Method for synergistically promoting wound healing by utilizing medium-intensity pulse ultrasonic waves and zinc oxide nanoparticle-type I collagen hydrogel

Abstract

A method for synergistically promoting wound healing by means of moderate-intensity pulse ultrasonic waves and zinc oxide nanoparticle-type-I collagen hydrogel comprises the following steps that zinc oxide nanoparticles and type-I collagen hydrogel are physically mixed and crosslinked in advance and applied to a chronic infected wound, and then the wound is accelerated to heal through multiple times of MIPUS stimulation. The effect of the ZNP-COL hydrogel is enhanced through MIPUS, immune cell polarization can be activated, and the proportion of anti-inflammatory M2 type macrophages is increased; wherein ZNP is capable of releasing zinc ions and reactive oxygen substances and has antibacterial activity; meanwhile, ZNP can generate current and an electric field under stimulation of MIPUS, calcium ions on the surface of a cell membrane are activated to flow inwards, and proliferation of anti-inflammatory M2 type macrophages is promoted; the COL hydrogel is used as a medium to transmit ultrasonic waves and fix the ZNP, and the function of the ZNP at the local position is enhanced. The method solves the problems of bacterial infection and chronic inflammation at the same time, is safe, simple and reusable, and can be widely applied to infection of other parts besides the application.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a method for activating an immune response and promoting the healing of chronically infected wounds using medium-intensity pulsed ultrasound (MIPUS) through hydrogels and nanoparticles as media. Background Technology

[0002] Chronic wounds, especially those associated with diabetes, pose significant challenges to clinical care due to delayed healing and a high risk of infection. Traditional wound care methods, including non-hydrogel and hydrogel dressings, often lack sufficient antibacterial properties and fail to actively promote tissue regeneration.

[0003] Zinc oxide nanoparticles (ZNPs) possess antibacterial and anti-inflammatory potential, but their direct application is limited by poor dispersibility and bioavailability. Although ZNPs are used for their antibacterial properties, their cytotoxicity at high concentrations and ineffective delivery to wound sites limit their application.

[0004] Type I collagen (COL) hydrogel is a commonly used biodegradable biocompatible scaffold. Although it has bioactivity that is beneficial to cell adhesion and proliferation, it lacks antibacterial function and cannot effectively promote the repair of infected wounds.

[0005] Ultrasound therapy has a wide range of applications in the biomedical field, and has been explored for enhancing drug delivery and tissue repair, but its efficacy in wound healing remains limited. The efficacy and application of ultrasound are closely related to its parameters; for example, based on intensity, it can be divided into low intensity (0.03-0.1 W / cm²). 2 Medium strength (0.6-2.0 W / cm) 2 ) and high strength (10-100 W / cm) 2 There are three ranges; the mode of action can be continuous or pulsed; the action time is divided into high duty cycle (80-100%) or low duty cycle (0.5-20%). Low duty cycle does not generate high temperature and is safer for biological tissues. Therefore, selecting appropriate ultrasound parameters is crucial for wound repair.

[0006] In summary, although various biomaterials have been attempted for wound repair, their simple properties have limited their effectiveness in promoting healing. Existing composite wound dressings, such as ZNP encapsulated in type I collagen hydrogel (COL), still suffer from problems including lack of antibacterial activity, insufficient tissue repair capacity, poor bioavailability, high cytotoxicity of nanoparticle additives at high concentrations, and poor diffusion at the wound site. To address these issues, this invention discloses a method for synergistically promoting wound healing using medium-intensity pulsed ultrasound (MIPUS) and zinc oxide nanoparticle-type I collagen hydrogel. This method utilizes MIPUS to enhance the antibacterial and anti-inflammatory functions of the ZNP-COL hydrogel wound dressing and elucidates the underlying biological mechanisms, ultimately promoting the healing of chronically infected wounds. Summary of the Invention

[0007] The purpose of this invention is to provide a method for rapid healing of diabetic infected wounds. This method overcomes the limitations of traditional wound treatment methods by applying a type I collagen (COL) hydrogel encapsulating ZNPs and then utilizing repeated MIPUS stimulation. While both ultrasound therapy and ZNP-COL hydrogel have limited effectiveness in promoting wound healing, their combination introduces complementary biochemical and biophysical modalities. ZNPs provide strong antibacterial and anti-inflammatory effects, while the COL hydrogel, as a biocompatible carrier matrix, positions the nanoparticles and transmits ultrasound waves, promoting the retention of the entire material at the wound site. Furthermore, repeated application of MIPUS enhances the interaction between nanoparticles and immune cells, promoting cell migration and modulating inflammation. This comprehensive approach effectively accelerates the healing of infected diabetic wounds by addressing the issues of microbial infection and inflamed tissue regeneration.

