A thermoelectric hydrogel composite dressing for promoting repair of diabetic ulcers and loading of targeted modified exosomes and a preparation method thereof

Abstract

The application provides a thermoelectric hydrogel composite dressing for promoting repair of diabetic ulcers and loading of targeted modified exosomes and a preparation method, which comprises the following steps: preparing methacrylated hyaluronic acid; preparing polyether F127 diacrylate; co-incubating exosomes with palmitic acid grafted targeting peptides to prepare targeted modified exosomes; mixing the methacrylated hyaluronic acid, the polyether F127 diacrylate, a photosensitizer, the targeted modified exosomes and an electrolyte, and crosslinking under ultraviolet light to form a composite gel. The composite dressing is a novel composite dressing for thermoelectric stimulation and exosome treatment. On one hand, the dressing generates a wireless and passive electric field to simulate the endogenous electric field in the wound, promote directional migration of skin cells, accelerate reepithelialization and dermal formation; on the other hand, the dressing releases exosomes to precisely target blood vessels of a wound lesion, reverse endothelial dysfunction in a high-sugar environment and promote angiogenesis, and effectively solves the problems in repair and regeneration of diabetic ulcer wounds.

Description

Technical Field

[0001] The present invention belongs to the field of diabetic ulcer treatment, and relates to a thermoelectric composite dressing that promotes diabetic ulcer healing and is loaded with targeted traditional Chinese medicine exosomes, and a preparation method thereof. Background Art

[0002] Diabetic ulcers are one of the leading causes of amputation and death in diabetic patients, posing a major challenge to their treatment. Endogenous electric fields and growth factors are two important factors that promote wound healing. Mechanistically, the asymmetric distribution of ion channels in epithelial cells allows cations and anions to be transported to the basal and apical sides of the epidermis, respectively, thereby generating a transepithelial potential. When skin injury occurs, the current driven by the transepithelial potential accurately generates a lateral endogenous electric field from the wound edge to the center, guiding the migration of keratinocytes and fibroblasts, achieving re-epithelialization and dermal regeneration. At the same time, blood vessels in the tissue surrounding the wound transport nutrients, especially growth factors, which stimulate the proliferation and migration of vascular endothelial cells to promote wound angiogenesis.

[0003] However, in the pathological microenvironment of diabetic ulcers, severe electrolyte loss often weakens the transepithelial potential, resulting in a lack of endogenous electric fields in the wound surface. At the same time, glucose metabolism disorders and oxidative stress damage caused by hyperglycemia can lead to endothelial cell dysfunction, thereby impairing the angiogenesis process of diabetic ulcers. In summary, the wound's endogenous electric field and growth factors cannot be activated at the site of diabetic ulcers, and the re-epithelialization, dermal regeneration, and angiogenesis processes mediated by both cannot be initiated, resulting in poor healing of diabetic ulcers. Although various technical means, including hyperbaric oxygen therapy, surgical sutures, and local application of growth factors, have shown certain therapeutic effects, these interventions still show quite limited efficiency in clinical practice.

[0004] Electrical stimulation is an effective means of reshaping the endogenous electric field within a wound. Traditional electrical stimulation methods generally use an external power source and metal electrodes to directly apply direct or alternating current to the wound site to promote healing. However, the rigid electrode interface can easily cause secondary wound damage, and the complex operation and reliance on an external power source can lead to reduced patient compliance. Currently, most research focuses on incorporating conductive materials into flexible carriers such as hydrogels as soft electrodes, which are then assembled with self-powered layers formed by triboelectric nanogenerators or piezoelectric materials to deliver electrical stimulation to the wound site. However, the inherent limitations of these systems still pose numerous challenges to clinical translation and optimal therapeutic efficacy. First, metallic or inorganic conductive fillers are generally non-degradable, resulting in limited biocompatibility. Furthermore, the electrical stimulation generated by triboelectric nanogenerators or piezoelectric materials is unstable or anisotropic, making it difficult to accurately mimic the directionality of the endogenous electric field, thereby reducing the controllability and efficiency of the electrical stimulation.

