Medium-entropy nano-enzyme with targeting effect as well as preparation method and application of medium-entropy nano-enzyme
By preparing medium-entropy nanoenzymes with particle size 2-3 nm, combined with cancer cell membrane coating technology, the catalytic activity limitation and targeted delivery of metal-based nanoenzymes were solved, and efficient tumor treatment effect and biosafety were achieved.
Patent Information
- Application Number
- CN202510380361.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-28
- Publication Date
- 2025-07-08
AI Technical Summary
The existing metal-based nanoenzymes have limited catalytic activity, easy agglomeration, and lack targeted delivery capabilities, resulting in low drug enrichment efficiency in tumor sites and may cause toxic damage to normal tissues, limiting their clinical transformation and application in the field of biomedical science.
Metal-ligand cross-linking strategy and homologous cancer cell membrane coating technology were used to prepare medium-entropy nanoentropy nanozymes with particle size 2-3nm. Through high-temperature calcination and self-assembly, a targeting medium-entropy nanozyme was formed, and biomimetic modification of cancer cell membranes was used to improve biocompatibility and tumor targeting.
It realizes the efficient catalytic activity and targeted delivery of medium-entropy nanoenzymes in the tumor microenvironment, which can efficiently catalyze the formation of highly toxic hydroxyl radicals, achieving precise tumor inhibition effect, and at the same time, it has high biosafety, improving the tumor treatment effect.
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Figure CN120267696A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of functional materials, and more specifically, to a targeted medium-entropy nanozyme and its preparation method and application. Background Art
[0002] In recent years, biomimetic materials have shown great potential in the biomedical field, and among them, 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 and the catalytic functions of biological enzymes, since Fe3O4 nanoparticles were first demonstrated to have peroxidase-like activity, important progress has been made in fields such as tumor catalytic therapy and environmental remediation.
[0003] Currently, metal-based nanozymes are regarded as ideal candidate materials to replace natural enzymes due to their high catalytic performance. However, existing metal-based nanozymes still have significant defects: firstly, the catalytic activity is limited by the single electronic structure of single-metal or dual-metal components, making it difficult to achieve efficient substrate activation; secondly, the nanoparticles are prone to agglomeration and inactivation due to high surface energy; thirdly, the lack of targeted delivery ability leads to low drug enrichment efficiency at the tumor site and may cause toxic damage to normal tissues at the same time. These problems seriously restrict the clinical translational application of nanozymes.
[0004] Medium / high-entropy alloy materials are composed of three or more metal elements in a near-equimolar ratio. Their multi-component synergistic effect can significantly optimize the electronic structure of the material, form abundant catalytic active sites, and at the same time, the high-entropy effect can enhance the thermodynamic stability of the material. Therefore, medium / high-entropy alloy materials have performance advantages over traditional single-metal or dual-metal materials.
[0005] However, in the biomedical field, the application of medium / high-entropy alloys 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 biocompatibility modification, the stable synthesis technology of ultra-small-sized (<5 nm) medium-entropy nanozymes has not been broken through, etc., which greatly limits their catalytic efficiency and in vivo application potential.
[0006] Therefore, how to develop a targeted medium / high-entropy alloy nanozyme is an urgent problem 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 targeted medium-entropy nanozyme and its preparation method and application to solve the deficiencies in the prior art.
[0008] To achieve the above purpose, the present invention adopts the following technical solutions:
[0009] A preparation method of a targeted medium-entropy nanozyme specifically includes the following steps:
[0010] (1) Mix water, absolute ethanol, and concentrated ammonia water, stir, add an aqueous solution of polyphenolic substances, stir again, add an ethanol solution of medium-entropy alloy, continue stirring, centrifuge, collect the precipitate, wash, and freeze-dry to obtain a medium-entropy nanozyme precursor;
[0011] (2) Calcinate the medium-entropy nanozyme precursor in sequence, and then cool it to obtain a medium-entropy nanozyme for standby;
[0012] (3) Digest tumor cells in sequence, centrifuge, collect the precipitate, wash, resuspend, lyse, and lyse again to obtain a cell lysate;
[0013] (4) Centrifuge the cell lysate in sequence, take the supernatant, centrifuge again, take the supernatant again, and ultracentrifuge to obtain a cell membrane precipitate for standby;
[0014] (5) Prepare the medium-entropy nanozyme and the cell membrane precipitate into an aqueous solution of medium-entropy nanozyme and an aqueous solution of cell membrane precipitate respectively, mix them in sequence, perform ultrasonic treatment, centrifuge, collect the precipitate, and wash to obtain a medium-entropy nanozyme with a targeting effect.
