Preparation and application of polyphosphazene-based hydrogel with inherent antibacterial property

Abstract

The application provides preparation and application of a polyphosphazene-based hydrogel with inherent antibacterial property, which is prepared by modifying polyphosphazene with ethyl bromoacetate to obtain a quaternary ammonium salt grafted polymer PZBA, and is formed by crosslinking of the quaternary ammonium salt grafted polyphosphazene-based polymer (PZBA) and polyvinyl alcohol (PVA). The polyphosphazene-based hydrogel (PZBA-PVA hydrogel) of the application can down-regulate pro-inflammatory cytokines, has an anti-inflammatory effect, and up-regulates the expression level of growth factors to promote tissue regeneration. The hydrogel prepared by the application has good tissue adhesion, biocompatibility and antibacterial property, plays an antibacterial and wound healing promoting role on infected wounds, and can be used for preparing antibacterial and wound healing promoting drugs.

Description

Technical Field

[0001] This invention belongs to the field of biomaterials application and relates to the preparation and application of a polyphosphazene-based hydrogel with inherent antibacterial properties. Background Technology

[0002] Wound infection is caused by microbial overload in wound tissue, which interferes with the wound healing process, prolongs wound repair time, and leads to chronic wounds that are difficult to heal, or even death. Clinically, antibiotics are widely used to eradicate harmful bacteria, but with the increasing number of drug-resistant bacteria, antibiotic resistance and overuse have become major concerns regarding antibiotic use. As an alternative, metal-based materials such as silver and gold nanoparticles have good bactericidal effects. Although these metal nanoparticles are used in low quantities, their toxicity to cells and tissues should not be underestimated. Silver nanoparticles have a destructive effect on DNA, mitochondria, and erythrocytes. Gold nanoparticles impair cell viability by inducing ROS production and affect cell proliferation and migration by disrupting the cytoskeleton. Biogenic natural antibacterial polymers have been widely developed in recent years due to their non-toxicity and good biocompatibility. Chitosan and its derivatives are among the most well-known natural antibacterial polymers. However, chitosan is sensitive to pH; its antibacterial activity disappears when the pH reaches 7.0. Therefore, developing synthetic polymers with inherent antibacterial properties, non-toxicity, and minimal side effects is another research hotspot. Polymers containing antibacterial components, including peptides and zwitterions, can be endowed with inherent antibacterial activity through synthesis. Quaternary ammonium salts (QA) are commonly used amphoteric antibacterial ions that have been widely applied in synthetic polymers, exhibiting good antibacterial activity against both Gram-positive and Gram-negative bacteria. The electrostatic interaction between the positively charged QA and the negatively charged bacterial membrane leads to membrane rupture and bacterial death.

[0003] Polyphosphazene is a unique synthetic polymer with a controllable structure, allowing for the introduction of functional groups. It exhibits excellent biocompatibility and bioavailability, and its degradation products are non-toxic, making it a promising candidate for biomedical materials applications. To stably apply polymers to wounds, adhesive hydrogels, due to their stretchability, adhesiveness, breathability, and ability to maintain a moist environment for cell growth, have been widely used as carriers for wound dressings. Compared to traditional wound dressings such as bandages that require secondary dressing support, adhesive hydrogels with inherent antibacterial properties adhere more easily to and conform to irregular wounds, while providing antibacterial activity more effectively.

[0004] Therefore, developing a synthetic polymer hydrogel with inherent antibacterial properties is of great significance for accelerating the healing of infected wounds. Summary of the Invention

[0005] To overcome the shortcomings of existing technologies, this invention provides a method for preparing a polyphosphazene-based hydrogel with inherent antibacterial properties, achieved through the following steps:

[0006] (1) Hexachlorotriphosphazene (HCCP, 4002.1 mg) was thermally ring-opened at 250 °C. The resulting product was then dissolved in toluene, and n-hexane was injected into it. The resulting precipitate was dried under vacuum to obtain polydichlorophosphazene (PDCP).

