Medicinal preparation for promoting healing of chronic wound of diabetes mellitus as well as preparation method and application of medicinal preparation

Abstract

The invention discloses a medicinal preparation for promoting healing of chronic diabetic wounds as well as a preparation method and application of the medicinal preparation, and relates to the technical field of medicinal preparations. According to the invention, a thrombin solution containing Se-CQDs and a fibrinogen solution containing GOX are mixed and reacted to prepare the pharmaceutical preparation for promoting healing of chronic wounds of diabetes. The pharmaceutical preparation for promoting healing of the chronic wound of diabetes can effectively remove excessive ROS at the chronic wound of diabetes and reduce the pH, glucose content and M1 / M2 macrophage proportion at the chronic wound, so that the high-glucose alkaline inflammation microenvironment of the chronic wound of diabetes is reversed, collagen deposition, revascularization and skin tissue reepithelization are promoted, and the healing of the chronic wound of diabetes is promoted. Therefore, healing of chronic diabetic wounds is promoted. Meanwhile, Se-CQDs and GOX are combined to prepare the pharmaceutical preparation, and the pharmaceutical preparation has a synergistic effect on promoting healing of chronic wounds of diabetes mellitus.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical preparations, and particularly to a pharmaceutical preparation for promoting the healing of diabetic chronic wounds, its preparation method and application. Background Art

[0002] Diabetes, as an endocrine disease characterized by persistent hyperglycemia, seriously threatens the health and quality of life of patients due to its various complications. Among them, diabetic chronic wounds are one of the most typical complications of diabetes. Due to the oxidative stress caused by long-term systemic hyperglycemia in the pathological wounds of diabetic patients, which disrupts the normal redox signaling, the excessive production of reactive oxygen species (ROS) in the wound area will induce oxidative damage and hinder wound healing. In view of this, removing the excessive ROS in the diabetic wound area and reducing oxidative stress to protect cells from damage can be considered an effective targeted strategy for promoting the healing of diabetic chronic wounds.

[0003] In the prior art, the paper "In Vitro Antioxidant and Anti-Inflammatory Properties of Selenium-Doped Carbon Quantum Dots" (Ou Yanzhen, Jilin University, May 2022) discloses that selenium-doped carbon quantum dots can reduce the intracellular ROS generation induced by H 2 O 2 However, the wound surface of diabetic patients has a complex local microenvironment of hyperglycemia, alkalinity (pH 7.0 - 8.9), excessive ROS and excessive inflammation. The selenium-doped carbon quantum dots prepared in the above paper only rely on scavenging excessive ROS to avoid oxidative damage, and the effect on promoting wound healing is not good. The inherent hyperglycemic and alkaline microenvironment of diabetic chronic wounds will still induce excessive protein glycosylation and advanced glycation end products (AGEs). Subsequently, AGEs hinder the polarization of macrophages from pro-inflammatory M1 type to anti-inflammatory M2 type by stimulating the production of pro-inflammatory cytokines, leading to macrophage dysfunction and blocking the processes of collagen deposition, granulation tissue formation, angiogenesis and cell migration in wound repair.

[0004] Therefore, preparing a pharmaceutical preparation that can not only effectively remove the excessive ROS in the diabetic chronic wound area but also reverse the hyperglycemic and alkaline inflammatory microenvironment on the surface of chronic wounds, thereby promoting the healing of diabetic chronic wounds has great potential for clinical application. Summary of the Invention

[0005] In view of the above-mentioned prior art, the purpose of the present invention is to provide a pharmaceutical preparation for promoting the healing of diabetic chronic wounds, its preparation method and application. The present invention uses a thrombin solution containing Se-CQDs and a fibrinogen solution containing GOX to prepare a pharmaceutical preparation that can be used to promote the healing of diabetic chronic wounds. The pharmaceutical preparation for promoting the healing of diabetic chronic wounds can effectively remove excessive ROS at the site of diabetic chronic wounds, reduce the pH, glucose content and M1 / M2 macrophage ratio at the chronic wound site, so as to reverse the hyperglycemic alkaline inflammatory microenvironment of diabetic chronic wounds, promote collagen deposition, angiogenesis and re-epithelialization of skin tissue, thereby promoting the healing of diabetic chronic wounds.

