Preparation and application of photo-thermal antibacterial casein hydrogel for in-situ synthesis of nano-silver

Abstract

The invention relates to the technical field of biomedical materials, in particular to a preparation method of in-situ biomineralized nano-silver loaded photo-thermal antibacterial casein hydrogel and application of the in-situ biomineralized nano-silver loaded photo-thermal antibacterial casein hydrogel in the field of biomedical materials. The invention discloses a preparation method of an antibacterial casein hydrogel precursor solution. The preparation method comprises the following steps: S1, preparing modified casein; and S2, preparing an antibacterial casein hydrogel precursor solution for in-situ synthesis of nano-silver. The antibacterial casein hydrogel precursor solution is irradiated under blue light (405 nm), and the antibacterial casein hydrogel is obtained and can be used as a gel type medical antibacterial material.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical materials, and in particular to a method for preparing a photothermal antibacterial casein hydrogel loaded with in-situ biomineralized nanosilver and its application in the field of biomedical materials. Background Art

[0002] Bacterial infection and skin wound healing have always been common and unavoidable problems in daily life. Wound dressings can serve as a partial skin barrier to avoid secondary injury and provide a favorable microenvironment for wound healing. At present, a variety of wound dressings have been designed, such as hydrogels, foams, skin patches, films, nanoparticles, hydrocolloids, nanofibers, and membranes. Among them, hydrogel dressings are a new type of wound dressing developed in recent years. They have excellent flexibility, adjustability, hygroscopicity, and degradability, and have become one of the most competitive candidate materials for wound dressings. Studies have shown that mixed infections of multiple pathogenic microorganisms are observed in most infected wounds, which has promoted the development of multifunctional hydrogels targeting common pathogenic microorganisms in wounds. However, most hydrogel dressings do not have antibacterial properties, which limits their clinical application. Therefore, by effectively combining antibacterial materials with hydrogel dressings, the ability to resist bacterial infection can be effectively given to antibacterial materials.

[0003] In order to prepare multifunctional hydrogel dressings, antibiotics are embedded into the hydrogel three-dimensional network structure to achieve sustained and controlled release. However, the abuse of antibiotics will lead to the emergence of drug-resistant microorganisms. In view of the above situation, metal nanoparticles silver nanoparticles are introduced into hydrogel dressings to achieve the purpose of effective antibacterial. AgNPs (silver nanoparticles) have multiple antibacterial mechanisms: (i) inhibiting bacterial reproduction by interfering with DNA replication and RNA production, (ii) destroying cell membranes, (iii) attacking the reaction chain in bacterial mitochondria, and (iv) inactivating enzyme activity. Generally, 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 damage the environment and human health.

[0004] The traditional method of mixing AgNPs with pre-gel polymers has several limitations: i) the carryover of potentially toxic agents from the AgNPs synthesis process ii) uneven distribution of AgNPs during mixing iii) the risk of AgNPs aggregation and oxidation leading to efficacy loss during daily storage. Therefore, there is a need for improvements to the existing technology.

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

[0006] It has long been known that milk-derived proteins can promote bone mineralization, but there have been no reports on the synthesis of nanosilver from casein mineralization. As one of the main milk proteins, bovine casein contains most of the tryptophan, tyrosine, histidine and proline residues, which give it a certain reducibility. In addition, casein has good biocompatibility and biodegradability, so casein is considered to be an ideal biomedical material, but pure casein powder, like most proteins, is prone to become a breeding ground for bacteria in a humid environment. These problems greatly limit the application range and market competitiveness of casein products. Therefore, the development of casein antibacterial materials with good biocompatibility, certain mechanical properties, shape and stability, and giving full play to the advantages of casein, has important economic benefits and social significance.

[0007] CN113694250 discloses a method for preparing a curdlan antibacterial hydrogel loaded with nanosilver particles, which significantly enhances the antibacterial effect of the gel; by improving the gelling method, the mechanical strength of the gel is improved, solving the problem that the antibacterial gel dressing cannot fit the skin. However, the preparation process is relatively cumbersome, and sodium borohydride is used as a reducing agent in the synthesis of nanosilver. Sodium borohydride is a dangerous chemical substance with certain toxicity. Prefabricated hydrogel dressings are difficult to adapt to wound types with various shapes and depths.

