Self-healing hydrogel for skin repair and preparation method thereof

Abstract

The invention provides a self-healing hydrogel for skin repair and a preparation method thereof, and the preparation method comprises the following steps: adopting an aprotic polar solvent as a solvent, and providing an anhydrous environment to modify dipotassium glycyrrhizinate serving as a glycyrrhizic acid derivative; the defect that the grafting degree is low due to the fact that glycyrrhizic acid and glycyrrhizic acid derivative molecules wrap hydrophilic group carboxyl under the self-assembly or hydrophobic association effect in a traditional aqueous solvent is overcome, and the self-healing efficiency of the glycyrrhizic acid and glycyrrhizic acid derivative hydrogel is remarkably improved; a hydrazide group is introduced through a carboxyl coupling method, and a triterpenoid skeleton and a glycosyl structure of dipotassium glycyrrhizinate are completely reserved by controlling a reaction feeding ratio and reaction pH conditions, so that the problem that the bioactivity of dipotassium glycyrrhizinate is weakened or lost due to damage of a dipotassium glycyrrhizinate skeleton structure is avoided; the biological activities of dipotassium glycyrrhizinate, such as inflammation resistance and oxidation resistance, can be exerted to the greatest extent, and wound healing is remarkably promoted.

Description

Technical Field

[0001] The present invention relates to the technical field of hydrogel materials, and particularly to a self-healing hydrogel for skin repair and a preparation method thereof. Background Art

[0002] Hydrogels have good biocompatibility and great application prospects in wound repair. However, due to the irreversibility of their chemical cross-linking, traditional hydrogels are easily damaged under external stress, which limits their application environment.

[0003] As a triterpenoid saponin, glycyrrhizic acid has anti-inflammatory, antibacterial, and antiviral effects. At the same time, its special molecular structure can self-assemble into micelles, which can regulate the microstructure of the system, can be used for drug release, and is beneficial to wound repair.

[0004] Currently, the use of glycyrrhizic acid and its derivatives in hydrogels can be roughly divided into the following two categories: one is to utilize the special molecular structure of glycyrrhizic acid, add metal ions to achieve the self-assembly of glycyrrhizic acid molecules, or utilize temperature to regulate its self-assembly behavior, with glycyrrhizic acid as the hydrogel skeleton. The other is to mainly exert its drug activity, add it to the hydrogel network, and at the same time utilize the structural characteristics of glycyrrhizic acid to assist in the formation of the hydrogel.

[0005] The patent with the publication number CN118340934A discloses a preparation method and application of a self-adhesive glycyrrhizic acid hydrogel. The raw materials of this hydrogel include glycyrrhizic acid hydrophilic or water-soluble salts and their modifiers, metal ions, aldehyde-modified polysaccharides, and acylhydrazide, hydroxylamine, or primary amine polymer derivatives. The prepared hydrogel has good biocompatibility, biological functional activity, and biodegradability, and plays a promoting role in the wound healing of biological tissues. However, glycyrrhizic acid is not used as the dominant force participating in gel formation, but mainly as an active ingredient, and at the same time, its gel forming time is very long.

[0006] The patent with the publication number CN110585122A discloses an injectable natural triterpenoid antibacterial hydrogel and a preparation method thereof. The hydrogel disclosed in this invention uses a mixture of glycyrrhizic acid and pentacyclic triterpenoid compounds or tetracyclic triterpenoid compounds as gel molecules, uses phosphate buffered saline (PBS) as the gel solvent, and is prepared by a small molecule self-assembly mechanism using the non-covalent bond forces formed between the mixtures. However, the mechanical properties of the hydrogel obtained solely by the non-covalent bonds between small molecules are poor.

[0007] The patent with the publication number 115671376A discloses an injectable self-healing glycyrrhizic acid hydrogel dressing and its preparation method and application. In this preparation method, a glycyrrhizic acid or glycyrrhizic acid derivative solution is first reacted with an oxidant to obtain an oxidation product; then the oxidation product is mixed with a chitosan derivative, and a multifunctional hydrogel is constructed by using the non-covalent bonds and covalent bonds between the two. The prepared hydrogel has the properties of injectability, self-healing, high adhesion, high mechanical strength, and significant antibacterial properties, and has the ability to promote skin wound healing. This method breaks the carbon-carbon bond at the 2,3 positions of the glycyrrhizic acid sugar ring through oxidation, forming a linear structure, completely destroying the cyclic structure of the glycosyl group, and affecting the physiological activity and metabolism of glycyrrhizic acid, a triterpenoid saponin. Research shows that the glycosyl group is an important constituent structure of saponins, and the presence of the glycosyl group is crucial for the biological activities of many saponins. (Mskhiladze L, Legault J, Lavoie S, Mshvildadze V, Kuchukhidze J, Elias R, Pichette A. Cytotoxic steroidal saponins from the flowers of Alliumleucanthum. Molecules. 2008 Nov 26;13(12):2925 - 34.).

[0008] The gel obtained by the above method has either low strength and poor toughness, or a long gelation time, or poor self-healing performance and is easily damaged when subjected to external mechanical stress, or it destroys the cyclic structure of the glycosyl group, affecting the biological activity of glycyrrhizic acid.

