A core-shell nanozyme and a preparation method and application thereof

Abstract

The present application relates to a kind of core-shell nanometer enzyme and its preparation method and application, the core-shell nanometer enzyme by CeO2 Nanoparticle core and zeolite imidazolate shell material shell, and tannin acid being covered on the shell composition.The nanometer enzyme of the present application can gradually release CeO2 with the decomposition of metal organic framework shell, exert SOD and nanometer enzyme activity, eliminate the excess ROS generated in wound, framework outside also includes tannin acid, improve the biocompatibility of material, while also can significantly improve the dispersibility of nanoparticle, the nanometer enzyme of the present application is suitable for wound healing, especially suitable for diabetic wound healing.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of pharmaceutical preparations, in particular to a core-shell nanoscale enzyme and a preparation method and application thereof. BACKGROUND

[0002] Nanoscale enzyme is a kind of nanomaterial with enzyme-like catalytic activity. Since it was reported in 2007, the research on nanoscale enzyme has entered a period of rapid development. The antibacterial effect of nanoscale enzyme is based on its peroxidase activity: nanoscale enzyme catalyzes hydrogen peroxide (H2O2) to generate highly active hydroxyl radicals (·OH), which in turn destroys the structure of bacteria and achieves antibacterial effect. However, studies have shown that the activity of nanoscale enzyme in diabetic chronic wounds is limited. In addition, under the premise of maintaining the high catalytic activity of nanoscale enzyme, how to improve the targeting of antibacterial materials and reduce the damage of nanoscale enzyme to normal human cells is also an important scientific problem that must be solved in the research of nanoscale enzyme treatment, which is of great significance to the rapid healing of wounds.

[0003] In the field of nanoscale enzyme, cerium-based nanoscale enzyme has been widely concerned at home and abroad due to its unique physicochemical properties. Among them, CeO2 has mixed valence of Ce 3+ and Ce 4+ and oxygen vacancies. Different valences can switch with each other to obtain better catalytic activity. It has oxidation and antioxidant properties for different reaction substrates under different reaction conditions. This feature makes CeO2 have catalytic properties and various artificial enzyme activities, including oxidase, catalase, peroxidase, superoxide dismutase, and even phosphatase activity. It has been reported that cerium oxide nanoparticles with a diameter of less than 5 nm exhibit high ROS scavenging activity. However, common CeO2 nanoscale enzyme preparations are combined with noble metals and CeO2 nanoparticles, such as the Pt / CeO2 nanoscale enzyme particles disclosed in Chinese patent document CN118304433A. It includes single-atom Pt and CeO2 nanoparticles combined therewith. The Pt / CeO2 nanoparticles are modified by PEG. The nanoscale enzyme has unique catalytic properties, especially in ROS scavenging and inflammation inhibition. SUMMARY

[0004] In order to overcome at least one of the problems existing in the prior art, the present application provides a CeO2 core-shell nanoscale enzyme and a preparation method and application thereof. The nanoscale enzyme is particularly suitable for ROS scavenging and inflammation inhibition in diabetic wounds.

[0005] In one aspect of the present application, a core-shell nanoscale enzyme is provided, which is composed of a spherical CeO2 nanoparticle core, a zeolitic imidazolate framework material ZIF-8 shell, and tannic acid TA coated on the shell.

[0006] Zeolite imidazole ester framework material, ZIF-8, is a metal-organic framework (MOF) with non-toxicity and good biocompatibility. Furthermore, the tannic acid (TA) on the ZIF-8 shell can further endow nanozymes with additional bioactivity, such as antibacterial, anti-inflammatory, and antioxidant properties. CeO2 surface Ce 3+ and Ce 4+ Rapid interconversion between these components exhibits excellent redox and self-regeneration properties. Furthermore, it possesses various enzymatic activities, including oxidase, catalase, peroxidase, superoxide dismutase, and even phosphatase activities. Spherical CeO2 nanoparticles have abundant surface oxygen vacancies, which can be converted through Ce... 4+ / Ce 3+ The reversible process between these processes is used to regulate the activity, endowing it with strong antioxidant capabilities. Simultaneously, the spherical CeO2 nanoparticles, due to their regular morphology and nanoscale size, possess extremely high specific surface area, resulting in more efficient reactivity. The spherical CeO2 nanoparticles are tightly bonded to the shell material, ultimately causing the resulting core-shell nanozyme to also exhibit a spherical shape. The spherical CeO2, as the core component, is encapsulated within ZIF-8, temporarily restricting its SOD and various enzyme activities. However, as the ZIF-8 structure gradually decomposes in the acidic microenvironment, the CeO2 core is gradually released, exerting its SOD and nanozyme activities, eliminating excess ROS generated at the wound site, and exhibiting a sustained-release effect. Coating with tannins can improve the biocompatibility of the nanozyme and also enhance the dispersibility of the nanoparticles.

