Glycosaminoglycan hydrogel with physical and chemical double crosslinking mechanism as well as preparation method and application of glycosaminoglycan hydrogel

Abstract

The invention provides glycosaminoglycan hydrogel with a physical and chemical double crosslinking mechanism as well as a preparation method and application thereof, and relates to the technical field of biological materials. The glycosaminoglycan hydrogel is prepared from the following raw materials: oxidized sodium hyaluronate, chondroitin sulfate, epsilon-polylysine hydrochloride, a polydopamine-titanium dioxide compound and a stabilizer. The glycosaminoglycan hydrogel is prepared through a physical and chemical double-crosslinking mechanism under the specific raw materials and proportion, the problems of cytotoxicity and the like caused by use of a crosslinking agent or introduction of other solvents are avoided, a stable covalent network is constructed through chemical crosslinking, the gel is endowed with the self-repairing characteristic through physical crosslinking, and the self-repairing performance of the hydrogel is improved. And the viscosity of the product is remarkably improved, so that the hydrogel has stronger mechanical support performance, is suitable for wound repair, wound healing and the like, and has good antibacterial activity and stability.

Description

Technical Field

[0001] This invention relates to the field of biomaterials technology, specifically to a glycosaminoglycan hydrogel with a physicochemical dual crosslinking mechanism, its preparation method, and its application. Background Technology

[0002] Hydrogels are based on a network of polymeric materials in which the polymer chains are highly hydrophilic, allowing them to bind with large amounts of water without dissolving. Water can be tightly bound to the polymer network or move freely within it. Due to their high water content, hydrogels possess many unique properties, such as swelling / deswelling, stimuli responsiveness, shock absorption, and low sliding friction. These unique properties enable hydrogels to be used in a wide range of applications, including tissue scaffolds, drug delivery, contact lenses, corneal implants, and as cartilage.

[0003] Glycosaminoglycans (such as hyaluronic acid and chondroitin sulfate) are a class of natural anionic polysaccharides with good biocompatibility, biodegradability, and cell-interacting ability, and are widely used in the biomedical field. However, the low mechanical strength and poor stability of mono-glycosaminoglycan hydrogels limit their application in dynamic physiological environments (such as articular cartilage and skin repair).

[0004] Currently, the preparation of glycosaminoglycan hydrogels mainly relies on a single cross-linking mechanism: I. Chemical crosslinking (e.g., covalent crosslinking): Stable networks are formed through glutaraldehyde, carbodiimide (EDC / NHS), or click chemical reactions, but this may introduce toxic crosslinking agents, affecting biocompatibility, etc. For example, Chinese patent application CN120118376A discloses a method for preparing hyaluronic acid dual-network hydrogel and sponge dressing, belonging to the field of biomedical materials technology. Its key technical points include the following steps: Step 1: Wet squid capitol is dissolved in sodium hydroxide solution, then reacted at high temperature for 120 minutes and filtered. The filtrate is washed with deionized water until the pH value is neutral. Step 2: Dilute hydrochloric acid is added, and the reaction is carried out at room temperature for 24 hours. After filtration, washing, and washing the filter residue with deionized water until the pH value is neutral, wet β-chitosan is obtained. Step 3: The β-chitosan is dispersed in ultrasonically... Hyaluronic acid and glutaraldehyde, a crosslinking agent, were added to the chitin fiber dispersion. After crosslinking, freezing, and demolding, the mixture underwent low-temperature solvent replacement and freeze-drying to obtain a sponge.

[0005] II. Physical cross-linking (e.g., ionic bonds, hydrogen bonds, or hydrophobic interactions): Reports indicate that cross-linking can be achieved through metal ions, temperature-responsive self-assembly, or electrostatic interactions, but these methods result in low gel strength and are prone to swelling and degradation. For example, Chinese patent application CN120459018A discloses a method for preparing curcumin-loaded pectin-sodium alginate gel, comprising the following steps: mixing curcumin, ajramyl acid, o-aminophenol, and an organic solvent; adding a polyvinyl alcohol aqueous solution, sodium alginate solution, and pectin solution; mixing thoroughly; ultrasonicating; adding calcium chloride and stirring; adding oxidized hyaluronic acid and continuing stirring; and washing the hydrogel with deionized water to obtain the final product. This invention achieves physical cross-linking between calcium ions and sodium alginate.

