Multi-response self-healing gelatin hydrogel

Abstract

The invention relates to a multi-response self-healing gelatin hydrogel. The hydrogel is prepared from phenylboronic acid modified pullulan and gelatin in a mass ratio of (1: 4)-(2: 3), wherein the phenylboronic acid modified pullulan is prepared by the following steps: mixing and dissolving 4-formyl phenylboronic acid and pullulan according to the mass ratio of 4-formyl phenylboronic acid to pullulan being (1-4): 5, heating to 50-70 DEG C, reacting for 18-36 hours, and freeze-drying to obtain the phenylboronic acid modified pullulan. The gelatin hydrogel provided by the invention not only can be self-healed, but also has good multiple responses of temperature, pH, ROS and the like.

Description

Technical Field

[0001] This invention relates to hydrogels, and more specifically to gelatin hydrogels, which are not only self-healing but also exhibit good temperature, pH, and ROS responsiveness. Background Technology

[0002] Gelatin is a natural biopolymer derived from the partial hydrolysis of collagen. Due to its excellent biocompatibility, biodegradability, low immunogenicity, and ease of acquisition, it is widely used in biomedical engineering, particularly in tissue engineering, drug delivery, and wound dressings. Gelatin hydrogels are widely used due to their high water retention, non-toxicity, and negative antigenicity in living organisms. However, gelatin hydrogels suffer from poor mechanical properties, low self-healing ability, and low environmental responsiveness, limiting their application in complex biological environments. To overcome these bottlenecks, various strategies have been developed to improve the performance of gelatin hydrogels. For example, Xiang Siyuan et al. from Dalian University of Technology prepared tyramine-modified hyaluronic acid backbone and vanillin / NaH-3BCN-modified gelatin backbone respectively, and mixed them under the catalysis of horseradish peroxidase and hydrogen peroxide to finally obtain a gelatin-based composite hydrogel with self-healing ability (Chinese Invention Patent 202411312959.9); Liu Dahai et al. from Guangdong Andao Technology Co., Ltd. prepared a gelatin-based hydrogel with good biocompatibility and temperature-sensitive properties by blending gelatin with a chitosan solution functionalized by α-ketoglutarate and protocatechuic acid (Chinese Invention Patent 202510115329.0); Wang Zhiyu et al. from the School of Materials Design and Engineering of Beijing Institute of Fashion Technology mixed sodium alginate with gelatin and introduced borax as a crosslinking agent to strengthen the network structure, and successfully prepared a gelatin-based composite hydrogel with self-healing properties. Gel (Wang Zhiyu, Liu Hongru, Wang Xiaochun, et al. Preparation and performance study of self-healing sodium alginate / gelatin hydrogel [J]. New Chemical Materials, 2025, 53(09): 165-169); Wang Xiaoyuan et al. of South China University of Technology successfully developed a gelatin-based hydrogel with pH-responsive properties by dissolving chitosan in citric acid environment and mixing it with gelatin, and then using glutaraldehyde for cross-linking and curing (Wang Xiaoyuan, Yang Xiaoquan. Preparation and performance of novel pH-sensitive chitosan / gelatin hydrogel [J]. Chemical Industry and Engineering Progress, 2009, 28(10): 1781-1786); However, the above hydrogels lack a gelatin hydrogel that can simultaneously integrate self-healing, good mechanical properties and multiple environmental responses (temperature, pH, ROS), which greatly limits its intelligent application in complex biological environments. Summary of the Invention

[0003] The technical problem to be solved by the present invention is to provide a multi-responsive self-healing gelatin hydrogel, which has high swelling degree, good rheological properties and multi-responsive properties such as temperature, pH, and ROS.

[0004] The technical solution of the present invention to solve the above problems is:

[0005] A multi-responsive, self-healing gelatin hydrogel is made of phenylboronic acid-modified pullulan and gelatin in a mass ratio of 1:4 to 2:3.

[0006] The phenylboronic acid-modified pullulan polysaccharide is prepared by the following method: 4-formylphenylboronic acid and pullulan polysaccharide are mixed and dissolved in a mass ratio of 4-formylphenylboronic acid: pullulan polysaccharide = 1~4:5, heated to 50~70℃ and reacted for 18~36h, and then freeze-dried to obtain the product.

[0007] In the above scheme, the preferred mass ratio of phenylboronic acid-modified pullulan to gelatin is 3:7.

[0008] In the above scheme, the reaction temperature is preferably 60°C and the reaction time is preferably 24h.

