Photodynamic therapy composition, medical instrument thereof and application of photodynamic therapy composition in wound repair

Abstract

The photodynamic therapy composition comprises salvianolic acid B and fibrin, and the salvianolic acid B and the fibrin are in weak interaction to form an interlaced or interpenetrating network so as to achieve photodynamic therapy synergism. Photodynamic therapy active components are loaded in an acidic environment provided by salvianolic acid B and fibrin, so that O2 <-> is further converted into hydrogen peroxide (H2O2) and hydroxyl radicals (. OH), the dependence on oxygen is reduced, and the efficiency of the photodynamic therapy is improved. Through verification, the composition disclosed by the invention maintains strong bactericidal activity in resisting infection of methicillin-resistant staphylococcus aureus (MRSA), pseudomonas aeruginosa (Pa) and acinetobacter baumannii (Ab), enhances angiogenesis and collagen deposition, remarkably improves the healing effect, and shows a remarkable treatment advantage in wound management of chronic diabetes mellitus.

Description

TECHNICAL FIELD

[0001] The present application relates to a biomaterial, in particular to a composite gel, and the application of the medical device in assisting phototherapy. BACKGROUND

[0002] Chronic diabetic wounds are a type of disease with great challenges in clinical practice, which are usually accompanied by complex pathological characteristics such as persistent infection, impaired angiogenesis and damaged healing microenvironment. Its pathogenesis involves decreased blood perfusion, prolonged chronic inflammation and massive proliferation of drug-resistant pathogenic bacteria (such as methicillin-resistant Staphylococcus aureus MRSA, Pseudomonas aeruginosa Pa and Acinetobacter baumannii Ab). Due to the rising of antimicrobial resistance and the limitation of local hypoxic conditions in wounds, the efficacy of traditional antibiotics and conventional healing mechanisms is limited, and existing antimicrobial therapy is facing significant bottlenecks.

[0003] Photodynamic therapy (PDT) and sonodynamic therapy (SDT) are new methods developed in recent years. PDT, as a new antibacterial and tissue regeneration promoting method, has attracted increasing attention due to its advantages such as minimal invasiveness, good selectivity and low risk of drug resistance. However, most traditional photosensitizers (PS) rely on oxygen to generate singlet oxygen (1O2, type II PDT), which is limited in hypoxic wounds. Especially in the hypoxic and nutrient-deficient microenvironment of diabetic wounds, oxygen-dependent treatments such as photodynamic therapy (PDT) and sonodynamic therapy (SDT) are greatly compromised, so there is an urgent need for new treatment strategies that can still be effective under harsh conditions. SUMMARY

[0004] One object of the present application is to provide a composition comprising the active components of photodynamic therapy to enhance the effect of photodynamic antibacterial therapy.

[0005] Another object of the present application is to provide a composition comprising a composite gel to enhance the antibacterial performance of photodynamic antibacterial therapy and promote wound repair and healing.

[0006] Still another object of the present application is to provide a composite gel as a medical device for assisting phototherapy to improve the antibacterial performance of wounds and promote wound repair and healing.

[0007] Yet another object of the present application is to provide a medical device combining a device for phototherapy and a composite gel for wound repair and healing.

[0008] The wound referred to in the present application more refers to chronic wounds, including but not limited to senile wounds, diabetic wounds, pressure wounds, venous ulcers and burns, etc.

[0009] In molecular structure, salvianolic acid B (SAB) has multiple hydroxyl groups and rigid aromatic skeleton, which not only endows it with biological activity, but also implies its potential to self-assemble into supramolecular structure. The abundant hydroxyl groups can form extensive hydrogen bonding, while the aromatic rings can mediate π-π stacking interactions, which are essential for the formation of ordered network structure. In addition, there is a hydrophobic region in SAB molecule, which can drive hydrophobic interaction and further promote the self-assembly process under suitable conditions. The surface charge simulation results of SAB molecule show that the charge distribution is partitioned, which is conducive to the formation of hydrogen bonds.

[0010] SAB self-assembles into acidic hydrogel, which has good biocompatibility, biodegradability and permeability, and forms an acidic microenvironment locally on the wound surface, which helps to regulate inflammatory response, stimulate angiogenesis and promote tissue regeneration. The dosage is, for example, 5 mg / mL to 50 mg / mL, preferably 10 mg / mL to 30 mg / mL, and especially 18 mg / mL to 22 mg / mL.

[0011] To improve the mechanical properties of SAB-gel and obtain biocompatible hydrogel that is easy to apply, such as sprayable, the present application also adds fibrin to complex with SAB (SAB / F-gel). The polyphenol groups of SAB molecules can form interwoven or interpenetrating networks with fibrin fibers through hydrogen bonding, π-π interactions and potential electrostatic interactions, so that the SAB network is stably anchored in the fibrin scaffold. These molecular-scale interactions endow the hydrogel with superior mechanical properties and biological activity.

[0012] SAB fibrin includes at least three high-affinity binding sites. At these main binding sites, hydrogen bonds are formed between SAB and fibrin amino acid residues, providing a strong and stable bonding effect for the complex.

[0013] The complex gel of the present application includes SAB and fibrin in a weight ratio of 1:3 to 3:1. Preferably, the weight ratio is 1:1 to 3:1, for example, 2:1.

[0014] SAB solution (20 mg / mL, prepared with PBS) is mixed with thrombin (10 U / mL) to form solution A. Fibrinogen solution (10 mg / mL) is used as solution B. The mixture of solution A and solution B is quickly mixed and gels immediately to form SAB / fibrin gel.

[0015] The photodynamic therapy active component is loaded into the complex gel to obtain a photodynamic therapy composition.

[0016] The photodynamic therapy active component is, for example, but not limited to, verteporfin, hematoxylin and 5-amino ketoglutaric acid, etc. These components are used alone or in combination in the present application.

[0017] A photodynamic therapy active component for the present application consists of TQ / Ce6. Under the mediation of TQ, Ce6 can produce superoxide anion (O2 - ) via the type I PDT pathway, which is further converted into H2O2 and ·OH in the acidic SAB / F-gel environment, thus still maintaining strong bactericidal ability under hypoxic conditions.

[0018] Another photodynamic therapy active component for the present application is formed by TQ / Ce6 microparticles with a hydration diameter of 90 nm ± 8 nm. TQ and Ce6 can be self-assembled into TQ / Ce6 nanoparticles (TQ / Ce6 NPs) via hydrophobicity, constituting a stable and efficient PDT delivery system.

[0019] Another photodynamic therapy composition includes TQ / Ce6 and SAB / F-gel, and TQ / Ce6 is loaded in SAB / F-gel. For example, SAB solution (20 mg / mL, prepared with PBS) is mixed with thrombin (10 U / mL) to form solution A. Fibrinogen solution (10 mg / mL) is mixed with TQ / Ce6 nanoparticles (10 μg / mL) to form solution B. After rapid mixing of solution A and solution B, the SAB / fibrin double network hydrogel is immediately gelled.

[0020] The composition of the present application also contains calcium ions to promote the activity of thrombin, facilitate the formation of fibrin, and enhance the mechanical properties of the gel.

[0021] By utilizing the intrinsic antioxidant and pro-angiogenic properties of SAB and constructing it in a fibrin-based double network hydrogel structure, a scaffold with excellent biocompatibility and mechanical strength is prepared, which can effectively regulate inflammatory response and promote angiogenesis.

[0022] TQ and Ce6 are co-assembled into nanoparticles, which endow the system with type I photodynamic therapy (PDT) mechanism, i.e., active oxygen (ROS) can be continuously produced even in the typical hypoxic environment of diabetic wounds. This scheme not only can eliminate drug-resistant pathogenic bacteria (such as MRSA, Pa, and Ab), but also can enhance cell proliferation and tissue regeneration in vivo.

[0023] By encapsulating TQ / Ce6 NPs in SAB / fibrin double network hydrogel (TQ / Ce6@SAB / F-gel), the formation of TQ / Ce6 nanoparticles and SAB / F-gel is synergistically integrated, and the acidic environment provided by SAB / F-gel provides synergistic effect for photodynamic therapy, improving the antibacterial performance. Under hypoxic conditions, red light (RL) irradiation efficiently produces O2 - , which is converted into H2O2 and ·OH in the acidic environment, forming a bio-inspired type I PDT antibacterial system.

[0024] In one aspect, in vitro experiments show that under red light irradiation, TQ / Ce6@SAB / F-gel can regulate macrophage M2 polarization through the immune-metabolic pathway, promote endothelial cell proliferation, migration and tube formation, accelerate wound closure, enhance collagen deposition and angiogenesis, and at the same time, the SAB / F-gel matrix regulates the immune-metabolic pathway to play an anti-inflammatory, pro-vascular and excellent mechanical support role, and promotes rapid healing and tissue remodeling.

