CN116139166A - Biocatalyst integrating SOD/CAT and preparation method and application thereof - Google Patents

Abstract

The present invention relates toA biocatalyst integrating SOD/CAT, a preparation method and application thereof belong to the field of biocatalysis materials. The invention uses iridium salt, cobalt salt, urea and NH ₄ F undergoes a precipitation reaction to prepare a CoO-Ir catalyst, wherein the CoO-Ir catalyst is a clustered branched sphere with Ir clusters uniformly distributed on a CoO substrate, and the catalyst has Ir-O bonds. CoO-Ir shows higher efficiency in terms of SOD and CAT activity, and integrates the activity of SOD/CAT. Further, coO-Ir synergistically enhances ROS scavenging activity, protects bone marrow mesenchymal stem cells and osteoblast precursor cells from ROS attack, and promotes in vitro osteogenic differentiation. In addition, coO-Ir shows excellent stepwise treatment effect of periodontitis in vivo, exerts anti-inflammatory effect by scavenging ROS early and realizes tissue regeneration by promoting bone later.

Description

Biocatalyst integrating SOD/CAT and preparation method and application thereof

Technical Field

The invention relates to a biocatalyst integrating SOD/CAT, a preparation method and application thereof, belonging to the field of biocatalysis materials.

Background

Periodontitis is a chronic inflammatory disease with a worldwide incidence of over 40% as one of the most common oral diseases. Periodontitis is considered to be the leading cause of tooth loss and is also associated with several systemic diseases such as atherosclerosis and diabetes. Currently, the primary strategy for clinical periodontal treatment is to prevent the progression of the disease, including mechanical cleaning, antibiotics and anti-inflammatory drugs, and periodontal surgery. However, reconstruction of periodontal tissue structure is difficult to achieve because of the persistent exposure to the oral bacterial environment, and local inflammation is difficult to eliminate. Thus, there is a need for effective alternatives to periodontal treatment that are non-invasive with minimal side effects.

Over the whole inflammatory periodontal tissueIn the course, bacteria in subgingival plaques lead to recruitment of immune cells (mainly polymorphonuclear neutrophils). While these "overactive" immune cells were originally designed to protect against bacterial pathogens, they overproduce Reactive Oxygen Species (ROS) and cause pathological changes. ROS are a class of free radicals (especially O ₂ ^- 、H ₂ O ₂ And OH (OH) ^- ) An imbalance between excessive ROS and antioxidant defense systems, originating from respiratory bursts of mitochondria, leads to oxidative stress. Direct tissue damage by ROS is mainly through lipid peroxidation, but also causes damage to proteins and DNA. In addition, ROS mediate many physiological and pathological signal transduction, thereby promoting inflammation and osteoclast formation. The ROS-initiated inflammatory environment will inhibit the expression of osteogenic markers such as bone morphogenic protein-2 (BMP-2), runt-related transcription factor 2 (Runx 2), and alkaline phosphatase (ALP) of stem cells. In addition, ROS promote RANKL-induced osteoclastogenesis that will break the equilibrium relationship between osteoclastogenesis and bone resorption. Thus, ROS can trigger pro-inflammation and lead to bone loss, consistent with clinical symptoms in patients with periodontitis. Accordingly, ROS in the local microenvironment is regulated to protect stem cells and osteoblasts from oxidative stress damage and to enhance their osteogenic capacity, which is critical for the regenerative treatment of periodontitis.

Effective ROS scavenging can alter the local microenvironment and inhibit pathological progression under inflammatory conditions. Various antioxidant defense strategies based on natural enzymes, nanoenzymes and antioxidants have been developed to maintain intracellular redox balance. Natural enzymes in biological systems, such as superoxide dismutase (SOD) and Catalase (CAT), perform well in eliminating ROS. The closed cascade of SOD and CAT is advantageous over traditional multi-step reactions in that it encloses multiple enzymes within subcellular organelles to ensure amplification and efficient metabolism of the cascade signal. It starts with SOD and adds-O ₂ Conversion to H ₂ O ₂ (typically mitochondrial or cell membrane) and then H is removed by CAT ₂ O ₂ Conversion to water and O ₂ (typically in the peroxo). However, the efficiency of this natural system is limited by SOD and CAAdditional transport steps between T, because they are not within the same subcellular organelle. In addition, native enzymes are limited by numerous self-deficiencies, such as high cost, low stability, and potential immunogenicity. To address these limitations, the need for artificial biocatalysts has increased dramatically. Although most nanoezymes require mutually incompatible reaction conditions and typically exhibit only one or two antioxidant enzyme activities. Inspired by the cascade catalytic reaction system in organisms, we report here a single-component cascade nanoenzyme with high overall activity, and demonstrate for the first time its ability to efficiently scavenge ROS in periodontitis diseases.

Cobalt-based biomaterials have a positive effect on vascular formation in promoting regeneration of stem cells and osteoblasts, coupling osteogenesis and angiogenesis together by activating the hypoxia-inducible factor 1-alpha (HIF-1 a) pathway and upregulating Vascular Endothelial Growth Factor (VEGF). Cobalt has been reported to exert excellent osteogenic activity in combination with bioactive glass, hydroxyapatite, BMP-2 and stem cells. Cobalt-based biomaterials are of interest in the field of bone regeneration as a promising alternative due to their low cost and ease of handling.

Disclosure of Invention

In view of the above drawbacks, the present invention is directed to a process for preparing a catalyst comprising iridium salt, cobalt salt, urea and NH ₄ F undergoes a precipitation reaction to prepare a CoO-Ir catalyst, wherein the CoO-Ir catalyst is a clustered branched sphere with Ir clusters uniformly distributed on a CoO substrate, and the catalyst has Ir-O bonds. CoO-Ir shows higher efficiency in terms of SOD and CAT activity, and integrates the activity of SOD/CAT. Further, coO-Ir synergistically enhances scavenging ROS activity, protects bone marrow mesenchymal stem cells (BMSCs) and osteogenic precursor cells (MC 3T 3-E1) from ROS attack, and promotes osteogenic differentiation in vitro. In addition, coO-Ir shows excellent stepwise treatment effect of periodontitis in vivo, exerts anti-inflammatory effect by scavenging ROS early and realizes tissue regeneration by promoting bone later.

