Preparation and application of photo-thermal antibacterial casein hydrogel synthesized in situ with silver nanoparticles

Abstract

The present application relates to the technical field of biomedical materials, and particularly relates to a preparation method of a photo-thermal antibacterial casein hydrogel loaded with in-situ biomimetic nanosilver and application thereof in the field of biomedical materials.The present application discloses a preparation method of an antibacterial casein hydrogel precursor solution, which comprises the following steps: S1, preparation of modified casein; S2, antibacterial casein hydrogel precursor solution for in-situ synthesis of nanosilver.The antibacterial casein hydrogel precursor solution is irradiated under blue light (405 nm) to obtain an antibacterial casein hydrogel, which can be used as a gel-type medical antibacterial material.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, specifically to a method for preparing a photothermal antibacterial casein hydrogel loaded with in-situ biomineralized silver nanoparticles and its application in the field of biomedical materials. Background Technology

[0002] Bacterial infection and skin wound healing are common and unavoidable problems in daily life. Wound dressings can act as a partial skin barrier to prevent secondary damage and provide a favorable microenvironment for wound healing. Currently, various wound dressings have been designed, such as hydrogels, foams, skin patches, films, nanoparticles, hydrocolloids, nanofibers, and membranes. Among them, hydrogel dressings are a novel type of wound dressing developed in recent years, possessing excellent flexibility, adjustability, hygroscopicity, and biodegradability, making them one of the most competitive candidate materials for wound dressings. Studies have shown that mixed infections of multiple pathogens are observed in most infected wounds, which has promoted the development of multifunctional hydrogels targeting common pathogens in wounds. However, most hydrogel dressings lack antibacterial properties, limiting their clinical application. Therefore, by effectively combining antibacterial materials with hydrogel dressings, the ability of antibacterial materials to resist bacterial infection can be effectively endowed.

[0003] To prepare multifunctional hydrogel dressings, antibiotics are encapsulated within a three-dimensional network structure of the hydrogel to achieve sustained and controlled release. However, the overuse of antibiotics leads to the emergence of drug-resistant microorganisms. In light of this, silver nanoparticles are introduced into hydrogel dressings to achieve effective antibacterial effects. AgNPs (silver nanoparticles) possess multiple antibacterial mechanisms: (i) inhibiting bacterial reproduction by interfering with DNA replication and RNA production, (ii) disrupting cell membranes, (iii) attacking reaction chains in bacterial mitochondria, and (iv) inactivating enzyme activity. Typically, reducing agents such as NaBH4, citrate, glucose, hydrazine, and ascorbate are used to prepare AgNPs, and stabilizers are required to prevent AgNP aggregation. However, these preparation methods have high potential hazards and may harm the environment and human health.

[0004] Traditional methods of mixing AgNPs with pregelated polymers have several limitations: i) residual potentially toxic agents from the AgNP synthesis process; ii) uneven distribution of AgNPs during mixing; and iii) the risk of efficacy loss due to AgNP aggregation and oxidation during routine storage. Therefore, improvements to existing techniques are needed.

[0005] In-situ biomineralization is an efficient and environmentally friendly process that promotes the bonding of inorganic materials with organic macromolecules. In particular, the synthesis of metallic nanostructures using biomineralizing agents (such as proteins and other hydrophilic polymers) has attracted considerable interest due to their excellent biocompatibility, conformal interfaces, uniform distribution, and high stability. Strategies including repeated freeze-thaw cycles, ultraviolet (UV) irradiation, and alkaline conditions have been integrated to promote the biomineralization of various proteins in AgNP synthesis.

[0006] For a long time, it has been known that milk-derived proteins can promote bone mineralization, but there have been no reports of casein mineralization for the synthesis of silver nanoparticles. As one of the main milk proteins, bovine casein contains a large amount of tryptophan, tyrosine, histidine, and proline residues, which endows it with certain reducing properties. In addition, casein has good biocompatibility and biodegradability, thus it is considered an ideal biomedical material. However, like most proteins, pure casein powder easily becomes a breeding ground for bacteria in humid environments. These problems greatly limit the application range and market competitiveness of casein products. Therefore, developing casein antibacterial materials with good biocompatibility, certain mechanical properties, shape, and stability, and fully utilizing the advantages of casein, has significant economic and social benefits.