[0007] (2) Dissolve PDCP (1.8037 g) in tetrahydrofuran (THF, 100 mL), dissolve N,N-dimethylethylenediamine (DMAEA, 4.1135 g) in THF (50 mL), and add triethylamine (TEA, 6.5 mL). Stir at 40 °C for 2 h, then add to the PDCP solution and stir at 0 °C for 30 min. The resulting mixture is heated to 30 °C and reacted for 48 h. After centrifugation at 3000 rpm for 30 min, the precipitate is dissolved in deionized water and dialyzed (5 days, cutoff 3500). After drying, poly(N,N-dimethylethylenediaminephosphazene) (PDAP) is obtained.

[0008] (3) Dissolve PDAP (1.0031 g) in anhydrous methanol (MeOH, 200 ml), then dissolve 3-bromomethylphenylboronic acid (BPBA, 788.2 mg) in MeOH (20 ml), add the BPBA solution to the PDAP solution, stir at 40 °C for 48 h, dialyze (5 days, cutoff 3500), freeze dry to obtain poly[(N,N-dimethylethylenediamine)-g-(N,N,N,N-dimethylaminoethyl p-methylphenylboronic acid ammonium bromide)]phosphazene (PPBA).

[0009] (4) Dissolve PPBA (2.0003 g) in MeOH (200 ml), dissolve ethyl bromoacetate (3.0689 g) in MeOH (20 ml), add the ethyl bromoacetate solution to the PPBA solution, stir at 40 °C for 48 h, dialyze (5 days, cutoff 3500), freeze dry to obtain poly[(N,N-dimethylethylenediamine)-g-(N,N,N,N-dimethylaminoethyl p-methylphenylboronic acid ammonium bromide)-g-(N,N,N,N-dimethylaminoethyl ethyl acetate ammonium bromide)]phosphazene (PZBA).

[0010] (5) Dissolve PZBA (30 mg) in deionized water (1 mL) and polyvinyl alcohol (PVA) (30 mg) in deionized water (1 mL). Mix 3 wt% PZBA and 3 wt% PVA aqueous solution in a volume ratio of 1:1 to obtain PZBA-PVA hydrogel.

[0011] Another object of the present invention is to provide the application of the polyphosphazene-based hydrogel in the preparation of antibacterial and wound-healing drugs. The polyphosphazene-based hydrogel (PZBA-PVA hydrogel) of the present invention can downregulate pro-inflammatory cytokines, exhibiting anti-inflammatory effects, and upregulate the expression levels of growth factors, promoting tissue regeneration. The hydrogel prepared by the present invention has good tissue adhesion, biocompatibility, and antibacterial properties, and plays a role in antibacterial treatment and wound healing promotion for infected wounds.

[0012] The quaternary ammonium salt-grafted polyphosphazene-based polymer PZBA and the PZBA-PVA hydrogel formed by crosslinking with polyvinyl alcohol in this invention exhibit good antibacterial activity, biocompatibility, injectability, and adhesiveness. Wound healing is significantly accelerated after treatment with PZBA-PVA hydrogel. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) and total RNA sequencing (RNA-seq) of the hydrogel-treated wound tissue showed that PZBA-PVA hydrogel can downregulate pro-inflammatory cytokines, exhibiting anti-inflammatory effects, and upregulate the expression levels of growth factors, promoting tissue regeneration. In summary, the quaternary ammonium salt-grafted polyphosphazene-based hydrogel has been verified to possess strong antibacterial properties and accelerate tissue repair, showing promising application prospects in promoting the healing of infected wounds. Attached Figure Description

[0013] Figure 1 This is a schematic diagram of the synthesis of polymer PZBA and hydrogel PZBA-PVA.

[0014] Figure 2 For Fourier transform infrared spectroscopy analysis.

[0015] Figure 3 For thermogravimetric analysis.

[0016] Figure 4 The swelling rate of PZBA-PVA in PBS.