[0009] In view of this, it is necessary to design an improved self-healing hydrogel for skin repair and its preparation method to solve the above problems. Summary of the Invention

[0010] The object of the present invention is to provide a self-healing hydrogel for skin repair and its preparation method. In this preparation method, aprotic polar solvents (dimethyl sulfoxide, DMF, acetonitrile) are used as solvents to provide an anhydrous environment for modifying dipotassium glycyrrhizate, overcoming the defect that the grafting degree is low due to the self-assembly or hydrophobic association of dipotassium glycyrrhizate molecules in traditional aqueous solvents, which wrap the hydrophilic group carboxyl group, and significantly improving the self-healing efficiency of the dipotassium glycyrrhizate hydrogel; the hydrazide group is introduced by the method of carboxyl coupling, completely retaining the triterpene skeleton and glycosyl structure of dipotassium glycyrrhizate, avoiding the problem of weakening or loss of its biological activity due to the destruction of the dipotassium glycyrrhizate skeleton structure, and being able to maximize the anti-inflammatory, antioxidant and other biological activities of dipotassium glycyrrhizate, significantly promoting wound healing.

[0011] To achieve the above-mentioned invention object, the present invention provides a preparation method of a self-healing hydrogel for skin repair, comprising the following steps:

[0012] S1, Preparation of aldehyde-functionalized hyaluronic acid: Dissolve hyaluronic acid in PBS buffer, then add sodium periodate and react at room temperature in the dark for 4-6 h. Then add ethylene glycol and stir for 10-30 min to end the reaction. Next, dialyze, freeze, and freeze-dry the reaction solution to obtain aldehyde-functionalized hyaluronic acid solid;

[0013] S2, Preparation of hydrazide-functionalized dipotassium glycyrrhizinate: Completely dissolve dipotassium glycyrrhizinate in an aprotic polar solvent, then add N-hydroxysuccinimide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and 3,3'-dithiobis(propionyl hydrazide), adjust the solution pH to 5.0 - 6.5, and react at 25 - 35 °C for 10 - 48 h; Then, dialyze the reaction solution for 48 - 72 h, and then filter and freeze-dry to obtain hydrazide-functionalized dipotassium glycyrrhizinate;

[0014] S3, Prepare solution A with the aldehyde-functionalized hyaluronic acid solid prepared in step S1 at a concentration of 5% - 15%, prepare solution B with the hydrazide-functionalized dipotassium glycyrrhizinate prepared in step S2 at a concentration of 8% - 20%, and extrude and blend solution A and solution B through a double-barrel syringe to obtain a self-healing hydrogel for skin repair.

[0015] As a further improvement of the present invention, the aprotic polar solvent is one of dimethyl sulfoxide, DMF, and acetonitrile.

[0016] As a further improvement of the present invention, in step S2, the molar ratio of carboxyl to hydrazide in the hydrazide-functionalized dipotassium glycyrrhizinate is carboxyl / hydrazide = 3:(3 - 4).

[0017] As a further improvement of the present invention, in step S2, the mass-volume ratio of dipotassium glycyrrhizinate to the aprotic polar solvent is 1:(30 - 60).

[0018] As a further improvement of the present invention, in step S2, adjust the pH of the solution with HCl with a concentration higher than 1 mol / L or NaOH with a concentration higher than 1 mol / L.

[0019] As a further improvement of the present invention, in step S2, the molar ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide, and dipotassium glycyrrhizinate is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride:N-hydroxysuccinimide:dipotassium glycyrrhizinate = (1 - 1.4):(1 - 1.4):1.

[0020] As a further improvement of the present invention, in step S2, the preparation method of 3,3'-dithiobis(propionyl hydrazide) is as follows: hydrazine hydrate is added to absolute ethanol and stirred, the temperature is stabilized at 30 °C, and then dimethyl dithiodipropionate is added and stirred rapidly for reaction for 10-14 h. After the reaction is completed, it is filtered and washed with absolute ethanol, and finally vacuum dried for 5-10 h to obtain 3,3'-dithiobis(propionyl hydrazide).

[0021] As a further improvement of the present invention, in step S1, the oxidation degree of the aldehyde group in the aldehyde-functionalized hyaluronic acid is 0.2-0.6; the mass-volume ratio of the hyaluronic acid to the PBS buffer solution is 1:(150-250), the mass ratio of the hyaluronic acid to the sodium periodate is 1:(1-3), and the volume ratio of the ethylene glycol to the PBS buffer solution is 1:(150-250).

[0022] In the self-healing hydrogel prepared by the foregoing preparation method, dipotassium glycyrrhizinate still retains its original triterpene skeleton and glycosyl structure, making the hydrogel have pH responsiveness.

[0023] As a further improvement of the present invention, when the pH value is 5.4-6.8, the gel time of the self-healing hydrogel is 10-200 s; when the pH value is 7.0-7.4, the gel time of the self-healing hydrogel is 300-3600 s.

[0024] The self-healing hydrogel prepared by the present invention can not only be used for wound repair, but also for anti-allergic repair of intact skin.

