Nanometer magnetic targeted sustained-release delivery system and preparation method and application thereof, nanometer magnetic targeted sustained-release pharmaceutical preparation and preparation method and application thereof, and pharmaceutical composition

Abstract

The invention provides a nano magnetic targeted sustained-release delivery system, a preparation method and application thereof, a nano magnetic targeted sustained-release pharmaceutical preparation, a preparation method and application thereof, and a pharmaceutical composition, and relates to the technical field of medicines. The invention provides a nano magnetic targeted slow-release delivery system. The nano magnetic targeted slow-release delivery system comprises magnetic ferroferric oxide-hydroxyapatite and polysuccinimide, wherein the surface of the ferroferric oxide-hydroxyapatite is coated with the polysuccinimide. The nano magnetic targeted slow-release delivery system provided by the invention can quickly and intensively deliver therapeutic drugs, especially anti-cancer drugs, to diseased regions, so that damage to other tissues is avoided, and the dosage and toxic and side effects of the drugs are reduced; due to the guiding effect of magnetism, the drug-containing nano preparation can be detained in pathological tissues for a long time to form drug accumulation, and the drug-containing nano preparation can easily penetrate through tumor tissues to intensively input the drug into the tumor tissues to achieve the effect of sustained release, so that the drug-containing nano preparation has the treatment effects of enhancing efficacy and reducing toxicity, and the bioavailability of the drug is improved.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and in particular to a nanomagnetic targeted sustained-release delivery system and a preparation method and application thereof, a nanomagnetic targeted sustained-release pharmaceutical preparation and a preparation method and application thereof, and a pharmaceutical composition. Background Art

[0002] At present, the incidence and mortality of malignant tumors continue to rise, and cancer remains a major public health problem in China. With the continuous advancement of science and technology, a variety of new and efficient anti-cancer drugs have emerged, including small molecule drugs, protein and peptide drugs, nucleic acid gene drugs, etc. However, how to efficiently deliver these drugs to tumor cells to exert their efficacy is a problem that reduces the occurrence of systemic toxic and side effects of current anti-tumor drugs and solves the problems faced by the clinical application of protein, peptide and nucleic acid biological drugs.

[0003] Nanocarrier-based drug delivery systems (NDDSs) are drug delivery systems composed of nanomaterials with a diameter of less than 1000nm. Due to the specific spatial size of NDDSs, this drug delivery system has the characteristics of surface effect and small size effect. For example, typical nanomaterials have the following characteristics: high surface-to-volume ratio, strong conductivity, superparamagnetic behavior, spectral shift and unique fluorescence characteristics, etc. Among them, the high surface-to-volume ratio of nanomaterials determines that it can be assembled with biomolecules or protein groups, thereby enhancing the targeting of nanodrug delivery systems in tumor treatment, ultimately improving the overall tumor treatment effect and reducing its toxicity to normal cells. The targeting effect of NDDSs can be divided into active targeting and passive targeting. Passive targeting refers to the high permeability and retention effect (Enhanced permeability and retention effect, EPR effect) of tumor tissue to macromolecular substances (such as liposomes, nanoparticles and some macromolecular drugs), which makes NDDSs easier to penetrate into tumor tissue. Active targeting refers to modifying the corresponding ligands, antibodies, lectins, etc. on the surface of NDDSs to target some overexpressed receptors, antigens, and glycoproteins on tumor cells, thereby achieving the effect of actively targeting tumor cells. However, the problem with existing NDDSs is that due to the high interstitial fluid pressure in the tumor microenvironment, both passively targeted and actively targeted NDDSs systems limit their accumulation around tumor tissues. Summary of the invention

[0004] In view of this, the object of the present invention is to provide a nanomagnetic targeted sustained-release delivery system and its preparation method and application, a nanomagnetic targeted sustained-release drug preparation and its preparation method and application, and a pharmaceutical composition. The nanomagnetic targeted sustained-release delivery system provided by the present invention has strong targeting, can accumulate around tumor tissue, and improve the anti-tumor therapeutic effect of the drug.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The invention provides a nano magnetic targeted sustained-release delivery system (PSI-Fe3O4-HAP), comprising magnetic ferroferric oxide-hydroxyapatite (Fe3O4-HAP) and polysuccinimide (PSI) coated on the surface of the ferroferric oxide-hydroxyapatite.

[0007] Preferably, the mass content of ferroferric oxide in the magnetic ferroferric oxide-hydroxyapatite is 5 to 80%;

[0008] The mass content of polysuccinimide in the nano-magnetic targeted sustained-release delivery system is 10-90%.

[0009] The present invention also provides a method for preparing the nano-magnetic targeted sustained-release delivery system described in the above technical solution, comprising the following steps: mixing magnetic ferroferric oxide-hydroxyapatite, polysuccinimide and a solvent, and coating to obtain the nano-magnetic targeted sustained-release delivery system.

[0010] Preferably, the solvent includes one or more of water, methanol, ethanol, chloroform, dimethylformamide, dimethylacetamide and dimethyl sulfoxide.

[0011] Preferably, the preparation method of magnetic ferroferric oxide-hydroxyapatite comprises the following steps: mixing CaCO3, magnetic nano-Fe3O4, Na2HPO4 and water, adjusting the pH value to 7-12 with alkali metal hydroxide, and performing double decomposition reaction to obtain magnetic ferroferric oxide-hydroxyapatite.

[0012] The present invention also provides a nanomagnetic targeted sustained-release drug preparation, comprising a nanomagnetic targeted sustained-release delivery system and a drug loaded on the nanomagnetic targeted sustained-release delivery system; the nanomagnetic targeted sustained-release delivery system is the nanomagnetic targeted sustained-release delivery system described in the above technical scheme or the nanomagnetic targeted sustained-release delivery system prepared by the preparation method described in the above technical scheme.

[0013] Preferably, the drug comprises an anti-tumor drug;

[0014] The mass content of the drug in the nano-magnetic targeted sustained-release drug preparation is 0.01-99%.

[0015] The present invention also provides a method for preparing the nano-magnetic targeted sustained-release drug preparation described in the above technical solution, comprising the following steps: mixing the drug, magnetic ferroferric oxide-hydroxyapatite and a solvent for adsorption, and then adding polysuccinimide for coating to obtain the nano-magnetic targeted sustained-release drug preparation.

[0016] The present invention also provides the nanomagnetic targeted sustained-release delivery system described in the above technical scheme, the nanomagnetic targeted sustained-release delivery system prepared by the preparation method described in the above technical scheme, the nanomagnetic targeted sustained-release drug preparation described in the above technical scheme, or the use of the nanomagnetic targeted sustained-release drug preparation prepared by the preparation method described in the above technical scheme in preparing drugs.

[0017] The present invention also provides a pharmaceutical composition, comprising an active component and a pharmaceutically acceptable excipient; the active component comprises the nanomagnetic targeted sustained-release pharmaceutical preparation described in the above technical solution or the nanomagnetic targeted sustained-release pharmaceutical preparation prepared by the preparation method described in the above technical solution.

[0018] The present invention provides a nano-magnetic targeted sustained-release delivery system, comprising magnetic ferroferric oxide-hydroxyapatite and polysuccinimide coated on the surface of the ferroferric oxide-hydroxyapatite. After the nano-magnetic targeted sustained-release delivery system provided by the present invention enters the body, a magnetic field is applied to a specific part in vitro, and under the action of magnetic nano-Fe3O4, the drug (for example, an anti-tumor drug) is quickly guided to be transported to the lesion site to exert its efficacy, thereby achieving the effect of magnetic targeting, thereby avoiding damage to other tissues, reducing the dosage and toxic and side effects of the drug; the guiding effect of magnetic Fe3O4 can also make the nano-magnetic targeted sustained-release delivery system (i.e., nano-magnetic targeted sustained-release drug preparation) with added drugs stay in the lesion tissue for a long time, forming drug accumulation. The nano-magnetic targeted sustained-release delivery system provided by the present invention has the advantage of being easy to penetrate tumor tissue, further concentrates the drug into the tumor tissue, achieves the effect of continuous release (slow release), and further achieves the therapeutic effect of increasing efficacy and reducing toxicity. It can simultaneously solve the two problems of toxic and side effects caused by unreasonable distribution of drugs in the body and the difficulty of nano-systems to accumulate around tumor tissues, thereby improving the bioavailability of drugs. Under the same dosage, the concentration and retention time of the nanomagnetic targeted sustained-release delivery system provided by the present invention in the target area are significantly improved, and the toxicity of the drug to non-target organs is reduced, thereby greatly improving the efficacy and safety of the drug. The system can be used for the in vivo delivery of small molecule drugs, proteins, peptides, nucleic acids and other gene anti-tumor drugs, has strong versatility, and is suitable for multi-pathway administration in vivo, and can solve the bottleneck problem that protein drugs, peptide drugs and nucleic acid drugs are difficult to use clinically due to their poor stability and easy degradation in vivo.

