CeVO4-TPP nano particle and preparation method and application thereof

Abstract

The invention belongs to the technical field of nano materials, and particularly relates to CeVO4-TPP nano particles as well as a preparation method and application thereof. The CeVO4-TPP nano particle comprises an aminated CeVO4 nano particle and (3-carboxypropyl) triphenylphosphonium bromide, and the aminated CeVO4 nano particle and the (3-carboxypropyl) triphenylphosphonium bromide are bonded through an amido bond. CeVO4 nano particles have superoxide dismutase-like activity, superoxide anion free radicals can be converted into hydrogen peroxide, and the level of H2O2 in cells is greatly improved. CeVO4 nanoparticles have a mixed valence state of Ce ions (Ce < 3 + > / Ce < 4 + >) and vanadium ions (V < 4 + > / V < 5 + >), and can be used as a Fenton-like reaction catalyst to convert hydrogen peroxide in cells into toxic hydroxyl free radicals to cause cell death. According to the invention, the TPP for targeting mitochondria is modified on the CeVO4 nano particles, so that the targeting property on the mitochondria is enhanced. By increasing the generation strategy of H2O2 and. OH, tumor cells are effectively killed.

Description

Technical Field

[0001] The invention belongs to the technical field of nanomaterials, and specifically relates to CeVO4-TPP nanoparticles and a preparation method and application thereof. Background Art

[0002] Tumors are new organisms formed by the proliferation of local tissue cells. Traditional methods for treating tumors include radiotherapy and chemotherapy, but traditional radiotherapy and chemotherapy are often accompanied by side effects such as hair loss, loss of appetite, radiation inflammation, and immunosuppression. In order to solve these problems, chemodynamic therapy has been developed. Chemodynamic therapy uses the in situ catalytic conversion of endogenous hydrogen peroxide (H2O2) in tumor cells into highly toxic hydroxyl radicals (·OH) to induce cell apoptosis and necrosis. Although the H2O2 level in the tumor microenvironment is higher than that in normal cells, the current chemodynamic therapy has the problem of low sustainability after administration. Summary of the invention

[0003] In view of this, the purpose of the present invention is to provide a CeVO4-TPP nanoparticle and its preparation method and application. The CeVO4-TPP nanoparticle of the present invention can convert ·O2 - It is converted into H2O2, increasing the H2O2 level in the tumor microenvironment; it then catalyzes H2O2 to further generate ·OH, ultimately causing tumor cell death and improving the durability of chemodynamic therapy.

[0004] The invention provides a CeVO4-TPP nanoparticle, comprising an amination CeVO4 nanoparticle and (3-carboxypropyl) triphenylphosphonium bromide combined with the amination CeVO4 nanoparticle through an amide bond.

[0005] Preferably, the mass ratio of the amino CeVO4 nanoparticles to (3-carboxypropyl)triphenylphosphonium bromide is (2-3):(20-30).

[0006] The present invention also provides a method for preparing CeVO4-TPP nanoparticles described in the above technical solution, comprising the following steps:

[0007] aminating CeVO4 nanoparticles to obtain amination CeVO4 nanoparticles;

[0008] The aminated CeVO4 nanoparticles are mixed with (3-carboxypropyl)triphenylphosphonium bromide to carry out an amidation reaction to obtain CeVO4-TPP nanoparticles.

[0009] Preferably, the method for preparing CeVO4 nanoparticles comprises the following steps:

[0010] The aqueous solution containing cerium salt is added dropwise to the aqueous solution of vanadate, and the obtained mixed solution is adjusted to be alkaline, and a precipitation reaction is performed to obtain CeVO4 nanoparticles.

[0011] Preferably, the alkaline pH value is 9-12.

[0012] Preferably, the amination comprises the following steps:

[0013] CeVO4 nanoparticles, alkaline solution and silicate compounds are mixed to perform sol-gel reaction to obtain CeVO4-SiO2;

[0014] The CeVO4-SiO2, silane coupling agent and organic solvent are mixed to carry out silanization reaction to obtain amino CeVO4 nanoparticles.

[0015] Preferably, the alkaline solution comprises aqueous ammonia; the usage ratio of the CeVO4 nanoparticles, the alkaline solution and the silicate compound is (0.05-1.5) g: (0.12-0.2) mL: (0.15-0.25) mL.

[0016] Preferably, the temperature of the sol-gel reaction is 40-50° C. and the time is 4-5 hours.

[0017] Preferably, the usage ratio of CeVO4-SiO2 and silane coupling agent is (28-32) mg: (2.8-3.2) mL.

[0018] The present invention also provides the use of the CeVO4-TPP nanoparticles described in the above technical solution or the CeVO4-TPP nanoparticles obtained by the above preparation method in the preparation of drugs for treating tumors.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The invention provides a CeVO4-TPP nanoparticle, comprising an amination CeVO4 nanoparticle and (3-carboxypropyl) triphenylphosphonium bromide combined with the amination CeVO4 nanoparticle through an amide bond.

