Application of bifunctional bivalent platinum complex in medicament for promoting healing of diabetic wound

Abstract

The invention discloses an application of a bifunctional bivalent platinum complex in a medicine for promoting healing of a diabetic wound, and the bifunctional bivalent platinum complex is proved to have excellent biological safety on mouse skin layer fibrocytes (L929) in an in-vitro high-glucose environment, and can remarkably promote cell migration and enhance the wound repair capability. Meanwhile, the bifunctional bivalent platinum complex can effectively accelerate the wound healing process of a diabetic mouse, relieve diabetic wound inflammatory response, promote mouse skin angiogenesis and remarkably shorten the chronic wound healing period. Based on the multiple action mechanisms, the preparation is suitable for treating various complex wounds, including but not limited to diabetes related ulcers, refractory wounds, surgical incisions, obstetrical injuries and the like, a novel efficient solution is provided for treating chronic refractory wounds, and the preparation has wide clinical application prospects.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to the application of a bifunctionalized divalent platinum complex in a drug for promoting diabetic wound healing. Background Art

[0002] Diabetes, a global chronic disease, has been designated by the United Nations as one of the four major non-communicable diseases. Studies have shown that over half of diabetic wounds develop into chronic, difficult-to-heal wounds. The inability of diabetic patients to heal increases their risk of morbidity, deep infection, ulceration, non-traumatic amputation, and even early death. Studies have shown that approximately 50% of diabetic patients develop diabetic foot as a result of trauma, with amputation rates significantly increasing following infection. At least 68% of diabetic foot patients will die within five years. The causes of delayed wound healing in diabetic patients are not fully understood, but are generally believed to be related to high glucose levels, a diverse range of bacterial infections, chronic inflammation, impaired angiogenesis, and limited therapeutic options. Current clinical treatments for diabetic wounds primarily rely on antibiotics, debridement, vacuum drainage, and traditional dressings. However, these treatments are associated with antibiotic resistance, complex procedures, cumbersome procedures, limited efficacy, and limited clinical effectiveness.

[0003] Related studies have shown that platinum-derived materials have been shown to have promising therapeutic capabilities in chronic wound management due to their unique biophysical properties and catalytic functions. However, there are still major gaps in their clinical translation and effectiveness. Bifunctional divalent platinum complexes (cis-BBP) are organic photosensitizers with fluoroboron dipyrrole as the parent and divalent platinum as the ligand, and have been shown to have good antibacterial effects. However, there are currently no reports on bifunctional divalent platinum complexes promoting the healing of chronic infectious wounds such as diabetic wounds. Therefore, the use of bifunctional divalent platinum complexes (cis-BBP) in drugs to promote diabetic wound healing has broad clinical application prospects. Summary of the Invention

[0004] The purpose of the present invention is to overcome the defects of the prior art and provide a bifunctionalized divalent platinum complex for use in a drug for promoting diabetic wound healing. The bifunctionalized divalent platinum complex can significantly promote cell migration and enhance wound repair ability.

[0005] The object of the present invention is achieved by: using a bifunctionalized divalent platinum complex in a drug that promotes diabetic wound healing, wherein the bifunctionalized divalent platinum complex is an organic photosensitizer cis-BBP with fluoroboron dipyrrole as a matrix and divalent platinum as a ligand, and using the organic photosensitizer cis-BBP in a drug that promotes diabetic wound healing; the molecular structure of the cis-BBP is as follows:

[0006]

[0007] Furthermore, the diabetes is at least one of type 1 diabetes, type 2 diabetes, gestational diabetes, special diabetes and diabetic complications.

[0008] Furthermore, the diabetic wounds include wounds generated on the skin during diabetic drug administration and wounds caused by diabetes-related complications.

[0009] Furthermore, the wounds caused by the diabetes-related complications include ulcers, erosions, gangrene or diabetic foot.

[0010] Furthermore, the method promotes diabetic wound healing by promoting cell migration.

