Directional spiral stent with tendon-promoting and ossification-inhibiting functions, and its preparation method and application

Abstract

The present invention belongs to the field of biomedicine technology, and specifically relates to a directional spiral stent with the functions of promoting tendon formation and inhibiting ossification, as well as its preparation method and application. The preparation method of the directional spiral stent includes: using ZIF-8 as a delivery carrier of rapamycin to prepare ZIF-8@RAPA; the outer layer uses hexafluoroisopropanol as a solvent, polycaprolactone and type I collagen as solutes; the inner layer uses a mixed solution of dichloromethane and DMF as a solvent, polycaprolactone and ZIF-8@RAPA as solutes; a nanofiber membrane is prepared by electrospinning technology; the nanofiber membrane is mechanically rotated to prepare a directional spiral stent. The directional spiral stent of the present invention can effectively repair tendon damage and inhibit the occurrence of heterotopic ossification. The present invention provides a new option for tendon repair.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to a directional spiral stent with tendon-promoting and ossification-inhibiting functions, and a preparation method and application thereof. Background Art

[0002] Tendon injury refers to damage caused by rapid contraction or excessive stretching of the tendon during exercise, accounting for approximately 30% of musculoskeletal system diseases. Due to the characteristics of tendons such as low cells, lack of blood vessels and low metabolism, their endogenous repair capacity is poor. At present, the methods for treating tendon injuries mainly include surgical suturing, autologous (allogeneic / xenogeneic) transplantation, etc. However, in clinical applications, these methods are often limited by problems such as insufficient repair, limited sources, donor site complications, and immune rejection. Therefore, it is necessary to find new methods to overcome the defects of existing treatment methods and achieve the treatment of tendon injuries.

[0003] Scaffolds, a biomimetic extracellular matrix (ECM), provide structural support for stem cells and create a physicochemical microenvironment that can regulate their fate. With the advancement of tissue engineering technology, numerous researchers have attempted to construct tissue-engineered tendons by mimicking the composition, topology, and mechanical properties of native tendon ECM. This approach provides a biomimetic microenvironment for the directional differentiation of stem cells into the tendon lineage, thereby accelerating the endogenous healing process of the tendon. Existing studies have shown that tendon ECM is primarily composed of micro-curled, parallel collagen fibers; parallel nanofibers promote stem cell differentiation into the tendon lineage, while randomly arranged nanofibers promote stem cell differentiation into the bone lineage. On the one hand, helical scaffolds can reduce the local maximum strain on cells during stretching, maintaining high cell viability and promoting dynamic repair of tendon tissue. Specifically, oriented helical scaffolds can induce tendon stem cells (TSCs) to differentiate into the tendon lineage. On the other hand, scaffolds can serve as effective drug delivery systems for the release of drugs at the site of tissue injury, where their chemical microenvironment can induce stem cell differentiation. Numerous studies have shown that heterotopic ossification (HO) is a common histological feature of tendon repair in the later stages of repair, resulting in a significant difference in mechanical properties and repair quality compared to native tendon ECM. Therefore, timely and appropriate inhibition of the osteogenic differentiation of TSCs during tendon repair can prevent HO. Therefore, oriented helical scaffolds, which can serve as drug delivery vehicles, are ideal tissue engineering scaffolds for biomimetic tendon ECM.

[0004] Patent CN112023060B discloses a dual-drug-loaded nanoparticle targeting cartilage with photothermal responsiveness. Using small-particle mesoporous polydopamine as a matrix, it undergoes a cyclic reaction with FeCl3·6H2O and H3BTC, modifying the mesoporous polydopamine surface to form a metal-organic framework. The resulting MPDA-MOF nanoparticles are then reacted with EDC and NHS solutions to activate the carboxyl groups on the MOF surface. These activated groups then connect to the amino groups in the type II collagen targeting peptide, forming composite nanoparticles loaded with the cartilage-targeting peptide. Bilirubin and rapamycin are then loaded into the MOF shell and the pores of the mesoporous polydopamine, ultimately yielding RB@MPMW. This invention utilizes mesoporous polydopamine to load bilirubin and rapamycin for the targeted treatment of cartilage degeneration in osteoarthritis. Summary of the Invention

[0005] In light of this, the present invention addresses the problem of heterotopic ossification in tendon repair by developing a directional helical scaffold, its preparation method, and its application, designed to promote tendon healing and inhibit heterotopic ossification. Utilizing the synergistic effects of osteogenesis inhibition and bond promotion, the present invention utilizes electrospinning technology to co-spin ZIF-8@RAPA and PCL / COLⅠ into directional micro-curled nanofibers, creating a functionalized biomimetic scaffold capable of specifically delivering RAPA to pathological tendons. This scaffold effectively repairs damaged Achilles tendons while simultaneously inhibiting heterotopic ossification during the repair process.

[0006] One of the objectives of the present invention is to provide a composition for preparing a directional helical stent.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] A composition for preparing an oriented helical stent, the composition comprising composition A for preparing an outer layer of a nanofiber membrane and composition B for preparing an inner layer of a nanofiber membrane;

[0009] The composition A comprises the following components: polycaprolactone and type I collagen at a concentration of 10% to 20%; in the composition A, the mass ratio of type I collagen to polycaprolactone is 15 to 25:100;

[0010] The composition B comprises the following components: polycaprolactone at a concentration of 10% to 20% and a drug; in the composition B, the mass ratio of the drug to the polycaprolactone is 5 to 10:100; the drug is rapamycin with ZIF-8 as a carrier;

[0011] The nanofiber membrane is made into a directional spiral stent by mechanical rotation.

[0012] Preferably, the composition A comprises polycaprolactone and type I collagen at a concentration of 15%; in the composition A, the mass ratio of type I collagen to polycaprolactone is 20:100.

[0013] Preferably, the composition B comprises polycaprolactone at a concentration of 15% and a drug; in the composition B, the mass ratio of the drug to the polycaprolactone is 7:100.

[0014] Most preferably, the drug is ZIF-8@RAPA.

[0015] Furthermore, the composition A further comprises hexafluoroisopropanol; the composition B further comprises dichloromethane and DMF, and the volume ratio of the dichloromethane to DMF is 2 to 5:1.

[0016] Preferably, the volume ratio of dichloromethane to DMF is 3:1.

[0017] Furthermore, the nanofiber membrane has a sandwich structure.

[0018] A second object of the present invention is to provide a method for preparing a directional helical stent using the above composition.

