A multifunctional hydrogel dressing based on hydrogen sulfide gas therapy and its preparation method

Abstract

This invention belongs to the field of biomedical polymer technology, and specifically relates to a multifunctional hydrogel dressing based on hydrogen sulfide gas therapy and its preparation method. The multifunctional hydrogel dressing is prepared based on a dynamic Schiff base reaction between PHMG-modified aldehyde-based F108 (PFC) and carboxymethyl chitosan (CMC). The beneficial effects of this invention are: the wound dressing system can rapidly degrade in the low pH and high oxidative stress microenvironment of diabetic wounds, thereby achieving rapid H2S release. The PHMG modified on the surface of the wound dressing can exert excellent antibacterial effects in combination with DATS; while the released H2S can fully exert its highly efficient anti-inflammatory, antioxidant, and cell proliferation, migration, and angiogenesis-promoting effects, thereby improving the microenvironment of diabetic wounds and accelerating wound healing.

Description

Technical Field

[0001] This invention relates to the field of biomedical polymer technology, and in particular to a multifunctional hydrogel dressing based on hydrogen sulfide gas therapy and its preparation method. Background Technology

[0002] Diabetes mellitus is a common chronic endocrine disorder with extremely high morbidity, mortality, and disability rates. Chronic wounds, particularly diabetic ulcers, are among the most serious complications of diabetes. Due to lack of exercise and high fat intake in modern society, the number of patients is rapidly increasing. Expensive and cumbersome treatments bring immense suffering and a heavy economic burden to patients and society. Bacterial growth, inflammatory imbalance, and abnormal angiogenesis are the main reasons for its difficulty in healing.

[0003] Current clinical models for treating chronic diabetic wounds include glycemic control, wound debridement surgery, antibiotic treatment, and wound dressings. While these treatments can alleviate pain and help prevent infection, they still face many shortcomings. For example, long-term insulin injections can lead to serious complications; non-professional debridement can cause ulcers to enlarge and worsen infection; long-term use of antibiotics can lead to serious side effects and the development of multidrug resistance; and traditional wound dressings have limited functionality and require frequent changes

[26] . These problems are detrimental to the treatment and healing of diabetic wounds, and there is an urgent need for safe, simple, and effective treatment methods.

[0004] Hydrogels are polymer materials with a 3D network structure, used in the preparation of multifunctional hydrogel dressings based on hydrogen sulfide gas therapy. Due to their superior flexibility, biocompatibility, excellent fluid absorption properties, and ability to provide a moist environment conducive to tissue regeneration, they effectively prevent secondary damage caused by wound adhesion. Furthermore, by adjusting the structural design, hydrogel dressings can be endowed with state-of-the-art physical and chemical properties, such as injection capability, adhesion, self-healing, intelligent responsive degradation, and excellent antibacterial properties. Notably, hydrogels prepared from dynamic Schiff base bonds not only possess good injectability and self-healing properties but also can rapidly degrade and release drugs under weakly acidic and highly oxidizing conditions, exerting a therapeutic effect on the loaded drug. These excellent physicochemical properties are important factors for their suitability as ideal wound dressings. Therefore, multifunctional hydrogel dressings with properties such as antibacterial, anti-inflammatory, antioxidant, and pro-angiogenic effects have been researched and developed for the treatment of diabetic wounds.

[0005] However, single antibacterial, anti-inflammatory, or antioxidant treatments often fall short in addressing the complex microenvironment of diabetic wounds, failing to achieve the desired therapeutic effect. Therefore, treatment strategies that comprehensively address the microenvironment of diabetic wounds urgently need to be developed.

[0006] The applicant discovered the significant role of hydrogen sulfide (H2S) in anti-inflammatory, antioxidant, and pro-angiogenic effects. Diallyl trisulfide (DATS) can release H2S in the presence of glutathione (GSH) to exert its therapeutic effects. However, its oil solubility limits its application. Hydrogels possess excellent fluid absorption properties and good structural modification properties, expanding their application range. Therefore, the combination of hydrogels and H2S gas therapy is expected to be highly effective in treating diabetic wounds. Summary of the Invention

[0007] In view of the shortcomings of the existing technology, the purpose of this invention is to provide a multifunctional hydrogel dressing based on hydrogen sulfide gas therapy and its preparation method, so as to solve the above problems.

[0008] The technical solution of the present invention is achieved as follows: a multifunctional hydrogel dressing based on hydrogen sulfide gas therapy, wherein the multifunctional hydrogel dressing is prepared based on the dynamic Schiff base reaction between PHMG-modified aldehyde-based F108 (PFC) and carboxymethyl chitosan CMC.

[0009] Furthermore, the grafting rate of PFC was 1.4%.

[0010] Furthermore, the concentration of PFC was 100 mg / mL.

[0011] Further steps include the following:

[0012] S1. First, add 10g of chitosan CS and 13.5g of sodium hydroxide NaOH to a 500mL flask. Then, add a mixed solution containing 50mL of water and 50mL of isopropanol to the flask and stir thoroughly to form a mixture.

[0013] S2. Then, the mixture is placed in an oil bath at 50°C to swell and alkalize for 1 hour.

[0014] S3. Dissolve 15g of chloroacetic acid in 20mL of isopropanol and add it dropwise to the mixture over 30min. Stir vigorously and maintain the reaction in a 50℃ oil bath for 4h. After the reaction is complete, stop the reaction by adding 200mL of 70% (v / v) methanol.

[0015] S4. Filter the resulting mixture and collect the filter residue. Wash the filter residue three times with 90% (v / v) methanol. After filtration, place the filter residue in a vacuum drying oven at 50°C and dry it for one day. The collected solid product is carboxymethyl chitosan (CMC).

[0016] Furthermore, it also includes the following steps:

[0017] a. Add 5.8g F108 (0.4mmol), 0.6g 4-formylbenzoic acid (4mmol) and 30mg 4-dimethylaminopyridine (DMAP) to a 500mL pear-shaped flask with a side arm;

[0018] b. Under N2 protection, add 70 mL of anhydrous dichloromethane (DCM) and dissolve it completely. Then, add 1.01 g (4.89 mmol) of dicyclohexylcarbodiimide (DCC) dissolved in dichloromethane dropwise to the above reaction system under ice bath conditions and react at room temperature for 2 days.

[0019] c. After the reaction is complete, filter out the solids generated in the reaction, concentrate the obtained organic phase by rotary evaporator, and wash it three times with saturated NaCl.

[0020] d. After washing, remove all DCM by rotary evaporation, vacuum dry for 5 h, disperse the obtained white solid in 100 mL of pure water, stir vigorously for 2 h, remove the precipitate by high-speed centrifugation (9000 r / min, 10 min), and freeze dry the obtained supernatant to obtain product F108-CHO (FC).

[0021] e. Dissolve the obtained dry 1g FC and 10mg PHMG in DMSO and stir for 4h. After the reaction is complete, add 0.62mg sodium cyanoborohydride NaBH3CN to the reaction solution and continue the reaction at room temperature for 12h.

[0022] f. After the reaction was completed, the mixture was dialyzed for 24 hours using a wide-range dialysis bag (Spectrum, MWCO 3500Da) and then freeze-dried to obtain a white product (PHMG-F108-CHO, PFC).

[0023] Furthermore, it also includes the following steps:

[0024] g. Dissolve 200mg F108-CHO and 20mg DATS in 2mL DCM, stir thoroughly for 1h, and then remove all DCM using a rotary evaporator to form a uniform yellow film.

[0025] h, add pure water to hydrate the membrane for 8 hours;

[0026] i. Centrifuge the mixture obtained in step b (10000 rpm, 10 min) to remove insoluble products and larger micelles;

[0027] j. The filtrate obtained in step c is freeze-dried to obtain drug-loaded micelles DATS@PFCs;

[0028] k. By adjusting the DATS / PFC mass feed ratio to 0.1, 0.2 and 0.5, three drug-loaded micelles with different drug loading amounts, namely DATS@PFC-1, DATS@PFC-2 and DATS@PFC-3, were obtained.