[0007] CN113694250 discloses a method for preparing a gellan gum antibacterial hydrogel loaded with silver nanoparticles, which significantly enhances the antibacterial effect of the gel and improves its mechanical strength by modifying the gelation method, thus solving the problem of antibacterial gel dressings not adhering to the skin. However, the preparation process is relatively cumbersome. Sodium borohydride is used as a reducing agent in the synthesis of silver nanoparticles, and sodium borohydride is a hazardous chemical with a certain degree of toxicity. Pre-made hydrogel dressings are difficult to adapt to wounds of various shapes and depths.

[0008] CN105903057 discloses a method for preparing a nano-silver hybrid sericin porous gel antibacterial material. While the method is simple, uses mild conditions, and is environmentally friendly, the sericin porous gel requires at least 10 minutes of sealed irradiation under ultraviolet light to form nano-silver, followed by a prolonged drying process to obtain the nano-silver hybrid sericin porous gel antibacterial material. The entire preparation process is time-consuming, and sericin protein is expensive and difficult to extract. The final form is a dry, porous, sponge-like dressing, which is difficult to adhere to a wound.

[0009] CN106270548 discloses a method for in-situ green synthesis of sericin-nano silver using sericin protein. The method involves adding silver nitrate solution to a sericin solution and then reacting it under natural light until the solution turns yellow, thus obtaining sericin-nano silver. However, the reaction time is 24-36 hours, which is quite time-consuming. Summary of the Invention

[0010] The technical problem to be solved by the present invention is to provide a method for preparing and applying photothermal antibacterial casein hydrogels with in-situ synthesized nanosilver.

[0011] To address the aforementioned technical problems, this invention provides a method for preparing an antibacterial casein hydrogel precursor solution, comprising the following steps:

[0012] S1. Preparation of modified casein:

[0013] Methacrylic anhydride and casein were reacted in PBS buffer at room temperature (20-25°C) for 20-28 hours, with a ratio of methacrylic anhydride to casein of 1 ± 0.1 ml / 5 g. The resulting solution was filtered (or diluted and then filtered) to remove impurities, and then lyophilized to obtain modified casein powder (i.e., methacrylamide casein).

[0014] The filtration is ultrafiltration;

[0015] S2, Antibacterial casein hydrogel precursor solution for in-situ synthesis of silver nanoparticles:

[0016] The modified casein powder obtained in S1 was dissolved in PBS buffer to prepare a protein solution with a concentration of 8-15 g / 100 ml. Then, under light-protected conditions, photoinitiator LAP, resveratrol, and silver nitrate were added until the concentration of LAP was 0.01-0.5 g / 100 ml, the concentration of resveratrol was 0.1-1 mg / ml, and the concentration of silver nitrate was 1-2 mmol / L, thus obtaining an antibacterial casein hydrogel precursor solution (i.e., a photothermal antibacterial casein hydrogel precursor solution for in-situ synthesis of silver nanoparticles).

[0017] As an improvement to the preparation method of the antibacterial casein hydrogel precursor solution of the present invention, step S1 is:

[0018] Casein powder was dissolved in 1×PBS buffer (0.01M, pH 7-8) at room temperature (20-25℃) to prepare a casein powder stock solution with a concentration of 5±0.5g / 100mL.

[0019] According to the ratio of methacrylic anhydride to casein powder of 1±0.1ml / 5g, methacrylic anhydride was added to the casein powder stock solution, the pH was adjusted to 8.0±0.2, and the reaction was carried out by stirring at room temperature for 22-26h. The resulting solution was diluted with 2±0.2 times the volume of deionized water and then ultrafiltered. The retentate after ultrafiltration was freeze-dried (dried at -45 to -55℃ for 16-24h). The resulting freeze-dried powder is modified casein powder.

[0020] As a further improvement to the preparation method of the antibacterial casein hydrogel precursor solution of the present invention, step S1 is:

[0021] The ultrafiltration rejection capacity is 2000 Da;

[0022] pH was adjusted using a sodium hydroxide solution filtered through a 0.22 μm filter membrane (5 mol / L sodium hydroxide solution filtered through a 0.22 μm filter membrane was used to adjust the pH).

