Hydrogel composite material based on hydrogenase photohydrogen production and biomedical application thereof
Through a hydrogel composite composed of hydrogenase, AgNCs and FmocKYF polypeptides, light energy is used to generate hydrogen to remove reactive oxygen, solving the problem of hydrogenase sensitivity to oxygen, and achieving wound repair and anti-inflammatory effects.
Patent Information
- Application Number
- CN202410028127.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-01-09
- Publication Date
- 2025-07-11
AI Technical Summary
Existing hydrogenases are sensitive to oxygen, resulting in limitations in biomedical applications, and skin wounds are prone to reactive oxygen production, which leads to inflammation and difficulty in healing, and lacks effective wound repair materials.
A hydrogel composite composed of hydrogenase, AgNCs and FmocKYF polypeptides is used to form a hydrogenase-nano-silver cluster-FmocKYF polypeptide hydrogel through electrostatic and coordination, and light energy is used to generate hydrogen to eliminate reactive oxygen species and protect hydrogenase activity.
Effectively protect hydrogenase from oxygen inactivation, continuously produces and removes reactive oxygen species, promotes wound healing, and is widely used and biosafe.
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Figure CN120285893A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of biotechnology, and in particular relates to a hydrogenase photocatalytic hydrogen production hydrogel composite material and its biomedical applications. Background Art
[0002] Hydrogen (H2) is a colorless and odorless gas with a small molecular weight and the lightest mass, consisting of one electron and one proton. It has low solubility under normal temperature and pressure, and has not been widely used in the biomedical field due to limitations in application technologies. In recent years, in-depth studies have been conducted on the reducing and anti-free radical effects of H2, and it has been found that H2 can directly react with reactive free radicals such as hydroxyl radicals and peroxynitrite in cells, inhibit oxidative stress, reduce inflammation, inhibit apoptosis, and reduce fibrosis. At the same time, H2 does not interfere with other normal metabolic pathways while exerting its antioxidant effect. Therefore, the therapeutic effects of H2 in various fields have received attention.
[0003] Hydrogenase is an efficient catalyst that can reversibly catalyze the conversion between protons and hydrogen molecules. Hydrogenase has almost perfect catalytic activity - fast, specific, mild reaction conditions, environmentally friendly, and low energy consumption. Taking the [FeFe]-hydrogenase from Desulfovibrio desulfuricans as an example, the turnover frequencies (TOF) of its H2 oxidation and proton reduction respectively exceed 100,000 s -1 and 10,000 s -1 , which cannot be compared with existing synthetic catalysts. However, hydrogenase is sensitive to oxygen, and trace amounts of oxygen can inactivate hydrogenase, severely limiting its wide application. Developing oxygen-tolerant hydrogenase or hydrogenase oxygen-tolerant protection strategies is of great significance for exploring the potential biomedical applications of hydrogenase.
[0004] The skin is the largest organ of the human body and plays a crucial role in protecting the body from pathogen invasion and maintaining biological functions. However, the exposed skin is easily traumatized and damaged, and skin wounds can produce a large amount of reactive oxygen species (ROS), forming oxidative stress, leading to excessive accumulation of inflammatory cells, apoptosis, and delaying wound healing. In particular, chronic inflammatory wounds such as diabetic wounds are even more difficult to heal. Therefore, safe, effective, and highly applicable wound repair materials are urgently needed clinically. Summary of the Invention
[0005] To solve the above technical problems, the present invention provides a hydrogenase photocatalytic hydrogen production hydrogel composite material and its biomedical applications.
[0006] The technical solution adopted by the present invention is as follows: A hydrogenase photocatalytic hydrogen production hydrogel composite material is composed of hydrogenase, AgNCs, and FmocKYF polypeptide. The hydrogenase is connected to AgNCs through coordination, AgNCs is connected to FmocKYF polypeptide through electrostatic interaction, and the FmocKYF polypeptide self-assembles to form a hydrogel complex.
[0007] Preferably, the FmocKYF polypeptide is a 9-fluorenylmethoxycarbonyl-lysine-tyrosine-phenylalanine tripeptide;
[0008] For the preparation method of the hydrogenase photocatalytic hydrogen production hydrogel composite material, first mix and incubate the hydrogenase with AgNCs to obtain hydrogenase-AgNCs, and then add the hydrogenase-AgNCs to the aqueous solution of FmocKYF polypeptide. After mixing evenly, let it stand to obtain a hydrogenase-nanocluster silver-FmocKYF polypeptide hydrogel complex.
