Hollow cobalt hydroxide-iron nanoszyme as well as preparation method and application thereof

Abstract

The application discloses a kind of hollow cobalt hydroxide iron nanoscale enzyme and its preparation method and application, belong to medical technology field.The nanoscale enzyme preparation method includes: first, prepare silica nanoparticle template, then add cobalt chloride hexahydrate and iron chloride hexahydrate to template suspension, then add hexamethylene tetramine and sodium citrate, under the condition of heating stirring, grow cobalt iron hydroxide layer on the surface of silica particle template;Finally, using ammonia etching silica particle template under heating condition, obtain hollow cobalt iron hydroxide nanoscale enzyme.The hollow cobalt iron hydroxide nanoscale enzyme prepared by the application has very strong peroxidase-like activity, so that it has good application potential in the chemical kinetics therapy of tumor.At the same time, hollow structure and hydroxide characteristics provide support for the nanomaterial as drug carrier to realize efficient oncolytic peptide loading and acid response release.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medicine, in particular to a hollow cobalt hydroxide iron nanoscale enzyme and a preparation method and application thereof. BACKGROUND

[0002] Chemotherapy as a systemic treatment for cancer has problems such as poor water solubility of drugs, large side effects, and low bioavailability, which limits the application of cancer chemotherapy treatment agents. In recent years, with the in-depth study of the biochemical characteristics of tumors, our understanding of the tumor tissue microenvironment has become clearer. For example, compared with normal tissues, tumor tissues exhibit a weak acid environment; tumor cells overexpress hydrogen peroxide and other characteristics. These characteristics of the tumor tissue microenvironment provide ideas for the development of a new generation of precise treatment agents based on the tumor tissue microenvironment response.

[0003] Chemodynamic therapy (CDT) is a treatment method that uses Fe-based functional nanoparticles to initiate Fenton or Fenton-like reactions in situ in the tumor microenvironment (TME) under acidic and hydrogen peroxide conditions, thereby producing strong oxidative hydroxyl radicals to induce cancer cell death. The essence of chemodynamic therapy is catalysis. Catalytic metal ions (such as Fe 2+ , Mn 2+ , etc.) released by functional nanoparticles in response to tumor tissue acid or nanoscale enzymes with peroxidase activity can catalyze the hydrogen peroxide overexpressed by tumor cells into strong cytotoxic hydroxyl radicals, achieving tumor killing. However, for cells in normal tissues, there is no serious impact because the pH is not suitable and the hydrogen peroxide concentration is insufficient.

[0004] However, traditional nanoscale enzymes such as Fe3O4 have poor peroxidase activity in neutral and slightly acidic environments, and can only function in lysosomal environments with a pH of 5, which results in their weak tumor treatment ability. Developing nanoscale enzymes that exhibit high peroxidase activity near neutral pH is crucial for improving CDT based on peroxidase activity.

[0005] Layered double hydroxide (LDH) is a biodegradable two-dimensional nanomaterial that is widely used as a carrier for chemotherapy drugs. This material has good biocompatibility and acid responsiveness, and can be degraded into low-toxicity ions in an acidic physiological environment, which helps to minimize the biological safety risks (such as inflammation) caused by its long-term accumulation in the body. Some hydroxide materials containing catalytically active metals have peroxidase-like activity, which can catalyze hydrogen peroxide to hydroxyl radicals to achieve chemodynamic therapy. At the same time, some metal ions with reducing ability (such as Co 2+ , Ni2+ The incorporation of the CDT (or the like) can enhance the CDT effect of the nano-enzyme with peroxidase-like activity by changing the valence of the catalytically active metal ions.

[0006] Based on the above background, if cobalt-iron hydroxide nanoparticles with high specific surface area can be prepared, it is very likely that the nanoparticles will exhibit extremely strong peroxidase-like activity and high drug loading capacity in slightly acidic or even neutral environments, thereby effectively achieving tumor killing based on tumor microenvironment response. SUMMARY

[0007] The purpose of the present application is to construct a nano-enzyme with high peroxidase activity in neutral and slightly acidic environments through structure and component design, so that the nanoparticles can achieve efficient chemical kinetic therapy and acid-responsive drug release.