[0019] The raw materials for preparing the nanomagnetic targeted sustained-release delivery system provided by the present invention are PSI (soft film) and Fe3O4-HAP (hard core). The raw materials have low cost and are biodegradable in vivo, and have high raw material safety. The two can be rapidly and spontaneously formed into a non-aggregating, uniform-size nanomagnetic targeted sustained-release delivery system with a "hard core and soft film" structure by simply mixing with a drug in a solution. The preparation process is simple and suitable for industrial production. The preparation process does not require expensive materials, production equipment and harsh reaction conditions, almost no impurities are generated, and the sterilization operation can be completed during production. The production process has a certain ambiguity (that is, the feed amount does not affect the quality of the nanomagnetic targeted sustained-release delivery system within a certain error range), and has great production convertibility, which solves the problem that the current nano delivery system is difficult to convert into production. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 PSI-HAP prepared in Comparative Example 1 and PSI-Fe3O4-HAP prepared in Example 1 20 , PSI-Fe3O4-HAP prepared in Example 2 30 and PSI-Fe3O4-HAP prepared in Example 3 40 SEM images of

[0021] Figure 2 5-FU-PSI-HAP prepared in Comparative Example 1 and 5-FU-PSI-Fe3O4-HAP prepared in Example 1 20 5-FU-PSI-Fe3O4-HAP prepared in Example 2 30 and 5-FU-PSI-Fe3O4-HAP prepared in Example 3 40 SEM images of

[0022] Figure 3 5-FU-PSI-HAP (a) prepared in Comparative Example 1 and 5-FU-PSI-Fe3O4-HAP prepared in Example 1 20 (b) 5-FU-PSI-Fe3O4-HAP prepared in Example 2 30 (c) 5-FU-PSI-Fe3O4-HAP prepared in Example 3 40 (d) Particle size distribution diagram;

[0023] Figure 4 5-FU-PSI-HAP (a) prepared in Comparative Example 1 and 5-FU-PSI-Fe3O4-HAP prepared in Example 1 20 (b) 5-FU-PSI-Fe3O4-HAP prepared in Example 2 30 (c) 5-FU-PSI-Fe3O4-HAP prepared in Example 340 (d) Zeta potential test spectrum;

[0024] Figure 5 5-FU-PSI-HAP prepared in Comparative Example 1 and 5-FU-PSI-Fe3O4-HAP prepared in Example 1 20 5-FU-PSI-Fe3O4-HAP prepared in Example 2 30 and 5-FU-PSI-Fe3O4-HAP prepared in Example 3 40 Distribution map of tissues (heart) in animals;

[0025] Figure 6 5-FU-PSI-HAP prepared in Comparative Example 1 and 5-FU-PSI-Fe3O4-HAP prepared in Example 1 20 5-FU-PSI-Fe3O4-HAP prepared in Example 2 30 and 5-FU-PSI-Fe3O4-HAP prepared in Example 3 40 Distribution map of tissues (liver) in animals;

[0026] Figure 7 5-FU-PSI-HAP prepared in Comparative Example 1 and 5-FU-PSI-Fe3O4-HAP prepared in Example 1 20 5-FU-PSI-Fe3O4-HAP prepared in Example 2 30 and 5-FU-PSI-Fe3O4-HAP prepared in Example 3 40 Distribution map of tissues (spleen) in animals;

[0027] Figure 8 5-FU-PSI-HAP prepared in Comparative Example 1 and 5-FU-PSI-Fe3O4-HAP prepared in Example 1 20 5-FU-PSI-Fe3O4-HAP prepared in Example 2 30 and 5-FU-PSI-Fe3O4-HAP prepared in Example 3 40 Distribution map of tissues (lungs) in animals;

[0028] Fig. 9 5-FU-PSI-HAP prepared in Comparative Example 1 and 5-FU-PSI-Fe3O4-HAP prepared in Example 1 20 5-FU-PSI-Fe3O4-HAP prepared in Example 2 30 and 5-FU-PSI-Fe3O4-HAP prepared in Example 3 40 Distribution map of tissues (kidney) in animals;

[0029] Fig.10 5-FU, 5-FU-PSI-HAP prepared in Comparative Example 1, 5-FU-PSI-Fe3O4-HAP prepared in Example 1 20 5-FU-PSI-Fe3O4-HAP prepared in Example 2 30 and 5-FU-PSI-Fe3O4-HAP prepared in Example 3 40 The figure shows the inhibition of Huh-7 cell proliferation;

[0030] Fig.11 5-FU, 5-FU-PSI-HAP prepared in Comparative Example 1, 5-FU-PSI-Fe3O4-HAP prepared in Example 1 20 5-FU-PSI-Fe3O4-HAP prepared in Example 2 30 and 5-FU-PSI-Fe3O4-HAP prepared in Example 3 40 24h IC of Huh-7 cells 50 Value result graph;

[0031] Fig.12 The 5-FU-PSI-HAP prepared in Comparative Example 1 and the 5-FU-PSI-Fe3O4-HAP prepared in Example 1 were used for Huh-7 cells. 20 5-FU-PSI-Fe3O4-HAP prepared in Example 2 30 and 5-FU-PSI-Fe3O4-HAP prepared in Example 3 40 Uptake diagram. DETAILED DESCRIPTION

[0032] The invention provides a nanometer magnetic targeted sustained-release delivery system, comprising magnetic ferroferric oxide-hydroxyapatite and polysuccinimide coated on the surface of the ferroferric oxide-hydroxyapatite.

[0033] In the present invention, the mass content of ferroferric oxide in the magnetic ferroferric oxide-hydroxyapatite is preferably 5-80%, and in specific embodiments can be 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or 80%. In the present invention, the Fe3O4 in the magnetic ferroferric oxide-hydroxyapatite is fixed in the hydroxyapatite in a nanostructure.

[0034] In the present invention, the mass content of polysuccinimide in the nanomagnetic targeted sustained-release delivery system is 10-90%, and in specific embodiments it can be 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90%.

[0035] After the nano magnetic targeted sustained-release delivery system provided by the present invention enters the body, a magnetic field is applied to a specific part in vitro, and under the action of magnetic Fe3O4, the drug (for example, an anti-tumor drug) is quickly guided to be transported to the lesion site to exert its efficacy, thereby achieving the effect of magnetic targeting, thereby avoiding damage to other tissues, reducing the dosage and toxic and side effects of the drug; the guiding effect of magnetic Fe3O4 can also make the nano magnetic targeted sustained-release delivery system (i.e., nano magnetic targeted sustained-release drug preparation) with added drugs stay in the lesion tissue for a long time, forming drug accumulation. The nano magnetic targeted sustained-release delivery system provided by the present invention has the advantage of being easy to penetrate tumor tissue, further concentrates the drug into the tumor tissue, achieves the effect of continuous release (slow release), and further achieves the therapeutic effect of increasing efficacy and reducing toxicity. It can simultaneously solve the two problems of toxic and side effects caused by unreasonable distribution of drugs in the body and the difficulty of nano systems to accumulate around tumor tissues, thereby improving the bioavailability of drugs. Under the same dosage, the concentration and retention time of the nanomagnetic targeted sustained-release delivery system provided by the present invention in the target area are significantly improved, and the toxicity of the drug to non-target organs is reduced, thereby greatly improving the efficacy and safety of the drug. The system can be used for the in vivo delivery of small molecule drugs, proteins, peptides, nucleic acids and other gene anti-tumor drugs, has strong versatility, and is suitable for multi-pathway administration in vivo, and can solve the bottleneck problem that protein drugs, peptide drugs and nucleic acid drugs are difficult to use clinically due to their poor stability and easy degradation in vivo.

