Thermoelectric bionic skin for promoting healing of diabetic wound by recovering biological electric field microenvironment

Abstract

The invention relates to the technical field of biomedical materials, in particular to a thermoelectric bionic skin for promoting healing of diabetic wounds by restoring a biological electric field microenvironment, and a preparation method comprises the following steps: adding silver selenide nanoparticles into a 10% methacryloyl gelatin solution, putting the precursor solution into a mold, adding 0.25% phenyl (2, 4, 6-trimethyl-1, 3-pentanedione), and stirring for 20-30 minutes to obtain the thermoelectric bionic skin for promoting healing of diabetic wounds by restoring a biological electric field microenvironment. 2, 6-trimethylbenzoyl) lithium phosphate (LAP) photoinitiator is added, ultraviolet light curing forming is carried out, and after demolding, soaking is carried out to remove residual monomers and the initiator; wherein the drug-containing group is prepared by replacing water with an artemisinin solution, and Ag2Se-coated GelMA or Ag2Se-coated GelMA / ART composite hydrogel is obtained. The electric field generated by the thermoelectric effect in the invention activates a voltage-gated calcium ion channel, inhibits PHD2, and up-regulates an HIF-1 / VEGF-A signal axis, thereby promoting angiogenesis. Meanwhile, the artemisinin also plays an anti-inflammatory role.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medical materials, in particular to a pyroelectric biomimetic skin for promoting diabetic wound healing by restoring bioelectric field microenvironment. BACKGROUND

[0002] Diabetes mellitus is a major global health challenge, with an estimated 500 million patients worldwide. About one-third of these patients suffer from chronic skin wounds, a serious complication of diabetes. In healthy skin tissue, the asymmetric distribution of ion channels in epithelial cells drives the directional transport of cations (Na + ) to the basal side and anions (Cl - ) to the apical side, generating a bioelectric field. Once the skin is damaged, an endogenous electric field of 40-200 mV mm -1 can be generated in healthy tissues to guide wound healing in the damaged microenvironment. However, relevant studies have shown that in a microenvironment characterized by hypoxia, hyperglycemia, and inflammation, the endogenous electric field is significantly weakened, leading to the formation of chronic wounds. Thus, the introduction of an external electric field to repair the bioelectric field microenvironment as a method to treat diabetic wounds has emerged. However, in clinical practice, traditional electrical stimulation relies on bulky external equipment, which needs to be improved in terms of comfort.

[0003] In recent years, self-powered electronic skin and smart dressings have attracted much attention due to their integration and the advantage of not requiring batteries. However, most of them are based on piezoelectric and triboelectric technology, which are difficult to simulate the directional bioelectric field of natural skin. Thermoelectric materials can directly convert the temperature difference between the human body and the environment into electrical energy. Moreover, the direction of the generated bioelectric field can be determined by the properties of the semiconductor. Recent studies have also shown that hydrogels based on thermoelectric effects can effectively reconstruct endogenous electric fields and accelerate wound healing. However, existing hydrogels need to be improved in terms of thermoelectric performance, biocompatibility, response to wound inflammatory reactions, and mechanisms for promoting chronic wound healing. Therefore, it is of great significance to explore a hydrogel with high thermoelectric performance and multifunctional synergistic promotion of chronic wound healing.

[0004] In summary, traditional technology relies on external battery power. Related technology cannot simulate the directional bioelectric field of natural skin; biocompatibility is difficult to guarantee; the mechanism for accelerating wound healing needs to be further explored; therefore, further research is still needed. SUMMARY

[0005] The application provides a thermoelectric bionic skin (Ag2Se@GelMA / ART) based on silver selenide (Ag2Se) nanoparticles loaded in a gelatin methacrylamide (GelMA) hydrogel matrix, to solve the problem that the endogenous bioelectric field is weakened due to the hypoxia, hyperglycemia and inflammatory state of the microenvironment of a chronic wound of diabetes, leading to slow wound healing, and the problem that a traditional electric stimulation device is bulky, power-dependent on an external power supply, and cannot dynamically restore and regulate the electric field microenvironment of the wound.

[0006] In order to achieve the above-mentioned purpose, the application is realized by the following technical scheme, A thermoelectric bionic skin for promoting diabetic wound healing by restoring a bioelectric field microenvironment, characterized by comprising the following steps: S1: preparing silver selenide (Ag2Se) nanoparticles, specifically, generating silver selenide (Ag2Se) nanoparticles by reacting selenium dioxide, ascorbic acid and silver nitrate in an aqueous phase; S2: dissolving artemisinin (ART) in dimethyl sulfoxide, adding physiological saline to adjust the volume, oscillating and mixing to obtain a concentrated mother liquor, and diluting to a required concentration for standby; S3: adding silver selenide (Ag2Se) nanoparticles to a 10% methacryl gelatin solution, placing the previous precursor solution in a mold, adding 0.25% lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (LAP) photoinitiator, ultraviolet light curing, and soaking after demolding to remove residual monomers and initiators; wherein a drug-containing group is prepared by replacing water with an artemisinin solution to obtain Ag2Se@GelMA or Ag2Se@GelMA / ART composite hydrogel.

[0007] Preferably, the average particle size of the Ag2Se nanoparticles is 50-200 nanometers, which is uniformly dispersed and has excellent room-temperature thermoelectric performance.

[0008] Preferably, the concentration of the GelMA hydrogel is 8%-12% (mass / volume ratio), so that the hydrogel has both mechanical strength and flexibility, and is suitable for skin fitting.

[0009] Preferably, the concentration of the artemisinin solution is 25-150 μM, which endows the hydrogel with anti-inflammatory function, so as to promote the biological repair of the wound in cooperation with the thermoelectric effect.

