Cerium oxide nano-enzyme with anti-oxidation and anti-inflammatory functions and preparation method of cerium oxide nano-enzyme

Abstract

The invention relates to the technical field of biological medicine, in particular to a cerium oxide nano-enzyme with antioxidant and anti-inflammatory functions and a preparation method thereof.The nano-enzyme is of a core-shell structure formed by self-assembly of sialic acid, protocatechualdehyde and an amphiphilic polymer through coordination, the inner core of the core-shell structure is cerium oxide nano-particles, and the outer core of the core-shell structure is nano-particles; according to the cerium oxide nano-enzyme with the anti-oxidation and anti-inflammatory functions and the preparation method of the cerium oxide nano-enzyme, a lipoic acid zwitterionic polymer coating technology is adopted, so that the blood stability and the cell uptake efficiency of nano-particles are remarkably improved, the immune clearance rate is reduced, and the anti-oxidation and anti-inflammatory functions are achieved; and dual optimization of bioavailability is realized.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and specifically to a cerium oxide nanozyme with antioxidant and anti-inflammatory functions and a preparation method thereof. Background Art

[0002] As a neurovascular disease with the highest disability rate globally, ischemic stroke treatment faces multiple challenges. Traditional thrombolytic therapies are limited by a narrow time window and prone to reperfusion injury, leading to the burst of reactive oxygen species (ROS) and inflammatory cascade reactions. Existing antioxidants have problems such as easy inactivation of enzymes, single scavenging spectrum of small molecules, and lack of targeting. Although cerium oxide nanozymes have SOD / CAT-like activities, their poor in vivo stability, insufficient lesion targeting, and potential pro-inflammatory side effects limit their applications. Biomaterial delivery systems face bottlenecks such as low blood-brain barrier penetration efficiency (<0.1%), rapid blood clearance, and insufficient multi-enzyme synergistic effects. Therefore, in view of the above situation, there is an urgent need to develop a cerium oxide nanozyme with antioxidant and anti-inflammatory functions and a preparation method thereof to overcome the deficiencies in current practical applications. Summary of the Invention

[0003] The purpose of the present invention is to provide a cerium oxide nanozyme with antioxidant and anti-inflammatory functions and a preparation method thereof to solve the problems raised in the above background art.

[0004] To achieve the above purpose, the present invention provides the following technical solutions:

[0005] A cerium oxide nanozyme with antioxidant and anti-inflammatory functions, wherein the nanozyme self-assembles into a core-shell structure through coordination by sialic acid, protocatechuic aldehyde, and an amphiphilic polymer. The inner core of the core-shell structure is a cerium oxide nanoparticle, and the outer layer is a polymer-protocatechuic aldehyde-sialic acid composite layer;

[0006] The amphiphilic polymer is:

[0007] A copolymer of (R)-2-((3-(5-(1,2-dithiocyclopent-3-yl)pentylamino)propyl)dimethylamino)ethane-1-sulfonic acid and (R)-5-(1,2-dithiocyclopent-3-yl)-N-(2-hydroxyethyl)pentanamide.

[0008] As a further aspect of the present invention: in the amphiphilic polymer;

[0009] The molar ratio of (R)-2-((3-(5-(1,2-dithiocyclopent-3-yl)pentylamino)propyl)dimethylamino)ethane-1-sulfonic acid to (R)-5-(1,2-dithiocyclopent-3-yl)-N-(2-hydroxyethyl)pentanamide is 3:1.

[0010] As a further aspect of the present invention: the average particle size of the cerium oxide nanoparticles is 9.65 ± 0.64 nm, the hydrodynamic diameter of the core-shell structure is 138.60 ± 28.06 nm, and the zeta potential of the composite layer is -10 to -30 mV.

[0011] A method for preparing the cerium oxide nanozyme according to the above, comprising the following steps:

[0012] Step 1, synthesize an amphiphilic polymer:

[0013] (R)-2-((3-(5-(1,2-dithiocyclopent-3-yl)pentylamino)propyl)dimethylamino)ethane-1-sulfonic acid and (R)-5-(1,2-dithiocyclopent-3-yl)-N-(2-hydroxyethyl)pentanamide are dissolved in a methanol-water mixed solvent in a molar ratio of 3:1, an initiator 2-aminoethanethiol is added for polymerization reaction, and then the reaction is terminated with iodoacetamide, and the copolymer is obtained by dialysis purification;

[0014] Step 2, prepare the nanozyme by a one-pot method: mix the sialic acid solution, protocatechuic aldehyde solution and the copolymer solution obtained in Step 1, stir at 60 °C for 30 minutes, then dropwise add the cerium nitrate solution, adjust the pH to 12, and react in the dark for 8 hours, and the cerium oxide nanozyme is obtained by dialysis purification.

