Multifunctional polyrotaxane hydrogel FDS and application thereof

Abstract

The invention provides a multifunctional polyrotaxane hydrogel FDS. NO loaded on the FDS hydrogel has the capability of promoting cell migration, proliferation and angiogenesis at the same time. The FDS hydrogel can inhibit the activation of a downstream pathway by adsorbing extracellular DNA (cfDNA), thereby reducing the release of inflammatory factors. Animal experiments further prove that the FDS hydrogel not only can sterilize and promote cell proliferation and reconstruction of a diabetic wound area, but also can promote angiogenesis of a diabetic rat wound. Therefore, the FDS hydrogel has remarkable advantages in diabetes wound management.

Description

Technical Field

[0001] The present invention relates to the fields of medicine and molecular biology, and in particular to a multifunctional polyrotaxane hydrogel FDS and its application in diabetic wound management. Background Art

[0002] Difficult-to-heal infected wounds in diabetic patients are a common and difficult-to-treat clinical problem, significantly impacting patients' quality of life. Hyperglycemia is associated with insulin dysfunction, and abnormal glucose metabolism inhibits antioxidant expression and promotes oxidative stress. Oxidative stress triggers the massive production of reactive oxygen species (ROS), overexpression of proinflammatory cytokines, and bacterial infection, which together create a persistent inflammatory microenvironment around the wound, further exacerbating the inflammatory response. Furthermore, bacterial infection in wounds further exacerbates this problem, and the resulting inflammatory response complicates wound healing. Traditional treatments rely on antibiotics and wound debridement. However, with the overuse of antibiotics and the chronicity of wounds, the incidence of drug-resistant infections continues to increase, and conventional antibiotics often show limited effectiveness against these difficult-to-heal wounds. Traditional dressings such as gauze and bandages offer limited protection, fail to dynamically respond to changes in the wound microenvironment, and may even cause secondary damage. Therefore, the search for novel treatment strategies is urgent for addressing the problem of difficult-to-heal infected wounds in diabetic patients.

[0003] Hydrogels are considered an excellent platform for promoting wound healing due to their ability to absorb wound fluids, maintain wound moisture, and possess a modulus consistent with soft tissue. In recent years, multifunctional hydrogels have been designed to regulate the wound microenvironment, with functions including ROS scavenging, acid-responsive antimicrobial activity, immune cell regulation, and angiogenesis regulation. pH changes are one of the most prominent characteristics of skin wounds. The microenvironment of normal skin tissue is weakly acidic, while that of damaged skin tissue becomes alkaline. Wounds caused by bacterial infection have a low pH (4.5–6.5) due to the production of various acidic metabolites by bacteria. In the prior art, Lee et al. constructed a bilayer hydrogel surface with acid-responsive properties, in which the inner layer consisted of polyacrylic acid (PAA) and the outer layer consisted of chitosan (CS). Under physiological conditions, PAA is anionic, providing the surface with excellent biocompatibility. Upon bacterial invasion, as the pH decreases, PAA becomes protonated, losing its ability to adsorb tobramycin (Tob). Simultaneously, the outer layer of CS converts to a cationic NH under acidic conditions. 3+state, causing the molecular chains to repel each other and stretch, facilitating the release of tobramycin, which leads to the killing of almost all Staphylococcus aureus colonies. Furthermore, Gannimani et al. introduced a novel pH-responsive nanosystem that utilizes a dendritic lipid amphiphilic substance based on oleic acid (OLA). This dendritic lipid amphiphilic substance, namely oleic acid-sodium propionate dendritic amphiphilic substance (OLA-SPDA), exhibits faster release of vancomycin under acidic conditions. This phenomenon may be due to the protonation of the tertiary amine groups in OLA-SPDA under acidic conditions, leading to the rearrangement, expansion, or decomposition of the micelles, thereby promoting the release of vancomycin.

[0004] However, the antibiotics released by these existing technologies can induce drug resistance, so there is an urgent need to develop new methods to achieve antibacterial properties in diabetic difficult-to-heal infected wounds without inducing drug resistance. Summary of the Invention

[0005] In order to address the deficiencies in the prior art, the present invention synthesized a multifunctional polyrotaxane hydrogel, named FDS hydrogel. In this hydrogel, the tertiary amine structure of DMAPMA is protonated in an acidic environment, thereby having a bactericidal effect. The nitric oxide (NO) released by the -SNO bond that works synergistically with it has powerful bactericidal and biofilm dissipation functions. The synergistic effect of the two enables it to effectively inhibit the growth of drug-resistant bacteria MRSA. The NO loaded on the FDS hydrogel also has the ability to promote cell migration, proliferation and angiogenesis. By adsorbing extracellular DNA (cfDNA), the FDS hydrogel can inhibit the activation of downstream pathways, thereby reducing the release of inflammatory factors. Animal experiments further confirmed that FDS hydrogel can not only kill bacteria, promote cell proliferation and reconstruction in the diabetic wound area, but also promote angiogenesis in the wounds of diabetic rats. This shows that FDS hydrogel has significant advantages in diabetic wound management.

[0006] To this end, the present invention provides a multifunctional polyrotaxane hydrogel FDS, the preparation method of which is as follows:

[0007] (1) Synthesis of F-127DA: Dissolve the nonionic surfactant Pluronic F127 in dichloromethane under ice bath to obtain a Pluronic F127 solution. Slowly inject acryloyl chloride and triethylamine into the Pluronic F127 solution under nitrogen protection. Continue the reaction for a period of time. After purification, obtain pure F-127DA.

[0008] (2) Synthesis of β-CD-SNO: The 6-hydroxyl group of β-cyclodextrin was sulfonyl chlorided by p-toluenesulfonyl chloride, and then the 6-sulfonyl chloride group was replaced by thiourea to obtain the monothiol-substituted cyclodextrin β-CD-SH. Finally, the 6-thiol group of β-CD-SH was modified with sodium nitrite under acidic conditions to obtain the NO donor β-CD-SNO.

[0009] (3) Synthesis of multifunctional polyrotaxane hydrogel FDS: Dimethylaminopropyl methacrylamide (DMAPMA) and β-CD-SNO prepared in step (2) were added to the pure F-127DA prepared in step (1) to obtain a precursor solution. After mixing evenly, a photoinitiator was added and the solution was irradiated under ultraviolet light to achieve ultraviolet-induced free radical cross-linking polymerization to prepare a multifunctional polyrotaxane hydrogel FDS.

[0010] Furthermore, the photoinitiator is 2,2-dimethoxy-2-phenylacetophenone (DMPA) and N-vinyl pyrrolidone (NVP).

[0011] Furthermore, the photoinitiator is prepared by dissolving DMPA in NVP and making the concentration of DMPA be 300 mg / mL.

[0012] Furthermore, the specific steps of step (1) are as follows: 10.01 g of nonionic surfactant Pluronic F127 (M = 12600, PEG 99 -PPO 65 -PEG 99 ) was dissolved in 60 mL of dry dichloromethane. Under nitrogen protection, 0.9 mL of acryloyl chloride and 0.8 mL of triethylamine were slowly injected into the solution for 30 min. After reacting at room temperature for 24 h, the product was diluted with 200 mL of dichloromethane. 50 mL of 0.2% sodium bicarbonate solution and pure water were added to the solution in a separatory funnel, and the organic phase was collected for rotary evaporation and freeze-dried to obtain pure F-127DA.

[0013] Furthermore, the specific steps of step (2) are as follows: first, under ice bath conditions, 50.15 g of β-cyclodextrin is dissolved in 300 mL of deionized water, and 5.5 g of sodium hydroxide solid is added and stirred to dissolve until the solution becomes clear; then, 1.47 mmol / mL of p-toluenesulfonyl chloride acetonitrile solution is slowly added dropwise using a syringe pump; the ice bath is removed and the reaction is allowed to proceed at room temperature for 4 h; then, the pH value of the solution is adjusted to 9.0 using concentrated hydrochloric acid, and the solution is placed in a refrigerator at 4°C overnight; thereafter, the precipitate is collected by centrifugation and vacuum dried to obtain mono-6-p-toluenesulfonyl-β-cyclodextrin (β-CD-OTs); 2.124 g of β-CD-OTs and 2.012 g of thiourea are dissolved in 100 mL The reaction mixture was heated to reflux for 48 h in an 80% methanol-water (v / v) solution. After the reaction, the solvent was removed using a rotary evaporator. Then, 30 mL of methanol was added to the spin-dried bottle and stirred at room temperature for 1 h. The precipitate was collected by filtration, dissolved in 69 mL of 10% sodium hydroxide solution, and stirred at 50°C for 5 h. After the reaction, the pH value of the system was adjusted to 2 with 10% hydrochloric acid solution. Then, 5 mL of trichloroethylene was added to the light yellow solution and stirred at room temperature overnight. The white precipitate was recovered by filtration, washed three times with water, dried in a vacuum drying oven overnight, and recrystallized three times with ethanol aqueous solution to obtain a white solid monothiol-substituted cyclodextrin β-CD-SH. 0.115 g of β-CD-SH was dissolved in 1 mL of DMSO and 10 μL of DMSO was added. 6 M hydrochloric acid solution, vortex for 1 min, then add 10 μL of 4 M sodium nitrite solution, and the solution can be observed to change from light yellow to bright red and vortex for 5 min, add 10 mL of cold acetone to precipitate, and centrifuge to obtain pink solid β-CD-SNO.

