Photothermal composite hydrogel patch and preparation method and application thereof

Abstract

The application discloses a kind of photothermal composite hydrogel patches and its preparation method and application, the hydrogel patch is with methacrylated gelatin (GelMA) and methacrylated hyaluronic acid (HAMA) as matrix, load carbonized niobium nanoparticles (Nb2CNPs) and verteporfin (VP), and is formed by 3D printing or casting forming three-dimensional grid porous structure, is abbreviated as VP-Nb2C@GelMA / HAMA (VNGH) hydrogel patch.The VNGH has good photo-thermal antibacterial, antioxidant, and re-epithelialization function, can significantly improve the quality of mouse diabetic wound healing, and provides a new idea and strategy for realizing scarless healing of diabetic wound.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of materials, in particular to a photothermal composite hydrogel patch and a preparation method and application thereof. BACKGROUND

[0002] Wounds that fail to heal completely, achieve anatomical and functional integrity, and exceed 1 month are generally defined as chronic wounds. Common types of chronic wounds include venous ulcers, arterial ulcers, pressure ulcers, and diabetic foot ulcers. Delayed healing of diabetic wounds can be attributed to high local blood glucose concentration, vascular lesions, neuropathy, bacterial infection, and tissue hypoxia. The main factors related to delayed wound healing in diabetic patients include sustained high blood glucose, impaired angiogenesis, imbalance of extracellular matrix metabolism, infection, and inflammation. A long-term high glucose environment increases the accumulation of advanced glycation end products (AGEs) in tissues. AGEs bind to receptors to activate the NF-κB pathway, triggering an inflammatory response and exacerbating tissue damage. High glucose levels downregulate VEGF expression by inhibiting the HIF-1α pathway, leading to impaired angiogenesis, insufficient oxygen and nutrient supply to the wound, and thus exacerbating poor healing of diabetic wounds. In addition, bacterial infection is one of the biggest challenges for chronic wound healing. The proliferation of microorganisms can induce increased tissue damage, prolonged inflammation, and delayed wound healing. To prevent wound infection, antibiotics are widely used in clinical practice. However, overuse or misuse of antibiotics has accelerated the development of bacterial resistance and the emergence of multi-drug resistant pathogens. The development speed of antibiotics lags far behind the speed of bacterial resistance mutations, so it is necessary to develop new antibacterial agents to prevent bacterial resistance.

[0003] A safe and effective antibacterial treatment that is less likely to lead to bacterial resistance has become a global focus.

[0004] To solve the above problems in the prior art, it is necessary to develop a hydrogel patch for repairing diabetic wounds. SUMMARY

[0005] The present application aims to provide a preparation method and application of a photothermal composite hydrogel patch, which has good photothermal antibacterial properties, antioxidant properties, and re-epithelialization promotion function, can significantly improve the quality of mouse diabetic wound healing, and provides a new idea and strategy for achieving scarless healing of diabetic wounds.

[0006] To achieve the above-mentioned purpose, the present application adopts the following technical solutions:

[0007] In a first aspect of the present application, a photothermal composite hydrogel patch is provided, comprising:

[0008] A three-dimensional grid-shaped porous structure, referred to as VP-Nb2C@GelMA / HAMA (VNGH) hydrogel patch, is formed by 3D printing or casting molding using methacrylated gelatin (GelMA) and methacrylated hyaluronic acid (HAMA) as the matrix, loading carbonized niobium nanoparticles (Nb2CNPs) and verteporfin (VP).

[0009] Further, the pore size of the photothermal composite hydrogel patch is 150-250 μm, and the porosity is 30%-50%.

[0010] Further, the mass ratio of GelMA to HAMA is 350-450:1.

[0011] In the second aspect of the present application, a preparation method of the photothermal composite hydrogel patch is provided, which comprises:

[0012] Dissolve GelMA, HAMA, and photoinitiator LAP in PBS, then add Nb2CNPs dispersion and VP solution, mix and filter; dissolve GelMA in a solution containing photoinitiator LAP, sequentially add HAMA, Nb2CNPs dispersion, and VP solution, mix and filter.

[0013] Through 3D printing or casting molding, a VP-Nb2C@GelMA / HAMA (VNGH) hydrogel patch is obtained.

[0014] Further, the photoinitiator LAP (full name Lithium Phenyl-2,4,6-trimethylbenzoylphosphinate) is a photosensitive compound used to initiate photocuring reaction in hydrogel preparation. Its specific role is to generate active free radicals under specific wavelength light, promoting crosslinking polymerization of GelMA and HAMA matrix to form a stable three-dimensional structure.

[0015] Further, the concentration of GelMA dissolved in PBS is 5%-15% (w / v), the concentration of HAMA dissolved in PBS is 0.01%-0.05% (w / v), and the volume ratio of GelMA to HAMA is 350-450:1.

[0016] As a preferred embodiment, the concentration of GelMA dissolved in PBS is 5%-15% (w / v), which can balance the flowability and printing precision within this range.

[0017] More preferably, the concentration of the GelMA after dissolved in PBS is 10%, and the 3D printing mixed hydrogel patch with a GelMA concentration of 10% has certain adhesion and ductility, and is not limited by the location of the wound and the flatness of the skin when applied to the wound. After the inventors of the application prepared various groups of hydrogel solutions required for 3D printing, the printing performance of the hydrogel solutions under different GelMA concentrations was observed. The 3D printing parameters were set to a square with a side length of 10 mm, and the filling density was 40%. The test printing results showed that the hydrogel mixed solution with a GelMA concentration of 5% and 8% had low viscosity and good fluidity, but the material in the syringe was easy to overflow from the nozzle under the action of air pressure, and it was difficult to control the line width. The hydrogel mixed solution with a GelMA concentration of 15% had high viscosity and poor fluidity, and it was easy to appear uneven extrusion or nozzle blockage during printing, which was manifested as intermittent lines and reduced printing accuracy. Therefore, considering the fluidity and printing precision, the hydrogel mixed solution with a GelMA concentration of 10% was selected for subsequent 3D printing and experiments.

[0018] Further, the working concentration of Nb2CNPs in the Nb2CNPs dispersion liquid is 500-1500 μg / mL.

[0019] Further, in the VP solution, the working concentration of VP is 3-500 μg / mL.

[0020] Further, 405 nm light is used for 3D printing.

