Protein hydrogel for reversible controlled release and real-time monitoring of copper ions as well as preparation method and application of protein hydrogel

Abstract

The invention relates to protein hydrogel with reversible controlled release and real-time monitoring of copper ions as well as a preparation method and application of the protein hydrogel, the hydrogel is assembled by a one-step method, the copper ions are used as a unique cross-linking agent, bovine serum albumin and gelatin with opposite thermal gelation behaviors are synergistically integrated, and the hydrogel is prepared. And a thermal / ROS dual response injection type protein hydrogel system is constructed. The copper ions are simultaneously used as a structural crosslinking center and a therapeutic functional carrier of the hydrogel, so that the integration of the carrier and the medicine is realized, the preparation process is simplified, and the biological safety is improved. And meanwhile, by utilizing the synergistic thermal responsiveness of BSA and gelatin, reversible gel-sol conversion which is stable at physiological temperature, liquefied under slight heating, cooled and then gelatinized again is realized, so that the copper ion release can be subjected to multi-time and bidirectional on-demand switch control, and burst release and system toxicity are effectively avoided.

Description

Technical Field

[0001] The technical solution of this invention belongs to the field of biomedical materials and drug delivery, specifically relating to a smart responsive protein hydrogel for controllable delivery and real-time monitoring of therapeutic metal ions, and its preparation method and application. Background Technology

[0002] Copper ions (Cu) 2+ Copper ions, as an essential trace element for the human body, possess broad-spectrum antibacterial, anti-inflammatory, osteogenic, and angiogenic activities, demonstrating promising application potential in the repair of infected tissue damage. Especially in the treatment of refractory lesions such as periodontitis and infected skin wounds, copper ions can simultaneously address multiple pathological aspects, including microbial infection, inflammatory response, and tissue regeneration disorders, showing potential as a multifunctional therapeutic agent. However, the clinical application of copper ions has always been limited by its narrow therapeutic window: in vitro studies have shown that its effective antibacterial concentration is typically in the range of 1-20 µM, while the difference between the effective dose and the toxic dose in vivo is less than twofold. This strong dose-dependent toxicity issue poses a significant safety challenge to the clinical translation of copper ions.

[0003] To control the release of copper ions and improve their safety, existing technologies have primarily developed two strategies. The first is sustained-release delivery systems, such as loading copper ions into bioglass, polymer microspheres, or traditional hydrogels, relying on the material's own degradation to achieve slow ion release. The release behavior of these systems is entirely predetermined by the material's degradation kinetics; once implanted and release begins, the process cannot be interrupted or regulated. If local concentrations become excessively high or systemic toxicity occurs during treatment, active intervention is impossible, posing a potential safety hazard. The second is stimulus-responsive delivery materials, designed to trigger copper ion release using endogenous or exogenous stimuli. While this design provides a certain "switch" to the release behavior, most existing systems exhibit a "one-off" or "irreversible" response. That is, after stimulation, the release process is initiated, but it is difficult to effectively stop the release by withdrawing the stimulation, lacking reversible, bidirectional, fine-grained "switch" control. This makes dose control still insufficiently precise, unable to completely avoid the risks of burst release or continuous exposure.

[0004] Furthermore, both sustained-release and existing stimulus-response systems generally lack means for real-time, non-invasive monitoring of drug release kinetics in vivo. During treatment, the actual release concentration, release rate, and spatial distribution of copper ions cannot be obtained in a timely manner, making the entire treatment almost "blind." Doctors cannot adjust treatment plans based on real-time release data, making it difficult to achieve truly individualized, precise treatment and ensure safety.