[0014] Furthermore, the specific steps of step (3) are as follows: F-127DA (final concentration 10%) and DMAPMA solution (final concentration 1%) are mixed to form a precursor solution, β-CD-SNO is added to the precursor solution and then ultrasonicated for 30 minutes, 20 μL of DMPA / NVP photoinitiator is added to 1 mL of the precursor solution containing β-CD-SNO, and the precursor solution is exposed to ultraviolet light for 1 minute to form an FDS hydrogel, and then the FDS hydrogel is soaked in water for 3 days, the water is changed every day, and then dried to obtain the FDS hydrogel.

[0015] Furthermore, in step (3), the feed ratio of F-127DA to β-CD-SNO is 1:16.

[0016] Furthermore, the multifunctional polyrotaxane hydrogel FDS prepared by the present invention is used in diabetic wound management.

[0017] Furthermore, the multifunctional polyrotaxane hydrogel FDS prepared by the present invention is used to promote the healing of infectious diabetic wounds.

[0018] Furthermore, the multifunctional polyrotaxane hydrogel FDS prepared by the present invention is used in the preparation of products for promoting the healing of infectious diabetic wounds.

[0019] The present invention successfully synthesized a multifunctional polyrotaxane hydrogel (FDS) based on F-127DA by assembling an NO donor (-SNO)-modified β-cyclodextrin into the PPO structure of the F-127DA axle. Dimethylaminopropyl methacrylamide (DMAPMA) was then added, mixed thoroughly, and a photoinitiator was added. The terminal carbon-carbon double bond was capped by free radical polymerization via UV curing. The tertiary amine structure in the DMAPMA molecule passively responds to the acidic bacterial infection microenvironment by protonating, forming a bactericidal tertiary amine structure. The NO released through the synergistic -SNO bond has a strong biofilm dissipation capability. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Pluronic F127 and F-127DA 1 H-NMR spectrum.

[0021] Figure 2 Structural characterization of β-CD-SNO: (A) Image of β-CD-SNO mixed with Griess reagent for 5 minutes; (B) Reaction of β-CD-SNO and β-CD-SH 1 H-NMR comparison chart.

[0022] Figure 3 . FTIR spectra of freeze-dried F-127DA hydrogel, pure β-CD-SNO powder, and freeze-dried FDS hydrogel.

[0023] Figure 4 .Swelling and in vitro degradation evaluation of FDS hydrogels: (A) Swelling characteristics of hydrogels in PBS (pH 7.4); (B) Mass loss of hydrogels in PBS.

[0024] Figure 5 Rheological properties of FDS hydrogels: (A) Strain amplitude sweep of FDS hydrogels from 0.1% to 20% (1 Hz); (B) Rheological analysis of hydrogels in oscillating frequency sweep mode at 25°C; (C) Histogram of storage modulus of hydrogels.

[0025] Figure 6.Evaluation of the antibacterial performance of FD hydrogels in response to pH microenvironment: (A) Images of Staphylococcus aureus colonies on agar plates of FD hydrogels prepared with PBS at different pH values; (B) Statistical bar graph of the bactericidal effect of FD hydrogels prepared with PBS at different pH values ​​on Staphylococcus aureus; Data represent mean ± SD, and one-way ANOVA was used to analyze the different groups (****p < 0.0001).

[0026] Figure 7 Study on the effect of different spindle-ring ratios on the antibacterial properties of FS hydrogels: (A) Images of Staphylococcus aureus colonies on agar plates using FS hydrogels at different spindle-ring ratios; (B) Statistical bar graph of the bactericidal effects of FS hydrogels at different spindle-ring ratios on Staphylococcus aureus. Data represent mean ± SD, and statistical analysis of different groups was performed using one-way analysis of variance (****p < 0.0001).

[0027] Figure 8 Evaluation of FDS hydrogel against Gram-positive bacteria: (A) Photographs of Staphylococcus aureus and MRSA colonies after co-culture with hydrogel; (B) Survival rate of Staphylococcus aureus bacteria after co-culture with hydrogel; (C) Survival rate of MRSA bacteria after co-culture with hydrogel; Data represent mean ± SD, and one-way ANOVA was used to analyze the different groups statistically (*p < 0.05, ***p < 0.001, ****p < 0.0001).

[0028] Figure 9 .Study on the biofilm dissipation performance of FDS hydrogel: (A) Photographs of MRSA biofilms stained with crystal violet after co-culture with different hydrogels (F-127DA, FS, FD, and FDS); (B) Biofilms with an optical density of 570 stained with crystal violet.

[0029] Figure 10 . Expression of (A) TNF-α and (B) IL-6 in RAW264.7 cells stimulated by CpG solution after different hydrogel treatments.

[0030] Figure 11 Scratch assay results on FDS hydrogels: (A) Images of wound scratch migration assays and (B) quantification of HUVEC cell migration on F-127DA, FS, FD, and FDS hydrogels (n = 3) (Scale bar = 200 μm); Data represent mean ± SD. Statistical analysis of different groups was performed using one-way ANOVA (*p < 0.05, **p < 0.01).

[0031] Figure 12.Tube formation experimental results of FDS hydrogels: (A) Optical images of HUVEC cell tube formation treated with different hydrogels, and quantified by counting the number of (B) nodes (n=3) and (C) grids (n=3) in each group (scale bar = 200 μm).

[0032] Figure 13 In vivo diabetic wound healing evaluation of hydrogels: (A) Treatment regimen for diabetic wounds using different hydrogel formulations; (B) Representative images of the wound healing process in diabetic rats; (C) Quantitative data of relative wound area at different time points (n=3); (D) Quantitative data of relative wound area at different time points (n=3); (E) Blood glucose level recording curves of rats in different groups; (F) Photograph of LB agar plate 12 hours after plating of wound pus on day 3; Data represent mean ± SD; *p < 0.05, **p < 0.01.

[0033] Figure 14 (A) H&E staining and (B) Masson trichrome staining of wound tissues healed on days 7 and 14 (scale bar, 200 μm, green arrows: hair follicles; black arrows: blood vessels; black dashed line: epidermis); (C) epidermal thickness, (D) granulation tissue thickness, and (E) collagen deposition of different wound tissues on day 14.

[0034] Figure 15 . Immunofluorescence staining of wound tissues on days 7 and 14 after treatment: (A) α-smooth muscle actin (αSMA, green), platelet-endothelial cell adhesion molecule (CD31, red), and cell nuclei (DAPI, blue) (Scale bar = 100 μm); (B) Statistics of CD31-positive blood vessels in wound tissues (n = 3); (C) Statistics of αSMA in wound tissues (n = 3); Data represent mean ± SD; *p < 0.05, **p < 0.01.

[0035] Figure 16 Immunofluorescence staining of wound tissues on days 7 and 14 after treatment: (A) tumor necrosis factor-α (TNFα, green), interleukin 6 (IL-6, red), and cell nuclei (DAPI, blue) (scale bar = 100 μm); (B) statistical data of TNF-α expression in wound tissues (n = 3); (C) statistical data of IL-6 in wound tissues (n = 3); data represent mean ± SD; *p < 0.05, **p < 0.01, ****p < 0.0001.

[0036] Figure 17Immunohistochemistry results of wound tissues on the seventh day after treatment with different hydrogels: (A) Immunohistochemistry images of interleukin 10 (IL-10) and vascular endothelial growth factor (VEGF); (B) Statistical data of IL-10 expression in wound tissues (n=3); (C) Statistical data of VEGF in wound tissues (n=3); Data represent mean ± SD; (*p<0.05, **p<0.01, ****p<0.0001).

[0037] Figure 18 Biocompatibility of FDS hydrogels: (A) Fluorescence images of live / dead staining of 3T3 cells after 3-day incubation with F-127DA, FS, FD, and FDS hydrogel extracts; (B) cell viability of 3T3 and (C) HUVEC cells after 1 and 3-day incubation with F-127DA, FS, FD, and FDS hydrogel extracts; (D) photographs and (E) hemolysis rates of F-127DA, FD, and FDS hydrogels.

