Carbon monoxide releasing hydrogel and preparation method and use thereof

Abstract

The application discloses carbon monoxide slow-release hydrogel and a preparation method and application thereof, and belongs to the field of biomaterials. + And a manganese carbonyl; a sulfydryl on the sulfydryl-modified hyaluronic acid is combined with Ag + To form a hydrogel through metal coordination and load the manganese carbonyl as a drug loading platform, the sulfydryl-modified hyaluronic acid is prepared from hyaluronic acid and 3,3'-dithiodi(propionic hydrazine) as raw materials.The preparation method comprises preparing sulfydryl-containing hyaluronic acid and preparing carbon monoxide slow-release hydrogel material.The carbon monoxide slow-release hydrogel material has good plasticity, antibacterial property, self-healing property and antioxidant property, can continuously release carbon monoxide, can effectively inhibit excessive or continuous inflammation, promote new blood vessel formation and promote the repair of diabetic skin wounds, and has good clinical application potential.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a carbon monoxide sustained-release hydrogel, its preparation method, and its uses. Background Technology

[0002] In recent years, the incidence of diabetes has been gradually increasing. It is estimated that by 2025, the number of diabetic patients worldwide will increase from 177 million in 2000 to approximately 300 million. Diabetes is a heterogeneous syndrome that can cause various complications such as heart disease, kidney disease, brain disease, and diabetic ulcers. The high blood sugar and high cholesterol associated with diabetes impair the body's natural healing process, often resulting in wounds characterized by: difficult wound healing, prolonged inflammation, susceptibility to infection, and difficulty in re-epithelialization. 15% of diabetic patients develop lower extremity ulcers, known as diabetic foot ulcers (DFU). Due to reduced blood supply to the lower extremities, these ulcers are prone to necrosis and infection, leading to a lower extremity amputation rate 10-20 times higher than in non-diabetic individuals. Therefore, there is an urgent need for a wound dressing to promote the healing of diabetic wounds.

[0003] Carbon monoxide (CO) is a signal-regulating gas molecule in the body, possessing multiple important biological functions such as anti-inflammation, antibacterial activity, and anti-apoptosis. Low concentrations of carbon monoxide can shorten the inflammatory response period, downregulate the expression of M1 macrophages and upregulate the expression of M2 macrophages during the proliferation phase, aiming to regulate the inflammatory microenvironment at diabetic wound sites. Due to its excellent anti-inflammatory, antibacterial, and cell-protective effects, carbon monoxide can address the problems of frequent inflammation and bacterial infection at diabetic wound sites, while avoiding side effects and drug resistance. However, the supply of carbon monoxide still faces challenges such as poor controllability of release and potential toxicity. Based on the above, to reduce adverse reactions during CO therapy and improve therapeutic efficacy, it is necessary to prepare and develop a novel carbon monoxide sustained-release platform. Hydrogel materials, due to their high water content, plasticity, good biocompatibility, biodegradability, drug loading capacity, and controllable release, can serve as a good platform for sustained-release CO. Mn2(CO) 10 It is a molecular prodrug of carbon monoxide that can undergo a Fenton-like reaction to release low concentrations of carbon monoxide under the stimulation of ROS such as H2O2 and hydroxyl radicals (•OH). Considering the high levels of ROS in difficult-to-heal wounds, Mn2(CO) 10 This provides us with a good option for molecular prodrugs.

[0004] Based on this, this application designs a method for loading Mn2(CO). 10 A carbon monoxide slow-release platform. Summary of the Invention

[0005] The technical problem to be solved by this invention is to overcome the shortcomings of the prior art and provide a carbon monoxide sustained-release hydrogel, its preparation method and uses, in which the molecular prodrug Mn2(CO) of carbon monoxide is loaded onto the hydrogel. 10 This hydrogel, which locally releases sustained-release CO2 to regulate the inflammatory response and angiogenesis of wounds, promotes wound repair. It is injectable, self-healing, and responsive to the wound microenvironment, releasing carbon monoxide in a controlled manner. The preparation method is convenient and produces no harmful byproducts. It can be used not only for healing diabetic wounds but also for tissue regeneration and repair involving the regulation of the immune microenvironment, such as spinal cord repair and bone repair.

[0006] To solve the above-mentioned technical problems, the present invention adopts the following technical solution.