[0036] The present invention also provides a method for preparing the nano-magnetic targeted sustained-release delivery system described in the above technical solution, comprising the following steps: mixing magnetic ferroferric oxide-hydroxyapatite, polysuccinimide and a solvent, and coating to obtain the nano-magnetic targeted sustained-release delivery system.

[0037] Unless otherwise specified, the materials and equipment used in the present invention are all commercially available products in the art.

[0038] In the present invention, the solvent preferably includes one or more of water, methanol, ethanol, chloroform, dimethylformamide, dimethylacetamide and dimethyl sulfoxide.

[0039] In the present invention, the mixing preferably comprises: adding magnetic ferroferric oxide-hydroxyapatite to an organic solvent for premixing to obtain a magnetic ferroferric oxide-hydroxyapatite suspension; and dropwise adding a polysuccinimide solution into the magnetic ferroferric oxide-hydroxyapatite suspension.

[0040] In the present invention, the premixing is preferably carried out under ultrasound and / or stirring conditions, and the premixing temperature is preferably room temperature; the premixing time is preferably 1 to 60 minutes, and in specific embodiments it can be 1 minute, 5 minutes, 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes or 60 minutes; the stirring speed is preferably 100 to 10000 r / min, and in specific embodiments it can be 100 r / min, 500 r / min, 1000 r / min, 2000r / min, 3000r / min, 4000r / min, 5000r / min, 6000r / min, 7000r / min, 8000r / min, 9000r / min or 10000r / min; the power of the ultrasound is preferably 10-5000W, and in specific embodiments it can be 10W, 50W, 100W, 500W, 1000W, 2000W, 3000W, 4000W or 5000W.

[0041] In the present invention, the concentration of the magnetic ferroferric oxide-hydroxyapatite suspension is preferably 0.1-100 mg / mL, and in specific embodiments it can be 0.1 mg / mL, 0.5 mg / mL, 1 mg / mL, 5 mg / mL, 10 mg / mL, 20 mg / mL, 30 mg / mL, 40 mg / mL, 50 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL or 100 mg / mL.

[0042] In the present invention, the concentration of the polysuccinimide solution is preferably 0.1-200 mg / mL, and in specific embodiments can be 0.1 mg / mL, 0.5 mg / mL, 1 mg / mL, 5 mg / mL, 10 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 40 mg / mL, 50 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL, 100 mg / mL, 110 mg / mL, 120 mg / mL, 130 mg / mL, 140 mg / mL, 150 mg / mL, 160 mg / mL, 170 mg / mL, 180 mg / mL, 190 mg / mL or 200 mg / mL; the solvent in the polysuccinimide solution preferably includes one or more of dimethylformamide, dimethylacetamide and dimethyl sulfoxide.

[0043] In the present invention, the dropping is preferably carried out under ultrasound and / or stirring conditions; the dropping temperature is preferably room temperature; the ultrasound power is preferably 10-5000W, and in specific embodiments it can be 10W, 50W, 100W, 500W, 1000W, 2000W, 3000W, 4000W or 5000W; the stirring speed is preferably 100-10000r / min, and in specific embodiments it can be 100r / min, 500r / min, 1000r / min, 2000r / min, 3000r / min, 4000r / min, 5000r / min, 6000r / min, 7000r / min, 8000r / min, 9000r / min or 10000r / min.

[0044] In the present invention, during the addition of the polysuccinimide solution, the polysuccinimide breaks the aggregation state of the magnetic ferroferric oxide-hydroxyapatite and is coated on the surface of the magnetic ferroferric oxide-hydroxyapatite to form a nano-magnetic targeted sustained-release drug delivery system. The coating is completed when the polysuccinimide solution is added dropwise.

[0045] After the coating is completed, the present invention preferably further comprises solid-liquid separation, and the obtained solid component is a nanomagnetic targeted sustained-release delivery system. In the present invention, the solid-liquid separation preferably comprises filtration, suction filtration or centrifugal separation.

[0046] In the present invention, the preparation method of magnetic ferroferric oxide-hydroxyapatite preferably includes the following steps: mixing CaCO3, magnetic nano-Fe3O4, Na2HPO4 and water, adjusting the pH value to 7-12 with alkali metal hydroxide, and performing double decomposition reaction to obtain magnetic ferroferric oxide-hydroxyapatite.

[0047] In the present invention, the mass ratio of the magnetic nano-Fe3O4 to CaCO3 (powder) is preferably 1:0.01-200, and in specific embodiments can be 1:0.01, 1:0.1, 1:0.125, 1:0.15, 1:0.2, 1:0.25, 1:0.3, 1:0.35, 1:0.4, 1:0.45, 1:0.5, 1:0.55, 1:0.6, 1:0.65, 1:0.7, 1:0.75, 1:0.8, 1:0.85, 1:0.9, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:9.5, 1:10, 1:15, 1:19, 1:20, 1:25, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, 1:100, 1:110, 1:120, 1:130, 1:140, 1:150, 1:160, 1:170, 1:180, 1:190, or 1:200.

[0048] In the present invention, the molar ratio of CaCO3 to Na2HPO4 is preferably 1:0.6-6, and in specific embodiments it can be 1:0.6, 1:0.8, 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, 1:5, 1:5.5 or 1:6.

[0049] In the present invention, the mixing of CaCO3, magnetic nano-Fe3O4, Na2HPO4 and water preferably includes: adding CaCO3 to part of the water to obtain a CaCO3 aqueous suspension; adding magnetic nano-Fe3O4 to the CaCO3 suspension to obtain a mixed solution; dissolving Na2HPO4 in water to obtain a Na2HPO4 aqueous solution; and adding the Na2HPO4 aqueous solution dropwise to the mixed solution under stirring.

[0050] In the present invention, the ratio of the mass of CaCO3 to the volume of part of the water is preferably 1g:1-2000mL. In specific embodiments, it can be 1g:1mL, 1g:5mL, 1g:10mL, 1g:50mL, 1g:100mL, 1g:150mL, 1g:200mL, 1g:250mL, 1g:300mL, 1g:400mL, 1g:500mL, 1g:600mL, 1g:700mL, 1g:800mL, 1g:900mL, 1g:1000mL, 1g:1100mL, 1g:1200mL, 1g:1300mL, 1g:1400mL, 1g:1500mL, 1g:1600mL, 1g:1700mL, 1g:1800mL, 1g:1900mL or 1g:2000mL.

[0051] In the present invention, the concentration of the Na2HPO4 aqueous solution is preferably 0.001 to 0.54 mol / L, and in specific embodiments, it can be 0.001 mol / L, 0.01 mol / L, 0.02 mol / L, 0.03 mol / L, 0.04 mol / L, 0.05 mol / L, 0.06 mol / L, 0.07 mol / L, 0.08 mol / L, 0.09 mol / L, 0.1 mol / L, 0.15 mol / L, 0.2 mol / L, 0.25 mol / L, 0.3 mol / L, 0.35 mol / L, 0.4 mol / L, 0.45 mol / L, 0.5 mol / L or 0.54 mol / L.

[0052] In the present invention, the alkali metal hydroxide preferably includes NaOH and / or KOH; the alkali metal hydroxide is preferably used in the form of an alkali metal hydroxide aqueous solution, and the mass concentration of the alkali metal hydroxide aqueous solution is preferably 0.01-52%, and in specific embodiments it can be 0.01%, 0.1%, 0.5%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or 52%.

[0053] In specific embodiments, the pH value can be 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5 or 12.

[0054] In the present invention, the temperature of the metathesis reaction is preferably 0-100°C, and in specific embodiments can be 0°C, 10°C, 20°C, 30°C, 40°C, 50°C, 60°C, 70°C, 80°C, 90°C or 100°C; the time of the metathesis reaction is preferably 2-24h, and in specific embodiments can be 2h, 3h, 4h, 5h, 6h, 7h, 8h, 9h, 10h, 11h, 12h, 13h, 14h, 15h, 16h, 17h, 18h, 19h, 20h, 21h, 22h, 23h or 24h.

[0055] After completing the double decomposition reaction, the present invention preferably further comprises: subjecting the reaction system obtained by the double decomposition reaction to solid-liquid separation, washing the obtained solid component with water until neutral, washing with ethanol, washing with dichloromethane, and drying to obtain magnetic ferroferric oxide-hydroxyapatite. In the present invention, the solid-liquid separation preferably comprises filtration, suction filtration or centrifugal separation. The present invention does not specifically limit the method and conditions of the drying, and drying to constant weight is sufficient; in a specific embodiment, the drying method can be drying, spray drying, boiling drying or fluidized drying; specifically, such as drying in a drying oven at 80°C for 12 hours.