[0017] Figure 5 Electron micrograph of PZBA-PVA hydrogel.

[0018] Figure 6 PZBA-PVA hydrogel is used to adhere to latex gloves.

[0019] Figure 7 Adhesion strength analysis of PZBA-PVA hydrogel.

[0020] Figure 8 PZBA-PVA hydrogel was used to adhere to pigskin.

[0021] Figure 9 PZBA-PVA hydrogel was used to adhere to different organs.

[0022] Figure 10Photographs showing the self-healing properties of PZBA-PVA hydrogel.

[0023] Figure 11 Photographs showing the injectability properties of PZBA-PVA hydrogel.

[0024] Figure 12 For CCK-8 cell viability analysis.

[0025] Figure 13 The hemolysis rate of PZBA-PVA hydrogel.

[0026] Figure 14 The inhibition rate of PZBA polymer against Staphylococcus aureus is shown.

[0027] Figure 15 The inhibition rate of PZBA polymer against Escherichia coli is shown.

[0028] Figure 16 The antibacterial rate of PZBA-PVA hydrogel against Staphylococcus aureus.

[0029] Figure 17 The antibacterial rate of PZBA-PVA hydrogel against Escherichia coli.

[0030] Figure 18 Photographs showing the application of PZBA-PVA hydrogel to an infected wound.

[0031] Figure 19 The healing rate of infected wounds.

[0032] Figure 20 This is a count of bacteria in infected wounds.

[0033] Figure 21 Analysis of TNF-α expression levels in infected wounds.

[0034] Figure 22 Analysis of VEGF expression levels in infected wounds. Detailed Implementation

[0035] The present invention will be further described in conjunction with the accompanying drawings and embodiments.

[0036] Example 1: Synthesis of PZBA polymer

[0037] Synthesis of polydichlorophosphazene (PDCP): Hexachlorotriphosphazene (HCCP) (400 2.1 mg, 11.51 mmol) was placed in an anhydrous and oxygen-free vacuum tube and subjected to thermal ring-opening polymerization at 250 °C with aluminum chloride (80.2 mg, 0.60 mmol) as a catalyst. After 5 h, a light brown, transparent, viscous liquid was obtained, which was then dissolved in toluene to obtain a PDCP toluene solution. Hexane was injected into the PDCP toluene solution using a syringe, resulting in a light brown precipitate. The precipitate was then dried under vacuum to obtain high molecular weight PDCP.

[0038] Synthesis of poly(N,N-dimethylethylenediaminephosphazene) (PDAP): First, 1.8037 g of PDCP was dissolved in 100 mL of tetrahydrofuran (THF) to obtain a PDCP solution. 4.1135 g (46.7 mmol) of N,N-dimethylethylenediamine (DMAEA) was dissolved in 50 mL of THF, and then 6.5 mL of triethylamine (TEA) (42.25 mmol) was added. The mixture was stirred at 40 °C for 2 h to obtain a THF solution containing DMAEA and TEA. This solution was added dropwise to the PDCP solution in an ice-water bath under argon protection, and stirred at 0 °C for 30 min. The resulting mixture was gradually heated to 30 °C and reacted for 48 h. Subsequently, the mixture was centrifuged at 3000 rpm for 30 min, and the white precipitate was dissolved in deionized water. The white precipitate solution was placed in a dialysis bag with a cutoff of 3500 mg / L, and the dialysis bag was then immersed in a large amount of deionized water for dialyzing. The deionized water was changed every 12 hours, and the dialyzing was continued for 5 days. Finally, the solution in the dialysis bag was dried to obtain a pale yellow solid PDAP.

