A Triple-Responsive Hydrogel, Its Preparation Method and Application

Abstract

This invention provides a triple-responsive hydrogel, which is prepared by mixing the following raw materials: FeCO-PBA, tannic acid, carboxymethyl chitosan, terephthalic acid, and a first solvent; wherein, the FeCO-PBA is Fe3(CO). 12 The invention relates to a complex formed by a metal coordination reaction between 4-carboxyphenylboronic acid-SH and iron-containing carbonyl units and phenylboronic acid groups, wherein the iron-carbonyl units and phenylboronic acid groups are linked by iron-sulfur bonds; 4-carboxyphenylboronic acid-SH is a thiol-containing 4-carboxyphenylboronic acid derivative formed by the amide bond linking 4-carboxyphenylboronic acid and mercaptoethylamine. In the hydrogel of this invention, FeCO-PBA can release CO gas in response to ROS, and the dynamic covalent bonds of catechol-boronic acid esters can rapidly break down in response to high glucose, high ROS, and slightly acidic environments, promoting hydrogel degradation, thus forming a triple response mechanism targeting the microenvironment of diabetic wounds. This invention improves the safety of CO gas molecule use and maximizes therapeutic efficacy through intelligent targeting and on-demand drug delivery.

Description

Technical Field

[0001] This invention belongs to the field of medical hydrogel dressing technology, specifically relating to a triple-responsive hydrogel, its preparation method, and its application. Background Technology

[0002] Diabetic wounds, due to impaired blood flow and insufficient angiogenesis, are chronically hypoxic, resulting in reactive oxygen species (ROS) levels far exceeding those of physiological wound environments. Excessive ROS disrupts homeostasis, exacerbating oxidative stress and inflammatory dysregulation. In this environment, macrophages in the wound remain in a pro-inflammatory M1 state, unable to transition to a reparative M2 state. They continuously secrete inflammatory factors, damaging new tissue and hindering skin remodeling and re-epithelialization. The abundant carbon source in the hyperglycemic environment also provides fertile ground for bacterial growth. These bacteria form microbial clusters at the wound site and colonize the tissue, secreting large amounts of extracellular aggregates to form a dense biofilm. Hyperglycemia also weakens the chemotaxis and phagocytosis of leukocytes, making it difficult for the immune system to effectively clear bacteria. Furthermore, long-term hyperglycemia damages vascular endothelial cells, increasing vascular fragility and decreasing platelet function. Toxins and inflammatory factors released by bacteria also significantly disrupt vascular integrity, leading to recurrent bleeding. Therefore, developing novel, multidimensional, antibacterial, anti-inflammatory, antioxidant, and hemostatic dressings that promote healing is of great significance for the treatment of chronic diabetic wounds.

[0003] Hydrogels, with their porous structure and high similarity to human tissue, can serve as drug delivery carriers, enabling the sustained release of therapeutic molecules. Current research primarily utilizes hydrogels to deliver antibacterial or anti-inflammatory drugs to improve diabetic wound healing, but it overlooks three significant limitations. First, antibacterial resistance is an increasingly prominent issue. With the increasing overuse of antibiotics, the emergence of ESKAPE-resistant bacteria has significantly reduced the efficacy of traditional antibiotics. Second, biofilm penetration is insufficient. The physical barriers and chemical defense mechanisms formed by biofilms greatly hinder the penetration of antibacterial drugs. Third, simple bactericidal strategies ignore the complex wound microenvironment. Diabetic wound healing requires not only infection control but also the comprehensive regulation of multiple parallel processes such as inflammation, oxidative stress, angiogenesis, and ECM remodeling. Simple bactericidal action may even exacerbate the inflammatory response due to the release of endotoxins from the lysis of large numbers of bacteria.

[0004] The introduction of gas therapy has opened up new avenues for the treatment of diabetic wounds. CO, as an endogenous gaseous signaling molecule, possesses multiple therapeutic effects, including antibacterial, anti-inflammatory, and antioxidant properties, under certain physiological and pathological conditions. First, CO can bind to bacterial terminal oxidases, inhibiting bacterial respiration by blocking the electron transport chain. Compared to antibiotics, CO, as a small molecule, is more easily diffused and penetrates bacterial biofilms, promoting biofilm degradation. Due to the diverse targets of CO, bacteria rarely develop resistance through single gene mutations. Second, CO, with its immunomodulatory function, can block the NF-κB pathway, regulate the MAPK pathway, reduce the release of pro-inflammatory factors, and simultaneously activate the heme oxygenase-1 (HO-1) and cGMP-PKG pathways. Through its protective effect against oxidative stress, it maintains cell integrity and tissue homeostasis, improving wound microcirculation. CO can also help reprogram macrophages by inhibiting the expression of pro-inflammatory M1 macrophage markers, inducing them to polarize towards the anti-inflammatory M2 type, and promoting tissue repair and regeneration. However, the short-acting nature of CO as a gas molecule, its high concentration of toxicity, and the challenges of controlled release make its clinical translation still very challenging. At the same time, a single responsive drug delivery mode cannot precisely target the lesion area to ensure the safety of normal tissues.