[0010] The application also includes a diabetic wound individualized repair method of a thermoelectric bionic skin for promoting diabetic wound healing by restoring a bioelectric field microenvironment, and the specific steps include: S1: attaching the Ag2Se@GelMA / ART hydrogel bionic skin to a chronic wound of diabetes of a patient; S2: Utilize the temperature difference between the patient's skin and the outside world to stimulate the thermoelectric effect of Ag2Se nanoparticles in the hydrogel, generate a stable and directional bioelectric field, simulate and restore the damaged endogenous bioelectric field; S3: The hydrogel releases ART drugs to inhibit local inflammation, while the thermoelectric bioelectric field activates the voltage-gated calcium channel of endothelial cells, inhibits PHD2 protein expression, activates the HIF-1α / VEGF-A signal pathway, promotes neovascularization and collagen deposition, and accelerates wound healing.

[0011] Compared with the prior art, the thermoelectric bionic skin for promoting diabetic wound healing by restoring the bioelectric field microenvironment has the advantages that: The Ag2Se@GelMA / ART bionic skin can generate a bioelectric field through the temperature gradient between the skin and the air by using the thermoelectric effect, thereby providing a new method for restoring the endogenous electric field. In vitro studies show that the Ag2Se@GelMA / ART bionic skin can promote the proliferation, migration and vascularization of human umbilical vein endothelial cells. In in vivo experiments, the bionic skin accelerates diabetic wound healing, promotes neovascularization and collagen deposition. Mechanism studies show that the electric field generated by the thermoelectric effect activates the voltage-gated calcium ion channel, inhibits PHD2, and up-regulates the HIF-1 / VEGF-A signal axis, thereby promoting angiogenesis. At the same time, artemisinin also plays an anti-inflammatory role. This thermoelectric bionic skin introduces a new treatment strategy for diabetic care, and provides a new direction for tissue engineering and regenerative medicine. BRIEF DESCRIPTION OF DRAWINGS

[0012] Figure 1 The Ag2Se nanomaterial and the characterization results of the bionic electronic skin based on Ag2Se@GelMA / ART hydrogel are shown in the figure; Figure 2 The thermoelectric properties and functional characterization of Ag2Se@GelMA / ART hydrogel are shown in the figure; Figure 3 The effect of Ag2Se@GelMA / ART and its electric field (EF) system on the biological behavior of human umbilical vein endothelial cells (HUVECs) is shown in the figure; Figure 4 The mechanism of Ag2Se@GelMA / ART / EF promoting angiogenesis by activating the HIF-1α / VEGF-A signal axis is shown in the figure; Figure 5 The research figure of Ag2Se@GelMA / ART thermoelectric bionic skin promoting diabetic mouse skin defect healing is shown in the figure; Figure 6To show the effect of Ag2Se@GelMA / ART thermoelectric bionic skin on promoting angiogenesis at the histological level; Figure 7 To show the effect of Ag2Se@GelMA / ART thermoelectric bionic skin on promoting angiogenesis and accelerating wound healing through the HIF-1α / VEGF-A signal axis; Figure 8 To show the effect of artemisinin on promoting wound healing; Figure 9 To show the elemental imaging of the distribution of carbon, selenium and silver elements in the hydrogel by energy spectrum analysis technology; Figure 10 To show the adhesion ability of Ag2Se@GelMA / ART thermoelectric bionic skin to different tissues and substances; Figure 11 To show the experimental device for generating bioelectric field by hydrogel for in vitro experiment; Figure 12 To show the device for measuring the voltage of mouse skin wound; Figure 13 To show the H&E staining of various organ tissues in each experimental group after 15 days of treatment; Figure 14 To show the blood routine examination of mice in each group after 15 days of treatment. The data is expressed as mean ± standard deviation; Figure 15 To show the blood biochemical test results of mice in each group after 15 days of treatment. The data is expressed as mean ± standard deviation. DETAILED DESCRIPTION

[0013] The embodiments of the present application are described in detail below, examples of which are shown in the accompanying drawings, wherein the same or similar reference numerals represent the same or similar elements or elements with the same or similar functions throughout. The embodiments described below by reference to the accompanying drawings are exemplary and are intended to explain the present application, and cannot be understood as a limitation of the present application.