[0021] In the third aspect of the application, the application of the composite hydrogel in a dressing for treating diabetic wounds is provided.

[0022] The one or more technical solutions in the embodiments of the application have at least the following technical effects or advantages:

[0023] 1. The photothermal composite hydrogel patch provided by the application, the VNGH hydrogel scaffold obtained by 3D printing has good swelling, water retention and degradation performance, and can absorb 10 times the weight of liquid within 24 hours, and the loose and porous three-dimensional network structure can meet the characteristics required by ideal wound dressings, such as good air permeability, maintaining the wet environment of the wound, absorbing excess exudate, and appropriate degradation capacity. At the same time, the drug release curve results show that about 55% of VP is released within 48 hours, and VP can still be continuously released until the 8th day.

[0024] 2. Invention Innovation: This invention is the first to integrate Nb2C NPs and VP into a dual-matrix mesh structure, resolving the vicious cycle of "infection-oxidative stress-fibrosis" in diabetic wounds through triple synergy. Photothermal antibacterial action and controlled drug release are synergistically achieved: Nb2C NPs photothermal effect: Under 808 nm laser irradiation, the VNGH group heats up to 65.5℃ in 3 minutes (…). Figure 7 -B), which improved the antibacterial rate by 35% compared to the NGH group (48.6℃), achieving a >99% antibacterial rate (Staphylococcus aureus inhibition zone 18 mm, Figure 11 Animal experiments showed that VNGH can effectively promote wound healing in diabetic mice. Hair follicle regeneration density: 40 follicles / mm² 2 ( Figure 16 -B), far exceeding the current level of treatment for diabetic wounds. Attached Figure Description

[0025] Figure 1 Morphological characteristics and elemental mapping of Nb₂C NPs. A. Representative TEM image of Nb₂C; B. Representative SEM image of Nb₂C; C. EDS energy dispersive spectroscopy analysis of Nb₂C.

[0026] Figure 2 The structural characteristics of Nb₂C NPs are as follows: A. X-ray diffraction pattern of Nb₂C; B. Zeta potential of Nb₂C; C. Particle size analysis of Nb₂C.

[0027] Figure 3 The photothermal heating effect of Nb₂C NPs. A. In vitro thermal imaging of Nb₂C; B. Photothermal heating curve of Nb₂C.

[0028] Figure 4 The morphological characteristics of each group of hydrogel patches are shown.

[0029] Figure 5 The following are the FTIR spectra of each group of hydrogels.

[0030] Figure 6 Rheological properties characterization of each group of hydrogels: A. Strain scan of GH and VNGH; B. Dynamic step strain of GH and VNGH; C. Frequency scan of GH and VNGH; D. Shear viscosity test of GH and VNGH.

[0031] Figure 7 Photothermal heating effect of Nb2C and each group of hydrogels. A. In vitro thermal imaging of Nb2C solution (1000 μg / mL) and each group of hydrogel solutions; B. Photothermal heating curves of Nb2C solution (1000 μg / mL) and each group of hydrogel solutions.

[0032] Figure 8 This is the drug release curve of VNGH.

[0033] Figure 9 Cell survival rate of NIH-3T3 cells after being treated with different concentrations of VP for 24 h.

[0034] Figure 10 In vitro healing-promoting ability of each group of hydrogels; A. Cell migration of NIH-3T3 cells after being treated with each group of hydrogel extracts (5 μg / mL) for 0, 24, and 48 h; B. Quantitative statistical analysis results after being treated for 24 h, *P<0.05, **P<0.01.

[0035] Figure 11 In vitro antibacterial ability of each group of hydrogels (filter paper diameter: 6 mm).

[0036] Figure 12 Schematic diagram of grouping of treatment methods for wounds of diabetic mice.

[0037] Figure 13 Representative gross images (A) and wound healing pattern diagrams (B) of wounds of diabetic mice on days 0, 3, 7, 11, and 14 under different treatment methods; the inner diameter of the white circle in the diagram is 15 mm, and the scale bar is 5 mm.

[0038] Figure 14 Semi-quantitative analysis of wounds of diabetic mice under different treatment methods. A. Change in residual wound area; B. Wound healing rate (n=5; ****P<0.0001; ns, no statistically significant difference).

[0039] Figure 15 Photothermal effect of hydrogels on wounds of diabetic mice, A. Infrared thermal imaging; B. Local temperature rise curve of hydrogels.

[0040] Figure 16 Representative HE staining results of wounds of diabetic mice on day 14 after modeling: (A) important organs; (B) wound tissue.

[0041] Figure 17 Representative Masson staining results of wound tissue of diabetic mice on day 14 after modeling of wounds. DETAILED DESCRIPTION

[0042] The advantages and various effects of the embodiments of the present application will be more clearly presented by the following specific embodiments and examples. Those skilled in the art should understand that these specific embodiments and examples are used to illustrate the embodiments of the present application, rather than limit the embodiments of the present application.

[0043] Throughout the specification, unless otherwise specifically indicated otherwise, the terms used herein are understood to be as commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the embodiments of the present application belong. If there is a conflict, the present specification takes precedence.

[0044] Unless otherwise specifically indicated, the various raw materials, reagents, instruments and equipment used in the embodiments of the present application can be purchased on the market or obtained by existing methods.

[0045] The overall idea of the present application to solve the above technical problems is as follows:

[0046] We successfully synthesized Nb2C NPs with uniform particle size, good photothermal stability and uniform and stable dispersion in the medium. Through 3D printing performance test, it is determined that the concentration of GelMA is 10%, the concentration of VNGH is 10% and the concentration of VP is 1000 μg / mL.

[0047] GelMA: HAMA = 400:1 as the basic mixed hydrogel. Through infrared, ultraviolet, thermogravimetric and other tests, it is confirmed that the composition and structure of VNGH hydrogel are stable, and it has good thermal stability. The results of rheological test show that VNGH has good self-healing, adhesion and injectability. Through swelling, water retention and degradation property test, the feasibility of hydrogel patch as diabetic wound dressing has been verified. Through photothermal performance characterization, it is determined that the best experimental condition is to irradiate the Nb2C NPs hydrogel patch containing 1000 μg / mL with near-infrared laser with a wavelength of 808 nm and a power of 1.0 W / cm 2 The drug release curve shows that the hydrogel system can achieve VP slow release. In addition, the in vitro biocompatibility, wound healing ability and antibacterial ability of the hydrogel patch have also been verified. In summary, we successfully prepared a composite hydrogel scaffold patch (VNGH) loaded with VP and with good photothermal antibacterial properties.

