Monatomic nano-enzyme of in-situ derived functional cluster as well as preparation method and application of monatomic nano-enzyme
By preparing iron-zinc single-atom nanozymes with functional clusters, the stability and catalytic efficiency problems of traditional nanozymes in tumor therapy have been solved, achieving highly efficient tumor catalytic therapy and biosafety, with magnetic targeting and photothermal enhanced therapeutic effects.
Patent Information
- Application Number
- CN202510449870.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-11
- Publication Date
- 2025-11-25
AI Technical Summary
Traditional nanozymes suffer from problems such as low atom utilization, poor stability, low catalytic efficiency, and insufficient biocompatibility in the field of tumor therapy, making it difficult to achieve precise targeting and efficient catalysis.
Using mesoporous silica as a template, iron-zinc single-atom nanozymes were prepared via a hydrothermal method, and then calcined at low temperature to form nanozymes with functional clusters, FeZnSA-NC. Combined with mesoporous silica, iron acetylacetone, and zinc chloride as raw materials, single-atom nanozymes with functional clusters were prepared using hydrothermal reaction and low-temperature calcination technology.
It achieves highly efficient biocatalytic reactions, possesses significant magnetic and photothermal properties, and can achieve safe and efficient tumor catalytic therapy by magnetically targeting tumor sites, while enhancing the catalytic effect through photothermal stimulation.
Smart Images
Figure CN121003697A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the field of nanometer enzyme preparation, in particular to a single atom nanometer enzyme in situ derived functional cluster, a preparation method and application thereof. BACKGROUND
[0002] With the rapid development of nanotechnology, nanometer enzymes, as a kind of artificial enzyme simulating the function of natural enzymes, show broad application prospects in the field of biomedicine. However, traditional nanometer enzymes still face many challenges: on the one hand, traditional nanometer enzymes often rely on a single active center or structural unit, resulting in low atom utilization rate; on the other hand, the stability, catalytic efficiency and biocompatibility of traditional nanometer enzymes have not been fully solved. Especially in the field of tumor treatment, how to achieve precise targeting, efficient catalysis and good biological safety is still a technical bottleneck to be broken through. The emergence of single atom nanometer enzyme provides a new solution to break through the existing technical bottleneck of nanometer enzyme. Single atom nanometer enzyme has atomically dispersed metal active centers, compared with traditional nanometer enzyme, it has rich electronic structure, which can maximize the utilization rate of metal atoms, and is expected to significantly improve the catalytic activity and selectivity. In the preparation of single atom nanometer enzyme, we in situ derive functional clusters from it, which have magnetic properties and unique magnetic response characteristics, which will further expand its application range. Iron and zinc are two common and important biological active metal elements, which have unique advantages in enzyme catalysis. Iron ions act as key active sites in many redox enzymes, participating in electron transfer and substrate activation process. Zinc ions play an important role in many hydrolytic enzymes and synthetic enzymes, which can stabilize the structure of the enzyme and promote substrate binding. Constructing iron and zinc into iron-zinc single atom nanometer enzyme and in situ deriving functional clusters from it is expected to integrate the catalytic advantages of both and achieve more efficient biological catalytic reaction. In summary, it is of great scientific significance to develop a preparation method of single atom nanometer enzyme in situ derived functional cluster and further explore its application in the field of tumor catalytic therapy. SUMMARY
[0003] The purpose of the present application is to provide a single atom nanometer enzyme in situ derived functional cluster, a preparation method and application thereof, so as to solve the aforementioned problems existing in the prior art.
[0004] In order to achieve the above purpose, the technical scheme adopted by the present application is as follows:
[0005] A preparation method of a single atom nanometer enzyme in situ derived functional cluster, comprising the following steps:
[0006] S1, using mesoporous silica and iron ions and zinc ions as raw materials, an iron-zinc single atom nanometer enzyme FeZn is prepared by hydrothermal method SA ;
[0007] S2, low-temperature calcination of the hydrothermally prepared iron-zinc single-atom nanoszyme to obtain a nanoszyme FeZn with functional clusters SA -NC.
