Ultrasonic activation type piezoelectric gel dressing as well as preparation method and application thereof

Abstract

The invention discloses an ultrasonic activation type piezoelectric gel dressing and a preparation method and application thereof.The surface of a system is an antibacterial hydrogel layer, the antibacterial hydrogel layer is composed of polyethyleneimine (PEI) and polyvinyl alcohol (PVA) cross-linked hydrogel, and ultrasound and current can be transmitted to a deep area; bT-dNO piezoelectric nanoparticles are packaged inside, and BT-dNO is prepared by coating a nitric oxide (NO) donor on the surface of piezoelectric barium titanate (BT) subjected to hydroxylation and dopamine modification. Under the stimulation of low intensity focused ultrasound (LIFU), the donor can release NO as required, and the BT nanoparticles can generate reactive oxygen species (ROS) and output piezoelectric current. The dressing system has an effective combined repair effect, excellent biocompatibility and good anti-inflammatory ability.

Description

Technical Field

[0001] This invention relates to biomedical materials and methods, specifically to an ultrasound-activated piezoelectric gel dressing, its preparation method, and its application. Background Technology

[0002] In recent years, nanobiomaterials and technologies utilizing antibacterial, anti-inflammatory, or antioxidant strategies to promote wound healing in diabetic patients, such as stem cell tissue engineering, nano-wound dressings, and autologous skin transplantation, have shown great promise in wound management. Research has found that wound repair is closely related to the wound's endogenous electric field, which promotes cell proliferation and migration and is crucial for skin regeneration. Therefore, applying an external power source to the wound can biomimic or enhance the wound's endogenous electric field, thereby accelerating wound healing. Piezoelectric materials can immediately generate an internal electric field and surface pressure potential under mechanical stress or an external electric field, producing direct current output. Therefore, using piezoelectric materials as self-powered wound dressings, acting as an external electric field during wound healing, is a promising approach.

[0003] Current treatments for diabetic foot ulcers (DFU) primarily focus on systemic glycemic control, microcirculation recanalization, infection prevention, and wound repair, but their efficacy is quite limited. Electrical stimulation therapy typically requires an external power source, resulting in poor portability and comfort; furthermore, implanted electrodes are limited in application due to side effects such as invasiveness, infection, and electrode breakage. Considering that the expression, transport, and phosphorylation status of ion channels within sensory neurons directly affect functional abnormalities, targeted modulation of specific ion channels may reshape perception in diabetic peripheral nerves. Transient receptor potential vanillin-1 (TRPV1) is a highly expressed receptor in dorsal root ganglion neurons and is closely related to the pain process. Increasing research confirms the long-term analgesic effects of TRPV1 agonists or antagonists in preclinical chronic pain models. However, currently available selective TRPV1 agonists, such as capsaicin, have limited clinical efficacy due to the risk of local irritation.

[0004] Therefore, to achieve better therapeutic effects, it is necessary to actively and directly regulate the microenvironment and neural function to reshape neural perception. Developing self-powered flexible electrical stimulation devices and integrating them with wound dressings is of great significance. Summary of the Invention

[0005] Purpose of the invention: The purpose of this invention is to provide an ultrasound-activated piezoelectric gel dressing, its preparation method, and its application.

[0006] A novel LIFU-mediated immunoreceptor modulation strategy was established and demonstrated for the first time. This strategy utilizes a piezoelectric gel dressing system (BT-dNO@Gel) activated by low-intensity focused ultrasound to accelerate the healing process of diabetic wounds and increase the pain threshold. After the dressing adheres locally to the wound, the antibacterial outer gel layer adapts to changes in wound shape and protects the wound from pathogen infection, while simultaneously promoting the propagation of exogenous ultrasound. Subsequently, the piezoelectric nanoparticles embedded within the dressing release nitric oxide under LIFU stimulation, accelerating tissue repair by promoting immune modulation, angiogenesis, and endothelial cell migration. Simultaneously, the embedded piezoelectric particles deform under the mechanical force of ultrasound, generating free radicals to combat bacterial infection and outputting direct current to inhibit neuronal signal transduction.

[0007] Wound repair is closely related to the local electric field at the wound site, which promotes cell proliferation and migration and is crucial for skin regeneration. Modified piezoelectric nanoparticles possess the ability to output direct current, generate ROS, and release NO, thus simultaneously exerting anti-inflammatory, antibacterial, and repair-promoting effects. The nanoparticles are coated with an antibacterial hydrogel, which can fill complex wound areas, effectively transmitting ultrasound and current to deeper regions; on the other hand, it can resist pathogen invasion, absorb wound exudate, and protect the wound from infection.

[0008] Technical solution: In one aspect, the present invention provides an ultrasonically activated piezoelectric gel dressing, the surface of which is an antibacterial hydrogel layer and the interior is encapsulated with BT-dNO piezoelectric nanoparticles.

[0009] Furthermore, the antibacterial hydrogel layer is composed of cross-linked hydrogels of polyethyleneimine and polyvinyl alcohol; BT-dNO is a nitric oxide donor for the surface coating of piezoelectric barium titanate after hydroxylation and dopamine modification.

