Multifunctional low-frequency low-energy piezoelectric hydrogel based on natural triterpenoids assembly and preparation method thereof

Abstract

This invention belongs to the field of biomedical materials technology, specifically relating to a multifunctional low-frequency, low-energy piezoelectric hydrogel based on natural triterpenoid assembly and its preparation method. The hydrogel is formed by the self-assembly of small natural triterpenoid molecules with both antibacterial and anti-inflammatory activities, with a uniformly loaded low-frequency, low-energy piezoelectric material ZnO-CuO inside. On the one hand, it overcomes the shortcomings of existing piezoelectric hydrogels that require high-frequency ultrasound actuation and lack bioactivity in the gel matrix; on the other hand, it achieves the simultaneous generation of piezoelectrically catalytic active oxygen under low-frequency mechanical stimulation to exert piezoelectric antibacterial effects and generate piezoelectric microcurrents to promote angiogenesis. This hydrogel achieves highly efficient synergistic antibacterial and anti-inflammatory properties by utilizing endogenous bioenergy stimulation. Furthermore, this hydrogel has good injectability and tissue adhesion, showing broad clinical application prospects in the preparation of repair dressings for the treatment of complex wound infections such as diabetic foot ulcers and tissue regeneration.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, specifically relating to a multifunctional low-frequency, low-energy piezoelectric hydrogel based on natural triterpenoid assembly and its preparation method. Background Technology

[0002] Diabetic foot ulcer (DFU) is one of the most serious and prevalent chronic complications of diabetes, with a global prevalence of approximately 6.3% and a 5-year mortality rate as high as 30%. DFU wound healing is influenced by multiple factors, primarily persistent bacterial infection and chronic inflammation, leading to prolonged inflammatory stagnation and difficulty in healing. Currently, clinical treatments for DFU mainly include glycemic control, surgical debridement, routine topical dressings, and anti-infective therapy. Traditional dry dressings such as gauze and cotton pads tend to adhere to wound tissue, causing secondary damage, and are difficult to effectively regulate the wound microenvironment. In recent years, hydrogel dressings with high water content and good biocompatibility have attracted widespread attention. Among them, piezoelectric hydrogels, as a smart material, can convert the mechanical energy generated by limb activity into local electrical signals. On the one hand, they regulate cell behavior through electrical stimulation to promote angiogenesis; on the other hand, they utilize piezoelectric catalysis to generate reactive oxygen species for antibacterial effects, showing broad application prospects in antibacterial and tissue regeneration promotion.

[0003] Despite the promising prospects of piezoelectric hydrogels in the treatment of chronic wounds such as DFU, their clinical translation still faces several technical bottlenecks. First, existing piezoelectric material systems (such as BaTiO3, PZT, and PVDF) typically require high-frequency ultrasound or high-intensity mechanical impact as external excitation sources to effectively stimulate the piezoelectric effect and generate therapeutic microcurrents or reactive oxygen species. This strong dependence on external devices not only increases the complexity of clinical procedures and the burden on patients but also severely limits patients' continuous and convenient self-care outside of hospitals or at home, significantly falling short of the actual needs of long-term chronic wound management. Second, most reported piezoelectric hydrogels use synthetic polymers (such as PVA and PEG) as the gel matrix. While these materials possess good mechanical strength, processability, and biocompatibility, they lack pharmacological activities such as antibacterial, anti-inflammatory, or angiogenesis-promoting effects. Therefore, functionally, they primarily serve as physical scaffolds and piezoelectric material carriers, making it difficult for them to actively participate in the regulation of the wound's pathological microenvironment. This means that the overall therapeutic efficacy of hydrogels mainly relies on the piezoelectric effect itself, lacking synergistic effects with active pharmaceutical ingredients, which to some extent limits the further improvement of its comprehensive therapeutic effect.

[0004] Therefore, developing a multifunctional smart hydrogel dressing that can respond to low-frequency mechanical stimulation, utilizes the body's own activity for self-drive, and whose hydrogel matrix itself has both antibacterial and anti-inflammatory activities is of great practical significance for improving the clinical treatment effect of DFU. Summary of the Invention

[0005] One objective of this invention is to provide a method for preparing a multifunctional low-frequency, low-energy piezoelectric hydrogel based on natural triterpenoid assembly. ZnO-based piezoelectric materials have been extensively studied in the field of piezoelectric catalysis due to their excellent biocompatibility. Natural triterpenoids, possessing both excellent self-assembly properties and antibacterial and anti-inflammatory bioactivities, have attracted considerable attention in drug delivery and have the potential to serve as functional hydrogel matrices for synergistic therapy with piezoelectric catalytic materials. Combining these two methods to construct a low-frequency, low-energy, stimulus-responsive piezoelectric hydrogel achieves synergistic antibacterial, anti-inflammatory, and angiogenesis-promoting effects.

[0006] The second objective of this invention is to provide a multifunctional low-frequency, low-energy piezoelectric hydrogel based on natural triterpenes. This invention overcomes the shortcomings of existing piezoelectric hydrogels, which require high-frequency ultrasound actuation and whose corresponding hydrogel matrices lack bioactivity. The ZnO-CuO piezoelectric material possesses urchin-like tentacles; this unique structure can respond to endogenous biomechanical movements such as daily bodily activities. Under low-frequency mechanical stimulation, it simultaneously generates piezoelectrically catalyzed reactive oxygen species for synergistic antibacterial effects and produces piezoelectric microcurrents to promote angiogenesis. Through the synergistic effect of natural triterpenes and the piezoelectric material, highly efficient antibacterial, anti-inflammatory, and angiogenesis-promoting effects are achieved. Furthermore, this hydrogel exhibits good injectability and tissue adhesion, showing broad clinical application prospects in the treatment of complex infected wounds such as diabetic foot ulcers and in tissue regeneration.

