A hydrogel dressing, its preparation method and use

Abstract

The present application relates to the field of hydrogel materials, in particular to a hydrogel dressing and its preparation method and application. The present application loads deferoxamine mesylate on the gelatin microsphere material constructed by gelatin and genipin, which not only helps the dispersion of deferoxamine mesylate, but also the added gelatin can contact and degrade with matrix metalloproteinase-9 in the microenvironment of diabetic foot ulcer to induce the release of deferoxamine mesylate. And the present application forms a first crosslinking network by modifying chitosan with 3-carboxyphenylboronic acid and oxidized hyaluronic acid, and the first crosslinking network coats the 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, to solve the technical problem of uncontrollable release of deferoxamine mesylate.

Description

TECHNICAL FIELD

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

[0002] Diabetic foot ulcer is a serious complication of diabetes, mainly refers to the foot neuropathy and vascular lesions caused by diabetes, leading to foot skin damage, infection and ulcer formation. Diabetic foot ulcer is easy to infect, and is extremely difficult to cure, and can lead to amputation in severe cases. The site of diabetic foot ulcer has poor blood supply, impaired angiogenesis, insufficient microcirculation at the end of the foot, and long-term non-healing of the wound.

[0003] The common dressing for wound treatment at present generally has the functions of moisturizing and antibiosis, and can have certain therapeutic effect on common wounds, but it is difficult to achieve effective therapeutic effect on diabetic foot ulcer.

[0004] Deferoxamine mesylate is a kind of angiogenic agent for ischemia and wound healing. In ischemic disease models, deferoxamine mesylate can achieve the effects of up-regulating the expression of vascular endothelial growth factor, stimulating blood perfusion and promoting blood vessel growth. However, when deferoxamine mesylate is applied to actual treatment, due to the uncontrollable release of deferoxamine mesylate, the drug has a short action time, and it is difficult to achieve effective therapeutic effect on diabetic foot ulcer. SUMMARY

[0005] The present application aims at overcoming the shortcomings of the prior art, and provides a hydrogel dressing and a preparation method and application thereof, which solve the problem of uncontrollable release of deferoxamine mesylate.

[0006] To achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows:

[0007] A preparation method of a hydrogel dressing, comprising the following steps:

[0008] The gelatin microsphere precursor is prepared by emulsifying a gelatin and an aqueous solution of deferoxamine mesylate as an aqueous phase in an oil phase environment, the gelatin microsphere precursor and genipin are mixed, the gelatin and the genipin are crosslinked to prepare the gelatin microsphere material loaded with deferoxamine mesylate; epigallocatechin gallate is mixed with an oxidized hyaluronic acid solution to prepare a first pre-polymerization solution; the gelatin microsphere material loaded with deferoxamine mesylate is mixed with a 3-carboxyphenylboronic acid modified chitosan solution to prepare a second pre-polymerization solution; the first pre-polymerization solution and the second pre-polymerization solution are mixed, the oxidized hyaluronic acid and the 3-carboxyphenylboronic acid modified chitosan are subjected to a Schiff base reaction to form a first crosslinking network containing a Schiff base bond, and the first crosslinking network is wrapped around the gelatin microsphere material and the epigallocatechin gallate, at the same time, the epigallocatechin gallate and the 3-carboxyphenylboronic acid modified chitosan are subjected to an esterification reaction to form a second crosslinking network containing a borate ester bond; and the hydrogel dressing is prepared.

[0009] A large amount of MMP-9 exists in the microenvironment of a diabetic foot ulcer, and gelatin is an inherent degradation substrate of MMP-9, so the gelatin microspheres in the gelatin microsphere material loaded with deferoxamine mesylate will be degraded after contacting with MMP-9.

[0010] The epigallocatechin gallate can promote the expression of vascular endothelial growth factor by reducing the expression of active oxygen, so as to promote angiogenesis.

