A hydrogel and a preparation method and application thereof

Abstract

The application belongs to the technical field of biological medical material preparation, and provides a hydrogel, a preparation method and application thereof. The E-A powder is obtained by mixing the epigallocatechin gallate solution and the arginine solution, and then reacting under saturated water vapor at 121 DEG C to 134 DEG C, and drying the reaction solution. Then the E-A solution, the gelatin solution and the CMC solution are mixed, and then the copper salt is added, and the in-situ cross-linking is carried out to obtain the hydrogel. The hydrogel prepared by the application can realize long-acting and controllable release, reduce the nursing burden, and has a triple response phased function output. Meanwhile, the strong wet adhesion and self-healing of the hydrogel are more suitable for the skin and mucosa and other high-activity and high-humidity environments. In addition, the hydrogel can in-situ gel under physiological conditions, and has multi-form application. In application, the hydrogel has comprehensive comfort and economy, and has good popularization and economic feasibility in the clinical path.

Description

Technical Field

[0001] This invention belongs to the field of biomedical material preparation technology, specifically relating to a multi-corresponding hydrogel and its applications. Background Technology

[0002] The information disclosed in this background section is intended to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Perioperative and delivery-related wounds in obstetrics and gynecology (such as cesarean section skin / subcutaneous incisions, episiotomy or laceration suture sites, cervical conization / LEEP surgery wounds, and vaginal wall repair wounds) share common characteristics: moist location, high bacterial content, easy exudation, and significant impact from changes in position and friction. Common complications include infection, exudate imbalance, wound dehiscence, and scar formation. Biofilms and continuous exudation on the wound surface weaken dressing adhesion, increasing the frequency of dressing changes and the risk of secondary contamination. Mucosa and irregular incisions place higher demands on dressings for strong wet adhesion, smooth fit, and the ability to gel in situ. Traditional passive barrier dressings (gauze, conventional films / patches, etc.) struggle to maintain long-term adhesion and stable functional output in moist environments and irregular wounds, and their response to the combined repair needs of antibacterial, antioxidant, and angiogenic factors is insufficient, affecting repair quality and comfort.

[0004] While some existing functional hydrogels can provide a moist healing environment, their functions are limited and they lack the ability to synergistically regulate the microenvironment at different stages, such as infection / acidic exudation in the early perioperative period, tissue proliferation / neutral environment in the middle stage, and remodeling / microalkaline fluctuations in the later stage. At the same time, the problem of uncontrolled drug release remains prominent: single pH or single temperature response systems are prone to burst release or ineffective release during the infection period, resulting in local toxicity or insufficient efficacy; systems containing metal ions such as silver may also have side effects such as burst release of ions and inhibition of cell proliferation, and have limited support for angiogenesis and high-quality remodeling.

[0005] For example, Wu et al. prepared a DP7-ODEx single pH-responsive antibacterial hydrogel, which uses oxidized dextran (ODEX) and antimicrobial peptide DP7 to form a pH-sensitive network via a Schiff base reaction. This network can accelerate drug release in an acidic environment during the infection period, resulting in a prominent early antibacterial effect. However, in the synovial fluid / exudate fluctuations of soft tissues around bones and joints and in the later neutral to slightly alkaline environment, there is a tendency for early "burst" release followed by insufficient supply. At the same time, strong wet adhesion and adhesion retention after stress are insufficient, making it difficult to support long-term homeostasis management of sites such as portal incisions or synovectomy (Wu S, Yang Y, Wang S, et al. Dextran andpeptide-based pH-sensitive hydrogel boosts healing process in multidrug-resistant bacteria-infected wounds[J]. Carbohydrate polymers, 2022, 278:118994.). Zhang et al. reported a photocurable GelMA dressing, which is formed by mixing 10 wt% GelMA and 0.25 wt% LAP and then irradiating with 405 nm light for 60-90 s. This type of material belongs to a single phototriggered system, which requires a light source and a photoinitiator. It is inconvenient to operate on deep curved surfaces / narrow incisions or intraoperative / bedside conditions in soft tissues around bones and joints. It is acceptable for one-time application to moist surfaces, but secondary in-situ gelation and staged controlled release often require additional structural / process design, which is difficult to meet the combined requirements of long-term erosion resistance and staged drug delivery (Zhang J, Liu C, Li X, et al. Application of photo-crosslinkable gelatin methacryloyl in wound healing[J]. Frontiers in Bioengineering and Biotechnology, 2023, 11: 1303709.). Zhang et al. reported a sodium alginate-Ca²⁺ ion-crosslinked hydrogel, which was formed by spraying / soaking in a 2wt% sodium alginate solution to achieve crosslinking. Its advantages lie in liquid absorption and gel formation, as well as its passive barrier properties. However, in periarticular wounds with high exudation, synovial fluid infiltration, and repeated friction, it is prone to degradation due to Na₂O₃ ion crosslinking. + / Ca 2+Softening and detachment due to ion exchange, with insufficient strong wet adhesion and long-term steady-state output; lacking multi-stimulus adaptive and staged controlled release, and limited adhesion to irregular / stressed surfaces (Zhang M, Zhao X. Alginate hydrogel dressings for advanced wound management[J]. International Journal of Biological Macromolecules,2020, 162: 1414-1428.).

