Manganese peroxidase-loaded manganese-doped silicon dioxide nano material as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of nano materials, and particularly relates to a manganese peroxidase-loaded manganese-doped silicon dioxide nano material as well as a preparation method and application thereof. The preparation method comprises the following steps: doping manganese into silicon dioxide nano-particles, and adding a manganese peroxidase solution into the silicon dioxide nano-particles serving as a carrier to prepare the manganese-doped silicon dioxide nano-material loaded with manganese peroxidase. The synthesized nano material can be used for preparing an anti-tumor drug reagent, and by inducing ferroptosis of tumor cells, after ferroptosis of the tumor cells occurs, integrity of cell membranes is destroyed, finally cell membrane rupture and tumor cell death are caused, the tumor cells can be efficiently removed, and the tumor volume is reduced.

Description

Technical Field

[0001] This invention belongs to the technical field of nanomaterials, specifically relating to a manganese-doped silica nanomaterial loaded with manganese peroxidase, its preparation method, and its application. Background Technology

[0002] Silica nanomaterials, due to their high specific surface area, tunable pore structure, and good biocompatibility, have become ideal carriers for drug delivery systems. Many researchers have enhanced their targeting and therapeutic functions through meticulous design, such as constructing hollow structures, elemental doping (e.g., manganese / iron / copper doping), or surface functionalization (e.g., camouflage of cancer cell membranes), thus demonstrating broad application prospects in multiple fields.

[0003] Manganese-doped silica nanomaterials exert their anti-tumor effects primarily through catalytic generation of reactive oxygen species, consumption of glutathione, and activation of immune responses. Manganese peroxidase, a microbial oxidoreductase, derives its anti-tumor effect mainly from its powerful catalytic ability. In the presence of manganese ions, it can utilize H₂O₂ as a substrate to catalyze the oxidation of various organic compounds (such as unsaturated fatty acids), generating lipid free radicals, initiating lipid peroxidation, and leading to tumor cell death.

[0004] Patent CN 110028072 A, entitled "A Method for Preparing Manganese-Doped Mesoporous Silica Nanoparticles," discloses a method for preparing manganese-doped mesoporous silica nanoparticles using a "one-pot method" to incorporate manganese into the silica nanoparticle framework, ensuring uniform doping of manganese within the nanoparticles. Patent CN 113493223 A, entitled "A Method for Preparing and Applying Hollow Manganese Dioxide Nanospheres," discloses a method for preparing hollow manganese dioxide nanospheres and their application in tumor therapeutics and imaging. While there are numerous reports on manganese-doped silica nanoparticles, there are no reports on loading manganese peroxidase onto manganese-doped silica nanomaterials.

[0005] Therefore, in this invention, manganese peroxidase is loaded onto manganese-doped silica nanomaterials to construct a multifunctional nanoplatform, achieving synergistic anti-tumor effects. The dual catalysis, with both catalyzing in synergy, catalyzes more lipid peroxidation, significantly enhances the oxidative stress damage to tumor cells, and efficiently induces ferroptosis. Summary of the Invention

[0006] To address the existing technical problems, this invention provides a manganese-doped silica nanomaterial loaded with manganese peroxidase, its preparation method, and its application. Manganese is doped into silica nanoparticles and simultaneously used as a carrier to add manganese peroxidase, thus preparing manganese-doped silica nanoparticles loaded with manganese peroxidase. This constructs a multifunctional nanoplatform where the dual catalysis of manganese ions and manganese peroxidase synergistically achieves anti-tumor effects, realizing efficient and targeted ferroptosis-based anti-tumor therapy.

[0007] To achieve the purpose of the invention, the following technical solution is provided: This invention discloses a method for preparing manganese-doped silica nanomaterials loaded with manganese peroxidase. Manganese is doped into silica nanoparticles and used as a carrier. A manganese peroxidase solution is added to obtain manganese-doped silica nanomaterials loaded with manganese peroxidase.

