COFs-based nano implant material with exosome controlled release driven by biotin-avidin and preparation method of COFs-based nano implant material

Abstract

The invention discloses a COFs (covalent organic frameworks)-based nano implant material with exosome controlled release driven by biotin-avidin and a preparation method of the COFs-based nano implant material. The preparation method comprises the following steps: (1) synthesizing a COFs nano material; (2) grafting biotin on the surface of the COFs material to obtain a biotinylated COFs nano material; (3) extracting an exosome from a BMSCs (bone marrow mesenchymal stem cells) source; (4) performing biotinylation on the EXs to obtain biotinylated EXs; and (4) combining the Bi < o >-EXs with the Bi < o >-COFs, so as to obtain the COFs-based nano implant material. According to the invention, efficient loading of the MSC-EXs is realized, and the MSC-EXs can effectively resist biodegradation and immune clearance in an in-vivo environment by virtue of high affinity and stability of a biotin-avidin system, so that the problems of poor stability, low delivery efficiency, low targeting property and the like after administration of the MSC-EXs are effectively solved.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical materials, and in particular to a COFs-based nano implant material for biotin-avidin driven exosome controlled release and a preparation method thereof. Background Art

[0002] Exosomes (EXs) are biological nanoscale spherical lipid bilayer vesicles secreted by cells. They are released into the environment by donor cells in the form of exocytosis, and then fused by recipient cells in the form of endocytosis, thereby transferring biologically active RNA, proteins, lipids, cytokines, transcription factor receptors and other metabolites from donor cells to recipient cells, and affecting the biological characteristics of the latter, playing an important role in intercellular communication, cell proliferation, differentiation, anti-inflammatory, anti-tumor, etc. Among them, stem cell-derived EXs (MSC-EXs) can effectively replace stem cells to play a repair role. Compared with stem cell transplantation, MSC-EXs have better biosafety and are not prone to immune rejection. At the same time, they have good biocompatibility and a longer circulation time. These advantages make MSC-EXs a superior treatment option in many biomedical and bioengineering fields.

[0003] Although MSC-EXs have shown broad application prospects in regulating inflammation and promoting osteogenesis, clinical transformation still faces some challenges, among which the stability and retention of MSC-EXs after administration are one of the main obstacles to its clinical transformation. First, MSC-EXs are susceptible to the biological environment in the body, and their structure may be unstable due to enzymatic hydrolysis, pH changes and interference from serum proteins. This instability may cause MSC-EXs to be degraded before reaching the target tissue, weakening the therapeutic effect of the drug. Secondly, the retention problem of MSC-EXs is mainly reflected in its weak targeting. After entering the body, MSC-EXs are prone to diffuse and accumulate in non-target tissues, resulting in low efficiency in their effective delivery to the lesion site. Therefore, it is urgent to develop new MSC-EXs delivery methods to enhance their stability, prolong their half-life in the body and improve the efficiency of targeted delivery. Summary of the invention

[0004] In order to solve the above technical problems, the purpose of the present invention is to provide a biotin-avidin driven exosome controlled release COFs-based nano-implant material and a preparation method thereof, COFs are combined with MSC-EXs, and a biotin-avidin system is introduced to achieve efficient loading of MSC-EXs. At the same time, with the help of the high affinity and stability of the biotin-avidin system, MSC-EXs can effectively resist biodegradation and immune clearance in the in vivo environment, thereby maintaining the stable release and sustained effect of MSC-EXs, effectively solving the problems of poor stability of MSC-EXs after administration, low delivery efficiency and targeting.

[0005] The technical solution of the present invention to solve the above technical problems is as follows: a method for preparing a COFs-based nano-implant material for biotin-avidin driven exosome controlled release is provided, comprising the following steps:

[0006] (1) COFs synthesis: 1,3,5-tris(4-aminophenyl)benzene and 2,5-dimethoxybenzene-1,4-dicarboxaldehyde are ultrasonically dissolved in a solvent, and a Schiff base reaction is performed under the action of a catalyst, followed by elution, centrifugation and vacuum drying to obtain COFs nanomaterials;

[0007] (2) COFs biotinylation: The COFs nanomaterial obtained in step (1) is added to a reaction solution containing biotin, and the mixture is sonicated and incubated overnight at room temperature to graft biotin onto the surface of the COFs nanomaterial through an acylation reaction. The ungrafted biotin is then removed by dialysis, and the mixture is centrifuged and freeze-dried to obtain a biotinylated COFs nanomaterial, i.e., Bio-COFs.