[0006] For those containing Ag + There are many reports on antibacterial hydrogels, which usually form a network by physical freeze-thaw of PVA / acidification and dissolution of chitosan, and then introduce AgNO3 or AgNP to endow it with broad-spectrum antibacterial activity (Aldakheel FM, Mohsen D, El Sayed MM, et al. Silver nanoparticles loaded on chitosan-g-PVA hydrogel for the wound-healing applications[J]. Molecules, 2023, 28(7): 3241.). This type of dressing can rapidly reduce bacterial load in the early stages of infection, but literature shows that the burst release of silver ions may lead to fibroblast / keratinocyte toxicity and delayed repair, and the long-term benefits of using it on clean / closed incisions are limited; at the same time, most of the systems are single-function antibacterial and lack staged controlled release and multi-axis adaptive capabilities (Khansa I, Schoenbrunner AR, Kraft CT, et al. Silver in woundcare—friend or foe?: a comprehensive review[J]. Plastic and ReconstructiveSurgery–Global Open, 2019, 7(8): e2390;Nešporová K, Pavlík V, Šafránková B, et al. Effects of wound dressings containing silver on skin and immune cells[J]. Scientific reports, 2020, 10(1): 15216.).

[0007] The above-mentioned shortcomings are particularly prominent in mucosa and high exudative sites. Clinically, there is still an urgent need for a new type of dressing that can adhere stably in a moist environment, can be injected to form gel in situ, and can provide "staged functional output and controlled release" according to the stage of the wound and changes in pH / ion / temperature.

[0008] Related studies have also shown that injectable high-strength / strong wet adhesion hydrogel adhesives can be used for suture replacement, antibacterial and low-scar repair; gels with antioxidant / ROS scavenging or oxygen supply capabilities can improve the infection-inflammatory microenvironment; and adhesive hydrogels with conductive or ion-regulating capabilities can also promote neurovascular regeneration when combined with physical therapy as needed. Summary of the Invention

[0009] To address the problems of existing dressings in obstetric and gynecological perioperative and childbirth-related wounds, such as limited functionality, insufficient adhesion in moist environments, uncontrolled drug release, and nursing burden caused by frequent dressing changes, this invention provides a hydrogel with triple responses to temperature, metal ions, and pH, enabling precise, controllable, and phased functional output and drug release regulation.

[0010] Another objective of this invention is to provide the above-mentioned hydrogel for use in various scenarios of perioperative and childbirth-related wounds in obstetrics and gynecology, which still possesses characteristics such as injectable in-situ gelation, strong wet adhesion, self-healing, and long-term stable controlled release in moist and bacterial environments.

[0011] To achieve the above objectives, the present invention adopts the following technical solution.

[0012] A method for preparing a hydrogel includes the following steps: (1) After mixing epigallocatechin gallate (EGCG) solution and arginine (Arg) solution, react them under saturated water vapor at 121℃-134℃. After drying the reaction solution, EGCG-arginine complex (EA) powder is obtained. (2) After mixing EA solution, gelatin solution and carboxymethyl cellulose (CMC) solution, copper salt is added and crosslinked in situ to obtain hydrogel.

