Preparation method and application of piezoelectric material hydrogel for wound healing

Abstract

The invention discloses a preparation method and application of piezoelectric material hydrogel for wound healing, and relates to the field of preparation methods and application of hydrogel. The invention aims to solve the technical problem that the existing piezoelectric material is poor in biocompatibility and bioavailability. The method comprises the following steps: 1, preparing a bismuth-erbium solution from bismuth nitrate pentahydrate and erbium acetate hydrate; 2, adding a chlorinated potassium chloride ethylene glycol solution; 3, transferring into a high-pressure kettle for reaction, and collecting a precipitate; 4, cleaning, centrifugally washing and drying; 5, uniformly grinding, and calcining in a muffle furnace to obtain EBCL powder; 6, adding into a chitosan aqueous solution, and stirring to obtain a mixed solution; and 7, dissolving tetraethyl orthosilicate into absolute ethyl alcohol, adding the mixed solution, stirring, and reacting to complete gelation. The hydrogel prepared by the invention has a rapid sterilization effect and a wound protection function of the hydrogel, so that reinfection is effectively prevented, and the healing process is accelerated. The hydrogel prepared by the invention is applied to the field of wound treatment.

Description

TECHNICAL FIELD

[0001] The present application relates to a preparation method of piezoelectric material hydrogel for wound healing and its application field. BACKGROUND

[0002] Wounds such as infected wounds, burns, abscesses, and diabetic wounds can severely damage the barrier function of the skin, forming a moist microenvironment that promotes bacterial colonization and infection. At the same time, the lack of physical protection and the inhibition of immune function further exacerbate the invasion and reproduction of pathogens, leading to delayed wound healing and chronic inflammation. To address this issue, wound dressings based on functional biomaterials show potential for multi-modal therapy. Traditional dressings (such as gauze) can only passively cover the wound and lack biological activity and anti-infection ability, making it difficult to promote tissue regeneration; while the new generation of dressings (such as hydrogels, bioadhesive patches, and engineered membranes) can form a dynamic interactive interface with the wound, combining the advantages of moisture retention, oxygen permeability, and drug controlled release, thereby more effectively supporting wound repair.

[0003] Hydrogels are outstanding in wound treatment due to their unique properties: they can closely adhere to the wound surface, achieve sustained drug release, and can load bioactive molecules. Their mechanical flexibility, bioadhesion, and adjustable properties make them an ideal choice for irregular wound treatment, while significantly reducing drug waste. To enhance functionality, hydrogels can integrate responsive nanomaterials to achieve precise treatment triggered by external stimuli (such as light, sound, and electricity). In recent years, reactive oxygen species (ROS)-related therapies (including photodynamic therapy PDT, photothermal therapy PTT, and sonodynamic therapy SDT) have shown potential in antibacterial therapy, but are still limited by insufficient penetration depth, potential toxicity, slow reaction speed, and poor controllability. Therefore, piezoelectric dynamic therapy (PZDT) as a new strategy has attracted attention - by activating piezoelectric materials through mechanical stimulation such as ultrasound, using the electromechanical coupling effect to efficiently generate ROS in a hypoxic infected microenvironment, achieving rapid sterilization. However, piezoelectric materials have poor biocompatibility and bioavailability, and their application in wound healing is still challenging. Currently, the application of piezoelectric hydrogels in bacterial infected wound healing has not been systematically explored, and there is a clear research gap in this field, which needs further research. SUMMARY

[0004] The present application is to solve the technical problem of poor biocompatibility and bioavailability of piezoelectric materials, and provides a preparation method of piezoelectric material hydrogel for wound healing and its application.

[0005] A preparation method of piezoelectric material hydrogel for wound healing, specifically as follows:

[0006] I. Dissolve bismuth salt and erbium salt in ethylene glycol, ultrasonic treatment, and obtain bismuth-erbium solution;

[0007] II. Dissolve potassium chloride in ethylene glycol, stir to obtain a potassium chloride ethylene glycol solution, stir the bismuth-erbium solution obtained in step I, and add the potassium chloride ethylene glycol solution; adjust the pH of the solution to 8.5, form a precipitate, and then continue to stir the reaction to obtain a reaction liquid;

[0008] III. Transfer the reaction liquid obtained in step II to a stainless steel autoclave with a polytetrafluoroethylene liner, place it in a dust-free hot air oven, control the temperature to be 158-162 DEG C for 12 h, after the reaction is completed, naturally cool to room temperature, and collect the precipitate;

[0009] IV. Wash the precipitate collected in step III with deionized water, centrifuge and wash, and then dry to obtain a blocky product;

[0010] V. Grind the blocky product obtained in step IV uniformly, place it in a muffle furnace for calcination, and obtain EBCL powder;

[0011] VI. Disperse the EBCL powder obtained in step V in deionized water, ultrasonic treatment, then add to a chitosan aqueous solution, stir at 48-52 DEG C to obtain a mixed liquid;

[0012] VII. Dissolve tetraethyl orthosilicate in anhydrous ethanol, then add the mixed liquid obtained in step VI, stir at room temperature, then transfer to an oil bath, react at 78-82 DEG C for 6 h, and then react at 100-101 DEG C for 4 h; complete gelation at room temperature to obtain a piezoelectric material hydrogel for wound healing, and the preparation is completed.