[0007] A carbon monoxide sustained-release hydrogel comprising hyaluronic acid modified with thiol groups and Ag + And carbonyl manganese (Mn2(CO)) 10 The thiol group on the modified hyaluronic acid reacts with Ag. + Hydrogels are formed through metal coordination and used as drug delivery platforms to load the carbonyl manganese.

[0008] Further, in the hydrogel: the concentration of carbonyl manganese is 13 mM-78 mM; the concentration of thiol-modified hyaluronic acid is 25 mM-100 mM; Ag + The concentration is 10mM-100mM.

[0009] Preferably, the method for preparing the thiol-modified hyaluronic acid is as follows: 1) Dissolve sodium hyaluronate in water to prepare a sodium hyaluronate solution, then add 3,3'-dithiodi(hydrazine propionate) to obtain a mixed solution; 2) Dissolve 1-hydroxybenzotriazole in a mixture of acetonitrile and water, and then add it to the mixed solution obtained in step 1); 3) Adjust the pH of the solution obtained in step 2) to 3.5-6, add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and stir magnetically for 6-24 hours at a temperature of 15-40℃; 4) Add dithiothreitol to the solution obtained in 3) and adjust the pH value to 8-9. Stir magnetically for 6-24 hours at a temperature of 15-40℃. 5) Dialyze the solution obtained in step 4) to remove unreacted reagents; 6) Pre-freeze the solution obtained in step 5) at -80℃, and then transfer it to a vacuum freeze dryer for freeze drying to obtain hyaluronic acid modified with thiol groups.

[0010] Preferably, the mass ratio of sodium hyaluronate, 3,3'-dithiobis(propionylhydrazine), 1-hydroxybenzotriazole, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and dithiothreitol is 2-10:0.1-0.5:1-3:0.5-5:0.5-5.

[0011] Preferably, in step 5), the dialysis is performed using a dialysis bag, the dialysis bag retains molecules of 3500 Da, the number of dialysis cycles is 3-6, and the dialysis temperature is 4-25℃.

[0012] Preferably, in step 6), the freeze-drying time is 1 to 5 days and the freeze-drying temperature is -50°C.

[0013] As a general technical concept, the present invention also provides a method for preparing the above-mentioned carbon monoxide sustained-release hydrogel, comprising the following steps: 1) Disperse the carbonyl manganese powder evenly in water; 2) Dissolve the thiol-modified hyaluronic acid in the mixed solution obtained in step 1), and adjust the pH to neutral; 3) Add Ag to the solution obtained in 2). + The solution was rapidly mixed to obtain a carbon monoxide slow-release hydrogel.

[0014] Preferably, in step 2), the pH adjustment is achieved by using a NaOH solution with a concentration of 0.1-2 mol / L.

[0015] Preferably, the Ag + The solution is a silver nitrate solution.

[0016] Preferably, the pH adjustment is achieved by using a NaOH solution with a concentration of 0.1-2 mol / L.

[0017] As a general technical concept, the present invention also provides the use of the above-mentioned carbon monoxide sustained-release hydrogel as a novel wound dressing in the field of medical technology.

[0018] Compared with the prior art, the advantages of the present invention are as follows: The present invention provides a carbon monoxide sustained-release hydrogel loaded with carbonyl manganese. The hydrogel is cross-linked through metal coordination and used for wound healing treatment.

[0019] (1) Carbon monoxide slow-release hydrogel (CO@HAG) has good self-healing, injectability and plasticity.

[0020] (2) CO@HAG has excellent broad-spectrum antibacterial activity and good antibacterial properties against both Gram-positive and Gram-negative bacteria.

[0021] (3) CO@HAG is ROS responsive and can release CO in the presence of H2O2.

[0022] (4) CO@HAG can promote rapid healing of diabetic wounds, promote granulation tissue formation, promote collagen deposition, and promote re-epithelialization.

[0023] (5) CO@HAG can reduce the inflammatory response, accelerate the transition of traumatic tissue from the inflammatory phase to the proliferative phase, and may also have the ability to promote polarization to M2 phenotype macrophages.

[0024] (6) CO@HAG can promote the regeneration of new blood vessels in regenerating tissues and accelerate the maturation of functional blood vessels.

