Composite hydrogel dressing with keratin loaded with silver ion rhein and application of composite hydrogel dressing

Abstract

The invention discloses a composite hydrogel dressing with keratin loaded with silver ion rhein. The composite hydrogel dressing is prepared from keratin, silver nitrate and rhein. According to the composite hydrogel dressing with the keratin loaded with the silver ions and the rheinic acid, the Ker is loaded with the silver ions and the rheinic acid at the same time, and as a novel antibacterial drug composition, compared with previously reported hydrogel, the composite hydrogel dressing has a strong inhibition effect on drug-resistant bacteria # imgabs0 # / # imgabs1 # floating bacteria and biofilm bacteria and can be applied as a wound dressing for diabetes mellitus.

Description

Technical Field

[0001] The present invention belongs to the technical field of dressing preparation, and specifically relates to a composite hydrogel dressing loaded with keratin and silver ion rhein and its application. Background Technique

[0002] In healthy hosts, acute trauma triggers the wound healing response and activates the innate antimicrobial system. However, these processes are impaired in wounds of diabetic foot ulcers (DFUs) [A.S. MacLeod, Bad “Staph” in the Wound Environment of Diabetic Foot Ulcers, Cell Host&Microbe 25(5) (2019) 638-640.]. Compared with diabetic patients without foot infections, the risk of hospitalization in patients with diabetic foot infection (DFI) is 50 times higher, and the risk of lower limb amputation is 150 times higher [L.A. Lavery, D.G. Armstrong, R.P. Wunderlich, M.J. Mohler, C.S. Wendel, B.A. Lipsky, Risk factors for foot infections in individuals with diabetes, Diabetes Care 29(6) (2006) 1288-93..]. Antimicrobial Resistance (AMR) is one of the most urgent threats to health in the 21st century, and The Lancet predicts that the number of AMR deaths will increase in every region of the world from 2022 to 2050 [Global burden of bacterial antimicrobial resistance 1990–2021: a systematic analysis with forecasts to 2050, The Lancet 404(10459)(2024) 1199-1226.]. According to the surveillance data of CHINET, the detection rates of Methicillin-resistant Staphylococcus aureus (MRSA) and carbapenem-resistant Gram-negative bacilli in China remain high [Guo Yan et al., CHINET China Bacterial Resistance Surveillance in 2023, Chinese Journal of Infection and Chemotherapy 24(06) (2024) 627-637..].The infection rate of multidrug-resistant bacteria in DFI wounds is over 80% [R. Gadepalli, B. Dhawan, V. Sreenivas, A. Kapil, A.C. Ammini, R. Chaudhry, A clinico-microbiological study of diabetic foot ulcers in an Indian tertiary care hospital, Diabetes Care 29(8) (2006) 1727-32.], and methicillin-resistant Staphylococcus aureus (MRSA) [N.A. Turner, Methicillin-resistant Staphylococcus aureus: an overview of basic and clinical research, Nat Rev Microbiol 17(4) (2019) 203-218.] and carbapenem-resistant Escherichia coli (CRE) [S.S. Jean, Hsueh, Off-label use versus formal recommendations of conventional and novel antibiotics for the treatment of infections caused by multidrug-resistant bacteria, Int J Antimicrob Agents 61(5) (2023) 106763.] are the main drug-resistant bacteria in chronic non-healing wounds.

[0003] A biofilm is a microbial community residing in extracellular polymeric substances (EPS) (polysaccharides, nucleic acids, proteins, and microbial metabolites) [Extracellular Polymeric Substance Production and Aggregated Bacteria Colonization Influence the Competition of Microbes in Biofilms, Front Microbiol 8 (2017) 1865..]. Bacterial drug resistance is significantly associated with biofilm formation [Association between biofilm and multi / extensive drug resistance in diabetic foot infection, Int J Clin Pract 72(3) (2018) e13060. ], and is a common cause leading to the intractability of DFI [Biofilms in Diabetic Foot Ulcers: Impact, Risk Factors and Control Strategies, Int J Mol Sci 22(15) (2021). Redefining the Chronic-Wound Microbiome: Fungal Communities Are Prevalent, Dynamic, and Associated with Delayed Healing, Mbio 7(5) (2016). Chronic Pseudomonas aeruginosa biofilm infection impairs murine S100A8 / A9 and neutrophil effector cytokines-implications for delayed wound closure? (vol 75, ftx068, 2017), Pathogens and disease[electronic] (8) (2017) 75.].Compared with planktonic bacteria, the antibiotic resistance of biofilm bacteria is 100 to 1000 times higher [Biofilm-specific antibiotic tolerance and resistance, Eur J Clin Microbiol Infect Dis 34(5) (2015) 877-86..]. This is because the wound surface infected with biofilm usually has a shiny, translucent mucus layer, which can prevent antibacterial agents from penetrating into the interior of the colony.On the other hand, biofilms can also prevent nutrients and oxygen from entering [Molecular mechanisms of antibiotic resistance, Nat Rev Microbiol 13(1) (2015) 42-51..]. Due to the lack of nutrients and oxygen, the metabolic activity of the internal bacteria decreases, and their sensitivity to most antibiotics targeting bacterial proliferation declines [New Perspectives on Old and New Therapies of Staphylococcal Skin Infections: The Role of Biofilm Targeting in Wound Healing, Antibiotics (Basel) 10(11) (2021). Biofilm is a Major Virulence Determinant in Bacterial Colonization of Chronic Skin Ulcers Independently from the Multidrug Resistant Phenotype, Int J Mol Sci 18(5) (2017). Antimicrobial Resistance and Prevalence of MDR / XDR Organisms in Patients With Diabetic Foot Infection in an Indian Tertiary Care Hospital, Int J Low Extrem Wounds (2021) 15347346211038090.]. Although the biofilm structure has species diversity, regardless of species and conditions, the formation and development of biofilms conform to general characteristics: following the cycle of "planktonic bacteria adhesion, microcolony formation, biofilm maturation, and biofilm-dispersed planktonic bacteria" [Biofilm dispersion, Nature Reviews Microbiology 18(10) (2020) 571-586.].In this cycle, quorum sensing (QS) helps microorganisms communicate with each other, exhibit collective behavior, and regulate biofilm formation and virulence traits [The biofilm life cycle: expanding the conceptual model of biofilm formation, Nat Rev Microbiol 20(10) (2022) 608-620.].

[0004] The disposal methods recommended in the guidelines for biofilms are thorough debridement and covering with functional dressings after debridement [ESCMID guideline for the diagnosis and treatment of biofilm infections 2014, ClinMicrobiol Infect 21 Suppl 1 (2015) S1-25. Consensus guidelines for the identification and treatment of biofilms in chronic nonhealing wounds, Wound Repair Regen 25(5) (2017) 744-757.]. However, biofilms will reform and mature within 2 to 3 days after debridement [Biofilm maturity studies indicate sharp debridement opens a time-dependent therapeutic window, J Wound Care 19(8) (2010) 320-8.]. Clinicians cannot accurately identify biofilm aggregates visually, and it is difficult to remove all biofilms. There is no strongly recommended antimicrobial dressing in the current guidelines. In current new drug research and development, the combination strategy of bioactive components in natural products is determined through database mining [A Data-Driven Approach for Identifying Medicinal Combinations of Natural Products, IEEE Access 6 (2018) 58106-58118.] and repeated phenotypic screening in the laboratory [Phenotypic screens as a renewed approach for drug discovery, Drug Discovery Today 18(21) (2013) 1067-1073.], which may be inefficient. Researchers believe [Why is cancer drug discovery so difficult?, Nature Reviews Drug Discovery 6(2) (2007) 115-120.] that a wide range of combinations can be established from 200 anti-cancer drugs approved for clinical use, and there are great opportunities for new drug discovery with simple experiments. This simple, convenient, and efficient new drug discovery strategy is also applicable in the field of wound repair.To prevent bacterial resistance and reduce the dosage of individual drugs to avoid potential toxicity, traditional strategies recommend the combination of antimicrobials with other therapies that disrupt biofilms to release planktonic bacteria [Biofilm Management in Wound Care, Plast Reconstr Surg 148(2) (2021) 275e-288e.], such as the use of the cocktail method (using more than one antibiotic) for antibiotic resistance [Free radical-releasing systems for targeting biofilms, J Control Release 322 (2020) 248-273.]. Silver ions (Ag+) are the most commonly used antibacterial agents for infected wounds but are ineffective against biofilms [Prospective randomised placebo-controlled trial assessing the efficacy of silver dressings to enhance healing of acute diabetes-related foot ulcers, Diabetologia 66(4) (2023) 768-776.]. Developing antimicrobial combinations in combination with Ag+ to inhibit biofilm formation and disperse biofilms holds promise for achieving an anti-biofilm-anti-planktonic bacteria cascade function.