[0038] Figure 19 Liver and kidney function indicators of healthy rats and rats treated with different hydrogels: (A) alanine aminotransferase ALT, (B) aspartate aminotransferase AST, (C) alkaline phosphatase ALP (D) γ-glutamyl transpeptidase γ-GT, (E) serum urea nitrogen UREA, and (F) blood creatinine CREA.

[0039] Figure 20 .H&E staining images of the internal organs of rats in each group after treatment with different hydrogels (scale bar = 100 μm).

[0040] Figure 21 .Record of body weight changes of rats in each group after treatment with different hydrogels for different days (n=3).

[0041] Figure 22 Changes in the blood routine indicators of rats in each group after treatment with different hydrogels, including (A) red blood cell count, (B) white blood cell count, (C) platelet count, (D) hemoglobin, (E) lymphocyte count, (F) lymphocyte percentage, (G) neutrophil count, (H) neutrophil percentage, (I) monocyte count, (J) monocyte percentage, (K) mean platelet volume, (L) platelet distribution width, (M) hematocrit, (N) mean corpuscular volume, (O) mean corpuscular hemoglobin content, and (P) mean corpuscular hemoglobin concentration. DETAILED DESCRIPTION

[0042] Example 1 Preparation of multifunctional polyrotaxane hydrogel FDS

[0043] (1) Synthesis of F-127DA

[0044] The nonionic surfactant Pluronic F127 (M=12600, PEG 99 -PPO 65 -PEG 99 , 10.01 g, 0.8 mmol, purchased from Sigma-Aldrich Chemical Company, USA) was dissolved in 60 mL of dry dichloromethane. Under nitrogen, 0.9 mL of acryloyl chloride and 0.8 mL of triethylamine were slowly injected into the solution over a period of 30 minutes. After reacting at room temperature for 24 hours, the product was diluted with 200 mL of dichloromethane. 50 mL of 0.2% sodium bicarbonate solution and purified water were added to the solution in a separatory funnel. The organic phase was collected and rotary evaporated, followed by freeze-drying to yield pure F-127DA.

[0045] (2) Synthesis of β-CD-SNO

[0046] Generally speaking, the synthesis of β-CD-SNO is divided into three steps: first, the 6-hydroxyl group of β-cyclodextrin is sulfonyl-chlorinated using p-toluenesulfonyl chloride; second, the 6-sulfonyl chloride group is replaced by thiourea to obtain a monothiol-substituted cyclodextrin (β-CD-SH); finally, under acidic conditions, the 6-sulfhydryl group is modified using sodium nitrite to obtain the NO donor β-CD-SNO. The detailed synthesis steps are as follows:

[0047] First, under ice bath conditions, 50.15g of β-cyclodextrin was dissolved in 300mL of deionized water, and 5.5g of sodium hydroxide solid was added and stirred until the solution became clear. Then, a 1.47mmol / mL solution of p-toluenesulfonyl chloride in acetonitrile was slowly added dropwise using a syringe pump. The ice bath was removed and the reaction was allowed to proceed at room temperature for 4h. Subsequently, the pH value of the solution was adjusted to 9.0 using concentrated hydrochloric acid, and the solution was placed in a refrigerator at 4°C overnight. Afterwards, the precipitate was collected by centrifugation and vacuum dried to obtain mono-6-p-toluenesulfonyl-β-cyclodextrin (β-CD-OTs, yield: 30.34%).

[0048] 2.124 g of β-CD-OTs and 2.012 g of thiourea were dissolved in 100 mL of 80% methanol / water (v / v) and heated under reflux for 48 hours. After the reaction, the solvent was removed using a rotary evaporator. 30 mL of methanol was then added to the spin-dried bottle and stirred at room temperature for 1 hour. The precipitate was collected by filtration, dissolved in 69 mL of 10% sodium hydroxide solution, and stirred at 50°C for 5 hours. After the reaction, the pH was adjusted to 2 with 10% hydrochloric acid. 5 mL of trichloroethylene was added to the pale yellow solution and stirred at room temperature overnight. The white precipitate was recovered by filtration, washed three times with water, dried in a vacuum oven overnight, and recrystallized three times from aqueous ethanol. This yielded β-CD-SH, a white solid with monothiol-substituted cyclodextrin (yield: 14.17%).

[0049] β-CD-SH (0.115 g) was dissolved in 1 mL of DMSO, and 6 M hydrochloric acid solution (10 μL) was added. After vortexing for 1 min, 10 μL of 4 M sodium nitrite solution was added. The solution was observed to change from light yellow to bright red and vortexed for 5 min. 10 mL of cold acetone was added for precipitation, and pink solid β-CD-SNO was obtained by centrifugation (yield: 64.53%).

[0050] (3) Synthesis of multifunctional polyrotaxane hydrogel

[0051] By adding different monomers (DMAPMA and β-CD-SNO) to the prepared pure F-127DA, the present invention successfully synthesized a novel nanostructured ...

[0052] In the experiment, 2,2-dimethoxy-2-phenylacetophenone (DMPA) / N-vinylpyrrolidone (NVP) was used as a photoinitiator, and the final concentration of F-127DA was 10%.

[0053] According to the description in Table 1, the present invention obtained different groups of hydrogel samples. The first is Group F, which is pure F-127DA hydrogel. Using F-127DA as the axis, the hydrogel was prepared through a gelation process. Next is the preparation of FS hydrogel. On the F-127DA axis, β-CD-SNO was self-assembled onto the PPO unit (feed axis to ring ratio = 1:16), and then ultrasonicated for 30 minutes. Then, the precursor solution was exposed to ultraviolet light for 1 minute to form a polyrotaxane hydrogel. The preparation process of FD hydrogel is to mix F-127DA solution (final concentration 10%) and 2,2-dimethoxy-2-phenylacetophenone (DMAPMA) solution (final concentration 1%), add the photoinitiator DMPA / NVP (DMPA was dissolved in NVP to a DMPA concentration of 300 mg / mL), and irradiate the precursor solution under ultraviolet light for 1 minute to form a cationic hydrogel. Finally, the FDS hydrogel was prepared using a similar process to the FD hydrogel, except that β-CD-SNO was added to the precursor solution and sonicated for 30 minutes before UV irradiation. The precursor solution was then exposed to UV light for 1 minute to form the FDS hydrogel. The hydrogel was then immersed in water for 3 days, with the water changed daily. The gel was then dried for later use.

[0054] Table 1. Hydrogel name abbreviations and chemical composition

[0055]

[0056] Example 2 Structure Verification

[0057] (1) 1 H-NMR spectrum

[0058] In order to perform nuclear magnetic resonance (NMR) spectroscopy analysis, the present invention prepared F-127DA and β-CD-SNO samples according to Example 1, each sample weighing about 10 mg, and dissolved them in 500 μL of heavy water (D2O). Then, the dissolved samples were transferred to an NMR tube, ensuring that the operation was carried out at room temperature. Subsequently, the NMR spectra were measured using a NMR spectrometer (Bruker-300, Bruker, Germany) at room temperature, and the obtained NMR spectral data were further analyzed using MestReNova software. 1 The H-NMR spectra are Figure 1 and Figure 2 As shown in B.

[0059] according to Figure 1The results presented show that the peak at 1.07 ppm corresponds to the CH3 proton signal in the PPO block, while the peaks between 5.91 and 6.35 ppm represent the proton signals of the acrylate terminals of the F-127DA molecule. This analysis strongly confirms the successful synthesis of the F-127DA molecule.

[0060] according to Figure 2 The results presented in B compare the resonance peaks (δ = 3.1–2.7 ppm) of the diastereoisomers H6b H6′b of the in situ nitrosated β-CD-SH in C and D2O with the SNO resonance peaks of H6b H6′b where the SH group is strongly deshielded, confirming the conversion of the sulfhydryl (SH) group to the nitroso (SNO) group. Subsequently, the synthesis of β-CD-SNO was verified by using a Griess reagent. It was observed that after the addition of the Griess reagent, the β-CD-SNO solution showed a purple-red color change (e.g., Figure 2 A), which strongly confirmed the successful synthesis of β-CD-SNO.

[0061] (2) Infrared spectrum

[0062] In order to carry out sample testing, it is first necessary to prepare the working area and ensure that the agate mortar is clean. Then, take an appropriate amount of β-CD-SNO powder, F-127DA hydrogel and FDS hydrogel samples prepared in Example 1 and place them in the agate mortar respectively. Next, add dry potassium bromide powder to the sample and then grind it finely to ensure that the sample and potassium bromide powder are evenly mixed and the particle size is fine. Subsequently, take out an appropriate amount of powder from the mixture and place it in a device with a vacuum pressure of 20 mmHg for tableting, and the tableting time is 3 to 6 minutes. Through this step, a transparent thin sheet sample can be obtained. Set the scanning range of the Fourier infrared spectrometer (VERTEX70, Bruker, Germany) to 4000 to 400 cm -1 The prepared thin slice sample was placed in a Fourier transform infrared spectrometer for spectrum detection. The infrared spectrum obtained is shown in the figure below. Figure 3 shown.