[0005] Protein hydrogels, such as those constructed from bovine serum albumin (BSA) and gelatin, are considered ideal drug carriers due to their excellent biocompatibility, biodegradability, and similarity to the human extracellular matrix. However, the formation of traditional protein hydrogels typically relies on the addition of exogenous chemical cross-linking agents or complex enzymatic cross-linking systems. These additives not only increase the complexity of the preparation process and batch-to-batch instability, but their potential cytotoxicity may also introduce new biosafety issues, and lead to a separation between the carrier structure and therapeutic function, resulting in an impractical system design. Although some studies have explored cross-linking using the coordination of copper ions with amino acid residues in proteins, thereby avoiding the use of exogenous cross-linking agents, these systems often only focus on gel formation, and their release behavior still mainly relies on simple diffusion or passive degradation of the gel network, failing to integrate the aforementioned intelligent functions such as reversible control and real-time monitoring.

[0006] Therefore, there is an urgent need in this field to develop a novel copper ion delivery platform that can collaboratively address the following key issues: First, it should achieve reversible, on-demand "on / off" control of copper ion release, enabling interruption or restart at any time during the release process as needed for treatment, to precisely match complex in vivo treatment dynamics and minimize toxicity risks; second, it should possess the ability to monitor the release process in real time and non-invasively, providing feedback information for dynamic adjustments to treatment regimens and achieving precise drug delivery; third, it should construct a streamlined and safe delivery system that avoids the use of exogenous cross-linking agents, achieving a unity of carrier structure and therapeutic function, and possessing good clinical translational feasibility. Currently, no comprehensive solution has been found that simultaneously meets all these requirements. Summary of the Invention

[0007] Technical problem to be solved: This invention aims to overcome the key defects in existing copper ion delivery technologies and provide a protein hydrogel with reversible controlled release and real-time monitoring of copper ions, its preparation method, and its application. It can achieve multiple, reversible on / off control of copper ion release to cope with complex changes in the in vivo environment. At the same time, the platform can achieve real-time, non-invasive monitoring of the release process by means of integrated fluorescence tracing function. Finally, by eliminating exogenous cross-linking agents and simplifying the preparation process, a safe, efficient, and potentially therapeutic novel delivery system is constructed, thereby truly broadening the clinical application window of copper ions.

[0008] Technical solution: A smart protein hydrogel with reversible controlled release of copper ions, formed by coordination crosslinking of the following components: (a) bovine serum albumin; (b) gelatin; (c) divalent copper ions; wherein the mass ratio of bovine serum albumin to gelatin is (0.5-1.5):1, the concentration of divalent copper ions is 1mM-12M, and the hydrogel can maintain a gel state at 37℃ and undergo a reversible gel-sol transition under temperature control or photothermal action at 40-45℃.

[0009] The aforementioned divalent copper ions are provided by copper salts, including copper chloride, copper sulfate, or copper acetate, with a concentration of 4 mM-40 mM for the divalent copper ions.

[0010] The hydrogel is also loaded with indocyanine green as a photothermal conversion agent, and the content of indocyanine green is 0.0625-1 mg / mL.

[0011] Under near-infrared light irradiation at a wavelength of 808 nm, the photothermal effect generated by the indocyanine green can trigger the hydrogel to undergo the gel-sol transition.

[0012] The intensity of the fluorescence signal generated during the release of indocyanine green is positively correlated with the concentration of divalent copper ions released.

[0013] The method for preparing the intelligent protein hydrogel includes the following steps: S1: mixing bovine serum albumin solution with gelatin solution to obtain a protein mixture; S2: adding copper salt solution to the protein mixture and mixing well; S3: heating the mixture obtained in step S2 at 80-90℃ for 1-5 minutes, and then cooling to obtain the intelligent protein hydrogel.

[0014] Before or during step S1, indocyanine green is added to the bovine serum albumin solution and mixed well.

[0015] The above-mentioned intelligent protein hydrogel is used in the preparation of drugs for treating bacterial infections.

[0016] The above-mentioned intelligent protein hydrogel is used in the preparation of drugs for treating infectious tissue injuries, including periodontitis or infected skin wounds.

[0017] The above-mentioned intelligent protein hydrogel is used in the preparation of drugs for promoting tissue regeneration, including bone tissue regeneration or angiogenesis.

