Antibacterial hydrogel wound dressing with photothermal effect and preparation method and application thereof

Abstract

The invention provides an antibacterial hydrogel wound dressing with a photothermal effect as well as a preparation method and application of the antibacterial hydrogel wound dressing, and aims to solve the problems that the traditional wound dressing is insufficient in antibacterial ability and limited in inhibition of drug-resistant bacteria, and the hydrogel prepared by the invention has good functions of resisting bacteria, drug-resistant bacteria, inflammation and oxidation and promoting wound healing. The preparation method comprises the following steps: synthesizing the silver-polydopamine nanoparticles, preparing an extracellular matrix hydrogel prepolymer, and combining the silver-polydopamine nanoparticles with the polymyxin B to form the final hydrogel. The hydrogel disclosed by the invention can realize sterilization treatment through a photothermal effect under the irradiation of near-infrared light, has relatively good biocompatibility, temperature-sensitive characteristic and wound healing promotion effect, and is suitable for treating a wound infected by drug-resistant pseudomonas aeruginosa. Experimental results show that the hydrogel disclosed by the invention has good mechanical properties, degradability and adhesion, and shows remarkable antibacterial and healing-promoting effects in vitro and in mouse models.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials, and more specifically, to an antibacterial hydrogel wound dressing with photothermal effect, its preparation method, and its application. Background Technology

[0002] Chronic wounds, characterized by slow healing due to pathogen colonization, have become a major global public health challenge and place a heavy burden on healthcare systems. Once the skin barrier is lost, wounds are highly susceptible to bacterial colonization, biofilm formation, and persistent infection. Among these, *Pseudomonas aeruginosa* (…) is a particularly prevalent pathogen. Pseudomonas aeruginosa Polymyxin B (PMBB) is a common and difficult-to-treat pathogen, with an infection rate of approximately 21% in Asia. This bacterium not only exhibits strong intrinsic drug resistance and biofilm formation capabilities, but also demonstrates high resistance to a variety of antibiotics clinically, including last-line drugs such as polymyxin B. This situation severely hinders the healing of infected wounds, urgently requiring new treatment strategies.

[0003] Traditional wound dressings such as gauze and bandages only serve as a barrier, lacking antibacterial and tissue repair functions. Existing treatments include systemic antibiotics, collagen dressings, epidermal growth factor, and stem cell therapy, but these are mostly single-function and difficult to combat drug-resistant bacterial infections. In recent years, novel antibacterial methods such as chemokinetic therapy (CDT), photodynamic therapy (PDT), sonodynamic therapy (SDT), and photothermal therapy (PTT) have attracted attention. Among them, PTT has advantages such as minimal invasiveness, high spatiotemporal controllability, and low likelihood of inducing drug resistance, and is considered to have potential in the treatment of infected wounds.

[0004] To achieve multifunctional treatment, hydrogels, due to their high water content, porous three-dimensional structure, and good biocompatibility, have been widely explored as therapeutic platforms. By controlling the hydrogel matrix and loading functional components, they can be endowed with comprehensive properties such as antibacterial, anti-inflammatory, hemostatic, and sustained drug release. However, existing hydrogels generally suffer from insufficient mechanical properties and limited inhibition of drug-resistant bacteria, restricting their clinical application. Therefore, there is an urgent need to develop a novel multifunctional hydrogel that combines mechanical strength, good biocompatibility, and highly effective anti-drug-resistant bacteria capabilities to address the treatment challenges of drug-resistant Pseudomonas aeruginosa-infected wounds and promote tissue regeneration and wound repair. Summary of the Invention

[0005] Based on the above problems, the present invention provides an antibacterial hydrogel wound dressing with photothermal effect, its preparation method and application, which has good antibacterial properties, especially against drug-resistant bacteria, while also being safe and accelerating wound healing.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides an antibacterial hydrogel wound dressing with photothermal effect, comprising the following raw materials: 2% by mass of extracellular matrix, 0.025% by mass of polymyxin B, 0.02% by mass of silver-polydopamine nanoparticles, and the balance being water.

[0007] Secondly, the present invention provides a method for preparing an antibacterial hydrogel wound dressing with photothermal effect, comprising the following steps: S1. Preparation of silver-polydopamine nanoparticles: Dopamine hydrochloride powder was dissolved in ultrapure water. The dopamine hydrochloride aqueous solution was mixed with sodium hydroxide aqueous solution and stirred at 50°C and 800 rpm for 5 hours. After centrifugation at 4000 rpm for 25 minutes, the nanoparticles were washed three times with ultrapure water to obtain polydopamine nanoparticles. The polydopamine nanoparticles were dispersed in ultrapure water to obtain a polydopamine nanoparticle dispersion with a mass concentration of 2 g / mL. The dispersion was mixed with silver ammonia solution and stirred at room temperature in the dark for 1 hour. After centrifugation at 4000 rpm for 25 minutes, the nanoparticles were washed three times with ultrapure water to obtain silver-polydopamine nanoparticles. S2. Preparation of extracellular matrix hydrogel prepolymer solution: Pork heart with connective tissue and adipose tissue removed was chopped into myocardial fragments with a volume not exceeding 2 mm3. The fragments were soaked in decellularization solution for 7 days, with the decellularization solution being changed every 24 hours to obtain decellularized myocardial fragments. After washing with ultrapure water 3 times, the fragments were freeze-dried for 24 hours. The freeze-dried myocardial fragments were added to the degradation solution at a ratio of 40 mg: 1 mL, and the reaction was stirred at 800 rpm for 56 hours. The pH of the reaction system was adjusted to 7.4 by adding 1 mol / L sodium hydroxide aqueous solution dropwise to obtain an extracellular matrix hydrogel prepolymer with a mass concentration of 4%. S3. Silver-polydopamine nanoparticles were dispersed in ultrapure water. The silver-polydopamine dispersion was mixed with 4% extracellular matrix hydrogel prepolymer, and then polymyxin B raw material was added to it. The mixture was stirred and mixed evenly, and heated at 37°C for 5 min to prepare PmB / Ag-PDA@ECM hydrogel.

