Thermoelectric hydrogel composite dressing for promoting diabetic ulcer repair and loading targeted modified exosome and preparation method thereof

Abstract

The invention provides a thermoelectric hydrogel composite dressing for promoting diabetic ulcer repair and loading a targeted modification exosome and a preparation method thereof. The preparation method comprises the following steps: preparing methacrylated hyaluronic acid; preparing polyether F127 diacrylate; co-incubating the exosome and the palmitic acid grafted targeting peptide to prepare a targeting modified exosome; the preparation method comprises the following steps: mixing methacrylated hyaluronic acid, polyether F127 diacrylate, a photosensitizer, a targeted modified exosome and an electrolyte, and carrying out ultraviolet crosslinking to form the 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 a wound endogenous electric field to promote directional migration of skin cells and accelerate reepithelization and dermis formation, and on the other hand, the dressing slowly releases exosome to precisely target blood vessels with wound lesions, so that the wound healing effect is improved. Endothelial dysfunction in a high-glucose environment is reversed, angiogenesis is promoted, and the problems in diabetic ulcer wound repair and regeneration are effectively solved.

Description

Technical Field

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

[0002] Diabetic ulcers are one of the main causes of amputation and death in diabetic patients, posing a major challenge to 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 damage occurs, the current driven by the transepithelial potential accurately generates a lateral endogenous electric field from the edge of the wound to the center, guiding the migration of keratinocytes and fibroblasts to achieve re-epithelialization and dermal regeneration. At the same time, blood vessels in the tissue surrounding the wound transport nutrients, especially growth factors, to stimulate the proliferation and migration of vascular endothelial cells for 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 field in the wound. 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 endogenous electric field and growth factors of the wound cannot be activated at the site of diabetic ulcers, and the re-epithelialization, dermal regeneration and angiogenesis processes mediated by the two cannot be initiated, resulting in the difficulty of healing 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 of the wound. Traditional electrical stimulation methods generally use external power supplies and metal electrodes to directly apply direct current or alternating current to the wound site to promote its healing. However, the rigid interface of the electrode is prone to secondary injury to the wound surface, and the complex operation and dependence on external power supplies can easily reduce patient compliance. At present, most studies focus on incorporating conductive materials into flexible carriers such as hydrogels as soft electrodes, and then assembling them with self-powered layers formed by friction nanogenerators or piezoelectric materials to deliver electrical stimulation to the wound site. However, the inherent defects of these systems still face many challenges in achieving clinical translation and optimal therapeutic effects. First, metal or inorganic conductive fillers are usually non-degradable, resulting in limited biocompatibility. In addition, the electrical stimulation generated by friction nanogenerators or piezoelectric materials is unstable or anisotropic, making it difficult to accurately simulate the directionality of the endogenous electric field, reducing the controllability and efficiency of 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. They have shown extraordinary potential in the fields 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 wound repair. This is because the commonly used raw materials for synthesizing thermoelectric hydrogels are mostly polyacrylamide, polyvinyl alcohol, cellulose, etc. These materials degrade very slowly in the body. In addition, additives that induce thermoelectric effects, such as K3[Fe(CN)6], NaOH, Fe(ClO4)3, etc., have redox toxicity or biological toxicity, which hinders the biomedical transformation of thermoelectric hydrogels. Therefore, it is urgent to construct a directional electric field to reconstruct the wound surface to promote re-epithelialization and dermis formation, and ensure a new generation of thermoelectric medical hydrogels that are precisely controllable, biodegradable and highly biocompatible. However, research in this field is still blank.

[0006] On the other hand, oxidative stress damage caused by glucose metabolism disorders in the diabetic ulcer site causes impaired angiogenesis, which is also an important cause of non-healing of diabetic ulcers. At present, most angiogenic drugs, such as recombinant vascular endothelial growth factor, generally have disadvantages such as high cost, low stability, short half-life, potential biological risks, and are difficult to reverse endothelial dysfunction. Ginseng was first recorded in Shennong's Herbal Classic. It has the advantages of "replenishing qi to promote blood circulation and removing blood stasis without hurting the body". It is a commonly used Chinese medicine for promoting blood circulation, removing blood stasis and promoting muscle regeneration in clinical Chinese medicine. Modern pharmacological studies have shown that ginseng can dilate blood vessels, improve local microcirculation, and maintain the stability of vascular endothelial function. Ginseng exosomes have been shown to be a new type of nanomedicine to reverse endothelial cell dysfunction in a hyperglycemic environment and promote angiogenesis. However, in the local microenvironment of diabetic ulcers involving multiple cell types, competitive uptake by other cells may lead to reduced bioavailability of ginseng exosomes, and the repulsive effect between the surface negative charge of exosomes and the cell membrane also limits their internalization by vascular endothelial cells.

