An ultrasonic-activated piezoelectric-conductive composite hydrogel loaded with dasatinib and quercetin, and a preparation method and application thereof

Abstract

The application discloses a kind of load dasatinib and quercetin ultrasonic excitation piezoelectric-conductive composite hydrogel and its preparation method and application, belong to biomedical hydrogel, wound repair material, piezoelectric biomaterial, conductive composite hydrogel and local drug delivery material technical field.The composite hydrogel contains sodium hyaluronate / PAMAM G3-NH2 amide crosslinking network, FF / PEDOT:PSS / PVA conductive-piezoelectric composite phase and dasatinib and quercetin.The composite hydrogel PAMAM G3-NH2 in it has crosslinking node and double drug dispersion carrier function, make the electric signal generated by FF piezoelectric phase can be transmitted by PEDOT:PSS conductive phase.Under low-intensity pulsed ultrasound stimulation, the hydrogel can produce local electromechanical response, and combine dasatinib / quercetin to regulate cell senescence-related phenotype and inflammatory microenvironment, promote wound closure and tissue reconstruction.

Description

Technical Field

[0001] This invention belongs to the technical fields of biomedical hydrogels, wound repair materials, piezoelectric biomaterials, conductive composite hydrogels, and local drug delivery materials. Specifically, it relates to an ultrasonically excited piezoelectric-conductive composite hydrogel that integrates a sodium hyaluronate / PAMAM G3-NH2 hydrogel network, diphenylalanine piezoelectric nanofibers, a PEDOT:PSS conductive phase, and dasatinib / quercetin dual drugs, as well as its preparation method and its application in wound repair materials. Background Technology

[0002] Chronic wounds, diabetic wounds, age-related wounds, and other refractory tissue injuries are often accompanied by persistent inflammation, oxidative stress, decreased cell migration and proliferation, abnormal collagen remodeling, and delayed re-epithelialization. The abnormal accumulation of senescent cells and the continuous release of senescence-related secreted phenotypic factors at the wound site can maintain chronic inflammation and prevent the wound from transitioning from the inflammatory phase to the proliferative and remodeling phases. IL-6, IL-1β, TNF-α, chemokines, and matrix metalloproteinases released by senescent cells can further affect the function of surrounding cells, collagen deposition, and tissue regeneration.

[0003] Current wound dressings mainly include gauze, films, foam dressings, hydrocolloid dressings, ordinary hydrogel dressings, antibacterial dressings, growth factor dressings, and negative pressure wound therapy systems. While these materials can protect the wound, maintain a moist environment, or reduce bacterial load to some extent, they are insufficient for directly regulating the abnormal accumulation of senescent cells and the associated inflammatory microenvironment. Therefore, for refractory wounds involving both cellular senescence and chronic inflammation, relying solely on protective coverings or single antibacterial components often fails to achieve stable, rapid, and high-quality tissue reconstruction.

[0004] Dasatinib and quercetin are commonly used senolytics. Dasatinib can affect survival signals associated with senescent cells, while quercetin has antioxidant, anti-inflammatory, and cell fate-regulating effects. However, direct local administration of these two drugs presents challenges such as limited water solubility, short wound retention time, early burst release, and potential systemic exposure. Therefore, it is necessary to develop local delivery carriers to improve their dispersibility and retention in the wound area.

[0005] There is also the problem of weakened or disordered endogenous bioelectrical signals during wound repair. Exogenous electrical stimulation can promote the migration, proliferation and matrix secretion of fibroblasts, keratinocytes and endothelial cells, but traditional electrical stimulation usually relies on external power sources and electrodes, which has problems such as complicated operation, poor wound adaptability and inconvenience of long-term use.

[0006] Diphenylalanine dipeptide can self-assemble into ordered one-dimensional nanofibers and exhibit a piezoelectric response, but its charge collection and conduction efficiency is limited when used alone. PEDOT:PSS has good conductivity and flexibility, which can promote charge transfer, but it does not have the ability to convert mechanical energy into electrical signals. Therefore, it is necessary to combine the short peptide piezoelectric phase, the conductive phase, and the flexible hydrogel network in a suitable manner.

