CN116603096A

CN116603096A - Multifunctional hydrogel dressing based on hydrogen sulfide gas therapy and preparation method thereof

Info

Publication number: CN116603096A
Application number: CN202310556799.1A
Authority: CN
Inventors: 蔡晓军; 陈佳乐; 李林; 陈冬帆
Original assignee: SCHOOL & HOSPITAL OF STOMATOLOGY WENZHOU MEDICAL UNIVERSITY
Current assignee: SCHOOL & HOSPITAL OF STOMATOLOGY WENZHOU MEDICAL UNIVERSITY
Priority date: 2023-05-17
Filing date: 2023-05-17
Publication date: 2023-08-18

Abstract

The invention belongs to the technical field of biomedical polymers, and particularly relates to a multifunctional hydrogel dressing based on hydrogen sulfide gas therapy and a preparation method thereof. The beneficial effects of the invention are as follows: the wound dressing system can be rapidly degraded in the microenvironment of low pH and high oxidative stress of the diabetic wound, so that the effect of rapidly releasing H2S is achieved. The PHMG with the surface modified by the wound dressing can play an excellent antibacterial role in combination with DATS; the released H2S can fully play the roles of high-efficiency anti-inflammatory, antioxidation, cell proliferation promotion, migration and vascularization, thereby improving the microenvironment of the diabetes wound and achieving the effect of accelerating the healing of the diabetes wound.

Description

Multifunctional hydrogel dressing based on hydrogen sulfide gas therapy and preparation method thereof

Technical Field

The invention relates to the technical field of biomedical polymers, in particular to a multifunctional hydrogel dressing based on hydrogen sulfide gas therapy and a preparation method thereof.

Background

Diabetes mellitus is a common chronic endocrine system disease of a preparation method of a multifunctional hydrogel dressing based on hydrogen sulfide gas therapy, and has extremely high morbidity, mortality and disability rate. Whereas chronic wounds are one of the most serious complications of diabetes mellitus as diabetic ulcers. Due to lack of exercise and intake of large amounts of grease by modern social people, the number of patients is rapidly increased, and expensive and cumbersome treatment forms bring great pain and heavy economic burden to patients and society, and bacterial growth, unbalanced inflammation and abnormal angiogenesis are main causes of difficult healing.

Current clinical modalities for treating chronic diabetic wounds include glycemic control, wound debridement, antibiotic therapy, wound dressing, and the like. While these treatments may reduce pain and help prevent infection, they still suffer from a number of deficiencies. For example, prolonged insulin injections can lead to serious complications in the body; non-professional debridement can lead to enlargement of ulcer wounds, exacerbating infection; the long-term use of antibiotics can lead to serious side effects and the formation of multi-drug resistance; traditional wound dressings are single-function and require frequent replacement [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.

The hydrogel is a polymer material with a 3D network structure, and the preparation method of the multifunctional hydrogel dressing based on hydrogen sulfide gas therapy has the advantages of excellent flexibility, biocompatibility and fluid absorption performance, can provide a moist environment favorable for tissue regeneration, and effectively avoids secondary damage caused by wound adhesion. In addition, by adjusting the structural design, the hydrogel dressing can also be endowed with the most advanced physical and chemical properties, such as injection, adhesion, self-healing, smart response degradation, excellent antibacterial properties and the like. It is notable that hydrogels prepared from dynamic Schiff base bonds not only have good injectability and self-healing properties, but also can rapidly degrade and release drugs under weak acid and high oxidation conditions, exerting therapeutic effects of loaded drugs. These excellent physicochemical properties are important factors as ideal wound dressings. Accordingly, a variety of functionalized hydrogel dressings, such as antibacterial, anti-inflammatory, antioxidant, and pro-vascularization, have been researched and developed for the treatment of diabetic wounds.

However, single antibacterial, anti-inflammatory or antioxidant treatment regimens, facing complex diabetic wound microenvironments, often fail to achieve the desired therapeutic effect. Therefore, development of therapeutic strategies capable of comprehensively coping with diabetic wound microenvironments is urgently required.

Applicants have found that hydrogen sulfide (H ₂ S) has a great role in anti-inflammatory, antioxidant and pro-vascularization. Diallyl trisulfide (DATS) is capable of releasing H in the presence of Glutathione (GSH) ₂ S, exert the therapeutic effect thereof. However, oil solubility limits its application. The hydrogel has excellent fluid absorption performance and good structural transformation performance, and the application range of the hydrogel is widened. Thus, hydrogels are associated with H ₂ S-gas therapy in combination is expected to be highly effective in treating diabetic wounds.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide a multifunctional hydrogel dressing based on hydrogen sulfide gas therapy and a preparation method thereof so as to solve the problems.

The technical scheme of the invention is realized as follows: a multifunctional hydrogel dressing based on hydrogen sulfide gas therapy is prepared based on dynamic Schiff base reaction between PHMG modified aldehyde F108 (PFC) and carboxymethyl chitosan CMC.

Further, the grafting ratio of PFC was 1.4%

Further, the concentration of PFC was 100mg/mL.

Further, the method comprises the following steps:

s1, firstly adding 10g of chitosan CS and 13.5g of sodium hydroxide NaOH into a 500mL flask, then adding a mixed solution containing 50mL of water and 50mL of isopropanol into the flask, and fully stirring to form a mixed solution;

S2, then placing the mixed solution into an oil bath pot at 50 ℃ for swelling and alkalizing for 1h;

s3, uniformly dropwise adding 15g of chloroacetic acid dissolved in 20mL of isopropanol into the mixed solution within 30min, vigorously stirring, and keeping the solution in an oil bath at 50 ℃ for reaction for 4h. After the reaction was completed, the reaction was stopped by adding 200mL of 70% (v/v) methanol;

and S4, filtering the obtained mixed solution, and collecting filter residues. The resulting residue was washed 3 times with 90% (v/v) methanol. After filtration, the residue was dried in vacuo in a vacuum oven at 50℃for 1 day. The collected solid product carboxymethyl chitosan CMC

Further, the method also comprises the following steps:

a. 5.8g F108 (0.4 mmol), 0.6g 4-formylbenzoic acid (4 mmol) and 30mg 4-dimethylaminopyridine DMAP were added to a 500mL branched pear-shaped bottle;

b. 70mL of anhydrous dichloromethane DCM was added under the protection of N2 to be fully dissolved, then 1.01g (4.89 mmol) of dicyclohexylcarbodiimide DCC dissolved in dichloromethane was added dropwise to the reaction system under the ice bath condition, and the reaction was carried out at room temperature for 2 days;

c. after the reaction is finished, removing solid generated in the reaction by suction filtration, concentrating the obtained organic phase by a rotary evaporator, and washing with saturated NaCl for three times;

d. After the washing was completed, all DCM was removed by a rotary evaporator, and after drying in vacuo for 5 hours, the resulting white solid was dispersed in 100mL of pure water, vigorously stirred for 2 hours, the precipitate was removed by high-speed centrifugation (9000 r/min,10 min), and the resulting supernatant was freeze-dried to give the product F108-CHO (FC).

e. Fully dissolving the obtained dried 1g FC and 10mg PHMG in DMSO, stirring for 4 hours, adding 0.62mg sodium cyanoborohydride NaBH3CN into the reaction solution after the reaction is finished, and continuing to react for 12 hours at room temperature;

f. after the reaction was completed, the mixture was dialyzed with a wide dialysis bag (Spectrum, MWCO 3500 Da) for 24 hours, and freeze-dried to give a white product (PHMG-F108-CHO, PFC).

