CN112294751B - Preparation method and application of a calcium peroxide-loaded metal-organic framework pharmaceutical composition - Google Patents

Abstract

The invention relates to a preparation method and application of a metal organic framework pharmaceutical composition loaded with calcium peroxide, which can effectively solve the preparation of the metal organic framework pharmaceutical composition loaded with calcium peroxide, and realize cascade catalysis for treating tumors and ion interference for tumor cells, and promote apoptosis of tumor cells. The metal organic frame is a cerium-based synthesized organic frame, and is carbonized into the cerium-based metal organic frame with the grain diameter of 80-180nm and the aperture of 6-8nm under high-temperature calcination, and then the micromolecular chemotherapeutic drug is connected to CaO through chemical bonds ₂ Surface to be loaded with CaO ₂ The small particles are adhered to the holes of the cerium-based metal organic framework through physical adsorption, so that the metal organic framework pharmaceutical composition with the particle size of 100-200nm and loaded with calcium peroxide is obtained, the drug loading weight is 20% -60%, the preparation method is stable and reliable, the cost is low, and the prepared metal organic framework pharmaceutical composition loaded with calcium peroxide is used for preparing antitumor drugsIn the aspect, the effects of cascade catalysis and ion interference can be exerted, and the anti-tumor effect is enhanced.

Description

Preparation method of a calcium peroxide-loaded metal-organic framework pharmaceutical composition and its application

Technical field

The invention relates to the field of biomedicine, in particular to a preparation method and application of a calcium peroxide-loaded metal-organic framework pharmaceutical composition.

Background technique

The hot metal-based frameworks (MOFs) are an emerging nanocarrier, which have the advantages of large pore size, high loading and high catalysis, and are widely used in fields such as catalysis, energy storage, separation and medicine. In addition, the cerium-based metal organic framework (CeMOF) contains a large number of small cerium oxide particles with strong peroxidase activity, which can produce Fenton-like reactions with H ₂ O ₂ in acidic tumor environments to generate highly toxic reactive oxygen species ( ROS). However, this reaction is limited by the low concentration of H ₂ O ₂ in the cells. Providing exogenous H ₂ O ₂ will undoubtedly improve the anti-tumor efficacy.

Interestingly, peroxide can produce H ₂ O ₂ through a catalytic reaction in the acidic environment of tumors, solving the above-mentioned problem of CeMOF performing Fenton-like reactions. Among them, while calcium peroxide nanoparticles produce H ₂ O ₂ , they are also disintegrated to produce a large amount of Ca ²⁺ . As is known to all, Ca ²⁺ is involved in apoptosis signaling as a second messenger in cells, and calcium-based ion interference therapy has become a focus of researchers. Studies have shown that the influx of extracellular Ca ²⁺ or the release of intracellular calcium stores can lead to intracellular calcium overload, thereby activating the Caspase-3 pathway and inducing cell apoptosis; in addition, abnormal accumulation of Ca ²⁺ can also promote the development of tumor lesions. development of calcification. Therefore, loading small and highly active CaO ₂ particles into a cerium-based metal organic framework, and then adding anti-tumor small molecule drugs, can achieve the triple-hit interactive comprehensive treatment effect of Fenton-like catalytic oxidation effect, ion interference therapy, and chemotherapy. .

Contents of the invention

In view of the above situation, in order to solve the deficiencies of the prior art, the purpose of the present invention is to provide a preparation method and application of a calcium peroxide-loaded metal organic framework pharmaceutical composition, which can effectively solve the problem of calcium peroxide-loaded metal organic framework pharmaceutical compositions. The preparation of the composition realizes the drug problem of cascade catalysis for treating tumors, ion interference on tumor cells, and promoting apoptosis of tumor cells.

The technical solution solved by the present invention is a method for preparing a calcium peroxide-loaded metal-organic framework pharmaceutical composition. The metal-organic framework is a cerium-based synthesized organic framework, which is carbonized under high-temperature calcination into a particle size of 80-180 nm. A cerium-based metal organic framework with a pore size of 6-8 nm is used, and then small molecule chemotherapy drugs are connected to the CaO ₂ surface through chemical bonds, and finally the drug-loaded CaO ₂ small particles are attached to the pores of the cerium-based metal organic framework through physical adsorption to obtain particles. A calcium peroxide-loaded metal-organic framework pharmaceutical composition with a diameter of 100-200 nm and a drug-loading weight of 20% to 60%. The specific steps are as follows:

(1) Dissolve 450-470 mg of ceric ammonium nitrate in 2-4 mL of ultrapure water to form a ceric ammonium nitrate aqueous solution, and at the same time dissolve 130-150 mg of terephthalic acid in 2-4 mL of N,N-dimethyl In methyl formamide, the N,N-dimethylformamide solution of terephthalic acid and the cerium ammonium nitrate aqueous solution are mixed and sealed, stirred in an oil bath at 95-115°C for 20-40 minutes, and then centrifuged at 8000-12000 rpm for 10 minutes. Take the precipitate, wash it 3 times with N,N-dimethylformamide, the amount used each time is 2-3 times the weight volume of the precipitation, and then wash it 3 times with acetone, the amount used each time is 2-3 times the weight volume of the precipitation. times, the weight volume refers to the liquid in mL and the solid in g (the same below). After vacuum drying, grind it into a white powder that passes through a 100-120 mesh sieve to obtain a cerium-based metal organic framework (CeMOF); The cerium-based metal organic framework (CeMOF) is calcined in a muffle furnace, heated to 350-400°C at 5-10°C/min, calcined for 3-5 hours, and cooled to below 20°C to obtain the calcined carbonized cerium-based metal organic framework. Framework(CeMOF);

