Traditional Chinese medicine compound superfine powder hydrogel as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biomedical materials, and particularly relates to traditional Chinese medicine compound superfine powder-hydrogel as well as a preparation method and application thereof. According to the application, the external treatment empirical prescription aiming at the diabetic wound is crushed into superfine powder of 2.5-5 microns, and then the traditional Chinese medicine superfine powder is loaded through the hydrogel composed of the chitosan, the sodium alginate, the calcium carbonate and the gluconic acid delta lactone, so that a local sustained and controlled release system of the medicine is constructed, the defects of a traditional Chinese medicine external treatment dosage form are overcome, and the external treatment function of the traditional Chinese medicine is better exerted. According to the traditional Chinese medicine compound superfine powder-multifunctional hydrogel system, the defects of traditional Chinese medicine external dosage forms are overcome, and a new solution is provided for treating diabetic refractory wounds through efficient utilization of traditional Chinese medicines and local sustained and controlled release.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, specifically relating to a traditional Chinese medicine compound ultrafine powder-hydrogel, its preparation method and application. Background Technology

[0002] Diabetic ulcers that are difficult to heal (such as diabetic foot) are chronic and prone to spreading, worsening of the condition, and even amputation. Treatment requires a multi-pronged approach, including blood sugar control, infection control, wound care, improvement of blood circulation, and treatment of peripheral neuropathy. Even with a combination of these measures, many patients' conditions remain difficult to control or improve. Traditional Chinese medicine (TCM) has proven efficacy and advantages in treating this condition. However, current external treatments for diabetic wounds utilize traditional TCM topical formulations such as ointments and elixirs, which have several drawbacks, including increased risk of infection and difficulty in complete removal after application.

[0003] Therefore, a more efficient and convenient method of medication administration is needed in this field. Summary of the Invention

[0004] Based on the above problems, the technical solution of this application discloses a method for preparing a compound ultrafine powder hydrogel of traditional Chinese medicine, including adding ultrafine powder of traditional Chinese medicine to a gel material, heating and mixing, and then cooling to form a gel; wherein, the ultrafine powder of traditional Chinese medicine is a mixture of external treatment formulas for diabetic wounds with a particle size of 2.5-5μm; the gel material includes chitosan, sodium alginate, calcium carbonate, and glucono-delta-lactone.

[0005] Furthermore, the mass ratio of chitosan to sodium alginate is 1:2, the mass ratio of calcium carbonate to chitosan is 1:4.5, and the mass ratio of calcium carbonate to glucono-delta-lactone is 1:6.3.

[0006] Furthermore, the gel material has a drug loading concentration of 9-36 mg / mL for the ultrafine powder of traditional Chinese medicine.

[0007] Furthermore, the gel material has a drug loading concentration of 18 mg / mL for the traditional Chinese medicine ultrafine powder.

[0008] Furthermore, the heating and mixing temperature is 37 degrees Celsius, and the heating and mixing time is 20 minutes.

[0009] Furthermore, the traditional Chinese medicine compound ultrafine powder hydrogel obtained according to the above preparation method and its application in the preparation of topical materials for diabetic wounds.

[0010] Compared with existing technologies, this invention has the following beneficial effects: This application pulverizes a traditional Chinese medicine formula for external treatment of diabetic wounds into ultrafine powder of 2.5-5 μm. Then, the ultrafine powder is loaded into a hydrogel composed of chitosan, sodium alginate, calcium carbonate, and glucono-delta-lactone, constructing a local sustained-release drug system. This overcomes the shortcomings of traditional Chinese medicine external treatment dosage forms and better utilizes the functions of external Chinese medicine treatment. This compound Chinese medicine ultrafine powder-multifunctional hydrogel system overcomes the shortcomings of traditional Chinese medicine external dosage forms and provides a new solution for the efficient utilization and local sustained-release treatment of refractory diabetic wounds using traditional Chinese medicine. Attached Figure Description

[0011] Figure 1 The hydrogel formation results are for Example 1;

[0012] Figure 2 Images of ultrafine hydrogels containing different drug loading concentrations of traditional Chinese medicine compound powders.

[0013] Figure 3 SEM images of ultrafine hydrogels of traditional Chinese medicine compound powders with different drug loading concentrations;

[0014] Figure 4 Cell viability after 72 hours of incubation with ultrafine powder hydrogel extracts of different traditional Chinese medicine compound preparations;

[0015] Figure 5 This is the result of the hemolysis test;

[0016] Figure 6 The water content and swelling properties of the hydrogel;

[0017] Figure 7 Stress-strain curves for a drug-loaded hydrogel with a concentration of 18 mg / mL;

[0018] Figure 8 The rats' blood glucose and body weight levels;

[0019] Figure 9 The healing rate at each intervention stage;

[0020] Figure 10 To intervene in blood perfusion at each stage;

[0021] Figure 11 HE staining results 7 and 14 days after intervention;

[0022] Figure 12 Masson staining results 7 and 14 days after intervention;

[0023] Figure 13 To detect the expression level of COL-Ⅰ in wound tissue using immunohistochemistry;

[0024] Figure 14To detect the expression level of COL-III in wound tissue by immunohistochemistry

[0025] Figure 15 To detect CD31 expression levels in wound tissue using immunofluorescence;

[0026] Figure 16 To detect CD86 expression levels in wound tissue using immunofluorescence

[0027] Figure 17 To detect CD206 expression levels in wound tissue using immunofluorescence

[0028] Figure 18 14. ELISA was used to detect the concentration levels of TGF-β, VEGF, MMP-9, IL-1β, IL-6, IL-10, and TNF-α in wound tissue;

[0029] Figure 19 The results are the transcriptome RNA sequencing and analysis results;

[0030] Figure 20 To validate differentially expressed gene levels using RT-qPCR;

[0031] Figure 21 17. Western Blot method was used to verify the levels of related pathway proteins. Detailed Implementation

[0032] The present invention will now be described in detail with reference to embodiments to facilitate understanding of the invention by those skilled in the art. It is particularly important to note that the embodiments are merely illustrative of the invention and should not be construed as limiting the scope of protection of the invention. Non-essential improvements and adjustments made to the invention by those skilled in the art based on the above description should still fall within the scope of protection of the invention. Furthermore, unless otherwise specified, the raw materials are commercially available products; and any process steps or preparation methods not mentioned in detail are process steps or preparation methods known to those skilled in the art.