[0010] A second aspect of the present invention provides a method for preparing the aforementioned ultrasound-activated piezoelectric gel dressing, comprising the following steps:

[0011] (1) BT nanoparticle hydroxylation modification (BT-OH);

[0012] (2) BT-OH polydopamine modification (BT-OH-pDA);

[0013] (3) BT-OH-pDA surface coated with NO donor (BT-dNO);

[0014] (4) Preparation of branched polyethyleneimine (PEI-BA) gel precursor;

[0015] (5) Preparation of BT-dNO@Gel.

[0016] Further, the method for hydroxylating BT-OH nanoparticles in step (1) is as follows: BT is refluxed in a 30% hydrogen peroxide aqueous solution, a white dispersion is collected, centrifuged and washed with deionized water to collect the BT-OH precipitate, and then dried to obtain BT-OH nanoparticles; the ratio of BT to hydrogen peroxide aqueous solution is 0.5g-3g:20mL-60mL.

[0017] Further, the method for modifying BT-OH-pDA with BT-OH polydopamine in step (2) is as follows: the obtained BT-OH nanoparticles are dispersed in a mixed solution of deionized water and anhydrous ethanol and stirred. During the stirring process, dopamine hydrochloride is added and ammonia is added to adjust the pH of the solution. The mixture is stirred overnight at room temperature. The BT-OH of the pDA coating is collected by centrifugation the next day and dried. The mass ratio of the BT-OH nanoparticles to dopamine hydrochloride is 10-30:2-5.

[0018] Further, the method for coating the surface of BT-OH-pDA with NO donor BT-dNO in step (3) is as follows: the dried product is dissolved in deionized water to prepare a BT-OH-pDA solution, an aqueous solution of NONOate is added, BT-dNO is obtained by π-π attraction, and then dried.

[0019] Further, the method for preparing the branched polyethyleneimine (PEI-BA) gel precursor in step (4) is as follows: PEI is weighed and dissolved in methanol solution; EDC is weighed and dissolved in methanol solution; 3-(carboxy)phenylboronic acid is weighed and dissolved in DMF solution. The above three solutions are mixed, dialyzed, and dried to obtain the PEI-BA gel precursor. The ratio of PEI to methanol solution is 1g-3g:5mL-15mL; the ratio of EDC to methanol solution is 2g-6g:5mL-15mL; and the ratio of 3-(carboxy)phenylboronic acid to DMF solution is 1g-3g:5mL-15mL.

[0020] Further, the preparation method of step (5) BT-dNO@Gel is as follows: the obtained PEI-BA gel precursor is dissolved in deionized water, followed by the addition of BT-dNO aqueous solution, and then polyvinyl alcohol aqueous solution. The volume ratio of PEI-BA gel precursor solution: BT-dNO aqueous solution: polyvinyl alcohol aqueous solution is 2-4: 1-2: 2-4. After mixing, BT-dNO@Gel gel dressing is formed.

[0021] Another aspect of the present invention provides the use of the aforementioned ultrasound-activated piezoelectric gel dressing in wound repair.

[0022] Furthermore, the wound is a diabetic ulcer.

[0023] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0024] 1. It has excellent biocompatibility. At therapeutic doses, all components have been fully validated, and their toxicity is negligible.

[0025] 2. It has an effective combined repair effect. The external gel has the characteristics of tight adhesion, high self-healing ability, strong swelling and strong hemostasis, and can prevent pathogen invasion and promote the spread of exogenous LIFU; the NO release mode triggered by local LIFU in the wound overcomes the challenge of low delivery effectiveness due to the short half-life of NO; ​​the loaded piezoelectric barium titanate can convert the mechanical force generated by LIFU into microcurrent, and at the same time generate ROS, which can perform self-powered, precise and safe neuromodulation and anti-infection.

[0026] 3. It has good anti-inflammatory capabilities. The rational combination of immune homeostasis remodeling and pain receptor desensitization alters the previously existing pro-inflammatory transcriptomic landscape of DFU, making it an anti-inflammatory niche, and promotes wound repair by upregulating the expression of genes related to immune regulation, cell proliferation and nociceptive regulation. Attached Figure Description

[0027] Figure 1 This is a schematic diagram illustrating the synthesis of the piezoelectric hydrogel used in this invention; BT-dNO nanoparticles with piezoelectric and NO-releasing properties were prepared through a stepwise surface modification process and NO donor loading.

[0028] BT-dNO aqueous solution was mixed with PEI-PBA gel precursor solution, and then PVA aqueous solution was added. After mixing, BT-dNO@Gel dressing was formed.

[0029] Figure 2 The images show the transmission electron microscope (TEM) images and corresponding elemental distribution maps of the BT-dNO nanoparticles used in this invention. The first two are TEM images, showing that the BT-dNO nanoparticles are polygonal with a thin polymer layer on the surface. The latter is an energy-dispersive spectroscopy (EDS) elemental mapping image, showing that Bi, Ti, N, O and C elements are uniformly distributed.