[0005] "Shennong Ben Cao Jing" records that Chinese herbal medicine rhubarb has the effects of clearing heat and purging fire, cooling blood and detoxifying, removing stasis and dredging meridians, and can treat heat-toxic sores and burns. Rhein (Rh) is one of the main components of rhubarb. A number of studies have shown that rhein is an anthraquinone compound with pharmacological effects such as inhibiting Staphylococcus aureus [Aramid Nanofibers-Reinforced Rhein Fibrous Hydrogelsas Antibacterial and Anti-Inflammatory Burn Wound Dressings, ACS Appl MaterInterfaces 14(40) (2022) 45167-45177. Rhein incorporated silk fibroinhydrogels with antibacterial and anti-inflammatory efficacy to promotehealing of bacteria-infected burn wounds, Int J Biol Macromol 201 (2022) 14-19.], anti-inflammatory [ Rhein attenuates inflammation through inhibition of NF-κB andNALP3 inflammasome in vivo and in vitro, Drug Des Devel Ther 11 (2017) 1663-1671.], and antioxidant [Rhein protects the myocardiac cells against hypoxia / reoxygention-induced injury by suppressing GSK3β activity, Phytomedicine 51(2018) 1-6.]. However, the solubility of rhein is very low. In order to improve its bioavailability, some researchers have prepared nanoparticles containing rhein. However, the premature and massive release of the drug will cause cytotoxicity [Building Nanostructures with Drugs, Nano Today 11(1) (2016) 13-30.].Furthermore, a study [Directed self-assembly of herbal small molecules into sustainedrelease hydrogels for treating neural inflammation, Nat Commun 10(1) (2019)1604.] prepared rhein self-assembled hydrogels, which can reduce neural inflammation compared with free drugs equivalent in vitro, stably release drugs without cytotoxicity. However, the pH (8.0 - 9.4) of this hydrogel is not suitable for chronic wounds.

[0006] Gel-based wound dressings can provide a moist healing environment, increase cell-tissue contact, and help autolytic enzymes play a role in autolytic debridement and softening of eschar. A systematic review and Meta-analysis showed that functional hydrogels accelerated the healing process of DFU and alleviated the symptoms of patients [The Promising Hydrogel Candidates for Preclinically Treating Diabetic Foot Ulcer: A Systematic Review and Meta-Analysis, Adv Wound Care (New Rochelle) (2022)]. Keratin (Ker) is an intermediate filament-forming protein expressed in epithelial cells, responsible for keratinocyte proliferation and maintaining its integrity in the epithelium [Convergent Evolution of Cysteine-Rich Keratins in Hard Skin Appendages of Terrestrial Vertebrates, Mol Biol Evol 37(4) (2020) 982-993.], and is involved in various signal transduction processes such as cell movement, apoptosis, and proliferation [The expanding significance of keratin intermediate filaments in normal and diseased epithelia, Curr Opin Cell Biol 25(1) (2013) 47-56.]. It has a content of over 90% in human hair, has good biocompatibility, and can meet various requirements in the biomedical field [Comparative study of keratin extraction from human hair, International Journal of Biological Macromolecules 133 (2019) 382-390.].Among them, the keratin-based hydrogel provides moderate mechanical properties and strong liquid absorption capacity [pH and Glucose Dual-Responsive Injectable Hydrogels with Insulin and Fibroblasts as Bioactive Dressings for Diabetic Wound Healing, ACS Appl Mater Interfaces 9(43) (2017) 37563-37574. Moldable and Removable Wound Dressing Based on Dynamic Covalent Cross-Linking of Thiol-Aldehyde Addition, ACS Biomater Sci Eng 5(8) (2019) 4048-4053.], becoming one of the most promising forms of wound dressings [An injectable self-healing hydrogel with adhesive and antibacterial properties effectively promotes wound healing, Carbohydr Polym 201 (2018) 522-531. Naturally derived dual dynamic crosslinked multifunctional hydrogel for diabetic wound healing, Composites Part B: Engineering 257 (2023) 110687.]. Summary of the Invention

[0007] The object of the present invention is to provide a composite hydrogel dressing loaded with silver ions and rhein by keratin.

[0008] Another object of the present invention is to provide a preparation method of the composite hydrogel dressing loaded with silver ions and rhein by keratin.

[0009] Still another object of the present invention is to provide an application of the composite hydrogel dressing loaded with silver ions and rhein by keratin in the preparation of a wound healing dressing.

[0010] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0011] In the first aspect of the present invention, a composite hydrogel dressing loaded with keratin, silver ions and rhein is provided, which is made of keratin, silver nitrate and rhein with a mass ratio of 800 - 1500:1:1 - 10 (preferably 1059:1:8.8, 1059:1:5.9, 1323:1:3.8).

[0012] In the second aspect of the present invention, a preparation method of the composite hydrogel dressing loaded with keratin, silver ions and rhein is provided, comprising the following steps:

[0013] Dissolve keratin powder (Ker) in PBS solution, add silver nitrate solution with a concentration of 1 - 10 mmol / L (preferably 10, 5, 2.5 mmol / L), vortex to dissolve, add dilute hydrochloric acid to adjust the pH to 7.0, add rhein solution with a concentration of 1 - 20 mg / mL (preferably 6 mg / mL). The mass ratio of keratin, silver nitrate and rhein is 800 - 1500:1:1 - 10 (preferably 1059:1:8.8, 1059:1:5.9, 1323:1:3.8). Let the solution stand at room temperature for 1 - 12 h (preferably 6, 8 h) to obtain a composite hydrogel loaded with keratin, silver ions and rhein (Ker / Ag + / Rh hydrogel).

[0014] The concentration of the PBS solution is 0.001 - 0.1 mol / L (preferably 0.01 mol / L), and the pH is 6 - 8 (preferably 7.4).

[0015] The preparation method of the rhein solution comprises the following steps: dissolve rhein in sodium bicarbonate solution, heat in a water bath at 70 - 90 °C (preferably 80 °C) for 5 - 30 min (preferably 15 min) to obtain a rhein solution with a concentration of 1 - 20 mg / mL (preferably 6 mg / mL).

[0016] The concentration of the sodium bicarbonate solution is 0.1 - 1 mol / L (preferably 0.2 mol / L), and the pH is 7 - 9 (preferably 8.3).

[0017] The preparation method of the keratin powder (Ker) comprises the following steps:

[0018] Dissolve a urea solution with a concentration of 6 - 10 mol / L (preferably 8 mol / L), a sodium dodecyl sulfate (SDS) solution with a concentration of 0.1 - 0.3 mol / L (preferably 0.2 mol / L), and a sodium metabisulfite solution with a concentration of 0.1 - 1 mol / L (preferably 0.5 mol / L) in deionized water. The mass ratio of urea, sodium dodecyl sulfate (SDS), and sodium metabisulfite is 1 - 20:1:1 - 5 (preferably 8.3:1:1.6). Heat and stir at 60 - 80 °C (preferably 70 °C) for 1 h, add feathers, and the mass ratio of feathers to sodium dodecyl sulfate is 1 - 5:1 (preferably 1.7:1). Process until there are no lumps of feathers, then perform suction filtration and dialysis (Mw = 8 - 14 kDa) for at least three days, concentrate, and freeze-dry step by step to obtain keratin powder.

[0019] The conditions for the step-by-step freeze-drying are: freeze at -20 °C for 3 - 4 h and freeze at -80 °C for more than 8 h.

[0020] The feathers are white raw duck feathers.

[0021] In the third aspect of the present invention, there is provided an application of the keratin-loaded silver ion rhein composite hydrogel dressing in the preparation of a wound healing dressing.

[0022] The wound healing refers to ulcer healing, especially diabetic foot ulcer infection (DFI) healing.

[0023] In the fourth aspect of the present invention, there is provided an application of the keratin-loaded silver ion rhein composite hydrogel dressing in the preparation of an antibacterial reagent.

[0024] The antibacterial reagent refers to anti-Methicillin-resistant Staphylococcus aureus (USA300), carbapenem-resistant Escherichia coli (RJ112).

[0025] Due to the adoption of the above technical solutions, the present invention has the following advantages and beneficial effects:

[0026] The Ker / Ag + / Rh hydrogel prepared by the present invention makes full use of the characteristic of high thiol group content in keratin, forms Ag-S dynamic cross-linking bonds between keratin and silver ions, uses Ker / Ag + as the main network, and rhein self-assembles into the secondary network, and adjusts the overall gel concentration with the concentration of keratin.

[0027] The composite hydrogel dressing loaded with silver ions and rhein by keratin provided by the present invention has simple and mild preparation conditions, without the need for chemical modification of the matrix material or addition of chemical cross-linking agents, ensuring good biocompatibility of the material.

[0028] The present invention utilizes the sulfhydryl groups in keratin to form Ag-S dynamic bonds with Ag + , endowing the hydrogel with various functions such as injectability and self-healing, and being able to meet the application requirements for filling irregular wounds.

[0029] The composite hydrogel dressing loaded with silver ions and rhein by keratin provided by the present invention, Ker simultaneously loads silver ions and rhein. As a new combination of antibacterial drugs, compared with the previously reported hydrogels, it has a strong inhibitory effect on / planktonic bacteria and biofilm bacteria, and can be used as a diabetic wound dressing. Brief Description of the Drawings

[0030] Figure 1 It is a physical characterization result diagram of the keratin-based injectable hydrogel.

[0031] Figure 2 It is a result diagram of in vitro drug release behavior, hemolysis test, and cell compatibility evaluation of the hydrogel.

[0032] Figure 3 It is a result diagram of the in vitro inhibitory effect of the composite hydrogel loaded with silver ions and rhein by keratin on biofilm formation and cascade antibacterial efficacy.

[0033] Figure 4 For Ker / Ag + / Rh hydrogel to promote / the ulcer healing of biofilm-infected T2DM mice.

[0034] Figure 5 For Ker / Ag + / Rh hydrogel in vivo promoting collagen deposition and angiogenesis result diagram.

[0035] Figure 6 For Ker / Ag + / Rh hydrogel in vivo inhibiting / biofilm formation antibacterial and anti-inflammatory experimental result diagram.

