Preparation method of self-powered thermoelectric hydrogel material for wound healing

Abstract

The invention discloses a preparation method of a self-powered thermoelectric hydrogel material for wound healing. The preparation method comprises the following steps: S1, preparing an Ag2Se semiconductor nanomaterial; s2, combining an Ag2Se nano material with a gelatin methacrylate (GelMA) hydrogel to form an Ag2Se-coated GelMA thermoelectric hydrogel; and S3, applying the prepared thermoelectric hydrogel to a wound, and generating electric energy by using the temperature difference between the wound and the environment. The thermoelectric hydrogel has a Seebeck coefficient up to-1080 [mu] V / K under the mass concentration of 1.0 wt%, can effectively up-regulate the intracellular calcium ion level by electrically activating a voltage-gated calcium ion channel (VGCCs) on a cell membrane, enhances the mitochondrial function through a CaMKK [beta] / AMPK / Nrf2 signal channel, remarkably promotes angiogenesis through mitochondrial regulation, and has the effect of promoting the angiogenesis. The comprehensive effects finally promote the wound healing process and contribute to successful reconstruction of skin tissues.

Description

Technical Field

[0001] The present invention relates to the technical field of wound healing, and in particular to a preparation method of a self-powered thermoelectric hydrogel material for wound healing. Background Art

[0002] The skin is the largest organ of the human body and has multiple key functions such as protection, immunity, body temperature regulation, sensation, and excretion of waste. Skin wounds caused by surgical operations, trauma, thermal injuries, or metabolic disorders such as diabetes impose a significant burden on human health.

[0003] The endogenous electric field in the skin and the biological electrical stimulation regulated by it play a key role in a series of physiological processes such as cell signal transduction, nerve regeneration, and wound repair through ion transmembrane transport. When a wound occurs, due to the polarized distribution of sodium (Na + ) and chloride (Cl - ) ion channels in epithelial cells, the generated transepithelial potential results in an endogenous electric field in the wound, which guides cell proliferation, migration, adhesion, and promotes angiogenesis, thereby promoting the skin's self-repair mechanism. However, its self-repair speed is slow. Therefore, various types of hydrogels have emerged on the market to assist in repair, but their effects are not good and are not conducive to rapid repair. For this reason, we have proposed a preparation method of a self-powered thermoelectric hydrogel material for wound healing to solve the above problems. Summary of the Invention

[0004] The purpose of the present invention is to solve the deficiencies existing in the prior art and to propose a preparation method of a self-powered thermoelectric hydrogel material for wound healing.

[0005] To achieve the above purpose, the present invention adopts the following technical solutions:

[0006] A preparation method of a self-powered thermoelectric hydrogel material for wound healing includes the following steps:

[0007] S1. Prepare Ag2Se semiconductor nanomaterials;

[0008] S2. Combine the Ag2Se nanomaterials with a gelatin methacrylate (GelMA) hydrogel to form an Ag2Se@GelMA thermoelectric hydrogel;

[0009] S3. Apply the prepared thermoelectric hydrogel to the wound to generate electric energy using the temperature difference between the wound and the environment;

[0010] S4. The electrical stimulation generated by the thermoelectric hydrogel activates VGCCs through electricity, up-regulates the intracellular calcium ion level, and enhances mitochondrial function through the CaMKKβ / AMPK / Nrf2 signaling pathway;

[0011] S5. Angiogenesis is significantly enhanced, accelerating the wound healing process.

[0012] Preferably, according to step S1, the Ag2Se particles are prepared by existing mature methods.

