Conductive hydrogel with ROS (reactive oxygen species) response as well as preparation method and application of conductive hydrogel

Abstract

The invention discloses conductive hydrogel with ROS responsiveness and a preparation method and application thereof, and belongs to the technical field of medical biological materials. The preparation method provided by the invention comprises the following steps: dissolving sulfhydrylated hyaluronic acid and 5-hydroxymethylfurfural in water, then adding trifluoroacetic acid for catalysis to carry out reaction, and after the reaction is finished, carrying out dialysis purification to obtain a compound containing a thioacetal bond; and carrying out thermal cross-linking reaction on the compound containing the thioacetal bond, acrylamide, N, N '-methylene bisacrylamide, ammonium persulfate and a conductive medium to obtain the conductive hydrogel with ROS responsiveness. The prepared conductive hydrogel has good ROS responsiveness, the release rate of the medicine is in positive correlation with the ROS concentration, real-time monitoring of a wound surface and diagnosis and treatment integration of a chronic wound surface can be achieved, inflammatory response can be relieved, wound healing is promoted, meanwhile, the conductive hydrogel has excellent mechanical performance and biocompatibility, and the conductive hydrogel can be applied to the field of wound healing. The method has a wide application prospect in the field of preparation of biomedical materials.

Description

Technical Field

[0001] The present application belongs to the technical field of medical biomaterials, and in particular relates to a conductive hydrogel with ROS response and a preparation method and application thereof. Background Art

[0002] Wound healing is a complex and delicate pathophysiological process, which can be divided into four typical stages: coagulation / hemostasis stage, inflammation stage, new tissue formation stage and wound remodeling stage. However, chronic wounds often stagnate in the inflammatory stage, accompanied by problems such as large amounts of exudate, severe infection, pain and tissue necrosis. Once a chronic wound is formed, the combined effects of factors such as persistent inflammatory response, oxidative stress, hypoxia-reoxidation and fibrosis process cause its reactive oxygen species (ROS) level to be much higher than that of normal skin tissue, which in turn triggers chronic inflammation and forms a vicious cycle. Therefore, it is necessary to develop biomedical materials with excellent comprehensive performance to achieve efficient wound healing.

[0003] The existing ROS-responsive biomaterials are usually developed by utilizing the transformation characteristics of ROS signals to promote wound healing. For example, Professor Guo Baolin of Xi'an Jiaotong University (ROS-responsive hydrogels with spatiotemporallysequential delivery of antibacterial and anti-inflammatory drugs for the pair of MRSA-infected wounds, Regenerative Biomaterials, Volume 11, 2024, rbad110) prepared ROS-responsive hydrogel dressings by cross-linking the phenylboronic acid bond between 3-aminophenylboronic acid grafted with hyaluronic acid and polyvinyl alcohol, achieving the timed release of the two drugs for wound repair. However, these widely used ROS-responsive chemical bonds have the defect of multiple responsiveness and cannot respond precisely to ROS. Summary of the invention

[0004] The present application discloses a ROS-responsive conductive hydrogel and a preparation method and application thereof, aiming to solve the technical problem that existing ROS-responsive hydrogel dressings have multiple responses and cannot accurately respond to ROS.

[0005] In order to achieve the above purpose, the technical solution of this application is:

[0006] The first aspect of the present application provides a method for preparing a conductive hydrogel having ROS response, the preparation method comprising:

[0007] The thiolated hyaluronic acid, 5-hydroxymethylfurfural and trifluoroacetic acid are dissolved in water and reacted, and after the reaction is completed, the dialysis is performed for purification to obtain a compound containing a thioacetal bond;

[0008] The compound containing thioacetal bond, acrylamide, N,N'-methylenebisacrylamide, ammonium persulfate and conductive medium are cross-linked to obtain conductive hydrogel with ROS response.

