Manganese-doped cerium oxide nanomaterial, and preparation method and application thereof

Abstract

The application discloses a manganese-doped cerium oxide nanomaterial and a preparation method and application thereof. 2+ :Mn 3+ :Mn 4+ :) is smaller than the ionic radius of cerium ions (Ce 3+ :Ce 4+ :), when manganese ions are doped into the cerium oxide crystal lattice, the oxygen dissociation can be promoted to form surface oxygen vacancies, thereby increasing the number of surface catalytic sites. The manganese-doped cerium oxide nanomaterial in the application can exhibit excellent enzyme-like activity, including phosphatase-like activity, catalase-like activity, superoxide dismutase-like activity and hydroxyl radical scavenging activity. The manganese-doped cerium oxide nanomaterial provided by the application can regulate abnormal PTMs of neuron proteins by using phosphatase-like activity, and restore the phosphorylation level thereof, thereby treating neurodegenerative diseases. In addition, the manganese-doped cerium oxide nanomaterial provided by the application can relieve oxidative stress by using catalase-like activity, superoxide dismutase-like activity and hydroxyl radical scavengng activity to scavenge active oxygen, thereby treating active oxygen-related diseases.

Description

Technical Field

[0001] This invention relates to the field of medical nanomaterials technology, specifically to a manganese-doped cerium oxide nanomaterial, its preparation method, and its applications. Background Technology

[0002] Proteins play a variety of biological functions in living systems, such as catalysts, structural basis, and signaling messengers. Reversible protein phosphorylation, as a major form of post-translational modification (PTM), plays a crucial role in coordinating protein function. In vivo, protein phosphatases work in tandem with kinases to precisely regulate the protein phosphorylation-dephosphorylation cycle. However, in pathological microenvironments, the catalytic activity of protein phosphatases is impaired, leading to irreversible phosphorylation of proteins, particularly neuronal proteins, including α-synuclein (α-syn) and tau protein. These dysregulated PTMs disrupt the structure and biological function of these neuronal proteins, severely impairing synaptic function and contributing to the occurrence and progression of neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease, and Lewy body dementia. Although strategies exist to recruit protein phosphatases to dephosphorylate phosphorylated proteins and untangle or eliminate hyperphosphorylated protein aggregates, the imbalance between kinase and protein phosphatase systems remains an unavoidable challenge, irreversibly exacerbating abnormal protein PTMs.

[0003] Reactive oxygen species (ROS) play a crucial role in cell signal transduction and homeostasis. However, when the body's antioxidant system is impaired, such as by enzymes like catalase (CAT) and superoxide dismutase (SOD), ROS homeostasis is disrupted, leading to oxidative stress. Excessive ROS production oxidizes many cellular substances (such as proteins and DNA), causing abnormal cell function, cell death, and promoting disease development. Furthermore, excessive ROS production can also cause local inflammation, inhibiting disease recovery. Therefore, clearing ROS from lesion sites to alleviate oxidative stress is of great significance for treating ROS-related diseases (such as Parkinson's disease, ischemic stroke, epilepsy, kidney failure, liver damage, and allergic diseases).

[0004] In recent years, cerium oxide nanomaterials have exhibited various enzyme-like activities, such as phosphatase-like, CAT-like, and SOD-like activities, due to their unique surface oxygen vacancies and the coexistence of trivalent and tetravalent cerium redox pairs. These activities hold great promise for regulating protein micromolars (PTMs), scavenging reactive oxygen species (ROS), alleviating oxidative stress, and treating diseases. However, the application of cerium oxide nanomaterials in the biomedical field still faces many challenges. One significant bottleneck is how to enhance the enzyme-like activity of cerium oxide nanomaterials to further improve their therapeutic effects. Therefore, it is urgent to modify cerium oxide nanomaterials and develop cerium oxide-based nanomaterials with higher enzyme-like activity. Summary of the Invention

[0005] This invention addresses the problem of weak enzyme-like activity in cerium oxide nanomaterials by providing a manganese-doped cerium oxide nanomaterial, its preparation method, and its applications.

[0006] The technical solution provided by this invention is as follows:

[0007] A manganese-doped cerium oxide nanomaterial is obtained by doping manganese into the unit cell of cerium oxide nanomaterial, and the manganese-doped cerium oxide nanomaterial has an asymmetric catalytic center, in which a catalytic center composed of cerium, manganese and oxygen vacancies coexist at the same catalytic site.

[0008] In a preferred embodiment, the elemental ratio of cerium to manganese in the manganese-doped cerium oxide nanomaterial is 9-0.5:1, more preferably 2.5-3.5:1. At a ratio of 9-0.5:1, the resulting manganese-doped cerium oxide nanomaterial can form asymmetric catalytic centers, exhibiting higher activity compared to standard cerium oxide nanomaterials. At a ratio of 2.5-3.5:1, the resulting manganese-doped cerium oxide nanomaterial has the highest number of oxygen vacancies, thus also the highest number of asymmetric catalytic centers, resulting in higher activity.