(2) Dissolve 0.4-0.6 mg calcium chloride particles in 4-6 mL of ultrapure water, stir at 2000-2400 rpm to completely dissolve, add 2-3 mL of 0.5-1.5 M ammonia water with stirring at room temperature, and then Add 30-50 mL of polyethylene glycol 200 (PEG200), continue stirring to completely mix it into a uniform solution, then add 2-3 mL of hydrogen peroxide with a mass concentration of 30%, continue stirring for 5-7 hours, and then use sodium hydroxide solution Gradually adjust the pH to 11.5, centrifuge at 8000-12000 rpm for 10 minutes, discard the supernatant, and obtain a white precipitate. The white precipitate is washed 3 times with absolute ethanol, and the amount used each time is 2-3 times the weight and volume of the white precipitate. Dry under vacuum to obtain 2 Calcium oxide (CaO ₂ );

(3) Disperse 1-3 mg of calcium dioxide (CaO ₂ ) and 1 mg of small molecule chemotherapy drug in a mixed solution of 3-5 mL of ethanol and water with an equal volume ratio, and stir at room temperature at 2000-2400 rpm in the dark for 48 hours. Then centrifuge at 8000-12000rpm for 10 minutes, discard the supernatant, and obtain a precipitate. The precipitate is washed three times with a mixed solution of ethanol-water with an equal volume ratio, and the amount used each time is 2-3 times the weight and volume of the white precipitate to obtain small-molecule drugs. of calcium peroxide;

The small molecule chemotherapy drugs are L-arginine, doxorubicin, paclitaxel, docetaxel, hydroxycamptothecin or mitoxantrone;

(4) Dissolve 5-10 mg of the calcined carbonized cerium-based metal organic framework (CeMOF) prepared in step (2) in 5-10 mL of solvent, disperse evenly using ultrasound, and take another portion of step (3). 3-5mg of the prepared calcium peroxide loaded with small molecule drugs is dissolved in 3-5mL of solvent; add the two solutions together into a round-bottomed flask, stir and react at 2000-2400rpm at room temperature for 20-40min in the dark, and then 8000 Centrifuge at -12000rpm for 10 minutes, discard the supernatant, and obtain a precipitate. Wash the precipitate 3 times with PBS at pH 7.2-7.4. The amount used each time is 2-3 times the weight and volume of the precipitate. Dry under vacuum and grind to 100-120 mesh. Sieve the powder to obtain a calcium peroxide-loaded metal-organic framework pharmaceutical composition;

The solvent is a mixture of ethanol and water in an equal volume ratio, PBS or MES with a pH of 7.2-7.4.

Application of the calcium peroxide-loaded metal organic framework pharmaceutical composition in the preparation of anti-tumor drugs combined with pharmaceutically active or pharmacologically active molecules, the pharmaceutically active or pharmacologically active molecules being L-arginine, doxorubicin , paclitaxel, docetaxel, hydroxycamptothecin, or mitoxantrone.

The calcium peroxide-loaded metal-organic framework pharmaceutical composition prepared by the method is used in the preparation of anti-tumor injections, oral dosage forms or implantable dosage forms.

The calcium peroxide-loaded metal-organic framework pharmaceutical composition prepared by the method is used in the preparation of triple-hit interactive comprehensive treatment of tumor drugs with Fenton-like catalytic oxidation effect, ion interference therapy, and chemotherapy.

The preparation method of the present invention is stable and reliable, and has low cost. The prepared metal-organic framework pharmaceutical composition loaded with calcium peroxide can play the role of cascade catalysis and ion interference in the preparation of anti-tumor drugs, enhance the anti-tumor effect, and is a tumor treatment drug. The innovation has brought huge economic and social benefits.

Detailed ways

The specific implementation modes of the present invention will be described in detail below with reference to examples and specific situations.

The specific implementation of the present invention is given by the following examples.

Example 1:

In a specific implementation of the present invention, a method for preparing a calcium peroxide-loaded metal-organic framework pharmaceutical composition includes the following steps:

(1) Dissolve 460 mg of ceric ammonium nitrate in 3 mL of ultrapure water to form a ceric ammonium nitrate aqueous solution. At the same time, dissolve 140 mg of terephthalic acid in 3 mL of N,N-dimethylformamide. The N,N-dimethylformamide solution of diformic acid and the ceric ammonium nitrate aqueous solution were mixed and sealed, stirred in an oil bath at 100°C for 30 minutes, and then centrifuged at 10,000rpm for 10 minutes, and the precipitate was taken and washed with N,N-dimethylformamide. Wash with amide 3 times, each time using an amount of 2-3 times the weight and volume of the precipitation, then wash 3 times with acetone, each time using an amount of 2-3 times the weight and volume of the precipitation, vacuum dry and then grind to a white powder that passes through a 110 mesh sieve. , to obtain a cerium-based metal organic framework (CeMOF); the prepared cerium-based metal organic framework (CeMOF) is calcined in a muffle furnace, heated to 370°C at 7°C/min, calcined for 4 hours, and cooled to below 20°C. , to obtain the calcined carbonized cerium-based metal organic framework (CeMOF);

(2) Dissolve 0.5 mg of calcium chloride particles in 5 mL of ultrapure water, stir at 2200 rpm to completely dissolve, add 2.5 mL of 1M ammonia water with stirring at room temperature, and then add 40 mL of polyethylene glycol 200 (PEG200) , continue stirring to completely mix it into a uniform solution, then add 2.5 mL of hydrogen peroxide with a mass concentration of 30%, continue stirring for 6 hours, then gradually adjust the pH to 11.5 with sodium hydroxide solution, centrifuge at 10000 rpm for 10 min, discard the supernatant, and obtain White precipitate, wash the white precipitate 3 times with absolute ethanol, the amount used each time is 2-3 times the weight volume of the white precipitate, and vacuum dry to obtain calcium dioxide (CaO ₂ );

