Nanoprotease catalytic double-network hydrogel, preparation method and application thereof

Abstract

The present application relates to the technical field of wound repair, in particular to a nano-enzyme catalytic double-network hydrogel, a preparation method and application. The hydrogel comprises biomimetic nano-enzyme CaP@TGnase, gamma-polyglutamic acid, epsilon-polylysine and gelatin, and the hydrogel GPP@VP loaded with photosensitizer verteporfin can be applied to the preparation of a wound scarless repair material. The hydrogel has potential transformation application value in realizing scarless healing and regenerative repair in a complex infection and fibrosis environment. Through integration of ion-covalent double-network structure and photodynamic antibacterial and immune regulation anti-fibrosis functions, the hydrogel exhibits good transformation application potential in infection wound repair and postoperative adhesion prevention.

Description

Technical Field

[0001] This invention relates to the field of wound repair technology, and more specifically, to a nanozyme-catalyzed dual-network hydrogel, its preparation method, and its application. Background Technology

[0002] Burn wounds remain a significant challenge in clinical treatment due to their susceptibility to infection, delayed healing, and high risks of fibrosis and scarring. Methicillin-resistant Staphylococcus aureus (MRSA) exhibits strong biofilm-forming capabilities and multidrug resistance, often significantly exacerbating the wound repair process, increasing the risk of systemic infection, and leading to adverse long-term outcomes. These intertwined pathological processes highlight the urgent need to develop advanced wound treatment strategies that can simultaneously regulate infection, immune responses, and fibrotic remodeling.

[0003] Hydrogels based on natural polymers have attracted widespread attention in the field of wound repair due to their good biocompatibility, biodegradability, and similarity to the extracellular matrix (ECM) structure.

[0004] However, traditional single-network hydrogels often suffer from insufficient mechanical properties, poor injectability, and limited immunomodulatory capabilities. In the complex stress environment of a wound, Engrailed-1 positive fibroblasts (ENFs) can be activated through the Yes-associated protein (YAP) / TEAD signaling pathway, inducing excessive matrix deposition and promoting hypertrophic scar formation. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a nanozyme-catalyzed dual-network hydrogel, its preparation method and application.

[0006] The technical solution of the present invention to solve the above-mentioned technical problems is as follows: This invention provides a method for preparing biomimetic nanozymes, using transglutaminase as a protein template and inducing calcium phosphate nucleation through biomimetic mineralization to obtain the biomimetic nanozymes.

[0007] Based on the above technical solution, the present invention can be further improved as follows.

[0008] Furthermore, glutaminase was mixed with a cell culture medium containing phosphate, calcium ion solution was added, and after incubation, the mixture was centrifuged and freeze-dried to obtain the biomimetic nanozyme.

[0009] Furthermore, the cell culture medium is high-glucose DMEM with a phosphate concentration of 0.1-1.0 g / L, and the calcium ion solution is calcium chloride solution with a calcium ion concentration of 0.5-2 M; the mass-volume ratio of glutaminase, high-glucose DMEM, and calcium chloride solution is 100:10:0.1.

[0010] The present invention also provides a biomimetic nanozyme, which is prepared by the method described above.

[0011] The present invention also provides a method for preparing a nanozyme-catalyzed dual-network hydrogel, wherein the components in the hydrogel raw material are mixed and incubated to obtain the hydrogel; wherein the hydrogel raw material includes the above-mentioned biomimetic nanozyme, γ-polyglutamic acid, ε-polylysine and gelatin.

[0012] Furthermore, the mass ratio of the biomimetic nanozyme, γ-polyglutamic acid, ε-polylysine, and gelatin is 1:10:10:100.

[0013] Further, the method includes the following steps: dissolving gelatin, γ-polyglutamic acid, and ε-polylysine in PBS to obtain solution A; dispersing the biomimetic nanozyme in PBS to obtain solution B; mixing solution A and solution B; and incubating at 37°C and 5% CO2 (volume percentage) for 1-20 minutes to obtain the hydrogel.

[0014] Furthermore, the hydrogel raw material also includes the photosensitizer vertiporfin.

[0015] The present invention also provides a nanozyme-catalyzed dual-network hydrogel, which is prepared by the method described above.

[0016] This invention also provides the application of the nanozyme-catalyzed dual-network hydrogel described above in the preparation of scarless wound repair materials.

[0017] The present invention also provides a scarless wound repair material, including the nanoenzyme catalyzed dual-network hydrogel as described above.

[0018] Furthermore, the scarless wound repair material is a scarless burn wound repair material.

[0019] The beneficial effects of this invention are as follows: (1) The preparation method of the biomimetic nanozyme of the present invention constructs a biomimetic nanozyme (CaP@TGnase) through a template-induced calcium phosphate mineralization strategy, which not only improves the environmental stability of the enzyme, but also introduces bioactive Ca 2+ To promote blood clotting and tissue repair; (2) The biomimetic nanozyme of the present invention can catalyze the covalent cross-linking of gelatin in hydrogel, so that the system has both the structural stability of covalent network and the reversible repair ability of ionic network, thereby achieving excellent mechanical properties, injectability and self-repair characteristics. (3) The method for preparing nanoenzyme-catalyzed dual-network hydrogel of the present invention is based on biocompatible natural polymers such as γ-PGA, ε-PLL and gelatin, and constructs a dual-network (DN) structure hydrogel GPP formed by ionic bonds and enzyme-catalyzed crosslinking; (4) The nanozyme catalyzed dual-network hydrogel of the present invention synergistically integrates multiple functions such as antibacterial, hemostatic, immune regulation, anti-fibrosis and regeneration promotion of ε-PLL, VP, CaP@TGnase and dual-network matrix, and realizes effective intervention on infected burn wounds and postoperative abdominal adhesions. (5) The nanozyme catalyzed dual-network hydrogel of the present invention integrates the ion-covalent dual-network structure with photodynamic antibacterial and immunomodulatory antifibrotic functions. This hydrogel shows good potential for transformational application in the repair of infected wounds and the prevention of postoperative adhesions. Attached Figure Description