[0005] Thermoelectric hydrogels can directly convert thermal energy into electrical energy and generate a stable directional electric field at both ends with a temperature difference. At present, it has shown great potential in the field of energy and batteries. The flexible stretchable, high water content and adjustable mechanical properties of hydrogel materials are very suitable for wound treatment. However, it is challenging to apply thermoelectric hydrogels to the field of wound repair, because the current commonly used synthetic materials of thermoelectric hydrogels are polyacrylamide, polyvinyl alcohol, cellulose and the like, which degrade very slowly in the body. In addition, the additives such as K3[Fe(CN)6], NaOH, Fe(ClO4)3 and the like to induce thermoelectric effect have oxidation-reduction toxicity or biological toxicity, which hinder the biomedical conversion of thermoelectric hydrogels. Therefore, it is urgent to construct a new generation of thermoelectric medical hydrogel which can reconstruct the directional electric field of the wound surface to promote reepithelialization and dermal formation, and ensure precise control, biodegradation and high biocompatibility. However, research in this field is still blank.

[0006] On the other hand, oxidative stress damage caused by sugar metabolism disorder at the site of diabetic ulcer causes impaired angiogenesis, which is an important reason for the non-healing of diabetic ulcers. Most of the current pro-angiogenic drugs, such as recombinant vascular endothelial growth factor, have the disadvantages of high cost, low stability, short half-life and potential biological risks, and are difficult to reverse endothelial dysfunction. Ginseng was first recorded in Shennong's Herbal Classic, and has the advantages of "benefiting qi to promote blood circulation and removing blood stasis without damaging normal qi". It is a commonly used traditional Chinese medicine for promoting blood circulation and removing blood stasis in clinical practice. Modern pharmacological studies have shown that ginseng can dilate blood vessels, improve local microcirculation, maintain the stability of vascular endothelial function, and the like. Ginseng exosomes have been proven to be a new type of nanomedicine to reverse endothelial dysfunction in a hyperglycemic environment and promote angiogenesis. However, in the local microenvironment of diabetic ulcers involving multiple cell types, the competitive uptake of other cells may reduce the bioavailability of ginseng exosomes, and the repulsion effect between the negative charge on the surface of the exosomes and the cell membrane also limits the internalization of the exosomes by vascular endothelial cells.

[0007] The three-dimensional pores of the thermoelectric hydrogel can well load and release drugs such as exosomes, so how to use the thermoelectric hydrogel to apply artificial electric stimulation and deliver exosome drugs to promote angiogenesis is the key to promoting the overall repair of diabetic ulcers. However, research in this field is still blank. SUMMARY

[0008] The purpose of the present application is to overcome the shortcomings of the prior art and provide a thermoelectric hydrogel dressing for promoting the repair of diabetic ulcers and loading targeted modified traditional Chinese medicine exosomes and a preparation method thereof.

[0009] The technical scheme of the present application is as follows:

[0010] In a first aspect, the present invention provides a method for preparing a thermoelectric hydrogel composite dressing that promotes diabetic ulcer repair and is loaded with targeted modified exosomes, comprising the following steps:

[0011] A. reacting hyaluronic acid with methacrylic anhydride to prepare methacrylated hyaluronic acid;

[0012] B. reacting polyether F127 with acryloyl chloride to prepare polyether F127 diacrylate;

[0013] C. preparing and extracting Chinese herbal exosomes by differential centrifugation and ultracentrifugation; in step C, the source of the Chinese herbal exosomes is ginseng;

[0014] D. co-incubating the TCM exosomes with the targeting peptide at 37° C., and ultracentrifuging to obtain targeted-modified TCM exosomes; in step D, the targeting peptide is a vascular targeting peptide, which may be one or more of RGD peptide, NGR peptide, VHPK peptide, REDV peptide, CREKA peptide, F3 peptide, GX1 peptide, and RGP peptide;

[0015] E. Dissolving methacryloylated hyaluronic acid, polyether F127 diacrylate, sodium chloride, and LAP photosensitizer in ultrapure water, and adding targeted modified traditional Chinese medicine exosomes to form a pregel, which is cross-linked by ultraviolet light to prepare a thermoelectric composite dressing; in step E, the mass percentage of methacryloylated hyaluronic acid in the thermoelectric composite dressing is 0.1%-3.5%, and the mass percentage of polyether F127 diacrylate is 6.5%-9.9%; in the pregel, the concentration of sodium chloride is 0M-2.0M, and the concentration of targeted modified exosomes is 1 μg / mL-5000 μg / mL; the photosensitizer is one or both of LAP and I2959; and the mass percentage of the photosensitizer in the pregel is 0.01-5%.