(3) Disperse 2 mg of calcium dioxide (CaO ₂ ) and 1 mg of doxorubicin in 4 mL of a mixed solution of equal volume ratio of ethanol and water, stir at room temperature at 2200 rpm in the dark for 48 h, then centrifuge at 10000 rpm for 10 min, and discard The supernatant liquid is used to obtain a precipitate. The precipitate is washed three times with a mixed solution of ethanol and water of equal volume ratio, and the amount used each time is 2-3 times the weight volume of the white precipitate, to obtain calcium peroxide loaded with doxorubicin;

(4) Dissolve 7.5 mg of the calcined carbonized cerium-based metal organic framework (CeMOF) prepared in step (2) in 7.5 mL of a mixture of ethanol and water with an equal volume ratio, and disperse it evenly using ultrasound. Take 4 mg of the doxorubicin-loaded calcium peroxide prepared in step (3) and dissolve it in 4 mL of a mixture of ethanol and water with an equal volume ratio; add the two solutions together into a round-bottomed flask and keep it at room temperature at 2200 rpm in the dark. Stir the reaction for 30 minutes, and then centrifuge at 10,000 rpm for 10 minutes. Discard the supernatant to obtain a precipitate. The precipitate is washed three times with PBS at pH 7.3. The amount used each time is 2-3 times the weight and volume of the precipitate. Dry in vacuum and grind to a size of 110 mesh. Sieve the powder to obtain a calcium peroxide-loaded metal-organic framework pharmaceutical composition.

Example 2:

In a specific implementation of the present invention, a method for preparing a calcium peroxide-loaded metal-organic framework pharmaceutical composition includes the following steps:

(1) Dissolve 452 mg of ceric ammonium nitrate in 2 mL of ultrapure water to form a ceric ammonium nitrate aqueous solution. At the same time, dissolve 132 mg of terephthalic acid in 2 mL of N,N-dimethylformamide. The N,N-dimethylformamide solution of dicarboxylic acid and the ceric ammonium nitrate aqueous solution were mixed and sealed, stirred in an oil bath at 97°C for 38 minutes, and then centrifuged at 9000 rpm for 10 minutes to remove the precipitate, which was washed with N,N-dimethylformamide. Wash with amide 3 times, each time using an amount of 2-3 times the weight and volume of the precipitation, then wash 3 times with acetone, each time using an amount of 2-3 times the weight and volume of the precipitation, vacuum dry and then grind to a white powder that passes through a 100 mesh sieve. , to obtain a cerium-based metal organic framework (CeMOF); the prepared cerium-based metal organic framework (CeMOF) is calcined in a muffle furnace, heated to 355°C at 6°C/min, calcined for 4.5h, and cooled to 20°C. In the following, the calcined and carbonized cerium-based metal organic framework (CeMOF) is obtained;

(2) Dissolve 0.4 mg of calcium chloride particles in 4 mL of ultrapure water, stir at 2000 rpm to completely dissolve, add 2 mL of 1.5 M ammonia water with stirring at room temperature, and then add 30 mL of polyethylene glycol 200 (PEG200 ), continue stirring to completely mix it into a uniform solution, then add 2 mL of hydrogen peroxide with a mass concentration of 30%, continue stirring for 5 hours, then gradually adjust the pH to 11.5 with sodium hydroxide solution, centrifuge at 9000 rpm for 10 minutes, discard the supernatant, and obtain White precipitate, wash the white precipitate 3 times with absolute ethanol, the amount used each time is 2-3 times the weight volume of the white precipitate, and vacuum dry to obtain calcium dioxide (CaO ₂ );

(3) Disperse 1 mg of calcium dioxide (CaO ₂ ) and 1 mg of paclitaxel in 3 mL of a mixed solution of equal volume ratio of ethanol and water, stir at room temperature at 2000 rpm in the dark for 48 h, then centrifuge at 9000 rpm for 10 min, discard the supernatant liquid to obtain a precipitate. The precipitate is washed three times with a mixed solution of ethanol-water with an equal volume ratio, and the amount used each time is 2-3 times the weight volume of the white precipitate, to obtain calcium peroxide loaded with paclitaxel;

(4) Dissolve 6 mg of the calcined and carbonized cerium-based metal organic framework (CeMOF) prepared in step (2) in 6 mL of PBS with a pH of 7.2-7.4, and disperse it evenly using ultrasound. Take another step (3). 3 mg of paclitaxel-loaded calcium peroxide prepared in ) was dissolved in 3 mL of PBS with a pH of 7.2-7.4; add the two solutions together into a round-bottomed flask, stir and react at 2000 rpm for 38 min at room temperature in the dark, and then centrifuge at 9000 rpm for 10 min. , discard the supernatant, and obtain a precipitate. The precipitate is washed three times with PBS at pH 7.2, and the amount used each time is 2-3 times the weight and volume of the precipitate. It is vacuum dried and ground into powder that passes through a 100-mesh sieve to obtain loaded peroxide. Calcium metal-organic framework pharmaceutical compositions.

