Nano-cellular delivery system of bioactive macromolecule and application thereof in preparation of bone repair material

Abstract

The application belongs to the technical field of biological medicine, and discloses a nano intracellular delivery system of a bioactive macromolecule, a preparation method of the nano intracellular delivery system, and a drug-loaded nano composite material based on the nano intracellular delivery system and application of the drug-loaded nano composite material in preparation of bone repair materials. The application provides a nano delivery system of a bioactive macromolecule, which comprises a metal organic framework carrier and an amorphous calcium phosphate coating layer. The application also provides a drug-loaded nano composite material based on the nano delivery system, which can load a large amount of bioactive macromolecules, can penetrate a cell membrane to enter a cell to realize intracellular drug delivery, and can enhance the stability of the material through the interaction of the core-shell structure, further load bioactive macromolecules, delay drug release, greatly prolong the drug release time to 21 days, and achieve a long-lasting drug release effect, so that the drug-loaded nano composite material can be applied to preparation of bone repair materials. The preparation process of the composite material only uses water as a solvent, is simple to operate, is safe, and is suitable for industrial production.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and specifically relates to a nano-intracellular delivery system for bioactive macromolecules, its preparation method, and drug-loaded nanocomposites based thereon, and their application in the preparation of bone repair materials. Background Technology

[0002] Altered or absent intracellular protein function can trigger numerous diseases. Protein therapy involves delivering proteins or equivalent biomolecules into cells to replace dysfunctional proteins, producing highly specific, low-toxicity transient modulation. The rational use of protein-based bioactive macromolecules can alter cellular life activities and treat diseases. However, natural proteins and other bioactive macromolecules have poor cell membrane permeability and low utilization rates. Furthermore, bioactive macromolecules directly exposed to body fluids are prone to denaturation and degradation by proteolytic enzymes. Therefore, a delivery system is needed to load bioactive macromolecules and allow them to cross the cell membrane for release into the cell.

[0003] Combining bioactive macromolecules with cell-penetrating molecules or nanomaterials is one of the effective ways to achieve intracellular delivery of bioactive macromolecules. CN116589524A discloses an amphiphilic small molecule, its preparation method, and its application as a protein drug delivery carrier. CN113648288A discloses the preparation and application of a nanocomposite of a functional molecule coated on a erythrocyte membrane. Other researchers have used lipid nanoparticles to achieve intracellular drug delivery (Nature Communications, 11(2020), 983-996). However, the loading rates of existing materials are generally low.

[0004] Metal-organic frameworks (MOFs) are novel porous materials composed of metal ions or metal clusters and organic ligands linked by coordination bonds. MOFs possess high specific surface areas and abundant framework structures, enabling them to load and store large quantities of drugs. Studies have used ZIF-8 as a protein carrier, achieving a high drug loading rate (Journal of the American Chemical Society, 140(2018), 9912-9920). However, ZIF-8 decomposes rapidly, failing to achieve long-term drug release, and its PVP coating is organic, limiting its application in organisms. Therefore, there is an urgent need to develop an intracellular delivery system with high drug loading rates suitable for use in organisms. Summary of the Invention

[0005] In order to overcome the shortcomings and deficiencies of the prior art, the primary objective of this invention is to provide a nano-delivery system for bioactive macromolecules.

[0006] Another object of the present invention is to provide a method for preparing the above-mentioned nanodelivery system.

[0007] Another object of the present invention is to provide a drug-loaded nanocomposite material based on the above-described nanodelivery system.

[0008] Another object of the present invention is to provide the application of the above-mentioned nanodelivery system and drug-loaded nanocomposite materials.

[0009] The objective of this invention is achieved through the following solution:

[0010] The first aspect is a nanodelivery system for bioactive macromolecules, comprising a metal-organic framework (MOF) support and an amorphous calcium phosphate (ACP) encapsulation layer.

[0011] In the nanodelivery system of this invention, the metal-organic framework (MOF) support comprises a framework material formed by coordination bonds between metal ions or metal clusters and organic ligands. For example, it may include zeolite imidazole framework-8 (ZIF-8).

[0012] In the nanodelivery system of this invention, the amorphous calcium phosphate (ACP) coating layer comprises any material containing Ca. 2+ and PO4 3- The calcium phosphate material formed by the combination.