[0008] In a first aspect, a method is provided for synergistically promoting wound healing using medium-intensity pulsed ultrasound and zinc oxide nanoparticle (ZNP)-type collagen water (COL) gel, the method comprising the following steps: Applying ZNP-COL hydrogel to chronically infected wounds accelerates healing through MIPUS stimulation.

[0009] Based on the above technical solution, the preparation method of the ZNP-COL hydrogel includes the following steps: The ZNP solution and COL solution were mixed and incubated to form a ZNP-COL hydrogel.

[0010] Based on the above technical solution, the concentration of the COL solution is 1-3 mg / mL, preferably 2 mg / mL; The average diameter of ZNPs in the ZNP solution is 50-200 nm, preferably 100 nm; The concentration of ZNP in the ZNP and COL mixed solution is 80–150 µg / mL, preferably 125 µg / mL.

[0011] Based on the above technical solution, the incubation conditions are as follows: The incubation temperature is 32-37℃, preferably 37℃; The incubation time is 0.5-2 hours, preferably 1 hour.

[0012] Based on the above technical solution, the conditions for MIPUS stimulation are as follows: The strength of the MIPUS is 0.6-2.0 W / cm². 2 The preferred value is 1.2 W / cm. 2 ; The frequency of the MIPUS is 0.5-3 MHz, preferably 1 MHz; The duty cycle of the MIPUS is 1-25%, preferably 20%; The MIPUS time is 7-15 minutes, preferably 10 minutes.

[0013] Based on the above technical solution, the chronically infected wound is a wound model of type 1 diabetes mellitus complicated with bacterial infection, and the bacteria are at least one of Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Proteus mirabilis, and Klebsiella pneumoniae, preferably Escherichia coli.

[0014] Based on the above technical solution, ZNP-COL hydrogel is applied to the wound multiple times, and MIPUS stimulation is applied 1-6 times per week, preferably 3 times per week.

[0015] Based on the above technical solution, the ratio of M2 / M1 macrophages around the wound that accelerates healing is increased to 1.0-1.91 times, preferably 1.39 times; The wound area repair rate of the accelerated healing is increased by 5%-15%, preferably 8.7%; the wound area of ​​the accelerated healing is reduced by 10%-40% compared with the wound area of ​​ZNP-COL hydrogel alone, preferably 21.3%.

[0016] Based on the above technical solution, the surface temperature of ZNP-COL hydrogel at the wound site during MIPUS stimulation is between 30-37℃, preferably 34.3℃.

[0017] Secondly, a method for promoting wound healing by synergistically combining medium-intensity pulsed ultrasound with zinc oxide nanoparticles-type collagen hydrogel is provided for the application of this method in the treatment of common wound infections, skin and soft tissue infections, respiratory tract infections, eye infections, digestive tract infections (oral and intestinal), urinary tract infections, reproductive tract infections, surgical site infections, and peri-implantation infections.

[0018] In the in vitro temperature test, the MIPUS strength was 1.8 W / cm². 2 Temperature measurements were performed on 500 µL of medium containing ZNP (15 µg / mL) and medium without ZNP under a 50% duty cycle treatment; the MIPUS intensity was 1.2 W / cm². 2 Temperature tests were conducted on 500 µL of medium containing ZNP (15 µg / mL) and medium without ZNP at a duty cycle of 50%.

[0019] In the in vitro antibacterial test, *E. coli* culture dishes were prepared in advance. Experimental group: Medium-intensity pulsed ultrasound stimulation (MIPUS: 0.6-2.0 W / cm²) was used. 2 Zinc oxide nanoparticles (ZNP: 250-500 µg / mL) were used to treat E. coli with different concentrations of ZNP nanoparticles and different parameters of MIPUS. The control group differed from the experimental group in that it did not use MIPUS stimulation.

[0020] Beneficial effects (1) The technical solution disclosed in this invention activates ZNP-COL hydrogel through MIPUS, enhances the antibacterial activity of ZNP through a non-thermal mechanism, and increases the proportion of anti-inflammatory M2 phenotype macrophages. At the same time, it solves the pathological infection of chronic wounds and reduces the symptoms of inflammation, creating a more favorable environment for wound healing. This dual-action technology surpasses traditional methods.

[0021] (2) The technical solution disclosed in this invention realizes the controllable distribution of biological information transmission. Collagen makes ZNPs evenly distributed in the three-dimensional space of the entire wound, overcoming the problems of poor dispersion and cytotoxicity caused by direct use of ZNPs. At the same time, collagen, as a medium for ultrasound, effectively activates the piezoelectric properties of ZNPs, increases the proportion of repair macrophages, and thus accelerates wound healing.