[0015] Further, in the above step (1), the dosage ratio of water, absolute ethanol, concentrated ammonia water, the aqueous solution of polyphenolic substances, and the ethanol solution of medium-entropy alloy is 90 mL: 40 mL: 3 mL: 10 mL: 10 mL; the aqueous solution of polyphenolic substances is an aqueous solution of dopamine hydrochloride with a concentration of 50 mg / mL; the ethanol solution of medium-entropy alloy is an ethanol solution of medium-entropy alloy; the medium-entropy alloy is at least two of ruthenium acetylacetonate, iridium acetylacetonate, platinum acetylacetonate, molybdenum acetylacetonate, rhodium acetylacetonate, palladium acetylacetonate, iron acetylacetonate, and copper acetylacetonate, preferably 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 (0.02 - 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] Further, in the above step (1), the stirring temperature is room temperature, the rotation speed is 600 - 800 r / min, and the time is 15 min; the time for the second stirring is 1 - 2 h; the time for continuous stirring is 12 - 24 h; the rotation speed for centrifugation is 12000 r / min, and the time is 10 - 20 min; the washing reagent is ethanol, and the number of times is 3; the time for freeze-drying is 24 - 36 h.
[0017] Further, in the above step (2), the calcination equipment is a tube furnace, the atmosphere is nitrogen, the temperature is 900 °C, and the time is 2 h.
[0018] Further, in the above step (3), the enzyme for digestion is trypsin; the reagent for washing is PBS buffer solution, and the number of times is 2; the temperature for lysis is 4°C, and the time is 1 h; the condition for re-lysis is ice bath, the equipment is a cell disruptor, the power is 60 W, and the time is 5 min.
[0019] Further, in the above step (4), the rotation speed for centrifugation is 4000 rpm, and the time is 10 min; the rotation speed for re-centrifugation is 12000 rpm, and the time is 15 min; the rotation speed for ultracentrifugation is 120000 rpm, and the time is 1 h.
[0020] Further, in the above step (5), the concentration of the aqueous solution of the medium-entropy nanozyme is 1 mg / mL; the concentration of the aqueous solution of the cell membrane precipitate is 1 mg / mL; the volume ratio of the aqueous solution of the medium-entropy nanozyme to the aqueous solution of the cell membrane precipitate is 1:2.
[0021] Further, in the above step (5), the ultrasonic power for ultrasonic treatment is 250 W, and the time is 20 min; the rotation speed for centrifugation is 12000 rpm, and the time is 15 min; the reagent for washing is deionized water, and the number of times is 3.
[0022] The present invention also claims protection for a medium-entropy nanozyme with a targeting effect prepared by the above preparation method.
[0023] The present invention also claims protection for the application of a medium-entropy nanozyme with a targeting effect prepared by the above preparation method in the preparation of anti-tumor nano-drugs.
[0024] As can be seen from the above technical solutions, compared with the prior art, the beneficial effects of the present invention are as follows:
[0025] 1. The present invention constructs a super-small targeted medium-entropy nanozyme system through a metal-ligand crosslinking strategy combined with a homologous cancer cell membrane coating technology, and for the first time realizes the atomic-level uniform dispersion of ruthenium, platinum, iridium, and molybdenum quaternary metals, forming a stable medium-entropy nanozyme with a particle size of 2-3 nm. Its high specific surface area and multi-metal synergistic effect significantly improve the enzyme-like catalytic activity. Further, through homologous cancer cell membrane biomimetic modification, the nanozyme is endowed with biocompatibility and the ability to actively target tumors. The targeted medium-entropy nanozyme wrapped by the homologous cell membrane of the present invention can efficiently catalyze the conversion of hydrogen peroxide in the tumor microenvironment into highly toxic hydroxyl radicals, and achieve precise tumor suppression through ROS-mediated DNA damage, apoptosis, and autophagy pathways, and the systemic toxicity is controllable. This technological breakthrough not only overcomes the dual problems of controllable preparation and targeted delivery of medium-entropy nanozymes, but also provides a new technical path for the catalytic treatment of malignant tumors.
[0026] 2. The present invention uses dopamine hydrochloride, metal acetylacetonate salts, and cancer cell membranes as raw materials, dopamine nanozymes as carriers, generates medium-entropy nanozyme precursors through oxidative self-polymerization, and then obtains quaternary metal medium-entropy nanozymes with ultra-small active centers by means of high-temperature calcination. Subsequently, the cancer cell membranes are uniformly wrapped on the surface of the medium-entropy nanozymes through self-assembly. The medium-entropy nanozymes prepared thereby have good peroxidase-like activity, can effectively catalyze hydrogen peroxide in the tumor microenvironment to generate highly toxic hydroxyl radicals, and kill cancer cells. In addition, the nanozymes are modified by biomimetic homologous cancer cell membranes, have good biocompatibility and the ability to actively target tumor tissues, effectively improve the accumulation of nanozymes at the tumor site, can quickly and efficiently exert therapeutic effects in the tumor microenvironment, and the nanozymes have high biosafety and have good prospects for tumor treatment. BRIEF DESCRIPTION OF THE DRAWINGS
[0027] Figure 1 It is the high-resolution transmission electron microscope (HR-TEM) image, high-angle annular dark field scanning-transmission electron microscope (HADDF-STEM) image, and X-ray energy spectrum analysis (EDS-mapping) image of the targeted medium-entropy nanozyme prepared in Example 1 of the present invention;
[0028] Figure 2 It is the peroxidase-like activity of the targeted medium-entropy nanozyme prepared in Example 1 of the present invention. DETAILED DESCRIPTION OF THE INVENTION
[0029] The technical solutions in the embodiments of the present invention are clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.
