Sacran-based thermosensitive injection hydrogel and application thereof in full-thickness skin wound treatment

Abstract

The invention relates to the technical field of polymer hydrogel, in particular to a heat-sensitive injectable hydrogel material based on natural polysaccharide Sacran and poloxamer 407 and application of the heat-sensitive injectable hydrogel material in wound treatment. The hydrogel is prepared by a physical blending method, wherein P407 endows the material with excellent thermosensitive gelling property and injectability. The Sacran chain penetrates through a P407 network through physical entanglement and hydrogen-bond interaction to form a stable semi-interpenetrating structure, so that the mechanical strength, the water absorption swelling capacity and the biocompatibility of the hydrogel are enhanced. Furthermore, two medicines of tannic acid and dipotassium glycyrrhizinate are synergistically loaded, so that multiple functions of resisting bacteria, resisting oxidation, resisting inflammation and the like are integrated, and wound healing can be effectively promoted. The hydrogel prepared by the invention not only shows excellent heat sensitivity, mechanical property and cytocompatibility, but also can continuously release drugs, so that the treatment effect is further improved. The preparation process of the hydrogel is simple, convenient, efficient, green and environment-friendly. Wide clinical application prospects are realized in the fields of wound dressings and tissue engineering.

Description

Technical Field

[0001] This invention relates to the field of polymer hydrogel technology, specifically to a thermosensitive injectable hydrogel material based on natural polysaccharides Sacran and poloxamer 407 and its application in wound treatment. Background Technology

[0002] Sacran is a natural high-molecular-weight polysaccharide extracted from *Aphanothece sacrum*, possessing an extremely high molecular weight (over 10). 7 (Da), and contains abundant hydroxyl, carboxylic acid and sulfate groups.

[0003] Hydrogels, as three-dimensional network structures formed by physical or chemical cross-linking, possess excellent hydrophilicity, good biocompatibility, and tunable hydration, leading to their widespread application in drug delivery, tissue engineering, and wound dressings. Injectable hydrogels have attracted significant attention due to their ability to seamlessly fill irregular wounds and form protective barriers in situ. Poloxamer is a commonly used material for constructing such hydrogels, but it has significant limitations when used alone: ​​firstly, its gel network has low strength and is prone to disintegration; secondly, drug release is mostly rapid burst release, failing to achieve long-term therapeutic effects; and thirdly, it lacks the biological function of actively promoting wound healing.

[0004] While existing technologies employ methods to enhance poloxamer performance by mixing natural polymers (such as chitosan), these methods often face challenges such as complex modification processes, questionable biosafety, or low functional integration. For instance, simple physical blending struggles to achieve stable drug encapsulation and synergistic release within a gel network; while complex chemical modifications often involve intricate chemical cross-linking reactions, potentially introducing cytotoxic agents, and the network structure's ability to regulate drug release behavior remains insufficient, hindering the achievement of synergistic and controlled release of multiple functions such as antibacterial, antioxidant, and anti-inflammatory effects.

[0005] Therefore, existing technologies lack an ideal wound dressing that can be prepared through a simple and safe process, integrating excellent injection gelation properties, mechanical strength, and synergistic sustained release of multiple functions such as antibacterial, antioxidant, and anti-inflammatory properties. Summary of the Invention

[0006] To overcome the problems of uncontrollable drug release rate and insufficient antibacterial and antioxidant properties in existing hydrogels during wound healing, this invention provides a thermosensitive injectable hydrogel material based on the natural polysaccharide Sacran and poloxamer P407. Based on the natural polymer Sacran, P407 provides thermosensitive support, effectively enhancing the hydration and biostability of the hydrogel, thereby improving the sustained-release effect of the drug and enhancing its application potential in treating complex skin wounds. The hydrogel material of this invention possesses excellent hydration, high mechanical strength, antioxidant and antibacterial properties, and good cell compatibility, effectively promoting wound healing, especially in the treatment of complex skin wounds. The thermosensitivity, shear-thinning properties, and excellent mechanical properties of this hydrogel make it suitable for injection therapy, allowing it to adapt to the morphology of the wound bed and promote wound healing.

[0007] This invention provides a thermosensitive injectable hydrogel material based on the natural polysaccharide Sacran and poloxamer P407, which is formed by physically mixing Sacran and P407.