[0020] Figure 1 This refers to the design, characterization, and functional evaluation of the CaP@TGnase nanozyme in Example 1 of the present invention. Figure 1 A in the diagram represents the formation of CaP@TGnase through TGnase-mediated biomimetic mineralization. Figure 1 In section B, the Tyndall effect is used to verify the formation of nanoparticles. Figure 1 C represents the dispersion and sedimentation behavior of CaP@TGnase; Figure 1 D represents the Fourier transform infrared (FTIR) spectra of CaP (a), CaP@TGnase (b), and TGnase (c). Figure 1 E in the middle represents the X-ray diffraction (XRD) patterns of CaP and CaP@TGnase; Figure 1 In the middle, F–G are scanning electron microscope (SEM) images of CaP and CaP@TGnase, respectively; Figure 1 H–I in the image represents a transmission electron microscope (TEM) image of CaP@TGnase. Figure 1 J represents the thermogravimetric-differential scanning calorimetry (TGA-DSC) curve of CaP@TGnase; Figure 1 K is the SEM image of CaP@TGnase; Figure 1 L in the diagram represents the elemental distribution of CaP@TGnase energy dispersive spectroscopy (EDS). Figure 1 M represents the catalytic activity of CaP@TGnase over 10 cycles; Figure 1 N–P represent the enzyme stability of TGnase and CaP@TGnase under different conditions; Figure 2 The construction and characterization of GPP hydrogel in Example 2 of the present invention; Figure 2 A in the diagram is a schematic diagram of the formation of a double-network hydrogel by CaP@TGnase catalyzing γ-PGA, ε-PLL and gelatin; Figure 2 In section B, the sol-gel transition process of the precursor solution is represented. Figure 2 C in the image represents the SEM image of the CaP@TGnase crosslinked hydrogel. Figure 2D represents the Fourier transform infrared (FTIR) spectra of CaP@TGnase crosslinked gelatin, GPP, and GPP@VP hydrogel. Figure 2 E represents the self-healing behavior of the GPP hydrogel cutting interface; Figure 2 In the middle F, the macroscopic adhesion of the hydrogel to rat skin before and after PBS immersion is shown. Figure 2 G is a SEM image of the hydrogel-tissue interface after soaking in PBS for 24 h; Figure 2 In the middle, H is a photograph showing the morphological recovery of the GPP hydrogel after being subjected to pressure; Figure 2 In Figure I, the compressive stress-strain curves of gelatin and GPP hydrogel are shown. Figure 2 Photograph J shows the morphology of the GPP hydrogel before and after tensile deformation and recovery. Figure 2 K represents the tensile stress-strain curve of gelatin and GPP hydrogel; Figure 2 L represents the frequency scan of the GPP hydrogel; Figure 2 M represents the amplitude-scanning rheological curve; Figure 2 N represents the brittle fracture of gelatin hydrogel during the folding process; Figure 2 The O in GPP hydrogel can resist fracture and restore its shape after deformation; Figure 3 The physicochemical properties, drug release, biocompatibility, and mechanotransduction regulation of GPP@VP hydrogel in Example 4 of the present invention; Figure 3 Image A shows the injectability and shape retention of the GPP@VP hydrogel after CaP@TGnase-mediated crosslinking. Figure 3 B represents the standard calibration curve for VP at 442 nm; Figure 3 C represents the cumulative VP release curve of GPP@VP hydrogel over 12 days; Figure 3 D represents the degradation behavior of GPP and GPP@VP hydrogel in PBS at 37°C; Figure 3 E represents the swelling behavior of the hydrogel within 12 h; Figure 3 F represents cell viability after treatment with different concentrations of VP (1–6 μM), n = 3; *p<0.05; Figure 3 In the middle G, the Live / Dead staining results were obtained after 1 day and 3 days of culture on different hydrogel substrates. Figure 3 H represents the CCK-8 test results after 24, 48, and 72 h of different material treatments, n = 3; *p<0.05; Figure 3 Image I in the middle is an immunofluorescence image; Figure 3 J represents the proportion of intranuclear YAP-positive cells based on immunofluorescence images, n = 9; *p<0.05, **p<0.01, ***p<0.001; Figure 4This is an evaluation of the antibacterial properties of GPP@VP hydrogel in Example 5 of the present invention; Figure 4 Image A shows representative images of MRSA colonies after GPP@VP treatment at different NIR irradiation times; Figure 4 Image B shows representative images of MRSA colonies after 2 hours of treatment with different materials; Figure 4 Image C in the middle is a representative image of the inhibition zone in the plate diffusion experiment; Figure 4 D is with Figure 4 Quantitative analysis of colony forming units (CFU) corresponding to A in the sample; Figure 4 E in the middle is with Figure 4 Quantitative analysis of CFU corresponding to B in the formula; Figure 4 F is with Figure 4 Quantitative analysis of the inhibition zone area corresponding to C in the middle section; Figure 4 G represents the representative fluorescence image of MRSA live / dead staining, n = 3; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; Figure 5 Evaluation of the in vitro and in vivo hemostatic properties of GPP and GPP@VP hydrogels in Examples 6 and 7 of the present invention; Figure 5 Image A in the image represents a hemolysis experiment after incubation with different materials; Figure 5 Image B is a SEM image of erythrocyte adhesion on the surface of gauze, gelatin sponge (GS), GPP and GPP@VP; Figure 5 The diagram in C is a schematic of a tail amputation hemostasis model; Figure 5 The middle image (D) shows representative images of hemostasis after different material treatments in the SD rat tail amputation model. Figure 5 E represents the quantitative analysis of hemolysis rate; Figure 5 In the middle F, the whole blood coagulation index (BCI) is represented by different treatment groups. Figure 5 G represents the quantitative analysis of blood loss after different material treatments in the tail amputation model; Figure 5 In the middle H, the bleeding time after different material treatments in the tail-severance model is quantitatively analyzed; Figure 5 In the middle section (I), a quantitative analysis of blood loss after treatment with different materials was performed in a liver injury model. Figure 5 J represents a quantitative analysis of bleeding time after different material treatments in a liver injury model; Figure 5 The diagram in K shows a liver hemorrhage model in SD rats. Figure 5 In the middle L, there are representative images of liver hemorrhage after treatment with different materials; where n=5; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; Figure 6 This is an evaluation of the in vivo therapeutic effect of GPP@VP in an MRSA-infected burn wound model in Example 8 of the present invention. Figure 6A in the diagram represents the animal experiment process. Figure 6 Image B shows representative wound healing in each treatment group from postoperative day 0 to day 14 (POD 0–14); Figure 6 C is a pseudo-color diagram illustrating the changes in wound area; Figure 6 D represents the quantitative analysis of residual wound area (%) at each time point, n = 3; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; Figure 6 In the middle E, the H&E and Masson staining histological analysis at POD 7 and POD 14 is shown. The black arrows indicate the wound edge location, and the dashed circles mark the regeneration-related areas, including granulation tissue, new epidermis, and skin appendages. Figure 7 This refers to the immunomodulatory and antifibrotic effects of GPP@VP and GPP@VP NIR in the healing process of MRSA-infected burn wounds as described in Examples 9-11 of this invention. Figure 7 Image A shows an immunofluorescence image of MPO expression in the POD 3 wound tissue; Figure 7 In Figure B, the quantitative analysis of the MPO positive area in each treatment group is presented. Figure 7 In the middle C, we see the co-localization of YAP (red) and Vimentin (green) in POD 14 fibroblasts. The cell nuclei are stained with DAPI (blue), and the yellow signal indicates the intranuclear localization of YAP. Figure 7 Image D in the middle is an immunofluorescence staining image of POD14 α-SMA; Figure 7 E represents a quantitative analysis of the proportion of YAP-positive fibroblasts in the nucleus; Figure 7 In the middle F, the positive area of ​​α-SMA in each group is quantitatively analyzed; Figure 7 The GH in the POD 7 (G) and POD 14 (H) wound tissues is a double immunofluorescence staining of iNOS (red) and Arg-1 (green), and the cell nuclei are labeled with DAPI (blue). Figure 7 The expression of Arg-1 (I) and iNOS (J) in POD 7 was quantitatively analyzed. Figure 7 Quantitative analysis of Arg-1 (K) and iNOS (L) expression in KL at POD 14; n = 9; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; Figure 8 This refers to the multi-level evaluation of GPP@VP NIR in the abdominal adhesion model in Examples 15 and 16 of the present invention. Figure 8 A is a schematic diagram of a double-injury abdominal adhesion model; Figure 8 Image B is a macroscopic representative image of the adhesion tissue in each treatment group on postoperative day 14 (POD 14). Figure 8In the middle C, the results of H&E staining and Masson's trichrome staining of the adhesion sites in each group are shown. Figure 8 D represents a quantitative analysis of adhesion scores based on a standardized 0–5 scoring system (n = 3). Figure 8 E represents the GO enrichment analysis of differentially expressed genes (DEGs) between the GPP@VP NIR group and the control group; Figure 8 F represents KEGG pathway enrichment analysis; Figure 8 The G–J section is a heatmap of the top 15 genes with the most significant differential expression in the following pathways: cell adhesion (G), ECM–receptor interaction (H), adhesion plaques (I), and antigen processing and presentation (J). Figure 8 In the middle K, a radar chart shows the upregulated differentially expressed genes enriched in the “wound healing” GO item; G1–G5 represent samples from the GPP@VP+NIR treatment group; C1–C3 represent samples from the control group. Figure 9 This is a schematic diagram illustrating the mechanism and therapeutic effect of the γ-PGA / ε-PLL / gelatin dual-network hydrogel loaded with verteporfen (VP) of the present invention in achieving dual-mode antibacterial, immunomodulatory, hemostatic, tissue regeneration and anti-adhesion effects in infected burn wounds. Figure 10 This is a schematic diagram of the reformation of a continuous gel from the dried and ground GPP hydrogel in Embodiment 2 of the present invention; Figure 11 This is a graph showing the absorbance test results of VP in Embodiment 3 of the present invention; Figure 12 This is an image showing the immunofluorescence staining results in Example 8 of the present invention; Figure 13 This is a Sirius red staining result diagram in Example 12 of the present invention; Figure 14 This is a graph showing the results of quantitative analysis of the type I / type III collagen ratio using Sirius red staining in Example 12 of the present invention. Figure 15 This is a macroscopic comparison image of scar formation in each group in Embodiment 13 of the present invention; Figure 16 This is an image showing the immunofluorescence staining results of keratin 14-positive hair follicles in each group of healing tissue sections in Example 13 of the present invention. Figure 17 This is a graph showing the quantitative results of keratin 14-positive hair follicle density in each group of healing tissue sections in Example 13 of the present invention. Figure 18 This is a diagram showing the H&E staining analysis results of the main organs in each group in Example 14 of the present invention; Figure 19 This is a graph showing the results of routine blood tests on the major organs in each group in Embodiment 14 of the present invention; Figure 20 This refers to the principal component analysis results of the control group and the GPP@VP NIR treatment group in the transcriptome sequencing analysis of Example 16 of the present invention. Figure 21 This refers to the consistency of gene expression distribution between the control group and the GPP@VP NIR treatment group in transcriptome sequencing analysis in Example 16 of the present invention. Figure 22 This refers to the intragroup Pearson correlation results of each group in the transcriptome sequencing analysis of Example 16 of the present invention. Figure 23 This refers to the differential expression analysis results of the GPP@VP NIR treatment group in the transcriptome sequencing analysis of Example 16 of the present invention. Figure 24 This refers to the hierarchical clustering analysis results of each group in the transcriptome sequencing analysis of Example 16 of the present invention. Figure 25 In Embodiment 16 of the present invention, multi-node expression clustering analysis is performed in the MAPK signaling pathway; Figure 26 This is a comparison diagram of the expression of various related genes in the MAPK signaling pathway in Example 16 of the present invention. Detailed Implementation

[0021] The principles and features of the present invention are described below. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0022] The method for preparing biomimetic nanozymes of the present invention uses transglutaminase as a protein template and induces calcium phosphate nucleation through biomimetic mineralization to obtain biomimetic nanozymes.

[0023] The method for preparing biomimetic nanozymes of the present invention constructs biomimetic nanozymes (CaP@TGnase) through a template-induced calcium phosphate mineralization strategy, which not only improves the environmental stability of the enzyme but also introduces bioactive Ca. 2+ To promote blood clotting and tissue repair.

[0024] Preferably, the preparation method involves mixing glutaminase with a cell culture medium containing phosphate, adding calcium ion solution, incubating, centrifuging, and freeze-drying to obtain a biomimetic nanozyme.

[0025] Specifically, the cell culture medium is high-glucose DMEM with a phosphate concentration of 0.1–1.0 g / L, and the calcium ion solution is calcium chloride solution with a calcium ion concentration of 1 M; the mass-volume ratio of glutaminase, high-glucose DMEM, and calcium chloride solution is 100:10:0.1.

[0026] The biomimetic nanozyme of the present invention is prepared by the above method; the nanozyme can catalyze covalent cross-linking of gelatin in hydrogel, so that the system has both the structural stability of covalent network and the reversible repair ability of ionic network, thereby achieving excellent mechanical properties, injectability and self-healing characteristics.

[0027] The method for preparing the nanozyme-catalyzed dual-network hydrogel of the present invention involves mixing and incubating the components of the hydrogel raw material to obtain the hydrogel; wherein the hydrogel raw material includes the biomimetic nanozyme, γ-polyglutamic acid, ε-polylysine and gelatin as described above.

[0028] This invention utilizes biocompatible natural polymers such as γ-PGA, ε-PLL, and gelatin to construct a dual-network (DN) structure hydrogel (GPP) formed through ionic bonds and enzymatic crosslinking. γ-PGA is a hydrophilic, biodegradable anionic polypeptide with excellent water retention and drug loading capacity; ε-PLL, as a cationic antimicrobial peptide, exhibits good safety and membrane-disrupting antibacterial activity. The two interact electrostatically to form a dynamic ionic network, endowing the system with antibacterial protection, energy dissipation, and self-healing capabilities.

[0029] Preferably, to further enhance the therapeutic effect, the hydrogel material also includes the photosensitizer verteporfin (VP), and the VP-loaded hydrogel is GPP@VP.

[0030] The hydrogel GPP system of this invention synergistically integrates multiple functions such as antibacterial, hemostatic, immunomodulatory, antifibrotic and regeneration-promoting properties of ε-PLL, VP, CaP@TGnase and dual network matrix, achieving effective intervention for infected burn wounds and postoperative abdominal adhesions.