[0030] Example 1
[0031] A preparation method of a targeted medium-entropy nanozyme specifically includes the following steps:
[0032] (1) Mix 90 mL of water, 40 mL of absolute ethanol, and 3 mL of concentrated ammonia water, stir at a speed of 600 r / min for 15 min at room temperature, add 10 mL of an aqueous dopamine hydrochloride solution with a concentration of 50 mg / mL, stir for another 2 h, dropwise add 10 mL of ethanol solutions of ruthenium acetylacetonate, iridium acetylacetonate, platinum acetylacetonate, and molybdenum acetylacetonate with a concentration of 5 mg / mL for each metal salt, continue to stir for 12 h, centrifuge at a speed of 12000 r / min for 15 min, collect the precipitate, wash it repeatedly with ethanol 3 times, and freeze-dry for 24 h to obtain a medium-entropy nanozyme precursor;
[0033] (2) Transfer the medium-entropy nanozyme precursor into a tube furnace, heat it to 900 °C at a rate of 10 °C / min in a nitrogen atmosphere, calcine for 2 h, and then cool to obtain the medium-entropy nanozyme for standby;
[0034] (3) Digest the cultured tumor cells with trypsin, centrifuge, collect the precipitate, wash it twice with PBS buffer, add lysis buffer to resuspend the tumor cells, first lyse at 4 °C for 1 h, and then use a cell disruptor to fully lyse the cells at a power of 60 W in an ice bath for 5 min to obtain a cell lysate;
[0035] (4) Centrifuge the cell lysate at 4000 rpm for 10 min, take the supernatant, centrifuge again at 12000 rpm for 15 min, then take the supernatant, and ultracentrifuge at 120000 rpm for 1 h to obtain a cell membrane precipitate for standby;
[0036] (5) Prepare a medium-entropy nanozyme aqueous solution with a concentration of 1 mg / mL and a cell membrane precipitate aqueous solution with a concentration of 1 mg / mL from the medium-entropy nanozyme and the cell membrane precipitate respectively, mix them at a volume ratio of 1:2, perform ultrasonic treatment at a ultrasonic power of 250 W for 20 min, then centrifuge at 12000 rpm for 15 min, collect the precipitate, and wash it three times with deionized water to obtain the medium-entropy nanozyme with targeting effect.
[0037] Performance Test
[0038] 1. HR-TEM, HADDF-STEM and EDS-mapping Characterization
[0039] The high-resolution transmission electron microscope (HR-TEM) image of the medium-entropy nanozyme with targeting effect prepared in Example 1 is as shown in Figure 1 Figure A, the high-angle annular dark-field scanning-transmission electron microscope (HADDF-STEM) image is as shown in Figure 1 Figures B-C, and the X-ray energy spectrum analysis (EDS-mapping) image is as shown in Figure 1 Figure D.
[0040] It can be seen from Figure 1 that the cancer cell membrane uniformly wraps on the surface of the medium-entropy nanozyme, and the four metal elements are uniformly distributed, with a size of about 2-3 nm.
[0041] 2. Study on Peroxidase-like Activity
[0042] Take the medium-entropy nanozyme with targeting effect prepared in Example 1 and conduct a study on 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 sonicate it for 30 min with a sonication power of 250 W to obtain an aqueous solution of the medium-entropy nanozyme;
[0044] (2) Uniformly mix 2 μL of the aqueous solution of the medium-entropy nanozyme, 20 μL of the hydrogen peroxide solution, 20 μL of the TMB ethanol solution with a concentration of 5 mg / mL, 2 mL of the HAc-NaAc buffer solution (pH = 4.5), and 2 μL of the DMPO solution with a concentration of 1 M. After vortexing and shaking the centrifuge tube, wait for 5 min to obtain a mixed solution;
[0045] (3) Aspirate the mixed solution into a quartz capillary for electron paramagnetic resonance spectroscopy (EPR) analysis to detect peroxidase-like activity.
[0046] The results are as Figure 2 shown.