[0006] In recent years, physical-chemical dual crosslinked hydrogels have attracted attention due to their synergistic advantages. For example, hyaluronic acid-gelatin dual-network hydrogels: chemical crosslinking of hyaluronic acid enhances stability, while physical crosslinking of gelatin provides dynamic reversibility, but the immunogenicity of gelatin limits its application for long-term implantation; chondroitin sulfate-polyacrylamide gels: chemical crosslinking of the acrylamide network combines with ionically crosslinked glycosaminoglycans, but residual synthetic monomers may trigger inflammatory responses.

[0007] However, existing technologies still have the following problems: low cross-linking efficiency: the synergistic effect of physical and chemical cross-linking is not fully optimized, resulting in uneven gel performance; limited biological function: lack of targeted preservation of the inherent biological activities of glycosaminoglycans (such as binding with growth factors and anti-inflammatory effects); complex preparation process: multi-step cross-linking reaction increases production costs and makes it difficult to control the porosity and degradation rate of the gel.

[0008] To address the aforementioned issues, there is an urgent need to develop a novel double-crosslinked glycosaminoglycan hydrogel. This hydrogel would, on the one hand, significantly improve viscosity and provide stronger mechanical support by constructing a stable covalent network through chemical crosslinking and providing an energy dissipation mechanism through physical crosslinking; on the other hand, it would better leverage the natural biological activities of glycosaminoglycans, such as promoting cell adhesion or regulating inflammatory responses. Summary of the Invention

[0009] To address the aforementioned problems, this invention provides a glycosaminoglycan hydrogel with a physicochemical dual crosslinking mechanism, its preparation method, and its application. The prepared glycosaminoglycan hydrogel exhibits excellent wound care, adhesion, non-wetting properties, and antibacterial activity.

[0010] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides a glycosaminoglycan hydrogel with a physicochemical dual crosslinking mechanism, comprising the following raw materials: sodium hyaluronate oxide, chondroitin sulfate, ε-polylysine hydrochloride, polydopamine-titanium dioxide complex, and a stabilizer.

[0011] Preferably, the mass ratio of sodium oxyhyaluronate, chondroitin sulfate and ε-polylysine hydrochloride is (1-10):(1-10):(3.5-6.5).

[0012] More preferably, the mass ratio of sodium oxyhyaluronate, chondroitin sulfate and ε-polylysine hydrochloride is (1-9): (1-9): (3.9-6.2).

[0013] More preferably, the mass ratio of sodium oxyhyaluronate, chondroitin sulfate and ε-polylysine hydrochloride is 4:6:5.5.

[0014] Preferably, the raw materials include, by weight parts: 5-55 parts sodium hyaluronate oxide, 5-48 parts chondroitin sulfate, 15-35 parts ε-polylysine hydrochloride, 0.1-10 parts polydopamine-titanium dioxide complex, and 0.1-5 parts stabilizer.

[0015] Preferably, the raw materials include the following by weight: 5-50 parts sodium hyaluronate oxide, 5-45 parts chondroitin sulfate, 19-31 parts ε-polylysine hydrochloride, 0.1-5 parts polydopamine-titanium dioxide complex, and 1-5 parts stabilizer.

[0016] More preferably, the raw materials include the following by weight: 20 parts sodium hyaluronate oxide, 30 parts chondroitin sulfate, 27.5 parts ε-polylysine hydrochloride, 2 parts polydopamine-titanium dioxide complex, and 3 parts stabilizer.

[0017] Preferably, the stabilizer is a polyethylene glycol-gallic acid complex.

[0018] Preferably, the polyethylene glycol-gallic acid complex is prepared as follows: polyethylene glycol-acrylate is dissolved in phosphate buffer, and then 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) are added. The mixture is stirred for 20-40 min, and then gallic acid is added. The mixture is stirred at 20-30°C for 10-14 h.

[0019] Preferably, the mass ratio of polyethylene glycol-acrylate, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide and gallic acid is (80-120):(5-15):(3-10):(10-30).

[0020] More preferably, the mass ratio of polyethylene glycol-acrylate, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide and gallic acid is 100:8:8:20.