The technical proposal of the invention

The first technical problem to be solved by the invention is to provide a biocatalyst integrating SOD/CAT, wherein the catalyst is formed by uniformly distributing Ir clusters on a CoO substrate, and the catalyst has Ir-O bonds.

Further, the catalyst has a crystal structure.

Further, the catalyst has SOD and CAT activities.

Further, the catalyst also has the activity of scavenging ROS.

The second technical problem to be solved by the invention is to provide a preparation method of the biocatalyst, which comprises the following steps: iridium salt, cobalt salt, urea and NH ₄ F, performing coprecipitation reaction and calcining to obtain the catalyst.

Further, the iridium salt is at least one selected from iridium chloride and iridium acetylacetonate; the cobalt salt is at least one selected from cobalt nitrate, cobalt acetate and cobalt chloride.

Further, the iridium salt is preferably iridium chloride, and the cobalt salt is preferably cobalt nitrate.

Further, in the above preparation method, urea is used as a mineralizer to provide an alkali source for the whole reaction. The urea reacts at high temperature and high pressure to produce ammonium and carbonate, the ammonium provides alkaline environment to precipitate iridium and cobalt to form hydroxide, and the carbonate serves as intercalation anion, so that iridium/cobalt-carbonate hydroxide is prepared through one-step reaction.

Further, in the above preparation method, NH ₄ F is a complexing agent, is a stabilizer for the whole hydrothermal reaction, and is favorable for crystallization of the product.

In the preparation method, the mixture ratio of the raw materials is cobalt salt, iridium salt and NH ₄ The molar ratio of the urea to the urea is 5-20:0.5-2:15-60:25-100, preferably 10:1:30:50.

Further, the conditions of the coprecipitation reaction are as follows: iridium salt, cobalt salt, urea and NH ₄ And F, dispersing the mixture into water, stirring, reacting for 7-10 hours (preferably 8 hours) at 110-130 ℃ (preferably 120 ℃), cooling, centrifuging and washing.

Further, the calcination conditions are: in nitrogen or inert atmosphere, heating at 350-450 ℃ (preferably 450 ℃) for 2-5 ℃ min ⁻¹ (preferably 2 ℃ C. Min ⁻¹ ) Under the condition of (2)Calcining for 2-3 h (preferably 2 h) to obtain the catalyst.

Further, the inert atmosphere is at least one of helium, neon, argon, krypton, xenon and radon.

The third technical problem to be solved by the present invention is to provide the use of the SOD/CAT integrated biocatalyst for scavenging ROS.

The fourth technical problem to be solved by the invention is to provide the application of the biocatalyst integrating SOD/CAT in preparing medicines for treating inflammatory diseases.

Further, the inflammatory diseases refer to orthopedic inflammatory diseases such as acute osteomyelitis, skull defect, fracture, osteoarthritis and the like, and/or other inflammatory diseases such as periodontitis, atherosclerosis, acute kidney injury, acute liver injury, reperfusion injury of myocardial ischemia and the like.

The fifth technical problem to be solved by the present invention is to provide the use of the SOD/CAT integrated biocatalyst for cytoprotection and promotion of cell differentiation, wherein the cells are stem cells and differentiated cells. The biocatalyst integrating SOD/CAT can ensure that the stem cells and the precursor cells are not stimulated by inflammation in the cell differentiation process, and can promote the cell differentiation.

Further, the stem cells are bone marrow-derived, umbilical cord-derived or adipose-derived mesenchymal stem cells, and the differentiated cells are osteoblast precursor cells, endothelial precursor cells or neural precursor cells.

The beneficial effects of the invention are that

The biocatalyst of the invention integrates SOD/CAT activity, and realizes excellent ROS cascade scavenging activity in monomer systems. While protecting stem cells and precursor cells, differentiation of stem cells and precursor cells is promoted. The biocatalyst realizes good step-by-step treatment of periodontitis through early anti-inflammatory and late osteogenesis, and provides an effective new way for inflammatory diseases.

Drawings

FIG. 1 is a schematic diagram of the synthesis of CoO-Ir.

FIG. 2a is an SEM image of CoO-Ir, FIG. 2b is a TEM image of CoO-Ir, FIG. 2c is an HR-TEM image of CoO-Ir, and FIGS. 2d and 2e are SEM images of CoO under different fields of view; FIG. 2f is the XRD pattern for CoO and CoO-Ir.

FIG. 3a is a full scan of XPS spectra for CoO and CoO-Ir; FIG. 3b shows XPS spectrum of Co 2p structure; FIG. 3c shows XPS spectrum of Ir 4f structure; FIG. 3d is an XPS spectrum of O1 s structure.

FIG. 4 is a graph showing SOD performance of CoO-Ir, wherein FIG. 4a shows CoO and CoO-Ir elimination.O ^2- FIG. 4b shows the SOD activity of the samples at different concentrations ^2- FIG. 4c is a chart showing the activity of the KSCN for detecting CoO-Ir.

FIG. 5 is a graph of CAT performance of CoO-Ir, FIG. 5a is H at various times ₂ O ₂ Dynamic elimination Activity map, FIG. 5b is O at various times ₂ Dynamic generation of Activity map, FIG. 5c is a graph of H ₂ O ₂ Typical Michaelis-Menten curves and birefringence maps for the kinetic constants of the substrate CoO-Ir; FIG. 5d is CoO and CoO-Ir with H ₂ O ₂ Vmax and Km values of (c); FIG. 5e is a graph comparing Vmax and TON values for CoO-Ir and other recently reported ROS-eliminating biocatalysts.

FIG. 6a is CoO decomposition H ₂ O ₂ Is O ₂ FIG. 6b shows Michaelis-Menten plots of different concentrations H ₂ O ₂ Double reciprocal plot of CoO kinetic constants.

FIG. 7 is a graph comparing CAT performance at different calcination temperatures and under different metal doping conditions.

FIG. 8a is a graph of the biocompatibility of CoO and CoO-Ir for BMSC cells, and FIG. 8b is a graph of the biocompatibility of CoO and CoO-Ir for MC3T3-E1 cells.