[0009] In a preferred embodiment, the manganese-doped cerium oxide nanomaterial is prepared by a non-hydrolyzed sol-gel method. The ratio of cerium to manganese in the manganese-doped cerium oxide nanomaterial can be controlled by changing the feed ratio of cerium and manganese precursor compounds, the reaction time, and the reaction temperature. In other words, the feed ratio of cerium and manganese precursor compounds, the reaction time, and the reaction temperature all have interrelated effects on the cerium to manganese ratio in the manganese-doped cerium oxide nanomaterial. For example, when the molar ratio of cerium to manganese precursor compounds is 7:3, and the reaction temperature is 80 degrees Celsius and the reaction time is 24 hours, the resulting manganese-doped cerium oxide nanomaterial has a cerium / manganese elemental ratio in the range of 2.5-3.5:1. At this point, the nanomaterial has the highest number of oxygen vacancies and asymmetric catalytic centers, resulting in high activity. If the molar ratio of cerium precursor compound to manganese precursor compound is selected as 5:5, a product with a cerium / manganese element ratio of 2.5-3.5:1 in manganese-doped cerium oxide nanomaterials can be obtained by shortening the reaction time.

[0010] In a preferred embodiment, the surface of the manganese-doped cerium oxide nanomaterial is modified with ligands to ensure its dispersibility in an aqueous medium.

[0011] In a preferred embodiment, the ligand is selected from at least one reagent having a polyethylene glycol tail. The presence of polyethylene glycol at the tail of the ligand can increase systemic circulation time, thereby increasing the probability of reaching the lesion and contributing to better therapeutic effects.

[0012] In one exemplary embodiment, the ligand is selected from at least one of distearate phosphatidylethanolamine-polyethylene glycol, citric acid, and aminopolyethylene glycol.

[0013] In a preferred embodiment, the manganese-doped cerium oxide nanomaterials possess various enzyme activities, including phosphatase-like activity, catalase-like activity, superoxide dismutase-like activity, and hydroxyl radical scavenging activity.

[0014] This invention also provides a method for preparing manganese-doped cerium oxide nanomaterials, comprising: dissolving a cerium precursor compound and a manganese precursor compound in an oxygen-supplying alkyl oxide solvent; heating the reaction system under an inert atmosphere and continuously stirring the reaction; and then precipitating the manganese-doped cerium oxide nanoparticles with a poor solvent. This method belongs to the non-hydrolytic sol-gel method. A specific example is as follows: adding a cerium precursor compound (e.g., cerium acetylacetone hydrate, or cerium acetate hydrate) and a manganese precursor compound (e.g., manganese acetylacetone, or manganese acetate) to an oxygen-supplying alkyl oxide solvent (e.g., oleylamine) and dispersing and dissolving them (preferably by ultrasonication, for example); then heating and aging the reaction system under an inert atmosphere; and then precipitating the manganese-doped cerium oxide nanomaterials with a poor solvent (e.g., acetone or ethanol).

[0015] Preferably, in the preparation method of the manganese-doped cerium oxide nanomaterial, the cerium precursor compound is cerium acetylacetone hydrate, the manganese precursor compound is manganese acetylacetone, and the total amount of cerium acetylacetone hydrate and manganese acetylacetone is 0.5 g. The molar ratio of cerium acetylacetone hydrate to manganese acetylacetone is (9-0.3):1. The oxygen-supplying alkyl oxide solvent is oleylamine, and the amount of oleylamine can be 10-20 ml. When the molar ratio of cerium acetylacetone hydrate to manganese acetylacetone changes, the reaction temperature and reaction time can be adjusted appropriately. The reaction temperature affects the doping rate; the higher the temperature, the faster the doping rate. For example, when the molar ratio of cerium precursor compound to manganese precursor compound is 7:3, and the reaction temperature is 80 degrees Celsius with a reaction time of 24 hours, the resulting manganese-doped cerium oxide nanomaterials have a cerium / manganese elemental ratio of 2.5-3.5:1. At this ratio, the nanomaterials exhibit the highest number of oxygen vacancies and asymmetric catalytic centers, resulting in high activity. If a molar ratio of cerium precursor compound to manganese precursor compound of 5:5 is chosen, the reaction time can be shortened to obtain manganese-doped cerium oxide nanomaterials with a cerium / manganese elemental ratio of 2.5-3.5:1.

[0016] Preferably, the preparation method of the manganese-doped cerium oxide nanomaterial is as follows: cerium acetylacetone and manganese acetylacetone are added to oleylamine, subjected to thorough sonication, and then the reaction system is heated to 70–150 degrees Celsius and aged for 0–48 hours under an inert atmosphere. The aging time is further preferably 12–36 hours, and even more preferably 12–24 hours. The heating temperature is further preferably 70–80 degrees Celsius, or 80–150 degrees Celsius. More preferably, the heating rate is 1–8 degrees Celsius / min, more preferably 1–2 degrees Celsius / min, or 2–8 degrees Celsius / min.

[0017] The method for preparing manganese-doped cerium oxide nanomaterials further includes: modifying the surface of the prepared manganese-doped cerium oxide nanomaterials with ligands to obtain manganese-doped cerium oxide nanomaterials with good water dispersibility.

[0018] A specific example is as follows: Manganese-doped cerium oxide nanomaterials are dispersed in a polar solvent (e.g., chloroform), and disteaaroylphosphatidylethanolamine-polyethylene glycol 2000 (1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000], DSPE-PEG2000) is added for further dispersion. Subsequently, the solvent is removed by rotary evaporation to obtain manganese-doped cerium oxide nanomaterials modified with DSPE-PEG2000.