[0021] The CeVO4-TPP nanoparticles of the present invention include aminated CeVO4 nanoparticles and (3-carboxypropyl) triphenylphosphine bromide (TPP). The CeVO4 nanoparticles in the aminated CeVO4 nanoparticles of the present invention have superoxide dismutase (SOD) enzyme activity and can convert superoxide anion free radicals (·O2 - ) is converted into hydrogen peroxide (H2O2), greatly increasing the intracellular H2O2 level. CeVO4 nanoparticles contain Ce ions (Ce 3+ / Ce 4+) and vanadium ions (V 4+ / V 5+ )The mixed valence state of CeVO4 nanoparticles allows them to act as Fenton-like catalysts to convert intracellular hydrogen peroxide (H2O2) into toxic hydroxyl radicals (·OH), causing cell death and improving the durability of chemodynamic therapy. Mitochondria are the main source of cellular reactive oxygen species (ROS), including ·O2 - The present invention modifies CeVO4 nanoparticles with (3-carboxypropyl) triphenylphosphonium bromide (TPP) targeting mitochondria, thereby enhancing the targeting of mitochondria. The present invention effectively and continuously kills tumor cells by increasing the generation strategy of H2O2 and ·OH. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0023] Figure 1 TEM image of cerium vanadate prepared in Example 1;

[0024] Figure 2 This is the XPS analysis spectrum of the cerium vanadate prepared in Example 1;

[0025] Figure 3 Zeta potential of CeVO4, CeVO4-NH2 and CeVO4-TPP prepared in Example 1;

[0026] Figure 4 Detection of CeVO4 nanozyme on O2 by NBT colorimetric experiment - The clearing ability;

[0027] Figure 5 To test the H2O2 generation ability of CeVO4 nanozyme for Amplex Red;

[0028] Figure 6 The ability of CeVO4 to produce ·OH by Fenton-like reaction was tested for MB;

[0029] Figure 7 Cell survival rates of MCF-7 cells after incubation with different concentrations of CeVO4 and CeVO4-TPP;

[0030] Figure 8 Co-localization of Mito-Tracker Green and CeVO4-TPP nanoparticles, scale bars: 10 μm;

[0031] Fig. 9 DHE probe for detecting intracellular O2 - Clearance, scale bars are: 10 μm;

[0032] Fig.10 The Amplex Red probe is used to detect the generation of intracellular H2O2. The scale bars are 50 μm.

[0033] Fig.11 The DCFH-DA probe detects the generation of intracellular ·OH. The scale bar is 20 μm. DETAILED DESCRIPTION

[0034] The invention provides a CeVO4-TPP nanoparticle, comprising an amination CeVO4 nanoparticle and (3-carboxypropyl) triphenylphosphonium bromide combined with the amination CeVO4 nanoparticle through an amide bond.

[0035] In the present invention, the CeVO4-TPP nanoparticles are amination cerium vanadate nanoparticles whose surfaces are modified with (3-carboxypropyl)triphenylphosphine bromide.

[0036] In the present invention, the mass ratio of the aminated CeVO4 nanoparticles to (3-carboxypropyl)triphenylphosphine bromide is preferably (2-3):(20-30). The ratio of the aminated CeVO4 nanoparticles to (3-carboxypropyl)triphenylphosphine bromide is preferably 5 mg:120 μmol.

[0037] In the present invention, the preparation method of CeVO4 nanoparticles in the aminated CeVO4 nanoparticles preferably comprises the following steps:

[0038] The aqueous solution containing cerium salt is added dropwise to the aqueous solution of vanadate, and the obtained mixed solution is adjusted to be alkaline, and a precipitation reaction is performed to obtain CeVO4 nanoparticles.

[0039] In the present invention, the cerium salt in the aqueous solution containing cerium salt is preferably cerium nitrate, specifically Ce(NO3)3·6H2O; the concentration of the cerium nitrate is preferably 0.067mmol / L. The aqueous solution containing cerium salt preferably also contains ethylenediaminetetraacetic acid, and the concentration of the ethylenediaminetetraacetic acid is preferably 0.067mmol / L. The ethylenediaminetetraacetic acid can form a stable chelate with cerium ions, which helps to prevent cerium ions from reacting with ions in the solution to form insoluble precipitates, thereby maintaining the stability and uniformity of the solution.

[0040] In the present invention, the vanadate in the vanadate aqueous solution is preferably sodium orthovanadate (Na3VO4), and the concentration of the sodium orthovanadate is preferably 0.067 mmol / L.

[0041] In the present invention, the molar ratio of the cerium salt in the aqueous solution containing the cerium salt to the vanadate in the aqueous solution containing the vanadate is preferably 1:1.

