A meso-entropy nano-enzyme with targeting effect and a preparation method and application thereof

Abstract

The application discloses a kind of medium-entropy nano-enzyme with targeting effect and its preparation method and application, and preparation method specifically includes the following steps: (1) water, anhydrous ethanol and concentrated ammonia are mixed, stirring, adding polyphenol aqueous solution, stirring again, adding medium-entropy alloy ethanol solution, continue stirring, centrifugation, collect precipitate, wash, freeze-drying;(2) calcination, cooling, standby;(3) tumor cells are sequentially digested, centrifugation, collect precipitate, wash, resuspend, lysis, lysis again;(4) centrifugation, take supernatant, centrifugation again, take supernatant again, ultracentrifugation, obtain cell membrane precipitate, standby;(5) preparation of aqueous solution, mixing, ultrasonic treatment, centrifugation, collect precipitate, wash, obtain.The medium-entropy nano-enzyme has good peroxidase-like activity, can effectively catalyze hydrogen peroxide in tumor microenvironment to generate highly toxic hydroxyl radical, kill cancer cells, and has good tumor treatment prospect.

Description

Technical Field

[0001] This invention relates to the field of functional materials technology, and more specifically to a medium-entropy nanozyme with targeting function, its preparation method, and its application. Background Technology

[0002] In recent years, biomimetic materials have shown great potential in the biomedical field, among which artificial enzyme technology has attracted much attention due to its unique advantage of being able to replace natural enzymes. As a type of artificial enzyme that combines the physicochemical properties of nanomaterials with the catalytic function of biological enzymes, nanozymes have made significant progress in fields such as tumor catalytic therapy and environmental remediation since Fe3O4 nanoparticles were first shown to have peroxidase-like activity.

[0003] Currently, metal-based nanozymes are considered ideal candidates to replace natural enzymes due to their highly efficient catalytic properties. However, existing metal-based nanozymes still have significant drawbacks: firstly, their catalytic activity is limited by the singular electronic structure of the single metal or bimetallic component, making it difficult to achieve efficient substrate activation; secondly, nanoparticles are prone to aggregation and inactivation due to excessively high surface energy; and thirdly, they lack targeted delivery capabilities, resulting in low drug enrichment efficiency at tumor sites and potentially causing toxic damage to normal tissues. These problems severely restrict the clinical translational application of nanozymes.

[0004] Medium / high entropy alloys are composed of three or more metallic elements in near equimolar ratios. Their multi-component synergistic effect can significantly optimize the electronic structure of the material, forming abundant catalytic active sites. At the same time, the high entropy effect can enhance the thermodynamic stability of the material. Therefore, medium / high entropy alloys have performance advantages that surpass those of traditional single-metal or bimetallic materials.

[0005] However, the application of medium / high entropy alloys in the biomedical field still faces multiple challenges: for example, the preparation process is complex and it is difficult to control the particle size; conventional medium / high entropy alloys lack biocompatible modifications to achieve targeted delivery; and the stable synthesis technology of ultra-small (<5nm) medium / high entropy nanozymes has not yet been broken through, which greatly limits their catalytic efficiency and potential for in vivo application.

[0006] Therefore, how to develop a medium / high entropy alloy nanozyme with targeting function is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0007] In view of this, the purpose of the present invention is to provide a medium-entropy nanozyme with targeting function, its preparation method and application, so as to overcome the shortcomings of the prior art.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] A method for preparing a medium-entropy nanozyme with targeting function specifically includes the following steps:

[0010] (1) Mix water, anhydrous ethanol and concentrated ammonia, stir, add polyphenolic aqueous solution, stir again, add medium entropy alloy ethanol solution, continue stirring, centrifuge, collect precipitate, wash, freeze dry to obtain medium entropy nanozyme precursor.

[0011] (2) The medium-entropy nanozyme precursor was calcined and cooled sequentially to obtain medium-entropy nanozyme for later use.

[0012] (3) The tumor cells were digested, centrifuged, the precipitate was collected, washed, resuspended, lysed, and lysed again to obtain cell lysate;

[0013] (4) Centrifuge the cell lysate sequentially, take the supernatant, centrifuge again, take the supernatant again, and ultracentrifuge to obtain cell membrane precipitate for later use.

[0014] (5) Medium-entropy nanozymes and cell membrane precipitates were prepared into medium-entropy nanozyme aqueous solution and cell membrane precipitate aqueous solution, respectively. They were mixed sequentially, ultrasonically treated, centrifuged, the precipitates were collected and washed to obtain medium-entropy nanozymes with targeting effects.