[0031] Specifically, the covalent-ionic dual crosslinking in GPP@VP endows the hydrogel with excellent mechanical properties, combining injectability and in-situ gelation ability, making it adaptable to irregular wound morphologies; the ε-PLL within it provides intrinsic antibacterial activity against MRSA; and the CaP@TGnase nanozyme utilizes Ca... 2+ Mediated coagulation and enzymatic cross-linking further enhance hemostasis and structural stability.

[0032] GPP@VP can inhibit fibroblast activation and myofibroblast differentiation by suppressing the Yes-associated protein (YAP) mechanotransduction pathway. Simultaneously, GPP@VP can regulate macrophage polarization, reshaping the immune microenvironment. In infected wounds, GPP@VP can reduce inflammation and fibrosis, inhibit pathological scar formation, and promote the establishment of a regenerative microenvironment. Furthermore, it can effectively prevent abdominal adhesions by reducing ECM deposition and downregulating adhesion-related signaling pathways.

[0033] Overall, the results of in vitro and in vivo experiments together indicate that GPP@VP has potential translational application value for achieving scarless healing and regenerative repair in complex infection and fibrotic environments.

[0034] Preferably, the mass ratio of biomimetic nanozyme, γ-polyglutamic acid, ε-polylysine and gelatin is 1:10:10:100.

[0035] The preparation method of the nanozyme-catalyzed dual-network hydrogel of the present invention specifically includes the following steps: dissolving gelatin, γ-polyglutamic acid, and ε-polylysine in PBS to obtain solution A, dispersing the biomimetic nanozyme in PBS to obtain solution B, mixing solution A and solution B, and incubating at 37°C and 5% CO2 volume for 1-20 minutes to obtain hydrogel.

[0036] The nanozyme-catalyzed dual-network hydrogel of the present invention is prepared by the method described above; the nanozyme-catalyzed dual-network hydrogel can be applied to the preparation of scarless wound repair materials; by integrating the ion-covalent dual-network structure with photodynamic antibacterial and immunomodulatory antifibrotic functions, the hydrogel shows good potential for translational application in the repair of infected wounds and the prevention of postoperative adhesions.

[0037] The scarless wound repair material of the present invention includes the nanozyme-catalyzed dual-network hydrogel as described above.

[0038] Preferably, the scarless wound repair material is a scarless burn wound repair material.

[0039] The effects of the present invention will be illustrated below through specific embodiments.

[0040] The materials involved in the following embodiments are: γ-PGA (molecular weight approximately 1000 kDa) was purchased from CITES Biotechnology Co., Ltd. (China).

[0041] ε-PLL (molecular weight 2000–5000 Da) was purchased from Aladdin (China).

[0042] TGnase was purchased from Solarbio Biotechnology Co., Ltd. (China).

[0043] Gelatin (derived from pigskin) was purchased from Sigma-Aldrich (USA).

[0044] High-glucose Dulbecco modified Eagle medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco (Carlsbad, California, USA).

[0045] NIH-3T3 fibroblasts and RAW 264.7 macrophages were cultured in high-glucose DMEM complete medium containing 10% FBS and 1% penicillin-streptomycin.

[0046] All cells were cultured in a constant temperature and humidity incubator at 37°C and 5% CO2.

[0047] Example 1 Synthesis and Characterization of CaP@TGnase Nanozyme To construct a hybrid nanostructure with biological functions, this embodiment employs a biomimetic mineralization strategy using transglutaminase (TGnase) as a protein template to induce calcium phosphate (CaP) nucleation.

[0048] The specific preparation steps of CaP nanoparticles and CaP@TGnase nanozymes in this embodiment are as follows: 500 μL of 1 M CaCl2 was added to 50 mL of high-sugar DMEM, and the mixture was incubated at 37 °C and 5% CO2 for 24 h. The precipitate was then collected by centrifugation at 10,000 rpm at 4 °C for 20 min and lyophilized to obtain CaP nanoparticles.

[0049] 500 mg of TGnase was dissolved in 50 mL of high-glucose DMEM, filtered through a 0.22 μm filter membrane, and then 500 μL of 1M CaCl2 was added. After incubation at 37 °C and 5% CO2 for 24 h, the mixture was centrifuged at 10,000 rpm for 20 min (44 °C), and the resulting precipitate was lyophilized to obtain the CaP@TGnase nanozyme.

[0050] like Figure 1 As shown in Figure A, the negatively charged structural domains on the TGnase surface can promote Ca2+ uptake. 2+ and PO4 3- Local enrichment of the enzyme triggers a surface-restricted nucleation process, forming enzyme-inorganic composite nanoparticles (CaP@TGnase).

[0051] Through the Tyndall effect ( Figure 1 (B) Preliminary verification of the formation of colloidal nanoassemblies: After TGnase was mixed with calcium and phosphorus precursors, obvious light scattering was observed, while the solution of TGnase in DMEM remained transparent, indicating that the nanocolloids were successfully formed and the mineralization process was realized.

[0052] The dispersibility of CaP@TGnase was further evaluated by visual inspection. The freshly prepared suspension showed good dispersion. Figure 1 (Ci), while after standing for 24 hours, some sedimentation occurred ( Figure 1The presence of C-ii indicates good initial colloidal stability, but gravity-driven aggregation occurs over time. This sedimentation characteristic facilitates particle recovery without ultracentrifugation, thereby improving the material's reusability.

[0053] X-ray diffraction (XRD) analysis revealed the regulatory role of the TGnase template on CaP crystal formation. For example... Figure 1 As shown in Figure D, pure CaP exhibits seven clear diffraction peaks, displaying a multiphase, polycrystalline structure; while CaP@TGnase shows only three main diffraction peaks, some of which overlap with CaP, suggesting that TGnase selectively regulates crystal growth, forming a simpler crystal phase structure. This phenomenon is consistent with the protein-regulated biomimetic mineralization mechanism.

[0054] Fourier transform infrared spectroscopy (FTIR) further verified the introduction of TGnase. Figure 1 (E). CaP@TGnase also exhibits a phosphate characteristic peak (approximately 1035 cm⁻¹). -1 and 563 cm -1 ) and protein-associated amide I and II bands (approximately 1650 cm) -1 and 1540 cm -1 ), and at approximately 3400 cm -1 A broad O–H / N–H stretching vibration peak appears. The coexistence of these signals indicates that mineral and enzyme components are present simultaneously, suggesting the possible presence of hydrogen bonds or electrostatic interactions between the two phases, while the enzyme conformation is preserved to a certain extent.

[0055] Scanning electron microscopy (SEM) revealed significant morphological differences between CaP and CaP@TGnase. Pure CaP exhibited a uniform spherical structure (50–100 nm). Figure 1 (F), while CaP@TGnase exhibits a rough polyhedral structure (F). Figure 1 The presence of G indicates that TGnase regulates the mineral growth direction and surface topology during nucleation.

[0056] Transmission electron microscopy (TEM) further revealed its microstructural features. Low magnification image ( Figure 1 The middle (H) image shows that the nanoparticles exist in clusters, and their size is consistent with the SEM results; the high-resolution image ( Figure 1 The image (I) shows that individual particles are composed of densely arranged fibrous or rod-shaped substructures, suggesting that primary nanocrystals undergo directional assembly to form a structure with hierarchical anisotropy, further demonstrating its biomimetic mineralization characteristics.

[0057] This embodiment tested the enzyme activity, stability, and reusability of CaP@TGnase. The specific testing methods are as follows: (1) Determination of CaP@TGnase enzyme activity: The CaP@TGnase activity was determined by colorimetric method using N-CBZ-Gln-Gly and hydroxylamine as substrates, in the same manner as free TGnase. 20 μL of enzyme solution was mixed with 200 μL of substrate buffer (30 mM N-CBZ-Gln-Gly, 100 mM hydroxylamine hydrochloride, 10 mM glutathione, 200 mM Tris-HCl, pH 6.0) and incubated at 37℃ for 10 min. 200 μL of stop solution (3 M HCl, 12% TCA, 5% FeCl3·6H2O) was added to terminate the reaction, and the absorbance was measured at 525 nm. Enzyme activity units were defined as the amount of enzyme that catalyzes the production of 1 μmol of hydroxyxamic acid per minute at 37℃.

[0058] (2) Stability evaluation of CaP@TGnase: To assess stability, free TGnase and CaP@TGnase with equal enzyme activity units were treated under different environmental conditions: pH stability: Residual activity was determined after incubation in PBS buffer at pH 2–10 (37°C, 15 min); Thermal stability: Activity was measured after incubation in PBS at different temperatures (37℃, 40℃, 45℃, 50℃, 55℃, 60℃) for 30 min. Solvent stability: Activity was determined after incubation in PBS containing different concentrations of ethanol (0–100%) (room temperature, 15 min).

[0059] Residual enzyme activity is expressed as a percentage relative to initial activity.

[0060] (3) Reusability of CaP@TGnase: CaP@TGnase was subjected to 10 consecutive cycles. After each reaction, the nanozyme was recovered by centrifugation, washed with PBS, and reused. The residual activity was measured under the same conditions and normalized based on the activity of the first cycle.

[0061] The test results are as follows: Thermogravimetric-differential scanning calorimetry (TG-DSC) results showed that CaP@TGnase experienced a mass loss of approximately 30% in the temperature range of 200–600℃, corresponding to approximately 24 wt% of organic components, while pure CaP showed almost no decomposition. Figure 1 (J), confirming that the enzyme successfully embedded in the mineral matrix.

[0062] Elemental composition and spatial distribution were mapped using SEM-EDS ( Figure 1The K–L (K-L) assay was validated, and Ca, P, C, N and O elements were uniformly distributed in the nanoparticles. The C and N signals (not present in pure CaP) further proved the successful introduction and uniform dispersion of the protein components.

[0063] The catalytic reusability of CaP@TGnase was evaluated over 10 reaction cycles. Figure 1 As shown in Figure M, the nanozyme retained more than 55% of its initial activity after 10 cycles. The decrease in activity was mainly attributed to physical loss during the washing process rather than enzyme structure inactivation, indicating that it has good structural and operational stability.

