Melatonin-loaded bi-crosslinked hydrogel and application thereof

Abstract

The invention relates to a melatonin-loaded bi-crosslinking hydrogel and an application thereof. According to the invention, polylysine (PL-GA) grafted with gallic acid is used as an intelligent carrier, hyaluronic acid (HA-PBA-GMA) grafted with phenylboronic acid and glycidyl methacrylate is used as a responsive network skeleton and a stable skeleton precursor, and melatonin is loaded on the PL-GA to form a drug-loading functional unit (MT at PL-GA); mT < PL-GA > and an HA-PBA-GMA solution are mixed, and the bi-crosslinked hydrogel with environmental responsiveness and structural stability is prepared through'dynamic boric acid ester bond crosslinking 'and'photo-initiated free radical polymerization'. The melatonin loading rate of the double-crosslinking hydrogel is high, the double-crosslinking hydrogel has excellent stability and self-healing performance, meanwhile, the mechanical property of the double-crosslinking hydrogel is matched with the mechanical environment of a wound surface, and the double-crosslinking hydrogel can release drugs in a responsive mode according to the wound microenvironment, so that the double-crosslinking hydrogel is very suitable for treatment of local wounds.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical formulation technology and relates to hydrogels, specifically to a melatonin-loaded double cross-linked hydrogel and its application in wound healing and scar inhibition. Background Technology

[0002] Melatonin (N-acetyl-5-methoxytryptamine), a pleiotropic indoleamine hormone secreted by the pineal gland, has physiological functions far beyond regulating circadian rhythms. Recent studies have found that melatonin and its metabolites are powerful endogenous antioxidants and free radical scavengers, effectively neutralizing reactive oxygen species (ROS) and reactive nitrogen species (RNS), and enhancing the cell's endogenous antioxidant defense system by activating pathways such as nuclear factor E2-related factor 2 (Nrf2). In the complex pathophysiology of wound healing, excessive oxidative stress is a key factor leading to persistent inflammation, abnormal cell proliferation, and disordered collagen deposition, ultimately contributing to the formation of pathological scars (such as hypertrophic scars and keloids). Therefore, topical application of melatonin, acting directly on the wound microenvironment, can precisely regulate the healing process: inhibiting excessive inflammation and oxidative damage during the inflammatory phase, promoting angiogenesis and re-epithelialization during the proliferative phase, and regulating the balance between collagen synthesis and degradation during the remodeling phase, thereby accelerating healing while inhibiting scarring.

[0003] However, melatonin faces several challenges in practical applications: First, chemical instability. Its indole ring structure is sensitive to light, heat, and oxidative environments, leading to easy inactivation upon direct application. Second, low bioavailability. Simple creams or solutions are difficult to retain effectively on moist wounds and have limited permeability. Third, lack of spatiotemporal controllability. The demand for melatonin varies dynamically at different stages of wound healing, and conventional formulations cannot achieve precise, on-demand release, potentially resulting in insufficient efficacy or wasted resources. These issues severely limit its effectiveness in local wound treatment.

[0004] Therefore, there is a need to develop a new melatonin formulation that is suitable for the treatment of local wounds, thereby improving its actual effects on wound healing and inhibiting scar formation. Summary of the Invention

[0005] Based on this, the present invention provides a melatonin-loaded double cross-linked hydrogel precursor solution and the double cross-linked hydrogel thereof. The double cross-linked hydrogel has excellent stability and self-healing properties, while its mechanical properties match the mechanical environment of the wound and the drug release is controllable, making it very suitable for the treatment of local wounds.

[0006] The specific technical solutions are as follows.

[0007] In a first aspect, the present invention provides a modified hyaluronic acid, which is obtained by reacting hyaluronic acid grafted with phenylboronic acid groups with glycidyl methacrylate.

[0008] The grafting rate of phenylboronic acid groups in the modified hyaluronic acid is 35%-55%;

[0009] The grafting rate of glycidyl methacrylate groups in the modified hyaluronic acid is 15%-35%.

[0010] Secondly, the present invention provides a method for preparing the modified hyaluronic acid, comprising the following steps:

[0011] Hyaluronic acid undergoes an amidation reaction with 3-aminophenylboronic acid to obtain hyaluronic acid grafted with phenylboronic acid groups;

[0012] The hyaluronic acid grafted with phenylboronic acid groups undergoes an epoxy ring-opening reaction with glycidyl methacrylate to obtain the modified hyaluronic acid.

[0013] Thirdly, the present invention provides a melatonin-loaded bi-crosslinked hydrogel precursor solution, comprising solution A and solution B;

[0014] Solution A is composed of the modified hyaluronic acid, photoinitiator and solvent described in this invention;

[0015] Solution B consists of a melatonin-loaded polymer and a solvent;

[0016] The melatonin-loaded polymer is obtained by loading melatonin onto modified polylysine.

[0017] The modified polylysine is obtained by amidation reaction of ε-polylysine and gallic acid.

[0018] Fourthly, the present invention provides a method for preparing the melatonin-loaded double cross-linked hydrogel precursor solution, comprising the following steps:

[0019] Preparation of solution A: Dissolve the modified hyaluronic acid in a solvent, add the photoinitiator, stir and dissolve in the dark to obtain the solution;

[0020] Preparation of Solution B: Melatonin is dissolved in ethanol to obtain a melatonin ethanol solution; the modified polylysine is dissolved in water or a buffer solution to obtain a polymer solution; the melatonin ethanol solution is added dropwise to the polymer solution, and after the addition is complete, stirring is continued, and the solution is allowed to stand to remove the ethanol, thus obtaining the final solution.

[0021] Fifthly, the present invention provides a method for preparing a melatonin-loaded double cross-linked hydrogel, comprising the following steps: mixing solution A and solution B in the melatonin-loaded double cross-linked hydrogel precursor solution of the present invention evenly, allowing it to stand, and irradiating it with ultraviolet light to obtain the melatonin-loaded double cross-linked hydrogel.

[0022] In a sixth aspect, the present invention provides a melatonin-loaded double crosslinked hydrogel prepared by the preparation method described in the present invention.

[0023] In a seventh aspect, the present invention provides the use of the melatonin-loaded bicrosslinked hydrogel precursor solution and / or the melatonin-loaded bicrosslinked hydrogel in the preparation of a drug, said drug being an anti-inflammatory drug, an antioxidant drug, a drug that promotes wound healing and / or inhibits scar formation.

[0024] The present invention has the following beneficial effects:

[0025] The double crosslinked hydrogel precursor solution and its hydrogel prepared from the modified polylysine, modified hyaluronic acid and melatonin of the present invention can significantly improve the loading rate of melatonin in the hydrogel (reaching 10% (w / w) and the encapsulation rate (over 90%), and can significantly improve the stability of melatonin, making it less prone to decomposition during use and storage, thereby effectively improving its therapeutic effect.

[0026] The double-crosslinked hydrogel precursor solution of this invention features controllable gelation time, adjustable mechanical properties, and adjustable drug release characteristics. The precursor solution completes the first stage of dynamic crosslinking within 3-5 minutes at room temperature, forming an injectable pregel, facilitating clinical operation. The second stage of photocrosslinking is completed within 60 seconds, significantly shortening dressing formation time. Furthermore, by adjusting the photocrosslinking time, the mechanical properties and drug release characteristics of the hydrogel can be precisely controlled within a certain range, effectively matching the different wound mechanical environments from soft mucous membranes to relatively tough skin, as well as the drug release requirements of different wounds.

[0027] The dual-crosslinked hydrogel formed by the dual-crosslinked hydrogel precursor solution of the present invention has excellent stability and self-healing properties. After being soaked in PBS at 37°C for 7 days, the mass retention rate of the formed dual-network hydrogel is still higher than 80%, ensuring the long-term integrity and functionality of the dressing on the body surface.

[0028] The bicrosslinked hydrogel formed from the bicrosslinked hydrogel precursor solution of this invention exhibits spatiotemporal tunable release of melatonin. For the critical first 48 hours of acute wound healing, the bicrosslinked hydrogel provided by this invention is an intelligent hydrogel system with "low leakage, high response, and full coverage" characteristics. Under normal physiological conditions (pH 7.4), this hydrogel effectively overcomes the "burst release" defects of traditional materials, with a melatonin release rate of less than 40% within 12 hours, ensuring drug safety in non-target areas. At the lesion site, the hydrogel can sensitively sense changes in the wound microenvironment. Once it detects an acidic environment (pH 6.5), high blood glucose concentration, and / or oxidative stress (ROS) signals (which usually indicate bacterial infection or severe inflammation), the hydrogel immediately initiates an accelerated release mechanism (melatonin release exceeding 70% within 12 hours), achieving "on-demand drug delivery." Furthermore, even under the strongest stimulation conditions, the hydrogel can maintain effective release for 48 hours, showing good periodicity matching with the critical first 48 hours of acute wound healing. Under mild stimulation conditions, this can continue for up to 96 hours, which helps reduce the frequency of dressing changes and improve patient compliance in clinical applications. This design not only ensures that the continuous drug supply fully covers the most critical inflammation clearance and early proliferation phases of wound healing but also avoids ineffective residues after drug depletion, laying the foundation for rapid subsequent tissue repair.

[0029] The double crosslinked hydrogel of this invention can significantly reduce intracellular ROS levels, inhibit the expression of pro-inflammatory factors, and improve the cellular microenvironment through the synergistic effect of hydrogel and melatonin, thereby significantly restoring and promoting cell migration ability. Therefore, it exhibits excellent migration-promoting potential under oxidative stress conditions, which can effectively promote wound healing and tissue repair and inhibit scar formation. Attached Figure Description

[0030] Figure 1 The 1H NMR spectra of polylysine and gallic acid-grafted polylysine prepared in Example 1.

[0031] Figure 2 The 1H NMR spectra of hyaluronic acid and the hyaluronic acid grafted with phenylboronic acid and GMA prepared in Example 2.

[0032] Figure 3 To investigate the effect of different grafting ratios on the release behavior of melatonin (MT) in hydrogels.

[0033] Figure 4 Strain scans of double crosslinked hydrogels with different UV crosslinking times.