[0008] Preferably, in step S1, the mass concentration of the dopamine hydrochloride aqueous solution is 2 mg / mL, the molar concentration of the sodium hydroxide aqueous solution is 1 mol / L, the mass concentration of the silver ammonia solution is 1.05 mg / mL, and the volume ratio of the polydopamine nanoparticle dispersion to the silver ammonia solution is 2 mL:1 mL; the volume of the sodium hydroxide aqueous solution added is 0.84% ​​of the volume of the dopamine hydrochloride aqueous solution.

[0009] Preferably, the decellularization solution in step S2 is a 1xPBS solution containing sodium dodecyl sulfate and penicillin / streptomycin, wherein the mass concentration of sodium dodecyl sulfate is 1% and the mass concentration of penicillin / streptomycin is 0.1%.

[0010] Preferably, the degradation solution in step S2 is a hydrochloric acid solution containing pepsin, wherein the mass concentration of pepsin is 1 mg / mL and the solvent is a 0.1 mol / L hydrochloric acid solution.

[0011] Preferably, in step S3, the concentration of the silver-polydopamine nanoparticle dispersion is 400 μg / mL, the silver-polydopamine dispersion and the extracellular matrix hydrogel prepolymer are mixed at a volume ratio of 1:1, and polymyxin B raw material is added at a ratio of 2.31 mg: 1 mL.

[0012] Thirdly, the present invention provides the application of an antibacterial hydrogel wound dressing with photothermal effect in the preparation of a wound dressing for treating drug-resistant Pseudomonas aeruginosa infection.

[0013] The beneficial effects of this invention are as follows: (1) The nanoparticle composite material in this invention has a stable photothermal conversion efficiency, which can effectively utilize photothermal therapy to treat bacterial infected wounds. It combines the antibacterial effect of silver nanoparticles with the anti-inflammatory and antioxidant functions of PDA. It can not only overcome the drug resistance of Pseudomonas aeruginosa, but also reduce oxidative stress damage, improve wound immune response, and enhance biosafety.

[0014] (2) The extracellular matrix hydrogel used in this invention has good biocompatibility and temperature-sensitive properties. It can form a hydrogel at body temperature, simulate the cell survival environment and provide a suitable pore structure, thereby effectively promoting wound regeneration. Attached Figure Description

