A nanosphere drug delivery system for preventing tendon adhesion, and its preparation method and application

Abstract

The present invention proposes a nanosphere drug delivery system for preventing tendon adhesion, and its preparation method and application, which belong to the field of medical technology. Exosomes are extracted after incubation and culture of bone marrow mesenchymal stem cells, embedded in mesoporous silica microspheres, modified by tannic acid and magnetic particles, adsorbed with 6-fructose disodium phosphate and bone morphogenetic protein composition, and the surface of the obtained microspheres is coated with hyaluronic acid-silk protein peptide to obtain a nanosphere drug delivery system for preventing tendon adhesion. The nanospheres prepared by the present invention have adjustable particle size, good dispersibility, uniform particle size, and good mechanical properties, biocompatibility and degradability. In terms of structure, it has a good hollow structure and a large specific surface area effect, can efficiently load drugs, promote targeted release and sustained-controlled release of drugs, and has a good effect on promoting healing and preventing and reducing tendon adhesion.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a nanosphere drug-carrying system for preventing tendon adhesion, and a preparation method and application thereof. Background Art

[0002] Tendon injury caused by trauma or excessive exercise is a common occupational and sports disease. Tendon injury is prone to adhesion with surrounding tissues after repair surgery, which seriously affects the patient's limb function recovery and has become a difficult problem that needs to be solved in the field of surgery.

[0003] Wrapping absorbable anti-adhesion barrier materials around tendons is a widely used method in clinical practice to prevent tendon adhesion. Currently, the absorbable membranes that are widely studied include poly-DL-lactic acid medical membranes, hyaluronic acid membranes, chitosan membranes, etc. However, these absorbable anti-adhesion membranes still cannot achieve satisfactory results in clinical use, which may be related to their poor tensile strength in the body, easy rupture, inability to deliver drugs in a targeted manner, low drug loading rate and limited therapeutic effect. Summary of the invention

[0004] The purpose of the present invention is to provide a nanosphere drug delivery system for preventing tendon adhesion, and its preparation method and application, with adjustable particle size, good dispersibility, uniform particle size, and good mechanical properties, biocompatibility and degradability. In terms of structure, it has a good hollow structure and a large specific surface area effect, can efficiently load drugs, promote targeted release and slow-release drugs, and has a good effect on promoting healing and preventing and reducing tendon adhesion.

[0005] The technical solution of the present invention is achieved in this way:

[0006] The present invention provides a preparation method of a nanosphere drug delivery system for preventing tendon adhesion. Bone marrow mesenchymal stem cells are incubated and cultured to obtain exosomes, which are then embedded in mesoporous silica microspheres, modified with tannic acid and magnetic particles, and adsorbed with a composition of disodium fructose 6-phosphate and a bone morphogenetic protein. The surfaces of the obtained microspheres are coated with hyaluronic acid-silk fibroin peptides to obtain a nanosphere drug delivery system for preventing tendon adhesion.

[0007] As a further improvement of the present invention, the following steps are included:

[0008] S1. Acquisition of exosomes: Bone marrow mesenchymal stem cells were added into MSCM-sf medium and the density was adjusted to 10 6 -10 7 / mL and mixed evenly. After incubation for 7-10 days, ultra-high-speed centrifugation combined with ultrafiltration tube method was used to obtain exosome fluid.

[0009] S2. Preparation of mesoporous embedded microspheres: dissolving tetraethyl orthosilicate, a porogen and an oil-soluble emulsifier in ethyl acetate, dropping the exosome fluid in step S1, emulsifying, adjusting the pH value of the solution, stirring the reaction, centrifuging, washing, and drying at room temperature to obtain mesoporous embedded microspheres;

[0010] S3. Preparation of magnetic particles: adding ferric chloride and ferrous chloride to water, adding aqueous ammonia dropwise under inert gas protection, heating and stirring the reaction, centrifuging, washing, drying, and calcining to obtain magnetic particles;

[0011] S4 modification: the mesoporous embedded microspheres prepared in step S2 are added to water, the magnetic particles prepared in step S3, tannic acid and a catalyst are added, the reaction is heated and stirred, centrifuged, washed, and dried to obtain modified microspheres;

[0012] S5. Preparation of a drug solution: Disodium fructose 6-phosphate and a bone morphogenetic protein composition were dissolved in water and stirred to mix well to obtain a drug solution;

[0013] S6 drug loading: The modified microspheres prepared in step S4 are added to the drug solution prepared in step S5, and the solvent is evaporated to obtain drug-loaded microspheres;

[0014] S7. Sodium hyaluronate-silk fibroin peptide coating: dissolve hyaluronic acid in water, add NHS and EDC, stir to activate the reaction, add silk fibroin peptide and step S6 to prepare drug-loaded microspheres under the protection of inert gas, stir to react, centrifuge, wash, and dry to prepare a nanosphere drug-loaded system for preventing tendon adhesion.

[0015] As a further improvement of the present invention, the incubation and culturing conditions in step S1 are incubation and culturing in a constant temperature box containing 4-6v / v% CO2 at a temperature of 36-38°C, and the ultrahigh-speed centrifugation method combined with the ultrafiltration tube method is as follows: the cell fluid is centrifuged at 3-5°C and 500-1000r / min for 10-15min to remove cells, and then centrifuged at 3-5°C and 3000-4000r / min for 15-20min to remove cell debris, etc., the supernatant is concentrated by ultrafiltration tube, and centrifuged at 3-5°C and 12000-15000r / min for 20-30min to remove large vesicles and proteins, the supernatant is filtered with a 0.22μm filter membrane, and finally centrifuged at 3-5°C and 30000-35000r / min for 60-70min, and resuspended in PBS with pH=6.5-6.7 The mixture was centrifuged again at 30000-35000 r / min for 60-70 min at 3-5 ° C. The obtained precipitate was exosomes, which were resuspended in a PBS solution with a pH of 6.5-6.7 to obtain exosome fluid. In step S2, the mass ratio of tetraethyl orthosilicate, porogen, oil-soluble emulsifier and exosome fluid was 12-15:0.5-1:0.3-0.5:50, the pH value of the adjusted solution was 7.3-7.7, the stirring reaction time was 10-12 h, the porogen was selected from at least one of hexadecyldimethylbenzyl sodium chloride, hexadecyltrimethylammonium bromide, hexadecyltrimethyl sodium chloride and hexadecyltrimethylammonium chloride, and the oil-soluble emulsifier was selected from at least one of Span-20, Span-40, Span-60, Span-80 and Span-85.

[0016] As a further improvement of the present invention, the mass ratio of ferric chloride, ferrous chloride and ammonia water in step S3 is 3.2-3.3:1.2-1.3:2-4, the temperature of the heating and stirring reaction is 60-70°C, the time is 3-5h, and the temperature of the calcination is 700-800°C, and the time is 1-2h.

[0017] As a further improvement of the present invention, the mass ratio of the mesoporous embedded microspheres, magnetic particles, tannic acid and catalyst in step S4 is 10:1-2:2-4:0.1-0.2, the catalyst is a Tris-HCl solution with a pH of 8.5-9.5, the temperature of the heating and stirring reaction is 40-50°C, and the time is 1-2h.