[0008] CN105903057 discloses a method for preparing a nano-silver hybrid sericin porous gel antibacterial material, which is simple to operate, mild in conditions, and environmentally friendly. The sericin porous gel can only form nano-silver after being sealed and irradiated under ultraviolet light for at least 10 minutes, and then it still needs to be dried for a long time to obtain the nano-silver hybrid sericin porous gel antibacterial material. The entire preparation process is time-consuming, and the sericin protein cost is high and is not easy to extract. The final form is a dry porous sponge dressing, which is difficult to fit the wound.

[0009] CN106270548 discloses a method for in-situ green synthesis of sericin-nanosilver using sericin protein, wherein a silver nitrate solution is added to a sericin solution, and then reacted under natural light conditions until the solution turns yellow to obtain sericin-nanosilver. However, the reaction time is 24-36 hours, which is relatively long. Summary of the invention

[0010] The technical problem to be solved by the present invention is to provide a photothermal antibacterial casein hydrogel preparation method for in-situ synthesis of nanosilver and its application.

[0011] In order to solve the above technical problems, the present 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 are reacted in PBS buffer at room temperature (20-25° C.) for 20-28 hours, with the ratio of methacrylic anhydride to casein being 1±0.1 ml / 5 g; the reaction solution is filtered (or filtered after dilution) to remove impurities, and freeze-dried to obtain modified casein powder (i.e., methacrylylated casein);

[0014] The filtration is ultrafiltration;

[0015] S2. Antibacterial casein hydrogel precursor solution for in situ synthesis of nanosilver:

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

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

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

[0019] According to the dosage ratio of methacrylic anhydride to casein powder of 1±0.1 ml / 5 g, methacrylic anhydride is added to the casein powder stock solution, the pH is adjusted to 8.0±0.2, and the reaction is continuously stirred at room temperature for 22 to 26 hours; 2±0.2 times the volume of deionized water is added to the reaction solution for dilution and then ultrafiltration is performed, and the retentate after ultrafiltration is freeze-dried (dried at -45 to -55°C for 16 to 24 hours), and the obtained freeze-dried powder is modified casein powder.

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

[0021] The cut-off of ultrafiltration is 2000Da;

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

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

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

[0025] The present invention also provides a method for preparing a photothermal antibacterial casein hydrogel of in-situ biomineralized nanosilver, using the antibacterial casein hydrogel precursor solution as described above;

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

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

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

[0029] Based on the technical difficulties such as the limitations of the current traditional method of mixing AgNPs with pregel polymers, the present invention can promote the combination of AgNPs and casein through in situ biomineralization, so that the AgNPs have excellent biocompatibility, conformal interface, uniform distribution and high stability.

[0030] The photothermal antibacterial casein hydrogel with in-situ biomineralized nano-silver of the present invention has modified casein as a structural skeleton, and the nano-silver is in-situ synthesized and distributed in the hydrogel.

[0031] The present invention can provide an efficient, in-situ, sequentially light-activated antibacterial hydrogel wound dressing based on protein biomineralization. In order to screen suitable proteins, several commonly used protein materials are used as candidate materials, and casein has excellent light-activated biomineralization ability for AgNP generation. Resveratrol is introduced to further increase the mineralization rate of in-situ AgNP generation. Casein is methacrylated to enhance the mechanical properties of the generated hydrogel. In addition, the photosensitive free radical generator LAP has the potential to further accelerate the formation of AgNP. Under the first blue light irradiation, the cross-linking polymerization of the modified casein and the formation of AgNPs are completed simultaneously, and the second NIR irradiation further enhances the bactericidal effect. The sequentially light-activated nanosilver casein hydrogel prepared by the present invention has good application prospects as an infected wound dressing.

[0032] The invention prepares an antibacterial hydrogel with in-situ synthesized nanosilver using methacrylic acid-modified casein as a matrix by means of biomineralization and the like, and converts the nanosilver into a medical material with biological activity, which can be used as a gel-type medical antibacterial material.

[0033] By preparing the S1 modified casein of the present invention, methacrylylated casein can be prepared on a large scale.

[0034] The hydrogel preparation process of the present invention is simple and easy, the reaction conditions are mild, the product properties are stable, and it is suitable for industrial production.

[0035] Photothermal properties of the antibacterial casein hydrogel synthesized with nanosilver in situ: The antibacterial casein hydrogel synthesized with nanosilver in situ was irradiated with a near-infrared (NIR) laser (808 nm, 3 W cm -2 ), after 10 minutes, the temperature rises to above 50°C. This photothermal effect has the function of auxiliary sterilization.

