Photosensitive spray gel and application thereof in preparation of medicine for treating diabetic wounds

Abstract

The invention belongs to the technical field of biological medicine, and particularly discloses MnO2 (at) COF loaded glucose oxidase photosensitive spray gel and application thereof in preparation of a treatment system for diabetic wounds, MnO2 nanosheets are coated with COF to obtain a MnO2 (at) COF compound, then glucose oxidase is connected with COF on the surface of the compound through covalent bonds, and the MnO2 (at) COF-GOx compound is obtained. And finally, the MnO2 (at) COF-GOx / Gel compound is added into sodium alginate gel which is polymerized under the promotion of a photosensitizer, and the MnO2 (at) COF-GOx / Gel compound is obtained. The traditional Chinese medicine composition can specifically aim at various pathophysiological mechanisms of diabetic wounds, improve microcirculation and play a good role in promoting regeneration of the wounds, so that the traditional Chinese medicine composition can be used for treating the diabetic wounds, especially diabetic skin ulcers.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to a photosensitive spray gel and application of the photosensitive spray gel in preparing a medicine for treating diabetic wounds. Background Art

[0002] According to the "Chinese Diabetic Foot Diagnosis and Treatment Clinical Pathway (2023 Edition)", diabetic foot (DF) and lower limb complications are a serious chronic disease. It affects about 15% of diabetic patients worldwide, of which 5% of diabetic patients are at high risk of DF and are experiencing the pain of DF. Diabetic foot ulcers and amputations are one of the main causes of significantly reduced quality of life and even premature death in patients. In China, the number of people with diabetes is currently huge, resulting in millions of people diagnosed with DF. The wounds of diabetic patients can be treated with nutritional support therapy, blood sugar control, wound debridement, anti-infection treatment, microcirculation improvement and other treatment measures. At present, the clinical treatment of diabetic skin ulcers is mainly prevention-oriented, including regular foot examinations, keeping feet clean, choosing appropriate shoes and socks, and cultivating healthy living habits. In clinical practice, hyperbaric oxygen therapy and surgical debridement are mainly used for treatment. However, these methods have problems such as inconsistent efficacy and poor controllability, and there is an urgent need to seek a safer and more efficient treatment method.

[0003] Researchers believe that the pathophysiological mechanism of chronic diabetic wounds is very complex, caused by many factors such as persistent high-sugar environment, aggregation of glycosylation products, cellular oxidative stress, vascular regeneration disorders, hypoxia, neuropathy, bacterial susceptibility, etc. These factors circulate back and forth, making diabetic wounds difficult to heal and circulate for a long time, causing wound rupture and easy recurrence. Among them, the persistent high-sugar state of diabetic wounds, the long transition period from inflammation to proliferation, and bacterial infection are key factors affecting wound healing. The persistent inflammatory response and limited tissue regeneration pose major challenges to treatment.

[0004] Nanotechnology and biochemistry have flourished in recent decades, including artificial materials with diverse applications. Certain nanomaterials have enzyme-like characteristics and substrate specificity and are often referred to as "nanozymes". Although the substrate specificity of nanozymes is rarely as high as that of natural enzymes, their multiple active sites are conducive to more efficient and stable catalytic activity. Manganese dioxide nanosheets, a new type of nanozyme, have been shown to catalyze endogenous ROS (hydrogen peroxide) and decompose it into O 2, which is a candidate material that is expected to alleviate oxidative stress and hypoxia. Covalent organic framework materials COFs are a class of crystalline porous materials formed by lightweight organic molecular units connected by all covalent bonds. It has the following characteristics: ① High crystallinity. The molecular structure of COFs materials is very regular and shows excellent crystallinity. This long-range ordered lattice structure makes COFs highly regular and predictable in structure. ② Porosity. COFs have rich pore structures and uniform pore sizes, providing an ideal platform for the adsorption and separation of molecules. ③ Structural tailorability and functional adjustability. COFs have very high structural diversity and designability. Their pore size and chemical function can be regulated by selecting different monomers and polymerization strategies. At the same time, by introducing functional groups on monomers or polymers, COFs can be given specific chemical and physical properties, such as pH response, photocatalysis, electrocatalysis, etc. ④ High thermal stability and chemical stability. Since the covalent bonds constituting COFs are relatively stable, this type of material is not easily attacked by chemical reagents or dissociated at high temperatures. This stability enables COFs to maintain stable structure and properties over a wide range of temperature and chemical environments.