[0019] Preferably, in the above examples, the dosage ratio of manganese-doped cerium oxide nanomaterials, DSPE-PEG2000, and chloroform is 5–20 mg: 5–30 mg: 5–10 ml. The rotary evaporation temperature is 40–80 degrees Celsius.

[0020] Another specific example is as follows: Manganese-doped cerium oxide nanomaterials were dispersed in a mixed solution of N,N-dimethylformamide and chloroform, and citric acid (CA) was added and stirred for 12–36 h. The resulting product was activated with N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, and then reacted with a solution of methoxy(polyethylene glycol)2000amine (mPEG2000-NH2) to obtain CA-PEG2000 modified manganese-doped cerium oxide nanomaterials.

[0021] Preferably, in the above examples, the amounts of manganese-doped cerium oxide nanomaterials, citric acid, N,N-dimethylformamide, and chloroform are 5–20 mg, 50–600 mg, 4–10 ml, and 4–10 ml, respectively; the amounts of N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride are 5–50 mg and 5–50 mg, respectively; and the amount of mPEG2000-NH2 is 10–50 mg.

[0022] In the above preferred embodiments, the surface of the manganese-doped cerium oxide nanomaterials of the present invention is modified with hydrophilic ligands such as CA-PEG2000 and DSPE-PEG2000, which can be dispersed in aqueous solution and have good in vivo dispersibility and stability. They can exhibit excellent enzyme-like activities, including phosphatase-like activities, CAT-like activities, SOD-like activities, and hydroxyl radical scavenging activities.

[0023] Based on the same inventive concept, the present invention also provides a nano-therapeutic preparation comprising the above-mentioned manganese-doped cerium oxide nanomaterials.

[0024] In this invention, due to the size of surface oxygen cavities The larger size of the manganese ion can promote cation exchange between manganese ions and cerium ions next to surface oxygen vacancies, thereby forming a doped structure. Furthermore, due to the smaller ionic radius of the manganese ion... smaller than the ionic radius of the cerium ion When manganese ions are doped into the cerium oxide lattice, they can promote oxygen dissociation and form surface oxygen vacancies, thereby increasing the number of surface catalytic sites. The manganese-doped cerium oxide nanomaterial of the present invention has an asymmetric catalytic center, in which cerium, manganese, and oxygen vacancies coexist as a catalytic center (or reaction center) at the same catalytic site. In this structure, the dopant element also participates in the reaction, which can significantly improve the activation effect.

[0025] Therefore, the manganese-doped cerium oxide nanomaterials of the present invention can exhibit excellent enzyme-like activity, which can be used to reduce the phosphorylation level of hyperphosphorylated proteins and restore protein function, thereby treating diseases related to abnormal protein phosphorylation; and can be used to clear excessive ROS at lesion sites by utilizing antioxidant enzyme activity, thereby alleviating oxidative stress and treating ROS-related diseases.

[0026] Furthermore, this invention has demonstrated through material characterization, in vitro cell experiments, and animal experiments that manganese-doped cerium oxide nanomaterials possess excellent enzyme-like activity and can be used to treat diseases related to abnormal protein phosphorylation and ROS-related diseases, providing a promising nanotherapeutic agent for clinical use.

[0027] Compared with the prior art, the beneficial effects of the present invention are reflected in the following aspects:

[0028] (1) The preparation method of manganese-doped cerium oxide nanomaterials in this invention has a mild reaction system and controllable conditions. The synthesized manganese-doped cerium oxide nanomaterials have controllable nanoparticle size and morphology, and manganese can be uniformly doped into cerium oxide nanocells. The prepared materials have good biocompatibility and good clinical translation potential.

[0029] (2) The manganese-doped cerium oxide nanomaterials in this invention have a large number of surface catalytic sites, and the surface catalytic sites exhibit an asymmetric structure (asymmetric catalytic center) due to manganese ion doping, which can exert excellent enzyme-like activities, including phosphatase-like activities, CAT-like activities, SOD-like activities, and hydroxyl radical scavenging activities.

[0030] (3) The manganese-doped cerium oxide nanomaterials in this invention can utilize phosphatase-like activity to hydrolyze phosphorylated proteins, reduce the phosphorylation level of hyperphosphorylated proteins, restore protein function, and thus treat diseases related to abnormal protein phosphorylation; they can also utilize antioxidant enzyme activity to remove excessive ROS at the lesion site, thereby alleviating oxidative stress and treating ROS-related diseases.

[0031] Of course, any product implementing this invention does not necessarily need to achieve all of the advantages described above at the same time. Attached Figure Description

[0032] Figure 1 The following are density functional theory simulation results of manganese-doped cerium oxide nanomaterials in the embodiments of the present invention. Figures a and b show the energy changes as Ce and Mn move to the oxygen-free cerium oxide crystal plane (a) and the oxygen-containing cerium oxide crystal plane (b); Figure c shows the energy changes during the cation exchange reaction driven by surface oxygen vacancies; Figure d shows the formation energy of the oxygen-free cerium oxide crystal plane, the oxygen-containing cerium oxide crystal plane, and the cerium oxide crystal planes with different manganese doping; Figure e shows a schematic diagram of the formation of manganese-doped cerium oxide nanomaterials through the cation exchange reaction driven by surface oxygen vacancies.