[0056] In the present invention, the CaCO3 is preferably a commercial product or self-made. In the present invention, the preparation method of the CaCO3 preferably comprises the following steps: mixing a surfactant, an alkali metal carbonate, a calcium ion source and a solvent, and performing a precipitation reaction to obtain CaCO3.

[0057] In the present invention, the surfactant preferably includes one or more of a nonionic surfactant, a double-chain ionic surfactant, an anionic surfactant and a cationic surfactant; the nonionic surfactant preferably includes Tween and polyoxyethylene ether surfactants, more preferably includes Tween-80 (Tween-80) and / or TritonX series surfactants; the double-chain ionic surfactant preferably includes sodium dioctyl sulfosuccinate (AOT); the anionic surfactant preferably includes sodium dodecyl sulfonate (SDS) and / or sodium dodecylbenzene sulfonate (DBS); the cationic surfactant preferably includes hexadecyltrimethylammonium bromide (CTAB).

[0058] In the present invention, the solvent preferably includes an organic solvent and water. In the present invention, the organic solvent preferably includes one or more of an ester solvent, a straight-chain hydrocarbon solvent and a cycloalkane solvent; the ester solvent preferably includes butyl acetate; the straight-chain hydrocarbon solvent preferably includes one or more of hexane, heptane and octane; the cycloalkane solvent preferably includes one or more of toluene, cyclohexane and methylcyclohexane.

[0059] In the present invention, the alkali metal carbonate preferably includes Na2CO3 and / or KCO3.

[0060] In the present invention, the calcium ion source preferably includes a water-soluble calcium salt, more preferably includes one or more of CaCl2, CaBr2, CaI2, Ca(NO3)2, Ca(ClO3)2, Ca(ClO4)2 and Ca(HCO3)2. In the present invention, the molar ratio of the alkali metal carbonate to the calcium ion source is preferably 1:0.1-10, and in specific embodiments can be 1:0.1, 1:0.5, 1:0.8, 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, 1:5, 1:5.5, 1:6, 1:6.5, 1:7, 1:7.5, 1:8, 1:8.5, 1:9, 1:9.5 or 1:10.

[0061] In the present invention, the mixing of the surfactant, alkali metal carbonate, calcium ion source and solvent preferably comprises: mixing the surfactant solution and the alkali metal carbonate aqueous solution to obtain an emulsion; and dropwise adding the emulsion into the calcium ion source aqueous solution.

[0062] In the present invention, the solvent in the surfactant solution is preferably the organic solvent. In the present invention, the ratio of the mass of the surfactant to the volume of the solvent in the surfactant solution is preferably 5.5g:1-100mL, and in specific embodiments can be 5.5g:1mL, 5.5g:5mL, 5.5g:10mL, 5.5g:15mL, 5.5g:20mL, 5.5g:25mL, 5.5g:30mL, 5.5g:35mL, 5.5g:40mL, 5.5g:45mL, 5.5g:50mL, 5.5g:55mL, 5.5g:60mL, 5.5g:65mL, 5.5g:70mL, 5.5g:75mL, 5.5g:80mL, 5.5g:85mL, 5.5g:90mL, 5.5g:95mL or 5.5g:100mL.

[0063] In the present invention, the concentration of the alkali metal carbonate aqueous solution is preferably 0.01-2 mol / L, and in specific embodiments it can be 0.01 mol / L, 0.05 mol / L, 0.1 mol / L, 0.2 mol / L, 0.3 mol / L, 0.4 mol / L, 0.5 mol / L, 0.6 mol / L, 0.7 mol / L, 0.8 mol / L, 0.9 mol / L, 1 mol / L, 1.1 mol / L, 1.2 mol / L, 1.3 mol / L, 1.4 mol / L, 1.5 mol / L, 1.6 mol / L, 1.7 mol / L, 1.8 mol / L, 1.9 mol / L or 2 mol / L.

[0064] In the present invention, the concentration of the calcium ion source aqueous solution is preferably 0.01 to 1.5 mol / L, and in specific embodiments it can be 0.01 mol / L, 0.05 mol / L, 0.1 mol / L, 0.2 mol / L, 0.3 mol / L, 0.4 mol / L, 0.5 mol / L, 0.6 mol / L, 0.7 mol / L, 0.8 mol / L, 0.9 mol / L, 1 mol / L, 1.1 mol / L, 1.2 mol / L, 1.3 mol / L, 1.4 mol / L or 1.5 mol / L.

[0065] In the present invention, the surfactant solution and the alkali metal carbonate aqueous solution are preferably mixed by stirring or ultrasonic mixing. The present invention has no special restrictions on the conditions of stirring and ultrasonic mixing, as long as the raw materials can be mixed uniformly.

[0066] In the present invention, the temperature of the precipitation reaction is preferably room temperature. The present invention has no special limitation on the time of the precipitation reaction, and the reaction can be continued until the precipitation no longer increases, specifically 1 hour.

[0067] After the precipitation reaction is completed, the present invention preferably further comprises: solid-liquid separation, washing the obtained solid component with water, washing with anhydrous ethanol and drying in sequence to obtain CaCO3. In the present invention, the solid-liquid separation preferably comprises filtration, suction filtration or centrifugal separation. The present invention does not specifically limit the method and conditions of the drying, and drying to constant weight is sufficient; in a specific embodiment, the drying method can be drying, spray drying, boiling drying or fluidized drying; specifically, drying in a drying oven at 80°C for 12 hours.

[0068] In the present invention, the magnetic nano-Fe3O4 is preferably prepared by coprecipitation method. In the present invention, the preparation method of the magnetic nano-Fe3O4 preferably comprises the following steps: mixing an iron source, a ferrous source, ammonia water and water, and performing an oxidation-reduction reaction to obtain magnetic nano-Fe3O4.

[0069] In the present invention, the iron source preferably includes a water-soluble iron salt, more preferably ferric chloride and / or ferric sulfate. In the present invention, the ferrous source preferably includes a water-soluble ferrous salt, more preferably ferrous chloride and / or ferric sulfate. In the present invention, the molar ratio of the iron ions in the iron source to the ferrous ions in the ferrous source is preferably 0.5 to 5:1, and in specific embodiments can be 0.5:1, 1:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1 or 5:1.

[0070] In the present invention, the mass concentration of the ammonia water is preferably 5-25%, and in specific embodiments, it can be 5%, 10%, 15%, 20% or 25%. In the present invention, the volume ratio of the amount of the ferrous source to the ammonia water is preferably 1mmol:3-10mL, and in specific embodiments, it can be 1mmol:3mL, 1mmol:3.6mL, 1mmol:4mL, 1mmol:5mL, 1mmol:6mL, 1mmol:7mL, 1mmol:8mL, 1mmol:9mL or 1mmol:10mL.

[0071] In the present invention, the mixing of the iron source, the ferrous source, the ammonia and the water preferably comprises: mixing the iron source aqueous solution, the ferrous source aqueous solution and the ammonia. In the present invention, the concentration of the iron source aqueous solution is preferably 0.001 to 5.670 mol / L, and in a specific embodiment can be 0.001 mol / L, 0.005 mol / L, 0.01 mol / L, 0.02 mol / L, 0.03 mol / L, 0.04 mol / L, 0.05 mol / L, 0.1 mol / L, 0.5 mol / L, 1 mol / L, 1.5 mol / L, 2 mol / L, 2.5 mol / L, 3 mol / L, 3.5 mol / L, 4 mol / L, 4.5 mol / L or 5 mol / L. In the present invention, the concentration of the ferrous source aqueous solution is preferably 0.001-7.4 mol / L. In specific embodiments, it can be 0.001 mol / L, 0.005 mol / L, 0.01 mol / L, 0.02 mol / L, 0.03 mol / L, 0.04 mol / L, 0.05 mol / L, 0.1 mol / L, 0.5 mol / L, 1 mol / L, 1.5 mol / L, 2 mol / L, 2.5 mol / L, 3 mol / L, 3.5 mol / L, 4 mol / L, 4.5 mol / L, 5 mol / L, 5.5 mol / L, 6 mol / L, 6.5 mol / L, 7 mol / L or 7.4 mol / L.