[0034] Figure 5 Frequency scans of double crosslinked hydrogels with different UV crosslinking times.

[0035] Figure 6Self-healing properties of double crosslinked hydrogels with different UV crosslinking times.

[0036] Figure 7 Release curves of double crosslinked hydrogels with different UV crosslinking times.

[0037] Figure 8 The figures show the release curves of the double crosslinked hydrogel under different conditions.

[0038] Figure 9 This is a degradation diagram (mass retention rate) of the double crosslinked hydrogel under simulated physiological conditions.

[0039] Figure 10 The effect of double cross-linked hydrogels on TNF-α levels in HUVEC cells under an oxidation model.

[0040] Figure 11 The effect of double cross-linked hydrogels on IL-6 levels in HUVEC cells under an oxidation model.

[0041] Figure 12 The effect of double cross-linked hydrogels on IL-10 levels in HUVEC cells under an oxidation model.

[0042] Figure 13 The effect of double cross-linked hydrogels on ROS levels in HUVEC cells under an oxidation model.

[0043] Figure 14 The effect of double cross-linked hydrogels on the migration ability of HUVEC cells under an oxidation model. Detailed Implementation

[0044] To facilitate understanding of the present invention, a more complete description will be provided below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0045] Unless otherwise specified, experimental methods in the following examples are generally performed under standard conditions or as recommended by the manufacturer. All commonly used chemical reagents used in the examples are commercially available products.

[0046] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention. The term "and / or" as used in this invention includes any and all combinations of one or more of the associated listed items.

[0047] Furthermore, as used herein, the term "or" is an inclusive "or" sign and is equivalent to the term "and / or" unless the context clearly specifies otherwise. The term "based on" is not exclusive and allows for basing on other factors not described unless the context clearly specifies otherwise. Additionally, throughout the specification, the meanings of "an," "a," and "the" include plural indicators. The meaning of "in" includes both "in" and "on."

[0048] Hydrogel dressings, due to their high water content, good biocompatibility, and ability to load drugs, can be used for local drug delivery. To develop a novel melatonin formulation suitable for treating local wounds and improve its effectiveness in wound healing and scar inhibition, the inventors conceived of developing a melatonin-loaded hydrogel dressing. However, during the research process, it was found that using existing hydrogel dressings as carriers resulted in very low melatonin loading rates (often below 5%), and the loaded melatonin experienced severe burst release, failing to maintain a long-term therapeutic concentration. Furthermore, the gel network was unresponsive to changes in the wound microenvironment such as pH and ROS, leading to a disconnect between drug release and the healing process. Additionally, it suffered from low gel strength and poor stability, making it difficult to meet the mechanical requirements of dynamic wound healing. To overcome these shortcomings, the inventors of this invention further discovered that by using polylysine grafted with gallic acid (GA) (PL-GA) as a smart carrier and hyaluronic acid grafted with phenylboronic acid (PBA) and glycidyl methacrylate (GMA) (HA-PBA-GMA) as a responsive network framework and stabilizing framework precursor, melatonin (MT) is loaded onto PL-GA to form a drug-loaded functional unit (MT@PL-GA). Then, by mixing MT@PL-GA with HA-PBA-GMA solution and employing a two-step method of "dynamic borate ester crosslinking" and "photo-initiated free radical polymerization," a dual-crosslinked hydrogel (MT@EGHP) with both environmental responsiveness and structural stability can be formed. This dual-crosslinked hydrogel has a high melatonin loading rate, excellent stability and self-healing properties, and its mechanical properties match the mechanical environment of the wound. Furthermore, it can release drugs in response to pH, ROS, and other factors in the wound microenvironment, perfectly matching drug release with the healing process, making it highly suitable for the treatment of local wounds.

[0049] Based on this, some embodiments of the present invention involve a modified hyaluronic acid, which is obtained by reacting hyaluronic acid grafted with phenylboronic acid groups with glycidyl methacrylate.

[0050] The grafting rate of phenylboronic acid groups in the modified hyaluronic acid is 35%-55%;

[0051] The grafting rate of glycidyl methacrylate groups in the modified hyaluronic acid is 15%-35%.

[0052] In some embodiments, the weight-average molecular weight of the hyaluronic acid is 400kDa-800kDa.

[0053] In some embodiments, the grafting rate of phenylboronic acid groups in the modified hyaluronic acid is 40%-50%, more preferably 43%-50%.

[0054] In some embodiments, the grafting rate of glycidyl methacrylate groups in the modified hyaluronic acid is 20%-32%, more preferably 23%-31%.

[0055] Some embodiments of the present invention relate to a method for preparing the modified hyaluronic acid, comprising the following steps:

[0056] Hyaluronic acid undergoes an amidation reaction with 3-aminophenylboronic acid to obtain hyaluronic acid grafted with phenylboronic acid groups;

[0057] The hyaluronic acid grafted with phenylboronic acid groups undergoes an epoxy ring-opening reaction with glycidyl methacrylate to obtain the modified hyaluronic acid.

[0058] In some embodiments, the molar ratio of sugar units in the hyaluronic acid to 3-aminophenylboronic acid is 1:0.7-0.85, preferably 1:0.8.

[0059] In some embodiments, the molar ratio of sugar units in the hyaluronic acid to glycidyl methacrylate is 1:0.5-0.7, preferably 1:0.6.

[0060] Some embodiments of the present invention relate to a melatonin-loaded bi-crosslinked hydrogel precursor solution, comprising solution A and solution B;

[0061] Solution A is composed of the modified hyaluronic acid, photoinitiator, and solvent as described in claim 1 or 2;

[0062] Solution B consists of a melatonin-loaded polymer and a solvent;

[0063] The melatonin-loaded polymer is obtained by loading melatonin onto modified polylysine.

[0064] The modified polylysine is obtained by amidation reaction of ε-polylysine and gallic acid.

[0065] In some embodiments, solution A and solution B are mixed evenly at a volume ratio of 1:0.9-1.1 before use.

[0066] In some embodiments, the grafting rate of gallic acid in the modified polylysine is 2.8%-23%, preferably 12%-18%, and more preferably 14%-17%.

[0067] In some embodiments, the ε-polylysine and gallic acid react in the following molar ratio: the molar ratio of the amino group in ε-polylysine to gallic acid is 1:0.8-2, preferably 1:1.2-1.8, and more preferably 1:1.4-1.6.

[0068] In some embodiments, the weight-average molecular weight of the ε-polylysine is 3000-5000.

[0069] In some embodiments, the melatonin loading in the melatonin-loaded polymer is 4%-15%, preferably 9%-12%.

[0070] In some embodiments, the solvent in solution B is water or a buffer solution, preferably a PBS buffer with a pH of 7.4.

[0071] In some embodiments, the concentration of solution B, based on the mass of modified polylysine, is 0.25 g / mL to 0.35 g / mL, preferably 0.28 g / mL to 0.32 g / mL.

[0072] In some embodiments, the photoinitiator is lithium phenyl (2,4,6-trimethylbenzoyl) phosphate.

[0073] In some embodiments, the solvent in solution A is water or a buffer solution, preferably a PBS buffer with a pH of 7.4.

[0074] In some embodiments, the concentration of the modified hyaluronic acid in solution A is 90 mg / mL to 110 mg / mL.

[0075] In some embodiments, the concentration of the photoinitiator in solution A is 0.5 mg / mL to 2 mg / mL.

[0076] Some embodiments of the present invention relate to a method for preparing the melatonin-loaded double crosslinked hydrogel precursor solution, comprising the following steps:

[0077] Preparation of solution A: Dissolve the modified hyaluronic acid in a solvent, add the photoinitiator, stir and dissolve in the dark to obtain the solution;

[0078] Preparation of Solution B: Melatonin is dissolved in ethanol to obtain a melatonin ethanol solution; the modified polylysine is dissolved in water or a buffer solution to obtain a polymer solution; the melatonin ethanol solution is added dropwise to the polymer solution, and after the addition is complete, stirring is continued, and the solution is allowed to stand to remove the ethanol, thus obtaining the final solution.

[0079] In some embodiments, the mass ratio of melatonin to modified polylysine is 1:5-15, preferably 1:7-10, and more preferably 1:8-9.

[0080] In some embodiments, the melatonin concentration in the melatonin ethanol solution is 15 mg / mL to 25 mg / mL.

[0081] In some embodiments, the buffer is a PBS buffer with a pH of 7.4.

[0082] In some embodiments, the concentration of modified polylysine in the polymer solution is 0.25 g / mL to 0.35 g / mL, preferably 0.28 g / mL to 0.32 g / mL.

[0083] In some embodiments, the melatonin ethanol solution is added dropwise to the polymer solution at a rate of 0.4 mL / min to 0.6 mL / min.

[0084] In some embodiments, the melatonin ethanol solution is added dropwise to the polymer solution while stirring at a speed of 120 rpm to 180 rpm.

[0085] In some implementations, stirring continues for 3-6 hours after the dripping is complete.

[0086] In some implementations, the plant is left to stand for 10 to 16 hours.

[0087] Some embodiments of the present invention relate to a method for preparing a melatonin-loaded dual crosslinked hydrogel, comprising the following steps: mixing solution A and solution B in the melatonin-loaded dual crosslinked hydrogel precursor solution evenly, allowing it to stand, and irradiating it with ultraviolet light for 0-60 seconds to obtain the melatonin-loaded dual crosslinked hydrogel.

[0088] In some embodiments, solution A and solution B are mixed evenly at a volume ratio of 1:0.9-1.1.

[0089] In some embodiments, the irradiation time with ultraviolet light is 0-60 seconds, preferably 28-32 seconds.

[0090] In some embodiments, the settling period is 3-5 minutes at 35°C-40°C.

[0091] In some embodiments, the ultraviolet light has a wavelength of 365 nm and an intensity of 4 mW / cm²-6 mW / cm².

[0092] Some embodiments of the present invention relate to melatonin-loaded double crosslinked hydrogels prepared by the preparation method described in the present invention.