[0005] Therefore, there is an urgent need to develop a smart, responsive CO2 injectable hydrogel dressing to solve the above problems. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a triple-responsive hydrogel that addresses the shortcomings of the prior art.

[0007] Another technical problem to be solved by the present invention is to provide a method for preparing the triple responsive hydrogel.

[0008] The final technical problem to be solved by the present invention is to provide the application of the triple-responsive hydrogel in the treatment of chronic wounds in diabetes.

[0009] To solve the above-mentioned technical problems, the technical solution provided by the present invention is as follows:

[0010] The first aspect of the present invention provides a triple-responsive hydrogel (FeCO@CS / TA hydrogel), said hydrogel being prepared by mixing the following raw materials: FeCO-PBA, tannic acid (TA), carboxymethyl chitosan (CMCS), terephthalic acid (BDBA) and a first solvent;

[0011] Wherein, FeCO-PBA is Fe3(CO). 12A complex formed by metal coordination reaction with 4-carboxyphenylboronic acid-SH, wherein an iron carbonyl unit and a phenylboronic acid group are linked by an iron-sulfur bond; wherein the 4-carboxyphenylboronic acid-SH is a thiol-containing 4-carboxyphenylboronic acid derivative formed by the amide bond linking 4-carboxyphenylboronic acid and mercaptoethylamine.

[0012] The first solvent is a mixture of ethanol and water.

[0013] The preparation method of the 4-carboxyphenylboronic acid-SH includes the following steps:

[0014] Step 1: 4-Carboxyphenylboronic acid is activated by carboxyl group. The activated 4-carboxyphenylboronic acid undergoes an amidation reaction with cystamine dihydrochloride to obtain the first reaction solution. The solution is then subjected to the first separation and purification to obtain cystamine-phenylboronic acid monoamide.

[0015] Step 2: The cystamine-phenylboronic acid monoamide obtained in Step 1 is reduced with sodium borohydride to obtain a second reaction solution, which is then subjected to a second separation and purification to obtain the final product.

[0016] In step 1, the carboxyl activation of the 4-carboxyphenylboronic acid is carried out under the action of 1-hydroxybenzotriazole (HOBt) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI); the mass ratio of the 4-carboxyphenylboronic acid, 1-hydroxybenzotriazole, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 376~400 : 380~400 : 540~560; both the carboxyl activation and amidation reactions are carried out in the solvent N,N-dimethylformamide (DMF); the carboxyl activation is carried out with stirring at 0~4°C for 10~30 min; during the amidation reaction, triethylamine is used to neutralize the hydrochloric acid in cystamine dihydrochloride; the volume ratio of the triethylamine used to the mass of the cystamine dihydrochloride is 1.25~1.40 mL : 260~270 mL. mg; the mass ratio of cystamine dihydrochloride to 4-carboxyphenylboronic acid is 260~270 : 376~400; the amidation reaction is carried out by stirring at room temperature for 5~7 h.

[0017] In some embodiments, in step 1, the mass ratio of 4-carboxyphenylboronic acid, 1-hydroxybenzotriazole and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 388:380:540.

[0018] In some embodiments, in step 1, the ratio of the volume of triethylamine used to the mass of cystamine dihydrochloride is 1.27 mL : 262 mg.

[0019] In some embodiments, in step 1, the mass ratio of cystamine dihydrochloride to 4-carboxyphenylboronic acid is 262:388.

[0020] In some embodiments, in step 1, there are no special requirements for the amount of N,N-dimethylformamide used as the solvent in the carboxyl activation and amidation reactions; it is sufficient to dissolve and / or disperse the raw materials evenly.

[0021] In some embodiments, step 1, the first separation and purification method includes the following steps: adding water to the first reaction solution to adjust the pH to 6 to 7.4, performing solid-liquid separation, collecting the solid (i.e., cystamine-phenylboronic acid monoamide), washing it with water, and so on.

[0022] In step 2, the molar ratio of sodium borohydride to cystamine-phenylboronic acid monoamide is 10-15:1; the reduction reaction is carried out in methanol solvent; and the reduction reaction is carried out at room temperature for 1-2 hours.

[0023] In some embodiments, in step 2, the molar ratio of sodium borohydride to cystamine-phenylboronic acid monoamide is 13:1.

[0024] In some embodiments, in step 2, there are no special requirements for the amount of methanol used as a solvent in the reduction reaction; it is sufficient to dissolve and / or disperse the raw materials evenly.

[0025] In some embodiments, step 2, the second separation and purification method includes the following steps: purifying the second reaction solution by column chromatography to obtain the 4-carboxyphenylboronic acid-SH.

[0026] The preparation method of FeCO-PBA includes the following steps:

[0027] Fe3(CO) 12 The 4-carboxyphenylboronic acid-SH is dissolved in a second solvent to carry out a metal coordination reaction, resulting in a third reaction solution. After a third separation and purification, the final product is obtained.

