Tacrolimus / Zif-8 airway covering stent and its application in inhibiting airway cicatricial stenosis

Abstract

The application belongs to the technical field of covered stents, and relates to a tacrolimus / ZIF-8 airway covered stent and application of the tacrolimus / ZIF-8 airway covered stent in inhibiting airway cicatricial stenosis. Preparation of the tacrolimus / ZIF-8 airway covered stent comprises the following steps: loading tacrolimus on ZIF-8 to obtain tacrolimus@ZIF-8 particles; dissolving PLLA in hexafluoroisopropanol to prepare a PLLA solution; dissolving the tacrolimus@ZIF-8 particles in the PLLA solution to obtain a tacrolimus@ZIF-8 / PLLA solution; and electrospinning the tacrolimus@ZIF-8 / PLLA solution and spraying the solution on a metal bare stent to obtain the tacrolimus / ZIF-8 airway covered stent. The obtained tacrolimus / ZIF-8 airway covered stent has good biocompatibility, a low degree of foreign body reaction, a broad-spectrum antibacterial effect and a multi-pathway inhibition of fibrous tissue proliferation, and can be used for benign airway cicatricial stenosis and prevention of airway restenosis.

Description

Technical Field

[0001] This invention belongs to the field of covered stent technology, specifically relating to the tacrolimus / ZIF-8 airway covered stent and its application in inhibiting airway scarring stenosis. Background Technology

[0002] Benign cicatricial airway stenosis (BCAS) is caused by various endogenous or exogenous factors that damage the airway mucosa, such as endotracheal intubation, tracheotomy, tracheobronchial tuberculosis, chest trauma, and post-lung transplantation. These factors stimulate granulation tissue proliferation, which then organizes into fibrotic scar tissue, leading to airway stenosis. BCAS primarily manifests as cough and sputum production, often accompanied by recurrent respiratory infections. When the stenosis is ≥70%, it results in severe insufficiency of lung ventilation, causing respiratory distress and potentially threatening the patient's life. Current treatments for BCAS vary. Surgical resection of the thickened, stenotic tissue remains the preferred option for most benign airway stenosis cases. However, severe complications, the location of the stenosis (e.g., high subglottic), or an excessively long longitudinal extent (>4-6 cm) preclude open surgery. Some new techniques, such as airway cartilage grafting, have been reported, but extensive evidence is still needed before their application.

[0003] Interventional treatments for airway stenosis are gradually replacing traditional surgical procedures, such as airway stent placement and thermal ablation therapy.

[0004] Airway stent placement can rapidly relieve airway stenosis, improve ventilation in patients with benign airway scarring syndrome (BCAS), and help them quickly recover from life-threatening conditions. Airway stent placement is inexpensive, minimally invasive, and well-tolerated by patients, making it a preferred palliative treatment option for BCAS patients who are not suitable for surgery. However, bare-metal stents can cause airway restenosis due to foreign body rejection, infection, and other issues that may stimulate further granulation tissue growth. Currently, silicone stents are widely used clinically, significantly reducing restenosis rates compared to traditional bare-metal stents. However, even with long-term stent placement, varying degrees of respiratory distress still occur. Therefore, developing novel functional airway stents with antibacterial properties, inhibiting inflammatory responses, and regulating the airway immune microenvironment, and elucidating their biological mechanisms for inhibiting stent restenosis, are urgent medical challenges to be addressed in the field of benign airway scarring.

[0005] To reduce friction between bare metal stents and the body wall, and to seal fistulas, metal-coated stents are being used more and more widely in clinical practice. Summary of the Invention

[0006] To address the problems of foreign body rejection and infection caused by bare metal stents, this invention proposes a tacrolimus / ZIF-8 airway covered stent, which has good biocompatibility, low degree of foreign body reaction, broad-spectrum antibacterial effect, and multi-pathway inhibition of fibrous tissue proliferation. It can be used for benign airway scar stenosis and to prevent airway restenosis.