Example 3:

In a specific implementation of the present invention, a method for preparing a calcium peroxide-loaded metal-organic framework pharmaceutical composition includes the following steps:

(1) Dissolve 468 mg of ceric ammonium nitrate in 4 mL of ultrapure water to form a ceric ammonium nitrate aqueous solution. At the same time, dissolve 148 mg of terephthalic acid in 4 mL of N,N-dimethylformamide. The N,N-dimethylformamide solution of dicarboxylic acid and the ceric ammonium nitrate aqueous solution were mixed and sealed, stirred in an oil bath at 115°C for 22 minutes, and then centrifuged at 11000 rpm for 10 minutes, and the precipitate was taken, and the precipitate was washed with N,N-dimethylformamide. Wash with amide 3 times, each time using an amount of 2-3 times the weight and volume of the precipitation, then wash 3 times with acetone, each time using an amount of 2-3 times the weight and volume of the precipitation, vacuum dry and then grind to a white powder that passes through a 120 mesh sieve. , to obtain a cerium-based metal organic framework (CeMOF); the prepared cerium-based metal organic framework (CeMOF) is calcined in a muffle furnace, heated to 390°C at 9°C/min, calcined for 3 hours, and cooled to below 20°C. , to obtain the calcined carbonized cerium-based metal organic framework (CeMOF);

(2) Dissolve 0.6 mg calcium chloride particles in 6 mL of ultrapure water, stir at 2400 rpm to completely dissolve, add 3 mL of 0.5 M ammonia water with stirring at room temperature, and then add 50 mL of polyethylene glycol 200 (PEG200 ), continue stirring to completely mix it into a uniform solution, then add 3 mL of hydrogen peroxide with a mass concentration of 30%, continue stirring for 7 hours, then gradually adjust the pH to 11.5 with sodium hydroxide solution, centrifuge at 11000 rpm for 10 minutes, discard the supernatant, and obtain White precipitate, wash the white precipitate 3 times with absolute ethanol, the amount used each time is 2-3 times the weight volume of the white precipitate, and vacuum dry to obtain calcium dioxide (CaO ₂ );

(3) Disperse 3 mg of calcium dioxide (CaO ₂ ) and 1 mg of docetaxel in 5 mL of a mixed solution of equal volume ratio of ethanol and water, stir at room temperature at 2400 rpm in the dark for 48 h, then centrifuge at 11000 rpm for 10 min, and discard The supernatant liquid is used to obtain a precipitate. The precipitate is washed three times with a mixed solution of ethanol and water with an equal volume ratio, and the amount used each time is 2-3 times the weight volume of the white precipitate, to obtain docetaxel-loaded calcium peroxide;

(4) Dissolve 10 mg of the calcined and carbonized cerium-based metal organic framework (CeMOF) prepared in step (2) in 10 mL of MES with a pH of 7.2-7.4, and disperse it evenly using ultrasound. Take another step (3). 5 mg of docetaxel-loaded calcium peroxide prepared in ) was dissolved in 5 mL of MES with a pH of 7.2-7.4; add the two solutions together into a round-bottomed flask, stir and react at 2400 rpm at room temperature in the dark for 22 min, and then 11000 rpm Centrifuge for 10 minutes, discard the supernatant, and obtain a precipitate. The precipitate is washed three times with PBS at pH 7.4, and the amount used each time is 2-3 times the weight and volume of the precipitate. Dry under vacuum, and grind to a powder that passes a 120-mesh sieve to obtain the load. Metal-organic framework pharmaceutical compositions of calcium peroxide.

It should be noted that the above are only examples of the present invention and are used to illustrate the specific implementation of the present invention and are not intended to limit the scope of protection of the present invention. Any equivalent or equivalent modifications and substitutions that are essentially the same as the technical solution of the present invention are included in the protection scope of the present invention.

The preparation method of the present invention is scientific and reasonable, the raw materials are easily available, easy to produce and prepare, the method is stable and reliable, and the product quality is good. After scientific testing, the calcium peroxide-loaded metal organic framework pharmaceutical composition prepared by the present invention, calcium peroxide provides H ₂ O ₂ enhances the Fenton-like reaction to generate reactive oxygen species and simultaneously increases intracellular Ca ²⁺ , while the addition of small molecule chemotherapy drugs greatly enhances the anti-tumor effect. In terms of preparing anti-tumor drugs, it can exert the anti-tumor effects of cascade catalysis and ion interference. Compared with traditional chemotherapy, it has the advantages of high efficiency and low toxic and side effects. The relevant experimental data are as follows (taking Example 1 as an example):

1. Characterization experiments of calcium peroxide-loaded metal organic framework pharmaceutical compositions

1. Determination of doxorubicin content in calcium peroxide-loaded metal organic framework pharmaceutical composition:

Use ultraviolet spectrophotometry to measure the content of doxorubicin at a wavelength of 482 nm. Use formula (1) to calculate the drug loading capacity of the sample, and the drug loading capacity is 50%.

2. Determination of particle size and potential of calcium peroxide-loaded metal organic framework pharmaceutical composition:

An appropriate amount of the metal organic framework pharmaceutical composition loaded with doxorubicin and calcium peroxide was dispersed in water, and the particle size and potential of CeMOF and CeMOF-CaO ₂ /DOX were measured with a Nano-ZS90 laser nanoparticle size analyzer to be 116 nm and 132 nm respectively. and -20.1±1.1mV, -24.7±2.0mV.

3. Determination of pore size of H ₂ O ₂ self-supplying Ca ²⁺ generator pharmaceutical composition:

Take an appropriate amount of CeMOF powder and disperse it evenly in water, and use the ASAP 2020 fully automatic specific surface and porosity analyzer and the BJH algorithm to measure the pore diameter to be 7.3nm.