[0036] In summary, the present invention prepares an antibacterial hydrogel with in situ synthesized nanosilver using methacrylic acid-modified casein as a matrix by means of biomineralization and the like, and converts the nanosilver into a medical material with biological activity, which can be used as a gel-type medical antibacterial material.

[0037] The present invention has the following advantages:

[0038] 1) The present invention utilizes the high reducing property of resveratrol to enhance the biomineralization ability of casein, thereby forming AgNPs in situ. The highly active 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 property, resveratrol was introduced into the biomineralization system to assist the synthesis of AgNPs. The addition of resveratrol makes the antibacterial casein hydrogel of in situ biomineralized nanosilver have good antioxidant and anti-oxidation capabilities, which helps to clear inflammation in wound tissues.

[0040] 2) As an inorganic antibacterial agent, nanosilver particles are widely used due to their high efficiency, safety and broad-spectrum antibacterial effects. However, the traditional synthesis process of nanosilver has the disadvantages of residual potential toxic agents and easy oxidation. The present invention utilizes the biomineralization of casein to synthesize nanosilver in situ, which significantly reduces the biological toxicity of silver ions and gives the hydrogel antibacterial properties.

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

[0042] In summary, the purpose of the present invention is to provide an efficient, in-situ, sequentially photoactivated antibacterial hydrogel wound dressing based on casein biomineralization. Under blue light irradiation, casein hydrogel and silver nanoparticles (AgNPs) are formed simultaneously, and the whole process only takes about 5 minutes. Photocuring gelation can be applied to irregular wound shapes; the photothermal antibacterial casein hydrogel of in-situ biomineralized nanosilver can be photothermally converted under near-infrared irradiation, which greatly improves the antibacterial effect. The entire preparation process has strong operability, green synthesis, fast biomineralization, and low material and equipment costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] The specific implementation modes of the present invention are further described in detail below with reference to the accompanying drawings.

[0044] Figure 1 This is a photograph of the antibacterial casein hydrogel of in situ biomineralized nanosilver of Example 1;

[0045] Figure 2 This is the UV-visible absorption spectrum of the in situ biomineralized silver nanoparticles in Experiment 1;

[0046] Figure 3 This is the transmission electron microscopy image of the in situ biomineralized silver nanoparticles in Experiment 2;

[0047] Figure 4 The microstructure of the antibacterial casein hydrogel with in situ biomineralized nanosilver in Experiment 3;

[0048] Figure 5 The antioxidant activity of the antibacterial casein hydrogel with in situ biomineralized silver nanoparticles in Experiment 4;

[0049] Figure 6 The cell compatibility of the antibacterial casein hydrogel with in situ biomineralized silver nanoparticles in Experiment 5;

[0050] Figure 7The in vitro photothermal properties of the photothermal antibacterial casein hydrogel with in situ biomineralized silver nanoparticles in Experiment 6;

[0051] Figure 8 The in vivo photothermal performance of the photothermal antibacterial casein hydrogel with in situ biomineralized nanosilver in Experiment 7;

[0052] Fig. 9 The antibacterial properties of the photothermal antibacterial casein hydrogel with in situ biomineralized nanosilver from experiment 8. DETAILED DESCRIPTION

[0053] The present invention is further described below in conjunction with embodiments, but the present invention is not limited thereto.

[0054] The PBS in the present invention refers to 1×PBS buffer (0.01 M, pH 7-8).

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

[0056] Example 1: A method for preparing a photothermal antibacterial casein hydrogel containing in situ biomineralized nanosilver:

[0057] S1. Preparation of modified casein:

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

[0059] Add methacrylic anhydride to the casein powder stock solution, the ratio of methacrylic anhydride to casein powder (v / w) is 1 ml / 5 g, and adjust the pH to 8.0 with a 5 mol / L sodium hydroxide solution filtered with a 0.22 μm filter membrane, and continue stirring and reacting at 20-25° C. for 24 hours; add 2 volumes of deionized water to the resulting reaction solution for dilution and ultrafiltration (retention capacity is 2000 Da);

[0060] The retentate after ultrafiltration is freeze-dried (-45 to -55°C for 16 to 24 hours), and the obtained freeze-dried powder is modified casein powder.

