Preparation method and application of multi-enzyme mimetic active ni-fe-mn-cu-lDH

Abstract

The application discloses a preparation method of multi-enzyme mimetic active NiFeMnCu-LDH and application thereof, and the preparation method of the multi-enzyme mimetic active NiFeMnCu-LDH nanomaterial comprises the following steps: S1, dissolving soluble inorganic salts of Ni, Fe, Mn and Cu in deionized water to prepare solution A; S2, adjusting the pH of the solution A to a set range by using an alkaline solution B; S3, stirring the reaction under a set condition, and after the reaction is completed, washing the residual inorganic salts with deionized water and ethanol, and drying to obtain the multi-enzyme mimetic active NiFeMnCu-LDH nanomaterial. 2+ 3+ 2+ 2+ The size of the NiFeMnCu-LDH nanomaterial prepared by the method is about 50 nm, and the nanomaterial can target tumor tissues by enhancing permeability and retention (EPR) effect. The NiFeMnCu-LDH material prepared by the application can provide multi-enzyme mimetic activities of POD, OXD, CAT and GPx, can significantly catalyze H2O2 to produce reactive oxygen species (ROS) for resisting tumors, can also catalyze H2O2 to produce O2 for solving hypoxia in tumor sites, and can consume glutathione (GSH) for solving antioxidant effect in tumor sites. Therefore, the NiFeMnCu-LDH nanomaterial with the activities of POD, OXD, GPx and CAT has potential application in the field of resisting tumors.​​​

Description

Technical Field

[0001] This invention belongs to the field of two-dimensional metal nanomaterial preparation and biomedicine, specifically, it relates to a method for preparing multi-enzyme-mimicking NiFeMnCu-LDH nanomaterials and their applications. Background Technology

[0002] In recent years, the correlation between the regulation of various chemical indicators in the tumor microenvironment (TME) and the efficacy of tumor treatment has attracted much attention and research. Recent advances indicate that rationally designed nanomaterials can not only directly induce tumor cell death, but also achieve synergistic effects with radiotherapy, chemotherapy, and immunotherapy by precisely regulating chemical indicators such as oxygen partial pressure, pH, and redox state in the TME. Therefore, TME chemical regulation strategies based on nanomaterials show broad clinical application prospects in improving tumor hypoxia, neutralizing the tumor microacidic environment, reshaping redox balance, and regulating immune cell function. However, developing novel nanomaterials with multi-enzyme mimicry activity responsive to the TME still faces significant challenges.

[0003] Layered double hydroxides (LDHs) are a class of two-dimensional layered materials composed of positively charged metal hydroxide layers and anions located between the layers. Due to their diverse chemical compositions, simple preparation, structural stability, and excellent biocompatibility, they hold broad application prospects in biomedical fields such as drug delivery, bioimaging, and cancer therapy. Furthermore, their low cost, ease of large-scale synthesis, and environmental friendliness also contribute to their practical application potential. Therefore, by adjusting the composition of metal elements in LDHs, it is hoped to develop nanomaterials with multi-enzyme mimicry activities. These activities mainly include peroxidase (POD), catalase (CAT), oxidase (OXD), and glutathione peroxidase (GPx). Summary of the Invention

[0004] The technical problem to be solved by the present invention is to overcome the shortcomings of the prior art and provide a method for preparing multi-enzyme mimicry active NiFeMnCu-LDH and its application.

[0005] To achieve the above objectives, the technical solution of the present invention is as follows:

[0006] In a first aspect, the present invention provides a method for preparing multi-enzyme-mimicking NiFeMnCu-LDH nanomaterials, comprising the following steps:

[0007] S1, Ni 2+Fe 3+ Mn 2+ Cu 2+ Soluble inorganic salts are dissolved in deionized water to prepare solution A;

[0008] S2. Adjust the pH of solution A to the set range using alkaline solution B;

[0009] S3. Stir the reaction under the set conditions. After the reaction is complete, wash off the excess inorganic salt with deionized water and ethanol, and dry to obtain NiFeMnCu-LDH nanomaterials with multi-enzyme mimicry activity.

[0010] Furthermore, in step S1, based on the molar ratio of metal ions, Ni 2+ Fe 3+ Mn 2+ Cu 2+ The amount of soluble inorganic salt added is (Ni 2+ +Cu 2+ ): (Fe 3+ +Mn 2+ =3:1.