[0048] In the in vivo experiment, the HE staining results of the important organs of mice heart, liver, spleen, lung and kidney show that the hydrogel material has reliable biological safety. The animal experiment results show that VNGH can effectively promote the wound healing of diabetic mice, and has excellent photothermal antibacterial ability under 808 nm laser, which can effectively promote the re-epithelialization, granulation tissue regeneration and the appearance and formation of skin appendages (hair follicles and sebaceous glands) of diabetic wounds, and realize the rapid recovery of wound structure and function.

[0049] The photothermal composite hydrogel patch of the present application will be described in detail below in combination with examples and experimental data.

[0050] Example 1, synthesis of photothermal composite hydrogel patch

[0051] 1. Preparation of Nb2C NPs

[0052] 1) 1 g of Nb2AlC powder was immersed in 6 ml of 50% hydrofluoric acid (HF) aqueous solution to etch the middle Al layer, and after 2 days, the dispersion was collected by centrifugation, and then washed with deionized water. After repeated centrifugal washing, the etched Nb2C precipitate was obtained;

[0053] 2) The Nb2C precipitate was ultrasonically dispersed in 6 ml of TPAOH, and stirred for dispersion for 3 days to continue to peel off and reduce the particle size of Nb2C. After repeated washing and resuspension with deionized water, centrifugation was performed to obtain Nb2C nanoparticles with good dispersity. The Nb2C nanoparticles were ultrasonically dispersed in deionized water to obtain 500, 1000 and 1500 μg / mL Nb2C solutions.

[0054] 2. Characterization of Nb2C NPs

[0055] 1) Morphological characteristics:

[0056] (1) Transmission electron microscopy:

[0057] The washed and centrifuged Nb2C precipitate was ultrasonically dispersed in deionized water, and the suspension was dropped on the transmission electron microscope grid film. After the droplet was completely dry, the morphology was observed using transmission electron microscopy.

[0058] (2) Scanning electron microscopy:

[0059] The washed and centrifuged Nb2C precipitate was ultrasonically dispersed in deionized water, and the suspension was dropped on the scanning electron microscope sample stage. After the droplet was completely dry, surface gold spraying treatment was performed, and the surface morphology of the Nb2C NPs was observed and collected.

[0060] (3) Atomic force microscopy:

[0061] The probe was installed, the prepared Nb2C suspension was added to the sample holder, the probe and sample positions were adjusted, the microscope lens was brought close to the sample, and the sample surface was focused to collect the three-dimensional surface morphology of the Nb2C NPs.

[0062] TEM and SEM images are shown in Figure 1 A and B, the Nb2C NPs are dispersed particles, with uniform size and a length of about 200 nm. Further use of a spectrometer combined with SEM images ( Figure 1Nb2C NPs, EDS elemental mapping images can be seen in the Nb2C NPs, niobium element (red), carbon element (yellow) and oxygen element (green) uniform distribution, the content of niobium element is about 2 times of carbon element, in line with our target product Nb2C element ratio, indicating that we successfully prepared Nb2C.

[0063] 2) ingredient analysis:

[0064] (1) EDS spectroscopy: the sample is placed in the scanning electron microscope, after preliminary observation, the energy dispersive spectrometer scanning is selected in the area of interest, and the element mapping image is obtained.

[0065] (2) X-ray diffraction pattern: using X-ray diffractometer to determine the crystal structure, size and crystallization characteristics of the sample.

[0066] X-ray diffraction pattern as Figure 2 shown, the prepared Nb2C main diffraction peak appears at 2θ about 34.4°, 40.2°, 58.4°, 69.8° position, respectively, corresponding to (111), (200), (220) and (311) crystal face. The shape of the diffraction peak is sharp, indicating that the crystal structure of the detected sample is regular, and has higher crystallinity. The intensity of these peaks and the corresponding crystal face position indicate that there is Nb2C phase in the sample, and the particle crystal structure is relatively complete. The particle size and zeta potential analyzer is used to further evaluate the surface characteristics and average particle size of Nb2C. When the zeta potential peak value is less than-30 mV or greater than 30 mV, it indicates that there is enough electrostatic repulsion between the detected particles so that they can be dispersed in the medium.

[0067] As Figure 2 shown in the middle B, the zeta potential peak value of Nb2C appears near-25 mV, and the shape of the peak is sharp, indicating that the potential distribution of Nb2C particles is relatively concentrated, and the detected sample has good uniformity and can maintain a relatively stable dispersion state in the solution system. The particle size analysis diagram of Nb2C Figure 2 in the middle C) shows that the particle size distribution is concentrated between 200-300 nm, and the average particle size is 261.5 nm. This is consistent with the line scanning result of atomic force microscope, and the above results prove that the Nb2C particles synthesized by us have uniform size and can be stably and uniformly dispersed in the medium, which can be used for the subsequent preparation of hydrogel.

[0068] (3) photothermal properties of Nb2C

[0069] We selected Nb2C suspension with concentrations of 500, 1000 and 1500 μg / mL, and used a handheld thermal imager to take in vitro thermal imaging pictures Figure 3(A) Temperature data were collected and Nb₂C was plotted at a wavelength of 808 nm and a power of 1.0 W / cm². 2 The photothermal heating curves under near-infrared laser irradiation are shown in Figure 3B. Analysis reveals that under 808 nm near-infrared light irradiation, Nb₂C suspensions of 500, 1000, and 1500 μg / mL all rapidly heated up to a stable temperature after approximately 3 min, reaching 39.2, 44.6, and 54.4 ℃ respectively after 5 min of irradiation. With prolonged irradiation time, the temperature of Nb₂C at different concentrations gradually increased, stabilizing after 3 min. At the same irradiation time, higher Nb₂C concentrations resulted in higher peak temperatures. This indicates that the photothermal effect of Nb₂C is concentration-dependent and light-time-dependent.