[0009] In combination with the first aspect, preferably, the method for preparing the thiolated hyaluronic acid comprises:

[0010] Hyaluronic acid, cystamine dihydrochloride and 1-hydroxybenzotriazole hydrate are dissolved in polybutylene succinate, the pH is adjusted, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and dithiothreitol are added to react, and the reaction is performed by dialysis purification to obtain thiolated hyaluronic acid.

[0011] In combination with the first aspect, preferably, the pH value is adjusted to a range of 7.5-8.5.

[0012] In combination with the first aspect, preferably, when the thiolated hyaluronic acid, 5-hydroxymethylfurfural and trifluoroacetic acid are dissolved in water and reacted, the reaction is carried out at room temperature and away from light.

[0013] In combination with the first aspect, preferably, the percentage of acrylamide added is 16-26%.

[0014] In combination with the first aspect, preferably, the added amount of the N,N'-methylenebisacrylamide is 10% of the acrylamide monomer.

[0015] In combination with the first aspect, preferably, the conductive medium is poly(3,4-ethylenedioxythiophene):polystyrene sulfonate, and the added amount is 0.2-1%.

[0016] In combination with the first aspect, preferably, when the compound containing thioacetal bond, acrylamide, N,N'-methylenebisacrylamide, ammonium persulfate and the conductive medium are cross-linked, the reaction temperature is 60-80° C. and the reaction time is 1.5-4 h.

[0017] The second aspect of the present application provides a conductive hydrogel prepared by the preparation method described in the first aspect.

[0018] The third aspect of the present application provides the use of the conductive hydrogel prepared by the preparation method described in the first aspect or the conductive hydrogel described in the second aspect in the preparation of biomedical materials.

[0019] Compared with the prior art, the advantages or beneficial effects of the embodiments of the present application include at least:

[0020] The preparation method provided by the present application is prepared by cross-linking a compound containing a thioacetal bond with a single-layer hydrogel network composed of thiolated hyaluronic acid and 5-hydroxymethylfurfural, and then reacting with acrylamide, N,N'-methylenebisacrylamide, ammonium persulfate and a conductive medium; on the one hand, it can achieve accurate response to ROS. When in a high ROS environment, the sulfur atoms in the thioacetal chemical bonds in the conductive hydrogel will lose electrons and be oxidized to sulfur oxides to form electron transfer. The movement of these electrons forms a current signal, which can realize real-time monitoring of the wound surface and realize the integrated diagnosis and treatment of chronic wounds. At the same time, the thioacetal chemical bonds that respond to ROS will respond to ROS breakage during the inflammatory period of the wound surface, and release 5-hydroxymethylfurfural to relieve the inflammatory response and promote wound healing; on the other hand, the cross-linking reaction can give the hydrogel excellent mechanical properties and biocompatibility, ensuring that the hydrogel structure will not be destroyed while exerting its performance; at the same time, the preparation process of the present application is simple, does not require special equipment, and is easy to achieve mass production and application promotion. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments recorded in the present application, and for ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0022] Figure 1 The H NMR spectra of HA, HA-SH and HA-TA-HMF provided in the examples of the present application;

[0023] Figure 2 Infrared spectra of HA, HA-SH and HA-TA-HMF provided in the examples of the present application;

[0024] Figure 3 This is a nuclear magnetic resonance hydrogen spectrum of the responsiveness of the thioacetal bond-containing compound HA-TA-HMF to ROS according to an embodiment of the present application;

[0025] Figure 4 A graph showing the responsive release of 5-hydroxymethylfurfural in a conductive hydrogel provided in an embodiment of the present application;

[0026] Figure 5 The tensile stress-strain curve and compressive stress-strain curve of the conductive hydrogel provided in the embodiments of the present application;

[0027] Figure 6 The electrical signal diagram of the conductive hydrogel of group B provided in the embodiment of the present application as the ROS concentration changes;

[0028] Figure 7Biocompatibility diagram of the conductive hydrogel provided in the embodiments of the present application. DETAILED DESCRIPTION

[0029] The technical solutions in the embodiments of the present application will be described clearly and completely below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, rather than all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

[0030] In the following description of this embodiment, the term "and / or" is used to describe the association relationship of associated objects, indicating that there may be three relationships. For example, A and / or B can represent: A exists alone, B exists alone, and A and B exist at the same time. A and B can be singular or plural. The character " / " generally indicates that the associated objects before and after are in an "or" relationship.

