An amorphous biomimetic calcified protein nanocarrier material for disease tissue penetration administration, and a preparation method and application thereof

Abstract

The application provides a preparation method of an amorphous biomimetic calcified protein nanocarrier material for disease tissue penetration administration, and is characterized by comprising the following steps: (1) adding calcium salt and magnesium salt reagents, active protein molecules and streptomycin sulfate into a cell culture solution, stirring uniformly to prepare a mixed solution; (2) adding the cell culture solution containing dissolved phosphate into the mixed solution prepared in step (1), stirring and incubating the generated emulsion at room temperature to obtain a suspension; (3) washing the suspension obtained in step (2) through a dialysis bag, then sterilizing through a filter membrane and freeze-drying, so as to obtain the amorphous biomimetic calcified protein nanocarrier material for disease tissue penetration administration. The material can load fluorescent reagents or tumor drug molecules, so as to achieve the purposes of disease treatment and fluorescent imaging.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, specifically relating to an amorphous biomimetic calcified protein nanocarrier material for infiltration drug delivery in diseased tissues, its preparation method, and its application. Background Technology

[0002] Calcified protein particles in the blood are a type of colloidal nanoparticles composed of protein molecules and inorganic calcium salts. Their main components are calcium phosphate, fetuin-A, and albumin, etc. [Reference 1: Smith et al., Phosphorylated fetuin-A-containing calciprotein particles are associated with aortic stiffness and a procalcific milieu in patients with pre-dialysis CKD. NephrolDial. Transplant 2012, 27:p.1957-1966.]. In normal individuals, calcified protein particles bind with calcium ions through acidic proteins to form amorphous calcium phosphate, thereby hindering further biomineralization. Ultimately, the calcified protein particles are cleared through metabolism by macrophages and the kidneys [Reference 2: Pasch et al., Phosphate, calcification in blood, and mineral stress: the physiologic blood mineral buffering system and its association with cardiovascular risk. Int. J. Nephrol. 2018, 2018:918-2078.]. As a real, endogenous nanomaterial existing in the body, calcified protein particles have better biocompatibility, but their application in the field of drug delivery has not yet been reported.

[0003] Traditional drug carriers mainly include silica and carbon nanospheres, which are difficult to degrade and metabolize rapidly in vivo [Reference 3: Li et al., Mesoporous carbon nanospheres featured fluorescent aptasensor for multiple diagnosis of cancer in vitro and in vivo. ACS Nano. 2015, 9(12): p. 12096-12103.]. At the same time, tumors and other disease tissues often lack capillary networks, have very dense extracellular matrix and very high cell density, making it difficult for traditional nanomedicine carrier materials to penetrate deep into disease tissues and achieve effective treatment [Reference 4: Matsumoto et al., Vascular bursts enhance permeability of tumor blood vessels and improve nanoparticle delivery. Nature Nanotechnology. 2016, 11: 533-538.]. Domestic and foreign scholars have reduced the penetration resistance of nanomedicines in tumor and other disease tissues by reducing the size of nanomedicines, but it is still difficult to solve the problems of weak penetration and diffusion ability of nanomedicine carriers [Reference 5: Zhou et al., Enzyme-activatable polymer-drug conjugate augmentstumour penetration and treatment efficacy. Nature Nanotechnology. 2019, 14(8): 799-809.]. Summary of the Invention

[0004] The purpose of this invention is to address the bottleneck problem in the field of drug carriers, which have poor in vivo degradation performance and biosafety, making it difficult to penetrate deep into disease tissues and achieve effective treatment. The invention proposes an amorphous biomimetic calcified protein nanocarrier material for infiltration drug delivery into disease tissues, as well as its preparation method and application.

[0005] This invention mimics the microscopic principle of protein molecules inhibiting biomineralization in the human body. It prepares a composite material of protein molecules and amorphous calcium phosphate using a wet chemical precipitation method, forming a complex nanonetwork structure that enhances the amorphous stability of the hybrid material, ensuring its degradation performance in vivo and exhibiting better biosafety and stability. The complex nanonetwork structure can efficiently dope fluorescent molecules and other markers, as well as tumor therapeutic drugs, and relies on its small nanoparticle size and the synergistic effect of the hybrid protein molecules to achieve effective penetration and diffusion deep into diseased tissues. Compared with existing calcium phosphate drug carriers, the biomimetic calcified protein nanomaterials have a composition and microstructure similar to calcified protein particles present in human blood, thus exhibiting better biosafety and degradation performance than existing drug carriers.