[0009] Preferably, dissolve the FmocKYF polypeptide in ultrapure water to prepare a 5 mg / mL solution, add hydrogenase-AgNCs thereto, and mix evenly; add 0.1 M sodium salt solution, and let it stand to obtain a hydrogenase-AgNCs-FmocKYF polypeptide hydrogel complex;
[0010] Preferably, the sodium salt is NaNO3 or NaCl.
[0011] Preferably, the specific steps are as follows:
[0012] Step 1: Prepare hydrogenase, AgNCs, and FmocKYF polypeptide respectively;
[0013] The hydrogenase Hyd2 is prepared by a genetically engineered bacterium capable of expressing Hyd2. After culturing the engineered bacterium, the hydrogenase Hyd2 is separated and purified;
[0014] Mix silver nitrate and polymethacrylic acid solution in a molar ratio of 2:1, remove oxygen with nitrogen, and generate an aqueous solution of AgNCs after illumination. Purify it with 15-30% tetrahydrofuran to obtain AgNCs;
[0015] Adopt solid-phase peptide synthesis method, cleave the peptide from the resin with a mixture of trifluoroacetic acid, ultrapure water, and triisopropylsilane, where the proportions of trifluoroacetic acid, ultrapure water, and triisopropylsilane are 95%, 2.5%, and 2.5% respectively; evaporate the mixture and precipitate with ice-cold ether. Wash the precipitate with ice-cold ether, dissolve it in dimethyl sulfoxide, and purify it by high-performance liquid chromatography to obtain the FmocKYF polypeptide;
[0016] Step 2: Mix the hydrogenase Hyd2 and AgNCs in a molar ratio of 1:1, incubate, dialyze, and concentrate to obtain hydrogenase-AgNCs;
[0017] Step 3: Dissolve the mocKYF polypeptide in ultrapure water to prepare a 5 mg / mL solution, add the hydrogenase-AgNCs complex thereto, and mix evenly; then add a 0.1 M NaNO3 solution, and let stand to obtain a hydrogenase-AgNCs-FmocKYF polypeptide hydrogel complex.
[0018] Use of a hydrogenase photocatalytic hydrogen-producing hydrogel composite material in an anti-inflammatory drug or anti-inflammatory preparation.
[0019] Use of a hydrogenase photocatalytic hydrogen-producing hydrogel composite material in an antioxidant preparation.
[0020] Use of a hydrogenase photocatalytic hydrogen-producing hydrogel composite material in a wound repair drug or preparation.
[0021] Preferably, the hydrogenase-nanoscale silver clusters-FmocKYF polypeptide hydrogel complex is made into a dressing or an injection, or used as an additive for cell culture.
[0022] Preferably, it is used in a repair drug or repair preparation for diabetic wounds, surgical or acute traumatic wounds, burn or frostbite wounds, or pressure injury wounds.
[0023] The advantages and positive effects of the present invention are as follows: The hydrogenase-nanoscale silver clusters-FmocKYF polypeptide hydrogel complex effectively protects the activity of hydrogenase by adopting the strategy of using a hydrogel to block oxygen; photoexciting electrons, effectively utilizing clean solar energy, and converting it into hydrogen that can effectively scavenge reactive oxygen species, with high biosafety, non-toxic and harmless to wounds, and having an obvious effect of promoting the healing of various wounds, wide application scenarios, and good market promotion potential;
[0024] The preparation of the hydrogenase-AgNCs-FmocKYF polypeptide hydrogel is simple, effectively protects hydrogenase from inactivation by oxygen, and uses light energy to in-situ green hydrogen production, slow-release hydrogen, and continuously exerts the efficacy of anti-inflammatory and promoting healing, with strong novelty, great application potential, and good market promotion potential;
[0025] This composite material is simple to prepare, has good biocompatibility, low cost, and strong plasticity, and shows great application potential in inflammation, apoptosis, autophagy, tumorigenesis, etc. involving various reactive oxygen species (ROS), and can be developed into new antibacterial materials, wound dressings, health products and other products. Description of the Drawings
[0026] Figure 1 Molecular structure of the FmocKYF polypeptide;
[0027] Figure 2 Mass spectrometry analysis results of the FmocKYF polypeptide prepared in Example 1;
[0028] Figure 3 The HPLC analysis result of the FmocKYF polypeptide prepared in Example 1;
[0029] Figure 4 Hydrogenase-AgNCs-FmocKYF polypeptide hydrogel;
[0030] Figure 5 Preparation result diagram of the self-assembled hydrogel; A, white light; B, irradiated with ultraviolet light at 365 nm;
[0031] Figure 6 Oxygen penetration resistance performance of the hydrogenase-AgNCs-FmocKYF polypeptide hydrogel complex;
[0032] Figure 7 Gas-phase analysis of gas products after illumination of the hydrogenase-AgNCs-FmocKYF polypeptide hydrogel complex;
[0033] Figure 8 Hydrogen production effect of the hydrogenase-AgNCs-FmocKYF polypeptide hydrogel complex;
[0034] Figure 9 Anti-inflammatory cell verification effect of the hydrogenase-AgNCs-FmocKYF polypeptide hydrogel complex;
[0035] Figure 10 Repair effect of the hydrogenase-AgNCs-FmocKYF polypeptide hydrogel complex in diabetic wounds;
[0036] Figure 11 Histological detection of the hydrogenase-AgNCs-FmocKYF polypeptide hydrogel complex at the wound healing site. Detailed implementation manners
[0037] The embodiments of the present invention will be described below with reference to the accompanying drawings.