[0011] The gelatin microsphere material loaded with deferoxamine mesylate can not only benefit the dispersion of deferoxamine mesylate, but also the added gelatin can be degraded by contacting with matrix metalloproteinase-9 in the microenvironment of a diabetic foot ulcer to induce the release of deferoxamine mesylate.

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

[0013] The gelatin microsphere precursor is prepared by mixing and emulsifying an aqueous phase of deferoxamine mesylate, gelatin and water and an oil phase of Span-80 and vegetable oil, the gelatin microsphere precursor is mixed with a phosphate buffer solution, and then mixed with genipin, the gelatin and the genipin are reacted to form a third crosslinking 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, hemp oil, olive oil. The main role of the vegetable oil is as a solvent of Span-80, and the type of vegetable oil has no significant effect on the solubility of Span-80, so common types of vegetable oil are within the protection scope of the present application.

[0015] Optionally, the mass ratio of the gelatin to the deferoxamine 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 45-60℃. 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 15-30min, and the cooling temperature is 0-7℃. After the gelatin microsphere precursor is mixed with the phosphate buffer solution, the concentration of the gelatin microsphere precursor is 10-15mg·mL -1 . -1 The mass fraction of the genipin in the deferoxamine mesylate-loaded gelatin microsphere material is 0.5%-1.5%. Preferably, the mass ratio of the gelatin to the deferoxamine mesylate is 1000-2000:1.

[0016] Optionally, before the first pre-polymerization liquid and the second pre-polymerization liquid are prepared, the method further comprises:

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

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

[0019] The hyaluronic acid solution, the sodium periodate solution, and the ethylene glycol are mixed to react in the dark, the reaction is terminated after stirring, and the oxidized hyaluronic acid precursor is prepared; the oxidized hyaluronic acid precursor is subjected to freeze-drying dialysis treatment, and the oxidized hyaluronic acid is prepared.

[0020] Optionally, in the hyaluronic acid solution, the mass ratio of the solvent to the hyaluronic acid is 40-200:1. 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 10-30:1, 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 5-20:1.

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

[0022] The chitosan is mixed with an acetic acid aqueous solution to prepare a chitosan solution, 3-carboxyphenylboronic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and N-hydroxysuccinimide are mixed with anhydrous methanol to prepare 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 prepared.

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

[0024] The application provides a hydrogel dressing prepared by the preparation method.

[0025] The application provides application of the hydrogel dressing in preparation of a dressing for diabetic foot ulcers.

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

[0027] 1、The gelatin microsphere material on which the methanesulfonic acid deferoxamine is loaded is constructed by gelatin and genipin, which is beneficial to the loading and dispersion of the methanesulfonic acid deferoxamine, and the added gelatin can contact and degrade matrix metalloproteinase-9 in the microenvironment of diabetic foot ulcers to induce the release of the methanesulfonic acid deferoxamine.

[0028] 2、The water gel dressing prepared by the deoxycholate and epigallocatechin gallate can have good effects of promoting angiogenesis and improving microcirculation, so that the treatment effect on diabetic foot ulcers can be achieved.

[0029] 3、The first prepolymer liquid and the second prepolymer liquid of the present application can be prepared and used at the same time, and the water gel dressing prepared by coating / injecting the first prepolymer liquid and the second prepolymer liquid on the wound at the same time can be more fitted to cover the wound, so that better treatment effect can be achieved.

[0030] 4、The water gel dressing prepared by the present application does not add an additional crosslinking agent, and the epigallocatechin gallate can form a second crosslinking network as a crosslinking agent, so that the toxicity and side effects caused by the additional crosslinking agent can be avoided, and the toxicity and side effects of the water gel dressing can be reduced. The first crosslinking network containing a Schiff base bond and the second crosslinking network containing a borate bond make the water gel dressing form a double crosslinking structure, so that the water gel dressing can have good self-healing property, mechanical strength and adhesion. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 The scanning electron microscope image of the gelatin microsphere material loaded with deoxycholate prepared in Example 1 of the present application.

