A pharmaceutical preparation for promoting healing of chronic diabetic wounds, and its preparation method and application

Abstract

The present invention discloses a pharmaceutical preparation for promoting the healing of diabetic chronic wounds, and its preparation method and application, and relates to the technical field of pharmaceutical preparations. The present invention adopts a mixed reaction of 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 excess ROS at the diabetic chronic wound, reduce the pH, glucose content and M1 / M2 macrophage ratio at the chronic wound, so as to achieve reversal of the high sugar-alkaline inflammatory microenvironment of the diabetic chronic wound, promote collagen deposition, angiogenesis and re-epithelialization of skin tissue, thereby achieving the promotion of the healing of diabetic chronic wounds. At the same time, the present invention adopts a combination of Se-CQDs and GOX for the preparation of pharmaceutical preparations, which has a synergistic effect in promoting the healing of diabetic chronic wounds.

Description

Technical Field

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

[0002] Diabetes, an endocrine disease characterized by persistent hyperglycemia, presents a variety of complications that seriously threaten patients' health and quality of life. Among them, chronic diabetic wounds are one of the most typical complications of diabetes. Because oxidative stress caused by long-term systemic hyperglycemia in pathological wounds of diabetic patients disrupts normal redox signaling, excessive production of reactive oxygen species (ROS) in the wound area induces oxidative damage and hinders wound healing. Therefore, clearing excess ROS in diabetic wounds, reducing oxidative stress, and thus protecting cells from damage, can be considered an effective and targeted strategy to promote the healing of chronic diabetic wounds.

[0003] Prior art, the paper "In Vitro Antioxidant and Anti-inflammatory Properties of Selenium-Doped Carbon Quantum Dots" (Ou Yanzhen, Jilin University, May 2022) disclosed that selenium-doped carbon quantum dots can reduce H2O2-induced intracellular ROS production. However, diabetic wounds possess a complex local microenvironment characterized by hyperglycemia, alkalinity (pH 7.0-8.9), excessive ROS production, and excessive inflammation. The selenium-doped carbon quantum dots prepared in the aforementioned paper, which rely solely on scavenging excess ROS to prevent oxidative damage, are ineffective in promoting wound healing. The inherently hyperglycemic and alkaline microenvironment of chronic diabetic wounds still induces excessive protein glycation and advanced glycation end products (AGEs). AGEs subsequently stimulate the production of proinflammatory cytokines, hindering the polarization of macrophages from the pro-inflammatory M1 phenotype to the anti-inflammatory M2 phenotype, leading to macrophage dysfunction and hindering collagen deposition, granulation tissue formation, angiogenesis, and cell migration during wound repair.

[0004] Therefore, the preparation of a drug preparation that can not only effectively remove excess ROS in the diabetic chronic wound area, but also reverse the high sugar-alkaline inflammatory microenvironment on the surface of the chronic wound, thereby promoting the healing of diabetic chronic wounds has great clinical application potential. 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, as well as its preparation method and application. The present invention adopts a mixture of 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 excess ROS in diabetic chronic wounds, reduce the pH, glucose content and M1 / M2 macrophage ratio in chronic wounds, thereby reversing the high sugar-alkaline inflammatory microenvironment of diabetic chronic wounds, promoting collagen deposition, angiogenesis and skin tissue re-epithelialization, and thus promoting the healing of diabetic chronic wounds.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] The first aspect of the present invention provides a method for preparing a pharmaceutical preparation for promoting healing of diabetic chronic wounds, comprising the following steps:

[0008] 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 the first mixed solution and the second mixed solution are mixed and reacted to obtain a pharmaceutical preparation for promoting the healing of chronic wounds in diabetes.

[0009] Preferably, the Se-CQDs are prepared by the following method:

[0010] Alkali solution is added to the L-selenocystine dispersion to adjust the pH, and the mixture is heated for reaction. After the reaction is completed, the mixture is centrifuged, the supernatant is collected, the supernatant is dialyzed, the dialyzate is collected, and dried to obtain Se-CQDs.