[0013] In step (1), the solvent for the EGCG solution or Arg solution is water or a buffer solution. The pH of the buffer solution is 3.8-8.0; preferably 4.5-7.5; more preferably 4.5-5.5 or 6.5-7.5; most preferably 6.8-7.4. The concentration of the EGCG solution is 0.5%w / v-1.0%w / v; preferably 0.7wt%. The concentration of the Arg solution is 0.1%w / v-1.0%w / v; preferably 0.3wt%. The volume ratio of the EGCG solution to the Arg solution is 1:2-2:1, such as 1:2, 1:1, 2:1; preferably 1:1.

[0014] In step (1), the pressure of saturated water vapor at 121℃-134℃ is 0.10-0.15 MPa; the reaction time is 20-30 min.

[0015] In step (1), the drying method is preferably freeze drying.

[0016] In step (2), the mass ratio of EA to gelatin is 1:150-1:400, preferably 1:200-1:300, and more preferably about 1:200. Cu 2+ The mass ratio of EA to EA is 1:2-2:1, such as 1:2, 1:1, 2:1; preferably 2:1.

[0017] In step (2), the concentration of the EA solution is 0.3% w / v to 1.2% w / v; preferably 1.0% w / v. The concentration of the gelatin solution is 10% w / v to 30% w / v; preferably 20% w / v. The concentration of the CMC solution is 1% w / v to 3% w / v, preferably 2% w / v.

[0018] In step (2), the copper salt can be added as a solid or as a solution. The concentration of the copper sulfate solution is 0.2% w / v to 1.0% w / v; preferably 0.6% w / v.

[0019] A hydrogel obtained by the above preparation method. The gelatin segments of this hydrogel undergo a reversible triple-helix-coil transition near physiological temperatures, endowing it with thermosensitive mechanical regulation and morphological reconstruction capabilities; secondly, Cu... 2+ It forms dynamic coordination bonds with the coordination sites on the EGCG-arginine complex (EA) and CMC / gelatin, and can quickly rebuild the network after external disturbances (such as changes in body position, friction, and exudate flushing), achieving self-healing and adhesion retention. In addition, the hydrogen bonds and electrostatic interactions between EA and CMC / gelatin are synergistically coupled with the above coordination network to jointly construct a multi-point reversible cross-linked structure.

[0020] This invention also provides that the above-mentioned hydrogel can be used to prepare patches or gels for use on skin or mucous membrane surfaces. The patches or gels have temperature, metal ion concentration, and pH responsive characteristics, and can also serve as carriers or matrices for other active ingredients, providing sustained drug release. When used as a carrier or matrix, the active ingredients can be added and mixed evenly during the in-situ crosslinking process of the raw materials; the crosslinking reaction is complete when the product is obtained.

[0021] The present invention also provides drugs or medical devices prepared from the above-described hydrogel. These drugs or medical devices can be used on the surface of skin or mucous membranes or wounds, and have antibacterial, antioxidant, and anti-inflammatory effects, promoting wound healing.

[0022] The mechanism of the multi-response hydrogel of the present invention is as follows: (1) Temperature response: The thermosensitive transition of gelatin is related to the network viscoelasticity. Body surface / mucosal temperature helps in-situ gelation and adhesion, and restores strength after shear release; (2) Metal ion concentration response (Cu) 2+ Coordination bond density and strength vary with Cu 2+ Horizontal changes thereby regulate network reconstruction rate, adhesion strength, and exudation adaptability; (3) pH response: Local pH affects the dissociation and coordination of phenolic hydroxyl / amine groups and hydrogen bonding, thereby finely regulating the drug release rate and adhesion / barrier effectiveness.

[0023] The present invention has the following advantages: The hydrogel prepared by this invention can achieve long-lasting and controlled release, reducing the burden of care: through "EA prepolymer oligomer particles + Cu 2+ The dual sustained-release and stabilization mechanism of "dynamic coordination nodes" significantly prolongs the release time and inhibits burst release, reducing the risk of cell stimulation caused by local peak concentrations, thereby reducing the frequency of dressing changes and improving nursing efficiency.

[0024] This hydrogel has a triple-response phased functional output: temperature-metal ion-pH synergistic regulation, which allows the hydrogel to output antibacterial / antioxidant / angiogenic functions according to the phased changes in the microenvironment of the obstetric and gynecological wound, taking into account the continuous needs of early bacterial control and anti-inflammatory and mid-to-late-stage tissue reconstruction.

