阅读说明：本技术 一种具有原位调节肿瘤微环境及抗肿瘤效应的双金属纳米酶复合材料的制备方法 (Preparation method of bimetallic nano-enzyme composite material with in-situ tumor microenvironment regulation and anti-tumor effect ) 是由 丁鹤 杨飘萍 贺飞 盖世丽 杨丹 刘志亮 冯莉莉 常金虎 于 2021-08-13 设计创作，主要内容包括：一种具有原位调节肿瘤微环境及抗肿瘤效应的双金属纳米酶复合材料的制备方法,它涉及一种纳米酶复合材料的制备方法。本发明的目的是要解决现有肿瘤治疗中纳米酶催化效率低,肿瘤微环境响应敏感度低的问题。方法：一、合成双金属纳米粒子CoFe-(2)O-(4)；二、制备双金属纳米酶复合材料；三、表面修饰,得到具有原位调节肿瘤微环境及抗肿瘤效应的双金属纳米酶复合材料。本发明利用GOD酶活性和纳米酶的多元类酶活性原位调节肿瘤微环境,优化酶促反应条件,最大化地利用肿瘤微环境的内源性动力,提高肿瘤治疗效率。本发明可获得一种具有原位调节肿瘤微环境及抗肿瘤效应的双金属纳米酶复合材料。(A preparation method of a bimetallic nano-enzyme composite material with in-situ adjustment of a tumor microenvironment and an anti-tumor effect relates to a preparation method of a nano-enzyme composite material. The purpose of the invention isThe method aims to solve the problems of low nano-enzyme catalysis efficiency and low tumor microenvironment response sensitivity in the existing tumor treatment. The method comprises the following steps: firstly, synthesizing bimetal nano particle CoFe 2 O 4 (ii) a Secondly, preparing a bimetal nano enzyme composite material; and thirdly, surface modification is carried out to obtain the bimetallic nano-enzyme composite material with the functions of in-situ adjustment of the tumor microenvironment and anti-tumor effect. According to the invention, the tumor microenvironment is regulated in situ by utilizing GOD enzyme activity and the activity of multiple enzymes of nano enzyme, the enzymatic reaction condition is optimized, the endogenous power of the tumor microenvironment is utilized to the maximum extent, and the tumor treatment efficiency is improved. The invention can obtain the bimetal nano enzyme composite material with the functions of in-situ regulating the tumor microenvironment and resisting the tumor effect.)

1. A preparation method of a bimetallic nano-enzyme composite material with in-situ regulation of tumor microenvironment and anti-tumor effect is characterized by comprising the following steps:

firstly, synthesizing bimetal nano particle CoFe₂O₄：

Firstly, respectively adding a cobalt source and an iron source into a solvent, and uniformly stirring to obtain a mixed solution A;

secondly, placing the mixed solution A into a three-neck round-bottom flask, vacuumizing, heating the mixed solution A to 90-120 ℃ under the condition of magnetic stirring, and reacting at 90-120 ℃ under the condition of magnetic stirring to obtain a mixed solution B;

thirdly, introducing nitrogen into the mixed solution B, heating the mixed solution B to 260-298 ℃, reacting at 260-298 ℃, and cooling to room temperature to obtain a reaction product I; centrifuging the reaction product I, and removing centrifugate to obtain a solid substance; cleaning the solid substance, and drying to obtain the bimetal nano particle CoFe₂O₄；

Secondly, preparing the bimetal nano enzyme composite material:

firstly, dissolving triethanolamine into deionized water, then magnetically stirring for 20-40 min at 75-80 ℃, then adding cetyl trimethyl ammonium bromide, magnetically stirring for 0.8-1.2 h, then dropwise adding tetraethyl orthosilicate, and magnetically stirring for 10-12 h to obtain a reaction product II; centrifuging the reaction product II, cleaning, and drying to obtain branched mesoporous silicon;

② the bimetal nano particle CoFe₂O₄Uniformly dispersing into n-hexane, adding branched mesoporous silicon, and stirring at 30-60 ℃ for reaction to obtain a reaction product III; centrifuging the reaction product III, and collecting solid substances to obtain DMSN @ CoFe₂O₄Nanoparticles;

③ mixing DMSN @ CoFe₂O₄Dispersing the nano particles in phosphate buffer solution, and adding 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochlorideStirring for 0.4-0.6 h at room temperature, then adding glucose oxidase, and stirring for 10-12 h at room temperature in a dark place to obtain a reaction product IV; centrifuging the reaction product IV, and collecting solid substances to obtain DMSN @ CoFe₂O₄a/GOD nanocomposite;

thirdly, surface modification:

mixing DMSN @ CoFe₂O₄Adding the/GOD nano composite material and absolute ethyl alcohol into tetradecanol, and stirring at the temperature of 30-40 ℃ for 10-12 hours to obtain a reaction product V; and centrifuging the reaction product V to obtain the bimetallic nano-enzyme composite material with the functions of in-situ regulating the tumor microenvironment and resisting the tumor effect.

2. The method for preparing the bimetallic nano-enzyme composite material with the functions of in-situ regulating the tumor microenvironment and resisting the tumor effect according to claim 1, wherein the cobalt source in the first step is cobalt acetylacetonate; the iron source in the first step is iron acetylacetonate; the solvent in the first step is dibenzyl ether, tetraethylene glycol or octadecene.

3. The preparation method of the bimetallic nanoenzyme composite material with the functions of in-situ adjustment of the tumor microenvironment and anti-tumor effect according to claim 1, wherein the mass ratio of the cobalt source to the iron source in the first step is 1 (0.5-2); the mass ratio of the total mass of the cobalt source and the iron source to the solvent in the first step is 1 (50-80).

4. The method for preparing the bimetal nano enzyme composite material with the functions of in-situ adjustment of the tumor microenvironment and the anti-tumor effect according to claim 1, wherein the reaction time in the first step is 20-60 min; the temperature rising speed in the first step is 3-10 ℃/min; the rotating speed of the magnetic stirring in the first step is 200 r/min-400 r/min.