[0030] Figure 3 The images show the X-ray photoelectron spectroscopy (XPS), ultraviolet (UV-vis), and thermogravimetric (TGA) spectra of BT-dNO nanoparticles in this invention. a) is the XPS spectrum, and b) is the UV-vis spectrum, both confirming the successful loading of the NO donor (NONOate). c) is the thermogravimetric analysis, showing that the NONOate mass content is 3.62 wt%, which is consistent with the loading content (3.95 ± 0.14%) determined by the absorbance of NONOate at 253 nm in the UV spectrum.

[0031] Figure 4The diagram shows the particle size distribution and dispersion stability of BT-dNO nanoparticles in this invention. In the diagram, a is the particle size distribution, showing that the average hydrated particle size of BT-dNO is 80.49±12.11 nm; b is the dispersion stability diagram, showing that BT-dNO has high colloidal stability and is well dispersed in various solutions within one week.

[0032] Figure 5 This is a digital image of the piezoelectric hydrogel used in this invention; BT-dNO nanoparticles are added...

[0033] The precursor solution is formed in the PBA-PEI solution and then mixed with the PVA solution, undergoing a solution-gel transition under the interaction of imine, boronic ester and hydrogen bonding without additional stimulation.

[0034] Figure 6 These are cryo-scanning electron microscopy (Cryo-SEM) and fluorescence microscopy images of the piezoelectric hydrogel used in this invention. Image a is a Cryo-SEM image showing the typical porous microstructure of BT-dNO@Gel, which may facilitate exudate absorption and oxygen diffusion. Image b is a fluorescence microscopy image showing a uniform distribution of the red Cy3 signal from BT-dNO in the FITC-labeled gel.

[0035] Figure 7 The graphs show the self-healing ability, swelling, and degradation performance of the piezoelectric hydrogel used in this invention, where a represents the macroscopic and microscopic images of self-healing, b represents the swelling rate, and c represents the degradation percentage. Macroscopic healing tests using Rhodamine B (red) and methylene blue (blue) staining showed that two independently contacting gels could heal rapidly without any external interference, and the healed gel could be lifted by its own weight without cracking. Further continuous-step strain tests yielded the same results. When a high dynamic strain (500%) exceeding the critical value was applied, the value of G′ decreased from ~100 Pa to ~10 Pa, lower than G″. However, once a lower strain (1%) was applied, the values ​​of G′ and G″ immediately recovered to almost their original values ​​even after four alternating cycles, demonstrating the self-healing behavior of BT-dNO@Gel. Furthermore, BT-dNO@Gel exhibited time-dependent swelling behavior in PBS at 37°C, with a swelling rate of approximately 800% within 1 hour, reaching an equilibrium swelling state within ~2 hours, indicating that the prepared dressing is sufficient to effectively absorb wound exudate. Furthermore, BT-dNO@Gel is almost completely degraded in physiological culture medium within approximately 10 days.

[0036] Figure 8The rheological and viscosity tests of the piezoelectric hydrogel used in this invention are shown in the figures: a) rheological dynamic frequency sweep experiment, b) time-based Young's modulus, c) strain amplitude sweep experiment, and d) composite viscosity. The dynamic rheological tests show that within a 30-minute timeframe of 0.1-100% Hz, the storage modulus (G′) is significantly higher than the loss modulus (G”), indicating successful gel formation of BT-dNO@Gel. Both G′ and G” values ​​are relatively stable within the strain range of 0.1% to 100%, indicating that BT-dNO@Gel is in the linear viscoelastic region and possesses good viscosity.

[0037] Figure 9 To illustrate the NO release capacity of the piezoelectric hydrogel used in this invention, we show: a) NO release with and without LIFU, b) NO release at different BT-dNO concentrations, c) NO release at different ultrasonic durations, and d) NO release at different acoustic intensities. The LIFU-responsive NO production of BT-dNO@Gel was evaluated under physiological conditions using the classic Griess assay. Figure a shows that grafting NONOate onto the surface of BT NPs under LIFU irradiation produced a large amount of NO, and the responsive NO release characteristics remained unchanged even after further encapsulation into a PEI / PVA gel. Figures b and d show that NO production is not only positively linearly related to the concentration of BT-dNO in the gel but also closely related to the intensity or duration of LIFU.

[0038] Figure 10 The piezoelectric properties of the piezoelectric hydrogel used in this invention were tested, where a is an X-ray diffraction (XRD) pattern, b is the piezoelectric response phase and amplitude curve, c is the voltage signal output by the electrochemical workstation, and d is the current signal output by the electrochemical workstation. XRD shows that the BT-dNO nanoparticles undergo peak splitting near 2θ = 45°, resulting in separate (002) and (200) reflections, verifying the preservation of tetragonality to generate piezoelectricity. Figure b shows the typical butterfly amplitude curve observed in atomic force microscopy (AFM), with a ramp voltage ranging from -10 to +10V, indicating a continuous change in strain induced by the applied external electric field; on the phase diagram, a ~180° phase switch exists in the local circuit of the piezoelectric hysteresis, demonstrating the ideal piezoelectric efficiency of BT-dNO. Figure c shows the output voltage measured by a precision benchtop digital multimeter; without a LIFU, the BT NP produces almost no voltage. In contrast, at 1.5W cm⁻¹... 2Under LIFU stimulation, the output voltage of BT or BT-dNO NPs increased significantly, indicating that surface modification and drug loading processes had no effect on the piezoelectric properties of BT NPs. More importantly, the voltage transfer capability of BT-dNO NPs was amplified to hundreds of millivolts after encapsulation in a gel, suggesting that BT-dNO@Gel can achieve a higher electrical signal output under the same LIFU parameters. Similar to the piezoelectric voltage output results, the real-time current generated by the gel dressing under LIFU stimulation showed that BT-dNO produced 0.12 ± 0.04 μA cm⁻¹. 2 The peak current density, especially after encapsulation in a gel matrix, amplifies the current transport capability of BT-dNO nanoparticles by about 10 times.

