A low-irritation cerium oxide nanoscale enzyme solution and a preparation method thereof

Abstract

The application discloses a low-irritation cerium oxide nanoscale enzyme solution and a preparation method thereof, the solvent of the solution is medium-chain triglyceride, isopropyl myristate or liquid paraffin, the content of the cerium oxide nanoscale enzyme in the solution is 1-100 mg / ml, and the average particle size of the cerium oxide nanoscale enzyme is less than 10 nm; the cerium oxide nanoscale enzyme is prepared by using an improved reverse micelle method, first, cerium acetate is used to prepare a cerium complex which is dispersed into xylene to form a uniform, stable and clear solution, then the cerium complex is hydrolyzed to generate the cerium oxide nanoscale enzyme in a water-oil two-phase microemulsion reverse micelle, and finally, the cerium oxide nanoscale enzyme is dispersed into a liquid matrix solvent to obtain the cerium oxide nanoscale enzyme solution.The liquid matrix solvent used in the cerium oxide nanoscale enzyme solution is safe, low-irritation and biologically friendly, the solution has good stability, and the cerium oxide nanoscale enzyme activity can be maintained for a long time.

Description

[0001] This patent claims priority to the entirety of the following patent, patent application number 202510747332.4, patent application date 2025-06-05, patent title: A Low-Irritation Cerium Oxide Nanoenzyme Solution and Its Preparation Method. Technical Field

[0002] This invention belongs to the field of nanomaterials technology, specifically relating to a low-irritant cerium oxide nanoenzyme solution and its preparation method. Background Technology

[0003] Metal nanomaterials were among the first inorganic chemical materials discovered to exhibit biocatalytic reactions similar to those of natural enzymes within the body. These materials possess outstanding optical, electrical, and catalytic properties and have gained widespread acceptance in various fields. Cerium oxide nanoparticles (CeO2NPs) are a novel type of bio-antioxidant that has emerged in recent years. They are nano-rare earth metal oxides with a fluorite cubic crystal structure, containing one Ce element per unit cell. 4+ It coordinates with 8 nearby O2- groups, and each O2- group coordinates with 4 nearby Ce- groups. 4+ Coordination; the escape and entry of oxygen atoms in the unit cell cause the generation and elimination of oxygen vacancies, allowing cerium ions to reversibly switch between the +3 and +4 valence states, forming a redox cycle. Therefore, cerium oxide nanoparticles can serve as mimics of peroxidase (POD), oxidase (OXD), superoxide dismutase (SOD), catalase (CAT), phosphatase, and photolyase, etc. They have functions such as inducing tumor cell apoptosis, inhibiting abnormal angiogenesis, destroying bacterial cell walls, and scavenging reactive oxygen species. They have great application potential in drug delivery, cancer treatment, prevention and treatment of cardiovascular and cerebrovascular diseases, and antioxidation. Literature (Small, 2024:2405417) reports a ROS-responsive cerium oxide nanozyme coated with an RBC membrane and loaded with resatorvid, which releases cerium oxide nanozyme and resatorvid upon reaching the lesion area to treat renal calcium oxalate stones; literature (Adv. Funct. Mater. 2024:2313198) reports a cerium oxide nanozyme loaded with minocycline for the comprehensive treatment of intracerebral hemorrhage (ICH); patent CN114053300 reports a cerium oxide nanozyme with the ability to eliminate reactive oxygen species, which can improve the hair follicle microenvironment in the hair loss area and promote hair follicle repair to achieve the purpose of treating hair loss.

[0004] Numerous methods exist for preparing cerium oxide nanozymes, which can be broadly categorized into two types: high-temperature decomposition and low-temperature synthesis. High-temperature decomposition methods include thermal decomposition, flame synthesis, and thermal plasma synthesis. Generally, organometallic compounds are dissolved in combustible organic matter, heated, atomized, and fed into a combustion chamber, where the precursor is rapidly combusted to generate the product. While high-temperature decomposition methods can synthesize nanoparticles on a large scale, they often cause oxidative stress in cells, making them unsuitable for in vivo antioxidant applications. Low-temperature synthesis methods include co-precipitation, electrochemical methods, hydrothermal methods, microemulsions, sol-gel methods, and reverse micelle methods. These methods typically use oxidants to convert trivalent cerium into sparingly soluble tetravalent cerium, allowing nanoparticles to nucleate and grow in a supersaturated solution. Low-temperature synthesis methods allow for better control of the shape and size of cerium oxide nanoparticles through reactant concentrations and auxiliary stabilizers. Cerium oxide nanozymes used in the pharmaceutical and biological fields are generally prepared using low-temperature synthesis methods.

[0005] In the field of nanoscience, the smaller the size of nanocrystals, the larger their specific surface area, and therefore the more active sites are exposed. Thus, the size and shape of cerium oxide nanoparticles play a crucial role in the transformation between redox pairs on their surface. Smaller nanoparticle sizes allow for higher oxygen vacancy concentrations and Ce2+ ionization. 3+ Compared to other large-scale cerium oxide nanoparticles, this method can express a variety of enhanced catalytic activities mimicking natural enzymes. Literature (Angew. Chem. Int. Ed. 2012, 51, 1-6) uses oleylamine as a surfactant and employs the reverse micelle method to prepare uniformly sized cerium oxide nanoparticles smaller than 10 nm in a microemulsion environment formed by water, xylene, and oleylamine. Patent (CN109806278) also uses this method to synthesize bioactive ultra-small cerium oxide nanoparticles ranging from 0.1 to 10 nm, which can generate more oxygen vacancies and achieve faster trivalent and tetravalent cerium conversion on the nanoparticle surface. Although this method allows for controllable particle size preparation of cerium oxide nanozymes, it consumes a large amount of oleylamine and solvent, resulting in high costs. Furthermore, after scaling up to mass production, the reverse micelle method often fails to guarantee the uniformity and stability of the reaction system, easily leading to the aggregation of nano-ions, resulting in larger particle sizes, and sometimes even preventing the production of nanoparticles altogether. This type of small-particle cerium oxide nanozyme has high activity, but it is easy to aggregate and clump together after losing the protection of the organic solvent on the particle surface, resulting in increased particle size and inactivation. At the same time, because the surface of cerium oxide nanozyme is hydrophobic, it cannot be dissolved in water or low-toxicity solvents such as alcohol. Currently, it is preserved by dissolving in highly toxic chlorinated solvents or low-polarity aprotic solvents (Nature Communications (2021) 12: 1436), which limits its direct application in the biomedical field. Summary of the Invention