[0031] In the following description of this embodiment, the term "at least one" refers to one or more, and "plurality" refers to two or more. "At least one of the following" or similar expressions refers to any combination of these items, including any combination of single items or plural items. For example, "at least one of a, b or c", or "at least one of a, b and c", can all represent: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, c can be single or multiple, respectively.

[0032] Those skilled in the art should understand that in the following description of the embodiments of the present application, the order of serial numbers does not mean the order of execution, some or all of the steps can be executed in parallel or sequentially, and the execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0033] The terms used in the embodiments of the present application are only for the purpose of describing specific embodiments, and are not intended to limit the present application. The singular forms "a", "an" and "the" used in the embodiments of the present application and the appended claims are also intended to include plural forms, unless the context clearly indicates other meanings.

[0034] Those skilled in the art will appreciate that the numerical ranges in the examples of the present application are to be understood as also specifically disclosing each intermediate value between the upper and lower limits of the range. Each smaller range between the intermediate values ​​in any stated value and stated range and any other stated value or intermediate value in the range is also included in the present application. The upper and lower limits of these smaller ranges may be independently included or excluded in the scope.

[0035] Unless otherwise stated, the technical / scientific terms used herein have the same meanings as those generally understood by those skilled in the art in the field described in the present application. Although the present application only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the embodiments or test cases of the present application. All documents mentioned in this specification are generally incorporated by reference to disclose and describe the methods and / or materials related to the documents. In the event of a conflict with any incorporated document, the content of the present application shall prevail.

[0036] It should be noted that all raw materials and reagents in the examples of the present application were purchased on the market or prepared according to conventional methods well known to those skilled in the art.

[0037] In a first aspect, an embodiment of the present application provides a method for preparing a conductive hydrogel having ROS response, the preparation method comprising:

[0038] The thiolated hyaluronic acid, 5-hydroxymethylfurfural and trifluoroacetic acid are dissolved in water and reacted, and after the reaction is completed, the dialysis is performed for purification to obtain a compound containing a thioacetal bond;

[0039] The compound containing thioacetal bond, acrylamide, N,N'-methylenebisacrylamide, ammonium persulfate and conductive medium are cross-linked to obtain conductive hydrogel with ROS response.

[0040] Among them, on the one hand, it can achieve accurate response to ROS. When in a high ROS environment, the sulfur atoms in the thioacetal chemical bonds in the conductive hydrogel will lose electrons and be oxidized to sulfur oxides to form electron transfer. The movement of these electrons forms a current signal, which can realize real-time monitoring of the wound surface and realize the integrated diagnosis and treatment of chronic wounds. At the same time, the thioacetal chemical bonds that respond to ROS will respond to ROS breakage during the inflammatory period of the wound surface, and release 5-hydroxymethylfurfural, which relieves the inflammatory response and promotes wound healing; on the other hand, the cross-linking reaction can give the hydrogel excellent mechanical properties and biocompatibility, ensuring that the hydrogel structure will not be destroyed while exerting its performance; at the same time, the preparation process of this application is simple, does not require special equipment, and is easy to achieve mass production and application promotion.

[0041] It should be noted that the 5-hydroxymethylfurfural used in this application exerts its anti-inflammatory effect by inhibiting MAPK, NF-κB and Akt / mTOR pathway-related proteins; it also has the ability to promote the proliferation and migration of skin fibroblasts, giving the conductive hydrogel good biocompatibility, excellent physicochemical properties, and the effect of accelerating wound healing.