[0025] On the other hand, in the MRSA infected diabetic mouse wound model, the antibacterial system combined with red light irradiation completely removes bacteria, promotes collagen deposition and angiogenesis, and significantly accelerates wound closure, and the histological and transcriptome sequencing results verify its repair effect, and these effects have been confirmed by systematic in vitro and in vivo experiments. The multifunctional hydrogel can effectively remove drug-resistant bacteria, enhance angiogenesis and collagen deposition, significantly improve healing effect, and show significant therapeutic advantages in chronic diabetic wound management.

[0026] In addition, transcriptome analysis reveals the down-regulation of pro-inflammatory factors and the activation of anti-inflammatory pathways, further highlighting the immunoregulatory potential of the hydrogel system. Overall, the bifunctional hydrogel system proposed in the present application provides an innovative treatment platform with transformational potential for refractory and infected wounds, and is expected to solve the key problems of antibiotic resistance and healing disorders in diabetic wound repair.

[0027] The photodynamic therapy composition of the present application, combined with a phototherapy device (device), is used to enhance the efficiency of wound repair, improve the antibacterial ability of the wound, and the repair and healing speed of the wound.

[0028] A medical device comprising the photodynamic therapy composition provided by the present application.

[0029] Another medical device comprising a device for implementing phototherapy and the photodynamic therapy composition provided by the present application, the photodynamic therapy composition being located in the light path of the outgoing light.

[0030] The device for implementing phototherapy can emit red light, such as red light (605nm-700nm) emitted by an LED as a light source. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1Figures for characterization results of SAB / F-gel of the present application; wherein a is the structural fluctuation graph of SAB / F-gel in 100 ns molecular dynamics simulation, b is the area dynamic change curve graph of SAB / F-gel during 100 ns simulation, c is the radius dynamic change curve graph of SAB / F-gel during 100 ns simulation, d is the root mean square deviation (RMSD) dynamic change curve graph of SAB / F-gel during 100 ns simulation, e is the hydrogen bond number dynamic change curve graph of SAB / F-gel during 100 ns simulation, f is the clear field graph of gel state of SAB-gel, F-gel and SAB / F-gel in the bottle at each sampling period, g is the spray film forming performance clear field graph of SAB-gel, F-gel and SAB / F-gel, h is the rheological performance comparison curve graph of F-gel, i is the rheological performance comparison curve graph of SAB / F-gel, j is the compression curve graph of F-gel and SAB / F-gel, k is the compression stress (Compression Stress) and Young's modulus (Young's modulus) statistics graph of F-gel and SAB / F-gel, l is the scanning electron microscope (SEM) field graph of SAB / F-gel, m is the assembly interaction graph between SAB and fibrin based on energy minimization molecular docking simulation;

[0032] Figure 2 Figures for characterization results of TQ / Ce6 nanoparticles; wherein a is the simulated electrostatic potential distribution graph of TQ / Ce6 complex, b is the transmission electron microscope (TEM) field graph of TQ / Ce6 complex, c is the hydrated particle size distribution statistics graph of TQ / Ce6 nanoparticles measured by dynamic light scattering (DLS) in PBS, d is the Fourier transform infrared spectrum (FTIR) graph of TQ, Ce6 and TQ / Ce6 complex, e is the absorption spectrum graph of TQ, Ce6 and TQ / Ce6 complex, f is the fluorescence intensity change statistics graph of DCF probe at each time point under red light irradiation (20 mW / cm 2 ) of TQ / Ce6@SAB / F-gel (TQ 7.5 μg / mL, Ce6 2.5 μg / mL), Ce6@SAB / F-gel (2.5 μg / mL) and TQ@SAB / F-gel (7.5 μg / mL), g is the fluorescence intensity change statistics graph of SOSG probe at each time point under red light irradiation (20 mW / cm 2) fluorescence intensity change statistical chart of each time point, h is the DHR 123 probe in TQ / Ce6@SAB / F-gel (TQ 7.5 μg / mL, Ce6 2.5 μg / mL), Ce6@SAB / F-gel (2.5 μg / mL) and TQ@SAB / F-gel (7.5 μg / mL) three groups under red light irradiation (20 mW / cm 2 ) fluorescence intensity change statistical chart of each time point, i is the Amplex Red probe in TQ / Ce6@SAB / F-gel (TQ 7.5 μg / mL, Ce6 2.5 μg / mL), Ce6@SAB / F-gel (2.5 μg / mL) and TQ@SAB / F-gel (7.5 μg / mL) three groups under red light irradiation (20 mW / cm 2 ) fluorescence intensity change statistical chart of each time point, j is the HPF probe in TQ / Ce6@SAB / F-gel (TQ 7.5 μg / mL, Ce6 2.5 μg / mL), Ce6@SAB / F-gel (2.5 μg / mL) and TQ@SAB / F-gel (7.5 μg / mL) three groups under red light irradiation (20 mW / cm 2 ) fluorescence intensity change statistical chart of each time point, k is the HOMO and LUMO energy level diagram of Ce6 and TQ, l is the emission spectrum change diagram of Ce6@SAB / F-gel (2.5 μg / mL) and TQ / Ce6@SAB / F-gel (Ce6 2.5 μg / mL, TQ 25 μg / mL) under excitation wavelength 405 nm in DMSO, m is the fluorescence lifetime decay curve of Ce6@SAB / F-gel (2.5 μg / mL) and TQ / Ce6@SAB / F-gel (Ce6 2.5 μg / mL, TQ 25 μg / mL), n is the schematic diagram of TQ / Ce6 triggering type I photodynamic reaction under red light irradiation;

[0033] Figure 3 is the in vitro antibacterial effect and mechanism verification result diagram of RL combined with TQ / Ce6@SAB / F-gel; wherein, a is the experimental design and the schematic diagram of the antibacterial mechanism, b is the bactericidal curve of planktonic bacteria under each group treatment, c is the SEM image of MRSA in each experimental group (the arrow indicates that the bacterial envelope is damaged, and the asterisk indicates that the intracellular vacuole and bubble structure appear), d is the SEM image of Pa in each experimental group (the arrow indicates that the bacterial envelope is damaged, and the asterisk indicates that the intracellular vacuole and bubble structure appear), e is the CLSM live / dead bacteria staining image of MRSA in each experimental group (SYTO TM 9 (green) marks all cells, and PI (red) marks dead bacteria), f is the CLSM live / dead bacteria staining image of Pa in each experimental group (SYTO TM9 (green) marks all cells, PI (red) marks dead bacteria, g is the CLSM images of planktonic MRSA treated with RL (15 J / cm 2 ) combined with TQ / Ce6@SAB / F-gel (final concentration: TQ 7.5 μg / mL; Ce6 2.5 μg / mL) (HPF staining detects the increase of intracellular hydroxyl radicals (·OH), PI staining reflects the decrease of bacterial viability), h is the CLSM images of planktonic Pa treated with RL (15 J / cm 2 ) combined with TQ / Ce6@SAB / F-gel (final concentration: TQ 7.5 μg / mL; Ce6 2.5 μg / mL) (HPF staining detects the increase of intracellular hydroxyl radicals (·OH), PI staining reflects the decrease of bacterial viability), i is the statistical chart of the antibacterial effect of each experimental group under hypoxic conditions, j is the bright field images of mature biofilm stained with crystal violet after treatment, k is the gene expression heat map of MRSA treated with PBS and RL (6 J / cm 2 ) combined with TQ / Ce6@SAB / F-gel (fold change≥2, q<0.05), l is the GO enrichment analysis chart, m is the gene set enrichment analysis (GSEA) chart;

[0034] Figure 4Figure for TQ / Ce6@SAB / F-gel regulating macrophage polarization; Wherein, a is the schematic diagram of experimental design (G1: control group; G2: LPS; G3: LPS+TQ / Ce6@SAB-gel; G4: LPS+TQ / Ce6@F-gel; G5: LPS+TQ / Ce6@SAB / F-gel), b is the CLSM image of NF-κB p65 nuclear translocation in THP-1 and RAW264.7 cells under LPS and each group of TQ / Ce6@SAB / F-gel treatment, c is the quantitative analysis statistical diagram of p65 fluorescence intensity in the nucleus of THP-1 and RAW264.7 cells under LPS and each group of TQ / Ce6@SAB / F-gel treatment, d is the CLSM image of iNOS expression in THP-1 and RAW264.7 cells in each experimental group, e is the quantitative statistical diagram of the average fluorescence intensity of iNOS expression in THP-1 and RAW264.7 cells in each experimental group, f is the CLSM image of CD86 expression in THP-1 and RAW264.7 cells in each experimental group, g is the quantitative statistical diagram of CD86 fluorescence intensity in THP-1 and RAW264.7 cells in each experimental group, h is the CLSM image of CD206 expression in THP-1 and RAW264.7 cells in each experimental group, i is the quantitative statistical diagram of CD206 expression fluorescence intensity in THP-1 and RAW264.7 cells in each experimental group, j is the statistical diagram of ELISA determination of TNF-α, IL-6 and IL-1β in THP-1 cell culture supernatant, k is the statistical diagram of ELISA determination of TGF-β and IL-10 in THP-1 cell culture supernatant;