FIG. 9 is a graph showing the cytocompatibility of biocatalysts at various concentrations, wherein FIG. 9a is a quantitative analysis of live/dead double-stained fluorescence of BMSCs and MC3T3-E1, and FIGS. 9b and 9c are graphs showing cell proliferation assays of BMSCs and MC3T3-E1 cells after incubation with CoO and CoO-Ir at various concentrations.

FIG. 10 is a graph of the apoptosis rate detected by Annexin V-FITC/PI staining, wherein numerals 1 and 5 represent concentrations of 1 and 5 μg mL ^-1 。

FIG. 11 is a graph showing intracellular ROS scavenging activity of various concentrations of biocatalysts, wherein FIG. 11a is a graph showing the oxidative stress modeling of BMSCs cells, FIG. 11b is a graph showing the oxidative stress modeling of MC3T3-E1 cells, FIG. 11c is a microscopic field plot showing ROS elimination of BMSCs and MC3T3-E1 after various treatments, and FIG. 11d is a representative graph showing CFH-DA fluorescence. Control: cells without treatment; h ₂ O ₂ : with 150 mu M H ₂ O ₂ Pretreated cells; coO and CoO-Ir: cells pretreated with the corresponding biocatalyst (1 μg/mL) and then associated with 150 μg M H ₂ O ₂ Co-cultivation is performed.

FIG. 12 is a graph showing the effect of CoO-Ir on protecting BMSCs from ROS and promoting osteogenesis, wherein FIG. 12a is a graph showing the ratio of dead cells calculated by overactivity/dead staining, FIG. 12b is a graph showing the number of cell branches calculated by F-actin staining, FIG. 12c is a graph showing F-actin staining with phalloidin, FIG. 12d is an ALP assay on day 7 of osteogenesis induction, FIG. 12e is an ARS staining on day 10, FIG. 12F is a graph showing quantitative analysis of ALP activity, and FIGS. 12g and 12h are semi-quantitative analysis of BMP-2 and Runx2 on day 7 of osteogenesis induction.

FIG. 13 is a graph showing the BMSC cytoprotective effect of CoO-Ir by scavenging active oxygen.

FIG. 14 is a graph showing the inhibition of BMSC apoptosis by CoO-Ir by scavenging active oxygen.

FIG. 15 is a graph showing the recovery of MC3T3-E1 cell activity and promotion of osteogenesis in oxidative stress by CoO-Ir, wherein FIG. 15a is a graph showing the dead cell ratio calculated by live/dead staining, FIG. 15b is a graph showing the number of cell branches calculated by F-actin staining, FIG. 15c is an ALP detection graph on day 7, FIG. 15 d is an ARS staining graph on day 14, and FIG. 15E is a graph showing the expression of osteogenesis-related genes and osteogenesis markers.

FIG. 16 is a graph showing the protective effect of CoO-Ir on MC3T3-E1 cells by scavenging active oxygen.

FIG. 17 is a graph showing the inhibition of MC3T3-E1 apoptosis by CoO-Ir by scavenging active oxygen.

FIG. 18 is a cytoskeletal staining chart of MC3T3-E1 cells.

Fig. 19 is a view of CoO-Ir scavenging periodontitis active oxygen to save alveolar bone loss, wherein fig. 19a is a view of maxillary micro-CT scan (upper layer) and longitudinal 3D reconstruction (lower layer), fig. 19b is a view of periodontal tissue DHE fluorescent staining, fig. 19c is a view of micro-CT total bone mass loss, fig. 19D is a view of CEJ to ABC distance (mm), fig. 19e is a graph of percentage of bone trabecular volume (BV/TV), and fig. 19f is a view of bone trabecular thickness (tb.th).

FIG. 20 is a graph of in vivo safety tests of CoO-Ir.

Detailed Description

The following describes the invention in further detail with reference to examples, which are not intended to limit the invention thereto.

In the examples of the present invention, cobalt nitrate hexahydrate (Co (NO ₃ ) ₂ ·6H ₂ O), ammonium fluoride (NH) ₄ F) And urea from aladine (Shanghai, china), iridium (III) chloride hydrate (IrCl) ₃ •xH ₂ O) was purchased from ampoul gei chemistry (Shanghai, china). All other chemicals were obtained from a company of acla Ding Shiji and used as received materials without further purification, and deionized pure water (18.2M Ω -cm) used in the experiments was produced by Milli-Q academy system (Millipore corp., billerica, MA, USA). All chemicals used were analytical grade without any further purification.

Example 1

First, 2mmol Co (NO) ₃ ) ₂ ·6H ₂ O、0.2mmol IrCl ₃ •xH ₂ O、6mmol NH ₄ F and 10mmol urea were dispersed in 70mL deionized water and stirred at room temperature for 30 minutes. The prepared solution was then sealed in a 100 ml teflon lined autoclave and placed in an oven at 120 ℃ for 8 hours. After natural cooling to room temperature, the precursor was collected by centrifugation and washed with deionized water and alcohol to remove impurities. Finally, under the action of argon gas flow, the heating rate is 2 ℃ for min at 450 DEG C ⁻¹ Is calcined for 2 hours under the conditions of (a) to finally convert the precursor into Ir ⁰ /Ir ⁴⁺ Co-doped CoO.

Comparative examples 1 to 5

IrCl was not added under the same conditions as in example 1 ₃ •xH ₂ O synthesis control CoO (comparative example 1), and addition of the corresponding metal salt, coO-Cu (comparative example 2), coO-Cu/Ru (comparative example 3), coO-Ru (comparative example 4), coO-Rh (comparative example 5) were prepared.

Test example 1 structural characterization

1. Test method

Scanning Electron Microscope (SEM) images were obtained by using Thermo Fisher Scientific (FEI) Apreo S HiVoc. Transmission Electron Microscopy (TEM) and Energy Dispersive Spectroscopy (EDS) images were obtained by Talos F200x TEM microscopy (FEI ltd., usa) operating at 200kV and analyzing by GMS-free analysis. Powder X-ray diffraction Pattern (PXRD) showed crystalline phase by copper radiation in the 2θ range of 40 kV, 5-80 ° by DX-2700BH multipurpose X-ray diffractometer (XRD, a source of a zebra, china). X-ray photoelectron spectroscopy (XPS) was measured on a K-Alpha ™ +X-ray photoelectron spectrometer system (Thermo Scientific) equipped with a hemispherical 180 DEG bifocal analyzer and a 128 channel detector to detect the valence state and electronic structure of the compound.

