Cascade nano-enzyme with metabolic reprogramming function as well as preparation method and application of cascade nano-enzyme

Abstract

The invention discloses a cascaded nano-enzyme with metabolic reprogramming as well as a preparation method and application of the cascaded nano-enzyme. The method comprises the following steps: firstly, synthesizing manganese and cobalt-based Prussian blue nanoparticles by adopting a precipitation method, and then carrying out calcination treatment in an ammonia gas atmosphere to finally obtain the target nano-enzyme. The nano-enzyme has the catalytic activity of lactate oxidase (LOX) and catalase (CAT) at the same time, and continuous cascade reactions of oxygen, lactic acid, pyruvic acid and hydrogen peroxide and hydrogen peroxide, oxygen and water can be achieved. Compared with a natural LOX enzyme with the same mass concentration, the nano enzyme shows more excellent catalytic activity and temperature stability in an acid environment (pH = 5). The nano enzyme has the following advantages: 1) metabolism regulation is realized through cascade catalytic reaction, and lactic acid is effectively consumed; 2) an anti-oxidation treatment effect is achieved by removing hydrogen peroxide; and 3) the pathological microenvironment of inflammatory tissues can be obviously improved. The invention provides a novel nano-enzyme solution for metabolism regulation and anti-oxidation treatment.

Description

Technical Field

[0001] This invention belongs to the fields of biomaterials, tissue engineering and regenerative medicine, specifically relating to a cascade nanozyme with metabolic reprogramming function, its preparation method and application. Background Technology

[0002] Under pathological conditions (such as inflammation and tumors), cells undergo metabolic pathway alterations to adapt to changes in the microenvironment, with enhanced glycolysis and lactate accumulation being key characteristics. Lactic acid is not only the end product of glycolysis but also a crucial molecule regulating cellular metabolism and the microenvironment. Studies have shown that reducing lactate concentration can effectively inhibit glycolysis in damaged tissues, thereby mitigating the fibrotic tendency of damaged skin and myocardial tissues through metabolic reprogramming. Therefore, reducing lactate concentration has become an important target for regulating glucose metabolism and has potential applications in inflammation treatment and tissue repair.

[0003] However, inflamed tissues are often in a hypoxic state, and oxygen is an essential substrate for lactate oxidase (LOX) to convert lactate to pyruvate. Under hypoxic conditions, the catalytic activity of LOX is significantly limited, leading to ineffective lactate removal and further exacerbating the pathological microenvironment of the tissue. Furthermore, hydrogen peroxide generated during LOX catalysis accumulates and triggers oxidative stress, worsening tissue damage. Therefore, developing a catalytic system capable of efficiently removing lactate and simultaneously eliminating hydrogen peroxide under hypoxic conditions is crucial to solving this problem.

[0004] In recent years, nanozymes have attracted widespread attention due to their high stability, tunable catalytic activity, and multifunctionality. By designing nanozymes with cascade catalytic functions, lactate oxidation and hydrogen peroxide scavenging can be achieved simultaneously, thereby improving the pathological microenvironment of inflamed tissues. These nanozymes can not only efficiently catalyze lactate under hypoxic conditions but also exert antioxidant effects by scavenging hydrogen peroxide, providing a new therapeutic strategy for metabolic reprogramming and tissue repair.

[0005] Based on the above background, this invention proposes a cascaded nanozyme with metabolic reprogramming function, which aims to solve the problems of low lactate clearance efficiency and hydrogen peroxide accumulation under hypoxic conditions, and provide a new technical means for inflammation treatment and tissue repair. Summary of the Invention

[0006] This invention addresses the following problems in existing technologies: Inflammatory tissues are typically in a hypoxic state, while oxygen is a necessary substrate for lactate oxidase (LOX) to convert lactate to pyruvate, leading to low lactate clearance efficiency under hypoxic conditions; hydrogen peroxide produced during LOX catalysis accumulates and triggers oxidative stress, exacerbating tissue damage; furthermore, natural LOX exhibits low catalytic activity and poor stability under acidic conditions (e.g., pH=5), making it difficult to adapt to the typical acidic microenvironment of inflamed tissues. Considering that the pH of inflamed tissues is often lower than normal physiological levels, typically around 5.0, evaluating LOX performance at pH=5 more accurately reflects its application effect under pathological conditions. To solve the above technical problems, this invention provides a cascade nanozyme with metabolic reprogramming function, its preparation method, and its application. The technical solution is as follows:

[0007] The cascaded nanozyme of the present invention is prepared by means of the following steps:

[0008] a) Manganese and cobalt-based Prussian blue nanoparticles Mn3[Co(CN)6]2 were synthesized by precipitation method;

[0009] b) The nanoparticles obtained in a) are calcined under an ammonia atmosphere. Preferably, the calcination temperature is 400-550℃ and the calcination time is 2-5 hours to obtain the cascaded nanozyme with a porous structure.

[0010] The nanozyme possesses dual catalytic activity as both lactate oxidase (LOX) and catalase (CAT), enabling it to catalyze cascade reactions of "oxygen + lactic acid → pyruvate + hydrogen peroxide" and "hydrogen peroxide → oxygen + water." Under acidic conditions at pH 5, its catalytic activity is significantly higher than that of natural LOX enzymes of the same mass concentration, reaching 2.1 times, 1.5 times, and 2.5 times that of natural LOX enzymes at 25℃, 37℃, and 60℃, respectively.

[0011] Further, the precipitation method described in step a) specifically includes: dissolving potassium cobalt cyanate and manganese acetate in deionized water containing citric acid, stirring, letting stand overnight, centrifuging, and vacuum drying to obtain Mn3[Co(CN)6]2 nanoparticles. Preferably, the ratio of potassium cobalt cyanate, manganese acetate, and citric acid is 0.6 mmol: 1.13 mmol: 0.5 mmol.

[0012] Furthermore, the optimized conditions for the calcination process in step b) are as follows: heating to 550℃ under a nitrogen atmosphere, holding at 550℃ for 3 hours under an ammonia atmosphere, then replacing with a nitrogen atmosphere and allowing natural cooling; the particle size of the calcined cascade nanozymes is close to that of the nanoparticles before calcination, and the calcined nanozymes exhibit a porous structure. This calcination scheme, through segmented control of the atmosphere during the heat treatment process, is significantly superior to direct ammonia calcination in multiple aspects such as particle size maintenance, porous structure construction, heteroelement doping, and specific surface area control, which is beneficial for obtaining cascade nanozyme materials with better catalytic activity and stability.

[0013] Furthermore, the lactate catalytic kinetic constant of the cascaded nanozyme is Vmax = 0.87 mM / min, Km = 27.96 mM; the hydrogen peroxide catalytic kinetic constant is Vmax = 0.18 mM / min, Km = 9.44 mM.

[0014] Furthermore, the application effects of the cascaded nanozyme are as follows: it achieves metabolic regulation by consuming lactic acid, inhibits glycolysis, and reduces the tendency of fibrosis; it exerts an antioxidant effect by scavenging hydrogen peroxide, alleviating the damage of oxidative stress to tissues; and it improves the pathological microenvironment of inflamed tissues, making it suitable for inflammation treatment and tissue repair.

[0015] The cascaded nanozymes provided by this invention have the following beneficial effects: (1) They still have high catalytic activity under low oxygen and acidic conditions, which solves the limitations of natural LOX enzymes in inflamed tissues; (2) They simultaneously achieve lactic acid oxidation and hydrogen peroxide removal, avoiding oxidative stress caused by hydrogen peroxide accumulation; (3) They can be widely used in the treatment of inflamed or tumor tissues through injection, local application or transdermal administration. Attached Figure Description

[0016] Figure 1 Scanning electron microscopy morphology characterization of nanoparticles in Example 1 before and after calcination;

[0017] Figure 2 The lactate catalytic activity of the cascaded nanozymes obtained in Example 1 and Comparative Examples 1-3 (pH = 7.2-7.4, temperature at room temperature);

[0018] Figure 3 This is a comparison of the catalytic activity of the cascaded nanozyme obtained in Example 1 with that of the natural LOX enzyme under acidic (pH=5) and different temperature conditions;

[0019] Figure 4 The lactate catalytic kinetics of the cascaded nanozyme obtained in Example 1;

[0020] Figure 5 The hydrogen peroxide catalytic kinetics of the cascaded nanozyme obtained in Example 1;

[0021] Figure 6This describes the regulatory effect of the cascaded nanozyme obtained in Example 1 on lactate concentration and inflammation level in fibroblasts. Detailed Implementation Plan

[0022] The technical solution of the present invention will be further described below with reference to specific embodiments and accompanying drawings.