[0042] In the present invention, the alkaline pH value is preferably 9 to 12. The present invention adjusts the pH value of the mixed solution to alkaline. Under a higher pH value (alkaline conditions), Ce 4+ Will be restored to Ce 3+ , leading to Ce 4+ The CeVO4 nanoparticles prepared by the present invention have a lower content. 4+ Low content and good SOD-like enzyme activity.

[0043] In the present invention, the temperature of the precipitation reaction is preferably 170-190° C., specifically 180° C., and the time is preferably 13-15 h, specifically 14 h.

[0044] In the present invention, the precipitation reaction preferably further includes solid-liquid separation, and the obtained solid is washed and dried; the solid-liquid separation method is preferably centrifugation; the washing preferably includes water washing and alcohol washing, the water washing preferably uses deionized water, and the alcohol washing preferably uses ethanol; the drying temperature is preferably 80°C, and the time is preferably 10 hours.

[0045] The present invention utilizes the SOD-like enzyme activity of CeVO4 nanoparticles and the mitochondrial targeting effect of TPP to convert the oxygen in the mitochondria into - Converted into H2O2, increasing the H2O2 level in the tumor microenvironment; then using Ce 3+ / Ce 4+ With V 4+ / V 5+ It has Fenton-like reaction catalytic activity, catalyzing H2O2 to further generate ·OH, ultimately causing cell death.

[0046] The present invention also provides a method for preparing CeVO4-TPP nanoparticles described in the above technical solution, comprising the following steps:

[0047] aminating CeVO4 nanoparticles to obtain amination CeVO4 nanoparticles;

[0048] The aminated CeVO4 nanoparticles are mixed with (3-carboxypropyl)triphenylphosphonium bromide to carry out an amidation reaction to obtain CeVO4-TPP nanoparticles.

[0049] In the present invention, unless otherwise specified, the materials and equipment used are commercially available products in the art.

[0050] The present invention performs amination on CeVO4 nanoparticles to obtain CeVO4-NH2 nanoparticles, wherein the CeVO4-NH2 nanoparticles are amination CeVO4 nanoparticles.

[0051] In the present invention, the amination preferably comprises the following steps:

[0052] CeVO4 nanoparticles, alkaline solution and silicate compounds are mixed to perform sol-gel reaction to obtain CeVO4-SiO2;

[0053] The CeVO4-SiO2, silane coupling agent and organic solvent are mixed to carry out silanization reaction to obtain amino CeVO4 nanoparticles.

[0054] In the present invention, the CeVO4 nanoparticles are preferably used in the form of a solution, the solvent of the solution preferably includes ethanol and water, the volume ratio of the ethanol to water is preferably 4:1, and the concentration of the CeVO4 nanoparticles in the solution is preferably 0.01 g / mL.

[0055] In the present invention, the alkaline solution preferably includes aqueous ammonia, and the concentration of the aqueous ammonia is preferably 10 mol / L.

[0056] In the present invention, the silicate compound preferably includes tetraethyl silicate (TEOS) or methyl orthosilicate.

[0057] In the present invention, the usage ratio of the CeVO4 nanoparticles, the alkaline solution and the silicate compound is preferably (0.05-1.5) g: (0.12-0.2) mL: (0.15-0.25) mL, and more preferably 0.1 g: 0.16 mL: 0.2 mL.

[0058] In the present invention, the sol-gel reaction is a sol-gel reaction of silicate, preferably carried out in a water bath, the temperature of the sol-gel reaction is preferably 40-50°C, and the time is preferably 4-5 hours; the sol-gel reaction preferably also includes washing and drying; the washing is preferably washing with pure water and anhydrous ethanol alternately, and the drying is preferably drying at room temperature. During the sol-gel reaction of silicate, Ce and V ions on the surface of CeVO4 can serve as coordination sites and bind to the hydroxyl groups on the surface of SiO2 generated by hydrolysis through coordination bonds.

[0059] In the present invention, the silane coupling agent preferably includes 3-aminopropyltrimethoxysilane (APTES) or 3-aminopropyltriethoxysilane.

[0060] In the present invention, the organic solvent preferably includes toluene.

[0061] In the present invention, the CeVO4-SiO2 and the organic solvent are preferably mixed first and then the silane coupling agent is added dropwise. The dosage ratio of the CeVO4-SiO2 and the organic solvent is preferably 30 mg:60 mL. The dosage ratio of the CeVO4-SiO2 and the silane coupling agent is preferably (28-32) mg:(2.8-3.2) mL, and more preferably 30 mg:3 mL.

[0062] In the present invention, the temperature of the silanization reaction is preferably room temperature, and the time is preferably 24 hours. During the silanization reaction, the ethoxy groups in the silane coupling agent react with trace water molecules in the solvent to generate silanol groups; the silanol groups generated by hydrolysis react with the silanol groups on the surface of the CeVO4-SiO2 nanoparticles to form Si-O-Si bonds, thereby covalently grafting the silane coupling agent to the surface of the CeVO4-SiO2 nanoparticles; the silane coupling agent APTES has amino (-NH2) functional groups to form CeVO4-NH2.