[0033] Figure 2 The figures shown are test results of manganese-doped cerium oxide nanomaterials in Example 1 of the present invention. Figure a is a transmission electron microscope (TEM) image of manganese-doped cerium oxide nanomaterials, Figure b is a spherical aberration TEM image of manganese-doped cerium oxide nanomaterials, Figure c is an X-ray diffraction pattern, and Figure d is an X-ray photoelectron spectroscopy pattern.

[0034] Figure 3 Figure 1 shows the test results of manganese-doped cerium oxide nanomaterials at different reaction times in Example 1 of the present invention. Figure 1a is a spherical aberration transmission electron microscope image of manganese-doped cerium oxide nanomaterials with a curing time of 15 minutes. Figure 2b is a graph showing the change in manganese content in manganese-doped cerium oxide nanomaterials as the curing time increases. Figure 3c is a time-of-flight secondary ion mass spectrometer image of manganese-doped cerium oxide nanomaterials with a curing time of 24 hours.

[0035] Figure 4 Transmission electron microscope (TEM) image (a) and hydrodynamic diameter distribution map (b) of the manganese-doped cerium oxide nanomaterial modified with CA-PEG2000 in Example 1 of this invention;

[0036] Figure 5 The image shows the characterization of phosphatase-like activity, CAT-like activity, SOD-like activity, and hydroxyl radical scavenging activity of the CA-PEG2000 modified manganese-doped cerium oxide nanomaterial in Example 1 of this invention.

[0037] Figure 6 This is a transmission electron microscope (TEM) image of the manganese-doped cerium oxide nanomaterial modified with DSPE-PEG2000 in Example 2 of this invention.

[0038] Figure 7 This is a laser confocal image of the intracellular p-α-syn level in SH-SY5Y cells after various treatments in Application Example 1 of this invention;

[0039] Figure 8 This is a laser confocal image of the p-α-syn level in the brain of mice after various treatments in Application Example 1 of this invention;

[0040] Figure 9 This is a laser confocal image of the intracellular ROS levels of SH-SY5Y cells after various treatments in Application Example 2 of this invention;

[0041] Figure 10 This is a laser confocal image showing the intracellular mitochondrial damage in SH-SY5Y cells after various treatments in Application Example 2 of this invention;

[0042] Figure 11 Figure a shows the free energy diagram (CAT-like activity) of H2O2 decomposition on manganese-doped cerium oxide nanomaterials and cerium oxide nanomaterials. Figure b shows a schematic diagram of the mechanism of H2O2 decomposition catalyzed by the asymmetric catalytic center of the present invention. Detailed Implementation

[0043] This invention discloses a manganese-doped cerium oxide nanomaterial, its preparation method, and its application. The manganese-doped cerium oxide nanomaterial of this invention is obtained by uniformly doping manganese into the unit cell of cerium oxide nanomaterials through a surface oxygen vacancy-driven cation exchange strategy. Furthermore, the manganese-doped cerium oxide nanomaterial possesses asymmetric catalytic centers, with catalytic centers composed of cerium, manganese, and oxygen vacancies coexisting at the same catalytic site. The manganese-doped cerium oxide nanomaterial of this invention is prepared by a non-hydrolyzed sol-gel method. In a preferred embodiment, the preparation method is as follows: 1) Using oleylamine as a solvent, and cerium acetylacetone hydrate and manganese acetylacetone as precursor compounds, cerium acetylacetone hydrate and manganese acetylacetone are added to oleylamine and fully dispersed. Then, the reaction system is heated and matured under an inert atmosphere. Specifically, cerium oxide nanonuclei are first synthesized under heating conditions, and then heating is continued. Manganese undergoes cation exchange at the surface oxygen vacancy sites of the synthesized cerium oxide nanonuclei to obtain manganese-doped cerium oxide nanoparticles uniformly doped with manganese. 2) The prepared manganese-doped cerium oxide nanomaterials were modified with hydrophilic ligands to obtain manganese-doped cerium oxide nanomaterials with better water dispersibility.

[0044] In the manganese-doped cerium oxide nanomaterials of the present invention, due to the size of surface oxygen holes... The relatively large size of the manganese ion promotes cation exchange between manganese ions and cerium ions adjacent to surface oxygen vacancies, thereby forming a doped structure. Furthermore, due to the relatively large ionic radius of the manganese ion... smaller than the ionic radius of the cerium ion When manganese ions are doped into the cerium oxide lattice, they can promote oxygen dissociation and form surface oxygen vacancies, thereby increasing the number of surface catalytic sites.

[0045] See Figure 1 Figures a and b show the energy changes as Ce and Mn migrate to cerium oxide crystal faces (a) without oxygen vacancies and (b) with oxygen vacancies. The results show that Mn is more likely to contact the material surface than Ce when migrating into the bulk phase, and its migration barrier is lower, especially in cerium oxide crystal faces with oxygen vacancies. Therefore, Mn preferentially enters the bulk phase and does not migrate into the metal vacancies. Figure c shows the energy changes during the cation exchange reaction driven by surface oxygen vacancies. The results show that Mn has a lower migration barrier inside the vacancies and can migrate inward to form uniform doping. Figure d shows the formation energy of cerium oxide crystal faces without oxygen vacancies, cerium oxide crystal faces with oxygen vacancies, and cerium oxide crystal faces with different manganese doping. The results show that manganese preferentially performs single doping in the lattice, which confirms that manganese doping is controllable and uniform. Figure e is a schematic diagram of manganese-doped cerium oxide nanomaterials formed by cation exchange reaction driven by surface oxygen vacancies.