[0025] Meanwhile, the hydrogel's strong wet adhesion and self-healing properties make it more suitable for skin and mucous membranes: the material can still adhere stably under moist exudation and bacterial conditions; under shearing action, it is an injectable low-viscosity fluid, and after shearing stops, it quickly reconstructs into a stable network; microcracks / microdamage can be self-repaired through dynamic bonds, extending the service life of the dressing and maintaining barrier integrity, making it suitable for high-activity and high-humidity environments such as the perineum, cervix and vaginal mucosa.

[0026] In addition, this hydrogel can form in situ under physiological conditions and has multiple applications: it can form in situ under physiological conditions without the need for toxic chemical cross-linking agents or external irradiation; the system can be prepared as an injection type, film type or surface coating type as needed for wound management in multiple sites such as cesarean section incision, perineal incision / laceration, and cervical / vaginal surgery.

[0027] In terms of application, this hydrogel offers a combination of comfort and economy: its stable fit, reduced dressing changes, and phased release work together to help reduce the risk of secondary contamination caused by exudation, extravasation, and edge lifting, thereby improving patient comfort and demonstrating good scalability and economic feasibility in clinical pathways. Attached Figure Description

[0028] Figure 1 The infrared spectrum (FT-IR) of the EA complex is shown below. Figure 2 The image shows a scanning electron microscope (SEM) image of the hydrogel. Figure 3 This diagram illustrates the injectable and in-situ gelation of hydrogels. Figure 4 This is a schematic diagram of hydrogel self-healing. Figure 5 Visual representation of the pH response of the hydrogel; Figure 6 The repair process of the wound model; Figure 7 This represents the percentage of the healed area in the wound model. Figure 8 Image of hematoxylin-eosin (H&E) staining at the wound site; Figure 9 Masson staining image of the wound; Figure 10 The images show the in vitro release curves of the active components in CMC / Gel / Cu hydrogels with different EA contents; where CMC@Gel represents the blank matrix without added EA active components; 0.002%CE, 0.004%CE, 0.006%CE and 0.008%CE represent samples with EA concentrations of 0.002%, 0.004%, 0.006% and 0.008%, respectively. Detailed Implementation

[0029] The present invention will be further described below with reference to the embodiments and accompanying drawings, but the present invention is not limited to the following embodiments.

[0030] Example 1: Preparation of Multi-responsive Hydrogels 0.50 g of EGCG was dissolved in PBS buffer and the volume was adjusted to 100 mL (0.5%); 0.20 g of arginine was dissolved in PBS buffer and the volume was adjusted to 200 mL (0.1%). The two mixtures were combined at a volume ratio of 1:2, reacted under high pressure at 121 °C for 20-30 min, cooled, rapidly frozen in liquid nitrogen, and lyophilized to obtain EA powder. Its Fourier transform infrared spectrum (FT-IR, wavenumber range 4000-400 cm⁻¹) was analyzed. -1 4 cm resolution -1 (32 scans average) Figure 1 As shown: Comparing the spectra of EGCG and arginine, EA powder at 3300 cm⁻¹... -1 The absorption peak of phenol-OH at 1500 cm⁻¹ weakens and undergoes a red shift. -1 The decreased peak intensity of the left and right aromatic rings (C=C) suggests the formation of the EA complex; 1700 cm⁻¹ -1 The slight red shift and decreased intensity of the nearby ester / ketone C=O stretching peak further corroborate that EGCG reacted with arginine. Weigh out CMC and add it to water. Heat in a water bath at 60-70℃ for 0.5-1 h to obtain 10 mL of 1% solution A. Weigh out gelatin and add it to water. Heat in a water bath at 50-60℃ until completely dissolved to obtain 10 mL of 10% solution B. Weigh out EA powder and dissolve it completely in water to obtain 2 mL of 0.3% solution C. Measure out 5.9 mL of 0.2% CuSO4·5H2O solution (Cu... 2+ Approximately 3.0 mg, causing Cu 2+ Solution D was prepared by mixing solutions A, B, C, and D (EA to gelatin mass ratio of 1:2) at room temperature, and then performing an in-situ crosslinking reaction to obtain a hydrogel.