[0015] Figure 1 This is a schematic diagram of the preparation method of the antibacterial hydrogel wound dressing with photothermal effect of the present invention; Figure 2 These are transmission electron microscope (TEM) images of PDA and Ag-PDA obtained in step one of the embodiments of the present invention, wherein image a is a TEM image of PDA and image b is a TEM image of Ag-PDA. Figure 3 Images of H&E staining and Masson staining of myocardial tissue before and after decellularization are shown. Image a shows the H&E staining of myocardial tissue before and after decellularization, and image b shows the Masson staining of myocardial tissue before and after decellularization. Figure 4 The images show cross-sectional scanning electron microscope (SEM) images of the PmB / Ag-PDA@ECM hydrogel, where image a is a low-magnification SEM image (scale bar 50 μm) and image b is a high-magnification SEM image (scale bar 500 nm). Figure 5 The swelling ratio characterization diagram is shown for the PmB / Ag-PDA@ECM hydrogel prepared in the embodiments of the present invention. Figure 6The image shows the characterization of the erosion rate of the PmB / Ag-PDA@ECM hydrogel prepared in an embodiment of the present invention. Figure 7 The in vitro degradation rate characterization diagram of the PmB / Ag-PDA@ECM hydrogel prepared in the embodiments of the present invention is shown. Figure 8 The image shows the adhesion performance characterization of the PmB / Ag-PDA@ECM hydrogel prepared in the embodiments of the present invention. The image shows the test results of the prepared PmB / Ag-PDA@ECM hydrogel being bonded to the heart, liver, spleen, lung, and kidney of a mouse, pig skin, and gloves and skin under different conditions such as torsion, stretching, and bending. Figure 9 This is a schematic diagram of the adhesion test of the universal testing machine used in an embodiment of the present invention; Figure 10 The adhesion strength curves of the ECM hydrogel and PmB / Ag-PDA@ECM hydrogel prepared in the embodiments of the present invention are shown. Figure 11 Figure 1 shows the rheological properties of the PmB / Ag-PDA@ECM hydrogel prepared in the embodiments of the present invention. Figure 2a shows the changes in storage modulus (G') and loss modulus (G") of the hydrogel under strain scanning, Figure 3b shows the changes in composite viscosity of the hydrogel under strain scanning, and Figure 4c shows the changes in storage modulus (G') and loss modulus (G") under frequency scanning. Figure 12 This is a cumulative drug release curve of the PmB / Ag-PDA@ECM hydrogel prepared in an embodiment of the present invention. Figure 13 Figure 1 shows the in vitro cytotoxicity test results of the PmB / Ag-PDA@ECM hydrogel prepared in the embodiments of the present invention. Figure 2a shows the survival rate of HaCaT (human immortalized keratinocytes) after co-incubation with different hydrogels for 24 hours and 48 hours, and Figure 3b shows the survival rate of HUVEC (human umbilical vein endothelial cells) after co-incubation with different hydrogels for 24 hours and 48 hours. Figure 14 Figure 1 shows a comparison of the in vitro antibacterial effects of the antibacterial hydrogel wound dressings prepared according to embodiments of the present invention. Figure 2a shows the results of the plate inhibition experiment of different hydrogels against sensitive Pseudomonas aeruginosa (ATCC 27853) and drug-resistant Pseudomonas aeruginosa (P2550). Figure 3b shows the bacterial survival rate statistics of the inhibition experiment of different hydrogels against sensitive Pseudomonas aeruginosa (ATCC 27853) and drug-resistant Pseudomonas aeruginosa (P2550). NIR(-) represents no infrared light irradiation, and NIR(+) represents near-infrared light irradiation. Figure 15The figures show a comparison of the in vitro healing effects of the antibacterial hydrogel wound dressing prepared according to the embodiments of the present invention. Figure a shows the results of the HaCaT cell scratch experiment, and Figure b shows the results of the semi-quantitative analysis of cell migration. The migration rate is represented by the migration rate. Figure 16 The above figures show a comparison of the therapeutic effects of the hydrogel dressings prepared according to the embodiments of the present invention on a mouse skin wound infection model. Figure a shows the experimental effect of different hydrogels in promoting the healing of mouse drug-resistant Pseudomonas aeruginosa infection wounds, and Figure b shows the bacterial survival rate of infected wounds under different hydrogel treatments at different time periods. Figure 17 Figure 1 shows the photothermal effect characterization of PDA and Ag-PDA prepared in the embodiments of the present invention under 808 nm near-infrared light irradiation. Figure 2a shows the temperature change of PDA suspension with different concentrations, Figure 3b shows the temperature change of PDA suspension with different near-infrared light power, Figure 4c shows the temperature change of Ag-PDA suspension with different concentrations, and Figure 5d shows the temperature change of Ag-PDA suspension with different near-infrared light power. Detailed Implementation

[0016] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0017] The silver-polydopamine nanoparticles used in this invention are composite nanomaterials obtained by combining dopamine polymers with silver nanoparticles. They possess stable photothermal conversion efficiency and can be used to treat bacterial-infected wounds by stimulating photothermal effects with near-infrared light (NIR). Silver nanoparticles are one of the most widely used metal nanomaterials, possessing advantages such as broad antibacterial spectrum and high antibacterial efficiency. PDA can effectively reduce the generation of reactive oxygen species at the wound site, which is beneficial for improving oxidative stress damage, while downregulating various inflammatory factors and regulating the wound immune response. Therefore, the combination of silver nanoparticles and PDA not only overcomes bacterial drug resistance through the combined Ag+ and PDA photothermal therapy, but also constructs a multifunctional hybrid material with anti-inflammatory and antioxidant properties. At the same time, it avoids the high-concentration aggregation of silver nanoparticles, thus improving biocompatibility.

[0018] The extracellular matrix hydrogel used in this invention is a naturally extracted exogenous ECM with temperature-sensitive properties. It can undergo collagen self-assembly at 37°C to form a hydrogel. It can simulate the cell survival environment and has advantages such as suitable pore structure, sufficient binding sites, and high biocompatibility, which can effectively promote wound regeneration.

[0019] The polymyxin B used in this invention is a cationic antimicrobial peptide. Multiple studies have shown that when other antibiotics have limited efficacy against Pseudomonas aeruginosa, PmB still has good therapeutic effects. In recent years, the large-scale and excessive use of PmB has led to drug resistance in Pseudomonas aeruginosa through various mechanisms, which has greatly reduced the therapeutic effect.

[0020] Pepsin has a certain degree of irritation, but its optimal enzyme activity is at pH=1. The final pH of this invention is 7.4. Furthermore, pepsin will undergo irreversible inactivation under neutral conditions, so it has no adverse effect on the hydrogel wound dressing that is finally prepared.

[0021] The antibacterial hydrogel wound dressing with photothermal effect prepared by this invention consists of 2% by mass of extracellular matrix, 0.025% by mass of polymyxin B, 0.02% by mass of silver-polydopamine nanoparticles, and the balance being water.