[0006] To address the aforementioned issues, this application proposes a low-irritant cerium oxide nanozyme solution and its preparation method. The self-made cerium oxide nanozyme is dispersed in a safe, low-irritant, and bio-friendly liquid matrix, which not only preserves the bioactivity of the cerium oxide nanozyme but also improves its stability, thus broadening its application range. The preparation method is simple, low-cost, and suitable for industrial production.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] The present invention provides a low-irritant cerium oxide nanoenzyme solution, wherein the solvent of the solution is one or more of medium-chain triglycerides, isopropyl myristate, and liquid paraffin, and the content of cerium oxide nanoenzyme in the solution is 1-100 mg / ml.

[0009] Preferably, the content of cerium oxide nanozyme in the solution is 30-100 mg / ml.

[0010] Preferably, the liquid paraffin is a lightweight liquid paraffin.

[0011] Furthermore, in the above solution, the average particle size of the cerium oxide nanozyme is less than 10 nm.

[0012] Furthermore, the preparation method of the cerium oxide nanozyme solution is as follows:

[0013] S1: Vacuum drying of cerium acetate yields anhydrous cerium acetate;

[0014] S2: Add anhydrous cerium acetate, oleylamine, oleic acid and xylene to the flask in sequence, then add glacial acetic acid dropwise to adjust the pH of the reaction solution to 6-7, stir the reaction under nitrogen protection to obtain a homogeneous clear solution, and then slowly heat to 85℃-95℃.

[0015] S3: Add purified water to the reaction solution, and then introduce a constant nitrogen flow into the bottom of the reaction solution to make the reaction solution a microemulsion. Continue to stir the reaction at 85℃~95℃. The solid suspension in the reaction solution gradually disappears and an orange clear solution is obtained. Cool, let stand and separate the lower water layer. Add the organic layer to anhydrous ethanol under stirring to precipitate a pale yellow solid powder. Centrifuge and wash with anhydrous ethanol to obtain cerium oxide nanozyme wet product.

[0016] S4: Add liquid matrix solvent to the wet cerium oxide nanozyme, then concentrate under reduced pressure to remove low-boiling-point solvent, and obtain cerium oxide nanozyme solution.

[0017] Furthermore, in step S1, the vacuum drying temperature is 80℃~130℃, and the vacuum drying time is 0.5h~4h.

[0018] Furthermore, in step S2, the molar ratio of anhydrous cerium acetate, oleylamine, and oleic acid is 1:(3-12):(0.5-6), and the weight ratio of the solvent xylene to anhydrous cerium acetate is (10-30):1.

[0019] Furthermore, in step S3, the weight ratio of purified water to xylene is (0.05-0.15):1.

[0020] Furthermore, in step S4, the liquid matrix solvent is one or more of medium-chain triglycerides, isopropyl myristate, and liquid paraffin.

[0021] Preferably, the nitrogen flow rate in step S3 is 60-100 mL / min.

[0022] Furthermore, the oleic acid in step S2 is modified to obtain an oleic acid-polyethylene glycol coupling, prepared as follows:

[0023] (1) Oleic acid, N,N'-dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide (NHS) were dissolved in anhydrous dichloromethane (DCM), stirred in an ice bath for 1-3 hours, reacted at room temperature for 2-4 hours, and then filtered to obtain activated oleic acid.

[0024] (2) Dissolve polyethylene glycol in anhydrous dimethylformamide (DMF), add activated oleic acid and 4-dimethylaminopyridine (DMAP), react at room temperature for 12-30 hours under nitrogen protection, filter the reaction solution and rotary evaporate, precipitate the crude product with diethyl ether 3-5 times and then vacuum dry to obtain oleic acid-polyethylene glycol coupling.

[0025] Oleic acid contains a carboxyl group, which, under the action of DCC and NHS, generates an activated ester. The specific reaction mechanism is as follows: DCC reacts with the carboxyl group of oleic acid to form a highly active O-acyl isourea intermediate; NHS nucleophilically attacks the intermediate to generate a more stable oleic acid-NHS ester. This activated ester can react efficiently with amino or hydroxyl groups in subsequent reactions, avoiding side reactions.

[0026] PEG can be capped with a monomethoxy group (mPEG-OH) or a dihydroxy group (HO-PEG-OH). Under DMAP catalysis, the terminal hydroxyl group of PEG nucleophilically attacks the carbonyl carbon of oleic acid-NHS ester to form an ester bond.

[0027] Furthermore, polyethylene glycol is a polyethylene glycol compound with an amino group attached to one end and a carboxyl group attached to the other end.

[0028] When NH2-PEG-COOH is selected as the polyethylene glycol, the amino group in it attacks the carbonyl carbon in the oleic acid NHS ester to form an amide bond, and the resulting oleic acid-polyethylene glycol coupling has the structure R-CO-NH-PEG-COOH.

[0029] Preferably, the molar ratio of oleic acid, DCC, and NHS in step (1) is 1:(1.1-1.3):(1.1-1.3).

