A reduced single-atom nanomedicine, its preparation method and application

Abstract

This invention provides a reduced single-atom nanomedicine, its preparation method, and its application, belonging to the field of nanobiomedicine technology. The single-atom nanomedicine prepared by this invention is a cerium single-atom-anchored phosphomolybdic acid nanocluster, namely Ce-POM. The preparation method includes using activated carbon as a substrate, adding a phosphomolybdic acid solution for loading, subsequently introducing cerium(III) acetylacetone hydrate for mixing and stirring, and finally calcining and reducing with hydrogen in a tubular furnace to obtain the Ce-POM single-atom nanomedicine. The Ce-POM nanomedicine of this invention possesses peroxidase-like and catalase-like activities. Through highly efficient multi-enzyme activity and charge migration, it promotes the consumption of reactive oxygen species (ROS), reduces cellular oxidative stress damage, inhibits ferroptosis, improves the microenvironment of neural tissue in cerebral hemorrhage, and promotes neural tissue repair. Simultaneously, the decrease in ROS levels further inhibits the activation of the intracellular immune signaling pathway cGAS-STING, regulates immune system function, protects neural cell repair, and achieves the ideal therapeutic effect for cerebral hemorrhage.

Description

Technical Field

[0001] This invention belongs to the field of nanobiomaterials and neuroimmunotherapy technology, specifically relating to a reduced single-atom nanomedicine, its preparation method and application. Background Technology

[0002] The brain, as a vital organ, is susceptible to various diseases, leading to corresponding deficits in the nervous system. A major study published in *The Lancet Neurology* indicates that over 3 billion people worldwide are affected by neurological disorders, resulting in disability, illness, and premature death. Stroke is one of the most serious diseases, including two types: transient ischemic attack (TIA) and intracerebral hemorrhage (ICH). ICH accounts for approximately 20-30% of acute cerebrovascular diseases. Its primary causes are arteriosclerosis and hypertension, which damage cerebral blood vessels, causing blood to leak out and resulting in oxidative stress in peripheral brain tissue. Immune cells and inflammatory factors from the peripheral blood system enter the central nervous system, exacerbating nerve tissue damage and producing focal lesions. This oxidative stress is mainly due to iron overload in the body, leading to an imbalance between oxidation and antioxidation, making oxidation more prevalent. This results in inflammatory infiltration of neutrophils, increased secretion of proteases, and the production of large amounts of oxidative intermediates, causing lipid peroxidation of cell membranes and inducing ferrodeogenesis. Currently, the clinical treatment of intracerebral hemorrhage is mainly divided into conservative treatment and surgical treatment. Therefore, designing and constructing a highly effective drug to regulate the physiological microenvironment in response to oxidative stress-induced inflammatory damage to the nervous system in cerebral hemorrhage, and improving the limitations of clinical treatment for cerebral hemorrhage, has important scientific guiding significance and practical value.

[0003] Oxidative stress and inflammatory diseases are closely linked. When cells and tissues are damaged by oxidative stress and generate a large number of reactive free radicals, the autoimmune system is activated, secreting inflammatory factors to protect its own health. The signaling pathway composed of cyclic guanosine monophosphate-adenosine synthase (cGAS) and interferon gene stimulator (STING) can regulate cellular function and is associated with various diseases. In cerebral hemorrhage, the rupture of red blood cells releases hemoglobin and iron ions, leading to intracellular iron overload and the generation of reactive oxygen species (ROS). This induces the massive oxidation and accumulation of polyunsaturated fatty acids (PUFAs) in the cell membrane, resulting in non-apoptotic cell death. cGAS specifically recognizes abnormally present double-stranded DNA (dsDNA) in the cytoplasm, generating cyclic GMP-AMP (cGAMP) and activating the STING-stimulating gene, recruiting TBK1 and activating IRF3. This induces the expression and secretion of type I interferon and inflammatory factors, establishing an immune response. The STING protein can regulate PUFA metabolism by inhibiting the rate-limiting enzyme function of fatty acid desaturase 2 (FADS2), creating a negative feedback regulatory loop between the two. Therefore, excessive activation of STING disrupts PUFA metabolism, leading to oxidative stress and ferrodeogenesis. This oxidative stress not only causes cellular damage but also damages double-stranded DNA and mitochondria, such as the upregulation of proteins related to mitochondrial apoptosis, including the lymphoma-2 family protein (Bcl-2).