[0006] The technical solution of the present invention is as follows:

[0007] This invention provides a method for preparing an amorphous biomimetic calcified protein nanocarrier material for infiltration drug delivery to diseased tissues, comprising the following steps:

[0008] (1) Add calcium and magnesium salt reagents, active protein molecules, and penicillin-streptomycin mixture to cell culture medium, stir well, and prepare a mixed solution;

[0009] (2) Add the cell culture medium containing dissolved phosphate dropwise to the mixed solution prepared in step (1), and stir and incubate the resulting emulsion at room temperature to obtain a suspension;

[0010] (3) The suspension obtained in step (2) is washed through a dialysis bag, then sterilized by a filter membrane and freeze-dried to obtain an amorphous biomimetic calcified protein nanocarrier material for infiltration drug delivery to disease tissues.

[0011] Preferably, in step (1), the penicillin-streptomycin mixture contains 10,000 U / ml of penicillin and 10 mg / ml of streptomycin; the volume of the penicillin-streptomycin mixture added is 0-3% of the cell culture medium volume; and / or

[0012] The concentration of the calcium salt in the cell culture medium is 0.1-2 g / L; and / or

[0013] The concentration of the magnesium salt in the cell culture medium is 0-0.5 g / L; and / or

[0014] After stirring well, adjust the pH to 8-10.

[0015] Preferably, the cell culture medium includes DMEM cell culture medium, 1640 cell culture medium; and / or

[0016] The active protein molecules include fetal bovine serum, fetal globulin, and albumin; the volume of the active protein molecules added is 5-50% of the cell culture medium volume.

[0017] Preferably, in step (2), phosphate is first dissolved in cell culture medium and the pH of the solution is adjusted by sodium hydroxide. Then, the resulting solution is added dropwise to an equal volume of the mixed solution prepared in step (1) and stirred at room temperature to generate an emulsion.

[0018] Preferably, in step (2), the concentration of the phosphate in the cell culture medium is 0.1-2 g / L; and / or

[0019] The drop rate is 1-20 mL / min; and / or

[0020] The pH of the solution is adjusted to 8-10; and / or

[0021] The stirring and incubation time is 1-4 hours.

[0022] Preferably, in step (3), the molecular weight cutoff of the dialysis bag is 8000-18000; and / or

[0023] The freeze-drying temperature is -60 to (-40)℃.

[0024] The present invention also provides an amorphous biomimetic calcified protein nanocarrier material for infiltration drug delivery to diseased tissues, which is prepared by the above-described method.

[0025] This invention also provides the application of an amorphous biomimetic calcified protein nanocarrier material for infiltration drug delivery into disease tissues in loading fluorescent reagents or tumor drug molecules.

[0026] Preferably, fluorescent reagents or tumor drug molecules are added to the mixed solution in step (1).

[0027] Preferably, the concentration of the fluorescent reagent is 0.05-0.2 g / L; the concentration of the tumor drug molecule is 0.05-0.2 g / L.

[0028] Compared with the prior art, the beneficial effects of the present invention are reflected in:

[0029] 1. Inspired by the microscopic principle of protein molecules inhibiting biomineralization in the real human body, a composite material of protein molecules and amorphous calcium phosphate was prepared by wet chemical precipitation. The protein molecules in the composite material can bind to the calcium ions of amorphous calcium phosphate clusters to form a complex nano-network structure, which effectively inhibits the crystallization of amorphous calcium phosphate, thereby significantly improving the degradation performance and biosafety of the material in vivo.

[0030] 2. Compared with existing nanomedicine carriers, the amorphous biomimetic calcified protein nanocarrier material of the present invention has a smaller particle size and hybrid protein molecules. The two work synergistically to effectively disrupt the connections between cells, which helps to penetrate and pass through the deep tissues of tumors that lack capillaries, thus achieving effective penetration and diffusion into the deep tissues of tumors and other diseases that are difficult for blood to reach.