[0015] Furthermore, in step (1) above, the ratio of water, anhydrous ethanol, concentrated ammonia, polyphenolic aqueous solution, and medium-entropy alloy ethanol solution is 90 mL: 40 mL: 3 mL: 10 mL: 10 mL; the polyphenolic aqueous solution is an aqueous solution of dopamine hydrochloride with a concentration of 50 mg / mL; the medium-entropy alloy ethanol solution is an ethanol solution of medium-entropy alloy; the medium-entropy alloy is ruthenium acetylacetonate, iridium acetylacetonate, platinum acetylacetonate, molybdenum acetylacetonate, and rhodium acetylacetonate. The mixture contains at least two of palladium acetylacetone, iron acetylacetone, and copper acetylacetone, preferably ruthenium acetylacetone, iridium acetylacetone, platinum acetylacetone, and molybdenum acetylacetone; the mass ratio of ruthenium acetylacetone, iridium acetylacetone, platinum acetylacetone, molybdenum acetylacetone, and dopamine hydrochloride is (0.02-0.1):(0.02-0.1):(0.02-0.1):(0.02-0.1):1, preferably 0.1:0.1:0.1:0.1:1.

[0016] Furthermore, in step (1) above, the stirring temperature is room temperature, the stirring speed is 600-800 r / min, and the time is 15 min; the stirring time is 1-2 h; the stirring time is 12-24 h; the centrifugation speed is 12000 r / min, and the time is 10-20 min; the washing reagent is ethanol, and the number of times is 3; the freeze-drying time is 24-36 h.

[0017] Furthermore, in step (2) above, the calcination equipment is a tube furnace, the atmosphere is nitrogen, the temperature is 900℃, and the time is 2h.

[0018] Furthermore, in step (3) above, the digesting enzyme is trypsin; the washing reagent is PBS buffer, and the number of times is 2; the lysis temperature is 4℃ and the time is 1h; the conditions for the second lysis are ice bath, the equipment is a cell disruptor with a power of 60W, and the time is 5min.

[0019] Furthermore, in step (4) above, the centrifugation speed is 4000 rpm and the time is 10 min; the centrifugation speed is 12000 rpm and the time is 15 min; the ultracentrifugation speed is 120000 rpm and the time is 1 h.

[0020] Furthermore, in step (5) above, the concentration of the medium-entropy nanozyme aqueous solution is 1 mg / mL; the concentration of the cell membrane precipitation aqueous solution is 1 mg / mL; and the volume ratio of the medium-entropy nanozyme aqueous solution to the cell membrane precipitation aqueous solution is 1:2.

[0021] Furthermore, in step (5) above, the ultrasonic power of the ultrasonic treatment is 250W and the time is 20min; the centrifugation speed is 12000rpm and the time is 15min; the washing reagent is deionized water and the number of times is 3.

[0022] The present invention also claims protection for a medium-entropy nanozyme with targeting function prepared by the above preparation method.

[0023] This invention also claims the application of a targeted medium-entropy nanozyme prepared by the above-described method in the preparation of antitumor nanomedicines.

[0024] As can be seen from the above technical solution, compared with the prior art, the beneficial effects of the present invention are as follows:

[0025] 1. This invention constructs an ultra-small targeted medium-entropy nanozyme system by combining a metal-ligand crosslinking strategy with homologous cancer cell membrane encapsulation technology. For the first time, it achieves atomic-level uniform dispersion of ruthenium, platinum, iridium, and molybdenum, forming stable medium-entropy nanozymes with a particle size of 2-3 nm. Their high specific surface area and multi-metal synergistic effect significantly enhance enzyme-like catalytic activity. Furthermore, through biomimetic modification with homologous cancer cell membranes, the nanozymes are endowed with biocompatibility and the ability to actively target tumors. The targeted medium-entropy nanozymes encapsulated in homologous cell membranes of this invention can efficiently catalyze the conversion of hydrogen peroxide in the tumor microenvironment into highly toxic hydroxyl radicals, achieving precise tumor suppression through ROS-mediated DNA damage, apoptosis, and autophagy pathways, while maintaining controllable system toxicity. This technological breakthrough not only overcomes the dual challenges of controllable preparation and targeted delivery of medium-entropy nanozymes but also provides a novel technical pathway for the catalytic therapy of malignant tumors.

