An antibacterial and antioxidant hydrogel and a preparation method thereof

Abstract

The present application relates to an antibacterial and antioxidant hydrogel and its preparation method and application, and belongs to the technical field of biomedical materials. The matrix of the hydrogel is formed by agarose and polyvinyl alcohol, and has an interpenetrating network structure; the active ingredients are tetramethylthionine and silk fibroin, and the active ingredients are uniformly dispersed in the matrix. The pore size distribution of the hydrogel is uniform, and the hydrogel has excellent mechanical properties. The hydrogel effectively prevents infection, removes free radicals, reduces oxidative stress level and reduces inflammatory response through the synergistic effect of antibacterial and antioxidant properties, thereby accelerating wound healing. In the preparation process, tetramethylthionine and silk fibroin are uniformly dispersed in the matrix, and the hydrogel with antibacterial and antioxidant properties is prepared through freeze-thaw cycle reaction. The preparation process is simple, convenient to operate, and low in cost, and is suitable for large-scale production.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, and in particular to an antibacterial and antioxidant hydrogel and its preparation method. Background Technology

[0002] In the traditional silk reeling industry, sericin is usually considered a byproduct and discharged along with silk reeling wastewater, leading to low resource utilization efficiency and serious environmental pollution. As a natural macromolecule, sericin possesses various beneficial properties, including antioxidant activity, biocompatibility, and the ability to promote cell proliferation.

[0003] Functionalizing sericin can effectively address the problem of resource waste. In recent years, researchers have combined sericin with other natural or synthetic polymers to develop a series of biomaterials for tissue engineering. However, the extraction process often leads to the degradation of sericin, thus limiting its autonomous cross-linking ability.

[0004] Hydrogels are ideal wound dressing materials due to their stable three-dimensional network structure and high water content. Current techniques typically incorporate antibiotics (such as gentamicin, vancomycin, and amoxicillin) into hydrogels to impart antibacterial properties. However, this method carries the risk of increased bacterial resistance and failure to eradicate biofilm bacteria. Alternatively, inorganic metal materials (such as metals or metal oxides) are introduced into hydrogels to enhance antibacterial properties, but this also carries the potential risk of metallocytotoxicity.

[0005] Existing hydrogel materials face certain challenges in dealing with infection and high oxidative stress, and cannot simultaneously achieve excellent antibacterial and antioxidant capabilities.

[0006] Therefore, developing a new hydrogel with antibacterial and antioxidant capabilities is of significant practical importance. Summary of the Invention

[0007] This invention aims to address the dual challenges of infection and high oxidative stress during wound healing by providing an antibacterial and antioxidant hydrogel and its preparation method. This hydrogel, through the synergistic effect of antibacterial and antioxidant properties, effectively prevents infection, scavenges free radicals, reduces oxidative stress levels, weakens inflammatory responses, creates a favorable physiological environment for damaged tissue, promotes cell proliferation and tissue repair, thereby improving wound healing efficiency.

[0008] To address the aforementioned technical problems, the first aspect of this invention provides an antibacterial and antioxidant hydrogel, wherein the matrix of the hydrogel comprises polyvinyl alcohol and agarose; the active ingredients are tetramethylol phosphate sulfate and sericin, and the active ingredients are uniformly dispersed in the matrix;

[0009] The proportion of tetramethylol phosphate in the hydrogel is no higher than 0.08 wt%.

[0010] Furthermore, the proportion of sericin in the hydrogel is no higher than 5 wt%.

[0011] In some embodiments, the hydrogel comprises the following components: 5-15% polyvinyl alcohol, 0.5-3% agarose, 0.01-0.08% tetramethylolphosphine sulfate and 0.01-5% sericin, with the balance being deionized water.

[0012] In some preferred embodiments, the hydrogel comprises the following components: 10% polyvinyl alcohol, 1% agarose, 0.08% tetramethylolphosphine sulfate and 5% sericin, with the balance being deionized water.

