Preparation method and application of cerium oxide-based nanoparticles

Abstract

The invention relates to a preparation method and application of cerium oxide-based nanoparticles, belongs to the technical field of medical biological materials, and particularly provides a preparation method and application of regulating cerium oxide Ce < 3 + >-Ce < 4 + > redox couple. According to the method, an albumin-mediated biomimetic synthesis method is adopted, Ce < 3 + > ions are used as a reaction precursor, Cu < 2 + > ions are used as auxiliary cations, and the cerium oxide-based nanoparticles are prepared. By regulating the pH value to 10-12, synthesis of cerium oxide and regulation of Ce < 3 + >-Ce < 4 + > redox pairs are realized. The preparation process is mild and controllable, and industrial popularization is facilitated; the proportion of Ce < 3 + > / Ce < 4 + > of the prepared cerium oxide-based nanoparticles is higher than 90%, the oxygen vacancy content is higher than 40%, and the cerium oxide-based nanoparticles show excellent hydrogen peroxide, hydroxyl radical and superoxide anion scavenging activity and can be applied to the biomedical fields of oxidative stress regulation and control and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of medical biomaterials, and in particular relates to a preparation method and application of cerium oxide-based nanoparticles. Background Art

[0002] Oxidative stress is a significant feature shared by many clinical diseases. In this state, excessive reactive oxygen species (ROS), such as hydrogen peroxide, hydroxyl radicals, and superoxide anions, will attack proteins, lipids, and DNA in cells, causing fatal damage to cells and inducing a series of pathological processes, such as neurodegenerative diseases, kidney damage, and liver damage. Acute liver injury is characterized by a rapid deterioration of liver cell function in a short period of time, and its causes include drug poisoning, alcohol abuse, viral infection, etc. In the absence of timely and effective intervention, acute liver injury can further deteriorate into acute liver failure, ultimately leading to the death of the patient. At present, a large amount of research evidence has revealed the core role of oxidative stress in various forms of liver damage and liver failure. Therefore, the development of antioxidants with efficient ROS scavenging ability has become an urgent need to meet this challenge.

[0003] As a nano antioxidant, cerium oxide nanoparticles have been widely used in the biomedical field in recent years. 3+ -Ce 4+ Redox couple, Ce 3+ The presence of is beneficial to the scavenging of superoxide anions and hydroxyl radicals, while Ce 4+ The presence of helps to remove hydrogen peroxide. ROS substances in organisms can undergo a chain reaction, such as superoxide anions can first be converted into hydrogen peroxide, and then into hydroxyl free radicals with stronger cytotoxicity. Therefore, the preparation of high Ce 3+ / Ce 4+ Cerium oxide-based nanoparticles with a high ratio will be more conducive to cutting off chain reactions and efficiently removing ROS. However, the currently reported methods for preparing cerium oxide nanoparticles usually require high temperature, high pressure or a specific atmosphere, and the synthesis steps are cumbersome and time-consuming, resulting in high production difficulty and high cost. In addition, the prepared cerium oxide nanoparticles often contain toxic solvents that are difficult to remove, and they also need to undergo complex surface modification to improve biocompatibility. More importantly, the Ce in the prepared cerium oxide nanoparticles is 3+ / Ce 4+ The ratio is usually less than 60%, and there is a lack of simple and effective means to regulate its valence ratio. These factors severely limit the effectiveness of cerium oxide in scavenging ROS, treating diseases and biological applications. Therefore, it is necessary to develop a simpler and more efficient synthesis method to prepare cerium oxide nanoparticles with good biocompatibility and improve its Ce 3+ / Ce 4+ The ratio is of great significance for expanding its application in the biomedical field. Summary of the invention

[0004] The first aspect of the present invention provides a method for preparing cerium oxide-based nanoparticles, the main purpose of which is to simply and efficiently control the cerium oxide Ce 3+ -Ce 4+ Redox couple, which makes it have good biocompatibility and excellent hydrogen peroxide, hydroxyl radical and superoxide anion scavenging activity.

