A hollow Prussian blue complex, preparation method and application thereof

Abstract

The present invention belongs to the field of medical technology and relates to a hollow Prussian blue complex. The hollow Prussian blue complex comprises hollow Prussian blue composite nanoparticles and a protease loaded on the hollow Prussian blue composite nanoparticles. The hollow Prussian blue composite nanoparticles are composite materials formed by coating one or more hollow Prussian blue nanoparticles with a mesoporous Prussian blue analog. The hollow Prussian blue complex provided by the present invention comprises hollow Prussian blue composite nanoparticles and a protease. The loaded protease can increase the hydrogen peroxide content in tumor tissues, synergistically achieving cascade catalysis with the catalase activity of the hollow Prussian blue composite nanoparticles.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology and relates to a hollow Prussian blue complex, a preparation method and an application thereof. Background Art

[0002] Malignant tumors pose a serious threat to human life and health. In recent years, breakthroughs have been made in novel treatments based on reactive oxygen species that inhibit tumor growth and metastasis by directly inducing tumor cell apoptosis and necrosis. Chemodynamic therapy utilizes in situ Fenton / Fenton-like reactions to convert hydrogen peroxide into hydroxyl radicals, which has high specificity and selectivity. However, due to limited catalytic conditions in the tumor microenvironment and insufficient hydrogen peroxide levels, chemodynamic therapy is severely limited and urgently needs to be combined with other anti-tumor methods to achieve better therapeutic effects. The combined use of self-supplied hydrogen peroxide protease to enhance chemodynamic performance is expected to be an effective method to improve the poor therapeutic effect of chemodynamic therapy.

[0003] However, since proteases that self-generate hydrogen peroxide may require a high oxygen demand, the hypoxic tumor microenvironment struggles to provide a sustained oxygen supply for hydrogen peroxide production. Therefore, self-supply of oxygen is crucial. Studies on hollow Prussian blue complexes encapsulating proteases that also generate oxygen without sacrificing peroxidase-like performance have yet to be reported. Summary of the Invention

[0004] In response to the deficiencies in the prior art, the present invention provides a hollow Prussian blue complex, a preparation method, and an application thereof. The hollow Prussian blue complex comprises hollow Prussian blue composite nanoparticles and a protease. The loaded protease can increase the hydrogen peroxide content in tumor tissues and synergize with the catalase activity of the hollow Prussian blue composite nanoparticles to achieve the purpose of cascade catalysis.

[0005] One object of the present invention is achieved by the following technical solutions:

[0006] A hollow Prussian blue complex comprises hollow Prussian blue complex nanoparticles and a protease loaded on the hollow Prussian blue complex nanoparticles.

[0007] Preferably, the hollow Prussian blue composite nanoparticles are composite materials formed by coating a mesoporous Prussian blue analog on the surface of one or more hollow Prussian blue nanoparticles.

[0008] The hollow Prussian blue nanoparticles refer to Prussian blue nanoparticles with a hollow structure inside. The average particle size of the hollow Prussian blue nanoparticles is preferably 20 to 280 nanometers.

[0009] The mesoporous Prussian blue analogue has a pore structure with a pore size between 1 and 50 nanometers. The Prussian blue analogue is formed by replacing the iron element of Prussian blue with a dopant element. Preferably, the dopant element of the mesoporous Prussian blue analogue is one or more of Cu, Co, Cd, Au, Ag, Ce, Ni, and Mn.

[0010] Preferably, the hollow Prussian blue composite nanoparticles have peroxidase-like activity and / or catalase-like activity, which can convert hydrogen peroxide in the tumor area into hydroxyl radicals and oxygen, improve the hypoxic environment of the tumor, and thus promote tumor cell apoptosis.

[0011] Preferably, the average particle size of the hollow Prussian blue composite nanoparticles is 20 to 300 nanometers.

[0012] Preferably, the protease is one or more of plasma amine oxidase, laccase, lactate oxidase, chloroperoxidase, monoamine oxidase, and glucose oxidase.

[0013] The protease loaded on the hollow Prussian blue composite nanoparticles can increase the hydrogen peroxide content in tumor tissue and improve the current situation of insufficient endogenous hydrogen peroxide supply during chemodynamic therapy.