5. The method for preparing the bimetal nano enzyme composite material with the functions of in-situ regulating the tumor microenvironment and the anti-tumor effect according to claim 1, wherein the temperature rise speed in the first step is 3 ℃/min to 10 ℃/min; the reaction time in the step one is 20min to 60 min; the centrifugation speed in the third step is 12000r/min, and the centrifugation time is 10 min-15 min; firstly, washing the solid substance for 3 to 5 times by using deionized water, and then washing the solid substance for 3 to 5 times by using absolute ethyl alcohol; the drying temperature in the step one is 80 ℃, and the drying time is 8-12 h.

6. The method for preparing the bimetal nano enzyme composite material with the functions of in-situ regulating the tumor microenvironment and resisting the tumor effect according to claim 1, which is characterized in that the bimetal nano particles CoFe in the step one₂O₄The particle size of (A) is 3nm to 5 nm.

7. The method for preparing the bimetal nano enzyme composite material with the functions of in-situ adjusting the tumor microenvironment and the anti-tumor effect according to claim 1, wherein the volume ratio of the triethanolamine to the deionized water in the second step is (0.06 g-0.10 g) to (5 mL-10 mL); the mass ratio of the hexadecyl trimethyl ammonium bromide to the deionized water in the second step is (350 mg-400 mg): 10 mL-20 mL; the mass ratio of the tetraethyl orthosilicate to the deionized water in the second step is (1 mg-5 mg) to (5 mL-10 mL); the speed of the magnetic stirring in the second step is 300 r/min; the centrifugation speed in the second step is 12000r/min, and the centrifugation time is 10 min-15 min; firstly, washing a reaction product II for 3-5 times by using deionized water, and then washing a solid substance for 3-5 times by using absolute ethyl alcohol; and the drying temperature in the second step is 80 ℃, and the drying time is 8-12 h.

8. The method for preparing the bimetal nano enzyme composite material with the functions of in-situ regulating the tumor microenvironment and the anti-tumor effect according to claim 1, wherein the bimetal nano particle CoFe in the second step₂O₄The mass ratio of the (5 mg-10 mg) to the volume ratio of the n-hexane is (5 mL-10 mL); the mass ratio of the branched mesoporous silicon to the normal hexane in the second step is (10 mg-20 mg): 5 mL-10 mL; the stirring reaction time in the second step is 7-8 h, and the stirring reaction speed is 300 r/min.

9. The method for preparing the bimetal nano enzyme composite material with the functions of in-situ regulating the tumor microenvironment and resisting the tumor effect according to claim 1, wherein the DMSN @ CoFe in the second step and the third step₂O₄The ratio of the mass of the nano particles to the volume of the phosphate buffer solution is (5 mg-10 mg) to (5 mL-10 mL); the mass ratio of the 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine hydrochloride to the volume ratio of the phosphate buffer solution in the second step (III) is (2 mg-5 mg) to (5 mL-10 mL); the ratio of the mass of the glucose oxidase to the volume of the phosphate buffer solution in the second step is (10 mg-20 mg): 5 mL-10 mL; the stirring speed in the second step and the third step is 300 r/min.

10. The method for preparing the bimetallic nano-enzyme composite material with the functions of in-situ regulating the tumor microenvironment and resisting the tumor effect of claim 1, wherein the DMSN @ CoFe in the third step₂O₄The ratio of the mass of the/GOD nano composite material to the volume of the tetradecanol is (10 mg-20 mg) to (20 mL-40 mL); the volume ratio of the absolute ethyl alcohol to the tetradecanol in the third step is (5-10) to (20-40); the stirring speed in the third step is 300 r/min.

Technical Field

The invention relates to a preparation method of a nano enzyme composite material.

Background

The traditional tumor treatment means, such as operation treatment, chemical treatment and radiotherapy, has poor selectivity and great side effect, and the drug resistance generated by continuous medication is great. The development of nanotechnology provides a new idea for solving the above problems. The finely adjustable structure, the morphology and the modifiable surface characteristics of the nano material directly determine the interaction mode of the nano material with biomolecules, cells, tissues, organs and individuals, and therefore, a unique biological effect, namely a nano biological effect, is generated. The intensive research on nano-biological effects at the individual, cellular and molecular level and the elucidation of the precise mechanism thereof have become a very challenging hot frontier. In 2004, Pasquato et al named the nanomaterial with enzymatic activity-nanoenzyme for the first time. The nano enzyme is used as a novel artificial material, benefits from the continuous development and mutual fusion of nanotechnology and biotechnology, and has controllable catalytic activity and high stability in severe environment. At present, nanoenzymes having activities of Peroxidase (POD), Catalase (CAT), Superoxide dismutase (SOD), Oxidase (Oxidase), and Glutathione Peroxidase (GPx) have been reported. However, due to the limitations of the autocatalytic activity of the nanoenzyme and the special physicochemical properties of the Tumor Microenvironment (TME), the catalytic treatment effect of the Tumor involving the nanoenzyme is still not ideal.

Characteristics of TME include, in contrast to normal tissue, microacid, hypoxia, over-expressed Glutathione (GSH) and hydrogen peroxide (H)₂O₂). Although the extreme nature and complexity of TME prevent the application of nanoenzyme to a certain extent, TME can be effectively regulated by nanoenzyme activity, and the tumor treatment efficiency is improved. E.g. catalysis of high concentrations of H in TME by Nanocatalysis with POD activity₂O₂Decomposing to generate hydroxyl free radicals, thereby inducing the death of tumor cells; the nano enzyme with GPx activity is used for reducing the concentration of GSH in TME, avoiding the consumption of Reactive Oxygen Species (ROS) and simultaneously generating O₂And can relieve anoxia. In addition, the biological toxicity of the nano enzyme material can be utilized to induce protein denaturation and DNA damage to promote cell death, so that the treatment effect is achieved. Therefore, the development of the nano enzyme complex system which has the functions of in-situ tumor microenvironment regulation, high enzymatic reaction efficiency and multi-mode cooperative treatment for tumor catalytic treatment has great significance.