[0007] Current technologies still lack a complete technical solution that uses sodium hyaluronate and PAMAM G3-NH2 to form a flexible hydrophilic network, allowing PAMAM G3-NH2 to simultaneously serve as a crosslinking node and a pre-dispersion carrier for dasatinib / quercetin, and introduces a pre-constructed conductive-piezoelectric composite phase of FF / PEDOT:PSS / PVA, thereby achieving local retention of dual drugs, ultrasonic excitation of electrical signals, and regulation of the microenvironment related to cell senescence in the same hydrogel. Summary of the Invention

[0008] The present invention aims to provide an ultrasonically excited piezoelectric-conductive composite hydrogel loaded with dasatinib and quercetin, its preparation method and application, in order to solve the problem that existing wound materials cannot simultaneously achieve local retention of poorly soluble dual drugs, conversion of mechanical energy to electrical signals, charge conduction and regulation of the microenvironment related to cell senescence.

[0009] The core of this invention lies in the sequential construction of FF nanofibers, PF conductive-piezoelectric composite phase, and drug-loaded G3 pre-dispersion system, followed by the formation of an amide crosslinking network by activating sodium hyaluronate and PAMAM G3-NH2, so that the drug, conductive-piezoelectric phase, and hydrogel network can coexist stably in the same flexible three-dimensional structure.

[0010] The present invention employs the following technical solutions to achieve the above objectives: An ultrasonically excited piezoelectric-conductive composite hydrogel loaded with dasatinib and quercetin comprises a sodium hyaluronate / PAMAM G3-NH2 amide crosslinking network, an FF / PEDOT:PSS / PVA conductive-piezoelectric composite phase, and dasatinib and quercetin.

[0011] PAMAM G3-NH2 is an amino-terminated third-generation polyamide-amine dendritic macromolecule, preferably with ethylenediamine as the core, and the molecular surface has multiple primary amino groups.

[0012] This invention provides a method for preparing the above-mentioned composite hydrogel, which mainly involves: dissolving FF in HFIP and forming FF nanofibers through solvent displacement; mixing PEDOT:PSS dispersion with PVA solution and then adding FF nanofibers to form a PF conductive-piezoelectric composite phase; pre-dispersing dasatinib and quercetin in PAMAM G3-NH2 solution to obtain a drug-loaded G3 solution; activating sodium hyaluronate solution by adding EDC·HCl and NHS to obtain an activated sodium hyaluronate solution; adding the PF conductive-piezoelectric composite phase to the drug-loaded G3 solution and reacting it with the activated sodium hyaluronate solution to obtain the HGPF-DQ composite hydrogel.

[0013] Specifically, the preparation method includes the following steps: Step 1, Preparation of FF nanofibers: Dissolve FF in HFIP to obtain an FF / HFIP solution; add the FF / HFIP solution to water under stirring, continue stirring and let stand, and then dry to obtain FF nanofibers; Step 2, Preparation of PEDOT:PSS / PVA conductive dispersion: PVA is added to water and heated and stirred until dissolved to obtain a PVA solution; the PEDOT:PSS dispersion is mixed with the PVA solution to obtain the PEDOT:PSS / PVA conductive dispersion. Step 3, Preparation of PF conductive-piezoelectric composite phase: FF nanofibers are added to PEDOT:PSS / PVA conductive dispersion, and dispersed by stirring and intermittent ultrasonication to obtain PF conductive-piezoelectric composite phase; Step 4, Preparation of drug-loaded G3 solution: Dasatinib and quercetin were prepared as DMSO stock solutions and then added to PAMAM G3-NH2 aqueous solution. The mixture was stirred in the dark to obtain the drug-loaded G3 solution. Step 5, Preparation of HGPF-DQ composite hydrogel: The PF conductive-piezoelectric composite phase is added to the drug-loaded G3 solution to form a drug-loaded G3 / PF mixture; sodium hyaluronate solution is activated by adding EDC·HCl and NHS to obtain an activated sodium hyaluronate solution; the activated sodium hyaluronate solution is added to the drug-loaded G3 / PF mixture, and the reaction is carried out at pH 5.0–5.8 to form the HGPF-DQ composite hydrogel. Here, D represents dasatinib, and Q represents quercetin.

[0014] Furthermore, in step 1, the concentration of FF in the FF / HFIP solution is 5–20 mg / mL, preferably 8–12 mg / mL.

[0015] Furthermore, in step 1, the volume ratio of FF / HFIP solution to water is 1:8 to 1:12, preferably 1:9 to 1:11.

