Epigallocatechin-3-gallate loaded guanosine borate hydrogel as well as preparation method and application thereof

Abstract

The invention relates to the field of biomedical materials, in particular to catechin loaded guanosine borate hydrogel as well as a preparation method and application thereof. The preparation method comprises the following steps: putting guanosine and KOH into a round-bottom flask, adding deionized water, heating and stirring until guanosine is completely dissolved; then adding boric acid and epigallocatechin-3-gallate (EGCG), continuously heating and stirring until the reaction is completed, and cooling to room temperature, so as to prepare the EGCG loaded guanosine borate hydrogel. According to the hydrogel disclosed by the invention, guanosine is self-assembled to form a G-quadrubplex network, and boric acid ester bonds are introduced to strengthen crosslinking, so that the stability and the mechanical strength of the gel are improved. PH / ROS dual response is achieved, and precise controlled release is achieved; the epigallocatechin-3-gallate is stably loaded, so that the stability of the epigallocatechin-3-gallate is improved, the bioavailability is increased, the biocompatibility is good, and the epigallocatechin-3-gallate is free from tissue toxicity; angiogenesis is promoted, and diabetes wound healing is accelerated.

Description

Technical Field

[0001] The present invention relates to the field of biomedical materials, and particularly relates to an epigallocatechin-3-gallate-loaded guanosine borate hydrogel, its preparation method and application. Background Art

[0002] Diabetes is a common chronic metabolic disease, mainly characterized by persistent hyperglycemia. According to statistics, about 19–34% of diabetic patients may develop chronic diabetic wounds, which are not only common and complex but also accompanied by high treatment costs. Wound healing failure is usually attributed to a variety of internal and external factors. Among them, neuropathy, vascular abnormalities and systemic pathological changes caused by diabetes belong to the internal pathogenic mechanisms, while wound infection, blocked formation of healing tissue and excessive local mechanical pressure are external influencing factors. Although the specific mechanism of diabetic wound healing disorder has not been fully elucidated, studies have shown that the chronic inflammatory state is the core feature of this pathological process, which is mainly driven by factors such as oxidative stress, impaired angiogenesis, abnormal expression of pro-inflammatory cytokines and bacterial infection.

[0003] In response to this medical problem, the scientific community has devoted a great deal of effort to finding effective intervention strategies. At present, some bioactive substances (such as antibiotics, anti-inflammatory agents, exosomes and growth factors) are considered to be able to inhibit chronic inflammation and promote wound repair. However, these treatment methods also have certain limitations, including the development of drug resistance, adverse reactions and high economic burden. As a non-antibiotic antibacterial strategy, natural antibacterial compounds have received extensive attention in recent years, and they show good prospects in inhibiting bacterial growth, reducing the risk of antibiotic abuse and reducing drug resistance problems.

[0004] Wound dressings are considered an efficient means of locally delivering therapeutic drugs to the wound area. To date, various types of wound dressings have been designed to promote wound repair, including porous foams, biocompatible films, rubber-based materials, electrospun nanofibers, and functional hydrogels. Among these materials, hydrogels have received extensive attention due to their excellent physical and chemical properties, such as high porosity, soft elasticity, and good water retention capacity. In addition, hydrogels can effectively absorb excess exudate, maintain a moist microenvironment, and provide a cooling effect to relieve pain. In recent years, researchers have been committed to introducing various bioactive functions into the hydrogel system to regulate the local wound microenvironment and accelerate the healing process, such as strategies for reactive oxygen species scavenging, photothermal / photodynamic therapy, and immune cell regulation. Although these hydrogel-based dressings have shown good therapeutic potential in experiments on different animal models, their insufficient mechanical strength and lack of self-healing ability remain the main obstacles to clinical application. Normal human activities and local pressure changes may cause the dressing to rupture, thus weakening its protective effect on the wound surface and increasing the risk of infection. Therefore, endowing hydrogels with adjustable mechanical properties and self-healing ability is crucial for optimizing the clinical application of wound dressings.

[0005] Introducing multiple reversible cross-linking bonds into the hydrogel system is considered an effective strategy to improve the mechanical properties and self-healing ability of hydrogels. Such hydrogels have been widely used in fields such as tissue engineering, flexible electronic devices, and electronic skin. Due to the dynamic nature of reversible cross-linking, they not only have excellent mechanical strength but also possess good shape recovery and self-healing properties, which are particularly important for the construction of ideal wound dressings. Currently, hydrogels based on cross-linking systems such as polydopamine, Schiff base, and ureidopyrimidinone have been developed and applied in the field of wound healing. Although these hydrogels have shown good antibacterial, antioxidant, and wound-healing effects in the repair of ordinary skin wounds, they still face challenges in the treatment of chronic diabetic wounds, mainly due to the lack of effective immune regulation and angiogenesis-promoting abilities.

