Hydrogel dressing as well as preparation method and application thereof

Abstract

The invention relates to the field of hydrogel materials, in particular to a hydrogel dressing as well as a preparation method and application thereof. The deferoxamine mesylate is loaded on the gelatin microsphere material constructed by the gelatin and the genipin, so that the loading dispersion of the deferoxamine mesylate can be benefited, and the added gelatin can be in contact with matrix proteinase-9 in a diabetic foot ulcer microenvironment for degradation so as to induce the release of the deferoxamine mesylate. A first cross-linked network is formed by the 3-carboxyphenylboronic acid modified chitosan and the oxidized hyaluronic acid, and the gelatin microsphere material loaded with the deferoxamine mesylate and the epigallocatechin gallate are coated with the first cross-linked network. The hydrogel dressing realizes controllable release of the deferoxamine mesylate and the epigallocatechin gallate, so that the technical problem of uncontrollable release of the deferoxamine mesylate is solved.

Description

Technical Field

[0001] The invention relates to the field of hydrogel materials, and in particular to a hydrogel dressing and a preparation method and application thereof. Background Art

[0002] Diabetic foot ulcer is a serious complication of diabetes, mainly refers to the foot neuropathy and vascular disease caused by diabetes, which leads to foot skin damage, infection and ulcer formation. Diabetic foot ulcer is easy to get infected, very difficult to cure, and may lead to amputation in severe cases. The blood supply of diabetic foot ulcer is poor, angiogenesis is blocked, the microcirculation of the foot end is insufficient, and the wound surface does not heal for a long time.

[0003] Currently, the common dressings used for wound treatment generally have moisturizing and antibacterial effects, and can have a certain therapeutic effect on common wounds, but it is difficult to achieve effective therapeutic effects on diabetic foot ulcers.

[0004] Deferoxamine mesylate is an angiogenic agent used for ischemia and wound healing. In ischemic disease models, deferoxamine mesylate can upregulate the expression of vascular endothelial growth factor, stimulate blood perfusion, and promote blood vessel growth. However, when deferoxamine mesylate is used in actual treatment, due to the problem of uncontrolled release of deferoxamine mesylate, its drug action time is short, and it is difficult to achieve effective treatment effects on diabetic foot ulcers. Summary of the invention

[0005] The purpose of the present invention is to overcome the deficiencies of the prior art, provide a hydrogel dressing and a preparation method and application thereof, and solve the problem of uncontrolled release of deferoxamine mesylate.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] A method for preparing a hydrogel dressing comprises the following steps:

[0008] The gelatin and deferoxamine mesylate aqueous solution are used as the aqueous phase for emulsification in an oil phase environment to obtain a gelatin microsphere precursor, the gelatin microsphere precursor and genipin are mixed, wherein the gelatin and genipin undergo a cross-linking reaction to obtain a gelatin microsphere material loaded with deferoxamine mesylate; epigallocatechin gallate is mixed with an oxidized hyaluronic acid solution to obtain a first prepolymer solution; the gelatin microsphere material loaded with deferoxamine mesylate is mixed with a 3-carboxyphenylboronic acid modified chitosan solution to obtain a second prepolymer solution; the second prepolymer solution is prepared by mixing the gelatin microsphere material loaded with deferoxamine mesylate with a 3-carboxyphenylboronic acid modified chitosan solution. A prepolymer liquid and a second prepolymer liquid are mixed, wherein the oxidized hyaluronic acid reacts with the 3-carboxyphenylboronic acid modified chitosan to form a first cross-linked network containing Schiff base bonds, and the first cross-linked network is coated with the gelatin microsphere material and the epigallocatechin gallate, and at the same time, the epigallocatechin gallate reacts with the 3-carboxyphenylboronic acid modified chitosan to form a second cross-linked network containing borate ester bonds; and a hydrogel dressing is prepared.

[0009] There is a large amount of MMP-9 in the microenvironment of diabetic foot ulcers, and gelatin is an inherent degradation substrate of MMP-9, so the gelatin microspheres in the gelatin microsphere material loaded with deferoxamine mesylate will degrade after contact with MMP-9. Among them, MMP-9 refers to matrix proteinase-9, which belongs to the matrix proteinase family.

[0010] Among them, epigallocatechin gallate can promote the expression of vascular endothelial growth factor by reducing the expression of reactive oxygen species, thereby promoting angiogenesis.

[0011] The present invention not only facilitates the load dispersion of deferoxamine mesylate by loading deferoxamine mesylate on a gelatin microsphere material constructed of gelatin and genipin, but also the added gelatin can contact and degrade with matrix proteinase-9 in the microenvironment of diabetic foot ulcers to induce the release of deferoxamine mesylate. In addition, the present invention forms a first cross-linked network by 3-carboxyphenylboronic acid-modified chitosan and oxidized hyaluronic acid, and the first cross-linked network is coated with a gelatin microsphere material loaded with deferoxamine mesylate and epigallocatechin gallate, so that the hydrogel dressing realizes the controlled release of deferoxamine mesylate and epigallocatechin gallate, so as to solve the technical problem of uncontrolled release of deferoxamine mesylate.