[0025] The beneficial effects of the present invention are as follows:

[0026] 1. The present invention provides a preparation method of a self-healing hydrogel for skin repair. By using aprotic polar solvents (dimethyl sulfoxide, DMF, acetonitrile) as solvents, an anhydrous environment is provided to modify dipotassium glycyrrhizinate. The solvent acts on the dipotassium glycyrrhizinate molecules to produce a solvation effect, reducing the activation energy of the reaction, thereby accelerating the reaction rate and increasing the substitution degree of the dipotassium glycyrrhizinate molecules grafted with acyl hydrazide groups. It overcomes the defect that the dipotassium glycyrrhizinate molecules wrap the hydrophilic group carboxyl due to self-assembly or hydrophobic association in traditional aqueous solvents, resulting in low grafting degree, and significantly improves the self-healing efficiency of the dipotassium glycyrrhizinate hydrogel.

[0027] 2. The present invention modifies dipotassium glycyrrhizinate by introducing a hydrazide group through a carboxyl coupling method, completely retaining the triterpene skeleton and glycosyl structure of dipotassium glycyrrhizinate. Without destroying the main skeleton structure of dipotassium glycyrrhizinate, it can participate in chemical crosslinking to form a gel, avoiding the problem of weakening or loss of biological activity caused by the destruction of the skeleton structure of dipotassium glycyrrhizinate (in the prior art, the carbon-carbon bond at the 2,3 positions of the glycyrrhizic acid sugar ring is broken by an oxidation method, completely destroying the cyclic structure of the glycosyl group), and can maximize the anti-inflammatory, antioxidant and other biological activities of dipotassium glycyrrhizinate, significantly promoting wound healing.

[0028] 3. The polymer skeleton of the self-healing hydrogel of the present invention is high-oxidation-degree high-molecular-weight hyaluronic acid, and the crosslinking agent is dipotassium glycyrrhizinate modified with 3,3'-dithiobis(propionyl hydrazide) (DTP). The modified dipotassium glycyrrhizinate not only participates in chemical crosslinking but also exerts its pharmacological activity as a drug. At the same time, the skeleton structure of dipotassium glycyrrhizinate (one molecule of hydrophobic glycyrrhetinic acid and two molecules of hydrophilic glucuronic acid) is not changed during the reaction process, so it still has a certain pH responsiveness, and the formation rate of the gel can be regulated by controlling the pH of the solution, realizing the improvement of the self-healing efficiency and mechanical properties of the hydrogel.

[0029] 4. The present invention forms a dynamic acylhydrazone bond between an aldehyde group and a hydrazide as the main force, and the hydrophilic-hydrophobic interaction (dipotassium glycyrrhizinate is composed of one molecule of hydrophobic glycyrrhetinic acid and two molecules of hydrophilic glucuronic acid) as the auxiliary force, significantly shortening the gel time and improving the mechanical properties of the hydrogel. Since the formed bond is a dynamic bond, the obtained hydrogel has good self-healing properties, and during the wound repair process, dipotassium glycyrrhizinate can exert its drug activity as the dynamic bond dissociates.

[0030] 5. The present invention mixes by a dual-tube injection method, which is simple to operate and the solution is evenly mixed; crosslinking and forming are carried out by dual-tube injection using the formation of dynamic acylhydrazone bonds, significantly shortening the gel time and self-healing time, and having excellent mechanical properties. The gel also has good biocompatibility, and the degradation products will not cause harm to the human body. At the same time, due to the drug activity of dipotassium glycyrrhizinate, the hydrogel has a certain antibacterial function, which is beneficial to wound repair.

[0031] 6. The present invention uses natural high-molecular-weight hyaluronic acid as the skeleton to improve the mechanical properties of the hydrogel, and selects small-molecule dipotassium glycyrrhizinate with more easily movable molecular chains to increase the collision probability between aldehyde groups and hydrazide groups, enabling the hydrogel to gel and self-heal quickly while having excellent mechanical properties; when applied to wound repair, the moist environment, self-healing properties and drug activity of dipotassium glycyrrhizinate of the hydrogel are beneficial for the wound to resist external damage and thus accelerate wound repair. At the same time, the natural polymer has good biocompatibility and degradability and will not cause harm to organisms. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a schematic diagram of the synthesis process of aldehyde - modified hyaluronic acid in the present invention.

[0033] Figure 2 This is a schematic diagram of the synthesis process of hydrazide - modified dipotassium glycyrrhizinate in the present invention.

[0034] Figure 3 This is the gel process diagram in the present invention.

[0035] Figure 4 This is the NMR spectra of DTP, dipotassium glycyrrhizinate modified by DTP (GD), and dipotassium glycyrrhizinate (G) in Example 1 of the present invention.

[0036] Figure 5 This is the IR spectra of DTP, dipotassium glycyrrhizinate modified by DTP (GD), and dipotassium glycyrrhizinate (G) in Example 1 of the present invention.

[0037] Figure 6 This is the rheological diagram of the gelation time of the hydrogel in Example 1 of the present invention.

[0038] Figure 7 This is the curve graph showing the change of the viscosity of the hydrogel prepared in Example 1 of the present invention with the shear rate.

[0039] Figure 8 This is the frequency - sweep graph of the hydrogel prepared in Example 1 of the present invention.

[0040] Figure 9 This is the rheological property - oscillatory strain graph of the hydrogel prepared in Example 1 of the present invention.