[0004] In recent years, with the emerging development of nanotechnology, nanozymes have been widely applied in the research and application of inflammatory diseases. As a new generation of artificial enzymes, nanozymes, due to their stable structure and abundant catalytic sites, exhibit various enzyme-like activities involved in the regulation of ROS levels in vivo. Single-atom nanozymes (SAzymes), as a type of nanozyme with high catalytic activity, have independent metal atoms stably fixed on a support in a monodisperse form, and their active sites are composed of independent metal atoms, maximizing metal utilization. Under physiological conditions, they can catalyze enzyme substrate reactions, exhibiting catalytic efficiency and enzymatic reaction kinetic properties similar to natural metalloenzymes. Single-atom nanozymes, with their abundant and efficient enzyme-like activity, are widely used in the diagnosis and treatment of biomedical diseases. However, their extremely high surface energy makes them prone to atomic aggregation during preparation and application, leading to a sharp drop in catalytic activity. Therefore, designing and constructing a single-atom nanozyme with highly efficient anti-inflammatory properties is crucial. Furthermore, using this nanomaterial to treat neuroinflammatory damage caused by cerebral hemorrhage has significant clinical implications. Summary of the Invention

[0005] This invention aims to...

[0006] To achieve the above-mentioned technical objectives and effects, the present invention provides the following technical solution:

[0007] A reduced single-atom nanomedicine (Ce-POM) is derived from activated carbon, phosphomolybdic acid (POM), and cerium(III) acetylacetone. The nanomedicine is a nanomaterial in which cerium (Ce) single atoms are anchored to the structure of phosphomolybdic acid (POM). The Ce element is mainly in the reduced valence state, and the particle size is 600-700 nm. The irregularly shaped particulate aggregates are loaded on an activated carbon substrate.

[0008] Furthermore, the electrode potential of the nanomedicine is -10mV to -12.5mV.

[0009] On the other hand, the present invention also provides a method for preparing the above-mentioned reduced single-atom nanomedicine (Ce-POM), comprising the following steps:

[0010] (1) Add phosphomolybdic acid solution to activated carbon powder and mix thoroughly. After stirring, sonicate to disperse phosphomolybdic acid on activated carbon.

[0011] (2) The solution obtained in step (1) is centrifuged and dried sequentially to obtain POM nanomaterials;

[0012] (3) Add cerium acetylacetone (III) hydrate solution to the POM nanomaterial obtained in step (2), stir and then ultrasonically disperse.

[0013] (4) After drying the mixture obtained in step (3), it is calcined at low temperature in a hydrogen atmosphere to obtain Ce-POM single-atom nanomedicine.

[0014] Further, in step (1), the mass-to-volume ratio of activated carbon solid to phosphomolybdic acid solution is 1 g: 10 mL; the concentration of the phosphomolybdic acid solution is 0.01 g / mL, which is prepared by dissolving phosphomolybdic acid in acetone.

[0015] Further, in step (1), the stirring conditions are a rotation speed of 500-700 r / min, a temperature of 37℃, a stirring time of 3h, and an ultrasonic treatment time of 10min.

[0016] Furthermore, in steps (2) and (3), the centrifugation conditions are both 7000 rpm / min for 4 min; the drying conditions are both 70℃ for 30 min.

[0017] Further, in step (3), the mass-to-volume ratio of POM solid to cerium acetylacetone (III) solution is 1 g: 1 mL; the concentration of the cerium acetylacetone (III) solution is 0.0265 g / mL, and it is prepared by dissolving cerium acetylacetone (III) hydrate in acetone.

[0018] Furthermore, in step (4), the calcination temperature under a hydrogen atmosphere is 160–170 °C.

[0019] On the other hand, the present invention also provides the application of the above-mentioned reduced single-atom nanomedicine (Ce-POM) in the preparation of neuroimmunotherapy drugs for cerebral hemorrhage.

[0020] Furthermore, the nanomedicine exerts its therapeutic effect by consuming reactive oxygen species (ROS), inhibiting ferroptosis, regulating the cGAS-STING immune signaling pathway, and promoting the polarization of macrophages from M1 to M2.

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

[0022] 1. The method for preparing the reduced single-atom nanomedicine Ce-POM of the present invention is simple, has low synthesis cost, and good catalytic performance. It utilizes the reversible mixed redox properties of anionic polyoxometalate nanoclusters and the high-density active sites of cerium single atoms to promote the consumption of reactive oxygen species (ROS) through efficient multi-enzyme activity and charge migration, thereby reducing cellular oxidative stress damage and improving the microenvironment of neural tissue in cerebral hemorrhage.