[0016] According to a preferred embodiment of the present invention, the targeting peptide sequences are shown in the following table:

[0017] Targeting peptides Amino acid sequence RGD peptide RGD NGR peptide NGR VHPK peptide VHPK REDV peptide REDV CREKA peptide CREKA F3 peptide KDEPQRRSARLSAKPAPPKPEPPKPKKAPAKK GX1 peptide CNGRCVSGCAGRC RGP peptide RWRNMGGGGIVRRADRAAVP

[0018] According to a preferred embodiment of the present invention, step A specifically comprises: adding methacrylic anhydride dropwise to a sodium hyaluronate aqueous solution, stirring at 4°C for 1-24 hours, and continuously adding 10M NaOH to maintain the pH at 8; adding methacrylic anhydride again, reacting at 4°C for 6-36 hours, and then maintaining for 2-12 hours. The resulting final product is dialyzed against deionized water and then freeze-dried to obtain the prepared methacrylated hyaluronic acid; the mass ratio of methacrylic anhydride added initially to sodium hyaluronate is 0.1:1-10:1, and the mass ratio of methacrylic anhydride added again to sodium hyaluronate is 0.1:1-10:1.

[0019] According to a preferred embodiment of the present invention, step B specifically comprises: dissolving polyether F127 and triethylamine in anhydrous dichloromethane under a nitrogen atmosphere, and then slowly adding acryloyl chloride; stirring under a nitrogen atmosphere for 6-48 hours, filtering the precipitate, and adding the filtrate to diethyl ether to precipitate the remaining filtrate; and drying the resulting product under vacuum to prepare the obtained polyether F127 diacrylate; wherein the molar ratio of polyether F127 to triethylamine is 1:0.5-1:40, and the molar ratio of polyether F127 to acryloyl chloride is 1:0.5-1:50.

[0020] According to a preferred embodiment of the present invention, step C is specifically as follows: washing the medicinal materials and squeezing the juice; passing the squeezed juice through a 100-300 mesh sieve to remove the residue; differentially centrifuging the sieved juice at 1000-10000 g for 10-90 min, taking the supernatant; ultracentrifuging at 30000-250000 g for 0.5-4 h, and taking the precipitate to obtain the prepared traditional Chinese medicine exosomes.

[0021] According to a preferred embodiment of the present invention, in step D, the mass ratio of Chinese medicine exosomes to targeting peptide is 0.5:1-4:1, and step D is incubated at 37°C for 0.1-5 hours and ultracentrifuged at 30,000-250,000g for 0.5-4 hours to obtain targeted modified Chinese medicine exosomes.

[0022] According to a preferred embodiment of the present invention, in step E, the pre-gel is cross-linked by ultraviolet light by irradiating the pre-gel under a 405 nm blue-violet laser for 10-100 seconds.

[0023] In a second aspect, the present invention provides a composite dressing prepared according to the above method. This composite dressing is a novel composite dressing that combines thermoelectric stimulation with exosome therapy. On the one hand, the dressing generates a wireless, passive electric field that simulates the wound's endogenous electric field, promoting directional migration of skin cells and accelerating re-epithelialization and dermal formation. On the other hand, the dressing slowly releases exosomes that precisely target the blood vessels of wound lesions, reversing endothelial dysfunction in a high-glucose environment and promoting angiogenesis, effectively addressing the challenges of wound repair and regeneration in diabetic ulcers.

[0024] In a third aspect, the present invention provides use of the above-mentioned composite dressing in preparing a diabetic ulcer repair preparation.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] (1) The present invention uses fully degradable materials to prepare biocompatible thermoelectric hydrogels that can output directional electric fields, overcoming the difficulty of clinical translation of existing electrical stimulation systems, which are difficult to degrade. This achieves the effect of promoting electric field stimulation healing of diabetic ulcer reconstruction wounds with high biosafety.

[0027] (2) The present application uses a target-modified ginseng-derived exosome as a pro-angiogenic factor, which overcomes the problems of high cost, low stability, short half-life and potential carcinogenic risk of growth factor drugs in the prior art, thereby achieving the effect of reversing endothelial dysfunction of diabetic ulcers and promoting angiogenesis.

