A CeO2@Pd nanozyme and its preparation method and application

Abstract

The present invention provides a CeO2@Pd nanozyme and a preparation method thereof, the preparation method comprising the following steps: S1. adding ascorbic acid to ultrapure water, ultrasonically dispersing 5 15min to obtain an ascorbic acid solution, S2. adding cerium acetate powder to the ascorbic acid solution obtained in step S1, stirring the reaction at room temperature for 1 3h to obtain a mixed solution; S3. adding potassium hexachloropalladate powder to the mixed solution obtained in step S2, stirring the reaction at room temperature for 1 3h to obtain a reaction solution; S4. centrifuging the reaction solution obtained in step S3 for 10 20min to obtain a precipitate; S5. washing the precipitate obtained in step S4 with anhydrous ethanol and centrifuging 3 times, collecting the precipitate and placing it in a vacuum drying oven to vacuum dry to obtain CeO2@Pd nanozyme. The present invention loads metal Pd on CeO2 nanoparticles, and the obtained CeO2@Pd nanozyme has excellent antioxidant and anti-inflammatory abilities, good stability and biosafety, and organ targeting, and can be used to remove excessive ROS when sepsis-related organ function is damaged.

Description

Technical Field

[0001] The present invention relates to a nanozyme, and in particular to a CeO2@Pd nanozyme and a preparation method and application thereof. Background Art

[0002] Sepsis is a syndrome characterized by a dysregulated host response to infection, leading to organ dysfunction. Sepsis-related organ damage is the primary cause of its high mortality. Physiologically, reactive oxygen species (ROS), such as hydroxyl radicals (·OH), hydrogen peroxide (H2O2), and superoxide anions (·O2-), maintain cellular energy metabolism and homeostasis. Oxidative stress is a manifestation of an imbalance between oxidants and antioxidants in pathological conditions. Excessive ROS production is key to the dysregulated state of many inflammatory diseases. Excessive inflammatory responses and oxidative stress have been shown to be important contributors to sepsis-related liver injury.

[0003] Nanozymes are nanomaterials with similar kinetics and catalytic mechanisms to natural enzymatic reactions. They have the ability to regulate ROS levels and are currently widely used in the medical field. Cerium oxide nanoparticles (CeO2NPs) are a rare earth oxide nanomaterial with free radical scavenging, antioxidant, anti-tumor and antibacterial properties. Nanoparticles have high permeability and retention effect (ERP effect) in tumors and inflammatory tissues, making them more effective in damaged tissues. They have gradually been applied to the protection of cells, tissues and organs. The anti-inflammatory effect of CeO2 NPs mainly relies on its powerful antioxidant effect. CeO2 NPs can be used in Ce 3+ and Ce 4+ The rapid conversion between valence states, its surface has both trivalent cerium ions (Ce 3+ ), there are also tetravalent cerium ions (Ce 4+ ) and has a typical fluorite structure. The large number of oxygen vacancies on its surface determine its strong oxygen storage and release capacity, which can provide redox binding sites for superoxide and hydroxide, realizing the function of scavenging free radicals. Studies have shown that CeO2NPs have superoxide dismutase (SOD) and catalase (CAT) mimicking activities, and can therefore effectively remove ROS pollution. However, pure CeO2 has low activity in catalytic reactions, poor specificity and selectivity, and lacks targeting.

[0004] Since metal-metal oxide heterostructures can produce a synergistic effect by changing the electronic structure, loaded nanoparticles exhibit better activity than unloaded nanoparticles. Taking advantage of the strong synergistic effect between CeO2 and precious metals, loading precious metals on the CeO2 surface to enhance its catalytic activity is one of the key feasible strategies, which can achieve comprehensive optimization of the catalytic activity and selectivity of composite catalytic materials. Nano-palladium (Pd), as a precious metal with enzyme-like activity, also has functions similar to catalase and superoxide dismutase, and can scavenge ROS broadly. At the same time, the precious metal Pd has a strong localized surface plasmon resonance (LSPR) effect, resulting in good light absorption capacity and high photothermal conversion efficiency. Precious metal nanomaterials have been well studied and applied in fields such as photothermal sterilization. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a method for preparing CeO2@Pd nanozyme, in which metal Pd is loaded on CeO2 nanoparticles. The prepared CeO2@Pd nanozyme has excellent antioxidant and anti-inflammatory capabilities, good stability and biosafety, and organ targeting, and can be used to eliminate excessive ROS when sepsis-related organ function is damaged.