[0014] The specific data in each figure is explained as follows: Figure 1a-b are the scanning electron microscope (SEM) images of Ag2Se nanomaterials and their silver and selenium element distribution maps; c is the energy dispersive X-ray spectroscopy (XRD) of Ag2Se nanomaterials; d is the X-ray diffraction (XRD) spectrum of Ag2Se nanomaterials and its standard spectrum; e-f are the transmission electron microscope (TEM) and high-resolution transmission electron microscope (HRTEM) images of Ag2Se nanomaterials; g is the fast Fourier transform (FFT) image corresponding to the image f; h is the atomic structure schematic diagram of the image f; i-k are the X-ray photoelectron spectroscopy (XRD) scanning and amplified silver and selenium peaks of Ag2Se nanomaterial powder; l is the SEM image of GelMA hydrogel, GelMA / ART hydrogel, Ag2Se@GelMA hydrogel and Ag2Se@GelMA / ART hydrogel; Figure 2 a are the Seebeck coefficients (S) S ), conductivities (σ) σ ) and power factors (PF) PF ) of GelMA hydrogel, GelMA / ART hydrogel, Ag2Se@GelMA hydrogel and Ag2Se@GelMA / ART hydrogel; b is the Seebeck coefficient curve of Ag2Se@GelMA / ART hydrogel with time and its digital photo of ultraviolet curing process; c is the release curve of silver ions and Ag2Se in Ag2Se@GelMA / ART hydrogel with time; d is the release curve of artemisinin molecules from Ag2Se@GelMA / ART hydrogel with time; e is the expansion rate curve of GelMA, GelMA / ART, Ag2Se@GelMA and Ag2Se@GelMA / ART hydrogel with time; f is the voltage response of Ag2Se@GelMA / ART hydrogel to environmental airflow, showing its environmental response type electric stimulation function; g is the digital photo of the adhesion of Ag2Se@GelMA / ART hydrogel on different substrates; Figure 3a The optimal concentration of Ag2Se@GelMA extract and artemisinin solution was determined; b The relative viability of HUVECs under lipopolysaccharide (LPS) conditions; c The live-dead cell fluorescence staining image at 24 hours, the scale bar is 200 pm; d Quantitative analysis of the proportion of live cells at 24 hours; e The live-dead cell fluorescence staining image at 48 hours, the scale bar is 200 pm; f Quantitative analysis of the proportion of live cells at 48 hours; g The microscope image of scratch test, showing the cell migration at 0 hours, 24 hours and 48 hours, the scale bar is 200 pm; h Quantitative analysis of scratch residual area; i The process of tubular network formation of human umbilical vein endothelial cells, the scale bar is 200 pm; j Quantitative analysis of the total length of tubular network and the number of junctions. The figure data is expressed as mean ± standard deviation (SD) (n = 3). n p p p Figure 4 a Immunofluorescence staining of PHD2. The heat map image showing the expression level of PHD2; b Quantitative analysis of PHD2 fluorescence intensity; c Immunofluorescence staining of HIF-1a. The heat map image showing the expression level of HIF-1a; d Quantitative analysis of HIF-1a fluorescence intensity; e Immunofluorescence staining of VEGF-A; f Quantitative analysis of VEGF-A fluorescence intensity. The figure data is expressed as mean ± standard deviation (SD) (n = 3). n p p p Figure 5 ​​​​​​​​Image a shows a schematic diagram of the establishment and healing treatment process of a full-thickness skin defect model in diabetic mice; image b shows a thermal imaging of the temperature gradient on the cross-section of the mouse's back using thermoelectric bionic skin; image c shows typical potential curves of skin wounds in healthy mice, diabetic mice, and diabetic mice in the treatment group; image d shows representative images of wounds in diabetic mice at different healing time points; image e shows a combined overlay of wound contours at each time point, displaying the healing process; image f shows a quantitative analysis of wound contraction rate, with data expressed as mean ± standard deviation (n = 5); image g shows H&E stained sections of wound tissue on day 15; image h shows Masson stained sections of wound tissue on day 15; image i shows a quantitative statistical analysis of wound edge distance, with data expressed as mean ± standard deviation (n = 5), ***p < 0.001, **p < 0.01, *p < 0.05 compared with the Ag2Se@GelMA / ART hydrogel group; image j shows a quantitative analysis of collagen deposition on day 15 based on Masson staining, with data expressed as mean ± standard deviation (n = 5). n = 3) indicates that ***p < 0.001, **p < 0.01, *p < 0.05 are compared with the Ag2Se@GelMA / ART hydrogel group; Figure 6 Images a, c, and e show immunofluorescence staining images of the angiogenesis markers CD31, VE-cadherin, and α-smooth muscle actin (α-SMA), respectively, with a scale bar of 100 μm. Images b, d, and f show the quantitative analysis of the corresponding CD31, VE-cadherin, and α-SMA protein expression. Data are presented as mean ± standard deviation (n = 5), with ***p < 0.001, **p < 0.01, and *p < 0.05, compared with the Ag2Se@GelMA / ART hydrogel group. Thermal imaging showed the expression levels of the corresponding proteins. Figure 7 Images a, c, and e show immunofluorescence staining images of PHD2, HIF-1α, VEGF-A, and VEGF-R2, respectively, with a scale bar of 100 μm. Images b, d, and f show quantitative analyses of the corresponding PHD2, HIF-1α, VEGF-A, and VEGF-R2 protein expression. Data are expressed as mean ± standard deviation (SD) (n = 5), with ***p < 0.001, **p < 0.01, and *p < 0.05. Comparison with the Ag2Se@GelMA / ART hydrogel group was also included. Thermal imaging showed the expression levels of the corresponding proteins. Figure 8Fig. 1. Immunofluorescence staining of TNF-a and IL-1 b in the Ag2Se@GelMA / ART hydrogel with and without ART. Scale bar, 100 pm. Fig. 2. Quantitative analysis of TNF-a and IL-1 b protein expression in the Ag2Se@GelMA / ART hydrogel with and without ART. Data are presented as mean ± standard deviation (n = 5). ***p < 0.001, **p < 0.01, *p < 0.05 compared with the Ag2Se@GelMA / ART hydrogel group. Thermal imaging shows the expression level of the corresponding protein. Figure 13 Scale bar, 200 pm.

[0015] Example 1. Synthesis and characterization of Ag2Se@GelMA / ART hydrogel S1) Synthesis of silver selenide (Ag2Se) nanoparticles Dissolve 1.0 gram of selenium dioxide (Se02) in 200 milliliters of distilled water in a glass beaker and continuously stir to form solution A. At the same time, dissolve 4.0 grams of ascorbic acid in another beaker in 200 milliliters of distilled water to prepare solution B. Slowly add solution B to solution A while maintaining continuous stirring. After about 5 minutes of stirring, for the synthesis of Ag2Se nanomaterials, an appropriate amount of silver nitrate (AgN03) solution is slowly added to the above mixture and continue stirring for 1 hour. Centrifuge the mixture at 4000 revolutions per minute for 5 minutes, discard the supernatant, and collect the Ag2Se nanomaterials. Subsequently, the particles are purified by repeated cycles of washing with deionized water and centrifugation.