[0023] 2. The reduced single-atom nanomedicine Ce-POM of this invention targets the inflammatory microenvironment of cerebral hemorrhage, clearing overexpressed ROS in the microenvironment, repairing mitochondrial functional damage, inhibiting ferroptosis, and suppressing the expression of inflammatory cytokine TNF-α and related protein NF-κB by inhibiting the cGAS-STING immune signaling pathway. Furthermore, Ce-POM can further regulate macrophage polarization from M1 to M2, promoting immune system response, protecting nerve cell repair, and achieving ideal therapeutic effects for cerebral hemorrhage. Attached Figure Description

[0024] To more clearly illustrate the technical solution of the present invention, the accompanying drawings used in the description of the specific embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 Transmission electron microscopy image of Ce-POM prepared in Example 1;

[0026] Figure 2 The hydrated particle size analysis diagram of Ce-POM prepared in Example 1;

[0027] Figure 3 A comparison diagram of the electrode potentials of C, POM, and Ce-POM prepared in Example 1;

[0028] Figure 4 The elemental mapping energy spectrum of Ce-POM prepared in Example 1;

[0029] Figure 5 The infrared spectrum of Ce-POM prepared in Example 1;

[0030] Figure 6 X-ray energy spectrum of Ce-POM prepared in Example 1;

[0031] Figure 7 The graph shows the evaluation results of Ce-POM scavenging hydroxyl radicals prepared in Example 1;

[0032] Figure 8 The graph shows the superoxide dismutase activity evaluation results of Ce-POM prepared in Example 1;

[0033] Figure 9 The graph shows the catalase activity evaluation results of Ce-POM prepared in Example 1;

[0034] Figure 10 The image shows the results of the cytotoxicity verification of Ce-POM prepared in Example 1 against SHSY5Y cells;

[0035] Figure 11 The image shows the results of Ce-POM prepared in Example 1 repairing damaged SHSY5Y cells.

[0036] Figure 12 The graph shows the evaluation results of the hemolytic effect of Ce-POM prepared in Example 1;

[0037] Figure 13 Flow cytometry results of ROS removal in H2O2-stimulated SHSY5Y cells using Ce-POM prepared in Example 1. I: Control, II: H2O2 stimulation, III: H2O2 stimulation + 40 μg / mL Ce-POM, IV: H2O2 stimulation + 80 μg / mL Ce-POM.

[0038] Figure 14 The image shows the fluorescence verification results of mitochondrial membrane potential in H2O2-stimulated SHSY5Y cells using Ce-POM prepared in Example 1. I: Control, II: H2O2 stimulation, III: H2O2 stimulation + 40 μg / mL Ce-POM, IV: H2O2 stimulation + 80 μg / mL Ce-POM.

[0039] Figure 15 The figure shows the verification results of MDA content in H2O2-stimulated SHSY5Y cells using Ce-POM prepared in Example 1. I: Control, II: H2O2 stimulation, III: H2O2 stimulation + 40 μg / mL Ce-POM, IV: H2O2 stimulation + 80 μg / mL Ce-POM.

[0040] Figure 16 The image shows the Western blot results of the expression of STING, TBK1, TNF-α and IL-10 proteins in H2O2-stimulated SHSY5Y cells using Ce-POM prepared in Example 1.

[0041] Figure 17 The flow cytometry results of LPS-stimulated RAW264.7 cells prepared with Ce-POM in Example 1 are shown.

[0042] Figure 18 Image showing the Evans blue staining results of brain tissue from a mouse with cerebral hemorrhage;

[0043] Figure 19 A graph showing the evaluation and analysis of neurological deficit scores in mice with cerebral hemorrhage;

[0044] Figure 20 Images of brain tissue sections stained with hematoxylin and eosin (H&E) and Prussian blue from mice with cerebral hemorrhage. Detailed Implementation

[0045] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0046] To address existing clinical technical challenges, the present invention aims to provide a reduced single-atom nanomedicine, its preparation method, and its application. Based on its highly efficient multi-enzyme activity and charge migration, it promotes the consumption of reactive oxygen species (ROS), improves the microenvironment of neural tissue in cerebral hemorrhage, and enhances the efficacy of neuroimmunotherapy for cerebral hemorrhage.