2. Experimental results

By IrCl ₃ 、Cu(NO ₃ ) ₂ Urea and NH ₄ The coprecipitation reaction between F prepares iridium/cobalt-hydroxide precursor. Then annealing the precursor in argon environment to generate metals Ir and Ir ⁴⁺ Doped CoO, named CoO-Ir, is synthesized schematically as shown in FIG. 1.

The morphology and distribution of CoO-Ir was further studied using SEM and TEM, the sample being clustered branched spheres of uniform size (fig. 2 a), ir nanoclusters being uniformly distributed throughout the CoO matrix (fig. 2 b). CoO-Ir was more directly observed using HR-TEM, showing clear CoO lattice fringes and Ir clusters (FIG. 2 c). If IrCl is not added ₃ A large rod-like CoO is obtained (fig. 2 d) and the CoO assumes a broken rod-like structure after further reduction of the field of view (fig. 2 e). To understand the effect of iridium on the CoO crystal structure, the resulting iridium/cobalt-hydroxide precursor was calcined at the same temperature of 450 ℃ and passed through XRThe crystal structure of the samples obtained by D analysis showed similar XRD patterns as shown in fig. 2f, both samples had a face-centered cubic CoO (PDF No. 97-000-9865) with no detectable Ir diffraction peak, confirming that Ir clusters were uniformly distributed on the CoO substrate. Meanwhile, due to the introduction of Ir species, the diffraction peak of CoO-Ir is slightly offset by an angle from that of CoO, indicating local structure shrinkage.

After verifying the crystal structure, we further explored the chemical composition and valence state of CoO-Ir using X-ray photoelectron spectroscopy (XPS). XPS showed the presence of C, O, co and Ir elements in the CoO-Ir composite (FIG. 3 a), confirming successful modification of Ir species on the CoO substrate. To understand the detailed binding information for each element, we analyzed high resolution XPS spectra. First, we studied the chemical state of cobalt in CoO-Ir. FIG. 3b shows Co 2p spectra, co 2p _3/2 The binding energy of the catalyst is 780.65/783.87 eV, co 2p _1/2 The binding energy of (a) was 796.44/799.66 eV, and two distinct satellite peaks (labeled "sat.") could be ascribed to Co ²⁺ Co was not observed ³⁺ Is a peak of (c). These results indicate that only CoO is present in the sample. The high resolution Ir 4p spectrum of FIG. 3c shows that the main peaks of 61.06 eV and 64.3 eV are from Ir 4f of Ir0 _2/7 And Ir 4f _2/5 The remaining peaks are from Ir ⁴⁺ Indicating that the Ir precursor has been successfully reduced to metallic Ir. It should be noted that the peaks at 59.65 eV and 63.05 eV correspond to Co 3p. Furthermore, the O1 s spectrum of FIG. 3d shows three characteristic peaks at 529.85 eV, 531.56 eV and 532.93 eV, which belong to the M-O-M, M-O-H and H-O-H bonds, respectively.

Test example 2 enzyme Activity test

2.1 test methods

CAT-like enzyme assay

H ₂ O ₂ And (3) clearing: first, 20 mu L H ₂ O ₂ (1M), 1.97mL of PBS (pH=7.4) and 10. Mu.L of CoO-Ir (10 mg/mL) were mixed. Then 50. Mu.L of the solution was added to 100. Mu.L of Ti (SO ₄ ) ₂ Solution (13.9 mM, allatin, shanghai, china) measured at 40 at 2, 4, 6, 8, 10, 12 and 15 minutesAbsorbance at 5nm to evaluate biocatalyst H ₂ O ₂ Scavenging ability.

O ₂ Generating and measuring: will be 200 mu L H ₂ O ₂ (10M), 20. Mu.L CoO-Ir (10 mg/mL), 20 mL PBS (pH=7.4) were mixed and then O was measured every 5 s using an oxygen dissolving meter (INESA, JPSJ-605F) ₂ Concentration up to 300 s.

To analyze O ₂ The biocatalytic kinetics of the formation are modified by H ₂ O ₂ To evaluate steady state kinetic detection of catalase activity. H at different concentrations ₂ O ₂ (50-1200-mM) was mixed with PBS solution containing 10. Mu.g/mL CoO-Ir to finally obtain 20. 20 mL, and then O was measured every 5 seconds ₂ Concentration up to 300 s. According to the corresponding H ₂ O ₂ The reaction rate was plotted for concentration and then fitted with Michaelis-Menten curve (equation (1)). Furthermore, a linear double reciprocal graph (Lineweaver-Burk graph, equation (2)) was used to determine the maximum reaction rate (V) _max ) And Michaelis-Menten constant (Km). Further, the number of revolutions (TON, the maximum number of conversion substrates per unit active catalytic center) was calculated according to equation (3). [ E ₀ ]Represents the molar concentration of metal in the material.

(1)

(2)

(3)

SOD-like enzyme assay

O ₂ ^- And (3) clearing: 1mg KO ₂ Dissolved in 1mL of dimethyl sulfoxide solution (DMSO, containing 3 mg/mL of 18-crown-6-ether). Then, coO-Ir was dispersed in the above KO at a final concentration of 50. Mu.g/mL ₂ In DMSO solution. After 5 minutes of reaction, residual. O ₂ ⁻ Will be detected by Nitro Blue Tetrazolium (NBT) -DMSO solution (10. Mu.L, 10 mg/mL). The absorbance of the solution at 680 and nm is measured and then compared with. O ₂ ⁻ Comparative evaluation of the original concentration of (A) O ₂ ⁻ Scavenging ability.

