An injectable hydrogel, its preparation method, and its application in skin damage repair.

Abstract

This invention discloses an injectable hydrogel, its preparation method, and its application in skin injury repair, belonging to the technical field of hydrogel preparation and application. The preparation method includes the following steps: 1. Preparation of tea protein oligopeptides (TPP); 2. Preparation of hyaluronic acid-grafted tea protein oligopeptides (TPP-HA); 3. Preparation of APTES-TPP-HA hydrogel. Beneficial effects: This invention combines organosilicon materials with TPP and HA through chemical grafting to prepare a novel hydrogel with excellent bioactivity and physical properties. The resulting hydrogel has certain application potential in skin wound healing, possessing both good biocompatibility and specific bioactivity. It provides a suitable microenvironment in the wound environment, promoting cell adhesion, migration, and proliferation, thereby potentially accelerating the wound healing process.

Description

Technical Field

[0001] This invention belongs to the field of hydrogel preparation and application technology, specifically relating to an injectable hydrogel, its preparation method, and its application in skin damage repair. Background Technology

[0002] Tea protein is a plant-based protein, mainly found in tea leaves. Over 80% is insoluble, with only 1-2% being water-soluble. Tea protein is primarily extracted from tea leaves through water or enzymatic hydrolysis. It possesses high nutritional value and various bioactivities, with an amino acid composition higher than soybeans and only slightly lower than cow's milk. It exhibits a prominent free radical scavenging effect. Further hydrolysis yields polypeptides with specific bioactivities. Tea protein-derived oligopeptides (TPPs) are a class of bioactive peptides obtained from tea residue protein through enzymatic hydrolysis. These peptides have a concentrated length of 6-10 amino acids and are abundant, highly active, and safe and non-toxic. Studies have shown that small-molecule bioactive peptides possess higher activity than proteins, and their lower molecular weight makes them easier for the human body to absorb and utilize. As a naturally sourced bioactive substance, TPP has been proven to have significant antihypertensive, antioxidant, and antibacterial activities, demonstrating broad application potential. These properties make TPP an ideal choice for preparing biomedical materials. At present, the resources of tea protein have not been fully utilized, and research on the development of functional TPP is still in its early stages. As a potential active substance for repairing cellular oxidative damage, the related repair mechanism of TPP needs to be further elucidated.

[0003] Data shows that in developed countries, approximately 60% of the population experiences at least one skin inflammatory reaction annually caused by environmental pollutants. In Europe alone, the number of people seeking medical attention for skin diseases due to air pollution exceeds one million each year. Research data in my country shows that about 75% of urban residents have significantly elevated levels of skin oxidative stress, with 35% exhibiting marked skin barrier dysfunction. Typical skin damage is primarily induced by external environmental stressors such as ultraviolet radiation, air pollutants, and pathogenic microorganisms. Meanwhile, my country sees approximately 5 million new cases of skin barrier dysfunction each year, bringing the cumulative total to over 30 million, with 25%–40% of these cases accompanied by complications such as skin sensitivity and inflammation. The National Health Commission of China has included skin barrier function repair in its key areas of chronic skin disease prevention and control. Therefore, there is a significant market demand for safe and effective skin repair products.

[0004] As the largest organ in the human body, the skin plays a crucial role not only in physical barrier function but also in immune defense and repair regulation through a complex network of cells and molecules. However, long-term exposure to environmental stressors such as ultraviolet radiation, pollutants, and pathogens can lead to a significant increase in oxidative stress levels. Oxidative stress refers to an imbalance between intracellular reactive oxygen species (ROS) and the antioxidant system, directly affecting cellular homeostasis and damaging skin structure and function. The main sources of ROS include electron leakage during mitochondrial oxidative phosphorylation, the catalytic action of NADPH oxidase, and oxidation reactions induced by external environmental stimuli such as ultraviolet radiation. Its chemical reactivity is extremely high, capable of directly reacting with cell membrane lipids, inducing lipid peroxidation, and resulting in impaired cell membrane integrity and fluidity. Furthermore, it can attack DNA bases through oxidation, leading to breaks and mutations in genetic material; this damage may further trigger apoptosis or necrosis.

[0005] Currently, the main treatment for mechanical skin injuries is bandages, but compared to traditional excipients, hydrogels are ideal biomaterials for wound dressings. Traditional excipients have diverse compositions, relatively simple chemical structures, and significant differences in solubility, mechanical properties, and breathability. Biocompatibility varies depending on the material, and some synthetic excipients have potential safety issues. They are mainly used in pharmaceuticals, food, and cosmetics in conventional formulations. Hydrogels, on the other hand, are high-molecular-weight polymers containing numerous hydrophilic groups and a three-dimensional network structure. They are insoluble in water but can swell, possess good elasticity and flexibility, and have some breathability. They also exhibit good biocompatibility and have unique and wide applications in biomedical fields such as tissue engineering, as well as in cosmetic moisturizing products. Hydrogels possess high water content and a porous structure, making them ideal materials for wound repair and carrying bioactive molecules. They can absorb exudate and blood, maintain moisture, and promote debridement, making them suitable for various types of wounds.