[0021] Preferably, the phosphate in the phosphate buffer solution is at least one of calcium dihydrogen phosphate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, sodium polyphosphate, sodium pyrophosphate, disodium hydrogen phosphate, and disodium hydrogen phosphate.

[0022] Preferably, the raw material for the glycosaminoglycan hydrogel further includes a phosphate buffer solution with a pH of 5.0-6.0.

[0023] Preferably, the phosphate in the phosphate buffer solution is at least one of calcium dihydrogen phosphate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, sodium polyphosphate, sodium pyrophosphate, disodium hydrogen phosphate, and disodium hydrogen phosphate.

[0024] More preferably, the phosphate in the phosphate buffer solution is a mixture of sodium dihydrogen phosphate and disodium hydrogen phosphate.

[0025] Preferably, the phosphate buffer solution is prepared by taking 2.5-3.0 g of disodium hydrogen phosphate and 2.5-3.0 g of sodium dihydrogen phosphate to prepare 100 mL of aqueous solution, and mixing 1.36-12.3 mL of the aqueous solution of disodium hydrogen phosphate and 87.70-98.64 mL of the aqueous solution of sodium dihydrogen phosphate at 18-25°C for 10-20 min.

[0026] Preferably, the polydopamine-titanium dioxide complex is prepared by mixing titanium dioxide with Tris-HCl buffer, adding dopamine hydrochloride, and stirring to obtain the polydopamine-titanium dioxide complex.

[0027] Preferably, the mass ratio of titanium dioxide to dopamine hydrochloride is 1:4-6. More preferably, the mass ratio of titanium dioxide to dopamine hydrochloride is 1:5.

[0028] Secondly, the present invention provides a method for preparing the above-mentioned glycosaminoglycan hydrogel, comprising the following steps: S1: Sodium hyaluronate oxide, chondroitin sulfate and ε-polylysine hydrochloride are mixed to obtain a mixed powder; S2: Mix the powder with phosphate buffer and stabilizer to form a pregel solution; S3: Pregel solution, let stand, add polydopamine-titanium dioxide complex, irradiate with ultraviolet light to obtain glycosaminoglycan hydrogel.

[0029] Preferably, in step S2, the mass-to-volume ratio of the mixed powder to the phosphate buffer solution is 0.2-0.5 g: 1 mL.

[0030] Preferably, in step S2, the mixing is carried out by stirring at 30-40°C for 0.5-1.5 hours.

[0031] More preferably, in step S2, the mixing is performed by stirring at 35°C for 1 hour.

[0032] Preferably, in step S3, the settling time is 1-4 hours.

[0033] More preferably, in step S3, the settling time is 3 hours.

[0034] Preferably, in step S3, the parameters of the ultraviolet light irradiation are: wavelength of 365 nm and radiation intensity of 4-6 mW / cm². 2 The time is 2-4 minutes.

[0035] Thirdly, the present invention provides the application of the above-described glycosaminoglycan hydrogel in the preparation of skin and tissue scar repair products.

[0036] Compared with the prior art, the present invention has the following beneficial effects: 1. The present invention provides a glycosaminoglycan hydrogel prepared by a specific ratio of sodium oxyhyaluronate, chondroitin sulfate, and ε-polylysine hydrochloride. Through a physicochemical double cross-linking process, the carboxyl and aldehyde groups of sodium oxyhyaluronate and the carboxyl group of chondroitin sulfate form a chemical cross-link with the amino group of ε-polylysine hydrochloride; sodium oxyhyaluronate and ε-polylysine hydrochloride form a physical cross-link through electrostatic interaction. This dual cross-linking mechanism avoids problems such as cytotoxicity caused by the use of cross-linking agents or the introduction of other solvents. On the other hand, it constructs a stable covalent network through chemical cross-linking, and the reversibility of physical cross-linking endows the gel with self-repairing properties, adapts to the dynamic microenvironment of tissues, significantly improves the viscosity of the product, and thus makes the hydrogel have stronger mechanical support. It is suitable for wound repair, wound healing, and especially for the repair of chronic and refractory wounds and wound healing in dermatology.

[0037] 2. This invention significantly enhances the antibacterial properties of the hydrogel by adding a polydopamine-titanium dioxide complex to the three components: sodium hyaluronate-chondroitin sulfate-ε-polylysine hydrochloride. Furthermore, the addition of a stabilizer slows down the degradation of the hydrogel and improves its stability.