[0039] Synthesis of poly[(N,N-dimethylethylenediamine)-g-(N,N,N,N-dimethylaminoethyl p-methylphenylboronic acid ammonium bromide)]phosphazene (PPBA): 1.0031 g of PDAP was dissolved in 200 mL of anhydrous methanol (MeOH), and stirred thoroughly until the PDAP was completely dissolved. Then, 788.2 mg (3.664 mmol) of 3-bromomethylphenylboronic acid (BPBA) was dissolved in 20 mL of anhydrous methanol (MeOH). The BPBA solution was then slowly added dropwise to the PDAP solution, and a rotor (1000 rpm) was added. The reaction was carried out at 40 °C for 48 h. Finally, the resulting solution was placed in a dialysis bag with a cutoff of 3500 mg / L, dialyzed against deionized water for 5 days, and then freeze-dried to obtain PPBA.

[0040] Synthesis of poly[(N,N-dimethylethylenediamine)-g-(N,N,N,N-dimethylaminoethyl p-methylphenylboronic acid ammonium bromide)-g-(N,N,N,N-dimethylaminoethyl ethyl acetate ammonium bromide)]phosphazene (PZBA): 2.0003 g of PPBA was dissolved in 200 ml of MeOH and stirred thoroughly until completely dissolved. 3.0689 g of ethyl bromoacetate (18.36 mmol) was dissolved in 20 ml of MeOH. The ethyl bromoacetate solution was then slowly added dropwise to the PPBA solution, and a rotor (1000 rpm) was added. The reaction was carried out at 40 °C for 48 h. Finally, the resulting solution was placed in a dialysis bag with a cutoff of 3500 mg / L and dialyzed against deionized water for 5 days, followed by freeze-drying to obtain the polymer PZBA. (See Appendix) Figure 1 .

[0041] Example 2: Preparation of PZBA-PVA hydrogel

[0042] PZBA-PVA hydrogels were prepared by reacting PZBA and PVA solutions to form dynamic borate ester bonds. First, 30 mg of PZBA and 30 mg of PVA were dissolved in 1 mL of deionized water (3 wt%). Then, the 3 wt% PZBA and 3 wt% PVA aqueous solutions were mixed at a 1:1 volume ratio and stirred for 10 min to obtain the PZBA-PVA hydrogel. (See attached image) Figure 1 .

[0043] Example 3: Characterization of polyphosphazene-based hydrogel PZBA-PVA

[0044] Chemical structure analysis of polymers and hydrogels was performed using near-infrared spectroscopy. Figure 2 It can be seen that the strong absorption peaks at 2964 cm⁻¹ and 3015 cm⁻¹ are attributed to the stretching vibrations of the quaternary ammonium (QA) compound (-(CH₃)N quat), indicating that 3-bromomethylphenylboronic acid and ethyl bromoacetate were successfully introduced into PPBA and PZBA, respectively. Furthermore, the PPBA spectrum shows strong absorption peaks at 2768 cm⁻¹ and 2857 cm⁻¹, which are related to the tertiary amine group (-(CH₃)N). However, due to the high grafting rate of QAs in PZBA, no tertiary amine peaks were found in the PZBA spectrum. Figure 2 In the study, the spectral absorption peak of the hydrogel at 1320 cm⁻¹ is related to the dynamic boron ester bond (-BO) formed by the dihydroxy group of PVA and the phenylboronic acid group of PZBA.

[0045] Thermogravimetric analysis (TGA) showed that the polymer and hydrogel began to pyrolyze at approximately 200°C, confirming that all materials exhibited good thermal stability. Figure 3 ).

[0046] The swelling ratio of the hydrogel was measured by immersion in phosphate buffered solution (PBS 1X, Biosharp) at 37°C for different times. Figure 4 The PZBA-PVA retained 28% of its mass at 8 hours because the cationic QA compound (-(CH3)N quat) reacted with ions in PBS, causing water to precipitate from the hydrogel and resulting in a decrease in mass.

[0047] The morphology of the hydrogel was observed using scanning electron microscopy. The hydrogel for scanning electron microscopy was prepared using lyophilization and gold sputtering methods. (e.g.,...) Figure 5 As shown in the figure, PZBA-PVA hydrogel has a porous structure.