[0031] 3. The amorphous biomimetic calcified protein nanocarrier material prepared by the method of the present invention has a complex nano-network structure, which can efficiently dop with fluorescent molecules and other markers and tumor drug molecules. Relying on its small particle size, it can effectively penetrate and diffuse deep into disease tissues, and then completely degrade and release drugs and fluorescent molecules, thereby achieving effective penetration and diffusion deep into disease tissues, and thus achieving the purpose of disease treatment and fluorescence imaging. Attached Figure Description

[0032] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used in conjunction with embodiments of the invention to explain the invention and do not constitute a limitation thereof. In the drawings:

[0033] Figure 1 The XRD results are for the amorphous biomimetic calcified protein nanocarrier material.

[0034] Figure 2 The TEM morphology results are for the amorphous biomimetic calcified protein nanocarrier material.

[0035] Figure 3 The results of in vitro cytotoxicity studies of amorphous biomimetic calcified protein nanocarrier materials at concentrations of (a) 0 μg / mL, (b) 800 μg / mL, and (c) 1600 μg / mL.

[0036] Figure 4 The TG / DSC results are for amorphous biomimetic calcified protein nanocarrier materials.

[0037] Figure 5 Immunofluorescence staining images of VE-cadherin transecting the endothelial cell layer for (a) 0 μg / mL (control group) and (b) 445 μg / mL concentrations of amorphous biomimetic calcified protein nanocarrier materials.

[0038] Figure 6 This image shows the morphology of an amorphous biomimetic calcified protein nanocarrier material loaded with fluorescent molecules co-cultured with cells. Detailed Implementation

[0039] The following examples are provided to better understand the present invention, but do not limit the invention. Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were purchased from conventional biochemical reagent companies.

[0040] This invention simulates the microscopic principle of protein molecules inhibiting biomineralization in the real human body, and prepares a composite material of protein molecules and amorphous calcium phosphate, namely calcified protein nanomaterials, through a wet chemical precipitation method.

[0041] The preparation method of the amorphous biomimetic calcified protein nanocarrier material for tissue permeation drug delivery of the present invention is as follows:

[0042] (1) Add calcium and magnesium salt reagents, active protein molecules, and penicillin-streptomycin mixture to cell culture medium, stir well, and prepare a mixed solution;

[0043] (2) Add the cell culture medium containing dissolved phosphate dropwise to the mixed solution prepared in step (1), and stir and incubate the resulting emulsion at room temperature to obtain a suspension;

[0044] (3) The suspension obtained in step (2) is washed through a dialysis bag, then sterilized by a filter membrane and freeze-dried to obtain an amorphous biomimetic calcified protein nanocarrier material for infiltration drug delivery to disease tissues.

[0045] As some embodiments of the present invention, in step (1), calcium salt and magnesium salt are first dissolved in cell culture medium, then active protein molecules and penicillin-streptomycin mixture are added, and the pH is adjusted by sodium hydroxide to obtain a mixed solution.

[0046] As some embodiments of the present invention, in step (1), the penicillin content in the penicillin-streptomycin mixture is 10000 U / ml, and the streptomycin content is 10 mg / ml; the volume of the penicillin-streptomycin mixture added is 0-3% of the volume of the cell culture medium. Compared with adding the penicillin-streptomycin mixture, if the penicillin-streptomycin mixture is not added, the material will be easily contaminated with bacteria during use.

[0047] As some embodiments of the present invention, in step (1), the concentration of the calcium salt in the cell culture medium is 0.1-2 g / L.

[0048] Calcium salts in cell culture medium with a concentration greater than 2 g / L will rapidly crystallize after the addition of phosphate and cannot stabilize in an amorphous state; concentrations less than 0.1 g / L will make it difficult to form a precipitate.

[0049] As some embodiments of the present invention, in step (1), the concentration of the magnesium salt in the cell culture medium is 0-0.5 g / L.

[0050] In some embodiments of the present invention, in step (1), the stirring rate after mixing is 100-500 rpm. This stirring rate range is selected to ensure uniform mixing.

[0051] As some embodiments of the present invention, in step (1), after stirring evenly, the pH is adjusted to 8-10.

[0052] As some embodiments of the present invention, in steps (1) and (2), the cell culture medium includes, but is not limited to, DMEM cell culture medium, 1640 cell culture medium, etc.

[0053] As some embodiments of the present invention, in step (1), the calcium salt and magnesium salt include, but are not limited to, calcium chloride and magnesium chloride, calcium nitrate and magnesium nitrate.