[0023] As a further improvement to the preparation method of the antibacterial casein hydrogel precursor solution of the present invention, step S2 is:

[0024] Modified casein powder was dissolved in 1×PBS buffer (0.01M, pH 7-8) to prepare a protein solution with a concentration of 10g / 100ml, LAP concentration of 0.05g / 100ml, resveratrol concentration of 0.5mg / mL, and silver nitrate concentration of 1-1.5mmol / L.

[0025] This invention also provides a method for preparing photothermal antibacterial casein hydrogel of in-situ biomineralized silver nanoparticles, using the antibacterial casein hydrogel precursor solution as described above.

[0026] The antibacterial casein hydrogel precursor solution was placed under blue light (405 nm) for 4–6 min to obtain the antibacterial casein hydrogel.

[0027] An improvement to the preparation method of the in-situ biomineralized nanosilver photothermal antibacterial casein hydrogel of the present invention: blue light intensity of 60±10mW / cm 2 .

[0028] The present invention also provides the use of the above-mentioned photothermal antibacterial casein hydrogel as a gel-type medical antibacterial material (e.g., a hydrogel wound dressing).

[0029] Addressing the limitations and technical challenges of traditional methods for mixing AgNPs with pregelated polymers, this invention promotes the binding of AgNPs to casein through in-situ biomineralization. This results in AgNPs exhibiting excellent biocompatibility, conformal interface, uniform distribution, and high stability.

[0030] The present invention relates to an in-situ biomineralized nanosilver photothermal antibacterial casein hydrogel, wherein the hydrogel uses modified casein as a structural framework and nanosilver is synthesized and distributed in-situ within the hydrogel.

[0031] This invention provides a highly efficient, in-situ, sequential photoactivated antibacterial hydrogel wound dressing based on protein biomineralization. To screen suitable proteins, several commonly used protein materials were considered as candidates. Casein exhibits excellent photoactivated biomineralization capabilities for AgNP formation. Resveratrol was introduced to further enhance the mineralization rate of in-situ AgNP formation. Casein was methacrylated to enhance the mechanical properties of the resulting hydrogel. Furthermore, a photosensitive free radical generator (LAP) has the potential to further accelerate AgNP formation. Under the first blue light irradiation, the cross-linking polymerization of modified casein and the formation of AgNPs are completed simultaneously, and the second NIR irradiation further enhances the bactericidal efficacy. The sequentially photoactivated nano-silver casein hydrogel prepared by this invention shows promising application prospects as an infected wound dressing.

[0032] This invention prepares an in-situ synthesized antibacterial hydrogel of nano-silver based on methacrylic acid modified casein through biomineralization and other methods, transforming it into a bioactive medical material that can be used as a gel-type medical antibacterial material.

[0033] The S1 modified casein of this invention enables the large-scale preparation of methacrylated casein.

[0034] The hydrogel preparation process involved in this invention is simple and easy to implement, with mild reaction conditions, stable product properties, and is suitable for industrial production.

[0035] The photothermal properties of the in-situ synthesized antibacterial casein hydrogel with nano-silver: The in-situ synthesized antibacterial casein hydrogel with nano-silver was irradiated with a near-infrared (NIR) laser (808 nm, 3 W cm⁻¹). -2 After 10 minutes, the temperature rises to over 50℃. This photothermal effect has an auxiliary bactericidal effect.

[0036] In summary, this invention prepares an in-situ synthesized antibacterial hydrogel of nano-silver based on methacrylic acid-modified casein through biomineralization and other methods, transforming it into a bioactive medical material that can be used as a gel-type medical antibacterial material.

[0037] This invention has the following advantages:

[0038] 1) This invention utilizes the high reducing power of resveratrol to enhance the biomineralization ability of casein, thereby forming AgNPs in situ. The highly reactive free radicals generated by the homolytic cleavage of the photoinitiator Lap can also accelerate the synthesis of AgNPs in the hydrogel.

[0039] Due to its strong reducing properties, resveratrol is introduced into the biomineralization system to assist in the synthesis of AgNP. The addition of resveratrol endows the in-situ biomineralized silver nanoparticle antibacterial casein hydrogel with good antioxidant and antioxidant capabilities, which helps to clear inflammation in wound tissue.