[0047] To solve the aforementioned technical problems and achieve the objective of this invention, a method for preparing reduced single-atom nanomedicines is provided, comprising the following preparation technical route:

[0048] (1) Add phosphomolybdic acid solution to activated carbon powder and mix thoroughly. After stirring evenly, ultrasonic treatment is performed to disperse phosphomolybdic acid on activated carbon.

[0049] (2) The solution obtained in step (1) is centrifuged and dried sequentially to obtain POM nanomaterials;

[0050] (3) Add cerium acetylacetone (III) hydrate solution to the POM obtained in step (2) and stir to mix, then perform ultrasonic dispersion treatment;

[0051] (4) After drying the mixture obtained in step (3), it is calcined at low temperature with hydrogen to obtain Ce-POM single-atom nanomedicine;

[0052] (5) The product obtained in step (4) is subjected to index detection.

[0053] As further described above based on the preparation technique, the Ce-POM nanomedicine mainly includes the following preparation steps:

[0054] (1) Add 1 g of activated carbon solid powder to a 15 mL centrifuge tube, weigh 0.1 g of phosphomolybdic acid and dissolve it in 10 mL of acetone solution to obtain a phosphomolybdic acid solution with a concentration of 0.01 g / mL, add it to the centrifuge tube, stir it thoroughly for 3 h at a speed of 500-700 r / min and 37℃, and then sonicate it for 10 min to obtain solution A;

[0055] (2) Place solution A in an ultracentrifuge and centrifuge at 7000 rpm / min for 4 min. Then place the precipitate after centrifugation in an electric heating drying oven and dry it at 70℃ for 30 min to obtain solid B.

[0056] (3) Add solid B to a centrifuge tube, weigh 0.0265 g of cerium acetylacetone (III) hydrate and dissolve it in 1 mL of acetone solution to obtain a cerium acetylacetone solution with a concentration of 0.0265 g / mL. Add it to a centrifuge tube and stir at 37°C with a speed of 500-700 r / min. Mix it thoroughly with solid B and then sonicate it for 10 min to obtain solution C.

[0057] (4) Place solution C in an ultracentrifuge and centrifuge at 7000 rpm / min for 4 min. Then place the precipitate after centrifugation in an electric heating drying oven and dry it at 70℃ for 30 min. The resulting solid D is placed in a tube furnace and heated to 160-170℃ in a hydrogen atmosphere to obtain Ce-POM single-atom nanomedicine.

[0058] (5) The product obtained in step (4) is subjected to index detection.

[0059] As a further description of the above preparation steps:

[0060] In the specific preparation method of Ce-POM single-atom nanomedicine, in step (1), the mass-volume ratio of activated carbon solid to phosphomolybdic acid solution is 1 g: 10 mL; in step (3), the mass-volume ratio of POM solid to cerium acetylacetone (III) solution is 1 g: 1 mL.

[0061] As a further description of the above preparation steps:

[0062] The single-atom nanomedicine Ce-POM has a particle size of 600–700 nm; the electrode potential of Ce-POM single-atom nanomedicine is -10 mV to -12.5 mV; Ce-POM single-atom nanomedicine presents as amorphous particulate aggregates loaded on an activated carbon substrate.

[0063] The single-atom nanomedicine Ce-POM prepared by this invention has a rigid structure of phosphomolybdic acid polyoxometalate, which has an accurate molecular structure and can achieve precise control over the selectivity of single-atom catalysts. In addition, the presence of oxygen vacancy defects in the phosphomolybdic acid structure is conducive to the insertion of Ce single atoms, thereby forming a stable metal single-atom nanomaterial that can effectively transfer electrons and improve catalytic efficiency.

[0064] Based on the further description of the above preparation steps:

[0065] The reduced single-atom nanomedicine Ce-POM reagent prepared in this invention is applied to the immunotherapy of neuroinflammatory diseases caused by cerebral hemorrhage.

[0066] The invention will now be further described with reference to the accompanying drawings.

[0067] Example 1

[0068] A novel reduced Ce-POM single-atom nanomedicine, comprising the following main raw materials: activated carbon, phosphomolybdic acid and cerium acetylacetone (III), wherein the Ce-POM single-atom nanomedicine is prepared by using activated carbon as a material substrate, loading phosphomolybdic acid onto activated carbon, adding cerium acetylacetone (III) solution, and reducing it with hydrogen.