[0033] Example 1: Preparation and performance optimization of ultrafine hydrogels made from traditional Chinese medicine compound powders

[0034] 1. Hydrogel Formation: A one-pot method was used to prepare the hydrogel, with heating at 37°C for 20 minutes. The experiment consisted of a control group and an experimental group. The experimental group contained 10 mg / mL chitosan, 20 mg / mL sodium alginate, 2.22 mg / mL calcium carbonate, and 14 mg / mL glucono-delta-lactone (GDL); the control group contained 10 mg / mL chitosan, 20 mg / mL sodium alginate, and 2.22 mg / mL calcium carbonate. After heating, the mixture was cooled to room temperature for 25 minutes to obtain the hydrogel. Figure 1The gelation results are shown; where A is the control group and B and C are the experimental groups. It can be seen that after cooling at room temperature for 25 min, the hydrogel containing glucono delta-lactone (GDL) completes gelation at room temperature within 25 min through the sustained release of calcium ions. Inverting the vial verifies that the precursor solution has formed a stable hydrogel, while the hydrogel without GDL does not gel. Therefore, subsequent experiments in this application will use chitosan, sodium alginate, calcium carbonate, and glucono delta-lactone (GDL) as raw materials to prepare hydrogels.

[0035] 2. Effect of drug loading concentration on the properties of hydrogels

[0036] The components of ultrafine powders of traditional Chinese medicine are quite complex. The polysaccharides, phenolic compounds and metal ions contained therein may interact with the polysaccharide chains in the hydrogel, affecting its cross-linking density and physical properties.

[0037] A mixture of external treatment formulas for diabetic wounds with a particle size of 2.5-5 μm was added to the chitosan, sodium alginate, calcium carbonate, and glucono delta-lactone (GDL) described in Examples 1-1. The external treatment formula for diabetic wounds was obtained from the Affiliated Hospital of Chengdu University of Traditional Chinese Medicine and has been proven to have a clear therapeutic effect on the healing of diabetic wounds. The drug loading concentrations of the hydrogels were 0 mg / mL, 9 mg / mL, 18 mg / mL, 27 mg / mL, and 36 mg / mL, respectively. The hydrogels were prepared using a one-pot method, and after heating, they were cooled at room temperature for 25 min to obtain ultrafine powder hydrogels of traditional Chinese medicine compound with different drug loading concentrations.

[0038] like Figure 2 The images shown are of the appearance of hydrogels at various concentrations. Figure 3 The images are SEM images, where A represents 0 mg / mL, B represents 9 mg / mL, C represents 18 mg / mL, D represents 27 mg / mL, and E represents 36 mg / mL. It can be seen that all four dosage groups can stably form a gel. Since the ultrafine powder of *Lithospermum erythrorhizon* contains pigments, the color of the hydrogel indicates an increase in drug dosage. Figure 2 The addition of ultrafine powder of traditional Chinese medicine accelerated the gelation of hydrogels, and with equal amounts of sodium alginate, chitosan, calcium carbonate, and GDL, the higher the drug concentration, the greater the hardness of the hydrogel. Scanning electron microscopy analysis was performed on each group of hydrogels. Figure 3The porosity was statistically analyzed, showing 52.51% for the 9 mg / mL group, 48.92% for the 18 mg / mL group, 40.17% for the 27 mg / mL group, and 45.95% for the 36 mg / mL group. Overall, this demonstrates that the cross-linking density of the hydrogel slightly increases with increasing drug concentration. Analysis of the water content of the hydrogels in each group showed that the water content was 94.61% in the untreated group, 93.89% in the 9 mg / mL group, 93.36% in the 18 mg / mL group, 92.53% in the 27 mg / mL group, and 91.58% in the 36 mg / mL group. The water content decreased slightly with increasing drug concentration, possibly due to the more dense cross-linking of the hydrogel after drug introduction. However, all groups maintained a relatively high water content, which is beneficial for maintaining a moist wound environment.

[0039] Biocompatibility: The cell viability of L929 cells after incubation with different traditional Chinese medicine compound ultrafine powder hydrogel extracts for 72 h was detected by CCK8 assay. Figure 4 After co-incubation with the hydrogel extract for 72 hours, there was no significant difference in cell viability among the groups. At the highest concentration of 36 mg / mL, cell activity decreased slightly but remained above 90%, indicating that even at this concentration, the drug-loaded hydrogel had relatively low cytotoxicity. The sodium alginate / chitosan composite hydrogel showed good biocompatibility, and the addition of the drug had no significant effect on cell viability.

[0040] Blood compatibility: Hemolysis tests were performed on each group of materials using rat erythrocyte suspension. The positive control, ddH2O, showed a high hemolysis rate, indicating complete erythrocyte lysis. The negative control, physiological saline, showed minimal or no hemolysis. The hemolysis test results showed ( Figure 5 The hemolysis rate of the materials with different drug concentrations was far below the safety standard of 5%, demonstrating good biocompatibility and meeting the low requirements of wound dressings for red blood cell lysis rate.

[0041] The above analysis shows that the ultrafine hydrogels of traditional Chinese medicine compound powder prepared within the drug loading concentration range of 9-36 mg / mL all have good hydrogel properties. Considering the effect of the drug on hydrogel formation and cell survival, the hydrogel with a dosage of 18 mg / mL was selected for subsequent experiments.