[0015] As a further aspect of the present invention: in Step 2, the concentration of the cerium nitrate solution is 43.7 mg / mL, and the dropping rate is 0.15 mL / min.

[0016] As a further aspect of the present invention: in Step 2, ammonium hydroxide is used to adjust the pH, and the reaction system is carried out under nitrogen protection.

[0017] As a further aspect of the present invention: in Step 2, dialysis is carried out using a dialysis bag with a cut-off molecular weight of 1000 Da, and the dialysis time is 48 hours.

[0018] A use of the cerium oxide nanozyme according to the above for preparing an antioxidant for scavenging hydroxyl radicals, superoxide anion radicals or DPPH radicals.

[0019] A use of the cerium oxide nanozyme according to the above for preparing an anti-inflammatory drug for inhibiting the expression of inflammatory factors IL-6, TNF-α or promoting the expression of anti-inflammatory factors IL-10, TGF-β.

[0020] A pharmaceutical composition comprising the cerium oxide nanozyme according to the above and a pharmaceutically acceptable carrier, and the pharmaceutical composition is used for treating stroke, neurodegenerative diseases or oxidative stress-related inflammation.

[0021] Compared with the prior art, the beneficial effects of the present invention are:

[0022] The present invention adopts the technology of coating with lipoic acid zwitterionic polymer, significantly improving the blood stability and cell uptake efficiency of nanoparticles, reducing the immune clearance rate, and realizing the dual optimization of bioavailability. Brief Description of the Drawings

[0023] Figure 1 It is a schematic diagram of the particle size and potential characterization of cerium oxide nanoparticles and composite nanozymes in the embodiments of the present invention;

[0024] Among them, (a) is the particle size distribution of CeO2 nanoparticles, (b) is the hydrated particle size distribution of PLSP@CeO2 composite nanozymes, (c) is the comparison of zeta potentials of PLA, CeO2 and PLSP@CeO2, and (d) is the comparison of hydrated particle sizes of PLA, CeO2 and PLSP@CeO2.

[0025] Figure 2 It is a schematic diagram of fluorescence imaging of cell uptake behavior in the embodiments of the present invention.

[0026] Figure 3 It is a schematic diagram for evaluating the brain targeting ability of nanozymes in the embodiments of the present invention;

[0027] Among them, (a) is the quantitative analysis of the fluorescence intensity of neutrophils taking up PLSP@CeO2, (b) is the ratio of PLSP@CeO2 crossing the blood-brain barrier (BBB), and (c) is the quantitative analysis of the fluorescence intensity of ICG-labeled PLSP@CeO2 in the ischemic hemisphere.

[0028] Figure 4 It is a schematic diagram of the endothelial cell function repair experiment in the embodiments of the present invention;

[0029] Among them, (a) is the fluorescence image of the tube formation ability of endothelial cells in different treatment groups, (b) is the image of the migration ability after the cell scratch experiment, and (c) is the quantitative analysis of the scratch healing area.

[0030] Figure 5 It is a schematic diagram for the pathological and safety evaluation of brain tissue in the embodiments of the present invention;

[0031] Among them, (a) is the survival of neurons in the hippocampal CA region shown by Nissl staining, and (b) are the H&E staining results of heart, liver, spleen, lung and kidney tissues.

[0032] Figure 6 It is a schematic diagram for the study of the inflammation regulation mechanism in the embodiments of the present invention;

[0033] Among them, (a) is the immunofluorescence staining of Iba-1 (red) and CD206 (green) in cortical microglia (DAPI labels the cell nucleus), (b) is the quantification of the fluorescence intensity of CD86 (a marker of M1 microglia), (c) is the quantification of the fluorescence intensity of CD206 (a marker of M2 microglia), and (d)-(g) are the levels of inflammatory factors IL-6, TNF-α, TGF-β, and IL-10 in the brain tissue. Detailed implementation manners

[0034] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0035] The following describes the specific implementation of the present invention in detail in conjunction with specific embodiments.

[0036] Please refer to Figures 1-6 A cerium oxide nanozyme with antioxidant and anti-inflammatory functions provided by an embodiment of the present invention (the nanozyme is a nano-particle with enzyme activity), the nanozyme is self-assembled into a core-shell structure by the coordination of sialic acid, protocatechuic aldehyde, and an amphiphilic polymer, the inner core of the core-shell structure is a cerium oxide nano-particle, and the outer layer is a polymer-protocatechuic aldehyde-sialic acid composite layer;

[0037] The amphiphilic polymer is:

[0038] A copolymer of (R)-2-((3-(5-(1,2-dithiocyclopent-3-yl)pentylamino)propyl)dimethylamino)ethane-1-sulfonic acid and (R)-5-(1,2-dithiocyclopent-3-yl)-N-(2-hydroxyethyl)pentanamide.