[0006] In order to solve the above technical problems, the technical solution of the present invention is:

[0007] A method for preparing CeO2@Pd nanozyme comprises the following steps:

[0008] S1. Add ascorbic acid to ultrapure water and ultrasonically disperse for 5-15 minutes to obtain an ascorbic acid solution.

[0009] S2. The cerium acetate powder was added to the ascorbic acid solution obtained in step S1, and the reaction was stirred at room temperature for 1-3h to obtain a mixed solution;

[0010] S3. The potassium hexachloropalladate powder was added to the mixed solution obtained in step S2, and the reaction was stirred at room temperature for 1-3h to obtain a reaction solution;

[0011] S4. The reaction solution obtained in step S3 was centrifuged for 10-20 min to obtain a precipitate;

[0012] S5. The precipitate obtained in step S4 was washed with anhydrous ethanol and centrifuged three times. The precipitate was collected and placed in a vacuum drying oven for vacuum drying to obtain CeO2@Pd nanozyme.

[0013] Furthermore, the ratio of ascorbic acid, ultrapure water, cerium acetate powder, and potassium hexachloropalladate powder of the present invention is (85-90) mg:35 mL:(6-10) mg:(8-12) mg.

[0014] Furthermore, in step S1 of the present invention, the ultrasonic frequency of the ultrasonic dispersion is 40 Hz and the temperature is 35°C.

[0015] Furthermore, in step S2 of the present invention, the stirring speed of the reaction at room temperature is 400-600 rpm.

[0016] Furthermore, in step S3 of the present invention, the stirring speed of the reaction at room temperature is 400-600 rpm.

[0017] Furthermore, in step S4 of the present invention, the centrifugal speed is 12000 rpm.

[0018] Furthermore, in step S5 of the present invention, the centrifugal speed is 6000 rpm; the vacuum drying temperature is room temperature, and the time is 24-48 hours.

[0019] Another technical problem to be solved by the present invention is to provide CeO2@Pd nanozyme prepared by the above preparation method.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] 1) Metal nanoparticles can be loaded onto CeO2 nanoparticles. Interaction exists between the metal nanoparticles and CeO2, stabilizing the precious metal particles and reducing clustering. CeO2 can also enhance the catalytic activity of the metal nanoparticles by regulating their electronic and coordination structures. Furthermore, the material can tightly grasp the metal nanoparticles, thereby improving stability. This invention uniformly loads enzymatically active precious metal Pd nanoparticles onto CeO2 nanoparticles, resulting in a CeO2@Pd nanozyme with a suitable shape and particle size. This enhances the catalytic performance and targeting of the CeO2 nanozyme, while also improving the stability and dispersibility of the Pd nanoparticles.

[0022] 2) The CeO2@Pd nanozyme constructed in the present invention has excellent photothermal properties and can be targeted to the mouse liver. Under NIR irradiation, its local temperature can be increased to a suitable photothermal treatment temperature, thereby increasing the therapeutic concentration of the local target organ and achieving a better ROS removal effect.

[0023] 3) In the CeO2@Pd nanozyme constructed in the present invention, both CeO2 and the loaded Pd nanoparticles have strong enzyme-like activity, and Pd has good photothermal conversion performance. The combination of the two can synergistically enhance photothermal therapy and enzyme-like activity, achieve better ROS removal effect, and have better dispersibility. Compared with CeO2, it shows better stability in different culture medium environments.

[0024] 4) The raw materials used in the present invention are economical, cheap and easy to synthesize, and the prepared CeO2@Pd nanozyme has good catalytic activity, chemical and thermal stability, excellent near-infrared (NIR) absorbance, as well as biocompatibility and biodegradability. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The drawings described herein are used to provide a further understanding of the present invention, constitute a part of this application, and do not constitute an improper limitation of the present invention. In the drawings:

[0026] Figure 1 This is a transmission electron microscopy (TEM) image of the CeO2@Pd nanozyme prepared in Example 1 of the present invention;

[0027] Figure 2 This is a photothermal effect diagram of the CeO2@Pd nanozyme prepared in Example 1 of the present invention;

[0028] Figure 3 This is a photothermal effect diagram of the CeO2@Pd nanozyme prepared in Example 1 of the present invention;

[0029] Figure 4 This is the ESR result of CeO2@Pd nanozyme prepared in Example 1 of the present invention to remove superoxide anions;

[0030] Figure 5 This is the ESR result of CeO2@Pd nanozyme prepared in Example 1 of the present invention to remove singlet oxygen;

[0031] Figure 6 This is the ESR result of CeO2@Pd nanozyme prepared in Example 1 of the present invention to scavenge hydroxyl radicals;

[0032] Figure 7 This is a diagram showing the cytotoxicity test results of the CeO2@Pd nanozyme prepared in Example 1 of the present invention. DETAILED DESCRIPTION

[0033] The present invention will be described in detail below with reference to specific embodiments. The exemplary embodiments and descriptions of the present invention are used to explain the present invention but are not intended to limit the present invention.