2. Experiment on the generation of reactive oxygen species in vitro by calcium peroxide-loaded metal-organic framework pharmaceutical compositions

Select MCF-7 human breast cancer cells in the logarithmic growth phase, adjust the number of cells to 3×10 ⁵ /ml and inoculate them into a 6-well culture plate, 2 ml per well. Add the drug after 24 hours of adherent growth of the cells, followed by the blank group and CeMOF. group, CeMOF-CaO ₂ group, and CeMOF-CaO ₂ /DOX group, in which the final concentration of CeMOF is set to 150 μg/ml. After culturing for 24 hours, the old culture medium is discarded, and each well is washed 2 to 3 times with fresh PBS. Add 1 ml of reactive oxygen species probe medium containing DCFH-DA, incubate at 37°C for 30 minutes, wash 2 to 3 times with fresh PBS, and observe under a microscope. The results showed that except the blank group and CeMOF group, all the other groups were detected Green DCFH-DA fluorescence signal. This result is consistent with literature reports. CeMOF can generate a large amount of reactive oxygen species through a Fenton-like reaction with H ₂ O ₂ produced by calcium peroxide.

3. Cellular uptake experiment of calcium peroxide-loaded metal-organic framework pharmaceutical compositions

Select MCF-7 human breast cancer cells in the logarithmic growth phase, adjust the number of cells to 3×10 ⁵ /ml and inoculate them into a 6-well culture plate, 2 ml per well, add drugs after 24 hours of adherent growth, and then divide into DOX group, CeMOF- In the CaO ₂ /DOX group, the final concentration of the drug was set to 5 μg/ml, and the incubation time was set to 0.5 h, 1 h, and 2 h. After adding the drug, the culture medium was cultured at 37°C and 5% CO ₂ , and the drug-containing culture medium in the wells was discarded. Remove, wash each well 2 to 3 times with 1 ml PBS, add 500 μl EDTA-free trypsin to digest the cells, add 1 ml fresh culture medium to terminate digestion, until the cells are separated from the wall, move the cell suspension into a 10 ml centrifuge tube, centrifuge and discard Remove the supernatant, resuspend in PBS, and measure by flow cytometry. It is found that the uptake of DOX and CeMOF-CaO ₂ /DOX by MCF-7 human breast cancer cells at 0.5h is: 47.1% and 24.1% respectively; at 1h The intake amounts were: 83.8% and 54.4% respectively; the intake amounts at 2 hours were: 99.0% and 98.4% respectively. The results show that the water-soluble small molecule API DOX is uptaken by cells the fastest, and the uptake by cells is almost saturated at 1 hour; while the CeMOF-CaO ₂ /DOX nanopreparation has a larger molecular weight, and the cell uptake is slower, with MCF-7 at 2 hours. The uptake of DOX and CeMOF-CaO ₂ /DOX by human breast cancer cells is basically the same.

4. Cell proliferation inhibition experiment of calcium peroxide-loaded metal-organic framework pharmaceutical compositions

Using the MTT method, MCF-7 human breast cancer cells in the logarithmic growth phase were selected, and the number of cells was adjusted to 5×10 ⁴ /ml and inoculated in a 96-well culture plate. The cells were grown adherently for 24 hours before adding the drug. They were divided into blank group and CeMOF group in sequence. group, CaO ₂ group, DOX group, CeMOF-CaO ₂ group, and CeMOF-CaO ₂ /DOX group, in which the final concentration of the drug was set to 70 μg/ml; the cells added with the drug were incubated in an environment of 37°C and 5% CO ₂ After 24 hours, add 25 μl of freshly prepared and filtered 5 mg/mL MTT to each well, place it in an incubator and incubate in the dark for 4 hours, then take it out, suck out all the liquid in the well and shake it dry, add 150 μl of DMSO to each well, and place it on a shaker. Shake, shake slightly at 100 rpm for 10 minutes at 37°C, measure the OD value of each well at a wavelength of 490 nm on a microplate reader, and calculate the tumor cell growth inhibition rate (%) = (1-OD value of the experimental group/OD value of the control group) _{) _ _} %. The results show that the CeMOF, CaO ₂ and DOX groups have a certain degree of toxicity to cells, but loading DOX-modified CaO ₂ in CeMOF increases the anti-tumor effect. This experiment shows that the CeMOF-CaO ₂ /DOX group exerts a great anti-tumor effect The effect is the result of synergistic treatment of cascade catalysis, ion interference, and chemotherapy.

5. Pharmacodynamic research experiments on calcium peroxide-loaded metal-organic framework pharmaceutical compositions

Purchase 4-6 week old female nude mice, inoculate MCF-7 human breast cancer cells subcutaneously on the back of the right upper limb of the mouse, measure the tumor volume 7 days later, and select mice with tumor volume ^≥100mm3 and similar tumor volume and body weight. They were randomly divided into 6 groups, with 6 mice in each group. The specific groups are as follows: normal saline group, CeMOF group, CaO ₂ group, DOX group, CeMOF-CaO ₂ group, and CeMOF-CaO ₂ /DOX group; the mice in each group The administration method was tail vein injection, once every two days, for a total of 7 administrations. During the entire experiment, the mice were ensured to eat a normal diet every day. The weight of each mouse was weighed every two days and measured with a digital vernier caliper. The long diameter (A) and short diameter (B) of sarcoma in tumor-bearing mice, and the tumor volume is calculated according to the formula tumor volume V = A × B ² /2. The recorded data showed that the tumor inhibition rates of the CeMOF group, CaO ₂ group, DOX group, CeMOF-CaO ₂ group, and CeMOF-CaO ₂ /DOX group were 25.32%, 26.71%, 24.18%, 71.32%, and 85.91% respectively. The results showed that the CeMOF-CaO ₂ /DOX group had significant efficacy and could greatly inhibit tumor growth. This experiment demonstrated the synergistic therapeutic effect of this tumor-targeted drug delivery system based on the combination of cascade catalysis, ion interference and chemotherapy.