[0022] Example 1 This invention prepares an antibacterial hydrogel wound dressing with photothermal effect. Figure 1 This is a schematic diagram of the process for preparing the hydrogel according to the present invention, which specifically includes the following steps: 1. Preparation of silver-polydopamine nanoparticles Accurately weigh 360 mg of dopamine hydrochloride and dissolve it in 180 mL of ultrapure water. Add 1520 μL of 1 mol / L sodium hydroxide solution dropwise at 50 °C with vigorous stirring. After reacting for 5 hours, centrifuge the product at 4000 rpm for 25 min. The resulting precipitate is PDA nanoparticles. Weigh 200 mg of PDA nanoparticles and sonicate them uniformly in 10 mL of ultrapure water at 800 W for 30 min at 0 °C. Dissolve 100 mg of silver nitrate solid in 2 mL of ultrapure water and add 1 M ammonia solution dropwise under ice bath conditions until the solution is clear to obtain a silver ammonia solution. Mix the PDA nanoparticle dispersion with the silver ammonia solution and stir at room temperature in the dark for 1 hour at a stirring rate of 800 rpm. Centrifuge at 4000 rpm for 25 min. Wash with ultrapure water to obtain the product, which is Ag-PDA nanoparticles.

[0023] Figure 2 The images show transmission electron microscopy (TEM) images of PDA and Ag-PDA. Image a shows that the prepared PDA nanoparticles are uniformly sized spherical structures. Image b shows that silver is bonded to the surface of the PDA nanoparticles in the form of nanoparticles to form Ag-PDA, which has a uniform particle size of about 160 nm.

[0024] 2. Preparation of extracellular matrix hydrogel prepolymer solution Pig hearts, after the removal of connective and adipose tissue, were minced into myocardial fragments no larger than 2 mm³. These fragments were then soaked in 10 times their volume of decellularization solution for 7 days. The decellularization solution contained sodium dodecyl sulfate (1%) and penicillin / streptomycin in 1xPBS, with the sodium dodecyl sulfate concentration at 1% and the penicillin / streptomycin concentration at 0.1%. The decellularization solution was changed every 24 hours until the myocardial tissue was completely decellularized. Myocardial tissue samples before and after decellularization were then soaked in 4% paraformaldehyde. The degree of decellularization was assessed using H&E staining, and changes in collagen content before and after decellularization were detected using Masson staining to investigate the main components of the extracellular matrix. After washing three times with ultrapure water, the myocardial fragments were freeze-dried for 24 hours. The freeze-dried myocardial fragments were then added to the degradation solution at a ratio of 40 mg: 1 mL. The degradation solution was a 0.1 M hydrochloric acid solution containing 1 mg / mL pepsin. The mixture was stirred at 800 rpm for 56 hours. The pH of the reaction system was adjusted to 7.4 by adding 1 mol / L sodium hydroxide aqueous solution. ECM (Extracellular Matrix hydrogel) prepolymer was obtained, and after freeze-drying, it was stored at -80℃.

[0025] Figure 3 Images of H&E staining and Masson staining of myocardial tissue before and after decellularization. Figure 3 In a Masson staining image, the uncellular myocardial tissue contained numerous red-stained myofiber cells, while blue collagen was sparsely distributed and constituted a relatively small proportion. After decellularization, the tissue was mainly composed of blue collagen, with no red myofiber cells observed, further indicating complete decellularization of the myocardial tissue and that the extracellular matrix was primarily composed of collagen. Figure 3 In the b H&E staining images, numerous blue-green cell nuclei were clearly observed in the myocardial tissue before decellularization, while no cell nuclei were observed in the decellularized tissue, only the extracellular matrix stained red. The staining results indicate that the cells in the myocardial tissue have been completely washed away. 3. Preparation of antibacterial hydrogel wound dressing containing polymyxin B and photothermal conversion agent. 400 μg of Ag-PDA nanoparticles were dispersed in 1 mL of ultrapure water. 1 mL of Ag-PDA dispersion was mixed with 1 mL of 4% (w / w) extracellular matrix hydrogel prepolymer (ECM prepolymer). 4.61 mg of polymyxin B active pharmaceutical ingredient was then added to the mixture. The mixture was stirred until homogeneous and heated at 37 °C for 5 minutes to prepare PmB / Ag-PDA@ECM hydrogel.

[0026] Figure 4The image shows a cross-sectional scanning electron microscope image of the PmB / Ag-PDA@ECM hydrogel. The porosity is 49.24% and the average pore size is 28.3 μm. It can be observed that Ag-PDA is uniformly embedded in the hydrogel.

[0027] Example 2 1. Characterization of swelling properties of PmB / Ag-PDA@ECM hydrogel After freeze-drying the PmB / Ag-PDA@ECM hydrogel, the freeze-dried mass of the PmB / Ag-PDA@ECM hydrogel was weighed and recorded. Twenty times the volume of purified water was added to the freeze-dried ECM hydrogel solid, and the gel was placed in a 37℃ incubator. At the set time point, the supernatant was discarded, the liquid on the gel surface was aspirated, and the gel mass was weighed at this point. The above operation was repeated until the mass of the hydrogel approached a constant, with three parallel sets. A graph was plotted with swelling time on the x-axis and swelling rate on the y-axis to investigate the swelling behavior of the PmB / Ag-PDA@ECM hydrogel.