[0013] A second aspect of the present invention provides a method for preparing the aforementioned antibacterial and antioxidant hydrogel, comprising the following steps:

[0014] S1. Extraction of sericin: After cleaning the silkworm cocoons, add deionized water and degumming them under high temperature and high pressure; filter and separate, collect the filtrate, then centrifuge and collect the supernatant, and freeze-dry to obtain sericin;

[0015] S2. Preparation of hydrogel solution: Dissolve polyvinyl alcohol and agarose in deionized water, heat and stir until completely dissolved; then add tetramethylolphosphine sulfate and sericin, continue heating and stirring to mix thoroughly to obtain hydrogel solution;

[0016] S3. Freeze-thaw cycle treatment: Pour the hydrogel solution obtained in step S2 into the mold while it is still hot, and perform a freeze-thaw cycle treatment.

[0017] In some embodiments, in step S1, the degumming process is carried out at a temperature of 100-140°C for a processing time of 0.5-8 hours.

[0018] In a preferred embodiment, in step S1, the degumming process involves a high-temperature and high-pressure degumming treatment at 120°C for 4 hours; the reaction is carried out in a high-temperature and high-pressure sterilizer.

[0019] In some embodiments, in step S1, the centrifugation process is carried out at a speed of 6000-12000 rpm. -1 The processing time is 5-15 minutes.

[0020] In a preferred embodiment, the centrifugation process is carried out at 8000 rpm. -1 Centrifuge for 10 minutes to obtain the supernatant.

[0021] In some embodiments, in step S2, the heating and dissolving process is carried out at a temperature of 70-100°C and a stirring time of 2-3 hours.

[0022] In a preferred embodiment, the heating and dissolving process involves mechanical stirring in a 90°C water bath for 2-3 hours until complete dissolution.

[0023] In some embodiments, in step S2, the heating and mixing process is carried out at a temperature of 70-100°C for 1-3 hours.

[0024] In a preferred embodiment, the heating and mixing process involves mechanical stirring in a 90°C water bath for 1-2 hours until the mixture is completely dissolved.

[0025] In a preferred embodiment, in step S2, the polyvinyl alcohol is type 1799 with a degree of alcoholysis of 98%-99%.

[0026] In a preferred embodiment, in step S2, the tetrahydroxymethylphosphoric acid is a 75% aqueous solution.

[0027] In some implementations, the freeze-thaw cycle is repeated 2-5 times in step S3;

[0028] In some embodiments, in step S3, the cyclic freeze-thaw process involves freezing at ≤-20°C for 3-6 hours and then thawing at room temperature for 0.5-2 hours.

[0029] In a preferred embodiment, the mixture is frozen at -20°C for 4 hours, then thawed at room temperature for 1 hour; this process is repeated three times.

[0030] In some embodiments, in step S1, the protein content of the sericin is 87.23%-95.37%;

[0031] In some embodiments, the silkworm cocoons used in step S1 are dried silkworm cocoons with the pupa removed.

[0032] In some preferred embodiments, in step S1, the sericin extraction rate is 17.33%-17.74%.

[0033] In a preferred embodiment, step S1, extracting sericin: 10g of silkworm cocoons are cleaned and then added to 200mL of deionized water. The cocoons are degummed under high temperature and pressure at 120℃ for 4 hours. Solid residues are then removed by filtration, and the filtrate is collected. The filtrate is then extracted at 8000rpm. -1Centrifuge for 10 min to obtain the supernatant, and finally freeze-dry the supernatant to obtain sericin. Step S2: Prepare hydrogel solution: Dissolve 10 wt% polyvinyl alcohol and 1 wt% agarose in 83.92 wt% deionized water, and mechanically stir in a 90℃ water bath for 2-3 h until completely dissolved; then add 0.08 wt% tetramethylol phosphate sulfate and 5 wt% sericin to the above solution, and continue to mechanically stir in a 90℃ water bath for 1-2 h until fully mixed to obtain the hydrogel solution. Step S3: Perform freeze-thaw cycle treatment: Pour the hydrogel solution into a mold while hot, and perform 3 cycles of freeze-thaw treatment, i.e., freeze at -20℃ for 4 h, and then thaw at room temperature for 1 h to obtain the antibacterial / antioxidant hydrogel.