[0007] The third objective of this invention is to provide an application of a multifunctional low-frequency, low-energy piezoelectric hydrogel based on natural triterpenoid assembly.

[0008] To achieve the first objective of this invention, the following technical solution is adopted: A method for preparing a multifunctional low-frequency, low-energy piezoelectric hydrogel based on natural triterpenoid assembly includes the following steps: S1. Triterpenoids and choline hydroxide are added to a solvent and refluxed to obtain triterpenoid choline salts. S2. Add sea urchin-shaped ZnO-CuO to water to obtain a uniform suspension; add triterpenoid choline salts to the suspension, heat and then induce self-assembly by ultrasound to obtain the final product.

[0009] Further, the triterpenoid compound in step S1 is any one of ursolic acid, oleanolic acid, and betulinic acid, and the solvent is methanol.

[0010] Further, in step S1, the molar ratio of the triterpenoid compound to choline hydroxide is 1:1.2, the molar ratio of the triterpenoid compound to the solvent is 1-40 mg:1 mL, and the solvent is methanol.

[0011] Furthermore, the reflux reaction in step S1 is carried out at a temperature of 30-80°C for 2-24 hours.

[0012] Furthermore, the reflux reaction in step S1 is carried out at a temperature of 65°C for 10 hours.

[0013] Further, in step S2, the ratio of sea urchin-like ZnO-CuO, triterpenoid choline salt, and water is 1-4 mg: 50-64 mg: 1 mL.

[0014] Furthermore, the heating temperature in step S2 is 60℃-90℃, and the heating time is 3 min-15 min.

[0015] Furthermore, the heating temperature in step S2 is 65°C, and the heating time is 5 minutes.

[0016] Further, the preparation method of the sea urchin-shaped ZnO-CuO in step S2 is as follows: weigh zinc acetate dihydrate and copper acetate and dissolve them in deionized water to obtain a mixed solution; add the obtained mixed solution to deionized water containing DMSO, stir for 1 hour and then add ammonia solution, heat at 60°C for 4 hours, centrifuge to collect the precipitate, and wash to obtain the product.

[0017] Furthermore, the time for ultrasound-induced self-assembly in step S2 is 3-5 minutes, and the power of ultrasound-induced self-assembly is 80-200 MHz.

[0018] Furthermore, the frequency of the ultrasound-induced self-assembly is 120 MHz.

[0019] To achieve the second objective of this invention, the following technical solution is adopted: A multifunctional low-frequency, low-energy piezoelectric hydrogel based on natural triterpenoid assembly was prepared according to the aforementioned preparation method.

[0020] To achieve the third objective of this invention, the following technical solution is adopted: Application of a multifunctional low-frequency, low-energy piezoelectric hydrogel based on natural triterpenoid assembly in the preparation of dressings for diabetic foot wound healing.

[0021] Compared with the prior art, the beneficial effects of this invention are as follows: 1. This invention provides a multifunctional low-frequency, low-energy piezoelectric hydrogel based on natural triterpenoid assembly. The hydrogel is composed of a hydrogel matrix formed by the self-assembly of natural triterpenoid small molecules with antibacterial and anti-inflammatory activities, and a low-frequency, low-energy piezoelectric material ZnO-CuO uniformly loaded therein.

[0022] 2. The ZnO-CuO piezoelectric material loaded in the hydrogel has urchin-like tentacles. This special structure can respond to endogenous biomechanical movements such as daily bodily activities. Under low-frequency mechanical stimulation, it can simultaneously generate piezoelectric catalytic active oxygen to achieve synergistic antibacterial effect and generate piezoelectric microcurrent to promote angiogenesis.

[0023] 3. The ZnO-CuO piezoelectric material and triterpenoids in this hydrogel can work synergistically. The piezoelectric hydrogel constructed by ZnO-CuO (ZC) and triterpenoids achieves efficient antibacterial, anti-inflammatory and angiogenesis-promoting effects. In addition, the hydrogel has good injectability and tissue adhesion, and has broad clinical application prospects in the treatment of complex infected wounds such as diabetic foot ulcers and tissue regeneration. Attached Figure Description

[0024] Figure 1 The UA obtained in Embodiment 1 of the present invention 1 H NMR spectrum; Figure 2 The BA obtained in Embodiment 2 of the present invention 1 H NMR spectrum; Figure 3 The OA obtained in Embodiment 3 of the present invention 1 H NMR spectrum; Figure 4 The GA obtained in Example 4 of this invention 1 H NMR spectrum; Figure 5 A represents the chemical structural formulas of four triterpenoid choline salts: UA, BA, OA, and GA. Figure 5 B is a schematic diagram of the preparation of the obtained UA, BA, OA and GA hydrogels; Figure 6 The preparation process of UAZC piezoelectric hydrogel, BAZC piezoelectric hydrogel, OAZC piezoelectric hydrogel, and GAZC piezoelectric hydrogel; Figure 7 This diagram shows the molecular formula of glycyrrhizic acid GL, the preparation of GL hydrogel and GLZC piezoelectric composite hydrogel.