[0046] Therefore, the manganese-doped cerium oxide nanomaterials of the present invention can exhibit excellent enzyme-like activities, including phosphatase-like activities, catalase-like activities, superoxide dismutase-like activities, and hydroxyl radical scavenging activities. The manganese-doped cerium oxide nanomaterials provided by the present invention can utilize phosphatase-like activities to regulate abnormal PTMs of neuronal proteins and restore their phosphorylation levels, thereby potentially treating neurodegenerative diseases. Furthermore, the manganese-doped cerium oxide nanomaterials provided by the present invention can utilize catalase-like activities, superoxide dismutase-like activities, and hydroxyl radical scavenging activities to scavenge reactive oxygen species and alleviate oxidative stress, thereby potentially treating reactive oxygen species-related diseases.

[0047] The present invention will be further illustrated below with reference to specific embodiments, application examples, and the accompanying drawings.

[0048] Example 1

[0049] Synthesis of CA-PEG2000-modified manganese-doped cerium oxide nanomaterials

[0050] (1) Synthesis of manganese-doped cerium oxide nanoparticles

[0051] A total of 0.5 g of cerium acetylacetone hydrate and manganese acetylacetone (0.372 g of cerium acetylacetone hydrate and 0.128 g of manganese acetylacetone) were dissolved in 15 ml of oleylamine and sonicated until fully dissolved. The reaction system was then heated to 80°C under an inert atmosphere and stirred continuously for 24 h. The product was precipitated with acetone and washed several times to remove impurities, then collected by centrifugation. The collected product was dispersed in 10 ml of chloroform for later use.

[0052] The prepared manganese-doped cerium oxide nanoparticles were tested, and the results are shown in the figure. Figure 2 Figure 1 shows the transmission electron microscope (TEM) image of manganese-doped cerium oxide nanoparticles, Figure 2 shows the aberration-corrected TEM image, Figure 3 shows the X-ray diffraction pattern, and Figure 4 shows the X-ray photoelectron spectroscopy (XPS) pattern. The results show that the manganese-doped cerium oxide nanoparticles have a small size, with manganese uniformly incorporated into the cerium oxide unit cell without forming manganese aggregates or particles (Figure 1). The surface of the manganese-doped cerium oxide nanoparticles has numerous catalytic sites and asymmetric catalytic centers, with catalytic centers composed of cerium, manganese, and oxygen vacancies coexisting at the same catalytic site (Figure 1). The manganese-doped cerium oxide nanoparticles exhibit only a cubic fluorite structure, demonstrating that the original crystal form was maintained during doping, and no other crystal forms were formed (Figure 1). The surface of the manganese-doped cerium oxide nanoparticles is rich in oxygen vacancies (Figure 4), laying the chemical foundation for their excellent enzyme-like activity.

[0053] The reaction time was also investigated in this study; the effect of reaction time on doping is detailed in [link to study]. Figure 3Figure a shows a spherical aberration transmission electron microscope (TEM) image of manganese-doped cerium oxide nanomaterials with a reaction aging time of 15 minutes. The results in the figure indicate that manganese ions preferentially dope at oxygen vacancy sites. Figure b shows the change in manganese content in the manganese-doped cerium oxide nanomaterials with the extension of reaction aging time. The results in the figure show that, on the one hand, the manganese-doped cerium oxide nanomaterials are first formed as cerium oxide nanomaterials and then doped on the cerium oxide nanomaterials. On the other hand, it shows that this doping is controllable and slow. Figure c shows a time-of-flight secondary ion mass spectrometry (TOF-MS) image of manganese-doped cerium oxide nanomaterials with a reaction aging time of 24 hours. The results in the figure show that manganese ions are uniformly doped into the cerium oxide nanomaterials.

[0054] (2) Synthesis of CA-PEG2000 modified manganese-doped cerium oxide nanomaterials

[0055] 200 mg of CA and 10 mg of manganese-doped cerium oxide nanoparticles were added to a mixture of 7.5 ml N,N-dimethylformamide and 7.5 ml chloroform, and the mixture was stirred at room temperature for 24 h. CA-modified manganese-doped cerium oxide nanomaterials were precipitated with diethyl ether and washed several times with acetone. The obtained CA-modified manganese-doped cerium oxide nanomaterials were then dispersed in 5 ml of MES buffer (pH = 5.6), and 15 mg of N-hydroxysuccinimide and 15 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride were added for activation for half an hour. After centrifugation, the precipitate was dispersed in a solution containing 20 mg NH2-PEG2000 and stirred overnight. Ultrafiltration yielded CA-PEG2000-modified manganese-doped cerium oxide nanomaterials.

[0056] The prepared CA-PEG2000 modified manganese-doped cerium oxide nanomaterials were tested, and the results are shown in the figure. Figure 4 Figure a shows a transmission electron microscope (TEM) image of the CA-PEG2000-modified manganese-doped cerium oxide nanomaterial, and figure b shows its hydrodynamic diameter distribution. The results in the figures show that the CA-PEG2000 manganese-doped cerium oxide nanomaterial has good water dispersibility (Figures a and b).