[0011] Furthermore, in step S1, Ni 2+ Fe 3+ Mn 2+ Cu 2+ The soluble inorganic salts are selected from nitrates, sulfates and chlorides that are soluble in water.

[0012] Furthermore, in step S2, the pH setting range is 8 to 11.

[0013] Furthermore, in step S2, alkaline solution B is prepared by dissolving an excess of intercalated anionic salt and alkali in deionized water, wherein...

[0014] The intercalated anionic salt includes at least one of Na2CO3 and K2CO3;

[0015] The alkali includes at least one of NaOH and KOH.

[0016] Furthermore, in step S3, the set conditions include a reaction temperature of 25~100℃ and a reaction time of 1~24 hours.

[0017] Secondly, the present invention also provides a multi-enzyme-mimicking NiFeMnCu-LDH nanomaterial prepared by the preparation method described above.

[0018] Thirdly, the present invention also provides the application of the multi-enzyme-mimicking active NiFeMnCu-LDH nanomaterials as described above in anti-liver cancer drugs.

[0019] By adopting the above technical solution, the present invention has the following beneficial effects compared with the prior art:

[0020] (1) This invention is the first to use the water bath method to prepare nano-sized NiFeMnCu-LDH, and the synthesis method is simple, the particle size is small and the energy consumption is low.

[0021] (2) The NiFeMnCu-LDH nanomaterials prepared by the method of this invention have a size of approximately 50 nm and can target tumor tissues through enhanced permeability and retention (EPR). The NiFeMnCu-LDH materials prepared by this invention can provide multi-enzyme mimicry activities such as POD, OXD, CAT, and GPx. They can significantly catalyze the production of reactive oxygen species (ROS) from H2O2 for anti-tumor purposes, and can also catalyze the production of O2 from H2O2 to address hypoxia at the tumor site, as well as consume glutathione (GSH) to address antioxidant effects at the tumor site. Therefore, these NiFeMnCu-LDH nanomaterials with POD, OXD, GPx, and CAT activities have potential applications in the field of anti-tumor therapy. Attached Figure Description

[0022] The accompanying drawings, as part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments and descriptions of the invention are used to explain the invention, but do not constitute an undue limitation of the invention. Obviously, the drawings described below are merely some embodiments, and those skilled in the art can obtain other drawings based on these drawings without creative effort. In the drawings:

[0023] Figure 1 These are the XRD patterns of NiFeMnCu-LDH, NiFeMn-LDH, NiFeCu-LDH, and NiFe-LDH in the embodiments of the present invention.

[0024] Figure 2 This is an AFM height profile of NiFeMnCu-LDH in an embodiment of the present invention.

[0025] Figure 3 This is a hydrodynamic diameter distribution diagram of NiFeMnCu-LDH in an embodiment of the present invention.

[0026] Figure 4 a is a TEM image of NiFeMnCu-LDH in an embodiment of the present invention.

[0027] Figure 4b is the HRTEM image of NiFeMnCu-LDH in the embodiment of the present invention.

[0028] Figure 4 c is the SAED diagram of NiFeMnCu-LDH in the embodiment of the present invention.

[0029] Figure 4 di is the elemental distribution diagram of NiFeMnCu-LDH in the embodiment of the present invention.

[0030] Figure 5 ab represents the oxidation capacity of NiFeMnCu-LDH against 3,3',5,5'-tetramethylbenzidine (TMB) in 0.2 M NaAc / HAc buffer solutions at different pH values ​​in the embodiments of this invention. The experimental conditions were 10 µg / mL. -1 NiFeMnCu-LDH, 1 mMMTMB, 1 mM H2O2, reaction time 5 min.

[0031] Figure 5 c represents the TMB content in this embodiment of the invention, with or without 10 µg / mL of 0.2 M NaAc / HAc buffer (pH=3.5), 1 mM TMB. -1 Absorbance spectra of LDH in different reaction systems with and without 1 mM H2O2.

[0032] Figure 5 de is the Michaelis-Menten curve of NiFeMnCu-LDH against H2O2 and TMB substrates in the embodiments of the present invention.

[0033] Figure 5 f represents the specific POD activity (U mg) of NiFeMnCu-LDH at different pH values ​​in the embodiments of the present invention. -1 The determination is as follows: One nanozyme activity unit (U) is defined as the amount of nanozyme that catalyzes 1 μmol of product per minute.