[0008] (3) EXs extraction: extract bone marrow mesenchymal stem cells and purify them to the 3rd to 5th generation, enrich bone marrow mesenchymal stem cells, replace the exosome-free culture medium and culture them, collect the supernatant, filter and concentrate it with an ultrafiltration tube, extract it with ultracentrifugation or extract it with a kit to obtain an EXs suspension;

[0009] (4) EXs biotinylation: biotin is completely dissolved in an organic solvent to obtain a mother solution, and then the mother solution is diluted and mixed with the EXs suspension obtained in step (3), and incubated to obtain biotinylated exosomes, i.e., Bio-Exs;

[0010] (5) Bio-COFs loaded with Bio-Exs: The Bio-Exs obtained in step (4) and streptavidin are mixed to react to obtain an Avidin@Bio-EXs complex, which is then mixed with the Bio-COFs obtained in step (2) to form a Bio-EXs@Bio-COFs complex, which is then centrifuged and freeze-dried to obtain a biotin-avidin driven exosome controlled release COFs-based nanoimplant material, i.e., Bio-EXs@Bio-COFs.

[0011] Furthermore, in step (1), the molar ratio of 1,3,5-tris(4-aminophenyl)benzene to 2,5-dimethoxybenzene-1,4-dicarbaldehyde is 1:1-3.

[0012] Furthermore, in step (1), the molar volume ratio of 1,3,5-tris(4-aminophenyl)benzene, 2,5-dimethoxybenzene-1,4-dicarbaldehyde, solvent and catalyst is 0.1 mmol: 0.15 mmol: 16 mL: 1 mL.

[0013] Furthermore, in step (1), the solvent is acetonitrile solution and the catalyst is glacial acetic acid.

[0014] Furthermore, in step (1), the reaction is carried out at a temperature of 0-120° C. for 3 days.

[0015] Furthermore, in step (1), tetrahydrofuran is used for elution 2-4 times, centrifuged at 5000-10000 r / min for 5-10 min, and vacuum dried at 40-60° C. and minus 0.02-0.08 MPa for 6-12 h.

[0016] Further, in step (2), the reaction solution comprises the following components in mass percentage: 2-4% sodium chloride, 1-3% 2-(N-morpholinyl)ethanesulfonic acid, 0.3-0.6% 1-ethyl-(3-dimethylaminopropyl)-carbodiimide and 0.5-0.8% N-hydroxysuccinimide, and the balance is deionized water; the mass volume ratio of the added amount of biotin to the reaction solution is 5 mg: 4 mL; and the pH value of the reaction solution is 7-7.5.

[0017] Furthermore, the reaction solution was prepared by the following method: 97.5 mg of 2-(N-morpholinyl)ethanesulfonic acid and 290 mg of sodium chloride were dissolved in 5 mL of deionized water, and the pH value was adjusted to 6.2 to obtain a MES activation buffer; then 10 mg of 1-ethyl-(3-dimethylaminopropyl)-carbodiimide, 10 mg of N-hydroxysuccinimide and 5 mg of biotin were added to 4 mL of the MES activation buffer and mixed to obtain a reaction solution.

[0018] Furthermore, in step (2), the product is dialyzed with a 8-15 kDa dialysis bag for 24-48 h, the volume ratio of the reaction solution to deionized water is 1:100-400, centrifuged at 5000-10000 r / min for 10-30 min, and freeze-dried at -30-50° C. and -0.4-0.8 bar for 12-24 h.

[0019] Furthermore, in step (3), the culture medium without exosomes is replaced and then cultured for 24-72 hours, filtered with a 0.22 μm filter head and concentrated with a 10-100 kDa ultrafiltration tube, with a concentration multiple of 2-4 times.

[0020] Furthermore, in step (3), when ultracentrifuging, the extraction is carried out at 100,000-120,000×g and 4°C for 1-10 hours; when extracting using the kit, the volume ratio of the extraction reagent to the concentrated solution is 1:3-6, the mixture is allowed to stand for 2-24 hours, and then centrifuged at 8,000-10,000×g and 4°C for 1-2 hours.

