CeO2-coated Pd nano-enzyme as well as preparation method and application thereof

Abstract

The invention provides a CeO2-coated Pd nano-enzyme and a preparation method thereof, the preparation method comprises the following steps: S1, adding ascorbic acid into ultrapure water, and carrying out ultrasonic dispersion for 5-15 min to obtain an ascorbic acid solution, S2, adding cerium acetate powder into the ascorbic acid solution obtained in the step S1, and carrying out a stirring reaction at room temperature for 1-3 h to obtain a mixed solution; s3, adding potassium hexachloropalladate powder into the mixed solution obtained in the step S2, and stirring and reacting at room temperature for 1-3 hours to obtain a reaction solution; s4, centrifuging the reaction liquid obtained in the step S3 for 10-20 minutes to obtain a precipitate; s5, the precipitate obtained in the step S4 is washed with absolute ethyl alcohol and then centrifuged three times, the precipitate is collected and placed in a vacuum drying box to be subjected to vacuum drying, and the CeO2-coated Pd nano-enzyme is obtained. According to the preparation method disclosed by the invention, the metal Pd is loaded on the CeO2 nano particles, and the prepared CeO2-coated Pd nano enzyme has excellent anti-oxidation and anti-inflammatory capacities, relatively good stability, relatively good biological safety and relatively good organ targeting property, and can be used for removing excessive ROS (Reactive Oxygen Species) when septiemia-related organ functions are damaged.

Description

Technical Field

[0001] The present invention relates to a nanozyme, in particular to a CeO 2 @Pd nanozyme and its preparation method and application. Background Art

[0002] Sepsis is a syndrome in which infection causes host response disorders and organ dysfunction. Sepsis-related organ dysfunction is the main cause of its high mortality rate. Reactive oxygen species (ROS) under physiological conditions, such as hydroxyl radical (·OH), hydrogen peroxide (Hydrogen peroxide, H 2 O 2 ) and superoxide anion (Superoxide anion, ·O 2 -) can maintain cellular energy metabolism and stability. Oxidative stress is a manifestation of imbalance between oxidants and antioxidants under pathological conditions. Excessive ROS production is the key to the disordered state of many inflammatory diseases. Excessive inflammatory response and oxidative stress have been proven to be important causes of sepsis-related liver damage.

[0003] Nanozymes are nanomaterials with similar kinetics and catalytic mechanisms to natural enzyme-catalyzed reactions. They have the ability to regulate ROS levels and are currently widely used in the medical field. 2 CeO NPs are a rare earth oxide nanomaterial with the properties of free radical scavenging, anti-oxidation, anti-tumor and antibacterial. Nanoparticles have high permeability and retention effect (ERP effect) in tumor and inflammatory tissues, and are more likely to take effect in damaged tissues. They have gradually been applied to the protection of cells, tissues and organs. 2 The anti-inflammatory effect of NPs mainly relies on its strong antioxidant effect. 2 NPs can 3+ and Ce 4+ The surface of the cerium ion (Ce 3+ ), there are also tetravalent cerium ions (Ce 4+ ) and has a typical fluorite structure. The large amount of oxygen vacancies on the surface determine its strong oxygen storage and release capacity, which can provide redox binding sites for superoxide and hydroxide to achieve the function of scavenging free radicals. Studies have shown that CeO 2 NPs have the mimetic activity of superoxide dismutase (SOD) and catalase (CAT), so they can effectively remove ROS pollution.2 It 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, the supported nanoparticles show better activity than the unsupported nanoparticles. 2 There is a strong synergistic effect between CeO 2 One of the feasible and important strategies is to load precious metals on the surface to improve their catalytic activity, 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 remove ROS broadly. At the same time, the precious metal Pd has a strong localized surface plasmon resonance (LSPR) effect, so it has good light absorption ability 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 CeO 2 Preparation method of @Pd nanozyme, loading metal Pd on CeO 2 CeO nanoparticles 2 @Pd nanozymes have excellent antioxidant and anti-inflammatory capabilities, good stability, biosafety, and organ targeting, and can be used to remove excess 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 CeO 2 The preparation method of @Pd nanozyme comprises the following steps:

[0008] S1. Add ascorbic acid into ultrapure water and disperse by ultrasonic 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 is added to the mixed solution obtained in step S2, and the reaction is stirred at room temperature for 1-3h to obtain a reaction solution;

[0011] S4. The reaction solution obtained in step S3 is 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, and the precipitate was collected and placed in a vacuum drying oven to obtain CeO 2 @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 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 room temperature stirring reaction is 400-600 rpm.

[0016] Furthermore, in step S3 of the present invention, the stirring speed of the room temperature stirring reaction 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 CeO 2 @Pd nanozyme.