[0093] Some embodiments of the present invention relate to the use of the melatonin-loaded bicrosslinked hydrogel precursor solution and / or the melatonin-loaded bicrosslinked hydrogel of the present invention in the preparation of a drug, said drug being an anti-inflammatory drug, an antioxidant drug, a drug that promotes wound healing and / or inhibits scar formation.

[0094] The present invention will be further described in detail below with reference to specific embodiments.

[0095] Example 1: Synthesis of gallic acid-grafted polylysine (PL-GA)

[0096] Raw materials: ε-polylysine hydrochloride (ε-PL·HCl, Mw 3000-5000); gallic acid (GA, purity ≥98%); 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl); N-hydroxysuccinimide (NHS); 2-morpholinoethanesulfonic acid (MES) buffer (0.1 M, pH 5.0); dialysis bag (molecular weight cutoff MWCO: 3.5 kDa).

[0097] Synthetic principle: The carboxyl group of GA is covalently linked to the primary amino group of the PL side chain via an EDC-mediated amidation reaction. The catechol structure of GA provides binding sites for the subsequent formation of dynamic borate ester bonds, while also endowing the polymer with antioxidant activity.

[0098] Synthesis steps: Dissolve ε-PL·HCl in MES buffer to prepare a PL solution with a concentration of 20 mg / mL. Weigh GA and dissolve it in an appropriate amount of ethanol, according to a molar ratio of amino group to GA of 1:1.5 in ε-PL, and then add it dropwise to the PL solution. Under stirring at 45℃, add EDC·HCl and NHS sequentially (molar ratio EDC:GA:NHS = 1.2:1:1.2) to activate the carboxyl group of GA. Stir the reaction in the dark for 24 hours. After the reaction is complete, transfer the mixture to a dialysis bag and dialyze against deionized water for 3 days, changing the water 3 times a day. Freeze-dry to obtain a white, spongy solid PL-GA, and store at -20℃ in the dark.

[0099] The chemical structures of polylysine (PL) and gallic acid-grafted polylysine (PL-GA) were characterized by proton nuclear magnetic resonance (¹H NMR). Approximately 5-10 mg of sample was weighed and dissolved in 0.55 mL of deuterated water (D₂O). After complete dissolution at room temperature, the solution was transferred to a 5 mm NMR tube. ¹H NMR measurements were performed on a 400 MHz NMR spectrometer at 25 °C. The ¹H NMR results showed (…). Figure 1 Polylysine exhibits typical polypeptide structural features in D2O, with methylene protons in the lysine side chain and α-protons in the main chain distributed in the δ 1.2–1.8 ppm, δ 2.8–3.1 ppm, and δ 4.1–4.4 ppm regions, respectively. Compared with pure PL, the ¹H NMR spectrum of gallic acid-grafted polylysine (PL-GA) retains the above characteristic peaks while showing a new aromatic proton signal at δ 6.8–7.2 ppm. This signal can be clearly attributed to the benzene ring structure of gallic acid, indicating that gallic acid has been successfully grafted onto the polylysine molecular chain. Furthermore, the characteristic peaks of the PL main chain did not undergo significant shifts or disappearance before and after grafting, indicating that the grafting reaction did not destroy the polypeptide backbone structure of polylysine.

[0100] By comparing the NMR integrals of polylysine (PL) and gallic acid-grafted polylysine (PL-GA), specifically based on the area ratio of the methylene proton peak of the lysine side chain (δ 1.2-1.8 ppm) to the characteristic proton peak of the gallic acid benzene ring (δ 6.8-7.2 ppm), the following formula was used for calculation:

[0101] GA grafting rate (%) = (A GA / n GA ) / (A PL / n PL )

[0102] Among them, A GA and A PL These are the integral areas of the benzene ring hydrogens of gallic acid and the characteristic hydrogens of lysine, respectively; n GA and n PL The number of protons in the corresponding group.

[0103] The GA grafting rate of the PL-GA prepared in this embodiment was 15.4 ± 1.16%.

[0104] Example 2: Synthesis of hyaluronic acid grafted with phenylboronic acid and glycidyl methacrylate (HA-PBA-GMA)

[0105] Raw materials: Hyaluronic acid (HA, Mw: 400kDa-800kDa); 3-aminophenylboronic acid (3-PBA); Glycidyl methacrylate (GMA); 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl); N-hydroxysuccinimide (NHS); 2-morpholinoethanesulfonic acid (MES) buffer (0.1 M, pH 5.0); dialysis bag (MWCO: 8 Da).

[0106] Synthetic principle: The first step involves an amidation reaction between the amino group in 3-PBA and the carboxyl group in HA to introduce a phenylboronic acid (PBA) group into the HA chain. This introduced phenylboronic acid group is crucial for forming dynamic bonds. The second step, under acidic conditions, involves a ring-opening reaction between the epoxy group of GMA and the hydroxyl group of HA, grafting a methacryloyl group containing a double bond onto HA, providing reaction sites for photocrosslinking.

[0107] Synthesis steps:

[0108] (1) Synthesis of HA-PBA: HA was dissolved in MES to prepare a 10 mg / mL HA solution. 3-PBA (molar ratio of HA sugar units: 3-PBA = 1:0.8) was added and stirred to dissolve. EDC·HCl and NHS (molar ratio of EDC: 3-PBA: NHS = 1.2:1:1.2) were added, and the reaction was carried out at 45 °C in the dark for 24 hours. The reaction solution was dialyzed (with deionized water) and then lyophilized to obtain HA-PBA.

[0109] (2) GMA grafting of HA-PBA: Dissolve HA-PBA in deionized water (10 mg / mL). Adjust the pH to 3.5 with hydrochloric acid. Slowly add GMA (molar ratio of HA sugar units:GMA = 1:0.6) in an ice bath with stirring. Continue the reaction at room temperature for 48 hours. Dialyze and freeze dry to obtain the final product HA-PBA-GMA.

[0110] The chemical structures of hyaluronic acid (HA) and its graft product (HA-PBA-GMA) were characterized by proton nuclear magnetic resonance (¹H NMR). Approximately 5–10 mg of sample was weighed and dissolved in 0.55 mL of deuterated water (D₂O). After complete dissolution at room temperature, the solution was transferred to a 5 mm NMR tube. ¹H NMR measurements were performed on a 400 MHz NMR spectrometer at 25 °C. The ¹H NMR results showed that hyaluronic acid in D₂O exhibited typical polysaccharide characteristic peaks, including an N-acetylmethyl peak at δ 1.9 ppm and a proton signal in the sugar ring range of δ 3.0–4.2 ppm. In contrast, the ¹H NMR spectrum of HA-PBA-GMA retained the characteristic peaks of the HA backbone while showing new chemical shift signals. Specifically, the aromatic proton peak at δ 7.3–7.9 ppm was attributed to the phenylboronic acid structural unit, indicating that the phenylboronic acid group had been successfully grafted onto the HA molecular chain. Furthermore, the signals observed at δ 5.6–6.2 ppm and δ 1.2–1.4 ppm can be attributed to olefinic and methyl protons in GMA, respectively, confirming the successful introduction of GMA into the hyaluronic acid backbone. Therefore, the ¹H NMR results clearly demonstrate that phenylboronic acid and GMA have been successfully grafted onto the hyaluronic acid molecular chain.

[0111] The grafting ratio of PBA and GMA in HA-PBA-GMA was determined by proton nuclear magnetic resonance spectroscopy (NMR). 1 Calculations were performed using ¹H NMR, and the proton signals of the sugar rings on the modified sodium hyaluronate backbone (δ 3.0-4.0 ppm) were compared with the characteristic signals of the introduced groups, following a method similar to Example 1. The GMA grafting rate was determined by the integral ratio of the proton signals of the double bonds on the methacrylate groups (δ 5.6-6.2 ppm); the PBA grafting rate was determined by the integral ratio of the proton signals of the benzene rings on the phenylboronic acid groups (δ 7.3-7.9 ppm). The calculated grafting rates were 46.5 ± 2.80% for PBA and 26.8 ± 3.51% for GMA.

[0112] Example 3: Preparation of a melatonin-loaded double cross-linked hydrogel

[0113] (1) Preparation of PL-GA@MT pre-assembled solution

[0114] Process Principle: Melatonin, as a hydrophobic molecule, tends to interact hydrophobically with GA-modified polylysine chains, which also possess some hydrophobicity. Furthermore, hydrogen bonds may exist between the amino group of PL and the amide / methoxy group of MT, thereby achieving efficient MT drug loading.

[0115] Melatonin (MT) was dissolved in anhydrous ethanol to prepare a high-concentration (20 mg / mL) melatonin ethanol solution. PL-GA synthesized in Example 1 was dissolved in PBS (pH 7.4) to prepare a 30% w / v (i.e., 0.3 g / mL) PL-GA solution. Under vigorous stirring at 150 rpm, the melatonin ethanol solution was added dropwise to the PL-GA solution at a rate of 0.5 mL / min (maintaining a MT to PL-GA mass ratio of 1:8). After the addition was complete, stirring was continued at 150 rpm for 4 hours. The mixture was then placed in a 4°C refrigerator and allowed to stand for 12 hours to allow melatonin and PL-GA to fully bind through hydrophobic interactions and possible hydrogen bonds. Ethanol was removed by rotary evaporation to obtain a homogeneous MT@PL-GA pre-assembled solution.

[0116] (2) In-situ construction of double crosslinked hydrogels

[0117] (2.1) Preparation of hydrogel precursor solution

[0118] Solution A: Dissolve HA-PBA-GMA in PBS (pH 7.4) at a concentration of 10% (w / v). Add a photoinitiator (lithium phenyl (2,4,6-trimethylbenzoyl)phosphate, lithium salt, LAP) at a concentration of 0.1% (w / v) and stir to dissolve in the dark.

[0119] Solution B: MT@PL-GA pre-assembled solution (concentration of 30% w / v PL-GA) prepared in (1).

[0120] Solution A and solution B are gently mixed in a sterile container at a volume ratio of 1:1 to obtain a homogeneous hydrogel precursor solution. This hydrogel precursor solution remains liquid for several minutes at room temperature and can be used for injection or topical application.

