CN117443410B - ROS scavenging biocatalysis material and preparation and application thereof - Google Patents

ROS scavenging biocatalysis material and preparation and application thereof Download PDF

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CN117443410B
CN117443410B CN202311780966.7A CN202311780966A CN117443410B CN 117443410 B CN117443410 B CN 117443410B CN 202311780966 A CN202311780966 A CN 202311780966A CN 117443410 B CN117443410 B CN 117443410B
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feooh
scavenging
ros
active oxygen
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CN117443410A (en
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程冲
谢蓝
邱逦
黄颂雅
容逍
文琴龙
白明茹
任显诚
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Sichuan University
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    • B22F9/24—Making metallic powder or suspensions thereof using chemical processes with reduction of metal compounds starting from liquid metal compounds, e.g. solutions
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    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
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    • A61P19/00—Drugs for skeletal disorders
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    • A—HUMAN NECESSITIES
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    • B01J23/8906—Iron and noble metals

Abstract

The invention belongs to the field of biocatalysis, and particularly relates to a ROS scavenging biocatalysis material, and preparation and application thereof. The invention provides a ROS scavenging biocatalytic material, which is prepared by constructing ruthenium clusters on the surface of ferric oxide hydroxide in situ and is denoted as Ru@FeOOH. The Ru@FeOOH obtained by the method can remove excessive active oxygen in a neutral environment, and can generate active oxygen in an acidic environment, and has CAT-like enzyme and SOD-like enzyme activities required for removing active oxygen and POD-like enzyme activities required for generating active oxygen. Ru@FeOOH has excellent active oxygen scavenging ability and is resistant to H 2 O 2 The scavenging capacity can reach 100% in 12 min, and 80% -95% of superoxide anions can be scavenged in 10 min. Meanwhile, ru@FeOOH also shows very excellent effect in RA treatment modelPotential for treating RA.

Description

ROS scavenging biocatalysis material and preparation and application thereof
Technical Field
The invention belongs to the field of biocatalysis, and particularly relates to a ROS scavenging biocatalysis material, and preparation and application thereof.
Background
Rheumatoid Arthritis (RA) is an autoimmune disease characterized by synovial inflammation and pannus, skeletal and cartilage damage. Its global prevalence is about 0.5-1%, with females being more common than males. Inheritance is a key factor in the development of RA, and sex, smoking and environmental factors can influence the development of RA. In rheumatoid arthritis, permanent T-cell and monocyte mediated synovitis is the root cause of disease progression. Flaccidity is a characteristic pathological product of rheumatoid arthritis, has tumor-like properties, can drive synovial hyperplasia and bone erosion, ultimately leading to disability and severely affecting the quality of life of the patient. The onset of RA involves not only joints but also cardiovascular and interstitial lung diseases, which are serious complications leading to shortened life expectancy in RA patients. Mitochondria are a significant source of Reactive Oxygen Species (ROS) in most mammalian cells. However, ROS accumulation may activate mitochondrial permeability transition pore (mPTP) and intimal anion channel (IMAC) openings. Longer mPTP openings may release ROS bursts, resulting in mitochondrial damage. ROS and mitochondrial damage are indispensible from several key pathological processes of RA, modulation of mitochondrial function, clearance of ROS, and relief of oxidative stress are hot targets for current RA therapies.
In inflamed joints of RA patients, the cells may be living in the cartilage and synoviumActivated neutrophils in the activated macrophages or synovium form reactive species. The NADPH oxidase system catalyzes the conversion of molecular oxygen to superoxide anion radicals. Lipoproteins, lipopolysaccharides and cytokines (e.g., TNF- α, IL-1β and IFN- γ) drive the activation of the NADPH oxidase complex. Active substances are also formed in mitochondria by oxidative phosphorylation processes. ROS formation within mitochondria increases with decoupling of electron transfer. In mitochondria, superoxide anions are produced by single electron reduction of oxygen. Superoxide anions are one of the major ROS involved in inflammation in RA patients. Superoxide anions, along with other oxygen and nitrogen radicals, destroy cartilage and extracellular matrix components either directly or indirectly by reducing synthesis of matrix components such as collagen and proteoglycans. Reactive superoxide radicals are disproportionated to hydrogen peroxide by superoxide dismutase. The hydrogen peroxide is then converted to water by catalase and glutathione reductase. In neutrophils H 2 O 2 Is converted into hypochlorous acid. Hypochlorous acid and H 2 O 2 Further reaction forms singlet oxygen, which is also a very damaging radical. H 2 O 2 In the presence of iron ions or other transition metals, are converted to hydroxyl radicals by the Fenton reaction. Hydroxyl radicals react very rapidly with nearby molecules, which may be proteins, lipids and nucleic acids, thereby destroying them and leading to serious consequences of RA pathogenesis.
Under physiological conditions, intracellular ROS scavenging is mainly dependent on rapid reactions of antioxidant enzymes and antioxidant enzymes such as superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT), glutathione Reductase (GR), etc. However, the direct use of these natural enzymes for the treatment of RA patients appears to be undesirable because they are readily degraded and cleared in vivo, and thus it is difficult to achieve sustained ROS clearance. With the continuous development and application of nanomaterials, the research of utilizing nanomaterials to remove active oxygen in vivo is gradually increasing, and the nanomaterials are expected to make great contribution in the field of treating and preventing RA.
Disclosure of Invention
In view of the above problems, the present invention has developed a novel enzyme having SOD-like activityROS scavenging material that is reactive and CAT-like enzymatic activity. The invention constructs ruthenium cluster active site on the lamellar structure of ferric hydroxide by one-pot synthesis technology, the synthesized material is denoted as Ru@FeOOH, the obtained Ru@FeOOH can remove excessive active oxygen in a neutral environment (pH: 7-8), and can generate active oxygen in an acidic environment (pH: 4-6), and the active oxygen has CAT-like enzyme and SOD-like enzyme activities required for removing active oxygen and POD-like enzyme activities required for generating active oxygen. Experiments show that Ru@FeOOH has excellent active oxygen scavenging capacity and is specific to H 2 O 2 The scavenging capacity can reach 100% in 12 min, and meanwhile 80% -95% of superoxide anions (O) can be scavenged in 10min 2 - ). Meanwhile, ru@FeOOH also shows very excellent potential for treating RA in an RA treatment model.