[0007] Preferably, the core-shell nanozyme C@Z@T has a particle size of 20-700 nm and a zeta potential of -26.9 ± 0.721 mV, exhibiting better dispersibility and a more stable system.

[0008] Another aspect of the present invention provides a method for preparing the aforementioned core-shell nanozyme, comprising the steps of:

[0009] Preparation of S1 CeO2@ZIF-8 nanoparticles;

[0010] Spherical CeO2 nanoparticles and PVP were dissolved separately in organic solvents and mixed evenly. The product was then centrifuged and immediately redispersed in a 2-methylimidazole / organic solvent solution until completely mixed. Subsequently, zinc salt was added and the mixture was reacted to obtain the CeO2@ZIF-8 nanoparticles.

[0011] Preparation of S2 CeO2@ZIF-8@TA nanozymes;

[0012] The prepared CeO2@ZIF-8 nanoparticles were evenly dispersed in water, and then tannic acid (TA) was added and evenly dispersed. After centrifugation, washing, and drying, the core-shell nanozyme was obtained.

[0013] Preferably, in the step S1, the CeO2 nanoparticles: PVP = 30-40 mg: 0.5-1.5 g.

[0014] Preferably, in the step S2, the CeO2@ZIF-8 nanoparticles: tannic acid = 0.1-0.5 g: 0.05-0.15 g.

[0015] Preferably, the reaction temperature in the step S1 is 24-37℃, and the reaction temperature in the step S2 is 25-35℃.

[0016] Preferably, the spherical CeO2 nanoparticles are prepared by a hydrothermal method.

[0017] Further preferably, the preparation method of the spherical CeO2 nanoparticles comprises the following steps: adding cerium nitrate salt into ethylene glycol, adding ultra-pure water dropwise, dispersing at room temperature, then hydrothermal synthesis at 180℃ for 8-12h, centrifugal washing, drying at 110℃, and then calcining the dried product at 450-600℃, wherein Ce ion: ethylene glycol: ultra-pure water = 4-5mmol: 75-100mL: 2-6mL.

[0018] In still another aspect of the present application, the use of the core-shell nanoscale enzyme in the preparation of wound dressings is also provided, which is particularly suitable for diabetic wounds. Preferably, the concentration of the core-shell nanoscale enzyme in the wound dressing is 100-300μg / mL, and further preferably, 200μg / mL.

[0019] Compared with the prior art, the present application has the following beneficial effects:

[0020] The CeO2 nanoscale enzyme of the present application uses a metal organic framework material as a framework, and the spherical CeO2 nanoparticles in the core are placed therein and can be gradually released with the decomposition of the metal organic framework, thereby exerting its SOD and nanoscale enzyme activity to eliminate the excess ROS generated at the wound surface. The tannic acid coated on the outside of the framework can improve the biocompatibility of the material, and also can significantly improve the dispersibility of the nanoscale enzyme. The nanoscale enzyme of the present application can spontaneously activate the production and removal function of ROS, balance the steady state of ROS, and is suitable for wound healing, and particularly suitable for diabetic wound healing. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 TEM image of C@Z@T of the embodiment of the present application;

[0022] Figure 2 Particle size distribution graph of C@Z@T of the embodiment of the present application;

[0023] Figure 3 Zeta potential of each nanoparticle of the embodiment of the present application. DETAILED DESCRIPTION

[0024] For the purposes of this disclosure, reference will be made to the accompanying drawings which form a part of the disclosure. The drawings are not necessarily to scale of proportion and are exemplary of numerous embodiments of the disclosure. It is to be understood that the examples are illustrative of various embodiments of the present application and are not intended to limit the scope of the present application, as claimed.

[0025] In the description of the present application, unless otherwise explicitly defined, the words heating, cleaning, weighing, freezing, etc. should be understood in a broad sense, and those skilled in the art can reasonably determine the specific meaning of the above words in the present application in combination with the specific content of the technical solution.