[0008] To achieve the above purpose, the technical scheme adopted by the present application is as follows:

[0009] The present application provides a preparation method of hollow cobalt-iron hydroxide nano-enzyme, comprising the following steps:

[0010] (1) preparing a silica nanoparticle template;

[0011] (2) adding cobalt chloride hexahydrate and iron chloride hexahydrate to the silica nanoparticle template suspension, and then adding hexamethylenetetramine and sodium citrate, and growing a cobalt-iron hydroxide layer on the surface of the silica particle template under the condition of heating and stirring;

[0012] (3) etching the silica particle template using ammonia water under heating conditions to obtain hollow cobalt-iron hydroxide nano-enzyme.

[0013] In the present application, a cobalt-iron hydroxide layer is prepared on the surface of the silica nanoparticle template, and then the silica nanoparticle template is etched to obtain a hollow cobalt-iron hydroxide material. The nano-material contains catalytically active metals, which endow it with peroxidase-like activity. The hollow structure increases the specific surface area of the material and improves the peroxidase-like activity of the material. Studies have shown that the material has high peroxidase activity in neutral and slightly acidic environments. In addition, the high specific surface area of the hollow material can provide space for effective drug loading.

[0014] In the preparation of nanoparticles, the present application first uses tetraethyl orthosilicate as a silicon source to prepare a silica nanoparticle template by hydrolysis and polycondensation reaction of tetraethyl orthosilicate in an ethanol solution using ammonia water. Specifically, the silica particle template is obtained by crosslinking tetraethyl orthosilicate in an ethanol solution using ammonia water at a reaction temperature of 24-30℃.

[0015] Further, the volume ratio of water, tetraethyl orthosilicate and ammonia in the reaction system is 1:2-2.5:4-5.

[0016] In step (2), iron chloride hexahydrate and cobalt chloride hexahydrate are added to the aqueous solution of the silica particle template, and metal ions are adsorbed on the surface of the silica particles by virtue of the negative charge of the silica particle surface. Then, hexamethylenetetramine capable of generating hydroxyl ions by hydrolysis and sodium citrate capable of complexing metal ions to prevent the reaction from being too fast are added to the solution, and a hydroxide layer is grown on the surface of the silica particle template under heating.

[0017] Further, the silica particle template is dispersed in water by ultrasonic treatment, and then iron chloride hexahydrate and cobalt chloride hexahydrate are added, and the positively charged metal ions are fully adsorbed on the surface of the negatively charged silica nanoparticle template by continuing the ultrasonic treatment.

[0018] Further, the molar ratio of cobalt chloride hexahydrate to iron chloride hexahydrate is 2-4:1, and the total metal ion to silica nanoparticle template ratio is 0.1-0.2 mmol:10 mg.

[0019] Further, the concentration of hexamethylenetetramine in the reaction system is 0.5-1 mg / mL, and the concentration of sodium citrate is 0.3-0.5 mg / mL.

[0020] Further, the reaction condition is 80-100 DEG C for 4 h.

[0021] In step (3), the silica particle template is etched by ammonia under hydrothermal conditions to obtain a hollow cobalt-iron hydroxide nanoscale enzyme.

[0022] Further, the volume ratio of ethanol to aqueous solution in the reaction liquid system is 1.5-2.5:1, and the aqueous phase is composed of water and ammonia, and the volume ratio of water to ammonia is 2-5:1.

[0023] Further, the reaction condition is 120 DEG C for 18-30 h.

[0024] The present application provides a hollow cobalt-iron hydroxide nanoscale enzyme prepared by the preparation method.

[0025] The present application provides the use of the hollow cobalt-iron hydroxide nanoscale enzyme in the preparation of a tumor treatment drug.

