A copper-manganese loaded calcium carbonate composite material, its preparation method and application

Abstract

The present invention belongs to the technical field of anti-cancer drugs, and specifically relates to a copper-manganese loaded calcium carbonate composite material, a preparation method thereof, and an application thereof. In the present invention, gallic acid, calcium carbonate nanoparticles, a water-soluble manganese salt, and a water-soluble copper salt are mixed to obtain copper-manganese loaded calcium carbonate nanoparticles; the copper-manganese loaded calcium carbonate nanoparticles are surface-modified with sodium dioleoyl phosphatide, cholesterol, 1,2-distearoyl-sn-glycero-3-phosphocholine, phospholipid polyethylene glycol, and (2,3-dioleoyloxypropyl) trimethyl ammonium chloride to obtain a copper-manganese loaded calcium carbonate composite material. In the present invention, the coated nanoparticles loaded with copper ions and manganese ions accumulate at the tumor site through the enhanced permeability and retention effect, realizing an efficient Fenton reaction. A large amount of ROS generated during the Fenton reaction will disrupt the intracellular Ca<supgt;2+< / supgt; balance, achieve calcium overload, and further cause mitochondrial dysfunction in tumor cells, realizing an efficient killing effect on tumor cells.

Description

Technical Field

[0001] The invention belongs to the technical field of anticancer drugs, and particularly relates to a copper-manganese-loaded calcium carbonate composite material, a preparation method and an application thereof. Background Art

[0002] Ca 2+ Ca is a ubiquitous second messenger in cells and is reported to be involved in regulating a variety of intracellular activities by regulating mitochondrial dehydrogenases, inducing the production of reactive oxygen species (ROS) and opening the mitochondrial permeability transition pore. 2+ It plays an important role in mediating mitochondrial events by disrupting intracellular Ca 2+ It is feasible that the balance of intracellular Ca levels can lead to cell death. However, through multiple organelle functions, such as transmembrane uptake and efflux, mitochondrial and endoplasmic reticulum buffering functions, intracellular Ca 2+ The concentration is always stable (10.7 mol / L). Therefore, the introduction of excess Ca 2+ , through intracellular Ca 2+ Overloading that disrupts the balance and leads to cell death is a viable treatment approach.

[0003] In recent years, calcium-based nanomaterials, such as calcium carbonate, calcium phosphate, hydroxyapatite, calcium peroxide, and tricalcium phosphate, have been widely developed in biomedical applications due to their biocompatibility and biodegradability. Among them, CaCO3, as one of the main inorganic substances in natural organisms, has been widely used as a nanocarrier for tumor microenvironment-responsive drug delivery.

[0004] Although the decomposition of CaCO3 can increase Ca 2+ levels, but Ca 2+ The toxicity induced by the mitochondrial membrane was still limited, which may be due to the presence of Ca in the mitochondrial membrane. 2+ Uptake and Ca 2+ Specific mechanisms of release. Summary of the Invention

[0005] The purpose of the present invention is to provide a copper-manganese-loaded calcium carbonate composite material, a preparation method and application thereof. The copper-manganese-loaded calcium carbonate composite material provided by the present invention can effectively disrupt the calcium balance in tumor cells, achieve calcium overload, and thereby cause mitochondrial dysfunction in tumor cells, thereby achieving a highly efficient killing effect on tumor cells.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] The present invention provides a method for preparing a copper-manganese-loaded calcium carbonate composite material, comprising the following steps:

[0008] Mix gallic acid, triethylamine, an organic dispersant, calcium carbonate nanoparticles, water, an alcohol solvent, a water-soluble manganese salt, and a water-soluble copper salt to obtain copper and manganese loaded calcium carbonate nanoparticles;

[0009] Mix the copper and manganese loaded calcium carbonate nanoparticles, sodium dioleoyl phosphatidate, and an organic solvent for the first surface modification to obtain pre-surface modified nanoparticles;

[0010] Mix the pre-surface modified nanoparticles, cholesterol, 1,2-distearoyl-sn-glycero-3-phosphocholine, phospholipid polyethylene glycol, (2,3-dioleyloxypropyl) trimethyl ammonium chloride, and an organic solvent for the second surface modification to obtain a copper and manganese loaded calcium carbonate composite material.

[0011] Preferably, the organic dispersant is polyvinylpyrrolidone; the water-soluble manganese salt is a water-soluble divalent manganese salt; the water-soluble copper salt is CuSO4.

[0012] Preferably, the mass ratio of gallic acid to calcium carbonate nanoparticles is 1:5 to 15; the mass ratio of the organic dispersant to the calcium carbonate nanoparticles is 1 to 3:1.

[0013] Preferably, the water-soluble divalent manganese salt is MnCl2; the mass ratio of gallic acid, the water-soluble manganese salt, and the water-soluble copper salt is (1 to 3):(3 to 5):(4 to 7).

[0014] Preferably, the mass ratio of the copper and manganese loaded calcium carbonate nanoparticles to sodium dioleoyl phosphatidate is 5 to 15:1.

[0015] Preferably, the phospholipid polyethylene glycol is distearoyl phosphatidylethanolamine-polyethylene glycol 50,000 or distearoyl phosphatidylethanolamine-polyethylene glycol 20,000.