[0064] Enzyme activity tests under different pH conditions showed that CaP@TGnase maintained high residual activity in both acidic (pH 2–3) and alkaline (pH 8–10) environments. Figure 1 The presence of N indicates that mineral coating can effectively buffer the effects of pH changes on enzyme activity, which is of great significance for complex physiological environments.

[0065] Thermal stability tests showed that both TGnase and CaP@TGnase exhibited optimal activity at 37℃, with activity gradually decreasing at 50℃. Figure 1 (O). It is worth noting that CaP@TGnase retains about 20% of its activity at 55℃, while TGnase is almost completely deactivated, indicating that mineralization coating significantly improves its thermal stability.

[0066] Finally, the tolerance test in an environment containing ethanol solvent ( Figure 1 The results (P) show that CaP@TGnase retains over 90% of its enzyme activity even at ethanol concentrations below 70%, while TGnase activity fluctuates significantly and exhibits an abnormal increase in 80% ethanol, possibly related to conformational changes. In contrast, the mineral matrix of CaP@TGnase maintains stable enzyme function under solvent stress, further demonstrating its application potential in complex biological environments or sterilization conditions.

[0067] Example 2 Construction and characterization of GPP dual-network hydrogel like Figure 2 As shown in Figure A, this embodiment integrates γ-polyglutamic acid (γ-PGA), ε-polylysine (ε-PLL) with gelatin through CaP@TGnase nanozyme-mediated crosslinking to construct a structure-enhanced dual-network (DN) hydrogel (GPP).

[0068] In this system, γ-PGA and ε-PLL form a dynamic ion-consuming network through electrostatic interactions between carboxyl and amino groups. Lacking suitable glutamine and lysine residues, neither can serve as a substrate for TGnase, forming a network structure only through reversible non-covalent interactions. In contrast, gelatin, rich in glutamine and lysine residues, can form a stable covalently cross-linked network under TGnase catalysis. CaP@TGnase nanozymes, acting as biomimetic catalytic nodes, achieve hierarchical integration of the two networks. This dual-mode cross-linking strategy endows GPP with excellent mechanical toughness, flexibility, and tissue adhesion, meeting the dual requirements of structural support and dynamic adaptability in biomedical applications.

[0069] In this embodiment, the specific preparation steps of the GPP hydrogel and powder are as follows: Gelatin (4 g), γ-PGA (400 mg), and ε-PLL (400 mg) were dissolved in 15 mL of PBS (solution A). CaP@TGnase (40 mg) was dispersed in 5 mL of PBS (solution B). After mixing solutions A and B, the mixture was incubated at 37°C and 5% CO2 for several minutes to form a GPP hydrogel. The above process was carried out under light-protected conditions. After obtaining the hydrogel, it was lyophilized and ground into powder.

[0070] like Figure 2 As shown in Figure B, the precursor solution is white and opaque with good fluidity, mainly attributed to the colloidal dispersion of CaP@TGnase and the formation of the γ-PGA / ε-PLL complex. Under incubation conditions of 37°C, the system can rapidly undergo a sol-gel transition to form a self-supporting hydrogel.

[0071] Scanning electron microscopy (SEM) results ( Figure 2 As shown in Figure C), the different systems exhibit significantly different microstructures. Gelatin hydrogels display a regular porous structure and a beam-like internal scaffold, while GPP and GPP@VP exhibit disordered and heterogeneous porous networks lacking distinct structural units. This structural variation may stem from the interference of ionic interactions on the gelatin assembly process. Despite the reduced orderliness, the complex, entangled structure is beneficial for energy dissipation, morphological adaptation, and adhesion to irregular tissues.

[0072] Fourier transform infrared spectroscopy (FTIR) Figure 2 The results from the D-scan show that each hydrogel has typical protein absorption peaks, and the spectral differences between different systems indicate that both enzyme-catalyzed crosslinking and the introduction of VP lead to structural changes.

[0073] It is worth noting that, such as Figure 2As shown in Figure E, two separate GPP hydrogel fragments can reconnect within minutes under physiological conditions, which may be attributed to residual TGnase activity and reversible ionic interactions between γ-PGA / ε-PLL. Although this process is not typical of dynamic self-healing, it reflects a certain degree of structural repairability of the material.

[0074] Furthermore, GPP hydrogels can rapidly absorb water and reform into continuous gels after drying and grinding. Figure 10 This further demonstrates its excellent rehydration capability and structural adaptability.

[0075] Adhesion performance test ( Figure 2 The results showed that the newly formed GPP hydrogel could adhere firmly to the surface of rat skin and remained stable after being soaked in PBS for 24 h.

[0076] SEM images ( Figure 2 The results (G) show that the hydrogel is in close contact with the tissue interface, with no obvious interface separation, proving that it has good interface integration ability.

[0077] The GPP hydrogel was subjected to compression and tensile property tests, and the specific test methods were as follows: (1) Compression performance test: The gelatin and GPP hydrogel were subjected to compression test using an Instron 68SC-1 universal testing machine at a loading rate of 1 mm / min. The stress-strain curves were recorded and the compressive modulus was calculated from the linear region.

[0078] (2) Tensile property test: Tensile tests were performed on gelatin and GPP hydrogel using an Instron 68SC-1 universal testing machine at a loading rate of 10 mm / min. The stress-strain curves were recorded, and the maximum stress before fracture was taken as the tensile strength.

[0079] In compression tests ( Figure 2 (H), GPP hydrogels can recover their original shape without damage after being subjected to pressure. Mechanical test results ( Figure 2 The results (I) show that the yield stress of GPP is 7675 kPa, significantly higher than that of gelatin (165 kPa, an increase of approximately 47 times), while the compressive moduli of both are similar, indicating that its mechanical reinforcement mainly comes from the synergistic effect of the dual network rather than a simple increase in stiffness. Tensile property tests ( Figure 2 Further studies using J and K models confirmed GPP's superior elasticity. GPP exhibited a fracture stress of 0.04 MPa and a fracture strain of 235.84%, significantly better than gelatin (0.02 MPa, 12.78%). Although GPP had a lower tensile modulus (0.03 MPa vs. 0.07 MPa), its higher ductility and recovery demonstrated superior elastic characteristics.

[0080] Rheological tests further validated its gel properties. Frequency scan results ( Figure 2 The data from the L-wavelength (L) shows that the storage modulus G′ (1583.6 Pa) is significantly higher than the loss modulus G″ (109.5 Pa), and tanδ < 0.07 in the 0.1–100 Hz range, indicating that the system exhibits a predominantly elastic response. Amplitude scan ( Figure 2 The results show that G′ remains stable (1637.3 Pa) in the small strain range (<2.02%), but gradually decreases with increasing strain, while G″ increases to 284.6 Pa (100% strain), indicating that the network structure is damaged and accompanied by viscous energy dissipation.

[0081] Fracture performance testing further highlights the advantages of the dual-network structure. For example... Figure 2 As shown in Figure N, gelatin hydrogel breaks under slight bending, while GPP ( Figure 2 The ability of ionized oxygen (O2) to recover its original shape without crack formation under strong folding conditions demonstrates its excellent fracture resistance. This characteristic stems from the synergistic effect of the ion network and covalent network, giving it good application potential in dynamic stress environments.

[0082] Example 3: Physicochemical properties and drug release behavior of GPP@VP hydrogel In this embodiment, the photosensitizer verteporfin (VP) is loaded onto a GPP hydrogel to obtain GPP@VP. The specific preparation process is as follows: Gelatin (4 g), γ-PGA (400 mg), ε-PLL (400 mg), and VP (20 μg) were dissolved in 15 mL of PBS (solution A), then mixed with solution B containing CaP@TGnase (40 mg), and incubated under the same conditions to form a hydrogel. The above process was carried out under light-protected conditions.

[0083] This embodiment first evaluated the injectability and shape adaptability of the GPP@VP hydrogel to verify its potential in minimally invasive applications. Figure 3 As shown in Figure A, the precursor solution can be smoothly extruded using a standard syringe and gelled in situ under the catalysis of CaP@TGnase nanozyme. The resulting hydrogel can conform to a predetermined shape, indicating that it has good injectability and spatial adaptability.

[0084] To quantitatively analyze the content and release behavior of verteporfen (VP), this example measures the UV-Vis absorption spectrum of VP in the range of 300–800 nm to determine characteristic peaks and characterize its UV-Vis absorption properties.

[0085] Specifically, VP was prepared in a series of concentrations of 1–10 μg / mL (PBS containing 10% DMSO), the absorbance was measured at 442 nm, and an absorbance-concentration standard curve was plotted.

[0086] Test results show that a characteristic absorption peak was observed at approximately 442 nm. Figure 11 Based on this, in 1–10 μg·mL -1 Establish a standard curve within the range ( Figure 3 (B)

[0087] Based on the above calibration relationship, the release behavior of VP in the hydrogel was monitored. The monitoring method was as follows: the GPP@VP hydrogel was placed in PBS (2 mL) containing 10% DMSO and incubated at 37°C with gentle shaking. At set time points (days 1, 2, 3, 4, 5, 7, 9, and 12), 300 μL of the release solution was collected, and an equal volume of fresh buffer was added. The absorbance was measured at 442 nm, and the cumulative release of VP was calculated based on the standard curve.

[0088] like Figure 3 As shown in Figure C, VP exhibits a continuous and time-dependent release pattern, and its release kinetics conform to a transport mechanism mediated by diffusion through the hydrogel network.

[0089] This embodiment tested the degradation performance of the GPP and GPP@VP hydrogels prepared in Example 2. The specific testing method was as follows: GPP and GPP@VP hydrogels were prepared into cylindrical shapes (6 mm in diameter, 5 mm in height). Immediately after gel formation, the surface moisture was gently aspirated and the samples were weighed, recording the initial mass (W0). The samples were then placed in 1 mL of simulated body fluid (SBF) and incubated with gentle shaking at 37°C, with the culture medium changed daily. At preset time points (1, 3, 5, 7, 14, 24, 40, and 60 days), parallel samples were taken, gently aspirated (10 s), and weighed (Wt). The degradation rate was calculated using the following formula: Degradation rate (%) = 100 × Wt / W0 like Figure 3 As shown in Figure D, on day 1, GPP and GPP@VP retained 91.28% and 93.30% of their initial mass, respectively; on day 14, they retained 81.43% and 86.72%; on day 40, they retained 67.59% and 74.44%; and by day 60, they retained 66.36% and 67.79%. At each time point, GPP@VP showed a higher mass retention rate, suggesting that the introduction of VP may slow down the degradation process to some extent by increasing the crosslinking density or altering water interactions.