[0018] As a further improvement of the present invention, the mass ratio of fructose 6-phosphate and the bone morphogenetic protein composition in step S5 is 10:4-7, the bone morphogenetic protein composition is a mixture of bone morphogenetic protein-2, bone morphogenetic protein-7, and bone morphogenetic protein-14, and the mass ratio is 5-7:3-5:1-2; the mass ratio of the modified microspheres and the drug solution in step S6 is 10:15-18.

[0019] As a further improvement of the present invention, the mass ratio of hyaluronic acid, NHS, EDC, silk fibroin peptide and drug-loaded microspheres in step S7 is 15-20:5-7:3-6:7-10:20, the activation reaction time is 0.5-1.5h, and the stirring reaction time is 17-20h.

[0020] As a further improvement of the present invention, the present invention specifically comprises the following steps:

[0021] S1. Acquisition of exosomes: Bone marrow mesenchymal stem cells were added into MSCM-sf medium and the density was adjusted to 10 6 -10 7 / mL and mix well, incubate in a constant temperature incubator at 36-38°C containing 4-6v / v% CO2 for 7-10 days, and obtain exosome fluid by ultra-high speed centrifugation combined with ultrafiltration tube method;

[0022] The ultra-high speed centrifugation method combined with the ultrafiltration tube method is as follows: the cell fluid is centrifuged at 3-5°C and 500-1000r / min for 10-15min to remove cells, and then centrifuged at 3-5°C and 3000-4000r / min for 15-20min to remove cell debris, etc., the supernatant is concentrated by ultrafiltration tube, and the large vesicles and proteins are removed by centrifugation at 3-5°C and 12000-15000r / min for 20-30min, and the supernatant is filtered with a 0.22μm filter membrane, and finally centrifuged at 3-5°C and 30000-35000r / min for 60-70min, and the precipitate is resuspended in PBS with pH=6.5-6.7, and centrifuged again at 3-5°C and 30000-35000r / min for 60-70min. The obtained precipitate is exosomes, which is resuspended with a PBS solution with pH=6.5-6.7 to obtain exosome fluid;

[0023] S2. Preparation of mesoporous embedded microspheres: 12-15 parts by weight of tetraethyl orthosilicate, 0.5-1 parts by weight of a porogen and 0.3-0.5 parts by weight of an oil-soluble emulsifier are dissolved in ethyl acetate, 50 parts by weight of the exosome fluid in step S1 are added dropwise, emulsified, the pH value of the solution is adjusted to 7.3-7.7, the reaction is stirred for 10-12 hours, centrifuged, washed, and dried at room temperature to obtain mesoporous embedded microspheres;

[0024] S3. Preparation of magnetic particles: 3.2-3.3 parts by weight of ferric chloride and 1.2-1.3 parts by weight of ferrous chloride were added to water, and 2-4 parts by weight of aqueous ammonia were added dropwise under inert gas protection, heated to 60-70 ° C, stirred for 3-5h, centrifuged, washed, dried, and calcined at 700-800 ° C for 1-2h to obtain magnetic particles;

[0025] S4 modification: 10 parts by weight of the mesoporous embedded microspheres prepared in step S2 were added to water, 1-2 parts by weight of the magnetic particles prepared in step S3, 2-4 parts by weight of tannic acid and 0.1-0.2 parts by weight of the catalyst were added, heated to 40-50 ° C, stirred for 1-2h, centrifuged, washed, and dried to obtain modified microspheres;

[0026] The catalyst is a Tris-HCl solution with a pH of 8.5-9.5;

[0027] S5. Preparation of a drug solution: 10 parts by weight of disodium fructose 6-phosphate, 4-7 parts by weight of a bone morphogenetic protein composition were dissolved in 100 parts by weight of water, and stirred to mix well to obtain a drug solution;

[0028] The bone morphogenetic protein composition is a mixture of bone morphogenetic protein-2, bone morphogenetic protein-7, and bone morphogenetic protein-14, with a mass ratio of 5-7:3-5:1-2;

[0029] S6 drug loading: 10 parts by weight of the modified microspheres prepared in step S4 are added to 15-18 parts by weight of the drug solution prepared in step S5, and the solvent is evaporated to obtain drug loaded microspheres;

[0030] S7. Sodium hyaluronate-silk fibroin peptide coating: dissolve 15-20 parts by weight of hyaluronic acid in water, add 5-7 parts by weight of NHS and 3-6 parts by weight of EDC, stir and activate the reaction for 0.5-1.5 hours, under the protection of inert gas, add 7-10 parts by weight of silk fibroin peptide and 20 parts by weight of the drug-loaded microspheres prepared in step S6, stir and react for 17-20 hours, centrifuge, wash, and dry to obtain a nanosphere drug-loaded system for preventing tendon adhesion.

[0031] The present invention further protects a nanosphere drug-carrying system for preventing tendon adhesion, which is prepared by the above-mentioned preparation method.

[0032] The present invention further protects the use of the above-mentioned nanosphere drug-carrying system for preventing tendon adhesion in the preparation of a medicine for preventing tendon adhesion and promoting healing after tendon injury repair surgery.

[0033] The present invention has the following beneficial effects:

[0034] Bone marrow mesenchymal stem cells exist in a variety of tissues and organs of the human body. They are multipotent stem cells that can rapidly proliferate and differentiate into other cell tissues, and can construct tissue engineering. The exosomes obtained from them can provide nutrition, promote bone tissue repair, induce bone differentiation activity through microRNA regulation, and reduce postoperative tendon adhesion. The present invention resuscitates and incubates them, obtains exosome fluid through ultra-high-speed centrifugation combined with ultrafiltration tube method, and embeds them in mesoporous hollow silica nanospheres. The mesoporous and nanosphere structures have a large specific surface area. Through chemical deposition of magnetic ferroferric oxide, the drug-loaded nanospheres can be directed to move to the repair site under the action of an external magnetic field, and the drug is released in a targeted controlled release, which has the characteristics of high efficiency and low toxicity. At the same time, it can generate a magnetic field to promote healing.

[0035] The surface of the magnetic mesoporous embedded microspheres prepared by the present invention is modified by loading with tannic acid. Tannic acid is a molecule rich in catechol groups, which exhibits good adhesion to drug molecules, can promote the adhesion of disodium fructose 6-phosphate and bone morphogenetic protein composition and adsorb it into the pores of the microspheres, achieving a sustained and controlled release effect.

[0036] Among them, disodium fructose 6-phosphate can significantly reduce the production of type I collagen in tendon cells, thereby reducing the adhesion of injured tendons after surgery. At the same time, it will not affect the healing speed and quality of injured tendons.

[0037] Bone morphogenetic protein has the ability to promote the rapid occurrence of bone, cartilage and musculoskeletal system-related connective tissue. Among them, bone morphogenetic protein-2 has a strong osteogenic ability, which can induce cells to rapidly proliferate and differentiate into other cells, has a positive effect on bone growth and repair, and helps precursor cells to differentiate into osteoblasts; bone morphogenetic protein-7 enhances the ability of periosteal cells to express alkaline phosphatase, and can induce periosteal cells to transform into osteoblasts; bone morphogenetic protein-14 can promote the formation of chondrocytes, help adipose stem cells to secrete proteoglycans and collagen fibers, so that the tensile strength of the broken ends of the injured tendons of patients is enhanced, and the thickness of the tendons of patients is thickened. Therefore, the bone morphogenetic protein composition of the present invention can promote osteogenesis, promote chondrocyte formation, promote stem cell tendon formation, reduce adhesion, and promote rapid healing and rehabilitation of patients, with a synergistic effect.