[0011] Furthermore, the L-selenocystine dispersion is prepared by mixing L-selenocystine and water in a material-liquid ratio of (150-250) mg:10 mL.

[0012] Furthermore, the alkali solution is a NaOH solution, and the concentration of the NaOH solution is 0.5M.

[0013] Furthermore, the pH is adjusted to 8.5-9.5.

[0014] Furthermore, the heating temperature is 55-65° C. and the reaction time is 20-30 h.

[0015] Furthermore, the molecular weight cut-off of the dialysis bag used in the dialysis treatment process is 500-1000.

[0016] 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.

[0017] Preferably, the material-liquid ratio of Se-CQDs and thrombin solution is 10 μg: (15-25) μL.

[0018] Preferably, the material-to-liquid ratio of GOX and fibrinogen solution is 10 μg: (15-25) μL.

[0019] Preferably, the volume ratio of the first mixed liquid to the second mixed liquid is (0.8-1.2):1.

[0020] Preferably, a double-barreled syringe is used to mix the first mixed liquid and the second mixed liquid.

[0021] The second aspect of the present invention provides a pharmaceutical preparation for promoting the healing of diabetic chronic wounds prepared by the above preparation method.

[0022] The third aspect of the present invention provides the use of the above-mentioned pharmaceutical preparation for promoting healing of diabetic chronic wounds in any one of the following 1) to 3):

[0023] 1) Promote the healing of chronic diabetic wounds;

[0024] 2) Promote collagen deposition, angiogenesis and skin tissue re-epithelialization;

[0025] 3) Clear excess ROS in chronic wounds, reduce pH, glucose content and M1 / M2 macrophage ratio in chronic wounds.

[0026] Beneficial effects of the present invention:

[0027] The present invention utilizes a mixture of 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 excess ROS at diabetic chronic wounds, reduce the pH, glucose content, and M1 / M2 macrophage ratio at the chronic wounds, so as to achieve reversal of the high sugar-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.

[0028] The present invention uses a combination of Se-CQDs and GOX to prepare a pharmaceutical preparation that has a synergistic effect in promoting the healing of chronic diabetic wounds. Specifically, after 8 days of treatment with the pharmaceutical preparation prepared by the present invention, the residual wound area rate was only 24.6%. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 : TEM characterization diagram, zeta potential diagram, XRD characterization diagram and FT-IR characterization diagram of Se-CQDs prepared in Example 1; wherein A is a TEM characterization diagram, B is a zeta potential diagram, C is an XRD characterization diagram, and D is an FT-IR characterization diagram;

[0030] Figure 2 : XPS spectrum of Se-CQDs prepared in Example 1, high-resolution spectra of C 1s, N 1s, and Se 3d; wherein A is the XPS spectrum, B is the high-resolution spectrum of C 1s, C is the high-resolution spectrum of N 1s, and D is the high-resolution spectrum of Se 3d;

[0031] Figure 3 : UV-visible spectrum, fluorescence spectrum and excitation light-dependent fluorescence spectrum of Se-CQDs prepared in Example 1; wherein A is the UV-visible spectrum, B is the fluorescence spectrum, and C is the excitation light-dependent fluorescence spectrum;

[0032] Figure 4 : Preparation flow chart, SEM characterization diagram, and rheological behavior diagrams of the second mixed solution and the pharmaceutical preparation prepared in Example 1; wherein 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 mixed solution, 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;

[0033] Figure 5 : Schematic diagram of wounds, residual wound area ratio, and wound healing traces corresponding to different treatment times for mice in groups G1-G5; A is the schematic diagram of wounds, B is the residual wound area ratio, and C is the wound healing trace;

[0034] Figure 6 : H&E, Masson's trichrome, and Sirius red-stained sections of the wound surfaces of mice in groups G1 to G5 14 days after treatment; in the figures, the purple scale bar is 200 μm, and the black scale bar is 50 μm;

[0035] Figure 7 : Immunofluorescence staining of CK14 and CK10 in the wounds of mice in groups G1 to G5 after 14 days of treatment; the scale bar is 50 μm;

[0036] Figure 8 : Immunofluorescence staining of Ki67, CD31 (red) + α-SMA (green), and CD86 (red) + CD206 (green) on the wound surfaces of mice in groups G1 to G5 after 14 days of treatment; the scale bar is 50 μm.