[0029] Furthermore, the process includes the following steps: PFC is mixed with three different concentrations of CMC (2.0% w / v, 2.5% w / v and 3.0% w / v) at a volume ratio of 1:1. After thorough mixing, the mixture is placed in a 37°C oven to prepare hydrogels PFC&CMC2.0, PFC&CMC2.5 and PFC&CMC3.0 respectively. Then, the PFC micelle solution is replaced with DATS@PFC-1, DATS@PFC-2 and DATS@PFC-3 micelle solutions of the same concentration to prepare drug-loaded hydrogels DATS@PFC&CMC-1, DATS@PFC&CMC-2 and DATS@PFC&CMC-3.

[0030] The beneficial effects of this invention are as follows:

[0031] 1. A hydrogel dressing, DATS@PFC&CMC, was prepared by combining H2S with hydrogel.

[0032] 2. This wound dressing system can rapidly degrade in the low pH and high oxidative stress microenvironment of diabetic wounds, thereby achieving the effect of rapid H2S release;

[0033] 3. The PHMG modified on the surface of this wound dressing can work in combination with DATS to exert excellent antibacterial effects;

[0034] 4. The released H2S can fully exert its highly effective anti-inflammatory, antioxidant, and cell proliferation, migration, and angiogenesis-promoting effects;

[0035] 5. Effectively improves the microenvironment of diabetic wounds, thereby accelerating wound healing. Attached Figure Description

[0036] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0037] Figure 1 This is a schematic diagram illustrating the preparation of DATS@PFC&CMC and its mechanism of action in promoting the healing of diabetic wounds infected with MRSA, as described in a specific embodiment of the present invention.

[0038] Figure 2 The hemolysis rates of PFC micelles at different PHMG grafting rates in specific embodiments of the present invention are shown in 1h (a) and 24h (b).

[0039] Figure 3 This is a photograph of the sol-gel transition of PFC & CMC according to a specific embodiment of the present invention.

[0040] Figure 4 These are transmission electron microscope (TEM) images of PFC(a) and DATS@PFC(b) according to specific embodiments of the present invention;

[0041] Figure 5 Scanning electron microscope image of PFC & CMCs hydrogel according to a specific embodiment of the present invention;

[0042] Figure 6 The porosity of the PFC & CMCs hydrogel is shown in the specific embodiment of the present invention.

[0043] Figure 7 The swelling ratio of PFC & CMCs hydrogel in a specific embodiment of the present invention;

[0044] Figure 8 The adhesion strength of PFC & CMCs hydrogel in a specific embodiment of the present invention;

[0045] Figure 9 The image shows the actual adhesion strength of PFC&CMC3.0 hydrogel to pigskin, as per a specific embodiment of the present invention.

[0046] Figure 10 The graph shows the relationship between the apparent viscosity and shear rate of the PFC&CMC3.0 hydrogel in a specific embodiment of the present invention.

[0047] Figure 11 This is a photograph of the injectability of the PFC&CMC3.0 hydrogel, a specific embodiment of the present invention.

[0048] Figure 12 The storage modulus (G') and loss modulus (G") of PFC&CMCs hydrogel as a function of strain are shown in the specific embodiments of the present invention.

[0049] Figure 13 The rheological properties of PFC & CMCs hydrogels vary with strain from 0.1% to 300% in a specific embodiment of the present invention.

[0050] Figure 14 This is a photograph of the self-healing properties of the PFC&CMC3.0 hydrogel, a specific embodiment of the present invention.

[0051] Figure 15This is a diagram illustrating the oxidative degradation behavior of the PFC&CMC3.0 hydrogel according to a specific embodiment of the present invention.

[0052] Figure 16 This is a diagram illustrating the H2S release behavior of the DATS@PFC&CMC hydrogel in a specific embodiment of the present invention.

[0053] Figure 17 Images of (a) live / dead staining and (b) CCK-8 assay used to evaluate the cytotoxicity of PFC&CMCs and DATS@PFC&CMCs to HUVECs cells according to specific embodiments of the present invention;

[0054] Figure 18 Flat plate images (a) and survival rates (b, c) of E. coli and MRSA after processing with FC&CMC, PFC&CMC and DATS@PFC&CMC according to specific embodiments of the present invention;

[0055] Figure 19 The images show the live and dead staining of E. coli (a) and MRSA (b) after processing with FC&CMC, PFC&CMC and DATS@PFC&CMC according to a specific embodiment of the present invention.

[0056] Figure 20 SEM images of E. coli and MRSA after processing with FC&CMC, PFC&CMC and DATS@PFC&CMC according to specific embodiments of the present invention;

[0057] Figure 21 Images showing (a) leakage of proteins and (b) changes in ATP synthesis levels in E. coli and MRSA cells after treatment with FC&CMC, PFC&CMC, and DATS@PFC&CMC, according to specific embodiments of the present invention;

[0058] Figure 22 Fluorescence imaging of intracellular H2S (a) and corresponding fluorescence intensity (b) images are shown in the specific embodiments of the present invention.

[0059] Figure 23 The changes in the expression levels of intracellular inflammatory factors (a) TNF-α, (b) IL-1β and (c) IL-6 after treatment with DATS, PFC&CMC and DATS@PFC&CMC are shown in the specific embodiments of the present invention.

[0060] Figure 24 The changes in the expression levels of intracellular inflammatory factors (a) IL-10 and (b) IL-4 after treatment with DATS, PFC&CMC and DATS@PFC&CMC are shown in the specific embodiments of the present invention.

[0061] Figure 25The expression levels of p-STAT3 and p-ERK proteins in activated macrophages after treatment with DATS, PFC&CMC, and DATS@PFC&CMC are shown in the specific embodiments of the present invention.

[0062] Figure 26 The expression levels of HO-1 mRNA and protein in activated macrophages after treatment with DATS, PFC&CMC, and DATS@PFC&CMC are shown in the specific embodiments of the present invention.

[0063] Figure 27 This is a comparative diagram showing the effects of DATS@PFC&CMC hydrogel on scratch healing and proliferation behavior of HUVECs cells according to a specific embodiment of the present invention.

[0064] Figure 28 For specific embodiments of the present invention, (a) duct formation of endothelial cells, (b) branching points and capillary lengths of the network formed by HUVECs, and (c) expression levels of p-ERK1 / 2 and p-p38 in HUVECs after different treatments.

[0065] Figure 29 This is a schematic diagram of the mouse treatment process according to a specific embodiment of the present invention;

[0066] Figure 30 For specific implementation of the present invention, (a) wound healing images, (b) wound tracking, and (c) wound healing rate comparison charts are provided.

[0067] Figure 31 The images show (a) H&E and (b) Masson staining of wound tissue at 15 days after the presentation of this invention.

[0068] Figure 32 This is a bacterial count image of a diabetic mouse wound after 5 days of treatment, according to a specific embodiment of the present invention.

[0069] Figure 33 The graph shows the expression levels of inflammatory factors IL-1β, IL-6, and TNF-α in mouse blood according to a specific embodiment of the present invention.

[0070] Figure 34 This is an imaging and quantitative analysis diagram of iNOS and CD163 in wound tissue according to a specific embodiment of the present invention.

[0071] Figure 35 This is an imaging and quantitative analysis diagram of HO-1 in wound tissue according to a specific embodiment of the present invention;

[0072] Figure 36 This is an immunohistochemical (CD31) analysis of wound tissue at 15 days, according to a specific embodiment of the present invention.

[0073] Figure 37The following images show the blood routine analysis of mice in each group on day 15, as described in the specific embodiments of the present invention: (a) red blood cell count (RBC), (b) white blood cell count (WBC), and (c) hemoglobin (HGB) images.