[0013] The piezoelectric material hydrogel for wound healing prepared in the application is applied as a piezoelectric material in the field of wound treatment.

[0014] The application provides a multifunctional hydrogel dressing based on a novel piezoelectric nanomaterial, chitosan-coated erbium-doped bismuth oxychloride (CEBCL), which is used for ultrasound-driven antibacterial treatment. BiOCl (BCL) is a two-dimensional nanostructure with excellent piezoelectric properties, and its sheet-like morphology makes it have wider coverage and better charge transport capacity in the nano form. Doping Er 3+Afterwards, the defects introduced in the crystal can significantly enhance its piezoelectric response, thereby improving the ROS generation capacity under ultrasonic stimulation to achieve antibacterial effect. In addition, the chitosan coating not only improves the dispersibility and biocompatibility of the material, but also promotes the formation of hydrogel system, providing a moist healing environment for the wound. Notably, the subtle deformation polarization effect of chitosan can further enhance the piezoelectric performance of the material, making it have a stronger polarization response under mechanical stimulation. The hydrogel dressing not only serves as a physical barrier to protect the wound, but also as a local drug release platform for piezoelectric dynamic therapy. Under ultrasonic stimulation, CEBCL can rapidly generate reactive oxygen species to rapidly inactivate bacteria. Its piezoelectric enhanced ROS generation capacity is verified by electron paramagnetic resonance (EPR) spectroscopy and rhodamine B degradation chemical detection methods. The hydrogel system shows excellent antibacterial effect in vitro and in a mouse burn infection model, significantly reducing bacterial numbers, inhibiting inflammatory response, and promoting tissue regeneration. The rapid bactericidal effect combined with the wound protection function of the hydrogel effectively prevents reinfection and accelerates the healing process. 3+ As a doping element, it plays a key role in enhancing the piezoelectric activity of BiOCl. The crystal defects introduced by it increase the lattice asymmetry and dipole polarization, thereby enhancing the ROS generation capacity of the material under mechanical stimulation. By combining mechanical flexibility and electromechanical functionality, the CEBCL hydrogel provides a new treatment strategy for infected burn wounds without relying on antibiotics or phototherapy, and provides a promising candidate for a new generation of wound treatment platform in the context of drug-resistant infections.

[0015] The hydrogel prepared by the present application is used in the field of wound treatment. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 X-ray diffraction pattern (XRD) of the sample synthesized for the example;

[0017] Figure 2 Fourier transform infrared spectrogram (FTIR) of the sample synthesized for the example;

[0018] Figure 3 Zeta potential analysis chart of the sample synthesized for the example;

[0019] Figure 4 Field emission scanning electron microscope (FESEM) image of the sample synthesized for the example;

[0020] Figure 5 Field emission scanning electron microscope (FESEM) image of the sample synthesized for the example;

[0021] Figure 6 Bandgap diagram of BCL and EBCL plotted by Tauc equation;

[0022] Figure 7 Rheological property test results of CEBCL hydrogel synthesized for the example;

[0023] Figure 8 Real-time photos of CEBCL hydrogel synthesized for the example;

[0024] Figure 9 Photos of CEBCL hydrogel applied in vivo in a mouse model;

[0025] Figure 10 Photos of CEBCL hydrogel applied in vitro in a mouse model;

[0026] Figure 11 Piezoresponse force microscopy (PFM) amplitude loop map of the hydrogel prepared for the example;

[0027] Figure 12 PFM phase map of the hydrogel showing 180° phase inversion map (corresponding to Figure 11 );

[0028] Figure 13 Electron paramagnetic resonance (EPR) experimental results of the hydrogel prepared for the example under water condition;

[0029] Figure 14 Electron paramagnetic resonance (EPR) experimental results of the hydrogel prepared for the example under dimethyl sulfoxide (DMSO) condition;

[0030] Figure 15 Graph of optical density change of the dye degraded by the hydrogel prepared for the example under different reaction time conditions;

[0031] Figure 16 Efficiency graph of the dye degraded by the hydrogel prepared for the example;

[0032] Figure 17 Piezoelectric voltage generated by the hydrogel prepared for the example under ultrasonic action;

[0033] Figure 18 Graph showing electron-hole separation of the hydrogel prepared for the example under short-time ultrasonic pulse using COMSOL simulation;