[0013] Secondly, the present invention also provides a method for preparing the above-mentioned nanodelivery system of bioactive macromolecules, comprising the following steps: reacting 2-methylimidazole with a zinc source compound in water to obtain a metal-organic framework (MOF) support; dispersing the support, calcium source compound, and phosphate in water, and reacting to obtain the nanodelivery system.

[0014] Furthermore, the molar ratio of the 2-methylimidazole to the zinc ions in the zinc source compound can be 40:1 to 100:1.

[0015] Furthermore, the calcium source compound contains Ca 2+ PO4 in phosphate 3- The molar ratio can be 0.5:1 to 2:1.

[0016] Furthermore, the calcium source compound in the reaction system contains Ca... 2+ The concentration can be 0.001–0.1 mol / L.

[0017] Furthermore, Ca in calcium source compounds 2+ Zn in metal-organic frameworks 2+ The molar ratio can be 0.222:1 to 1.11:1.

[0018] Furthermore, in the process of dispersing the support, calcium source compound, and phosphate in water, it is preferable to first disperse the support and calcium source compound in water, and then add the phosphate.

[0019] Furthermore, the phosphate is soluble in water to obtain a phosphate solution, which is then slowly added to the system for reaction. The phosphate solution can be added over a period of 0.5 to 4 hours. The reaction rate can be controlled by adjusting the timing of phosphate addition.

[0020] Furthermore, the zinc source compound includes water-soluble zinc salts, such as zinc chloride, zinc nitrate, zinc acetate, zinc sulfate, etc.

[0021] Furthermore, the calcium source compound includes water-soluble calcium salts, such as calcium chloride, calcium nitrate, and calcium acetate.

[0022] Furthermore, the phosphate may include at least one of diammonium hydrogen phosphate, disodium hydrogen phosphate, dipotassium hydrogen phosphate, ammonium dihydrogen phosphate, potassium dihydrogen phosphate, and sodium dihydrogen phosphate.

[0023] Furthermore, the temperature of reaction A can be 15–37°C; the reaction time can be 5–60 min.

[0024] Furthermore, after each reaction step in the preparation process, solid-liquid separation can be achieved through centrifugation or other methods to obtain the product. The separated product can then be post-processed through conventional operations such as washing, freeze-drying, and grinding.

[0025] Thirdly, the present invention provides a drug-loaded nanocomposite material based on the above-mentioned nanodelivery system, specifically comprising a bioactive macromolecule, a metal-organic framework (MOF) support, and an amorphous calcium phosphate (ACP) encapsulation layer.

[0026] Furthermore, the bioactive macromolecules include bioactive macromolecular materials such as peptides and proteins, such as human bone morphogenetic protein-2 (BMP-2).

[0027] The mass ratio of bioactive macromolecules to the support is 0.005:1 to 0.2:1.

[0028] In the drug-loaded nanocomposite material of the present invention, bioactive macromolecules provide the bioactivity of the material. The high specific surface area and rich, ordered framework structure of the MOF support can effectively and efficiently load bioactive macromolecules, and the bioactive macromolecules are released as the MOF structure decomposes. The ACP encapsulation layer enhances the biocompatibility of the material, enabling the material to enter cells for drug delivery. Furthermore, the ACP layer enhances the stability of the material by reacting with the MOF structure during decomposition, delaying drug release and greatly extending the drug release time to achieve a sustained drug release effect.

[0029] Fourthly, the present invention provides a method for preparing the above-mentioned drug-loaded nanocomposite material, which differs from the preparation method of the nanodelivery system in that bioactive macromolecules are added to the loading reaction system to prepare a loading body loaded with bioactive macromolecules, and then subsequent reactions are carried out.

[0030] Specifically, this involves heating and reacting bioactive macromolecules, 2-methylimidazole, and zinc source compounds in water to obtain a support containing bioactive macromolecules; then dispersing the support containing bioactive macromolecules, calcium source compounds, and phosphates in water to obtain a nanodelivery system.

[0031] Furthermore, the concentration of bioactive macromolecules in water can be 0.1–2.5 mg / mL.

[0032] Furthermore, the bioactive macromolecule can be added to the 2-methylimidazole aqueous solution first and stirred until dissolved before adding the zinc source compound for reaction; the stirring time can be 1 to 30 minutes.

[0033] Furthermore, the carrier containing the bioactive macromolecule and the calcium source compound can be first dispersed in water, ultrasonically dispersed, and then phosphate can be added for reaction. The ultrasonic dispersion time can be 10–60 min.