[0028] Figure 5 This is a characterization diagram of the swelling rate of PmB / Ag-PDA@ECM hydrogel. After immersion in water for 6 hours, the final swelling rate of PmB / Ag-PDA@ECM hydrogel was 1068.21±59.24%. The results of PmB / Ag-PDA@ECM hydrogel indicate that ECM hydrogel can absorb a large amount of water in a short time, quickly reach the swelling equilibrium point, and remain stable. It can efficiently and rapidly absorb wound exudate around the wound, creating a moist wound repair environment that is beneficial to cell proliferation and tissue regeneration.

[0029] 2. Characterization of the erosion properties of PmB / Ag-PDA@ECM hydrogel Using 1xPBS solutions with pH=7.4 and pH=5.5 as etching media, different pH etching media were added to the PmB / Ag-PDA@ECM hydrogels to completely immerse the gel. The gel was then incubated at 37℃ with shaking for 5 minutes to allow for full swelling. The supernatant of etching media was discarded, the surface liquid was aspirated, and the gel mass was measured. The appropriate etching media were then added to the gel, and the mixture was incubated at 37℃ with shaking. The remaining mass of the hydrogel was measured at each set time point. This process was repeated until the hydrogel was completely etched, with three parallel sets. A graph was plotted with etching time on the x-axis and etching rate on the y-axis to investigate the etching of the PmB / Ag-PDA@ECM hydrogel in PBS media of different pH values.

[0030] Figure 6The figure shows the characterization of the erosion rate of PmB / Ag-PDA@ECM hydrogel. At 360 hours, 97.93±0.77% of the PmB / Ag-PDA@ECM hydrogel eroded in the medium at pH=5.5, and 49.16±0.91% of the PmB / Ag-PDA@ECM hydrogel eroded in the medium at pH=7.4. The experimental results indicate that the PmB / Ag-PDA@ECM hydrogel has good stability in vitro, which is beneficial for long-term storage after gelation and maintaining the original gel properties. In addition, the stability of PmB / Ag-PDA@ECM hydrogel is lower in a weakly acidic environment than in a neutral environment.

[0031] 3. Characterization of in vitro degradation properties of PmB / Ag-PDA@ECM hydrogel Type I collagenase solutions with concentrations of 5 U / mL and 10 U / mL were used as enzyme degradation solutions. 5 mL of the same enzyme solution was added to each PmB / Ag-PDA@ECM hydrogel, completely immersing 1 g of PmB / Ag-PDA@ECM hydrogel, and the gel was placed in a 37℃ incubator. At the set time points, the upper layer of collagenase solution was discarded, the liquid on the gel surface was aspirated, and the remaining mass of the hydrogel was weighed. The above operation was repeated until the PmB / Ag-PDA@ECM hydrogel was completely degraded, with three parallel groups. A graph was plotted with degradation time on the x-axis and degradation rate on the y-axis to investigate the degradation of PmB / Ag-PDA@ECM hydrogel in collagenase solutions of different concentrations.

[0032] Figure 7 The graph shows the degradation rate characterization of PmB / Ag-PDA@ECM hydrogel. Under the action of 10 U / mL collagenase, 96.98±0.47% of the PmB / Ag-PDA@ECM hydrogel degraded within approximately 78 hours; under the action of 5 U / mL collagenase, the degradation rate reached 89.63±4.01% after 78 hours. These results indicate that PmB / Ag-PDA@ECM hydrogel can be degraded under the action of type I collagenase. Collagenase and matrix metalloenzymes are ubiquitous in the human body, especially during wound healing, when collagenase levels increase; therefore, extracellular matrix hydrogels can be biodegraded in vivo.

[0033] 4. Characterization of the adhesion properties of PmB / Ag-PDA@ECM hydrogel The prepared PmB / Ag-PDA@ECM hydrogel was tested under different conditions, including torsion, stretching, and bending, by bonding it to the heart, liver, spleen, lung, and kidney of mice, pig skin, gloves, and skin.

[0034] Figure 8Adhesion performance characterization diagram of PmB / Ag-PDA@ECM hydrogel. PmB / Ag-PDA@ECM hydrogel maintains good adhesion on different material surfaces and under different conditions, meeting the adhesion requirements of wound dressings.

[0035] Cut the degreased pigskin into strips of 5.0cm x 1.5cm. Place two strips of pigskin overlapping each other, with the overlapping area measuring 1.5cm x 1.5cm. Add the PmB / Ag-PDA@ECM hydrogel prepolymer between the two strips of pigskin, filling the overlapping area. (Example:) Figure 9 As shown, the adhesion properties of PmB / Ag-PDA@ECM hydrogel on pigskin were tested using a universal testing machine.