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

[0033] Figure 3 The Fourier transform infrared absorption spectrum, the nuclear magnetic resonance hydrogen spectrum and the synthesis route diagram of the oxidized hyaluronic acid prepared in Example 1 of the present application. Among them, a is the synthesis route diagram 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 gel formation diagram of the water gel dressing prepared in Example 1 of the present application. Among them, a is the actual schematic diagram of the second prepolymer liquid; b is the actual schematic diagram of the second prepolymer liquid; c is the actual schematic diagram of the water gel dressing.

[0035] Figure 5 The scanning electron microscope image of the hydrogel dressing prepared in Example 1. Wherein, (a) is the scanning electron microscope image of the hydrogel dressing prepared in Example 1; (b) is a local enlarged view of the marked part in Figure (a).

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

[0037] Figure 7 A cell morphology comparison chart provided by the present application. Wherein, a is the cell morphology observation chart of the blank control group; b is the cell morphology observation chart of the hydrogel dressing group of Comparative Example 1; c is the cell morphology observation chart of the hydrogel dressing group of Example 1.

[0038] Figure 8 A cell compatibility comparison column chart provided by the present application. DETAILED DESCRIPTION

[0039] To solve the above technical problems, the present application provides a hydrogel dressing, a preparation method and application thereof, and the technical solutions and examples of the present application will be described in detail in combination with the drawings.

[0040] The present application will be described in detail below through specific examples, which are exemplary and intended to explain the present application, and cannot be understood as a limitation of the present application.

[0041] Example 1

[0042] A preparation method of a hydrogel dressing, comprising the following steps:

[0043] Step one, preparation of oxidized hyaluronic acid.

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

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

[0046] Mix 100mL of hyaluronic acid solution and 10mL of NaIO4 solution, react in the dark for 3h, add 22mL of ethylene glycol, stir for 1h to terminate the reaction, and prepare an oxidized hyaluronic acid precursor.

[0047] The oxidized hyaluronic acid is prepared by dialysis treatment and freeze-drying treatment, and the dialysis treatment time is 3 days.

[0048] Step two, preparation of 3-carboxyphenyl boronic acid modified chitosan.

[0049] 1g of chitosan is dissolved in 400mL of 0.3% acetic acid aqueous solution to prepare a uniform and transparent chitosan solution.

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

[0051] The chitosan solution and the reaction activation solution are mixed for 24h to obtain a final reaction solution, which is then subjected to dialysis treatment and freeze-drying treatment to prepare 3-carboxyphenyl boronic acid modified chitosan, and the dialysis treatment time is 3 days.

[0052] Step three, preparation of gelatin microsphere material loaded with deferoxamine mesylate.

[0053] 0.001g of deferoxamine mesylate, 1.5g of gelatin powder and 10mL of deionized water are configured as an aqueous phase at a temperature of 60℃.

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

[0055] The oil phase is added dropwise to the aqueous phase, and after 30min of mixing and emulsification, the emulsion is cooled at 7℃ and continuously stirred for 30min. Then, 150mL of acetone is added for dehydration for 30min, followed by suction filtration and drying to prepare a gelatin microsphere precursor.

[0056] 50mg of the gelatin microsphere precursor is dissolved in 4mL of a phosphate buffer solution, and then mixed with 1mL of a 1wt% genipin solution. After 12h of crosslinking reaction, the gelatin microsphere material loaded with deferoxamine mesylate is prepared by repeated centrifugal washing with ethanol for 3 times, rotary evaporation, and freeze-drying. The phosphate buffer solution contains 0.15g of sodium dihydrogen phosphate and 0.02g of disodium hydrogen phosphate.

[0057] Step four, preparation of hydrogel dressing.