[0070] The photothermal stability of a 1000 μg / mL Nb₂C dispersion was further investigated using a power of 1.0 W / cm². 2 After irradiation with a near-infrared laser at a wavelength of 808 nm for 5 min, the near-infrared light was turned off, and the Nb₂C dispersion was allowed to cool to room temperature. The temperature of the dispersion was recorded every 30 s, with the start of laser irradiation as the zero point, and this cycle was repeated four times. The results showed that after 5 min of near-infrared laser irradiation, the 1000 μg / mL Nb₂C dispersion rapidly heated from approximately 22°C to 45–48°C, and then naturally cooled to room temperature after 10 min. In the four continuous irradiation-off cycles, the Nb₂C dispersion exhibited a regular heating and cooling pattern, with consistent amplitude and trend, indicating that the 1000 μg / mL Nb₂C dispersion possesses good stability and repeatability under repeated photothermal processes, and can be used at a power of 1.0 W / cm². 2 The material maintained stable performance under repeated irradiation with a near-infrared laser at a wavelength of 808 nm, providing an experimental basis for subsequent photothermal therapy. In the subsequent hydrogel preparation, Nb₂C NPs at a concentration of 1000 μg / mL were selected for the experiment.

[0071] 2. Preparation and printing of each group of hydrogel mixtures

[0072] 1) Preparation of basic solution:

[0073] (1) LAP standard solution: Dissolve 50 mg LAP powder in 10 mL PBS solution, shake thoroughly and dissolve evenly to obtain a 0.5% (W / V) LAP standard solution.

[0074] (2) HAMA solution: Dissolve 25 mg HAMA in 1 mL of 0.5% (W / V) LAP standard solution to obtain a 2.5% (W / V) HAMA solution.

[0075] (3) VP solution: Dissolve 10 mg of VP powder in 0.5 mL of DMSO solution, shake thoroughly and mix evenly to obtain a VP solution with a concentration of 2% (W / V).

[0076] (4) Nb2C solution: 100 mg of the previously prepared Nb2C powder was ultrasonically mixed in 1 mL of deionized water to obtain a 10% (W / V) Nb2C solution. This Nb2C solution refers to a solution loaded with niobium carbide nanoparticles (Nb2CNPs).

[0077] (5) VP-Nb2C-GelMA-HAMA (VNGH) mixed solution: 100, 160, 200 and 300 mg of GelMA were dissolved in 1.91 mL of 0.5% (W / V) LAP standard solution, and then 20 μL of 2.5% (W / V) HAMA solution, 20 μL of 10% (W / V) Nb2C solution and 50 μL of 2% (W / V) VP solution were added. The solution was thoroughly mixed and filtered through a 0.22 μm filter to obtain VNGH mixed solutions with GelMA concentrations of 5%, 8%, 10% and 15% (W / V).

[0078] 2) 3D printing:

[0079] (1) Preparation before printing: After heating the prepared VNGH mixture in a 37 ℃ constant temperature water bath for 6 h, refrigerate it in a 4 ℃ refrigerator for 10 min. Then, add the prepared mixed hydrogel solutions of each group into the sample tube of the extrusion bio-3D printer for printing. Avoid light throughout the process.

[0080] (2) Printing parameter settings: layer height 0.2 mm, trace width 0.4 mm, fill density 40%, print grid-shaped cylindrical hydrogel patches with a diameter of 8 mm. After printing, the hydrogel patches are irradiated with a 405 nm handheld photocuring tube for 210 s to complete the photocuring of the hydrogel.

[0081] Comparative Example 1: GelMA-HAMA (GH) hydrogel patch

[0082] GelMA-HAMA (GH) mixed solution: 100 and 200 mg of GelMA were dissolved in 1.98 mL of 0.5% (w / v) LAP standard solution, respectively. Then, 20 μL of 2.5% (w / v) HAMA solution was added, and the mixture was thoroughly mixed. The solution was filtered through a 0.22 μm filter to obtain a GH mixed solution with GelMA concentrations of 5% and 10% (w / v). The 3D printing steps were the same as in Example 1 to obtain GelMA-HAMA (GH) hydrogel patches.

[0083] Comparative Example 2: Nb2C-GelMA-HAMA (NGH) hydrogel patch

[0084] Nb2C-GelMA-HAMA (NGH) mixed solution: Add 20 μL of 10% (w / v) Nb2C solution to 1.98 mL of 10% (w / v) GelMA GH mixed solution, mix thoroughly, and filter the solution using a 0.22 μm filter to obtain the NGH mixed solution. The 3D printing steps are the same as in Example 1 to obtain Nb2C-GelMA-HAMA (NGH) hydrogel patches.

[0085] Comparative Example 3: VP-GelMA-HAMA (VGH) hydrogel patch

[0086] VP-GelMA-HAMA (VGH) mixed solution: Add 50 μL of 2% (w / v) VP solution to 1.95 mL of 10% (w / v) GelMA GH mixed solution, mix thoroughly, and filter the solution using a 0.22 μm filter to obtain the VGH mixed solution. The 3D printing steps are the same as in Example 1 to obtain VP-GelMA-HAMA (VGH) hydrogel patches.

[0087] Experimental Example 1

[0088] The preparation of the hydrogel mixture solutions in Examples 1 and 3 is shown in Table 1, and the performance of each group was tested.

[0089] Table 1. Preparation of hydrogel mixtures for each group

[0090]

[0091] 1. Morphological observation of each group of hydrogel patches

[0092] Each hydrogel mixture was 3D printed into cylindrical patches with a diameter of 8 mm and a thickness of 2 mm. The GH group was transparent; compared with the GH group, the NGH group had decreased transmittance and appeared white due to the incorporation of 1000 μg / mL Nb2C NPs; the VGH and VNGH groups contained VP and appeared black as VP powder. After freeze-drying each hydrogel patch for 48 h and surface sputtering with gold, the samples were placed on a scanning electron microscope stage for morphological observation. Figure 4 All hydrogel patches exhibited an ordered mesh-like arrangement with uniform pore size and a uniform network scaffold thickness. In the NGH group, Nb₂C NPs were observed to be uniformly distributed throughout the network scaffold. In the VNGH group, Nb₂C NPs formed a ring-like structure and were uniformly distributed, which may be related to the interaction between Nb₂C and specific chemical groups of the VP molecule, thus tending to form stable conjugated rings.