[0038] The present invention relates to a hydrogenase photocatalytic hydrogen production hydrogel composite material and its biomedical applications, and provides a hydrogenase-AgNCs-FmocKYF polypeptide hydrogel complex with simple process, rapid reaction, biocompatibility, controllability and adjustability, which can protect hydrogenase through FmocKYF polypeptide hydrogel, block oxygen, and achieve the purpose of hydrogen release. The hydrogenase photocatalytic hydrogen production hydrogel composite material is composed of hydrogenase, silver nanoclusters, and 9-fluorenylmethoxycarbonyl-lysine-tyrosine-phenylalanine (FmocKYF) tripeptide, and forms a hydrogenase-AgNCs-FmocKYF polypeptide hydrogel complex through electrostatic interaction, coordination interaction, and self-assembly. The hydrogenase-AgNCs-FmocKYF polypeptide hydrogel complex has adjustable, controllable, and stable hydrogen production performance; at the same time, the composite material has a controlled release effect on the generated hydrogen; enabling hydrogen to be used in situ for antioxidant, anti-inflammatory, and wound repair.
[0039] When preparing the hydrogenase-silver nanocluster-FmocKYF polypeptide hydrogel complex, hydrogenase-silver nanocluster-FmocKYF polypeptide is first prepared separately. Hydrogenase Hyd2 can first construct an engineered bacterium capable of expressing hydrogenase. After culturing the engineered bacterium, the hydrogenase expressed by it is extracted and purified, and stored in liquid nitrogen. An aqueous solution of polymethacrylic acid PMAA is added to an aqueous solution of silver nitrate, and the molar ratio of silver nitrate to PMAA is 2:1. After deoxygenation with nitrogen, the formation of AgNCs is induced by solar irradiation, and the final product is purified with a 15% - 35% (v / v) aqueous solution of tetrahydrofuran to obtain AgNCs. Using solid-phase peptide synthesis (SPPS), the peptide is cleaved from the resin with a mixture of trifluoroacetic acid (TFA), ultrapure water, and triisopropylsilane. The mixture is evaporated and precipitated with ice-cold diethyl ether, and the FmocKYF polypeptide is obtained after washing and purification.
[0040] Hydrogenase Hyd2 and AgNCs are incubated at a molar ratio of 1:1, dialyzed and concentrated to obtain the required hydrogenase-AgNCs complex; the FmocKYF polypeptide is dissolved in ultrapure water to prepare a 5 mg / mL solution, and the hydrogenase-AgNCs complex is added thereto and mixed evenly. Then, 0.1 M NaNO3 solution is added, and the mixture is allowed to stand for 20 minutes to produce the hydrogenase-AgNCs-FmocKYF polypeptide hydrogel complex.
[0041] Polypeptide hydrogels are excellent biomatrix materials that combine the advantages of synthetic and naturally derived hydrogel-forming materials. They are easy to prepare and modify, have good biocompatibility, and provide a superstructure for the design and assembly of enzymes and new materials. Polypeptide hydrogels can form a physical barrier to oxygen through their backbone network, and their hierarchical assembly structure is conducive to binding hydrogen and has the property of slowly releasing hydrogen. In the hydrogenase-AgNCs-FmocKYF polypeptide hydrogel complex, the FmocKYF polypeptide hydrogel forms a complex framework that completely encapsulates the hydrogenase and isolates it from oxygen.