Further, the method also comprises the following steps:

g. 200mg of F108-CHO and 20mg of DATS were dissolved in 2mL of DCM and stirred well for 1h, then all DCM was removed using a rotary evaporator to form a uniform yellow film;

h. adding pure water to hydrate the film for 8 hours;

i. centrifuging (10000 rpm,10 min) the mixed solution obtained in the step b to remove insoluble products and larger micelles;

j. c, freeze-drying the filtrate obtained in the step c to obtain drug-loaded micelles DATS@PFCs;

k. the three drug-loaded micelles with different drug loading rates of DATS@PFC-1, DATS@PFC-2 and DATS@PFC-3 are obtained by adjusting the mass feeding ratio of DATS/PFC to 0.1,0.2 and 0.5.

Further, the method also comprises the following steps: PFC and CMC (2.0% w/v,2.5% w/v and 3.0% w/v) with three different concentrations are mixed according to the volume ratio of 1:1, after the mixing is uniform, the mixed solution is placed in a baking oven at 37 ℃ to prepare hydrogel PFC & CMC2.0, PFC & CMC2.5 and PFC & CMC3.0 respectively, and then the PFC micelle solution is replaced by DATS@PFC-1, DATS@PFC-2 and DATS@PFC-3 micelle solution with the same concentration and the carrier liquid gels DATS@PFC & CMC-1, DATS@PFC & CMC-2 and DATS@PFC-3 are prepared.

The beneficial effects of the invention are as follows:

1. will H ₂ S and hydrogel are combined to prepare the hydrogel dressing DATS@PFC&CMC；

2. The wound dressing system can be rapidly degraded in the microenvironment of low pH and high oxidative stress of the diabetic wound, thereby achieving H ₂ S, an effect of quick release;

3. the PHMG with the surface modified by the wound dressing can play an excellent antibacterial role in combination with DATS;

4. released H ₂ S can fully exert the high-efficiency anti-inflammatory, antioxidant, cell proliferation promoting, migration and vascularization effects;

5. effectively improves the microenvironment of the diabetes wound and achieves the effect of accelerating the healing of the diabetes wound.

Drawings

In order to more clearly illustrate the embodiments of the invention or the technical solutions of the prior art, the drawings which are used in the description of the embodiments or the prior art will be briefly described, it being obvious that the drawings in the description below are only some embodiments of the invention, and that other drawings can be obtained according to these drawings without inventive faculty for a person skilled in the art.

FIG. 1 is a schematic diagram showing the mechanism action of the preparation of DATS@PFC & CMC according to the specific embodiment of the invention, wherein the preparation has the functions of efficient antibiosis, anti-inflammation, angiogenesis and promoting the healing of a diabetes wound infected by MRSA;

FIG. 2 shows the hemolysis rates of PFCs micelles 1h (a) and 24h (b) with different PHMG grafting rates according to the embodiment of the invention;

FIG. 3 is a sol-gel transition diagram of PFC & CMC according to an embodiment of the present invention;

FIG. 4 is a Transmission Electron Microscope (TEM) photograph of PFC (a) and DATS@PFC (b) according to a specific embodiment of the present invention;

FIG. 5 is a scanning electron microscope image of PFC & CMCs hydrogels according to an embodiment of the present invention;

FIG. 6 is a graph showing the porosity of PFC & CMC hydrogels in accordance with embodiments of the present invention;

FIG. 7 is a graph showing the swelling ratio of PFC & CMC hydrogels in accordance with embodiments of the present invention;

FIG. 8 is an illustration of the adhesive strength of PFC & CMCs hydrogels according to an embodiment of the present invention;

FIG. 9 is a graph showing the physical adhesive strength of PFC & CMC3.0 hydrogel to pigskin according to an embodiment of the present invention;

FIG. 10 is a graph of apparent viscosity versus shear rate for PFC & CMC3.0 hydrogels in accordance with embodiments of the present invention;

FIG. 11 is an injectable physical view of a PFC & CMC3.0 hydrogel according to an embodiment of the present invention;

FIG. 12 is a graph showing the change in storage modulus (G ') and loss modulus (G') as a function of strain for PFC & CMCs hydrogels according to embodiments of the present invention;

FIG. 13 is a graph showing the rheological properties of PFC & CMCs hydrogels according to embodiments of the present invention as a function of strain from 0.1% to 300%;

FIG. 14 is a graph showing the self-healing properties of PFC & CMC3.0 hydrogels according to embodiments of the present invention;

FIG. 15 is a graph showing oxidative degradation behavior of PFC & CMC3.0 hydrogels in accordance with embodiments of the present invention;

FIG. 16 is the presentDescription of the invention DATS@PFC&H of CMC hydrogel ₂ S, releasing a behavior diagram;

FIG. 17 is an image of the evaluation of PFC & CMC and DATS@PFC & CMCs cytotoxicity on HUVECs cells by (a) live/dead staining and (b) CCK-8 according to embodiments of the present invention;

FIG. 18 is a flat panel image (a) and survival rate (b, c) of E.coli and MRSA after treatment of FC & CMC, PFC & CMC and DATS@PFC & CMC in accordance with embodiments of the present invention;

FIG. 19 is a live-dead stained image of E.coli (a) and MRSA (b) after treatment with FC & CMC, PFC & CMC and DATS@PFC & CMC in accordance with embodiments of the present invention;

FIG. 20 is an SEM photograph of E.coli and MRSA after treatment of FC & CMC, PFC & CMC and DATS@PFC & CMC in accordance with embodiments of the present invention;

FIG. 21 is a graph showing leakage of E.coli and MRSA cell proteins (a) and variation in ATP synthesis levels (b) after treatment with FC & CMC, PFC & CMC and DATS@PFC & CMC in accordance with embodiments of the present invention;

FIG. 22 is a fluorescence image of H2S (a) and corresponding fluorescence intensity (b) image within a cell according to an embodiment of the present invention;

FIG. 23 shows changes in the expression levels of intracellular inflammatory factors (a) TNF- α, (b) IL-1β and (c) IL-6 following treatment with DATS, PFC & CMC and DATS@PFC & CMC in accordance with embodiments of the present invention;

FIG. 24 shows changes in the expression levels of intracellular inflammatory factors (a) IL-10 and (b) IL-4 after DATS, PFC & CMC and DATS@PFC & CMC treatment in accordance with embodiments of the present invention;

FIG. 25 shows the expression levels of p-STAT3 and p-ERK proteins in activated macrophages after DATS, PFC & CMC and DATS@PFC & CMC treatment in accordance with embodiments of the present invention;

FIG. 26 shows the expression levels of mRNA and protein of HO-1 in activated macrophages after DATS, PFC & CMC and DATS@PFC & CMC treatment according to a specific embodiment of the present invention;

FIG. 27 is a graph comparing the effect of DATS@PFC & CMC hydrogels of the present invention on HUVECs cell scratch healing and proliferation behavior;

FIG. 28 shows (a) endothelial cell catheterization, (b) branching points and capillary lengths of the network of HUVECs, and (c) expression levels of p-ERK1/2 and p-p38 in HUVECs after various treatments, according to embodiments of the present invention;

FIG. 29 is a schematic representation of a mouse treatment protocol in accordance with one embodiment of the present invention;

FIG. 30 is a graph comparing wound healing rates of (a), wound tracking, and (c) for an embodiment of the present invention;

FIG. 31 is a chart of Masson staining of wound tissue at 15 days according to an embodiment of the present invention (a) H & E and (b);

FIG. 32 is a graph showing bacterial counts after 5 days of diabetic wound treatment in mice in accordance with an embodiment of the present invention;

FIG. 33 is a graph showing the expression levels of inflammatory factors IL-1. Beta., IL-6 and TNF-. Alpha.in the blood of mice in accordance with an embodiment of the present invention;

FIG. 34 is a graph of imaging and quantitative analysis of iNOS and CD163 in wound tissue in accordance with an embodiment of the present invention;

FIG. 35 is a graph of the imaging and quantitative analysis of HO-1 in wound tissue in accordance with an embodiment of the invention;

FIG. 36 is a chart of a wound tissue immunohistochemical analysis (CD 31) at 15 days according to the embodiment of the present invention;

FIG. 37 is a photograph of (a) red blood cell count (RBC), (b) white blood cell count (WBC) and (c) Hemoglobin (HGB) of a day 15 blood routine analysis of groups of mice according to embodiments of the present invention;

FIG. 38 is a graph of H & E staining of heart, liver, spleen, lung, kidney of a control group and DATS@PFC & CMC group according to an embodiment of the present invention.