[0025] (7) CO@HAG has enriched the treatment methods for difficult-to-heal wounds in clinical applications and broadened its application prospects. Attached Figure Description

[0026] Figure 1 These are scanning electron microscope (SEM) images of CO@HAG prepared in Example 1 of this invention and HAG prepared in Comparative Example 2.

[0027] Figure 2 This is a rheological data graph of HA and AgNO3 mixed in Comparative Example 1 of the present invention under frequency scanning.

[0028] Figure 3 The rheological data of CO@HAG prepared in Example 1 and HAG prepared in Comparative Example 2 under frequency scanning are shown.

[0029] Figure 4 This is a rheological data graph of CO@HAG under simulated injection state obtained in Example 1 of the present invention under time scanning.

[0030] Figure 5 Visual representation of the self-healing properties (A) and plasticity (B) of carbon monoxide slow-release hydrogel (CO@HAG).

[0031] Figure 6 The image shows the antibacterial results of carbon monoxide slow-release hydrogel (CO@HAG) against Escherichia coli and Staphylococcus aureus. In this image, A shows a photograph of bacterial colonies; B shows a comparison of antibacterial rates.

[0032] Figure 7 This is a diagram showing the in vitro CO release results of carbon monoxide slow-release hydrogel (CO@HAG) in the presence of H2O2.

[0033] Figure 8 Representative images (A) and wound closure rates (B) at different time points during the healing process of diabetic chronic wounds treated with different methods.

[0034] Figure 9 Expression of M1 macrophage-associated protein (iNOS) and M2 macrophage-associated protein (CD206) after different treatments for diabetic chronic wounds.

[0035] Among them, A. iNOS staining of regenerated wound tissue on days 3 and 7; B. Quantitative analysis of iNOS; C. CD206 staining of regenerated wound tissue on days 3 and 7; D. Quantitative analysis of CD206; Scale bar: 50 μm.

[0036] Figure 10 The expression of vascular-associated protein (CD31) after different treatment methods for diabetic chronic wounds.

[0037] Among them, A, CD31 staining of regenerated wound tissue on days 10 and 16 (positive signal: green); B, quantitative analysis of CD31; scale bar: 50 μm. Detailed Implementation

[0038] The present invention will be further described below with reference to the accompanying drawings and specific preferred embodiments, but this does not limit the scope of protection of the present invention. All materials and instruments used in the following embodiments are commercially available. Example 1

[0039] This embodiment provides a carbon monoxide sustained-release hydrogel, comprising hyaluronic acid modified with thiol groups and Ag. + and carbonyl manganese; the thiol group on the thiol-modified hyaluronic acid and Ag + Hydrogels are formed through metal coordination and used as drug delivery platforms to load the carbonyl manganese.

[0040] In the hydrogel, the concentration of manganese carbonyl is 45 mM; the concentration of hyaluronic acid modified with thiol groups is 70 mM; Ag + The concentration is 60 mM.

[0041] Its preparation method is as follows: 1. Preparation of hyaluronic acid modified with thiol groups: 1) Dissolve 400 mg of sodium hyaluronate in water to prepare a sodium hyaluronate solution, then add 16.7 mg of 3,3'-dithiodi(hydrazine propionate) to obtain a mixed solution; 2) Dissolve 135 mg of 1-hydroxybenzotriazole in a mixture of 1.5 mL of acetonitrile and 1.5 mL of deionized water, and then add it to the mixed solution obtained in step 1).

[0042] 3) Adjust the pH of the solution obtained in step 2) to 4.7, add 57.5 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and stir magnetically for 10 h at room temperature; 4) Add 54.5 mg of dithiothreitol to the solution obtained in 3) to adjust the pH to 8, and stir magnetically for 10 h at room temperature; 5) Place the solution obtained in step 4) into a 3500 Da dialysis bag and dialyze it 4 times to remove unreacted reagents; 6) Place the solution obtained in step 5) in a -80℃ freezer for pre-freezing, and then transfer it to a vacuum freeze dryer for freeze drying to obtain hyaluronic acid modified with thiol groups.

[0043] 2. Preparation of carbon monoxide sustained-release hydrogel (CO@HAG): 1) Disperse the carbonyl manganese powder evenly in water.

[0044] 2) Dissolve the hyaluronic acid modified with thiol groups in the mixed solution obtained in step 1).

[0045] 3) Adjust the pH of the solution obtained in step 2) to neutral using 1 mol / L NaOH solution.