[0121] (2.2) Implementation of the double crosslinking process

[0122] The principle of dual crosslinking: The first step, dynamic crosslinking, is based on the high affinity and rapid reversible binding of phenylboronic acid and catechol to form a borate ester bond, which is sensitive to pH and ROS. The second step, photocrosslinking, is based on the free radical polymerization of acrylate double bonds to form stable C-C covalent bonds.

[0123] First crosslinking (formation of a dynamic borate ester bond network): The hydrogel precursor solution is allowed to stand at 37°C for 4 minutes. During this period, the phenylboronic acid groups on HA-PBA-GMA in solution A rapidly complex with the catechol groups (from GA) on MT@PL-GA in solution B, forming a large number of dynamically reversible borate ester bonds. The solution viscosity increases sharply, forming a primary hydrogel with a certain shape-retention ability. This process occurs autonomously and requires no external triggering.

[0124] Second crosslinking (photoinitiated covalent network formation): The primary hydrogel was irradiated with ultraviolet light at a wavelength of 365 nm (5 mW / cm²) for 30 seconds. Under these conditions, the double bonds of the GMA units on HA-PBA-GMA underwent free radical polymerization, constructing an interpenetrating, permanent covalent polymer network within the already formed dynamic network, thus obtaining the final double-crosslinked hydrogel (MT@EGHP).

[0125] Example 4: Effect of different ε-PL and GA feed ratios on the properties of the double crosslinked hydrogel and melatonin loading capacity

[0126] By changing the ratio of ε-polylysine hydrochloride to gallic acid (as shown in Table 1), and keeping the other steps, raw materials, and reaction conditions the same as in Examples 1-3, double crosslinked hydrogels loaded with melatonin with different GA grafting rates were prepared, and the following performance tests and characterizations were performed. The test results are shown in Table 1.

[0127] 1. GA grafting rate: The result was obtained by comparing the NMR integrals of polylysine and gallic acid grafted polylysine, as described in Example 1.

[0128] 2. Crosslinking density (G', storage modulus): Tested using a rotational rheometer at 37 °C. A parallel rotor with a diameter of 20 mm was used, and the gap was set to 0.3 μm. Frequency sweep tests were performed at a fixed strain (LVE linear viscoelastic region, such as 1%), and the storage modulus (G') was recorded in the frequency range of 0.1–100 rad / s. The values ​​recorded in Table 1 are the G' values ​​at a frequency of 10 rad / s.

[0129] 3. Quantitative determination of self-healing efficiency: The self-healing efficiency of the hydrogel was evaluated through tensile testing. Hydrogel strips, after being cut and healed for a certain period, were subjected to tensile testing. The fracture stress was recorded and compared with the fracture stress of the uncut original strip. The stress recovery rate was calculated as the self-healing efficiency. The specific method is as follows:

[0130] The mechanical properties and self-healing efficiency of the hydrogels were tested using a universal testing machine. The hydrogel precursor solutions for each group were injected into dumbbell-shaped molds, molded according to the method described in Example 3, and then removed for later use. Referring to standard GB / T 528-2009, the tensile speed was set to 50 mm / min, and the original specimens were tested until they fractured, with the fracture stress recorded.

[0131] Self-healing test: The dumbbell-shaped specimen was cut at the middle of the gauge length, and then the two cut surfaces were brought back into close contact and placed in a constant temperature and humidity chamber at 37 ℃ and 60% relative humidity for 24 hours to heal. After healing, a tensile test was performed on the healed specimen under the same test conditions, and its fracture stress was recorded.

[0132] Self-healing efficiency calculation: The self-healing efficiency of the hydrogel is calculated using the following formula:

[0133] Self-healing efficiency = (fracture stress of the healed spline) / (fracture stress of the original spline) × 100%.

[0134] 4. Determination of antioxidant activity: The antioxidant activity of the hydrogel was evaluated using the DPPH free radical scavenging method. One mL of the hydrogel sample was immersed in 2 mL of 0.1 mM DPPH ethanol solution and reacted in the dark for 30 minutes. The absorbance of the supernatant at 517 nm was then measured. Pure DPPH solution was used as a blank control, and the free radical scavenging rate was calculated based on the change in absorbance.

[0135] 5. Determination of Melatonin Drug Loading Content (DLC): The prepared MT@PL-GA pre-assembled solution was freeze-dried to obtain MT@PL-GA drug-loaded polymer powder. A certain amount of the powder was accurately weighed, and an appropriate amount of dimethyl sulfoxide (DMSO) was added. The mixture was ultrasonically treated to fully destroy the polymer structure and promote complete dissolution of melatonin. The powder was then filtered through a 0.22 μm microporous membrane for later use. The melatonin content in the sample was determined by high-performance liquid chromatography (HPLC). The chromatographic conditions were as follows: a C18 reversed-phase column (250 mm × 4.6 mm, 5 μm) was used, with methanol / 0.1% trifluoroacetic acid as the mobile phase for isocratic elution (volume ratio 45:55), a flow rate of 0.8 mL / min, a column temperature of 30 ℃, a detection wavelength of 222 nm, and an injection volume of 10 μL. Under these conditions, the mass content of melatonin in the sample was calculated based on a pre-established melatonin standard curve. Drug loading is calculated using the following formula: Drug loading (DLC, %) = (mass of drug detected in the polymer / total mass of drug-loaded polymer) × 100%.

[0136] 6. Determination of melatonin encapsulation efficiency:

[0137] Encapsulation Efficiency (EE): The percentage of drug actually encapsulated in the hydrogel relative to the initial total drug dosage. The prepared MT@PL-GA pre-assembled solution was freeze-dried to obtain a drug-loaded polymer powder. 10 mg of the powder was accurately weighed and added to 5 mL of dimethyl sulfoxide (DMSO). The mixture was sonicated for 15 minutes to fully disrupt the polymer structure and promote complete dissolution of melatonin. The powder was then filtered through a 0.22 μm microporous membrane for later use. EE was measured under the HPLC detection conditions described above. EE (%) = (detected drug mass) / (initial total drug dosage) × 100%

[0138] Table 1. Effects of different ε-PL and GA feed ratios on the physicochemical properties of the hydrogel and melatonin loading.

[0139]

[0140] Experimental results show that the GA grafting rate in the PL-GA polymer has a certain influence on the melatonin loading capacity and the overall performance of the hydrogel. With the increase of the GA grafting rate, the drug loading capacity and encapsulation efficiency show a significant upward trend. This may be because during solvent exchange, the organic solvent gradually diffuses into the aqueous phase, causing a change in system polarity. Driven by thermodynamics, hydrophobic melatonin molecules preferentially accumulate on the hydrophobic GA groups on the PL-GA polymer chain and are stably captured under the synergistic effect of hydrophobic interactions and π–π stacking interactions, thus forming a self-assembled micro-region structure with melatonin as the core and the polymer as the shell. As the number of GA sites that can participate in this molecular interaction increases, the number of melatonin molecules that can be effectively encapsulated and stably exist also increases, resulting in a drug loading capacity increase from about 5% in systems with low grafting rates to a highly efficient range of over 10%. However, as the GA grafting rate further increased, both drug loading and encapsulation efficiency showed a decreasing trend. This may be because the system entered a "diminishing marginal effect" stage dominated by steric hindrance. Simultaneously, the excessively high density of hydrophobic groups significantly increased the crosslinking density of the hydrogel, making the network structure more rigid and brittle, restricting chain segment movement, and significantly reducing self-healing efficiency, which is detrimental to structural recovery under dynamic biological environments. When the molar ratio of amino groups to GA in ε-PL was 1:1.5, the system maintained a high storage modulus and near-complete self-healing performance while ensuring a melatonin drug loading of over 10%, achieving the optimal balance between drug loading capacity and the mechanical and dynamic properties of the hydrogel.

[0141] Example 5: Effect of PBA and GMA grafting rates on the properties of the double crosslinked hydrogel and melatonin release behavior

[0142] By varying the feed ratios of HA, 3-PBA, and GMA (as shown in Table 1), while maintaining the same steps, raw materials, and reaction conditions as in Examples 1-3, melatonin-loaded bicrosslinked hydrogels with different grafting rates of PBA and GMA were prepared. The gelation state of the obtained bicrosslinked hydrogels was observed, and their performance was tested and characterized as follows. The test results are shown in Table 2 and... Figure 3 As shown.

[0143] 1. Grafting rates of PBA and GMA: The grafting rates were calculated by comparing the proton signals (δ 3.2-4.0 ppm) of the sugar rings on the modified sodium hyaluronate backbone with the characteristic signals of the introduced groups, using the same method as in Example 2.

[0144] 2. Polymer Solubility: 100 mg of the modified polymer HA-PBA-GMA powder, which had been vacuum freeze-dried, was accurately weighed and added to 10 mL of phosphate-buffered saline (PBS) at pH 7.4. The solution was magnetically stirred continuously at 300 rpm for 24 hours at 37 ℃ to ensure sufficient swelling and diffusion of the polymer. After the dissolution process was completed, the appearance of the solution was first observed and recorded visually: if the solution was completely clear and transparent with no insoluble matter, it was rated as "excellent"; if the solution was basically clear but showed a weak Tyndall effect, it was rated as "good"; if obvious turbidity, suspended particles, or bottom precipitation appeared, it was rated as "poor". Subsequently, the transmittance (T%) of the solution was measured at a wavelength of 600 nm using a UV-Vis spectrophotometer, with pure PBS buffer as a blank control (T = 100%), as a quantitative evaluation index of polymer solubility.

[0145] 3. Melatonin Release Characteristics: The release was determined using dialysis. 1 mL of drug-loaded hydrogel was placed in a dialysis bag (molecular weight cutoff 3500 Da), then added to 20 mL of PBS buffer (pH 7.4), and released by constant temperature shaking at 37 ℃ and 100 rpm. 1 mL samples were taken at preset time points, and an equal volume of fresh medium was added. The MT concentration in the released solution was determined by HPLC, the cumulative release percentage was calculated, and a release curve was plotted. Figure 3 ).