Detailed Description

The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

In the description of the present application, it is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments in accordance with the present application. For ease of description, the dimensions of the various features shown in the drawings are not drawn to actual scale. Techniques, methods, and apparatus known to one of ordinary skill in the relevant art may not be discussed in detail, but should be considered part of the specification where appropriate. In all examples shown and discussed herein, any specific values should be construed as merely illustrative, and not a limitation. Thus, other examples of the exemplary embodiments may have different values. It should be noted that: like reference numerals and letters denote like items in the following figures, and thus once an item is defined in one figure, no further discussion thereof is necessary in subsequent figures.

Example 1:

since chitosan can only be dissolved into an acidic solution, its application is severely limited. And the introduction of carboxymethyl breaks the regularity of chitosan, thereby increasing the water solubility and expanding the application of chitosan. The CMC is synthesized by the following steps: first, chitosan (CS, 10 g) and sodium hydroxide (NaOH, 13.5 g) were added to a 500mL flask, and then a mixed solution containing 50mL of water and 50mL of isopropyl alcohol was added to the flask, followed by stirring well to form a mixed solution. The mixture was then placed in an oil bath at 50 ℃ to swell and alkalize for 1h. After that, the process is performed. 15g chloroacetic acid dissolved in 20mL isopropanol was added dropwise to the mixture uniformly over 30min, vigorously stirred, and kept at 50℃for 4h under an oil bath. After the reaction was completed, the reaction was stopped by adding 200mL of 70% (v/v) methanol. The resulting mixture was filtered, and the filter residue was collected. The resulting residue was washed 3 times with 90% (v/v) methanol. After filtration, the residue was dried in vacuo in a vacuum oven at 50℃for 1 day. The collected solid product is CMC. The structure of CS and CMC was characterized using fourier infrared spectroscopy (FT-IR) and nuclear magnetic resonance spectroscopy (1 HNMR). The degree of substitution of carboxymethyl is calculated by the relative integral area of the nuclear magnetic hydrogen spectrogram.

Example 2:

f108 is subjected to hydroformylation modification by utilizing an esterification reaction. 5.8g F108 (0.4 mmol), 0.6g 4-formylbenzoic acid (4 mmol) and 30mg 4-Dimethylaminopyridine (DMAP) were added to a 500mL branched pear-shaped bottle and 70mL anhydrous Dichloromethane (DCM) was added under N2 protection to be sufficiently dissolved, and then 1.01g (4.89 mmol) Dicyclohexylcarbodiimide (DCC) dissolved in dichloromethane was added dropwise to the above reaction system under ice bath conditions and reacted at room temperature for 2 days. After the reaction was completed, the solid formed in the reaction was removed by suction filtration, and the resulting organic phase was concentrated by a rotary evaporator and washed three times with saturated NaCl. After the washing was completed, all DCM was removed by a rotary evaporator, and after drying in vacuo for 5 hours, the resulting white solid was dispersed in 100mL of pure water, vigorously stirred for 2 hours, the precipitate was removed by high-speed centrifugation (9000 r/min,10 min), and the resulting supernatant was freeze-dried to give the product F108-CHO (FC). The obtained dried 1g of FC and 10mg of PHMG were sufficiently dissolved in DMSO, stirred for 4 hours, and after completion of the reaction, 0.62mg of sodium cyanoborohydride (NaBH 3 CN) was added to the reaction solution, and the reaction was continued at room temperature for 12 hours. After the reaction was completed, the mixture was dialyzed with a wide dialysis bag (Spectrum, MWCO 3500 Da) for 24 hours, and freeze-dried to give a white product (PHMG-F108-CHO, PFC). The PFC with three different grafting rates is obtained by adjusting the mass ratio of PHMG/FC to 0.01,0.02 and 0.03, and the PFC is named PFC-1, PFC-1 and PFC-3 respectively. And the structures of F108 and FC were characterized using Fourier infrared spectroscopy (FT-IR) and nuclear magnetic resonance spectroscopy (1H NMR).

The grafting rate of PHMG is calculated by titration of aldehyde groups, and the specific experimental steps are as follows: a methanol solution (20 g/L) of hydroxylamine hydrochloride in which 5mg of FC and 5mg of PFC were dissolved was accurately weighed, and then 80. Mu.L of thymol blue ethanol solution (0.1%) was added thereto as an indicator. The mixture is fully stirred and dissolved uniformly, and hydrochloric acid is generated due to the reaction of aldehyde groups and hydroxylamine hydrochloride, so that the solution is acidic, and the indicator is pink. Standard sodium hydroxide solution (0.01 mol/L) was then added dropwise until the solution turned yellow and did not fade at 30s, and the experiment was repeated three times. The grafting ratio of PHMG was calculated using the following formula (2-1):

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

Since PHMG has a large amount of guanidine groups, brings about excellent antibacterial ability and causes huge cytotoxicity, the grafting rate and concentration of PFC are screened through a hemolysis experiment. The specific experimental steps are as follows: mu.L of mouse erythrocytes and 12. Mu.L of PFC of different concentrations or different PHMG grafting degrees were first mixed homogeneously in sterile tubes with physiological saline and incubated for 1 or 24h at 37℃and the mixture was centrifuged (3000 rpm,4 ℃) and the absorbance of the supernatant at 545nm was measured with a microplate reader. 0.9% sodium chloride and ultrapure water were used as negative control and positive control, respectively. The hemolysis ratio was calculated according to the formula (2-2).

Example 3:

the PHMF content was determined by titration and is shown in tables 2-3. The above experiments show that FC and PFC are successfully synthesized.

Tables 2 to 3 grafting ratio and aldehyde group content of PHMG in three micelles

Table2-3 Graft rate of PHMG andaldehyde content in three types of micelles

As shown in FIG. 2 below, the high concentration of PFC and high grafting ratio of PHMG resulted in great hemolysis, and after 1h, the hemolysis ratio of PFC-2 and PFC-3 even at 100mg/mL had reached 26% and 54%, which was far higher than the safe hemolysis ratio (5%). Notably, although the 1h hemolysis rate of PFC-1 at the concentrations of 140mg/mL and 180mg/mL was 2.4% and 4.5%, the hemolysis rate after 24h increased to 8.1% and 14.5%, indicating that PFC-1 micelles at these two concentrations still caused relatively large hemolysis and could not be used. While the hemolysis rates of 100mg/mL PFC-1 at 1h and 24h are 0.3% and 2.8% (< 5%), respectively, indicating that the PFC-1 still has good biosafety at a concentration of 100 mg/mL. The following experiments used 100mg/mL of PFC-1 material as a subject and were named PFC.