[0028] Among them, Fe3(CO) 12 The mass ratio of the metal to 4-carboxyphenylboronic acid-SH is 120~140 : 220~280; the second solvent is tetrahydrofuran; the metal coordination reaction is carried out under reflux at 80°C with stirring for 4 h.

[0029] In some embodiments, the Fe3(CO) 12 The mass ratio of 4-carboxyphenylboronic acid-SH to 4-carboxyphenylboronic acid-SH is 125:225.

[0030] In some embodiments, the amount of the second solvent is not particularly required; it is sufficient to dissolve and / or disperse the raw materials evenly.

[0031] In some embodiments, the third separation and purification includes the following steps: purifying the third reaction solution by column chromatography to obtain the FeCO-PBA.

[0032] A second aspect of this invention provides a method for preparing the aforementioned triple-responsive hydrogel, comprising the following steps:

[0033] The FeCO-PBA is dissolved in a third solvent to obtain a FeCO-PBA solution, which is then mixed evenly with a tannic acid solution, a terephthalic acid solution, and a hydroxymethyl chitosan solution to obtain the final product.

[0034] The method for mixing the FeCO-PBA solution with the tannic acid solution, the terephthalic acid solution, and the hydroxymethyl chitosan solution is as follows: the FeCO-PBA solution and the tannic acid solution are mixed evenly to obtain the FeCO-PBA / TA mixture, and then the mixture is mixed evenly with the terephthalic acid and hydroxymethyl chitosan mixture.

[0035] Wherein, the third solvent is ethanol; the solvent for the tannic acid solution is water; the solvent for the mixed solution of terephthalic acid and hydroxymethyl chitosan is water; the concentration of the FeCO-PBA solution is 8-10 mg / mL; the concentration of the tannic acid solution is 20-25 mg / mL; the concentrations of terephthalic acid and hydroxymethyl chitosan in the mixed solution of terephthalic acid and hydroxymethyl chitosan are 1.95-2.3 mg / mL and 60-70 mg / mL, respectively; the volume ratio of the FeCO-PBA solution to the tannic acid solution is 1-1.2:9-10; and the volume ratio of the FeCO-PBA / TA mixture to the mixed solution of terephthalic acid and hydroxymethyl chitosan is 1:1.

[0036] In some embodiments, the concentration of the FeCO-PBA solution is 8 mg / mL; the concentration of the tannic acid solution is 20 mg / mL; the concentrations of phenylboronic acid and hydroxymethyl chitosan in the mixed solution of terephthalic acid and hydroxymethyl chitosan are 1.95 mg / mL and 60 mg / mL, respectively; and the volume ratio of the FeCO-PBA solution to the tannic acid solution is 1:9.

[0037] A third aspect of the present invention provides the application of the aforementioned triple-responsive hydrogel in the treatment of chronic wounds in diabetes.

[0038] In the triple-responsive hydrogel provided by this invention, tannic acid and carboxymethyl chitosan form a hydrogel framework through hydrogen bonding and electrostatic interactions. Terephthaloboric acid acts as a "molecular glue," with its boric acid functional groups forming dynamic covalent bonds with the polyphenolic structure of tannic acid—a catechol-boronate ester. This enhances the mechanical stability of the hydrogel and endows the system with triple responsiveness to high ROS, high glucose, and slightly acidic environments. Since diabetic infectious wounds are typically accompanied by elevated ROS levels, high glucose, and an acidic microenvironment, the dynamic covalent bonds of the catechol-boronate ester can selectively break at the lesion site, thereby achieving precise degradation of the hydrogel and controlled release of the drug tannic acid. Furthermore, FeCO-PBA, as a CO donor incorporating boric acid groups, can also be stably loaded into the hydrogel network through dynamic covalent bonds of catechol-boronate ester with tannic acid. Compared to traditional physical embedding methods, this chemical bond-based crosslinking strategy significantly improves the stability of the CO donor and reduces premature leakage of gas molecules. Simultaneously, FeCO-PBA can further respond to high ROS environments by releasing CO, enabling on-demand release for gas therapy. Additionally, the hydrogen bonding and electrostatic interactions between tannic acid and carboxymethyl chitosan can also rapidly respond to ROS at diabetic wound sites, further releasing tannic acid.

[0039] Therefore, the FeCO@CS / TA hydrogel provided by this invention achieves a synergistic mechanism with the CO donor through cross-linking bonds, targeting the microenvironment of diabetic wounds. This enables precise delivery and targeted release of CO at the lesion site, solving the problems of uncontrollable release, insufficient targeting, and limited safety of traditional CO donors. It achieves precise delivery and on-demand release of CO gas molecules, which not only improves the therapeutic effect but also effectively reduces the toxic risks caused by the systemic diffusion of CO.

[0040] Beneficial effects:

[0041] (1) The triple-response mechanism of the triple-responsive hydrogel of the present invention ensures that the release of CO is strictly limited to the wound area with the most severe pathological environment (i.e., the diabetic wound environment with the highest ROS and blood glucose levels), with minimal impact on normal tissue, greatly improving the safety of gas therapy. The more severe the wound infection and inflammation, the higher the ROS and glucose concentrations, and the faster and more CO is released. This "positive feedback" on-demand release ensures a perfect match between the treatment intensity and the severity of the condition, realizing a precise control mode of accelerating drug release when blood glucose is high and slowing down release when blood glucose is stable, significantly improving the accuracy and efficacy of treatment.