[0034] Example 1

[0035] The CeO2@Pd nanozyme was prepared by the following steps:

[0036] S1. Add ascorbic acid to ultrapure water and disperse it ultrasonically at 40 Hz frequency and 35°C for 10 min to obtain an ascorbic acid solution.

[0037] S2. The cerium acetate powder was added to the ascorbic acid solution obtained in step S1, and the reaction was stirred at room temperature for 2h at a stirring speed of 500 rpm to obtain a mixed solution;

[0038] S3. The potassium hexachloropalladate powder was added to the mixed solution obtained in step S2, and the reaction was stirred at room temperature for 2h at a stirring speed of 500rpm to obtain a reaction solution; ascorbic acid, ultrapure water, cerium acetate powder, potassium hexachloropalladate powder ratio of 88mg:35mL:8mg:10mg;

[0039] S4. The reaction solution obtained in step S3 was centrifuged at 12000 rpm for 15 min to obtain a precipitate;

[0040] S5. The precipitate obtained in step S4 was washed with anhydrous ethanol and centrifuged at 6000 rpm for 3 times. The precipitate was collected and placed in a vacuum drying oven at room temperature for 36 hours to obtain CeO2@Pd nanozyme.

[0041] Example 2

[0042] The CeO2@Pd nanozyme was prepared by the following steps:

[0043] S1. Add ascorbic acid to ultrapure water and disperse it ultrasonically at 40 Hz frequency and 35°C for 5 min to obtain an ascorbic acid solution.

[0044] S2. The cerium acetate powder was added to the ascorbic acid solution obtained in step S1, and the reaction was stirred at room temperature for 3h at a stirring speed of 400 rpm to obtain a mixed solution;

[0045] S3. The potassium hexachloropalladate powder was added to the mixed solution obtained in step S2, and the reaction was stirred at room temperature for 3h at a stirring speed of 400rpm to obtain a reaction solution; ascorbic acid, ultrapure water, cerium acetate powder, potassium hexachloropalladate powder ratio of 85mg:35mL:6mg:8mg;

[0046] S4. The reaction solution obtained in step S3 was centrifuged at 12000 rpm for 20 min to obtain a precipitate;

[0047] S5. The precipitate obtained in step S4 was washed with anhydrous ethanol and centrifuged at 6000 rpm for 3 times. The precipitate was collected and placed in a vacuum drying oven at room temperature for 48 hours to obtain CeO2@Pd nanozyme.

[0048] Example 3

[0049] The CeO2@Pd nanozyme was prepared by the following steps:

[0050] S1. Add ascorbic acid to ultrapure water and disperse it ultrasonically at 40 Hz frequency and 35°C for 15 min to obtain an ascorbic acid solution.

[0051] S2. The cerium acetate powder was added to the ascorbic acid solution obtained in step S1, and the reaction was stirred at room temperature for 1h at a stirring speed of 600 rpm to obtain a mixed solution;

[0052] S3. The potassium hexachloropalladate powder was added to the mixed solution obtained in step S2, and the reaction was stirred at room temperature for 1 h at a stirring speed of 600 rpm to obtain a reaction solution; ascorbic acid, ultrapure water, cerium acetate powder, potassium hexachloropalladate powder ratio of 87 mg: 35 mL: 9 mg: 9 mg;

[0053] S4. The reaction solution obtained in step S3 was centrifuged at 12000 rpm for 12 min to obtain a precipitate;

[0054] S5. The precipitate obtained in step S4 was washed with anhydrous ethanol and centrifuged at 6000 rpm for 3 times. The precipitate was collected and placed in a vacuum drying oven at room temperature for 40 h to obtain CeO2@Pd nanozyme.

