Application of conductive hydrogel combined with electrical stimulation in treatment of diabetic foot ulcer caused by neuropathy

Abstract

The invention discloses application of conductive hydrogel combined with electrical stimulation in treatment of diabetic foot ulcer caused by neuropathy, and belongs to the field of biological medicine. The conductive hydrogel is obtained by mixing chitosan, polyvinyl alcohol, MXene and gelatin; the chitosan is catechol group modified chitosan, the MXene is polyphenol and Mg ion modified MXene, the gelatin is phenylboronic acid modified gelatin, the electrical stimulation condition is 100 mV cm <-1 >, and the diabetic foot ulcer comprises diabetic foot ulcer caused by neuropathy. Experiments prove that the prepared conductive hydrogel can promote cell migration in combination with electrical stimulation, wound healing of the diabetic foot ulcer can be accelerated, functional repair can be achieved, and a new thought is provided for clinical treatment of the diabetic foot ulcer caused by neuropathy.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to the application of conductive hydrogel combined with electrical stimulation in treating diabetic foot ulcers caused by neuropathy. Background Art

[0002] Diabetic neuropathy is the most common complication, which occurs in about 50% of diabetic patients. It is mainly manifested by impaired nerve regeneration and sensory motor disorders. If not treated properly, it will eventually lead to ulcers, infections and gangrene in the lower limbs. On the other hand, about 34% of diabetic patients will develop diabetic foot ulcers (DFUs), and patients with neuropathy are more likely to have DFUs. After the wound appears, impaired blood supply and ROS accumulation will immediately trigger an inflammatory response, further aggravating vascular lesions and neuropathy. Diabetic foot ulcers caused by diabetic neuropathy are difficult to heal and are at risk of amputation and death, which greatly reduces the overall health level and quality of life of patients. Although a variety of treatments for diabetic foot ulcers have emerged in recent years, their efficacy for diabetic foot ulcers caused by neuropathy is still limited. Therefore, an effective treatment strategy is urgently needed for DFUs caused by neuropathy. Summary of the invention

[0003] The purpose of the present invention is to provide an application of a conductive hydrogel combined with electrical stimulation in the treatment of diabetic foot ulcers caused by neuropathy, so as to solve the problems existing in the above-mentioned prior art. The conductive hydrogel prepared by the present invention combined with electrical stimulation can promote cell migration, accelerate wound healing of diabetic foot ulcers and achieve functional repair. The present invention provides a new idea for the clinical treatment of diabetic foot ulcers caused by neuropathy.

[0004] To achieve the above object, the present invention provides the following solutions:

[0005] The present invention provides an application of a conductive hydrogel combined with electrical stimulation in the preparation of a drug for treating diabetic foot ulcers, wherein the conductive hydrogel is obtained by mixing chitosan, polyvinyl alcohol, MXene and gelatin;

[0006] The chitosan is chitosan modified with catechol groups, the MXene is MXene modified with polyphenols and Mg ions, and the gelatin is gelatin modified with phenylboronic acid.

[0007] Optionally, the chitosan modified with catechol groups is prepared by the following method: dissolving chitosan to obtain a chitosan solution, adding dihydrocaffeic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysulfosuccinimide, reacting in an argon environment, and dialyzing, dialysis, and freeze-drying to obtain chitosan modified with catechol groups; the mass ratio of chitosan, dihydrocaffeic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysulfosuccinimide is (50-80):(26-45.5):(24-30):(15-20);

[0008] The polyphenol and Mg ion modified MXene is prepared by the following method: epigallocatechin gallate or gallic acid is added to the MXene dispersion, and then MgCl 2 6H 2 O is stirred for reaction, washed with deionized water and centrifuged, and the precipitate is collected to obtain MXene modified with polyphenol and Mg ion; the epigallocatechin gallate or gallic acid, MXene and MgCl 2 6H 2 The mass ratio of O is (1-5):1:(5-6);

[0009] The phenylboronic acid modified gelatin is prepared by the following method: N-hydroxysulfosuccinimide and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride are added to gelatin and stirred evenly, and then N-phenylbenzamide is added and stirred in a water bath, and the phenylboronic acid modified gelatin is obtained by dialysis and freeze drying; the mass ratio of the gelatin, N-hydroxysulfosuccinimide, N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and N-phenylbenzamide is (4-6):(7-10):(2-3):(3.5-5);

[0010] The conductive hydrogel is prepared by mixing chitosan modified with catechol groups, polyvinyl alcohol and water in a ratio of 0.1 g: 1 g: 10 mL to obtain solution A; MXene modified with polyphenol and Mg ions and gelatin modified with phenylboronic acid in a mass ratio of (0.8-1.6): 12.5 to obtain solution B; and solutions A and B are mixed in a volume ratio of 1:1 to obtain solution B.