[0042] As an excellent photosensitizer, AgNC generates photogenerated electrons under visible light. The nickel-iron hydrogenase Hyd2 uses the photoelectrons generated by AgNC to convert protons in the solution into hydrogen. Positively charged polypeptides can bind to negatively charged AgNCs through electrostatic interactions. AgNCs are interspersed in the hydrogel, enhancing the conductivity. The hydrophobic pockets and hydrogen bond networks of FmocKYF polypeptide molecules can effectively block oxygen and protect the catalytic activity of hydrogenase. The FmocKYF polypeptide hydrogel has an enrichment and retention effect on hydrogen, and the hydrogen generated under light can be slowly released to achieve the long-term elimination of ROS.
[0043] When the hydrogen production system based on nickel-iron hydrogenase is used in the biomedical field, a developed hydrogenase-AgNCs-FmocKYF polypeptide hydrogel for wound repair can effectively scavenge ROS in the wound surface through biological photocatalytic hydrogen production, achieving the purposes of anti-inflammatory and promoting wound healing.
[0044] The hydrogenase-AgNCs-FmocKYF polypeptide hydrogel complex can be used in the field of hydrogen therapy, such as for clearing cell inflammation, repairing diabetic wounds, repairing surgical or acute trauma wounds, repairing burns or frostbite wounds, repairing pressure injury wounds, or in anti-inflammatory drugs or dressings for the aforementioned wound repair drugs or dressings. The hydrogenase-AgNCs-FmocKYF polypeptide hydrogel complex also has antioxidant function and can be added to antioxidants. When in use, the hydrogenase-AgNCs-FmocKYF polypeptide hydrogel complex can be made into forms such as wound dressings, cell culture additives, and injections.
[0045] The following describes the solution of the present invention in conjunction with the accompanying drawings. Among them, for the experimental methods without specific operation steps, they are all carried out according to the corresponding product instructions. For the instruments, reagents, and consumables used in the examples, if not otherwise specified, they can all be purchased from commercial companies.
[0046] Example 1: Preparation of hydrogenase-AgNCs-FmocKYF polypeptide hydrogel complex
[0047] 1.1 Preparation of FmocKYF polypeptide
[0048] The 9-fluorenylmethoxycarbonyl-lysine-tyrosine-phenylalanine (FmocKYF) tripeptide was synthesized on CLEAR amide resin by standard solid phase peptide synthesis (SPPS) with the following structure: Figure 1 As shown. In anhydrous N,N'-dimethylformamide (DMF), HATU and DIPEA were selected as coupling agents. The peptide derivative was cleaved from the resin with a mixture of TFA, ultrapure water and triisopropylsilane at room temperature for 3 hours. The mixture was then evaporated and precipitated with ice-cold ether. The resulting precipitate was washed with cold ether, dissolved in DMSO and purified by high performance liquid chromatography. The purity and molecular weight of the product were characterized by HPLC and MALDI-TOF MS, and the results are shown in Figures 2 - 3 shown.
[0049] 1.2 Preparation of silver clusters (AgNCs)
[0050] First, weigh 1.0 g of PMAA, add 80 mL of ultrapure water, adjust the pH to 3.0, and make up to 100 mL. Then accurately weigh 0.16 g of silver nitrate solid, add 10 mL of ultrapure water, and store in the dark. Use a measuring cylinder to measure 10 mL of PMAA solution and silver nitrate solution, seal the film to isolate the air, stir in a round-bottom flask, and pass nitrogen for 15 minutes to eliminate dissolved oxygen in the solution. Fix it on an iron stand and irradiate the above reaction system with sunlight while stirring with a magnetic stirrer. This is to fully exclude oxygen. The time can be appropriately extended. Solar irradiation can induce the formation of AgNCs until the color of the solution changes from colorless to magenta. Centrifuge to remove tiny black silver oxide particles in the solution. Slowly transfer the supernatant to a new centrifuge tube to obtain an "as-prepared AgNCs" solution. The AgNCs solution obtained in the previous step was quickly added with tetrahydrofuran to 30 mL of the centrifuge tube scale line in a fume hood (the tetrahydrofuran content was 15% to 35% (v / v)), and the solution was shaken rapidly and violently, and the solution changed from magenta to misty liquid. After balancing again, centrifuge at 3600 rpm for 15 minutes. The supernatant was discarded and dried in a fume hood. AgNC powder was collected after 24 hours.
[0051] 1.3 Preparation of hydrogenase Hyd2
[0052] The engineered bacteria capable of expressing hydrogenase Hyd2 were cultured for preparing hydrogenase Hyd2. The Escherichia coli expression bacteria used in this example were stored in the laboratory.