[0063] Depend on Figure 3 It can be seen that in the spectrum of pure β-CD-SNO, a broad band can be observed, located at 3100 cm -1 and 3600cm -1 The broadband is generated by the symmetric and antisymmetric OH stretching vibration modes. It is worth noting that the 3288 cm -1 The hydroxyl band at the position shifts to a higher frequency of 3372 cm -1This frequency shift may be due to the non-covalent interaction between β-CD-SNO and the macromolecular monomer backbone containing the PPO block. In addition, in the spectrum of F-127DA hydrogel, a peak at 2,879 cm -1 The CH stretching vibration mode in β-CD is located at approximately 2,924 cm -1 In addition, a peak at 2,879 cm -1 The absorption band is slightly shifted to low frequency, which may be caused by the macromolecular monomer chains contained in the inclusion complex.

[0064] Example 3 Hydrogel swelling and in vitro degradation experiments

[0065] Water swelling is one of the most important properties of hydrogel materials. Swelling experiments can be used to evaluate their ability to absorb and swell under different environmental conditions, as well as their sensitivity to water. Meanwhile, in vitro degradation experiments are designed to simulate the degradation of hydrogel materials in an in vitro biological environment to understand their degradation rate and stability.

[0066] Swelling experiment details: To measure the swelling ratio, a pre-weighed (Wf) freeze-dried hydrogel was placed in a 37°C PBS solution (pH = 7.4). At different time points, the weight of the hydrogel (Wa) was remeasured. The swelling ratio (SR) was calculated as follows: SR = (Wa - Wf) / Wf × 100%.

[0067] In vitro degradation assay steps: 6 mm diameter hydrogels (200 μL) were prepared in a 48-well plate and formed under UV light. After 1 hour, 2 mL of PBS was added as a degradation medium. The plate was then placed in a constant temperature shaking incubator (DPX-9052B-2, Shanghai Fuma Laboratory Equipment Co., Ltd.) at 60 rpm and 37°C. At various time points, the PBS was removed and the weight of the hydrogel was measured, followed by the addition of 2 mL of fresh PBS. Three replicates were analyzed for each group.

[0068] For the three hydrogels (F-127DA, FD and FDS) prepared in Example 1, their swelling behaviors showed different characteristics during immersion in PBS. The experimental results are as follows: Figure 4 As shown in A. Figure 4 As shown in Figure 1, all three hydrogels reached a state of equilibrium after water absorption and swelling within about 1 hour. However, the swelling rates of F-127DA and FD hydrogels were slightly lower than those of FDS hydrogel. In addition, in vitro degradation experiments revealed the degradation characteristics of these hydrogel materials in a simulated in vitro environment. The experimental results are shown in Figure 1. Figure 4 As shown in B. Figure 4As shown in Figure 2, after 7 days of PBS immersion, the FDS hydrogel completely degraded, while the F-127DA and FD hydrogels degraded relatively slowly, with residual masses of 37.5% and 21.7%, respectively. This difference may be attributed to the release of NO in the FDS hydrogel, which resulted in a higher swelling ratio and degradation rate compared to the F-127DA and FD hydrogels.

[0069] Example 4 Verification of the mechanical properties of hydrogel

[0070] Rheological experiments evaluate the rheological properties and deformation ability of materials, as well as their responses under different shear stresses, by applying external shear forces. The present invention uses a rotational rheometer (Kinexus pro, Malvern, UK) to measure the rheological properties of several supramolecular hydrogels prepared in Example 1. During the amplitude scanning stage, the storage modulus (G') remains higher than the loss modulus (G") across the varying strain range (0.1% to 10%), indicating that the FDS hydrogel has excellent viscoelasticity and good resistance to large strains, such as Figure 5 As shown in A.

[0071] In addition, the values ​​of G' and G" remain relatively stable in the strain range from 0.1% to 5%, which indicates that the FDS hydrogel is in the linear viscoelastic region. Further frequency sweep experiments selected the strain value (1%) in the linear viscoelastic region, as shown in Figure 5. Figure 5 As shown in Figure B, G' and G" show obvious frequency dependence in the frequency range of 1 to 10 Hz, maintaining the hydrogel state (G'>G"), indicating that the integrity of the fiber network is maintained in a wide frequency range. At the same time, the other three gels (F-127DA, FS, and FD) also maintain the hydrogel state (G'>G") in the same frequency range. By comparing the storage modulus, it was observed that with the introduction of β-CD-SNO and DMAPMA, the storage modulus gradually decreased, especially when β-CD-SNO and DMAPMA were introduced at the same time, the decrease in the storage modulus was more significant, as shown in Figure 2. Figure 5 As shown in C.

[0072] Example 5 Verification of the antibacterial and biofilm dissipation effects of the hydrogel

[0073] (1) Antibacterial activity test

[0074] To evaluate the antibacterial properties of FDS hydrogels, Staphylococcus aureus (S. aureus) and methicillin-resistant Staphylococcus aureus (MRSA) were first cultured in LB liquid culture medium and incubated overnight at 37°C with continuous stirring at 150 rpm. Subsequently, 100 μL of the bacterial suspension was transferred to 10 mL of fresh LB broth and incubated for another 6 hours to reach the logarithmic growth phase. The bacterial culture was then centrifuged at 5000 rpm for 5 minutes to remove the LB liquid culture medium and then washed twice with PBS (pH 7.4) to remove excess culture medium. Finally, the bacteria were suspended in PBS (pH 7.4) at the desired concentration.

[0075] 200 μL of the hydrogel prepared in Example 1 was added to each well of a 48-well plate and sterilized by ultraviolet irradiation to ensure the aseptic conditions of the experiment. 20 μL of a bacterial suspension of Staphylococcus aureus (S. aureus) and methicillin-resistant Staphylococcus aureus (MRSA) (concentration of 10 7 CFU / mL, suspended in PBS solution) was added to the surface of the hydrogel disc. The 48-well plate was placed in an incubator and cultured at 37°C for 3 hours to provide suitable growth conditions. The bacteria surviving on the hydrogel were resuspended with sterile PBS solution, and the resuspended bacteria were suspended with 1 mL of sterile PBS solution, and 100 μL of the bacterial solution was added to the LB agar. At the same time, 20 μL of bacterial suspension (concentration of 10 7 CFU / mL, suspended in PBS solution) was added to 1 mL of PBS. After incubation for 18-24 hours, the culture dish was photographed.

[0076] By observing the growth, the antibacterial effect of the hydrogel on Staphylococcus aureus and methicillin-resistant Staphylococcus aureus can be preliminarily evaluated. In addition, 100 μL of resuspended bacterial suspension was added to a test tube containing 10 mL of culture medium, and the mixture was then placed in a shaker (150 rpm) and cultured for 8 hours. The absorbance of the solution at 600 nm was measured using a microplate reader (MULTISKAN MK3, Thermo Fisher Scientific), and the bacterial survival rate was calculated. By comparing the absorbance values ​​of the test group and the control group, the bactericidal effect of the hydrogel on bacteria can be evaluated.

[0077] (2) Biofilm dissipation experiment

[0078] Prepare the hydrogel in a 48-well plate, add 200 μL of hydrogel to each well, and sterilize using ultraviolet light to ensure sterile conditions. 7CFU / mL, suspended with PBS solution) was added to each well, 20 μL was added to each well. The plate was placed in an incubator for incubation for 3 hours. The surviving bacteria on the hydrogel were resuspended with sterile PBS, and the surviving bacteria were suspended with 1 mL of sterile PBS solution. At the same time, 20 μL of bacterial suspension was added to 1 mL of PBS in the control group. The treated bacterial solution was moved into a 24-well plate, 1 mL of culture medium was added, and the mixture was then incubated for 36 hours. The formed biofilm was washed twice with PBS and then stained with crystal violet (1 mg / mL) for 20 minutes. The biofilm was washed again twice with PBS and then photographed. Finally, the biofilm was dissolved in 100 μL of ethanol (95%), and the absorbance of the solution at OD570 wavelength was measured using a microplate reader (MULTISKANMK3, Thermo Fisher Scientific).

[0079] (3) Antibacterial activity test results

[0080] Bacterial metabolites form a complex ecosystem within the infection microenvironment. Organic acids produced during metabolism, such as acetic acid, lactic acid, and malic acid, lower the pH of the microenvironment to 4.5-6.5, creating an acidic environment suitable for bacterial reproduction. The acidic environment triggered by bacterial metabolites can affect the performance of materials. For example, bacterial microenvironment-responsive materials can adapt to changes in the infection microenvironment and exert antibacterial activity. This helps to produce reversible antibacterial activity that can be regulated during the progression and suppression of bacterial infection, and reduces toxicity to normal tissues during recovery. The DMAPMA structure is rich in tertiary amine groups. Under acidic conditions, these groups will be protonated to form tertiary amine structures with bactericidal effects.