[0016] S2) Preparation of artemisinin solution Dissolve artemisinin (Macklin China) in dimethyl sulfoxide (DMSO, 2 milliliters, ACMEC, China) and mix well by shaking. Then add normal saline to adjust the total volume to 50 milliliters, followed by vigorous shaking for 30 minutes to prepare a 1000-fold concentrated stock solution. This stock solution is then diluted stepwise to obtain artemisinin (ACT) solutions with concentrations of 25, 50, 75, 100, and 150 micromolar (pM), respectively, and stored at 4 °C for subsequent experiments.

[0017] S3) Synthesis of Ag2Se@GelMA / ART hydrogel (with and without artemisinin) Selenium silver (Ag2Se) nanomaterials were added to 10% GelMA solution (Suzhou Smart Manufacturing Research Institute, China) at a concentration of 1%. After thorough mixing, 0.25% of the photoinitiator, phenyl-2,4,6-trimethylbenzoylphosphine oxide lithium (LAP) (Everlight, China) was added. The mixture was then exposed to UV light for curing, i.e. photo-crosslinking via UV light initiation, to load the selenium silver (Ag2Se) nanomaterials (and / or ART) into the GelMA. The cured hydrogel was carefully demolded and soaked in Milli-Q water to remove residual monomers and initiators. For the Artemisinin-containing hydrogel group, when preparing the GelMA hydrogel precursor, the corresponding concentration of Artemisinin solution was used to replace water according to the operating procedure to prepare GelMA / ART or Ag2Se@GelMA / ART hydrogels.

[0018] S4) Ag2Se@GelMA / ART hydrogel biomimetic skin characterization and characterization results To observe the morphology of the prepared selenium silver (Ag2Se) nanomaterials, we performed scanning electron microscopy (SEM) analysis (Figure 1). Figure 1 a). The results showed that the synthesized selenium silver (Ag2Se) nanomaterials had uniform microstructure and particle size.

[0019] Subsequently, we further performed chemical composition analysis using energy dispersive X-ray spectroscopy (EDS) technology to verify its chemical properties. Figure 1 b shows the overlapping elemental distribution map of silver (Ag) and selenium (Se) elements, Figure 1 c shows the corresponding energy dispersive X-ray spectroscopy (EDS) spectrum. Subsequently, we performed X-ray photoelectron spectroscopy (XPS) analysis to verify the chemical composition of the selenium silver (Ag2Se) nanomaterials (Figure 2). Figure 1 i). As shown in Figure 1 k, the characteristic doublet of Ag 3d 5 / 2 and Ag 3d 3 / 2 corresponds to the silver element, while the doublet of Se 3d 5 / 2 and Se 3d 3 / 2 belongs to the selenium element. The identification of these peaks directly confirms the presence of silver and selenium elements in the sample, which is consistent with the chemical formula of selenium silver (Ag2Se).

[0020] In addition, we used X-ray powder diffraction (XRD) technology to detect the phase composition of the selenium silver (Ag2Se) nanomaterials (Figure 3). Figure 1d), and simultaneously using the detected intermediate product (selenium powder) and the standard diffraction pattern of β-Ag2Se (PDF #00-024-1041) as references. The diffraction pattern matched the standard diffraction pattern, further confirming that the silver selenide (Ag2Se) nanomaterials are composed of β-Ag2Se.

[0021] To study the structural characteristics of silver selenide (Ag2Se) nanomaterials, Figure 1 Images e and 1f show bright-field transmission electron microscopy (TEM) and high-resolution transmission electron microscopy (HRTEM) images, respectively, revealing lattice fringes corresponding to 2.6 Å and 4.3 Å. Subsequently, Figure 1 g displays the corresponding Fast Fourier Transform (FFT) pattern. Based on this pattern, Figure 1 The atomic structure shown in f was further drawn. Figure 1 In g, it matches the previous theoretical structure.

[0022] Subsequently, we acquired scanning electron microscope (SEM) images of GelMA hydrogel, GelMA / ART hydrogel, Ag2Se@GelMA hydrogel, and Ag2Se@GelMA / ART hydrogel. Figure 1 l) and its corresponding distribution map of C, Ag and Se elements ( Figure 9 The results indicate that the Ag2Se nanomaterials are uniformly distributed within the hydrogel matrix after introduction. This porous structure facilitates the absorption of wound exudate, promotes drug release, and facilitates nutrient exchange, making it suitable for wound dressing materials.

[0023] Example 2: Identification of the thermoelectric properties, release properties, and material stability of the Ag2Se@GelMA / ART hydrogel biomimetic skin of the present invention. 1) Thermoelectric performance testing To evaluate the thermoelectric properties of hydrogels loaded with silver selenide (Ag₂Se) nanomaterials, we measured and analyzed several key parameters. The measurements revealed that GelMA hydrogels without silver selenide (Ag₂Se) nanomaterials exhibited a lower Seebeck coefficient. S ) and conductivity ( σ GelMA significantly improved performance by loading silver selenide (Ag2Se) nanomaterials. S , σ and power factor ( PF ), reaching a level sufficient to jointly establish a bioelectric microenvironment with the endogenous electric field ( Figure 2 a).

[0024] When used as a wound dressing, the thermoelectric hydrogel must maintain relatively stable thermoelectric properties within a clinically relevant time period. Therefore, we monitored the time-dependent changes in the thermoelectric properties of Ag2Se@GelMA / ART hydrogels. The measurements showed that the thermoelectric output gradually decreased over time, but remained at a high level within 7 days Figure 2 b), suitable for clinical use, such as replacing the dressing every 3 days.