[0021] Further, in step (2) and step (4), biotin is unmodified biotin Biotin, PEG-modified biotin PEG n-Biotin, DSPE and PEG modified biotin DSPE-PEG n -At least one of Biotin; n=200-20000.

[0022] Furthermore, in step (4), the organic solvent is at least one of methanol, ethanol, dimethyl sulfoxide and chloroform.

[0023] Furthermore, in step (4), the mother solution is diluted to 10-100 μmol / L and incubated at 37° C. for 1 h.

[0024] Furthermore, in step (5), the molar ratio of Bio-Exs to streptavidin is 1-3:1, and the mass ratio of Avidin@Bio-EXs complex to Bio-COFs is 1:1.

[0025] Furthermore, in step (5), centrifugation is performed at 5000-10000 r / min for 10-30 min, and freeze-drying is performed at -30-50° C. and -0.4-0.8 bar for 12-24 h.

[0026] The present invention also provides a COFs-based nano implant material for controlled release of exosomes driven by biotin-avidin prepared by the above preparation method.

[0027] The present invention has the following beneficial effects:

[0028] 1. The biotin-avidin driven exosome controlled release COFs-based nano-implant material of the present invention uses COFs material as a carrier, can provide more drug loading space, improve the overall efficiency and safety of the drug delivery system, and provide a new and efficient way to treat various diseases.

[0029] 2. The COFs-based nano-implant material of the present invention uses MSC-EXs as the therapeutic unit, has good biosafety, low immunogenicity and better targeted modifiability, which makes the material have a broader application prospect in the biomedical field.

[0030] 3. The present invention introduces a biotin-avidin system, the high affinity and stability of which enable MSC-EXs to effectively resist biodegradation and immune clearance in the in vivo environment, thereby maintaining the stable release and sustained action of MSC-EXs and improving the accuracy of MSC-EXs delivery and the therapeutic effect.

[0031] 4. The COFs-based nano-implant material of the present invention can provide more loading space, improve the overall efficiency and safety of the EXs delivery system, and can also effectively resist the biodegradation and immune clearance of EXs in the body, thereby maintaining the stable release and sustained action of EXs, improving the accuracy and therapeutic effect of the delivery system, and providing new ideas for the application of EXs in the biomedical field. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a scanning electron microscope image of the COFs nanomaterial obtained in Example 1;

[0033] Figure 2 This is a fluorescence verification image of the successful synthesis of Bio-PCOFs in Example 1;

[0034] Figure 3 This is a fluorescence verification image of the binding of DSPE-PEG2000-Biotin of Example 1 to the cell membrane phospholipid bilayer;

[0035] Figure 4 The biocompatibility verification results of COFs and Bio-COFs in Example 1 are shown. DETAILED DESCRIPTION

[0036] The principles and features of the present invention are described below, and the examples are only used to explain the present invention and are not used to limit the scope of the present invention. If no specific conditions are specified in the embodiments, they are carried out according to normal conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased commercially.

[0037] Example 1

[0038] A biotin-avidin driven exosome controlled release COFs-based nano implant material, the preparation method of which comprises the following steps:

[0039] (1) COFs synthesis: 20 mg of 1,3,5-tris(4-aminophenyl)benzene and 40 mg of 2,5-dimethoxybenzene-1,4-dicarboxaldehyde were ultrasonically dissolved in 20 mL of acetonitrile solution, reacted at room temperature for 3 days under the action of 1 mL of glacial acetic acid catalyst, eluted with tetrahydrofuran three times, centrifuged at 10000 r / min for 5 min, and vacuum dried at 40°C and -0.02 MPa for 12 h to obtain COFs nanomaterials;

[0040] (2) COFs biotinylation: 10 mg of the COFs nanomaterial obtained in step (1) was added to the reaction solution containing biotin, and then ultrasonicated for 10 min at room temperature overnight to graft biotin onto the surface of the COFs nanomaterial through an acylation reaction. The next day, the material was dialyzed for 24 h using an 8-12 kDa dialysis bag, with the volume ratio of the reaction solution to deionized water being 1:200. The material was centrifuged at 8000 r / min for 10 min, and freeze-dried at -50°C and -0.6 bar for 12 h to obtain biotinylated COFs nanomaterial, i.e., Bio-COFs.