[0034] Figure 5 g refers to the amount of 10 µg / mL added or not added in different reaction systems containing 0.2 M NaAc / HAc buffer (pH=3.5) and 1 mM TMB in the embodiments of the present invention. -1 The absorbance spectrum of TMB was measured after reacting with LDH for 5 min.

[0035] Figure 5 h is the Michaelis-Menten curve of NiFeMnCu-LDH against the TMB substrate in an embodiment of the present invention.

[0036] Figure 5i represents the OXD specific activity (U mg) of NiFeMnCu-LDH at different pH values ​​in the embodiments of the present invention. -1 ) Measurement.

[0037] Figure 6 'a' represents the dissolved oxygen concentration detected during the reaction process using a specific oxygen electrode in this embodiment of the invention. The reaction conditions are: 10 µg mL -1 LDH, 40 mM H2O2, 0.2 M NaAc / HAc buffer (pH=7.5).

[0038] Figure 6 b illustrates the effect of different concentrations of H2O2 on dissolved oxygen production in the embodiments of this invention. Reaction conditions: 10 µg mL -1 NiFeMnCu-LDH, 0 ~ 100 mM H2O2, 0.2 M NaAc / HAc buffer (pH=7.5).

[0039] Figure 6 c is the Michaelis-Menten curve of NiFeMnCu-LDH on H2O2 substrate in an embodiment of the present invention.

[0040] Figure 6 d represents the CAT specific activity (U mg) of NiFeMnCu-LDH at different pH values ​​in the embodiments of the present invention. -1 ) Measurement.

[0041] Figure 6 e refers to the addition of 10 µg mL to different reaction systems containing 0.2 M NaAc / HAc buffer (pH=7.5) and 0.2 mM GSH in the embodiments of the present invention. -1 The absorbance spectra of NiFeMnCu-LDH with and without 0.2 mM H2O2 at different reaction times were obtained. DTNB was used as the index.

[0042] Figure 6 f represents a comparison of the GPx activities of different samples in the embodiments of the present invention.

[0043] Figure 6 gh is the Michaelis-Menten curve of NiFeMnCu-LDH against H2O2 and GSH substrates in the embodiments of the present invention.

[0044] Figure 6 i represents the GPx specific activity (U mg) of NiFeMnCu-LDH at different pH values ​​in the embodiments of the present invention. -1 ) Measurement.

[0045] Figure 7a is a schematic diagram of LDH treatment for hepatocellular carcinoma in an embodiment of the present invention.

[0046] Figure 7 b is the tumor volume of mice after different LDH treatments in this embodiment of the invention (data are expressed as mean ± standard deviation, n=5, and the p value is determined by two-way ANOVA combined with Tukey post-hoc test).

[0047] Figure 7 c is the body weight of mice after different LDH treatments in this embodiment of the invention (data are expressed as mean ± standard deviation, n=5, p value is determined by two-way ANOVA combined with Tukey post-hoc test).

[0048] Figure 7 d represents the tumor weight of mice after different LDH treatments in this embodiment of the invention (data are expressed as mean ± standard deviation, n=5, and p-value is determined by two-way ANOVA combined with Tukey post-hoc test).

[0049] Figure 7 e is a gross image of mouse tumors after different LDH treatments in this embodiment of the invention (n=5).

[0050] Figure 7 f shows the H&E staining of mouse tumors after different LDH treatments in this embodiment of the invention. Scale bar = 50 µm.

[0051] Figure 7 g represents Ki-67 staining of mouse tumors after different LDH treatments in this embodiment of the invention. Scale bar = 50 µm.

[0052] *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0053] Figure 8 This is an analysis of liver (alanine aminotransferase, aspartate aminotransferase and albumin) and kidney function (creatinine) of mice in different LDH treatment groups in this invention (data are expressed as mean ± standard deviation, n=5).

[0054] Figure 9 These are H&E staining images of major organs in mice from different LDH treatment groups in this embodiment of the invention. Scale bar = 50µm.

[0055] Figure 10 This is a schematic diagram of the planar structure and anti-cancer effect of the NiFeMnCu-LDH nanomaterial of this invention.