[0005] This is achieved specifically through the following technical solutions: A method for preparing cerium oxide-based nanoparticles, comprising the following steps: S1. Under vigorous stirring conditions, low-endotoxin bovine serum or human serum albumin, cerium chloride or cerium nitrate, copper chloride or copper sulfate aqueous solution are mixed evenly, and the rich active functional groups in albumin are used to bind Ce 3+ Ions and Cu 2+ ion; S2, adding potassium hydroxide or sodium hydroxide solution to adjust the reaction pH to trigger the redox reaction, promoting the metal ions to form stably bound oxides with albumin; S3, starting the oil bath to heat the reaction, and then performing a series of purification treatments to obtain a cerium oxide-based nanoparticle product; Preferably, in S1, the source of the albumin is bovine serum or human serum, and the type of protein used is low endotoxin protein.

[0006] Preferably, in S1, Ce is first added to the albumin aqueous solution. 3+ ions as reaction precursors, and then Cu 2+ ions as auxiliary cations, Ce 3+ With Cu 2+ The molar ratio of the ions is 2:1.

[0007] Preferably, in S1, the albumin aqueous solution has a concentration of 25-50 mg / mL, Ce 3+ The concentration of the ion precursor aqueous solution is 50~100 mM, Cu 2+ The concentration of the ion precursor aqueous solution is 25~50 mM.

[0008] Preferably, in S2, the concentration of the potassium hydroxide or sodium hydroxide aqueous solution is 1-2 M, the reaction pH is adjusted to 10-12, and the reaction time is 3-5 min.

[0009] Preferably, in S3, the oil bath heating temperature is 30-50°C, and the heating reaction duration is 3-6 h.

[0010] Preferably, in S3, the purification treatment is to first remove free ions by ultrafiltration, the ultrafiltration tube specification is 30-50 kDa, and the centrifugation time is not less than 20 min; then remove protein aggregates by centrifugation, the centrifugation speed is 6000-8000 rpm, the centrifugation time is 5-10 min, and the supernatant is collected as the cerium oxide-based nanoparticle solution.

[0011] The above scheme successfully prepared a cerium oxide-based nanoparticle, which corresponds to CeO 2 PDF#34-0394 crystalline phase, particle size 12~18 nm, Ce 3+ / Ce 4+ The ratio is not less than 90%, the oxygen vacancy content is not less than 40%, and it has good biocompatibility and efficient active oxygen scavenging ability.

[0012] The second aspect of the present invention provides the use of the cerium oxide-based nanoparticles prepared above in treating acute liver failure.

[0013] Compared with the prior art, the present invention has the following beneficial effects: (1) The preparation process of the present invention is simple and easy to operate, which is conducive to industrial promotion and application; (2) The present invention uses albumin in plasma with good biocompatibility and low immunogenicity as a carrier to prepare cerium oxide-based nanoparticles, thereby ensuring that the prepared nanoparticles have good biocompatibility, avoiding cumbersome nanoparticle surface modification and purification steps, and simplifying the synthesis method; (3) The cerium oxide-based nanoparticles prepared by the present invention have high particle size uniformity and excellent dispersibility; (4) The present invention only introduces auxiliary cation Cu into the reaction process. 2+ ions, which can increase the Ce content in cerium oxide 3 + / Ce 4+ The ratio of cerium oxide to cerium oxide makes the prepared cerium oxide-based nanoparticles have efficient active oxygen scavenging ability, thus achieving efficient treatment of liver failure. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 is a transmission electron micrograph of cerium oxide-based nanoparticles prepared in the example; Figure 2 is a transmission electron microscope image of cerium oxide nanoparticles prepared in the comparative example; Figure 3 The X-ray diffraction spectra of the cerium oxide-based nanoparticles prepared in the embodiment and the cerium oxide nanoparticles prepared in the comparative example; Figure 4The high-resolution X-ray photoelectron spectra of Ce element of the cerium oxide-based nanoparticles prepared in the embodiment and the cerium oxide nanoparticles prepared in the comparative example; Figure 5 The high-resolution X-ray photoelectron spectra of the O element of the cerium oxide-based nanoparticles prepared in the example and the cerium oxide nanoparticles prepared in the comparative example; Figure 6 The cerium oxide nanoparticles prepared in the examples and the cerium oxide nanoparticles prepared in the comparative examples are tested for their ability to scavenge active oxygen (including hydroxyl radicals, superoxide anions and hydrogen peroxide); Figure 7 The invention shows the application of cerium oxide-based nanoparticles prepared in the embodiment in the efficient treatment of mice with acute liver failure. Specific implementation plan