[0007] The present invention specifically adopts the following technical solution:

[0008] The preparation method of the tacrolimus@ZIF-8 / PLLA (Tac@ZIF-8 / PLLA) electrospun fiber coating of the present invention includes:

[0009] Step 1: Load tacrolimus onto ZIF-8 to obtain tacrolimus@ZIF-8 particles; this step specifically includes:

[0010] Step 1.1: Preparation of ZIF-8: Mix 2-methylimidazole solution and zinc nitrate hexahydrate aqueous solution, stir at room temperature, and react to obtain a white precipitate, which is ZIF-8 particles; the molar ratio of 2-methylimidazole to zinc nitrate hexahydrate is 10:1.

[0011] Step 1.2: Tacrolimus loading: Tacrolimus and ZIF-8 particles were added to water and stirred in the dark at room temperature to obtain tacrolimus@ZIF-8 particles.

[0012] The mass ratio of tacrolimus to ZIF-8 particles is 1:(2.5-3), preferably 1:2.9.

[0013] Step 2: Dissolve PLLA in hexafluoroisopropanol to prepare a PLLA solution; the mass ratio of PLLA to hexafluoroisopropanol is 1:10.

[0014] Tacrolimus@ZIF-8 granules were dissolved in PLLA solution to obtain a tacrolimus@ZIF-8 / PLLA solution; the volume ratio of tacrolimus@ZIF-8 granules to PLLA solution was 2 mg: 1 mL. That is, every 2 mg of tacrolimus@ZIF-8 granules was dissolved in 1 mL of PLLA solution.

[0015] Step 3: Electrospinning was performed on the tacrolimus@ZIF-8 / PLLA solution to obtain electrospun fiber coating. The electrospinning voltage was 15 kV and the flow rate was 0.5 mL / h.

[0016] The present invention also provides a tacrolimus / ZIF-8 airway membrane stent, the stent comprising a bare metal stent and a tacrolimus@ZIF-8 / PLLA electrospun fiber membrane covering the metal stent.

[0017] The method for preparing the tacrolimus / ZIF-8 airway membrane stent includes:

[0018] Step 1: Load tacrolimus onto ZIF-8 to obtain Tac@ZIF-8;

[0019] Step 2: Dissolve PLLA in hexafluoroisopropanol to prepare a PLLA solution; dissolve tacrolimus@ZIF-8 in the PLLA solution to obtain a tacrolimus@ZIF-8 / PLLA solution;

[0020] Step 3: The tacrolimus@ZIF-8 / PLLA solution is electrospun and sprayed onto a bare metal scaffold to obtain a tacrolimus / ZIF-8 airway membrane scaffold. The bare metal scaffold is a self-expanding bare metal scaffold.

[0021] This invention further provides the application of tacrolimus@ZIF-8 / PLLA electrospun fiber membrane, or the tacrolimus / ZIF-8 airway membrane stent, in inhibiting airway scarring stenosis. Application of the membrane stent of this invention in rabbits has shown that it can inhibit tracheal mucosal hyperplasia, reduce fibrous tissue infiltration of the airway wall, and alleviate the degree of airway inflammation.

[0022] The beneficial effects of this invention are as follows:

[0023] Poly-L-lactic acid (PLLA) is a semi-crystalline polymer with good mechanical stability and processing properties, as well as good biocompatibility and non-toxic degradation products.

[0024] Tacrolimus (Tac, also known as FK506) is a water-insoluble macrolide immunosuppressant with antiproliferative and anti-inflammatory activities.

[0025] ZIF-8 (Zeolitic imidazolate framework-8) is a non-toxic and biocompatible metal-organic framework (MOF) constructed from zinc ions and 2-methylimidazolium via coordination. Its large pore size allows it to accommodate drugs and biomolecules with large molecular structures. In the airway microenvironment or the acidic environment of lysosomes, the 2-methylimidazolium chain in ZIF-8 can undergo protonation, disrupting the coordination between zinc ions and the imidazolium ring, leading to the gradual degradation of the ZIF-8 structure and the release of the drug. The Zn released from the decomposition of ZIF-8 is partly due to this degradation. 2+ Zn exhibits excellent antibacterial effects. 2+It can disrupt the integrity of bacterial cysts and catalyze the production of oxygen free radicals, leading to bacterial death. Furthermore, the rough surface of ZIF-8 nanoparticles increases the contact area between bacteria, thereby enhancing antibacterial activity.