Metal-based nanomaterials have been extensively studied for their nanoenzyme activity due to the presence of mixed-valence metal ions. However, the catalytic activity of the single metal ion nano material is lower, and the enzymatic reaction efficiency in the special TME is more limited.

Disclosure of Invention

The invention aims to solve the problems of low catalytic efficiency of the nano enzyme and low response sensitivity of a tumor microenvironment in the conventional tumor treatment, and provides a preparation method of a bimetallic nano enzyme composite material with the functions of in-situ adjustment of the tumor microenvironment and anti-tumor effect.

A preparation method of a bimetallic nano-enzyme composite material with in-situ regulation of a tumor microenvironment and an anti-tumor effect is specifically completed according to the following steps:

firstly, synthesizing bimetal nano particle CoFe₂O₄：

Firstly, respectively adding a cobalt source and an iron source into a solvent, and uniformly stirring to obtain a mixed solution A;

secondly, placing the mixed solution A into a three-neck round-bottom flask, vacuumizing, heating the mixed solution A to 90-120 ℃ under the condition of magnetic stirring, and reacting at 90-120 ℃ under the condition of magnetic stirring to obtain a mixed solution B;

thirdly, introducing nitrogen into the mixed solution B, heating the mixed solution B to 260-298 ℃, reacting at 260-298 ℃, and cooling to room temperature to obtain a reaction product I; centrifuging the reaction product I, and removing centrifugate to obtain a solid substance; cleaning the solid substance, and drying to obtain the bimetal nano particle CoFe₂O₄；

Secondly, preparing the bimetal nano enzyme composite material:

firstly, dissolving triethanolamine into deionized water, then magnetically stirring for 20-40 min at 75-80 ℃, then adding cetyl trimethyl ammonium bromide, magnetically stirring for 0.8-1.2 h, then dropwise adding tetraethyl orthosilicate, and magnetically stirring for 10-12 h to obtain a reaction product II; centrifuging the reaction product II, cleaning, and drying to obtain branched mesoporous silicon;

② the bimetal nano particle CoFe₂O₄Uniformly dispersing into n-hexane, adding branched mesoporous silicon, and stirring at 30-60 ℃ for reaction to obtain a reaction product III; centrifuging the reaction product III, and collecting solid substances to obtain DMSN @ CoFe₂O₄Nanoparticles;

③ mixing DMSN @ CoFe₂O₄Dispersing the nano particles in a phosphate buffer solution, adding 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride, stirring for 0.4-0.6 h at room temperature, adding glucose oxidase, and stirring for 10-12 h at the dark room temperature to obtain a reaction product IV; centrifuging the reaction product IV, and collecting solid substances to obtainTo DMSN @ CoFe₂O₄a/GOD nanocomposite;

thirdly, surface modification:

mixing DMSN @ CoFe₂O₄Adding the/GOD nano composite material and absolute ethyl alcohol into tetradecanol, and stirring at the temperature of 30-40 ℃ for 10-12 hours to obtain a reaction product V; and centrifuging the reaction product V to obtain the bimetallic nano-enzyme composite material with the functions of in-situ regulating the tumor microenvironment and resisting the tumor effect.

The principle of the invention is as follows:

the invention provides a method for constructing a nano-composite treatment system taking cobalt/iron double-cycle nanoenzyme as a core aiming at physicochemical properties such as subacidity, high-concentration hydrogen peroxide and the like which are peculiar to TME, and a multi-mode treatment platform is constructed by utilizing a cobalt/iron double-cycle enzymatic reaction in a near-infrared light trigger system and cooperating with a photothermal effect and a chemotherapy effect of a cobalt-based material. Meanwhile, the tumor microenvironment is regulated in situ by utilizing the activity of the multielement enzymes of the nanoenzyme, the enzymatic reaction condition is optimized, and the endogenous power of the tumor microenvironment is utilized to the maximum extent, so that the in-situ tumor microenvironment is regulated.

The invention has the beneficial effects that:

CoFe synthesized by high-temperature pyrolysis method₂O₄Compared with the conventional synthesis method, the nano-particle size is small (the particle size is 3 nm-5 nm), and the size is uniform; the prepared bimetallic nanoenzyme composite material (DCGP nanocomposite) with the functions of in-situ regulation of the tumor microenvironment and the anti-tumor effect can utilize cobalt/iron double-cycle enzymatic reaction in a near-infrared light trigger system to cooperate with the photothermal effect and the chemotherapy effect of a cobalt-based material to form a multi-mode treatment platform.

The invention can obtain the bimetal nano enzyme composite material with the functions of in-situ regulating the tumor microenvironment and resisting the tumor effect.

Drawings

FIG. 1 is an XRD pattern, in which 1 is an XRD curve of CFOs prepared in example one, and 2 is an XRD characteristic diffraction peak of JCPDS Card No. 003-0864;

FIG. 2 is a TEM image of CFOs prepared in example one;

FIG. 3 is a statistical plot of the particle size distribution of CFOs prepared in example one;

FIG. 4 is a SAED map of CFOs prepared in example one;

FIG. 5 is a Mapping chart of CFOs prepared in example one;

FIG. 6 is a TEM image of DMSN prepared in the first example;

FIG. 7 is a TEM image of DCGP prepared in the first example;

FIG. 8 is a Mapping chart of DCGP prepared in example one;

FIG. 9 is an XPS survey of CFOs prepared according to example one;

FIG. 10 is an XPS fine scan of Co in CFOs prepared according to example one;

FIG. 11 is an XPS fine scan of Fe in CFOs prepared in example one;

FIG. 12 is an XPS fine scan of O in CFOs prepared in example one;

FIG. 13 shows an ultraviolet absorption spectrum in which 1 is TMB group and 2 is TMB + H₂O₂Group, 3 is TMB + CFOs group, 4 is TMB + H₂O₂+ CFOs group, 5 is TMB + H₂O₂+ CFOs +50 ℃ and 6 is TMB + H₂O₂+ CFOs +1064 nm;

FIG. 14 shows 500. mu.g mL at different times^-1CFOs+300μL H₂O₂+ TMB ultraviolet absorption spectrum, in which 1 is reaction time 1min, 2 is reaction time 2min, 3 is reaction time 3min, 4 is reaction time 4min, and 5 is reaction time 5 min;

