10⁶ cells per well. 4Preferably, in step (B02), the amount of complete culture medium added to each well is 0.9 mL to 1.1 mL. More preferably, in step (B02), the amount of complete culture medium added to each well is 1 mL. Preferably, the adherent culture temperature in step (B02) is 35°C to 40°C. More preferably, the adherent culture temperature in step (B02) is 37°C to 38°C. Preferably, the adherent culture time in step (B02) is 18 h to 30 h. More preferably, the adherent culture time in step (B02) is 24 h. Preferably, the CO2 concentration in the CO2 incubator in step (B02) is 5%. Preferably, in step (B03), the hydrogel product is extracted in physiological saline at a ratio of 0.1 g / mL. Preferably, the extraction temperature in step (B03) is 35°C to 40°C. More preferably, the extraction temperature in step (B03) is 37°C to 38°C. Preferably, the extraction time in step (B03) is 18h to 30h. More preferably, the extraction time in step (B03) is 21h to 27h. More preferably, the extraction time in step (B03) is 24h. Preferably, the pore size of the filter membrane in step (B03) is 0.2μm to 0.25μm. More preferably, the pore size of the filter membrane in step (B03) is 0.22μm to 0.24μm. Preferably, the amount of extract and fresh complete culture medium added to the upper chamber of each of the two transwell chambers in step (B03) is 0.4mL to 0.6mL. More preferably, the amount of extract and fresh complete culture medium added to the upper chamber of each of the two transwell chambers in step (B03) is 0.5mL. Preferably, the co-culture time in step (B03) is 18h to 30h. More preferably, the co-culture time in step (B03) is 24h. Preferably, in step (B04), the cells in the lower chamber are gently washed 2 to 3 times with preheated PBS solution. Preferably, the staining working solution in step (B04) consists of MCalcein-AM prepared with PBS and propidium iodide, wherein the amount of MCalcein-AM is 2 μM and the amount of propidium iodide is 4 μM. Preferably, the amount of staining working solution added to each well in step (B04) is 200 μL to 300 μL. More preferably, the amount of staining working solution added to each well in step (B04) is 250 μL. Preferably, the culture temperature in step (B04) is 35°C to 39°C. More preferably, the culture temperature in step (B04) is 37°C. Preferably, the culture time in step (B04) is 15 min to 25 min. More preferably, the culture time in step (B04) is 20 min. Preferably, in step (B05), after removing the staining working solution, the cells are washed with PBS 2 to 3 times.Preferably, the amount of PBS solution added to each well in step (B05) is 450 μL to 550 μL. More preferably, the amount of PBS solution added to each well in step (B05) is 500 μL.

[0021] Preferably, the hydrogel product is used for human umbilical vein endothelial cell toxicity experiments, comprising the following steps: (C01) Human umbilical vein endothelial cells are collected, digested, and prepared into a cell suspension, and cell counting is performed; (C02) The cells from step (C01) are added to the wells of a plate, and an appropriate amount of complete culture medium is added to each well. The cells are then dispersed evenly by pipetting the medium into each well; (C03) The plate from step (C02) is placed in a CO2 incubator for culture. When all cells have adhered and begun to divide, the culture medium in each well is removed, and the wells are divided into six groups. The following solutions are added to each group: 100% hydrogel product extract, 50% hydrogel product extract, 25% hydrogel product extract, cell-free fresh culture medium, fresh culture medium containing cells, and culture medium containing 10% hydrogel product extract, respectively. DMSO culture medium; (CO4) The solution after drug addition in step (CO3) is placed in a CO2 incubator for further culture, and CCK-8 assay is performed on the proliferated cells at time points of 12H, 24H and 48H respectively.

[0022] Preferably, the human umbilical vein endothelial cells in step (C01) are human umbilical vein endothelial cells of the fifth generation or less in good growth condition.

[0023] Preferably, the plate in step (CO2) is a 24-well plate. Preferably, the number of cells in the wells of the plate in step (CO2) is 4.5 × 10⁻⁶. 4 ~5.5×10 4 More preferably, the number of cells in the wells during step (CO2) is 5 × 10⁶. 4 Preferably, the amount of complete culture medium added to the wells in step (CO2) is 0.9 mL to 1.1 mL. More preferably, the amount of complete culture medium added to the wells in step (CO2) is 1 mL.

[0024] Preferably, the culture temperature in step (CO3) is 35℃~40℃. More preferably, the culture temperature in step (CO3) is 37℃~38℃. Preferably, the culture time in step (CO3) is 18h~30h. More preferably, the culture time in step (CO3) is 24h. Preferably, the CO2 concentration in the CO2 incubator in step (CO3) is 5%. Preferably, the amount of 100% hydrogel extract, 50% hydrogel extract, 25% hydrogel extract, cell-free fresh culture medium, cell-containing fresh culture medium, and culture medium containing 10% DMSO added in step (CO3) is 1mL each. Preferably, the number of wells in each group in step (CO3) is 3 to 6. More preferably, the number of wells in each group in step (CO3) is 4 to 5. The preparation of the hydrogel extract in step (C03) includes the following steps: (C03-1) the hydrogel product is cut into small pieces under aseptic conditions, added to complete endothelial cell culture medium, and then cultured in a CO2 incubator; (C03-2) the supernatant after culture in step (C03-1) is collected, filtered through a sterile filter, and the hydrogel extract is obtained; (C03-3) the hydrogel extract from step (C03-2) is diluted to 50% and 25% concentrations respectively using complete culture medium, and then temporarily stored under cold. Preferably, the mass-to-volume ratio of the hydrogel product to the complete endothelial cell culture medium in step (C03-1) is 0.1 g / mL. Preferably, the culture temperature in step (C03-1) is 35℃~40℃. More preferably, the culture temperature in step (C03-1) is 37℃~38℃. Preferably, the culture time in step (C03-1) is 18h~30h. More preferably, the incubation time in step (C03-1) is 24 hours. Preferably, the CO2 concentration in the CO2 incubator in step (C03-1) is 5%. Preferably, the pore size of the filter membrane in the sterile filter in step (C03-2) is 0.2 μm to 0.25 μm. More preferably, the pore size of the filter membrane in the sterile filter in step (C03-2) is 0.22 μm to 0.24 μm. Preferably, the refrigeration temperature in step (C03-3) is 3°C to 5°C. More preferably, the refrigeration temperature in step (C03-3) is 4°C.Preferably, at each time point in step (C04), CCK-8 reagent (10% of the original culture medium volume) is added to the test wells, and the plates are returned to the incubator for further incubation in the dark for 1.5-2.5 hours. After incubation, the liquid in each well is gently mixed with a pipette, and 140-160 μL is transferred to a 96-well plate. The absorbance (OD) value at 450 nm is measured using a microplate reader. The relative cell viability is calculated using the following formula: Cell viability (%) = [(OD experimental group - OD blank group) / (OD negative control group - OD blank group)] × 100%, where the OD blank group is the cell-free fresh culture medium group, and the OD negative control group is the cell-containing fresh culture medium group. If the cell viability is ≥70%, it indicates that the material has no potential cytotoxicity. Preferably, the incubation time in the dark is 2 hours. More preferably, 150 μL is transferred to a 96-well plate.

[0025] Preferably, a hydrogel product is used for the human umbilical vein endothelial cell scratch assay, comprising the following steps: (D01) Take a well plate and mark the bottom of the well plate with a marker line; (D02) Take human umbilical vein endothelial cells, and after the cells grow to 85%–90% confluence, digest them to prepare a cell suspension and count the cells; (D03) Distribute the cells from step (D02) evenly into the wells of the well plate, add complete culture medium to the wells until the cells form a dense monolayer, and use a pipette tip to draw several perpendicular lines along the marker line. The scratches were aligned with the marking lines, and the resulting intersections were used as fixed detection points. (D04) The culture medium from step (D03) was removed, and the plate was rinsed with sterile PBS. (D05) The scratches from step (D04) were divided into three groups. For each group, appropriate amounts of serum-free culture medium, serum-free culture medium containing VEGF, and serum-free culture medium containing 50% hydrogel extract were added to the wells of the plate. Images were recorded in a fluorescence imaging microscope at 0H, 12H, and 24H of incubation in a CO2 incubator. The width of the scratches in each field of view was measured using ImageJ software, the scratch healing rate was calculated, and statistical analysis was performed.

[0026] Preferably, the well plate in step (D01) is a 6-well plate. Preferably, the marking lines in step (D01) are two horizontal lines drawn with a marker along a ruler. Preferably, the human umbilical vein endothelial cells in step (D02) are human umbilical vein endothelial cells in good growth condition within the last 5 generations. Preferably, the cell density in the wells of the plate in step (D03) is 5.6 × 10⁻⁶. 5 ~6.5×10 5 More preferably, the cell density in the wells of the plate in step (D03) is 6 × 10⁶. 5Preferably, in step (D03), the amount of complete culture medium added to the well is 1.5 mL to 2.5 mL. More preferably, in step (D03), the amount of complete culture medium added to the well is 2 mL. Preferably, in step (D03), the incubation time after adding the complete culture medium to the well is 18 h to 24 h. More preferably, in step (D03), the incubation time after adding the complete culture medium to the well is 20 h to 22 h. Preferably, the pipette tip in step (D03) is a sterile 150 μL to 250 μL pipette tip. More preferably, the pipette tip in step (D03) is a sterile 200 μL pipette tip. Preferably, the vertical lines in step (D03) are parallel. Preferably, there are 2 to 4 vertical lines in step (D03). More preferably, there are 3 vertical lines in step (D03).

[0027] Preferably, the rinsing in step (D04) is performed 2 to 3 times. The preparation of the hydrogel extract in step (D05) includes the following steps: (D05-1) the hydrogel product is aseptically cut into small pieces, added to serum-free culture medium, and then incubated in a CO2 incubator; (D05-2) the supernatant from step (D05-1) is collected, filtered through a sterile filter to obtain the hydrogel extract; (D05-3) the hydrogel extract from step (D05-2) is diluted to a 50% concentration using complete culture medium and temporarily stored under cold. Preferably, the mass-to-volume ratio of the hydrogel product to the serum-free culture medium in step (D05-1) is 0.1 g / mL. Preferably, the incubation temperature in step (D05-1) is 35℃ to 40℃. More preferably, the incubation temperature in step (D05-1) is 37℃ to 38℃.

[0028] Preferably, the incubation time in step (D05-1) is 18h to 30h. More preferably, the incubation time in step (D05-1) is 24h. Preferably, the CO2 concentration in the CO2 incubator in step (D05-1) is 5%. Preferably, the pore size of the filter membrane in the sterile filter in step (D05-2) is 0.2μm to 0.25μm. More preferably, the pore size of the filter membrane in the sterile filter in step (D05-2) is 0.22μm to 0.24μm. Preferably, the refrigeration temperature in step (D05-3) is 3℃ to 5℃. More preferably, the refrigeration temperature in step (D05-3) is 4℃. Preferably, the serum-free culture medium containing VEGF in step (D05) is a serum-free culture medium containing 20ng / mL VEGF. Preferably, in step (D05), the amount of serum-free culture medium, serum-free culture medium containing VEGF, and serum-free culture medium containing 50% hydrogel extract added is 2 mL.