[0019] To achieve the above object, the present invention adopts the following technical solutions:

[0020] The method for preparing a directional helical stent using the above composition comprises the following steps:

[0021] (1) ZIF-8 was used as a delivery vector for rapamycin to prepare ZIF-8@RAPA;

[0022] (2) The outer layer uses hexafluoroisopropanol as solvent, polycaprolactone and type I collagen as solutes; the inner layer uses a mixed solution of dichloromethane and DMF as solvent, polycaprolactone and ZIF-8@RAPA obtained in step (1) as solutes; and a nanofiber membrane is prepared by electrospinning technology;

[0023] (3) The nanofiber membrane obtained in step (2) is mechanically stirred to prepare a directional spiral stent.

[0024] Furthermore, the particle size of the ZIF-8@RAPA is 190 nm to 300 nm.

[0025] Furthermore, step (1) specifically includes the following steps:

[0026] 1) Dissolving zinc nitrate hexahydrate and adding RAPA methanol solution to prepare a zinc-based solution;

[0027] 2) Prepare an imidazole solution using 2-methylimidazole as the solute and methanol as the solvent;

[0028] 3) adding the zinc-based solution obtained in step 1) to the imidazole solution obtained in step 2) to react and obtain ZIF-8@RAPA.

[0029] Preferably, the solvent of the zinc nitrate hexahydrate is methanol.

[0030] Preferably, in step 1), the final concentration of the RAPA methanol solution is 730 μg / mL.

[0031] Preferably, in step 3), the reaction conditions are as follows: stirring for 3 to 10 minutes, standing for 50 to 57 minutes, and the total reaction time is 60 minutes.

[0032] Most preferably, in step 3), the reaction conditions are: stirring for 5 minutes and standing for 55 minutes.

[0033] Preferably, the final concentration of zinc ions is 0.015 to 0.025 mol / L; 2-mIm - The final concentration of the zinc nitrate hexahydrate is 0.10-0.2 mol / L; the concentration ratio of the zinc nitrate hexahydrate to the 2-methylimidazole is 1:7-10.

[0034] As the most preferred, the final concentration of zinc ion is 0.0175mol / L; 2-mIm - The final concentration is 0.14 mol / L; the concentration ratio of the zinc nitrate hexahydrate to the 2-methylimidazole is 1:8.

[0035] Furthermore, after the reaction in step 3) is completed, the resulting solution is centrifuged, washed, and dried to obtain ZIF-8@RAPA;

[0036] Preferably, the centrifugal speed is 8000 rpm and the time is 10 min;

[0037] Preferably, the washing is performed three times with methanol.

[0038] Preferably, the drying is carried out in a vacuum drying oven at 25° C. for 24 hours.

[0039] Furthermore, in step (2), the preparation parameters of electrospinning are: shaft speed 3500-4500 rpm, voltage 20-30 kV, injection speed 0.4-0.7 mL / h, and receiving distance 15-20 cm.

[0040] Preferably, the preparation parameters of the electrospinning are: shaft speed 4000 rpm, voltage 25 kV, injection speed 0.5 mL / h, and receiving distance 18 cm.

[0041] Furthermore, the nanofiber membrane obtained in step (2) has a sandwich structure.

[0042] Furthermore, step (3) specifically includes: cutting the nanofiber membrane into 12 mm×80 mm, fixing it on an improved mechanical stirring shaft for rotation treatment, setting the rotation speed to 30 rpm, and preparing the directional spiral stent.

[0043] A third object of the present invention is to provide a directional helical stent produced by the above-mentioned preparation method.

[0044] Furthermore, the number of spiral turns of the directional spiral stent is evenly distributed, and the width of a single turn is about 500 μm.

[0045] Furthermore, the fibers in the oriented helical stent exhibit obvious orientation distribution, and there is a porous structure between the fibers.

[0046] A fourth object of the present invention is to provide a nanoformulation for treating and repairing tendon injuries and / or inhibiting the occurrence of heterotopic ossification.

[0047] To achieve the above object, the present invention adopts the following technical solutions:

[0048] A nanoformulation for treating and repairing tendon injuries and / or inhibiting heterotopic ossification consists of a drug and a carrier; the drug is RAPA; the carrier is ZIF-8; and the nanoformulation is prepared using a pharmaceutically acceptable method.

[0049] A fifth object of the present invention is to provide a use of the aforementioned composition, the aforementioned directional helical stent and / or the aforementioned nanoformulation in the preparation of a product for repairing tendon damage and / or inhibiting the occurrence of heterotopic ossification.

[0050] The beneficial effects of the present invention are:

[0051] 1. This study leverages the synergistic effects of osteogenesis inhibition and tendon bonding. Using electrospinning technology, ZIF-8@RAPA and PCL / COLⅠ are co-spun into oriented micro-curled nanofibers to construct a functionalized biomimetic scaffold capable of specifically delivering RAPA to pathological tendons. This oriented helical scaffold effectively repairs damaged Achilles tendons while simultaneously inhibiting heterotopic ossification during tendon repair, demonstrating broad application prospects.

[0052] 2. The oriented helical scaffold (aNFs / Z@R helical scaffold) of the present invention has a uniform distribution of helical turns, the fibers in the scaffold show a clear orientation distribution, and there is a porous structure between the fibers, which is conducive to cell migration.

[0053] 3. The directional helical stent of the present invention has an elastic modulus of approximately 20.64±2.25 MPa and a breaking strength of approximately 51.92±9.02 MPa, and has excellent mechanical properties.

[0054] 4. The present invention's oriented helical scaffold (aNFs / Z@R(helical)) demonstrated superior mechanical properties and heterotopic ossification inhibition compared to aNFs(helical) without ZIF-8@RAPA. This demonstrates that both the oriented helical fiber topology and ZIF-8@RAPA can inhibit heterotopic ossification to a certain extent, with the two acting synergistically.

[0055] 5. The present invention uses ZIF-8 as a delivery vector for rapamycin. The constructed directional spiral stent containing ZIF-8@RAPA has good long-term drug sustained-release effect, pH sensitivity and cell compatibility. It can continuously release RAPA in the inflammatory environment in the body and inhibit the formation of heterotopic ossification. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 The results of ZIF-8 characterization are shown in Figure 1, where: Figure 1 -A is the pH monitoring graph of Z1-Z4; Figure 1 -B is the pH monitoring graph of Z5-Z8; Figure 1 -C is the test result diagram of the effect of different stirring times on particle size; Figure 1 -D is the particle size distribution diagram of Z1-Z4; Figure 1 -E is the particle size distribution diagram of Z5-Z8; Figure 1 -F is the XRD pattern of Z1-Z8;

[0057] Figure 2 The morphology and particle size characterization results of ZIF-8@RAPA and ZIF-8 are shown in Figure 1. Figure 2 -A is the SEM image of ZIF-8; Figure 2 -B is the SEM image of ZIF-8@RAPA; Figure 2 -C is the particle size distribution diagram of ZIF-8 and ZIF-8@RAPA;