[0011] The present invention adopts the above technical solution, and has the following beneficial effects compared with the prior art:

[0012] The present invention discloses the use of a bifunctionalized divalent platinum complex in a drug that promotes diabetic wound healing; the complex has been confirmed by in vitro experiments to have excellent biosafety for mouse skin fibroblasts L929 under high sugar conditions, and can significantly promote cell migration and enhance wound repair ability. Animal experiments further show that the bifunctionalized divalent platinum complex can effectively accelerate the wound healing process of diabetic mice, significantly shorten the diabetic wound healing cycle, improve the inflammatory response of mice, and promote angiogenesis in mouse skin. Based on its multiple mechanisms of action, the complex is suitable for the treatment of a variety of complex wounds, including but not limited to diabetes-related ulcers, refractory wounds, surgical incisions and obstetric injuries, and has important clinical value for wound healing disorders in diabetic patients. The present invention provides a new and efficient solution for the treatment of chronic refractory wounds, and has broad clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 The figure shows the experimental results of cis-BBP's cytotoxicity in high-glucose environment and mouse skin fibroblast L929.

[0014] Figure 2 This is a diagram showing the effect of cis-BBP on high glucose environment and migration of mouse skin fibroblasts L929.

[0015] Figure 3 The statistical results of the effects of cis-BBP on high glucose environment and the migration of mouse skin fibroblasts L929.

[0016] Figure 4 These are actual photos of the wound healing status of mice in the WT group, T2DM group, T2DM-2μg / ml cis-BBP group, and T2DM-4μg / ml cis-BBP group at different times after cis-BBP administration.

[0017] Figure 5These are the statistical results of the wounds of mice in the WT group, T2DM group, T2DM-2μg / ml cis-BBP group, and T2DM-4μg / ml cis-BBP group at different times after cis-BBP administration.

[0018] Figure 6 These are the immunofluorescence results of CD68 and CD163 in the wound skin tissues of mice in the WT group, T2DM group, T2DM-2μg / ml cis-BBP group, and T2DM-4μg / ml cis-BBP group at different times after cis-BBP administration.

[0019] Figure 7 These are the results of VEGF immunofluorescence in the wound skin tissues of mice in the WT group, T2DM group, T2DM-2μg / ml cis-BBP group, and T2DM-4μg / ml cis-BBP group at different times after cis-BBP administration. DETAILED DESCRIPTION

[0020] The present invention will be further described below with reference to specific embodiments.

[0021] Example 1: Effect of cis-BBP on the Growth Toxicity of L929 Cells in a High Glucose Environment in Vitro

[0022] 1. Cells were cultured in a 37°C cell culture incubator containing 5% carbon dioxide. The culture medium was DMEM supplemented with 10% fetal bovine serum and 1% double-streptomycin. All operations in the experimental group were performed in a clean bench pre-sterilized with ultraviolet light. Cells in good growth condition were taken to prepare a cell suspension (5×10 3 The cell suspension was inoculated into a 96-well plate (100 μL / well) at a concentration of 10 cells / 100 μL, and the plate was pre-cultured in an incubator for 24 h.

[0023] 2. The experimental groups of this study are:

[0024] (1) Blank control group (Control group): L929 cells were incubated with 5.5 mM glucose medium for 24 h;

[0025] (2) Diabetes model group (Model group): L929 cells were incubated with 33.3 mM glucose medium for 24 h;

[0026] (3) 0.0250 μg / ml cis-BBP group: L929 cells were incubated with 33.3 mM glucose medium + 0.0250 μg / ml cis-BBP for 24 h;

[0027] (4) 0.050 μg / ml cis-BBP group: L929 cells were incubated with 33.3 mM glucose medium + 0.050 μg / ml cis-BBP for 24 h;

[0028] (5) 0.010 μg / ml cis-BBP group: L929 cells were incubated with 33.3 mM glucose medium + 0.010 μg / ml cis-BBP for 24 h.

[0029] 3. After observing that the cells have adhered well, aspirate the culture medium. For each group of 4 replicate wells, add 10 μL of cis-BBP at different concentrations of 0.025, 0.05, and 0.10 μg / ml to the plate and culture for 24 hours. The light source is 30 mW / cm 2 Irradiate with LED light for 15 minutes. A dark control group was subjected to a light-protected experiment. 10 μL of CCK8 solution was added to each well. The plate was incubated in an incubator for 4 hours. The absorbance at 450 nm was measured using a microplate reader.