[0009] The multi-responsive, self-healing gelatin hydrogel of this invention is prepared by the following method:

[0010] First, gelatin and phenylboronic acid-modified pullulan were dissolved in water to prepare a gelatin solution with a concentration of 8-12% (w / v) and a pullulan solution with a concentration of 4-6% (w / v). Then, the gelatin solution and the pullulan solution were mixed evenly, the pH was adjusted to 8-9, and the mixture was allowed to stand for 10-16 hours to obtain the final product.

[0011] The self-healing gelatin hydrogel of this invention is composed of phenylboronic acid-modified pullulan and gelatin. The phenylboronic acid in the phenylboronic acid-modified pullulan is a Lewis acid, which can combine with the vicinal diol-carrying gelatin to form borate ester bonds, creating a reversible dynamic network structure. These borate ester bonds have a dynamic cross-linking effect, facilitating the formation of a self-healing gel. Simultaneously, the borate ester bonds dissociate under acidic conditions or in the presence of ROS (reactive oxygen species), resulting in a gel-solution transition. This causes the hydrogel to degrade under acidic conditions or ROS, changing from a gel state to a liquid state, achieving a shear-induced "gel-sol" flow transition and a "sol-gel" self-healing transition. Furthermore, in the phenylboronic acid-modified pullulan of this invention, the aldehyde group of 4-formylphenylboronic acid and the amine group on the pullulan react to form a shiff base. The shiff base dissociates under acidic conditions, causing the gel to undergo a gel-solution transition, achieving a shear-induced "gel-sol" flow transition and a "sol-gel" self-healing transition. Furthermore, gelatin possesses excellent thermosensitivity, endowing the self-healing gelatin hydrogel with good temperature responsiveness. Therefore, the multi-responsive self-healing gelatin hydrogel of this invention exhibits good temperature, pH, and ROS sensitivity. The multi-responsive self-healing gelatin hydrogel of this application simultaneously possesses the advantages of high swelling degree, good rheological properties, and multi-responsive properties such as temperature, pH, and ROS. Attached Figure Description

[0012] Figure 1 The 1H NMR spectra of 4-formylphenylboronic acid, pullulan, and phenylboronic acid-modified pullulan are shown.

[0013] Figure 2 Infrared spectra of 4-formylphenylboronic acid, pullulan, phenylboronic acid-modified pullulan, gelatin, and the hydrogel.

[0014] Figure 3 With a fixed frequency of 10 rad / s, an oscillating shear strain with continuously increasing amplitude was applied, ranging from 0.1% to 1000%. The amplitude of the sample was scanned, and the curves showing the changes in the storage modulus (G') and loss modulus (G'') of the hydrogel were obtained.

[0015] Figure 4 With the strain set at 4% and the oscillation frequency ranging from 100 rad / s to 0.1 rad / s, frequency scanning was performed on the sample, and the curves showing the changes in the storage modulus (G') and loss modulus (G'') of the hydrogel were obtained.

[0016] Figure 5 The graph shows the changes in storage modulus (G') and loss modulus (G'') of the hydrogel at different scanning times under alternating strain (4% and 100%). Detailed Implementation

[0017] The following specific embodiments will be used to further describe in detail the hydrogel, its preparation method, and its beneficial effects described in this invention.

[0018] Example 1

[0019] 1. Preparation of pullulan modified with phenylboronic acid (mass ratio of pullulan to 4-formylphenylboronic acid = 10:3)

[0020] 1000 mg of pullulan was dissolved in 150 mL of deionized water to obtain an aqueous pullulan solution; 300 mg of 4-formylphenylboronic acid was dissolved in 2 mL of dimethyl sulfoxide. The 4-formylphenylboronic acid solution was then added to the pullulan solution. The pH of the mixture was adjusted to 8 using sodium hydroxide, stirred at 50 °C for 18 h, dialyzed, and freeze-dried to obtain 525 mg of phenylboronic acid-modified pullulan.

[0021] use 1 The phenylboronic acid-modified pullulan polysaccharide obtained by 1H NMR analysis. For example... Figure 1 As shown, a signal of phenylboronic acid was observed between 7.46 and 7.73 ppm.

[0022] Based on the above data and preparation method, the phenylboronic acid-modified pullulan polysaccharide was successfully synthesized.