[0038] The present invention coats the surface of the drug-loaded microspheres by reacting hyaluronic acid and silk protein peptides. After being activated by NHS (N-hydroxysuccinimide) and EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide), the carboxyl group of the hyaluronic acid reacts with the amino group of the silk protein peptide to form an amide bond, thereby achieving cross-linking and coating the surface of the microspheres to form a shell layer. At the same time, after the degradation of the shell material hyaluronic acid, it can inhibit the migration and phagocytosis of granulocytes, reduce the body's inflammatory mediators, and reduce the occurrence of tendon adhesion; at the same time, hyaluronic acid can also inhibit the activity of fibroblasts, increase vascular endothelial cells, regulate the human body's microvessels, participate in the endogenous repair process of the tendon, and promote the recovery of the tendon. The silk protein peptide and hyaluronic acid also have a lubricating effect, can inhibit inflammatory mediators, reduce the body's pain, and provide favorable help for the recovery of the activity function of the joint.

[0039] The nanospheres prepared by the present invention have adjustable particle size, good dispersibility, uniform particle size, good mechanical properties, biocompatibility and degradability. In terms of structure, they have a good hollow structure and large specific surface area effect, can efficiently load drugs, promote targeted release and slow-release drugs, and have a good effect on promoting healing and preventing and reducing tendon adhesion. DETAILED DESCRIPTION

[0040] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0041] Mesenchymal stem cell culture medium (MSCM-sf) was purchased from Beijing Yuhengfeng Technology Co., Ltd. and the brand is American Sciencell.

[0042] Bone marrow mesenchymal stem cells were freeze-dried and purchased from Qingdao Aoke Biological Development Co., Ltd.

[0043] Hyaluronic acid, molecular weight 50,000-100,000, was purchased from Shaanxi Rankang Biotechnology Co., Ltd.

[0044] Silk fibroin peptide, with a protein content of >90%, was purchased from Wuhan Huaxiang Kejie Biotechnology Co., Ltd.

[0045] Example 1

[0046] This embodiment provides a method for preparing a nanosphere drug delivery system for preventing tendon adhesion, which specifically includes the following steps:

[0047] S1. Acquisition of exosomes: Bone marrow mesenchymal stem cells were added into MSCM-sf medium and the density was adjusted to 10 6 -107 / mL and mixed evenly, incubated in a constant temperature incubator containing 4v / v% CO2 at 36°C for 7 days, and then exosome fluid was obtained by ultra-high speed centrifugation combined with ultrafiltration tube method;

[0048] The ultra-high speed centrifugation method combined with the ultrafiltration tube method is as follows: the cell fluid is centrifuged at 4°C and 500 r / min for 10 min to remove cells, and then centrifuged at 4°C and 3000 r / min for 15 min to remove cell debris, etc., the supernatant is concentrated by ultrafiltration tube, and the large vesicles and proteins are removed by centrifugation at 4°C and 12000 r / min for 20 min, and the supernatant is filtered with a 0.22 μm filter membrane, and finally centrifuged at 4°C and 30000 r / min for 60 min, and the precipitate is resuspended in PBS with pH=6.5, and centrifuged again at 4°C and 30000 r / min for 60 min. The obtained precipitate is exosomes, which is resuspended with a PBS solution with pH=6.5 to obtain exosome fluid;

[0049] S2. Preparation of mesoporous embedded microspheres: 12 g of tetraethyl orthosilicate, 0.5 g of hexadecyl dimethyl benzyl sodium chloride and 0.3 g of Span-20 were dissolved in 200 mL of ethyl acetate, and 50 g of the exosome fluid in step S1 was added dropwise, emulsified at 10000 r / min for 15 min, the pH value of the solution was adjusted to 7.3, the reaction was stirred for 10 h, centrifuged, washed, and dried at room temperature to obtain mesoporous embedded microspheres;

[0050] S3. Preparation of magnetic particles: 3.2 g of ferric chloride and 1.2 g of ferrous chloride were added to 100 mL of water, 2 g of ammonia was added dropwise under nitrogen protection, heated to 60 ° C, stirred for 3 h, centrifuged, washed, dried, and calcined at 700 ° C for 1 h to obtain magnetic particles;

[0051] S4. Modification: 10 g of the mesoporous embedded microspheres prepared in step S2 were added to 100 mL of water, 1 g of the magnetic particles prepared in step S3, 2 g of tannic acid and 0.1 g of the catalyst were added, heated to 40 ° C, stirred for 1 h, centrifuged, washed, and dried to obtain modified microspheres;

[0052] The catalyst is a Tris-HCl solution with a pH of 8.5;

[0053] S5. Preparation of drug solution: 10 g of disodium fructose 6-phosphate and 4 g of bone morphogenetic protein composition were dissolved in 100 mL of water and stirred for 10 min to obtain a drug solution;

[0054] The bone morphogenetic protein composition is a mixture of bone morphogenetic protein-2, bone morphogenetic protein-7, and bone morphogenetic protein-14, with a mass ratio of 5:3:1;

[0055] S6 drug loading: 10g of the modified microspheres prepared in step S4 were added to 15g of the drug solution prepared in step S5, and the solvent was evaporated to obtain drug-loaded microspheres;

[0056] S7. Sodium hyaluronate-silk fibroin peptide coating: Dissolve 15g hyaluronic acid in 500mL water, add 5g NHS and 3g EDC, stir and activate the reaction for 0.5h, add 7g silk fibroin peptide and 20g drug-loaded microspheres prepared in step S6 under nitrogen protection, stir and react for 17h, centrifuge, wash and dry to obtain a nanosphere drug-loaded system for preventing tendon adhesion.

[0057] Example 2

[0058] This embodiment provides a method for preparing a nanosphere drug delivery system for preventing tendon adhesion, which specifically includes the following steps:

[0059] S1. Acquisition of exosomes: Bone marrow mesenchymal stem cells were added into MSCM-sf medium and the density was adjusted to 10 6 -10 7 / mL and mixed evenly, incubated in a constant temperature incubator containing 6v / v% CO2 at 38°C for 10 days, and then the exosome fluid was obtained by ultra-high speed centrifugation combined with ultrafiltration tube method;

[0060] The ultra-high speed centrifugation method combined with the ultrafiltration tube method is as follows: the cell fluid is centrifuged at 4°C and 1000 r / min for 15 min to remove cells, and then centrifuged at 4°C and 4000 r / min for 20 min to remove cell debris, etc., the supernatant is concentrated by ultrafiltration tube, and the large vesicles and proteins are removed by centrifugation at 4°C and 15000 r / min for 30 min, and the supernatant is filtered with a 0.22 μm filter membrane, and finally centrifuged at 4°C and 35000 r / min for 70 min, and the precipitate is resuspended in PBS with pH=6.7, and centrifuged again at 4°C and 35000 r / min for 70 min. The obtained precipitate is exosomes, which is resuspended with a PBS solution with pH=6.7 to obtain exosome fluid;