[0014] Another object of the present invention is achieved through the following technical solutions:

[0015] A method for preparing a hollow Prussian blue complex comprises the following steps:

[0016] The hollow Prussian blue composite nanoparticles are dispersed in water, and then a cationic polymer is added. After stirring for 1 to 4 hours, protease is added and stirred for 4 to 12 hours. The mixture is centrifuged, washed with water, and dried to obtain a hollow Prussian blue composite.

[0017] Cationic polymers act as an intermediate "bridge", which is beneficial for the loading of proteases on hollow Prussian blue composite nanoparticles, thereby increasing the loading capacity and loading firmness.

[0018] Preferably, the cationic polymer is one or more of polyacrylamine hydrochloride (PAH), cationic polyacrylamide (CPAM), polyhexamethylenedimethylammonium bromide, amino-functionalized polyethylene glycol (NH2-PEG), and polyethyleneimine (PEI).

[0019] Preferably, the mass ratio of the hollow Prussian blue composite nanoparticles to the cationic polymer is 1:0.5-2.

[0020] Preferably, the added amount of protease is 5-40% of the mass of the hollow Prussian blue composite nanoparticles.

[0021] Preferably, the centrifugal speed is 7000 to 13000 rpm, and the centrifugal time is 5 to 20 minutes.

[0022] Preferably, the preparation method of hollow Prussian blue composite nanoparticles comprises the following steps:

[0023] S1. Preparation of Prussian blue nanoparticles: mixing a dispersant, potassium ferrocyanide, and a 0.005-0.05 mol / L hydrochloric acid aqueous solution, reacting at 60-90° C. for 18-30 hours, centrifuging, washing, and drying to obtain Prussian blue nanoparticles;

[0024] S2. Preparation of hollow Prussian blue nanoparticles: Prussian blue nanoparticles, a dispersant, and a 0.5-5 mol / L hydrochloric acid aqueous solution are mixed, stirred for 1-5 hours, reacted at 120-150° C. for 1-5 hours, centrifuged, washed with water, and dried to obtain hollow Prussian blue nanoparticles;

[0025] S3. Preparation of hollow Prussian blue composite nanoparticles: Disperse the hollow Prussian blue nanoparticles, doping source, and dispersant in water, stir for 1 to 5 hours, then add potassium ferrocyanide, continue stirring for 1 to 5 hours, and then stand at room temperature for 10 to 36 hours. After centrifugation, washing with water, and drying, hollow Prussian blue composite nanoparticles are obtained.

[0026] Preferably, the dispersant is one or more of citric acid, sodium citrate, polyvinyl pyrrolidone, polyethyleneimine, human serum albumin, and bovine serum albumin.

[0027] Preferably, in step S1, the ratio of the mass mg of the dispersant, the mass mg of potassium ferricyanide and the volume ml of the hydrochloric acid aqueous solution is (50-100):(1-6):1.

[0028] Preferably, in step S2, the ratio of the mass mg of the Prussian blue nanoparticles, the mass mg of the dispersant, and the volume ml of the hydrochloric acid aqueous solution is 1:(2-10):(0.8-2).

[0029] Preferably, the doping source is acetate of one or more elements selected from the group consisting of Cu, Co, Cd, Au, Ag, Ce, Ni, and Mn.

[0030] When the doping source added in step S3 is manganese acetate tetrahydrate, the doping element of the shell of the mesoporous Prussian blue analog in the obtained hollow Prussian blue composite nanoparticles is Mn.

[0031] Preferably, in step S3, the mass ratio of the hollow Prussian blue nanoparticles, the doping source, the dispersant, and the potassium ferrocyanide is (1-2): (0.5-1): (7-15): (0.6-1.5).

[0032] The third object of the present invention is achieved through the following technical solutions:

[0033] Application of a hollow Prussian blue complex in self-enhanced chemodynamic therapy.