[0008] The first aspect of the present invention provides a thermosensitive injectable hydrogel based on the natural polysaccharide Sacran and poloxamer P407, the hydrogel material being obtained by physically mixing a Sacran solution and a poloxamer P407 solution; In the hydrogel, Sacran molecular chains form a three-dimensional network structure with poloxamer P407 through physical entanglement and hydrogen bonding, and the Sacran polymer chains provide network support. The mass ratio of Sacran to poloxamer P407 in the hydrogel material is 1:100-1:300.

[0009] Furthermore, the hydrogel has a gel point of 30-39℃, preferably 30-36℃. Based on this characteristic, the hydrogel can remain in a solution state at low temperatures and rapidly gel at body temperature to form a dense three-dimensional network structure; it can be directly injected and gelled in situ to cover the wound.

[0010] Furthermore, this hydrogel is composed of a mixture of the natural polysaccharide Sacran and the thermosensitive polymer P407 in a specific ratio. P407 imparts excellent thermal response behavior and injection gelation properties to the material, while Sacran provides the network support of the polymer chains and excellent hydrophilicity, biocompatibility, and drug loading capacity. This hydrogel exhibits good flowability, shear-thinning properties, and thermosensitive gelling performance, enabling it to rapidly form a stable gel in wound environments and adhere to the wound surface, effectively isolating it from external contamination.

[0011] In some embodiments, the water absorption rate of the hydrogel is ≥300%, preferably ≥500%. A second aspect of the present invention provides a method for preparing the injectable hydrogel, comprising the following steps: S1. Preparation of Sacran solution: Provide Sacran and dissolve it completely in deionized water at 70-90°C with mechanical stirring to obtain a Sacran solution; S2. Preparation of poloxamer P407 solution: Prepare a 25-35% (w / w) P407 solution by cold water method; S3. Mix the Sacran solution obtained in step S1 with the P407 solution obtained in step S2 to obtain a hydrogel pre-solution. Let it stand at low temperature for 12-48 h to obtain an injectable hydrogel.

[0012] In some embodiments, in step S1, the mass concentration of Sacran in the Sacran solution is 0.3%-0.5wt%; preferably 0.4wt%. In one embodiment, in step S1, a magnetic stirrer is used to dissolve the Sacran solution at 80 °C to obtain solutions of different concentrations.

[0013] In some embodiments, in step S2, the cold water method preparation conditions are: stirring and dissolving at 4±2 °C; In one embodiment, in step S2, 3g of P407 powder is dissolved in 7mL of cold water to prepare a 30% (w / w) P407 solution.

[0014] In some embodiments, in step S3, the volume ratio of the Sacran solution to the P407 solution is 1:1 to 1:3; preferably 1:2 (v / v).

[0015] In some embodiments, in step S3, the mixing method is vortex mixing for 5-20 minutes, followed by reaction at 4°C for 12-48 hours; preferably, vortex mixing for 10 minutes.

[0016] In some preferred embodiments, the mass concentration of P407 is 30-35 wt%; more preferably 30 wt%.

[0017] A third aspect of this invention provides a composite hydrogel for wound healing, comprising the injectable hydrogel and tannic acid (TA) and dipotassium glycyrrhizinate (DG) loaded therein; wherein the mass ratio of tannic acid to dipotassium glycyrrhizinate in the composite hydrogel is 1:3 to 3:1. The drug is dissolved in the hydrogel network structure, exhibiting excellent sustained-release properties, enabling continuous and stable drug release in vivo. Both tannic acid and dipotassium glycyrrhizinate are water-soluble, allowing for convenient adjustment of their loading in the hydrogel system of this invention, which can be easily adjusted according to the needs of different application scenarios.

[0018] In this invention, tannic acid (TA) and dipotassium glycyrrhizate (DG) are co-loaded in a Sacran / P407-based hydrogel. Tannic acid, as a natural antioxidant and antibacterial agent, enhances the antibacterial and antioxidant properties of the hydrogel and promotes wound healing; dipotassium glycyrrhizate has anti-inflammatory effects and can effectively reduce inflammatory responses at the wound site. Through this synergistic effect, the hydrogel of this invention can provide more lasting and multi-effect therapeutic effects, and is particularly suitable for the treatment of complex skin wounds.