[0058] Figure 2 -D is the TEM image of ZIF-8; Figure 2 -E is the TEM image of ZIF-8@RAPA; Figure 2 -F is the statistical diagram of the average particle size of ZIF-8 and ZIF-8@RAPA;

[0059] Figure 3 The results of characterization of the structure and physicochemical properties of ZIF-8@RAPA and ZIF-8 are shown in Figure 2. Figure 3 -A is the XRD pattern of ZIF-8@RAPA and ZIF-8; Figure 3 -B is the FTIR images of ZIF-8@RAPA and ZIF-8; Figure 3 -C is the UV-vis image of ZIF-8@RAPA and ZIF-8; Figure 3-D is the TGA graph of ZIF-8@RAPA and ZIF-8; Figure 3 -E is the zeta potential diagram of ZIF-8@RAPA and ZIF-8; Figure 3 -F is a graph showing the cumulative release rate of RAPA from ZIF-8@RAPA under different pH conditions;

[0060] Figure 4 The figure is a statistical graph of drug loading rate at different RAPA addition amounts;

[0061] Figure 5 Figure 2 shows the characterization results of the morphology, fiber orientation, and fiber diameter of aNFs / Z@R and rNFs / Z@R. Figure 5 -A is the SEM image of aNFs / Z@R; Figure 5 -B is the fiber direction distribution statistics of aNFs / Z@R; Figure 5 -C is the statistical diagram of fiber diameter distribution of aNFs / Z@R; Figure 5 -D is the SEM image of rNFs / Z@R; Figure 5 -E is the statistical diagram of fiber orientation distribution of rNFs / Z@R; Figure 5 -F is the statistical diagram of fiber diameter distribution of rNFs / Z@R;

[0062] Figure 6 TEM image of ZIF-8@RAPA in a single fiber;

[0063] Figure 7 The stress-strain curves of aNFs / Z@R and rNFs / Z@R nanofiber membranes;

[0064] Figure 8 The graph shows the test results of drug sustained release behavior of aNFs / Z@R nanofiber membrane in PBS (pH 6.5 and pH 7.2) within 14 days;

[0065] Figure 9 Figure 2 is the cell compatibility test result of aNFs / Z@R nanofiber membrane, where: Figure 9 -A is the SEM image of aNFs / Z@R nanofiber membrane; Figure 9 -B is the Live / Dead staining image of aNFs / Z@R nanofiber membrane; Figure 9 -C is the EdU staining image of aNFs / Z@R nanofiber membrane;

[0066] Figure 10 The macroscopic morphology of aNFs / Z@R helical scaffold;

[0067] Figure 11 A magnified microscopic image of the aNFs / Z@R helical scaffold;

[0068] Figure 12 The macroscopic morphology of rNFs / Z@R helical scaffold;

[0069] Figure 13 This is a magnified microscopic image of the rNFs / Z@R helical scaffold;

[0070] Figure 14 Diagram of the surgical procedure for establishing the Achilles tendon defect model in SD rats;

[0071] Figure 15 The following are representative micro-CT three-dimensional reconstruction images of the Achilles tendon in each group 6 weeks after surgery. Figure 15 -A is a representative micro-CT three-dimensional reconstruction image of the Achilles tendon in the aNFs / Z@R(spiral) group 6 weeks later; Figure 15 -B is a representative micro-CT three-dimensional reconstruction of the Achilles tendon in the aNFs (spiral) group 6 weeks later; Figure 15 -C is a representative micro-CT three-dimensional reconstruction image of the Achilles tendon in the rNFs / Z@R(helical) group 6 weeks later;

[0072] Figure 16 The following are representative HE staining images of the Achilles tendons of each group 6 weeks after surgery. Figure 16 -A is a representative HE staining image of the Achilles tendon in the aNFs / Z@R(spiral) group 6 weeks after surgery; Figure 16 -B is a representative HE staining image of the Achilles tendon in the aNFs (spiral) group 6 weeks after surgery;

[0073] Figure 16 -C is a representative HE staining image of the Achilles tendon in the rNFs / Z@R(helical) group 6 weeks after surgery. DETAILED DESCRIPTION

[0074] The technical solutions of the present invention will be further described in detail below with reference to specific embodiments. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Therefore, all other embodiments derived by those skilled in the art based on the embodiments of the present invention without inventive effort are intended to fall within the scope of protection of the present invention.

[0075] The pathogenesis of tendon HO remains unclear, but it may be related to the mTOR signaling pathway. Existing studies have found that mTOR inhibition can slow tendon ossification after Achilles tenotomy in rats. Rapamycin (RAPA) is an inhibitor of the mTOR signaling pathway, and its targets regulate HO in multiple tissues. Therefore, RAPA may prevent tendon HO by regulating the abnormal differentiation of TSCs. However, RAPA has low in vivo bioavailability, which can lead to failure before reaching the diseased site. Metal-organic framework (MOF) materials, such as zeolitic imidazolate framework-8 (ZIF-8), have demonstrated excellent drug delivery capabilities, including sustained and pH-responsive release. ZIF-8 is formed by self-assembly of zinc ions and 2-methylimidazole. Its crystallization process is similar to that of zeolites, with stable ZIF-8 crystals formed through stages such as nucleation and crystal growth. Because ZIF-8 has advantages such as high surface area, high porosity, adjustable pore size and shape, and stable physical and chemical properties, and the presence of active binding sites within the framework that can adsorb various molecules (genes, drugs, peptides, nucleic acids, proteins, etc.), it can be used as a carrier for drug delivery systems to achieve the sustained release function of loaded drugs. Therefore, to address the problem of heterotopic ossification in tendon injury repair, the present invention takes the synergistic effect of inhibiting osteogenesis and promoting bond formation as the starting point, uses ZIF-8 as a RAPA delivery carrier (ZIF-8@RAPA), and uses electrospinning technology to co-spin ZIF-8@RAPA with PCL / COLⅠ to form oriented micro-curled nanofibers, constructing a functionalized biomimetic scaffold that can specifically deliver RAPA to pathological tendons for the repair of damaged Achilles tendons.

[0076] In the present invention, it is considered that particle size is an important parameter of drug delivery carrier, which determines its biodistribution, cellular internalization, tissue clearance and metabolism in vivo, which plays a vital role in therapeutic effect. Studies have shown that micron-scale ZIF-8 usually does not enter the interior of the cell, has poor biocompatibility, and may cause a certain immune response. Nanoscale ZIF-8 can pass through the cell membrane to achieve effective distribution of drugs in the body, and has good biocompatibility and low toxicity. Specifically, the nanocarrier size should not exceed 200nm. Particles of 20-30nm can be absorbed and eliminated by the renal system, while particles of 30-300nm are absorbed by mononuclear phagocytes and subsequently stored in organs such as the liver, spleen or bone marrow. Accordingly, the present invention selects to encapsulate RAPA using 100-200nm ZIF-8, using a directional spiral stent as a delivery system, so that RAPA is achieved by local sustained sustained release at the tendon injury site to regulate and control the differentiation of TSCs. In the present invention, the particle size of ZIF-8 is controlled to be 100-200 nm by optimizing the synthesis conditions, such as reaction time, stirring time, standing time, etc.