[0037] Figure 9 : Immunohistochemical staining of IL-1β, IL-6, and TNF-α in the wound surfaces of mice in groups G1 to G5 after 14 days of treatment; scale bar is 50 μm;

[0038] Figure 10 : H&E staining of the main organs (heart, liver, spleen, lung, and kidney) of mice in groups G1 to G5; the scale bar is 50 μm.

[0039] Figure 11 : Whole blood routine and serum biochemistry data graphs of mice in groups G1-G5; among them, A is the whole blood routine index concentration graph, B is the serum ALT concentration graph, C is the serum AST concentration graph, D is the serum ALB concentration graph, E is the ALP concentration graph, F is the CREA concentration graph, and G is the UREA concentration graph. DETAILED DESCRIPTION

[0040] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present application belongs.

[0041] 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 with reference to specific embodiments.

[0042] The experimental materials used in the examples of the present invention are all conventional experimental materials in the art and can be purchased through commercial channels.

[0043] In the present invention, L-selenocystine (98%) was purchased from Shanghai MacLean Biochemical Technology Co., Ltd., glucose oxidase (GOX) was purchased from Sigma-Aldrich, USA, thrombin was purchased from Shanghai Yuanye Biotechnology Co., Ltd., and fibrinogen was purchased from Beijing Solaibao Technology Co., Ltd. C57BL / 6 mice and diabetic (db / db) mice were purchased from Hunan Slake SJA Animal Co., Ltd.

[0044] Example 1: Preparation of a pharmaceutical preparation for promoting healing of chronic diabetic wounds

[0045] (1) L-selenocystine and ultrapure water were mixed at a material-liquid ratio of 200 mg:10 mL to obtain an L-selenocystine dispersion; 0.5 M NaOH solution was added to the L-selenocystine dispersion to adjust the pH to 9.0, and then heated to 60 ° C for reaction for 24 h. After the reaction was completed, the reaction product was placed at 12000 rpm for centrifugation, and the supernatant after centrifugation was collected and dialyzed using a dialysis bag with a molecular weight cutoff of 500-1000. The dialyzate was collected and freeze-dried to obtain a brown-red powder, which is Se-CQDs;

[0046] (2) Mixing fibrinogen and saline to prepare a fibrinogen solution with a concentration of 20 mg / mL; mixing thrombin and saline to prepare a thrombin solution with a concentration of 50 U / mL;

[0047] Se-CQDs are added to a thrombin solution to obtain a first mixed solution, and GOX is added to a fibrinogen solution to obtain a second mixed solution. The first mixed solution and the second mixed solution are mixed and injected using a double-barreled syringe in a volume ratio of 1:1 to obtain a pharmaceutical preparation for promoting the healing of chronic wounds in diabetes.

[0048] Example 2: Preparation of a pharmaceutical preparation for promoting healing of chronic diabetic wounds

[0049] (1) L-selenocystine and ultrapure water were mixed at a material-liquid ratio of 150 mg:10 mL to obtain an L-selenocystine dispersion; 0.5 M NaOH solution was added to the L-selenocystine dispersion to adjust the pH to 8.5, and then heated to 55 ° C for 30 h. After the reaction was completed, the reaction product was centrifuged at 12000 rpm, the supernatant after centrifugation was collected and dialyzed using a dialysis bag with a molecular weight cutoff of 500-1000, and the dialyzate was collected and freeze-dried to obtain a brown-red powder, namely Se-CQDs;

[0050] (2) Mixing fibrinogen and saline to prepare a fibrinogen solution with a concentration of 15 mg / mL; mixing thrombin and saline to prepare a thrombin solution with a concentration of 45 U / mL;

[0051] Se-CQDs are added to a thrombin solution to obtain a first mixed solution, and GOX is added to a fibrinogen solution to obtain a second mixed solution. The first mixed solution and the second mixed solution are mixed and injected using a double-barreled syringe at a volume ratio of 0.8:1 to obtain a pharmaceutical preparation for promoting the healing of chronic wounds in diabetes.

