Fibrin gel, medical device thereof and application of fibrin gel in auxiliary phototherapy

Abstract

In a composition, at least one of TQ and NADH is loaded in a gel. The composition disclosed by the invention is loaded on a gel carrier formed by mixing fibrinogen and thrombin, the gel carrier is smeared on a wound, and then the gel carrier, red light and blue light have a synergistic effect, so that the dual functions of resisting infection and accelerating wound healing are realized under the condition that the illumination time is remarkably shortened. In this way, ramp is used; the combination of bLED and the fibrin gel provides an effective scheme for managing chronic wounds, and is expected to be used for clinical intervention of family chronic diabetic wounds.

Description

Technical Field

[0001] The invention relates to a biomaterial, in particular to a biogel and application of the biogel in medical devices for auxiliary phototherapy. Background Art

[0002] Chronic wounds are a favorable environment for the reproduction of pathogenic microorganisms. The toxic enzymes secreted by these microorganisms can harm host tissues and interfere with the wound healing process, leading to increased inflammation, delayed wound healing, and potentially serious complications. Infected wounds require frequent visits to the hospital for treatment, which places tremendous financial and psychological pressure on patients.

[0003] Non-healing chronic wounds significantly reduce the quality of life of patients and put a strain on the healthcare system, and the U.S. Food and Drug Administration has identified them as a top research priority. The incidence and associated costs of chronic wounds (including serious consequences such as amputation and death) continue to rise, and the 5-year mortality rate is similar to that of cancer. However, unlike diseases such as cancer, where treatment strategies are well-established, the treatment of chronic wounds remains a "trial and error" approach, with expensive and ineffective treatment processes due to their diverse etiologies, including infection with various pathogens, biofilms, and pro-inflammatory bacterial toxins. These factors, coupled with the heterogeneity of patient wounds, lead to treatment challenges, and wounds continue to progress to advanced stages despite standard care measures. Despite a large number of clinical trials, there has been no significant improvement in efficacy. In the past 26 years, only one agent has been approved by the U.S. Food and Drug Administration (FDA) for the treatment of chronic wounds, becaplermin, despite the risk of carcinogenesis and safety warning labels. The treatment of chronic wounds includes various interventions such as debridement, negative pressure wound therapy, topical growth factors, and skin substitute coverage. Traditional methods often have long treatment times, suboptimal outcomes, and are expensive, so new approaches are needed for effective and cost-effective chronic wound management, especially for low-income populations.

[0004] Safe and non-invasive phototherapy, including photodynamic therapy (PDT) and photobiomodulation (PBM), is gaining increasing attention. Taking chronic wound care as an example, PDT and PBM are becoming effective methods to fight microbial infection and promote wound healing, respectively. Blue light is widely used in photodynamic therapy due to its safe, efficient, and broad-spectrum antimicrobial properties, and can effectively kill a variety of bacteria. In addition, PBM uses red or near-infrared (NIR) light to stimulate the dissociation of NO from complex IV (CIV, cytochrome c oxidase), a key rate-limiting enzyme of the electron transport chain (ETC), resulting in changes in oxidative stress in mitochondria. This process enhances oxidative phosphorylation (OXPHOS) and accelerates the mitochondrial respiratory chain (MRC) of cells under stress, thereby improving mitochondrial function and wound healing. For decades, various phototherapy methods have been used in hospital settings and have shown beneficial effects. However, the equipment currently used to implement phototherapy is not only bulky and relies on professional operators, but also has problems such as high treatment costs, inconvenient treatment processes for users, and inability to adapt to various shapes of wounds during treatment, all of which have burdened phototherapy equipment. On the other hand, the effectiveness of phototherapy for wounds still needs to be improved to adapt to these limitations, which hinders their widespread use in the home. Summary of the invention

[0005] One object of the present invention is to provide a composition for enhancing the effect of wound repair.

[0006] Another object of the present invention is to provide a fibrin gel that is combined with phototherapy to improve antibacterial properties and promote wound repair and healing.

[0007] Another object of the present invention is to provide a fibrin gel for use as a medical device in auxiliary phototherapy to improve the antibacterial properties of wounds and promote wound repair and healing.

[0008] Another object of the present invention is to provide a medical device that combines a device for implementing phototherapy and fibrin gel for wound repair and healing.

[0009] The wounds referred to in the present invention refer more to chronic wounds, including but not limited to elderly wounds, diabetic wounds, pressure wounds, venous ulcers and burns.

[0010] A composition includes at least one of thymoquinone (TQ) and NADH.

[0011] Another composition uses a gel material as a carrier, which carries at least one of TQ and NADH.

[0012] Another composition is a gel carrier formed by mixing fibrinogen and thrombin, and at least one of TQ and NADH is loaded in the gel.

[0013] Another composition is a fibrin gel, which includes fibrinogen, thrombin, TQ and NADH. Fibrinogen and thrombin are mixed to form a gel carrier, and TQ and NADH are loaded in the gel.

[0014] The fibrin gel of the present invention is prepared by mixing a fibrinogen solution (fibrinogen concentration, for example, 2-10 mg / mL) and a thrombin solution (thrombin content, for example, 1-10 U / mL) in equal volumes to obtain a gel carrier.

[0015] The fibrin gel of the present invention also contains calcium ions to promote the activity of thrombin, facilitate the formation of fibrin and enhance the mechanical properties of the gel.

[0016] The fibrin gel of the present invention is prepared by mixing a fibrinogen solution and a thrombin solution, wherein the TQ loaded in each milliliter of the gel is 50 μg to 500 μg, especially 50 μg to 300 μg, such as 60±10 μg, 70±10 μg, 80±10 μg, 90±10 μg, 100±10 μg, 110±10 μg, 120±10 μg, 130±10 μg, 140±10 μg, 150±10 μg, 160±10 μg, 170±10 μg, 180±10 μg, 190±10 μg, 200±10 μg, 210±10 μg, 220±10 μg, 230±10 μg, 240±10 μg, 250±10 μg, 260±10 μg, 270±10 μg, 280±10 μg, 290±10 μg, 300±10 μg, 310±10 μg, 320±10 μg, 330±10 μg, 340±10 μg, 350±10 μg, 360±10 μg, 370±10 μg, 380±10 μg, 390±10 μg, 400±10 μg, 410±10 μg, 420±10 μg, 430±10 μg, 440±10 μg, 450±10 μg, 460±10 μg, 470±10 μg, 480±10 μg, 490±10 μg, 50 0±10μg, 150±10μg, 160±10μg, 170±10μg, 180±10μg, 190±10μg, 200±10μg, 210±10μg, 220±10μg, 230±10μg, 240±10μg, 250±10μg, 260±10μg, 270±10μg, 280±10μg, 290±10μg. Specifically: 45±5μg, 50±5μg, 55±5μg, 60±5μg, 65±5μg, 70±5μg, 75±5μg, 80±5μg, 85±5μg, 90±5μg, 95±5μg, 100±5μg, 105±5μg, 110±5μg and 115±5μg.

[0017] The fibrin gel of the present invention is prepared by mixing a fibrinogen solution and a thrombin solution, wherein the NADH loaded in each milliliter of the gel is 10 μg to 100 μg, especially 40 μg to 80 μg, such as: 45±5 μg, 50±5 μg, 55±5 μg, 60±5 μg, 65±5 μg, 70±5 μg and 75±5 μg. Specifically, such as: 60±1 μg, 61±1 μg, 62±1 μg, 63±1 μg, 64±1 μg, 65±1 μg, 66±1 μg, 67±1 μg, 68±1 μg and 69±1 μg.

[0018] The fibrin gel of the present invention further contains mesoporous silica nanoparticles (MSN) for loading NADH, that is, the NADH loaded in the gel is loaded in the MSN nanoparticles.

[0019] The fibrin gel of the present invention is combined with a phototherapy device (apparatus) to enhance the efficiency of wound repair, thereby improving the antibacterial ability of the wound, as well as the repair and healing speed of the wound.

[0020] A medical device comprises various fibrin gels provided by the invention.