[0061] S2. Preparation of mesoporous embedded microspheres: 15 g of tetraethyl orthosilicate, 1 g of hexadecyltrimethylammonium bromide and 0.5 g of Span-40 were dissolved in 200 mL of ethyl acetate, and 50 g of the exosome fluid in step S1 was added dropwise, emulsified at 10000 r / min for 15 min, the pH value of the solution was adjusted to 7.7, the reaction was stirred for 12 h, centrifuged, washed, and dried at room temperature to obtain mesoporous embedded microspheres;

[0062] S3. Preparation of magnetic particles: 3.3 g of ferric chloride and 1.3 g of ferrous chloride were added to 100 mL of water, 4 g of ammonia was added dropwise under nitrogen protection, heated to 70 ° C, stirred for 5 h, centrifuged, washed, dried, and calcined at 800 ° C for 2 h to obtain magnetic particles;

[0063] S4. Modification: 10 g of the mesoporous embedded microspheres prepared in step S2 were added to 100 mL of water, 2 g of the magnetic particles prepared in step S3, 4 g of tannic acid and 0.2 g of the catalyst were added, heated to 50 ° C, stirred for 2 h, centrifuged, washed, and dried to obtain modified microspheres;

[0064] The catalyst is a Tris-HCl solution with a pH of 9.5;

[0065] S5. Preparation of drug solution: 10 g of disodium fructose 6-phosphate and 7 g of bone morphogenetic protein composition were dissolved in 100 mL of water and stirred for 10 min to obtain a drug solution;

[0066] The bone morphogenetic protein composition is a mixture of bone morphogenetic protein-2, bone morphogenetic protein-7, and bone morphogenetic protein-14, with a mass ratio of 7:5:2;

[0067] S6 drug loading: 10g of the modified microspheres prepared in step S4 were added to 18g of the drug solution prepared in step S5, and the solvent was evaporated to obtain drug-loaded microspheres;

[0068] S7. Sodium hyaluronate-silk fibroin peptide coating: Dissolve 20g of hyaluronic acid in 500mL of water, add 7g of NHS and 6g of EDC, stir and activate the reaction for 1.5h, add 10g of silk fibroin peptide and 20g of the drug-loaded microspheres prepared in step S6 under nitrogen protection, stir and react for 20h, centrifuge, wash, and dry to obtain a nanosphere drug-loaded system for preventing tendon adhesion.

[0069] Example 3

[0070] This embodiment provides a method for preparing a nanosphere drug delivery system for preventing tendon adhesion, which specifically includes the following steps:

[0071] S1. Acquisition of exosomes: Bone marrow mesenchymal stem cells were added into MSCM-sf medium and the density was adjusted to 10 6 -10 7 / mL and mixed evenly, incubated in a constant temperature incubator containing 5v / v% CO2 at 37°C for 8 days, and then exosome fluid was obtained by ultra-high speed centrifugation combined with ultrafiltration tube method;

[0072] The ultra-high speed centrifugation method combined with the ultrafiltration tube method is as follows: the cell fluid is centrifuged at 4°C and 700 r / min for 15 min to remove cells, and then centrifuged at 4°C and 3500 r / min for 20 min to remove cell debris, etc., the supernatant is concentrated by ultrafiltration tube, and the large vesicles and proteins are removed by centrifugation at 4°C and 13500 r / min for 25 min, and the supernatant is filtered with a 0.22 μm filter membrane, and finally centrifuged at 4°C and 32000 r / min for 65 min, and the precipitate is resuspended in PBS with pH=6.6, and centrifuged again at 4°C and 32000 r / min for 65 min. The obtained precipitate is exosomes, which is resuspended with a PBS solution with pH=6.6 to obtain exosome fluid;

[0073] S2. Preparation of mesoporous embedded microspheres: 13.5 g of tetraethyl orthosilicate, 0.7 g of hexadecyltrimethyl sodium chloride and 0.4 g of Span-85 were dissolved in 200 mL of ethyl acetate, and 50 g of the exosome fluid in step S1 was added dropwise, emulsified at 10000 r / min for 15 min, the pH value of the solution was adjusted to 7.5, the reaction was stirred for 11 h, centrifuged, washed, and dried at room temperature to obtain mesoporous embedded microspheres;

[0074] S3. Preparation of magnetic particles: 3.24 g of ferric chloride and 1.26 g of ferrous chloride were added to 100 mL of water, 3 g of aqueous ammonia was added dropwise under nitrogen protection, heated to 65 ° C, stirred for 4 h, centrifuged, washed, dried, and calcined at 750 ° C for 1.5 h to obtain magnetic particles;

[0075] S4. Modification: 10 g of the mesoporous embedded microspheres prepared in step S2 were added to 100 mL of water, 1.5 g of the magnetic particles prepared in step S3, 3 g of tannic acid and 0.15 g of the catalyst were added, heated to 45 ° C, stirred for 1.5 h, centrifuged, washed, and dried to obtain modified microspheres;

[0076] The catalyst is a Tris-HCl solution with a pH of 9;

[0077] S5. Preparation of drug solution: 10 g of disodium fructose 6-phosphate and 5.5 g of bone morphogenetic protein composition were dissolved in 100 mL of water and stirred for 10 min to obtain a drug solution;

[0078] The bone morphogenetic protein composition is a mixture of bone morphogenetic protein-2, bone morphogenetic protein-7, and bone morphogenetic protein-14, with a mass ratio of 6:4:1.5;

[0079] S6 drug loading: 10g of the modified microspheres prepared in step S4 were added to 17g of the drug solution prepared in step S5, and the solvent was evaporated to obtain drug-loaded microspheres;

[0080] S7. Sodium hyaluronate-silk fibroin peptide coating: Dissolve 17 g of hyaluronic acid in 500 mL of water, add 6 g of NHS and 4.5 g of EDC, stir and activate the reaction for 1 hour, add 8.4 g of silk fibroin peptide and 20 g of the drug-loaded microspheres prepared in step S6 under nitrogen protection, stir and react for 18.5 hours, centrifuge, wash, and dry to obtain a nanosphere drug-loaded system for preventing tendon adhesion.

[0081] Comparative Example 1

[0082] Compared with Example 3, the difference is that the porogen hexadecyltrimethyl sodium chloride is not added in step S2.

[0083] The details are as follows:

[0084] S2. Preparation of embedded microspheres: 13.5 g of tetraethyl orthosilicate and 0.4 g of Span-85 were dissolved in 200 mL of ethyl acetate, and 50 g of the exosome fluid in step S1 was added dropwise. The mixture was emulsified at 10,000 r / min for 15 min, the pH value of the solution was adjusted to 7.5, and the reaction was stirred for 11 h. The mixture was centrifuged, washed, and dried at room temperature to obtain embedded microspheres.

[0085] Comparative Example 2

[0086] Compared with Example 3, the difference is that step S3 is not performed.