[0013] Preferably, according to step S2, the base materials of the thermoelectric hydrogel include inorganic thermoelectric materials such as silver selenide, silicon germanium alloy, bismuth antimony alloy, bismuth selenide, bismuth telluride, cadmium selenide, bismuth antimony selenide, germanium tin alloy, copper indium selenide, indium antimonide, bismuth indium selenide, bismuth telluride, etc., and also include organic thermoelectric materials such as Pedot:PSS, polyaniline, polytriphenylamine, poly-p-phenylenediamine, polythiophene, polyfluorene, polystyrene, polyacetylene, polypropyne, polymer fibers, graphene, etc., and also include hydrogel thermoelectric materials such as lithium ion thermoelectric hydrogel, bismuth ion thermoelectric hydrogel, sodium ion thermoelectric hydrogel, manganese ion thermoelectric hydrogel, vanadium ion thermoelectric hydrogel, copper ion thermoelectric hydrogel, iron ion thermoelectric hydrogel, titanium ion thermoelectric hydrogel, nickel ion thermoelectric hydrogel, iron ion thermoelectric hydrogel, etc.

[0014] Preferably, according to step S2, Ag2SeNPs are mixed with the GelMA hydrogel precursor by photopolymerization technology to obtain the Ag2Se@GelMA composite hydrogel.

[0015] The present invention proposes a self-powered silver selenide gelatin methacrylate thermoelectric hydrogel dressing, which utilizes the natural temperature difference between the wound and the surrounding environment to convert the thermal gradient into electrical energy, thereby providing a sufficient potential difference to enhance the endogenous electric field and promote cell proliferation and migration.

[0016] The thermoelectric hydrogel of the present invention has a Seebeck coefficient as high as ~1080 μV / K (at a mass concentration of 1.0 wt%), which can effectively activate the voltage-gated calcium channels (VGCCs) on the cell membrane through electricity, up-regulate the intracellular calcium level, and enhance mitochondrial function through the CaMKKβ / AMPK / Nrf2 signaling pathway, significantly promoting angiogenesis through mitochondrial regulation. These combined effects ultimately promote the wound healing process and contribute to the successful reconstruction of skin tissue. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 It is a schematic diagram of the preparation of the Ag2Se@GelMA hydrogel-based electrical stimulation (ES) device and its mechanism for wound healing in the present invention. (a) The preparation process of the Ag2Se@GelMA hydrogel electrical stimulation device, showing the photo-crosslinking molding process; (b) The intracellular and extracellular mechanisms of the Ag2Se@GelMA hydrogel promoting wound healing under electrical stimulation, including enhancing the endogenous electric field (EEF) and mitochondrial function through voltage-gated calcium channels (VGCCs), and activating the CaMKKβ / AMPK / Nrf2 signaling pathway.

[0018] Figure 2 This is the characterization and property diagram of the Ag2Se@GelMA hydrogel in the present invention. Characterization and properties of the Ag2Se@GelMA hydrogel material: (a) Low-magnification SEM image of Ag2Se nanoparticles and C / Ag / Se element distribution map; (b) Bright-field TEM and high-resolution lattice images of Ag2Se nanoparticles; (c) XRD diffraction pattern and (d) XPS full-spectrum scan; (e-f) XPS peak-fitting spectra of Ag3d and Se3d orbitals; (g) GelMA precursor solution containing Ag2Se NPs and physical diagram of the photocured hydrogel; (h) SEM morphology and EDS element distribution of composite hydrogels with different Ag2Se contents (0.0 - 1.5 wt%); (i) Wet adhesion performance test of the hydrogel on biological tissues such as skin / liver / kidney; (j-k) Seebeck coefficient, conductivity, and power factor of hydrogels with different Ag2Se contents; (l-m) Voltage output characteristics of the material under air flow and contact stimulation to verify its environment-responsive electrical stimulation ability.

[0019] Figure 3 This is the in vitro mechanism of the Ag2Se@GelMA hydrogel combined with electrical stimulation to regulate mitochondrial function. (a) Schematic diagram of the activation mechanism of the CaMKKβ / AMPK / Nrf2 signaling pathway; (b) Fluo-3AM staining shows the change in intracellular calcium ion levels in HUVECs and quantitative analysis (n = 6); (c) Western blot detects the protein expression of CaMKKβ, AMPK, p-AMPK, and Nrf2; (d) Statistical analysis of protein expression levels (n = 3).