Example 4:

the lipid solubility of DATS severely limits its application, so we load DATS into PFC micelles by thin film dispersion. The specific experimental steps are as follows: 200mg of FC and 20mg of DATS were dissolved in 2mL of DCM and stirred well for 1h, then all DCM was removed using a rotary evaporator to form a uniform yellow film. Then, a small amount of pure water was added to hydrate the film for 8 hours. During hydration, PFC micelles will self-assemble to form micelles to load DATS. The resulting mixture was centrifuged (10000 rpm,10 min) to remove insoluble products and larger micelles. And freeze-drying the obtained filtrate to obtain drug-loaded micelles (DATS@PFCs). The three drug-loaded micelles with different drug loading rates of DATS@PFC-1, DATS@PFC-2 and DATS@PFC-3 are obtained by adjusting the mass feeding ratio of DATS/PFC to 0.1,0.2 and 0.5. And calculating the drug loading quantity and the drug loading efficiency of the DATS@PFC by using an organic element analyzer. The specific experimental steps are as follows [67]: the DATS@PFCs were accurately weighed, burned with tungsten oxide at 1150 ℃ with excess oxygen in a helium stream, and then reduced with metallic copper at 850 ℃ to obtain sulfur dioxide. And calculating the content of the S element by measuring the mass of sulfur dioxide, thereby obtaining the mass of the DATS. The drug loading and drug loading rate of the DATS were calculated using the following formulas:

F108 is an amphiphilic block copolymer that self-assembles into micelles in water and is thus widely studied in terms of drug loading. As shown in fig. 3, direct mixing of the oily DATS with water causes emulsification, and the solution is a white emulsion. The prepared aqueous solution of the drug-loaded micelle is yellow clear liquid (DATS is yellow oily matter), which indicates that the DATS is successfully loaded into the PFC micelle, and the obtained drug-loaded micelle can be uniformly dispersed in water.

Example 5:

particle size and potential of PFC and dats@pfc were determined by malvern particle size (DLS), and morphology and the like of micelles were further studied by TEM. The results are shown in FIG. 4. The particle size of dats@pfc is smaller than that of PFC, which may be that micelle-loaded fat-soluble DATs makes the comfortable core of the original micelle more compact, thereby reducing the size of the micelle. The Transmission Electron Microscope (TEM) detects that the micelles of PFC and DATS@PFC have definite three-dimensional structures, and the micelles are uniformly dispersed.

Example 6:

after successful preparation of the drug-loaded micelle DATS@PFCs is determined, the content of S element in the drug-loaded micelle is detected by a high-temperature combustion method, so that the drug-loading capacity and the drug-loading efficiency in the drug-loaded micelle are calculated. Taking DATS@PFC-1 as an example, 5.254mg of DATS@PFC-1 has a content of 1.545% of S element. The mass of S in 5.254mg is 5.254 ×1.545% = 0.0812mg. While one DATS has three sulfur atoms, the relative molecular mass of S is 32g/mol, and the relative molecular mass of DATS is 178.34g/mol. Therefore, according to the proportional relationship, the mass of dats=mass of S element/(relative molecular mass of S element×3) ×relative molecular mass of dats=0.151 mg in dats@pfc-1 is calculated. It can be calculated that the drug loading of DATS in dats@pfc-1= (mass of DATS in micelle)/(mass of dats@pfc-1) ×100% = (0.151/5.254) ×100% = 2.88%. DATS drug delivery efficiency = (mass of DATS in micelle)/(mass of DATS) ×100% = (0.151/5.254) ×100% = 31.63% in dats@pfc-1. The drug loading capacity and the drug loading efficiency of the other two drug loading micelles can be obtained by the same method, and the calculation results are shown in tables 2-3 respectively.

Tables 2-3 drug loading and drug loading rates of DATS in three micelles

In summary, the components CMC and PFC of the hydrogel were synthesized, the structure thereof was characterized by FT-IT and 1H NMR, and the grafting ratio and PFC concentration of PHMG were selected by hemolysis experiments. And then loading the DATS into a hydrophobic inner core of the PFC, preparing the drug-loaded micelle DATS@PFC, and calculating the drug loading capacity and the drug loading efficiency of the drug-loaded micelle with three different feeding ratios. The particle size and the morphology of the prepared blank micelle and drug-loaded nano micelle are also characterized. The following can be concluded: 1) Synthesizing CMC and PFC, and determining that the substitution degree of carboxymethyl of CMC is 74%, and obtaining the safe grafting rate of PFC of 1.4% and the safe concentration of PFC of 100mg/mL through hemolysis screening; 2) The DATS is successfully loaded into the PFC micelle, and the prepared drug-loaded micelle has the particle size of 198nm and uniform structure; 3) Three kinds of nano-micelles with different drug loading rates are prepared through different feeding ratios, and the drug loading rates and the drug loading efficiencies of the three kinds of drug loading micelles are detected through a high-temperature combustion method.

Example 7:

in the above examples, safe PFC concentrations (10%, w/v) were screened by hemolysis experiments, so we prepared three different CMC concentrations (2.0%, 2.5% and 3.0%, w/v) to prepare three different hydrogels. Firstly, mixing PFC and CMC with different concentrations according to the volume ratio of 1:1, and after the PFC and CMC are uniformly mixed, placing the mixed solution in a 37 ℃ oven to prepare three hydrogels: PFC & CMC2.0, PFC & CMC2.5 and PFC & CMC3.0. The PFC micelle solution is replaced by DATS@PFC-1, DATS@PFC-2 and DATS@PFC-3 micelle solutions with the same concentration to prepare three carrier liquid gels, and the three carrier liquid gels are named DATS@PFC & CMC-1, DATS@PFC & CMC-2 and DATS@PFC & CMC-3 respectively.

Example 8:

the gel time of the hydrogels was determined by the inverted bottle method. The specific experimental method is as follows, 100 mu L of 10% (w/v) PFC aqueous solution and 100 mu L of CMC solution with different concentrations are respectively added into a 1.5mL centrifuge tube, and after uniform mixing, the centrifuge tube is placed into a water bath kettle with constant temperature of 37 ℃. And taking out each 10s to inversely observe the state of the liquid in the centrifugal tube until the observed liquid does not flow any more, and recording the time at the moment, namely the gel forming time.

The results are shown in Table 3-1. It can be found that the gel forming time of the hydrogel gradually decreases with increasing concentration of CMC, because the increase of CMC concentration increases the crosslinking density and hydrogen bonding between hydrogels, accelerating the formation of hydrogels. The gel forming time of the hydrogel is beneficial for application on wounds.

TABLE 3-1 gelation time of PFC & CMC hydrogels of different mass ratios

Example 9:

the section sample is prepared from the freeze-dried hydrogel sample by a liquid nitrogen crisp surface method, and the specific steps are as follows: the resulting hydrogel was placed in liquid nitrogen, cooled sufficiently, then taken out with forceps, cut open with a blade, and the morphology of the cross section was observed with a high resolution transmission electron microscope (SEM), and finally the porosity of the hydrogel was analyzed by Image J software.

