Method for cross-linking riboflavin and mediating guanosine to prepare casein hydrogel and application thereof

Abstract

The application discloses a method for preparing casein hydrogel by cross-linking riboflavin and mediating guanosine and application thereof. The method comprises the following steps: stirring and dissolving riboflavin suspension, guanosine suspension and sodium persulfate mother liquor in a casein solution to obtain a mixed solution, and then irradiating the mixed solution by light to ensure gelation, thereby obtaining casein hydrogel. The concentration of the casein solution is 5-15% w / v; the concentration of the riboflavin suspension is 1-5 mM; the concentration of the guanosine suspension is 0.02-0.04 mM; and the concentration of the sodium persulfate mother liquor is 45-150 mM. The application is that the casein hydrogel is used in wound dressings. The casein hydrogel prepared by the method has good antibacterial effect and suitable mechanical properties, and can be used as a wound material without causing compression to the wound and affecting the recovery of the wound.

Description

Technical Field

[0001] This invention relates to the field of hydrogel preparation technology, and in particular to a method and application of riboflavin crosslinking and guanosine-mediated preparation of casein hydrogels. Background Technology

[0002] In recent years, casein hydrogels have seen further applications. Casein gels have been used in the food industry for decades and are an excellent material for forming functional dairy products. Because casein readily cross-links with other substances to form stable polymer networks, it is suitable for film preparation. Casein is a multifunctional protein derived from milk; its excellent water retention, emulsifying properties, and stability allow it to form a colloid with a specific viscosity in water. Like general protein hydrogels, casein hydrogels possess good biocompatibility, biodegradability, and low toxicity. Furthermore, casein hydrogels, with casein as their main component, are readily available and inexpensive. In general, the application of casein hydrogels in the food industry is relatively mature, but its application in biomedical fields has not yet yielded ideal results. Summary of the Invention

[0003] The purpose of this invention is to provide a method and application for preparing casein hydrogels by riboflavin cross-linking and mediating guanosine. The casein hydrogel prepared by this invention has good antibacterial effects and suitable mechanical properties, allowing it to be used as a wound healing material without causing pressure and affecting wound healing.

[0004] The technical solution provided by this invention is as follows: A method for preparing casein hydrogel by riboflavin cross-linking and mediating guanosine, comprising: dissolving riboflavin suspension, guanosine suspension and sodium persulfate mother liquor in casein solution by stirring to obtain a mixture; then irradiating the mixture with light to ensure gelation, thereby obtaining casein hydrogel; wherein:

[0005] The concentration of the casein solution is 5-15% w / v;

[0006] The concentration of the riboflavin suspension is 1-5 mM;

[0007] The concentration of the guanosine suspension is 0.02-0.04 mM;

[0008] The concentration of the sodium persulfate mother liquor is 45-150 mM.

[0009] In the above-described method for preparing casein hydrogels by riboflavin cross-linking and mediating guanosine, the concentration of the casein solution is 10% w / v.

[0010] The aforementioned method for preparing casein hydrogel by riboflavin cross-linking and mediating guanosine cross-linking involves preparing the casein solution by adding casein to a 0.1 mol / L NaOH solution and then stirring it on a magnetic stirrer for 18 hours.

[0011] In the aforementioned method for preparing casein hydrogels by cross-linking riboflavin and mediating guanosine, the concentration of the riboflavin suspension is 3 mM.

[0012] In the aforementioned method for preparing casein hydrogels by riboflavin cross-linking and mediating guanosine, the concentration of the guanosine suspension is 0.035 mM.

[0013] In the aforementioned method for preparing casein hydrogels by riboflavin cross-linking and mediating guanosine preparation, the concentration of the sodium persulfate mother liquor is 75 mM.

[0014] In the aforementioned method for preparing casein hydrogels via riboflavin cross-linking and guanosine-mediated irradiation, the wavelength of the light irradiation is 450 nm, the irradiation duration is 3 min, and the irradiation intensity is 1.5 mW cm⁻¹. -2 .