[0005] Glucose oxidase (GOx) is an endogenous oxidoreductase widely distributed in organisms. In recent years, GOx has attracted increasing attention in the biomedical field due to its inherent biocompatibility, non-toxicity and unique catalytic effect on β-d-glucose. GOx can effectively catalyze the oxidation of glucose into gluconic acid and hydrogen peroxide (H 2 O 2 ), plays an important role in lowering glucose.

[0006] At the same time, in order to overcome the limitations of clinical diabetic wound dressings, a large number of studies are developing smart responsive hydrogels that respond to specific diabetic wound environments (such as low pH, high ROS, high glucose, and overexpressed enzymes) or external conditions (such as temperature, light, and magnetism). These smart responsive hydrogels can achieve precise and on-demand controlled drug release, avoid drug resistance caused by high-dose antibiotic use, and improve the harsh microenvironment of diabetic wound sites. Summary of the invention

[0007] The present invention develops a specific treatment measure for the complex immune microenvironment of diabetic wounds, namely, a MnO 2 @COF loaded with glucose oxidase (GOx) and photosensitive spray gel prepared therefrom. The present invention obtains MnO by a solvent thermal synthesis method. 2 @COF, and further loaded with GOx, and then added to the sodium alginate gel burst by the photoinitiator to obtain the gel spray. The present invention utilizes the dual response of the borate COF coating (response to ROS and glucose) to release MnO 2and GOx, which play multiple roles such as reducing blood sugar, reducing reactive oxygen species ROS, supplying oxygen and antibacterial, and are used to treat diabetic wounds, especially diabetic skin ulcers. Specifically, the present invention adopts the following technical solutions: First, the present invention provides a composite MnO 2 @COF-GOx, where MnO 2 The structure is nanosheet, MnO 2 @COF is borate COFs monomer and MnO 2 GOx was loaded on MnO by solvothermal reaction. 2 @COF.

[0008] Secondly, the present invention also provides the composite MnO 2 The preparation method of @COF-GOx is to combine boron-containing COFs monomers with MnO 2 MnO was synthesized by solvothermal reaction 2 @COF, then MnO 2 @COF, GOx mixed reaction. During the synthesis process, COF is synthesized on the surface of MnO2, that is, MnO2 is synthesized first, and then COF is loaded, such as Figure 2 As shown in the electron microscope image.

[0009] For the above-mentioned preparation method, preferably, the MnO 2 Obtained by the reaction of tetramethylammonium pentahydrate and manganese chloride.

[0010] For the preparation method described above, it is preferred that the borate COFs monomer is a boric acid monomer, including at least one of phenylboric acid and 1,4-phenylboronic acid. The monomer does not require the use of an acidic catalyst during the synthesis process and will not affect the MnO2 structure.

[0011] For the above-mentioned preparation method, preferably, the solvent thermal reaction is heated at 100-140° C. for 50-80 hours, preferably at 110-130° C. for 65-75 hours.

[0012] For the above-mentioned preparation method, preferably, MnO 2 @COF, GOx mixed reaction is to convert MnO 2 @COF, GOx and 1,4-dioxane (1,4-dioxane) were mixed in a round bottom flask, sonicated, and then assembled using a microfluidic syringe. The microfluidic syringe injected pure water into the flask, and a magnetic rotor was installed at the bottom of the flask to stir overnight. The sample was then freeze-dried to obtain the product. 2 @COF, GOx mass ratio 1-2:1-2.

[0013] For the above-mentioned preparation method, COF is coated on MnO 2 surface, forming a MnO with COF as coating 2 Composite material MnO 2 @COF, and then NH 3 GOx was loaded onto MnO via covalent bonding of BN bonds with borate bonds on the COF surface. 2 @COF, thus obtaining MnO 2 @COF-GOx.

[0014] Thirdly, the present invention provides a wound repair gel, which comprises the above-mentioned composite MnO 2 @COF-Gox and sodium alginate.