[0006] Among the existing treatment methods for diabetic wounds, traditional dressings and conventional hydrogels have many deficiencies: Traditional dressings (such as gauze, foam, and fiber dressings) have certain advantages in aspects such as moisture absorption, antibacterial properties, and wound adhesion. However, their functions are single, they cannot achieve precise drug release according to the dynamic changes in the wound microenvironment (such as local low pH and high reactive oxygen species levels), and they are prone to cause secondary injuries during dressing changes; Conventional chemically cross-linked macromolecular hydrogels, although having a good moist environment, generally have poor mechanical properties, cannot adapt to the dynamic stress at the wound site, and have a single drug release mode, lacking responsive regulation to changes in the wound environment; Although the supramolecular hydrogels constructed by the self-assembly of guanosine currently exhibit excellent performance in terms of self-healing, injectability, and biocompatibility, they still suffer from problems such as insufficient gel structure stability and limited controlled-release precision. At the same time, the loading and protection effects on natural drugs such as catechins are limited, which easily leads to a decrease in the activity of the drugs during delivery.

[0007] Catechin compounds are the main functional components in tea leaves, mainly including 8 monomers. Epigallocatechin-3-gallate (EGCG) is the catechin monomer with the most prominent antioxidant properties among them and has gradually become a research hotspot due to its extensive pharmacological effects. Research shows that EGCG has various biological activities such as antibacterial, anti-inflammatory, antioxidant, anti-aging, pro-angiogenic, and anti-tumor effects, and has been deeply explored in multiple medical fields. However, its application is still limited by defects such as low bioavailability and rapid metabolism. Therefore, developing a suitable drug delivery system to accurately and efficiently deliver EGCG to the lesion site and achieve controlled release is of great significance for the treatment of diabetic wounds.

[0008] As a natural polyphenol, epigallocatechin-3-gallate not only has biological activities such as antibacterial, anti-inflammatory, antioxidant, and pro-angiogenic effects, but also the catechol group in its molecular structure can form reversible borate ester bonds with boric acid (BA), thus it can be used to construct a dynamic cross-linked hydrogel. Therefore, it is envisioned to use boric acid (BA) and epigallocatechin-3-gallate to form a dynamic cross-linked system to prepare a hydrogel with excellent mechanical properties and therapeutic functions in a simple manner. In addition, this hydrogel can achieve the controlled release of epigallocatechin-3-gallate, thereby effectively exerting its therapeutic effect of promoting wound healing locally. Summary of the Invention

[0009] The present invention aims to overcome the deficiencies of traditional dressings and conventional hydrogels in terms of dynamic response, mechanical strength, and drug controlled release, and provides a method for preparing an epigallocatechin-3-gallate-loaded guanosine borate hydrogel. The epigallocatechin-3-gallate-loaded guanosine borate hydrogel of the present invention not only has better performance than the prior art in terms of mechanical properties, responsiveness, drug release control, and biological safety, but also can achieve the precise release of drugs in the low-pH and high-ROS environment at the local diabetic wound surface, thereby improving the wound microenvironment, promoting the rapid healing of the wound surface, and ensuring good biological safety and operational convenience.

[0010] Epigallocatechin-3-gallate, as a natural polyphenolic compound, has anti-inflammatory and antibacterial properties. However, its stability is affected by the environment and it is prone to oxidation reactions, leading to the degradation of active ingredients and low bioavailability. These limitations restrict its clinical application. The present invention designs and prepares a supramolecular hydrogel of epigallocatechin-3-gallate based on guanosine (GBE hydrogel or also known as EGCG hydrogel), forms a stable G-quadruplex nanofiber network, and realizes the precise loading and responsive release of epigallocatechin-3-gallate by combining dynamic borate bonds. This system accelerates drug release in acidic and oxidative environments, and at the same time has mechanical strength, self-healing ability and injectability, providing a new type of dressing option for solving problems such as out-of-control inflammation and slow tissue regeneration in diabetic wound repair.

[0011] To solve the above technical problems, the technical solution adopted in the present invention is: a preparation method of a catechin-loaded guanosine borate hydrogel. Put guanosine and KOH in a round-bottom flask, add deionized water, heat and stir until the guanosine is completely dissolved; then add boric acid and epigallocatechin-3-gallate, continue to heat and stir until the reaction is completed and then cool to room temperature, and the epigallocatechin-3-gallate-loaded guanosine borate hydrogel is prepared.

[0012] As a further limitation of the technical solution of the present invention, the molar ratio of guanosine, boric acid, KOH and epigallocatechin-3-gallate is: 1.2:1:2.4: 0.3-0.8.

[0013] As a further limitation of the technical solution of the present invention, the molar ratio of guanosine, boric acid, KOH and epigallocatechin-3-gallate is: 1.2:1:2.4:0.8.

[0014] As a further limitation of the technical solution of the present invention, the temperature of heating and stirring is 95 °C.

[0015] The present invention provides an epigallocatechin-3-gallate-loaded guanosine borate hydrogel prepared by the above preparation method.

[0016] As a further limitation of the technical solution of the present invention, the hydrogel is formed by self-assembly of guanosine to form a G-quadruplex network, and borate bonds are introduced to strengthen cross-linking. Epigallocatechin-3-gallate is stably embedded in the gel network through dynamic covalent coupling, hydrogen bonds and π-π stacking interactions.

[0017] In addition, the present invention also provides the application of the above epigallocatechin-3-gallate-loaded guanosine borate hydrogel in diabetic wound repair.