[0012] Optionally, the preparation method of the gelatin microsphere material loaded with deferoxamine mesylate comprises:

[0013] Deferoxamine mesylate, gelatin and water are used as the aqueous phase, and Span-80 and vegetable oil are used as the oil phase. The aqueous phase and the oil phase are mixed and emulsified to prepare a gelatin microsphere precursor; the gelatin microsphere precursor is mixed with a phosphate buffer solution and then mixed with genipin, wherein the gelatin and the genipin react to form a third cross-linked network, and the gelatin microsphere material loaded with deferoxamine mesylate is prepared.

[0014] Optionally, the vegetable oil can be any one of corn oil, castor oil, sesame oil and olive oil. The main function of vegetable oil is to serve as a solvent for Span-80. The type of vegetable oil has no significant effect on the dissolution effect of Span-80, so common types of vegetable oils all fall within the scope of protection of the present invention.

[0015] Optionally, the mass ratio of the gelatin to the ammonium mesylate is 1000-3000:1, the mass ratio of the water to the gelatin is 5-10:1, and the reaction temperature of the aqueous phase is 45°C-60°C. The mass ratio of the vegetable oil to the Span-80 is 50-200:1, the volume ratio of the aqueous phase to the oil phase is 1:5-20, the reaction time of the emulsification reaction is 15min-30min, and the cooling temperature is 0°C-7°C. After the gelatin microsphere precursor is mixed with the phosphate buffer solution, the concentration of the gelatin microsphere precursor is 10mg·mL -1 ~15 mg mL -1 The mass fraction of genipin in the gelatin microsphere material loaded with deferoxamine mesylate is 0.5% to 1.5%. Preferably, the mass ratio of the gelatin to the deferoxamine mesylate is 1000 to 2000:1.

[0016] Optionally, before the preparation of the first prepolymer solution and the second prepolymer solution, the process further comprises:

[0017] The epigallocatechin gallate, the oxidized hyaluronic acid, the gelatin microsphere material loaded with deferoxamine mesylate, and the 3-carboxyphenylboronic acid modified chitosan are sterilized.

[0018] Optionally, the preparation method of the oxidized hyaluronic acid comprises:

[0019] The hyaluronic acid solution and the sodium periodate solution are mixed, and then mixed with ethylene glycol to react in the dark, and the reaction is terminated after stirring to obtain an oxidized hyaluronic acid precursor; the oxidized hyaluronic acid precursor is freeze-dried and dialyzed to obtain oxidized hyaluronic acid.

[0020] Optionally, in the hyaluronic acid solution, the mass ratio of the solvent to the hyaluronic acid is 40 to 200: 1. In the sodium periodate solution, the mass ratio of the solvent to the sodium periodate is 4.5 to 5.5: 1. The volume ratio of the hyaluronic acid solution to the sodium periodate solution is 10 to 30: 1, and the ratio of the sum of the volumes of the hyaluronic acid solution and the sodium periodate solution to the volume of the ethylene glycol is 5 to 20: 1.

[0021] Optionally, the preparation method of the 3-carboxyphenylboronic acid modified chitosan comprises:

[0022] Chitosan is mixed with an acetic acid aqueous solution to obtain a chitosan solution, 3-carboxyphenylboronic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide and anhydrous methanol are mixed to obtain a reaction activation solution; the chitosan solution and the reaction activation solution are mixed and reacted, and after dialysis and freeze-drying treatment, 3-carboxyphenylboronic acid modified chitosan is obtained.

[0023] Optionally, in the acetic acid aqueous solution, the mass ratio of the water to the acetic acid is 200:1 to 500:1. The mass ratio of the chitosan in the chitosan solution is 0.2% to 0.7%, the molar ratio of the 3-carboxyphenylboric acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and N-hydroxysuccinimide is 2:1 to 5:1 to 5, and the pH of the reaction activation solution is 4.5 to 6.5. The mass ratio of the chitosan solution to the reaction activation solution is 5 to 10:1. Preferably, the mass ratio of the chitosan in the chitosan solution is 0.2% to 0.5%, and the molar ratio of the 3-carboxyphenylboric acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and N-hydroxysuccinimide is 2:2 to 3:2 to 4.

[0024] The invention provides a hydrogel dressing, which is prepared by the above-mentioned preparation method.

[0025] The present invention provides use of the above-mentioned hydrogel dressing in preparing a dressing for diabetic foot ulcers.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] 1. The present invention not only facilitates the load dispersion of deferoxamine mesylate by loading deferoxamine mesylate on a gelatin microsphere material constructed of gelatin and genipin, but also the added gelatin can contact and degrade with matrix proteinase-9 in the microenvironment of diabetic foot ulcers to induce the release of deferoxamine mesylate. In addition, the present invention coats the gelatin microsphere material loaded with deferoxamine mesylate and epigallocatechin gallate through a first cross-linked network formed by 3-carboxyphenylboronic acid-modified chitosan and oxidized hyaluronic acid, so that the hydrogel dressing realizes the controlled release of deferoxamine mesylate and epigallocatechin gallate, prolongs the drug action time of deferoxamine mesylate, and solves the technical problem of uncontrolled release of deferoxamine mesylate.

