A red light excitable photoelectric response heterostructure nanotube, a hydrogel and a preparation method and use thereof

Abstract

The application provides a red light excitable photoelectric response heterostructure nanotube, a hydrogel and a preparation method and application thereof, and belongs to the technical field of nanomaterials. By integrating a heterostructure, a quantum confinement effect and morphological modification, a one-dimensional TiO2 / Bi2S3 quantum dot nanotube with visible red light excitation and high photoelectric conversion efficiency is prepared, which is combined with a hydrogel precursor to simulate an extracellular matrix, so that a high-performance red light response photoelectric hydrogel dressing is developed. The hydrogel dressing has excellent ability in promoting nerve ending reinnervation, promoting skin substructure repair and regulating immune response in a deep burn model, and has wide application prospects in overall skin repair.

Description

Technical Field

[0001] This invention belongs to the field of nanomaterials technology, specifically relating to a red light-excited heterojunction nanotube, hydrogel, its preparation method and application. Background Technology

[0002] As the outermost layer of the human body, the skin is highly susceptible to burns from fire, hot liquids, and chemicals, leading to damage to the dermal matrix and the skin's epithelial neural network. Therefore, it is crucial to develop advanced wound dressings that can accelerate wound closure and enhance the reinnervation of muscle nerve endings.

[0003] Compared to traditional drug therapy, physical signal-based electrical stimulation therapy avoids potential side effects and drug tolerance issues, and its therapeutic effects are more lasting compared to drug-only treatments. Electrical stimulation not only helps wound closure but also helps restore the function of damaged tissues. In recent years, with a deeper understanding of the endogenous electric field in the human body, the role of exogenous electrical stimulation in wound healing and nerve regeneration has become increasingly recognized. However, due to the complexity of external power supply devices and the continuous foreign body reaction at the implantation site, battery-powered stimulation faces challenges that are difficult for patients to accept. Therefore, passive remote electrical stimulation has become the focus of electrical stimulation therapy and has opened up a new avenue in medical and health management. However, common photoelectric conversion materials have many drawbacks in tissue engineering applications, including heavy metal toxicity (such as Cd and Pb quantum dots), short excitation wavelengths (such as Si, TiO2, and P3HT), resulting in poor tissue penetration and biosafety, low photoelectric conversion efficiency, and complex manufacturing costs. While red light-driven photoelectric stimulation can avoid the biohazards associated with short-wavelength excitation materials, the lower energy carried by long-wavelength light makes it difficult to design materials for efficient photoelectric conversion.

[0004] Chinese patent application CN202211068883.0 discloses a biomimetic hydrogel constructed from a red-light-responsive nano-optoelectronic composite semiconductor and its preparation method. The method constructs Bi₂S₃ / TiO₂ / rGO(BTG) nanoparticles with a pn heterostructure, achieving wider light absorption and higher light conversion than conventional UV excitation. The photoconductive biomimetic hydrogel formed by these Bi₂S₃ / TiO₂ / rGO(BTG) nanoparticles and a biomimetic hydrogel matrix provides a light-driven electrostimulation platform, applicable to fields such as bone integration, nerve regeneration, electronic skin, and wound healing. However, the current response of the photoconductive biomimetic hydrogel prepared from these Bi₂S₃ / TiO₂ / rGO(BTG) nanoparticles is limited to the pA level, and the photoelectric conversion efficiency needs further improvement. Summary of the Invention

[0005] To address the aforementioned problems in the prior art, the first objective of this invention is to provide a heterostructured nanotube with improved photoelectric conversion efficiency, its preparation method, and its applications. The second objective of this invention is to provide a red light-excited heterostructured nanotube hydrogel, its preparation method, and its applications.

[0006] This invention provides a heterostructured nanotube, which is a product prepared from titanium dioxide nanotubes, bismuth salts or their hydrates and sulfides.

[0007] Furthermore, the bismuth salt is Bi(NO3)3, and the sulfide is a metal sulfide; the titanium dioxide nanotubes are prepared by a two-step anodic oxidation method using titanium as the working electrode and platinum as the counter electrode.

[0008] Further, the metal sulfide is Na2S; the electrolyte used in the two-step anodizing method is a solution composed of HF, NH4F, water and a polar organic solvent, wherein the concentration of HF is 1-3 wt.%, the concentration of NH4F is 0.1-1 wt.%, and the concentration of water is 0.5-2 wt.%; the two-step anodizing method includes two steps: pre-oxidation and oxidation, wherein the pre-oxidation conditions are: pre-oxidation at 45-55V for 1-3 hours, and the oxidation conditions are: oxidation at 25-35V for 20-40 minutes.

[0009] Further, the concentration of HF is 2 wt.%, the concentration of NH4F is 0.3 wt.%, the concentration of water is 1 wt.%, and the polar organic solvent is dimethyl sulfoxide.

[0010] Furthermore, the pre-oxidation conditions are: pre-oxidation at 50V for 2 hours, and oxidation conditions are: oxidation at 30V for 30 minutes.

[0011] Furthermore, the diameter of the heterostructured nanotube is 80-120 nm, preferably 100 nm, and the wall thickness is 2-12 nm, preferably 5-10 nm.

[0012] The present invention also provides a method for preparing the above-mentioned heterostructured nanotubes, the method comprising the following steps: first soaking titanium dioxide nanotubes in an aqueous solution of bismuth salt or its hydrate, and then soaking them in an aqueous solution of sulfide; repeating this process 1-20 times to obtain heterostructured nanotubes.