[0146] Table 2: Effects of different grafting ratios on the physicochemical properties of hydrogels and melatonin (MT) release behavior

[0147]

[0148] Experimental results show that the grafting rates of PBA and GMA significantly affect the physicochemical properties, processability, and melatonin release characteristics of the resulting hydrogel. Grafting only GMA without PBA results in a brittle, hard gel that fails to meet the basic flexibility requirements of wound dressings, and the drug release rate is too rapid. When the PBA grafting rate exceeds 60%, the water solubility of modified hyaluronic acid significantly decreases under physiological conditions, making it difficult to form a uniform and stable drug-loaded hydrogel. Similarly, an excessively high GMA grafting rate (>40%) leads to an overly rigid and brittle photocrosslinked network, failing to meet the basic flexibility requirements of wound dressings. On the other hand, excessively low grafting rates (e.g., 25.1% PBA and 10.2% GMA) fail to construct an effective dual-release network: low GMA results in a loose cross-linking network and insufficient physical barrier capacity; low PBA weakens its π–π interaction with melatonin, significantly reducing the chemical anchoring effect, thus exhibiting a significant burst release phenomenon in in vitro release experiments (release exceeding 80% within 24 hours), making it difficult to cover the key inflammatory regulation stage of wound healing. When the grafting rate of PBA is within the range of 40-50% and the grafting rate of GMA is within the range of 20-30%, a dense and stable dual-cross-linking network and sufficient molecular interaction sites can be constructed simultaneously, achieving long-term, controllable release of melatonin, effectively prolonging its action time in the wound microenvironment, and ensuring the continuous clearance of ROS and the full realization of its healing-promoting effects from the inflammatory phase to the repair phase.

[0149] Example 6: Effect of melatonin addition method on drug loading efficiency and stability

[0150] 1. Preparation of drug-loaded polymer solutions using the "solvent exchange-self-assembly" method

[0151] First, a certain amount of melatonin (MT) was weighed and dissolved in anhydrous ethanol to prepare a melatonin organic solution with a mass concentration of 20 mg / mL. Simultaneously, the PL-GA polymer synthesized and purified in Example 1 was dissolved in PBS (pH 7.2-7.4) to prepare a 30% (w / v) PL-GA solution, which was then fully dissolved under magnetic stirring to obtain a homogeneous and transparent solution. Subsequently, under vigorous stirring at 150 rpm, the melatonin organic solution was added dropwise to the PL-GA solution at a rate of 0.5 mL / min using a microinjection pump, controlling the MT to PL-GA dosage ratio at 1:8. After the addition was complete, the system was stirred at 150 rpm for 4 hours. Subsequently, the resulting mixture was placed in a 4°C refrigerator and allowed to stand for 12 hours. During this process, as the organic solvent continued to diffuse into the aqueous phase, the solvent polarity of the system gradually changed. Under thermodynamic drive, hydrophobic melatonin molecules preferentially accumulated onto the hydrophobic catechol groups on the PL-GA polymer chain. Under the synergistic effect of hydrophobic interactions and π–π stacking, they spontaneously formed nanoscale micro-regions or micellar self-assembled structures with melatonin as the hydrophobic core and PL-GA polymer as the shell, thereby achieving stable capture of melatonin. After the reaction was completed, the resulting mixed solution was transferred to a dialysis bag with a molecular weight cutoff of 3500 Da and dialyzed in deionized water for 48 hours, with the dialysis medium being changed every 8 hours to fully remove residual organic solvent and unloaded free melatonin, resulting in a stable drug-loaded polymer solution (i.e., MT@PL-GA pre-assembled solution).

[0152] 2. Preparation of drug-loaded precursor solution by physical mixing method

[0153] As a control, a direct physical mixing method was used: an equivalent amount of melatonin powder as described in the "solvent exchange-self-assembly" method was weighed and directly added to a 30% (w / v) PL-GA PBS solution. The solution was vigorously stirred at 150 rpm for 12 hours at room temperature. Due to the low solubility of melatonin in the aqueous phase, obvious undissolved particles were observed in the solution. These undissolved drug particles were then removed by filtration through a 0.45 μm filter membrane, yielding a physically mixed drug-loaded solution.

[0154] 3. The drug loading and encapsulation efficiency of melatonin in the obtained MT@PL-GA were tested according to the method in Example 4.

[0155] Test results showed that the drug loading of MT@PL-GA prepared by the "solvent exchange-self-assembly" method reached 10.5%, and the encapsulation efficiency was as high as 94.5%, while the drug loading of the sample prepared by the physical mixing method was less than 2%, and there was obvious drug crystal precipitation.

[0156] 4. Stability test of MT@PL-GA dry powder

[0157] After storing the melatonin-loaded polymer powder at 4°C in the dark for 30 days, the structure was destroyed by ultrasonication with DMSO according to the method in Example 4. The melatonin content before and after storage was determined by high-performance liquid chromatography (HPLC), and the retention rate of the active ingredient was calculated. The results showed that the MT activity retention rate of the sample prepared by the physical mixing method was only 62.5%, while the activity retention rate of the MT@PL-GA powder prepared by the "solvent exchange-self-assembly" method remained above 98%, thus solving the problem of melatonin instability.

[0158] Example 7: Rheological characterization of double crosslinked hydrogels

[0159] In this embodiment, the illumination time of the second crosslinking was changed, while the other steps, raw materials, and reaction conditions were the same as those in Examples 1-3. MT@EGHP hydrogels under different illumination times were prepared, and their rheological characterization as double-crosslinked hydrogels was performed.

[0160] The mechanical properties of the MT@EGHP hydrogel were characterized using a rotational rheometer under isothermal conditions of 37 °C to simulate the physiological environment in vivo. All tests were performed after the gel had completely gelled to ensure the stability of the hydrogel structure during the testing process. During the tests, the storage modulus (G′) characterizing the elastic behavior and the loss modulus (G″) characterizing the viscous behavior were recorded.

[0161] 7.1 Determination of the Linear Viscoelastic Region (LVR)

[0162] To determine the linear viscoelastic region of the MT@EGHP hydrogel, strain scanning tests were performed on the sample. The strain was measured at a fixed angular frequency of 1 rad·s. -1 Under varying strains ranging from 0.01% to 1000%, oscillatory shear strains were applied, and the changes in G′ and G″ were recorded. When G′ and G″ remained essentially constant within a certain strain range, this interval was defined as the linear viscoelastic region, indicating that the gel network structure remained intact. As the strain further increased, when G′ decreased significantly and gradually approached or fell below G″, it indicated that the internal network of the gel began to collapse, and the system transitioned from a solid to a liquid state. The corresponding strain value was defined as the yield strain, used to reflect the structural stability and deformation resistance of the hydrogel network. By analyzing the variation trends of G′ and G″, the mechanical stability and network collapse behavior of the MT@EGHP hydrogel under different illumination times were evaluated.

[0163] 7.2 Frequency Scan Test

[0164] Within the aforementioned defined linear viscoelastic region, frequency sweep tests were performed on the MT@EGHP hydrogel to evaluate its viscoelastic behavior under different dynamic conditions. During the tests, the shear strain was fixed at 0.1% (within the LVR), and the angular frequency range was set from 0.1 to 100 rad·s. -1 By recording the trends of G′ and G″ with frequency, the influence of dynamic borate ester bonds and covalent crosslinking networks on the time-dependent mechanical behavior of the hydrogel was analyzed. When G′ is consistently higher than G″ throughout the frequency range and is insensitive to frequency, it indicates that the hydrogel exhibits stable solid-state characteristics, possesses good network structure integrity and mechanical stability, and is suitable for withstanding dynamic mechanical stimuli in vivo.

[0165] 7.3 Self-healing behavior test

[0166] The self-healing properties of EGHP hydrogels were evaluated using a step-strain test. During the test, alternating low and high strains were applied to simulate repeated mechanical damage experienced by the gel in real-world applications. The specific test procedure was as follows: first, a low shear strain of 0.1% (within the LVR) was applied to monitor the initial stable state of the gel; then, a high shear strain of 1000% was applied to artificially disrupt the internal network structure of the gel; subsequently, the strain was restored to 0.1%, and the recovery processes of G′ and G″ were recorded. This low-high-low strain cycle was repeated multiple times to evaluate the reversible reconstruction capability of the gel network.

[0167] After the high strain is removed, G′ can rapidly recover to near its initial value, indicating that MT@EGHP hydrogel possesses excellent self-healing ability and thixotropic recovery properties. This property is of great significance for maintaining the structural stability and long-term function of hydrogels in complex mechanical environments such as skin and soft tissues.

[0168] 7.4 Experimental Results:

[0169] The MT@EGHP hydrogel constructed in this invention employs a stepwise dual crosslinking strategy. First, the phenylboronic acid group (HA-PBA) introduced onto the hyaluronic acid backbone undergoes a reversible borate ester bond reaction with the ortho-bisphenol structure in the gallic acid-grafted polylysine (PL-GA), forming the first dynamic crosslinking network. This network exhibits reversibility and environmental sensitivity, endowing the hydrogel with excellent shear-thinning, self-healing, and microenvironment-responsive capabilities.

[0170] Subsequently, in the presence of a photoinitiator, the GMA double bonds in HA-PBA-GMA undergo free radical polymerization under ultraviolet light irradiation, forming a second permanent covalent cross-linked network. This network acts as a stable framework, significantly improving the mechanical strength and structural stability of the hydrogel. By adjusting the irradiation time (0 s, 30 s, 60 s), a gradual evolution from a single dynamic network to a dynamic / covalent dual-network structure can be achieved.

[0171] Within the low strain range (approximately 0.1%–100%), the storage modulus (G′) and loss modulus (G″) of the three hydrogel samples all exhibited stable plateau regions, with G′ consistently higher than G″. This indicates that the hydrogel network structure remained intact within this strain range, exhibiting overall solid characteristics dominated by elastic response.

[0172] For the unilluminated sample (0 s, orange), the hydrogel mainly relies on dynamic borate ester bonds to maintain its network structure, exhibiting a relatively low storage modulus and a narrow linear viscoelastic region (LVER). This indicates that although dynamic borate ester bonds can effectively dissipate external strain energy through rapid and reversible breakage and recombination, thereby maintaining network integrity, under large strain, this type of dynamically cross-linked network is still relatively prone to structural relaxation and failure, exhibiting limited strain resistance. Figure 4 ).