[0023] 2. Synthesis of the gelatin hydrogel described in this application (mass ratio of gelatin to phenylboronic acid-modified pullulan polysaccharide is 8:2)

[0024] A gelatin solution with a concentration of 8% (w / v) was prepared by dissolving gelatin in water; a phenylboronic acid-modified pullulan solution with a concentration of 4% (w / v) was prepared by dissolving phenylboronic acid-modified pullulan in water. 20 mL of the gelatin solution and 10 mL of the phenylboronic acid-modified pullulan solution were taken, mixed thoroughly, and the pH was adjusted to 8. The mixture was defoamed by ultrasonication, transferred to a mold, and reacted for 10 hours to obtain the gelatin hydrogel described in this application.

[0025] 3. Performance testing of the gelatin hydrogel described in this application

[0026] 3.1. Detection of phenyl borate bonds and imine bonds

[0027] The infrared spectra of 4-formylphenylboronic acid, pullulan, phenylboronic acid-modified pullulan, gelatin, and the gelatin hydrogel obtained in step 2 above were detected using a Thermo Scientific iS20 Fourier transform infrared spectrometer. Figure 2 As shown. By Figure 2 It can be seen that the 1330 cm section belongs to the asymmetric extension of the BOC. -1The peak at 1650 cm⁻¹ confirms the formation of the phenyl borate structure, while the peak at 1650 cm⁻¹... -1 The peak at that point confirmed the formation of the imine bond.

[0028] 3.2. Swelling Degree Test

[0029] At room temperature, take the gelatin hydrogel obtained in step 2 above, weigh it precisely, and obtain the weight W. o The hydrogel was then immersed in water at room temperature until it reached swelling equilibrium. Excess water was wiped off the surface of the support with filter paper, and the mass of the hydrogel was measured again to obtain the weight W. e Then, the swelling ratio of the gelatin hydrogel obtained in step 2 above is calculated using the following formula: Swelling ratio (%) = (We - W0) / W0. The swelling ratio of the gelatin hydrogel obtained in this example is 227.14%, which indicates that the formed hydrogels all have a high degree of swelling.

[0030] 3.3. Temperature, pH, and ROS sensitivity tests

[0031] Take the gelatin hydrogel obtained in step 2 above, immerse it in water with a pH of 7 at room temperature until it reaches swelling equilibrium, wipe off excess water from the surface of the hydrogel with filter paper, and measure the mass of the hydrogel to obtain mass W1; then take the gelatin hydrogel prepared in step 2 above, and place it in deionized aqueous solutions with pH of 4.5 and 8.5, as well as deionized aqueous solutions with hydrogen peroxide concentrations of 0 mM, 400 mM, and 800 mM, respectively, at room temperature, and incubate for 24 h, remove the solution, wipe off excess solution from the surface of the hydrogel with filter paper, and measure the mass of the hydrogel to obtain mass W2. Then calculate the residual rate of the self-healing gelatin hydrogel according to the following formula: residual rate (%) = W2 / W1*100%, and the results are shown in Table 1 below.

[0032] Table 1. Residual rate of hydrogel at different temperatures, pH levels, and hydrogen peroxide concentrations.

[0033] [H2O2]=0mM 39% [H2O2] = 400mM 18% [H2O2] = 800mM 11% pH=4.5 20% pH=7.2 39% pH=8.5 28% Temperature = 25℃ 39% Temperature = 37℃ 0%

[0034] The gelatin hydrogel prepared in this example contains a borate ester obtained by reacting phenylboronic acid and vicinal diol. The borate ester is pH sensitive and dissociates under acidic conditions, resulting in a gel-solution transition and causing hydrogel degradation under acidic conditions, leading to a significant change in hydrogel mass. Furthermore, in the phenylboronic acid-modified pullulan polysaccharide described in this invention, the aldehyde group of 4-formylphenylboronic acid and the amine group on pullulan polysaccharide react to form a shiff base. The shiff base dissociates under acidic conditions, leading to a gel-solution transition and a significant change in hydrogel mass. Table 1 shows that the hydrogel prepared in this example exhibits a large mass change under acidic conditions, indicating a gel-solution transition; while under neutral conditions, the mass change is smaller. Simultaneously, the borate ester is sensitive to ROS and dissociates in the presence of ROS, achieving a gel-solution transition and causing hydrogel degradation under ROS, resulting in a significant change in mass. Table 1 also shows that the hydrogel prepared in this example exhibits a large mass change after reacting with hydrogen peroxide, indicating a gel-solution transition. This demonstrates that gelatin possesses good temperature sensitivity, imparting excellent temperature responsiveness to the gelatin hydrogel. As shown in Table 1 above, the hydrogel prepared in this example exhibits significant mass change at 37°C. Therefore, the gelatin hydrogel described in this invention possesses good temperature, pH, and ROS sensitivity.