[0025] In addition, the airflow in the living environment can adjust the temperature difference between the upper and lower surfaces of the thermoelectric hydrogel, thereby generating an additional pulsed bioelectric field to promote wound healing. As shown in Fig. f, the Ag2Se@GelMA / ART hydrogel exhibits a clear voltage response under the action of airflow. Figure 2

[0026] 2) Release performance test To understand the dynamic release behavior of silver selenide (Ag2Se) nanomaterials from the hydrogel matrix, ICP-MS analysis was performed to determine the Ag2Se content in the Ag2Se@GelMA / ART hydrogel Figure 2 c) During the 13-day period, there was almost no leakage of nanoparticles from the Ag2Se@GelMA and Ag2Se@GelMA / ART hydrogels, ensuring the stability of their performance as self-powered biomaterials. In addition, the release curve of artemisinin from the hydrogel was determined by ultraviolet spectrophotometry Figure 2 d) The corresponding calibration curve is shown in Fig. S2. The results show that artemisinin is released rapidly within the first 24 hours of immersion in water, and then the release rate tends to be flat after 96 hours. In contrast, the release rate of Ag2Se@GelMA / ART hydrogel is slightly lower than that of GelMA / ART hydrogel, which may be attributed to the difference in the swelling properties of the two.

[0027] 3) Swelling and adhesion test Hydrogels have the ability to absorb wound exudates and maintain a moist environment conducive to healing, and their absorption and water retention properties are reflected in the swelling behavior. To test the material stability of Ag2Se@GelMA / ART hydrogels, we performed a swelling test. Figure 2 The swelling curves shown in Fig. e show that the hydrogels in each group rapidly swell within a few hours after immersion and tend to be balanced after 12 hours. In addition, the thermoelectric biomimetic skin of the present application exhibits strong adhesion to a variety of substrates including fingers, skin, plastic, glass, paper, internal organs, and small intestines Figure 2 g、 Figure 10 ), which reflects its good potential for clinical application.

[0028] Example 3 Evaluation of the effect of the Ag2Se@GelMA / ART / EF system on the behavior of HUVECs cells.

[0029] ​To clarify the role of thermoelectric biomimetic skin in wound healing, and human umbilical vein endothelial cells (HUVECs) play a key role in the repair of diabetic wounds, this example selected HUVECs as the in vitro experimental object. To simulate the microbioelectric field generated by the skin and the environment temperature difference when the hydrogel dressing works, and in view of the small temperature difference in vitro, we created an artificial electric field (EF) to simulate the possible electric stimulation (ES). The experiment was divided into five groups: control group, Ag2Se@GelMA group, Ag2Se@GelMA / ART group, Ag2Se@GelMA / EF group and Ag2Se@GelMA / ART / EF group. The control group was cultured in standard medium as the baseline. The Ag2Se@GelMA group and the Ag2Se@GelMA / ART group were cultured in Ag2Se@GelMA extract and Ag2Se@GelMA extract containing artemisinin, respectively, to simulate the biochemical microenvironment. In contrast, the Ag2Se@GelMA / EF group and the Ag2Se@GelMA / ART / EF group were cultured in the corresponding solution, and the external electric field was provided by the external electric device.

[0030] 1) Preparation of Ag2Se@GelMA extract and Ag2Se@GelMA extract containing artemisinin The prepared Ag2Se@GelMA hydrogel was treated with ultraviolet sterilization. Then, it was immersed in the freshly prepared complete medium (DMEM) at a ratio of 1 cm 3 The hydrogel was immersed in the freshly prepared complete medium (DMEM) at a ratio of 1 cm

[0031] 2) Determination of the optimal concentration of Ag2Se@GelMA extract and artemisinin solution Since the biocompatibility of hydrogel wound dressings is crucial, we first studied the optimal concentration of Ag2Se@GelMA extract and artemisinin solution (a), and used these concentrations in subsequent in vitro experiments. The experimental device of in vitro experiment is shown in Figure 3 a), and used these concentrations in subsequent in vitro experiments. The experimental device of in vitro experiment is shown in Figure 11 Subsequently, the CCK8 method was used to evaluate cell viability. Under non-inflammatory conditions, after 12 hours of treatment, the relative viability of HUVECs in the Ag2Se@GelMA and Ag2Se@GelMA / ART treatment groups did not decrease significantly compared with the control group, while the relative viability of the Ag2Se@GelMA / EF and Ag2Se@GelMA / ART / EF groups was slightly improved (b). Figure 3b). This trend remained at 24 hours and 48 hours, indicating that the synthesized hydrogels not only have no cytotoxicity, but also help cell proliferation under the action of bioelectric field.

[0032] 3) In vitro, simulate inflammatory wound microenvironment by HUVECs to explore the influence of each group of hydrogels on the proliferation ability of the cells HUVECs were induced to be in an inflammatory state by lipopolysaccharide (LPS), and then treated with the corresponding components. Then, Calcein / PI kit was used to evaluate cell viability and cytotoxicity at 24 hours and 48 hours. As shown in Figure 3 c, 3d, 3f, within 24 hours, the ratio of live cells to dead cells in Ag2Se@GelMA group and Ag2Se@GelMA / ART group showed no significant difference from the control group. However, Ag2Se@GelMA / EF and Ag2Se@GelMA / ART / EF groups showed a significantly higher proportion of living cells, and the survival / death cell ratio of Ag2Se@GelMA / ART / EF group was the highest. As shown in Figure 3 d, 3e, 3f and the figure, this trend remained basically unchanged at 48 hours. These results further prove that the synthesized Ag2Se@GelMA and Ag2Se@GelMA / ART are almost non-toxic, and under the action of bioelectric field, they are conducive to the proliferation of HUVECs. Especially in the presence of artemisinin, inflammation is inhibited and cell proliferation is enhanced.