[0039] In an embodiment of the present invention, in the amphiphilic polymer;

[0040] The molar ratio of (R)-2-((3-(5-(1,2-dithiocyclopent-3-yl)pentylamino)propyl)dimethylamino)ethane-1-sulfonic acid to (R)-5-(1,2-dithiocyclopent-3-yl)-N-(2-hydroxyethyl)pentanamide is 3:1.

[0041] The average particle size of the cerium oxide nano-particle is 9.65±0.64 nm, the hydrated particle size of the core-shell structure is 138.60±28.06 nm, and the zeta potential of the composite layer is -10 to -30 mV.

[0042] In one embodiment of the present invention, a method for preparing the above-mentioned cerium oxide nanozyme includes the following steps:

[0043] Step 1: Synthesize amphiphilic polymer:

[0044] (R)-2-((3-(5-(1,2-dithiocyclopent-3-yl)pentylamino)propyl)dimethylamino)ethane-1-sulfonic acid and (R)-5-(1,2-dithiocyclopent-3-yl)-N-(2-hydroxyethyl)pentanamide are dissolved in a methanol-water mixed solvent in a molar ratio of 3:1. An initiator, 2-aminoethanethiol, is added for polymerization reaction, and then the reaction is terminated with iodoacetamide. The copolymer is obtained by dialysis purification.

[0045] Step 2: Prepare the nanozyme by a one-pot method: Mix the sialic acid solution, protocatechuic aldehyde solution and the copolymer solution obtained in Step 1. After stirring at 60 °C for 30 minutes, add cerium nitrate solution dropwise, adjust the pH to 12, and react in the dark for 8 hours. The cerium oxide nanozyme is obtained by dialysis purification.

[0046] In Step 2, the concentration of the cerium nitrate solution is 43.7 mg / mL, and the dropping rate is 0.15 mL / min.

[0047] In Step 2, ammonium hydroxide is used to adjust the pH, and the reaction system is carried out under nitrogen protection.

[0048] In Step 2, dialysis is carried out using a dialysis bag with a molecular weight cut-off of 1000 Da, and the dialysis time is 48 hours.

[0049] A use of the above-mentioned cerium oxide nanozyme for preparing an antioxidant for scavenging hydroxyl radicals, superoxide anion radicals or DPPH radicals.

[0050] A use of the above-mentioned cerium oxide nanozyme for preparing an anti-inflammatory drug for inhibiting the expression of inflammatory factors IL-6 and TNF-α or promoting the expression of anti-inflammatory factors IL-10 and TGF-β.

[0051] A pharmaceutical composition comprising the above-mentioned cerium oxide nanozyme and a pharmaceutically acceptable carrier, and the pharmaceutical composition is used for treating stroke, neurodegenerative diseases or oxidative stress-related inflammation.

[0052] Example 1:

[0053] Synthesis of 2,5-dioxopyrrolidin-1-yl (R)-5-(1,2-dithiocyclopent-3-yl)pentanoate (LA-NHS);

[0054] Take 24 mmol of lipoic acid and 28.8 mmol of N,N'-disuccinimidyl carbonate, dissolve them in 200 mL of acetonitrile, stir well, add 10 mL of triethylamine thereto, stir and react at room temperature for 4 h, then evaporate 2 / 3 of the solvent, and pour the remaining solvent into 275 mL of saturated sodium bicarbonate solution. A yellow precipitate is formed. After filtration, add dichloromethane to the precipitate to dissolve it, extract the yellow clear solution, and dry it under vacuum to obtain the purified product.

[0055] Example 2:

[0056] Synthesis of (R)-N-(3-(dimethylamino)propyl)-5-(1,2-dithiolan-3-yl)pentanamide (LA-PA);

[0057] Dissolve 8.89 g of LA-NHS in 80 mL of ultra-dry dichloromethane, add dropwise 3-(dimethylamino)-1-propylamine (10 mL), and stir the reaction overnight at room temperature. Evaporate under reduced pressure. Dissolve the obtained viscous oil in 1 M HCl (80 mL) and stir for one hour, filter, and then add the aqueous solution to chloroform (CHCl3, 100 mL). Adjust the pH value of the mixture to 12 with NaOH, and then stir the resulting two-phase system thoroughly for 30 min. Separate the two phases, and extract the aqueous layer with CH2Cl2 (2×40 mL). Combine the organic phases. After the solution is dried (Na2SO4), filtered, and concentrated under reduced pressure, a thick yellow oily derivative is obtained.