[0016] Preferably, the mass ratio of the copper and manganese loaded calcium carbonate nanoparticles to the phospholipid polyethylene glycol is 3 to 8:2.

[0017] Preferably, the mass ratio of the copper and manganese loaded calcium carbonate nanoparticles to cholesterol is 5 to 15:1; the mass ratio of the copper and manganese loaded calcium carbonate nanoparticles to 1,2-distearoyl-sn-glycero-3-phosphocholine is 2 to 6:1; the mass ratio of the copper and manganese loaded calcium carbonate nanoparticles to (2,3-dioleyloxypropyl) trimethyl ammonium chloride is 5 to 15:6.

[0018] The present invention provides a copper-manganese-loaded calcium carbonate composite material prepared by the preparation method described in the above technical solution, comprising copper-manganese-loaded calcium carbonate nanoparticles and an organic layer chemically modified on the surface of the copper-manganese-loaded calcium carbonate nanoparticles; the copper-manganese-loaded calcium carbonate nanoparticles comprise a calcium carbonate nanoparticle carrier and a manganese gallate metal complex and a copper gallate metal complex loaded on the surface of the calcium carbonate nanoparticle carrier; the organic layer contains polyethylene glycol.

[0019] The present invention provides the use of the copper-manganese-loaded calcium carbonate composite material described in the above technical solution in the preparation of anticancer drugs.

[0020] The present invention provides a method for preparing a copper-manganese-loaded calcium carbonate composite material, comprising the following steps: mixing gallic acid, triethylamine, an organic dispersant, calcium carbonate nanoparticles, water, an alcohol solvent, a water-soluble manganese salt, and a water-soluble copper salt to obtain copper-manganese-loaded calcium carbonate nanoparticles; mixing the copper-manganese-loaded calcium carbonate nanoparticles, dioleoylphosphatidic acid sodium salt, and an organic solvent to perform a first surface modification to obtain pre-surface-modified nanoparticles; and mixing the pre-surface-modified nanoparticles, cholesterol, 1,2-distearoyl-sn-glyceride-3-phosphocholine, phospholipid polyethylene glycol, (2,3-dioleyloxypropyl)trimethylammonium chloride, and an organic solvent to perform a second surface modification to obtain the copper-manganese-loaded calcium carbonate composite material. The method first simultaneously grafts manganese metal ions and copper metal ions onto the surface of the calcium carbonate nanoparticles through the compatibility of gallic acid, then coats the calcium carbonate nanoparticles grafted with the metal ions, and coats the surface of the nanoparticles with polyethylene glycol. The coated nanoparticles loaded with copper and manganese ions (denoted as Cu / Mn@CaCO3-PEG) prepared by the present invention can accumulate in the tumor site through enhanced permeability and retention (EPR). Under the triggering of the acidic tumor microenvironment (TME), CaCO3 nanoparticles can decompose and release Mn 4+ / Mn 2+ 、Cu 2+ and a large amount of Ca 2+ .Mn 2+ 、Cu 2+ It can catalyze the overexpressed H2O2 to generate ·OH, thus alleviating tumor hypoxia. At the same time, high-valent Mn ions and Cu ions can downregulate glutathione in the tumor site, changing the reducing environment. Its reduction product Mn 2+ With Cu + A large amount of ROS generated during the Fenton reaction will destroy the intracellular Ca 2+ Balance (i.e., mitochondrial Ca 2+ The buffering capacity is destroyed by exogenous ROS), and with the opening of mitochondrial calcium channels, sufficient Ca is decomposed in CaCO3. 2+ In the presence of mitochondrial Ca2+ The concentration rapidly increases, leading to mitochondrial Ca 2+ overload, which in turn causes mitochondrial dysfunction, inhibits ATP synthesis, and accelerates apoptosis. In summary, the Cu / Mn@CaCO3-PEG prepared in this invention generates ·OH and ROS through the Fenton reaction, disrupts the calcium balance in tumor cells to achieve this calcium overload, and then causes mitochondrial dysfunction in tumor cells, realizing an efficient killing effect on tumor cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 TEM images and DLS data spectra of the CaCO3 nanoparticles prepared in Example 1;

[0022] Figure 2 TEM images and DLS data spectra of the CMC nanoparticles prepared in Example 1;

[0023] Figure 3 TEM images of the CMC-PEG nanoparticles prepared in Example 1, zeta potential diagrams of CaCO3, CMC, and CMC-PEG;

[0024] Figure 4 DLS data diagrams of CaCO3, CMC, and CMC-PEG prepared in Example 1, TEM energy spectrum diagram of CMC-PEG, and elemental composition diagram of CMC-PEG;

[0025] Figure 5 pH neutralization curves of the CMC-PEG nanoparticles prepared in Example 1 at pH values of 7.2, 6.5, and 5.5 respectively, pH-time curve for neutralization to neutral at pH 6, ultraviolet absorption curves of GA at different pH values, and TEM images of CMC-PEG at pH 7.2, 6.5, and 5.5;

[0026] Figure 6 Ultraviolet absorption curves after different contents of CMC-PEG nanoparticles are incubated with GSH and then DTNB is added, and ultraviolet absorption curves after reaction with GSH when the sample concentration is 200 μg·mL -1 ;