[0041] Figure 10 This is the rheological property - alternating strain graph of the hydrogel prepared in Example 1 of the present invention.

[0042] Figure 11 This is the self - healing process graph of the hydrogel in Example 1 of the present invention.

[0043] Figure 12 This is the gel morphology of the hydrogel prepared in the present invention at different pH values. DETAILED DESCRIPTION OF THE INVENTION

[0044] In order to make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0045] Here, it should also be noted that in order to avoid obscuring the present invention with unnecessary details, only the structures and / or processing steps closely related to the solution of the present invention are shown in the drawings, while other details less relevant to the present invention are omitted.

[0046] In addition, it should be noted that the term "comprising", "including" or any other variant thereof is intended to cover non-exclusive inclusion, such that a process, method, article or apparatus comprising a series of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article or apparatus.

[0047] In the prior art, dipotassium glycyrrhizinate self-healing hydrogel is prepared by an oxidation method. This method breaks the carbon-carbon bond at the 2,3 positions of the sugar ring of dipotassium glycyrrhizinate, completely destroying the cyclic structure of the glycosyl group and affecting the biological activity of dipotassium glycyrrhizinate.

[0048] In view of the above problems, the present invention provides a method for preparing a self-healing hydrogel for skin repair, comprising the following steps:

[0049] S1, Preparation of aldehyde-functionalized hyaluronic acid: Dissolve hyaluronic acid in PBS buffer, then add sodium periodate and react at room temperature in the dark for 4-6 h, then add ethylene glycol and stir for 10-30 min to end the reaction. Then, dialyze, freeze and freeze-dry the reaction solution to obtain aldehyde-functionalized hyaluronic acid solid;

[0050] Among them, the oxidation degree of aldehyde groups in aldehyde-functionalized hyaluronic acid is 0.2-0.6.

[0051] The mass-volume ratio of hyaluronic acid to PBS buffer is 1:(150-250), the mass ratio of hyaluronic acid to sodium periodate is 1:(1-3), and the volume ratio of ethylene glycol to PBS buffer is 1:(150-250).

[0052] S2, Preparation of hydrazide-functionalized dipotassium glycyrrhizinate: Completely dissolve dipotassium glycyrrhizinate in an aprotic polar solvent, then add N-hydroxysuccinimide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 3,3'-dithiobis(propionyl hydrazide), adjust the solution pH to 5.0-6.5, and react at 25-35 °C for 10-48 h; then, dialyze the reaction solution for 48-72 h, and then filter and freeze-dry to obtain hydrazide-functionalized dipotassium glycyrrhizinate;

[0053] Among them, the aprotic polar solvent is one of dimethyl sulfoxide, DMF, and acetonitrile.

[0054] The mass-volume ratio of dipotassium glycyrrhizinate to the aprotic polar solvent is 1:(30-60).

[0055] Among them, the molar ratio of carboxyl groups to hydrazide groups in hydrazide-functionalized dipotassium glycyrrhizinate is carboxyl / hydrazide = 3:(3-4).

[0056] Among them, the molar ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide, and dipotassium glycyrrhizinate is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride:N-hydroxysuccinimide:dipotassium glycyrrhizinate = (1 to 1.4):(1 to 1.4):1.

[0057] In step S2, the pH of the solution is adjusted using HCl with a concentration higher than 1 mol / L or NaOH with a concentration higher than 1 mol / L.

[0058] In step S2, the preparation method of 3,3'-dithiobis(propionylhydrazide) is as follows: Hydrazine hydrate is added to absolute ethanol and stirred, the temperature is stabilized at 30 °C, and then dimethyl dithiodipropionate is added and stirred rapidly for reaction for 10 to 14 h. After the reaction is completed, it is filtered and washed with absolute ethanol, and finally vacuum dried for 5 to 10 h to obtain 3,3'-dithiobis(propionylhydrazide).

[0059] S3. The aldehyde-functionalized hyaluronic acid solid prepared in step S1 is formulated into solution A with a concentration of 5% to 15%, and the hydrazide-functionalized dipotassium glycyrrhizinate prepared in step S2 is formulated into solution B with a concentration of 8% to 20%. Solution A and solution B are extruded and blended through a double-barreled syringe to obtain a self-healing hydrogel for skin repair.

[0060] In the self-healing hydrogel prepared by the present invention, the modified dipotassium glycyrrhizinate still maintains its original triterpene skeleton and glycosyl structure, making the hydrogel have pH responsiveness. When the pH value is 5.4 to 6.8, the gelation time of the self-healing hydrogel is 10 to 200 s; when the pH value is 7.0 to 7.4, the gelation time of the self-healing hydrogel is 300 to 3600 s. That is, the gelation time and mechanical strength of the hydrogel can be adjusted by regulating the pH, which is beneficial to the dynamic monitoring of wound infection.