[0013] Further, the concentration of the aqueous solution of the bismuth salt or its hydrate is 3-7 mM, the soaking time in the aqueous solution of the bismuth salt or its hydrate is 0.5-5 min, the concentration of the aqueous solution of the sulfide is 20-30 mM, the soaking time in the aqueous solution of the sulfide is 0.5-5 min, and the number of repetitions is 5-15 times.

[0014] Furthermore, the concentration of the aqueous solution of the bismuth salt or its hydrate is 5 mM, the soaking time in the aqueous solution of the bismuth salt or its hydrate is 1 min, the concentration of the aqueous solution of the sulfide is 25 mM, the soaking time in the aqueous solution of the sulfide is 1 min, and the number of repetitions is 10.

[0015] This invention also provides the use of the above-mentioned heterostructured nanotubes in the preparation of photoelectric conversion biomaterials.

[0016] The present invention also provides a heterostructured nanotube hydrogel, which is a product prepared by the above-mentioned heterostructured nanotubes and hydrogel precursor.

[0017] Furthermore, the preparation method of the hydrogel precursor includes the following steps: reacting collagen with hyaluronic acid or its derivative under the action of a photoinitiator to obtain the hydrogel precursor; the mass ratio of collagen, hyaluronic acid or its derivative and photoinitiator is 1:(1-3):(0.1-0.5).

[0018] Furthermore, the collagen is type I collagen, the hyaluronic acid derivative is methacrylate-modified hyaluronic acid, and the photoinitiator is I2959; the mass ratio of the collagen, hyaluronic acid or its derivative, and the photoinitiator is 1:2:0.25.

[0019] The present invention also provides a method for preparing the above-mentioned heterostructured nanotube hydrogel, the method comprising the following steps: adding the heterostructured nanotubes into a solution of the hydrogel precursor, incubating, and irradiating with light to obtain the heterostructured nanotube hydrogel.

[0020] Furthermore, the incubation temperature is 30-40℃, and the time is 10-20 minutes; the light is ultraviolet light.

[0021] Furthermore, the mass-to-volume ratio of the heterostructured nanotubes to the hydrogel precursor solution is (30-70) mg:100 mL, preferably 50 mg:100 mL.

[0022] This invention also provides the use of the above-mentioned heterostructured nanotube hydrogel in the preparation of skin repair dressings.

[0023] The present invention has achieved the following beneficial effects:

[0024] (1) Compared with the Bi2S3 / TiO2 / rGO(BTG) nanoparticles reported in Chinese patent application CN202211068883.0, the heterostructured nanotubes TiO2 / Bi2S3(TBNTs) provided by the present invention have a nanotube structure, which enhances the light absorption efficiency and quantum dot effect. It not only has excellent red light excitable photoelectric properties, but also the photocurrent curve amplitude under illumination period reaches the μA level.

[0025] (2) The current response of the photoelectric conductive biomimetic hydrogel prepared from Bi2S3 / TiO2 / rGO(BTG) nanoparticles reported in Chinese patent application CN202211068883.0 is only at the pA level. However, the photocurrent curve amplitude of the hydrogel prepared by this invention using TBNTs and hydrogel precursors as raw materials reaches the μA level under illumination cycle, and the photoelectric conversion efficiency is significantly improved.

[0026] (3) The hydrogel dressing prepared by the present invention using TBNTs and hydrogel precursors as raw materials has excellent red light responsiveness and outstanding ability to promote nerve ending reinnervation, promote skin substructure repair and regulate immune response in deep burn models. It has broad application prospects in comprehensive skin repair.

[0027] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions, or alterations can be made without departing from the basic technical concept of the present invention.

[0028] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following embodiments. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0029] Figure 1 Preparation and characterization of TBNTs. (a) Schematic diagram of the preparation process; (b) SEM images of TBNTs: (b1) top view, (b2) bottom view, (b3) monodisperse TBNTs; (c) TEM elemental mapping of TBNTs; (d) TEM images of TBNTs; (e) High-resolution TEM image of TBNTs with SAED; (f) XPS images before and after compositing with Bi2S3; (g) XRD patterns during the preparation of TBNTs; (h) The nanotube structure increases the incident optical path and enhances light absorption.

[0030] Figure 2 The pre-oxidation process in the two-step anodizing strategy produces a titanium foil with a rough surface (left) to form a titanium foil with regular shallow pits (right).

[0031] Figure 3 The tightly packed TiO2 nanoarray (top) was ultrasonically exfoliated into aggregated nanotubes with low recombination efficiency, while the spaced TiO2 nanoarray (bottom) was exfoliated into monodisperse nanotubes with high recombination efficiency.

[0032] Figure 4 TEM image of monodisperse nanotubes after being composited with Bi2S3 quantum dots.

[0033] Figure 5 XPS fine spectra of Ti2p and Bi4f, S2p orbitals in TBNTs.

[0034] Figure 6 Electrochemical performance characterization. (a) XPS valence band spectrum; (b) UV-Vis absorption spectrum; (c) Photoluminescence spectrum; (d) Impedance spectrum; (e) Phase angle spectrum; (f) Photocurrent curves of TBNTs and TNTs under illumination cycle; (g) CV curves; (h) Charge storage capacity statistics; (i) TBNT heterojunction reduces semiconductor band gap and promotes electron-hole pair separation; (j) Photocurrent curves of TBCHA under illumination cycle.

[0035] Figure 7 Cell compatibility testing of TBNTs. (a) Live and dead stained fibroblasts cultured on titanium foil for 3 days; (b) Live and dead stained fibroblasts cultured on TBNTs for 3 days; (c) Cell viability assessed using a CCK-8 assay kit; (d) SEM images of cells cultured on TBNTs for 3 days.

[0036] Figure 8 Effects of photoelectric stimulation on the proliferation activity of NIH-3T3 cells. (a) Live and dead cell staining after 3 days of culture. (b) Cell viability assay using a CCK-8 assay kit.