[0008] 1. Preferably, the preparation method of the single-atom nanoszyme with in-situ derived functional clusters in step S1 is characterized in that the mesoporous silica in step S1 is prepared by using tetraethyl orthosilicate as a silicon source, hexadecyl trimethyl ammonium p-toluenesulfonate as a surfactant, adding triethanolamine and 1-butyl-3-methylimidazolium trifluoromethanesulfonate. The addition amounts are as follows: 7-12 mL of tetraethyl orthosilicate, 0.8-2 g of hexadecyl trimethyl ammonium p-toluenesulfonate; 0.1-0.2 g of triethanolamine and 10-20 mg of 1-butyl-3-methylimidazolium trifluoromethanesulfonate. After mixing the above substances, stirring reaction is carried out at 80°C for 2-3 h, and then the product is centrifuged and washed.
[0009] The centrifuged and washed product is dispersed in 100-200 mL of anhydrous ethanol, and after stirring for 20-24 h, the reaction product is again centrifuged, washed and dried, and the white solid powder obtained is mesoporous silica.
[0010] 2. Preferably, step S1 specifically comprises: adding 80-200 mg of acetylacetone iron and 40-80 mg of zinc chloride, 1-2 mL of ammonia water and 0.5-1 g of ammonium chloride into 50-100 mg of mesoporous silica, and stirring uniformly to obtain a mixed solution system; the above solution is sealed in an autoclave, and kept at 180-200°C for 12-18 h; after the autoclave is naturally cooled to room temperature, the obtained product is washed and centrifuged multiple times to completely remove residual metal ions, and finally an iron-zinc single-atom nanoszyme FeZn is obtained. SA ;
[0011] Preferably, the low-temperature calcination in step S2 specifically comprises: placing the prepared FeZn SA in a tube furnace, and calcining at a temperature increasing rate of 3-5°C / min, 400-500°C, under N2 protection for 3-4 h to obtain FeZn SA -NC.
[0012] Preferably, the centrifugation, washing and drying of the reaction product are specifically as follows: first, the reaction product is placed in a centrifuge and centrifuged at a speed of 8000-10000 r / min. After centrifugation, the product is washed twice with deionized water, and then washed twice with anhydrous ethanol. After the washing step is completed, the centrifuged product is transferred to a vacuum oven with a temperature setting of 40-60°C for drying treatment.
[0013] Another object of the present application is to provide a preparation method of a single-atom nanoscale enzyme with a functional group cluster derived in situ, denoted as FeZn SA -NC.
[0014] A final object of the present application is to provide an application of the single-atom nanoscale enzyme with a functional group cluster derived in situ in terms of enzyme catalytic performance and photothermal performance.
[0015] The present application has the following beneficial effects:
[0016] The present application provides a preparation method of a single-atom nanoscale enzyme with a functional group cluster derived in situ and an application in the field of tumor catalytic therapy. Mesoporous silica is used as a template, acetylacetone iron is used as an iron source, and zinc chloride is used as a zinc source. After hydrothermal reaction for 12 h, the nanoscale enzyme with coexistence of functional group clusters and iron-zinc single atoms is successfully prepared by calcination in a tube furnace at 500 DEG C under a nitrogen atmosphere for 3 h. The designed single-atom nanoscale enzyme with a functional group cluster exhibits the largest atomic utilization rate, significant magnetism, higher peroxidase-like activity, and excellent photothermal performance. These four advantages endow it with higher catalytic activity. In addition, the nanoscale enzyme can be precisely positioned to the tumor site through magnetic targeting, which not only ensures the biosafety of the nanoscale enzyme, but also ensures the tumor specificity. Finally, due to the efficient near-infrared two-zone photothermal conversion capability, photothermal enhanced catalytic therapy can be achieved. BRIEF DESCRIPTION OF DRAWINGS
[0017] Figure 1 Transmission electron microscope picture of mesoporous silica provided in Example 1;
[0018] Figure 2 Transmission electron microscope picture of FeZn SA -NC prepared in Example 1;
[0019] Figure 3 Corresponding element mapping image of FeZn SA -NC prepared in Example 1;
[0020] Figure 4 X-ray diffraction spectrum of FeZn SA -NC prepared in Example 1;
[0021] Figure 5 Magnetic hysteresis loop of FeZn SA -NC prepared in Example 1;
[0022] Figure 6 Nitrogen adsorption and desorption curve of FeZn SA -NC prepared in Example 1;
[0023] Figure 7 FeZnSA - the pore size distribution curve of the NCs;
[0024] Figure 8 FeZn SA - the enzymatic performance test of the NCs. DETAILED DESCRIPTION
[0025] In order to make the objects, technical solutions and advantages of the present application clearer, the present application will be further described in detail below with reference to the drawings. It should be understood that the specific embodiments described herein are only used to explain the present application and do not limit the present application.