[0016] Furthermore, in step 2, the concentration of the PVA solution is 0.3% to 0.8% (w / v), preferably 0.4% to 0.6% (w / v).

[0017] Furthermore, in step 2, the volume ratio of PEDOT:PSS dispersion to PVA solution is 7:1 to 11:1, preferably 8:1 to 10:1.

[0018] Furthermore, in step 3, the concentration of FF in the PF conductive-piezoelectric composite phase is 0.6–1.5 mg / mL, preferably 0.8–1.2 mg / mL.

[0019] Furthermore, in step 4, the concentration of the PAMAM G3-NH2 solution is 0.8–1.5 mg / mL, preferably 0.9–1.1 mg / mL.

[0020] Furthermore, in step 4, the concentration of dasatinib in the drug-loaded G3 solution is 180–320 μg / mL, the concentration of quercetin is 550–950 μg / mL, preferably 220–280 μg / mL for dasatinib and 650–850 μg / mL for quercetin; and the final volume fraction of DMSO in the drug-loaded G3 solution is 0.5%–2%.

[0021] Furthermore, in step 5, the concentration of the sodium hyaluronate solution is 0.8–1.5 mg / mL, preferably 0.9–1.1 mg / mL.

[0022] Furthermore, in step 5, the volume ratio of sodium hyaluronate solution to drug-loaded G3 solution is 1:3 to 1:5, preferably 1:3.5 to 1:4.5.

[0023] Furthermore, in the preparation of the activated sodium hyaluronate solution in step 5, the molar ratio of carboxyl groups, EDC and NHS in sodium hyaluronate is 1:(1.5-3):(1.5-3), preferably 1:(1.8-2.2):(1.8-2.2).

[0024] Furthermore, in step 5, the volume fraction of the PF conductive-piezoelectric composite phase in the gelation system is 7% to 12%, preferably 8.5% to 10%.

[0025] In a preferred embodiment, the concentration of FF in the FF / HFIP solution is 10 mg / mL, the volume ratio of FF / HFIP solution to water is 1:10, the concentration of PVA solution is 0.5% (w / v), the volume ratio of PEDOT:PSS dispersion to PVA solution is 9:1, the concentration of FF in the PF conductive-piezoelectric composite phase is 1.0 mg / mL, the concentration of PAMAM G3-NH2 solution is 1.0 mg / mL, the concentration of dasatinib in the drug-loaded G3 solution is 250 μg / mL, the concentration of quercetin is 750 μg / mL, the concentration of sodium hyaluronate solution is 1.0 mg / mL, the volume ratio of sodium hyaluronate solution to drug-loaded G3 solution is 1:4, the molar ratio of carboxyl groups, EDC, and NHS in sodium hyaluronate is 1:2:2, and the volume fraction of PF conductive-piezoelectric composite phase in the gelling system is 9-10%. This embodiment is only for illustrating one of the preferred component compositions, and the scope of protection is limited accordingly.

[0026] The HGPF-DQ composite hydrogel can be made into sheet, film, block, patch or coating dressing; used to prepare cell senescence-related wound repair materials, wound dressings or wound patches.

[0027] Based on the above applications of composite hydrogels, this invention provides a wound repair material system composed of composite hydrogels and low-intensity pulsed ultrasound, mainly comprising: When used in conjunction with low-intensity pulsed ultrasound, the ultrasound frequency is 0.7–1.3 MHz, the power density is 0.3–0.8 W / cm², the duty cycle is 15%–30%, and the single stimulation time is 3–8 min. Preferably, the ultrasound frequency is 0.9–1.1 MHz, the power density is 0.4–0.6 W / cm², the duty cycle is 18%–25%, and the single stimulation time is 4–6 min.

[0028] In the composite hydrogel and its preparation method of this invention: FF is diphenylalanine dipeptide, HFIP is hexafluoroisopropanol, PVA is polyvinyl alcohol, EDC·HCl is 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, NHS is N-hydroxysuccinimide, DMSO is dimethyl sulfoxide, and PEDOT:PSS is a commercially available poly(3,4-ethylenedioxythiophene):polystyrene sulfonic acid aqueous dispersion.