[0008] Furthermore, the preparation method of the manganese-doped silica nanomaterial loaded with manganese peroxidase includes the following steps: (1) Preparation of silica nanoparticles; (2) Add anhydrous manganese chloride and disodium maleate to deionized water and stir to dissolve. Add the silica nanoparticles prepared in step (1) and prepare manganese-doped silica nanoparticles by hydrothermal method. (3) Add the manganese-doped silica nanoparticles synthesized in step (2) and polyethylene glycol to deionized water, stir and incubate at low temperature for at least 8 hours, centrifuge, collect the precipitate and dry to obtain MnMSN-PEG nanoparticles, disperse the MnMSN-PEG nanoparticles in 1 mg / mL manganese peroxidase solution, sonicate to uniform dispersion, incubate the mixture at low temperature overnight, collect the product by centrifugation, wash with deionized water to obtain the final manganese-doped silica (MnMSN@MnP-PEG) nanomaterial loaded with manganese peroxidase.

[0009] Furthermore, the mass ratio of anhydrous manganese chloride to disodium maleate is 3:5.

[0010] Furthermore, the mass ratio of anhydrous manganese chloride to silica nanoparticles is 2:1.

[0011] Furthermore, in step (3), the mass ratio of manganese-doped silica nanoparticles to polyethylene glycol is 1:1.

[0012] Furthermore, in step (3), the mass ratio of MnMSN-PEG nanoparticles to manganese peroxidase is 1:1.

[0013] The present invention also discloses a manganese-doped silica nanomaterial loaded with manganese peroxidase.

[0014] This invention also discloses the application of manganese-doped silica nanomaterials loaded with manganese peroxidase in antitumor drug reagents.

[0015] Furthermore, the application involves inducing ferroptosis in tumor cells. After ferroptosis occurs, the cell membrane ruptures, leading to tumor cell death.

[0016] Compared with existing technologies, the advantages of this invention are: 1. The manganese-doped silica nanomaterials prepared in this invention are hollow mesoporous manganese-doped silica nanoparticles with the solid silica core removed. The pores in the uniform mesoporous shell serve as channels for enzyme molecules to enter the hollow interior, while the hollow cavity also provides a large internal space for subsequent loading of large amounts of manganese peroxidase. Loading MnP enzyme into the hollow interior of the nanoparticles places it in a relatively closed and stable microenvironment, avoiding direct contact with complex biomolecules in the bloodstream and effectively shielding it from protease degradation and pH fluctuations. This protects enzyme activity and achieves efficient loading.

[0017] 2. This invention loads manganese peroxidase onto manganese-doped silica nanomaterials to construct a multifunctional nanoplatform. The prepared manganese-doped nanomaterials loaded with manganese peroxidase can achieve synergistic effects and controllable release. The MnMSN@MnP-PEG nanoparticles contain divalent manganese (Mn... 2+ ), trivalent manganese ions (Mn 3+ MnMSN@MnP-PEG exhibits microacidic responsiveness and high-concentration glutathione responsiveness in the tumor microenvironment, enabling it to release Mn in a responsive manner. 2+ Mn 3 + And MnP, thereby achieving controlled release. In tumor cells, the released Mn 3+ It can catalyze the production of ROS from H2O2, releasing MnP and Mn. 2+ It can catalyze the generation of lipid free radicals, triggering lipid peroxidation. Therefore, the dual catalytic synergy of the two increases tumor cell death.

[0018] 3. Ferroptosis is an iron-dependent, programmed cell death caused by the excessive accumulation of lipid peroxides. Its core is the collapse of the cell's antioxidant defense system, and our invented MnMSN@MnP-PEG nanoparticles precisely target this core system. Firstly, GPX4 (glutathione peroxidase 4) is a key enzyme in the intracellular repair of lipid peroxidation damage, using glutathione (GSH) as a cofactor to protect the cell membrane from oxidative damage. Mn... 3+ After entering the cell, it can undergo redox reactions with high concentrations of GSH, consuming a large amount of GSH and reducing GPX4 levels. Secondly, Mn...3+ and Mn 3+ The / MnP dual-catalytic system acts like an amplifier, significantly promoting the generation of lipid peroxides. This, in turn, drives ferroptosis in tumor cells. Ferroptosis disrupts the integrity of the cell membrane, ultimately leading to membrane rupture and cell death. This efficient elimination of tumor cells results in tumor shrinkage. Furthermore, ferroptosis is a highly immunogenic form of cell death. Dying tumor cells release damage-associated molecular patterns (DAMPs), such as ATP (which attracts antigen-presenting cells), HMGB-1 (which promotes dendritic cell maturation), and CRT (which promotes macrophage phagocytosis). These DAMPs work together to efficiently activate dendritic cells, thereby initiating an anti-tumor immune response and inhibiting tumor cell metastasis and invasion. Attached Figure Description