[0063] In the present invention, the silanization reaction preferably further includes washing and drying; the washing agent is preferably anhydrous ethanol, and the number of washings is preferably 2 to 3 times; and the drying is preferably oven drying.

[0064] After obtaining CeVO4-NH2 nanoparticles, the CeVO4-NH2 nanoparticles are mixed with (3-carboxypropyl)triphenylphosphonium bromide to carry out an amidation reaction to obtain CeVO4-TPP nanoparticles.

[0065] The present invention preferably disperses the CeVO4-NH2 into a PBS buffer solution, activates (3-carboxypropyl)triphenylphosphonium bromide, and then performs the mixing;

[0066] The dosage ratio of CeVO4-NH2 to PBS buffer is preferably 5 mg:6 mL, and the pH value of the PBS buffer is preferably 6;

[0067] The (3-carboxypropyl) triphenylphosphonium bromide activation preferably comprises: mixing 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride, N-hydroxysulfosuccinimide sodium salt, (3-carboxypropyl) triphenylphosphonium bromide and dimethyl sulfoxide to carry out a carboxyl activation reaction. The molar ratio of the 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), N-hydroxysulfosuccinimide sodium salt (NHS) and (3-carboxypropyl) triphenylphosphonium bromide (TPP-COOH) is preferably 3:1:1. The carboxyl activation reaction is preferably carried out at room temperature in the dark, and the reaction time is preferably 30 to 40 minutes.

[0068] In the present invention, the usage ratio of the CeVO4-NH2 nanoparticles to (3-carboxypropyl)triphenylphosphonium bromide is preferably 5 mg:120 μmol.

[0069] In the present invention, the amidation reaction is preferably carried out in a dark place with stirring, and the time is preferably 24 hours. After the amidation reaction, it is preferred to further include solid-liquid separation, and the obtained solid is washed and dried; the solid-liquid separation method is preferably centrifugation; the washing preferably includes water washing and alcohol washing, the water washing is preferably carried out with deionized water, and the alcohol washing is preferably carried out with ethanol; the water washing and alcohol washing are preferably carried out alternately.

[0070] The present invention also provides the use of the CeVO4-TPP nanoparticles described in the above technical solution or the CeVO4-TPP nanoparticles obtained by the above preparation method in the preparation of drugs for treating tumors.

[0071] The CeVO4-TPP nanoparticles of the present invention have good targeting properties and can effectively reach the tumor site by utilizing the enhanced permeability and retention (EPR) effect of tumor tissue, thereby reducing damage to normal cells.

[0072] In order to further illustrate the present invention, the CeVO4-TPP nanoparticles provided by the present invention and the preparation method and application thereof are described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0073] In the embodiments of the present invention, the materials and reagents used include:

[0074] (3-Carboxypropyl)triphenylphosphonium bromide (TPP-COOH), ammonium metavanadate (NH4VO3), cerium nitrate hexahydrate (Ce(NO3)3·6H2O), disodium hydrogen phosphate (Na2HPO4), sodium dihydrogen phosphate (NaH2PO4), L-methionine, riboflavin, nitro blue tetrazolium chloride (NBT), xanthine, xanthine oxidase, hydrogen peroxide (H2O2), dimethyl sulfoxide (DMSO), tetraethyl silicate (TEOS), 3-aminopropyltrimethoxysilane (APTMS), 5,5-dithio-bis-(2-nitrobenzoic acid) (DTNB), methylene blue (MB), ethylenediaminetetraacetic acid (EDTA), sodium orthovanadate (Na3VO4) were purchased from Macklin. N-hydroxysulfosuccinimide sodium salt (NHS) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) were purchased from Sangon Biotech (Shanghai) Co., Ltd. Anhydrous ethanol was purchased from Sinopharm. DAPI, Amplex Red, horseradish peroxidase (HRP), dihydroethidium (DHE), mitochondrial probe (Mito-Tracker Green), and 2.7-dichlorofluorescein dihydrogen diacetate (DCFH-DA) were purchased from Bio-Tech. MTT cell proliferation and cytotoxicity assay kits were purchased from Sangon Biotech. Fetal bovine serum was provided by Solebol. MCF-7 breast cancer cells were from Wuhan Pronocell Life Science Co., Ltd.

[0075] Instruments include:

[0076] The morphology of the materials was characterized by transmission electron microscopy (JEM2100). X-ray photoelectron spectroscopy analysis was performed on an X-ray photoelectron spectrometer (ESCALABXi+). UV-visible spectra were recorded on a UV-visible spectrophotometer (Agilent Cary60). MTT assays were performed on a microplate reader (K3 touch). Fluorescence imaging of cells was performed by fluorescence microscopy (Nikon Ti2-E).