[0006] Hyaluronic acid (HA) is a natural linear polysaccharide found in human tissues, widely present in skin, synovial fluid, and cartilage. Due to its excellent moisturizing properties, superior biocompatibility, and skin-repairing functions, it has become an important ingredient in skin care products. The HA molecule contains abundant hydroxyl groups; these hydrophilic functional groups give it strong moisturizing capabilities, forming hydrogen bonds with water molecules to prevent moisture loss. However, its use alone still has certain limitations, especially in the areas of skin repair and anti-aging. HA is highly biodegradable and easily decomposes and is lost in aqueous environments, thus limiting its long-term effects. Furthermore, although it has good moisturizing properties, it lacks other bioactive ingredients to consolidate its repairing effects. Therefore, esterification is used to combine HA with TPP to construct a stable composite system, aiming to improve the problems of TPP's easy degradation and low bioavailability. The ester bond not only provides a strong chemical bond, ensuring the stability and durability of HA and TPP, but also effectively prevents their separation and dissociation in aqueous environments. HA-based hydrogels can provide cells with a microenvironment that mimics the natural extracellular matrix, which helps cells adhere, proliferate, and migrate, thereby promoting wound healing.

[0007] Secondly, the mechanical properties and stability were optimized by combining silane coupling agents. The synthesis of hydrogels using organosilicon grafted macromolecules is a mild preparation method that can be carried out at room temperature, avoiding the damage to bioactive molecules caused by harsh conditions such as high temperatures. The silane coupling agent g-aminopropyltriethoxysilane (APTES, KH550) was introduced into the hydrogel, and a tight three-dimensional network structure was constructed through the cross-linking reaction between its siloxy groups and the HA-TPP complex. This effectively improved the mechanical strength of the hydrogel and also enhanced its durability and biocompatibility in aqueous environments. Summary of the Invention

[0008] The technical problem to be solved by this invention is how to provide a method for preparing a hydrogel suitable for repairing mechanical skin damage. It mainly addresses the problem of excessive oxidative stress in skin wound healing and designs an injectable hydrogel that has both antioxidant and cell migration-promoting properties.

[0009] The present invention solves the above-mentioned technical problems through the following technical means:

[0010] The first aspect of this invention provides a method for preparing an injectable hydrogel, comprising the following steps: (1) Preparation of tea protein source oligopeptides (TPP) The tea leaves were soaked in water, filtered, and the tea residue was retained. Alkaline solution was added for further soaking, and the supernatant was retained. The pH was adjusted to acidic, the protein precipitate was collected, and dried to obtain crude tea protein extract. The crude extract of tea protein was dispersed in water, the pH was adjusted to alkaline, alkaline protease was added for enzymatic hydrolysis, enzyme inactivation was performed, centrifugation was carried out, ultrafiltration was performed, and desalting was performed to obtain tea protein-derived oligopeptides (TPP). (2) Preparation of hyaluronic acid grafted tea protein oligopeptide (TPP-HA) Hyaluronic acid (HA) is dissolved in formamide to obtain an HA solution; Mix the TPP, water, DMAP and EDC obtained in (1) to obtain a TPP mixed solution; The HA solution was added dropwise to the TPP mixed solution, mixed well, and purified to obtain TPP-HA; (3) Preparation of APTES-TPP-HA hydrogel The TPP-HA obtained in (2) is mixed with water to obtain a TPP-HA solution. EDC, NHS, MES, APTES, TEOS and PDMS are added and mixed well to obtain injectable APTES-TPP-HA hydrogel.

[0011] DMAP: N,N-dimethyl-4-pyridineamine; EDC: 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride NHS: N-hydroxysuccinimide MES: 2-Modolinoethanesulfonic acid APTES: γ-aminopropyltriethoxysilane TEOS: Tetraethoxysilane PDMS: Polydimethylsiloxane Preferably, in step (1), the ratio of tea leaves to water is 1g:30~50mL; more preferably, it is 1g:40mL.

[0012] Preferably, in step (1), the tea leaves include, but are not limited to, any one or more of the following: Huangda tea, Huangshan Maofeng tea, Liuan Guapian tea, Anji white tea, Taiping Houkui tea, and Qimen black tea.

[0013] Preferably, in step (1), the extraction specifically involves adding tea leaves to water and heating it to boiling for 10-30 minutes; more preferably, it is 20 minutes.

[0014] Preferably, in step (1), the tea residue is further crushed and sieved, and the sieving is through a 60-100 mesh sieve; more preferably, it is through an 80 mesh sieve.

[0015] Preferably, in step (1), the ratio of tea residue to alkaline solution is 1g:15~35mL; more preferably, it is 1g:25mL. More preferably, the alkaline solution is a 0.3 mol / L NaOH solution.

[0016] Preferably, in step (1), the alkaline extraction specifically involves adding tea residue powder to an alkaline solution, mixing, and then sonicating at 40-60°C for 20-40 minutes. More preferably, it is 50°C for 30 minutes.

[0017] Preferably, in step (1), adjusting the pH to acidity specifically means adjusting the pH to 3.5~5.5; more preferably 4.5.