[0034] A third aspect of the present invention provides the application of the aforementioned antibacterial and antioxidant hydrogel in the preparation of wound dressings.

[0035] Compared with the prior art, the beneficial effects of the present invention are at least as follows:

[0036] (1) The present invention firstly provides a hydrogel with antibacterial and antioxidant functions; the matrix of the hydrogel is composed of polyvinyl alcohol and agarose, has a rich cross-linked pore structure, and the pore size distribution in the hydrogel is uniform, which endows the hydrogel with excellent mechanical properties.

[0037] (2) The hydrogel effectively prevents infection, removes free radicals, reduces oxidative stress, and weakens inflammatory response through the synergistic effect of antibacterial and antioxidant properties, thereby improving wound healing efficiency. The hydrogel prepared by this invention has a significant antibacterial effect on Escherichia coli and Staphylococcus aureus. It also has a high scavenging effect on highly active free radicals (·OH) and stable free radicals (DPPH free radicals).

[0038] (3) The present invention also provides a method for preparing the hydrogel, which is simple, convenient to operate and low in cost, and suitable for large-scale production. Attached Figure Description

[0039] The accompanying drawings are provided to further illustrate the present application and form part of the specification. They are used together with the embodiments of the present application to explain the present application, but do not constitute a limitation thereof. In the drawings:

[0040] Figure 1 Scanning electron microscope images of the antibacterial and antioxidant hydrogel prepared in Example 1;

[0041] Figure 2 The results of the inhibition zone experiment for Escherichia coli and Staphylococcus aureus;

[0042] Figure 3 OD of Escherichia coli (A) and Staphylococcus aureus (B)600 Experimental results;

[0043] Figure 4 Results of DPPH free radical scavenging experiments;

[0044] Figure 5 The results are from the ·OH free radical scavenging experiment. Detailed Implementation

[0045] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0046] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0047] Unless otherwise defined, the technical or scientific terms used in this invention should be interpreted in the manner of their ordinary meaning as understood by one of ordinary skill in the art. Techniques, methods, and apparatus known to those skilled in the art may not be discussed in detail, but where appropriate, such techniques, methods, and apparatus should be considered part of the specification.

[0048] All prior art documents cited in this specification are incorporated herein by reference in their entirety and are therefore part of the disclosure of this invention.

[0049] Example 1

[0050] An antibacterial and antioxidant hydrogel and its preparation method, comprising the following steps:

[0051] 1. Extraction of sericin: After cleaning 100g of silkworm cocoons, add 2000mL of deionized water and degumming at 120℃ for 4 hours. Then filter to remove solid residue and collect the filtrate. Next, extract the filtrate at 8000rpm. -1 Centrifuge for 10 min to obtain the supernatant, and finally freeze-dry the supernatant to obtain sericin.

[0052] 2. Dissolve 10g of polyvinyl alcohol and 1g of agarose in 83.92g of deionized water, and mechanically stir in a 90℃ water bath for 2-3 hours until completely dissolved. Then add 0.08g of tetramethylol phosphate and 5g of sericin to the above solution, and continue to mechanically stir in a 90℃ water bath for 1-2 hours until fully mixed to obtain a hydrogel solution;

[0053] 3. Pour the hydrogel solution into the mold while it is still hot, and perform 3 cycles of freeze-thaw treatment (each freeze-thaw cycle is frozen at -20℃ for 4 hours, and then thawed at room temperature for 1 hour) to obtain antibacterial and antioxidant hydrogel.

[0054] Scanning electron microscopy (SEM) analysis of the hydrogel prepared in Example 1 is as follows: Figure 1 As shown, the hydrogel prepared in Example 1 has a rich cross-linked porous structure, which is attributed to the cross-linking of polyvinyl alcohol and agarose through hydrogen bonding between hydroxyl groups, forming a matrix material with an interpenetrating network structure. Furthermore, the pore size distribution in the hydrogel is uniform, thus ensuring excellent mechanical properties.