[0028] 2. The present invention can make the hydrogel dressing have good effects of promoting angiogenesis and improving microcirculation through deferoxamine mesylate and epigallocatechin gallate, so as to produce a therapeutic effect on diabetic foot ulcers. The present invention can form a three-dimensional network structure of biomimetic extracellular matrix through the first cross-linked network. The structure has excellent biocompatibility and can promote cell migration and cell proliferation functions, thereby promoting re-epithelialization of diabetic foot ulcer wounds, and then the hydrogel dressing prepared by the present invention can achieve better treatment of diabetic foot ulcers.

[0029] 3. The first prepolymer liquid and the second prepolymer liquid of the present invention can be prepared and used immediately. The first prepolymer liquid and the second prepolymer liquid can be simultaneously coated / injected into the wound, so that the prepared hydrogel dressing can better fit the wound and achieve a better treatment effect.

[0030] 4. The present invention does not add additional crosslinking agents when preparing the hydrogel dressing. Epigallocatechin gallate can be used as a crosslinking agent to form a second crosslinking network, which can avoid the toxicity and side effects caused by the additional addition of crosslinking agents and reduce the toxicity and side effects of the hydrogel dressing. The first crosslinking network containing Schiff base bonds and the second crosslinking network containing borate bonds enable the hydrogel dressing to form a double crosslinking structure, which can make the hydrogel dressing have better self-healing properties, mechanical strength and adhesion properties. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is a scanning electron microscope image of the gelatin microsphere material loaded with deferoxamine mesylate prepared in Example 1 of the present invention.

[0032] Figure 2 The Fourier transform infrared absorption spectrum, nuclear magnetic resonance hydrogen spectrum and synthesis route diagram of 3-carboxyphenylboronic acid modified chitosan prepared in Example 1 of the present invention are shown in Figure 1. Among them, a is the synthesis route diagram of 3-carboxyphenylboronic acid modified chitosan; b is the Fourier transform infrared absorption spectrum diagram of 3-carboxyphenylboronic acid modified chitosan prepared in Example 1; c is the nuclear magnetic resonance hydrogen spectrum diagram of 3-carboxyphenylboronic acid modified chitosan prepared in Example 1.

[0033] Figure 3 The Fourier transform infrared absorption spectrum, nuclear magnetic resonance hydrogen spectrum and synthesis route of the oxidized hyaluronic acid prepared in Example 1 of the present invention are shown in Figure 1. Among them, a is the synthesis route of the oxidized hyaluronic acid; b is the Fourier transform infrared absorption spectrum of the oxidized hyaluronic acid prepared in Example 1; c is the nuclear magnetic resonance hydrogen spectrum of the oxidized hyaluronic acid prepared in Example 1.

[0034] Figure 4 The gelation diagram of the hydrogel dressing prepared in Example 1 of the present invention. Among them, a is a physical schematic diagram of the second prepolymer solution; b is a physical schematic diagram of the second prepolymer solution; and c is a physical schematic diagram of the hydrogel dressing.

[0035] Figure 5 The scanning electron microscope images of the hydrogel dressing prepared in Example 1 of the present invention are shown in FIG. (a) is a scanning electron microscope image of the hydrogel dressing prepared in Example 1; and (b) is a partial enlarged image of the marked portion in FIG. (a).

[0036] Figure 6 The figure is a comparison chart of the therapeutic effect of a diabetic wound. Among them, a~d are the observation charts of the therapeutic effect of the blank control group at different times; e~h are the observation charts of the therapeutic effect of the commercially available hydrogel group at different times; i~l are the observation charts of the therapeutic effect of the hydrogel dressing group of Example 1 at different times.

[0037] Figure 7 A cell morphology comparison diagram provided by the present invention. Among them, a is a cell morphology observation diagram of the blank control group; b is a cell morphology observation diagram of the hydrogel dressing group of comparative example 1; and c is a cell morphology observation diagram of the hydrogel dressing group of example 1.

[0038] Figure 8 A cell compatibility comparison bar graph provided by the present invention. DETAILED DESCRIPTION

[0039] In order to solve the above technical problems, the present invention provides a hydrogel dressing and a preparation method and application thereof. The technical scheme and embodiments of the present invention are now described in detail in conjunction with the accompanying drawings.

[0040] The present invention is described in detail below through specific examples. The examples are exemplary and intended to be used to explain the present invention, but should not be understood as limiting the present invention.

[0041] Example 1

[0042] A method for preparing a hydrogel dressing comprises the following steps:

[0043] Step 1: Preparation of oxidized hyaluronic acid.

[0044] Dissolve 0.5 g of hyaluronic acid in 100 mL of deionized water to prepare a uniform and transparent hyaluronic acid solution.

[0045] Under light-proof conditions, dissolve 2 g of NaIO4 in 10 mL of deionized water to prepare a uniform and transparent NaIO4 solution.

[0046] 100 mL of hyaluronic acid solution and 10 mL of NaIO4 solution were mixed, reacted in the dark for 3 h, 22 mL of ethylene glycol was added, and the reaction was terminated after stirring for 1 h to obtain an oxidized hyaluronic acid precursor.

[0047] The oxidized hyaluronic acid precursor is first dialyzed and then freeze-dried to obtain the oxidized hyaluronic acid, wherein the dialysis time is 3 days.

[0048] Step 2: Preparation of 3-carboxyphenylboronic acid modified chitosan.

[0049] 1 g of chitosan was dissolved in 400 mL of deionized water with a mass fraction of 0.3% acetic acid aqueous solution to prepare a uniform and transparent chitosan solution.