[0018] Beneficial Effects: The intelligent protein hydrogel platform provided by this invention successfully unifies the carrier and drug by using copper ions as the sole network cross-linking center and therapeutic carrier, thereby simplifying the system and fundamentally improving biosafety. This design eliminates the need for any exogenous chemical cross-linking agents in the preparation process, allowing for completion through a single-step self-assembly, significantly reducing process complexity and potential toxicity risks. More importantly, by combining bovine serum albumin and gelatin, which exhibit opposite thermogelation behaviors, the hydrogel maintains a stable gel state at physiological temperatures (37°C), while reversibly transforming into a sol upon slight heating (e.g., above 41°C), and recovering its gel structure upon cooling. This unique reversible gel-sol transition capability enables multiple, bidirectional, on-demand switching control of the copper ions loaded within, effectively avoiding the burst release problem of traditional materials. This results in an approximately 10-fold increase in its therapeutic index in animal experiments, significantly broadening its safe application window. Furthermore, the co-loaded photothermal converter indocyanine green (ICG) not only enables the hydrogel to respond to near-infrared light-triggered release, but the fluorescence signal generated during release also shows a positive correlation with the copper ion concentration. This enables, for the first time, real-time, non-invasive optical monitoring of the in vivo copper ion release kinetics, providing a direct basis for precise dosage control. The components of this platform exhibit significant synergistic effects: copper ions exert multiple therapeutic functions, including antibacterial and osteogenic effects, while their coordination and cross-linking constitute the structural basis of the hydrogel; ICG provides a monitoring signal, and its photothermal effect triggers and regulates the release process; while the protein matrix not only provides good biocompatibility and degradability, but its synergistic thermal response characteristics are key to achieving reversible controlled release. Ultimately, this integrated system synergistically promoted efficient bacterial clearance, inflammation reduction, bone tissue formation, and angiogenesis in various animal models, including periodontitis and infected skin wounds. Attached Figure Description

[0019] Figure 1 A schematic diagram comparing the gelation effects of gelatin and BSA at different concentrations.

[0020] Figure 2 Synthesis and characterization of PICu hydrogel. A shows the effect of temperature on the sol-gel state of PICu hydrogels with different BSA / gelatin ratios. B shows the sol-gel phase transition mechanism. C shows the effect of copper ions on the injectability of the hydrogel and the sol-gel phase transition. D shows SEM micrographs and elemental distribution maps of PICu and PI. E shows XPS analysis of PICu and PI. F shows the viscoelastic properties of gel, BSA, PI, and PICu measured by temperature.

[0021] Figure 3 For the release and monitoring of copper ions in vivo and in vitro. A represents the power intensity at 1 W / cm². 2The temperature changes of PICu, free ICG, and PBS with irradiation time under continuous laser irradiation are shown in Figure B. Figure B represents the near-infrared stimulus response characteristics of the PICu hydrogel. Figure C represents the dithiothreitol (DTT) stimulus response characteristics of the PICu hydrogel. Figure D represents the Cu released by the hydrogel under laser irradiation and DTT stimulation. 2+ Concentration. E represents the functional relationship between copper ion release concentration and ICG fluorescence intensity. F represents the reversible thermosensitive response characteristics of the PICu hydrogel. G represents real-time in vivo monitoring of the hydrogel via ICG fluorescence signal. H represents the serum copper concentration after subcutaneous injection of PICu and free copper.

[0022] Figure 4 To assess the safety of PICu hydrogel. A represents the survival rate of bone marrow mesenchymal stem cells after treatment with free copper and PICu hydrogel. B represents the median lethal dose (LD50) of PICu hydrogel and free copper in mice. C represents the copper ion concentration in the major organs and peripheral blood of mice. D represents the HE staining results of the major organs.