[0055] Comparative Example 1

[0056] A hydrogel without antibacterial and antioxidant properties and its preparation method, comprising the following steps:

[0057] The steps for extracting sericin are the same as in Example 1;

[0058] 1. Dissolve 10g of polyvinyl alcohol and 1g of agarose in 89g of deionized water, and mechanically stir in a 90℃ water bath for 2-3 hours until completely dissolved to obtain a hydrogel solution.

[0059] 2. Pour the hydrogel solution into the mold while it is still hot, and perform 3 cycles of freeze-thaw treatment (each freeze-thaw cycle is frozen at -20℃ for 4 hours, and then thawed at room temperature for 1 hour) to obtain antibacterial and antioxidant hydrogel.

[0060] Comparative Example 2

[0061] A hydrogel without antibacterial and antioxidant properties and its preparation method, comprising the following steps:

[0062] The steps for extracting sericin are the same as in Example 1;

[0063] 1. Dissolve 10g of polyvinyl alcohol and 1g of agarose in 88.92g of deionized water and mechanically stir in a 90℃ water bath for 2-3 hours until completely dissolved. Then add 0.08g of tetramethylolphosphine sulfate to the above solution and continue mechanically stirring in a 90℃ water bath for 1-2 hours until fully mixed to obtain a hydrogel solution.

[0064] 2. Pour the hydrogel solution into the mold while it is still hot, and perform 3 cycles of freeze-thaw treatment (each freeze-thaw cycle is frozen at -20℃ for 4 hours, and then thawed at room temperature for 1 hour) to obtain antibacterial and antioxidant hydrogel.

[0065] Comparative Example 3

[0066] A hydrogel without antibacterial and antioxidant properties and its preparation method, comprising the following steps:

[0067] The steps for extracting sericin are the same as in Example 1;

[0068] 1. Dissolve 10g of polyvinyl alcohol and 1g of agarose in 84g of deionized water, and mechanically stir in a 90℃ water bath for 2-3 hours until completely dissolved. Then add 5g of sericin to the above solution and continue mechanically stirring in a 90℃ water bath for 1-2 hours until fully mixed to obtain a hydrogel solution.

[0069] 2. Pour the hydrogel solution into the mold while it is still hot, and perform 3 cycles of freeze-thaw treatment (each freeze-thaw cycle is frozen at -20℃ for 4 hours, and then thawed at room temperature for 1 hour) to obtain antibacterial and antioxidant hydrogel.

[0070] Test Example - Antibacterial Test

[0071] The antimicrobial properties of the hydrogel were evaluated using *Escherichia coli* and *Staphylococcus aureus*. Bacteria were inoculated into liquid LB medium and incubated at 37°C for 24 h. The cultured bacteria were centrifuged at 6000 rpm for 10 min, the supernatant was removed, and the bacteria were washed twice with sterile PBS (0.01 M, pH 7.3) to remove residual culture medium components. The collected bacteria were then resuspended in LB liquid medium to achieve a colony concentration of 1 × 10⁻⁶. 6 CFU mL -1 The experimental groups were: a) Comparative Example 1; b) Comparative Example 2; c) Comparative Example 3; d) Example 1. 100 μL of bacterial suspension was evenly spread on LB solid medium, and the hydrogel sample was placed in the center of the medium. The samples were incubated at 37°C for 24 h. The diameter of the inhibition zone was measured to assess the antibacterial activity of the hydrogel. Furthermore, bacterial growth was observed by measuring the absorbance at 600 nm at specified time intervals.

[0072] The antibacterial properties of the hydrogels obtained in the examples and comparative examples were tested under the same experimental conditions.

[0073] like Figure 2 As shown, Example 1 exhibited inhibition zones of 4.00 mm and 6.88 mm in diameter against *Escherichia coli* and *Staphylococcus aureus*, respectively, demonstrating a good antibacterial effect. Comparative Example 2 also showed inhibition zones, but no inhibition zones were observed in Comparative Examples 1 and 3. This indicates that the antibacterial effect of Example 1 is superior to that of the comparative examples. Further combining... Figure 3 It was also observed that Example 1 significantly inhibited the growth of both bacterial strains. This is likely because tetramethylol phosphate exerts a significant antibacterial effect by disrupting the bacterial cell membrane and damaging its internal structure.