[0042] In the present application embodiment, the preparation method of the thiolated hyaluronic acid includes: hyaluronic acid (HA), cystamine dihydrochloride (CA) and 1-hydroxybenzotriazole hydrate (HOBT) are dissolved in polybutylene succinate (PBS), pH is adjusted, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and dithiothreitol (DTT) are added to react, and dialysis purification is performed to obtain thiolated hyaluronic acid (HA-SH). Wherein, by carrying out thiolation modification to high molecular weight hyaluronic acid, reacting the amino group in cystamine dihydrochloride with the carboxyl group in hyaluronic acid, then forming thiolated hyaluronic acid after the disulfide bond is broken, the introduction of sulfhydryl can promote the cross-linking reaction of hyaluronic acid and subsequent acrylamide. Specific reaction process is as shown in formula (1):

[0043]

[0044] It should be noted that thiolated hyaluronic acid (HA-SH), 5-hydroxymethylfurfural (5-HMF) and trifluoroacetic acid (TFA) are dissolved in water and reacted to obtain a compound containing a thioacetal bond (HA-TA-HMF). The specific reaction process is shown in formula (2):

[0045]

[0046] It should be noted that the molecular structure of the conductive hydrogel prepared in this application is shown in formula (3):

[0047]

[0048] It should be noted that the anti-inflammatory properties of the conductive hydrogel are mainly achieved through hyaluronic acid and 5-hydroxymethylfurfural, and the ROS responsiveness of the gel system is achieved by the thioacetal bond. The drug release rate is positively correlated with the ROS concentration, causing changes in different electrical signals, thereby achieving stable monitoring and realizing the integrated diagnosis and treatment of chronic wounds.

[0049] In the examples of the present application, the thiolated hyaluronic acid, 5-hydroxymethylfurfural and trifluoroacetic acid are dissolved in water and reacted under room temperature and light-proof conditions. The room temperature and light-proof conditions can simulate the environment in the body, allowing the reaction to proceed under optimal conditions, thereby maintaining a high reaction rate and efficiency.

[0050] In the embodiment of the present application, the percentage of acrylamide added is preferably 16-26%. Among them, by controlling the amount of acrylamide added, excellent mechanical properties can be effectively imparted to the hydrogel.

[0051] In the embodiment of the present application, the amount of N,N'-methylenebisacrylamide added is preferably 10% of the acrylamide monomer. As a crosslinking agent, N,N'-methylenebisacrylamide can react with the active sites on the linear polymer chain through its two acrylamide groups to form a three-dimensional network structure, thereby improving the mechanical strength and stability of the material, and can enhance mechanical properties, control porosity and swelling, and improve thermal stability and chemical stability.

[0052] In the embodiment of the present application, the conductive medium is poly(3,4-ethylenedioxythiophene):polystyrene sulfonate (PEDOT:PSS); the added amount is preferably 0.2-1%. By controlling the added amount of the conductive medium, the hydrogel can be endowed with excellent electrochemical properties while having excellent mechanical properties.

[0053] In the embodiment of the present application, when the compound containing thioacetal bond, acrylamide, N,N'-methylenebisacrylamide, ammonium persulfate and conductive medium are cross-linked, the reaction temperature is 60-80°C and the reaction time is 1.5-4h. Among them, by controlling the time and temperature of the cross-linking reaction to ensure that the reactants are fully in contact and chemical bonds are formed, the hydrogel is given excellent mechanical properties.

[0054] In the second aspect, the embodiment of the present application also provides a conductive hydrogel prepared by the preparation method described in the first aspect. Among them, the preparation method of the present application can give the conductive hydrogel good ROS responsiveness, and its drug release rate is positively correlated with the ROS concentration, causing changes in different electrical signals, thereby achieving stable monitoring, realizing the integration of diagnosis and treatment of chronic wounds, and being able to alleviate inflammatory reactions and promote wound healing, while having excellent mechanical properties and biocompatibility.