The technical scheme of the invention is as follows:
the first technical problem to be solved by the invention is to provide a ROS scavenging biocatalytic material with SOD-like enzyme activity and CAT-like enzyme activity, wherein the biocatalytic material is prepared by constructing ruthenium clusters on the surface of ferric oxide hydroxide in situ, and is denoted as Ru@FeOOH.
Further, the biocatalytic material has a sheet-like structure.
Further, in the microstructure of the biocatalytic material, highly dispersed Ru cluster active sites are formed on the surface of the FeOOH sheet-like substrate.
Further, the biocatalysis material has POD-like enzyme activity, CAT-like enzyme activity and SOD-like enzyme activity at the same time.
Further, the biocatalysis material Ru@FeOOH can remove excessive active oxygen in a neutral environment (pH: 7-8), and can generate active oxygen in an acidic environment (pH: 4-6).
The second technical problem to be solved by the invention is to provide a preparation method of the ROS scavenging biocatalytic material, which comprises the following steps: the biocatalytic material (Ru@FeOOH) is prepared by solvothermal reaction of ferric salt, ruthenium salt and hexamethylenetetramine or urea by a one-pot method.
Further, the iron salt is selected from: feCl 3 ·6H 2 O。
Further, the ruthenium salt is selected from: ruCl 3 ·xH 2 O。
Further, the mass ratio of the ferric salt to the ruthenium salt is as follows: 1: 1-20: 1, preferably 1:1 to 10:1, a step of; preferably 2:1, 5:1, 10:1 or 20:1. The Ru@FeOOH series material without Ru proportion is prepared by adjusting the content of Ru added in the synthesis process: ru@FeOOH-1:2, ru@FeOOH-1:5, ru@FeOOH-1:10 and Ru@FeOOH-1:20 are not particularly described in the invention, and Ru@FeOOH refers to Ru@FeOOH-1:5.
Further, the preparation method comprises the following steps: firstly, dissolving hexamethylenetetramine or urea in a solvent to prepare a transparent colorless solution, then adding ferric salt and ruthenium salt, stirring for 0-15min (preferably 5 min), and placing the obtained solution in a corrosion-resistant autoclave to react for 3-6 h (preferably 4 h) at 80-110 ℃ (preferably 95 ℃); cooling to room temperature, and collecting the obtained yellow brown precipitate; washing and drying the obtained precipitate to obtain the biocatalysis material Ru@FeOOH.
Further, in the above preparation method, the solvent is deionized water.
The third technical problem to be solved by the invention is to point out the application of the ROS scavenging biocatalytic material in preparing the reactive oxygen species scavenging material.
The fourth technical problem to be solved by the present invention is to indicate the use of the ROS scavenging biocatalytic material described above for the preparation of stem cell protective materials or materials for the treatment of periodontitis.
The fifth technical problem to be solved by the present invention is to point out the use of the above-mentioned ROS scavenging biocatalyst material for preparing a biocatalyst material having both POD-like enzyme activity, CAT-like enzyme activity and SOD-like enzyme activity.
The invention has the beneficial effects that:
the invention constructs ruthenium cluster active site on the lamellar structure of ferric oxide hydroxide by one-pot synthesis technology to synthesize a new biocatalytic material Ru@FeOOH, the obtained Ru@FeOOH can remove excessive active oxygen in a neutral environment (pH: 7-8), and can generate active oxygen in an acidic environment (pH: 4-6), namely, the invention has the function of removingCAT-like enzyme and SOD-like enzyme activities required for active oxygen and POD-like enzyme activities required for active oxygen production. Experiments show that Ru@FeOOH has excellent active oxygen scavenging capacity and is specific to H 2 O 2 The scavenging capacity can reach 100% in 30min, and meanwhile 80% -95% of superoxide anions (O) can be scavenged in 10min 2 - ). Meanwhile, ru@FeOOH also shows very excellent potential for treating RA in an RA treatment model.
Drawings
FIG. 1 is a schematic diagram of the synthesis of Ru@FeOOH.
Fig. 2 is an SEM image of a different embodiment: (a) Ru@FeOOH-1:20; (b) Ru@FeOOH-1:10; (c) Ru@FeOOH-1:5; (d) Ru@FeOOH-1:2; (e) FeOOH-Ru; (f) FeNiOH-Ru.
FIG. 3 is an XRD pattern for Ru@FeOOH obtained in the different examples.
Fig. 4 is a TEM image of ru@feooh.
FIG. 5 is an HR-TEM lattice diagram of Ru@FeOOH.
Fig. 6 is an energy dispersive spectroscopy element map of ru@feooh.
Fig. 7 is a high angle annular dark field scanning transmission electron microscope image of ru@feooh.
Fig. 8 is CAT performance test: (a) H in different material groups 2 O 2 A concentration map; (b) Different material pairs H 2 O 2 Is a clean-up rate of (a).
FIG. 9 is SOD performance test: different material pairs O 2 - Is a graph of the clearance results of (1).
Fig. 10 is POD performance detection: absorbance intensity plot at 652nm for different materials.
Fig. 11 is a TEM image after 24 h co-incubations of RAW264.7 and RAW 264.7+ Lipopolysaccharide (LPS) with ru@feooh, respectively.
FIG. 12 is a graph of viable/dead cell staining after 24 h co-incubation of simple Human Umbilical Vein Endothelial Cells (HUVECs)/RAW 264.7 macrophages and human umbilical vein endothelial cells/RAW 264.7 macrophages with varying concentrations of Ru@FeOOH.
FIG. 13 is a fluorescent image of active oxygen probe (dichlorofluorescein-acetoacetate) staining after RAW264.7 (normal control) incubated for 12 h after RAW 264.7+ lipopolysaccharide and 12 h after RAW264.7+ lipopolysaccharide+Ru@FeOOH.