[0033] 4) HUVECs scratch test In the inflammatory microenvironment, the scratch test of 100% confluence of HUVECs was carried out for each group, Figure 3 g and 3h show the migration ability of HUVECs treated with different hydrogel extracts. Ag2Se@GelMA group and Ag2Se@GelMA / ART group slightly promote the migration of HUVECs. However, after 48 hours, there is still a clear scratch in Ag2Se@GelMA group and Ag2Se@GelMA / ART group. In contrast, the scratch healing rate of Ag2Se@GelMA / EF group and Ag2Se@GelMA / ART / EF group is significantly improved, and the effect of Ag2Se@GelMA / ART / EF group is better than that of Ag2Se@GelMA / EF group. After 48 hours, the scratch of Ag2Se@GelMA / ART / EF group is almost completely closed. These results show that the hydrogels we prepared have the potential to promote the healing of inflammatory wounds, and the anti-inflammatory effect of artemisinin further enhances this potential.

[0034] 5) Tube formation experiment Tube formation assay is a widely used technique to assess angiogenesis in vitro. HUVECs are seeded on a gel matrix consisting of low growth factor basement membrane extract to evaluate their ability to organize into three-dimensional capillary-like structures. It was observed that HUVECs undergo differentiation and directional migration, eventually forming branched tubular polygonal networks that reflect the angiogenic potential of different stimulatory factors. We obtained tube formation images after 6 hours for different treatment groups under inflammatory conditions and calculated the total length, node number, mesh number, and mesh area using ImageJ software. The results show that Figure 3 i、 Figure 3 j), the tube formation ability of Ag2Se@GelMA group and Ag2Se@GelMA / ART group is comparable to the control group. In contrast, the Ag2Se@GelMA / EF group exhibits a significantly increased tube density, and the tube density of the Ag2Se@GelMA / ART / EF group is further enhanced. These data confirm the angiogenic potential of our thermoelectric hydrogel and further enhance this potential when artemisinin exerts its anti-inflammatory effect.

[0035] Example 4 The Ag2Se@GelMA / ART hydrogel of the present application modulates the biological properties of HUVECs to perform HIF-1α / VEGF-A signal activation and inflammation suppression by Ag2Se@GelMA / ART / EF system During the wound healing process, the formation of new blood vessels is achieved through the branching, branching, and extension of existing capillaries, which is crucial for the successful repair of wounds. Angiogenesis (or neovascularization) is a physiological process driven by key growth factors such as vascular endothelial growth factor (VEGF). Chronic wounds in diabetic patients often have angiogenesis and vascular formation disorders, the main causes of which include high blood sugar and chronic inflammation leading to dysfunction and recruitment of bone marrow-derived endothelial progenitor cells (EPCs). In detail, under normal oxygen conditions, prolyl hydroxylase domain (PHD) enzymes mark HIF-1α protein for proteasomal degradation by hydroxylating it, maintaining low activity. Under hypoxic conditions, as the activity of PHD enzymes depends on oxygen, their hydroxylation function is weakened, leading to the stable presence of HIF-1α and its translocation into the nucleus, where it forms a heterodimer with HIF-1β, which in turn induces the transcriptional expression of target genes such as VEGF, promoting angiogenesis. In a high-glucose environment, HIF-1α function is inhibited, impairing the hypoxic adaptation response and thus delaying wound repair. In this example, we promote the release of VEGF at the wound site by applying electrical stimulation to promote the formation of blood vessels.

[0036] 1) Inducing HUVECs to analyze HIF-1α and VEGF-A expression by immunofluorescence staining under a simulated inflammatory microenvironment In this example, we used lipopolysaccharide to induce HUVECs to simulate the inflammatory microenvironment of diabetic wounds. After treating the cells with the corresponding components, we analyzed the expression of HIF-1a and VEGF-A by immunofluorescence staining (Fig. 4a, 4b). Figure 4 Corresponding thermal imaging and quantitative fluorescence analysis (Fig. 4d, 4f) showed that, compared with the control group, Ag2Se@GelMA and Ag2Se@GelMA / ART groups did not significantly increase the expression of HIF-1a and VEGF-A; while Ag2Se@GelMA / EF and Ag2Se@GelMA / ART / EF groups significantly enhanced the expression levels of the two. This result indicates that the local electric field generated by electrical stimulation activates the HIF-1a / VEGF-A signaling axis, thereby affecting the behavior of HUVECs in the inflammatory microenvironment. Figure 4

[0037] 2) Immunofluorescence staining and quantitative analysis of PHD2 Since the HIF-1a / VEGF-A signaling axis is regulated by prolyl hydroxylase (PHD), and PHD2 is a key oxygen sensor in the HIF degradation pathway, we further performed immunofluorescence staining and quantitative analysis of PHD2 (Fig. 4a, 4b). The results showed that, compared with the control group and Ag2Se@GelMA and Ag2Se@GelMA / ART groups, the expression of PHD2 in Ag2Se@GelMA / EF and Ag2Se@GelMA / ART / EF groups was significantly reduced, indicating that the introduced electric field activated the HIF-1a / VEGF-A signaling axis by down-regulating PHD2 expression. Figure 4

[0038] Example 5 Healing of skin damage in diabetic mice by Ag2Se@GelMA / ART hydrogel of the present application To accurately evaluate and compare the therapeutic effect of Ag2Se@GelMA / ART biomimetic skin, rats were randomly divided into five groups: (I) control group, (II) GelMA group, (III) GelMA / ART group, (IV) Ag2Se@GelMA group, and (V) Ag2Se@GelMA / ART group. The experimental process is shown in Fig. 5a. Figure 5

[0039] 1) Establishment of full-thickness skin defect model in diabetic miceA high blood glucose mouse model was induced by daily injection of streptozotocin (STZ) for five consecutive days; mice with blood glucose levels ≥20 mmol / L were included in the study. A full-thickness skin defect model was established in diabetic mice as an in vivo experimental model. Figure 5 b shows the temperature gradient of the thermoelectric biomimetic skin cross-section.