[0035] Figure 5 Figure for TQ / Ce6@SAB / F-gel promoting angiogenesis by improving mitochondrial function; Wherein, a is the schematic diagram of the mechanism of TQ / Ce6@SAB / F-gel improving mitochondrial function, b is the EdU staining diagram of HUVECs in each experimental group, c is the quantitative statistical diagram of the proportion of EdU staining positive cells of HUVECs in each experimental group, d is the result diagram of observing HUVECs scratch healing under optical microscope, e is the statistical diagram of measuring the relative scratch closure area of HUVECs under optical microscope, f is the field diagram of HUVECs lumen formation experiment, g is the statistical diagram of capillary-like structure length in HUVECs treated by each experimental group, h is the CLSM image of reactive oxygen species (ROS) in HUVECs in each experimental group, i is the CLSM image of mitochondrial membrane potential (ΔΨM) in HUVECs in each experimental group, j is the CLSM image of NF-κB p65 nuclear translocation in HUVECs in each experimental group, k is the NAD(H) concentration statistical diagram in HUVECs in each experimental group, l is the NADPH concentration statistical diagram in HUVECs in each experimental group, m is the statistical diagram of mitochondrial DNA (mtDNA) content in HUVECs in each experimental group, n is the statistical diagram of mitochondrial biogenesis in HUVECs in each experimental group, o is the statistical diagram of mitochondrial fission in HUVECs in each experimental group, p is the statistical diagram of mitochondrial fusion in HUVECs in each experimental group, q is the statistical diagram of mitochondrial autophagy in HUVECs in each experimental group, r is the statistical diagram of mitochondrial apoptosis in HUVECs in each experimental group, s is the statistical diagram of mitochondrial oxidative stress in HUVECs in each experimental group, t is the statistical diagram of mitochondrial calcium overload in HUVECs in each experimental group, u is the statistical diagram of mitochondrial calcium overload in HUVECs in each experimental group, v is the statistical diagram of mitochondrial calcium overload in HUVECs in each experimental group, w is the statistical diagram of mitochondrial calcium overload in HUVECs in each experimental group, x is the statistical diagram of mitochondrial calcium overload in HUVECs in each experimental group, y is the statistical diagram of mitochondrial calcium overload in HUVECs in each experimental group, z is the statistical diagram of mitochondrial calcium overload in HUVECs in each experimental group, aa is the statistical diagram of mitochondrial calcium overload in HUVECs in each experimental group, bb is the statistical diagram of mitochondrial calcium overload in HUVECs in each experimental group, cc is the statistical diagram of mitochondrial calcium overload in HUVECs in each experimental group, dd is the statistical diagram of mitochondrial calcium overload in HUVECs in each experimental group, ee is the statistical diagram of mitochondrial calcium overload in HUVECs in each experimental group, ff is the statistical diagram of mitochondrial calcium overload in HUVECs in each experimental group, gg is the statistical diagram of mitochondrial calcium overload in HUVECs in each experimental group, hh is the statistical diagram of mitochondrial calcium overload in HUVECs in each experimental group, ii is the statistical diagram of mitochondrial calcium overload in HUVECs in each experimental group, jj is the statistical diagram of mitochondrial calcium overload in HUVECs in each experimental group, kk is the statistical diagram of mitochondrial calcium overload in HUVECs in each experimental group, and LL is the statistical diagram of mitochondrial calcium overload in HUVECs in each experimental group. +Figure 1 shows the NADH ratio histogram, m is the ATP concentration histogram in HUVECs of each experimental group;

[0036] Figure 6 Figure 1 shows the NADH ratio histogram, m is the ATP concentration histogram in HUVECs of each experimental group; 2 ; TQ concentration 15 μg / mL; Ce6 concentration 5 μg / mL), b is the bacterial fluorescence imaging and plate culture photos of the acute infected wounds of each experimental group, c is the statistical curve of bacterial fluorescence intensity expressed in logarithmic relative fluorescence units (lg RLU) (normalized to the starting time point), d is the GO enrichment analysis of down-regulated genes, e is the statistical curve of bacterial load of the wound after inoculation of each experimental group, f is the change curve of the wound healing rate of each experimental group within 16 days, g is the H&E staining chart of each experimental group at 8th and 16th day after treatment, h is the statistical chart of granulation tissue thickness of each experimental group at 8th and 16th day after treatment, i is the Masson trichrome staining chart of each experimental group at 8th and 16th day after treatment, j is the statistical chart of collagen deposition ratio of each experimental group at 8th and 16th day after treatment, k is the CD31 and α-SMA immunofluorescence staining chart of each experimental group at 8th and 16th day after treatment; l is the statistical chart of blood vessel number of each experimental group at 8th and 16th day after treatment, m is the NADH content statistical chart of each experimental group, n is the NADH content statistical chart of each experimental group, o is the AMP content statistical chart of each experimental group, p is the ATP content statistical chart of each experimental group; + ; TQ concentration 15 μg / mL; Ce6 concentration 5 μg / mL), b is the bacterial fluorescence imaging and plate culture photos of the acute infected wounds of each experimental group, c is the statistical curve of bacterial fluorescence intensity expressed in logarithmic relative fluorescence units (lg RLU) (normalized to the starting time point), d is the GO enrichment analysis of down-regulated genes, e is the statistical curve of bacterial load of the wound after inoculation of each experimental group, f is the change curve of the wound healing rate of each experimental group within 16 days, g is the H&E staining chart of each experimental group at 8th and 16th day after treatment, h is the statistical chart of granulation tissue thickness of each experimental group at 8th and 16th day after treatment, i is the Masson trichrome staining chart of each experimental group at 8th and 16th day after treatment, j is the statistical chart of collagen deposition ratio of each experimental group at 8th and 16th day after treatment, k is the CD31 and α-SMA immunofluorescence staining chart of each experimental group at 8th and 16th day after treatment; l is the statistical chart of blood vessel number of each experimental group at 8th and 16th day after treatment, m is the NADH content statistical chart of each experimental group, n is the NADH content statistical chart of each experimental group, o is the AMP content statistical chart of each experimental group, p is the ATP content statistical chart of each experimental group;

[0037] Figure 7Figure 8 is a result diagram of the TQ / Ce6@SAB / F-gel combined with RL in vivo to regulate inflammatory response; wherein a is the immunofluorescence image of the wound tissue of each experimental group on the 8th day and the 16th day, b is the IL-6 expression amount statistical diagram of the wound tissue of each experimental group on the 8th day and the 16th day, c is the TNF-α expression amount statistical diagram of the wound tissue of each experimental group on the 8th day and the 16th day, d is the immunofluorescence image of the wound tissue of each experimental group on the 8th day and the 16th day, e is the CD86 statistical diagram of the wound tissue of each experimental group on the 8th day and the 16th day, f is the CD206 statistical diagram of the wound tissue of each experimental group on the 8th day and the 16th day, g is the heat map and functional classification of the differentially expressed genes in the wound tissue of the control group (G1) and the treatment group (G5) (fold change≥2, q<0.05), h is the GO enrichment analysis diagram of the up-regulated genes, i is the KEGG pathway enrichment analysis diagram of the up-regulated genes, j is the GESA enrichment analysis diagram of angiogenesis, k is the GESA enrichment analysis diagram of collagen fiber tissue, l is the GESA enrichment analysis diagram of mitochondrial respiratory chain complex I, and m is the GESA enrichment analysis diagram of oxidative phosphorylation. DETAILED DESCRIPTION

[0038] The technical solutions of the present application are described in detail below. The embodiments of the present application are only used to illustrate the technical solutions of the present application and not to limit. Although the present application is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solutions of the present application can be modified or replaced equivalently without departing from the spirit and scope of the technical solutions of the present application, which should be covered in the scope of the claims of the present application.

[0039] All solvents and reagents used in the following examples of the present application are analytical pure or higher grade, and the specific sources of the main materials are as follows:

[0040] Chlorin e6 (Ce6, purity≥98%) and thymoquinone (TQ, purity≥98%) were purchased from Sigma-Aldrich and used directly without further purification.

[0041] Salvianolic acid B (SAB, purity≥98%) was purchased from Adamas.

[0042] Fibrinogen (human, lyophilized, high purity) and thrombin (human, activity consistent with the supplier's specifications) were purchased from Sigma-Aldrich.

[0043] Phosphate buffered saline solution (PBS, pH 7.4) and sterile deionized water were prepared in the laboratory.