FIG. 15 shows CFOs absolute ethanol solutions + 300. mu. L H at different concentrations₂O₂+ TMB ultraviolet absorption spectrum, where 1 is TMB dye and 2 is 37% H by mass₂O₂And 3 is 500. mu.g mL^-1The CFOs absolute ethanol solution of (4) is TMB + 100. mu.g mL^-1+CFOs+H₂O₂And 5 is TMB + 200. mu.g mL^-1+CFOs+H₂O₂And 6 is TMB + 300. mu. gmL^-1+CFOs+H₂O₂And 7 is TMB + 400. mu.gmL^-1+CFOs+H₂O₂And 8 is TMB + 500. mu.g mL^-1+CFOs+H₂O₂；

FIG. 16 shows different contents of H₂O₂+ CFOs + TMB UV absorption spectrum, 1 in the figure is 100 μ LH₂O₂+ TMB + CFOs, 2 is 200 μ LH₂O₂+ TMB + CFOs, 3 is 300 μ LH₂O₂+TMB+CFOs；

FIG. 17 is an ESR spectrum of DCGP catalyzed active oxygen generation prepared in example one, where 1 is DMPO + H₂O₂+ DCGP, 2 is DMPO + H₂O₂；

FIG. 18 is a graph showing UV absorption spectra of DCGP absolute ethanol solutions of different concentrations, wherein 1 is 100. mu.g mL^-12 is 200. mu.g mL^-1And 3 is 300. mu.g mL^-1And 4 is 400. mu.g mL^-15 is 500. mu.g mL^-1；

FIG. 19 is a diagram of the photothermal conversion mechanism of DCGP nanocomposites;

FIG. 20 shows the different concentrations of DCGP absolute ethanol solutions (100, 200, 300, 400 and 500. mu.g mL^-1) At 1.7W cm^-2Thermal infrared imaging pictures obtained under 1064nm light irradiation;

FIG. 21 shows DCGP absolute ethanol solutions with different concentrations and H₂O is 1.7W cm^-2Temperature rise curve under 1064nm light irradiation, wherein 1 is 100. mu.g mL^-12 is 200. mu.g mL^-1And 3 is 300. mu.g mL^-1And 4 is 400. mu.g mL^-15 is 500. mu.g mL^-16 is H₂O；

FIG. 22 shows different powers (1.0, 1.3, 1.4, 1.7 and 1.8W cm^-2) 300 μ g mL under 1064nm light excitation^{- 1}Temperature rise curve of DCGP absolute ethyl alcohol solution, wherein the power of 1 in the graph is 1.0W cm^-22 power of 1.3W cm^-2And the power of 3 is 1.4W cm^-24 power of 1.7W cm^-2And 5 at a power of 1.8W cm^-2；

FIG. 23 is a graph of temperature versus time;

FIG. 24 shows the signal at 1.7W cm^-2500. mu.g mL under 1064nm light irradiation^-1Temperature change curve of DCGP absolute ethyl alcohol solution for 3 heating and cooling cycles.

Detailed Description

The following examples further illustrate the present invention but are not to be construed as limiting the invention. Modifications and substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit of the invention.

The first embodiment is as follows: the embodiment of the invention relates to a preparation method of a bimetallic nano-enzyme composite material with in-situ adjustment of a tumor microenvironment and an anti-tumor effect, which is characterized by comprising the following steps:

firstly, synthesizing bimetal nano particle CoFe₂O₄：

Firstly, respectively adding a cobalt source and an iron source into a solvent, and uniformly stirring to obtain a mixed solution A;

secondly, placing the mixed solution A into a three-neck round-bottom flask, vacuumizing, heating the mixed solution A to 90-120 ℃ under the condition of magnetic stirring, and reacting at 90-120 ℃ under the condition of magnetic stirring to obtain a mixed solution B;

thirdly, introducing nitrogen into the mixed solution B, heating the mixed solution B to 260-298 ℃, reacting at 260-298 ℃, and cooling to room temperature to obtain a reaction product I; centrifuging the reaction product I, and removing centrifugate to obtain a solid substance; cleaning the solid substance, and drying to obtain the bimetal nano particle CoFe₂O₄；

Secondly, preparing the bimetal nano enzyme composite material:

firstly, dissolving Triethanolamine (TEA) into deionized water, then magnetically stirring at 75-80 ℃ for 20-40 min, then adding Cetyl Trimethyl Ammonium Bromide (CTAB), magnetically stirring for 0.8-1.2 h, then dropwise adding tetraethyl orthosilicate (TEOS), and magnetically stirring for 10-12 h to obtain a reaction product II; centrifuging the reaction product II, cleaning, and drying to obtain branched mesoporous silicon (DMSN);

② the bimetal nano particle CoFe₂O₄Uniformly dispersing into n-hexane, adding branched mesoporous silicon (DMSN), and stirring at 30-60 ℃ for reaction to obtain a reaction product III; centrifuging the reaction product III, and collecting solid substances to obtain DMSN @ CoFe₂O₄Nanoparticles;

③ mixing DMSN @ CoFe₂O₄Dispersing the nano particles in a phosphate buffer solution, adding 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), stirring for 0.4-0.6 h at room temperature, adding Glucose Oxidase (GOD), and stirring for 10-12 h at dark room temperature to obtain a reaction product IV; centrifuging the reaction product IV, and collecting solid substances to obtain DMSN @ CoFe₂O₄a/GOD nanocomposite;

thirdly, surface modification:

mixing DMSN @ CoFe₂O₄Adding the/GOD nano composite material and absolute ethyl alcohol into tetradecanol (PCM), and stirring at the temperature of 30-40 ℃ for 10-12 h to obtain a reaction product V; and centrifuging the reaction product V to obtain the bimetallic nano-enzyme composite material (DCGP) with the functions of in-situ regulating the tumor microenvironment and resisting the tumor effect.