[0056] It should be noted that these accompanying drawings and textual descriptions are not intended to limit the scope of the invention in any way, but rather to illustrate the concept of the invention to those skilled in the art by referring to specific embodiments. Detailed Implementation

[0057] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments will be clearly and completely described below with reference to the accompanying drawings. The following embodiments are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0058] This invention provides a novel NiFeMnCu-LDH prepared by a water bath method, which can provide enzyme activities such as POD, OXD, CAT and GPx in the weakly acidic environment of tumors, and therefore has potential applications in tumor treatment.

[0059] In this embodiment, the LDH material with POD, OXD, CAT and GPx activity is NiFeMnCu-LDH powder material, and the raw materials are Ni(NO3)2·6H2O, Fe(NO3)3·9H2O, Cu(NO3)2·3H2O, Mn(NO3)2, and the inorganic salts are Na2CO3 and NaOH. These raw materials can be obtained through commercial channels.

[0060] In this embodiment, the synthesis method of NiFeMnCu-LDH is as follows:

[0061] Solution A is prepared by dissolving 1.3 mmol Ni(NO3)2·6H2O, 0.3 mmol Fe(NO3)3·9H2O, 0.2 mmol Cu(NO3)2·3H2O, and 0.2 mmol Mn(NO3)2 in 100 mL of deionized water. Solution B is prepared by dissolving 12.5 mmol NaOH and 2.5 mmol Na2CO3 in 50 mL of deionized water. Solution B is then added dropwise to solution A. After adjusting the pH of the solution to 10, the mixture is stirred at 65 °C for 12 hours.

[0062] Subsequently, the sample was washed five times by centrifugation with deionized water and twice by centrifugation with ethanol.

[0063] Finally, the sample was dried in an oven at 60°C to obtain NiFeMnCu-LDH powder.

[0064] In comparison, this embodiment also provides methods for synthesizing NiFeCu-LDH, NiFeMn-LDH, and NiFe-LDH. Compared with the synthesis method of NiFeMnCu-LDH, only the molar amount of the metal nitrate is different while keeping other preparation conditions unchanged. Specifically:

[0065] For NiFeCu-LDH, the molar composition is Ni(NO3)2·6H2O (1.3 mmol), Fe(NO3)3·9H2O (0.5 mmol) and Cu(NO3)2·3H2O (0.2 mmol);

[0066] For NiFeMn-LDH, the molar composition is Ni(NO3)2·6H2O (1.5 mmol), Fe(NO3)3·9H2O (0.3 mmol) and Mn(NO3)2 (0.2 mmol);

[0067] For NiFe-LDH, the molar composition is Ni(NO3)2·6H2O (1.5 mmol) and Fe(NO3)3·9H2O (0.5 mmol).

[0068] It should be noted that in the above preparation process, the Ni source, Fe source, Mn source, and Cu source include, but are not limited to, water-soluble inorganic salts such as nitrates, sulfates, and chlorides. The Ni source is determined by the molar ratio of metal ions. 2+ Fe 3+ Mn 2+ Cu 2+ The amount of soluble inorganic salt added is (Ni 2+ +Cu 2+ ): (Fe 3+ +Mn 2+ =3:1;

[0069] Solution B contains an excess of intercalated anionic salt and base. The intercalated anion is selected from any one or a combination of two of Na2CO3 and K2CO3, and the base is selected from any one or a combination of two or more of NaOH and KOH.

[0070] Figure 1 The XRD patterns of the NiFeMnCu-LDH, NiFeCu-LDH, NiFeMn-LDH, and NiFe-LDH materials prepared in this embodiment are shown. As can be seen from the XRD patterns, all synthesized samples exhibit crystal diffraction characteristics highly consistent with the NiFe-LDH standard card (JCPDS 40-0215). Obvious diffraction peaks were observed at characteristic diffraction angles of 11.4°, 22.9°, and 34.4°, with peak position shifts less than 0.2° and relative intensities perfectly matching the standard card data. This confirms that the samples successfully maintained a typical layered double hydroxide (LDH) crystal structure. Notably, the sharp morphology of the (003) and (006) diffraction peaks indicates a highly ordered interlayer stacking structure, while the broadened (110) diffraction peak near 60° reveals the short-range ordered arrangement of Ni / Fe-O octahedra within the layers.