[0036] Figure 7 For the composite hydrogel dressing loaded with silver ions and rhein by keratin with different concentrations of rhein on and the treatment effect diagram of the ulcer of biofilm-infected T2DM mice. Detailed Implementation Modes

[0037] To more clearly illustrate the present invention, the present invention will be further described below in conjunction with preferred embodiments. Those skilled in the art should understand that the content specifically described below is illustrative rather than restrictive, and should not be used to limit the protection scope of the present invention.

[0038] Example 1

[0039] Extraction of keratin (Ker): Dissolve 48 g of urea solution with a concentration of 8 mol / L (the solvent is deionized water), 5.76 g of sodium dodecyl sulfate (SDS) solution with a concentration of 0.2 mol / L (the solvent is deionized water), and 9.5 g of sodium metabisulfite solution with a concentration of 0.5 mol / L (the solvent is deionized water) in 100 mL of deionized water. The mass ratio of urea, sodium dodecyl sulfate, and sodium metabisulfite is 8.3:1:1.6. Heat and stir for 1 h at a temperature of 70 °C, and add 10 g of duck feathers (white raw duck feathers: Hangzhou Slip Home Textiles Co., Ltd.). Filter the dissolved solution until there are no obvious impurities, dialyze (Mw = 8 - 14 kDa) for three days, rotary evaporate and concentrate, and freeze-dry step by step (-20 °C for 3 - 4 h, -80 °C for more than 8 h) to obtain 6.5 g of keratin powder with a yield of 65%, and store it at -20 °C.

[0040] Preparation of silver nitrate solution: Dissolve silver nitrate in deionized water to obtain a silver nitrate solution with a concentration of 0.1 mol / L, and sequentially dilute it into three gradient concentrations of silver nitrate solutions of 10, 5, and 2.5 mmol / L.

[0041] Preparation of keratin hydrogel (Ker hydrogel): Dissolve 0.09 g of keratin powder (Ker) in 500 μL of PBS (concentration is 0.01 mol / L, pH is 7.4) solution. The keratin concentration is 18% g / mL. Vortex to dissolve, add dilute hydrochloric acid with a concentration of 0.1 mol / L to adjust the pH to 7.0, and let it stand to obtain keratin hydrogel. Determine the gelling time of the keratin hydrogel by the inversion method. Observe the state of the sample at regular intervals. If the sample in the sample bottle does not flow for 30 s after inversion, it is determined that the sample has gelled, and record the gelling time of the keratin hydrogel. In this example, let it stand for 6 h to obtain the keratin hydrogel.

[0042] Preparation of keratin-based injectable hydrogel (Ker / Ag + hydrogel):

[0043] Dissolve 0.09 g of keratin powder (Ker) in 400 μL of PBS (concentration 0.01 mol / L, pH 7.4), add 100 μL of silver nitrate solutions with different concentrations so that the silver ion concentrations in the final hydrogel system are 0.5 mmol / L, 1 mmol / L, and 2 mmol / L respectively, and the keratin concentration in the final hydrogel system is 18% g / mL. After vortex dissolution, add dilute hydrochloric acid with a concentration of 0.1 mol / L to adjust the pH to 7.0, then let it stand for 6 h to form a gel (if the sample in the sample bottle does not flow for 30 s after inversion, it is determined that the sample has formed a gel). Record the gelation time of the hydrogel by the inversion method and select the optimal silver ion concentration for preparing the hydrogel.

[0044] Table 1 Ker / Ag + Formulation of hydrogel

[0045]

[0046] Preparation of composite hydrogel of keratin-loaded silver ion rhein (Ker / Ag + / Rh hydrogel):

[0047] Dissolve 3 mg of rhein (Rh) in 500 μL of sodium bicarbonate (concentration 0.2 mol / L, pH 8.3) solution, heat it in a water bath at 80 °C for 15 min to dissolve it completely, and obtain a rhein solution with a concentration of 6 mg / mL.

[0048] Dissolve 0.09 g of keratin powder (Ker) in 275 μL of PBS (concentration 0.01 mol / L, pH 7.4) solution, add 100 μL of silver nitrate solution with a concentration of 5 mmol / L, vortex to dissolve, add dilute hydrochloric acid with a concentration of 0.1 mol / L to adjust the pH to 7.0, then add 125 μL of rhein solution with a concentration of 6 mg / mL, and let the solution stand at room temperature for 6 h to obtain a composite hydrogel of keratin-loaded silver ion rhein (Ker / Ag + / Rh hydrogel). In the composite hydrogel of keratin-loaded silver ion rhein, the loaded rhein (Rh) concentration is 1.5 mg / mL.

[0049] Preparation method of hydrogel extract: Lyophilize the prepared composite hydrogel of keratin-loaded silver ion rhein (Ker / Ag + / Rh hydrogel), then sterilize the lyophilized hydrogel by ultraviolet light for 24 h, soak it in cell culture medium (containing 10% serum and 1% double antibody) at a dose of 2 mg / mL for 24 h to obtain a hydrogel extract, filter it through a sterile filter (filter head diameter 0.22 μm) to prepare a hydrogel extract, and store it in a 4 °C refrigerator for later use.

[0050] I. Hydrogel Microscopic Morphology Analysis: The hydrogel sample was freeze-dried and sliced using a freeze dryer. The sliced sample was adhered to conductive adhesive, and a pipette bulb was used to blow it several times to prevent the sample from falling during vacuum pumping. The sample was sputter-coated with gold, and then the microscopic morphological characteristics of the hydrogel were observed using a scanning electron microscope (HITACHI / TM-1000). Finally, the pore size data was processed using Image J software.

[0051] II. Hydrogel Swelling Property Test: The mass of the wet hydrogel was weighed and denoted as M0. Then, the hydrogel sample was immersed in 10 mL of phosphate buffer solution (PBS, concentration 0.01 mol / L, pH 7.4). The hydrogel sample was taken out at regular intervals, and the water on its surface was blotted off with filter paper. The mass of the swollen hydrogel was weighed and recorded as M1. When the final mass of the hydrogel remained unchanged, the swelling ratio was calculated. The swelling ratio of the hydrogel was (M1 - M0) / M0×100%. The experiment was repeated three times, and finally, the swelling ratio data was processed using Image J software.

[0052] III. Hydrogel Rheological Property Test:

[0053] A cylindrical hydrogel sample with a diameter of 25 mm and a height of 3 mm, prepared using a mold (a set of multifunctional anti-sticking hydrogel molds disclosed in the patent application with publication number CN221717551U), was placed on a 25-mm parallel plate, and a strain amplitude sweep test was conducted to determine the linear viscoelastic region with a strain range of 0.01% - 1000%. To test the shear thinning property of the hydrogel, the viscosity of the hydrogel was measured using a continuous ramp shear rate (0 - 100 s -1 ) The elastic modulus G’ and viscous modulus G’’ of the hydrogel were tested using a dynamic frequency sweep mode. The test conditions for the frequency sweep were: 1% strain, a frequency of 1 Hz, a temperature of 25°C, and a frequency sweep range of 0.1 - 100 rad / s.

[0054] IV. Hydrogel Mechanical Property Test: At room temperature, the hydrogel sample was tested using an electronic universal testing machine (Instron 5567, Norwood, MA). The sample was cylindrical, with a diameter of 15 mm and a length of 12 mm, a compression rate of 5 mm / min, and a strain range of 0 - 85%.

[0055] V. Hydrogel Compressibility Test: The hydrogel sample was subjected to a compression test to explore the mechanical toughness of the hydrogel.

[0056] VI. Injectability test of hydrogel: To more intuitively observe the injectability of the hydrogel, the hydrogel sample was stained with Coomassie Brilliant Blue, placed in a 1 mL syringe, and by squeezing the syringe, it was observed whether the hydrogel could pass through a 26-gauge needle and the state of the hydrogel in water was observed.

[0057] VII. Self-healing performance test of hydrogel: The hydrogel was stained, and then the separated hydrogel samples were placed together at room temperature. After a period of time, the state of the two hydrogels was observed and pulled with forceps to observe the self-healing effect of the hydrogel.

[0058] VIII. Reciprocal rheological behavior test of hydrogel: The hydrogel was subjected to reciprocal rheological testing by continuous cyclic strain tests (1% strain → 1000% strain → 1% strain) to further evaluate the self-healing performance of the hydrogel.

[0059] IX. Study on the in vitro drug release behavior of hydrogel:

[0060] Determination of the standard curve of Rhein: Rhein was dissolved in methanol solution, and standard solutions with concentrations of 10, 20, 30, 40, 50, 60, and 70 μg / mL were prepared respectively, and they were detected by high performance liquid chromatography (HPLC). The test conditions were as follows: the chromatographic column was Kromasil C18 (250 mm × 4.6 mm, 5 μm), the mobile phase was methanol - 0.1% phosphoric acid (85:15) aqueous solution, the flow rate was 1.0 mL / min, the column temperature was 30 °C, the detection wavelength was 254 nm, and the injection volume was 10 μL. With the Rhein concentration as the abscissa and the peak area as the ordinate, the Rhein standard curve was as shown in Figure 2 Figure B. When the Rhein concentration was 10 - 70 μg / mL, the measured peak area showed a good linear relationship with the Rhein concentration, and the linear regression equation was: y = 62789x - 62140, R 2 = 0.9985.

[0061] In vitro drug release: The hydrogel was placed in a 5 mL PBS (concentration 0.01 mol / L, pH 7.4) solution, and it was placed in an incubator at 37 °C. Samples of 1 mL were taken at different time points, and 1 mL of fresh PBS (concentration 0.01 mol / L, pH 7.4) was added to the original system. The peak area of the release solution at 254 nm was measured by HPLC, and the release amount of Rhein was calculated through the Rhein standard curve.