[0026] In the description of the present application, the description of the terms "some embodiments", "examples", etc. means that the specific methods, materials described in combination with the embodiments or examples are included in at least one embodiment or example of the present application. In the present specification, the illustrative description of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific methods, materials described can be combined in any one or more embodiments or examples in a suitable manner.

[0027] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description of the application herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

[0028] In the following examples and comparative examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are commercially available unless otherwise specified.

[0029] Example 1

[0030] The present embodiment provides a core-shell nanozyme composed of a spherical CeO2 nanoparticle core and a zeolitic imidazolate framework material ZIF-8 shell, and tannic acid TA coated on the shell. The specific preparation method includes the following steps:

[0031] (1) Preparation of CeO2@ZIF-8 nanoparticles

[0032] CeO2@ZIF-8@TA nanoparticles were prepared by adding 0.3 g of prepared CeO2@ZIF-8 into 100 mL of water and stirring for 30 min to uniformly disperse. 0.1 g of tannic acid (TA) was added into the solution and stirred for 10 min at 30 °C. The precipitate was centrifuged and washed with deionized water for several times to remove the residual TA. Then, the wet CeO2@ZIF-8@TA nanoparticles were dried at 60 °C for 12 h to obtain the CeO2@ZIF-8@TA nanoparticles, denoted as C@Z@T.

[0033] (2) Preparation of CeO2@ZIF-8@TA nanoparticles

[0034] CeO2@ZIF-8@TA nanoparticles were prepared by adding 0.3 g of prepared CeO2@ZIF-8 into 100 mL of water and stirring for 30 min to uniformly disperse. 0.1 g of tannic acid (TA) was added into the solution and stirred for 10 min at 30 °C. The precipitate was centrifuged and washed with deionized water for several times to remove the residual TA. Then, the wet CeO2@ZIF-8@TA nanoparticles were dried at 60 °C for 12 h to obtain the CeO2@ZIF-8@TA nanoparticles, denoted as C@Z@T.

[0035] The preparation methods of the spherical CeO2 and ZIF-8 nanoparticles used in this example are as follows:

[0036] (1) Preparation of spherical CeO2

[0037] Ce(NO3)2.6H2O (2 g) was added into 80 mL of ethylene glycol, and 4 mL of ultrapure water was added dropwise. The mixture was stirred vigorously at room temperature for 30 min. The mixed solution was transferred to a polytetrafluoroethylene liner, and a stainless steel crystallization kettle was sealed with a cover. The solution was hydrothermally synthesized at 180 °C for 8 h. The solution was centrifuged and washed repeatedly with ultrapure water, and then dried in an oven at 110 °C overnight. The solid was ground into powder and placed in a muffle furnace. The temperature was raised to 500 °C at a rate of 2 °C / min in air, and calcined for 6 h to obtain spherical CeO2 nanoparticles.

[0038] (2) Preparation of ZIF-8 nanoparticles

[0039] 3.3 g of 2-methylimidazole was dissolved in 70 mL of methanol solution to form solution A. Then, 1.5 g of Zn(NO3)2.6H2O was dissolved in 70 mL of methanol solution to form solution B. Solution A was slowly added to solution B to form a mixed solution, which was stirred at room temperature for 24 h and centrifuged. The obtained solid was washed with methanol solution for 3 times and dried at 80 °C overnight. The obtained sample was denoted as ZIF-8.

[0040] Figure 1The transmission electron microscope image of the prepared C@Z@T nanoparticles can be seen, since the core cerium oxide is spherical, the prepared C@Z@T nanoparticles also present uniform spherical shape, and the size is uniform. Figure 2 The particle size distribution of the C@Z@T nanoparticles can be seen, the particle size is between 20-700nm, and the average particle size is 214±8.246nm.

[0041] Figure 3 For the Zeta potential statistics of each nanoparticle in the example, including CeO2, ZIF-8, C@Z, C@Z@T, it can be seen that the potentials of the separate CeO2 and ZIF-8 nanoparticles are-7.56±1.58mV and 23.8±1.65mV respectively. The composite nanoparticles C@Z@T of the embodiment of the application present negative charge as a whole due to the negative charge of tannic acid, and the Zeta potential is significantly improved to-26.9±0.721mV after coating with tannic acid, which shows that the electrostatic repulsion between particles is increased, and the stability is significantly improved. The C@Z@T nanoparticles of the application have good dispersibility.

[0042] Comparative Example 1

[0043] A nano-enzyme, which is the spherical CeO2 nanoparticle in Example 1.