[0034] Furthermore, the Ca in amorphous calcium phosphate 2+ Zn in metal-organic framework support 2+ The molar ratio can be 0.222:1 to 1.11:1.

[0035] The drug-loaded nanocomposite material of this invention uses MOF as a carrier for bioactive macromolecules, which not only loads a large number of bioactive macromolecules but also provides protection against degradation by protein hydrolytic enzymes. The outer coating of ACP greatly enhances the biocompatibility of the composite material, enabling it to penetrate the cell membrane and enter the cell for intracellular drug delivery. As the MOF structure decomposes and releases bioactive macromolecules, ACP can further react with the MOF structure during the decomposition process to enhance the stability of the material, further load bioactive macromolecules, delay drug release, and greatly prolong the drug release time to achieve a sustained drug release effect and achieve effective utilization. The preparation process of the drug-loaded nanocomposite material of this invention uses only aqueous solution as a solvent, avoiding the use of organic solvents, reducing environmental pollution and harm to human health. The reaction time is short (2-3 hours), the operation is simple and easy to control, the conditions are mild (can be carried out at room temperature), the reaction conditions are simple, the safety is high, and it is suitable for industrial production.

[0036] The preparation method of this invention can control the size of the synthesized nanoparticles (<150nm) by controlling the reaction conditions, which is beneficial for the nanoparticles to enter cells to deliver drugs (the diameter of the nanoparticles that are phagocytosed by cells is within 150nm). It can be applied to fields such as the delivery of bioactive macromolecular drugs.

[0037] Fifthly, the present invention provides the application of the above-mentioned nanodelivery system of bioactive macromolecules in the preparation of bone repair materials.

[0038] In a sixth aspect, the present invention provides the application of the above-mentioned drug-loaded nanocomposite material in the preparation of bone repair materials.

[0039] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0040] 1. This invention combines a high-load MOF with a deceptive ACP to form a core-shell structure, which can efficiently load bioactive macromolecules for intracellular delivery.

[0041] 2. The synthesis process of the nanoparticles of this invention is carried out in aqueous solution, which avoids the use of organic solvents, reduces environmental pollution and harm to the human body, and lowers costs.

[0042] 3. This invention has a fast synthesis speed, mild synthesis conditions, and low equipment requirements, making it suitable for industrial production.

[0043] 4. The size of the nanoparticles in this invention can be controlled by the reaction conditions (<150nm), which is beneficial for the nanoparticles to enter the cell to deliver drugs (the diameter of the nanoparticles that are phagocytosed by the cell is within 150nm). Attached Figure Description

[0044] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0045] Figure 1 The infrared spectra of BMP-2@ZIF-8 and BMP-2@ZIF-8@ACP of the present invention are shown.

[0046] Figure 2 The XRD pattern of BMP-2@ZIF-8@ACP of this invention is shown.

[0047] Figure 3The images show the TEM spectra and particle size distributions of ZIF-8, BMP-2@ZIF-8, and BMP-2@ZIF-8@ACP of this invention.

[0048] Figure 4 The UV-Vis spectra of BMP-2@ZIF-8 and BMP-2@ZIF-8@ACP of this invention are shown.

[0049] Figure 5 The drug release curves of BMP-2@ZIF-8 and BMP-2@ZIF-8@ACP of the present invention are shown.

[0050] Figure 6 Biocompatibility testing of BMP-2@ZIF-8@ACP of this invention.

[0051] Figure 7 This invention relates to the intracellular delivery assay of BMP-2@ZIF-8@ACP.

[0052] Figures 8-11 This invention relates to the osteogenic properties testing of BMP-2@ZIF-8@ACP. Figure 8 and Figure 9 These are the results of qualitative and quantitative tests for alkaline phosphatase. Figure 10 and Figure 11 The results are qualitative and quantitative test results for calcium nodules. Detailed Implementation

[0053] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto. Unless otherwise specified, all materials involved in the following embodiments are commercially available. Unless otherwise specified, all methods described are conventional methods.

[0054] In the following examples, human bone morphogenetic protein-2 (BMP-2) is used as an example of a bioactive macromolecule, and zeolite imidazole framework-8 (ZIF-8) is used as an example of a MOF support to further illustrate the technical solution of the present invention. The amounts of each component are expressed in parts by mass and parts by volume, in g and mL.