[0069] The specific synthesis process of the nanomedicine includes the following steps:

[0070] (1) Add 1 g of activated carbon solid powder to a 15 mL centrifuge tube, weigh 0.1 g of phosphomolybdic acid and dissolve it in 10 mL of acetone solution to obtain a phosphomolybdic acid solution with a concentration of 0.01 g / mL, add it to the centrifuge tube, stir it thoroughly for 3 h at a speed of 500-700 r / min and 37℃, and then sonicate it for 10 min to obtain solution A;

[0071] (2) Place solution A in an ultracentrifuge and centrifuge at 7000 rpm / min for 4 min. Then place the precipitate after centrifugation in an electric heating drying oven and dry it at 70℃ for 30 min to obtain solid B.

[0072] (3) Add solid B to a centrifuge tube, weigh 0.0265 g of cerium acetylacetone (III) hydrate and dissolve it in 1 mL of acetone solution to obtain a cerium acetylacetone solution with a concentration of 0.0265 g / mL. Add it to a centrifuge tube and stir at 37°C with a speed of 500-700 r / min. Mix it thoroughly with solid B and then sonicate it for 10 min to obtain solution C.

[0073] (4) Place solution C in an ultracentrifuge and centrifuge at 7000 rpm / min for 4 min. Then place the precipitate after centrifugation in an electric heating drying oven and dry it at 70℃ for 30 min. The resulting solid D is placed in a tube furnace and heated to 160-170℃ in a hydrogen atmosphere to obtain Ce-POM single-atom nanomedicine.

[0074] (5) The product obtained in step (4) is subjected to index detection.

[0075] Morphology and structural characterization evaluation of nanomedicines:

[0076] The reduced single-atom nanomedicine (Ce-POM) prepared in Example 1 was characterized using transmission electron microscopy, and the results are as follows: Figure 1 As shown in the figure, Ce-POM nanomedicine exhibits an irregularly shaped aggregate of particles, with the nanoparticles loaded on an activated carbon substrate.

[0077] The nanomedicine Ce-POM prepared in Example 1 was characterized using a Malvern particle size potential analyzer, and the results are as follows: Figure 2-3 As shown, from Figure 2 It can be seen that the Ce-POM nanoparticles loaded on activated carbon have a particle size between 600-700 nm. From... Figure 3 The results show that the electrode potential of activated carbon is between -11 and -14 mV, that of POM is between -11 and -13 mV, and that of Ce-POM is between -10 mV and -12.5 mV, indicating that they have better stability and biocompatibility.

[0078] The energy-dispersive spectrum of the nanomedicine Ce-POM was characterized by elemental analysis using characteristic X-rays, and the results are as follows: Figure 4 As shown in the figure, Ce-POM contains C, O, P, Mo and Ce elements, indicating that Ce element is introduced into phosphomolybdic acid.

[0079] The nanomedicine Ce-POM prepared in Example 1 was characterized using Fourier transform infrared spectroscopy, and the results are as follows: Figure 5 As shown, at 1064 cm -1 850 cm -1 and 789 cm-1 Three characteristic absorption peaks representing the Keggin structure of polyoxometalates were observed at the specified wavelength, belonging to the stretching vibrations of the PO and Mo-O bonds, respectively. The elemental valence distribution in the Ce-POM nanomedicine was characterized using X-ray photoelectron spectroscopy, and the results are as follows: Figure 6 As shown in the figure, in the Ce3d XPS fine spectrum, Ce 4+ 3d3 / 2 and Ce 3+ The ratio of 3d5 / 2 is 43:57.

[0080] Evaluation of enzyme activity detection results:

[0081] (1) The ability to inhibit ·OH was detected using the TMB colorimetric reaction. FeSO4 (1 mM) was first added to H2O2 (2 mM) and allowed to react fully. Then, different concentrations of Ce-POM (5, 25, 50, 75, 100, and 150 μg / mL) were prepared and added to the mixture. The mixture was reacted in the dark for 5 min, and finally, TMB (250 μM) was added for the colorimetric reaction. The results are as follows: Figure 7 As shown, the characteristic absorption peak of oxidized TMB at 652 nm decreases with increasing Ce-POM material concentration, indicating that Ce-POM's ability to scavenge ·OH is concentration-dependent.

[0082] (2) O2- scavenging capacity was determined using a superoxide anion exchange kit. Different concentrations of Ce-POM (25, 50, 75, 100, 150, 200 μg / mL) were prepared and mixed with the superoxide anion exchange kit reagents. The absorbance at 550 nm was measured, and superoxide dismutase (SOD) activity was calculated according to the kit instructions. Results are as follows: Figure 8 As shown, the characteristic absorption peak at 550 nm decreases with increasing Ce-POM material concentration, indicating that Ce-POM's ability to scavenge ·O2- is concentration-dependent and it has good superoxide dismutase-like activity.

