Double-enzyme characteristic single-atom nanoscale enzyme with fe-si n3 coordination structure and preparation method and application thereof

Abstract

The application belongs to the field of non-noble metal catalysts, and particularly relates to a Fe-S1N3 coordination structure double-enzyme characteristic single-atom nanozyme as well as a preparation method and application thereof. The preparation method comprises the following steps: taking Hemin / ZIF-8 as a template, taking trithiocyanic acid TTCA as a sulfur source, forming a Hemin / ZIF-8@TTCA core-shell precursor, and then further carbonizing at high temperature to obtain the Fe-S1N3 coordination structure double-enzyme characteristic single-atom nanozyme. The asymmetric Fe-S1N3 coordination configuration and electronic structure make the Fe-S / N-C have higher peroxidase-like activity and glutathione oxidase activity compared with a typical Fe-N4 configuration Fe-N-C SAzyme.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of non-noble metal catalysts, and particularly relates to a Fe-SiN3 coordination structure double-enzyme characteristic single-atom nanozyme as well as a preparation method and application thereof. BACKGROUND

[0002] The emerging nanozyme-based nanocatalytic drugs are widely used in tumor treatment by locally inducing the production of reactive oxygen species (ROS) at the tumor site. Specifically, nanozymes can catalyze the decomposition of hydrogen peroxide (H2O2) to generate ROS, which can cause irreversible oxidative damage to tumor cells by oxidizing proteins and destroying DNA structure. However, as an essential substance for maintaining the redox balance, the overexpressed endogenous reducing glutathione (GSH) in the tumor microenvironment (TME) can effectively eliminate ROS at the tumor site, thereby reducing the tumor cell killing effect of the ROS elevation-related strategy. In addition, the low level of H2O2 in the TME further limits the generation of ROS, making it difficult to achieve satisfactory therapeutic effect. Therefore, it is an urgent and challenging task to rationally design high-performance nanozymes that can effectively increase the level of active oxygen, consume cellular GSH, and self-supply H2O2.

[0003] As a new and popular subcategory of nanozymes, single-atom nanozymes (SAzymes) have the largest atom utilization rate and a clear coordination structure. Due to their excellent catalytic activity and kinetic behavior, they have attracted widespread attention in biomedical applications in recent years. However, these SAzymes with metal-nitrogen 4 (M-N4) coordination configuration have insufficient adsorption capacity for substrates due to their structural and electronic symmetry and high chemical inertness, and their catalytic activity is also not ideal. Therefore, precise optimization of the local coordination configuration of SAzymes is a necessary condition for further improving their zymelike activity and tumor treatment effect.

[0004] Recent studies have shown that two heteroatom doping strategies can effectively optimize the electronic structure of M-N4 active centers, improve catalytic activity and therapeutic effect. One strategy is to dope heteroatoms (such as phosphorus (P), sulfur (S), boron (B) and other elements) into the carbon carrier to change its electronic structure, thereby indirectly affecting the electron density of the M-N4 site through metal-support interaction. The other method is to directly change the geometry and electronic structure of the M-N4 site by replacing one of the first coordination shell N atoms with other heteroatoms (called proximal coordination). Many studies have shown that the latter is more effective because it can fundamentally destroy the symmetrical charge distribution around the metal center, thereby improving its catalytic activity. S has weaker electronegativity and larger atomic radius than N atoms, so S-mediated proximal coordination is considered an effective method to break the symmetrical M-N4 site, adjust the adsorption strength of reaction intermediates, and thus improve catalytic performance. However, if the precursor is not carefully constructed, the S atom will often evaporate or penetrate into the carbon matrix during pyrolysis, rather than directly coordinating with the metal center. Therefore, it is highly desirable to precisely construct the S proximal coordination structure of SAzymes, but it is still challenging. In addition, most S proximal coordination strategies mainly focus on promoting peroxidase-like (POD) activity to increase ROS levels, and little attention is paid to enhancing other enzyme activities related to TME-highly expressed antioxidant molecules such as GSH. SUMMARY

[0005] The purpose of the present application is to overcome the shortcomings in the prior art, and to provide a Fe-S1N3 coordination structure double-enzyme characteristic single-atom nanoscale enzyme and a preparation method and application thereof.