[0028] (3) The present application uses a thermoelectric hydrogel loaded with target-modified ginseng exosomes to prepare a multifunctional integrated composite dressing, which overcomes the problem of limited efficacy and regeneration caused by single electrical stimulation or drug therapy in the prior art, thereby achieving the effect of promoting the overall repair of diabetic ulcers. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 is the nuclear magnetic resonance hydrogen spectrum of methacrylated hyaluronic acid, polyether F127 diacrylate;

[0030] Figure 2 is the thermoelectric coefficient quantitative graph of the thermoelectric hydrogel under different concentrations of sodium chloride;

[0031] Figure 3 is a schematic diagram of the mechanism of the thermoelectric hydrogel generating a directional electric field;

[0032] Figure 4 is a transmission electron microscopy result graph of the target-modified exosome;

[0033] Figure 5 is a cell-specific uptake result graph of the target-modified exosome;

[0034] Figure 6 is a potential output-temperature difference result graph of the composite dressing;

[0035] Figure 7 is a curve graph of the composite dressing controlling the release of exosomes;

[0036] Figure 8 is a wound healing result graph of the composite dressing in diabetic mice;

[0037] Figure 9 is a body weight result graph of the composite dressing in diabetic mice;

[0038] Figure 10 is a skin keratin (CK14) immunofluorescence result graph of the composite dressing in diabetic mice;

[0039] Figure 11 is a skin vimentin (Vimentin) immunofluorescence result graph of the composite dressing in diabetic mice;

[0040] Figure 12 is a skin endothelial cell adhesion molecule (CD31) immunofluorescence result graph of the composite dressing in diabetic mice. DETAILED DESCRIPTION

[0041] The present invention will be described in detail below with reference to the accompanying drawings.

[0042] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below with reference to the accompanying drawings and embodiments. The specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0043] Example 1: Synthesis, characterization and thermoelectric effect testing of thermoelectric hydrogel, including the following steps:

[0044] 1) Add 2.5 mL of methacrylic anhydride dropwise to 100 mL of an aqueous solution containing 1 wt% sodium hyaluronate, stir at 4°C for 8 hours, and continuously add 10 M NaOH solution to maintain the pH of the system at 8. Add 1 mL of methacrylic anhydride again and react at 4°C for 12 hours. The final mixture is dialyzed against deionized water and lyophilized at -80°C to obtain methacryloylated hyaluronic acid.

[0045] After preparing methacryloyl hyaluronic acid, it was dissolved in deuterated water and then tested by nuclear magnetic resonance spectroscopy to obtain 1 H-NMR spectrum, see Figure 1 In the H spectrum of methacryloyl hyaluronic acid, there is a peak with a chemical shift between 5.2 and 5.6 ppm (shown in the black solid line box), which represents the hydrogen atoms on the double bond carbon.

[0046] 2) Under a nitrogen atmosphere, 50 g of polyether F127 and 4 mL of triethylamine were dissolved in dichloromethane. 3.2 mL of acryloyl chloride was then added using a dropping funnel. The mixture was stirred at 4°C under a nitrogen atmosphere for 24 h. The precipitate was filtered off and anhydrous ether was added to the filtrate to precipitate the filtrate. The resulting product was vacuum dried to obtain polyether F127 diacrylate.

[0047] After preparing polyether F127 diacrylate, it was dissolved in deuterated water and then tested by nuclear magnetic resonance spectroscopy to obtain 1 H-NMR spectrum, see Figure 1 In the H spectrum of polyether F127 diacrylate, there is a peak with a chemical shift between 5.2 and 5.6 ppm (shown in the black solid line box), which represents the hydrogen atoms on the double bond carbon.

[0048] 3) Methacryloylated hyaluronic acid, polyether F127 diacrylate, and sodium chloride were dissolved in an aqueous solution containing a LAP photosensitizer to form a pregel, wherein the methacryloylated hyaluronic acid content was 2 wt %, the polyether F127 diacrylate content was 8 wt %, and the LAP photosensitizer content was 0.05 wt %. The pregel was then exposed to 405 nm violet light for 45 seconds to crosslink and produce a thermoelectric hydrogel.