[0035] 3.4. Rheological property testing

[0036] The rheological behavior of the hydrogel obtained in step 2 above was tested using a dynamic rheology analyzer (MCR 301, Anton Paar GmbH, Austria) at a test temperature of 25℃ and a mold plate spacing of 1mm. 1) An oscillating shear strain with continuously increasing amplitude was applied at a fixed frequency of 10 rad / s, ranging from 0.1% to 1000%, and the sample was subjected to amplitude scanning to characterize its mechanical strength and stability; 2) The strain was set to 4%, and the oscillation frequency was changed from 100 rad / s to 0.1 rad / s, and the sample was subjected to frequency scanning to further characterize its mechanical strength and stability; 3) The small strain was set to 4%, and the large strain was set to 100%, and the sample was subjected to alternating strain scanning to characterize its self-healing characteristics.

[0037] The mechanical strength, stability, and self-healing behavior of the hydrogel were analyzed using dynamic rheology through frequency scanning, amplitude scanning, and alternating strain scanning. Figure 3 , 4 As shown, G' of this hydrogel material is always greater than G'', indicating that it exhibits a stable gel-like structure, mainly attributed to the effect of dynamic borate esters in the gel network. The self-healing properties of the dynamic hydrogel were further investigated using alternating strains, i.e., small strains of 4% and large strains of 100%. Figure 3It can be seen that under small strain, G'>G''; while under large strain, G'<G''. This transition can be rapid, and the modulus does not decrease significantly, indicating that the gelatin hydrogel described in this invention has rapid self-healing properties.

[0038] Example 2

[0039] 1. Preparation of pullulan modified with 4-formylphenylboronic acid (mass ratio of pullulan to 4-formylphenylboronic acid = 5:4)

[0040] 1000 mg of pullulan was dissolved in 150 mL of deionized water to obtain an aqueous pullulan solution; 800 mg of 4-formylphenylboronic acid was dissolved in 2 mL of dimethyl sulfoxide. The 4-formylphenylboronic acid solution was then added to the pullulan solution. The pH of the mixture was adjusted to 8 using sodium hydroxide, stirred at 50 °C for 36 h, dialyzed, and freeze-dried to obtain 656 mg of phenylboronic acid-modified pullulan.

[0041] 2. Synthesis of the gelatin hydrogel described in this application (mass ratio of gelatin to phenylboronic acid-modified pullulan polysaccharide is 7:3)

[0042] A 10% (w / v) gelatin solution was prepared by dissolving gelatin in water; a 6% (w / v) phenylboronic acid-modified pullulan solution was prepared by dissolving pullulan in water. 70 mL of the gelatin solution and 50 mL of the phenylboronic acid-modified pullulan solution were mixed thoroughly, and the pH was adjusted to 9. The mixture was defoamed by ultrasonication, transferred to a mold, and reacted for 16 hours to obtain the gelatin hydrogel described in this application.

[0043] 3. Performance testing of gelatin hydrogels

[0044] 3.1. Detection of phenyl borate bonds and imine bonds

[0045] The infrared spectra of 4-formylphenylboronic acid, pullulan, phenylboronic acid-modified pullulan, gelatin, and the gelatin hydrogel obtained in step 2 above can also be obtained by detection using a Thermo Scientific iS20 Fourier transform infrared spectrometer according to the method described in Example 1.

[0046] 3.2. Swelling Degree Test

[0047] The swelling ratio of the hydrogel prepared in this example was tested according to the method described in Example 1 and found to be 221.08%, which indicates that the hydrogels prepared in this example all have a high degree of swelling.

[0048] 3.3. Temperature, pH, and ROS sensitivity tests

[0049] The residual rate of the hydrogel prepared in this example was tested according to the method described in Example 1, and the results are shown in Table 2 below.

[0050] Table 2. Residual rate of hydrogel at different temperatures, pH levels, and hydrogen peroxide concentrations.