[0093] 2. Spectral analysis of each group of hydrogel patches

[0094] The chemical structures of the printed hydrogel patches were further examined using FTIR spectroscopy (Figure 5). It was observed that the GH and NGH groups did not exhibit significant absorption peaks, while the FTIR spectra of the VGH and VNGH groups were quite consistent, with the main absorption peaks appearing at 3281, 3060, 1630, and 1013 cm⁻¹. -1 Location. Among them, 3281cm -1 The absorption peak at 3060 cm⁻¹ is usually related to the stretching vibration of the NH bond, indicating that amide bonds (-CONH) or amino groups (=NH) may exist in VGH and VNGH; -1 The absorption peak at this point is usually related to the stretching vibration of the CH bond, indicating that a carboxyl group (-COOH) may be present in VGH and VNGH; 1630 cm⁻¹ -1 The absorption peak at this point is usually related to the stretching vibration of the C=C double bond, indicating that unsaturated bonds may exist in VGH and VNGH; 10¹³ cm⁻¹ -1 The absorption peaks at these peaks are typically associated with the stretching vibrations of the COC bond, indicating the possible presence of ether bonds in VGH and VNGH. The chemical structure of VP shows that, as a benzoporphyrin derivative, its core structure is a porphyrin ring (containing two -NH- groups), and its characteristic chemical groups include vinyl (-CH=CH-), methoxycarbonyl (-O-CO-CH3), and propionate (-COO-). The characteristic chemical groups detected by the FTIR spectra of VGH and VNGH are consistent with the chemical structure of VP; therefore, we can conclude that the synthesized hydrogel system contains VP.

[0095] 3. Rheological properties of GH and VNGH

[0096] Strain scan results (Figure 6 A) The elastic and viscous behaviors of GH and VNGH under different strain conditions are shown. In the low strain region (0–10%) and the medium strain region (10–100%), the G' value of GH and VNGH is greater than the G'' value, indicating that the elastic characteristics of the hydrogel are greater than the viscous characteristics, and they can recover their original shape after being subjected to stress. In the high strain region (100–1000%), the G' value and G'' value decrease sharply, and the decrease in G' value is greater than that in G'' value, indicating that GH and VNGH undergo significant structural damage under high strain, and the viscous characteristics become dominant. The hydrogel exhibits viscous flow behavior or plasticity.

[0097] The intersection of the G' curve and the G'' curve represents the critical point at which the hydrogel material transitions from elastic dominance to viscous dominance, often referred to as the "flow point." The G' curve and G'' curve intersect at approximately 120% stress in the GH group, while they intersect at approximately 180% stress in the VNGH group. Furthermore, the downward trend of the G' curve and G'' curve after the flow point is significantly less in the VNGH group than in the GH group, indicating that the addition of Nb2C NPs and VP can improve the structural stability of the hydrogel.

[0098] Based on the strain scan results, we selected strain levels switching between 1% and 400%, maintaining each level for 60 seconds to test the dynamic step strain of GH and VNGH. In the strain scan test, at 1% strain, both materials were elastically dominated, while at 400% strain (more than three times the flow point), both materials were viscous-dominated. Similarly, in Figure 6 As shown in Figure B, at a strain of 1%, the G' values ​​of both GH and VNGH are consistently higher than the G'' value, exhibiting elastic properties. When the strain increases to 400%, the G' values ​​of GH and VNGH decrease rapidly, while the G'' value increases rapidly and exceeds the G' value, exhibiting viscous properties and indicating network structure disruption. When the strain returns to 1%, the G' values ​​of GH and VNGH increase sharply again and momentarily exceed the G'' value, indicating that both have recovered to the hydrogel network structure state. Notably, after three cycles of alternating strains of 1% and 400%, the G' and G'' values ​​of GH and VNGH can quickly recover to their initial values, proving that the addition of Nb2C NPs and VP does not affect the outstanding self-healing ability of the hydrogel.

[0099] Further comparison of the stability of GH and VNGH under different frequency scans ( Figure 6 Both the GH and VNGH groups exhibited good stability in the 0.1–10 Hz range, with G' values ​​greater than G'' values, indicating that both were fully gelled and structurally stable. The G'' value of the GH group showed a partial shift in the high-frequency range, suggesting that the GH hydrogel structure was less stable than the VNGH structure at high frequencies.

[0100] In the process of bio-3D printing, hydrogel materials need to maintain a low viscosity under high shear rates when passing through the printing nozzle to ensure smooth flow, and a high viscosity under low shear rates after being extruded onto the substrate to maintain structural stability.

[0101] Figure 6 Figure D shows the viscosity changes of GH and VNGH at different shear rates. VNGH and GH exhibit similar shear-thinning behavior, with viscosity gradually decreasing as the shear rate increases. GH shows viscosity decreasing at a shear rate of 5.5 s⁻¹. -1 At this point, the viscosity decreases sharply, possibly because the shear stress reaches the yield stress of GH, causing a change in the hydrogel structure and initiation of flow. Shear viscosity test results demonstrate that both GH and VNGH possess excellent injectability, and also further prove that VNGH has superior structural stability compared to GH.

[0102] The above experimental results demonstrate that the dual matrix ratio (GelMA 10% + HAMA 0.025%) gives VNGH the following characteristics: (1) high structural stability (flow point 180%, G' recovery >95% after strain 400%). Figure 6 (2) Excellent injectability (shear thinning index n=0.35), meeting the requirements of 3D printing.

[0103] 4. Characterization of the photothermal properties of each group of hydrogel solutions