[0031] After hydrogelling, the gel was pre-frozen at -80℃ for 4 h and then freeze-dried under vacuum for 48 h to obtain a dry gel. The gel was then observed using a field emission scanning electron microscope (accelerating voltage 5-10 kV, working distance 8-12 mm). Figure 2 As shown: The hydrogel exhibits a porous structure with interconnected channels. This structure has a large specific surface area, which on the one hand is conducive to increasing drug loading and supporting the two-stage release of early acceleration and late steady state. On the other hand, the interconnected channels provide a three-dimensional scaffold for cell migration and angiogenesis. At the same time, it also helps to disperse external forces, conform to irregular incisions and drain exudates / metabolic waste, thus making it suitable for sites with a large amount of exudate, such as cesarean sections and perineum.

[0032] Example 2 Preparation of Injectable Hydrogel The hydrogel prepared in Example 1 was used directly as an injection hydrogel and pre-filled into a 5 mL syringe.

[0033] The apparent viscosity of the above-mentioned injectable hydrogel was measured at different shear rates. The results showed that at 0.1 s⁻¹... -1 At the shear rate, the apparent viscosity is 1.2 Pa·s (=1200 mPa·s) at 100 s⁻¹. -1 At the shear rate, the apparent viscosity is 0.06 Pa·s (=60 mPa·s), which is about 20 times lower than the viscosity, proving that the system has obvious shear-thinning characteristics.

[0034] The aforementioned injectable hydrogel was injected onto glass slides at 25°C and 37°C, respectively. The results showed that a shear-thinning-shear-relief-rapid thickening process, characteristic of injectable hydrogels, could be directly observed between 25-37°C, and temperature significantly affected the gelation rate: at 37°C, gelation occurred within approximately 60 seconds due to the rapid recovery of the gel's triple helix structure, while at 25°C, it took approximately 120 seconds for complete solidification. This indicates that increasing the temperature is beneficial for the recovery and strength enhancement of the gel's triple helix. These results demonstrate that the injectable hydrogel exhibits excellent temperature response, rapidly gelling near human body temperature, adhering to wounds or lesions, and providing support.

[0035] The above-mentioned injectable hydrogel was subjected to injection speed changes at 37°C to observe its ability to form continuous gels. The results showed that continuous gel filaments could be formed when the injection speed was controlled within the range of 0.2-1.0 mL / min. If the speed was too fast, the local shear heat would increase and the network would be slightly damaged. If the speed was too slow, the filaments would be uneven. The best gel formation and molding effect could be obtained at around 0.5 mL / min.

[0036] The pre-filled hydrogel was fitted with a 25G needle and continuously injected into PBS (pH 7.4) at 37°C at a bolus rate of 0.5 mL / min. Figure 3 It can be seen that continuous gel filaments can be formed in water, and gel solidification can be completed in about 60 seconds after shearing stops.

[0037] The above experimental results show that the prepared hydrogel exhibits shear thinning during injection / push, while maintaining continuity and coatability during the push process. After entering the wound, it can quickly gel and rebuild strength as the shear is released, achieving precise adhesion and sealing of irregular or deep gaps. It can adhere closely to moist mucous membranes and irregular wounds without the need for UV / additional crosslinking agents, providing wet adhesion sealing, self-healing, and long-lasting controllable release.

[0038] Example 3: Preparation of hydrogel films Dissolve 0.70 g of EGCG in PBS buffer and bring the volume to 100 mL (0.7%); dissolve 0.30 g of arginine in PBS buffer and bring the volume to 100 mL (0.3%). Mix them at a volume ratio of 1:1, autoclave at 121℃ for 20-30 min, cool, freeze quickly in liquid nitrogen, and lyophilize to obtain EA powder. Weigh out CMC and add it to water. Heat in a water bath at 60-70℃ for 0.5-1 h to obtain 10 mL of 2% solution A; weigh out gelatin and add it to water. Heat in a water bath at 50-60℃ until completely dissolved to obtain 10 mL of 20% solution B; weigh out EA powder and completely dissolve it in water to obtain 1 mL of 1% solution C; measure out 6.55 mL of 0.6% CuSO4·5H2O solution (Cu 2+ Approximately 10.0 mg, causing Cu 2+ Solution D was prepared by rapidly mixing solutions A, B, C, and D at room temperature (EA to gelatin mass ratio of 1:400), pouring the mixture into a silicone mold, and coating it into a film at a uniform speed of 0.2 mm / s. The film was then placed horizontally at 32°C for 6 h to allow for slow evaporation, and subsequently dried under vacuum at 40°C for 2 h to obtain a self-supporting film.