[0074] In summary, the antibacterial and antioxidant hydrogel obtained by this invention has superior antibacterial properties and can effectively inhibit the growth of common bacteria such as Escherichia coli and Staphylococcus aureus.

[0075] Test Example - Antioxidant Experiment

[0076] DPPH and ·OH radical scavenging capacity are important indicators for assessing antioxidant capacity.

[0077] The hydrogel sample extract was prepared in accordance with Part 12 of GB / T16886 "Biological Evaluation of Medical Devices": Sample Preparation and Reference Materials.

[0078] For the DPPH free radical scavenging experiment, blank, sample determination, and sample control were set up according to Table 1. The solutions were added in sequence, mixed well, and allowed to stand at room temperature in the dark for 30 minutes. The solutions from each tube were then added to cuvettes in sequence, and the absorbance of each tube at 517 nm was measured using a spectrophotometer, with the instrument zeroed using anhydrous ethanol.

[0079] Table 1. Amounts of each component added

[0080]

[0081] DPPH clearance rate (%) = [A 空白 -(A 样品测定 -A 样品对照 )] / A 空白 ×100%

[0082] For the ·OH radical scavenging experiment, 0.5 mL of 9 mM salicylic acid, 0.5 mL of 9 mM ferrous sulfate, and 0.5 mL of 8.8 mM hydrogen peroxide were added to 1 mL of the extract, mixed well, and allowed to stand at room temperature in the dark for 30 min. The absorbance was then measured at 510 nm. Distilled water was used instead of the extract in the control group, and distilled water was used instead of salicylic acid in the blank group.

[0083] ·OH scavenging rate (%) = [A 对照 -(A 样品 -A 空白 )] / A 对照 ×100%

[0084] like Figure 4 As shown, the DPPH scavenging ability of Example 1 was 95.35%, significantly higher than that of Comparative Example 1 (53.98%), while Comparative Example 2 also showed DPPH scavenging ability. This may be because tetrahydroxymethylphosphonic acid contains multiple hydroxyl groups, which have a strong hydrogen donor capacity and can effectively release hydrogen atoms during the reaction with DPPH free radicals, thereby neutralizing the free radicals.

[0085] like Figure 5As shown, Example 1 exhibits a 57.44% scavenging capacity for ·OH. The antioxidant properties of sericin primarily stem from its amino acid composition, particularly its rich content of sulfur-containing amino acids, which can directly react with free radicals, effectively neutralizing oxidants. Simultaneously, the presence of polar amino acids increases the water solubility of sericin and its interaction with water molecules, thereby enhancing its antioxidant capacity. Therefore, the superior antioxidant capacity of Example 1 is mainly attributed to the synergistic effect of tetramethylolpropionate sulfate and sericin.

[0086] Example 1 demonstrates superior overall performance compared to Comparative Examples 1-3. In terms of antibacterial activity, it exhibits significant inhibitory effects against both *Escherichia coli* and *Staphylococcus aureus*. Regarding antioxidant activity, it shows high scavenging effects against both highly reactive free radicals (·OH) and stable free radicals (DPPH radicals). In contrast, while Comparative Example 2 possesses certain antibacterial properties and a relatively high scavenging effect against stable free radicals (DPPH radicals), its scavenging effect against highly reactive free radicals (·OH) is poor. Comparative Example 3 exhibits poor antibacterial activity, and its antioxidant indicators are not particularly outstanding.