[0052] Example 3: Preparation of a pharmaceutical preparation for promoting healing of chronic diabetic wounds

[0053] (1) L-selenocystine and ultrapure water were mixed at a material-liquid ratio of 250 mg:10 mL to obtain an L-selenocystine dispersion; 0.5 M NaOH solution was added to the L-selenocystine dispersion to adjust the pH to 10.0, and then heated to 65 ° C for 20 h. After the reaction was completed, the reaction product was centrifuged at 12000 rpm, the supernatant after centrifugation was collected and dialyzed using a dialysis bag with a molecular weight cutoff of 500-1000, and the dialyzate was collected and freeze-dried to obtain a brown-red powder, namely Se-CQDs;

[0054] (2) Mixing fibrinogen and saline to prepare a fibrinogen solution with a concentration of 25 mg / mL; mixing thrombin and saline to prepare a thrombin solution with a concentration of 55 U / mL;

[0055] Se-CQDs are added to a thrombin solution to obtain a first mixed solution, and GOX is added to a fibrinogen solution to obtain a second mixed solution. The first mixed solution and the second mixed solution are mixed and injected using a double-barreled syringe at a volume ratio of 1.2:1 to obtain a pharmaceutical preparation for promoting the healing of chronic wounds in diabetes.

[0056] Comparative Example 1:

[0057] The difference between this comparative example and Example 1 is that GOX and Se-CQDs are not used in the preparation of the pharmaceutical preparation for promoting the healing of chronic diabetic wounds. The specific preparation method is as follows:

[0058] A fibrinogen solution with a concentration of 20 mg / mL and a thrombin solution with a concentration of 50 U / mL were prepared according to the method in Example 1; the fibrinogen solution and the thrombin solution were mixed and injected at a volume ratio of 1:1 using a double-barreled syringe to obtain a pharmaceutical preparation for promoting the healing of chronic diabetic wounds.

[0059] Comparative Example 2:

[0060] The difference between this comparative example and Example 1 is that GOX is not used in the preparation of the pharmaceutical preparation for promoting the healing of chronic diabetic wounds. The specific preparation method is as follows:

[0061] A fibrinogen solution with a concentration of 20 mg / mL, a thrombin solution with a concentration of 50 U / mL, and Se-CQDs were prepared according to the method of Example 1. Se-CQDs were added to the thrombin solution to obtain a mixed solution. The mixed solution and the thrombin solution were mixed and injected at a volume ratio of 1:1 using a double-barreled syringe to obtain a pharmaceutical preparation for promoting the healing of chronic wounds in diabetes.

[0062] Comparative Example 3:

[0063] The difference between this comparative example and Example 1 is that Se-CQDs are not used in the preparation of the pharmaceutical preparation for promoting the healing of chronic diabetic wounds. The specific preparation method is as follows:

[0064] A fibrinogen solution with a concentration of 20 mg / mL and a thrombin solution with a concentration of 50 U / mL were prepared according to the method in Example 1; GOX was added to the fibrinogen solution to obtain a mixed solution; and the fibrinogen solution and the mixed solution were mixed and injected using a double-barreled syringe at a volume ratio of 1:1 to obtain a pharmaceutical preparation for promoting the healing of chronic wounds in diabetes.

[0065] Experimental Example 1: Structural Characterization

[0066] 1. The Se-CQDs prepared in step (1) of Example 1 were characterized. Figure 1-Figure 3 shown.

[0067] Depend on Figure 1 As can be seen from Figure A, the diameter of Se-CQDs prepared by the present invention is 14.03±4.48 nm. Figure 1 As can be seen from Figure B, due to the presence of carboxyl and hydroxyl groups on the surface of Se-CQDs, its surface potential is negative, which is -21.83 ± 1.94 mV. Figure 1 As can be seen in C, there is a broad diffraction peak at about 22°, indicating that this is an amorphous structure, which matches that of graphite. Figure 1 As can be seen from the D, 3600-3100 cm -1 The characteristic broadband between NH and OH corresponds to the stretching vibration at 1692 cm -1 and 1394 cm -1 The two peaks at 1200-900 cm correspond to the stretching vibrations of the C=O and C=C bonds in the aromatic domain. -1 The peaks between the regions are mainly related to the stretching vibrations of CO, CN and C-Se.