[0030] 4. Calculation of experimental results: In this example, a blank control group was used as the culture medium without cells, a control group was used as the cell culture medium without cis-BBP solution, and experimental groups were used as the cells containing different concentrations of cis-BBP solution. The calculation formula for this example is as follows:

[0031] Cell survival rate = (OD value of experimental group - OD value of blank group) / (OD value of control group - OD value of blank group) × 100%.

[0032] 5. Result analysis: Figure 1 High glucose levels inhibited L929 cell viability. Within the concentration range of 0.025, 0.05, and 0.10 μg / mL, cis-BBP showed no significant dark toxicity to L929 cells within 24 hours. After 15 minutes of illumination, cell viability at different concentrations of cis-BBP was not statistically different from that of the model group (P>0.05). These results indicate that cis-BBP has good biosafety.

[0033] Example 2: Effect of cis-BBP on L929 cell migration in a high glucose environment in vitro

[0034] 1. The cells were cultured in a 37°C cell culture incubator containing 5% carbon dioxide. The culture medium was DMEM supplemented with 10% fetal bovine serum and 1% double-streptomycin. All operations were performed in a clean bench pre-sterilized with ultraviolet light. Cells in good growth condition were taken to prepare a cell suspension (5×10 5 The cell suspension was inoculated into 6 plates with 3 replicate wells for each group of cells, and the plates were pre-cultured in an incubator for 24 h.

[0035] 2. The experimental groups of this study are:

[0036] (1) Blank control group (Control group): L929 cells were incubated with 5.5 mM glucose medium for 24 h;

[0037] (2) Diabetes model group (Model group): L929 cells were incubated with 33.3 mM glucose medium for 24 h;

[0038] (3) 0.0250 μg / ml cis-BBP group: L929 cells were incubated with 33.3 mM glucose medium + 0.0250 μg / ml cis-BBP for 24 h;

[0039] (4) 0.050 μg / ml cis-BBP group: L929 cells were incubated with 33.3 mM glucose medium + 0.050 μg / ml cis-BBP for 24 h;

[0040] (5) 0.010 μg / ml cis-BBP group: L929 cells were incubated with 33.3 mM glucose medium + 0.010 μg / ml cis-BBP for 24 h.

[0041] 3. Use a marker to draw black horizontal lines on the bottom of the 6-well plate, keeping the spacing between the horizontal lines at 0.8 cm, and ensure that there are at least 3 horizontal lines passing through each well. When the 6-well plate is filled with fibroblasts, use a 200μL pipette tip to draw a straight line from one end of the well to the other end with the same force. Do not tilt the pipette tip during the marking process to ensure that it is perpendicular to the bottom plane of the six-well plate and the horizontal line on the back. After the scratching is completed, discard the old culture medium and wash the scratched cells with PBS. Serum-free culture medium was added as the negative control group, and serum-free culture medium containing different concentrations of cis-BBP was used as the drug stimulation group. Three replicates were set up for each group of cells. Cultured in a 37°C 5% CO2 incubator. Take photos of the scratches at different culture time periods (0 hours and 24 hours), and finally calculate the cell migration rate and migration area.

[0042] 4. Calculation of experimental results: Image J was used to compare the speed of L929 cells recovering from scratches at different concentrations of cis-BBP, expressed as cell migration rate. The calculation formula for this example is as follows:

[0043] Cell migration rate (%) = (0 h scratch area - 24 h scratch area) / 0 h scratch area × 100%.

[0044] 5. Result analysis: Figure 2 and Figure 3The results showed that the wound healing rates of the control, model, 0.025μg / ml cis-BBP, 0.05μg / ml cis-BBP, and 0.10μg / ml cis-BBP groups after 24 hours of intervention were 35.00%, 16.50%, 27.48%, 28.39%, and 29.96%, respectively. Compared with the model group, the wound healing rates of the control, 0.025μg / ml cis-BBP, 0.05μg / ml cis-BBP, and 0.10μg / ml cis-BBP groups were significantly increased (P < 0.05), indicating that cis-BBP can promote the migration of L929 cells in a high glucose environment.

[0045] Example 3: Study on the effect of cis-BBP on promoting wound healing in diabetic mice

[0046] 1. Animal Preparation: For this example, 40 healthy male C57BL / 6J mice were selected. They were housed in an environment with fresh air circulation and a suitable temperature. Prior to the experiment, the mice were acclimated in the animal room for two weeks, with free access to water and food. Each mouse was observed 24 hours prior to the experiment to eliminate any potential stressors.