[0038] 3. The preparation method of the present invention is simple to operate, the raw materials are widely available, and the reaction conditions are mild; the hydrogel has good adhesion properties and can effectively prevent the dressing from falling off the wound; the gel is non-wetting, which means that the hydrogel dressing is not easily soluble in water and can prevent external aqueous solutions from entering the wound and infecting bacteria to a certain extent; it has good antibacterial ability and has a good inhibitory effect on both Gram-positive and Gram-negative bacteria, promoting wound repair.

[0039] 4. The hydrogel prepared in this invention is used in medical dressing hydrogels for the repair of scarred wounds on the skin and tissues. As a dressing, it promotes skin wound care, exhibiting good adhesion, non-wetting properties, and antibacterial properties, thus promoting wound healing. Detailed Implementation

[0040] To make the technical means, creative features, achieved objectives, and effects of this invention readily understandable, the invention is further illustrated below with specific embodiments. However, these embodiments are merely preferred embodiments and not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments described herein without creative effort are all within the scope of protection of this invention. It is worth noting that the raw materials used in this invention are all common commercially available products, and their sources are not specifically limited. The technical and scientific terms used in the embodiments have the meanings commonly understood by those skilled in the art to which this invention pertains.

[0041] Sodium oxidized hyaluronic acid: It is produced by our company according to the production process. Small quantities can be produced by our company in the laboratory according to the same production process. Chondroitin sulfate: supplied by Chongqing Aoli Biopharmaceutical Co., Ltd., pharmaceutical grade; ε-Polylysine Hydrochloride: supplied by Jiangsu Yiming Biotechnology Co., Ltd.; The source of disodium hydrogen phosphate is anhydrous disodium hydrogen phosphate: purchased from Nanjing Chemical Reagent Co., Ltd., with a molecular weight of 141.96; Sodium dihydrogen phosphate was sourced from sodium dihydrogen phosphate monohydrate, purchased from Nanjing Chemical Reagent Co., Ltd., with a molecular weight of 137.99.

[0042] The preparation methods of the phosphate buffer solutions in the embodiments and comparative examples of this invention are as follows: Prepare 100 mL aqueous solutions of 2.84 g disodium hydrogen phosphate and 2.76 g sodium dihydrogen phosphate respectively. Mix 6.15 mL of the disodium hydrogen phosphate aqueous solution and 43.85 mL of the sodium dihydrogen phosphate aqueous solution at 18-25℃ for 10-20 min, with a pH of 5.0-6.0.

[0043] Example 1 A glycosaminoglycan hydrogel with a physicochemical dual cross-linking mechanism: (1) The ingredients include the following raw materials by mass: 20 parts sodium hyaluronate, 30 parts chondroitin sulfate, 27.5 parts ε-polylysine hydrochloride, 2 parts polydopamine-titanium dioxide complex and 3 parts polyethylene glycol-gallic acid complex.

[0044] The preparation method of polydopamine-titanium dioxide complex is as follows: titanium dioxide is mixed with Tris-HCl buffer, dopamine hydrochloride is added, and the mixture is stirred to obtain polydopamine-titanium dioxide complex; wherein the mass ratio of titanium dioxide to dopamine hydrochloride is 1:5.

[0045] The preparation method of the polyethylene glycol-gallic acid complex is as follows: 100g of polyethylene glycol-acrylate is dissolved in 1000mL of phosphate buffer, and then 8g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 8g of N-hydroxysuccinimide (NHS) are added. The mixture is stirred for 20-40 min, and then 20g of gallic acid is added. The mixture is stirred at 20-30℃ for 10-14 h.

[0046] (2) The preparation method of the glycosaminoglycan hydrogel includes the following steps: S1: Sodium hyaluronate oxide, chondroitin sulfate and ε-polylysine hydrochloride are mixed to obtain a mixed powder; S2: Mix the powder and phosphate buffer at a mass-volume ratio of 0.3g:1mL, then add the polyethylene glycol-gallic acid complex and stir at 35°C for 1h to form a pregel solution; S3: Pregel solution, allowed to stand for 3 hours, then polydopamine-titanium dioxide complex added, and irradiated with ultraviolet light at a wavelength of 365 nm and a radiation intensity of 5 mW / cm². 2 The reaction time was 3 minutes, and glycosaminoglycan hydrogel was obtained.