[0011] Optionally, the electrical stimulation condition is 100 mV cm -1 .

[0012] Optionally, the diabetic foot ulcer includes diabetic foot ulcer caused by neuropathy.

[0013] Optionally, the conductive hydrogel plays a role in treating diabetic foot ulcers by promoting cell migration.

[0014] The present invention also provides a hydrogel preparation for treating diabetic foot ulcers, comprising the conductive hydrogel;

[0015] The hydrogel preparation is combined with electrical stimulation to treat diabetic foot ulcers.

[0016] Optionally, the electrical stimulation condition is 100 mV cm -1 .

[0017] Optionally, the diabetic foot ulcer includes diabetic foot ulcer caused by neuropathy.

[0018] Optionally, the conductive hydrogel plays a role in treating diabetic foot ulcers by promoting cell migration.

[0019] The present invention discloses the following technical effects:

[0020] The main raw materials of the conductive hydrogel synthesized by the present invention have wide sources, good biocompatibility, and are non-toxic, which is conducive to the commercialization of the product; and the hydrogel gelation process is easy and the gelation speed is fast.

[0021] Experimental verification shows that the conductive hydrogel prepared by the present invention combined with electrical stimulation can promote cell migration, accelerate wound healing of diabetic foot ulcers and achieve functional repair. It can be seen that the conductive hydrogel prepared by the present invention combined with electrical stimulation can treat diabetic foot ulcers caused by neuropathy. The present invention provides a new idea for the clinical treatment of diabetic foot ulcers caused by neuropathy. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0023] Figure 1 This is a diagram of the steps of forming the conductive hydrogel in Example 1;

[0024] Figure 2 The figure is a graph showing the results of the cell migration experiment within 24 hours of Example 1; the left figure is a picture of the cell migration experiment; the right figure is a statistical graph of the migration rate;

[0025] Figure 3 The left picture is a wound image; the right picture is a statistical chart of the residual wound area;

[0026] Figure 4 This is the Masson staining picture of the wound surface in Example 1. DETAILED DESCRIPTION

[0027] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0028] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0029] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0030] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0031] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0032] Example 1

[0033] 1. Preparation of conductive hydrogel

[0034] Preparation steps: Figure 1 As shown, the modified chitosan (CSDA) and polyvinyl alcohol (PVA) are mixed to obtain solution A, and the nanosheets modified with polyphenol and Mg ions (MXene@EGCG-Mg) and modified gelatin (GPBA) are mixed to obtain solution B. After mixing solutions A and B in a certain ratio and refrigerating at low temperature, a conductive hydrogel is obtained.

[0035] 1.1 Preparation of modified MXene nanosheets

[0036] First, add 1g of LiF to 20mL of 9M hydrochloric acid solution and stir thoroughly to react. Then slowly add 1g of Ti 3 AlC 2 The reaction was stirred at 35-45°C for 26 hours. The precipitate was collected and resuspended in deionized water and centrifuged at 4500rpm for 5 minutes, and washed several times until the pH value of the supernatant reached 6. Then the precipitate was collected and added to 60mL of deionized water for ultrasonic dispersion under Ar environment for 1 hour, and the supernatant was collected by centrifugation to obtain the MXene nanosheet dispersion. 10mg of EGCG (epigallocatechin gallate) was dissolved in Tris at pH=8.5, and then slowly added to the 10mg / mL MXene nanosheet dispersion and stirred for 2 hours in the dark. Then 50mg of MgCl 2 6H 2 O was added to 2 mL of Tris solution and then added dropwise to the above MXene nanosheet dispersion containing EGCG and stirred for 1 h. Finally, the mixture was centrifuged at 8000 rpm for 5 min and the precipitate MXene@EGCG-Mg was collected by washing with deionized water.