[0021] Another medical device includes a device for implementing phototherapy and various fibrin gels provided by the present invention, wherein the fibrin gel is located in the light path of the outgoing light.

[0022] Devices for implementing light therapy can emit blue light and red light, for example: red light (605nm~700nm) or blue light (405nm~470nm) emitted by LED as the light source.

[0023] It has been demonstrated that thymequinone (TQ), which is sensitive to blue light, and reduced nicotinamide adenine dinucleotide (NADH), which cooperates with red light, are added to the sprayable fibrin gel, which is highly compatible with the skin and can easily form a thin film on the skin.

[0024] Fluorescence probes and EPR confirmed that F-gel can produce ROS, including ·OH, H2O2 and O2 under bLED irradiation. - , and showed the ability to eliminate a variety of microorganisms, including multidrug-resistant MRSA, Pa, Ab, and common clinical fungal infections Ca. In addition, the NADH contained in F-gel can reduce ROS damage caused by PDT to healthy tissues. This combination therapy provides a practical, economical and convenient home treatment for infected wounds. That is, F-gel is applied to the wound, and then acts on red and blue light with the light turned on, achieving the dual functions of anti-infection and accelerated wound healing with significantly shortened lighting time.

[0025] The study also showed that the combination of PBM and F-gel containing NADH can synergistically improve the mitochondrial function of HUVEC under inflammatory stress, thereby synergistically promoting wound vascularization through energy supplementation. In addition, the combination of F-gel and phototherapy can also 1) improve the mitochondrial function of macrophages under LPS stimulation, thereby enhancing OXPHOS, and 2) induce metabolic reprogramming of macrophages, directing them to the anti-inflammatory M2 phenotype through the energy metabolism-immunomodulation pathway. All of these have been verified in the diabetic infection wound model of mice and mini pigs, that is, the combination of the fibrin gel of the present invention and F-gel is effective in eliminating microbial infection and accelerating wound healing. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1The figure is a characterization result diagram of the fibrin gel prepared by the present invention; wherein, a is a bright field photograph of the sprayability and film-forming properties of F-gel, b is a photograph of F-gel under the field of view of a scanning electron microscope, c is a time curve of G′ / G″ of F-gel, d is a curve of the angular frequency of G′ / G″ of F-gel, e is a curve of the release of TQ and NADH from F-gel, f is a spectrum diagram of TQ, NADH, rLED and bLED. g is the excitation spectrum and emission spectrum related to TQ and NADH, h is the excitation spectrum and emission spectrum of F-gel, rLED and bLED, i is a statistical diagram of H2O2 generated by F-gel combined with bLED and rLED, j is a statistical diagram of -OH generated by F-gel combined with bLED and rLED, k is a statistical diagram of DMPO-OH, DMPO-O2 and TEMPO- 1 The results of the identification and measurement of O2 adducts by EPR, m is a bright field photograph of an embodiment of the adhesion detection of F-gel, n is a bright field photograph of another embodiment of the adhesion detection of F-gel, o is a schematic diagram of the principle of TQ and NADH anti-oxidation and cell apoptosis after exposure to light, p is a statistical diagram of the survival rate of HUVECs cells after being acted on in various ways, and r is a statistical diagram of the intracellular ROS fluorescence signal of HUVECs cells after being acted on in various ways;

[0027] Figure 2 The results of the in vitro antibacterial effect of the combination of r&bLED and F-gel; a is a schematic diagram of the experimental design and antibacterial mechanism, b is the killing curve of various bacterial strains in planktonic culture, c is a heat map of the synergistic effect of bLED and F-gel on the specified bacterial strains in planktonic cells, d is a distribution diagram of S values, and e is a representative scanning electron microscopic field of view of MRSA and Pa treated in each group (bLED is 30 J / cm 2 , F-gel (final TQ concentration was 100 μg / mL), arrows indicate damaged bacterial envelope, asterisks indicate vacuoles and bubbles in cells, f is the transmission electron microscopic view of MRSA and Pa treated in each group (bLED was 30 J / cm 2 , F-gel (final TQ concentration was 100 μg / mL), arrows indicate damaged bacterial envelope, asterisks indicate vacuoles and bubbles in cells, g is bLED (30 J / cm 2 Representative DCF fluorescence images of MRSA and Pa planktonic cells after co-treatment with F-gel (final TQ concentration of 100 μg / mL), f is bLED (30 J / cm 2 Representative HPF fluorescence images of MRSA and Pa planktonic cells after combined treatment with F-gel (final TQ concentration of 100 μg / mL), i represents various modes (bLED, 60 J / cm 2; F-Final TQ concentration of gel, 100 μg / mL) after SYTO TM Confocal images and corresponding 3D images of mature biofilms stained with 9 (green) and PI (red), j is Pa after PBS and bLED (15 J / cm 2 ) and F-gel (final TQ concentration, 100 μg / mL) treatments, heatmap (fold change ≥ 2, q < 0.05), k is the functional classification map of differentially expressed genes between the PBS group and the bLED and F-gel groups by GO enrichment analysis;

[0028] Figure 3 The results of r&bLED and F-gel improving mitochondrial function and promoting angiogenesis synergistically; a is a schematic diagram of the mechanism, and b is the interaction between HUVECs and LPS, F-gel (NADH concentration 100 μM) and rLED of various energy densities (rLED-L: 1 J / cm 2 ,rLED-M:3J / cm 2 ,rLED-H:5J / cm 2 ) after co-culture, the statistical graphs of intracellular NAD(H) levels, CⅣ activity and ATP levels (mean ± sd, n = 5), c is the microphotographs of EdU staining in each experimental group, d is the visual field of the wound scratch test of HUVEC in each experimental group observed by optical microscope, e is the fluorescence image of tube formation in each experimental group, f is the statistical graph of EdU staining in each experimental group (mean ± sd, n = 5), g is the statistical graph of the wound scratch test of HUVEC in each experimental group (mean ± sd, n = 5), h is the statistical graph of the length of the tube formed in each experimental group (mean ± sd, n = 5), i is the visual field of intracellular ROS in each experimental group; j is the statistical graph of DCF fluorescence value in each experimental group (mean ± sd, n = 5), k is the graph of mitochondrial membrane potential (ΔΨM) in each experimental group, l is the statistical graph of mitochondrial membrane potential (ΔΨM) value in each experimental group (mean ± sd, n = 5), m is the NF-κB in each experimental group p65 nuclear translocation field of view, n is the statistical diagram of NF-κB p65 nuclear translocation value in each experimental group (mean ± sd, n = 5), o is the result of Annexin V / PI double staining analysis of HUVECs cell apoptosis in each group induced by LPS (100 ng / mL, 48 h), p is the proportion of HUVECs cell apoptosis in each group induced by LPS (100 ng / mL, 48 h) by Annexin V / PI double staining analysis, q is the result of Annexin V / PI double staining analysis of HUVECs cell apoptosis in each group induced by TNF-α (80 ng / mL, 48 h), r is the proportion of HUVECs cell apoptosis in each group induced by TNF-α (80 ng / mL, 48 h) by Annexin V / PI double staining analysis;

[0029] Figure 4 The results of r&bLED combined with F-gel regulating macrophage polarization by reprogramming; a is a schematic diagram of the scheme (G1, control group; G2, LPS; G3, LPS+F-gel; G4, LPS+rLED; G5, LPS+F-gel+rLED) (rLED, 3J / cm 2 a is a schematic diagram of the mechanism by which rLED combined with F-gel regulates macrophage polarization through metabolic reprogramming, c is a real-time measurement of the ECAR results and OCR results of THP-1 cells that were not stimulated (control group) or stimulated with LPS (mean ± sd, n = 5), d is a statistical graph of the NAD(H) content and the NAD+ / NAD(H) ratio in THP-1 cells of each experimental group (mean ± sd, n = 5), e is a statistical graph of the NOx level and CⅣ activity in THP-1 cells of each experimental group (mean ± sd, n = 5), f is the NF-κB observed by CLSM in THP-1 and RAW264.7 cells in each experimental group p65 nuclear translocation field of view, g is the quantitative analysis statistical graph of P65 intensity in the nucleus of THP-1 and RAW264.7 cells in each experimental group observed by CLSM (mean ± sd, n = 5), h is the iNOS field of view of THP-1 and RAW264.7 cells observed by CLSM, i is the statistical graph of iNOS observed by CLSM in THP-1 and RAW264.7 cells (mean ± sd, n = 5), j is the statistical graph of pro-inflammatory cytokines in THP-1 cell culture supernatant quantitatively analyzed by ELISA (mean ± sd, n = 5), k is the flow cytometry analysis result of CD206 and CD86 expression in THP-1 cells, l is the CD86 - / CD206 - 、CD86 + / CD206 - and CD86 - / CD206 + Quantitative analysis ratio graph of cells;

