Preparation method and application of an ultrasound-excited antibacterial fiber wound repair material

Abstract

The present invention designs and develops a sound-activated antibacterial fiber wound repair material containing a barium titanate (BTO) and liquid metal (GaIn) composite material for ultrasonic-responsive biomaterials to combat wound bacterial infections. The present invention also provides a preparation method for the above material: first, construct a BTO / GaIn composite material and a PLGA film respectively, and load the BTO / GaIn composite material on the PLGA film by surface spin coating. Under ultrasonic action, the BTO / GaIn composite material can generate sufficient reactive oxygen species (ROS) to replace traditional antibiotic treatment to combat bacterial infections, and at the same time promote wound regeneration during the repair process. The material has excellent biocompatibility, low cytotoxicity and persistent antibacterial properties, and can effectively combat the lack of anti-microbial infection in dressings during skin repair.

Description

Technical Field

[0001] The present invention belongs to the field of biomaterials, and a sound-excited antibacterial fiber wound repair material which is flexible, biodegradable, has good biocompatibility, and contains barium titanate and liquid metal gallium-indium alloy is prepared by electrospinning technology and surface spin coating technology. Background Art

[0002] The skin is the body's first line of defense against external damage and internal water loss. When the integrity of the skin is breached, pathogens can invade the body, leading to various complications, including pain, bleeding, inflammation, infection, delayed healing, and scar formation. Additionally, in severe cases, without proper clinical management, infection and chronic inflammation will inevitably result in chronic stagnant wounds, which typically do not progress through the normal stages of healing and cause significant suffering to the affected patients. It has been reported that nanofiber membranes can serve as excellent dressing platforms for accelerating wound healing by promoting cell adhesion and proliferation. Electrospinning is a simple, versatile, and cost-effective method for producing nanofiber membranes with controllable composition and microstructure, a large specific surface area, high porosity, excellent mechanical properties, and the ability to mimic the extracellular matrix. As a wound dressing, electrospun nanofiber membranes can not only provide a physical barrier against bacterial penetration and biofilm formation but also facilitate cell adhesion, proliferation, and gas exchange required for wound healing. Poly(lactic-co-glycolic acid) (PLGA) is a copolymer approved by the US Food and Drug Administration and is considered one of the most attractive biomaterials due to its biodegradability and biocompatibility. Electrospun PLGA fiber membranes have been shown to support the growth of many cell types, including endothelial cells, fibroblasts, chondrocytes, and osteocytes. The structure in the electrospun fiber membranes is similar to that in the natural extracellular matrix, where the high microporosity and large specific surface area provide a favorable site for cell adhesion and oxygen. PLGA is widely used as a raw material for electrospinning, especially in the field of wound dressings, because of its biodegradability and relatively low cost, as well as its biocompatibility and excellent mechanical properties. Therefore, they are an ideal choice for chronic wound dressings. However, a single PLGA membrane cannot directly kill the bacteria in contact, so it needs to be modified accordingly. As a new treatment modality, sonodynamic therapy has attracted extensive attention due to its high spatiotemporal selectivity, deep tissue penetration ability, non-invasiveness, and the fact that it does not cause bacterial resistance, making it a potentially effective means for treating related microbial infections. Similar but different from the principle of photodynamic therapy, under the action of the periodic mechanical stress generated by ultrasound, the sonosensitizer deforms to generate a piezoelectric surface potential, which induces the polarization separation of charge carriers inside the sonosensitizer and reacts with the surrounding oxygen-containing substances to release reactive oxygen species (ROS), causing oxidative damage to cell components and attacking multiple targets in bacteria, such as cell membranes, respiratory enzymes, proteins, DNA, etc. Among various piezoelectric materials, barium titanate (BTO) has become a classic material due to its high piezoelectric performance, environmental friendliness, and simple structure. However, due to the relatively weak piezoelectric performance of BTO itself and its narrow bandgap, which limit the efficient separation of polarized charge carriers, resulting in a low production yield of ROS and an unsatisfactory bacterial clearance effect.In previous studies, constructing composites with metals has been proven to promote the recombination of interfacial charges, thereby achieving effective charge separation at the interface, making it a potential strategy to improve the sonodynamic antibacterial performance of BTO. Research has shown that gallium indium alloy (GaIn) is a promising material due to its high conductivity, large specific surface area, and excellent biocompatibility. Therefore, constructing BTO / GaIn composites is expected to achieve satisfactory therapeutic effects in the treatment of bacterial infections. Here, the present invention prepares BTO / GaIn composites by a solvothermal method. The BTO / GaIn is loaded onto the surface of an electrospun PLGA membrane through a surface spin-coating technique. Under ultrasonic action, the energy band of BTO shifts, and at the same time, a piezoelectric surface potential is generated to drive the separation of carriers. At this time, GaIn exerts its efficient charge separation ability due to its high conductivity, realizing the efficient separation of carriers across space, thereby promoting the explosive generation of ROS and achieving the rapid clearance of bacterial infections. Summary of the Invention