FIG. 14 is a confocal immunofluorescence staining chart of hypoxia inducible factor 1-alpha (HIF-1 alpha) after RAW264.7 (normal control), after another anaerobic incubation for 12 h after RAW 264.7+lipopolysaccharide, and after another anaerobic incubation for 12 h after RAW 264.7+lipopolysaccharide+Ru@FeOOH.
Fig. 15 is a view of RAW264.7 (normal control), 12 h after RAW 264.7+ lipopolysaccharide and 12 h after raw264.7+ lipopolysaccharide+ru@feooh after anoxia incubation: (a) a PCR assay for M1 (iNOS) markers; (b) PCR detection map of M2 (Arg-1) marker.
FIG. 16 is a visual score of arthritis in non-molded mice (Negative Control group), RA-molded mice (Model group) and post-molded Ru@FeOOH treated mice (Ru@FeOOH group).
FIG. 17 is a general image of the Negative Control group, model group and Ru@FeOOH group mice.
FIG. 18 is a graph of hematoxylin-eosin staining (H & E staining) of ankle joints of mice in the Negative Control group, model group and Ru@FeOOH group.
FIG. 19 is a schematic Control group, model group and Ru@FeOOH group mouse ankle joint (a): m1 (iNOS) marker PCR detection diagram; (b) PCR detection map of M2 (Arg-1) marker.
FIG. 20 is a graph showing the results of ELISA detection of inflammatory factors in mice in the Negative Control group, model group and Ru@FeOOH group: (a) IL-1 beta content; (b) TNF- α content.
FIG. 21 is an H & E staining pattern of visceral tissue sections (heart, liver, spleen, lung, kidney) of experimental mice 12 days after treatment.
Description of the embodiments
The invention develops a ROS scavenging biocatalysis material with SOD-like enzyme activity and CAT-like enzyme activity. The invention constructs ruthenium cluster active site on the lamellar structure of ferric oxide hydroxide by one-pot synthesis technique, synthesizes a new biocatalysis material Ru@FeOOH, ru@FeOOH which can remove excessive active oxygen in neutral environment (pH: 7-8) and in acid environmentpH:4 to 6) can generate active oxygen, and has CAT-like enzyme and SOD-like enzyme activities required for scavenging active oxygen and POD-like enzyme activities required for generating active oxygen. Experiments show that Ru@FeOOH has excellent active oxygen scavenging capacity and is specific to H 2 O 2 The scavenging capacity can reach 100% in 30min, and meanwhile 80% -95% of superoxide anions (O) can be scavenged in 10min 2 - ). Meanwhile, ru@FeOOH also shows very excellent potential for treating RA in an RA treatment model.
The following describes the invention in further detail with reference to examples, which are not intended to limit the invention thereto.
The raw materials used in the experiments of the examples of the present invention are shown in Table 1.
Table 1 raw materials table
Raw materials Purity of Source
Hydrated ruthenium trichloride (RuCl) 3 ·xH 2 O） 99% "Bailingwei
Ferric trichloride hexahydrate (FeCl) 3 ·6H 2 O） 99.5% Aladdin
Hexamethylene tetramine 99% An Naiji chemistry
Nickel chloride hydrate (NiCl) 2 ·xH 2 O） 99% Aladdin
The raw materials are not treated, and are directly used after being purchased. The remaining reagents, unless specifically mentioned, were all supplied by Aladin China.
Example 1 preparation of Ru@FeOOH
The invention constructs ruthenium cluster active sites on a lamellar structure of ferric oxide hydroxide in situ by a one-pot synthesis technology, and synthesizes the obtained catalytic material Ru@FeOOH, which comprises the following specific steps:
first 3.154g of hexamethylenetetramine is dissolved in deionized water to prepare a transparent colorless solution, and then 135.2 mg of FeCl is added 3 ·6H 2 O and 27.04 mg RuCl 3 ·xH 2 O（FeCl 3 ·6H 2 O and RuCl 3 ·xH 2 O in a mass ratio of 5:1), stirring for 5min, and placing the obtained solution in a corrosion-resistant autoclave to react at 95 ℃ for 4 h; cooling to room temperature, and collecting the obtained yellow brown precipitate; the precipitate obtained was washed with deionized water and then placed in a vacuum oven at 60 ℃. The solid powder finally obtained is a catalyst Ru@FeOOH-1:5 (Ru@FeOOH is specifically referred to as Ru@FeOOH-1:5 hereinafter).
Example 2 preparation of Ru@FeOOH-1:2
First 3.154g of hexamethylenetetramine is dissolved in deionized water to prepare a transparent colorless solution, and then 135.2 mg of FeCl is added 3 ·6H 2 O and 67.6 mg RuCl 3 ·xH 2 O（FeCl 3 ·6H 2 O and RuCl 3 ·xH 2 O in a mass ratio of 2:1), stirring for 5min, and placing the obtained solution in a corrosion-resistant autoclave to react at 95 ℃ for 4 h; cooling to room temperature, and collecting the obtained yellow brown precipitate; the precipitate obtained was washed with deionized water and then placed in a vacuum oven at 60 ℃. Finally, the obtained solid powder is the biocatalyst Ru@FeOOH-1:2.
Example 3 preparation of Ru@FeOOH-1:10
First 3.154g of hexamethylenetetramine is dissolved in deionized water to prepare a transparent colorless solution, and then 135.2 mg of FeCl is added 3 ·6H 2 O and 13.5 mg RuCl 3 ·xH 2 O（FeCl 3 ·6H 2 O and RuCl 3 ·xH 2 O in the mass ratio of 10:1), stirring for 5min, and placing the obtained solution in a corrosion-resistant autoclave to react at 95 ℃ for 4 h; cooling to room temperature, and collecting the obtained yellow brown precipitate; the precipitate obtained was washed with deionized water and then placed in a vacuum oven at 60 ℃. Finally, the obtained solid powder is the biocatalyst Ru@FeOOH-1:10.