[0058] Epigallocatechin gallate, oxidized hyaluronic acid, gelatin microsphere material loaded with deferoxamine mesylate, and 3-carboxyphenyl boronic acid modified chitosan are subjected to sterilization treatment by cobalt-60 irradiation sterilization for 24h.

[0059] The first pre-polymer solution was prepared by dissolving 0.01 g of sterilized epigallocatechin gallate and 0.1 g of sterilized oxidized hyaluronic acid in 10 mL of water.

[0060] The second pre-polymer solution was prepared by dissolving 0.2 g of sterilized gelatin microsphere material loaded with deferoxamine mesylate and 0.2 g of sterilized 3-carboxyphenylboronic acid modified chitosan in 10 mL of water.

[0061] The hydrogel dressing was prepared by mixing the first pre-polymer solution and the second pre-polymer solution and stirring until uniform.

[0062] Example 2

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

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

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

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

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

[0068] The oxidized hyaluronic acid precursor was first subjected to dialysis treatment and then freeze-drying treatment to prepare the oxidized hyaluronic acid, wherein the dialysis treatment time was 3 days.

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

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

[0071] 0.02 mol of 3-carboxyphenylboronic 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 to prepare a reaction activation solution, and the pH of the solution was adjusted to 4.5.

[0072] The chitosan solution and the reaction activation solution were mixed and reacted for 24 h to obtain a final reaction solution. The 3-carboxyphenylboronic acid modified chitosan was prepared by first subjecting to dialysis treatment and then freeze-drying treatment, wherein the dialysis treatment time was 3 days.

[0073] Step three, preparation of gelatin microspheres material loaded with deferoxamine mesylate.

[0074] At 60℃, 0.75mg deferoxamine mesylate, 1.5g gelatin powder and 10mL deionized water were configured as an aqueous phase.

[0075] 1g Span-80 was added to 50mL corn oil to configure an oil phase.

[0076] The oil phase was added dropwise to the aqueous phase, and after 20min of mixing and emulsification, the emulsion was cooled at 5℃ and continuously stirred for 40min. Then 180mL of acetone was added for dehydration for 40min, followed by suction filtration and drying to obtain a gelatin microsphere precursor.

[0077] After 40mg of the gelatin microsphere precursor was dissolved in 4mL of a phosphate buffer solution, 1mL of a 1.5wt% genipin solution was added and mixed, and the crosslinking reaction was carried out for 12h. After repeated centrifugal 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 contains 0.15g of sodium dihydrogen phosphate and 0.02g of disodium hydrogen phosphate.

[0078] Step four, preparation of hydrogel dressing.

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

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

[0081] 0.2g of sterilized gelatin microspheres material loaded with deferoxamine mesylate and 0.3g of sterilized 3-carboxyphenylboric acid modified chitosan were dissolved in 10mL of water to obtain a second pre-polymer solution.

[0082] The first pre-polymer solution and the second pre-polymer solution were mixed and stirred uniformly to obtain a hydrogel dressing.

[0083] Example 3

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

[0085] Step one, preparation of oxidized hyaluronic acid.

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

[0087] Dissolve 2.2 g NaIO4 in 10 mL deionized water to prepare a uniform and transparent NaIO4 solution under light shielding condition.

[0088] Mix 100 mL hyaluronic acid solution and 10 mL NaIO4 solution, and react under light shielding condition for 4.5 h. Add 20 mL ethylene glycol, and terminate the reaction after stirring for 2 h to prepare an oxidized hyaluronic acid precursor.

[0089] Dialyze the oxidized hyaluronic acid precursor, and then freeze-dry to prepare the oxidized hyaluronic acid. The dialysis time is 3 d.

[0090] Step two, preparation of 3-carboxyphenylboronic acid modified chitosan.

[0091] Dissolve 1.5 g chitosan in 400 mL deionized water to prepare a uniform and transparent chitosan solution.

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

[0093] Mix the chitosan solution and the reaction activation solution to react for 24 h to obtain a final reaction solution. Dialyze and then freeze-dry to prepare 3-carboxyphenylboronic acid modified chitosan. The dialysis time is 4 d.