[0003] The object of the present invention is to design and develop a sonodynamic composite material composed of BTO and GaIn for combating infections related to infectious wounds and promoting tissue repair in view of the fact that electrospun PLGA membranes cannot effectively combat and clear bacterial infections. The present invention also provides a preparation method of the above material: first, gallium indium alloy sheets are prepared by a liquid phase method, and then BTO / GaIn is prepared by hydrothermal synthesis, and the BTO / GaIn composite material is loaded onto the electrospun PLGA membrane by spin coating. The material has good biocompatibility and high antibacterial performance, and can reshape infectious chronic wounds into regenerative wounds.

[0004] The antibacterial membrane provided by the present invention can be prepared by including the following process steps:

[0005] In the above technical solution of the present invention, BTO and GaIn are combined to form a piezoelectric composite material. Through the close interfacial contact between GaIn with metallic properties and BTO, the redistribution of interfacial charges is regulated, the piezoelectric performance of BTO is enhanced, the yield of ROS is promoted, and its antibacterial performance is strengthened. At the same time, the tissue repair process is shortened by rapid antibacterial.

[0006] In summary, the present invention overcomes the lack of effective means for anti-microbial infection in existing dressings. Antibacterial fibers excited by ultrasound of P-BTO / GaIn are prepared through a simple solvothermal method and electrospinning technology for treating infectious wounds. The construction of the BTO / GaIn composite material not only strengthens the sonodynamic performance of BTO but also promotes the effective separation of carriers. Under the synergistic action of multiple reactive oxygen species and carriers, it effectively promotes bacterial oxidative stress and interferes with bacterial energy metabolism, ultimately achieving efficient bacterial clearance. In addition, the P-BTO / GaIn piezoelectric bioheterojunction has good biocompatibility. Therefore, the ultrasound-excited antibacterial fibers have great potential in the repair of wound infections. Description of the Drawings

[0007] Figure 1 XPS full spectra of the materials PLGA and P-BTO / GaIn prepared in Example 1.

[0008] Figure 2 Sonodynamic performance diagrams of the materials prepared in Example 1, where A is the production amount of ·O 2- produced by BTO / GaIn detected by an electron spin resonance instrument (ESR) at different times under ultrasound; B is the production amount of ·OH produced by BTO / GaIn detected by ESR at different times under ultrasound; C is the ultraviolet-visible absorption spectrum of the consumption of MB by ROS produced by BTO / GaIn under ultrasound with different powers.

[0009] Figure 3 SEM morphology of cells after co-culturing the materials prepared in Example 1 with L929 cells.

[0010] Figure 4 Bacteriostatic effects of different samples before and after ultrasound treatment, where A is the bacteriostatic effect diagram of Escherichia coli and B is the bacteriostatic effect diagram of Staphylococcus aureus.