[0018] Preferably, in step (1), the crude tea protein extract is dispersed in water to obtain a tea protein solution with a concentration of 1-3% (w / v). More preferably, it is 2% (w / v).

[0019] Preferably, in step (1), adjusting the pH to alkaline means adjusting the pH to 9-11; more preferably 10.

[0020] Preferably, in step (1), the enzymatic hydrolysis specifically involves adding alkaline protease and hydrolyzing for 2-4 hours, during which the temperature is maintained at 40-60°C, and adding alkaline solution to maintain the pH at 9-11. More preferably, it involves 3 hours, 50°C, and pH at 10.

[0021] Preferably, in step (1), the enzyme inactivation is specifically achieved by heating to 80~100℃ for 5~20 min. More preferably, it is achieved at 90℃ for 10 min.

[0022] Preferably, in step (1), the centrifugation is performed at 5000~7000 r / min for 10~30 min. More preferably, it is performed at 6000 r / min for 20 min.

[0023] Preferably, in step (1), the ultrafiltration specifically involves passing the material through ultrafiltration membranes of 0.2 μm, 10 kDa, 5 kDa, 3 kDa, and 1 kDa in sequence.

[0024] Preferably, in step (1), the desalination specifically involves passing the solution through a 150 Da ultrafiltration membrane, retaining the unfiltered portion. (The ultrafiltered liquid is a solution containing salts, and the unfiltered portion is the desired sample.) Preferably, in step (1), the molecular weight of the components in the tea protein source oligopeptide (TPP) is 150 Da ~ 1 kDa.

[0025] Preferably, in step (2), the ratio of hyaluronic acid (HA) to formamide is (80~120) mg: (7~15) mL; more preferably, it is 100 mg: 10 mL.

[0026] Preferably, in step (2), the ratio of the amount of TPP, water, DMAP and EDC is (45~55) mg: (3~8) mL: (6~12) mg: (55~65) mg; more preferably, it is 50 mg: 5 mL: 9.1 mg: 58 mg.

[0027] Preferably, in step (2), the purification specifically involves dialysis of the obtained mixture with excess water for two days, with a molecular weight cutoff of 3000~4000 Da.

[0028] Preferably, in step (3), the ratio of TPP-HA to water is (25~38) mg: (2~7) mL; more preferably, it is 32 mg: 4 mL.

[0029] Preferably, in step (3), the ratio of the amount of TPP-HA to EDC, NHS, MES, APTES, TEOS, and PDMS is (25~38) mg: (18~27) mg: (6.5~15) mg: (32~45) mg: (16~25) mL: (16~25) mL: (145~160) mL; more preferably, it is 32 mg: 22.4 mg: 9.6 mg: 38.4 mg: 19.2 mL: 19.2 mL: 153.6 mL.

[0030] A second aspect of the present invention provides an injectable hydrogel prepared by the above-described preparation method.

[0031] A third aspect of this invention proposes the application of the injectable hydrogel prepared by the above-described method in the preparation of products for skin damage repair. (For skin cells, mimicking the cell migration-promoting effect of the hydrogel.) The beneficial effects of this invention are as follows: This invention combines organosilicon materials with TPP and HA through chemical grafting to prepare a novel hydrogel with excellent bioactivity and physical properties. The resulting hydrogel has certain application potential in skin wound healing, possessing both good biocompatibility and specific bioactivity. It provides a suitable microenvironment in the wound environment, promoting cell adhesion, migration and proliferation, thereby having the potential to accelerate the wound healing process.

[0032] (1) HA-based hydrogels can provide cells with a microenvironment that mimics the natural extracellular matrix, which helps cells adhere, proliferate and migrate, thereby promoting wound healing.

[0033] (2) TPP, as a naturally derived bioactive substance, has been proven to have multiple biological functions such as anti-inflammatory, antioxidant, and cell proliferation promotion. The esterification reaction between HA and TPP forms a stable ester bond structure, providing a strong chemical bond that ensures the stability and durability of the two, effectively preventing separation and dissociation in an aqueous environment. Furthermore, the stability of the ester bond allows TPP to be slowly released into the hydrogel, exerting its effects continuously and thus prolonging the bioactivity of the hydrogel.

[0034] (3) Using organosilicon grafted macromolecules to synthesize hydrogels is a mild preparation method that can be carried out at room temperature, avoiding the damage to bioactive molecules caused by harsh conditions such as high temperature.

[0035] Of course, implementing any product or method of the present invention does not necessarily require achieving all of the advantages described above at the same time. Attached Figure Description

[0036] Figure 1 This is a schematic diagram of the process for preparing the dynamic hydrogel in Example 1 of the present invention; Figure 2 The infrared spectrum of the dynamic hydrogel prepared in Example 2 of this invention; Figure 3 This is a transmission scanning electron microscope image of the hydrogel prepared in Example 3 of the present invention; Figure 4 This is a dynamic rheological diagram of the hydrogel prepared in Example 4 of the present invention; Figure 5 This is a graph showing the in vitro antioxidant activity results of the hydrogel prepared in Example 5 of the present invention; Figure 6 The figure shows the in vitro degradation results of the hydrogel prepared in Example 6 of this invention. Figure 7 This is a diagram showing the cell scratch results of the hydrogel prepared in Example 7 of the present invention; Figure 8 This is a cell senescence staining result image of the hydrogel prepared in Example 8 of the present invention; Figure 9 The figures show the results of wound healing in mice on days 0, 3, 7, and 14 after use of the hydrogel prepared in Example 9 of this invention.