[0048] Example 4: Determination of the Adhesion of Hydrogels

[0049] Using pigskin as a substrate, the adhesion of the hydrogel to skin tissue was tested. Pigskin was cut into rectangles (10mm × 30mm). The hydrogel was applied to the pigskin, and then another piece of pigskin was placed on top, forming an adhesion area of ​​10mm × 10mm. A tensile shear test was performed on a 500N mechanical element to determine the adhesion strength of the hydrogel to skin tissue. The test speed was 50mm / min, and the test was repeated 5 times. Macroscopically, the PZBA-PVA hydrogel adhered easily to latex gloves. Figure 6 In tensile shear tests, the adhesion strength of the further cationized PZBA-PVA hydrogel (20.84±4.2 kPa) was higher than that of the PPBA-PVA hydrogel (15.2±2.0 kPa). The results indicate that increasing the positive charge of the hydrogel is beneficial to improving its adhesion strength. Figure 7 ).

[0050] Furthermore, no deformation of the PZBA-PVA hydrogel was observed when it was adhered to pigskin and subjected to bending, twisting, lifting, and immersion in water. Figure 8 ).like Figure 9 As shown, the hydrogel also exhibited strong adhesion in multiple organs of mice.

[0051] Example 5: Determination of the self-healing and injectability of the hydrogel

[0052] Two PZBA-PVA hydrogels were prepared; one was stained with Rhodamine B, and the other was not stained. After the two materials were in contact for 10 minutes, they were stacked together, and the healing performance of the hydrogel interface was observed. Figure 10 As shown, the two hydrogel pieces fused together within 10 minutes, and tweezers could not separate them again. This self-healing property is crucial for maintaining the flexibility and integrity of wound dressings when applied to wounds requiring frequent stretching or subjected to strong external mechanical forces. Figure 11In this study, PZBA-PVA hydrogel can be written with the letters abc using a 20g syringe (0.60mm inner diameter), and this injectability is beneficial for application to irregular wounds.

[0053] Example 6: Cell compatibility test of hydrogel

[0054] The cell compatibility of the hydrogel was determined using the CCK-8 staining method. The hydrogel was soaked in DMEM cell culture medium for 24 h, and the extract was obtained by filtration through a 0.22 μm filter. The extract was then diluted with DMEM to 50, 100, 200, and 400 μg / mL, respectively.

[0055] For CCK-8 assay, mouse myoblast C2C12 cells were seeded in 96-well plates and incubated for 24 hours. The cell culture medium was then replaced with different concentrations of extract solution for another 24 hours. Control group cells were treated with DMEM. Blank group cells were treated with cell-free DMEM. CCK-8 reagent (10 μL) was added to each well, and the cells were incubated at 37°C for 1 hour. The absorbance of the solution was measured at 450 nm using a microplate reader. Cell viability was calculated using the formula: Cell viability = (Sample OD - Blank OD) / (Control OD - Blank OD), where OD is absorbance. Compared with the control group (0 μg / mL), the cell viability of the PZBA-PVA hydrogel extract solution treatment group was not significantly affected. Figure 12 Compared with the control group, no significant changes in cell viability were observed when the concentration of the hydrogel leachate increased.

[0056] Example 7: Hemolysis test of hydrogel

[0057] The hemocompatibility of the hydrogel was verified using a hemolysis test with porcine whole blood. The hydrogel was soaked in PBS solution for 24 h to obtain a hydrogel extract. The extract was diluted to 50, 100, 200, and 400 μg / mL. To obtain red blood cells, porcine whole blood was centrifuged at 1000 rpm for 10 min and washed three times with PBS. Red blood cells were diluted with PBS to a final concentration of 5% v / v (volume / volume). 100 μL of the red blood cell solution and 100 μL of the hydrogel suspension were added to 96-well plates. The positive control was mixed with sterile water, and the negative control was mixed with PBS. The 96-well plates were incubated at 37°C with shaking at 180 rpm for 1 h. Microplates were centrifuged at 1000 rpm for 10 min, and 100 μL of supernatant was added to each 96-well plate. The absorbance was measured at 540 nm using a microplate reader. The hemolysis rate was calculated using the following formula: Hemolysis rate = (Sample OD - Negative control group OD) / (Positive control group OD - Negative control group OD) × 100%, where OD is absorbance. Compared to the control group (sterile water), no excessive hemolysis was observed in any sample, indicating that no significant exudates from PZBA-PVA interfered with the normal function of blood cells. The hemolysis rate of PZBA-PVA was 1.1 ± 0.3%, which is within the acceptable range (5%). Figure 13 ).