2.2 experimental results

We further tested and compared their catalytic ROS scavenging activity, including. O ^2- And H ₂ O ₂ . Due to removal of O ^2- As the first step in the anti-ROS cascade, we first studied the SOD-like activities of CoO and CoO-Ir, using tetrazolium sodium nitrate blue chloride (NBT) as the sensitive. O ^2- Indicators monitor their elimination O ^2- Is provided). As shown in FIG. 4a, both CoO and CoO-Ir showed strong SOD-like activity, indicating that Co in CoO has a key effect on the simulated SOD-like activity. The slightly higher SOD-like activity of CoO-Ir is probably due to the synergistic effect of Ir clusters. Meanwhile, as can be seen from FIG. 4b, two sample pairs. O ^2- Is dose-dependent, at very low concentrations (10 μg mL ⁻¹ ) CoO-Ir pair. O ^2- The removal rate of (2) can reach about 50%. Active site toxicity was also employed to verify the importance of Ir clusters in biocatalysts by using potassium thiocyanate reagent (KSCN, which can bind to metal centers to form inactive chelates). The CoO-Ir biocatalyst showed a significant decrease in catalytic activity upon addition of KSCN (fig. 4 c), confirming that the Ir center is the active site for ROS elimination.

H ₂ O ₂ Is O. ^2- Downstream product of (C) with. O ^2- As well as a strong oxidizing agent. CAT catalyzed H in vivo ₂ O ₂ Decomposition into H ₂ O and O ₂ This is the second key step in scavenging ROS cascade systems. We monitored for H ₂ O ₂ Decomposition and O ₂ CAT-like performance was generated that evaluated for CoO and CoO-Ir. At time-varying H ₂ O ₂ In the scavenging test, coO-Ir showed significant H compared to the original CoO ₂ O ₂ Abrogating activity (86.3%) (fig. 5 a). O (O) ₂ The generation experiments also confirm that CoO-Ir can rapidly convert H ₂ O ₂ Decomposition into O ₂ . FIG. 5b shows different concentrations of H in the presence of CoO and CoO-Ir ₂ O ₂ Decomposition into O ₂ Rate. At the same concentration, coO-Ir showed better CAT-like activity. Subsequently we calculated and compared the maximum reaction rate (Vmax), michaelis constant (Km) and number of revolutions (TON, maximum number of substrates per unit active catalytic center dialog) with CoO (vmax= 3.646mg L ^-1 min ^-1 CoO-Ir shows a higher vmax= 76.923 mg L than km= 73.914 mM) (fig. 6a and 6 b) ^-1 min ^-1 And ton=152.2 s ^-1 (FIGS. 5c and 5 d), demonstrating that Ir clusters exhibit the kinetics of rapid ROS elimination. We then systematically compared CoO-Ir with the most recently reported most advanced ROS scavenging materials in Vmax and TON (fig. 5 e), which found CoO-Ir to exhibit the best CAT-like activity in these mature biocatalysts.

In addition, as can be seen from FIG. 7, by comparing the decomposition H of CoO materials doped with different metals ₂ O ₂ O generation ₂ After the process of (2) CoO-Ir has been found to exhibit significant advantages.

Experimental example 3 cell experiment

3.1 test methods

Cell culture

Mesenchymal Stem Cells (BMSCs) were isolated from Sprague Dawley rats (Dossy laboratory animal limited, chinese adult) at 2 weeks of age and third to fifth generation cells were selected for subsequent experiments. MC3T3-E1 cells (mouse osteoblast cell line) were purchased from the China Shanghai cell bank. Cells were cultured in basal medium MEM-alpha (Gibco, USA) plus 10% fetal bovine serum (Gibco, australia) and 1% diabody at 37℃under 5% carbon dioxide. Prior to use, the biocatalyst was sterilized using ethanol and rinsed thoroughly with phosphate buffered saline (PBS, gibco) solution.

Cytotoxicity study

Cell proliferation assay: BMSCs and MC3T3-E1 cells were seeded in 96-well plates and replaced 8h later with fresh medium containing 0, 1, 5, 10, 25, 50 and 100. Mu.g/mL CoO and CoO-Ir. After 24 hours of incubation, cell viability was assessed using enhanced CCK-8 (#C0041, beyotime, china) according to the manufacturer's instructions.

Live/dead staining: cells were placed in 24-well plates and incubated with 1 and 5. Mu.g/mL CoO and CoO-Ir for 24 hours and stained for CalceinAM/PI (#C2015M, beyotime, china) according to the manufacturer's instructions. Live cells and dead cells were stained with CalceinAM and PI solutions, respectively. Fluorescence images were acquired on a fluorescence microscope (#ix 83, olympus, japan) and cell numbers were calculated by ImageJ program (Media Cybernetics, rockville, usa).

Apoptosis detection: annexin V-FITC/PI apoptosis detection kit (# 40302ES60, YEASEN, china) was used to evaluate apoptosis after 24 hours incubation with 1 and 5 μg/mL CoO and CoO-Ir. Cell samples were first harvested, washed and centrifuged three times, resuspended in binding buffer and stained with FITC Annexin V and PI for 15 minutes in the dark for detection by flow cytometry (CytoFLEX, bechman, usa).

Intracellular Reactive Oxygen Species (ROS) scavenging assay

And (3) establishing an oxidation stress model: cells were first treated with 100, 200, 300, 400, 500. Mu.M H ₂ O ₂ Culturing for 2 hours, and then culturing for 24 hours by changing to a normal culture medium. Based on the cell viability of CCK-8 assay, it was determined that the oxidative stress model was established using 150. Mu.M H ₂ O ₂ Concentration stimulation was performed for 2 hours.

Intracellular ROS clearance: cells were inoculated into 24-well plates, ensuring approximately 70% confluency, and 1 and 5. Mu.g-mL-1 CoO and CoO-Ir medium were used for 24 hours. Then washed three times with PBS containing 150. Mu. M H ₂ O ₂ For 2 hours, incubated with DCFH-DA probe (#D6883, sigma, USA) for 30 minutes and observed under a fluorescent microscope.

Cell protection assay

Cell viability assay: cells were pretreated with biocatalyst (1. Mu.g/mL) for 24 hours, then 150. Mu.M H was added ₂ O ₂ Co-cultivation was performed for 2 hours. Cells without any treatment were considered as a control group,with 150. Mu.M H ₂ O ₂ Treatment but no biocatalyst was considered H ₂ O ₂ A group. Following stimulation, cell proliferation rate was measured using Cell Counting Kit-8, live/dead ratio was measured using Calcein-AM/PI Kit, and apoptosis was measured using Annexin V-FITC/PI Kit.