[0026] Example 1
[0027] The present embodiment provides a single-atom nanoscale enzyme with a functional group cluster derived in situ, and the preparation method comprises the following steps:
[0028] (1) Preparation of mesoporous silica (mSiO2): first, 0.96 g of cetyltrimethylammonium p-toluenesulfonate, 0.105 g of triethanolamine and 10 mg of 1-butyl 3-methyl-imidazole trifluoromethanesulfonate were mixed in 50 mL of deionized water, and stirred at 80°C for 1 h. Then, 7.8 mL of tetraethyl orthosilicate was quickly added to the mixture, and the reaction was stirred at 80°C for another 2 h. After that, the product was centrifuged at 8000 r / min for 5 min and washed with anhydrous ethanol and water twice.
[0029] In order to remove the template cetyltrimethylammonium p-toluenesulfonate, the centrifuged and washed product was dispersed in 150 mL of anhydrous ethanol, stirred for 24 h, centrifuged at 10000 r / min for 10 min and washed with ethanol twice. Finally, the centrifuged product was transferred to a 50°C vacuum oven for drying, and the obtained white solid powder was mesoporous silica.
[0030] The structure of the prepared mesoporous silica was confirmed, and the observation was carried out by transmission electron microscopy. Figure 1 It can be seen from the figure that the particle size of the mesoporous silica is about 120 nm, and the mesoporous silica has a clear dendritic structure inside and uniform particle size distribution.
[0031] (2) Iron-zinc single-atom nanoscale enzyme (FeZn SAPreparation of FeZn single-atom nanozyme: First, the mesoporous silica (50 mg) obtained in the first step was dispersed in 10 mL of deionized water and ultrasonically treated to ensure uniform dispersion. Then, 90 mg of ferric acetylacetone and 35 mg of zinc chloride were added and stirred to ensure uniform dispersion. Next, 1 mL of ammonia water and 0.5 g of ammonium chloride were added to the above mixed solution, and the mixture was stirred at room temperature for 20 min. The solution was then sealed in a 100 mL high-pressure reactor lined with polytetrafluoroethylene and kept at 200 °C for 12 h. After the reactor cooled to room temperature, the resulting product was centrifuged and washed several times to remove residual metal ions. The final product was placed in an oven and dried overnight at 50 °C to obtain the FeZn single-atom nanozyme. SA .
[0032] (3) In-situ derived functional clusters of single-atom nanozymes (FeZn) SA Preparation of FeZn(NC): SA The sample was placed in a tube furnace and calcined at 500°C for 3 hours under N2 protection at a heating rate of 5°C / min. After cooling to room temperature, FeZn was obtained. SA -NC.
[0033] FeZn SA -NC was used for morphological and structural characterization, and transmission electron microscopy images were obtained, such as Figure 2 As shown in the figure, FeZn SA -NC has a spherical structure and uniform particle size. Figure 3 This is an elemental mapping image of an iron-zinc single-atom nanozyme with functional clusters. The image shows that different elements are uniformly distributed on the nanoparticles.
[0034] Figure 4 FeZn was obtained by X-ray diffraction testing. SA -More details on the crystal structure of NC were found, revealing FeZn SA The X-ray diffraction image of -NC corresponds to the standard card (PDF#22-1012) of ZnFe2O4.
[0035] To verify the magnetic type of nanozymes with functional clusters, hysteresis loop tests were performed, such as... Figure 5 FeZn was obtained SA -NC is a superparamagnetic material with a saturation magnetization of 7.89 emu / g.
[0036] FeZn prepared by the above method SA -NC performed nitrogen adsorption and desorption tests, and the results are as follows: Figure 6 As shown in the figure, FeZn can be obtained. SA The specific surface area of -NC is 138.50 m². 2 / g;Figure 7 FeZn SA - The pore size distribution curve of NC shows an average particle size of 13.70 nm.
[0037] Figure 8 To detect the peroxidase-like activity of single-atom nanozymes with functional clusters.