[0055] Example 1

[0056] Dissolve 2.58 parts by mass of 2-methylimidazole in 9 parts by volume of water, then add BMP-2 and stir to dissolve it to a concentration of 0.5 mg / mL. Set the stirring time A to 10 min. Then add 1 part by volume of zinc nitrate aqueous solution to make the molar ratio of 2-methylimidazole to zinc ions 70:1. Control the temperature B to 30℃ and stir the reaction C for 10 min. After the reaction is completed, separate the solid and liquid, wash with water, and dry to obtain BMP-2@ZIF-8.

[0057] Disperse 0.1 parts by mass of BMP-2@ZIF-8 in 10 parts by volume of water and sonicate for 30 min. Add 0.033 parts by mass of calcium chloride and stir to dissolve. Slowly add 5 parts by volume of an aqueous solution containing 0.0264 parts by mass of diammonium hydrogen phosphate, controlling the addition to be completed within 2 h. After the reaction is complete, separate the solid and liquid, wash with water, and dry to obtain BMP-2@ZIF-8@ACP.

[0058] In this embodiment, the theoretical mass of amorphous calcium phosphate contains Ca. 2+ Zn in metal-organic framework support 2+ The molar ratio was determined to be 0.667:1.

[0059] The particle size of the prepared BMP-2@ZIF-8@ACP was analyzed and found to be 120 nm.

[0060] The reaction equation is shown below:

[0061] 2-Methylimidazole + Zn 2+ +BMP-2→BMP-2@ZIF-8

[0062] BMP-2@ZIF-8+Ca 2+ +P04 3- →BMP-2@ZIF-8@ACP

[0063] Example 2

[0064] The difference between this embodiment and Embodiment 1 is that the stirring time A is set to 1 min and 30 min respectively. The particle sizes of BMP-2@ZIF-8@ACP obtained with different stirring times are 113 nm and 124 nm respectively, which proves that the present invention can obtain a composite material with a particle size of about 120 nm under stirring conditions of 1 to 30 min.

[0065] Example 3

[0066] The difference between this embodiment and Example 1 is that the amount of BMP-2 added was adjusted to make its concentration 0.1 mg / mL and 2.5 mg / mL, respectively. The resulting BMP-2@ZIF-8@ACP particle sizes were 103 nm and 132 nm, respectively, proving that the present invention can obtain a composite material with a particle size of about 120 nm under the condition that the concentration of biomacromolecules is 0.1 to 2.5 mg / mL.

[0067] Example 4

[0068] The difference between this embodiment and Example 1 is that 2-methylimidazole was dissolved in 99 parts by volume and 0.9 parts by volume of water, respectively, and the amount of BMP-2 added was adjusted accordingly to maintain its concentration at 2.5 mg / mL. The concentration and amount of zinc ion solution added were also adjusted to maintain the same molar ratio of 2-methylimidazole to zinc ions as in Example 1. The resulting BMP-2@ZIF-8@ACP particle sizes were 102 nm and 122 nm, respectively, demonstrating that the concentration of biomacromolecules in the reaction system remained within the range of this invention, and that changes in the amount added did not affect the size of the resulting particles.

[0069] Example 5

[0070] The difference between this embodiment and Example 1 is that the amount of 2-methylimidazole added was changed so that its molar ratio with zinc ions was 40:1 and 100:1, respectively. The resulting BMP-2@ZIF-8@ACP particle sizes were 103 nm and 123 nm, respectively, proving that a composite material with a particle size of about 120 nm can be obtained by varying the molar ratio of 2-methylimidazole to zinc ions within the range of the present invention.

[0071] Example 6

[0072] The difference between this embodiment and Embodiment 1 is that the control temperature B was changed to 15℃ and 37℃ respectively, and the resulting BMP-2@ZIF-8@ACP particle sizes were 104nm and 131nm respectively. This proves that the composite material with a particle size of about 120nm can be obtained by changing the temperature of the metal-organic framework (MOF) support reaction process within the range of the present invention.

[0073] Example 7

[0074] The difference between this embodiment and Example 1 is that the stirring reaction time C was changed to 5 min and 60 min, and the resulting BMP-2@ZIF-8@ACP particle sizes were 88 nm and 135 nm, respectively. This proves that a composite material with a particle size of about 80 to 140 nm can be obtained by varying the reaction time within the range of the present invention.