[0047] Example 2 A glycosaminoglycan hydrogel with a physicochemical dual cross-linking mechanism: (1) The ingredients include the following raw materials by mass: 50 parts sodium hyaluronate oxide, 5 parts chondroitin sulfate, 19.5 parts ε-polylysine hydrochloride, 0.1 parts polydopamine-titanium dioxide complex and 1 part polyethylene glycol-gallic acid complex.

[0048] The preparation method of polydopamine-titanium dioxide complex is as follows: titanium dioxide is mixed with Tris-HCl buffer, dopamine hydrochloride is added, and the mixture is stirred to obtain polydopamine-titanium dioxide complex; wherein the mass ratio of titanium dioxide to dopamine hydrochloride is 1:6.

[0049] The preparation method of the polyethylene glycol-gallic acid complex is as follows: 80g of polyethylene glycol-acrylate is dissolved in 1000mL of phosphate buffer, followed by the addition of 5g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 3g of N-hydroxysuccinimide (NHS). The mixture is stirred for 20-40 minutes, then 10g of gallic acid is added, and the mixture is stirred at 20-30℃ for 10-14 hours.

[0050] (2) The preparation method of the glycosaminoglycan hydrogel includes the following steps: S1: Sodium hyaluronate oxide, chondroitin sulfate and ε-polylysine hydrochloride are mixed to obtain a mixed powder; S2: Mix the powder and phosphate buffer at a mass-volume ratio of 0.5g:1mL, then add the polyethylene glycol-gallic acid complex and stir at 30℃ for 1.5h to form a pregel solution; S3: Pregel solution, allowed to stand for 1 hour, then polydopamine-titanium dioxide complex added, and irradiated with ultraviolet light at a wavelength of 365 nm and a radiation intensity of 6 mW / cm². 2 The reaction time was 2 minutes, and glycosaminoglycan hydrogel was obtained.

[0051] Example 3 A glycosaminoglycan hydrogel with a physicochemical dual cross-linking mechanism: (1) The ingredients include the following raw materials by mass: 5 parts sodium hyaluronate, 45 parts chondroitin sulfate, 31 parts ε-polylysine hydrochloride, 5 parts polydopamine-titanium dioxide complex and 5 parts polyethylene glycol-gallic acid complex.

[0052] The preparation method of polydopamine-titanium dioxide complex is as follows: titanium dioxide is mixed with Tris-HCl buffer, dopamine hydrochloride is added, and the mixture is stirred to obtain polydopamine-titanium dioxide complex; wherein the mass ratio of titanium dioxide to dopamine hydrochloride is 1:4.

[0053] The preparation method of the polyethylene glycol-gallic acid complex is as follows: 120g of polyethylene glycol-acrylate is dissolved in 1200mL of phosphate buffer, and then 15g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 10g of N-hydroxysuccinimide (NHS) are added. The mixture is stirred for 20-40min, and then 10g of gallic acid is added. The mixture is stirred at 20-30℃ for 10-14h.

[0054] (2) The preparation method of the glycosaminoglycan hydrogel includes the following steps: S1: Sodium hyaluronate oxide, chondroitin sulfate and ε-polylysine hydrochloride are mixed to obtain a mixed powder; S2: Mix the powder and phosphate buffer at a mass-volume ratio of 0.2g:1mL, then add the polyethylene glycol-gallic acid complex and stir at 40℃ for 0.5h to form a pregel solution; S3: Pregel solution, allowed to stand for 4 hours, then polydopamine-titanium dioxide complex added, and irradiated with ultraviolet light at a wavelength of 365 nm and a radiation intensity of 4 mW / cm². 2 The reaction time was 4 minutes, and glycosaminoglycan hydrogel was obtained.

[0055] Comparative Example 1 A glycosaminoglycan hydrogel with a physicochemical dual crosslinking mechanism: Compared with Example 1, the amount of raw materials was changed, specifically: The product comprises the following raw materials by weight: 70 parts sodium hyaluronate oxide, 3.75 parts chondroitin sulfate, 3.75 parts ε-polylysine hydrochloride, 2 parts polydopamine-titanium dioxide complex, and 3 parts polyethylene glycol-gallic acid complex.