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

[0062] S2. In situ synthesis of antibacterial casein hydrogel precursor solution of nanosilver:

[0063] The modified casein powder obtained in the above step S1 is dissolved in 1×PBS buffer (0.01M, pH7-8) to obtain a protein solution with a concentration of 10 g / 100 ml; then, under light-proof conditions, lithium phenyl 2,4,6-trimethylbenzoylphosphinate (LAP) is added as a photoinitiator to a concentration of 0.05 g / 100 ml, resveratrol is added to a concentration of 0.5 mg / mL, silver nitrate is added to a concentration of 1 mM, and then mixed; a photothermal antibacterial casein hydrogel precursor solution for in situ synthesis of nanosilver is obtained; referred to as the antibacterial casein hydrogel precursor solution.

[0064] S3. Preparation of antibacterial casein hydrogel with in situ synthesis of nanosilver:

[0065] After irradiating the antibacterial casein hydrogel precursor solution obtained by S2 with blue light (405 nm) for 5 min, the casein hydrogel with in situ biomineralized nanosilver was prepared, referred to as antibacterial casein hydrogel (CASMA-Ag1). The blue light intensity was 60 mW / cm 2 The morphology of the obtained 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 was 1 cm.

[0067] Embodiment 2: With respect to Embodiment 1, the following changes are made:

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

[0069] Embodiment 3, compared with embodiment 1, makes the following changes:

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

[0071] Blank comparative example 1, with respect to Example 1, the following changes were made:

[0072] Eliminate the use of silver nitrate, that is, change the concentration of silver nitrate from 1 mM to 0 mM, and the rest is the same as Example 1. The obtained product is named casein hydrogel (CASMA-Ag0).

[0073] Blank comparative example 2, with respect to Example 1, the following changes were made:

[0074] Eliminate the use of silver nitrate and resveratrol, that is, change the concentration of silver nitrate from 1 mM to 0 mM, change the concentration of resveratrol from 0.5 mg / mL to 0 mg / mL, and the rest is the same as Example 1. The obtained product is named casein hydrogel (Casein-Ma).

[0075] Experiment 1: UV-visible spectrum of photothermal antibacterial casein hydrogel with in situ biomineralized nanosilver:

[0076] The antibacterial casein hydrogel precursor solution (100 μL) obtained in S2 of Examples 1 to 3 was added to a 96-well plate, and then according to S3, antibacterial casein hydrogel (CASMA-Ag0.5, CASMA-Ag1, CASMA-Ag1.5) was obtained and the adsorption spectrum was scanned by an ELISA instrument at a wavelength of 300-700 nm. Figure 2 It shows that the characteristic peak of nanosilver appears at 410nm, and the intensity of the peak reflects the content of synthesized nanosilver to a certain extent. The content of nanosilver in CASMA-Ag1 and CASMA-Ag1.5 hydrogels is 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 to 3 were hydrolyzed with proteinase K to release AgNPs. The hydrolysis reaction was as follows: 1 g of the hydrogel sample was added to 5 mL of proteinase K solution (200 μg / mL), incubated at 37° C. for 24 hours, and a hydrogel hydrolyzate was obtained.

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

[0080] Experiment 3: Microstructure of photothermal antibacterial casein hydrogel with in situ biomineralized nanosilver:

[0081] The hydrogel samples (CASMA-Ag0.5, CASMA-Ag1, CASMA-Ag1.5) obtained in Examples 1 to 3 were frozen with liquid nitrogen, dried in a freeze dryer, sprayed with platinum on the cross section, and observed with a field emission scanning electron microscope. The obtained scanning electron microscope image is as follows: Figure 4 As shown, there is a uniform porous structure inside the gel.

[0082] Experiment 4: Antioxidant activity of antibacterial casein hydrogel with in situ biomineralized nanosilver:

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

[0084] In addition, the casein hydrogel (Casein-Ma) without adding silver and resveratrol obtained in Blank Comparative Example 2 and the casein hydrogel without adding silver (CASMA-Ag0) obtained in Blank Comparative Example 1 were used as controls.

[0085] The antioxidant activity of these extracts was evaluated using the total antioxidant capacity assay kit (Beyotime, China) using the ABTS method. The assay results were expressed as Trolox equivalent (mM). Figure 5 The results show that the antioxidant activity of CASMA-Ag0, CASMA-Ag0.5, CASMA-Ag1, and CASMA-Ag1.5 is significantly higher than that of Casein-Ma, indicating that the introduction of resveratrol significantly enhances the antioxidant capacity. With the addition of Ag+, the antioxidant activity of the hydrogel decreases, indicating that the in situ synthesis of AgNPs consumes the antioxidant activity of the hydrogel. However, the above results show that the nanosilver antibacterial casein hydrogel still has good antioxidant activity.