Example 4 preparation of Ru@FeOOH-1:20
First 3.154g of hexamethylenetetramine is dissolved in deionized water to prepare a transparent colorless solution, and then 135.2 mg of FeCl is added 3 ·6H 2 O and 6.8 mg RuCl 3 ·xH 2 O（FeCl 3 ·6H 2 O and RuCl 3 ·xH 2 O in a mass ratio of 20:1), stirring for 5min, and placing the obtained solution in a corrosion-resistant autoclave to react at 95 ℃ for 4 h; cooling to room temperature, and collecting the obtained yellow brown precipitate; the precipitate obtained was washed with deionized water and then placed in a vacuum oven at 60 ℃. Finally, the obtained solid powder is the biocatalyst Ru@FeOOH-1:20.
The synthesis process of the Ru@FeOOH series material is shown in figure 1.
Comparative example 1 Synthesis of FeOOH-Ru
First 3.154g of hexamethylenetetramine is dissolved in deionized water to prepare a transparent colorless solution, and then 135.2 mg of FeCl is added 3 ·6H 2 O, stirring for 5min, and placing the obtained solution in a corrosion-resistant autoclave to react at 95 ℃ for 4 h; cooling to room temperature, and collecting the obtained yellow brown precipitate; washing the obtained precipitate with deionized water, and then placing the washed precipitate into a vacuum drying oven at 60 ℃ to obtain FeOOH after drying. After dissolving the FeOOH in deionized water, 27.04 mg of RuCl was added 3 ·xH 2 O, stirring at normal temperature for 24 and h, collecting the obtained precipitate, washing the obtained precipitate with deionized water, and placing into a vacuum drying oven at 60 ℃ to obtain FeOOH-Ru。
Comparative example 2 Synthesis of FeNiOH-Ru
First 3.154g of hexamethylenetetramine is dissolved in deionized water to prepare a transparent colorless solution, and then 135.2 mg of FeCl is added 3 ·6H 2 O and 357 mg NiCl 2 ·xH 2 O, stirring for 5min, and placing the obtained solution in a corrosion-resistant autoclave to react at 95 ℃ for 4 h; cooling to room temperature, and collecting the obtained yellow brown precipitate; washing the obtained precipitate with deionized water, and then placing the washed precipitate into a vacuum drying oven at 60 ℃ to obtain FeNiOH after drying. After the obtained FeNiOH was dissolved in deionized water, 27.04 mg of RuCl was added 3 ·xH 2 O, stirring at normal temperature for 24 and h, collecting the obtained precipitate, washing the obtained precipitate by deionized water, and then placing the washed precipitate into a vacuum drying oven at 60 ℃ to obtain FeNiOH-Ru.
Experimental example 1 structural characterization of nano biocatalysts
1. The detection method comprises the following steps:
a morphological image of the material was obtained by using a Scanning Electron Microscope (SEM) of regulatory 8220 (japanese hitachi). The material crystal information was obtained by using a DX-2700BH (source instrument) X-ray diffractometer. The morphology image and the elemental mapping image of the material were obtained by Transmission Electron Microscopy (TEM) using a Talos F200x TEM (FEI company, usa) at an operating voltage of 200 kV. Atomic resolution images of the material were obtained at an operating voltage of 200 kV using a JEM-ARM 200F Scanning Transmission Electron Microscope (STEM) from JEOL, which is equipped with a cold field emission electron source and a DCOR probe corrector (CEOS GmbH), one 100 mm 2 JEOL Centurio EDX detector of (a), and a Gatan GIF quantum ERS electron energy loss spectrometer.
2. Detection result:
the SEM properties of the catalytic materials obtained in examples 1-4 and comparative examples 1, 2 are shown in fig. 2a-f, and the results show that the catalytic material ru@feooh obtained by the method in example 1 has the best morphology (smaller particle size and uniform scale).
As shown in FIG. 3, the present invention first performed an X-ray diffraction experiment on the crystal information obtained in example 1, and the experiment confirmed that the crystal structure was FeOOH. To further investigate the fine structure of Ru@FeOOH, HR-TEM images (FIG. 4) showed a typical platelet morphology of Ru@FeOOH, with a size of about 100 nm. As shown in fig. 5, the lattice fringes with the interplanar spacing were 0.368 nm, corresponding to FeOOH (0 1 2) crystal planes. It is clearly observed from STEM images at atomic resolution that ru@feooh is composed of FeOOH crystals (relatively bright arrays of atoms) and that no significant lattice deformation is observed around the FeOOH crystals, indicating that Ru is likely to be uniformly dispersed on the surface of FeOOH crystals as single atoms or as very small clusters. It is believed that the catalyst surface participates in the catalytic process and that the active sites that cause CAT, SOD performance may be concentrated primarily in the surface area of the material. The invention utilizes energy dispersive spectroscopy (EDX) element mapping to verify the distribution of Ru elements loaded on the surface of Ru@FeOOH (figure 6), wherein the Ru elements are uniformly distributed on the outer surface of Ru@FeOOH. In order to confirm the existence form of Ru, the invention performs high-angle annular dark field scanning transmission electron microscope imaging (HAADF-STEM) on the atomic scale, and the image shows that Ru is uniformly distributed on the surface of lamellar FeOOH in a cluster form of about 2nm (figure 7).
The data show that Ru@FeOOH is a type of Fe-based hydroxide lamellar material with Ru cluster loading, and can form highly dispersed Ru cluster active sites on a lamellar substrate of FeOOH, thereby providing great possibility for further researching the catalytic behavior of the FeOOH.
Test example 2 catalytic Properties of the nanocatalyst
1. Detection method
Catalase (CAT) like catalytic activity assay:
by evaluating hydrogen peroxide (H) 2 O 2 ) Is evaluated by the clearance of (a). H 2 O 2 The clearance of (c) is evaluated as follows: will total 10 mM H 2 O 2 (Aladin, shanghai, china) and 50 μg mL -1 Is mixed in PBS to 2 mL. Then, 50. Mu.L of the solution was added to 100. Mu.L of Ti (SO 4 ） 2 Solutions (13.9 mM, aladin, shanghai, china) at 5, 10, 15, 20, 25 and 30min, respectively. Measuring the absorbance of the solution at 405nmDegree to evaluate residual H 2 O 2 Is a concentration of (3).