[0018] Compared with the prior art, the present invention has the following beneficial effects: The catechin-loaded guanosine borate supramolecular hydrogel of the present invention is superior to the prior art in terms of mechanical properties, responsiveness, drug release control, and biosafety. The functions and beneficial effects of each technical feature are as follows: (1) Stable structure, excellent mechanical properties, and possess self-healing and injectability This hydrogel uses guanosine to self-assemble to form a G-quadruplex network, and introduces borate ester bonds to strengthen cross-linking, improving the stability and mechanical strength of the gel. Rheological tests show that in time sweep and frequency sweep, the storage modulus (G′) of the hydrogel is always higher than the loss modulus (G″), indicating its stable solid-like characteristics. The dynamic strain sweep results show that when the shear strain reaches 23.5%, the hydrogel changes from the gel state to the sol state and can then quickly recover, possessing good self-healing ability. When the shear rate increases, the viscosity of the hydrogel decreases significantly, showing a shear-thinning effect, and can smoothly pass through the syringe needle without clogging, indicating its excellent injectability and better conformability to the wound surface.

[0019] (2) Possess dual pH / ROS responsiveness and achieve precise controlled release Utilizing dynamic borate ester bonds, the gel disintegrates in the unique low pH and high reactive oxygen species (ROS) environment of diabetic wounds, achieving precise drug release. At pH = 7.4 (physiological environment), the release rate of epigallocatechin-3-gallate is 57% (36 hours); in the presence of pH = 5.8 (diabetic wound environment) and 1 mM H2O2, the drug release rate increases significantly. This characteristic enables the hydrogel to regulate drug release according to the pathological environment of the wound, improving the treatment efficiency while reducing systemic side effects.

[0020] (3) Stable loading of epigallocatechin-3-gallate, improving its stability and increasing bioavailability Epigallocatechin-3-gallate is stably embedded in the gel network through dynamic covalent coupling, hydrogen bonding, and π-π stacking interactions, avoiding its rapid degradation in vivo and in vitro and improving the drug bioavailability. Ultraviolet spectroscopy and nuclear magnetic resonance show that epigallocatechin-3-gallate is successfully embedded in the hydrogel structure and significantly improves the solubility.

[0021] (4) Good biocompatibility and no tissue toxicity The results of H&E staining show that the hydrogel has no obvious toxicity to the heart, liver, spleen, lungs, and kidneys of mice, does not affect liver and kidney functions, and ensures safety.

[0022] (5) Promote angiogenesis and accelerate the healing of diabetic wounds Hydrogels can reduce the levels of inflammatory factors, promote angiogenesis, and accelerate wound repair. In the group of mice treated with EGCG hydrogel (or called GBE hydrogel), the wound was almost completely closed within 8 days, and the healing rate was significantly higher than that of the control group. Western blot analysis showed that the expressions of IL-1β and TNF-α in the EGCG hydrogel group were significantly reduced, indicating its good anti-inflammatory performance. Brief Description of the Drawings

[0023] Figure 1 It is a schematic diagram of the assembly of epigallocatechin-3-gallate-loaded guanosine borate ester hydrogel of the present invention.

[0024] Figure 2 They are inverted vials of different proportion gel systems (A is different gel concentrations (counted by the concentration of BA); B is different boric acid equivalents; C is different KOH equivalents; D is different epigallocatechin-3-gallate equivalents).

[0025] Figure 3 It is the 1 1H NMR spectrum of epigallocatechin-3-gallate-loaded guanosine borate ester GBE hydrogel prepared by the present invention.

[0026] Figure 4 It is for GBG.Na + CD spectrogram of solution, GBG and GBE hydrogel.

[0027] Figure 5 It is the PXRD pattern of GBG and GBE hydrogel.

[0028] Figure 6 It is the infrared (FTIR) spectrogram of EGCG and GBE hydrogel.

[0029] Figure 7 It is the SEM image of GBE hydrogel.

[0030] Figure 8 It is the rheological mechanical test of GBE hydrogel (A is the time sweep plot; B is the angular frequency sweep plot; C is the dynamic strain sweep plot; D is the thixotropy sweep plot).

[0031] Figure 9 It is the viscosity determination graph of GBE hydrogel.

[0032] Figure 10 Test on the injectable property and self-healing behavior of GBE hydrogel.

[0033] Figure 11 It is the in vitro release graph of the stimulus-responsive GBE hydrogel.

[0034] Figure 12It is the hematoxylin-eosin staining diagram of the main organs of mice.

[0035] Figure 13 It shows the healing of diabetic chronic wounds in different treatment groups (scale bar = 100 μm).

[0036] Figure 14 It is a schematic diagram of the wound healing rate of each treatment group at different time points after treatment.

[0037] Figure 15 It is the hematoxylin-eosin staining of the wound surface (the blue double-headed arrow indicates the quantification of the thickness of granulation tissue; the blue, purple, green, yellow, and orange arrows represent sebaceous glands, nerve bundles, sweat gland ducts, blood vessels, and hair follicles respectively) (scale bar = 100 μm).

[0038] Figure 16 It is the immunofluorescence of macrophages in wound tissue with CD31 (red), and the cell nuclei are stained with DAPI (blue) (scale bar = 50 μm).