[0029] Compared with the prior art, the present invention has the following beneficial effects: First, the working principle of the composite hydrogel of this invention is as follows: The sodium hyaluronate / PAMAM G3-NH2 network provides a flexible hydrophilic matrix for the wound site. PAMAM G3-NH2 also serves as a crosslinking node for sodium hyaluronate and a pre-dispersion carrier for dasatinib / quercetin, which is beneficial for the uniform distribution and local retention of the drug within the hydrogel.

[0030] FF nanofibers generate an electromechanical response under ultrasound or other mechanical stimulation. The PEDOT:PSS conductive phase improves the charge transfer inside the hydrogel, enabling the electrical signals generated by the PF composite phase to act more effectively on the wound area.

[0031] After release from the hydrogel, dasatinib and quercetin act on oxidative stress-induced aging-related phenotype cells, reducing the proportion of SA-β-gal positive cells and the expression of SASP-related inflammatory factors. Ultrasound-induced local electrical stimulation and the dual-drug microenvironment regulation jointly promote wound closure and tissue remodeling.

[0032] Secondly, the present invention has the following beneficial effects: 1. This invention integrates local delivery of dual drugs, ultrasonically excited piezoelectric response, and conductive transport into the same flexible hydrogel material.

[0033] 2. PAMAM G3-NH2 combines the functions of a hydrogel crosslinking node and a dasatinib / quercetin predispersant, which is beneficial for improving the dispersion and local retention of poorly soluble drugs.

[0034] 3. FF first self-assembles into nanofibers and forms a PF composite phase with PEDOT:PSS / PVA in advance, which can reduce the problem of uneven dispersion caused by the separate addition of functional components.

[0035] 4. Under low-intensity ultrasound stimulation, HGPF-DQ can generate measurable voltage, current and piezoelectric coefficient, and reduce oxidative stress-induced cellular senescence-related phenotypes and inflammatory protein expression.

[0036] 5. The composite hydrogel has good cell compatibility and blood compatibility, and can promote the closure of full-thickness skin defects in mice. Attached Figure Description

[0037] Figure 1 : Structural characterization of FF nanofibers and PF composite phase, where (a) shows the low-magnification and high-magnification microstructure of FF nanofibers, and (b) shows the XRD patterns of FF and PEDOT / FF composite phases; Figure 2 Hydrogel formation and chemical structure characterization, where (a) is a sol-gel transition photograph before and after EDC / NHS activation, and (b) is the FTIR spectrum of HG, HGPF, HG-DQ and HGPF-DQ; Figure 3: Gel rheological curves of HG, HGPF and HGPF-DQ, where (a), (b) and (c) are the curves of G′ and G″ of the three hydrogels as a function of time, respectively; Figure 4 Release behavior of dasatinib and quercetin, where (a) is the cumulative release rate of dasatinib at different loading concentrations, (b) is the cumulative release rate of quercetin at different loading concentrations, and (c) is the release amount at different time points at the preferred drug concentration; Figure 5 Electrical output of different hydrogels under ultrasonic stimulation, where (a) is voltage waveform, (b) is average output voltage, (c) is current waveform, (d) is average output current, (e) is response of positive and negative wiring, and (f) is piezoelectric coefficient d33. Figure 6 Biocompatibility of different hydrogels, where (a) cell viability at 24 h and 48 h, (b) Live / Dead staining, and (c) hemolysis rate; Figure 7 The results of H2O2-induced senescence-related cells after different treatments are shown in (a) SA-β-gal staining, (b) SA-β-gal positive area statistics, (c) SASP-related inflammatory protein bands, and (d) IL-1 related expression quantification. Figure 8 The results of full-thickness skin defect repair in mice under different treatment groups are shown in (a) and (b) on day 1, 3, 5 and 10. The results are shown in (c) and (d) on (e) on (f) on (g ... Detailed Implementation

[0038] The present invention will be further illustrated below with reference to specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of protection of the invention. Unless otherwise specified, all reagents used are conventional commercial reagents in the art, and the water is deionized water or equivalent purified water.

[0039] Example 1 (1) FF nanofibers: Weigh 100 mg of FF and add 10 mL of HFIP. Stir at room temperature until completely dissolved to obtain a 10 mg / mL FF / HFIP solution. Take 1 mL of this solution and slowly add 10 mL of deionized water under continuous stirring. Continue stirring for 30 min and let stand for 1–2 h. FF nanofibers are obtained by freeze drying.