[0027] Figure 7 Absorption curves of MB after CMC-PEG is treated with PBS at pH values of 7.2, 6.5, and 5.5 respectively and then co-incubated with H2O2 (10 mM) and MB (10 μgmL -1 ), and fluorescence intensity of the product 7-hydroxycoumarin after reaction with coumarin;

[0028] Figure 8 Cell survival rates after MCF-7 cells are co-incubated with CaCO3, CMC, and CMC-PEG respectively. Detailed implementation mode

[0029] The present invention provides a preparation method of a copper-manganese loaded calcium carbonate composite material, comprising the following steps:

[0030] Mix gallic acid, triethylamine, an organic dispersant, calcium carbonate nanoparticles, water, an alcohol solvent, a water-soluble manganese salt and a water-soluble copper salt to obtain copper-manganese loaded calcium carbonate nanoparticles;

[0031] Mix the copper-manganese loaded calcium carbonate nanoparticles, sodium dioleoyl phosphatide and an organic solvent for the first surface modification to obtain pre-surface modified nanoparticles;

[0032] Mix the pre-surface modified nanoparticles, cholesterol, 1,2-distearoyl-sn-glycero-3-phosphocholine, phospholipid polyethylene glycol, (2,3-dioleoyloxypropyl) trimethyl ammonium chloride and an organic solvent for the second surface modification to obtain a copper-manganese loaded calcium carbonate composite material.

[0033] In the present invention, unless otherwise specified, all preparation raw materials / components are commercially available products well known to those skilled in the art.

[0034] In the present invention, gallic acid (GA), triethylamine, an organic dispersant, calcium carbonate nanoparticles, water, an alcohol solvent, a water-soluble manganese salt and a water-soluble copper salt are mixed (hereinafter referred to as the first mixing) to obtain copper-manganese loaded calcium carbonate nanoparticles. In the present invention, the organic dispersant is preferably polyvinylpyrrolidone; the water-soluble manganese salt is preferably a water-soluble divalent manganese salt; the water-soluble divalent manganese salt is preferably MnCl2. The water-soluble copper salt is preferably CuSO4. The alcohol solvent is preferably ethanol. The particle size of the calcium carbonate nanoparticles is preferably 84-85 nm. The mass ratio of gallic acid to calcium carbonate nanoparticles is preferably 1:5-15, more preferably 1:10; the mass ratio of gallic acid, the water-soluble manganese salt and the water-soluble copper salt is preferably (1-3):(3-5):(4-7), more preferably 2:4:5. The mass ratio of the organic dispersant to the calcium carbonate nanoparticles is preferably 1-3:1, more preferably 2:1. The mass ratio of the mass of gallic acid to the volume of triethylamine is preferably 2 mg:10 μL.

[0035] In the present invention, the calcium carbonate nanoparticles are preferably prepared by the gas diffusion method. The specific preparation method of the calcium carbonate preferably includes the following steps: placing an organic solution of calcium chloride and solid NH4HCO3 together in a sealed vacuum environment and allowing it to stand to obtain the calcium carbonate nanoparticles. The organic solution of calcium chloride preferably includes calcium chloride and absolute ethanol, and the calcium chloride is specifically preferably CaCl2·2H2O. The mass ratio of CaCl2·2H2O to the volume of absolute ethanol is preferably 150 mg: 100 mL. In the present invention, the organic solution of calcium chloride is preferably placed in an open container, and the opening of the open container is covered with a layer of aluminum foil with holes punched in it. The mass of the solid NH4HCO3 is preferably 5 g. The sealed vacuum environment is preferably a vacuum drying oven. The standing time is preferably 24 hours. After the standing is completed, in the present invention, the obtained slurry is preferably subjected to solid-liquid separation to obtain calcium carbonate nanoparticles. The solid-liquid separation is preferably centrifugal separation, and the rotation speed of the centrifugal separation is preferably 8000 rpm. After obtaining the calcium carbonate nanoparticles, in the present invention, the calcium carbonate nanoparticles are preferably resuspended in absolute ethanol for storage.

[0036] In the present invention, the first mixing preferably includes the following steps: dissolving gallic acid in a part of water to obtain an aqueous gallic acid solution; mixing triethylamine and the aqueous gallic acid solution to obtain a first mixed solution; dispersing calcium carbonate nanoparticles in an alcohol solvent to obtain a calcium carbonate solution; mixing an organic dispersant and the calcium carbonate solution to obtain a second mixed solution; first stirring and mixing the first mixed solution and the second mixed solution to obtain a third mixed solution; dissolving a water-soluble manganese salt in another part of water to obtain a manganese salt solution; dissolving a water-soluble copper salt in the remaining water to obtain a copper salt solution; second stirring and mixing the third mixed solution, the manganese salt solution and the copper salt solution to obtain copper and manganese loaded calcium carbonate nanoparticles. The mass concentration of the aqueous gallic acid solution is preferably 20 mg·mL -1 . The mass concentration of the calcium carbonate solution is preferably 5 mg·mL -1 . The mass concentration of the manganese salt solution is preferably 20 mg·mL -1 . The mass concentration of the copper salt solution is preferably 20 mg·mL -1 . The temperature of the first stirring and mixing is preferably room temperature, and the time is preferably 5 min. The temperature of the second stirring and mixing is preferably room temperature, and the time is preferably 1 h. After the second stirring and mixing, in the present invention, the obtained reaction slurry is preferably centrifugally separated, and the obtained solid product is washed with ethanol to obtain copper and manganese loaded calcium carbonate nanoparticles. The rotation speed of the centrifugal separation is preferably 12000 rpm.