[0006] To achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows:

[0007] A preparation method of a Fe-S1N3 coordination structure double-enzyme characteristic single-atom nanoscale enzyme, comprising the following steps: using Hemin / ZIF-8 as a template, TTCA trithiocyanic acid as a sulfur source, forming a Hemin / ZIF-8@TTCA core-shell precursor, and then further carbonizing at high temperature to obtain.

[0008] The Hemin / ZIF-8 is prepared in the following manner: a methanol solution of zinc nitrate hexahydrate is mixed with a methanol solution of dimethyl imidazole and hemin to obtain a precipitate of hemin-doped ZIF-8, which is Hemin / ZIF-8.

[0009] The mass ratio of the zinc nitrate hexahydrate, dimethyl imidazole, and hemin is 1.7:2.35:0.05.

[0010] Specifically comprising the following steps: 1) adding a methanol solution of TTCA into a methanol dispersion of Hemin / ZIF-8 to obtain a product Hemin / ZIF-8@TTCA; 2) calcining the Hemin / ZIF-8@TTCA under an inert atmosphere to obtain a single-atom nano-enzyme Fe-S / N-C with Fe-S1N3 coordination structure anchored on a carbon base.

[0011] The mass ratio of Hemin / ZIF-8 to TTCA is 300:70-300:10.

[0012] The calcination temperature in step 2) is 800-1000 DEG C, preferably 920 DEG C; the calcination time is 1-3 h; preferably 3 h.

[0013] The application also includes a Fe-S1N3 coordination structure double-enzyme characteristic single-atom nano-enzyme prepared by the preparation method.

[0014] The application also includes an application of the Fe-S1N3 coordination structure double-enzyme characteristic single-atom nano-enzyme, which is applied to the preparation of a tumor drug.

[0015] Compared with the prior art, the application has the following beneficial effects:

[0016] The technical scheme of the application aims at the problems of limited catalytic activity of typical M-N4 nano-enzymes and inaccurate positioning of hetero-element doping, takes Hemin / ZIF-8 doped with hemin as a template and TTCA as a sulfur source, the mercapto group in TTCA can form a Fe-S coordination bond with Fe in Hemin / ZIF-8, so that the S atom can be directly coordinated with the Fe atom to form a Hemin / ZIF-8@TTCA core@shell precursor, then the Fe-S bond can be preserved through further carbonization at high temperature to obtain a single-atom nano-enzyme (Fe-S / N-C) with a Fe-S1N3 coordination structure.

[0017] The asymmetric Fe-S1N3 coordination configuration and electronic structure make the Fe-S / N-C have higher peroxidase-like activity and glutathione oxidase activity compared with the typical Fe-N4 configuration Fe-N-CSAzyme. The Fe-S / N-C can catalyze the tumor cells to produce more toxic ROS from H2O2, and at the same time, can efficiently consume endogenous GSH, thereby doubling the local oxidative stress and improving the in-vitro ferroptosis tumor treatment. In addition, the Fe-S / N-C consumes GSH while generating H2O2 in situ, to some extent, making up for the defect of insufficient H2O2 content in the tumor microenvironment.

[0018] In summary, the preparation method of the Fe-S1N3 coordination structure of the dual-enzyme characteristic single-atom nanoscale enzyme of the application is specific:

[0019] (1) The method can realize precise regulation of the near-end coordination of sulfur atoms of the metal center active site, which is conducive to fundamentally destroying the symmetrical charge distribution around the metal center, thereby improving the catalytic activity.

[0020] (2) The nanoscale enzyme involved in the method is a carbon material, which has good biocompatibility, low price, high stability, and can be prepared in large quantities.

[0021] (3) The method can produce more toxic ROS by catalyzing H2O2 in cells, and can also efficiently consume GSH and generate H2O2 in situ, double-amplifying local oxidative stress, and to some extent, making up for the defect of insufficient H2O2 content in the tumor microenvironment, thereby improving the tumor treatment effect. DETAILED DESCRIPTION

[0022] In order for those skilled in the art to better understand the technical solutions of the present application, the present application will be further described in detail below in combination with the drawings and the best embodiments.