[0042] (2) The hydrogel system provided by this invention has excellent physical properties, including rapid gelation, self-healing, tissue adhesion, and injectability. It can achieve in-situ coverage and long-term adhesion of irregular wounds and effectively isolate external bacteria from invasion. At the same time, its high water content and good swelling properties can continuously maintain a moist environment on the wound, absorb exudate and blood, reduce the risk of secondary infection, and provide a stable microenvironment for wound repair.

[0043] (3) The carbon monoxide released by the hydrogel provided by the present invention has multiple therapeutic effects, including antibacterial, anti-biofilm, anti-inflammatory and antioxidant effects. It can effectively inhibit the proliferation of MRSA (methicillin-resistant Staphylococcus aureus) and promote biofilm clearance. At the same time, it regulates oxidative stress damage, improves cell activity, and promotes the transformation of macrophages from pro-inflammatory M1 phenotype to repair-type M2 phenotype, thereby systematically improving the long-term inflammatory state of diabetic wounds.

[0044] (4) The carrier material of the present invention is composed of biocompatible components such as tannic acid and carboxymethyl chitosan, and has good biodegradability, tissue compatibility and hemostatic properties, which can rapidly promote wound coagulation and reduce blood loss. Animal experimental results show that the hydrogel provided by the present invention can significantly accelerate the closure speed of diabetic infected wounds and promote wound regeneration and repair, and has good prospects for clinical translation. Attached Figure Description

[0045] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0046] Figure 1 The FeCO@CS / TA hydrogel prepared in Example 1.

[0047] Figure 2 The image shows a scanning electron microscope (SEM) image of the cross-section of the FeCO@CS / TA hydrogel prepared in Example 1.

[0048] Figure 3 The swelling curve of the FeCO@CS / TA hydrogel in Example 2 is shown.

[0049] Figure 4 The image shows the self-healing properties of the FeCO@CS / TA hydrogel in Example 3.

[0050] Figure 5 The image shows the tissue adhesion properties of the FeCO@CS / TA hydrogel in Example 3.

[0051] Figure 6 The image shows the injectability properties of the FeCO@CS / TA hydrogel in Example 3.

[0052] Figure 7UV-Vis absorption spectra of each group of reaction solutions in Example 4.

[0053] Figure 8 The images show the colony and biofilm conditions of the MRSA bacterial suspension after treatment in each treatment group in Example 5.

[0054] Figure 9 The figures are statistical graphs of bacterial survival rate and biofilm ablation rate after the MRSA bacterial suspension was treated by each treatment group in Example 5; where Figure a is the bacterial survival rate and Figure b is the biofilm ablation rate.

[0055] Figure 10 The cell viability of L929 cells after treatment in each treatment group in Example 6.

[0056] Figure 11 The images show the blood loss of mice with different treatment groups 6 minutes after the liver wound was opened, along with a statistical chart of the blood loss.

[0057] Figure 12 This is a statistical graph showing the change in wound area over time in diabetic mice under different treatment groups in Example 8. Detailed Implementation

[0058] The present invention will be further described below with reference to the following embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the present invention.

[0059] Example 1: Preparation of FeCO@CS / TA hydrogel

[0060] (1) Preparation of 4-carboxyphenylboronic acid-SH

[0061] 388 mg of 4-carboxyphenylboronic acid was dissolved in 5 mL of anhydrous DMF to prepare a 4-carboxyphenylboronic acid solution. Then, 380 mg of HOBt and 540 mg of EDCI were added to this solution as activators, and the mixture was stirred at 0 °C for 10 min to activate it. After activation, 262 mg of cystamine dihydrochloride and 1.27 mL of triethylamine were added, and the reaction was continued at room temperature for 6 h to carry out the amidation reaction, yielding an amidation reaction solution. Pure water was added to adjust the pH to 7.4, causing solid cystamine-phenylboronic acid monoamide to precipitate. After filtration, the solid was washed with pure water to obtain cystamine-phenylboronic acid monoamide.

[0062] 140 mg of cystamine-phenylboronic acid monoamide was dissolved in 5 mL of methanol, and then 230 mg of sodium borohydride was added. The reaction was carried out at room temperature for 1 h to obtain a reduced reaction solution. 4-Carboxyphenylboronic acid-SH was obtained by separation and purification from the reduced reaction solution by column chromatography.

[0063] (2) Preparation of FeCO-PBA

[0064] 125 mg Fe3(CO) 12 FeCO-PBA was dissolved in 20 mL of tetrahydrofuran along with 225 mg of 4-carboxyphenylboronic acid-SH and reacted under reflux at 80 °C for 4 h with stirring. After the reaction was complete, the reaction solution was separated and purified by column chromatography to obtain FeCO-PBA.