[0029] As a preferred method, the human umbilical vein endothelial cell tube formation assay is performed using a pre-prepared hydrogel, comprising the following steps: (E01) Thawing frozen Matrigel overnight in a refrigerator, and dispensing the required amount for later use; (E02) Wrapping well plates, EP tubes, and sterilization pipette tips in aluminum foil and pre-cooling them in a refrigerator; (E03) Spreading the liquid Matrigel dispensed in step (E01) onto the well plates from step (E02); (E04) Incubating the well plates from step (E03) in a CO2 incubator; (E05) Collecting human umbilical vein endothelial cells, passaged them when they reach logarithmic growth, and collecting the post-growth cells. The cells were prepared into a single-cell suspension and counted; (E06) The cell suspension from step (E05) was added to the wells of the plate after incubation in step (E04), and the plate was placed back into the CO2 incubator to allow the cells to adhere; (E07) The supernatant from each well in step (E06) was removed, and the wells were divided into three groups. An appropriate amount of EBM-2 basal medium, EBM-2 basal medium containing VEGF, and EBM-2 basal medium containing 50% hydrogel extract were added to each group of wells, respectively. The plate was then placed in the CO2 incubator for further culture; (E08) The plate cultured in step (E07) was observed and recorded under a fluorescence imaging microscope.

[0030] Preferably, the temperature in the refrigerator in step (E01) is 2°C to 6°C. More preferably, the temperature in the refrigerator in step (E01) is 4°C.

[0031] Preferably, the pre-cooling in step (E02) is performed 0.5 to 1.5 hours before the experiment. More preferably, the pre-cooling in step (E03) is performed 1 hour before the experiment. Preferably, the well plate in step (E02) is a sterile 96-well plate. Preferably, the pre-cooling temperature in step (E02) is -25°C to -15°C. More preferably, the pre-cooling temperature in step (E02) is -20°C. Preferably, the pre-cooling time in step (E02) is 0.3 to 1 hour. More preferably, the pre-cooling time in step (E02) is 0.5 hours. Preferably, the adhesive application in step (E03) is performed by vertically inserting the pre-cooled, sterilized pipette tip into the bottom of the wells of the well plate to add the adhesive without air bubbles. Preferably, the amount of liquid adhesive drawn by the sterilized pipette tip is 40 to 60 μL. More preferably, the amount of liquid adhesive drawn by the sterilized pipette tip is 50 μL.

[0032] Preferably, the incubation time in step (E04) is 30 min to 60 min. More preferably, the incubation time in step (E05) is 40 min to 50 min. More preferably, the incubation time in step (E05) is 45 min. Preferably, the incubation temperature in step (E04) is 35°C to 40°C. More preferably, the incubation temperature in step (E04) is 37°C to 38°C. Preferably, the CO2 concentration in the CO2 incubator in step (E04) is 5%. Preferably, the human umbilical vein endothelial cells in step (E05) are human umbilical vein endothelial cells of passage 5 or less in good growth condition. Preferably, the number of cells in each well of the plate in step (E06) is 1.5 × 10⁶ cells / well. 4 ~2.5×10 4 More preferably, in step (E06), the number of cells in each well of the plate is 2 × 10⁶. 4Preferably, the cell adhesion time in step (E06) is 0.5 h to 1.5 h. More preferably, the cell adhesion time in step (E06) is 1 h. The preparation of the hydrogel product extract in step (E07) includes the following steps: (E07-1) the hydrogel product is cut into small pieces under sterile conditions, added to serum-free culture medium, and then placed in a CO2 incubator for culture; (E07-2) the supernatant after culture in step (E07-1) is collected, filtered through a sterile filter, and the hydrogel product extract is obtained; (E07-3) the hydrogel product extract from step (E07-2) is diluted to a 50% concentration using complete culture medium and then temporarily stored under cold. Preferably, the mass-to-volume ratio of the hydrogel product to the serum-free culture medium in step (E07-1) is 0.1 g / mL. Preferably, the culture temperature in step (E07-1) is 35℃ to 40℃. More preferably, the culture temperature in step (E07-1) is 37℃~38℃. Preferably, the culture time in step (E07-1) is 18h~30h. More preferably, the culture time in step (E07-1) is 24h. Preferably, the CO2 concentration in the CO2 incubator in step (E07-1) is 5%. Preferably, the pore size of the filter membrane in the sterile filter in step (E07-2) is 0.2μm~0.25μm. More preferably, the pore size of the filter membrane in the sterile filter in step (E07-2) is 0.22μm~0.24μm. Preferably, the refrigeration temperature in step (E07-3) is 3℃~5℃. More preferably, the refrigeration temperature in step (E07-3) is 4℃. Preferably, in step (E07), the amount of EBM-2 basal medium, VEGF-containing EBM-2 basal medium, and 50% hydrogel extract-containing EBM-2 basal medium added to each well is 100 µL. Preferably, the VEGF-containing EBM-2 basal medium in step (E07) is EBM-2 basal medium containing 20 ng / mL VEGF. Preferably, the incubation time in step (E07) is 4 h to 8 h. More preferably, the incubation time in step (E07) is 5 h to 7 h. More preferably, the incubation time in step (E07) is 6 h. Preferably, in step (E08), ImageJ software is used to analyze the images, calculate the total tubular length, number of branch points, and number of grids formed in each field of view, perform statistical analysis on the data, and compare the differences between groups.

[0033] Preferably, the modulus test of the hydrogel product includes the following steps: (F01) Take an appropriate amount of the hydrogel product and use a solvent trap to perform dynamic oscillation amplitude balancing in the strain range of 0.01% to 100% using a rheometer equipped with multiple parallel plates, and record the storage modulus and loss modulus; (F02) Take the strain range where the storage modulus and loss modulus recorded in step (F01) remain stable, and perform angular frequency scanning at 1% strain; (F03) Use Origin plotting software to obtain the test diagram based on the scanning results in step (F02).

[0034] Preferably, in step (F01), the width of the parallel plates is 15mm to 25mm, and the spacing between adjacent parallel plates is 0.8mm to 1.2mm. More preferably, in step (F01), the width of the parallel plates is 20mm, and the spacing between adjacent parallel plates is 1mm. Preferably, in step (F01), the testing frequency of the rheometer is 0.5Hz to 1.5Hz. More preferably, in step (F01), the testing frequency of the rheometer is 1Hz. Preferably, in step (F01), the testing temperature of the rheometer is 20℃ to 30℃. More preferably, in step (F01), the testing temperature of the rheometer is 25℃. Preferably, in step (F01), the balancing time is 3min to 8min. More preferably, in step (F01), the balancing time is 5min. Preferably, in step (F02), the angular frequency range is 0.1rad / s to 100rad / s. Preferably, in step (F02), the modulus stability is monitored by scanning at a scanning temperature of 35℃ to 40℃ for 0.5h to 1.5h.

[0035] Preferably, the viscosity test of the hydrogel product includes the following steps: (G01) Take an appropriate amount of the hydrogel product and use a solvent trap to perform dynamic oscillation amplitude balancing in the strain range of 0.01% to 100% using a rheometer equipped with multiple parallel plates, and record the storage modulus and loss modulus; (G02) Take the strain range where the storage modulus and loss modulus are stable as recorded in step (G02), and gradually increase the temperature in the temperature range of 10℃ to 50℃ at 1% strain and constant shear rate, and record the changes in storage modulus and loss modulus. The temperature corresponding to the intersection point of storage modulus and loss modulus, or the inflection point of storage modulus, is taken as the sol-gel transition temperature; (G03) Use Origin plotting software to generate a test diagram based on the test results in step (G02).

[0036] Preferably, in step (G01), the width of the parallel plates is 15mm to 25mm, and the spacing between adjacent parallel plates is 0.8mm to 1.2mm. More preferably, in step (G01), the width of the parallel plates is 20mm, and the spacing between adjacent parallel plates is 1mm. Preferably, in step (G01), the testing frequency of the rheometer is 0.5Hz to 1.5Hz. More preferably, in step (G01), the testing frequency of the rheometer is 1Hz. Preferably, in step (G01), the testing temperature of the rheometer is 20℃ to 30℃. More preferably, in step (G01), the testing temperature of the rheometer is 25℃. Preferably, in step (G01), the balancing time is 3min to 8min. More preferably, in step (G01), the balancing time is 5min. Preferably, in step (G02), the constant shear rate is 40s. -1 ~60s -1 More preferably, the constant shear rate in step (G02) is 50 s. -1 Preferably, the heating rate during step (G02) is 0.5℃ / min to 1.5℃ / min. More preferably, the heating rate during step (G02) is 1℃ / min.

[0037] The present invention has the following beneficial effects: (1) After end modification of Pluronic F127, it is combined with hyaluronic acid (HA) and copper sulfide nanodots (CuS-NDs) to form a multifunctional hydrogel with good physicochemical properties and biological functions; Pluronic F127 is a thermosensitive block copolymer, and its solubility in water is closely related to temperature. The end-modified Pluronic F127 can effectively form hydrogen bonds with HA, so that the hydrogel can form a gel at body temperature after injection and can undergo sol-gel transition near body temperature. Using a chemical synthesis method, Pluronic F127 is end-modified by the reaction of N,N′-disuccinimidyl carbonate and β-alanine to transform Pluronic F127 into a multifunctional hydrogel with good physicochemical properties and biological functions. The terminal hydroxyl group of F127 is converted into a carboxyl group, which enables it to chemically crosslink with the amino or hydroxyl groups of HA to form a network, thereby enhancing the stability of the hydrogel. Hyaluronic acid, as a natural polysaccharide, has biocompatibility, moisturizing properties and cell adhesion. In hydrogel, it not only enhances the biological properties of the material, but also enhances the mechanical properties of the hydrogel. The introduction of CuS-NDs provides new functionality for hydrogel. Copper sulfide nanoparticles exert antibacterial effects by generating reactive oxygen species (ROS), releasing copper ions, destroying cell membranes and photothermal effects. They have significant inhibitory effects on a variety of pathogens. Their anti-inflammatory effects are manifested in clearing ROS, inhibiting the release of inflammatory factors, regulating macrophage polarization and protecting tissue cells, thus enabling the hydrogel to have photothermal therapy, imaging and antibacterial functions. (2) Using N,N′-disuccinimidyl carbonate to activate the terminal hydroxyl group of Pluronic F127 and then reacting it with β-alanine to introduce a carboxyl group can enhance the stability of Pluronic F127. F127 was modified with good carboxylation at the end, and CuS nanoparticles were synthesized by co-precipitation method. The steps were relatively simple. The combination of modified polymer, natural polysaccharide and functional nanomaterial can achieve complementary material functions. (4) In the preparation process, a simple physical synthesis method was adopted. By adjusting the amount of synthetic raw materials, temperature and reaction time, the structure and performance of hydrogel were optimized. Through physicochemical characterization, the successful synthesis and superior performance of hydrogel were verified. The results showed that the prepared hydrogel had good mechanical strength and viscosity, and at the same time showed excellent biodegradability and biocompatibility. The cell compatibility test of the hydrogel showed that it had no obvious toxicity to normal cells, indicating that the material has good safety in biomedical applications.(5) A method for preparing thermosensitive hydrogels was prepared by simply physical mixing hyaluronic acid and modified Pluronic F127 in an aqueous solution of CuS-NDs. By modifying the ends of Pluronic F127, the raw material for synthesizing hydrogels, carboxylic acids and other molecules were introduced into the ends of the polymer. These molecules exhibited stronger hydrogen bonding than Pluronic F127. The modified hydrogel (Pluronic F127) had higher viscosity and storage modulus. CuS-NDs were also introduced. The nanoparticles had certain antibacterial effects. In addition, Cu; 2+ It is a basic element for human health, present in various foods and drinking water, and can promote angiogenesis. First, Pluronic F127 was chemically modified at the end to introduce carboxyl groups, which enhanced the viscosity and storage modulus of the original Pluronic F127. Then, copper sulfide nanoparticles were synthesized. A certain amount of copper chloride dihydrate and sodium citrate were dissolved in a certain amount of pure water, stirred, and then a certain amount of sodium sulfide nonahydrate was added to the solution. The solution was heated to 90°C until it turned dark green. After centrifugation, washing, and freeze-drying, the powder was obtained for later use. A certain amount of Pluronic F127, Pluronic CF127, hyaluronic acid, and copper sulfide nanoparticle solutions were taken to prepare hydrogels with different compositions. The modulus and viscosity of the prepared hydrogels were tested, and related cell compatibility experiments were performed. Attached Figure Description