[0037] Statistical analysis of results: All results are expressed as mean ± standard deviation (Mean ± SEM). T-tests were used to compare differences between two groups, and one-way ANOVA was used to compare differences among multiple groups. p <0.05, p <0.01. Figure 1 , 7 The letters "a ~ d" in 8 represent significant differences. Detailed Implementation

[0038] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise defined, the technical terms used below have the same meaning as understood by those skilled in the art.

[0039] Unless otherwise specified, the test materials and reagents used in the following examples are commercially available or prepared by known methods.

[0040] Unless otherwise specified, all techniques or conditions described in the embodiments can be performed in accordance with the techniques or conditions described in the literature in this field or in the product manual. Unless otherwise specified, the quantitative experiments in the following embodiments are all repeated three times or more, and the results are averaged.

[0041] Example 1: Preparation method of APTES-TPP-HA hydrogel (see schematic diagram of preparation process) Figure 1 (As shown), including the following steps: (1) Preparation of TPP: Weigh an appropriate amount of dried Huangda tea leaves, add distilled water and dried tea leaves to a container at a material-to-liquid ratio of 1 g:40 mL, heat to boiling for 20 min, filter, retain the tea residue, and repeat this operation twice. Then freeze-dry the tea residue, pulverize and sieve (through an 80-mesh sieve). Mix the tea residue powder with 0.3 mol / L NaOH solution at a material-to-liquid ratio of 1 g:25 mL, and sonicate at 50 ºC for 30 min. Centrifuge the extract at 4000 r / min for 20 min, remove the residue, and adjust the pH of the supernatant to 4.5 with hydrochloric acid solution to precipitate the protein. After standing at room temperature until the protein precipitation is complete, centrifuge again at 4000 r / min for 20 min. Collect the protein precipitate, freeze-dry, and obtain the crude tea protein extract. Prepare a tea protein solution with a substrate concentration of 2% (w / v), sonicate for 10 min, adjust the pH of the solution to 10.0, and stabilize in a 50ºC constant temperature water bath for 10 min. The tea protein solution was maintained at 50ºC and pH 10.0. Alkaline protease was added for enzymatic hydrolysis for 3 h, and the solution was heated to 90ºC for 10 min to inactivate the enzyme. After rapid cooling, the solution was centrifuged at 6000 rpm for 20 min to obtain a mixed peptide solution. The mixed peptide solution was sequentially passed through ultrafiltration membranes of 0.2 μm, 10 kDa, 5 kDa, 3 kDa, and 1 kDa, and desalted (the solution was passed through a 150 Da ultrafiltration membrane, retaining the unfiltered portion). Fractions with molecular weights between 150 Da and 1 kDa were separated, then concentrated under reduced pressure and dried to obtain TPP powder.

[0042] (2) Preparation of TPP-HA conjugate 100 mg of water-soluble HA was dissolved in 10 mL of anhydrous formamide and heated to 50 °C with stirring for 10 min to prepare an HA solution for later use. Under light-protected conditions at room temperature, 50 mg of TPP was dissolved in 5 mL of deionized water, and 9.1 mg of DMAP and 58 mg of EDC were added with stirring for 1 h to activate the carboxylic acid groups. The HA solution was then added dropwise to the activated TPP solution with stirring, and the mixture was stirred in the dark at ambient temperature for 43.1 h. The resulting mixture was then purified by dialyzing with excess distilled water for two days, with a molecular weight cutoff of 3,500 Da, to remove unreacted reagents. Finally, the product was lyophilized to obtain TPP-HA.

[0043] (3) Preparation of hydrogels Weigh 32 mg of TPP-HA from (2) and dissolve it in 4 mL of pure water. Stir for 30 min to dissolve. First, add 22.4 mg of EDC and 9.6 mg of NHS and stir for 30 min. Then, add 38.4 mg of MES powder and adjust the pH to 6-6.5. Add 19.2 mL of APTES and mix well. Then, add 19.2 mL of TEOS and 153.6 mL of PDMS and stir for 1 h to obtain injectable TPP-HA hydrogel. Hydrogel samples prepared with TPP-HA concentrations of 15 mg / mL, 12.5 mg / mL, 10 mg / mL, 8 mg / mL, 6 mg / mL, and 4 mg / mL are GEL-15, GEL-12.5, GEL-10, GEL-8, GEL-6, and GEL-4, respectively.