[0071] Figure 2 This is an AFM high-resolution cross-sectional view of NiFeMnCu-LDH in this embodiment. AFM morphology analysis shows that the synthesized nanosheets exhibit a regular two-dimensional layered structure with a statistically estimated thickness of approximately 2.5 nm and a lateral width distribution of around 50 nm. This narrow particle size exhibits a good EPR effect and can accumulate in tumor tissue.

[0072] Figure 3 This figure shows the dynamic light scattering (DLS) hydrodynamic diameter distribution of NiFeMnCu-LDH in aqueous phase in this embodiment. Data shows that the material exhibits a single-peak distribution in its dispersed state, with the main peak located around 300 nm, and the overall size concentrated between 200 and 600 nm. Notably, this value is significantly larger than the size of a single nanosheet observed by AFM / TEM (approximately 50 nm), which is attributed to the apparent size expansion caused by the hydration layer on the nanosheet surface and slight edge curling during DLS detection. Nevertheless, the absence of micron-sized aggregates (such as secondary peaks >1 μm) in the system indicates that the material achieves good colloidal stability through the electrostatic repulsion of surface hydroxyl groups or adsorbed ions, without significant aggregation. This characteristic is crucial for maintaining circulation time in vivo and preventing vascular embolism, making it beneficial for in vivo experiments.

[0073] Figure 4 a is a TEM image of NiFeMnCu-LDH in this embodiment. The results show that the average size of a single nanosheet is about 50 nm, which is in high agreement with the AFM measurement results, indicating that the material did not undergo significant structural breakage or aggregation during liquid-phase dispersion and sample preparation.

[0074] Figure 4 b is an HRTEM image of NiFeMnCu-LDH in this embodiment. The image clearly shows lattice fringes with a spacing of 0.23 nm, corresponding to the (015) crystal plane (JCPDS 40-0215) of NiFe-LDH. This result is corroborated by the (015) diffraction peak at 34.4° in the XRD analysis, confirming that the typical layered crystal structure of LDH is maintained after quaternary metal doping. Furthermore, the continuity and distortion-free nature of the lattice fringes indicate that the material has high crystallinity.

[0075] Figure 4 c represents the SAED pattern of NiFeMnCu-LDH in this embodiment. The SAED pattern shows a series of concentric diffraction rings, which, after calibration, correspond to the (012) and (113) crystal planes of LDH, respectively. The continuity of the diffraction rings indicates that the material has a polycrystalline structure, while the ring distribution characteristics reflect the random orientation arrangement of the grains within the nanosheets. This structural characteristic originates from the self-assembly growth mechanism of the layers during hydrothermal synthesis.

[0076] Figure 4 di is the elemental distribution map of NiFeMnCu-LDH in this embodiment. The spatial distribution of five elements (Ni, Fe, Mn, Cu, and O) was characterized by an elemental surface mapping (EDSmapping) system. The results show that all metal elements (Ni, Fe, Mn, Cu) exhibit a highly uniform distribution within the nanosheet region, completely overlapping with the oxygen distribution, and no elemental segregation or independent phase formation was observed. This phenomenon confirms the presence of transition metal ions (Mn, Fe, Mn, Cu) in the nanosheet region. 2+ , Cu2+ The LDH layer was successfully embedded through isomorphous substitution, forming a stable solid solution structure. The uniform elemental distribution will help to exert the synergistic effect.

[0077] Figure 5 The pH-dependent peroxidase (POD) activity of NiFeMnCu-LDH was investigated using the ab system, specifically the oxidation capacity of NiFeMnCu-LDH on 3,3′,5,5′-tetramethylbenzidine (TMB) in 0.2 M NaAc / HAc buffer solutions at different pH values. The experimental conditions were 10 µg / mL... -1 NiFeMnCu-LDH, 1 mM TMB, 1 mM H2O2, reaction time 5 min. The results showed that the POD activity of the material reached its peak under acidic conditions at pH 3.5. 0.183 U mg-1 Its kinetic curve exhibits a typical bell-shaped distribution. This phenomenon can be attributed to the protonation regulation of metal active sites in the LDH layer: under acidic conditions, Cu2+ / Mn3+ The hydroxyl groups (-OH) on the site surface are protonated to -H2O+ Significantly enhanced H2O2 The adsorption and heterolytic cleavage of TMB generate highly reactive hydroxyl radicals (·OH), thereby driving the oxidation and color development of TMB. However, under weakly acidic conditions (pH 6.5), although the activity is significantly reduced (…), the reaction proceeds smoothly. 0.0051 U mg-1 Although a weak catalytic ability was still detected, this suggests that the material still has the potential to trigger the generation of reactive oxygen species (ROS) in the tumor microenvironment (pH about 6.5), providing a chemical basis for subsequent anti-tumor applications.