[0006] To achieve the above purpose, the present invention adopts the following technical solutions: In the first aspect of the present invention, a preparation method of a pharmaceutical preparation for promoting the healing of diabetic chronic wounds is provided, including the following steps: Add Se-CQDs to the thrombin solution to obtain a first mixed solution; add GOX to the fibrinogen solution to obtain a second mixed solution; after mixing and reacting the first mixed solution and the second mixed solution, the pharmaceutical preparation for promoting the healing of diabetic chronic wounds is obtained.

[0007] Preferably, the Se-CQDs are prepared by the following method: Add an alkali solution to the L-selenocysteine dispersion to adjust the pH, heat for reaction, after the reaction is completed, centrifuge, collect the supernatant after centrifugation, perform dialysis treatment on the supernatant, collect the dialysis solution, and dry to obtain Se-CQDs.

[0008] Further, the L-selenocysteine dispersion is prepared by mixing L-selenocysteine and water according to a material-liquid ratio of (150-250) mg: 10 mL.

[0009] Further, the alkali solution is a NaOH solution, and the concentration of the NaOH solution is 0.5 M.

[0010] Further, adjust the pH to 8.5-9.5.

[0011] Further, the heating temperature is 55-65 °C, and the reaction time is 20-30 h.

[0012] Further, the cut-off molecular weight of the dialysis bag used in the dialysis treatment process is 500-1000.

[0013] Preferably, the fibrinogen solution is prepared from fibrinogen and physiological saline, with a concentration of 15-25 mg / mL; the thrombin solution is prepared from thrombin and physiological saline, with a concentration of 45-55 U / mL.

[0014] Preferably, the material ratio of Se-CQDs to thrombin solution is 10 μg : (15 - 25) μL.

[0015] Preferably, the material ratio of GOX to fibrinogen solution is 10 μg : (15 - 25) μL.

[0016] Preferably, the volume ratio of the first mixture to the second mixture is (0.8 - 1.2) : 1.

[0017] Preferably, a double-barrel syringe is used to mix the first mixture and the second mixture.

[0018] In the second aspect of the present invention, there is provided a pharmaceutical preparation for promoting the healing of diabetic chronic wounds prepared by the above preparation method.

[0019] In the third aspect of the present invention, there is provided the application of the above pharmaceutical preparation for promoting the healing of diabetic chronic wounds in any one of the following 1) - 3): 1) Promoting the healing of diabetic chronic wounds; 2) Promoting collagen deposition, angiogenesis and re-epithelialization of skin tissue; 3) Removing excessive ROS at the chronic wound site, reducing the pH, glucose content and M1 / M2 macrophage ratio at the chronic wound site.

[0020] Advantages of the present invention: The present invention uses a thrombin solution containing Se-CQDs and a fibrinogen solution containing GOX to prepare a pharmaceutical preparation that can be used to promote the healing of diabetic chronic wounds. The pharmaceutical preparation for promoting the healing of diabetic chronic wounds can effectively remove excessive ROS at the diabetic chronic wound site, reduce the pH, glucose content and M1 / M2 macrophage ratio at the chronic wound site, so as to reverse the hyperglycemic alkaline inflammatory microenvironment of diabetic chronic wounds, promote collagen deposition, angiogenesis and re-epithelialization of skin tissue, thereby promoting the healing of diabetic chronic wounds. At the same time, through experimental verification, the pharmaceutical preparation prepared by the present invention has good biosafety and does not cause systemic toxicity. Therefore, the pharmaceutical preparation prepared by the present invention shows great application potential in the healing of diabetic chronic wounds.