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

[0021] 1) Metal nanoparticles can be loaded on CeO 2 On the nanoparticles, metal nanoparticles and CeO 2 There is an interaction force between CeO 2 It can stabilize precious metal particles, reduce clusters, and improve the catalytic activity of metal nanoparticles by adjusting the electronic and coordination structures of the loaded metal nanoparticles. It can also firmly grasp the metal nanoparticles, thereby improving stability. The present invention uniformly loads the precious metal Pd nanoparticles, which also have enzymatic reactions, on CeO 2 On the nanoparticles, a CeO with suitable shape and particle size was synthesized. 2 @Pd nanozymes, enhanced CeO 2 The catalytic performance and targeting of nanozymes also improve the stability and dispersibility of Pd.

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

[0023] 3) CeO constructed by the present invention 2 @CeO in Pd nanozyme 2 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 CeO in different culture medium environments, 2 Better stability.

[0024] 4) The raw materials used in the present invention are economical, cheap and easy to synthesize. 2 The @Pd nanozymes possess 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 the present application, and do not constitute an improper limitation of the present invention. In the drawings:

[0026] Figure 1 The CeO prepared in Example 1 of the present invention 2 @TEM image of Pd nanozyme;

[0027] Figure 2 The CeO prepared in Example 1 of the present invention 2 @In vitro photothermal effect of Pd nanozyme;

[0028] Figure 3 The CeO prepared in Example 1 of the present invention 2 @Image of the in vivo photothermal effect of Pd nanozyme;

[0029] Figure 4 The CeO prepared in Example 1 of the present invention 2 @ESR results of Pd nanozyme removing superoxide anions;

[0030] Figure 5 The CeO prepared in Example 1 of the present invention 2 @ESR results of Pd nanozyme removing singlet oxygen;

[0031] Figure 6 The CeO prepared in Example 1 of the present invention 2 @ESR results of Pd nanozyme removing hydroxyl radicals;

[0032] Figure 7 The CeO prepared in Example 1 of the present invention 2 @Chart of cytotoxicity test results of Pd nanozyme. DETAILED DESCRIPTION

[0033] The present invention will be described in detail below in conjunction with specific embodiments. The illustrative 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] CeO was prepared as follows: 2 @Pd nanozyme:

[0036] S1. Add ascorbic acid to ultrapure water, and disperse by ultrasonic at 40 Hz 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 at a stirring speed of 500 rpm for 2 h 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 2 h at a stirring speed of 500 rpm to obtain a reaction solution; ascorbic acid, ultrapure water, cerium acetate powder, potassium hexachloropalladate powder ratio of 88 mg: 35 mL: 8 mg: 10 mg;

[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 h to obtain CeO 2 @Pd nanozyme.

[0041] Example 2

[0042] CeO was prepared as follows: 2 @Pd nanozyme:

[0043] S1. Add ascorbic acid to ultrapure water, and disperse by ultrasound at 40 Hz 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 400rpm 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 3 h at a stirring speed of 400 rpm to obtain a reaction solution; ascorbic acid, ultrapure water, cerium acetate powder, potassium hexachloropalladate powder ratio of 85 mg: 35 mL: 6 mg: 8 mg;

[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 h to obtain CeO 2 @Pd nanozyme.

[0048] Example 3

[0049] CeO was prepared as follows: 2 @Pd nanozyme:

[0050] S1. Add ascorbic acid to ultrapure water, and disperse by ultrasound at 40 Hz 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 1 h 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 CeO 2 @Pd nanozyme.

[0055] Example 4

[0056] CeO was prepared as follows: 2 @Pd nanozyme:

[0057] S1. Add ascorbic acid to ultrapure water, and disperse by ultrasonic at 40 Hz 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 at a stirring speed of 500 rpm for 1.5 h 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 2.5 h at a stirring speed of 500 rpm to obtain a reaction solution; ascorbic acid, ultrapure water, cerium acetate powder, potassium hexachloropalladate powder ratio of 90 mg: 35 mL: 10 mg: 12 mg;

[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 CeO 2 @Pd nanozyme.

[0062] Experimental Example 1

[0063] The irradiation was performed using an 808 nm infrared laser emitter with an excitation power of 1.5 W / cm 2 PBS and 100 μg / mL CeO 2 solution and CeO with a concentration of 100 μg / mL 2 The Pd nanozyme solution (prepared in Example 1) was 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 Shown: PBS group, CeO 2 After 10 minutes of 808nm near-infrared laser irradiation, the temperature of the groups increased slowly, and the temperature increase was less than 5℃. The highest temperature in the PBS group was 26.3℃, and the temperature in the CeO 2 The highest temperature in the CeO group was 26.2℃. 2 The temperature of the @Pd nanozyme group rose rapidly after near-infrared laser irradiation, reaching a maximum of 57.8°C after 10 minutes, proving that it has an obvious photothermal conversion effect.