[0039] Figure 11 The ROS catalytic degradation performance of the piezoelectric hydrogel in this invention embodiment is shown in the figures, where a represents the ROS degradation with and without LIFU, b represents the ROS degradation at different BT-dNO concentrations, c represents the ROS degradation at different ultrasonic times, d represents the ROS degradation at different acoustic intensities, and e represents the ROS degradation at different O2 concentrations. - Electron spin resonance (ESR) spectroscopy and f-spin resonance (f) of ·OH electron spin resonance spectra were performed. Rhodamine B degradation experiments revealed a positive correlation between ROS yield and BT-dNO concentration, LIFU power, and duration. ESR technology was used to systematically detect and quantitatively analyze the types of reactive oxygen species generated by BT-dNO@Gel during LIFU. Under LIFU irradiation conditions, both the BT-dNO group and the BT-dNO@Gel group exhibited characteristic 1:2:2:1 and 1:1:1:1 quartet signals, respectively confirming the generation of hydroxyl radicals and superoxide anions.

[0040] Figure 12 To illustrate the in vitro repair-promoting properties of the piezoelectric hydrogel used in this invention, where a represents the cell scratch assay, b represents the cell tube formation assay, c represents the migration rate (quantitative of a), d represents the total tube length in the cell tube formation assay, and e represents the number of network cells in the cell tube formation assay. The scratch assay showed that, compared with LIFU irradiation alone, the combination of LIFU and BT@Gel or BT-dNO@Gel significantly promoted the migration of HUVECs, especially after BT-dNO@Gel+LIFU treatment, the 48-hour migration rate of HUVECs reached 84.7% (approximately 2.5 times higher than the control group). Furthermore, compared with other groups, the BT-dNO@Gel+LIFU group exhibited the best HUVEC angiogenesis, characterized by a larger total tube length and more network cells.

[0041] Figure 13To illustrate the in vitro antibacterial properties of the piezoelectric hydrogel used in this invention, the following figures are presented: a) SEM images of Staphylococcus aureus after different treatments; b) bacterial ROS staining; c) ROS quantitative analysis; d) colony growth after different treatments; e) bacterial survival rate in d); f) bacterial live / dead staining; and g) bacterial protein leakage after different treatments. SEM observation showed that the bacteria in the control group had intact morphology and smooth surfaces, while some cytoplasm leaked from the damaged bacterial membrane in the gel group and the BT@Gel group. Notably, the bacterial membrane structure in the BT@Gel+LIFU and BT-dNO@Gel+LIFU groups was severely wrinkled and ruptured, leading to leakage of internal substances and significantly aggravated morphological damage. Similarly, bacterial ROS staining showed strong green ROS fluorescence in Staphylococcus aureus treated with BT@Gel and BT-dNO@Gel after LIFU irradiation, especially in the BT-dNO@Gel+LIFU group. Agar plate assays and bacterial live / dead staining also confirmed that, in the presence of LIFU, both BT@Gel and BT-dNO@Gel treatments significantly reduced the survival rate of Staphylococcus aureus.

[0042] Figure 14 To evaluate the in vitro and in vivo safety of the piezoelectric hydrogel used in this invention, the following parameters were used: a) cytotoxicity of different concentrations of BT-dNO nanoparticles; b) cytotoxicity of different concentrations of hydrogel; c) hemolysis test at different nanoparticle concentrations; and d) HE staining of the heart, liver, spleen, lungs, and kidneys at 7 and 14 days post-treatment. CCK-8 assay showed that BT-dNO (less than 0.3 mg / mL)... -1 ) and PEI / PVA gel (less than 3 mg / mL) -1 The effect of BT-dNO on HUVEC activity is negligible. Furthermore, hemolysis tests showed that the hemolysis rate of BT-dNO was less than 5% within the safe range, indicating good blood compatibility. Post-treatment H&E staining of major organs also showed no abnormalities.

[0043] Figure 15 This diagram illustrates the repair of diabetic rat wounds with piezoelectric hydrogels according to the present invention, showing the treatment process (a), wound closure from day 0 to day 14 (b), and wound closure rate from day 0 to day 14 (c). Wound healing analysis showed that, compared with control rats exhibiting significantly delayed healing, all gel groups containing different formulations promoted the healing process to some extent. Furthermore, the wound closure rate treated with BT@Gel was significantly higher when combined with LIFU than with BT@Gel alone, indicating that piezoelectric stimulation may promote endothelial cell migration. As expected, the BT-dNO@Gel+LIFU group achieved optimal wound repair, with a wound closure rate of approximately 93% compared to the control group.