[0026] 2. This invention uses dopamine hydrochloride, acetylacetone metal salt, and cancer cell membranes as raw materials, and dopamine nanozymes as a carrier. A medium-entropy nanozyme precursor is generated through oxidative self-polymerization, followed by high-temperature calcination to obtain a quaternary metal medium-entropy nanozyme with ultra-small active centers. Subsequently, the cancer cell membrane is uniformly coated onto the surface of the medium-entropy nanozyme through self-assembly. The resulting medium-entropy nanozyme exhibits excellent peroxidase-like activity, effectively catalyzing the generation of highly toxic hydroxyl radicals from hydrogen peroxide in the tumor microenvironment, thereby killing cancer cells. Furthermore, this nanozyme, through biomimetic modification with homologous cancer cell membranes, possesses excellent biocompatibility and the ability to actively target tumor tissue, effectively enhancing nanozyme accumulation at the tumor site. It can rapidly and efficiently exert therapeutic effects in the tumor microenvironment, and the nanozyme exhibits high biosafety, showing great promise for tumor treatment. Attached Figure Description

[0027] Figure 1 The images show high-resolution transmission electron microscopy (HR-TEM), high-angle annular dark-field scanning transmission electron microscopy (HADDF-STEM), and X-ray energy dispersive spectroscopy (EDS-mapping) images of the targeted medium-entropy nanozyme prepared in Example 1 of this invention.

[0028] Figure 2 This refers to the peroxidase-like activity of the medium-entropy nanozyme with targeting function prepared in Example 1 of the present invention. Detailed Implementation

[0029] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0030] Example 1

[0031] The preparation method of a targeted medium-entropy nanozyme specifically includes the following steps:

[0032] (1) Mix 90 mL of water, 40 mL of anhydrous ethanol and 3 mL of concentrated ammonia, and stir at 600 r / min for 15 min at room temperature. Add 10 mL of 50 mg / mL dopamine hydrochloride aqueous solution, stir again for 2 h, and add dropwise 10 mL of 5 mg / mL ethanol solutions of ruthenium acetylacetonate, iridium acetylacetonate, platinum acetylacetonate and molybdenum acetylacetonate. Continue stirring for 12 h, centrifuge at 12000 r / min for 15 min, collect the precipitate, wash three times with ethanol, freeze dry for 24 h to obtain the intermediate entropy nanozyme precursor.

[0033] (2) The medium-entropy nanozyme precursor was transferred to a tube furnace and calcined at 900℃ for 2 hours at a rate of 10℃ / min in a nitrogen atmosphere. After cooling, the medium-entropy nanozyme was obtained and set aside.

[0034] (3) The cultured tumor cells were digested with trypsin, centrifuged, the precipitate was collected, washed twice with PBS buffer, and the tumor cells were resuspended in lysis buffer. The cells were first lysed at 4°C for 1 h, and then the cells were fully lysed at 60 W for 5 min on ice to obtain cell lysis buffer.

[0035] (4) Centrifuge the cell lysis buffer at 4000 rpm for 10 min, take the supernatant, centrifuge again at 12000 rpm for 15 min, take the supernatant again, and ultracentrifuge at 120000 rpm for 1 h to obtain cell membrane precipitate for later use.

[0036] (5) Prepare medium-entropy nanozymes and cell membrane precipitates into aqueous solutions of medium-entropy nanozymes with a concentration of 1 mg / mL and aqueous solutions of cell membrane precipitates with a concentration of 1 mg / mL, respectively. Mix them at a volume ratio of 1:2, sonicate them with an ultrasonic power of 250W for 20 min, and then centrifuge them at a speed of 12000 rpm for 15 min. Collect the precipitates and wash them three times with deionized water to obtain medium-entropy nanozymes with targeting effects.

[0037] Performance testing

[0038] 1. Characterization using HR-TEM, HADDF-STEM, and EDS-mapping

[0039] The high-resolution transmission electron microscope (HR-TEM) image of the targeted medium-entropy nanozyme prepared in Example 1 is shown below. Figure 1 As shown in Figure A, the high-angle annular dark-field scanning-transmission electron microscope (HADDF-STEM) image is as follows. Figure 1 As shown in BC, the X-ray energy dispersive spectroscopy (EDS-mapping) diagram is as follows. Figure 1 As shown in D.

[0040] Depend on Figure 1 It can be seen that the cancer cell membrane is uniformly wrapped on the surface of the medium-entropy nanoenzyme, and the four metal elements are evenly distributed with a size of about 2-3 nm.

[0041] 2. Study on peroxidase-like activity

[0042] The targeted medium-entropy nanozyme prepared in Example 1 was used to study its peroxidase-like activity. The steps are as follows:

[0043] (1) Disperse the medium-entropy nanozyme in deionized water to a concentration of 2 mg / mL, and treat it with ultrasonic power of 250 W for 30 min to obtain a medium-entropy nanozyme aqueous solution.

[0044] (2) Mix 2 μL of medium entropy nanozyme aqueous solution, 20 μL of hydrogen peroxide solution, 20 μL of TMB ethanol solution with a concentration of 5 mg / mL, 2 mL of HAc-NaAc buffer (pH=4.5) and 2 μL of DMPO solution with a concentration of 1 M evenly. After vortexing the centrifuge tube, wait for 5 min to obtain the mixed solution.