[0053] The specific preparation steps are as follows:
[0054] (1) A single clone with good growth was selected and inoculated into 5 mL of LB liquid culture medium containing ampicillin resistance, and cultured at 37°C with shaking at 200 rpm for 6 hours.
[0055] (2) Inoculate the small tube of bacterial liquid into an enlarged culture flask, supplement with glycerol and sodium fumarate, and place it in an anaerobic incubator at 37 °C for static culture for 4 hours. Then add isopropyl β-D-thiogalactopyranoside (IPTG) with a final concentration of 1 mM to induce enzyme expression. After 12 hours, centrifuge at 5000 rpm for 15 minutes to collect the bacteria.
[0056] (3) Resuspend the bacterial pellet with Tris Buffer, and add 0.003% deoxyribonuclease and 0.012% lysozyme thereto. Disrupt at a pressure of 1200 kPa for 3 minutes. Then centrifuge at 30000 rpm and 4 °C for 50 minutes, and filter through a 0.22 μm microporous filter membrane.
[0057] (4) The filtrate is purified by AKTA protein NTA affinity chromatography, identified by gel electrophoresis, and the protein sample is desalted using a desalting column. The prepared hydrogenase Hyd2 is aliquoted and stored in liquid nitrogen.
[0058] 1.4 Preparation of hydrogenase-AgNCs complex
[0059] Mix 30 μL of hydrogenase Hyd2 with a concentration of 1 mg / mL and 50 μL of AgNCs solution (10% ethanol) with a concentration of 5 mg / mL. After incubating in the dark in a 4 °C refrigerator for 1 hour, centrifuge at 12000 rpm for 20 minutes using a concentrator tube with a cut-off molecular weight of 50 kDa to remove unbound free AgNCs, and obtain the hydrogenase-AgNCs complex.
[0060] 1.5 Preparation of hydrogenase-AgNCs-FmocKYF polypeptide hydrogel
[0061] Dissolve 5 mg of the above-prepared FmocKYF polypeptide in 1 mL of ultrapure water, add 50 μL of the hydrogenase-AgNCs complex thereto, and mix evenly. Then add 100 μL of a 1 M NaNO3 solution and let it stand for 20 minutes to complete the preparation of the hydrogenase-AgNCs-FmocKYF polypeptide hydrogel. The preparation process is carried out in an anaerobic glove box. The hydrogel-forming effect is as Figure 4 shown
[0062] 1.6 Preparation of control FmocKYF polypeptide hydrogel
[0063] Dissolve 5 mg of the FmocKYF polypeptide prepared in Step 1.1 above in 1 mL of ultrapure water, and sequentially prepare experimental groups that only contain the FmocKYF polypeptide, contain the FmocKYF polypeptide and NaNO3 (add 100 μL of a 1 M NaNO3 solution), include the FmocKYF polypeptide and AgNCs (add 50 μL of a 5 mg / mL AgNCs solution), and only contain AgNCs. Let stand for 20 minutes, and the preparation process is carried out in an anaerobic glove box. The hydrogel gelation effects of each group are as Figure 5 shown.
[0064] It can be seen from the figure that the experimental group that only includes the FmocKYF polypeptide and AgNC cannot form a gel state, and the experimental groups added with NaNO3 or AgNCs can induce the FmocKYF polypeptide to form a gel; by irradiating the figure with 365 nm ultraviolet light, it can be seen that after AgNCs induce the FmocKYF polypeptide to form a gel, AgNCs are evenly dispersed in the gel, indicating the strong interaction between AgNCs and FmocKYF molecules.
[0065] Example 2: Verification that the FmocKYF polypeptide hydrogel can protect hydrogenase from oxygen attack
[0066] Prepare three groups of glass bottles respectively, and prepare the experimental groups according to the following methods;
[0067] Experimental group 1 (aqueous solution control group): Add 100 mM methyl viologen and 100 mM sodium dithionite to 2 mL of ultrapure water that has been deoxygenated by passing nitrogen;
[0068] Experimental group 2 (blank polypeptide hydrogel): Dissolve 10 mg of the FmocKYF polypeptide in 2 mL of ultrapure water, add 100 mM methyl viologen and 100 mM sodium dithionite to it, and then add 200 μL of a 1 M NaNO3 solution, and let stand for 20 minutes;
[0069] Experimental group 3 (hydrogenase-AgNCs-FmocKYF polypeptide hydrogel): Dissolve 10 mg of the FmocKYF polypeptide in 2 mL of ultrapure water, add 100 μL of the hydrogenase-AgNC complex to it, and then add 100 mM methyl viologen and 100 mM sodium dithionite, and mix evenly. Then add 200 μL of a 1 M NaNO3 solution, and let stand for 20 minutes;
[0070] After the preparation is completed, take out the above three groups of glass bottles from the glove box, expose them to the air environment, and take pictures at regular intervals to record the color changes, and the changes are as Figure 5 shown, and from left to right in the figure are Experimental group 1, Experimental group 2, and Experimental group 3.