[0015] Fourthly, the present invention provides a photosensitive spray gel, which comprises the above-mentioned composite MnO 2 @COF-Gox, further comprises a photoinitiator (photosensitizer or photocrosslinker) and methacrylated sodium alginate AlgMA. The photoinitiator can be selected from LAP, L2959, and the like.

[0016] Fifthly, the present invention also provides a method for preparing the above-mentioned photosensitive spray gel, which comprises preparing the composite MnO 2 @COF-Gox, photoinitiator and methacrylated sodium alginate (AlgMA) are mixed and dissolved. When used, it can be sprayed onto the wound surface and irradiated with blue-violet light (405-425 nanometers) to form a gel in situ.

[0017] Sixth, the composite MnO 2 @COF-GOx, wound repair gel or photosensitive spray gel can be further used to prevent and / or treat diabetic wounds or chronic diabetic wounds. The diabetic wounds or chronic diabetic wounds include but are not limited to diabetic skin wounds or ulcers, diabetic foot wounds or ulcers, and the like.

[0018] When the photosensitive spray gel of the present invention is used, a photoinitiator and methacrylated sodium alginate are mixed to obtain a gel precursor, and the composite material MnO 2 @COF-Gox is mixed with the gel precursor and put into a spray bottle. After spraying it onto the wound surface, it is irradiated with blue-violet light (405-450 nanometers) to form a gel in situ.

[0019] For the MnO 2@COF-GOx hydrogel spray. The methacryloyl sodium alginate gel precursor is a commercially available product, which forms a uniform and stable liquid state after loading the drug. After illumination, the gel forms a three-dimensional mesh structure inside, with a uniform and dense network distribution, and has the function of adhering to and slowly releasing drugs; on the other hand, the structure of MnO2@COF is generally a few hundred nanometers after ultrasonic homogenization. In this way, the drug material can effectively reach and fill the wound surface before forming a gel, and the drug can continue to exert a therapeutic effect after forming a gel. At the same time, the design of this spray gel is more convenient to use and has better effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 MnO 2 @Schematic diagram of the preparation process of COF-GOx gel composite.

[0021] Figure 2 MnO 2 @SEM image of COF powder, showing a large number of circular structures (COF) attached to the surface of the block (aggregated MnO2 nanosheets).

[0022] Figure 3 The particle size distribution diagram is shown in the figure. 2 The particle size of @COF is 517 nm, and the particle size after loading GOx is 744 nm, confirming the loading of GOx.

[0023] Figure 4 The results of biocompatibility test of composite materials are shown in the figure. The left figure is the hemolysis experiment, and the right figure is the CCK-8 test of L929 cells.

[0024] Figure 5 This is a graph showing the changes in the wound surface over time after different gel complexes were applied to the wound surface of diabetic mice.

[0025] Figure 6 The effects of different material compositions on the treatment of skin wounds in diabetic mice (HE staining). DETAILED DESCRIPTION

[0026] The following embodiments are further descriptions of the content of the present invention as an explanation of the technical content of the present invention, but the essential content of the present invention is not limited to the following embodiments. Ordinary technicians in this field can and should know that any simple changes or replacements based on the essential spirit of the present invention should fall within the scope of protection required by the present invention.

[0027] Instruments and reagents: Transmission electron microscopy (TEM) and SAED images were obtained on a Talos L120C G2 electron microscope (ThermoFisher, America) at an accelerating voltage of 120 kV. Laser scanning confocal images were recorded on an LSM-800 (Zeiss Inc., Germany) microscope. Fluorescence spectra were recorded using a FluoroMax-4 fluorescence spectrophotometer (HORIBA Scientific, Japan) with an excitation and emission slit width of 5 nm. Cell experimental results were photographed using an inverted fluorescence microscope (Axio Observer 3, Zeiss, Germany). Sections were scanned using a slide scanner (PANNORAMIC MIDI). 1,4-Benzenediboric acid, 1,3,5-trimethylbenzene, 1,4-dioxane, tetramethylammonium pentahydrate, manganese chloride, hydrogen peroxide, and glucose oxidase were purchased from Shanghai MacLean Biochemical Technology Co., Ltd., and streptozotocin (STZ) was purchased from Shanghai Aladdin Reagent Co., Ltd. Photoinitiator and sodium alginate gel matrix were purchased from EFL.