[0030] Figure 5 The results of promoting the healing of MRSA-infected diabetic wounds in mice model; a is a schematic diagram of the experimental scheme (G1: control group, G2: F-gel, G3: rLED+F-gel, G4: bLED+F-gel and G5: r&bLED+F-gel; rLED: 3J / cm 2 , bLED36 J / cm 2, NADH concentration in F-gel: 100 μM; TQ concentration in F-gel: 200 μg / mL), b is the bacterial luminescence image of the acutely infected wound and the bright field photo of the microbial agar plate, c is the statistical graph of the average luminescence of each experimental group (in logarithmic relative luminescence unit of each model relative to time zero, lg RLU, mean ± sd, n = 5), d is the bioluminescence graph of the mouse wound surface changing over time, e is the photo of the mouse wound surface at each time point, f is the statistical graph of the wound healing rate, g is the H&E staining image of the wound on the 8th and 16th day of each experimental group, h is the statistical graph of the granulation tissue thickness on the 8th and 16th day of each experimental group (mean ± sd, n = 5), i is the Masson staining image of the wound on the 8th and 16th day of each experimental group, j is the statistical graph of the collagen deposition ratio on the 8th and 16th day of each experimental group (mean ± sd, n =5), k is the CD31 / αSMA immunofluorescence staining diagram of the wounds of each experimental group on the 8th and 16th days, l is the statistical diagram of the number of blood vessels of each experimental group on the 8th and 16th days (mean ± sd, n = 5), m is the statistical diagram of the NAD+ / NAD(H) ratio in the wound tissue of each experimental group (mean ± sd, n = 5), n is the statistical diagram of the CⅣ activity in the wound tissue of each experimental group (mean ± sd, n = 5), o is the statistical diagram of the ATP level ratio in the wound tissue of each experimental group (mean ± sd, n = 5);

[0031] Figure 6 Figure 2 is the result of r&bLED combined with F-gel regulating inflammation in vivo; wherein, a is the IL-6 / TNFα immunofluorescence staining of the wounds of each experimental group on the 8th and 16th days, b is the CD86 / CD206 immunofluorescence staining of the wounds of each experimental group on the 8th and 16th days, c is the heat map and functional classification of differentially expressed genes in wound tissues after treatment with PBS or F-gel and r&bLED (converted change ≥ 2, q < 0.05), d is the GO enrichment analysis of down-regulated genes, e is the result of GSEA enrichment analysis of cell respiration obtained by cellular respiration, f is the result of GSEA enrichment analysis of macrophage activity obtained by macrophage activity, g is the result of GSEA enrichment analysis of TNF signaling pathway obtained by TNF signaling pathway, h is the result of GSEA enrichment analysis of IL-4 family signaling pathway, i is the result of GSEA enrichment analysis of NF-κB pathway, j is the result of GSEA enrichment analysis of HIF pathway;

[0032] Figure 7 The results of r&bLED and F-gel promoting wound healing in infected diabetic piglets are shown in Figure 2; a is a schematic diagram of the experimental scheme (G1: control group, G2: F-gel, G3: rLED+F-gel, G4: bLED+F-gel and G5: r&bLED+F-gel; rLED: 3J / cm2 , bLED: 36J / cm 2 , NADH concentration in F-gel: 100 μM; TQ concentration in F-gel: 200 μg / mL), b is a bright field photo of pig wounds. C is a statistical graph of NAD+ / NAD(H) in wound tissues of each experimental group, d is a statistical graph of CⅣ activity in wound tissues of each experimental group, e is a H&E staining image of wounds on day 8 of each experimental group, f is a Masson staining image of wounds on day 8 of each experimental group, g is a CD31 / αSMA immunofluorescence staining image of wounds on days 8 and 20, h is a heat map and functional classification map of differentially expressed genes in wound tissues of G1 and G5 phases (converted change ≥ 2 and q < 0.05), i is a GO enrichment analysis map of down-regulated genes, and j is a GO enrichment analysis map of up-regulated genes. DETAILED DESCRIPTION

[0033] The technical solution of the present invention is described in detail below. The embodiments of the present invention are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the invention can be modified or replaced by equivalents without departing from the spirit and scope of the technical solution of the present invention, which should be included in the scope of the claims of the present invention.

[0034] The various test methods used in the following examples of the present invention are specifically described as follows:

[0035] 1) Preparation of gel

[0036] Add NADH aqueous solution (e.g. 100 μg / mL) to MSN solution (e.g. mass ratio 1:10), stir and incubate at 25°C for 2 hours to induce the formation of NADH@MSN complex, and use UV-visible spectrophotometer to detect the amount of NADH remaining in the solution to calculate the NADH loading rate. Dry at 37°C for at least 3 hours to remove the residual solvent in the particles, and obtain NADH-loaded MSN nanoparticles, recorded as: NADH@MSN.

[0037] Dissolve fibrinogen in a buffer (e.g. PBS or saline, pH 6.8-7.4) at a concentration of 10-20 mg / mL. Ensure complete dissolution and filter to remove insoluble impurities.

[0038] NADH@MSN and TQ were added to the fibrin solution to make the NADH concentration be 50-200 μg / mL and the TQ concentration be 50-500 μg / mL, and mixed evenly.

[0039] The concentration of thrombin solution is adjusted according to actual needs, usually 1 to 10 units / mL. The concentration of thrombin determines the speed of conversion of fibrinogen to fibrin, which in turn affects the speed of gel formation and the final gel structure.

[0040] In addition, calcium ions are optionally added to the thrombin solution to promote the activity of thrombin, which not only supports the formation of fibrin but also regulates the Ca 2 + Concentration affects the mechanical properties of the gel. Add calcium ion carriers such as, but not limited to, CaCl2 solution (10-50 mM).

[0041] Finally, the prepared fibrinogen solution (containing at least one of NADH@MSN and TQ) and thrombin solution are mixed in a volume ratio of 1:1 or adjusted according to experimental requirements. The mixture is poured into a mold or a predetermined position (such as a tissue culture dish, microplate, etc.). As thrombin catalyzes the conversion of fibrinogen into fibrin, the fibers self-assemble to form a network structure, thereby generating a fibrin gel, denoted as: F-gel or F-gel, in which: the loading efficiency and content of NADH@MSNs are 53.6% and 8.9%, respectively, and the TQ content is 50-500μg / mL.

[0042] The mesoporous silica nanoparticles (MSN) used in the present invention can be realized by referring to the prior art, which is briefly described as follows:

[0043] Add an alkaline catalyst (such as NaOH or ammonia water) to the hexadecyltrimethylammonium bromide (CTAB) solution and continue stirring to ensure that the system is uniform. Then, after adding tetraethoxysilane, silica nanoparticles are formed. The reaction is usually carried out at room temperature or a slightly higher temperature (30-50°C), and stirring is maintained for at least 2-6 hours to ensure the full generation of silica and the formation of a mesoporous structure. After the reaction is completed, the generated mesoporous silica nanoparticles are collected by centrifugation or filtration. The nanoparticles are washed with deionized water and ethanol several times to remove residual templates and unreacted silicon sources. After removing the template, the particles are centrifuged again and dried under vacuum drying or air drying conditions to obtain the final mesoporous silica nanoparticles.