[0044] Figure 16 This invention presents schematic diagrams and effect images of piezoelectric hydrogel repairing infected wounds in diabetic rats. Figure a shows a treatment diagram, figure b shows wound closure from day 0 to day 14, figure c shows the wound closure rate from day 0 to day 14, figure d shows the bacterial infection status of the wound on days 7 and 14, and figure e shows the quantitative bacterial survival rate in figure d. Results showed that all groups containing gel dressings promoted wound healing to some extent, especially the BT-dNO@Gel+LIFU treatment, which achieved a 90% wound healing rate on day 14. Simultaneously, fluid samples were collected from the wound edges of each group on days 7 and 14 after different interventions. Although significant bacterial colonies were observed in all groups on day 7, it was clear that the colony counts in the BT@Gel+LIFU and BT-dNO@Gel+LIFU groups were slightly lower than in the other groups. The colony survival rate in the BT-dNO@Gel+LIFU group on day 14 was only 7% of the control group, significantly lower than the BT@Gel+LIFU group, indicating that the antibacterial effect was caused by the combined action of generated ROS and NO.

[0045] Figure 17 This diagram illustrates the effect of piezoelectric hydrogel dressing in alleviating diabetic neuropathy in rats during the implementation of this invention. Figure a shows a treatment diagram; figure b shows changes in mechanical pain in rats during diabetic neuropathy modeling; figure c shows changes in thermal pain in rats during modeling; figure d shows changes in mechanical pain in rats during treatment; and figure e shows changes in thermal pain in rats during treatment. The Von Frey filament test and hot plate assay were used to test mechanical and thermal pain behaviors, respectively. Successfully modeled DNP rats were randomly divided into two groups and received different treatments, including PBS and BT-dNO@Gel+LIFU. Compared with the PBS control treatment, the BT-dNO@Gel+LIFU treatment significantly increased the pain threshold of rats, as evidenced by a 50% increase in PWT and PWL. Detailed Implementation

[0046] To make the objectives, technical solutions, and advantages of the present invention clearer, the technical solutions of the present invention will be further described below.

[0047] The ultrasonically activated piezoelectric gel dressing and its preparation method in this embodiment include the following steps:

[0048] 1. Hydroxylation modification of BT nanoparticles (BT-OH). Weigh 1 g of BT and reflux it in 50 mL (30 wt%) of hydrogen peroxide aqueous solution at 105 °C for 2-3 days, collecting the white dispersion. Centrifuge (9000 rpm, 5 min) three times and wash with deionized water to collect the BT-OH precipitate. Dry in a vacuum oven to obtain BT-OH nanoparticles, weigh them, and set aside for later use.

[0049] 2. BT-OH Polydopamine Modification (BT-OH-pDA). The BT-OH nanoparticles (12.5 mg) obtained in step 1 were dispersed in a mixed solution of 10 mL deionized water and 10 mL anhydrous ethanol, and homogenized using an ultrasonicator before stirring. Fresh dopamine hydrochloride (2.5 mg) was rapidly added during stirring, and 20 μL of ammonia was added to adjust the pH of the solution. The mixture was stirred overnight at room temperature. The next day, the BT-OH-pDA coated with pDA was collected three times by centrifugation (10000 rpm, 10 min), dried in a vacuum oven, and weighed for later use.

[0050] 3. BT-OH-pDA surface coated with NO donor (BT-dNO). Prepare a BT-OH-pDA solution (1 mg / mL) by dissolving 2 in deionized water. 1 During stirring, add NONOate aqueous solution (1 mg / mL) while protecting from light. -1 Continue stirring in the dark overnight to obtain BT-dNO through π-π attraction. The next day, wash three times with water, dry in a vacuum oven, and weigh for later use.

[0051] 4. Preparation of branched polyethyleneimine (PEI-BA) gel precursor. Weigh 1 g of PEI and dissolve it in 5 mL of methanol solution; weigh 2 g of EDC and dissolve it in 5 mL of methanol solution; weigh 1 g of 3-(carboxy)phenylboronic acid and dissolve it in 5 mL of DMF solution. Mix the three solutions and stir overnight, then dialyze for 3 days. After freeze-drying, obtain the PEI-BA gel precursor and weigh it for later use.

[0052] 5. Preparation of BT-dNO@Gel. The PEI-BA gel precursor obtained in step 4 was dissolved in deionized water (60 mg / mL). -1 Then add the BT-dNO aqueous solution obtained in step 3 (2 mg / mL) -1 After stirring evenly, add polyvinyl alcohol (40 mg / mL). -1 The three components, in an aqueous solution with a volume ratio of 2:1:2, can form a BT-dNO@Gel dressing within minutes of mixing.

[0053] 6. LIFU Response Implementation Method. The gel dressing can simulate the transmission of acoustic energy by ultrasound coupling agent. After receiving the LIFU signal, the piezoelectric nanoparticles inside the gel dressing undergo mechanical deformation, converting the mechanical force generated by LIFU into microcurrent and generating ROS. At the same time, the NO donor loaded on the surface of the nanoparticles decomposes and releases NO, synergistically performing gas therapy.