[0173] In contrast, the hydrogels treated with light for 30 s (red) and 60 s (blue) still maintained good LVER stability, while the difference between G′ and G″ increased significantly, and the overall storage modulus was significantly improved. This result indicates that the covalent crosslinking network formed after photo-initiated free radical polymerization significantly enhances the mechanical stiffness and deformation resistance of the hydrogel. Notably, photocrosslinking did not lead to significant embrittlement; on the contrary, the rigid covalent framework and the dynamic borate ester network worked synergistically, allowing the hydrogel to maintain structural integrity even under greater strain. Figure 4 ).

[0174] Furthermore, as the illumination time increased from 30 s to 60 s, the storage modulus of the hydrogel further improved, and the stability and strain resistance of the linear viscoelastic region continued to increase, indicating that the covalent crosslinking density increased with increasing illumination time, thus providing stronger mechanical support for the network. These results collectively demonstrate that illumination time can be used as an effective control method to achieve a gradual enhancement of the mechanical strength and strain resistance of the hydrogel. Figure 4 ).

[0175] Frequency scan test ( Figure 5This study further revealed the regulatory mechanism of UV irradiation time on the viscoelastic behavior of MT@EGHP hydrogel. Within the test frequency range (0.1-10 Hz), the storage modulus (G′) of all experimental groups (0 s, 30 s, 60 s) was always higher than the loss modulus (G″), and no crossover point was observed, indicating that the hydrogel maintains stable elastic solid characteristics under dynamic shear conditions.

[0176] Notably, the mechanical strength of the hydrogel exhibits a significant light-time dependence. The un-illuminated sample (0s, orange) maintains its network structure solely through dynamic borate ester bonds, with its G′ remaining at a low level (approximately 0.5-1 kPa), exhibiting a soft and compliant texture. With prolonged light exposure, UV light induces free radical polymerization of the GMA double bonds on HA-PBA-GMA, resulting in a stepwise increase in storage modulus: after 30 s (red) light exposure, G′ increases to approximately 2-3 kPa; after 60 s (blue) light exposure, G′ further increases to approximately 5-10 kPa. Compared to the un-illuminated sample, 60 s light treatment increases the hydrogel's storage modulus by nearly an order of magnitude. This result demonstrates that by controlling the light exposure time, the density of covalent crosslinking points in the double-crosslinked network can be precisely controlled, thereby achieving on-demand adjustment of the hydrogel's stiffness.

[0177] This ability to flexibly adjust the modulus through light exposure time has significant clinical value for wound dressing applications. The elastic modulus of human skin and subcutaneous soft tissue is typically in the range of 1-10 kPa (depending on skin layer and location). In its initial state (0 s), the hydrogel modulus is approximately 1 kPa, exhibiting excellent softness and compliance, effectively filling irregular wound beds and establishing close contact with tissue at the microscopic level. After 30-60 s of in-situ light curing, the hydrogel modulus rapidly increases to 5-10 kPa (60 s group), highly matching the modulus of human dermis and epidermis, achieving a biomimetic mechanical effect. This "mechanical matching" characteristic not only provides sufficient shear and friction resistance, effectively protecting the wound, but also avoids interfacial stress concentration caused by modulus mismatch between the dressing and skin, thereby reducing patient discomfort during movement and promoting scarless wound healing.

[0178] To evaluate the structural stability and self-healing ability of MT@EGHP hydrogel under dynamic mechanical conditions, this embodiment conducted cyclic testing at a fixed frequency, alternating between high strain (1000%, simulating severe damage) and low strain (1%, simulating resting state). Figure 6 The experimental results visually reveal the trade-off between photocrosslinking density and the mechanical strength and self-healing properties of hydrogels.

[0179] 0s Group: Exceptional rapid self-healing properties

[0180] The 0 s sample (orange) without light exposure has a low initial storage modulus (G′), but after experiencing a 1000% strain that causes network collapse, G′ recovers almost instantaneously to its initial level upon returning to a 1% strain. In multiple failure-recovery cycles, its recovery efficiency remains close to 100%. This indicates that the supramolecular interactions between the sample network, maintained entirely by dynamic borate ester bonds, can rapidly break and reassemble, giving the hydrogel significant "liquid-solid reversibility." It can flow and fill like a liquid, and also rapidly reshape like a solid, endowing the material with excellent self-healing properties.

[0181] 60-second set: High intensity but loss of self-healing ability

[0182] The initial G′ of the sample (blue) after 60 s of UV irradiation reached approximately 10. 6 Pa, significantly higher than other groups, indicates that the covalent network formed by complete photopolymerization provides strong mechanical support. However, after the first 1000% strain failure, when the strain returned to 1%, G′ failed to recover to the initial level, instead plummeting to near 0 s or even lower, and failing to recover in subsequent cycles. This shows that prolonged light exposure constructs a high-density GMA covalently cross-linked network (rigid framework), significantly enhancing hardness and resistance to deformation. However, once covalent bonds break under severe deformation, they cannot reform, causing the original dual-network system to degenerate into a single network or fragmented gel maintained only by dynamic bonds, thus losing the ability to recover its original high strength.

[0183] 30-second set: The fulcrum of balancing strength and self-healing

[0184] The 30 s sample (red) exhibits properties intermediate between the two groups, with a significantly higher initial G′ than the 0 s group, while still partially recovering its modulus after failure. Although the recovery efficiency does not reach the perfect level of the 0 s group, most of the structure can be reconstructed.

[0185] Example 8: In vitro drug release behavior of double cross-linked hydrogels

[0186] In this embodiment, the light exposure time for the second crosslinking was changed, while the other steps, raw materials, and reaction conditions were the same as those in Examples 1-3. MT@EGHP hydrogels under different light exposure times were prepared, and their in vitro drug release behavior was tested.

[0187] Prepare a 1 mL hydrogel sample in a centrifuge tube and add 20 mL of skin exudate simulation solution (PBS 6.5, 5 mg / mL glucose, and 1 mM H2O2). Place the tube in a 37°C shaking incubator to simulate dynamic conditions under physiological conditions. At predetermined time points, 1 mL of the solution is taken for analysis, and an equal volume of fresh skin exudate simulation solution is added to maintain a constant total volume. Drug concentration is determined by high-performance liquid chromatography (HPLC) and quantified according to a standard curve. The cumulative release rate is then calculated based on the measured concentration and the following formula:

[0188]

[0189] Among them, Q n Cumulative release rate (%); C n V: Concentration measured at the nth sampling point (μg / mL); V: Volume of release medium (mL); V0: Sampling volume (mL); C i : Concentration measured at the i-th sampling point (μg / mL); A: Total drug content of the double cross-linked hydrogel (μg).

[0190] Experimental results show that ( Figure 7 As the duration of UV irradiation increases, the degree of free radical polymerization of GMA groups within the hydrogel system increases, forming a denser covalently cross-linked network, which significantly affects the release characteristics of melatonin. Low-crosslinked (0 s) hydrogels exhibit typical burst release effects, with rapid initial drug release and almost complete release by 12 hours. This is due to the loose network structure maintained by dynamic borate ester bonds and the larger pore size, which facilitates rapid dissolution of the drug through swelling and diffusion. Medium-crosslinked (30 s) hydrogels effectively inhibit initial burst release, exhibiting a more stable release curve and achieving a balance between rapid onset and long-lasting effect, suitable for mid-term treatment coverage. High-crosslinked (60 s) hydrogels exhibit excellent long-acting sustained-release characteristics, with a significantly slower initial release followed by a near-linear sustained release pattern, maintaining a stable drug supply for up to 96 hours. This mechanism can be attributed to the reduced mesh size due to network densification, enhanced steric hindrance, and restricted relaxation of polymer segments, thereby delaying the diffusion and precipitation of drug molecules. This long-acting, sustained-release behavior has significant clinical implications in wound treatment: it not only avoids peak-valley effects and the cytotoxicity that may result from excessively high local drug concentrations, but also maintains effective drug concentrations for extended periods, covering the critical treatment window from the acute inflammatory phase to the early proliferative phase of acute wounds. This reduces the frequency of dressing changes, alleviates patient pain and secondary injury, and lightens the workload of healthcare professionals. In summary, MT@EGHP hydrogel achieves a controlled release transition from burst release to long-acting sustained release by regulating photocrosslinking density, demonstrating highly tunable drug delivery capabilities and significant clinical application potential.

[0191] Considering the combined effects of different UV crosslinking times on the rheological properties and in vitro release behavior of the materials, the MT@EGHP hydrogel prepared with an irradiation time of 30 s not only has a mechanical modulus that is more compatible with skin tissue (the hydrogel G' is stable at 1.5-4.0 kPa, which is closest to the median modulus of newly formed granulation tissue and subcutaneous soft tissue. Compared with the rigidity of the 60 s group, which is close to the upper limit of the range, the 30 s group can better adapt to the daily deformation of the skin and reduce interfacial shear stress), but also, in terms of release behavior, the double crosslinking network constructed in the 30 s group achieves a balance between diffusion resistance and pharmacokinetics, avoiding the excessive burst release of the 0 s group and ensuring the continuous effective concentration of melatonin throughout the inflammatory regulation period; at the same time, the 30 s group also retains good self-healing properties. Therefore, 30 s of irradiation is the optimal irradiation condition.

[0192] Example 9: In vitro drug release behavior of MT@EGHP hydrogel under different conditions

[0193] The in vitro drug release behavior of the bicrosslinked hydrogel was tested according to the method in Example 8, using PBS 7.4, PBS 6.5, PBS 6.5 + 5 mg / mL, and PBS 6.5 + 5 mg / mL glucose + 1 mM H2O2 as release media. The bicrosslinked hydrogel used was an MT@EGHP hydrogel prepared according to the methods in Examples 1-3 (illumination time 30 s).