[0026] This invention achieves both superior antibacterial properties and increased Tac loading rate by loading Tac onto ZIF-8. First, the Tac loaded onto ZIF-8 is placed in a narrow airway, and Zn is released as ZIF-8 decomposes. 2+ In synergy with Tac, Tac exerts its antibacterial effect. Tac induces fibroblast apoptosis through the JNK / ERK pathway, and locally released Tac exhibits a strong anti-inflammatory effect in the airway. On the other hand, Tac can also directly target the JAK2 / STAT3 signaling pathway to inhibit macrophage polarization towards the M2 phenotype, reduce the induction of fibroblast transformation into myofibroblasts, and thus reduce the proliferation of fibrous tissue.

[0027] In summary, the Tac@ZIF-8 / PLLA electrospun fiber-coated scaffold (i.e., tacrolimus / ZIF-8 airway-coated scaffold) constructed in this invention has good biocompatibility, low degree of foreign body reaction, broad-spectrum antibacterial activity, and multiple pathways to inhibit fibrous tissue proliferation and prevent airway restenosis after scaffold placement. Attached Figure Description

[0028] Figure 1 Structure and chemical composition of each group of electrospun fiber coatings. (a) Microscopic images of PLLA, ZIF-8 / PLLA, Tac / PLLA, and Tac@ZIF-8 / PLLA films under SEM. (b) FT-IR spectra of PLLA, ZIF-8 / PLLA, Tac / PLLA, and Tac@ZIF-8 / PLLA films. (c) Raman spectra of PLLA, ZIF-8 / PLLA, Tac / PLLA, and Tac@ZIF-8 / PLLA films.

[0029] Figure 2 Mechanical analysis of electrospun fiber coatings in each group and Zn 2+ Ion release behavior analysis. (a) Strain-stress curves of PLLA, ZIF-8 / PLLA, Tac / PLLA, and Tac@ZIF-8 / PLLA films. (b) Zn content of ZIF-8 / PLLA and Tac@ZIF-8 / PLLA films. 2+ Ion release curve.

[0030] Figure 3Cytotoxic effects of electrospun fiber coating extracts on HBE and HPF. (a) Statistical analysis of HBE cytotoxicity rates of PLLA, ZIF-8 / PLLA, Tac / PLLA, and Tac@ZIF-8 / PLLA electrospun fiber coating extracts. (b) Statistical analysis of HPF cytotoxicity rates of PLLA, ZIF-8 / PLLA, Tac / PLLA, and Tac@ZIF-8 / PLLA electrospun fiber coating extracts. BC in the figure represents group BC.

[0031] Figure 4 Inhibitory effects of electrospun fiber coating extracts on MRSA bacteria. (a) Plate colony experiment of MRSA bacteria by PLLA, ZIF-8 / PLLA, and Tac@ZIF-8 / PLLA electrospun fiber coating extracts. (b) Statistical analysis of bacterial inhibition rates of MRSA bacteria by PLLA, ZIF-8 / PLLA, and Tac@ZIF-8 / PLLA electrospun fiber coating extracts. (c) MRSA bacterial proliferation curves under the action of PLLA, ZIF-8 / PLLA, and Tac@ZIF-8 / PLLA electrospun fiber coating extracts.

[0032] Figure 5 A visual image of a drug-eluting metal-coated stent.

[0033] Figure 6 : DSA-guided airway stent deployment process, with the white arrow indicating the airway stent deployment position.

[0034] Figure 7 HE staining, Masson staining, and immunohistochemical IL-6 staining of rabbit airway tissues 4 weeks after airway stent treatment in each group. Detailed Implementation

[0035] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of the embodiments of this invention will be clearly described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0036] The normal tracheal epithelial cells (HBE) or fibroblasts (HPF) used in the examples were purchased from the Center for Excellence in Molecular Cell Science (Institute of Biochemistry and Cell Biology), Chinese Academy of Sciences, and the New Zealand rabbits were purchased from Henan Xincheng Youkang Biotechnology Co., Ltd.