[0044] 2) Fabrication and characterization of red / blue LED (r&bLED) patches

[0045] Thermoplastic polyurethane (TPU) substrates were used as the initial substrates, and holes were opened to connect the bottom and top layers. The typical sizes of the TPU substrates were 4 cm × 4 cm and 2 cm × 2 cm, and the thickness of the TPU pattern was set to 20 μm. The packaged rLED chips (wavelength = 630 nm; REFOND, China) and bLED chips (wavelength = 470 nm; REFOND, China) were manually placed on the contact pads together with the temperature sensor (M117Z, Mysentech) individually, and then coated with high-density conductive silver paste (Shanghai Mifang Technology, China) and reflowed at 90 °C with a melting interval of 3 min. PDMS was then coated onto the surface-mounted LEDs using a dip coating process (mixing ratio of prepolymer and curing agent was 10:1; SYLGARD 184, Dow, USA) and cured in an oven at 70 °C overnight. Finally, PDMS mixed with reflective paint was coated on the back of the substrate to prevent light from leaking out from the back.

[0046] The optical power density of the r&bLED patch was measured with an optical power meter (PM100D, Thorlabs, USA), and the mechanical durability of the r&bLED patch was repeatedly tested by load pressure in a folded state (Mifang Technology Co., Ltd., Shanghai, China). The heat generation of the r&bLED patch was measured with an infrared thermal imager (E54, FLIR, Shanghai, China).

[0047] The control module consists of a rigid FR4 printed circuit board (PCB), surface mount electronic components, a battery, and a package. The double-layer custom PCB was designed using JLCEDA (Professional Edition) and manufactured by an ISO 9001:2015 certified supplier (JLCPCB.com). The device uses a 0.4 mm thick FR4 PCB with solder mask and electroless nickel immersion gold (ENIG) treatment on the exposed pads. The electronic components including the ESPSoC and peripherals are attached to the PCB using lead-free solder paste (AZ50 solder paste, Anrecson). The ESPSoC is programmed using a custom embedded firmware developed in Arduino IDE 2.3.2.

[0048] The device housing was made using a DLP light-curing 3D printer (Anycubic Photon D2) and designed using SolidWorks 2019 (Education Edition). The .stl file was sliced ​​using Anycubic Slicer, then 3D printed and post-processed using various photosensitive resins (Wash and Cure 2.0, Anycubic).

[0049] The printed circuit board and lithium battery were assembled in the device housing and then encapsulated using PDMS (prepolymer and curing agent mixed in a ratio of 10:1; SYLGARD 184, Dow, USA). Subsequently, the assembly was cured in an oven at 70 °C to form a soft and smooth protective layer.

[0050] 3) Detection of antibacterial activity of planktonic cells

[0051] Add PBS for about 10 7 CFU / mL of microbial suspensions were added to 35 mm dishes together with different volumes of F-gel (TQ concentration, 500 μg / mL) to explore final TQ concentrations of 25, 50, 75, and 100 μg / mL in solution and incubated for 20 min before being irradiated with LED blue light (bLED) at the bottom of the dish while stirring at 60 rpm. 2 bLED irradiation) was performed and 50 μL of the suspension was collected. The evaluation of microbial activity was similar to that when bLED or F-gel was used alone. Colony forming units (CFU) were determined by 10-fold serial dilutions on BHI or YM agar plates and counted after 24 h of incubation and were expressed as lg CFU / mL throughout the study unless otherwise stated. The detection limit was 100 CFU / mL. SEM (JSM-7800F; JEOL) and TEM (Talos L120C G2, Thermofisher) were used to observe the morphological changes of microorganisms after combined treatment.

[0052] 4) Treatment of formed biofilm

[0053] Prepare 1 × 10 cells in tryptic soy broth (TSB) containing 0.1% glucose. 6 CFU / mL of mid-logarithmic growth phase culture. The biofilm was rinsed twice with PBS and then immersed in F-gel (TQ concentration of 500 μg / mL) and PBS solution to make the final concentration of TQ 100 μg / mL and incubated for 20 minutes before exposure to 60 J / cm bLED. PI and SYTO TM 9 (Thermo Fisher Scientific) staining to verify the bacterial survival rate under CLSM.

[0054] 5) Bacterial transcriptome sequencing

[0055] The stationary cultured Pa were transferred into a solution of PBS and F-gel (TQ concentration, 500 μg / mL) to make the final TQ concentration 100 μg / mL. Then, the Pa were exposed to a sublethal dose of bLED for 5 min (50 mW, 15 J / cm2 ), and then incubated for 2 hours. Finally, the cells were harvested by centrifugation and analyzed by high-throughput sequencing.

[0056] 6) Cell culture

[0057] HUVECs, THP-1 and RAW264.7 cell lines were obtained from ATCC. HUVECs were cultured in endothelial cell medium (ECM; ScienCell) supplemented with 10% fetal bovine serum (FBS). RAW264.7 cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM; Gibco) supplemented with 10% FBS; THP-1 cells were cultured in RPMI 1640 medium (Gibco) supplemented with 10% FBS.

[0058] 7) Determination of cellular NAD(H), CIV activity and ATP levels

[0059] HUVECs or THP-1 cells were plated at 2 × 10 5 The cells were seeded at a density of 1.5 μg / mL into 24-well plates and cultured overnight. Afterwards, the cells were treated with one-tenth the volume of culture medium, rLED, or a combination of F-gel and red LED (rLED) for 2 h, and then cultured with LPS (100 ng / mL) for 12 h. Subsequently, the cells were rinsed three times with PBS and lysed with 100 μL of lysis buffer. Cellular NAD(H) levels and NAD were assessed using the NAD / NADH assay kit (ab65348, Abcam). + The cellular ATP level was detected using a luminescent ATP assay kit (ab113849, Abcam), and the cellular complex IV (CIV) activity was assessed using a complex IV enzyme activity microplate assay kit (ab109909, Abcam).

[0060] 8) Measurement of inflammation-related cytokine levels

[0061] 2×10 per well 4 THP-1 or RAW264.7 cells were cultured in 96-well plates at a density of 100 μg / mL and treated as described above. After 24 hours of treatment, the cell culture medium was collected and cytokine production was assessed using an ELISA kit purchased from Thermo Fisher Scientific according to the manufacturer's instructions. Data were obtained by measuring the difference in absorbance at a wavelength of 450 nm using a microplate reader.

[0062] 9) In vitro macrophage phenotype regulation

[0063] THP-1 and Raw 264.7 cells were seeded into 12-well plates and incubated at 37°C, 5% CO2 for 24 hours. The cells were then treated as above and incubated with LPS (100 ng / mL) for 24 hours. Subsequently, the cells were stained with PE-labeled anti-CD86 antibody and APC-labeled anti-CD86 antibody according to the manufacturer's instructions and analyzed by flow cytometry.

[0064] 10) In vivo wound healing study in diabetic mice infected with

[0065] All procedures for the in vivo diabetic mouse skin wound healing study were approved by the Institutional Animal Care and Use Committee (IACUC) of Shanghai Jiao Tong University. Male BALB / c mice (6 weeks old) were obtained from Slac Biotechnology. To establish the diabetic mouse model, mice were intraperitoneally (ip) injected with streptozotocin (STZ) at a dose of 50 mg / kg for five consecutive days. The blood glucose level of mice was monitored using a glucometer. Mice with blood glucose levels <16.7 mmol were excluded, and the remaining mice were confirmed as diabetic mice.