[0050] 0.025 mol of 3-carboxyphenylboronic acid, 0.03 mol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and 0.03 mol of N-hydroxysuccinimide were mixed with 40 mL of anhydrous methanol, and the pH of the solution was adjusted to 4.5 to prepare a reaction activation solution.

[0051] The chitosan solution and the reaction activation solution were mixed and reacted for 24 hours to obtain the final reaction solution, which was first dialyzed and then freeze-dried to obtain 3-carboxyphenylboronic acid modified chitosan, wherein the dialysis treatment time was 3 days.

[0052] Step 3: Preparation of gelatin microsphere material loaded with deferoxamine mesylate.

[0053] At 60°C, 0.001 g of deferoxamine mesylate, 1.5 g of gelatin powder and 10 mL of deionized water were prepared as an aqueous phase.

[0054] 1 g of Span-80 was added to 100 mL of corn oil to prepare the oil phase.

[0055] The oil phase was added dropwise into the water phase, mixed and emulsified for 30 minutes, the emulsion was cooled at 7°C and stirred for 30 minutes, and then 150 mL of acetone was added for dehydration for 30 minutes, filtered and dried to obtain a gelatin microsphere precursor.

[0056] After dissolving 50 mg of gelatin microsphere precursor in 4 mL of phosphate buffer solution, it was mixed with 1 mL of 1 wt% genipin solution, and cross-linked for 12 hours. After repeated centrifugation and washing with ethanol for 3 times, rotary evaporation, and freeze-drying, the gelatin microsphere material loaded with deferoxamine mesylate was obtained. The phosphate buffer solution contained 0.15 g of sodium dihydrogen phosphate and 0.02 g of disodium hydrogen phosphate.

[0057] Step 4: Preparation of hydrogel dressing.

[0058] Epigallocatechin gallate, oxidized hyaluronic acid, gelatin microspheres loaded with deferoxamine mesylate, and 3-carboxyphenylboronic acid modified chitosan were sterilized by cobalt 60 irradiation for 24 hours.

[0059] 0.01 g of sterilized epigallocatechin gallate and 0.1 g of sterilized oxidized hyaluronic acid were dissolved in 10 mL of water to prepare a first prepolymer solution.

[0060] 0.2 g of sterilized gelatin microsphere material loaded with deferoxamine mesylate and 0.2 g of sterilized 3-carboxyphenylboronic acid-modified chitosan were dissolved in 10 mL of water to prepare a second prepolymer solution.

[0061] The first prepolymer liquid and the second prepolymer liquid are mixed and stirred evenly to prepare a hydrogel dressing.

[0062] Example 2

[0063] A method for preparing a hydrogel dressing comprises the following steps:

[0064] Step 1: Preparation of oxidized hyaluronic acid.

[0065] Dissolve 1 g of hyaluronic acid in 100 mL of deionized water to prepare a uniform and transparent hyaluronic acid solution.

[0066] Under light-proof conditions, 1.8 g of NaIO4 was dissolved in 10 mL of deionized water to prepare a uniform and transparent NaIO4 solution.

[0067] 100 mL of hyaluronic acid solution and 10 mL of NaIO4 solution were mixed, reacted in the dark for 3 h, 20 mL of ethylene glycol was added, and the reaction was terminated after stirring for 1 h to obtain an oxidized hyaluronic acid precursor.

[0068] The oxidized hyaluronic acid precursor is first dialyzed and then freeze-dried to obtain the oxidized hyaluronic acid, wherein the dialysis time is 3 days.

[0069] Step 2: Preparation of 3-carboxyphenylboronic acid modified chitosan.

[0070] 2 g of chitosan was dissolved in 400 mL of acetic acid aqueous solution containing 0.3% by mass of deionized water to prepare a uniform and transparent chitosan solution.

[0071] 0.02 mol of 3-carboxyphenylboric acid, 0.04 mol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 0.04 mol of N-hydroxysuccinimide and 60 mL of anhydrous methanol were mixed, and the pH of the solution was adjusted to 4.5 to prepare a reaction activation solution.

[0072] The chitosan solution and the reaction activation solution were mixed and reacted for 24 hours to obtain the final reaction solution, which was first dialyzed and then freeze-dried to obtain 3-carboxyphenylboronic acid modified chitosan, wherein the dialysis treatment time was 3 days.

[0073] Step 3: Preparation of gelatin microsphere material loaded with deferoxamine mesylate.

[0074] At 60°C, 0.75 mg of deferoxamine mesylate, 1.5 g of gelatin powder and 10 mL of deionized water were prepared as the aqueous phase.

[0075] Add 1 g of Span-80 into 50 mL of corn oil to prepare the oil phase.

[0076] The oil phase was added dropwise into the water phase, mixed and emulsified for 20 minutes, and then the emulsion was cooled at 5°C and stirred for 40 minutes. Then, 180 mL of acetone was added to dehydrate for 40 minutes, filtered, and dried to obtain a gelatin microsphere precursor.