[0074] Figure 38 The images show H&E staining of the heart, liver, spleen, lungs, and kidneys in the control group and the DATS@PFC&CMC group, respectively, according to a specific embodiment of the present invention. Detailed Implementation

[0075] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0076] In the description of this application, it should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments according to this application. For ease of description, the dimensions of the various parts shown in the drawings are not drawn to actual scale. Techniques, methods, and devices known to those skilled in the art may not be discussed in detail, but where appropriate, such techniques, methods, and devices should be considered part of the specification. In all examples shown and discussed herein, any specific values ​​should be interpreted as merely exemplary and not as limitations. Therefore, other examples of exemplary embodiments may have different values. It should be noted that similar reference numerals and letters in the following drawings denote similar items; therefore, once an item is defined in one drawing, it need not be further discussed in subsequent drawings.

[0077] Example 1:

[0078] Chitosan's ability to dissolve only in acidic solutions severely limits its applications. The introduction of carboxymethyl groups breaks the regularity of chitosan, increasing its water solubility and expanding its applications. The specific synthesis steps of CMC are as follows: First, chitosan (CS, 10g) and sodium hydroxide (NaOH, 13.5g) are added to a 500mL flask. Then, a mixed solution containing 50mL water and 50mL isopropanol is added to the flask, and the mixture is stirred thoroughly to form a solution. The mixture is then placed in an oil bath at 50℃ to swell and alkalize for 1 hour. Afterward, 15g of chloroacetic acid dissolved in 20mL isopropanol is added dropwise to the mixture over 30 minutes, with vigorous stirring, and the reaction is maintained at 50℃ in an oil bath for 4 hours. After the reaction is complete, the reaction is stopped by adding 200mL of 70% (v / v) methanol. The resulting mixture is filtered, and the residue is collected. The residue is washed three times with 90% (v / v) methanol. After filtration, the filter residue was placed in a vacuum drying oven at 50℃ and dried for 1 day. The collected solid product was CMC. The structures of CS and CMC were characterized using Fourier transform infrared spectroscopy (FT-IR) and nuclear magnetic resonance spectroscopy (1H NMR). The degree of substitution of carboxymethyl groups was calculated by the relative integrated area of ​​the 1H NMR spectrum.

[0079] Example 2:

[0080] F108 was modified with an aldehyde by esterification. 5.8 g of F108 (0.4 mmol), 0.6 g of 4-formylbenzoic acid (4 mmol), and 30 mg of 4-dimethylaminopyridine (DMAP) were added to a 500 mL pear-shaped flask with a side arm. Under N2 protection, 70 mL of anhydrous dichloromethane (DCM) was added and the mixture was completely dissolved. Then, 1.01 g (4.89 mmol) of dicyclohexylcarbodiimide (DCC) dissolved in dichloromethane was added dropwise to the reaction system under ice bath conditions, and the reaction was carried out at room temperature for 2 days. After the reaction was completed, the solids generated in the reaction were removed by filtration, and the resulting organic phase was concentrated by rotary evaporation and washed three times with saturated NaCl. After washing, all DCM was removed by rotary evaporation. After vacuum drying for 5 hours, the resulting white solid was dispersed in 100 mL of pure water and stirred vigorously for 2 hours. The precipitate was removed by high-speed centrifugation (9000 r / min, 10 min). The supernatant was then freeze-dried to obtain the product F108-CHO (FC). 1 g of the dried FC was dissolved in 10 mg of PHMG in DMSO and stirred for 4 hours. After the reaction was complete, 0.62 mg of sodium cyanoborohydride (NaBH3CN) was added to the reaction solution, and the reaction was continued at room temperature for 12 hours. After the reaction was complete, the solution was dialyzed for 24 hours using a wide-range dialysis bag (Spectrum, MWCO 3500 Da) and freeze-dried to obtain the white product (PHMG-F108-CHO, PFC). By adjusting the PHMG / FC mass ratio to 0.01, 0.02, and 0.03, three PFCs with different grafting rates were obtained and named PFC-1, PFC-2, and PFC-3, respectively. The structures of F108 and FC were characterized using Fourier transform infrared spectroscopy (FT-IR) and nuclear magnetic resonance spectroscopy (1H NMR).

[0081] The grafting rate of PHMG was calculated by titrating the aldehyde group. The specific experimental steps are as follows: Accurately weigh 5 mg FC and 5 mg PFC and dissolve them in 8 mL of hydroxylamine hydrochloride methanol solution (20 g / L). Then add 80 μL of thymol blue ethanol solution (0.1%) as an indicator. Stir thoroughly to dissolve evenly. Since the reaction of the aldehyde group with hydroxylamine hydrochloride produces hydrochloric acid, the solution is acidic at this time, and the indicator is pink. Then add standard sodium hydroxide solution (0.01 mol / L) dropwise until the solution turns yellow and does not fade for 30 seconds. Repeat the experiment three times. Calculate the grafting rate of PHMG using the following formula (2-1):

[0082]

[0083] In the formula, c (mol / L) is the concentration of the standard sodium hydroxide solution, V1 and V2 (mL) are the volumes of sodium hydroxide solution consumed in the titration of FC and PFC, respectively, M is the relative molecular mass of PHMG (3000 g / mol), and m (mg) is the mass of PFC.

[0084] Given that PHMG contains a large number of guanidinyl groups, which provides excellent antibacterial activity but also causes significant cytotoxicity, we screened the grafting rate and concentration of PFC using a hemolysis experiment. The specific experimental steps are as follows: First, 100 μL of mouse erythrocytes and 12 μL of PFC with different concentrations or different PHMG grafting degrees were mixed thoroughly with physiological saline in a sterile tube and incubated at 37°C for 1 or 24 h. Then, the mixture was centrifuged (3000 rpm, 4°C), and the absorbance of the supernatant at 545 nm was measured using a microplate reader. 0.9% sodium chloride and ultrapure water were used as negative and positive controls, respectively. The hemolysis rate was calculated according to formula (2-2).

[0085]

[0086] Example 3:

[0087] The content of PHMF was determined by titration, as shown in Table 2-3. These experiments demonstrate the successful synthesis of FC and PFC.

[0088] Table 2-3 Grafting rate and aldehyde content of PHMG in three types of micelles

[0089] Table2-3 Graft rate of PHMG andaldehyde content in three types ofmicelles

[0090]

[0091] The results of the hemolysis test are as follows: Figure 2As shown, high concentrations of PFC and PFC with high PHMG grafting rates caused significant hemolysis. After 1 hour, even at 100 mg / mL, the hemolysis rates of PFC-2 and PFC-3 reached 26% and 54%, respectively, far exceeding the safe hemolysis rate (5%). Notably, although the hemolysis rates of PFC-1 at 140 mg / mL and 180 mg / mL concentrations were 2.4% and 4.5% after 1 hour, the rates increased to 8.1% and 14.5% after 24 hours, indicating that PFC-1 micelles at these two concentrations still caused significant hemolysis and were unusable. In contrast, the hemolysis rates of 100 mg / mL PFC-1 at 1 hour and 24 hours were 0.3% and 2.8% (<5%), respectively, indicating that PFC-1 at a concentration of 100 mg / mL still exhibited good biocompatibility. Therefore, the following experiments all used materials with 100 mg / mL PFC-1 as the research object, and named them PFC.