Superoxide dismutase (SOD) -like activity assay:
·O 2 - from 1mg KO 2 (Sigma-Aldrich, MO, USA) in 1 mL of 18-crown-6-dimethyl sulfoxide (DMSO) solution (Aladin, shanghai, china) (3 mg mL) -1 ) Is generated in the process. Then the catalyst was dispersed to the above final concentration of 50. Mu.g mL, respectively -1 KO of (2) 2 In DMSO solution. After 5min of reaction, 10. Mu.L of nitroblue tetrazolium (NBT) -DMSO solution (10 mg mL) was added -1 NBT, aladin, shanghai, china) to detect remaining. O 2- . The absorbance of the solution at 680 nm was then measured and its clearance O was quantitatively assessed 2- Is provided).
Detection of active oxygen free radicals:
ROS production was detected using 3, 3', 5, 5' -Tetramethylbenzidine (TMB). 25. Mu.L FeOMo 6 @WO x Dispersion (4 mg mL) -1 ) 100 mu L of H 2 O 2 Solution (0.1M) and 24. Mu.L of TMB solution (10 mg. Mu.L) -1 ) Added to NaOAc-HOAc buffer (100 mM, ph=4.5) and the final volume of the mixed system was adjusted to 2mL with NaOAc-HOAc buffer. Then, 200. Mu.L of the liquid was aspirated and the absorbance at 652nm was measured under a microplate reader.
2. Detection result:
after successfully verifying the structure of Ru@FeOOH, the catalytic performance of the catalyst is further studied. First, the antioxidant activity was tested. In the biologically relevant active oxygen, hydrogen peroxide (H 2 O 2 ) Is extremely important because it has higher membrane permeability and specific O 2 - And a longer half-life of OH and a higher intracellular concentration. Thus, the present invention focuses primarily on CAT performance (H 2 O 2 Decomposing to form oxygen (O) 2 )). First, with Ti (SO) 4 ） 2 CAT-like activity was studied as a probe in a time-dependent manner by measuring the absorbance of the solution at 405 nm. As shown in fig. 8a, H increases with time 2 O 2 The concentration gradually decreases. Notably, at 12 minutes, H of Ru@FeOOH-1:5 and Ru@FeOOH-1:2 2 O 2 The concentration ratio is 13.2% -15.5%, which is far lower than Ru@FeOOH-1:10 and Ru@FeOOH-1:20, and the concentration ratio shows that Ru@FeOOH-1:5 and Ru@FeOOH-1:2 have better CAT-like enzyme activity. Then, H of LDHs and Ru-LDHs at 12 minutes was calculated 2 O 2 The cancellation ratio is shown in fig. 8 b. H after Ru@FeOOH-1:5 is added 2 O 2 Has a clearance of 85% and H of the group Ru@FeOOH-1:2 2 O 2 The clearance rate (83.8%) is relatively close to that of Ru@FeOOH-1:10 (69.7%), ru@FeOOH-1:20 (40.8%) and FeNiOH-Ru (50.68%). At the same time greatly exceeds the H of FeOOH-Ru at the same concentration 2 O 2 Clearance (13.4%).
Ru@FeOOH has SOD enzyme-like activity in addition to catalytic activity similar to CAT enzyme by scavenging superoxide anion free radical (. O) 2 - ) But plays a critical role in the balance between oxidation and antioxidation. As shown in FIG. 9, from the absorbance of the obtained solution at 680 and nm, O can be calculated 2 - Is a purge amount of (a). The results show that 50. Mu.g/ml Ru@FeOOH (example 1) vs. O 2 - Is better than Ru@FeOOH-1:2 (69.46%), ru@FeOOH-1:10 (50.12%), ru@FeOOH-1:20 (43.9%), feNiOH-Ru (1.75%) and FeOOH-Ru (1.814%).
Materials with peroxidase-like (POD) activity can catalyze H 2 O 2 Generating ROS (e.g.,. OH,. O) 2 − And 1 O 2 etc.). The POD-like catalytic activity of the catalytic materials obtained in example 1 and comparative example of the present invention was evaluated by typical colorimetric probes 3, 3', 5, 5' -Tetramethylbenzidine (TMB). As shown in fig. 10, the absorbance value at 652nm was negligible for example 2, indicating that FeOOH-Ru has little POD-like catalytic performance. Whereas biocatalysts with multi-metal doping showed slightly increased absorption intensity at 652nm, indicating that FeNiOH-Ru has weak POD-like activity. Notably, ru@FeOOH-1:2 has the highest intensity absorption intensity at 652nm, ru@FeThe absorption intensity of OOH-1:5 is only slightly lower than Ru@FeOOH-1:2. As shown in FIG. 10, the different FeCls of the present invention 3 ·6H 2 O/RuCl 3 ·xH 2 The POD performance of Ru@FeOOH-1:5 and Ru@FeOOH-1:2 materials is the best in the Ru@FeOOH series materials synthesized by O mass ratio. The experimental result proves that the Ru@FeOOH catalyst synthesized by the one-pot method can achieve the highest efficient ROS catalytic performance while simplifying experimental steps.
And combining all catalytic performance results, and optimizing the Ru@FeOOH-1:5 catalytic material with the optimal catalytic performance.
Test example 3 cellular uptake and in vitro biocompatibility of nanocatalysts
The invention assumes that the synthesized Ru@FeOOH catalyst can be taken in by RAW264.7 macrophages (RAW 264.7) and has better biocompatibility.