[0071] According to Figure 6It can be seen that the aqueous solution control group turned from blue to colorless after being exposed to air for 3 hours, indicating that the reducing methyl viologen has been completely oxidized by oxygen. Compared with the aqueous solution control group, both the blank polypeptide hydrogel and the hydrogenase-AgNCs-FmocKYF polypeptide hydrogel showed good oxygen barrier ability. It is proved that the FmocKYF polypeptide hydrogel can produce the ability to block oxygen and can protect the hydrogenase encapsulated therein; the FmocKYF polypeptide hydrogel can block oxygen and has antioxidant effect.
[0072] Example 3: Verification of the hydrogen production effect of hydrogenase-AgNCs-FmocKYF polypeptide hydrogel
[0073] According to the method of Example 1, 2 mL of hydrogenase-AgNCs-FmocKYF polypeptide hydrogel was prepared in a 10 mL photocatalytic reaction flask. Add 1 mL of hydrogen production buffer (0.1 M NaNO3, 0.1 M TEOA, pH 7.0) to the reaction flask and seal it with a rubber stopper and an aluminum cap. Use a Newport arc lamp (300 W) for irradiation, install a 420 nm filter, and irradiate the reaction system in the reaction flask at a distance of 5 cm from the solution for photocatalysis.
[0074] After 2 hours of irradiation, take 50 μL of the headspace gas for gas phase analysis. The results are as Figure 7 shown. The hydrogen production device and effect are as Figure 8 shown. The hydrogenase-AgNCs-FmocKYF polypeptide hydrogel showed good hydrogen production performance under light irradiation. Bubbles were formed in the gel after light irradiation, and these bubbles were hydrogen bubbles, and the hydrogen bubbles still existed after 2 h, proving that the hydrogenase-AgNCs-FmocKYF polypeptide hydrogel complex has a storage and slow release effect on hydrogen.
[0075] Example 4 Cellular verification of the anti-inflammatory effect of hydrogenase-AgNCs-FmocKYF polypeptide hydrogel
[0076] (1) Culture RAW264.7 mouse macrophages in DMEM supplemented with 10% fetal bovine serum, and the culture conditions are 5% carbon dioxide and 37 °C.
[0077] (2) Prepare 500 μL of blank polypeptide hydrogel or hydrogenase-AgNCs-FmocKYF polypeptide hydrogel in a six-well plate according to the method in Example 1, and add 500 μL of 1×PBS buffer to the anhydrous gel control group.
[0078] (3) Place a cover glass on the surface of the gel or at the bottom of the well plate, add 2.5 mL of medium (pH 7.3) supplemented with 50 mM TEOA, and inoculate RAW264.7 mouse macrophages at an inoculation density of 1×10 4 cells / well.
[0079] (4) Inflammatory induction process: Lipopolysaccharide (LPS) with a final concentration of 100 ng / mL and interferon γ (INF-γ) with a final concentration of 2.5 ng / mL were added to the culture medium and induced for 12 hours. The hydrogenase-AgNCs-FmocKYF polypeptide hydrogel group was divided into two groups: dark culture and light culture.
[0080] (5) After 12 hours, the original culture medium was discarded, and the ROS stain 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) with a final concentration of 5 μM was added to the new culture medium, and the culture was continued for 30 minutes.
[0081] (6) The stained culture medium was discarded, washed three times with 1×PBS, and 1 mL of 4% tissue fixative was added and fixed for 20 minutes.
[0082] (7) Washed three times with 1×PBS, 10 μL of mounting medium containing DAPI was added in the center of the glass slide, and the cell climbing slice was covered on the mounting medium to avoid the appearance of bubbles. After solidification, it was photographed with a fluorescence confocal microscope.
[0083] The experimental results are as Figure 9 shown. The green signal of ROS in the cells added with LPS and INF-γ was extremely strong, while the green signal of ROS in the hydrogenase-AgNCs-FmocKYF polypeptide hydrogel group under light culture was basically absent, indicating that the hydrogenase-AgNCs-FmocKYF polypeptide hydrogel group under light culture showed good anti-inflammatory effects.