[0077] In the examples of the present invention, the basic information of the key reagents / materials involved is shown in Table 1.

[0078] Table 1. Basic information of key reagents / materials

[0079] Reagents / Materials factory effect <![CDATA[Zinc nitrate hexahydrate (Zn(NO3)2·6H2O, 99%)]]> Chengdu Kelong Chemicals Co., Ltd. Preparation of ZIF-8 drug carrier <![CDATA[2-Methylimidazole (C4H6N2, 98%)]]> MACKLIN Preparation of ZIF-8 drug carrier Rapamycin (98%) Aladdin Drugs to repair tendon injuries Methanol (99.5%) Aladdin Reaction solvent Polycaprolactone (PCL) SIGMA-ALDRICH Construction of nanofiber scaffolds Type I collagen (COLI) MACKLIN Construction of nanofiber scaffolds Hexafluoroisopropanol Aladdin Dissolve PCL and collagen dichloromethane Chongqing Chuandong Chemical Dissolved PCL Dimethylformamide (DMF) Chongqing Chuandong Chemical Dissolved PCL Phosphate-buffered saline (PBS) Anhui Biosharp Simulated body fluids

[0080] In the embodiment of the present invention, the basic information of the key instruments involved is shown in Table 2.

[0081] Table 2. Basic information of key instruments

[0082] equipment factory effect centrifuge Xiangyi Centrifugation of ZIF-8 electrospinning machine Qingdao Pansi Technology Preparation of nanofiber scaffolds electronic balance ThermoFisherScientific Weighing Ultrapure water system ThermoFisherScientific purified water Field emission scanning electron microscopy JEOL Morphology observation Transmission electron microscopy FEITecnaiG212 Morphology observation microplate reader ThermoFisherScientific Absorbance measurement Universal Mechanical Stretching Machine Zhuhai Sansi Taijie Fiber membrane mechanical properties test

[0083] In the present invention, the preparation method of the biomimetic spiral scaffold (aNFs / Z@R spiral scaffold) comprises the following steps:

[0084] (1) Preparation of ZIF-8@RAPA: Dissolve 0.3 g of zinc nitrate hexahydrate in 14.3 mL of methanol, add 3.5 mL of 6 mg / mL RAPA methanol solution (final concentration is 730 μg / mL), and stir for 5 min to obtain a zinc-based solution (zinc ion concentration is 0.0175 mol / L). Dissolve 0.66 g of 2-methylimidazole solution in 14.3 mL of methanol to obtain an imidazole solution (2-mIm - The zinc-based solution was added to the imidazole solution, stirred for 5 minutes, and allowed to stand for 55 minutes. The mixture was centrifuged at 8000 rpm for 10 minutes, washed three times with methanol, and then dried in a vacuum drying oven at 25°C for 24 hours to obtain ZIF-8@RAPA.

[0085] (2) Preparation of nanofiber membrane (NFs / Z@R)

[0086] The outer layer consisted of hexafluoroisopropanol (HFIP) as the solvent, 15% (w / v) polycaprolactone (PCL) and 18.75% (w / w, based on the mass ratio of PCL) type I collagen as the solutes, while the inner layer consisted of dichloromethane:DMF (3:1) as the solvent, 15% (w / v) PCL and 7% (w / w, based on the mass ratio of PCL) ZIF-8@RAPA as the solutes. Electrospinning was used to create a highly oriented nanofiber membrane with a "sandwich structure." The parameters for preparing the membrane were: spindle speed of 4000 rpm, voltage of 25 kV, injection rate of 0.5 mL / h, and receiving distance of 18 cm.

[0087] (3) Preparation of directional helical stents

[0088] The sandwich-structured nanofiber membrane was cut into 12 mm × 80 mm pieces, fixed on an improved mechanical stirring shaft, and the rotation speed was set to 30 rpm to prepare the NFs / Z@R spiral scaffold.

[0089] Example 1. Synthesis and characterization of ZIF-8

[0090] (1) Synthesis of ZIF-8

[0091] Dissolve zinc nitrate hexahydrate in 14.3 mL of methanol and sonicate for 10 minutes to obtain a zinc nitrate solution. Dissolve 2-methylimidazole in 14.3 mL of methanol and sonicate for 10 minutes to obtain a 2-methylimidazole solution. Quickly add the zinc nitrate solution to the 2-methylimidazole solution and stir for a period of time to obtain a white suspension, which is ZIF-8. Collect the product by centrifugation at 8000 rpm for 10 minutes, wash it three times with methanol to fully remove unreacted reactants, and then dry it in a vacuum oven at 75°C for 24 hours to collect the ZIF-8 particles.

[0092] (2) Characterization of ZIF-8

[0093] Further investigate the influence of synthesis conditions on ZIF-8 particle diameter and yield. Because the pH of the reaction system can change along with the change of synthesis time, the change of pH value reflects the evolution of ZIF-8 crystalline structure to a certain extent. Therefore, the present embodiment first carries out pH monitoring to 8 groups of solutions of different raw material proportions and different concentrations. Secondly, stirring time is a key parameter in the ZIF-8 building-up process, which affects the degree of mixing, nucleation process and crystal growth between the reactants. Insufficient stirring may cause uneven crystal nucleation, and excessive stirring may cause too many nuclei to form. Therefore, further explore appropriate stirring time to obtain nanoparticles with little and uniform distribution of particle diameter. Specific experimental method and result are as follows:

[0094] 1) Grouping

[0095] The concentrations of zinc nitrate hexahydrate and 2-methylimidazole were changed, and two concentration ratios of 1:8 and 1:16 were set, with four concentration gradients totaling 8 groups, as shown in Table 3.

[0096] Table 3. Initial concentration, final reactant concentration, and reactant concentration ratio for the synthesis of ZIF-8 particles

[0097]

[0098] 2) Condition optimization

[0099] The pH of the solution was monitored at 10-minute intervals until the pH stabilized for 30 minutes. Based on the pH monitoring results, the total reaction time was kept constant, and only the stirring and resting times were varied to explore the effect of stirring time on ZIF-8 particle size (see Table 4 for details). Finally, based on the stirring time results, the effect of reactant concentration on ZIF-8 particle size and yield was investigated.