[0056] The rest is the same as in Example 1.

[0057] Comparative Example 2 A glycosaminoglycan hydrogel with a physicochemical dual crosslinking mechanism: compared with Example 1, chitosan is used instead of ε-polylysine hydrochloride, and the other raw materials are the same as in Example 1.

[0058] (2) The preparation method of the glycosaminoglycan hydrogel includes the following steps: S1: Oxygenated sodium hyaluronate, chondroitin sulfate and chitosan are mixed to obtain a mixed powder; S2: Mix the powder and phosphate buffer at a mass-volume ratio of 0.2g:1mL, then add the polyethylene glycol-gallic acid complex and stir at 40℃ for 0.5h to form a pregel solution; S3: Pregel solution, allowed to stand for 4 hours, then polydopamine-titanium dioxide complex added, and irradiated with ultraviolet light at a wavelength of 365 nm and a radiation intensity of 4 mW / cm². 2 The reaction time was 4 minutes, and glycosaminoglycan hydrogel was obtained.

[0059] Comparative Example 3 A glycosaminoglycan hydrogel with a physicochemical dual crosslinking mechanism: Compared with Example 1, polydopamine was used to replace the polydopamine-titanium dioxide complex, and the other raw materials were the same as in Example 1.

[0060] (2) The preparation method of the glycosaminoglycan hydrogel includes the following steps: S1: Oxygenated sodium hyaluronate, chondroitin sulfate and chitosan are mixed to obtain a mixed powder; S2: Mix the powder and phosphate buffer at a mass-volume ratio of 0.2g:1mL, then add the polyethylene glycol-gallic acid complex and stir at 40℃ for 0.5h to form a pregel solution; S3: Pregel solution, allowed to stand for 4 hours, then polydopamine added, and irradiated with ultraviolet light at a wavelength of 365 nm and a radiation intensity of 4 mW / cm². 2 The reaction time was 4 minutes, and glycosaminoglycan hydrogel was obtained.

[0061] Comparative Example 4 A glycosaminoglycan hydrogel with a physicochemical dual crosslinking mechanism: Compared with Example 1, gallic acid was used to replace the polyethylene glycol-gallic acid complex, and the other raw materials were the same as in Example 1.

[0062] (2) The preparation method of the glycosaminoglycan hydrogel includes the following steps: S1: Oxygenated sodium hyaluronate, chondroitin sulfate and chitosan are mixed to obtain a mixed powder; S2: Mix the powder and phosphate buffer at a mass-to-volume ratio of 0.2 g: 1 mL, add gallic acid, and stir at 40 °C for 0.5 h to form a pre-gel solution; S3: Pregel solution, allowed to stand for 4 hours, then polydopamine added, and irradiated with ultraviolet light at a wavelength of 365 nm and a radiation intensity of 4 mW / cm². 2 The reaction time was 4 minutes, and glycosaminoglycan hydrogel was obtained.

[0063] Test Example 1 Antibacterial rate test: (1) Test method: Weigh 25 mL of each of the medium glycosaminoglycan hydrogels prepared in Examples 1-3 and Comparative Examples 1-4, add 500 μL of physiological saline for injection and mix well to obtain a thin paste. The physiological saline for injection is a commercially available product that meets the sterility requirements.

[0064] Escherichia coli was selected as the experimental bacteria. 200 μL of a thin paste was added to each well of a 48-well plate. After 10 minutes of incubation until the paste solidified to a certain extent, 10 μL of E. coli bacterial suspension (10⁶ CFU / mL) was added to the surface of the paste in each well. The plate was incubated at 37°C for 2 hours. Then, 1 mL of PBS was added to each well to resuspend the bacteria. A control group was prepared by adding 10 μL of bacterial suspension (10⁶ CFU / mL) to 1 mL of PBS. The plates were then incubated for 24 hours. Subsequently, 40 μL of E. coli bacterial suspension was taken from each well and incubated on an agar plate at 37°C for 24 hours. Bacterial growth on the agar plate surface was observed, and the results were recorded. Three replicates were performed for each group, and the average value was calculated. The results are summarized in Table 1.

[0065] Table 1

[0066] As shown in Table 1, the antibacterial rate of the glycosaminoglycan hydrogel prepared in the embodiments of the present invention is significantly higher than that of the comparative examples, especially compared with comparative examples 3 and 4.