[0042] Hydrogel water content and swelling properties: The water content and swelling properties of the 18 mg / mL hydrogels before and after drug loading were characterized. Results are as follows... Figure 6Figures A and C show the water content of the hydrogel before and after drug loading (A), swelling characteristics after lyophilization (B), and wet swelling characteristics (C). The water content of the unloaded hydrogel was 95.43%, and the water content of the 18 mg / mL group was 93.65%, as shown in Figure A. The swelling properties of the hydrogel in distilled water are shown in Figures B and C. The swelling behavior of the hydrogel is an important indicator for evaluating wound dressings. The experiment showed that the lyophilized hydrogel swelled rapidly, exceeding 400% within 5 minutes, and reached swelling equilibrium after approximately 120 minutes. The hydrogel without a drug carrier had a higher swelling rate, reaching 884.48% at 120 minutes, indicating that drug loading may affect the water absorption capacity of the hydrogel network. The wet hydrogel also exhibited a certain swelling capacity; both the unloaded and drug-loaded hydrogels reached swelling equilibrium within 180 minutes, with swelling rates of 137.50% and 133.89%, respectively.

[0043] Hydrogel stress-strain curves: Experiments were conducted using a dynamic thermomechanical analyzer (DMA) in compression mode to determine and compare the stress-strain curves of the untreated hydrogel and the drug-loaded hydrogel with an ultrafine powder concentration of 18 mg / mL. Figure 7 According to the fitting results, the Young's modulus of the untreated hydrogel was 56.1 Pa, and the Young's modulus of the ultrafine powder hydrogel was 63.5 Pa. The results further confirm that the current drug loading concentrations did not have a significant impact on the mechanical properties and cross-linking degree of the hydrogel.

[0044] Example 2: Study on the treatment of diabetic chronic wounds by traditional Chinese medicine compound ultrafine powder hydrogel.

[0045] Grouping and intervention methods:

[0046] Normal group (C): A skin ulcer model in non-diabetic rats was established. The wound was treated with 4 layers of saline gauze (containing 2 ml of saline) and fixed with a breathable sterile dressing for 14 days.

[0047] Model group (M): Diabetic rat skin ulcer model, the wound was wet-dressed with 4 layers of saline gauze (containing 2ml of saline), fixed with breathable sterile dressing, and the intervention lasted for 14 days;

[0048] Growth factor group (S): Diabetic rat skin ulcer model, administered human epidermal growth factor (Efflu) (10g / 100cm). 2 Apply evenly, cover and fix with a breathable sterile dressing, and intervene for 14 days;

[0049] Blank hydrogel group (K): Diabetic rat skin ulcer model, 2 mL / rat of blank hydrogel was applied to cover the ulcer, and the ulcer was fixed with blank Sanfu patch for 14 days;

[0050] Drug-loaded hydrogel group (Z): Diabetic rat skin ulcer model, 2 mL of drug-loaded hydrogel per rat was applied to cover the ulcer, and the ulcer was fixed with blank Sanfu patch for 14 days.

[0051] like Figure 8 The following table shows the blood glucose and weight levels of rats: (Rats' weight and fasting blood glucose were monitored weekly at various stages of modeling and intervention).

[0052] The weight of rats in the normal group did not change significantly; the weight of rats in the diabetic group increased after being fed a high-fat, high-sugar diet, and decreased significantly after intraperitoneal injection of STZ. The weight did not change significantly during the intervention period. The fasting blood glucose of rats in the normal group did not change significantly and was below 6.1 mmol / L. After intraperitoneal injection of streptozotocin (STZ), the blood glucose of rats in the diabetic group gradually increased. On the third day after injection, the fasting blood glucose was higher than 11.1 mmol / L, reaching the standard of diabetic model, and the hyperglycemic level was maintained stably until the end of the experiment.

[0053] 1. Changes in wound healing rate and blood perfusion

[0054] Experimental methods: (1) Wound healing rate: At the end of the intervention on days 0, 7, and 14 of treatment, after gas anesthesia, rats were placed prone on a table, and a right-angle measuring ruler was placed at the lower left edge of the wound to record the scale. A camera was taken vertically above the wound to record the skin wound repair status of each rat. The size, color, secretions, and tissue hyperplasia of the wounds in each group of rats were observed. The wound size was automatically calculated using ImageJ software, and the wound healing rate of each group of rats was calculated. Wound healing rate = (initial wound area - current wound area) / initial wound area × 100%.

[0055] (2) Blood perfusion: At the end of the intervention on days 0, 7, and 14 of treatment, after gas anesthesia, rats were placed prone on the animal fixation table of the laser speckle blood flow imaging system, with their mouths and noses aligned with the anesthesia mask, ensuring an open airway. Subsequent procedures were performed under low-flow continuous inhalation anesthesia. The laser speckle blood flow imaging system was turned on, the camera was adjusted, the laser was focused on the center point of the wound, the resolution and recording time were set (15 seconds for all tests), and the start button was clicked. The test was then completed, and the results were saved and exported. Experimental results: Figure 9 The figure shows the healing rate at each stage of intervention. Figure 10The figure shows the blood perfusion volume at each stage of the intervention. It can be seen that: (1) Wound healing rate on days 7 and 14: At the end of day 7 of the intervention, the wound healing rate of each group was significantly lower than that of the normal group (P<0.01), and the wound healing rate of the blank hydrogel group and the drug-loaded hydrogel group was significantly higher than that of the model group (P<0.01); At the end of day 14 of the intervention, the wound healing rate of the model group and the blank hydrogel group was significantly lower than that of the normal group (P<0.01), and the wound healing rate of the blank hydrogel group was significantly higher than that of the model group (P<0.05), and the wound healing rate of the drug-loaded hydrogel group was significantly higher (P<0.01).