[0051] [H2O2]=0mM 37% [H2O2] = 400mM 16% [H2O2] = 800mM 9% pH=4.5 22% pH=7.2 37% pH=8.5 26% Temperature = 25℃ 37% Temperature = 37℃ 0%

[0052] As shown in Table 2 above, the hydrogel prepared in this example exhibits a significant mass change under acidic conditions, resulting in a gel-solution transition; while under neutral conditions, the mass change is smaller. Furthermore, the hydrogel prepared in this example shows a significant mass change after reacting with hydrogen peroxide, also undergoing a gel-solution transition. Additionally, the hydrogel prepared in this example shows a significant mass change at 37°C. Therefore, the gelatin hydrogel described in this invention exhibits good temperature, pH, and ROS sensitivity.

[0053] 3.3. Rheological property testing

[0054] Following the method described in Example 1, the mechanical strength, stability, and self-healing behavior of the hydrogel were analyzed using dynamic rheology through frequency scanning, amplitude scanning, and alternating strain scanning. The G' of this hydrogel material was consistently greater than G'', indicating its stable gel-like structure, primarily attributed to the effect of the dynamic borate ester within the gel network. Further investigation of the self-healing properties of the dynamic hydrogel was conducted using alternating strain, i.e., small strain of 4% and large strain of 100%. Under small strain, G'>G''; while under large strain, G'<G''. This transition was rapid, and the modulus did not decrease significantly, indicating that the gelatin hydrogel in this example possesses rapid self-healing properties.

[0055] Example 3

[0056] 1. Preparation of pullulan modified with 4-formylphenylboronic acid (mass ratio of pullulan to 4-formylphenylboronic acid = 10:4)

[0057] 1000 mg of pullulan was dissolved in 150 mL of deionized water to obtain an aqueous pullulan solution; 400 mg of 4-formylphenylboronic acid was dissolved in 2 mL of dimethyl sulfoxide. The 4-formylphenylboronic acid solution was then added to the pullulan solution. The pH of the mixture was adjusted to 8 using sodium hydroxide, stirred at 70 °C for 30 h, dialyzed, and freeze-dried to obtain 625 mg of phenylboronic acid-modified pullulan.

[0058] 2. Synthesis of self-healing gelatin hydrogel (mass ratio of gelatin to phenylboronic acid-modified pullulan polysaccharide is 6:4)

[0059] A 12% (w / v) gelatin solution was prepared by dissolving gelatin in water; a 5% (w / v) phenylboronic acid-modified pullulan solution was prepared by dissolving pullulan in water. 10 mL of the gelatin solution and 19 mL of the phenylboronic acid-modified pullulan solution were mixed thoroughly, and the pH was adjusted to 9. The mixture was defoamed by ultrasonication, transferred to a mold, and reacted for 12 hours to obtain the gelatin hydrogel described in this application.

[0060] 3. Performance testing of gelatin hydrogels

[0061] 3.1. Detection of phenyl borate bonds and imine bonds

[0062] The infrared spectra of 4-formylphenylboronic acid, pullulan, phenylboronic acid-modified pullulan, gelatin, and the hydrogel can also be obtained by detection using a Thermo Scientific iS20 Fourier transform infrared spectrometer according to the method described in Example 1.

[0063] 3.2. Swelling Degree Test

[0064] The swelling ratio of the hydrogel prepared in this example was tested according to the method described in Example 1 and found to be 218.92%, which indicates that the formed hydrogels all have a high degree of swelling.

[0065] 3.2. Temperature, pH, and ROS sensitivity tests

[0066] The residual rate of the hydrogel prepared in this example was tested according to the method described in Example 1, and the results are shown in Table 3 below.

[0067] Table 3. Residual rate of hydrogel at different temperatures, pH levels, and hydrogen peroxide concentrations.

[0068] [H2O2]=0mM 41% [H2O2] = 400mM 20% [H2O2] = 800mM 13% pH=4.5 18% pH=7.2 41% pH=8.5 30% Temperature = 25℃ 41% Temperature = 37℃ 0%

[0069] As shown in Table 3 above, the hydrogel prepared in this example exhibits a significant mass change under acidic conditions, resulting in a gel-solution transition; while under neutral conditions, the mass change is smaller. Furthermore, the hydrogel prepared in this example shows a significant mass change after reacting with hydrogen peroxide, also undergoing a gel-solution transition. Additionally, the hydrogel prepared in this example shows a significant mass change at 37°C. Therefore, the gelatin hydrogel described in this invention exhibits good temperature, pH, and ROS sensitivity.