[0077] Cells were cultured as follows:

[0078] The MCF-7 breast cancer cells involved in the experiment were from Wuhan Pronocell Life Science Co., Ltd. and cultured in a constant temperature and humidity chamber at 37°C, with a humidity of 95% and a CO2 concentration of 5%. The culture medium used for the cells was DMEM containing 10% fetal bovine serum and 1% double antibody (penicillin-streptomycin).

[0079] Example 1 Preparation of Nanoparticles

[0080] The first step is to prepare CeVO4 nanoparticles. Ce(NO3)3·6H2O (1.0mmol) and EDTA (1.0mmol) were dissolved in 15mL of H2O, the solution was vigorously stirred for 15min, and then Na3VO4 solution (1.0mmol Na3VO4 dissolved in 15mL H2O) was added dropwise. The pH value of the reaction mixture was adjusted to 12.0 with ammonia water and stirred for 15min. The obtained reaction mixture was sealed in a 50mL stainless steel autoclave lined with Teflon and heated at 180℃ for 14h. The obtained precipitate was centrifuged, washed with deionized water and ethanol, and dried at 80℃ for 10h to obtain CeVO4 nanoparticles.

[0081] The second step is to prepare CeVO4-NH2. Dissolve 0.1g CeVO4 nanoparticles in 8mL anhydrous ethanol and 2mL distilled water, add ammonia water (10mol / L, 0.16mL) dropwise, add 0.2mL TEOS, react in a 40℃ water bath for 4h, wash alternately with pure water and anhydrous ethanol, and dry at room temperature to obtain the product CeVO4@SiO2; weigh 30mg CeVO4@SiO2 nanoparticles and ultrasonically disperse them in 60mL toluene, add 3.0mL APTES dropwise, react at room temperature for 24h, wash the product 3 times with anhydrous ethanol, and dry to obtain CeVO4-NH2 nanoparticles.

[0082] The third step is to prepare CeVO4-TPP. CeVO4-NH2 (5 mg) was dispersed in 6 mL PBS buffer (10 mM, pH 6.0). EDC (180 μM, 2 mL), NHS (60 μM, 2 mL) and TPP-COOH (60 μM, 2 mL) dissolved in DMSO were mixed and reacted for 30 minutes at room temperature in the dark. Afterwards, the mixed solution of the above three was added to the CeVO4-NH2 solution and stirred in the dark for 24 hours. Finally, centrifuged, washed alternately with anhydrous ethanol and deionized water to remove the residual solvent, and collected CeVO4-TPP.

[0083] Test Example 1

[0084] 1. Test methods

[0085] 1) UV detection·O2 - The cleaning ability

[0086] UV measurement of the SOD-like activity of CeVO4 was performed to investigate the scavenging of ·O2 by superoxide dismutase based on CeVO4 - CeVO4 nanoparticles were synthesized according to the steps of Example 1, and CeVO4 nanoparticles with concentrations of 25, 50, 75, 100, and 125 μg / mL were prepared for subsequent detection. ·O2 -The absorption spectra were recorded from 350 nm to 800 nm using an Agilent Cary 60 UV-visible spectrophotometer.

[0087] 2) UV detection of H2O2 generation capacity

[0088] The SOD-like activity of CeVO4 was measured by UV to study the superoxide dismutase-catalyzed ·O2 - The ability to further convert into H2O2. CeVO4 was synthesized and prepared according to the steps of Example 1, and CeVO4 nanoparticles with concentrations of 50, 100, 150, 200, and 250 μg / mL were prepared for subsequent detection. The determination of H2O2 was performed using an Agilent Cary60 UV-visible spectrophotometer to record the absorption spectrum from 350nm to 800nm.

[0089] 3) UV detection of the ability to generate OH in a Fenton-like reaction

[0090] The Fenton-like catalytic activity of CeVO4 was measured by UV to study the ability of CeVO4 to catalyze H2O2 to generate ·OH in a Fenton-like reaction. CeVO4 was synthesized according to the steps of Example 1, and PBS buffer solution was used as the solvent to prepare 100 μg mL -1 CeVO4 nanoparticles were added to MB solution (3 mL, 10 μg mL -1 ), the supernatant was collected after centrifugation at different incubation time intervals (0, 2, 4, 6, 8, 10, 20 and 30 min) for subsequent detection. The absorption spectrum from 350 nm to 800 nm was recorded using an Agilent Cary 60 UV-visible spectrophotometer.