[0020] Figure 4 This is the in vivo effect diagram of the Ag2Se@GelMA hydrogel dressing accelerating wound healing and decomposition through electrical stimulation (ES). (a) Design of the 15-day animal experiment process; (b) Thermal infrared imaging of the mouse wound shows the hydrogel temperature gradient; (c) Physical diagrams of the wound healing process in different treatment groups (0 - 15 days); (d) Schematic diagram of the pseudo-color analysis of the wound area; (e) Quantitative statistics of the wound closure rate (n = 5); (f-g) H&E and Masson staining on the 15th day show new hair follicles (red triangles) and angiogenesis (yellow circles); (h-k) Quantitative analysis of wound length, epidermal thickness, collagen deposition, and granulation thickness (n = 5). The data were analyzed by one-way ANOVA, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns indicates no statistical difference. Detailed implementation manners

[0021] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments.

[0022] Example 1

[0023] Preparation process of Ag2Se@GelMA thermoelectric hydrogel

[0024] The preparation process of Ag2Se@GelMA thermoelectric hydrogel is described in detail in this example. This hydrogel was subsequently used as a self-powered wound dressing to enhance wound healing in an electrical stimulation (ES) device. First, Ag2Se nanoparticles (NPs) were synthesized by a known method and their morphology was regulated. Then, Ag2Se NPs were mixed with the GelMA hydrogel precursor through a photocuring technique to obtain the Ag2Se@GelMA composite hydrogel. The specific synthesis method is detailed in the experimental method section.

[0025] During the wound healing process, due to the n-type semiconductor properties of Ag2Se, a directional electron movement from the warm wound to the cold hydrogel surface was achieved through the Seebeck effect, thus generating an electron pumping effect and realizing an enhanced endogenous electric field (EEF). The increased influx of electrically stimulated Ca2 + increased the intracellular Ca2 + concentration, thereby upregulating the expression of CaMKKβ, which regulates the phosphorylation of AMPK and induces downstream signaling pathways involved in enhancing mitochondrial function.

[0026] When applied as an ES device, these hydrogels promoted the acceleration of cell proliferation and migration, angiogenesis, and the improvement of mitochondrial function by activating the CaMKKβ / AMPK / Nrf2 pathway. In summary, these factors jointly promoted wound healing.

[0027] Example 2

[0028] Characterization and properties of Ag2Se@GelMA hydrogel

[0029] Preparation of Ag2Se nanoparticles (NPs)

[0030] First, Ag2Se NPs were prepared according to the mature method described in the literature. It was observed from the SEM images that the Ag2Se NPs had a uniform morphology with an average diameter of 150 nm ( Figure 2 a). The corresponding EDS analysis showed a uniform distribution of Ag, Se, and C elements, confirming the pure chemical composition of Ag2Se, which was also confirmed by EDS quantitative analysis. Bright-field TEM and HRTEM images ( Figure 2 b) showed and The lattice fringes, corresponding to the lattice spacings of the {02-2} and {100} crystal planes respectively, were observed along the [01-1] direction, confirming the crystal structure of β-Ag2Se.

[0031] Phase structure confirmation of Ag2SeNPs

[0032] To further confirm the phase structure of the obtained Ag2SeNPs, the typical XRD pattern of Ag2Se prepared at room temperature was analyzed. The XRD pattern confirmed the polycrystalline nature of Ag2Se, with relatively strong (001) and (121) peaks, which is consistent with the crystallographic characteristics of orthorhombic Ag2Se, and the lattice parameters are ( Figure 2 c).

[0033] Chemical property analysis of Ag2SeNPs

[0034] To further examine the chemical properties of the obtained Ag2Se thin film, XPS was applied. The survey spectrum of XPS and the high-resolution XPS spectra of the corresponding Ag2SeNPs showed peaks of Ag3d3 / 2, Ag3d5 / 2, Se3d3 / 2, and Se3d5 / 2 ( Figure 2 e and Figure 2 f), confirming that the prepared Ag2SeNPs are pristine Ag2Se, which is consistent with the EDS analysis results.