[0083] (3) The activity of catalase was verified using a portable dissolved oxygen meter. Different concentrations of reduced single-atom nanoparticles (Ce-POM) were added to a 2 mM H₂O₂ solution, and the change in oxygen levels in the solution was measured to evaluate the catalase activity. The results are as follows: Figure 9 As shown, the oxygen production rate gradually increases with the increase of Ce-POM nanomedicine concentration, indicating that Ce-POM nanomedicine has good catalase-like activity and promotes the decomposition of H2O2 into O2.

[0084] Biosafety assessment:

[0085] The biosafety of Ce-POM was assessed using the CCK-8 assay, a cell proliferation and toxicity assay. Neuroblastoma subclonal cells (SHSY5Y) were selected as the research subject. When the cells reached 80-90% confluence in culture flasks, the culture medium was discarded, and trypsin was added to digest the cells into a cell suspension. The suspension was then transferred to centrifuge tubes, centrifuged, resuspended, and counted. Based on the counting results, the cell density was adjusted to 1×10⁻⁶ cells / mL. 5 / ml, 100μL / well, seeded into 96-well plates, with 6 replicates. After 24 hours of incubation, the old culture medium was removed, and fresh Ce-POM medium of different concentrations without bovine serum albumin (FBS) was added and incubated for another 24 hours. After removing the medium, the cells were washed three times with PBS, and CCK-8 medium solution without FBS was added to each 96-well plate. After incubation for 30 minutes, the absorbance at 450 nm was measured using a microplate reader, and the survival rate of SHSY5Y cells treated with different concentrations of Ce-POM nanomedicine was calculated. Results are as follows. Figure 10 As shown, Ce-POM nanomedicines maintain relative safety in SHSY5Y cells at low doses.

[0086] Following the same experimental procedures as described above, after SHSY5Y cells were plated for 24 hours, the old culture medium was removed. First, 200 mM H2O2 was added to stimulate the cells for 12 hours, followed by co-incubation with fresh Ce-POM culture medium of different concentrations. Subsequent experimental procedures were the same as described above. Results are as follows: Figure 11 As shown, H2O2 stimulation damages SHSY5Y cells and inhibits their growth and proliferation, while Ce-POM exhibits repair capabilities on damaged SHSY5Y cells.

[0087] The biocompatibility of Ce-POM was assessed using a hemolysis assay. Whole blood was collected from the orbital portion of C57 mice and placed in anticoagulant tubes containing EDTA. After thorough mixing, the cells were centrifuged at 3000 rpm for 15 min to obtain blood cells. Ce-POM was diluted with PBS to different concentrations. 1 mL of the nanomaterial solution (25, 50, 75, 100, 150 μg / mL), 1 mL of ultrapure water (ddH2O), and 1 mL of PBS were mixed with 20 μL of blood cells. The mixtures were incubated at 37°C for 4 h, then centrifuged at 3000 rpm for 15 min. The supernatant was pipetted into 96-well plates, and the absorbance at 542 nm was measured using a microplate reader. Three replicates were used for each group. Results are shown below. Figure 12 As shown, the hemolysis rate of Ce-POM is far below the international safety standard of 5%, indicating that Ce-POM has excellent blood compatibility and biocompatibility.

[0088] Evaluation of cell experiment results:

[0089] Evaluation of inhibition of ferroptosis:

[0090] SHSY5Y cells were used at a rate of 2 × 10 5 Cells were cultured at a density of 100 cells / mL, with 2 mL per well, evenly distributed in 6-well cell culture plates and incubated until complete cell adhesion. The old culture medium was then removed, and the cells were stimulated with 200 μM H2O2 for 12 h. Then, 40 μg / mL and 80 μg / mL Ce-POM were added to the damaged cells, and the cells were incubated at 37°C with 5% CO2 for 24 h. The DCFH-DA fluorescent probe was diluted 1:1000 with serum-free medium to prepare a 10 μM working solution. The cell supernatant was removed, and the cells were washed three times with PBS. 1 mL of diluted DCFH-DA was added, and the cells were co-incubated for 20 min. The cells were washed three times with serum-free cell culture medium to thoroughly remove any untreated DCFH-DA, and then analyzed by flow cytometry. I: Control, II: H2O2 stimulation, III: H2O2 stimulation + 40 μg / mL Ce-POM, IV: H2O2 stimulation + 80 μg / mL Ce-POM. Results are shown below. Figure 13 As shown, Ce-POM has a significant promoting effect on the recovery of oxidatively damaged SHSY5Y cells.