[0058] Example 8: Minimum inhibitory concentration (MIC) test of polyphosphazene-based polymer PZBA

[0059] The polymer was diluted with sterile water to different concentrations (0.117, 0.235, 0.489, 0.938, 1.875, 3.75, 7.5, and 15 mg / mL). Staphylococcus aureus and Escherichia coli were first amplified overnight in LB liquid medium at 37°C with shaking. The bacterial solutions were centrifuged and diluted with PBS to a final concentration of 10⁸ CFU. 200 μL of polymer solution was mixed with 200 μL of bacterial solution in a test tube and incubated at 37°C with shaking for 12 h. The bacterial solution mixed with PBS served as a control. The absorbance of the control group was diluted to 10⁴ CFU. The remaining samples were diluted proportionally. 100 μL of the diluted solution was spread onto LB solid medium and incubated at 37°C for 24 h. The number of colonies in each petri dish was counted using ImageJ software. The inhibition rate of the polymer was calculated using the following formula:

[0060] Antibacterial rate = (number of control colonies - number of sample colonies) / number of control colonies × 100%.

[0061] like Figure 14As shown, at a concentration of 0.024 mg / mL, PPBA exhibited an antibacterial rate of 82.17 ± 0.05% against Staphylococcus aureus, while PZBA showed an antibacterial rate of 90.29 ± 0.07%. At a concentration of 0.469 mg / mL, PZBA achieved a 100% antibacterial rate, while PPBA showed an antibacterial rate of 93.20 ± 1.10%. PZBA demonstrated superior antibacterial efficacy compared to PPBA. In addition to Gram-positive bacteria, we also evaluated the antibacterial efficacy of the PZBA polymer against Gram-negative bacteria. Figure 15 At a concentration of 7.5 mg / mL, PPBA showed an antibacterial rate of only 70.35 ± 0.61% against Escherichia coli, while PZBA showed an antibacterial rate of 98.95 ± 0.57%. When the polymer concentration was 15 mg / mL, the antibacterial rates of PPBA and PZBA were 77.53 ± 2.51% and 99.54 ± 0.42%, respectively.

[0062] Example 9: In vitro antibacterial test of hydrogel

[0063] The antibacterial activity of the hydrogel was determined using a hydrogel contact antimicrobial test. Staphylococcus aureus and Escherichia coli were initially amplified by incubating overnight in LB liquid at 37°C with a shaker. The bacterial solutions were then centrifuged and diluted with PBS to a final concentration of 10. 6 CFU / mL. Add 400 μL of hydrogel to a test tube and centrifuge at 8000 rpm for 1 min to smooth the surface. Add 10 μL of 10 CFU / mL to the surface of the hydrogel. 6 CFU / mL bacterial suspension was incubated at 37℃ on a shaking table (180 rpm) for 0.5, 1, 2, and 4 h, respectively. The control group consisted of 10 μL of 10⁶ CFU bacterial suspension in an empty tube. After incubation, 1 mL of PBS was added to each tube, and the mixture was sonicated for 10 min. Then, 100 μL of the solution was coated onto LB agar plates and incubated at 37℃ for 24 h. Colonies were counted using ImageJ software. The antibacterial rate was calculated using the following formula.