[0015] In the aforementioned method for preparing casein hydrogel by riboflavin cross-linking and mediating guanosine, the light source is positioned 1 cm above the mixture.

[0016] The casein hydrogel prepared by the aforementioned method is used in wound dressings.

[0017] Compared with existing technologies, this invention uses riboflavin as a cross-linking agent and sodium persulfate as a catalyst to prepare casein hydrogels via light irradiation. During the gel preparation process, guanosine is added to mediate its introduction into the hydrogel. In this invention, the casein hydrogel is cross-linked through the reaction between riboflavin and tyrosine residues during light irradiation. Simultaneously, guanosine reacts with riboflavin to produce hydrogen peroxide, thus exhibiting good antibacterial effects while possessing suitable mechanical properties. This allows it to be used as a wound healing material without causing pressure and hindering wound healing. Attached Figure Description

[0018] Figure 1 It is a casein hydrogel with a 5mM riboflavin concentration;

[0019] Figure 2 It is a casein hydrogel with a 3mM riboflavin concentration;

[0020] Figure 3 A comparison graph showing the storage modulus and loss modulus of casein gels at different concentrations of SPS is presented.

[0021] Figure 4 This is a schematic diagram showing the amount of hydrogen peroxide produced by gels with different guanosine contents;

[0022] Figure 5 This is a schematic diagram showing the amount of hydrogen peroxide produced by the gel under different light exposure times;

[0023] Figure 6 This is a schematic diagram illustrating the mass change of the hydrogel during swelling.

[0024] Figure 7 This is a schematic diagram showing the change in the swelling fraction of the hydrogel;

[0025] Figure 8 This is a diagram showing the results of an E. coli antibacterial experiment;

[0026] Figure 9 This is a diagram showing the results of an antibacterial experiment against Staphylococcus aureus.

[0027] Figure 10 The results are the colony measurements of the blank bacterial culture group and the colony measurement results after 4 hours of hydrogel incubation.

[0028] Figure 11 The following are the results of the cytotoxicity test: live / dead cell culture images of cells cultured for 24 h in the extracts of blank group (culture medium), control group (guanosine-free gel extract), and experimental group (hydrogen peroxide-producing gel extract);

[0029] Figure 12 This represents the percentage of live cells measured in each group;

[0030] Figure 13 These are the hemolysis results of gel extracts at various mass concentrations;

[0031] Figure 14 The hemolysis rate of the gel extract was measured by spectrophotometry. Detailed Implementation

[0032] The present invention will be further described below with reference to the accompanying drawings and embodiments, but this should not be construed as limiting the present invention.

[0033] Example 1: A method for preparing casein hydrogels by riboflavin cross-linking and mediating guanosine. Gel preparation is carried out in a casein solution. Under sodium persulfate (SPS) catalysis, casein gel is formed through riboflavin (RB) cross-linking while simultaneously mediating guanosine. The casein solution and reagent stock solution need to be prepared in advance. Hydrogel formation requires mild, weakly alkaline conditions. First, a 0.1 M NaOH solution is prepared. 0.1 g of casein is placed in a glass bottle and 10 ml of NaOH is added to prepare a 10% w / v casein solution, which is then stirred on a magnetic stirrer for 18 h. A 3 mM riboflavin (RB) suspension, a 75 M sodium persulfate (SPS) stock solution, and a 0.035 mM guanosine suspension are prepared in ultrapure water. The riboflavin and guanosine suspensions are stored in the dark. On a magnetic stirrer, riboflavin suspension, guanosine suspension, and sodium persulfate stock solution were dissolved in 1 ml of casein sodium hydroxide solution. The mixture was then dispensed into cell culture plates using a syringe. The samples were irradiated with a 450 nm wavelength lamp at 1 cm intervals for 3 min to ensure gelation. The irradiation intensity was 1.5 mW / cm². -2 This yields the casein hydrogel product.