[0025] Figure 8 XRD crystal structure comparison of ZnO-CuO, UA hydrogel obtained in Comparative Example 1, and UAZC piezoelectric hydrogel obtained in Example 1; Figure 9 The images show the SEM morphology and EDS elemental distribution of the UAZC piezoelectric hydrogel obtained in Example 1; where... Figure 9 A is a SEM image of the UAZC piezoelectric hydrogel obtained in Example 1. Figure 9 B is the elemental surface scan of the UAZC piezoelectric hydrogel EDS obtained in Example 1; Figure 10The rheological properties of the UAZC piezoelectric hydrogel obtained in Example 1 and the UA hydrogel obtained in Comparative Example 1 are shown in the graphs. Figure 10 A shows the frequency scan results of the two groups of hydrogels. Figure 10 B shows the strain scanning test results of the two groups of hydrogels. Figure 10 C shows the results of continuous cyclic step strain tests on two groups of hydrogels; Figure 11 The images show in vitro antibacterial plate plots and bacterial survival rate bar charts for each group of materials; among them... Figure 11 A and Figure 11 Figure B shows the bacterial plating results of Staphylococcus aureus with ZnO-CuO (ZC) dispersion and UAZC piezoelectric hydrogel under shaking and static culture conditions. Figure 11 C is a statistical graph showing the survival rate of Staphylococcus aureus after treatment with UAZC piezoelectric hydrogel obtained in Example 1, UA hydrogel obtained in Comparative Example 1, and ZnO-CuO (ZC) dispersion. Figure 11 D is a statistical graph showing the survival rate of Escherichia coli after treatment with UAZC piezoelectric hydrogel obtained in Example 1, UA hydrogel obtained in Comparative Example 1, and ZnO-CuO (ZC) dispersion. Figure 12 The images show fluorescence staining of endothelial cells forming tubes and quantitative statistical graphs of tube-forming nodes; among which... Figure 12 A shows representative images of angiogenesis in HUVECs cells after different treatment conditions and images stained with calcein-AM (scale bar: 200 μm). Figure 12 B is a statistical result graph of angiogenesis capacity.

[0026] Figure 13 The images show real-life photos of DFU wound healing in rats and a statistical chart of quantitative recovery of wound area; among them Figure 13 A and Figure 13 B represents the recovery trajectory of DFU wounds in rats of different treatment groups at days 0, 3, 7, 10, and 14, respectively; and the corresponding wound area statistics. Detailed Implementation

[0027] The technical solution of the present invention will be further explained below with reference to specific embodiments, comparative examples, experimental examples and accompanying drawings.

[0028] Unless otherwise specified, the raw materials and preparation methods used in the following examples, comparative examples, and experimental cases are all conventional materials and techniques in the art.

[0029] The preparation method of sea urchin-shaped ZnO-CuO in this invention refers to Example 1 in CN 118324176 B, and the specific steps are as follows: Accurately weigh 13 mg of zinc acetate dihydrate and 1.3 mg of copper acetate, dissolve them in 1 mL of deionized water, and ultrasonically disperse them evenly. Then, add the mixture to 20 mL of deionized water containing 1 mL of DMSO, ultrasonically disperse it evenly, and stir (600 rpm) for 1 h. Subsequently, add 25 μL of ammonia solution (1 mol / L), heat the mixture at 60 °C for 4 h, and centrifuge and wash to obtain a light brown solid powder, which is the sea urchin-shaped ZnO-CuO.

[0030] Example 1 A method for preparing a multifunctional low-frequency, low-energy piezoelectric hydrogel based on natural triterpenoid assembly includes the following steps: S1. Ursolic acid and choline hydroxide were added to methanol at a molar ratio of 1:1.2, with a volume ratio of 20 mg ursolic acid to 1 mL methanol; the mixture was then refluxed at 65 °C for 10 h; after the reaction was completed, the solvent was removed by rotary evaporation to obtain ursolic acid choline salt (UA); UA 1 H NMR spectrum as follows Figure 1 As shown.

[0031] S2. Sea urchin-like ZnO-CuO (ZC) was ultrasonically dispersed in deionized water to obtain a uniform suspension (ZC concentrations in deionized water were 1 mg / mL, 2 mg / mL, 3 mg / mL, and 4 mg / mL, respectively); then 64 mg of ursolic acid choline salt (UA) was added to the suspension; then it was heated at 65°C for 5 min to ensure complete dissolution, followed by ultrasonic treatment (120 MHz) for 3 min to induce self-assembly and form a stable three-dimensional hydrogel network, namely the multifunctional low-frequency low-energy piezoelectric hydrogel UA@ZnO–CuO (UAZC).

[0032] Example 2 A method for preparing a multifunctional low-frequency, low-energy piezoelectric hydrogel based on natural triterpenoid assembly includes the following steps: S1. Betulinic acid and choline hydroxide were added to methanol at a molar ratio of 1:1.2, with a betulinic acid to methanol ratio of 20 mg: 1 mL; the mixture was then refluxed at 65 °C for 10 h; after the reaction was complete, the solvent was removed by rotary evaporation to obtain betulinic acid choline salt (BA); BA 1 H NMR spectrum as follows Figure 2 As shown.