[0065] (3) Preparation of FeCO@CS / TA hydrogel

[0066] First, a mixed aqueous solution of CMCS and BDBA was prepared, with CMCS concentration of 60 mg / mL and BDBA concentration of 1.95 mg / mL. Then, a 20 mg / mL TA aqueous solution and an 8 mg / mL FeCO-PBA ethanol solution were prepared. The TA aqueous solution and FeCO-PBA ethanol solution were thoroughly mixed at a volume ratio of 9:1. Finally, the FeCO-PBA / TA mixed solution was mixed with the CMCS / BDBA mixed solution under vortex conditions at a volume ratio of 1:1 to obtain the FeCO@CS / TA hydrogel. Figure 1 The FeCO@CS / TA hydrogel prepared in this embodiment is shown.

[0067] The FeCO@CS / TA hydrogel prepared in Example 1 was pre-frozen at −80℃ for 30 min, and then transferred to a freeze dryer for freeze-drying. After freeze-drying, a cross-section of the hydrogel was cut with a scalpel and sputter-coated with gold. The microstructure of the cross-section was then observed using a scanning electron microscope (SEM). The results are as follows: Figure 2 As shown, the FeCO@CS / TA hydrogel exhibits a distinct three-dimensional porous network structure, indicating that the hydrogel has good water storage capacity.

[0068] Example 2: Determination of swelling properties of FeCO@CS / TA

[0069] A certain mass of FeCO@CS / TA hydrogel sample was weighed and immersed in an appropriate amount of PBS buffer, then incubated at 37°C. At predetermined time points, the sample was removed, excess liquid was gently absorbed using filter paper, and its mass was measured. When the mass of the hydrogel essentially no longer changed, it was considered to have reached a fully swollen state. The swelling rate at each time point was calculated using the following formula, and a swelling curve was plotted. The results are shown in [Figure number missing]. Figure 3 Experimental results show that the swelling ratio of FeCO@CS / TA hydrogel can reach 33.3%, indicating that the hydrogel has good water absorption and retention capacity, which can provide experimental basis for its subsequent application in the field of medical hemostasis.

[0070] Swelling ratio (%) = (W s - W w ) / W w ×100%

[0071] Among them, W w W represents the initial mass of the hydrogel. s This indicates the mass of the hydrogel after swelling at each time point.

[0072] Example 3: Self-healing properties, tissue adhesion, and injectability of FeCO@CS / TA hydrogel

[0073] (1) Self-healing properties of FeCO@CS / TA hydrogel

[0074] The FeCO@CS / TA hydrogel prepared in Example 1 was stained with methylene blue. The stained and unstained hydrogels were then cut into two parts, and half of each part was swapped before being reassembled. After standing for a certain period, the self-healing process was observed using tweezers. The macroscopic self-healing experimental results are as follows: Figure 4 As shown, the FeCO@CS / TA hydrogel re-adheres at the cut interface after being cut and re-contacted for about 4 minutes, and can be lifted and suspended by tweezers without separating, indicating that the hydrogel has good self-healing ability.

[0075] (2) Tissue adhesion of FeCO@CS / TA hydrogel

[0076] The FeCO@CS / TA hydrogel prepared in Example 1 was applied to the finger joints, and joint mobility tests were performed. The results are as follows: Figure 5 As shown, the hydrogel has a soft texture, which can effectively reduce friction during activities. Even under conditions of frequent joint movement, it can still maintain complete adhesion without falling off, indicating that it has suitable tissue adhesion properties.

[0077] (3) Injectability properties of FeCO@CS / TA hydrogel

[0078] Injection tests were performed on the FeCO@CS / TA hydrogel prepared in Example 1 using a 23G fine needle. During the experiment, the hydrogel was successfully extruded through the needle, forming the preset lettering while maintaining its gel state without significant dissolution. No needle blockage occurred during the injection process. The results are as follows: Figure 6 As shown, this demonstrates that the FeCO@CS / TA hydrogel has good injectability.

[0079] Example 4: CO release performance of FeCO@CS / TA hydrogel

[0080] The CO release behavior of FeCO@CS / TA hydrogel was detected using the hemoglobin method. 2 mg of hemoglobin was dissolved in 3.75 mL of PBS buffer, and 1.6 mg of sodium hyposulfite was added for reduction under N2 protection. Three groups were set up: Group 1 was a blank control group with no added substances; Group 2 contained 100 μL of FeCO@CS / TA hydrogel; and Group 3 contained 100 μL of FeCO@CS / TA hydrogel and 500 μL of 10 mM H2O2 solution. The addition of H2O2 simulated the high ROS microenvironment of diabetic wounds to verify whether FeCO@CS / TA hydrogel possessed H2O2-responsive CO release ability.

[0081] Each group of reaction solutions was sealed in quartz test tubes, and the changes in their absorption spectra in the range of 350–650 nm were detected using a UV-Vis spectrophotometer. Simultaneously, the changes in the absorption peaks of the third group of reaction solutions at different reaction times (10–60 min) were further analyzed.