[0056] (2) Wound blood perfusion on days 0, 7 and 14: At the end of day 0 of the intervention, the blood perfusion of each group was significantly lower than that of the normal group (P<0.01), and there was no significant difference in blood perfusion among the groups compared with the model group; At the end of day 7 of the intervention, the blood perfusion of each group was significantly lower than that of the normal group (P<0.01), and the blood perfusion of each group was significantly higher than that of the model group (P<0.01); At the end of day 14 of the intervention, the blood perfusion of the model group, growth factor group and blank hydrogel group was significantly lower than that of the normal group (P<0.01), and the blood perfusion of each group was significantly higher than that of the model group (P<0.01).

[0057] 2. Staining experiment

[0058] (1) HE staining

[0059] Experimental methods: Fixed tissues were dehydrated using a fully automatic dehydrator (dehydration time: 75% alcohol 2 h, 85% alcohol 1 h, 95% alcohol 1 h, 100% alcohol I 20 min, 100% alcohol II 20 min, 100% alcohol III 20 min, 100% alcohol IV 20 min, clearing agent I 25 min, clearing agent II 30 min, paraffin I 30 min, paraffin II 1 h, paraffin III 1 h), embedded, and sectioned as follows: (1) Dewaxing the sections to water, specifically: dewaxing solution I 30 min, dewaxing solution II 30 min, anhydrous ethanol I 5 min, anhydrous ethanol II 5 min, 95% alcohol 5 min, 85% alcohol 5 min, 75% alcohol 5 min, and rinsing with running tap water for 5 min; (2) Hematoxylin staining for 5-10 min, and rinsing with running tap water until colorless; (3) Differentiation with hydrochloric acid alcohol solution for about 3 minutes. s, rinse with tap water; (4) turn blue with weak alkaline aqueous solution, rinse with tap water; (5) immerse in alcohol-soluble eosin for 3 min; (6) dehydrate with gradient alcohol; (7) clear with clearing agent; (8) seal with neutral resin; (9) use a slide scanning imaging system to acquire images of the slides. Each slide is first observed under low magnification of all tissues, and then 40x and 200x microscopic images are acquired to observe specific lesions.

[0060] Experimental results: such as Figure 11 The above describes the HE staining results on days 7 and 14 of the intervention. At the end of day 7 of the intervention, compared with the normal group, the model group, growth factor group, and blank hydrogel group showed more severe epidermal defects, superficial dermal degeneration and necrosis, and collagen fiber degeneration and necrosis. The cytoplasm was lightly stained, and the nuclei were shrunken or disappeared. A large number of inflammatory cells were infiltrated, and there was less fibroblast proliferation and new blood vessels. Compared with the model group, the growth factor group and blank hydrogel group showed no significant improvement, while the drug-loaded hydrogel group showed milder epidermal defects and inflammatory cell infiltration.

[0061] At the end of day 14 of the intervention, compared with the normal group, the epidermal defects persisted in the model group, growth factor group, and blank hydrogel group, with inflammatory infiltration still present, and a small amount of fibrous tissue hyperplasia and new blood vessel formation were visible. Compared with the model group, the inflammatory infiltration in the drug-loaded hydrogel group was significantly reduced, the epidermis was thickened, the fibrous tissue proliferated densely, and new blood vessels were formed in an orderly manner.

[0062] (2) Masson staining

[0063] Experimental methods: (1) Dewax the sections to water (the specific operation is the same as HE staining (1)); (2) Incubate overnight with potassium dichromate; (3) Heat the sections in a 63°C oven for 1 hour; (4) Stain with Ponceau S and Fuchsia for 10 minutes, and rinse slightly with distilled water; (5) Treat with phosphomolybdic acid solution for a few seconds to 2 minutes until the collagen fibers fade; (6) Stain with aniline blue for about 2 minutes until the collagen fibers are stained; (7) Dehydrate with gradient alcohol; (8) Clear with clearing agent; (9) Mount with neutral resin; (10) Use a slide scanning imaging system to acquire images of the sections. First, observe all tissues under low magnification on each section, and then acquire 3 microscopic images at 400x magnification. The area of ​​fibrous tissue in the acquired images was measured using the Image-ProPlus 6.0 image analysis system (Media Cybernetics, USA), and the percentage of fibrous tissue area was calculated as: fibrous tissue area / field of view (pixel area).

[0064] Experimental results: such as Figure 12 Masson staining results on days 7 and 14 of the intervention showed that at the end of day 7, compared with the normal group, collagen deposition levels were decreased in the model group, growth factor group, and blank hydrogel group (P>0.05), while compared with the model group, collagen deposition levels were increased in the drug-loaded hydrogel group (P>0.05). At the end of day 14, compared with the normal group, collagen deposition levels were increased in the model group, growth factor group, and drug-loaded hydrogel group (P>0.05), and decreased in the blank hydrogel group (P>0.05). Compared with the model group, collagen deposition levels were decreased in the growth factor group and blank hydrogel group (P>0.05), while increased in the drug-loaded hydrogel group (P>0.05).