[0037] After obtaining the copper-manganese loaded calcium carbonate nanoparticles, the present invention mixes the copper-manganese loaded calcium carbonate nanoparticles, sodium dioleoyl phosphatidate (DOPA), and an organic solvent (the first organic solvent) (hereinafter referred to as the second mixing) for the first surface modification to obtain pre-surface modified nanoparticles. The first organic solvent is preferably ethanol and chloroform. The mass ratio of the copper-manganese loaded calcium carbonate nanoparticles to sodium dioleoyl phosphatidate is preferably 5-15:1, more preferably 10:1. The second mixing preferably includes the following steps: dispersing the copper-manganese loaded calcium carbonate nanoparticles in a part of the first organic solvent to obtain a copper-manganese loaded calcium carbonate nanoparticle solution; dissolving DOPA in the remaining first organic solvent to obtain a DOPA solution; and mixing the copper-manganese loaded calcium carbonate nanoparticle solution and the DOPA solution. The part of the first organic solvent is preferably ethanol, and the remaining first organic solvent is preferably chloroform. The mass concentration of the copper-manganese loaded calcium carbonate nanoparticle solution is preferably 4 mg·mL -1 . The mass concentration of the DOPA solution is preferably 2 mg·mL -1 . The first surface modification is carried out under ultrasonic conditions, and the ultrasonic time is preferably 30 min. After the first surface modification is completed, the present invention preferably centrifuges the slurry obtained from the first surface modification, washes the obtained solid product, and obtains pre-surface modified nanoparticles. The pre-surface modified nanoparticles are DOPA-modified nanoparticles.

[0038] After obtaining the pre-surface modified nanoparticles, the present invention mixes the pre-surface modified nanoparticles, cholesterol, 1,2-distearoyl-sn-glycero-3-phosphocholine (DPPC), phospholipid-polyethylene glycol, (2,3-dioleyloxypropyl) trimethylammonium chloride (DOTAP), and an organic solvent (hereinafter referred to as the second solvent) (hereinafter referred to as the third mixing) for the second surface modification to obtain a copper-manganese loaded calcium carbonate composite material. In the present invention, the phospholipid-polyethylene glycol is preferably distearoyl phosphatidylethanolamine-polyethylene glycol 5000 (DSPE-PEG 5k ) or distearoyl phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG 2k ), more preferably DSPE-PEG 5kThe second organic solvent is preferably chloroform. The mass ratio of the copper-manganese loaded calcium carbonate nanoparticles to the phospholipid-polyethylene glycol is preferably 3-8:2, more preferably 5:2. The mass ratio of the copper-manganese loaded calcium carbonate nanoparticles to cholesterol is preferably 5-15:1, preferably 10:1. The mass ratio of the copper-manganese loaded calcium carbonate nanoparticles to 1,2-distearoyl-sn-glycero-3-phosphocholine is preferably 2-6:1, more preferably 5:1. The mass ratio of the copper-manganese loaded calcium carbonate nanoparticles to (2,3-dioleyloxypropyl) trimethylammonium chloride is preferably 5-15:6, more preferably 10:6. The third mixing is preferably as follows: dissolving cholesterol, DPPC, phospholipid-polyethylene glycol and DOTAP in the second organic solvent to obtain a mixed solution; mixing the pre-surface modified nanoparticles with the mixed solution. The temperature of the second surface modification is preferably room temperature, and the second surface modification is carried out under stirring conditions. After the second surface modification, the present invention preferably removes the organic solvent from the obtained slurry to obtain a copper-manganese loaded calcium carbonate composite material. The present invention preferably mixes the copper-manganese loaded calcium carbonate composite material with water for ultrasonic dispersion, and then stores the copper-manganese loaded calcium carbonate composite material solution at 4°C. The removal of the organic solvent is preferably carried out in a rotary evaporator.

[0039] The present invention provides a copper-manganese loaded calcium carbonate composite material prepared by the preparation method described in the above technical solution, which includes copper-manganese loaded calcium carbonate nanoparticles and an organic layer chemically modified on the surface of the copper-manganese loaded calcium carbonate nanoparticles; the copper-manganese loaded calcium carbonate nanoparticles include a calcium carbonate nanoparticle carrier and a manganese gallate metal complex and a copper gallate metal complex loaded on the surface of the calcium carbonate nanoparticles; the organic layer contains polyethylene glycol.