[0038] Figure 1 This is the nuclear magnetic resonance H-spectrum of PluronicCF127 of this invention; Figure 2 These are TEM images of CuS-NDs of the present invention; Figure 3 These are physical images of the three hydrogels of the present invention: F127 / HA, CF127 / HA, and CuS@CF127 / HA. Figure 4 This is a hemolysis experiment diagram of the three hydrogels of the present invention and an equal volume of 0.5 mg / mL CuS-NDs solution; Figure 5 This is an experimental diagram of a live-dead cell staining experiment performed by co-culturing three types of hydrogels and equal volumes of 0.5 mg / mL CuS-NDs solution with human umbilical vein endothelial cells (HUVECs) according to the present invention. Figure 6 This is a diagram of a cytotoxicity experiment (CCK-8) conducted by co-culturing three types of hydrogels and equal volumes of 0.5 mg / mL CuS-NDs solution with different concentrations of CuS-NDs solution and human umbilical vein endothelial cells (HUVECs) according to the present invention. Figure 7 This is an experimental diagram of a cell scratch assay performed by co-culturing three types of hydrogels and equal volumes of 0.5 mg / mL CuS-NDs solution with human umbilical vein endothelial cells (HUVECs) according to the present invention. Figure 8This is an experimental diagram of a cell tube formation experiment in which three types of hydrogels and equal volumes of 0.5 mg / mL CuS-NDs solution and human umbilical vein endothelial cells (HUVECs) were co-cultured. Figure 9 This is an experimental diagram showing the modulus test of two hydrogels synthesized by adding a small amount of hyaluronic acid to unmodified Pluronic F127 and modified Pluronic CF127, respectively. Figure 10 This is an experimental diagram showing the viscosity of two hydrogels synthesized by adding a small amount of hyaluronic acid to unmodified Pluronic F127 and modified Pluronic CF127, respectively. Detailed Implementation

[0039] The present invention will be further described below with reference to the accompanying drawings and embodiments, but this should not be construed as limiting the present invention.

[0040] Example 1: A method for preparing a hydrogel formed by end modification of Pluronic F127 combined with HA and CuS-NDs, comprising the following steps:

[0041] (S01) Take an appropriate amount of anhydrous tetrahydrofuran and place it in a container. Then, take an appropriate amount of Pluronic F127, N,N′-disuccinimidyl carbonate, and 4-dimethylaminopyridine and place them together in the container and stir to mix, obtaining mixture I. The container used in step (S01) is a 100ml glass bottle. The amount of anhydrous tetrahydrofuran used in step (S01) is 30ml. The specifications of the anhydrous tetrahydrofuran in step (S01) are CAS: 109-99-9, purity ≥99.9%. The amount of Pluronic F127 used in step (S01) is 12.6g. The specifications of Pluronic F127 in step (S01) are MW=12,600Da. The amount of N,N′-disuccinimidyl carbonate used in step (S01) is 1... 0.025g; the specification of N,N′-disuccinimidyl carbonate in step (S01) is CAS: 74124-79-1, purity ≥98%; the amount of 4-dimethylaminopyridine used in step (S01) is 0.489g; the specification of 4-dimethylaminopyridine in step (S01) is CAS: 1122-58-3, purity ≥99%; the stirring and mixing in step (S01) is magnetic stirring; the magnetic stirring speed is 400rpm; the stirring and mixing temperature in step (S01) is 24℃; the stirring and mixing time in step (S01) is 20min; in step (S01), the container is pre-dried and the operation is carried out under nitrogen or argon protection; in step (S01), based on the terminal hydroxyl group of Pluronic F127, the Pluronic... The molar ratio of F127, N,N′-disuccinimidyl carbonate, and 4-dimethylaminopyridine is 1:1.5:0.15; in step (S01), the volume ratio of 4-dimethylaminopyridine to anhydrous tetrahydrofuran is 1:5; Pluronic CF127 is synthesized using a two-step acetylation procedure; N,N′-disuccinimidyl carbonate (DSC) and 4-dimethylaminopyridine (DMAP) can activate Pluronic F1 27; N,N′-disuccinimidyl carbonate can activate hydroxyl groups to form succinimidyl carbonate derivatives because it has a very strong reactivity to nucleophiles; (S02) Add an appropriate amount of anhydrous dimethyl sulfoxide to the mixture I in step (S01) and stir to obtain mixture II; the amount of anhydrous dimethyl sulfoxide added in step (S02) is 5 ml; the specifications of the anhydrous dimethyl sulfoxide in step (S02) are CAS: 67-68-5, purity ≥99%.8%; the stirring and mixing in step (S02) is magnetic stirring; the magnetic stirring speed is 400 rpm; the stirring and mixing temperature in step (S02) is 24℃; the stirring and mixing time in step (S02) is 36 h; (S03) an appropriate amount of β-alanine is added to ultrapure water, the pH is adjusted to 8-9 and then stirred to dissolve, obtaining a solution; the amount of β-alanine used in step (S03) is 0.3564 g; the specification of β-alanine in step (S03) is CAS: 107-95-9, purity ≥99%; the amount of ultrapure water used in step (S03) is 5 ml; in step (S03), the pH is adjusted to 8 by using a 1M NaOH solution; the dissolution process in step (S03) is carried out in a vortex mixer.

[0042] (S04) The solution from step (S03) is added dropwise to mixture II from step (S02) and stirred to obtain a reaction solution; the solution in step (S04) is added slowly dropwise; the stirring in step (S04) is magnetic stirring; the reaction temperature in step (S04) is 24℃; the reaction time in step (S04) is 60h; in step (S04), the reaction endpoint is monitored by TLC; (S05) The reaction solution from step (S04) is placed in a dialysis bag and dialyzed with ultrapure water, and the retentate is freeze-dried to constant weight to obtain the modified Pluronic F127, as shown below. Figure 1As shown, it is designated as PluronicCF127; the molecular weight cutoff of the dialysis bag in step (S05) is 950 Da; the ultrapure water in step (S05) is replaced every 6 hours until the conductivity of the dialysate is <5 μS / cm; the dialysis time with ultrapure water in step (S05) is 3 days to remove unreacted components; the lyophilization in step (S05) is carried out in a lyophilizer; the lyophilization time in step (S05) is 2 days; after lyophilization in step (S05), the lyophilized product is sealed and stored at 24°C; an appropriate amount of the lyophilized product from step (S05) is taken for nuclear magnetic resonance H2S analysis. The spectrum was analyzed using MestReNova software to determine if the synthesis was successful. If successful, the sample was stored for future use; otherwise, steps (S01) to (S04) were repeated. 10 mg of the lyophilized product from step (S05) was dissolved in D2O to generate an NMR 1H NMR spectrum. The spectrum was analyzed using MestReNova software to determine if the synthesis was successful. If a characteristic peak of -CH2CH2COOH appeared at δ2.5 ppm–3.0 ppm, the synthesis was considered successful. The sample was stored in a sealed, light-protected desiccator. If unsuccessful, steps (S01) to (S04) were repeated. Compared to the original Pluronic F127 spectrum, new characteristic peaks appeared at δ2.5 ppm–3.0 ppm and δ3.3 ppm–3.4 ppm, indicating that Pluronic synthesis was successfully achieved. The terminal carboxylation modification of F127 showed peaks at δ 2.5 ppm to 3.0 ppm, which were attributed to the protons of the methylene group in the added β-alanine, and peaks at δ 3.3 ppm to 3.4 ppm, which were attributed to the protons of the methylene group linked to the amide bond, indicating successful chemical modification. (S06) Appropriate amounts of CuCl2·2H2O and sodium citrate were added to ultrapure water, the pH was adjusted to 7, and the solution was stirred to dissolve, yielding solution I. The amount of CuCl2·2H2O used in step (S06) was 0.252 mmol. The specifications of CuCl2·2H2O in step (S06) were CAS: 10125-13-0, purity ≥99%. In step (S06), the amount of sodium citrate used is 0.4 mmol; the specification of sodium citrate in step (S06) is CAS: 68-04-2, purity ≥99%; the amount of ultrapure water used in step (S06) is 20 ml; the stirring temperature in step (S06) is 24℃; the stirring time in step (S06) is 40 min; in step (S06), the pH is adjusted to 7 using 0.1 M NaOH solution; (S07) an appropriate amount of sodium sulfide nonahydrate is added to ultrapure water and stirred to dissolve under nitrogen protection to obtain solution II; the amount of sodium sulfide nonahydrate used in step (S07) is 0.258 mmol; the sodium sulfide nonahydrate in step (S07) has the specification CAS: 1313-84-4 and a purity ≥98%; the amount of ultrapure water used in step (S07) is 20 mL; solution II in step (S07) is an aqueous solution of sodium sulfide nonahydrate; the concentration of the aqueous solution of sodium sulfide nonahydrate is 0.0129 mol / L.