[0045] (3) The mixed solution was drawn into a quartz capillary tube for electron paramagnetic resonance (EPR) spectroscopy analysis to detect peroxidase-like activity.

[0046] The results are as follows Figure 2 As shown.

[0047] Depend on Figure 2 It is evident that the targeted medium-entropy nanozyme prepared in Example 1 exhibits highly efficient peroxidase-like activity, capable of rapidly catalyzing hydrogen peroxide to generate hydroxyl radicals, thereby killing cancer cells.

[0048] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing a medium-entropy nanozyme with targeting effect, characterized in that, Specifically, the following steps are included: (1) Mix water, anhydrous ethanol and concentrated ammonia, stir, add polyphenolic aqueous solution, stir again, add medium entropy alloy ethanol solution, continue stirring, centrifuge, collect precipitate, wash, freeze dry to obtain medium entropy nanozyme precursor. The volume ratio of water, anhydrous ethanol, concentrated ammonia, polyphenol aqueous solution, and medium entropy alloy ethanol solution is 90 mL: 40 mL: 3 mL: 10 mL: 10 mL; The polyphenolic substance aqueous solution is an aqueous solution of dopamine hydrochloride with a concentration of 50 mg / mL; The medium-entropy alloy ethanol solution is an ethanol solution of a medium-entropy alloy; The medium-entropy alloy is ruthenium acetylacetonate, iridium acetylacetonate, platinum acetylacetonate, and molybdenum acetylacetonate; the mass ratio of ruthenium acetylacetonate, iridium acetylacetonate, platinum acetylacetonate, molybdenum acetylacetonate, and dopamine hydrochloride is 0.1:0.1:0.1:0.1:

1. (2) The medium-entropy nanozyme precursor was calcined and cooled sequentially to obtain medium-entropy nanozyme for later use. (3) The tumor cells were digested, centrifuged, the precipitate was collected, washed, resuspended, lysed, and lysed again to obtain cell lysate; (4) Centrifuge the cell lysate sequentially, take the supernatant, centrifuge again, take the supernatant again, and ultracentrifuge to obtain cell membrane precipitate for later use. (5) The medium-entropy nanozyme and cell membrane precipitate were respectively prepared into medium-entropy nanozyme aqueous solution and cell membrane precipitate aqueous solution, respectively, and then mixed, sonicated, centrifuged, collected, and washed to obtain the medium-entropy nanozyme with targeting effect. The concentration of the medium-entropy nanoenzyme aqueous solution is 1 mg / mL; the concentration of the cell membrane precipitation aqueous solution is 1 mg / mL; the volume ratio of the medium-entropy nanoenzyme aqueous solution to the cell membrane precipitation aqueous solution is 1:2; The particle size of the targeted medium-entropy nanozyme is 2-3 nm.

2. The method for preparing a targeted medium-entropy nanozyme according to claim 1, characterized in that, In step (1), the stirring temperature is room temperature, the stirring speed is 600-800 r / min, and the time is 15 min; the stirring time again is 1-2 h; the stirring time is 12-24 h; the centrifugation speed is 12000 r / min, and the time is 10-20 min; the washing reagent is ethanol, and the washing is performed 3 times; the freeze-drying time is 24-36 h.

3. The method for preparing a targeted medium-entropy nanozyme according to claim 1, characterized in that, In step (2), the calcination equipment is a tube furnace, the atmosphere is nitrogen, the heating rate is 10℃ / min, the temperature is 900℃, and the time is 2h.

4. The method for preparing a targeted medium-entropy nanozyme according to claim 1, characterized in that, In step (3), the digesting enzyme is trypsin; the washing reagent is PBS buffer, and the washing is performed twice; the lysis temperature is 4°C and the time is 1 hour; the conditions for the second lysis are an ice bath, the equipment is a cell disruptor with a power of 60W, and the time is 5 minutes.

5. The method for preparing a targeted medium-entropy nanozyme according to claim 1, characterized in that, In step (4), the centrifugation speed is 4000 rpm and the time is 10 min; the second centrifugation speed is 12000 rpm and the time is 15 min; the ultracentrifugation speed is 120000 rpm and the time is 1 h.

6. The method for preparing a targeted medium-entropy nanozyme according to claim 1, characterized in that, In step (5), the ultrasonic power of the ultrasonic treatment is 250W and the time is 20min; the centrifugation speed is 12000rpm and the time is 15min; the washing reagent is deionized water and the number of times is 3.

7. A medium-entropy nanozyme with targeting function prepared by the preparation method according to any one of claims 1-6.

8. The application of a targeted medium-entropy nanozyme prepared by the preparation method according to any one of claims 1-6 in the preparation of antitumor nanomedicines.

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