[0040] (2) PEDOT:PSS / PVA conductive dispersion: Weigh 50 mg PVA and add 10 mL of deionized water. Heat and stir at 80 °C until completely dissolved. After cooling, a 0.5% (w / v) PVA solution is obtained. Mix 9 mL of PEDOT:PSS dispersion with 1 mL of PVA solution and stir at room temperature for 1 h.

[0041] (3) PF composite phase: FF nanofibers were added to PEDOT:PSS / PVA dispersion to make the FF concentration 1.0 mg / mL. After stirring at low speed for 30 min, intermittent ultrasonic dispersion was performed. The ultrasonic dispersion was repeated 10 times for 30 s and 30 s intervals to obtain the PF conductive-piezoelectric composite phase.

[0042] (4) Drug-loaded G3 solutions: Prepare 50 mg / mL dasatinib stock solutions and 50 mg / mL quercetin stock solutions using DMSO. Take 20 μL of dasatinib stock solution and 60 μL of quercetin stock solution, and make up to 4 mL with 1.0 mg / mL PAMAM G3-NH2 solution to make the concentrations of dasatinib and quercetin 250 μg / mL and 750 μg / mL, respectively. The final volume fraction of DMSO is 2%. Stir for 2 h in the dark.

[0043] (5) Gel formation: Add 0.5 mL of PF composite phase to 4 mL of drug-loaded G3 solution and stir for 10–20 min. Take another 1 mL of 1.0 mg / mL sodium hyaluronate solution, add approximately 0.96 mg of EDC·HCl and approximately 0.58 mg of NHS (the molar ratio of carboxyl groups, EDC, and NHS in sodium hyaluronate is 1:2:2), and activate at pH 5.0–5.8 for 30 min to obtain activated sodium hyaluronate solution; then add the drug-loaded G3 / PF mixture and react at room temperature for 12 h to obtain HGPF-DQ hydrogel. The volume fraction of PF in the gel formation system is approximately 9.1%.

[0044] Example 2 The preparation method is as described in Example 1, wherein: The concentration of the FF / HFIP solution was set to 5 mg / mL, and the volume ratio of the FF / HFIP solution to water was 1:8. The PVA solution concentration was 0.3% (w / v), and the volume ratio of PEDOT:PSS dispersion to PVA solution was 7:1. The concentration of FF in the PF composite phase was 0.6 mg / mL; The concentration of PAMAM G3-NH2 solution was 0.8 mg / mL. Taking 4 mL of drug-loaded G3 solution as an example, 14.4 μL of 50 mg / mL dasatinib stock solution and 44 μL of 50 mg / mL quercetin stock solution were added to make the concentrations of dasatinib and quercetin 180 μg / mL and 550 μg / mL, respectively, and the final volume fraction of DMSO was approximately 1.46%.

[0045] Add 0.40 mL of PF composite phase to 4 mL of drug-loaded G3 solution. Take 1.33 mL of 0.8 mg / mL sodium hyaluronate solution, add approximately 0.77 mg of EDC·HCl and approximately 0.46 mg of NHS (the molar ratio of carboxyl groups, EDC, and NHS in sodium hyaluronate is 1:1.5:1.5), and activate at pH 5.0–5.8 for 30 min to obtain activated sodium hyaluronate solution; then add the drug-loaded G3 / PF mixture and react at room temperature for 12 h. The volume ratio of sodium hyaluronate solution to drug-loaded G3 solution in the resulting system is approximately 1:3, and the volume fraction of PF is approximately 7%.

[0046] Example 3 The preparation method is as described in Example 1, wherein: The concentration of the FF / HFIP solution was set to 20 mg / mL, and the volume ratio of the FF / HFIP solution to water was 1:12. The PVA solution concentration was 0.8% (w / v), and the volume ratio of PEDOT:PSS dispersion to PVA solution was 11:1. The concentration of FF in the PF composite phase was 1.5 mg / mL; The concentration of PAMAM G3-NH2 solution was 1.5 mg / mL. Taking 4 mL of drug-loaded G3 solution as an example, 12.8 μL and 38 μL of 100 mg / mL dasatinib and quercetin DMSO stock solutions were added, respectively, to make the concentrations of dasatinib and quercetin 320 μg / mL and 950 μg / mL, respectively, with a final DMSO volume fraction of approximately 1.27%.