[0007] The three-dimensional pores of thermoelectric hydrogels can load and release drugs such as exosomes very well. Therefore, how to use thermoelectric hydrogels to apply artificial electrical stimulation to synergistically deliver exosome drugs to promote angiogenesis is the key to promoting the comprehensive repair of diabetic ulcers. However, research in this field is still blank. Summary of the invention

[0008] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a thermoelectric hydrogel dressing that promotes the repair of diabetic ulcers and carries targeted modified traditional Chinese medicine exosomes and a preparation method thereof.

[0009] The technical solution of the present invention 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 carries targeted modified exosomes, which comprises the following steps:

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

[0012] B. preparing polyether F127 diacrylate by reacting polyether F127 and acryloyl chloride;

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

[0014] D. co-incubating the Chinese medicine exosomes with the targeting peptide at 37° C. and ultracentrifuging to obtain the targeted modified Chinese medicine exosomes; in the 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. Methacryloyl hyaluronic acid, polyether F127 diacrylate, sodium chloride, and LAP photosensitizer are dissolved in ultrapure water, and targeted modified Chinese medicine exosomes are added to form a pregel, and the pregel is cross-linked by ultraviolet light to prepare a thermoelectric composite dressing; in the step E, the mass percentage of methacryloyl 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 1μg / mL-5000μg / mL; the photosensitizer is one or both of LAP and I2959; 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 sequence is shown in the following table:

[0017] Targeting peptides Amino acid sequence RGD peptide RGD NGR peptide NGR VHPK peptide VxP 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, the step A is specifically as follows: methacrylic anhydride is dripped into a sodium hyaluronate aqueous solution, stirred at 4°C for 1-24h, and 10M NaOH is continuously added to maintain pH=8; methacrylic anhydride is added again, reacted at 4°C for 6-36h, and maintained for 2-12h, and the obtained final product is dialyzed under deionized water, and then freeze-dried to obtain the prepared methacryloyl 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.

[0019] According to a preferred embodiment of the present invention, the step B is specifically as follows: dissolving polyether F127 and triethylamine in anhydrous dichloromethane under a nitrogen atmosphere, and then slowly adding acryloyl chloride; stirring for 6-48 hours under a nitrogen atmosphere, filtering and precipitating, taking the filtrate and adding it into diethyl ether to precipitate the remaining filtrate; and drying the obtained 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; subjecting the sieved juice to differential centrifugation, the centrifugation sequence is 1000-10000g for 10-90min, and taking the supernatant; ultracentrifuging at 30000-250000g for 0.5-4h, and taking the precipitate as the prepared Chinese medicine exosomes.

[0021] According to a preferred embodiment of the present invention, in the step D, the mass ratio of the Chinese medicine exosomes to the targeting peptide is 0.5:1-4:1, and the step D is incubated at 37°C for 0.1-5 hours, and ultracentrifuged at 30000-250000g for 0.5-4h to obtain targeted modified Chinese medicine exosomes.

[0022] According to a preferred embodiment of the present invention, in step E, the condition for ultraviolet cross-linking of the pre-gel is irradiation with 405 nm blue-violet laser for 10-100 seconds.

[0023] In the second aspect, the present invention provides a composite dressing prepared according to the above method. The composite dressing is a new type of composite dressing for thermoelectric stimulation and exosome therapy. On the one hand, the dressing generates a wireless passive electric field to simulate the endogenous electric field of the wound to promote the directional migration of skin cells, accelerate re-epithelialization and dermal formation, and on the other hand, the dressing slowly releases exosomes to accurately target the blood vessels of wound lesions, reverse endothelial dysfunction in a high-sugar environment, and promote angiogenesis, effectively solving the problem of diabetic ulcer wound repair and regeneration.