[0037] 1.2 Preparation of chitosan modified with catechol groups (CSDA)

[0038] 0.5g chitosan was dissolved in deionized water, pH was adjusted to 5.0 by HCl, and stirred in a water bath at 60℃ for 20min. Then 260mg dihydrocaffeic acid was dissolved in 5.0mL deionized water, stirred thoroughly and dissolved, and then added to the chitosan solution. 240mg 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC) and 150mg N-hydroxysulfosuccinimide (NHS) were dissolved in an ethanol solution (50.0mL ethanol and deionized water were mixed in a ratio of 1:1). Then it was added to the chitosan solution containing HCA, and reacted at room temperature for 12h after passing Ar. The solution was then dialyzed in acidified deionized water for 2d, then dialyzed in deionized water for another half a day, and finally freeze-dried in a freeze dryer to obtain a yellow solid (CSDA).

[0039] 1.3 Preparation of phenylboronic acid-modified gelatin (GPBA)

[0040] 4g of gelatin was added to 400mL of deionized water and stirred at 70℃. 7g of N-hydroxysulfosuccinimide (NHS) and 2g of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) powder were added to the above solution and stirred evenly, and the pH was adjusted to 5 with 1M hydrochloric acid solution. Then 3.5g of N-phenylbenzamide (PBA) powder was added and stirred in a water bath at 37℃ for 48h, and the obtained solution was dialyzed in deionized water for 3 days and then freeze-dried to obtain GPBA.

[0041] 1g of PVA powder was added to 10mL of deionized water and dissolved at 95℃. 0.1g of CSDA was added to the dissolved PVA solution and stirred for 1h to obtain solution A. Then GPBA was prepared into a 12.5wt% solution, 0.25mL was taken, and 20μL of 10mg / mL MXene@EGCG-Mg was added to it to obtain solution B. Solutions A and B were mixed in a volume ratio of 1:1 and placed in a -80℃ refrigerator for 12h, and thawed at room temperature to obtain a conductive hydrogel (PCPM2).

[0042] 2. Cell migration assay

[0043] PC12 cells were plated at 8×10 5 The cells were plated at a density of 100 cells / mL in 6-well plates and incubated at 37°C in 5% CO 2 Incubate in the incubator for 8 hours. Use a 10 μL pipette tip to make a direct cell scratch on each well of the plate to thoroughly rinse off the floating cells. Mark this moment as time zero, and observe and photograph the sample under a confocal microscope. Then, the cells were electrically stimulated with the hydrogel extract (external electric field was applied, and the parameters of the electrical stimulation were: 100 mV cm -1 ) for 30 minutes, and a control group without adding anything was set up. After incubation for 12 and 24 hours, the cells were observed and photographed under a confocal microscope.

[0044] The results are as follows Figure 2 As shown, the results showed that conductive hydrogel combined with electrical stimulation could accelerate cell migration compared with the control group.

[0045] 3. Diabetic foot ulcer wound model experiment

[0046] Male Sprague-Dawley rats weighing about 350 g were used to establish a diabetes model. After fasting for 12 hours, streptozotocin (50 mg / kg) dissolved in 2% citric acid solution was intraperitoneally injected. After 7 days, random samples were taken from the rat tail vein to test blood glucose levels. The diabetes model was effectively established when the random blood glucose level exceeded 16.7 mmol / L. After 4 weeks, diabetic rats with neuropathy were screened out by mechanical withdrawal threshold. After anesthesia, a circular skin wound (diameter = 5 mm) was established on the dorsum of the foot of all diabetic rats with neuropathy using a skin punch to obtain a diabetic foot ulcer wound model. A total of 12 diabetic rats with neuropathy were randomly assigned to two different groups: a control group and an electrical stimulation group (exogenous electric field was applied, and the parameters of the electrical stimulation were: 100 mV cm -1 ) in the PCPM2 group, and the wound healing process was carefully observed and photographed on days 0, 4, 8, 12, and 16.

[0047] On the 16th day after surgery, the animals were killed, and the foot skin wounds and surrounding tissues were collected and fixed with 4.0% paraformaldehyde buffer solution. After 1 hour, the samples were embedded in paraffin and prepared into 5 μm thick sections with a tissue slicer for further Masson staining.

[0048] Wound healing Figure 3 As shown in the figure, it can be seen that conductive hydrogel combined with electrical stimulation can promote the repair of diabetic foot ulcers within 16 days, and the wound can be completely healed on the 16th day. Figure 4 , it was found that conductive hydrogel combined with electrical stimulation can increase the collagen density of tissues and promote hair follicle generation.