[0044] (MES is used to adjust the pH of the solution; PDMS and TEOS are used together as silicon-based crosslinking agents; TPP-HA undergoes an amidation reaction with APTES; after introducing silanol groups into the TPP-HA chain, the silanol groups undergo hydrolysis and condensation with PDMS and TEOS to form a silicon network support structure. Other solvents cannot be used to replace this.) Example 2: Fourier transform infrared spectroscopy determination of hydrogels The spectrometer used the transmission method to obtain the near-infrared FTIR spectra of TPP, HA, TPP-HA conjugates, and composite hydrogel freeze-dried samples, and performed resolution measurements (results are shown in the figure). Figure 2 (As shown). Infrared resolution is 4 cm. -1 The wave number is 4000–400 cm⁻¹ -1 During the testing process, the sample was ground into powder, mixed with KBr powder, compressed into a pellet, and then scanned. The measurement environment was kept at a constant temperature and humidity: 25 ℃ and 60% relative humidity. The structural characteristics of the sample were analyzed, such as the formation of ester or amide bonds, to gain a deeper understanding of the sample's chemical composition and functional group information.

[0045] Example 3: TEM observation of hydrogels The microstructure of lyophilized hydrogel samples prepared with different TPP-HA conjugates was observed using a Hitachi HT7700 transmission electron microscope. Before TEM observation, the dehydrated lyophilized samples were rapidly frozen in liquid nitrogen, quickly cut, and then subjected to gold-palladium sputtering coating treatment before scanning observation. (Results are shown below.) Figure 3 (As shown) Example 4: Rheological testing of hydrogels Hydrogels prepared with different concentrations of TPP-HA conjugate were placed on a test plate. The gap between the test plate and the rotor was set to 0.1 mm, and the rotor diameter was 40 mm. The test temperature was set to 37 ℃. First, the storage modulus (G′) and loss modulus (G″) of all hydrogels were measured in the strain range of 1%-1000% using the oscillation amplitude scanning method at a fixed frequency of 1 Hz. Subsequently, with the strain fixed, the modulus change of the composite hydrogel in the angular frequency range of 0.1-100 rad / s was measured using the oscillation amplitude scanning method. (Results are shown in the figure.) Figure 4 (As shown) Example 5: In vitro antioxidant experiments of hydrogels 10 mg of DPPH (2,2-Diphenyl-1-picrylhydrazyl) was added to 50 mL of methanol and sonicated for 20 min to fully dissolve, preparing a 0.25 mM DPPH solution for later use. Then, GEL-15, GEL-12.5, GEL-10, GEL-8, GEL-6, and GEL-4 were prepared according to a concentration gradient, with 100 mg weighed and placed in centrifuge tubes respectively. 1 mL of the diluted 0.25 mM DPPH solution was added to each hydrogel sample, and the samples were then placed in a 37°C oven for 30 min. The co-incubated solutions were then added to 96-well cell culture plates, 100 mL per well. The DPPH solution without hydrogel samples served as the control group, and the DPPH solution co-incubated with the hydrogel served as the experimental group. The reaction was conducted in the dark. The OD value was measured at 517 nm, with four replicates per group. The DPPH free radical scavenging rate formula is:

[0046] Where As represents the absorbance value of the sample group. c represents the absorbance value of the control.

[0047] ABTS+ radicals were generated using the reaction system of ABTS and K2S2O8. ABTS working solution preparation: Equal volumes of 7.00 mmol / L ABTS solution and 2.45 mmol / L K2S2O8 aqueous solution were mixed and incubated in the dark for 12 h. Before use, the solution was diluted with ethanol to an absorbance of 0.70 ± 0.02 at 734 nm. For use, 0.5 mL of sample was mixed with 1 mL of ABTS working solution and reacted at room temperature in the dark for 10 min. The assay was performed according to the kit method. The sample group was treated as above, and the control group was prepared using 0.5 mL of double-distilled water instead of the sample. Each group was tested in triplicate. (Results are as follows...) Figure 5 (As shown) Example 6: In vitro degradation experiment of hydrogel The composite hydrogel (20 mg), labeled W0, was immersed in 10 mL of PBS buffer. It was then placed in a 37°C shaking incubator at a rotation speed of 100 rad / min. At predetermined time points, an equal volume of hydrogel was lyophilized, weighed, and labeled W. i Each group was tested three times (n=3). The remaining weight percentage of the hydrogel was calculated using the following formula:

[0048] Where W0 is the initial weight of the hydrogel, W i This represents the weight of the hydrogel at the end of the oscillation. (Results are as follows) Figure 6 (As shown) Example 7: Cell scratch assay with hydrogels Draw a line in the center of the outer side of the 6-well plate using a marker (to determine the field of view for photography). Then, use a density of 1×10⁻⁶. 4 HaCaT cells were seeded into 6-well tissue culture dishes and incubated at 37°C and 5% CO2 for 24 h. When the cell density reached approximately 80%, a straight "wound" was made on the bottom of the cell culture dish using a 200 mL pipette tip, perpendicular to the direction of the marker mark. The scratching was performed as vertically as possible, and the dish was rinsed with culture medium to remove all cell debris. The culture medium was discarded, and complete culture medium containing different concentrations of GEL-6 and GEL-8 hydrogel extracts and the corresponding TPP was added. After adding the hydrogel extracts, the dish was photographed at a predetermined time point under an inverted microscope (0 h). The cells were then cultured for another 24 h and 48 h, and photographed again. Cell migration around the scratch was recorded at 24 h and 48 h and compared with cells at the same location at 0 h. Cell proliferation rate in the damaged area was detected and photographed under an inverted microscope. (Results are shown below.) Figure 7 (As shown) Example 8: Cell senescence staining experiment with hydrogels β-galactosidase staining is used to detect cellular senescence. HaCaT is stained at 1×10⁻⁶. 5 Cells were seeded in 6-well plates and divided into control, model, GEL-6, GEL-8, and corresponding TPP groups. After treatment, cells were cultured for 24 h, the cell culture medium was aspirated, and the cells were washed once with PBS. 1 mL of b-Gal fixative was added, and the cells were fixed at room temperature for 15 min. The fixative was then aspirated, and the cells were washed three times with PBS for 3 min each time. Staining working solution was prepared according to the specified ratio, and 1 mL of staining working solution was added to each well. The cells were incubated overnight at 37°C and observed under an optical microscope. (Results are as follows...) Figure 8 (As shown) Example 9: Hydrogels promote wound healing in mice SPF-grade male mice weighing 18... 22 g mice were randomly assigned to groups. A skin defect model was created on the back of mice using a sterile biopsy punch with a diameter of 5 mm. The wound was treated with the hydrogel from Example 1, and the hydrogel dressing was changed daily. On days 1, 3, 5, 7, and 14 after the wound model was established, the wound site was photographed, and the wound area was measured using ImageJ software. (Results are as follows) Figure 9 (As shown) Results and Analysis: 1. Fourier transform infrared spectroscopy results of hydrogels The Fourier transform infrared spectra of each substance are as follows: Figure 2 The infrared curve of HA is at 3424.73 cm⁻¹. -1 There is a broad and strong peak at 2923.35 cm⁻¹, attributed to the -OH vibration absorption peak. -1 The peak is the -CH3 absorption peak at 1412.99 cm⁻¹. -1 The -CN- absorption peak is at 1152.33 cm⁻¹. -1 The peak is the stretching vibration peak of COC, at 1042.22 cm⁻¹. -1 These are -CN stretching vibration peaks, and all are characteristic absorption peaks of HA. 1046.68 cm⁻¹ -1 1151.29 cm -1 The presence of an ester bond is indicated by the stretching vibration peak of the -COC- bond. The increased intensity of -COC- in the infrared spectra of HA and TPP-HA can be interpreted as the hydroxyl group of HA combining with the carboxyl group of TPP to form an ester bond, indicating that HA and TPP are well grafted together.

[0049] 1567.44 cm -1 The band at 975 cm⁻¹ belongs to the amide bond formed between the carboxyl-COO group in HA and the amino group in APTES, indicating amidation grafting between APTES and TPP. This band is mainly attributed to the bending vibration of the amide group forming a single hydrogen bond. -1 and 895 cm -1 The bands at 850 cm⁻¹ belong to Si-OH groups and Si-OH from PDMS, respectively, indicating that a hydrolysis reaction occurred; -1 The band at that location is attributed to (TEOS) Si-O-Si (PDMS) bonds, corresponding to hybrid cross-linked SiO2-PDMS, indicating that PDMS was successfully incorporated into the 3D SiO2 network.

[0050] 2. Observation results of hydrogel by transmission electron microscopy The results of TEM observation of the microstructure of lyophilized hydrogel samples of different concentrations are as follows: Figure 3 All hydrogels possess an interconnected three-dimensional porous network structure. As the concentration of the coupling agent increases, the material structure is relatively regular in the early stages of the reaction; as the concentration increases, cracks and depressions appear on the material surface, the roughness increases, and a complex dendritic network structure is formed, which has a high specific surface area and porous structure; if the concentration of reactants is too high, the reaction will be excessive, and the material will gradually develop pores and depressions of various sizes and shapes, and the overall structure will become loose, with irregular protrusions and depressions.

[0051] 3. Dynamic rheological experimental results of hydrogels Hydrogel rheological results are as follows Figure 4 Most concentrations of hydrogels, under constant oscillation, show a reaction time of 1–100 s. -1 The storage modulus (G') is consistently higher than the loss modulus (G") within the angular frequency range, and G' and G" have no intersection, indicating that most hydrogels have stable network structures and maintain good elastic characteristics. However, GEL-4 exhibits G">G', suggesting that GEL-4 did not form a dense cross-linked network, resulting in its inability to support the gel state and structural damage. Furthermore, the frequency sweep results show that all hydrogel samples exhibit frequency-dependent viscoelasticity, highlighting the dynamic reversible network characteristics of the hydrogels.

[0052] The injectability of the hydrogel was tested by shear rate. Due to the correlation between the gel structure and the applied force, the hydrogel was able to break down its internal cross-linked structure when the shear force increased, thus reducing the shear viscosity and eventually approaching a liquid state, exhibiting shear-thinning properties, which further demonstrates the excellent injectability of the hydrogel.