1. The detection method comprises the following steps:
the uptake of material by RAW264.7 cells was first observed using TEM. Culturing RAW264.7 macrophage, discarding culture solution when the RAW is grown to 80-90%, washing with PBS solution for 3 times, adding Lipopolysaccharide (LPS) into cell dish to stimulate cell differentiation, and adding culture solution containing Ru@FeOOH. Another dish was added with a culture solution containing ru@feooh alone, and placed in a 37 ℃ incubator for continuous incubation 24 h. Taking out the cell dish, discarding the supernatant, washing 3 times with PBS solution, and adding 0.25% trypsin (containing 1 mM EDTA) to digest the cells; cells were collected in centrifuge tubes by conventional centrifugation (1000 rpm, 5 min). The supernatant was discarded, 2mL of 3% glutaraldehyde fixative, which was diluted 1:5, was slowly added along the tube wall with a pipette, and the cells were resuspended and allowed to stand at 4℃for 5 min. The cell suspension was transferred to a 1.5mL tip EP tube, centrifuged at high speed (12000 rpm,10 min), the supernatant was discarded, the cell pellet sample was retained, 3% glutaraldehyde fixative was slowly added along the tube wall with a pipette and stored at 4 ℃. Then 1% osmium tetroxide is fixed again, acetone is dehydrated step by step, and 100% concentration is changed for 3 times. The dehydrating agent and Epon812 are embedded, ultrathin slices of about 60-90 nm are prepared, and the slices are fished to a copper net after being spread. Uranium acetate is firstly dyed for 10-15min at room temperature, and then is dyed for 1-2min by lead citrate. And (5) observing the copper mesh by adopting a Transmission Electron Microscope (TEM) and acquiring images.
The cytocompatibility of the materials was verified by Human Umbilical Vein Endothelial Cells (HUVEC) and RAW 264.7. HUVEC and RAW264.7 cells were cultured in plates containing 10% Fetal Bovine Serum (FBS) (Bioind, israel) and 1% antibiotic mixtures (10000U penicillin and 10 mg streptomycin) respectively, when they were grown to 80-90%, the culture solution was discarded, PBS solution was washed 3 times, culture solutions (RAW 264.7-10. Mu.g/mL, 20. Mu.g/mL, 40. Mu.g/mL; HUVEC-20. Mu.g/mL, 40. Mu.g/mL, 60. Mu.g/mL) containing different concentrations of Ru@FeOOH were added to the different wells, and incubation was continued in a 37℃incubator for 24 h, followed by cell viability/death staining. Treated cells were stained with Calcein-AM (labeled live cells) for 30min and propidium iodide (labeled dead cells) for 2-5 min. The cells were then immediately observed by an inverted fluorescence microscope (Olympus, japan).
2. Detection result:
fig. 11 is a TEM image of RAW264.7 and RAW 264.7+lps incubated with ru@feooh (example 1) for 24 h, respectively, showing that RAW264.7 cells can take up ru@feooh catalyst regardless of whether there is stimulation by inflammatory factor LPS, while RAW264.7 cells can take up more ru@feooh after LPS stimulation, indicating that ru@feooh can reach the therapeutic target, and RAW264.7 cells are more beneficial to the material generation under inflammatory conditions.
FIG. 12 is a fluorescence image of co-cultured Ru@FeOOH materials with different concentrations, HUVECs and RAW264.7 cells, wherein most of the cells are living for pure HUVECs and RAW264.7 groups, and after Ru@FeOOH catalysts with different concentrations are added, a small amount of cells die, and the number of dead cells is not obviously increased compared with that of pure HUVECs and RAW264.7 groups, so that the Ru@FeOOH catalysts have better biocompatibility, and the results indicate that the biocatalyst can be used for in vivo and in vitro experiments.
Test example 4 ROS scavenging, hypoxia mitigation and M1 and M2 polarization and anti-inflammatory Properties of nanocatalysts
The invention assumes that the synthesized Ru@FeOOH catalyst can utilize the CAT enzyme activity and SOD enzyme-like activity of the catalyst, remove ROS generated in inflammatory cells, generate oxygen, improve the hypoxic environment of the cells and achieve the anti-inflammatory purpose.
1. The detection method comprises the following steps:
1.1 For detection of ROS scavenging performance, log phase cells were seeded in 24-well plates and placed in 5% CO 2 Incubate for one day at 37 ℃. After the cells are fully adhered, sucking out the culture medium in the pore plate, carefully washing the culture medium for 1 time by using a PBS solution, adding LPS to stimulate the cells, adding the culture medium containing Ru@FeOOH materials according to groups, and placing the culture medium into a hypoxia incubator for incubation of 12 h. Subsequently, the incubated cells were removed, the medium in the well plate was aspirated, washed 1-2 times with PBS solution, and the prepared active oxygen detection probe, dichlorofluorescein-acetoacetate (DCFH-DA), was added to the well plate and incubated at 37℃for 30 min. Finally, the working solution was aspirated, washed twice with PBS solution, and observed with a fluorescence microscope with an appropriate amount of PBS solution at 4 ℃. At the maximum excitation wavelength of 480 nm, fluorescence signals are detected by using a fluorescence microscope to indirectly reflect the amount of ROS generated in the cell, and the stronger the fluorescence is, the more ROS are contained in the cell, so as to judge the ROS scavenging performance of the material.
1.2 Regarding the performance test for reducing hypoxia, the lower the HIF-1α content, the more reduced the intracellular hypoxia condition was, by utilizing the performance of the test reaction to hypoxia-inducible factor-1α (HIF-1α) in cells.
RAW264.7 cells in logarithmic growth phase were seeded into copolymer Jiao Kongban, 5% CO 2 Incubating at 37 ℃, adding LPS (LPS) into two groups except untreated (control) groups after cells grow fully, adding Ru@FeOOH material into one group, co-culturing in a low-oxygen cell incubator for 12 h, sucking out culture medium, washing with PBS, adding 4% paraformaldehyde for room temperature fixation, adding 0.5% Triton X-100 solution, and allowing room temperature permeation. After blocking with donkey serum, primary antibody incubation (rabbit HIF-1. Alpha. Antibody) was performed followed by secondary antibody (donkey anti-rabbit IgG H)&L (Alexa Fluor 574), red fluorescent staining. Staining cytoskeleton (action) with phalloidin (green fluorescence), incubating at room temperature, and finally dripping 2-3 drops of DAPI-containing sealing liquid on each hole, and observing with confocal microscope (CLSM).
1.3 detection of anti-inflammatory Properties the properties were reflected by PCR detection of the expression of the M1/M2 markers of the cells.