[0040] 2) Nano-silver particles, as inorganic antibacterial agents, are widely used due to their high efficiency, safety, and broad-spectrum antibacterial effects. However, traditional nano-silver synthesis processes suffer from drawbacks such as residual potentially toxic agents and easy oxidation. This invention utilizes the biomineralization of casein to synthesize nano-silver in situ, significantly reducing the biotoxicity of silver ions while simultaneously endowing the hydrogel with antibacterial properties.

[0041] 3) Nanosilver has a high absorption rate for near-infrared light, which can be effectively converted into heat. The introduction of nanosilver provides satisfactory photothermal behavior for in-situ biomineralized nanosilver antibacterial casein hydrogels, which helps to inactivate bacteria.

[0042] In summary, the purpose of this invention is to provide a highly efficient, in-situ, sequential photoactivated antibacterial hydrogel wound dressing based on casein biomineralization. Under blue light irradiation, casein hydrogel and silver nanoparticles (AgNPs) form simultaneously, with the entire process taking only about 5 minutes. The photocuring gelation is suitable for irregular wound shapes. The in-situ biomineralized silver nanoparticles in the photothermal antibacterial casein hydrogel can undergo photothermal conversion under near-infrared irradiation, greatly enhancing its antibacterial effect. The entire preparation process is highly operable, involves green synthesis, features rapid biomineralization, and has low material and equipment costs. Attached Figure Description

[0043] The specific embodiments of the present invention will be further described in detail below with reference to the accompanying drawings.

[0044] Figure 1 Photograph of the in-situ biomineralized silver nanoparticles antibacterial casein hydrogel of Example 1;

[0045] Figure 2 The UV-Vis absorption spectrum of in-situ biomineralized silver nanoparticles from Experiment 1 is shown.

[0046] Figure 3 Transmission electron microscope image of in-situ biomineralized silver nanoparticles from Experiment 2;

[0047] Figure 4 The image shows the microstructure of the in-situ biomineralized silver nanoparticle antibacterial casein hydrogel from Experiment 3.

[0048] Figure 5 The antioxidant properties of the in-situ biomineralized silver nanoparticle antibacterial casein hydrogel in Experiment 4;

[0049] Figure 6 Cell compatibility of the in-situ biomineralized silver nanoparticle antibacterial casein hydrogel in Experiment 5;

[0050] Figure 7The in vitro photothermal properties of the in-situ biomineralized silver nanoparticle antibacterial casein hydrogel in Experiment 6 were studied.

[0051] Figure 8 The in vivo photothermal properties of the in-situ biomineralized silver nanoparticle antibacterial casein hydrogel in Experiment 7.

[0052] Figure 9 To investigate the antibacterial properties of the in-situ biomineralized nanosilver photothermal antibacterial casein hydrogel in Experiment 8. Detailed Implementation

[0053] The present invention will be further described below with reference to embodiments, but the content of the present invention is not limited thereto.

[0054] In this invention, PBS refers to 1×PBS buffer (0.01M, pH 7-8).

[0055] The casein powder is food-grade casein powder (purity ≥90%).

[0056] Example 1: A method for preparing an in-situ biomineralized silver nanoparticle photothermal antibacterial casein hydrogel:

[0057] S1. Preparation of modified casein:

[0058] Dissolve casein powder thoroughly in 1×PBS buffer (0.01M, pH 7-8) at room temperature (20-25℃) to prepare a casein powder stock solution with a concentration of 5g / 100mL.

[0059] Methacrylic anhydride was added to the casein powder stock solution at a ratio of 1 ml / 5 g (v / w). The pH was adjusted to 8.0 using a 5 mol / L sodium hydroxide solution filtered through a 0.22 μm filter membrane. The mixture was stirred continuously at 20–25 °C for 24 h. The resulting solution was diluted with two volumes of deionized water and then ultrafiltered (with a cutoff of 2000 Da).

[0060] The retentate after ultrafiltration was freeze-dried (drying at -45 to -55°C for 16 to 24 hours) to obtain a modified casein powder.

[0061] Note: The purpose of ultrafiltration is to remove residual impurities (including unreacted modifiers, solvents, or byproducts).