[0100] ZIF-8 particle yield = ZIF-8 actual yield / ZIF-8 theoretical yield × 100%.

[0101] Table 4. Changes in stirring time under the same reactant concentration

[0102] ZIF-8 number Mixing time (minutes) Standing time (minutes) J5 5 55 J15 15 45 J30 30 30 J60 60 0

[0103] (3) Results and Discussion

[0104] The pH monitoring results of Z1-Z4 are as follows Figure 1 -A, the pH monitoring results of Z5-Z8 are as follows Figure 1 -B. In general, when zinc nitrate hexahydrate (Zn 2+ ) and 2-methylimidazole (2-mIm - ) ratio remains unchanged, with the increase of ion concentration, the initial pH and corresponding stable pH of the reaction system tend to increase. Similarly, when Zn2+ The concentration remains constant, with the 2-mIm - With the increase of concentration, the initial pH and stable pH still showed an upward trend. It is worth noting that the pH of each group showed a similar trend with the synthesis time, including three obvious stages, namely S Ⅰ :When the crystallization time (t) is less than 10min, the pH drops suddenly; S Ⅱ : When 10min<t<50min, the pH decrease rate slows down significantly; S Ⅲ When t≥60min, the pH value is basically stable. Therefore, in order to synthesize ZIF-8 nanocarriers with small particle size and high crystallinity, the synthesis time is selected as 60min.

[0105] The test results of the effect of different stirring times on particle size are as follows: Figure 1 -C. The present invention takes Z1 as an example to explore the effect of stirring time on the particle size of ZIF-8. After the two reactants are mixed evenly, the total reaction time is kept unchanged, and the stirring time and standing time are changed. The results show that with the increase of stirring time, the particle size gradually increases. When the stirring time is increased to 60min, large-particle crystals are formed, and the particle size distribution is bimodal. The minimum particle size is 163nm when stirring for 5min and standing for 55min. Therefore, the reaction conditions optimized according to the above results are: total reaction time 60min, stirring for 5min, and standing for 55min.

[0106] Based on the above optimized conditions, Z1-Z8 were synthesized. Figure 1 -D and Figure 1 -E, when [Zn 2+ ] / [2-mIm - ] is constant, and with the increase of ion concentration, the particle size gradually increases. In a certain concentration range, when the reaction system keeps the concentration of zinc nitrate hexahydrate constant, increasing [2-mIm - ] can reduce the particle size of ZIF-8 (Z2-Z4 vs. Z6-Z8), which may be because the excess imidazole can provide more ligands to promote nucleation, while the number of zinc ions available for each nucleus is relatively reduced, thus forming smaller crystals. However, when [Zn 2+ ] is 0.0175 mol / L, and its trend is just the opposite (D Avg (Z1)=163.8nm±3.1nmvs.D Avg (Z5) = 245.7 nm ± 5.5 nm). This may be because when the ion concentration is too low, the nucleation rate of ZIF-8 is low, and the increase of [2-mIm -] may increase the probability of random nucleation, thereby increasing the particle size. Further, in order to explore the effects of different concentrations and ratios on the crystal structure of ZIF-8, the present invention conducted X-ray diffraction (XRD) analysis on the solid products of Z1-Z8. The results are as follows Figure 1 -F shows that the characteristic peaks of each group are narrow and sharp and the peak positions are basically the same. The main diffraction peaks such as (011), (002), (112), (022), (013), and (222) are observed, which is consistent with the existing literature reports. This result shows that changing the concentration and ratio of the reactants within a certain range has no effect on the main lattice of ZIF-8. However, it can be observed that the (011) peak intensity of the Z1 group is higher than the (112) peak intensity, which is consistent with the theory, while the trends of the two crystal planes in other groups are just the opposite. Literature reports that before the crystal grows mature, the relative crystallinity of ZIF-8 increases with the increase of synthesis time. At this stage, the most important (011) peak of ZIF-8 will increase with the extension of time and become the strongest peak. Therefore, the above phenomenon may be because Z2-Z8 is still in the intermediate stage of crystal growth, the (011) crystal plane has not yet fully grown, and the crystal has not reached maturity.

[0107] The yields of the products in groups Z1-Z8 were calculated, and the results are shown in Table 5. Overall, the solid yield was higher when the reactant molar ratio was 1:8 than when it was 1:16. When the reactant molar ratio was fixed, the ZIF-8 yield decreased with increasing ion concentration. Z1 had a yield of 51.20%, the highest among the eight groups, and this yield is close to that reported by Beh et al.

[0108] Based on the above experimental results, the optimal synthesis condition of ZIF-8 is Z1, that is, [Zn 2+ ]=0.0175mol / L,[2-mIm - ] = 0.14 mol / L, stirred at room temperature for 5 min, and allowed to stand for 55 min. Under these conditions, nano-ZIF-8 with high crystallinity, good uniformity, and good dispersibility can be prepared, which is suitable for use as a drug delivery carrier.

[0109] Table 5. Different Zn 2+ and 2-mIm - Particle size and solid phase yield of ZIF-8 at different concentrations

[0110]

[0111]

[0112] Example 2. Preparation and characterization of ZIF-8@RAPA

[0113] The optimal synthesis conditions of ZIF-8 obtained according to Example 1 are: [Zn 2+]=0.0175mol / L,[2-mIm - ] = 0.14 mol / L, stirred at room temperature for 5 minutes, and allowed to stand for 55 minutes. ZIF-8@RAPA nanoparticles were further synthesized. The structure and physicochemical properties of ZIF-8@RAPA were characterized using ZIF-8 as a control group. The specific methods and results are as follows:

[0114] (1) Preparation of ZIF-8@RAPA

[0115] Dissolve 0.3 g of zinc nitrate hexahydrate in 14.3 mL of methanol. Add 1 mL, 2 mL, 3 mL, 3.5 mL, and 4 mL of a 6 mg / mL RAPA methanol solution, respectively, and stir for 5 minutes to obtain a zinc-based solution. Dissolve 0.66 g of 2-methylimidazole solution in 14.3 mL of methanol to obtain an imidazole solution. Add the zinc-based solution to the imidazole solution, stir for 5 minutes, and let it stand for 55 minutes. Separate the resulting particles by centrifugation at 8000 rpm for 10 minutes and wash three times with methanol. Dry in a vacuum oven at 25°C for 24 hours to obtain ZIF-8@RAPA.

[0116] (2) Characterization of the structure and physicochemical properties of ZIF-8@RAPA

[0117] 1) Dynamic light scattering analysis (DLS): Using a Malvern Zetasizer Nano ZS laser particle size analyzer, an appropriate amount of sample was dispersed in water and diluted to an appropriate concentration and ultrasonicated for 15 min. Each ZIF-8 sample was measured at least three times to obtain the average data.