[0062] X. Antibacterial performance test of hydrogel: Using the film-forming strains methicillin-resistant Staphylococcus aureus (USA300), carbapenem-resistant Escherichia coli (RJ112) was used as the representative strain.

[0063] Add 200 μL of the bacterial suspension (1×10 5 CFU / mL) to a 48-well plate, and then add 100 μL of different treatment drugs. A blank control group (treated with PBS), a positive control group (10 μg / mL vancomycin (VAN) / cefixime (CFM)), and experimental groups (Ker hydrogel, Ker / Ag + hydrogel, Ker / Ag + / Rh hydrogel extract) were set up. After incubation at 37 °C for 24 h, add pre-cooled termination solution (0.1% Tween 80 + 0.9% NaCl) to terminate the reaction. Dilute serially to 10 -3 and then take 100 μL to coat TSA plates. Colony counting was performed using a fine-point camera automatic colony counter. The calculation formula for the bactericidal rate: (CFU of the blank group - CFU of the treatment group) / CFU of the blank group × 100%.

[0064] Crystal violet staining method: Adjust the concentration of the overnight-cultured amplified bacterial suspension to OD 590 = 0.1 ± 0.02 with the medium under an ultraviolet spectrophotometer. Place a 6-mm circular transparent silicone sheet at the bottom of each well of a 96-well plate as a carrier. Add 100 μL of the adjusted bacterial suspension to each well, and then add 100 μL of PBS and hydrogel extract respectively and place them in a constant temperature and humidity biochemical incubator at 37 °C for continuous static culture for 72 h. A relatively stable and mature 、 biofilm can be formed on the silicone sheet carrier in the Control group. Take out the silicone sheet cultured for 72 h, gently rinse the planktonic bacteria on the surface of the carrier with sterile PBS (pH = 7.4), and then fix it with 2.5% glutaraldehyde solution for 2 h. After sucking out the glutaraldehyde, air-dry it at room temperature. Add 1 mL of 0.5% crystal violet solution to a 24-well cell culture plate for staining for 25 min. Gently rinse each carrier with PBS to remove the unbound crystal violet staining solution until the rinsing solution sucked out is a colorless liquid. Dry the well plate at room temperature, add 1 mL of 95% ethanol, repeatedly pipette each well and decolorize for 15 min. Pipette 100 μL of the decolorized solution into a 96-well cell culture plate, and measure the OD value (OD 590 ) of each well at a wavelength of 590 nm with a multifunctional microplate reader. Each strain was cultured with 3 carriers each time, and the experiment was independently repeated in parallel 3 times, and the results were averaged. The determination of the biofilm inhibition rate was performed using the formula: (OD of the negative control group - OD of the drug group) / (OD of the negative control group - OD of the blank group) × 100%.

[0065] Bio-scanning electron microscopy (SEM): The samples were fixed with 2.5% glutaraldehyde for 24 h. After pouring out the fixative, the samples were rinsed three times with 0.1 M phosphate buffer solution at pH 7.2 for 15 min each time. Then the samples were fixed with 1% osmium tetroxide solution for 2 h. The osmium tetroxide waste liquid was carefully removed, and the samples were rinsed three times with 0.1 M phosphate buffer solution for 15 min each time. Gradient dehydration: The samples were dehydrated with gradient concentration ethanol solutions, with each concentration treatment for 15 min, then treated with 100% ethanol for 20 min, and finally, fresh 100% ethanol was used. Drying: Drying was carried out in a carbon dioxide critical point dryer. Gold spraying: The samples were fixed on the sample stage with conductive carbon glue, and gold was sprayed with an ion sputtering instrument for 60 s. Observation was carried out under a scanning electron microscope, and 3 random fields of view were taken for each sample for photography.

[0066] Observation method of microbial live / dead staining by confocal laser scanning microscope (CLSM): The samples were taken out and placed in sterile disposable small dishes, and washed 3 times with sterile normal saline solution. The staining working solution was prepared with the Live-dead ® Baclight™ bacterial viability kit (the working concentration of SYTO-9 was 5 μM, and the working concentration of PI was 2 μg / mL). 500 μL of each staining working solution was gently dropped on the surface of the samples, and stained in the dark at 37 °C for 15 min. After staining was completed, the staining solution was discarded. The staining solution was washed away with sterile normal saline. The samples were placed face-down for photography in a confocal dish and placed on a confocal laser scanning microscope (200 times) for photography and 3D image reconstruction.

[0067] XI. Hydrogel hemolysis test: All experimental mice were approved by the Ethics Committee of Yueyang Hospital of Integrated Traditional Chinese and Western Medicine Affiliated to Shanghai University of Traditional Chinese Medicine (Ethical Approval No. 231932). SPF-grade C57BL / 6J mice, 10 weeks old, with half males and half females, were purchased from Shanghai Slake Laboratory Animal Co., Ltd. (License No.: SCXK (Shanghai) 2022-0004; Certificate No.: 20220004024199).

[0068] Fix the mice and collect blood from the retro-orbital venous plexus. Place the fresh blood of the mice in an anticoagulation tube, centrifuge the collected blood 3 times at 1500 rpm for 10 min each time. At the same time, wash the obtained red blood cells 3 times with PBS (concentration 0.01 mol / L, pH 7.4) solution, and dilute the red blood cells with PBS solution to a 5% (v / v) red blood cell suspension. Then add 500 μL of the hydrogel sample to 500 μL of the red blood cell suspension, place it in a shaker at 37 °C and 100 rpm for incubation for 1 h. Select deionized water as the positive control and PBS (concentration 0.01 mol / L, pH 7.4) as the negative control. After incubation, centrifuge the solution in the 24-well plate at 1000 rpm for 10 min, take 100 μL of the supernatant and add it to the 96-well plate, and measure the OD value of the solution with an enzyme-linked immunosorbent assay (ELISA) reader at 540 nm.

[0069] The formula for calculating the hemolysis rate is: Hemolysis rate (%) = [(As - Al - Ap) / (Aw - Ap)] × 100%.

[0070] As is the absorbance value of the supernatant treated with the hydrogel, Al is the absorbance value of the leaching solution of the hydrogel in PBS (concentration 0.01 mol / L, pH 7.4) solution, Aw is the absorbance value of the supernatant treated with deionized water, and Ap is the absorbance value of the supernatant treated with PBS.

[0071] XII. Evaluation of the cytocompatibility of the hydrogel:

[0072] Culture and seeding of L929 fibroblasts: Culture L929 cells with DMEM high-glucose medium (containing 10% fetal bovine serum and 1% double antibody), place the L929 cells in a cell culture incubator (37 °C, 5% CO2) for culture, observe the number and morphology of the cells with a microscope. When the cells grow to about 80 - 90% of the culture flask, digest the cells with trypsin. The method is as follows: Aspirate the cell culture medium, add an appropriate amount of trypsin for digestion, place it in the cell culture incubator for incubation for about 1 min, then place it under the microscope for observation. When most cells are observed to float, add cell culture medium with twice the volume of trypsin to stop digestion. Then transfer the cells to a sterile centrifuge tube, centrifuge and remove the supernatant, add an appropriate amount of cell culture medium, and pipette the suspended cells with a sterile pipette tip to make a cell suspension. Subsequently, perform cell counting and dilute the cell suspension with cell culture medium to the required concentration. Finally, inoculate L929 cells into a 48-well plate containing medium at a density of 10000 cells / well, then culture the cells in a 37 °C incubator for 24 h, aspirate the medium and add the hydrogel extract. Perform subsequent experiments 1, 3, and 5 days after cell culture, and set three parallels for the experiment.

[0073] Cell proliferation was detected by CCK-8 assay:

[0074] Principle of detection: The CCK-8 reagent contains WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt), which is reduced by dehydrogenases in mitochondria to form an orange-yellow formazan product. The more viable cells there are, the darker the color; the greater the cytotoxicity, the lighter the color. Therefore, the CCK-8 kit can be used to detect cell proliferation.

[0075] Detection method: After culturing the cells for 1, 3, and 5 days, the cell culture medium in the well plate was aspirated, and 200 μL of DMEM medium containing 10% CCK-8 staining solution was added, followed by incubation at 37 °C for 1 h. The experiment needed to be carried out under light protection. After incubation, 100 μL of the liquid was taken into a 96-well plate, and the OD value at a wavelength of 450 nm was measured using a full-wavelength microplate reader.

[0076] Calcein-AM / PI dual staining method for live and dead cells:

[0077] Principle of detection: Calcein-AM is a substance that can fluorescently label live cells and emit green fluorescence. Because it introduces an acetoxymethyl (AM) group on the basis of Calcein (calcein), its hydrophobicity is enhanced, enabling it to more easily cross the live cell membrane. When it enters the cell, Calcein-AM, which does not fluoresce itself, can be cleaved by esterases in the cell to form Calcein, which then remains in the cell and emits strong green fluorescence. Compared with other similar reagents (such as BCECF-AM and CFDA), Calcein-AM has extremely low toxicity and is therefore the most suitable fluorescent probe for live cell staining. Propidium iodide (PI) cannot cross the cell membrane of live cells and can only penetrate the cell membrane of dead cells and intercalate into the DNA double helix of the nucleus to produce red fluorescence. Therefore, PI only stains dead cells. Since both Calcein and PI can be excited at 490 nm, fluorescence microscopy can be used to observe live and dead cells simultaneously.