[0023] Figure 5 This study illustrates the effect of PICu hydrogel on BMSCs function. A shows the migration of cells at 0 and 24 hours after scratching. PICu+light indicates a power intensity of 1 W / cm². 2 PICu hydrogel was irradiated with an 808 nm laser. B is a bright-field image of BMSCs Transwell migration assay (scale bar: 100 μm). C is alkaline phosphatase (ALP) staining of BMSCs co-cultured with different drugs after mineralization induction (scale bar: 1 mm). D is alizarin red (ARS) staining of BMSCs co-cultured with different drugs after mineralization induction (scale bar: 1 mm).

[0024] Figure 6 PICu promotes angiogenesis in the chorioallantoic membrane of chicken embryos (**** p<0.001).

[0025] Figure 7 To demonstrate the anti-inflammatory effect of PICu hydrogel. A is a flow cytometry image showing M1 (CD86 positive) and M2 (CD206 positive) macrophage populations in different treatment groups. B is an immunofluorescence (IF) image of M1 (CD86) and M2 (CD206) macrophage markers (scale bar = 100 μm).

[0026] Figure 8 The antibacterial effect of PICu hydrogel is shown in Figure A. Colony forming units of *Porphyromonas gingivalis* were determined by different treatment groups. Figure B shows the live / dead staining of *Porphyromonas gingivalis* treated with different treatment groups (scale bar: 20 μm). Figure C shows the effect of different treatment groups on biofilm formation of *Porphyromonas gingivalis*. Figure D shows the quantitative statistical results of Figure B. Figure E shows the quantitative statistical results of Figure C.

[0027] Figure 9 This section describes the therapeutic effect of PICu hydrogel on periodontitis in vivo. A shows the 3D reconstruction, coronal, and sagittal images from micro-CT scans. The red area represents alveolar bone resorption induced by ligation. B shows the distance between the cementoenamel junction (CEJ) and the alveolar ridge crest (ABC). C shows the quantitative analysis of bone morphological parameters (including bone volume fraction BV / TV, trabecular thickness Tb.Th, and trabecular spacing Tb.Sp).

[0028] Figure 10 This study analyzes in vivo wound healing in a rat model of skin infection. A shows the colony-forming units (CFU) of Staphylococcus aureus in different treatment groups. B shows the live / dead staining of Staphylococcus aureus after different treatment groups (scale bar: 20 μm). C shows photographs of the wound healing process on days 0, 1, 3, 5, 7, and 10 (scale bar: 1 mm). D shows a schematic diagram of the wound area in different groups. E shows the quantitative analysis of the skin wound area of ​​rats at different time points. F shows photographs of Staphylococcus aureus colonies grown on TSB plates from different wound sites on day 3. G shows the quantitative analysis of Staphylococcus aureus colonies. Detailed Implementation

[0029] The following embodiments are provided to enable those skilled in the art to more fully understand the present invention, but do not limit the invention in any way.

[0030] Comparative Example 1: Single Protein Control Group Weigh 5 mg of bovine serum albumin (BSA) and 0.5 mg of intracellular glucose (ICG), mix thoroughly, and add 10 μL of 400 mM CuCl2 solution to trigger self-assembly into nanoparticles. Subsequently, add 0.5 mL of a 10% BSA aqueous solution or 0.5 mL of a 10% gelatin aqueous solution to the solution, mix well, and heat at 85 °C for 3 minutes. Figure 1 As shown, BSA hydrogels without injectability and thermo-reversible properties can be obtained; the gelatin-only group cannot form a gel after heating at 85°C for 3 minutes. Figure 1 As shown.

[0031] Example 1: Preparation of PICu hydrogel 5 mg of bovine serum albumin (BSA) and 0.5 mg of intracellular glucose (ICG) were weighed and mixed thoroughly. 10 μL of 400 mM CuCl2 solution was added to trigger self-assembly into nanoparticles. Subsequently, 0.5 mL of a 10% BSA aqueous solution and 0.5 mL of a 10% gelatin aqueous solution were added to the solution, mixed thoroughly, and heated at 85 °C for 3 minutes. The final product was a PICu hydrogel crosslinked with BSA, gelatin, and copper ions. Figure 2Subsequent examples all used the PICu hydrogel from Example 1 for experiments.