[0067] Test Example 2 Rotational viscosity measurement Take 10-20 mL of the glycosaminoglycan hydrogels prepared in Examples 1-3 and Comparative Examples 1-4 for later use. Test preparation: Calibrate the instrument to meet standard requirements; select a suitable rotor according to the viscosity range of the sample; select rotor number 2 according to the sample; place the sample to be tested into the test cup of the viscometer, ensuring the rotor is immersed in the solution to an appropriate depth; set parameters: set the rotational speed (rpm) of the rotational viscometer according to the sample condition; start the rotational viscometer and record the viscosity reading at the set speed. Record the viscosity value according to the device display. The viscosities of the obtained samples are shown in Table 2 below: Table 2

[0068] As shown in Table 2, the glycosaminoglycan hydrogel prepared in this embodiment of the invention has good viscosity and stronger mechanical support, making it suitable for wound repair and healing, especially for chronic and refractory wound repair and healing in dermatology. Changing the mass ratio of sodium oxidized hyaluronic acid, chondroitin sulfate, and ε-polylysine hydrochloride (Comparative Example 1) significantly reduces its viscosity.

[0069] Test Example 3 Adhesion test experiment The adhesive properties of the glycosaminoglycan hydrogels prepared in Examples 1-3 and Comparative Examples 1-4 were evaluated using glass slides. 7.5cm × 2.5cm glass slides were ultrasonically cleaned with ethanol for 5 min and then naturally dried. 0.5 mL of the sample was evenly spread on one end of a 2.5cm × 2.5cm glass slide. Another glass slide was then attached to the area where the hydrogel was evenly spread. It was observed that the gel stably adhered to both slides without falling off, demonstrating that the gel can adhere well to skin wounds without detaching. The two adhered slides were left to stand for 30 min. Then, the gel-bonded slides were fixed to the two clamps of a tensile testing machine. The machine speed was set to 100 mm / min, and the slides were peeled at a 180° angle. The maximum force (N) was recorded.

[0070] The maximum peel force of the obtained samples is shown in Table 3 below: Table 3

[0071] As can be seen from the data in Table 3, the glycosaminoglycan hydrogel prepared in the embodiments of the present invention has stronger adhesion.

[0072] Test Example 4 In vitro degradation performance: Weigh 20 mL of the dressing (mass after freeze-drying is m0), soak it in 100 mL of PBS (pH = 7.4) with collagenase content of 12.5 U / mL, and digest it in a constant temperature incubator at 37℃ for 7 days. Filter to remove the solution, wash 3 times with deionized water, freeze-dry, and weigh (m0). t The in vitro degradation rate of the dressing is: Degradation rate = (m0) m t ) / m0×100%.

[0073] The results are shown in Table 4: Table 4

[0074] As can be seen from the data in Table 4, the glycosaminoglycan hydrogel prepared in the embodiments of the present invention has better stability.

[0075] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A glycosaminoglycan hydrogel with a physicochemical dual crosslinking mechanism, characterized in that, It includes the following raw materials: sodium hyaluronate oxide, chondroitin sulfate, ε-polylysine hydrochloride, polydopamine-titanium dioxide complex, and stabilizers.

2. The glycosaminoglycan hydrogel according to claim 1, characterized in that, The mass ratio of sodium oxyhyaluronate, chondroitin sulfate and ε-polylysine hydrochloride is (1-10): (1-10): (3.5-6.5).

3. The glycosaminoglycan hydrogel according to claim 2, characterized in that, The mass ratio of sodium oxyhyaluronate, chondroitin sulfate and ε-polylysine hydrochloride is (1-9): (1-9): (3.9-6.2).

4. The glycosaminoglycan hydrogel according to claim 3, characterized in that, The mass ratio of sodium oxyhyaluronate, chondroitin sulfate, and ε-polylysine hydrochloride is 4:6:5.

5.

5. The glycosaminoglycan hydrogel according to claim 1, characterized in that, The product comprises the following raw materials by weight: 5-55 parts sodium hyaluronate oxide, 5-48 parts chondroitin sulfate, 15-35 parts ε-polylysine hydrochloride, 0.1-10 parts polydopamine-titanium dioxide complex, and 0.1-5 parts stabilizer.