[0015] The present invention is further described below in conjunction with specific embodiments to facilitate a better understanding of the present technical solution. Example

[0016] A method for preparing cerium oxide-based nanoparticles, the preparation method adding Cu 2+ Ions are auxiliary cations used to regulate the Ce content of cerium oxide. 3+ -Ce 4+ Redox couple, the specific process is as follows: S1. Mix 6 mL of 40 mg / mL low-endotoxin bovine serum albumin, 2 mL of 50 mM cerium chloride aqueous solution, and 2 mL of 25 mM copper chloride aqueous solution under vigorous stirring; S2, add 2 M sodium hydroxide solution to adjust the reaction pH to 11, and react for 4 min; S3. Heat the reaction in a 37°C oil bath for 3 h, then remove free ions by ultrafiltration with an ultrafiltration tube specification of 30 kDa and a centrifugation time of 25 min. Then remove protein aggregates by centrifugation with a centrifugal speed of 6000 rpm and a centrifugation time of 10 min. Collect the supernatant, which is the cerium oxide-based nanoparticle solution.

[0017] Comparative Example A method for preparing cerium oxide nanoparticles without adding Cu 2+ Ions are auxiliary cations, and the specific process is as follows: S1. Mix 6 mL of 40 mg / mL low-endotoxin bovine serum albumin, 2 mL of 50 mM cerium chloride aqueous solution, and 2 mL of biological pure water under vigorous stirring. S2, add 2 M sodium hydroxide solution to adjust the reaction pH to 11, and react for 4 min; S3. Heat the reaction in a 37°C oil bath for 3 h, then remove free ions by ultrafiltration with an ultrafiltration tube specification of 30 kDa and a centrifugation time of 25 min. Then remove protein aggregates by centrifugation with a centrifugal speed of 6000 rpm and a centrifugation time of 10 min. Collect the supernatant, which is the cerium oxide-based nanoparticle solution.

[0018] The following describes the embodiments, comparative examples and application examples: Figure 1 3 is a transmission electron microscope image of the cerium oxide-based nanoparticles prepared in the example, and nanoparticles with uniform particles and uniform size were synthesized.

[0019] Figure 2 It is a transmission electron microscope picture of the cerium oxide nanoparticles prepared in the comparative example, and the particle size is slightly smaller than the cerium oxide-based nanoparticles in the example.

[0020] Figure 3 The X-ray diffraction spectra of the cerium oxide-based nanoparticles prepared in the example and the cerium oxide nanoparticles prepared in the comparative example, both of which have peak positions corresponding to PDF#34-0394 of cerium oxide, confirming that the cerium oxide lattice configuration is still maintained in the cerium oxide-based nanoparticles after being regulated by the redox pair.

[0021] Figure 4 The Ce spectra of the X-ray photoelectron spectroscopy of the cerium oxide-based nanoparticles prepared in the example and the cerium oxide nanoparticles prepared in the comparative example are shown in Table 1. 3+ ions as reaction precursors, with Cu 2+ ions as auxiliary cations to achieve Ce 3+ -Ce 4+ After the regulation of the redox pair, Ce 3+ / Ce 4+ The ratio was successfully increased from 10% to over 90%, thereby successfully enhancing the ability to remove active oxygen.

[0022] Figure 5 The O spectra of the X-ray photoelectron spectrum of the cerium oxide-based nanoparticles prepared in the embodiment and the cerium oxide nanoparticles prepared in the comparative example successfully increase the ratio of oxygen vacancies after being regulated by the redox pair, which is beneficial to the adsorption and removal of free active oxygen, and further enhances the ability to remove active oxygen.

[0023] Figure 6The cerium oxide-based nanoparticles prepared in the example and the cerium oxide nanoparticles prepared in the comparative example were tested for their ability to scavenge various reactive oxygen species (including hydroxyl radicals, superoxide anions and hydrogen peroxide). By comparison, it was found that the product cerium oxide-based nanomaterials significantly enhanced their ability to scavenge reactive oxygen species after being regulated by the redox pair, thereby achieving a more efficient reactive oxygen scavenging effect. Application Examples Based on the fact that acute liver failure caused by overdose of acetaminophen in clinical practice is an oxidative stress disease model, the application of cerium oxide-based nanoparticles in the preparation of nano-antioxidants for the efficient treatment of acute liver failure is described. The specific implementation process is as follows: Twenty BALB / C mice, each weighing about 20 g, were prepared. Each mouse was intraperitoneally injected with 500 mg / kg of acetaminophen to establish an acute liver failure model. Two hours after the disease model was established, the 20 mice with acute liver failure were randomly divided into two groups, with 10 mice in each group. One group was not treated (i.e., the untreated group), and the other group was treated with cerium oxide-based nanoparticles by tail vein injection (i.e., the treated group). The survival of the BALB / C mice in the untreated group and the cerium oxide-based nanoparticles treated group was counted.