[0077] After dissolving 40 mg of gelatin microsphere precursor in 4 mL of phosphate buffer solution, it was mixed with 1 mL of 1.5 wt% genipin solution, and cross-linked for 12 h. After repeated centrifugation and washing with ethanol for 3 times, rotary evaporation, and freeze-drying, the gelatin microsphere material loaded with deferoxamine mesylate was obtained. The phosphate buffer solution contained 0.15 g of sodium dihydrogen phosphate and 0.02 g of disodium hydrogen phosphate.

[0078] Step 4: Preparation of hydrogel dressing.

[0079] Epigallocatechin gallate, oxidized hyaluronic acid, gelatin microspheres loaded with deferoxamine mesylate, and 3-carboxyphenylboronic acid modified chitosan were sterilized by cobalt 60 irradiation for 24 hours.

[0080] 0.01 g of sterilized epigallocatechin gallate and 0.15 g of sterilized oxidized hyaluronic acid were dissolved in 10 mL of water to prepare a first prepolymer solution.

[0081] 0.2 g of sterilized gelatin microsphere material loaded with deferoxamine mesylate and 0.3 g of sterilized 3-carboxyphenylboronic acid-modified chitosan were dissolved in 10 mL of water to prepare a second prepolymer solution.

[0082] The first prepolymer liquid and the second prepolymer liquid are mixed and stirred evenly to prepare a hydrogel dressing.

[0083] Example 3

[0084] A method for preparing a hydrogel dressing comprises the following steps:

[0085] Step 1: Preparation of oxidized hyaluronic acid.

[0086] Dissolve 2 g of hyaluronic acid in 100 mL of deionized water to prepare a uniform and transparent hyaluronic acid solution.

[0087] Under light-proof conditions, 2.2 g of NaIO4 was dissolved in 10 mL of deionized water to prepare a uniform and transparent NaIO4 solution.

[0088] 100 mL of hyaluronic acid solution and 10 mL of NaIO4 solution were mixed, reacted in the dark for 4.5 h, 20 mL of ethylene glycol was added, and the reaction was terminated after stirring for 2 h to obtain an oxidized hyaluronic acid precursor.

[0089] The oxidized hyaluronic acid precursor is first dialyzed and then freeze-dried to obtain the oxidized hyaluronic acid, wherein the dialysis time is 3 days.

[0090] Step 2: Preparation of 3-carboxyphenylboronic acid modified chitosan.

[0091] 1.5 g of chitosan was dissolved in 400 mL of deionized water with a mass fraction of 0.3% acetic acid aqueous solution to prepare a uniform and transparent chitosan solution.

[0092] 0.02 mol of 3-carboxyphenylboronic acid, 0.05 mol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 0.05 mol of N-hydroxysuccinimide and 65 mL of anhydrous methanol were mixed, and the pH of the solution was adjusted to 4.5 to prepare a reaction activation solution.

[0093] The chitosan solution and the reaction activation solution were mixed and reacted for 24 hours to obtain the final reaction solution, which was first dialyzed and then freeze-dried to obtain 3-carboxyphenylboronic acid modified chitosan, wherein the dialysis treatment time was 4 days.

[0094] Step 3: Preparation of gelatin microsphere material loaded with deferoxamine mesylate.

[0095] At 60°C, 0.5 mg of deferoxamine mesylate, 1.5 g of gelatin powder and 10 mL of deionized water were prepared as the aqueous phase.

[0096] 1 g of Span-80 was added to 80 mL of corn oil to prepare the oil phase.

[0097] The oil phase was added dropwise into the water phase, mixed and emulsified for 30 minutes, and then the emulsion was cooled at 3°C ​​and stirred continuously for 40 minutes. Then, 200 mL of acetone was added to dehydrate for 50 minutes, filtered, and dried to obtain a gelatin microsphere precursor.

[0098] After dissolving 60 mg of gelatin microsphere precursor in 4 mL of phosphate buffer solution, it was mixed with 1.5 mL of 1.5 wt% genipin solution, cross-linked for 12 h, and washed by repeated centrifugation with ethanol for 3 times, rotary evaporation, and freeze-dried to obtain the gelatin microsphere material loaded with deferoxamine mesylate. The phosphate buffer solution contained 0.15 g of sodium dihydrogen phosphate and 0.02 g of disodium hydrogen phosphate.

[0099] Step 4: Preparation of hydrogel dressing.

[0100] Epigallocatechin gallate, oxidized hyaluronic acid, gelatin microspheres loaded with deferoxamine mesylate, and 3-carboxyphenylboronic acid modified chitosan were sterilized by cobalt 60 irradiation for 36 hours.

[0101] 0.01 g of sterilized epigallocatechin gallate and 0.2 g of sterilized oxidized hyaluronic acid were dissolved in 10 mL of water to prepare a first prepolymer solution.

[0102] 0.3 g of sterilized gelatin microsphere material loaded with deferoxamine mesylate and 0.4 g of sterilized 3-carboxyphenylboronic acid-modified chitosan were dissolved in 10 mL of water to prepare a second prepolymer solution.

[0103] The first prepolymer liquid and the second prepolymer liquid are mixed and stirred evenly to prepare a hydrogel dressing.

[0104] The present invention also provides embodiments 4 to 6. Compared with embodiment 1, the difference of embodiment 4 is that the corn oil used in step three is replaced by castor oil; compared with embodiment 1, the difference of embodiment 5 is that the corn oil used in step three is replaced by sesame oil; compared with embodiment 1, the difference of embodiment 6 is that the corn oil used in step three is replaced by olive oil.