[0070] 3.3. Rheological property testing

[0071] Following the method described in Example 1, the mechanical strength, stability, and self-healing behavior of the hydrogel prepared in this example were analyzed using dynamic rheology through frequency scanning, amplitude scanning, and alternating strain scanning. The G' of this hydrogel material was consistently greater than G'', indicating its stable gel-like structure, primarily attributed to the effect of the dynamic borate ester in the gel network. Further investigation of the self-healing properties of the dynamic hydrogel was conducted using alternating strain, i.e., small strain of 4% and large strain of 100%. Under small strain, G'>G''; while under large strain, G'<G''. This transition was rapid, and the modulus did not decrease significantly, indicating that the gelatin hydrogel in this example possesses rapid self-healing properties.

[0072] Example 4

[0073] 1. Preparation of pullulan modified with 4-formylphenylboronic acid (mass ratio of pullulan to 4-formylphenylboronic acid = 10:6)

[0074] 1000 mg of pullulan was dissolved in 150 mL of deionized water to obtain an aqueous pullulan solution; 600 mg of 4-formylphenylboronic acid was dissolved in 2 mL of dimethyl sulfoxide. The 4-formylphenylboronic acid solution was then added to the pullulan solution. The pH of the mixture was adjusted to 8 using sodium hydroxide, stirred at 60 °C for 24 h, dialyzed, and freeze-dried to obtain 623 mg of phenylboronic acid-modified pullulan.

[0075] 2. Synthesis of self-healing gelatin hydrogel (mass ratio of gelatin to phenylboronic acid-modified pullulan polysaccharide is 8:2)

[0076] A 10% (w / v) gelatin solution was prepared by dissolving gelatin in water; a 5% (w / v) phenylboronic acid-modified pullulan solution was prepared by dissolving pullulan in water. 8 mL of the gelatin solution and 4 mL of the phenylboronic acid-modified pullulan solution were mixed thoroughly, and the pH was adjusted to 9. The mixture was defoamed by ultrasonication, transferred to a mold, and reacted for 13 hours to obtain the gelatin hydrogel described in this application.

[0077] 3. Performance testing of gelatin hydrogels

[0078] 3.1. Detection of phenyl borate bonds and imine bonds

[0079] The infrared spectra of 4-formylphenylboronic acid, pullulan, phenylboronic acid-modified pullulan, gelatin, and the hydrogel obtained in step 2 above can also be obtained by detection using a Thermo Scientific iS20 Fourier transform infrared spectrometer according to the method described in Example 1.

[0080] 3.2. Swelling Degree Test

[0081] The swelling ratio of the hydrogel prepared in this example was tested according to the method described in Example 1 and found to be 208.35%, which indicates that the hydrogels of this application all have a high degree of swelling.

[0082] 3.2. Temperature, pH, and ROS sensitivity tests

[0083] The residual rate of the hydrogel prepared in this example was tested according to the method described in Example 1, and the results are shown in Table 4 below.

[0084] Table 4. Residual rate of hydrogel at different temperatures, pH levels, and hydrogen peroxide concentrations.

[0085] [H2O2]=0mM 35% [H2O2] = 400mM 17% [H2O2] = 800mM 12% pH=4.5 24% pH=7.2 35% pH=8.5 27% Temperature = 25℃ 35% Temperature = 37℃ 0%

[0086] As shown in Table 4 above, the hydrogel prepared in this example exhibits a significant mass change under acidic conditions, resulting in a gel-solution transition; while under neutral conditions, the mass change is smaller. Furthermore, the hydrogel prepared in this example shows a significant mass change after reacting with hydrogen peroxide, also undergoing a gel-solution transition. Additionally, the hydrogel prepared in this example shows a significant mass change at 37°C. Therefore, the self-healing gelatin hydrogel described in this invention exhibits good temperature, pH, and ROS sensitivity.

[0087] 3.3. Rheological property testing

[0088] Following the method described in Example 1, the mechanical strength, stability, and self-healing behavior of the hydrogel in this example were analyzed using dynamic rheology through frequency scanning, amplitude scanning, and alternating strain scanning. The G' of this hydrogel material was consistently greater than G'', indicating its stable gel-like structure, primarily attributed to the effect of the dynamic borate ester within the gel network. Furthermore, the self-healing properties of the dynamic hydrogel were investigated using alternating strain, i.e., small strain of 4% and large strain of 100%. Under small strain, G'>G''; while under large strain, G'<G''. This transition was rapid, and the modulus did not decrease significantly, indicating that the gelatin hydrogel in this example possesses rapid self-healing properties.