[0023] The following details the steps for synthesizing Prussian blue-like nanoparticles via coprecipitation, using Mn3[Co(CN)6]2 as an example. Solution A is prepared by dissolving 0.6 mmol of potassium cobalt cyanate in 25 mL of deionized water. Solution B is prepared by dissolving 1.13 mmol of manganese acetate and 0.5 mmol of citric acid in 25 mL of deionized water. Solution A is added dropwise to solution B, and the mixture is stirred continuously for 10 min to ensure thorough mixing. Stirring is then stopped, and the mixture is allowed to stand overnight. The precipitate is collected by centrifugation and washed 3-5 times with deionized water. The precipitate is then dried under vacuum to obtain a white powder, which is Mn3[Co(CN)6]2.

[0024] The following details the steps for synthesizing cascade nanozymes using the calcination method. The prepared Mn3[Co(CN)6]2 nanoparticles were placed in a ceramic boat and stably positioned in a tube furnace. Nitrogen gas was first purged for 20 minutes, then the air inside the tube was purged. The furnace was then slowly heated to 550°C at a rate of 5°C / min under a nitrogen atmosphere. The atmosphere was then replaced with ammonia gas and held at this temperature for 3 hours. After 3 hours, the atmosphere was replaced with nitrogen gas and the furnace was allowed to cool naturally to room temperature, yielding a black powder, which is the cascade nanozyme described in this invention.

[0025] To illustrate the beneficial effects of the present invention, the calcination process of the Prussian blue-like nanoparticles in the following comparative examples is exactly the same. The only difference is that manganese acetate is replaced with the same amount of cobalt acetate, zinc acetate, and copper acetate in the preparation of the Prussian blue-like nanoparticles. This is only used to explain the technical solution and effects of the present invention in conjunction with the following examples, and is not intended to limit the scope of protection of the present invention. The samples of different embodiments and comparative examples are described in detail below.

[0026] Example 1

[0027] Solution A was prepared by dissolving 0.6 mmol of potassium cobalt cyanate in 25 mL of deionized water. Solution B was prepared by dissolving 1.13 mmol of manganese acetate and 0.5 mmol of citric acid in 25 mL of deionized water. Solution A was added dropwise to solution B, and the mixture was stirred continuously for 10 min to ensure thorough mixing. After stirring was stopped, the mixture was allowed to stand overnight. The precipitate was collected by centrifugation and washed 3-5 times with deionized water. The precipitate was then dried under vacuum to obtain a white powder, which was Mn3[Co(CN)6]2. The precipitate was calcined at 550 °C for 3 hours under an ammonia atmosphere to obtain a black powder, which was the cascaded nanozyme. The scanning electron microscope morphology of the nanoparticles before and after calcination is shown in the figure. Figure 1As shown, the particles exhibit a cubic structure before calcination and a porous structure after calcination.

[0028] The following details the characterization of the lactate catalytic activity of the prepared cascade nanozyme. 100 μg / mL of the cascade nanozyme from Example 1 was added to a 5 mM lactate solution and incubated for 30 min under different temperatures or pH conditions. The lactate concentration in the supernatant before and after incubation was measured using a lactate kit to obtain the amount of lactate consumed within 30 min. Figure 2 As shown, the cascade nanozyme of Example 1 consumed 2.49 ± 0.13 mM of lactic acid within 30 min under conditions of pH = 7.2-7.4 and room temperature. 100 μg / mL of the cascade nanozyme of Example 1 and 100 μg / mL of natural LOX were added separately to 5 mM lactic acid solution (pH = 5) and incubated at 25°C, 37°C, or 60°C for 30 min, respectively, and the lactic acid consumption was measured. Figure 3 As shown, the lactate consumption of natural LOX at 25℃, 37℃, and 60℃ was 1.74±0.53mM, 2.71±0.30mM, and 1.64±0.38mM, respectively; the lactate consumption of the cascade nanozyme in Example 1 at 25℃, 37℃, and 60℃ was 3.73±0.49mM, 3.97±0.03mM, and 4.04±0.31mM, respectively, maintaining stable catalytic activity at different temperatures, and the catalytic ability at 25℃, 37℃, and 60℃ was 2.1 times, 1.5 times, and 2.5 times that of natural LOX enzyme, respectively.