[0065] 3. Immunohistochemical detection of COL-Ⅰ and COL-Ⅲ expression levels in wound tissue

[0066] Experimental methods: (1) Dewaxing paraffin sections to water: Place the sections in dewaxing solution I for 15 min, dewaxing solution II for 15 min, dewaxing solution III for 15 min, anhydrous ethanol I for 5 min; anhydrous ethanol II for 5 min, 85% ethanol for 5 min, 75% ethanol for 5 min, and wash with distilled water. (2) Antigen retrieval: Immerse the sections in retrieval solution and microwave for 20 min; after cooling, wash with PBS 3 times, 5 min each time. (3) Blocking endogenous peroxidase: Place the sections in 3% hydrogen peroxide and incubate at room temperature in the dark for 25 min; place the slides in PBS and shake and wash 3 times on a decolorizing shaker, 5 min each time. (4) Serum blocking: Add bovine serum albumin (BSA) and incubate at room temperature for 20 min. (5) Add the prepared primary antibody and incubate the sections flat in a humidified box at 4°C overnight. (6) Wash with PBS 3 times, 5 min each time; add secondary antibody and incubate at 37°C for 30 min; wash with PBS 3 times, 5 min each time. (7) DAB staining: Prepare fresh DAB staining solution, add it to the tissue, and stain at room temperature. Control the staining time under a microscope. The positive result is brownish-yellow. Wash the section with distilled water to stop the staining. (8) Counterstaining cell nuclei: Counterstain with hematoxylin for 3 min, wash with tap water, and rinse with running water after hematoxylin blueing solution. (9) Dehydration and mounting: Soak the sections in 75%, 85%, 95%, anhydrous ethanol, and xylene for 10 min respectively, and mount with neutral resin. (10) Use a microscopic imaging system (Macody Industrial Group Co., Ltd., BA400Digital) to acquire images of the sections. Observe the entire tissue under low magnification for each section first, and then acquire 400x microscopic images for a total of 3 images. The percentage of positive area per image (% DABPositive Tissue) was calculated using the Halo data analysis system (Indica Labs (USA), Halo 101-WL-HALO-1). Hematoxylin staining of cell nuclei appeared blue, while positive expression of COL-I and COL-III appeared brownish-yellow. Experimental results: [The text abruptly ends here, likely due to an incomplete sentence or missing information.] Figure 13 Immunohistochemical detection of COL-I expression level in wound tissue Figure 14 Immunohistochemical staining of COL-III expression levels in wound tissue showed that: 1) COL-I: At the end of day 7 of intervention, compared with the normal group, the expression levels in the model group and the blank hydrogel group were significantly decreased (P < 0.01), compared with the model group, the expression level in the blank hydrogel group was increased (P < 0.05), and the expression level in the drug-loaded hydrogel group was significantly increased (P < 0.01); At the end of day 14 of intervention, compared with the normal group, the expression levels in the growth factor group and the blank hydrogel group were significantly decreased (P < 0.01), and compared with the model group, the expression level in the drug-loaded hydrogel group was significantly increased (P < 0.01).

[0067] (2) COL-III: At the end of day 7 of intervention, compared with the normal group, the expression levels of the model group and the growth factor group were significantly decreased (P<0.01), and compared with the model group, the expression levels of each group were significantly increased (P<0.01); at the end of day 14 of intervention, compared with the normal group, the expression levels of the model group, the growth factor group and the blank hydrogel group were decreased (P<0.05), and compared with the model group, the expression level of the drug-loaded hydrogel group was increased (P<0.05).

[0068] 4. Immunofluorescence detection of CD31, CD86, and CD206 expression levels in wound tissue

[0069] Experimental methods: (1) Dewaxing paraffin sections to water: Place the sections in dewaxing solution I for 15 min, dewaxing solution II for 15 min, dewaxing solution III for 15 min, anhydrous ethanol I for 5 min; anhydrous ethanol II for 5 min, 85% ethanol for 5 min, 75% ethanol for 5 min, and wash with distilled water. (2) Antigen retrieval: Immerse the sections in retrieval solution and microwave for 20 min; after cooling, wash with PBS 3 times, 5 min each time. (3) Blocking endogenous peroxidase: Place the sections in 3% hydrogen peroxide and incubate at room temperature in the dark for 25 min; place the slides in PBS and shake and wash 3 times on a decolorizing shaker, 5 min each time. (4) Serum blocking: After slightly drying the sections, draw circles around the tissue with a histochemical pen, add bovine serum albumin (BSA), and incubate at room temperature for more than 30 min. (5) Adding primary antibody: Add the prepared primary antibody, place the sections flat in a humidified box and incubate overnight at 4°C. (6) Add secondary antibody: Place the slide in PBS buffer and wash three times on a decolorizing shaker for 5 min each time; add secondary antibody and incubate at 37℃ for 30 min; wash three times with PBS for 5 min each time. (7) Counterstain cell nuclei with DAPI: Add DAPI and incubate at room temperature for 10 min; wash three times with PBS for 5 min each time. (8) Mount the slide: Mount the slide with anti-fluorescence quenching mounting medium. (9) Use a scanning imaging system (OLYMPUS (JAPAN), VS200) to acquire images of the slides. For each slide, first observe all tissues at low magnification, and then acquire 100x and 200x microscopic images respectively, for a total of 3 fields of view. Use the Halo data analysis system to calculate the percentage of positive area (% Positive Tissue) for each image. DAPI staining of cell nuclei is blue, and positive expression of CD31 is green.

[0070] Experimental results: Figure 15 Immunofluorescence detection of CD31 expression levels in wound tissue Figure 16 Immunofluorescence detection of CD86 expression level in wound tissue Figure 17Immunofluorescence assays showed the expression levels of CD206 in wound tissue. CD31: At the end of day 7 of intervention, compared with the normal group, the expression levels in the model group, growth factor group, and blank hydrogel group were significantly decreased (P < 0.01), while compared with the model group, the expression levels in the growth factor group and drug-loaded hydrogel group were significantly increased (P < 0.01). At the end of day 14 of intervention, compared with the normal group, the expression levels in the model group, growth factor group, and blank hydrogel group were decreased (P < 0.05), while compared with the model group, the expression levels in the growth factor group were increased (P < 0.05) and the expression level in the drug-loaded hydrogel group was significantly increased (P < 0.01).