[0035] Preparation of Ag2Se@GelMA hydrogel

[0036] After the successful synthesis of uniform Ag2SeNPs, the Ag2Se@GelMA hydrogel was prepared by a photocrosslinking process under ultraviolet light irradiation. Figure 2 g shows the precursor and the finished product of the Ag2Se@GelMA hydrogel obtained by the photocuring method. The SEM images ( Figure 2 h) revealed the morphological characteristics of the freeze-dried hydrogels with different Ag2Se mass ratios (0.0 - 1.5 wt%). A large number of micropores were observed in the structures of all hydrogel samples, indicating the existence of pathways for the easy migration of water molecules and ions in the hydrogel. Subsequent EDS analysis showed that the Ag2SeNPs were uniformly distributed in the hydrogel matrix.

[0037] Adhesion test of Ag2Se@GelMA hydrogel

[0038] As Figure 2 shown in i, the Ag2Se@GelMA hydrogel can effectively adhere to tissue interfaces such as skin, liver, kidney, intestine, and spleen, indicating that the Ag2Se@GelMA hydrogel has good adhesion as a wound dressing material.

[0039] Thermoelectric Performance Evaluation of Ag2Se@GelMA Hydrogel To evaluate the thermoelectric performance of the obtained Ag2Se@GelMA hydrogel, the key thermoelectric parameters of Ag2Se@GelMA hydrogels with different concentrations (0.25 wt% to 1.50 wt%) at room temperature were measured, including the Seebeck coefficient S and the electrical conductivity σ( Figure 2 ). Since the original GelMA hydrogel is an ionic conductor with weak σ, it is expected that the conductivity of our conductive hydrogel comes from the combination of electronic and ionic mechanisms, where the electronic conductivity promoted by Ag2Se semiconductors is the main mode. The low S of the GelMA hydrogel also supports that the introduction of Ag2Se in the Ag2Se@GelMA hydrogel is a key component for establishing the conductive network, and the obtained thermoelectric hydrogel mainly conducts charge carriers through electronic conduction.

[0040] Calculation of the Power Factor of Ag2Se@GelMA Hydrogel

[0041] As Figure 2 shown in j, as the concentration increases, S and σ also increase, and when the mass content of Ag2Se reaches 1.0 wt%, their values become relatively stable. This observation indicates that adding 1.0 wt% of Ag2Se can effectively construct the conductive network in the Ag2Se@GelMA hydrogel. Therefore, as Figure 2 shown in j, the power factor (PF) values of the Ag2Se@GelMA hydrogel at 300 K were calculated, PF = S^2σ, and it was found that as the loading amount of Ag2Se NPs in the hydrogel increased from 0 to 1.5 wt%, the PF value also increased, indicating that the prepared composite hydrogel has potential in the field of self-powered generators. It is worth noting that the S of 1.0 wt% Ag2Se@GelMA can reach 1080.72 μV / K. Figure 2 (k)-(l) Characterize the voltage response characteristics of the Ag2Se@GelMA hydrogel to hydrodynamic stimuli and contact pressure, and its significant temperature gradient sensing performance verifies the ability of this material to

[0042] maintain a stable electrical stimulation output in a dynamic environment, confirming the technical feasibility of promoting wound repair through continuous electrical stimulation therapy (ES).

[0043] Example 3 In Vitro Experimental Verification of the Regulation of Mitochondrial Function by the Ag2Se@GelMA Composite Hydrogel Electrical Stimulation System