[0062] S2, In-situ synthesis of antibacterial casein hydrogel precursor solution for nano-silver:

[0063] The modified casein powder obtained in step S1 above was dissolved in 1×PBS buffer (0.01M, pH 7-8) to obtain a protein solution with a concentration of 10g / 100ml. Then, under light-protected conditions, lithium phenyl 2,4,6-trimethylbenzoylphosphinic acid (LAP) was added as a photoinitiator to a concentration of 0.05g / 100ml, resveratrol was added to a concentration of 0.5mg / mL, and silver nitrate was added to a concentration of 1mM. The mixture was then stirred to obtain a photothermal antibacterial casein hydrogel precursor solution for in-situ synthesis of nano-silver; referred to as antibacterial casein hydrogel precursor solution.

[0064] S3. Preparation of antibacterial casein hydrogel with in-situ synthesized nano-silver:

[0065] Irradiating the antibacterial casein hydrogel precursor solution obtained from S2 with blue light (405nm) for 5 minutes yields an in-situ biomineralized casein hydrogel of nano-silver, abbreviated as antibacterial casein hydrogel (CASMA-Ag1), with a blue light intensity of 60mW / cm². 2 The morphology of the resulting antibacterial casein hydrogel (CASMA-Ag1) is as follows: Figure 1 .

[0066] The distance between the blue light source and the antibacterial casein hydrogel precursor solution is 1 cm.

[0067] Example 2, compared to Example 1, makes the following changes:

[0068] The concentration of silver nitrate was changed from 1 mM to 0.5 mM, while the rest remained the same as in Example 1. The resulting product was named antibacterial casein hydrogel (CASMA-Ag0.5).

[0069] Example 3: The following changes are made compared to Example 1:

[0070] The concentration of silver nitrate was changed from 1 mM to 1.5 mM, while the rest remained the same as in Example 1. The resulting product was named antibacterial casein hydrogel (CASMA-Ag1.5).

[0071] Blank Comparative Example 1: The following changes were made compared to Example 1:

[0072] The use of silver nitrate was omitted; that is, the concentration of silver nitrate was changed from 1 mM to 0 mM, while the rest remained the same as in Example 1. The resulting product was named casein hydrogel (CASMA-Ag0).

[0073] Blank Comparative Example 2, with the following changes compared to Example 1:

[0074] The use of silver nitrate and resveratrol was omitted; that is, the concentration of silver nitrate was changed from 1 mM to 0 mM, and the concentration of resveratrol was changed from 0.5 mg / mL to 0 mg / mL, while the rest remained the same as in Example 1. The resulting product was named casein hydrogel (Casein-Ma).

[0075] Experiment 1. UV-Vis spectrum of photothermal antibacterial casein hydrogel of in-situ biomineralized silver nanoparticles:

[0076] The antibacterial casein hydrogel precursor solution (100 μL) obtained in S2 of Examples 1-3 was added to a 96-well plate, and then according to S3, the antibacterial casein hydrogel (CASMA-Ag0.5, CASMA-Ag1, CASMA-Ag1.5) was obtained by scanning the adsorption spectrum of the microplate reader at a wavelength of 300-700 nm. Figure 2 The results showed that a characteristic peak of silver nanoparticles appeared at 410 nm, and the intensity of the peak reflected the content of synthesized silver nanoparticles to some extent. The content of silver nanoparticles in CASMA-Ag1 and CASMA-Ag1.5 hydrogels was significantly higher than that in CASMA-Ag0.5 hydrogel.

[0077] Experiment 2. Size and morphology of in-situ biomineralized silver nanoparticles:

[0078] The hydrogel samples (CASMA-Ag0.5, CASMA-Ag1, CASMA-Ag1.5) obtained in Examples 1-3 were hydrolyzed with proteinase K to release AgNPs. The specific hydrolysis reaction was as follows: 1g of hydrogel sample was added to 5mL of proteinase K solution (200μg / mL) and incubated at 37℃ for 24 hours to obtain hydrogel hydrolysate.

[0079] Centrifuge the hydrolyzed solution (12000 g min). -1 The precipitate was retained for 20 min and washed three times with ultrapure water. The size and morphology of AgNPs in the hydrogel were determined by transmission electron microscopy at a working voltage of 200 kV. Figure 3 As shown, the silver nanoparticles synthesized by in-situ biomineralization have uniform morphology. The AgNPs prepared by CASMAAg0.5 have a size of 5–50 nm, the AgNPs prepared by CASMA-Ag1 have a size of 10–70 nm, and the AgNPs prepared by CASMA-Ag1.5 have a size of 5–90 nm.