[0032] Example 2: Stimulus-responsive drug release behavior of PICu hydrogel Take 1 mL of the hydrogel and place it in a dialysis bag, then immerse it in 10 mL of PBS release medium (pH=7.4). The experimental group used a power density of 1.0 W / cm². 2 An 808 nm near-infrared laser was used to irradiate the samples for 5 minutes every hour. The control group was not irradiated. Samples were taken at predetermined time points, and an equal volume of fresh PBS was added. The Cu in the released medium was determined using inductively coupled plasma optical emission spectrometry (ICP-OES) or atomic absorption spectrometry. 2+ Concentration was determined by measuring the ICG fluorescence intensity using a UV spectrophotometer.

[0033] The results are as follows Figure 3 A and Figure 3 As shown in Figure B, the ICG in the PICu hydrogel can effectively convert light energy into heat energy, resulting in a local temperature rise in the gel. Figure 3 (A), thereby accelerating the release of copper ions ( Figure 3 (B). The temperature rise was not significant in the samples without ICG or the PBS control group.

[0034] Reduced stimulus (DTT) responsiveness The procedure was the same as in 3.1, but the release medium was replaced with a PBS solution containing 10 mM dithiothreitol (DTT) to simulate a highly reducing tumor microenvironment. The control group used PBS without DTT. Results were as follows: Figure 3 C and Figure 3 As shown in Figure D, DTT can disrupt structures such as disulfide bonds in hydrogels, leading to hydrogel degradation and the release of copper ions.

[0035] Fluorescence self-monitoring release behavior In experiments 3.1 and 3.2, the determination of Cu 2+ Simultaneously, the fluorescence intensity of ICG in the release medium at the emission wavelength of 810 nm (excitation wavelength 780 nm) was detected using a fluorescence spectrophotometer.

[0036] The results are as follows Figure 3 As shown in E, there is a good linear relationship between the release concentration of copper ions and the fluorescence intensity of ICG (y = 0.00964x + 37.85, R0). 2 =0.9514). This indicates that the release kinetics of the therapeutic agent (copper ions) in the hydrogel can be monitored in real time and non-invasively by detecting the fluorescence signal of ICG. Figure 3 As shown in Figure F, the PICu hydrogel has reversible temperature-sensitive properties, enabling repeated "on / off" cycles.

[0037] Example 3: In vivo experiments with PICu hydrogel Healthy SD rats were selected and 100 μL of the PICu hydrogel prepared in Example 1 was injected subcutaneously into their backs. A control group was also injected with an equal volume of free CuCl2 solution.

[0038] Real-time monitoring: ICG fluorescence signals at the injection site were monitored at different time points using a small animal in vivo optical imaging system. Results are as follows: Figure 3 As shown in Figure G, the location and degradation of hydrogels in vivo can be tracked over a long period of time using fluorescence signals.

[0039] Pharmacokinetics: Tail vein blood was collected from mice at different time points after injection (e.g., 1 h, 6 h, 24 h, 72 h), serum was separated, and the concentration of copper ions in the serum was detected using ICP-OES. Results are as follows: Figure 3 As shown in Figure H, compared with free copper ions that rapidly enter the bloodstream and are quickly metabolized, the PICu hydrogel group can maintain a more stable blood copper concentration for a longer period of time, exhibiting sustained-release properties.

[0040] Example 4: Biosafety evaluation of PICu hydrogel Cytotoxicity test Bone marrow mesenchymal stem cells (BMSCs) viability was assessed using the CCK-8 assay. BMSCs were seeded in 96-well plates and reacted with different concentrations of free copper ions (Cu). 2+ The solution and the PICu hydrogel extract prepared in Example 1 were co-cultured for 24 hours. The results are as follows: Figure 4 As shown in Figure A, the cell activity of the PICu hydrogel-treated group was significantly higher than that of free copper ions, indicating that the hydrogel form can effectively reduce the direct toxicity of copper ions and has excellent biocompatibility.