[0041] The reaction solution was prepared by the following method: 97.5 mg of 2-(N-morpholinyl)ethanesulfonic acid and 290 mg of sodium chloride were dissolved in 5 mL of deionized water, and the pH value was adjusted to 6.2 to obtain a MES activation buffer; then 10 mg of 1-ethyl-(3-dimethylaminopropyl)-carbodiimide, 10 mg of N-hydroxysuccinimide and 5 mg of biotin were added to 4 mL of the MES activation buffer and mixed to obtain a reaction solution;

[0042] (3) EXs extraction: Bone marrow mesenchymal stem cells (BMSCs) were extracted and purified to the 3rd to 5th generations. About 60% of BMSCs were grown in 10 dishes (10 cm). After replacement with exosome-free culture medium, about 10 mL was added to each large dish. After 48 h of culture, about 100 mL of cell supernatant was collected, filtered with a 0.22 μm filter head, and concentrated to about 50 mL using a 100 kDa ultrafiltration tube. The EXs suspension was extracted at 120,000 × g and 4 °C for 2 h.

[0043] (4) EXs biotinylation: 9.048 mg of biotin was completely dissolved in 1 mL of anhydrous ethanol to obtain a mother solution, and then the mother solution was diluted to 50 μmol / L and mixed with the EXs suspension obtained in step (3), and incubated at 37°C for 1 h to obtain biotinylated exosomes, i.e., Bio-Exs;

[0044] (5) Bio-COFs loaded with Bio-Exs: The Bio-Exs obtained in step (4) and streptavidin were mixed at a molar ratio of 2:1 to obtain an Avidin@Bio-EXs complex, which was then mixed with the Bio-COFs obtained in step (2) at a mass ratio of 1:1 to form a Bio-EXs@Bio-COFs complex, which was centrifuged at 8000 r / min for 10 min and freeze-dried at -50°C and -0.6 bar for 10 h to obtain a COFs-based nanoimplant material for controlled release of biotin-avidin driven exosomes, namely Bio-EXs@Bio-COFs.

[0045] Example 2

[0046] A biotin-avidin driven exosome controlled release COFs-based nano implant material, the preparation method of which comprises the following steps:

[0047] (1) COFs synthesis: 20 mg of 1,3,5-tris(4-aminophenyl)benzene and 40 mg of 2,5-dimethoxybenzene-1,4-dicarboxaldehyde were ultrasonically dissolved in 20 mL of acetonitrile solution, reacted at room temperature for 3 days under the action of 1 mL of glacial acetic acid catalyst, eluted with tetrahydrofuran three times, centrifuged at 8000 r / min for 5 min, and vacuum dried at 40°C and -0.02 MPa for 6-12 h to obtain COFs nanomaterials;

[0048] (2) COFs biotinylation: 10 mg of the COFs nanomaterial obtained in step (1) was added to the reaction solution containing biotin, and then ultrasonicated for 10 min at room temperature overnight to graft biotin onto the surface of the COFs nanomaterial through an acylation reaction. The next day, the material was dialyzed for 24 h using an 8-12 kDa dialysis bag, with the volume ratio of the reaction solution to deionized water being 1:200. The material was centrifuged at 8000 r / min for 10 min, and freeze-dried at -50°C and -0.6 bar for 10 h to obtain biotinylated COFs nanomaterial, i.e., Bio-COFs.

[0049] The reaction solution was prepared by the following method: 97.5 mg of 2-(N-morpholinyl)ethanesulfonic acid and 290 mg of sodium chloride were dissolved in 5 mL of deionized water, and the pH value was adjusted to 6.2 to obtain MES activation buffer; then 10 mg of 1-ethyl-(3-dimethylaminopropyl)-carbodiimide, 10 mg of N-hydroxysuccinimide and 5 mg of biotin PEG were added. 200 -Biotin was added into 4 mL of MES activation buffer and mixed to obtain a reaction solution;

[0050] (3) EXs extraction: BMSCs were extracted and purified to the 3rd to 5th generation. About 60% of BMSCs were grown in 10 dishes (10 cm). After replacement with exosome-free culture medium, about 10 mL of culture medium was added to each large dish. After 48 h of culture, about 100 mL of cell supernatant was collected, filtered with a 0.22 μm filter head and concentrated to about 50 mL using a 100 kDa ultrafiltration tube. 5 mL of extraction reagent was added and mixed. The mixture was allowed to stand at 4°C for 16 h. The mixture was centrifuged at 10,000 × g and 4°C for 2 h to obtain an EXs suspension.