[0044] Methicillin-resistant Staphylococcus aureus (MRSA, ATCC strain), Pseudomonas aeruginosa (Pa, BNCC358235, BeNa Culture Collection) and Acinetobacter baumannii (Ab, ATCC strain) were cultured according to the standard culture method.

[0045] Diabetic mouse models (BALB / c, male, 6-8 weeks old) were induced by streptozotocin (STZ) injection, and were operated in accordance with the institutional animal experiment ethics standards, and all animal experiments were approved by the institutional animal ethics committee.

[0046] The test methods used in the following examples of the present application are specifically described as follows:

[0047] 1) Molecular dynamics simulation and molecular docking

[0048] Molecular dynamics simulation was performed using Gromacs version 2024.2 (Software X 1, 19-25 (2015)). The simulation calculation was completed on a computing platform configured with an Intel Core i9-12900K processor, an NVIDIA 3080 Ti GPU (CUDA 12.3.103), and 64 GB of memory. The topology file of the SAB molecule was generated by the Sobtop tool (Tian Lu, Sobtop, Version 1.0 (dev4), http: / / sobereva.com / soft / Sobtop, accessed January 6, 2025). The SAB was dissolved in solution at a concentration of 2% by mass, and the force field used was the Amber force field, and the solvent water model was TIP3P (Journal of computational chemistry 25, 1157-1174 (2004), The Journal of Physical Chemistry A 105, 9954-9960 (2001)). After pre-equilibration, 100 ns of molecular dynamics simulation was performed with a 2 fs step, and a snapshot was recorded every 10 ns.

[0049] 2) Molecular docking simulation

[0050] The structural model of Salvianolic acid B was downloaded from PubChem (CID: 6451084), and the structural model of fibrinogen was obtained from the AlphaFold protein structure database (Entry: P02679). Hydrogen atoms were added, residue charges were assigned, and the protonated state was generated using the Molecular Operating Environment (MOE2022) software for subsequent molecular docking simulation. After the protein structure was prepared, CB-DOCK2 tool (Nucleic Acids Res 50, W159-W164 (2022)) was used for blind docking, and the binding site with the most appropriate cavity volume and the lowest Vina score was selected as the most possible binding site of SAB in fibrinogen.

[0051] 3) Preparation of SAB / fibrin double network hydrogel

[0052] SAB solution (20 mg / mL, prepared in PBS) was mixed with thrombin (10 U / mL) to form solution A. Fibrinogen solution (10 mg / mL) was mixed with TQ / Ce6 nanoparticles (10 pg / mL) to form solution B. Solution A and B were mixed rapidly and gelled immediately to form SAB / fibrin double network hydrogel. The rheological properties of the hydrogel were measured by rotational rheometer (DHR10, TA Instruments), including storage modulus (G’), loss modulus (G”), gelation time and shear-thinning behavior. Compression modulus test was performed using mechanical tester (Instron) to evaluate the load bearing capacity. Scanning electron microscope (SEM, JSM-7800F, JEOL) was used to observe the microstructure and fiber arrangement of the hydrogel.

[0053] 4) Preparation of TQ / Ce6 nanoparticles

[0054] Ce6 and TQ were dissolved in DMSO to prepare 2 mg / mL stock solution, respectively. Then, 20 pL of Ce6 / DMSO solution (denoted as PS1 / DMSO) was mixed with 60 pL of TQ / DMSO solution at room temperature, and the resulting mixture was diluted with 10 mL of distilled water to obtain a TQ / PS1 composite solution with a final concentration of 4 pg / mL for Ce6 (PS1) and 12 pg / mL for TQ. Dynamic light scattering (DLS, omni, Brookhaven) was used to determine the particle size; transmission electron microscopy (TEM, Talos L120C, Thermo Fisher) was used to observe the morphology; ultraviolet-visible spectroscopy (UV–Vis, Uy-3600iplus, SHIMADZU) and Fourier-transform infrared spectroscopy (FTIR, Nicolet 6700, Thermo Fisher) were used to characterize the optical properties and molecular interactions. Fluorescence emission spectra and lifetime measurements were performed using a fluorescence spectrometer (FLS1000, Edinburgh Instruments) to confirm energy transfer and reactive oxygen species generation ability.

[0055] 5) Quantum mechanical simulation

[0056] The calculation of molecular structure, energy and electrostatic potential map was accomplished by Gaussian 16w software (Gaussian Inc., 340 Quinnipiac Street, Building 40, Wallingford, CT 06392, licensed)

[62] . The B3LYP / 6-31G* hybrid exchange-correlation functional was used to ensure the accuracy of the electronic interaction [63, 64]. The solvent effect was considered by the density-based solvent model (SMD) using water as the solvent environment

[65] . The calculation was run on a computing platform configured with an Intel Core i9-12900K processor and 64 GB of memory to meet the performance requirements of complex calculations. The resulting calculation data was visualized and analyzed using Multiwfn and VMD software [66, 67] to analyze the molecular properties and interaction characteristics in detail.

[0057] 6) ROS generation and evaluation of photodynamic performance

[0058] The TQ / Ce6 nanoparticle dispersion and its hydrogel encapsulation system were irradiated under red light LED (650 nm, 20 mW / cm 2 ). Different fluorescent probes were used to distinguish between type I and type II photodynamic pathways: DCF was used to detect total ROS, SOSG was used to detect singlet oxygen ( 1 O2), DHR 123 was used to detect superoxide anion (O2 - ), Amplex Red was used to detect hydrogen peroxide (H2O2), and HPF was used to detect hydroxyl radicals (·OH). The Ce6 single-component system was used as a control.

[0059] 7) Antibacterial experiments, biofilm analysis and transcriptomic analysis

[0060] MRSA, Pa and Ab strains were treated with hydrogels combined with red light irradiation (650 nm, 20 mW / cm 2 ). Bacterial activity was evaluated by colony-forming unit (CFU) counting and scanning electron microscopy (SEM, JSM-7800F, JEOL) imaging. Live / dead bacterial staining (SYTO9 TM , Invitrogen) was observed using a laser confocal microscope (STELLARIS 5, Leica). Biofilm formation was detected by crystal violet staining. RNA was extracted from the treated bacterial samples for transcriptome sequencing (Illumina platform) to screen for differentially expressed genes.

[0061] 8) Antibacterial experiments under hypoxic conditions

[0062] MRSA, Pa and Ab strains were cultured to mid-log phase, washed and resuspended in anaerobic PBS (02< 5%). TQ / Ce6 loaded hydrogels were placed on sterile membrane scaffolds and contacted with bacterial suspensions and irradiated with red light (650 nm) for 10 min. After incubation, samples were gradient diluted and plated on LB agar plates for CFU counting. Ce6 alone hydrogels and untreated bacteria were used as controls. All experiments were repeated independently 5 times.

[0063] 9) Macrophage polarization and immunomodulation experiments

[0064] THP-1 and RAW264.7 cells were stimulated with LPS and treated with hydrogels. P65, iNOS, CD86 and CD206 were detected by immunofluorescence staining to evaluate M1 / M2 polarization status (Leica SP8 confocal microscope). TNF-a, IL-6, IL-1 b, TGF-b and IL-10 levels were measured by ELISA kits (R&D Systems) according to the manufacturer’s instructions.

[0065] 10) Endothelial cell angiogenic activity evaluation

[0066] HUVECs were cultured on hydrogels and subjected to EdU proliferation assay (Beyotime EdU kit), scratch wound healing assay and Matrigel tube formation assay. ROS detection, JC-1 mitochondrial membrane potential detection and P65 immunofluorescence were used to evaluate redox balance and NF-kB pathway activity to comprehensively evaluate the pro-angiogenic effect of hydrogels.

[0067] 11) Intracellular NAD(H) and ATP content determination

[0068] HUVECs were seeded in 24-well plates at a density of 2 x 105 5 cells / well and cultured overnight. The next day, SAB-gel, F-gel or SAB / F-gel (volume of 1 / 10 of culture medium) were added and incubated for 2 hours. After treatment, cells were treated with TNF-a (100 ng / mL) for 24 hours. After incubation, cells were washed with PBS three times and lysed with 100 pL lysis buffer. NAD(H) levels and NAD + / NADH ratio were determined using NAD / NADH assay kit (ab65348, Abeam) and ATP content was detected using luminescence ATP assay kit (ab113849, Abeam).

[0069] 12) Male C57BL / 6 mice (blood glucose >16.7 mM) with a diabetes model induced by streptozotocin (STZ) were anesthetized, and an 8 mm full-thickness skin defect model was created on their backs. MRSA suspension was inoculated into the wound to establish an infectious diabetic wound model. Mice were randomly divided into five groups:

[0070] • G1 (control group);

[0071] • G2 (TQ / Ce6@F-gel, TQ concentration 15 μg / mL, Ce6 concentration 5 μg / mL);

[0072] • G3 (TQ / Ce6@SAB / F-gel, TQ concentration 15 μg / mL, Ce6 concentration 5 μg / mL);

[0073] • G4(RL+Ce6@SAB / F-gel, RL dose 14.4 J / cm 2 (Ce6 concentration 5 μg / mL);

[0074] • G5(RL+TQ / Ce6@SAB / F-gel, RL dose 14.4 J / cm 2 (TQ concentration 15 μg / mL, Ce6 concentration 5 μg / mL).