[0037] Example 1

[0038] 1. Preparation of coating on electrospun fibers

[0039] (1) Preparation of tacrolimus@ZIF-8 particles

[0040] Tac@ZIF-8 was prepared by physical blending, specifically including the following steps:

[0041] ① Preparation of ZIF-8: 85.1 mg of 2-methylimidazole was added to 4.2 mL of sterile deionized water and stirred until dissolved. Then, 1 mL of 0.1 M Zn(NO3)2·6H2O aqueous solution (zinc nitrate hexahydrate aqueous solution) was added dropwise. After mixing thoroughly at room temperature, a white precipitate was obtained. Finally, the product was separated by centrifugation at 10000 rpm for 10 min, washed three times with distilled water, and freeze-dried until white ZIF-8 solid particles were obtained and stored at -20℃ for later use.

[0042] ② Tacrolimus loading: 6.4 mg tacrolimus and 18.6 mg ZIF-8 particles were added to 5 mL of deionized water and stirred vigorously for 12 h in the dark at room temperature. The mixture was then centrifuged at 12000 rpm for 10 minutes to separate the product. The product was washed twice with distilled water and then freeze-dried to obtain tacrolimus@ZIF-8 particles.

[0043] (2) Preparation of electrospun fiber coating

[0044] The preparation of fibrous membranes or membrane-coated scaffolds based on PLLA as the basic biomaterial (divided into four groups: PLLA, Tac / PLLA, ZIF-8 / PLLA, and Tac@ZIF-8) specifically includes the following steps:

[0045] First, PLLA was dissolved in hexafluoroisopropanol (HFIP) to prepare a PLLA solution (PLLA to HFIP mass ratio of 1:10). Then, 20 mg of Tac, ZIF-8, and Tac@ZIF-8 were dissolved in 10 mL of the PLLA solution to prepare PLLA, Tac / PLLA, ZIF-8 / PLLA, and Tac@ZIF-8 / PLLA solutions, respectively. These solutions were then electrospun under a 15 kV high-voltage power supply at a flow rate of 0.5 mL / h, with the collector positioned 20 cm from the needle tip, resulting in a 0.1 mm thick electrospun fiber coating.

[0046] 2. Structure and chemical composition of electrospun fiber coating

[0047] The microstructure of electrospun fiber-coated PLLA, ZIF-8 / PLLA, Tac / PLLA, and Tac@ZIF-8 / PLLA was observed using scanning electron microscopy (SEM). Before observation, the samples were sputter-coated with gold in a vacuum coating machine. The surfaces of the four fiber films were imaged at a magnification of 300 nm. At least four images were taken for each sample. The average diameter of the electrospun fibers was measured using image analysis software from at least 50 randomly selected fibers in each image.

[0048] like Figure 1 As shown in Figure a, we can observe that the fiber structures of the four groups of electrospun fiber coatings prepared by electrospinning—PLLA, ZIF-8 / PLLA, Tac / PLLA, and Tac@ZIF-8 / PLLA—are all randomly and uniformly distributed. However, the fiber diameters of the ZIF-8 / PLLA and Tac@ZIF-8 / PLLA groups are significantly finer than those of the PLLA and Tac / PLLA groups. Measurements show that the fiber diameters of the PLLA and Tac / PLLA groups are mostly around 2000 nm, while the fiber diameters of the ZIF-8 / PLLA and Tac@ZIF-8 / PLLA groups are mostly around 700 and 800 nm. This may be related to the addition of ZIF-8.

[0049] Fourier transform infrared spectroscopy (FTIR) and Raman spectroscopy were used to preliminarily determine whether the composition and chemical structure of Tac and ZIF-8 in electrospun fibers coated with PLLA, Tac / PLLA, ZIF-8 / PLLA, and Tac@ZIF-8 / PLLA had changed after electrospinning. The measurement range was 4000-500 cm⁻¹. -1 The spectrum was collected in reflectance mode at 4 cm⁻¹. -1 The resolution was adjusted. For each spectrum, 32 scans were performed, and data analysis was conducted using Origin software (Origin 9.1).