[0082] Experimental results are as follows Figure 7 As shown, reduced Hb exhibits a characteristic absorption peak at 427 nm. When only FeCO@CS / TA hydrogel is added, the position of its absorption peak remains essentially unchanged, indicating that no significant CO release is detected in the system. However, after the addition of H2O2, the absorption peak at 427 nm gradually weakens and shifts towards 404 nm, which is the characteristic absorption peak of HbCO. This indicates that the FeCO@CS / TA hydrogel can release CO and bind to Hb in the presence of H2O2. With prolonged reaction time, the intensity of the absorption peak at 404 nm continuously increases, further demonstrating that this hydrogel can continuously release CO and exhibits good H2O2 response characteristics.

[0083] Comparative Example 1: Preparation of CS / TA Hydrogel

[0084] First, a mixed aqueous solution of CMCS and BDBA was prepared, with a concentration of 60 mg / mL for CMCS and 1.95 mg / mL for BDBA. Then, a 20 mg / mL TA aqueous solution was prepared. The TA aqueous solution and the CMCS / BDBA mixed aqueous solution were mixed at a 1:1 volume ratio under vortex conditions to obtain the CS / TA hydrogel.

[0085] Example 5: Colony and biofilm conditions after FeCO@CS / TA treatment

[0086] The antibacterial properties of FeCO@CS / TA hydrogel were evaluated using a colony counting method. 100 µL of freshly prepared hydrogel was added to 1 mL of MRSA bacterial suspension (approximately 1 × 10⁻⁶). 7The bacterial culture was incubated at 37°C with shaking for 6 h in CFU / mL solution. The treated bacterial culture was then serially diluted and added dropwise to the surface of LB solid medium. After incubation at 37°C for 18 h, appropriately diluted bacterial cultures were spread on a plate and incubated for another 18 h. Finally, the colonies were counted and the bacterial survival rate was calculated. Separate CS / TA hydrogel treatment groups and PBS treatment groups were also included, with the PBS treatment group serving as a control group.

[0087] The biofilm scavenging ability of FeCO@CS / TA hydrogel was evaluated using crystal violet staining. 200 µL of MRSA bacterial suspension (approximately 1 × 10⁻⁶) was added to each well of a 24-well plate. 8 Add 800 µL of TSB medium (CFU / mL) and mix thoroughly. Incubate at 37°C for 48 h, changing the medium once during this period to promote biofilm formation. After biofilm formation, slowly wash with PBS to remove residual TSB medium, then add 1 mL of hydrogel extraction buffer (1 g of hydrogel added to 5 mL of PBS, extracted at 37°C for 24 h) and continue incubation for 24 h. After treatment, wash the sample again with PBS. Then add 500 µL of 0.1% crystal violet staining solution to stain the biofilm for 30 min, and wash to remove unbound dye. After the sample is air-dried, add 500 µL of 95% ethanol to decolorize the residual biofilm for 30 min. Dilute the decolorizing solution and measure its OD value at 570 nm, and calculate the biofilm ablation rate. A CS / TA hydrogel treatment group and a non-gel treatment group were also set up, with the non-gel treatment group serving as a control group.

[0088] The results of the colony counting experiment are as follows Figure 8 and Figure 9 As shown in figure a, the bacterial survival rate of the CS / TA group was 61.8%, with a corresponding inhibition rate of 38.2%; while the number of colonies in the FeCO@CS / TA group plate was significantly reduced, with the bacterial survival rate decreasing to 34.2% and the inhibition rate reaching 65.8%, showing superior antibacterial activity.

[0089] Crystal violet staining results as follows Figure 8 and Figure 9 As shown in b, the biofilm structure in the control group remained intact, while both the CS / TA hydrogel group and the FeCO@CS / TA hydrogel group exhibited varying degrees of biofilm removal. Among them, the FeCO@CS / TA hydrogel group showed the most significant ablation effect on the biofilm. The biofilm ablation rate was 23.1% in the CS / TA hydrogel group and reached 38.8% in the FeCO@CS / TA hydrogel group. These results indicate that FeCO@CS / TA hydrogel possesses good antibacterial activity and strong biofilm penetration and removal capabilities.

[0090] Example 6: Detection of Improvement in Cell Viability Due to H2O2 Oxidation Damage

[0091] A H2O2-induced oxidative damage model of L929 cells was constructed to evaluate the ability of FeCO@CS / TA hydrogel to repair cell damage and improve cell viability. L929 cells were seeded in 96-well plates at a seeding density of 8 × 10⁶ cells / well. 3 Cells were cultured in wells for 24 h, then exposed to basal medium containing 400 μM H2O2 and different concentrations of hydrogel extract (0.5 mg / mL, 1 mg / mL, 2.5 mg / mL, 5 mg / mL) for 1 h. After treatment, cells were washed twice with PBS buffer, and cell viability was detected using the CCK-8 assay.

[0092] Experimental results are as follows Figure 10 As shown, after L929 cells were co-incubated with H2O2 for 1 h, cell viability decreased to 75.06%, indicating that the oxidative damage model was successfully constructed. After the addition of hydrogel extraction solution, cell viability gradually recovered with increasing extraction solution concentration. The FeCO@CS / TA hydrogel group showed a more significant improvement in cell viability, demonstrating stronger cell repair capabilities.