According to the method of the embodiment, the DCGP nano composite material can be obtained, a new design thought and a new process flow are provided, and the bimetal nano enzyme composite material which has an anti-tumor effect and can adjust the tumor microenvironment is reasonably designed. The novel scientific research idea and application prospect are shown through the aspects of good treatment efficiency, organism-friendly performance and the like.

CoFe synthesized by high temperature pyrolysis in step one of the present embodiment₂O₄The nano material has uniform size and obvious spherical structure.

In the first step of the embodiment, before the reaction, vacuum pumping is performed, so that water and oxygen in the solution can be removed.

The first step and the first step of the embodiment are carried out according to the temperature rise speed of 3-10 ℃/min, so that the bumping phenomenon is avoided, the reaction can be fully carried out, and the timely observation and control are facilitated.

In the first step of the implementation mode, nitrogen is introduced as protective gas, so that the introduction of external oxygen to generate impurities is avoided, the interference of air on a reaction system is avoided, and the purity of the product is improved.

The stirring in this embodiment is for dispersing the reagent more favorably, and the purpose of complete reaction, uniform temperature rise, and the like is achieved.

In the first step of the embodiment, a high-temperature pyrolysis method is adopted, and the product synthesized by the method has smaller particles compared with the product synthesized by a solvothermal method and better dispersibility compared with the material synthesized by a coprecipitation method, so that the method is more suitable for being applied to organisms.

In the third step of the embodiment, the product is cleaned in absolute ethyl alcohol, so that a pure product can be obtained more effectively and stably.

The method has the advantages of stable process, mature method and no pollution to the environment of a reaction system, and the provided process route can successfully synthesize the target composite material and has good application prospect. Small-sized CoFe synthesized by high-temperature pyrolysis₂O₄The nano-particles have excellent morphology, and the small size is beneficial to the uptake of cells. The prepared composite material has the characteristics of good hydrophilicity, low toxicity and good biocompatibility. The embodiment discloses a preparation method of a bimetallic nanoenzyme composite material with in-situ adjustment of a tumor microenvironment and an anti-tumor effect, wherein a multi-mode cooperative treatment platform is constructed through adjustment of the in-situ tumor microenvironment and optical triggering of enzymatic reaction enhanced by bimetallic nanoenzyme, the tumor treatment effect is remarkably enhanced, and the preparation method has good application prospect and innovative guidance of development.

The second embodiment is as follows: the present embodiment differs from the present embodiment in that: the cobalt source in the first step is cobalt acetylacetonate; the iron source in the first step is iron acetylacetonate; the solvent in the first step is dibenzyl ether, tetraethylene glycol or octadecene. Other steps are the same as in the first embodiment.

The third concrete implementation mode: the present embodiment differs from the first or second embodiment in that: the mass ratio of the cobalt source to the iron source in the first step is 1 (0.5-2); the mass ratio of the total mass of the cobalt source and the iron source to the solvent in the first step is 1 (50-80). The other steps are the same as in the first or second embodiment.

The fourth concrete implementation mode: the difference between this embodiment and one of the first to third embodiments is as follows: the reaction time in the first step is 20-60 min; the temperature rising speed in the first step is 3-10 ℃/min; the rotating speed of the magnetic stirring in the first step is 200 r/min-400 r/min. The other steps are the same as those in the first to third embodiments.

The fifth concrete implementation mode: the difference between this embodiment and one of the first to fourth embodiments is: the temperature rising speed in the third step is 3-10 ℃/min; the reaction time in the step one is 20min to 60 min; the centrifugation speed in the third step is 12000r/min, and the centrifugation time is 10 min-15 min; firstly, washing the solid substance for 3 to 5 times by using deionized water, and then washing the solid substance for 3 to 5 times by using absolute ethyl alcohol; the drying temperature in the step one is 80 ℃, and the drying time is 8-12 h. The other steps are the same as those in the first to fourth embodiments.

The sixth specific implementation mode: the difference between this embodiment and one of the first to fifth embodiments is as follows: the bimetal nano particle CoFe in the step one₂O₄The particle size of (A) is 3nm to 5 nm. The other steps are the same as those in the first to fifth embodiments.

The seventh embodiment: the difference between this embodiment and one of the first to sixth embodiments is: the mass ratio of the triethanolamine to the deionized water in the second step is (0.06 g-0.10 g) to (5 mL-10 mL); the mass ratio of the hexadecyl trimethyl ammonium bromide to the deionized water in the second step is (350 mg-400 mg): 10 mL-20 mL; the mass ratio of the tetraethyl orthosilicate to the deionized water in the second step is (1 mg-5 mg) to (5 mL-10 mL); the speed of the magnetic stirring in the second step is 300 r/min; the centrifugation speed in the second step is 12000r/min, and the centrifugation time is 10 min-15 min; firstly, washing a reaction product II for 3-5 times by using deionized water, and then washing a solid substance for 3-5 times by using absolute ethyl alcohol; and the drying temperature in the second step is 80 ℃, and the drying time is 8-12 h. The other steps are the same as those in the first to sixth embodiments.

The specific implementation mode is eight: the difference between this embodiment and one of the first to seventh embodiments is: the bimetal nano particle CoFe in the second step₂O₄The mass ratio of the (5 mg-10 mg) to the volume ratio of the n-hexane is (5 mL-10 mL); the mass ratio of the branched mesoporous silicon to the normal hexane in the second step is (10 mg-20 mg): 5 mL-10 mL; the stirring reaction time in the second step is 7-8 h, and the stirring reaction speed is 300 r/min. The other steps are the same as those in the first to seventh embodiments.

The specific implementation method nine: the difference between this embodiment and the first to eighth embodiments is: the DMSN @ CoFe in the second step and the third step₂O₄The ratio of the mass of the nano particles to the volume of the phosphate buffer solution is (5 mg-10 mg) to (5 mL-10 mL); the mass ratio of the 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine hydrochloride to the volume ratio of the phosphate buffer solution in the second step (III) is (2 mg-5 mg) to (5 mL-10 mL); the ratio of the mass of the glucose oxidase to the volume of the phosphate buffer solution in the second step is (10 mg-20 mg): 5 mL-10 mL; the stirring speed in the second step and the third step is 300 r/min. The other steps are the same as those in the first to eighth embodiments.