[0036] Figure 10 The adhesion strength curves of ECM hydrogel and PmB / Ag-PDA@ECM hydrogel are shown. PmB / Ag-PDA@ECM hydrogel can produce an adhesion strength of 6376.30±334.97 Pa, which is significantly better than that of ECM hydrogel (2281.82±103.54 Pa), indicating that Ag-PDA can effectively improve the adhesion performance of hydrogels to meet the adhesion requirements of wound dressings.

[0037] 5. Rheological property characterization of PmB / Ag-PDA@ECM hydrogel An appropriate amount of PmB / Ag-PDA@ECM hydrogel was placed in a rheometer. The storage modulus (G'), loss modulus (G"), and composite viscosity of the ECM hydrogel were measured using the rheometer. Strain scanning was performed with a fixed frequency of 1 Hz and a strain range of 0.1-100%. Dynamic frequency scanning was performed with a fixed strain of 1% and a frequency range of 0.1-100% Hz. The mechanical properties of the PmB / Ag-PDA@ECM hydrogel were investigated by observing the changes in G', G"), and composite viscosity under different strains and frequencies.

[0038] Figure 11 Figure 1 shows the rheological results and composite viscosity of the PmB / Ag-PDA@ECM hydrogel during strain scanning and dynamic frequency scanning. Figure a shows that, during strain scanning at a fixed frequency of 1 Hz, both G' and G'' values ​​are relatively stable within the strain range of 0.1% to 100%, indicating that the PmB / Ag-PDA@ECM hydrogel is in the linear viscoelastic region, and G' reaches 1 × 10⁴ Pa. Furthermore, Figure b shows that the composite viscosity of the PmB / Ag-PDA@ECM hydrogel remains stable within the strain range of 0.1% to 1%. Figure c shows that, during dynamic frequency scanning at a fixed strain of 1%, within the range of 0.1–100 Hz, G' is significantly higher than G'', representing the formation of an elastic hydrogel network.

[0039] Example 3 1. In vitro drug release characterization of PmB / Ag-PDA@ECM hydrogel 1 g of PmB / Ag-PDA@ECM hydrogel was soaked in 30 mL of 1xPBS solution (pH=5.5) and shaken at 37 °C. Samples were taken at fixed time points, and an equal volume of 1xPBS solution at 37 °C was added. The absorbance of PmB aqueous solutions with concentrations of 0.001 mg / mL, 0.005 mg / mL, 0.010 mg / mL, 0.050 mg / mL, and 0.100 mg / mL was measured using a UV spectrophotometer at a wavelength of 215 nm. A standard curve was plotted between concentration and absorbance, and the curve equation was: Absorbance = 0.0452 × sample concentration + 0.0031, with a correlation coefficient of 0.9993. The absorbance of the samples at each time point was measured, and the sample concentration at each time point was calculated according to the standard curve. The release amount was also calculated, and a drug release curve was plotted, where the release amount (100%) = (30 mL × sample concentration) / 2.31 mg × 100%.

[0040] Figure 12 The image shows the drug release curve of PmB / Ag-PDA@ECM hydrogel in PBS at pH 5.5. After 6 hours, the PmB release reached 80%, indicating that PmB can quickly reach an effective antibacterial concentration at the infected wound site.

[0041] 2. Biocompatibility testing of PmB / Ag-PDA@ECM hydrogel The biocompatibility of PmB / Ag-PDA@ECM hydrogels was tested using the CCK8 assay. Five experimental groups were set up, including a blank control group, ECM hydrogel, PmB@ECM hydrogel, Ag-PDA@ECM hydrogel, and PmB / Ag-PDA@ECM hydrogel. Immortalized human keratinocytes (HaCaT) and human umbilical vein endothelial cells (HUVECs) were added to 24-well plates at a density of 2 × 10⁴ cells per well. The hydrogels were placed in Transwell chambers and immersed in culture medium. After irradiation with near-infrared light (808 nm, 2.0 W / cm²) for 5 minutes, the plates were incubated at 37°C. At 24 and 48 hours, 100 μL of CCK8 reagent was added, and the absorbance at 450 nm was measured using a microplate reader. Each group consisted of three replicates, and cell viability was calculated.

[0042] Figure 13 The graph shows the cell viability statistics after co-incubation with different hydrogels for 24 hours and 48 hours. The results indicate that the components in PmB / Ag-PDA@ECM have good biocompatibility, and the photothermal effect excited by NIR irradiation does not have adverse effects on the cells.

[0043] 3. In vitro photothermal antibacterial properties of PmB / Ag-PDA@ECM hydrogel The polymyxin B-sensitive Pseudomonas aeruginosa used in the examples is Pseudomonas aeruginosa ATCC 27853, and the polymyxin B-resistant Pseudomonas aeruginosa is Pseudomonas aeruginosa P2550.