[0019] In some embodiments, the total drug loading is 0.01-20 mg / mL. In some embodiments, the tannic acid loading is 0.01-10 mg / mL; in some embodiments, the dipotassium glycyrrhizinate loading is 0.01-10 mg / mL.

[0020] In some preferred embodiments, the drug concentration (tannic acid + dipotassium glycyrrhizinate) is 0.5-2 mg / mL.

[0021] Furthermore, the preparation method of the composite hydrogel for wound healing is as follows: The injectable hydrogel pre-solution is provided, and tannic acid and dipotassium glycyrrhizinate are added and loaded at low temperature to obtain the composite hydrogel. In some embodiments, the composite hydrogel is obtained by storing the mixed solution at 4°C for 1 day.

[0022] Another aspect of the invention provides the application of the injectable hydrogel in wound dressings, bio-dresses, and related therapeutic products. In the drug-loaded hydrogel system, the Sacran / P407-based thermosensitive hydrogel combines good injectability, gelling properties, and tissue compatibility, making it suitable for in-situ injection or application to wounds, forming a soft, highly adhesive gel barrier that effectively isolates external contaminants. The multifunctional chemical group structure of Sacran helps to construct a hydrogel microenvironment with antibacterial barrier and free radical scavenging functions, thereby optimizing overall wound healing conditions.

[0023] Furthermore, the present invention provides the application of the composite hydrogel in a full-thickness skin trauma treatment product, wherein the full-thickness skin trauma includes superficial wounds, burns, postoperative incisions, and diabetic foot ulcers.

[0024] In some embodiments, the material can be used to prepare various types of products such as medicated wound dressings, spray dressings, and injectable wound repair agents, suitable for various wound repair scenarios such as superficial wounds, burns, postoperative incisions, and diabetic foot ulcers.

[0025] Beneficial effects: (1) This invention provides a thermosensitive injectable hydrogel material based on the synergistic construction of natural polymers Sacran and P407, which overcomes the shortcomings of traditional hydrogels in terms of biological properties and therapeutic effects, endows the material with good thermal responsiveness and convenient injection, and significantly improves its application potential in the treatment of acute and chronic wounds. (2) The present invention further provides a composite hydrogel co-loaded with tannic acid and dipotassium glycyrrhizate, which effectively improves the wound microenvironment through synergistic antibacterial, antioxidant and anti-inflammatory effects, promotes the migration and proliferation of endothelial cells, thereby accelerating tissue regeneration and wound closure, and has excellent healing effect; (3) The hydrogel material prepared by the present invention has good rheological properties, injectability and thermosensitive gel transformation behavior, can achieve rapid gelation at body temperature, and can achieve in-situ wound shaping without the aid of additional auxiliary instruments. It has strong adhesion, is convenient to use, and significantly improves the feasibility and operability of clinical application. (4) The preparation process of this invention is mild and simple, green and environmentally friendly, requires no complicated equipment, the raw materials used are safe and available, green and environmentally friendly, and the entire preparation process is non-toxic and pollution-free, with good biosafety and sustainable development prospects, and is suitable for large-scale production and industrialization. Attached Figure Description

[0026] The accompanying drawings are provided to further illustrate the present application and form part of the specification. They are used together with the embodiments of the present application to explain the application and do not constitute a limitation thereof. In the drawings: Figure 1 The image shows the Fourier Transform Infrared (FTIR) spectrum of the hydrogel prepared according to this invention. Figure 2 The rheological properties of the hydrogels prepared in Examples 1-3 of this invention are shown, where A is the rheological property curve of the hydrogel at different temperatures; B is the viscosity change of the hydrogel at different shear rates, reflecting its shear thinning characteristics, and the inset shows that the letter "JNU" can be injected in Example 2; C is the strain-dependent oscillatory rheological curve of the hydrogel.