Cytoskeletal staining: cell morphology was detected by cytoskeletal staining. Cells were seeded at low density, fixed with 4% paraformaldehyde (#BL 539A, biosharp, china) at room temperature for 15 min, and permeabilized with 0.1% Triton X-100 (#T8200, solarbio, china). Rhodaminephulloid (#C2207S, beyotidme, china) was used for F-actin (red) staining for 30 minutes, and DAPI (#P0131, beyotidme, jiangsu, china) was used for DNA (blue) staining for 5 minutes. The fluorescence photograph is taken with a fluorescence microscope.

3.2 Test results:

we then carefully evaluate their potential for use in antioxidant stress and restoring cellular function in the inflammatory microenvironment. Before conducting the cell experiments, we first studied the cell biocompatibility of the biocatalyst for stem cells (rat BMSCs) and osteoblasts (mouse MC3T3-E1 cell line) (FIG. 8a and FIG. 8 b), and live-dead staining showed no apparent dead cells at working concentrations (1 and 5. Mu.g/mL). After semi-quantitative analysis, coO and CoO-Ir were found to show no significant cytotoxicity for cell viability and apoptosis at working concentrations (1 and 5 μg/mL) (fig. 9 a). At concentrations of 1-10 μg/mL, the biocatalyst has very limited toxic effects on cell proliferation, and at higher concentrations (25-100 μg/mL) it shows some toxicity (fig. 9b and 9 c). Subsequent flow cytometry found that apoptosis was not significantly affected at working concentrations (fig. 10), and finally we confirmed the cellular safety of CoO and CoO-Ir from three levels of cellular activity, proliferation, apoptosis.

Next we used 1 and 5 μg/mL as safe concentrations in 2, 7-dichlorofluorescein diacetate (DCFH-DA) staining to analyze H ₂ O ₂ Efficiency of intracellular ROS clearance in the presence. By measuring BMSCs and MC3T3-E1 cells at 100-500 [ mu ] m M H ₂ O ₂ Cell viability under stimulation, determined for 2 hours using a concentration of 150 μm to model oxidative stress (fig. 11a and 11 b). Microscopic bright field determines the different morphologies of BMSCs and MC3T3-E cells after different treatments (fig. 11 c). DCFH staining detects H ₂ O ₂ The intracellular ROS induced by pretreatment can be effectively eliminated by CoO-Ir, and has no obvious difference from a control group; and at H ₂ O ₂ And a clear green signal was observed in the CoO group, indicating that intracellular ROS levels were high (fig. 11 d). Since 1 and 5 μg/mL of CoO-Ir showed equally excellent antioxidant activity, we selected 1 μg/mL for the next study. The addition of iridium clusters enhances antioxidant activity by directly targeting excess ROS, thereby protecting the cells from further damage.

Test example 4 in vitro osteogenic differentiation assessment

4.1 test method:

osteogenesis induction: cells were seeded in 12-well plates and treated as before. After stimulation, the medium was changed to osteoinductive medium supplemented with 50 μg-mL-1L-ascorbic acid (#a8960, sigma, usa), 10mM β -glycerophosphate (#g9422, sigma, usa) and 0.1 μg M dexamethasone (Sigma, usa). Early osteogenic related markers for both cells were observed on day 7, and late markers on day 10 of BMSCs and day 14 of MC3T3-E1 cells.

ALP and ARS staining: after fixation with 4% paraformaldehyde, ALP activity was quantified using the ALP activity assay and NBT/BCIP ALP staining kit (#C3206, beyotidme, china) according to the manufacturer's instructions. Calcium nodules were measured by alizarin red-S (ARS) staining (#alir-10001, cyagen, china). The bright field image is obtained by an optical microscope.

RT-qPCR: RT-PCR analysis was used to evaluate the gene expression levels of osteogenic related markers, including alkaline phosphatase Activity (ALP), run-related transcription factor 2 (Runx 2), osteocalcin (OCN) and bone cell protein (OPN), and GAPDH was used as a reference gene. Total RNA from MC3T3-E1 cells was extracted on day 7 with TRIzol reagent (# 15596026, invitrogen, USA). The cDNA was inverted with PrimeScript RT Master Mix (#RR036A, takara, japan) and RT-qPCR was performed using specific primers.

4.2 test results

Oxidative stress is formed when intracellular antioxidants are not effective in eliminating excessive ROS production, and ROS accumulation can lead to cell death or inflammatory chain reactions. To explore the use of CoO-Ir in the treatment of periodontitis, we studied the use of CoO-Ir in H ₂ O ₂ Protective effect on stem cells in induced oxidative stress microenvironment. A double-staining fluorescence experiment was first performed to examine the cell membrane rupture associated with ROS. As shown in FIG. 12a, a semi-quantitative analysis of live-dead staining and a representative image of staining shown in FIG. 13, H ₂ O ₂ The number of dead cells in the (58%) and CoO (57%) groups was significantly reduced by CoO-Ir (2%) to achieve a similar proportion of death to the control group. Furthermore, annexin V-FITC/PI staining demonstrated that CoO-Ir significantly reduced the apoptosis rate (FIG. 14).

In addition, the morphology of the cell is closely related to its function, which is also applicable to stem cells and osteoblasts. Bone cells have a highly branched cell morphology, and radiate multiple branches along the osseous tubules. Thus, we next assessed the cell shape and cytoskeletal structure of BMSCs by cytoskeletal staining. By comparing morphology and calculating cell fraction between the different groups, we found that BMSCs cultured with CoO-Ir showed a distinct spindle-like "osteoblast-like" shape with the largest cell expansion area and the largest protrusion. Conversely, H ₂ O ₂ Induced intracellular ROS lead to abnormalities and atrophy of cell morphology, whereas CoO is unable to restore these morphologies (fig. 12b and 12 c). Our results indicate that CoO-Ir reverses the adverse effects of oxidative stress on BMSCs morphology to normal levels and thereby changes the fate of the dried cells. The highly branched morphology and stress fiber formation resulting from cytoskeletal protein recombination are believed to be closely related to cell differentiation into osteoblasts. In contrast, excessive ROS can disrupt the cytoskeleton and thus inhibit bone deposition, leading to inflammatory destruction of periodontal tissue.