[0094] Step three, preparation of gelatin microsphere material loaded with deferoxamine mesylate.

[0095] At a temperature of 60°C, 0.5 mg deferoxamine mesylate, 1.5 g gelatin powder and 10 mL deionized water are configured as an aqueous phase.

[0096] Add 1 g Span-80 to 80 mL corn oil to prepare an oil phase.

[0097] Drop the oil phase into the aqueous phase, emulsify for 30 min, cool the emulsion at 3°C and continuously stir for 40 min. Add 200 mL acetone to dehydrate for 50 min, and then perform suction filtration and drying to prepare a gelatin microsphere precursor.

[0098] Dissolve 60 mg gelatin microsphere precursor in 4 mL phosphate buffer solution, and then mix with 1.5 mL genipin solution with a mass percentage of 1.5 wt%. Crosslink for 12 h, repeatedly centrifuge and wash with ethanol for 3 times, rotary evaporate, and freeze-dry to prepare the gelatin microsphere material loaded with deferoxamine mesylate. The phosphate buffer solution contains 0.15 g sodium dihydrogen phosphate and 0.02 g disodium hydrogen phosphate.

[0099] Step four, preparation of the hydrogel dressing.

[0100] The epigallocatechin gallate, oxidized hyaluronic acid, gelatin microsphere material loaded with deferoxamine mesylate, and 3-carboxyphenylboronic acid modified chitosan are subjected to sterilization treatment, and the sterilization method is Co60 irradiation sterilization for 36 h.

[0101] 0.01 g of the sterilized epigallocatechin gallate and 0.2 g of the sterilized oxidized hyaluronic acid are dissolved in 10 mL of water to prepare a first pre-polymerization liquid.

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

[0103] The first pre-polymerization liquid and the second pre-polymerization liquid are mixed and uniformly stirred to prepare the hydrogel dressing.

[0104] The application also provides Examples 4-6, wherein, compared with Example 1, the difference of Example 4 lies in that the corn oil used in step three is replaced by castor oil; compared with Example 1, the difference of Example 5 lies in that the corn oil used in step three is replaced by hemp oil; and compared with Example 1, the difference of Example 6 lies in that the corn oil used in step three is replaced by olive oil.

[0105] Comparative Example 1

[0106] A preparation method of a hydrogel dressing, comprising the following steps:

[0107] Step one, preparation of oxidized hyaluronic acid.

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

[0109] 2.2 g of NaIO4 is dissolved in 10 mL of deionized water under light shielding conditions to prepare a uniform and transparent NaIO4 solution.

[0110] 100 mL of the hyaluronic acid solution and 10 mL of the NaIO4 solution are mixed, and the reaction is carried out in the dark for 4.5 h, 20 mL of ethylene glycol is added, the reaction is terminated after stirring for 2 h, and an oxidized hyaluronic acid precursor is prepared.

[0111] The oxidized hyaluronic acid precursor is first subjected to dialysis treatment, and then subjected to freeze-drying treatment to prepare the oxidized hyaluronic acid, wherein the dialysis treatment time is 3 d.

[0112] Step two, preparation of 3-carboxyphenylboronic acid modified chitosan.

[0113] Dissolve 1.5 g of chitosan in 400 mL of 0.3% acetic acid aqueous solution to prepare a uniform and transparent chitosan solution.

[0114] Mix 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 to prepare a reaction activation solution, and adjust the pH of the solution to 4.5.

[0115] Mix the chitosan solution and the reaction activation solution to react for 24 h to obtain a final reaction solution. Then, the final reaction solution is treated by dialysis for 4 d and freeze-drying to obtain 3-carboxyphenylboronic acid modified chitosan.

[0116] Step three, preparation of the hydrogel dressing.