[0039] Figure 17 It is the semi-quantitative analysis of CD31. Compared with the blank group, P < 0.05.

[0040] Figure 18 It is the immunohistochemical staining results of tumor necrosis factor TNF-α and interleukin IL-1β in wound tissue (scale bar = 50 μm).

[0041] Figure 19 It is the analysis diagram of the positive staining result of tumor necrosis factor TNF-α. Compared with the blank group, P < 0.05.

[0042] Figure 20 It is the analysis diagram of the positive staining result of interleukin IL-1β. Compared with the blank group, P < 0.05.

[0043] Figure 21 It is the protein expression of IL-1β in wound tissue.

[0044] Figure 22 It is the protein expression of TNF-α in wound tissue.

[0045] Figure 23 It is the semi-quantitative analysis of IL-1β protein, P < 0.01.

[0046] Figure 24 It is the semi-quantitative analysis of TNF-α protein, P < 0.05. Specific implementation manners

[0047] The present invention will be further described below in conjunction with specific embodiments. Embodiment 1

[0048] Preparation of Epigallocatechin-3-gallate Loaded Guanosine Borate Hydrogel Place 170 mg of guanosine (G, 0.6 mmol, 1.2 eq.) and 67 mg of KOH (1.2 mmol, 2.4 eq.) in a round-bottom flask, add 20 mL of deionized water, and heat and stir at 95 °C until the guanosine is completely dissolved. Then add 31 mg of boric acid (BA, 0.5 mmol, 1.0 eq.) and 183 mg of epigallocatechin-3-gallate (E, 0.4 mmol, 0.8 eq.), and continue to stir at 95 °C for 20 min. After the reaction is completed, cool to room temperature to form a stable brown self-assembled hydrogel with a concentration of 25 mM (based on BA) and a drug loading of 0.8 eq. (as shown in Figure 1 ). GBG (without epigallocatechin-3-gallate) and GBE (containing epigallocatechin-3-gallate) hydrogels with different component ratios can be prepared by the same method.

[0049] Optimization of Assembly Conditions Based on the above assembly conditions, the inverted bottle experiment was further used to test the effects of gel concentration and the stoichiometric ratio of each component on the formation and stability of the hydrogel (as shown in Figure 2 ). According to the above preparation method, first fix the stoichiometric ratio (G:BA:KOH:E = 1.2:1:2.4:0.8) and change the gel concentration (based on BA). The gel formed at a gel concentration of 25 mM is the most stable and transparent. By changing the concentration of a certain component (taking 25 mM as 1 equivalent) and keeping the concentrations of other components unchanged, the optimal component ratio can be found. Finally, it was found that a stable gel can be formed when BA is 1.0 equivalent and the amount of KOH used is 2.4 equivalents. In addition, the drug loading equivalent was also tested. Under the condition of loading 0.3 - 1.2 equivalents of epigallocatechin-3-gallate, a stable hydrogel can be formed in the gel system. However, when the loading amount of epigallocatechin-3-gallate increases to 1.2 equivalents, part of the epigallocatechin-3-gallate cannot be completely dissolved, resulting in a turbid state of the system. Within the test range, the maximum drug loading of the gel system is 0.8 equivalents. Example 2

[0050] NMR Structural Characterization of 25 mM GBE Hydrogel Prepared in Example 1 Through 1 1H NMR experiment to study the substances in the gel solution phase, and indirectly determine the structural information of the gel phase composition. To better assign proton signals and clarify the quantity or ratio of each component, the1 For the \(^1H\) NMR spectrum, 2,2,3,3-(d4)-3-(trimethylsilyl) (TMSP) was added as an internal standard to the sample. As Figure 3 shown, in the 25 mM GBE system, the multiplets at δ 3.12 ppm - δ 2.56 ppm correspond to H-5′ and H-6′ of EGCG, while the multiplets at δ 4.43 ppm - δ 4.16 ppm correspond to H-3 and H-4 of G. By integrating the above peaks, in the 25 mM GBE hydrogel, only 0.74 mM of total G and 3.5 mM of EGCG are visible by nuclear magnetic resonance, indicating that the self-assembly in the hydrogel consists of 16.5 mM of GBE (20 - 3.5 mM) and 6.4 mM of GBG ((30 - 16.5 - 0.74 mM) / 2). The assembly rate of the GBE gel is 82.5% (16.5 / 20 mM), and the drug composition of GBE / GBG is approximately 2.6:1. Example 3

[0051] Circular dichroism and infrared spectroscopy characterization of the 25 mM GBE hydrogel prepared in Example 1 The circular dichroism spectrum (CD spectrum) of the 25 mM GBE hydrogel prepared in Example 1 showed a positive peak at 298 nm and a negative peak at 326 nm; while the characteristic signal of the 25 mM GBE hydrogel showed a negative peak at 328 nm ( Figure 4 ). These signals indicate the presence of a stacked structure of G-quadruplex in the hydrogel, while verifying the incorporation of epigallocatechin-3-gallate in the GBE system. After introducing epigallocatechin-3-gallate, the CD spectrum of the hydrogel changed significantly, suggesting that it may affect the conformation of the molecule, thus changing the assembly process. In addition, no CD signal was detected in the 25 mM GBE.Na + solution, further proving that these signals originate from the formation of G-quadruplex.