[0050] like Figure 1 As shown in b, the FT-IR results of the electrospun fiber coatings show that the characteristic peaks of Tac and ZIF-8 are masked by PLLA. The PLLA, ZIF-8 / PLLA, Tac / PLLA, and Tac@ZIF-8 / PLLA fiber membranes all exhibit characteristic peaks similar to those of the PLLA fiber membrane. The raw material PLLA has characteristic peaks at 2995.43, 1751.84, 1181.99, and 1085.23 cm⁻¹. -1The characteristic peaks at these locations are generated by the stretching vibrations of methyl (-CH3), carbonyl (C=O), COC, and O-CH-CH3, respectively. ZIF-8 / PLLA, Tac / PLLA, and Tac@ZIF-8 / PLLA, made from PLLA, also exhibit these characteristic peaks with slight shifts. Figure 1 As shown in c, the characteristic peak results of the Raman spectra are roughly similar to those of FT-IR. All four film structures showed characteristic peaks roughly the same as those of PLLA, at 870.05, 1453.46, and 1768.70 cm⁻¹. -1 The characteristic peaks at these locations are generated by the stretching vibrations of COC, -CH3, and C=O, respectively.

[0051] 3. Mechanical analysis of electrospun fiber coating and Zn 2+ Analysis of ion release behavior

[0052] Mechanical analysis was performed on the four groups of electrospun fiber coatings. The electrospun fiber coatings of PLLA, ZIF-8 / PLLA, Tac / PLLA and Tac@ZIF-8 / PLLA were made into 5.0×0.5cm pieces with a thickness of 0.1mm. The tensile mechanical properties were measured using a universal testing machine at room temperature and 50% humidity. The tensile strength was calculated and analyzed by stress-strain curves.

[0053] like Figure 2 As shown in Figure a, the stress-strain curves of the electrospun fiber membranes in each group reveal that the maximum tensile strength of PLLA, Tac / PLLA, ZIF-8 / PLLA, and Tac@ZIF-8 / PLLA fiber membranes is between 6 and 8 MPa. Compared to PLLA and Tac / PLLA fiber membranes, the maximum strain capacity of ZIF-8 / PLLA and Tac@ZIF-8 / PLLA fiber membranes is around 0.4%, while that of PLLA and Tac / PLLA fiber membranes is above 2.0%. This is due to the smaller fiber diameter of ZIF-8 / PLLA and Tac@ZIF-8 / PLLA fiber membranes after the addition of ZIF-8.

[0054] To evaluate the Zn content of electrospun fiber-coated ZIF-8 / PLLA and Tac@ZIF-8 / PLLA... 2+To investigate ion release behavior, electrospun fiber coatings were first cut into small cubes weighing approximately 10 mg each, and then immersed in extraction flasks containing 5 mL of PBS. The suspension was maintained in a temperature-controlled water bath and shaken at 50 rpm and 37 °C. At 0 h, 1 h, 6 h, 12 h, 1 day, 3 days, 5 days, and 7 days, 1.0 mL of the sustained-release solution was collected from the extraction flasks for analysis. 1.0 mL of fresh PBS was then added to the extraction flasks. The Zn concentration in the release medium (PBS, pH 7.4) at different time points was measured using a zinc ion colorimetric method. 2+ The content of ions.

[0055] like Figure 2 As shown in b, Zn from ZIF-8 / PLLA and Tac@ZIF-8 / PLLA 2+ The release curve shows that Zn 2+ The Zn content of ZIF-8 / PLLA and Tac@ZIF-8 / PLLA gradually reaches equilibrium within 0–7 days. 2+ There was no significant difference in release on day 7, which were 0.34±0.021 μg / mL and 0.37±0.01 μg / mL, respectively.

[0056] 4. Cytotoxicity test of electrospun fiber coating

[0057] The experiments in this section used the CCK-8 kit.

[0058] Normal tracheal epithelial cells (HBE) or fibroblasts (HPF) (1×10 3 Cells were seeded overnight in 96-well plates with 100 μL of PBS in the BC group (blank control group), and 100 μL of electrospun fiber-coated PLLA, Tac / PLLA, ZIF-8 / PLLA, and Tac@ZIF-8 / PLLA extracts were added to each well in the experimental groups, respectively. On days 1, 2, and 3, CCK-8 reagent was added to each well, and the plates were incubated at 37°C and 5% CO2 for 3 hours for color development. The OD value at 450 nm was measured using a microplate reader. The relative cell proliferation rate was calculated using the following formula: Relative cell proliferation rate = [(ODc-ODs) / (ODc-ODb)] × 100%. ODb is the OD value of the blank well; ODc is the OD value of the control group on day 1; ODs is the OD value of the sample groups each day. The electrospun fiber-coated extract was prepared as follows: the electrospun fiber coating was cut into small cubes and then immersed in an extraction bottle containing PBS. The suspension was kept in a temperature-controlled water bath and shaken at 50 rpm and 37°C for 7 days to obtain the electrospun fiber coating extract. Each 10 mg of electrospun fiber coating was extracted with 5 mL of PBS.