[0090] This embodiment also tested the swelling properties of GPP and GPP@VP hydrogels. The hydrogels were cylindrical (6 mm in diameter and 5 mm in height). The specific testing method was as follows: After the hydrogel formed, gently aspirate and weigh it, recording the initial wet weight (W0). Place the hydrogel in PBS at pH 7.4 and incubate with gentle shaking at 37°C. At set time points (10, 20, 30, 40, 50, 60, 90, 120, 150, and 720 min, until equilibrium is reached), remove the sample, aspirate, and weigh it (Wt).

[0091] Swelling rate (%) = Wt / W0 × 100 like Figure 3 As shown in Figure E, at 10 min, the swelling ratios of GPP and GPP@VP were 114.86% and 110.92%, respectively; at 60 min, they increased to 134.25% and 127.18%, respectively; and at 720 min, GPP reached 151.34%, while GPP@VP was 137.97%. The lower swelling degree of GPP@VP may be due to the introduction of VP reducing network porosity or enhancing hydrophobic interactions, thereby helping to improve structural stability and achieve more sustained drug release.

[0092] Example 4: Biocompatibility and Antifibrotic Signal Regulation of GPP@VP Hydrogel In this embodiment, the GPP@VP hydrogel prepared in Example 3 was used for cytotoxicity experiments, live / dead staining, and cell immunofluorescence staining.

[0093] (1) Cytotoxicity assay: The cytocompatibility of verteporfen was assessed in NIH-3T3 fibroblasts using the CCK-8 assay.

[0094] NIH-3T3 cells at 5×10 3 Cells / wells were seeded in 96-well plates and cultured for 4 h until adherence. They were then cultured for 12 h in serum-free medium to synchronize the cell cycle. Afterward, they were treated with different concentrations of VP (1–6 μg / mL) for 24 h. After treatment, the culture medium was discarded, and the cells were washed three times with PBS. CCK-8 reagent and complete culture medium (1:9 v / v) were added, and the cells were incubated at 37°C for 1 h, with absorbance measured at 450 nm. RAW 264.7 cells were treated with VP, GPP, and GPP@VP extracts for 24, 48, and 72 h, respectively. Cell viability was assessed using CCK-8 reagent and calculated using the following formula: Cell viability (%) = (A_sample − A_blank) / (A_control − A_blank) × 100% like Figure 3As shown in F, at 1–5 μg·mL -1 Within the specified range, cell viability was not significantly different from the control group (p>0.05), especially at 2 μg·mL⁻¹. -1 It reached its highest level (101.18%) when the concentration increased to 6 μg·mL. -1 At that time, cell viability decreased significantly to 64.00% (*p<0.01), indicating dose-dependent cytotoxicity.

[0095] Therefore, 2 μg·mL was chosen for subsequent experiments. -1 As the working concentration.

[0096] (2) Live / Dead Cell Staining: In this embodiment, the effects of different hydrogels on RAW264.7 macrophages were further evaluated through live / dead cell staining. The live / dead cell staining process was as follows: Gelatin, GPP, and GPP@VP were coated into 6-well plates, RAW 264.7 cells were seeded and cultured. At 24 h and 72 h, live / dead staining was performed according to the kit instructions (Beyotime, China) (GPP@VP was treated in the dark throughout the process), and the cells were observed using a fluorescence microscope.

[0097] like Figure 3 As shown in Figure G, on days 1 and 3 of culture, all groups exhibited strong green fluorescence signals while showing very little red fluorescence, indicating high cell viability and no significant cytotoxicity. No significant differences were observed in cell density and morphology among different material groups or at different time points, indicating that all types of hydrogel systems have good cell compatibility.

[0098] CCK-8 quantitative analysis results ( Figure 3 The above conclusions were further confirmed by the H-cell assay. At 24 h of culture, the cell viability of the VP, GPP, and GPP@VP groups were 98.71%, 100.77%, and 99.90%, respectively. At 48 h, the viability of the GPP and GPP@VP groups increased to 110.43% and 110.98%, respectively, while that of the VP group was 101.40%. At 72 h, the GPP and GPP@VP groups maintained high viability (103.86% and 105.30%), while the viability of the VP group slightly decreased to 95.42%. These results indicate that this hydrogel system can support the proliferation and growth of macrophages over a relatively long culture period.

[0099] (3) Immunofluorescence staining: The effect of hydrogel on YAP-mediated mechanotransduction signaling was evaluated by immunofluorescence staining. The specific experimental procedure was as follows: NIH-3T3 cells were seeded on slides placed in 12-well plates and cultured overnight. Then, they were cultured in serum-free medium for 12 h and YAP activation was induced by TGF-β1 (10 ng / mL, 24 h). After induction, the cells were treated with control medium, VP, GPP extract, or GPP@VP extract for 24 h (the extract was obtained by incubating the hydrogel in complete medium and filtering it through 0.22 μm; the VP-containing sample was kept in the dark throughout the process). The cells were fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.1% Triton X-100 for 10 min, and blocked with QuickBlock™ for 10 min. Then, anti-YAP primary antibody (1:250) was added and incubated at 37℃ for 1 h. After washing with PBS, Alexa Fluor-labeled secondary antibody (1:500) was added and incubated in the dark for 30 min. The slides were mounted using a mounting medium containing DAPI to inhibit fluorescence quenching, and then imaged using a fluorescence microscope.

[0100] like Figure 3 As shown in Figure I, compared with the gelatin and GPP groups, the intranuclear localization of YAP in the GPP@VP group was significantly reduced. Quantitative analysis ( Figure 3 The results showed that the proportion of YAP-positive cells in the nucleus was 60.4% in the control group, 34.1% in the GPP group, 24.4% in the VP group, and 25.3% in the GPP@VP group, which was about 58.1% lower than the control group. This result suggests that GPP@VP hydrogel can inhibit YAP nuclear translocation, thereby regulating the mechanosensitive signaling pathway, which may be related to its anti-fibrotic effect.

[0101] In summary, GPP@VP hydrogel maintains good biocompatibility while possessing injectability, sustained drug release, and structural stability, and can effectively inhibit YAP nuclear translocation, suggesting its potential application in drug delivery and fibrosis-related mechanoregulation.

[0102] Example 5: Photodynamic bactericidal and intrinsic antibacterial mechanism of GPP@VP hydrogel against MRSA To elucidate the antibacterial mechanism of GPP@VP hydrogel against methicillin-resistant Staphylococcus aureus (MRSA), this embodiment conducted a series of in vitro experiments, including colony counting, inhibition zone assay, and bacterial live / dead staining.

[0103] (1) Antibacterial test: MRSA bacterial solution (1×10 6 200 μL of CFU / mL was added to each well of a 96-well plate coated with GPP@VP hydrogel, and incubated at 37 °C for 2 h. Subsequently, the plates were incubated under 790 nm near-infrared light (160 mW·cm⁻¹). -2Irradiation was performed for 0, 30 s, 1, 2, 4, and 6 min. After irradiation, 100 μL of bacterial suspension was spread onto LB agar plates and incubated at 37°C for 24 h. Colonies were recorded and counted. Comparative experiments were conducted on the Control, CTS, VP, GPP, GPP@VP, and GPP@VP NIR groups (the non-light-illuminated groups were kept in the dark throughout the process). The antibacterial properties were further evaluated using the agar diffusion method. MRSA was evenly spread on LB agar plates, and 6 mm diameter holes were punched in the agar. 50 μL of each group's sample was added. The GPP@VP NIR group underwent NIR irradiation before incubation, while the other groups were kept in the dark. Inhibition zones were observed after incubation at 37°C for 24 h.

[0104] like Figure 4 As shown in Figure A, irradiation at 790 nm near-infrared (NIR) (160 mW·cm⁻¹) -2 Under these conditions, the hydrogel exhibited a significant bactericidal effect. With prolonged irradiation time, the number of bacterial colonies gradually decreased, and complete elimination was achieved at 6 minutes.

[0105] Quantitative results of colony forming units (CFU) Figure 4 The results (D) showed that the colony count gradually decreased from 293 in the control group to 230, 183, 116, and 20 at 30s, 1 min, 2 min, and 4 min, respectively, and no colonies were detectable at 6 min. Statistical differences appeared between the groups starting from 1 min, indicating that VP effectively induced bacterial death by generating reactive singlet oxygen after photoactivation.

[0106] Limited antibacterial activity was observed under conditions without NIR irradiation. Figure 4 (B) The CFU values ​​for the CTS (positive control), VP, GPP, and GPP@VP groups were 318.7 ± 8.0, 387.3 ± 20.0, 299.3 ± 22.3, and 293.0 ± 43.5, respectively. Figure 4 (E). Among them, the GPP and GPP@VP groups showed relatively more obvious antibacterial effects, which was mainly attributed to the presence of ε-PLL.

[0107] Although previous studies have reported that ε-PLL has bactericidal activity against Escherichia coli and Staphylococcus aureus, in this example it only showed an inhibitory effect on MRSA, suggesting that the effect of antimicrobial materials is strain-dependent and requires targeted verification.

[0108] Its dual antibacterial mechanism was further verified by plate diffusion experiments. Figure 4 (C, F). No obvious inhibition zone (approximately 28.27 mm) was observed in the control group and VP group. 2The CTS, GPP, and GPP@VP groups formed areas of 133.26 ± 58.80, 355.43 ± 87.43, and 427.38 ± 90.26 mm, respectively. 2 The inhibition zone was observed. GPP@VP exhibited the largest inhibition zone (485.07 ± 245.10 mm) under NIR irradiation (GPP@VP NIR group). 2 (), significantly better than the other groups.

[0109] This enhancement effect can be attributed to the synergistic effect of ε-PLL's disruptive effect on bacterial membrane structure and VP's photodynamic bactericidal effect.

[0110] (2) Staining of live / dead bacteria: Staining was performed using the Live / Dead Bacterial Viability Detection Kit (Beyotime, China). After following the instructions, the bacteria were observed under a fluorescence microscope.