[0105] Comparative Example 1

[0106] A method for preparing a hydrogel dressing comprises the following steps:

[0107] Step 1: Preparation of oxidized hyaluronic acid.

[0108] Dissolve 2 g of hyaluronic acid in 100 mL of deionized water to prepare a uniform and transparent hyaluronic acid solution.

[0109] Under light-proof conditions, 2.2 g of NaIO4 was dissolved in 10 mL of deionized water to prepare a uniform and transparent NaIO4 solution.

[0110] 100 mL of hyaluronic acid solution and 10 mL of NaIO4 solution were mixed, reacted in the dark for 4.5 h, 20 mL of ethylene glycol was added, and the reaction was terminated after stirring for 2 h to obtain an oxidized hyaluronic acid precursor.

[0111] The oxidized hyaluronic acid precursor is first dialyzed and then freeze-dried to obtain the oxidized hyaluronic acid, wherein the dialysis time is 3 days.

[0112] Step 2: Preparation of 3-carboxyphenylboronic acid modified chitosan.

[0113] 1.5 g of chitosan was dissolved in 400 mL of deionized water with a mass fraction of 0.3% acetic acid aqueous solution to prepare a uniform and transparent chitosan solution.

[0114] 0.02 mol of 3-carboxyphenylboronic acid, 0.05 mol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 0.05 mol of N-hydroxysuccinimide and 65 mL of anhydrous methanol were mixed, and the pH of the solution was adjusted to 4.5 to prepare a reaction activation solution.

[0115] The chitosan solution and the reaction activation solution were mixed and reacted for 24 hours to obtain the final reaction solution, which was first dialyzed and then freeze-dried to obtain 3-carboxyphenylboronic acid modified chitosan, wherein the dialysis treatment time was 4 days.

[0116] Step 3: Preparation of hydrogel dressing.

[0117] Epigallocatechin gallate, oxidized hyaluronic acid, and 3-carboxyphenylboronic acid-modified chitosan were sterilized by cobalt 60 irradiation for 36 hours.

[0118] 0.01 g of sterilized epigallocatechin gallate and 0.2 g of sterilized oxidized hyaluronic acid were dissolved in 10 mL of water to prepare a first prepolymer solution.

[0119] 0.4 g of sterilized 3-carboxyphenylboronic acid-modified chitosan was dissolved in 10 mL of water to prepare a second prepolymer solution.

[0120] The first prepolymer liquid and the second prepolymer liquid are mixed and stirred evenly to prepare a hydrogel dressing.

[0121] Test 1: Microstructural analysis.

[0122] Figure 1 This is a scanning electron microscope image of the gelatin microsphere material loaded with deferoxamine mesylate prepared in Example 1 of the present invention. It can be seen from the image that the gelatin microsphere material loaded with deferoxamine mesylate is uniformly spherical, uniform in size, and complete in structure. The average diameter of the gelatin microsphere material calculated by the particle size software is 8.51±0.86μm.

[0123] Figure 2 The Fourier transform infrared absorption spectrum, nuclear magnetic resonance hydrogen spectrum and synthesis route diagram of 3-carboxyphenylboronic acid modified chitosan prepared in Example 1 of the present invention. Figure 2 b: 3-Carboxyphenylboronic acid modified chitosan at 770 cm -1 A new peak appears at , which represents the CH bending vibration peak outside the aromatic ring. Figure 2As shown in Figure c: 3-carboxyphenylboronic acid modified chitosan has peaks at 7.8 and 8.2 ppm, representing phenyl protons. The Fourier transform infrared absorption spectrum and nuclear magnetic resonance hydrogen spectrum together prove the successful preparation of 3-carboxyphenylboronic acid modified chitosan.

[0124] Figure 3 The Fourier transform infrared absorption spectrum, nuclear magnetic resonance hydrogen spectrum and synthesis route diagram of the oxidized hyaluronic acid prepared in Example 1 of the present invention. Figure 3 b shows: Oxidized hyaluronic acid at 1734 cm -1 There is a weak C=O bending vibration peak at . This may be due to the formation of hemiacetal, so it is difficult to detect the signal of the aldehyde group. The H NMR spectra of oxidized hyaluronic acid and hyaluronic acid are shown in Figure 3 As shown in Figure c, a new peak was observed at 4.9ppm to 5.0ppm in the spectrum of oxidized hyaluronic acid, corresponding to the hemiacetal proton formed by the aldehyde group and the adjacent hydroxyl group. The above results indicate that the adjacent dihydroxyl group of hyaluronic acid was successfully oxidized to the aldehyde group.

[0125] Figure 4 This is a gelation diagram of the hydrogel dressing prepared in Example 1 of the present invention. The gelation performance of the hydrogel dressing is measured by the inverted no-flow method. As shown in the figure, after the first prepolymer liquid and the second prepolymer liquid are mixed, the vial is inverted and the mixed liquid does not flow within 57.6±2.5s, that is, the hydrogel dressing is formed.

[0126] Figure 5 This is a scanning electron microscope image of the hydrogel dressing prepared in Example 1 of the present invention. As can be seen from the image, the hydrogel dressing has a three-dimensional pore structure inside, and gelatin microspheres loaded with deferoxamine mesylate are distributed in the pores.