According to the above method, experiments were conducted on Examples 2-3 of the present invention, and the same or similar results were obtained, so they will not be listed one by one here.

From the above, it can be clearly seen that the method of the present invention is stable and reliable, the product quality is good, and the drug loading capacity is as high as 35%. Compared with the existing technology, it has the following outstanding beneficial technical effects:

1. The calcium peroxide-loaded metal organic framework pharmaceutical composition provided by the present invention is carbonized by high-temperature calcination, which improves the activity of CeMOF. The product has good quality, the drug loading capacity is as high as about 35%, and the particle size is small, only 100- 200nm (nano), which facilitates drug absorption and improves utilization and efficacy;

2. For the calcium peroxide-loaded metal-organic framework pharmaceutical composition provided by the present invention, the selection of the metal-organic framework is conducive to fixing CaO ₂ , maintaining the activity of CaO ₂ , and improving the Fenton-like reaction with H ₂ O ₂ produced by CaO ₂ The content of reactive oxygen species, while the calcium ions generated by calcium peroxide perform ion interference therapy;

3. The calcium peroxide-loaded metal-organic framework pharmaceutical composition provided by the present invention combines the Fenton-like catalytic oxidation effect of CeMOF, CaO ₂ ion interference therapy, and chemotherapy to carry out triple-strike interactive comprehensive treatment against tumors and enhance anti-tumor resistance. The tumor treatment effect is an innovation in tumor treatment drugs, with huge economic and social benefits.

Claims (7)

1. A preparation method of a calcium peroxide-loaded metal-organic framework pharmaceutical composition is characterized in that the metal-organic framework is a cerium-based synthesized organic framework, the cerium-based metal-organic framework with the particle size of 80-180nm and the pore diameter of 6-8nm is carbonized under high-temperature calcination, and then a small molecular chemotherapeutic drug is connected to CaO through a chemical bond ₂ Surface, finally, caO loaded with medicine ₂ The small particles are adhered to the holes of the cerium-based metal organic framework through physical adsorption, so that the metal organic framework pharmaceutical composition with the particle size of 100-200nm and loaded with calcium peroxide is obtained, and the drug loading weight is 20% -60%, and the specific steps are as follows:

(1) Dissolving 450-470mg of ceric ammonium nitrate in 2-4mL of ultrapure water to form ceric ammonium nitrate aqueous solution, dissolving 130-150mg of terephthalic acid in 2-4mL of N, N-dimethylformamide, mixing and sealing the N, N-dimethylformamide solution of terephthalic acid and the ceric ammonium nitrate aqueous solution, stirring for 20-40min in an oil bath at 95-115 ℃, centrifuging for 10min at 8000-12000rpm, taking precipitate, washing the precipitate with N, N-dimethylformamide for 3 times, wherein each time is 2-3 times of the volume of the precipitate by weight, washing with acetone for 3 times, each time is 2-3 times of the volume of the precipitate by weight, wherein the volume by weight is calculated as mL of liquid, the solid is calculated as g, and grinding into white powder which passes through a 100-120 mesh sieve after vacuum drying to obtain a cerium-based metal organic framework (CeMOF); calcining the prepared cerium-based metal organic framework (CeMOF) in a muffle furnace, heating to 350-400 ℃ at 5-10 ℃/min, calcining for 3-5h, and cooling to below 20 ℃ to obtain a carbonized cerium-based metal organic framework (CeMOF);

(2) Dissolving 0.4-0.6mg of calcium chloride particles in 4-6mL of ultrapure water, stirring at 2000-2400rpm to dissolve completely, adding 2-3mL of 0.5-1.5M ammonia water under stirring at room temperature, then adding 30-50mL of polyethylene glycol 200 (PEG 200), continuing stirring to mix completely into a uniform solution, adding 2-3mL of hydrogen peroxide with the mass concentration of 30%, continuing stirring for 5-7h, then gradually adjusting the pH to 11.5 with sodium hydroxide solution, centrifuging at 8000-12000rpm for 10min, discarding the supernatant to obtain white precipitate, washing the white precipitate with absolute ethyl alcohol for 3 times, wherein the dosage of each time is 2-3 times of the weight volume of the white precipitate, and drying in vacuum to obtain calcium dioxide;

(3) Dispersing 1-3mg of calcium dioxide and 1mg of small molecule chemotherapeutic drugs in a mixed solution with the equal volume ratio of 3-5mL of ethanol and water, stirring at room temperature at 2000-2400rpm in the dark for 48h, centrifuging at 8000-12000rpm for 10min, discarding supernatant to obtain a precipitate, washing the precipitate with the mixed solution with the equal volume ratio of ethanol-water for three times, wherein the dosage is 2-3 times of the weight volume of the white precipitate, and obtaining the calcium peroxide loaded with the small molecule drugs;

the small molecule chemotherapeutic medicine is doxorubicin, paclitaxel, docetaxel, hydroxycamptothecin or mitoxantrone;

(4) Dissolving 5-10mg of the calcined carbonized cerium-based metal organic framework (CeMOF) prepared in the step (1) in 5-10mL of a solvent, uniformly dispersing by utilizing ultrasonic, and dissolving 3-5mg of the calcium peroxide loaded with the micromolecule drug prepared in the step (3) in 3-5mL of the solvent; adding the two solutions into a round bottom flask together, stirring at 2000-2400rpm at dark room temperature for reaction for 20-40min, centrifuging at 8000-12000rpm for 10min, discarding supernatant to obtain precipitate, washing the precipitate with PBS with pH of 7.2-7.4 for 3 times, each time with 2-3 times of the weight volume of the precipitate, vacuum drying, and grinding into 100-120 mesh powder to obtain metal organic frame pharmaceutical composition loaded with calcium peroxide;

the solvent is one of PBS or MES with the pH of 7.2-7.4 and a mixed solution of ethanol and water in an equal volume ratio.