[0033] S2. 1 mg of sea urchin-shaped ZnO-CuO (ZC) was ultrasonically dispersed in 1 mL of deionized water to obtain a uniform suspension; then 50 mg of betulinic acid choline salt (BA) was added to the suspension; then it was heated at 65°C for 5 min to ensure complete dissolution, followed by ultrasonic treatment (120 MHz) for 5 min to induce self-assembly, thus forming a multifunctional low-frequency low-energy piezoelectric hydrogel BA@ZnO–CuO (BAZC).

[0034] Example 3 A method for preparing a multifunctional low-frequency, low-energy piezoelectric hydrogel based on natural triterpenoid assembly includes the following steps: S1. Oleanolic acid and choline hydroxide were added to methanol at a molar ratio of 1:1.2, with an oleanolic acid to methanol ratio of 20 mg: 1 mL; the mixture was then refluxed at 65 °C for 10 h; after the reaction was complete, the solvent was removed by rotary evaporation to obtain oleanolic acid choline salt (OA); OA... 1 H NMR spectrum as follows Figure 3 As shown.

[0035] S2. 1 mg of sea urchin-shaped ZnO-CuO (ZC) was ultrasonically dispersed in 1 mL of deionized water to obtain a uniform suspension; then 60 mg of oleanolic acid choline salt (OA) was added to the suspension; then heated at 65°C for 5 min to ensure complete dissolution, followed by ultrasonic treatment (120 MHz) for 5 min to induce self-assembly, thus forming a multifunctional low-frequency low-energy piezoelectric hydrogel OA@ZnO–CuO (OAZC).

[0036] Example 4 A method for preparing a multifunctional low-frequency, low-energy piezoelectric hydrogel based on natural triterpenoid assembly includes the following steps: S1. Glycyrrhetinic acid and choline hydroxide were added to methanol at a molar ratio of 1:1.2, with a glycyrrhetinic acid to methanol ratio of 20 mg: 1 mL; the mixture was then refluxed at 65 °C for 10 h; after the reaction was completed, the solvent was removed by rotary evaporation to obtain glycyrrhetinic choline salt (GA); GA... 1 H NMR spectrum as follows Figure 4 As shown.

[0037] S2. 1 mg of sea urchin-shaped ZnO-CuO (ZC) was ultrasonically dispersed in 1 mL of deionized water to obtain a uniform suspension; then 240 mg of glycyrrhetinic choline salt (GA) was added to the suspension; then it was heated at 65°C for 5 min to ensure complete dissolution, followed by ultrasonic treatment (120 MHz) for 5 min, but GAZC piezoelectric hydrogel was not successfully prepared. Furthermore, despite attempts under various conditions, piezoelectric hydrogels (GAZC hydrogels) could not be successfully prepared.

[0038] Comparative Example 1 This comparative example provides a method for preparing UA hydrogel, comprising the following steps: 64 mg of ursolic acid choline salt (UA, obtained in step S1 of Example 1) is added to 1 mL of deionized water and heated at 65°C for 5 min to completely dissolve and uniformly disperse it. The hydrogel is then treated with ultrasound (120 MHz) for 5 min to induce self-assembly, forming a stable three-dimensional hydrogel network, denoted as UA hydrogel.

[0039] Comparative Example 2 This comparative example provides a method for preparing BA hydrogel, comprising the following steps: adding 50 mg of betulinic acid choline salt (BA, obtained in step S1 of Example 2) to 1 mL of deionized water. The mixture is heated at 80°C for 5 min to completely dissolve and uniformly disperse it. After standing at room temperature for 10 min, it is induced to self-assemble and form a stable three-dimensional hydrogel network, denoted as BA hydrogel.

[0040] Comparative Example 3 This comparative example provides a method for preparing OA hydrogel, comprising the following steps: adding 60 mg of oleanolic acid choline salt (OA, obtained in step S1 of Example 3) to 1 mL of deionized water, heating at 80°C for 5 min to completely dissolve and uniformly disperse it, and then letting it stand at room temperature for 10 min to induce self-assembly and form a stable three-dimensional hydrogel network, denoted as OA hydrogel.

[0041] Comparative Example 4 This comparative example provides a method for preparing GA hydrogel, comprising the following steps: 240 mg of glycyrrhetinic choline salt (GA, obtained in step S1 of Example 4) is added to 1 mL of deionized water. The mixture is heated at 80°C for 5 min to completely dissolve and uniformly disperse it. It is then allowed to stand at 4°C for 10 min to induce self-assembly, forming a stable three-dimensional hydrogel network, denoted as GA hydrogel.

[0042] Comparative Example 5 This comparative example provides a method for preparing glycyrrhizic acid (GL) hydrogel, comprising the following steps: adding 10 mg of glycyrrhizic acid to 1 mL of deionized water, heating the mixture at 70°C for 5 min to completely dissolve and uniformly disperse it, and then inducing self-assembly to form a stable three-dimensional hydrogel network, denoted as GL hydrogel, by ultrasonic treatment (120 MHz) for 5 min.

[0043] Comparative Example 6 This comparative example provides a method for preparing glycyrrhizic acid piezoelectric hydrogel (GLZC), comprising the following steps: 0.5 mg of ZnO-CuO (ZC) is ultrasonically dispersed in 1 mL of deionized water to obtain a uniform suspension; then 10 mg of glycyrrhizic acid (GL) is added to the system. The mixture is heated at 70 °C for 5 min to ensure complete dissolution, followed by ultrasonic treatment (120 MHz) for 3 min to induce self-assembly, thereby obtaining the GLZC piezoelectric composite hydrogel, denoted as GLZC hydrogel.