[0051] (4) EXs biotinylation: 9.048 mg biotin PEG 5000 -Biotin is completely dissolved in 1 mL of anhydrous ethanol to obtain a mother solution, and then the mother solution is diluted to 50 μmol / L and mixed with the EXs suspension obtained in step (3), and incubated at 37°C for 1 h to obtain biotinylated exosomes, namely Bio-Exs;

[0052] (5) Bio-COFs loaded with Bio-Exs: The Bio-Exs obtained in step (4) and streptavidin were mixed at a molar ratio of 2:1 to obtain an Avidin@Bio-EXs complex, which was then mixed with the Bio-COFs obtained in step (2) at a mass ratio of 1:1 to form a Bio-EXs@Bio-COFs complex, which was centrifuged at 8000 r / min for 10 min and freeze-dried at -50°C and -0.6 bar for 10 h to obtain a COFs-based nanoimplant material for controlled release of biotin-avidin driven exosomes, namely Bio-EXs@Bio-COFs.

[0053] Example 3

[0054] A biotin-avidin driven exosome controlled release COFs-based nano implant material, the preparation method of which comprises the following steps:

[0055] (1) COFs synthesis: 20 mg of 1,3,5-tris(4-aminophenyl)benzene and 40 mg of 2,5-dimethoxybenzene-1,4-dicarboxaldehyde were ultrasonically dissolved in 20 mL of acetonitrile solution, reacted at room temperature for 3 days under the action of 1 mL of glacial acetic acid catalyst, eluted with tetrahydrofuran three times, centrifuged at 7000 r / min for 5 min, and vacuum dried at 40°C and -0.02 MPa for 12 h to obtain COFs nanomaterials;

[0056] (2) COFs biotinylation: 10 mg of the COFs nanomaterial obtained in step (1) was added to the reaction solution containing biotin, and then ultrasonicated for 10 min at room temperature overnight to graft biotin onto the surface of the COFs nanomaterial through an acylation reaction. The next day, the material was dialyzed for 24 h using an 8-12 kDa dialysis bag, with the volume ratio of the reaction solution to deionized water being 1:200. The material was centrifuged at 8000 r / min for 10 min, and freeze-dried at -50°C and -0.5 bar for 12 h to obtain biotinylated COFs nanomaterial, i.e., Bio-COFs.

[0057] The reaction solution was prepared by the following method: 97.5 mg of 2-(N-morpholinyl)ethanesulfonic acid and 290 mg of sodium chloride were dissolved in 5 mL of deionized water, and the pH value was adjusted to 6.2 to obtain MES activation buffer; then 10 mg of 1-ethyl-(3-dimethylaminopropyl)-carbodiimide, 10 mg of N-hydroxysuccinimide and 5 mg of biotin PEG were added. 5000 -Biotin was added into 4 mL of MES activation buffer and mixed to obtain a reaction solution;

[0058] (3) EXs extraction: BMSCs were extracted and purified to the 3rd to 5th generation. About 60% of BMSCs were grown in 10 dishes (10 cm). After replacement with exosome-free culture medium, about 10 mL of culture medium was added to each large dish. After 48 h of culture, about 100 mL of cell supernatant was collected, filtered with a 0.22 μm filter head and concentrated to about 50 mL using a 100 kDa ultrafiltration tube. 5 mL of extraction reagent was added and mixed. The mixture was allowed to stand at 4°C for 16 h. The mixture was centrifuged at 10,000 × g and 4°C for 2 h to obtain an EXs suspension.

[0059] (4) EXs biotinylation: 9.048 mg of biotin DSPE-PEG 20000 -Biotin is completely dissolved in 1 mL of anhydrous ethanol to obtain a mother solution, and then the mother solution is diluted to 50 μmol / L and mixed with the EXs suspension obtained in step (3), and incubated at 37°C for 1 h to obtain biotinylated exosomes, namely Bio-Exs;

[0060] (5) Bio-COFs loaded with Bio-Exs: The Bio-Exs obtained in step (4) and streptavidin were mixed at a molar ratio of 3:1 to obtain an Avidin@Bio-EXs complex, which was then mixed with the Bio-COFs obtained in step (2) at a mass ratio of 1:1 to form a Bio-EXs@Bio-COFs complex, which was centrifuged at 8000 r / min for 10 min and freeze-dried at -50°C and -0.5 bar for 12 h to obtain a COFs-based nanoimplant material for controlled release of biotin-avidin driven exosomes, namely Bio-EXs@Bio-COFs.