[0027] Figure 3 This is a scanning electron microscope image of the hydrogel prepared in Example 2 of the present invention; Figure 4 The swelling properties of the hydrogels prepared in Examples 1-3 of this invention; Figure 5 The drug release curves of the hydrogels prepared in Examples 1-3 of the present invention are shown below; where A is the drug release curve of g(D) releasing DG, B is the drug release curve of g(T) releasing TA, C is the drug release curve of g(TD) releasing DG, and D is the drug release curve of g(TD) releasing TA. Figure 6The antioxidant properties of the hydrogel prepared in Example 2 of the present invention are shown in Figure A, where A is a visual representation of the hydrogel's scavenging of DPPH free radicals; and B is a diagram showing the scavenging efficiency of the hydrogel against DPPH free radicals. Figure 7 This is a comparative diagram of the antibacterial activity of the hydrogel prepared in Example 2 of the present invention, where A represents Escherichia coli and B represents Staphylococcus aureus. Figure 8 Cell compatibility of the hydrogel prepared in Example 2 of this invention; Figure 9 This invention relates to wound healing using the hydrogel prepared in Example 2 of the present invention. Detailed Implementation

[0028] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the examples in the specification. Many specific details are set forth in the following description to provide a thorough understanding of the present invention; however, the present invention may also be implemented in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below. Unless otherwise specified, the experimental methods described in the following embodiments are conventional methods; the reagents and instruments described, unless otherwise specified, are commercially available.

[0029] The technical solution of the present invention will be further described in detail below through specific embodiments and in conjunction with the accompanying drawings.

[0030] In this invention, unless otherwise specified, all equipment and raw materials are available from the market or commonly used in the industry. The methods in the following embodiments are conventional methods in the field unless otherwise specified.

[0031] Sacran was provided by Green Science Material Co., Ltd. (Kumamoto, Japan).

[0032] In the examples, the drug-loaded hydrogel samples were named g(T), g(D), and g(TD), representing tannic acid (TA), dipotassium glycyrrhizinate (DG), and a hydrogel co-loaded with both, respectively.

[0033] The testing methods used in the embodiments include the following aspects: (1) Fourier transform infrared spectroscopy characterization: Take an appropriate amount of sample and place it on the ATR accessory equipped with the Fourier transform infrared spectrometer (FTIR), and measure the temperature at 4000–400 cm⁻¹. -1 Infrared spectra of the Sacran / P407 thermosensitive injectable hydrogel and its purified components were obtained by scanning within the wavenumber range.

[0034] (2) Rheological property testing: In temperature scanning mode, the temperature was increased from 0 ℃ to 50 ℃ at a rate of 1 ℃ / min, and the storage modulus (G′) and loss modulus (G″) were recorded to analyze the change of viscoelasticity of the hydrogel with temperature. In the viscosity test, a viscosity of 0.1–100 s was applied at 37 ℃. -1 The shear rate was measured to investigate the flow properties of the hydrogel under different shear conditions. To determine the linear viscoelastic region (LVR) of the hydrogel, an amplitude scan was performed at a frequency of 1 Hz, and the strain range was gradually increased (0.01%–100%) to evaluate the mechanical response of the material under different strains.

[0035] (3) Scanning electron microscopy test: After sputtering gold onto the cross-section of the freeze-dried hydrogel sample, the cross-section was observed using a scanning electron microscope (SEM) to analyze its pore size distribution and microstructure.

[0036] (4) Swelling rate test: The initial mass W0 of the completely dried hydrogel sample was obtained by weighing, and then placed in a pH 7.4 buffer solution at 37 °C. The sample was taken out at set time intervals, the surface moisture was removed, and then the sample was weighed to obtain the mass W. t The swelling ratio of the sample is calculated using the following formula: (1) (5) Drug release curves: Hydrogel samples g(T) loaded with tannic acid, g(D) loaded with dipotassium glycyrrhizate, and g(TD) co-loaded with both drugs were placed in 20 mL of pH 7.4 buffer solution and subjected to in vitro release experiments under constant temperature water bath conditions at 37 ℃. At predetermined time intervals, 2 mL of solution samples were taken out, and their absorbance was measured at 278 nm and 254 nm using a UV-Vis spectrophotometer to plot the cumulative drug release curves.

[0037] (6) Antioxidant test: An equal volume of 5 mg / mL hydrogel extract was mixed with 200 μM DPPH methanol solution and allowed to stand for 30 minutes in the dark. After the reaction was completed, 100 μL of the supernatant was taken and the absorbance was measured at 517 nm. The free radical scavenging ability and antioxidant activity of the hydrogel were evaluated based on the decrease in absorbance and the change in color of the DPPH solution from dark purple to light purple.