[0037] Figure 9 .TBCHA photoelectric dressing in vitro cell characteristics study. (a) Photoelectric stimulation-induced Ca 2+ (a) Flow diagram; (b) Cellular Ca2+ recorded by time-series confocal microscopy 2+3D plot of flow curve, below are exposure images before and after illumination (arrows indicate cells with changes in fluorescence intensity); (c) Staining results of α-SMA and phalloidin expression after co-culturing NIH-3T3 and PC12 cells; (d) Statistical analysis of the average fluorescence intensity of α-SMA; (e) Staining results of β3-tubulin and phalloidin expression; (f) Statistical analysis of the average fluorescence intensity of β3-Tubulin; (g) Effect of photoelectric stimulation on RAW264.7 cell polarization; (h) Statistical analysis of positive regions co-localized with IL-10 and iNOS and cell nuclei (confocal images were taken with a 40x lens, scale bar = 20 μm).

[0038] Figure 10 Phototherapy repair model of burned skin. (a) Digital photographs of the wound on days 0, 7, and 14; (b) H&E stained images (black arrows indicate new skin appendages, blue arrows indicate necrosis (scale bar = 500 μm)); (c) Statistical graph of wound area change over time; (d) Statistics on wound healing rate.

[0039] Figure 11 Digital photographs of burns treated with phototherapy dressings.

[0040] Figure 12 Effects of phototherapy on collagen deposition and ratio regulation in wounds. (a) Masson's trichrome staining (scale bar = 200 μm); (b) Picrosirius Red staining (green represents COL3, red represents COL1, scale bar = 100 μm); (c) Percentage of collagen-positive area calculated by Masson's staining method; (d) Statistical analysis of the ratio of COL1 to COL3.

[0041] Figure 13 Fluorescent immunohistochemistry of burned skin tissue. (a) Macrophage polarization markers on day 7; (b) Inflammatory cytokines TNF-α (green) and VEGF (yellow) on day 7; (c) Fibroblast activation (green) and nerve ending maturation (purple) on day 14 (scale bar = 500 μm), statistically analyzed by ELISA fluorescence quantitative analysis; (d) IL-10; (e) TNF-α; (f) VEGF. Detailed Implementation

[0042] The raw materials and equipment used in this invention are all known products, obtained by purchasing commercially available products.

[0043] Titanium foil (0.127 mm, 99.6%) was purchased from Haiyuan Scientific Metals. Bismuth nitrate (Bi(NO3)3·5H2O, 99%), sodium sulfide (Na2S, 95%), and ammonium fluoride (NH4F, 99.9%) were purchased from Aladdin Reagent Co., Ltd. Hydrogen fluoride (HF, 49%), nitric acid (HNO3, 68%), and dimethyl sulfoxide (DMSO, 99.9%) were purchased from Kron Reagent Co., Ltd. Type I collagen (Col) was prepared from calf hide. Sodium hyaluronate (Mw = 20 kDa) was purchased from Bloomage Biotechnology. 2-Hydroxy-4'-(2-hydroxyethoxy)-2-methylphenylacetone (I2959) and methacrylic anhydride (MA) were purchased from Sigma-Aldrich. Cell counting kit-8 (CCK-8), fluorescein diacetate (FDA), and propidium iodide (PI) were purchased from Beyotime Biotechnology. Primary antibodies against IL-10, iNOS, TNF-α, VEGF, α-SMA, and β3-tubulin, and their corresponding secondary antibodies, were purchased from Abcam. All reagents were used according to the manufacturer's instructions and were not further purified at the recommended dilutions.

[0044] Red light exposure at 625nm wavelength was performed using a DPSSL driver (VLP-2000-30W). Output power and pulse generation were regulated by a DDS signal generator / counter (FY1100, FeelElec). The modulation light source settings included 13.6mW / mm². 2 The receiving power, frequency of 0.5Hz and duty cycle of 50%.

[0045] Example 1: Preparation of heterostructured nanotubes

[0046] Titanium foil was sequentially degreased by ultrasonication in acetone, alcohol, and distilled water (250 W, 25°C, 15 min each time), and then dried in nitrogen. TiO2 nanotubes (TNTs) were then synthesized using a two-step anodic oxidation strategy. Specifically, a solution of 2 wt.% HF, 0.3 wt.% NH4F, and 1 wt.% deionized water (DIW) in DMSO was used as the electrolyte solution. Titanium was used as the working electrode, and platinum as the counter electrode, with a 2 cm gap between the electrodes. First, pre-oxidation was performed at 50 V and 25°C for 2 h, followed by ultrasonication in a 1 M HCl aqueous solution (250 W, 25°C, 10 min) to remove the oxide layer. The titanium substrate was rinsed with DIW to ensure a clean and uniform surface. Finally, oxidation was performed in a fresh electrolyte solution at 30 V and 25°C for 30 min to obtain the TNTs.

[0047] Subsequently, a 5 mM Bi(NO3)3 aqueous solution was prepared, and 0.1 mM HNO3 was added to promote dissolution. The TNT samples were immersed in the prepared Bi(NO3)3 aqueous solution for 1 min, removed and air-dried, and then soaked in a 25 mM Na2S aqueous solution for 1 min. The samples were then removed and air-dried again. The above soaking process was repeated 10 times to form heterostructured nanotubes (TiO2 / Bi2S3 quantum dot nanotubes, abbreviated as TBNTs).