During PCR detection, cell culture and treatment are carried out in the same ROS scavenging performance detection process, then the culture medium is sucked out, PBS is used for washing 3 times, cells are digested by pancreatin, centrifugation (800 rpm,4 min) is carried out, and PBS solution is used for resuspension; after RNA is extracted according to the instructions of the RNA extraction kit, the RNA is reversely transcribed into cDNA according to the requirements of the reverse transcription kit; detection was performed on a fluorescent quantitative PCR instrument by the step of denaturation-annealing-extension-dissolution curve; the experiment uses beta-action as an internal reference gene, and finally obtains Ct values of each gene and the internal reference gene, and analyzes the expression condition of mRNA, wherein M1 type markers: iNOS, type M2 markers: arg-1. The quantitative PCR system configuration of each gene is shown in Table 2, and the basic conditions of the PCR primer sequences are shown in Table 3.
TABLE 2 System configuration for quantitative PCR
2×SYBR qPCR Master Mix 10 μL
Primer Foward (10μM) 0.4 μL
Primer Reverse (10μM) 0.4 μL
Double distilled water 7.2 μL
cDNA 2 μL
TABLE 3 PCR primer sequences
Target gene Direction Sequence
iNOS Forward ACCATGAGGCTGAAATCCCA
Reverse TCCACAACTCGCTCCAAGAT
Arg-1 Forward GTAGACAAGCTGGGGATTGG
Reverse TCAAAGCTCAGGTGAATCGG
2. Detection result:
firstly, the invention detects the ROS scavenging performance of the Ru@FeOOH catalyst obtained in the embodiment 1, DCF green fluorescence in FIG. 13 represents the stained oxidized cells, and it can be seen that the amount of ROS in the pure RAW264.7 cells is the least, the amount of ROS in the group of the LPS added with only inflammatory factors is the most, and the ROS generated in the group of the Ru@FeOOH catalyst added with the other components except the LPS is obviously reduced compared with the single LPS, namely the expression level of ROS is obviously reduced, which indicates the strong ROS scavenging performance of the Ru@FeOOH catalyst.
In addition, the invention also detects the capacity of Ru@FeOOH catalyst to improve Hypoxia, reduce the intracellular HIF-1 alpha by oxygen production, the intracellular HIF-1 alpha is mainly expressed at the cell nucleus position and marked as red fluorescence, CLSM shows that the red fluorescence in untreated (Control) group cells is less, the maximum intensity of the red fluorescence of LPS+Hypoxia group is highest, and the red fluorescence expression of Ru@FeOOH treatment group is lower than that of LPS+Hypoxia group (FIG. 14). It is shown that the HIF-1 alpha expression of RAW264.7 cells is higher in the anoxic environment, and oxygen generated after Ru@FeOOH treatment can relieve the condition of hypoxia of the cells, so that the expression of the HIF-1 alpha in the cells is down-regulated.
The invention also proves the anti-inflammatory performance of Ru@FeOOH through detecting the expression of M1 and M2 markers by PCR. The PCR results in FIGS. 15a and b suggest that LPS stimulated group has the highest expression of the M1 type marker iNOS, the M2 type marker Arg-1 has lower expression, while Ru@FeOOH treated group has reduced expression of iNOS and increased expression of Arg-1, demonstrating the ability of the material to promote macrophage M2 type polarization and anti-inflammatory properties.
Experimental example 5 RA treatment experiment and results for nanocatalysts
According to the method, potential feasibility of treating RA by Ru@FeOOH is further evaluated through animal RA modeling, ru@FeOOH treatment and a series of treatment effect detection experiments after treatment.
1. The detection method comprises the following steps:
male DBA/1 mice with the body weight of 15-20 g and the age of 6-8 weeks are selected as animal experiment modeling objects, all animal experiment procedures are approved by the ethical committee of animals of university of Sichuan, and the animal experiment procedures conform to the nursing principle of experimental animals formulated by national medical research institute. The CIA mouse model was established according to standard methods, and first an emulsion for immunization was prepared. Mixing 2mg/mL of bovine type II collagen acetic acid solution (CII) with an equal volume of Freund's complete adjuvant (CFA) under ice water bath by a high-speed shearing machine, emulsifying, continuously stirring for 2 minutes at 20000 r/min, cooling for 5 minutes at 0 ℃, repeating for 2-3 times, and preparing the emulsion successfully when the emulsion is completely dripped into water and is not stunned. Two or more points at approximately 1-2 cm near the root of the tail of the mouse were injected intradermally with 150. Mu.L of the emulsion (CII content: 150. Mu.g). Boosting injections were performed 21 days after the first immunization, and an equal volume of Freund's incomplete adjuvant (IFA) and bovine type II collagen acetic acid solution (CII, 2 mg/mL) were used to prepare an emulsion, and 100. Mu.L of the emulsion (CII content: 100. Mu.g) was injected intradermally at two or more points about 1-2 cm near the root of the mouse tail. The progress of arthritis was monitored daily, and at 28 days after primary immunization, the joints of mice had been significantly red and swollen, and the symptoms of lameness and anorexia were seen as successful modeling. Firstly, taking 3 normal mice as a group (1) of positive Control, and additionally selecting 6 successfully modeled mice, wherein 3 mice in each group are randomly divided into the following two groups: (2) a Control group; (3) Ru@FeOOH group. (3) The Ru@FeOOH group is injected with the medicine through the tail vein of the mouse, the injection dose of each reagent is 50 mu L, the concentration is 2mg/mL, and the PBS liquid is injected into the groups (1) and (2). The mice were sacrificed on days 35, 37, 39, 41 and 43 after primary immunization for a total of 5 treatments, and were visually scored for arthritis prior to each treatment, and specific scoring criteria for paw arthritis in the mice are shown in table 4. On day 45, taking eyeball blood of the mice before killing, centrifuging to obtain serum of the mice, and expressing serum inflammatory factors (including IL-1 beta and TNF-alpha) according to an ELISA kit; immediately after sacrifice, the ankle and viscera of the mice were taken and fixed with 10% formaldehyde solution for histological H & E staining analysis and tissue PCR detection (PCR detection method as before, detection of M1 type markers iNOS, M2 type markers Arg-1.).