[0046] 4) Add 1.5M AgNO3 solution to the solution obtained in 3), mix quickly to obtain a carbon monoxide slow-release hydrogel. Example 2

[0047] This embodiment provides a carbon monoxide sustained-release hydrogel, comprising hyaluronic acid modified with thiol groups and Ag. + and carbonyl manganese; the thiol group on the thiol-modified hyaluronic acid and Ag + Hydrogels are formed through metal coordination and used as drug delivery platforms to load the carbonyl manganese.

[0048] In the hydrogel, the concentration of manganese carbonyl is 13 mM; the concentration of hyaluronic acid modified with thiol groups is 25 mM; Ag... + The concentration is 10 mM.

[0049] Its preparation method is as follows: 1. Preparation of hyaluronic acid modified with thiol groups: 1) Dissolve 400 mg of sodium hyaluronate in water to prepare a sodium hyaluronate solution, then add 16.7 mg of 3,3'-dithiodi(hydrazine propionate) to obtain a mixed solution; 2) Dissolve 135 mg of 1-hydroxybenzotriazole in a mixture of 1.5 mL of acetonitrile and 1.5 mL of deionized water, and then add it to the mixed solution obtained in step 1).

[0050] 3) Adjust the pH of the solution obtained in step 2) to 4.7, add 57.5 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and stir magnetically for 10 h at room temperature; 4) Add 54.5 mg of dithiothreitol to the solution obtained in 3) to adjust the pH to 8, and stir magnetically for 10 h at room temperature; 5) Place the solution obtained in step 4) into a 3500 Da dialysis bag and dialyze it 4 times to remove unreacted reagents; 6) Place the solution obtained in step 5) in a freezer at -80°C for pre-freezing, and then transfer it to a vacuum freeze dryer for freeze drying to obtain hyaluronic acid modified with thiol groups.

[0051] 2. Preparation of carbon monoxide sustained-release hydrogel (CO@HAG): 1) Disperse the carbonyl manganese powder evenly in water.

[0052] 2) Dissolve the hyaluronic acid modified with thiol groups in the mixed solution obtained in step 1).

[0053] 3) Adjust the pH of the solution obtained in step 2) to neutral using 1 mol / L NaOH solution.

[0054] 4) Add 1.5M AgNO3 solution to the solution obtained in 3), mix quickly to obtain a carbon monoxide slow-release hydrogel. Example 3

[0055] This embodiment provides a carbon monoxide sustained-release hydrogel, comprising hyaluronic acid modified with thiol groups and Ag. + and carbonyl manganese; the thiol group on the thiol-modified hyaluronic acid and Ag + Hydrogels are formed through metal coordination and used as drug delivery platforms to load the carbonyl manganese.

[0056] The hydrogel contains: manganese carbonyl at a concentration of 78 mM; hyaluronic acid modified with thiol groups at a concentration of 100 mM; and Ag. + Concentration of 100mM Its preparation method is as follows: 1. Preparation of hyaluronic acid modified with thiol groups: 1) Dissolve 400 mg of sodium hyaluronate in water to prepare a sodium hyaluronate solution, then add 16.7 mg of 3,3'-dithiodi(hydrazine propionate) to obtain a mixed solution; 2) Dissolve 135 mg of 1-hydroxybenzotriazole in a mixture of 1.5 mL of acetonitrile and 1.5 mL of deionized water, and then add it to the mixed solution obtained in step 1).

[0057] 3) Adjust the pH of the solution obtained in step 2) to 4.7, add 57.5 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and stir magnetically for 10 h at room temperature; 4) Add 54.5 mg of dithiothreitol to the solution obtained in 3) to adjust the pH to 8, and stir magnetically for 10 h at room temperature; 5) Place the solution obtained in step 4) into a 3500 Da dialysis bag and dialyze it 4 times to remove unreacted reagents; 6) Place the solution obtained in step 5) in a freezer at -80°C for pre-freezing, and then transfer it to a vacuum freeze dryer for freeze drying to obtain hyaluronic acid modified with thiol groups.

[0058] 2. Preparation of carbon monoxide sustained-release hydrogel (CO@HAG): 1) Disperse the carbonyl manganese powder evenly in water.

[0059] 2) Dissolve the hyaluronic acid modified with thiol groups in the mixed solution obtained in step 1).

