DESCRIPTION LU600291
POLYLACTIC ACID-SILVER NANOPARTICLES-ANLOTINIB HYDROCHLORIDE
NANOFIBER FILM-COVERED TRACHEA STENT AND PREPARATION METHOD
THEREOF
TECHNICAL FIELDThe present invention relates to the field of biomedical polymer materials, and in particular to a polylactic acid (PLA)-silver nanoparticles (AgNPs)-anlotinib hydrochloride (AL) nanofiber film-covered trachea stent and a preparation method thereof.
BACKGROUNDTrachea stent implantation is an effective and timely method to restore the trachea patency and relieve the clinical symptoms of patients with tracheal stenosis. Currently, silicone stents and self-expandable metallic stents (SEMS) are the two main stent types commonly used in clinical practice. However, the high incidence of tracheal in-stent restenosis (TISR) prevents the long-term effectiveness and durability of stent implantation.
As a continuously expanding foreign body in the trachea, trachea stent may induce trachea microenvironment disorder, which is manifested as persistent inflammatory reaction, uncontrolled angiogenesis and high fibroblast activation. To make matters worse, this cascade of events can eventually lead to granulation tissue hyperplasia and worsening of TISR. Abnormal angiogenesis plays a key role in the promotion of granulation tissue proliferation. In addition, placement of trachea stents may increase microbial colonization, exacerbating the local inflammatory response. In order to relieve trachea inflammation after trachea stent implantation, various trachea stents loaded with anti-inflammatory or antibacterial drugs (e.g. indomethacin, dexamethasone, doxycycline, vancomycin) are used for trachea stent implantation. The construction of trachea stents capable of exerting anti-inflammatory and anti-angiogenic effects to remodel the trachea microenvironment may be an ideal strategy. LU600291
Advances in nanotechnology have highlighted the potential applications of AgNPs in the biomedical field. AgNPs has broad-spectrum bactericidal properties, enhanced safety and no antibiotic resistance properties. AgNPs can directly destroy bacterial cell wall, and also release Ag+ to stimulate the production of reactive oxygen species (ROS), destroy nucleic acid and protein in bacteria, and finally result in bacterial lysis. In addition, AQNPs exerts anti-inflammatory effects by down-regulating the expression of inflammatory factors including interleukin (IL)-6, IL-8 and tumor necrosis factor-a (TNF-a). Therefore, AgNPs is expected to be an ideal anti-microbial and anti-inflammatory agent to reduce the host's response.
In the abnormal microenvironment of trachea, vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF) and transforming growth factor-B (TGF-B) and other abnormal growth factors can stimulate the migration of vascular endothelial cells and uncontrolled angiogenesis through mediated signaling pathways thereof. Aberrant neovascularization is critical for the recruitment of inflammatory cells, the infiltration and activation of fibroblasts, and the transport of nutrients. More importantly, activated fibroblasts proliferate wildly and overexpress collagen, leading to extracellular matrix accumulation that exacerbates local granulation tissue hyperplasia. Only the trachea stent with anti-inflammatory effect could not effectively reduce the occurrence of tracheal stenosis. Therefore, further development of a stent for effectively reducing tracheal stenosis has been a crucial objective.
SUMMARYTo overcome the deficiencies in the prior art, the objective of the present invention is to provide a PLA-AgNPs-AL nanofiber film-covered trachea stent and a preparation method thereof.
To achieve the objective, the present invention provides the following technical solutions.
According to a first aspect of the present invention, there is provided a drug-loaded nanofiber film-covered trachea stent, including a SEMS. A nanofiber film outside the
SEMS is loaded with AgNPs and AL drugs.
According to a second aspect of the present invention, there is provided a preparatiqn,600294 method for the drug-loaded nanofiber film-covered trachea stent of the first aspect, including the following steps:
S1: dispersing AgNPs in a PLA solution to prepare a PLA/AgNPs solution, and sufficiently dispersing the AgNPs in the PLA;
S2: dissolving AL in the PLA solution to prepare a PLA/AL solution;
S3: mixing the PLA/AgNPs solution and the PLA/AL solution in the PLA solution to prepare a PLA/AgNPs/AL solution; and
S4: allowing the PLA/AgNPs/AL solution to be used as an electrospinning solution for electrospinning, and forming a nanofiber film on a surface of SEMS, and obtaining a
PLA/AgNPs/AL nanofiber film-covered trachea stent loaded with the AgNPs and Al drugs, namely a PAGL (PLA/AgNPs/AL) nanofiber film-covered trachea stent.
Preferably, the PLA is dissolved in hexafluoroisopropyl alcohol to produce the PLA solution.
More preferably, the PLA is present in the PLA solution in an amount of 0.12-0.13 g/ml.
Preferably, a mass of AgNPs in the PLA/AgNPs/AL solution is 3-5% of a mass of PLA.