TABLE 4 visual scoring criteria for CIA arthritis in mice
Scoring of Clinical manifestations
0 No red and swelling
1 Redness and swelling of joints
2 Two types of joint redness and swelling
3 Redness and swelling occurred in all three types of joints
4 The whole paw is red and swollen, and the appearance is free of anatomical marks
2. Detection result:
in the invention, in the treatment process of 5 times, the visual grade of arthritis is carried out on mice before each treatment (the visual grade of the single paw arthritis of each mouse is 0-4 minutes, the visual grade of the arthritis is 0-16 minutes, and the paw of each mouse is provided with a model), as shown in fig. 16, the grade of the arthritis in a Control group gradually rises along with time and reaches a peak on the 45 th day, the grade of the arthritis in a Ru@FeOOH group initially gradually increases along with time, the grade result at the 43 th day is lower than the previous grade and tends to be stable on the 45 th day, the treatment is prompted to have a certain inhibition effect on the inflammation progress, and the anti-inflammatory performance of the material is illustrated. After 5 treatments were completed, the joints of the mice were photographed before being sacrificed (fig. 17), and the general images showed that the joints of the mice in the different groups were swollen to different extents, the Control group was swollen most significantly and the ru@feooh group was swollen to a relatively light extent, and scored visually.
The invention takes the ankle joint of the mice to carry out pathological H & E staining (figure 18), and the result indicates that Ru@FeOOH treatment group mice show lighter synovitis in ankle joint cavity than Control group, and the treatment has a certain anti-inflammatory effect. In the invention, the ankle joint of the mouse is additionally taken for tissue PCR detection, as shown in figures 19a and b, the expression of the M1 type marker iNOS in the Ru@FeOOH treatment group is obviously reduced compared with that in the Control group, and the expression of the M2 type marker Arg-1 is increased compared with that in the Control group, so that the capacity of the Ru@FeOOH catalyst for promoting the M2 type polarization of macrophages is further verified in vivo experiments.
In addition, the serum inflammation index can reflect the systemic inflammation status of CIA mice. According to ELISA detection method, the content of IL-1 beta and TNF-alpha in the serum is detected, and the result shows (as shown in figures 20a and b), the IL-1 beta and TNF-alpha of the simple model serum are obviously increased, and the IL-1 beta and TNF-alpha levels of the mice are obviously reduced to be close to the normal mice after being treated by Ru@FeOOH catalyst.
Meanwhile, in order to further verify the biocompatibility of the material, the invention further performs H & E staining on main organs (heart, liver, spleen, lung and kidney) of experimental mice, and no obvious damage or abnormality is found in the tissue sections (figure 21), which indicates that the method for treating RA by using Ru@FeOOH has biological safety.
The results prove that the Ru@FeOOH catalyst can achieve a better RA treatment effect by promoting the polarization of M2 macrophages, reducing inflammatory factors and the like.
In conclusion, the nano biocatalyst Ru@FeOOH obtained by the method has good enzyme-like activity. Ru@FeOOH can remove excessive active oxygen in a neutral environment (pH: 7-8), and can generate active oxygen in an acidic environment (pH: 4-6), and has CAT-like enzyme and SOD-like enzyme activities required for removing active oxygen and POD-like enzyme activities required for generating active oxygen. Experiments show that Ru@FeOOH has excellent active oxygen scavenging capacity and is specific to H 2 O 2 The cleaning capacity can reach 100% in 12 min, and meanwhile, 80% -95% of O can be cleaned in 10min 2 - . The in vitro cell experiment result shows that Ru@FeOOH has better biocompatibility, and can relieve the hypoxia environment in the joint cavity by removing excessive ROS in cells and generating oxygen, so that M2 macrophage polarization is promoted to jointly achieve the anti-inflammatory effect, and the in vivo experiments of mice further verify the biosafety of Ru@FeOOH and the effectiveness of anti-inflammatory treatment by evaluating the performance of arthritis, detecting inflammatory factors and the like. Therefore, the material design and performance of the Ru@FeOOH catalyst are developed to provide a new strategy for scavenging ROS and regulating macrophage polarization so as to treat rheumatoid arthritis.

Claims (9)

1. A method for preparing a ROS scavenging biocatalytic material, the method comprising: the iron salt, ruthenium salt and hexamethylenetetramine or urea are subjected to one-pot solvothermal reaction to prepare the biocatalysis material; the biocatalysis material is prepared by constructing ruthenium clusters on the surface of ferric hydroxide in situ, and is denoted as Ru@FeOOH.
2. The method of claim 1, wherein the iron salt is selected from the group consisting of: feCl 3 ·6H 2 O; the ruthenium salt is selected from: ruCl 3 ·xH 2 O。
3. The method for preparing the ROS scavenging biocatalytic material according to claim 2, wherein the mass ratio of the iron salt to the ruthenium salt is: 1: 1-20: 1.
4. a method of preparing a ROS scavenging biocatalytic material according to claim 3, wherein the mass ratio of the iron salt and the ruthenium salt is 2:1, 5:1, 10:1 or 20:1.
5. The method of claim 1, wherein the method of preparing comprises: firstly, dissolving hexamethylenetetramine or urea in a solvent to prepare a transparent colorless solution, then adding ferric salt and ruthenium salt, stirring for 0-15min, and placing the obtained solution in a corrosion-resistant autoclave to react at 80-110 ℃ for 3-6 h; cooling to room temperature, and collecting the obtained yellow brown precipitate; washing and drying the obtained precipitate to obtain the biocatalysis material.
6. The method of claim 1, wherein the biocatalytic material has a sheet-like structure.
7. Use of a ROS scavenging biocatalytic material prepared by the method of any one of claims 1-6 for the preparation of a scavenging active oxygen material.
8. Use of a ROS scavenging biocatalytic material prepared by the method of any one of claims 1-6 for the preparation of stem cell protective materials or for the preparation of materials for the treatment of periodontitis.
9. Use of a ROS scavenging biocatalytic material prepared by the method of any one of claims 1-6 for the preparation of a biocatalytic material having both POD-like enzymatic activity, CAT-like enzymatic activity and SOD-like enzymatic activity.
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