[0064] Antibacterial rate = (control colony count - sample colony count) / control colony count × 100%. After Staphylococcus aureus contact with hydrogel for 0.5 h, the antibacterial rate of PZBA-PVA group was 99.81±0.04%, while that of PPBA-PVA group was 98.66±0.05%. After 1 h of contact with Staphylococcus aureus, the antibacterial rate of all groups reached over 99%, with the antibacterial rate of PZBA-PVA group being 99.99±0.05%, which was higher than that of PPBA-PVA group (99.48±0.08%). Figure 16 The antibacterial rate of PZBA-PVA against Escherichia coli at 0.5 h was 98.80 ± 2.16%, which was higher than that of the PPBA-PVA group. Figure 17After 1 hour of hydrogel treatment, the antibacterial rate of the PZBA-PVA group (99.87±0.06%) was significantly higher than that of the PPBA-PVA group (90.26±2.39%). Overall, the in vitro antibacterial effect of PZBA-PVA hydrogel was superior to that of PPBA-PVA hydrogel.

[0065] Example 10: Experiment on the application of hydrogel in infected wounds

[0066] Infected wound experiments were conducted using male SD rats (200–220 g). Rats were fasted for 8 hours prior to the experiment. Anesthesia was administered via intramuscular injection of zoletil-50 (50–75 mg / kg). Four full-thickness circular skin excision wounds were created on the back using an 8 mm diameter skin puncturist. Staphylococcus aureus (20 μL, 10 ml) was added to the wounds. 8 CFU / mL). After 10 minutes, Tegaderm TM Membranes and hydrogels were applied to the wound. The control group received only Tegaderm. TM Treatment. On days 3, 7, 10, and 14 of the experiment, rats were euthanized due to cervical dislocation. Wound area was measured, and tissue was collected in sterile centrifuge tubes. For wound bacterial count testing, wound tissue was immersed in sterile PBS and sonicated for 10 minutes to resuspend surviving bacteria. The PBS solution was then diluted 1000-fold, and 100 μL of the diluted solution was coated onto LB agar plates. The LB plates were incubated at 37°C for 24 hours, and colony counts were performed. Wound healing rate and antibacterial rate were calculated using the following formulas:

[0067] Wound healing rate = (Wound area on day 0 - Wound area on day n) / Wound area on day 0 × 100%

[0068] Antibacterial rate = (number of control colonies - number of sample colonies) / number of control colonies × 100%.

[0069] Compared with the control group, the healing rate of the PZBA-PVA hydrogel treatment group was significantly higher. Figure 18 and 19 On day 3, the healing rate in the PZBA-PVA group was 41.78±5.08%, while that in the control group was 18.29±4.98%. On day 7, the healing rate in the PZBA-PVA group was 62.85±1.06%, while that in the control group was 36.25±4.36%. On day 10, the healing rate in the PZBA-PVA treatment group exceeded 80%, while the healing rate in the control group was approximately 60%. On day 14, over 90% of the wounds in the PZBA-PVA treatment group healed without significant scab formation, while significant scab formation was observed in the control group, with a healing rate of 88.25±5.35%.

[0070] On days 3 and 10, compared with the control group (1.57 × 10⁻⁶), 5 and 1.65×104 Compared with the CFU / mL group, the PZBA-PVA group had a lower number of Staphylococcus aureus (2.83 × 10⁻⁶ CFU / mL). 4 and 3.39×10 3 CFU / mL). This trend is consistent with the healing results on days 3 and 10. Figure 20 ).

[0071] During wound healing, excessively high levels of the pro-inflammatory TNF-α are associated with impaired healing and prolonged inflammation. Therefore, qRT-PCR was used to detect the expression levels of TNF-α and vascular endothelial growth factor (VEGF) in wound tissue. Figure 21 and 22 On days 3, 7, and 14, the relative expression level of TNF-α in the control group was higher than that in the PZBA-PVA group, indicating that PZBA-PVA hydrogel has the potential to reduce the inflammatory response during wound infection. On the other hand, the relative expression level of VEGF in the PZBA-PVA group was significantly higher than that in the control group on days 3, 7, and 14, which was associated with faster healing. PZBA-PVA hydrogel can effectively reduce the number of surviving microorganisms, promptly reduce the inflammatory response, accelerate the regeneration process, and thus speed up wound healing. These results indicate that PZBA-PVA hydrogel is an excellent candidate material for antibacterial wound dressings.