[0048] Example 2: Preparation of Red Light Responsive Photoelectric Hydrogel Dressing

[0049] (1) Preparation of active hydrogel precursor solution

[0050] 2 g of hyaluronic acid (HA) powder was dissolved in 200 mL of deionized water (DIW) to prepare a 1% (w / v) concentration, followed by the dropwise addition of 16 mL of methacrylate (MA). The reaction mixture was maintained at pH 8 and stirred at 4 °C for 24 hours. Subsequently, a white flocculent substance was precipitated using a large amount of ethanol, centrifuged, and HAMA was obtained. The HAMA was then dialyzed and lyophilized, and stored in the dark at -20 °C. Next, 1% (w / v) collagen (Col) was dissolved in 0.5 M acetic acid and neutralized with NaOH to approximately pH 7.4 under ice bath conditions. This was then thoroughly mixed with a solution containing 2% (w / v) HAMA and 0.25% (w / v) I2959 at a 1:1 volume ratio to obtain the CHA precursor solution.

[0051] (2) Preparation of red light responsive photoelectric hydrogel dressing

[0052] Take 50 mg of TBNTs from Example 1 and add it to 100 mL of the CHA precursor solution from step (1), and mix thoroughly. Then transfer the mixture to a mold and incubate it in a 37°C incubator for 15 minutes. After incubation, expose the mold to ultraviolet light (365 nm, 3 W / cm²). 2 After 30 seconds, remove the product from the mold to obtain the red light responsive photoelectric hydrogel dressing (TBCHA).

[0053] The following experimental examples demonstrate the beneficial effects of the present invention.

[0054] All numerical data are reported as mean and standard deviation (mean ± SD). All experimental data were obtained from at least three replicates. Data analysis was performed using GraphPad Prism 9 (GraphPad Inc., USA) and Origin 2019 (OriginLab, USA). Image processing and analysis were performed using ImageJ, CaseViewer 2.4 (3DHISTECH Ltd.), Image-Pro Plus 6.0 (Media Cybernetics), and ZEN blue edition (Zeiss). Student's t-test was used to analyze differences between two groups, and one-way ANOVA was used to compare differences between three or more groups. (*p<0.05,**p<0.01,***p<0.001,****p<0.0001). p<0.05 was considered statistically significant.

[0055] Experimental Example 1: Characterization of TBNTs

[0056] 1. Experimental Methods

[0057] The morphology of TBNTs was examined using a FESEM (S-4800, Hitachi, Japan) transmission electron microscope (TEM, JEM-2100F, NEC). XRD analysis was performed using XRD (X'Pert Pro MPD, Philips, Netherlands). Bonding energy and valence band spectra were measured using X-ray photoelectron spectroscopy (XPS, AXIS Ultra DLD). Photoluminescence was measured using a fluorescence spectrometer (Hitachi U-3900, Japan). Electrochemical tests and photocurrents were collected using an electrochemical workstation (Gamry Instruments). Samples were prepared into thin films measuring 20 mm × 20 mm × 0.008 mm, held in place by clip electrodes to form the working electrode. A platinum sheet electrode served as the counter electrode, and a silver chloride electrode as the reference electrode, forming a three-electrode testing system. The working electrode was excited using a 625 nm red LED light source, with a cycle of 30 seconds on and 30 seconds off, and 10 cycles were performed.

[0058] 2. Experimental Results

[0059] (1) Structural characterization of TBNTs

[0060] Figure 1 The preparation and characterization of TBNTs are demonstrated. The preparation process of TiO2 / Bi2S3 quantum dot nanotubes (TBNTs) is as follows: Figure 1 As shown in figure a. This invention synthesizes ordered, well-spaced TiO2 nanoarrays using a two-step anodic oxidation strategy. Figure 2 The gap structure of the nanoarray is beneficial to the monodispersity of the nanotubes and the deposition efficiency of Bi2S3 quantum dots. Figure 3 SEM analysis revealed that the TiO2 nanoarrays possess a vertically oriented hollow tube structure. Furthermore, bottom-up analysis indicated that these nanoarrays are periodically arranged throughout the region. Figure 1 b1, b2). After ultrasonic ablation, the TNTs exhibited good homogeneity and monodispersity, with a length of approximately 2-4 μm. Figure 1 b3). Then. Figure 1 TEM element mapping in c shows that TBNTs are composed of Ti, O, Bi, S, and trace amounts of F remaining from electrolysis. Figure 1 c1, c2, c3, c4). In addition, Figure 1 The TEM images in d show that the TBNTs are approximately 100 nm in diameter, with a wall thickness between 5 and 10 nm, and maintain an intact tubular structure. Figure 4 It has a large number of Bi2S3 quantum dot deposits on its inner and outer surfaces. In addition, Figure 1 The HRTEM image in the image shows that the Bi2S3 quantum dots are approximately 5 nm in size and consist of (130) crystal planes. From Figure 1 The XPS spectrum in f shows that after Bi2S3 quantum dots are composited on TBNTs, characteristic peaks of Ti2p, Bi4f, and S2p appear, and their corresponding detailed spectra are shown in Figure f. Figure 5 As shown. Furthermore, Figure 1 The XRD pattern in g showed that the electrolyzed TiO2 (PDF#89-2762) was transformed into anatase (PDF#86-1157) after annealing. All samples underwent annealing in subsequent reports. Due to the extremely small size of Bi2S3 quantum dots, only the crystalline peaks of TiO2 were observed in the TBNT spectra, making the quantum dots indistinguishable. However, Bi2S3 quantum dots synthesized individually in the same manner showed characteristic peaks of Bi2S3 in the spectra (PDF#17-0320), confirming the feasibility of this method. Because nanotubes possess unique light absorption properties, and the deposition efficiency of heterojunctions was improved, when nanotubes are irradiated, light is trapped inside the tube, undergoing continuous reflection and scattering, thus increasing the optical path. This significantly improves the absorption rate (e.g., ...). Figure 1 (as shown in h), which enhances the photovoltaic effect of the heterojunction.