[0071] CD86: At the end of day 7 of intervention, compared with the normal group, the expression levels of CD86 in the model group, growth factor group, and blank hydrogel group were significantly increased (P < 0.01), while compared with the model group, the expression levels of CD86 in the growth factor group, blank hydrogel group, and drug-loaded hydrogel group were significantly decreased (P < 0.01). At the end of day 14 of intervention, compared with the normal group, the expression levels of CD86 in the model group, growth factor group, and blank hydrogel group were significantly increased (P < 0.01), while compared with the model group, the expression levels of CD86 in the blank hydrogel group and drug-loaded hydrogel group were significantly decreased (P < 0.05) (P < 0.01).

[0072] CD206: At the end of the intervention on days 7 and 14, the expression levels of CD206 in the model group, growth factor group and blank hydrogel group were significantly lower than those in the normal group (P < 0.01), while the expression level of CD206 in the drug-loaded hydrogel group was significantly higher than that in the model group (P < 0.01).

[0073] 5. ELISA was used to detect the concentration levels of TGF-β, VEGF, MMP-9, IL-1β, IL-6, IL-10, and TNF-α in wound tissue.

[0074] Experimental methods: 1) Tissue sample processing: Accurately weigh the tissue, remove residual blood, add PBS at a mass-to-volume ratio of 1:9, homogenize the tissue using a tissue homogenizer, centrifuge the homogenate at 5000 g for 10 min, and take the supernatant for testing. (2) Preparation: Equilibrate the kit at room temperature for 30 min, and then take out the required strips from the aluminum foil bag. (3) Adding standards: Set up standard wells and sample wells, and add 50 μL of different concentrations of standards to each standard well. (4) Adding samples: Add 50 μL of the sample to be tested to the sample wells, and do not add any to the blank wells. (5) Adding enzymes: Except for the blank wells, add 100 μL of horseradish peroxidase (HRP) labeled detection antibody to each standard well and sample well, seal the reaction wells with sealing film, and incubate at 37 ℃ in a water bath or incubator for 60 min. (6) Washing: Discard the liquid, pat dry on absorbent paper, fill each well with washing solution, let stand for 1 min, shake off the washing solution, pat dry on absorbent paper, and repeat this washing process 5 times. (7) Color development: Add 50 μL of substrate A and B to each well, and incubate at 37 ℃ in the dark for 15 min. (8) Termination: Add 50 μL of stop solution to each well, and within 10 min, measure the OD value of each well at a wavelength of 450 nm. (10) Calculation: Subtract the OD value of the blank well from the OD value of each standard and sample to obtain the ΔOD value. Calculate the ΔOD value of the replicate wells, take the average value, and use the concentration of the standard and the ΔOD value to calculate the linear regression equation of the standard curve. Substitute the ΔOD value of the sample into the equation to calculate the sample concentration, and then multiply by the dilution factor to obtain the actual concentration of the sample.

[0075] Experimental results: Figure 18 The concentrations of TGF-β, VEGF, MMP-9, IL-1β, IL-6, IL-10, and TNF-α in wound tissue were detected by ELISA. (1) TGF-β: At the end of day 7 of the intervention, the concentrations in all groups decreased compared with the normal group (P < 0.05); there was no significant difference between the groups compared with the model group (P > 0.05). At the end of day 14 of the intervention, the concentrations in all groups decreased compared with the normal group (P < 0.05), and the concentrations in the growth factor group and the drug-loaded hydrogel group increased significantly compared with the model group (P < 0.01).

[0076] (2) VEGF: At the end of day 7 of the intervention, the concentrations in all groups decreased compared with the normal group (P < 0.05); compared with the model group, there was no significant difference among the groups (P > 0.05). At the end of day 14 of the intervention, compared with the normal group, the concentrations in the model group and the blank hydrogel group decreased significantly (P < 0.01), and the concentration in the drug-loaded hydrogel group decreased (P < 0.05). Compared with the model group, the concentrations in the growth factor group and the drug-loaded hydrogel group increased significantly (P < 0.01).

[0077] (3) MMP-9: At the end of day 7 of intervention, compared with the normal group, the concentration of MMP-9 in the model group, growth factor group and blank hydrogel group increased (P<0.05), while the concentration of MMP-9 in the drug-loaded hydrogel group decreased (P<0.05) compared with the model group. At the end of day 14 of intervention, compared with the normal group, the concentration of MMP-9 in the model group and blank hydrogel group increased significantly (P<0.01), while the concentration of MMP-9 in the growth factor group and drug-loaded hydrogel group decreased significantly (P<0.01) compared with the model group.

[0078] (4) IL-1β: At the end of day 7 of intervention, the concentration of each group was higher than that of the normal group (P<0.05); at the end of day 14 of intervention, the concentration of the model group, growth factor group and blank hydrogel group was higher than that of the normal group (P<0.05), while the concentration of the growth factor group and drug-loaded hydrogel group was lower than that of the model group (P<0.01).

[0079] (5) IL-6: At the end of day 7 of intervention, compared with the normal group, the concentration of IL-6 in the model group and the blank hydrogel group increased (P<0.05), while the concentration of IL-6 in the growth factor group decreased (P<0.05) compared with the model group. At the end of day 14 of intervention, compared with the normal group, the concentration of IL-6 in each group increased significantly (P<0.01), while the concentration of IL-6 in the growth factor group decreased significantly (P<0.01) compared with the model group, and the concentration of IL-6 in the drug-loaded hydrogel group decreased (P<0.05).

[0080] (6) IL-10: At the end of day 7 of the intervention, the concentration of each group was significantly lower than that of the normal group (P<0.01), while the concentration of the growth factor group and the drug-loaded hydrogel group was higher than that of the model group (P<0.05). At the end of day 14 of the intervention, the concentration of the model group and the blank hydrogel group was significantly lower than that of the normal group (P<0.01), while the concentration of the growth factor group and the drug-loaded hydrogel group was significantly higher than that of the model group (P<0.01).

[0081] (7) TNF-α: At the end of the 7th day of intervention, the concentration in each group was significantly higher than that in the normal group (P<0.01).