[0034] Example 2: The preparation process in this example is the same as in Example 1, wherein the concentration of the casein solution is 5% w / v; the concentration of the riboflavin suspension is 1 mM; the concentration of the guanosine suspension is 0.02 mM; and the concentration of the sodium persulfate mother liquor is 40 mM.

[0035] Example 3: The preparation process in this example is the same as in Example 1, wherein the concentration of the casein solution is 15% w / v; the concentration of the riboflavin suspension is 5 mM; the concentration of the guanosine suspension is 0.04 mM; and the concentration of the sodium persulfate mother liquor is 150 mM.

[0036] Example 4: The preparation process in this example is the same as in Example 1, wherein the concentration of the casein solution is 10% w / v; the concentration of the riboflavin suspension is 4 mM; the concentration of the guanosine suspension is 0.03 mM; and the concentration of the sodium persulfate mother liquor is 100 mM.

[0037] Example 5: The casein hydrogel prepared in Examples 1-4 was used in wound dressings.

[0038] In this invention, to explain the optimal process parameters and their corresponding effects in the preparation method of casein hydrogel, the following experiments and verifications were conducted using the following materials and methods.

[0039] 1. The materials are as follows:

[0040] Casein (Fonterra), 98% riboflavin (Aladdin R104137), 99% guanosine (Maclean G6043), sodium persulfate (Shanghai Test 7775-27-1), hydrogen peroxide content test kit (Solarbio BC3590-50T / 48S), sodium hydroxide, PBS buffer, ultrapure water (ddH2O), LB solid medium, LB liquid medium, Staphylococcus aureus, Escherichia coli, and fresh mouse blood.

[0041] Centrifuge (Xiangyi TGL-16M), spectrophotometer (Spark 30086376), rheometer (MCR302, Anton Paar, Austria), cell counting kit-8 (Yeasen Biotech, China), live / dead staining kit (Yeasen Biotech, China), microplate reader (Tecan M200 PRO, Tecan Company, Switzerland), fluorescence microscope (Leica DMI8, Germany), autoclave, 450nm blue light source.

[0042] 2. Optimization of various parameters during the preparation of casein hydrogels;

[0043] 2.1 Selection of Riboflavin Suspension Concentration:

[0044] The appearance of the casein hydrogel prepared in the experiment is related to riboflavin and the properties of casein hydrogel. Figure 1 The appearance of the protein hydrogel formed by riboflavin cross-linking casein via dityrosine bonds under light illumination is shown. Figure 1 Casein hydrogel with a 5mM riboflavin concentration. Figure 2 The casein hydrogel was prepared with a 3 mM riboflavin concentration. The hydrogel prepared with 5 mM riboflavin was not fully gelled, had a darker yellow color, and while the surface was fully gelled, liquid substances remained inside. The casein hydrogel prepared with 3 mM riboflavin was fully gelled and had a lighter color. Therefore, 3 mM riboflavin is preferred.

[0045] 2.2 Selection of casein solution concentration and sodium persulfate mother liquor concentration:

[0046] The rheological properties of casein hydrogels were examined under light irradiation to evaluate the effect of SPS on gelation time and storage modulus (G'). During rheological measurements, the storage modulus of the gel initially increased to a certain extent after exposure to visible light, until it stabilized. The study showed that a 10% w / v casein concentration resulted in a higher storage modulus and a faster gelation time. Therefore, subsequent experiments used 10% w / v casein gels.

[0047] Figure 3 A comparison of the storage modulus (1) and loss modulus (2) of casein gels at different concentrations of SPS is shown. It can be seen that there are significant differences in the rheological results at 10% w / v for different concentrations of SPS. 100 mM and 150 mM SPS lead to an increase in storage modulus, reaching 3600 and 2800 respectively, with gelation occurring in approximately 60 s. The storage moduli of 45 mM and 75 mM SPS are 850 and 950 respectively, with gelation time of approximately 160 s for 45 mM SPS and approximately 150 s for 75 mM SPS. Considering that the gel used as a wound material should not have excessively high mechanical strength to avoid tissue compression, and also requires a catalyst concentration that maximizes gel formation, as well as the possibility that rapid gelation due to the color of riboflavin may result in incomplete gelation within the material, the gelation time should not be too fast, and the strength should not be too high. In summary, 75 mM SPS is used as the catalyst.