[0061] (2) Electrochemical performance characterization of TBNTs

[0062] exist Figure 6In the valence band XPS spectrum shown in Figure a, the incorporation of Bi₂S₃ quantum dots significantly reduces the band gap width from 2.6 eV in the original TNTs to 0.3 eV in TBNTs. This significant reduction in band gap width indicates a lower energy barrier for electron transitions from the valence band to the conduction band. Figure 6 The UV-Vis spectrum shown in b indicates that TNTs absorb light at 300 nm in the UV spectrum. The absorption of TBNTs extends to the visible red light region at approximately 600 nm. This broadening of the absorption spectrum demonstrates an enhanced ability of TBNTs to utilize a wider range of the solar spectrum, thus confirming their potential to improve light absorption and energy conversion efficiency. Furthermore, Figure 6 The PL spectra presented in Figure c indicate that TBNTs improve the separation efficiency of photogenerated electron-hole pairs. These results are consistent with the previously discussed expansion of the absorption range and reduction of the band gap, collectively supporting the potential of TBNTs to possess highly efficient photoelectrochemical activity. This invention also provides electrochemical characterization of TBNTs. Electrochemical impedance spectroscopy (EIS) data ( Figure 6 (d) and (6e) confirm that under low-frequency operating conditions, the impedance of TBNTs decreases when the phase angle approaches -90°. This capacitive characteristic provides a favorable environment for the formation and sustainability of charge carriers.

[0063] In photocurrent testing ( Figure 6 f) It was observed that TBNTs produced an amplitude of 9.22 μA / cm under pulsed illumination at 625 nm. 2 The photocurrent indicates that they have excellent response to red light, while TNTs exhibit slight periodic fluctuations with an amplitude of approximately 0.14 μA / cm. 2 .

[0064] Meanwhile, the CV curves show that TBNTs have a higher charge storage capacity. Figure 6 g). The specific capacitance obtained from quantitative evaluation is approximately 0.04 F / cm. 2 It is 8 times that of TNTs. Figure 6 These results demonstrate that the light-harvesting efficiency and charge separation capability of TBNTs are improved due to the effective bridging of the band gap and the promotion of longer-wavelength electronic excitation by Bi2S3 quantum dots. Figure 6 i).

[0065] The above experimental results show that the heterostructured nanotubes TBNTs of the present invention not only have excellent red light excitable photoelectric properties, but also the photocurrent curve amplitude under illumination period reaches the μA level, and the photoelectric conversion efficiency is high.

[0066] Example 2: Electrochemical performance characterization of TBCHA

[0067] 1. Experimental Methods

[0068] Following the method in Example 1, electrochemical measurements and photocurrents were collected using an electrochemical workstation (Gamry instrument), and photocurrent curves were measured under TBCHA illumination cycles. The working electrode was replaced with a platinum sheet electrode with TBCHA fixed to it.

[0069] 2. Experimental Results

[0070] In photocurrent testing ( Figure 6 j) It was observed that TBCHA produced an amplitude of 5.14 μA / cm under pulsed illumination at 625 nm. 2 The photocurrent indicates that it has excellent responsiveness to red light.

[0071] The TBCHA of this invention not only exhibits excellent responsiveness to red light, but also achieves photocurrent curve amplitudes at the μA level under illumination cycles, resulting in a significant improvement in photoelectric conversion efficiency.

[0072] Experiment Example 3: In vitro experiment

[0073] 1. Experimental Methods

[0074] (1) Cell Culture

[0075] NIH-3T3, PC12, and RAW 264.7 cell lines were obtained from the Chinese Academy of Sciences Culture Bank (Shanghai, China). These cells were cultured in DMEM high-glucose medium (Hyclone, China) containing 10% fetal bovine serum (TBD Science, China) and 1% penicillin-streptomycin (Hyclone). For neural differentiation experiments, the medium was additionally supplemented with 5% donor horse serum (Hyclone). Cells were cultured under optimal conditions of 37°C, 5% CO2, and 95% humidity. Cells were passaged every 3 days using 0.25% v / v trypsin-EDTA solution. For all experimental groups requiring illumination, LED light sources were sterilized with 75% alcohol and connected to a DDS signal generator to produce pulsed light signals. Designated samples were illuminated for 30 minutes daily.

[0076] (2) Methods for detecting cell viability

[0077] 200 μl of hydrogel prepolymer was added to the bottom of each well of a 48-well plate. After gelation, NIH-3T3 cells were seeded on the gel surface at a density of 5 × 10³ cells per well. In vitro cell viability was measured on days 1, 3, and 5 using the standard CCK-8 assay and FDA / PI staining. Optical density (OD) values ​​(absorbance at 450 nm) were measured using a Multiskan FC (ThermoScientific, USA). FDA / PI staining was performed according to the provided instructions, followed by imaging using a confocal laser scanning microscope (CLSM, Leica-TCS-SP5).

[0078] (3) Methods for assessing macrophage polarization

[0079] RAW264.7 macrophages were seeded on a hydrogel at a density of 1 × 10⁵ cells per well. After 4 hours of culture, lipopolysaccharide (LPS, 1 μg / ml) was added to each well to induce M1 polarization. Subsequently, the group receiving light exposure was stimulated for 30 minutes daily. After 48 hours of incubation, cells were fixed and stained with markers IL-10 and iNOS to observe changes in macrophage polarization phenotype.