[0059] like Figure 3As shown in a, the cell activity of HBEs treated with electrospun fiber coated PLLA, Tac / PLLA, ZIF-8 / PLLA and Tac@ZIF-8 / PLLA extracts did not change significantly.

[0060] like Figure 3 As shown in b, compared with the BC, PLLA, and ZIF-8 / PLLA groups, the Tac / PLLA and Tac@ZIF-8 / PLLA membrane extracts showed a significant inhibitory effect from day 1, and the inhibitory effect was even more obvious on day 4. This is because the addition of Tac to these two membranes can significantly inhibit the growth of HPF.

[0061] 5. Antibacterial test of electrospun fiber coating

[0062] In the experimental groups, 1 mL each of sterile electrospun fiber-coated PLLA, ZIF-8 / PLLA, and Tac@ZIF-8 / PLLA extracts were placed in three wells of a 6-well plate. In group BC, 1 mL of PBS was added to each well, and in group PC, 1 mL of vancomycin solution (5 mg / mL) was added to each well. Then, 2 mL of MRSA suspension (1 × 10⁻⁶) was added to each well. 8 The bacterial culture (CFU / mL) was incubated in a relatively humid environment for 5 h, and the culture was then gradually diluted to obtain a specific dilution range of 10. -4 Up to 10 -6 Then, 100 μL of diluted bacterial culture was inoculated onto Luria-Bertani (LB) culture dishes. After incubation overnight at 37°C, images of LB culture dishes from different groups were captured, and the colony-forming units (CFU) on the culture dishes were counted. The antimicrobial activity was calculated as the percentage reduction in bacteria using the following formula: Inhibition rate IR (%) = (NC - NS) / NC × 100, where NC and NS represent the average colony count in the blank control and the sample, respectively.

[0063] like Figure 4 As shown in Figure a, the bacterial colonies in the Tac@ZIF-8 / PLLA group were significantly lower than those in the other experimental groups after treatment with different electrospun fiber coating extracts. Compared with the BC and PLLA groups, the ZIF-8 / PLLA group showed a more significant inhibitory effect on bacterial proliferation, and the inhibition of bacteria was even more pronounced in the Tac@ZIF-8 / PLLA group with added Tac, with an inhibition rate of 92.61±4.02%. Figure 4 b).

[0064] Sterile electrospun fiber films (PLLA, ZIF-8 / PLLA, and Tac@ZIF-8 / PLLA) measuring 1cm x 1cm and 0.1mm in thickness were immersed in 3 mL of LB liquid medium as experimental groups. Groups BC contained only 3 mL of LB liquid medium, while Group PC contained 3 mL of LB liquid medium with 15 mg of vancomycin added. Then, 10 μL of LMRSA bacterial suspension (1 × 10⁻⁶) was added to the liquid medium of each group. 6 (CFU / mL). All samples were incubated at 37°C in a shaker for 24 h. At 0, 1, 2, 3, 4, 5, and 6 h, 200 μL of bacterial suspension was transferred to sterile 96-well plates, and the absorbance at 600 nm was measured using a microplate reader. Figure 4 As shown in c, the OD value of the Tac@ZIF-8 / PLLA bacterial culture at 600 nm decreased significantly after 6 h, specifically to 0.195±0.038, indicating that Tac@ZIF-8 / PLLA showed a significant inhibitory effect on MRSA.