[0054] Figure 1This invention demonstrates the step-by-step synthesis of the piezoelectric hydrogel. First, BT-dNO nanoparticles with piezoelectric and NO-releasing properties were prepared through a step-by-step surface modification process and NO donor loading. Then, an aqueous solution of BT-dNO was mixed with a PEI-PBA gel precursor solution, followed by the addition of an aqueous solution of polyvinyl alcohol, resulting in a BT-dNO@Gel gel dressing.

[0055] Figure 2 Display: Transmission electron microscopy (TEM) images and corresponding elemental distribution maps of BT-dNO nanoparticles in this embodiment of the invention. TEM images and energy-dispersive elemental mapping (EDS) show that the BT-dNO nanoparticles are polygonal in shape with a thin polymer layer on the surface, and that Bi, Ti, N, O, and C elements are uniformly distributed.

[0056] Figure 3 Display: X-ray photoelectron spectroscopy (XPS), ultraviolet (UV-vis), and thermogravimetric analysis (TGA) spectra of BT-dNO nanoparticles, where a is X-ray photoelectron spectroscopy, b is ultraviolet spectroscopy, and c is thermogravimetric analysis. XPS and UV-vis confirmed the successful loading of the NO donor (NONOate). TGA showed that the mass content of NONOate was 3.62 wt%, which is consistent with the loading content (3.95 ± 0.14%) determined by the absorbance of NONOate at 253 nm in the ultraviolet spectrum.

[0057] Figure 4 The results showed that dynamic light scattering (DLS) revealed an average hydrated particle size of BT-dNO of 80.49 ± 12.11 nm, indicating high colloidal stability and good dispersion in various solutions within one week.

[0058] Figure 5 Display: A digital image of the piezoelectric hydrogel in this embodiment of the invention. BT-dNO nanoparticles are added to a PBA-PEI solution to form a precursor solution, which is then mixed with a PVA solution, undergoing a solution-gel transition through the interactions of imine, boronic ester, and hydrogen bonding without additional stimulation.

[0059] Figure 6 The results show that cryo-scanning electron microscopy (Cryo-SEM) reveals the typical porous microstructure of BT-dNO@Gel, which may facilitate exudate absorption and oxygen diffusion. Fluorescence microscopy observations show that the red Cy3 signal from BT-dNO is uniformly distributed in the FITC-labeled gel.

[0060] Figure 7The self-healing ability of BT-dNO@Gel was first tested. Macroscopic healing assays using Rhodamine B (red) and methylene blue (blue) staining showed that two independently contacting gels could heal rapidly without any external interference, and the healed gel could be lifted by its own weight without cracking. Further continuous-step strain tests yielded the same results. When a high dynamic strain (500%) exceeding the critical value was applied, the value of G′ decreased from ~100 Pa to ~10 Pa, below G″. However, once a lower strain (1%) was applied, the values ​​of G′ and G″ immediately recovered to almost their original values ​​even after four alternating cycles, demonstrating the autonomous repair behavior of BT-dNO@Gel. Furthermore, BT-dNO@Gel exhibited time-dependent swelling behavior in PBS at 37°C, with a swelling rate of approximately 800% within 1 hour and reaching equilibrium swelling within ~2 hours, indicating that the prepared dressing was sufficient to effectively absorb wound exudate. Additionally, BT-dNO@Gel almost completely degraded in physiological culture medium within approximately 10 days. The swelling rate and degradation rate were calculated as follows: The hydrogel sample was freeze-dried, and its dry weight (W0) was recorded. To simulate physiological conditions, the hydrogel was immersed in 5 mL of PBS at a constant temperature (37°C) and shaken at 60 rpm. At predetermined time intervals, the sample was removed and surface moisture was removed using filter paper. For the swelling test, the hydrogel was weighed as Wts, and the swelling ratio (%) was calculated using the following formula: (Wts - W0) / W0 × 100. For the degradation test, the hydrogel sample was freeze-dried and weighed as Wtd. The degradation percentage of the hydrogel was calculated using the following formula: Degradation percentage (%) = (W0 - Wtd) / Wtd × 100.

[0061] Figure 8 The results show that dynamic rheological tests indicate that within 30 minutes of a frequency range of 0.1-100% Hz, the storage modulus (G′) is significantly higher than the loss modulus (G″), indicating successful gel formation of BT-dNO@Gel. Both G′ and G″ values ​​are relatively stable within the strain range of 0.1% to 100%, suggesting that BT-dNO@Gel is in the linear viscoelastic region and possesses good viscosity.

[0062] Figure 9 Display: The LIFU-responsive NO production curve of BT-dNO@Gel was evaluated using the classic Griess assay under physiological conditions. Under LIFU irradiation, grafting NONOate onto the surface of BT NPs generated a large amount of NO, and the responsive NO release characteristics remained unchanged even after further encapsulation into a PEI / PVA gel. NO production was not only positively linearly related to the concentration of BT-dNO in the gel, but also closely related to the intensity or duration of LIFU.