[0066] Diabetic mice were anesthetized with isoflurane, and a circular wound with a diameter of 7 mm was made on the back of each mouse using a skinner. Then 50 μL of bioluminescent Staphylococcus aureus (MRSA) or Pseudomonas aeruginosa (Pa) was applied to the wound at a concentration of 1×10 per ml. 9 CFUs were used to induce diabetic skin infection. At the same time, the mice were divided into five groups: G1, control group; G2, F-gel group (NADH concentration 100μM; TQ concentration 200μg / mL); G3, rLED+F-gel group; G4, bLED+F-gel group; G5, r&bLED+F-gel group. On the second day, 100μL F gel was sprayed onto the wounds of G2, G3, G4 and G5 groups using a double-barrel sprayer and incubated for 20 minutes. The wounds were treated with rLED (630nm, 10mW / cm 2 ) irradiated the wounds of mice in group G3 for 5 minutes, and covered the wounds with LED patches. For groups G4 and G5, mice were irradiated with bLED (470 nm, 50 mW / cm 2 ) for 12 minutes. After 30 minutes, the cells were illuminated with rLED (630 nm, 10 mW / cm 2) irradiated G5 group mice for 5 minutes. The patch was removed after treatment. The Lumina in vivo imaging system (IVIS, PerkinElmer) was used to collect bacterial bioluminescence at different times after the start of treatment (10 seconds exposure, medium binning). Before the 8th day, treatment and bacterial bioluminescence detection were performed every 2 days, and wound photos were taken every 4 days, and the length and width were recorded to calculate the area. On the 8th and 16th days, representative mice in each group were killed, and the injured skin was collected for histology and immunofluorescence staining.

[0067] 11) In vivo study on wound healing of infected pig skin

[0068] All procedures were approved by the IACUC of Shanghai Jiao Tong University. Two Panamanian miniature pigs weighing 12–15 kg were used in this study. The piglets were allowed to acclimate for 7 days before wounding. During the experiment, the hair on the back of the piglets was plucked, and the back area was disinfected with iodine and 70% ethanol. Surgery was then performed under general anesthesia. Ten full-thickness wounds with a diameter of 15 mm were made on both sides of the piglets using a skinner, with a distance of 2 cm between the two wounds. Then, 100 μL of MRSA suspension at a concentration of 1 × 10 per ml was applied to each wound. 9 CFUs were used to induce diabetic skin infection. Meanwhile, the wounds were divided into five groups: G1, control group; G2, F-gel group (NADH concentration 100 μM; TQ concentration 200 μg / mL); G3, rLED+F-gel group; G4, bLED+F-gel group; G5, r&bLED+F-gel group.

[0069] On the second day, 200 μL of F-gel was sprayed on the wounds of G2, G3, G4, and G5 groups using a double-barrel sprayer and incubated for 20 min. 2 ) irradiated the wound of G3 group for 5 minutes, and covered the wound with LED patch. For G4 and G5 groups, bLED (470 nm, 50 mW / cm 2 ) irradiated the wound for 12 minutes and covered the wound with an LED patch. 30 minutes later, rLED (630 nm, 10 mW / cm 2 ) irradiated the mice in group G5 for 5 minutes. After treatment, the patch was removed and 3M TM Tegaderm TM A transparent dressing was placed over the wound and secured with skin staples. In addition, the wound was covered with sterile cotton gauze to further support and protect the wound.

[0070] After the surgery, the mini pigs were monitored for any signs of discomfort and provided with feed and water. Each mini pig was housed individually and monitored daily. Treatments were administered every four days. Wound images were collected every four days. At predetermined intervals, the piglets were euthanized and wound tissues were collected for further histological analysis.

[0071] 12) Histological analysis

[0072] Wound tissues were collected on days 8 and 16 after model induction and fixed with 4% paraformaldehyde (mouse wound tissues) or neutral formalin (minipig wound tissues). Subsequently, the tissues were embedded in paraffin and cut into 5 μm sections for subsequent H&E staining, Masson trichrome staining (MTTS) and immunofluorescence staining.

[0073] H&E staining and immunofluorescence staining followed the standard protocols provided by the manufacturer. Sections were deparaffinized in xylene and rehydrated in graded ethanol. After microwave in 10 mM sodium citrate, sections were incubated with primary antibodies at 4 °C overnight and then with secondary antibodies at 37 °C for 30 min. Wound tissues were fixed with 4% formaldehyde and embedded in paraffin. Tissue sections (5 μm) were mounted on slides for histological analysis. Wound closure area was measured by H&E staining. Collagen deposition was quantified using MTTS. Immunofluorescence staining of CD86 and CD206 was performed on days 8 and 16 after model establishment to observe the infiltration and differentiation of macrophages in the wound.

[0074] 13) Transcriptome analysis

[0075] RNA was extracted from wound tissues of diabetic mice and mini pigs on day 8 using TRIzol reagent (Invitrogen). For library preparation, 2 μg of total RNA was treated with Ribo-off rRNA Depletion Kit (human / mouse / rat) (Illumina, USA), and then RNA sequencing libraries were prepared using KCTM Stranded mRNA Library Prep Kit for Illumina (Wuhan Saike Health Technology Co., Ltd., China) according to the manufacturer's method. The library products of 200 to 500 bp were enriched, quantified, and sequenced using the PE150 model NovaSeq 6000 sequencer (Illumina, USA).

[0076] RNA-seq data were analyzed using bioinformatics tools. Differential expression analysis was performed using edge R software. Heatmaps were generated using TBtools software (https: / / github.com / CJ-Chen / TBtools / releases). Gene Ontology (GO) terms and Kyoto Encyclopedia of Genomes (KEGG) pathways were determined using KOBAS2.0. The hypergeometric test and Benjamini-Hochberg false discovery rate (FDR) control procedure were used to determine the enrichment of each term.

[0077] 14) Statistical analysis

[0078] GraphPad Prism (version 9.50) was used to analyze the statistical significance of all comparisons in this study. Parametric tests assumed normality of data distribution and were not formally tested. One-way analysis of variance and Tukey's multiple comparison test were used for comparisons between groups. Statistical significance for all analyses was based on P < 0.05.

[0079] Example 1 Preparation and characterization of sprayable and in situ forming fibrin gels

[0080] F-gel was prepared by spraying equal volumes of fibrinogen (10 mg / mL) and thrombin (10 U / mL), and a film-like hydrogel was rapidly formed in a dish or on the palm of the hand within 120 seconds ( Figure 1 a). The overall formation of the hand gel indicated that the skin adhesion was very ideal. The characteristic C=O and PO peaks in the Fourier transform infrared spectrum of the F-gel sample confirmed that TQ and NADH were successfully co-loaded with drugs. Scanning electron microscopy (SEM) showed that the F-gel had a distinct porous fibrin network structure ( Figure 1 b), indicating that it has good air permeability and drug loading capacity. The rheological test results show that the storage modulus (G′) and loss modulus (G″) of F-gel increase significantly over time, indicating that fibrinogen and thrombin are successfully gelled ( Figure 1 c). In addition, in the entire frequency domain, the G′ of F-gel is always greater than G″, indicating that the hydrogel has good properties ( Figure 1 d). From the release time curves of TQ and NADH in F-gel, it can be seen that about 75% of TQ was rapidly released within 20 minutes, while NADH was released from NADH@MSNs in F-gel prepared with PBS within 4 hours, indicating that it has a sustained release effect ( Figure 1 e).

[0081] The absorption spectrum of TQ is mainly in the blue and ultraviolet regions ( Figure 1f), which has a good intersection with the bLED chip, so the bLED and TQ can be combined. At the same time, the absorption spectrum of NADH does not overlap with the spectrum of either the rLED or bLED chip, ensuring that it will not be quenched and inactivated after irradiation by r&bLED ( Figure 1 f). In addition, the excitation spectrum of TQ shows a peak at 481nm, which is close to the peak of the bLED chip at 470nm, while the excitation spectrum of NADH is not observed in the range from blue light to near-infrared region ( Figure 1 g). The prepared F-gel still retains the fluorescence properties of TQ, which can be specifically excited by bLED but not by rLED ( Figure 1 h).

[0082] The generation of hydroxyl radicals (-OH) and hydrogen peroxide (H2O2) was further identified as the main causes of the bactericidal activity. Figure 1 i) and -OH( Figure 1 j) is significantly increased when exposed to blue light than when not exposed to blue light. Blue light may also induce HO 22 Photolysis further increases the generation of -OH, thereby improving the bactericidal effect, because -OH is more toxic to bacteria than HO. 22 20,25 With the increase of H2O2 concentration and the extension of bLED exposure time, the level of -OH increased significantly. Electron paramagnetic resonance (EPR) further detected the generation of various ROS, -OH and superoxide radicals (O2- - ) show obvious signals, while singlet oxygen ( 1 O2) has no obvious signal ( Figure 1 k), indicating that a predominantly hypoxia-tolerant type I photodynamic response occurred.