[0034] Figure 19 Comparison graph of cell viability of methicillin-resistant Staphylococcus aureus under different ultrasonic exposure times and different treatment groups;

[0035] Figure 20 Comparison graph of cell viability of Enterococcus faecalis under different ultrasonic exposure times and different treatment groups;

[0036] Figure 21 Figure of hemolysis test results of CEBCL hydrogel prepared for the example;

[0037] Figure 22 Figure of MRSA survival test results of hydrogels prepared for the example by agar diffusion method;

[0038] Figure 23 Figure of E. faecalis survival test results of hydrogels prepared for the example by agar diffusion method;

[0039] Figure 24 Figure of in vivo healing of mouse MRSA infected burn wounds after 8 days of treatment in different treatment groups;

[0040] Figure 25 Figure of agar plates of collected tissues after treatment;

[0041] Figure 26 Figure of bacterial infection analysis;

[0042] Figure 27 Figure of biological safety test results of CEBCL hydrogel prepared for the example on macrophages (CCK-8 cell viability assay);

[0043] Figure 28 Figure of schematic diagram of treatment process of CEBCL hydrogel prepared for the example in mice;

[0044] Figure 29 Figure of hemoglobin (Hb) test results in blood of mice in different treatment groups and control group;

[0045] Figure 30 Figure of red blood cell count (RBC) test results in blood of mice in different treatment groups and control group;

[0046] Figure 31 Figure of C-reactive protein (CRP) test results in blood of mice in different treatment groups and control group;

[0047] Figure 32 Figure of platelet count test results in blood of mice in different treatment groups and control group;

[0048] Figure 33 Figure of superoxide dismutase (SOD) activity test results in blood of mice in different treatment groups and control group;

[0049] Figure 34 Figure of lactate dehydrogenase (LDH) level test results in blood of mice in different treatment groups and control group. DETAILED DESCRIPTION

[0050] Specific embodiment one: the preparation method of the piezoelectric material hydrogel for wound healing, which is specifically carried out according to the following steps:

[0051] I. Dissolve bismuth salt and erbium salt in ethylene glycol, and ultrasonic treatment to obtain a bismuth-erbium solution;

[0052] II. Dissolve potassium chloride in ethylene glycol, and stir to obtain a potassium chloride ethylene glycol solution. Stir the bismuth-erbium solution obtained in step I, and add the potassium chloride ethylene glycol solution. Adjust the pH of the solution to 8.5 to form a precipitate, and then continuously stir the reaction to obtain a reaction liquid;

[0053] III. Transfer the reaction liquid obtained in step II to a stainless steel autoclave lined with polytetrafluoroethylene, and place it in a dust-free hot air oven. Control the temperature to be 158-162℃ and react for 12 h. After the reaction is completed, naturally cool to room temperature, and collect the precipitate;

[0054] IV. Wash the precipitate collected in step III with deionized water, and then centrifuge and wash. Dry to obtain a block-shaped product;

[0055] V. Grind the block-shaped product obtained in step IV uniformly, and place it in a muffle furnace for calcination to obtain an EBCL powder;

[0056] VI. Disperse the EBCL powder obtained in step V in deionized water, and ultrasonic treatment. Then, add it to a chitosan aqueous solution, and stir at 48-52℃ to obtain a mixed liquid;

[0057] VII. Dissolve tetraethyl orthosilicate in anhydrous ethanol, and then add the mixed liquid obtained in step VI. Stir at room temperature, and then transfer it to an oil bath. React at 78-82℃ for 6 h, and then react at 100-101℃ for 4 h. Complete the gelation at room temperature to obtain a piezoelectric material hydrogel for wound healing, and the preparation is completed.

[0058] Specific embodiment two: the difference between this embodiment and specific embodiment one is that the amount ratio of the bismuth salt, the erbium salt and the ethylene glycol in step I is 1-3 g: 0.02-0.06 g: 60 mL. The others are the same as specific embodiment one.

[0059] Specific embodiment three: the difference between this embodiment and specific embodiment one or two is that the amount ratio of the potassium chloride and the ethylene glycol in step II is 0.6 g: 10 mL.

[0060] The stirring speed of the bismuth-erbium solution in step II is 600-700 rpm. The others are the same as specific embodiment one or two.

[0061] Specific implementation four: the difference between this embodiment and one of the specific implementations one to three is that: step two uses a sodium hydroxide solution with a concentration of 12.5 mM to adjust the pH. The others are the same as one of the specific implementations one to three.

[0062] Specific implementation five: the difference between this embodiment and one of the specific implementations one to four is that: after the formation of the precipitate in step two, the reaction is continuously stirred for 4 h, and the stirring speed is controlled at 900 rpm. The others are the same as one of the specific implementations one to four.