[0117] The epigallocatechin gallate, oxidized hyaluronic acid and 3-carboxyphenylboronic acid modified chitosan are sterilized by Co60 irradiation for 36 h.

[0118] Dissolve 0.01 g of the sterilized epigallocatechin gallate and 0.2 g of the sterilized oxidized hyaluronic acid in 10 mL of water to prepare a first pre-polymerization solution.

[0119] Dissolve 0.4 g of the sterilized 3-carboxyphenylboronic acid modified chitosan in 10 mL of water to prepare a second pre-polymerization solution.

[0120] Mix the first pre-polymerization solution and the second pre-polymerization solution, and stir until uniform to obtain the hydrogel dressing.

[0121] Test 1: microstructure analysis.

[0122] Figure 1 The scanning electron microscope image of the gelatin microsphere material loaded with deferoxamine mesylate prepared in Example 1 of the present application shows that the gelatin microsphere material loaded with deferoxamine mesylate is uniform in spherical shape, uniform in size and complete in structure. The average diameter of the gelatin microsphere material is calculated to be 8.51 ± 0.86 μm by using a particle size software.

[0123] Figure 2 The Fourier transform infrared absorption spectrum, the nuclear magnetic resonance hydrogen spectrum and the synthetic route diagram of the 3-carboxyphenylboronic acid modified chitosan prepared in Example 1 of the present application are shown in the figure. Figure 2 b shows that the 3-carboxyphenylboronic acid modified chitosan appears a new peak at 770 cm -1 which represents the C-H bending vibration peak outside the aromatic ring. Figure 2c shows: 3-carboxyphenylboronic acid modified chitosan peaks at 7.8 and 8.2 ppm, representing phenyl protons. 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 synthetic route diagram of hyaluronic acid oxidized prepared in Example 1 of the present application. Figure 3 b shows: hyaluronic acid has a weak C=O bending vibration peak at 1734 cm -1 This may be due to the formation of hemiacetal, so it is difficult to detect the signal of aldehyde group. The nuclear magnetic resonance hydrogen spectrum of hyaluronic acid and hyaluronic acid oxidized is shown in Figure 3 c, a new peak is observed at 4.9 ppm-5.0 ppm in the spectrum of hyaluronic acid oxidized, corresponding to the hemiacetal proton formed by the aldehyde group and the adjacent hydroxyl group. The above results show that the ortho-dihydroxy group of hyaluronic acid is successfully oxidized to aldehyde group.

[0125] Figure 4 The gelation diagram of the hydrogel dressing prepared in Example 1 of the present application. The gelation performance of the hydrogel dressing is measured by the inverted non-flow method. As shown in the figure, after the first pre-polymer solution and the second pre-polymer solution are mixed, the mixed solution does not flow within 57.6±2.5s after the vial is inverted, and the hydrogel dressing is formed.

[0126] Figure 5 The scanning electron microscope image of the hydrogel dressing prepared in Example 1 of the present application. As can be seen from the figure, the hydrogel dressing has a three-dimensional porous structure inside, and the gelatin microsphere material loaded with deferoxamine mesylate is distributed in the pores.

[0127] Test 2: analysis of therapeutic effect.

[0128] Test material: SPF level 8-week-old male SD rats, weighing 200-250g.

[0129] Establishment of diabetic SD rat model: 60 SPF level 8-week-old male SD rats weighing 200-250g were selected and adaptively fed at 25℃ for 1 week; the rats were fasted before operation, and 1% streptozotocin solution was injected according to the standard of 65mg / kg of rat body weight; 72h later, the blood glucose of the SD rats was continuously observed for 3 weeks; when the random blood glucose of the rats was stable at more than 16.7mmol / L within 3 weeks, it was considered that the model was established.

[0130] Test method: after the diabetic SD rat model was successfully established, a full-thickness skin defect model was established on the feet of the rats. The diabetic rats after modeling were randomly divided into a blank control group, a commercial hydrogel dressing group and an Example 1 hydrogel dressing group, 20 rats in each group. Among them, the commercial hydrogel dressing is HeraDerm.