[0023] Example 1

[0024] (1) Preparation and characterization of Fe-S1N3 site anchored single-atom nanoscale enzyme on carbon-based material:

[0025] ① Preparation of Hemin / ZIF-8

[0026] 1.07g Zn(NO3)2·6H2O was added to 40mL methanol, and the obtained mixed solution was marked as A; then 2.35g dimethyl imidazole and 50mg hemin were added to 40mL methanol to obtain a mixed solution marked as B; then solution A was quickly added to solution B under vigorous stirring, and the reaction was carried out at room temperature for 24h; finally, the obtained product was centrifuged and washed several times, and vacuum dried at 70℃ for a night to obtain Hemin / ZIF-8;

[0027] ② Preparation of Hemin / ZIF-8@TTCA

[0028] 300mg Hemin / ZIF-8 was redispersed in 60mL methanol to obtain a mixed solution marked as C; then 30mg TTCA was dissolved in 40mL methanol to obtain a mixed solution marked as D, and then solution D was quickly added to solution C under vigorous stirring, and the reaction was carried out at room temperature for 30 minutes, followed by centrifugation and washing several times, and vacuum drying at 70℃ for a night, and the obtained product was marked as Hemin / ZIF-8@TTCA.

[0029] ③ Preparation of Fe-S / N-C

[0030] The Hemin / ZIF-8@TTCA product is transferred to a porcelain boat and placed in a tube furnace, heated to 920℃ (can be adjusted to 800℃, 1000℃ as needed, with 920℃ as the preferred embodiment) at a heating rate of 5℃ per second in a nitrogen atmosphere, and kept for 3h (can be adjusted to 1h, 2h as needed, with 3h as the preferred embodiment), to obtain a single-atom nanoscale enzyme with Fe-S1N3 sites anchored on a carbon base, denoted as Fe-S / N-C 300：30 .

[0031] Example 2

[0032] Example 2 differs from Example 1 in that the mass ratio of Hemin / ZIF-8 to TTCA in step ② is 300mg:10mg, to obtain a single-atom nanoscale enzyme with Fe-S1N3 sites anchored on a carbon base, denoted as Fe-S / N-C 300：10 .

[0033] Example 3

[0034] Example 3 differs from Example 1 in that the mass ratio of Hemin / ZIF-8 to TTCA in step ② is 300mg:50mg, to obtain a single-atom nanoscale enzyme with Fe-S1N3 sites anchored on a carbon base, denoted as Fe-S / N-C 300：50 .

[0035] Example 4

[0036] Example 4 differs from Example 1 in that the mass ratio of Hemin / ZIF-8 to TTCA in step ② is 300mg:70mg, to obtain a single-atom nanoscale enzyme with Fe-S1N3 sites anchored on a carbon base, denoted as Fe-S / N-C 300：70 .

[0037] Comparative Example

[0038] The preparation method of Fe-N-C adopts the following steps:

[0039] 1.07g Zn(NO3)2·6H2O is added to 40mL of methanol to obtain a mixed solution denoted as A; then 2.35g of dimethyl imidazole and 50mg of hemin are added to 40mL of methanol to obtain a mixed solution denoted as B; then solution A is quickly added to solution B under vigorous stirring, and the reaction is carried out at room temperature for 24h; finally, the obtained product is centrifuged and washed several times, and vacuum dried at 70℃ for one night to obtain Hemin / ZIF-8;

[0040] The Hemin / ZIF-8 product above was transferred to a porcelain boat and placed in a tube furnace, heated to 920℃ at a heating rate of 5℃ / s in a nitrogen atmosphere for 3h, to obtain Fe-N-C site anchored on carbon-based single atom nanozyme Fe-N-C.

[0041] Test characterization:

[0042] The prepared Fe-S / N-C SAzymes precursor Hemin / ZIF-8@TTCA was characterized by X-ray energy spectrometer, Fourier infrared spectrometer and ultraviolet-visible near infrared photometer;

[0043] The prepared Fe-S / N-C SAzymes was characterized by scanning electron microscope, transmission electron microscope, spherical aberration corrected transmission electron microscope and X-ray absorption spectrum.

[0044] Figure 1 The characterization maps of Hemin / ZIF-8 and Hemin / ZIF-8@TTCA in Example 1, wherein A is the transmission electron microscope image and element mapping image of Hemin / ZIF-8@TTCA, Figure 1 B is the Fourier infrared spectrum of Hemin / ZIF-8 and Hemin / ZIF-8@TTCA; and C is the ultraviolet spectrum of Hemin / ZIF-8 and Hemin / ZIF-8@TTCA, Figure 1 A indicates that the S element is successfully doped in the Hemin / ZIF-8@TTCA precursor; Figure 1 B and Figure 1 C prove the formation of Fe-S coordination bond.