[0080] (4)Ca 2+ Imaging methods

[0081] Plasma cells were seeded at a density of 5 × 10⁴ cells per well on TBCHA plates and cultured for 24 hours. Then, they were incubated at 37°C for 30 minutes with Fluo-8 AM (2.5 μM, diluted in medium, Biyuntian). Subsequently, the samples were washed and artificial cerebrospinal fluid (ACSF) was added. Images were acquired using a confocal laser scanning microscope (LSM 880Zeiss) with time-series mode and processed using ZEN software.

[0082] 2. Experimental Results

[0083] This invention demonstrates that co-culturing TBNTs with NIH-3T3 cells exhibits excellent cell compatibility, and SEM images show that cells spread well on TBNTs. Figure 7 Furthermore, to investigate the effect of photoelectric stimulation on the regulation of nerve regeneration by subsequent wound dressings, a bioactive hydrogel (CHA) was synthesized by photocrosslinking exfoliated TBNTs with grafted collagen / hyaluronic acid at room temperature using methacrylic anhydride. The excellent light absorption capacity of the nanotube structure endowed the hydrogel with significant photoelectric properties, providing electrostimulation to enhance cell proliferation. Figure 8 ).

[0084] As is well known in the art, Ca 2+ Ca2+ plays an important secondary messenger role in cell signaling, particularly in nerve transmission, muscle contraction, and hormone secretion. Ca2+ is distributed on the cell membrane. 2+ Voltage-gated channels (L-VGCCs) can be activated by electrical signals, promoting extracellular calcium absorption. 2 +Inflow( Figure 9 a). For example Figure 9 As shown in b, Ca is performed using Fluo-8AM. 2 Imaging experiments showed that the photoinduced current induced by TBCHA reached the activation threshold of the less electroactive NIH-3T3, triggering Ca2+. 2A slight influx of +. Furthermore, the co-cultured NIH-3T3 and PC12 cell groups exhibited broad cellular responses and significant fluorescence enhancement, indicating that the interaction between activated neurons and fibroblasts promotes electrical reactivity and epithelial-neural function.

[0085] Subsequently, this invention designed a co-culture experiment of NIH-3T3 and PC12 cells to further explore the synergistic cell behavior of neural cell maturation and fibroblast activation under photoelectric stimulation. On the seventh day of co-culture (during co-culture: daily exposure to red light for 30 minutes), cell nuclei were stained with DAPI (blue), the cytoskeleton was highlighted with rhodamine-phalloidin (red), and α-SMA and β3-tubulin were labeled with green fluorescence, respectively. Figure 9 As shown in c and 9d, the increased mean fluorescence intensity and cell density of α-SMA indicate enhanced fibroblast activity, promoting muscle contraction during wound healing. Furthermore, photoelectric stimulation enhanced β3-tubulin expression (…). Figure 9 (e, 9f) indicates that PC12 differentiates into mature neurons while simultaneously enhancing cytoskeleton structure, emphasizing the effective differentiation-inducing effect of electrical signals on excitable nerve cells. Therefore, the experimental results highlight that photostimulation is beneficial for the functional establishment of fibroblasts and nerve cells in skin tissue.

[0086] Finally, the polarization phenotypes of macrophages under the photoelectric effect were characterized. For example... Figure 9 As shown in the confocal fluorescence images in g, RAW 264.7 cells, induced by 1 μg / mL LPS, polarized from the M0 phenotype to the M1 pro-inflammatory phenotype, characterized by abundant expression of inducible nitric oxide synthase (iNOS, marked by red fluorescence) and almost no expression of the green fluorescent marker M2 phenotype characteristic cytokine IL-10. This occurred in both the CHA and TBNT-L groups. In contrast, under photoelectric stimulation in the TBNT-L group, red fluorescence almost disappeared, replaced by significant green fluorescence, indicating that the photoelectric effect can effectively promote the polarization of macrophages from the M1 pro-inflammatory phenotype to the M2 anti-inflammatory phenotype. Figure 9 The positive nuclear colocalization region shown in h provides more intuitive evidence that TBNT L can induce a shift from the M1 to the M2 phenotype in already polarized macrophages.

[0087] The above experimental results demonstrate that the multifaceted biological interactions introduced by the TBCHA hydrogel of this invention can promote the second messenger Ca2+ by activating L-VGCC. 2+ The influx of these cells triggers downstream signals for potential neural regeneration and muscle contraction, while simultaneously promoting macrophage polarization toward an anti-inflammatory phenotype.

[0088] Experiment Example 4: In vivo experiment

[0089] 1. Experimental Methods

[0090] (1) Establishment of animal models

[0091] Male SD rats (8 weeks old, 220±10g) were purchased from Chengdu Dossy Laboratory Animal Co., Ltd. This study was approved and conducted under the supervision of the Animal Experimentation and Use Committee of Sichuan University, in accordance with institutional and NIH guidelines on the care and use of research animals (Approval No. KS2022864).

[0092] SD rats were first anesthetized using an animal anesthesia ventilator (RWD Life Sciences, Ltd.) containing 1.5% isoflurane and 60% oxygen. After shaving and disinfecting the backs of the rats with povidone-iodine, each rat's back was scalded at 100°C for 10 seconds with a 10mm diameter copper rod to establish a third-degree burn model. The burned rats were randomly assigned to the following groups (n=3): (1) untreated burned skin (BLK group); (2) receiving CHA without phototherapy (CHA-L group); (3) receiving CHA and phototherapy (CHA+L group); (4) receiving TBCHA without phototherapy (TBCHA-L group); (5) receiving TBCHA and phototherapy (TBCHA+L group). Rats were housed in phototherapy and non-phototherapy groups, and their dressings were changed daily throughout the treatment period. For the phototherapy group, rats received 30 minutes of 620nm red light irradiation daily. After 7 or 14 days of treatment, the rats were euthanized and the skin from the burned areas was collected.