[0082] At the end of day 14 of the intervention, compared with the normal group, the concentrations of growth factor in the model group, growth factor group, and blank hydrogel group were increased (P < 0.05), while compared with the model group, the concentrations of growth factor in the growth factor group and drug-loaded hydrogel group were significantly decreased (P < 0.01).

[0083] 6. Transcriptome RNA sequencing and analysis results

[0084] Experimental methods: (1) RNA extraction and library construction

[0085] Total RNA was extracted using TRIzol reagent according to the manufacturer's instructions. RNA purity and quantification were determined using a NanoDrop 2000 spectrophotometer (Thermo Scientific, USA), and RNA integrity was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Transcriptome libraries were constructed using the VAHTSUniversal V5 RNA-seq Library Prep kit according to the manufacturer's instructions.

[0086] (2) RNA sequencing and differentially expressed gene analysis

[0087] Library sequencing was performed using the Ilumina Novaseq 6000 sequencing platform, generating 150 bp paired-end reads. Approximately 45 raw reads were obtained per sample. The raw reads in FASTQ format were processed using FASTP software to remove low-quality reads, resulting in clean reads for subsequent data analysis. Reference genome alignment was performed using HISAT2 software, and gene expression levels (FPKM) were calculated. Read counts for each gene were obtained using HTSeq-count. PCA analysis and plotting of gene counts were performed using R (v3.2.0) to assess sample biological replicates. Differentially expressed genes (DEGs) were analyzed using DESeq2 software, where genes meeting the threshold of q-value < 0.05 and foldchange > 2 or foldchange < 0.5 were defined as differentially expressed genes (DEGs). Hierarchical cluster analysis of DEGs was performed using R (v3.2.0) to demonstrate gene expression patterns across different groups and samples. The R package `ggradar` was used to create a radar chart of the top 30 genes to show changes in the expression of upregulated or downregulated genes. Subsequently, GO and KEGG enrichment analyses were performed on differentially expressed genes using the hypergeometric distribution algorithm to screen for significantly enriched functional items. R (v3.2.0) was used to create bar charts for the significantly enriched functional items. Gene set enrichment analysis was performed using GSEA software. Using a predefined gene set, genes were ranked according to their differential expression levels in the two classes of samples, and then it was examined whether the predefined gene set was enriched at the top or bottom of this ranking list.

[0088] Experimental results: such as Figure 19The results of transcriptome RNA sequencing and analysis are as follows: (1) Number of differentially expressed genes: At the end of day 7 of intervention, compared with the normal group, the model group upregulated 139 genes and downregulated 847 genes. Compared with the model group, the drug-loaded hydrogel group upregulated 86 genes and downregulated 45 genes. At the end of day 14 of intervention, compared with the normal group, the model group upregulated 846 genes and downregulated 1092 genes. Compared with the model group, the drug-loaded hydrogel group upregulated 405 genes and downregulated 132 genes.

[0089] (2) GO enrichment analysis of differentially expressed genes: The differentially expressed genes among the three groups were mainly enriched in terms of inflammatory response and epidermal repair. Compared with the normal group, the model group showed upregulated inflammatory response and downregulated epidermal repair. Compared with the model group, the drug-loaded hydrogel group showed downregulated inflammatory response and upregulated epidermal repair.

[0090] (3) KEGG enrichment analysis of differentially expressed genes: The differentially expressed genes among the three groups were mainly enriched in pathways such as oxidative stress, inflammatory response, cell proliferation, and tissue repair. After screening the most relevant pathways among the groups, it was found that the Wnt signaling pathway and the TNF-α / NF-κB signaling pathway were closely related on the 7th and 14th days of treatment. Compared with the normal group, the TNF-α / NF-κB signaling pathway was upregulated and the Wnt signaling pathway was downregulated in the model group. Compared with the model group, the TNF-α / NF-κB signaling pathway was downregulated and the Wnt signaling pathway was upregulated in the drug-loaded hydrogel group.

[0091] (4) Screening of related genes: Based on the GO analysis results and biological significance, genes with differential structural and functional characteristics, such as inflammatory response, cellular response to lipopolysaccharide, intermediate filament organization, epithelial cell differentiation, keratinization, bicellular tight junction, establishment of skin barrier, structural constituent of skin epidermis, and structural molecule activity, were screened out. These genes include Il1b, Il6, Cxcl12, Cldn1, Cldn4, Krt75, and Krt83. GO analysis showed that, compared with the normal group, the genes Cldn1, Cldn4, Krt75, and Krt83 in the model group were downregulated, while the genes in the drug-loaded hydrogel group were upregulated. Compared with the normal group, the genes Il1b, Il6, and Cxcl12 in the model group were upregulated, while the genes in the drug-loaded hydrogel group were downregulated.

[0092] 7. RT-qPCR to validate differentially expressed gene levels

[0093] Experimental methods: Total RNA was extracted from wound skin tissues of each group using the TRIzol method. Following the instructions of the Real-time PCR kit, reverse transcription was performed, followed by PCR amplification. The reaction conditions (20 μL system) were: pre-denaturation at 95℃ for 30 s, denaturation at 95℃ for 5 s, annealing at 55℃ for 30 s, and extension at 72℃ for 30 s, for a total of 45 cycles. The relative expression level of the target gene was calculated using the 2-ΔΔct formula.

[0094] Experimental results: such as Figure 20 RT-qPCR verification of differential gene expression levels showed that: (1) Cldn1: Compared with the normal group, the expression level of the model group was significantly decreased (P<0.01), while the expression level of the drug-loaded hydrogel group was increased (P<0.05).

[0095] (2) Cldn4: Compared with the normal group, the expression level in the model group decreased (P<0.05), while the expression level in the drug-loaded hydrogel group increased (P<0.05).

[0096] (3) Krt75: Compared with the normal group, the expression level in the model group was significantly decreased (P<0.01), while the expression level in the drug-loaded hydrogel group was increased (P<0.05).