[0055] In the third aspect, the embodiment of the present application also provides the use of the conductive hydrogel prepared by the preparation method described in the first aspect or the conductive hydrogel described in the second aspect in the preparation of biomedical materials. Based on the above-prepared conductive hydrogel having good ROS responsiveness, and its drug release rate is positively correlated with the ROS concentration, it can relieve inflammatory response and promote wound healing, and at the same time has excellent mechanical properties and biocompatibility, and has broad application prospects in the preparation of biomedical materials.

[0056] The technical solution of the present application will be further described below in conjunction with specific embodiments.

[0057] Example 1

[0058] This embodiment provides a method for preparing thiolated hyaluronic acid (HA-HS), which specifically comprises:

[0059] Hyaluronic acid (HA) was dissolved in PBS, and then cystamine dihydrochloride (CA) was added to the solution (added in excess molar amount relative to -COOH), and 1-hydroxybenzotriazole hydrate (HOBT) was added after full dissolution (also added in excess molar amount relative to -COOH), and the pH of the solution was immediately adjusted from 4.1 to 7.5 with 1M NaOH. 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) was added to the solution, and stirred overnight at room temperature in the dark. The reaction solution was then dialyzed with deionized water for 3 days, the solution pH was adjusted to 8.5, dithiothreitol (DTT) was added to the solution, and the reaction was stirred overnight at room temperature in the dark. The reaction solution was adjusted to pH 3.5, dialyzed with deionized water for 3 days, and stored in nitrogen in the dark after freeze drying to obtain thiolated hyaluronic acid (HA-SH).

[0060] Example 2

[0061] This embodiment provides a method for preparing a compound containing a thioacetal bond (HA-TA-HMF), which specifically includes:

[0062] Dissolve thiolated hyaluronic acid (HA-SH) in 100 mL of deionized water, add 5-hydroxymethylfurfural (5-HMF) and fully dissolve it, add trifluoroacetic acid (TFA) to the reaction solution, and stir overnight at room temperature in a dark place. Then, dialyze the reaction solution for 3 days, freeze-dry it, and store it in nitrogen in a dark place to obtain a compound containing a thioacetal bond (HA-TA-HMF).

[0063] In order to verify the structural properties of the compound containing thioacetal bonds (HA-TA-HMF), the compound was characterized by NMR and IR tests. The results are as follows Figure 1-2 shown.

[0064] according to Figure 1 It can be seen that the NMR hydrogen spectra of HA, HA-SH and HA-TA-HMF, among which a, b: double bond peaks in 5-HMF, double bond peaks appear in the range of 5.5-6.3ppm, which are double bond peaks of aldehyde groups in pentahydroxymethylfurfural, and the superposition of methine appears at 4.3-4.7ppm, proving that the thioacetal bond is successfully synthesized. Since the connected 5-hydroxymethylfurfural has a double bond, the phenomenon of judging that it is connected is the appearance of this double bond peak in the NMR. Figure 1In the marked part, it can be seen that there is an obvious small peak. Because the small molecule is connected to the macromolecule, the peak is not very high, which is a normal phenomenon; c: characteristic peak of thioacetal bond (4.3-4.7ppm peak shape is superimposed and increased); d, e: methylene peak of modified thiolated hyaluronic acid. By comparing the chemical formula of HA, it can be seen that the thiolated hyaluronic acid has two more methylene groups and one HS, so the peak position of the methylene group should be found on the NMR to prove the success of the modification, that is, the triple peaks at positions d and e are the characteristic peaks of the methylene group, which can be clearly seen by comparing the NMR images of HA and HA-HS.

[0065] according to Figure 2 It can be seen that the infrared spectra of HA, HA-SH and HA-TA-HMF are as follows, wherein a) is 500-4000cm -1 Spectrum in the range of b) 500-2000cm -1 The chemical bonds C=C, CO, etc. on the small molecule 5-pentahydroxymethylfurfural can be found in the spectrum: 1600-1750cm -1 The stretching vibration of C=C appears, and the stretching vibration of CO appears at 1200-1000cm -1 ; Deformation vibration of double bond carbon and hydrogen, benzene ring aromatic hydrogen: 1000-630cm -1 , can be obtained in the corresponding interval.