[0060] 3) Adjust the pH of the solution obtained in step 2) to neutral using 1 mol / L NaOH solution.

[0061] 4) Add 1.5M AgNO3 solution to the solution obtained in 3), mix quickly to obtain a carbon monoxide slow-release hydrogel. Comparative Example 1

[0062] 40 mg of hyaluronic acid was dissolved in 990 μL of water to obtain a hyaluronic acid solution (HA); 10 μL of 1.5 mg NO3 solution was added to the hyaluronic acid solution to obtain a mixed solution. Comparative Example 2

[0063] Preparation of metal coordination hydrogels (HAG): 1) Dissolve hyaluronic acid modified with thiol groups in water.

[0064] 2) Adjust the pH of the solution obtained in step 1) to neutral using 1 mol / L NaOH solution.

[0065] 3) Add 1.5M AgNO3 solution to the solution obtained in 2), mix quickly to obtain a metal coordination hydrogel. Performance testing

[0066] 1. Appearance Figure 1The images show scanning electron microscope (SEM) images of CO@HAG prepared in Example 1 and HAG prepared in Comparative Example 2. As can be seen from the images, both the freeze-dried HAG and CO@HAG exhibit a continuous porous structure, but only the inner wall of CO@HAG has a large amount of particulate manganese carbonyl attached to it.

[0067] 2. Rheology The changes in storage modulus (G') and loss modulus (G") of HAG and CO@HAG after gelation for 24 hours were tested under both frequency and time scanning modes. During the tests, the time-scan strain alternated between 1% and 100% at a constant frequency of 1 Hz; the frequency scan ranged from 1 to 100 rad / s, with a fixed strain of 0.5%. All rheological tests were conducted at 25°C using a 15 mm diameter rotor.

[0068] Figure 2 This is a rheological data graph of HA and AgNO3 mixed in Comparative Example 1 under frequency scanning. Figure 3 The rheological data of CO@HAG prepared in Example 1 and HAG prepared in Comparative Example 2 under frequency scanning are shown. Figure 4 This is a rheological data graph of CO@HAG prepared in the simulated injection state under time scanning, as shown in Example 1 of the present invention. Figure 2 , 3 As shown in section 4, HA without thiol modification does not exhibit gel properties when mixed with AgNO3, while the storage modulus of the mixture of HA-SH and AgNO3 is much greater than the loss modulus, indicating a hydrogel. Furthermore, rheological analysis under simulated injection conditions demonstrates its excellent injectability. This result proves that the thiol groups in thiol-modified hyaluronic acid act as cross-linking points in the hydrogel network, and the coordination between silver ions and thiol groups is one of the key factors in material preparation.

[0069] 3. Self-healing and plasticity The self-healing and plasticity properties of the CO@HAG hydrogel material of the present invention were investigated, specifically using the CO@HAG prepared in Example 1, including the following steps: The prepared cylindrical CO@HAG hydrogel was cut in half with a blade, then the two halves were placed together. After a period of time, the two pieces of hydrogel were observed to see if they had healed into one. Alternatively, the two prepared cylindrical hydrogels were crushed with a blade and placed into a rectangular / pentagonal mold. After a period of time, the shape of the hydrogel was observed to verify its plasticity.

[0070] Figure 5This document presents the self-healing and plasticity results of the CO@HAG hydrogel material prepared in Example 1 of this invention. The results show that when two halves of CO@HAG are gently placed together, after a period of time, the two separate hydrogels heal to form a single, continuous hydrogel, demonstrating the self-healing properties of the CO@HAG hydrogel material. Both hydrogels can still be reshaped after being crushed with a blade, transforming from a cylindrical shape into a five-pointed star shape, proving the hydrogel's good plasticity and providing possibilities for its application to wounds of different shapes.

[0071] 4. Antibacterial properties The antibacterial properties of the CO@HAG hydrogel material of the present invention were investigated, specifically using the CO@HAG prepared in Example 1 and the HAG prepared in Comparative Example 2, including the following steps: The antibacterial properties of the blank control group, HAG hydrogel, and CO@HAG hydrogel were evaluated using Escherichia coli (ATCC 25922, Gram-negative strain) and Staphylococcus aureus (ATCC 25923, Gram-positive strain), respectively, by colony counting. 1 mL of diluted bacterial suspension was co-cultured with HAG hydrogel and CO@HAG hydrogel in 24-well plates for 12 hours at 37°C and 120 rpm. After 12 hours, the bacterial suspensions were diluted and evenly spread onto plates. The plates were incubated at 37°C for 12 hours. After 12 hours, photographs were taken, colonies were counted, and the inhibition rate of each sample was calculated.