[0086] Note: The synthesis of silver nanoparticles will consume part of the reducing capacity of the original hydrogel, but the oxidizing properties of CASMA-Ag0.5, CASMA-Ag1, and CASMA-Ag1.5 are still significantly higher than that of the casein-Ma group.

[0087] Experiment 5: Cytocompatibility of antibacterial casein hydrogel with in situ biomineralized nanosilver:

[0088] The photothermal antibacterial casein hydrogel (CASMA-Ag0.5, CASMA-Ag1, CASMA-Ag1.5) of in situ biomineralized nanosilver prepared in Examples 1 to 3 was extracted with DMEM standard culture medium, the mass 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; a methacryloyl whey protein hydrogel extract was prepared.

[0089] Mouse epidermal fibroblasts were selected for culture, and the cells were counted after obtaining the cell suspension. The cell suspension was diluted to 50,000 / mL according to the number of cells. The cell suspension was inoculated in a 96-well plate, 100 μL was inoculated in each well, and the cells were cultured for 12 hours to allow the cells to adhere to the bottom of the well plate. The original culture medium was removed, and 100 μL of hydrogel extract was added to each well of the experimental group. 100 μL of DMEM culture medium was added to each well of the control group (Norml), and 100 μL of DMEM culture medium containing 1 mM silver nitrate was added to each well of the experimental group (1mM Ag+). The culture plate was placed in an incubator and incubated for 24 hours at a temperature of 37°C. DMEM culture medium containing 10% CCK-8 was prepared and added to the well plate in the form of liquid replacement. The culture plate was placed in an incubator and incubated for 2 hours at a temperature of 37°C. The absorbance (OD) at 450nm was measured using an enzyme marker. Figure 6 As shown in the figure, the introduction of 1mM Ag+ significantly reduced cell activity, and the cell compatibility 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, the cytotoxicity was significantly reduced, and AgNPs were successfully embedded in the casein hydrogel skeleton, further improving the biosafety of AgNPs.

[0090] Experiment 6: In vitro photothermal properties of in situ biomineralized nanosilver antibacterial casein hydrogel:

[0091] The antibacterial casein hydrogel precursor solution (200 μL) obtained in S2 of Examples 1 to 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 for in vitro photothermal performance testing;

[0092] The antibacterial casein hydrogel precursor solution (200 μL) obtained in S2 of the blank comparative example 1 was added to a 48-well plate, and then the casein hydrogel (CASMA-Ag0) was obtained according to S3 for in vitro photothermal performance testing;

[0093] The hydrogel was placed under 808 nm NIR laser (3 W cm -2 At the same time, infrared imaging equipment was used to monitor temperature changes and obtain photothermal images. Figure 7 It is shown that the temperature of CASMA-Ag0 and CASMA-Ag0.5 hydrogels increases with near-infrared radiation (3 W cm -2 ), which is related to the content of nanosilver in the hydrogel. The temperature of CASMA-Ag1 hydrogel increased by 37.6°C in 10 minutes, and the temperature of CASMA-Ag1.5 hydrogel increased by 40.6°C in 10 minutes, indicating that the introduction of AgNPs provides satisfactory photothermal behavior for the hydrogel.

[0094] Experiment 8: In vivo photothermal performance of in situ biomineralized nanosilver antibacterial casein hydrogel:

[0095] Mice (C57BL) were anesthetized with pentobarbital (30 mg / kg body weight) and the back hair was removed. A full-thickness circular wound with a diameter of 6 mm was formed on the back skin. The antibacterial casein hydrogel precursor solution (50 μL) obtained in step 2) of Example 1 was dripped onto the wound. After irradiation with blue light (405 nm) for 5 min, the casein hydrogel with in situ biomineralized nanosilver was prepared. The wound was placed under 808 nm NIR laser (3 W cm -2 , 10min). At the same time, infrared imaging equipment was used to monitor temperature changes and obtain photothermal images. 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: The blue light intensity is about 60mW / cm 2 The distance between the blue light source and the antibacterial casein hydrogel precursor solution was about 1 cm.