[0011] Figure 5 Effect diagrams of in vivo animal antibacterial experiments of the materials prepared in Example 1 and the recovery of animal infectious wounds after treatment with the materials.

[0012] The correspondence between the symbols and the materials in the above figures is as follows: BTO - barium titanate, GaIn - gallium indium alloy, BTO / GaIn - composite material formed by the combination of BTO and GaIn, S.aureus - Staphylococcus aureus, E.coli - Escherichia coli, P-BTO / GaIn - BTO / GaIn composite material modified on the surface of the PLGA membrane, US(+) - 1.5 W / cm 2 Ultrasound treatment, US(-) - no ultrasound treatment. Detailed Implementation Modes

[0013] The preparation method of the acoustically excited antibacterial fiber applied to the repair of wound-related bacterial infection wounds of the present invention will be further described below through specific embodiments, but the protection scope of the present invention is not limited to the following embodiments. Example 1

[0014] (1) Preparation of BTO: Using Ba(OH)2·H2O (barium hydroxide) and tetrabutyl titanate (Ti[O(CH2)3CH3]4) as starting materials based on the solvothermal method. Mix 17.018 g (50 mmol) of tetrabutyl titanate with 20 mL of ethanol (analytical pure), and then add 7 mL of ammonium hydroxide solution (25% NH3 in H2O solution) to the solution mixture. At the same time, dissolve 14.204 g (75 mmol) of Ba(OH)2·H2O in 25 mL of deionized water to prepare a barium hydroxide solution, and then add the barium hydroxide aqueous solution to the above solution mixture. Transfer the final mixed solution to a 100 mL polytetrafluoroethylene-lined stainless steel autoclave and perform hydrothermal treatment at 200 °C for 48 hours. After the reaction, wash the obtained product repeatedly with acetic acid and ethanol, and then dry it in an oven at 80 °C for 24 hours.

[0015] (2) Preparation of BTO / GaIn composite: Mix 17.018 g (50 mmol) of tetrabutyl titanate with 20 mL of ethanol (analytical pure), and then add 7 mL of ammonium hydroxide solution (25% NH3 in H2O solution) to the solution mixture. At the same time, dissolve 14.204 g (75 mmol) of Ba(OH)2·H2O in 25 mL of deionized water to prepare a barium hydroxide solution, then add GaIn (2.7 mmol) dispersed in an ethanol solution, and then add the barium hydroxide aqueous solution to the above solution mixture. Transfer the final mixed solution to a 100 mL polytetrafluoroethylene-lined stainless steel autoclave and perform hydrothermal treatment at 200 °C for 48 hours. After the reaction, wash the obtained product repeatedly with acetic acid and ethanol, and then dry it in an oven at 80 °C for 24 hours.

[0016] (3) Preparation of PLGA fibers: Dissolve 1 g of PLGA in 10 mL of hexafluoroisopropanol (HFIP) solvent and stir ultrasonically overnight to obtain a spinning solution. Then add 5 mL of the spinning solution to a spinning syringe for electrospinning, and finally let the sample dry naturally after spinning. The spinning uses a 24G needle, the rate is 1 mL / h, and the voltage is 15 kV.

[0017] (4) Preparation of P-BTO / GaIn fibers: Add 50 μL of BTO / GaIn solution (2 mg / mL) to the surface of a polydopamine (PDA)-modified PLGA membrane (1 cm × 1 cm) to obtain a BTO / GaIn-modified P-BTO / GaIn fabric (P-BTO / GaIn). Example 2

[0018] (1) Preparation of BTO: Using Ba(OH)2·H2O (barium hydroxide) and tetrabutyl titanate (Ti[O(CH2)3CH3]4) as starting materials based on the solvothermal method. Mix 34.036 g (100 mmol) of tetrabutyl titanate with 20 mL of ethanol (analytical grade), and then add 14 mL of ammonium hydroxide solution (25% NH3 in H2O solution) to the solution mixture. At the same time, dissolve 14.204 g (75 mmol) of Ba(OH)2·H2O in 25 mL of deionized water to prepare a barium hydroxide solution, and then add the barium hydroxide aqueous solution to the above solution mixture. Transfer the final mixed solution to a 100 mL stainless steel autoclave lined with polytetrafluoroethylene, and perform hydrothermal treatment at 200 °C for 24 hours. After the reaction, repeatedly wash the obtained product with acetic acid and ethanol, and then dry it in an oven at 80 °C for 12 hours.