[0118] 2) Scanning electron microscopy (SEM): An appropriate amount of sample was dispersed on the conductive adhesive, sprayed with gold for 60 seconds, and its morphology was observed and photographed using a JEOL JSM-7800F scanning electron microscope.

[0119] 3) X-ray Diffraction (XRD): The dried nanoparticles were ground and placed on a sample stage, ensuring that the sample and the edge of the stage were aligned. Measurements were made using a Rigaku SmartLab-9 X-ray diffractometer. Test conditions: Scan angles of 5°-60° and a speed of 5° / min.

[0120] 4) Transmission electron microscopy (TEM): The dried ZIF-8 was dispersed in ethanol solution and added dropwise onto the carbon film copper grid using a disposable dropper. After drying, the morphology was observed using a FEI Tecnai G212 transmission electron microscope.

[0121] 5) Infrared spectroscopy (FTIR): 2 mg of sample was mixed with 200 mg of KBr powder, ground into powder in an agate mortar and pressed into a pellet (16 MPa, 2 min). The wavelength was recorded at 4000-400 cm -1 Spectrum within the range.

[0122] 6) Thermogravimetric analysis (TG): The temperature range was 25°C to 700°C in a nitrogen atmosphere with a constant heating rate of 10°C / min.

[0123] 7) Ultraviolet absorption spectroscopy (UV): RAPA, ZIF-8, and ZIF@RAPA were dispersed in methanol solutions, respectively, and their absorption curves in the wavelength range of 200-400 nm were measured using a UV spectrophotometer.

[0124] (3) Results and analysis

[0125] The material morphology was observed using SEM and TEM. Figure 2 -A. Figure 2 -B, the synthesized ZIF-8@RAPA has a similar morphology to ZIF-8 and a relatively uniform particle size. Figure 2 -D, Figure 2 -E, the crystals are regular hexagons, which are the projections of the {110} face in the rhombic dodecahedron, consistent with the literature reports. The particle size distribution of the two nanoparticles in aqueous solution was measured using a Malvern particle size analyzer. The results are shown in Figure 2 -C. Figure 2 -F, the measured particle size distributions of ZIF-8@RAPA and ZIF-8 were narrow (190.1nm-295.3nm vs. 122.4nm-190.2nm), and their average particle sizes were 257.4±4.6nm and 163.8±3.1nm, respectively.

[0126] XRD, FTIR, and UV-vis were used to further characterize the structures of ZIF-8@RAPA and ZIF-8. Figure 3 As shown in Figure 1-A, the main peak positions of the diffraction peak spectra of ZIF-8@RAPA and ZIF-8 are basically consistent with the previously reported ZIF-8 XRD curve. At the same time, no obvious RAPA characteristic peaks appear in the XRD peak spectrum of ZIF-8@PAPA, which can be inferred that RAPA is successfully encapsulated in ZIF-8. The FTIR test results are shown in Figure 1-A. Figure 3 -B, similar to ZIF-8, ZIF-8@RAPA has a peak at 3128 cm -1 、2935cm -1 、1585cm -1The absorption peak at 420cm is attributed to the stretching vibration of aromatic CH bonds, aliphatic CH bonds and CN bonds in 2-mIm; -1 The absorption peak at is the stretching vibration peak of Zn-N bond, which is caused by Zn 2+ The coordination with 2-mIm indicates that ZIF-8@RAPA has the same complete framework structure as ZIF-8. It is worth noting that ZIF-8@RAPA has the same complete framework structure as ZIF-8. -1 The smaller absorption peak that appears at the ZIF-8 corresponds to the carbonyl stretching vibration peak in RAPA, indicating that most of RAPA is successfully loaded in the ZIF-8 framework structure, and a small amount is adsorbed on its surface. From the UV-vis spectrum, we can see that the characteristic absorption peak of Zn in ZIF-8 is around 223nm, and the characteristic absorption peak of RAPA is 278nm. ZIF-8@RAPA has both of the above characteristic absorption peaks (225nm, 278nm), indicating that RAPA is loaded in the ZIF-8 crystal. Figure 3 -C.

[0127] The thermal stability of ZIF-8@RAPA and ZIF-8 was further tested by TGA. Figure 3 As shown in Figure 1-D, as the temperature rises, ZIF-8@RAPA and ZIF-8 begin to lose weight and collapse at 420°C, and complete the first weight loss at around 520°C. At this temperature, ZIF-8@RAPA loses 15% more weight than ZIF-8, which is presumably due to the loaded RAPA, indirectly proving that RAPA is loaded in ZIF-8. In addition, the surface Zeta potentials of ZIF-8@RAPA and ZIF-8 are 26.8±0.8mV and 21.8±0.3mV, respectively. Figure 3 The results indicate that the loading of RAPA reduces the electromotive force of ZIF-8, which may be related to the presence of negatively charged groups such as hydroxyl groups in RAPA.

[0128] The present invention also investigated the effect of different addition amounts of RAPA methanol solution on drug loading rate, including final concentrations of 210μg / mL, 420μg / mL, 630μg / mL, 730μg / mL and 840μg / mL. Figure 4 As shown in the figure, when the final concentration of RAPA methanol solution was 730 μg / mL, it had a better drug loading rate DLC%.

[0129] The above results show that RAPA is successfully loaded into the framework structure of ZIF-8 without changing the crystal structure of ZIF-8.

[0130] Example 3. In vitro drug release experiment of ZIF-8@RAPA

[0131] The drug delivery rate and time are closely related to the medium microenvironment, and play an important role in the efficacy of RAPA and the effective biological utilization. Therefore, this example monitors the pH-responsive release of RAPA in ZIF-8@RAPA nanoparticles under PBS (pH 5.5, pH 6.5 and pH 7.2). 20 mg of nanoparticles were dispersed in 5 mL of deionized water and placed in a blocked dialysis membrane, and then immersed in 40 mL of release medium. Since RAPA is a hydrophobic drug, 1% polyoxyethylene ether was added to the release medium. Gently shake at 120 rpm in a constant temperature shaker at 37°C. At predetermined time intervals, 1 mL of release buffer was taken, measured using an enzyme reader, and the same amount of fresh buffer was added.

[0132] The results are as follows Figure 3 -F shows that PARA is released rapidly in acidic environments (pH 5.5 and pH 6.5), but is released relatively slowly in neutral environments. RAPA is released rapidly in the first 24 hours in all three groups, and the cumulative release rate gradually changes from 24 hours to 96 hours, basically reaching stability at 96 hours and decreasing with increasing pH, reaching 68.23%, 45.67%, and 35.96%, respectively. This result shows that ZIF-8 synthesized under the aforementioned optimized conditions is an effective drug delivery carrier that can achieve long-term sustained release and has broad application prospects.