[0021] The present invention uses the combination of Se-CQDs and GOX for preparing a pharmaceutical preparation, which has a synergistic effect in promoting the healing of diabetic chronic wounds. Specifically, after treating with the pharmaceutical preparation prepared by the present invention for 8 days, the residual wound area rate is only 24.6%. Description of the Drawings

[0022] Figure 1: TEM characterization diagram, zeta potential diagram, XRD characterization diagram and FT-IR characterization diagram of Se-CQDs prepared in Example 1; where A is the TEM characterization diagram, B is the zeta potential diagram, C is the XRD characterization diagram, and D is the FT-IR characterization diagram; Figure 2 : XPS spectrogram of Se-CQDs prepared in Example 1, high-resolution spectrograms of C 1s, N 1s and Se 3d; where A is the XPS spectrogram, B is the high-resolution spectrogram of C 1s, C is the high-resolution spectrogram of N 1s, and D is the high-resolution spectrogram of Se 3d; Figure 3 : UV-visible spectrogram, fluorescence spectrogram and excitation light-dependent fluorescence spectrogram of Se-CQDs prepared in Example 1; where A is the UV-visible spectrogram, B is the fluorescence spectrogram, and C is the excitation light-dependent fluorescence spectrogram; Figure 4 : Preparation flow chart, SEM characterization diagram, rheological behavior diagrams of the second mixture and the pharmaceutical preparation of the pharmaceutical preparation prepared in Example 1; where A is the preparation flow chart of the pharmaceutical preparation, B is the SEM characterization diagram of the pharmaceutical preparation, C is the frequency-dependent rheological behavior diagram of the second mixture, D is the frequency-dependent rheological behavior diagram of the pharmaceutical preparation, and E is the strain-dependent rheological behavior diagram of the pharmaceutical preparation; Figure 5 : Wound schematic diagrams, wound residual area ratio diagrams and wound healing trace diagrams corresponding to different treatment times of mice in groups G1-G5; where A is the wound schematic diagram, B is the wound residual rate diagram, and C is the wound healing trace diagram; Figure 6 : H&E staining, Masson's trichrome staining and Sirius red staining section diagrams of the wounds of mice in groups G1-G5 after 14 days of treatment; in the figure, the purple scale bar is 200 μm and the black scale bar is 50 μm; Figure 7 : Immunofluorescence staining diagrams of CK14 and CK10 of the wounds of mice in groups G1-G5 after 14 days of treatment; in the figure, the scale bar is 50 μm; Figure 8 : Immunofluorescence staining diagrams of Ki67, CD31 (red) + α-SMA (green) and CD86 (red) + CD206 (green) of the wounds of mice in groups G1-G5 after 14 days of treatment; in the figure, the scale bar is 50 μm; Figure 9 : Immunohistochemical staining diagrams of IL-1β, IL-6 and TNF-α of the wounds of mice in groups G1-G5 after 14 days of treatment; in the figure, the scale bar is 50 μm; Figure 10: H&E staining images of the major organs (heart, liver, spleen, lung, kidney) of mice in groups G1 - G5; in the figure, the scale bar is 50 μm; Figure 11 : Blood routine and serum biochemical data graphs of mice in groups G1 - G5; among them, A is the concentration graph of blood routine indexes, B is the ALT concentration graph in serum, C is the AST concentration graph in serum, D is the ALB concentration graph in serum, E is the ALP concentration graph, F is the CREA concentration graph, and G is the UREA concentration graph. Detailed implementation manners

[0023] It should be noted that the following detailed description is illustrative and aims to provide further explanation for this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which this application belongs.

[0024] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in combination with specific embodiments.

[0025] The experimental materials used in the embodiments of the present invention are all conventional experimental materials in the art and can be obtained through commercial channels.

[0026] In the present invention, L-selenocysteine (98%) was purchased from Shanghai Macklin Biochemical Co., Ltd., glucose oxidase (GOX) was purchased from Sigma-Aldrich Company in the United States, thrombin was purchased from Shanghai Yuanye Bio-Technology Co., Ltd., and fibrinogen was purchased from Beijing Solarbio Science & Technology Co., Ltd. C57BL / 6 mice and diabetic (db / db) mice were purchased from Hunan Slack SJA Animal Co., Ltd.