[0044] Experimental Example 1 like Figures 1-4 As shown, the four triterpenoid choline salts (corresponding to UA, BA, OA, and GA, respectively) prepared in Examples 1-4 1 The H NMR spectra all showed characteristic signals consistent with the theoretical structure, confirming the successful synthesis of the target product. In UA... 1 H NMR spectrum ( Figure 1 The signal at δH 4.96 corresponds to the CH=C bond at the C-13 position of ursolic acid, while the signal at δH 3.85 is attributed to the methylene group at the C-2 position of the choline group. In BA... 1 H NMR spectrum ( Figure 2 In the OA, the characteristic signals at δH 4.66 and 4.42 are attributed to the terminal CH=C bond at C-29 of betulinic acid, while the signal at δH 3.86 corresponds to the methylene group at C-2 of the choline group. 1 H NMR spectrum ( Figure 3 The signal at δH 4.99 is attributed to the CH=C position at C-12 of oleanolic acid, and the signal at δH 3.85 is the characteristic absorption of the methylene group at the C-2 position of the choline group. (GA) 1 H NMR spectrum ( Figure 4 The signal at δH 5.42 is attributed to the CH=C position at C-12 of glycyrrhetinic acid, and the signal at δH 3.87 is attributed to the methylene group at C-2 of the choline group. The above... 1 The clear assignment of the H NMR characteristic peaks indicates that the choline group has been successfully introduced into the triterpenoid nucleus structure, confirming the successful preparation of a series of triterpenoid choline derivatives (UA, BA, OA, GA).

[0045] Experimental Example 2 The self-assembly behavior of four choline-modified triterpenoids (UA, OA, BA, and GA) in aqueous medium was systematically studied, and the formation of hydrogels obtained in Comparative Examples 1-4 was observed. The results are as follows: Figure 5 As shown.

[0046] Figure 5 A represents the chemical structural formulas of four triterpenoid choline salts: UA, BA, OA, and GA. Figure 5 As shown in B, choline modification did not alter their inherent self-assembly capabilities. Instead, the enhanced hydrophilicity enabled all four compounds (UA, OA, BA, and GA) to form homogeneous hydrogels. UA formed an ultrasound-responsive hydrogel, while OA, BA, and GA exhibited thermoresponsive gelation behavior. All three systems required heating to 80°C to dissolve, and subsequently gelled upon cooling to room temperature or 4°C.

[0047] Experimental Example 3 The UAZC piezoelectric hydrogel obtained in Example 1 and the BAZC piezoelectric hydrogel (BA, 50 mg / mL) obtained in Example 2 were observed. -1 ZC, 1 mg mL -1 ), the OAZC piezoelectric hydrogel (OA, 60 mg / mL) obtained in Example 3 -1 ZC, 1 mg mL -1 Example 4: Preparation of GAZC piezoelectric hydrogel (GA, 240 mg / mL) -1 ZC, 1 mg mL -1 The preparation processes of the GL hydrogel (10 mg / mL) obtained in Comparative Example 5 and the GLZC hydrogel (GL, 10 mg; ZC, 0.5 mg) obtained in Comparative Example 6 were described. Then, the gels obtained in each group were inverted, and their state was observed. The results are as follows: Figure 6-7 As shown, where Figure 6 The left side shows the process of preparing UAZC piezoelectric hydrogels with different concentrations of ZC in Example 1 and the inverted photographs. Figure 6 The right side shows the preparation processes of BAZC piezoelectric hydrogel in Example 2, OAZC piezoelectric hydrogel in Example 3, and GAZC piezoelectric hydrogel in Example 4. Figure 7 The left side shows the molecular formula of glycyrrhizic acid (GL), and the right side shows the preparation process of GL hydrogel (10 mg / mL) and GLZC (GL, 10 mg; ZC, 0.5 mg) piezoelectric hydrogel.

[0048] observe Figure 6It was found that the UAZC system underwent rapid ultrasonic-induced gelation when the ZC concentration was in the range of 1 to 4 mg / mL. BAZC and OAZC piezoelectric hydrogels were also successfully prepared under similar conditions. However, despite repeated optimization, the GAZC system failed to form a stable piezoelectric hydrogel. In summary, these results indicate that the proposed triterpenoid-based piezoelectric hydrogel strategy has a certain degree of universality, but still strongly depends on the inherent molecular structure of the triterpenoids.

[0049] like Figure 7 As shown, glycyrrhizic acid (GL) hydrogel and glycyrrhizic acid piezoelectric hydrogel (GLZC) were successfully prepared under specific conditions.

[0050] Test Example 4 X-ray diffraction (XRD) was used to analyze the ZnO-CuO (ZC), UA hydrogel, and UAZC piezoelectric hydrogel (UA, 64 mg / mL). -1 ZC, 3 mg mL -1 The structure of ) was characterized, and the results are as follows Figure 8 As shown.

[0051] In the characterization of crystal structures, X-ray diffraction (XRD) patterns ( Figure 8 The results showed that both pure ZnO-CuO (ZC) and UAZC samples exhibited sharp diffraction peaks at 32.1°, 34.7°, 36.5°, 56.8°, 63.1°, and 68.2°, which is in high agreement with the standard card (JCPDS 36-1451) for hexagonal wurtzite ZnO. Although the CuO phase content in the system was low, characteristic diffraction peaks attributable to CuO (35.5°, 38.9°, and 68.1°, JCPDS 48–1548) were still detected in the spectrum, confirming the successful construction of the ZnO-CuO piezoelectric material and its loading in the UA gel matrix.