[0063] Specific implementation six: the difference between this embodiment and one of the specific implementations one to five is that: the centrifugal washing speed in step four is 12000 rpm, and the washing time is 10 min. The others are the same as one of the specific implementations one to five.

[0064] Specific implementation seven: the difference between this embodiment and one of the specific implementations one to six is that: the calcination temperature in step five is controlled at 250℃, and the calcination time is 2 h. The others are the same as one of the specific implementations one to six.

[0065] Specific implementation eight: the difference between this embodiment and one of the specific implementations one to seven is that: the use amount ratio of the EBCL powder to the chitosan aqueous solution in step six is 30 mg:30 mL, and the chitosan aqueous solution contains 100 mg of chitosan. The others are the same as one of the specific implementations one to seven.

[0066] Specific implementation nine: the difference between this embodiment and one of the specific implementations one to eight is that: the use amount ratio of the tetraethyl orthosilicate to the anhydrous ethanol in step seven is 20 μL:5 mL. The others are the same as one of the specific implementations one to eight.

[0067] Specific implementation ten: an application of a piezoelectric material hydrogel for wound healing, which is applied in the field of wound treatment as a piezoelectric material.

[0068] The content of the present application is not limited to the content of each of the above embodiments, and a combination of one or more specific implementations can also achieve the purpose of the application.

[0069] Example:

[0070] The chemicals used in this example were purchased from Sigma-Aldrich, Innochem and Lumiprobe companies, and were directly used without further purification. The deionized water used in the experiment had a resistivity of not less than 18.2 MΩ·cm. The glassware used was first cleaned with chromic acid and then rinsed with ultrapure water.

[0071] Bacterial culture and colony forming unit (CFU) plating experiment related reagents were purchased from MacLin Biotechnology Co., Ltd. Live / dead bacteria double staining kit was purchased from Thermo Fisher Scientific Company. Cell culture related reagents include Dulbecco's modified Eagle's medium (DMEM), Roswell Park Memorial Institute 1640 medium (RPMI 1640), fetal bovine serum (FBS), penicillin and streptomycin, which were purchased from Gibco Life Technologies Company. Interleukin-6 (IL-6) and interleukin-1β (IL-1β) enzyme-linked immunosorbent kit were purchased from Jiangsu Meilian Industry Co., Ltd. All reagents were used according to the original packaging of the manufacturer, without further purification.

[0072] The preparation method of the piezoelectric material hydrogel for wound healing in this embodiment is chitosan-coated erbium-doped bismuth oxychloride hydrogel, which is specifically carried out according to the following steps:

[0073] I. 1.892 g of bismuth nitrate pentahydrate (Bi (NO3) 3·5H2O) and 0.035 g of erbium acetate hydrate (Er (CH3COO) 3·xH2O) were dissolved in 60 mL of ethylene glycol, and ultrasonic treatment was performed for at least 30 minutes to obtain a bismuth-erbium solution;

[0074] II. 0.6 g of potassium chloride (KCl) was dissolved in 10 mL of ethylene glycol, and stirring was performed until the solution was uniform to obtain a potassium chloride ethylene glycol solution. The bismuth-erbium solution obtained in step I was stirred at a speed of 600-700 rpm, and the potassium chloride ethylene glycol solution was added dropwise. A sodium hydroxide solution with a concentration of 12.5 mM was added dropwise to adjust the pH of the solution to 8.5, a precipitate was formed, and then the stirring speed was controlled at 900 rpm, and the stirring reaction was continued for 4 h to obtain a reaction liquid;

[0075] III. The reaction liquid obtained in step II was transferred to a polytetrafluoroethylene-lined stainless steel autoclave, placed in a dust-free hot air oven, and the temperature was controlled at 160℃ for 12 h to promote crystal growth. After the reaction was completed, the precipitate was collected after natural cooling to room temperature;

[0076] IV. The precipitate collected in step III was washed with deionized water, and centrifugal washing was performed at a speed of 12000 rpm for 10 min. The washing was repeated three times, and then a 60℃ hot air oven was used for drying for 8 h to obtain a block-shaped product;

[0077] V. The block-shaped product obtained in step IV was ground uniformly and placed in a muffle furnace for calcination. The calcination temperature was 250℃, and the calcination time was 2 h to obtain an EBCL powder;

[0078] Six, 30 mg of the EBCL powder obtained in step five was dispersed in 20 mL of deionized water and ultrasonically treated for 30 min. Then, the mixture was slowly added to 30 mL of a chitosan aqueous solution containing 100 mg of chitosan, and stirred at 50°C for 30 min to obtain a mixed solution;

[0079] Seven, 20 μL of tetraethyl orthosilicate (TOES) was dissolved in 5 mL of anhydrous ethanol, and then added to the mixed solution obtained in step six. The mixture was stirred at room temperature for 1 h, and then transferred to an oil bath and reacted at 80°C for 6 h and at 100°C for 4 h. The gelation was completed at room temperature to obtain a piezoelectric material hydrogel for wound healing, i.e., chitosan-coated EBCL hydrogel (CEBCL), thus completing the preparation.