[0078] Furthermore, we further evaluated the POD activity of different samples under optimal pH conditions. Figure 5 c represents the TMB content in this embodiment of the invention, with or without 10 µg / mL of 0.2 M NaAc / HAc buffer (pH=3.5), 1 mM TMB. -1 Absorbance spectra of LDH in different reaction systems with and without 1 mM H2O2. Control group (TMB+H2O2 The absence of the characteristic blue-violet color (no change in absorbance at 650 nm) indicates that... H2O2 TMB oxidation cannot be driven independently. Notably, the color intensity of the quaternary doped NiFeMnCu-LDH is significantly higher than that of the binary (NiFe-LDH) and ternary (NiFeMn / Cu-LDH) systems, with the POD activity in the order of NiFeMnCu-LDH > NiFeCu-LDH > NiFeMn-LDH > NiFe-LDH. This result confirms... Cu2+ The introduction of [a specific ingredient] plays a dominant role in enhancing POD activity (Δ activity increase of approximately 70%), while Mn [another ingredient]... 2+ It may exert a synergistic effect by adjusting the electronic structure of the LDH layer or stabilizing the oxidation state of the Cu active center.

[0079] Figure 5 The graphs (de) of NiFeMnCu-LDH with H2O2 and TMB substrates in this embodiment of the invention are Michaelis-Menten curves. This analysis further reveals the catalytic mechanism. H2O2 When TMB is used as the substrate, the Michaelis constant of NiFeMnCu-LDH ( K M ) are respectively 0.10×10-3 M and 3.11×10-3 M This indicates that the material has a significantly higher affinity for H2O2 than TMB. H2O2 Affinity (1 / K M The substrate selectivity was approximately 31 times that of TMB. This difference in substrate selectivity may stem from... H2O2 The smaller molecular size and steric hindrance make it easier to access the metal active sites in the interlaminar spaces. Simultaneously, the high affinity for H₂O₂ indicates that the material can efficiently utilize the low concentration of H₂O₂ in the tumor microenvironment. H2O2 (Approximately 10-100 µM), which is of great significance for the treatment of solid tumors.

[0080] Figure 5 f represents the specific activity (U mg) of NiFeMnCu-LDH at different pH values ​​in this embodiment. -1 The specific activity of NiFeMnCu-LDH at pH 3.5 and 6.5 was determined to be 0.183 U mg. One nanozyme activity unit (U) is defined as the amount of nanozyme that catalyzes 1 μmol of product per minute. -1 and 0.0051 U mg -1 Although the decrease in acidity reduces POD activity, NiFeMnCu-LDH still maintains a certain level of POD activity at pH 6.5, with its residual activity significantly higher than that of traditional metal oxide catalysts (such as Fe3O4, typically around 0.0012 U mg). -1This pH adaptability indicates that NiFeMnCu-LDH can continuously generate reactive oxygen species (ROS) through the weakly acidic environment (pH 6.5-7.0) specific to tumor tissue, thus enabling its anti-tumor effects.

[0081] We systematically evaluated the oxidase (OXD) activity of different doping systems. Figure 5 g represents the amount of 10 µg / mL added or not added in different reaction systems containing 0.2 M NaAc / HAc buffer (pH=3.5) and 1 mM TMB, as described in the embodiments of the present invention. -1 The absorbance spectrum of TMB was measured for LDH with a reaction time of 5 min. The results showed that the OXD activity of NiFeMnCu-LDH was significantly higher than that of Mn-doped and Cu-doped NiFe-LDH, indicating that Cu and Mn sites have a synergistic effect on O2 catalysis.

[0082] Figure 5 h represents the steady-state kinetic analysis of the TMB substrate for NiFeMnCu-LDH in this embodiment. K M The value is 0.02 × 10 -3 M indicates that the material has ultra-high sensitivity to chromogenic substrates, making it suitable for in vitro detection or in vivo real-time imaging.