More preferably, a mass of AgNPs in the PLA/AgNPs/AL solution is 4% of a mass of PLA.
Preferably, a mass of AL in the PLA/AgNPs/AL solution is between 0.5 and 1.5% of a mass of PLA.
More preferably, a mass of AL in the PLA/AgNPs/AL solution is 1% of a mass of PLA.
Preferably, a thickness of the nanofiber film in S4 is 0.2-1000 mum.
Preferably, the electrospinning process is carried out using a 15 kV high voltage power supply, a flow rate of 0.5 mL/h and a collector located 20 cm from a needle tip.
Preferably, an average diameter of the nanofibers formed after electrospinning of the
PLA/AgNPs/AL solution ranges from 314.10-525.60 nm.
According to a third aspect of the present invention, there is provided an application of the drug-loaded nanofiber film-covered trachea stent of the first aspect in medical materials.
According to a fourth aspect of the present invention, there is provided an application of the drug-loaded nanofiber film-covered trachea stent of the first aspect in treatment,500291 materials for tracheal stenosis.
The present invention has the following advantageous effects. (1) According to the preparation method provided by the present invention, AgNPs and
AL are introduced into a PAGL nanofiber film at the same time, and the prepared PAGL nanofiber film-covered trachea stent has good hydrophobicity, high mechanical strength and good mechanical properties. The hydrophobicity makes the stent beneficial to prevent sputum adhesion and improve trachea cleanliness. At the same time, the AgNPs and AL are not only effective on PAGL nanofiber film, but also have suitable drug release properties. Burst release of AgNPs and AL within 0-7 days to exert drug effect rapidly, release drug rapidly within a short time to exert drug effect, and achieve an objective of anti-inflammation and anti-proliferation; and slow and stable release of AgNPs and AL in 8-21 days, local maintenance of a certain drug concentration, continued to play the drug effect. In addition, although there is a sudden release process of AgNPs and/or AL in the nanofiber film, an almost closed environment is formed between the drug-loaded nanofiber film-covered trachea stent and the trachea, which is not eroded by the flowing liquid and air stream, and the released drug is still locally concentrated in the trachea, thereby maintaining an effective high drug concentration. (2) The nanofiber film loaded with AL alone has no antibacterial activity, but the nanofiber film loaded with AgNPs alone and the nanofiber film loaded with AgNPs and AL drugs significantly inhibit the growth of methicillin-resistant Staphylococcus aureus (MRSA), and the nanofiber film loaded with AgNPs and AL drugs simultaneously inhibit the growth of
MRSA by 83.81 + 8.32%, significantly higher than the nanofiber film loaded with AgNPs alone. The nanofiber film loaded with AgNPs and AL drugs provided by the present invention has excellent antibacterial properties. At the same time, the nanofiber film- covered trachea stent prepared in the present application can effectively inhibit the proliferation of microorganisms in the trachea, has the ability to maintain the microecology of the trachea, can reduce the incidence of TISR, and has a positive effect on improving the patency of trachea stent.
(3) In an abnormal microenvironment of the trachea, various abnormal growth factofs,690291 including vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF) and transforming growth factor-B (TGF-B) can stimulate the migration of vascular endothelial cells and uncontrolled angiogenesis through their mediated signaling pathways. Aberrant neovascularization is critical for the recruitment of inflammatory cells, the infiltration and activation of fibroblasts, and the transport of nutrients. Activated fibroblasts proliferate wildly and overexpress collagen, leading to extracellular matrix accumulation, which exacerbates local granulation tissue hyperplasia. The drug-loaded nanofiber film-covered trachea stent prepared in the present application can significantly inhibit the proliferation activity and migration ability of human umbilical vein endothelial cells (HUVECS), especially when loaded with AgNPs and AL at the same time, the inhibitory effect is the strongest, and the number of migrated cells can be reduced from 568.33 + 51.93 to 56.67 + 5.69, thereby enhancing the anti-angiogenesis ability of the nanofiber film, reducing the recruitment of inflammatory cells, infiltration and activation of fibroblasts and the transport effect of nutrients by the new blood vessels in the trachea, and further reducing local granulation tissue proliferation, reducing trachea tissue inflammation, reducing the degree of granulation tissue proliferation and collagen deposition, thereby improving
TISR. At the same time, the synergistic effect of AgNPs and AL is further proved to enhance the anti-angiogenic effect of PAGL nanofiber film-covered trachea stent by quantitative detection of protein Caspase-3, protein Ki67 and marker CD31.
In summary, according to the PLA-AgNPs-AL nanofiber film-covered trachea stent prepared in the present application, PLA is used as a drug delivery platform, loaded with
AgNPs and AL, and a synergistic strategy is adopted to impart excellent anti-inflammatory, anti-vascular, anti-bacterial, anti-angiogenesis and anti-granulation properties to the trachea stent, which can effectively reduce the incidence of TISR, and has a positive effect on improving the patency of the trachea stent.