[0048] 2.3 Hydrogen peroxide generation in gels with different guanosine contents;

[0049] In the prepared gel system, riboflavin reacts with guanosine under light irradiation to generate H2O2. This invention determines the optimal guanosine concentration by preparing casein hydrogels with different guanosine contents and measuring their H2O2 production. 1 ml of a 10% w / v casein solution with 3 mM riboflavin, 75 mM SPS, and 0 g, 0.005 g, 0.01 g, and 0.02 g guanosine contents was prepared and added with a magnetic stirrer. 300 μL of each solution was aliquoted into 48-well cell culture plates, with three replicates (as shown in Table 1). The plates were irradiated at 450 nm for 3 min to form hydrogels. After gelation, 1 ml of ultrapure water was added to the gel surface for absorption for 15 min.

[0050] Group Casein (ml) SPS (mM) RB (mM) Guanosin (g) Light intensity (min) 0 1 75 3 0 3 1 1 75 3 0.001 3 2 1 75 3 0.003 3 3 1 75 3 0.005 3 4 1 75 3 0.01 3 5 1 75 3 0.02 3

[0051] Table 1

[0052] The H2O2 content generated by the hydrogel was detected by the titanium sulfate precipitation method, based on the fact that H2O2 reacts with titanium sulfate to form a yellow titanium peroxide complex, which has an absorption peak at 415 nm. The hydrogen peroxide content was detected by a kit, which included Reagent 1 (acetone not provided), Reagent 2 (dissolved in 6 ml concentrated hydrochloric acid), Reagent 3, Reagent 4, and 1 M H2O2 standard solution.

[0053] Dilute the 1M standard solution to prepare a 1mM H2O2 standard solution. First, transfer 1ml of ultrapure water absorption solution (as a test tube), 1ml of 1mM H2O2 standard solution (as a standard tube), and 1ml of reagent one (as a blank tube) to each 2ml centrifuge tube. Then, add 100ul of reagent two and 200ul of reagent three to each centrifuge tube, mix well, centrifuge at 4000g at room temperature for 10 min, discard the supernatant, and retain the precipitate. Wash away excess pigment with a small amount of acetone, then add 1000ul of reagent four, shake thoroughly until the precipitate dissolves, and let stand at room temperature (20℃) for 5 min. Add 100ul of each solution to a 96-well cell plate, setting up three replicates. Measure the absorbance at 415nm using a preheated spectrophotometer and record the data. The amount of hydrogen peroxide released by the hydrogel is calculated based on serum volume.

[0054] H2O2 content (umol / ml) = 10 × ΔA (determined) / ΔA (standard);

[0055] Where, △A_determination = A_determination_tube - A_blank_tube, △A_standard = A_standard_tube - A_blank_tube.

[0056] like Figure 4 The concentration of hydrogen peroxide in hydrogels containing 0.001 g / ml and 0.003 g / ml guanosine was very low, with a significant increase occurring at 0.005 g and then rising. Related studies have shown that a hydrogen peroxide concentration of 20 μM can produce an antibacterial effect, meeting a basic requirement for photodynamic therapy (PDT). In this experiment, the 0.01 g / ml guanosine hydrogel produced hydrogen peroxide levels close to this value. However, excessively high concentrations of guanosine (0.02 g / ml) would affect light exposure, reducing light transmittance and thus affecting gelation; therefore, a 0.01 g / ml guanosine concentration was chosen.