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] (1) The hollow Prussian blue complex provided by the present invention is easy to prepare, has a relatively uniform particle size, and has the advantages of a large specific surface area, good catalytic performance, and good biocompatibility;

[0036] (2) The hollow Prussian blue complex provided by the present invention has excellent peroxidase-like activity and / or catalase-like activity, which can convert hydrogen peroxide in the tumor area into hydroxyl radicals and oxygen, improve the hypoxic environment of the tumor, and thus promote tumor cell apoptosis;

[0037] (3) The hollow Prussian blue composite provided by the present invention is loaded with protease, which can convert glucose in the tumor area into hydrogen peroxide, improving the current situation of insufficient hydrogen peroxide supply in chemodynamic therapy. It also cooperates with the catalase activity of the hollow Prussian blue composite nanoparticles to achieve the purpose of cascade catalysis, significantly improving the anti-tumor treatment effect, and has good biological application prospects;

[0038] (4) The hollow Prussian blue nanoparticles prepared by the present invention and the hollow Prussian blue composite nanoparticles coated with mesoporous Prussian blue analogs have better peroxidase-like activity and / or catalase-like activity than hollow Prussian blue nanoparticles, and have a more excellent therapeutic effect of inhibiting tumor growth after loading with protease;

[0039] (5) The hollow Prussian blue composite nanoparticles of the present invention load protease via cationic polymer, thereby increasing the loading amount and loading firmness of the protease. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 is a transmission electron micrograph of compound C1 prepared in Example 1;

[0041] Figure 2 is the oxygen production of Mn-HMPB and HMPB in Example 1;

[0042] Figure 3 The figure shows the cytotoxicity test results of compound C1 and Mn-HMPB prepared in Example 1;

[0043] Figure 4 This is a graph showing the results of treating breast cancer in mice with compound C1 prepared in Example 1. DETAILED DESCRIPTION

[0044] The technical solutions of the present invention are further described below by means of specific examples and accompanying drawings. It should be understood that the specific examples described herein are only intended to help understand the present invention and are not intended to limit the present invention. Unless otherwise specified, the raw materials used in the embodiments of the present invention are all commonly used raw materials in the art, the methods used in the embodiments are all conventional methods in the art, and all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art.

[0045] In the following embodiments, the reagents involved are as follows:

[0046]

[0047]

[0048] In the following embodiments, the cells MCF-7, U87mg, 786-O, HEC-1-B, a2780, and SK-ES-1 were purchased from the American Cell Bank and cultured according to their instructions before conducting experiments.

[0049] Example 1

[0050] The hollow Prussian blue complex of this embodiment was prepared by the following method:

[0051] S1. Stir 1.5 g of polyvinyl pyrrolidone, 66 mg of potassium ferricyanide, and 20 ml of a 0.01 mol / L hydrochloric acid aqueous solution until clear, place in a glass bottle, and react in an oven at 80° C. for 24 hours. Wash the product three times with water by centrifugation at 13,000 rpm, and then dry in a vacuum drying oven to obtain Prussian blue nanoparticles.

[0052] S2. 20 mg of the prepared Prussian blue nanoparticles, 100 mg of polyvinylpyrrolidone, and 20 ml of a 1 mol / L hydrochloric acid aqueous solution were placed in a beaker, stirred for 3 hours, and then placed in a reactor. The mixture was reacted in an oven at 140° C. for 3 hours. The product was washed three times by centrifugation at 13,000 rpm and air-dried at room temperature for 24 hours to obtain hollow Prussian blue nanoparticles HMPB.

[0053] S3. Weigh 25 mg of hollow Prussian blue nanoparticles, add 11 mg of manganese acetate tetrahydrate, 33 mg of sodium citrate dihydrate, and 125 mg of polyvinyl pyrrolidone, and dissolve the mixture in 5 ml of ultrapure water. Stir for 3 hours, then add 5 ml of a 3.3 mg / ml potassium ferrocyanide aqueous solution, and continue stirring for 2 hours. After the product is allowed to stand at room temperature for 24 hours, it is washed three times with ultrapure water at 11,000 rpm to obtain hollow Prussian blue composite nanoparticles Mn-HMPB.

[0054] S4. Weigh 5 mg of hollow Prussian blue composite nanoparticles, disperse them in 10 ml of water, and sonicate for 10 minutes. Slowly add 1 ml of a 5 mg / ml polyacrylamine hydrochloride aqueous solution dropwise thereto, and stir for 3 hours. Then, add 1 mg of glucose oxidase to the above solution, and stir for 8 hours. The product is washed three times by centrifugation at 10,000 rpm with water, and then naturally air-dried to obtain the hollow Prussian blue complex C1.