[0038] (7) The antibacterial properties of the hydrogel were evaluated using the colony counting method: Staphylococcus aureus and Escherichia coli were prepared into concentrations of 10. 6CFU / mL bacterial suspension. After gelling the hydrogel samples at 37 °C and sterilizing them under UV light, 10 μL of bacterial suspension was added to each well and incubated at 37 °C for 2 hours to promote bacterial contact with the hydrogel surface. Subsequently, sterile PBS was added and eluted by shaking. 10 μL of the resulting bacterial suspension was spread onto LB agar plates and incubated at 37 °C for 24 hours. The number of colonies was counted, and the inhibition rate was calculated to evaluate the antibacterial effect of the hydrogel.

[0039] (8) Assessing the cell compatibility of the hydrogel using human umbilical vein endothelial cells (HUVECs): HUVECs were prepared at a concentration of 5 × 10⁻⁶ cells / mL. 3 Seeded at a density of [number] samples per well in 96-well plates, and incubated for 24 h, a hydrogel sample suspension with a final concentration of 600 μg / mL was added for further incubation. After 24, 48, and 72 h of incubation, 100 μL of DMEM medium containing 10% CCK-8 reagent was added to each well, and incubation continued for 2 h. The absorbance was then measured at 450 nm using a microplate reader (Infinite 200Pro, Tecan, Austria). (2) In equation (2), OD sample This represents the absorbance value measured after cells are co-incubated with the hydrogel extract; OD control This indicates the absorbance value of cells after incubation in anhydrous gel extract medium; OD blank This represents the absorbance value of the culture medium measured under conditions where neither cells nor hydrogel extract were present. All samples were measured independently in six parallel wells.

[0040] (9) Wound healing test of hydrogel: Male ICR mice aged 5-6 weeks and weighing 30-40 g were selected, and a full-thickness skin defect model with a diameter of approximately 8 mm was prepared on the back. The control group was covered with Tegaderm. TM Protective film; in the experimental group, SA / P407, g(T), g(D), and g(TD) hydrogels were applied to the wound, respectively. Wound images were recorded and the wound area was measured on postoperative days 0, 3, 7, and 14. The wound healing rate was calculated using the following formula: (3) In equation (3), S0 represents the wound area measured on day 0, S n This represents the wound area recorded on day n.

[0041] The raw material usage amounts for each embodiment of the present invention are shown in Table 1.

[0042] Each of Examples 1-3 includes one experiment, and the hydrogels obtained are numbered as shown in Table 1; It should be further noted that, in the performance tests of the hydrogels and drug-loaded systems in the embodiments, the hydrogel samples used for testing the mechanical / physical properties of the hydrogels were all blank hydrogels. However, drug-loaded hydrogels were used for testing the sustained-release performance and antibacterial and antioxidant properties.

[0043] In each embodiment of the present invention, 1 mg / mL of tannic acid or dipotassium glycyrrhizate was added in the individual drug loading step to conduct drug loading experiments; when loading compound drugs, the mass ratio of tannic acid and dipotassium glycyrrhizate was 1:1, and the total amount was 1 mg / mL.

[0044] Table 1. Amounts of each raw material used in Example 13 Example 1 The present invention discloses a method for preparing Sacran / P407 thermosensitive injectable hydrogel, comprising the following steps: 1) Dissolve 0.1g Sacran at 80 °C using a magnetic stirrer, then add water and stir until the total volume is 100ml; 2) Dissolve 3g of P407 powder in 7mL of cold water to prepare a 30% (w / vol) P407 solution; 3) Mix the Sacran solution obtained in step 1 with the P407 solution in step 2 at a ratio of 1:2 (v / v) and vortex mix for 10 minutes to ensure thorough mixing and obtain the pre-solution; 4) Add the drug to the pre-solution obtained in step 3 to obtain the drug-loaded hydrogel material; 5) Store the mixed solution at 4°C for 1 day to obtain the composite hydrogel.