[0091] Mitochondrial lipid peroxidation levels were investigated using the BODIPY 581 / 591 C11 fluorescent probe. Cells were subjected to oxidative stress for 12 h by stimulating them with 200 mM H2O2 in DMEM / F12 medium, followed by Ce-POM treatment for 24 h. The prepared BODIPY 581 / 591 C11 staining solution was then added to the Ce-POM-treated cells, and the confocal culture dishes were placed in a cell culture incubator for approximately 30 min. After incubation, the cells were washed twice with PBS and photographed under a confocal microscope. Reduced lipids mainly exhibited red fluorescence, while oxidized lipids mainly showed green fluorescence. Results are as follows: Figure 14 As shown, the mitochondria of H2O2-stimulated SHSY5Y cells mainly exhibited a lipid oxidized state, while the mitochondria of H2O2-stimulated SHSY5Y cells treated with Ce-POM mainly exhibited a lipid reduced state, indicating that Ce-POM has a pro-repair function for damaged SHSY5Y cells. Malondialdehyde (MDA), a byproduct of cellular lipid peroxidation, was detected using an MDA kit. [The text abruptly ends here, likely due to an incomplete sentence or missing information.] 7 Add 1 mL of extraction buffer to each cell, centrifuge, and retain the supernatant. Add MDA working solution to the supernatant, mix thoroughly, and measure the absorbance at 532 nm using a microplate reader. The results are as follows: Figure 15 As shown, Ce-POM can reduce the oxidative byproduct MDA in damaged SHSY5Y cells and inhibit ferroptosis.

[0092] Evaluation of the inhibition of the cGAS-STING immune pathway in cells:

[0093] Total protein was extracted from mouse hippocampus, lysed using RIPA shear buffer, and quantified using a BCA protein assay kit. Proteins were separated by SDS-PAGE prepared with 30% acrylamide solution, transferred to a PVDF membrane, blocked with skim milk powder, and then detected with the following primary antibodies: STING, TBK1, TNF-α, and IL-10. After washing, the membrane was incubated with HRP-linked secondary antibodies. The signal was detected using enhanced chemiluminescence and captured using a UVITEC alliancemini HD6 imaging system. Results are shown below. Figure 16 As shown, Ce-POM can inhibit the activation of STING and TBK1 induced by H2O2 stimulation, and inhibit the expression of pro-inflammatory factor TNF-α while promoting the expression of anti-inflammatory factor IL-10.

[0094] Evaluation of macrophage immune regulation:

[0095] Using RAW264.7 macrophages as the research subject, the experiment was divided into a blank control group, a lipopolysaccharide (LPS) stimulation group, and an LPS+Ce-POM treatment group. After co-incubation of the cells, CD86 and CD206 antibodies were added, and the macrophage expression was detected by flow cytometry. Figure 17 As shown, the level of CD206 in the Ce-POM treatment group was higher than that of CD86, indicating that it can promote the transformation of damaged macrophages from M1 to M2 and improve the level of cellular inflammation.

[0096] Evaluation of animal test results:

[0097] SPF-grade C57BL / 6 mice were used as experimental animals and divided into four groups: sham-operated group, ICH + saline group, ICH + 0.5 times therapeutic concentration Ce-POM group, and ICH + therapeutic concentration Ce-POM group. Surgical models were established in all groups. Except for the sham-operated group, other experimental animals were used to establish a brain hemorrhage model using the Rynkowski method of autologous blood injection. Appropriate drug interventions were administered to each group of mice 6 h and 24 h after model establishment. The experimental period was 7 days. 0.5 h before dissection on day 7, one animal from each group was injected via the tail vein with 0.4 mL / kg of 2% Evans blue. After dissection, intact brain tissue was perfused and photographed. Neurological deficit scoring in ICH mice was performed daily from day 1 to day 7 after ICH modeling using a neurological deficit scoring scale to assess the mice's neurological function. For mice that underwent modeling intervention for 3 days, one mouse from each group was selected for formaldehyde fixation, followed by paraffin embedding and sectioning, and then subjected to H&E staining and Prussian blue staining, respectively. Results are as follows: Figure 18As shown, Evans blue staining was significant in the ICH group on day 7 after modeling, while Evans blue staining was significantly reduced in the Ce-POM treatment group. Figure 19 The results showed that as the treatment time with Ce-POM gradually increased, the score of neurological deficit in mice decreased, indicating that Ce-POM can promote the repair of brain hemorrhage damage in mice. Figure 20 As shown, H&E and Prussian blue staining of brain tissue sites indicates that Ce-POM treatment can promote the repair of brain hemorrhage damage.