[0044] The in vitro photothermal antibacterial properties of PmB / Ag-PDA@ECM hydrogel were evaluated using the plate count method. Ten experimental groups were set up, including a blank control group, ECM hydrogel, PmB@ECM hydrogel, Ag-PDA@ECM hydrogel, PmB / Ag-PDA@ECM hydrogel, blank control group + NIR, ECM hydrogel + NIR, PmB@ECM hydrogel + NIR, Ag-PDA@ECM hydrogel + NIR, and PmB / Ag-PDA@ECM hydrogel + NIR. 1 mL of hydrogel was added to 1 mL of *Pseudomonas aeruginosa* suspension (1×10⁵ CFU / mL). The samples requiring NIR irradiation were then subjected to NIR (808 nm, 2.0 W / cm²) irradiation for 5 minutes, incubated at 37°C for 16 hours, and the bacterial suspension was plated onto MHA agar plates. After incubation at 37°C for 12 hours, colony counting was performed. The plate results are shown below. Figure 14 As shown in a, the bacterial survival rate is calculated, where the bacterial survival rate (%) = (colony forming units in the experimental group / colony forming units in the control group) × 100%.

[0045] Figure 14 The results show the antibacterial characterization of each experimental group against susceptible and drug-resistant Pseudomonas aeruginosa. Figure 14 The results showed that, compared with the control group, the survival rate of both sensitive and drug-resistant Pseudomonas aeruginosa was as low as 0% after near-infrared light irradiation of PmB / Ag-PDA@ECM hydrogel, indicating that PmB / Ag-PDA@ECM hydrogel has excellent antibacterial properties against both sensitive and drug-resistant Pseudomonas aeruginosa.

[0046] 4. In vitro healing-promoting performance test of PmB / Ag-PDA@ECM hydrogel HaCaT cells were used as experimental cells, and the in vitro healing-promoting performance of PmB / Ag-PDA@ECM hydrogel was tested using the cell scratch assay. Five experimental groups were set up, including a blank control group, ECM hydrogel, PmB@ECM hydrogel, Ag-PDA@ECM hydrogel, and PmB / Ag-PDA@ECM hydrogel. HaCaT cells in the logarithmic growth phase were trypsinized and seeded into 12-well plates at a density of 5 × 10⁵ cells per well and cultured in a cell culture incubator. When the cells reached the confluence state, vertical scratches were made with a sterile 200 μL pipette tip, the culture medium was discarded, and the cells were washed three times with sterile PBS to remove cell debris. Fresh culture medium was added, and Transwell chambers containing the hydrogels of each group were added to the well plates. The plates were irradiated with NIR (808 nm, 2.0 W / cm²) for 5 minutes and incubated at 37°C for 24 hours. After incubation, the cells were observed and photographed under a microscope to analyze cell migration.

[0047] Figure 15 The results show the in vitro healing-promoting properties of PmB / Ag-PDA@ECM hydrogel. Compared with the blank control group, PmB / Ag-PDA@ECM hydrogel effectively promoted HaCaT cell migration, indicating that PmB / Ag-PDA@ECM hydrogel has excellent healing-promoting properties.

[0048] Example 4 An experiment on the promotion of wound healing by PmB / Ag-PDA@ECM hydrogel in mice infected with drug-resistant Pseudomonas aeruginosa. To clarify the actual antibacterial effect of the antibacterial amorphous hydrogel dressing prepared in this invention, an experiment was conducted in a mouse animal model to clear drug-resistant Pseudomonas aeruginosa infection and promote wound healing. Specifically, infected wounds were treated, wound healing was observed, and the degree of infection was observed by agar plate culture of the bacteria on the wound.

[0049] Male Balb / c mice weighing approximately 20g were used as experimental subjects. Before the experiment, the mice's backs were shaved, and a 10mm diameter circular full-thickness skin defect was cut into the back. A rubber gasket was used to fix the wound. 100μL of drug-resistant Pseudomonas aeruginosa suspension (1×10⁷ CFU / mL) was added to the wound. After the bacterial suspension dried, the wound was sealed with a sterile dressing and incubated for 24 hours to allow bacterial infection. The model was then established. Mice were randomly divided into five experimental groups: a blank control group, ECM hydrogel, PmB@ECM hydrogel, Ag-PDA@ECM hydrogel, and PmB / Ag-PDA@ECM hydrogel. During treatment, the gel sample prepared according to this invention was applied to the entire wound area to a thickness of approximately 1.5mm, and the hydrogel was replaced every 48 hours. Before each application of hydrogel, bacterial colonies were scraped from the wound surface using a cotton swab to assess the bacterial load. After application, the wound was irradiated with near-infrared light for 5 minutes (808nm, 2.0W / cm²). The treatment cycle was 14 days. Results were... Figure 16 As shown in a.

[0050] Figure 16 Figure showing the experimental results of PmB / Ag-PDA@ECM hydrogel promoting wound healing in mice infected with drug-resistant Pseudomonas aeruginosa. Figure 16 The bacterial activity statistical chart showed that, compared with the control group, PmB / Ag-PDA@ECM hydrogel could effectively promote the healing of infected wounds; on day 4, the bacterial survival rate at the wound site had decreased to 3.17%, indicating that PmB / Ag-PDA@ECM hydrogel has excellent anti-drug-resistant bacterial properties.

[0051] Comparative Example 1 The photothermal conversion performance of PDA and Ag-PDA was tested.