[0093] The excellent antioxidant and repair effects of FeCO@CS / TA hydrogel mainly come from the free radical scavenging ability of the polyphenol structure in TA, while the released CO has a regulatory effect on cell function. The two work together to endow the material with stronger antioxidant activity and cell protection ability.

[0094] Example 7 Evaluation of hemostatic performance of mouse liver wound

[0095] The in vivo hemostatic performance of FeCO@CS / TA hydrogel was evaluated using a mouse liver hemorrhage model. Male ICR mice (7–8 weeks old, weighing 25–30 g) were anesthetized preoperatively. After abdominal incision, the liver tissue was carefully exposed, and surface tissue fluid was absorbed using absorbent paper. Pre-weighed filter paper was placed under the liver to collect blood. Subsequently, an approximately 8 mm wound was made on the liver surface using a scalpel to induce bleeding, and 200 µL of FeCO@CS / TA hydrogel, 200 µL of CS / TA hydrogel, and gauze were immediately applied for hemostasis. Six minutes after bleeding, the filter paper was reweighed, and the blood loss was calculated. The group without any wound treatment served as a blank control group.

[0096] Experimental results are as follows Figure 11As shown, in the mouse liver hemostasis model, the blood loss in the control group (untreated group) was approximately 267.7 mg; the blood loss in the gauze group was 119.6 mg; the blood loss in the CS / TA hydrogel group was further reduced to 48.4 mg; while the blood loss on the filter paper in the FeCO@CS / TA hydrogel group was not significantly different at 0 min, 3 min and 6 min, indicating that it can quickly achieve hemostasis and has the lowest final blood loss of only 35.8 mg, showing the best hemostatic effect.

[0097] The hemostatic mechanism of FeCO@CS / TA hydrogel mainly includes the following aspects: the abundant positively charged groups in CMCS (such as -NH3) + FeCO@CS / TA hydrogel can adsorb negatively charged red blood cells and platelets, promoting their rapid aggregation at the wound site and the formation of initial thrombi. Simultaneously, the hydrophilic groups (such as -OH and -COOH) in the CMCS molecular chain and the phenolic hydroxyl groups (-OH) in TA can form insoluble complex precipitates with amino groups (-NH2) and thiol groups (-SH) in proteins through hydrogen bonding and hydrophobic interactions, thereby sealing the wound. Furthermore, swelling experiments show that FeCO@CS / TA hydrogel can rapidly absorb blood in the early stages and specifically bind to blood components using more borate ester bonds, thus exhibiting superior hemostatic properties.

[0098] Example 8 Evaluation of Diabetic Wound Treatment Capability

[0099] Male ICR mice (5-6 weeks old, 20-25 g) were acclimatized for one week and then intraperitoneally injected with STZ (streptozotocin) at a dose of 120 mg / kg for 5 consecutive days to induce a diabetic model. Blood glucose levels were continuously monitored during the modeling period, and the diabetic model was considered successfully established when blood glucose levels were above 16.7 mM for 3 consecutive days. To establish an infected wound model, diabetic mice were anesthetized, their dorsal hair was shaved, and a circular full-thickness skin defect with a diameter of approximately 8 mm was prepared on their backs. Subsequently, 10 μL of MRSA bacterial suspension (approximately 1×10⁻⁶ mcg) was inoculated onto the wound surface. 8 (CFU / mL) After the bacterial solution dried naturally, a stable infection model was established in the wound. Then, 200 μL of FeCO@CS / TA hydrogel and 200 μL of CS / TA hydrogel were injected and applied to the wound surface, respectively, and bandaged with sterile dressings. The untreated group served as a blank control group, and chitosan medical biogel served as a commercial control group. This was recorded as day 0 of the experiment. During the experiment, the mouse wounds were photographed and the dressings were changed every 3 days. ImageJ software was used to measure the wound area of ​​each group of mice, and the percentage change in wound area was calculated.

[0100] Experimental results are as follows Figure 12As shown, the control group (untreated group) exhibited the slowest wound healing, with wound areas reaching 77.7%, 45.2%, 27.9%, and 11.7% of the initial area on days 3, 6, 9, and 12, respectively. In contrast, the FeCO@CS / TA hydrogel group demonstrated the fastest wound healing rate, with the wound shrinking to 47.8% of its initial area by day 3 and essentially healed by day 9. Both the CHITOSAN and CS / TA hydrogel groups showed better wound healing effects than the untreated group, but their overall healing speed was slower than that of the FeCO@CS / TA hydrogel group. Specifically, the CHITOSAN group showed faster early healing on day 3; while the CS / TA group showed significantly faster wound healing after day 6, achieving complete healing by day 12. These results indicate that FeCO@CS / TA hydrogel can significantly promote the repair of infectious diabetic wounds, accelerate the wound closure process, and demonstrate excellent wound treatment capabilities.