[0057] Figure 5This diagram shows a comparison of the enzyme-like activity characterization of the CA-PEG2000-modified manganese-doped cerium oxide nanomaterials and undoped cerium oxide nanomaterials in this embodiment. In the diagram, a represents the phosphatase-like activity characterization of the CA-PEG2000-modified manganese-doped cerium oxide nanomaterials and undoped cerium oxide nanomaterials in this embodiment; b represents the CAT-like activity characterization of the CA-PEG2000-modified manganese-doped cerium oxide nanomaterials and undoped cerium oxide nanomaterials in this embodiment; c represents the SOD-like activity characterization of the CA-PEG2000-modified manganese-doped cerium oxide nanomaterials and undoped cerium oxide nanomaterials in this embodiment; and d represents the hydroxyl radical scavenging ability detection results of the CA-PEG2000-modified manganese-doped cerium oxide nanomaterials and undoped cerium oxide nanomaterials in this embodiment. The test results show that the CA-PEG2000 manganese-doped cerium oxide nanomaterials prepared in this embodiment have better enzyme-like activity, exhibiting superior phosphatase-like activity, CAT-like activity, SOD-like activity, and hydroxyl radical scavenging activity compared to undoped cerium oxide nanomaterials.

[0058] Example 2

[0059] Synthesis of DSPE-PEG2000-modified manganese-doped cerium oxide nanomaterials

[0060] (1) Synthesis of manganese-doped cerium oxide nanoparticles

[0061] A total of 0.5 g of cerium acetylacetone hydrate and manganese acetylacetone (0.372 g of cerium acetylacetone hydrate and 0.128 g of manganese acetylacetone) were dissolved in 15 ml of oleylamine and sonicated until fully dissolved. The reaction mixture was then heated to 80°C under an inert atmosphere and stirred continuously for 24 h. Impurities were removed by washing several times with acetone, and the product was collected by centrifugation. The collected product was dispersed in 10 ml of chloroform for later use.

[0062] (2) Synthesis of manganese-doped cerium oxide nanomaterials modified with DSPE-PEG2000

[0063] 20 mg of DSPE-PEG2000 and 10 mg of manganese-doped cerium oxide nanoparticles were added to 5 ml of chloroform and sonicated in a water bath ultrasonic cooker for 10 min. The mixture was then rotary evaporated at 60°C for 1 hour, followed by hydration with 1 ml of deionized water to obtain DSPE-PEG2000-modified manganese-doped cerium oxide nanomaterials.

[0064] Figure 6 This is a transmission electron microscope (TEM) image of the manganese-doped cerium oxide nanomaterial modified with DSPE-PEG2000 in this embodiment. The results show that the DSPE-PEG2000 manganese-doped cerium oxide nanomaterial has good water dispersibility.

[0065] Application Example 1

[0066] Application of manganese-doped cerium oxide nanomaterials in the treatment of diseases related to abnormal protein phosphorylation

[0067] (1) CA-PEG2000 modified manganese-doped cerium oxide nanomaterials reduce the level of intracellular phosphorylated α-syn (p-α-syn).

[0068] Drug preparation: The CA-PEG2000 modified manganese-doped cerium oxide nanomaterials prepared in Example 1 were diluted with deionized water to prepare a concentrated stock solution.

[0069] Cell model establishment: SH-SY5Y cells were incubated with N-methyl-4-phenylpyridinium iodide (MPP+) to increase the level of p-α-syn in SH-SY5Y cells.

[0070] Group setup: a. Control group: Normal cells were given fresh culture medium. b. Model group: SH-SY5Y cells were incubated in culture medium containing 1 mM MPP+. c. Treatment group: SH-SY5Y cells were incubated in culture medium containing 1 mM MPP+ and 20 μg / mL CA-PEG2000 modified manganese-doped cerium oxide nanomaterials.

[0071] After 24 h of incubation, SH-SY5Y cells were fixed with 4% paraformaldehyde for 20 min. After washing three times with phosphate-buffered saline (PBS), the cells were treated with 0.2% Triton X-100 for 20 min. After washing three times with PBS, the cells were blocked with 5% bovine serum albumin for 1 h. Subsequently, the cells were incubated with anti-p-α-syn antibody (1:500) at 37 °C for 2 h. After washing three times with PBS, the cells were incubated with secondary antibody for 1 h. Cell nuclei were stained with DAPI and mounted with anti-fluorescence attenuation mounting medium. Laser confocal microscopy was then used to evaluate the ability of CA-PEG2000-modified manganese-doped cerium oxide nanomaterials to reduce p-α-syn levels at the cellular level.

[0072] Figure 7 Laser confocal microscopy images show the levels of p-α-syn in SH-SY5Y cells from different groups. The results in the images show that manganese-doped cerium oxide nanomaterials modified with CA-PEG2000 can hydrolyze abnormally phosphorylated p-α-syn in cells, restoring it to α-syn.

[0073] (2) Treatment of Parkinson's Disease with Manganese-Doped Cerium Oxide Nanomaterials Modified with CA-PEG2000

[0074] Drug preparation: The CA-PEG2000 modified manganese-doped cerium oxide nanomaterials prepared in Example 1 were diluted with physiological saline to prepare a concentrated stock solution.

[0075] Establishment of animal model: 7-8 week old male C57BL / c mice were injected subcutaneously into the brain with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) (20 mg / kg) once every 2 hours for a total of 4 injections to establish a Parkinson's disease mouse model.