[0063] Figure 10 Display: X-ray diffraction (XRD) showed that BT-dNO nanoparticles exhibited peak splitting near 2θ = 45°, resulting in separate (002) and (200) reflections, verifying the preservation of tetragonality to generate piezoelectricity. A typical butterfly-shaped amplitude curve observed in atomic force microscopy (AFM), with a ramp voltage ranging from -10 to +10 V, indicates a continuous change in strain induced by the applied external electric field; on the phase diagram, a ~180° phase switch exists in the local circuit of the piezoelectric hysteresis, demonstrating the ideal piezoelectric efficiency of BT-dNO. The LIFU response output voltage of BT-dNO@Gel was measured using a high-precision benchtop digital multimeter. Without the LIFU, the BT NP produces almost no voltage. In contrast, at 1.5 W cm⁻¹… 2 Under LIFU stimulation, the output voltage of BT or BT-dNO NPs increased significantly, indicating that surface modification and drug loading processes had no effect on the piezoelectric properties of BT NPs. More importantly, the voltage transfer capability of BT-dNO NPs was amplified to hundreds of millivolts after encapsulation in a gel, suggesting that BT-dNO@Gel can achieve a higher electrical signal output under the same LIFU parameters. Similar to the piezoelectric voltage output results, the real-time current generated by the gel dressing under LIFU stimulation showed that BT-dNO produced 0.12 ± 0.04 μA cm⁻¹. -2 The peak current density, especially after encapsulation in a gel matrix, amplifies the current transport capability of BT-dNO nanoparticles by about 10 times.

[0064] Figure 11 The experiment demonstrated the ROS generation capacity of BT-dNO@Gel under LIFU activation. Rhodamine B degradation experiments revealed a positive correlation between ROS yield and BT-dNO concentration, LIFU power, and duration. Electron spin resonance (ESR) technology was used to systematically detect and quantitatively analyze the types of reactive oxygen species generated by BT-dNO@Gel during LIFU. Experimental data showed that under LIFU irradiation conditions, both the BT-dNO group and the BT-dNO@Gel group exhibited characteristic 1:2:2:1 and 1:1:1:1 quartet signals, respectively confirming the generation of hydroxyl radicals and superoxide anions.

[0065] Figure 12The results showed that scratch assays demonstrated that, compared to LIFU irradiation alone, the combination of LIFU with BT@Gel or BT-dNO@Gel significantly promoted HUVEC migration, particularly with BT-dNO@Gel + LIFU treatment achieving a 48-hour migration rate of 84.7% (approximately 2.5 times higher than the control group). Furthermore, compared to other groups, the BT-dNO@Gel + LIFU group exhibited the best HUVEC angiogenesis, characterized by a larger total vessel length and a greater network of vessels.

[0066] Figure 13 The study evaluated the antibacterial properties of BT-dNO@Gel against Staphylococcus aureus. Scanning electron microscopy revealed that bacteria in the control group maintained intact morphology and had smooth surfaces, while some cytoplasm leaked from the damaged bacterial membranes in the gel and BT@Gel groups. The bacterial membrane structures in the BT@Gel+LIFU and BT-dNO@Gel+LIFU groups showed severe wrinkling and rupture, leading to leakage of internal substances and significantly exacerbating morphological damage. Similarly, bacterial ROS staining showed strong ROS green fluorescence in Staphylococcus aureus treated with BT@Gel and BT-dNO@Gel after LIFU irradiation, especially in the BT-dNO@Gel+LIFU group. Agar plate assays and bacterial viability / deadness staining also confirmed that both BT@Gel and BT-dNO@Gel treatments significantly reduced the survival rate of Staphylococcus aureus in the presence of LIFU.

[0067] Figure 14 The results showed that the CCK-8 assay showed BT-dNO (less than 0.3 mg / mL). -1 ) and PEI / PVA gel (less than 3 mg / mL) -1 The effect of BT-dNO on HUVEC activity is negligible. Furthermore, hemolysis tests showed that the hemolysis rate of BT-dNO was less than 5% within the safe range, indicating good blood compatibility. Post-treatment H&E staining of major organs also showed no abnormalities.

[0068] Figure 15 The study demonstrated the therapeutic effect of BT-dNO@Gel in a streptozotocin (STZ)-induced diabetic rat full-thickness skin wound model. Figure a shows the schematic diagram of the study design. Wound healing analysis showed that all gel groups containing different formulations promoted the healing process to some extent compared to control rats with significantly delayed healing. Furthermore, the wound closure rate of BT@Gel-treated wounds was significantly higher when combined with LIFU than when treated with BT@Gel alone, indicating that piezoelectric stimulation may promote endothelial cell migration. As expected, the BT-dNO@Gel+LIFU group achieved optimal wound repair, with a wound closure rate of ≈93% compared to the control group.

[0069] Figure 16 The study demonstrated a diabetic rat model with a full-thickness infected wound, as previously described. Figure 'a' shows the schematic diagram of the study design. Results showed that all groups receiving gel dressings promoted wound healing to some extent, with the BT-dNO@Gel+LIFU treatment achieving a 90% wound healing rate by day 14. Fluid samples were collected from the wound edges of each group on days 7 and 14 after each intervention. Although significant bacterial colonies were observed in all groups on day 7, the colony counts in the BT@Gel+LIFU and BT-dNO@Gel+LIFU groups were slightly lower than in the other groups. The colony survival rate in the BT-dNO@Gel+LIFU group on day 14 was only 7% of that in the control group, significantly lower than that in the BT@Gel+LIFU group, indicating that the antibacterial effect was caused by the combined action of generated ROS and NO.