[0075] Example 8

[0076] The difference between this embodiment and Embodiment 1 is that the ultrasonic D dispersion time was changed to 10 min and 60 min, and the resulting BMP-2@ZIF-8@ACP particle sizes were 139 nm and 121 nm, respectively, proving that a composite material with a particle size of about 120 nm can be obtained by varying the dispersion time within the range of the present invention.

[0077] Example 9

[0078] The difference between this embodiment and Example 1 is that the time for adding diammonium hydrogen phosphate aqueous solution to control E was changed to 0.5h and 4h. The resulting BMP-2@ZIF-8@ACP particle sizes were 133nm and 115nm, respectively, proving that a composite material with a particle size of about 120nm can be obtained by varying the addition time within the range of the present invention.

[0079] Example 10

[0080] The difference between this embodiment and Embodiment 1 is that Ca is changed. 2+ PO4 3- The molar ratios of Ca were 0.5:1 and 2:1, and the resulting BMP-2@ZIF-8@ACP particle sizes were 134 nm and 112 nm, respectively, proving that Ca 2+ PO4 3- The molar ratio can be varied within the range of the present invention to obtain a composite material with a particle size of approximately 120 nm.

[0081] Example 11

[0082] The difference between this embodiment and Example 1 is that the theoretical mass of amorphous calcium phosphate produced contains Ca. 2+ Zn in metal-organic framework support 2+ The molar ratios were 0.222:1 and 1.11:1, and the resulting BMP-2@ZIF-8@ACP particle sizes were 101 nm and 139 nm, respectively, demonstrating that the theoretical mass of amorphous calcium phosphate contained in the Ca content... 2+ Zn in metal-organic framework support 2+ Composite materials with a particle size of approximately 120 nm can be obtained when the molar ratio is between 0.222:1 and 1.11:1.

[0083] Comparative Example 1

[0084] Dissolve 2.58 parts by mass of 2-methylimidazole in 9 parts by volume of water, then add BMP-2 and stir to dissolve it to a concentration of 0.5 mg / mL. The stirring time A is set to 10 min. Then add 1 part by volume of zinc ion solution to make the molar ratio of 2-methylimidazole to zinc ions 7:1. Control the temperature B at 30℃ and stir the reaction C for 10 min. After the reaction is completed, the solid and liquid are separated, washed, and dried to obtain BMP-2@ZIF-8.

[0085] Disperse 0.1 parts by mass of BMP-2@ZIF-8 in 10 parts by volume of water and sonicate for 30 min. Add 0.055 parts by mass of calcium chloride and stir to dissolve. Slowly add 5 parts by volume of an aqueous solution containing 0.044 parts by mass of diammonium hydrogen phosphate, controlling the addition to be completed within 2 h. After the reaction is complete, separate the solid and liquid, wash, and dry to obtain BMP-2@ZIF-8@ACP.

[0086] In this embodiment, the theoretical mass of amorphous calcium phosphate contains Ca. 2+ Zn in metal-organic framework support 2+ The molar ratio is 0.667:1.

[0087] The particle size of the prepared BMP-2@ZIF-8@ACP was analyzed and found to be 310 nm.

[0088] Material performance testing

[0089] (1) Infrared spectroscopy was performed on BMP-2@ZIF-8@ACP from Example 1, and the results are shown below. Figure 1 As shown in the figure, the BMP-2@ZIF-8@ACP of the present invention has a chromaticity of 1666 cm⁻¹. -1 The characteristic absorption peak at 1070 cm⁻¹ corresponds to the C=O peptide bond in the protein, which is absent in ZIF-8, indicating that ZIF-8 has successfully loaded BMP-2; -1 The characteristic absorption peak at [location] corresponds to the stretching vibration absorption peak of PO, indicating the formation of calcium phosphate compounds. The difference in the preparation of ZIF-8 is that BMP-2 is not added to the reaction system, while other conditions remain unchanged.

[0090] (2) XRD pattern analysis was performed on BMP-2@ZIF-8@ACP from Example 1. The results are shown in […]. Figure 2 As shown in the figure, characteristic diffraction peaks of ZIF-8 appear between 5-30°, and the crystal form of ZIF-8 does not change after loading BMP-2. After coating with a layer of ACP, a relatively broad diffraction peak appears at around 2θ=30°, indicating that calcium phosphate has an amorphous structure. It can be inferred that the surface of BMP-2@ZIF-8 is coated with a layer of amorphous calcium phosphate.