[0054] In some embodiments of the present invention, the active protein molecules in step (1) include, but are not limited to, fetal bovine serum, fetal globulin, albumin, etc. The volume of the active protein molecules added is 5-50% of the volume of the cell culture medium.

[0055] When the amount of active protein molecules added is greater than 50% of the cell culture medium volume, protein precipitation will occur; when it is less than 5% of the cell culture medium volume, rapid crystallization will occur, making it impossible to stabilize the amorphous state.

[0056] As some embodiments of the present invention, in step (2), phosphate is first dissolved in cell culture medium and the pH of the solution is adjusted by sodium hydroxide. Then the resulting solution is added dropwise to an equal volume of the mixed solution prepared in step (1) and stirred at room temperature to generate an emulsion.

[0057] As some embodiments of the present invention, in step (2), the concentration of phosphate in the cell culture medium is 0.1-2 g / L.

[0058] Phosphate concentrations above 2 g / L in cell culture medium will rapidly crystallize and become unstable in an amorphous state, while concentrations below 0.1 g / L will make it difficult to form a precipitate.

[0059] In some embodiments of the present invention, in step (2), the dropping rate is 1-20 mL / min.

[0060] As some embodiments of the present invention, in step (2), the pH of the solution is adjusted to 8-10.

[0061] Alkaline byproducts will form when the pH is above 10, and it is difficult to form a precipitate when the pH is below 8.

[0062] In some embodiments of the present invention, in step (2), the stirring and incubation time is 1-4 hours.

[0063] In some embodiments of the present invention, in step (2), the stirring rate after mixing is 100-500 rpm. This stirring rate range is selected to ensure uniform mixing.

[0064] As some embodiments of the present invention, in step (2), the phosphate includes, but is not limited to, sodium dihydrogen phosphate, disodium hydrogen phosphate, ammonium dihydrogen phosphate and diammonium hydrogen phosphate, and may be one or more of them.

[0065] As some embodiments of the present invention, in step (3), the suspension obtained in step (2) is added to a dialysis bag, and after stirring and washing with 50-200 times the volume of deionized water for 24 hours and changing the liquid three times, it is freeze-dried to obtain an amorphous biomimetic calcified protein nanocarrier material for infiltration drug delivery to diseased tissues; wherein, the molecular weight cutoff of the dialysis bag is 8000-18000.

[0066] As some embodiments of the present invention, in step (3), the freeze-drying temperature is (-60℃)-(-40)℃.

[0067] The prepared amorphous biomimetic calcified protein nanocarrier material is amorphous with a particle size range of 10-100 nm and a protein content of 5-30 wt.%.

[0068] The amorphous biomimetic calcified protein nanocarrier material can disrupt intercellular connections in tissues, thereby penetrating deep into diseased tissues such as tumors that lack capillaries, achieving effective penetration and diffusion into deep diseased tissues. The effective penetration concentration of the calcified protein nanocarrier material is 10-445 μg / mL.

[0069] In some embodiments of the present invention, in step (1), a fluorescent reagent is added to the mixed solution of step (1) to give the finally obtained amorphous biomimetic calcified protein nanocarrier material a fluorescent effect. The initial concentration of the fluorescent reagent used is 0.05-0.2 g / L, where the initial concentration refers to the concentration of the fluorescent reagent dissolved in the cell culture medium. The fluorescent reagent includes, but is not limited to, indocyanine green (ICG, IR-820) and fluorescein isothiocyanate (FITC).

[0070] In some embodiments of the present invention, in step (1), drug molecules are added to the mixed solution of step (1) to give the finally obtained amorphous biomimetic calcified protein nanocarrier material a therapeutic effect. The initial concentration of the drug molecules used is 0.05-0.2 g / L, where the initial concentration refers to the concentration of the fluorescent reagent dissolved in the cell culture medium. The drug molecules include, but are not limited to, doxorubicin (DOX) and fluorouracil (5-FU).

[0071] The various embodiments will be described in more detail with reference to the following examples. The following examples are for illustrative purposes only and are not intended to limit the scope of the embodiments.