[0022] (3) The technical solution disclosed in this invention is a non-invasive method, namely, using external MIPUS to activate the action of biomaterials to accelerate wound healing, which has the potential for application in areas such as bruises and abrasions.

[0023] (4) The technical method disclosed in this invention can be used for a long time and at home. That is, ZNP-COL hydrogel is applied to the wound every day and external MIPUS is used to accelerate wound healing. It can be used for wounds and sites with various bacterial infections, including but not limited to, common wound infections, skin and soft tissue infections, respiratory tract infections, eye infections, digestive tract infections (oral cavity, intestines), urinary tract infections, reproductive tract infections, surgical site infections and peri-implant infections, etc., and has clinical practical effects. Attached Figure Description

[0024] Figure 1 Flowchart of the preparation and animal experiment of the ZNP-COL hydrogel described in Example 1 of this invention; Figure 2 SEM images of the ZNP-COL hydrogel prepared in Example 1 of this invention and the control group (left: 1000x magnification, COL hydrogel; middle: 1000x magnification, ZNP-COL hydrogel; right: 20000x magnification, ZNP-COL hydrogel). Figure 3 The viscoelastic properties and frequency-dependent viscosity of the ZNP-COL hydrogel prepared in Example 1 of this invention are complex compared with those of the control group. Figure 4 A schematic diagram of the rat wound treatment process described in Example 1 of this invention; Figure 5 The wound healing status and area closure rate of rats in Example 1 of this invention (**, p<0.01, Student's t statistical analysis); Figure 6 The present invention applies the tissue sections and staining analysis of rats one week after wound repair in Example 1 (a: macrophage immunofluorescence staining, b: masson staining, c: H&E staining); Figure 7 Immunofluorescence staining and M2 / M1 macrophage ratio analysis of tissue sections from rats one week after wound repair in Example 1 of this invention (*, p<0.05, Student's t statistical analysis); Figure 8 The present invention applies the MIPUS treatment in Example 1 to monitor temperature changes of ZNP-COL hydrogel in animal wound areas; Figure 9 The application example 2 of this invention monitors the temperature changes of cell culture medium (alpha-MEM) with different MIPUS treatment parameters and whether or not ZNP is present; Figure 10 The in vitro antibacterial test and result analysis in the test examples of this invention. Detailed Implementation

[0025] The present invention will be further described below with reference to specific embodiments, but this does not limit the present invention in any way.

[0026] Unless otherwise specified, the instruments, reagents, and materials used in the following embodiments are all conventional instruments, reagents, and materials already available in the prior art and can be obtained through legitimate commercial channels. Unless otherwise specified, the experimental methods and detection methods used in the following embodiments are all conventional experimental methods and detection methods already available in the prior art.

[0027] The composition of the 1X phosphate-buffered saline (PBS) in the examples is as follows: 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4, purchased from Servicebio. The composition of the PBS in the 10X example is as follows: 1.37 M NaCl, 27 mM KCl, 100 mM Na2HPO4, and 20 mM KH2PO4 were purchased from Servicebio.

[0028] Example 1 Figure 1 Preparation of ZNP-COL hydrogel: 1 mg ZNP (100 nm) was dissolved in 1 mL of 1X phosphate buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4) to prepare a ZNP stock solution with a concentration of 1 mg / mL. A ZNP-collagen mixture with a final concentration of 125 μg / mL ZNP and 2.0 mg / mL collagen was prepared as follows: The collagen stock solution was stored on ice. 478 μL of commercially available type I collagen stock solution (3.17 mg / mL, Corning) was added to a 1.5 mL centrifuge tube. Then, 125 μL of the 1 mg / mL ZNP stock solution was added, followed by 100 μL of 10X PBS solution. Finally, 294.5 µL of 1X PBS solution was added until the total solution volume reached 1 mL. Add 2.5 µL of 1 mol / L sodium hydroxide solution to mL of the solution, adjust the pH of the solution to 7.5, and shake up and down until the solution is mixed to obtain a ZNP-COL mixed solution; incubate the ZNP-collagen mixed solution at 37℃ for 1 h to obtain ZNP-COL hydrogel-1.

[0029] The SEM characterization results of the ZNP-COL hydrogel-1 are as follows: Figure 2 As shown (middle: 1000x, ZNP-collagen hydrogel; right: 20000x, ZNP-collagen hydrogel), its viscoelastic properties and frequency dependence are complex, and its viscosity is as follows: Figure 3 As shown in the figure (left figure, showing storage modulus G' and loss modulus G"; right figure: frequency-dependent complex viscosity), the results confirm the successful preparation of ZNP-collagen hydrogel and its excellent mechanical properties.