[0089] Example 5

[0090] 1. Preparation of pullulan modified with 4-formylphenylboronic acid (mass ratio of pullulan to 4-formylphenylboronic acid = 5:1)

[0091] 1000 mg of pullulan was dissolved in 150 mL of deionized water to obtain an aqueous pullulan solution; 200 mg of 4-formylphenylboronic acid was dissolved in 2 mL of dimethyl sulfoxide. The 4-formylphenylboronic acid solution was then added to the pullulan solution. The pH of the mixture was adjusted to 9 using sodium hydroxide, stirred at 70 °C for 18 h, dialyzed, and freeze-dried to obtain 350 mg of phenylboronic acid-modified pullulan.

[0092] 2. Synthesis of gelatin hydrogel (mass ratio of gelatin to phenylboronic acid-modified pullulan polysaccharide is 7:3)

[0093] A 10% (w / v) gelatin solution was prepared by dissolving gelatin in water; a 6% (w / v) phenylboronic acid-modified pullulan solution was prepared by dissolving pullulan in water. 14 mL of the gelatin solution and 10 mL of the phenylboronic acid-modified pullulan solution were mixed thoroughly, and the pH was adjusted to 9. The mixture was defoamed by ultrasonication, transferred to a mold, and reacted for 14 hours to obtain the gelatin hydrogel.

[0094] 3. Performance testing of gelatin hydrogels

[0095] 3.1. Detection of phenyl borate bonds and imine bonds

[0096] The infrared spectra of 4-formylphenylboronic acid, pullulan, phenylboronic acid-modified pullulan, gelatin, and the hydrogel prepared in this example can also be obtained by detection using a Thermo Scientific iS20 Fourier transform infrared spectrometer according to the method described in Example 1.

[0097] 3.2. Swelling Degree Test

[0098] The swelling ratio of the hydrogel prepared according to the method described in Example 1 was 201.47%, which indicates that the hydrogels obtained in this example all have a high degree of swelling.

[0099] 3.3. Temperature, pH, and ROS sensitivity tests

[0100] The residual rate of the hydrogel prepared in this example was tested according to the method described in Example 1, and the results are shown in Table 5 below.

[0101] Table 5. Residual rate of hydrogel at different temperatures, pH levels, and hydrogen peroxide concentrations.

[0102] [H2O2]=0mM 39% [H2O2] = 400mM 19% [H2O2] = 800mM 10% pH=4.5 21% pH=7.2 39% pH=8.5 29% Temperature = 25℃ 39% Temperature = 37℃ 0%

[0103] As shown in Table 5 above, the hydrogel prepared in this example exhibits a significant mass change under acidic conditions, resulting in a gel-solution transition; while under neutral conditions, the mass change is smaller. Furthermore, the hydrogel prepared in this example shows a significant mass change after reacting with hydrogen peroxide, also undergoing a gel-solution transition. Additionally, the hydrogel prepared in this example shows a significant mass change at 37°C. Therefore, the gelatin hydrogel described in this invention exhibits good temperature, pH, and ROS sensitivity.

[0104] 3.3. Rheological property testing

[0105] Following the method described in Example 1, the mechanical strength, stability, and self-healing behavior of the hydrogel prepared in this example were analyzed using dynamic rheology through frequency scanning, amplitude scanning, and alternating strain scanning. The G' of this hydrogel material was consistently greater than G'', indicating its stable gel-like structure, primarily attributed to the effect of the dynamic borate ester in the gel network. Furthermore, the self-healing properties of the dynamic hydrogel were investigated using alternating strain, i.e., small strain of 4% and large strain of 100%. Under small strain, G'>G''; while under large strain, G'<G''. This transition was rapid, and the modulus did not decrease significantly, indicating that the gelatin hydrogel in this example possesses rapid self-healing properties.

[0106] Example 6

[0107] 1. Preparation of pullulan modified with 4-formylphenylboronic acid (mass ratio of pullulan to 4-formylphenylboronic acid = 10:7)

[0108] 1000 mg of pullulan was dissolved in 150 mL of deionized water to obtain an aqueous pullulan solution; 700 mg of 4-formylphenylboronic acid was dissolved in 2 mL of dimethyl sulfoxide. The 4-formylphenylboronic acid solution was then added to the pullulan solution. The pH of the mixture was adjusted to 9 using sodium hydroxide, stirred at 60 °C for 20 h, dialyzed, and freeze-dried to obtain 636 mg of phenylboronic acid-modified pullulan.