[0080] Experiment 3. Microstructure of photothermal antibacterial casein hydrogel of in-situ biomineralized silver nanoparticles:

[0081] The hydrogel samples (CASMA-Ag0.5, CASMA-Ag1, CASMA-Ag1.5) obtained in Examples 1-3 were frozen with liquid nitrogen, dried in a freeze dryer, and then sprayed with platinum on the cross-sections. The samples were observed using a field emission scanning electron microscope (SEM). The resulting SEM images are shown below. Figure 4 As shown, the gel has a uniform porous structure inside.

[0082] Experiment 4. Antioxidant properties of in-situ biomineralized silver nanoparticles and antibacterial casein hydrogels:

[0083] 0.2 g of the in-situ biomineralized nanosilver photothermal antibacterial casein hydrogels (CASMA-Ag0.5, CASMA-Ag1, CASMA-Ag1.5) obtained in Examples 1-3 were soaked in 1 ml of PBS and incubated at 37°C for 12 h to obtain the extract.

[0084] In addition, the casein hydrogel without silver and resveratrol (Casein-Ma) obtained from blank comparative example 2 and the casein hydrogel without silver (CASMA-Ag0) obtained from blank comparative example 1 were used as controls.

[0085] The antioxidant activity of these extracts was assessed using the Total Antioxidant Capacity Assay Kit (Beyotime, China) based on the ABTS method. Results are expressed as Trolox equivalents (mM). Figure 5 The results showed that the antioxidant activities of CASMA-Ag0, CASMA-Ag0.5, CASMA-Ag1, and CASMA-Ag1.5 were significantly higher than those of Casein-Ma, indicating that the introduction of resveratrol significantly enhanced the antioxidant capacity. With the addition of Ag+, the antioxidant activity of the hydrogel decreased, indicating that the in-situ synthesis of AgNPs consumed the antioxidant activity of the hydrogel. However, these results indicate that the nano-silver antibacterial casein hydrogel still possesses good antioxidant activity.

[0086] Note: The synthesis of nano-silver consumes some of the reducing power of the original hydrogel, but the oxidizing power of CASMA-Ag0.5, CASMA-Ag1, and CASMA-Ag1.5 is still significantly higher than that of the casein-Ma group.

[0087] Experiment 5. Cell compatibility of in-situ biomineralized silver nanoparticles with antibacterial casein hydrogels:

[0088] The in-situ biomineralized silver nanoparticles photothermal antibacterial casein hydrogels (CASMA-Ag0.5, CASMA-Ag1, CASMA-Ag1.5) prepared in Examples 1-3 were extracted using DMEM standard culture medium. The mass-to-volume ratio of hydrogel to culture medium was 1 g / 20 ml, the extraction time was 24 h, and the extraction temperature was 37 °C. This yielded a methacrylamide whey protein hydrogel extract.

[0089] Mouse epidermal fibroblasts were cultured, and cell suspensions were obtained. Cell counts were performed, and the cell suspensions were diluted to 50,000 / mL according to the cell count. 100 μL of the cell suspension was seeded into each well of a 96-well plate and cultured for 12 h to allow cell adhesion to the bottom of the plate. The original culture medium was removed, and 100 μL of hydrogel extraction buffer was added to each well of the experimental group. 100 μL of DMEM medium was added to each well of the control group (Norml), and 100 μL of DMEM medium containing 1 mM silver nitrate was added to each well of the experimental group (1 mM Ag+). The culture plates were incubated at 37°C for 24 h. DMEM medium containing 10% CCK-8 was prepared and added to the wells via medium replacement. The culture plates were incubated at 37°C for 2 h. The absorbance (OD) at 450 nm was measured using a microplate reader. Figure 6 As shown, the introduction of 1 mM Ag+ significantly reduced cell viability, and the cytocompatibility of CASMA-Ag0.5 and CASMA-Ag1 was significantly stronger than that of CASMA-Ag1.5. After Ag+ was mineralized in situ to form AgNPs, cytotoxicity was significantly reduced, and AgNPs were successfully embedded in the casein hydrogel backbone, further improving the biosafety of AgNPs.