[0019] Figure 1 The images shown are transmission electron microscope (TEM) images of silica (MSN) and manganese-doped silica (MnMSN) nanomaterials from Example 1. Figure 1 a is a transmission electron microscope image of MSN. Figure 1 b is a transmission electron microscope image of MnMSN; Figure 2 The image shows the UV absorption spectrum of manganese peroxidase (MnP) in Example 1. Figure 3 The UV absorption spectra of the MnMSN, MnMSN-PEG, MnP, and MnMSN@MnP-PEG solutions in Example 1 are shown below. Figure 4 This is a comparison diagram of the application of different concentrations of MnP, MnMSN-PEG and MnMSN@MnP-PEG in the in vitro culture of mouse breast cancer cells 4T1, as shown in Example 1. Figure 5 The manganese-doped silica loaded with manganese peroxidase prepared in Example 1 was used to treat subcutaneous breast tumors in mice. The tumor growth curves in mice after different treatment groups were shown. Figure 6 A comparison of tumor growth curves and survival rates in mice during the treatment of lung metastasis with manganese-doped silica loaded with manganese peroxidase prepared in Example 1. Detailed Implementation

[0020] The present invention will be further described below with reference to the accompanying drawings and preferred embodiments. The embodiments given are only for illustrating the present invention and are not intended to limit the scope of the present invention.

[0021] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0022] Unless otherwise specified, the experimental methods described in the following examples are conventional methods.

[0023] Example 1 This embodiment provides a method for preparing manganese-doped silica-supported manganese peroxidase nanomaterials, including the following steps: (1) Preparation of silica nanoparticles (MSN): 500 mg of cetyltrimethylammonium bromide and 0.4 mL of 0.1 mol / L sodium hydroxide were dissolved in 25 mL of deionized water and gently stirred at 60 °C for 2 h; 10 mL of a mixed solution containing tetraethoxysilane and cyclohexane in a volume ratio of 1:4 was added to the reaction system, and the reaction was continuously stirred at 60 °C for 72 h; finally, the obtained product was collected by centrifugation, thoroughly washed with anhydrous ethanol and deionized water, the precipitate was redispersed in 50 mL of acetone, refluxed at 60 °C for 12 h, and finally washed twice with ethanol and vacuum dried at 45 °C for 8 h to obtain silica nanoparticles (MSN), with the morphology as shown in the figure. Figure 1 As shown in a; (2) Preparation of manganese-doped silica nanoparticles (MnMSN): 60 mg of anhydrous manganese chloride and 100 mg of disodium maleate were dissolved in 10 mL of deionized water by stirring. Subsequently, 30 mg of MSN was added to the above solution, and the mixture was stirred continuously and ultrasonically to ensure uniform dispersion. The resulting mixture was then transferred to a reaction vessel and heated at 120 °C for 4 h. Finally, the product was collected by centrifugation and thoroughly washed with anhydrous ethanol and deionized water to remove residual reactants. After drying, the final manganese-doped silica nanoparticles (MnMSN) were obtained, with the morphology shown in the figure. Figure 1 As shown in b; (3) Preparation of manganese-doped silica nanomaterials loaded with manganese peroxidase (MnMSN@MnP-PEG): 1 mg of the synthesized MnMSN nanoparticles was weighed and mixed with 1 mg of polyethylene glycol (DSPE-mPEG) in 1 mL of deionized water. After incubation at 4℃ for 8 h with stirring, the mixture was centrifuged, the precipitate (MnMSN-PEG) was collected, and dried. Then, 1 mg of the obtained MnMSN-PEG nanoparticles was weighed and dispersed in 1 mL of 1 The manganese peroxidase (MnP) solution was prepared by sonication to ensure uniform dispersion, and the mixture was incubated overnight at 4°C. The resulting product was collected by centrifugation and thoroughly washed with deionized water to obtain the final manganese-doped silica (MnMSN@MnP-PEG) nanocomposite material loaded with manganese peroxidase. The encapsulation efficiency (EE) of MnP in the MnMSN@MnP-PEG nanocomposite material was approximately 74.6%, calculated as the ratio of the weight of encapsulated MnP to the total weight of MnP multiplied by 100%. The UV absorption of MnP is 280 nm, and its standard curve is shown below. Figure 2 As shown.