[0052] Experimental Example 5 The structure of the prepared UAZC piezoelectric hydrogel was analyzed using scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDS). The results... Figure 9 As shown; where Figure 9 A is the UAZC piezoelectric hydrogel (UA, 64 mg / mL) obtained in Example 1. -1 ZC, 3 mg mL -1 Scanning electron microscope (SEM) topography of ) (scale bar: 10 μm). Figure 9 B is the elemental surface scan of the energy dispersive X-ray spectroscopy (EDS) of the UAZC piezoelectric hydrogel obtained in Example 1.

[0053] like Figure 9As shown, the UAZC piezoelectric hydrogel has a loose and porous three-dimensional structure, and the ZnO-CuO (ZC) material is uniformly distributed in the hydrogel.

[0054] Experimental Example 6 The UA hydrogel prepared in Comparative Example 1 and the UAZC piezoelectric hydrogel prepared in Example 1 (UA, 64 mg / mL) were taken respectively. -1 ZC, 3 mg mL -1 Rheological performance tests were conducted, and the test results are as follows: Figure 10 As shown; where Figure 10 A shows the frequency scan results of the two groups of hydrogels. Figure 10 B shows the strain scanning test results of the two groups of hydrogels. Figure 10 Figure C shows the results of continuous cyclic step strain tests on two groups of hydrogels.

[0055] Rheological test results show that ( Figure 10 In the angular frequency scanning range of 0.1–100 rad / s, the storage modulus (G') of both UA hydrogel and UAZC piezoelectric hydrogel was consistently significantly higher than their loss modulus (G''), exhibiting solid-state viscoelastic behavior, indicating that their supramolecular crosslinked network possesses good structural stability. Strain scanning tests showed that the yield strain of the UAZC piezoelectric hydrogel network was approximately 6.7%. When the applied shear strain exceeded this critical threshold, G'' became greater than G', and the system underwent a phase transition from a gel state to a sol state. Figure 10 B). Continuous cyclic step strain testing revealed the thixotropic nature of the material: when the shear strain increased to 200%, the gel network was disrupted and transformed into a sol; when the strain recovered to 1%, G' and G'' rapidly recovered to their initial levels. Figure 10 C). Even after undergoing multiple cycles of strain, UA and UAZC piezoelectric hydrogels can still undergo rapid and reversible sol-gel transitions, exhibiting excellent structural self-healing capabilities.

[0056] Experimental Example 7 This experiment aims to evaluate the synergistic in vitro antibacterial effect of piezoelectric hydrogels against Staphylococcus aureus and Escherichia coli. The effects of material component concentration and static / low-frequency mechanical conditions on the antibacterial effect were investigated. Specific experimental procedures are as follows: Collect Staphylococcus aureus suspension and Escherichia coli culture in the logarithmic growth phase, and adjust the bacterial concentration to 10. 6 CFU / mL. The UAZC piezoelectric hydrogel (UA, 64 mg / mL) obtained in Example 1 was used. -1 ZC, 3 mg mL -1The UA hydrogel obtained in Comparative Example 1 and the ZnO-CuO (ZC) suspension (prepared by dispersing ZnO-CuO in deionized water) were added to the two bacterial cultures mentioned above and incubated together to obtain the following groups: ZC group: The final concentrations of ZC in the system were 0 μg / mL, 0.09 μg / mL, 0.38 μg / mL, 0.75 μg / mL, 1.5 μg / mL, 3 μg / mL, and 6 μg / mL, respectively. UAZC group: The final concentrations of UA in the system were 0 μg / mL, 2 μg / mL, 8 μg / mL, 16 μg / mL, 32 μg / mL, 64 μg / mL, and 128 μg / mL, respectively; the corresponding final ZC concentrations for each UA group were 0 μg / mL, 0.09 μg / mL, 0.38 μg / mL, 0.75 μg / mL, 1.5 μg / mL, 3 μg / mL, and 6 μg / mL, respectively. UA group: The final concentrations of UA in the system were 0 μg / mL, 2 μg / mL, 8 μg / mL, 16 μg / mL, 32 μg / mL, 64 μg / mL, and 128 μg / mL, respectively.

[0057] The experiments were then conducted under static and low-frequency mechanical shaking conditions, respectively, and incubated at 37°C for 7 hours. Afterward, each group of bacterial suspensions was serially diluted and plated onto LB agar plates. All LB agar plates were incubated at 37°C for 24 hours, and colony counts were performed to calculate the sterilization rate. Results are as follows: Figure 11 As shown. Figure 11 A and Figure 11 Figure B shows the bacterial plating results after Staphylococcus aureus was incubated with ZnO-CuO (ZC) dispersion and UAZC piezoelectric hydrogel for 7 h under shaking and static conditions. Figure 11 C is a statistical graph showing the survival rate of Staphylococcus aureus after treatment with UAZC piezoelectric hydrogel obtained in Example 1, UA hydrogel obtained in Comparative Example 1, and ZnO-CuO (ZC) dispersion. Figure 11 D is a statistical graph showing the survival rate of Escherichia coli after treatment with UAZC piezoelectric hydrogel obtained in Example 1, UA hydrogel obtained in Comparative Example 1, and ZnO-CuO (ZC) dispersion; data are expressed as mean ± standard deviation (SD) (n=3), *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 indicate statistical significance, and ns indicates no significant difference.