[0194] The results show that ( Figure 8All experimental groups exhibited a gradual increase in cumulative release over time, demonstrating a generally good sustained release characteristic. However, the drug release rate of the hydrogel varied significantly in different simulated microenvironments, showing a clear stepwise effect in stimulus response. The release rates were as follows: PBS7.4 < PBS 6.5 < PBS 6.5 + Glucose < PBS 6.5 + Glucose + H2O2, indicating that the system has progressively increasing sensitivity to pathological stimuli. Under PBS 7.4 conditions, the drug release from the hydrogel was the slowest, with approximately 40% released at 12 h and a cumulative release of approximately 90% by 96 h. This condition simulates a normal physiological fluid environment, where the hydrogel's cross-linked network structure is stable and relatively dense. Drug release is mainly controlled by a concentration gradient-driven diffusion process, demonstrating good drug retention and "drug preservation" capabilities, which helps avoid excessively rapid release in non-lesion areas. Under weakly acidic PBS 6.5 conditions, the drug release rate increased significantly, with the cumulative release increasing to approximately 55% at 12 h. This environment was designed to simulate an acidic microenvironment associated with infection or inflammation. This is likely due to the partial dissociation or network swelling of acid-sensitive crosslinks in the system under low pH conditions, which increases the hydrogel pore size and promotes melatonin diffusion. Further addition of glucose under PBS 6.5 conditions further accelerated drug release, with a cumulative release of approximately 65% ​​after 12 hours. This result is closely related to the phenylboronic acid (PBA) groups introduced into the system, which can form a dynamically reversible borate ester complex with glucose molecules, thereby weakening the original polymer chain interactions or crosslinking point stability, leading to gel network relaxation and responsive release in response to a high-glucose environment. Notably, the hydrogel exhibited the fastest drug release behavior under conditions of weak acidity, high glucose, and hydrogen peroxide (H2O2), with over 70% release within 12 hours and nearing complete release after 48 hours. This condition was used to simulate the typical high-oxidative-stress microenvironment in chronic, difficult-to-heal wounds, where H2O2, as a strong oxidant, can induce oxidative breakage of phenylboronic acid-related structures, leading to rapid disintegration or severe swelling of the hydrogel network, thus triggering rapid drug release. The above results indicate that the hydrogel system can achieve "emergency" drug delivery during the most severe stage of oxidative stress, enabling the antioxidant melatonin to be released in large quantities during the time window when the removal of reactive oxygen species is most needed.

[0195] From an application perspective, this release behavior is highly compatible with the pathological microenvironment of diabetic chronic wounds. Under normal physiological conditions, the hydrogel maintains a slow release to reduce drug waste; however, once exposed to the unique weakly acidic, high-glucose, and high-ROS microenvironment of diabetic wounds, the system can significantly accelerate drug release through multiple stimulus-response mechanisms, achieving true on-demand delivery. Simultaneously, melatonin itself possesses potent antioxidant activity, and it is rapidly released when ROS levels are highest, further enhancing the synergistic therapeutic model of "stimulus enhancement - drug enhancement." Furthermore, even under the strongest stimulus conditions, the system can maintain effective release for more than 48 hours, and under mild stimulus conditions, it can continue for up to 96 hours, which helps reduce the frequency of dressing changes and improve patient compliance in clinical applications.

[0196] Example 10: Stability of MT@EGHP hydrogel

[0197] MT@EGHP hydrogels were prepared according to the methods in Examples 1-3 (illumination time of 30 s), and their structural stability and in vitro degradation behavior under physiological conditions were tested.

[0198] After removing surface free water from the prepared hydrogel, its initial mass (W0) was measured. It was then placed in a pH 7.4 PBS solution and incubated continuously in a shaker at 37 °C. At predetermined time points (Days 1, 3, 5, and 7), the hydrogel was removed, surface water was gently blotted dry, and its mass (W0) was measured. t ), through formula Calculate the quality changes at different time points.

[0199] The results show that ( Figure 9 The hydrogel exhibited a slow and stable decline in mass under simulated physiological conditions, without sudden degradation or structural collapse. During the initial incubation period, slight swelling occurred due to the presence of hydrophilic groups in the network, but the overall structure remained intact. By day 7, the mass retention rate of the hydrogel remained above 80% (approximately 85.93 ± 2.73%), indicating that the MT@EGHP hydrogel prepared in this invention possesses excellent hydrolytic stability and can maintain structural integrity within the time window required for wound healing.

[0200] Example 11: In vitro pharmacodynamic testing of double cross-linked hydrogels

[0201] (1) Anti-inflammatory test - cytokine secretion level measurement (ELISA)

[0202] To test the regulatory effect of MT@EGHP hydrogel on the secretion of inflammatory factors under oxidative stress microenvironment, the expression levels of pro-inflammatory factors tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and anti-inflammatory factor interleukin-10 (IL-10) in cell culture supernatant were detected by enzyme-linked immunosorbent assay (ELISA).

[0203] Hydrogels containing or without melatonin were prepared according to the methods in Examples 1-3, namely MT@EGHP hydrogel and EGHP hydrogel, respectively.

[0204] Preparation of the extract: The EGHP and MT@EGHP hydrogel extracts used in the experiment were prepared according to ISO 10993-12 standard. The prepared sterile hydrogel samples (already UV cross-linked) were immersed at a ratio of 0.1 g / mL in DMEM / F12 intact medium containing 1% penicillin and no serum, and extracted continuously at 37 ℃ for 24 h. After extraction, the supernatant was filtered through a 0.22 μm microporous membrane for sterilization to obtain the hydrogel extract.

[0205] Human umbilical vein endothelial cells (HUVECs) in the logarithmic growth phase were seeded in 24-well plates at a density of 5 × 10⁶ cells / well. 4 Cells were cultured at 37 °C and 5% CO2 for 24 h to allow for full cell adhesion. Cells were then divided into several groups: Control group (cultured in complete culture medium), Model group (containing ROSUP, 1:200, to induce an oxidative stress model), EGHP group (containing EGHP hydrogel extract and ROSUP), melatonin-only MT group (containing 10 μM free melatonin), and MT@EGHP group (containing MT@EGHP hydrogel extract and ROSUP; the melatonin content in MT@EGHP was the same as in the MT group). After culturing for another 24 h, the culture supernatant was collected and centrifuged at 4 °C and 4000 rpm for 10 min to remove cell debris. The clear supernatant was used for subsequent analysis. The ELISA assay was performed strictly according to the kit instructions, including sample addition, incubation, plate washing, biotinylated antibody reaction, horseradish peroxidase (HRP) conjugate incubation, TMB color development, and reaction termination. The absorbance (OD) was measured at 450 nm. The concentrations of each cytokine were calculated based on the standard curve. All experiments were independently repeated at least three times. Experimental data are expressed as Mean ± SD, and differences between groups were statistically analyzed using one-way ANOVA.

[0206] Oxidative stress is often accompanied by a significant inflammatory response, characterized by increased secretion of pro-inflammatory factors and suppressed expression of anti-inflammatory factors. The levels of TNF-α, IL-6, and IL-10 in the cell culture supernatant of different treatment groups were detected by ELISA to systematically evaluate the regulatory effect of MT@EGHP hydrogel on the oxidative stress-related inflammatory microenvironment. The results showed ( Figures 10-12 (Table 3) Compared with the Control group, the Model group showed significantly increased levels of pro-inflammatory factors TNF-α and IL-6 under ROSUP stimulation, while the secretion of anti-inflammatory factor IL-10 was significantly reduced, indicating that the oxidative stress model was successfully constructed. Compared with the Model group, the melatonin-only MT group showed significantly decreased levels of TNF-α and IL-6, and a significant rebound in IL-10 secretion, essentially returning to the Control group level. This confirms that melatonin, as a potent antioxidant, has clear anti-inflammatory and immunomodulatory capabilities in in vitro cell experiments. The EGHP group also showed partial inhibition of pro-inflammatory factors, which may be due to the basic antioxidant capacity conferred by gallic acid and phenylboronic acid groups in the hydrogel backbone, which alleviates the inflammatory response to some extent by partially scavenging ROS and weakening oxidative stress-related signaling pathways. In contrast, the MT@EGHP group showed the most significant immunomodulatory effect, with significantly inhibited levels of TNF-α and IL-6, and significantly upregulated IL-10 secretion, which was significantly better than the MT and EGHP groups alone. The above results indicate a synergistic anti-inflammatory mechanism between melatonin and the hydrogel scaffold: the EGHP hydrogel network first improves the oxidative stress microenvironment by continuously scavenging excess ROS, while the loaded and slowly released melatonin further exerts stable and long-lasting antioxidant and immunomodulatory effects, thereby more effectively inhibiting the transcription of pro-inflammatory factors and promoting the activation of anti-inflammatory signals, ultimately restoring the dynamic balance between pro-inflammatory and anti-inflammatory factors. Therefore, MT@EGHP gel can reshape the imbalanced inflammatory microenvironment under oxidative stress conditions, reduce tissue damage, shorten the inflammatory phase, and promote subsequent tissue repair, with significantly better effects than free melatonin or blank hydrogel.

[0207] Table 3. Content of TNF-α, IL-6 and IL-10 in cell culture supernatant of different treatment groups

[0208]

[0209] (2) Detection of intracellular ROS levels.

[0210] To test the effect of MT@EGHP hydrogel on the regulation of intracellular reactive oxygen species (ROS) levels and inflammatory factors under oxidative stress, the generation of intracellular ROS was detected using the 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) probe, and the results were combined with changes in inflammatory factor levels for comprehensive analysis.

[0211] HUVEC cells in the logarithmic growth phase were seeded in 24-well plates at a density of approximately 5 × 10⁶ cells / well. 4 Cells were cultured at 37°C and 5% CO2 for 24 h before being grouped for treatment. The experiments included: a Control group (cultured in complete medium), a Model group (with ROSUP added at a 1:200 dilution to induce oxidative stress), an EGHP group (with EGHP hydrogel extract and simultaneous ROSUP stimulation; EGHP was prepared according to Examples 1-3, except without melatonin), a melatonin-only MT group (with melatonin at a final concentration of 10 μM), and an MT@EGHP group (with MT@EGHP hydrogel extract and simultaneous ROSUP stimulation; MT@EGHP was prepared according to Examples 1-3). After 24 h of treatment, the culture medium was discarded, and the cells were slowly washed three times with PBS. Then, DCFH-DA probe working solution (final concentration 10 μM) prepared in serum-free medium was added, and the cells were incubated at 37°C in the dark for 30 min. After incubation, the cells were washed again with PBS to remove probes that did not enter the cells. Fluorescence signals (excitation wavelength 488 nm, emission wavelength 525 nm) were detected using a fluorescence microscope or ELISA reader to reflect changes in intracellular ROS levels by fluorescence intensity.