[0075] Forty-eight hours later, 100 μL of hydrogel was applied to the wounds of groups G2-G5 using a dual-channel spray device and incubated for 15 minutes. Groups G4 and G5 were subsequently irradiated with red LED light (650 nm, 30 mW / cm²). 2 For 8 minutes, an LED light source was placed directly above the wound area. The wound area was photographed and recorded daily, and the wound healing rate was measured and analyzed using ImageJ software.

[0076] Tissue samples were collected at predetermined time points on days 8 and 16. The tissues were used for histological analysis (H&E staining, Masson trichrome staining) and immunofluorescence detection (CD31 and α-SMA were used as markers of angiogenesis, IL-6 and TNF-α were used for inflammation detection, and CD86 and CD206 were used for macrophage phenotype detection).

[0077] 13) Transcriptome analysis

[0078] Wound tissue from diabetic mice was collected on day 8 post-injury. Total RNA was extracted using TRIzol reagent (Invitrogen, USA) according to the manufacturer's instructions. RNA purity and integrity were verified using Nanodrop. TM The A260 / A280 ratio was measured using a One spectrophotometer (Thermo Fisher Scientific, USA), while using a Qubit spectrophotometer. TM3.0 fluorometer and Qubit TM RNA concentration was determined by Qubit 4 Fluorometer (Thermo Fisher Scientific, USA) and RNA Broad Range kit (Thermo Fisher Scientific, USA).

[0079] Library construction used Ribo-off TM rRNA was removed by Ribo-Gone rRNA Removal Kit (Human / Mouse / Rat, Illumina, USA), followed by KCTM Stranded mRNA Library Prep Kit (Wuhan SeqHealth Co., Ltd., China) according to the manufacturer’s instructions to construct the strand-specific RNA sequencing library. The library fragment length was 200-500 bp, and enriched and quantified by qPCR. High-throughput sequencing was performed on the Illumina NovaSeq 6000 platform (Illumina, USA) with double-end 150 bp sequencing (PE150).

[0080] RNA-Seq data analysis was completed using multiple bioinformatics tools. Differential expression gene analysis was completed by the edgeR software package, and gene expression heat maps were drawn by TBtools software (https: / / github.com / CJ-Chen / TBtools / releases). GO enrichment analysis and KEGG pathway annotation were completed using the KOBAS2.0 platform. Enrichment analysis used hypergeometric testing, and P values were corrected for false discovery rate (FDR) by the Benjamini-Hochberg method.

[0081] 14) Statistical analysis

[0082] All data were expressed as mean ± standard deviation. Statistical differences were analyzed by one-way ANOVA with Tukey’s post-hoc test or two-tailed Student’s t-test (GraphPad Prism 9.5 software). Differences were statistically significant when P < 0.05.

[0083] Example 1 Preparation and characterization of SAB / F-gel

[0084] To verify the self-assembly ability of SAB, 100 nanosecond scale molecular dynamics simulation was used, and the results showed that there were significant structural fluctuations in the system ( Figure 1 a), including changes in molecular area, radius, root mean square deviation (RMSD), and number of hydrogen bonds formed ( Figure 1 b, Figure 1 c, Figure 1 d and Figure 1e), indicating that the self-assembly process was actively ongoing. These dynamic interactions revealed a robust and adaptive molecular network within the hydrogel matrix.

[0085] In addition, the gelation process could be smoothly completed in a vial, and SAB at 2% (w / w) concentration could spontaneously transform from a liquid to a solid in a short time (SAB-gel) Figure 1 f). However, the mechanical strength of SAB-gel itself was low and easy to be destroyed, which made it difficult to be applied alone to the wound surface.

[0086] In fact, the addition of SAB not only did not weaken the ability of fibrin to form a gel (F-gel), but also accelerated the gelation rate of F-gel Figure 1 f). Further photographic evidence confirmed the excellent spraying and film-forming ability of this hydrogel on the surface of human and pig skin, which was particularly crucial for biomedical applications such as wound dressings Figure 1 g).

[0087] Rheological evaluation showed that SAB / F-gel had higher storage modulus G' and loss modulus G" than F-gel alone, indicating that its fluidity and deformation characteristics were improved, making it more suitable for application and adhesion to irregular wound surfaces Figure 1 h, Figure 1 i). The mechanical test results of the compression curve further showed that SAB / F-gel exhibited higher compressive stress (P<0.0001) and Young's modulus (P=0.0002) under physiological stress conditions Figure 1 j, Figure 1 k), reflecting its excellent mechanical stability. Scanning electron microscope (SEM) images revealed that its microstructure presented a good filamentous and porous structure, which was conducive to nutrient transfer and cell infiltration, thus supporting tissue regeneration Figure 1 l).

[0088] In addition, this embodiment carried out a comprehensive molecular docking simulation based on energy minimization technology to explore the assembly mechanism between SAB and fibrin. The CB-DOCK2 tool was used to predict the optimal binding conformation and orientation of SAB in the fibrin matrix, and the results showed that there were multiple binding sites between SAB and fibrin, with the first three high-affinity sites as shown in Figure 1 m. At these main binding sites, SAB and fibrin amino acid residues formed significant hydrogen bonds, providing a directional and stable bonding effect for the complex.

[0089] At the same time, π-π stacking interactions were formed between the aromatic rings of SAB and the phenylalanine residues in fibrin, further enhancing the binding affinity and molecular compatibility. These interactions played an important role in maintaining the structural integrity and functional performance of SAB / F-gel.

[0090] Overall, these results demonstrate that SAB / F-gel can not only be formed in situ efficiently and uniformly, but also possess the required structure and mechanical properties to meet the needs of advanced biomedical applications (e.g. wound repair and tissue engineering).

[0091] Example 2 Characterization of TQ / Ce6 nanoparticles

[0092] The self-assembly of thymoquinone (TQ) and chlorin e6 (Ce6) into nanoparticles is mainly driven by their strong non-covalent interactions Figure 2 a) TQ has a hydrophobic aromatic skeleton and two carbonyl groups, which can form stable synergistic assemblies with Ce6 molecules through hydrophobic interactions, π-π stacking and hydrogen bonding. These molecular properties promote the stable co-assembly of TQ and Ce6, forming uniform nanoparticle structures. Figure 2 a shows the stable structure and electrostatic potential map of the TQ / Ce6 complex, visualizing the electrostatic interactions after intermolecular adsorption. The results show that there is an attractive electrostatic interaction between the negative potential region with high electron density (blue) and the positive potential region with low electron density (red). The electron tends to migrate from the negative region to the positive region, promoting the electron transfer process, thereby enhancing the type I photodynamic reaction pathway.

[0093] Transmission electron microscopy (TEM) observation confirmed the uniform morphology and consistent size of the TQ / Ce6 nanoparticles, exhibiting reliable structural characteristics Figure 2 b). Dynamic light scattering (DLS) detection showed that the hydration diameter of the particles in phosphate buffered saline (PBS) was about 90 nm, consistent with the TEM results Figure 2 c).

[0094] Fourier transform infrared (FTIR) spectroscopy showed the characteristic functional groups of TQ (C=O stretching vibration at 1657 cm -1 ) and Ce6 (C=O stretching vibration at 1711 cm -1 ), and the TQ / Ce6 complex showed the same characteristic peaks, confirming the successful formation of the complex Figure 2 d). Ultraviolet-visible (UV-Vis) absorption spectroscopy further confirmed the formation of the complex, with the absorption peaks of TQ (257 nm) and Ce6 (405 nm) both present in the TQ / Ce6 nanoparticle Figure 2 e).

[0095] To facilitate clinical application, TQ / Ce6 nanoparticles were encapsulated in an acidic SAB / F-gel matrix, forming a TQ / Ce6@SAB / F-gel composite hydrogel. The cell viability of HUVECs treated with SAB-gel, SAB / F-gel, and TQ / Ce6@SAB / F-gel was evaluated using a live / dead cell staining method. The results showed that none of the three materials had a significant negative impact on cell viability.