[0058] Example 3:

[0059] Synthesis of (R)-2-((3-(5-(1,2-dithiolan-3-yl)pentylamino)propyl)dimethylamino)ethane-1-sulfonic acid (LA-SB);

[0060] Weigh LA-PA (1.16 g, 4.00 mmol) and dissolve it in dry tetrahydrofuran THF (8 mL). Slowly add dropwise a THF solution of 2 mL of 1,3-propane sultone (540 mg, 4.40 mmol) under stirring, and then stir the reaction mixture overnight at room temperature. The formed white suspension is separated by centrifugation, and the supernatant is discarded. Mix the residue with ether and acetone, and dry it under vacuum to obtain the white solid of LA-SB.

[0061] Example 4: Synthesis of (R)-5-(1,2-dithiolan-3-yl)-N-(2-hydroxyethyl)pentanamide (LA-OH);

[0062] React 4.85 mmol of LA-NHS and 5.82 mmol of 2-aminoethanol in dry dichloromethane (125 mL) to obtain the crude product. Then, extract with dichloromethane and saturated brine for multiple times, collect and dry the organic phase, and concentrate it under vacuum to obtain a pale yellow viscous liquid.

[0063] Example 5: Synthesis of Amphiphilic Polymer;

[0064] Take 3 mM LLA-SB monomer and 1 mM LLA-OH monomer, and dissolve them with 4.5 mL of reaction solvent (methanol:water = 1:1). Add an appropriate amount of initiator 2-aminoethanethiol (1 eq) under a nitrogen atmosphere, and polymerize at room temperature in the dark for 4 h. Subsequently, add the terminator iodoacetamide (2 eq) and stir for 30 min to terminate the polymerization reaction. Transfer the solution to a 3500 Da dialysis bag, dialyze for 48 h, and lyophilize to obtain the product.

[0065] The degree of polymerization is controlled by the molar ratio of the compound to the initiator. After the reaction, the mixture is quenched with iodoacetamide, dialyzed with deionized water, and then lyophilized to obtain the copolymer.

[0066] Example 6: One-pot Preparation of PLSP@CeO2 Nanoparticles;

[0067] First, add sialic acid (10 mL, 10 mg / mL) to a single-neck flask, and add protocatechuic aldehyde (10 mL, 10 mg / mL) and polymer (10 mL, 5 mg / mL) under stirring. After stirring at 60 °C for 30 min, slowly add cerium nitrate (1.5 mL, 43.7 mg / mL), add ammonium hydroxide to adjust the pH to 12, and react under dark N2 for at least 8 h. Subsequently, dialyze with a 1000 Da dialysis bag for 48 h, and lyophilize to obtain the nanoparticles.

[0068] Example 7: Acid-responsive Charge Change and Complex Disassembly Test;

[0069] To evaluate the responsive release characteristics of cerium oxide, 1 mg of cerium oxide was resuspended in phosphate buffer solutions (PB) under four different conditions: PB (pH 7.4), PB (pH 6.5), PB (pH 6.0), and PB (pH 5.5). These suspensions were incubated at 37 °C for 2 h. Subsequently, the reacted solutions were subjected to UV scanning and particle size potential testing.

[0070] Material Characterization: Comprehensive characterization was carried out through various microscopic and spectroscopic analyses. Specifically, a Malvern particle size analyzer was used to determine the particle size distribution and zeta potential, while the morphology and elemental composition were evaluated by TEM (100 kV), high-resolution transmission electron microscopy (HRTEM) (JEOL2010), and energy-dispersive spectroscopy (EDS). The structural information and surface valence distribution of CeNZs were obtained by X-ray diffraction (XRD) and X-ray photoelectron spectroscopy (XPS).

[0071] Example 8: Antioxidant Performance Test;

[0072] ·OH scavenging evaluation: ·OH was generated by mixing 20 μL of FeSO4·7H2O (1.8 mM) with 20 μL of H2O2 (4.9 mM), and then 20 μL of each sample to be tested (0.1 mg / mL) was added. After incubation at room temperature for 3 min, 10 μL of DMPO scavenger (0.2 M) was added, and the EPR was used to capture the ·OH signal to evaluate the scavenging performance of antioxidants on ·OH.

[0073] In addition, the ability of each composite antioxidant to scavenge ·OH was further evaluated by UV-vis. The ·OH generated by the Fenton reaction was effectively captured by salicylic acid and reacted with it to form a colored substance, 2,3-dihydroxybenzoic acid, which has a maximum absorption peak at 510 nm. After adding the composite antioxidant, the absorbance at this wavelength decreased. 100 μL of salicylic acid (6 mM), 700 μL of the sample to be tested, 100 μL of FeSO4·7H2O (2 mM) and 100 μL of H2O2 (6 mM) were mixed, and after incubation for 15 min, the absorbance of the solution was measured.