[0083] The transparency of F-gel is an important parameter for phototherapy effect. About 93% of red light and 85% of blue light can penetrate through F-gel with a thickness of 4 mm, showing good light transmittance ( Figure 1 l). In addition, F-gel can evenly cover the surface of r&bLED patch and can be easily peeled off as a whole, which proves its compatibility with r&bLED patch in joint application ( Figure 1 m and Figure 1 n).

[0084] The ROS generated by TQ under bLED excitation may cause harm to normal tissues. In order to mitigate this harmful effect, NADH was added to F-gel to prevent cell dysfunction and cell death caused by inflammation. Figure 1In fact, bLED irradiation significantly reduced cell viability in the presence of TQ@F-gel, whereas cell viability was well preserved when NADH was added to F-gel and cultured before bLED irradiation ( Figure 1 p). ROS detection further confirmed the antioxidant effect of NADH on cells during PDT, thereby minimizing the damage to normal tissues during treatment ( Figure 1 q and Figure 1 r). F-gel is easy to use, has a large drug loading capacity, good light transmittance, and can be sprayed and molded, so it has great potential for home wound care when used in conjunction with r&bLED patches.

[0085] Example 2 Verification of the in vitro antibacterial effect of the combination of r&bLED and F-gel

[0086] Four common strains associated with chronic wounds were used, including G + MRSA (Methicillin-resistant Staphylococcus aureus), G - Three multidrug-resistant bacteria, Pa (Pseudomonas aeruginosa) and Ab (Acinetobacter baumannii), and Ca (Candida albicans) from the ATCC cell bank. Figure 4 As shown in a, PBS was added for 10 7 CFU / mL of microbial suspension was added to a 35 mm dish along with TQ F-gel at final concentrations of 25, 50, 75, and 100 μg / mL, and then incubated for 20 minutes before irradiating the bottom of the dish with an r&bLED spot. - It is generated in large quantities under bLED irradiation and further reacts to generate HO2 · and H2 O2, which are then photolyzed into highly toxic -OH( Figure 2 a). Figure 2 As shown in b, when the TQ concentration was 100 μg / mL and the LED irradiation intensity was 30 J / cm 2 When the concentration is 10 7MRSA, Pa, and Ab were completely eradicated at CFU / mL, while Ca was reduced by 6.6lg CFU / mL. The Bliss independence model further verified the synergistic eradication of MRSA, Ab, Pa, and Ca, with S values ​​of all combinations exceeding 0, and the synergistic effect level increased with increasing bLED doses and TQ concentrations ( Figure 2 c). Among the 80 combinations tested, the mean and median values ​​were 0.410 and 0.334, respectively, indicating that the combination therapy has a synergistic effect ( Figure 2 d).

[0087] According to SYTO TM 9 and propidium iodide (PI) staining, the combination of bLED and F-gel was more effective in disrupting the cell envelope than either method alone. For MRSA, Pa, Ab, and Ca, PI-positive (PI + ) cells increased to 96%, 97%, 99% and 93%, respectively. In contrast, the PI + Scanning electron microscopy and transmission electron microscopy (TEM) confirmed the ultrastructural changes of the cells, showing that the cell envelope of MRSA, Pa, Ab, and Ca was broken or damaged ( Figure 2 e and Figure 2 f). Cell envelope damage (arrows) and cytoplasmic leakage (asterisks) of a large number of MRSA and Pa indicate that both the cell wall and cell membrane may be damaged. In contrast, the cell envelopes of untreated MRSA, Pa, Ab, and Ca were intact and smooth. In addition, ROS-specific 2',7'-dichlorofluorescein (DCF) ( Figure 2 g) or -OH-specific hydroxyphenylfluorescein (HPF) ( Figure 2 h) The combined yellow color produced by the combination with PI staining provides evidence for the formation of ROS or -OH inside MRSA and Pa, followed by a lethal dose of the combined treatment, indicating that cell death was mainly caused by ROS generated by the combined treatment. More importantly, in the presence of the -OH scavenger DMSO or the O2- scavenger Tiron, the bactericidal ability of the combined treatment was significantly reduced, while as 1 The presence of NaN3, an O2 scavenger, did not significantly affect the antibacterial effect, further demonstrating that the antibacterial effect is generally triggered by a type I photodynamic response. Similarly, mature biofilms of MRSA and Pa were completely eliminated after 20 minutes of bLED irradiation in combination with F-gel ( Figure 2 i), HPF and PI staining in the biofilms of MRSA and Pa further demonstrated the generation of -OH under bLED irradiation.

[0088] Transcriptome sequencing (RNA-seq) analysis was performed on Pa, a typical skin infection bacterium, to further elucidate the mechanism of synergistic antibacterial effect. A total of 1055 genes were significantly expressed in both groups (fold change ≥ 2 and q < 0.05) ( Figure 2 j). Figure 2 As shown in k, the Gene Ontology (GO) database was used to identify genes with significant differential expression levels of biological functions, and it was found that terms related to cellular components such as plasma membrane, cytoplasm and ribosomes were increased (green rectangles), indicating that the combined treatment had a significant effect on the integrity of cellular components. In addition, the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and gene set enrichment analysis (GSEA) showed that genes related to bacterial chemotaxis (such as nahY, STM3577, EAE_15525, PP_1228, PA0175, PA1459), flagellar assembly (such as PA1104, STM1183, b1942, BSU13690, BSU29720) and biofilm formation (such as MXAN_5788, PA3622, PA3105, PA3103, TTHA1437) were downregulated, indicating that the pathogenicity of Pa was reduced after the combined treatment. Therefore, the combination of r&bLED and F-gel provides a convenient, rapid and effective sterilization method for treating infected wound healing at home.

[0089] Example 3: Verification of the combination of r&bLED and F-gel to improve MRC and accelerate angiogenesis

[0090] Red or near-infrared light can activate complex IV (CIV) on the mitochondrial respiratory chain (MRC), increasing the production of adenosine triphosphate (ATP) in a manner that is less dependent on oxygen levels. NADH is a hydrogen donor that plays a vital role in maintaining the integrity of the electron transport chain (ETC) and MRC. PBM and NADH have been shown to work synergistically in improving mitochondrial function. In fact, the combination of rLED and NADH can synergistically enhance ETC flux and MRC by rebuilding the NAD(H) pool and increasing CIV activity, further increasing ATP production, thereby promoting tube formation, migration and proliferation of endothelial cells ( Figure 3 a). In addition, under inflammatory stress conditions, combined treatment can alleviate cellular oxidative stress, reduce the cellular NAD / NADH ratio, and prevent the loss of mitochondrial membrane potential (MMP), thereby avoiding cell apoptosis.

[0091] The energy parameter selection of PBM therapy is crucial to its efficacy. The application of low energy density optical dose often leads to reduced efficacy, while too high energy may lead to adverse treatment effects. 36,38 To determine the ideal energy density, we performed energy gradient experiments. 3 J / cm 2rLED irradiation of (rLED-M) enhanced the activity of CⅣ and the production of ATP, while when the power density was 5 J / cm 2 (rLED-H), the efficacy decreased significantly, indicating that 3 J / cm 2 is the optimal power density for PBM. In addition, F-gel can effectively replenish the cellular NAD(H) pool and increase the ATP production of HUVECs, while the activity of CIV does not change significantly ( Figure 3 b). rLED-M and F-gels can significantly restore the NAD(H) pool of cells, increase the enzyme activity of CⅣ in MRC, and increase NAD + / NADH and ATP production, indicating that r&bLED patch and F-gel can produce excellent synergistic effects on the mitochondrial function of HUVECs regardless of the presence or absence of LPS.

[0092] To further confirm the synergistic effect of rLED-M and F-gel, we performed EdU proliferation assay and wound scratch assay after different treatments ( Figure 3 c and Figure 3 d) EdU in the rLED-M combined with F-gel group + The cell ratio and wound healing rate were significantly the highest in each group, indicating that cell proliferation and migration abilities were enhanced ( Figure 3 f and Figure 3 g). Figure 3 e and Figure 3 As shown in h, the capillary length increased significantly after combined treatment, indicating enhanced tube formation ability.