[0058] like Figure 11As shown in A and C, under static conditions, pure ZnO-CuO (ZC) showed weak inhibitory effects on both tested bacterial strains, with a relatively high bacterial survival rate even at a ZC concentration of 6 μg / mL. However, after incubation with mechanical shaking at 150 rpm for 7 h, the inhibitory effect on Staphylococcus aureus was significantly enhanced and showed a concentration-dependent effect: at an extremely low ZC concentration of 0.09 μg / mL, the inhibition rate reached 54.58%; and when the ZC concentration increased to 1.5 μg / mL, the inhibition rate further increased to 86.3%. Figure 11 A and C). Although ZC's inhibitory effect on Escherichia coli is relatively weaker than that on Staphylococcus aureus ( Figure 11 D), but the antibacterial rate also reached 69.1% at a concentration of 6 μg / mL ( Figure 11 D).

[0059] Under the same oscillation conditions, the UAZC piezoelectric hydrogel achieved an inhibition rate of 98.2% against Staphylococcus aureus at extremely low concentrations (UA: 2 μg / mL; ZC: 0.09 μg / mL); even under static conditions, it maintained an inhibition rate of 61.3%. Figure 11 (C), which is significantly superior to the single UA hydrogel or the pure ZnO-CuO (ZC) group. This result indicates that the UAZC piezoelectric hydrogel constructed by combining ZnO-CuO (ZC) with the UA matrix exhibits a synergistic antibacterial effect.

[0060] Experiments targeting Escherichia coli also support this synergistic mechanism: UA alone showed almost no antibacterial activity at a concentration of 128 μg / mL ( Figure 11 D), the antibacterial rate of pure ZnO-CuO (ZC, 6 μg / mL) after shaking incubation for 7 h was 69.1%; while under the same concentration and shaking conditions, the antibacterial rate of UAZC composite piezoelectric hydrogel was significantly increased to 74.9% (D). Figure 11 D). The above results indicate a synergistic antibacterial effect between the UA matrix and the ROS generated by the piezoelectricity of ZnO-CuO (ZC).

[0061] Experimental Example 8 Evaluation of in vitro angiogenesis-promoting properties: This experiment evaluated the in vitro angiogenesis-promoting ability of the piezoelectric hydrogel using a lumen formation assay with human umbilical vein endothelial cells (HUVECs). The specific implementation steps are as follows: Human umbilical vein endothelial cells (HUVECs) in logarithmic growth phase and in good condition were incubated with PBS, UA hydrogel, and UAZC piezoelectric hydrogel, respectively. Control group: sterile PBS buffer; UA hydrogel: final UA concentration of 16 μg / mL; UAZC piezoelectric hydrogel (UA, 64 mg / mL). -1ZC, 3 mg mL -1 The final concentrations of UA and ZC in the system were 16 μg / mL and 0.75 μg / mL, respectively. Static incubation and shaking incubation conditions were set. Human umbilical vein endothelial cells (HUVECs) were collected after treatment and cultured at 1 × 10⁻⁶ cells per well. 4 HUVECs were seeded at a density of [number] cells per well on polymerized Matrigel. At 4h and 8h of culture, microscopic imaging was used to observe the formation of tubular structures and the generation of vascular networks in each group of HUVECs, recording the number of nodes in the tubular network. After observation, HUVECs in each well were stained with calcein-AM fluorescent dye for live-cell specificity. After staining in the dark, fluorescence images of the cells in each group were acquired. Results are as follows: Figure 12 As shown. Figure 12 A shows representative images of angiogenesis in HUVECs cells after different treatment conditions and images stained with calcein-AM (scale bar: 200 μm). Figure 12 B represents the statistical results of angiogenesis capacity (Nb nodes represent the formation of cell nodes in the initial process of new angiogenesis); data are expressed as mean ± standard deviation (SD) (n=3), and **p < 0.01, ***p < 0.001 and ****p < 0.0001 indicate statistical significance.

[0062] like Figure 12 As shown in Figure A, compared with the blank control group, both UA hydrogel and UAZC piezoelectric hydrogel exhibited certain pro-angiogenic activities. Among them, UAZC piezoelectric hydrogel showed the most significant pro-angiogenic effect under dynamic mechanical stimulation. After 4 h of culture, the UAZC group treated with shaking induced the formation of a dense capillary-like network with 150 tube-forming nodes; under static conditions, the number of nodes in this group also reached 126, both significantly more than the blank control group (90 nodes) and the UA hydrogel group (111 nodes) under shaking conditions. Figure 12 (B) As the culture time was extended to 8 h, the in vitro tubular networks in all groups exhibited physiological shrinkage due to local nutrient consumption and matrix degradation. At this point, the number of nodes in the UAZC oscillation (3 times) group decreased slightly to approximately 142, but its three-dimensional lumen structure still maintained good integrity, with a significantly higher number of nodes than other treatment groups, demonstrating good lumen maintenance ability. This result is consistent with the cell migration experiment results, indicating that the biomimetic micro-electric field generated by the UAZC piezoelectric hydrogel under low-frequency mechanical stimulation can promote angiogenesis in endothelial cells.