[0049] 4) Construct a test platform to perform thermoelectric testing on the prepared hydrogel samples. Two Peltier plates are placed in parallel and connected to two independent power supplies, with the cooling surface of one Peltier facing up and the heating surface of the other Peltier facing up. Hydrogel samples containing sodium chloride concentrations of 0M, 0.1M, 0.15M, 0.2M, 0.3M, and 0.4M are placed on two Peltier plates, and direct current is passed to establish a temperature difference at both ends of the hydrogel. Copper sheets are then used to connect the two ends of the hydrogel to the electrochemical workstation, and the change in potential difference with temperature difference is monitored in real time. The results are shown. The thermoelectric coefficients of the thermoelectric hydrogels containing 0M, 0.1M, 0.15M, 0.2M, 0.3M, and 0.4M sodium chloride are -1.715mV / K, -1.393mV / K, -0.6450mV / K, 0.6265mV / K, 1.442mV / K, and 1.000mV / K, respectively, indicating that when the temperature difference is 12°C, the potential difference outputs at the cold end and the hot end of the hydrogel are 20.58mV, 16.716mV, 7.74mV, -7.518mV, -17.304mV, and -12.00mV, respectively. Figure 2 shown.

[0050] The mechanism by which thermoelectric hydrogels generate directional potential differences at different sodium chloride concentrations is as follows: At low electrolyte concentrations, the hydrogel's methacryloylated hyaluronic acid component contains negatively charged carboxyl groups, which can use electrostatic adsorption to hinder the migration of cations, while anions quickly migrate along the negatively charged surface toward the cold end, causing the cold end potential to decrease. In the presence of a large amount of electrolyte, some cations tend to "aggregate" along the negatively charged methacryloylated hyaluronic acid chains. These fixed cations create resistance to the migration of anions, while the remaining free cations exhibit a strong migration rate and can reach the cold end faster, resulting in an increase in the cold end potential, see Figure 3 shown.

[0051] Example 2: Surface modification of ginseng exosomes and targeting evaluation of endothelial cells, comprising the following steps:

[0052] 1) Wash the ginseng with deionized water, peel, cut into pieces, and juice the resulting juice. Sieve the resulting juice to remove large pieces of plant tissue. Centrifuge the juice at 2000-6000g for 20-50 minutes in a tabletop centrifuge to remove larger plant debris. Centrifuge the supernatant at 7000-15000g for 1 hour to remove small plant fibers and debris. Centrifuge the supernatant at 1000000-200000g for 1-3 hours in a floor-standing ultracentrifuge. Resuspend the bottom precipitate in ultrapure water to obtain the ginseng exosome solution.

[0053] 2) Incubate the ginseng exosomes with REDV-Pal peptide at 30-45 °C under shaking condition for 0.25-1 hour, use a bench top ultracentrifuge to centrifuge at 1000000-200000 for 1-3h to obtain the targeted modified ginseng exosomes. Then filter sterilization with 0.22 pm filter, finally store the suspension at -80 °C until use.

[0054] Drop the exosome solution on carbon film coated copper grid, then counterstain with uranyl acetate, observe under transmission electron microscope, the targeted modified exosomes show standard pie structure, size is about 100-150 nm, see Figure 4

[0055] 3) This example uses human umbilical vein endothelial cells (HUVECs) and human keratinocytes (HaCats) as cell models to explore the targeting of the targeted modified exosomes. HUVECs were cultured in RPMI 1640 medium containing 10% fetal bovine serum (Gibco BRL), l-glutamine, penicillin (50 U / ml) and streptomycin (50 U / ml); HaCats were cultured in DMEM high glucose medium containing 10% fetal bovine serum (Gibco BRL), l-glutamine, penicillin (50 U / ml) and streptomycin (50 U / ml). Cells were stored at 37 °C, 5% CO2.

[0056] In order to explore the targeting of the targeted modified exosomes to vascular endothelial cells, HUVCEs and HaCats were seeded in the well plate respectively and allowed to adhere overnight. The next day, the cells were cultured with 8 pg / mL of unmodified ginseng exosomes and targeted modified ginseng exosomes for 12 hours, then the intracellular fluorescence intensity was detected using flow cytometry, and the data was analyzed by FlowJo software. The results showed that the ratio of HUVECs to HaCats for unmodified ginseng exosome uptake efficiency was 0.71, and the ratio of targeted modified ginseng exosome uptake efficiency was 1.46. It showed that the targeted modified ginseng exosomes not only significantly improved the uptake efficiency of HUVECs, but also showed significant specific targeting effect on HUVECs, see Figure 5