[0066] In order to verify the responsiveness of the compound containing thioacetal bond (HA-TA-HMF) to ROS, the compound was characterized by nuclear magnetic resonance testing. The freeze-dried HA-TA-HMF was dissolved in heavy water, and H2O2 solutions of different proportions and concentrations were added to it for incubation for 6h, 12h and 24h, respectively. The nuclear magnetic hydrogen spectrum was used to analyze whether there was a responsive characteristic peak of thioacetal. The specific proportions are shown in Table 1 below.

[0067] Table 1 HA-TA-HMF in different proportions

[0068]

[0069] 10 mg of HA-TA-HMF freeze-dried sample was weighed and dissolved in different proportions of heavy water and H2O2. They were control group (no H2O2 solution), 0.55 mL of heavy water mixed with 1 mM H2O2, and 0.55 mL of heavy water mixed with 10 mM H2O2, and were named HA-TA-HMF0, HA-TA-HMF1, and HA-TA-HMF2, respectively.

[0070] according to Figure 3It can be seen from the chemical formula that there is an aldehyde group on 5-HMF, namely at a. When it is connected to the polymer, the aldehyde group will react, namely disappear, and the corresponding characteristic peak of the aldehyde group will not appear on the NMR, namely the NMR image of -H2O2. In order to prove that the connected chemical bond is ROS responsive (ie H2O2), after adding a certain concentration of H2O2 solution, if it is responsive, the chemical bond will break, that is, 5-HMF will return to its original state, that is, the aldehyde group will be restored, and the aldehyde peak will appear on the NMR, that is, at a in +H2O2 in the figure, 9-11ppm, which is the aldehyde peak. At the same time, at the thioacetal bond b, the peak shape will be weakened accordingly. These two places can simultaneously prove that the ROS response was successfully carried out.

[0071] Example 3

[0072] This embodiment provides a method for preparing a conductive hydrogel, which specifically includes:

[0073] A compound containing a thioacetal bond (HA-TA-HMF), acrylamide, N,N'-methylenebisacrylamide, ammonium persulfate and PEDOT:PSS are cross-linked to obtain a conductive hydrogel.

[0074] Take 50 mg of freeze-dried sample, dissolve it in acrylamide solution with different solid contents, add a certain proportion of cross-linking agent and conductive medium, observe the cross-linking of acrylamide solution with different proportions and sample, name them as A, B, C, D, add them to the mold after uniform stirring, heat cross-link at 80℃ for about 2h, seal and store at low temperature or room temperature after gelation. See Table 2 for specific proportions:

[0075] Table 2 Prepared different conductive hydrogels

[0076]

[0077] In order to verify the responsiveness of the conductive hydrogel to ROS, the conductive hydrogel was characterized accordingly. The test results are as follows: Figure 4 shown.

[0078] according to Figure 4It can be seen that the prepared conductive hydrogel has a responsive release of 5-hydroxymethylfurfural under the condition of ROS through in vitro release within 72h. Take 12 identical hydrogels and divide them into four groups: PBS, 0.1mM H2O2, 1mMH2O2, and 10mM H2O2. Three parallel samples are set in each group, for a total of 12 groups. Put them into 45mL medium for in vitro release experiment, take 1min, 30min, 2h, 6h, 12h, 24h, 48h and 72h as samples, and measure their OD values. According to the standard curve and the known absorption peak position, there is a maximum absorption peak at 384nm. The cumulative release amount is calculated by the formula. It can be seen that with the increase of H2O2 concentration, the release amount of 5-HMF in the hydrogel increases significantly, and the release rate is accelerated, while there is no obvious release in the PBS control group, which proves the responsiveness of the conductive hydrogel under the condition of ROS. And as the H2O2 concentration increases, the responsiveness of the chemical bonds is enhanced and more drugs are released, which is in line with the expected trend.