[0045] Figure 2 The characterization results of Fe-S / N-C SAzymes in Example 1 are shown in the following figure: Figure 2 A is a scanning electron microscope image, Figure 2 B is a transmission electron microscope image, from Figure 2 From the characterization results of A and B, it can be seen that the prepared nanozyme is a hollow structure, the surface is invaginated, but basically maintains the rhombohedral dodecahedron structure of ZIF-8, no agglomerated iron particles are generated, and the size is about 200nm.

[0046] Figure 2 C is a spherical aberration corrected transmission electron microscope image, from Figure 2 From C, it can be clearly observed that the bright spots in the region are single Fe atoms. Further analysis of the coordination environment of Fe atoms by X-ray absorption spectrum shows that Fe atoms exist in the form of single Fe-S1N4 in Fe-S / N-C.

[0047] Figure 2D is a near-edge X-ray absorption fine structure diagram; in Figure 2 In D, the near-edge structure spectrum curve of Fe-S / N-C is located between FeO and Fe2O3, indicating that Fe δ+ (2<δ<3) is the oxidation state.

[0048] Figure 2 E is an extended X-ray absorption fine structure diagram; in Figure 2 In E, no Fe-Fe bond is detected in the FT-EXAFS spectrum of Fe K-edge, indicating the existence of single-atom Fe sites in Fe-S / N-C.

[0049] Figure 2 F is a k 3 -weighted Fe K-edge extended X-ray absorption fine structure Fourier transform diagram, from Figure 2 In F, the EXAFS best fitting result shows that each Fe atom is coordinated with 3 N atoms and 1 S atom. These characterizations show that a single-atom nanoscale enzyme (Fe-S / N-C) with Fe-S1N3 sites anchored on a carbon base is successfully prepared.

[0050] Figure 3 、 4 is a characterization result diagram of Fe-S / N-C of Example 1 and Fe-N-C of the comparative example; Figure 3 A is a UV-visible light absorption spectrum diagram of Fe-S / N-C and Fe-N-C of the comparative example catalyzing H2O2 to oxidize TMB, Figure 3 In A, Fe-S / N-C can catalyze H2O2 to oxidize colorless TMB into blue oxidized TMB (TMBox), and a characteristic absorption peak appears at 652 nm, proving that Fe-S / N-C has peroxidase-like activity and is higher than Fe-N4 Fe-N-C.

[0051] In addition, Figure 3 B is an ESR spectrum diagram, from Figure 3 In B, compared with Fe-N-C, Fe-S / N-C can catalyze H2O2 to produce more hydroxyl radicals.

[0052] Figure 3 C and Figure 4 D show that Fe-S / N-C can consume GSH faster and produce more H2O2 than Fe-N-C.

[0053] Figure 4 A is a UV light absorption spectrum diagram, Figure 4 In A, it is shown that the high-activity Fe-S / N-C prepared by the application has high biocompatibility.

[0054] In addition, Figure 4B is an electron paramagnetic resonance spectrum, Figure 4 B shows that Fe-S / N-C has a stronger killing effect on MCF-7 cells than Fe-N-C. Figure 4 C is a GSH consumption rate constant graph, Figure 4 C shows that the high-activity Fe-S / N-C prepared in the application can consume GSH in MCF-7 cancer cells faster than Fe-N-C.

[0055] Figure 4 D is a 2',7'-Dichlorodihydrofluorescein diacetate (DCFH-DA) laser confocal microscope image of MCF-7 cancer cells after treatment with Fe-S / N-C and Fe-N-C, Figure 4 D shows that the high-activity Fe-S / N-C prepared in the application can induce more ROS production in MCF-7 cancer cells.

[0056] In addition, relative to Example 1, the electron microscope results of the precursors Hemin / ZIF-8@TTCA of Examples 2, 3 and 4 are also tested (shown in Figure 5 , the electron microscope results of Fe-S / N-C (shown in Figure 6 ) and its ultraviolet-visible absorption spectrum (shown in Figure 7 ) are also shown, and the results show that the product Fe-S / N-C obtained in Example 1 300：30 has the highest activity, so the characterization of the examples is exemplarily illustrated with it as a preferred example.