[0087] In summary, this invention provides an antibacterial and antioxidant hydrogel. The matrix of this hydrogel is polyvinyl alcohol and agarose, which together endow it with mechanical properties. The active ingredients, tetramethylolpropionate sulfate (TMS) and sericin, are uniformly dispersed in the matrix. By combining the unique properties of sericin with innovative material design, a more efficient and safer biomaterial has been developed to meet the needs of modern medicine for wound dressings. The antibacterial activity of TMS mainly occurs by disrupting the bacterial cell membrane structure, damaging bacterial proteins and nucleic acids, and it also possesses anti-inflammatory capabilities against skin wounds. Sericin effectively scavenge free radicals such as DPPH and ·OH, reducing oxidative stress levels and thus enhancing the antioxidant properties of the hydrogel. Using the prepared antibacterial and antioxidant hydrogel as a dressing can effectively prevent bacterial infection, scavenge free radicals in wounds, reduce oxidative stress levels, alleviate inflammatory responses, and thus accelerate wound healing. The preparation method of this hydrogel is simple, convenient, and low-cost, making it suitable for large-scale production.

[0088] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. An antibacterial and antioxidant hydrogel, characterized in that, The matrix of the hydrogel includes polyvinyl alcohol and agarose; the active ingredients are tetramethylol phosphate sulfate and sericin, and the active ingredients are uniformly dispersed in the matrix. In the hydrogel, tetrahydroxymethyl phosphate and sericin work synergistically to exert antioxidant functions; The hydrogel comprises the following components: 5-15% polyvinyl alcohol, 0.5-3% agarose, 0.01-0.08% tetramethylolphosphine sulfate and 0.01-5% sericin, with the balance being deionized water.

2. The method for preparing the antibacterial and antioxidant hydrogel according to claim 1, characterized in that, The preparation method includes the following steps: S1. Extraction of sericin: After cleaning the silkworm cocoons, add deionized water and degumming them under high temperature and high pressure; filter and separate, collect the filtrate, then centrifuge and collect the supernatant, and freeze-dry to obtain sericin; S2. Preparation of hydrogel solution: Dissolve polyvinyl alcohol and agarose in deionized water, heat and stir until completely dissolved; then add tetramethylolphosphine sulfate and sericin, continue heating and stirring to mix thoroughly to obtain hydrogel solution; S3. Freeze-thaw cycle treatment: Pour the hydrogel solution obtained in step S2 into the mold while it is still hot, and perform a freeze-thaw cycle treatment.

3. The method for preparing the antibacterial and antioxidant hydrogel according to claim 2, characterized in that, In step S1, the degumming process is carried out at a temperature of 100-140℃ for a time of 0.5-8 hours. And / or, in step S1, the centrifugation process is carried out at a speed of 6000-12000 rpm for a processing time of 5-15 min.

4. The method for preparing the antibacterial and antioxidant hydrogel according to claim 2, characterized in that, In step S2, the heating and dissolving process is carried out at a temperature of 70-100℃ for 2-3 hours. And / or, in step S2, the heating and mixing process is carried out at a temperature of 70-100℃ and a stirring time of 1-3 hours.

5. The method for preparing the antibacterial and antioxidant hydrogel according to claim 2, characterized in that, In step S2, the polyvinyl alcohol is type 1799 with a degree of alcoholysis of 98%-99%. And / or, in step S2, the tetrahydroxymethylphosphonic acid is a 75% aqueous solution.

6. The method for preparing the antibacterial and antioxidant hydrogel according to claim 2, characterized in that, In step S3, the freeze-thaw cycle is repeated 2-5 times; And / or, in step S3, the cyclic freeze-thaw treatment step is to freeze at ≤-20℃ for 3-6 hours, and then thaw at room temperature for 0.5-2 hours.

7. The method for preparing the antibacterial and antioxidant hydrogel according to claim 2, characterized in that, In step S1, the protein content of the sericin is 87.23%-95.37%.

8. The method for preparing the antibacterial and antioxidant hydrogel according to claim 2, characterized in that, The silkworm cocoons used in step S1 are the dried outer shells of silkworm cocoons after the cicada pupae have been removed.

9. The use of the antibacterial and antioxidant hydrogel of claim 1, or the antibacterial and antioxidant hydrogel prepared by any one of claims 2-8, in the preparation of wound dressings.

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