[0212] Experimental results show that ( Figure 13 The melatonin-loaded double-crosslinked hydrogel (MT@EGHP) prepared in this invention exhibits excellent ROS scavenging ability. In the HUVEC cell oxidative damage model, the blank hydrogel carrier (EGHP) showed a certain ROS downregulation ability, indicating that the carrier material itself has certain antioxidant functions. However, after loading melatonin (MT@EGHP), this composite system showed a superior synergistic antioxidant effect, reversing the intracellular ROS level from a high oxidative state (strong green fluorescence in the Model group) to a very low level. This demonstrates that the MT@EGHP hydrogel can not only serve as a drug delivery carrier but also as an active antioxidant dressing, creating a favorable low-oxidative stress microenvironment for tissue repair by clearing excess ROS in the wound microenvironment.

[0213] 3.3 Cell migration ability detection

[0214] To test the effect of MT@EGHP hydrogel on cell migration under oxidative stress, the Transwell assay was used to detect the migration behavior of cells in different treatment groups.

[0215] HUVEC cells in the logarithmic growth phase were selected, digested, resuspended in serum-free medium, and the cell density was adjusted to approximately 2 × 10⁻⁶. 5Cells / mL. 200 μL of cell suspension was added to the upper chamber (8 μm pore size) of the Transwell, and 500 μL of complete culture medium containing 5% fetal bovine serum was added to the lower chamber as a source of chemokines. The experiment was divided into a Control group (normal culture conditions), a Model group (ROSUP, 1:200, added to the culture medium to induce oxidative stress), an EGHP group (EGHP hydrogel extract added and simultaneously stimulated with ROSUP; EGHP was prepared according to the methods in Examples 1-3, except that melatonin was not added), a melatonin-only MT group (melatonin added to a final concentration of 10 μM), and an MT@EGHP group (MT@EGHP hydrogel extract added and simultaneously stimulated with ROSUP; MT@EGHP was prepared according to the methods in Examples 1-3). After incubating the Transwell chambers at 37°C in a 5% CO2 incubator for 24 h, the chambers were removed, and unmigrated cells in the upper chamber were gently wiped away with a cotton swab. Cells were then fixed with 4% paraformaldehyde for 20 min and stained with 0.1% crystal violet solution for 15 min. After washing with PBS, several fields of view were randomly selected under an inverted microscope to count and quantitatively analyze the cells that migrated across the membrane to the lower surface.

[0216] Transwell experimental results show that ( Figure 14 Under ROSUP (1:200) stimulation, the number of cells migrating across the membrane in the Model group was significantly reduced compared to the Control group, confirming that oxidative stress severely inhibited cell migration. Compared to the Model group, the number of migrating cells in the EGHP group significantly increased, returning to a level comparable to the Control group, indicating that the ROS scavenging ability of the hydrogel matrix itself and the polyphenol components can effectively neutralize the negative interference of oxidative stress on cell migration, restoring it to normal. Among all treatment groups, the MT@EGHP group showed the most outstanding migration-promoting effect, with its number of cells migrating across the membrane not only far exceeding that of the Model and EGHP groups, but also significantly surpassing the normal Control group level. This result fully demonstrates that the MT@EGHP hydrogel is not a simple functional additive, but rather generates "beyond homeostasis" migration-promoting potential by synergistically reducing intracellular ROS levels, reshaping the inflammatory microenvironment, and utilizing the sustained release effect of melatonin and the hydrogel matrix. In summary, MT@EGHP hydrogel can strongly drive cell migration to the wound area. Under complex oxidative stress conditions, it can not only ensure the basic activity of cells, but also significantly accelerate the wound healing and tissue repair process and effectively inhibit scar formation.

[0217] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A modified hyaluronic acid, characterized in that, It is obtained by reacting hyaluronic acid grafted with phenylboronic acid groups with glycidyl methacrylate. The grafting rate of phenylboronic acid groups in the modified hyaluronic acid is 35%-55%; The grafting rate of glycidyl methacrylate groups in the modified hyaluronic acid is 15%-35%.

2. The modified hyaluronic acid according to claim 1, characterized in that, The weight-average molecular weight of the hyaluronic acid is 400kDa-800kDa; And / or, the grafting rate of phenylboronic acid groups in the modified hyaluronic acid is 40%-50%, more preferably 43%-50%; And / or, the grafting rate of glycidyl methacrylate groups in the modified hyaluronic acid is 20%-32%, more preferably 23%-31%.

3. A method for preparing the modified hyaluronic acid according to claim 1 or 2, characterized in that, Includes the following steps: Hyaluronic acid undergoes an amidation reaction with 3-aminophenylboronic acid to obtain hyaluronic acid grafted with phenylboronic acid groups; The hyaluronic acid grafted with phenylboronic acid groups undergoes an epoxy ring-opening reaction with glycidyl methacrylate to obtain the modified hyaluronic acid. Preferably, the molar ratio of sugar units to 3-aminophenylboronic acid in the hyaluronic acid is 1:0.7-0.85, more preferably 1:0.8; Preferably, the molar ratio of sugar units in the hyaluronic acid to glycidyl methacrylate is 1:0.5-0.7, more preferably 1:0.

6.

4. A melatonin-loaded, double-crosslinked hydrogel precursor solution, characterized in that, Includes solution A and solution B; Solution A is composed of the modified hyaluronic acid, photoinitiator, and solvent as described in claim 1 or 2; Solution B consists of a melatonin-loaded polymer and a solvent; The melatonin-loaded polymer is obtained by loading melatonin onto modified polylysine. The modified polylysine is obtained by amidation reaction of ε-polylysine and gallic acid; Preferably, solution A and solution B are mixed evenly at a volume ratio of 1:0.9-1.1 before use.

5. The melatonin-loaded double cross-linked hydrogel precursor solution according to claim 4, characterized in that, The grafting rate of gallic acid in the modified polylysine is 2.8%-23%, preferably 12%-18%, and more preferably 14%-17%. And / or, the ε-polylysine and gallic acid react in the following molar ratio: the molar ratio of the amino group in ε-polylysine to gallic acid is 1:0.8-2, preferably 1:1.2-1.8, more preferably 1:1.4-1.6; And / or, the weight-average molecular weight of the ε-polylysine is 3000-5000; And / or, the melatonin loading in the melatonin-loaded polymer is 4%-15%, preferably 9%-12%; And / or, the solvent in solution B is water or a buffer solution, preferably a PBS buffer with a pH of 7.4; And / or, based on the mass of modified polylysine, the concentration of solution B is 0.25 g / mL to 0.35 g / mL, preferably 0.28 g / mL to 0.32 g / mL.

6. The melatonin-loaded bicrosslinked hydrogel precursor solution according to claim 4 or 5, characterized in that, The photoinitiator is lithium phenyl (2,4,6-trimethylbenzoyl) phosphate; And / or, the solvent in solution A is water or a buffer solution, preferably a PBS buffer with pH 7.4; And / or, the concentration of the modified hyaluronic acid in solution A is 90 mg / mL-110 mg / mL; And / or, the concentration of the photoinitiator in solution A is 0.5 mg / mL to 2 mg / mL.

7. A method for preparing a melatonin-loaded bicrosslinked hydrogel precursor solution according to any one of claims 4-6, characterized in that, Includes the following steps: Preparation of solution A: Dissolve the modified hyaluronic acid in a solvent, add the photoinitiator, stir and dissolve in the dark to obtain the solution; Preparation of Solution B: Melatonin is dissolved in ethanol to obtain a melatonin ethanol solution; the modified polylysine is dissolved in water or a buffer solution to obtain a polymer solution; the melatonin ethanol solution is added dropwise to the polymer solution, and after the addition is complete, stirring is continued, and the solution is allowed to stand to remove the ethanol, thus obtaining the final solution. Preferably, the mass ratio of melatonin to modified polylysine is 1:5-15, more preferably 1:7-10, and even more preferably 1:8-9; Preferably, the melatonin concentration in the melatonin ethanol solution is 15 mg / mL to 25 mg / mL; Preferably, the buffer solution is a PBS buffer with a pH of 7.4; Preferably, the concentration of modified polylysine in the polymer solution is 0.25 g / mL to 0.35 g / mL, and more preferably 0.28 g / mL to 0.32 g / mL; Preferably, the melatonin ethanol solution is added dropwise to the polymer solution at a rate of 0.4 mL / min to 0.6 mL / min; Preferably, the melatonin ethanol solution is added dropwise to the polymer solution while stirring at a speed of 120 rpm to 180 rpm; Preferably, stirring should continue for 3-6 hours after the dripping is complete; Preferably, let it stand for 10 to 16 hours.

8. A method for preparing a melatonin-loaded double cross-linked hydrogel, characterized in that, The process includes the following steps: mixing solution A and solution B in the melatonin-loaded double cross-linked hydrogel precursor solution according to any one of claims 4-6, allowing it to stand, and irradiating it with ultraviolet light to obtain the melatonin-loaded double cross-linked hydrogel. Preferably, solution A and solution B are mixed evenly at a volume ratio of 1:0.9-1.1; Preferably, the irradiation time with ultraviolet light is 0-60 seconds, more preferably 28-32 seconds; Preferably, the standing period is 3-5 minutes at 35°C-40°C; Preferably, the wavelength of the ultraviolet light is 365 nm and the light intensity is 4 mW / cm²-6 mW / cm².

9. A melatonin-loaded double crosslinked hydrogel prepared by the preparation method of claim 8.

10. The use of the melatonin-loaded bicrosslinked hydrogel precursor solution according to any one of claims 4-6 and / or the melatonin-loaded bicrosslinked hydrogel according to claim 9 in the preparation of a drug. The drug is an anti-inflammatory drug, an antioxidant drug, or a drug that promotes wound healing and / or inhibits scar formation.

Images