[0091] 4) MTT assay to determine the cytotoxicity of nanoparticles

[0092] MCF-7 breast cancer cells (100 μL, 2×10 6 cells mL -1 ) were inoculated in a 96-well plate and cultured for 24 h. -1 ) for 24 h, with PBS buffer as solvent. Then, 10 μL MTT solution (5 mg mL -1 ) for 4 hours. After rinsing with PBS to remove the nanoparticles, 100 μL DMSO was used to dissolve the formaldehyde crystal deposits in each well, and the cells were detected at 490 nm by an ELISA reader, and the cell survival rate was calculated based on the detection data.

[0093] 5) Fluorescence microscopy to detect mitochondrial targeting of nanoparticles

[0094] MCF-7 breast cancer cells were cultured in confocal microplates for 24 hours for mitochondrial targeting analysis. CeVO4-TPP nanoparticles were added for 8 hours and Mito-TrackerGreen mitochondrial probe was added for 30 minutes. After washing the cells three times with PBS to remove excess Mito-Tracker Green mitochondrial probe, the fluorescence intensity was observed using a Nikon Ti2-E fluorescence microscope.

[0095] 6) Fluorescence microscopy to detect the consumption of O2 by nanoparticles in cells - Capabilities

[0096] MCF-7 breast cancer cells were cultured in confocal microplates for 24 h to consume intracellular O2 - Different nanoparticles were added and cultured for 24 h. Cells were incubated with 30 μM DHE for 40 min. After washing the cells three times with PBS to remove excess DHE, the fluorescence intensity was observed using a Nikon Ti2-E fluorescence microscope.

[0097] 7) Fluorescence microscopy to detect the ability of nanoparticles to generate H2O2 in cells

[0098] MCF-7 breast cancer cells were cultured in a confocal dish for 24 hours for the detection and analysis of intracellular H2O2 production. CeVO4-TPP nanoparticles were added and cultured for 0, 0.5, and 2 hours. The cells were incubated with 200μM Amplex Red dye for 50 minutes. After washing the cells three times with PBS to remove excess Amplex Red, the fluorescence intensity was observed using a Nikon Ti2-E fluorescence microscope.

[0099] 8) Fluorescence microscopy to detect the ability of nanoparticles to generate ·OH in cells

[0100] MCF-7 breast cancer cells were cultured in confocal microplates for 24 hours for the detection and analysis of intracellular ·OH generation. Different nanoparticles were added and cultured for 24 hours. The cells were incubated with 10μM DCFH-DA probe for 20 minutes. After washing the cells three times with PBS to remove excess DCFH-DA, the fluorescence intensity was observed using a Nikon Ti2-E fluorescence microscope.

[0101] 2. Results and Discussion

[0102] 1) Design and synthesis of CeVO4-TPP

[0103] The nanomaterials were characterized by transmission electron microscopy (TEM), X-ray photoelectron spectroscopy (XPS) and Zeta potential. Figure 1 TEM image of cerium vanadate nanoparticles. Figure 2This is the XPS analysis spectrum of cerium vanadate nanoparticles. Figure 3 is the Zeta potential of CeVO4, CeVO4-NH2, and CeVO4-TPP. The results show that CeVO4-TPP nanoparticles were successfully synthesized. First, CeVO4-NH2 was synthesized on the basis of synthesizing CeVO4 nanoparticles, and CeVO4-TPP was obtained by modifying its surface with TPP-COOH. Figure 1 In the TEM data, the synthesized cerium vanadate nanoparticles are rod-shaped. In the XPS data, Figure 2 A is shown on the Ce 3d orbital, and the signal can be fitted to the Ce 3+ and Ce 4+ , indicating that it has mixed valence characteristics and the surface Ce 4+ The amount of V 2p orbital ( Figure 2 B) shows V 5+ and V 4+ existence. Figure 3 The Zeta potential results show that the Zeta potential of CeVO4 in aqueous solution is negative, at -6.47. After amination, the potential of the material changes significantly, showing -1.13. Since TPP-COOH has a negative potential, the Zeta potential value is -13.1 after connecting TPP-COOH, and the potential value decreases. The above data show that CeVO4-TPP is successfully prepared.

[0104] 2) Detection of O2 - The cleaning ability

[0105] The SOD activity of CeVO4 nanozymes was investigated by UV / visible spectroscopy. The SOD activity of CeVO4 nanozymes was evaluated by a typical NBT colorimetric assay, in which L-methionine and riboflavin produced ·O2 under light irradiation. - , NBT is reduced to formazan, and an absorption peak appears at 500nm~600nm. Figure 4 Detection of CeVO4 nanozyme on O2 by NBT colorimetric experiment - The removal ability of Figure 4 As shown, when there is ·O2 - When CeVO4 nanozyme is absent, it has a strong absorption peak in this range. As the concentration of CeVO4 nanozyme increases, its absorbance intensity gradually decreases, which indicates that CeVO4 nanozyme has the ability to catalyze the consumption of O2 - ability, indicating that CeVO4 nanozymes have effective SOD-like activity.