[0092] Example 4:

[0093] The lipophilicity of DATS severely limits its application; therefore, we loaded DATS into PFC micelles using a thin-film dispersion method. The specific experimental steps are as follows: 200 mg of FC and 20 mg of DATS were dissolved in 2 mL of DCM and stirred thoroughly for 1 h. Then, all DCM was removed using a rotary evaporator to form a uniform yellow film. A small amount of pure water was then added to hydrate the film for 8 h. During hydration, the PFC micelles self-assembled to form micelles loaded with DATS. The resulting mixture was centrifuged (10000 rpm, 10 min) to remove insoluble products and larger micelles. The resulting filtrate was freeze-dried to obtain drug-loaded micelles (DATS@PFCs). Three drug-loaded micelles with different loading capacities (DATS@PFC-1, DATS@PFC-2, and DATS@PFC-3) were obtained by adjusting the DATS / PFC mass ratio to 0.1, 0.2, and 0.5. The drug loading capacity and efficiency of DATS@PFCs were calculated using an organic elemental analyzer. The specific experimental steps are as follows

[67] : Accurately weigh DATS@PFCs, burn tungsten oxide in excess oxygen at 1150℃ in a helium gas flow, and then reduce it with metallic copper at 850℃ to obtain sulfur dioxide. Calculate the S element content by measuring the mass of sulfur dioxide, thereby obtaining the mass of DATS. The drug loading and drug loading efficiency of DATS are calculated using the following formula:

[0094]

[0095]

[0096] F108 is an amphiphilic block copolymer that can self-assemble into micelles in water, and has therefore been extensively studied for its drug loading applications. Figure 3 As shown, direct mixing of oily DATS with water induces emulsification, resulting in a white emulsion. In contrast, the aqueous solution of the prepared drug-loaded micelles is a clear yellow liquid (DATS is a yellow oil), indicating that DATS was successfully loaded into the PFC micelles and that the obtained drug-loaded micelles were uniformly dispersed in water.

[0097] Example 5:

[0098] The particle size and potential of PFC and DATS@PFC were determined using a Malvern particle size analyzer (DLS), and the morphology and other characteristics of the micelles were further investigated using TEM. The results are as follows: Figure 4 As shown, compared to PFC, DATS@PFC has a smaller particle size. This is likely because the lipophilic DATS loaded onto the micelles makes the original micelle core more compact, thus reducing the micelle size. Transmission electron microscopy (TEM) revealed that both PFC and DATS@PFC micelles have a well-defined three-dimensional structure and are uniformly dispersed.

[0099] Example 6:

[0100] After confirming the successful preparation of drug-loaded micelles DATS@PFCs, the sulfur (S) content in the micelles was determined using a high-temperature combustion method, allowing for the calculation of drug loading and efficiency. Taking DATS@PFC-1 as an example, the S content in 5.254 mg of DATS@PFC-1 is 1.545%. Therefore, the mass of S in 5.254 mg is 5.254 × 1.545% = 0.0812 mg. Since one DATS atom has three sulfur atoms, the relative molecular mass of S is 32 g / mol, and the relative molecular mass of DATS is 178.34 g / mol. Therefore, based on the proportional relationship, the mass of DATS in DATS@PFC-1 is calculated as: Mass of S / (Relative molecular mass of S × 3) × Relative molecular mass of DATS = 0.151 mg. Therefore, the drug loading of DATS in DATS@PFC-1 can be calculated as follows: (mass of DATS in micelles) / (mass of DATS@PFC-1) × 100% = (0.151 / 5.254) × 100% = 2.88%. The drug loading efficiency of DATS in DATS@PFC-1 can be calculated as follows: (mass of DATS in micelles) / (mass of DATS) × 100% = (0.151 / 5.254) × 100% = 31.63%. Similarly, the drug loading and loading efficiency of the other two types of drug-loaded micelles can be obtained, and the calculation results are shown in Table 2-3.

[0101] Table 2-3 DATS loading and loading efficiency in three types of micelles

[0102]

[0103] In summary, the hydrogel components CMC and PFC were synthesized, and their structures were characterized by FT-IT and 1H NMR. The grafting rate of PHMG and the concentration of PFC were screened using a hemolysis experiment. DATS was then loaded into the hydrophobic core of PFC to prepare drug-loaded micelles DATS@PFC, and the drug loading capacity and efficiency of three different feed ratios were calculated. The particle size and morphology of the prepared blank micelles and drug-loaded nanomicelles were also characterized. The following conclusions can be drawn: 1) CMC and PFC were synthesized, and the degree of carboxymethyl substitution of CMC was determined to be 74%. After hemolysis screening, the safe grafting rate of PFC was 1.4% and the safe concentration was 100 mg / mL; 2) DATS was successfully loaded into PFC micelles, and the prepared drug-loaded micelles had a particle size of 198 nm and a uniform structure; 3) Three types of nanomicelles with different drug loading were prepared by different feed ratios, and the drug loading capacity and drug loading efficiency of the three types of drug-loaded micelles were detected by high-temperature combustion method.

[0104] Example 7:

[0105] In the above embodiments, a safe PFC concentration (10%, w / v) was screened through a hemolysis experiment. Therefore, we prepared three different CMC concentrations (2.0%, 2.5%, and 3.0%, w / v) to prepare three different hydrogels. First, PFC and different concentrations of CMC were mixed at a 1:1 volume ratio. After thorough mixing, the mixture was placed in a 37°C oven to prepare three hydrogels: PFC&CMC2.0, PFC&CMC2.5, and PFC&CMC3.0. The PFC micelle solution was then replaced with DATS@PFC-1, DATS@PFC-2, and DATS@PFC-3 micelle solutions of equal concentration to prepare three drug-loaded hydrogels, named DATS@PFC&CMC-1, DATS@PFC&CMC-2, and DATS@PFC&CMC-3, respectively.

[0106] Example 8:

[0107] The gelation time of the hydrogel was determined using the inverted bottle method. The specific experimental procedure is as follows: 100 μL of a 10% (w / v) PFC aqueous solution and 100 μL of CMC solutions of different concentrations were added to 1.5 mL centrifuge tubes, mixed thoroughly, and then the centrifuge tubes were placed in a water bath at 37°C. The tubes were removed and inverted every 10 seconds to observe the state of the liquid inside until the liquid stopped flowing; this time was recorded as the gelation time.

[0108] The results are shown in Table 3-1. It can be observed that the gelation time of the hydrogel gradually decreases with increasing CMC concentration. This is because the increased CMC concentration leads to increased cross-linking density and enhanced hydrogen bonding between hydrogels, thus accelerating hydrogel formation. This shorter gelation time is beneficial for application in wounds.

[0109] Table 3-1 Gelation time of PFC & CMC hydrogels with different mass ratios

[0110]

[0111] Example 9:

[0112] Cross-sectional samples were prepared from freeze-dried hydrogel samples using the liquid nitrogen brittle surface method. The specific steps are as follows: the obtained hydrogel was placed in liquid nitrogen and cooled completely. Then, it was removed with tweezers, the hydrogel was cut open with a blade, and the morphology of the cross-section was observed using a high-resolution transmission electron microscope (SEM). Finally, the porosity of the hydrogel was analyzed using ImageJ software.

[0113] The numerous pores of hydrogels endow them with excellent water absorption properties, so a swelling experiment is used to test these properties. The specific experimental steps are as follows: Accurately weigh dried hydrogels of the same size and record the mass as W0. Then place them in pure water and weigh them every 1 hour until the mass of the hydrogel no longer changes, at which point swelling equilibrium can be considered reached. Weigh the hydrogel at this point and record it as Wt. The swelling rate of the hydrogel is calculated using formula (3-1):

[0114]

[0115] We used SEM to characterize the cross-sectional morphology of three dried hydrogels, PFC&CMC2.0, PFC&CMC2.5, and PFC&CMC3.0, and calculated the porosity of these three hydrogels using ImageJ software. The results are shown below. Figure 5 As shown, all hydrogels exhibit a porous structure, and the network pores in the hydrogel cross-section become smaller with increasing CMC concentration. Furthermore, the porosity also increases with increasing CMC concentration. Figure 6 The percentage decreased from 43.1% to 24.8%. This reduction in pore size and porosity is also reflected in the swelling ratio. Figure 7As the CMC concentration increased, the swelling ratio of the hydrogel decreased from 17.6 to 16.1. This is because the compact network structure reduces the space for water storage. However, even the CMC & PFC3.0 with the lowest swelling ratio can absorb more than 15 times its own weight in water, due to the large number of pores and hydrogen bonds that effectively lock in moisture. These results demonstrate the excellent water absorption properties of the hydrogel, which can effectively absorb exudate from wound tissue and maintain a moist wound environment, thus promoting rapid wound healing.