[0029] The following details the characterization of the lactate catalytic kinetics of the prepared cascade nanozyme. 100 μg / mL of the cascade nanozyme from Example 1 was added to a 1-80 mM lactate solution and incubated for 10 min at pH 7.2-7.4 and 25°C. The lactate consumption ΔC in the supernatant was measured, and the initial reaction rate v = ΔC / 10 (mM / min) was calculated. Figure 4 As shown, the effects of lactic acid concentration (S) and initial reaction rate (V0), S0, and S0 were respectively applied. -1 and V0 -1 Plotting the graphs, we calculated the catalytic kinetic constants in the Michaelis equation: Vmax = 0.87 mM / min, Km = 27.96 mM.

[0030] The following details the characterization of the hydrogen peroxide catalytic kinetics of the prepared cascade nanozyme. 100 μg / mL of the cascade nanozyme from Example 1 was added to a 0.5-20 mM lactic acid solution and incubated for 10 min at pH 7.2-7.4 and 25°C. The hydrogen peroxide consumption ΔC was measured, and the initial reaction rate v = ΔC / 10 (mM / min) was calculated. Figure 5As shown, the effects of hydrogen peroxide concentration (S) and initial reaction rate (V0), S0, and S0 are respectively applied. -1 and V -1 Plotting the graphs, we obtained the catalytic kinetic constants in the Michaelis equation: Vmax = 0.18 mM / min, Km = 9.44 mM.

[0031] The following details the regulatory effects of the prepared cascade nanozyme on lactate concentration and inflammation levels in fibroblasts. Mouse L929 fibroblasts were cultured at 5 × 10⁻⁶ cells... 5 CFU / well was seeded in the lower layer of a 24-well Transwell plate. A cascade nanozyme was added to the upper chamber to a final concentration of 20 μg / mL. After 24 hours of inflammatory stimulation with 400 ng / mL lipopolysaccharide (LPS), the cell supernatant was collected to detect lactate and IL-6 concentrations. Unstimulated normal cultured cells served as a negative control, and cells stimulated with LPS but without the cascade nanozyme served as a positive control. Figure 6 As shown, the supernatant lactate concentrations were 2.99±0.18 mM (negative control group), 3.79±0.10 mM (positive control group), and 3.00±0.06 mM (Example 1). The supernatant IL-6 concentrations were 11.41±2.09 pg / mL (negative control group), 23.10±1.19 pg / mL (positive control group), and 12.49±0.69 pg / mL (Example 1).

[0032] Comparative Example 1

[0033] The only difference in the preparation of Prussian blue-like nanoparticles was that 1.13 mmol of manganese acetate was replaced with 1.13 mmol of cobalt acetate; the rest of the preparation method was the same as in Example 1.

[0034] The lactic acid catalytic ability of Comparative Example 1 was characterized by the same method as that of Example 1. 100 μg / mL of the nanozyme of Comparative Example 1 was added to a 5 mM lactic acid solution. Under the conditions of pH = 7.2-7.4 and room temperature, the amount of lactic acid consumed within 30 min was 1.55 ± 0.27 mM.

[0035] Comparative Example 2

[0036] The only difference in the preparation of Prussian blue-like nanoparticles was that 1.13 mmol of manganese acetate was replaced with 1.13 mmol of zinc acetate; the rest of the preparation method was the same as in Example 1.

[0037] The lactic acid catalytic ability of Comparative Example 1 was characterized by the same method as that of Example 1. 100 μg / mL of the nanozyme of Comparative Example 2 was added to a 5 mM lactic acid solution. Under the conditions of pH = 7.2-7.4 and room temperature, the amount of lactic acid consumed within 30 min was 1.62 ± 0.22 mM.

[0038] Comparative Example 3

[0039] The only difference in the preparation of Prussian blue-like nanoparticles was that 1.13 mmol of manganese acetate was replaced with 1.13 mmol of copper acetate; the rest of the preparation method was the same as in Example 1.