To explore the impact of different treatments on the osteogenic behaviour of BMSCs, we subsequently found in an oxidative stress microenvironmentIs subjected to osteogenesis. And H is ₂ O ₂ The CoO-Ir treated group showed more expression of alkaline phosphatase (ALP, early osteoblast markers) and mineralized nodules (expressed as alizarin red S, late osteoblast expression) in the medium at high ROS levels than the CoO group (fig. 12d and fig. 12 e). Quantitative detection of ALP showed that CoO-Ir was advantageous over the control (FIG. 12 f). In the mineralization of osteoblasts, the early stages involve increased secretion of collagen I (Col I) matrix, bone morphogenic protein-2 (BMP-2) and Runt related transcription factors (Runx 2), and the later stages are characterized by elevated levels of extracellular matrix protein, osteocalcin (OCN) and Osteocalcin (OPN). BMP-2 and Runx2 are essential conditions for bone formation that can regulate differentiation of BMSCs into osteoblasts, thereby accelerating repair of bone defects. As shown by immunofluorescent staining, increased signaling of BMP-2 and Runx2 was observed in CoO-Ir, while a significant decrease in signaling of CoO was observed in high ROS level medium. Semi-quantitative analysis further demonstrated that CoO-Ir significantly promoted Runx2 secretion in BMSCs, even better than control (fig. 12g and 12 h). Runx2, a specific transcription factor necessary for bone formation (especially chondrocyte maturation), can up-regulate the expression of matrix genes (OCN, col I, etc.). Runx2 is closely related to energy metabolism during osteogenic differentiation and bone formation. Mitochondria are the most important organelles for energy metabolism, ROS are mainly derived from the electron transport chain of mitochondria in the body. This suggests that CoO-Ir can eliminate excessive ROS produced by abnormal metabolism, directly promoting osteoblast differentiation of stem cells.

To confirm the antioxidant and osteogenic properties of CoO-Ir, we further studied another cell type, namely the osteoblast cell line MC3T3-E1, in addition to BMSCs. MC3T3-E1 is a well-characterized mouse osteoblast line, a mature osteoblast model. MC3T3-E1 cells and BMSCs differ in morphology and function, and are all commonly used and commonly represented cell types in bone research. After cell culture and staining, we performed the same measurement procedure on MC3T3-E1 cells. Similar to the results of BMSCs, coO-Ir was able to move more than half of MC3T3-E1 cells from H ₂ O ₂ Rescue of induced ROS attackFig. 15a, fig. 16 and 17). The CoO-Ir treated group and the control group exhibited abduction and applanation with good structure compared to the rounded morphology exhibited by MC3T3-E1 under ROS conditions and reduced adverse structure of the cytoskeleton (fig. 18). The quantitative results of F-actin staining also showed that the CoO-Ir group possessed a larger spreading area and more cell protrusions than the ROS conditions, and was similar to the control group (FIG. 15 b).

Next, we also evaluated the effect of CoO-Ir on the osteogenic activity of MC3T3-E1 cells under oxidative stress. The highest expression levels of ALP (FIG. 15 c) and ARS (FIG. 15 d) were observed in the CoO-Ir treated group, over the other groups. Next, we performed quantitative polymerase chain reaction (qPCR) to analyze the relative expression of osteogenesis-related genes ALP, col-1, runnx2 and OCN on day 7 of osteogenesis induction. Interestingly, with H ₂ O ₂ Compared to the CoO group, coO-Ir not only exerts cytoprotective effects by eliminating ROS, but also confers a higher osteogenic differentiation performance on cells than the control group (fig. 15 e). The osteoinductive properties of CoO-Ir may be attributed to cobalt, which is considered a possible alternative to growth factors and genetic methods in tissue engineering, because of their significant progressive effects on angiogenesis and osteogenesis, wound healing and antibacterial. One of the important findings is that cobalt ions have a positive effect on the formation of blood vessels and can couple osteogenesis and angiogenesis together. Therefore, the cobalt-based biomaterial has great potential in the application of bone tissue engineering.

Test example 5 in vivo treatment of periodontitis model

5.1 Test method

Establishing a periodontitis model: sprague-Dawley rats (8 weeks old, male, 200-250 g, duosi laboratory animal Co., ltd., chinese adults) were fed for one week to acclimatize, and were randomized into four groups: control, saline periodontitis (P+Saline), coO periodontitis (P+CoO), and CoO-Ir periodontitis (P+CoO-Ir). After anesthetizing the rats with sodium pentobarbital, 4-0 silk thread was ligated under the left maxillary second molar gingiva. After 1 week, biocatalysts (10 microliters, 1 mg/mL) or physiological saline (10 microliters) were injected into the palatine gingival sulcus of the second molar of the upper jaw every other day. After 3 weeks, the animal's maxilla and major organs were harvested. All studies on these animals were performed according to the animal ethics standards of the ethics committee of the oral hospital of university Hua Xi, and all animal protocols were allowed and on demand.

Micro-CT and bone mass analysis: the maxilla with teeth was collected and fixed with 4% paraformaldehyde for one week. The samples were then scanned using a microct system (#μct 50,SCANCO Medical AG, switzerland). Scan data was evaluated and reconstructed using SCANCO medical evaluation software (SCANCO medical company, switzerland). From the three-dimensional digitized image, the alveolar bone resorption is evaluated by measuring the distance and area between the cementum kingdom and the alveolar ridge crest (CEJ-ABC). The alveolar bone between the first and second molars was drawn as a region of interest (ROI) for measuring bone mass, the slice thickness was 10 microns, and consisted of 50 two-dimensional slices.

Scavenging ROS in vivo: ROS levels of periodontal tissue surrounding the left maxillary second molar were measured with a Dihydroethidium (DHE) staining kit (#bb-470515, bestbio, china) according to the manufacturer's instructions. Images were acquired on a fluorescence microscope (#ix 83, olympus, tokyo, japan).

