PH-sensitive antioxidant hydrogel as well as preparation and application thereof

Abstract

The invention discloses pH-sensitive antioxidant hydrogel as well as preparation and application thereof. The pH-sensitive antioxidant hydrogel comprises a component A, a component B and a gelatinizing solution, wherein the component A is one of chitosan, a chitosan derivative, epsilon-polylysine, hyaluronic acid and collagen; and the component B is one of oligomeric proanthocyanidins and epigallocatechin gallate, and polyvinyl alcohol. The pH-sensitive antioxidant hydrogel can also comprise a drug. The medicine comprises deferoxamine or an insulin growth factor 1. The hydrogel continuously releases effective components to promote angiogenesis, remove ROS (reactive oxygen species) to control inflammation, promote polarization of M2 macrophages and accelerate healing of wounds, especially healing of wounds of diabetic patients, by responding to a low-pH environment at diabetic wounds.

Description

Technical Field

[0001] The present invention relates to the field of biomedical materials, and in particular to a pH-sensitive antioxidant hydrogel and a preparation method and application thereof. Background Art

[0002] With the high incidence of diabetes worldwide, diabetic chronic wound complications have seriously threatened the physical health and quality of life of patients. The process of normal wound healing can be divided into four stages, including hemostasis, inflammation, proliferation, and remodeling, and this process of diabetic wounds is disturbed due to the high systemic blood glucose concentration in diabetic patients. Hyperglycemia leads to excessive glycosylation of proteins, resulting in the expression of proinflammatory cytokines and oxidative damage. Studies have found that the inflammatory phase is the most dysregulated phase in diabetic wound healing. Due to the high-sugar environment unique to diabetes, the polarization of macrophages from M1 (proinflammatory phenotype) to M2 (anti-inflammatory phenotype) is hindered. The accumulation of M1 macrophages leads to the release of proinflammatory cytokines and the production of reactive oxygen species (ROS), resulting in oxidative stress and cell damage, leading to prolonged inflammatory phase and vascular lesions. Therefore, regulating the inflammatory microenvironment of diabetic wounds is a prospective strategy for diabetic wound healing.

[0003] The use of antioxidants to scavenge reactive oxygen species is a common treatment for regulating inflammation in diabetic wound healing. Repeated tissue damage stimulates excessive accumulation of inflammatory cells into the wound, producing high levels of ROS, leading to destruction of the extracellular matrix (ECM). Proteolytic destruction of the ECM in turn recruits more inflammatory cells to the wound, thereby amplifying the inflammatory response and promoting inflammation into a harmful vicious cycle. Therefore, scavenging excessive ROS or enhancing the expression of ROS detoxifying enzymes by antioxidants can reduce inflammation and promote chronic wound healing. In addition, excessive ROS concentrations are also associated with impaired angiogenesis, because ROS-mediated covalent modification of HIF-1α coactivators impairs HIF-1 signaling. Therefore, promoting angiogenesis can effectively regulate the ischemic microenvironment of diabetic wounds, and the formation of new blood vessels can provide nutrients, oxygen, and metabolite exchange to the wound, which is of great significance for wound healing.

[0004] Hydrogels are considered to be one of the most promising types of wound dressings due to their good biocompatibility and moisture retention. However, existing hydrogel dressings have limited efficacy in regulating chronic inflammation and vascular damage. Therefore, there is an urgent need to develop a multifunctional hydrogel that can regulate the chronic inflammatory environment at the diabetic wound, promote angiogenesis, and accelerate the healing rate of diabetic wounds. Summary of the invention

[0005] In view of the problem that diabetic wounds are difficult to heal due to oxidative stress, persistent inflammation and impaired angiogenesis, the purpose of the present invention is to provide a pH-sensitive antioxidant hydrogel that can effectively promote the healing and repair of diabetic wounds. The hydrogel responds to the low pH environment of the diabetic wound through its specific components and the unique hydrogel network structure formed by the specific components, continuously releases active ingredients to promote angiogenesis, removes ROS to control inflammation, promotes the polarization of M2 macrophages, and accelerates the healing of diabetic wounds.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] A pH-sensitive antioxidant hydrogel comprises component A, component B and a gelling liquid. Component A is one of chitosan, chitosan derivatives, ε-polylysine (εPL), hyaluronic acid and collagen (Col). Component B is one of oligomeric proanthocyanidins (OPC), epigallocatechin gallate (EGCG) and polyvinyl alcohol (PVA).

[0008] The pH-sensitive antioxidant hydrogel of the present invention uses a polymer with both biocompatibility and physiological activity as a basic raw material, combined with oligomeric proanthocyanidins or epigallocatechin gallate through bio-friendly chemical crosslinking and physical crosslinking (Schiff base and hydrogen bond crosslinking, etc.), and promotes the accelerated healing of wounds, especially the accelerated healing of diabetic wounds, with its specific components and the unique structure formed by its specific components.

[0009] In order to achieve better technical effects, the following optimization is performed:

[0010] The chitosan derivative can be selected from one of carboxymethyl chitosan (CMCS), hydroxyethyl chitosan, etc. Further preferably: the weight average molecular weight (Mw) of the chitosan is 10kDa-20kDa. The Mw of the carboxymethyl chitosan is 15kDa-20kDa. The Mw of the hydroxyethyl chitosan is 100kDa. The Mw of the ε-polylysine is 2000Da-5000Da. The Mw of the hyaluronic acid is 400kDa-2500kDa. The Mw of the collagen is 1000Da-10000Da. The Mw of the polyvinyl alcohol is 50kDa-100kDa. The oligomeric proanthocyanidins refer to proanthocyanidins with a small molecular weight, usually one or more of dimers to tetramers.

[0011] Preferably, in the pH sensitive antioxidant hydrogel, the weight percentage of component A is 2%-5%, the weight percentage of OPC or EGCG is 0.25%-2%, and the weight percentage of PVA is 0.5%-2%. Further preferably, the balance in the pH sensitive antioxidant hydrogel is gelling liquid and an appropriate amount of pH regulator.