[0019] (2) Preparation of BTO / GaIn composite: Mix 34.036 g (100 mmol) of tetrabutyl titanate with 20 mL of ethanol (analytical grade), and then add 14 mL of ammonium hydroxide solution (25% NH3 in H2O solution) to the solution mixture. At the same time, dissolve 14.204 g (75 mmol) of Ba(OH)2·H2O in 25 mL of deionized water to prepare a barium hydroxide solution, then add GaIn (2.7 mmol) dispersed in ethanol solution, and then add the barium hydroxide aqueous solution to the above solution mixture. Transfer the final mixed solution to a 100 mL stainless steel autoclave lined with polytetrafluoroethylene, and perform hydrothermal treatment at 200 °C for 24 hours. After the reaction, repeatedly wash the obtained product with acetic acid and ethanol, and then dry it in an oven at 80 °C for 12 hours.

[0020] (3) Preparation of PLGA fibers: Dissolve 1 g of PLGA in 10 mL of hexafluoroisopropanol (HFIP) solvent and stir ultrasonically overnight to obtain a spinning solution. Then add 5 mL of the spinning solution into a spinning syringe for electrospinning, and finally let the sample dry naturally after spinning. The spinning uses a 24G needle, with a rate of 1 mL / h and a voltage of 15 kV.

[0021] (4) Preparation of P-BTO / GaIn fibers: Add 50 μL of BTO / GaIn solution (2 mg / mL) to the surface of a polydopamine (PDA)-modified PLGA membrane (1 cm × 1 cm) to obtain a BTO / GaIn-modified P-BTO / GaIn fabric (P-BTO / GaIn). Example 3

[0022] (1) Preparation of BTO: Using Ba(OH)2·H2O (barium hydroxide) and tetrabutyl titanate (Ti[O(CH2)3CH3]4) as starting materials based on the solvothermal method. Mix 34.036 g (100 mmol) of tetrabutyl titanate with 20 mL of ethanol (analytical pure), and then add 14 mL of ammonium hydroxide solution (25% NH3 in H2O solution) to the solution mixture. Meanwhile, dissolve 28.408 g (150 mmol) of Ba(OH)2·H2O in 25 mL of deionized water to prepare a barium hydroxide solution, and then add the barium hydroxide aqueous solution to the above solution mixture. Transfer the final mixed solution to a 100 mL Teflon-lined stainless steel autoclave and perform hydrothermal treatment at 200 °C for 60 hours. After the reaction, wash the obtained product repeatedly with acetic acid and ethanol, and then dry it in an oven at 80 °C for 12 hours.

[0023] (2) Preparation of BTO / GaIn composite: Mix 34.036 g (100 mmol) of tetrabutyl titanate with 20 mL of ethanol (analytical pure), and then add 14 mL of ammonium hydroxide solution (25% NH3 in H2O solution) to the solution mixture. Meanwhile, dissolve 28.408 g (150 mmol) of Ba(OH)2·H2O in 25 mL of deionized water to prepare a barium hydroxide solution, and then add GaIn (5.4 mmol) dispersed in an ethanol solution. Subsequently, add the barium hydroxide aqueous solution to the above solution mixture. Transfer the final mixed solution to a 100 mL Teflon-lined stainless steel autoclave and perform hydrothermal treatment at 200 °C for 48 hours. After the reaction, wash the obtained product repeatedly with acetic acid and ethanol, and then dry it in an oven at 80 °C for 24 hours.