[0024] The absorption spectra of MnMSN, MnMSN-PEG, MnP, and MnMSN@MnP-PEG solutions were measured using a UV spectrophotometer, respectively. Figure 3 As shown, both MnP and MnMSN@MnP-PEG have an absorption peak at 280 nm, indicating that MnP has been encapsulated into MnMSN@MnP-PEG.

[0025] Example 2 In this embodiment, the manganese-doped silicon dioxide nanomaterial loaded with manganese peroxidase prepared in Example 1 is applied to mouse breast cancer cells 4T1 in vitro.

[0026] 4T1 cells were seeded into 96-well plates and cultured for 24 h. Cytotoxicity was analyzed by adding different concentrations (0, 10, 25, 50, 100, and 200 μg / mL) of MnP or different concentrations (0, 1, 5, 10, 25, 50, 100, and 200 μg / mL) of MnMSN-PEG. Additionally, 4T1 cells were incubated with different concentrations (10, 20, and 50 μg / mL) of MnMSN@MnP-PEG (with MnP concentration of 25 μg / mL in all cases). After 24 h, CCK-8 assay was performed to detect 4T1 cell viability. Absorbance at 450 nm was measured using a microplate reader. Figure 4 As shown, 50 μg / mL MnMSN@MnP-PEG (with a MnP concentration of 25 μg / mL) demonstrates excellent cell-killing activity. Therefore, 25 μg / mL MnP, 50 μg / mL MnMSN-PEG, and 50 μg / mL MnMSN@MnP-PEG (with a MnP concentration of 25 μg / mL) were selected for subsequent experiments.

[0027] Example 3 In this embodiment, the manganese-doped silicon dioxide nanomaterial loaded with manganese peroxidase prepared in Example 1 is applied to the treatment of subcutaneous breast tumors in mice.

[0028] Balb / c female mice (4 weeks old) were purchased from Shanghai Slack Laboratory Animal Co., Ltd., and the experiment was conducted according to the protocol approved by the Animal Experiment Ethics Committee of Fujian Normal University. 100 μL of 4T1 cells (1 × 10⁻⁶) were subcutaneously injected into the right hind leg of the female Balb / c mice. 6 (100 cells) to establish an animal model of breast cancer. When the tumor reached approximately 100 mm... 3Mice were randomly divided into four groups (n = 5): PBS, 5 mg / kg MnP, 10 mg / kg MnMSN-PEG, and 10 mg / kg MnMSN@MnP-PEG (MnP was 5 mg / kg). Each mouse received a 100 μL injection via the tail vein. Figure 5 As shown, the growth rate of mouse tumors was observed over 22 days. It was found that the tumors in the PBS group grew rapidly, while the MnP and MnMSN-PEG groups slowed down the growth of mouse tumors. The MnMSN@MnP-PEG group significantly inhibited the development of mouse tumors and showed good therapeutic effects.