[0106] 3) Detection of H2O2 generation capacity

[0107] The SOD-like activity of CeVO4 nanozymes can also be verified by detecting the generation of H2O2. - , when CeVO4 nanozymes are present, catalyzes·O2 - Generate H2O2. Amplex Red, as a fluorescent probe, can react with H2O2 to generate red resorcinol, with a typical absorption peak at 571nm. Figure 5 Amplex Red was used to detect the ability of CeVO4 nanozymes to generate H2O2. Figure 5 As shown, when there is ·O2 - When CeVO4 nanozyme is absent, it has a lower absorption peak at 571nm. As the concentration of CeVO4 nanozyme increases, its absorbance intensity gradually increases, which indicates that CeVO4 nanozyme can catalyze the oxidation of ·O2 - The generation of H2O2 further verified that CeVO4 nanozyme had effective SOD-like activity.

[0108] 4) Detection of the ability to generate OH by Fenton-like reaction

[0109] UV / visible spectroscopy was used to study the catalytic ability of CeVO4 in the Fenton-like reaction. The ability of CeVO4 in catalyzing H2O2 to produce ·OH in the Fenton-like reaction was studied by MB decolorization experiment, in which MB has an absorption peak at 665nm. After MB reacts with ·OH, its absorbance intensity at 665nm decreases. In the presence of CeVO4 nanozymes, the MB concentration at different time points was observed. Figure 6 MB was used to detect the ability of CeVO4 to produce ·OH by Fenton-like reaction. Figure 6 As shown, with the continuous extension of incubation time, the absorption peak of MB at 665nm gradually decreased, indicating that the MB content gradually decreased, which shows that CeVO4 nanoparticles played a Fenton-like reaction catalytic activity to produce more ·OH.

[0110] 5) MTT assay to determine the cytotoxicity of nanoparticles

[0111] The cytotoxic effects of nanomaterials on cancer cells were studied, and the cytotoxicity of CeVO4 and CeVO4-TPP was detected and recorded using the MTT detection kit. Figure 7 Cell survival rates of MCF-7 cells after incubation with different concentrations of CeVO4 and CeVO4-TPP. Figure 7The data in the figure show that after adding different concentrations of CeVO4 (0, 12.5, 25, 50, 100, 200 μg / mL), the cell viability gradually decreases with the increase of CeVO4 concentration. This is because CeVO4 nanoparticles exert SOD enzyme activity and Fenton-like reaction catalytic activity in cells, thereby producing more ·OH in cells and killing tumor cells. When adding different concentrations of CeVO4-TPP (0, 12.5, 25, 50, 100, 200 μg / mL), the cell viability significantly decreases with the continuous increase of its concentration. This is because TPP has a mitochondrial targeting effect, which increases the probability of nanoparticles entering mitochondria, thereby killing tumor cells to a greater extent.

[0112] 6) Fluorescence microscopy to detect mitochondrial targeting of CeVO4-TPP nanoparticles

[0113] The targeting of CeVO4-TPP nanoparticles to mitochondria was studied, and Mito-Tracker Green probe was used as a control group. Aminated CeVO4 nanoparticles were prepared and synthesized according to the steps of Example 1, and carboxylated Cy5.5 red fluorescent probe was simultaneously connected to study the mitochondrial targeting of the nanoparticles. Figure 8 The co-localization of Mito-Tracker Green and CeVO4-TPP nanoparticles, the scale bar is 10 μm. Figure 8 As shown, it can be clearly observed that the green fluorescence of Mito-TrackerGreen highly overlaps with the bright red fluorescence in the cells, confirming that CeVO4-TPP nanoparticles are mainly anchored on mitochondria, indicating that CeVO4-TPP nanoparticles have good mitochondrial targeting.

[0114] 7) Fluorescence microscopy to detect intracellular O2 removal by nanoparticles - Capabilities

[0115] Study on the effect of nanomaterials on oxygen in cancer cells - To investigate the clearance of DHE, different nanomaterials were used to treat MCF-7 cells and the results were judged based on the fluorescence of DHE. Fig. 9 DHE probe for detecting intracellular O2 - The scale bars are 10 μm. Fig. 9 The fluorescence data show that when PBS is co-incubated with MCF-7 cells, a stronger DHE red fluorescence intensity is displayed, indicating that there is more O2 in the cells. - When CeVO4 and CeVO4-TPP nanoparticles were co-incubated with MCF-7 cells, the red fluorescence intensity of DHE was significantly reduced, indicating that the intracellular O2 -The CeVO4-TPP nanoparticles can significantly reduce the amount of O2 in cells. - Effective cleaning.