[0028] The structure of the gel composite of the present invention is as follows Figure 1 As shown, the preparation method is generally as follows: 1) Preparation of MnO from tetramethylammonium pentahydrate and manganese chloride 2 Nanosheets; 2) Using the solvothermal synthesis method, 1,4-phenyldiboronic acid and MnO 2 Chemical synthesis to obtain MnO 2 @COF; 3) Use a microfluidic syringe to load glucose oxidase on MnO 2 @COF, we get MnO 2 @COF-GOx; 4) mixing the photoinitiator and the sodium alginate preparation to obtain a gel precursor; 5) Mix the above product with the hydrogel precursor to obtain the final MnO 2 @COF-GOx gel precursor, liquid is put into a spray bottle; 6) The MnO 2 The gel precursor of @COF-GOx is sprayed onto diabetic skin ulcers and then irradiated with 408-nanometer blue light to form a gel at the ulcer, thereby achieving the purpose of treating diabetic skin ulcers. Example 1

[0029] The preparation of relevant materials and gel is as follows: 0.755 g of manganese chloride was dissolved in 20 ml of deionized water by ultrasonication. At the same time, 4.4 g of tetramethylammonium pentahydrate (TMAOH·5H 2 O) was dissolved in 40 ml 3wt.% hydrogen peroxide solution. Then the TMAOH solution was quickly added to the manganese chloride solution, stirred, centrifuged and ultrasonicated, and then washed with ethanol and deionized water respectively to obtain MnO 2 Finally, the black product is dried by drying.

[0030] MnO 2 Preparation of @COF: 1,4-phenylenediboronic acid (BDBA) and the above-synthesized MnO were added to a heat-resistant glass tube. 2 (1,4-phenylenediboronic acid and MnO 2 The mass ratio is 2:5), and the volume ratio is 1:1 of 1,3,5-trimethylbenzene (0.5 mL): dioxane (0.5 ml). The glass tube was frozen at 77K (liquid nitrogen bath), the inside was evacuated and the glass tube was sealed with a flame. After the reaction mixture was heated at 120°C for 72 hours in a sealed state, an off-white solid was generated at the bottom of the tube. The solid was separated by filtration, washed with acetone (30 mL) 3 times, and vacuum dried for 24 hours to obtain the final product.

[0031] MnO 2 Preparation of @COF-GOx: MnO 2 @COF (30 mg), GOx (30 mg) and 2 ml of 1,4-dioxane (1,4-dioxane) were mixed in a round bottom flask and sonicated for 20 minutes. Then the assembly was completed with a microfluidic syringe. The microfluidic syringe injected 7 mL of pure water into the bottle, and after stirring overnight with a magnetic rotor at the bottom of the bottle, the sample was lyophilized to obtain the product.

[0032] Preparation of gel precursor: Add 10 ml PBS to a light-proof brown bottle containing photoinitiator LAP (0.025 g); heat in a 40-50°C water bath to dissolve for 15 minutes, stirring while dissolving. Take 500 mg of AlgMA and put it into the above brown bottle, stir and dissolve at room temperature for 0.5-1 h, and filter to obtain the gel precursor.

[0033] MnO 2 @Preparation of COF-GOx gel: Evenly mix the above freeze-dried product with the gel precursor and put it into a spray bottle for use. Example 2

[0034] 1) Electron microscopy characterization: Take samples and freeze-dried MnO 2@COF powder was directly adhered to the conductive glue for photography, and the sample was observed and photographed under a scanning electron microscope (ZEISS GeminiSEM 300). Figure 2 As shown, MnO 2 @COF structure. It proves that COF in MnO 2 The surface was able to be synthesized and the structure was intact, maintaining the old structure. At the same time, the diameter of the COF sphere was about 400nm.