[0080] The phase identification of all the prepared samples was performed by an X-ray powder diffractometer (PANalytical Aris type) with a scanning range of 2θ = 15° to 70°, an instrument operating voltage of 35 kV, and a current of 35 mA. A Cu-Ka radiation source with a wavelength of 1.5418 Å was used, and the scanning speed was 0.1 s per step.

[0081] To confirm the different phases and functional group vibrations in the samples, a Thermo Scientific Nicolet iS50 Fourier transform infrared spectrometer (FT-IR) was used for testing, and the test wave number range was 650-4000 cm -1 The infrared test was performed using an attenuated total reflection accessory (ATR, model ITR ATR Diamond, Thermo Fisher Scientific), and only a small amount of sample was placed on the ATR platform for analysis.

[0082] The surface charge of the samples was tested by a Malvern Instruments Zetasizer instrument in an aqueous solution. The surface morphology of the samples was obtained by a field emission scanning electron microscope (FESEM, model FEI F-50) with an operating voltage of 20 kV. The samples were first placed on a glass slide and subjected to metal spraying treatment to improve the electrical conductivity, and then imaged. The band gap energy of the samples was calculated by the Tauc relationship, and the equation used was as follows:

[0083] αE = A(E -E g ) n (1)

[0084] wherein E is the photon energy, Eg is the optical band gap energy (unit: eV), a is the absorption coefficient, and A is a constant.

[0085] By drawing the Tauc plot, i.e. plotting (ahv) 2 The photonic energy hv was plotted, and the intersection of the linear part of the graph with the energy axis was extrapolated to obtain the band gap energy of the sample. The rheological properties of the hydrogel were tested by an MCR302e rheometer produced by Anton Paar Company.

[0086] Measurement of piezoelectric properties

[0087] The piezoelectric response force microscopy (PFM) test was performed on the prepared sample by using an atomic force microscope (AFM, Bruker Dimension Icon) equipped with a ferroelectric system to evaluate the piezoelectric properties thereof.

[0088] In addition, finite element analysis (FEA) was performed on the prepared sample by using COMSOL Multiphysics software to simulate and calculate the piezoelectric polarization effect generated on the surface of the sample. The two-dimensional structure of CEBCL under vacuum conditions was used as the basis for model construction, and the simulation system was built in the model builder. The material properties of the related nanosheet were also set in the model, including density (p = 7720 kg / m 3 ), elastic matrix (cE), piezoelectric coupling matrix (eEs), and relative dielectric constant (e r ). The main parameters are as follows:

[0089] ;

[0090] ;

[0091] ;

[0092] Under the condition of ultrasonic vibration (frequency of 10 6 Hz), the simulated pressure on the nanoparticles was regulated in the form of a sine wave (range of 10 5 Pa to 10 8 Pa), and the surface polarization behavior of the sample was measured along the three coordinate axes (x, y, z). The transient piezoelectric voltage of the sample was recorded by a Keysight X3012A digital storage oscilloscope combined with an Agilent N2862B 10:1 passive probe system. During the test, the sample was immersed in a 10 kHz ultrasonic water bath (protected by a soft plastic layer) and measured under the condition of ultrasonic radiation.

[0093] To detect the ability of piezoelectric materials to induce the production of reactive oxygen species (ROS) under ultrasonic stimulation, experiments were performed using a Bruker ELEXSYS E500 electron spin resonance (ESR) spectrometer. 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) was used as a spin-trapping agent to detect hydroxyl radicals (·OH) and superoxide anion radicals (·O2 - ). The ESR experiment set three groups of samples: the control group (DMPO), the CEBCL group (DMPO + CEBCL), and the ultrasonic treatment group (DMPO + CEBCL + ultrasound), all under standard conditions.

[0094] The piezocatalytic degradation of dyes experiment used Rhodamine B (RhB) as the target dye and a Telesonic ultrasonic instrument with a frequency of 40 kHz for processing. In the experiment, 20 mL of RhB dye solution with an initial concentration of 15 ppm was placed in a glass bottle, and a fixed concentration of 1 mg / mL (w / v) of the sample was added. Each group was subjected to ultrasonic stimulation for 20 minutes. Every 4 minutes, a small amount of dye solution was removed, and the remaining particulate matter was separated by centrifugation. Then, the supernatant was measured for optical density at a wavelength of 554 nm using a UV-Vis spectrophotometer to evaluate the piezocatalytic degradation efficiency and reaction kinetics. In addition, the degradation efficiency (η) and the first-order kinetic constant (k) were calculated according to the following formulas:

[0095] ;

[0096] C = C0 exp(-kt);

[0097] where C0 is the initial dye concentration, and C is the dye concentration at a specific time t.