[0030] Preferably, in step (2), the molar ratio of polyethylene glycol, activated oleic acid, and DMAP is 1:(1-3):(0.01-0.1).

[0031] Preferably, the molecular weight of polyethylene glycol in step (2) is 200-2000;

[0032] Preferably, the molar ratio of oleic acid-polyethylene glycol coupling compound to oleylamine is 1:(1-6).

[0033] This invention provides a low-irritant cerium oxide nanozyme solution and its preparation method. This solution can maintain the activity of cerium oxide nanozymes for a long time, and the liquid matrix solvent used in the solution is low in irritation and bio-friendly, making it directly applicable to the biomedical field. The preparation method uses a modified reverse micelle method to obtain the cerium oxide nanozyme solution. First, a cerium complex prepared from cerium acetate is dispersed in xylene to form a homogeneous and stable clear solution. Then, in a two-phase microemulsion reverse micelle of water and oil, the cerium complex undergoes hydrolysis to generate cerium oxide nanozymes. Finally, the nanozymes are dispersed in a liquid matrix solvent to obtain the cerium oxide nanozyme solution. The preparation method is simple, without harsh or violent reaction conditions, does not require high-temperature reactions, has low requirements for production equipment, and is suitable for industrial-scale production.

[0034] The beneficial effects of the present invention include, but are not limited to:

[0035] 1. The present invention provides a low-irritation cerium oxide nanoenzyme solution, wherein the liquid matrix solvent used is medium-chain triglycerides, isopropyl myristate, or liquid paraffin. These are generally widely used in the food and biopharmaceutical fields as nutritional supplements, lubricants, moisturizers, or drug carriers. They are safe, non-toxic or low-toxic, bio-friendly, and have low irritation to the skin and mucous membranes.

[0036] 2. The low-irritant cerium oxide nanozyme solution of the present invention has good stability and can maintain the valence state cycling ability of cerium oxide nanozymes for a long time, i.e., mimicking the activity of biological enzymes. Small-particle-size cerium oxide nanozymes are prone to agglomeration and loss of biological activity when there is no dispersant on their surface. Furthermore, the surface of cerium oxide nanozymes is hydrophobic and insoluble in solvents such as water and ethanol. Existing technologies all preserve them in chlorinated solvents (such as dichloromethane or chloroform) or low-polarity aprotic solvents (such as n-hexane, n-heptane, toluene, xylene, and trimethylbenzene). These solvents are all carcinogenic solvents. According to the ICH guidelines for pharmaceuticals, the residual concentration of n-heptane, which has the lowest toxicity, should be below 5000 ppm. In addition, these solvents have low boiling points and are easily volatile, which cannot maintain the activity of cerium oxide nanozymes for a long time. Therefore, such cerium oxide nanozyme solutions cannot be used directly in biomedicine. Existing technologies generally chemically modify the surface of cerium oxide nanozymes to make them soluble in safe and low-toxicity solvents such as water or alcohols, and then release cerium oxide nanoparticles in vivo to exert their role as mimicking biological enzymes. The liquid matrix solvent of this invention consists of medium-chain triglycerides, isopropyl myristate, or liquid paraffin, which have high boiling points and are not easily volatile, exhibiting good biocompatibility. Their long-chain aliphatic hydrocarbon structure can better maintain the stability of the oxygen vacancy structure on the surface of cerium oxide nanoenzymes, thus maintaining the bioactivity of cerium oxide nanoenzymes for a long time. The resulting cerium oxide nanoenzyme solution can be directly applied in the biomedical field.

[0037] 3. This invention uses an improved reverse micelle method to prepare cerium oxide nanoenzyme solution. Acetic acid is used to maintain a slightly acidic environment (pH 6-7) in the reaction solution, which promotes the rapid formation of cerium coordination compounds from oleylamine, oleic acid and cerium acetate and their uniform dispersion in xylene solvent. Subsequently, when hydrolyzing to cerium oxide in an aqueous and oil-based microemulsion, the aggregation of cerium oxide nanoparticles due to changes in solution pH will not cause the particle size to be too large. Existing technologies use oleylamine or a combination of oleylamine and oleic acid as surfactants to form cerium-surfactant complexes with cerium precursors. However, the pH of the reaction solution is not controlled, and oleic acid is weakly acidic with weak ionization of its carboxyl hydrogen, failing to effectively regulate pH. This reverse micelle method for preparing cerium oxide nanoenzyme solutions differs from conventional micelle methods (where hydrophilic groups are on the outside and hydrophobic groups are on the inside). It forms reverse micelles with hydrophilic groups facing inward and hydrophobic groups facing outward in a non-aqueous solvent like xylene. Within the micelles, the aqueous phase undergoes hydrolysis of cerium coordination compounds, generating cerium oxide nanoparticles which are then rapidly transferred to the external xylene solvent. This invention adds acetic acid, which is both water-soluble and lipid-soluble. Besides forming a buffer salt with oleylamine in the initial reaction stage to regulate the pH of the reaction solution and accelerate the formation of the cerium complex, acetic acid also helps the nanoparticles reach the aqueous phase inside the reverse micelles to achieve internal and external acid-base balance during the growth stage. This results in controllable and uniformly distributed particle sizes, all below 10 nm.

[0038] 4. This invention employs an improved reverse micelle method to prepare cerium oxide nanoenzyme solution. During the hydrolysis of cerium complexes to generate nanoparticles, a constant nitrogen flow is introduced into the bottom of the reaction solution, promoting a microemulsion state and resulting in finer reverse micelles and more uniform nanoparticle size. Existing reverse micelle methods for preparing cerium oxide nanoenzymes, after scale-up to mass production, fail to form a stable microemulsion reverse micelle system from the xylene and water mixture using only mechanical stirring, leading to prolonged reaction time or failure in nanoparticle preparation. This invention introduces a constant nitrogen flow in conjunction with mechanical stirring, ensuring thorough emulsification of the solution and preventing the effects of scale-up reactions.