[0097] Experiment 7: Antibacterial properties of photothermal antibacterial casein hydrogel with in situ biomineralized nanosilver:

[0098] To evaluate the antibacterial activity with or without NIR irradiation, 200 μL of the antibacterial casein hydrogel precursor solution obtained in step 2) of Examples 1 to 3 was added to a 48-well plate and irradiated with blue light for 5 min to prepare casein hydrogels with in situ biomineralized nanosilver (CASMA-Ag0.5, CASMA-Ag1, and CASMA-Ag1.5). 50 μL of bacterial suspensions of Escherichia coli (ATCC8099) or Staphylococcus aureus (ATCC 25923) diluted with PBS (10 7 CFU mL -1 ) was added to each well and in the presence or absence of NIR (808 nm, 3 W cm -2, 10min) in each well. PBS was used as a blank control. After incubation at 37°C for 4 hours, the bacteria were resuspended and diluted 10 -2 The cells were then inoculated on LB agar for 24 hours. The colony forming units per milliliter (CFU / mL) value of each group was calculated. Fig. 9 As shown in the figure, compared with the PBS control group, the nanosilver casein hydrogel had a significant bactericidal effect on both Escherichia coli and Staphylococcus aureus. -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 the photothermal hydrogel. These results indicate that the designed nanosilver hydrogel exhibits strong antibacterial activity under the synergistic effect of continuous UV and NIR light activation.

[0099] In summary, compared with CASMA-Ag0.5 and CASMA-Ag1.5, CASMA-Ag1 has antioxidant properties, biosafety and antibacterial activity, which can be converted into a bioactive medical material and used as a gel-type medical antibacterial material.

[0100] Comparative Example 1: The casein powder in the above-mentioned Example 1 was replaced with the protein materials described in Table 1 below, and the dosage remained unchanged, so as to obtain the corresponding modified protein materials for preparing the hydrogel precursor solution, and the rest was the same as Example 1.

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

[0102] Table 1

[0103]

[0104] Comparative Example 2: The resveratrol in the above Example 1 was replaced with the reducing agents described in Table 2 below, and the dosage remained unchanged, and the rest was the same as in Example 1. The obtained properties were compared as shown in Table 2 below.

[0105] Table 2

[0106]

[0107]

[0108] Finally, it should be noted that the above examples are only some specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments, and there are many variations. All variations that can be directly derived or associated with the content disclosed by a person skilled in the art should be considered as the protection scope of the present invention.

Claims

1. A method for preparing an antibacterial casein hydrogel precursor solution, characterized in that The following steps are involved: S1. Preparation of modified casein: Methacrylic anhydride and casein are reacted in PBS buffer at room temperature for 20 to 28 hours, and the ratio of methacrylic anhydride to casein is 1±0.1 ml / 5 g; the reaction solution is filtered to remove impurities, and freeze-dried to obtain modified casein powder; S2. Antibacterial casein hydrogel precursor solution for in situ synthesis of nanosilver: The modified casein powder obtained in S1 is dissolved in PBS buffer to prepare a protein solution with a concentration of 8-15 g / 100 ml, and then a photoinitiator LAP, resveratrol and silver nitrate are added under light-proof conditions to a concentration of LAP of 0.01-0.5 g / 100 ml, a concentration of resveratrol of 0.1-1 mg / ml and a concentration of silver nitrate of 1-2 mmol / L to obtain an antibacterial casein hydrogel precursor solution.

2. The method for preparing an antibacterial casein hydrogel precursor solution according to claim 1, characterized in that The 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 dosage ratio of methacrylic anhydride to casein powder of 1±0.1 ml / 5 g, methacrylic anhydride is added to the casein powder stock solution, the pH is adjusted to 8.0±0.2, and the reaction is continuously stirred at room temperature for 22 to 26 hours; 2±0.2 times the volume of deionized water is added to the reaction solution for dilution and then ultrafiltration is performed, and the retentate after ultrafiltration is freeze-dried to obtain a modified casein powder.

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

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

5. A method for preparing a photothermal antibacterial casein hydrogel with in situ biomineralized nanosilver, characterized in that: Utilizing the antibacterial casein hydrogel precursor solution as described in any one of claims 1 to 4; The antibacterial casein hydrogel precursor solution is placed under blue light for 4 to 6 minutes to obtain the antibacterial casein hydrogel.

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

7. Use of the photothermal antibacterial casein hydrogel prepared by the method of claim 5 or 6, characterized in that: As a gel-type medical antibacterial material.

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