[0098] Biological experiments

[0099] Bacterial culture

[0100] Methicillin-resistant Staphylococcus aureus (MRSA) was inoculated in LB liquid medium (Luria-Bertani Broth) and cultured at 37°C with shaking at 120 rpm for 10 hours. Then, the culture was washed three times with 1x PBS buffer (4500 rpm, centrifugation for 10 minutes), and the optical density at 600 nm (OD600) was measured.

[0101] Evaluation of antibacterial effect in vitro

[0102] To evaluate the bactericidal effect of the prepared samples, a spread plate method was used. 50 μL of E. coli, P. aeruginosa (PA) and methicillin-resistant Staphylococcus aureus (MRSA) bacterial solution (bacterial concentration of 1 x 10 8 CFU / mL) was taken respectively, mixed with 50 μL of PBS or CEBCL (concentration of 20 μg / mL) for 3 hours of treatment. Subsequently, the mixed solution was treated with 10 minutes of ultrasonic stimulation at 1 W / cm 2 power using a small animal portable ultrasonic treatment instrument (Intelect). Then, the treated mixed solution was diluted by 10 times gradient, and 100 μL of each group of diluent was evenly dropped on the LB agar plate for coating. After 24 hours of incubation in a 37°C incubator, the colony forming units (CFUs) were observed, and the number of surviving bacteria was calculated.

[0103] Wound healing effect evaluation

[0104] To evaluate the wound healing effect of the prepared samples, 6-week-old female BALB / c mice (purchased from Guangdong Experimental Animal Center) were selected. The mice were anesthetized with chloral hydrate (10%, 0.3 mL / 100 g), and the back was depilated (using a depilator and depilatory cream combined treatment). The wound device was preheated to 95°C (for 5 minutes), and then it was attached to the mouse back skin for 10 seconds to cause a burn wound with a diameter of about 1 cm. Then, 20 μL of MRSA suspension (concentration of 10 8 CFU / mL) was dropped on the wound to establish an infection model. Treatment was started after the appearance of pus on the wound (2nd or 3rd day). The mice were sacrificed after 12 days of treatment, and the infected tissue was collected and homogenized using a tissue grinder. The obtained homogenate was diluted by 10 times gradient, and 100 μL of each diluent was coated on the LB agar medium, which was incubated at 37°C for 24-36 hours. The number of bacterial colonies was observed and recorded to evaluate the bacterial quantity.

[0105] Hemolytic test

[0106] Red blood cells were isolated from adult mouse blood and prepared into a 2% red blood cell suspension. Different experimental groups were mixed with red blood cells and incubated at 37°C for 1 hour. After incubation, the supernatant was collected by centrifugation at 800 x g for 10 minutes, and the absorbance was measured at 540 nm wavelength using a microplate reader (BioTek Epoch) to evaluate the amount of hemoglobin released. The Triton X-100 treated group was used as a positive control, and the PBS treated group was used as a negative control. Samples with a hemolysis rate below 10% were considered to have good hemocompatibility. The hemolysis rate was calculated according to the following formula:

[0107] Hemolysis (%) = [(A positive - A negative ) / (A sample - A negative )] x 100

[0108] Cell viability assay

[0109] The effect of CEBCL on cells was evaluated using a cell viability assay. Cells were seeded in a 96-well plate at a density of 5 x 10 3 cells / well. After 24 hours of adherent culture, cells were treated with different concentrations of CEBCL and incubated for another 24 hours. After incubation, the culture medium was discarded, and 100 µL of fresh culture medium containing 10 µL of MTT solution (Solarbio, China) was added and incubated at 37°C for 4 hours. After incubation, the culture medium was removed, and 100 µL of dimethyl sulfoxide (DMSO) was added to dissolve the purple formazan crystals formed. Finally, the absorbance was measured at 490 nm wavelength using a SpectraMax M2 microplate reader (Molecular Devices, USA) to evaluate cell viability.

[0110] Figure 1 X-ray diffraction pattern (XRD) of the example synthesized sample; Figure 2 Fourier transform infrared spectrogram (FTIR) of the example synthesized sample; Figure 3 Zeta potential analysis chart of the example synthesized sample; Figure 4 Field emission scanning electron microscope (FESEM) image of the example synthesized EBCL; Figure 5 Field emission scanning electron microscope (FESEM) image of the example synthesized CEBCL; Figure 6 Bandgap diagram of BCL and EBCL plotted by Tauc equation; Figure 7 Rheological property test result chart of the example synthesized CEBCL hydrogel; Figure 8Real-time photographs of the synthesized CEBCL hydrogel for this example; Figure 9 Photographs showing the application of CEBCL hydrogel in a mouse model; Figure 10 Photographs showing the application of CEBCL hydrogel in vitro in a mouse model.