[0024] Figure 7 The statistical results of the survival of BALB / C mice in the untreated group and the cerium oxide-based nanoparticle treatment group in the application example showed that the mortality rate of mice in the untreated group reached 100% within two days, while the mice treated with cerium oxide-based nanoparticles significantly inhibited the development of the disease, effectively increasing the survival rate of mice to 70%, achieving timely and efficient treatment effects.

Claims

1. A method for preparing cerium oxide-based nanoparticles, characterized in that: Albumin was used as a carrier, and Ce 3+ ions as reaction precursors, with Cu 2+ Ions are auxiliary cations, and the synthesis of cerium oxide and its Ce are achieved by adjusting the reaction pH value. 3+ -Ce 4+ The regulation of redox pairs, the preparation method thereof comprises the following steps: S1. Under vigorous stirring conditions, low-endotoxin bovine serum or human serum albumin, cerium chloride or cerium nitrate, copper chloride or copper sulfate aqueous solution are mixed evenly, and the rich active functional groups in albumin are used to bind Ce 3+ Ions and Cu 2+ ion; S2. Using potassium hydroxide or sodium hydroxide solution to adjust the reaction pH to trigger the redox reaction, promoting the formation of stably bound oxides between metal ions and albumin; S3, start the oil bath to heat the reaction, and then obtain the cerium oxide-based nanoparticle product through a series of purification treatments.

2. The method for preparing cerium oxide-based nanoparticles according to claim 1, characterized in that: Add Ce to the albumin aqueous solution 3+ ions as reaction precursors, and then Cu 2+ ions as auxiliary cations, Ce 3+ Ions and Cu 2+ The molar ratio of the ions is 2:

1.

3. The method for preparing cerium oxide-based nanoparticles according to claim 1, characterized in that In step S1, the concentration of the albumin aqueous solution is 25-50 mg / mL, Ce 3+ The concentration of the ion precursor aqueous solution is 50~100 mM, Cu 2+ The concentration of the ion precursor aqueous solution is 25~50 mM.

4. The method for preparing cerium oxide-based nanoparticles according to claim 1, characterized in that In step S2, the concentration of the potassium hydroxide or sodium hydroxide aqueous solution is 1-2 M, the reaction pH is adjusted to 10-12, and the reaction time is 3-5 min.

5. The method for preparing cerium oxide-based nanoparticles according to claim 1, characterized in that In step S3, the oil bath heating temperature is 30-50° C., and the heating reaction duration is 3-6 h.

6. The method for preparing cerium oxide-based nanoparticles according to claim 1, characterized in that: The purification process is to first remove free ions by ultrafiltration, the ultrafiltration tube specification is 30~50 kDa, and the centrifugation time is not less than 20 min; The protein aggregates are then removed by centrifugation at a speed of 6000-8000 rpm for 5-10 min, and the supernatant is collected as the cerium oxide-based nanoparticle solution.

7. The cerium oxide-based nanoparticles prepared by the preparation method according to any one of claims 1 to 6, characterized in that The cerium oxide-based nanoparticles correspond to the CeO2 PDF#34-0394 crystalline phase.

8. The cerium oxide-based nanoparticles according to claim 7, characterized in that The particle size of the cerium oxide-based nanoparticles is 12 to 16 nm.

9. The cerium oxide-based nanoparticles according to claim 7, characterized in that The cerium oxide-based nanoparticles Ce 3+ / Ce 4+ The ratio is not less than 90%, the oxygen vacancy content is not less than 40%, and it exhibits excellent hydrogen peroxide, hydroxyl radical and superoxide anion scavenging activity.

10. Use of the cerium oxide-based nanoparticles according to any one of claims 7 to 9 in the preparation of nano antioxidants for the efficient treatment of acute liver failure.

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