[0072] In the present invention, the temperature of the redox reaction is preferably room temperature, the time of the redox reaction is preferably 10 to 120 min, and in specific embodiments it can be 10 min, 20 min, 30 min, 40 min, 50 min, 60 min, 70 min, 80 min, 90 min, 100 min, 110 min or 120 min; the redox reaction is preferably carried out under stirring conditions, and the stirring speed is preferably 100 to 5000 r / min, and in specific embodiments it can be 100 r / min, 500 r / min, 1000 r / min, 1500 r / min, 2000 r / min, 2500 r / min, 3000 r / min, 3500 r / min, 4000 r / min, 4500 r / min or 5000 r / min.

[0073] After the redox reaction is completed, the present invention preferably further comprises: subjecting the reaction solution obtained by the redox reaction to magnetic adsorption, washing the obtained solid component with water and then drying it to obtain magnetic nano-Fe3O4. The present invention has no special restrictions on the drying method and conditions, and drying to constant weight is sufficient; in a specific embodiment, the drying method can be drying, spray drying, boiling drying or fluidized drying; specifically, drying to constant weight in a drying oven at 80°C.

[0074] In the present invention, the method for preparing polysuccinimide preferably comprises the following steps: mixing L-aspartic acid, acid and water, and performing polycondensation reaction to obtain polysuccinimide.

[0075] In the present invention, the ratio of the mass of L-aspartic acid to the volume of water is preferably 10g:0-20mL, and in specific embodiments it can be 10g:0mL, 10g:1mL, 10g:2mL, 10g:3mL, 10g:4mL, 10g:5mL, 10g:6mL, 10g:7mL, 10g:8mL, 10g:9mL, 10g:10mL, 10g:15mL or 10g:20mL.

[0076] In the present invention, the acid preferably includes phosphoric acid, sulfuric acid, p-toluenesulfonic acid or trifluoromethanesulfonic acid; the acid preferably includes an acid aqueous solution, and the mass concentration of the acid aqueous solution is preferably 50-98%, and in specific embodiments, it can be 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98%. In the present invention, the mass ratio of the L-aspartic acid to the volume of the acid aqueous solution is preferably 10g:1-20mL, and in specific embodiments, it can be 10g:1mL, 10g:5mL, 10g:6mL, 10g:10mL, 10g:15mL or 10g:20mL.

[0077] In the present invention, the temperature of the polycondensation reaction is preferably 160-200°C, and in specific embodiments it can be 160°C, 170°C, 180°C, 190°C or 200°C; the time of the polycondensation reaction is preferably 1-6h, and in specific embodiments it can be 1h, 2h, 3h, 4h, 5h or 6h.

[0078] After completing the polycondensation reaction, the present invention preferably further comprises: cooling the reaction solution obtained by the polycondensation reaction to, adding an organic solvent to dissolve the residue to obtain a residue solution, dropping the residue solution into stirring-ultrasonic water, solid-liquid separation, and washing the obtained solid component with water, washing with dichloromethane and drying in sequence to obtain polysuccinimide. In the present invention, the temperature after cooling is preferably 30 to 150°C, and in specific embodiments can be 30°C, 40°C, 50°C, 60°C, 70°C, 80°C, 90°C, 100°C, 110°C, 120°C, 130°C, 140°C or 150°C. In the present invention, the organic solvent preferably includes N,N-dimethylformamide and / or N,N-dimethylacetamide. In the present invention, the ratio of the mass of the L-aspartic acid to the volume of the organic solvent is preferably 10g:20-200mL, and in specific embodiments can be 10g:20mL, 10g:30mL, 10g:40mL, 10g:50mL, 10g:60mL, 10g:70mL, 10g:80mL, 10g:90mL, 10g:100mL, 10g:110mL, 10g:120mL, 10g:130mL, 10g:140mL, 10g:150mL, 10g:160mL, 10g:170mL, 10g:180mL, 10g:190mL or 10g:200mL. In the present invention, the solid-liquid separation preferably includes filtration, suction filtration or centrifugal separation. The present invention has no special limitation on the drying method and conditions, and the drying may be performed to a constant weight. In a specific embodiment, the drying method may be drying, spray drying, boiling drying or fluidized bed drying. Specifically, the drying may be performed in a drying oven at 80° C. for 12 hours.

[0079] The raw materials for preparing the nanomagnetic targeted sustained-release delivery system provided by the present invention are PSI (soft film) and Fe3O4-HAP (hard core). The raw materials have low cost and are biodegradable in vivo, and have high raw material safety. The two can be rapidly and spontaneously formed into a non-aggregating, uniform-size nanomagnetic targeted sustained-release delivery system with a "hard core and soft film" structure by simply mixing with a drug in a solution. The preparation process is simple and suitable for industrial production. The preparation process does not require expensive materials, production equipment and harsh reaction conditions, almost no impurities are generated, and the sterilization operation can be completed during production. The production process has a certain ambiguity (that is, the feed amount does not affect the quality of the nanomagnetic targeted sustained-release delivery system within a certain error range), and has great production convertibility, which solves the problem that the current nano delivery system is difficult to convert into production.

[0080] The present invention also provides a nanomagnetic targeted sustained-release drug preparation, comprising a nanomagnetic targeted sustained-release delivery system and a drug loaded on the nanomagnetic targeted sustained-release delivery system; the nanomagnetic targeted sustained-release delivery system is the nanomagnetic targeted sustained-release delivery system described in the above technical scheme or the nanomagnetic targeted sustained-release delivery system prepared by the preparation method described in the above technical scheme.

[0081] In the present invention, the drug preferably includes an anti-tumor drug. The present invention has no special limitation on the anti-tumor drug. The anti-tumor drug well known to those skilled in the art can be used. In specific embodiments, it can be a protein drug, a polypeptide drug, a nucleic acid drug, nimustine, carmustine, cyclophosphamide, ifosfamide, gromustine, doxorubicin, doceflurane, 5-fluorouracil (5-FU), mercaptopurine, thioguanine, fluguanidine, tegafur, gemcitabine, actinomycin D, doxorubicin, adriamycin, One or more of daunorubicin, daunorubicin, epirubicin, epirubicin, mitomycin, autismycin, peplomycin, pyromycin, pingyangmycin, pirarubicin, pyranorubicin, irinotecan, vinorelbine, paclitaxel, docetaxel, topotecan, vincristine, aldehyde vinblastine, vindesine, vincamide, vinblastine, vinblastine, tenipoxi, etopoxi, atamestane, anastrozole, aminoglutethimide, letrozole, formestane, megestrol, tamoxifen, carboplatin, cisplatin, dacarbazine and asparaginase. The nanomagnetic targeted sustained-release delivery system provided by the present invention can realize the delivery and protection of various anti-tumor drugs, especially protein drugs, polypeptide drugs and nucleic acid drugs and other unstable drugs in vivo, and improve the bioavailability of drugs.

[0082] In the present invention, the mass content of the drug in the nanomagnetic targeted sustained-release drug preparation is 0.01-99%, and in specific embodiments it can be 0.01%, 0.05%, 0.1%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99%.

[0083] The present invention also provides a method for preparing the nano-magnetic targeted sustained-release drug preparation described in the above technical solution, comprising the following steps: mixing the drug, magnetic ferroferric oxide-hydroxyapatite and a solvent for adsorption, and then adding polysuccinimide for coating to obtain the nano-magnetic targeted sustained-release drug preparation.

[0084] In the present invention, the solvent preferably includes one or more of water, methanol, ethanol, chloroform, dimethylformamide, dimethylacetamide and dimethyl sulfoxide.

[0085] In the present invention, the mixing preferably comprises: adding the drug and magnetic ferroferric oxide-hydroxyapatite into an organic solvent for premixing to obtain a mixed suspension; and dropping a polysuccinimide solution into the mixed suspension.

[0086] In the present invention, the concentration of magnetic ferroferric oxide-hydroxyapatite in the mixed suspension is preferably 0.1-100 mg / mL, and in specific embodiments it can be 0.1 mg / mL, 0.5 mg / mL, 1 mg / mL, 5 mg / mL, 10 mg / mL, 20 mg / mL, 30 mg / mL, 40 mg / mL, 50 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL or 100 mg / mL.