[0047] Add 0.65 mL of PF composite phase to 4 mL of drug-loaded G3 solution. Take 0.80 mL of 1.5 mg / mL sodium hyaluronate solution, add approximately 1.73 mg of EDC·HCl and approximately 1.04 mg of NHS (the molar ratio of carboxyl groups, EDC, and NHS in sodium hyaluronate is 1:3:3), and activate at pH 5.0–5.8 for 30 min to obtain activated sodium hyaluronate solution; then add the drug-loaded G3 / PF mixture and react at room temperature for 12 h. The resulting system has a volume ratio of sodium hyaluronate solution to drug-loaded G3 solution of 1:5, and a PF volume fraction of approximately 11.9%.

[0048] Example 4 The preparation method is as described in Example 1, wherein: The concentration of the FF / HFIP solution was set to 12 mg / mL, and the volume ratio of the FF / HFIP solution to water was 1:11. The PVA solution concentration was 0.6% (w / v), and the volume ratio of PEDOT:PSS dispersion to PVA solution was 10:1. The concentration of FF in the PF composite phase was 1.2 mg / mL; The concentration of PAMAM G3-NH2 solution was 1.1 mg / mL. Taking 4 mL of drug-loaded G3 solution as an example, 11.2 μL and 34 μL of 100 mg / mL dasatinib and quercetin DMSO stock solutions were added to make the concentrations of dasatinib and quercetin 280 μg / mL and 850 μg / mL, respectively, and the final volume fraction of DMSO was approximately 1.13%.

[0049] Add 0.54 mL of PF composite phase to 4 mL of drug-loaded G3 solution. Take 0.89 mL of 1.1 mg / mL sodium hyaluronate solution, add approximately 1.03 mg of EDC·HCl and approximately 0.62 mg of NHS (the molar ratio of carboxyl groups, EDC, and NHS in sodium hyaluronate is 1:2.2:2.2), and activate at pH 5.0–5.8 for 30 min to obtain activated sodium hyaluronate solution; then add the drug-loaded G3 / PF mixture and react at room temperature for 12 h. The volume ratio of sodium hyaluronate solution to drug-loaded G3 solution in the resulting system is 1:4.5, and the volume fraction of PF is approximately 10%.

[0050] Example 5 The preparation method is as described in Example 1, wherein: The concentration of the FF / HFIP solution was set to 8 mg / mL, and the volume ratio of the FF / HFIP solution to water was 1:9. The PVA solution concentration was 0.4% (w / v), and the volume ratio of PEDOT:PSS dispersion to PVA solution was 8:1. The concentration of FF in the PF composite phase is 0.8 mg / mL; The concentration of PAMAM G3-NH2 solution was 0.9 mg / mL. Taking 4 mL of drug-loaded G3 solution as an example, 8.8 μL and 26 μL of 100 mg / mL dasatinib and quercetin DMSO stock solutions were added, respectively, to make the concentrations of dasatinib and quercetin 220 μg / mL and 650 μg / mL, respectively, with a final DMSO volume fraction of approximately 0.87%.

[0051] Add 0.48 mL of PF composite phase to 4 mL of drug-loaded G3 solution. Take 1.14 mL of 0.9 mg / mL sodium hyaluronate solution, add approximately 0.88 mg of EDC·HCl and approximately 0.53 mg of NHS (the molar ratio of carboxyl groups, EDC, and NHS in sodium hyaluronate is 1:1.8:1.8), and activate at pH 5.0–5.8 for 30 min to obtain activated sodium hyaluronate solution; then add the drug-loaded G3 / PF mixture and react at room temperature for 12 h. The volume ratio of sodium hyaluronate solution to drug-loaded G3 solution in the resulting system is 1:3.5, and the volume fraction of PF is approximately 8.5%.

[0052] Example 6 Preparation of HG hydrogel A 1.0 mg / mL sodium hyaluronate solution and a 1.0 mg / mL PAMAM G3-NH2 solution were prepared separately. 1 mL of sodium hyaluronate solution was taken, and approximately 0.96 mg of EDC·HCl and 0.58 mg of NHS were added. The solution was activated at pH 5.0–5.8 for 30 min to obtain an activated sodium hyaluronate solution. Subsequently, 4 mL of PAMAM G3-NH2 solution was added, and the reaction was carried out at room temperature for 12 h to obtain HG hydrogel.