[0090] Experiment 6. In vitro photothermal properties of in-situ biomineralized silver nanoparticles with antibacterial properties of casein hydrogel:

[0091] The antibacterial casein hydrogel precursor solution (200 μL) obtained in S2 of Examples 1-3 was added to a 48-well plate, and then according to S3, antibacterial casein hydrogels (CASMA-Ag0.5, CASMA-Ag1, CASMA-Ag1.5) were obtained and their in vitro photothermal properties were tested.

[0092] The antibacterial casein hydrogel precursor solution (200 μL) obtained in S2 of blank comparative example 1 was added to a 48-well plate, and then casein hydrogel (CASMA-Ag0) was obtained according to S3 and its in vitro photothermal properties were tested.

[0093] The above hydrogel was placed in an 808nm NIR laser (3W cm⁻¹). -2 (10 min). At the same time, infrared imaging equipment was used to monitor temperature changes and acquire photothermal images. Figure 7 The results show that the temperatures of CASMA-Ag0 and CASMA-Ag0.5 hydrogels in the near-infrared radiation (3W cm⁻¹) are... -2 The temperature did not increase significantly, which is related to the content of nano-silver in the hydrogel. The temperature of CASMA-Ag1 hydrogel increased by 37.6℃ within 10 minutes, and the temperature of CASMA-Ag1.5 hydrogel increased by 40.6℃ within 10 minutes, indicating that the introduction of AgNPs provides satisfactory photothermal behavior for the hydrogel.

[0094] Experiment 8. In vivo photothermal properties of in-situ biomineralized silver nanoparticles with antibacterial properties of casein hydrogel:

[0095] Mice (C57BL) were anesthetized with pentobarbital (30 mg / kg body weight) and their back hair was removed. A full-thickness circular wound with a diameter of 6 mm was formed on the skin of the back. The antibacterial casein hydrogel precursor solution (50 μL) obtained in step 2) of Example 1 was dropped onto the wound. After irradiation with blue light (405 nm) for 5 min, an in-situ biomineralized silver nanoparticle casein hydrogel was formed. The wound was then placed under an 808 nm NIR laser (3W cm⁻¹). -2 (10 min). Simultaneously, infrared imaging equipment was used to monitor temperature changes and acquire photothermal images. For example... Figure 8 As shown, CASMA-Ag1 hydrogel increased the temperature of the mouse wound area from 30.4°C to 53.7°C within 10 minutes under NIR.

[0096] Note: Blue light intensity is approximately 60mW / cm². 2 The distance between the blue light source and the antibacterial casein hydrogel precursor solution is approximately 1 cm.

[0097] Experiment 7. Antibacterial properties of photothermal antibacterial casein hydrogels of in-situ biomineralized silver nanoparticles:

[0098] To evaluate the antibacterial activity with and without NIR irradiation, 200 μL of the antibacterial casein hydrogel precursor solution obtained in step 2) of Examples 1-3 was added to a 48-well plate and irradiated with blue light for 5 min to prepare in-situ biomineralized silver nanoparticle casein hydrogels (CASMA-Ag0.5, CASMA-Ag1, CASMA-Ag1.5). 50 μL of Escherichia coli (ATCC8099) or Staphylococcus aureus (ATCC 25923) bacterial suspension diluted with PBS (10 7 CFU mL -1 Add to each well, and with or without NIR (808nm, 3W cm⁻¹) -2Incubate in each well for 10 min. Using PBS as a blank control, incubate at 37°C for 4 hours, then resuspend the bacteria and dilute 10⁻⁶ in PBS. -2 The cells were then inoculated onto LB agar for 24 hours. The colony-forming units (CFU / mL) value per milliliter was calculated for each group. Figure 9 As shown, compared with the PBS control group, the nano-silver casein hydrogel exhibited significant bactericidal effects against both *Escherichia coli* and *Staphylococcus aureus*. Near-infrared light irradiation (3W cm⁻¹) -2 After 10 min, almost all Escherichia coli (99.1%) and Staphylococcus aureus (97.9%) in the CASMA-Ag1 and CASMA-Ag1.5 treatment groups were inactivated by photothermal hydrogel. These results indicate that the designed nanosilver hydrogel exhibits strong antibacterial activity under the synergistic effect of continuous UV and near-infrared light activation.