[0040] The lactic acid catalytic ability of Comparative Example 1 was characterized by the same method as that of Example 1. 100 μg / mL of the nanozyme of Comparative Example 2 was added to a 5 mM lactic acid solution. Under the conditions of pH = 7.2-7.4 and room temperature, the amount of lactic acid consumed within 30 min was 0.49 ± 0.28 mM.

[0041] The embodiments described above are merely some preferred embodiments of the present invention, and are not intended to limit the invention. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the invention. Therefore, all technical solutions obtained by equivalent substitution or equivalent transformation fall within the protection scope of the present invention.

Claims

1. A cascade nanozyme with metabolic reprogramming function, characterized in that, The nanozyme is obtained by calcining manganese and cobalt-based Prussian blue nanoparticles under an ammonia atmosphere. It has dual catalytic activity of lactate oxidase (LOX) and catalase (CAT). The nanozyme can catalyze the cascade reactions of "oxygen + lactic acid → pyruvate + hydrogen peroxide" and "hydrogen peroxide → oxygen + water". In an acidic environment with pH=5, its catalytic activity is higher than that of natural LOX enzyme of the same mass concentration.

2. The cascaded nanozyme according to claim 1, characterized in that, The preparation method includes the following steps: a) synthesizing manganese and cobalt-based Prussian blue nanoparticles by precipitation method, with the chemical formula Mn3[Co(CN)6]2; b) calcining the nanoparticles obtained in step a) under an ammonia atmosphere at a temperature of 400-550℃ for 2-5 hours to obtain the cascaded nanozyme.

3. The cascaded nanozyme according to claim 2, characterized in that, The precipitation method described in step a) specifically includes: dissolving potassium cobalt cyanate and manganese acetate in deionized water containing citric acid, stirring and letting stand overnight, centrifuging and vacuum drying to obtain Mn3[Co(CN)6]2 nanoparticles.

4. The cascaded nanozyme according to claim 2, characterized in that, The optimized conditions for the calcination process in step b) are: heating to 550°C under a nitrogen atmosphere, holding at 550°C for 3 hours under an ammonia atmosphere, then replacing with a nitrogen atmosphere and cooling naturally; the cascaded nanozymes after calcination have a particle size close to that of the nanoparticles before calcination, and the calcined nanozymes exhibit a porous structure.

5. The cascaded nanozyme according to claim 1, characterized in that, The lactate catalytic kinetic constants of the cascaded nanozymes are Vmax = 0.87 mM / min and Km = 27.96 mM; the hydrogen peroxide catalytic kinetic constants are Vmax = 0.18 mM / min and Km = 9.44 mM.

6. The cascaded nanozyme according to claim 1, characterized in that, The cascaded nanozyme exhibits lactate catalytic activity that is 2.1 times (25℃), 1.5 times (37℃), and 2.5 times (60℃) of the same mass concentration of natural LOX enzyme under acidic conditions (pH=5), and also demonstrates excellent thermal stability.

7. The method for preparing cascaded nanozymes according to any one of claims 1-6, characterized in that, include: a) Manganese and cobalt-based Prussian blue nanoparticles with the chemical formula Mn3[Co(CN)6]2 were synthesized by precipitation method; b) The nanoparticles obtained in step a) were calcined under an ammonia atmosphere at a temperature of 400-550℃ for 2-5 hours to obtain the cascaded nanozyme.

8. The method for preparing cascaded nanozymes according to claim 7, characterized in that, The precipitation method involves dissolving potassium cobalt cyanate and manganese acetate in deionized water containing citric acid, stirring, letting stand overnight, centrifuging, and vacuum drying to obtain Mn3[Co(CN)6]2 nanoparticles. The calcination method involves heating to the calcination temperature under a nitrogen atmosphere, then holding at that temperature under an ammonia atmosphere, followed by replacement with a nitrogen atmosphere and natural cooling.

9. The use of the cascaded nanozyme according to any one of claims 1-6 in the preparation of drugs for treating inflammation, tissue repair, or antitumor purposes.

10. The application according to claim 9, characterized in that, The nanozyme is applied to the target tissue via injection, topical application, or transdermal delivery.

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