[0043] (S08) Under nitrogen protection, solution II from step (S07) is added dropwise to solution I from step (S06) and stirred until mixed. After the addition is complete, stirring continues, followed by constant temperature heating to obtain a CuS-NDs solution. The stirring in step (S08) is magnetic stirring; the stirring speed in step (S08) is 450 rpm; the stirring time in step (S08) is 20 min; the constant temperature heating in step (S08) is an oil bath; the constant temperature heating temperature in step (S08) is 80℃; the constant temperature heating time in step (S08) is 1.5 h; the constant temperature heating in step (S08) continues until the solution color changes from brown to dark green. (S09) The CuS-NDs solution from step (S08) is sequentially cooled with ice water, collected by centrifugation, washed, and freeze-dried to obtain freeze-dried CuS-NDs powder, such as... Figure 2As shown; the ice-water cooling temperature in step (S09) is 3℃; the centrifugation speed for collection in step (S09) is 11000 rpm, and the centrifugation time is 40 min; the washing in step (S09) involves ultrasonically redispersing the collected precipitate with an ethanol-water solution containing 1 mM sodium citrate, followed by centrifugation; the volume ratio of ethanol to water is 1:1; the washing is performed twice; the freeze-drying is carried out in a vacuum freeze dryer; the image shows uniformly dispersed and approximately spherical nanoparticles with a narrow particle size distribution, indicating the successful synthesis of copper sulfide nanoparticles with uniform size and good dispersion, and a clear crystal lattice. The stripes further demonstrate its good crystallinity; (S10) Pluronic CF127 from step (S05) is prepared into a PBS solution of Pluronic CF127, hyaluronic acid is prepared into a PBS solution of hyaluronic acid, CuS-NDs powder from step (S08) is prepared into a PBS solution of CuS-NDs powder, appropriate amounts of Pluronic CF127 solution, hyaluronic acid solution and CuS-NDs solution are mixed and stirred in an ice-water bath, and then allowed to stand to obtain the hydrogel product, denoted as CuS@CF127 / HA; Pluronic CF127 from step (S10) The concentration of solution 7 is 20% w / v, the concentration of the hyaluronic acid solution is 2% w / v, and the concentration of the CuS-NDs solution is 0.5 mg / mL; the volume of Pluronic CF127 solution used is 3 mL, the volume of the hyaluronic acid solution used is 1.5 mL, and the volume of the CuS-NDs solution used is 2.37 mL; the stirring time is 20 min; the mixing container in step (S10) is a 5 mL glass bottle; the mixing and stirring in step (S10) is performed on a vortex mixer; the solution mixed and stirred in step (S10) is sealed and shaken; the shaking is performed on a shaker. The shaking temperature is 3℃; the shaking time is overnight; the standing temperature is 3℃, and the standing time is 1h; Pluronic F127 is prepared into a PBS solution of Pluronic F127; appropriate amounts of Pluronic F127 solution, hyaluronic acid solution, and pure water are mixed and stirred in an ice-water bath, and then allowed to stand to obtain the hydrogel product, denoted as F127 / HA; the concentration of Pluronic F127 solution is 20% w / v; the volume of Pluronic F127 solution used is 3 mL; the volume of hyaluronic acid solution used is 1.5 mL; and the volume of water used is 2 mL.37 mL; Take appropriate amounts of Pluronic F127 solution, hyaluronic acid solution, and CuS-NDs solution, mix and stir in an ice-water bath, and then let stand to obtain the hydrogel product, denoted as CF127 / HA; The concentration of the Pluronic F127 solution is 20% w / v, the amount of the Pluronic F127 solution used is 3 mL, the amount of the hyaluronic acid solution used is 1.5 mL, and the amount of the CuS-NDs solution used is 2.37 mL, as shown. Figure 3 As shown, three inverted test tubes or vials contain F127 / HA (with strong fluidity), CF127 / HA (forming a more stable gel), and CuS@CF127 / HA (its color is deepened by CuS, forming a uniform dark gel). This visually demonstrates the formation state of the hydrogels, indicating that the carboxylation modification of Pluronic F127 and the combination of CF127 and hyaluronic acid (HA) enhance the stability of the gel. The addition of CuS-NDs successfully composited the gel without damaging its structure, resulting in a uniform gel.

[0044] Example 2: This example is basically the same as Example 1, except that the magnetic stirring speed is 600 rpm; the stirring and mixing temperature in step (S01) is 26℃; the stirring and mixing time in step (S01) is 10 min; in step (S01), based on the terminal hydroxyl group of Pluronic F127, the molar ratio of Pluronic F127, N,N′-disuccinimidyl carbonate, and 4-dimethylaminopyridine is 1:2.5:0.25; in step (S01), the volume ratio of 4-dimethylaminopyridine to anhydrous tetrahydrofuran is 1:7; the magnetic stirring speed is 600 rpm; the stirring and mixing temperature in step (S02) is 26℃; the stirring and mixing time in step (S02) is 12 h; (S03) Take an appropriate amount of β-alanine and add it to ultrapure water, adjust the pH to 9, and stir to dissolve to obtain a solution; In step (S03), 15 ml of ultrapure water is used; in step (S03), the pH is adjusted to 9 using a 1M NaOH solution; in step (S04), the stirring reaction temperature is 26°C; in step (S04), the stirring reaction time is 36 h; in step (S05), the molecular weight cutoff of the dialysis bag is 1050 Da; in step (S05), the ultrapure water is replaced every 10 h until the conductivity of the dialysate is <5 μS / cm; in step (S05), the dialysis time with ultrapure water is... The interval is 7 days; the freeze-drying time in step (S05) is 4 days; after freeze-drying in step (S05), the freeze-dried product is sealed and stored at 26℃; (S06) take an appropriate amount of CuCl2·2H2O and sodium citrate and add them to ultrapure water, adjust the pH to 8 and stir to dissolve, to obtain solution I; the stirring temperature in step (S06) is 26℃; the stirring time in step (S06) is 20 min; the stirring speed in step (S08) is 550 rpm; the stirring time in step (S08) is... 10 min; the constant temperature heating temperature in step (S08) is 95℃; the constant temperature heating time in step (S08) is 0.5 h; the ice water cooling temperature in step (S09) is 5℃; the centrifugation speed for centrifugation collection in step (S09) is 13000 rpm, and the centrifugation time for centrifugation collection is 20 min; the number of washing cycles is four; the stirring time is 40 min; the shaking is performed on a shaker; the shaking temperature is 5℃; the settling temperature is 5℃, and the settling time is 1 h;

[0045] Example 3: This example is basically the same as Example 1, except that the magnetic stirring speed is 500 rpm; the stirring and mixing temperature in step (S02) is 25°C; the stirring and mixing time in step (S01) is 15 min; in step (S01), based on the terminal hydroxyl group of Pluronic F127, the molar ratio of Pluronic F127, N,N′-disuccinimidyl carbonate, and 4-dimethylaminopyridine is 1:2:0.2; the volume ratio of 4-dimethylaminopyridine to anhydrous tetrahydrofuran is 1:6; the magnetic stirring speed is 500 rpm; the stirring and mixing temperature in step (S02) is 25°C; the stirring and mixing time in step (S02) is 24 h; the amount of ultrapure water used in step (S03) is 10 ml; in step (S03), the water is passed through a 1 M NaO solution. The pH of the solution was adjusted to 8.5; the dissolution process in step (S03) was carried out in a vortex mixer; the stirring reaction temperature in step (S04) was 25℃; the stirring reaction time in step (S04) was 48h; the molecular weight cutoff of the dialysis bag in step (S05) was 1000Da; the ultrapure water was replaced every 8h in step (S05) until the conductivity of the dialysate was <5μS / cm; the dialysis time with ultrapure water in step (S05) was 5 days; the process in step (S04) was... 5) The freeze-drying time is 3 days; after freeze-drying in step (S05), the freeze-dried product is sealed and stored at 25°C; the stirring temperature in step (S06) is 25°C; the stirring time in step (S06) is 30 min; in step (S06), the pH is adjusted to 7.5 using 0.1M NaOH solution; the stirring speed in step (S08) is 500 rpm; the stirring time in step (S08) is 15 min; the continued... The stirring time is 10 min; the constant temperature heating temperature in step (S08) is 85℃; the constant temperature heating time in step (S08) is 1 h; the ice water cooling temperature in step (S09) is 4℃; the centrifugation speed for centrifugation collection in step (S09) is 12000 rpm, and the centrifugation time for centrifugation collection is 30 min; the washing number is three times; the stirring time is 30 min; the shaking temperature is 4℃; the settling temperature is 4℃, and the settling time is 2 h.

[0046] Example 4: A method for preparing a hydrogel formed by end modification of Pluronic F127 combined with HA and CuS-NDs, comprising the following steps:

[0047] (S01) Take an appropriate amount of anhydrous tetrahydrofuran and place it in a container. Then, take an appropriate amount of Pluronic F127, N,N′-disuccinimidyl carbonate, and 4-dimethylaminopyridine and place them together in the container and stir to mix, obtaining mixture I. The container used in step (S01) is a 100L metering vessel. In step (S01), the container is pre-dried and the operation is carried out under nitrogen or argon protection. The amount of anhydrous tetrahydrofuran used in step (S01) is 30L. The amount of Pluronic F127 in step (S01) is... The amount of 27 used is 12.6 kg; the amount of N,N′-disuccinimidyl carbonate used in step (S01) is 1.025 kg; the amount of 4-dimethylaminopyridine used in step (S01) is 0.489 kg; the stirring and mixing in step (S01) is mechanical stirring co-mixing; the speed of the mechanical stirring is 200 rpm; the stirring and mixing time in step (S01) is 20 min; (S02) an appropriate amount of anhydrous dimethyl sulfoxide is added to the mixture I in step (S01) and stirred and mixed. The mixture was prepared as follows: Mixture II was obtained; the amount of anhydrous dimethyl sulfoxide added in step (S02) was 5 L; the stirring and mixing in step (S02) was mechanical stirring; the stirring and mixing temperature in step (S02) was 24 °C; the mechanical stirring speed was 200 rpm; the stirring and mixing time in step (S02) was 36 h; the mixture was stirred and mixed until the solid was completely dissolved and the reaction solution was homogeneous and clear; TLC monitoring showed that N,N′-disuccinimidyl carbonate was completely consumed, resulting in a clear mixture. Liquid II; (S03) Add an appropriate amount of β-alanine to ultrapure water, adjust the pH to 8, and stir to dissolve, obtaining a solution; the amount of β-alanine used in step (S03) is 0.3564 kg; the amount of ultrapure water used in step (S03) is 50 ml; the dissolution process in step (S03) is carried out in a vortex mixer; (S04) Add the solution from step (S03) dropwise into the mixture II from step (S02) and stir to react, obtaining a reaction solution; the stirring in step (S04) is mechanical stirring;

[0048] (S05) The reaction solution from step (S04) is placed in a dialysis bag and dialyzed with ultrapure water. The retentate is lyophilized to constant weight to obtain the modified Pluronic F127, denoted as Pluronic CF127; (S06) An appropriate amount of CuCl2·2H2O and sodium citrate are added to ultrapure water, the pH is adjusted to 7, and the solution is stirred to dissolve, yielding solution I; the amount of CuCl2·2H2O used in step (S06) is 0.252 mol; the amount of sodium citrate used in step (S06) is 0.4 mol; the amount of ultrapure water used in step (S06) is 20 L; (S07) An appropriate amount of sodium sulfide nonahydrate is added to ultrapure water, and the solution is dissolved in water. Dissolved under nitrogen protection by stirring to obtain solution II; the amount of sodium sulfide nonahydrate used in step (S07) is 0.252 mol; the amount of ultrapure water used in step (S07) is 20 L; (S08) under nitrogen protection, solution II from step (S07) is added dropwise to solution I from step (S06) and stirred until mixed. After the addition is complete, stirring continues, followed by constant temperature heating to obtain CuS-NDs solution; the stirring in step (S08) is mechanical stirring; (S09) the CuS-NDs solution from step (S08) is successively cooled with ice water, collected by centrifugation, washed, and freeze-dried to obtain freeze-dried CuS-NDs powder; (S10) the steps (S05) The Pluronic CF127 solution was prepared into a PBS solution, and the hyaluronic acid solution was prepared into a PBS solution. The CuS-NDs powder from step (S08) was prepared into a PBS solution. Appropriate amounts of the Pluronic CF127 solution, hyaluronic acid solution, and CuS-NDs solution were mixed and stirred in an ice-water bath, and then allowed to stand to obtain the hydrogel product. In step (S10), the amount of Pluronic CF127 solution used was 3 L, the amount of hyaluronic acid solution used was 1.5 L, and the amount of CuS-NDs solution used was 2.37 L. The mixing container in step (S10) is a 10L stainless steel mechanically stirred tank with a cooling jacket; the mixing and stirring in step (S10) is carried out on a vortex mixer; Pluronic F127 is prepared into a PBS solution of Pluronic F127; appropriate amounts of Pluronic F127 solution, hyaluronic acid solution and pure water are taken, mixed and stirred in an ice-water bath, and then allowed to stand to obtain the hydrogel product, denoted as F127 / HA; the concentration of the Pluronic F127 solution is 20% w / v, the amount of Pluronic F127 solution used is 3L, the amount of hyaluronic acid solution used is 1.5L, and the amount of water used is 2L.37L; Take appropriate amounts of Pluronic F127 solution, hyaluronic acid solution, and CuS-NDs solution, mix and stir in an ice-water bath, and then let stand to obtain the hydrogel product, denoted as CF127 / HA; The concentration of the Pluronic F127 solution is 20% w / v, the amount of the Pluronic F127 solution used is 3L, the amount of the hyaluronic acid solution used is 1.5L, and the amount of the CuS-NDs solution used is 2.37L.