[0026] Another object of the present application is to provide a composite nanoparticle composed of the hollow cobalt-iron hydroxide nanoscale enzyme and oncolytic peptide.

[0027] In the present application, the hollow cobalt-iron hydroxide nanoscale enzyme and the oncolytic peptide can be combined by stirring in an aqueous solution at room temperature. The oncolytic peptide is derived from an antibacterial peptide and is a peptide that can specifically destroy the cell membrane of tumor cells. The oncolytic peptide usually has positive electricity and can realize structural transformation against the high negative potential on the surface of tumor cells, thereby destroying the structure of the tumor cell membrane. This process provides a basis for promoting the entry of the nanoscale enzyme into tumor cells, thereby enhancing the therapeutic effect of the nanoscale enzyme.

[0028] Further, the preparation method of the composite nanoparticle comprises: adding an oncolytic peptide to a hollow cobalt-iron hydroxide nanoscale enzyme aqueous solution, stirring at room temperature, and then washing with ultrapure water and collecting the product by centrifugation to obtain the composite nanoparticle.

[0029] Further, the oncolytic peptide can be, but is not limited to, the 13-peptide Peptide C2 with the amino acid sequence GLKKLLGLLKKLL.

[0030] The present application also provides the use of the above-mentioned composite nanoparticle in the preparation of a tumor treatment drug.

[0031] Further, the tumor is a solid tumor. The solid tumor can be, but is not limited to, breast cancer.

[0032] The present application has the following beneficial effects:

[0033] (1) The present application prepares a hollow cobalt-iron hydroxide nanoscale enzyme through process design. Compared with cobalt hydroxide and iron hydroxide with the same metal content, this material exhibits stronger hydrogen peroxide catalytic capacity. At the same time, this material also exhibits extremely strong peroxidase-like activity in a neutral environment, so that the material can exert the desired catalytic function in the tumor microenvironment and the tumor cytoplasm. In addition, the hydroxide characteristics also provide a basis for the nanoscale enzyme to carry drugs and release them in an acidic environment.

[0034] (2) Compared with ordinary cancer treatment drugs, the nanomaterial provided by the present application has the following advantages: (I) based on the characteristics of the tumor microenvironment, the nanomaterial can realize precise treatment of tumors; (II) based on the functional design, the nanomaterial is combined with an antitumor peptide, and the composite of the two exhibits extremely strong tumor growth inhibition capacity. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 The transmission electron microscope photo of the silica nanoparticle template prepared in Example 1.

[0036] Figure 2Transmission electron microscopy image of SiO2@CoFe(OH)x prepared in Example 1.

[0037] Figure 3 Transmission electron microscopy image of H-CF prepared in Example 1.

[0038] Figure 4 Elemental mapping results of H-CF prepared in Example 1.

[0039] Figure 5 Spectra results of H-CF prepared in Example 1 exhibiting peroxidase-like activity catalyzing the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) by hydrogen peroxide.

[0040] Figure 6 Spectra results of H-CF prepared in Example 1 catalyzing the oxidation of TMB by hydrogen peroxide compared with hollow cobalt hydroxide, iron hydroxide, where a is compared with hollow iron hydroxide, b is compared with hollow cobalt hydroxide.

[0041] Figure 7 Transmission electron microscopy images of H-CF prepared in Example 1 after different time in PBS buffer with pH=5, a is 10 min, b is 1 h, c is 6 h.

[0042] Figure 8 Transmission electron microscopy image of P-CF prepared in Example 2.

[0043] Figure 9 Transmission electron microscopy image of oncolytic peptide used in Example 2.

[0044] Figure 10 Hydrodynamic radius results of P-CF and its components in Example 2.

[0045] Figure 11 Zeta-potential characterization results of P-CF and its components in Example 2.

[0046] Figure 12 Thermal analysis results of P-CF and its components in Example 2.

[0047] Figure 13 Hemolysis results of P-CF and its components in Example 2

[0048] Figure 14 Cytotoxicity of H-CF or P-CF on 4T1 cells under neutral (a), acidic (b) conditions in Example 2.