The detailed implementation mode is ten: the difference between this embodiment and one of the first to ninth embodiments is as follows: DMSN @ CoFe described in step three₂O₄The ratio of the mass of the/GOD nano composite material to the volume of the tetradecanol is (10 mg-20 mg) to (20 mL-40 mL); the volume ratio of the absolute ethyl alcohol to the tetradecanol in the third step is (5-10) to (20-40); the stirring speed in the third step is 300 r/min. The other steps are the same as those in the first to ninth embodiments.

The present invention will be described in detail below with reference to the accompanying drawings and examples.

The first embodiment is as follows: a preparation method of a bimetallic nano-enzyme composite material with in-situ regulation of a tumor microenvironment and an anti-tumor effect is specifically completed according to the following steps:

firstly, synthesizing bimetal nano particle CoFe₂O₄：

Firstly, respectively adding a cobalt source and an iron source into a solvent, and uniformly stirring to obtain a mixed solution A;

the cobalt source in the first step is cobalt acetylacetonate;

the iron source in the first step is iron acetylacetonate;

the solvent in the first step is dibenzyl ether;

the mass ratio of the cobalt source to the iron source in the first step is 1: 0.5;

the mass ratio of the total mass of the cobalt source and the iron source to the solvent in the first step is 1: 70;

secondly, placing the mixed solution A into a three-neck round-bottom flask, vacuumizing, and heating the mixed solution A to 120 ℃ under the condition of magnetic stirring at a heating speed of 10 ℃/min; then the mixture is stirred magnetically and reacts for 60min at the temperature of 120 ℃ to obtain a mixed solution B;

the rotating speed of the magnetic stirring in the first step is 300 r/min;

thirdly, introducing nitrogen into the mixed solution B, and then heating the mixed solution B to 298 ℃ at a heating speed of 10 ℃/min; reacting at 298 ℃ for 60min, and cooling to room temperature to obtain a reaction product I; centrifuging the reaction product I, and removing centrifugate to obtain a solid substance; cleaning the solid substance, and drying to obtain the bimetal nano particle CoFe₂O₄(CFOs)；

The centrifugal speed in the third step is 12000r/min, and the centrifugal time is 15 min;

firstly, washing the solid substance for 3 times by using deionized water, and then washing the solid substance for 3 times by using absolute ethyl alcohol;

the drying temperature in the third step is 80 ℃, and the drying time is 10 hours;

the bimetal nano particle CoFe in the step one₂O₄The particle size of the (B) is 3 nm-5 nm;

secondly, preparing the bimetal nano enzyme composite material:

firstly, dissolving 0.068g of Triethanolamine (TEA) into 25mL of deionized water, then magnetically stirring for 30min at 80 ℃, then adding 380mg of Cetyl Trimethyl Ammonium Bromide (CTAB), magnetically stirring for 1h, then dropwise adding 2mg of tetraethyl orthosilicate (TEOS), and magnetically stirring for 12h to obtain a reaction product II; centrifuging the reaction product II, cleaning, and drying to obtain branched mesoporous silicon (DMSN);

the speed of the magnetic stirring in the second step is 300 r/min;

the centrifugation speed in the second step is 12000r/min, and the centrifugation time is 10 min-15 min;

firstly, washing a reaction product II for 3 times by using deionized water, and then washing a solid substance for 3 times by using absolute ethyl alcohol;

the drying temperature in the second step is 80 ℃, and the drying time is 10 hours;

② 10mg of double-metal nano particle CoFe₂O₄Uniformly dispersing into 10mL of n-hexane, adding 20mg of branched mesoporous silicon (DMSN), and stirring at 30 ℃ for reacting for 8 hours to obtain a reaction product III; centrifuging the reaction product III, and collecting solid substances to obtain DMSN @ CoFe₂O₄Nanoparticles;

the stirring reaction speed in the second step is 300 r/min;

③ mixing 10mg of DMSN @ CoFe₂O₄Dispersing the nano particles in 10mL of phosphate buffer solution, adding 5mg of 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine hydrochloride (EDC), stirring for 0.5h at room temperature, adding 20mg of Glucose Oxidase (GOD), and stirring for 12h at room temperature in a dark place to obtain a reaction product IV; centrifuging the reaction product IV, and collecting solid substances to obtain DMSN @ CoFe₂O₄a/GOD nanocomposite;

the stirring speed in the second step is 300 r/min;

thirdly, surface modification:

mixing 20mgDMSN @ CoFe₂O₄the/GOD nanocomposite and 10mL of absolute ethanol were added to 40mL of tetradecanol (PCM)Stirring for 12 hours at the temperature of 40 ℃ to obtain a reaction product V; centrifuging the reaction product V to obtain a bimetallic nano-enzyme composite material (DCGP) with the functions of in-situ regulating the tumor microenvironment and resisting the tumor effect;

the stirring speed in the third step is 300 r/min.

FIG. 1 is an XRD pattern, in which 1 is an XRD curve of CFOs prepared in example one, and 2 is an XRD characteristic diffraction peak of JCPDS Card No. 003-0864;

as can be seen from FIG. 1, the first step of the example is that the bimetallic nanoparticles CoFe prepared by the third step₂O₄The diffraction peaks of (CFOs) correspond to the (220), (311), (400), (422), (511), and (440) crystal planes at 2 θ values of 30.2 °,35.48 °,43.11 °,53.49 °,57.02 °, and 62.61 °, respectively. XRD characterization confirms that the synthesized bimetallic nano-particle CoFe₂O₄(CFOs) is a single-phase spherical structure with space group Fd3m and unit cell parameter aIn accordance with the standard PDF card (JCPDS No. 003-.

FIG. 2 is a TEM image of CFOs prepared in example one;

as can be seen from FIG. 2, the CFOs nanoparticles have an ultra-small size, a particle size of about (4. + -.1 nm), and a uniform size.

FIG. 3 is a statistical plot of the particle size distribution of CFOs prepared in example one;

as can be seen from FIG. 3, the CFOs prepared in example one had an average diameter of 3nm to 5 nm.