[0053] Example 7 Preparation of HGPF hydrogel Add 0.5 mL of the PF composite phase prepared in Example 1 to 4 mL of 1.0 mg / mL PAMAM G3-NH2 solution and stir for 10–20 min. Take 1 mL of 1.0 mg / mL sodium hyaluronate solution, add approximately 0.96 mg of EDC·HCl and approximately 0.58 mg of NHS, and activate at pH 5.0–5.8 for 30 min to obtain activated sodium hyaluronate solution; then add G3 / PF mixture and react at room temperature for 12 h to obtain HGPF hydrogel.

[0054] Example 8 Preparation of HG-DQ hydrogel A drug-loaded G3 solution with a dasatinib concentration of 250 μg / mL and a quercetin concentration of 750 μg / mL was prepared according to Example 1. 1 mL of a 1.0 mg / mL sodium hyaluronate solution was taken, and approximately 0.96 mg of EDC·HCl and approximately 0.58 mg of NHS were added. The solution was activated at pH 5.0–5.8 for 30 min to obtain an activated sodium hyaluronate solution. Subsequently, 4 mL of the drug-loaded G3 solution was added, and the reaction was carried out at room temperature for 12 h to obtain HG-DQ hydrogel.

[0055] Effect verification test The FF nanofibers, PF composite phase and HGPF-DQ hydrogel obtained in Example 1, the HG hydrogel obtained in Example 6, the HGPF hydrogel obtained in Example 7 and the HG-DQ hydrogel obtained in Example 8 were subjected to relevant index or performance tests, as detailed below.

[0056] Experimental Example 1: Structural Composition and Microstructure The morphology, XRD, FTIR, SEM, and elemental distribution of the FF nanofibers, PF composite phase, and HGPF-DQ hydrogel obtained in Example 1 are characterized. The results are shown in the figure. Figures 1-2 FF self-assembles into one-dimensional nanofibers with a large aspect ratio. The PF composite phase retains the main diffraction characteristics of FF. The sodium hyaluronate / G3 precursor system changes from a fluid state to a gel state after activation by EDC / NHS; FTIR results support amide bond formation and retain the PEDOT:PSS and FF related absorption characteristics.

[0057] Experimental Example 2: Rheological and Drug Release Properties Time-scan rheological tests were performed on HG, HGPF, and HGPF-DQ. The results are shown in [Figure number missing]. Figure 3 After mixing, the storage modulus G′ of each group gradually exceeds the loss modulus G″ and a stable gel is formed. The final G′ of HGPF and HGPF-DQ is higher than that of HG.

[0058] The release behavior of dasatinib and quercetin was evaluated using different loading concentrations, and the results are shown in [Figure number missing]. Figure 4 The cumulative release rate of dasatinib within 24 hours decreased with increasing loading concentration; quercetin release was relatively rapid. In the preferred embodiment, the differentiated release of D 250 μg / mL and Q 750 μg / mL facilitates the sustained local action of the two drugs on the wound.

[0059] Experimental Example 3: Electrical Output Excited by Ultrasonic Excitation HG, HGPF, HG-DQ, and HGPF-DQ were fabricated into thin slices, and their electrical signals were recorded under 1 MHz, 0.5 W / cm² ultrasound stimulation. The results are shown in [Figure number missing]. Figure 5 The output voltages for each group are approximately 0.02, 0.37, 0.02, and 0.44 V, and the output currents are approximately 38, 83, 55, and 91 nA; the piezoelectric coefficients d33 are approximately 0.3, 6.5, 0.4, and 7.4 pC / N. The signal polarity is reversed when the HGPF-DQ is connected in both directions, indicating that the PF composite phase has a measurable electromechanical response.

[0060] Experimental Example 4: Cell Compatibility and Blood Compatibility The safety of the material was evaluated using L929 fibroblast and hemolysis assays; the results are shown in […]. Figure 6After 24 h and 48 h of culture, the cell viability of each hydrogel group was approximately 94%–100%, and Live / Dead staining showed predominantly green viable cells. The hemolysis rates of HG, HGPF, HG-DQ, and HGPF-DQ were approximately 1.2%, 1.6%, 2.9%, and 2.8%, respectively, indicating that the materials have good cell compatibility and blood compatibility.