[0070] Figure 17 To test the anti-nociceptive effect of BT-dNO@Gel on LIFU irradiation, a diabetic neuropathic pain model was established. The Von Frey filament test and hot plate assay were used to test mechanical and thermal pain behaviors, respectively. Successfully modeled DNP rats were randomly divided into two groups, receiving different treatments: PBS and BT-dNO@Gel + LIFU. Compared with the PBS control treatment, BT-dNO@Gel + LIFU therapy significantly increased the pain threshold in rats, as evidenced by a 50% increase in PWT and PWL.

[0071] The above are merely preferred embodiments of the present invention and do not constitute any limitation on the present invention. Any equivalent substitutions or modifications made by those skilled in the art to the technical solutions and content disclosed in the present invention without departing from the scope of the present invention shall be deemed to have remained within the protection scope of the present invention.

Claims

1. An ultrasonically activated piezoelectric gel dressing, characterized in that, The dressing has an antibacterial hydrogel layer on its surface and encapsulates BT-dNO piezoelectric nanoparticles inside.

2. The ultrasonically activated piezoelectric gel dressing according to claim 1, characterized in that, The antibacterial hydrogel layer is composed of cross-linked hydrogels of polyethyleneimine and polyvinyl alcohol; BT-dNO is a nitric oxide donor coated on the surface of barium piezoelectric titanate after hydroxylation and dopamine modification.

3. The method for preparing the ultrasonically activated piezoelectric gel dressing according to claim 1 or 2, characterized in that, Includes the following steps: (1) BT nanoparticles were hydroxylated to modify BT-OH; (2) BT-OH-pDA modified with BT-OH polydopamine; (3) The NO donor BT-dNO is coated on the surface of BT-OH-pDA; (4) Preparation of branched polyethyleneimine (PEI-BA) gel precursor; (5) Preparation of BT-dNO@Gel.

4. The method for preparing the ultrasonically activated piezoelectric gel dressing according to claim 3, characterized in that, The method for hydroxylating BT-OH nanoparticles in step (1) is as follows: BT is refluxed in hydrogen peroxide aqueous solution, a white dispersion is collected, centrifuged and washed with deionized water to collect BT-OH precipitate, and then dried to obtain BT-OH nanoparticles; the ratio of BT to hydrogen peroxide aqueous solution is 0.5g-3g:20mL-60mL.

5. The method for preparing the ultrasonically activated piezoelectric gel dressing according to claim 4, characterized in that, The method for modifying BT-OH-pDA with BT-OH polydopamine in step (2) is as follows: the obtained BT-OH nanoparticles are dispersed in a mixed solution of deionized water and anhydrous ethanol, dopamine hydrochloride is added, and ammonia is added to adjust the pH of the solution. The BT-OH of the pDA coating is collected by centrifugation and dried. The mass ratio of the BT-OH nanoparticles to dopamine hydrochloride is 10-30:2-5.

6. The method for preparing the ultrasonically activated piezoelectric gel dressing according to claim 5, characterized in that, The method for coating the surface of BT-OH-pDA with NO donor BT-dNO in step (3) is as follows: the dried product is dissolved in deionized water to prepare a BT-OH-pDA solution, an aqueous solution of NONOate is added, BT-dNO is obtained by π-π attraction, and then dried.

7. The method for preparing the ultrasonically activated piezoelectric gel dressing according to claim 6, characterized in that, The method for preparing branched polyethyleneimine (PEI-BA) gel precursor in step (4) is as follows: PEI is weighed and dissolved in methanol solution; EDC is weighed and dissolved in methanol solution; 3-(carboxy)phenylboronic acid is weighed and dissolved in DMF solution. The above three solutions are mixed, dialyzed, and dried to obtain PEI-BA gel precursor. The ratio of PEI to methanol solution is 1g-3g:5mL-15mL; the ratio of EDC to methanol solution is 2g-6g:5mL-15mL; and the ratio of 3-(carboxy)phenylboronic acid to DMF solution is 1g-3g:5mL-15mL.

8. The method for preparing the ultrasonically activated piezoelectric gel dressing according to claim 7, characterized in that, The preparation method of step (5) BT-dNO@Gel is as follows: the obtained PEI-BA gel precursor is dissolved in deionized water, followed by the addition of BT-dNO aqueous solution, and then polyvinyl alcohol aqueous solution. The volume ratio of PEI-BA gel precursor solution: BT-dNO aqueous solution: polyvinyl alcohol aqueous solution is 2-4: 1-2: 2-4. After mixing, BT-dNO@Gel gel dressing is formed.

9. Use of the ultrasound-activated piezoelectric gel dressing according to claim 1 or 2 in wound repair.

10. The use according to claim 9, wherein the wound is a wound caused by a diabetic ulcer.

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