2. The method for preparing a calcium peroxide-loaded metal-organic framework pharmaceutical composition according to claim 1, comprising the steps of:

(1) Dissolving 460mg of ceric ammonium nitrate in 3mL of ultrapure water to form ceric ammonium nitrate aqueous solution, dissolving 140mg of terephthalic acid in 3mL of N, N-dimethylformamide at the same time, mixing and sealing the N, N-dimethylformamide solution of terephthalic acid and the ceric ammonium nitrate aqueous solution, stirring for 30min at 100 ℃ in an oil bath, centrifuging for 10min at 10000rpm, taking precipitate, washing the precipitate with N, N-dimethylformamide for 3 times, wherein the amount of the precipitate is 2-3 times of the weight volume of the precipitate, washing the precipitate with acetone for 3 times, wherein the amount of the precipitate is 2-3 times of the weight volume of the precipitate, and grinding the precipitate into white powder which passes through a 110-mesh sieve after vacuum drying to obtain a cerium-based metal organic frame (CeMOF); calcining the prepared cerium-based metal organic framework (CeMOF) in a muffle furnace, heating to 370 ℃ at 7 ℃/min, calcining for 4 hours, and cooling to below 20 ℃ to obtain a carbonized cerium-based metal organic framework (CeMOF);

(2) Dissolving 0.5mg of calcium chloride particles in 5mL of ultrapure water, stirring at 2200rpm to dissolve completely, adding 2.5mL of 1M ammonia water under stirring at room temperature, then adding 40mL of polyethylene glycol 200 (PEG 200), continuing stirring to mix completely to form a uniform solution, adding 2.5mL of hydrogen peroxide with the mass concentration of 30%, continuing stirring for 6 hours, then gradually adjusting the pH to 11.5 by using a sodium hydroxide solution, centrifuging at 10000rpm for 10 minutes, discarding the supernatant to obtain white precipitate, washing the white precipitate with absolute ethyl alcohol for 3 times, wherein the dosage of each time is 2-3 times the weight volume of the white precipitate, and drying in vacuum to obtain calcium dioxide;

(3) Dispersing 2mg of calcium dioxide and 1mg of doxorubicin in 4mL of mixed solution with equal volume ratio of ethanol and water, stirring at room temperature at 2200rpm in the dark for 48h, centrifuging at 10000rpm for 10min, discarding supernatant to obtain precipitate, washing the precipitate with the mixed solution of ethanol and water with equal volume ratio for three times, wherein the dosage is 2-3 times of the weight volume of the white precipitate, and obtaining doxorubicin-loaded calcium peroxide;

(4) Dissolving 7.5mg of the calcined carbonized cerium-based metal organic framework (CeMOF) prepared in the step (1) in 7.5mL of an ethanol-water mixed solution with an equal volume ratio, uniformly dispersing by utilizing ultrasonic, and dissolving 4mg of the doxorubicin-loaded calcium peroxide prepared in the step (3) in 4mL of the ethanol-water mixed solution with an equal volume ratio; adding the two solutions into a round bottom flask together, stirring and reacting for 30min at room temperature under dark condition at 2200rpm, centrifuging at 10000rpm for 10min, discarding supernatant to obtain precipitate, washing the precipitate with PBS with pH of 7.3 for 3 times, each time with 2-3 times of the weight volume of the precipitate, vacuum drying, and grinding into powder with 110 mesh sieve to obtain the metal organic frame pharmaceutical composition loaded with calcium peroxide.

3. The method for preparing a calcium peroxide-loaded metal-organic framework pharmaceutical composition according to claim 1, comprising the steps of:

(1) Dissolving 452mg of ceric ammonium nitrate in 2mL of ultrapure water to form ceric ammonium nitrate aqueous solution, dissolving 132mg of terephthalic acid in 2mL of N, N-dimethylformamide at the same time, mixing and sealing the N, N-dimethylformamide solution of terephthalic acid and the ceric ammonium nitrate aqueous solution, stirring for 38min at an oil bath of 97 ℃, centrifuging for 10min at 9000rpm, taking precipitate, washing the precipitate with N, N-dimethylformamide for 3 times, wherein the amount of the precipitate is 2-3 times of the weight volume of the precipitate, washing the precipitate with acetone for 3 times, wherein the amount of the precipitate is 2-3 times of the weight volume of the precipitate, and grinding the precipitate into white powder which passes through a 100-mesh sieve after vacuum drying to obtain a cerium-based metal organic framework (CeMOF); calcining the prepared cerium-based metal organic framework (CeMOF) in a muffle furnace, heating to 355 ℃ at a speed of 6 ℃/min, calcining for 4.5 hours, and cooling to below 20 ℃ to obtain a carbonized cerium-based metal organic framework (CeMOF);

(2) Dissolving 0.4mg of calcium chloride particles in 4mL of ultrapure water, stirring at 2000rpm to dissolve completely, adding 2mL of 1.5M ammonia water under stirring at room temperature, then adding 30mL of polyethylene glycol 200 (PEG 200), continuing stirring to mix completely to form a uniform solution, adding 2mL of hydrogen peroxide with the mass concentration of 30%, continuing stirring for 5 hours, then gradually adjusting the pH to 11.5 by using a sodium hydroxide solution, centrifuging at 9000rpm for 10 minutes, discarding the supernatant to obtain white precipitate, washing the white precipitate with absolute ethyl alcohol for 3 times, wherein the dosage of each time is 2-3 times the weight volume of the white precipitate, and drying in vacuum to obtain calcium dioxide;