[0061] Example 4

[0062] A biotin-avidin driven exosome controlled release COFs-based nano implant material, the preparation method of which comprises the following steps:

[0063] (1) COFs synthesis: 20 mg of 1,3,5-tris(4-aminophenyl)benzene and 40 mg of 2,5-dimethoxybenzene-1,4-dicarboxaldehyde were ultrasonically dissolved in 20 mL of acetonitrile solution, reacted at room temperature for 3 days under the action of 1 mL of glacial acetic acid catalyst, eluted with tetrahydrofuran three times, centrifuged at 6000 r / min for 5 min, and vacuum dried at 40°C and -0.02 MPa for 12 h to obtain COFs nanomaterials;

[0064] (2) COFs biotinylation: 10 mg of the COFs nanomaterial obtained in step (1) was added to the reaction solution containing biotin, and then ultrasonicated for 10 min at room temperature overnight to graft biotin onto the surface of the COFs nanomaterial through an acylation reaction. The next day, the material was dialyzed for 24 h using an 8-12 kDa dialysis bag, with the volume ratio of the reaction solution to deionized water being 1:200. The material was centrifuged at 8000 r / min for 10 min, and freeze-dried at -50°C and -0.5 bar for 12 h to obtain biotinylated COFs nanomaterial, i.e., Bio-COFs.

[0065] The reaction solution was prepared by the following method: 97.5 mg of 2-(N-morpholinyl)ethanesulfonic acid and 290 mg of sodium chloride were dissolved in 5 mL of deionized water, and the pH value was adjusted to 6.2 to obtain a MES activation buffer; then 10 mg of 1-ethyl-(3-dimethylaminopropyl)-carbodiimide, 10 mg of N-hydroxysuccinimide and 5 mg of biotin were added to 4 mL of the MES activation buffer and mixed to obtain a reaction solution;

[0066] (3) EXs extraction: BMSCs were extracted and purified to the 3rd to 5th generation. About 60% of BMSCs were grown in 10 dishes (10 cm). After replacement with exosome-free culture medium, about 10 mL of culture medium was added to each large dish. After 48 h of culture, about 100 mL of cell supernatant was collected, filtered with a 0.22 μm filter head and concentrated to about 50 mL using a 100 kDa ultrafiltration tube. 5 mL of extraction reagent was added and mixed. The mixture was allowed to stand at 4°C for 16 h. The mixture was centrifuged at 10,000 × g and 4°C for 2 h to obtain an EXs suspension.

[0067] (4) EXs biotinylation: 9.048 mg of biotin DSPE-PEG 2000 -Biotin is completely dissolved in 1 mL of anhydrous ethanol to obtain a mother solution, and then the mother solution is diluted to 50 μmol / L and mixed with the EXs suspension obtained in step (3), and incubated at 37°C for 1 h to obtain biotinylated exosomes, namely Bio-Exs;

[0068] (5) Bio-COFs loaded with Bio-Exs: The Bio-Exs obtained in step (4) and streptavidin were mixed at a molar ratio of 2:1 to obtain an Avidin@Bio-EXs complex, which was then mixed with the Bio-COFs obtained in step (2) at a mass ratio of 1:1 to form a Bio-EXs@Bio-COFs complex, which was centrifuged at 8000 r / min for 10 min and freeze-dried at -50°C and -0.5 bar for 12 h to obtain a COFs-based nanoimplant material for controlled release of biotin-avidin driven exosomes, namely Bio-EXs@Bio-COFs.