[0030] Example 2 The difference from Example 1 is that the amount of ZNP added is different. Specifically, 100 μL of ZNP stock solution with a concentration of 1 mg / mL is added and mixed with collagen stock solution. The rest of the process is the same as in Example 1 to obtain ZNP-COL hydrogel-2.

[0031] Example 3 The difference from Example 1 is that the amount of ZNP added is different. Specifically, 150 μL of ZNP stock solution with a concentration of 1 mg / mL is added and mixed with collagen stock solution. The rest of the process is the same as in Example 1 to obtain ZNP-COL hydrogel-3.

[0032] Example 4 The difference from Example 1 is that ZNP was not added; the rest of the process remained the same as in Example 1, resulting in a collagen hydrogel. The SEM image of the collagen hydrogel is shown below. Figure 2 As shown (left side: 1000 times); its viscoelastic properties and frequency dependence are complex, such as viscosity. Figure 3 As shown in the figure (left figure, showing storage modulus G' and loss modulus G"; right figure: frequency-dependent complex viscosity), the results indicate that the mechanical properties of collagen hydrogels are partially improved after the addition of ZNP.

[0033] Application Example 1 Control group: Female rats were anesthetized with 2.5% isoflurane (rat rats were intraperitoneally injected with streptozotocin, STZ, 55 mg / kg and maintained for 1-1.5 months to induce type 1 diabetes in 6-8 weeks of Sprague Dawley rats, and blood glucose levels were continuously monitored). The fur on the backs of the rats was shaved and disinfected with 70% ethanol. Two full-thickness circular incisions with a diameter of 1.5 cm were made on the backs using surgical scissors. Each incision was treated with 100 µL of 10% isoflurane. 5 / mL E. coli suspension infection. 2 mL of ZNP-COL hydrogel prepared in Example 1 was applied to the wound area, the hydrogel was fixed with an adhesive bandage, and the torso was wrapped with a bandage.

[0034] Experimental group: The difference from the control group was that ultrasound equipment (Sonovitro and LIPUSTIM, SXUltrasonic) was used for assisted treatment. The relevant parameters of the ultrasound equipment were as follows: frequency 1 MHz, intensity 1.2 W / cm². 2The duty cycle was 20%, and the duration was 10 minutes per wound. Ultrasound treatment was performed every few days, specifically 10 minutes per wound on day 0, day 2, and day 5. The contact medium was the ZNP-collagen hydrogel prepared in Example 1. During the ultrasound treatment, the rats were anesthetized with 2.5% isoflurane. The specific wound treatment process is as follows: Figure 4 As shown.

[0035] The absorption of the ZNP-COL hydrogel was observed. On the third day, most of the hydrogel was absorbed or degraded. 2 mL of the ZNP-COL hydrogel prepared in Example 1 was replaced for each wound. The wound recovery of the experimental and control groups was as follows: Figure 5 As shown, the results indicate that, compared to the control group, the wound area in the experimental group decreased from 1.5 cm² on day 7. 2 Reduced to 0.48 cm 2 Compared to the control group, the size was 1.5 cm. 2 Reduced to 0.61 cm 2 It accelerated the healing rate by approximately 8.7% and reduced the area by approximately 21.3%, which was statistically significant (n = 6 per group, **, p < 0.01).

[0036] The inflammatory status of wound tissue sections from rats in the control and experimental groups was analyzed. Immunofluorescence staining of wound tissue sections was performed using Solarbio's Masson trichrome staining kit and HE staining kit, with antibodies against F4 / 80 (pan-macrophages), CD86 (M1 marker), and CD206 (M2 marker). The test results are as follows: Figure 6-7 As shown, the results indicated that the mean fluorescence intensity measurement showed that more macrophages in the experimental group were converted to the M2 anti-inflammatory phenotype, i.e., experimental group (M1: 1.18, M2: 1.60) and control group (M1: 1.44, M2: 1.32).

[0037] The effect of ultrasonic treatment on the temperature of ZNP-COL hydrogel dressing at the wound site; results are as follows Figure 8 As shown, the surface temperature of ZNP-COL hydrogel was approximately 25°C before ultrasonic treatment, and remained below 35°C after ultrasonic treatment, confirming that this method is a non-thermal healing method.