[0039] 5. This application modifies oleic acid to obtain an oleic acid-polyethylene glycol (PEG) conjugate. This PEG conjugate is introduced into the preparation of cerium oxide nanozymes. The ether bonds, terminal -OH groups, or carboxyl groups in the conjugate can coordinate cerium particles and are stabilized by oleylamine. Due to the steric hindrance effect of PEG, excessive aggregation of nanoparticles is more effectively suppressed, resulting in more uniform nucleation and a narrower particle size distribution, thus obtaining cerium oxide nanocrystals with better monodispersity. The ether bonds in the conjugate form hydrogen bonds with water molecules, enhancing the dispersion of the aqueous phase. During high-temperature reactions, small particles easily dissolve and redeposit onto larger particles, leading to uneven particle size. The steric hindrance of the PEG chains can hinder particle fusion. In the step of adding water to form a microemulsion, the amphiphilic properties of the oleic acid-PEG conjugate (hydrophilic PEG segments and hydrophobic oleic acid segments) can more effectively stabilize the water / oil interface and promote Ce2O3 production. 3+ The uniformity of the hydrolysis reaction reduces the random aggregation of nanoparticles and avoids the formation of large-sized precipitates. The oleic acid-polyethylene glycol coupling is more hydrophilic and easier to elute, resulting in less residue on the surface of the cerium oxide nanozyme, which has higher surface activity and lower cytotoxicity. Even if it is not completely eluted, the anchoring effect of polyethylene glycol will reduce the release of free oleic acid, and the steric hindrance effect of the long polyethylene glycol chain will make the cerium oxide nanozyme more stably dispersed in nonpolar solvents.

[0040] 6. In this application, NH2-PEG-COOH is used for polyethylene glycol. The amino-containing polyethylene glycol derivative can inhibit the Ostwald ripening of nanoparticles and reduce the particle size distribution. The reserved active carboxyl group facilitates subsequent bio-coupling and expands the functionalization potential. The resulting oleic acid-polyethylene glycol conjugate contains carboxyl groups that can participate in pH regulation to help stabilize the acid-base environment in the reaction system and promote the controllability of cerium acetate hydrolysis nucleation.

[0041] 7. The process of this invention is simple, requires no special high-temperature and high-pressure equipment, the raw materials are abundant and inexpensive, the production cost is low, and it is suitable for mass production.

[0042] 8. The cerium oxide nanozyme obtained by the process of the present invention can be well dispersed in liquid matrix solvents such as medium-chain triglycerides, isopropyl myristate, or liquid paraffin without further surface modification or the addition of dispersants, and has high dispersion stability. Attached Figure Description

[0043] The accompanying drawings, which are included to provide a further understanding of this application and form part of this application, illustrate exemplary embodiments and are used to explain this application, but do not constitute an undue limitation of this application. In the drawings:

[0044] Figure 1 This is a transmission electron microscope (TEM) image of the cerium oxide nanozyme prepared in Example 1 of this application.

[0045] Figure 2 The image shows the dynamic light scattering (DLS) nanoparticle size distribution of the cerium oxide nanozyme prepared in Example 1 of this application. Detailed Implementation

[0046] The present application is described in detail below with reference to the embodiments, but the present application is not limited to these embodiments.

[0047] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of skill in the art. The reagents and raw materials used in this invention are readily available through conventional means, and unless otherwise specified, they shall be used in accordance with conventional methods in the art or as per the product instructions. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of this invention. The preferred embodiments and materials described in this patent are for illustrative purposes only.

[0048] Example 1: Preparation of medium-chain triglyceride solution of cerium oxide nanozyme

[0049] (1) Measure 300g of cerium acetate, place it in a 2L single-necked bottle, and dry it under reduced pressure at 100℃ for 2 hours to remove water, and then cool it down.

[0050] (2) Weigh anhydrous cerium acetate (190.35 g, 0.6 mol) and add it to a 5 L three-necked flask. Then add oleylamine (963 g, 3.6 mol), oleic acid (169.5 g, 0.6 mol) and xylene (2.6 kg) in sequence. While stirring, slowly add glacial acetic acid (43.6 g, 0.73 mol) until the pH of the reaction solution is 6. Replace the nitrogen gas three times and stir the reaction at room temperature (25 °C) for 72 h to obtain a clear orange-red solution. Then slowly heat the solution to 95 °C.

[0051] (3) Add 390 ml of purified water to the reaction solution, and then introduce a constant nitrogen flow into the bottom of the reaction solution at a flow rate of 60 mL / min to make the reaction solution a microemulsion. Continue stirring at 95 °C for 48 h. The solid suspension in the reaction solution gradually disappears and an orange clear solution is obtained. Cool the reaction solution to room temperature and let it stand to separate the layers. Add the organic layer to 6 L of anhydrous ethanol under stirring to precipitate a pale yellow solid powder. Centrifuge and wash three times with anhydrous ethanol to obtain a pale yellow solid powder wet product.

[0052] (4) Add 1.8L of medium-chain triglyceride to the above pale yellow solid, stir for 0.5h, and then evaporate under reduced pressure at 50℃ for 1h to obtain a medium-chain triglyceride solution of cerium oxide nanozyme with a concentration of 50mg / ml.

[0053] The transmission electron microscope (TEM) image and dynamic light scattering (DLS) nanoparticle size distribution map of the cerium oxide nanozyme obtained in this embodiment are shown below. Figure 1 and Figure 2 .like Figure 1 As shown, TEM results indicate that the cerium oxide nanozymes have a regular and uniform morphology with a particle size of less than 10 nm; Figure 2 As shown, the cerium oxide nanozyme has a uniform particle size distribution.