[0072] Example 1

[0073] The preparation method of the amorphous biomimetic calcified protein nanocarrier material in this embodiment is as follows:

[0074] Solution preparation: Weigh 0.042 g of anhydrous calcium chloride and dissolve it in 50 mL of DMEM medium. Add 10 mL of fetal bovine serum and 1 mL of penicillin-streptomycin mixture and stir until homogeneous at 400 rpm. Adjust the pH of the solution to 9.0 with sodium hydroxide to obtain solution A. Weigh 0.038 g of disodium hydrogen phosphate dodecahydrate and 0.028 g of sodium dihydrogen phosphate dihydrate and dissolve them in 50 mL of DMEM medium. Adjust the pH of the solution to 9.0 with sodium hydroxide and stir until homogeneous at 400 rpm to obtain solution B.

[0075] The penicillin-streptomycin mixture contains 10,000 U / ml of penicillin and 10 mg / ml of streptomycin.

[0076] Wet chemical precipitation: Add 50 mL of solution B prepared in the previous step dropwise to 50 mL of solution A at a rate of 5 mL / min, and stir and incubate at room temperature for 4 hours at a speed of 500 rpm.

[0077] Cleaning: After the reaction is complete, add the resulting suspension to an 8000 molecular weight dialysis bag, use 5L of deionized water to stir and clean for 24 hours, change the solution three times, and sterilize the filter membrane.

[0078] Freeze-drying: The cleaned product was placed in a -20°C freezer for 6 hours, and then placed in a freeze dryer at -55°C for freeze-drying to obtain amorphous biomimetic calcified protein nanocarrier material.

[0079] The prepared amorphous biomimetic calcified protein nanocarrier material was tested:

[0080] 1. XRD test results prove that the amorphous biomimetic calcified protein nanocarrier material is amorphous. Figure 1 ).

[0081] Figure 1 The XRD results are for the amorphous biomimetic calcified protein nanocarrier material.

[0082] 2. TEM results confirm that the particle size of the amorphous biomimetic calcified protein nanocarrier material is 20-50 nm. Figure 2 ).

[0083] Figure 2The TEM morphology results are for the amorphous biomimetic calcified protein nanocarrier material.

[0084] 3. In vitro culture experiments of human endothelial cells in DMEM cell culture medium containing different concentrations of nanomaterials showed that the cells grew well after 24 hours, the materials were non-cytotoxic, and exhibited good biocompatibility. Figure 3 ).

[0085] Figure 3 The results of in vitro cytotoxicity studies of amorphous biomimetic calcified protein nanocarrier materials at concentrations of (a) 0 μg / mL, (b) 800 μg / mL, and (c) 1600 μg / mL.

[0086] 4. TG / DSC analysis confirmed that the protein content in the amorphous biomimetic calcified protein nanocarrier material was 18.9 wt.%. Figure 4 ).

[0087] Figure 4 The TG / DSC results are for amorphous biomimetic calcified protein nanocarrier materials.

[0088] 5. After co-culturing human endothelial cells with amorphous biomimetic calcified protein nanocarrier material for 4 hours, the morphology of VE-cadherin in the cells under different concentrations of S-CPP1 was observed using a fluorescence inverted microscope. Compared with the control group, the addition of amorphous biomimetic calcified protein nanocarrier material disrupted calcium adhesion proteins, thereby enabling them to cross the cell barrier between tissues.

[0089] Figure 5 Immunofluorescence staining images of VE-cadherin transecting the endothelial cell layer for (a) 0 μg / mL (control group) and (b) 445 μg / mL concentrations of amorphous biomimetic calcified protein nanocarrier materials.

[0090] Example 2

[0091] The preparation method of the active magnesium-doped amorphous biomimetic calcified protein nanocarrier material in this embodiment is as follows:

[0092] Solution preparation: Weigh 0.038 g of anhydrous calcium chloride and 0.008 g of magnesium chloride hexahydrate, dissolve them in 50 mL of DMEM medium, add 10 mL of fetal bovine serum and 1 mL of penicillin-streptomycin mixture, stir well at 400 rpm, and adjust the pH of the solution to 9.0 with sodium hydroxide to obtain solution A. Weigh 0.038 g of disodium hydrogen phosphate dodecahydrate and 0.028 g of sodium dihydrogen phosphate dihydrate, dissolve them in 50 mL of DMEM medium, adjust the pH of the solution to 9.0 with sodium hydroxide, stir well at 400 rpm to obtain solution B.