[0081] In order to accurately evaluate the acid-responsive antibacterial effect of DMAPMA, PBS buffer solutions with different pH values ​​(5.5, 6.5, 7.4, and 8.5) were used to prepare hydrogel precursor solutions, and the antibacterial activity experiments of this example were used to verify the bactericidal effects of some hydrogels prepared in Example 1. Figure 6 、 7 and 8.

[0082] exist Figure 6 The results presented in A show that compared with the pH = 7.4 and pH = 8.5 groups, after 12 hours of co-culture with the FD hydrogels in the pH = 5.5 and 6.5 groups, a large number of bacterial colonies appeared on the agar plates of the former two groups, while no obvious bacterial growth was observed on the plates of the latter two groups. When the culture experiment was extended to 24 hours, a large number of bacterial colonies also appeared on the agar plates of the pH = 6.5 group. Interestingly, there were almost no bacterial colonies in the pH = 5.5 group at 12 hours and 24 hours. Figure 6In Figure B, it can be clearly observed that the survival rate of Staphylococcus aureus in the pH 5.5 group is the lowest, highlighting the strong bactericidal ability of the FD hydrogel prepared in PBS buffer at pH 5.5. This result shows that the FD hydrogel has antibacterial effects under the acidic microenvironment of bacterial infection, highlighting its prospects and potential application in treating diabetic wound infections.

[0083] Pluronic F-127 contains 65 PPO units, and every two PPO units can form an inclusion complex with at most one β-cyclodextrin molecule, which means that 1 mol of F-127DA can accommodate at most 32 mol of β-cyclodextrin. As a supramolecular polyrotaxane hydrogel, β-CD-SNO that can be effectively assembled onto the copolymer axis can play a corresponding role. In this example, FS hydrogels were prepared by adjusting different feed ratios, and antibacterial experiments were performed on these hydrogels to determine the feed ratio with the best antibacterial performance for subsequent research.

[0084] according to Figure 7 The results showed that after 16 hours of incubation on the culture plate, a large number of bacterial colonies were observed on the LB agar plates with feed ratios of 1:8 and 1:32, and there was no significant difference compared with the control group. However, on the agar plate of the group with a feed ratio of 1:16, only a small number of bacterial colonies appeared, indicating that the FS hydrogel with a feed ratio of 1:16 has good antibacterial properties. This phenomenon may be due to the fact that neither too low nor too high a feed ratio can effectively assemble β-cyclodextrin on the F-127DA molecular axis, ultimately causing it to be free in the hydrogel and dissolve after soaking in deionized water. The subsequent feed ratio of polyrotaxane hydrogels was F-127DA:β-CD-SNO=1:16.

[0085] Diabetic patients experience prolonged elevated blood sugar levels, which hinder wound healing. Even long-term wounds that don't heal can become susceptible to severe bacterial infections, particularly those caused by drug-resistant bacteria, which can further impair wound healing and endanger the patient's life. Therefore, antimicrobial properties have become a key indicator for wound dressings. This study aimed to evaluate the antimicrobial activity of FDS hydrogels, testing their antimicrobial properties against Staphylococcus aureus and methicillin-resistant Staphylococcus aureus (MRSA), pathogens commonly found in chronic wounds.

[0086] like Figure 8The results presented in A show that a large number of bacterial colonies were observed on the culture dishes of the F-127DA hydrogel group that lacks antibacterial functional groups. The FS group only has independent nitric oxide (NO) antibacterial properties, and the FD group has an acid-responsive tertiary amine antibacterial structure. When these two are used alone, bacterial colonies can be seen on the agar plate regardless of Staphylococcus aureus or MRSA. However, in the FDS group, the synergistic NO and DMAPMA showed excellent antibacterial effects, and there was almost no obvious bacterial colony formation on the agar culture dish. Further statistical analysis of bacterial survival rate ( Figure 8 B, Figure 8 C) confirmed these results. Therefore, the present experimental results confirm that the synergistic effect of acid-responsive tertiary amine structure and NO in FDS hydrogels imparts excellent antibacterial properties to them.

[0087] (4) Biofilm dissipation experimental results

[0088] Biofilm is an adhesive structure formed by the aggregation of microorganisms on a solid surface. It is highly resistant to drugs and difficult to treat. In the study of antibacterial materials, biofilm dissipation experiments are used to evaluate whether the material can destroy the biofilm that has been formed, thereby reducing the drug resistance of bacteria and improving the antibacterial effect. Biofilm is one of the main reasons for the aggravation of bacterial resistance and wound infection. In order to explore the inhibitory effect of hydrogel on biofilm formation, according to the biofilm dissipation experiment of this embodiment, the bacterial biofilm produced by methicillin-resistant Staphylococcus aureus (MRSA) was used for experimental evaluation. The experimental results are as follows Figure 9 shown.

[0089] Depend on Figure 9 As shown in A, the biofilm formation in the FDS group was extremely limited, while the control group, F-127DA group, and FD group all had a large amount of biofilm. Although the FS group also had slight biofilm formation, the absorbance measurement at 570 nm for the FDS and FS groups ( Figure 9 B) shows a prominent biofilm inhibition effect. This strongly suggests that nitric oxide (NO) plays a key role in biofilm dissipation, further supporting the existence of anti-biofilm properties and the significant effect of FDS hydrogel in inhibiting biofilm formation.

[0090] Example 6 In vitro anti-inflammatory evaluation

[0091] (1) In vitro anti-inflammatory experiment

[0092] RAW 264.7 cells (mouse macrophages) were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin. At the beginning of the experiment, RAW 264.7 macrophages were plated at 2×10 5The cells were seeded into a 24-well culture plate at a density of 10 cells / mL and the culture plate was placed in an incubator at 37°C to allow the cells to attach to the surface of the culture plate, which usually takes 30 minutes. Subsequently, 2 μL of CpG (at a concentration of 1 mg / mL) was added to the culture. Over the next 30 minutes, Transwell chambers containing different hydrogels were added to the well plate. The well plate was then incubated, usually in an incubator at 37°C for 24 hours. Finally, the human TNF-α ELISA kit and the human IL-6 ELISA kit (Shenzhen Xinbosheng Biotechnology Co., Ltd.) were used to analyze the cells. Human TNF-α ELISA kit, The expression levels of TNF-α and IL-6 in the supernatant were measured using the Human IL-6 ELISA kit. The detailed steps are as follows:

[0093] 1) One day before running the ELISA, dilute the capture antibody in 1x coating buffer. Add 100 μL of this capture antibody solution to all wells of a 96-well plate. Seal the plate and incubate overnight (16-18 hours) between 2°C and 8°C.

[0094] 2) Bring all reagents to room temperature (RT) before use. Wash the plate four times with at least 300 μL of wash buffer per well, and vigorously tap the remaining buffer by inverting the plate on absorbent paper. All subsequent washes should be performed in a similar manner.

[0095] 3) To block nonspecific binding and reduce background, add 200 μL of 1× Assay Diluent A to each well;

[0096] 4) Seal the plate and incubate at room temperature for 1 hour while shaking on a plate shaker (e.g., 500 rpm, 0.3 cm circular orbit). All subsequent shaking incubations should be performed in a similar manner.

[0097] 5) While blocking the plate, prepare appropriate sample dilutions (if necessary) and standards. Wash the plate 4 times with wash buffer.

[0098] 6) Add 100 μL / well of standard or sample to the appropriate wells. If dilution is necessary, dilute the sample with 1× Assay Diluent A before adding to the wells. Seal the plate and incubate at room temperature with shaking for 2 hours. Wash the plate four times with wash buffer.

[0099] 7) Add 100 μL of diluted detection antibody solution to each well, seal the plate and incubate with shaking at room temperature for 1 hour, and wash the plate four times with wash buffer;

[0100] 8) Add 100 μL of diluted Avidin-HRP solution to each well, seal the plate and incubate with shaking at room temperature for 30 min. Wash the plate five times with wash buffer.

[0101] 9) Add 100 μL of freshly mixed TMB substrate (A+B; ratio is 1:1) and incubate in the dark for 15 min.

[0102] Positive wells should turn blue;

[0103] 10) Stop the reaction by adding 100 μL of stop solution to each well. Positive wells should turn from blue to yellow.

[0104] 11) Read the absorbance at 450 nm within 15 min using a microplate reader (MULTISKAN MK3, Thermo Fisher Scientific). If the microplate reader can read at 570 nm, subtract the absorbance at 570 nm from the absorbance at 450 nm.