[0049] Example 2

[0050] 1. Preparation of conductive hydrogel

[0051] 1.1 Preparation of modified MXene nanosheets

[0052] First, add 2g of LiF to 40mL of 10M hydrochloric acid solution and stir thoroughly to react. 3 AlC 2 Slowly add and stir the reaction at 35°C for 28h. Collect the precipitate, resuspend it in deionized water and centrifuge it at 4500rpm for 8min, wash it several times until the pH value of the supernatant reaches 6. Then collect the precipitate, add 100mL deionized water and ultrasonically disperse it in Ar environment for 1.5h, and collect the supernatant by centrifugation to obtain the MXene nanosheet dispersion. Dissolve 50mg of gallic acid (GA) in Tris at pH=8.5, and then slowly add it dropwise to the 10mg / mL MXene nanosheet dispersion and stir it in the dark for 2h. Then add 60mg of MgCl 2 6H 2 O was added to 2 mL of Tris solution and then added dropwise to the previous solution, and the reaction was continued under stirring for 1 h. Finally, the mixture was centrifuged at 10,000 rpm with deionized water for 5 min, and the precipitate MXene@GA-Mg was collected by washing with deionized water.

[0053] 1.2 Preparation of chitosan modified with catechol groups (CSDA)

[0054] 0.8g chitosan was dissolved in deionized water, the pH was adjusted to 5.0 by HCl, and stirred in a water bath at 80℃ for 30min. Then 455mg of dihydrocaffeic acid was dissolved in 8.0mL deionized water, stirred thoroughly and dissolved, and then added to the chitosan solution. 300mg 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC) and 200mg N-hydroxysulfosuccinimide (NHS) were dissolved in an ethanol solution (50.0mL ethanol and deionized water were mixed in a ratio of 1:1). Then it was added to the chitosan solution containing HCA, and reacted at room temperature for 12h after passing Ar. The solution was then dialyzed in acidified deionized water for 3 days, then dialyzed in deionized water for another half a day, and finally placed in a freeze dryer for freeze drying to obtain a yellow solid (CSDA).

[0055] 1.3 Preparation of phenylboronic acid-modified gelatin (GPBA)

[0056] 6g of gelatin was added to 600mL of deionized water and stirred at 80℃. 10g of N-hydroxysulfosuccinimide (NHS) and 3g of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) powder were added to the above solution and stirred evenly, and the pH was adjusted to 5 with 2M hydrochloric acid solution. Then 5g of N-phenylbenzamide (PBA) powder was added and stirred in a water bath at 45℃ for 48h, and the obtained solution was dialyzed in deionized water for 3 days and then freeze-dried.

[0057] 2g of PVA powder was added to 20mL of deionized water solution and dissolved at 100℃. 0.2g of CSDA was added to the dissolved PVA solution and stirred for 2h to obtain solution A. Then GPBA was prepared into a 12.5wt% solution, 0.25mL was taken, and 40μL of 10mg / mL MXene@GA-Mg was added to it to obtain solution B. After the solution A and B were mixed in a volume ratio of 1:1, they were placed in a -80℃ refrigerator for 48h and thawed at room temperature to obtain a conductive hydrogel (PCPM2).

[0058] 2. Cell migration assay

[0059] PC12 cells were plated at 8×10 5 The cells were plated at a density of 100 cells / mL in 6-well plates and incubated at 37°C in 5% CO 2 Incubate in the incubator for 8 hours. Use a 10 μL pipette tip to make a direct cell scratch on each well of the plate to thoroughly rinse off the floating cells. Mark this moment as time zero, and observe and photograph the sample under a confocal microscope. Then, the cells were electrically stimulated with the hydrogel extract (external electric field was applied, and the parameters of the electrical stimulation were: 100 mV cm -1) for 30 minutes, and a control group without adding anything was set up. After incubation for 12 and 24 hours, the cells were observed and photographed under a confocal microscope.

[0060] Results and Figure 2 Similarly, it is shown that the conductive hydrogel prepared in this example combined with electrical stimulation can accelerate cell migration.