[0076] Group setup: a. Blank group: Subcutaneous injection of an equal volume of physiological saline into the brain. b. Model group: Subcutaneous injection of MPTP (20 mg / kg) into the brain, once every 2 hours, for a total of 4 injections. c. Treatment group: Tail vein injection of CA-PEG2000 modified manganese-doped cerium oxide nanomaterials (2 mg / kg), followed by subcutaneous injection of MPTP (20 mg / kg) into the brain 24 hours later, once every 2 hours, for a total of 4 injections. CA-PEG2000 modified manganese-doped cerium oxide nanomaterials (2 mg / kg) were injected again 24 hours later.

[0077] Mice were sacrificed 24 hours later, and brain tissue from different treatment groups was collected and fixed in 10% formalin for 24 hours, followed by further processing and paraffin embedding. Paraffin-embedded tissue sections were analyzed by p-α-syn staining.

[0078] Figure 8 Laser confocal microscopy images show the levels of p-α-syn in the brains of mice from different groups. The results in the images show that manganese-doped cerium oxide nanomaterials modified with CA-PEG2000 can hydrolyze the abnormally phosphorylated p-α-syn in the brains of Parkinson's disease patients, restoring it to α-syn.

[0079] Application Example 2

[0080] Therapeutic applications of manganese-doped cerium oxide nanomaterials in ROS-related diseases

[0081] (1) Evaluation of the ability of CA-PEG2000 modified manganese-doped cerium oxide nanomaterials to scavenge ROS in vitro

[0082] Drug preparation: The CA-PEG2000 modified manganese-doped cerium oxide nanomaterials prepared in Example 1 were diluted with deionized water to prepare a concentrated stock solution.

[0083] Cell model establishment: SH-SY5Y cells were incubated with hydrogen peroxide to increase the ROS level in SH-SY5Y cells.

[0084] Group setup: a. Control group: Normal cells were given fresh culture medium. b. Model group: SH-SY5Y cells were incubated in culture medium containing hydrogen peroxide. c. Treatment group: SH-SY5Y cells were incubated in culture medium containing hydrogen peroxide and 20 μg / mL CA-PEG2000 modified manganese-doped cerium oxide nanomaterials.

[0085] After 24 h of incubation, the cells were washed three times with PBS. The DCFH-DA probe (10 μmol / L) was diluted 1:1000 with serum-free DMEM medium and added to a confocal dish for 20 min of incubation. The confocal dish was washed with serum-free DMEM medium to remove excess DCFH-DA probe. SH-SY5Y cells were fixed with 4% paraformaldehyde for 20 min. After three washes with PBS, the cell nuclei were stained with DAPI, and the cells were mounted with anti-fluorescence attenuation mounting medium. Subsequently, laser confocal microscopy was used to image and evaluate the ROS scavenging effect of manganese-doped cerium oxide nanomaterials at the cellular level.

[0086] Figure 9 Laser confocal microscopy images show the intracellular ROS levels in SH-SY5Y cells from different groups. The results indicate that CA-PEG2000-modified manganese-doped cerium oxide nanomaterials can scavenge excess ROS in cells and alleviate oxidative stress.

[0087] (2) Evaluation of the protective effect of CA-PEG2000 modified manganese-doped cerium oxide nanomaterials against oxidative damage to mitochondria.

[0088] Drug preparation: The CA-PEG2000 modified manganese-doped cerium oxide nanomaterials prepared in Example 1 were diluted with deionized water to prepare a concentrated stock solution.

[0089] Cell model establishment: SH-SY5Y cells were incubated with hydrogen peroxide to increase the ROS level in SH-SY5Y cells.

[0090] Group setup: a. Control group: Normal cells were given fresh culture medium. b. Model group: SH-SY5Y cells were incubated in culture medium containing hydrogen peroxide. c. Treatment group: SH-SY5Y cells were incubated in culture medium containing hydrogen peroxide and 20 μg / mL CA-PEG2000 modified manganese-doped cerium oxide nanomaterials.

[0091] After 24 hours of incubation, the cells were washed three times with PBS. JC-1 working solution was added and incubated for 20 minutes, followed by removal of the JC-1 working solution and washing twice with washing buffer. The cell nuclei were then stained with DAPI and mounted with anti-fluorescence attenuation mounting medium. Subsequently, laser confocal microscopy was used to evaluate the protective effect of manganese-doped cerium oxide nanomaterials against mitochondrial oxidative damage at the cellular level.

[0092] Figure 10 Laser confocal microscopy images show mitochondrial damage in SH-SY5Y cells from different groups. The results indicate that CA-PEG2000-modified manganese-doped cerium oxide nanomaterials can protect mitochondria and prevent oxidative damage.

[0093] Figure 11 Figure a shows the free energy diagram (CAT-like activity) of H2O2 decomposition on manganese-doped cerium oxide nanomaterials and cerium oxide nanomaterials, while Figure b shows a schematic diagram of the mechanism of H2O2 decomposition catalyzed by the asymmetric catalytic center of the present invention.

[0094] In this document, the range expressed as "from one value to another" is a concise way of representing a range to avoid listing all the values ​​in that range in the specification. Therefore, the description of a particular range of values ​​covers any value within that range as well as the smaller range of values ​​defined by that value, just as if the arbitrary value and the smaller range of values ​​were explicitly stated in the specification.

[0095] Under the guidance of the present invention and the above embodiments, those skilled in the art will readily foresee that all the raw materials or their equivalents, processing methods or their equivalents listed or exemplified in the present invention can achieve the present invention, and that the upper and lower limits and range values ​​of the parameters of each raw material and processing method can also achieve the present invention. Examples are not listed one by one here.