[0047] As Figure 2 can be seen, the medium-entropy nanozyme with targeting effect prepared in Example 1 has high peroxidase-like activity, can rapidly catalyze hydrogen peroxide to generate hydroxyl radicals, and kill cancer cells.
[0048] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be obvious to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to these embodiments shown herein, but will conform to the widest scope consistent with the principles and novel features disclosed herein.
Claims
1. A preparation method of a targeted medium-entropy nanozyme, characterized in that, Specifically, it includes the following steps: (1) Mix water, absolute ethanol and concentrated ammonia water, stir, add an aqueous solution of polyphenolic substances, stir again, add an ethanol solution of medium-entropy alloy, continue to stir, centrifuge, collect the precipitate, wash, and freeze-dry to obtain a medium-entropy nanozyme precursor; (2) Calcinate the medium-entropy nanozyme precursor in sequence, and cool to obtain a medium-entropy nanozyme for standby; (3) Digest tumor cells in sequence, centrifuge, collect the precipitate, wash, resuspend, lyse, and lyse again to obtain a cell lysate; (4) Centrifuge the cell lysate in sequence, take the supernatant, centrifuge again, take the supernatant again, and ultracentrifuge to obtain a cell membrane precipitate for standby; (5) Prepare the medium-entropy nanozyme and the cell membrane precipitate into an aqueous solution of medium-entropy nanozyme and an aqueous solution of cell membrane precipitate respectively, mix them in sequence, perform ultrasonic treatment, centrifuge, collect the precipitate, and wash to obtain the medium-entropy nanozyme with targeting effect.
2. The preparation method of a targeted medium-entropy nanozyme according to claim 1, characterized in that, In step (1), the dosage ratio of the water, absolute ethanol, concentrated ammonia water, aqueous solution of polyphenolic substances and ethanol solution of medium-entropy alloy is 90 mL: 40 mL: 3 mL: 10 mL: 10 mL; the aqueous solution of polyphenolic substances is an aqueous solution of dopamine hydrochloride with a concentration of 50 mg / mL; the ethanol solution of medium-entropy alloy is an ethanol solution of medium-entropy alloy; the medium-entropy alloy is at least two of ruthenium acetylacetonate, iridium acetylacetonate, platinum acetylacetonate, molybdenum acetylacetonate, rhodium acetylacetonate, palladium acetylacetonate, iron acetylacetonate and copper acetylacetonate.
3. The preparation method of a meso-entropy nanozyme with a targeting effect according to claim 1, characterized in that, In step (1), the temperature of the stirring is room temperature, the rotation speed is 600 - 800 r / min, and the time is 15 min; the time of the second stirring is 1 - 2 h; the time of the continuous stirring is 12 - 24 h; the rotation speed of the centrifuge is 12000 r / min, and the time is 10 - 20 min; the washing reagent is ethanol, and the number of times is 3; the time of freeze-drying is 24 - 36 h.
4. The preparation method of a meso-entropy nanozyme with a targeting effect 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 °C / min, the temperature is 900 °C, and the time is 2 h.
5. The preparation method of a targeted medium-entropy nanozyme according to claim 1, wherein, In step (3), the enzyme for digestion is trypsin; the washing reagent is PBS buffer solution, and the number of times is 2; the temperature of lysis is 4 °C, and the time is 1 h; the conditions for the second lysis are ice bath, the equipment is a cell disruptor with a power of 60 W, and the time is 5 min.
6. The preparation method of a targeted medium-entropy nanozyme according to claim 1, characterized in that, In step (4), the rotation speed of the centrifuge is 4000 rpm, and the time is 10 min; the rotation speed of the second centrifuge is 12000 rpm, and the time is 15 min; the rotation speed of the ultracentrifuge is 120000 rpm, and the time is 1 h.
7. The preparation method of a meso-entropy nanozyme with a targeting effect according to claim 1, characterized in that, In step (5), the concentration of the aqueous solution of medium-entropy nanozyme is 1 mg / mL; the concentration of the aqueous solution of cell membrane precipitate is 1 mg / mL; the volume ratio of the aqueous solution of medium-entropy nanozyme to the aqueous solution of cell membrane precipitate is 1:
2.
8. The preparation method of a meso-entropy nanozyme with a targeting effect according to claim 1, characterized in that, In step (5), the ultrasonic power of the ultrasonic treatment is 250 W, and the time is 20 min; the rotation speed of the centrifuge is 12000 rpm, and the time is 15 min; the washing reagent is deionized water, and the number of times is 3.
9. A targeted medium-entropy nanozyme prepared by the preparation method according to any one of claims 1-8.
10. Use of a targeted medium-entropy nanozyme prepared by the preparation method according to any one of claims 1-8 in the preparation of anti-tumor nano-drugs.