[0087] In the present invention, the concentration of the polysuccinimide solution is preferably 0.1-200 mg / mL, and in specific embodiments can be 0.1 mg / mL, 0.5 mg / mL, 1 mg / mL, 5 mg / mL, 10 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 40 mg / mL, 50 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL, 100 mg / mL, 110 mg / mL, 120 mg / mL, 130 mg / mL, 140 mg / mL, 150 mg / mL, 160 mg / mL, 170 mg / mL, 180 mg / mL, 190 mg / mL or 200 mg / mL; the solvent in the polysuccinimide solution preferably includes N,N dimethylformamide and / or N N dimethylacetamide.

[0088] In the present invention, the premixing is preferably carried out under ultrasound and / or stirring conditions, and the premixing temperature is preferably room temperature; the premixing time is preferably 1 to 60 minutes, and in specific embodiments it can be 1 minute, 5 minutes, 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes or 60 minutes; the stirring speed is preferably 100 to 10000 r / min, and in specific embodiments it can be 100 r / min, 500 r / min, 10 The ultrasonic power is preferably 50 to 5000 W, and in a specific embodiment may be 50 W, 100 W, 500 W, 1000 W, 2000 W, 3000 W, 4000 W or 5000 W.

[0089] In the present invention, the dropping is preferably carried out under ultrasound and / or stirring conditions; the dropping temperature is preferably room temperature; the ultrasound power is preferably 50-5000W, and in specific embodiments it can be 50W, 100W, 500W, 1000W, 2000W, 3000W, 4000W or 5000W; the stirring speed is preferably 100-10000r / min, and in specific embodiments it can be 100r / min, 500r / min, 1000r / min, 2000r / min, 3000r / min, 4000r / min, 5000r / min, 6000r / min, 7000r / min, 8000r / min, 9000r / min or 10000r / min.

[0090] In the present invention, the adsorption is preferably static adsorption, the adsorption temperature is preferably room temperature, and the adsorption time is preferably 1 to 120 min. In specific embodiments, it can be 1 min, 5 min, 10 min, 20 min, 30 min, 40 min, 50 min, 60 min, 70 min, 80 min, 90 min, 100 min, 110 min or 120 min.

[0091] In the present invention, during the addition of the polysuccinimide solution, the polysuccinimide breaks the aggregation state of the magnetic ferroferric oxide-hydroxyapatite and coats the surface thereof to form a nano-magnetic targeted sustained-release drug preparation. The coating is completed after the addition of the polysuccinimide solution is completed.

[0092] After the coating is completed, the present invention preferably further comprises solid-liquid separation, and the obtained solid component is a nanomagnetic targeted sustained-release pharmaceutical preparation. In the present invention, the solid-liquid separation preferably comprises filtration, suction filtration or centrifugal separation.

[0093] The present invention also provides the nanomagnetic targeted sustained-release delivery system described in the above technical scheme, the nanomagnetic targeted sustained-release delivery system prepared by the preparation method described in the above technical scheme, the nanomagnetic targeted sustained-release drug preparation described in the above technical scheme, or the use of the nanomagnetic targeted sustained-release drug preparation prepared by the preparation method described in the above technical scheme in preparing drugs.

[0094] In the present invention, the administration of the nanomagnetic targeted sustained-release delivery system and the nanomagnetic targeted sustained-release pharmaceutical preparation independently preferably includes oral administration or injection administration. When administered orally, the PSI-Fe3O4-HAP system will not release the drug in gastric acid. After being guided by the magnetic field, the drug will be released when the pH rises after reaching the target tissue site. Oral administration can also reduce the difficulty and cost of preparation production, improve patient compliance, and reduce the safety hazards caused by injection administration.

[0095] Under the same dosage, the nano-magnetic targeted sustained-release delivery system provided by the present invention has magnetic targeting, the concentration and retention time in the target area are significantly improved, and the toxicity of the drug to non-target organs is reduced, which greatly improves the efficacy and safety of the drug. The system can be used for the in vivo delivery of small molecule drugs, proteins, peptides, nucleic acids and other gene anti-tumor drugs, has strong versatility, and is suitable for multi-pathway administration in vivo, and can solve the bottleneck problem that protein drugs, polypeptide drugs and nucleic acid drugs are difficult to use clinically due to poor stability and easy degradation in the body.

[0096] The present invention also provides a pharmaceutical composition, comprising an active ingredient and a pharmaceutically acceptable excipient; the active ingredient comprises the nanomagnetic targeted sustained-release pharmaceutical preparation described in the above technical solution or the nanomagnetic targeted sustained-release pharmaceutical preparation prepared by the preparation method described in the above technical solution. The present invention has no special limitation on the pharmaceutically acceptable excipient, and the pharmaceutically acceptable excipients well known to those skilled in the art can be used. In the present invention, the administration method of the pharmaceutical composition preferably includes oral administration or injection administration.

[0097] In order to further illustrate the present invention, the nanomagnetic targeted sustained-release delivery system and its preparation method and application, the nanomagnetic targeted sustained-release drug preparation and its preparation method and application, and the pharmaceutical composition provided by the present invention are described in detail below in combination with the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0098] Example 1

[0099] Preparation of magnetic nano-PSI-Fe3O4-HAP containing 5-fluorouracil

[0100] (1) Preparation of CaCO3

[0101] Dissolve 5 mL of Tween-80 in 20 mL of butyl acetate to obtain a Tween-80 solution, add the Tween-80 solution to 60 mL of a 1 mol / L Na2CO3 aqueous solution, and stir magnetically for 20 min to obtain an emulsion. Drop the emulsion into a 0.2 mol / L CaCl2 (0.06 mol) aqueous solution, stir for 1 h, and filter. Wash the obtained solid component with deionized water and anhydrous ethanol to remove the organic solvent, and dry it in an oven at 80°C for 12 h to obtain CaCO3 powder.

[0102] (2) Preparation of magnetic nano-iron tetroxide (Fe3O4)

[0103] Magnetic nanoparticles were prepared by coprecipitation. FeCl3·6H2O was dissolved in 50 mL of deionized water to prepare a 0.050 mol / L FeCl3 aqueous solution, and FeCl2·4H2O was dissolved in 50 mL of deionized water to prepare a 0.028 mol / L FeCl2 aqueous solution. The FeCl3 aqueous solution and the FeCl2 aqueous solution were mixed, and the iron ions in the mixed solution were slightly higher than the stoichiometric molar ratio of magnetite (Fe 3+ / Fe 2+ =2:1), add 5mL25wt% ammonia solution, stir vigorously at 2000r / min for 20min, and obtain a black suspension, separate magnetically, wash the obtained solid component with water for 5 times, and dry in a drying oven at 80℃ to obtain magnetic nano-Fe3O4.

[0104] (3) Preparation of magnetic hydroxyapatite (Fe3O4-HAP)

[0105] 1 g of the CaCO3 powder prepared in step (1) was added to 200 mL of water to prepare a CaCO3 aqueous suspension, and 0.0500 g of magnetic nano-Fe3O4 was added. Under stirring, 200 mL of 0.03 mol / L Na2HPO4 aqueous solution was added dropwise at a rate of 2 mL / min. The pH value was adjusted to 11 with a 20 wt% NaOH aqueous solution in a constant temperature water bath at 60°C under normal pressure. The reaction was carried out for 2 h, and the solid component was filtered. The solid component was washed with deionized water until the solution was neutral, washed with anhydrous ethanol 3 times, washed with dichloromethane 3 times, and dried in a drying oven at 80°C for 12 h to obtain magnetic Fe3O4-HAP. 20 .

[0106] (4) Preparation of polysuccinimide (PSI)

[0107] L-aspartic acid (10.0 g), phosphoric acid (85 wt%, 6 mL) and deionized water (1 mL) were added, and the mixture was heated to 170°C under reduced pressure in a rotary evaporator and kept warm for 1 h, and the temperature was lowered to 100°C, and 60 mL of N,N-dimethylformamide was added to dissolve the residue to obtain a residue solution. The residue solution was dripped into 300 mL of deionized water at a rate of 2 mL / min under mechanical stirring (700 r / min) and ultrasonic conditions (60°C, 1500 W), filtered, and the obtained solid component was washed with deionized water, washed with dichloromethane, and dried in an oven at 80°C for 12 h to obtain PSI.