FIG. 4 is a SAED map of CFOs prepared in example one;

as can be seen from fig. 4, the surface material of CFOs prepared in the first example was polycrystalline.

FIG. 5 is a Mapping chart of CFOs prepared in example one;

as can be seen from FIG. 5, the elements Co, Fe and O in CFOs are uniformly distributed.

FIG. 6 is a TEM image of DMSN prepared in the first example;

fig. 6 shows that DMSN has a dendritic spherical structure.

FIG. 7 is a TEM image of DCGP prepared in the first example;

fig. 7 shows that CFOs have been loaded into DMSN mesostructures.

FIG. 8 is a Mapping chart of DCGP prepared in example one;

FIG. 8 shows that the distribution of Co, Fe, O and Si elements in DCGP is uniform. .

FIG. 9 is an XPS survey of CFOs prepared according to example one;

fig. 9 demonstrates the presence of Co, Fe, O in CFOs samples.

FIG. 10 is an XPS fine scan of Co in CFOs prepared according to example one;

FIG. 10 shows characteristic peaks for Co2p3/2(689.50, 700.36 and 705.21eV) and Co2p1/2(683.12eV), respectively, demonstrating the presence of multivalent Co in CFOs.

FIG. 11 is an XPS fine scan of Fe in CFOs prepared in example one;

FIG. 11 shows characteristic peaks for Fe2p3/2(761.53, 770.16 and 774.95eV) and Fe2p1/2(752.50eV), respectively, demonstrating the presence of multi-valence Fe in CFOs.

FIG. 12 is an XPS fine scan of O in CFOs prepared in example one;

FIG. 12 shows different valence assignments for oxygen.

Example one prepared CFOs oxidized TMB dye with uv absorption spectra, grouped as: TMB + H₂O₂+CFOs+1064nm、TMB+H₂O₂+CFOs+50℃、TMB+H₂O₂+CFOs、TMB+CFOs、TMB+H₂O₂And TMB, the specific operation process is as follows:

TMB group is: the TMB dye has a characteristic absorption peak at 652 nm;

TMB+H₂O₂group (2): 20 mu L of TMB dye absolute ethyl alcohol solution with the concentration of 1mmol/mL and 300 mu L of H with the mass fraction of 37 percent₂O₂Adding the solution into 2mL of deionized water, reacting for 5min, and testing that a characteristic absorption peak exists at 652 nm;

TMB + CFOs group: adding 20 mu L of TMB dye absolute ethyl alcohol solution with the concentration of 1mmol/mL and 300 mu L of CFOs absolute ethyl alcohol solution with the concentration of 500 mu g/mL into 2mL of deionized water, reacting for 5min, and testing that characteristic absorption peaks exist at 652 nm;

TMB+H₂O₂+ CFOs group: 20 mu.L of TMB dye absolute ethyl alcohol solution with the concentration of 1mmol/mL, 200 mu.L of CFOs absolute ethyl alcohol solution with the concentration of 500 mu g/mL and 300 mu.L of H with the mass fraction of 37 percent₂O₂Adding the solution into 2mL of deionized water, reacting for 5min, and testing that a characteristic absorption peak exists at 652 nm;

TMB+H₂O₂+ CFOs +50 ℃ group: 20 mu.L of TMB dye absolute ethyl alcohol solution with the concentration of 1mmol/mL, 200 mu.L of CFOs absolute ethyl alcohol solution with the concentration of 500 mu g/mL and 300 mu.L of H with the mass fraction of 37 percent₂O₂Adding the solution into 2mL of deionized water, reacting for 5min under the heating of water bath at 50 ℃, and testing that a characteristic absorption peak exists at 652 nm;

TMB+H₂O₂+ CFOs +1064nm group: 20 mu.L of TMB dye absolute ethyl alcohol solution with the concentration of 1mmol/mL, 200 mu.L of CFOs absolute ethyl alcohol solution with the concentration of 500 mu g/mL and 300 mu.L of H with the mass fraction of 37 percent₂O₂Adding the solution into 2mL of deionized water, carrying out laser irradiation at 1064nm for 5min to enable the mixed solution to reach about 50 ℃, and testing that a characteristic absorption peak exists at 652 nm;

the above ultraviolet absorption spectrum is shown in FIG. 13;

FIG. 13 shows an ultraviolet absorption spectrum in which 1 is TMB group and 2 is TMB + H₂O₂Group, 3 is TMB + CFOs group, 4 is TMB + H₂O₂+ CFOs group, 5 is TMB + H₂O₂+ CFOs +50 ℃ and 6 is TMB + H₂O₂+ CFOs +1064 nm;

as can be seen in FIG. 13, TMB alone and CFOs or H₂O₂Upon mixing, negligible absorbance values indicated that no oxidation reaction occurred in the mixed solution. And in TMB and H₂O₂After CFOs is added into the mixed solution, the maximum absorbance value is increased, and the fact that CFOs can catalyze H is proved₂O₂Generating ROS. TMB + H₂O₂+ CFOs +50 ℃ is compared and verified by heating in a 50 ℃ water bath, and the active oxygen generating capacity is enhanced when the temperature is increased. TMB + H₂O₂+ CFOs +1064nm is laser irradiated at 1064nm for 5min to make the mixed solution reach 50 deg.C or so to develop color and absorb obviouslyAnd (4) increasing. These results indicate that the high temperature effect produced by 1064nm laser irradiation indeed promotes the generation of reactive oxygen species, i.e., the temperature increase promotes the activity of CFOs nanoenzymes.

200. mu.L of 500. mu.g mL^-1Example one CFOs absolute ethanol solution prepared, 300 μ L of 37% by mass fraction H₂O₂Reacting the solution with 20 mu L of TMB dye absolute ethyl alcohol solution with the concentration of 1mmol/mL for 1-5 min, wherein ultraviolet absorption spectra under different reaction times are shown in figure 14;

FIG. 14 shows 500. mu.g mL at different times^-1CFOs+300μL H₂O₂+ TMB ultraviolet absorption spectrum, in which 1 is reaction time 1min, 2 is reaction time 2min, 3 is reaction time 3min, 4 is reaction time 4min, and 5 is reaction time 5 min;

the results in FIG. 14 show that the active oxygen content in the mixed solution gradually increases as the reaction time increases.