[0111] like Figure 4 As shown in Figure G, the bacterial live / dead staining results further support the above conclusions. The control group and VP group showed predominantly green fluorescence, indicating high bacterial activity; the GPP and GPP@VP groups showed increased red fluorescence, indicating cell membrane damage and inhibited growth; while the GPP@VP NIR group showed almost entirely red fluorescence, indicating widespread bacterial membrane disruption and death.

[0112] In summary, GPP@VP hydrogel achieves effective removal of MRSA through a dual antibacterial mechanism: on the one hand, ε-PLL provides intrinsic, strain-dependent antibacterial activity; on the other hand, VP generates singlet oxygen under NIR activation, achieving a rapid and irreversible photodynamic bactericidal effect.

[0113] Example 6: Evaluation of in vitro blood compatibility and hemostatic properties of GPP@VP hydrogel In this embodiment, the blood compatibility of the material was assessed by red blood cell (RBC) hemolysis test, and the procoagulant properties of the material were further assessed by whole blood coagulation index (BCI).

[0114] (1) The specific procedure for the hemolysis experiment is as follows: Fresh anticoagulated blood was washed with PBS and then prepared into a 2% red blood cell suspension. CTS, GPP, and GPP@VP powder were dispersed in PBS (10 mg / mL). 0.8 mL of sample solution was mixed with 0.2 mL of red blood cells for each group and incubated at 37℃ for 1 h. The positive control was 0.1% Triton X-100, and the negative control was PBS. After centrifugation, the absorbance of the supernatant at 540 nm was measured, and the hemolysis rate was calculated according to the following formula: Hemolysis rate (%) = (A_sample − A_PBS) / (A_Triton − A_PBS) × 100 like Figure 5 As shown in Figure A, the hemolysis rates of GPP (1.04±0.88%) and GPP@VP (2.05±0.88%) were both low, comparable to the control groups of PBS (0.00003±1.15%) and CTS (0.96±1.40%), and significantly lower than the 5% safety threshold specified in ISO 10993-4. Figure 5 The presence of E in the blood indicates that it has good blood compatibility.

[0115] SEM observation results showed that only a small number of round red blood cells were attached to the surface of commonly used clinical gauze and gelatin sponge (GS), while GPP and GPP@VP surfaces showed a large number of red blood cells adhering and obvious deformation. Figure 5 (B) suggests that it has a stronger blood-material interaction capability. This phenomenon may be attributed to the hydrophilic porous structure of the hydrogel and Ca. 2+ The presence of [something] can promote thrombus formation.

[0116] (2) Blood Coagulation Index (BCI) Test: CS, gauze, GPP, and GPP@VP powder (10 mg) were placed in a 50 mL centrifuge tube and preheated to 37°C. 20 μL of 0.2 M CaCl2 was added to 200 μL of anticoagulated whole blood for activation, and then immediately added to the test material (37°C, 10 min). Subsequently, 25 mL of deionized water was added, and the non-coagulated red blood cells were lysed by gentle shaking at 37°C for 20 min. After centrifugation, the absorbance of the supernatant was measured (540 nm), and the BCI was calculated. BCI (%)=A_sample / A_control×100 like Figure 5 As shown in Figure F, the BCI values ​​of GPP and GPP@VP were 32.81±0.63% and 31.85±0.45%, respectively, which were significantly lower than those of the control group (99.99±2.08%). They were also approximately 2.2 times and 2.5 times lower than those of medical chitosan (CTS, 71.52±0.54%) and gauze (81.81±0.36%), respectively.

[0117] The above results indicate that the introduction of VP did not weaken the coagulation properties of the hydrogel; its excellent hemostatic effect mainly stems from the hydrogel's structural characteristics and Ca. 2+ Activation of the mediated coagulation cascade.

[0118] Example 7: Evaluation of the in vivo hemostatic properties of GPP@VP hydrogel This embodiment evaluates the in vivo hemostatic performance of GPP@VP hydrogel through in vivo hemostasis experiments.

[0119] The in vivo hemostasis experiment was conducted as follows: Hemostasis performance was assessed using an SD rat tail amputation model. After anesthesia, the midpoint of the tail was severed, and the rats were treated with Control, CTS, GPP, and GPP@VP, respectively. Hemostasis time was recorded, and blood loss was calculated by weighing the rats using filter paper. A liver injury model was further employed, with a standardized wound (2 mm in diameter, 2 mm in depth) established in the left lobe of the liver, to assess hemostasis time and blood loss.

[0120] like Figure 5 As shown in C and D, in the Sprague–Dawley (SD) rat tail-bark model, GPP and GPP@VP reduced blood loss to 0.60±0.20 g and 0.62±0.08 g, respectively, significantly lower than the control group's 1.86±1.02 g. Figure 5 (G) Bleeding time was significantly shortened from 276.2±23.0 s to 41.2±14.1 s (GPP) and 34.0±5.7 s (GPP@VP). Figure 5 The hemostatic effect was comparable to that of chitosan (CTS, 0.56±0.18 g, 43.4±31.7 s), indicating that the hydrogel has good hemostatic ability in the peripheral injury model.

[0121] In a liver hemorrhage model ( Figure 5 The addition of K, L), GPP, and GPP@VP further reduced blood loss to 0.06±0.05 g and 0.04±0.05 g, respectively, which were approximately 24-fold and 35-fold lower than the control group (1.42±0.13 g). Figure 5 (Middle I). Bleeding time was also significantly shortened from 260.6±46.2 s to 5.4±1.8 s and 5.2±1.5 s, respectively, a reduction of approximately 48-fold and 50-fold. Figure 5 (J).

[0122] The above results were superior to those of the CTS group (0.36±0.15 g, 45.2±24.4 s), indicating that this material has a better hemostatic effect in high-bleeding environments. The more significant hemostatic effect in the liver model may be related to the temperature-dependent activation of CaP@TGnase.

[0123] TGnase exhibits high catalytic activity at physiological temperatures, and the warm and moist environment (approximately 37°C) on the liver surface is conducive to enzymatic cross-linking and rapid gelation, thereby enhancing coagulation stability. In contrast, the lower ambient temperature at the tail severance site may reduce enzyme activity, resulting in relatively lower tissue sealing efficiency. These results suggest that CaP@TGnase hydrogels can respond to changes in the local physiological environment, and their in vivo properties depend on the temperature-activated characteristics of TGnase.

[0124] Example 8: Evaluation of GPP@VP in vivo treatment of MRSA-infected burn wound model In this embodiment, a full-thickness burn model was established using SD rats, and the in vivo therapeutic effect of GPP@VP was evaluated using this model.

[0125] The MRSA-infected burn model was established as follows: a heated copper rod (120℃) was applied to the skin on the back for 5 seconds to form a 1 cm third-degree burn wound. The eschar was removed after 24 hours. 50 μL of MRSA bacterial solution (1 × 10⁻⁶) was then dripped into the wound. 8 CFU / mL was used to simulate clinically infected full-thickness skin defects. To reduce the interference of rodent-specific wound contraction on the results and to more closely resemble the human wound healing process, fixation was achieved by attaching rubber bands to the wound edges and supplementing with interrupted sutures. Figure 6 (A) Two hours after infection, different hydrogels were applied locally, and the patients were divided into Control, CTS, VP, GPP, GPP@VP, and GPP@VP NIR groups. The wound healing process was recorded by photographs on postoperative days 0, 1, 3, 5, 7, 9, 12, and 14.

[0126] like Figure 6 As shown in Figure B, there were significant differences in the appearance of the wounds among the groups. The control group and VP group showed persistent necrotic tissue and black eschar throughout the observation period, with limited signs of healing. The CTS and GPP groups showed some improvement from postoperative day 5 (POD 5). In contrast, the GPP@VP group, especially the GPP@VP NIR group, showed a significant reduction in wound area from POD 7 and essentially closed by POD 14.

[0127] Changes in wound boundaries, such as Figure 6 As shown in Figure C, the quantitative analysis results ( Figure 6 The wound area showed a consistent trend at POD 0. All groups had 100% wound area at POD 0; by POD 14, the GPP@VP NIR group achieved complete healing (0.00 ± 0.00%), the GPP@VP group had only 3.94 ± 4.78% of the initial wound area remaining, while the CTS, VP, and control groups had 24.72 ± 6.82%, 76.67 ± 9.30%, and 72.91 ± 28.16%, respectively, with statistically significant differences. Histological analysis further revealed the dynamic changes in the healing process.

[0128] The procedure was performed at POD 3, including histological and immunofluorescence staining (H&E staining). Specifically, the tissue was fixed, dehydrated, paraffin-embedded, and sectioned (5 μm). H&E, Masson, and Sirius Red staining were used to assess tissue structure and collagen distribution. Immunofluorescence staining included markers such as MPO, Arg-1, Vimentin, iNOS, and K14. Images were acquired using confocal microscopy, and quantitative analysis was performed using ImageJ.

[0129] H&E staining ( Figure 12 The results showed that the control group, CTS group and VP group had obvious inflammatory cell infiltration, edema and dermal structure destruction; the GPP group had reduced inflammation and early granulation tissue formation, while the GPP@VP and GPP@VP NIR groups had further relief of inflammation and more orderly arrangement of extracellular matrix, suggesting that they entered the proliferation stage more quickly.

[0130] H&E and Masson staining results for POD 7 and POD 14 ( Figure 6 The results (E) showed that collagen deposition was sparse and disordered in the control group, CTS group, and VP group; the GPP group improved matrix deposition to some extent; while the GPP@VP group, especially the GPP@VPNIR group, showed denser and more regularly arranged collagen fibers, and clearer restoration of dermal structure. By POD 14, the GPP@VP NIR group showed thicker collagen bundles, clear dermal structure, and hair follicle-like structures, which were basically not observed in the control group.

[0131] The above results indicate that the superior therapeutic effect of the GPP@VP NIR group stems from its synergistic antibacterial and regenerative functions. The photodynamic bactericidal effect combined with the intrinsic antibacterial effect mediated by ε-PLL effectively reduces MRSA load and alleviates excessive inflammation, thereby accelerating the transition of the wound from the inflammatory phase to the remodeling phase and promoting the regeneration of skin appendages. This suggests that it can drive the transformation of wound healing from scar repair to regenerative repair.