[0049] Figure 15 Volume changes of tumors of mice treated with different therapeutic agents in Example 2.

[0050] Figure 16 Hollow cobalt-iron hydroxide nanoszyme with cobalt-iron ratio of 2:1 in Example 3.

[0051] Figure 17 Transmission electron microscope image of nanoparticles in Comparative Example 1 in which the hydroxide layer was too thick, resulting in the failure of the silica particle template to be successfully etched. DETAILED DESCRIPTION

[0052] The application will be further described in conjunction with specific examples. The following examples are intended to illustrate the application and not to limit the scope of the application. Modifications or substitutions of the methods, steps or conditions of the application, which do not depart from the spirit and essence of the application, are intended to fall within the scope of the application.

[0053] The test methods used in the following examples are conventional unless otherwise specified; the materials, reagents, etc. used are commercially available reagents and materials unless otherwise specified.

[0054] Example 1: Preparation and performance characterization of hollow cobalt-iron hydroxide nanoszyme

[0055] 1. Preparation and morphology characterization of hollow cobalt-iron hydroxide nanoszyme

[0056] Into a beaker containing 30 mL of ethanol, 0.5 mL of deionized water, 1.15 mL of tetraethyl orthosilicate and 2.25 mL of ammonia water were sequentially added. The cup opening was sealed with a plastic film to prevent the evaporation of ethanol and ammonia water. After reaction at 26°C and 400 rpm for 4 h, centrifugation was performed, and washing with deionized water was performed once to obtain silica nanoparticle templates, the morphology of which is shown in FIG. 1. Figure 1

[0057] Subsequently, 20 mg of the silica nanoparticle templates were suspended in 180 mL of deionized water under ultrasonication at a power of 100 W. Subsequently, 10 mL of an aqueous solution in which 0.3 mmol of cobalt chloride hexahydrate and 0.1 mmol of iron chloride hexahydrate were dissolved were added. Ultrasonication was continued for 10 min to allow the positively charged metal ions to be sufficiently adsorbed on the surface of the negatively charged silica nanoparticle templates. Subsequently, 10 mL of an aqueous solution in which 200 mg of methenamine and 73.5 mg of sodium citrate were dissolved were added to the solution system. After ultrasonication was continued for 10 min, reaction was performed at 90°C and 400 rpm for 4 h. Centrifugation was performed, and washing with a mixture of water, ethanol and acetone at a ratio of 4:2:1 was performed twice to obtain core-shell structure nanoparticles SiO2@CoFe(OH)x in which cobalt-iron hydroxide was grown on the surface of silica. The morphology thereof is shown in FIG. 2. Figure 2

[0058] ​​Subsequently, 5 mg of the prepared SiO2@CoFe(OH)x was suspended in 14 mL of ethanol, and 2.22 mL of ammonia and 4.78 mL of deionized water were added. The mixture was then reacted in a hydrothermal reactor at 120 °C for 24 hours to obtain hollow cobalt-iron hydroxide nanozymes H-CF, the morphology of which is shown in the attached figure. Figure 3 As shown.

[0059] The elemental composition of the above products was analyzed using X-ray energy dispersive spectroscopy (EDS) with transmission electron microscopy. Figure 4 The elemental distribution shown demonstrates that the elemental composition of the prepared hollow particles meets expectations.

[0060] 2. Peroxidase activity analysis of H-CF:

[0061] Iron-based materials are widely recognized as being able to catalyze the generation of hydroxyl radicals from hydrogen peroxide, which have the second highest redox potential in nature—a peroxidase-like activity. This process is widely used in organic matter treatment, wastewater treatment, and other fields. It is generally believed that Fe... 2+ Fe 3+ It exhibits good peroxidase-like activity. Based on Co... 2+ Fe in cobalt-iron layered bimetallic hydroxide 3+ Through valence state compensation, we demonstrated that 25 μg / mL H-CF exhibits extremely high POD enzyme activity even under neutral conditions. The experiment was conducted using a 3,3',5,5'-tetramethylbenzidine (TMB) colorimetric reaction. TMB is oxidized by hydroxyl radicals generated from hydrogen peroxide catalyzed by H-CF to form blue TMB-ox, which shows an absorption peak at 655 nm.