[0047] 2. Construction of diabetic trauma model: There was no statistically significant difference in the average body weight between the mice in each group. The mice in the blank control group were fed with ordinary feed, and the mice in the experimental group were fed with high-fat feed. After 3 weeks of feeding, they were fasted but not watered for 12 hours. The high-fat-fed mice were intraperitoneally injected with 100 mg / kg streptozotocin to destroy the pancreatic β cells of the mice to construct a T2DM animal model. The mice in the blank control group were intraperitoneally injected with an equal volume of sodium citrate buffer. 72 hours after modeling, blood was collected from the tail vein and the fasting blood pressure was determined to be ≥7.8 mmol / L, indicating a successful model. If unsuccessful, the injection was repeated once. After the diabetic mouse model was successfully established, a diabetic trauma model was constructed. In order to ensure the smooth completion of the wound model, 24 hours before modeling, the hair on an appropriate area of the mouse's back was shaved with a shaver, and an appropriate amount of depilatory cream was evenly applied to the mouse's back. After three minutes, the depilatory cream was gently scraped off and wiped clean with a cotton ball. Select 1.5% sodium pentobarbital as the anesthetic, injecting the mouse intraperitoneally at a dose of 3.2 mL / kg. After injection, return the mouse to its cage and observe its condition. Modeling experiments can only be performed after it is fully anesthetized. Use sterile scissors and forceps to remove full-thickness skin tissue from the mouse's back, creating a circular full-thickness excision wound with a diameter of 6 mm ± 1 mm. After the mouse regains consciousness, return it to its cage for observation.

[0048] 3. Grouping and dosing: 40 13-week-old C57BL / 6J mice of similar weight were selected and grouped and dosed as follows:

[0049] Table 1 Grouping and drug administration

[0050]

[0051] The diabetic mouse trauma model was successfully established, and the drug solution was evenly applied to the wound site according to the group. The drug was given daily before the wound scabs formed. After the drug was given, the mice were placed under 30mW / cm 2 Illuminate the mice under LED light for 15 minutes. Different groups were identified by hanging labels on the outside of the cages, and mice in the same group were identified by marking with a marker pen.

[0052] 4. Observation indicators: Take photos of the wound with a mobile phone on days 1, 3, 5, 7, and 14, compare the wound healing status and record them, and calculate the relative wound area of the wound. The calculation formula is as follows:

[0053] Relative wound area = wound area on the day of measurement / wound area on day 0 × 100%.

[0054] 5. Result analysis: Figure 4 and Figure 5 The T2DM group showed the slowest wound healing. After administration for 1, 3, 5, 7, and 14 days, the wound areas of the WT, T2DM-2μg / ml cis-BBP, and T2DM-4μg / ml cis-BBP groups were significantly smaller than those of the T2DM group (P < 0.0001). Compared with the WT group, the wound areas of the T2DM-2μg / ml cis-BBP and T2DM-4μg / ml cis-BBP groups at the same time points of 1, 3, 5, 7, and 14 days were not statistically significant (P > 0.05). This suggests that wound healing in diabetic mice treated with cis-BBP is similar to that in normal mice, and that cis-BBP significantly promotes wound healing in diabetic mice.

[0055] Example 4: Tissue Immunofluorescence of CD68, CD163 and VEGF in Mouse Skin Wound Tissue Sections

[0056] 1. Place sections in environmentally friendly deparaffinization solutions I and II for 20 minutes each, followed by 5 minutes each in anhydrous ethanol I, anhydrous ethanol II, 95% ethanol, 85% ethanol, and 75% ethanol. Wash sections twice in ddH2O for 5 minutes each. For CD68 and CD163, heat sections in a 95°C water bath using sodium citrate buffer (0.01M, pH 6.0) for 5 minutes and allow to cool for 30 minutes. For VEGF, microwave-recover sections using EDTA buffer (pH 8.0) (medium heat for 8 minutes, then off heat for 8 minutes, then medium-low heat for 7 minutes) to prevent drying. After repair, wash sections three times with PBS (5 minutes each on a shaker).

[0057] 2. Using conventional immunofluorescence staining method:

[0058] (1) Circle sealing: After the slices are slightly dried, use a histochemical pen to draw a circle around the tissue. Add a small amount of goat serum to the circle of the histochemical pen until the tissue area is completely covered. Block at 37°C for 30 minutes.