[0104] To verify the photothermal effect of Nb2C, we used a 1000 μg / mL Nb2C NPs solution as a control and tested the photothermal properties of GH, NGH, VGH, and VNGH solutions. In vitro thermal images were captured using a handheld thermal imager. Figure 7 (A) Temperature data were collected and plotted at a wavelength of 808 nm and a power of 1.0 W / cm². 2 The photothermal heating curves of each group of solutions under near-infrared laser irradiation are shown. Analysis reveals (...). Figure 7Under 808 nm near-infrared irradiation, 1000 μg / mL Nb₂C NPs, NGH, and VNGH solutions all rapidly heated up and then stabilized after about 3 min, reaching average temperatures of 41.6, 46.2, and 58.9 °C, respectively, at 3 min, and 44.6, 48.6, and 65.5 °C, respectively, after 5 min of irradiation. With prolonged irradiation time, the temperatures of all three solutions gradually increased. The temperature increases of Nb₂C NPs and VNGH solutions stabilized after 3 min, while the NGH solution reached a near-constant temperature after 2 min. The VNGH solution showed the most significant temperature increase after 3 min, approximately 4 °C / min. At the same irradiation time, the VNGH solution had a higher temperature than the NGH solution, which in turn had a higher temperature than the Nb₂C NPs solution. The NGH hydrogel solution reached its final stable temperature earlier than the Nb₂C NPs deionized aqueous solution, and its final temperature was higher than that of the Nb₂C NPs solution. This is likely due to the more uniform and stable distribution of Nb₂C NPs in the GH mixed hydrogel system, thus improving the photothermal conversion efficiency. The VNGH solution exhibited the largest temperature rise and the highest final temperature among the three, likely because the hydrogel turned black after the addition of VP, and scanning electron microscopy revealed that the Nb₂C particles formed a ring structure after the addition of VP, which improved the light absorption and photothermal conversion efficiency of the VNGH hydrogel. To achieve the photothermal antibacterial effect while maintaining a safe temperature range to avoid thermal damage to normal cells and healthy tissues, a wavelength of 808 nm and a power of 1.0 W / cm² were selected for subsequent animal experiments. 2 Irradiate with near-infrared laser for 3 minutes.

[0105] 5. Drug release characteristics of VNGH hydrogel patches

[0106] During the fabrication of the hydrogel patch, VP molecules are uniformly encapsulated within the pores of the three-dimensional network structure of the GH hydrogel. As the hydrogel absorbs liquid and swells, the pores gradually increase in size, and it gradually degrades over time, the drug molecules inside are continuously released. To clarify the drug release characteristics of VP in the VNGH hydrogel patch, we incubated the VNGH hydrogel patch in PBS solution. At set time intervals, quantitative amounts of solution were aspirated, and the VP concentration at 689 nm was measured using a spectrophotometer. Based on the measured VP concentration and the set time, a VP release curve was plotted. Figure 8 ).

[0107] like Figure 8As shown, approximately 55% of the VP was released into the PBS solution within 48 hours, and about 88% of the VP was released after 288 hours, indicating that the hydrogel system can achieve sustained release of VP. The VP release rate significantly increased after 192 hours, possibly due to hydrogel degradation. Sustained and continuous release of VP helps reduce the frequency of drug administration and can prolong the duration of action of a single drug dose, thereby improving the convenience of diabetic wound treatment and patient compliance.

[0108] 6. Biocompatibility of each group of hydrogel patches

[0109] VP was initially used in ophthalmological clinical treatment of age-related macular degeneration, and in recent years has begun to be used in various oncological diseases such as leukemia, breast cancer, and neuroblastoma. High concentrations of VP can inhibit tumor cell proliferation and migration, therefore we need to evaluate the safety of VP in subsequent experimental use. VP solutions were mixed with prepared complete cell culture medium to obtain cell culture media with VP concentrations ranging from 1 to 10 μg / mL. Using complete cell culture medium (VP concentration of 0 μg / mL) as a control, cell culture media containing different concentration gradients of VP were used to culture NIH-3T3 cells. After 24 h of treatment, CCK8 reagent was added proportionally, and the OD value of the supernatant at 450 nm was measured to calculate cell viability.

[0110] Figure 9 The results showed that when the VP concentration did not exceed 5 μg / mL, the survival rate of NIH-3T3 cells was not significantly different from that of the control group. However, when the VP concentration was greater than or equal to 6 μg / mL, the survival rate of NIH-3T3 cells was inversely proportional to the VP concentration. After 24 h of treatment with VP at a concentration of 8 μg / mL, the survival rate of NIH-3T3 cells was only 75.8%. Therefore, if VP solution is applied directly to the wound, the concentration must be strictly controlled to ensure the safety of normal cells.

[0111] To verify and explore the safe concentration range for subsequent cell experiments, we immersed each group of hydrogels in complete cell culture medium and shook them in a shaker at 37 ℃ and 200 rpm for 24 h to obtain the corresponding hydrogel extracts. We then diluted them into extracts of different concentration gradients for CCK8 assay.

[0112] When the extract concentration was 5 μg / mL, cell viability remained above 91% after 24 h of treatment with the hydrogel extract in all groups. At an extract concentration of 10 μg / mL, cell viability in the VGH and VNGH groups decreased to below 70%. At an extract concentration of 20 μg / mL, cell viability in the NGH group decreased to below 70%. Based on the CCK8 assay results, a hydrogel extract concentration of 5 μg / mL was selected for subsequent experiments. NIH-3T3 cells were treated with each group of hydrogel extracts at a concentration of 5 μg / mL for 0, 24, 48, and 72 h, and cell viability was measured at the corresponding time points. It was observed that there was no significant difference in cell viability between the hydrogel groups and the control group, and the cell viability consistently remained above 93%, confirming that the prepared hydrogel has good cell safety.

[0113] 7. In vitro healing ability of each group of hydrogel patches

[0114] The high glucose environment in diabetes can affect local cell viability and migration ability at the wound site, thus delaying wound healing. We used serum-free high glucose cell culture medium to conduct a scratch assay to detect the ability of hydrogel patches to promote wound healing in vitro. Direct observation under a microscope showed that at 24 h, there was no significant difference in cell migration rate between the GH group and the NGH group and the control group, while the cell migration rate of the VGH group and the VNGH group was higher than that of the control group. Figure 10 (A). According to Figure 10 As shown in Figure B, the migration rate of the VGH group at 24 h was approximately 5.03%, that of the VNGH group was approximately 4.80%, and that of the control group was approximately 3.08%. At 48 h, the scratches in the VNGH group were almost completely closed under the microscope, and the cell migration and proliferation abilities were significantly better than those of other groups, indicating that the VNGH hydrogel patch has good in vitro healing-promoting ability.

[0115] 8. In vitro antibacterial activity of each group of hydrogel patches

[0116] Previous studies have reported that Nb2C, in addition to ablating bacteria through photothermal effects under 808 nm laser irradiation, can inhibit bacterial energy metabolism pathways, thereby disrupting biofilm formation and directly eliminating bacteria. The ability of VP to disrupt bacterial structure is dose-dependent; its antibacterial activity is evident at concentrations exceeding 4 μg / mL. Under 689 nm laser irradiation, VP can be activated to produce singlet oxygen and ROS to kill bacteria, and the antibacterial effect is enhanced by photoactivation. To verify the in vitro antibacterial effect of the hydrogel patch, we used an inhibition zone assay to test the inhibitory effects of each hydrogel group on *S. aureus* (representing Gram-positive bacteria) and *E. coli* (representing Gram-negative bacteria).