[0039] The thickness was measured at five locations on a single film, including the center and four corners, using a digital thickness gauge or micrometer. The measured thickness of the film at different locations ranged from 0.5 to 1.0 mm.

[0040] The above-mentioned film was cut into two halves at room temperature (23℃±2℃, relative humidity 45-60%) and then joined together. After standing for 1-2 hours, a macroscopic evaluation was performed. The macroscopic self-healing behavior was that the sample could be lifted and bent without breaking. The results are as follows: Figure 4 As shown: the complete membrane on the left is cut into two segments as shown in the middle image; then, without applying continuous external force, the membrane can complete the macroscopic self-healing process shown in the middle image, where it bends without breaking, as shown in the right image. This demonstrates that after the material is subjected to external force or spatial friction that generates microcracks, it can rely on a dynamic coordination / hydrogen bond network to achieve structural self-repair and restore adhesion / barrier properties, reducing replacement frequency and maintaining continuous sealing and stable release.

[0041] The above-mentioned film was cut into dumbbell-shaped specimens prepared according to ASTM D412 Method A at room temperature (23±2℃, relative humidity 45%-60%), and the thickness of the measured part was the actual thickness of the film. The original film and the self-healed film were subjected to uniaxial tensile testing until fracture, and their fracture strength σ was recorded, denoted as σ0. pristine and σ healed During the test, the fixture separation rate was 500 mm / min. The fracture strength of the self-healed film was significantly restored compared to the original film. The self-healing efficiency η = σ healed / σ pristine × 100%.

[0042] Table 1 Mechanical strength of thin films The results in Table 1 show that the fracture strength of the self-healing film is significantly restored compared to the original film, indicating that the dynamic coordination bond and hydrogen bond network in the material can be reconstructed at the fracture interface, thereby restoring the continuity and load-bearing capacity of the film.

[0043] Example 4: Preparation of wound dressing 1. Preparation of Fiber Membranes Preparation of bromothymol blue-chitosan electrostatic fibers by electrospinning: Chitosan was dissolved in a 2% (v / v) aqueous acetic acid solution to prepare a 2wt%-3wt% spinning solution. 0.05wt%-0.1wt% bromothymol blue was added and stirred until homogeneous. After ultrasonic degassing, the solution was loaded into a syringe and electrospun at a flow rate of 0.4 mL / h, a voltage of 15 kV-18 kV, and a collection distance of 15 cm under conditions of 25℃±2℃ and relative humidity of 40%-50%. The resulting fiber membrane was vacuum dried to remove acid before use.

[0044] 2. Preparation of pH-responsive wound dressings Solutions A, B, C, and D were prepared according to the method in Example 3, quickly mixed, poured into a silicone mold, and uniformly coated at a speed of 0.2 mm / s. Then, bromothymol blue-chitosan electrostatic fibers were laid on the wet film surface and bonded in situ to obtain a pH-visually responsive wound dressing.

[0045] The above-mentioned wound dressing was prepared into small, identical samples, which were then immersed in different buffer solutions with pH values ​​ranging from 2 to 12, and the color changes were observed. The results are as follows: Figure 5 As shown: the sample exhibits a color change from yellow to green to blue within the pH range of 5.0-8.0.

[0046] The above experimental results show that the composite membrane exhibits a reversible color change from yellow to green to blue within the pH range of 5.0-8.0, which can reflect the changes in pH of wound exudate or local environment in real time: it is yellow-green when acidic (infection or inflammation period), and gradually turns blue as the wound environment returns to neutral or slightly alkaline (proliferation and remodeling period), thereby realizing the visual monitoring of wound status and providing intuitive basis for dressing change, infection early warning and healing stage assessment.

[0047] In addition, by replacing or compounding the acid-base indicator in the fiber membrane (such as bromothymol blue / methyl red, etc.), the response range can be extended to pH 2-12, which can be used for visual indication of exudation and pH fluctuations, guiding dressing changes and care.