The large number of pores of the hydrogel gives the hydrogel good water absorption properties, so the water absorption properties of the hydrogel were examined by the swelling experiment. The specific experimental steps are as follows: the dried hydrogels of the same size were accurately weighed, the mass at this time was recorded as W0, then placed in pure water, and weighing was taken out every 1h until the hydrogel mass was no longer changed, which can be considered to be in equilibrium with swelling at this time, and the hydrogels at this time were weighed and recorded as Wt. The swelling ratio of the hydrogel was calculated by the formula (3-1):

we characterized the cross-sectional morphology of the dried three hydrogels PFC & CMC2.0, PFC & CMC2.5 and PFC & CMC3.0 using SEM and calculated the porosities of the three hydrogels using Image J software, the results are shown in fig. 5. All hydrogels had a porous structure and as CMC concentration increased, the network pores of the hydrogel cross-section became smaller. And porosity also decreased from 43.1% to 24.8% with increasing CMC concentration (fig. 6). This reduction in pore size and porosity is also reflected in the swelling ratio. As shown in fig. 7, the swelling ratio of the hydrogel was reduced from 17.6 to 16.1 as the CMC concentration was increased, because the space for storing water was reduced due to the compact network structure, but even CMC & PFC3.0, which had the lowest swelling ratio, was able to absorb water 15 times more than its own mass because of the large pore size and the large hydrogen bonding effect, which was able to well lock water. The results show that the hydrogel has excellent water absorption performance, can effectively absorb exudates of wound tissues, maintains the moist environment of the wound, and is beneficial to rapid healing of the wound tissues.

Example 10:

as shown in fig. 8, as the CMC concentration increases, the adhesion strengths of PFC & CMC2.0, PFC & CMC2.5 and PFC & CMC3.0 are 4.4,7.6 and 13.8kPa, respectively. The adhesion strength of PFC & CMC3.0 is far higher than that of the other two groups, and the adhesion strength of the hydrogel to tissues is studied through a lap shear test. The experimental procedure was as follows: experiment with fresh pigskin to simulate normal skin tissue, firstly soaking the pigskin in PBS for one day to remove surface grease, then cutting the pigskin into 10×40 (mm 2) long strips, uniformly mixing 50 μl of CMC solution with different concentrations with 50 μl of PFC solution, uniformly smearing on one pigskin, closely adhering the other pigskin at the position coated with the adhesive, and then standing at 37deg.C for 4 hr. The adhesion strength of the hydrogel to the tissues was measured by a universal tester and set at 20N and the pressure measurement rate at 1mm/min. We further verified the adhesion of the hydrogel to the tissue by hanging a 100g weight under the bonded pigskin.

As can be seen from fig. 9, two pigskins adhered together with PFC & CMC3.0 hydrogel can withstand a weight of 100g without separating for a long period of time. This adhesion of the hydrogel is due to the strong electrostatic interaction of the charge on the hydrogel surface with the phospholipid molecules on the cells, and can be greatly improved with increasing CMC concentration. The strong adhesion strength can ensure that the hydrogel can be firmly adhered to wound tissues, and ensure good treatment effect. The conclusion can clearly prove the excellent adhesion performance of the hydrogel.

Example 11:

the apparent viscosity of PFC & CMC3.0 hydrogels varies with shear rate. As shown in fig. 10, the apparent viscosity of PFC & CMC3.0 hydrogels decreased rapidly from 3000 Pa-s to 0.55 Pa-s as the shear rate increased. Indicating that the hydrogels have good shear thinning properties. In addition, the hydrogel can be extruded from the syringe force and form a continuous strip in the water (fig. 11). Indicating that the hydrogels have good injectability. This excellent injectability results from the gel-forming manner of the hydrogels: schiff base reactions based on dynamic imine bonds, which can spontaneously proceed without external forces. Thus, when the hydrogel is extruded from the syringe, the hydrogel is subjected to a strong shearing force, and at this time, the crosslinked network of the hydrogel is broken, the hydrogel is converted to a fluid state, and thus the hydrogel can be extruded from the syringe; the extruded hydrogel is quickly connected together by the shearing force, and the original connection state is restored, so that the hydrogel is restored to the gel state again. The conclusion proves that the PFC & CMC hydrogel has good injectability, and the good injectability enables the PFC & CMC hydrogel to be applied to various places, thereby greatly expanding the application range of the PFC & CMC hydrogel

Example 12:

the self-healing properties of PFC & CMC were studied by rheometry. Samples were prepared as in the previous procedure, and the change in storage modulus G' and loss modulus G "of the hydrogel over time was studied using a time sweep mode, with strain set at 0.1% -1500% and frequency set at 1Hz. The self-healing properties of the hydrogels were then studied using an alternating scan pattern, with an angular frequency set at 6rad/s and strain set at 0.1% (60 s) and 300% (60 s) switching alternately three times, and the changes in storage modulus G' and loss modulus G "were recorded.

As shown in fig. 12, at a small strain of 0.1%, the values and magnitude of the storage modulus G ' and the loss modulus G "of PFC & CMC2.0, PFC & CMC2.5 and PFC & CMC3.0 can be kept unchanged (G ' is greater than G"), and it is noted that the storage modulus G ' of PFC & CMC2.0, PFC & CMC2.5 and PFC & CMC3.0 are 150, 4000 and 5300kPa, respectively, which indicates that PFC & CMC3.0 has better mechanical strength due to the increase of CMC concentration, so that PFC & CMC3.0 has a greater crosslink density. However, as the strain increases, the G 'and G "also change, and eventually an intersection is created, and the magnitude relationship of G' and G" after this intersection is reversed, which change in magnitude relationship indicates that the hydrogel infrastructure is destroyed, while in the flow regime. Notably, the strain value corresponding to the PFC & CMC3.0 (270.23)% intersection point is much greater than the strain value corresponding to the PFC & CMC2.0 (68.2%) and (160.2%) PFC & CMC2.5 intersection point. This is because the hydrogel structure becomes stronger due to the increase in the crosslink density, and thus has more excellent strain resistance.

Example 12:

after determining the strains corresponding to critical damage of the three hydrogels by the above examples, the cyclic process of damage-repair of the hydrogels was studied by the time cycle scan mode of the rheometer, as shown in fig. 13, taking PFC & CMC3.0 hydrogels as an example, G' and G "of the hydrogels were 5300Pa and 2000Pa respectively under the strain of 0.1%, and such strain sizes could be maintained unchanged at 60 s. In contrast, G "rapidly exceeded G' when strain reached 300% (> 270.23%) and this size relationship was able to inhibit holding for 60s without significant change, indicating that the stable structure of PFC & CMC3.0 was destroyed. However, when the strain was recovered to 0.1%, G' and G "were able to quickly recover to the original levels, and the values were almost identical to the previous values, indicating that the PFC & CMC3.0 hydrogel was restored to the original stable structure. Such results are reproducible in two cycles, and such conclusion indicates that hydrogels have good self-healing properties.

Two hydrogels of different colors are cut into semicircle and placed together, and the two hydrogels can be well connected together, as can be seen from fig. 14, the connection part turns black due to the successful connection of the two hydrogels, indicating the successful connection of the hydrogels. Notably, the connected hydrogels were able to withstand some stretching without breaking, indicating that the hydrogels had been well repaired together. Therefore, the results clearly show that the PFC & CMC3.0 hydrogel has good self-repairing performance, and the self-repairing can ensure that the hydrogel can be quickly repaired even if damaged in the application process, so that the hydrogel has more excellent dressing performance.