[0117] Using hydrogel extracts diluted to 5 μg / mL for each group, and with cell culture medium without penicillin-streptomycin solution as a control, blank sterile antimicrobial susceptibility testing paper discs were soaked. After soaking for 24 h, the filter paper discs were placed in a 37 ℃ oven until completely dried. The dried filter paper discs were then irradiated with UV light and placed on bacterial-inoculated plates, incubated at 37 ℃ for 24 h, and the formation of transparent inhibition zones was observed. The diameter of the inhibition zone directly reflects the degree of bacterial inhibition by the hydrogel extract; a larger diameter inhibition zone indicates that the corresponding hydrogel can effectively inhibit bacterial proliferation and spread, exhibiting strong antibacterial properties.

[0118] Figure 11 It is evident that NGH, VGH, and VNGH all exhibit some bactericidal effects against Escherichia coli and Staphylococcus aureus, with the inhibition zone diameter for Staphylococcus aureus being significantly larger than that for Escherichia coli. Nb2C and VP demonstrate strong antibacterial effects against Gram-positive Staphylococcus aureus, but weaker effects against Gram-negative Escherichia coli, possibly due to the hydrophobic lipopolysaccharide component of the outer membrane of Gram-negative bacteria acting as a drug barrier. In the absence of laser irradiation, Nb2C shows stronger antibacterial activity than VP (NGH vs VGH). The combined use of both (VNGH) has a synergistic effect in inhibiting Gram-negative bacteria, showing a more significant antibacterial effect than either NGH or VGH alone.

[0119] Experimental Example 2: VNGH hydrogel patches promote wound healing in diabetic mice

[0120] I. Construction and Grouping of Diabetic Mouse Models

[0121] 1. Molding steps:

[0122] (1) Prepare a 1% (W / V) STZ sodium citrate mixed solution (i.e., 0.1 g STZ dissolved in 10 mL sodium citrate buffer) under light-protected conditions and use it within 15 min.

[0123] (2) Mice were injected intraperitoneally with 150 mg / kg STZ. One 20 g mouse was injected intraperitoneally with 0.3 mL of 1% (W / V) STZ sodium citrate mixture.

[0124] (3) Change the bedding frequently after STZ injection to ensure adequate food and water. Measure fasting blood glucose using the tail clipping method one week later. Then measure random blood glucose every 7 days. After one month of observation, if the fasting blood glucose level is >11.1 mmol / L and the random blood glucose level is >16.7 mmol / L for three consecutive measurements, the mouse diabetes model is considered successfully established, and wound modeling experiments can be carried out.

[0125] 2. Blood glucose levels were measured in 50 male C57BL mice one week after intraperitoneal injection of STZ. Fasting blood glucose levels in 47 mice were >11.1 mmol / L. Random blood glucose levels were measured every 7 days thereafter. After one month of observation, 5 mice died and 45 mice survived. Among the surviving mice, 40 mice had random blood glucose levels >16.7 mmol / L for three consecutive measurements, and 5 mice had random blood glucose levels <16.7 mmol / L. Therefore, these 40 successfully modeled diabetic mice were selected for the next step of wound modeling.

[0126] Overall, compared to pre-modeling conditions, successfully modeled diabetic mice exhibited typical diabetic symptoms such as gradual weight loss, rough and easily falling hair, lethargy, and polydipsia, polyphagia, and polyuria. A full-thickness skin defect diabetic mouse model was established by creating a 1 cm diameter circular skin incision on the back of the diabetic mice. These 40 mice were randomly divided into 5 groups and treated with different methods before being fed a power source of 1 W / cm². 2 The effects of near-infrared light with a wavelength of 808 nm on promoting the healing of diabetic wounds were compared. The specific groupings were as follows: sterile PBS treatment group (hereinafter referred to as PBS group), GH patch treatment group (hereinafter referred to as GH group), NGH patch treatment group (hereinafter referred to as NGH group), VGH patch treatment group (hereinafter referred to as VGH group), and VNGH patch treatment group (hereinafter referred to as VNGH group).

[0127] II. Wound healing in diabetic mice

[0128] We photographed the wound healing status of diabetic mice in each group on the day of wound modeling and on days 3, 7, 11, and 14 after modeling. The skin wound area and healing process of each group are shown in the figure. (The wound area healing diagram is used to illustrate this.) Figure 13 As observed, the wound area of ​​diabetic mice in each group gradually decreased over time. Since mice possess a certain degree of wound self-healing ability, although the PBS, GH, and NGH groups showed no effect from VP, and the wounds of the mice underwent some repair, the VGH and VNGH groups exhibited a faster wound healing rate. Furthermore, in the PBS, GH, and NGH groups, the healed portion showed visible epidermis, while at the same time point, the epidermis in the VGH and VNGH groups was almost completely covered by hair. Figure 13 Figure B shows a schematic diagram of overlapping wound areas at different time intervals. The results show that the degree of wound repair increases in a time-dependent manner, with the VNGH and VGH groups exhibiting the highest wound healing efficiency. This is consistent with... Figure 13The wound healing rate in the A group was consistent with that in the B group. From the general images, we can conclude that hydrogel patches containing VP can promote faster healing of diabetic wounds and accelerate hair growth after wound healing.

[0129] III. Analysis of Wound Healing Results in Diabetic Mice

[0130] After treating diabetic mouse wounds with five different methods, semi-quantitative analysis (Figure 14) showed that the wounds treated with VNGH had healed by more than 70% on day 3 and were almost completely healed (95%) by day 11, while the wounds in the PBS and GH groups healed more slowly. Furthermore, the NGH group initially healed faster than the PBS and GH groups, but by day 14, there was no significant difference in healing between the NGH, GH, and PBS groups. This demonstrates that the photothermal effect of Nb2C NPs plays a role in the early stages of wound healing, but it is not a key factor significantly affecting the final healing efficiency of diabetic wounds. In addition, there was no significant difference in the final healing efficiency between the VNGH and VGH groups, but the healing-promoting effect of the VNGH group was significantly better than that of the VGH group on day 7. This may be due to the photothermal antibacterial effect and antioxidant properties of Nb2C NPs, which also reflects the unique advantages of the dual-use Nb2C NPs and VP hydrogel system.