[0061] The present invention selects aprotic polar solvents (dimethyl sulfoxide, DMF, acetonitrile) as solvents to provide an anhydrous environment for modifying dipotassium glycyrrhizinate, overcoming the defect that the grafting degree is low due to the self-assembly or hydrophobic association of dipotassium glycyrrhizinate molecules in traditional aqueous solvents to wrap the hydrophilic group carboxyl. In the present invention, while not destroying the main body skeleton structure of dipotassium glycyrrhizinate, it is modified so that it can participate in chemical cross-linking to form a gel. At the same time, because the formed bonds are dynamic bonds, the hydrogel has good self-healing performance. During the wound repair process, as the dynamic bonds dissociate, dipotassium glycyrrhizinate can exert its drug activity.

[0062] In the self-healing hydrogel prepared by the present invention, the polymer backbone is high-molecular-weight hyaluronic acid with a high degree of oxidation, and the cross-linking agent is dipotassium glycyrrhizinate modified with 3,3'-dithiobis(propionyl hydrazide) (DTP). The modified dipotassium glycyrrhizinate not only participates in chemical cross-linking but also exerts its pharmacological activity as a drug. At the same time, since the present invention does not change the backbone structure of dipotassium glycyrrhizinate (one molecule of hydrophobic glycyrrhetinic acid and two molecules of hydrophilic glucuronic acid), the prepared hydrogel still has a certain pH responsiveness, enabling the control of the gel formation rate by controlling the pH of the solution, and improving the self-healing efficiency and mechanical properties of the hydrogel.

[0063] In the present invention, cross-linking and forming are carried out by double-tube injection using the formation of dynamic hydrazone bonds, which significantly shortens the gel time and self-healing time and has excellent mechanical properties. The gel also has good biocompatibility, and the degradation products will not cause harm to the human body. At the same time, due to the drug activity of dipotassium glycyrrhizinate, the hydrogel has a certain antibacterial function, which is beneficial to wound repair.

[0064] The following describes the preparation method of the self-healing hydrogel for skin repair provided by the present invention in combination with specific examples. Unless otherwise specified, the raw materials and reagents in the examples of this application are purchased through commercial channels.

[0065] Example 1

[0066] This example provides a preparation method of a self-healing hydrogel for skin repair, including the following steps:

[0067] S1, Preparation of aldehyde-functionalized hyaluronic acid (AHA):

[0068] Weigh 2 g of hyaluronic acid (HA) powder and put it into a 250 ml three-necked flask. Add 200 ml of PBS 5.0 buffer solution, stir at room temperature until completely dissolved and stabilize the temperature at 25 °C. Then add 2 g of sodium periodate (NaIO 4 ) and react in the dark for 5 h. Finally, add 2 mL of ethylene glycol and stir for 15 min to end the reaction. Then dialyze the obtained reaction solution for 48 h, and finally freeze and freeze-dry the dialysate to obtain solid aldehyde-functionalized hyaluronic acid.

[0069] Among them, the synthesis process of AHA is as Figure 1 shown, the aldehyde oxidation degree is 0.5, and the relative molecular weight of hyaluronic acid is not less than 8 million;

[0070] S2, Preparation of hydrazide-functionalized dipotassium glycyrrhizinate (anhydrous condition);

[0071] Take 2 g of dipotassium glycyrrhizinate and completely dissolve it in 100 ml of aprotic polar solvent dimethyl sulfoxide (DMSO). Then add 0.46 g of N-hydroxysuccinimide (NHS), 0.77 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), and 0.8 g of 3,3'-dithiobis(propionyl hydrazide) (DTP). The pH of the solution is 5.6, and the reaction is carried out at 30 °C for 24 h. Pour the reacted liquid into a dialysis bag with a cut-off molecular weight of 500 and dialyze for 48 h. Finally, filter the dialysate and freeze-dry it to obtain hydrazide-modified dipotassium glycyrrhizinate (GD).

[0072] The synthesis process of hydrazide-modified dipotassium glycyrrhizinate is as Figure 2 shown.

[0073] Among them, the molar ratio of carboxyl group to hydrazide in hydrazide-modified dipotassium glycyrrhizinate is carboxyl / hydrazide = 3:3.2.

[0074] Among them, the synthesis method of 3,3'-dithiobis(propionyl hydrazide) (DTP) is as follows:

[0075] Measure 300 mL of absolute ethanol into a 500 mL three-necked flask, add 18 mL of 85% hydrazine hydrate, stabilize the temperature at 30 °C, add 13.44 mL of dimethyl dithiodipropionate and stir rapidly for 12 h. After the reaction, filter and wash with 2000 - 3000 mL of absolute ethanol, and finally vacuum dry for 8 h to obtain 3,3'-dithiobis(propionyl hydrazide) (DTP) solid, and its chemical structural formula is as follows:

[0076]

[0077] S3. Prepare solution A with the aldehyde-functionalized hyaluronic acid prepared in step S1 at a mass percentage concentration of 8% (m / v), and prepare solution B with the hydrazide-modified dipotassium glycyrrhizinate prepared in step S2 at a mass percentage concentration of 15% (m / v). Extrude and blend solution A and solution B (volume ratio 1:1) through a double-tube syringe to obtain a self-healing hydrogel (AG) for skin repair. The gel process diagram is as Figure 3 shown.

[0078] Please refer to Figures 4 to 5 shown Figure 4 and Figure 5 are the NMR spectra and IR spectra of DTP, DTP-modified dipotassium glycyrrhizinate (GD), and dipotassium glycyrrhizinate (G) in Example 1, respectively.