[0097] (4) Krt83: Compared with the normal group, the expression level of the model group and the drug-loaded hydrogel group decreased (P<0.05), while the expression level of the drug-loaded hydrogel group increased (P<0.05) compared with the model group.

[0098] (5) Il1b: Compared with the normal group, the expression level of the model group and the drug-loaded hydrogel group was increased (P<0.05), while the expression level of the drug-loaded hydrogel group was decreased compared with the model group (P<0.05).

[0099] (6) Il6: Compared with the normal group, the expression level in the model group was significantly increased (P<0.01), while the expression level in the drug-loaded hydrogel group was decreased (P<0.05).

[0100] (7) Cxcl12: Compared with the normal group, the expression level in the model group was significantly increased (P<0.01), while the expression level in the drug-loaded hydrogel group was significantly decreased (P<0.05).

[0101] 8. Western blotting method to verify the levels of related pathway proteins

[0102] Experimental Methods: Proteins were extracted from skin tissues of each group. Protein concentrations were determined according to the BCA protein quantification kit instructions. Precast Protein Plus Gel precast gels were used; proteins were loaded, electrophoresed, and then transferred to PVDF membranes. The membranes were blocked with 5% skim milk diluted in TBST buffer for 2 h, then primary antibody was added, and the membranes were gently shaken and incubated overnight at 4°C. The membranes were washed three times with TBST buffer. Secondary antibody was added, and the membranes were incubated for 2 h with shaking, followed by three washes with TBST buffer. The membranes were developed using ECL, and the gray values ​​of the target bands were analyzed using ImageJ.

[0103] Experimental results: such as Figure 21 Western Blot analysis of relevant pathway protein levels showed that: (1) GSK-3β: Compared with the normal group, the expression level of the model group was increased (P<0.05), and compared with the model group, the expression level of the drug-loaded hydrogel group was decreased (P>0.05).

[0104] (2) β-Catenin: Compared with the normal group, the expression level of the model group and the drug-loaded hydrogel group was significantly decreased (P<0.01), while the expression level of the drug-loaded hydrogel group was significantly increased (P<0.01) compared with the model group.

[0105] (3) c-Myc: Compared with the normal group, the expression level of the model group and the drug-loaded hydrogel group was significantly decreased (P<0.01), while the expression level of the drug-loaded hydrogel group was significantly increased (P<0.01) compared with the model group.

[0106] (4) TNF-α: Compared with the normal group, the expression levels in the model group and the drug-loaded hydrogel group were significantly increased (P<0.01), while the expression level in the drug-loaded hydrogel group was decreased compared with the model group (P<0.05).

[0107] (5) p-p65 / p65: Compared with the normal group, the expression level of the model group and the drug-loaded hydrogel group was increased (P<0.05), while the expression level of the drug-loaded hydrogel group was decreased compared with the model group (P<0.05).

[0108] In summary, 1. Drug-loaded hydrogels can reduce inflammatory responses and shorten the inflammatory period by downregulating pro-inflammatory factors IL-1β, IL-6, and TNF-α, upregulating anti-inflammatory factor IL-10, and regulating M2 macrophage polarization. They can also promote cell proliferation and angiogenesis by upregulating TGF-β and VEGF levels and increasing CD31 expression. Furthermore, they can increase collagen deposition, reduce collagen degradation, and enhance extracellular matrix remodeling by upregulating COL-I and COL-III expression levels and downregulating MMP-9 levels. These effects can shorten wound healing time and improve the healing rate of refractory skin ulcers in diabetic rats.

[0109] 2. Drug-loaded hydrogels promote the healing of refractory skin ulcers in diabetic rats by upregulating the expression of Cldn1, Cldn4, Krt75, and Krt83, downregulating Il1b, Il6, and Cxcl12, and activating the TNF-α / NF-κB signaling pathway and the classical Wnt signaling pathway.

[0110] This document uses specific examples to illustrate the principles and implementation methods of the present invention. The descriptions of the above embodiments are only for the purpose of helping to understand the method and core ideas of the present invention. Furthermore, those skilled in the art will recognize that, based on the ideas of the present invention, there will be changes in the specific implementation methods and application scope. Therefore, the content of this specification should not be construed as a limitation of the present invention.

Claims

1. A preparation method of a traditional Chinese medicine compound ultrafine powder hydrogel, characterized in that, The method comprises adding traditional Chinese medicine ultra-fine powder into gel material, heating, mixing, cooling and forming gel; wherein the traditional Chinese medicine ultra-fine powder is a mixture of a diabetes wound external treatment formula with a particle size of 2.5-5 μm; the gel material comprises chitosan, sodium alginate, calcium carbonate and gluconic acid δ-lactone.

2. The method of claim 1, wherein, The mass ratio of chitosan to sodium alginate is 1:2, the mass ratio of calcium carbonate to chitosan is 1:4.5, and the mass ratio of calcium carbonate to gluconic acid δ-lactone is 1:6.

3.

3. The preparation method according to claim 1, characterized in that, The drug loading concentration of the gel material to the traditional Chinese medicine ultra-fine powder is 9-36 mg / mL.

4. The preparation method according to claim 3, characterized in that, The drug loading concentration of the gel material to the traditional Chinese medicine ultra-fine powder is 18 mg / mL.

5. The method of claim 1, wherein the step of forming the first and second layers is performed by a process selected from the group consisting of: sputtering, evaporation, and chemical vapor deposition. The heating and mixing temperature is 37 ℃, and the heating and mixing time is 20 min.

6. The traditional Chinese medicine compound ultra-fine powder hydrogel obtained by the preparation method in any one of claims 1-5.

7. The application of the traditional Chinese medicine compound ultra-fine powder hydrogel in claim 6 in the preparation of a diabetes wound external material.

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