[0012] Preferably, the pH-sensitive antioxidant hydrogel can simultaneously encapsulate active drug molecules to obtain a drug-loaded pH-sensitive antioxidant hydrogel. The drug-loaded pH-sensitive antioxidant hydrogel comprises component A, component B, a drug and a gelling liquid. Further preferably, the drug comprises deferoxamine (DFO) or insulin growth factor 1 (IGF-1).

[0013] Preferably, in the pH sensitive antioxidant hydrogel, the weight percentage of component A is 2%-5%, the weight percentage of OPC or EGCG is 0.25%-2%, the weight percentage of PVA is 0.5%-2%, and the weight percentage of the drug is 0.1%-1%. Further preferably, the balance in the pH sensitive antioxidant hydrogel is a gelling liquid and an appropriate amount of a pH regulator.

[0014] The gelling solution is a hydrogel gelling solution commonly used in the art, and can be deionized water or a buffer solution with a pH value of 7-8.

[0015] The raw materials are all of medical grade.

[0016] The present invention also provides a method for preparing the pH sensitive antioxidant hydrogel, comprising the steps of:

[0017] (1) dissolving component A in a gelling liquid to prepare a component A solution;

[0018] (2) dissolving the oligomeric proanthocyanidins or epigallocatechin gallate in component B in a gelling liquid to prepare a first solution; dissolving the polyvinyl alcohol in component B in a gelling liquid to prepare a second solution; mixing the first solution and the second solution evenly, adjusting the pH value to 7.0-8.0, and obtaining a component B solution;

[0019] (3) The component A solution and the component B solution are mixed evenly and allowed to stand to form a pH-sensitive antioxidant hydrogel.

[0020] The components selected by the present invention can prepare hydrogels in a simple A+B mixing mode, which is simple to operate, environmentally friendly, free of by-products, friendly to the environment and organisms, and suitable for large-scale industrial promotion.

[0021] Preferably, the concentration of component A in the component A solution is 10 mg / mL-100 mg / mL. Further preferably, the concentration of component A is 60 mg / mL-100 mg / mL.

[0022] Preferably, the concentration of OPC or EGCG in the first solution is 10 mg / mL-80 mg / mL (more preferably 20 mg / mL). The concentration of PVA in the second solution is 20 mg / mL-80 mg / mL (more preferably 40 mg / mL).

[0023] When preparing the drug-loaded pH-sensitive antioxidant hydrogel, in step (1), the drug is dissolved in a gel-forming solution to form a drug solution, component A is added to the drug solution, dissolved, and a component A solution containing the drug is obtained. Preferably: the concentration of the drug in the component A solution is 1 mg / mL-10 mg / mL (further preferably 6 mg / mL), and the concentration of component A is 10 mg / mL-100 mg / mL (further preferably 60 mg / mL-100 mg / mL). The concentration of OPC or EGCG in the first solution is 10 mg / mL-80 mg / mL (further preferably 20 mg / mL). The concentration of PVA in the second solution is 20 mg / mL-80 mg / mL (further preferably 40 mg / mL).

[0024] In step (2), the pH value is adjusted by using a common pH adjusting agent such as a NaOH aqueous solution or a HCl aqueous solution. For example, a 2M (mol / L) NaOH aqueous solution or a HCl aqueous solution can be used.

[0025] The volume usage of the component A solution / component A solution containing drugs, the volume usage of the first solution, and the volume usage of the second solution are selected according to their respective concentrations and the content of each component in the pH-sensitive antioxidant hydrogel and the drug-loaded pH-sensitive antioxidant hydrogel, without any other special restrictions.

[0026] The present invention also provides the use of the pH-sensitive antioxidant hydrogel in the preparation of a medicine or medical dressing for promoting wound healing and wound repair, especially in the preparation of a medicine or medical dressing for promoting wound healing and wound repair in patients with diabetes.

[0027] The pH-sensitive antioxidant hydrogel is applied to the wound surface when in use. Under the triggering of the low pH value of the diabetic wound surface, the hydrogel continuously releases drugs, removes free radicals, controls the inflammatory environment at the wound surface, promotes angiogenesis, and accelerates the healing and repair of the diabetic wound surface.

[0028] The raw materials, reagents, etc. of the present invention can all be commercially available products.

[0029] The present invention has the following technical effects:

[0030] (1) The pH-sensitive antioxidant hydrogel of the present invention has pH responsiveness and can respond to the low pH value of the diabetic wound surface and continuously release the active ingredients.

[0031] (2) The pH-sensitive antioxidant hydrogel of the present invention has excellent free radical scavenging ability and excellent antioxidant properties.

[0032] (3) The pH-sensitive antioxidant hydrogel of the present invention can effectively protect cells from oxidative damage.

[0033] (4) The pH-sensitive antioxidant hydrogel of the present invention has good anti-inflammatory ability.

[0034] (5) The pH-sensitive antioxidant hydrogel of the present invention has good biocompatibility.

[0035] (6) The pH-sensitive antioxidant hydrogel of the present invention has the ability to promote cell migration.

[0036] (7) The pH-sensitive antioxidant hydrogel of the present invention has the ability to promote angiogenesis.

[0037] (8) The pH-sensitive antioxidant hydrogel of the present invention can effectively promote wound healing and repair wounds, especially can effectively promote the healing and repair of diabetic wounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 It is the in vitro cumulative release curve of DFO in hydrogel under different pH conditions; wherein, Cumulative release is the cumulative release amount, and Time (h) is the time (hour).

[0039] Figure 2 a is the evaluation diagram of the scavenging efficiency of DPPH free radicals by different hydrogel groups; Figure 2 b is an evaluation diagram of the scavenging efficiency of ABTS free radicals by different hydrogel groups; Scavenging activity is the scavenging rate.