[0024] In a third aspect, the present invention provides use of the above-mentioned composite dressing in the preparation of 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 completely degradable materials to prepare biocompatible thermoelectric hydrogels that can output directional electric fields, overcoming the difficulty of clinical transformation caused by the difficulty of degradation of the electric stimulation system in the prior art, thereby achieving the effect of promoting electric field stimulation healing of diabetic ulcer reconstruction wounds with high biosafety;

[0027] (2) The present invention uses targeted modified ginseng-derived exosomes as pro-angiogenic factors, overcoming 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 in diabetic ulcers and promoting their angiogenesis.

[0028] (3) The present invention uses thermoelectric hydrogel loaded with targeted 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 treatment in the prior art, thereby achieving the effect of promoting the comprehensive repair of diabetic ulcers. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 It is the H NMR spectrum of methacryloyl hyaluronic acid and polyether F127 diacrylate;

[0030] Figure 2 It is a quantitative diagram of the thermoelectric coefficient of thermoelectric hydrogel at different sodium chloride concentrations;

[0031] Figure 3 Schematic diagram of the mechanism for generating a directional electric field for thermoelectric hydrogels;

[0032] Figure 4 This is the transmission electron microscopy result of targeted modification of exosomes;

[0033] Figure 5 This is a diagram showing the cell-specific uptake results of targeted modified exosomes;

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

[0035] Figure 7 The graph of the controlled release of exosomes from the composite dressing;

[0036] Figure 8 This is a picture of the wound healing results of diabetic mice with composite dressing;

[0037] Fig. 9 This is the body weight result of diabetic mice treated with composite dressing;

[0038] Fig.10 This is the immunofluorescence result of keratin (CK14) in the skin of diabetic mice with composite dressing;

[0039] Fig.11 This is the immunofluorescence result of Vimentin in the skin of diabetic mice with composite dressing;

[0040] Fig.12 This is the immunofluorescence result of endothelial cell adhesion molecule (CD31) in the skin of diabetic mice with composite dressing. Specific implementation plan

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

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

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

[0044] 1) 2.5 mL of methacrylic anhydride was dripped into 100 mL of an aqueous solution containing 1 wt% sodium hyaluronate, stirred at 4°C for 8 h, and 10 M NaOH solution was continuously dripped to maintain the system pH = 8. 1 mL of methacrylic anhydride was added again and reacted at 4°C for 12 h. The final mixture was dialyzed in deionized water and freeze-dried at -80°C to obtain methacrylylated hyaluronic acid;

[0045] After preparing the 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 methacryloylated 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 nitrogen atmosphere, 50 g of polyether F127 and 4 mL of triethylamine were dissolved in dichloromethane, and then 3.2 mL of acryloyl chloride was added using a dropping funnel. The mixture was stirred at 4 °C under nitrogen atmosphere for 24 h, and the precipitate was filtered out. Anhydrous ether was added to the filtrate to precipitate the filtrate. The obtained 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) dissolving methacryloyl hyaluronic acid, polyether F127 diacrylate and sodium chloride in an aqueous solution containing LAP photosensitizer to form a pre-gel, wherein the content of methacryloyl hyaluronic acid is 2wt%, the content of polyether F127 diacrylate is 8wt% and the content of LAP photosensitizer is 0.05wt%. The pre-gel is then exposed to 405nm blue-violet light for 45 seconds to cross-link and obtain a thermoelectric hydrogel.

[0049] 4) Construct a test platform to perform thermoelectric tests 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. Then a copper sheet is used to connect the two ends of the hydrogel and the electrochemical workstation to monitor the change of the potential difference with the temperature difference 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 output of the cold end minus the hot end of the hydrogel is 20.58mV, 16.716mV, 7.74mV, -7.518mV, -17.304mV, and -12.00mV, respectively, see Figure 2 shown.

[0050] The mechanism by which the thermoelectric hydrogel generates a directional potential difference at different sodium chloride concentrations is as follows: At low electrolyte concentrations, the hydrogel methacryloyl 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 to the cold end, resulting in a decrease in the potential at the cold end. In the presence of a large amount of electrolyte, some cations tend to "agglomerate" along the negatively charged methacryloyl hyaluronic acid chain. 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 potential at the cold end, see Figure 3 shown.