[0052] In addition, powder X-ray diffraction (PXRD) analysis was performed on the freeze-dried powder of the 25 mM GBE hydrogel. The results are as Figure 5 shown. A well-resolved diffraction peak was detected at 2θ ≈ 28.4º in the wide-angle region, and its corresponding interplanar spacing was d = 3.07 Å, which is consistent with the distance between G-quartet planes (usually 3.0 - 3.4 Å). This result further confirms the presence of G-quadruplex in the gel system.

[0053] Fourier transform infrared spectroscopy (FTIR) further provides evidence for the formation of borate ester bonds and self-assembly ( Figure 6). The ν(B-O-C) vibration peak was not observed in the infrared spectrum of EGCG, while in the GBE system, a ν(B-O-C) vibration peak appeared at 1088 cm⁻¹, indicating the formation of borate ester bonds. This shows that EGCG binds to guanosine through BA as a linking molecule and is incorporated into the G-quadruplex structure, ultimately forming a hydrogel. Example 4

[0054] Morphological study of the 25 mM GBE hydrogel prepared in Example 1 As Figure 7 shown, the scanning electron microscope (SEM) test results of the GBE hydrogel show that there are a large number of nanofibers in the hydrogel system, which are dense spatial network fiber structures formed by the interweaving of multiple nanowires. This is because guanosine (G) interacts with each other through hydrogen bonds, and the nanowires cross-link to form a tight three-dimensional network structure. Example 5

[0055] Rheological study of the 25 mM GBE hydrogel prepared in Example 1 To evaluate the mechanical properties of the hydrogel, time sweep and frequency sweep experiments were carried out on it using a rheometer. The test range was 0.1~100 rad·s⁻¹, and the constant strain was set at 1%. The results show that the storage modulus (G′) was always higher than the loss modulus (G″) ( Figure 8 A, B), indicating that the hydrogel was successfully formed, with stable solid-like behavior and good mechanical strength. Further evaluation of the self-healing performance of the hydrogel was carried out through dynamic strain sweep and thixotropic sweep experiments. When the shear strain exceeded 1.3%, G′ began to decrease; when the shear strain reached 23.5%, the values of G′ and G″ coincided, and then G′ was less than G″, indicating that the system changed from the gel state to the sol state ( Figure 8 C). In the thixotropic sweep experiment, strains of 0.1% and 1000% were alternately applied, the test frequency was 10 rad·s⁻¹, and the continuous test was 900 seconds, experiencing a total of four cycles ( Figure 8 D). Under the condition of low strain (1%), G′ was greater than G″, and the hydrogel remained in a solid-like state; when the strain increased to 1000%, G′ was less than G″, and the system changed to the sol state. However, when the strain returned to the small strain condition, the G′ of the hydrogel quickly recovered to be greater than G″, indicating that the system could quickly reconstruct the gel state and had good self-healing ability.

[0056] To evaluate the injectability of the hydrogel, the relationship between viscosity and shear rate was tested using a rheometer ( Figure 9). The results showed that as the shear rate increased, the viscosity decreased significantly, showing a typical shear-thinning effect. At higher shear rates, the hydrogen bonds and π−π stacking interactions within the hydrogel decreased, and the physical network was disrupted, resulting in the deformation of the assembled structure and a decrease in viscosity. This shear-thinning behavior indicates that the hydrogel has good injectability and can smoothly pass through the needle when a certain shear stress is applied by the syringe.

[0057] The injectability was further verified by visual inspection. The GBE hydrogel was injected into water using a syringe, and the formed red filaments indicated that the hydrogel could smoothly pass through the needle without clogging. In addition, the extruded hydrogel immediately recovered its viscoelastic properties and reformed into a gel state. This indicates that the hydrogel has good structural recovery ability while maintaining injection performance. To further verify the self-healing performance of the hydrogel system, two pieces of GBE hydrogels of different colors and one piece of GBG hydrogel without epigallocatechin-3-gallate were closely contacted at room temperature. After 4 hours, these hydrogels fused into a complete gel body, and the interface was invisible, indicating its excellent self-healing ability ( Figure 10 ). Example 6

[0058] Stimulus Responsiveness and Release Kinetics of the 25 mM GBE Hydrogel Drug Prepared in Example 1 To study the release responsiveness of the GBE hydrogel under different environments, it was placed in PBS buffers under different conditions, and the cumulative release amount was calculated by detecting the absorbance of epigallocatechin-3-gallate in the system by ultraviolet ( Figure 11 ). In the simulated healthy tissue environment (pH = 7.4 PBS buffer), the hydrogel released 32% of epigallocatechin-3-gallate within 8 h, and the cumulative release amount reached 49% after 24 h. In this environment, the hydrogel swelled, and some unreacted epigallocatechin-3-gallate was released from the gel into the buffer medium. In the simulated weak acidic environment of inflammation (pH = 5.8 PBS buffer), the release rate of the hydrogel increased significantly. The cumulative release amount reached 37% after 8 h and increased to 78% after 24 h. Further, the same release experiment was carried out in the simulated environment of overexpressed ROS and weak acidity in inflammation (pH = 5.8 PBS buffer containing 10 μM hydrogen peroxide). The results showed that more than 35% of epigallocatechin-3-gallate was released within 8 h, and the cumulative release amount was 74% after 24 h, showing obvious rapid release behavior. These results indicate that the presence of dynamic borate ester bonds in the hydrogel makes it sensitive to environmental conditions. Under low pH and high ROS conditions, the borate ester bonds break, accelerating the degradation of the hydrogel and the release of epigallocatechin-3-gallate, thus endowing it with environmental responsiveness characteristics.