[0040] Figure 3 a is the ROS signal image in cells of each experimental group; Figure 3 b is Figure 3 a is a comparison chart of fluorescence intensity of each experimental group; the Blank group is the blank group: HSF cells without H2O2 stimulation were cultured in normal culture medium; the Control group is the control group: HSF cells stimulated by H2O2 were cultured in normal culture medium.

[0041] Figure 4 a-4d is a comparison of the expression levels of TNF-α, IL-6, IL-4, and IL-10 in the supernatant of RAW264.7 macrophages induced by each experimental group; Concentration is the concentration.

[0042] Figure 5 These are photos of the wound surface of full-thickness wounds in diabetic mice on days 0, 2, 7, and 11 after different treatments.

[0043] Figure 6 is the SEM image of the cross-sectional slice of Col / EGCG / PVA-IGF-1 freeze-dried hydrogel.

[0044] Figure 7 a is the ROS signal image in HSF cells of each experimental group; Figure 7 b is Figure 7 a Comparison of fluorescence intensity of each experimental group.

[0045] Figure 8 a is the effect diagram of HSF cell scratch test treated by each experimental group; Figure 8 b is a quantitative graph of the cell scratch area in each experimental group; Cell scratch area refers to the cell scratch area.

[0046] Fig. 9 It is a graph of the cell viability values ​​of HSF cells in each experimental group detected by CCK-8; wherein Cell Viablity is the cell viability value.

[0047] Fig.10 It is the effect diagram of HSF cell scratch test treated by each experimental group;

[0048] Fig.11 yes Fig.10 Quantification of the relative scratch area in the scratch test; Relative ScratchArea is the relative scratch area.

[0049] Fig.12 a is a representative image of HUVECs seeded on the matrix and formed into tubes after treatment in each experimental group;

[0050] Fig.12 b is a comparison chart of the number of pipe networks in each component;

[0051] Fig.12 c is a comparison chart of the number of nodes of each component tube;

[0052] Fig.12 d is a comparison chart of the number of tubes in each component;

[0053] Among them, Number of nodes is the number of networks that form a pipe, Number of junctions is the number of nodes that form a pipe, and Number of master segments is the number of pipes.

[0054] In the figure, * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001, n=3; letter marking is also used, and there is no significant difference between groups with the same letter, and there is a significant difference between groups with different letters. DETAILED DESCRIPTION

[0055] The present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention.

[0056] Example 1

[0057] Preparation of drug-loaded pH-sensitive antioxidant hydrogel (referred to as CMCS / OPC / PVA-DFO):

[0058] (1) DFO was dissolved in deionized water to prepare a DFO solution, and carboxymethyl chitosan (CMCS, Mw: 15 kDa) was added to the DFO solution to dissolve it to prepare a DFO / CMCS solution (component A solution containing the drug). The concentrations of DFO and CMCS in the solution were 6 mg / mL and 60 mg / mL, respectively.

[0059] (2) OPC (purchased from Pharmacopoeia Pharmaceutical Standards Research and Development (Shanghai) Co., Ltd.) and PVA (Mw: 80 kDa) were dissolved in deionized water to prepare a first solution with a concentration of 20 mg / mL and a second solution with a concentration of 40 mg / mL, respectively. The two solutions were mixed in equal volumes, and the pH value of the solution was adjusted to 7.4 with 2M HCl aqueous solution to form an OPC / PVA solution (component B solution).

[0060] (3) DFO / CMCS solution and OPC / PVA solution were mixed in equal volumes, gently stirred at room temperature to make them evenly mixed, and allowed to stand for about 1 hour to form a pH-sensitive antioxidant hydrogel: CMCS / OPC / PVA-DFO.

[0061] Example 2

[0062] Preparation of pH-sensitive antioxidant hydrogel without DFO (referred to as CMCS / OPC / PVA):

[0063] CMCS (Mw: 15 kDa) was dissolved in deionized water to prepare a 60 mg / mL CMCS solution; OPC (purchased from Pharmacopoeia Pharmaceutical Standard Material Research and Development (Shanghai) Co., Ltd.) and PVA (Mw: 80 kDa) were dissolved in deionized water to prepare a first solution with a concentration of 20 mg / mL and a second solution with a concentration of 40 mg / mL, respectively, and the two were mixed in equal volumes, and the pH value of the solution was adjusted to 7.4 with 2M HCl aqueous solution to form an OPC / PVA solution; CMCS solution and OPC / PVA solution were mixed in equal volumes, gently stirred at room temperature to mix evenly, and allowed to stand for about 1 hour to form a pH-sensitive antioxidant hydrogel: CMCS / OPC / PVA.

[0064] Comparative Example 1

[0065] Preparation of hydrogel without DFO and OPC (referred to as CMCS / PVA):

[0066] CMCS (Mw: 15 kDa) was dissolved in deionized water to prepare a 60 mg / mL CMCS solution; PVA (Mw: 80 kDa) was dissolved in deionized water to prepare a 40 mg / mL PVA solution, and the pH value of the solution was adjusted to 7.4 with a 2M HCl aqueous solution; the CMCS solution and the PVA solution were mixed in equal volumes, gently stirred at room temperature to mix evenly, and allowed to stand for about 1 hour to form a gel: CMCS / PVA.

[0067] Example 3: Properties of the gel

[0068] 1. pH-triggered drug release

[0069] The in vitro drug release behavior of CMCS / OPC / PVA-DFO of Example 1 was characterized under physiological conditions of pH 7.4 and weakly acidic pH 6.5. 1 mL of the prepared hydrogel sample was placed in a brown sample bottle containing 10 mL of PBS (pH 7.4 or 6.5), and the sample bottle was placed in a 37°C water bath to maintain a constant temperature. At the set time point, 2 mL of the release solution was taken out each time, and then 2 mL of fresh release solution was added to keep the total volume unchanged. The DFO content in the release solution was determined by high performance liquid chromatography. Figure 1 As shown, it can be observed that DFO is continuously released from the hydrogel without obvious burst release. Since the imine bond is unstable under weak acid conditions, the hydrogel is released faster under phosphate buffer (PBS) pH 6.5 conditions, showing a low pH-triggered drug release behavior. The data of the final cumulative release amount show that after 72 hours of continuous release, the hydrogel finally released 84.7% of DFO under PBS pH 7.4 conditions, and under PBS pH 6.5 conditions, the release of DFO in the hydrogel can reach 96.5%, indicating that the pH-sensitive antioxidant hydrogel of the present invention has pH responsiveness, can respond to the low pH value of diabetic wounds, continuously release drugs and significantly increase the cumulative release of drugs per unit time.