[0108] (5)PSI-Fe3O4-HAP 20 Preparation of sustained-release delivery systems

[0109] At room temperature, Fe3O4-HAP was added to 5 mL of anhydrous ethanol and ultrasonicated for 1 min. PSI solution (concentration of 25 mg / mL, PSI: Fe3O4-HAP) was added dropwise under room temperature, ultrasonication and stirring (1000 r / min). 20 The mass ratio is 5:1). After the addition is completed, high-speed centrifugation is performed at 12000r / min for 4min. The solid component is PSI-Fe3O4-HAP 20 . Among them, the ultrasonic power is 50W.

[0110] (6) 5-FU-PSI-Fe3O4-HAP 20 Preparation of nanoparticles

[0111] 5-Fluorouracil and Fe3O4-HAP 20 It was added to 5 mL of anhydrous ethanol at a mass ratio of 4:1 to obtain a mixed solution (5-fluorouracil concentration was 2 mg / mL). The mixed solution was ultrasonicated for 1 min, allowed to stand for adsorption for 30 min, and PSI solution (solvent was N,N-dimethylformamide, concentration was 25 mg / mL, PSI:Fe3O4-HAP mass ratio was 5:1) was added dropwise at room temperature, ultrasonicated, and stirred (1000 r / min). After the addition was completed, high-speed centrifugation was performed at 12000 r / min for 4 min, and the solid component was 5-FU-PSI-Fe3O4-HAP. 20 . Among them, the ultrasonic power is 50W.

[0112] Example 2

[0113] Prepare Fe3O4-HAP according to the method of Example 1 30 、PSI-Fe3O4-HAP 30 Sustained release delivery system and 5-FU-PSI-Fe3O4-HAP 30 The nanoformulation is different from Example 1 only in that the amount of magnetic Fe3O4 added in step (3) is 0.0333 g, and magnetic Fe3O4-HAP is obtained. 30 ; Fe3O4-HAP is used in step (5) 30 Raw materials, PSI-Fe3O4-HAP 30 In step (6), Fe3O4-HAP is used 30 Raw materials, 5-FU-PSI-Fe3O4-HAP 30 .

[0114] Example 3

[0115] Prepare Fe3O4-HAP according to the method of Example 1 40 、PSI-Fe3O4-HAP 40Sustained release delivery system and 5-FU-PSI-Fe3O4-HAP 40 The nanoformulation is different from Example 1 only in that the amount of magnetic Fe3O4 added in step (3) is 0.0250 g, and magnetic Fe3O4-HAP is obtained. 40 ; Fe3O4-HAP is used in step (5) 40 Raw materials, PSI-Fe3O4-HAP 40 In step (6), Fe3O4-HAP is used 40 Raw materials, 5-FU-PSI-Fe3O4-HAP 40 .

[0116] Comparative Example 1

[0117] (1) Preparation of hydroxyapatite (HAP)

[0118] 1 g of the CaCO3 powder prepared in Example 1 was added to 200 mL of water to prepare a suspension. Under stirring, 200 mL of a 0.03 mol / L Na2HPO4 aqueous solution was dropwise added to the suspension at a rate of 2 mL / min. The pH value was adjusted to 11 with a 20 wt% NaOH aqueous solution in a constant temperature water bath at 60°C under normal pressure. The reaction was carried out for 2 h. The product was separated by suction filtration, washed with deionized water until the solution was neutral, washed with anhydrous ethanol, washed with dichloromethane, and dried in an oven at 80°C for 12 h to obtain HAP.

[0119] The preparation reaction equation of HAP is as follows:

[0120] Na2CO3+CaCl2→CaCO3+ NaCl ;

[0121] CaCO3+Na2HPO4→Ca 10 (PO4)6(OH)2.

[0122] (2) Preparation of PSI-HAP sustained-release delivery system: 20 The preparation of the sustained-release delivery system differed only in that Fe3O4-HAP was replaced by HAP.

[0123] (3) Preparation of 5-FU-PSI-HAP nanoformulation: 5-FU-PSI-Fe3O4-HAP in Example 1 20 The preparation of the nanoformulation differed only in that Fe3O4-HAP was replaced by HAP.

[0124] Test Example 1

[0125] Figure 1 PSI-HAP, PSI-Fe3O4-HAP20 、PSI-Fe3O4-HAP 30 and PSI-Fe3O4-HAP 40 From the SEM images, we can see that PSI, HAP, and Fe3O4-HAP 20 、Fe3O4-HAP 30 and Fe3O4-HAP 40 Both can form spherical or nearly spherical nanoparticles with relatively uniform particle size.

[0126] Figure 2 For 5-FU-PSI-HAP, 5-FU-PSI-Fe3O4-HAP 20 、5-FU-PSI-Fe3O4-HAP 30 and 5-FU-PSI-Fe3O4-HAP 40 SEM images of Figure 3 For 5-FU-PSI-HAP(a), 5-FU-PSI-Fe3O4-HAP 20 (b), 5-FU-PSI-Fe3O4-HAP 30 (c) and 5-FU-PSI-Fe3O4-HAP 40 From the particle size distribution diagram (d), it can be seen that the nanoparticles containing 5-FU are all relatively uniform spherical nanoparticles, which can be taken up by tumor cells and enter the tumor cell membrane.

[0127] Figure 4 For 5-FU-PSI-HAP(a), 5-FU-PSI-Fe3O4-HAP 20 (b), 5-FU-PSI-Fe3O4-HAP 30 (c) and 5-FU-PSI-Fe3O4-HAP 40 (d) The zeta potential test spectrum shows that 5-FU-PSI-HAP and 5-FU-PSI-Fe3O4-HAP 20 、5-FU-PSI-Fe3O4-HAP 30 and 5-FU-PSI-Fe3O4-HAP 40 The zeta potentials are -19.5mV, -22.6mV, -23.2mV and -23.6mV, respectively, all of which are negative values, the same as the charge of human biological membranes, and the absolute value is less than 30, indicating that the nanoparticles can exist stably in solution.

[0128] Prepare Nile red solution (1 mg / mL), add HAP or Fe3O4-HAP and drugs (5-FU-PSI-HAP, 5-FU-PSI-Fe3O4-HAP) 20、5-FU-PSI-Fe3O4-HAP 30 、5-FU-PSI-Fe3O4-HAP 40 ), mixed well, and then dripped into the PSI solution. After the dripping was completed, the mixture was centrifuged, washed, and dried to prepare the Nile red preparations containing 5-FU, PSI-HAP, and PSI-Fe3O4-HAP. 84 mice, half male and half female, SPF grade, 4-6 weeks old, weighing 25-30g, were randomly divided into 5 groups: gavage blank group (12 mice), gavage 5-FU-PSI-HAP group (18 mice), gavage 5-FU-PSI-Fe3O4 ...). 20 Group (18 rats), gavage 5-FU-PSI-Fe3O4-HAP 30 Group (18 rats), gavage 5-FU-PSI-Fe3O4-HAP 40 The mice were divided into two groups (18 mice), and the drug group was given the mouse preparation (200 mg / kg) and a magnetic field (4000 Gs) was applied to the liver for 30 minutes after drug administration. The mice were fasted for 12 hours before drug administration, but not drinking. After drug administration, the mice were killed by cervical dislocation at different time points such as 1 hour, 3 hours, 6 hours, 9 hours, 12 hours, and 24 hours. The mouse organs (heart, liver, spleen, lung, and kidney) were taken for small animal in vivo imaging to observe the distribution and retention time of the drug in the body. The instrument parameters were set as follows: select the mode (fluorescence spectral imaging), fix the excitation wavelength (530 nm), and scan with the emission wavelength (600 nm). Figures 5 to 9 For 5-FU-PSI-HAP, 5-FU-PSI-Fe3O4-HAP 20 、5-FU-PSI-Fe3O4-HAP 30 and 5-FU-PSI-Fe3O4-HAP 40 The distribution map of the heart, liver, spleen, lung and kidney tissues in the animal body showed that compared with the nanoformulation group (5-FU-PSI-HAP) without Fe3O4 magnetic components, after applying the magnetic field for a certain period of time (for mice, after oral administration, the magnetic field was applied to the liver outside the animal body for 0.5h), the nanoformulation containing Fe3O4 (5-FU-PSI-Fe3O4-HAP) had significant liver tissue targeting.