[0084] Example 5 Verification of the repair effect of hydrogenase-AgNCs-FmocKYF polypeptide hydrogel in diabetic wounds
[0085] (1) Establishment of a diabetic mouse skin wound model: The mice used were 12-week-old male BABL / c (15 mice). Streptozotocin (STZ) was intraperitoneally injected at a dose of 50 mg / kg, injected every other day, and continuously injected 3 times. And the blood glucose was detected every other day. When the blood glucose was stably reached 20 mM, the diabetic model was successfully established. The above diabetic mice were anesthetized, depilated, and the back skin was disinfected with 2% iodophor. A puncher with a diameter of 7 mm was used to punch holes in the back skin.
[0086] (2) The above model mice were randomly divided into 3 groups, namely the control group, the blank polypeptide hydrogel group, and the hydrogenase-AgNCs-FmocKYF polypeptide hydrogel group.
[0087] (3) The control group added 50 μL of 1×PBS to the wound, the blank polypeptide hydrogel group added 50 μL of the blank polypeptide hydrogel in Example 1 to the wound, and the hydrogenase-AgNCs-FmocKYF polypeptide hydrogel group added 50 μL of the hydrogenase-AgNCs-FmocKYF polypeptide hydrogel in Example 1 to the wound. Subsequently, each group was bandaged with a conventional medical dressing. The hydrogenase-AgNCs-FmocKYF polypeptide hydrogel group was irradiated with LED light for 2 hours every day.
[0088] (4) The hydrogel and dressing were replaced every other day, and the wound healing degree was photographed and recorded continuously for 12 days. The specific healing situation is shown in Figure 10 .
[0089] The results showed that compared with the control group and the blank polypeptide hydrogel group, the wound in the hydrogenase-AgNCs-FmocKYF polypeptide hydrogel group healed the fastest.
[0090] (5) The mice were anesthetized and sacrificed on the 12th day of treatment. The wound and the surrounding skin were excised, washed 3 times with PBS, and then placed in 4% tissue fixative and fixed overnight at 4°C.
[0091] (6) Wash 3 times with PBS, dehydrate programmatically with a dehydrator, and embed in paraffin.
[0092] (7) The paraffin was sectioned into 5-μm thin layers, and then stained by hematoxylin-eosin staining (HE) for morphological structure and epidermis observation. The degree of collagen fiber formation was observed by Masson staining. The experimental results are as Figure 11 , and the morphological structure and epidermal recovery of the wound surface in the hydrogenase-AgNCs-FmocKYF polypeptide hydrogel group were the best. Obvious angiogenesis and pore neogenesis could be observed, and the collagen fiber generation was the most complete.
[0093] Example 6 Application of hydrogenase-AgNCs-FmocKYF polypeptide hydrogel in the repair of surgical wounds or acute trauma wounds
[0094] Establish a common wound model: The mice used were 12-week-old male BABL / c. The mice were anesthetized, depilated, and the back skin was disinfected with 2% iodophor. A punch with a diameter of 7 mm was used to punch holes in the back skin.
[0095] The usage method of the hydrogenase-AgNCs-FmocKYF polypeptide hydrogel was the same as that in Example 5. 50 μL of the hydrogenase-AgNCs-FmocKYF polypeptide hydrogel in Example 1 was added to the wound, bandaged with a conventional medical dressing, irradiated with LED light for 2 hours every day, and the hydrogel and dressing were replaced every other day.
[0096] Example 7 Application of Hydrogenase-AgNCs-FmocKYF Polypeptide Hydrogel in the Repair of Scald Wounds
[0097] Establish a scald wound model: The mice used were 12-week-old male BABL / c mice (15 in number). The mice were anesthetized, their hair was removed, and the back skin was disinfected with 2% iodophor. A 50-g weight was heated in boiling water for 10 minutes, and after being taken out, the surface water droplets were immediately wiped off, and it was brought into contact with the back skin for 10 seconds to cause a deep second-degree burn.
[0098] The usage method of the hydrogenase-AgNCs-FmocKYF polypeptide hydrogel was the same as that in Example 5. 50 μL of the hydrogenase-AgNCs-FmocKYF polypeptide hydrogel in Example 1 was added to the wound, and it was bandaged with a conventional medical dressing. It was irradiated with LED light for 2 hours every day, and the new hydrogel and dressing were replaced every other day.
[0099] Example 8 Application of Hydrogenase-AgNCs-FmocKYF Polypeptide Hydrogel in the Repair of Deep Tissue Pressure Injury Wounds
[0100] Establish a deep tissue pressure injury model: The mice used were 12-week-old male BABL / c mice (15 in number). The hair of the mice was removed, and the abdominal and back skin was disinfected with 2% iodophor. Magnets (diameter 12 mm, thickness 5 mm, mass 2.4 g, surface magnetic flux density 1000 Gs) were used to apply pressure on both sides of the back and abdomen of the ischial spine for 12 h, and then the magnets were removed.