[0096] In addition, the 2,7-dichlorodihydrofluorescein (DCFH) probe was used to detect RL irradiation (20 mW / cm²). 2 The total ROS generation in Ce6@SAB / F-gel, TQ@SAB / F-gel, and TQ / Ce6@SAB / F-gel was measured (DCFH can produce strong green fluorescence after being oxidized by ROS). The results showed that after 5 min of RL irradiation, the fluorescence intensity of the TQ / Ce6@SAB / F-gel group was significantly increased by more than 3 times compared to the Ce6@SAB / F-gel group (P<0.0001). Figure 2 f) indicates that TQ can effectively improve the overall ROS generation efficiency. On the other hand, TQ@SAB / F-gel failed to generate ROS because it could not absorb RL, which is consistent with the predicted results. In addition, there was no significant difference in ROS yield between free TQ / Ce6 and TQ / Ce6 encapsulated in SAB / F-gel, indicating that gel encapsulation does not affect ROS generation (Figure S4). These results highlight the synergistic effect of TQ in enhancing Ce6 ROS generation capability, both in solution and in the SAB / F-gel system.

[0097] To further explore the generation mechanisms of different types of ROS, singlet oxygen probe SOSG and dihydrorhodamine 123 (DHR123) were used to detect them respectively. 1 O2 and O2 - The yield was reduced. SOSG analysis results showed that the presence of TQ significantly inhibited the production of [product name] under RL irradiation. 1 O2 generation ( Figure 2 g). After 5 minutes of irradiation, the TQ / Ce6@SAB / F-gel group... 1 O2 production was only about one-third of that in the Ce6@SAB / F-gel group. Conversely, DHR123 analysis showed that O2 production in the TQ / Ce6@SAB / F-gel group was significantly higher. - The yield was significantly higher than that of the Ce6@SAB / F-gel group ( Figure 2 h).

[0098] Furthermore, other experiments have verified that free TQ / Ce6 generated under RL irradiation 1 There was no significant difference in O2 between the O2 group and the gel encapsulation group, but its O2 -The level of H2O2 and ·OH in TQ / Ce6@SAB / F-gel was significantly higher than that of TQ / Ce6 encapsulated in SAB / F-gel (P = 0.0142). The acidic environment can promote the conversion of H2O2 to ·OH. - H2O2 is further converted to ·OH.

[0099] Since SAB can provide an acidic microenvironment, this embodiment uses Amplex Red and hydroxyphenyl fluorescein (HPF) probes to detect H2O2 and ·OH production, respectively, to verify whether O2 - is effectively converted in SAB / F-gel. The results show that the level of H2O2 and ·OH in TQ / Ce6@SAB / F-gel group is significantly higher, while the detection value of Ce6@SAB / F-gel group is close to the background Figure 2 i and Figure 2 j).

[0100] Subsequent detection also shows that the production of H2O2 and ·OH by free TQ / Ce6 NPs is lower than that of TQ / Ce6 encapsulated in SAB / F-gel. When the pH of the solution is adjusted to 6, TQ / Ce6 can also generate more H2O2 and ·OH under RL irradiation, further confirming the promotion of H + by the acidic SAB / F-gel.

[0101] The above results show that the introduction of TQ effectively changes the photodynamic reaction mechanism from type II to type I, and promotes the production of H2O2 and ·OH in the acidic SAB / F-gel environment.

[0102] Analysis of the highest occupied molecular orbital (HOMO) and the lowest unoccupied orbital (LUMO) energy levels of the molecule shows that there is a favorable electronic energy level matching between Ce6 (–2.42 eV and –4.92 eV) and TQ (–3.24 eV and –7.06 eV), which is conducive to the occurrence of type I photodynamic electron transfer process Figure 2 k).

[0103] The fluorescence emission spectrum excited at 405 nm in dimethyl sulfoxide (DMSO) also shows that Ce6 fluorescence is quenched in the presence of TQ, suggesting that it promotes the intersystem crossing process and enhances ROS generation Figure 3 l).The fluorescence lifetime decay curve further reveals the changes in the photophysical behavior of TQ / Ce6 NPs, and the shortening of the lifetime suggests an improvement in energy transfer efficiency Figure 3 m).

[0104] Based on the above results, the reaction mechanism diagram of TQ / Ce6@SAB / F-gel system generating O2 - through type I path by electron transfer under RL irradiation, and further generating H2O2 and ·OH in the presence of H + Figure 3n) This mechanism comprehensively utilizes the structural stability, ROS generation ability and favorable electronic energy level characteristics of nanoparticles to achieve excellent antibacterial photodynamic therapy effect under hypoxic conditions.

[0105] Example 3 In vitro antibacterial efficacy of RL combined with TQ / Ce6@SAB / F-gel

[0106] Three common drug-resistant strains related to chronic wounds were selected, including gram-positive bacteria MRSA and gram-negative bacteria Pseudomonas aeruginosa (Pa) and Acinetobacter baumannii (Ab). The experimental design and antibacterial mechanism are as shown in Figure 3 a, wherein red light (RL) irradiation is achieved by a flexible red LED patch, and the light power density is adjusted by a smart control module to 20 mW / cm 2 .

[0107] Under RL irradiation, TQ / Ce6 can efficiently generate superoxide anion (O2 - ), which is further converted to hydrogen peroxide (H2O2) and hydroxyl radicals (·OH) in the acidic environment provided by SAB. This series of reactions can destroy bacterial membrane integrity, toxin activity and pathogenicity, ultimately leading to bacterial death. RL irradiation alone does not show any bactericidal effect on the three planktonic bacteria. However, when RL is combined with TQ / Ce6@SAB / F-gel, the bactericidal curve Figure 3 b) shows that the number of viable bacteria of the three strains decreases significantly.

[0108] Among them, the bactericidal activity of group G4 (Ce6@SAB / F-gel + RL) is more than 100 times higher than that of group G3 (Ce6@SAB / F-gel without RL), indicating that the addition of TQ indeed enhances the antibacterial effect of Ce6. In addition, as the concentration of TQ / Ce6 in SAB / F-gel increases, its bactericidal capacity also increases. When the concentration of TQ / Ce6 reaches 0.4‰ (w / w) and the RL irradiation dose is 15 J / cm 2 , MRSA, Pa and Ab are completely eliminated.

[0109] To verify the synergistic effect of combined therapy, the Bliss independence model was further used to evaluate the combined clearance effect of MRSA, Ab and Pa. When the S value is between 0 and 1, it indicates that there is a synergistic effect. The chessboard dilution method confirms that when the RL energy density and the TQ / Ce6 concentration increase simultaneously, the synergistic antibacterial effect is significantly enhanced. Among the 60 combinations, the mean and median of the S value are 0.449 and 0.383, respectively, indicating that the overall synergistic effect is clear.

[0110] The bacterial morphological changes were observed by scanning electron microscopy (SEM) (c and Figure 3 d). Figure 3d), obvious bacterial envelope damage (arrow) was observed in G4 group, while partial damage was also observed in G3 group, but lower than that in G4 group; the bacterial envelope was complete and smooth in the control groups G1 and G2. Laser confocal scanning microscopy (CLSM) live / dead staining images Figure 3 e and Figure 3 f) showed that the number of dead bacteria increased significantly: SYTO TM 9 (green) labeled all cells, and propidium iodide (PI, red) labeled dead bacteria. Under RL irradiation, the TQ / Ce6 group almost completely killed bacteria, and its bactericidal ability was much better than that of Ce6 alone and TQ alone.

[0111] To further elucidate the bactericidal mechanism, HPF fluorescent probes were used to detect the generation of hydroxyl radicals (·OH) in cells, and PI staining was used to evaluate bacterial activity. After RL (15 J / cm 2 ) combined with TQ / Ce6@SAB / F-gel (TQ / Ce6 concentration 0.1‰ w / w) treatment, the production of ·OH in MRSA and Pa cells increased significantly Figure 3 g and Figure 3 h). This indicates that superoxide anions have been converted to ·OH, which has stronger bactericidal efficacy, verifying the occurrence of type I photodynamic reaction-induced bacterial death.

[0112] Quantitative analysis results showed that in a 1% O2 environment, the combined treatment still had high bactericidal ability against MRSA, Pa, and Ab Figure 3 i). Compared with the Ce6@SAB / F-gel+RL group, the bactericidal activity of the combined treatment group increased by more than 100 times (P=0.0021, P=0.0002, P=0.0011). This advantage fully demonstrates the wide applicability of the system under hypoxic wound conditions, and can effectively overcome the treatment bottleneck caused by low oxygen.

[0113] Bacterial biofilm formation is an important mechanism for the refractory of chronic wound infection, and its dense hypoxic microenvironment often leads to the failure of conventional treatment. Therefore, the anti-biofilm efficiency of RL combined with TQ / Ce6@SAB / F-gel was further evaluated on a mature biofilm model. Crystal violet staining images Figure 3 j) showed that the combined treatment group significantly weakened the integrity of the biofilm, and the total amount of the biofilm was significantly reduced compared with the control group. The quantitative results also showed that the biofilm density and CFU number decreased significantly, verifying that RL combined with TQ / Ce6@SAB / F-gel has excellent destruction ability to mature biofilms under the mechanism of hypoxia-tolerant type I photodynamic reaction (P<0.0001).