[0131] Preoperative fasting, after successful anesthesia with 75% alcohol disinfect the rat paw, with skin sampler in the middle of the paw to cut off the full-thickness skin defect wound with a diameter of 6mm. The blank control group is not treated with any dressing, the commercial hydrogel dressing group is treated with HeraDerm dressing, and the hydrogel dressing group of example 1 is treated with the hydrogel dressing prepared in example 1. Observe the rats in each group daily to ensure that the wound is effectively covered with the dressing, and replace the dressing every 2 days.

[0132] Analysis of test results:

[0133] The treatment effect of diabetic wounds of diabetic SD rats is compared, as shown in Figure 6 The treatment speed of the hydrogel dressing group of example 1 is significantly faster than that of the blank control group and the commercial hydrogel dressing group, which is about 2.4 times that of the blank control group, and the wound healing time of the hydrogel dressing group of example 1 can be as short as 13 days, and the rats in the hydrogel dressing group of example 1 do not show obvious abnormalities. The animal experiment results show that the hydrogel dressing prepared in example 1 of the present application 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 application can have a good therapeutic effect on diabetic foot ulcers, and can solve the technical problem of the lack of dressings that can treat diabetic foot ulcers, and apply the hydrogel dressing to the treatment of diabetic foot ulcers.

[0134] In addition, the hydrogel dressing prepared in example 1 is replaced with the hydrogel dressings prepared in examples 4-6, and there is no significant difference in the treatment effect of diabetic wounds of diabetic SD rats, indicating that the type of vegetable oil does not significantly affect the performance of the hydrogel dressing, so the present application does not specifically limit the specific type of vegetable oil used.

[0135] Test 3: cell compatibility analysis.

[0136] Test material: L929 cell line, i.e. mouse fibroblast cell line.

[0137] Test method: The L929 cell line, i.e. mouse fibroblast cell line, is randomly divided into three groups, which are blank control group, example 1 hydrogel dressing group and comparative example 1 hydrogel dressing group. Among them, the mouse fibroblast cell line of the blank control group is incubated alone for 3 days and the incubation condition is observed and recorded daily; the mouse fibroblast cell line of the example 1 hydrogel dressing group is incubated with the hydrogel dressing prepared in example 1 for 3 days and the incubation condition is observed and recorded daily; the mouse fibroblast cell line of the comparative example 1 hydrogel dressing group is incubated with the hydrogel dressing prepared in example 1 for 3 days and the incubation condition is observed and recorded daily. The final experimental results are shown in Figure 7 and Figure 8 ​

[0138] Analysis of test results:

[0139] Figure 7 As can be seen from the above, the cell morphology of the three groups of mouse fibroblast cell lines is basically spindle-shaped and has no significant difference, indicating that the hydrogel dressing prepared in Example 1 does not have obvious toxic side effects on the growth of mouse fibroblast cell lines.

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

[0141] The above description is only the preferred embodiments of the present application, and the above specific embodiments are not a limitation on the present application. Various modifications and changes can occur within the scope of the technical idea of the present application, and any modification, change or equivalent replacement made by those skilled in the art according to the above description shall fall within the scope of the present application.