[0133] Example 4. Preparation of nanofiber membranes and oriented helical scaffolds

[0134] (1) Preparation of nanofiber membranes with a “sandwich structure” (NFs / Z@R)

[0135] The outer layer consisted of hexafluoroisopropanol (HFIP) as the solvent, polycaprolactone (15%), and type I collagen (18.75%) as the solutes. The inner layer consisted of dichloromethane:DMF (3:1) as the solvent, PCL (15%), and ZIF-8@RAPA (7%) as the solutes. The highly oriented spun-fiber membrane (aNFs / Z@R(M)) was prepared using the following parameters: spindle speed of 4000 rpm, voltage of 25 kV, injection rate of 0.5 mL / h, and receiving distance of 18 cm. A control group, the low-oriented spun-fiber membrane (rNFs / Z@R(M)), was prepared using the following parameters: spindle speed of 500 rpm, voltage of 25 kV, injection rate of 0.5 mL / h, and receiving distance of 18 cm.

[0136] (2) Preparation of directional helical scaffolds (NFs / Z@R helical scaffolds)

[0137] The sandwich-structured nanofiber membrane was cut into 12 mm × 80 mm pieces, fixed on an improved mechanical stirring shaft, and the rotation speed was set to 30 rpm to prepare spiral scaffolds (aNFs / Z@R spiral scaffold and rNFs / Z@R spiral scaffold).

[0138] Example 5. Characterization of Nanofiber Membrane (NFs / Z@R)

[0139] The morphology and distribution of aNFs / Z@R and rNFs / Z@R were observed using SEM and TEM, and their mechanical properties, drug release behavior, and cell compatibility were also tested. The experimental methods and results are as follows:

[0140] (1) Experimental methods

[0141] 1) SEM: The spun membrane was formed into a 0.5 cm × 0.5 cm sheet, fixed to a sample stage with conductive adhesive, and then gold-sprayed. The surface morphology and microstructure were observed and photographed using a SEM at an accelerating voltage of 5 kV and an emission current of 10 mA. Fiber orientation was also observed, and diameter distribution was analyzed.

[0142] 2) TEM: The electrospun fibers were spun onto a carbon film copper mesh. After drying, the morphology of the single fibers and the ZIF-8 therein were observed using TEM.

[0143] 3) Mechanical Properties Testing: The film was cut into 40 mm x 10 mm strips. The test procedure was set with a center effective distance of 20 mm and a stretching rate of 10 mm / min. The strips were stretched until they broke, and the displacement-force curve was recorded in real time. The mechanical properties of the fiber membrane were characterized by parameters such as breaking strength, elongation at break, and Young's modulus.

[0144] 4) In vitro drug release: The release of RAPA from aNFs / Z@R nanofibers was monitored in PBS (pH 6.5 and pH 7.2). 20 mg of nanofibers were placed in 5 mL of deionized water, placed in a capped dialysis membrane, and then immersed in 40 mL of release medium. Since RAPA is a hydrophobic drug, 1% polyoxyethylene ether was added to the release medium. The mixture was gently shaken at 120 rpm in a 37°C incubator. At predetermined time intervals, 1 mL of release buffer was removed, measured using a microplate reader, and the same amount of fresh buffer was added.

[0145] 5) Cytocompatibility Testing: Tendon stem cells (TSCs) at passages P3-P5 were seeded onto the scaffolds and cultured in a medium containing 10% fetal bovine serum and 1% penicillin / streptomycin (37°C, 5% CO2, 80% RH) for 24 hours. Cell viability was measured using a Live / Dead assay kit and cell proliferation assay using an EdU-555 assay kit. Following paraformaldehyde fixation and gradient dehydration, the morphology of the TSCs on the fibers was observed using scanning electron microscopy (SEM).

[0146] (2) Results and Discussion

[0147] The morphology of aNFs / Z@R and rNFs / Z@R was observed by SEM, and the fiber orientation and fiber diameter were statistically analyzed. Figure 5 As shown in the figure, the orientation of aNFs / Z@R fibers is concentrated between -50° and 50°, and the orientation of rNFs / Z@R fibers is concentrated between -100° and 100°. The diameter of aNFs / Z@R fibers is concentrated between 0.2-0.4μm, and the diameter of rNFs / Z@R fibers is concentrated between 0.2-0.5μm. The distribution and morphology of ZIF-8@RAPA in a single fiber were observed by TEM, and it was found that ZIF-8@RAPA was wrapped in PCL fibers with sharp edges and corners. The morphology of ZIF-8 particles was not affected by spinning. Figure 6 .

[0148] The mechanical tensile test results are shown in Table 6. Figure 7 As shown, aNFs / Z@R exhibits superior mechanical properties, with both breaking strength and elastic modulus superior to those of the control group, rNFs / Z@R. This is closely related to the fiber orientation distribution, indirectly demonstrating the successful preparation of the aligned fibers. Furthermore, the elongation at break of aNFs / Z@R is 33.4% ± 4.7%, which meets the 10% elongation strain requirement for tendon repair.

[0149] Table 6. Mechanical properties of aNFs / Z@R and rNFs / Z@R nanofiber membranes

[0150]

[0151] The long-term sustained release of RAPA is the prerequisite for it to exert its drug function, so the present invention tested the drug release behavior of aNFs / Z@R nanofiber membrane in vitro. Figure 8As shown in the results, RAPA can be slowly released in both pH 6.5 and pH 7.2 environments within a week, with the former releasing faster than the latter. This suggests that in an inflammatory environment in vivo (which can accelerate the formation of heterotopic ossification in tendons), RAPA release can be promoted and the formation of heterotopic ossification can be inhibited, indicating that it has pH sensitivity. SEM, Live / Dead staining, and EdU staining results show that the aNFs / Z@R nanofiber membrane has good cell compatibility. Figure 9 .

[0152] Example 6. Characterization of NFs / Z@R Helical Scaffold

[0153] The surface morphology of the NFs / Z@R helical scaffold was observed using SEM, and the mechanical tensile properties and cell compatibility of the scaffold were tested. The experimental methods and results are as follows:

[0154] (1) Experimental methods

[0155] 1) SEM and mechanical property testing: the methods are the same as those in Example 5.

[0156] 2) Cytocompatibility Testing: Tendon stem cells (TSCs) at passages P3-P5 were seeded onto the scaffolds and cultured in a medium containing 10% fetal bovine serum and 1% penicillin / streptomycin (37°C, 5% CO2, 80% RH) for 24 hours. Cell viability and proliferation were measured using a Live / Dead assay and an EdU-555 assay, respectively. The cells were then fixed with paraformaldehyde and dehydrated using a gradient elution method. The morphology of the TSCs on the fibers was observed using scanning electron microscopy (SEM).