[0101] The usage method of the hydrogenase-AgNCs-FmocKYF polypeptide hydrogel was the same as that in Example 5. 50 μL of the hydrogenase-AgNCs-FmocKYF polypeptide hydrogel in Example 1 was added to the wound, and it was bandaged with a conventional medical dressing. It was irradiated with LED light for 2 hours every day, and the new hydrogel and dressing were replaced every other day.
[0102] For the usage scenarios in Examples 6-8 respectively, control groups similar to those in Example 5 were constructed. Through observation, it was found that the hydrogenase-AgNCs-FmocKYF polypeptide hydrogel had the same healing trend as when used for diabetic wounds in surgical operations or acute traumas, scalds, and pressure injuries, and it was shown that the hydrogenase-AgNCs-FmocKYF polypeptide hydrogel could promote wound healing faster.
[0103] The above has described the embodiments of the present invention in detail, but the content described is only the preferred embodiments of the present invention and cannot be considered as limiting the implementation scope of the present invention. All equivalent changes and improvements made according to the scope of the application of the present invention should still fall within the scope covered by the patent of the present invention.
Claims
1. A hydrogenase-based photocatalytic hydrogen-producing hydrogel composite material, characterized in that: It consists of hydrogenase, AgNCs and FmocKYF polypeptide. Hydrogenase is connected to AgNCs through coordination, AgNCs is connected to FmocKYF polypeptide through electrostatic interaction, and FmocKYF polypeptide self-assembles to form a hydrogel complex.
2. The hydrogenase-based photocatalytic hydrogen-producing hydrogel composite material according to claim 1, wherein: FmocKYF polypeptide is a tripeptide of 9-fluorenylmethoxycarbonyl-lysine-tyrosine-phenylalanine.
3. The preparation method of the hydrogenase-based photocatalytic hydrogen production hydrogel composite material according to claim 1 or 2, characterized in that: First, hydrogenase and AgNCs are mixed and incubated to obtain hydrogenase-AgNCs. Then, hydrogenase-AgNCs is added to the aqueous solution of FmocKYF polypeptide. After mixing evenly and standing still, a hydrogenase-nanocluster-Ag-FmocKYF polypeptide hydrogel complex is obtained.
4. The preparation method of the hydrogenase-based photocatalytic hydrogen production hydrogel composite material according to claim 3, wherein: Dissolve FmocKYF polypeptide in ultrapure water to prepare a 5 mg / mL solution. Add hydrogenase-AgNCs to it and mix evenly. Add 0.1 M sodium salt solution and stand still to obtain a hydrogenase-AgNCs-FmocKYF polypeptide hydrogel complex. Preferably, the sodium salt is NaNO3 or NaCl.
5. The preparation method of the hydrogenase-based photocatalytic hydrogen-producing hydrogel composite material according to claim 3, characterized in that: The specific steps are as follows: Step 1: Prepare hydrogenase, AgNCs and FmocKYF polypeptide respectively. Step 2: Mix hydrogenase Hyd2 and AgNCs according to a molar ratio of 1:1, incubate, dialyze and concentrate to obtain hydrogenase-AgNCs. Step 3: Dissolve mocKYF polypeptide in ultrapure water to prepare a 5 mg / mL solution. Add the hydrogenase-AgNCs complex to it and mix evenly. Then add 0.1 M NaNO3 solution and stand still to obtain a hydrogenase-AgNCs-FmocKYF polypeptide hydrogel complex.
6. Use of the hydrogenase photocatalytic hydrogen production hydrogel composite material according to claim 1 or 2 in an anti-inflammatory drug or anti-inflammatory preparation.
7. Use of the hydrogenase photocatalytic hydrogen production hydrogel composite material according to claim 1 or 2 in an antioxidant preparation.
8. Use of the hydrogenase photocatalytic hydrogen production hydrogel composite material according to claim 1 or 2 in a wound repair drug or preparation.
9. The application according to claim 8, wherein: The hydrogenase-nanocluster-Ag-FmocKYF polypeptide hydrogel complex is made into a dressing or injection, or used as a cell culture additive.
10. The application according to claim 8, wherein: A repair drug or repair preparation for diabetic wounds, surgical or acute trauma wounds, burn or frostbite wounds, or pressure injury wounds.