[0063] Experimental Example 9 Evaluation of in vivo healing promotion of diabetic foot ulcers (DFU): This experiment used a diabetic rat model with Staphylococcus aureus infection to evaluate the in vivo tissue repair effect of composite piezoelectric hydrogel. The specific implementation steps are as follows: A diabetic rat model was established using intraperitoneal injection of streptozotocin (STZ). A DFU wound approximately 5 mm in diameter was created on the dorsum of the rat's paw and inoculated with 100 μL of Staphylococcus aureus bacterial suspension (1 × 10⁻⁶). 8 CFU mL -1 To simulate infectious diabetic foot ulcers, rats with successful modeling were randomly divided into groups, and each group received an in-situ injection of 80 μL of UAZC piezoelectric hydrogel (UA, 64 mg / mL) obtained in Example 1 into the wound. -1 ZC, 1 mg mL -1 Rats were given 80 μL LUA hydrogel and a blank control group. Natural friction and micro-strain generated during the rats' daily cage activities activated the low-frequency piezoelectric effect of the hydrogel. Wound area was recorded on days 0, 3, 7, and 14 of treatment, and wound healing was assessed. Results are as follows: Figure 13 As shown. Figure 13 A and Figure 13 B represents the recovery trajectory of DFU wounds in rats of different treatment groups at days 0, 3, 7, 10, and 14, respectively; and the corresponding wound area statistics. Data are expressed as mean ± standard deviation (SD) (n=3), and *p < 0.05, **p < 0.01, and ***p < 0.001 indicate statistical significance.

[0064] like Figure 13 As shown in Figure A, during the 14-day observation period, the UAZC piezoelectric hydrogel treatment group exhibited the most significant wound contraction. Quantitative analysis indicated that ( Figure 13 (B) By day 7 of treatment, the wound closure rate in the UAZC group had reached 66.33%, significantly higher than that in the UA hydrogel group and the blank control group at the same time. By the observation endpoint of day 14, the wound closure rate in the UAZC group was 87.67%, significantly higher than that in the UA hydrogel group (77.67%) and the blank control group (58%), indicating that it has a good healing-promoting effect in the foot ulcer model.

[0065] The above are merely preferred embodiments of the present invention and are not limited to the examples described above. Those skilled in the art will recognize that various modifications and variations can be made based on the principles of the present invention. Any modifications or improvements made should be considered within the scope of protection of the present invention.

[0066] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the protection scope of the present invention.

Claims

1. A method for preparing a multifunctional low-frequency, low-energy piezoelectric hydrogel based on natural triterpenoid assembly, characterized in that, Includes the following steps: S1. Triterpenoids and choline hydroxide are added to a solvent and refluxed to obtain triterpenoid choline salts. S2. Add sea urchin-shaped ZnO-CuO to water to obtain a uniform suspension; add triterpenoid choline salts to the suspension, heat and then induce self-assembly by ultrasound to obtain the final product.

2. The method for preparing the multifunctional low-frequency, low-energy piezoelectric hydrogel based on natural triterpenoid assembly according to claim 1, characterized in that, The triterpenoid compound in step S1 is any one of ursolic acid, oleanolic acid, and betulinic acid, and the solvent is methanol.

3. The method for preparing the multifunctional low-frequency, low-energy piezoelectric hydrogel based on natural triterpenoid assembly according to claim 2, characterized in that, In step S1, the molar ratio of the triterpenoid compound to choline hydroxide is 1:1.2, the molar ratio of the triterpenoid compound to the solvent is 1-40 mg: 1 mL, and the solvent is methanol.

4. The method for preparing the multifunctional low-frequency, low-energy piezoelectric hydrogel based on natural triterpenoid assembly according to claim 1, characterized in that, The reflux reaction in step S1 is carried out at a temperature of 30-80°C for 2-24 hours.

5. The method for preparing the multifunctional low-frequency, low-energy piezoelectric hydrogel based on natural triterpenoid assembly according to claim 1, characterized in that, In step S2, the ratio of sea urchin-like ZnO-CuO, triterpenoid choline salts, and water is 1-4 mg: 50-64 mg: 1 mL.

6. The method for preparing the multifunctional low-frequency, low-energy piezoelectric hydrogel based on natural triterpenoid assembly according to claim 1, characterized in that, The heating temperature in step S2 is 60℃-90℃, and the heating time is 3 min-15 min.

7. The method for preparing the multifunctional low-frequency, low-energy piezoelectric hydrogel based on natural triterpenoid assembly according to claim 5, characterized in that, The preparation method of the sea urchin-shaped ZnO-CuO in step S2 is as follows: weigh zinc acetate dihydrate and copper acetate and dissolve them in deionized water to obtain a mixed solution; add the obtained mixed solution to deionized water containing DMSO, stir for 1 h, add ammonia solution, heat at 60℃ for 4 h, centrifuge to collect the precipitate, and wash to obtain the final product.

8. The method for preparing the multifunctional low-frequency, low-energy piezoelectric hydrogel based on natural triterpenoid assembly according to claim 1, characterized in that, The time for ultrasound-induced self-assembly in step S2 is 3-5 minutes, and the frequency of ultrasound-induced self-assembly is 80-200 MHz.

9. A multifunctional low-frequency, low-energy piezoelectric hydrogel assembled based on natural triterpenes, characterized in that, It is prepared according to any one of claims 1-8.

10. The application of the multifunctional low-frequency low-energy piezoelectric hydrogel based on natural triterpenoid assembly according to claim 9 in the preparation of a dressing for healing diabetic foot wounds.

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