[0061] Experimental Example 5: Regulation of Cellular Senescence-Related Phenotypes and Inflammatory Factors H2O2 was used to induce senescence-related phenotypes in fibroblasts. Four groups were established: Control, H2O2, H2O2+HG-DQ, H2O2+HGPF, and H2O2+HGPF-DQ. The HGPF and HGPF-DQ groups were further stimulated with ultrasound. Results are shown below. Figure 7 The positive area of ​​SA-β-gal increased from about 5% in the Control group to about 39% in the H2O2 group, decreased to about 15% in the HG-DQ group, about 26% in the HGPF group, and further decreased to about 7% in the HGPF-DQ group. The expression of SASP-related inflammatory proteins decreased more significantly in the HGPF-DQ group.

[0062] Experimental Case 6: In vivo wound repair A full-thickness skin defect was created on the back of male Balb / c mice, and they were divided into Control, HG, HGPF, and HGPF-DQ groups. The HGPF and HGPF-DQ groups were excited by low-intensity pulsed ultrasound at 1 MHz, 0.5 W / cm², 20% duty cycle, and 5 min after hydrogel application. Results are shown below. Figure 8 On day 3, the relative wound areas of each group were approximately 86%, 80%, 64%, and 63%; on day 5, they were approximately 63%, 47%, 24%, and 17%; and on day 10, they were approximately 36%, 18%, 8%, and 3%, with the HGPF-DQ group having the smallest residual wound area.

Claims

1. An ultrasonically excited piezoelectric-conductive composite hydrogel loaded with dasatinib and quercetin, characterized in that, The composite hydrogel contains a sodium hyaluronate / PAMAM G3-NH2 amide crosslinking network, an FF / PEDOT:PSS / PVA conductive-piezoelectric composite phase, and dasatinib and quercetin; The method for preparing the composite hydrogel includes the following steps: FF was dissolved in HFIP and solvent displacement was used to form FF nanofibers; PEDOT:PSS dispersion was mixed with PVA solution and then added to FF nanofibers to form PF conductive-piezoelectric composite phase; dasatinib and quercetin were pre-dispersed in PAMAM G3-NH2 solution to obtain drug-loaded G3 solution; sodium hyaluronate solution was activated by adding EDC·HCl and NHS to obtain activated sodium hyaluronate solution; the PF conductive-piezoelectric composite phase was added to drug-loaded G3 solution and reacted with activated sodium hyaluronate solution to obtain HGPF-DQ composite hydrogel.

2. The composite hydrogel as described in claim 1, characterized in that, The concentration of the PVA solution is 0.3% to 0.8%, and the volume ratio of PEDOT:PSS dispersion to PVA solution is 7:1 to 11:

1.

3. The composite hydrogel as described in claim 1, characterized in that, The concentration of FF in the PF conductive-piezoelectric composite phase is 0.6–1.5 mg / mL.

4. The composite hydrogel as described in claim 1, characterized in that, The concentration of PAMAM G3-NH2 solution is 0.8–1.5 mg / mL, and the concentration of sodium hyaluronate solution is 0.8–1.5 mg / mL.

5. The composite hydrogel as described in claim 1, characterized in that, The concentrations of dasatinib in the drug-loaded G3 solution were 180–320 μg / mL, and the concentrations of quercetin were 550–950 μg / mL.

6. The composite hydrogel as described in claim 1, characterized in that, The volume ratio of sodium hyaluronate solution to drug-loaded G3 solution is 1:3 to 1:

5.

7. The composite hydrogel as described in claim 1, characterized in that, In the preparation of activated sodium hyaluronate solution, the molar ratio of carboxyl groups, EDC and NHS in sodium hyaluronate is 1:(1.5-3):(1.5-3).

8. The composite hydrogel as described in claim 1, characterized in that, The volume fraction of the PF conductive-piezoelectric composite phase in the gelation system is 7% to 12%.

9. The use of the composite hydrogel as described in any one of claims 1 to 8 in the preparation of cell senescence-related wound repair materials, wound dressings, or wound patches.

10. A wound repair material system composed of the composite hydrogel according to any one of claims 1 to 8 and low-intensity pulsed ultrasound, characterized in that, The ultrasound frequency is 0.7–1.3 MHz, the power density is 0.3–0.8 W / cm², the duty cycle is 15%–30%, and the single stimulation time is 3–8 min.

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