[0054] Example 2: Preparation of cerium oxide nanozyme with isopropyl myristate solution

[0055] (1) Take 300g of cerium acetate, place it in a 2L single-necked bottle, and dry it under reduced pressure at 130℃ for 0.5h to remove water, and then cool it down;

[0056] (2) Weigh anhydrous cerium acetate (190.35 g, 0.6 mol) and add it to a 5 L three-necked flask. Then add oleylamine (481.5 g, 1.8 mol), oleic acid (84.7 g, 0.3 mol) and xylene (1.9 kg) in sequence. While stirring, slowly add glacial acetic acid (27.7 g, 0.46 mol) until the pH of the reaction solution is 6. Replace the nitrogen gas three times and stir the reaction at room temperature (25 °C) for 72 h to obtain a clear orange-red solution. Then slowly heat the solution to 90 °C.

[0057] (3) Add 95 ml of purified water to the reaction solution, and then introduce a constant nitrogen flow into the bottom of the reaction solution at a flow rate of 80 mL / min to make the reaction solution a microemulsion. Continue stirring at 90 °C for 48 h to obtain a clear orange solution. Cool the reaction solution to room temperature and let it stand to separate the layers. Add the organic layer to 5 L of anhydrous ethanol under stirring to precipitate a pale yellow solid powder. Centrifuge and wash three times with anhydrous ethanol to obtain a wet product of pale yellow solid powder.

[0058] (4) Add 2.5L of isopropyl myristate to the pale yellow solid, stir for 0.5h, and then rotary evaporate at 50℃ under reduced pressure for 1h to obtain a solution of isopropyl myristate of cerium oxide nanozyme with a concentration of 37mg / ml.

[0059] Example 3: Preparation of liquid paraffin solution of cerium oxide nanozyme

[0060] (1) Take 300g of cerium acetate, place it in a 2L single-necked bottle, and dry it under reduced pressure at 80℃ for 4h to remove water, and then cool it down.

[0061] (2) Weigh anhydrous cerium acetate (190.35 g, 0.6 mol) and add it to a 10 L three-necked flask. Then add oleylamine (1926 g, 7.2 mol), oleic acid (1017 g, 3.6 mol) and xylene (5.7 kg) in sequence. While stirring, slowly add glacial acetic acid (180 g, 2.99 mol) until the pH of the reaction solution is 7. Replace the nitrogen gas three times and stir the reaction at room temperature (25 °C) for 48 h to obtain a clear orange-red solution. Then slowly heat the solution to 85 °C.

[0062] (3) Add 500 ml of purified water to the reaction solution, and then introduce a constant nitrogen flow into the bottom of the reaction solution at a flow rate of 100 mL / min to make the reaction solution a microemulsion. Continue stirring at 85 °C for 24 h to obtain a clear orange solution. Cool the reaction solution to room temperature and let it stand to separate the layers. Add the organic layer to 15 L of anhydrous ethanol under stirring to precipitate a pale yellow solid powder. Centrifuge and wash three times with anhydrous ethanol to obtain a wet product of pale yellow solid powder.

[0063] (4) Add 1.5L of liquid paraffin to the pale yellow solid solution, stir for 0.5h, and then evaporate under reduced pressure at 60℃ for 1h to obtain a liquid paraffin solution of cerium oxide nanozyme with a concentration of 63mg / ml.

[0064] Example 4

[0065] (1) Oleic acid, DCC, and NHS (the molar ratio of oleic acid, DCC, and NHS is 1:1.1:1.3) were dissolved in anhydrous DCM, stirred in an ice bath for 1 hour, reacted at room temperature for 4 hours, and the activated oleic acid was obtained by filtration to remove dicyclohexylurea.

[0066] (2) Dissolve PEG-1000 with single-hydroxyl end in anhydrous DMF, add activated oleic acid and DMAP (the molar ratio of polyethylene glycol with a molecular weight of 1000, activated oleic acid and DMAP is 1:1:0.01), react at room temperature for 12 hours under nitrogen protection, filter the reaction solution and rotary evaporate, precipitate the crude product with diethyl ether 3 times and then vacuum dry to obtain oleic acid-polyethylene glycol coupling;

[0067] (3) Replace the oleic acid in Example 1 with oleic acid-polyethylene glycol coupling (760g, 0.6mol), and repeat steps (2) and (3) in Example 1 to obtain cerium oxide nanozyme wet product. Add 1L of isopropyl myristate, stir for 0.5h, and then evaporate under reduced pressure to obtain cerium oxide nanozyme isopropyl myristate solution with a concentration of 90mg / ml.

[0068] Example 5

[0069] (1) Oleic acid, DCC, and NHS (the molar ratio of oleic acid, DCC, and NHS is 1:1.3:1.1) were dissolved in anhydrous DCM, stirred in an ice bath for 3 hours, reacted at room temperature for 2 hours, and the activated oleic acid was obtained by filtration to remove dicyclohexylurea.

[0070] (2) Dissolve PEG-400 with hydroxyl ends in anhydrous DMF, add activated oleic acid and DMAP (the molar ratio of polyethylene glycol with a molecular weight of 400, activated oleic acid and DMAP is 1:3:0.1), react at room temperature for 30 hours under nitrogen protection, filter the reaction solution and rotary evaporate, precipitate the crude product with diethyl ether 5 times and then vacuum dry to obtain oleic acid-polyethylene glycol coupling.

[0071] (3) Replace the oleic acid in Example 1 with oleic acid-polyethylene glycol coupling (835g, 0.9mol), and repeat steps (2) and (3) in Example 1 to obtain cerium oxide nanozyme wet product. Add 60L of medium chain triglyceride, stir for 0.5h, and evaporate under reduced pressure to obtain medium chain triglyceride solution of cerium oxide nanozyme. The concentration of cerium oxide nanozyme is 1.5mg / ml.