[0052] The PDA and Ag-PDA obtained in step 1 were prepared into dispersions of different concentrations (200 μg / mL, 400 μg / mL, 600 μg / mL, 800 μg / mL, and 1000 μg / mL). 200 μL of each concentration of PDA and Ag-PDA dispersion was placed in a 96-well plate, and the plate was irradiated with a fixed near-infrared light power of 1.0 W / cm² for 5 min, with the temperature recorded every 20 s. Similarly, 200 μL of a 200 μg / mL PDA and Ag-PDA dispersion were irradiated with near-infrared light at different powers (0.5 W / cm², 1.0 W / cm², 1.5 W / cm², and 2.0 W / cm²) for 5 min, with the temperature recorded every 20 s. Three samples were repeated for each group.

[0053] Figure 17These are the temperature-time curves of PDA and Ag-PDA dispersions under different conditions. The results show that the temperature increment increases with increasing concentration and near-infrared light power, and the temperature increment of the Ag-PDA dispersion is higher than that of the PDA dispersion under the same conditions, indicating that Ag-PDA has good photothermal conversion performance.

[0054] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. An antibacterial hydrogel wound dressing with photothermal effect, characterized in that, It includes the following raw materials: 2% by mass of extracellular matrix, 0.025% by mass of polymyxin B, 0.02% by mass of silver-polydopamine nanoparticles, and the balance being water.

2. A method for preparing an antibacterial hydrogel wound dressing with photothermal effect according to claim 1, characterized in that, Includes the following steps: S1. Preparation of silver-polydopamine nanoparticles: Dopamine hydrochloride powder was dissolved in ultrapure water. The dopamine hydrochloride aqueous solution was mixed with sodium hydroxide aqueous solution and stirred at 50°C and 800 rpm for 5 hours. After centrifugation at 4000 rpm for 25 minutes, the nanoparticles were washed three times with ultrapure water to obtain polydopamine nanoparticles. The polydopamine nanoparticles were dispersed in ultrapure water to obtain a polydopamine nanoparticle dispersion with a mass concentration of 2 g / mL. The dispersion was mixed with silver ammonia solution and stirred at room temperature in the dark for 1 hour. After centrifugation at 4000 rpm for 25 minutes, the nanoparticles were washed three times with ultrapure water to obtain silver-polydopamine nanoparticles. S2. Prepare the extracellular matrix hydrogel prepolymer solution by chopping the pig heart (after removing connective tissue and adipose tissue) into pieces no larger than 2 mm in volume. 3 Myocardial fragments were soaked in decellularization solution for 7 days, with the decellularization solution being changed every 24 hours to obtain decellularized myocardial fragments. After washing three times with ultrapure water, the fragments were freeze-dried for 24 hours. The freeze-dried myocardial fragments were added to the degradation solution at a ratio of 40 mg: 1 mL, and the reaction was stirred at 800 rpm for 56 hours. The pH of the reaction system was adjusted to 7.4 by adding 1 mol / L sodium hydroxide aqueous solution dropwise to obtain an extracellular matrix hydrogel prepolymer with a mass concentration of 4%. S3. Silver-polydopamine nanoparticles were dispersed in ultrapure water. The silver-polydopamine dispersion was mixed with 4% extracellular matrix hydrogel prepolymer, and then polymyxin B raw material was added to it. The mixture was stirred and mixed evenly, and heated at 37°C for 5 min to prepare PmB / Ag-PDA@ECM hydrogel.

3. The method for preparing the antibacterial hydrogel wound dressing with photothermal effect according to claim 2, characterized in that, In step S1, the mass concentration of the dopamine hydrochloride aqueous solution is 2 mg / mL, the molar concentration of the sodium hydroxide aqueous solution is 1 mol / L, the mass concentration of the silver ammonia solution is 1.05 mg / mL, and the volume ratio of the polydopamine nanoparticle dispersion to the silver ammonia solution is 2:1; the volume of the sodium hydroxide aqueous solution added is 0.84% ​​of the volume of the dopamine hydrochloride aqueous solution.

4. The method for preparing the antibacterial hydrogel wound dressing with photothermal effect according to claim 2, characterized in that, In step S2, the decellularization solution is a 1xPBS solution containing sodium dodecyl sulfate and penicillin / streptomycin, wherein the mass concentration of sodium dodecyl sulfate is 1% and the mass concentration of penicillin / streptomycin is 0.1%.

5. The method for preparing the antibacterial hydrogel wound dressing with photothermal effect according to claim 2, characterized in that, In step S2, the degradation solution is a hydrochloric acid solution containing pepsin, wherein the mass concentration of pepsin is 1 mg / mL and the solvent is a 0.1 mol / L hydrochloric acid solution.

6. The method for preparing the antibacterial hydrogel wound dressing with photothermal effect according to claim 2, characterized in that, In step S3, the concentration of the silver-polydopamine nanoparticle dispersion is 400 μg / mL. The silver-polydopamine dispersion and the extracellular matrix hydrogel prepolymer are mixed at a volume ratio of 1:1, and polymyxin B raw material is added at a ratio of 2.31 mg: 1 mL.

7. The use of the antibacterial hydrogel wound dressing with photothermal effect as described in claim 1 in the preparation of wound dressings for treating drug-resistant Pseudomonas aeruginosa infections.

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