[0065] 6. Preparation of metal-coated scaffolds

[0066] First, PLLA was dissolved in hexafluoroisopropanol (HFIP) to prepare a PLLA solution (PLLA to HFIP mass ratio of 1:10). Then, 20 mg of Tac, ZIF-8, and Tac@ZIF-8 were dissolved in 10 mL of the PLLA solution to prepare Tac / PLLA, ZIF-8 / PLLA, and Tac@ZIF-8 / PLLA solutions, respectively. The PLLA, Tac / PLLA, ZIF-8 / PLLA, and Tac@ZIF-8 / PLLA solutions were electrospun under a 15 kV high-voltage power supply at a flow rate of 0.5 mL / h. The spun fibers were then sprayed onto self-expandable metallic stents (SEMS) with a diameter of 0.8 mm and a length of 1.8 mm. After drying, the SEMS were stored in a sealed container at -4 °C for later use. The SEMS was aligned with a roller collector to collect the spun fibers.

[0067] like Figure 5 As shown, taking the Tac@ZIF-8 / PLLA metal-coated scaffold as an example, we can see that the electrospun fiber membrane was successfully coated onto the bare metal scaffold, and the fiber membrane was of uniform thickness and tightly adhered to the bare metal scaffold.

[0068] 7. Application of Tac@ZIF-8 / PLLA metal-coated stent

[0069] Under fluoroscopic guidance (Artis zee DSA system, SIEMENS, Germany), airway placement was performed using PLLA, Tac / PLLA, ZIF-8 / PLLA, and Tac@ZIF-8 / PLLA metal-coated stents. New Zealand rabbits were placed in a supine position with their necks hyperextended, and a 12F dilator (Cook Medical, USA) was used to dilate the rabbit's oral cavity. Then, a 0.035-inch guidewire (Terumo Corporation, Japan) and a 5F catheter (Terumo Corporation, Japan) were used. During this procedure, iodine contrast agent diluted with saline was injected into the airway via the catheter to perform tracheography, visualizing the tracheobronchial tree and establishing the path for stent placement. Referring to the airway path diagram, precise positioning was achieved under fluoroscopic guidance. When the stent was positioned approximately 1.5-2.0 cm above the carina at its distal end, the pusher lever of the stent pusher was fixed, and the outer sheath of the pusher was withdrawn to release the stent. Figure 6 As shown in the figure.

[0070] Four weeks later, the animals were euthanized under anesthesia with a high dose of xerazine hydrochloride injection. The trachea of ​​the rabbits was exposed, and the skin was prepared by cutting along the midline of the trachea. The neck tissue was dissected layer by layer to expose the tracheal stent site. The tracheal stent segment was cut along the transverse axis of the trachea, and the stent was immediately removed from the airway. The tracheal sample was rinsed with 0.9% sodium chloride solution and then fixed in 4% paraformaldehyde tissue fixative for 24 hours. The fixed tracheal tissue was dehydrated and then cleared. After dehydration, the tissue was cleared and immersed in paraffin (60℃) for 10 minutes to obtain tissue blocks. The obtained tissue blocks were embedded in paraffin and sectioned. The sections were flattened and floated on 40℃ warm water using a 3μm thick tissue microtome. The sections were quickly retrieved using a glass slide and dried in a 60℃ oven for 3 hours. HE staining was performed to observe airway narrowing under a microscope, and Masson staining was used to observe airway fibrosis.

[0071] To determine IL-6 expression, paraffin sections were dewaxed and hydrated for antigen retrieval. The tissue sections were placed in a retrieval box containing citrate buffer and microwaved to block endogenous peroxidase. The sections were then incubated in 3% hydrogen peroxide solution at room temperature in the dark for 25 min. The slides were washed three times with PBS (pH 7.4), 5 min each time. Serum blocking was performed by adding serum working solution and blocking at room temperature for 30 min. Primary and secondary antibodies were added for incubation, followed by DAB staining. Cell nuclei were counterstained with hematoxylin, and finally, the slides were dehydrated and mounted.