[0093] The penicillin-streptomycin mixture contains 10,000 U / ml of penicillin and 10 mg / ml of streptomycin.

[0094] Wet chemical precipitation: Add 50 mL of solution B prepared in the previous step dropwise to 50 mL of solution A at a rate of 5 mL / min, and stir at 500 rpm at room temperature for 4 hours.

[0095] Cleaning: After the reaction is complete, add the resulting suspension to an 8000 molecular weight dialysis bag, use 5L of deionized water to stir and clean for 24 hours, change the solution three times, and sterilize the filter membrane.

[0096] Freeze-drying: The cleaned product was placed in a -20℃ freezer for 6 hours, and then placed in a freeze dryer at -55℃ for freeze-drying to obtain an amorphous biomimetic calcified protein nanocarrier material doped with active magnesium element, with a particle size range of 10-100nm.

[0097] Example 3

[0098] The preparation method of the amorphous biomimetic calcified protein nanocarrier material in this embodiment is as follows:

[0099] Solution preparation: Weigh 0.042 g of anhydrous calcium chloride and 0.010 g of FITC fluorescent reagent, dissolve them in 50 mL of DMEM medium, add 10 mL of fetal bovine serum and 1 mL of penicillin-streptomycin mixture, stir well at 400 rpm, and adjust the pH of the solution to 9.0 with sodium hydroxide to obtain solution A. Weigh 0.038 g of disodium hydrogen phosphate dodecahydrate and 0.028 g of sodium dihydrogen phosphate dihydrate, dissolve them in 50 mL of DMEM medium, adjust the pH of the solution to 9.0 with sodium hydroxide, stir well at 400 rpm to obtain solution B.

[0100] The penicillin-streptomycin mixture contains 10,000 U / ml of penicillin and 10 mg / ml of streptomycin.

[0101] Wet chemical precipitation: Add 50 mL of solution B prepared in the previous step dropwise to 50 mL of solution A at a rate of 5 mL / min, and stir at 500 rpm at room temperature for 4 hours.

[0102] Cleaning: After the reaction is complete, add the resulting suspension to an 8000 molecular weight dialysis bag, use 5L of deionized water to stir and clean for 24 hours, change the solution three times, and sterilize the filter membrane.

[0103] Freeze-drying: The cleaned product was placed in a freezer at -20°C for 6 hours, and then placed in a freeze dryer at -55°C for freeze-drying to obtain amorphous biomimetic calcified protein nanocarrier material loaded with fluorescent molecules, with a particle size range of 10-100 nm.

[0104] Detection: Confocal microscopy images demonstrate that the amorphous biomimetic calcified protein nanomaterials loaded with fluorescent molecules possess good fluorescence imaging properties. Figure 6 ).

[0105] Figure 6 This image shows the morphology of an amorphous biomimetic calcified protein nanocarrier material loaded with fluorescent molecules co-cultured with cells.

[0106] Example 4

[0107] The preparation method of the amorphous biomimetic calcified protein nanocarrier material in this embodiment is as follows:

[0108] Solution preparation: Weigh 0.045 g of anhydrous calcium chloride and 0.010 g of DOX, dissolve them in 50 mL of DMEM medium, add 20 mL of fetal bovine serum and 1 mL of penicillin-streptomycin mixture, stir well at 300 rpm, and adjust the pH of the solution to 8.0 with sodium hydroxide to obtain solution A. Weigh 0.038 g of disodium hydrogen phosphate dodecahydrate and 0.028 g of sodium dihydrogen phosphate dihydrate, dissolve them in 50 mL of DMEM medium, adjust the pH of the solution to 8.0 with sodium hydroxide, stir well at 300 rpm to obtain solution B.

[0109] The penicillin-streptomycin mixture contains 10,000 U / ml of penicillin and 10 mg / ml of streptomycin.

[0110] Wet chemical precipitation: Add 50 mL of solution B prepared in the previous step dropwise to 50 mL of solution A at a rate of 1 mL / min, and stir at 300 rpm at room temperature for 4 hours.

[0111] Cleaning: After the reaction is complete, add the resulting suspension to a dialysis bag with a molecular weight of 18,000, and use 5L of deionized water to stir and clean for 24 hours, changing the solution three times. Sterilize the filter membrane.