[0049] Example 5: This example is basically the same as Example 4, except that the mechanical stirring speed is 300 rpm; the stirring and mixing time in step (S01) is 10 min; the stirring and mixing temperature in step (S02) is 26℃; the mechanical stirring speed is 300 rpm; the stirring and mixing time in step (S02) is 12 h; (S03) Take an appropriate amount of β-alanine and add it to ultrapure water, adjust the pH to 9 and stir to dissolve, to obtain a solution; the amount of ultrapure water used in step (S03) is 100 ml; (S06) Take an appropriate amount of CuCl2·2H2O and sodium citrate and add them to ultrapure water, adjust the pH to 8 and stir to dissolve, to obtain solution I.

[0050] Example 6: This example is basically the same as Example 4, except that the mechanical stirring speed is 250 rpm; the stirring and mixing time in step (S01) is 15 min; the stirring and mixing temperature in step (S02) is 25℃; the mechanical stirring speed is 250 rpm; the stirring and mixing time in step (S02) is 24 h; (S03) Take an appropriate amount of β-alanine and add it to ultrapure water, adjust the pH to 8.5 and stir to dissolve, to obtain a solution; the amount of ultrapure water used in step (S03) is 70 ml; (S06) Take an appropriate amount of CuCl2·2H2O and sodium citrate and add them to ultrapure water, adjust the pH to 7.5 and stir to dissolve, to obtain solution I.

[0051] Example 7: The biocompatibility of the hydrogel product was tested, including the following steps: (A01) An appropriate amount of rat blood was centrifuged and the supernatant was removed to obtain a collection; the amount of rat blood used in step (A01) was 0.8 ml; the centrifugation temperature in step (A01) was 23℃; the centrifugation speed in step (A01) was 3000 rpm; the centrifugation time in step (A01) was 6 min; (A02) After adding an appropriate amount of PBS solution to the collection in step (A01) for resuspending, the supernatant was removed by centrifugation again, and this was repeated twice to obtain the final resuspended solution; the amount of PBS solution added in step (A02) was 8 ml; the volume of the final resuspended solution in step (A02) was 8 ml; (A03) The final resuspended solution in step (A02) was divided into groups, and each group of resuspended solution was placed in the well of a plate; an appropriate amount of the hydrogel product was immersed in physiological saline, extracted, and then filtered to obtain an extract;

[0052] The wells of the plate were divided into three groups. Appropriate amounts of extraction buffer, PBS solution, and 1% Triton X-100 solution were added to the wells of each group, and the plates were then placed in an incubator for static incubation. In step (A03), the resuspension volume in each well was 0.4 ml. In step (A03), the hydrogel product was extracted in physiological saline at a ratio of 0.1 g / mL. The extraction temperature in step (A03) was 35℃. The extraction time in step (A03) was 30 h. The extraction buffer, PBS solution, and 1% Triton X-100 solution were used in step (A03). The volume of X-100 solution added was 0.4 ml; the culture temperature in step (A03) was 35℃; the culture time in step (A03) was 4 h; (A04) using a pipette, appropriate amounts of culture medium from different wells in step (A03) were transferred to PE tubes for centrifugation, and an appropriate amount of supernatant was taken to read the absorbance (OD) value at 545 nm using an ELISA reader; the volume of the pipette in step (A04) was 0.4 ml; the centrifugation temperature in step (A04) was 23℃; the centrifugation speed in step (A04) was 1500 rpm; the centrifugation time in step (A04) was 6 min; the volume of supernatant used in step (A04) was 90 μl;

[0053] (A05) Based on the different absorbance values ​​in step (A04), calculate the hemolysis rate of the hydrogel product extract. If the hemolysis rate is less than 5%, the hydrogel product is considered to have no potential hemolysis. The formula for calculating the hemolysis rate in step (A05) is: Hemolysis rate (%) = [(OD sample - OD negative) / (OD positive - OD negative)] × 100%.

[0054] Example 8: This example is basically the same as Example 7, except that the amount of rat blood used in step (A01) is 1.2 ml; the centrifugation temperature in step (A01) is 27°C; the centrifugation speed in step (A01) is 4000 rpm; the centrifugation time in step (A01) is 4 min; (A02) after adding an appropriate amount of PBS solution to the collected material for resuspending in step (A01), centrifuging again to remove the supernatant, repeating 3 times, and taking the final resuspended solution; the amount of PBS solution added in step (A02) is 12 ml; the volume of the final resuspended solution in step (A02) is 12 ml; the amount of resuspended solution in each well in step (A03) is 1 ml; the extraction temperature in step (A03) is 40°C; the extraction time in step (A03) is 18 h; and the extraction solution, PBS solution, and 1% Triton in step (A03) are used in step (A03). The amount of X-100 solution added was 1 ml; the culture temperature in step (A03) was 39℃; the culture time in step (A03) was 2 h; the volume of liquid aspirated by the pipette in step (A04) was 0.6 ml; the centrifugation temperature in step (A04) was 27℃; the centrifugation speed in step (A04) was 2500 rpm; the centrifugation time in step (A04) was 4 min; and the amount of supernatant used in step (A04) was 110 μl.

[0055] Example 9: As Figure 4As shown, this embodiment is basically the same as Embodiment 7, except that: in step (A01), the amount of rat blood used is 1 ml; the centrifugation temperature in step (A01) is 25°C; the centrifugation speed in step (A01) is 3500 rpm; the centrifugation time in step (A01) is 5 min; in step (A02), after adding an appropriate amount of PBS solution to the collected material for resuspending in step (A01), centrifugation is performed again to remove the supernatant, and this is repeated 3 times to collect the final resuspended solution; in step (A02), the amount of PBS solution added is 10 ml; the volume of the final resuspended solution in step (A02) is 10 ml; in step (A03), the amount of resuspended solution in each well is 0.5 ml; the extraction temperature in step (A03) is 37°C; the extraction time in step (A03) is 24 h; and the extract, PBS solution, and 1% Triton are used in step (A03). The amount of X-100 solution added was 0.5 ml; the culture temperature in step (A03) was 37℃; the culture time in step (A03) was 3 h; the volume of pipette aspirated in step (A04) was 0.5 ml; the centrifugation temperature in step (A04) was 25℃; the centrifugation speed in step (A04) was 2000 rpm; the centrifugation time in step (A04) was 5 min; the volume of supernatant used in step (A04) was 100 μl; a set of EP tube photographs after centrifugation showed that the supernatant of the positive control Triton X-100 was bright red, and the supernatant of the negative control physiological saline was clear. The supernatants of the three hydrogel extracts and CuS-NDs solutions in each experimental group should be as clear as the negative control or only very pale red, indicating that all hydrogel materials and CuS-NDs had no obvious hemolytic activity and good blood compatibility, indicating that the biomaterials are safe for use in contact with blood.

[0056] Example 10: Staining experiment of human umbilical vein endothelial cells using a hydrogel product, including the following steps: (B01) Human umbilical vein endothelial cells are collected, digested, and made into a cell suspension, and cell counting is performed; the human umbilical vein endothelial cells in step (B01) are human umbilical vein endothelial cells in good growth condition within the 5th generation; (B02) The cells in step (B01) are evenly distributed into the wells of a plate, and an appropriate amount of complete culture medium is added to each well before being placed in a CO2 incubator for adherent culture; the plate in step (B02) is a 24-well plate; the adherent culture in step (B02) is performed until the cell count in the well is 2.5 × 10⁶. 4The amount of complete culture medium added to each well in step (B02) is 0.9 mL; the adherent culture temperature in step (B02) is 35℃; the adherent culture time in step (B02) is 30 h; (B03) Take an appropriate amount of the hydrogel product and immerse it in physiological saline, centrifuge after extraction, collect the supernatant, and then filter through a membrane to obtain the extract; divide the wells on the plate in step (B02) into two groups, place transwell chambers on the corresponding wells of the two groups of wells respectively, and add to the upper chambers of the two transwell chambers respectively. After adding an appropriate amount of extract and fresh complete culture medium, continue co-culturing in a CO2 incubator; in step (B03), the hydrogel product is extracted in physiological saline at a ratio of 0.1 g / mL; the extraction temperature in step (B03) is 35℃; the extraction time in step (B03) is 30 h; the pore size of the filter membrane in step (B03) is 0.2 μm; the amount of extract and fresh complete culture medium added to the upper chamber of each of the two transwell chambers in step (B03) is 0.4 mL; The co-culture time in step (B03) is 18 hours; (B04) after the co-culture in step (B03) is completed, remove the Transwell chamber, aspirate the culture medium in the lower chamber, wash the cells in the lower chamber, add an appropriate amount of staining working solution to each well of the lower chamber, and continue to incubate in a CO2 incubator in the dark; in step (B04), gently wash the cells in the lower chamber twice with preheated PBS solution; the staining working solution in step (B04) is MCalcei-AM prepared with PBS and propidium iodide. The solution was prepared by adding 2 μM Calcein-AM and 4 μM propidium iodide to each well in step (B04). The culture temperature in step (B04) was 35 °C. After step (B04) was completed, the staining solution was removed and the cells were washed. Then, an appropriate amount of PBS solution was added to each well, and the cells were observed under a fluorescence imaging microscope. Calcein-AM and propidium iodide were imaged separately, and the cell morphology and the ratio of live to dead cells were recorded and analyzed.

[0057] Example 11: This example is basically the same as Example 10, except that in step (B02), the adherent cell number in the well is 3.5 × 10⁻⁶. 4The following steps were performed: Step (B02) – 1.1 mL of complete culture medium was added to each well; Step (B02) – Adherent culture temperature was 40°C; Step (B02) – Adherent culture time was 18 h; Step (B03) – Extraction temperature was 40°C; Step (B03) – Extraction time was 18 h; Step (B03) – 0.25 μm pore size of the filter membrane; Step (B03) – 0.6 mL of extract and fresh complete culture medium were added to the upper chamber of each of the two transwell chambers; Step (B03) – Co-culture time was 30 h; Step (B04) – Cells in the lower chamber were gently washed three times with preheated PBS solution; Step (B04) – 300 u of staining working solution was added to each well; Step (B04) – Culture temperature was 39°C; Step (B04) – Culture time was 15 min.