[0057] 2.4 Hydrogen peroxide generation in gels exposed to different light durations

[0058] Light exposure is a crucial condition for hydrogen peroxide production in the gel and needs to be analyzed as a single-factor variable. A casein solution with 3 mM riboflavin, 75 mM SPS, and 0.01 g guanosine was prepared and added with a magnetic stirrer. 300 μL of the solution was added to each 48-well cell culture plate, and two replicates were set up. The gels were formed under 450 nm light for 1 min, 2 min, 3 min, and 4 min (as shown in Table 2). 1 mL of ultrapure water was added to the gel for absorption for 15 min. The hydrogen peroxide content was detected by titanium sulfate precipitation. Figure 5 As shown.

[0059] Group Casein (ml) SPS (mM) RB (mM) Guanosin (g) Light intensity (min) 0 1 75 3 0.01 1.5 1 1 75 3 0.01 2 2 1 75 3 0.01 2.5 3 1 75 3 0.01 3 4 1 75 3 0.01 3.5 5 1 75 3 0.01 4

[0060] Table 2

[0061] from Figure 5 The results showed that after 1.5 minutes of complete gel formation, there was no significant difference in hydrogen peroxide content among the groups. Therefore, it can be determined that the reaction between riboflavin and guanosine was complete when gel formation occurred. By comparing the light exposure time required for complete hydrogen peroxide release with the gelation time of casein gel in the rheological results, it was finally determined that the time required to ensure complete gelation was sufficient to ensure the full release of hydrogen peroxide from the gel. Further extension of the light exposure time did not increase hydrogen peroxide release; therefore, a light exposure time of 3 minutes was determined.

[0062] The above experiments successfully optimized the formulation of the hydrogen peroxide-producing casein hydrogel, and the optimized hydrogel material was successfully prepared multiple times. The final determined formulation is 10% w / v casein solution with 3 mM riboflavin, 0.01 g / ml (0.035 mM) guanosine, 75 mM SPS, and an irradiation time of 3 min, which is the scheme in Example 1.

[0063] 3. Performance testing based on the casein hydrogel in Example 1.

[0064] 3.1 Swelling test:

[0065] The casein hydrogel (8 mm in diameter, 6 mm in height) prepared in Example 1 and the casein hydrogel without guanosine were used as controls. After complete gelation, their initial mass was measured. They were then completely immersed in 40 ml of PBS buffer (pH=7.4) and stored at room temperature. They were weighed periodically. The swelling fraction was calculated as follows:

[0066] Swelling fraction (%) = (m i -m0) / m0×100%

[0067] Where, m i m0 represents the mass of the sample after swelling and the initial mass before swelling.

[0068] Swelling of hydrogels can be detrimental to their biomedical applications, such as wound dressings. This is because swelling reduces the mechanical properties of the hydrogel and makes it more prone to compressing surrounding tissues. Hydrogel samples from both the control group (without guanosine) and the experimental group (with guanosine) were immersed in PBS for 48 hours. The gel mass was measured at different time points, and the swelling rate was calculated. The results are as follows: Figure 6 and Figure 7 As shown. Figure 6 and Figure 7 The changes in the swelling mass and swelling fraction of the hydrogel are shown separately. Figure 6 and Figure 7The results showed that neither the 10% w / v casein hydrogels with nor without guanosine showed significant swelling during the soaking process. This is because casein is a mixed protein composed of hydrophobic and hydrophilic segments, which leads to a micellar structure in water. Overall, the hydrogel mass increased slightly during the swelling process. The swelling characteristics of the two groups were similar before 30 h, but the swelling fraction of the experimental group decreased compared to the initial swelling at 48 h. This is because the casein gel in the guanosine-added experimental group had undergone partial degradation, and some substances entered the soaking solution, resulting in a decrease in the mass of the gel solid.