[0059] (2) Primary antibody incubation: Add diluted primary antibody (CD68 / CD163 / VEGF) dropwise and incubate in the dark at 4°C for 8 h;

[0060] (3) Secondary antibody incubation: Remove the sections from the refrigerator and place them at room temperature for 15 minutes to warm up. Wash with PBS three times, 5 minutes each time. Add the secondary antibody working solution to cover the tissue in the dark and incubate at 37°C for 2 hours.

[0061] (4) Nuclear staining: Add 1 μg / mL LDAPI staining solution and incubate at room temperature in a dark box for 10 min.

[0062] (5) Sealing: Add anti-fluorescence quenching agent and seal the slide with a coverslip.

[0063] 3. Experimental results: Microscopic examination and image acquisition using a Pannoramic panoramic slide scanner.

[0064] 4. Result analysis: Figure 6 As shown, the pro-inflammatory biomarker (CD68) remained highly expressed in the T2DM group on days 7 and 14. CD68 expression was significantly lower in the WT, T2DM-2μg / ml cis-BBP, and T2DM-4μg / ml cis-BBP groups than in the T2DM group, indicating increased macrophage infiltration in the T2DM group and chronic inflammatory responses in the skin tissues of diabetic mice. Cis-BBP significantly alleviated the inflammatory response in diabetic mice. CD68 expression in the T2DM-2μg / ml cis-BBP and T2DM-4μg / ml cis-BBP groups approached that of the WT group on day 14, indicating that cis-BBP can reduce macrophage infiltration in diabetic mice, with the effect increasing over time. The anti-inflammatory biomarker (CD163) was most strongly expressed on day 7 of light exposure in the WT, T2DM-2μg / ml cis-BBP, and T2DM-4μg / ml cis-BBP groups. On day 14, the expression of CD163 in the WT group, T2DM-2μg / ml cis-BBP group, and T2DM-4μg / ml cis-BBP group was low, indicating the end of the inflammatory response, indicating that cis-BBP promoted the polarization of M2 macrophages and synergistically inhibited inflammation. Figure 7 It can be seen that blood vessels began to form in the WT group, T2DM-2μg / ml cis-BBP group and T2DM-4μg / ml cis-BBP group on the 7th day, and were characterized by VEGF expression on the 14th day, indicating that cis-BBP drives the establishment of new blood vessels.

[0065] The present invention provides the use of a bifunctional divalent platinum complex (cis-BBP) in promoting diabetic wound healing. Cis-BBP has good biosafety in a high-sugar environment, significantly promotes cell migration, reduces inflammatory responses, and promotes angiogenesis, among other multi-pathway features that promote diabetic wound healing. It systematically optimizes the toxic side effects, drug resistance, and single functional limitations of traditional platinum drugs. Its application in diabetic wounds not only expands the clinical scenarios of platinum compounds, but also provides a new strategy that combines high efficiency and safety for the treatment of chronic, refractory wounds.

[0066] The present invention is not limited to the above-mentioned embodiments. On the basis of the technical solutions disclosed in the present invention, those skilled in the art can make some substitutions and modifications to some of the technical features therein according to the disclosed technical content without creative labor, and these substitutions and modifications are all within the protection scope of the present invention.

Claims

1. A bifunctionalized divalent platinum complex for use in a diabetic wound healing drug, wherein the bifunctionalized divalent platinum complex is an organic photosensitizer cis-BBP with fluoroboron dipyrrole as a matrix and divalent platinum as a ligand, characterized in that: The application of the organic photosensitizer cis-BBP in a drug for promoting diabetic wound healing; the molecular structure of the cis-BBP is shown below:

2. The use according to claim 1, characterized in that The diabetes is at least one of type 1 diabetes, type 2 diabetes, gestational diabetes, special diabetes and diabetic complications.

3. The use according to claim 1, characterized in that The diabetic wounds include wounds caused by the skin during diabetic drug administration and wounds caused by diabetes-related complications.

4. The use according to claim 3, characterized in that The wounds caused by the diabetes-related complications include ulcers, erosions, gangrene or diabetic foot.

5. The use according to claim 1, characterized in that The method promotes diabetic wound healing by promoting cell migration.

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