[0062] Test method: 100 μL of 3M hydrogen peroxide, 300 mL of 8mM TMB, and 100 μL of 0.75 mg / mL H-CF were added to 2.5 mL of PBS buffer at different pH values. The absorption peak intensity at 650 nm was recorded using a UV-Vis spectrometer. This intensity indicates the amount of hydroxyl radicals produced.

[0063] Test results: such as Figure 5 As shown, 25 μg / mL H-CF rapidly increased the absorption peak of TMB at 650 nm in environments with pH values ​​of 7.4, 5.8, and 4.7. At pH 7.4, H-CF rapidly catalyzed the production of hydroxyl radicals from hydrogen peroxide, achieving an absorbance of 0.5 within 100 s. This indicates that H-CF exhibits extremely high catalytic ability to produce hydroxyl radicals from hydrogen peroxide even in neutral environments, enabling rapid colorimetric oxidation of TMB by hydrogen peroxide.

[0064] Meanwhile, H-CF also has significant advantages in catalytic performance compared to equal amounts of hollow iron hydroxide and cobalt hydroxide.

[0065] Test method: 100 μL of 3M hydrogen peroxide, 300 mL of 8 mM TMB were added into 2.5 mL of PBS buffer with pH = 5.8. Then 100 μL of 0.75 mg / mL H-CF or different concentration of 100 μL of hollow cobalt hydroxide / hollow iron hydroxide were added into the solution. The intensity of the absorption peak at 650 nm of the system was recorded by UV-Vis spectrometer. The intensity indicates the amount of hydroxyl radical production.

[0066] Test results: As shown in Figure 6 , hollow iron hydroxide and hollow cobalt hydroxide do not show obvious hydrogen peroxide catalytic performance in the environment of pH = 5.8, while the H-CF prepared in this embodiment shows strong catalytic ability. This may be related to the valence compensation of cobalt ions to iron ions.

[0067] 3. Acid-responsive degradation experiment of H-CF:

[0068] The hydroxide characteristics endow H-CF with potential acid-degradation ability, which provides a basis for using H-CF as a drug carrier for releasing drugs against the acidic microenvironment of tumors and the lysosomal environment of tumor cells. To characterize the acid-responsive degradation characteristics of H-CF, we used PBS buffer with pH = 5 to simulate the pH of the lysosome of tumor cells. After dispersing H-CF in PBS buffer with pH = 5, the structural changes of H-CF over time were as shown in Figure 7 , which lost the original structure and became dispersed after 6 hours, which proved the acid-responsive drug release potential of H-CF.

[0069] Example 2: H-CF complexed with oncolytic peptide

[0070] 1. H-CF complexed with oncolytic peptide with sequence GLKKLLGLLKKLL

[0071] To improve the tumor treatment potential of H-CF, H-CF was complexed with Peptide C2 (G is glycine, K is lysine, and L is leucine) with sequence GLKKLLGLLKKLL developed by us with oncolytic activity to obtain Peptide C2 / H-CF complex, denoted as P-CF. The specific operation process is as follows: 10 mg of H-CF was mixed with 2 mg of Peptide C2 in pure water and stirred at room temperature overnight, and then centrifuged and washed once to obtain P-CF.

[0072] 2. Characterization of P-CF

[0073] The transmission electron microscope photo of P-CF obtained by complexing is as shown in Figure 8As shown, it is not significantly different from H-CF. This is because Peptide C2 is much smaller than H-CF and does not significantly affect the morphology of P-CF. Electron micrographs of Peptide C2 are shown below. Figure 9 As shown.

[0074] Before and after recombination, the hydrodynamic radii of each component were characterized by dynamic light scattering as follows: Figure 10 As shown, the results are consistent with those of the electron microscope images.