[0055] Example 4

[0056] The CeO2@Pd nanozyme was prepared by the following steps:

[0057] S1. Add ascorbic acid to ultrapure water and disperse it ultrasonically at 40 Hz frequency and 35°C for 8 min to obtain an ascorbic acid solution.

[0058] S2. The cerium acetate powder was added to the ascorbic acid solution obtained in step S1, and the reaction was stirred at room temperature for 1.5h at a stirring speed of 500rpm to obtain a mixed solution;

[0059] S3. The potassium hexachloropalladate powder was added to the mixed solution obtained in step S2, and the reaction was stirred at room temperature for 2.5h at a stirring speed of 500rpm to obtain a reaction solution; ascorbic acid, ultrapure water, cerium acetate powder, potassium hexachloropalladate powder ratio of 90mg: 35mL: 10mg: 12mg;

[0060] S4. The reaction solution obtained in step S3 was centrifuged at 12000 rpm for 10 min to obtain a precipitate;

[0061] S5. The precipitate obtained in step S4 was washed with anhydrous ethanol and centrifuged at 6000 rpm for 3 times. The precipitate was collected and placed in a vacuum drying oven at room temperature for 24 h to obtain CeO2@Pd nanozyme.

[0062] Experimental Example 1

[0063] An 808 nm infrared laser emitter was used for irradiation, with an excitation power of 1.5 W / cm 2PBS, a CeO2 solution with a concentration of 100 μg / mL, and a CeO2@Pd nanozyme solution with a concentration of 100 μg / mL (prepared in Example 1) were placed in a 1.5 mL EP tube and irradiated vertically with a laser for 10 min. The temperature was measured using a near-infrared thermal imager, and the thermal imaging and temperature data were recorded to conduct a photothermal effect experiment.

[0064] The experimental results are as follows Figure 2 As shown in the figure, after 10 minutes of 808nm near-infrared laser irradiation, the temperature of the PBS and CeO2 groups increased slowly, with the temperature increase amplitude less than 5°C. The PBS group rose to a maximum of 26.3°C, and the CeO2 group rose to a maximum of 26.2°C. However, the temperature of the CeO2@Pd nanozyme group rose rapidly after near-infrared laser irradiation, reaching a maximum of 57.8°C after 10 minutes, demonstrating its significant photothermal conversion effect.

[0065] Experimental Example 2

[0066] 100 μL of 100 μg / mL CeO2 solution, 100 μg / mL CeO2@Pd nanozyme (prepared in Example 1) solution, and 100 μL of PBS were injected into the tail vein of mice, respectively. The right upper abdomen of the mice was vertically irradiated with an 808 nm near-infrared laser emitter at an excitation power of 1.5 W / cm 2 , a near-infrared thermal imager was used to measure its temperature and record thermal imaging and temperature data to verify the in vivo photothermal effect.

[0067] The experimental results are as follows Figure 3 As shown: there was no significant temperature change in the PBS group and the CeO2 group after 10 minutes of laser irradiation, while the temperature of the CeO2@Pd nanozyme group was 42.6°C after 10 minutes of irradiation, proving that it has a photothermal effect in vivo. At the same time, no adverse reactions such as skin burns were observed in the near-infrared irradiation area of ​​mice.

[0068] Experimental Example 3

[0069] (1) Superoxide anion (·O2 -) Clearance ability test: Use PBS buffer as a solvent to prepare a xanthine solution with a concentration of 10mM and a xanthine oxidase solution with a concentration of 1U / mL. Take 100μL of xanthine solution and 100μL of xanthine oxidase solution, then add 20μL of DMPO solution and 180mL of PBS buffer. Incubate the reaction for 10 minutes and then take a sample for testing. For other sample test groups, replace 180μL of buffer with 180μL of sample solution. The sample solutions are a CeO2 solution with a concentration of 100μg / mL, a CeO2@Pd nanozyme (prepared in Example 1) solution with a concentration of 100μg / mL, and a CeO2@Pd nanozyme (prepared in Example 1) solution with a concentration of 100μg / mL after near-infrared irradiation.

[0070] (2) Singlet oxygen ( 1 O2) scavenging ability test: Take 50L of TMP stock solution, dissolve it in 5mL of pure water to obtain diluted TMP, take 1mL of diluted TMP and add it to a 0.5mL centrifuge tube containing a sample solution, the sample solution is a CeO2 solution with a concentration of 100μg / mL, a CeO2@Pd nanozyme (prepared in Example 1) solution with a concentration of 100μg / mL, and a CeO2@Pd nanozyme (prepared in Example 1) solution with a concentration of 100μg / mL after near-infrared irradiation. After shaking, directly illuminate.