Example 13:

to evaluate PFC&CMC in vitro degradation behavior, firstly accurately weighing PFC after freeze drying&CMC hydrogel, mass recorded as W1, was first brought to swelling equilibrium. The hydrogel was then soaked in PBS under different conditions: 1) ph=7.4; 2) ph=7.4, 100 μ M H2O2; 3) ph=6.5; 4) ph=6.5, 100 μ M H2O2. The hydrogel samples were taken every other day, freed of excess salt and dried, and the mass recorded as W2. The degradation behavior of the hydrogel was evaluated by calculating the remaining mass ratio of the hydrogel by the formula (3-2):

example 14:

the in vitro degradation behavior of the hydrogel PFC & CMC3.0 in the microenvironment of the diabetic wound is simulated through different conditions, as shown in figure 15, after 6 days, the residual mass of the PFC & CMC3.0 hydrogel is respectively reduced to 37% (pH=7.4), 23% (pH=7.4+100 mu M H2O 2), 20% (pH=6.5) and 10% (pH=6.5+100 mu M H O2), which shows that the PFC & CMC3.0 hydrogel realizes the quick response release of the drug in the diabetic wound in the low pH and high oxidation state, thereby well avoiding secondary injury possibly caused by frequent dressing replacement of the traditional dressing and seriously inhibiting the healing of the wound.

Example 15:

response of DATS to GSH by assessing H release by varying concentrations of GSH ₂ S behavior, as shown in FIG. 16, DATS@PFC treated in PBS&CMC-1 hydraulic settingThe glue did not detect any H within 80min ₂ S release, H with increasing GSH concentration ₂ The release rate of S gradually increases and the release amount increases. For example, under ph=7.4, dats@pfc&CMC-1 hydrogel at GSH concentration of 2.0mM, H ₂ The S release amount can reach 1.1. Mu. Mol, which is far higher than the same pH condition but the GSH concentration is 1.0Mm (0.5. Mu. Mol) and 1.5mM (0.6. Mu. Mol). Although acidic conditions accelerate the degradation of hydrogels, the rate of DATS release is instead decreased because GSH' S reducibility is mainly derived from thiol groups (-SH), whereas it is more difficult to deprotonate under acidic conditions to form reactive thiolate anions (-S-1). It is noted, however, that DATS@PFC&The CMC-1 hydrogel still can reach 0.9 mu mol in 80min, which shows that the influence of the slightly acid environment on the release rate and the release amount of the DATS is not great. Thus, DATS@FC&CMC-1 hydrogels are capable of releasing sufficient amounts of H in the presence of GSH ₂ S, thereby giving play to H ₂ Therapeutic action of S, which is beneficial to long-acting slow release of H ₂ S has chronic wound recovery effect on diabetic wounds.

To sum up: 1) First, a blank hydrogel PFC is prepared&CMC2.0，PFC&CMC2.5 and PFC&CMC3.0 and hydrogel carrier DATS@PFC&CMC-1，DATS@PFC&CMC-2 and DATS@PFC&CMC-3, and researches the gel forming time, swelling, adhesion, injectability, self-repairing and other relevant physical and chemical properties of the blank hydrogel, and discovers that the hydrogel has excellent physical and chemical properties; 2) Blank hydrogel PFC&CMC3.0 has good water absorption performance although the swelling rate is low, and besides, PFC&CMC3.0 has better adhesion, mechanical properties and self-repairing properties; 3) The hydrogel can be rapidly degraded in simulated diabetes mellitus microenvironment, release drug-loaded micelles, and rapidly release H in the presence of GSH ₂ S, S. In summary, in view of PFC&CMC3.0 has the most excellent physical and chemical properties, so the subsequent experiments were carried out by using the blank hydrogel, and the name is PFC&CMC。

Example 16:

human Umbilical Vein Endothelial Cells (HUVECs) and macrophage RAW264.7 were selected for cell experiments. HUVECs were prepared using DMEM as medium and 10% fetal bovine serum and 1% penicillin/streptomycin were added to complete medium. RAW264.7 was cultured using macrophage specific medium. Cells were cultured in a cell incubator at 37℃with 5% CO 2. Experiments the macrophages without any treatment were called normal macrophages, whereas the macrophages after 24h incubation with lipopolysaccharide (LPS, 5. Mu.g/mL) medium became activated macrophages.

HUVECs were cultured with the complete medium in the above procedure and cells were cultured in a cell incubator at 37℃with 5% CO 2. The experimental procedure was as follows: first, 24. Mu.L of the sterilized FC & CMC, PFC & CMC, DATS@PFC & CMC-1, DATS@PFC & CMC-2 and DATS@PFC & CMC-3 hydrogel liquids were added to a 96-well plate and left in an oven for 10min to gel. Then 1X 104 HUVECs cells per well were added and incubated at 37℃for 48h. After incubation, cells were washed with PBS, CCK-8 was added to the well plate and incubated in the dark for 30min. The absorbance of the cells at 450nm was detected by a multifunctional microplate reader detector. Furthermore, after HUNVECs were treated according to the above procedure, cells were stained with AM/PI for 30min, and the cells were observed for fluorescence with an inverted fluorescence microscope and photographed.

Excess H ₂ S causes relatively large cytotoxicity, and thus requires screening for safe drug loading, as shown in FIG. 17, FC&CMC，PFC&CMC and DATS@PFC&The survival rate of HUVECs after CMC-1 treatment is over 90%, so that the HUVECs have good biological safety. In contrast, DATS@PFC&CMC-2 and DATS@PFC&The viability of HUVECs after CMC-3 treatment was below 70%, giving rise to a large amount of cytotoxicity. The live dead staining experiments of the cells also confirmed this conclusion. As shown in fig. 17, FC &CMC，PFC&CMC and DATS@PFC&CMC-1 treated cells all had a large amount of green fluorescence (calcein AM-labeled viable cells), in contrast to DATS@PFC&CMC-2 and DATS@PFC&CMC-3 treated HUVECs exhibited a large amount of red fluorescence (PI-labeled dead cells), indicating the appearance of a large number of dead cells. This toxicity is mainly due to the excess H released by PHMG and DATS ₂ S, and S. In conclusion, the method comprises the steps of,PFC&CMC and DATS@PFC&CMC-1 hydrogels all have good biosafety. In view of the above, a DATS@PFC is selected&CMC-1 was subjected to subsequent experiments and was named DATS@PFC&CMC。

Example 17:

two bacteria, namely escherichia coli (E.coli) and methicillin-resistant staphylococcus aureus (MRSA), were selected to evaluate the in vitro antibacterial effect of the hydrogel by a colony counting method, and as shown in FIG. 18, DATS@PFC & CMC shows better antibacterial capacity than PFC & CMC, and the survival number of the bacteria after 6h treatment is reduced to 2.2 and 1.6log10 CFU/mL, because of the antibacterial capacity of DATS itself, good synergistic antibacterial effect with PHMG is generated, so that more excellent antibacterial performance is shown. E.coli and MRSA after PFC & CMC and DATS@PFC & CMC treatment showed a lot of red fluorescence, but almost no green fluorescence, indicating that PFC & CMC and DATS@PFC & CMC killed almost all bacteria.

Example 18:

to study the antibacterial mechanism of dats@pfc & CMC, after treatment of e.coli and MRSA, hydrogel-treated bacteria were collected by centrifugation at 4 ℃ and protein concentration was measured with the enhanced BCA protein assay kit and the change in ATP levels of the treated bacteria was measured with the enhanced ATP assay kit; as shown in fig. 20, PBS and FC & CMC treated e.coli and MRSA were still able to maintain intact bacterial structures. And E.coll after PFC & CMC and DATS@PFC & CMC are treated presents a large number of holes, MRSA is contracted and collapsed, and a large number of substances leak, which indicates that PFC & CMC and DATS@PFC & CMC can kill bacteria efficiently by destroying the complete structure of the cell membrane of the bacteria.