[0070] The cross-linked structure of the CMCS / OPC / PVA hydrogel in Example 2 is the same as that of the CMCS / OPC / PVA-DFO hydrogel in Example 1, and both are cross-linked through imine bonds. The imine bonds are pH-responsive and have the ability to trigger drug release by pH.

[0071] 2. Free radical scavenging ability

[0072] By DPPH and ABTS ·+ Experiments were performed to evaluate the free radical scavenging ability of hydrogels. Figure 2As shown in a and 2b, after 24 hours of incubation, the CMCS / OPC / PVA-DFO hydrogel (drug-loaded) in Example 1 and the CMCS / OPC / PVA hydrogel (drug-free) in Example 2 both had significant antioxidant properties; the DPPH free radical scavenging rate of the CMCS / OPC / PVA hydrogel in Example 2 was 84.2%±2.2%, and the ABTS ·+ The free radical scavenging rate was 72.6%±3.4%; the DPPH free radical scavenging rate of CMCS / OPC / PVA-DFO hydrogel in Example 1 was 77.9%±3.5%, and the ABTS ·+ The free radical scavenging rate was 69.5% ± 8.0%, which was significantly higher than that of the CMCS / PVA hydrogel sample in Comparative Example 1 (DPPH free radical scavenging rate was 14.3% ± 1.5%, ABTS ·+ The free radical scavenging rate was 28.4%±5.1%), and there were significant differences (DPPH free radical scavenging rate: CMCS / OPC / PVA group compared with CMCS / PVA group p<0.001, CMCS / OPC / PVA-DFO group compared with CMCS / PVA group p<0.001; ABTS ·+ Free radical scavenging rate: p<0.001 for the CMCS / OPC / PVA group compared with the CMCS / PVA group, p<0.001 for the CMCS / OPC / PVA-DFO group compared with the CMCS / PVA group); this shows that the pH-sensitive antioxidant hydrogel of the present invention has excellent free radical scavenging ability and excellent antioxidant properties.

[0073] 3. Ability to remove intracellular reactive oxygen species

[0074] The prepared hydrogel was immersed in a PBS solution (pH 7.4) at 37° C. for 24 h, with the volume ratio of hydrogel to PBS solution being 1:10, to obtain a hydrogel extract.

[0075] Human skin fibroblasts (HSF cells) were used to test the intracellular ROS scavenging ability of the hydrogel. HSF cells were cultured at a rate of 1×10 4Cells / well were seeded in a 24-well plate and cultured in a cell culture incubator with DMEM medium for 24 hours. The medium was removed and replaced with DMEM medium containing 100μM H2O2. After incubation for 2 hours, the medium was removed and replaced with different hydrogel extracts. The cells were co-cultured in a cell culture incubator for 24 hours. The hydrogel extract was removed and incubated with DCFH-DA (10μM, serum-free DMEM) medium for 20 minutes. The cells were observed under a fluorescence microscope at an excitation wavelength of 488nm. A blank group (not treated with H2O2, cultured with normal cell culture medium: DMEM medium) and a control group (cells treated with H2O2 were cultured with normal cell culture medium: DMEM medium instead of hydrogel extract) were designed as controls. Figure 3 a and Figure 3 As shown in b, after HSF cells were stimulated with H2O2, the level of ROS increased significantly, while only a small amount of green fluorescence was observed in the cells in the blank group that were not stimulated with H2O2. When the cells stimulated with H2O2 were treated with the hydrogel extract, the green fluorescence of HSF cells in the control group was significantly reduced, especially in the experimental group containing OPC components in the hydrogel: the ROS levels of CMCS / OPC / PVA-DFO hydrogel in Example 1 and CMCS / OPC / PVA hydrogel in Example 2 were significantly weakened and the difference was significant (CMCS / OPC / PVA-DFO compared with the control group p<0.01, CMCS / OPC / PVA compared with the control group p<0.01); compared with the CMCS / PVA hydrogel in Comparative Example 1, the CMCS / The ROS levels of OPC / PVA-DFO hydrogel and CMCS / OPC / PVA hydrogel in Example 2 were significantly weakened and the differences were significant (CMCS / OPC / PVA-DFO compared with the control group p<0.01, CMCS / OPC / PVA compared with the control group p<0.01); and the ROS levels of CMCS / OPC / PVA-DFO hydrogel in Example 1 and CMCS / OPC / PVA hydrogel in Example 2 were equivalent to those of the blank group; indicating that the hydrogel of the present invention can effectively remove intracellular ROS and protect cells from oxidative damage, which is consistent with the free radical scavenging efficiency results.