[0093] (2) Animal sample collection

[0094] Animals were euthanized on days 7 and 14 using an excess of 10% chloral hydrate for histological evaluation. Protein analysis samples were frozen in liquid nitrogen and transported. Animal tissues were then weighed and homogenized with 9 volumes of 0.9% saline. The homogenate was mechanically homogenized to a 10% homogenate under ice-water bath conditions, centrifuged at 2500-3000 RPM for 10 minutes, and the supernatant was used for analysis. Sections were completely fixed with 4% paraformaldehyde, embedded in paraffin, and then longitudinally sectioned to a thickness of 10 μm. Finally, H&E staining, Masson staining, Picrosirius red staining, and immunofluorescence staining were performed. Image acquisition and analysis were performed using an automated quantitative pathological imaging system (Vectra 3S6, PerkinElmer Inc.).

[0095] (3) Immunofluorescence staining method

[0096] First, cell / paraffin-embedded skin sections were fixed with 4% paraformaldehyde for 15 minutes, washed after removing the culture medium. Then, they were infiltrated with 0.2% Triton X-100 for 5 minutes and washed. Subsequently, non-specific proteins were blocked with 10% bovine serum albumin (BSA). Next, primary antibody was added at the appropriate dilution and incubated overnight at 4°C, followed by washing. Next, secondary antibody was added at the appropriate dilution and incubated in the dark at room temperature for 1 hour, followed by rapid washing away unbound secondary antibody. Finally, DAPI solution was added for staining for 15 minutes, followed by washing.

[0097] 2. Experimental Results

[0098] (1) Phototherapy repair model of burned skin

[0099] The therapeutic performance and potential mechanism of TBCHA hydrogel dressing were evaluated in a deep burn model. Clearly, wounds not treated with hydrogel dressings exhibited swelling and redness within the first three days, with a significant inflammatory response. Figure 10 a). Then, comparing the CHA-L and CHA+L groups, it was found that the bioactive hydrogel could reduce edema and inflammation, while red light irradiation alone had a negligible effect on reducing burn area. Among them, the TBCHA+L group demonstrated a key role of phototherapy in rapidly shrinking the wound and reducing scarring. Figure 10 c). Therefore, from a macroscopic perspective, the strategy of photoelectro-hydrogel can promote epithelialization and shorten healing time. Figure 11 ).

[0100] Then, H&E analysis was performed on the wound tissue to assess the repair effect of each treatment group. Figure 10 b). In the burn control group without hydrogel dressings, the entire wound healing process was accompanied by extensive inflammatory cell infiltration, resulting in severe pathological fibrosis. On day 7, the epidermis of the BLK group was significantly thinner, with large-area necrosis (blue arrows), and sub-organs of the skin such as epithelium, sweat glands, and hair follicles were sloughed off, impairing skin function. The CHA group showed slightly better repair than the BLK group, but a small amount of necrosis and subcutaneous vacuoles were present. In contrast, the TBCHA+L group showed enhanced granulation tissue proliferation and wound healing speed (…). Figure 10 d) accompanied by fibrous connective tissue hyperplasia and a small amount of granulocyte-lymphocyte infiltration. Furthermore, a significant increase in neodermal appendages (black arrows) was observed only in the TBCHA+L group.

[0101] (2) Effects of phototherapy on collagen deposition and proportion regulation in wounds

[0102] Secondly, based on the results of Masson's trichrome staining ( Figure 12a) In the early stage of the wound, collagen arrangement was sparse in the BLK group, while collagen deposition in the CHA group was disordered and lacked suborganization. In contrast, the TBCHA+L group showed abundant and dense collagen deposition with strong directional arrangement, indicating that photoelectric stimulation significantly improved the reconstruction of the extracellular matrix and tissue remodeling in the wound. Simultaneously, quantitative analysis ( Figure 12 c) shows that the TBCHA+L group exhibited significant collagen fiber positivity almost throughout the entire healing process. Red light irradiation had a certain promoting effect on early collagen deposition, while the repair effect of photoelectric stimulation was more significant.

[0103] Furthermore, the dynamic changes in the ratio of type I collagen (COL1) to type III collagen (COL3) are crucial physiological events in wound healing. Typically, COL3 synthesis dominates in the early stages of wound healing, but during the remodeling phase, COL3 is gradually replaced by COL1. This is because COL1 is tougher and more stable, providing greater tensile strength. Figure 12 As shown in b, the BLK and CHA groups were primarily characterized by green-stained COL3. In contrast, the TBCHA+L group showed an increased proportion of COL1, which not only promoted collagen deposition but also enhanced tissue stability. There were no statistically significant differences between the other three groups and the BLK group. Further analysis is needed. Figure 12 As can be seen from d, by day 14, the proportion of COL1 in the TBCHA+L group further increased, approaching the level observed in normal tissue (approximately 80%), which contrasts sharply with the continuous decrease in the proportion of COL1 in the BLK group.

[0104] (3) Fluorescent immunohistochemistry of burned skin tissue

[0105] first, Figure 13 The data in section a are closely related to the results of in vitro macrophage experiments, showing that phototherapy promoted the upregulation of the anti-inflammatory cytokine IL-10 and the downregulation of iNOS expression. This regulation indicates a shift in macrophage polarization towards the M2 phenotype. However, differences were observed in the BLK group during the early stages of wound healing, where deterioration and necrosis led to reduced macrophage recruitment. Furthermore, Figure 13 ELISA quantitative analysis of d showed that IL-10 expression was upregulated in the BLK group and relatively decreased in the TBCHA+L group during the later stages of wound healing. This phenomenon may be attributed to the accelerated healing process in the latter group, which allows for faster wound remodeling and leads to a reduction in macrophages.