[0040] The copper-manganese loaded calcium carbonate composite material provided by the present invention includes copper-manganese loaded calcium carbonate nanoparticles. The copper-manganese loaded calcium carbonate nanoparticles include a calcium carbonate nanoparticle carrier and a manganese gallate metal complex and a copper gallate metal complex loaded on the surface of the calcium carbonate nanoparticles. In the present invention, manganese (Mn) is an essential trace element in the organism and plays an important role in various cells and physiological processes. Incorporating manganese ions into nanoparticles can effectively enhance the therapeutic effect of manganese ions and enable them to be released more accurately at the tumor site. In the Fenton-like reagent, Cu 2+ has higher catalytic activity in a wider pH range, and its rate is 6 times and 132 times faster than that of Fe 2+ respectively. In addition to excellent Fenton-like activity, the copper-manganese loaded calcium carbonate nanoparticles provided by the present invention can also consume glutathione (GSH) as glutathione peroxidase, and at the same time generate higher catalytic activity Mn 2+ and Cu +, to achieve a more efficient Fenton catalytic reaction, generate more ·OH, and enhance the killing effect on tumor cells.

[0041] The copper-manganese-loaded calcium carbonate composite material provided by the present invention includes an organic layer chemically modified on the surface of the copper-manganese-loaded calcium carbonate nanoparticles, and the organic layer contains polyethylene glycol. By coating an organic layer on the surface of the copper-manganese-loaded calcium carbonate nanoparticles, the present invention effectively reduces the cytotoxicity of the copper-manganese-loaded calcium carbonate nanoparticles.

[0042] The present invention provides an application of the copper-manganese-loaded calcium carbonate composite material described in the above technical solution in the preparation of anti-cancer drugs.

[0043] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below in conjunction with embodiments, but they cannot be understood as limiting the protection scope of the present invention.

[0044] The sources of raw materials and reagents used in the following embodiments are as follows: Sodium bicarbonate, polyvinylpyrrolidone (PVPK30), calcium chloride dihydrate, gallic acid (GA) are purchased from Sangon Biotech, absolute ethanol is purchased from Sinopharm, dimethyl sulfoxide (DMSO), manganese chloride anhydrous, copper sulfate anhydrous, coumarin, sodium dioleoyl phosphatidylcholine, (DOPA), triethylamine, 1,2-distearoyl-sn-glycero-3-phosphocholine (DPPC), (2,3-dioleyloxypropyl) trimethylammonium chloride (DOTAP), distearoyl phosphatidylethanolamine-polyethylene glycol 5000 (DSPE-PEG 5k ) and cholesterol are purchased from Macklin, and the above reagents are all of analytical pure grade. Methylene blue (MB), 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) are purchased from J&K Scientific.

[0045] The MTT cell proliferation and cytotoxicity assay kit is purchased from Sangon Biotech. Sodium dihydrogen phosphate and disodium hydrogen phosphate are purchased from Aladdin. Fetal bovine serum is provided by Solarbio. MCF-7 breast cancer cells are from Wuhan Pure Science Life Technology Co., Ltd.

[0046] The instruments used in the following embodiments are:

[0047] The particle size and Zeta potential are measured by a laser particle size analyzer (Zetasizer Nano ZS90) at 25°C. The Nano-ZS90 zeta potential instrument (Malvern Instrument Ltd., UK) is used to measure the particle size of the nanomaterials.

[0048] Transmission electron microscope images --- JEM 2100.

[0049] Scanning electron microscope image ---- Hitachi Regulus8220.

[0050] Elemental content composition --- ICP-OES (Avio200).

[0051] pH value determination -- Shanghai Youke digital pH meter PHS-3C.

[0052] Record the ultraviolet (UV)-visible spectrum on a Cary 60 UV-vis-NIR spectrophotometer.

[0053] MTT assay was performed on a microplate reader (EP0CH2, China).

[0054] The cell culture method in the following examples is as follows:

[0055] The MCF-7 breast cancer cells participating in the experiment were from Wuhan Punosai Life Science Co., Ltd. and were cultured in a constant temperature and humidity incubator at 37 °C with a humidity of 95% and a CO2 concentration of 5%. The culture medium for the cells was DMEM containing 10% fetal bovine serum and 1% double antibody (penicillin-streptomycin).

[0056] Example 1

[0057] Disperse 150 mg of CaCl2·2H2O into a beaker containing 100 mL of absolute ethanol. Cover the beaker with a layer of aluminum foil with holes punched in it. Then, place the beaker in a vacuum drying oven containing 5 g of NH4HCO3. Keep the whole system in a vacuum environment for 24 hours to obtain CaCO3 nanoparticles, and centrifuge them at 8000 rpm. These nanoparticles were redispersed in absolute ethanol for further use.

[0058] Add 100 μL of GA aqueous solution (20 mg·mL -1 ) and 10 μL of triethylamine (TEA) to 4 mL of CaCO3 solution (5 mg·mL -1 ethanol) containing 40 mg of PVP polyvinylpyrrolidone, stir vigorously at room temperature for 5 min, and then sequentially add 200 μL of MnCl2 solution (20 mg·mL -1 ) and 250 μL of CuSO4 solution (20 mg·mL -1 ) to the reaction mixture, and continue to stir at room temperature for 1 h. Collect the obtained Cu / Mn@CaCO3 (abbreviated as CMC) nanoparticles and wash them with ethanol after centrifugation at 12000 rpm.