[0072] Example 6

[0073] (1) Oleic acid, DCC, and NHS (the molar ratio of oleic acid, DCC, and NHS is 1:1.2:1.2) were dissolved in anhydrous DCM, stirred in an ice bath for 2 hours, reacted at room temperature for 3 hours, and the activated oleic acid was obtained by filtration to remove dicyclohexylurea.

[0074] (2) Dissolve NH2-PEG-COOH with a molecular weight of 2000 in anhydrous DMF, add activated oleic acid and DMAP (the mass ratio of polyethylene glycol, activated oleic acid and DMAP is 1:2:0.05), react at room temperature for 20 hours under nitrogen protection, filter the reaction solution and rotary evaporate, precipitate the crude product with diethyl ether 5 times and then vacuum dry to obtain oleic acid-polyethylene glycol coupling.

[0075] (3) Replace the oleic acid in Example 1 with oleic acid-polyethylene glycol coupling (1358g, 0.6mol), and repeat steps (2) and (3) in Example 1 to obtain a light yellow wet powder. Add 5L of liquid paraffin, stir for 0.5h, and then evaporate under reduced pressure to obtain a liquid paraffin solution of cerium oxide nanozyme with a concentration of 18mg / ml.

[0076] Example 7

[0077] (1) Weigh 190.35g of anhydrous cerium acetate and add it to a 5L three-necked flask. Then add 963g of oleylamine, 169.5g of oleic acid and 2.6kg of xylene in sequence. Stir the reaction at room temperature (25℃) for 72h under nitrogen protection and slowly heat to 95℃.

[0078] (2) Add 390 ml of purified water to the reaction solution, and then introduce a constant nitrogen flow into the bottom of the reaction solution at a flow rate of 60 mL / min to make the reaction solution a microemulsion. Continue stirring at 95 °C for 48 h. Cool the reaction solution to room temperature and let it stand to separate the layers. Add the organic layer to 6 L of anhydrous ethanol under stirring to precipitate a pale yellow solid powder. Centrifuge and wash three times with anhydrous ethanol to obtain a wet product of pale yellow solid powder.

[0079] (3) Add 1.8 L of medium-chain triglyceride to the above pale yellow solid, stir for 0.5 h, and then rotary evaporate at 50 °C under reduced pressure for 1 h to obtain a medium-chain triglyceride solution of cerium oxide nanozyme.

[0080] Example 8

[0081] The difference from Example 4 is that oleylamine is not added in step (3), only anhydrous cerium acetate, oleic acid-polyethylene glycol coupling compound, and xylene are used. All other parameters are the same as in Example 4, and will not be repeated here.

[0082] Comparative Example 1

[0083] 0.4 g of cerium acetate hydrate was added to a 100 mL single-necked flask, along with 3.2 g of oleylamine and 15 mL of xylene. The mixture was stirred at room temperature for 12 h. Under vacuum and argon protection, the reactants were heated to 90 °C at a rate of 2 °C per minute. 1 mL of purified water was injected into the reaction system, and the mixture was stirred at 90 °C for 3 h. After cooling, 50 mL of acetone was added to precipitate a pale yellow solid. The solid was obtained by centrifugation and washing. A small amount of the product was stored at room temperature, while the remainder was dissolved in 20 mL of chloroform, 20 mL of n-hexane, and 20 mL of medium-chain triglycerides for storage.

[0084] The particle size distribution was detected by dynamic light scattering (DLS) nanoparticle size analyzer. The particle size distribution of cerium oxide nanozymes prepared by this method was not uniform. Solid cerium oxide nanozymes placed at room temperature turned into brown solids after 1 day and were insoluble in chloroform, n-hexane and medium-chain triglycerides, thus losing their activity.

[0085] Ce 3+ / 4+ Qualitative determination of valence cycling ability: Freshly prepared chloroform, n-hexane and medium-chain triglyceride solutions were found to have valence cycling ability. After being left at room temperature for 3 months, they no longer had valence cycling ability. Due to the uneven particle size distribution, large-sized nanoparticles are more likely to agglomerate. The aggregation process accelerates the aggregation rate of nanoparticles in the entire solution, causing the solution to lose its activity.

[0086] Comparative Example 2

[0087] 0.6 g of cerium acetate was added to a 100 mL three-necked flask, along with 3.8 g of oleylamine and 50 mL of xylene. The mixture was stirred at room temperature for 30 min, then heated to 90 °C, and 1 mL of purified water was added. The mixture was kept warm and stirred for 6 h. After cooling, the mixture was filtered and washed successively with chloroform and ethanol. The resulting solid was calcined in a muffle furnace at 700 °C for 6 h to obtain a light yellow cerium oxide nanoparticle solid powder.

[0088] This solid powder is soluble in chloroform, dichloromethane, n-hexane, n-heptane, toluene, and xylene, but insoluble in water, ethanol, and acetone. It is also insoluble in medium-chain triglycerides, isopropyl myristate, or liquid paraffin. This may be because the surface structure of the cerium oxide nanoparticles obtained by high-temperature calcination is different from that of nanoparticles prepared by the reverse micelle method, resulting in their insolubility in solvents such as medium-chain triglycerides, isopropyl myristate, or liquid paraffin.

[0089] Comparative Example 3

[0090] (1) Measure 300g of cerium acetate and place it in a 2L single-necked bottle. Dry it under reduced pressure at 100℃ for 2 hours to remove water, and then cool it down.