[0058] Example 12: As Figure 5 As shown, this embodiment is basically the same as Embodiment 10, except that in step (B02), the adherent cell culture is carried out until the number of cells in the well is 3 × 10⁻⁶. 4 The following steps were performed: In step (B02), 1 mL of complete culture medium was added to each well; the adherent culture temperature in step (B02) was 37°C; the adherent culture time in step (B02) was 24 h; the extraction temperature in step (B03) was 37°C; the extraction time in step (B03) was 24 h; the pore size of the filter membrane in step (B03) was 0.22 μm; in step (B03), 0.5 mL of extract and fresh complete culture medium were added to the upper chamber of each of the two transwell chambers; and the co-culture time in step (B03) was 24 h. In step (B04), the cells in the lower chamber are gently washed three times with preheated PBS solution; the amount of staining working solution added to each well in step (B04) is 250 μL; the culture temperature in step (B04) is 37 °C; the culture time in step (B04) is 20 min; in the combined image of fluorescence microscopy, the field of view should be filled with HUVECs emitting green fluorescence, with good morphology and only a few or no red fluorescent dots, indicating that the hydrogel extract has no acute cytotoxicity to HUVECs, the cell survival rate is high, and the morphology is normal, indicating that the material has good cell compatibility.

[0059] Example 13: A human umbilical vein endothelial cell toxicity experiment was conducted using a hydrogel product, comprising the following steps: (C01) Human umbilical vein endothelial cells were collected, digested, and prepared into a cell suspension, followed by cell counting; the human umbilical vein endothelial cells in step (C01) were healthy human umbilical vein endothelial cells within the 5th generation; (C02) The cells from step (C01) were added to the wells of a 24-well plate, and an appropriate amount of complete culture medium was added to each well. The cells were then dispersed evenly by pipetting each well; the 24-well plate in step (C02) contained 4.5 × 10⁻⁶ cells. 4 Each; the amount of complete culture medium added to the wells in step (CO2) is 0.9 mL;

[0060] (C03) Place the well plate from step (C02) in a CO2 incubator. Once all cells have adhered and begun to divide, aspirate the culture medium from each well and divide the wells into six groups. Add to each group the following concentrations of culture medium: 100% hydrogel extract, 50% hydrogel extract, 25% hydrogel extract, cell-free fresh culture medium, fresh culture medium containing cells, and culture medium containing 10%... The culture medium contains DMSO; the culture temperature in step (C03) is 35℃; the culture time in step (C03) is 30h; the CO2 concentration in the CO2 incubator in step (C03) is 5%; the addition volume of 100% hydrogel product extract, 50% hydrogel product extract, 25% hydrogel product extract, cell-free fresh culture medium, cell-containing fresh culture medium, and culture medium containing 10% DMSO in step (C03) is 1mL each; each group in step (C03) has 3 wells; the preparation of the hydrogel product extract in step (C03) includes the following steps: (C03-1) the hydrogel product is cut into small pieces under aseptic conditions, added to complete endothelial cell culture medium, and then placed in a CO2 incubator for culture; the mass-to-volume ratio of the hydrogel product to the complete endothelial cell culture medium in step (C03-1) is 0.1g / mL; the culture temperature in step (C03-1) is 35℃. The culture time in step (C03-1) is 30 hours; the CO2 concentration in the CO2 incubator in step (C03-1) is 5%; (C03-2) the supernatant after culture in step (C03-1) is collected and filtered through a sterile filter to obtain the hydrogel product extract; the pore size of the filter membrane in the sterile filter in step (C03-2) is 0.2 μm; (C03-3) the hydrogel product extract from step (C03-2) is diluted separately using complete culture medium. After reaching concentrations of 50% and 25%, the cells were temporarily stored under refrigeration; the refrigeration temperature in step (C03-3) was 3°C; (C04) the solution after drug addition in step (C03) was placed in a CO2 incubator for further culture, and CCK-8 assays were performed on the proliferated cells at time points of 12H, 24H, and 48H; at each time point in step (C04), CCK-8 reagent at 10% of the original culture medium volume was added to the well to be tested, and the cells were returned to the incubator for further incubation in the dark for 1 hour.After 5 hours of incubation, gently mix the liquid in each well with a pipette, then transfer 140 μL to a 96-well plate. Measure the absorbance (OD) at 450 nm using a microplate reader. Calculate the relative cell viability using the following formula: Cell viability (%) = [(OD experimental group - OD blank group) / (OD negative control group - OD blank group)] × 100%, where the OD blank group is the cell-free fresh culture medium group, and the OD negative control group is the cell-containing fresh culture medium group. If the cell viability is ≥70%, it indicates that the material has no potential cytotoxicity.

[0061] Example 14: This example is basically the same as Example 13, except that the number of cells in the wells during step (CO2) is 5.5 × 10⁻⁶. 4 The following steps are described: Step (CO2) involves adding 1.1 mL of complete culture medium to each well; Step (CO3) involves incubation at 40°C; Step (CO3) involves incubation for 18 hours; Each group in Step (CO3) contains 6 wells; Step (CO3-1) involves incubation at 40°C; Step (CO3-1) involves incubation for 18 hours; Step (CO3-2) involves using a sterile filter with a membrane pore size of 0.25 μm; Step (CO3-3) involves refrigeration at 5°C. ℃; at each time point in step (C04), add 10% of the original culture medium volume of CCK-8 reagent to the test wells, then return them to the incubator for further incubation in the dark for 2.5 h; after incubation, gently mix the liquid in each well with a pipette, then transfer 160 μL to a 96-well plate, and measure the absorbance (OD) value at 450 nm using a microplate reader; calculate the relative cell viability according to the following formula: cell viability (%) = [(OD experimental group - OD blank group) / (OD negative control group - OD blank group)] × 100%,

[0062] The OD blank group was a cell-free fresh culture medium group, and the OD negative control group was a cell-containing fresh culture medium group. If the cell survival rate was ≥70%, it indicated that the material had no potential cytotoxicity.

[0063] Example 15: As Figure 6 As shown, this embodiment is basically the same as embodiment 13, except that the number of cells in the wells in step (CO2) is 5 × 10⁻⁶. 4The following steps are described: Step (C02) involves adding 1 mL of complete culture medium to each well; Step (C03) involves incubation at 37°C; Step (C03) involves incubation for 24 hours; Each group in Step (C03) contains 4 wells; Step (C03-1) involves incubation at 37°C; Step (C03-1) involves incubation for 24 hours; Step (C03-2) involves using a sterile filter membrane with a pore size of 0.22 μm; Step (C03-3) involves refrigeration at 4°C; and Step (C04) involves adding 1 mL of complete culture medium to each well at each time point. After adding 10% of the original culture medium volume of CCK-8 reagent, the culture was returned to the incubator and incubated in the dark for another 2 hours. 150 μL was then transferred to a 96-well plate. The horizontal axis of the bar chart represents the concentration gradient of different material groups and CuS-NDs, and the vertical axis represents the cell survival rate (%). The survival rate of the negative control was close to 100%, while the survival rate of the positive control was very low. The height of the bars in each experimental group should be close to or even higher than that of the negative control group, and should not decrease significantly with the increase of CuS-NDs concentration. Quantitative analysis confirmed that the hydrogel and the CuS-NDs contained therein did not inhibit cell proliferation within the tested concentration range, indicating that the material has good safety.

[0064] Example 16: Scratch assay of human umbilical vein endothelial cells using a hydrogel product, including the following steps: (D01) Take a well plate and mark the bottom of the well plate with marking lines; the well plate in step (D01) is a 6-well plate; the marking lines in step (D01) are two horizontal lines drawn with a marker along a ruler; (D02) Take human umbilical vein endothelial cells, digest them after they grow to 85%-90% confluence, and then perform cell suspension and cell counting; the human umbilical vein endothelial cells in step (D02) are human umbilical vein endothelial cells of good growth condition within the 5th generation;

[0065] (D03) Distribute the cells from step (D02) evenly into the wells of the plate, and add complete culture medium to the wells until the cells form a dense monolayer. Use a pipette tip to draw several perpendicular lines along the marking line, so that the scribing lines intersect the marking line, and the resulting intersections are used as fixed detection points; the cell density in the wells of step (D03) is 5.6 × 10⁻⁶. 5The following steps were performed: (D03) 1.5 mL of complete culture medium was added to each well; (D04) the culture time after adding the complete culture medium was 18 h; (D05) the pipette tip used in step (D06) was sterile (150 μL); (D06) several vertical lines in step (D03) were parallel; (D07) there were two vertical lines in step (D08); (D08) the culture medium in step (D03) was removed and rinsed with sterile PBS; (D09) the rinsing was performed twice; (D000) the scratches in step (D04) were divided into three groups, and appropriate amounts of serum-free culture medium, serum-free culture medium containing VEGF, and serum-free culture medium containing 50% hydrogel extract were added to the wells of each group of scratches, respectively. The images were recorded in a fluorescence imaging microscope at 0 H, 12 H, and 24 H of incubation in a CO2 incubator. The J software measures the width of each scratch in each field of view, calculates the scratch healing rate, and performs statistical analysis. In step (D05), the serum-free culture medium containing VEGF is a serum-free culture medium containing 20 ng / mL VEGF. In step (D05), the addition volume of the serum-free culture medium, the serum-free culture medium containing VEGF, and the serum-free culture medium containing 50% hydrogel extract is 2 mL each. The preparation of the hydrogel extract in step (D05) includes the following steps: (D05-1) the hydrogel product is cut into small pieces under aseptic conditions, added to the serum-free culture medium, and then placed in a CO2 incubator for incubation. In step (D05-1), the mass-to-volume ratio of the hydrogel product to the serum-free culture medium is 0.1 g / mL. The incubation temperature in step (D05-1) is... 35℃; the culture time in step (D05-1) is 30h; the CO2 concentration in the CO2 incubator in step (D05-1) is 5%; (D05-2) collect the supernatant after the culture in step (D05-1), and then filter it through a sterile filter to obtain the hydrogel product extract; the pore size of the filter membrane in the sterile filter in step (D05-2) is 0.2μm; (D05-3) take the hydrogel product extract in step (D05-2), dilute it to a concentration of 50% with complete culture medium, and then refrigerate it temporarily; the refrigeration temperature in step (D05-3) is 3℃.

[0066] Example 17: This example is basically the same as Example 16, except that the cell density in the wells of step (D03) is 6.5 × 10⁻⁶. 5The following steps are described: Step (D03) involves adding 2.5 mL of complete culture medium to each well; Step (D03) involves incubating for 24 hours after adding the complete culture medium; Step (D03) uses sterile 250 μL pipette tips; Step (D03) includes four vertical lines; Step (D04) involves 3 rinses; Step (D05-1) involves an incubation temperature of 40°C; Step (D05-1) involves an incubation time of 18 hours; Step (D05-2) uses a sterile filter membrane with a pore size of 0.2 μm; and Step (D05-3) involves a refrigeration temperature of 5°C.

[0067] Example 18: As Figure 7 As shown, this embodiment is basically the same as embodiment 16, except that the cell density in the wells of step (D03) is 6 × 10⁻⁶. 5 The following steps are described: Step (D03) involves adding 2 mL of complete culture medium to each well; Step (D03) involves incubating for 20 hours after adding the complete culture medium; Step (D03) uses sterile 200 μL pipette tips; Step (D03) includes three vertical lines; Step (D04) involves three rinses; Step (D05-1) involves an incubation temperature of 37°C; Step (D05-1) involves an incubation time of 24 hours; Step (D05... -2) The pore size of the filter membrane in the sterile filter is 0.22 μm; the refrigeration temperature in step (D05-3) is 4℃; a series of bright-field micrographs at 0h, 12h and 24h show that, compared with the negative control group, the scratches in the pores containing the hydrogel extract closed faster and more completely at the same time point, indicating that the hydrogel extract, especially the composite hydrogel extract containing CuS-NDs, can significantly promote the migration ability of human umbilical vein endothelial cells, indicating that the material has the potential to promote wound healing or angiogenesis.