[0083] Figure 5 i represents the specific activity of NiFeMnCu-LDH in this embodiment at pH 3.5 and 6.5, which are 0.0895 Umg, respectively. -1 and 0.0145 U mg -1 Therefore, it can be inferred that NiFeMnCu-LDH has the ability to generate ROS using O2 in the tumor microenvironment, which could be used for anti-tumor purposes.

[0084] Figure 6 In this embodiment, 40 mM H2O2 did not decompose to produce oxygen within 15 minutes in a buffer solution at pH 7.5. However, after adding NiFeMnCu-LDH, we detected a significant increase in oxygen production. Furthermore, the CAT activity of NiFeMnCu-LDH was significantly higher than that of Cu-doped and Mn-doped NiFeMnCu-LDH, indicating that Cu and Mn sites have a synergistic catalytic effect in promoting H2O2 decomposition.

[0085] Figure 6 bc explored further H2O2 The effect of concentration on CAT activity. As the H2O2 concentration increases, the oxygen generation rate gradually increases. Nonlinear fitting shows its Michaelis constant (…). K M )for 78.8×10-3 M .

[0086] Figure 6 d represents the CAT specific activity of NiFeMnCu-LDH under different pH conditions in this embodiment. The activities at physiological pH 7.5 and tumor-microacidic pH 6.5 are respectively... 0.448 U mg-1 and 0.446 U mg-1 The differences were not significant. This indicates that the CAT activity of NiFeMnCu-LDH is insensitive to pH changes, maintaining high catalytic performance in both tumor tissue (pH approximately 6.5) and normal tissue (pH approximately 7.4). NiFeMnCu-LDH can effectively catalyze the decomposition of H2O2 in the tumor microenvironment to produce O2, thereby effectively alleviating tumor hypoxia. The generated O2 can further promote the oxidation reaction cascade, leading to an increase in ROS production.

[0087] Figure 6 In this embodiment, in a buffer solution at pH 7.5, the absorbance at 412 nm for the NiFeMnCu-LDH + GSH group showed no significant difference between 0 min and 20 min reaction times, indicating that the sample possesses negligible glutathione oxidase (GSHOx) activity. Interestingly, the GSH content decreased in the GSH+H2O2 group, indicating that H2O2 has an oxidizing effect on GSH. After adding NiFeMnCu-LDH, the absorbance at 412 nm decreased significantly, indicating that it possesses GPx-like activity.

[0088] Figure 6 f indicates that all samples used in this embodiment exhibit a certain level of GPx activity. However, the catalytic performance of NiFeMnCu-LDH is significantly better than that of samples doped with Cu and Mn alone, indicating that Cu and Mn sites have a synergistic effect in enhancing GPx activity.

[0089] Figure 6 gh represents the steady-state kinetic analysis of NiFeMnCu-LDH on H2O2 and GSH in this embodiment. The results show that NiFeMnCu-LDH on H2O2 and GSH... H2O2 and GSH K M The values ​​are respectively 0.20×10-3 M and 0.45×10-3 M Lower H2O2 K M The value (high affinity) indicates that the material has low affinity. H2O2 It can still efficiently start the catalytic cycle at high concentrations, while higher concentrations of GSH and K... MThe value suggests that it is more suitable for the GSH-enriched tumor cytoplasmic environment (GSH concentration of approximately 10 mM). This difference in substrate selectivity helps to specifically activate GPx-like activity in tumor cells, avoiding off-target effects on normal cells.

[0090] Figure 6 i shows that the GPx activity of NiFeMnCu-LDH is pH-dependent: the specific activity at pH 7.5 is 0.124 U mg-1 , while at pH 6.5 it drops to 0.0772 U mg-1 The material retains its basic activity in the tumor microenvironment and can synergistically consume GSH and H2O2 It disrupts the redox balance (the GSH / GSSG ratio decreases), inducing tumor cell death.

[0091] Figure 7 The in vivo antitumor efficacy and safety of the material were verified using a tumor-bearing mouse model. [The material will carry...] 50 mm3 Mice with tumors were randomly divided into 5 groups (n=5) and treated with PBS, NiFe-LDH, NiFeMn-LDH, NiFeCu-LDH, and NiFeMnCu-LDH (tail vein injection, 5 mg / kg, q2d × 3 weeks), respectively. Results showed:

[0092] Regarding the tumor volume growth rate, both NiFeMn-LDH and NiFeCu-LDH treatments could inhibit tumor growth to some extent, but this phenomenon was more significant in the NiFeMnCu-LDH group. Figure 7 b).