[0111] pass Figures 1-3 Characterization results showed that the synthesized erbium-doped bismuth oxychloride (EBCL) was successfully constructed as a piezoelectric hydrogel (CEBCL). Figure 6 The bandgap plots of BCL and EBCL are shown, illustrating their wide bandgap characteristics. Figure 7 The rheological properties of the CEBCL hydrogel showed a high storage modulus G' under low shear conditions, verifying its successful gelation. Figure 8 Real-time photographs of the hydrogel show its gel-state characteristics. Figure 9 and Figure 10 The material exhibits good skin adhesion properties under different physical disturbances (scale bar: 1 mm).

[0112] Figure 11 Piezoelectric response force microscopy (PFM) amplitude ring plot of the hydrogel prepared for the example; Figure 12 The PFM phase diagram of the hydrogel shows a 180° phase reversal (corresponding to...) Figure 11 ); Figure 13 The figure shows the experimental results of electron paramagnetic resonance (EPR) of the hydrogel prepared for the example under aqueous conditions; Figure 14 The figure shows the electron paramagnetic resonance (EPR) experimental results of the hydrogel prepared for the example under dimethyl sulfoxide (DMSO) conditions; Figure 15 The optical density variation of the hydrogel prepared for the example under different reaction time conditions for dye degradation is shown in the graph. Figure 16 The efficiency graph of the hydrogel-degraded dye prepared for the example; Figure 17 Piezoelectric voltage diagram of the hydrogel prepared in the example under ultrasonic treatment; Figure 18 The image shows the electron-hole separation of the hydrogel prepared in the example under short-duration ultrasonic pulses, using COMSOL simulation.

[0113] Depend on Figure 11 It can be seen that it presents a butterfly-shaped circular curve; from Figure 12 The corresponding 180° phase reversal verified the piezoelectric properties of the sample. (By...) Figure 13 and Figure 14 Electron paramagnetic resonance (EPR) experiments under different conditions in water and dimethyl sulfoxide (DMSO) demonstrated that the hydrogel generates hydroxyl radicals (•OH) and superoxide anion radicals (•O2) under ultrasonic irradiation. - (and other reactive oxygen species) Figure 15 andFigure 16 The degradation experiment of rhodamine B dye shows that the degradation rate of the dye is about 94% under the action of ultrasound (US), further verifying the piezoelectric catalytic performance of the prepared hydrogel.

[0114] Figure 19 A comparison chart of cell viability of methicillin-resistant Staphylococcus aureus under different ultrasound exposure times and different treatment groups; Figure 20 A comparison chart of cell viability of Enterococcus faecalis under different ultrasound exposure times and different treatment groups; Figure 21 A hemolytic test result chart of the CEBCL hydrogel prepared in the example; Figure 22 A test chart of evaluating the survival rate of methicillin-resistant Staphylococcus aureus (MRSA) of the hydrogel prepared in the example by agar diffusion method; Figure 23 A test chart of evaluating the survival rate of Enterococcus faecalis of the hydrogel prepared in the example by agar diffusion method; Figure 24 A chart of in vivo healing of mouse MRSA infected burn wounds after 8 days of treatment in different treatment groups; Figure 25 A chart of agar plate photos of tissues collected after treatment; Figure 26 A bacterial infection analysis chart.

[0115] Figure 19 And Figure 20 The piezoelectric dynamic therapy (PZDT) of the CEBCL hydrogel shows significant inhibition of bacteria. Figure 21 Through the hemolytic test results of the CEBCL hydrogel, it shows that the cell lysis rate is very low, verifying its good biological safety. Figure 22 And Figure 23 Through the evaluation of bacterial survival rate by agar diffusion method, the results show that the sample has a significant killing effect on MRSA and E. faecalis. Figure 24 The in vivo healing of mouse MRSA infected burn wounds after 8 days of treatment in different treatment groups, the ultrasound activated CEBCL group almost achieved complete tissue regeneration, significantly promoting the healing of burn wounds. Figure 25 And Figure 26 The agar plate photos of tissues collected after treatment and the bacterial infection analysis show that the bacterial infection of the ultrasound activated CEBCL group is significantly reduced. (Statistical significance: < 0.05, < 0.01, < 0.001)

[0116] Figure 27 A chart of biological safety test of macrophages by the CEBCL hydrogel prepared in the example (CCK-8 cell viability assay); Figure 28A schematic diagram of the treatment process of the CEBCL hydrogel prepared for the example in mice; Figure 29 A graph of the hemoglobin (Hb) detection results in the blood of mice in different treatment groups and control groups; Figure 30 A graph of the red blood cell count (RBC) detection results in the blood of mice in different treatment groups and control groups; Figure 31 A graph of the C-reactive protein (CRP) detection results in the blood of mice in different treatment groups and control groups; Figure 32 A graph of the platelet count detection results in the blood of mice in different treatment groups and control groups; Figure 33 A graph of the superoxide dismutase (SOD) activity detection results in the blood of mice in different treatment groups and control groups; Figure 34 A graph of the lactate dehydrogenase (LDH) level detection results in the blood of mice in different treatment groups and control groups; (statistical significance: < 0.05, < 0.01, < 0.001).