[0109] 2. Synthesis of self-healing gelatin hydrogel (mass ratio of gelatin to phenylboronic acid-modified pullulan polysaccharide is 3:2)

[0110] A 12% (w / v) gelatin solution was prepared by dissolving gelatin in water; a 6% (w / v) phenylboronic acid-modified pullulan solution was prepared by dissolving pullulan in water. 13 mL of the gelatin solution and 17 mL of the phenylboronic acid-modified pullulan solution were mixed thoroughly, and the pH was adjusted to 8. The mixture was defoamed by ultrasonication, transferred to a mold, and reacted for 10 hours to obtain the gelatin hydrogel.

[0111] 3. Performance testing of gelatin hydrogels

[0112] 3.1. Detection of phenyl borate bonds and imine bonds

[0113] The infrared spectra of 4-formylphenylboronic acid, pullulan, phenylboronic acid-modified pullulan, gelatin, and the hydrogel described in this example can also be obtained by detection using a Thermo Scientific iS20 Fourier transform infrared spectrometer according to the method described in Example 1.

[0114] 3.2. Swelling Degree Test

[0115] The swelling ratio of the hydrogel prepared in this example was tested according to the method described in Example 1 and found to be 195.63%, which indicates that the hydrogels described in this invention all have a high degree of swelling.

[0116] 3.3. Temperature, pH, and ROS sensitivity tests

[0117] The residual rate of the hydrogel prepared in this example was tested according to the method described in Example 1, and the results are shown in Table 6 below.

[0118] Table 6. Residual rate of hydrogel at different temperatures, pH levels, and hydrogen peroxide concentrations.

[0119] [H2O2]=0mM 42% [H2O2] = 400mM 15% [H2O2] = 800mM 14% pH=4.5 17% pH=7.2 42% pH=8.5 26% Temperature = 25℃ 42% Temperature = 37℃ 0%

[0120] As shown in Table 6 above, the hydrogel prepared in this example exhibits a significant mass change under acidic conditions, resulting in a gel-solution transition; while under neutral conditions, the mass change is smaller. Furthermore, the hydrogel prepared in this example shows a significant mass change after reacting with hydrogen peroxide, also undergoing a gel-solution transition. Additionally, the hydrogel prepared in this example shows a significant mass change at 37°C. Therefore, the gelatin hydrogel described in this invention exhibits good temperature, pH, and ROS sensitivity.

[0121] 3.4. Rheological property testing

[0122] Following the method described in Example 1, the mechanical strength, stability, and self-healing behavior of the hydrogel in this example were analyzed using dynamic rheology through frequency scanning, amplitude scanning, and alternating strain scanning. The G' of this hydrogel material was consistently greater than G'', indicating its stable gel-like structure, primarily attributed to the effect of the dynamic borate ester in the gel network. Further investigation of the self-healing properties of the dynamic hydrogel was conducted using alternating strain, i.e., small strain of 4% and large strain of 100%. Under small strain, G'>G''; while under large strain, G'<G''. This transition was rapid, and the modulus did not decrease significantly, indicating that the gelatin hydrogel in this example possesses rapid self-healing properties.

Claims

1. A multi-responsive, self-healing gelatin hydrogel, the hydrogel being made of phenylboronic acid-modified pullulan and gelatin in a mass ratio of 1:4 to 2:3; in, The phenylboronic acid-modified pullulan polysaccharide is prepared by the following method: 4-formylphenylboronic acid and pullulan polysaccharide are mixed and dissolved in a mass ratio of 4-formylphenylboronic acid: pullulan polysaccharide = 1~4:5, heated to 50~70℃ and reacted for 18~36h, and then freeze-dried to obtain the product.

2. The multi-responsive, self-healing gelatin hydrogel according to claim 1, characterized in that, The mass ratio of the phenylboronic acid-modified pullulan to gelatin is 3:7; the reaction temperature for preparing the phenylboronic acid-modified pullulan is 60℃, and the reaction time is 24h.

3. The multi-responsive, self-healing gelatin hydrogel according to claim 1 or 2 is prepared by the following method: First, gelatin and phenylboronic acid-modified pullulan were dissolved in water to prepare a gelatin solution with a concentration of 8-12% (w / v) and a phenylboronic acid-modified pullulan solution with a concentration of 4-6% (w / v). Then, the gelatin solution and the phenylboronic acid-modified pullulan solution were mixed evenly, the pH was adjusted to 8-9, and the mixture was allowed to stand for 10-16 hours to obtain the gelatin hydrogel.

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