[0091] (3) TEM and particle size analysis were performed on BMP-2@ZIF-8@ACP from Example 1. The results are shown in […]. Figure 3 As shown in the figure, ZIF-8 has a typical dodecahedral crystal structure with a particle size of approximately 73 nm. After loading BMP-2 onto ZIF-8, the microstructure changes, and the contrast becomes uneven, with the darker areas representing the loaded protein, which has a particle size of approximately 100 nm. The surface of BMP-2@ZIF-8@ACP consists of a layer of granular amorphous calcium phosphate with an increased particle size of approximately 120 nm.

[0092] (4) After acid decomposition of BMP-2@ZIF-8@ACP from Example 1, the protein concentration was measured using a UV spectrometer. The results are shown in [Figure 1]. Figure 4As shown in the figure, based on the standard curve, the drug encapsulation efficiency of BMP-2@ZIF-8 is 89.1%. The encapsulation efficiency of BMP-2@ZIF-8@ACP is 87.2%, with a loading of 40.0 μg / mg. BMP-2@ZIF-8@ACP can retain the drug in BMP-2@ZIF-8 with almost no loss.

[0093] (5) Drug release testing was performed on BMP-2@ZIF-8@ACP from Example 1. Specifically, BMP-2@ZIF and BMP-2@ZIF-8@ACP were dispersed in PBS (pH = 7.4) and placed in a constant temperature shaker (37°C). After the same time interval, the supernatant was centrifuged and the protein concentration was tested. Then, the same volume of PBS was added, and the mixture was redispersed and the release test was continued. The results are shown in […]. Figure 5 As shown in the figure, BMP-2@ZIF-8 decomposes rapidly in water (pH=7.4), releasing 80% of the loaded drug within 6 hours. However, the BMP-2@ZIF-8@ACP of this invention significantly prolongs the drug release time, achieving a BMP-2 release rate of 98.1% after 21 days.

[0094] Biological experiments with materials

[0095] (1) Biocompatibility test of BMP-2@ZIF-8@ACP

[0096] The BMP-2@ZIF-8@ACP prepared in Example 1 was used to prepare dispersions of 40, 80, and 120 μg / mL using cell culture medium (90% DMEM, 9% serum, 1% penicillin antibody). 10 μg / mL of the dispersion was added to each well of a 48-well plate. 4 Mouse bone marrow mesenchymal stem cells (extracted from mouse bone marrow) were added to 1.0 mL of the above dispersion and cultured for 5 days. Stem cell proliferation was tested using the CCK-8 assay and AM-PI live / dead staining was performed at days 1, 3, and 5. Results are shown in the figure. Figure 6 The co-culture of BMP-2@ZIF-8@ACP with mouse bone marrow mesenchymal stem cells, as demonstrated by CCK-8 assay, showed that the stem cells maintained normal proliferation even at a concentration of 40 μg / mL of BMP-2@ZIF-8@ACP. Furthermore, cell viability / deadness staining results indicated that the stem cells exhibited good growth morphology (tightly arranged in a whorl pattern, abundant cytoplasm, and smooth cell bodies), demonstrating the excellent biocompatibility of BMP-2@ZIF-8@ACP.

[0097] (2) Intracellular delivery assay of BMP-2@ZIF-8@ACP

[0098] BMP-2 was fluorescently labeled (green), and composite materials were prepared according to Example 1 and Comparative Example 1. The fluorescently labeled materials were prepared into 40 μg / mL dispersions using cell culture medium (90% DMEM, 9% serum, 1% penicillin-dextrose antibody) and co-cultured with mouse bone marrow mesenchymal stem cells for 12 h. After culture, the cells were fixed, and then the cytoskeleton (red) and nuclei (blue) were stained. The fluorescence of the cells was observed under a fluorescence microscope. The results are shown in [Figure 1]. Figure 7 As shown in the figure, the composite material of Comparative Example 1 cannot enter the cell and can only release the drug outside the cell, with only a small amount of protein entering the cell; while the BMP-2@ZIF-8@ACP of this invention has a suitable particle size and can enter the cell through endocytosis, directly delivering the protein drug into the cell, achieving intracellular protein delivery and releasing the loaded drug into the cell; it can be applied in fields such as the delivery of bioactive macromolecular drugs.