[0076] 4. Anti-Inflammatory Capabilities

[0077] Raw264.7 cells were seeded in a new 6-well plate and cultured with DMEM medium for 12 hours. After incubation for 24 hours, the cells were replaced with lipopolysaccharide (LPS) solution (1 mL, 1 μg / mL prepared in serum-free DMEM medium). The LPS solution was removed, the cells were collected and seeded on the 1 mL hydrogel surface in a 6-well plate. The cells treated with LPS for 24 hours were treated with fresh DMEM medium (1 mL) as a control group (Control). After 72 hours, the concentrations of pro-inflammatory cytokines (IL-6 and TNF-α) and anti-inflammatory cytokines (IL-4 and IL-10) in the supernatant of the cell culture medium were determined using ELISA kits. Figure 4As shown in a-4d, compared with the control group, the expression levels of IL-6 and TNF-α in the CMCS / PVA hydrogel group were decreased but without significant difference, and the expression levels of IL-4 and IL-10 were increased but without significant difference; the expression levels of IL-6 and TNF-α in the CMCS / OPC / PVA and CMCS / OPC / PVA-DFO hydrogel groups were significantly decreased (IL-6: CMCS / OPC / PVA compared with the control group p<0.05, CMCS / OPC / PVA-DFO compared with the control group p<0.05; TNF-α : CMCS / OPC / PVA compared with the control group p<0.05, CMCS / OPC / PVA-DFO compared with the control group p<0.05), the expression levels of IL-4 and IL-10 were significantly increased (IL-4: CMCS / OPC / PVA compared with the control group p<0.05, CMCS / OPC / PVA-DFO compared with the control group p<0.05; IL-10: CMCS / OPC / PVA compared with the control group p<0.05, CMCS / OPC / PVA-DFO compared with the control group p<0.05 ); compared with the CMCS / PVA group, the expression levels of IL-6 and TNF-α in the CMCS / OPC / PVA and CMCS / OPC / PVA-DFO hydrogel groups were significantly decreased, and the expression levels of IL-4 and IL-10 were significantly increased (TNF-α: CMCS / OPC / PVA compared with CMCS / PVA p<0.05, CMCS / OPC / PVA-DFO compared with CMCS / PVA p<0.05; IL-6: CMCS / OPC / PVA compared with CMCS / PVA p<0.05, C MCS / OPC / PVA-DFO compared with CMCS / PVA p<0.05; IL-4: CMCS / OPC / PVA compared with CMCS / PVA p<0.05, CMCS / OPC / PVA-DFO compared with CMCS / PVA p<0.05; IL-10: CMCS / OPC / PVA compared with CMCS / PVA p<0.05, CMCS / OPC / PVA-DFO compared with CMCS / PVA p<0.05;); indicating that the hydrogel system of the present invention has good anti-inflammatory activity.

[0078] 5. Evaluation of the ability to promote diabetic wound healing

[0079] The wound healing effect of the hydrogel was evaluated using a diabetic full-thickness wound mouse model. The STZ-induced diabetic animal model of BALB / c mice was established by intraperitoneal injection of streptozotocin (STZ) for 5 consecutive days. Mice with blood glucose levels above 300 mg / dL for two consecutive weeks were tested. The diabetic mice were randomly divided into 4 groups (n=7). The mice were anesthetized with chloral hydrate (5%, mass percentage) in saline solution, and the hair on the back of the mice was shaved. Then, a full-thickness skin wound with a diameter of 4 mm was made on the back of the mouse. The wound was immediately treated with PBS (pH 7.4, rinsing the wound) and different hydrogels (applying hydrogel on the wound). The wound healing was observed regularly and recorded with a camera. Figure 5 It is a representative image of the wound site during wound healing. As can be seen from the figure, the wound contraction speed of the CMCS / OPC / PVA and CMCS / OPC / PVA-DFO hydrogel-treated groups was significantly faster than that of the control group and the CMCS / PVA group; after 11 days of observation, the control group still had a large area of ​​dry scab and a larger wound area, the CMCS / PVA group had a smaller area of ​​dry scab and a smaller wound area than the control group, the CMCS / OPC / PVA group and the CMCS / OPC / PVA-DFO group were basically healed, and the healing of the CMCS / OPC / PVA-DFO group was faster and better than that of the CMCS / OPC / PVA group, indicating that the hydrogel of the present invention can significantly promote the healing of diabetic wounds, especially CMCS / OPC / PVA-DFO has a better ability to promote the healing of diabetic wounds.

[0080] Example 4: Preparation of drug-loaded pH-sensitive antioxidant hydrogel (denoted as Col / EGCG / PVA-IGF-1):

[0081] (1) Insulin-like growth factor 1 (IGF-1) was dissolved in deionized water to prepare an IGF-1 solution. Collagen (Col, Mw: 5000Da) was added to the IGF-1 solution to dissolve it and prepare an IGF-1 / Col solution. The concentrations of IGF-1 and Col in the solution were 6 mg / mL and 100 mg / mL, respectively.

[0082] (2) EGCG and PVA (Mw: 80 kDa) were dissolved in deionized water to prepare a first solution with a concentration of 20 mg / mL and a second solution with a concentration of 40 mg / mL, respectively. The two solutions were mixed in equal volumes, and the pH value of the solution was adjusted to 7.4 with a 2M HCl aqueous solution to form an EGCG / PVA solution.

[0083] (3) IGF-1 / Col solution and EGCG / PVA solution were mixed in equal volumes, gently stirred at room temperature to make them evenly mixed, and allowed to stand for about 1 hour to form a pH-sensitive antioxidant hydrogel: Col / EGCG / PVA-IGF-1.

[0084] Example 5

[0085] Preparation of pH-sensitive antioxidant hydrogel without IGF-1 (denoted as Col / EGCG / PVA):

[0086] Col (Mw: 5000Da) was dissolved in deionized water to prepare a 100 mg / mL Col solution; EGCG and PVA (Mw: 80kDa) were dissolved in deionized water to prepare a first solution with a concentration of 20 mg / mL and a second solution with a concentration of 40 mg / mL, respectively, and the two were mixed in equal volumes, and the pH value of the solution was adjusted to 7.4 with a 2M HCl aqueous solution to form an EGCG / PVA solution; the Col solution and the EGCG / PVA solution were mixed in equal volumes, gently stirred at room temperature to mix evenly, and allowed to stand for about 1 hour to form a pH-sensitive antioxidant hydrogel: Col / EGCG / PVA.