[0059] Surface modification of CMC. An ethanol solution of CMC (20 mg, 5 mL) was mixed with a chloroform solution of DOPA (2 mg, 1 mL), and then ultrasonicated for 30 min. Then, the obtained DOPA-modified nanoparticles were purified by centrifugation and resuspended in a chloroform solution (4 mL) containing cholesterol (2 mg), DPPC (4 mg), DSPE-PEG 5k (8 mg) and DOTAP (12 mg). After the solution was vigorously stirred overnight at room temperature, chloroform was removed using a rotary evaporator to collect the nanoparticles Cu / Mn@CaCO3-PEG (abbreviated as CMC-PEG), which were then dispersed in 2 mL of water. Finally, the sample solution was stored in a refrigerator at 4 °C for further use.

[0060] Test Example 1: pH responsiveness study of CMC-PEG

[0061] CMC-PEG was added to phosphate buffer solutions with pH values of 7.2, 6.5, and 5.5 respectively, and the pH changes of the solutions were measured with a pH meter within 180 min. Then, CMC-PEG was added to a phosphate buffer solution with a pH of 6, and the pH change was measured with a pH meter within 500 min. After CMC-PEG was added to phosphate buffer solutions with pH values of 7.2, 6.5, and 5.5 respectively and reacted for 20 min, the changes in the characteristic peaks of GA were measured by a UV-vis-NIR spectrophotometer.

[0062] Test Example 2: Generation of free radicals by CMC-PEG

[0063] After 20 mg of CMC-PEG was treated with PBS at pH values of 7.2, 6.5, and 5.5, it was added to a NaHCO3 solution (25 mM) containing H2O2 (10 mM) and MB (10 μg·mL -1 ), and then incubated in the dark at 37 °C. At different time points, the mixture was centrifuged, and the remaining MB in the supernatant was measured at 664 nm with a UV-vis-NIR spectrophotometer.

[0064] The content of ·OH was determined by the fluorescence intensity of the product 7-hydroxycoumarin of coumarin. Coumarin (4 mM), H2O2 (8 mM) and samples with different concentrations were fully mixed. After the samples reacted with PBS at pH values of 7.2, 6.5, and 5.5 were co-incubated with coumarin for 1 h, the fluorescence intensity of hydroxycoumarin was measured at 455 nm (λ ex = 332 nm) to evaluate the generation amount of ·OH.

[0065] Test Example 3: Study on the ability of CMC-PEG to consume glutathione (GSH)

[0066] To consume glutathione, CMC-PEG suspensions with different concentrations (50, 100, 150, 200 μg·mL -1 ) were incubated with 10 mM glutathione (GSH) at 37 °C for 20 min, and then 2.5 mg·mL -1 of 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) was added and mixed for 5 min. Its ultraviolet spectrum was measured by a UV-vis-NIR spectrophotometer, and the characteristic absorption peak at 412 nm was compared.

[0067] Test Example 4: MTT method for determining the cytotoxicity of nanoparticles

[0068] MCF-7 cells were incubated with CMC-PEG, CMC, and CaCO3 in a cell culture incubator for 6 h. The supernatant in each well was carefully aspirated, and then 10 μL of MTT and 90 μL of fresh medium were added to each well and kept in the dark at 37 °C for 4 hours. After 4 hours, the supernatant was aspirated, 100 μL of DMSO was added, and it was shaken at low speed on an oscillator for 10 min. It was detected at 490 nm by an enzyme-labeling instrument, and the cell survival rate was calculated based on the detection data.

[0069] Results and Discussion

[0070] (1) Characterization of Cu / Mn@CaCO3-PEG

[0071] The CaCO3 nanoparticles, Cu / Mn@CaCO3, and Cu / Mn@CaCO3-PEG prepared in the examples were characterized by transmission electron microscopy (TEM), dynamic light scattering (DLS), and ultraviolet-visible spectroscopy. In Example 1 of the present invention, Cu / Mn@CaCO3 was first synthesized on the basis of the synthesized CaCO3 nanoparticles, and CMC-PEG was obtained by surface modification of it.

[0072] Figure 1 A in Figure 1 is the TEM picture of CaCO3 nanoparticles, Figure 1 and B in Figure 2 is the DLS data of CaCO3 nanoparticles. In Figure 2 , the TEM data and EDS show that the synthesized calcium carbonate nanomaterial has a good spherical structure with a diameter of about 84.84 nm and good dispersibility. Figure 2 It can be seen from Figure 2 that after the calcium carbonate nanoparticles are loaded with Cu / Mn ions, the size does not change significantly, and the diameter is about 86.24 nm (

[0073] Figure 3A in it is the TEM image of CMC-PEG nanoparticles. From Figure 3 A in it, it can be seen that after surface modification with PEG, the size is about 90.33 nm ( Figure 3 A in it), and it still maintains good dispersibility.