[0038] Application Example 2 The effect of ultrasonic treatment on liquid temperature, the results are as follows: Figure 9 As shown, this indicates that the MIPUS strength is 1.8 W / cm². 2Compared to treatment with a 50% duty cycle, the temperature rise rate was faster in 500 µL of medium containing ZNP (15 µg / mL); at a MIPUS intensity of 1.2 W / cm², the temperature rise rate was also faster. 2 Compared with the 50% duty cycle treatment, the rate of temperature rise in 500 µL of medium containing ZNP (15 µg / mL) did not change.

[0039] Test case In vitro antibacterial test (effect of ultrasound intensity on zinc oxide nanoparticles): E. coli culture dishes were prepared beforehand. Experimental group: Medium-intensity pulsed ultrasound stimulation (MIPUS: 0.6-2.0 W / cm²) was used. 2 The combined treatment of Escherichia coli with zinc oxide nanoparticles (ZNP: 250-500µg / mL) was determined by ANOVA analysis and Student's t statistical analysis (*p<0.05, **p<0.01, ***p<0.001).

[0040] Control group: Unlike the experimental group, the control group did not use MIPUS stimulation, but only used different concentrations of ZNP (250-500µg / mL) to treat Escherichia coli.

[0041] The processing results under different conditions are as follows: Figure 10 As shown, the combined treatment with MIPUS stimulation and ZNP has a stronger bactericidal effect than either method alone. The conditions and results of the in vitro antibacterial experiments are summarized in Table 2.

[0042] Table 1 summarizes the wound treatment conditions and results one week later.

[0043] Table 2 summarizes the in vitro antibacterial test conditions and results.

[0044] The foregoing descriptions and embodiments are provided to enable those skilled in the art to understand and apply the present invention. It will be apparent to those skilled in the art that various modifications can be easily made to these contents, and the general principles described herein can be applied to other embodiments without creative effort. Therefore, the present invention is not limited to the foregoing descriptions and embodiments. Improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from its scope should be within the protection scope of the present invention.

Claims

1. A method for synergistically promoting wound healing using medium-intensity pulsed ultrasound and zinc oxide nanoparticle-type collagen hydrogel, characterized in that, The method includes the following steps: Applying ZNP-COL hydrogel to chronically infected wounds accelerates healing through MIPUS stimulation.

2. The method according to claim 1, characterized in that, The preparation method of the ZNP-COL hydrogel includes the following steps: The ZNP solution and COL solution were mixed and incubated to form a ZNP-COL hydrogel.

3. The method according to claim 2, characterized in that, The concentration of the COL solution is 1–3 mg / mL, preferably 2 mg / mL; The average diameter of ZNPs in the ZNP solution is 50-200 nm, preferably 100 nm; The concentration of ZNP in the ZNP and COL mixed solution is 80–150 µg / mL, preferably 125 µg / mL.

4. The method according to claim 2, characterized in that, The incubation conditions are as follows: The incubation temperature is 32-37℃, preferably 37℃; The incubation time is 0.5-2 hours, preferably 1 hour.

5. The method according to claim 1, characterized in that, The conditions for the MIPUS stimulation are as follows: The strength of the MIPUS is 0.6-2.0 W / cm². 2 The preferred value is 1.2 W / cm. 2 ; The frequency of the MIPUS is 0.5-3 MHz, preferably 1 MHz; The duty cycle of the MIPUS is 1-25%, preferably 20%; The MIPUS time is 7-15 minutes, preferably 10 minutes.

6. The method according to claim 1, characterized in that, The chronically infected wound is a wound model of type 1 diabetes mellitus complicated with bacterial infection. The bacteria are at least one of Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Proteus mirabilis, and Klebsiella pneumoniae, preferably Escherichia coli.

7. The method according to claim 1, characterized in that, Apply ZNP-COL hydrogel to the wound multiple times and apply MIPUS stimulation 1-6 times per week, with the conditions for MIPUS stimulation being the same as in claim 5, preferably 3 times per week.

8. The method according to claim 1, characterized in that, Around the wound where healing is accelerated, the ratio of M2 / M1 macrophages increases to 1.0-1.91 times, preferably 1.39 times; The wound area repair rate of the accelerated healing is increased by 5%-15%, preferably 8.7%; the wound area of ​​the accelerated healing is reduced by 10%-40% compared with the wound area of ​​ZNP-COL hydrogel alone, preferably 21.3%.

9. The method according to claim 1, characterized in that, When stimulated by MIPUS, the surface temperature of ZNP-COL hydrogel at the wound site is between 30-37°C, preferably 34.3°C.

10. The use of any one of the methods of claims 1-9 in the treatment of common wound infections, skin and soft tissue infections, respiratory tract infections, eye infections, digestive tract infections (oral cavity, intestines), urinary tract infections, reproductive tract infections, surgical site infections, and peri-implantation infections.

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