[0116] Example 10:

[0117] like Figure 8 As shown, with increasing CMC concentration, the adhesion strengths of PFC&CMC2.0, PFC&CMC2.5, and PFC&CMC3.0 were 4.4, 7.6, and 13.8 kPa, respectively. The adhesion strength of PFC&CMC3.0 was significantly higher than the other two groups. The adhesion strength of the hydrogel to tissue was investigated using an overlap shear test. The experimental procedure was as follows: Fresh pigskin was used to simulate normal skin tissue. The pigskin was first soaked in PBS for one day to remove surface grease. Then, the pigskin was cut into strips of 10×40 (mm²). 50 μL of CMC solution of different concentrations was mixed evenly with 50 μL of PFC solution and then evenly applied to one piece of pigskin. Another piece of pigskin was then placed tightly against the coated area, and the mixture was placed at 37°C for 4 hours. The adhesion strength of the hydrogel to tissue was measured using a universal testing machine, set at 20 N and a pressure measurement rate of 1 mm / min. We further verified the adhesion of the hydrogel to the tissue by suspending a 100g weight under the bonded pigskin.

[0118] from Figure 9 It can be seen that two pieces of pigskin bonded together with PFC & CMC3.0 hydrogel can withstand a 100g weight for an extended period without separating. This adhesion of the hydrogel originates from the strong electrostatic interaction between the surface charge of the hydrogel and the phospholipid molecules on the cells, and this adhesive strength increases significantly with increasing CMC concentration. This strong adhesive strength allows the hydrogel to firmly adhere to the wound tissue, ensuring good therapeutic effects. The above conclusions clearly demonstrate the excellent adhesive properties of this hydrogel.

[0119] Example 11:

[0120] The apparent viscosity of PFC&CMC3.0 hydrogel changes with shear rate. For example... Figure 10As shown, with increasing shear rate, the apparent viscosity of the PFC&CMC3.0 hydrogel rapidly decreased from 3000 Pa·s to 0.55 Pa·s. This indicates that the hydrogel exhibits good shear-thinning properties. Furthermore, the hydrogel can be extruded by syringe force and forms continuous strips in water. Figure 11 This indicates that the hydrogel possesses excellent injectability. This superior injectability stems from the hydrogel's gelation mechanism: a Schiff base reaction based on dynamic imine bonds. These reversible dynamic imine bonds can spontaneously form without external force. Therefore, when the hydrogel is extruded from the syringe, it is subjected to strong shear force. At this point, the cross-linked network of the hydrogel is disrupted, and the hydrogel transitions to a flow dynamic state, allowing it to be extruded from the syringe. After extrusion, due to the disappearance of the shear force, the dynamic imine bonds quickly reconnect, restoring the original connected state, and the hydrogel thus returns to its gel state. These conclusions demonstrate that PFC&CMC hydrogels possess excellent injectability. This excellent injectability allows PFC&CMC hydrogels to be applied in various fields, greatly expanding their application range.

[0121] Example 12:

[0122] The self-healing properties of PFC & CMC were investigated using a rheometer. Samples were prepared following the previous steps. The changes in storage modulus G' and loss modulus G” of the hydrogel over time were studied using time-scan mode, with strain ranging from 0.1% to 1500% and frequency set to 1 Hz. Subsequently, the self-healing properties of the hydrogel were investigated using alternating scan mode, with an angular frequency set to 6 rad / s and strain values ​​alternating between 0.1% (60 s) and 300% (60 s) three times, recording the changes in storage modulus G' and loss modulus G”.

[0123] like Figure 12As shown, under a small strain of 0.1%, the storage modulus G' and loss modulus G” of PFC&CMC2.0, PFC&CMC2.5, and PFC&CMC3.0 maintain their numerical values ​​and magnitudes (G' is greater than G”). Notably, the storage modulus G' of PFC&CMC2.0, PFC&CMC2.5, and PFC&CMC3.0 are 150, 4000, and 5300 kPa, respectively, indicating that PFC&CMC3.0 has better mechanical strength. This is due to the increased CMC concentration, which results in a higher crosslinking density in PFC&CMC3.0. However, with increasing strain, G' and G” also change and eventually intersect. After this intersection, the magnitudes of G' and G” reverse, indicating that the basic structure of the hydrogel is disrupted, and it is now in a flow-like state. It is worth noting that the strain value corresponding to the intersection of PFC&CMC3.0 (270.23%) is much greater than the strain value corresponding to the intersection of PFC&CMC2.0 (68.2%) and PFC&CMC2.5 (160.2%). This is because the increased crosslinking density makes the hydrogel structure more robust, thus possessing superior strain resistance.

[0124] Example 12:

[0125] After determining the critical strain corresponding to the failure of the three hydrogels through the above embodiments, the failure-repair cycle process of the hydrogels was studied using the time-cycle scanning mode of a rheometer. Figure 13 As shown, taking PFC&CMC3.0 hydrogel as an example, under a strain of 0.1%, the G' and G” of the hydrogel are 5300 Pa and 2000 Pa, respectively, and this strain magnitude remains unchanged for 60 s. Conversely, when the strain reaches 300% (>270.23%), G” rapidly exceeds G', and this relationship remains unchanged for 60 s, indicating that the stable structure of PFC&CMC3.0 is disrupted. However, when the strain recovers to 0.1%, G' and G” quickly return to their initial levels, and their values ​​are almost identical to before, indicating that the PFC&CMC3.0 hydrogel recovers to its original stable structure. These results are repeatable within two cycles, demonstrating that the hydrogel possesses good self-healing properties.

[0126] Cut two pieces of hydrogel of different colors into semicircles and place them together. The two pieces of hydrogel will adhere well to each other. Figure 14As can be seen, the joint turns black due to the successful bonding of the two hydrogels, indicating a successful connection. Notably, the bonded hydrogels can withstand a certain amount of tension without breaking, demonstrating that they have healed well together. Therefore, these results clearly demonstrate that PFC&CMC3.0 hydrogels possess excellent self-healing properties. This self-healing ensures that even if the hydrogel is damaged during application, it can quickly repair itself, resulting in superior dressing performance.

[0127] Example 13:

[0128] To evaluate the in vitro degradation behavior of PFC & CMC, the freeze-dried PFC & CMC hydrogel was first accurately weighed and recorded as W1, allowing it to reach swelling equilibrium. The hydrogel was then immersed in PBS under different conditions: 1) pH = 7.4; 2) pH = 7.4, 100 μM H2O2; 3) pH = 6.5; 4) pH = 6.5, 100 μM H2O2. The hydrogel sample was removed every other day, excess salt was removed, and the sample was dried; the mass was recorded as W2. The remaining mass ratio of the hydrogel was calculated using formula (3-2) to assess its degradation behavior.

[0129] Example 14:

[0130] The in vitro degradation behavior of hydrogel PFC&CMC3.0 in the microenvironment of diabetic wounds was simulated under different conditions, such as... Figure 15 As shown, after 6 days, the remaining mass of PFC&CMC3.0 hydrogel decreased to 37% (pH=7.4), 23% (pH=7.4+100μM H2O2), 20% (pH=6.5), and 10% (pH=6.5+100μM H2O2), respectively. This indicates that PFC&CMC3.0 hydrogel achieves rapid drug release in diabetic wounds under low pH and high oxidation conditions, effectively avoiding the secondary damage that may be caused by the frequent dressing changes required by traditional dressings and the severe inhibition of wound healing.