[0053] Stress-strain experiments showed that all hydrogels exhibited G'>G'' in the initial strain stage, indicating that the hydrogels could maintain the stability of their gel network structure. With further strain increase, the two values ​​intersected, and the G' value significantly decreased, falling below the G” value. This indicates that after the critical point, the hydrogel network structure was disrupted under increasing stress, leading to a significant decrease in internal cross-linking and a gradual transition from a gel state to a sol state. Specifically, the structural change points for GEL-6 and GEL-8 were both above 1000% strain. Within the 1000% strain range, G'>G'', indicating good internal cross-linking density and gel stability, demonstrating the hydrogels' excellent elasticity and resistance to deformation.

[0054] 4. Results of the in vitro antioxidant activity of the hydrogel When the body experiences a wound, its defense mechanisms are activated. During this process, various factors such as cell necrosis, acute tissue damage, and chronic inflammation can lead to excessive reactive oxygen species (ROS) at the wound site. Excessive ROS negatively impacts wound healing and exacerbates the inflammatory response. TPP is a plant protein peptide with excellent antioxidant activity, effectively scavenging free radicals and reducing intracellular ROS levels. The antioxidant properties of hydrogels prepared with different concentrations of TPP-HA conjugates were screened using ABTS and DPPH free radical scavenging assays. Figure 5 The results showed that GEL-6 and GEL-8 exhibited higher antioxidant activity compared to other concentrations, with ABTS scavenging rates reaching 65.95% ± 0.39 and 60.90% ± 0.23, respectively. Both evaluation methods demonstrated that GEL-6 and GEL-8 hydrogels possess significant potential for free radical scavenging.

[0055] 5. Results of in vitro degradation experiments of hydrogels All hydrogels exhibited swelling during the initial degradation phase after immersion in PBS, indicating good water absorption and the ability to effectively absorb exudate from the surrounding damaged tissue. Over time, the remaining weight of the hydrogel gradually decreased. Figure 6 All hydrogels exhibited varying degrees of degradation within 10 days. Both excessively high and low conjugate concentrations slowed the degradation rate, indicating that the degree of cross-linking affects the degradation rate. At day 10, the remaining amounts of GEL-8 and GEL-6 were 45.78% ± 0.92 and 47.45% ± 1.51, respectively, slightly higher than the other hydrogels. Considering the strain strength, adhesion strength, degradation rate, and antioxidant properties of TPP-HA hydrogels at different concentrations, GEL-6 and GEL-8 hydrogels were selected for subsequent material characterization and cell experiments.

[0056] 6. Results of hydrogel cell scratch assay The effect of hydrogels on skin cell migration ability was evaluated using a scratch assay. The results are as follows: Figure 7 The results showed that, compared with the Control group, hydrogel treatment effectively promoted the migration ability of HaCaT cells, with the highest migration rate reaching 67.67% ± 1.26. There was no significant difference in migration rate among different concentrations. Compared with the TPP treatment group, the migration rate of all hydrogel groups was improved, with the highest migration rate in the TPP group at 59.48% ± 1.38. The calculated degree of substitution in the hydrogel was 59.08%. Under the same mass concentration, the overall repair ability of the hydrogel group was higher than that of the TPP group, indicating that hydrogel can effectively promote skin cell migration and is beneficial to wound healing. Furthermore, the migration rate of GEL-6 was significantly better than that of GEL-8 (…). p <0.05).

[0057] 7. Cell senescence staining results of hydrogels Senescent cells exhibit β-galactosidase activity; therefore, SA-β-Gal staining was performed to count the number of positive cells. Figure 8 It was found that after hydrogen peroxide induction, 11.88% of HaCaT cells showed significant senescence. However, after treatment with hydrogel, the proportion of senescent cells decreased to approximately 9%. Notably, GEL-6 exhibited a superior anti-senescence effect compared to GEL-8.

[0058] 8. Effect of hydrogel on wound healing in mice like Figure 9 As shown, the wound treated with the TPP-HA hydrogel prepared in Example 1 of this invention was significantly smaller than that in the control group after 4 days; after 7 days of modeling, the wound healing rate exceeded 50%, and by day 14, the wound healing rate exceeded 90%, with the wound essentially healed. This demonstrates that the TPP-HA hydrogel prepared in Example 1 of this invention can significantly promote wound healing.

[0059] Example 10: The difference between this embodiment and embodiment 1 is that "Huangda Tea" is replaced with "Huangshan Maofeng", and the rest is the same as step 1.

[0060] Example 11: The difference between this embodiment and embodiment 1 is that "Huangda Tea" is replaced with "Liuan Melon Seed Tea", and the rest is the same as step 1.

[0061] Example 12: The difference between this embodiment and Embodiment 1 is that: In step (1), The ratio of tea leaves to water is 1g:30mL; heat to boiling for 10 minutes; sieve through a 60-mesh sieve; the ratio of tea residue to alkaline solution is 1g:15mL; alkaline extraction is performed by adding tea residue powder to alkaline solution, mixing, and then sonicating at 40℃ for 40 minutes; pH is adjusted to acidity to 5.5. The tea protein solution was prepared at a substrate concentration of 1% (w / v); the pH was adjusted to alkaline by adjusting the pH to 9; the enzymatic hydrolysis was carried out by adding alkaline protease and hydrolyzing for 2 hours, during which the temperature was maintained at 60℃; the enzyme inactivation was carried out by heating to 80℃ for 20 minutes; the centrifugation was carried out at 5000 r / min for 30 minutes. In step (2), The dosage of hyaluronic acid (HA) and formamide is 80 mg: 7 mL; The dosage of TPP, water, DMAP, and EDC is 45mg, 3mL, 6mg, and 55mg respectively. In step (3), The ratio of TPP-HA to water is 38 mg: 7 mL; The dosage of TPP-HA, EDC, NHS, MES, APTES, TEOS, and PDMS is 38mg:27mg:15mg:45mg:25mL:25mL:160mL.