[0045] Example 2 The present invention provides a method for preparing a semi-interpenetrating network hydrogel, comprising the following steps: 1) Dissolve 0.2g of Sacran at 80 °C using a magnetic stirrer, then add water and stir until the total volume is 100ml; 2) Dissolve 3g of P407 powder in 7mL of cold water to prepare a 30% (w / vol) P407 solution; 3) Mix the Sacran solution obtained in step 1 with the P407 solution in step 2 at a ratio of 1:2 (v / v) and vortex mix for 10 minutes to ensure thorough mixing and obtain the pre-solution; 4) Add the drug to the pre-solution obtained in step 3 to obtain the drug-loaded hydrogel material; 5) Store the mixed solution at 4°C for 1 day to obtain the composite hydrogel.

[0046] Example 3 The present invention provides a method for preparing a semi-interpenetrating network hydrogel, comprising the following steps: 1) Dissolve 0.3g Sacran at 80 °C using a magnetic stirrer, then add water and stir until the total volume is 100ml; 2) Dissolve 3g of P407 powder in 7mL of cold water to prepare a 30% (w / vol) P407 solution; 3) Mix the Sacran solution obtained in step 1 with the P407 solution in step 2 at a ratio of 1:2 (v / v) and vortex mix for 10 minutes to ensure thorough mixing and obtain the pre-solution; 4) Add the drug to the pre-solution obtained in step 3 to obtain the drug-loaded hydrogel material; 5) Store the mixed solution at 4°C for 1 day to obtain the composite hydrogel.

[0047] Performance testing: Figure 1 Fourier transform infrared (FT-IR) spectra of the hydrogel and its components prepared in Example 2. The infrared spectrum of Sacran is at 1638 cm⁻¹. -1 1419 cm -1 1243 cm -1 and 1040 cm -1 Characteristic absorption peaks appear at [values ​​missing], corresponding to the νCO stretching vibration, νCO vibration of amide I, and the νSO vibration and νCO vibration of the sulfate group (SO4), respectively. Furthermore, at 2930 cm⁻¹... -1 The absorption peak at [value missing] is attributed to the stretching vibration of the CH group in Sacran. The FTIR spectrum of P407 is at 2885 cm⁻¹. -1 and 1096 cm -1 Characteristic absorption peaks appear at the locations corresponding to the stretching vibrations of the CH2 group and the C–O–C group, respectively. When comparing the FTIR spectra of 0.3 Sacran / P407, 0.4 Sacran / P407, 0.5 Sacran / P407, g(T), g(D), and g(TD) hydrogels with those of each component, it can be observed that their characteristic absorption peaks are similar to those of P407. This is mainly because P407 is dominant in the final material, while the proportions of TA and DG are relatively low.

[0048] Figure 2 The rheological properties of the hydrogels prepared in Examples 1-3 of this invention were determined by temperature scanning tests. Figure 2-A shows that the gel points of the 0.3 Sacran / P407, 0.4 Sacran / P407, and 0.5 Sacran / P407 samples are 38.4 ℃, 36.6 ℃, and 31.3 ℃, respectively. This indicates that the phase transition temperature of the gel can be precisely controlled by adjusting the Sacran content, thus adapting to different clinical application scenarios. In particular, the 0.5 SA / P407 hydrogel shows a significant difference in storage modulus before and after the gel point, indicating that it can rapidly form a dense network structure of gel at near body temperature (31.3 ℃), exhibiting high mechanical properties and fast response speed.

[0049] Figure 2 The rheological test results of -B show that the viscosity of all hydrogel samples decreased significantly with increasing shear rate, exhibiting typical shear-thinning characteristics, which is consistent with the rheological behavior of injectable hydrogels. Figure 2 -C indicates that all samples exhibited a stable linear viscoelastic region (LVR) within the 1% oscillatory strain range, demonstrating good structural stability within this range. Throughout the LVR range, G′ consistently exceeded G″, a characteristic consistent with typical gel states, further proving that the hydrogel network structure is intact and dominated by elastic response.

[0050] Figure 3 This is a scanning electron microscope image of the hydrogel obtained in Example 2. As can be seen from the image, the hydrogel exhibits a uniformly distributed three-dimensional porous structure with large pore sizes and good connectivity, which facilitates rapid liquid penetration and absorption, thereby endowing the material with excellent water absorption properties.