[0093] There was no significant difference in mean weight change among all groups of mice, indicating the high safety and low toxicity of NiFe-LDH-based mice. Figure 7 c).

[0094] Figure 7 d represents tumor samples collected from mice in each group after 3 weeks of treatment in this embodiment. The results showed that the tumor weight of mice in the NiFeMnCu-LDH treatment group was significantly lower than that in the control group and other treatment groups, demonstrating the excellent anti-tumor effect of NiFeMnCu-LDH.

[0095] Figure 7 The image above shows gross images and H&E staining of mouse tumor tissues treated with different LDHs in this embodiment. Compared with the control group, tumors treated with NiFeMnCu-LDH showed significant tumor inhibition.

[0096] Figure 7Further immunohistochemical staining in this embodiment showed that the expression level of nuclear protein Ki67 was high in the control group and NiFe-LDH group, while its expression level was significantly downregulated after NiFeMnCu-LDH treatment.

[0097] Figure 8 In this embodiment, the liver and kidney function of mice was tested and analyzed. The results showed that there were no significant differences in the main liver and kidney function indicators among the groups, further highlighting the safety of the materials used.

[0098] Figure 9 Histological evaluation of the heart, liver, spleen, lungs and kidneys of mice in each group in this embodiment showed that, compared with the PBS group, the treatment group had no significant pathological damage, indicating consistent biocompatibility and low toxicity.

[0099] These results indicate that NiFeMnCu-LDH possesses reliable biosafety and potent in vivo antitumor activity, and is expected to become a new option for the clinical treatment of liver cancer.

[0100] In summary, this invention is the first discovery of NiFeMnCu-LDH nanomaterials with multiple enzyme activities including POD, OXD, CAT, and GPx. These enzyme activities can effectively improve tumor hypoxia, induce ROS production, and regulate GSH molecules, showing broad application prospects in the field of anti-tumor therapy.

[0101] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0102] It will be understood by those skilled in the art that, unless otherwise defined, all terms used herein (including technical and scientific terms) have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. It should also be understood that terms such as those defined in general dictionaries should be understood to have the same meaning as in the context of the prior art, and should not be interpreted in an idealized or overly formal sense unless defined as herein.

[0103] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-described technical content to create equivalent embodiments without departing from the scope of the present invention. The implementation schemes in the above embodiments can also be further combined or replaced. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A method for preparing multi-enzyme-mimicking NiFeMnCu-LDH nanomaterials, characterized in that, Includes the following steps: S1, Ni 2+ Fe 3+ Mn 2+ Cu 2+ Soluble inorganic salts are dissolved in deionized water to prepare solution A; S2. Adjust the pH of solution A to the set range using alkaline solution B; S3. Stir the reaction under the set conditions. After the reaction is complete, wash off the excess inorganic salt with deionized water and ethanol, and dry to obtain NiFeMnCu-LDH nanomaterials with multi-enzyme mimicry activity. In step S1, Ni is calculated based on the molar ratio of metal ions. 2+ Fe 3+ Mn 2+ Cu 2+ The amount of soluble inorganic salt added is (Ni 2+ +Cu 2+ ): (Fe 3+ +Mn 2+ =3:1; In step S1, Ni 2+ Fe 3+ Mn 2+ Cu 2+ The soluble inorganic salts are selected from nitrates, sulfates, and chlorides that are soluble in water; In step S2, the pH setting range is 8~11; In step S2, alkaline solution B is prepared by dissolving an excess of intercalated anionic salt and alkali in deionized water. The intercalated anionic salt includes at least one of Na2CO3 and K2CO3; The alkali includes at least one of NaOH and KOH; In step S3, the set conditions include a reaction temperature of 25~100℃ and a reaction time of 1~24 hours.

2. A multi-enzyme-mimicking NiFeMnCu-LDH nanomaterial prepared by the preparation method described in claim 1.

3. The application of the multi-enzyme-mimicking NiFeMnCu-LDH nanomaterial as described in claim 2 in the preparation of anti-liver cancer drugs.

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