[0127] Test 2: Analysis of treatment effect.

[0128] Test materials: SPF grade 8-week-old male SD rats weighing 200-250 g.

[0129] Establishment of diabetic SD rat model: 60 SPF-grade 8-week-old male SD rats weighing 200-250g were selected and adaptively raised at 25℃ for 1 week; fasting was performed before surgery, and 1% streptozotocin solution was injected according to the standard of 65mg / kg body weight of rats. After 72h, the blood glucose of SD rats was continuously observed for 3 weeks. When the random blood glucose of the rats was stably greater than 16.7mmol / L within 3 weeks, the model was considered to be established.

[0130] Experimental method: After the diabetic SD rat model was successfully established, a full-thickness skin defect model was established on the rat's foot. The diabetic rats were randomly divided into a blank control group, a commercially available hydrogel dressing group, and an Example 1 hydrogel dressing group, with 20 rats in each group. Among them, the commercially available hydrogel dressing was HeraDerm.

[0131] The rats were fasted before surgery. After successful anesthesia, the rat paws were disinfected with 75% alcohol, and a full-thickness skin defect with a diameter of 6 mm was excised in the middle of the paw using a skin sampler. The blank control group was not treated with any dressings, the commercially available hydrogel dressing group was treated with HeraDerm dressings, and the Example 1 hydrogel dressing group was treated with the hydrogel dressing prepared in Example 1. The rats in each group were observed daily to ensure that the wound was effectively covered by the dressings, and the dressings were changed every 2 days.

[0132] Analysis of test results:

[0133] Comparison of therapeutic effects on diabetic wounds in diabetic SD rats, such as Figure 6 As shown, the treatment speed of the hydrogel dressing group in Example 1 is significantly faster than that of the blank control group and the commercially available hydrogel dressing group, which is about 2.4 times that of the blank control group. The wound healing time of the hydrogel dressing group in Example 1 can reach 13 days at the fastest, and the rats in the hydrogel dressing group in Example 1 do not have obvious abnormalities. The animal experiment results show that the hydrogel dressing prepared in Example 1 of the present invention can significantly promote the healing of diabetic foot ulcers and shorten the healing time, indicating that the hydrogel material prepared by the preparation method of the present invention can have a good therapeutic effect on diabetic foot ulcers, can solve the technical problem of lack of dressings that can have a therapeutic effect on diabetic foot ulcers, and the hydrogel dressing is used in the treatment of diabetic foot ulcers.

[0134] In addition, when the hydrogel dressing prepared in Example 1 was replaced with the hydrogel dressing prepared in Examples 4 to 6, there was no significant difference in the therapeutic effect on the diabetic wounds of diabetic SD rats, indicating that the type of vegetable oil will not have a significant effect on the performance of the hydrogel dressing. Therefore, the present application does not specifically limit the specific type of vegetable oil used.

[0135] Test 3: Cytocompatibility analysis.

[0136] Experimental materials: L929 cell line, a mouse fibroblast cell line.

[0137] Experimental method: The L929 cell line, i.e., the mouse fibroblast cell line, was randomly divided into three groups, namely, a blank control group, an Example 1 hydrogel dressing group, and a comparative example 1 hydrogel dressing group. Among them, the mouse fibroblast cell line of the blank control group was incubated alone for 3 days, and the incubation conditions were observed and recorded daily; the mouse fibroblast cell line of the Example 1 hydrogel dressing group was incubated together with the hydrogel dressing prepared in Example 1 for 3 days, and the incubation conditions were observed and recorded daily; the mouse fibroblast cell line of the comparative example 1 hydrogel dressing group was incubated together with the hydrogel dressing prepared in Example 1 for 3 days, and the incubation conditions were observed and recorded daily. The final experimental results are as follows. Figure 7 and Figure 8 shown.

[0138] Analysis of test results:

[0139] Figure 7 It can be seen that the cell morphology of the three groups of mouse fibroblast cell lines is basically spindle-shaped and there is no significant difference, indicating that the hydrogel dressing prepared in Example 1 has no obvious toxic side effects on the growth of mouse fibroblast cell lines.

[0140] Figure 8 In the experiment, compared with the blank control group, the mouse fibroblast cell lines of the hydrogel dressing group of comparative example 1 and the hydrogel dressing group of embodiment 1 showed a significant proliferation trend during the incubation process. Among them, compared with the hydrogel dressing group of comparative example 1, the mouse fibroblast cell line of the hydrogel dressing group of embodiment 1 showed significantly better proliferation ability on the 2nd and 3rd days. This experimental result shows that compared with the hydrogel dressing group of comparative example 1 containing only epigallocatechin gallate, the hydrogel dressing of embodiment 1 of the present application has a better effect of promoting cell growth, indicating that the simultaneous presence of deferoxamine mesylate and epigallocatechin gallate can make the hydrogel dressing have a better effect of promoting angiogenesis and improving microcirculation.

[0141] The above description is only a preferred embodiment of the present invention, and the above specific embodiment is not intended to limit the present invention. Various deformations and modifications may occur within the scope of the technical concept of the present invention, and any modification, modification or equivalent replacement made by a person of ordinary skill in the art based on the above description shall fall within the scope of protection of the present invention.