[0062] Example 13: The difference between this embodiment and Embodiment 1 is that: In step (1), The ratio of tea leaves to water is 1g:50mL; heat to boiling for 30 minutes; sieve through a 100-mesh sieve; the ratio of tea residue to alkaline solution is 1g:35mL; alkaline extraction is performed by adding tea residue powder to alkaline solution, mixing, and then sonicating at 60℃ for 20 minutes; pH is adjusted to acidity to 4.5. The tea protein solution was prepared at a substrate concentration of 3% (w / v); the pH was adjusted to alkaline by adjusting the pH to 11; the enzymatic hydrolysis was carried out by adding alkaline protease and hydrolyzing for 2 hours, during which the temperature was maintained at 60℃; the enzyme inactivation was carried out by heating to 100℃ for 5 minutes; the centrifugation was carried out at 7000 r / min for 10 minutes. In step (2), The ratio of hyaluronic acid (HA) to formamide is 120 mg: 15 mL; The ratio of TPP, water, DMAP, and EDC is 55mg:8mL:12mg:65mg; In step (3), The ratio of TPP-HA to water is 25 mg: 2 mL; The dosage of TPP-HA, EDC, NHS, MES, APTES, TEOS, and PDMS is 25mg:18mg:6.5mg:32mg:16mL:16mL:145mL.

[0063] The injectable hydrogels prepared in Examples 10-13 have similar properties to the hydrogel prepared in Example 1.

[0064] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing an injectable hydrogel, characterized in that, Includes the following steps: (1) Preparation of tea protein source oligopeptides (TPP) Soak the tea leaves in water, filter, and keep the tea residue; Add alkaline solution for extraction, retain the supernatant, adjust the pH to acidic, collect the protein precipitate, dry it, and obtain crude tea protein extract; The crude extract of tea protein was dispersed in water, the pH was adjusted to alkaline, alkaline protease was added for enzymatic hydrolysis, the enzyme was inactivated, centrifuged, ultrafiltered, and desalted to obtain tea protein-derived oligopeptides (TPP). (2) Preparation of hyaluronic acid grafted tea protein oligopeptide (TPP-HA) Hyaluronic acid (HA) is dissolved in formamide to obtain an HA solution; Mix the TPP, water, DMAP and EDC obtained in (1) to obtain a TPP mixed solution; The HA solution was added dropwise to the TPP mixed solution, mixed well, and purified to obtain TPP-HA; (3) Preparation of APTES-TPP-HA hydrogel The TPP-HA obtained in (2) is mixed with water to obtain a TPP-HA solution. EDC, NHS, MES, APTES, TEOS and PDMS are added and mixed well to obtain injectable APTES-TPP-HA hydrogel.

2. The preparation method according to claim 1, characterized in that, In step (1), the ratio of tea leaves to water is 1g:30~50mL; the ratio of tea residue to alkali solution is 1g:15~35mL.

3. The preparation method according to claim 1, characterized in that, In step (1), adjusting the pH to acidic means adjusting the pH to 3.5~5.5; adjusting the pH to alkaline means adjusting the pH to 9~11.

4. The preparation method according to claim 1, characterized in that, In step (1), the conditions for enzymatic hydrolysis are: 40~60℃, 2~4h.

5. The preparation method according to claim 1, characterized in that, In step (1), the crude extract of tea protein is dispersed in water to obtain a tea protein solution with a concentration of 1~3% (w / v).

6. The preparation method according to claim 1, characterized in that, The ultrafiltration specifically involves passing the solution through ultrafiltration membranes of 0.2 μm, 10 kDa, 5 kDa, 3 kDa, and 1 kDa in sequence; the desalination specifically involves passing the solution through an ultrafiltration membrane of 150 Da, retaining the unfiltered portion.

7. The preparation method according to claim 1, characterized in that, In step (2), the ratio of hyaluronic acid (HA) to formamide is (80~120) mg: (7~15) mL; the ratio of TPP, water, DMAP and EDC is (45~55) mg: (3~8) mL: (6~12) mg: (55~65) mg.

8. The preparation method according to claim 1, characterized in that, In step (3), the ratio of TPP-HA to water is (25~38) mg: (2~7) mL; the ratio of TPP-HA to EDC, NHS, MES, APTES, TEOS, and PDMS is (25~38) mg: (18~27) mg: (6.5~15) mg: (32~45) mg: (16~25) mL: (16~25) mL: (145~160) mL.

9. An injectable hydrogel prepared by the preparation method according to any one of claims 1-8.

10. The use of the injectable hydrogel of claim 9 in the preparation of products for skin damage repair.

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