[0078] Detection method: The staining solution was prepared in advance. 1.5 μL of Calcein and 1.1 μL of PI were added to 2 mL of serum-free DMEM medium and mixed well. 200 μL of the staining solution was added to each well under light protection, and then the well plate was placed in a 37 °C incubator and incubated for 25 - 40 min. After incubation, an inverted fluorescence microscope (10×) was used to observe the cell staining morphology and take pictures.

[0079] XIII. Evaluation of the ability of hydrogel to promote wound healing:

[0080] Establishment of type 2 diabetes wound model: All experimental mice were approved by the Ethics Committee of Yueyang Hospital of Integrated Traditional Chinese and Western Medicine, Shanghai University of Traditional Chinese Medicine (Ethical Approval No. 231932). SPF-grade C57BL / 6J mice, 5-6 weeks old, with an equal number of males and females, were purchased from Shanghai Slake Laboratory Animal Co., Ltd. (License No.: SCXK (Shanghai) 2022-0004; Certificate No.: 20220004024199). After a 2-month high-fat diet, streptozotocin (STZ) prepared with 0.1 M sodium citrate buffer (pH 4.2) was intraperitoneally injected at a dose of 30 mg / kg once a day for one week. The mice were fasted for 8 h before each administration. The fasting blood glucose level of the mice was measured with a blood glucose meter. When the blood glucose level > 16.7 mmol / L and accompanied by symptoms such as polydipsia, polyphagia, polyuria, and weight loss, the modeling was considered successful. After co-housing males and females, they were grouped, and the hair in the modeling area on the back was shaved using a razor. The mice were anesthetized by inhaling isoflurane, and two 1-cm-diameter circular marks were made on the back. After skin preparation and disinfection, the skin was incised with surgical scissors deep into the fascia layer to create a skin defect wound. A silicone ring was sutured and fixed around the wound with suture thread, and a mixed bacterial solution was inoculated in the modeling area. ( and A cotton ball soaked with a bacterial solution with a concentration of 1×10 8 CFU / mL was placed on the wound surface, and the wound was covered with a 3M transparent dressing for 72 h). After 72 h, a mature biofilm was formed, and each group of drugs was applied to the wound surface, and the dressing was changed daily.

[0081] XIV. Masson staining: Paraffin sections were routinely dewaxed and hydrated, stained with hematoxylin solution for 10 minutes to stain the nuclei after washing with distilled water, and washed thoroughly. If overstained, differentiation could be carried out with hydrochloric acid alcohol. Then, stained with Ponceau acid fuchsin solution for 10 minutes, washed with 2% glacial acetic acid aqueous solution, and differentiated with 1% phosphomolybdic acid aqueous solution for 5 minutes. Then, without washing with water, directly stained with aniline blue or light green solution for 5 minutes, and washed with 0.2% glacial acetic acid aqueous solution. Finally, dehydrated with alcohol, cleared with xylene, and sealed with neutral gum. Observed and photographed under a light microscope, collagen fibers were blue or green, cytoplasm was red, and cell nuclei were black and blue.

[0082] XV. Western blot: Operate according to the standard configuration method of YaMei Universal SDS-PAGE Gel Kit, prepare 12% separating gel and 5% stacking gel. Add them into the glass plate, insert a comb at the top, and let it stand for 60 min. After the gel preparation is completed, remove the glass plate from the gel preparation rack, rinse the outer side of the glass plate with deionized water, then fix the glass plate in the inner electrophoresis tank, fill it with 1× electrophoresis buffer, pull out the comb, add protein molecular weight standard (Marker) and the protein sample to be tested into the comb holes in sequence, start the electrophoresis device, and continue electrophoresis until the bromophenol blue indicator runs out of the bottom of the separating gel to complete electrophoresis. Immerse the PVDF membrane in methanol solution for 30 s. Assemble the transfer mold clamp in the order of black transfer mold clamp (negative electrode) → sponge pad → three layers of filter paper → gel → PVDF membrane → three layers of filter paper → sponge pad → white transfer membrane clamp (positive electrode). After the transfer is completed, add blocking solution (5% skimmed milk powder prepared with 1× TBST solution) to the PVDF membrane, and block it on a horizontal shaker at the lowest speed at room temperature for 2 h. Incubate with the primary antibody overnight in a 4°C refrigerator. Incubate with the secondary antibody for 1 h at room temperature. Develop using the ECL chemiluminescence method. Use Image J 1.0 software to perform semi-quantitative analysis of the gray value of protein bands.

[0083] XVI. Real-time quantitative polymerase chain reaction (qRT-PCR): Extract total RNA from tissues and cells using the Trizol method, measure the concentration and purity, and reverse transcribe the RNA into complementary DNA (cDNA) according to the instructions of the reverse transcription kit. The sequences refer to the sequences of each target gene in the Gene Bank database, and the primers are designed by NCBI Primer-blast and synthesized by Beijing Tsingke Biotechnology Co., Ltd. Dissolve the primers in enzyme-free water and dilute them 10 times. Prepare a 10 μL PCR reaction system according to the instructions, then add it to an eight-well tube, mix well and centrifuge, and put it into a PCR instrument for amplification reaction.

[0084] XVII. Hematoxylin-eosin (H&E) staining: Paraffin sections are successively baked, dewaxed, stained with hematoxylin, stained with eosin, then dehydrated, cleared and sealed conventionally, and observed under a microscope.

[0085] XVIII. Immunofluorescence of tissue sections

[0086] The paraffin sections were routinely dewaxed. The sections were placed in a repair box filled with EDTA antigen repair buffer (pH 8.0) and antigen repair was carried out in a microwave oven. After natural cooling, the slides were placed in PBS (pH 7.4) and shaken and washed 3 times on a shaker for 5 minutes each time. After the sections were slightly dried by centrifugation, a histochemical pen was used to draw a circle around the tissue (to prevent the antibody from flowing away), and a spontaneous fluorescence quencher was added to the circle for 5 minutes, followed by rinsing with running water for 10 minutes. BSA was added dropwise to the circle and incubated for 30 minutes. The primary antibodies (Histone H3, Proteintech, Cat No. 13754-1-AP; MPO, ABclonal, A1374; LRP2, Sigma-Aldrich, HPA005980; CD29, ABclonal, A23497) were added and incubated overnight at 4°C. After the sections were slightly dried by centrifugation, the secondary antibody corresponding to the primary antibody was added dropwise to cover the tissue in the circle and incubated at room temperature in the dark for 1 hour. The slides were placed in PBS (pH 7.4) and shaken and washed 3 times on a shaker for 5 minutes each time. After the sections were slightly dried by centrifugation, DAPI staining solution was added dropwise to the circle and incubated at room temperature in the dark for 10 minutes. The sections were sealed with an anti-fluorescence quenching mounting medium.

[0087] Immunohistochemistry: Dewaxing was performed on 4-μm thick tissue sections fixed with 4% paraformaldehyde and embedded in paraffin, and dehydration and clearing were carried out with ethanol of gradient concentration. By using the heat repair method, the tissue sections were placed in a citric acid buffer (pH 6.0) at 95°C for 30 minutes for antigen repair. BSA was used for blocking and incubated at room temperature for 30 minutes, and then rinsed with PBS. The diluted primary antibodies (VEGF-A, Servicebio, GB15165-100; Hif-1α, Sigma-Aldrich, PLA0081; LDLR, ABclonal, A20808; LRP2, Sigma-Aldrich, HPA005980; LRPAP1, ABclonal, A13026) were added and incubated overnight at 4°C. After incubation with the primary antibody, the sections were washed with PBS, and a horseradish peroxidase (HRP)-specific secondary antibody was added and incubated at room temperature in the dark for 60 minutes. Finally, after staining with DAB chromogenic reagent and hematoxylin, dehydration and sealing were carried out. The staining was observed under a microscope and photographed. At 200-fold magnification, three random fields of view were photographed, and the average optical density value was analyzed using Image J software for statistics.

[0088] The experimental results are as follows:

[0089] Keratin-based injectable hydrogel (Ker / Ag + hydrogel) The performance evaluation results are as Figure 1 shown, Figure 1Figure showing the physical characterization results of the keratin-based injectable hydrogel. All data are from at least 3 independent experiments and are presented as mean ± standard deviation (SD).