[0059] By loading epigallocatechin-3-gallate into the G-quadruplex hydrogel, its uniform release on diabetic wounds can be achieved. This system can effectively reduce local inflammatory responses, promote cell proliferation, and thus accelerate the wound healing process. In addition, due to the three-dimensional network structure of the GBE hydrogel, epigallocatechin-3-gallate is encapsulated in the gel network, showing excellent drug sustained-release characteristics, which further enhances its therapeutic effect. Example 7

[0060] Biological tissue safety assessment - H&E staining of organ tissues Through the H&E staining experiment of mouse organ tissues, the potential toxicity of epigallocatechin-3-gallate hydrogel to organs such as the heart, liver, and kidney can be evaluated. Figure 12 The tissue morphology of the five major organs of the heart, liver, spleen, lung, and kidney of mice in the control group (Control), GBG group (25 mM, calculated as BA), EGCG aqueous solution group (20 mM), and EGCG hydrogel group (i.e., the 25 mM GBE hydrogel prepared in Example 1) is shown.

[0061] In the control group, the organizational structures of each organ were normal, the myocardial cells were arranged neatly, without swelling or necrosis; the hepatic cords were orderly, the hepatic sinusoids were clear, without inflammatory infiltration or fatty degeneration; the splenic corpuscles had intact structures, and the demarcation between the red pulp and white pulp was clear; the alveolar structures were intact, and the alveolar walls were not thickened or had inflammatory exudates; the glomeruli and renal tubules had normal morphologies, and there was no interstitial edema or aggregation of inflammatory cells. The organ tissue morphologies of the GBG group and the EGCG epigallocatechin-3-gallate aqueous solution group were consistent with those of the control group, and no cell damage, inflammation, or necrosis was observed, indicating that they had no obvious toxicity. No abnormalities were also found in the EGCG hydrogel group, and the structures such as myocardial cells, hepatocytes, and nephrons were all intact, without inflammation, necrosis, or abnormal hyperplasia.

[0062] The H&E staining results showed that the EGCG hydrogel and its components (GBG, EGCG aqueous solution) were non-toxic to the main organs, without accompanying aggregation of inflammatory cells or tissue damage. It was proved that the EGCG hydrogel had good biosafety in vivo, providing an important safety basis for its clinical application in diabetic wound dressings. Example 8

[0063] Wound healing rate Figure 13It shows the diabetic wound healing process and wound closure conditions of different treatment groups (Control group, GBG group (25 mM, calculated as BA), EGCG aqueous solution group (20 mM), and EGCG hydrogel group (i.e., the 25 mM GBE hydrogel prepared in Example 1) at 0, 2, 4, 6, and 8 days. Among them, the Control group serves as a natural healing control, the GBG group is used to exclude the influence of the hydrogel matrix, the EGCG aqueous solution group verifies the effect of free EGCG, and the EGCG hydrogel group evaluates its wound-healing promotion effect.

[0064] At 0 day, the wound areas of each group were similar. Between 2 and 8 days, the Control group healed slowly, and the wound was still clearly visible after 8 days; the GBG group healed slightly faster than the control group, but there were still large unclosed areas; the EGCG aqueous solution group healed better than the previous two groups, but the degree of closure was limited; the EGCG hydrogel group healed the fastest, and the wound was almost completely closed at 8 days, with only extremely small micro-wound areas remaining. The comparison of the wound area changes showed that the EGCG hydrogel was significantly superior to other groups in promoting diabetic wound healing.

[0065] Figure 14 The wound healing rates of each group at different time points (0, 2, 4, 6, 8 days) were further quantified. On the 2nd day of treatment, the healing rate of the Control group was the lowest, and the EGCG hydrogel group was significantly higher, showing superior initial closure ability. On the 4th day, the differences between the experimental groups increased. The healing rate of the control group was 26%, the GBG group was 30%, the EGCG aqueous solution group was 38%, while the EGCG hydrogel group was as high as 44%. On the 6th day, this trend continued. On the 8th day, the healing rate of the control group was 42%, the GBG group was 46%, the EGCG aqueous solution group was 51%, while the EGCG hydrogel group reached 68%, showing the best wound-healing promotion effect. The experimental results visually verified the significant promotion effect of the EGCG hydrogel on diabetic wound healing and highlighted its potential application value as a treatment material for diabetic wounds.