[0087] The details are as follows:

[0088] S1. Acquisition of exosomes: Bone marrow mesenchymal stem cells were added into MSCM-sf medium and the density was adjusted to 10 6 -10 7 / mL and mixed evenly, incubated in a constant temperature incubator containing 5v / v% CO2 at 37°C for 8 days, and then exosome fluid was obtained by ultra-high speed centrifugation combined with ultrafiltration tube method;

[0089] The ultra-high speed centrifugation method combined with the ultrafiltration tube method is as follows: the cell fluid is centrifuged at 4°C and 700 r / min for 15 min to remove cells, and then centrifuged at 4°C and 3500 r / min for 20 min to remove cell debris, etc., the supernatant is concentrated by ultrafiltration tube, and the large vesicles and proteins are removed by centrifugation at 4°C and 13500 r / min for 25 min, and the supernatant is filtered with a 0.22 μm filter membrane, and finally centrifuged at 4°C and 32000 r / min for 65 min, and the precipitate is resuspended in PBS with pH=6.6, and centrifuged again at 4°C and 32000 r / min for 65 min. The obtained precipitate is exosomes, which is resuspended with a PBS solution with pH=6.6 to obtain exosome fluid;

[0090] S2. Preparation of mesoporous embedded microspheres: 13.5 g of tetraethyl orthosilicate, 0.7 g of hexadecyltrimethyl sodium chloride and 0.4 g of Span-85 were dissolved in 200 mL of ethyl acetate, and 50 g of the exosome fluid in step S1 was added dropwise, emulsified at 10000 r / min for 15 min, the pH value of the solution was adjusted to 7.5, the reaction was stirred for 11 h, centrifuged, washed, and dried at room temperature to obtain mesoporous embedded microspheres;

[0091] S3 modification: 10g of the mesoporous embedded microspheres prepared in step S2 were added to 100mL of water, 4.5g of tannic acid and 0.15g of catalyst were added, heated to 45°C, stirred for 1.5h, centrifuged, washed, and dried to obtain modified microspheres;

[0092] The catalyst is a Tris-HCl solution with a pH of 9;

[0093] S4. Preparation of drug solution: 10 g of disodium fructose 6-phosphate and 5.5 g of bone morphogenetic protein composition were dissolved in 100 mL of water and stirred for 10 min to obtain a drug solution;

[0094] The bone morphogenetic protein composition is a mixture of bone morphogenetic protein-2, bone morphogenetic protein-7, and bone morphogenetic protein-14, with a mass ratio of 6:4:1.5;

[0095] S5 drug loading: 10g of the modified microspheres prepared in step S3 were added to 17g of the drug solution prepared in step S4, and the solvent was evaporated to obtain drug-loaded microspheres;

[0096] S6. Sodium hyaluronate-silk fibroin peptide coating: Dissolve 17 g of hyaluronic acid in 500 mL of water, add 6 g of NHS and 4.5 g of EDC, stir and activate the reaction for 1 hour, add 8.4 g of silk fibroin peptide and 20 g of the drug-loaded microspheres prepared in step S5 under nitrogen protection, stir and react for 18.5 hours, centrifuge, wash, and dry to obtain a nanosphere drug-loaded system for preventing tendon adhesion.

[0097] Comparative Example 3

[0098] Compared with Example 3, the difference is that no tannic acid modification is performed in step S4.

[0099] The details are as follows:

[0100] S1. Acquisition of exosomes: Bone marrow mesenchymal stem cells were added into MSCM-sf medium and the density was adjusted to 10 6 -10 7 / mL and mixed evenly, incubated in a constant temperature incubator containing 5v / v% CO2 at 37°C for 8 days, and then exosome fluid was obtained by ultra-high speed centrifugation combined with ultrafiltration tube method;

[0101] The ultra-high speed centrifugation method combined with the ultrafiltration tube method is as follows: the cell fluid is centrifuged at 4°C and 700 r / min for 15 min to remove cells, and then centrifuged at 4°C and 3500 r / min for 20 min to remove cell debris, etc., the supernatant is concentrated by ultrafiltration tube, and the large vesicles and proteins are removed by centrifugation at 4°C and 13500 r / min for 25 min, and the supernatant is filtered with a 0.22 μm filter membrane, and finally centrifuged at 4°C and 32000 r / min for 65 min, and the precipitate is resuspended in PBS with pH=6.6, and centrifuged again at 4°C and 32000 r / min for 65 min. The obtained precipitate is exosomes, which is resuspended with a PBS solution with pH=6.6 to obtain exosome fluid;

[0102] S2. Preparation of mesoporous embedded microspheres: 13.5 g of tetraethyl orthosilicate, 0.7 g of hexadecyltrimethyl sodium chloride and 0.4 g of Span-85 were dissolved in 200 mL of ethyl acetate, and 50 g of the exosome fluid in step S1 was added dropwise, emulsified at 10000 r / min for 15 min, the pH value of the solution was adjusted to 7.5, the reaction was stirred for 11 h, centrifuged, washed, and dried at room temperature to obtain mesoporous embedded microspheres;

[0103] S3. Preparation of magnetic particles: 3.24 g of ferric chloride and 1.26 g of ferrous chloride were added to 100 mL of water, 3 g of aqueous ammonia was added dropwise under nitrogen protection, heated to 65 ° C, stirred for 4 h, centrifuged, washed, dried, and calcined at 750 ° C for 1.5 h to obtain magnetic particles;

[0104] S4. Modification: 10 g of the mesoporous embedded microspheres prepared in step S2 were added to 100 mL of water, 4.5 g of the magnetic particles prepared in step S3 were added, heated to 45 ° C, stirred for 1.5 h, centrifuged, washed, and dried to obtain modified microspheres;

[0105] S5. Preparation of drug solution: 10 g of disodium fructose 6-phosphate and 5.5 g of bone morphogenetic protein composition were dissolved in 100 mL of water and stirred for 10 min to obtain a drug solution;

[0106] The bone morphogenetic protein composition is a mixture of bone morphogenetic protein-2, bone morphogenetic protein-7, and bone morphogenetic protein-14, with a mass ratio of 6:4:1.5;

[0107] S6 drug loading: 10g of the modified microspheres prepared in step S4 were added to 17g of the drug solution prepared in step S5, and the solvent was evaporated to obtain drug-loaded microspheres;

[0108] S7. Sodium hyaluronate-silk fibroin peptide coating: Dissolve 17 g of hyaluronic acid in 500 mL of water, add 6 g of NHS and 4.5 g of EDC, stir and activate the reaction for 1 hour, add 8.4 g of silk fibroin peptide and 20 g of the drug-loaded microspheres prepared in step S6 under nitrogen protection, stir and react for 18.5 hours, centrifuge, wash, and dry to obtain a nanosphere drug-loaded system for preventing tendon adhesion.

[0109] Comparative Example 4

[0110] Compared with Example 3, the difference is that disodium fructose 6-phosphate is not added in step S5.

[0111] The details are as follows:

[0112] S5. Preparation of drug solution: 15.5 g of the bone morphogenetic protein composition was dissolved in 100 mL of water and stirred for 10 min to obtain a drug solution;

[0113] The bone morphogenetic protein composition is a mixture of bone morphogenetic protein-2, bone morphogenetic protein-7 and bone morphogenetic protein-14, with a mass ratio of 6:4:1.5.

[0114] Comparative Example 5

[0115] Compared with Example 3, the difference is that no bone morphogenetic protein composition is added in step S5.