[0091] (2) Weigh anhydrous cerium acetate (190.35 g, 0.6 mol) and add it to a 5 L three-necked flask. Then add oleylamine (963 g, 3.6 mol), oleic acid (169.5 g, 0.6 mol) and xylene (2.6 kg) in sequence. While stirring, slowly add glacial acetic acid (43.6 g, 0.73 mol) until the pH of the reaction solution is 6. Replace the nitrogen gas three times and stir the reaction at room temperature (25 °C) for 72 h to obtain a clear orange-red solution. Then slowly heat the solution to 95 °C.

[0092] (3) Add 390 ml of purified water to the reaction solution, and then introduce a constant nitrogen flow into the bottom of the reaction solution at a flow rate of 60 mL / min to make the reaction solution a microemulsion. Continue stirring at 95 °C for 48 h. The solid suspension in the reaction solution gradually disappears and an orange clear solution is obtained. Cool the reaction solution to room temperature and let it stand to separate the layers. Add the organic layer to 6 L of anhydrous ethanol under stirring to precipitate a pale yellow solid powder. Centrifuge and wash three times with anhydrous ethanol to obtain a pale yellow solid powder wet product.

[0093] (4) Take 20 mg of the pale yellow solid powder wet product and dissolve it in chloroform. Then add 20 mg of polyethylene glycol (molecular weight 1000), stir evenly, sonicate, evaporate under reduced pressure, and then disperse in 1 ml of water.

[0094] [Characteristics]

[0095] 1. Solvent Screening and Comparison Experiment for Cerium Oxide Nanozyme Solution

[0096] The wet cerium oxide nanoenzyme prepared in Example 1 was subjected to solubility tests using water, glycerol, oleic acid, glyceryl monooleate, glyceryl dioleate, polyethylene glycol 400, propylene glycol, diethylene glycol, polysorbate 80, perfluorohexyloctane, medium-chain triglycerides, isopropyl myristate, and liquid paraffin, as well as common organic solvents such as dichloromethane, chloroform, toluene, xylene, trimethylbenzene, n-hexane, n-heptane, anhydrous ethanol, and acetone. Ce was also tested. 3+ / 4+ Qualitative determination of valence cycle capability, data are shown below:

[0097]

[0098]

[0099] + indicates valence cycling ability, - indicates no valence cycling ability; the solvent that does not dissolve cerium oxide nanozymes was not tested for valence cycling ability.

[0100] Ce 3+ / 4+ Qualitative determination method for valence cycling ability: Take an appropriate amount of cerium oxide nanozyme solution, dilute it to 3 mg / mL, take 2 ml and put it in a vial, add 1 ml of 5 mol / L hydrogen peroxide, observe the color change before and after the addition, let it stand at room temperature for 3 days, and continue to add 1 ml of 5 mol / L hydrogen peroxide, and observe the color change before and after the addition.

[0101] Ce 3+ / 4+ Qualitative determination principle and judgment method of valence cycling ability: due to Ce 3+ It is colorless, Ce 4+ It appears yellow; after adding H2O2, Ce... 3+ Oxidized to Ce 4+The solution color darkened visually, changing from pale yellow to orange-yellow; after being left at room temperature for 3 days, Ce... 4+ Excess H₂O₂ decomposes into O₂, which is then reduced to Ce. 3+ The solution color lightened to pale yellow. With continued addition of H₂O₂, Ce... 3+ Oxidized to Ce 4+ The solution color deepened upon visual observation, changing from pale yellow to orange-yellow, indicating the presence of cerium oxide nanozymes. 3+ and Ce 4+ The cerium oxide nanozyme exhibits good valence state cycling ability; if the solution color does not change after adding H2O2 or does not lighten after 3 days, it indicates that the cerium oxide nanozyme no longer has valence state cycling ability.

[0102] According to national food standards, LD 50 A concentration (mg / kg) greater than 5000 indicates a non-toxic or low-toxicity class. This includes medical liquid matrices such as water, glycerol, oleic acid, glyceryl monooleate, glyceryl dioleate, polyethylene glycol 400, propylene glycol, diethylene glycol, polysorbate 80, medium-chain triglycerides, isopropyl myristate, and liquid paraffin. Common organic solvents, according to the ICH (International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use) classification standards, include chloroform, dichloromethane, n-hexane, toluene, xylene, and trimethylbenzene. Heptane, ethanol, and acetone are classified as Class II solvents (carcinogenic to animals) and Class III solvents (with slightly lower toxicity). These solvents are used in pharmaceuticals, and their residual limits in pharmaceuticals are as follows: chloroform 60 ppm, dichloromethane 600 ppm, hexane 290 ppm, toluene 890 ppm, and xylene 2170 ppm. The limits for heptane, ethanol, and acetone are slightly higher, at 5000 ppm (0.05%). Therefore, their cerium oxide nanoenzyme solutions cannot be directly used in pharmaceutical and biological applications.

[0103] The data in the table above show that freshly prepared cerium oxide nanozymes, when dissolved in liquid, all exhibit valence cycling ability. However, only three solutions—medium-chain triglycerides, isopropyl myristate, and liquid paraffin—retain valence cycling ability after being left at room temperature for 6 months. Furthermore, these three liquid matrices have low toxicity, and the prepared cerium oxide nanozyme solutions can be directly used in pharmaceutical and biological fields.

[0104] 2. Polydispersity Index (PDI)

[0105] The PDI is obtained by dynamic light scattering (DLS). If PDI < 0.1, it indicates that the solution is highly monodisperse; if PDI is between 0.1 and 0.3, it indicates that the solution is moderately dispersible; if PDI > 0.3, it indicates that the distribution is relatively broad.

[0106] Comparative Example 1 was tested using a cerium oxide nanozyme solution dissolved in 20 ml of medium-chain triglycerides. Comparative Example 2 was not tested because it is insoluble in medium-chain triglycerides, isopropyl myristate, or liquid paraffin.