[0101] This invention provides a triple-responsive hydrogel, its preparation method, and its application. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. A triple-responsive hydrogel, characterized in that, The hydrogel is made by mixing the following raw materials: FeCO-PBA, tannic acid, carboxymethyl chitosan, terephthalic acid and a first solvent; Wherein, FeCO-PBA is Fe3(CO). 12 A complex containing an iron carbonyl unit and a phenylboronic acid group, generated by a metal coordination reaction with 4-carboxyphenylboronic acid-SH, wherein the iron carbonyl unit and the phenylboronic acid group are linked by an iron-sulfur bond; wherein 4-carboxyphenylboronic acid-SH is a thiol-containing 4-carboxyphenylboronic acid derivative formed by the linkage of 4-carboxyphenylboronic acid and mercaptoethylamine through an amide bond.

2. The triple-responsive hydrogel according to claim 1, characterized in that, The preparation method of the 4-carboxyphenylboronic acid-SH includes the following steps: Step 1: 4-Carboxyphenylboronic acid is activated by carboxyl group. The activated 4-carboxyphenylboronic acid undergoes an amidation reaction with cystamine dihydrochloride to obtain the first reaction solution. The solution is then subjected to the first separation and purification to obtain cystamine-phenylboronic acid monoamide. Step 2: The cystamine-phenylboronic acid monoamide obtained in Step 1 is reduced with sodium borohydride to obtain a second reaction solution, which is then subjected to a second separation and purification to obtain the final product.

3. The triple-responsive hydrogel according to claim 2, characterized in that, In step 1, the carboxyl activation of 4-carboxyphenylboronic acid is carried out under the action of 1-hydroxybenzotriazole and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride; the mass ratio of 4-carboxyphenylboronic acid, 1-hydroxybenzotriazole, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 376~400 : 380~400 : 540~560; both the carboxyl activation and amidation reactions are carried out in the solvent N,N-dimethylformamide; the carboxyl activation is carried out with stirring at 0~4℃ for 10~30 min; during the amidation reaction, triethylamine is used to neutralize the hydrochloric acid in cystamine dihydrochloride; the volume ratio of triethylamine used to the mass of cystamine dihydrochloride is 1.25~1.40 mL : 260~270 mg; the mass ratio of cystamine dihydrochloride to 4-carboxyphenylboronic acid is 260~ 270: 376 ~ 400; the amidation reaction was carried out with stirring at room temperature for 5 ~ 7 h.

4. The triple-responsive hydrogel according to claim 2, characterized in that, In step 2, the molar ratio of sodium borohydride to cystamine-phenylboronic acid monoamide is 10-15:1; the reduction reaction is carried out in methanol solvent; and the reduction reaction is carried out at room temperature for 1-2 hours.

5. The triple-responsive hydrogel according to claim 1, characterized in that, The preparation method of FeCO-PBA includes the following steps: Fe3(CO) 12 The 4-carboxyphenylboronic acid-SH is dissolved in a second solvent to carry out a metal coordination reaction, resulting in a third reaction solution. After a third separation and purification, the final product is obtained.

6. The triple-responsive hydrogel according to claim 5, characterized in that, The Fe3(CO) 12 The mass ratio of the metal to 4-carboxyphenylboronic acid-SH is 120~140 : 220~280; the second solvent is tetrahydrofuran; the metal coordination reaction is carried out under reflux at 80°C with stirring for 4 h.

7. A method for preparing the triple-responsive hydrogel according to any one of claims 1 to 6, characterized in that, Includes the following steps: The FeCO-PBA is dissolved in a third solvent to obtain a FeCO-PBA solution, which is then mixed evenly with a tannic acid solution, a terephthalic acid solution, and a hydroxymethyl chitosan solution to obtain the final product.

8. The preparation method according to claim 7, characterized in that, The method for mixing the FeCO-PBA solution with the tannic acid solution, the terephthalic acid solution, and the hydroxymethyl chitosan solution is as follows: the FeCO-PBA solution and the tannic acid solution are mixed evenly to obtain the FeCO-PBA / TA mixture, and then the mixture is mixed evenly with the terephthalic acid and hydroxymethyl chitosan mixture.

9. The preparation method according to claim 8, characterized in that, The third solvent is ethanol; the solvent for the tannic acid solution is water; the solvent for the mixed solution of terephthalic acid and hydroxymethyl chitosan is water; the concentration of the FeCO-PBA solution is 8-10 mg / mL; the concentration of the tannic acid solution is 20-25 mg / mL; the concentrations of terephthalic acid and hydroxymethyl chitosan in the mixed solution of terephthalic acid and hydroxymethyl chitosan are 1.95-2.3 mg / mL and 60-70 mg / mL, respectively; the volume ratio of the FeCO-PBA solution to the tannic acid solution is 1-1.2:9-10; the volume ratio of the FeCO-PBA / TA mixture to the mixed solution of terephthalic acid and hydroxymethyl chitosan is 1:

1.

10. The use of the triple-responsive hydrogel according to any one of claims 1 to 6 in the treatment of chronic wounds in diabetes.

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