[0051] Figure 4 The swelling properties of the hydrogels prepared in Examples 1-3 were compared, with water absorption rates ranging from approximately 300% to 520%. The 0.5 Sacran / P407 group exhibited the highest water absorption capacity, while the 0.3 Sacran / P407 group showed the lowest, indicating that the water absorption capacity of the hydrogel significantly improves with increasing Sacran content. This trend may be related to the abundant polar functional groups in the Sacran molecule, including hydroxyl, carboxyl, and sulfonate groups. These groups can form numerous hydrogen bonds with water molecules, thereby enhancing the water absorption and retention capacity of the hydrogel. Furthermore, the electrostatic repulsion between anionic groups on the Sacran backbone may further promote the expansion of the polymer network structure, which is beneficial for the diffusion and retention of water in the gel system.

[0052] Figure 5The figures show the drug release curves of the hydrogels prepared in Examples 1-3 over 48 hours at pH 7.4. As shown in Figures AD, the drug release of the three hydrogels (g(T), g(D), and g(TD)) increased significantly in the first 10 hours, followed by a gradual stabilization of the release rate. At 48 hours, the cumulative release rates of DG and TA were approximately 82.2%, 92.0%, 80.6%, and 89.1%, respectively. These results indicate that after the hydrogel comes into contact with the release medium, the DG and TA adsorbed on the hydrogel surface rapidly diffuse outwards, exhibiting a clear burst release phase in the initial stage. Furthermore, the drug release rate of the hydrogel gradually increases with increasing Sacran content. This trend is consistent with the changes in its swelling properties, further demonstrating that the microstructure of the hydrogel has a significant impact on drug release behavior.

[0053] Figure 6 Figure A shows the antioxidant properties of the hydrogels prepared in Example 2. Compared with the blank group, all hydrogel groups could decolorize DPPH, indicating antioxidant properties. Figure B shows that the hydrogels in groups g(T) and g(D) exhibited significant DPPH free radical scavenging ability. This high antioxidant activity is mainly attributed to the introduction of tannic acid (TA) and dipotassium glycyrrhizate (DG) into the hydrogels. TA contains abundant polyphenol structures, which can effectively capture and neutralize free radicals; while DG has excellent antioxidant properties, which can further enhance the scavenging ability of the hydrogels. The maximum scavenging rate of group g(TD) reached 94%, significantly higher than that of g(T) and g(D), indicating that tannic acid (TA) and dipotassium glycyrrhizate (DG) enhance antioxidant capacity through synergistic effects in the hydrogel system of this invention. By synergistically introducing these two components into the hydrogel system, not only is its antioxidant performance significantly improved, but it also has the potential to regulate the level of reactive oxygen species (ROS) in the wound microenvironment, thereby helping to promote wound healing.

[0054] The antibacterial effect of the hydrogel prepared in Example 2 is as follows: Figure 7 As shown, Sacran / P407, g(T), g(D), and g(TD) hydrogels all exhibited strong antibacterial effects against *Escherichia coli* and *Staphylococcus aureus*. Compared with the control group, g(T), g(D), and g(TD) hydrogels significantly inhibited the growth and proliferation of *Escherichia coli* and *Staphylococcus aureus*. Notably, g(TD) hydrogel showed the best antibacterial effect, which was enhanced by the synergistic effect of the combined addition of TA and DG.

[0055] The cell compatibility of the hydrogel prepared in Example 2 is as follows: Figure 8As shown, there was no significant difference between the hydrogel group and the control group after 24 hours. However, after 72 hours, the cell viability of the hydrogel group was significantly improved, especially in the g(TD) hydrogel group, where the cell viability reached a maximum of 279.3%, which was about 30% higher than that of the control group. These results indicate that the tested hydrogel has excellent biocompatibility and can significantly promote the growth and survival of HUVECs.

[0056] The hydrogel prepared in Example 2 is used for wound healing, such as Figure 9 As shown, the wound healing progress of different treatment groups (including the commonly used commercial dressings Tegaderm™ film, Sacran / P407 hydrogel, g(T) hydrogel, g(D) hydrogel, and g(TD) hydrogel) at days 0, 3, 7, and 14) was compared. Based on the 14-day wound healing data, the healing rate of the Tegaderm™ film group was 73.5%, while that of the Sacran / P407 hydrogel group was 84.2%. This result indicates that the Sacran / P407 hydrogel group is more effective than the Tegaderm™ film. Furthermore, the healing rates of the g(T) hydrogel group, g(D) hydrogel group, and g(TD) hydrogel group were 95.3%, 96.7%, and 99.8%, respectively, showing that hydrogel treatments are significantly more effective than Tegaderm™ film. Overall, the hydrogel groups showed significantly better overall healing than the Tegaderm™ film, especially the g(TD) hydrogel group, which exhibited almost complete healing, reaching a healing rate of 99.8%.