[0074] O2 ·- Scavenging evaluation: O2 ·- was generated by the reaction between xanthine and xanthine oxidase. 25 μL of xanthine solution (0.4 mM) was reacted with 5 μL of xanthine oxidase (0.1 U / mL) to generate O2 ·- , and then 20 μL of the composite antioxidant (0.1 mg / mL) was added. After incubation at room temperature for 3 min, 25 μL of BMPO scavenger (25 mM) was added, and the EPR was used to capture the O2 ·- signal to evaluate the scavenging performance of antioxidants on O2 ·-

[0075] DPPH· scavenging efficiency: 0.5 mL of the composite antioxidant (0.2 mg / mL) was added to an ethanol solution containing 3 mL of DPPH (0.05 mg / mL). After incubation of the mixed solution at room temperature for 30 min, the absorbance at 520 nm was measured by UV-vis.

[0076] ABTS +· Scavenging efficiency: 1.5 mL of K2S2O8 (2.45 mM) was mixed with 0.4 mL of ABTS (7 mM) and reacted in the dark for 12 h to obtain the ABTS +· working solution, which was diluted with PBS buffer (0.1 M, pH 7.4) to an absorbance of 0.7 ± 0.02 before use. Then the composite antioxidant was added to the above working solution. After incubation in the dark at 25 °C for 10 min, the absorbance at 734 nm was measured with a microplate reader.

[0077] Example 9: Simulated enzyme activity test;

[0078] Scavenging superoxide radical (O2​·- ) The efficiency was achieved by measuring the inhibition ratio of NBT photoreduction. A solution containing riboflavin (20 μM), methionine (12.5 mM), NBT (75 μM) and different concentrations of NPs (50 μg / mL) was placed in PBS. The mixture was irradiated with ultraviolet (UV) light of constant intensity at 25 °C for 5 min. The negative control group was the control group containing riboflavin, methionine and NBT, but placed in the dark. The positive control group was the sample group containing riboflavin, methionine and NBT, irradiated with UV light for 5 min. The treated sample group was the group containing riboflavin, methionine, NBT and NPs and irradiated with UV light. All experiments were carried out in a dark environment, and O2 ·- The clearance rate was calculated according to the following formula:

[0079] O2 ·- scavengingeffect (%) = (Ap - As) / (Ap - An) × 100%;

[0080] where As, An and Ap are the absorbances of the treated sample, negative control and positive control, respectively. They are the absorbances of the treated sample, negative control and positive control, respectively.

[0081] CAT can catalyze the decomposition of H2O2 into H2O and O2. The CAT mimetic activity was determined by detecting the change in O2 concentration with a dissolved oxygen tester. The reactions were all carried out at 25 °C in 0.1 M PBS (pH 7.4). In addition, we used a CAT detection kit to determine the CAT mimetic activity, and its principle is to detect by colorimetry. In the presence of a large amount of H2O2, CAT catalyzes the decomposition of H2O2, and the residual H2O2 can oxidize the chromogenic substrate to generate a red product with a maximum absorption wavelength of 520 nm, thereby reflecting the CAT activity in the sample.

[0082] H2O2 (1.2 M H2O2) and PLSP@CeO2 (3 mg) were mixed and 50 mL of PBS was added. The O2 concentration was measured with a dissolved oxygen meter (SMARTSENSOR, AR8406) for 10 min. O2 bubbles were observed in an Eppendorf tube. Under an anaerobic environment, H2O2 (1.2 M) and 500 μg of PLSP@CeO2 were mixed into PBS and photographed to record the O2 bubbles.

[0083] Example 10: Cell experiment;

[0084] Endothelial cells (HUVEC), neuronal cells (PC12), and macrophages (RAW264.7) were cultured in DMEM complete medium, which contained 89% simple medium, 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin double antibody. The cells were cultured in a constant temperature incubator at 37 °C and 5% CO2.

[0085] Cell uptake experiment: The cell uptake behavior was monitored by fluorescence microscopy. Briefly, PC12 cells were seeded in confocal dishes at a density of 1×10 5 cells per well and incubated for 12 h. Then, H2O2 (200 μM) was added to induce cell stress and the incubation was continued for 12 h. To study the characteristics of cell uptake, the cells were incubated with DMEM complete medium containing different concentrations of NPS for 4 h, and then washed three times with PBS. The cell nuclei were stained with Hoechst for 30 min. The cells were observed with a fluorescence microscope at specific time points.

[0086] Intracellular ROS: Briefly, PC12 cells were seeded in 6-well plates at a density of 1×10 5 cells per well and cultured at 37 °C and 5% CO2 for 24 h. Then, H2O2 (200 μM) was added to induce cell stress. After washing the cells three times with PBS buffer, fresh medium containing the drug was added and the cells were incubated for 3 - 4 h. Then the cells were washed three times and incubated with 10 μM DCFH-DA in the dark for 30 min. Then the fluorescent probe solution was removed and the cells were washed with PBS. After staining with Hoechst for 5 - 30 min, fluorescence imaging was obtained with an inverted fluorescence microscope.