[0048] Application Example 1: Wound dressing for skin wound healing in mice 1. Establishment of animal models Female ICR mice aged 6-8 weeks (purchased from Spiford (Beijing) Biotechnology Co., Ltd.) were shaved on their backs to create a full-thickness circular excision wound with a diameter of 8 mm. After cutting the wound, the diameter was measured with a ruler and found to be 8±1 mm, thus obtaining a full-thickness skin wound model mouse.

[0049] 2. Experimental grouping and treatment The model mice were randomly divided into groups of 3 mice each, and then treated as follows: Control group: No treatment received; Experimental group: The wound dressing used in Example 4 was applied.

[0050] The control and experimental groups were photographed at fixed points on days 0, 3, 6, 9, 12, and 14, and the wound area was quantified using ImageJ. The percentage of healed area was calculated, and the wound healing time was observed and recorded.

[0051] Regenerated tissue was harvested on day 14, routinely fixed, paraffin-embedded, sectioned, and stained with H&E and Masson staining; epidermal continuity and thickness, degree of inflammatory cell infiltration, and amount of collagen deposition were evaluated to comprehensively reflect the quality of tissue repair and remodeling.

[0052] Compared to the control group, the experimental group showed faster wound shrinkage in the early stages, and by day 14 of treatment, the wound was almost closed. Figure 6 and Figure 7 No infection or rejection reactions were observed throughout the experiment.

[0053] Figure 8 The results showed that the control group's wound was still covered with a large amount of blood clots, and the density of newly formed tissue was significantly lower than that of the experimental group. On day 14, the area of ​​inflammatory cell infiltration at the wound site in the experimental group was significantly reduced compared to the control group. Furthermore, the experimental group had the highest number of newly formed blood vessels. In conclusion, the experimental group showed better wound healing.

[0054] Figure 9 The results showed that the experimental group had a higher area and density of blue-stained areas (collagen fibers) than the control group, and was rich in coarse collagen fibers with the collagen arranged in a parallel and orderly manner. These results suggest that the wound dressing in Example 4 helps promote the synthesis, deposition, and remodeling of the extracellular matrix in wound cells.

[0055] Example 5 Preparation of drug-loaded hydrogel Dissolve 2.00 g of EGCG in PBS buffer and bring the volume to 200 mL (1.0%); dissolve 1.00 g of arginine in PBS buffer and bring the volume to 200 mL (1.0%). Mix them at a volume ratio of 2:1, autoclave at 121℃ for 20-30 min, cool, freeze quickly in liquid nitrogen, and lyophilize to obtain EA powder. Weigh out CMC and add it to water. Heat in a water bath at 60-70℃ for 0.5-1 h to obtain 10 mL of 3% solution A; weigh out gelatin and add it to water. Heat in a water bath at 50-60℃ until completely dissolved to obtain 10 mL of 30% solution B; weigh out EA powder and dissolve it completely in water to obtain 0.8 mL of 1.2% solution C; measure out 7.55 mL of 1.0% CuSO4·5H2O solution (Cu 2+ Approximately 19.2 mg, making Cu 2+ Solution D is prepared by mixing solutions A, B, C, and D at a mass ratio of 2:1 (EA). Then, 1.0 mL of a 2 mg / mL vitamin B complex is added. 12 A sol with an EA concentration of approximately 0.003% (EA to gelatin mass ratio of 1:312) was obtained. The resulting sol can be applied to the mucosal surface after cervical conization / LEEP or vaginal wall repair surgery using a soft brush / applying head, and left for 1-2 minutes to form a dense gel layer, achieving wet adhesion sealing, exudate adaptation, and phased controlled release.

[0056] A blank matrix without added EA active component was prepared according to the above method, denoted as CMC@Gel; simultaneously, samples with EA concentrations of approximately 0.002%, 0.004%, 0.006%, and 0.008% were prepared, denoted as 0.002%CE, 0.004%CE, 0.006%CE, and 0.008%CE (EA to gelatin ratios of 1:469, 1:234, 1:156, and 1:117, respectively); then, the above gel (0.2 g) was placed in a dialysis bag with a molecular weight cutoff of 100 kDa, 20 mL of PBS (pH 7.4) was added, and an in vitro release experiment was conducted at 37°C. Samples were taken and tested at preset time points using a "sampling-equal volume replenishment" method. The cumulative release amount M was calculated using the cumulative correction method. t And further calculate the cumulative release rate M t / M ∞ ×100%. Where, t 50 Defined as the time when the cumulative release rate reaches 50%.