[0061] 3. Diabetic foot ulcer wound model experiment

[0062] Male Sprague-Dawley rats weighing about 350 grams were used to establish a diabetes model. After fasting for 12 hours, streptozotocin (50 mg / kg) dissolved in 2% citric acid solution was intraperitoneally injected. After 7 days, random samples were taken from the rat tail vein to test blood glucose levels. The diabetes model was effectively established when the random blood glucose level exceeded 16.7 mmol / L. After 4 weeks, diabetic rats with neuropathy were screened out by mechanical withdrawal threshold. After anesthesia, a circular skin wound (diameter = 5 mm) was established on the dorsum of the foot of all diabetic rats with neuropathy using a skin punch to obtain a diabetic foot ulcer wound model. A total of 12 diabetic rats with neuropathy were randomly assigned to two different groups: a control group and an electrical stimulation group (exogenous electric field was applied, and the parameters of the electrical stimulation were: 100 mV cm -1 Finally, the wound healing process was carefully observed and photographed on days 0, 4, 8, 12, and 16.

[0063] On the 16th day after surgery, the animals were killed, and the foot skin wounds and surrounding tissues were collected and fixed with 4.0% paraformaldehyde buffer solution. After 1 hour, the samples were embedded in paraffin and prepared into 5 μm thick sections with a tissue slicer for further Masson staining.

[0064] Wound healing and staining results Figure 3 , Figure 4 Similarly, conductive hydrogel combined with electrical stimulation can promote the repair of diabetic foot ulcers until complete healing within 16 days; conductive hydrogel combined with electrical stimulation can significantly increase the collagen density of the tissue and generate hair follicles.

[0065] The embodiments described above are only descriptions of the preferred embodiments of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. Application of conductive hydrogel combined with electrical stimulation in the preparation of a drug for treating diabetic foot ulcers, characterized in that: The conductive hydrogel is obtained by mixing chitosan, polyvinyl alcohol, MXene and gelatin; The chitosan is chitosan modified with catechol groups, the MXene is MXene modified with polyphenols and Mg ions, and the gelatin is gelatin modified with phenylboronic acid.

2. The use according to claim 1, characterized in that The chitosan modified with catechol groups is prepared by the following method: dissolving chitosan to obtain a chitosan solution, adding dihydrocaffeic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysulfosuccinimide, reacting in an argon environment, and dialyzing, dialysis, and freeze-drying to obtain chitosan modified with catechol groups; the mass ratio of chitosan, dihydrocaffeic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysulfosuccinimide is (50-80):(26-45.5):(24-30):(15-20); The polyphenol and Mg ion modified MXene is prepared by the following method: epigallocatechin gallate or gallic acid is added to a MXene dispersion, and then MgCl2·6H2O is added for stirring reaction, and deionized water is centrifuged for washing, and the precipitate is collected to obtain the polyphenol and Mg ion modified MXene; the mass ratio of epigallocatechin gallate or gallic acid, MXene and MgCl2·6H2O is (1-5):1:(5-6); The phenylboronic acid modified gelatin is prepared by the following method: N-hydroxysulfosuccinimide and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride are added to gelatin and stirred evenly, and then N-phenylbenzamide is added to react in a water bath and stirred, and the phenylboronic acid modified gelatin is obtained by dialysis and freeze drying; the mass ratio of the gelatin, N-hydroxysulfosuccinimide, N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and N-phenylbenzamide is (4-6): (7-10): (2-3): (3.5-5); The conductive hydrogel is prepared by mixing chitosan modified with catechol groups, polyvinyl alcohol and water in a ratio of 0.1 g: 1 g: 10 mL to obtain solution A; MXene modified with polyphenol and Mg ions and gelatin modified with phenylboronic acid in a mass ratio of (0.8-1.6): 12.5 to obtain solution B; and solutions A and B are mixed in a volume ratio of 1:1 to obtain solution B.

3. The use according to claim 1, characterized in that The electrical stimulation condition was 100 mV cm -1 .

4. The use according to claim 1, characterized in that The diabetic foot ulcer includes diabetic foot ulcer caused by neuropathy.

5. The use according to any one of claims 1 to 4, characterized in that: The conductive hydrogel plays a role in treating diabetic foot ulcers by promoting cell migration.

6. A hydrogel preparation for treating diabetic foot ulcers, characterized in that: Comprising the conductive hydrogel as claimed in claim 1; The hydrogel preparation is combined with electrical stimulation to treat diabetic foot ulcers.

7. The hydrogel preparation according to claim 6, wherein The electrical stimulation was conditioned at 100 mV cm -1 .

8. The hydrogel preparation according to claim 6, wherein The diabetic foot ulcer includes diabetic foot ulcer caused by neuropathy.

9. The hydrogel preparation according to claim 6, wherein The conductive hydrogel plays a role in treating diabetic foot ulcers by promoting cell migration.

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