[0129] Select Huh-7 liver cancer cells in the logarithmic growth phase, add trypsin containing 25% EDTA for digestion for 1 minute, and then use a microscope to observe the cells in the culture flask. When the cells become round and the intercellular gap becomes larger, remove the trypsin digestion solution and add a certain amount of culture medium containing 10% FBS to terminate the digestion. Gently blow the pipette to make a single cell suspension, count it using a hemocytometer, and dilute it to a concentration of 1×10 6Take out the 96-well plate and add 100 μL of the above cell suspension to each well so that the well plate has 1×10 4 After the cells were placed on the operating table for 10 minutes, they were placed in a 37°C incubator for overnight culture (12 hours). After the cells adhered to the wall, the old culture medium was discarded, and after washing with PBS buffer, 100 μL of 5-FU-PSI-HAP, 5-FU-PSI-Fe3O4-HAP with drug concentrations of 5, 20, 40, 80, and 100 μg / mL were added to each well. 20 、5-FU-PSI-Fe3O4-HAP 30 and 5-FU-PSI-Fe3O4-HAP 40 Preparation, 5 replicates in each group, and set up a blank control group (only culture medium) and a solvent blank control group (without cells and drugs). After incubation for 24 hours, the drug-containing culture medium was discarded, and the cells were washed with PBS buffer, and culture medium was added. 10 μL of CCK-8 reagent was added to each well. After incubation for 2 hours, the microplate reader was used to read the readings at 450 nm to calculate the cell survival rate. Fig.10 For 5-FU, 5-FU-PSI-HAP, 5-FU-PSI-Fe3O4-HAP 20 、5-FU-PSI-Fe3O4-HAP 30 and 5-FU-PSI-Fe3O4-HAP 40 The figure shows the inhibition of Huh-7 cell proliferation (**p<0.01, ***p<0.001, ****p<0.0001). The experimental results show that in the range of 5-FU concentration of 5-100μg / mL, the activity of Huh-7 cells is significantly concentration-dependent for different drugs. 5-FU, 5-FU-PSI-HAP, 5-FU-PSI-Fe3O4-HAP 20 、5-FU-PSI-Fe3O4-HAP 30 and 5-FU-PSI-Fe3O4-HAP 40 All concentrations had significant inhibitory effects on Huh-7 cells.

[0130] IC calculated based on CCK-8 results 50 value. Fig.11 For 5-FU, 5-FU-PSI-HAP, 5-FU-PSI-Fe3O4-HAP 20 、5-FU-PSI-Fe3O4-HAP 30 and 5-FU-PSI-Fe3O4-HAP 40 24h IC of Huh-7 cells 50The experimental results show that compared with 5-FU, 5-FU-PSI-HAP and 5-FU-PSI-Fe3O4-HAP have significant differences (P<0.0001). Compared with 5-FU-PSI-HAP, the IC value of 5-FU-PSI-Fe3O4-HAP containing magnetic Fe3O4 is significantly higher than that of 5-FU-PSI-HAP. 50 The value is lower, and good anti-tumor effect can be achieved at a lower dose. 20 、5-FU-PSI-Fe3O4-HAP 30 and 5-FU-PSI-Fe3O4-HAP 40 There was no significant difference among the three groups (P>0.5).

[0131] Coumarin 6 solution (1 mg / mL) was prepared and Fe3O4-HAP or HAP and drugs (5-FU-PSI-HAP, 5-FU-PSI-Fe3O4-HAP) were added. 20 、5-FU-PSI-Fe3O4-HAP 30 、5-FU-PSI-Fe3O4-HAP 40 ), mixed well, and then dripped into the PSI solution. After the dripping was completed, the solution was centrifuged, washed, and dried to prepare the fluorescent preparations of PSI-HAP containing 5-FU and PSI-Fe3O4-HAP containing coumarin 6. Huh-7 cells were placed in a 24-well plate (2×10 5 After the cells adhered to the wall, 5-FU-PSI-HAP and 5-FU-PSI-Fe3O4-HAP with a drug concentration of 50 μmol / L were added. 20 、5-FU-PSI-Fe3O4-HAP 30 、5-FU-PSI-Fe3O4-HAP 40 The fluorescent preparations were co-cultured with the cells for 1 h, 2 h, and 4 h. After fixation, the cells were stained with DAPI for 5 min. The slides were placed on coverslips for sealing, and the uptake of different preparations at each time point was recorded using a fluorescence microscope. Fig.12 To investigate the effect of Huh-7 cells on 5-FU-PSI-HAP and 5-FU-PSI-Fe3O4-HAP 20 、5-FU-PSI-Fe3O4-HAP 30 and 5-FU-PSI-Fe3O4-HAP 40The experimental results show that after 1 hour of incubation, each group only had weak green fluorescence, indicating that the nanoparticles gradually began to enter the cells. At 2 hours, the fluorescence was stronger than that at 1 hour, and at 4 hours, the green fluorescence reached the maximum. Compared with the 5-FU-PSI-HAP group, the 5-FU-PSI-Fe3O4-HAP 20 、5-FU-PSI-Fe3O4-HAP 30 and 5-FU-PSI-Fe3O4-HAP 40 The green fluorescence of group 4 was significantly stronger, and the fluorescence was concentrated in the cytoplasm, indicating that the nanoparticles had already crossed the membrane and entered the cell in large quantities.

[0132] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A nano-magnetic targeted sustained-release delivery system, comprising magnetic ferroferric oxide-hydroxyapatite and polysuccinimide coated on the surface of the ferroferric oxide-hydroxyapatite.

2. The nanomagnetic targeted sustained-release delivery system according to claim 1, characterized in that: The mass content of ferroferric oxide in the magnetic ferroferric oxide-hydroxyapatite is 5 to 80%; The mass content of polysuccinimide in the nano-magnetic targeted sustained-release delivery system is 10-90%.

3. The method for preparing the nanomagnetic targeted sustained-release delivery system according to claim 1 or 2 comprises the following steps: The magnetic ferroferric oxide-hydroxyapatite, polysuccinimide and a solvent are mixed and coated to obtain the nano magnetic targeted sustained-release delivery system.

4. The preparation method according to claim 3, characterized in that: The solvent includes one or more of water, methanol, ethanol, chloroform, dimethylformamide, dimethylacetamide, and dimethyl sulfoxide.

5. The preparation method according to claim 3, characterized in that: The preparation method of magnetic ferroferric oxide-hydroxyapatite comprises the following steps: mixing CaCO3, magnetic nano-Fe3O4, Na2HPO4 and water, adjusting the pH value to 7-12 with alkali metal hydroxide, and performing double decomposition reaction to obtain magnetic ferroferric oxide-hydroxyapatite.

6. A nanomagnetic targeted sustained-release drug preparation, comprising a nanomagnetic targeted sustained-release delivery system and a drug loaded on the nanomagnetic targeted sustained-release delivery system; the nanomagnetic targeted sustained-release delivery system is the nanomagnetic targeted sustained-release delivery system described in any one of claims 1 to 2 or the nanomagnetic targeted sustained-release delivery system prepared by the preparation method described in any one of claims 3 to 5.

7. The nanomagnetic targeted sustained-release pharmaceutical preparation according to claim 6, characterized in that: The drugs include anti-tumor drugs; The mass content of the drug in the nano-magnetic targeted sustained-release drug preparation is 0.01-99%.

8. The method for preparing the nanomagnetic targeted sustained-release pharmaceutical preparation according to claim 6 or 7, comprising the following steps: The drug, magnetic ferroferric oxide-hydroxyapatite and a solvent are mixed and adsorbed, and then polysuccinimide is added for coating to obtain the nano magnetic targeted sustained-release drug preparation.

9. Use of the nanomagnetic targeted sustained-release delivery system according to any one of claims 1 to 2, the nanomagnetic targeted sustained-release delivery system prepared by the preparation method according to any one of claims 3 to 5, the nanomagnetic targeted sustained-release pharmaceutical preparation according to any one of claims 6 to 7, or the nanomagnetic targeted sustained-release pharmaceutical preparation prepared by the preparation method according to claim 8 in the preparation of drugs.

10. A pharmaceutical composition comprising an active ingredient and a pharmaceutically acceptable excipient; the active ingredient comprises the nanomagnetic targeted sustained-release pharmaceutical preparation according to any one of claims 6 to 7 or the nanomagnetic targeted sustained-release pharmaceutical preparation prepared by the preparation method according to claim 8.

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