[0068] Example 19: Human umbilical vein endothelial cell tube formation experiment using a pre-prepared hydrogel, including the following steps: (E01) Thawing frozen Matrigel overnight in a refrigerator, and dispensing the required amount; the temperature in the refrigerator in step (E01) is 2°C; (E02) Wrapping the well plate, EP tubes, and sterilization pipette tip in aluminum foil and pre-cooling them in a refrigerator; the pre-cooling in step (E02) is 0.5 hours before the experiment; the well plate in step (E02) is a sterile 96-well plate; the pre-cooling temperature in step (E02) is -25°C; the pre-cooling time in step (E02) is 0.3 hours; (E03) Spreading the liquid Matrigel dispensed in step (E01) onto the well plate in step (E02); the spreading in step (E03) is done by vertically inserting the liquid Matrigel into the bottom of the wells of the well plate using the pre-cooled sterilization pipette tip to add it without air bubbles; The amount of liquid matrix gel drawn by the sterilization pipette tip is 40 μL; (E04) The well plate after gel coating in step (E03) is placed in a CO2 incubator for incubation; the incubation time in step (E04) is 30 min; the incubation temperature in step (E04) is 40 °C; the CO2 concentration in the CO2 incubator in step (E04) is 5%; (E05) Human umbilical vein endothelial cells are taken, and when the cells reach the logarithmic growth state, they are passaged, and the grown cells are made into a single-cell suspension for cell counting; the human umbilical vein endothelial cells in step (E05) are human umbilical vein endothelial cells in good growth state within the 5th generation; (E06) The cell suspension in step (E05) is added to the wells of the plate after incubation in step (E04), and the plate is placed back in the CO2 incubator to allow the cells to adhere; the number of cells in each well of the plate in step (E06) is 1.5 × 10⁶. 4The cell adhesion time in step (E06) is 0.5 h; (E07) the supernatant from each well in step (E06) is removed, and the wells are divided into three groups. Appropriate amounts of EBM-2 basal medium, EBM-2 basal medium containing VEGF, and EBM-2 basal medium containing 50% hydrogel extract are added to each group of wells, respectively. The wells are then placed in a CO2 incubator for further culture; in step (E07), the amount of EBM-2 basal medium, EBM-2 basal medium containing VEGF, and EBM-2 basal medium containing 50% hydrogel extract added to each group of wells is 100 µL; the EBM-2 basal medium containing VEGF in step (E07) contains 20 ng / mL... The VEGF-based EBM-2 basal medium; the culture time in step (E07) is 4 hours; the preparation of the hydrogel extract in step (E07) includes the following steps: (E07-1) the hydrogel product is aseptically cut into small pieces, added to serum-free medium, and then placed in a CO2 incubator for culture; the mass-to-volume ratio of the hydrogel product to the serum-free medium in step (E07-1) is 0.1 g / mL; the culture temperature in step (E07-1) is 35℃; the culture time in step (E07-1) is 30 hours; the CO2 incubator in step (E07-1) The CO2 concentration is 5%; (E07-2) Collect the supernatant after the culture in step (E07-1), and then filter it through a sterile filter to obtain the hydrogel product extract; the pore size of the filter membrane in the sterile filter in step (E07-2) is 0.2 μm; (E07-3) Take the hydrogel product extract in step (E07-2), dilute it to a concentration of 50% with complete culture medium, and then refrigerate it temporarily; the refrigeration temperature in step (E07-3) is 3℃; (E08) Place the well plate after the culture in step (E07) in a fluorescence imaging microscope for observation and recording; in step (E08), use ImageJ software to analyze the images, calculate the total tubular length, the number of branch points and the number of grids formed in each field of view, perform statistical analysis on the data, and compare the differences between groups.

[0069] Example 20: This example is basically the same as Example 19, except that the temperature in the refrigerator in step (E01) is 6°C; the pre-cooling in step (E02) is performed 1.5 hours before the experiment; the pre-cooling temperature in step (E02) is -15°C; the pre-cooling time in step (E02) is 1 hour; the amount of liquid matrix gel drawn by the sterilization pipette tip is 60 μL; the incubation time in step (E04) is 60 minutes; the incubation temperature in step (E04) is 35°C; and the number of cells in each well of the plate in step (E06) is 2.5 × 10⁶. 4The cell adhesion time in step (E06) is 1.5 h; the culture time in step (E07) is 8 h; the culture temperature in step (E07-1) is 40 °C; the culture time in step (E07-1) is 18 h; the pore size of the filter membrane in the sterile filter in step (E07-2) is 0.25 μm; the refrigeration temperature in step (E07-3) is 5 °C.

[0070] Example 21: As Figure 8 As shown, this embodiment is basically the same as Embodiment 19, except that the temperature in the refrigerator in step (E01) is 4°C; the pre-cooling in step (E03) is performed 1 hour before the experiment; the pre-cooling temperature in step (E02) is -20°C; the pre-cooling time in step (E02) is 0.5 hours; the amount of liquid matrix gel drawn by the sterilization pipette tip is 50 μL; the incubation time in step (E05) is 45 minutes; the incubation temperature in step (E04) is 37°C; and the number of cells in each well of the plate in step (E06) is 2 × 10⁶ cells / well. 4 The cell adhesion time in step (E06) is 1 hour; the culture time in step (E07) is 5 hours; the incubation time in step (E07) is 6 hours; the culture temperature in step (E07-1) is 37°C; the culture time in step (E07-1) is 24 hours; the pore size of the filter membrane in the sterile filter in step (E07-2) is 0.22 μm; the refrigeration temperature in step (E07-3) is 4°C; bright-field microscopy images show that the negative control group forms a sparse and incomplete tubular network, while the positive control group forms a dense and complex tubular network. In the experimental group, the CuS@CF127 / HA group should form a more complex tubular structure with more branches and a longer total length than the negative control group, with an effect close to the positive control. This indicates that the hydrogel extract, especially CuS@CF127 / HA, can effectively induce endothelial cells to form vascular-like structures in vitro and has strong pro-angiogenic activity, indicating that this material can be well used in the field of tissue regeneration.

[0071] Example 22: Modulus testing of the hydrogel product includes the following steps: (F01) Take an appropriate amount of the hydrogel product and use a solvent trap. Using a rheometer equipped with multiple parallel plates, perform dynamic oscillation amplitude testing within a strain range of 0.01% to 100% to achieve equilibrium before the test, and record the storage modulus and loss modulus. In step (F01), the width of the parallel plates is 15 mm, and the spacing between adjacent parallel plates is 0.8 mm. In step (F01), the testing frequency of the rheometer is 0.5 Hz. In step (F01), the testing temperature of the rheometer is... The temperature is 20℃; the equilibration time in step (F01) is 3 min; (F02) take the strain range when the storage modulus and loss modulus are stable as recorded in step (F01), and select 1% strain for angular frequency scanning; the angular frequency range in step (F02) is 0.1 rad / s to 100 rad / s; in step (F02), scan at a scanning temperature of 35℃ for 0.5 h to monitor the stability of the modulus; (F03) use Origin plotting software to obtain the test diagram based on the scanning results in step (F02).

[0072] Example 23: This example is basically the same as Example 22, except that the width of the parallel plate in step (F01) is 25mm and the spacing between adjacent parallel plates is 1.2mm; the test frequency of the rheometer in step (F01) is 1.5Hz; the test temperature of the rheometer in step (F01) is 30℃; the equilibration time in step (F01) is 8min; and in step (F02), the stability of the modulus is monitored by scanning at a scanning temperature of 40℃ for 1.5h.

[0073] Example 24: As Figure 9 As shown, this embodiment is basically the same as embodiment 22, except that the modulus of the hydrogel product is tested, including the following steps: in step (F01), the width of the parallel plates is 20 mm and the spacing between adjacent parallel plates is 1 mm; in step (F01), the test frequency of the rheometer is 1 Hz; in step (F01), the test temperature of the rheometer is 25℃; in step (F01), the equilibration time is 5 min; in step (F02), the scanning temperature is 37℃ for 1 h to monitor the stability of the modulus; the amplitude scanning curve, with frequency on the horizontal axis and modulus Pa on the vertical axis, the two curves represent F127 / HA and CF127 / HA respectively. The storage modulus curve of CF127 / HA is significantly higher than that of F127 / HA, and its linear viscoelastic region may be wider, indicating that the carboxylation modification of Pluronic F127 and its interaction with HA significantly enhance the mechanical strength and structural stability of the hydrogel. A stronger gel is more conducive to maintaining its shape and supporting cells in vivo.

[0074] Example 25: Viscosity testing of the hydrogel product includes the following steps: (G01) Take an appropriate amount of the hydrogel product and use a solvent trap. Using a rheometer equipped with multiple parallel plates, perform dynamic oscillation amplitude testing within a strain range of 0.01% to 100% to achieve equilibrium before the test, and record the storage modulus and loss modulus. In step (G01), the width of the parallel plates is 15 mm, and the spacing between adjacent parallel plates is 0.8 mm. In step (G01), the testing frequency of the rheometer is 0.5 Hz. The rheometer test temperature is 20℃; the equilibrium time in step (G01) is 8 min; (G02) taking the strain range where the storage modulus and loss modulus remain stable as recorded in step (G02), and gradually increasing the temperature within the range of 10℃ to 50℃ under 1% strain and constant shear rate, the changes in storage modulus and loss modulus are recorded. The temperature corresponding to the intersection point of storage modulus and loss modulus, or the inflection point of storage modulus, is taken as the sol-gel transition temperature; the constant shear rate in step (G02) is 40 s. -1 The heating rate during the measurement process in step (G02) is 0.5℃ / min; (G03) The test graph is obtained using Origin plotting software based on the test results in step (G02).

[0075] Example 26: This example is basically the same as Example 26, except that the width of the parallel plate in step (G01) is 25mm and the cross-sectional area is 1.2mm; the test frequency of the rheometer in step (G01) is 1.5Hz; the test temperature of the rheometer in step (G01) is 30℃; the equilibration time in step (G01) is 3min; and the constant shear rate in step (G02) is 60s. -1 The heating rate during the measurement process in step (G02) is 1.5℃ / min.

[0076] Example 27: As Figure 10 As shown, this embodiment is basically the same as embodiment 26, except that the width of the parallel plates in step (G01) is 20mm and the spacing between adjacent parallel plates is 1mm; the test frequency of the rheometer in step (G01) is 1Hz; the test temperature of the rheometer in step (G01) is 25℃; the equilibration time in step (G01) is 5min; and the constant shear rate in step (G02) is 50s. -1The heating rate during the measurement process in step (G02) is 1℃ / min; the temperature scan curves, with temperature (℃) on the horizontal axis and viscosity (Pa·s) on the vertical axis, represent F127 / HA and CF127 / HA respectively. Both exhibit typical thermosensitive characteristics, with low viscosity at low temperature and a sharp increase in viscosity at a certain temperature point as the temperature rises. The phase transition temperature of CF127 / HA is closer to 37℃, and the plateau modulus after the phase transition is higher, indicating that the modified hydrogel retains thermosensitivity, and its gelation behavior, transition temperature, and final strength have been optimized, making it more suitable for in-situ gelation applications at body temperature.