[0038] Example 2
[0039] This embodiment provides a single-atom nanozyme with in-situ derived functional clusters, the preparation method of which includes the following steps:
[0040] (1) Preparation of mesoporous silica (mSiO2): First, 1.95 g of hexadecyltrimethyl-p-toluenesulfonate ammonium, 0.21 g of triethanolamine and 20 mg of 1-butyl-3-methyl-imidazolium trifluoromethanesulfonate were mixed in 100 mL of deionized water and stirred at 80 °C for 1 h. Then, 15 mL of tetraethyl orthosilicate was quickly added to the mixture, and the mixture was stirred at 80 °C for another 2 h. The product was then centrifuged at 8000 r / min for 5 min and washed twice with anhydrous ethanol and water, respectively.
[0041] To remove the template hexadecyltrimethyl-p-toluenesulfonate ammonium, the product after centrifugation and washing was dispersed in 300 mL of anhydrous ethanol, stirred for 24 h, centrifuged at 10000 r / min for 10 min and washed twice with ethanol. Finally, the centrifuged product was transferred to a vacuum oven at 50 °C and dried. The resulting white solid powder was mesoporous silica.
[0042] (2) Iron-zinc single-atom nanozyme (FeZn) SA Preparation of FeZn nanozyme: First, 75 mg of mesoporous silica obtained in the first step was dispersed in 15 mL of deionized water and ultrasonically treated to ensure uniform dispersion. Then, 135 mg of ferric acetylacetone and 55 mg of zinc chloride were added and stirred to ensure uniform dispersion. Next, 1.5 mL of ammonia and 0.8 g of ammonium chloride were added to the silica system, and the mixture was stirred at room temperature for 20 min. The solution was then sealed in a 100 mL high-pressure reactor lined with polytetrafluoroethylene and kept at 200 °C for 12 h. After the reactor cooled to room temperature, the product was centrifuged and washed several times to remove residual metal ions. The final product was then placed in an oven and dried overnight at 50 °C to obtain the FeZn nanozyme. SA .
[0043] (3) In-situ derived functional clusters of single-atom nanozymes (FeZn) SA Preparation of FeZn(NC): SA The sample was placed in a tube furnace and calcined at 400°C for 4 hours under N2 protection at a heating rate of 3°C / min. After cooling to room temperature, FeZn was obtained.SA -NC.
[0044] Example 3
[0045] This embodiment provides a single-atom nanozyme with in-situ derived functional clusters, the preparation method of which includes the following steps:
[0046] (1) Preparation of mesoporous silica (mSiO2): First, 1.5 g of hexadecyltrimethyl-p-toluenesulfonate, 0.15 g of triethanolamine and 20 mg of 1-butyl-3-methyl-imidazolium trifluoromethanesulfonate were mixed in 75 mL of deionized water and stirred at 80 °C for 1 h. Then, 11.5 mL of tetraethyl orthosilicate was quickly added to the mixture, and the mixture was stirred at 80 °C for another 2 h. The product was then centrifuged at 8000 r / min for 5 min and washed twice with anhydrous ethanol and water, respectively.
[0047] To remove the template hexadecyltrimethyl-p-toluenesulfonate ammonium, the product after centrifugation and washing was dispersed in 230 mL of anhydrous ethanol, stirred for 24 h, centrifuged at 10000 r / min for 10 min and washed twice with ethanol. Finally, the centrifuged product was transferred to a vacuum oven at 50 °C and dried. The resulting white solid powder was mesoporous silica.
[0048] (2) Iron-zinc single-atom nanozyme (FeZn) SA Preparation of FeZn single-atom nanozyme: First, 100 mg of mesoporous silica obtained in the first step was dispersed in 20 mL of deionized water and ultrasonically treated to ensure uniform dispersion. Then, 175 mg of ferric acetylacetone and 70 mg of zinc chloride were added and stirred to ensure uniform dispersion. Next, 2 mL of ammonia water and 1.1 g of ammonium chloride were added to the silica system, and the mixture was stirred at room temperature for 20 min. The solution was then sealed in a 100 mL high-pressure reactor lined with polytetrafluoroethylene and kept at 200 °C for 12 h. After the reactor cooled to room temperature, the product was centrifuged and washed several times to remove residual metal ions. The final product was placed in an oven and dried overnight at 50 °C to obtain the FeZn single-atom nanozyme. SA .
[0049] (3) In-situ derived functional clusters of single-atom nanozymes (FeZn) SA Preparation of FeZn(NC): SA The sample was placed in a tube furnace and calcined at 450°C for 3 hours under N2 protection at a heating rate of 5°C / min. After cooling to room temperature, FeZn was obtained. SA -NC.