[0027] Example 1: Preparation of a pharmaceutical preparation for promoting the healing of chronic diabetic wounds (1) Mix L-selenocysteine and ultrapure water at a material-liquid ratio of 200 mg:10 mL to obtain an L-selenocysteine dispersion; add 0.5 M NaOH solution to the L-selenocysteine dispersion to adjust the pH to 9.0, then heat to 60 °C and react for 24 h. After the reaction is completed, centrifuge the reaction product at 12,000 rpm, collect the supernatant after centrifugation and dialyze it using a dialysis bag with a molecular weight cut-off of 500 - 1000. Collect the dialysate and perform freeze-drying to obtain a brownish-red powder, which is Se-CQDs; (2) Mix fibrinogen and physiological saline to prepare a fibrinogen solution with a concentration of 20 mg / mL; mix thrombin and physiological saline to prepare a thrombin solution with a concentration of 50 U / mL; Add Se-CQDs to the thrombin solution to obtain a first mixture, and add GOX to the fibrinogen solution to obtain a second mixture; use a dual-tube syringe to mix and inject the first mixture and the second mixture at a volume ratio of 1:1 to obtain a pharmaceutical preparation for promoting the healing of chronic diabetic wounds.

[0028] Example 2: Preparation of a pharmaceutical preparation for promoting the healing of chronic diabetic wounds (1) Mix L-selenocysteine and ultrapure water at a material-liquid ratio of 150 mg: 10 mL to obtain an L-selenocysteine dispersion; add 0.5 M NaOH solution to the L-selenocysteine dispersion to adjust the pH to 8.5, then heat to 55 °C and react for 30 h. After the reaction is completed, centrifuge the reaction product at 12,000 rpm, collect the supernatant after centrifugation and dialyze it using a dialysis bag with a molecular weight cut-off of 500-1000. Collect the dialysate and perform freeze-drying to obtain a brownish-red powder, which is Se-CQDs; (2) Mix fibrinogen and physiological saline to prepare a fibrinogen solution with a concentration of 15 mg / mL; mix thrombin and physiological saline to prepare a thrombin solution with a concentration of 45 U / mL; Add Se-CQDs to the thrombin solution to obtain a first mixture, and add GOX to the fibrinogen solution to obtain a second mixture; use a dual-tube syringe to mix and inject the first mixture and the second mixture at a volume ratio of 0.8:1 to obtain a pharmaceutical preparation for promoting the healing of chronic diabetic wounds.

[0029] Example 3: Preparation of a pharmaceutical preparation for promoting the healing of chronic diabetic wounds (1) Mix L-selenocysteine and ultrapure water at a material-liquid ratio of 250 mg: 10 mL to obtain an L-selenocysteine dispersion; add 0.5 M NaOH solution to the L-selenocysteine dispersion to adjust the pH to 10.0, then heat to 65 °C and react for 20 h. After the reaction is completed, centrifuge the reaction product at 12,000 rpm, collect the supernatant after centrifugation and dialyze it using a dialysis bag with a molecular weight cut-off of 500-1000. Collect the dialysate and perform freeze-drying to obtain a brownish-red powder, which is Se-CQDs; (2) Mix fibrinogen and physiological saline to prepare a fibrinogen solution with a concentration of 25 mg / mL; mix thrombin and physiological saline to prepare a thrombin solution with a concentration of 55 U / mL; Add Se-CQDs to the thrombin solution to obtain a first mixture, and add GOX to the fibrinogen solution to obtain a second mixture; use a dual-syringe to mix and inject the first mixture and the second mixture at a volume ratio of 1.2:1, and the pharmaceutical preparation for promoting the healing of chronic diabetic wounds is obtained.

[0030] Comparative Example 1: The difference between this comparative example and Example 1 is that during the preparation of the pharmaceutical preparation for promoting the healing of chronic diabetic wounds, GOX and Se-CQDs are not used. The specific preparation method is as follows: Prepare a fibrinogen solution with a concentration of 20 mg / mL and a thrombin solution with a concentration of 50 U / mL according to the method in Example 1; use a dual-syringe to mix and inject the fibrinogen solution and the thrombin solution at a volume ratio of 1:1, and the pharmaceutical preparation for promoting the healing of chronic diabetic wounds is obtained.

[0031] Comparative Example 2: The difference between this comparative example and Example 1 is that during the preparation of the pharmaceutical preparation for promoting the healing of chronic diabetic wounds, GOX is not used. The specific preparation method is as follows: Prepare a fibrinogen solution with a concentration of 20 mg / mL, a thrombin solution with a concentration of 50 U / mL and Se-CQDs according to the method in Example 1, add Se-CQDs to the thrombin solution to obtain a mixture, and use a dual-syringe to mix and inject the mixture and the thrombin solution at a volume ratio of 1:1, and the pharmaceutical preparation for promoting the healing of chronic diabetic wounds is obtained.