[0087] Comparative Example 2

[0088] Preparation of hydrogel without IGF-1 and EGCG (denoted as Col / PVA):

[0089] Col (Mw: 5000Da) was dissolved in deionized water to prepare a 100 mg / mL Col solution; PVA (Mw: 80kDa) was dissolved in deionized water to prepare a 40 mg / mL solution, and the pH value of the solution was adjusted to 7.4 with a 2M HCl aqueous solution to form a PVA solution; the Col solution and the PVA solution were mixed in equal volumes, gently stirred at room temperature to mix evenly, and allowed to stand for about 1 hour to form a gel: Col / PVA.

[0090] Example 6: Properties of the gel

[0091] 1. pH-triggered drug release

[0092] The cross-linked structure of the Col / EGCG / PVA-IGF-1 hydrogel of Example 4 is the same as that of the Col / EGCG / PVA hydrogel of Example 5, and both are cross-linked by imine bonds, which are pH-responsive and have the ability to trigger drug release by pH. The in vitro drug release behavior of the Col / EGCG / PVA-IGF-1 hydrogel of Example 4 was characterized under physiological conditions of pH 7.4 and weakly acidic pH 6.5, and it can be observed that IGF-1 is continuously released from the hydrogel without obvious burst release. Since the imine bond is unstable under weak acid conditions, the hydrogel is released faster under phosphate buffer (PBS) pH 6.5 conditions, showing a drug release behavior triggered by low pH.

[0093] 2. SEM images of Col / EGCG / PVA-IGF-1

[0094] The Col / EGCG / PVA-IGF-1 hydrogel prepared in Example 4 was freeze-dried, and the cross-sectional morphology of the hydrogel was observed using a Hitachi S-4300 field emission scanning electron microscope (NovaNanoSEM 450, FEI, USA) at an operating voltage of 15 kV. The SEM image of the cross-sectional slice of the freeze-dried hydrogel (see Figure 6 ) shows that the hydrogel of the present invention has a continuous porous network structure, which is beneficial to the transfer and transportation of substances.

[0095] 3. Characterization of intracellular reactive oxygen species scavenging ability

[0096] HSF cells were plated at 5 × 10 4 Each well was inoculated in a 24-well plate, cultured with DMEM medium for 24 hours, then replaced with DMEM medium containing 100 μM H2O2, incubated at 37°C for 2 hours, and each well was washed 3 times with PBS (pH 7.4). The PBS (pH 7.4) extract of the hydrogel for 48 hours (hydrogel: PBS = 1:10, v / v) was mixed with complete medium (calf serum mass percentage 10%) in a ratio of 1:9 (v / v), and incubated at 37°C for 24 hours. Each well was incubated with 500 μL of serum-free DMEM medium containing 5 μM DCFH-DA at 37°C in the dark for 20 minutes, and the plate was washed 5 times with complete medium (calf serum mass percentage 10%) and observed with a fluorescence microscope at 488 nm. Figure 7 a and Figure 7 As shown in Figure b, bright fluorescence was observed in HSF cells treated with H2O2 (Control group), indicating the generation of intracellular ROS. Compared with the Control group, the fluorescence intensity of HSF cells treated with Col / PVA hydrogel was slightly weakened (p>0.05), the fluorescence intensity of HSF cells treated with Col / EGCG / PVA hydrogel was significantly weakened (p<0.001), and the fluorescence of HSF cells treated with Col / EGCG / PVA-IGF-1 hydrogel was significantly quenched (p<0.001); compared with the Col / PVA group, the fluorescence intensity of HSF cells treated with Col / EGCG / PVA hydrogel was significantly weakened (p<0.001), and the fluorescence of HSF cells treated with Col / EGCG / PVA-IGF-1 hydrogel was significantly quenched (p<0.001); indicating that the hydrogel of the present invention has excellent antioxidant ability and can protect cells from oxidative damage.

[0097] 4. Characterization of cell migration ability

[0098] The prepared hydrogel was immersed in a PBS solution (pH 7.4) at 37° C. for 48 h, with the volume ratio of hydrogel to PBS solution being 1:10, to obtain a hydrogel extract.

[0099] The cell scratch test was used to characterize the ability of the hydrogel to promote cell migration. A vertical scratch was made in a 6-well plate filled with HSF cells using a 10μL pipette tip, and different hydrogel extracts were added for culture. At the preset time points, the changes in the scratch area were observed under a microscope. Figure 8 As can be seen from Figures 8a and 8b, after 24 hours of incubation, compared with the control group (Control), the cell-free area of ​​the Col / PVA-treated group was only slightly reduced (p>0.05), the cell-free area of ​​the Col / EGCG / PVA-treated group was significantly reduced (p<0.05), and the cells of the Col / EGCG / PVA-IGF-1-treated group had basically covered the scratch area (p<0.01); compared with the Col / PVA-treated group, the cell-free area of ​​the Col / EGCG / PVA-treated group was significantly reduced (p<0.05), and the cells of the Col / EGCG / PVA-IGF-1-treated group had basically covered the scratch area (p<0.01); indicating that the hydrogel of the present invention has a good ability to promote HSF cell migration.

[0100] Example 7: Preparation of pH-sensitive antioxidant hydrogel (denoted as εPL / PVA / OPC-DFO):

[0101] (1) DFO was dissolved in deionized water to prepare a DFO solution, and ε-polylysine (εPL, Mw: 5000Da) was added to the DFO solution to dissolve it to prepare a DFO / εPL solution. The concentrations of DFO and εPL in the solution were 6 mg / mL and 60 mg / mL, respectively.

[0102] (3) OPC and PVA (Mw: 80 kDa) were dissolved in deionized water to prepare a first solution with a concentration of 20 mg / mL and a second solution with a concentration of 40 mg / mL, respectively. The two solutions were mixed in equal volumes, and the pH value of the solution was adjusted to 7.4 with a 2M HCl aqueous solution to form an OPC / PVA solution.