[0057] The "steady-state phase" refers to the release curve entering the late near-linear sustained-release range, while simultaneously meeting the following conditions: (1) The cumulative release-time data at least three consecutive time points can be linearly fitted; (2) The correlation coefficient R of the linear fit in this interval 2 ≥0.95; (3) The coefficient of variation (CV) of the release rate in adjacent time periods is ≤15%; (4) The relative change in slope between two adjacent segments at the beginning and end of the interval shall not exceed 20%.

[0058] When the above conditions are met, this interval is denoted as the zero-order or quasi-zero-order release stage, and its linear fitting slope k ss As a characterization parameter for sustained release performance.

[0059] Table 2 t 50 And the sustained-release slope in the later stages.

[0060] The results are as follows Figure 10 As shown in Table 2, the blank matrix CMC@Gel group showed almost no detectable release of active components; however, after the addition of EA, all experimental groups exhibited a two-stage characteristic of rapid release in the early stage followed by a slow-release and stabilizing stage. Samples with EA concentrations of 0.002%, 0.004%, 0.006%, and 0.008% showed the following t 50 The average release rates were approximately 1.91 h, 3.59 h, 3.27 h, and 3.37 h, respectively; the average release rates in the sustained-release phase from 24 to 48 h were approximately 0.80, 0.53, 0.75, and 0.70 μg·mL, respectively.-1 ·h -1 This indicates that as the EA concentration increases, the overall cumulative release level of the system improves, while maintaining a low but sustained release rate in the later stages, exhibiting good sustained-release characteristics. An appropriate EA concentration can be selected based on the drug release rate.

[0061] The above description is merely an embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principle of this application should be included within the scope of the claims of this application.

Claims

1. A method for preparing a hydrogel, characterized in that, Includes the following steps: (1) Mix EGCG solution and Arg solution and react them under saturated water vapor at 121℃-134℃. After the reaction solution is dried, EA powder is obtained. (2) After mixing EA solution, gelatin solution and CMC solution, copper salt is added and crosslinked in situ to obtain hydrogel.

2. The preparation method according to claim 1, characterized in that, In step (1), the solvent for the EGCG solution or Arg solution is water or a buffer solution; the pH of the buffer solution is 3.8-8.0; the concentration of the EGCG solution is 0.5%w / v-1.0%w / v; the concentration of the Arg solution is 0.1%w / v-1.0%w / v; and the volume ratio of the EGCG solution to the Arg solution is 1:2-2:

1. The mass ratio of EA to gelatin is 1:150-1:400; Cu 2+ The mass ratio of EA to EA is 1:2-2:1; In step (1), the reaction time is 20-30 min; The concentration of EA solution was 0.3% w / v-1.2% w / v; the concentration of gelatin solution was 10% w / v-30% w / v; and the concentration of CMC solution was 1% w / v-3% w / v.

3. The preparation method according to claim 2, characterized in that, The buffer solution has a pH of 4.5-7.5; the EGCG solution has a concentration of 0.7 wt%; the Arg solution has a concentration of 0.3 wt%; and the volume ratio of the EGCG solution to the Arg solution is 1:

1. The mass ratio of EA to gelatin is 1:200-1:300; Cu 2+ The mass ratio of EA to EA is 2:

1.

4. The preparation method according to claim 2, characterized in that, The buffer solution has a pH of 4.5-5.5 or 6.5-7.5; the EA solution has a concentration of 1.0% w / v; the gelatin solution has a concentration of 20% w / v; and the CMC solution has a concentration of 2% w / v.

5. The preparation method according to claim 2, characterized in that, The pH of the buffer solution is 6.8-7.

4.

6. The preparation method according to claim 1, characterized in that, Copper salts are added either as a solid or in a solution.

7. The preparation method according to claim 6, characterized in that, The copper salt was added as a solution; the concentration of copper sulfate was 0.2% w / v - 1.0% w / v.

8. A hydrogel obtained by the preparation method according to any one of claims 1-7.

9. The use of the hydrogel of claim 7 in the preparation of a patch or gel for use on a skin surface or mucosal surface.

10. A drug or medical device prepared from the hydrogel as described in claim 7.

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