[0114] Overall, these results fully demonstrated the synergistic advantage of TQ and Ce6 in antibacterial activity, providing a potential new therapeutic strategy for refractory infected wounds that can effectively destroy the biofilm structure and continuously exert antibacterial activity in adverse microenvironments.

[0115] MRSA was subjected to RNA-seq to further analyze the molecular mechanism of synergistic antibacterial activity. The transcriptome heat map ( Figure 4 k) showed that the differentially expressed genes of MRSA were significantly changed (fold change≥2 and q<0.05) after RL combined with TQ / Ce6@SAB / F-gel treatment. Gene ontology (GO) enrichment analysis ( Figure 4 l) and GSEA analysis ( Figure 4 m) revealed the activation of multiple biological pathways, including pathogenicity, membrane components, toxin activity, staphylococcal infection, quorum sensing, phosphotransfer system, and pyrimidine metabolism.

[0116] In summary, RL combined with SAB / F-gel effectively eliminates bacterial pathogens through multiple mechanisms such as cell membrane structure destruction, ROS generation, and gene expression regulation, showing a broad application prospect in the treatment of chronic and infected wounds.

[0117] Example 4 In vitro immunomodulatory effect of TQ / Ce6@SAB / F-gel

[0118] The results showed that TQ / Ce6@SAB / F-gel could effectively induce macrophages to polarize to M2 type. The evidence includes: the expression level of M2 marker CD206 was significantly increased, the M1 type markers iNOS and CD86 and NF-κB signaling pathway activity were decreased, pro-inflammatory factors were down-regulated and anti-inflammatory factors were up-regulated ( Figure 4 a).

[0119] CLSM analysis showed that LPS could induce NF-κB p65 to enter the nucleus and activate the inflammatory signaling pathway. After pretreatment with TQ / Ce6@SAB-gel or double network TQ / Ce6@SAB / F-gel, the nuclear translocation of p65 in THP-1 and RAW264.7 cells was significantly inhibited ( Figure 4 b). Quantitative analysis of nuclear p65 fluorescence intensity further confirmed the anti-inflammatory regulation effect of SAB / F-gel on the key signaling pathway ( Figure 4 c), in which the nuclear p65 intensity accounted for only 20% of the total intensity, which was much lower than the nearly 80% nuclear accumulation in G2 and G4 groups. The inhibition of NF-κB signaling pathway suggested that SAB / F-gel effectively intervened the pro-inflammatory transcriptional activity, which was crucial for immune regulation.

[0120] Further observation by CLSM and quantitative analysis of fluorescence intensity showed that the expression level of iNOS protein was significantly decreased after treatment with TQ / Ce6@SAB-gel or TQ / Ce6@SAB / F-gel (P < 0.0001) Figure 4 d and Figure 4 e) The down-regulation of iNOS suggests a decrease in the synthesis of nitric oxide, thereby reducing tissue inflammation and damage.

[0121] The expression of pro-inflammatory marker CD86 and anti-inflammatory marker CD206 in THP-1 and RAW264.7 cells was detected to further investigate the phenotype changes of macrophages. After treatment with TQ / Ce6@SAB-gel or TQ / Ce6@SAB / F-gel, the expression of CD86 decreased significantly while the expression of CD206 increased synchronously Figure 4 f and Figure 4 h); quantitative analysis of fluorescence intensity also supported the above-mentioned phenotype conversion Figure 4 g and Figure 5 i). This shift from M1 to M2 phenotype indicates that TQ / Ce6@SAB / F-gel has the potential to alleviate inflammation and promote tissue repair.

[0122] Enzyme-linked immunosorbent assay (ELISA) results further verified this observation: after treatment with SAB / F-gel, the levels of pro-inflammatory cytokines (TNF-α, IL-6 and IL-1β) in the supernatant of THP-1 cells decreased significantly, while the levels of anti-inflammatory factors (TGF-β and IL-10) increased Figure 5 j Figure 5 k). The decrease in the levels of TNF-α, IL-6 and IL-1β is consistent with the inhibition of NF-κB signaling and iNOS expression, creating an overall anti-inflammatory microenvironment more conducive to tissue repair.

[0123] In summary, TQ / Ce6@SAB / F-gel can effectively induce macrophages to transform into anti-inflammatory M2 phenotype and alleviate inflammatory response. The complex construction of SAB and fibrin may create a microenvironment conducive to M2 polarization, thereby enhancing its therapeutic potential in chronic inflammatory pathological conditions. Overall, TQ / Ce6@SAB / F-gel shows good prospects in tissue repair and regenerative medical applications that require precise regulation of macrophage activity.

[0124] Example 5 TQ / Ce6@SAB / F-gel promotes angiogenesis by improving mitochondrial function

[0125] As shown in Figure 5 a, SAB / F-gel significantly promotes angiogenesis by improving mitochondrial function.

[0126] EdU staining Figure 5b) and quantitative analysis of its fluorescence intensity Figure 5 c) The proliferation level of HUVECs (human umbilical vein endothelial cells) was significantly higher than that of the control group and TQ / Ce6@F-gel group (P < 0.05) after 24 hours of TQ / Ce6@SAB-gel or TQ / Ce6@SAB / F-gel treatment, suggesting that it promotes endothelial cell proliferation, which is particularly crucial for the formation of new blood vessels.

[0127] The scratch healing experiment further verified this conclusion: HUVECs treated with TQ / Ce6@SAB / F-gel showed accelerated migration and wound closure ability Figure 5 d and Figure 5 e), suggesting that the migration function necessary for angiogenesis is improved. The tube formation experiment results showed that the length of the capillary-like structure formed by HUVECs treated with SAB / F-gel was significantly better than that of the control group (P < 0.0001), SAB-gel group (P = 0.0117), and F-gel group (P = 0.0015) Figure 5 f and Figure 5 g), further confirming its potential for promoting angiogenesis.

[0128] Inflammation and its secondary oxidative stress can induce rapid consumption of intracellular NAD(H), which is a coenzyme necessary for maintaining multiple energy metabolism pathways such as glycolysis and oxidative phosphorylation. Its depletion can ultimately trigger apoptosis. Figure 5 h) showed that TNF-α stimulation for 48 hours can induce oxidative stress in HUVECs and cause a decrease in mitochondrial transmembrane potential (ΔΨM) Figure 5 h and Figure 5 i), suggesting that the mitochondrial function of endothelial cells is impaired. Treatment with SAB / F-gel can effectively alleviate TNF-α-induced oxidative stress and maintain ΔΨM stable, demonstrating its protective effect on endothelial cells under inflammatory conditions.

[0129] CLSM results showed that SAB / F-gel can significantly inhibit TNF-α-induced NF-κB p65 nuclear translocation Figure 6 j), thereby inhibiting the pro-inflammatory signaling pathway, which is consistent with the results in macrophages described earlier.

[0130] Intracellular NAD(H) and ATP levels were detected to further clarify the effects of each group treatment on mitochondrial function. The results showed that after treatment with TQ / Ce6@SAB / F-gel or TQ / Ce6@SAB-gel, the NAD(H) level and ATP generation capacity in TNF-α-treated HUVECs were significantly improved Figure 6 k, Figure 6 l and Figure 6m), indicating that mitochondrial function has improved.

[0131] The restoration of NAD(H) levels and the increase in ATP production indicate that SAB / F-gel can maintain cellular energy metabolism and provide energy support for the proliferation and migration of endothelial cells during angiogenesis.

[0132] Enhanced mitochondrial function (including maintaining ΔΨM stability and increasing ATP levels) helps inhibit endothelial cell apoptosis under inflammatory conditions. The ability of SAB / F-gel to maintain mitochondrial integrity under stress suggests its potential to promote cell survival and function.

[0133] These results indicate that TQ / Ce6@SAB / F-gel not only protects endothelial cells from oxidative stress and mitochondrial dysfunction, but also creates a metabolic microenvironment conducive to angiogenesis. Overall, TQ / Ce6@SAB / F-gel promotes angiogenesis by inducing macrophage M2 polarization, alleviating inflammation, protecting endothelial cells, improving mitochondrial function, and promoting energy metabolism, and is expected to accelerate wound healing through immune-metabolic regulatory mechanisms.

[0134] Example 6: In vivo healing-promoting effect of TQ / Ce6@SAB / F-gel in a diabetic mouse model of MRSA-infected wounds.

[0135] Based on the aforementioned in vitro study results, RL combined with TQ / Ce6@SAB / F-gel can effectively kill bacteria, promote angiogenesis, and regulate inflammatory responses. This embodiment further systematically evaluated its in vivo therapeutic effect in a diabetic mouse infection model. The effect of TQ / Ce6@SAB / F-gel on promoting the healing of MRSA-infected diabetic wounds was evaluated using a BALB / c mouse model. Figure 6 a).