[0093] like Figure 3 As shown in b, when exposed to LPS, cellular NAD(H) levels and CIV activity decreased dramatically, leading to oxidative stress ( Figure 3 i and Figure 3 j) and MMP (ΔΨM) decreased ( Figure 3 k and Figure 3 l), indicating mitochondrial dysfunction in HUVECs. The combined therapy prevented the depletion of cellular NAD(H) pool and energy and the decrease in ΔΨM under LPS stimulation, demonstrating its protective effect on HUVECs under inflammatory conditions ( Figure 3 i and Figure 3 l).

[0094] To investigate the effects of combined therapy on HUVECs inflammation, confocal laser scanning microscopy (CLSM) was used to observe the activation of NF-κB p65. Figure 3 m). After rLED-M combined with F-gel treatment, LPS-induced P65 nuclear translocation was significantly reduced, and the efficacy of the combined therapy was also improved ( Figure 3n). Improved mitochondrial function may prevent apoptosis, a common response of endothelial cells to inflammation. Figure 3 o and Figure 3 As shown in r, the combined therapy effectively inhibited LPS ( Figure 3 o and Figure 3 p) and TNF-α( Figure 3 q and Figure 3 r), indicating that its therapeutic efficacy can be extended to a variety of inflammatory mediators. The above results show that the combination of r&bLED and F-gel can synergistically promote tube formation, cell proliferation and migration, and prevent inflammation-induced cell apoptosis by improving the ETC and MRC of HUVECs, indicating that they have a positive role in accelerating angiogenesis in infected wounds.

[0095] Example 4 r&bLED combined with F-gel to regulate macrophage polarization

[0096] Monocytes / macrophages (MΦs) play a crucial role in the innate ability to restore tissue function after injury. Energy-based metabolic reprogramming regulates macrophage polarization. Figure 6 a and Figure 6 As shown in b.

[0097] Extracellular acidification rate (ECAR, a measure of lactate production reflecting the rate of glycolysis) and oxygen consumption (OCR, a readout of oxidative phosphorylation) are the main indicators reflecting macrophage metabolism. When THP-1 cells were incubated with LPS (100 ng / mL) without additional treatment, ECAR increased dramatically, indicating a glycolytic burst. However, when cells were pre-irradiated with rLED or F-gel 2 h before LPS incubation, ECAR did not rise as significantly as in the G2 group, while the combined treatment showed the most favorable results, reducing ECAR levels to almost the level of the control group (G1 group) ( Figure 4 c). Similarly, the OCR of the G2 group dropped sharply after incubation with LPS, indicating a reduction in OXPHOS, whereas the combined treatment enhanced OXPHOS and prevented mitochondrial dysfunction, restoring the OCR to normal or higher levels ( Figure 4 c). To further investigate the effect of combined treatment on mitochondrial function in macrophages, NAD(H) and NAD + / NADH ratio. Figure 4 As shown in d, F-gel can significantly prevent the depletion of cellular NAD(H) reservoirs caused by LPS stimulation, and the effect of combined treatment is even better. + / NADH ratio, indicating that the cellular redox state was improved. In addition, the NO content, which can inhibit the activity of CⅣ in ETC, showed that rLED and F-gel could significantly inhibit the generation of NO ( Figure 4 e, left) and enhance CⅣ activity ( Figure 4 e, right). These results suggest that combined treatment can achieve energy-based metabolic reprogramming of macrophages.

[0098] The nuclear translocation of NF-κB p65 and the expression of iNOS in THP-1 cells (up-regulated) and RAW264.7 cells (down-regulated) were observed by CLSM to further investigate the effects of the combination of the two on inflammasome gene transcription. Figure 6 f and Figure 6 g As shown in Figure 2, rLED and F-gel treatment significantly reduced LPS-induced p65 nuclear translocation in both cell lines. In addition, iNOS, a marker of M1 macrophages, 49 The expression of Figure 6 h and Figure 6 i), THP-1( Figure 6 j) and RAW264.7 cells, the expression of inflammatory factors such as IL-1β, IL-6 and TNF-α was also significantly reduced. The anti-inflammatory effect was further verified by detecting CD206 and CD86 in THP-1 and RAW264.7 cells by flow cytometry. These results indicate that the combined therapy regulates the repolarization of macrophages to the M2 phenotype and inhibits inflammation through the energy metabolism-immunomodulatory pathway, which is beneficial to the healing of chronic wounds.

[0099] Example 5 In vivo efficacy of enhancing wound healing in diabetic mice infected with MRSA

[0100] Verify the therapeutic effect of the combination therapy in wounds infected with diabetic mice. Figure 5 As shown in a, including STZ ip injection, wound treatment, BALB / c mice inoculation with lux MRSA or Pa, and then various treatments. The infected diabetic mice were randomly divided into five groups: G1, control group; G2, F-gel group; G3, rLED+F-gel group; G4, bLED+F-gel group; G5, r&bLED+F-gel group. The progression of skin infection was tracked in real time by bioluminescent MRSA and Pa using IVIS. The bioluminescent MRSA strain was from ATCC 33591, while the bioluminescent Pa was genetically modified with the lux pGEX-Apr operator to emit bioluminescence, and the expression of the operator and the linear correlation between bioluminescence intensity and CFU (R 2 =0.9800) was verified before the test. On the second day of wound infection, the wounds were treated according to their respective groups ( Figure 5 b). bLED exposure for 12 minutes (36J / cm 2 ) completely eliminated the 8.3lg luminescence of MRSA and the 8.0lg luminescence of Pa, while F-gel or F-gel containing rLED did not significantly reduce the luminescence ( Figure 5 b and Figure 5 c). At the end of the experiment, the bactericidal activity was further confirmed by observing the corresponding decrease in CFU in the infected wound ( Figure 5 b). In addition, we failed to detect any viable bacteria in the wounds after 12 days of combined treatment, implying that the synergistic effect of r&bLED and F-gel completely cured the acute MRSA infection ( Figure 5 d).

[0101] On days 8, 12, and 16, the wound healing progress of mice in group G5 was the most significant, with the smallest visible scars compared to the other groups. In contrast, the control group still had obvious scabs ( Figure 5 e). The wound healing rate curve further confirmed that the wound in the G5 group healed better than the other groups and was almost completely closed, especially when compared with the control group ( Figure 5 f), which suggests that the combined therapy can effectively promote the healing of diabetic infected wounds.

[0102] The formation of granulation tissue is key to effective wound healing. Histological examination using H&E staining showed that the thickness of granulation tissue increased significantly after combined therapy compared with the control group ( Figure 7 g and Figure 7 h). In addition, on days 8 and 16, the collagen deposition in the G5 group was higher than that in the other groups ( Figure 7 i and Figure 7 j), indicating better wound healing quality.

[0103] Histological analysis was also performed using immunofluorescence staining of CD31 and α-SMA to verify the effect of the combined treatment on angiogenesis in vivo. A significant increase in the number of blood vessels was observed in the G5 group on day 8 compared with the other groups ( Figure 5 k and Figure 5 l). These results suggest that the combined treatment can effectively promote angiogenesis and accelerate wound healing. In addition, the number of wound vessels decreased on day 16 compared with day 8, indicating that the typical angiogenesis pattern in the wound healing process transitions from the proliferation phase to the remodeling phase.

[0104] It was also found that on the 8th and 16th days, NAD +The ratio of NADH / NADH was significantly higher than that of the other groups, and CⅣ activity showed a similar trend, indicating that the combined therapy enhanced mitochondrial function in wound tissue. The enhancement of mitochondrial function was further verified by evaluating the ATP level in the tissue, indicating that the combined therapy promoted the healing of MRSA-infected diabetic wounds through energy supplementation.