Claims

1. A method for preparing a polyphosphazene-based hydrogel with inherent antibacterial properties, characterized in that, This can be achieved through the following steps: (1) Polymerize hexachlorotriphosphazene at 250°C, dissolve the resulting product in toluene, and then inject n-hexane into it. The resulting precipitate was dried under vacuum to obtain polydichlorophosphazene; (2) Dissolve polydichlorophosphazene in tetrahydrofuran, dissolve N,N-dimethylethylenediamine in tetrahydrofuran and add triethylamine, stir at 40°C for 2 h, then add to polydichlorophosphazene solution, stir at 0°C for 30 min, heat the resulting mixture to react, centrifuge, remove precipitate, dissolve and dialyze, dry to obtain polyN,N-dimethylethylenediaminephosphazene; (3) Dissolve N,N-dimethylethylenediamine phosphazene in anhydrous methanol, then dissolve 3-bromomethylphenylboronic acid in anhydrous methanol, add the 3-bromomethylphenylboronic acid solution to the N,N-dimethylethylenediamine phosphazene solution, stir at 40°C for 48 h, dialyze, and freeze dry to obtain poly[(N,N-dimethylethylenediamine)-g-(N,N,N,N-dimethylamineethyl-p-methylphenylboronic acid ammonium bromide)]phosphazene; (4) Dissolve poly[(N,N-dimethylethylenediamine)-g-(N,N,N,N-dimethylaminoethyl p-methylphenylboronic acid ammonium bromide)]phosphazene in anhydrous methanol, dissolve ethyl bromoacetate in anhydrous methanol, add the ethyl bromoacetate solution to the poly[(N,N-dimethylethylenediamine)-g-(N,N,N,N-dimethylaminoethyl p-methylphenylboronic acid ammonium bromide)]phosphazene solution, stir at 40℃ for 48 h, dialyze, and freeze dry to obtain poly[(N,N-dimethylethylenediamine)-g-(N,N,N,N-dimethylaminoethyl p-methylphenylboronic acid ammonium bromide)-g-(N,N,N,N-dimethylaminoethyl ethyl ethyl bromide ammonium bromide)]phosphazene; (5) Poly[(N,N-dimethylethylenediamine)-g-(N,N,N,N-dimethylaminoethyl p-methylphenylboronic acid ammonium bromide)-g-(N,N,N,N-dimethylaminoethyl ethyl acetate ammonium bromide)]phosphazene was dissolved in deionized water, and polyvinyl alcohol was dissolved in deionized water. 3wt% of poly[(N,N-dimethylethylenediamine)-g-(N,N,N,N-dimethylaminoethyl p-methylphenylboronic acid ammonium bromide)-g-(N,N,N,N-dimethylaminoethyl ethyl acetate ammonium bromide)]phosphazene and 3wt% of polyvinyl alcohol aqueous solution were mixed at a volume ratio of 1:1 to obtain the target polyphosphazene-based hydrogel.

2. The preparation method according to claim 1, characterized in that, In step (2), the mixture was heated to 30°C and reacted for 48 hours. It was then centrifuged at 3000 rpm for 30 minutes. The precipitate was dissolved in deionized water and dialyzed for 5 days with a cutoff of 3500.

3. The preparation method according to claim 1, characterized in that, In steps (2)-(4), the dialysis time is 5 days and the retention volume is 3500.

4. The use of the polyphosphazene-based hydrogel prepared according to claim 1 in the preparation of antibacterial and wound-healing drugs.

Images