[0032] Comparative Example 3: The difference between this comparative example and Example 1 is that during the preparation of the pharmaceutical preparation for promoting the healing of chronic diabetic wounds, Se-CQDs are not used. The specific preparation method is as follows: Prepare a fibrinogen solution with a concentration of 20 mg / mL and a thrombin solution with a concentration of 50 U / mL according to the method in Example 1; add GOX to the fibrinogen solution to obtain a mixture; use a dual-syringe to mix and inject the fibrinogen solution and the mixture at a volume ratio of 1:1, and the pharmaceutical preparation for promoting the healing of chronic diabetic wounds is obtained.

[0033] Test Example 1: Structural Characterization 1. Perform structural characterization on the Se-CQDs prepared in step (1) of Example 1, and the results are as Figures 1 - 3 shown.

[0034] As can be seen from Figure 1 A in, the diameter of the Se-CQDs prepared in the present invention is 14.03 ± 4.48 nm. As can be seen from Figure 1As can be seen from Figure B, since the surface of Se-CQDs has carboxyl and hydroxyl groups, its surface potential is negatively charged, at -21.83 ± 1.94 mV. From Figure 1 As can be seen from Figure C, there is a broad diffraction peak at about 22°, indicating that this is an amorphous structure, which matches the graphite phase. From Figure 1 As can be seen from Figure D, the characteristic broadband between 3600 - 3100 cm -1 corresponds to the stretching vibrations of N-H and O-H. The two peak positions at 1692 cm -1 and 1394 cm -1 correspond to the stretching vibrations of the aromatic domain C=O and C=C bonds. The peaks in the region between 1200 - 900 cm -1 are mainly related to the stretching vibrations of C-O, C-N, and C-Se.

[0035] From Figure 2 As can be seen from Figure A, the Se-CQDs prepared in this invention are mainly composed of four elements: C, O, N, and Se. From Figure 2 As can be seen from Figure B, the peak at 284.5 eV in the deconvoluted XPS spectrum of C 1s is C-C, and the peaks at 286.0 eV indicate the presence of C-Se, C-N, and C-O, and the peak at 288.0 eV belongs to C=O. From Figure 2 As can be seen from Figure C, the presence of pyridine nitrogen and pyrrole nitrogen is observed at 398.9 eV and 400.9 eV in the deconvoluted XPS spectrum of N 1s. From Figure 2 As can be seen from Figure D, the presence of C-Se-C in Se-CQDs is revealed by 55.3 eV in the deconvoluted XPS spectrum of Se 3d.

[0036] From Figure 3 As can be seen from Figure A, the aqueous solution of Se-CQDs has a typical absorption peak at about 280 nm, which comes from the π-π* transition of the aromatic sp 2 core domain structure of Se-CQDs. There is also a typical absorption peak at about 340 nm because of the n-π* (-COOH, C-N / or C-Se) transition on the surface of Se-CQDs. From Figure 3 As can be seen from Figure B, Se-CQDs shows a maximum emission peak at about 490 nm. From Figure 3 As can be seen from Figure C, Se-CQDs also shows obvious fluorescence characteristics of excitation light dependence (410 - 510 nm), which is caused by the optical selective absorption of the defect sites of Se-CQDs.

[0037] 2. The drug preparation for promoting the healing of diabetic chronic wounds prepared in Example 1 was analyzed by electron microscopy. At the same time, the rheological analysis of the second mixture and the drug preparation in Example 1 was carried out, and the results are asFigure 4 as shown

[0038] From Figure 4 as can be seen from A in the figure, after mixing the first mixture prepared from Se-CQDs and thrombin solution and the second mixture prepared from GOX and fibrinogen solution in equal volumes, the prepared pharmaceutical preparation for promoting the healing of chronic diabetic wounds is a hydrogel. From Figure 4 as can be seen from B in the figure, the hydrogel prepared in the present invention has a porous network structure. The first mixture is a thrombin solution containing Se-CQDs, and the second mixture is a fibrinogen solution containing GOX. From Figure 4 as can be seen from C, D and E in the figure, compared with the fibrinogen solution containing GOX, after adding the thrombin solution containing Se-CQDs, the value of the elastic modulus (G’) of the prepared product increases rapidly and finally exceeds the viscous modulus (G’’), indicating the successful preparation of the hydrogel.