Claims

1. A method for preparing hydrogels formed by end modification of Pluronic F127 combined with HA and CuS-NDs, characterized by: Including the following step, (S01) Take an appropriate amount of anhydrous tetrahydrofuran and place it in a container. Then take an appropriate amount of Pluronic F127, N,N′-disuccinimidyl carbonate and 4-dimethylaminopyridine and place them in the container together and stir to mix to obtain mixture I. (S02) Add an appropriate amount of anhydrous dimethyl sulfoxide to the mixture I in step (S01) and stir to obtain mixture II; (S03) Take an appropriate amount of β-alanine and add it to ultrapure water. Adjust the pH to 8-9 and stir to dissolve to obtain a solution. (S04) Add the solution from step (S03) dropwise into the mixture II from step (S02) and stir to react, to obtain the reaction solution; (S05) The reaction solution in step (S04) is placed in a dialysis bag and dialyzed with ultrapure water. The reflux is taken and freeze-dried to constant weight to obtain the modified Pluronic F127, which is denoted as Pluronic CF127. (S06) Take an appropriate amount of CuCl2·2H2O and sodium citrate and add them to ultrapure water. Adjust the pH to 7-8 and stir to dissolve to obtain solution I; (S07) Take an appropriate amount of sodium sulfide nonahydrate and add it to ultrapure water. Stir and dissolve it under nitrogen protection to obtain solution II; (S08) Under nitrogen protection, solution II from step (S07) is added dropwise to solution I from step (S06) and stirred until the mixture is completely added. After stirring, the mixture is heated at a constant temperature to obtain CuS-NDs solution. (S09) The CuS-NDs solution in step (S08) is successively cooled with ice water, collected by centrifugation, washed and freeze-dried to obtain freeze-dried CuS-NDs powder. (S10) Prepare a PBS solution of PluronicCF127 from step (S05), prepare a PBS solution of hyaluronic acid from hyaluronic acid, prepare a PBS solution of CuS-NDs powder from step (S08), take appropriate amounts of PluronicCF127 solution, hyaluronic acid solution and CuS-NDs solution, mix and stir in an ice water bath, and then let stand to obtain the hydrogel product.

2. The method for preparing the hydrogel formed by end modification of Pluronic F127 combined with HA and CuS-NDs according to claim 1, characterized in that: In step (S01), the amount of anhydrous tetrahydrofuran used is 30 ml; the amount of Pluronic F127 used in step (S01) is 12.6 g; the amount of N,N′-disuccinimidyl carbonate used in step (S01) is 1.025 g; the amount of 4-dimethylaminopyridine used in step (S01) is 0.489 g; the stirring and mixing in step (S01) is magnetic stirring; the magnetic stirring speed is 400 rpm to 600 rpm; the stirring and mixing temperature in step (S01) is 24℃ to 26℃; and the stirring and mixing time in step (S01) is 10 min to 20 min.

3. The method for preparing the hydrogel formed by end modification of Pluronic F127 combined with HA and CuS-NDs according to claim 1, characterized in that: In step (S01), the container is pre-dried and operated under nitrogen or argon protection; in step (S01), the molar ratio of Pluronic F127, N,N′-disuccinimidyl carbonate and 4-dimethylaminopyridine is 1:1.5-2.5:0.15-0.25, based on the terminal hydroxyl group of Pluronic F127; in step (S01), the volume ratio of 4-dimethylaminopyridine to anhydrous tetrahydrofuran is 1:5-7.

4. The method for preparing the hydrogel formed by end modification of Pluronic F127 combined with HA and CuS-NDs according to claim 1, characterized in that: In step (S02), the amount of anhydrous dimethyl sulfoxide added is 5 ml; the stirring and mixing in step (S02) is magnetic stirring; the magnetic stirring speed is 400 rpm to 600 rpm; the stirring and mixing temperature in step (S02) is 24℃ to 26℃; the stirring and mixing time in step (S02) is 12 h to 36 h; in step (S03), the amount of β-alanine used is 0.3564 g; the amount of ultrapure water used in step (S03) is 5 ml to 15 ml; in step (S03), the pH is adjusted to 8 to 9 using a 1 M NaOH solution; in step (S04), the solution is added slowly dropwise; the stirring reaction temperature in step (S04) is 24℃ to 26℃; the stirring reaction time in step (S04) is 36 h to 60 h; in step (S04), the reaction endpoint is monitored by TLC.

5. The method for preparing the hydrogel formed by end modification of Pluronic F127 combined with HA and CuS-NDs according to claim 1, characterized in that: In step (S05), the molecular weight cutoff of the dialysis bag is 950 Da to 1050 Da; in step (S05), the ultrapure water is replaced every 6 to 10 hours until the conductivity of the dialysate is <5 μS / cm; in step (S05), the dialysis time with ultrapure water is 3 to 7 days; in step (S05), the lyophilization time is 2 to 4 days; after lyophilization in step (S05), the lyophilized product is sealed and stored at 24℃ to 26℃; an appropriate amount of the lyophilized product from step (S05) is used to perform a nuclear magnetic resonance H-ray spectrum, and the synthesis is analyzed using MestReNova software. If successful, it is kept for later use; if unsuccessful, steps (S01) to (S04) are repeated.

6. The method for preparing the hydrogel formed by end modification of Pluronic F127 combined with HA and CuS-NDs according to claim 1, characterized in that: In step (S06), the amount of CuCl2·2H2O used is 0.252 mmol; the amount of sodium citrate used in step (S06) is 0.4 mmol; the amount of ultrapure water used in step (S06) is 20 ml; the stirring temperature in step (S06) is 24℃~26℃; the stirring time in step (S06) is 20 min~40 min; in step (S06), the pH is adjusted to 7~8 using 0.1M NaOH solution; in step (S07), the amount of sodium sulfide nonahydrate used is 0.258 mmol; the amount of ultrapure water used in step (S07) is... The volume is 20 mL; the stirring in step (S08) is magnetic stirring; the stirring speed in step (S08) is 450 rpm to 550 rpm; the stirring time in step (S08) is 10 min to 20 min; the continued stirring time in step (S08) is 8 min to 12 min; the constant temperature heating in step (S08) is an oil bath; the constant temperature heating temperature in step (S08) is 80℃ to 95℃; the constant temperature heating time in step (S08) is 0.5 h to 1.5 h; the constant temperature heating in step (S08) continues until the solution color changes from brown to dark green.

7. The method for preparing the hydrogel formed by end modification of Pluronic F127 combined with HA and CuS-NDs according to claim 1, characterized in that: In step (S09), the ice water cooling temperature is 3℃~5℃; the centrifugation speed for collection in step (S09) is 11000rpm~13000rpm, and the centrifugation time is 20min~40min; the washing in step (S09) involves ultrasonically redispersing the collected precipitate with an ethanol-water solution containing 1mM sodium citrate, followed by centrifugation; the volume ratio of ethanol to water is 1:1; the washing is performed two to four times; in step (S10), the concentration of the Pluronic CF127 solution is 20%w / v, the concentration of the hyaluronic acid solution is 2%w / v, and the concentration of the CuS-NDs solution... The concentration is 0.5 mg / mL; the volume of the Pluronic CF127 solution is 3 mL, the volume of the hyaluronic acid solution is 1.5 mL, and the volume of the CuS-NDs solution is 2.37 mL; the stirring time is 20 min to 40 min; the mixing container in step (S10) is a 5 mL glass bottle; the mixing and stirring in step (S10) is carried out on a vortex mixer; the solution mixed and stirred in step (S10) is sealed and shaken; the shaking is carried out on a shaker; the shaking temperature is 3℃ to 5℃; the shaking time is overnight; the standing temperature is 3℃ to 5℃, and the standing time is 1 h to 3 h.

8. The method for preparing the hydrogel formed by end modification of Pluronic F127 combined with HA and CuS-NDs according to claim 1, characterized in that: In step (S01), a 100L metering tank is used; in step (S01), the container is pre-dried and operated under nitrogen or argon protection; in step (S01), the amount of anhydrous tetrahydrofuran used is 30L; in step (S01), the amount of Pluronic F127 used is 12.6kg; in step (S01), the amount of N,N′-disuccinimidyl carbonate used is 1.025kg; in step (S01), the amount of 4-dimethylaminopyridine used is 0.489kg; the stirring and mixing in step (S01) is mechanical stirring; the speed of the mechanical stirring is 200rpm~300rpm. 00 rpm; the stirring and mixing time in step (S01) is 10 min to 20 min; the amount of anhydrous dimethyl sulfoxide added in step (S02) is 5 L; the stirring and mixing in step (S02) is mechanical stirring co-mixing; the stirring and mixing temperature in step (S02) is 24℃ to 26℃; the speed of the mechanical stirring is 200 rpm to 300 rpm; the stirring and mixing time in step (S02) is 12 h to 36 h; the stirring and mixing in step (S02) continues until the solid is completely dissolved and the reaction solution is in a homogeneous and clear state; the N,N′-disuccinimidyl carbonate is completely consumed by TLC monitoring, resulting in a clear mixture II.

9. The method for preparing the hydrogel formed by end modification of Pluronic F127 combined with HA and CuS-NDs according to claim 1, characterized in that: In step (S03), the amount of β-alanine used is 0.3564 kg; in step (S03), the amount of ultrapure water used is 50 ml to 100 ml; the dissolution process in step (S03) is carried out in a vortex mixer; the stirring in step (S04) is mechanical stirring; in step (S06), the amount of CuCl2·2H2O used is 0.252 mol; in step (S06), the amount of sodium citrate used is 0.4 mol; in step (S06), the amount of ultrapure water used is 20 L; in step (S07)... The amount of sodium sulfide hydrate used is 0.252 mol; the amount of ultrapure water used in step (S07) is 20 L; the stirring in step (S08) is mechanical stirring; the amount of Pluronic CF127 solution used in step (S10) is 3 L, the amount of hyaluronic acid solution used is 1.5 L, and the amount of CuS-NDs solution used is 2.37 L; the mixing container in step (S10) is a 10 L stainless steel mechanically stirred tank with a cooling jacket; the mixing in step (S10) is carried out on a vortex mixer.

10. The method for preparing the hydrogel formed by end modification of Pluronic F127 combined with HA and CuS-NDs according to claim 1, characterized in that: The biocompatibility of the finished hydrogel product was tested, including the following steps: (A01) Take an appropriate amount of rat blood, centrifuge it, remove the supernatant, and obtain the collected product; (A02) After adding an appropriate amount of PBS solution to the collected material in step (A01) for resuspending, centrifuge again to remove the supernatant. Repeat this process 2 to 3 times and take the final resuspended solution. (A03) Group the last resuspension from step (A02), and place each group of resuspension in the well of the orifice plate; Take an appropriate amount of the hydrogel product, immerse it in physiological saline, extract it, and then filter it to obtain the extract. The wells on the plate were divided into three groups. After adding appropriate amounts of extraction solution, PBS solution and 1% Triton X-100 solution to the wells of the three groups respectively, the plates were placed in an incubator for static incubation. (A04) Use a pipette to take an appropriate amount of culture medium from different wells in step (A03) into PE tubes for centrifugation, and take an appropriate amount of supernatant to read the absorbance OD value of the microplate reader at 545nm. (A05) Calculate the hemolysis rate of the hydrogel product extract according to the different absorbance values ​​in step (A04). If the hemolysis rate is less than 5%, the hydrogel product is considered to have no potential hemolysis.

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