[0057] Example 3: Construction and characterization of thermoelectric hydrogel composite dressing loaded with targeted modified ginseng exosomes, including the following steps:

[0058] ​​1) The methacryloylated hyaluronic acid and polyether F127 diacrylate prepared in Example 1, the targeted modified ginseng exosomes prepared in Example 2, and sodium chloride electrolyte were dispersed or dissolved in ultrapure water containing a LAP photosensitizer to form a drug-loaded pre-gel. The methacryloylated hyaluronic acid content was 2 wt%, the polyether F127 diacrylate content was 8 wt%, the concentration of the targeted modified exosomes was 200 μg / mL, the concentration of sodium chloride was 0.1 M, and the LAP photosensitizer content was 0.5 wt%. The drug-loaded pre-gel was then exposed to 405 nm blue-violet light for 45 seconds to crosslink, resulting in a thermoelectric hydrogel composite dressing loaded with the targeted modified exosomes.

[0059] 2) The thermoelectric signal generated by the composite dressing was tested using the thermoelectric test platform in Example 1. The results showed that the composite dressing had a thermoelectric coefficient of 1.163 mV / K, indicating that it could generate a potential difference of 13.96 mV at a temperature difference of 12°C, and the hot end was higher than the cold end, indicating that it has the potential to provide biomimetic electrical stimulation at the site of diabetic ulcer wounds. Figure 6 shown.

[0060] 3) To characterize the release profile of the composite dressing for controlled release of targeted exosomes, 1 mL of the hydrogel composite dressing loaded with 200 μg of targeted exosomes was added to a dialysis bag and then placed in a centrifuge tube containing 25 mL of PBS. The centrifuge tube was placed in a shaker at 37°C and 100 rpm. At pre-set intervals, 1 mL of the solution was sampled and the same volume of PBS was added. The released protein concentration was determined using the micro-BCA assay and used to calculate the percentage of released targeted exosomes.

[0061] Over time, the targeted modified exosomes were gradually and stably released from the composite dressing. The release percentages of the targeted modified exosomes reached 25.8% and 86.7% within the first 8 hours and the next 48 hours, respectively. Figure 7 shown.

[0062] Example 4: Effect of composite dressing on diabetic ulcer wound healing and biosafety evaluation, comprising the following steps:

[0063] 1) Establish an animal model of diabetic ulcer. This example uses diabetic mice as an in vivo animal model and creates a local full-skin tissue defect model, which represents an in vivo animal model of diabetic ulcers, and explores the in vivo efficacy of the composite dressing. The study used 8-12 week old male B6.BKS(D)-Lepr db / J(db / db) mice. The mice were generally anesthetized and the wound was excised on the shaved back skin with a scalpel. The wound diameter was 12 mm. The diabetic mice were randomly divided into two experimental groups. The wound was treated with 100 μL PBS or composite dressing, respectively, as a blank control group and a composite dressing group, once every other day.

[0064] For the composite dressing group, treatment was performed as follows: First, a drug-loaded pregel was applied to the surface of the diabetic ulcer wound. Then, an indocyanine green-loaded hydrogel was applied to the entire periphery of the drug-loaded pregel. Photocrosslinking was performed for 45 seconds to allow the pregel to adhere tightly to the wound site. To induce a directional thermoelectric field to mimic the wound's endogenous electric field, infrared light was irradiated at the edge of the dressing, triggering the photothermal effect of indocyanine green, generating a directional electric field from the wound edge (hot end) to the wound center (cold end).

[0065] 2) Evaluate the effect of composite dressings on the healing rate of diabetic ulcers. Observe wound healing carefully, take photos of the wounds every other day when changing the dressing, and calculate the healing rate.

[0066]

[0067] The healing rate of the composite dressing group was significantly higher than that of the blank control group, indicating that the composite dressing promoted the rapid healing of diabetic ulcers. Figure 8 shown.

[0068] 3) Evaluate the biosafety of the composite dressing during in vivo application. The body weight of each group of diabetic mice was recorded every other day and the average value was calculated. During the entire animal experiment, the body weight of the mice in the blank control group and the composite dressing group remained stable, with no significant increase or decrease. The results objectively demonstrated that the composite dressing had no harmful systemic effects when applied in vivo. Figure 9 shown.