Claims

1. A method of preparing a hydrogel dressing, characterized by, The method comprises the following steps: The gelatin and the aqueous solution of deferoxamine mesylate are emulsified in the oil phase environment to obtain a gelatin microsphere precursor, the gelatin microsphere precursor and genipin are mixed, the gelatin and the genipin are crosslinked to obtain the gelatin microsphere material loaded with deferoxamine mesylate; The epigallocatechin gallate is mixed with the oxidized hyaluronic acid solution to obtain a first pre-polymerization solution; The gelatin microsphere material loaded with deferoxamine mesylate is mixed with the 3-carboxyphenylboronic acid modified chitosan solution to obtain a second pre-polymerization solution; The first pre-polymerization solution and the second pre-polymerization solution are mixed, the oxidized hyaluronic acid and the 3-carboxyphenylboronic acid modified chitosan are subjected to a Schiff base reaction to form a first crosslinking network containing a Schiff base bond, the first crosslinking network is used to coat the gelatin microsphere material and the epigallocatechin gallate, meanwhile, the epigallocatechin gallate and the 3-carboxyphenylboronic acid modified chitosan are subjected to an esterification reaction to form a second crosslinking network containing a borate ester bond, and a hydrogel dressing is obtained; The preparation method of the gelatin microsphere material loaded with deferoxamine mesylate comprises: The deferoxamine mesylate, the gelatin and water are used as an aqueous phase, and the Span-80 and the vegetable oil are used as an oil phase, the aqueous phase and the oil phase are mixed and emulsified to obtain a gelatin microsphere precursor, the gelatin microsphere precursor is mixed with a phosphate buffer solution, and then mixed with genipin, the gelatin and the genipin are reacted to form a third crosslinking network, and the gelatin microsphere material loaded with deferoxamine mesylate is obtained; The mass ratio of the gelatin to the deferoxamine mesylate is 1000-3000:1, the mass ratio of water to the gelatin is 5-10:1, and the reaction temperature of the aqueous phase is 45-60 DEG 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, and the reaction time of the emulsification reaction is 15-30 min; The concentration of the gelatin microsphere precursor is 10mg·mL -1 ~15mg·mL -1 after mixing with the phosphate buffer solution. The mass fraction of the genipin in the gelatin microsphere material loaded with deferoxamine mesylate is 0.5%-1.5%; The hydrogel dressing is used for controlled release of deferoxamine mesylate and epigallocatechin gallate.

2. The method of claim 1, wherein the hydrogel dressing is prepared by, Before the first pre-polymerization solution and the second pre-polymerization solution are prepared, 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 subjected to sterilization treatment.

3. The method of claim 1, wherein the hydrogel dressing is prepared by, The preparation method of the oxidized hyaluronic acid comprises: The hyaluronic acid solution, the sodium periodate solution and the ethylene glycol are mixed and subjected to a light-proof reaction to obtain an oxidized hyaluronic acid precursor; The oxidized hyaluronic acid precursor is subjected to freeze-drying dialysis treatment to obtain the oxidized hyaluronic acid.

4. The method of claim 3, wherein the hydrogel dressing is prepared by, In the hyaluronic acid solution, the mass ratio of a solvent to the hyaluronic acid is 40-200:1; In the sodium periodate solution, the mass ratio of a 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 10-30:1, 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 5-20:

1.

5. The method of claim 1, wherein the hydrogel dressing is prepared by, The preparation method of the 3-carboxyphenyl boronic acid modified chitosan solution comprises the following steps: Chitosan is mixed with an aqueous acetic acid solution to prepare a chitosan solution, and 3-carboxyphenyl boronic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide are mixed with anhydrous methanol to prepare a reaction activation solution; The chitosan solution and the reaction activation solution are mixed and reacted, and then subjected to dialysis and lyophilization to prepare 3-carboxyphenyl boronic acid modified chitosan.

6. The method of claim 5, wherein the hydrogel dressing is prepared by, In the aqueous acetic acid solution, the mass ratio of water to the acetic acid is 200-500:1; The mass fraction of the chitosan in the chitosan solution is 0.2%-0.7%, the molar ratio of 3-carboxyphenyl boronic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide is 2:1-5:1-5, and the pH of the reaction activation solution is 4.5-6.5; The mass ratio of the chitosan solution to the reaction activation solution is 5-10:

1.

7. A hydrogel dressing, characterized in that, The hydrogel dressing is prepared by the preparation method of any one of claims 1-6.

8. Use of the hydrogel dressing of claim 7 in the preparation of a dressing for diabetic foot ulcers.

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