[0069] 3.2 Antibacterial test;

[0070] Hydrogen peroxide has antibacterial effects, and whether the amount of hydrogen peroxide produced by the gel is sufficient to reach a certain level needs to be evaluated through an antibacterial test. First, LB liquid medium was prepared with 0.5g NaCl, 1g tryptone, and 0.5g yeast extract powder per 100mL of deionized water. LB solid medium was also prepared with 0.5g NaCl, 1g tryptone, 0.5g yeast extract powder, and 2.5g agar per 100mL of deionized water. The bacteria (Staphylococcus aureus and Escherichia coli) were activated twice on plates two days in advance, and single colonies were picked and inoculated into LB liquid medium at 37℃ and cultured overnight. Then, the Staphylococcus aureus bacterial suspension was diluted 2, 4, 8, 16 and 32 times, and 100 μL was taken to measure the absorbance at 600 nm using a spectrophotometer. For Staphylococcus aureus, a dilution factor with an OD value of 0.1 was selected to prepare the bacterial suspension (10⁸ CFU / ml), and for Escherichia coli, a corresponding dilution factor with an OD value of 0.15 was selected to prepare the bacterial suspension (10⁸ CFU / ml).

[0071] A 300 μL casein hydrogel as described in Example 1 was prepared in a 24-well cell culture plate. The group without guanosine served as a control, and the group with pure bacterial suspension without gel served as a blank control; two replicates were set up. After gel formation, 100 μL of bacterial suspension was added to the gel surface, and the plate was shaken to ensure even distribution of the bacterial suspension. The plate was incubated at 37 °C for 4 h. After incubation, 50 μL of the bacterial suspension was diluted to a final concentration of 10 μL. 3 10 4 and 10 5 Dilute 100 μL of the solution and spread it onto LB agar plates. Set up three replicates and incubate overnight at 37°C. Measure and record the colony count using the ImageJ method. The antibacterial results for *E. coli* are as follows: Figure 8 As shown, the antibacterial results of Staphylococcus aureus are as follows: Figure 9 As shown in the figure, the hydrogel has a good sterilization effect on both Escherichia coli and Staphylococcus aureus. Figure 10The results of colony measurements in the blank bacterial culture group and after 4 hours of hydrogel incubation are shown (data are presented as mean ± standard deviation. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). Figure 10 As shown, the bacterial concentrations of Escherichia coli and Staphylococcus aureus were 10. 8 The CFU / ml level of E. coli in the experimental group incubated with a casein hydrogel that produces hydrogen peroxide was 6.3 × 10⁻⁶. 6 CFU / ml, Staphylococcus aureus 2.46×10 7 With CFU / ml, the antibacterial rates reached 93.7% and 75.4% respectively, demonstrating good antibacterial properties. This proves that the casein hydrogel prepared in this invention effectively kills pathogenic bacteria.

[0072] 3.3 Biocompatibility testing;

[0073] 3.3.1 Cytotoxicity of Casein Hydrogels

[0074] Casein hydrogels with a diameter of 8 mm and a height of 6 mm were irradiated with a 450 nm lamp at 1 cm for 3 min to form stable gels. Casein hydrogels containing different amounts (0 g, 0.01 g) of guanosine were immersed in DMEM medium containing 10% FBS with an extraction rate of 200 mg / ml and soaked at 37°C for 12 h to prepare casein gel extracts. L929 mouse fibroblasts (5 × 10³ cells per well) were seeded into 96-well cell culture plates, and 100 μL of the extract was used to replace the medium. Fresh medium was used for the control group. After 24 h of cell culture, cell viability was detected according to the instructions using a cell counting kit-8 (Yeasen Biotech, China) and a live / dead staining kit (Yeasen Biotech, China). In the CCK8 assay, the absorbance of the medium at 450 nm wavelength was measured after 2 h using a microplate reader (Tecan M200 PRO, Tecan Company, Switzerland). Images of live and dead cells were acquired using a fluorescence microscope (Leica DMI8, Germany) and analyzed using ImageJ software to calculate the proportion of live cells. Figure 11 As shown, the casein hydrogel of the RB group without guanosine showed almost no cytotoxicity, with a cell viability rate of over 98%, thus eliminating the influence of riboflavin on the results of the guanosine group. At the same time, the study also proved that the SPS used in the experiment was non-cytotoxic, so it could also be ruled out. Figure 12The percentage of viable cells measured in each group is presented (data is shown as mean ± standard deviation. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). According to... Figure 12 The hydrogen peroxide-producing gel (i.e., the guanosine group) exhibited low cytotoxicity, with cell viability slightly lower than the control and blank groups, possibly due to the presence of low concentrations of hydrogen peroxide. However, the cell viability after soaking in its extract still reached 85.2%, indicating that this concentration of hydrogen peroxide would not cause significant damage to normal cells. The casein hydrogel prepared by RB crosslinking and simultaneously mediating guanosine supported cell viability in in vitro culture.