[0098] In summary, the reduced single-atom nanomedicine Ce-POM prepared in this invention possesses diverse enzyme activities, exhibits excellent scavenging effects against reactive oxygen species, reduces cellular oxidative stress damage, inhibits ferroptosis, and suppresses the cGAS-STING immune signaling pathway, thereby improving the microenvironment of neural tissue in cerebral hemorrhage and promoting neural tissue repair. Furthermore, by inhibiting the immune signaling pathway, Ce-POM further promotes macrophage polarization towards the M2 type, enhancing the therapeutic effect of cerebral hemorrhage.

[0099] In the description of this specification, references to terms such as "an embodiment," "example," "specific example," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0100] The preferred embodiments of the present invention disclosed above are merely illustrative of the invention. These preferred embodiments do not exhaustively describe all details, nor do they limit the invention to the specific implementations described. Clearly, many modifications and variations can be made based on the content of this specification. This specification selects and specifically describes these embodiments to better explain the principles and practical applications of the invention, thereby enabling those skilled in the art to better understand and utilize the invention. The invention is limited only by the claims and their full scope and equivalents.

Claims

1. A reduced single-atom nanomedicine (Ce-POM), characterized in that, The raw materials include activated carbon, phosphomolybdic acid (POM), and cerium (III) acetylacetone; the nanomedicine is a nanomaterial in which cerium (Ce) single atoms are anchored to the structure of phosphomolybdic acid (POM), with Ce element in the reduced valence state as the main component, and the particle size is 600-700 nm. It is an irregularly shaped particulate aggregate loaded on an activated carbon substrate.

2. The reduced single-atom nanomedicine (Ce-POM) according to claim 1, characterized in that, The electrode potential of the nanomedicine is -10mV to -12.5mV.

3. A method for preparing a reduced single-atom nanomedicine (Ce-POM) as described in any one of claims 1 or 2, characterized in that, Includes the following steps: (1) Add phosphomolybdic acid solution to activated carbon powder and mix thoroughly. After stirring, sonicate to disperse phosphomolybdic acid on activated carbon. (2) The solution obtained in step (1) is centrifuged and dried sequentially to obtain POM nanomaterials; (3) Add cerium acetylacetone (III) hydrate solution to the POM nanomaterial obtained in step (2), stir and then ultrasonically disperse. (4) After drying the mixture obtained in step (3), it is calcined at low temperature in a hydrogen atmosphere to obtain Ce-POM single-atom nanomedicine.

4. The preparation method according to claim 3, characterized in that, In step (1), the mass-to-volume ratio of activated carbon solid to phosphomolybdic acid solution is 1 g: 10 mL; the concentration of the phosphomolybdic acid solution is 0.01 g / mL, which is prepared by dissolving phosphomolybdic acid in acetone.

5. The preparation method according to claim 3, characterized in that, In step (1), the stirring conditions are 500-700 r / min, 37℃, 3h stirring time, and 10min ultrasonic treatment time.

6. The preparation method according to claim 3, characterized in that, In steps (2) and (3), the centrifugation conditions were 7000 rpm / min for 4 min; the drying conditions were 70℃ for 30 min.

7. The preparation method according to claim 3, characterized in that, In step (3), the mass-to-volume ratio of POM solid to cerium acetylacetone (III) solution is 1 g: 1 mL; the concentration of cerium acetylacetone (III) solution is 0.0265 g / mL, which is prepared by dissolving cerium acetylacetone (III) hydrate in acetone.

8. The preparation method according to claim 3, characterized in that, In step (4), the calcination temperature under a hydrogen atmosphere is 160-170℃.

9. The use of the reduced single-atom nanomedicine (Ce-POM) according to claim 1 or 2 in the preparation of neuroimmunotherapy drugs for cerebral hemorrhage.

10. The application according to claim 9, characterized in that, The nanomedicine exerts its therapeutic effect by consuming reactive oxygen species (ROS), inhibiting ferroptosis, regulating the cGAS-STING immune signaling pathway, and promoting the polarization of macrophages from M1 to M2.

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