[0072] like Figure 7As shown, HE staining revealed that 4 weeks after implantation of PLLA, ZIF-8 / PLLA, Tac / PLLA, and Tac@ZIF-8 / PLLA metal-coated stents, varying degrees of significant granulation tissue proliferation were observed in the PLLA and ZIF-8 / PLLA groups, while the proliferation of tracheal mucosa in the Tac / PLLA group was weaker than that in the PLLA and ZIF-8 / PLLA groups. No significant proliferative tissue was found in the Tac@ZIF-8 / PLLA group, and its structure was similar to that of normal airways. Masson staining was used to assess the infiltration of fibrous tissue in the airway wall; varying degrees of fibrous tissue staining were observed in the PLLA, ZIF-8 / PLLA, and Tac / PLLA groups, while only a small amount of fibrous tissue staining was observed in the Tac@ZIF-8 / PLLA covered stent group. Immunohistochemistry was used to stain IL-6, and the area of ​​IL-6-positive tissue in the airway tissue of rabbits in different treatment groups was observed to reflect the degree of airway inflammation. The PLLA group had the highest IL-6 positive area, while the Tac@ZIF-8 / PLLA covered stent group had the smallest IL-6 positive area, demonstrating that the Tac@ZIF-8 / PLLA stent treatment resulted in the mildest airway tissue inflammation in rabbits.

[0073] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Tacrolimus@ZIF-8 / PLLA electrospun fiber coating, characterized in that, Its preparation methods include: Step 1: Load tacrolimus onto ZIF-8 to obtain tacrolimus@ZIF-8 particles; Step 2: Dissolve PLLA in hexafluoroisopropanol to prepare a PLLA solution; dissolve tacrolimus@ZIF-8 particles in the PLLA solution to obtain a tacrolimus@ZIF-8 / PLLA solution; Step 3: Electrospin the tacrolimus@ZIF-8 / PLLA solution to obtain electrospun fiber coating.

2. The tacrolimus@ZIF-8 / PLLA electrospun fiber coating according to claim 1, characterized in that, Step 1 includes: Step 1.1: Mix the 2-methylimidazole solution and the zinc nitrate hexahydrate aqueous solution, stir at room temperature, and the reaction yields a white precipitate, which is ZIF-8 particles; Step 1.2: Add tacrolimus and ZIF-8 particles to water and stir under dark and room temperature conditions to obtain tacrolimus@ZIF-8 particles.

3. The tacrolimus@ZIF-8 / PLLA electrospun fiber coating according to claim 2, characterized in that, The mass ratio of tacrolimus to ZIF-8 particles is 1:(2.5-3).

4. The tacrolimus@ZIF-8 / PLLA electrospun fiber coating according to claim 1, characterized in that, In step 2, the mass ratio of PLLA to hexafluoroisopropanol is 1:

10.

5. The tacrolimus@ZIF-8 / PLLA electrospun fiber coating according to claim 1, characterized in that, In step 2, the ratio of tacrolimus@ZIF-8 to PLLA solution is 2 mg: 1 mL.

6. The tacrolimus@ZIF-8 / PLLA electrospun fiber coating according to claim 1, characterized in that, In step 3, the voltage for electrospinning is 15kV and the flow rate is 0.5mL / h.

7. A tacrolimus / ZIF-8 airway membrane stent, characterized in that, The stent comprises a bare metal stent and a tacrolimus@ZIF-8 / PLLA electrospun fiber coating as described in claim 1, which is wrapped around the metal stent.

8. The tacrolimus / ZIF-8 airway membrane stent according to claim 7, characterized in that, The preparation method of the tacrolimus / ZIF-8 airway membrane stent includes the following steps: Step 1: Load tacrolimus onto ZIF-8 to obtain Tac@ZIF-8; Step 2: Dissolve PLLA in hexafluoroisopropanol to prepare a PLLA solution; dissolve tacrolimus@ZIF-8 in the PLLA solution to obtain a tacrolimus@ZIF-8 / PLLA solution; Step 3: Electrospin the tacrolimus@ZIF-8 / PLLA solution and spray it onto a bare metal scaffold to obtain a tacrolimus / ZIF-8 airway membrane scaffold.

9. The tacrolimus / ZIF-8 airway membrane stent according to claim 8, characterized in that, In step 3, the bare metal support is a self-expanding bare metal support.

10. The use of the tacrolimus@ZIF-8 / PLLA electrospun fiber membrane of claim 1 or the tacrolimus / ZIF-8 airway membrane stent of claim 2 in suppressing airway scarring stenosis.

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