[0075] The potential changes of H-CF and P-CF before and after recombination are as follows: Figure 11 As shown, the successful loading of Peptide C2 can be demonstrated. The high opposite charge between Peptide C2 and H-CF is the basis for the loading of Peptide C2 in H-CF, namely electrostatic adsorption.

[0076] To further determine the loading of Peptide C2, Peptide C2, H-CF, and P-CF were subjected to thermal analysis experiments. The results are as follows: Figure 12 As shown, Peptide C2 accounts for 20.6% of the mass in P-CF, which proves the good compatibility between H-CF and Peptide C2.

[0077] like Figure 13 As shown, after H-CF, Peptide C2, and P-CF were co-dispersed with 0.5 mL of mouse peripheral blood in PBS, P-CF significantly reduced the hemolysis caused by Peptide C2, which also proves the potential of H-CF as a good oncolytic peptide carrier.

[0078] 3. Responsive cytotoxicity of H-CF and P-CF

[0079] The acid-responsive release of Peptide C2 further disrupts tumor cell membranes, providing a basis for H-CF to enter cells and exert its effects on damaging important structures such as mitochondria and the nucleus, and is expected to enhance the antitumor properties of H-CF. The tumor cytotoxicity of H-CF and P-CF was analyzed using the mouse breast cancer cell line 4T1. 4T1 cells were subjected to in vitro experiments at 37°C and 5% CO2. The culture medium used was RPMI 1640 medium containing 10% fetal bovine serum. The acidic medium was obtained by adjusting the pH to 6.5 with hydrochloric acid to simulate the pH of the tumor microenvironment.

[0080] Specifically, 4T1 cells were digested and seeded into 96-well plates at a density of 4000 cells per well. After 12 hours of culture, the original culture medium was removed, and 125 μL of fresh culture medium containing different concentrations of materials was added to each well. After 24 h, the absorbance of each well was measured by CCK-8 method, and the survival rate of 4T1 cells co-cultured with H-CF or P-CF under each condition was obtained by taking the PBS group as a control.

[0081] As shown in FIG. 6, the cytotoxicity of H-CF and P-CF to 4T1 cells under neutral and acidic conditions was compared, and it was found that the cytotoxicity of both H-CF and P-CF was enhanced in an acidic environment. At the same time, the acidic cytotoxicity of P-CF was more intense, because P-CF released Peptide C2 to destroy the tumor cell membrane in an acidic environment, so that it could exert POD enzyme activity closer to important organelles such as mitochondria in tumor cells, making up for the short free path of hydroxyl radicals. Figure 14

[0082] 4. Anti-tumor performance test of H-CF and P-CF:

[0083] To verify the application prospect of H-CF and P-CF in the field of tumor treatment, an in vivo anti-tumor model was used to evaluate the tumor treatment ability of the hybrid. The application of the material in the tumor treatment direction was illustrated by the tumor growth inhibition at the animal (mouse) level.

[0084] 3-week-old balb / c female mice were raised in a 12h light / 12h dark environment for 1 week to adapt to the environment, and then 10 6 4T1 cancer cells were injected into the back to construct a 4T1 tumor model. When the tumor grew to about 100 mm 3 in diameter, the 4T1 tumor-bearing mice were randomly divided into four groups, and the day was recorded as day 0. On days 1 and 7, each group of mice was treated, the first group of mice was injected with PBS solution, the second group of mice was injected with 0.25 mg of H-CF particles, the third group of mice was injected with 0.05 mg of Peptide C2, and the fourth group of mice was injected with 0.25 mg of P-CF intravenously. The tumor size was recorded every day since the first injection, and the results are shown in Figure 15 .

[0085] As can be seen from Figure 15 , compared with the control group, the tumor volume of mice in the H-CF and Peptide C2 injection groups was inhibited to a certain extent, and the intravenous injection of P-CF had a stronger tumor inhibition performance. This indicates the potential of H-CF and P-CF for anti-tumor treatment.