5.2 Test results

Based on the positive effect of CoO-Ir on osteogenic differentiation of BMSCs and MC3T3-E1 in an oxidative environment, we constructed rat ligation-induced periodontitis as an inflammatory disease model to evaluate the therapeutic effect of CoO-Ir in vivo. Ligating the left maxillary second molar of SD rats to establish a periodontitis model, taking different treatment measures after 1 week and performing one month. micro-CT scanning and three-dimensional reconstruction images show the destruction of alveolar bone and the absorption at the bifurcation after ligation, indicating successful establishment of periodontitis model. After CoO injection, alveolar bone height was slightly restored, but not as much as the CoO-Ir group (fig. 19 a). In addition, measurement of the distance between alveolar ridge (ABC) and enamel dentin junction (CEJ) (an index of alveolar bone destruction) consistently demonstrated that CoO-Ir saved alveolar bone loss caused by inflammatory disease (fig. 19c and 19 d). Further analysis of bone mass also showed that the bone trabecular volume (BV/TV) and bone trabecular thickness (tb. Th) were greater around the second molar region after CoO-Ir injection, indicating that bone trabecular was also restored (fig. 19e and 19 f).

The destructive pattern of chronic periodontitis is a result of active connective tissue destruction and progressive bone resorption, resulting from different stimuli in the plasma, which originate from respiratory bursts of polymorphonuclear neutrophils (producing excessive ROS). Various strategies for preventing periodontitis, such as natural enzymes, nano-enzymes and antioxidants, have attracted attention by effectively eliminating intracellular excess ROS and maintaining intracellular redox balance, exerting anti-inflammatory effects. Along with the improvement of destructive periodontal tissue, we speculate that CoO-Ir has potential in vivo ROS scavenging ability in the inflammatory microenvironment. ROS-dependent Dihydroethidium (DHE) fluorescence demonstrated lower fluorescence in both control and p+coo-Ir treated groups, while p+saline and p+coo groups showed higher fluorescence signals, suggesting that our NPs could become efficient nano ROS scavengers in oxidative stress related diseases (fig. 19 b).

Furthermore, in the biochemical detection of serum, liver and kidney functions were not significantly impaired, and major organ tissues (heart, liver, spleen, lung and kidney) were not significantly abnormal between the different groups (FIG. 20), indicating CoO-Ir (1 mg mL) ^-1 ) Can be used as an effective and safe nano-drug. Based on consistent in vitro and in vivo results, we believe that CoO-Ir may be a promising biocatalyst for the treatment of periodontitis, having antioxidant effects in inflammation and osteogenic effects in regeneration.

By introducing CoO similar to superoxide dismutase (SOD) and Ir cluster similar to Catalase (CAT), an integrated SOD/CAT simulated cascade biocatalyst is designed and synthesized, and excellent ROS cascade scavenging activity is realized in a monomer system. CoO-Ir protects the activity and cell morphology of stem cells (BMSCs) and osteoblasts (MC 3T 3-E1) in oxidative stress environments, and activates the BMP-2-Runx2 signaling axis of the cells to promote in vitro osteogenic differentiation. Finally, coO-Ir is applied to periodontitis treatment, and good periodontitis stepwise treatment is realized through early anti-inflammatory and late osteogenesis. The invention provides an effective new way for inflammatory diseases through cascade catalytic active oxygen elimination.

Claims (10)

1. A biocatalyst integrating SOD/CAT, characterized by: the biocatalyst is a catalyst formed by uniformly distributing Ir clusters on a CoO substrate, and the catalyst has Ir-O bonds.

2. The SOD/CAT integrated biocatalyst of claim 1, wherein: the catalyst has a crystal structure.

3. The SOD/CAT integrated biocatalyst of claim 1, wherein: the catalyst has SOD and CAT activities.

4. A SOD/CAT integrated biocatalyst according to any one of claims 1-3, characterized in that: the catalyst has the activity of scavenging ROS.

5. The method for preparing the biocatalyst integrated with SOD/CAT according to any one of claims 1-4, which is characterized by comprising the following steps: the preparation method comprises the following steps: iridium salt, cobalt salt, urea and NH ₄ F, performing coprecipitation reaction and calcining to obtain the catalyst.

6. The method for preparing the SOD/CAT integrated biocatalyst according to claim 5, wherein the method comprises the following steps: the iridium salt is at least one selected from iridium chloride and iridium acetylacetonate; or:

the cobalt salt is at least one selected from cobalt nitrate, cobalt acetate and cobalt chloride; or:

the mixture ratio of the raw materials is that cobalt salt: iridium salt: NH (NH) ₄ F: the molar ratio of urea is 5-20:0.5-2:15-60:25-100.

7. The method for preparing the SOD/CAT integrated biocatalyst according to claim 6, wherein the method comprises the following steps: the co-precipitationThe reaction conditions are as follows: iridium salt, cobalt salt, urea and NH ₄ Dispersing the solution F into water, stirring, reacting for 7-10 hours at 110-130 ℃, cooling, centrifuging and washing; or:

the calcination conditions are as follows: in nitrogen or inert atmosphere, heating at 350-450 ℃ for 2-5 ℃ min ⁻¹ Calcining for 2-3 hours under the condition of (2) to obtain a catalyst; or:

the mixture ratio of the raw materials is that cobalt salt: iridium salt: NH (NH) ₄ F: the molar ratio of urea is 10:1:30:50; or:

the iridium salt is iridium chloride; or:

the cobalt salt is cobalt nitrate.

8. Use of a SOD/CAT integrated biocatalyst for scavenging ROS characterized by: the biocatalyst is the biocatalyst according to any one of claims 1 to 4 or the biocatalyst prepared by the method according to any one of claims 5 to 7.

9. Use of an SOD/CAT integrated biocatalyst according to claim 8 for scavenging ROS, characterized by: the application of the biocatalyst in preparing medicines for treating inflammatory diseases.

10. Use of a SOD/CAT integrated biocatalyst for cytoprotection and promotion of cell differentiation, characterized in that: the biocatalyst is the biocatalyst according to any one of claims 1 to 4 or the biocatalyst prepared by the method according to any one of claims 5 to 7.

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