[0131] Example 15:

[0132] The H2S release behavior of DATS in response to GSH was evaluated by using different concentrations of GSH, such as... Figure 16As shown, no H2S release was detected in the DATS@PFC&CMC-1 hydrogel treated with PBS within 80 min. However, with increasing GSH concentration, the H2S release rate and amount gradually increased. For example, at pH 7.4, the DATS@PFC&CMC-1 hydrogel released 1.1 μmol of H2S at a GSH concentration of 2.0 mM, which is significantly higher than the release amounts at the same pH conditions but with GSH concentrations of 1.0 mM (0.5 μmol) and 1.5 mM (0.6 μmol). Although acidic conditions accelerate the degradation of the hydrogel, the reducing power of GSH mainly originates from the thiol group (-SH), and it is relatively difficult to remove protons under acidic conditions to form reactive thiolate anions (-S-1), thus causing the DATS release rate to decrease. However, it is worth noting that the DATS@PFC&CMC-1 hydrogel still released 0.9 μmol at 80 minutes, indicating that the slightly acidic environment had little impact on the release rate and amount of DATS. Therefore, the DATS@FC&CMC-1 hydrogel can release sufficient H2S in the presence of GSH, thereby exerting the therapeutic effect of H2S. This long-acting sustained release is beneficial for the healing of chronic wounds such as diabetic lesions.

[0133] In summary: 1) Blank hydrogels PFC&CMC2.0, PFC&CMC2.5, and PFC&CMC3.0, as well as drug-loaded hydrogels DATS@PFC&CMC-1, DATS@PFC&CMC-2, and DATS@PFC&CMC-3, were prepared. The gelation time, swelling, adhesion, injectability, and self-healing properties of the blank hydrogels were investigated, revealing excellent physicochemical properties. 2) Although the blank hydrogel PFC&CMC3.0 had a low swelling rate, it still exhibited good water absorption. Furthermore, PFC&CMC3.0 demonstrated better adhesion, mechanical properties, and self-healing capabilities. 3) The hydrogel rapidly degraded within the simulated diabetic microenvironment, releasing drug-loaded micelles, and rapidly released H2S in the presence of GSH. Therefore, given the superior physicochemical properties of PFC&CMC3.0, it was selected as the blank hydrogel for subsequent experiments and named PFC&CMC.

[0134] Example 16:

[0135] Human umbilical vein endothelial cells (HUVECs) and RAW264.7 macrophages were used for cell experiments. HUVECs were cultured in DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin to prepare a complete medium. RAW264.7 cells were cultured in macrophage-specific medium. All cells were cultured in a cell culture incubator at 37°C and 5% CO2. Macrophages without any treatment were designated as normal macrophages, while macrophages incubated with lipopolysaccharide (LPS, 5 μg / mL) for 24 h were designated as activated macrophages.

[0136] HUVECs were cultured using the complete culture medium described in the above steps and incubated at 37°C in a 5% CO2 cell culture incubator. The experimental procedures were as follows: First, 24 μL of sterile FC&CMC, PFC&CMC, DATS@PFC&CMC-1, DATS@PFC&CMC-2, and DATS@PFC&CMC-3 hydrogels were added to 96-well plates and incubated for 10 min in an oven to allow gelation. Then, 1 × 10⁴ HUVECs cells were added to each well and incubated at 37°C for 48 h. After incubation, the cells were washed with PBS, and CCK-8 was added to the wells, incubating in the dark for 30 min. The absorbance of the cells at 450 nm was detected using a multi-mode microplate reader. Furthermore, after treating HUVECs according to the above steps, the cells were stained with AM / PI for 30 min, and the cell fluorescence was observed and photographed using an inverted fluorescence microscope.

[0137] Excessive H2S can lead to significant cytotoxicity, therefore it is necessary to screen for safe drug loading levels, such as... Figure 17 As shown, the cell viability of HUVECs treated with FC&CMC, PFC&CMC, and DATS@PFC&CMC-1 was all above 90%, indicating good biocompatibility. Conversely, the cell viability of HUVECs treated with DATS@PFC&CMC-2 and DATS@PFC&CMC-3 was below 70%, exhibiting significant cytotoxicity. Cell liveness / death staining experiments further validated this conclusion. Figure 17As shown, cells treated with FC&CMC, PFC&CMC, and DATS@PFC&CMC-1 exhibited abundant green fluorescence (calcein AM-labeled live cells). Conversely, HUVECs treated with DATS@PFC&CMC-2 and DATS@PFC&CMC-3 showed abundant red fluorescence (PI-labeled dead cells), indicating the presence of numerous dead cells. This toxicity primarily stems from the excessive H2S release from PHMG and DATS. In conclusion, both PFC&CMC and DATS@PFC&CMC-1 hydrogels demonstrate good biocompatibility. Based on these results, DATS@PFC&CMC-1 was selected for subsequent experiments and named DATS@PFC&CMC.

[0138] Example 17:

[0139] The in vitro antibacterial effect of the hydrogel was evaluated using colony counting methods against two bacteria: *Escherichia coli* and methicillin-resistant Staphylococcus aureus (MRSA). Figure 18 As shown, DATS@PFC&CMC exhibited better antibacterial activity than PFC&CMC, with bacterial survival counts decreasing to 2.2 and 1.6 log10 CFU / mL after 6 hours of treatment. This is attributed to the inherent antibacterial activity of DATS, which, in conjunction with PHMG, produced a good synergistic antibacterial effect, resulting in superior antibacterial performance. Figure 19 The results show that E. coli and MRSA treated with PFC&CMC and DATS@PFC&CMC exhibit abundant red fluorescence but almost no green fluorescence, indicating that PFC&CMC and DATS@PFC&CMC killed almost all bacteria.

[0140] Example 18:

[0141] To investigate the antibacterial mechanism of DATS@PFC&CMC, E. coli and MRSA were treated, and the bacteria were collected by centrifugation at 4°C. Protein concentration was measured using an enhanced BCA protein assay kit, and changes in bacterial ATP levels were measured using an enhanced ATP assay kit. Figure 20 As shown, E. coli and MRSA treated with PBS and FC&CMC maintained their intact bacterial structures. However, E. coli treated with PFC&CMC and DATS@PFC&CMC showed numerous pores, and MRSA showed shrinkage and collapse with significant leakage of material, indicating that PFC&CMC and DATS@PFC&CMC can efficiently kill bacteria by disrupting the integrity of the bacterial cell membrane.

[0142] Example 19:

[0143] Protein concentrations and ATP levels in E. coli and MRSA treated with PFC&CMC and DATS@PFC&CMC were investigated using BCA and ATP kits. Figure 21 It can be clearly shown that PFC&CMC and DATS@PFC&CMC can effectively eliminate bacteria by disrupting the bacterial cell membrane, causing protein leakage and inhibiting ATP levels.

[0144] Example 20:

[0145] After confirming that DATS@PFC&CMC could release H2S in vitro in the presence of GSH, we further investigated whether DATS@PFC&CMC could produce H2S intracellularly; Figure 22 The green fluorescence intensity of activated macrophages treated with DATS@PFC&CMC was much higher than that of the other groups, nearly three times that of the DATS group. This conclusion proves that DATS@PFC&CMC can generate a large amount of H2S in cells, laying the groundwork for the subsequent therapeutic effect of H2S.

[0146] Example 21:

[0147] The effects of DATS@PFC&CMC on inflammatory factors were investigated using an ELISA kit.

[0148] like Figure 23 and Figure 24 As shown, the levels of TNF-α, IL-1β, and IL-6 in activated macrophages treated with DATS@PFC&CMC were 1.2, 0.4, and 2.1 ng / mL, respectively, which were far lower than those in macrophages induced by LPS. The expression levels of IL-10 and IL-4 in activated macrophages treated with DATS@PFC&CMC were 79 and 68 pg / mL, respectively. Sufficient H2S greatly promoted the expression of anti-inflammatory factors IL-10 and IL-4, thereby exerting an anti-inflammatory effect.

[0149] Example 22:

[0150] The mRNA expression levels of HO-1, CD206, and iNOS in macrophages were detected by W RT-qPCR.

[0151] The protein expression levels of HO-1, ERK, p-ERK, STAT3, p-STAT3, CD163, and iNOS were detected by Western blot.