[0117] According to Figure 27 CEBCL shows good biosafety at a concentration of up to 120 μg / mL.

Claims

1. A method for preparing a piezoelectric material hydrogel for wound healing, characterized by The method is specifically performed according to the following steps: I. Dissolve bismuth salt and erbium salt in ethylene glycol, and obtain a bismuth-erbium solution by ultrasonic treatment; II. Dissolve potassium chloride in ethylene glycol to obtain a potassium chloride ethylene glycol solution, and stir the bismuth-erbium solution obtained in step I to add the potassium chloride ethylene glycol solution; adjust the pH of the solution to 8.5 to form a precipitate, and then continuously stir the reaction to obtain a reaction liquid; III. Transfer the reaction liquid obtained in step II to a polytetrafluoroethylene-lined stainless steel autoclave, place it in a dust-free hot air oven, control the temperature to be 158-162℃, and react for 12 h; after the reaction is completed, naturally cool to room temperature, and collect the precipitate; IV. Wash the precipitate collected in step III with deionized water, then centrifuge and wash, and then dry to obtain a block-shaped product; V. Grind the block-shaped product obtained in step IV uniformly, place it in a muffle furnace for calcination, and obtain an EBCL powder; VI. Disperse the EBCL powder obtained in step V in deionized water, ultrasonic treat, then add to a chitosan aqueous solution, and stir at 48-52℃ to obtain a mixed liquid; VII. Dissolve tetraethyl orthosilicate in anhydrous ethanol, then add the mixed liquid obtained in step VI, stir at room temperature, then transfer to an oil bath, react at 78-82℃ for 6 h, and then react at 100-101℃ for 4 h; complete gelation at room temperature to obtain a piezoelectric material hydrogel for wound healing, and the preparation is completed.

2. The method for preparing a piezoelectric hydrogel for wound healing according to claim 1, characterized in that... The amount ratio of the bismuth salt, the erbium salt and the ethylene glycol in step I is (1-3) g:(0.02-0.06) g:60 mL.

3. The method of claim 1, wherein the piezoelectric material hydrogel for wound healing is prepared by the steps of: (a) mixing a piezoelectric material with a polymer solution; (b) adding a crosslinking agent to the mixture; (c) crosslinking the mixture; and (d) washing the crosslinked mixture. The amount ratio of the potassium chloride and the ethylene glycol in step II is 0.6 g:10 mL. The stirring speed of the bismuth-erbium solution in step II is 600-700 rpm.

4. The method of claim 1, wherein the piezoelectric material hydrogel for wound healing is prepared by the steps of: (a) mixing a piezoelectric material with a polymer solution; (b) adding a crosslinking agent to the mixture; (c) crosslinking the mixture; and (d) washing the crosslinked mixture. The pH is adjusted by using a 12.5 mM sodium hydroxide solution in step II.

5. The method for preparing a piezoelectric hydrogel for wound healing according to claim 1, characterized in that... After the precipitate is formed in step II, continuously stir the reaction for 4 h, and control the stirring speed to be 900 rpm.

6. The method for preparing a piezoelectric hydrogel for wound healing according to claim 1, characterized in that... The centrifugal washing speed in step IV is 12000 rpm, and the washing time is 10 min.

7. The method of claim 1, wherein the piezoelectric material hydrogel for wound healing is prepared by the steps of: a) mixing a piezoelectric material with a polymer solution; b) adding a crosslinking agent to the mixture; c) crosslinking the mixture; and d) washing the crosslinked mixture with distilled water. The calcination temperature in step V is controlled to be 250℃, and the calcination time is 2 h.

8. The method for preparing a piezoelectric hydrogel for wound healing according to claim 1, characterized in that... The amount ratio of the EBCL powder and the chitosan aqueous solution in step VI is 30 mg:30 mL, and the chitosan aqueous solution contains 100 mg of chitosan.

9. The method of claim 1, wherein the piezoelectric material hydrogel for wound healing is prepared by the steps of: a) mixing a piezoelectric material with a polymer solution; b) adding a crosslinking agent to the mixture; c) crosslinking the mixture; and d) washing the crosslinked mixture with distilled water. The amount ratio of the tetraethyl orthosilicate and the anhydrous ethanol in step VII is 20 μL:5 mL.

10. Use of a piezoelectric material hydrogel for wound healing prepared according to claim 1, characterized in that The hydrogel is applied in the field of wound treatment as a piezoelectric material.

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