[0040] ​​​2) Temperature gradient and voltage measurement of the pyroelectric bionic skin on mice We measured the temperature gradient across the cross-section of the pyroelectric bionic skin on mice Figure 5 b). Subsequently, the voltage at the wound site of the mouse skin was measured using a special voltage measurement device (shown in Figure 12 ). Figure 5 c shows that the endogenous electric field at the wound site of diabetic mice is weaker than that of healthy mice. After covering the back wound for 6 hours, the Ag2Se@GelMA / ART bionic skin significantly restores the local endogenous electric field.

[0041] 3) Monitoring of the wound healing process in mice After treating the wounds with each group of dressings, respectively, photographs of the wounds were taken on days 0, 3, 6, 9, 12, and 15 (d) Figure 5 ), Figure 5 e Superimposes the wound area at different time points with pseudo-color to more intuitively show the healing process. Figure 5 f Quantitative analysis of the wound closure rate of rats in different treatment groups. Diabetic wounds usually stagnate during the inflammatory stage. In this example, the wound contraction rates of the control group, GelMA group, GelMA / ART group, Ag2Se@GelMA group, and Ag2Se@GelMA / ART group were 23.54%, 37.07%, 56.58%, 60.09%, and 60.15%, respectively, on day 3. The Ag2Se@GelMA and Ag2Se@GelMA / ART groups were significantly better than the other three groups, and the difference between the two groups was small at this time point. This trend continued until days 6, 9, and 12. By day 15, the wound contraction rates of the five groups were 90.50%, 95.20%, 97.56%, 98.83%, and 100%, respectively. At this time, the wound contraction rate of the control group was just over 90%, while the wound of the Ag2Se@GelMA / ART group was completely closed. The wound contraction rate of the Ag2Se@GelMA group was significantly higher than that of the control group and close to that of the Ag2Se@GelMA / ART group, but there was still a small amount of scab on the wound surface.

[0042] 4) Tissue section staining of the healing area to study inflammation cells, collagen, and blood vessel formation Tissue sections were taken from the healing area and stained. The results of hematoxylin-eosin (H&E) staining (a Figure 5g) showed that the wound surface of the control group and GelMA group had crusting, but no continuous epithelium had formed. The dermis contained a large number of inflammatory cells, but no blood vessels or skin appendages were formed. The wound surface of the GelMA / ART group was also covered with crusting, and there was a clear gap between the newly formed epithelium and the underlying granulation tissue. Due to the presence of artemisinin, the number of inflammatory cells in the dermis was significantly reduced, but no blood vessels or skin appendages were formed. The epithelialization of the Ag2Se@GelMA group was more complete, the epithelium and granulation tissue were tightly connected, and new blood vessels were clearly seen in the dermis. Based on the Ag2Se@GelMA group, the Ag2Se@GelMA / ART group showed more complete epithelial regeneration and more new blood vessel formation. Masson staining ( Figure 5 h) and collagen deposition analysis ( Figure 5 j) Further analysis showed that the control group and GelMA group had less collagen deposition. Only localized, irregular collagen deposition was observed in the GelMA / ART group. In contrast, collagen deposition was significantly increased in the Ag2Se@GelMA and Ag2Se@GelMA / ART groups, and the deposition was uniform and well-organized. During wound healing, angiogenesis and collagen deposition work synergistically; moreover, collagen containing the arginine-glycine-aspartic acid (RGD) structure provides an optimal microenvironment for blood vessels, promoting angiogenesis and jointly accelerating wound closure. These results indicate that this self-powered thermoelectric biomimetic skin can promote the healing of refractory diabetic wounds, and the presence of artemisinin further enhances this effect.

[0043] 5) Measurement of PHD2 expression at the tissue level To verify the results of the in vitro experiment in Example 4, immunofluorescence staining was used to detect PHD2 at the tissue level. Figure 7 a) HIF-1α Figure 7 c), VEGF-A ( Figure 7 e) and VEGF-R2 ( Figure 7 The expression of e) was analyzed quantitatively. Figure 7 (b), (7d), (7f). Consistent with the results of step 2), PHD2 expression in the Ag2Se@GelMA group and the Ag2Se@GelMA / ART group was significantly lower than in the other three groups. Conversely, HIF-1α expression in the GelMA / ART group was slightly higher than in the control group and the GelMA group, while it was significantly higher in the Ag2Se@GelMA group and the Ag2Se@GelMA / ART group than in the other groups, with the Ag2Se@GelMA / ART group exhibiting the highest expression level. The expression trends of VEGF-A and VEGF-R2 were consistent with those of HIF-1α, indicating that the expression of VEGF-A and VEGF-R2 was induced by HIF-1α. Further inference suggests that the changes in the HIF-1α / VEGF-A / VEGF-R2 signal axis were triggered by alterations in PHD2 expression, consistent with theoretical analysis.

[0044] 6) Biocompatibility test of hydrogel The biocompatibility of hydrogel is the key for its biomedical application. Therefore, the biocompatibility of Ag2Se@GelMA / ART biomimetic skin was systematically evaluated in this example. At the 15th day when the skin samples were taken, the heart, liver, spleen, lung, kidney and blood of mice were collected for H&E staining (Fig. 6a), whole blood cell count (Fig. 6b) and blood biochemical analysis (Fig. 6c), respectively. No histological differences were observed in the heart, liver, spleen, lung and kidney tissues among the groups, and no significant abnormalities were found in the whole blood cell count and biochemical indicators. This result confirmed the excellent biocompatibility of the Ag2Se@GelMA / ART thermoelectric biomimetic skin. Figure 13 Figure 14 Figure 15