[0041] Acute toxicity test (LD50) Healthy ICR mice were selected, randomly grouped, and then injected with different doses of PICu hydrogel or free Cu via tail vein in a single injection. 2+ The mice were treated with the solution and their survival was observed over 14 days. The results were as follows: Figure 4 As shown in Figure B, the LD50 value of the PICu hydrogel group is significantly higher than that of the free Cu. 2+ The study showed that its systemic acute toxicity was significantly lower than that of free copper ions.

[0042] Biodistribution and tissue compatibility in vivo After subcutaneous injection of PICu hydrogel into mice, major organs (heart, liver, spleen, lung, and kidney) and blood were collected at different time points. The copper ion content was detected using a serum copper assay kit. Figure 4:C), and the organs were sectioned in paraffin and stained with H&E for histopathological analysis ( Figure 4 :D). The results showed that copper ions in the PICu hydrogel group were mainly enriched at the injection site, and the distribution to major organs was much lower than that in the free copper group, and no obvious pathological damage was observed in any organ.

[0043] Example 5: Effect of PICu hydrogel on osteogenic function of BMSCs Cell migration ability Scratch assay: Scratches were created on BMSCs monolayers, and culture medium containing PICu hydrogel extract was added (PICu group and PICu + near-infrared light irradiation group). Scratch healing was observed at 0 and 24 hours. Results are as follows: Figure 5 As shown in Figure A, the PICu hydrogel, especially under near-infrared light stimulation, can significantly promote the migration of BMSCs.

[0044] Transwell experiment: Results are as follows Figure 5 As shown in Figure B, the PICu hydrogel can significantly increase the number of BMSCs that penetrate the membrane, further confirming its ability to promote migration.

[0045] Osteogenic differentiation capacity BMSCs were co-cultured with culture media from different treatment groups, and then alkaline phosphatase (ALP) staining was performed: ALP is an early marker of osteogenic development. Results are as follows... Figure 5 The results showed that ALP activity was significantly enhanced in the PICu group.

[0046] Alizarin Red (ARS) staining and quantification: used to detect calcium nodule formation, a marker of late-stage osteogenic development. Results are as follows... Figure 5 The results showed that PICu hydrogel significantly promoted the formation of mineralized nodules in BMSCs.

[0047] Example 6: Angiogenesis-promoting effect of PICu hydrogel The angiogenesis-promoting capacity of BMSCs in PICu hydrogel was evaluated using a chicken embryo chorioallantoic membrane (CAM) assay. Fertilized eggs were incubated at 37 °C under constant humidity, with the eggs turned twice daily. On day 9 (E9), a window was created in the eggshell to expose the CAM, and then PICu hydrogel was placed on the CAM surface, with the window sealed with sterile tape. On day 12 (E12), the chicken embryos were removed, and CAM angiogenesis was observed and photographed under a stereomicroscope (SMZ745T, Nikon, Japan). The vascular area (VA) within the eggshell window and the CAM area were measured using Image-ProPlus 7.0 software, and the vascular area ratio (VA / CAM) was calculated. Figure 6 As shown, hydrogels significantly increased angiogenesis.

[0048] Example 7: Anti-inflammatory effect of PICu hydrogel RAW264.7 macrophages were induced to polarize towards the M1 (pro-inflammatory) type using lipopolysaccharide (LPS) and further treated with PICu hydrogel extract. Flow cytometry results are as follows: Figure 7 As shown in Figure A, PICu hydrogel significantly reduced the proportion of CD86+ (M1 marker) cells and increased the proportion of CD206+ (M2 anti-inflammatory marker) cells. Immunofluorescence (IF): Figure 7 B visually shows a decrease in M1 macrophages and an increase in M2 macrophages.