[0057] The technical solution of the application aims to solve the problems of limited catalytic activity of typical M-N4 nanoscale enzymes and inaccurate positioning of hetero-element doping. Taking Hemin / ZIF-8 doped with hemin as a template and trithiocyanic acid (TTCA) as a sulfur source, the mercapto group in TTCA can form an Fe-S coordination bond with Fe in Hemin / ZIF-8, ensuring that S atoms can be directly coordinated with Fe atoms to form a core@shell precursor of Hemin / ZIF-8@TTCA. Then, further carbonization at high temperature can preserve the Fe-S bond, obtaining a single-atom nanoscale enzyme (Fe-S / N-C) with Fe-S1N3 coordination structure.

[0058] The asymmetric coordination configuration and electronic structure of Fe-S1N3 result in Fe-S / NC exhibiting higher peroxidase-like and glutathione oxidase activities compared to the typical Fe-N4 structured Fe-N-CSAzyme. Fe-S / NC can catalyze the production of more toxic ROS from H2O2 within tumor cells while simultaneously and efficiently consuming endogenous GSH, thus doubly amplifying local oxidative stress and enhancing in vitro ferroptosis-based tumor therapy. Furthermore, the in-situ generation of H2O2 accompanying the consumption of GSH by Fe-S / NC partially compensates for the insufficient H2O2 content in the tumor microenvironment.

[0059] The preparation method of the Fe-S1N3 coordinated dual-enzyme single-atom nanozyme with the properties described in this application is as follows:

[0060] (1) This method can achieve precise control of the proximal coordination of sulfur atoms at the active sites of the metal center, which is beneficial to fundamentally destroy the symmetrical charge distribution around the metal center, thereby improving its catalytic activity.

[0061] (2) The nanozymes involved in this method are carbon materials, which have good biocompatibility, are inexpensive, have high stability, and can be prepared in large quantities.

[0062] (3) This method can catalyze the production of more toxic ROS by H2O2 in cells, and can also efficiently consume GSH and generate H2O2 in situ, thus doubly amplifying local oxidative stress and making up for the deficiency of insufficient H2O2 content in the tumor microenvironment to a certain extent, thereby improving the tumor treatment effect.

[0063] 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 dual-enzyme properties and a Fe-SiN3 coordination structure, characterized in that, The process includes the following steps: using Hemin / ZIF-8 as a template and TTCA as a sulfur source, a core-shell precursor of Hemin / ZIF-8@TTCA is formed, which is then further carbonized at high temperature to obtain the final product.

2. The method for preparing the Fe-SiN3 coordinated dual-enzyme single-atom nanozyme with the properties described in claim 1, characterized in that, Hemin / ZIF-8 was prepared by mixing a methanol solution of zinc nitrate hexahydrate with a methanol solution of dimethylimidazole and hemolybdenum to obtain a precipitate of hemolybdenum-doped ZIF-8, which is Hemin / ZIF-8.

3. The method for preparing the Fe-SiN3 coordinated dual-enzyme single-atom nanozyme with special properties according to claim 2, characterized in that, The mass ratio of zinc nitrate hexahydrate, dimethylimidazole, and hemolybdenum is 1.7:2.35:0.

05.

4. The method for preparing the Fe-SiN3 coordinated dual-enzyme single-atom nanozyme with the properties described in claim 1, characterized in that, Specifically, the steps are as follows: 1) Add the methanol solution of TTCA to the methanol dispersion of Hemin / ZIF-8 to obtain the product Hemin / ZIF-8@TTCA; 2) Calcine Hemin / ZIF-8@TTCA at high temperature under an inert atmosphere to obtain the single-atom nanozyme Fe-S / NC with Fe-S1N3 sites anchored on the carbon matrix.

5. The method for preparing the Fe-SiN3 coordinated dual-enzyme single-atom nanozyme with the characteristics described in claim 4, characterized in that, The mass ratio of Hemin / ZIF-8 to TTCA is 300:70-300:

10.

6. The method for preparing the Fe-SiN3 coordinated dual-enzyme single-atom nanozyme with the characteristics described in claim 4, characterized in that, The calcination temperature in step 2) is 800℃-1000℃; the calcination time is 1-3h.

7. The method for preparing the Fe-SiN3 coordinated dual-enzyme single-atom nanozyme with the characteristics described in claim 4, characterized in that, The calcination temperature in step 2) is 920℃; the calcination time is 3h.

8. The application of a Fe-SiN3 coordination structure dual-enzyme single-atom nanozyme obtained by the preparation method according to any one of claims 1-7, characterized in that, It is used in the preparation of tumor drugs.

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