[0116] The details are as follows:

[0117] S5. Preparation of drug solution: Dissolve 15.5 g of disodium fructose 6-phosphate in 100 mL of water and stir for 10 minutes to prepare a drug solution.

[0118] Comparative Example 6

[0119] Compared with the embodiment 3, the difference is that steps S5 and S6 are not performed.

[0120] The details are as follows:

[0121] S1. Acquisition of exosomes: Bone marrow mesenchymal stem cells were added into MSCM-sf medium and the density was adjusted to 10 6 -10 7 / mL and mixed evenly, incubated in a constant temperature incubator containing 5v / v% CO2 at 37°C for 8 days, and then exosome fluid was obtained by ultra-high speed centrifugation combined with ultrafiltration tube method;

[0122] The ultra-high speed centrifugation method combined with the ultrafiltration tube method is as follows: the cell fluid is centrifuged at 4°C and 700 r / min for 15 min to remove cells, and then centrifuged at 4°C and 3500 r / min for 20 min to remove cell debris, etc., the supernatant is concentrated by ultrafiltration tube, and the large vesicles and proteins are removed by centrifugation at 4°C and 13500 r / min for 25 min, and the supernatant is filtered with a 0.22 μm filter membrane, and finally centrifuged at 4°C and 32000 r / min for 65 min, and the precipitate is resuspended in PBS with pH=6.6, and centrifuged again at 4°C and 32000 r / min for 65 min. The obtained precipitate is exosomes, which is resuspended with a PBS solution with pH=6.6 to obtain exosome fluid;

[0123] S2. Preparation of mesoporous embedded microspheres: 13.5 g of tetraethyl orthosilicate, 0.7 g of hexadecyltrimethyl sodium chloride and 0.4 g of Span-85 were dissolved in 200 mL of ethyl acetate, and 50 g of the exosome fluid in step S1 was added dropwise, emulsified at 10000 r / min for 15 min, the pH value of the solution was adjusted to 7.5, the reaction was stirred for 11 h, centrifuged, washed, and dried at room temperature to obtain mesoporous embedded microspheres;

[0124] S3. Preparation of magnetic particles: 3.24 g of ferric chloride and 1.26 g of ferrous chloride were added to 100 mL of water, 3 g of aqueous ammonia was added dropwise under nitrogen protection, heated to 65 ° C, stirred for 4 h, centrifuged, washed, dried, and calcined at 750 ° C for 1.5 h to obtain magnetic particles;

[0125] S4. Modification: 10 g of the mesoporous embedded microspheres prepared in step S2 were added to 100 mL of water, 1.5 g of the magnetic particles prepared in step S3, 3 g of tannic acid and 0.15 g of the catalyst were added, heated to 45 ° C, stirred for 1.5 h, centrifuged, washed, and dried to obtain modified microspheres;

[0126] The catalyst is a Tris-HCl solution with a pH of 9;

[0127] S5. Sodium hyaluronate-silk fibroin peptide coating: Dissolve 17 g of hyaluronic acid in 500 mL of water, add 6 g of NHS and 4.5 g of EDC, stir and activate the reaction for 1 hour, add 8.4 g of silk fibroin peptide and 20 g of modified microspheres prepared in step S4 under nitrogen protection, stir and react for 18.5 hours, centrifuge, wash, and dry to obtain a nanosphere drug delivery system for preventing tendon adhesion.

[0128] Comparative Example 7

[0129] Compared with the embodiment 3, the difference is that step S7 is not performed.

[0130] The details are as follows:

[0131] S1. Acquisition of exosomes: Bone marrow mesenchymal stem cells were added into MSCM-sf medium and the density was adjusted to 10 6 -10 7 / mL and mixed evenly, incubated in a constant temperature incubator containing 5v / v% CO2 at 37°C for 8 days, and then exosome fluid was obtained by ultra-high speed centrifugation combined with ultrafiltration tube method;

[0132] The ultra-high speed centrifugation method combined with the ultrafiltration tube method is as follows: the cell fluid is centrifuged at 4°C and 700 r / min for 15 min to remove cells, and then centrifuged at 4°C and 3500 r / min for 20 min to remove cell debris, etc., the supernatant is concentrated by ultrafiltration tube, and the large vesicles and proteins are removed by centrifugation at 4°C and 13500 r / min for 25 min, and the supernatant is filtered with a 0.22 μm filter membrane, and finally centrifuged at 4°C and 32000 r / min for 65 min, and the precipitate is resuspended in PBS with pH=6.6, and centrifuged again at 4°C and 32000 r / min for 65 min. The obtained precipitate is exosomes, which is resuspended with a PBS solution with pH=6.6 to obtain exosome fluid;

[0133] S2. Preparation of mesoporous embedded microspheres: 13.5 g of tetraethyl orthosilicate, 0.7 g of hexadecyltrimethyl sodium chloride and 0.4 g of Span-85 were dissolved in 200 mL of ethyl acetate, and 50 g of the exosome fluid in step S1 was added dropwise, emulsified at 10000 r / min for 15 min, the pH value of the solution was adjusted to 7.5, the reaction was stirred for 11 h, centrifuged, washed, and dried at room temperature to obtain mesoporous embedded microspheres;

[0134] S3. Preparation of magnetic particles: 3.24 g of ferric chloride and 1.26 g of ferrous chloride were added to 100 mL of water, 3 g of aqueous ammonia was added dropwise under nitrogen protection, heated to 65 ° C, stirred for 4 h, centrifuged, washed, dried, and calcined at 750 ° C for 1.5 h to obtain magnetic particles;

[0135] S4. Modification: 10 g of the mesoporous embedded microspheres prepared in step S2 were added to 100 mL of water, 1.5 g of the magnetic particles prepared in step S3, 3 g of tannic acid and 0.15 g of the catalyst were added, heated to 45 ° C, stirred for 1.5 h, centrifuged, washed, and dried to obtain modified microspheres;

[0136] The catalyst is a Tris-HCl solution with a pH of 9;

[0137] S5. Preparation of drug solution: 10 g of disodium fructose 6-phosphate and 5.5 g of bone morphogenetic protein composition were dissolved in 100 mL of water and stirred for 10 min to obtain a drug solution;

[0138] The bone morphogenetic protein composition is a mixture of bone morphogenetic protein-2, bone morphogenetic protein-7, and bone morphogenetic protein-14, with a mass ratio of 6:4:1.5;

[0139] S6. Drug loading: 10 g of the modified microspheres prepared in step S4 are added to 17 g of the drug solution prepared in step S5, and the solvent is evaporated to obtain drug-loaded microspheres, which are nanosphere drug loading systems for preventing tendon adhesion.

[0140] Test Example 1

[0141] Breast cancer cells (HeLa) were cultured in MEM medium containing 10 v / v% fetal bovine serum, 50 μL / mL penicillin and 50 μL / mL streptomycin. The cells were incubated at 37°C and 5% CO2. Cytotoxicity was determined by the standard MTT method. HeLa cells in the logarithmic growth phase were taken at 1×10 4 / well density was inoculated into a 96-well plate and cultured overnight. The cells were then incubated for 24 hours with the nanosphere drug delivery system for preventing tendon adhesion prepared in Examples 1-3 and Comparative Examples 1-7 (with blank culture medium as control). After removing the culture medium, the cells were washed with PBS and treated with a standard MTT assay. Fresh culture medium with a concentration of 0.5 mg / mL of MTT reagent was added to each well and incubated for 4 hours. After removing the culture medium, 150 μL of dimethyl sulfoxide was added to the microplate wells to dissolve the formazan crystals. The absorbance at 570 nm was measured using an ELISA instrument to determine cell viability. The results are shown in Table 1.