[0044] Comparative Example 2

[0045] A nano-enzyme, which is the ZIF-8 nanoparticle in Example 1.

[0046] Comparative Example 3

[0047] A nano-enzyme, which is the CeO2@ZIF-8 nanoparticle in Example 1.

[0048] Test Example:

[0049] The nano-enzymes prepared in the examples and comparative examples are respectively configured into a solution with a set concentration of 40-300μg / mL using PBS (pH=7.4) for subsequent testing.

[0050] Table 1 Test Example Nano-enzyme Concentration Table

[0051] Number Comparative Example 1 Comparative Example 2 Comparative Example 3 Example 1 Nanocatalyst type CeO2 ZIF-8 CeO2@ZIF-8 C@Z@T Test Example 1 / / / 40 pg / mL Test Example 2 / / / 80 pg / mL Test Example 3 / / / 100 pg / mL Test Example 4 / / / 200 pg / mL Test Example 5 300 pg / mL Test Example 6 40 pg / mL / / / Test Example 7 300 pg / mL / / / Test Example 8 / 40 pg / mL / / Test Example 9 / 300 pg / mL / / Test Example 10 / / 40 pg / mL / Test Example 11 / / 300 pg / mL /

[0052] 1. In vitro antioxidant effect analysis of nano-enzyme

[0053] Table 2 DPPH free radical scavenging rate (%)

[0054]

[0055] Test method: Prepare the sample according to the instructions of the commercially available DPPH free radical scavenging ability detection kit. Add the extract of the corresponding examples and comparative examples to the DPPH solution, and then incubate in the dark for 30 minutes. Detect the absorbance at 515 nm by ultraviolet-visible spectrophotometer, and calculate the free radical scavenging rate.

[0056] The results show that: due to the multiple phenolic hydroxyl groups (-OH) in the molecular structure of TA, it has strong hydrogen-donating ability, and therefore has outstanding performance in antioxidant activity of scavenging free radicals. CeO2 has suitable oxidation vacancies and the ability to quickly convert Ce ions between tetravalent and trivalent ions. Therefore, in an acidic environment, CeO2 can exert the activity of a peroxidase. Therefore, in the C@Z@T nanoparticles of the examples of the present application, TA and CeO2 jointly exhibit good free radical scavenging ability.

[0057] 2. Analysis of antibacterial performance of nanoscale enzyme

[0058] Table 3 Antibacterial rate (%)

[0059]

[0060] Test method: After recovering Escherichia coli and Staphylococcus aureus, they were cultured to the logarithmic growth phase, centrifuged, and then resuspended in physiological saline. The final concentration of the bacterial solution was 1×10 8 CFU / mL. 500 μL of the corresponding concentration of nanoscale enzyme solution was added to a 24-well plate. 500 μL of physiological saline was added to the control group. Then 100 μL of diluted bacterial suspension and 500 μL of sterile physiological saline were added to each well, and incubated at 37°C for 24 hours. After gradient dilution, LB agar plates were coated, and the number of colonies was counted.

[0061] The results show that: due to the unique mixed valence state, CeO2 nanoparticles have a unique antibacterial mechanism. The reversible conversion between the two states is a self-renewal cycle (Ce 4+ → Ce 3+ → Ce 4+ ) that continues on the surface of the nanoparticles. Therefore, CeO2 nanoparticles have broad-spectrum antibacterial activity against bacteria and fungi. In the C@Z@T of the examples of the present application, ZIF-8 can release Zn 2+ and CeO2 drugs in a slightly acidic environment, exerting antibacterial effects. In addition, the outer coating of TA is a natural polyphenol antibacterial small molecule with antioxidant, antibacterial, anti-inflammatory and biodegradable effects, and can be used for antibiotic-free antibacterial strategies. Its antibacterial activity can be exerted through multiple pathways, such as destroying the stability of the cell membrane, penetrating the cell membrane, or inhibiting enzyme activity. Therefore, C@Z@T has excellent and unique antibacterial effects.