[0105] (2) Experimental results

[0106] In order to explore whether cationic hydrogels have the potential to inhibit the activation of Toll-like receptor 9 (TLR9) stimulated by extracellular DNA (cfDNA), the present invention placed some cationic hydrogels prepared in Example 1 in Transwell chambers, and cultured RAW 264.7 cells in a medium containing CpG. The expression levels of inflammatory factors (such as TNF-α and IL-6) in the cells were detected according to the in vitro anti-inflammatory experiment of this example. The test results are as follows: Figure 10 shown.

[0107] according to Figure 10 The data presented show that treatment with cationic hydrogels FD and FDS significantly inhibited the expression of intracellular TNF-α and IL-6, further confirming the effectiveness of these cationic hydrogels, which can successfully capture negatively charged CpG molecules and block the stimulation of the intracellular TLR9 pathway. In contrast, since F-127DA lacks positively charged groups, it did not inhibit the expression of TNF-α and IL-6, which is consistent with its relatively low cfDNA adsorption capacity. At the same time, F-127DA did not activate cells to express TNF-α and IL-6, indicating that this hydrogel itself did not trigger macrophage activation. Based on the above data, it can be concluded that cationic FD and FDS hydrogels have the ability to capture extracellular cfDNA and show significant effects in further inhibiting the activation of TLR9 downstream pathways.

[0108] Example 7 Scratch test and angiogenesis-promoting test

[0109] (1) Scratch test

[0110] The scratch assay is a widely used technique in cell-based experiments, primarily to assess cell migration and responses during wound healing. Human umbilical vein endothelial cells (HUVECs) are seeded. A 200μL pipette tip is then used to create a linear indentation on the surface of a cell culture dish, creating a nick in the cell monolayer. Subsequently, a Transwell device containing different types of hydrogels is placed in the cell culture dish, and the system is incubated in an incubator for 12 hours.

[0111] The specific steps of the scratch test are as follows: HUVEC cells were plated at 2×10 5 Cells were seeded into 6-well plates at a density of 100 / well. After culturing for 24 hours, a 200μL pipette tip or other suitable tools was used to neatly scratch each cell well. Make sure the width of the scratches is the same. Then wash with PBS 3 times to remove cell debris. Place a nested plate with gels of different components (F-127DA, FS, FD and FDS), and then use an inverted microscope to take photos of the scratch area at 0h and 12h after the start of the experiment. Image processing software was used to calculate the initial area (A1, i.e. the area of ​​the scratch) and the area after 12h of culture (A2, i.e. the area after cell migration) based on the scratch area in the photo. The migration area was then calculated using the following formula: Migration area = [(A1-A2) / A1]×100%. The results of the scratch experiment are shown in the figure. Figure 11 shown.

[0112] When observing the image ( Figure 11 A) found that after 12 hours of culture, the unfilled part of the initial scratch area in the experimental groups with the addition of functional hydrogel (FS) and multifunctional polyrotaxane hydrogel (FDS) was reduced, and this phenomenon was statistically significant (**p<0.01) compared with the control group ( Figure 11 B) This suggests that FS and FDS hydrogels may promote HUVEC cell migration, thereby accelerating the cells' ability to fill the wound area during wound healing. This result demonstrates the potential positive effects of NO on wound healing and provides valuable insights for further research into its mechanisms and applications.

[0113] (2) Angiogenesis-promoting experiment

[0114] The purpose of angiogenesis-promoting experiments is to evaluate the regulatory effects of cells or biomaterials on the angiogenesis process. These experiments aim to mimic the in vivo formation of new blood vessels. These experiments typically involve culturing endothelial cells (e.g., human umbilical vein endothelial cells) or other relevant cell types on three-dimensional scaffolds to investigate their ability to form microvascular conduits and conduct detailed analysis.

[0115] The specific experimental steps of the angiogenesis-promoting experiment are as follows: HUVEC cells are digested and removed from the culture flask and transferred to a pre-prepared 24-well plate. Culture in culture medium for 12-24 hours to allow the cells to adapt to the new culture conditions. Place an appropriate amount of different groups of gels in each transwell well, nest the transwell in a 24-well plate, and co-culture with HUVEC cells. Place the 96-well plate, sterilized pipette tip, and Matrigel in a 4°C refrigerator overnight to ensure the stability of Matrigel. Dilute Matrigel 2-fold, take 50 μL and put it into each 96-well plate well, then place the 96-well plate in a 37°C incubator and incubate for 30 minutes to allow Matrigel to form a gel. The HUVEC cells in the 24-well plate are transferred to a 96-well plate pre-coated with Matrigel at a density of 104 cells / well. The cells are cultured in the 96-well plate for 3 hours to promote tube formation. Use a microscope to observe the tube formation of cells in the 96-well plate. The degree of tube formation is evaluated based on the number, length, and connection of the tubes formed. The results of the angiogenesis-promoting experiment are as follows. Figure 12 shown.

[0116] like Figure 12 As shown in A, HUVECs in the control and FD groups failed to form tubular structures, and only showed sporadic cell nodes and aggregation. In contrast, the FDS group significantly stimulated the tubular structure formation of HUVEC cells due to the presence of NO, forming 25 tubes and 60 nodes ( Figure 12 B) These results clearly indicate that FDS can significantly promote HUVEC cell proliferation and tube formation.

[0117] Example 8 Animal Experiment

[0118] (1) Construction of a wound model of diabetes combined with drug-resistant bacteria infection

[0119] Due to their high blood sugar status, diabetic patients are often accompanied by factors such as impaired immune function and limited blood supply, which makes it difficult for their wounds to heal effectively. Such difficult-to-heal wounds are prone to infection, biofilm formation, and may even lead to serious complications such as gangrene and granuloma. Therefore, studying the mechanism of wound healing in diabetic patients and finding effective treatment methods have important clinical application value. In order to evaluate the effect of the FDS hydrogel prepared in the example in promoting the difficult-to-heal wounds of diabetes combined with drug-resistant bacterial infection, this example uses streptozotocin (STZ) to induce the manufacture of a diabetic rat model, and at the same time uses a wound model infected with methicillin-resistant Staphylococcus aureus (MRSA) ( Figure 13 A).

[0120] according to Figure 13E data, daily STZ injection for one week resulted in a continuous increase in blood glucose levels, and the blood glucose level on the 7th day was significantly higher than 16.6mmol / L (compared with the healthy group), thus confirming the successful construction of a diabetic rat model. After the successful establishment of the diabetic rat model, a wound infected with drug-resistant bacteria was further constructed on their backs. First, the hair on the back of the diabetic rats was shaved, and a circular area with a diameter of 10mm was outlined using a template. Next, surgical scissors were used to construct the wound. Then, the wound was infected with methicillin-resistant Staphylococcus aureus (MRSA). The next day, pus was collected from the wound and smeared on an LB agar plate. The results showed that the formation of multiple bacterial colonies was observed in the modeling group, which was not observed in the healthy control group (see Figure 13 F). These results confirm the successful establishment of a diabetic, drug-resistant bacteria (MRSA) infection model of difficult-to-heal wounds.

[0121] On the third day of wound infection, the wounds in the control group showed obvious symptoms of suppuration, which further indicated that the wounds were infected ( Figure 13 B) In contrast, the wound area of ​​the FD and FDS groups was smaller and no obvious pus was present. Figure 13 It can also be seen from the LB agar plate of F that the total number of colonies generated in the FDS group is significantly lower than that in the control group and the FD group. This can be attributed to the excellent antibacterial properties of the FDS hydrogel, which is consistent with the results of the in vitro antibacterial experiment. The wounds of the rats in the healthy group healed on their own under natural conditions because of their normal physical health. After 14 days of observation, only the wounds of the control group had not yet completely healed, while the wounds of the healthy group, FD group and FDS group had reached a completely healed state ( Figure 13 C). Quantitative analysis further confirmed that the wound healing of the FD and FDS groups was no different from that of the healthy group ( Figure 13 Specifically, on day 5, the wound areas of the FD and FDS groups were smaller than those of the other two groups. Of particular note, the healing rate of the FDS group (61%) was 1.3 times, 2.0 times, and 1.1 times higher than that of the healthy group, control group, and FD group, respectively. By day 14, wounds in the healthy group, FD group, and FDS group were almost completely healed, while the wound closure rate in the control group was only 72%.

[0122] (2) Histological analysis

[0123] To further elucidate the effects of the hydrogels prepared in Example 1 on promoting wound healing in diabetic patients with concurrent infection, the present invention conducted a histological analysis of the regenerated skin. Seven and fourteen days after administration of the prepared hydrogels, rats were sacrificed and wound tissues from their backs were collected. After fixation with 4% paraformaldehyde, the tissues were embedded in paraffin and stained with hematoxylin and eosin (H&E) to observe and record the progress of wound healing and epithelialization.