[0045] Example 6 The Ag2Se@GelMA / ART hydrogel biomimetic skin of the present application accelerates the healing of skin wounds in diabetic mice by promoting angiogenesis Angiogenesis plays a crucial role in the wound healing process by ensuring sufficient oxygen and nutrients for the wound through blood flow, thereby promoting cell proliferation, migration and metabolic activity. In this example, we further investigated the relationship between angiogenesis and this accelerated repair at the histological level. Since platelet endothelial cell adhesion molecule-1 (CD31), a-smooth muscle actin (a-SMA) and vascular endothelial cadherin (VE-cadherin) are key markers of angiogenesis, we performed immunofluorescence staining of CD31 (Fig. 6a), VE-cadherin (Fig. 6c) and a-SMA (Fig. 6e), and also quantitatively analyzed the staining results using ImageJ (Fig. 6b, 6d, 6f). For CD31 and VE-cadherin, the expression levels were low in the control and GelMA groups, although there was a slight increase in the GelMA / ART group, but it was not significant. The expression levels were significantly higher in the Ag2Se@GelMA and Ag2Se@GelMA / ART groups, with the Ag2Se@GelMA / ART group showing the highest expression level. For a-SMA, the expression levels were significantly higher in the Ag2Se@GelMA and Ag2Se@GelMA / ART groups than in the other three groups, and the Ag2Se@GelMA / ART group still had the highest expression. Figure 6 Figure 6 Figure 6 Figure 6

[0046] Example 7 The Ag2Se@GelMA / ART hydrogel biomimetic skin of the present application inhibits pro-inflammatory cytokines to promote the healing of skin wounds in mice ​​​​​​​The proinflammatory cytokines associated with insulin resistance and metabolic syndrome include tumor necrosis factor-α (TNF-α), interleukin (IL)-6 and IL-1β. A large number of studies have shown that artemisinin can inhibit the secretion of TNF-α, IL-1β and other proinflammatory cytokines and chemokines. Therefore, we detected the expression of TNF-α (Fig. 8a) and IL-1β (Fig. 8c) in the collected skin tissues by immunofluorescence staining, and carried out fluorescence quantitative analysis Figure 8 b、8d}.

[0047] The results show that for TNF-α and IL-1β, the expression levels of the control group and the GelMA group are the highest, and the GelMA / ART group significantly inhibits the expression of the two factors. However, the expression level of the Ag2Se@GelMA group is similar to that of the control group and the GelMA group, indicating that its anti-inflammatory effect mainly comes from artemisinin, rather than Ag2Se@GelMA. Notably, the expression level of the Ag2Se@GelMA / ART group is significantly lower than that of the GelMA / ART group, indicating that under the experimental conditions adopted in this embodiment, the self-powered system further enhances the anti-inflammatory effect of artemisinin.

[0048] The above merely describes preferred specific embodiments of the present application, but the protection scope of the present application is not limited thereto, and any person skilled in the art can make equivalent replacements or changes to the technical solutions and inventive concepts of the present application within the technical scope disclosed by the present application, which should be covered within the protection scope of the present application.

Claims

1. A thermoelectric biomimetic skin that promotes diabetic wound healing by restoring the bioelectric field microenvironment, characterized in that, Includes the following steps: S1: Preparation of silver selenide (Ag2Se) nanoparticles, specifically by reacting selenium dioxide, ascorbic acid and silver nitrate in an aqueous phase to generate silver selenide (Ag2Se) nanoparticles; S2: Dissolve artemisinin (ART) in dimethyl sulfoxide, add physiological saline to adjust the volume, shake to mix well to obtain concentrated mother liquor, and dilute to the required concentration for later use; S3: Silver selenide (Ag2Se) nanoparticles were added to a 10% methacryloyl gelatin solution. The precursor solution was placed in a mold, and 0.25% lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (LAP) photoinitiator was added. The mixture was cured under ultraviolet light and then removed by soaking after demolding to remove residual monomers and initiators. The drug-containing group was prepared by using artemisinin solution instead of water to obtain Ag2Se@GelMA or Ag2Se@GelMA / ART composite hydrogels.

2. The thermoelectric biomimetic skin that promotes diabetic wound healing by restoring the bioelectric field microenvironment according to claim 1, characterized in that, The Ag2Se nanoparticles have an average particle size of 50~200 nanometers, are uniformly dispersed, and exhibit excellent room temperature thermoelectric properties.

3. The thermoelectric biomimetic skin according to claim 1, which promotes diabetic wound healing by restoring the bioelectric field microenvironment, is characterized in that... The concentration of the GelMA hydrogel is 8%~12% (by mass / volume), which gives the hydrogel both mechanical strength and flexibility, making it suitable for skin adhesion.

4. The thermoelectric biomimetic skin that promotes diabetic wound healing by restoring the bioelectric field microenvironment according to claim 1, characterized in that, The concentration of the artemisinin solution is 25~150 μM, which endows the hydrogel with anti-inflammatory function and promotes the biological repair of wounds in synergistic thermoelectric effect.

5. A method for using thermoelectric biomimetic skin that promotes diabetic wound healing by restoring the bioelectric field microenvironment, prepared by the method described in claims 1-4, comprising the following specific steps: S1: Apply the Ag2Se@GelMA / ART hydrogel biomimetic skin patch to the patient's diabetic chronic wound; S2: By utilizing the temperature difference between the patient's skin and the external environment, the thermoelectric effect of Ag2Se nanoparticles in the hydrogel is stimulated to generate a stable and directional bioelectric field, simulating and restoring the damaged endogenous bioelectric field. S3: The hydrogel releases artemisinin to inhibit local inflammation, while the thermoelectric bioelectric field activates voltage-gated calcium channels in endothelial cells, inhibits PHD2 protein expression, activates the HIF-1α / VEGF-A signaling pathway, promotes angiogenesis and collagen deposition, and accelerates wound healing.

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