[0103] (4) DFO / εPL solution and OPC / PVA solution were mixed in equal volumes, gently stirred at room temperature to make them evenly mixed, and allowed to stand for about 1 hour to form a pH-sensitive antioxidant hydrogel: εPL / PVA / OPC-DFO.

[0104] Example 8 Preparation of an empty hydrogel without DFO (referred to as εPL / PVA / OPC):

[0105] εPL (Mw: 5000Da) was dissolved in deionized water to prepare a 60 mg / mL εPL solution. OPC and PVA (Mw: 80 kDa) were dissolved in deionized water to prepare a first solution with a concentration of 20 mg / mL and a second solution with a concentration of 40 mg / mL, respectively. The two solutions were mixed in equal volumes, and the pH value of the solution was adjusted to 7.4 with a 2M HCl aqueous solution to form an OPC / PVA solution. The εPL solution and the OPC / PVA solution were mixed in equal volumes, gently stirred at room temperature to mix evenly, and allowed to stand for about 1 hour to form a pH-sensitive antioxidant hydrogel: εPL / PVA / OPC.

[0106] Comparative Example 3 Preparation of hydrogel (denoted as εPL / PVA) without DFO and OPC:

[0107] εPL (Mw: 5000Da) was dissolved in deionized water to prepare a 60 mg / mL εPL solution; PVA (Mw: 80kDa) was dissolved in deionized water to prepare a 40 mg / mL solution, and the pH value of the solution was adjusted to 7.4 with a 2M HCl aqueous solution to form a PVA solution; the εPL solution and the PVA solution were mixed in equal volumes, gently stirred at room temperature to mix evenly, and allowed to stand for about 1 hour to form a gel: εPL / PVA.

[0108] Example 9: Properties of the gel

[0109] 1. pH-triggered drug release

[0110] The εPL / PVA / OPC-DFO hydrogel of Example 7 has the same cross-linking structure as the εPL / PVA / OPC hydrogel of Example 8, and both are cross-linked by imine bonds, which are pH-responsive and have the ability to trigger drug release by pH. Under physiological conditions of pH 7.4 and weakly acidic pH 6.5, the in vitro drug release behavior of the εPL / PVA / OPC-DFO hydrogel of Example 7 was characterized, and it can be observed that DFO is continuously released from the hydrogel without obvious burst release. Since the imine bond is unstable under weak acid conditions, the hydrogel is released faster under phosphate buffer (PBS) pH 6.5 conditions, showing a drug release behavior triggered by low pH.

[0111] 2. Biocompatibility

[0112] Human skin fibroblasts (HSF) were used to evaluate the biocompatibility of the hydrogel. 1 mL of the prepared hydrogel was immersed in 2 mL of PBS solution (pH 7.4) at 37 °C for 24 h to obtain the hydrogel extract. HSF cells were cultured at a rate of 5 × 10 3The cells were inoculated in 96-wells at a ratio of 1 cell / well, and cultured normally with DMEM medium for 24 hours until the cells were completely attached to the wall. The DMEM medium was then removed, and an leaching medium consisting of 10 μL hydrogel leaching solution + 90 μL DMEM medium was added to each well for culture. HSF cells were cultured with the leaching medium for several days, and the leaching medium was replaced every other day. On the 1st, 3rd, and 5th days, the leaching medium was removed, and the cells were washed twice with phosphate buffer solution (PBS) (pH 7.4), and then 100 μL of DMEM medium containing 10 wt% (mass percentage) CCK-8 was added. After incubation for 2 hours, the absorbance of each well was recorded at 450 m using an ELISA reader. The leaching medium was replaced with DMEM medium as a control group (Control). The cell viability (CellViability) was calculated using the following formula:

[0113]

[0114] The results of cell viability assay using CCK-8 were as follows: Fig. 9 As shown. The cell survival rate of HSF cells continued to increase over time, and the εPL / PVA / OPC-DFO hydrogel showed good biocompatibility. The cell viability of HSF cells in the εPL / PVA / OPC-DFO group on the 5th day was 2.1 times that of the cell viability on the 1st day, the cell viability of HSF cells in the control group on the 5th day was only 1.8 times that of the cell viability on the 1st day, and the cell viability of HSF cells in the εPL / PVA group on the 5th day was only 1.8 times that of the cell viability on the 1st day; on the 5th day, the cell viability of HSF cells in the εPL / PVA / OPC-DFO group was significantly different from that of the εPL / PVA group and the control group (p<0.05); This shows that the hydrogel system of the present invention is conducive to cell growth and has a significant effect.

[0115] 2. Promote cell migration ability

[0116] The prepared hydrogel was immersed in a PBS solution (pH 7.4) at 37° C. for 48 h, with the volume ratio of hydrogel to PBS solution being 1:10, to obtain a hydrogel extract.

[0117] The effect of hydrogel on HSF cell migration was investigated by in vitro cell scratch test. A vertical scratch was made in a 6-well plate filled with HSF cells using a 10μL pipette tip, and different hydrogel extracts were added for culture. The changes in the scratch area were observed under a microscope at preset time points. Fig.10 and Fig.11It can be seen that after 24 hours, the relative scratch areas of the control group, εPL / PVA group, εPL / PVA / OPC group and εPL / PVA / OPC-DFO group were 52.9%, 50.2%, 47.2% and 45.4%, respectively. After 48 hours, the scratches in the control group and εPL / PVA group were still there and the area was large, the scratch area in the εPL / PVA / OPC group was small, and the scratches in the εPL / PVA / OPC-DFO group had disappeared. There were significant differences in the scratch area between the εPL / PVA / OPC-DFO group and the εPL / PVA / OPC group, and between the εPL / PVA group and the control group (p<0.001 between the εPL / PVA / OPC-DFO group and the εPL / PVA group; p<0.001 between the εPL / PVA / OPC-DFO group and the εPL / PVA group; p<0.001 between the εPL / PVA / OPC-DFO group and the control group), and there were significant differences in the scratch area between the εPL / PVA / OPC group and the εPL / PVA group and the control group (p<0.05 between the εPL / PVA / OPC group and the εPL / PVA group; p<0.01 between the εPL / PVA / OPC group and the control group). This indicates that the hydrogel of the present invention can effectively promote the migration of HSF cells and has a good in vitro repair-promoting effect.