[0124] like Figure 14 As shown in A, on the 7th day of wound infection, the epithelial tissue of all groups showed obvious defects, and bacterial infection caused the infiltration of a large number of inflammatory cells. As time went on, on the 14th day, the wound cells in each group proliferated rapidly and granulation tissue began to form. The wounds of the healthy group, FD group and FDS group completed healing and showed typical cortical structure and morphological characteristics. Among these groups, the epithelialization effect of the FDS treatment group was the best ( Figure 14 C). However, the control group healed more slowly, with defects still present in some areas of the wound surface, indicating incomplete healing. Notably, while the healthy and FD groups developed a small number of new hair follicles and blood vessels, the FDS-treated group exhibited significantly more new hair follicles (green arrows) and blood vessels (black arrows). Granulation tissue plays a crucial role in wound regeneration and repair accompanied by inflammation. Therefore, thicker granulation tissue is crucial for assessing wound healing. Figure 14 Figure D shows the results after 14 days. The thickness of the granulation tissue in the control group was thinner than that in the other hydrogel groups. In the hydrogel groups, the thickness of the granulation tissue produced by the FD and FDS hydrogels was similar to that of the healthy group, indicating that the wound repair effect in the hydrogel-treated group was better. In summary, the analysis results of the H&E staining technique clearly show that the FDS hydrogel has a significant promoting effect on the healing of chronic wounds in diabetic patients with infection, including improved epithelialization and proliferation of granulation tissue.

[0125] Collagen deposition plays an important role in promoting the formation of extracellular matrix (ECM). Figure 14 Results in B. As the wound healed, the collagen fibers in the wound tissues of each group gradually tended to be arranged in an orderly manner, and the color change gradually deepened. In the observation on the 14th day, compared with the control group, the subepidermal area after FDS hydrogel treatment showed a large amount of dense and orderly collagen deposition (see Figure 14 E), with a morphology similar to that of normal skin. These results clearly demonstrate that FDS hydrogel accelerates the skin regeneration process by promoting collagen deposition. This phenomenon emphasizes the ability of FDS hydrogel to improve the organization of the extracellular matrix, thereby contributing to the restoration and integration of the underlying wound structure.

[0126] (3) Immunofluorescence analysis

[0127] Angiogenesis plays a crucial role in the healing process of chronic wounds, and angiogenesis in neoepithelial tissue is a hallmark of benign wound healing. CD31, a transmembrane protein, is expressed during early angiogenesis. Therefore, assessing its expression in the wound area provides insight into the formation of new blood vessels. This study used CD31 immunofluorescence staining on wound tissue at days 7 and 14 to assess angiogenesis in diabetic wounds. Figure 15 A revealed that on day 7, the wounds treated with FDS hydrogel showed a more significant increase in CD31 expression compared with the control group (*p<0.05, see Figure 15 B), indicating a higher degree of neovascularization maturity in the FDS group. However, the difference was not significant compared to the healthy group. By day 14, CD31 expression in the wound surface of each experimental group gradually increased. However, compared with the FD group, CD31 density was significantly increased in the FDS hydrogel group (**p < 0.01), indicating that FDS is more effective than FD.

[0128] In the process of wound healing, α-smooth muscle actin (α-SMA) plays a variety of important roles, including key steps such as granulation tissue formation, scar repair, angiogenesis, wound contraction, and extracellular matrix remodeling, providing necessary support for wound repair and recovery. The expression of αSMA was quantitatively analyzed by immunofluorescence technology. Figure 15 A shows that the wound tissue treated with FDS hydrogel showed stronger green fluorescence in the expression of α-SMA compared with the control group and FD group. This result clearly shows that FDS treatment can significantly increase the expression level of α-SMA, further verifying the significance of its therapeutic effect. It is worth noting that compared with the healthy group, the expression level of α-SMA in the FDS group did not show significant difference (see Figure 15 B), consistent with the trend shown by CD31 staining results. This consistent result strengthens the potential of FDS hydrogel therapy in promoting angiogenesis and wound healing.

[0129] One of the main factors that hinder diabetic wound healing is persistent infection caused by excessive inflammatory factors. In order to further explore the inhibitory effect of FDS hydrogel on inflammatory response, the present invention uses immunofluorescence chemical staining method to quantitatively measure the levels of typical inflammatory cytokines in healing wound tissue. Figure 16 As shown in A, on the 7th day, the expression levels of inflammatory factors (TNF-α and IL-6) in the wounds of the control group were higher. In contrast, the expression of inflammatory factors in the hydrogel treatment group was significantly reduced, especially in the FDS group, where the expression of inflammatory factors such as TNF-α and IL-6 decreased significantly ( Figure 16B, C). By day 14, the control group still showed high levels of inflammatory cytokine expression, while the wounds in the FD and FDS-treated groups showed almost no signs of inflammation or infection. These results suggest that the inflammatory response in the control group was persistent and excessive, while hydrogel treatment was able to reduce the severity of inflammation in diabetic wounds. The hydrogel's significant anti-inflammatory effect may be due to its positively charged nature, which enables it to continuously adsorb free nucleic acids in diabetic wounds through electrostatic effects, thereby reducing the inflammatory response in the wound surface. This result is consistent with the results of in vitro anti-inflammatory experiments.

[0130] (4) Immunohistochemical analysis

[0131] IL-10, as an important anti-inflammatory cytokine, has the ability to shorten the inflammatory cycle and effectively inhibit effector T cell responses. The immunohistochemistry (IHC) results are shown in the figure. Compared with the blank control group, the expression of IL-10 in the FDS hydrogel treatment group was significantly increased (**p<0.01). Specifically, the positive expression reached 3.4%, which was also significantly higher than that in the FD hydrogel treatment group (*p<0.05). This finding further supports the significant effect of FDS hydrogel in promoting the production of the anti-inflammatory cytokine IL-10, emphasizing its positive role in regulating inflammatory immune responses.

[0132] In this example, the levels of angiogenic factors in the wound site were quantitatively assessed by immunohistochemical staining of vascular endothelial growth factor (VEGF) on day 7 of wound healing. Figure 17 A clearly shows that the wound tissue treated with the hydrogel containing FDS showed a more obvious brown-yellow color, which indicates that the VEGF level in the wound tissue was significantly increased. Figure 17 As shown in Figure C, on day 7, VEGF levels were significantly increased in the NO-loaded hydrogel-treated group compared to the control group (****p<0.0001). This observation emphasizes the significant advantage of NO-loaded hydrogels in promoting angiogenesis and accelerating wound closure. Therefore, this may indicate that NO-loaded hydrogels exert positive biological effects in wound healing, providing strong support for their potential as an effective therapeutic strategy to promote wound repair.

[0133] Example 9 Biocompatibility Experiment

[0134] In order to further study the potential application of hydrogels in treating diabetic wounds complicated by bacterial infection, the cell compatibility of hydrogels was further evaluated. The LIVE / DEAD Viability test kit was used to measure the interaction between different hydrogel extracts and NIH-3T3 cells to evaluate their cytotoxic effects. The results showed that ( Figure 18A), the four groups of hydrogel extracts had no obvious toxic effects on NIH-3T3 cells. Even after 3 days of culture, NIH-3T3 cells in both the hydrogel-treated and control groups showed healthy spindle morphology and green fluorescence, and similar cell viability. The results of the Cck-8 cell proliferation experiment also reached a similar conclusion ( Figure 18 B) For 3T3 cells, except for the FD group, the other three groups of hydrogels did not induce obvious toxic reactions. Compared with the control group, the activity of the hydrogel in the FD group was weakened (***p<0.001). However, for HUVEC cells, after one day of co-incubation, the hydrogel extracts of the FS and FDS groups showed an effect of promoting cell proliferation. Compared with the control group, after three days of co-culture, only the FDS group showed a significant effect of promoting cell proliferation (*p<0.05). This shows that the hydrogel has the potential to promote HUVEC proliferation and can improve wound healing by promoting angiogenesis.

[0135] In biomaterial research, blood compatibility is of great significance. In order to comprehensively evaluate the blood compatibility of hydrogels, a hemolysis test was conducted. The specific steps are as follows:

[0136] Red blood cells were collected by centrifuging rabbit blood (purchased from Nanjing Senbeijia Biotechnology Co., Ltd.) containing sodium citrate as an anticoagulant at 3000rpm for 15min. After washing 3 times with PBS, the red blood cell solution was further diluted with PBS to a final concentration of 10% (v / v). Afterwards, the three hydrogels (F-127DA, FD and FDS) prepared according to the method in Example 1 were ground dry with a tissue grinder to prepare three separate hydrogel dispersions (concentrations ranging from 2 to 5 and 10 mg / mL). 800 μL of hydrogel dispersion and 200 μL of red blood cell suspension were added to a 2mL EP tube and gently mixed by pipette. After incubation at 37°C for 1h, all samples were centrifuged and the absorbance of the supernatant at 541nm was measured with a microplate reader. For comparison, PBS and ultrapure water without hydrogel were used as negative and positive controls, respectively. The hemolysis rate (%) was calculated using the formula:

[0137] Hemolysis rate (%) = (Abs sample -Abs negative ) / (Abs positive -Abs negative )×100%, where Abs sample , Abs negative and Abs positive The absorbance values ​​in the hydrogel, PBS, and ultrapure water groups are shown. Each experiment was performed in triplicate.