[0035] 2) Particle size detection: MnO 2 @COF and freeze-dried MnO 2 @COF-GOx powder was dissolved in deionized water and tested in a nanoparticle size analyzer (Malvern Zetasizer Nano S90). The results are as follows Figure 3 As shown, the particle size of MnO2@COF after ultrasonic dispersion is 517nm, and the particle size after loading GOx is 744nm. Example 3

[0036] 1) Hemolysis test: Different concentrations (25, 50, 250, 500 μg mL-1) of MnO were prepared in PBS (pH = 7.4). 2 @COF-GOx gel. Collect whole blood from BALB / c mouse eyes and place in a sodium citrate anticoagulant tube. Centrifuge at 3000 rad for 15 minutes and take 20 μL of the lower layer of blood cells, and mix them with the following solutions: 1 mL deionized water (DW), 1 mL PBS, 25 μL MnO 2 @COF-GOx gel + 1mL PBS, 50μMnO 2 @COF-GOx gel + 1mL PBS, 75μMnO 2 @COF-GOx gel + 1 mL PBS, 100 μMnO 2 @COF-GOx gel + 1mL PBS. Incubate at 37°C for 4 hours and centrifuge at 3000rpm for 15 minutes. Transfer 100μL of the supernatant solution to a 96-well plate and use a multifunctional microplate reader to measure the absorbance of the sample at 542 nm. Hemolysis rate = (OD (sample group) - OD (PBS group)) / (OD (pure water group) - OD (PBS group)) × 100%. The results are as follows Figure 4 As shown, MnO 2 @The hemolysis rate of COF-GOx gel and the materials involved in its synthesis was very similar to that of the control group (PBS), and the hemolysis rate was below 3%, with almost no hemolysis phenomenon.

[0037] 2) CCK-8 experiment: Extraction of MnO with DMEM medium 2 @COF-GOx gel, the extract was obtained, and the extract was serially diluted with blank 100ul DMEM medium to obtain 24μL / 100μL, 12μL / 100μL, 8μL / 100μL, 4μL / 100μL, and 2μL / 100μL extracts. L929 cells (Wuhan Shanen Biotechnology) were inoculated in 96-well plates, intervened with different concentrations of extracts and cultured under light, and cell viability was detected according to the CCK-8 kit, as shown in Figure 4 The experimental results showed that compared with the control group (100%), the cell viability of L929 was around 90% when the drug concentration was below 8% (v / v), confirming that MnO 2 @COF-GOx gel does not affect the appreciation of L929. Example 4

[0038] In vivo experiments: Balb / C male mice were fed with a high-fat, high-sugar diet and maintained in a specific pathogen-free environment. STZ (100 mg / kg) was mixed with sodium citrate buffer and administered (intraperitoneally) for two consecutive days. Under normal conditions, mice with blood glucose levels ≥ 16.7 mM 2 weeks after the first injection of STZ were considered to have successfully established diabetes. A full-thickness skin wound model with a diameter of 5 mm was made on the back of the diabetic mice after successful modeling using a tissue biopsy and scissors. In order to evaluate the effect of each group of gel on wound repair, we mixed equal amounts of saline (control group), simple gel (Gel), MnO 2 @COF gel (MnO 2 @COF / Gel), GOx gel (GOx / Gel) and MnO 2 @COF-GOx gel (MnO 2 @COF-GOx / Gel) were applied to the wound surface of the corresponding groups of mice, and the gel dressing was changed every 3 days, and the wound area was recorded by taking pictures. Samples were taken on the 6th and 12th days for further tissue characterization. The remaining wound area was measured by ImageJ software, and the wound closure was calculated by the following equation.

[0039] Wound closure rate (%) = (A0-At) / A0×100, where A0 is the wound area on day 0, and At is the wound area at different time points, e.g. Figure 5 6 and 12 days after surgery, the mice were sacrificed, and the tissues at the wound site were collected and fixed with 4% paraformaldehyde for histological analysis and stained with hematoxylin and eosin (H&E). Figure 5 As shown. On the 6th and 12th days, MnO 2The wound healing rate of the COF-GOx / Gel group was significantly higher than that of the control group. Figure 6 , H&E staining results show (the upper and lower rows in the figure are the staining results on the 6th and 12th days respectively), MnO 2 @COF-GOx / Gel achieved almost complete re-epithelialization on the 12th day, and produced stable skin structures such as sebaceous glands and hair follicles (the surface in the lower right corner of the picture is concave because a more stable structure was produced, which caused the surface to be concave when the slices were made). The regeneration of skin appendages was significantly more than that of the other three groups, indicating that skin function was restored, while the control group showed delayed growth.