[0090] Silver nitrate solutions with different concentrations were mixed with the keratin solution, and hydrogels were formed through the Ag-S dynamic reaction between silver ions and sulfhydryl groups in keratin ( Figure 1 as shown in A of + ). Apparently, the hydrogels prepared under different conditions had different gelation times and different injectabilities. When the silver ion concentration in the hydrogel was 0, it took only about 0.5 h for keratin to form the keratin hydrogel, but the hydrogel was difficult to inject. When the silver ion concentration in the hydrogel was 0.5 mmol / L, the Ker / Ag + hydrogel took about 1.5 h to form, and the hydrogel could be injected, but the injected hydrogel concentrated at the needle tip. When the silver ion concentration in the hydrogel was 1 mmol / L, the Ker / Ag + hydrogel took about 4 h to form, and the Ker / Ag + hydrogel could be continuously injected and was in good condition. When the silver ion concentration in the hydrogel was 2 mmol / L, the Ker / Ag + hydrogel took about 6 h to form, and the hydrogel could be injected, but there was more injected hydrogel fluid. This is because when silver nitrate was added, the sulfhydryl groups in Ker would rapidly form Ag-S dynamic bonds with silver ions. Subsequently, the sulfhydryl groups enriched in Ker were oxidized to form disulfide bonds at pH = 7. Therefore, there were dynamically cross-linked Ag-S bonds and covalently cross-linked disulfide bonds in the hydrogel network. Scanning electron microscopy observed that all Ker / Ag Figure 1 hydrogels had an interconnected porous network microstructure ( + as shown in B of + , scale bar is 200 μm). As the silver ion concentration increased, the average pore size of the Ker / Ag Figure 1 hydrogel ranged from 10 to 85 μm, and the swelling ratio of the Ker / Ag Figure 1 hydrogel further confirmed the results of the microstructure ( + as shown in C and D of Figure 1 ). The higher the silver ion concentration, the higher the swelling ratio and the looser the network cross-linking structure. The rheological properties of the hydrogel were investigated by rheological tests. When the silver ion concentration was high ( Figure 1 as shown in E and F of+ The hydrogel healed without cracks after 1 h and was stretchable after healing. The self-healing performance of the Ker / Ag+ hydrogel was further evaluated by continuous cyclic strain tests (1% strain → 1000% strain → 1% strain) ( Figure 1 as shown in I). When a 1000% strain was applied, the G’ value of the hydrogel decreased and was less than G”, indicating that the hydrogel network collapsed. Once the strain decreased to 1%, G’ almost recovered to its original value, meaning the recovery of the network. This cycle of collapse and recovery was repeated at least twice. Rheological tests showed that as the shear rate increased, the viscosity of the Ker / Ag + hydrogel decreased significantly, indicating that the Ker / Ag + hydrogel had obvious shear-thinning behavior. This property ensured that the Ker / Ag + hydrogel had good injectability and could be continuously injected through a syringe with a 26-gauge needle in air or water ( Figure 1 as shown in J). This property helped the Ker / Ag + hydrogel maintain its integrity when damaged by external forces or applied to moving wounds. In summary, the Ker / Ag + hydrogel had elasticity, self-healing ability, and good injectability and could be used as a wound dressing to fill irregular skin wounds to promote wound healing.

[0091] The in vitro drug release behavior, hemolysis test, and cell compatibility evaluation results of the hydrogel are as Figure 2 shown, Figure 2 which are the graphs of the in vitro drug release behavior, hemolysis test, and cell compatibility evaluation results of the hydrogel. The Ker / Ag + hydrogel was immersed in PBS solution at 37 °C to measure the release curve of Ag + . In the PBS release system, at 20 h, the percentage of cumulatively released silver ions rapidly increased from 0 to 40%. From 20 h to 140 h, the percentage of cumulatively released silver ions continued to increase, but the growth rate slowed down. At this stage, the release of silver ions showed a relatively stable upward trend, with an approximately 10% - 20% increase in the cumulative release amount every 20 h. At about 140 h, the percentage of cumulatively released silver ions reached a relatively stable plateau, maintaining at about 85% - 90%, indicating that at this time point, the release of silver ions was basically saturated and most of the silver ions in the sample had been released ( Figure 2 as shown in A). The in vitro release of the Ker / Ag + / Rh hydrogel was determined by HPLC. From 0 to 144 h, the cumulative drug release amount gradually increased. After 72 h, the Ker / Ag +The cumulative release amount of rhein in Ker / Ag / Rh hydrogel was about 30%. After 144 h, the cumulative release amount of rhein in Ker / Ag + / Rh hydrogel reached about 40% ( Figure 2 as shown in B of ). To evaluate the wound irritation of the hydrogel, the hemocompatibility of the hydrogel was evaluated by in vitro hemolysis experiment ( Figure 2 as shown in C of ). The hemolysis rate of each hydrogel treatment group was lower than the reported threshold (hemolysis rate < 5%). To evaluate the cytotoxicity of the hydrogel, the effects of different treatment groups on the viability of L929 cells were evaluated by CCK-8 method and live / dead cell staining experiment. After the hydrogel was co-incubated with L929 for 1, 3, and 5 d, there was no difference in cell viability compared with the control group ( Figure 2 as shown in D of ). The live / dead cell staining experiment confirmed the results of the CCK-8 method. With the extension of the incubation time, there was no difference in the ratio of live cells (green) to dead cells (red) between groups ( Figure 2 as shown in E of ), indicating that the hydrogels of each group had no effect on cell viability.

[0092] Ker / Ag + / Rh hydrogel inhibited biofilm formation and cascade antibacterial in vitro:

[0093] Figure 3 It is the result diagram of the cascade antibacterial efficacy of the composite hydrogel of keratin loaded with silver ions and rhein in inhibiting biofilm formation in vitro.

[0094] To clarify the interaction between silver ions and Rh, and were used for in vitro antibacterial evaluation. First, the antibacterial effects of each group were compared by plate colony counting method ( Figure 3 as shown in B to E of ).

[0095] The grouping situation of in vitro antibacterial evaluation using is as follows:

[0096] The prepared Ker hydrogel, Ker / Ag + hydrogel, and Ker / Ag + / Rh hydrogel were freeze-dried. The freeze-dried hydrogel was sterilized by ultraviolet for 24 h, added to the cell culture medium (containing 10% FBS) at a dose of 2 mg / mL and soaked for 24 h to obtain the hydrogel extract. The extract was filtered through a sterile filter (the filter head diameter was 0.22 μm) to prepare the extracts of Ker hydrogel, Ker / Ag + hydrogel, and Ker / Ag + / Rh hydrogel, which were placed in a 4 °C refrigerator for later use.

[0097] The experimental method adopted the modified ISO 22196 standard. Medium modification: The pH of the medium was stabilized in the alkaline range of 7.5 - 8.5 by adding sodium bicarbonate (NaHCO3), glucose was added to 20 mM to simulate the hyperglycemic state of diabetic ulcers, and 10% FBS was added to simulate proteins and growth factors in host wound exudate (10% of the total liquid volume). In a 48-well plate, first add 200 μL of bacterial suspension (1×10 5 CFU / mL), then add 100 μL of different treatment drugs, set up a blank control group (treated with PBS, Control in the figure), a positive control group (30 μg / mL VAN, 1 mg / mL CFM), and experimental groups (Ker, Ker / Ag + 、Ker / Ag + / Rh extract). After incubation at 37°C for 24 h, add pre-cooled termination solution (0.1% Tween 80 + 0.9% NaCl) to terminate the reaction. Dilute serially to 10 -3 and then take 100 μL to coat TSA plates, and colony counting was performed using a Jingdian camera automatic colony counter. The formula for calculating the bactericidal rate: (CFU in the blank group - CFU in the treatment group) / CFU in the blank group × 100%.

[0098] Control group: Treated with PBS.

[0099] VAN group: 30 μg / mL VAN.

[0100] Ker hydrogel group: 2 mg / mL Ker-containing cell culture medium.

[0101] Ker / Ag + hydrogel group: 2 mg / mL Ker / Ag + -containing cell culture medium.

[0102] Ker / Ag + / Rh hydrogel group: 2 mg / mL Ker / Ag + / Rh-containing cell culture medium.

[0103] Compared with the blank control group, Ker hydrogel had no antibacterial effect on , while VAN, Ker / Ag + hydrogel, and Ker / Ag + / Rh hydrogel had antibacterial rates of 78.05%, 59.75%, and 98.79% on respectively. Ker / Ag + / Rh hydrogel had the best antibacterial rate effect (as shown in A and C in , and the upper part of Figure B). Figure 3 ​

[0104] Usage Grouping for in vitro antibacterial evaluation:

[0105] Control group: Treated with PBS.

[0106] CFM group: 1 mg / mL CFM.

[0107] Ker hydrogel group: 2 mg / mL Ker-containing cell culture medium.

[0108] Ker / Ag + hydrogel group: 2 mg / mL Ker / Ag + -containing cell culture medium.

[0109] Ker / Ag + / Rh hydrogel group: 2 mg / mL Ker / Ag + / Rh-containing cell culture medium.

[0110] Compared with the blank control group, Ker had no obvious antibacterial effect, and the antibacterial rates of CFM, Ker / Ag + hydrogel, and Ker / Ag + / Rh hydrogel against were 72.65%, 50.61%, and 97.73% respectively. The antibacterial rate of Ker / Ag + / Rh hydrogel against was the best ( Figure 3 the upper half of D and E as shown).

[0111] To observe the morphological changes of bacteria after drug administration, SEM was used to observe the , morphological changes of ( Figure 3 the lower half of B and the lower half of D as shown), and the blank control group / had a smooth cell membrane, a complete surface with clear edges, and the bacterial morphology was regular rod-shaped. Ker hydrogel had / no effect on the + morphology. The VAN / CFM group and Ker / Ag + hydrogel wrinkled the cell surface and deformed the bacteria. Ker / Ag / hydrogel caused the cell wall and cell membrane to rupture, and the cytoplasmic contents to flow out, resulting in stronger damage to the morphological structure of bacteria.