[0074] Figure 3 B in it is the zeta potential of CaCO3, CMC and CMC-PEG. From Figure 3 B in it, it can be seen that the zeta potentials of CaCO3 and Cu / Mn@CaCO3 in aqueous solution are both negative, -2.36667 and -10.5 respectively. After surface modification, due to the presence of DOTAP, the potential of the material shows +6.17, indicating the successful modification of the material by PEG. Figure 4 D in it is the TEM energy spectrum of CMC-PEG. From Figure 4 D in it, it can be seen that the CMC-PEG material is mainly composed of C, O, Ca, Mn, and Cu, indicating that the calcium carbonate nanoparticles have successfully carried Mn and Cu ions ( Figure 4 D in it). Figure 4 E in it is the elemental composition map of CMC-PEG. From Figure 4 The ICP-OES results of E in it show that the Ca element content is 35.76%, 3.108%, 3.3915% ( Figure 4 E in it).

[0075] (2) pH responsiveness of CMC-PEG

[0076] The neutralization ability of CMC-PEG to pH 5.5, 6.5, and 7.2 was measured by a pH meter respectively to reflect the pH responsiveness of the sample. Figure 5 A in it is the pH neutralization curve of CMC-PEG nanoparticles at pH 7.2, 6.5, and 5.5 respectively. Figure 5 B in it is the pH time curve of CMC-PEG nanoparticles neutralized to neutral at pH 6, Figure 5 C in it is the UV absorption curve of GA of CMC-PEG nanoparticles at different pH values, Figure 5 D in it is the TEM image of CMC-PEG nanoparticles under different pH conditions, where Figure 5 a in D in it is the TEM image of CMC-PEG nanoparticles at pH 7.2, where Figure 5 b in D in it is the TEM image of CMC-PEG nanoparticles at pH 6.5, where Figure 5The c in D is the TEM image of CMC-PEG nanoparticles at pH 5.5. As can be seen from Figure 5, CMC-PEG has the strongest neutralization ability at pH 5.5. There is also a relatively obvious increase in pH in the pH 6.5 group, while the pH value of the sample solution remains basically unchanged at pH 7.2. The above results indicate that CMC-PEG decomposes significantly at pH 5.5 and 6.5, while the sample is relatively stable and does not decompose at pH 7.2, demonstrating that the sample has pH responsiveness.

[0077] The characteristic absorption peak of gallic acid in the sample was measured by an ultraviolet spectrophotometer. Figure 5 C in it is the ultraviolet absorption curve of GA of CMC-PEG nanoparticles at different pH values. Figure 5 It can also be seen from C in it that the sample is relatively stable at pH 7.2, and there is basically no characteristic absorption peak of gallic acid. However, characteristic absorption peaks of gallic acid appear at pH 6.5 and 5.5, and the absorption peak at pH 5.5 is the strongest. Figure 5 The TEM results in D show that the nanomaterial has a stable particle shape at pH 7.2, the particle shape changes at pH 6.5, indicating the start of decomposition, and the morphological structure of the nanoparticles disappears at pH 5.5, indicating that the sample is completely decomposed.

[0078] (3) CMC-PEG's ability to consume glutathione (GSH)

[0079] To evaluate the ability of MC-PEG to consume glutathione (GSH), in this invention, CMC-PEG suspensions with different concentrations (50, 100, 150, 200 μg·mL -1 ) were incubated with 10 mM GSH at 37 °C for 20 min, and then 2.5 mg·mL -1 of DTNB was added and mixed for 5 min. Figure 6 A in it is the ultraviolet absorption curve after adding DTNB after incubation of CMC-PEG nanoparticles with different contents with GSH. Figure 6 B in it is the ultraviolet absorption curve after reacting with GSH when the sample concentration is 200 μg·mL -1 ; Figure 6 The ultraviolet absorption spectrum in it shows that as the concentration of CMC-PEG increases, the characteristic absorption peaks of the yellow 2-nitro-5-mercaptobenzoic acid and glutathione disulfide (GSSG), the reaction products of DTNB and GSH, at 412 nm gradually decrease. When the sample concentration is 200 μg·mL -1 , as the reaction time increases, the characteristic absorption of GSSG at 412 nm gradually decreases. The above results indicate that CMC-PEG has good glutathione consumption ability for GSH.

[0080] (4) Radical generation ability of CMC-PEG

[0081] To evaluate the radical generation ability of CMC-PEG, in this invention, after CMC-PEG was treated with PBS at pH 7.2, 6.5, and 5.5 respectively, it was added to a NaHCO3 solution (25 mM) containing H2O2 (10 mM) and MB (10 μg mL -1 ). The remaining MB in the supernatant was measured at 664 nm using a UV-vis-NIR spectrophotometer. Figure 7 In A, the absorption curve of MB after CMC-PEG was treated with PBS at pH 7.2, 6.5, and 5.5 respectively and co-incubated with H2O2 (10 mM) and MB (10 μg mL -1 ). The results of the ultraviolet absorption spectrum showed that at 664 nm, as the pH value decreased, the characteristic absorption peak of MB gradually decreased, indicating that at pH 5.5 and 6.5, the released Mn 2+ and Cu 2+ contents were higher than those at pH 7.2, with higher catalytic efficiency of the Fenton reaction and more ·OH generated.