[0079] In order to verify the mechanical properties of the conductive hydrogel, the stress-strain characterization of the hydrogel was performed. The test results are as follows: Figure 5 As shown. Conductive hydrogels were made into different shapes and subjected to tensile and compression tests to test the mechanical properties of the gels. Rod-shaped hydrogel samples (diameter: 15.6 mm, height: 12 mm) were used for static compression tests with a compression rate of 1 mm / min, and the test was stopped when the compression was 90%; rectangular samples (length: 16 mm, width: 10 mm, height: 1 mm) were used for tensile measurements with a tensile rate of 100 mm / min, and the stretching was terminated when a break occurred.

[0080] according to Figure 5 It can be seen that, among them, a) tensile stress-strain curve, b) compressive stress-strain curve. According to the tensile stress-strain curve, it can be seen that the first inflection point in the tensile strain is the place where it breaks, corresponding to the force on the ordinate, and the purpose is to explore the tensile force that different groups of hydrogels can withstand. According to the figure, when the percentage of PEDOT:PSS added is 0.5%, the tensile force it can withstand is the largest. According to the compressive stress-strain curve, it can be seen that the first inflection point in the compressive strain is the place where it breaks, corresponding to the force on the ordinate, and the purpose is to explore the pressure that different groups of hydrogels can withstand. According to the figure, when the percentage of PEDOT:PSS added is 0.5%, the pressure it can withstand is the largest. Therefore, the mechanical properties of group B made in this application are the best.

[0081] In order to verify the electrochemical activity of the conductive hydrogel, the hydrogel was characterized by electrical signals. The test results are as follows: Figure 6As shown. The prepared conductive hydrogel has sensitive electrochemical activity in the presence of H2O2. Changes in electrical signals All electrochemical measurements were performed at room temperature using an electrochemical workstation (CHI 760E, CH Instruments, USA). A traditional three-electrode system was used, with a gold (Au) electrode as the working electrode, a platinum (Pt) electrode as the counting electrode, and Ag / AgCl saturated KCl as the reference electrode. In order to determine the responsiveness to H2O2, the equilibrium-swollen conductive hydrogel was immersed in solutions of different concentrations of H2O2 (0, 60, 80, 100, 120, 160, 200), and DPV measurements from -0.5V to 0V were performed.

[0082] according to Figure 6 It can be seen that the simulated DPV curve is adjusted for noise and smooth tail. DPV measures the current mutation of the sample within a certain potential range. Therefore, when the H2O2 concentration increases, the intensity of its redox reaction will also increase, resulting in an increase in the amplitude of the current response. Figure 6 It can be clearly seen that with the increase of H2O2 concentration, the peak current of DPV signal will also increase accordingly. Therefore, different concentrations of H2O2 solution will lead to different peak currents, thus forming different response gradients.

[0083] In order to verify the biocompatibility of the conductive hydrogel, the cytotoxicity of the hydrogel was characterized. The test results are as follows: Figure 7 As shown. The various stages of the synthesized hydrogels were grouped into control group, HA Hydrogel group, HA-TA-HMF Hydrogel group and HTH-PEDOT:PSS Hydrogel group. The biocompatibility and toxicity of the hydrogels were investigated by CCK-8 cytotoxicity test. The specific steps are as follows:

[0084] Count the cells (about 10,000-25,000 cells per well), respin with complete medium and inoculate into a 24-well plate, 500 μL per well. Set up duplicate wells for each group (parallel samples to reduce errors) and culture in a 37°C incubator for 24 hours. Then aspirate the medium, add an equal volume of new medium, and place the pre-prepared and UV-sterilized hydrogel into the well plate for 24 hours. Finally, aspirate the old medium in the well plate, take out the material, add the diluted Cell Counting Kit-8 solution, 400 μL per well, put it in the incubator for 4 hours and take it out (keep away from light throughout the process), and aspirate the liquid into a 96-well plate according to the group, 100 μL per well, and measure its OD value at a wavelength of 450 nm.