[0107] 3. DLS monitoring of particle size changes

[0108] After storing the cerium oxide nanozyme solution at room temperature for 30 days, observe whether stratification or flocculation occurs. If stratification or flocculation occurs, observe again after ultrasonic treatment. Perform DLS test again on the liquid without stratification or flocculation or after ultrasonic treatment to resolve the above phenomena. If the PDI is always less than 0.25, it means that the cerium oxide nanozyme is relatively stable in the solvent.

[0109] Comparative Example 1 was tested using a cerium oxide nanozyme solution dissolved in 20 ml of medium-chain triglycerides. Comparative Example 2 was not tested because it is insoluble in medium-chain triglycerides, isopropyl myristate, or liquid paraffin.

[0110]

[0111]

[0112] The cerium oxide nanozymes prepared in this invention have an average particle size of less than 10 nm, and the TEM images show a highly uniform distribution of nanoparticles. Analysis of the DLS monitoring results reveals the following:

[0113] The cerium oxide nanozyme solution prepared in this application exhibits good dispersibility and excellent dispersion stability of the nanoparticles; the nanoparticles obtained after introducing the oleic acid-polyethylene glycol coupling compound into the system show even better monodispersity.

[0114] As can be seen from the results of Example 7, if the pH adjustment step is missing during the preparation process, it will affect the dispersibility of nanoparticles in the solvent, and the dispersion stability will also be greatly affected.

[0115] As can be seen from the results of Example 8, the oleic acid-polyethylene glycol coupling compound and oleylamine have a synergistic effect and work together.

[0116] The results of Comparative Example 1 show that oleylamine and oleic acid can play a good synergistic role in the reaction system, and the control of the preparation process also has a great influence on the dispersibility of cerium oxide nanozymes.

[0117] As can be seen from the results of Comparative Example 3, after preparing cerium oxide nanozymes and then dispersing them in water using polyethylene glycol for surface modification, the dispersibility of cerium oxide nanozymes in fresh solution is acceptable, but its stability is not high, and it becomes unstable after 30 days.

[0118] The various embodiments in this specification are described in a progressive manner. Similar or identical parts between embodiments can be referred to interchangeably. Each embodiment focuses on describing the differences from other embodiments. In particular, the system embodiments are basically similar to the method embodiments, so the description is relatively simple; relevant parts can be referred to the descriptions in the method embodiments.

[0119] The above description is merely an embodiment of this application and is not intended to limit the scope of this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the scope of the claims of this application.

Claims

1. A low-irritation cerium oxide nanoenzyme solution, characterized in that, The solvent of the solution is one or more of medium-chain triglycerides, isopropyl myristate, and liquid paraffin; the content of cerium oxide nanozymes in the solution is 1~100 mg / ml; and the average particle size of the cerium oxide nanozymes is less than 10 nm. The preparation method of the low-irritation cerium oxide nanozyme solution includes the following steps: S1: Add anhydrous cerium acetate, oleylamine, oleic acid and xylene in sequence, then add glacial acetic acid dropwise to adjust the pH of the reaction solution to 6-7. Stir under nitrogen protection to obtain a homogeneous clear solution, then heat to 85℃-95℃; the molar ratio of anhydrous cerium acetate, oleylamine and oleic acid is 1:(3-12):(0.5-6), and the weight ratio of xylene to anhydrous cerium acetate is (10-30):1; S2: Add purified water to the reaction solution, then introduce a constant nitrogen flow into the bottom of the reaction solution to make the reaction solution a microemulsion. Continue stirring at 85℃~95℃ until the solid suspension in the reaction solution gradually disappears and an orange clear solution is obtained. Cool, let stand and separate the lower aqueous layer, and add the organic layer to anhydrous ethanol under stirring to precipitate a pale yellow solid powder. Centrifuge and wash with anhydrous ethanol to obtain cerium oxide nanozyme wet product; the weight ratio of purified water to xylene is (0.05~0.15):

1. S3: Add liquid matrix solvent to the wet cerium oxide nanozyme, and concentrate under reduced pressure to obtain cerium oxide nanozyme solution.

2. The low-irritation cerium oxide nanoenzyme solution according to claim 1, characterized in that: The oleic acid in step S1 of the preparation method is modified to obtain an oleic acid-polyethylene glycol coupling. The preparation method is as follows: (1) Dissolve oleic acid, N,N'-dicyclohexylcarbodiimide and N-hydroxysuccinimide in anhydrous dichloromethane, stir in an ice bath for 1-3 hours, react at room temperature for 2-4 hours, and filter to obtain activated oleic acid; (2) Dissolve polyethylene glycol in anhydrous dimethylformamide, add activated oleic acid and 4-dimethylaminopyridine, react at room temperature for 12-30 hours under nitrogen protection, filter the reaction solution and rotary evaporate, precipitate the crude product with diethyl ether 3-5 times and then vacuum dry to obtain oleic acid-polyethylene glycol coupling.

3. The low-irritation cerium oxide nanoenzyme solution according to claim 2, characterized in that: Polyethylene glycol is a polyethylene glycol compound with an amino group attached to one end and a carboxyl group attached to the other end.

4. The low-irritation cerium oxide nanoenzyme solution according to claim 2, characterized in that: In step (1), the molar ratio of oleic acid, N,N'-dicyclohexylcarbodiimide, and N-hydroxysuccinimide is 1:(1.1~1.3):(1.1~1.3); In step (2), the molar ratio of polyethylene glycol, activated oleic acid, and 4-dimethylaminopyridine is 1:(1~3):(0.01~0.1).

5. The low-irritation cerium oxide nanoenzyme solution according to claim 2, characterized in that: In step (2), the molecular weight of polyethylene glycol is 200-2000; The molar ratio of oleic acid-polyethylene glycol coupling to oleylamine is 1:(1-6).

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