Claims

1. A method for preparing a hydrogel dressing, characterized in that: The following steps are involved: The gelatin and deferoxamine mesylate aqueous solution are emulsified as the aqueous phase in an oil phase environment to prepare a gelatin microsphere precursor, and the gelatin microsphere precursor and genipin are mixed, wherein the gelatin and genipin undergo a cross-linking reaction to prepare a gelatin microsphere material loaded with deferoxamine mesylate; mixing epigallocatechin gallate with an oxidized hyaluronic acid solution to prepare a first prepolymer solution; The gelatin microsphere material loaded with deferoxamine mesylate is mixed with a 3-carboxyphenylboronic acid modified chitosan solution to prepare a second prepolymer solution; The first prepolymer liquid and the second prepolymer liquid are mixed, wherein the oxidized hyaluronic acid undergoes a Schiff base reaction with the 3-carboxyphenylboronic acid modified chitosan to form a first cross-linked network containing Schiff base bonds, and the first cross-linked network is coated with the gelatin microsphere material and the epigallocatechin gallate, and at the same time, the epigallocatechin gallate undergoes an esterification reaction with the 3-carboxyphenylboronic acid modified chitosan to form a second cross-linked network containing borate ester bonds; a hydrogel dressing is prepared.

2. The method for preparing the hydrogel dressing according to claim 1, characterized in that: The preparation method of the gelatin microsphere material loaded with deferoxamine mesylate comprises: Deferoxamine mesylate, gelatin and water are used as the aqueous phase, and Span-80 and vegetable oil are used as the oil phase. The aqueous phase and the oil phase are mixed and emulsified to prepare a gelatin microsphere precursor; the gelatin microsphere precursor is mixed with a phosphate buffer solution and then mixed with genipin, wherein the gelatin and the genipin react to form a third cross-linked network, and the gelatin microsphere material loaded with deferoxamine mesylate is prepared.

3. The method for preparing the hydrogel dressing according to claim 2, characterized in that: The mass ratio of the gelatin to the deferoxammonium mesylate is 1000-3000:1, the mass ratio of the water to the gelatin is 5-10:1, and the reaction temperature of the water phase is 45°C-60°C; The mass ratio of the vegetable oil to the Span-80 is 50-200:1, the volume ratio of the water phase to the oil phase is 1:5-20, the reaction time of the emulsification reaction is 15-30 minutes, and the cooling temperature is 0°C-7°C; After the gelatin microsphere precursor is mixed with the phosphate buffer solution, the concentration of the gelatin microsphere precursor is 10 mg·mL -1 ~15 mg mL -1 ; The mass fraction of genipin in the gelatin microsphere material loaded with deferoxamine mesylate is 0.5% to 1.5%.

4. The method for preparing the hydrogel dressing according to claim 1, characterized in that: Before the preparation of the first prepolymer solution and the second prepolymer solution, the method further comprises: The epigallocatechin gallate, the oxidized hyaluronic acid, the gelatin microsphere material loaded with deferoxamine mesylate, and the 3-carboxyphenylboronic acid modified chitosan are sterilized.

5. The method for preparing the hydrogel dressing according to claim 1, characterized in that: The preparation method of the oxidized hyaluronic acid comprises: The hyaluronic acid solution and the sodium periodate solution are mixed, and then mixed with ethylene glycol for a light-protected reaction to obtain an oxidized hyaluronic acid precursor; The oxidized hyaluronic acid precursor is freeze-dried and dialyzed to obtain the oxidized hyaluronic acid.

6. The method for preparing the hydrogel dressing according to claim 5, characterized in that: In the hyaluronic acid solution, the mass ratio of the solvent to the hyaluronic acid is 40 to 200:1; In the sodium periodate solution, the mass ratio of the solvent to the sodium periodate is 4.5-5.5:1; The volume ratio of the hyaluronic acid solution to the sodium periodate solution is 10 to 30:1, and the ratio of the sum of the volumes of the hyaluronic acid solution and the sodium periodate solution to the volume of the ethylene glycol is 5 to 20:

1.

7. The method for preparing the hydrogel dressing according to claim 1, characterized in that: The preparation method of the 3-carboxyphenylboronic acid modified chitosan solution comprises: The chitosan solution is prepared by mixing chitosan with an acetic acid aqueous solution, and the reaction activation solution is prepared by mixing 3-carboxyphenylboronic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide and anhydrous methanol; The chitosan solution and the reaction activation solution are mixed for reaction, and after dialysis and freeze-drying, 3-carboxyphenylboronic acid modified chitosan is obtained.

8. The method for preparing the hydrogel dressing according to claim 7, characterized in that: In the acetic acid aqueous solution, the mass ratio of water to acetic acid is 200-500:1; The mass proportion of the chitosan in the chitosan solution is 0.2% to 0.7%, the molar ratio of the 3-carboxyphenylboronic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and N-hydroxysuccinimide is 2:1 to 5:1 to 5, and the pH of the reaction activation solution is 4.5 to 6.5; The mass ratio of the chitosan solution to the reaction activation solution is 5 to 10:

1.

9. A hydrogel dressing, characterized in that: The hydrogel dressing is prepared by the preparation method of any one of claims 1 to 8.

10. Use of the hydrogel dressing according to claim 9 in preparing a dressing for diabetic foot ulcers.

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