[0085] according to Figure 7It can be seen that the control group is a blank group without hydrogel, and the following three groups are hydrogels in different stages of synthesis. The final conductive hydrogel is the group on the far right (HTH-PEDOT:PSS, HA-TA-HMF is abbreviated as HTH). According to the test results, if the cell activity decreases by more than 30%, it is considered to be cytotoxic. Figure 7 It can be seen that the survival rate of the second group of cells with the lowest cell survival rate is also above 90%, and the survival rates of the rest are all around 98%. Therefore, it is believed that the conductive hydrogel prepared in this application has no cytotoxicity and meets the biocompatibility safety standards. In addition, through statistical analysis, it can be seen that there is no significant difference overall, and there is a significant difference between the two groups above the 95% level, which meets the statistical requirements.

[0086] Therefore, the preparation method provided in the present application is prepared by cross-linking a single-layer hydrogel network containing a thioacetal bond compound composed of thiolated hyaluronic acid and 5-hydroxymethylfurfural, and then reacting with acrylamide, N,N'-methylenebisacrylamide, ammonium persulfate and a conductive medium. The conductive hydrogel prepared in the present application has good ROS responsiveness, and its drug release rate is positively correlated with the ROS concentration. It can realize real-time monitoring of the wound surface, realize the integrated diagnosis and treatment of chronic wounds, relieve inflammatory reactions, and promote wound healing. It also has excellent mechanical properties and biocompatibility, and has broad application prospects in the preparation of biomedical materials.

[0087] The various embodiments in this specification are described in a progressive manner, and the same or similar parts between the various embodiments can be referenced to each other, and each embodiment focuses on the differences from other embodiments.

[0088] The above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit the present application. Although the present application has been described in detail with reference to the aforementioned embodiments, a person of ordinary skill in the art should understand that the technical solutions recorded in the aforementioned embodiments may still be modified, or some or all of the technical features therein may be replaced by equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the present application.

Claims

1. A method for preparing a conductive hydrogel with ROS response, characterized in that: The preparation method comprises: The thiolated hyaluronic acid, 5-hydroxymethylfurfural and trifluoroacetic acid are dissolved in water and reacted, and after the reaction is completed, the dialysis is performed for purification to obtain a compound containing a thioacetal bond; The compound containing thioacetal bond, acrylamide, N,N'-methylenebisacrylamide, ammonium persulfate and conductive medium are cross-linked to obtain conductive hydrogel with ROS response.

2. The preparation method according to claim 1, characterized in that: The preparation method of the thiolated hyaluronic acid comprises: Hyaluronic acid, cystamine dihydrochloride and 1-hydroxybenzotriazole hydrate are dissolved in polybutylene succinate, the pH is adjusted, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and dithiothreitol are added to react, and the reaction is performed by dialysis purification to obtain thiolated hyaluronic acid.

3. The preparation method according to claim 2, characterized in that: The pH value is adjusted to be in the range of 7.5-8.

5.

4. The preparation method according to claim 1, characterized in that: When the thiolated hyaluronic acid, 5-hydroxymethylfurfural and trifluoroacetic acid are dissolved in water and reacted, the reaction is carried out at room temperature and away from light.

5. The preparation method according to claim 1, characterized in that: The percentage of acrylamide added is 16-26%.

6. The preparation method according to claim 1, characterized in that: The added amount of the N,N'-methylenebisacrylamide is 10% of the acrylamide monomer.

7. The preparation method according to claim 1, characterized in that: The conductive medium is poly(3,4-ethylenedioxythiophene): polystyrene sulfonate: the added amount is 0.2-1%.

8. The preparation method according to claim 1, characterized in that: When the compound containing thioacetal bond, acrylamide, N,N'-methylenebisacrylamide, ammonium persulfate and conductive medium are cross-linked, the reaction temperature is 60-80° C. and the reaction time is 1.5-4 hours.

9. A conductive hydrogel prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the conductive hydrogel prepared by the preparation method according to any one of claims 1 to 8 or the conductive hydrogel according to claim 9 in the preparation of biomedical materials.

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