[0132] Example 9: Suppression of early neutrophilic inflammatory response Based on Example 8, in order to assess the early inflammatory status of the infected wound, myeloperoxidase (MPO) immunofluorescence staining was performed on postoperative day 3 (POD 3).

[0133] like Figure 7 As shown in Figure A, the control group and VP group showed strong MPO expression, indicating significant neutrophil infiltration; the inflammatory response was reduced in the CTS and GPP groups; while the GPP@VP and GPP@VP NIR groups showed only weak MPO signal, suggesting that they can effectively inhibit early neutrophil-mediated inflammatory response.

[0134] Quantitative analysis showed that the MPO positive area in the control group was 18.44 ± 4.80%, while it decreased to 11.43 ± 3.94% and 13.30 ± 2.72% in the CTS and VP groups, respectively. The GPP group further decreased to 6.07 ± 1.97% (approximately one-third of the control group), with the most significant decreases observed in the GPP@VP (5.08 ± 2.00%) and GPP@VP NIR (2.08 ± 1.12%) groups, decreasing by approximately 3.6 times and 8.9 times, respectively. Figure 7 (B)

[0135] The reduction in neutrophil infiltration was primarily attributed to the antibacterial effect of the materials. In vitro experiments showed that GPP, due to the presence of ε-PLL, exhibited antibacterial activity against MRSA, while GPP@VP NIR significantly enhanced antibacterial capacity through the synergistic effect of ε-PLL and VP photodynamic bactericidal action, thereby reducing the bacterial load on the wound, weakening the pathogen-induced inflammatory cascade response, and limiting excessive neutrophil recruitment. These results indicate that infection control plays a crucial role in regulating the early inflammatory microenvironment of the wound.

[0136] Example 10: Inhibition of fibroblast activation and myofibroblast differentiation Based on Example 8, in order to explore fibrotic remodeling during wound healing, this example evaluated fibroblast activation and differentiation on postoperative day 14 (POD 14) using YAP / Vimentin colocalization and α-smooth muscle actin (α-SMA) immunofluorescence staining.

[0137] like Figure 7 As shown in C and D, co-localization analysis of YAP (red) and Vimentin (green) revealed a higher proportion of YAP-positive fibroblasts in the control group (82.63 ± 8.11%), which decreased in the CTS (59.30 ± 10.38%), VP (33.55 ± 10.09%), and GPP (48.97 ± 5.79%) groups. In contrast, the GPP@VP (23.67 ± 9.35%) and GPP@VP NIR (13.84 ± 11.79%) groups showed more significant inhibition of YAP nuclear translocation, reducing it by approximately 3.5-fold and 6.0-fold, respectively. Figure 7 (E).

[0138] α-SMA staining results showed that the control group (32.64 ± 0.91%), CTS group (32.27 ± 5.01%), and GPP group (33.27 ± 0.96%) all exhibited high levels of myofibroblast activation. The VP group showed a slight decrease (27.04 ± 1.75%). Figure 7(Middle F). The GPP@VP and GPP@VP NIR groups further reduced the α-SMA positive area to 26.25 ± 1.73% and 23.46 ± 2.97%, respectively, which were approximately 1.24 times and 1.39 times lower than the control group.

[0139] The effect of VP alone is relatively limited, possibly due to its lack of antibacterial ability and low bioavailability resulting from a single dose. In contrast, GPP@VP hydrogel can achieve sustained release of VP, and combined with NIR-triggered photodynamic activation, it further enhances the antifibrotic effect by attenuating the inflammatory response and mechanotransduction signaling pathways such as YAP / TAZ.

[0140] The above results indicate that GPP@VP NIR, as a synergistic regulatory strategy, can effectively inhibit fibroblast activation and reduce pathological scar formation.

[0141] Example 11: Immune Microenvironment Remodeling through Macrophage Polarization Regulation To investigate the immunomodulatory mechanisms related to inflammation resolution and fibrosis regulation, this embodiment, based on Example 8, performed immunofluorescence staining for inducible nitric oxide synthase (iNOS, an M1 marker) and arginase-1 (Arg-1, an M2 marker) on postoperative days 7 and 14.

[0142] like Figure 7 As shown in G and H, on day 7, Arg-1 expression was significantly increased in the CTS (10.59 ± 3.31%), VP (13.45 ± 4.34%), and GPP (10.44 ± 4.48%) groups, while the levels were lower in the control group (1.87 ± 1.50%) and the GPP@VP NIR group (1.89 ± 0.78%). Figure 7 Middle I).

[0143] Conversely, iNOS expression was high in the control group (13.56 ± 2.41%) and the VP group (15.77 ± 3.95%), decreased in the CTS (6.16 ± 1.90%) and GPP (6.00 ± 1.81%) groups, and significantly downregulated in the GPP@VP (2.07 ± 0.84%) and GPP@VP NIR (1.28 ± 0.72%) groups, decreasing by approximately 6.5-fold and 10.6-fold respectively compared to the control group. Figure 7 (J).

[0144] By day 14, Arg-1 expression had decreased in all groups, with the control group at 0.66 ± 0.23%, the CTS, VP, and GPP groups ranging from 1.67% to 1.90%, and the GPP@VP group at 1.30 ± 0.52%. The GPP@VP NIR group further approached baseline levels (0.49 ± 0.20%). Figure 7 K). iNOS remained at a high level in the control group (4.99 ± 1.49%) and the VP group (4.14 ± 1.06%), but was significantly lower in the CTS (1.55 ± 0.55%), GPP (1.45 ± 0.51%), GPP@VP (0.58 ± 0.12%), and GPP@VP NIR (0.38 ± 0.10%) groups, with the GPP@VP NIR group showing a decrease of approximately 13.2 times compared to the control group. Figure 7 (L).

[0145] The above results indicate that GPP@VP and GPP@VP NIR can not only inhibit the early inflammatory response mediated by M1 macrophages, but also prevent the excessive accumulation of M2 macrophages during the tissue remodeling phase. GPP@VP NIR exhibits good regulatory ability in controlling macrophage polarization and limiting overall macrophage infiltration, suggesting that it helps to construct a controlled immune microenvironment, thereby promoting inflammation resolution and reducing fibrotic remodeling.

[0146] Example 12: Infection and inflammation control promote ECM remodeling and collagen transformation This embodiment assesses extracellular matrix (ECM) remodeling during wound healing. Building upon Example 8, Sirius red staining was performed on day 14 to analyze the type I / III collagen ratio. Figure 13 This indicator can serve as a histological marker for entering the remodeling phase.

[0147] Quantitative analysis results ( Figure 14 The results showed that the control group had the lowest type I / III collagen ratio (0.93 ± 0.27%), suggesting that persistent inflammation led to delayed ECM remodeling. The CTS (1.16 ± 0.30%) and GPP (1.20 ± 0.26%) groups showed increases of approximately 1.25-fold and 1.29-fold, respectively, while the VP group (0.94 ± 0.28%) showed limited improvement, possibly related to its insufficient antibacterial effect.

[0148] In contrast, the GPP@VP (1.38 ± 0.28%) and GPP@VP NIR (1.71 ± 0.38%) groups showed significantly higher type I / III collagen ratios, increasing by approximately 1.48 times and 1.84 times compared to the control group, respectively. These improvements may be attributed to the synergistic effect of infection control and inflammation resolution, thereby promoting timely wound remodeling and facilitating the orderly reconstruction of the stromal structure.

[0149] Example 13: Hair follicle regeneration and epidermal repair in the late healing stage Based on Example 8, during macroscopic observation on the 30th day post-surgery, the control group, CTS group, VP group, and GPP group all showed significant scar formation. Figure 15 In contrast, the GPP@VP group, especially the GPP@VP NIR group, showed a certain degree of hair growth, suggesting that it has better tissue regeneration ability.

[0150] To further assess skin appendage regeneration and epidermal reconstruction, immunofluorescence staining was performed on keratin 14 (K14)-positive hair follicles in the healed tissue sections. Figure 16 In the control group, the wound was mainly composed of scar tissue, and K14 was almost undetectable. + Hair follicle structure; CTS, VP and GPP groups also showed only a few hair follicle-like structures, suggesting that their repair process was mainly scar formation with limited new hair follicle growth.

[0151] In contrast, a certain degree of K14 could be observed in the subepidermal layer of the GPP@VP and GPP@VP NIR groups. + Hair follicle regeneration was observed, with the GPP@VP NIR group exhibiting a greater number of follicle-like structures that were more orderly arranged.

[0152] Quantitative analysis results ( Figure 17 This further confirms the above trend: K14 of the GPP@VP NIR group + The highest hair follicle density was (22.11 ± 6.33 follicles / mm²). 2 The number of samples was approximately the same as the control group (2.78 ± 2.64 samples / mm). 2 The concentrations were 8 times higher than those in the CTS (5.78 ± 5.02), VP (13.11 ± 3.79), and GPP (6.22 ± 4.94) groups; the GPP@VP group had 16.56 ± 3.57 particles / mm. 2 It also showed a significant improvement.

[0153] The above results indicate that traditional treatments primarily lead to a scar-dominated repair process, while the GPP@VP NIR system can enhance tissue regeneration potential. The antibacterial effect of ε-PLL and the photodynamic effect of VP under NIR irradiation synergistically inhibit infection and inflammation, thereby helping to restore a regenerative microenvironment conducive to hair follicle regeneration and epidermal reconstruction.

[0154] Example 14: In vivo biosafety evaluation of GPP@VP In this embodiment, H&E staining analysis was performed on the major organs (heart, liver, spleen, lungs, and kidneys) to assess its systemic biocompatibility.

[0155] The results showed that no obvious histological abnormalities, including inflammatory cell infiltration, necrosis, or tissue damage, were observed in any of the treatment groups. Figure 18 Meanwhile, routine blood counts (WBC, RBC, HGB, PLT, LYM, MCV) were all within the normal physiological range in each group, with no significant differences compared to the control group. Figure 19 ).

[0156] The above results indicate that the hydrogel system has good biocompatibility in vivo and no obvious systemic toxicity was observed.