[0075] 3.3.2 Hemolytic;

[0076] Good biocompatibility requires certain hemolytic properties. In this experiment, mouse whole blood was used to test the blood compatibility of casein hydrogels. PBS buffer was used as the negative control and ultrapure water as the positive control in the hemolysis test. First, casein hydrogel mixtures of different concentrations (20, 40, 60, 80, 100 mg / ml) were prepared by light irradiation in 1 ml of PBS (pH=7.4). 20 μl of fresh mouse blood was added to each group, and the mixture was incubated at 37 ℃ for 1 h, followed by centrifugation at 1000 rpm for 10 min. 100 μl of the supernatant from each group was added to a 96-well plate, and the absorbance of the supernatant was measured at 545 nm using a spectrophotometer. The hemolysis rate was calculated using the following formula:

[0077] Hemolysis rate (%) = (ODL - ODN) / ODP × 100%

[0078] Among them, ODL, ODN, and ODP are the absorbance of the experimental group, the negative control group (PBS), and the positive control group (ddH2O), respectively.

[0079] Hemolytic capacity was assessed by evaluating the hemolysis of mouse blood cells in gel extracts of different concentrations. Figure 13 Macroscopically, no obvious hemolysis was observed in the casein hydrogel extract. The hemolysis rate for each group was calculated using spectrophotometry and the hemolysis rate calculation formula, and the results are as follows. Figure 14 As shown in the figure (in the adjacent bar charts, the left bar chart indicates the absence of guanosine, and the right bar chart indicates the presence of guanosine). Figure 14 The results showed that even the highest hemolysis rate in the 80 mg / ml guanosine group was only 2.1%. Therefore, the casein hydrogel that produces hydrogen peroxide is non-hemolytic and will not affect the healing of tissue wounds. It exhibits good biocompatibility characteristics and meets the basic requirements for wound material.

[0080] In summary, this invention presents a novel casein hydrogel material that generates trace amounts of hydrogen peroxide while forming a gel upon light exposure, exhibiting excellent antibacterial effects. This material also possesses suitable mechanical properties, allowing it to be used as a wound dressing without causing pressure and hindering wound healing. Furthermore, its excellent biocompatibility is indispensable; its low cytotoxicity and hemolytic activity may make it more conducive to wound healing than commercial fiber dressings while protecting tissue. Based on these characteristics, the casein hydrogel prepared in this invention can achieve ideal results in wound dressing applications.

[0081] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing casein hydrogels by riboflavin cross-linking and mediating guanosine, characterized in that: Riboflavin suspension, guanosine suspension, and sodium persulfate stock solution were dissolved in casein solution by stirring to obtain a mixture. The mixture was then irradiated with light to ensure gelation, resulting in casein hydrogel. Wherein: The concentration of the casein solution is 10% w / v; The concentration of the riboflavin suspension was 3 mM; The concentration of the guanosine suspension was 0.035 mM; The concentration of the sodium persulfate mother liquor was 75 mM; The light wavelength was 450 nm, the irradiation time was 3 min, and the irradiation intensity was 1.5 mW / cm². -2 ; The light source is positioned 1 cm above the mixture.

2. The method for preparing casein hydrogel by riboflavin cross-linking and mediating guanosine according to claim 1, characterized in that: The casein solution was prepared by adding casein to a 0.1 mol / L NaOH solution and then stirring it on a magnetic stirrer for 18 h.

3. The casein hydrogel prepared according to claim 1 or 2 is used in the preparation of wound dressings.

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