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

[0052] 1) Wash ginseng with deionized water, peel, cut into pieces, and squeeze the juice. Sieve the resulting juice to remove large pieces of plant tissue. Then centrifuge the juice at 2000-6000g for 20-50min using a desktop centrifuge to remove larger plant residues; take the supernatant and centrifuge at 7000-15000g for 1h to remove tiny plant fibers and debris; take the supernatant and centrifuge at 1000000-200000g for 1-3h using a floor-standing ultracentrifuge, take the bottom sediment and resuspend it with ultrapure water to obtain ginseng exosome solution;

[0053] 2) Incubate the ginseng exosomes with REDV-Pal peptide at 30-45°C with shaking for 0.25-1 hour, and centrifuge at 1000000-200000 for 1-3 hours using a desktop ultracentrifuge to obtain targeted modified ginseng exosomes. Then filter and sterilize with a 0.22 μm filter, and finally store the suspension at -80°C until use.

[0054] The exosome solution was dropped onto a copper mesh coated with a carbon film, and then counterstained with uranyl acetate. Transmission electron microscopy showed that the exosomes after targeted modification had a standard pancake-like structure with a size of approximately 100-150 nm. Figure 4 shown.

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

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

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

[0058] 1) The methacryloyl hyaluronic acid and polyether F127 diacrylate prepared in Example 1, the targeted modified ginseng exosomes prepared in Example 2, and the sodium chloride electrolyte are dispersed or dissolved in ultrapure water containing LAP photosensitizer to form a drug-loaded pre-gel. The content of methacryloyl hyaluronic acid is 2wt%, the content of polyether F127 diacrylate is 8wt%; the concentration of targeted modified exosomes is 200μg / mL; the concentration of sodium chloride is 0.1M; the content of LAP photosensitizer is 0.5wt%. The drug-loaded pre-gel is then exposed to 405nm blue-violet light for 45 seconds to cross-link to obtain a thermoelectric hydrogel composite dressing loaded with 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 thermoelectric coefficient of the composite dressing was 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 bionic electrical stimulation at the site of diabetic ulcer wounds. Figure 6 shown.

[0060] 3) In order to characterize the release pattern of the composite dressing for controlled release of targeted modified exosomes, 1 mL of the hydrogel composite dressing containing 200 μg of targeted modified exosomes was added to the dialysis bag, and then placed in a centrifuge tube containing 25 mL of PBS. The centrifuge tube was placed in a shaker and oscillated at 37°C and 100 rpm. 1 mL of the solution was taken at a pre-set time interval and the same volume of PBS solution was added. The released protein concentration was determined by the micro BCA method to calculate the percentage of released targeted modified 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 ulcer, 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, which 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 2 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, the treatment was as follows: first, the drug-loaded pre-gel was applied to the surface of the diabetic ulcer wound, and then the indocyanine green-loaded hydrogel was applied to the entire edge of the drug-loaded pre-gel. Photocrosslinking for 45 seconds made it crosslinked and tightly adhered to the wound site. In order to induce a directional thermoelectric field to simulate the endogenous electric field of the wound, infrared light was used to irradiate the edge of the dressing to induce the photothermal effect of indocyanine green, thereby 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 and composite dressings. Pay attention to the healing of the wound surface, take pictures of the wound when changing the dressing every other day, and calculate the healing rate.

[0066]

[0067] The healing rates of the composite dressing groups were significantly higher than those of the blank control group, indicating that composite dressings promoted the rapid healing of diabetic ulcers as a whole. Figure 8 shown.

[0068] 3) Evaluate the biosafety of the composite dressing when used in vivo. 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 mice in the blank control group and the composite dressing group remained stable, with no significant increase or decrease. The results objectively proved that the composite dressing had no harmful systemic effects when used in vivo. Fig. 9 shown.