[0131] IV. Photothermal Effect of Hydrogels in Vivo

[0132] We used a wavelength of 808 nm and a power of 1.0 W / cm². 2 Near-infrared laser light was used to irradiate the hydrogel patch placed on the wound surface for 3 minutes. Figure 15 As shown, the VNGH group had the highest local terminal temperature of 52.5 ℃ among all groups. At 30 s, the VNGH and NGH groups reached temperatures of 41.9 ℃ and 40.5 ℃ respectively, while the VNGH group only reached 50 ℃ after 2 min. The experimental results indicate that under this illumination condition, the photothermal effect of Nb2C NPs in the hydrogel can be effectively activated, achieving antibacterial effects while avoiding high-temperature burns to skin tissue and coagulative necrosis of the surrounding tissue.

[0133] V. Biocompatibility and Wound Healing Promotion Ability of Hydrogels

[0134] The biocompatibility of hydrogels has been confirmed at the cellular level. To further confirm the biocompatibility of hydrogel patches in direct contact with wounds, we collected and fixed vital organs such as the heart, liver, spleen, lungs, and kidneys of diabetic mice on day 14 after wound modeling, prepared paraffin sections, and performed HE staining for observation.

[0135] Figure 16The results showed that HE staining of vital organs in mice from the PBS, GH, NGH, VGH, and VNGH groups revealed no obvious signs of damage or inflammation, and there were no significant differences between the groups. Therefore, we preliminarily conclude that the VNGH hydrogel patch has good biocompatibility, no potential toxicity to the body, and meets the basic requirements for future application in wound treatment.

[0136] In diabetic mice, the wound tissue in the VNGH group was completely epithelialized, with intact epidermal structure, tightly packed keratinocytes, orderly arranged collagen fibers in the dermis, no obvious inflammatory cell infiltration, and intact and uniformly distributed skin appendages such as hair follicles and sebaceous glands. The structure of the healed tissue was not significantly different from that of the normal tissue at the edge. Figure 16 (B) Compared to the PBS group, both the NGH and VGH groups showed improved epithelialization of the wound surface. However, the NGH group did not yet show the formation of skin appendages, while the VGH group did not show complete regeneration of skin appendages. In both the PBS and GH groups, the epidermis did not regenerate completely, granulation tissue maturation at the wound site was not obvious, collagen arrangement was disordered, and inflammatory cells remained abundant. In the PBS group, scab residue was still visible on the epidermis. Therefore, the VNGH group showed complete new epidermal coverage of the wound, with no significant difference in epidermal thickness compared to the surrounding normal tissue. Granulation tissue gradually matured, fibroblasts were fewer, inflammatory cells were significantly reduced, and skin appendage regeneration was good. The repair effect was more significant than in other groups, indicating that the VNGH hydrogel patch has a better ability to promote wound epidermal regeneration and tissue repair, and can effectively accelerate the structural and functional repair process of the wound.

[0137] VI. VNGH hydrogel patches promote tissue remodeling in diabetic wounds.

[0138] Masson staining uses various dyes to stain different components in tissue, making collagen fibers in wound tissue appear blue, muscle fibers appear red, and cell nuclei appear blue-black.

[0139] Figure 17Masson staining results showed that the PBS, GH, and NGH groups had thinner dermal thickness, disordered collagen fiber deposition, and a relatively large number of newly formed capillaries. The NGH group showed better inflammatory infiltration than the PBS and GH groups. The VGH group had increased dermal thickness, but skin appendages such as hair follicles were not yet fully developed. The VNGH group had significantly thicker dermal thickness than the other treatment groups, with regular and directional collagen fiber arrangement, mature skin appendages such as hair follicles and sebaceous glands, and gradually stabilizing and degenerating newly formed blood vessels. The remodeling process of the healed wound tissue in the VNGH group was basically complete, with a structure closer to normal skin tissue. The newly formed capillaries, which formed in large numbers in the early stages of wound healing to meet the high oxygen and nutrient demands of the wound, gradually degenerated, and the vascular density was basically consistent with normal skin tissue. For diabetic wounds, the use of VNGH hydrogel patches has a higher efficiency in extracellular matrix reconstruction and tissue remodeling than other treatments.

[0140] Finally, it should be noted that the terms “comprising,” “including,” or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0141] Although preferred embodiments of the present invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of the embodiments of the present invention.

[0142] Obviously, those skilled in the art can make various modifications and variations to the embodiments of the present invention without departing from the spirit and scope of the embodiments of the present invention. Therefore, if these modifications and variations to the embodiments of the present invention fall within the scope of the claims of the embodiments of the present invention and their equivalents, the embodiments of the present invention are also intended to include these modifications and variations.

Claims

1. A photothermal composite hydrogel patch, characterized in that, The photothermal composite hydrogel patch is: A three-dimensional grid-shaped porous structure formed by 3D printing or casting molding using methacrylated gelatin (GelMA) and methacrylated hyaluronic acid (HAMA) as the matrix, loaded with carbonized niobium nanoparticles (Nb2CNPs) and verteporfin (VP), and is abbreviated as VP-Nb2C@GelMA / HAMA (VNGH) hydrogel patch; The photothermal composite hydrogel patch has a pore size of 150-250 μm and a porosity of 30%-50%; The mass ratio of GelMA to HAMA is 350-450:

1.

2. A method for preparing the photo-thermal composite hydrogel patch of claim 1, characterized in that, The method comprises: Dissolve GelMA in a solution containing a photoinitiator LAP, sequentially add HAMA, Nb2CNPs dispersion and VP solution, mix and filter; Obtain the VP-Nb2C@GelMA / HAMA (VNGH) hydrogel patch by 3D printing or casting molding; The concentration of GelMA dissolved in PBS is 5%-15% w / v, and the concentration of HAMA dissolved in PBS is 0.01%-0.05% w / v; The working concentration of Nb2CNPs in the Nb2CNPs dispersion is 500-1500 μg / mL; The working concentration of VP in the DMSO solution of VP is 3-500 μg / mL.

3. The method of claim 2, wherein the method further comprises the step of: The 3D printing uses 405 nm light curing.

4. Use of the composite hydrogel patch of claim 1 in the preparation of a dressing for treating diabetic wounds.

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