[0057] In summary, this invention provides a biomimetic design of a temperature-sensitive hydrogel based on the combination of the natural polysaccharide Sacran and Poloxamer 407 (P407). By adjusting the Sacran content and optimizing the hydrogel structure, the prepared hydrogel exhibits excellent antibacterial, antioxidant, and anti-inflammatory effects, and can achieve controlled drug release, showing broad application prospects in wound repair and clinical treatment.

[0058] The experimental results above demonstrate that the present invention provides a temperature-sensitive hydrogel based on the combination of natural polysaccharides Sacran and P407, exhibiting excellent water absorption and gelling properties. Furthermore, when synergistically loaded with tannic acid and dipotassium glycyrrhizate for wound healing, this composite hydrogel displays significant antioxidant and antibacterial properties, as well as good cell compatibility, effectively promoting the wound healing process. The drug release characteristics of the hydrogel can be time-controlled to achieve slow drug release, thereby improving therapeutic efficacy. This hydrogel shows broad application potential in wound repair, particularly suitable for the treatment of chronic wounds and lesion repair.

[0059] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the present invention.

Claims

1. A thermosensitive injectable hydrogel based on the natural polysaccharides Sacran and poloxamer P407, characterized in that, The hydrogel material was obtained by physically mixing Sacran solution and poloxamer P407 solution; In the hydrogel, Sacran molecular chains form a hydrogel network with poloxamer P407 through physical entanglement and hydrogen bonding, and the Sacran polymer chains provide network support. The mass ratio of Sacran to poloxamer P407 in the hydrogel material is 1:100-1:

300.

2. The thermosensitive injectable hydrogel according to claim 1, characterized in that, The hydrogel has a gel point of 30-39℃, preferably 30-36℃.

3. The thermosensitive injectable hydrogel according to claim 1, characterized in that, The water absorption rate of the hydrogel is ≥300%, preferably ≥500%.

4. The method for preparing the injectable hydrogel according to any one of claims 1-3, characterized in that, Includes the following steps: S1. Preparation of Sacran solution: Provide Sacran and dissolve it completely in deionized water at 70-90°C with mechanical stirring to obtain a Sacran solution; S2. Preparation of poloxamer P407 solution: Prepare a 25-35% (w / w) P407 solution by cold water method; S3. Mix the Sacran solution obtained in step S1 with the P407 solution obtained in step S2 to obtain a hydrogel pre-solution. Let it stand at low temperature for 12-48 h to obtain an injectable hydrogel.

5. The preparation method according to claim 4, characterized in that, In step S1, the mass concentration of Sacran in the Sacran solution is 0.3%-0.5%; In step S2, the cold water method is prepared by stirring and dissolving at 4±2 °C; In step S3, the volume ratio of Sacran solution to P407 solution is 1:1 to 1:3; In step S3, the mixing method is vortex mixing for 5-20 minutes.

6. A composite hydrogel for wound healing, characterized in that, Includes the injectable hydrogel according to any one of claims 1-3 and tannic acid and dipotassium glycyrrhizinate loaded therein; The mass ratio of tannic acid to dipotassium glycyrrhizinate is 1:3 to 3:

1.

7. The composite hydrogel for wound healing according to claim 6, characterized in that, The loading of tannic acid is 0.01-10 mg / mL; And / or, the loading of the dipotassium glycyrrhizinate is 0.01-10 mg / mL.

8. The hydrogel for wound healing according to claim 6, characterized in that, The preparation method is as follows: the injectable hydrogel presol is provided, and the composite hydrogel is obtained by adding tannic acid and dipotassium glycyrrhizinate and loading at low temperature.

9. The use of the composite hydrogel according to any one of claims 6-8 in a full-thickness skin wound treatment product, characterized in that, Full-thickness skin trauma includes superficial wounds, burns, postoperative incisions, and diabetic foot ulcers.

10. The application according to claim 9, characterized in that, The products include bandages, spray dressings, and injectable wound healing agents.

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