[0157] (2) Results and analysis

[0158] SEM test results are as follows Figures 10 to 13 As shown, the scaffold's spiral turns are evenly distributed, with a single turn width of approximately 500 μm. Further magnification revealed that the spiral processing had no effect on the fiber orientation in the local microregion. The fibers in the aNFs / Z@R spiral scaffold exhibited a distinct orientation distribution, and the porous structure between the fibers facilitated cell migration.

[0159] The mechanical tensile properties test results of the scaffold are shown in Table 7. The elastic modulus and fracture strength of aNFs / Z@R(helical) are higher than those of rNFs / Z@R(helical). At the same time, the addition of Z@R can improve the various mechanical properties parameters of the scaffold.

[0160] Table 7. Mechanical properties of aNFs / Z@R(helical), aNFs(helical), and rNFs / Z@R(helical) scaffolds

[0161]

[0162]

[0163] Example 7. Tendon repair application

[0164] Heterotopic ossification is a common problem during tendon repair, significantly reducing tendon morphology, mechanical properties, and subsequent function. Therefore, to visually observe heterotopic ossification in repaired Achilles tendons, this example used micro-CT to analyze and perform three-dimensional reconstructions of repaired Achilles tendon specimens from rats six weeks after surgery. HE staining was also used to assess tendon repair efficacy. The specific experimental methods and results are as follows:

[0165] 1) Establishment of SD rat Achilles tendon injury model and scaffold implantation

[0166] The surgical procedure for establishing the Achilles tendon defect model in SD rats is as follows: Figure 14 Rats were anesthetized with an intraperitoneal injection of 1.5% sodium pentobarbital. The skin of the right hind limb was incised, the fascia was separated to expose the Achilles tendon, and a complete defect approximately 5 mm long was created in the Achilles tendon 0.5 cm proximal to the calcaneus. The spiral stent was sutured at both ends (approximately 10 turns) with 6-0 suture, and the skin tissue was then sutured sequentially with 4-0 suture.

[0167] 2) Characterization of tissue repair

[0168] Micro-CT imaging: Six weeks after surgery, rats were sacrificed and Achilles tendon specimens were collected. The specimens were immersed in paraformaldehyde for 48 hours, then replaced with 75% ethanol. After blotting the surface liquid with filter paper, the specimens were placed on a test bench and scanned using the following parameters: 10 μm resolution, 55 kV voltage, and 200 μA current.

[0169] HE staining: Six weeks after surgery, rats were sacrificed and Achilles tendon samples were collected. Following tissue fixation, gradient dehydration, paraffin embedding, sectioning, and dewaxing, the tissues were stained with hematoxylin and eosin (HE), observed under a microscope, and photographed.

[0170] 3) Results

[0171] Representative micro-CT three-dimensional reconstruction results of the Achilles tendon in each group 6 weeks after surgery are shown in the figure. Figure 15 As shown in the figure, the degree of heterotopic ossification in the three groups is aNFs / Z@R (helix) < aNFs (helix) < rNFs / Z@R (helix), indicating that the oriented helical fiber topology and Z@R can inhibit the occurrence of heterotopic ossification to a certain extent, and the two play a synergistic role. The HE staining results are shown in the figure. Figure 16As shown, compared with the control groups (aNFs(helical) and rNFs / Z@R(helical), the aNFs / Z@R(helical) scaffold showed denser ECM, lower cytoplasmic ratio, more ordered fiber arrangement, and better histological repair in the injured area of ​​the tendon. These results indicate that the aNFs / Z@R(helical) scaffold can effectively repair injured Achilles tendons and inhibit heterotopic ossification, suggesting broad application prospects.

Claims

1. A composition for preparing a directional helical stent, characterized in that: The composition includes composition A for preparing the outer layer of the nanofiber membrane and composition B for preparing the inner layer of the nanofiber membrane; The composition A comprises the following components: polycaprolactone and type I collagen at a concentration of 10% to 20%; in the composition A, the mass ratio of type I collagen to polycaprolactone is 15 to 25:100; The composition B comprises the following components: polycaprolactone at a concentration of 10% to 20% and a drug; in the composition B, the mass ratio of the drug to the polycaprolactone is 5 to 10:100; the drug is rapamycin with ZIF-8 as a carrier; The nanofiber membrane is made into a directional spiral stent by mechanical rotation.

2. The composition according to claim 1, characterized in that The composition A further comprises hexafluoroisopropanol; the composition B further comprises dichloromethane and DMF, and the volume ratio of the dichloromethane to DMF is 2-5:

1.

3. A method for preparing a directional helical stent using the composition according to any one of claims 1 to 2, characterized in that: The steps include: (1) ZIF-8 was used as a delivery vector for rapamycin to prepare ZIF-8@RAPA; (2) The outer layer uses hexafluoroisopropanol as solvent, polycaprolactone and type I collagen as solutes; the inner layer uses a mixed solution of dichloromethane and DMF as solvent, polycaprolactone and ZIF-8@RAPA obtained in step (1) as solutes; and the nanofiber membrane is prepared by electrospinning technology; (3) The nanofiber membrane obtained in step (2) is mechanically stirred to prepare a directional spiral stent.

4. The method according to claim 3, characterized in that Step (1) specifically includes the following steps: 1) Dissolve zinc nitrate hexahydrate and add RAPA methanol solution to prepare a zinc-based solution; 2) Prepare imidazole solution using 2-methylimidazole as solute and methanol as solvent; 3) The zinc-based solution obtained in step 1) is added to the imidazole solution obtained in step 2) to react and obtain ZIF-8@RAPA.

5. The method according to claim 4, characterized in that In step 3), the reaction conditions are as follows: stirring for 3 to 10 minutes, standing for 50 to 57 minutes, and the total reaction time is 60 minutes.

6. The method according to claim 4, characterized in that The final concentration of zinc ion is 0.015~0.025mol / L; 2-mIm - The final concentration of the zinc nitrate hexahydrate is 0.10-0.2 mol / L; the concentration ratio of the zinc nitrate hexahydrate to the 2-methylimidazole is 1:7-10.

7. The method according to claim 3, characterized in that In step (2), the preparation parameters of electrospinning are: shaft speed 3500~4500rpm, voltage 20~30kV, injection speed 0.4~0.7mL / h, and receiving distance 15~20cm.

8. A directional spiral stent prepared by the method according to any one of claims 3 to 7.

9. Use of the composition according to any one of claims 1 to 2 and / or the directional spiral stent according to claim 8 in the preparation of a product for repairing tendon damage and / or inhibiting heterotopic ossification.

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