[0136] After establishing a diabetic model by intraperitoneal injection of streptozotocin (STZ), wounds were prepared on the backs of mice and inoculated with MRSA. The mice were then given different treatments: control group (G1), TQ / Ce6@F-gel (G2), TQ / Ce6@SAB / F-gel (G3), Ce6@SAB / F-gel+RL (G4), and TQ / Ce6@SAB / F-gel+RL (G5).

[0137] The antibacterial effects of each group were evaluated using bacterial fluorescence imaging and plate culture methods. Figure 6 b) Both the bacterial load and culture plate results showed a significant decrease in bacterial load in all treatment groups, especially in group G5 after combining with RL, which showed the strongest antibacterial activity. In contrast, group G4 still had residual bacteria, suggesting that Ce6 has limited efficacy in hypoxic wounds. Figure 6 c).

[0138] These results highlight the advantage of combination therapy in typical chronic wound hypoxic environment. After 10 days of treatment, no viable bacteria were detected in G5 group wound, indicating that RL combined with TQ / Ce6@SAB / F-gel can completely eliminate acute infected MRSA( Figure 6 d).

[0139] Wound morphological observation Figure 6 c) showed that the wound closure in G5 group was significantly accelerated within 16 days( Figure 6 e and Figure 6 f), with a healing rate much better than other groups. At day 16, the healing rate of G5 group was close to 100%, while that of G1 group was less than 70%. G3 group showed better healing than G2 group, indicating the anti-inflammatory and pro-angiogenic advantages of SAB, verifying the multiple benefits of TQ / Ce6@SAB / F-gel in tissue regeneration.

[0140] Histological analysis further verified the above observations. H&E staining results at day 8 and day 16 of treatment showed that the granulation tissue thickness of G5 group increased significantly(P<0.05) Figure 6 g and Figure 6 h). Meanwhile, G5 group showed higher collagen deposition levels at both time points Figure 6 i and Figure 7 j), suggesting improved wound quality.

[0141] CD31 and a-SMA immunofluorescence staining were used to evaluate the effect of angiogenesis. The results showed that the vascular density of G5 group was significantly higher than that of other groups at day 8(P<0.05) Figure 7 k and Figure 7 l). In summary, TQ / Ce6@SAB / F-gel not only accelerated wound closure, but also promoted the formation of a solid extracellular matrix and functional vascular network, laying the foundation for sustained tissue regeneration. The synergistic effect of combined RL treatment on cell proliferation, collagen synthesis and angiogenesis highlights its potential application value in improving wound healing effect.

[0142] In terms of energy metabolism, TQ / Ce6@SAB / F-gel treatment significantly increased the levels of NADH and NAD + ( Figure 7 m), AMP and ATP Figure 7 n) in wound tissue, with the highest increase in G5 group.

[0143] NADH and NAD +The elevated levels of horizontal indicate that the cell redox balance and oxidative phosphorylation capacity are enhanced, while the elevated levels of AMP and ATP reflect the increased energy supply of the cells. This metabolic improvement provides the necessary energy for high-energy-consuming processes such as cell proliferation and tissue repair, helps to inhibit apoptosis in an inflammatory environment, and maintains continuous healing.

[0144] Based on the above-verified results, TQ / Ce6@SAB / F-gel combined with RL irradiation can overcome the hypoxic microenvironment through the type I photodynamic mechanism, significantly reduce the bacterial load of diabetic infected wounds, and has a much better effect than Ce6@SAB / F-gel. At the same time, the loading of SAB further accelerates tissue repair and angiogenesis. The simultaneous improvement of mitochondrial function and energy metabolism further highlights the multiple therapeutic potential of TQ / Ce6@SAB / F-gel through the immune-metabolic regulation pathway, providing a promising intervention strategy for diabetic chronic infected wounds.

[0145] Example 7 The role of TQ / Ce6@SAB / F-gel in regulating inflammation in vivo

[0146] SAB / F-gel showed a significant effect on inflammation regulation in diabetic MRSA infected wounds Figure 7 ). The immunofluorescence staining results Figure 7 a) at the 8th and 16th days of treatment showed that the expression levels of pro-inflammatory factors TNF-a and IL-6 in the wound were significantly reduced after RL combined with TQ / Ce6@SAB / F-gel (G5) treatment, which was significantly different from the control group (G1) and other treatment groups (G2-G4) Figure 4 b and Figure 7 c). The decrease of pro-inflammatory mediators highlights the dual effect of RL combined with TQ / Ce6@SAB / F-gel on sterilization and anti-inflammation.

[0147] In addition, immunofluorescence analysis showed that SAB / F-gel treatment can significantly down-regulate the pro-inflammatory macrophage marker CD86, while up-regulating the anti-inflammatory marker CD206 Figure 7 d, Figure 7 e and Figure 7 f), suggesting that macrophages are polarized from M1 to M2, which is consistent with the previous in vitro results (see Figure 7 ). This phenotype transformation is of key significance to inflammation relief and tissue repair.

[0148] Transcriptome analysis further reveals the anti-inflammatory molecular mechanism of TQ / Ce6@SAB / F-gel. The heat map Figure 7 g) and the functional classification of differentially expressed genes (fold change≥2 and q<0.05) show that there are extensive changes in gene expression in the G5 group compared with the G1 group.

[0149] GO enrichment analysis Figure 7 h) revealed multiple upregulated pathways related to anti-inflammatory response, cell metabolism and tissue regeneration, including: collagen fibril organization (e.g. AU021092, Adamts12, Adamts14, Apoa1, Apoa2, Arsl), positive regulation of cell migration (e.g. Acp5, Adam9, Adra2a, C3arl, Cass4, Ccl7, Ccnl) and positive regulation of blood vessel development (e.g. C5arl, Ccbe1, Cd40, Cdh5, Chill, Cx3cr1, Cxcr2, Cybb, Cyp1bl, Ddahl).

[0150] KEGG pathway analysis ​ i) showed that multiple signaling pathways related to immune regulation and wound repair were activated, such as ECM-receptor interaction pathway (e.g. Cd44, Chad, Col1a1, Col1a2, Col4a1) and PI3K-Akt signaling pathway (e.g. Akt3, Angpt2, Areg, Artn, Bcl2l11).

[0151] GSEA enrichment analysis ​ j、 ​ k、 ​ l and ​ m) further confirmed the above results, and the enriched pathways included blood vessel development (e.g. Scg2, Adam8, Aplnr, Itga5, Srpx2), collagen fibril organization (e.g. Col11a1, Loxl3, Sfrp2, Adamts7, Col5a1), mitochondrial respiratory chain complex I (e.g. Ndufa8, Tmem126a, Lyrm2, Ndufa3, Dmac1, Foxredl) and oxidative phosphorylation (e.g. Atp6v0d2, Tcirgl, Atp6v1b2, Cox4i2, Atp6v0c).

[0152] These pathways are crucial for inflammation relief and tissue regeneration, further supporting the multi-level healing promotion mechanism mediated by SAB / F-gel.

[0153] Based on the above verification results, TQ / Ce6@SAB / F-gel effectively inhibited the inflammatory response at the wound site by reducing the expression of pro-inflammatory factors and promoting the conversion of macrophages to an anti-inflammatory phenotype. The transcriptome data revealed that it activated tissue repair and regeneration pathways, providing a solid molecular basis for wound repair.

[0154] The dual role of SAB / F-gel in regulating cytokine levels and macrophage phenotypes highlights its multidimensional advantages in immune regulation and tissue healing. Therefore, TQ / Ce6@SAB / F-gel is expected to become an effective therapeutic candidate for the management of diabetic chronic infected wounds, with its comprehensive ability to target immune regulation and tissue regeneration, providing a new treatment strategy for chronic non-healing wounds.

Claims

1. A composition comprising salvianolic acid B and fibrin, the salvianolic acid B and fibrin being in weak interaction, forming interwoven or interpenetrating networks, and providing an acidic environment, to enhance the implementation of photodynamic therapy means.

2. The composition of claim 1, wherein The interaction between salvianolic acid B and fibrin comprises three high-affinity interaction sites, hydrogen bonds being formed between salvianolic acid B and amino acid residues of fibrin.

3. The composition of claim 1, wherein The salvianolic acid B self-assembles to form an acidic hydrogel.

4. The composition of claim 1, wherein The composition further comprises active components for implementing photodynamic therapy.

5. The composition of claim 1, wherein The composition further comprises chlorin e6 and thymoquinone.

6. Use of the composition according to claim 1 in the manufacture of a medical device for photodynamic therapy.

7. Use of the composition according to claim 1 in the manufacture of a medical device for promoting wound repair and healing.

8. A medical device, characterized by A device for implementing phototherapy and the composition according to claim 1 are included.

9. The medical device of claim 8, wherein The phototherapy device emits red light.

10. The medical device of claim 8, wherein The composition is located in the optical path of the light emitted by the phototherapy device.

Images