[0118] 3. Characterization of the ability to promote tubulogenesis in human umbilical vein endothelial cells (HUVEC)

[0119] The prepared hydrogel was immersed in a PBS solution (pH 7.4) at 37° C. for 48 h, with the volume ratio of hydrogel to PBS solution being 1:10, to obtain a hydrogel extract.

[0120] The ability of the hydrogel to promote tubule formation was characterized by treating HUVECs seeded on Matrigel with hydrogel extract; the control group was not treated with hydrogel extract and was directly incubated with DMEM medium. Fig.12 a It can be seen that HUVECs in all experimental groups initially formed a network of blood vessels after 24 hours of treatment, among which the densest tubular network was in the εPL / PVA / OPC-DFO group, which was significantly better than the other experimental groups with a smaller tubular network density (εPL / PVA / OPC group) and two relatively sparse experimental groups (Control group and εPL / PVA group). The number of tubular networks in each experimental group was quantitatively analyzed by image J software ( Fig.12 b) Number of nodes ( Fig.12 c) and the number of tubes ( Fig.12d). The results showed that εPL / PVA / OPC-DFO hydrogel could effectively enhance the ability of HUVEC to form capillary-like structures, which was significantly better than that of the Control group, εPL / PVA group and εPL / PVA / OPC group. The ability of εPL / PVA / OPC hydrogel to enhance the ability of HUVEC to form capillary-like structures was better than that of the Control group and εPL / PVA group.

[0121] The pH sensitive antioxidant hydrogel of the present invention is based on a polymer with both biocompatibility and physiological activity, combined with oligomeric proanthocyanidins or epigallocatechin gallate through bio-friendly chemical crosslinking and physical crosslinking (crosslinking such as Schiff base and hydrogen bond), and its specific components and the unique structure formed by its specific components promote the accelerated healing of wounds, especially the accelerated healing of wounds of diabetic patients. Therefore, the combination of the components described in the present invention can basically achieve the technical effect of the present invention. In order to achieve better technical effects, the preferred changes in parameters such as the molecular weight of the polymer component and the content of each component do not affect the acquisition of the hydrogel of the present invention. Therefore, any combination of the molecular weight of the polymer component and the content of each component described in the present invention can obtain a hydrogel that promotes the accelerated healing of wounds, especially the accelerated healing of wounds of diabetic patients. They will not be listed one by one here.

Claims

1. A pH-sensitive antioxidant hydrogel, characterized in that: The invention comprises component A, component B and a gelling liquid; the component A is one of chitosan, chitosan derivatives, ε-polylysine, hyaluronic acid and collagen; the component B is one of oligomeric proanthocyanidins, epigallocatechin gallate and polyvinyl alcohol.

2. The pH sensitive antioxidant hydrogel according to claim 1, characterized in that In the pH sensitive antioxidant hydrogel, the weight percentage of component A is 2%-5%; the weight percentage of oligomeric proanthocyanidins or epigallocatechin gallate is 0.25%-2%; and the weight percentage of polyvinyl alcohol is 0.5%-2%.

3. The pH sensitive antioxidant hydrogel according to claim 1, characterized in that The pH sensitive antioxidant hydrogel further comprises a drug, wherein the drug comprises deferoxamine or insulin growth factor-1.

4. The pH sensitive antioxidant hydrogel according to claim 3, characterized in that In the pH sensitive antioxidant hydrogel, the weight percentage of component A is 2%-5%; the weight percentage of oligomeric proanthocyanidins or epigallocatechin gallate is 0.25%-2%, the weight percentage of polyvinyl alcohol is 0.5%-2%; and the weight percentage of the drug is 0.1%-1%.

5. The method for preparing the pH sensitive antioxidant hydrogel according to any one of claims 1 to 4, characterized in that: Includes steps: (1) dissolving component A in a gelling liquid to prepare a component A solution; (2) dissolving the oligomeric proanthocyanidins or epigallocatechin gallate in component B in a gelling liquid to prepare a first solution; dissolving the polyvinyl alcohol in component B in a gelling liquid to prepare a second solution; The first solution and the second solution are mixed evenly, and the pH value is adjusted to 7.0-8.0 to obtain a component B solution; (3) The component A solution and the component B solution are mixed evenly and allowed to stand to form a pH-sensitive antioxidant hydrogel.

6. The preparation method according to claim 5, characterized in that: The concentration of component A in the component A solution is 10 mg / mL-100 mg / mL; The concentration of oligomeric proanthocyanidins or epigallocatechin gallate in the first solution is 10 mg / mL-80 mg / mL; the concentration of polyvinyl alcohol in the second solution is 20 mg / mL-80 mg / mL.

7. The preparation method according to claim 5, characterized in that: In step (1), the drug is dissolved in a gelling liquid to prepare a drug solution, and component A is added to the drug solution and dissolved to obtain a component A solution containing the drug.

8. The preparation method according to claim 7, characterized in that: The concentration of the drug in the component A solution is 1 mg / mL-10 mg / mL, and the concentration of component A is 10 mg / mL-100 mg / mL; The concentration of oligomeric proanthocyanidins or epigallocatechin gallate in the first solution is 10 mg / mL-80 mg / mL; the concentration of polyvinyl alcohol in the second solution is 20 mg / mL-80 mg / mL.

9. Use of the pH-sensitive antioxidant hydrogel according to any one of claims 1 to 4 in the preparation of a medicine or medical dressing for promoting wound healing or wound repair.

10. Use of the pH-sensitive antioxidant hydrogel according to any one of claims 1 to 4 in the preparation of a medicine or medical dressing for promoting wound healing or repairing of diabetic patients' wounds.

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