[0079] In the NMR spectrum, the two strong peaks around 2.6 and 2.9 on GD belong to the hydrogen on the methylene group of DTP; in the IR spectrum, the peak at 1640 cm -1 on GD belongs to the carbonyl peak connected to the N atom, and the peak at 3306 cm -1The peaks at [[]] become sharper (belonging to the stretching vibration of NH), all indicating the successful synthesis of dipotassium glycyrrhizinate hydrazide.

[0080] Please refer to Figure 6 As shown, it is the rheological diagram of the gel time of the hydrogel in Example 1, where G’ is the storage modulus and G” is the loss modulus. When G’ < G”, it indicates a sol state at this time; when t = 46 s, G’ = G”, and this is the gel point; as time increases, G’ > G” and gradually stabilizes, indicating that the formed gel also gradually stabilizes.

[0081] Continue to test the rheological properties - shear thinning and rheological properties - oscillatory strain of the hydrogel prepared in Example 1, and the test results are as Figures 7 to 9 shown.

[0082] From Figure 7 it can be seen that the viscosity of the prepared hydrogel (AG hydrogel) decreases with the increase of the shear rate, indicating that the formed gel has injectability.

[0083] From Figure 8 it can be seen that the AG hydrogel can still maintain the gel state (G’ > G”) under oscillations at different frequencies, indicating that the formed gel has good stability.

[0084] From Figure 9 it can be seen that the AG hydrogel can withstand a strain of 500%, indicating its good mechanical properties.

[0085] Test the rheological property - alternating strain of the hydrogel:

[0086] Dynamic self - healing test method:

[0087] Perform amplitude scanning at 1 Hz within the strain range of 0.1 - 1000% to determine the fracture strain of the AG hydrogel. Subsequently, an alternating step - strain test was carried out to measure the modulus at a constant frequency of 1 Hz, with the alternating strain between 1% and 700%, and each step of the test was carried out for 60 seconds. The test results are as Figure 10 shown.

[0088] It can be known that the AG hydrogel has certain self - healing properties. Alternating large and small strains, with G’ > G” in the small - strain state being the gel state and G’ < G” in the large - strain state being the sol state, it can recover to the gel state (storage modulus G’ greater than loss modulus G”) within 60 s after the gel structure is damaged.

[0089] Figure 11 It is the diagram of the self - healing process of the hydrogel in Example 1.

[0090] First, cut the gel into two parts, then stick them together, and stretch after 20 min. The gel does not break at the cut, indicating that the gel can self - heal within 20 min and has excellent self - healing performance.

[0091] Comparative Examples 1-5

[0092] Comparative Examples 1-5 provided a preparation method of hydrogel. Compared with Example 1, the difference was that in step S2, ultrapure water was used to replace dimethyl sulfoxide, that is, dipotassium glycyrrhizinate was modified under aqueous conditions, or the feeding ratio was changed, specifically, the molar ratio of carboxyl group to hydrazide in hydrazide-modified dipotassium glycyrrhizinate was changed; the rest was substantially the same as that of Example 1 and would not be elaborated here. The dialysis phenomenon and gel situation of the product obtained in step S2 were counted, and the results are shown in the following table.

[0093]

[0094] It can be known from the experiment that gels could not be formed in Comparative Examples 1-5.

[0095] Comparative Examples 6-8

[0096] Comparative Examples 6-8 provided a preparation method of hydrogel. Compared with Example 1, the difference was that in step S2, ultrapure water was used to replace dimethyl sulfoxide, that is, dipotassium glycyrrhizinate was modified under aqueous conditions, or the reaction pH was changed; the rest was substantially the same as that of Example 1 and would not be elaborated here.

[0097]

[0098] It can be seen from the above table that under aqueous conditions, hydrazide-modified dipotassium glycyrrhizinate could not be successfully synthesized under acidic, neutral or alkaline conditions.

[0099] Examples 2-3 and Comparative Examples 9-10

[0100] Examples 2-3 and Comparative Examples 9-10 provided a preparation method of hydrogel. Compared with Example 1, the difference was that in step S2, the reaction pH was changed; the rest was substantially the same as that of Example 1 and would not be elaborated here.

[0101]

[0102] It can be seen from the above table that when the reaction pH exceeded the range of 5.0-6.5, hydrazide-modified dipotassium glycyrrhizinate could not be successfully synthesized.

[0103] Examples 4-5 and Comparative Examples 11-13

[0104] Examples 4-5 and Comparative Examples 11-13 provided a preparation method of hydrogel. Compared with Example 1, the difference was that in step S2, the feeding ratio was changed, specifically, the molar ratio of carboxyl group to hydrazide in hydrazide-modified dipotassium glycyrrhizinate was changed; the rest was substantially the same as that of Example 1 and would not be elaborated here.

[0105]

[0106] As can be seen from the above table, when the molar ratio of carboxyl group to hydrazide in dipotassium glycyrrhizinate hydrazide exceeds the range of -COOH / -NH 2 -NH- = 3:(3 - 4), the synthesis of dipotassium glycyrrhizinate hydrazide cannot be successful.