[0040] The MnO of the present invention 2 @COF-GOx hydrogel spray releases MnO through dual response of COF to reactive oxygen species and glucose 2 and GO X It plays a role in promoting the repair of diabetic wounds. Among them, borate covalent organic frameworks (COFs) are a classic organic nanopolymer material containing a large number of borate bonds. In recent years, their application range has been gradually expanded due to their unique biological properties. The borate bond is a dynamic covalent bond formed by the reaction of diol and boronic acid groups. The hydrogel based on the borate bond can not only realize the glucose-responsive release of drugs, but also remove excess hydrogen peroxide (H 2 O 2 ) and other oxygen free radicals, improve inflammatory cell infiltration, and promote diabetic wound healing. Manganese dioxide is a highly efficient catalyst that can accelerate the decomposition reaction of reactive oxygen species such as hydrogen peroxide (H2O2). In this process, manganese dioxide can reduce the activation energy of the reaction, allowing hydrogen peroxide to decompose into water and oxygen (O 2 ). This reaction not only reduces the concentration of harmful reactive oxygen, but also generates beneficial oxygen, which helps to improve the local hypoxic environment. Glucose oxidase GO X It is an enzyme that mainly catalyzes glucose metabolism in the human body. It can convert glucose in the blood into glycogen and further promote the synthesis and storage of liver glycogen. This process helps to maintain blood sugar within a normal range, thereby preventing hyperglycemia. The present invention can specifically target multiple pathological and physiological mechanisms of diabetic wounds at the same time, improve microcirculation, and play a good role in promoting wound regeneration.

[0041] It should be noted that the above-mentioned technical contents of the present invention are only for explanation and clarification to enable those skilled in the art to understand the technical essence of the present invention, so the technical contents are not used to limit the substantial protection scope of the present invention. The substantial protection scope of the present invention shall be subject to the claims. Those skilled in the art should know that any modification, equivalent substitution and improvement based on the substantial spirit of the present invention shall be within the substantial protection scope of the present invention.

Claims

1. A composite MnO2@COF-GOx, wherein MnO2 is a nanosheet structure, MnO2@COF is obtained by solvothermal reaction of borate COFs monomer and MnO2, and GOx is loaded on MnO2@COF.

2. The method for preparing the composite MnO2@COF-GOx according to claim 1 comprises reacting borate COFs monomer with MnO2 by solvothermal reaction to obtain MnO2@COF, and then mixing MnO2@COF and GOx for reaction.

3. The preparation method according to claim 2, characterized in that: The MnO2 is obtained by reacting tetramethylammonium pentahydrate and manganese chloride.

4. The preparation method according to claim 2, characterized in that: The borate COFs monomer is a boric acid monomer, including at least one of phenylboric acid and 1,4-phenylenediboric acid.

5. The preparation method according to claim 2, characterized in that: The solvothermal reaction is heated at 100-140° C. for 50-80 hours, preferably at 110-130° C. for 65-75 hours.

6. The preparation method according to claim 2, characterized in that: The mass ratio of MnO2@COF and GOx is 1-2:1-2.

7. A wound repair gel comprising the composite MnO2@COF-Gox as described in claim 1 or obtained by the preparation method as described in any one of claims 2 to 6 and sodium alginate.

8. A photosensitive spray gel, comprising the composite MnO2@COF-Gox according to claim 1 or obtained by the preparation method according to any one of claims 2 to 6, and further comprising a photoinitiator and methacrylated sodium alginate.

9. The method for preparing the photosensitive spray gel according to claim 8, which is obtained by mixing and dissolving the composite MnO2@COF-Gox, a photoinitiator and methacrylated sodium alginate.

10. Use of the composite MnO2@COF-GOx according to claim 1, the wound repair gel according to claim 7, or the photosensitive spray gel according to claim 8 in the preparation of a medicament for treating diabetic wounds.

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