Example 19:

protein concentration and ATP levels of e.coli and MRSA after PFC & CMC and dats@pfc & CMC treatment were studied by BCA and ATP kit. As can be clearly shown in fig. 21, PFC & CMC and dats@pfc & CMC can achieve the effect of efficiently destroying bacteria by destroying the cell membrane of bacteria, causing protein leakage and inhibiting ATP levels.

Example 20:

after confirming that the DATS@PFC & CMC can release H2S in vitro in the presence of GSH, we further explored whether the DATS@PFC & CMC can generate H2S in cells; as shown in fig. 22, the green fluorescence intensity of the dats@pfc & CMC treated activated macrophages was much higher than that of the remaining groups, approximately three times the DATS group fluorescence intensity, which can be confirmed by dats@pfc & CMC to be able to produce a large amount of H2S in the cells, and to lay down the H2S therapeutic effect later.

Example 21:

the influence condition of DATS@PFC & CMC on inflammatory factors is studied by an Elisa kit;

as shown in FIGS. 23 and 24, the levels of TNF- α, IL-1β and IL-6 in the activated macrophages after DATS@PFC & CMC treatment were 1.2,0.4 and 2.1ng/mL, respectively, which were far lower than those in the LPS-induced macrophages, and the levels of IL-10 and IL-4 expression in the activated macrophages after DATS@PFC & CMC treatment were 79 and 68pg/mL, respectively, with sufficient amounts of H2S, which greatly promoted the expression of the anti-inflammatory factors IL-10 and IL-4, thereby exerting an anti-inflammatory effect.

Example 22:

mRNA expression levels of macrophages HO-1, CD206 and iNOS were detected by W RT-qPCR;

protein expression levels of HO-1, ERK, p-ERK, STAT3, p-STAT3, CD163 and iNOS were detected by Westernblot;

to observe the changes in the morphology of macrophages, the macrophages were treated as described above, and then the cell morphology was observed under an inverted fluorescence microscope and photographed. In addition, the anti-inflammatory mechanism of DATS@PFC & CMC is also studied through immunofluorescence experiments; as shown in fig. 25, dsta@pfc & CMC is capable of exerting an anti-inflammatory effect by inhibiting the phosphorylation levels of STAT3 and ERK; as shown in FIG. 26, DSTA@PFC & CMC treatment further specifically activates expression of heme oxygenase, indicating that DSTA@PFC & CMC also has excellent antioxidation.

Example 23:

the effect of DATS@PFC & CMC in promoting HUVECs cell proliferation and migration was evaluated by experiments on HUVECs proliferation; as shown in fig. 27, the result shows that dats@pfc & CMC can indeed promote rapid proliferation and migration of endothelial cells, and finally promote healing of scratches.

Example 24:

angiogenesis was simulated by HUVECs. We next studied that treatment with dats@pfc & CMC stimulated HUVECs to form a tubular network on matrigel; the expression levels of p-p38 and p-ERK1/2 at different time points were determined using ELISA kits; as shown in fig. 28, dats@pfc & CMC is shown to promote the formation of longer and denser blood vessels by HUVECs, which facilitates the transport of nutrients, thereby promoting wound healing; DATS@PFC & CMC can promote proliferation, migration and vascularization of HUVECs by maintaining continuous high expression of p-p38 and p-ERK1/2, and can finally accelerate wound healing.

Example 25:

animal experiment: male ICR mice (25-30 g,6-8 weeks) were acclimatized for three days, and STZ sodium citrate solution (80 mg/kg) was injected four consecutive days and blood glucose of the mice was monitored with a blood glucose meter, and when the blood glucose level was higher than 16.8mM, it was indicated that the mice were in a diabetic state at this time. After the mice were anesthetized with isoflurane, the mice were subjected to dehairing treatment, and the dehairing sites of the mice were perforated with a 1cm diameter punch, and then 10 μl of PBS solution (108 CFU/mL) of MRSA was dropped into the sites, to establish a diabetic wound model. Mice were randomized into 4 groups and wounds were treated with different materials: 1) PBS (50. Mu.L); 2) Dats@fc & CMC (50 μl); 3) PFC & CMC (50. Mu.L); 4) DATS@PFC & CMC (50. Mu.L). The wound area of the mice was measured with a ruler at fixed time and photographed. The closure rate of the wound was calculated using formula (5-1):

Wherein A0 is the area of the wound on day 0, and At is the area of the wound At the specified time point.

Collecting wound tissue of treated mice for 5 days, putting the wound tissue into PBS, carrying out ultrasonic treatment for 0.5h, carrying out gradient dilution on the obtained liquid by a factor of 10, then taking 100 mu L of liquid with different concentrations, uniformly coating the liquid on a solid LB plate, culturing the liquid for 20h at 37 ℃, observing the growth condition of bacteria, and photographing. Meanwhile, the above-mentioned gradient diluted liquid was inoculated onto a solid LB plate, cultured at 37℃for 20 hours, and the colony count of the bacteria was observed.

Blood from mice after 15 days of treatment was collected, centrifuged at 2000rpm at 4℃and the supernatant was collected and assayed for the expression levels of TNF- α, IL-6 and IL-1β using the Elisa kit.

Wound tissue after 15 days of treatment was collected, subjected to fixation, dehydration, wax impregnation, embedding, slicing, and the like, and then immunolabeled with HO-1, iNOS and CD163 antibodies, observed with an inverted fluorescence microscope, and photographed. The resulting sections were stained with H & E and Masson to observe wound healing.

Mouse venous blood after 15 days was collected and the safety of the treatment process was assessed by the wuhansai wiener biotechnology company by detecting the number of Red Blood Cells (RBC), white Blood Cells (WBC) and Hemoglobin (HGB) in the mouse blood.

Heart, liver, spleen, lung and kidney were H & E stained after 15 days of collection treatment to assess the biosafety of the hydrogels.

FIG. 29 shows the establishment and treatment of a wound model of MRSA-infected diabetic mice

As shown in fig. 30, the wound closure rates for PBS groups at days 5, 10 and 15 were 28.8%, 51.4% and 68.4%, respectively, significantly slower than for PFC & CMC groups and dats@fc & CMC groups, as the wound closure rates for dats@fc & CMC groups and PFC & CMC groups were as high as 79.0% and 80.4%, respectively. The wound healing rates of the DATS@FC & CMC group and the PFC & CMC group are obviously accelerated, and the wound healing rates can be respectively attributed to the antibacterial or anti-inflammatory effects of the PHMG and the DATS, so that bacteria of the wound are effectively killed or inflammation of the wound is inhibited, the microenvironment of the wound is partially improved, and the healing of the wound is promoted. However, the inability of a single antibacterial or anti-inflammatory effect to cope with complex diabetic microenvironments, often is not enough, and wound healing remains unsatisfactory. Fortunately, due to the integration of the high-efficiency sterilization, anti-inflammation, antioxidation and tissue regeneration promotion of the DATS@PFC & CMC, the microenvironment of the diabetic wound can be improved efficiently, the wound healing is accelerated obviously finally, the wound closure rate reaches 85.8% on the 10 th day, the wound closure rate reaches 97.5% on the 15 th day, and the wound is almost closed.