[0044] As Figure 3 shown, the present invention verified through in vitro experiments the effects of the Ag2Se@GelMA hydrogel combined with electrical stimulation (ES) on mitochondrial function and angiogenesis by regulating the Ca 2 + channels. As Figure 3(a) Schematic diagram of the mechanism by which Ag2Se@GelMA hydrogel combined with ES activates the CaMKKβ / AMPK / Nrf2 signaling pathway. To explore the effects of Ag2Se@GelMA hydrogel and ES on intracellular Ca 2 + levels, the Ca 2 + -sensitive fluorescent probe Fluo-3 acetoxymethyl ester (Fluo-3AM) was used to detect the difference in Ca 2 + concentration in human umbilical vein endothelial cells (HUVECs). The experimental results showed that compared with the control group (Control), GelMA group, and Ag2Se@GelMA group, the Ca 2 + concentration in HUVECs of the Ag2Se@GelMA + ES group was significantly increased (**p < 0.01), as shown in Supplementary Figure 3 (b). Further analysis by Western blot showed that the protein expression levels of CaMKKβ, AMPK, phosphorylated AMPK (P-AMPK), and Nrf2 in the Ag2Se@GelMA + ES group were significantly upregulated compared with other groups, as shown in Supplementary Figure 3 (c)-(d). The above results indicate that ES applied through the Ag2Se hydrogel thermoelectric system can effectively increase intracellular Ca 2 + levels, thereby activating the CaMKKβ / AMPK / Nrf2 signaling pathway.

[0045] Example 4

[0046] Experimental verification of the promotion of wound healing by Ag2Se@GelMA hydrogel in vivo

[0047] Experimental design

[0048] To further verify the efficacy of Ag2Se@GelMA hydrogel in wound healing, we designed an in vivo experiment. Figure 4 a shows a schematic diagram of the animal experiment process. In this study, a mouse model of skin wound healing was used.

[0049]

[0050] Experimental operation

[0051] Figure 4 b shows a photo of a mouse with an Ag2Se@GelMA hydrogel wound dressing on its back in an animal facility with a controlled room temperature of 25°C. In this experiment, an Ag2Se@GelMA hydrogel with an Ag2Se concentration of 1.0 wt% was used. The corresponding thermal infrared image shows the temperature gradient within the hydrogel matrix, confirming the temperature difference between the wound area and the hydrogel surface. This difference is attributed to the evaporative cooling effect of the hydrogel, which helps the dressing remain effective continuously.

[0052] Experimental grouping

[0053] To evaluate the therapeutic effect of the Ag2Se@GelMA hydrogel, mice were randomly divided into the following four groups: I) PBS control group, II) GelMA group, III) Ag2Se dispersion group (Ag2Se NPs suspended in the GelMA precursor solution), and IV) Ag2Se@GelMA group. After treatment under different conditions, the skin wounds were photographed on days 0, 3, 6, 9, and 12 after surgery ( Figure 4 c).

[0054] Wound healing observation and analysis

[0055] Figure 4 d The wound area at different time points was overlaid with pseudocolors to more clearly observe the healing process. Figure 4 e shows the quantitative analysis of the wound closure rate of mice in different treatment groups. Wound healing analysis showed that the Ag2Se@GelMA group, the Ag2Se dispersion group, and the GelMA group showed an obvious wound closure trend within the first 3 days, with a healing rate of about 30%, while the healing rate of the control group was less than 20%. By day 9, the wounds treated with Ag2Se@GelMA had made significant progress in healing, with a contraction rate of 84.97%, exceeding 69.45% of the control group. This trend continued until day 12, with the average remaining unhealed area of the Ag2Se@GelMA treatment group being 3.74%, while the healing effect of the control group did not exceed 90% until day 12. During the entire healing process, the wounds treated with Ag2Se@GelMA healed faster than the other treatment groups and the control group. By day 15, the wound area of the Ag2Se@GelMA group was almost completely closed.

[0056] Histological evaluation

[0057] According to the quantitative analysis, compared with the Ag2Se@GelMA group, the Ag2Se dispersion group did not show a significant positive effect on wound healing because individual Ag2Se NPs could not form an effective electric field and provide a hydrogel-like microenvironment to promote wound healing.

[0058] Figure 4Figures f and g show the hematoxylin-eosin (HE) staining and Masson staining of the regenerated skin on day 15 under different treatments. The results show that the trend of subcutaneous bleeding gradually decreases from the control group, GelMA group to the Ag2Se dispersion group and the Ag2Se@GelMA group. Diffuse distribution of red blood cells in the subcutaneous area can be observed in the tissues of the control group and the GelMA group, while in the Ag2Se@GelMA group, as indicated by the arrows for hair follicles and circles for new blood vessels, hair follicles and new blood vessel tissues are widely observed, achieving re-epithelialization at the wound center, showing a complete new epidermis that is firmly connected to the underlying granulation tissue, accompanied by capillary growth and recovery of skin appendages.