[0068] Depend on Figure 2 As can be seen from Figure A, the Se-CQDs prepared by the present invention are mainly composed of four elements: C, O, N and Se. Figure 2 As can be seen in Figure B, the peak at 284.5 eV in the deconvolution XPS spectrum of C 1s is CC, the peak at 286.0 eV indicates the presence of C-Se, CN and CO, and the peak at 288.0 eV is attributed to C=O. Figure 2 As can be seen from C, the presence of pyridinic nitrogen and pyrrolic nitrogen was observed at 398.9 eV and 400.9 eV in the deconvolution XPS spectrum of N 1s. Figure 2As can be seen in Figure 5D, the 55.3 eV in the Se 3d deconvolution XPS spectrum reveals the existence of C-Se-C in Se-CQDs.

[0069] Depend on Figure 3 As can be seen in Figure A, the aqueous solution of Se-CQDs has a typical absorption peak at about 280 nm, which comes from the aromatic sp 2 The core domain π-π* transition, and a typical absorption peak at about 340 nm is due to the n-π* (-COOH, CN / or C-Se) transition on the surface of Se-CQDs. Figure 3 As can be seen in Figure B, Se-CQDs have a maximum emission peak at about 490 nm. Figure 3 As can be seen in Figure C, Se-CQDs also show obvious excitation light-dependent (410-510 nm) fluorescence properties, which is caused by the optical selective absorption of Se-CQDs defect sites.

[0070] 2. Electron microscopic analysis was performed on the pharmaceutical preparation for promoting the healing of diabetic chronic wounds prepared in Example 1. At the same time, rheological analysis was performed on the second mixed solution and the pharmaceutical preparation in Example 1. The results were as follows: Figure 4 shown.

[0071] Depend on Figure 4 As can be seen in Figure A, after mixing equal volumes of the first mixed solution made of Se-CQDs and thrombin solution and the second mixed solution made of GOX and fibrinogen solution, the prepared pharmaceutical preparation for promoting the healing of chronic diabetic wounds is a hydrogel. Figure 4 As can be seen from B, the hydrogel prepared by the present invention has a porous network structure. The first mixed solution is a thrombin solution containing Se-CQDs, and the second mixed solution is a fibrinogen solution containing GOX. Figure 4 As can be seen from Figures C, D, and E, compared with the fibrinogen solution containing GOX, the elastic modulus (G') of the product increased rapidly after the addition of the thrombin solution containing Se-CQDs, and eventually exceeded the viscous modulus (G''), indicating the successful preparation of the hydrogel.

[0072] Test Example 2: Animal Experiment

[0073] Diabetic (db / db) mice were selected as experimental subjects for the experiment. The specific steps are as follows:

[0074] (1) Twenty-five mice were anesthetized with isoflurane, and their back hair was removed and disinfected. A 6-mm diameter biopsy punch was used to create a circular wound on the back of the db / db mice. Micro scissors were then used to create a full-thickness skin exfoliation wound according to the contour.

[0075] (2) The mice were randomly divided into 5 groups, and each treatment group was treated with a different drug preparation to fill the wound site. The treatment groups are as follows:

[0076] Group G1: Control group, no treatment was performed on the wound surface of mice;

[0077] Group G2: The wound surface was filled with the pharmaceutical preparation prepared in Comparative Example 1;

[0078] Group G3: The wound surface was filled with the pharmaceutical preparation prepared in Comparative Example 2;

[0079] Group G4: The wound surface was filled with the pharmaceutical preparation prepared in Comparative Example 3;

[0080] Group G5: The wound surface was filled with the pharmaceutical preparation prepared in Example 1.

[0081] In each treatment group, 40 μL of the drug formulation was used to fill the wound surface. After filling, the wound surface was covered with commercially available Tegaderm 3M film. Each mouse was housed individually and fed ad libitum to ensure consistent feeding management across all groups during the experiment.