[0138] In this experiment, SD rat blood diluted with deionized water was used as a positive control, and PBS was used as a negative control. Hemolysis rates were tested for different concentrations of F-127DA, FD, and FDS hydrogels. Figure 18 C shows that the supernatant after hydrogel treatment exhibited a transparent light red color, similar to the PBS control group, while the positive control group treated with deionized water exhibited a bright red color. The quantitative hemolysis rate of each hydrogel group remained below 5%, significantly lower than that of the positive control (****p<0.0001). This result clearly demonstrates that FDS hydrogel does not induce significant hemolysis, further verifying its excellent blood compatibility.

[0139] In the evaluation of the therapeutic effect and safety of biomedical materials, the investigation of liver function and kidney function is of great value. In order to gain a deeper understanding of the effects of different hydrogels in the treatment of wounds in diabetic rats, alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) and γ-glutamyl transpeptidase (γ-GT) were used as indicators to evaluate the degree of liver damage, while serum urea nitrogen and creatinine were selected as indicators to evaluate the degree of renal damage. Based on Figure 19 The results showed that there was no significant difference in liver and kidney function-related indicators between the rats in the hydrogel-treated group and the rats in the healthy group. This finding further confirmed the conclusion that the hydrogel had no adverse effects on the liver and kidney function of rats.

[0140] The safety of rats' internal organs after treatment with different gels was evaluated by histological H&E staining. Figure 20 ) can be clearly observed that after FDS hydrogel treatment, the major internal organs of rats, including the heart, liver, spleen, lungs and kidneys, showed no toxic reactions and any side effects.

[0141] During the model establishment and treatment period of rats, their body weights were recorded, as shown in Figure 21 As shown. Except for the healthy group, the control, FD and FDS groups showed continuous weight loss after injection in the STZ-induced diabetic model. However, on the 7th day after treatment with FD and FDS hydrogels, the weight loss trend slowed down and entered a relatively stable stage. This example further conducted safety-related biochemical index detection ( Figure 22), including routine blood biochemical parameters such as red blood cell count (RBC), white blood cell count (WBC), platelet count (PLT), neutrophil granulocyte (NEUT), and lymphocyte (LY). Compared with healthy rats, rats treated with FD and FDS hydrogels showed no significant changes in blood biochemical parameters. Therefore, these results demonstrate that the FDS multifunctional hydrogel exhibits good biosafety and has broad potential for clinical application.

Claims

1. A multifunctional polyrotaxane hydrogel FDS, characterized in that: The preparation method is as follows: (1) Synthesis of F-127DA: The nonionic surfactant Pluronic F127 was dissolved in dichloromethane in an ice bath to obtain a Pluronic F127 solution. Acryloyl chloride and triethylamine were slowly injected into the Pluronic F127 solution under nitrogen protection. The reaction was continued for a period of time. After purification, pure F-127DA was obtained. (2) Synthesis of β-CD-SNO: The 6-hydroxyl group of β-cyclodextrin was sulfonyl chlorided by p-toluenesulfonyl chloride, and then the 6-sulfonyl chloride group was replaced by thiourea to obtain the monothiol-substituted cyclodextrin β-CD-SH. Finally, the 6-thiol group of β-CD-SH was modified with sodium nitrite under acidic conditions to obtain the NO donor β-CD-SNO. (3) Synthesis of multifunctional polyrotaxane hydrogel FDS: Dimethylaminopropyl methacrylamide (DMAPMA) and β-CD-SNO prepared in step (2) were added to the pure F-127DA prepared in step (1) to obtain a precursor solution. After mixing evenly, a photoinitiator was added and the solution was irradiated under ultraviolet light to achieve ultraviolet-induced free radical cross-linking polymerization to prepare a multifunctional polyrotaxane hydrogel FDS.

2. The multifunctional polyrotaxane hydrogel FDS according to claim 1, characterized in that The photoinitiators are 2,2-dimethoxy-2-phenylacetophenone (DMPA) and N-vinyl pyrrolidone (NVP).

3. The multifunctional polyrotaxane hydrogel FDS according to claim 2, characterized in that The photoinitiator was prepared by dissolving DMPA in NVP to a DMPA concentration of 300 mg / mL.

4. The multifunctional polyrotaxane hydrogel FDS according to claim 1, characterized in that The specific steps of step (1) are as follows: 10.01 g of nonionic surfactant Pluronic F127 (M=12600, PEG 99 -PPO 65 -PEG 99 ) was dissolved in 60 mL of dry dichloromethane. Under nitrogen protection, 0.9 mL of acryloyl chloride and 0.8 mL of triethylamine were slowly injected into the solution for 30 min. After reacting at room temperature for 24 h, the product was diluted with 200 mL of dichloromethane. 50 mL of 0.2% sodium bicarbonate solution and pure water were added to the solution in a separatory funnel, and the organic phase was collected for rotary evaporation and freeze-dried to obtain pure F-127DA.

5. The multifunctional polyrotaxane hydrogel FDS according to claim 1, characterized in that The specific steps of step (2) are as follows: first, under ice bath conditions, 50.15g of β-cyclodextrin was dissolved in 300mL of deionized water, and 5.5g of sodium hydroxide solid was added and stirred to dissolve until the solution became clear; then, 1.47mmol / mL of p-toluenesulfonyl chloride acetonitrile solution was slowly added dropwise using a syringe pump; the ice bath was removed and the reaction was allowed to proceed at room temperature for 4h; then, the pH value of the solution was adjusted to 9.0 using concentrated hydrochloric acid, and the solution was placed in a refrigerator at 4°C overnight; thereafter, the precipitate was collected by centrifugation and vacuum dried to obtain mono-6-p-toluenesulfonyl-β-cyclodextrin (β-CD-OTs); 2.124g of β-CD-OTs and 2.012g of thiourea were dissolved in 100mL of 80% methanol water (v / v) solution, heated under reflux for 48h, and after the reaction was completed, the solvent was removed using a rotary evaporator, and then 30mL of methanol was added to the spin-dried bottle and stirred at room temperature for 1h; the precipitate was collected by filtration and 69mL The product was dissolved in 10% sodium hydroxide solution and stirred at 50°C for 5h. After the reaction, the pH value of the system was adjusted to 2 with 10% hydrochloric acid solution. Then, 5mL of trichloroethylene was added to the light yellow solution and stirred at room temperature overnight. The white precipitate was recovered by filtration, washed three times with water, dried in a vacuum drying oven overnight, and recrystallized three times with ethanol aqueous solution to obtain a white solid monothiol-substituted cyclodextrin β-CD-SH. 0.115g of β-CD-SH was dissolved in 1mL of DMSO, 10μL of 6M hydrochloric acid solution was added, vortexed for 1min, and 10μL of 4M sodium nitrite solution was added. It can be observed that the solution changes from light yellow to bright red and vortexed for 5min. 10mL of cold acetone was added for precipitation, and pink solid β-CD-SNO was obtained by centrifugation.

6. The multifunctional polyrotaxane hydrogel FDS according to claim 1, characterized in that The specific steps of step (3) are as follows: F-127DA (final concentration 10%) and DMAPMA solution (final concentration 1%) are mixed to form a precursor solution, β-CD-SNO is added to the precursor solution and then ultrasonicated for 30 minutes, 20 μL of DMPA / NVP photoinitiator is added to 1 mL of the precursor solution containing β-CD-SNO, and the precursor solution is exposed to ultraviolet light for 1 minute to form an FDS hydrogel, and the FDS hydrogel is then soaked in water for 3 days, with the water being changed every day, and then dried to obtain the FDS hydrogel.

7. The multifunctional polyrotaxane hydrogel FDS according to claim 1, characterized in that In step (3), the feed ratio of F-127DA to β-CD-SNO is 1:

16.

8. Use of the multifunctional polyrotaxane hydrogel FDS according to any one of claims 1 to 7 in diabetic wound management.

9. Use of the multifunctional polyrotaxane hydrogel FDS according to any one of claims 1 to 7 in promoting healing of infectious diabetic wounds.

10. Use of the multifunctional polyrotaxane hydrogel FDS according to any one of claims 1 to 7 in preparing a product for promoting healing of infectious diabetic wounds.

Images