[0039] Experimental Example 2: Animal experiment Diabetic (db / db) mice were selected as the experimental subjects for the experiment. The specific steps are as follows: (1) Twenty-five mice were taken, anesthetized with isoflurane, then the back hair was removed and disinfected. A circular wound was established on the back of the db / db mice with a biopsy punch with a diameter of 6 mm, and then a full-thickness skin exfoliation wound surgery was established using a micro-scissors according to the contour.

[0040] (2) The mice were randomly divided into 5 groups, and different pharmaceutical preparations were used to fill the wound sites in each treatment group. The specific details of each treatment group are as follows: Group G1: Control group, no treatment was given to the wound sites of the mice; Group G2: The wound sites were filled with the pharmaceutical preparation prepared in Comparative Example 1; Group G3: The wound sites were filled with the pharmaceutical preparation prepared in Comparative Example 2; Group G4: The wound sites were filled with the pharmaceutical preparation prepared in Comparative Example 3; Group G5: The wound sites were filled with the pharmaceutical preparation prepared in Example 1.

[0041] Among them, 40 μL of the pharmaceutical preparation was used in each treatment group to fill the wound sites. After filling, a commercially available Tegaderm, 3M film was used to cover the wound. Each mouse was raised separately and fed freely to ensure that the feeding management of the mice in each group was consistent during the experiment.

[0042] 1. Healing of chronic diabetic wounds The mice in each treatment group were photographed 0, 4, 8, and 14 days after wound treatment, and ImageJ software was used to analyze the wound size and calculate the wound area ratio. The results are as Figure 5As shown below. The formula for calculating the wound area rate is: Residual wound area rate (%) = [Actual wound area on day n / Original wound area] × 100%; In the formula, n represents the 0th, 4th, and 8th days after wound treatment.

[0043] From Figure 5 as can be seen from A, B, and C, after the wounds of groups G2 - G4 were treated with the pharmaceutical preparation for filling the wound, there were still certain areas of the wounds that were not healed after 14 days of treatment. However, when the pharmaceutical preparation prepared by the present invention (group G5) was used to treat the wound, after 14 days of treatment, the wound was almost completely healed. Thus, it can be seen that the pharmaceutical preparation prepared by the present invention using GOX and Se - CQDs has excellent effects in promoting the healing of chronic diabetic wounds.

[0044] In addition, from Figure 5 as can be seen from B, after the wounds of group G2 were treated with the pharmaceutical preparation prepared in Comparative Example 1 for 8 days, the residual wound area rate was 44.4%; after the wounds of group G3 were treated with the pharmaceutical preparation prepared in Comparative Example 2 for 8 days, the residual wound area rate was 37.3%; after the wounds of group G4 were treated with the pharmaceutical preparation prepared in Comparative Example 3 for 8 days, the residual wound area rate was 41.2%. However, after the wounds were treated with the pharmaceutical preparation prepared by the present invention (group G5) for 8 days, the residual wound area rate was only 24.6%. Thus, it can be seen that the combination of GOX and Se - CQDs to prepare the pharmaceutical preparation has a synergistic effect in the treatment of chronic diabetic wounds.

[0045] 2. Collagen deposition, angiogenesis, re - epithelialization of skin tissue, and reversal of the inflammatory microenvironment at chronic wounds (1) One mouse from each treatment group was selected 14 days after treatment. After sacrificing the mouse, the wound skin tissue of the mouse was collected, soaked in 4% paraformaldehyde by mass fraction for 24 h, embedded in paraffin blocks, and made into 5 - μm - thick sections for histological staining.

[0046] (2) Hematoxylin - eosin (H&E) and Masson's trichrome staining were used to detect the formation of granulation tissue and collagen during wound healing. Sirius red staining was used to evaluate the ratio of collagen I / III during wound healing. The results are as Figure 6 shown.