[0069] Comparative Example 1

[0070] A nano material, the preparation method of which comprises the following steps:

[0071] (1) COFs synthesis: At room temperature, weigh 36 mg of TPB and 30 mg of DMTP into a 50 mL centrifuge tube, add 16 mL of ACN as a solvent while sonicating, sonicate for 10 min to completely dissolve the monomers, then add 1 mL of HOAc as a catalyst, continue sonicating for 10 min, and react at room temperature for 3 days. Elute with THF three times, centrifuge at 6000 r / min to obtain a yellow precipitate; collect the product at 40°C, -0.02 MPa, and vacuum dry for 12 h to obtain yellow powdered COFs;

[0072] (2) EXs extraction: First, extract BMSCs, subculture and purify them to the 3rd to 5th generation for use. Cultivate 10 dishes (10 cm) to grow about 80% of BMSCs, replace the complete medium without exosomes, about 10 mL for each large dish, culture for 24 h, collect about 100 mL of cell supernatant, filter with a 0.22 μm filter head and concentrate to about 50 mL with a 100 kDa ultrafiltration tube, add 5 mL of extraction reagent, mix well, let stand at 4°C for 16 h, centrifuge at 10,000 × g, 4°C for 2 h to obtain EXs, and measure the BCA protein concentration for subsequent experiments;

[0073] (3) COFs loading EXs: EXs and COFs were mixed at a ratio of 1:1 (w / w) to form an EXs@COFs complex. The unbound EXs were removed by centrifugation at 6000 r / min, and the mixture was freeze-dried at -0.6 bar and -50 °C for 10 h to collect the EXs@COFs complex powder.

[0074] Test example

[0075] The scanning electron microscope image of the COFs nanomaterial obtained in Example 1 is obtained. Figure 1 shown.

[0076] Depend on Figure 1 It can be seen that the obtained COFs nanomaterials are spherical particles with a diameter of about 300nm.

[0077] Take an appropriate amount of the Bio-COFs obtained in Example 1 and resuspend them in PBS, mix them with AF568 and incubate them, then observe and take pictures under an upright fluorescence microscope; take an appropriate amount of DSPE-PEG 2000-Biotin was co-incubated with BMSCs and then with AF568, and the images were observed and collected under an upright fluorescence microscope; the sample extract (appropriate amount of COFs, Bio-COFs and DMEM basal culture were taken at a concentration of 1 mg / mL and shaken at 37°C for 24 hours) was prepared into a complete culture medium (10% FBS + 1% double antibody) and co-cultured with HUVECs for CCK-8 test. The Bio-EXs@Bio-COFs (2:1) complex was suspended in PBS buffer and incubated, and the supernatant was collected at different time points (1st, 4th, and 7th days). The cumulative release of EXs was detected using BCA protein quantitative analysis, and the cumulative release curve of EXs was recorded and plotted. The above results are shown as follows. Figure 2-4 shown.

[0078] Depend on Figure 2 It can be seen that by combining Bio-COFs with Alexa Fluor TM After binding with 568 streptavidin conjugate (AF568), red fluorescence was observed in Bio-COFs under a fluorescence microscope, proving that the biotinylation of COFs was successful.

[0079] Depend on Figure 3 It can be seen that by 2000 -Biotin was co-incubated with BMSCs and then mixed with AF568. Red fluorescence of BMSCs was observed under an upright fluorescence microscope, proving that DSPE-PEG 2000 -Biotin can successfully bind to the phospholipid bilayer of the cell membrane.

[0080] Depend on Figure 4 It can be seen that the results of the CCK-8 test using COFs and Bio-COFs extracts prepared into complete culture medium and co-cultured with human umbilical vein endothelial cells (HUVECs) showed that there was no statistical difference in cell survival rate between the COFs group and the Bio-COFs group compared with the control group, proving that COFs and Bio-COFs materials have no obvious cytotoxicity.