[0107] Examples 6 - 7

[0108] Examples 6 - 7 provide a method for preparing a hydrogel. Compared with Example 1, the difference lies in that in step S2, the aprotic polar solvent dimethyl sulfoxide is replaced by DMF or acetonitrile; the rest is substantially the same as in Example 1 and will not be elaborated here.

[0109] It can be known from the experiment that replacing dimethyl sulfoxide with DMF or acetonitrile can also successfully synthesize dipotassium glycyrrhizinate hydrazide.

[0110] The products prepared in Comparative Examples 1 - 5 cannot form gels, so the data of mechanical strength, gel time or self - healing time cannot be measured.

[0111] The hydrogels prepared in Examples 1 - 7 were tested for mechanical strength, gel time and self - healing performance, and the results are shown in the following table.

[0112]

[0113]

[0114] As can be seen from Table 1, the hydrogel of Example 1 has the highest mechanical strength and gels the fastest, while the gels formed in Examples 2 - 5 have lower mechanical strength and longer gel times. However, due to their lower mechanical strength and softer gels, they heal faster, but generally the healing time is not much different from that of Example 1.

[0115] Quantitative self - healing test:

[0116] The self - healing efficiency (HE) is evaluated by tensile measurement. The hydrogel (60 mm×15 mm×5 mm) is cut into two pieces and spliced together at room temperature for self - healing for 30 minutes. The original and healed hydrogels are stretched at a speed of 0.8 mm / min by a universal testing machine.

[0117] The self - healing efficiency of the hydrogel is determined according to the following formula: H E (%) = S H / S 0 x100%, where S H and S 0 are the fracture stresses of the healed hydrogel and the original hydrogel, respectively.

[0118] Examples 8-9

[0119] Examples 8-9 provide a method for preparing a hydrogel. Compared with Example 1, the difference lies in that in step S3, the mass ratio of AHA / GD is changed, that is, the mass fraction ratio of AHA / GD; the rest is substantially the same as that in Example 1 and will not be elaborated here. The test results of the self-healing performance of Examples 1, 8-9 are shown in the following table.

[0120]

[0121] As can be seen from the above table, after 30 minutes, the self-healing efficiency of Examples 1, 8-9 all reached 95% and above.

[0122] Animal experiment wound contraction rate test:

[0123] The effect of the hydrogel on wound healing was evaluated through a full-thickness skin defect model of rats. All procedures followed the guidelines for the use and management of laboratory animals issued by the National Institutes of Health. SD rats (280-300 g, 7-8 weeks) had free access to sterile water and standard food. Before the surgery, the rats were anesthetized with 10% chloral hydrate. After shaving the back of the rats in the prone position, the back was disinfected with iodophor. Subsequently, two full-thickness defect circular wounds with a diameter of 15 mm were created in the dorsal area using a biopsy punch. All rats were randomly divided into three groups, namely the gauze group, the 3MINu-Dermrm hydrocolloid dressing (abbreviated as 3M hydrocolloid) group, and the AG hydrogel group.

[0124] The wound regeneration process was evaluated by monitoring the wound area at specific times (0, 3, 7, 14 days), and the wound area was analyzed using ImageJ software.

[0125] The wound closure rate was determined according to the following formula: Wound healing efficiency (%) = (A 0 -A) / A 0 ×100%, where A 0 and A are the wound areas on day 0 and time point t, respectively.

[0126] The gel AG prepared with the mass ratio of AHA / GD of 8:15 in Example 1 was selected for wound repair. The test results are shown in the following table.

[0127] Time / (DAY) 3 7 14 Gauze group 42％ 76％ 90％ 3M hydrocolloid 48％ 80％ 92％ AG hydrogel group 59％ 85％ 97％

[0128] As can be seen from the above table, the wound healing efficiency of the hydrogel prepared by the present invention is higher than that of the gauze group and the 3M hydrocolloid group. On the 3rd day of the experiment, the wound healing rate of the AG gel group was significantly higher than that of the gauze group by 42% or the 3M hydrocolloid group; by the 14th day, the wound healing rate of the AG gel group reached 97%, while that of the gauze group was 90%, indicating that the ability of the AG hydrogel to promote wound healing was significantly higher than that of the other two groups. The same trend was observed on the 7th day, indicating that the AG hydrogel could significantly promote wound healing.

[0129] pH responsiveness test of the gel:

[0130] In step S3, deionized water, sodium hydroxide or PBS 7.4 was used as the solvent, and the pH value during gel formation was changed to test the gel forming situation at different pH values; the rest was roughly the same as in Example 1 and will not be elaborated here. The gel situations under different pH value conditions are shown in the following table and Figure 12 as shown.

[0131] pH value Gel time Modulus Equilibrium time 5.4～6.8 10～200s 1000 - 3000 Pa 10 min 7.0～7.4 300～3600s 600 - 1000 Pa 35 min

[0132] The equilibrium time refers to the time from double-tube extrusion to the formation of a stable gel state.

[0133] From the above table and Figure 12 it can be seen that the decrease in pH value is beneficial to the self-assembly of dipotassium glycyrrhizinate and will accelerate the formation of the gel. At the same time, the modulus of the formed gel will also change with the change of pH value.