H & E and Masson staining also confirmed this conclusion, as shown in fig. 31, 1) the number of newly formed granulation tissue and skin attachments (glands and hair follicles) and collagen deposition were significantly greater in the wound sites of the dats@pfc & CMC group than in the other groups, with more mature fibrous tissue; 2) The immature tissue width and the minimum epithelial keratinized layer thickness in the DATS@PFC & CMC group show that the DATS@PFC & CMC can accelerate tissue healing and realize faster maturation reconstruction.

The colony status of wound site tissue after 5 days is shown in fig. 32. Almost no colony formation in wound tissue after 5 days of dats@pfc & CMC treatment, indicating that dats@pfc & CMC is able to kill bacteria at the wound site with high efficiency. PFC & CMC also has little colony count of bacteria due to PHMG, however the remaining groups still have a large colony count, indicating that bacterial infection is still present. Colony counts also validated this conclusion. The tissue after DATS@PFC & CMC treatment had only 101.6log10 CFU/mL, which was higher than 103.5log10 CFU/mL of PFC & CMC. This is due to the synergistic bactericidal effect of DATS and PHMG, which is highly effective in destroying bacteria from wounds.

The levels of inflammatory factors in blood after 15 days of treatment of mice were examined using the Elisa kit, as shown in FIG. 33, the wound sites of PBS-treated diabetic mice still had severe inflammatory responses, because the expression levels of IL-1β, IL-6 and TNF- α were still as high as 310.9, 287.2 and 413.3pg/mL, respectively. However, the expression levels of IL-1β, IL-6 and TNF- α were reduced to some extent after the treatment of DATS@FC & CMC, PFC & CMC, mainly due to the anti-inflammatory effect of DATS or the antibacterial effect of PHMG, partially alleviating the inflammatory response of diabetic wounds. Notably, optimal anti-inflammatory effects were also achieved using DATS@PFC & CMC, with IL-1β, IL-6 and TNF- α expression levels significantly reduced to 139.5, 91.9 and 222.3pg/mL, respectively, far below the inflammatory level of diabetic mice, almost the same as secreted by normal healthy mice. These results demonstrate that the excellent antibacterial effect of PHMG and the excellent anti-inflammatory effect of DATS together are highly effective in alleviating the inflammatory response at the wound site.

Furthermore, the immunofluorescent staining results of the wound tissue with excellent anti-inflammatory effect of dats@pfc & CMC further demonstrate. As shown in fig. 34, only intense red fluorescence (iNOS labeled M1-type macrophages) was observed in PBS-treated wound tissue, indicating that wound tissue of diabetic mice still had severe inflammation, while a large amount of green fluorescence (CD 163 labeled M2 macrophages) was present in dats@pfc & CMC-treated wound tissue. Quantitative data for fluorescence intensity also led to the conclusion that this was true. The above conclusion shows that dats@pfc & CMC can exert anti-inflammatory effects by modulating macrophage phenotype.

Furthermore, as shown in fig. 35, dats@pfc & CMC treatment further significantly activated HO-1 expression in wound tissue compared to the remaining groups. The amount of green fluorescence was much higher than for the remaining groups, consistent with the quantitative fluorescence results. The DATS@PFC & CMC is shown to promote the expression of HO-1 so as to exert excellent antioxidation.

Notably, immunohistochemical staining results of wound tissue further demonstrated that dats@pfc & CMC treatment significantly promoted vascularization of wound tissue, as shown in fig. 36, the number of CD31 positive microvasculature in dats@pfc & CMC treated wound tissue was 5.3, 1.7 and 4 times higher than in PBS, dats@fc & CMC and PFC & CMC groups.

Taken together, these results clearly demonstrate that dats@pfc & CMC can significantly promote rapid healing of diabetic wounds through efficient antibacterial, anti-inflammatory and pro-angiogenic effects.

The above results indicate that dats@pfc & CMC has important potential in the effective management of diabetic wounds, but its successful application in clinical practice depends on its good biocompatibility. Thus, changes in body weight were measured periodically throughout the treatment period. In addition, blood and vital organs were collected at the end of treatment for routine blood analysis and H & E staining. Except that the diabetic mice treated with PBS experienced significant weight loss, the weight of all diabetic mice wound treated with three hydrogels did not significantly change (fig. 37), indicating that the prepared hydrogels had good biocompatibility. The results of blood routine analysis and H & E staining of heart, liver, spleen, lung and kidney of mice further confirm that DATS@PFC & CMC has good biocompatibility and is expressed as follows: 1) The values of the three representative hematological markers (red blood cell count (RBC), white blood cell count (WBC), hemoglobin (HGB)) were all within the normal range (fig. 37); 2) Dats@pfc & CMC treated mice compared to normal healthy mice, no significant tissue damage was observed in the H & E staining results of the heart, liver, spleen, lung, kidney (fig. 38). In summary, all the above results clearly demonstrate that dats@pfc & CMC has good biosafety.

In summary, we treated MRSA-infected diabetic mice wounds with different materials, leading to the following conclusions: 1) The DATS@PFC & CMC can promote the generation of micro blood vessels to accelerate the healing of wounds of diabetic mice, and the wound closure rate is 97.5% after 15 days; 2) DATS@PFC & CMC can effectively kill bacteria of wounds, promote macrophages to convert into M2 type to play an anti-inflammatory role, and up-regulate the expression of HO-1 to play an anti-oxidation role; 3) The DATS@PFC & CMC has good biological safety while having good therapeutic effect.

The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.

Claims

1. The multifunctional hydrogel dressing based on hydrogen sulfide gas therapy is characterized in that the multifunctional hydrogel dressing is prepared based on a dynamic Schiff base reaction between PHMG modified aldehyde F108 (PFC) and carboxymethyl chitosan CMC.

2. The hydrogen sulfide gas therapy-based multifunctional hydrogel dressing of claim 1, wherein: the grafting ratio of PFC was 1.4%.

3. The hydrogen sulfide gas therapy-based multifunctional hydrogel dressing of claim 1, wherein: the concentration of PFC was 100mg/mL.

4. The method for preparing the multifunctional hydrogel dressing based on hydrogen sulfide gas therapy according to claim 1, comprising the following steps:

and S4, filtering the obtained mixed solution, and collecting filter residues. The resulting residue was washed 3 times with 90% (v/v) methanol. After filtration, the residue was dried in vacuo in a vacuum oven at 50℃for 1 day. The collected solid product carboxymethyl chitosan CMC.

5. The method for preparing a multifunctional hydrogel dressing based on hydrogen sulfide gas therapy according to claim 1, further comprising the steps of:

6. The method for preparing a multifunctional hydrogel dressing based on hydrogen sulfide gas therapy according to claim 5, further comprising the steps of:

g. 200mg of F108-CHO and 20mg of DATS were dissolved in 2mL of DCM and stirred well for 1

h, then removing all DCM by using a rotary evaporator to form a uniform yellow film;

h. adding pure water to hydrate the film for 8 hours;

7. The method for preparing a multifunctional hydrogel dressing based on hydrogen sulfide gas therapy according to claim 6, further comprising the steps of: PFC and CMC (2.0% w/v,2.5% w/v and 3.0% w/v) with three different concentrations are mixed according to the volume ratio of 1:1, after the mixing is uniform, the mixed solution is placed in a baking oven at 37 ℃ to prepare hydrogel PFC & CMC2.0, PFC & CMC2.5 and PFC & CMC3.0 respectively, and then the PFC micelle solution is replaced by DATS@PFC-1, DATS@PFC-2 and DATS@PFC-3 micelle solution with the same concentration and the carrier liquid gels DATS@PFC & CMC-1, DATS@PFC & CMC-2 and DATS@PFC-3 are prepared.