[0024] (3) Preparation of PLGA fibers: Dissolve 1 g of PLGA in 10 mL of hexafluoroisopropanol (HFIP) solvent and stir ultrasonically overnight to obtain a spinning solution. Then add 5 mL of the spinning solution into a spinning syringe for electrospinning. Finally, let the sample dry naturally after spinning. The electrospinning uses a 24G needle, with a rate of 1 mL / h and a voltage of 15 kV.

[0025] (4) Preparation of P-BTO / GaIn fibers: Add 50 μL of BTO / GaIn solution (4 mg / mL) onto the surface of a poly(dopamine) (PDA)-modified PLGA membrane (1 cm × 1 cm) to obtain a BTO / GaIn-modified P-BTO / GaIn fabric (P-BTO / GaIn).

[0026] Reactive oxygen species release experiment

[0027] Experiment 1: Different groups of samples prepared in Example 1 were placed in a 48-well plate. 4,5,5-Dimethyl-1-pyrroline-N-oxide was used as a scavenger for reactive oxygen superoxide radical (·O 2- ). Each group was placed under ultrasonic stimulation (1.5 W / cm 2 ) for 10 minutes. The generation of reactive oxygen species was recorded using an electron spin resonance instrument every 5 minutes. The results are shown in Figure 2 A.

[0028] Experiment 2: Different groups of samples prepared in Example 1 were placed in a 48-well plate. 5,5-Dimethyl-1-pyrroline-N-oxide (DMPO) was used as a scavenger for reactive oxygen hydroxyl radical (·OH). Each group was placed under ultrasonic stimulation (1.5 W / cm 2 ) for 10 minutes. The generation of reactive oxygen species was recorded using an electron spin resonance instrument every 5 minutes. The results are shown in Figure 2 B.

[0029] Experiment 3: Different groups of samples prepared in Example 1 were placed in a 48-well plate. 200 μL of methylene blue solution (MB, 100 mg / L) was added. After ultrasonic treatment at different powers (0, 0.5, 1, 2 W / cm 2 ) for 10 min, the absorption spectrum of MB at 450 to 750 nm was detected. The results are shown in Figure 2 C.

[0030] Experimental results: As can be seen from Figure 2 , BTO / GaIn is more conducive to the generation of reactive oxygen species under ultrasonic excitation conditions. As the ultrasonic power increases, more ROS are generated, showing the potential for antibacterial activity.

[0031] Antibacterial experiment

[0032] Experiment 1: The materials prepared in Example 1 (P-BTO / GaIn; 1 cm × 1 cm) were placed in a 48-well plate. PBS was used as a control group. 100 μL of liquid medium and 100 μL of 10 6 CFU / mL Escherichia coli were added to each sample well. Three parallel experimental groups were set up. After culturing each group of samples under ultrasonic stimulation (1.5 W / cm 2 ) and non-ultrasonic conditions for 10 minutes, 50 μL of the bacterial solution was evenly coated on the solid medium. After culturing at 37 °C for 24 hours, the bactericidal effects of various materials were observed. The experimental results are shown in Figure 4 A.

[0033] Experiment 2: Escherichia coli was replaced with Staphylococcus aureus. According to the same method above, the antibacterial effects of each material on Staphylococcus aureus were obtained, as shown in Figure 4 B.

[0034] Experimental results: As can be seen from Figure 4 , P-BTO / GaIn can effectively generate reactive oxygen species under ultrasonic excitation to inhibit the growth of bacteria, while PLGA has limited inhibitory effect on the growth of bacteria. Therefore, P-BTO / GaIn has good antibacterial effect under ultrasonic excitation.

[0035] Evaluation experiment of biosafety (cytotoxicity)

[0036] L929 mouse fibroblasts were cultured with DMEM containing 10% fetal bovine serum. After the cells adhered and grew, the fresh medium was changed. When the cell aggregation reached 80%, the cells were seeded on a 48-well plate at a density of 10 4 / well and cultured for 24 h. Then the cells were seeded into the 48-well plate containing the material prepared in Example 1 (P-BTO / GaIn; 1 cm × 1 cm), with PLGA as the control group. The state of the cells on the membrane was observed using a scanning electron microscope (SEM), and the results are shown in Figure 3 .