[0062] 3. Nanoscale enzyme biocompatibility evaluation

[0063] Table 4 Cell survival rate (%)

[0064] Test Example 1 2 3 4 5 6 7 8 9 10 11 L929 cell % 99.99 99.15 99.06 98.59 97.56 99.52 97.55 99.58 97.1 99.88 98.71 293T cell % 99.56 98.45 98.16 95.17 94.59 99.35 96.68 99.55 97.31 99.25 94.38

[0065] Test method: The nanoscale enzyme was sterilized by irradiation with ultraviolet light for 24 hours in advance. Then different concentrations of nanoscale enzyme solution were prepared with culture medium for subsequent cell culture experiments. L929 / 293T cells were digested with 0.25% trypsin, resuspended in DMEM, and inoculated in a 48-well plate at a density of 10000 / cell per well, and incubated in a 37℃ constant temperature carbon dioxide incubator (containing 5% CO2) for 24 hours until the cells adhered. The culture medium was aspirated and washed with PBS, and the corresponding concentration of nanoscale enzyme solution was added for continuous culture for 24 hours. After the culture was completed, the culture medium was aspirated and washed with PBS for 3 times, 500 μL of CCK-8 working solution (containing 10% CCK-8 DMEM basic culture medium) was added to each well, and incubated in a 37℃ constant temperature carbon dioxide incubator (containing 5% CO2) for 30 min. Then the absorbance (OD) was measured at 450 nm wavelength by an enzyme marker, and the cell survival rate was calculated according to the formula: cell survival rate (%) = (OD 实验组 -OD Blank ) / (OD 对照组 -OD Blank ) x 100%.

[0066] The results show that the core-shell nanoscale enzyme C@Z@T prepared in the embodiments has acceptable cell biocompatibility.

[0067] As can be seen from the above, the core-shell nanoscale enzyme C@Z@T of the present application has good catalytic performance, good dispersibility, and stable system, and the components cooperate with each other, significantly improving the antibacterial and antioxidant effects of single CeO2 nanoparticles, and having good synergistic support effect between components. The core-shell nanoscale enzyme of the present application can be applied to wound dressings, and is especially suitable for diabetic chronic wounds.

[0068] The technical features of the above-described embodiments can be combined in any manner. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described, but as long as the combinations of the technical features do not exist contradictions, they should be considered as the scope of the present disclosure.

[0069] The above embodiments only express several implementation manners of the present application, and the description is more specific and detailed, but it should not be understood as a limitation on the scope of the patent. It should be noted that for ordinary skilled persons in the art, without departing from the concept of the present application, several modifications and improvements can be made, which are within the scope of protection of the present application. Therefore, the protection scope of the patent of the present application should be subject to the appended claims.

Claims

1. A core-shell nanoszyme, characterized in that, The core-shell nanoscale enzyme is composed of a spherical CeO2 nanoparticle core and a zeolitic imidazolate framework ZIF-8 shell, and tannic acid TA coated on the shell. The core-shell nanoscale enzyme is prepared by the following steps: S1. Preparation of CeO2@ZIF-8 nanoparticles; The spherical CeO2 nanoparticles and PVP are dissolved in an organic solvent respectively, mixed uniformly, then the product is centrifuged and immediately dispersed in a 2-methyl imidazole / organic solvent solution until completely mixed; then, zinc salt is added and mixed to react, to obtain the CeO2@ZIF-8 nanoparticles; S2. Preparation of CeO2@ZIF-8@TA nanoscale enzyme; The prepared CeO2@ZIF-8 nanoparticles are dispersed uniformly in water, then tannic acid TA is added and dispersed uniformly, then centrifuged, washed, dried, to obtain the core-shell nanoscale enzyme CeO2@ZIF-8@TA; In the step S1, the CeO2 nanoparticles: PVP = 30-40 mg: 0.5-1.5 g; In the step S2, the CeO2@ZIF-8 nanoparticles: tannic acid = 0.1-0.5 g: 0.05-0.15 g; In the step S1, the reaction temperature is 24-37℃, and in the step S2, the reaction temperature is 25-35℃; The particle size of the core-shell nanoscale enzyme is 20-700 nm, and the zeta potential is -26.9±0.721 mV. The preparation method of the spherical CeO2 nanoparticles comprises the following steps: adding cerium nitrate salt into ethylene glycol, adding ultrapure water dropwise, dispersing fully at room temperature, then hydrothermal synthesis at 180℃ for 8-12 h, centrifugation, washing, drying at 110℃, then calcining the dried product at 450-600℃, wherein Ce ion: ethylene glycol: ultrapure water = 4-5 mmol: 75-100 mL: 2-6 mL.

2. The use of the core-shell nanoscale enzyme according to claim 1 in the preparation of a wound dressing.

3. Use according to claim 2, characterized in that, The concentration of the core-shell nanoscale enzyme in the wound dressing is 100-300 μg / mL.

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