[0049] Example 8: Antibacterial properties of PICu hydrogel Inhibitory effect against Porphyromonas gingivalis Colony forming unit (CFU) count: such as Figure 8 A, Figure 8 B and Figure 8 :D, PICu hydrogel has a strong killing effect on planktonic P. gingivalis. Live / Dead staining: Figure 8 B shows that most of the bacteria in the PICu group are dead (red). This indicates biofilm damage: such as... Figure 8 C and Figure 8 E,PICu hydrogel can effectively penetrate and disrupt existing bacterial biofilms.

[0050] Example 9: Therapeutic effect of PICu hydrogel on a rat model of periodontitis A rat model of periodontitis was established, and PICu hydrogel was injected locally for treatment.

[0051] Micro-CT analysis: such as Figure 9 The alveolar bone resorption distance (CEJ-ABC) was significantly reduced in the AC and PICu treatment groups, while the bone volume fraction (BV / TV) and trabecular bone thickness (Tb.Th) increased, and the trabecular bone separation (Tb.Sp) decreased, indicating that alveolar bone loss was effectively inhibited.

[0052] Example 10: Therapeutic effect of PICu hydrogel on rat skin infection wound model like Figure 10 :A and 10:B, PICu hydrogel exhibits good killing activity against Staphylococcus aureus; a rat model of full-thickness skin defects infected with Staphylococcus aureus was established by topical application of PICu hydrogel. For example... Figure 10C, D, and E: PICu hydrogel effectively removes *S. aureus* from the wound surface, promoting wound contraction and the fastest healing speed; the PICu group showed more complete epidermal regeneration, more regular collagen deposition, and richer granulation tissue on day 10. Figure 10 :F and Figure 10 As shown in Figure G, on the third day after treatment, the bacteria in the PICu+light group were significantly reduced after culturing the exudate from the wound.

Claims

1. A smart protein hydrogel with reversible controlled release of copper ions, characterized in that, The hydrogel is formed by coordination crosslinking of the following components: (a) bovine serum albumin; (b) gelatin; and (c) divalent copper ions. The mass ratio of bovine serum albumin to gelatin is (0.5-1.5):1, the concentration of divalent copper ions is 1 mM-12 M, and the hydrogel can maintain a gel state at 37 °C and undergo a reversible gel-sol transition under temperature control or photothermal action at 40-45 °C.

2. The intelligent protein hydrogel according to claim 1, characterized in that, The divalent copper ions are provided by copper salts, including copper chloride, copper sulfate, or copper acetate, and the concentration of the divalent copper ions is 4 mM-40 mM.

3. The intelligent protein hydrogel according to claim 1, characterized in that, The hydrogel is also loaded with indocyanine green as a photothermal conversion agent, and the content of indocyanine green is 0.0625-1 mg / mL.

4. The intelligent protein hydrogel according to claim 3, characterized in that, Under near-infrared light irradiation at a wavelength of 808 nm, the photothermal effect generated by the indocyanine green can trigger the hydrogel to undergo the gel-sol transition.

5. The intelligent protein hydrogel according to claim 4, characterized in that, The intensity of the fluorescence signal generated during the release of indocyanine green is positively correlated with the concentration of divalent copper ions released.

6. A method for preparing the intelligent protein hydrogel according to any one of claims 1-5, characterized in that, Includes the following steps: S1: Mix bovine serum albumin solution with gelatin solution to obtain protein mixture; S2: Add copper salt solution to the protein mixture and mix well; S3: Heat the mixture obtained in step S2 at 80-90℃ for 1-5 minutes, then cool it to obtain the intelligent protein hydrogel.

7. The method according to claim 6, characterized in that, Before or during step S1, indocyanine green is added to the bovine serum albumin solution and mixed well.

8. Use of the smart protein hydrogel according to any one of claims 1-5 in the preparation of a medicament for treating bacterial infections.

9. The use of the smart protein hydrogel of any one of claims 1-5 in the preparation of a medicament for treating infectious tissue injury, said infectious tissue injury including periodontitis or infectious skin wound.

10. The use of the smart protein hydrogel of any one of claims 1-5 in the preparation of a medicament for promoting tissue regeneration, wherein the tissue regeneration includes bone tissue regeneration or angiogenesis.

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