[0086] Example 3: Preparation of hollow cobalt-iron hydroxide nanoscale enzyme with a cobalt-iron ratio of 2:1 ​

[0087] To a beaker containing 30 mL of ethanol, 0.5 mL of deionized water, 1.15 mL of tetraethyl orthosilicate and 2.25 mL of ammonia water were added in sequence. The cup was sealed with a plastic film to prevent the evaporation of ethanol and ammonia water. After reaction at 26 °C, 400 rpm for 4 h, centrifugation and washing with deionized water once, the silica nanoparticle template was obtained. Subsequently, 20 mg of the silica nanoparticle template was suspended in 180 mL of deionized water under ultrasonication. Then, 10 mL of an aqueous solution containing 0.26 mmol of cobalt chloride hexahydrate and 0.13 mmol of ferric chloride hexahydrate was added. Ultrasonication was continued for 10 min to allow the positively charged metal ions to be fully adsorbed on the surface of the negatively charged silica nanoparticle template. Subsequently, 10 mL of an aqueous solution containing 200 mg of hexamethylenetetramine and 120 mg of sodium citrate was added to the solution system. After ultrasonication for another 10 min, the reaction was carried out at 90 °C, 400 rpm for 4 h. After centrifugation and washing with a mixture of water, ethanol and acetone (4:2:1) twice, the core-shell structure nanoparticles SiO2@CoFe(OH)x were obtained, in which cobalt iron hydroxide was grown on the surface of silica.

[0088] Subsequently, 5 mg of the prepared SiO2@CoFe(OH)x was suspended in 14 mL of ethanol, 1.22 mL of ammonia water and 5.78 mL of deionized water, and the reaction was carried out in a hydrothermal kettle at 120 °C for 24 h to obtain a hollow cobalt iron hydroxide nanoscale enzyme with a cobalt iron ratio of 2:1. Its morphology is shown in FIG. 2B, which is not significantly different from the cobalt iron ratio of 3:1 hollow cobalt iron hydroxide nanoscale enzyme obtained in Example 1. Figure 16

[0089] Comparative Example 1

[0090] ​Add 0.5 mL of deionized water, 1.15 mL of tetraethyl orthosilicate, and 2.25 mL of ammonia to a beaker containing 30 mL of ethanol. Seal the beaker with a plastic film to prevent excessive evaporation of ethanol and ammonia. React at 26 °C and 400 rpm for 4 hours, then centrifuge and wash once with deionized water to obtain a silica nanoparticle template. Then, take 20 mg of the silica nanoparticle template and suspend it in 180 mL of deionized water under ultrasonic conditions. Next, add 10 mL of an aqueous solution containing 0.45 mmol of cobalt chloride hexahydrate and 0.15 mmol of ferric chloride hexahydrate. Continue ultrasonication for 10 min to allow positively charged metal ions to fully adsorb onto the surface of the negatively charged silica nanoparticle template. Then, add 10 mL of an aqueous solution containing 200 mg of hexamethylenetetramine and 120 mg of sodium citrate to the solution. Continue ultrasonication for 10 minutes, then react at 90 °C and 400 rpm for 4 hours. Centrifuge and wash twice with a mixture of water, ethanol and acetone in a 4:2:1 ratio to obtain core-shell structured nanoparticles SiO2@CoFe(OH)x grown on the surface of silica.

[0091] Subsequently, 5 mg of the prepared SiO2@CoFe(OH)x was suspended in 14 mL of ethanol, and 1.22 mL of ammonia and 5.78 mL of deionized water were added. The mixture was then reacted in a hydrothermal reactor at 120 °C for 24 hours. The morphology of the obtained nanoparticles is shown in the attached figure. Figure 17 As shown, the silica nanoparticle template was not successfully etched. This is because the increased metal ion content resulted in an excessively thick outer hydroxide layer, preventing ammonia from effectively contacting the central silica nanoparticle template and performing the etching process.