[0106] Figure 13b and 13e show a significant downregulation of the pro-inflammatory cytokine TNF-α in the TBCHA+L phototherapy group, indicating that phototherapy effectively stimulates the expression of an anti-inflammatory macrophage phenotype. This immunomodulatory effect is crucial for resolving the inflammatory response in deep burns and preventing excessive scar tissue formation. The reconstruction of the vascular network not only provides the wound with essential nutrients and oxygen but also accelerates the healing process by promoting the transport of immune cells, growth factors, and intercellular signaling molecules. Therefore, this invention investigates angiogenesis by labeling vascular endothelial growth factor (VEGF). Figure 13 As shown in b, the fluorescence intensity of VEGF in the TBCHA+L treatment group was significantly higher than that in other groups, and microvascular structures were observed. ELISA data ( Figure 13 f) This further confirmed the high expression of VEGF in the TBCHA+L treatment group on days 7 and 14 of the healing process.

[0107] Finally, the reconstruction of epithelial neural networks is one of the most critical challenges in repairing burn-induced skin damage, largely depending on the reinnervation of nerve endings into muscle tissue. Supported by the photoelectric effect, TBCHA hydrogel provides a favorable electrophysiological environment for neuronal differentiation and axonal growth. This not only accelerates nerve regeneration but also ensures fibroblast activation. Figure 13 c shows the expression of β3-tubulin and α-SMA markers in the TBCHA+L phototherapy group on day 14. Under continuous electrical stimulation, damaged nerve cells repair connections through the regeneration of nerve fibers. This process begins with axonal deterioration near the injury site, followed by the formation of new growth cones and axons. Simultaneously, fibroblast activation plays a crucial role in wound contraction and extracellular matrix synthesis. The synergistic promotion of these two processes enhances the quality and function of the repaired tissue.

[0108] In summary, this invention provides a red-light-excited photoelectroresponsive heterostructure nanotube, hydrogel, its preparation method, and its applications. By integrating heterostructure, quantum confinement effect, and morphological modification, this invention prepares one-dimensional TiO2 / Bi2S3 quantum dot nanotubes with visible-red light excitation and high photoelectric conversion efficiency. These nanotubes are then combined with a biomimetic hydrogel to simulate the extracellular matrix, thereby developing a high-performance red-light-responsive photoelectrolytic hydrogel dressing. This hydrogel dressing exhibits excellent capabilities in promoting nerve ending reinnervation, promoting skin substructure repair, and regulating immune responses in deep burn models, showing broad application prospects in comprehensive skin repair.

Claims

1. The use of heterostructured nanotubes in the preparation of photoelectric conversion biomaterials, wherein the heterostructured nanotubes are products prepared from titanium dioxide nanotubes, bismuth salts or their hydrates and sulfides; the sulfides are metal sulfides; the titanium dioxide nanotubes are prepared by a two-step anodic oxidation method using titanium as the working electrode and platinum as the counter electrode; the electrolyte used in the two-step anodic oxidation method is a solution composed of HF, NH4F, water and a polar organic solvent, wherein... The concentration of HF is 1-3 wt.%, the concentration of NH4F is 0.1-1 wt.%, and the concentration of water is 0.5-2 wt.%. The two-step anodizing method includes two steps: pre-oxidation and oxidation. The pre-oxidation conditions are: pre-oxidation at 45-55 V for 1-3 h, and oxidation conditions are: oxidation at 25-35 V for 20-40 min. The heterostructured nanotubes have a diameter of 80-120 nm and a wall thickness of 2-12 nm. The method for preparing the heterostructured nanotubes includes the following steps: first, immersing titanium dioxide nanotubes in an aqueous solution of bismuth salt or its hydrate, and then immersing them in an aqueous solution of sulfide; repeating this process 1-20 times to obtain heterostructured nanotubes.

2. The use according to claim 1, characterized in that, The bismuth salt is Bi(NO3)3.

3. The use according to claim 1, characterized in that, The metal sulfide is Na2S.

4. The use according to claim 1, characterized in that, The concentration of the aqueous solution of the bismuth salt or its hydrate is 3-7 mM, and the soaking time in the aqueous solution of the bismuth salt or its hydrate is 0.5-5 min. The concentration of the aqueous solution of the sulfide is 20-30 mM, and the soaking time in the aqueous solution of the sulfide is 0.5-5 min. The number of repetitions is 5-15 times.

5. The use according to claim 1, characterized in that, The heterostructured nanotubes have a diameter of 100 nm and a wall thickness of 5-10 nm.

6. The use of a heterogeneous nanotube hydrogel in the preparation of skin repair dressings, characterized in that, The heterostructured nanotube hydrogel is a product prepared from the heterostructured nanotubes and hydrogel precursors according to any one of claims 1-5.

7. The use according to claim 6, characterized in that, The preparation method of the hydrogel precursor includes the following steps: reacting collagen with hyaluronic acid or its derivative under the action of a photoinitiator to obtain the hydrogel precursor; the mass ratio of collagen, hyaluronic acid or its derivative and photoinitiator is 1:(1-3):(0.1-0.5).

8. The use according to claim 7, characterized in that, The collagen is type I collagen, the hyaluronic acid derivative is methacrylate-modified hyaluronic acid, and the photoinitiator is I2959; the mass ratio of the collagen, hyaluronic acid or its derivative, and the photoinitiator is 1:2:0.

25.

9. The use according to claim 7, characterized in that, The preparation method of the heterostructured nanotube hydrogel includes the following steps: adding heterostructured nanotubes into a hydrogel precursor solution, incubating, and irradiating with light to obtain heterostructured nanotube hydrogel.

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