[0142] Table 1

[0143]

[0144]

[0145] It can be seen from the above table that the nanosphere drug delivery system for preventing tendon adhesion prepared in the present invention has a higher cell survival rate and therefore has good biocompatibility.

[0146] Test Example 2

[0147] Thirty-one adult male New Zealand white rabbits with a body weight of 2-2.4 kg were selected and fed in a standardized manner. After 7 days, the experimental animals ate and moved normally before surgery. The experimental animals were divided into 11 groups, Example 1-3 groups and Comparative Example 1-7 groups, and a control group, with 10 rabbits in each group.

[0148] The experimental animals were anesthetized, and the palmar skin of the second toe of both hind limbs was prepared after the effect. The animals were fixed on the operating table in a prone position, and the skin was cut longitudinally on the plantar side of the proximal segment of the second toe of both hind limbs, separated layer by layer to expose the tendon, separated the deep and superficial flexor tendons of the toe, and the deep flexor tendons of the toe were picked up by vascular clamps. The deep flexor tendons of the toe were sharply cut off at the midpoint of the proximal segment using a blade, and the deep flexor tendons of the toe were repaired by the eight-character method. The anastomosis of the embodiment 1-3 and the comparative example 1-7 groups was applied with 1g / kg of the corresponding nanosphere drug delivery system for preventing tendon adhesion; the control group was not treated, the skin was sutured continuously, and the bilateral hind limbs were fixed with plaster. After the animal model was established, the animals were free to move in the cage, and antibiotics were injected intramuscularly every day for 3 days after surgery, and the eating and activity conditions were recorded.

[0149] All animals in the control group and the experimental group were killed by air embolism 6 weeks after surgery, the tendon sliding distance was measured, and the tensile strength test was performed after the tendon was separated.

[0150] Examination of adhesions around the tendon:

[0151] After the experimental animals were killed, the plasters of both hind limbs were removed, both feet were amputated, the skin was longitudinally incised along the palmar side of the second metatarsal bone, the subcutaneous tissue was separated, the tendon anastomosis was exposed, and the adhesion between the tendon and the surrounding tissue was observed. The score was scored according to the scoring criteria (Table 2), and the average total score of each group was taken.

[0152] Table 2

[0153]

[0154] Tendon slip distance measurement:

[0155] The hind limb was removed along the distal side of the ankle joint, the skin on the plantar side was cut, the deep flexor tendon of the digitorum profundus was exposed and the deep flexor tendon of the second toe was separated. The proximal end of the tendon was fixed on a tension-free suture, and the other end of the suture was fixed on a standardized weight (146g). The proximal end of the specimen was vertically fixed on a tendon tensioner, and the skin on both sides of the proximal segment of the second toe was fixed so that only the mid-distal segment of the second toe could move. The second toe was kept in an extended position, the deep flexor tendon of the second toe was slightly tense, and the standardized weight was released. After stabilization, the sliding distance of the deep flexor tendon of the second toe was measured.

[0156] Tensile strength test:

[0157] Fix both ends of the fresh tendon on a biomechanical measuring instrument, with the fixing point at least 1 cm away from the tendon anastomosis. Pull the tendon at a speed of 25 mm / min and record the tension value when the tendon ruptures.

[0158] The results are shown in Table 3.

[0159] Table 3

[0160] Group Adhesion score (points) Average tendon slip distance (mm) Average tensile strength of tendon (N) Control group 8.67±0.32 2.1±0.2 19.4±1.3 Example 1 2.97±0.13* 4.2±0.2* 25.4±1.1* Example 2 2.94±0.16* 4.4±0.3* 25.3±1.4* Example 3 2.92±0.11* 4.5±0.1* 25.9±1.0* Comparative Example 1 6.89±0.29 3.8±0.5 24.1±1.9 Comparative Example 2 6.13±0.19 4.0±0.4 25.0±1.7 Comparative Example 3 6.94±0.27 3.7±0.7 23.9±1.5 Comparative Example 4 7.10±0.23 3.2±0.6 22.4±1.6 Comparative Example 5 7.97±0.22 3.6±0.7 23.1±1.5 Comparative Example 6 8.35±0.20 2.9±0.6 21.8±1.7 Comparative Example 7 7.28±0.26 3.1±0.7 22.9±1.8

[0161] Note: * compared with the control group, P < 0.05.

[0162] It can be seen from the above table that the nanosphere drug delivery system for preventing tendon adhesion prepared in Examples 1-3 of the present invention has a good effect in preventing adhesion and improves the average sliding distance and average tensile strength of the tendon.

[0163] 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 nanosphere drug delivery system for preventing tendon adhesion, characterized in that: Exosomes are extracted from bone marrow mesenchymal stem cells after incubation and culture, and are embedded in mesoporous silica microspheres. The microspheres are modified with tannic acid and magnetic particles, and adsorbed with a combination of disodium fructose 6-phosphate and bone morphogenetic protein. The surface of the microspheres is coated with hyaluronic acid-silk fibroin peptides to prepare a nanosphere drug delivery system for preventing tendon adhesion. The preparation method steps are: S1. Acquisition of exosomes: Bone marrow mesenchymal stem cells were added into MSCM-sf medium and the density was adjusted to 10 6 -10 7 / mL and mixed evenly. After incubation for 7-10 days, ultra-high-speed centrifugation combined with ultrafiltration tube method was used to obtain exosome fluid. S2. Preparation of mesoporous embedded microspheres: dissolving tetraethyl orthosilicate, a porogen and an oil-soluble emulsifier in ethyl acetate, dropping the exosome fluid in step S1, emulsifying, adjusting the pH value of the solution, stirring the reaction, centrifuging, washing, and drying at room temperature to obtain mesoporous embedded microspheres; S3. Preparation of magnetic particles: adding ferric chloride and ferrous chloride to water, adding aqueous ammonia dropwise under inert gas protection, heating and stirring the reaction, centrifuging, washing, drying, and calcining to obtain magnetic particles; S4 modification: the mesoporous embedded microspheres prepared in step S2 are added to water, the magnetic particles prepared in step S3, tannic acid and a catalyst are added, the reaction is heated and stirred, centrifuged, washed, and dried to obtain modified microspheres; S5. Preparation of a drug solution: Disodium fructose 6-phosphate and a bone morphogenetic protein composition were dissolved in water and stirred to mix well to obtain a drug solution; S6 drug loading: The modified microspheres prepared in step S4 are added to the drug solution prepared in step S5, and the solvent is evaporated to obtain drug-loaded microspheres; S7. Sodium hyaluronate-silk fibroin peptide coating: dissolve hyaluronic acid in water, add NHS and EDC, stir to activate the reaction, add silk fibroin peptide and step S6 to prepare drug-loaded microspheres under the protection of inert gas, stir to react, centrifuge, wash, and dry to prepare a nanosphere drug-loaded system for preventing tendon adhesion.