[0066] H&E staining of the wound tissue Wound healing is a complex biological process, in which a complete epidermis is a key marker. It can not only physically isolate external pathogens and reduce the risk of infection, but also provide a stable microenvironment to accelerate the repair of deep tissues. Blood vessels promote the proliferation of fibroblasts and collagen synthesis by transporting oxygen, nutrients, and immune cells, and at the same time accelerate the excretion of metabolic wastes, providing material support for wound healing. The reconstruction of skin appendages helps to restore the normal physiological functions of the skin and improve the healing quality. Granulation tissue is rich in fibroblasts and new blood vessels, and its thickening reflects the enhanced tissue filling and repair ability, which helps to accelerate wound closure and reduce scar formation. Figure 15H&E staining was used to compare the histological structures of different treatment groups (Control group, GBG group (25 mM, calculated as BA), EGCG aqueous solution group (20 mM), and EGCG hydrogel group (i.e., the 25 mM GBE hydrogel prepared in Example 1)) to evaluate the wound repair effect. Among them, sebaceous glands were marked with blue arrows, nerve bundles were marked with purple arrows, sweat gland ducts were marked with green arrows, blood vessels were marked with yellow arrows, hair follicles were marked with orange arrows, and granulation tissue was marked with light blue areas to reflect the thickness differences.

[0067] The wound repair in the Control group was poor, the epidermal layer was incomplete, the cell arrangement was loose, the granulation tissue was thin (light blue area), the angiogenesis (yellow arrow) was less, the accessory structures such as sebaceous glands and hair follicles were absent, and the degree of tissue regeneration was low. The repair effect of the GBG group was slightly improved, but there were still defects in the epidermal layer, the cell arrangement was irregular, the thickness of the granulation tissue increased slightly, the number of blood vessels was limited, and although the sweat gland ducts (green arrows) and hair follicles were visible, they were not fully developed. The repair effect of the EGCG aqueous solution group was further improved. The epidermal layer was relatively complete but still uneven locally, the angiogenesis increased, the thickness of the granulation tissue was medium, the sweat gland ducts and hair follicles were visible but sparsely distributed, and the tissue maturity was insufficient. The EGCG hydrogel group had the best repair effect, and the tissue structure was close to normal. The epidermal layer was complete, and the cells were arranged tightly and regularly; the thickness of the granulation tissue (light blue area) increased significantly, the blood vessels (yellow arrows) were abundant, promoting nutrient delivery; the skin accessory structures such as sebaceous glands, hair follicles (orange arrows), and sweat gland ducts were clear and well-developed, indicating more complete tissue regeneration. Example 9

[0068] Immunofluorescence staining Macrophages play a key role in wound repair. In the early stage of acute wound inflammation, M1 macrophages are dominant, responsible for clearing pathogens and necrotic tissues, and activating the inflammatory response at the same time. As the wound healing process progresses, M1 gradually transforms into M2. M2 macrophages can inhibit inflammation, release growth factors, and promote tissue regeneration. Their phenotypic conversion is a key link in the transition of the wound from the inflammatory phase to the proliferative phase and is crucial for wound repair.

[0069] To further evaluate the effect of the 25 mM GBE hydrogel prepared in Example 1 on the related expression of macrophages in vivo, CD31 immunofluorescence staining was used to analyze the angiogenesis situation ( Figure 16 and 17 ). Red fluorescence signals of CD31 were also detected in the control group, but both the fluorescence intensity and the relative area were significantly lower than those in the EGCG hydrogel group. Compared with other groups, the CD31 expression level in the EGCG epigallocatechin-3-gallate hydrogel group was significantly increased during the treatment period, indicating that it could effectively promote angiogenesis and thus accelerate wound healing.

[0070] The quantitative results of CD31 immunofluorescence staining showed that the number of wound microvessels in the EGCG hydrogel group reached (47.06 ± 0.77) per field of view, which was significantly higher than that in the control group (29.45 ± 0.93) per field of view, the GBG gel group (25 mM, calculated as BA) (41.12 ± 0.74) per field of view, and the EGCG aqueous solution group (20 mM) (45.98 ± 0.39) per field of view, and the differences were all statistically significant (P < 0.05). This result indicates that EGCG hydrogel can significantly promote the smooth transformation of diabetic wounds from the inflammatory phase to the proliferative phase, accelerate angiogenesis in wound tissues, and provide important support for wound repair. Example 10

[0071] Immunohistochemistry (IHC) staining and Western blot detection Diabetic wound healing is affected by persistent inflammatory responses. Pro-inflammatory factors TNF-α and IL-1β can exacerbate inflammation, inhibit cell proliferation and angiogenesis, and delay repair. Therefore, inhibiting their expression is crucial. The results of tissue staining ( Figure 18 ) showed that TNF-α and IL-1β were highly expressed in the blank control group and the GBG group (25 mM, calculated as BA), while their expression in the EGCG aqueous solution group (20 mM) decreased, but was still relatively obvious. The EGCG hydrogel group (25 mM GBE hydrogel prepared in Example 1) had the least positive staining, indicating the strongest inhibitory effect on inflammatory factors and contributing to accelerating wound healing. Quantitative analysis ( Figure 19 、 20 ) further confirmed that the positive ratios of TNF-α and IL-1β in the EGCG hydrogel group decreased to 2% respectively, which were significantly lower than those in other groups (P < 0.05). Western blot ( Figures 21 - 24 ) verified the protein expression levels. The results showed that TNF-α and IL-1β were highly expressed in the blank control group, the GBG group, and the EGCG aqueous solution group, while their expression in the EGCG hydrogel (25 mM GBE hydrogel prepared in Example 1) group decreased significantly. Semi-quantitative analysis ( Figure 23 、 24 ) confirmed that EGCG hydrogel could specifically inhibit inflammatory factors (P < 0.05), effectively reduce local inflammation, promote fibroblast proliferation and collagen synthesis, maintain immune homeostasis, accelerate diabetic wound repair, and improve the healing quality.