[0072] Figure 6 The results show the antibacterial effects of CO@HAG prepared in Example 1 and HAG prepared in Comparative Example 2. The results indicate that the colonies on the blank control group culture dishes were relatively dense, with approximately 298 E. coli and 313 S. aureus colonies, respectively. In contrast, almost no colonies were present on the HAG and CO@HAG hydrogel culture dishes. Quantitative testing revealed that the inhibition rates of both hydrogels against E. coli and S. aureus were 99.31% and 98.23%, and 100% and 100%, respectively.

[0073] 5. CO emission performance The CO@HAG hydrogel of the present invention was investigated for its responsive CO release. Specifically, the CO@HAG prepared in Example 1 and the HAG prepared in Comparative Example 2 were used, and the following steps were included: Hemoglobin solutions containing different concentrations of H2O2 were placed in glass cuvettes with HAG or CO@HAG, respectively. The UV absorption curves of HAG and CO@HAG hydrogels at 350 nm-650 nm were measured using a UV spectrophotometer at 0, 100, 200, 300, 400, and 500 min. Based on the UV absorption curves under different H2O2 concentrations, the characteristic absorption peaks at 410 nm and 430 nm were obtained. The CO release rates of HAG and CO@HAG hydrogels under different conditions and time were calculated.

[0074] Figure 7 This paper presents the CO release from the CO@HAG hydrogel prepared in Example 1 of this invention under different concentrations of H2O2 solutions. The results show that as the H2O2 concentration decreases, the amount of CO released also decreases. The highest CO release values ​​in Hb buffer solutions containing 5 μM, 3.75 μM, 2.5 μM, and 0.05 μM H2O2 were 6.50 μM, 5.80 μM, 4.60 μM, and 0.29 μM, respectively, indicating that the CO release is dependent on the H2O2 concentration. However, the CO release from the HAG hydrogel in the Hb buffer solution containing 5 μM H2O2 was essentially zero. All these results indicate that Mn2(CO)... 10 It can indeed react with H2O2 in the presence of H2O2 and release CO.

[0075] 6. Promotes wound healing Taking diabetic wound repair as an example, the in vivo wound healing effect of the CO@HAG hydrogel of the present invention was investigated. Specifically, the CO@HAG prepared in Example 1 and the HAG prepared in Comparative Example 2 were used, including the following steps: Male SD rats weighing approximately 200 g were fasted for more than 12 hours (overnight fasting with free access to water) and injected with the appropriate dose of STZ (65 mg / kg) according to their fasting body weight to induce the model. Three days after the injection, tail vein blood was collected, and the changes in blood glucose levels of the rats were monitored and recorded using a portable blood glucose meter. If the blood glucose level was equal to or higher than 16.7 mmol / L and remained so for more than 7 days, the model of type 1 diabetes could be considered successfully established.

[0076] The successfully modeled diabetic SD rats were randomly divided into 4 groups of 6 rats each. Three circular full-thickness skin defects with a diameter of about 1.5 cm were made vertically at the midline of the spine. The defect sites were treated with CO@HAG hydrogel and HAG hydrogel, respectively, while the remaining wound was left untreated (blank control group). The wound healing was observed and photographed on days 0, 3, 7, 10 and 16, and samples were taken for relevant index detection.