(3) Dispersing 1mg of calcium dioxide and 1mg of taxol in 3mL of mixed solution with equal volume ratio of ethanol and water, stirring at 2000rpm at room temperature for 48 hours in a dark place, centrifuging at 9000rpm for 10min, discarding supernatant to obtain precipitate, washing the precipitate with the mixed solution of ethanol and water with equal volume ratio for three times, wherein the dosage of the mixed solution is 2-3 times of the weight volume of the white precipitate to obtain taxol-loaded calcium peroxide;

(4) Dissolving 6mg of the calcined carbonized cerium-based metal organic framework (CeMOF) prepared in the step (1) in 6mL of PBS with the pH of 7.2-7.4, uniformly dispersing by utilizing ultrasonic, and dissolving 3mg of the taxol-loaded calcium peroxide prepared in the step (3) in 3mL of PBS with the pH of 7.2-7.4; adding the two solutions into a round bottom flask together, stirring and reacting for 38min at 2000rpm at room temperature in the absence of light, centrifuging for 10min at 9000rpm, discarding supernatant to obtain precipitate, washing the precipitate with PBS with pH of 7.2 for 3 times, each time with 2-3 times of the weight volume of the precipitate, vacuum drying, and grinding into powder passing through a 100-mesh sieve to obtain the metal organic frame pharmaceutical composition loaded with calcium peroxide.

4. The method for preparing a calcium peroxide-loaded metal-organic framework pharmaceutical composition according to claim 1, comprising the steps of:

(1) Dissolving 468mg of ceric ammonium nitrate in 4mL of ultrapure water to form ceric ammonium nitrate aqueous solution, dissolving 148mg of terephthalic acid in 4mL of N, N-dimethylformamide at the same time, mixing and sealing the N, N-dimethylformamide solution of terephthalic acid and the ceric ammonium nitrate aqueous solution, stirring for 22min at 115 ℃ under an oil bath, centrifuging for 10min at 11000rpm, taking precipitate, washing the precipitate with N, N-dimethylformamide for 3 times, wherein the amount of the precipitate is 2-3 times of the weight volume of the precipitate, washing the precipitate with acetone for 3 times, wherein the amount of the precipitate is 2-3 times of the weight volume of the precipitate, and grinding the precipitate into white powder which passes through a 120-mesh sieve after vacuum drying to obtain a cerium-based metal organic framework (CeMOF); calcining the prepared cerium-based metal organic framework (CeMOF) in a muffle furnace, heating to 390 ℃ at 9 ℃/min, calcining for 3 hours, and cooling to below 20 ℃ to obtain a carbonized cerium-based metal organic framework (CeMOF);

(2) Dissolving 0.6mg of calcium chloride particles in 6mL of ultrapure water, stirring at 2400rpm to dissolve completely, adding 3mL of 0.5M ammonia water under stirring at room temperature, then adding 50mL of polyethylene glycol 200 (PEG 200), continuously stirring to mix completely into a uniform solution, then adding 3mL of hydrogen peroxide with the mass concentration of 30%, continuously stirring for 7h, then gradually adjusting the pH to 11.5 by using a sodium hydroxide solution, centrifuging at 11000rpm for 10min, discarding the supernatant to obtain white precipitate, washing the white precipitate with absolute ethyl alcohol for 3 times, wherein the dosage of each time is 2-3 times the weight volume of the white precipitate, and drying in vacuum to obtain calcium dioxide;

(3) Dispersing 3mg of calcium dioxide and 1mg of docetaxel in 5mL of mixed solution with equal volume ratio of ethanol and water, stirring at 2400rpm at room temperature for 48 hours in a dark place, centrifuging at 11000rpm for 10min, discarding supernatant to obtain precipitate, washing the precipitate with the mixed solution of ethanol and water with equal volume ratio for three times, wherein the dosage of the mixed solution is 2-3 times of the weight volume of the white precipitate to obtain calcium peroxide loaded with docetaxel;

(4) Dissolving 10mg of the calcined carbonized cerium-based metal organic framework (CeMOF) prepared in the step (1) in 10mL of MES with the pH of 7.2-7.4, uniformly dispersing by utilizing ultrasonic, and dissolving 5mg of calcium peroxide of the loaded docetaxel prepared in the step (3) in 5mL of MES with the pH of 7.2-7.4; adding the two solutions into a round bottom flask together, stirring at 2400rpm at dark room temperature for reaction for 22min, centrifuging at 11000rpm for 10min, discarding supernatant to obtain precipitate, washing the precipitate with PBS with pH of 7.4 for 3 times, each time 2-3 times of the weight volume of the precipitate, vacuum drying, and grinding into powder passing through 120 mesh sieve to obtain the metal organic frame pharmaceutical composition loaded with calcium peroxide.

5. Use of a calcium peroxide-loaded metal organic framework pharmaceutical composition prepared by the method of any one of claims 1-4 for the preparation of an antitumor drug in combination with a pharmaceutically or pharmacologically active molecule, said pharmaceutically or pharmacologically active molecule being doxorubicin, paclitaxel, docetaxel, hydroxycamptothecin or mitoxantrone.

6. Use of a calcium peroxide-loaded metal organic framework pharmaceutical composition prepared by the method of any one of claims 1-4 in the preparation of an anti-tumor injection, oral or implant.

7. Use of a calcium peroxide-loaded metal-organic framework pharmaceutical composition prepared by the method of any one of claims 1-4 in the preparation of a drug for the triple impact interactive comprehensive treatment of tumors like Fenton catalytic oxidation effect, ion interference therapy and chemotherapy.