2. The preparation method according to claim 1, characterized in that: The incubation conditions in step S1 are as follows: the incubation is carried out in a constant temperature incubator at 36-38°C and containing 4-6 v / v% CO2. The ultracentrifugation method combined with the ultrafiltration tube method is as follows: the cell fluid is centrifuged at 3-5°C and 500-1000 r / min for 10-15 min to remove cells, and then at 3-5°C and 3000-4000 r / min for 15-20 min to remove cell debris, and the supernatant is concentrated by ultrafiltration tube, and at 3-5°C and 12000-150 00r / min centrifugation for 20-30min to remove large vesicles and proteins, the supernatant was filtered with a 0.22μm filter membrane, and finally centrifuged at 3-5℃, 30000-35000r / min for 60-70min, the precipitate was resuspended in PBS with pH=6.5-6.7, and centrifuged again at 3-5℃, 30000-35000r / min for 60-70min. The precipitate obtained was exosomes, which was resuspended in PBS solution with pH=6.5-6.7 to obtain exosome fluid; In step S2, the mass ratio of tetraethyl orthosilicate, porogen, oil-soluble emulsifier and exosome fluid is 12-15:0.5-1:0.3-0.5:50, the pH value of the adjusted solution is 7.3-7.7, the stirring reaction time is 10-12h, the porogen is selected from at least one of hexadecyldimethylbenzyl sodium chloride, hexadecyltrimethylammonium bromide, hexadecyltrimethyl sodium chloride and hexadecyltrimethylammonium chloride, and the oil-soluble emulsifier is selected from at least one of Span-20, Span-40, Span-60, Span-80 and Span-85.

3. The preparation method according to claim 1, characterized in that: In step S3, the mass ratio of ferric chloride, ferrous chloride and ammonia water is 3.2-3.3:1.2-1.3:2-4, the temperature of the heating and stirring reaction is 60-70°C, the time is 3-5h, and the temperature of the calcination is 700-800°C, the time is 1-2h.

4. The preparation method according to claim 1, characterized in that: The mass ratio of the mesoporous embedded microspheres, magnetic particles, tannic acid and catalyst in step S4 is 10:1-2:2-4:0.1-0.2, the catalyst is a Tris-HCl solution with a pH of 8.5-9.5, the temperature of the heating and stirring reaction is 40-50°C, and the time is 1-2h.

5. The preparation method according to claim 1, characterized in that: The mass ratio of fructose 6-phosphate and bone morphogenetic protein composition in step S5 is 10:4-7, and the bone morphogenetic protein composition is a mixture of bone morphogenetic protein-2, bone morphogenetic protein-7, and bone morphogenetic protein-14, with a mass ratio of 5-7:3-5:1-2; the mass ratio of modified microspheres and drug solution in step S6 is 10:15-18.

6. The preparation method according to claim 1, characterized in that: In step S7, the mass ratio of hyaluronic acid, NHS, EDC, silk fibroin peptide and drug-loaded microspheres is 15-20:5-7:3-6:7-10:20, the activation reaction time is 0.5-1.5h, and the stirring reaction time is 17-20h.

7. The preparation method according to claim 1, characterized in that: The specific steps include: S1. Acquisition of exosomes: Bone marrow mesenchymal stem cells were added into MSCM-sf medium and the density was adjusted to 10 6 -10 7 / mL and mix well, incubate in a constant temperature incubator at 36-38°C containing 4-6v / v% CO2 for 7-10 days, and obtain exosome fluid by ultra-high speed centrifugation combined with ultrafiltration tube method; The ultra-high speed centrifugation method combined with the ultrafiltration tube method is as follows: the cell fluid is centrifuged at 3-5°C and 500-1000r / min for 10-15min to remove cells, and then centrifuged at 3-5°C and 3000-4000r / min for 15-20min to remove cell debris, the supernatant is concentrated by ultrafiltration tube, and the large vesicles and proteins are removed by centrifugation at 3-5°C and 12000-15000r / min for 20-30min, and the supernatant is filtered with a 0.22μm filter membrane, and finally centrifuged at 3-5°C and 30000-35000r / min for 60-70min, and the precipitate is resuspended in PBS with pH=6.5-6.7, and centrifuged again at 3-5°C and 30000-35000r / min for 60-70min. The obtained precipitate is exosomes, which is resuspended with a PBS solution with pH=6.5-6.7 to obtain exosome fluid; S2. Preparation of mesoporous embedded microspheres: 12-15 parts by weight of tetraethyl orthosilicate, 0.5-1 parts by weight of a porogen and 0.3-0.5 parts by weight of an oil-soluble emulsifier are dissolved in ethyl acetate, 50 parts by weight of the exosome fluid in step S1 are added dropwise, emulsified, the pH value of the solution is adjusted to 7.3-7.7, the reaction is stirred for 10-12 hours, centrifuged, washed, and dried at room temperature to obtain mesoporous embedded microspheres; S3. Preparation of magnetic particles: 3.2-3.3 parts by weight of ferric chloride and 1.2-1.3 parts by weight of ferrous chloride were added to water, and 2-4 parts by weight of aqueous ammonia were added dropwise under inert gas protection, heated to 60-70 ° C, stirred for 3-5h, centrifuged, washed, dried, and calcined at 700-800 ° C for 1-2h to obtain magnetic particles; S4 modification: 10 parts by weight of the mesoporous embedded microspheres prepared in step S2 were added to water, 1-2 parts by weight of the magnetic particles prepared in step S3, 2-4 parts by weight of tannic acid and 0.1-0.2 parts by weight of the catalyst were added, heated to 40-50 ° C, stirred for 1-2h, centrifuged, washed, and dried to obtain modified microspheres; The catalyst is a Tris-HCl solution with a pH of 8.5-9.5; S5. Preparation of a drug solution: 10 parts by weight of disodium fructose 6-phosphate, 4-7 parts by weight of a bone morphogenetic protein composition were dissolved in 100 parts by weight of water, and stirred to mix well to obtain a drug solution; The bone morphogenetic protein composition is a mixture of bone morphogenetic protein-2, bone morphogenetic protein-7, and bone morphogenetic protein-14, with a mass ratio of 5-7:3-5:1-2; S6 drug loading: 10 parts by weight of the modified microspheres prepared in step S4 are added to 15-18 parts by weight of the drug solution prepared in step S5, and the solvent is evaporated to obtain drug loaded microspheres; S7. Sodium hyaluronate-silk fibroin peptide coating: dissolve 15-20 parts by weight of hyaluronic acid in water, add 5-7 parts by weight of NHS and 3-6 parts by weight of EDC, stir and activate the reaction for 0.5-1.5 hours, under the protection of inert gas, add 7-10 parts by weight of silk fibroin peptide and 20 parts by weight of the drug-loaded microspheres prepared in step S6, stir and react for 17-20 hours, centrifuge, wash, and dry to obtain a nanosphere drug-loaded system for preventing tendon adhesion.

8. A nanosphere drug delivery system for preventing tendon adhesion prepared by the preparation method according to any one of claims 1 to 7.

9. Use of the nanosphere drug delivery system for preventing tendon adhesion as claimed in claim 8 in the preparation of a drug for preventing tendon adhesion and promoting healing after tendon injury repair surgery.

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