[0037] Experimental results: There was no significant difference in cell morphology between P-BTO / GaIn and the PLGA group, demonstrating its excellent biocompatibility.

[0038] In vivo experimental analysis:

[0039] Experiment 1: Establishment of implant-related bacterial infection model

[0040] After depilation of the back of the mouse, it was anesthetized by intraperitoneal injection with sodium pentobarbital solution (50 mg / kg). Then a full-thickness skin wound with a diameter of 1 cm was created 2 cm outside the spine using surgical scissors, etc. (cut to the superficial fascia only), and then 10 μL of Staphylococcus aureus bacterial solution (1 × 10 8 CFU / mL) was dropped, and finally a band-aid without special ingredients was applied or wrapped with a sterile gauze to prevent the mouse from licking and scratching the wound.

[0041] Experiment 2: Wound treatment analysis

[0042] The wound was photographed 24 hours after the establishment of the infection model, and the length and width of the wound were measured with a ruler. Materials were placed at the wound according to the group, and all the ultrasonic groups received ultrasonic treatment with an intensity of 1.5 W / cm 2 for 10 min. On the second day after the ultrasonic treatment, the exudate on the surface of the mouse wound was collected with a cotton swab moistened with physiological saline and placed in 1 mL of physiological saline for spread plate antibacterial test, and the results are shown in Figure 5 .

[0043] Experimental results: For the single PLGA group, after 7 days of treatment, the wound still showed obvious scabs, and there were a large number of bacteria around the wound surface; while in the P-BTO / GaIn group, the wound recovered well after ultrasound treatment, and the bacteria were basically killed.

[0044] In summary, the P-BTO / GaIn sonodynamic antibacterial film has excellent bactericidal effects under ultrasound stimulation, and promotes the regeneration of the wound surface during the repair process. The present invention solves the problem that dressings in clinical skin repair lack the ability to resist microbial infection.

[0045] The above are only some embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A preparation method of an ultrasound-excited antibacterial fiber wound repair material, characterized in that, The method includes the following steps: S10. Prepare BTO / GaIn: Mix 17.018 g of tetrabutyl titanate with 20 mL of ethanol, and then add 7 mL of ammonium hydroxide solution to the solution mixture. The ammonium hydroxide solution is specifically a 25% NH₃ in H₂O solution; dissolve 14.204 g of Ba(OH)₂·H₂O in 25 mL of deionized water to prepare a barium hydroxide solution, and then add GaIn dispersed in an ethanol solution. The amount of GaIn is 2.7 mmol. Subsequently, add the aqueous barium hydroxide solution to the above solution mixture; transfer the final mixed solution to a 100 mL stainless-steel autoclave lined with polytetrafluoroethylene, and perform hydrothermal treatment at 200 °C for 48 hours; after the reaction, wash the obtained product repeatedly with acetic acid and ethanol, and then dry it in an oven at 80 °C for 24 hours; S20. Prepare PLGA: Dissolve 1 g of PLGA in 10 mL of hexafluoroisopropanol solvent and stir ultrasonically overnight to obtain a spinning solution. Then add 5 mL of the spinning solution to a spinning syringe for electrospinning. Finally, let the sample dry naturally after spinning to obtain PLGA; use a 24 G needle for spinning, with a rate of 1 mL / h and a voltage of 15 kV; S30. Prepare P-BTO / GaIn: Add 50 μL of a 2 mg / mL BTO / GaIn solution to the surface of a 1 cm × 1 cm PLGA membrane modified with polydopamine to obtain P-BTO / GaIn modified with a BTO / GaIn composite material.

2. The ultrasonic excitation antibacterial fiber wound repair material prepared by the preparation method according to claim 1.

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