[0047] (3) Ki67 immunofluorescence staining was used to detect cell proliferation during wound healing. CD31+α - SMA immunofluorescence staining was used to evaluate angiogenesis during wound healing. Cytokeratin 10 (CK10) + cytokeratin 14 (CK14) immunofluorescence staining was used to evaluate the degree of epithelialization during wound healing. CD86+CD206 immunofluorescence staining was used to evaluate the content of M1 / M2 macrophages during wound healing. The results are asFigure 7 and Figure 8 as shown

[0048] (4) The expression of pro-inflammatory cytokines during wound healing was detected by immunohistochemical staining of IL-6, IL-1β and TNF-α, and the results are as Figure 9 shown

[0049] Figure 6 The blue and red arrows in refer to blood vessels and hair follicles respectively. It can be seen from Figure 6 that after hematoxylin and eosin (H&E) staining, the skin tissues of the G1 and G2 groups still showed rupture and hardly healed. Compared with the G3 and G4 groups, the drug preparation prepared by the present invention filled the wound surface, and the skin tissues of the wounds of diabetic mice showed significantly better healing effect, with a large number of new blood vessels and new hair follicles formed. At the same time, the ratio of collagen deposition (blue area of Masson staining) and mature type I collagen (orange-yellow or red refraction of Sirius red staining) to early type III collagen (green refraction of Sirius red staining) in the wound tissues treated with the drug preparation of the present invention was significantly higher than that of other groups. Thus, it can be seen that the drug preparation prepared by the present invention can effectively promote skin reconstruction and collagen deposition of the wound

[0050] CK10 and CK14, as markers of spinous keratinocytes and basal keratinocytes, were used to observe the epithelialization of skin wound tissues. It can be seen from Figure 7 that re-epithelialization of the corresponding skin tissues in the G3, G4 and G5 groups was completed, and the epithelial stratification was clear. However, the keratinocyte layer in the G1 and G2 groups was missing. This result was consistent with the results of H&E staining sections

[0051] It can be seen from Figure 8 that the amount of Ki67-positive cells in the wound skin treated with the drug preparation prepared by the present invention was the highest. The fluorescence staining results of CD31 and α-SMA also showed that the density of regenerated blood vessels in the wound tissues treated with the drug preparation prepared by the present invention was the highest. At the same time, the skin tissues were immunostained with CD86 to label M1 inflammatory macrophages and CD206 to label M2 anti-inflammatory macrophages. After treatment with the drug preparation prepared in the G3 and G4 groups, the number of M2 anti-inflammatory cells increased slightly compared with the G1 group; after treatment with the drug preparation prepared by the present invention, the number of M2 anti-inflammatory cells was much higher than that of other treatment groups. Thus, it can be seen that the drug preparation prepared by combining Se-CQDs and GOX in the present invention can not only scavenge excessive ROS at the wound, but also reduce the pH and glucose content at the chronic wound to reverse the hyperglycemic alkaline inflammatory microenvironment of the chronic wound, promote the polarization of macrophages from the M1 phenotype to the M2 phenotype, and reduce the M1 / M2 macrophage ratio

[0052] It can be seen fromFigure 9 It can be seen that the contents of pro-inflammatory factors IL-1β, IL-6, and TNF-α in the wound tissues treated with the pharmaceutical preparation prepared by the present invention are significantly less than those in other groups. Thus, it can be seen that the pharmaceutical preparation prepared by the present invention can inhibit the secretion of pro-inflammatory factors and has a positive anti-inflammatory effect.

[0053] In summary, the pharmaceutical preparation prepared by the present invention can not only promote cell proliferation, angiogenesis, and epithelialization, but also scavenge ROS and control glucose levels to exert anti-inflammatory activity, reduce the M1 / M2 macrophage ratio, reverse the alkaline hyperglycemic inflammatory environment of the wound into a stable anti-inflammatory environment, thereby accelerating the healing process of diabetic wounds.

[0054] 3. Toxicity and biosafety (1) Each mouse in each treatment group was sacrificed 14 days after treatment, and the main organs of the mouse (heart, liver, spleen, lung, kidney) were collected for histological analysis by H&E staining, and the results are as Figure 10 shown.