[0069] Example 5: Effects of composite dressing on re-epithelialization, dermal formation, and angiogenesis of diabetic ulcer wounds, comprising the following steps:

[0070] 1) Using the animal experimental protocol described in Example 3, a diabetic mouse skin ulcer model was constructed, and the composite dressing administration regimen described in Example 3 was used for treatment. On the 7th day of the animal experimental treatment, the skin of the blank control group and the composite dressing group mice at the wound healing site was collected. The skin tissue was fixed by immersion in 4% paraformaldehyde, then dehydrated with ethanol, embedded in paraffin and sliced ​​into 0.22 μm thick tissue sections. The tissue sections were subjected to immunofluorescence staining, and the epithelial cell keratin CK14 signal, fibroblast vimentin Vimentin signal, and endothelial cell adhesion molecule CD31 signal were detected to evaluate the effects of the composite dressing on diabetic ulcer wound re-epithelialization, dermal formation, and angiogenesis.

[0071] Specifically, the skin sections of the two groups of animals were placed in antigen retrieval buffer for antigen retrieval, and 10% goat serum was added to block at 37°C for 30 minutes. The blocking solution was gently shaken off, and the primary antibody dilutions of CK14 (1:200), Vimentin (1:300), and CD31 (1:1000) were added in proportion. The sections were placed flat in a humidified chamber and incubated overnight at 4°C. The primary antibody incubation solution was then removed, and the secondary antibody of the corresponding species to the primary antibody was added and incubated at room temperature in the dark for 50 minutes. The CK14 and CD31 signals were visualized with cy3-conjugated secondary antibodies, and the Vimentin signal was visualized with Alexa Fluor 488-conjugated secondary antibodies. The sections were then rinsed thoroughly with PBS three times, and DAPI staining solution was added to locate the cell nuclei. The sections were incubated at room temperature in the dark for 10 minutes. The sections were observed under a confocal microscope and images were collected.

[0072] 2) CK14 immunofluorescence staining of skin tissue showed that the epithelial tongue of diabetic ulcer wounds in the blank control group was very short and thicker, suggesting the formation of a scar-like structure. In contrast, the epithelial tongue length and re-epithelialization level in the composite dressing group increased significantly, and the wound width decreased significantly. This demonstrates that the composite dressing can effectively promote the re-epithelialization of diabetic ulcer wounds in vivo. Figure 10 shown.

[0073] Vimentin immunofluorescence staining of skin tissue showed that the blank control group had only a small amount of Vimentin-positive signals in the diabetic ulcer wound, indicating that only a few fibroblasts proliferated and migrated in the ulcer wound and the dermis was not well formed. In contrast, the composite dressing group had a large number of Vimentin-positive signals, indicating that a large number of fibroblasts proliferated and migrated, and the dermis was well formed. This proves that the composite dressing can effectively promote the formation of dermis in diabetic ulcer wounds in vivo. Figure 11 shown.

[0074] The skin tissue CD31 immunofluorescence staining shows that only a small amount of CD31 positive signal exists in the blank control group of diabetic ulcer wound, indicating that the phenomenon of angiogenesis is not obvious; in contrast, a large amount of CD31 positive signal exists in the composite dressing group, indicating that the phenomenon of angiogenesis is very significant. It is proved that the composite dressing can reverse the dysfunction of vascular endothelial cells in vivo, and efficiently promote the angiogenesis of diabetic ulcer, as shown in FIG. 8. Figure 12

[0075] The above-described embodiments only express several embodiments of the present application, which are described in a more specific and detailed manner, but should not be understood as a limitation on the scope of the patent of the present application. It should be noted that, for ordinary skilled persons in the art, several modifications and improvements can be made without departing from the concept of the present application, which all belong to the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the appended claims.​