[0092] Comparative Example 2

[0093] Add 0.5 mL of deionized water, 1.15 mL of tetraethyl orthosilicate, and 2.25 mL of ammonia to a beaker containing 30 mL of ethanol. Seal the beaker with a plastic film to prevent excessive evaporation of ethanol and ammonia. React at 16 °C and 400 rpm for 4 hours, then centrifuge and wash once with deionized water to obtain silica nanoparticle templates. The silica nanoparticle templates exhibit large particle size and severe agglomeration during low-temperature growth, failing to meet the application requirements.

[0094] Conclusion: H-CF is a simple synthetic chemodynamic therapy agent. The nanoparticle also has strong peroxidase activity in neutral environment, and the peroxidase activity will be enhanced in acidic environment. It shows acid-enhanced anti-tumor activity at the cellular level. At the same time, the material is proved to have acid-responsive drug release ability, and is also a good oncolytic peptide carrier. After the material is combined with oncolytic peptide, oncolytic peptide can help H-CF enter tumor cells and exert peroxidase activity in a position closer to important organelles. This makes the complex show significantly enhanced in vivo and in vitro anti-tumor performance compared with H-CF.

[0095] The above merely describes preferred embodiments of the present application but is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for preparing a hollow cobalt hydroxide-iron nanoszyme, characterized in that, The method comprises the following steps: (1) preparing a silica nanoparticle template; (2) adding cobalt chloride hexahydrate and ferric chloride hexahydrate to the silica nanoparticle template suspension, the molar ratio of cobalt chloride hexahydrate to ferric chloride hexahydrate being 2-4:1, and the ratio of total metal ions to silica nanoparticle template being 0.1-0.2 mmol:10 mg; then adding hexamethylenetetramine and sodium citrate, and growing a cobalt-iron hydroxide layer on the surface of the silica particle template under the condition of heating and stirring; (3) etching the silica particle template using ammonia water under heating to obtain a hollow cobalt-iron hydroxide nanoscale enzyme.

2. The preparation method of hollow cobalt-iron hydroxide nanozyme as described in claim 1, characterized in that, In step (1), the silica nanoparticle template is prepared by using tetraethyl orthosilicate as a silicon source, and by hydrolyzing and polycondensing the tetraethyl orthosilicate in an ethanol solution using ammonia water; the reaction temperature is 24-30°C.

3. The preparation method of hollow cobalt-iron hydroxide nanozyme as described in claim 1, characterized in that, In step (2), the concentration of hexamethylenetetramine in the reaction system is 0.5-1 mg / mL, and the concentration of sodium citrate is 0.3-0.5 mg / mL.

4. The preparation method of hollow cobalt-iron hydroxide nanozyme as described in claim 1, characterized in that, In step (2), the reaction conditions are 80-100°C for 4 h.

5. The method for preparing hollow cobalt-iron hydroxide nanozymes as described in claim 1, characterized in that, In step (3), the volume ratio of ethanol to aqueous solution in the reaction liquid system is 1.5-2.5:1, and the aqueous phase is composed of water and ammonia water in a volume ratio of 2-5:1; the reaction conditions are 120°C for 18-30 h.

6. A hollow cobalt-iron hydroxide nanoscale enzyme prepared by the method of any one of claims 1-5.

7. Use of the hollow cobalt-iron hydroxide nanoscale enzyme of claim 6 in the preparation of a tumor treatment drug.

8. A composite nanoparticle, characterized in that, The composite nanoparticle is composed of the hollow cobalt-iron hydroxide nanoscale enzyme of claim 6 and an oncolytic peptide.

9. The composite nanoparticle of claim 8, wherein, The preparation method of the composite nanoparticle comprises adding an oncolytic peptide to a hollow cobalt-iron hydroxide nanoscale enzyme aqueous solution, stirring at room temperature, and then washing with ultrapure water and centrifuging to collect the product to obtain the composite nanoparticle.

10. Use of the composite nanoparticle of claim 8 or 9 in the preparation of a tumor treatment drug.

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