[0082] 1. Healing of chronic wounds in diabetes

[0083] The mice in each treatment group were photographed 0, 4, 8, and 14 days after wound treatment. The wound size was analyzed using ImageJ software, and the wound area ratio was calculated. The results are shown in Figure 2. Figure 5 As shown. Among them, the calculation formula of wound area rate is:

[0084] Wound residual area rate (%) = [actual wound area on day n / original wound area] × 100%;

[0085] Where n represents the 0th, 4th and 8th days after wound treatment.

[0086] Depend on Figure 5 As shown in Figures A, B, and C, after 14 days of treatment with the pharmaceutical preparation filling the wound surface in groups G2-G4, a certain area of ​​the wound surface remained unhealed. However, after 14 days of treatment with the pharmaceutical preparation prepared in the present invention (group G5), the wound surface was almost completely healed. This shows that the pharmaceutical preparation prepared in the present invention using GOX and Se-CQDs has an excellent effect in promoting the healing of chronic diabetic wounds.

[0087] In addition, by Figure 5As shown in Figure B, after 8 days of treatment with the pharmaceutical preparation prepared in Comparative Example 1, Group G2 had a residual wound area rate of 44.4%; after 8 days of treatment with the pharmaceutical preparation prepared in Comparative Example 2, Group G3 had a residual wound area rate of 37.3%; and after 8 days of treatment with the pharmaceutical preparation prepared in Comparative Example 3, Group G4 had a residual wound area rate of 41.2%. However, after 8 days of treatment with the pharmaceutical preparation prepared in the present invention (Group G5), the residual wound area rate was only 24.6%. This demonstrates that the pharmaceutical preparation prepared using a combination of GOX and Se-CQDs has a synergistic effect in healing chronic diabetic wounds.

[0088] 2. Collagen deposition, angiogenesis, skin tissue re-epithelialization and reversal of the inflammatory microenvironment in chronic wounds

[0089] (1) One mouse was selected from each treatment group after 14 days of treatment. The wound skin tissue of the mouse was collected after being killed and soaked in 4% paraformaldehyde for 24 hours. After being embedded in wax blocks, 5 μm thick sections were made for histological staining.

[0090] (2) Hematoxylin and eosin (H&E) and Masson trichrome staining were used to detect the formation of granulation tissue and collagen during wound healing. Sirius red staining was used to evaluate the collagen I / III ratio during wound healing. Figure 6 shown.

[0091] (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 shown in Figure 3. Figure 7 and Figure 8 shown.

[0092] (4) Immunohistochemical staining of IL-6, IL-1β and TNF-α was used to detect the expression of proinflammatory cytokines during wound healing. Figure 9 shown.

[0093] Figure 6 The blue and red arrows in the figure point to blood vessels and hair follicles, respectively. Figure 6As can be seen after hematoxylin and eosin (H&E) staining, the skin tissue in groups G1 and G2 still showed signs of rupture and little healing. Compared to groups G3 and G4, the pharmaceutical preparation prepared by the present invention significantly improved wound healing in diabetic mice, with the formation of numerous new blood vessels and hair follicles. Furthermore, the collagen deposition (blue area as determined by Masson staining) and the ratio of mature type I collagen (orange-yellow or red refraction from Sirius red staining) to early type III collagen (green refraction from Sirius red staining) in wound tissue treated with the pharmaceutical preparation of the present invention were significantly higher than those in the other groups. This demonstrates that the pharmaceutical preparation prepared by the present invention effectively promotes skin remodeling and collagen deposition in wounds.

[0094] CK10 and CK14 are used as markers of spinous keratinocytes and basal keratinocytes to observe the epithelialization of skin wound tissue. Figure 7 As can be seen, re-epithelialization of the skin tissue in groups G3, G4, and G5 was complete, with clear epithelial stratification. However, the keratinocyte layer was absent in groups G1 and G2. This result is consistent with the H&E staining results.