[0071] (3) Hydroxyl radical (·OH) scavenging ability test: Prepare a FeSO4 solution with a concentration of 5 mg / mL, take 200 μL of this solution, add 20 μL of 100 mM DMPO solution and 150 μL of deionized water, then add 30 μL of 30% H2O2, mix and react for 10 minutes, take a sample and test, and use it as the control group data. For the other sample test groups, replace 150 μL of deionized water with 150 μL of sample solution. The sample solutions are a CeO2 solution with a concentration of 100 μg / mL, a CeO2@Pd nanozyme (prepared in Example 1) solution with a concentration of 100 μg / mL, and a CeO2@Pd nanozyme (prepared in Example 1) solution with a concentration of 100 μg / mL after near-infrared irradiation.

[0072] The experimental results are as follows Figure 4 、 Figure 5 、 Figure 6 As shown: CeO2@Pd nanozymes can effectively remove O2 - , OH and 1 O2, and its removal effect is significantly enhanced under the synergistic effect of light and heat, ·OH, ·O2 - and 1 The order of O2 scavenging ability is CeO2@Pd+NIR>CeO2@Pd>CeO2.

[0073] Experimental Example 4

[0074] When cells were adherent and at a density of 70%-80%, complete culture medium containing varying concentrations of CeO2 solution and CeO2@Pd nanozyme (prepared in Example 1) (0, 5, 10, 20, 50, 100, 200, and 500 μg / mL) was added according to a concentration gradient. The cells were incubated in an incubator for 24 hours. Gently washed three times with PBS, 90 μL of basal culture medium and 10 μL of CCK-8 solution were added to each well. The cells were incubated in the dark for 1-2 hours, and the absorbance (OD) at 450 nm was measured.

[0075] The experimental results are as follows Figure 7 As shown in the figure: the CeO2@Pd nanozyme and CeO2 prepared by the present invention have good cell compatibility, and both have low cytotoxicity.

[0076] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the present invention. Anyone skilled in the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by one of ordinary skill in the art without departing from the spirit and technical principles disclosed herein are intended to be covered by the claims of the present invention.

Claims

1. A method for preparing CeO2@Pd nanozyme, characterized by: The following steps are involved: S1. Add ascorbic acid to ultrapure water and disperse by ultrasonic for 5-15 minutes to obtain an ascorbic acid solution. S2. The cerium acetate powder was added to the ascorbic acid solution obtained in step S1, and the reaction was stirred at room temperature for 1-3h to obtain a mixed solution; S3. The potassium hexachloropalladate powder was added to the mixed solution obtained in step S2, and the reaction was stirred at room temperature for 1-3h to obtain a reaction solution; S4. The reaction solution obtained in step S3 was centrifuged for 10-20 min to obtain a precipitate; S5. The precipitate obtained in step S4 was washed with anhydrous ethanol and centrifuged three times. The precipitate was collected and placed in a vacuum drying oven for vacuum drying to obtain CeO2@Pd nanozyme.

2. The method for preparing a CeO2@Pd nanozyme according to claim 1, wherein: The ratio of ascorbic acid, ultrapure water, cerium acetate powder and potassium hexachloropalladate powder is (85-90) mg:35 mL: (6-10) mg: (8-12) mg.

3. The method for preparing a CeO2@Pd nanozyme according to claim 1, wherein: In step S1, the ultrasonic dispersion is performed at an ultrasonic frequency of 40 Hz and a temperature of 35°C.

4. The method for preparing a CeO2@Pd nanozyme according to claim 1, wherein: In step S2, the stirring speed of the reaction at room temperature is 400-600 rpm.

5. The method for preparing a CeO2@Pd nanozyme according to claim 1, wherein: In step S3, the stirring speed of the reaction at room temperature is 400-600 rpm.

6. The method for preparing a CeO2@Pd nanozyme according to claim 1, wherein: In step S4, the centrifugal speed is 12000 rpm.

7. The method for preparing a CeO2@Pd nanozyme according to claim 1, wherein: In step S5, the centrifugal speed is 6000 rpm; the vacuum drying temperature is room temperature, and the time is 24-48 hours.

8. CeO2@Pd nanozyme prepared according to the preparation method of CeO2@Pd nanozyme according to any one of claims 1 to 7.

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