[0112] Under the confocal laser scanning microscope (CLSM), the biofilms in the blank control group showed dense, uniform, and relatively intact aggregates of viable bacteria (green fluorescence). Each treatment group showed varying degrees of looseness and sparseness, with varying amounts of dead bacteria (red fluorescence) in the biofilm, and the number of dead bacteria increased while the number of viable bacteria decreased. Among them, in the biofilm, the viable bacteria density in the VAN, Ker / Ag + hydrogel and Ker / Ag + / Rh hydrogel groups was lower than that in the blank control group. The number of dead bacteria in the VAN, Ker / Ag + hydrogel and Ker / Ag + / Rh hydrogel groups gradually increased, and almost all of the Ker / Ag + / Rh hydrogel group was covered by dead bacteria ( Figure 3 as shown in the upper part of F in Figure 3 ). The fluorescence intensity quantification graph of live / dead bacteria staining is as shown in G in . The scanning electron microscope (SEM) images of the biofilms in each group also showed destruction of the biofilm structure, with a small amount of residual planktonic bacteria, which was consistent with the CLSM images ( Figure 3 as shown in the lower part of F in + . In the crystal violet staining experiment, the absorbance value of crystal violet of the Ker / Ag Figure 3 / Rh hydrogel was the lowest (

[0113] as shown in J in ), indicating that the number of viable bacteria in this group was the least. + In the biofilm, the viable bacteria density in the Ker / Ag + hydrogel, CFM, and Ker / Ag + / Rh hydrogel groups was lower than that in the blank control group. The number of dead bacteria in the Ker / Ag + hydrogel, CFM, and Ker / Ag + / Rh hydrogel groups gradually increased, and almost all of the Ker / Ag Figure 3 / Rh hydrogel group was covered by dead bacteria ( as shown in the upper part of H in Figure 3 . The SEM images of the biofilms in each group also showed destruction of the biofilm structure, with a small amount of residual planktonic bacteria and varying degrees of morphological changes in the cell membrane, which was consistent with the CLSM images ( + as shown in the lower part of H and I in Figure 3 ). In the crystal violet staining experiment, the absorbance value of crystal violet of the Ker / Ag + hydrogel, Ker / Ag + / Rh hydrogel was the lowest ( as shown in K in It has a certain ability to inhibit bacteria and degrade biofilms, but the Ker / Ag + / Rh hydrogel group has the strongest ability to inhibit bacteria and degrade biofilms.

[0114] Ker / Ag + / Rh hydrogel promotes / The results of the promotion of ulcer healing in mice with biofilm-infected T2DM (type 2 diabetic wound model) by Ker / Ag Figure 4 are shown as Figure 4 follows. For Ker / Ag + / Rh hydrogel promoting / the results graph of ulcer healing in biofilm-infected T2DM mice.

[0115] Grouping (6 mice in each group):

[0116] Each time during treatment, the wound was first cleaned with normal saline, and then the drugs of each group were applied to the wound surface once a day.

[0117] Normal group: Normal mice + non-infected wound + treated with urea ointment (UO).

[0118] Vehicle group: T2DM mice + infected wound + UO group.

[0119] MO group: T2DM mice + infected wound + (mupirocin ointment (MO)) group.

[0120] Ker hydrogel group: T2DM mice + infected wound + keratin hydrogel group.

[0121] Ker / Ag + Hydrogel group: T2DM mice + infected wound + Ker / Ag + hydrogel group.

[0122] Ker / Ag + / Rh hydrogel group: T2DM mice + infected wound + Ker / Ag + / Rh hydrogel group.

[0123] Figure 4 Figure A shows a schematic diagram of the whole process of the construction and treatment plan of the type 2 diabetic wound model. On the first day, the wound tissue of the T2DM group was red and swollen and covered with light yellow exudate. During the wound closure record from day 3 to day 12, it was observed that the area of each treatment group gradually decreased. The use of Ker hydrogel alone could not improve / the healing of biofilm-infected wounds. On the 12th day, the MO group and Ker / Ag+ There was no difference in wound area in the hydrogel group, and there was no difference in wound area between the Normal group and the Ker / Ag + / Rh hydrogel group. It is worth noting that the Ker / Ag + / Rh hydrogel significantly accelerated wound healing, reaching a wound closure rate of nearly 50% on the 3rd day, about 3 days earlier than the MO group and the Ker / Ag + hydrogel group, approaching the wound closure rate of the Normal group ( Figure 4 as shown in C and D).

[0124] To determine the contribution of the Ker / Ag + / Rh hydrogel to the re-epithelialization process, some morphological features of the wound area were compared. Figure 4 Figure B in Figure 4 shows a schematic diagram of the histological morphometric parameters analyzed. The results of the histological analysis by H&E staining were consistent with the wound healing rate ( Figure 4 as shown in E). On the 12th day, the diameter of the wound bed (including wound size, tissue characteristics, exudate, bacteria, etc.) in the Vehicle treatment group was the largest, and there was no new epidermis and dermis under the eschar, and there was less epidermis and dermis in the Ker hydrogel group. While in the Normal group, MO group, Ker / Ag + hydrogel group, Ker / Ag + / Rh hydrogel group, denser granulation tissue and new epidermis and dermis (blue dotted line) were observed in the wound surface. Figure 4 The left figure in F of + is a quantification graph. It can be seen from the graph that compared with other groups, the re-epithelialized tissue of the mice in the Ker / Ag + / Rh hydrogel group had a larger coverage area and epithelial tongue length, and this difference indicated a stronger cell migration ability during the healing process; on the other hand, in the newly formed epithelium, the epithelial tongue thickness of the mice in the Ker / Ag + / Rh hydrogel group was higher, which indicated more proliferation and differentiation of epithelial cells ( Figure 4 as shown in the right figure of F).

[0125] Immunofluorescence staining of tissue sections was used to detect hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF-A) in order to evaluate the status of new blood vessels in granulation tissue on the 12th day. The results are as Figure 5 shown Figure 5 is the result graph of promoting collagen deposition and angiogenesis in vivo by Ker / Ag + / Rh hydrogel. It can be seen from the graph that compared with Vehicle, in the Normal group, MO group, Ker / Ag + hydrogel group, Ker / Ag +In the wound sections of the Ker / Ag / Rh hydrogel group, the expression levels of HIF-1α were all decreased, among which, Ker / Ag + / Rh hydrogel group showed the lowest expression level ( Figure 5 as shown in A and B). It is worth noting that Ker / Ag + / Rh hydrogel group presented a similar level of VEGF-A expression to the Normal group, while no significant differences in VEGF-A expression were observed among the Vehicle group, MO group, Ker hydrogel group, and Ker / Ag + hydrogel group ( Figure 5 as shown in C and D). On the 7th and 12th days, Masson's trichrome staining was performed to observe the deposition of collagen fibers during the healing process. Among them, in the collagen-stained sections on the 7th day, it could be observed that the tissues of the Vehicle group and Ker hydrogel group had obvious tissue edema, a large number of inflammatory cell infiltrations, and almost no collagen (blue) production. In the Normal group, MO group, Ker / Ag + hydrogel group, and Ker / Ag + / Rh hydrogel group, different degrees of increased collagen deposition in the wound area were observed, and the collagen increase in the Normal group and Ker / Ag + / Rh hydrogel group was the most obvious. The collagen-stained sections on the 12th day showed that the Vehicle group and Ker hydrogel group were still in a state of a large amount of granulation tissue hyperplasia. In the MO group and Ker / Ag + hydrogel group, the collagen was uneven in thickness, with breaks and disordered arrangements visible. In the Ker / Ag + / Rh hydrogel group, the arrangement of collagen fibers could be seen to be regular, dense, and uniform, approaching the structural and staining characteristics of the skin tissue of the Normal group, and the quantification graph also showed a higher collagen volume fraction ( Figure 5 as shown in E-H). In summary, the wounds of the Ker / Ag + / Rh hydrogel group in mice almost healed after 12 days of treatment and showed a histological structure similar to that of the skin of Normal group mice. It seemed to have no scar and had the most skin appendages (yellow arrows), showing better wound healing ability compared with other groups.

[0126] The main challenge during the DFI healing process is the persistent inflammatory phase, which is due to the biofilm regulating the production of virulence factors through quorum sensing to induce host cells to activate neutrophils and release neutrophil extracellular traps (NETs), various chemokines, cytokines, antimicrobial peptides, etc. The aggregation of a large number of inflammatory factors will cause damage to host cells. The results of visual observation of the wound area and histological analysis have confirmed Ker / Ag +The / Rh treatment group can accelerate the inflammatory phase of DFI healing. This invention explores whether the healing process is related to virulence factors, NETs, and pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α) in the wound.

[0127] Figure 6 It is Ker / Ag + Inhibition of / Rh hydrogel in vivo / Experimental result graph of biofilm formation antibacterial and anti-inflammatory. The relationship between biofilm and wound inflammation is as Figure 6 shown in A. (Inflammatory phase) Immunohistochemical staining of activated neutrophil marker myeloperoxidase (MPO) and citrullinated histone H3 (Cit-H3) in wound tissue on the 3rd day of the wound surface was used to characterize the generation of NETs. Compared with the Vehicle group, MO group, Ker hydrogel group, and Ker / Ag + hydrogel group, after 3 days of treatment, the neutrophil infiltration and NETs level in the Ker / Ag + / Rh hydrogel group were significantly reduced ( Figure 6 shown in D and E). On the 7th day of treatment, the results of pro-inflammatory cytokine levels (IL-1β, IL-6, and TNF-α) were consistent with those of NETs ( Figure 6 shown in F and G). The above indicates that the Ker / Ag + / Rh hydrogel group showed an earlier transition through the inflammatory phase than the positive drug MO group and the Ker / Ag + hydrogel group.