[0157] Example 15: GPP@VP-mediated in vivo anti-adhesion and regenerative healing To evaluate the in vivo anti-adhesion effect of GPP@VP hydrogel, this embodiment established a clinically relevant double-injury peritoneal adhesion model in SD rats, including liver injury and cecal-abdominal wall friction injury. Figure 8 (A)

[0158] Macroscopic observation results 14 days post-surgery showed significant differences among the groups. Figure 8 (B) In the control group, dense fibrous adhesions were observed between the liver and surrounding peritoneal structures, as well as between the cecum and the abdominal wall; similar extensive adhesions were also observed in the CTS group. In contrast, only mild, thin-film-like adhesions were formed in the GPP group. No significant adhesions were observed in the GPP@VP and GPP@VP NIR groups; the organ surfaces were smooth, and the injured areas of the liver and abdominal wall showed continuous epithelial coverage and relatively orderly subcutaneous structures, suggesting that it was closer to regenerative repair than fibrotic healing.

[0159] Histological analysis further validated the above results. Figure 8(C) In the control group and CTS group, collagen-rich bridging tissue was visible at the injury interface, and the material commonly seen in the CTS group was encapsulated by dense fibrous tissue. In the GPP group, the collagen fibers were loosely and disordered, and local encapsulation of hydrogel residue was still visible. In contrast, no abnormal collagen deposition was observed in the GPP@VP and GPP@VP NIR groups, and the epithelial and subcutaneous tissue structures recovered well, suggesting that they have near-scarless tissue repair characteristics.

[0160] A standardized adhesion scoring system (0–5 points) was used for quantitative assessment. Figure 8 The results (D) showed that the degree of adhesion was significantly reduced in the GPP@VP treatment group. The scores of the control group and CTS group were mostly concentrated in the range of 4–5, while the GPP@VP NIR group was mainly distributed in the range of 0–1, suggesting a significant anti-adhesion effect. In conclusion, GPP@VP hydrogel, especially under photodynamic activation conditions, effectively inhibits postoperative adhesion formation and promotes tissue regenerative healing through synergistic antibacterial and anti-fibrotic effects.

[0161] Example 16: Transcriptome analysis reveals the anti-adhesion and regeneration mechanism of GPP@VP NIR. To elucidate the molecular mechanism of GPP@VP NIR in anti-adhesion and regeneration promotion, this example performed transcriptome sequencing analysis on the adhesion tissues of the control group and the GPP@VP NIR treatment group (n=3 and n=5).

[0162] Principal component analysis (PCA) showed a clear separation between the two groups at the transcriptional level, with PC1 explaining 70.24% of the variation, suggesting that hydrogel treatment caused significant transcriptomic changes. Figure 20 ).

[0163] Gene expression distribution uniformity ( Figure 21 ) and good Pearson correlation within the group ( Figure 22 This validated the reliability and comparability of the sequencing data. Differential expression analysis identified 4,500 significantly altered genes (|log2FC| ≥ 1, adjusted p < 0.05), of which 1,707 were upregulated and 2,793 were downregulated. Figure 23 ).

[0164] Hierarchical cluster analysis showed that the treatment group exhibited downregulation of pro-fibrosis and pro-inflammatory gene clusters, while upregulation of genes related to tissue regeneration and matrix remodeling. Figure 24 This suggests that GPP@VP NIR can remodel the fibrotic microenvironment at the transcriptional level.

[0165] To further elucidate its mechanism of action, this embodiment performs GO and KEGG enrichment analysis on the differentially expressed genes.

[0166] GO analysis showed that biological processes related to epithelial regeneration (including epithelial cell differentiation, proliferation, and morphogenesis), ECM tissue, and mucosal immune responses were significantly upregulated. Figure 8 (E).

[0167] KEGG analysis identified five pathways with high functional relevance: cell adhesion molecules, ECM–receptor interaction, adhesion plaques, MAPK signaling pathway, and antigen processing and presentation. Figure 8 (F), and further analysis was conducted on it.

[0168] In the cell adhesion molecular pathway, the heatmap of the top 15 differentially expressed genes showed that GPP@VP NIR was downregulated overall ( Figure 8 In the study of G), the levels of genes related to tight junctions and adhesion junctions (such as Cldn2, Cldn8, Cdh1, Ocln, and Madcam1) were significantly reduced, suggesting weakened cell-cell and cell-matrix adhesion, consistent with the reduced fiber bridging and adhesion inhibition observed in histology.

[0169] In the ECM–receptor interaction pathway, the expression of multiple fibrosis-related matrix components and their receptors (such as Lama2, Thbs4, Vtn, Itga10, and Col6a6) is downregulated. Figure 8 These genes (H) are typically involved in increasing matrix stiffness and maintaining adhesion, and their downregulation suggests that GPP@VP NIR promotes the transformation of the ECM into a regenerative microenvironment.

[0170] In the focal adhesion pathway, genes associated with cell-matrix anchoring and mechanical contraction are generally downregulated. Figure 8 (I) Decreased expression of structural ECM proteins, including Col6a4, Col6a5, Col4a6, and Lamb3, as well as integrins (Itga10, Itga3), suggests weakened cell adhesion and mechanotransduction. Simultaneously, downregulated expression of cytoskeleton regulatory factors (Myl2, Myl9, Myh6) and mechanotransduction-related molecules (Thbs4, Cav3) indicates inhibited myofibroblast activation and tension generation, crucial processes in fibrotic contraction.

[0171] In the antigen processing and presentation pathway, multiple MHC-related genes (such as RT1-CE16, RT1-Hb-ps1, RT1-T18, and RT1-M2) are significantly downregulated. Figure 8 The results suggest that the level of immune activation is reduced, which may alleviate chronic inflammation and promote the formation of a regenerative immune microenvironment.

[0172] In the MAPK signaling pathway, multi-node expression is suppressed ( Figure 25 , Figure 26The study included genes related to inflammation and abnormal proliferation (Il1a, Tgfa, Mapk13, Areg, Ereg, Fgfr2, and Erbb2). The key upstream kinase Map3k21 was almost completely silenced (log2FC≈–9.8), indicating significant suppression of MAPK inflammatory signaling. Simultaneously, some pro-regeneration genes (such as Fgf5, Fgf18, and Pla2g4e) were upregulated, suggesting selective repair-related signal activation in this pathway.

[0173] Further analysis of differentially expressed genes related to the GO entry "wound healing" ( Figure 8 The study found that several key molecules involved in matrix remodeling and angiogenesis (including Fgf5, Fgf18, Col22a1, Col19a1, Col26a1, and Fras1) were upregulated, supporting the active remodeling process of the ECM. Among them, Fgf5 was significantly upregulated (log2FC=6.77), suggesting that it may play an important role in the regeneration process.

[0174] Furthermore, transcriptome results showed that GPP@VP NIR regulates macrophage polarization. Downregulation of some typical M2-related genes (Pparg, Retnla, Greb1) suggests that avoiding prolonged M2 polarization helps reduce inflammation and prevent fibrosis. Simultaneously, downregulation of genes related to immune metabolism and stress adaptation (Hsd3b2, Prr15l, Ttc38) indicates a trend towards immune homeostasis.

[0175] In summary, transcriptomic analysis shows that GPP@VP NIR achieves its anti-adhesion and regeneration effects through multi-pathway synergistic regulation, including inhibiting fibrosis-related signals, weakening immune activation, regulating ECM remodeling, and selectively promoting the activation of tissue repair-related pathways.

[0176] In the above embodiments of the present invention, all data are expressed as mean ± standard deviation (SD) (n≥3). Two-group comparisons were performed using t-tests, and multiple-group comparisons were performed using one-way ANOVA and Tukey's post-hoc test. Statistical analysis was performed using SPSS 26.0, and P < 0.05 was considered statistically significant. Images were generated using OriginPro 2024b, and image analysis was performed using ImageJ.

[0177] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A method for preparing a biomimetic nanozyme, characterized in that, Using transglutaminase as a protein template, the biomimetic nanozyme was obtained by inducing calcium phosphate nucleation through biomimetic mineralization.

2. The method for preparing a biomimetic nanozyme according to claim 1, characterized in that, Glutaminase was mixed with a cell culture medium containing phosphate, and then calcium ion solution was added. After incubation, the mixture was centrifuged and freeze-dried to obtain the biomimetic nanozyme.

3. The method for preparing a biomimetic nanozyme according to claim 2, characterized in that, The cell culture medium is high-glucose DMEM with a phosphate concentration of 0.1-1.0 g / L, and the calcium ion solution is calcium chloride solution with a calcium ion concentration of 0.5-2 M; the mass-volume ratio of glutaminase, high-glucose DMEM, and calcium chloride solution is 100:10:0.

1.

4. A biomimetic nanoenzyme, characterized in that, It is prepared by the method described in any one of claims 1-3.

5. A method for preparing a nanozyme-catalyzed dual-network hydrogel, characterized in that, The components of the hydrogel raw material are mixed and incubated to obtain the hydrogel; wherein the hydrogel raw material includes the biomimetic nanozyme, γ-polyglutamic acid, ε-polylysine and gelatin as described in claim 4.

6. The method for preparing a nanozyme-catalyzed dual-network hydrogel according to claim 5, characterized in that, The biomimetic nanozyme, γ-polyglutamic acid, ε-polylysine and gelatin are in a mass ratio of 1:10:10:

100.

7. The method for preparing a nanozyme-catalyzed dual-network hydrogel according to claim 5, characterized in that, Includes the following steps: Gelatin, γ-polyglutamic acid, and ε-polylysine were dissolved in PBS to obtain solution A. The biomimetic nanozyme was dispersed in PBS to obtain solution B. Solution A and solution B were mixed and incubated at 37°C with 5% CO2 for 1-20 minutes to obtain the hydrogel.

8. The method for preparing a nanozyme-catalyzed dual-network hydrogel according to claim 5, characterized in that, The hydrogel raw material also includes the photosensitizer vertiporfin.

9. A nanozyme-catalyzed dual-network hydrogel, characterized in that, It is prepared by the method described in any one of claims 5-8.

10. The application of the nanozyme-catalyzed dual-network hydrogel as described in claim 9 in the preparation of scarless wound repair materials.

11. A scarless wound repair material, characterized in that, Including the nanozyme-catalyzed dual-network hydrogel as described in claim 9.

12. The scarless wound repair material according to claim 11, characterized in that, The scarless wound repair material is a scarless burn wound repair material.

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