[0072] Animal experiment results show that EGCG hydrogel (25 mM GBE hydrogel prepared in Example 1) has good biosafety, significant anti-inflammatory effects, and can accelerate diabetic wound healing by regulating the immune microenvironment and promoting tissue repair. These data lay a solid theoretical foundation for the application of EGCG hydrogel in the treatment of diabetic chronic wounds. This discovery further verifies the immunomodulatory effect of EGCG hydrogel in diabetic wound repair and shows its clinical application value as a potential wound repair material.

[0073] In summary, the present invention constructs a supramolecular hydrogel nanodrug delivery system loaded with epigallocatechin-3-gallate. The GBE hydrogel is prepared through specific steps and the assembly conditions are optimized to determine the appropriate guanosine concentration, the stoichiometric ratio of each component, and the maximum drug loading capacity. Its structure is characterized by NMR, confirming that the relevant components participate in gel formation. Circular dichroism shows the G-quadruplex stacking structure and the incorporation of epigallocatechin-3-gallate in the hydrogel. Infrared spectroscopy indicates the formation of borate ester bonds, and PXRD analysis confirms the existence of G-quadruplex. Electron microscopy images show its morphology such as intertwined nanowires. Rheological studies show that it has good mechanical properties, self-healing ability, and injectability. The drug release kinetics shows different release profiles in simulated healthy tissues, weakly acidic inflammatory environments, and inflammatory overexpressed environments containing hydrogen peroxide, reflecting its stimulus responsiveness to the environment. This system is used in a diabetic rat wound healing model, achieving wound anti-inflammatory, moisturizing, promoting granulation growth, etc., reducing secondary injuries caused during the dressing change process, and providing new strategies and methods for tissue regeneration.

[0074] The present invention innovatively constructs a supramolecular hydrogel system based on the self-assembly of guanosine, achieving dual improvements in gel dynamic response and structural stability by introducing borate ester bonds; for the first time, epigallocatechin-3-gallate is loaded into the guanosine borate ester gel. Using the similar ortho-dihydroxy structure of epigallocatechin-3-gallate and guanosine, the drug is loaded as the original drug through covalent coupling, hydrogen bonding, and π-π stacking of borate ester bonds, avoiding the degradation of active ingredients during drug loading; it is achieved that in the high-ROS and low-pH microenvironment of diabetic wounds, the gel can rapidly disintegrate and release drugs in a responsive manner, increasing the local drug concentration and treatment effect; the hydrogel simultaneously has excellent mechanical properties, self-healing ability, and injectability, which not only meets the clinical operation requirements but also can adapt to the changes in the dynamic physiological environment of the wound; the hydrogel provided by the present invention not only shows excellent performance in terms of in vivo and in vitro biosafety, but also proves its comprehensive advantages of promoting angiogenesis, reducing inflammation, and accelerating wound healing in animal wound models.

Claims

1. A method for preparing epigallocatechin-3-gallate loaded guanosine borate hydrogel, characterized in that: Guanosine and KOH are placed in a round-bottom flask, deionized water is added, and the mixture is heated and stirred until guanosine is completely dissolved; then boric acid and epigallocatechin-3-gallate are added, and heating and stirring are continued until the reaction is completed and then cooled to room temperature to prepare epigallocatechin-3-gallate-loaded guanosine borate hydrogel.

2. The method for preparing an epigallocatechin-3-gallate-loaded guanosine borate hydrogel according to claim 1, characterized in that: The molar ratio of guanosine, boric acid, KOH and epigallocatechin-3-gallate is: 1.2:1:2.4: 0.3-0.8。 3. The method for preparing an epigallocatechin-3-gallate-loaded guanosine borate hydrogel according to claim 1, characterized in that: The molar ratio of guanosine, boric acid, KOH and epigallocatechin-3-gallate is: 1.2:1:2.4:0.8。 4. The method for preparing an epigallocatechin-3-gallate-loaded guanosine borate hydrogel according to claim 1, characterized in that: The temperature for heating and stirring was 95°C.

5. The epigallocatechin-3-gallate-loaded guanosine borate hydrogel prepared according to the preparation method according to any one of claims 1 to 4.

6. The epigallocatechin-3-gallate-loaded guanosine borate hydrogel according to claim 5, characterized in that: The hydrogel uses guanosine self-assembly to form a G-quadruplex network, and introduces borate bonds to strengthen cross-linking. Epigallocatechin-3-gallate is stably embedded in the gel network through hydrogen bonds and π-π stacking.

7. Use of the epigallocatechin-3-gallate loaded guanosine borate hydrogel according to claim 5 in repairing diabetic wounds.

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