6. The glycosaminoglycan hydrogel according to claim 5, characterized in that, The product comprises the following raw materials by weight: 5-50 parts sodium hyaluronate oxide, 5-45 parts chondroitin sulfate, 19-31 parts ε-polylysine hydrochloride, 0.1-5 parts polydopamine-titanium dioxide complex, and 1-5 parts stabilizer.

7. The glycosaminoglycan hydrogel according to claim 1, characterized in that, The stabilizer is a polyethylene glycol-gallic acid complex.

8. The glycosaminoglycan hydrogel according to claim 7, characterized in that, The preparation method of the polyethylene glycol-gallic acid complex is as follows: after dissolving polyethylene glycol-acrylate in phosphate buffer, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide are added and mixed for 20-40 min. Then gallic acid is added and stirred at 20-30℃ for 10-14 h.

9. The glycosaminoglycan hydrogel according to claim 8, characterized in that, The mass ratio of polyethylene glycol-acrylate, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide and gallic acid is (80-120):(5-15):(5-15):(5-15).

10. The glycosaminoglycan hydrogel according to claim 9, characterized in that, The phosphate in the phosphate buffer solution is at least one of calcium dihydrogen phosphate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, sodium polyphosphate, sodium pyrophosphate, disodium hydrogen phosphate, and disodium hydrogen phosphate.

11. The glycosaminoglycan hydrogel according to claim 1, characterized in that, The raw materials for the glycosaminoglycan hydrogel also include a phosphate buffer solution with a pH of 5.0-6.

0.

12. The glycosaminoglycan hydrogel according to claim 11, characterized in that, The phosphate in the phosphate buffer solution is at least one of calcium dihydrogen phosphate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, sodium polyphosphate, sodium pyrophosphate, disodium hydrogen phosphate, and disodium hydrogen phosphate.

13. The glycosaminoglycan hydrogel according to claim 11, characterized in that, The phosphate in the phosphate buffer solution is a mixture of sodium dihydrogen phosphate and disodium hydrogen phosphate.

14. The glycosaminoglycan hydrogel according to claim 13, characterized in that, The phosphate buffer solution is prepared as follows: 2.5-3.0 g of disodium hydrogen phosphate and 2.5-3.0 g of sodium dihydrogen phosphate are used to prepare 100 mL of aqueous solution. 1.36-12.3 mL of the disodium hydrogen phosphate aqueous solution and 87.70-98.64 mL of the sodium dihydrogen phosphate aqueous solution are mixed at 18-25℃ for 10-20 min.

15. The glycosaminoglycan hydrogel according to claim 1, characterized in that, The preparation method of the polydopamine-titanium dioxide complex is as follows: titanium dioxide is mixed with Tris-HCl buffer, dopamine hydrochloride is added, and the mixture is stirred to obtain the polydopamine-titanium dioxide complex.

16. The glycosaminoglycan hydrogel according to claim 15, characterized in that, The mass ratio of titanium dioxide to dopamine hydrochloride is 1:4-6.

17. The method for preparing the glycosaminoglycan hydrogel according to any one of claims 1-16, characterized in that, Includes the following steps: S1: Sodium hyaluronate oxide, chondroitin sulfate and ε-polylysine hydrochloride are mixed to obtain a mixed powder; S2: Mix the powder with phosphate buffer and stabilizer to form a pregel solution; S3: Pregel solution, let stand, add polydopamine-titanium dioxide complex, irradiate with ultraviolet light to obtain glycosaminoglycan hydrogel.

18. The preparation method according to claim 17, characterized in that, In step S2, the mass-to-volume ratio of the mixed powder to the phosphate buffer solution is 0.2-0.5 g: 1 mL.

19. The preparation method according to claim 17, characterized in that, In step S2, the mixing is carried out by stirring at 30-40°C for 0.5-1.5 hours.

20. The preparation method according to claim 17, characterized in that, In step S3, the settling time is 1-4 hours.

21. The preparation method according to claim 17, characterized in that, In step S3, the parameters for the ultraviolet light irradiation are: wavelength of 365nm and radiation intensity of 4-6mW / cm². 2 The time is 2-4 minutes.

22. The use of the glycosaminoglycan hydrogel according to any one of claims 1-16 in the preparation of skin and tissue scar repair products.

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