[0099] (3) Bone-promoting property test of BMP-2@ZIF-8@ACP

[0100] The BMP-2@ZIF-8@ACP-120 cells prepared in Example 1 were used to prepare a 40 μg / mL dispersion in cell culture medium (90% DMEM, 9% serum, 1% penicillin-dextrose antibody). 5 × 10⁵ cells were added to each well of a 6-well plate. 4 Mouse bone marrow mesenchymal stem cells were co-cultured with 3 mL of dispersion medium. In the BMP-2 control group, BMP-2 (4.8 μg) was added to each well, and in the ZIF-8@ACP control group, 3 mL of ZIF-8@ACP dispersion medium (40 μg / mL) was added to each well. Alkaline phosphatase (ALP) expression was tested on days 7 and 14, and calcium nodule expression was tested on day 21. The results are shown in [Figure number missing]. Figures 8-11 ALP and calcium nodules are early markers of bone formation in cells; the better the expression, the better the osteogenic effect of the material. As shown in the figure, the BMP-2@ZIF-8@ACP of this invention can promote the differentiation of stem cells into osteoblasts (cells are slender and have more filopodia), significantly increase the expression level of alkaline phosphatase, and have better calcium nodule expression, thus exhibiting excellent osteogenic effects.

[0101] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A drug-loaded nanocomposite material based on a nanodelivery system of bioactive macromolecules, characterized in that... Specifically, it consists of bioactive macromolecules, metal-organic framework supports, and an amorphous calcium phosphate encapsulation layer; The preparation process includes the following steps: reacting a bioactive macromolecule, 2-methylimidazole, and a zinc source compound in water by heating to obtain a support containing the bioactive macromolecule; dispersing the support containing the bioactive macromolecule, a calcium source compound, and a phosphate in water and reacting to obtain a nano-delivery system. The zinc source compound includes a water-soluble zinc salt; the calcium source compound includes a water-soluble calcium salt; the phosphate includes at least one of diammonium hydrogen phosphate, disodium hydrogen phosphate, dipotassium hydrogen phosphate, ammonium dihydrogen phosphate, potassium dihydrogen phosphate, and sodium dihydrogen phosphate. The particle size of the nanodelivery system is <150 nm; The molar ratio of 2-methylimidazole to zinc ions in the zinc source compound is 40:1 to 100:1; the calcium source compound contains Ca... 2 + PO4 in phosphate 3- The molar ratio is 0.5:1~2:1; the calcium source compound contains Ca 2+ Zn in metal-organic frameworks 2+ The molar ratio is 0.222:1 to 1.11:1; The concentration of bioactive macromolecules in water is 0.1~2.5 mg / mL; The bioactive macromolecule is BMP-2.

2. The drug-loaded nanocomposite material according to claim 1, characterized in that: In the reaction system, the calcium source compound contains Ca 2 + The concentration is 0.001~0.1 mol / L.

3. The drug-loaded nanocomposite material according to claim 1, characterized in that: The mass ratio of the bioactive macromolecule to the support is 0.005:1 to 0.2:

1.

4. A method for preparing a drug-loaded nanocomposite material according to any one of claims 1-3, characterized in that... Specifically, this involves heating and reacting bioactive macromolecules, 2-methylimidazole, and zinc source compounds in water to obtain a support containing bioactive macromolecules; then dispersing the support containing bioactive macromolecules, calcium source compounds, and phosphates in water to obtain a nanodelivery system.

5. The preparation method according to claim 4, characterized in that: The heating reaction temperature is 15–37°C; the reaction time is 5–60 min.

6. The preparation method according to claim 4, characterized in that: First, add the bioactive macromolecule to the 2-methylimidazole aqueous solution and stir until dissolved, then add the zinc source compound to react; the stirring time is 1 to 30 minutes.

7. The preparation method according to claim 4, characterized in that: First, the carrier containing bioactive macromolecules and the calcium source compound are dispersed in water and ultrasonically dispersed. Then, phosphate is added to react. The ultrasonic dispersion time is 10-60 min.

8. The preparation method according to claim 4, characterized in that: The phosphate is dissolved in water to obtain a phosphate solution, which is then slowly added to the system for reaction; the phosphate solution is added over a period of 0.5 to 4 hours.

9. The use of the drug-loaded nanocomposite material according to any one of claims 1-3 in the preparation of bone repair materials.

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