[0116] 8) Fluorescence microscopy to detect the ability of nanoparticles to generate H2O2 in cells

[0117] To study the generation of H2O2 in cells, CeVO4-TPP nanoparticles were used to treat MCF-7 cells, and the fluorescence of Amplex Red was observed at different time points for judgment. Fig.10 The Amplex Red probe is used to detect the generation of H2O2 in cells. The scale bar is 50μm. Fig.10 The fluorescence data show that when CeVO4-TPP nanoparticles were co-incubated with MCF-7 cells for 0 h, the red fluorescence of Amplex Red was weak, and when CeVO4-TPP nanoparticles were co-incubated with MCF-7 cells for 0.5 h, the red fluorescence of Amplex Red was the strongest. This is because CeVO4-TPP nanoparticles convert ·O2 - Subsequently, when CeVO4-TPP nanoparticles were co-incubated with MCF-7 cells for 2 h, the red fluorescence intensity of Amplex Red decreased, confirming the decrease in the H2O2 content, which should be attributed to the subsequent consumption of H2O2 by CeVO4-TPP nanoparticles to produce ·OH.

[0118] 9) Fluorescence microscopy to detect the ability of nanoparticles to generate ·OH in cells

[0119] To study the generation of intracellular ·OH, different nanomaterials were used to treat MCF-7 cells. ·OH is an important ROS, so it was judged based on the fluorescence of the ROS probe DCFH-DA. Fig.11 The DCFH-DA probe detects the generation of intracellular ·OH. The scale bar is 20 μm. Fig.11 The fluorescence data show that when PBS is co-incubated with MCF-7 cells, a weak DCFH-DA green fluorescence intensity is displayed. When CeVO4 and CeVO4-TPP nanoparticles are co-incubated with MCF-7 cells, the DCFH-DA green fluorescence intensity is significantly increased, indicating that the ·OH content in the cells is significantly increased, that is, the ·OH level in the cells is increased under the action of CeVO4-TPP nanoparticles.

[0120] The present invention develops a multifunctional CeVO4-TPP nanoparticle, which specifically targets mitochondria through TPP-COOH, so that the nanoparticles are precisely anchored to mitochondria. On the one hand, CeVO4 nanoparticles have SOD-like enzyme activity, which can convert ·O2 - On the other hand, CeVO4 nanoparticles have Fenton-like catalytic activity, which can catalyze the converted H2O2 to further generate ·OH, eventually causing cell death.

[0121] Although the above-mentioned embodiments have made a detailed description of the present invention, they are only some embodiments of the present invention, rather than all embodiments. People can also obtain other embodiments based on the embodiments of the present invention without creative work, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A CeVO4-TPP nanoparticle, characterized in that: The invention comprises aminated CeVO4 nanoparticles and (3-carboxypropyl)triphenylphosphonium bromide which is combined with the aminated CeVO4 nanoparticles via amide bonds.

2. The CeVO4-TPP nanoparticles according to claim 1, characterized in that: The mass ratio of the amino CeVO4 nanoparticles to (3-carboxypropyl)triphenylphosphonium bromide is (2-3):(20-30).

3. The method for preparing CeVO4-TPP nanoparticles according to claim 1 or 2, characterized in that: The following steps are involved: aminating CeVO4 nanoparticles to obtain amination CeVO4 nanoparticles; The aminated CeVO4 nanoparticles are mixed with (3-carboxypropyl)triphenylphosphonium bromide to carry out an amidation reaction to obtain CeVO4-TPP nanoparticles.

4. The preparation method according to claim 3, characterized in that: The preparation method of the CeVO4 nanoparticles comprises the following steps: The aqueous solution containing cerium salt is added dropwise to the aqueous solution of vanadate, and the obtained mixed solution is adjusted to be alkaline, and a precipitation reaction is performed to obtain CeVO4 nanoparticles.

5. The preparation method according to claim 4, characterized in that: The alkaline pH value is 9-12.

6. The preparation method according to claim 3, characterized in that: The amination comprises the following steps: CeVO4 nanoparticles, alkaline solution and silicate compounds are mixed to perform sol-gel reaction to obtain CeVO4-SiO2; The CeVO4-SiO2, silane coupling agent and organic solvent are mixed to carry out silanization reaction to obtain amino CeVO4 nanoparticles.

7. The preparation method according to claim 6, characterized in that: The alkaline solution includes ammonia water; the usage ratio of the CeVO4 nanoparticles, the alkaline solution and the silicate compound is (0.05-1.5) g: (0.12-0.2) mL: (0.15-0.25) mL.

8. The preparation method according to claim 6 or 7, characterized in that: The temperature of the sol-gel reaction is 40-50° C. and the time is 4-5 hours.

9. The preparation method according to claim 6, characterized in that: The usage ratio of CeVO4-SiO2 and silane coupling agent is (28-32) mg: (2.8-3.2) mL.

10. Use of the CeVO4-TPP nanoparticles according to claim 1 or 2 or the CeVO4-TPP nanoparticles obtained by the preparation method according to any one of claims 3 to 9 in the preparation of drugs for treating tumors.

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