[0077] Figure 8The results show the in vivo wound healing promotion effects of CO@HAG prepared in Example 1 and HAG prepared in Comparative Example 2. The results indicate that, throughout the treatment process, HAG hydrogel promoted wound healing compared to the blank control group; however, CO@HAG hydrogel exhibited the best wound healing promotion effect compared to CO@HAG hydrogel. This demonstrates that CO released from manganese carbonyl has a synergistic effect with HAG in promoting wound healing. Figure 9 The results of immunofluorescence analysis of the in vivo inflammatory regulation of CO@HAG prepared in Example 1 and HAG prepared in Comparative Example 2 were presented. The results showed that on day 3, most macrophages in the blank control group and the HAG group maintained the M1 phenotype (iNOS). The CO@HAG group had fewer M1 macrophages in the wound tissue and a large number of M2 phenotype macrophages (CD206). Although the other two groups also showed a small amount of CD206 expression, the intensity and relative area were much lower than in the CO@HAG group, indicating that CO@HAG can promote CD206 expression and inhibit iNOS expression. Figure 10 The results of immunofluorescence assay were used to investigate the in vivo angiogenesis regulation of CO@HAG prepared in Example 1 and HAG prepared in Comparative Example 2. The results showed that on days 10 and 16 post-treatment, almost no fluorescence was observed in the blank control group, while fluorescence signals were observed in both hydrogel groups. However, the CO@HAG group showed a significant increase in positive areas (green) in the wound tissue, with the highest CD31 expression level, indicating that CO@HAG significantly promoted angiogenesis.

[0078] It should be noted that the CO@HAG prepared in the other embodiments of the present invention has the same or equivalent performance as the CO@HAG prepared in Example 1.

[0079] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make many possible variations and modifications to the technical solutions of the present invention using the methods and techniques disclosed above, or modify them into equivalent embodiments with equivalent changes, without departing from the spirit and technical essence of the present invention. Therefore, any simple modifications, equivalent substitutions, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solutions of the present invention shall still fall within the protection scope of the technical solutions of the present invention.

Claims

1. A carbon monoxide sustained-release hydrogel, characterized in that, Including hyaluronic acid modified with thiol groups, Ag + and carbonyl manganese; the thiol group on the thiol-modified hyaluronic acid and Ag + Hydrogels are formed through metal coordination and used as drug delivery platforms to load the carbonyl manganese. Its preparation method includes the following steps: 1) Disperse manganese carbonyl powder uniformly in water; the chemical formula of the manganese carbonyl is Mn2(CO). 10 ; 2) Dissolve the thiol-modified hyaluronic acid in the mixed solution obtained in step 1); 3) Adjust the pH of the solution obtained in step 2) to neutral; 4) Add Ag to the solution obtained in 3). + The solution was quickly mixed to obtain a carbon monoxide slow-release hydrogel. The concentration of the carbonyl manganese was 13 mM-78 mM; the concentration of the thiol-modified hyaluronic acid was 25 mM-100 mM; Ag + Concentrations range from 10 mM to 100 mM; The method for preparing the thiol-modified hyaluronic acid is as follows: 1) Dissolve sodium hyaluronate in water to prepare a sodium hyaluronate solution, then add 3,3'-dithiodi(hydrazine propionate) to obtain a mixed solution; 2) Dissolve 1-hydroxybenzotriazole in a mixture of acetonitrile and water, and then add it to the mixed solution obtained in step 1); 3) Adjust the pH of the solution obtained in step 2) to 3.5-6, add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and stir magnetically for 6-24 hours at a temperature of 15-40℃; 4) Add dithiothreitol to the solution obtained in 3) and adjust the pH value to 8-9. Stir magnetically for 6-24 hours at a temperature of 15-40℃. 5) Dialyze the solution obtained in step 4) to remove unreacted reagents; 6) Pre-freeze the solution obtained in step 5) at -80℃, and then transfer it to a vacuum freeze dryer for freeze drying to obtain hyaluronic acid modified with thiol groups.

2. The carbon monoxide sustained-release hydrogel according to claim 1, characterized in that, The mass ratio of sodium hyaluronate, 3,3'-dithiobis(propionylhydrazine), 1-hydroxybenzotriazole, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and dithiothreitol is 2-10:0.1-0.5:1-3:0.5-5:0.5-5.

3. The carbon monoxide sustained-release hydrogel according to claim 1, characterized in that, The dialysis described in step 5) is performed using a dialysis bag, which retains molecules up to 3500 Da, and the number of dialysis cycles is 3-6; the dialysis temperature is 4-25℃.

4. The carbon monoxide sustained-release hydrogel according to claim 1, characterized in that, The freeze-drying time in step 6) is 1 to 5 days, and the freeze-drying temperature is -10 to -80℃.

5. The carbon monoxide sustained-release hydrogel according to claim 1, characterized in that, The Ag + The solution is a silver nitrate solution.

6. The carbon monoxide slow-release hydrogel according to claim 1, wherein the pH adjustment is achieved by using a NaOH solution with a concentration of 0.1-2 mol / L.

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