[0134] In summary, for the preparation method of the self-healing hydrogel for skin repair provided by the present invention, a non-protic polar solvent is selected as the solvent to provide an anhydrous environment, and dipotassium glycyrrhizinate is modified, overcoming the defect that the grafting degree is low due to the self-assembly or hydrophobic association of dipotassium glycyrrhizinate molecules in traditional aqueous solvents to wrap the hydrophilic group carboxyl. In the present invention, while not destroying the main body skeleton structure of dipotassium glycyrrhizinate, it is modified so that it can participate in chemical cross-linking to form a gel. At the same time, since the formed bonds are dynamic bonds, the hydrogel has good self-healing performance. During the wound repair process, with the dissociation of the dynamic bonds, dipotassium glycyrrhizinate can exert its drug activity.

[0135] At the same time, the present invention does not change the skeleton structure of dipotassium glycyrrhizinate (one molecule of hydrophobic glycyrrhetinic acid and two molecules of hydrophilic glucuronic acid), so the prepared hydrogel still has a certain pH responsiveness, realizing that the forming speed of the gel can be regulated by controlling the pH of the solution, and improving the self-healing efficiency and mechanical properties of the hydrogel.

[0136] The above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for preparing a self-healing hydrogel for skin repair, characterized in that: The following steps are involved: S1, preparation of aldehyde-modified hyaluronic acid; dissolving hyaluronic acid in PBS buffer, then adding sodium periodate to react for 4 to 6 hours at room temperature in the dark, then adding ethylene glycol and stirring for 10 to 30 minutes to terminate the reaction, then dialyzing, freezing, and freeze-drying the reaction solution to obtain aldehyde-modified hyaluronic acid solid; S2, preparation of hydrazide dipotassium glycyrrhizinate; dipotassium glycyrrhizinate is completely dissolved in a non-protonic polar solvent, followed by addition of N-hydroxysuccinimide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and 3,3'-dithiobis(propionyl hydrazide), the pH of the solution is controlled to be 5.0-6.5, and the reaction is carried out at 25-35° C. for 10-48 hours; then, the reaction solution is dialyzed for 48-72 hours, filtered, and freeze-dried to obtain hydrazide dipotassium glycyrrhizinate; S3, preparing the aldehyde-modified hyaluronic acid solid prepared in step S1 into a solution A with a concentration of 5% to 15%, preparing the hydrazide-modified dipotassium glycyrrhizinate prepared in step S2 into a solution B with a concentration of 8% to 20%, and extruding and blending solution A and solution B through a double-barreled syringe to obtain a self-healing hydrogel for skin repair.

2. The method for preparing a self-healing hydrogel for skin repair according to claim 1, characterized in that: The aprotic polar solvent is one of dimethyl sulfoxide, DMF and acetonitrile.

3. The method for preparing the self-healing hydrogel for skin repair according to claim 1, characterized in that: In step S2, the molar ratio of carboxyl group to hydrazide in the hydrazide dipotassium glycyrrhizinate is carboxyl group / hydrazide=3:(3-4).

4. The method for preparing the self-healing hydrogel for skin repair according to claim 1, characterized in that: In step S2, the mass volume ratio of the dipotassium glycyrrhizinate to the aprotic polar solvent is 1:(30-60).

5. The method for preparing the self-healing hydrogel for skin repair according to claim 1, characterized in that: In step S2, the pH of the solution is adjusted using HCl with a concentration higher than 1 mol / L or NaOH with a concentration higher than 1 mol / L.

6. The method for preparing the self-healing hydrogel for skin repair according to claim 1, characterized in that: In step S2, the molar ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide and dipotassium glycyrrhizinate is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride:N-hydroxysuccinimide:dipotassium glycyrrhizinate=(1-1.4):(1-1.4):

1.

7. The method for preparing the self-healing hydrogel for skin repair according to claim 1, characterized in that: In step S2, the preparation method of 3,3'-dithiobis(propionyl hydrazide) is as follows: hydrazine hydrate is added to anhydrous ethanol and stirred, the temperature is stabilized at 30°C, dimethyl dithiodipropionate is added and rapidly stirred to react for 10 to 14 hours, after the reaction is completed, the mixture is filtered and washed with anhydrous ethanol, and finally vacuum dried for 5 to 10 hours to obtain 3,3'-dithiobis(propionyl hydrazide).

8. The method for preparing the self-healing hydrogel for skin repair according to claim 1, characterized in that: In step S1, the mass volume ratio of the hyaluronic acid to the PBS buffer is 1:(150-250), the mass ratio of the hyaluronic acid to the sodium periodate is 1:(1-3), and the volume ratio of the ethylene glycol to the PBS buffer is 1:(150-250).

9. A self-healing hydrogel for skin repair, characterized in that: The self-healing hydrogel is prepared according to the preparation method described in any one of claims 1 to 8; the self-healing hydrogel has a triterpene skeleton and a glycosyl structure of dipotassium glycyrrhizinate and is pH responsive.

10. The self-healing hydrogel for skin repair according to claim 9, characterized in that: When the pH value is 5.4 to 6.8, the gel time of the self-healing hydrogel is 10 to 200 s; when the pH value is 7.0 to 7.4, the gel time of the self-healing hydrogel is 300 to 3600 s.

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