[0087] According to the "intracellular ROS detection" procedure, after incubating the drug with PC12 for 4 h, the cells were treated with 10 μM DCFH-DA for 30 min, washed three times with PBS buffer, digested with trypsin (trace amount) for 1 min, 1 mL of complete medium was added, centrifuged, resuspended and pipetted with 1 mL of PBS, centrifuged again, and the cells were resuspended with PBS and collected. The cell concentration was adjusted to 2×10 6 cells / mL, and the fluorescence intensity was recorded by flow cytometry. FlowJo software was used for data analysis.

[0088] Cytotoxicity: PC12 cells and endothelial cells (HUVEC) were selected for the cytotoxicity evaluation of the nanozyme. The cells were seeded in 96-well plates at a density of 5000 cells / well and cultured at 37 °C and 5% CO2 for 24 h. Then, 180 μL of medium solution containing different concentrations of the nanomaterial was added to the 96-well plates. After co-incubating with the cells for 24 h, 20 μL of MTT (5 mg / mL) was added to each well and the incubation was continued in the incubator for 4 h. Finally, the absorbance value at a wavelength of 492 nm was measured with an enzyme-linked immunosorbent assay (ELISA) reader, and the relative cell viability was calculated.

[0089] Mitochondrial membrane potential: In addition, the mitochondrial membrane potential (ΔΨm) of each group was analyzed using the green fluorescent probe JC-1. When the mitochondrial membrane potential is high, JC-1 can easily penetrate healthy mitochondria and form red fluorescent "aggregates"; however, when the mitochondrial membrane potential is low, JC-1 cannot aggregate in mitochondria and exists in the form of green fluorescent monomers. The transformation of JC-1 from red fluorescence to green fluorescence is an indicator of early apoptosis. Briefly, after treatment with H2O2, the cells in each group were incubated with 10 μg / mL JC-1 at 37 °C for 20 min. Fluorescence images were visualized using a laser scanning confocal microscope.

[0090] Transmembrane: A monolayer BBB model was established in vitro. Brain endothelial cells were added to the upper chamber of Transwell and cultured for 7 days. When the TEER value reached 200 Ω·cm -2 or above, PC12 cells were seeded in the lower chamber. After co-incubation for 24 h, the integrity of the barrier was confirmed by TEER detection. After adding NPs (50 μg / mL) to the upper chamber and incubating for 4 h, the fluorescence intensity of RhB in PC12 cells was observed under an inverted fluorescence microscope.

[0091] Cell apoptosis: To analyze cell apoptosis, PC12 cells were seeded into a six-well culture plate at a concentration of 10 6 cells / well. After cell attachment, the cells were treated at 37 °C, adding 0.4 mM H2O2 and the test samples, and co-incubated for 24 h. After the treatment, the cell culture medium was removed, and the cells were washed with diluted 10× PBS. The blank control group was treated with fresh medium, and the model group was stimulated with H2O2 alone. The cells were collected, stained with the ANNEXI-FITC cell apoptosis detection kit, and analyzed by flow cytometry. Similarly, the anti-apoptotic activity of NPs was visually evaluated by the live / dead cell staining method (Calcein AM / PI).

[0092] Cell scratch assay: Approximately 5×10 5 endothelial cells were added to a 6-well plate. After incubation for 24 h, a pipette tip or sterile toothpick was used to scratch the cell layer perpendicular to the cell plane along the line drawn on the back of the plate. After scratching, the cells were washed 3 times with sterile PBS to remove non-adherent cells. Complete medium containing NPs was added for co-incubation, and the results were observed by microscope photography.

[0093] Angiogenesis: Matrigel (10 mg / ml) was added to a 24-well plate and spread evenly with a 1 ml pipette tip. It was placed in a cell culture incubator for 30 min to solidify. Subsequently, the endothelial cells treated with starvation for 12 h were digested with trypsin, resuspended, and seeded on Matrigel. Then, the cell status was observed using an inverted fluorescence microscope.

[0094] Example 11: Animal experiment.