[0099] In summary, compared to CASMA-Ag0.5 and CASMA-Ag1.5, CASMA-Ag1 combines antioxidant properties, biocompatibility, and antibacterial activity. When converted into a bioactive medical material, it can be used as a gel-type medical antibacterial material.

[0100] Comparative Example 1: The casein powder in Example 1 was replaced with the protein materials listed in Table 1 below, with the amount remaining unchanged, to obtain the corresponding modified protein materials for preparing hydrogel precursor solutions. The rest was the same as in Example 1.

[0101] The comparison of the ability of different proteins to synthesize silver nanoparticles is shown in Table 1 below.

[0102] Table 1

[0103]

[0104] Comparative Example 2: The resveratrol in Example 1 was replaced with the reducing agent listed in Table 2 below, with the amount remaining unchanged, and the rest being the same as in Example 1. The resulting performance comparison is shown in Table 2 below.

[0105] Table 2

[0106]

[0107]

[0108] Finally, it should be noted that the above examples are merely some specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments and many variations are possible. All variations that can be directly derived or conceived by those skilled in the art from the disclosure of the present invention should be considered within the scope of protection of the present invention.

Claims

1. A method for preparing an antibacterial casein hydrogel precursor solution, characterized in that... Includes the following steps: S1. Preparation of modified casein: Methacrylic anhydride and casein were reacted in PBS buffer at room temperature for 20-28 h, with a ratio of methacrylic anhydride to casein of 1 ± 0.1 ml / 5 g. The resulting solution was filtered to remove impurities and then lyophilized to obtain modified casein powder. S2, Antibacterial casein hydrogel precursor solution for in-situ synthesis of silver nanoparticles: The modified casein powder obtained in S1 was dissolved in PBS buffer to prepare a protein solution with a concentration of 8-15 g / 100 ml. Then, under light-protected conditions, photoinitiator LAP, resveratrol, and silver nitrate were added until the concentration of LAP was 0.01-0.5 g / 100 ml, the concentration of resveratrol was 0.1-1 mg / ml, and the concentration of silver nitrate was 1-2 mmol / L, thus obtaining an antibacterial casein hydrogel precursor solution.

2. The method for preparing an antibacterial casein hydrogel precursor solution according to claim 1, characterized in that... S1 is: Casein powder was dissolved in 1×PBS buffer at room temperature to prepare a casein powder stock solution with a concentration of 5±0.5 g / 100 mL; According to the ratio of methacrylic anhydride to casein powder of 1 ± 0.1 ml / 5 g, methacrylic anhydride was added to the casein powder stock solution, the pH was adjusted to 8.0 ± 0.2, and the reaction was carried out by stirring at room temperature for 22-26 h. The resulting solution was diluted with 2 ± 0.2 times the volume of deionized water and then ultrafiltered. The retentate after ultrafiltration was freeze-dried, and the resulting freeze-dried powder was modified casein powder.

3. The method for preparing an antibacterial casein hydrogel precursor solution according to claim 2, characterized in that... In S1: The ultrafiltration rejection capacity is 2000 Da; pH was adjusted using sodium hydroxide solution filtered through a 0.22 μm filter membrane.

4. A method for preparing an antibacterial casein hydrogel precursor solution according to any one of claims 1 to 3, characterized in that... S2 is: Modified casein powder was dissolved in 1×PBS buffer to prepare a protein solution with a concentration of 10 g / 100 ml, LAP concentration of 0.05 g / 100 ml, resveratrol concentration of 0.5 mg / mL, and silver nitrate concentration of 1~1.5 mmol / L.

5. A method for preparing photothermal antibacterial casein hydrogel of in-situ biomineralized silver nanoparticles, characterized in that: The antibacterial casein hydrogel precursor solution was prepared using the method described in any one of claims 1 to 4; The antibacterial casein hydrogel precursor solution was placed under blue light for 4-6 minutes to obtain the antibacterial casein hydrogel.

6. The method for preparing the photothermal antibacterial casein hydrogel of in-situ biomineralized silver nanoparticles according to claim 5, characterized in that: Blue light intensity is 60 ±10 mW / cm 2 .

7. The use of the photothermal antibacterial casein hydrogel prepared by the method described in claim 5 or 6, characterized in that: Used to prepare gel-type medical antibacterial materials.

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