[0128] To explore whether the Ker / Ag + / Rh hydrogel group affects inflammation by inhibiting the reformation of biofilm, the tissue debridement fluid of the wound on the 3rd day was cultured for bacteria, diluted and then spread on plates for counting, and the reduction of CFU / mL bacteria in the wound was evaluated. The sterilization rate of the Ker / Ag + / Rh hydrogel group > 98.87% ( Figure 6 shown in B and C), which was better than the antibacterial rates of the MO group and the Ker / Ag + hydrogel group. CLSM was used to observe the effects of the Vehicle group, Ker / Ag + hydrogel group, and Ker / Ag + / Rh hydrogel group on the permeability and dispersibility of the wound biofilm. The SYTO-9 bacterial live-dead dual-staining green fluorescence probe was used to stain the live bacteria in the biofilm, showing green fluorescence. Propidium iodide PI was used to stain the dead bacteria in the biofilm, showing red fluorescence. It can be observed from the figure that the Vehicle group, Ker / Ag +The biofilm of the hydrogel group was dense and showed a monolithic mass, while that of the Ker / Ag + / Rh hydrogel group had a significantly reduced green fluorescence, with live bacteria highly dispersed, a significantly increased red fluorescence, and a large number of bacteria dead ( Figure 6 as shown in I and J in

[0129] is the gene encoding bacterial outer membrane protein A, and outer membrane protein A binds to the host's fibronectin to help Escherichia coli adhere and colonize on the surface of host cells. The polysaccharide adhesion gene is the gene operon (intercellular adhesion operon, ), which affects the formation of biofilm by encoding the polysaccharide adhesin ( ). The fibronectin-binding proteins ( and and ) are also one of the important virulence factors, which can help bacteria adhere together and interact with the surrounding extracellular matrix components to promote the construction and stability of biofilm. To explore the differences in the above biofilm-related virulence factors among the Vehicle group, Ker / Ag + hydrogel group, and Ker / Ag + / Rh hydrogel group, the present invention used RT-qPCR to qualitatively and quantitatively analyze the level changes of the target molecule mRNA. The results showed that the expression levels of + / Rh hydrogel group , , were the lowest, and there were no differences among the groups ( Figure 6 as shown in H in + ). These results indicate that compared with the Vehicle group and Ker / Ag + hydrogel group, the treatment with Ker / Ag and / Rh hydrogel group significantly reduced the adhesion and regeneration of biofilm, forming an anti-biofilm-anti-planktonic bacteria-anti-inflammatory cascade reaction, which accelerated the transition of the wound from the inflammatory phase to the proliferative phase.

[0130] The present invention uses keratin, silver nitrate, and rhein as matrix materials to construct a hydrogel formed by a crosslinking network based on dynamic crosslinking of Ag-S bonds, covalent crosslinking of disulfide bonds, and self-assembly of rhein without the need for chemical group modification or the aid of chemical crosslinking agents. The present invention conducts microscopic morphology, pore size, mechanical properties, rheological properties, compressibility test, injectability test, and self-healing test on the hydrogel. The experimental results show that the crosslinking density of the hydrogel prepared by the present invention gradually decreases, the pore size gradually increases, the mechanical properties gradually decrease, and the fluidity increases with the increase of the anion concentration. And this hydrogel has good self-healing, injectability, and mechanical toughness.

[0131] The results of the in vitro drug release behavior study show that the hydrogel prepared by the present invention has an obvious sustained release effect on the rhein drug. Cell experiments and hemolysis experiments prove that the Ker / Ag + / Rh hydrogel has excellent biocompatibility.

[0132] The results of the antibacterial experiment show that the Ker / Ag + / Rh hydrogel has good antibacterial effects on and . The present invention evaluates the wound healing promotion ability of the hydrogel by establishing a type 2 diabetes wound model. The results show that the Ker / Ag + / Rh hydrogel can accelerate the wound healing process by dispersing the biofilm, inhibiting the regeneration of the biofilm, and inhibiting the growth of planktonic bacteria.

[0133] Example 2

[0134] Preparation of a composite hydrogel of keratin-loaded silver ion and rhein (Ker / Ag+ / Rh hydrogel):

[0135] Dissolve 0.09 g of keratin powder (Ker) in 400 μL of PBS solution (concentration 0.01 mol / L, pH 7.4), add 80 μL of silver nitrate solution with a concentration of 5 mmol / L, vortex to dissolve, add dilute hydrochloric acid with a concentration of 0.1 mol / L until the pH is adjusted to 7.0, add 43.4 μL of rhein solution with a concentration of 6 mg / mL, and let the solution stand at room temperature for 8 h to obtain a composite hydrogel of keratin-loaded silver ion and rhein (Ker / Ag + / Rh hydrogel). In the composite hydrogel of keratin-loaded silver ion and rhein, the concentration of loaded rhein (Rh) is 0.5 mg / mL.

[0136] Example 3

[0137] Preparation of a composite hydrogel of keratin-loaded silver ion and rhein (Ker / Ag+ / Rh hydrogel):

[0138] Dissolve 0.09 g of keratin powder (Ker) in 316.7 μL of PBS solution (concentration: 0.01 mol / L, pH = 7.4). Add 100 μL of silver nitrate solution with a concentration of 5 mmol / L, and vortex to dissolve. Add dilute hydrochloric acid with a concentration of 0.1 mol / L until the pH is adjusted to 7.0. Then add 83.3 μL of rhein solution with a concentration of 6 mg / mL. Let the solution stand at room temperature for 6 h to obtain a composite hydrogel of keratin loaded with silver ions and rhein (Ker / Ag + / Rh hydrogel). In the composite hydrogel of keratin loaded with silver ions and rhein, the concentration of loaded rhein (Rh) is 1 mg / mL.

[0139] Figure 7 For the composite hydrogel dressings of keratin loaded with silver ions and different concentrations of rhein against and the treatment effect diagrams of ulcers in biofilm-infected T2DM mice. The wound healing diagram ( Figure 7 shown as A in Figure 7 ) and the wound area quantification diagram ( Figure 7 shown as B in Figure 7 ) show that compared with the wound surfaces of mice in the urea ointment solvent control group (Vehicle group) and the uninfected urea ointment control group (Normal group), there is a delay in wound healing. The healing rate of the hydrogel dressing treatment group is proportional to the dose of rhein. The dressing loaded with 1.5 mg / mL of rhein in Ker / Ag + exhibits the optimal antibacterial activity in terms of antibacterial and inhibiting the regeneration of biofilm, thus shortening the healing time.

[0140] The above is only a preferred embodiment of the present invention and does not impose any form of limitation on the present invention. Although the present invention has been disclosed above with the preferred embodiment, it is not intended to limit the present invention. Any person skilled in the art can make some changes or modifications to it as equivalent embodiments with equivalent changes within the scope of the technical solution of the present invention. However, as long as it does not depart from the content of the technical solution of the present invention, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A composite hydrogel dressing loaded with keratin and silver ion rhein, characterized in that, It is made of keratin, silver nitrate, and rhein with a mass ratio of 800~1500:1:1~10.

2. A preparation method of the composite hydrogel dressing loaded with silver ions and rhein by keratin according to claim 1, characterized in that, It includes the following steps: Dissolve keratin powder in PBS solution, add silver nitrate solution with a concentration of 1~10 mmol / L, vortex to dissolve, add dilute hydrochloric acid to adjust the pH to 7.0, add rhein solution with a concentration of 1~20 mg / mL. The mass ratio of keratin, silver nitrate, and rhein is 800~1500:1:1~10. Let the solution stand at room temperature for 1~12 h to obtain a composite hydrogel of keratin-loaded silver ion rhein.

3. The preparation method of the composite hydrogel dressing loaded with keratin and silver ion rhein according to claim 2, wherein, The concentration of the PBS solution is 0.001~0.1 mol / L, and the pH is 6~8.

4. The preparation method of the composite hydrogel dressing loaded with keratin and silver ion rhein according to claim 2, characterized in that, The preparation method of the rhein solution includes the following steps: Dissolve rhein in sodium bicarbonate solution, heat in a water bath at 70~90 °C for 5~30 min to obtain a rhein solution with a concentration of 1~20 mg / mL.

5. The preparation method of the composite hydrogel dressing loaded with silver ions and rhein with keratin according to claim 2, wherein, The concentration of the sodium bicarbonate solution is 0.1~1 mol / L, and the pH is 7~9.

6. The preparation method of the composite hydrogel dressing loaded with silver ions and rhein by keratin according to claim 2, characterized in that, The preparation method of the keratin powder includes the following steps: Dissolve urea solution with a concentration of 6~10 mol / L, sodium dodecyl sulfate solution with a concentration of 0.1~0.3 mol / L, and sodium metabisulfite solution with a concentration of 0.1~1 mol / L in deionized water. The mass ratio of urea, sodium dodecyl sulfate, and sodium metabisulfite is 1~20:1:1~5. Heat and stir at 60~80 °C for 1 h, add feathers. The mass ratio of feathers to sodium dodecyl sulfate is 1~5:

1. Treat until there are no lumps of feathers, filter by suction, dialyze for at least three days, concentrate, and freeze-dry step by step to obtain keratin powder.

7. The preparation method of the composite hydrogel dressing loaded with keratin and silver ion rhein according to claim 6, characterized in that The conditions for step-by-step freeze-drying are: freeze at -20 °C for 3 - 4 h, freeze at -80 °C for more than 8 h; The feathers are white raw duck feathers.

8. Use of the composite hydrogel dressing of keratin-loaded silver ion rhein described in claim 1 in the preparation of a wound healing dressing.

9. Use of the composite hydrogel dressing of keratin-loaded silver ion rhein described in claim 1 in the preparation of an antibacterial reagent.

10. The application according to claim 9, wherein, The antibacterial reagent refers to anti-Methicillin-resistant Staphylococcus aureus and Carbapenem-resistant Escherichia coli.

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