[0059] Wound re-epithelialization and granulation tissue assessment

[0060] For histological assessment of wound re-epithelialization and granulation tissue, the wound length, epithelial thickness, collagen thickness, and granulation tissue thickness under different treatments were further quantified ( Figure 4 h to k). The results show that the wounds treated with Ag2Se@GelMA showed a multi-layered epithelial structure, a significant reduction in wound length, enhanced epithelial thickness, increased collagen deposition, and enhanced granulation tissue thickness, which was attributed to increased neovascularization within the wound bed. These results indicate that the electrical stimulation generated by Ag2Se@GelMA played a key role in enhancing wound healing.

[0061] Biocompatibility assessment

[0062] To evaluate the biocompatibility of the Ag2Se@GelMA hydrogel, a comprehensive assessment was conducted on the key aspects in its potential biomedical applications. The excellent biocompatibility of the Ag2Se@GelMA hydrogel was supported, which was further confirmed by hematoxylin-eosin (HE) staining of the main organs treated with control (PBS), GelMA, Ag2Se dispersion, and Ag2Se@GelMA, as well as the corresponding blood routine analysis and blood biochemical analysis.

[0063] As described above, it is only the preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention, according to the technical solution and inventive concept of the present invention, making equivalent substitutions or changes, should be covered within the protection scope of the present invention.

Claims

1. A method for preparing a self-powered thermoelectric hydrogel material for wound healing, characterized in that: The following steps are involved: S1. Preparation of Ag2Se semiconductor nanomaterials; S2, combining Ag2Se nanomaterials with gelatin methacrylate (GelMA) hydrogel to form Ag2Se@GelMA thermoelectric hydrogel; S3, applying the prepared thermoelectric hydrogel to the wound, and generating electrical energy by utilizing the temperature difference between the wound and the environment; S4. The electrical stimulation generated by the thermoelectric hydrogel electrically activates VGCCs, upregulates intracellular calcium ion levels, and enhances mitochondrial function through the CaMKKβ / AMPK / Nrf2 signaling pathway; S5. Angiogenesis is significantly enhanced, accelerating the wound healing process.

2. The method for preparing a self-powered thermoelectric hydrogel material for wound healing according to claim 1, characterized in that: According to step S1, Ag2Se particles are prepared using an existing mature method.

3. The method for preparing a self-powered thermoelectric hydrogel material for wound healing according to claim 1, characterized in that: According to step S2, the substrate material of the thermoelectric hydrogel includes but is not limited to inorganic thermoelectric materials such as silver selenide, silicon-germanium alloy, bismuth-antimony alloy, bismuth selenide, bismuth telluride, cadmium selenide, bismuth antimony selenide, germanium-tin alloy, copper indium selenide, indium antimonide, bismuth indium selenide, and bismuth telluride, and also includes but is not limited to organic thermoelectric materials such as Pedot: PSS, polyaniline, polytriphenylamine, polyparaphenylenediamine, polythiophene, polyfluorene, polystyrene, polyacetylene, polypropyne, polymer fiber, and graphene, and also includes hydrogel thermoelectric materials such as lithium ion thermoelectric hydrogel, bismuth ion thermoelectric hydrogel, sodium ion thermoelectric hydrogel, manganese ion thermoelectric hydrogel, vanadium ion thermoelectric hydrogel, copper ion thermoelectric hydrogel, iron ion thermoelectric hydrogel, titanium ion thermoelectric hydrogel, nickel ion thermoelectric hydrogel, and iron ion thermoelectric hydrogel.

4. The method for preparing a self-powered thermoelectric hydrogel material for wound healing according to claim 1, characterized in that: According to step S2, Ag2SeNPs are mixed with a GelMA hydrogel precursor by photocuring technology to obtain an Ag2Se@GelMA composite hydrogel.

Images