Claims

1. A method for preparing a thermoelectric hydrogel composite dressing that promotes diabetic ulcer repair and is loaded with targeted modified traditional Chinese medicine exosomes, characterized in that: The following steps are involved: A. reacting hyaluronic acid with methacrylic anhydride to prepare methacrylated hyaluronic acid; B. reacting polyether F127 with acryloyl chloride to prepare polyether F127 diacrylate; C. preparing and extracting Chinese herbal exosomes by differential centrifugation and ultracentrifugation; in step C, the source of the Chinese herbal exosomes is ginseng; D. incubating the TCM exosomes with the targeting peptide at 37° C., and ultracentrifuging to obtain targeted modified TCM exosomes; in step D, the targeting peptide is one or more of RGD peptide, NGR peptide, VHPK peptide, REDV peptide, CREKA peptide, F3 peptide, GX1 peptide, and RGP peptide; E. Methacryloylated hyaluronic acid, polyether F127 diacrylate, sodium chloride, and LAP photosensitizer are dissolved in ultrapure water and the targeted modified traditional Chinese medicine exosomes are added to form a pregel. The pregel is cross-linked by ultraviolet light to prepare a thermoelectric composite dressing. In step E, the mass percentage of methacryloylated hyaluronic acid in the thermoelectric composite dressing is 0.1%-3.5%, and the mass percentage of polyether F127 diacrylate is 6.5%-9.9%; the concentration of sodium chloride in the pregel is 0M-2.0M, and the concentration of the targeted modified exosomes is 1μg / mL-5000μg / mL; the photosensitizer is one or both of LAP and I2959; and the mass percentage of the photosensitizer in the pregel is 0.01-5%.

2. The method for preparing a thermoelectric hydrogel composite dressing that promotes diabetic ulcer repair and is loaded with targeted modified traditional Chinese medicine exosomes according to claim 1, characterized in that: Step A is specifically as follows: adding methacrylic anhydride dropwise to a sodium hyaluronate aqueous solution, stirring at 4°C for 1-24 hours, and continuously adding 10M NaOH to maintain the pH at 8; adding methacrylic anhydride again, reacting at 4°C for 6-36 hours, and continuing to maintain for 2-12 hours, dialyzing the resulting final product under deionized water, and then freeze-drying to obtain the prepared methacrylated hyaluronic acid; the mass ratio of methacrylic anhydride added for the first time to sodium hyaluronate is 0.1:1-10:1, and the mass ratio of methacrylic anhydride added again to sodium hyaluronate is 0.1:1-10:

1.

3. The method for preparing a thermoelectric hydrogel composite dressing that promotes diabetic ulcer repair and is loaded with targeted modified traditional Chinese medicine exosomes according to claim 1, characterized in that: Step B specifically comprises: dissolving polyether F127 and triethylamine in anhydrous dichloromethane under a nitrogen atmosphere, and then slowly adding acryloyl chloride; stirring under a nitrogen atmosphere for 6-48 hours, filtering the precipitate, taking the filtrate, and adding diethyl ether to precipitate the remaining filtrate; and drying the resulting product under vacuum to obtain the prepared polyether F127 diacrylate; wherein the molar ratio of polyether F127 to triethylamine is 1:0.5-1:40, and the molar ratio of polyether F127 to acryloyl chloride is 1:0.5-1:

50.

4. The method for preparing a thermoelectric hydrogel composite dressing that promotes diabetic ulcer repair and is loaded with targeted modified traditional Chinese medicine exosomes according to claim 1, characterized in that: Step C is specifically as follows: washing the medicinal materials and squeezing the juice; passing the juice through a 100-300 mesh sieve to remove residues; differentially centrifuging the sieved juice at 1000-10000g for 10-90 minutes, taking the supernatant; ultracentrifuging at 30000-250000g for 0.5-4 hours, and taking the precipitate to obtain the prepared Chinese medicine exosomes.

5. The method for preparing a thermoelectric hydrogel composite dressing that promotes diabetic ulcer repair and is loaded with targeted modified traditional Chinese medicine exosomes according to claim 1, characterized in that: In step D, the mass ratio of Chinese medicine exosomes to targeting peptide is 0.5:1-4:

1. Step D is incubated at 37° C. for 0.1-5 hours and ultracentrifuged at 30,000-250,000 g for 0.5-4 hours to obtain targeted modified Chinese medicine exosomes.

6. The method for preparing a thermoelectric hydrogel composite dressing that promotes diabetic ulcer repair and is loaded with targeted modified traditional Chinese medicine exosomes according to claim 1, characterized in that: In step E, the pregel is cross-linked by ultraviolet light by irradiating with a 405 nm blue-violet laser for 10-100 seconds.

7. A composite dressing prepared according to the preparation method according to any one of claims 1 to 6.

8. Use of the composite dressing according to claim 7 in preparing a diabetic ulcer repair preparation.

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