[0152] To observe changes in macrophage morphology, macrophages were treated according to the steps described above, and then cell morphology was observed and photographed under an inverted fluorescence microscope. Furthermore, the anti-inflammatory mechanism of DATS@PFC&CMC was investigated using immunofluorescence experiments; for example... Figure 25 As shown, DSTA@PFC&CMC can exert anti-inflammatory effects by inhibiting the phosphorylation levels of STAT3 and ERK; Figure 26 As shown, DSTA@PFC&CMC treatment further specifically activates the expression of heme oxygenase, indicating that DSTA@PFC&CMC also has excellent antioxidant effects.

[0153] Example 23:

[0154] The role of DATS@PFC&CMC in promoting HUVEC cell proliferation and migration was evaluated through experiments on HUVEC proliferation; Figure 27 As shown, the results indicate that DATS@PFC&CMC can indeed promote the rapid proliferation and migration of endothelial cells, ultimately promoting the healing of scratches.

[0155] Example 24:

[0156] We simulated angiogenesis using HUVECs. Next, we investigated how DATS@PFC&CMC treatment stimulated the formation of tubular networks of HUVECs on Matrice; we used an ELISA kit to measure the expression levels of p-p38 and p-ERK1 / 2 at different time points; Figure 28 As shown, DATS@PFC&CMC promotes the formation of longer and denser blood vessels in HUVECs, which is beneficial for the transport of nutrients and thus promotes wound healing. DATS@PFC&CMC can promote the proliferation, migration and angiogenesis of HUVECs by maintaining sustained high expression of p-p38 and p-ERK1 / 2, thereby ultimately accelerating wound healing.

[0157] Example 25:

[0158] Animal experiments: Male ICR mice (25-30g, 6-8 weeks old) were acclimatized for three days and then injected with STZ sodium citrate solution (80mg / kg) for four consecutive days. Blood glucose levels were monitored using a glucometer. A blood glucose level above 16.8mM indicated that the mouse was in a diabetic state. Mice were anesthetized with isoflurane, and their hair was removed. A 1cm diameter hole was punched in the hairless area, and 10μL of MRSA in PBS solution (10⁸ CFU / mL) was added to the hole to establish a diabetic wound model. Mice were randomly divided into four groups and treated with different materials: 1) PBS (50μL); 2) DATS@FC&CMC (50μL); 3) PFC&CMC (50μL); 4) DATS@PFC&CMC (50μL). The wound area was measured with a ruler and photographed at fixed times. The wound closure rate was calculated using formula (5-1).

[0159]

[0160] Where A0 is the area of ​​the wound on day 0, and At is the area of ​​the wound at the specified time point.

[0161] Wound tissue from mice was collected 5 days after treatment and placed in PBS. After sonication for 0.5 h, the resulting liquid was serially diluted 10-fold. 100 μL of each concentration was then evenly spread onto solid LB agar plates using a spreader and incubated at 37°C for 20 h. Bacterial growth was observed and photographed. Simultaneously, the serially diluted liquids were inoculated onto solid LB agar plates and incubated at 37°C for 20 h to observe bacterial colony counts.

[0162] Blood was collected from mice 15 days after treatment. The mice were centrifuged at 2000 rpm and 4°C. The supernatant was collected and the expression levels of TNF-α, IL-6 and IL-1β in the supernatant were detected using an ELISA kit.

[0163] Wound tissue was collected 15 days after treatment. After fixation, dehydration, paraffin embedding, embedding, and sectioning, it was immunolabeled with HO-1, iNOS, and CD163 antibodies. The tissue was then observed and photographed using an inverted fluorescence microscope. The resulting sections were stained with H&E and Masson staining to observe wound healing.

[0164] Venous blood was collected from mice 15 days later, and the number of red blood cells (RBC), white blood cells (WBC), and hemoglobin (HGB) in the mouse blood was measured by Wuhan Saiwei Biotechnology Co., Ltd. to assess the safety of the treatment process.

[0165] Heart, liver, spleen, lungs, and kidneys were collected 15 days after treatment for H&E staining to assess the biocompatibility of the hydrogel.

[0166] like Figure 29 This demonstrates the establishment and treatment process of a MRSA-infected diabetic mouse wound model.

[0167] like Figure 30 As shown, the wound closure rates in the PBS group on days 5, 10, and 15 were 28.8%, 51.4%, and 68.4%, respectively, significantly slower than those in the PFC&CMC and DATS@FC&CMC groups. This is because the wound closure rates in the DATS@FC&CMC and PFC&CMC groups were as high as 79.0% and 80.4% on day 15, respectively. The significantly faster wound healing rates in the DATS@FC&CMC and PFC&CMC groups can be attributed to the antibacterial or anti-inflammatory effects of PHMG and DATS, respectively, effectively killing bacteria or inhibiting inflammation in the wound, thus partially improving the wound microenvironment and promoting healing. However, single antibacterial or anti-inflammatory effects cannot address the complex diabetic microenvironment and often miss some aspects, therefore the wound healing results remain unsatisfactory. Fortunately, due to the integration of multiple regulatory effects of DATS@PFC&CMC, which have high efficiency in sterilization, anti-inflammation, anti-oxidation and tissue regeneration, it can effectively improve the microenvironment of diabetic wounds and ultimately significantly accelerate wound healing, achieving a wound closure rate of 85.8% on day 10 and 97.5% on day 15, with the wound almost completely closed.

[0168] H&E and Masson staining also confirmed this conclusion, such as Figure 31 As shown, 1) the number of newly formed granulation tissue and skin attachments (gland and hair follicle) and collagen deposition in the wound site of the DATS@PFC&CMC group was significantly greater than that of other groups, and there was more mature fibrous tissue; 2) the width of immature tissue and the thickness of epithelial keratinization layer were the smallest in the DATS@PFC&CMC group, indicating that DATS@PFC&CMC can accelerate tissue healing and faster maturation and remodeling.

[0169] like Figure 32 The results show the bacterial colony status of the wound tissue 5 days after treatment. Almost no colonies formed in the wound tissue after 5 days of DATS@PFC&CMC treatment, indicating that DATS@PFC&CMC can effectively kill bacteria at the wound site. Due to the effect of PHMG, the bacterial colony count in PFC&CMC was also very low; however, the other groups still showed a large number of colonies, indicating that bacterial infection still existed. Colony counts also confirmed this conclusion. The tissue treated with DATS@PFC&CMC had only 101.6 log10 CFU / mL, higher than the 103.5 log10 CFU / mL of PFC&CMC. This is due to the synergistic bactericidal effect of DATS and PHMG, which effectively eliminated bacteria in the wound.

[0170] The levels of inflammatory factors in the blood of mice were detected using an ELISA kit 15 days after treatment, such as... Figure 33 As shown, diabetic mice treated with PBS still exhibited severe inflammation at the wound site, with IL-1β, IL-6, and TNF-α expression levels remaining as high as 310.9, 287.2, and 413.3 pg / mL, respectively. However, treatment with DATS@FC&CMC and PFC&CMC resulted in a reduction in the expression levels of IL-1β, IL-6, and TNF-α, primarily due to the anti-inflammatory effects of DATS or the antibacterial effects of PHMG, which partially alleviated the inflammatory response in diabetic wounds. Notably, DATS@PFC&CMC also achieved the best anti-inflammatory effect, with IL-1β, IL-6, and TNF-α expression levels significantly decreasing to 139.5, 91.9, and 222.3 pg / mL, respectively, far lower than the inflammatory levels in diabetic mice and almost identical to those secreted in normal healthy mice. These results indicate that the excellent antibacterial activity of PHMG and the excellent anti-inflammatory activity of DATS effectively alleviated the inflammatory response at the wound site.