[0029] Example 4 This embodiment demonstrates the application of MnMSN@MnP-PEG nanomaterials in anti-lung metastasis, specifically the application of manganese-doped silica loaded with manganese peroxidase prepared in Example 1 to the treatment of subcutaneous mammary tumors in mice. Balb / c female mice (4 weeks old) were purchased from Shanghai Slack Laboratory Animal Co., Ltd., and the experiment was conducted according to the protocol approved by the Animal Experiment Ethics Committee of Fujian Normal University. 100 μL of 4T1 cells (1 × 10⁻⁶) were subcutaneously injected into the right hind leg of the Balb / c female mice. 6 (Number of cells) to establish an animal model of breast cancer. When the tumor reached approximately 100 mm... 3 Mice were randomly divided into four groups (n = 4): PBS, 5 mg / kg MnP, 10 mg / kg MnMSN-PEG, and 10 mg / kg MnMSN@MnP-PEG (MnP was 5 mg / kg). Each mouse received a 100 μL injection via tail vein. Nine days later, mice were injected via tail vein with 2 × 10⁻⁶ MnMSN@MnP-PEG. 5 Four T1-Luc cells were administered intraperitoneally at days 16, 22, 30, and 34. D-fluorescein potassium (3 mg per mouse) was injected intraperitoneally. Fluorescence signals were measured using an imaging spectroscopy system 15 minutes later. Tumor growth curves and survival rates were monitored throughout the treatment. Figure 6 As shown, after 34 days, no mice survived in the control group, with a survival rate of 0%, while the survival rate of the MnMSN@MnP-PEG group was the highest, reaching 75%, demonstrating that the MnMSN@MnP-PEG nanomaterial has a good anti-lung metastasis effect.

[0030] The above description is merely an embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural or procedural transformations made based on the content of the present invention specification, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. A method for preparing manganese-doped silica nanomaterials loaded with manganese peroxidase, characterized in that, Manganese-doped silica nanoparticles were doped with manganese and used as a carrier to add manganese peroxidase solution, thus obtaining manganese-doped silica nanomaterials loaded with manganese peroxidase.

2. The method for preparing manganese-doped silica nanomaterials loaded with manganese peroxidase as described in claim 1, characterized in that, The method includes the following steps: (1) Preparation of silica nanoparticles; (2) Add anhydrous manganese chloride and disodium maleate to deionized water and stir to dissolve. Add the silica nanoparticles prepared in step (1) and prepare manganese-doped silica nanoparticles by hydrothermal method. (3) Add the manganese-doped silica nanoparticles synthesized in step (2) and polyethylene glycol to deionized water, stir and incubate at low temperature for at least 8 hours, centrifuge, collect the precipitate and dry it to obtain MnMSN-PEG nanoparticles. Disperse the obtained MnMSN-PEG nanoparticles in 1 mg / mL manganese peroxidase solution, sonicate to disperse evenly, incubate the mixture at low temperature overnight, collect the product by centrifugation, wash with deionized water to obtain manganese-doped silica nanomaterials loaded with manganese peroxidase.

3. The method for preparing manganese-doped silica nanomaterials loaded with manganese peroxidase as described in claim 2, characterized in that, In step (2), the mass ratio of anhydrous manganese chloride to disodium maleate is 3:

5.

4. The method for preparing manganese-doped silica nanomaterials loaded with manganese peroxidase as described in claim 2, characterized in that, In step (2), the mass ratio of anhydrous manganese chloride to silica nanoparticles is 2:

1.

5. The method for preparing manganese-doped silica nanomaterials loaded with manganese peroxidase as described in claim 2, characterized in that, In step (3), the mass ratio of manganese-doped silica nanoparticles to polyethylene glycol is 1:

1.

6. The method for preparing manganese-doped silica nanomaterials loaded with manganese peroxidase as described in claim 2, characterized in that, In step (3), the mass ratio of MnMSN-PEG nanoparticles to manganese peroxidase is 1:

1.

7. Manganese-doped silica nanomaterials loaded with manganese peroxidase prepared by any one of the preparation methods described in claims 1-6.

8. The application of the manganese-doped silica nanomaterial loaded with manganese peroxidase as described in claim 7 in the preparation of antitumor drug reagents.

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