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

[0070] 1) The animal experiment scheme described in Example 3 was used to construct a diabetic mouse skin ulcer model, and the composite dressing administration scheme described in Example 3 was used for treatment. The skin of the blank control group and the composite dressing group was collected at the wound healing site on the 7th day of the animal experiment. The skin tissue was fixed in 4% paraformaldehyde, then dehydrated with ethanol, embedded in paraffin and sliced ​​into 0.22 μm thick tissue sections. The tissue sections were immunofluorescently stained to detect the epithelial cell keratin CK14 signal, the fibroblast vimentin Vimentin signal, and the endothelial cell adhesion molecule CD31 signal 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 box and incubated overnight at 4°C. Then the primary antibody incubation solution was 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. Among them, CK14 and CD31 signals were visualized with cy3-conjugated secondary antibodies, and Vimentin signals were visualized with Alexa Fluor 488-conjugated secondary antibodies. Then the sections were rinsed with PBS three times, and DAPI staining solution was added to locate the cell nucleus, and 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 length of the epithelial tongue of diabetic ulcer wounds in the blank control group was very short and the thickness increased, indicating the formation of scar-like structure; in contrast, the length of the epithelial tongue in the composite dressing group increased significantly, the level of re-epithelialization increased significantly, and the width of the wound decreased significantly. This proves that the composite dressing can effectively promote the re-epithelialization of diabetic ulcer wounds in vivo, see Fig.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 formed well; in contrast, a large number of Vimentin positive signals appeared in the composite dressing group, 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 dermis formation of diabetic ulcer wounds in vivo, see Fig.11 shown.

[0074] CD31 immunofluorescence staining of skin tissue showed that there were only a few CD31 positive signals in the blank control group, indicating that the angiogenesis was not obvious; in contrast, a large number of CD31 positive signals appeared in the composite dressing group, indicating that the angiogenesis was very significant. This proves that the composite dressing can reverse the dysfunction of vascular endothelial cells in vivo and effectively promote angiogenesis in diabetic ulcers. Fig.12 shown.

[0075] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the present invention. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the attached claims.

Claims

1. A method for preparing a thermoelectric hydrogel composite dressing that promotes diabetic ulcer repair and loads targeted modified 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 and acryloyl chloride to prepare polyether F127 diacrylate; C. preparing and extracting Chinese medicine exosomes by differential centrifugation and ultracentrifugation; in step C, the source of the Chinese medicine exosomes is ginseng; D. co-incubating the Chinese medicine exosomes with the targeting peptide at 37° C. and ultracentrifuging to obtain targeted modified Chinese medicine exosomes; in the 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. Methacryloyl hyaluronic acid, polyether F127 diacrylate, sodium chloride, and LAP photosensitizer are dissolved in ultrapure water, and targeted modified Chinese medicine exosomes are added to form a pregel, and the pregel is cross-linked by ultraviolet light to prepare a thermoelectric composite dressing; in the step E, the mass percentage of methacryloyl 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 1μg / mL-5000μg / mL; the photosensitizer is one or both of LAP and I2959; 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 carries targeted modified Chinese medicine exosomes according to claim 1, characterized in that: The step A specifically comprises: dropping methacrylic anhydride into a sodium hyaluronate aqueous solution, stirring at 4°C for 1-24h, and continuously adding 10M NaOH to maintain pH=8; adding methacrylic anhydride again, reacting at 4°C for 6-36h, and continuing to maintain for 2-12h, dialyzing the obtained 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 carries targeted modified Chinese medicine exosomes according to claim 1, characterized in that: The step B specifically comprises: dissolving polyether F127 and triethylamine in anhydrous dichloromethane under a nitrogen atmosphere, and then slowly adding acryloyl chloride; stirring for 6-48 hours under a nitrogen atmosphere, filtering the precipitate, taking the filtrate and adding it into diethyl ether to precipitate the remaining filtrate; and drying the obtained 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.

4. The method for preparing a thermoelectric hydrogel composite dressing that promotes diabetic ulcer repair and carries targeted modified Chinese medicine exosomes according to claim 1, characterized in that: The 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; subjecting the sieved juice to differential centrifugation at 1000-10000g for 10-90min, taking the supernatant; ultracentrifuging at 30000-250000g for 0.5-4h, and taking the precipitate as the prepared Chinese medicine exosomes.

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

1. In step D, the mixture 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 carries targeted modified Chinese medicine exosomes according to claim 1, characterized in that: In the step E, the condition for ultraviolet cross-linking of the pre-gel is irradiation with 405 nm blue-violet laser for 10-100 seconds.

7. A composite dressing prepared according to the 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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