[0095] Depend on Figure 8 It can be seen that the amount of Ki67-positive cells in the wound skin after treatment with the pharmaceutical preparation prepared by the present invention is the highest. The fluorescent staining results of CD31 and α-SMA also show that the density of regenerated blood vessels in the wound tissue treated with the pharmaceutical preparation prepared by the present invention is the highest. At the same time, the skin tissue was immunostained by CD86 to mark M1 inflammatory macrophages and CD206 to mark M2 anti-inflammatory macrophages. After treatment with the pharmaceutical preparations prepared by the G3 group and the G4 group, the number of M2 anti-inflammatory cells was slightly increased compared to the G1 group; after treatment with the pharmaceutical preparation prepared by the present invention, the number of M2 anti-inflammatory cells was much higher than that of the other treatment groups. It can be seen that the present invention uses Se-CQDs and GOX to prepare a pharmaceutical preparation in combination, which can not only remove excess ROS at the wound, but also reduce the pH and glucose content at the chronic wound, so as to reverse the high sugar-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.

[0096] Depend on Figure 9 It can be seen that the levels of pro-inflammatory factors IL-1β, IL-6, and TNF-α in wound tissue treated with the pharmaceutical preparation of the present invention were significantly lower than those in the other groups. This shows that the pharmaceutical preparation of the present invention can inhibit the secretion of pro-inflammatory factors and has a positive anti-inflammatory effect.

[0097] 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, and reverse the alkaline high-sugar inflammatory environment of the wound into a stable anti-inflammatory environment, thereby accelerating the healing process of diabetic wounds.

[0098] 3. Toxicity and biosafety

[0099] (1) Each mouse in each treatment group was killed after 14 days of treatment, and the main organs (heart, liver, spleen, lung, and kidney) of the mice were collected for histological analysis by H&E staining. The results are as follows: Figure 10 shown.

[0100] (2) Select healthy C57BL / 6 mice and subcutaneously inject PBS, the pharmaceutical preparations prepared in Comparative Examples 1-3, and the pharmaceutical preparation prepared in Example 1, respectively, with the injection volume being 40 μL. Each group of mice was housed individually, and the feeding management of each group of mice was ensured to be consistent;

[0101] After 14 days of feeding, whole blood was collected from mice in each group, and the toxicity of the hydrogel in the wound healing study 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). The results are shown in Figure 4. Figure 11 shown.

[0102] Depend on Figure 10 It can be seen that compared with group G1, there were no significant histological differences in the major organs of groups G2-G5. This shows that the pharmaceutical preparation prepared by the present invention has high biosafety. Figure 11 It can be seen that there is no significant difference in the various indicators of the mice in groups G1 to G5. This shows that the pharmaceutical preparation prepared by the present invention has no systemic toxicity.

[0103] In summary, the pharmaceutical preparation prepared by the present invention has no systemic toxicity and good biosafety.

[0104] The above description is merely a preferred embodiment of the present application and is not intended to limit the present application. Various modifications and variations are possible for those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present application shall be included within the scope of protection 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 and thrombin solution are mixed at a material-liquid ratio of 10 μg: (15-25) μL to obtain a first mixed solution; GOX and fibrinogen solution are mixed at a material-liquid ratio of 10 μg: (15-25) μL to obtain a second mixed solution; the first mixed solution and the second mixed solution are mixed at a volume ratio of (0.8-1.2): 1 to obtain a pharmaceutical preparation for promoting chronic wound healing in diabetes; 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.

2. The method for preparing the pharmaceutical preparation for promoting healing of diabetic chronic wounds according to claim 1, wherein: The Se-CQDs were prepared by the following method: L-selenocystine and water are mixed in a material-liquid ratio of (150-250) mg:10 mL to obtain an L-selenocystine dispersion; 0.5 M NaOH solution is added to the L-selenocystine dispersion to adjust the pH to 8.5-9.5, and the mixture is reacted at 55-65°C for 20-30 hours. After the reaction is completed, the mixture is centrifuged, the supernatant is collected, the supernatant is dialyzed, the dialyzate is collected, and dried to obtain Se-CQDs.

3. A pharmaceutical preparation for promoting the healing of diabetic chronic wounds prepared by the preparation method according to claim 1 or 2.

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