[0055] The preparation methods of the hollow Prussian blue composites of Examples 2-24 were the same as those of Example 1, except that the doping elements, cationic polymers, and proteases in the hollow Prussian blue composites C2-C24 of Examples 2-24 differed, as shown in Table 1 below. In Examples 7-12, when the doping element was Co, the doping source added in step S3 of the hollow Prussian blue composite preparation process was cobalt acetate tetrahydrate; in Examples 13-18, when the doping element was Cu, the doping source added in step S3 of the hollow Prussian blue composite preparation process was copper acetate monohydrate; and in Examples 19-24, when the doping element was Zn, the doping source added in step S3 of the hollow Prussian blue composite preparation process was zinc acetate dihydrate.

[0056] Table 1

[0057]

[0058]

[0059] 1. Transmission electron microscopy characterization of products

[0060] The hollow Prussian blue nanoparticles of Example 1 and C1 were characterized using a JEOL JEM 3010UHR transmission electron microscope. The transmission electron microscope images of the hollow Prussian blue nanoparticles showed uniform particle size of about 180 nanometers, and a clear hollow structure could be seen. Figure 1 As shown in the figure, there is no significant change in the morphology compared to the hollow Prussian blue nanoparticles. A clear hollow structure can be seen, and the particle size is uniform. C2-24 all have similar morphologies.

[0061] 2. Characterization of catalase activity in vitro

[0062] The hollow Prussian blue nanoparticles HMPB and hollow Prussian blue composite nanoparticles Mn-HMPB prepared in Example 1 were characterized for in vitro catalase activity. 100 mg / mL HMPB and Mn-HMPB dispersed in 5 mL of PBS buffer (pH 7.4) were placed in a glass bottle, and hydrogen peroxide was added to a final concentration of 10 mmol / mL. A dissolved oxygen detector was used to record the dissolved oxygen value every 10 seconds. The results are shown in Figure 2. Figure 2 It can be seen that the catalase activity of the hollow Prussian blue composite nanoparticles Mn-HMPB is significantly better than that of the hollow Prussian blue nanoparticles HMPB.

[0063] 3. Cytotoxicity Study

[0064] MCF-7 cells were digested with trypsin and diluted to a 100,000 / ml solution. 100 μl of this solution was added to each well of a 96-well plate and allowed to adhere for 24 hours. After 24 hours, the culture medium was aspirated and then a culture medium containing various concentrations of hollow Prussian blue composite nanoparticles (Mn-HMPB) and C1 was added. Twelve hours later, CCK-8 was added to each well. After a 2-hour incubation, the absorbance at 450 nm was read on a microplate reader. Figure 3 .

[0065] Depend on Figure 3 It can be seen that the hollow Prussian blue composite nanoparticles loaded with GOx have stronger cytotoxicity and exhibit stronger chemical kinetics, which proves its ability to use glucose to react to generate hydrogen peroxide and improve chemical kinetics.

[0066] The cytotoxicity of other compounds C2 to C24 was investigated. The specific operation was the same as the above method. The other conditions are shown in Table 2.

[0067] Table 2

[0068]

[0069] Note: When the drug concentration is 100 μg / ml, ++++ indicates a survival rate of less than 30%; +++ indicates a survival rate of 30-50%; ++ indicates a survival rate of 50%-70%; + indicates a survival rate of more than 70%.

[0070] C2-C24 all showed similar chemodynamic therapeutic effects.

[0071] 4. Investigation of the effect of chemokinetic inhibition on tumors

[0072] Disperse C1 in PBS to a 1 mg / ml solution. Ten mice were divided into two groups: the PBS group and the C1 group. 100 μl of the above material was injected into the tail vein of each group. The injections were performed on days 0, 3, and 5. The tumor volumes of the mice were observed and recorded on day 14. Figure 4 The data shown show that hollow Prussian blue composite nanoparticles loaded with GOx have good chemodynamic therapeutic properties.

[0073] The chemokinetic inhibition effect of compounds C2-C24 was investigated. The specific operation was the same as the above method; the remaining conditions are shown in Table 3.