[0105] Example 6 Effect of r&bLED combined with F-gel for in vivo inflammation regulation

[0106] Diabetic wounds are characterized by long-term low-grade inflammation, typically characterized by the presence of M1 macrophages and proinflammatory cytokines (including IL-6 and TNF-α), which impede the progress of wound healing. In relation to excessive wound inflammation, the secretion of proinflammatory cytokines TNF-α and IL-6 was higher in the control group wounds, while it decreased significantly in G3 and G5, but not significantly in G2, suggesting that the anti-inflammatory effect of the combined treatment was better ( Figure 6 a) In addition to pro-inflammatory cytokines, the phenotype of macrophages recruited to the injury site is also an important indicator of inflammatory response. CD86 and CD206 were selected as typical surface markers of M1 or M2 macrophages, respectively. Figure 6 As shown in b, the combination of r&bLED and F-gel significantly increased the number of M2 macrophages (CD206 + ), with greater effects observed on days 8 and 16 compared with the other groups. In contrast, combined treatment reduced M1 macrophages (CD86 + ) ratio.

[0107] Transcriptome analysis of wound tissues was performed on day 8, overlapping the inflammatory and proliferative phases to further evaluate the mitigation of chronic inflammation by the combination therapy. 376 differentially expressed genes (fold change ≥ 2 and q < 0.05) were identified between G1 and G5 ( Figure 6 c). GO enrichment analysis showed that there was a decrease in genes related to inflammatory response (such as CCL7, CCR1, CD14, CXCL1, CXCL3, CXCL5, Elf3, Fpr1, IL18rap), innate immune response (such as Cd14, Cfi, Clec4e, Ifitm1, Krt16, Mefv, Nlrp3, Oas3, Rarres2), cytokine activity (such as IL11, IL1rn, IL24, IL18rap, IL1rn, IL24, IL6, Inhba, Lif, Osm, Spp1, Timp1) and CXC chemokine receptor activity (such as CXCR1, CXCR2, Gpr35) which is mainly related to inflammation. ( Figure 6 d)

[0108] Genes related to positive regulation of cellular respiration (such as Oas1a, Oas1e, Oas1f, Oas1g) were enriched in both GO analysis and GSEA ( Figure 6 e), further demonstrating the enhancement of mitochondrial function in tissues. In addition, GSEA showed that genes associated with activated macrophages (such as Wnt5a, Cebpa, Il10, Pla2g4a, Kars, Lgals9) were downregulated ( Figure 6 f). In addition, GSEA and KEGG enrichment showed decreased expression of TNF-α (such as IL1b, Map3k14, CXCL3, Ptgs2, Map2k1, Nfkbia, Nod2, Casp3), IL-6 (such as IL6, Crlf1, Osm, CLCF1, Lif, IL11, Socs3) and NF-κb signaling pathways (such as Cd14, Cxcl1, Cxcl3, Nfkbia, Ptgs2, Tnfsf11, Tnfsf14) ( Figure 6 g. Figure 6 h and Figure 6 i), which is consistent with the results of histological examination and biochemical analysis ( Figure 5 GSEA also revealed downregulation of HIF signaling pathways (such as NOS2, NOS3, Hmox1, Map2k1, Pgk1, Hk2, HIF1a), indicating improved angiogenesis after combined treatment ( Figure 6 j). These results suggest that r&bLED combined with F-gel can effectively inhibit inflammation and promote wound healing in vivo via the energy metabolism-immunity pathway.

[0109] Example 7 r&bLED and F-gel promote wound healing in infected diabetic mice

[0110] A porcine wound healing model was used that closely resembles the human wound healing process. STZ was injected intravenously into Panamanian piglets to induce diabetes. After monitoring blood glucose for a week to confirm the diabetic state, a series of circular full-thickness wounds with a diameter of 1.5 cm were created on both sides of the pig's back ( Figure 7 a). Then, 100 μL of 1×10 9 CFU / mL of MRSA suspension was used to induce acute skin infection. The infected wounds were then randomly divided into five groups. On the second day of wound infection, the wounds were treated differently and then every two days until the eighth day.

[0111] The wounds of G1, G2, and G3 showed purulent exudate and delayed crust formation on day 4, consistent with infection ( Figure 7b). Meanwhile, wounds in G4 and G5 were dry and scab formation was accelerated, indicating that wounds healed faster with the help of bLED irradiation and F-gel. In addition, granulation tissue growth was accelerated in the G5 group, epithelialization progressed rapidly, and scab shedding was effortless, thus achieving complete wound healing earlier. In contrast, in the G1 group, epithelial migration was slow and scab adhered tightly, hindering its natural shedding. To further demonstrate the improvement of mitochondrial function after combined treatment, we expressed NAD in pig wound tissue. + The ratio of NAD / NADH and CIV activity were evaluated. + The ratio of NADH / NADH and CⅣ activity were significantly higher than those in the other groups, indicating that the combined therapy improved mitochondrial function in wound tissue ( Figure 7 c Figure 7 d).

[0112] In addition, wounds treated with F-Gel and r&bLED showed accelerated wound closure and the thickest granulation tissue at both time points examined ( Figure 7 e). On days 8 and 20, the extent of re-epithelialization in G5 wounds increased, and by day 20, all wounds were almost completely re-epithelialized.

[0113] Compared with the other groups, the collagen deposition ratio of the G5 group was higher on both days 8 and 20 ( Figure 7 f). On days 8 and 20, immunostaining of CD31 and α-SMA was enhanced, indicating that the combined treatment promoted angiogenesis, which was significantly different from G1 and G2 on day 8 ( Figure 7 g).

[0114] Transcriptome analysis of wound tissues was performed on day 8 to further investigate the mechanism by which the combination therapy promoted wound healing in the porcine infected wound model. 1754 differentially expressed genes (fold change ≥ 2, q < 0.05) were identified between G1 and G5 ( Figure 7 h). GO enrichment analysis revealed down-regulation in genes related to inflammation, including inflammatory response(like ACOD1,ADAM8,AMCF-II,C5AR1,CCL19,CCL2,CCL20),CC chemokine receptor activity(like ACKR3,CCR1,CCR2,CCR6,CCR7,CXCR4), NAD +Nucleotidases (such as BST1, CD38, IL18RAP, TLR10, TLR2) and CC chemokine binding (such as ACKR3, CCR1, CCR2, CCR6, CCR7) once again confirmed the anti-inflammatory effect of the combination therapy (green rectangle) at the molecular level ( Figure 7 i).

[0115] Genes related to tissue repair were upregulated, including keratinization genes (e.g., KRT1, KRT2, KRT3, KRT5, KRT74, KRT77, KRT79), extracellular matrix genes (e.g., ACAN, ADAMTS18, ADAMTS8, ANOS1-2, CCBE1), and skin epidermal structural component genes (e.g., KRT77, KRT79, KRT82, KRT84, LOC100511079, LOC1005240) (green rectangles)( Figure 7 j). This further demonstrated the enhancement of wound healing after combined treatment. In addition, GSEA enrichment showed that genes associated with inflammation were downregulated, while genes associated with tissue repair were upregulated, which was consistent with the results of GO enrichment. These findings in mini pigs provide convincing evidence that r&bLED and F-gel effectively fight microbial infection and promote diabetic wound healing by regulating the energy metabolism-immunity pathway.

Claims

1. A composition comprising at least one of TQ and NADH.

2. The composition according to claim 1, which is a fibrin gel, characterized in that The gel carrier is formed by mixing fibrinogen and thrombin.

3. The composition according to claim 2, characterized in that The TQ loaded in each milliliter of the gel is 50 μg to 500 μg.

4. The composition according to claim 2, characterized in that The NADH loaded in each milliliter of the gel is 10 μg to 100 μg.

5. The composition according to claim 2, characterized in that The NADH is loaded in the MSN nanoparticles.

6. The composition according to claim 2, characterized in that Ca ions are also included.

7. Use of the composition according to claim 1 in preparing medical devices for promoting wound repair and healing.

8. A medical device, characterized in that The invention comprises a device for implementing phototherapy and the composition according to claim 1.

9. The medical device according to claim 8, characterized in that The phototherapy device emits at least one of red light and blue light.

10. The medical device according to claim 8, characterized in that The composition is located on the optical path of the light emitted by the phototherapy device.

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