[0055] (2) Healthy C57BL / 6 mice were selected and subcutaneously injected with PBS, the pharmaceutical preparations prepared in Comparative Examples 1-3, and the pharmaceutical preparation prepared in Example 1, ensuring that the injection volume was 40 µL for all. The mice in each group were separately housed to ensure consistent feeding management for each group of mice; After 14 days of feeding, the whole blood of each group of mice was collected, and the toxicity of the hydrogel in wound healing research was evaluated by measuring the concentrations of white blood cells (WBC), red blood cells (RBC), hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet count (PLT), serum alanine aminotransferase (ALT), serum aspartate aminotransferase (AST), albumin (ALB), alkaline phosphatase (ALP), creatinine (CREA), and urea nitrogen (UREA), and the results are as Figure 11 shown.

[0056] It can be seen from Figure 10 that compared with Group G1, there were no obvious histological differences in the corresponding main organs of Groups G2-G5. Thus, it can be seen that the pharmaceutical preparation prepared by the present invention has high biosafety. It can be seen from Figure 11 that there were no obvious differences in the various indicators of the corresponding mice in Groups G1-G5. Thus, it can be seen that the pharmaceutical preparation prepared by the present invention has no systemic toxicity.

[0057] In summary, the pharmaceutical preparation prepared by the present invention has no systemic toxicity and has good biosafety.

[0058] The above are only the preferred embodiments of the present application and are not intended to limit the present application. For those skilled in the art, various modifications and variations can be made to the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included within the protection scope of the present application.

Claims

1. A method for preparing a pharmaceutical preparation for promoting healing of chronic diabetic wounds, characterized in that: The following steps are involved: Se-CQDs are added to a thrombin solution to obtain a first mixed solution; GOX is added to a fibrinogen solution to obtain a second mixed solution; and after the first mixed solution and the second mixed solution are mixed and reacted, a pharmaceutical preparation for promoting the healing of chronic wounds of diabetes is obtained.

2. The method for preparing the pharmaceutical preparation for promoting healing of diabetic chronic wounds according to claim 1, characterized in that: The Se-CQDs are prepared by the following method: Alkaline solution is added to the L-selenocystine dispersion to adjust the pH, and the dispersion is heated to react. After the reaction is completed, the dispersion is centrifuged, the supernatant is collected, the supernatant is dialyzed, the dialyzate is collected, and the dispersion is dried to obtain Se-CQDs.

3. The method for preparing the pharmaceutical preparation for promoting healing of diabetic chronic wounds as claimed in claim 2, characterized in that: The L-selenocystine dispersion is prepared by mixing L-selenocystine and water in a solid-liquid ratio of (150-250) mg:10 mL.

4. The method for preparing the pharmaceutical preparation for promoting healing of diabetic chronic wounds as claimed in claim 2, characterized in that: The alkali solution is a NaOH solution with a concentration of 0.5 M and a pH value adjusted to 8.5-9.

5.

5. The method for preparing the pharmaceutical preparation for promoting healing of diabetic chronic wounds according to claim 2, characterized in that: The heating temperature is 55-65°C and the reaction time is 20-30h.

6. The method for preparing the pharmaceutical preparation for promoting healing of diabetic chronic wounds according to claim 1, characterized in that: The fibrinogen solution is prepared from fibrinogen and normal saline with a concentration of 15-25 mg / mL; the thrombin solution is prepared from thrombin and normal saline with a concentration of 45-55 U / mL.

7. The method for preparing the pharmaceutical preparation for promoting healing of diabetic chronic wounds according to claim 1, characterized in that: The material-liquid ratio of Se-CQDs and thrombin solution was 10 μg: (15-25) μL; the material-liquid ratio of GOX and fibrinogen solution was 10 μg: (15-25) μL.

8. The method for preparing the pharmaceutical preparation for promoting healing of diabetic chronic wounds according to claim 1, characterized in that: The volume ratio of the first mixed liquid to the second mixed liquid is (0.8-1.2):

1.

9. A pharmaceutical preparation for promoting healing of diabetic chronic wounds prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the pharmaceutical preparation for promoting healing of diabetic chronic wounds according to claim 9 in any one of the following 1) to 3): 1) Promote the healing of chronic diabetic wounds; 2) Promote collagen deposition, angiogenesis and re-epithelialization of skin tissue; 3) Clear excess ROS in chronic wounds, reduce pH, glucose content and M1 / M2 macrophage ratio in chronic wounds.

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