[0074] Table 3

[0075]

[0076]

[0077] Note: When the drug concentration injected into the tail vein is 1 μg / ml, ++++ indicates that the tumor inhibition effect is greater than 70%; +++ indicates that the tumor inhibition effect is 50-70%; ++ indicates that the tumor inhibition effect is 30%-50%; + indicates that the tumor inhibition effect is less than 30%.

[0078] C2-C24 all showed similar chemokinetic effects in inhibiting tumor growth.

[0079] Finally, it should be noted that the specific embodiments described herein are merely illustrative of the spirit of the present invention and are not intended to limit the manner in which the present invention is intended to be implemented. Persons skilled in the art may make various modifications, additions, or substitutions to the described embodiments, and it is not necessary or possible to provide a comprehensive list of all possible embodiments. However, any obvious changes or modifications arising from the essential spirit of the present invention remain within the scope of protection of the present invention, and interpreting them as any additional limitations would be contrary to the spirit of the present invention.

Claims

1. A hollow Prussian blue complex, characterized in that The invention comprises hollow Prussian blue composite nanoparticles and a protease loaded on the hollow Prussian blue composite nanoparticles; The method for preparing hollow Prussian blue composite nanoparticles comprises the following steps: S1. Preparing Prussian blue nanoparticles: mixing a dispersant, potassium ferrocyanide, and a 0.005-0.05 mol / L hydrochloric acid aqueous solution, reacting at 60-90° C. for 18-30 hours, centrifuging, washing, and drying to obtain Prussian blue nanoparticles; S2. Preparation of hollow Prussian blue nanoparticles: Prussian blue nanoparticles, a dispersant, and a 0.5-5 mol / L hydrochloric acid aqueous solution are mixed, stirred for 1-5 hours, reacted at 120-150° C. for 1-5 hours, centrifuged, washed with water, and dried to obtain hollow Prussian blue nanoparticles; S3. Preparation of hollow Prussian blue composite nanoparticles: Dispersing hollow Prussian blue nanoparticles, a doping source, and a dispersant in water, stirring for 1 to 5 hours, then adding potassium ferrocyanide, continuing to stir for 1 to 5 hours, and then standing at room temperature for 10 to 36 hours, centrifuging, washing with water, and drying to obtain hollow Prussian blue composite nanoparticles; The doping source is acetate of one or more elements selected from Cu, Co, Cd, Au, Ag, Ce, Ni, and Mn; the hollow Prussian blue composite nanoparticles have peroxidase-like activity and / or catalase-like activity; The protease is one or more of plasma amine oxidase, laccase, lactate oxidase, chloroperoxidase, monoamine oxidase, and glucose oxidase.

2. The hollow Prussian blue composite according to claim 1, characterized in that The average particle size of the hollow Prussian blue composite nanoparticles is 20 to 300 nanometers.

3. The hollow Prussian blue composite according to claim 1, characterized in that The dispersant is one or more of citric acid, sodium citrate, polyvinyl pyrrolidone, polyethyleneimine, human serum albumin, and bovine serum albumin.

4. The hollow Prussian blue composite according to claim 1, characterized in that In step S3, the mass ratio of the hollow Prussian blue nanoparticles, the doping source, the dispersant, and the potassium ferrocyanide is (1-2): (0.5-1): (7-15): (0.6-1.5).

5. A method for preparing the hollow Prussian blue complex according to claim 1, characterized in that: The following steps are involved: The hollow Prussian blue composite nanoparticles are dispersed in water, and then a cationic polymer is added. After stirring for 1 to 4 hours, protease is added and stirred for 4 to 12 hours. The mixture is centrifuged, washed with water, and dried to obtain a hollow Prussian blue composite. The cationic polymer is one or more of polyacrylamine hydrochloride, cationic polyacrylamide, polyhexamethylenedimethylammonium bromide, amino-functionalized polyethylene glycol, and polyethyleneimine.

6. The preparation method according to claim 5, characterized in that The mass ratio of hollow Prussian blue composite nanoparticles to cationic polymer is 1:0.5~2.

7. The preparation method according to claim 5, characterized in that The amount of protease added is 5-40% of the mass of the hollow Prussian blue composite nanoparticles.

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