[0095] The present invention uses male SD rats weighing 220 - 250 g. The rats are raised in a constant environment with a 12 h light / dark cycle, a temperature of 20 - 26 °C, and a humidity of 40 - 70%. They have unrestricted access to food and water. A transient middle cerebral artery occlusion model is established according to the improved suture occlusion method. First, fast the rats for 12 h before surgery, without restricting water intake. Anesthetize the rats intraperitoneally with 20% urethane (5 mL / kg). Fix the rats in the supine position on a small animal operating table. After shaving the rats, make an incision on the right side of the middle of the neck. Separate the muscles, fascia, the right common carotid artery and the vagus nerve of the rats; separate the external carotid artery and the internal carotid artery, ligate the distal end of the external carotid artery (ECA), electrocoagulate the blood vessel branches to prevent bleeding, make a slipknot at the carotid artery bifurcation, and clamp the internal and common carotid arteries. Make an incision on the external carotid artery, insert a suture from the ECA to the carotid artery bifurcation into the ICA to about 18 - 20 mm, block the blood flow of the middle cerebral artery, and remove the suture after 2 h for reperfusion to establish a tMCAO model. The Sham group does not insert the suture. After removing the suture and reperfusion, inject NPs (20 mg / kg) into the tail vein. The Sham and tMCAO models are given the same dose of normal saline. During the whole surgical process, the body temperature is maintained at 37 ± 0.5 °C.

[0096] Accumulation of inflammation-directed specific ischemic lesions (in vivo imaging):

[0097] Evaluate the brain targeting ability of NPs in tMCAO rats. After reperfusion, inject near-infrared fluorescent probe ICG-labeled NPS (relative to ICG = 2 mg / kg) into the rats intravenously, and inject simultaneously. 22.5 h after injection, deeply anesthetize the rats, perfuse and remove the brain through the heart, make continuous coronal sections, and perform fluorescence imaging using a small animal fluorescence imaging system.

[0098] In addition, in order to more accurately observe the co-localization of NPS and neutrophils in the cerebral ischemia area, tMCAO rats are injected intravenously with FITC-labeled NPS equivalent to 2 mg / kg FITC simultaneously after reperfusion. 22.5 h after injection, the rats are deeply anesthetized, and the brain is removed by perfusion through the heart. Prepare brain cryosections and perform neutrophil immunofluorescence staining (primary antibody CD11b (1:200), secondary antibody (CY3-labeled goat anti-rabbit IgG, 1:300)). Observe the co-localization of FITC and CD11b in each group by fluorescence microscopy.

[0099] TTC staining:

[0100] TTC is converted to red by dehydrogenase in living cells. When cells die or are damaged, they cannot be stained, so that cerebral infarction can be identified according to the stained area. After 24 h of MCAO, the rat brains were removed, frozen at -20 °C for at least 30 min, and 2-mm coronal sections were cut from the frontal pole. Then the sections were immersed in 2% TTC staining solution and incubated at 37 °C for 30 min. The stained brain sections were photographed using a camera, and the infarct volume was calculated using ImageJ software.

[0101] Inflammatory factors in brain tissue:

[0102] ELISA kits were used to detect the expression of inflammatory cytokines such as IL-10, TGF-β, IL-6, and TNF-α in the brain tissues of each group. At 24 h after stroke, the MCAO rats were deeply anesthetized, and the brain tissues were collected by cardiac perfusion with normal saline. The collected brain tissues were homogenized using a tissue homogenizer and centrifuged at 12,000 g for 10 min to obtain the supernatant. Then the protein concentration was quantified using a BCA protein assay kit, and the levels of inflammatory cytokines IL-10, TGF-β, IL-6, and TNF-α were measured using ELISA kits, all according to the instructions provided by the kits.

[0103] Neurobehavioral tests:

[0104] At 24 h after MCAO, cerebral infarction and neurological deficits in each group were evaluated as described above. A single investigator blinded to the animal groups performed the behavioral tests. Any data from rats that died during the experiment were excluded. The internationally recognized Longa scale was used for scoring 1 , and the following details were used: 0 indicates no nerve injury, 1 indicates inability to fully extend the contralateral forelimb, 2 indicates that the body circles to the contralateral side during walking, 3 indicates that the body tilts to the contralateral side during walking, and 4 indicates inability to walk spontaneously and loss of consciousness.

[0105] Immunohistochemical analysis:

[0106] Brain tissue samples from all experimental groups were collected after taking the brain by cardiac perfusion with PBS and 4% paraformaldehyde. Frozen sections and paraffin sections of brain tissue were prepared respectively. The paraffin sections were stained with H&E to observe the histological changes of brain cells. The TUNEL staining method was used to detect apoptosis through TdT (terminal deoxynucleotidyl transferase)-mediated fluorescein-dUTP nick end labeling, and then the anti-apoptotic effect was evaluated. It was also observed that the number and morphology of Nissl vesicles directly reflected the nerve recovery in the hippocampal region of the brain. Nissl vesicles in the cytoplasm of neurons were stained with Nissl staining solution to determine the loss of cortical and hippocampal neurons. In addition, the neuronal marker (NeuN) was used for immunofluorescence staining to label neurons in brain sections. Microglial polarization in infarcted lesions was monitored by staining frozen sections with anti-CD86 and anti-CD206 primary antibodies to label M1 and M2 phenotype microglia respectively, as well as secondary antibodies of Alexa Fluor 647-labeled IgG (H+L) and FITC-labeled IgG (H+L). Paraffin sections of brain tissue were also stained with Iba-1 antibody to label microglia and detect anti-inflammatory effects.