[0082] The content of ·OH was determined by the fluorescence intensity of the product 7-hydroxycoumarin of coumarin. Coumarin (4 mM), H2O2 (8 mM), and different concentrations of the sample were fully mixed. After the sample reacted with PBS at pH 7.2, 6.5, and 5.5 was co-incubated with coumarin for 1 h, the fluorescence intensity of hydroxycoumarin was measured at 455 nm (λex = 332 nm below), Figure 7 In B, the fluorescence intensity of the product 7-hydroxycoumarin after reaction with coumarin. It was found that the fluorescence intensities at pH 5.5 and 6.5 were stronger than those of the sample at pH 7.2. The above results indicate that in a more acidic environment, the material can release more metal ions, thereby catalyzing the Fenton reaction to generate ·OH.

[0083] (5) Investigation of the cytotoxicity of CMC-PEG

[0084] To further study the cytotoxic effect of the nanomaterial on cancer cells, in this invention, an MTT detection kit was used to detect and record the cytotoxicity of CaCO3, CMC, and CMC-PEG on MCF-7 cells. Figure 8 In A, the cell viability of MCF-7 cells after co-incubation with CaCO3( Figure 8 ), CMC( Figure 8 in B), and CMC-PEG( Figure 8 in C). From Figure 8It can be seen from the data that as the sample concentration increases, the cytotoxicity of CaCO3, CMC, and CMC-PEG to cells gradually increases. Through data analysis, it is obvious that CMC-PEG is more cytotoxic to cells than CaCO3 and CMC.

[0085] The present invention develops a nanomaterial CMC-PEG loaded with Cu / Mn bimetallic elements, and utilizes the pH responsiveness of the CaCO3 carrier to the tumor microenvironment to achieve precise release of Cu 2+ and Mn*(Mn 2+ and Mn 4+ ) at the tumor site. The released high-valent metal ions can effectively consume the excessive glutathione in tumor tissues, and at the same time, the generated Mn 2+ and Cu + catalyze the generation of highly toxic hydroxyl radicals ([ . OH). This process alleviates tumor hypoxia, and at the same time, the generated reactive oxygen species disrupt the intracellular Ca 2 + balance, thereby causing mitochondrial dysfunction and resulting in an efficient killing effect on tumor cells.

[0086] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, not all embodiments. Other embodiments can be obtained based on this embodiment without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Application of a copper-manganese loaded calcium carbonate composite material in the preparation of an anti-breast cancer drug, characterized in that: The preparation method of the copper-manganese-loaded calcium carbonate composite material comprises the following steps: Gallic acid, triethylamine, an organic dispersant, calcium carbonate nanoparticles, water, an alcohol solvent, a water-soluble manganese salt, and a water-soluble copper salt are mixed to obtain copper-manganese-loaded calcium carbonate nanoparticles, wherein the organic dispersant is polyvinylpyrrolidone, the mass ratio of the gallic acid to the calcium carbonate nanoparticles is 1:5-15, the mass ratio of the organic dispersant to the calcium carbonate nanoparticles is 1-3:1, and the mass ratio of the gallic acid to the water-soluble manganese salt to the water-soluble copper salt is (1-3):(3-5):(4-7); Mixing the copper-manganese loaded calcium carbonate nanoparticles, dioleoylphosphatidic acid sodium salt and an organic solvent to perform a first surface modification to obtain pre-surface-modified nanoparticles; The pre-surface-modified nanoparticles, cholesterol, 1,2-distearoyl-sn-glyceride-3-phosphocholine, phospholipid polyethylene glycol, (2,3-dioleyloxypropyl)trimethylammonium chloride and an organic solvent are mixed for a second surface modification to obtain a copper-manganese-loaded calcium carbonate composite material; The copper-manganese-loaded calcium carbonate composite material comprises copper-manganese-loaded calcium carbonate nanoparticles and an organic layer chemically modified on the surface of the copper-manganese-loaded calcium carbonate nanoparticles; the copper-manganese-loaded calcium carbonate nanoparticles comprise a calcium carbonate nanoparticle carrier and a manganese gallate metal complex and a copper gallate metal complex loaded on the surface of the calcium carbonate nanoparticle carrier; the organic layer contains polyethylene glycol.

2. The use according to claim 1, characterized in that The water-soluble manganese salt is a water-soluble divalent manganese salt; the water-soluble copper salt is CuSO4.

3. The use according to claim 1 or 2, characterized in that The water-soluble divalent manganese salt is MnCl2.

4. The use according to claim 1, characterized in that The mass ratio of the copper-manganese loaded calcium carbonate nanoparticles to dioleoylphosphatidic acid sodium salt is 5-15:

1.

5. The use according to claim 1, characterized in that The phospholipid polyethylene glycol is distearoyl phosphatidylethanolamine-polyethylene glycol 5000 or distearoyl phosphatidylethanolamine-polyethylene glycol 2000.

6. The use according to claim 1 or 5, characterized in that The mass ratio of the copper-manganese loaded calcium carbonate nanoparticles to the phospholipid polyethylene glycol is 3-8:

2.

7. The use according to claim 1, characterized in that The mass ratio of the copper-manganese-loaded calcium carbonate nanoparticles to cholesterol is 5-15:1; the mass ratio of the copper-manganese-loaded calcium carbonate nanoparticles to 1,2-distearoyl-sn-glyceride-3-phosphocholine is 2-6:1; and the mass ratio of the copper-manganese-loaded calcium carbonate nanoparticles to (2,3-dioleyloxypropyl)trimethylammonium chloride is 5-15:6.

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