[0050] By adopting the above-disclosed technical solution of this invention, the following beneficial effects are obtained:
[0051] This invention discloses a method for preparing and applying single-atom nanozymes with in-situ derived functional clusters. These single-atom nanozymes possess several superior characteristics: First, the synthesis process is simple, easy to scale up, and uses inexpensive raw materials. Furthermore, the generated nanozymes exhibit uniform particle size distribution and good dispersibility. Second, these nanozymes possess high peroxidase-like activity, catalyzing the generation of ·OH from H₂O₂, further increasing reactive oxygen species levels and inducing apoptosis. Third, the presence of functional clusters endows them with significant magnetic properties, enabling them to precisely target tumor sites via magnetic targeting, achieving safe and efficient tumor catalytic therapy. Fourth, the prepared nanozymes can degrade in response to the tumor microenvironment, improving biosafety on the one hand; on the other hand, the degraded metal ions undergo Fenton / Fenton-like reactions, enhancing the therapeutic effect. Fifth, the synthesized nanozymes possess highly efficient photothermal conversion capabilities, which can further improve the efficiency of tumor catalytic therapy.
[0052] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.
Claims
1. A method for preparing a single-atom nanozyme with in-situ derived functional clusters, characterized in that, Includes the following steps: S1, using mesoporous silica, iron ions, and zinc ions as raw materials, prepared iron-zinc single-atom nanoenzymes (FeZn) via a hydrothermal method. SA ; S2, FeZn nanozymes with functional clusters were obtained by calcining iron-zinc single-atom nanozymes prepared by hydrothermal method at low temperature. SA -NC.
2. The method for preparing single-atom nanozymes with in-situ derived functional clusters according to claim 1, characterized in that, The mesoporous silica described in step S1 was prepared using tetraethyl orthosilicate as the silicon source, hexadecyltrimethyl-p-toluenesulfonate as the surfactant, and the addition of triethanolamine and 1-butyl-3-methylimidazolium trifluoromethanesulfonate. The amounts added were: 7–12 mL tetraethyl orthosilicate, 0.8–2 g hexadecyltrimethyl-p-toluenesulfonate, 0.1–0.2 g triethanolamine, and 10–20 mg 1-butyl-3-methylimidazolium trifluoromethanesulfonate. After mixing the above substances, the mixture was stirred at 80°C for 2–3 hours, followed by centrifugation and washing of the product. The product after centrifugation and washing is dispersed in 100-200 mL of anhydrous ethanol and stirred for 20-24 h. The reaction product is then centrifuged, washed, and dried again to obtain a white solid powder, which is mesoporous silica.
3. The method for preparing single-atom nanozymes with in-situ derived functional clusters according to claim 1, characterized in that, Step S1 specifically includes: adding 80-200 mg of acetylacetone iron, 40-80 mg of zinc chloride, 1-2 mL of ammonia water, and 0.5-1 g of ammonium chloride to 50-100 mg of mesoporous silica, and stirring until a mixed solution system is obtained; sealing the above solution in an autoclave and maintaining it at 180-200℃ for 12-18 h; after the autoclave has naturally cooled to room temperature, the obtained product is washed and centrifuged multiple times to completely remove residual metal ions, finally obtaining the iron-zinc single-atom nanozyme FeZn. SA。 4. The method for preparing single-atom nanozymes with in-situ derived functional clusters according to claim 1, characterized in that, The low-temperature calcination in step S2 specifically includes: calcining the prepared FeZn... SA Place it in a tube furnace and calcine it for 3-4 hours at a heating rate of 3-5℃ / min, 400-500℃, and under N2 protection.
5. The method for preparing nanozymes according to any one of claims 1, 2, 3, and 4, characterized in that, The specific steps for centrifuging, washing, and drying the reaction products are as follows: First, place the reaction products in a centrifuge and centrifuge at a speed of 8000–10000 r / min. After centrifugation, wash the products twice with deionized water, and then twice with anhydrous ethanol. After completing the washing steps, transfer the centrifuged products to a vacuum oven set at a temperature of 40–60°C for drying.
6. The nanozyme containing both clusters and single atoms, obtained by the method for preparing single-atom nanozymes of in-situ derived functional clusters according to any one of claims 1-5, is denoted as FeZn. SA -NC. Applications of the single-atom nanozymes with in-situ derived functional clusters as described in claim 6 in terms of enzyme catalytic performance and photothermal performance.