200. mu.L of 100. mu.g mL^-1、200μg mL^-1、300μg mL^-1、400μg mL^-1And 500. mu.g mL^-1Example one CFOs absolute ethanol solution prepared in the same manner as in example one was mixed with 300. mu.L of 37% H₂O₂Reacting the solution with 20 μ L of 1mmol/mL TMB dye absolute ethanol solution for 5min, wherein the ultraviolet absorption spectrum is shown in figure 15;

FIG. 15 shows CFOs absolute ethanol solutions + 300. mu. L H at different concentrations₂O₂+ TMB ultraviolet absorption spectrum, where 1 is TMB dye and 2 is 37% H by mass₂O₂And 3 is 500. mu.g mL^-1The CFOs absolute ethanol solution of (4) is TMB + 100. mu.g mL^-1+CFOs+H₂O₂And 5 is TMB + 200. mu.g mL^-1+CFOs+H₂O₂And 6 is TMB + 300. mu.g mL^-1+CFOs+H₂O₂And 7 is TMB + 400. mu.g mL^-1+CFOs+H₂O₂And 8 is TMB + 500. mu.g mL^-1+CFOs+H₂O₂；

The results in FIG. 15 show that the active oxygen content increases with increasing DCGP concentration.

100 mu LH₂O₂、200μLH₂O₂And 300. mu.LH₂O₂Respectively mixed with 200 mu L of 500 mu g mL^-1Reacting the CFOs absolute ethyl alcohol solution with 20 μ L of TMB dye absolute ethyl alcohol solution with concentration of 1mmol/mL for 5min, wherein the ultraviolet absorption spectrum is shown in figure 16;

FIG. 16 shows different contents of H₂O₂+ CFOs + TMB UV absorption spectrum, 1 in the figure is 100 μ LH₂O₂+ TMB + CFOs, 2 is 200 μ LH₂O₂+ TMB + CFOs, 3 is 300 μ LH₂O₂+TMB+CFOs；

The results in FIG. 16 show that the active oxygen content in the mixed solution is dependent on H₂O₂The content increases.

Using DMPO as capture reagent, the concentration of 200 μ L is 500 μ g mL^-1Example one DCGP Anhydrous ethanol solution prepared, 300 μ L of 37% by mass H₂O₂Reacting the solution with 20 μ L of DMPO anhydrous ethanol solution with concentration of 1mmol/mL for 5min, wherein ESR spectrum is shown as 1 in FIG. 17; 300 mul of H with mass fraction of 37%₂O₂Reacting the solution with 20 μ L of DMPO anhydrous ethanol solution with concentration of 1mmol/mL for 5min, wherein ESR spectrum is shown in 2 in FIG. 17;

FIG. 17 is an ESR spectrum of DCGP catalyzed active oxygen generation prepared in example one, where 1 is DMPO + H₂O₂+ DCGP, 2 is DMPO + H₂O₂；

As can be seen from fig. 17, the characteristic peak was detected at an OH intensity ratio of 1:2:2:1 using DMPO as a trapping agent.

FIG. 18 is a graph showing UV absorption spectra of DCGP absolute ethanol solutions of different concentrations, wherein 1 is 100. mu.g mL^-12 is 200. mu.g mL^-1And 3 is 300. mu.g mL^-1And 4 is 400. mu.g mL^-15 is 500. mu.g mL^-1；

Fig. 18 shows that DCGP has the ability to act as a light-to-heat converter.

FIG. 19 is a diagram of the photothermal conversion mechanism of DCGP nanocomposites;

FIG. 19 shows that DCGP prepared in the first example has strong absorption at 1064nm and can be used as a photothermal conversion agent.

FIG. 20 shows the different concentrations of DCGP absolute ethanol solutions (100, 200, 300, 400 and 500. mu.g mL^-1) At 1.7W cm^-2Thermal infrared imaging pictures obtained under 1064nm light irradiation;

the results in FIG. 20 show that the effect of temperature increase increases with increasing concentration of DCGP solution.

FIG. 21 shows DCGP absolute ethanol solutions with different concentrations and H₂O is 1.7W cm^-2Temperature rise curve under 1064nm light irradiation, wherein 1 is 100. mu.g mL^-12 is 200. mu.g mL^-1And 3 is 300. mu.g mL^-1And 4 is 400. mu.g mL^-15 is 500. mu.g mL^-16 is H₂O；

As can be seen from fig. 21, the temperature increase effect increases with the increase in the concentration of the DCGP solution;

FIG. 22 shows different powers (1.0, 1.3, 1.4, 1.7 and 1.8W cm^-2) 300 μ g mL under 1064nm light excitation^{- 1}Temperature rise curve of DCGP absolute ethyl alcohol solution, wherein the power of 1 in the graph is 1.0W cm^-22 power of 1.3W cm^-2And the power of 3 is 1.4W cm^-24 power of 1.7W cm^-2And 5 at a power of 1.8W cm^-2；

The results of FIG. 22 show that the temperature increasing effect is improved with the increase of the 1064nm irradiated light power.

At 1.7W cm^-2500. mu.g mL under 1064nm light irradiation^-1The single-cycle temperature rise and fall curve of the DCGP absolute ethyl alcohol solution is shown in figure 23, the laser is turned off after laser irradiation is carried out for 10min, and the change curve of the temperature along with the time is shown in figure 23;

FIG. 23 is a graph of temperature versus time;

as can be seen from fig. 23, the solution was naturally cooled to the initial temperature.

FIG. 24 shows the signal at 1.7W cm^-2500. mu.g mL under 1064nm light irradiation^-1A temperature change curve chart of DCGP absolute ethyl alcohol solution for 3 temperature rise and fall cycles;

figure 24 detects the photo-stability of DCGP, with no significant temperature rise or decay during cycling indicating good photo-thermal stability of the material.