Claims

1. A manganese-doped cerium oxide nanomaterial with enzyme-like activity, characterized in that, The manganese-doped cerium oxide nanomaterial is obtained by doping manganese into the unit cell of cerium oxide nanomaterial through a surface oxygen vacancy-driven cation exchange method. The manganese-doped cerium oxide nanomaterial has asymmetric catalytic centers, with catalytic centers composed of cerium, manganese, and oxygen vacancy coexisting at the same catalytic site. In the manganese-doped cerium oxide nanomaterial, the elemental ratio of cerium to manganese is 9-2.5:

1. In the surface oxygen vacancy-driven cation exchange method, the cerium precursor compound and the manganese precursor compound are dissolved in an oxygen-supplying alkyl oxide solvent.

2. The manganese-doped cerium oxide nanomaterial with enzyme-like activity according to claim 1, characterized in that, In the manganese-doped cerium oxide nanomaterials, the elemental ratio of cerium to manganese is 3.5-2.5:

1.

3. The manganese-doped cerium oxide nanomaterial with enzyme-like activity according to claim 1 or 2, characterized in that, It is prepared by a non-hydrolyzed sol-gel method. The ratio of cerium to manganese in the manganese-doped cerium oxide nanomaterial can be controlled by changing the feed ratio of cerium precursor compound and manganese precursor compound, reaction time, and reaction temperature.

4. The manganese-doped cerium oxide nanomaterial with enzyme-like activity according to claim 1 or 2, characterized in that, The surface of the manganese-doped cerium oxide nanomaterial is modified with ligands to ensure its dispersibility in an aqueous medium.

5. The manganese-doped cerium oxide nanomaterial with enzyme-like activity according to claim 4, characterized in that, The ligand is selected from at least one reagent whose tail contains polyethylene glycol.

6. The manganese-doped cerium oxide nanomaterial with enzyme-like activity according to claim 5, characterized in that, The ligand is selected from at least one of distearate phosphatidylethanolamine-polyethylene glycol, CA-PEG2000, and aminopolyethylene glycol.

7. The manganese-doped cerium oxide nanomaterial with enzyme-like activity according to claim 1, characterized in that, The manganese-doped cerium oxide nanomaterials described above possess various enzyme activities, including phosphatase-like activities, catalase-like activities, superoxide dismutase-like activities, and hydroxyl radical scavenging activities.

8. A method for preparing manganese-doped cerium oxide nanomaterials with enzyme-like activity as described in any one of claims 1 to 7, characterized in that, Includes the following steps: Cerium and manganese precursor compounds were dissolved in an oxygen-supplying alkyl oxide solvent. The reaction system was heated to 70-150 degrees Celsius and aged for no more than 48 hours under an inert atmosphere while continuously stirring. Then, manganese-doped cerium oxide nanoparticles were obtained by precipitation with a poor solvent.

9. The method for preparing manganese-doped cerium oxide nanomaterials with enzyme-like activity according to claim 8, characterized in that, It also includes the following steps: using ligands to modify the surface of the prepared manganese-doped cerium oxide nanomaterials to ensure their dispersibility in aqueous media.

10. The method for preparing manganese-doped cerium oxide nanomaterials with enzyme-like activity according to claim 9, characterized in that, The manganese-doped cerium oxide nanoparticles have a size of 0.5-5 nm, and the manganese-doped cerium oxide nanomaterials modified with ligands have a size of 5-30 nm.

11. The method for preparing manganese-doped cerium oxide nanomaterials with enzyme-like activity according to claim 8, characterized in that, The heating rate is 1-8 degrees Celsius per minute.

12. The application of a manganese-doped cerium oxide nanomaterial with enzyme-like activity as described in any one of claims 1 to 7 in the preparation of a medicament for treating diseases related to abnormal protein phosphorylation, characterized in that, The manganese-doped cerium oxide nanomaterials described herein utilize phosphatase-like activity to reduce the phosphorylation level of hyperphosphorylated proteins, restore protein function, and thus treat diseases related to abnormal protein phosphorylation.

13. The application of the manganese-doped cerium oxide nanomaterial with enzyme-like activity according to claim 12 in the preparation of drugs for treating diseases related to abnormal protein phosphorylation, characterized in that, The diseases associated with abnormal protein phosphorylation include at least one of Parkinson's disease, Alzheimer's disease, and Lewy body dementia.

14. The application of the manganese-doped cerium oxide nanomaterial with enzyme-like activity according to claim 12 in the preparation of drugs for treating diseases related to abnormal protein phosphorylation, characterized in that, The manganese-doped cerium oxide nanomaterials utilize antioxidant enzyme activity to remove excessive reactive oxygen species at the lesion site, thereby alleviating oxidative stress and treating reactive oxygen species-related diseases.

15. The application of the manganese-doped cerium oxide nanomaterial with enzyme-like activity according to claim 14 in the preparation of drugs for treating diseases related to abnormal protein phosphorylation, characterized in that, The aforementioned reactive oxygen species-related diseases include at least one of the following: neurodegenerative diseases, stroke, epilepsy, kidney failure, liver damage, inflammation, enteritis, and arthritis.

16. A nanotherapeutic formulation, characterized in that, Includes manganese-doped cerium oxide nanomaterials with enzyme-like activity as described in any of claims 1-7.

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