[0171] Furthermore, the excellent anti-inflammatory effects of DATS@PFC&CMC are further demonstrated by immunofluorescence staining results in wound tissue. For example... Figure 34 In PBS-treated wound tissue, only strong red fluorescence (iNOS-labeled M1 macrophages) was observed, indicating severe inflammation in the wound tissue of diabetic mice. In contrast, abundant green fluorescence (CD163-labeled M2 macrophages) appeared in wound tissue treated with DATS@PFC&CMC. Quantitative data on fluorescence intensity also corroborated this conclusion. These findings suggest that DATS@PFC&CMC can exert its anti-inflammatory effect by modulating macrophage phenotype.

[0172] In addition, such as Figure 35 As shown, compared with the other groups, DATS@PFC&CMC treatment significantly activated HO-1 expression in wound tissue. The amount of green fluorescence was much higher than that of the other groups, consistent with the quantitative fluorescence results. This indicates that DATS@PFC&CMC can promote HO-1 expression and thus exert its excellent antioxidant effect.

[0173] Notably, immunohistochemical staining of the wound tissue further confirmed that DATS@PFC&CMC treatment significantly promoted vascularization of the wound tissue, such as... Figure 36 As shown, the number of CD31-positive microvessels in wound tissue treated with DATS@PFC&CMC was 5.3 times, 1.7 times, and 4 times higher than that in wound tissue treated with PBS, DATS@FC&CMC, and PFC&CMC, respectively.

[0174] In summary, these results clearly demonstrate that DATS@PFC&CMC can significantly promote the rapid healing of diabetic wounds through its highly effective antibacterial, anti-inflammatory, and angiogenesis-promoting effects.

[0175] The above results indicate that DATS@PFC&CMC has significant potential for effective management of diabetic wounds, but its successful application in clinical practice depends on its good biocompatibility. Therefore, weight changes were measured regularly throughout the treatment period. Furthermore, blood and vital organs were collected at the end of treatment for routine blood analysis and H&E staining. Except for diabetic mice treated with PBS, which experienced significant weight loss, all diabetic mice treated with the three hydrogels showed no significant weight change. Figure 37 This indicates that the prepared hydrogel has good biocompatibility. Blood routine analysis and H&E staining results of the mouse heart, liver, spleen, lungs, and kidneys further confirmed that DATS@PFC&CMC has good biocompatibility, as shown in: 1) the values ​​of three representative hematological markers (red blood cell count (RBC), white blood cell count (WBC), and hemoglobin (HGB)) were all within the normal range. Figure 37 ); 2) Compared with normal healthy mice, mice treated with DATS@PFC&CMC showed no obvious tissue damage in the H&E staining results of the heart, liver, spleen, lungs, and kidneys. Figure 38 In summary, all the above results clearly demonstrate that DATS@PFC&CMC has good biocompatibility.

[0176] In summary, we treated MRSA-infected diabetic mouse wounds with different materials and obtained the following conclusions: 1) DATS@PFC&CMC can promote microangiogenesis and accelerate wound healing in diabetic mice, with a wound closure rate of 97.5% after 15 days; 2) DATS@PFC&CMC can effectively kill bacteria in wounds, promote the transformation of macrophages to M2 type to exert anti-inflammatory effects, and upregulate HO-1 expression to exert antioxidant effects; 3) DATS@PFC&CMC has good therapeutic effects and good biocompatibility.

[0177] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A multifunctional hydrogel dressing based on hydrogen sulfide gas therapy, characterized in that, This multifunctional hydrogel dressing is DATS@PFC&CMC hydrogel dressing. DATS@PFC&CMC hydrogel dressing is prepared based on the dynamic Schiff base reaction between PHMG-modified aldehyde F108, denoted as PFC, and carboxymethyl chitosan CMC. DATS@PFC&CMC hydrogel dressing can release H2S in the presence of glutathione (GSH); The grafting rate of PFC was 1.4%; The concentration of PFC is 100 mg / mL.

2. The method for preparing a multifunctional hydrogel dressing based on hydrogen sulfide gas therapy according to claim 1, characterized in that, Includes the following steps: S1. First, add 10g of chitosan CS and 13.5g of sodium hydroxide NaOH to a 500mL flask. Then, add a mixed solution containing 50mL of water and 50mL of isopropanol to the flask and stir thoroughly to form a mixture. S2. Then, the mixture is placed in an oil bath at 50°C to swell and alkalize for 1 hour. S3. Dissolve 15g of chloroacetic acid in 20mL of isopropanol and add it dropwise to the mixture over 30min. Stir vigorously and maintain the reaction in an oil bath at 50℃ for 4h. After the reaction is complete, stop the reaction by adding 200mL of 70% v / v methanol. S4. Filter the obtained mixture and collect the filter residue. Wash the filter residue three times with 90% v / v methanol. After filtration, place the filter residue in a vacuum drying oven at 50°C and vacuum dry for 1 day. Collect the solid product carboxymethyl chitosan (CMC).

3. The method for preparing a multifunctional hydrogel dressing based on hydrogen sulfide gas therapy according to claim 2, characterized in that, It also includes the following steps: a. Add 5.8g F108, 0.6g 4-formylbenzoic acid and 30mg 4-dimethylaminopyridine (DMAP) to a 500mL pear-shaped flask with a side arm; b. Under N2 protection, add 70 mL of anhydrous dichloromethane (DCM) and dissolve it completely. Then, add 1.01 g of dicyclohexylcarbodiimide (DCC) dissolved in dichloromethane dropwise to the above reaction system under ice bath conditions and react at room temperature for 2 days. c. After the reaction is complete, filter to remove the solids generated in the reaction, concentrate the obtained organic phase by rotary evaporator, and wash three times with saturated NaCl. d. After washing, remove all DCM by rotary evaporation, vacuum dry for 5 hours, disperse the obtained white solid in 100 mL of pure water, stir vigorously for 2 hours, remove the precipitate by high-speed centrifugation at 9000 r / min for 10 minutes, and freeze-dry the obtained supernatant to obtain product F108-CHO, denoted as FC. e. Dissolve the obtained dry 1g FC and 10mg PHMG in DMSO and stir for 4h. After the reaction is complete, add 0.62mg sodium cyanoborohydride NaBH3CN to the reaction solution and continue the reaction at room temperature for 12h. f. After the reaction is complete, dialyze the product using a Spectrum wide-range dialysis bag at MWCO 3500 Da for 24 hours, and freeze-dry to obtain a white product, namely PHMG-F108-CHO or PFC.

4. The method for preparing a multifunctional hydrogel dressing based on hydrogen sulfide gas therapy according to claim 3, characterized in that, It also includes the following steps: g. Dissolve 200mg F108-CHO and 20mg DATS in 2mL DCM, stir thoroughly for 1h, and then remove all DCM using a rotary evaporator to form a uniform yellow film. h, add pure water to hydrate the membrane for 8 hours; i. Centrifuge the mixture obtained in step h at 10,000 rpm for 10 min to remove insoluble products and larger micelles; j. The filtrate obtained in step i is freeze-dried to obtain drug-loaded micelles DATS@PFCs; k. By adjusting the mass feed ratio of DATS / PFC to 0.1, 0.2 and 0.5, three drug-loaded micelles with different drug loading amounts, namely DATS@PFC-1, DATS@PFC-2 and DATS@PFC-3, were obtained.

5. The method for preparing a multifunctional hydrogel dressing based on hydrogen sulfide gas therapy according to claim 4, characterized in that, It also includes the following steps: PFC was mixed with three different concentrations of CMC (2.0% w / v, 2.5% w / v, and 3.0% w / v) at a 1:1 volume ratio. After thorough mixing, the mixture was placed in a 37°C oven to prepare hydrogels PFC&CMC2.0, PFC&CMC2.5, and PFC&CMC3.0, respectively. Then, the PFC micelle solution was replaced with micelle solutions of DATS@PFC-1, DATS@PFC-2, and DATS@PFC-3 of the same concentration to prepare drug-loaded hydrogels DATS@PFC&CMC-1, DATS@PFC&CMC-2, and DATS@PFC&CMC-3.

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