[0107] References:

[0108] 1. Li, J.; Liu, Z.; Wang, L.; Xu, H.; Wang, Y., Thousand and one kinase 1 protects MCAO-induced cerebral ischemic stroke in rats by decreasing apoptosis and pro-inflammatory factors. Bioscience Reports 2019, 39(10), BSR20190749.

[0109] It should be noted that in the present invention, it should be understood that although this specification is described according to the embodiments, not each embodiment only contains an independent technical solution. This narrative way of the specification is only for clarity. Those skilled in the art should regard the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A cerium oxide nanozyme with antioxidant and anti-inflammatory functions, characterized in that: The nanozyme is self-assembled by sialic acid, protocatechuic aldehyde and an amphiphilic polymer through coordination to form a core-shell structure, wherein the core of the core-shell structure is a cerium oxide nanoparticle and the outer layer is a polymer-protocatechuic aldehyde-sialic acid composite layer; The amphiphilic polymer is: Copolymer of (R)-2-((3-(5-(1,2-dithiolan-3-yl)pentylamino)propyl)dimethylamino)ethane-1-sulfonic acid and (R)-5-(1,2-dithiolan-3-yl)-N-(2-hydroxyethyl)pentanamide.

2. The cerium oxide nanozyme with antioxidant and anti-inflammatory functions according to claim 1, characterized in that In the amphiphilic polymer; The molar ratio of (R)-2-((3-(5-(1,2-dithiolan-3-yl)pentylamino)propyl)dimethylamino)ethane-1-sulfonic acid to (R)-5-(1,2-dithiolan-3-yl)-N-(2-hydroxyethyl)pentanamide is 3:

1.

3. The cerium oxide nanozyme with antioxidant and anti-inflammatory functions according to claim 2, characterized in that The average particle size of the cerium oxide nanoparticles is 9.65±0.64 nm, the hydrated particle size of the core-shell structure is 138.60±28.06 nm, and the zeta potential of the composite layer is -10 to -30 mV.

4. A method for preparing cerium oxide nanozyme according to claim 1, characterized in that: The following steps are involved: Step 1: Synthesis of amphiphilic polymers: (R)-2-((3-(5-(1,2-dithiocyclopentan-3-yl)pentylamino)propyl)dimethylamino)ethane-1-sulfonic acid and (R)-5-(1,2-dithiocyclopentan-3-yl)-N-(2-hydroxyethyl)pentanamide were dissolved in a methanol-water mixed solvent at a molar ratio of 3:1, and an initiator 2-aminoethanethiol was added to carry out polymerization reaction, and then the reaction was terminated by iodoacetamide, and the copolymer was purified by dialysis; Step 2, one-pot method for preparing nanozyme: sialic acid solution, protocatechuic aldehyde solution and the copolymer solution obtained in step 1 were mixed, stirred at 60° C. for 30 minutes, cerium nitrate solution was added dropwise, the pH was adjusted to 12, reacted in the dark for 8 hours, and dialyzed and purified to obtain the cerium oxide nanozyme.

5. The method for preparing the cerium oxide nanozyme with antioxidant and anti-inflammatory functions according to claim 4, characterized in that: In step 2, the concentration of the cerium nitrate solution is 43.7 mg / mL, and the dropping rate is 0.15 mL / min.

6. The method for preparing the cerium oxide nanozyme with antioxidant and anti-inflammatory functions according to claim 5, characterized in that: In step 2, ammonium hydroxide is used to adjust the pH, and the reaction system is carried out under nitrogen protection.

7. The method for preparing the cerium oxide nanozyme with antioxidant and anti-inflammatory functions according to claim 6, characterized in that: In step 2, dialysis was performed using a dialysis bag with a molecular weight cutoff of 1000 Da, and the dialysis time was 48 hours.

8. A use of the cerium oxide nanozyme according to claim 1, characterized in that: Used for preparing antioxidants for scavenging hydroxyl free radicals, superoxide anion free radicals or DPPH free radicals.

9. A use of the cerium oxide nanozyme according to claim 1, characterized in that: Used to prepare anti-inflammatory drugs that inhibit the expression of inflammatory factors IL-6 and TNF-α or promote the expression of anti-inflammatory factors IL-10 and TGF-β.

10. A pharmaceutical composition comprising the cerium oxide nanozyme according to any one of claims 1 to 3 and a pharmaceutically acceptable carrier, wherein the pharmaceutical composition is used to treat stroke, neurodegenerative diseases or oxidative stress-related inflammation.

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