[0112] Freeze-drying: The cleaned product was placed in a freezer at -20°C for 6 hours, and then placed in a freeze dryer at -50°C for freeze-drying to obtain amorphous biomimetic calcified protein nanocarrier material loaded with fluorescent molecules, with a particle size range of 10-100 nm.

[0113] Example 5

[0114] The preparation method of the amorphous biomimetic calcified protein nanocarrier material in this embodiment is as follows:

[0115] Solution preparation: Weigh 0.1 g of anhydrous calcium chloride and dissolve it in 50 mL of DMEM medium. Add 3 mL of fetal bovine serum and 0.5 mL of a penicillin-streptomycin mixture and stir until homogeneous at 100 rpm. Adjust the pH of the solution to 10.0 with sodium hydroxide to obtain solution A. Weigh 0.1 g of disodium hydrogen phosphate dodecahydrate and dissolve it in 50 mL of DMEM medium. Adjust the pH of the solution to 10.0 with sodium hydroxide and stir until homogeneous at 500 rpm to obtain solution B.

[0116] The penicillin-streptomycin mixture contains 10,000 U / ml of penicillin and 10 mg / ml of streptomycin.

[0117] Wet chemical precipitation: Add 50 mL of solution B prepared in the previous step dropwise to 50 mL of solution A at a rate of 20 mL / min, and stir at 300 rpm at room temperature for 2 hours.

[0118] Cleaning: After the reaction is complete, add the resulting suspension to a dialysis bag with a molecular weight of 14,000, and use 5L of deionized water to stir and clean for 24 hours, changing the solution three times. Sterilize the filter membrane.

[0119] Freeze-drying: The cleaned product was placed in a freezer at -20°C for 6 hours, and then placed in a freeze dryer at -40°C for freeze-drying to obtain amorphous biomimetic calcified protein nanocarrier material loaded with fluorescent molecules, with a particle size range of 10-100 nm.

[0120] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing an amorphous biomimetic calcified protein nanocarrier material for tissue permeation drug delivery in diseases, characterized in that: Includes the following steps: (1) First, calcium and magnesium salts are dissolved in cell culture medium, then active protein molecules and penicillin-streptomycin mixture are added, and the pH is adjusted to 8-10 with sodium hydroxide to obtain a mixed solution; the concentration of calcium salt in cell culture medium is 0.1-2 g / L; the active protein molecules are fetal bovine serum, fetal globulin or albumin; the volume of active protein molecules added is 5-50% of the volume of cell culture medium; the cell culture medium is DMEM cell culture medium or 1640 cell culture medium; The penicillin-streptomycin mixture contains 10,000 U / ml of penicillin and 10 mg / ml of streptomycin; the volume of the penicillin-streptomycin mixture added is 0-3% of the cell culture medium volume. The concentration of the magnesium salt in the cell culture medium is 0-0.5 g / L; (2) First, dissolve the phosphate in the cell culture medium and adjust the pH of the solution to 8-10 with sodium hydroxide. Then, add the resulting solution dropwise to an equal volume of the mixed solution prepared in step (1). Stir at room temperature to generate an emulsion. Incubate the generated emulsion at room temperature to obtain a suspension. The concentration of the phosphate in the cell culture medium is 0.1-2 g / L. The dropping rate is 1-20 mL / min; The stirring and incubation time is 1-4 hours; (3) The suspension obtained in step (2) is washed through a dialysis bag, then sterilized by a filter membrane and freeze-dried to obtain an amorphous biomimetic calcified protein nanocarrier material for infiltration drug delivery to disease tissues. The molecular weight cutoff of the dialysis bag is 8000-18000; the freeze-drying temperature is -60 ~ (-40) ℃.

2. An amorphous biomimetic calcified protein nanocarrier material for infiltration drug delivery to diseased tissues, prepared by the method described in claim 1.

3. The application of the amorphous biomimetic calcified protein nanocarrier material for tissue permeation drug delivery as described in claim 2 in the preparation of drugs, characterized in that: The amorphous biomimetic calcified protein nanocarrier material is loaded with fluorescent reagents or tumor drug molecules.

4. The application according to claim 3, characterized in that: Add the fluorescent reagent or tumor drug molecule to the mixed solution in step (1).

5. The application according to claim 4, characterized in that: The concentration of the fluorescent reagent is 0.05-0.2 g / L; the concentration of the tumor drug molecule is 0.05-0.2 g / L.

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