[0081] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a COFs-based nano-implant material for biotin-avidin driven exosome controlled release, characterized in that: The following steps are involved: (1) COFs synthesis: 1,3,5-tris(4-aminophenyl)benzene and 2,5-dimethoxybenzene-1,4-dicarboxaldehyde are ultrasonically dissolved in a solvent, and a Schiff base reaction is performed under the action of a catalyst, followed by elution, centrifugation and vacuum drying to obtain COFs nanomaterials; (2) COFs biotinylation: The COFs nanomaterial obtained in step (1) is added to a reaction solution containing biotin, and the mixture is sonicated and incubated overnight at room temperature to graft biotin onto the surface of the COFs nanomaterial through an acylation reaction. The ungrafted biotin is then removed by dialysis, and the mixture is centrifuged and freeze-dried to obtain a biotinylated COFs nanomaterial, i.e., Bio-COFs. (3) EXs extraction: extract bone marrow mesenchymal stem cells and purify them to the 3rd to 5th generation, enrich bone marrow mesenchymal stem cells, replace the exosome-free culture medium and culture them, collect the supernatant, filter and concentrate it with an ultrafiltration tube, extract it with ultracentrifugation or extract it with a kit to obtain an EXs suspension; (4) EXs biotinylation: biotin is completely dissolved in an organic solvent to obtain a mother solution, and then the mother solution is diluted and mixed with the EXs suspension obtained in step (3), and incubated to obtain biotinylated exosomes, i.e., Bio-Exs; (5) Bio-COFs loaded with Bio-Exs: The Bio-Exs obtained in step (4) and streptavidin are mixed to react to obtain an Avidin@Bio-EXs complex, which is then mixed with the Bio-COFs obtained in step (2) to form a Bio-EXs@Bio-COFs complex, which is then centrifuged and freeze-dried to obtain a COFs-based nano-implant material for controlled release of exosomes driven by biotin-avidin.

2. The method for preparing the COFs-based nano-implant material for biotin-avidin driven exosome controlled release according to claim 1, characterized in that: In step (1), the molar ratio of 1,3,5-tris(4-aminophenyl)benzene to 2,5-dimethoxybenzene-1,4-dicarbaldehyde is 1:1-3.

3. The method for preparing the COFs-based nano-implant material for biotin-avidin driven exosome controlled release according to claim 1, characterized in that: In step (1), the molar volume ratio of 1,3,5-tris(4-aminophenyl)benzene, 2,5-dimethoxybenzene-1,4-dicarbaldehyde, solvent and catalyst is 0.1 mmol: 0.15 mmol: 16 mL: 1 mL.

4. The method for preparing a COFs-based nano-implant material for controlled release of exosomes driven by biotin-avidin as claimed in claim 1, characterized in that: In step (2), the reaction solution comprises the following components in mass percentage: 2-4% sodium chloride, 1-3% 2-(N-morpholinyl)ethanesulfonic acid, 0.3-0.6% 1-ethyl-(3-dimethylaminopropyl)-carbodiimide and 0.5-0.8% N-hydroxysuccinimide, and the balance is deionized water; the mass volume ratio of the added amount of biotin to the reaction solution is 5 mg: 4 mL; and the pH value of the reaction solution is 7-7.

5.

5. The method for preparing a COFs-based nano-implant material for biotin-avidin driven exosome controlled release according to claim 1, characterized in that: In step (2), dialyze with a 8-15 kDa dialysis bag for 24-48 hours, the volume ratio of the reaction solution to deionized water is 1:100-400, centrifuge at 5000-10000 r / min for 10-30 minutes, and freeze-dry at -30-50°C and -0.4-0.8 bar for 12-24 hours.

6. The method for preparing a COFs-based nano-implant material for controlled release of exosomes driven by biotin-avidin as claimed in claim 1, characterized in that: In step (3), the culture medium without exosomes is replaced and then cultured for 24-72 hours. After filtration with a 0.22 μm filter, the culture medium is concentrated using a 10-100 kDa ultrafiltration tube with a concentration multiple of 2-4 times.

7. The method for preparing a COFs-based nano-implant material for controlled release of exosomes driven by biotin-avidin as claimed in claim 1, characterized in that: In step (2) and step (4), biotin is unmodified biotin Biotin, PEG-modified biotin PEG n -Biotin, DSPE and PEG modified biotin DSPE-PEG n -At least one of Biotin; n=200-20000.

8. The method for preparing a COFs-based nano-implant material for controlled release of exosomes driven by biotin-avidin as claimed in claim 1, characterized in that: In step (5), the molar ratio of Bio-Exs to streptavidin is 1-3:1, and the mass ratio of Avidin@Bio-EXs complex to Bio-COFs is 1:

1.

9. The method for preparing a COFs-based nano-implant material for controlled release of exosomes driven by biotin-avidin as claimed in claim 1, characterized in that: In step (5), centrifuge at 5000-10000 r / min for 10-30 min, and freeze-dry at -30-50° C. and -0.4-0.8 bar for 12-24 h.

10. A COFs-based nano-implant material for controlled release of exosomes driven by biotin-avidin, obtained by the method for preparing a COFs-based nano-implant material for controlled release of exosomes driven by biotin-avidin according to any one of claims 1 to 9.

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