[0065] Experimental Example 2

[0066] CeO with a concentration of 100 μg / mL 2 Solution, CeO with a concentration of 100 μg / mL 2100 μL of Pd nanozyme (prepared in Example 1) solution and 100 μL of PBS were injected into the tail vein of mice, and an 808 nm near-infrared laser emitter was used to vertically irradiate the right upper abdomen of the mice at an excitation power of 1.5 W / cm 2 , a near-infrared thermal imager was used to measure the temperature and record the thermal imaging and temperature data to verify the in vivo photothermal effect.

[0067] The experimental results are as follows Figure 3 Shown: PBS group, CeO 2 The temperature of the group did not change significantly after 10 minutes of laser irradiation, while the temperature of CeO 2 The temperature of the @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 (·O 2 - ) Clearing ability test: Use PBS buffer as solvent to prepare 10mM xanthine solution and 1U / mL xanthine oxidase solution, take 100μL xanthine solution and 100μL xanthine oxidase solution, then add 20μL DMPO solution and 180mL PBS buffer, incubate for 10min and take samples for testing. For other sample test groups, replace 180μL buffer with 180μL sample solution, the sample solution is 100μg / mL CeO 2 Solution, CeO with a concentration of 100 μg / mL 2 @Pd nanozyme (prepared in Example 1) solution and CeO with a concentration of 100 μg / mL after near-infrared irradiation 2 @Pd nanozyme (prepared in Example 1) solution.

[0070] (2) Singlet oxygen ( 1 O 2 ) Clearance test: Take 50L TMP stock solution, dissolve it in 5mL pure water to obtain diluted TMP, take 1mL diluted TMP and add it into a centrifuge tube containing 0.5mL sample solution. The sample solution is CeO with a concentration of 100μg / mL. 2 Solution, CeO with a concentration of 100 μg / mL 2 @Pd nanozyme (prepared in Example 1) solution and CeO with a concentration of 100 μg / mL after near-infrared irradiation 2 @Pd nanozyme (prepared in Example 1) solution was shaken well and directly exposed to light.

[0071] (3) Hydroxyl radical (·OH) scavenging ability test: FeSO with a concentration of 5 mg / mL was prepared 4 200 μL of the solution was added with 20 μL of 100 mM DMPO solution and 150 μL of deionized water, and then 30 μL of 30% H 2 O 2 , mix and react for 10 minutes, take samples for testing, and use them as the control group data. For the other sample test groups, replace 150μL of deionized water with 150μL of sample solution, which is a CeO solution with a concentration of 100μg / mL. 2 Solution, CeO with a concentration of 100 μg / mL 2 @Pd nanozyme (prepared in Example 1) solution and CeO with a concentration of 100 μg / mL after near-infrared irradiation 2 @Pd nanozyme (prepared in Example 1) solution.

[0072] The experimental results are as follows Figure 4 , Figure 5 , Figure 6 Shown: CeO 2 @Pd nanozymes can effectively remove O 2 - , OH and 1 O 2 , and its removal effect is significantly enhanced under the synergistic effect of light and heat, ·OH, ·O 2 - and 1 O 2 The order of scavenging ability is CeO 2 @Pd+NIR>CeO 2 @Pd>CeO 2 .

[0073] Experimental Example 4

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

[0075] The experimental results are as follows Figure 7 As shown: CeO prepared by the present invention 2 @Pd nanozyme and CeO 2The cell compatibility of these two drugs is good, and both of them 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 familiar with 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 a person of ordinary skill in the art without departing from the spirit and technical concept disclosed by the present invention shall still be covered by the claims of the present invention.

Claims

1. A method for preparing CeO2@Pd nanozyme, characterized in that: The following steps are involved: S1. Add ascorbic acid into 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 is added to the mixed solution obtained in step S2, and the reaction is stirred at room temperature for 1-3h to obtain a reaction solution; S4. The reaction solution obtained in step S3 is centrifuged for 10-20 min to obtain a precipitate; S5. The precipitate obtained in step S4 is washed with anhydrous ethanol and centrifuged three times. The precipitate is collected and placed in a vacuum drying oven for vacuum drying to obtain CeO2@Pd nanozyme.

2. The method for preparing CeO2@Pd nanozyme according to claim 1, characterized in that: 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 CeO2@Pd nanozyme according to claim 1, characterized in that: In the step S1, the ultrasonic frequency of the ultrasonic dispersion is 40 Hz and the temperature is 35°C.

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

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

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

7. The method for preparing CeO2@Pd nanozyme according to claim 1, characterized in that: 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 according to claims 1 to 7.

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