BRIEF DESCRIPTION OF THE DRAWINGS LU600294
FIG. 1 shows the test results of the physicochemical properties, mechanical properties and drug release performance of the nanofiber film-covered on a surface of drug-loaded nanofiber film-covered trachea stent. Where a: transmission electron microscopy (TEM) images of AgNPs; b: Fourier transform infrared spectroscopy (FTIR) spectra of PLA nanofiber films, PAG nanofiber films, PAL nanofiber films, and PAGL nanofiber films; c:
Raman spectra of PLA nanofiber films, PAG nanofiber films, PAL nanofiber films, and
PAGL nanofiber films; d: scanning electron microscopy (SEM) images of PLA nanofiber films, PAG nanofiber films, PAL nanofiber films, and PAGL nanofiber films; e: element mapping results of energy dispersive spectroscopy analysis of PAGL nanofiber films; f: fitting curve of fiber diameter distribution of PLA nanofiber, PAG nanofiber, PAL nanofiber and PAGL nanofiber; g: energy dispersive spectrometry spectra of PAGL nanofiber film; h: water contact angle images and statistical analysis results of PLA nanofiber films, PAG nanofiber films, PAL nanofiber films and PAGL nanofiber films; i. typical stress-strain curves of PLA nanofiber films, PAG nanofiber films, PAL nanofiber films and PAGL nanofiber films; j: AgNPs release curves of PAG nanofiber films and PAGL nanofiber films at room temperature on day 0-21, with X axis representing time and Y axis representing
AgNPs release ratio; and k: AL release curves in PAL nanofiber films and PAGL nanofiber films at room temperature on day 0-21, with X axis representing time and Y axis representing AL release ratio. In the figure: PLA is a PLA nanofiber film without drug loading; PAG is a nanofiber film loaded with AgNPs drug; PAL is a nanofiber film loaded with AL drug; and PAGL is a nanofiber film loaded with AgNPs and AL drugs.
FIG. 2 shows the results of testing the antibacterial property of the nanofiber film-covered on the surface of drug-loaded nanofiber film-covered trachea stent. Where a: mechanism diagram of inhibiting the growth of methicillin-resistant Staphylococcus aureus (MRSA) by nanofiber film loaded with AgNPs drug, ROS being reactive oxygen species; b: MRSA co-incubated with PAG nanofiber film, PAL nanofiber film and PAGL nanofiber film at 600 nm optical density (OD) test result curve; and c: MRSA representative colony formation images incubated with PAG nanofiber films, PAL nanofiber films and PAGL nanofiber filmS;609291
In the figure: BC is the untreated group (Blank control); PC is the Positive control group supplemented with vancomycin. PAG is a nanofiber film loaded with AgNPs drug; PAL is a nanofiber film loaded with AL drug; and PAGL is a nanofiber film loaded with AgNPs and
AL drugs.
FIG. 3 shows the results of the in vitro anti-angiogenic effect test of the nanofiber film- covered on the surface of drug-loaded nanofiber film-covered trachea stent. The PAG nanofiber films, PAL nanofiber films and PAGL nanofiber films are incubated with
HUVECs; a: statistical analysis results of cell proliferation activity; b: cell OD value curve at 450 nm for 3 consecutive days; c: representative pictures of cell scratch experiment and Transwell experiment; d: quantitative results of wound healing rate in scratch test; and e: quantitative results of HUVECs migration in Transwell experiment. In the figure:
BC is untreated group (Blank control); PAG is the nanofiber film loaded with AgNPs drug;
PAL is the nanofiber film loaded with AL drug; and PAGL is the nanofiber film loaded with
AgNPs and AL drugs, * P <0.05, ** P <0.01 and * * * P <0.001.
FIG. 4 shows the results of anti-TISR evaluation of in vivo drug-loaded nanofiber film- covered trachea stent. Where a: schematic diagram of trachea stent implantation in New
Zealand rabbit model; b: fluoroscopic monitoring images of digital subtraction angiography (DSA) during trachea stent implantation into trachea of New Zealand rabbits; c: trachea volume rendering technique (VRT) images, vascular stent VRT images and sagittal CT (computed tomography) images after 4 weeks of trachea stent implantation; d: H&E and Masson staining results of trachea tissue; e: statistical diagram of trachea stent implantation time; f: statistical chart of tracheal ventilation rate of New Zealand rabbits in each group after 4 weeks of trachea stent implantation; g: statistical analysis of tracheal epithelial thickness; and h: statistical analysis of collagen deposition. In the figure:
Ctrl is a commercial trachea stent; PAG is the nanofiber film-covered trachea stent loaded with AgNPs drug; PAL is the nanofiber film-covered trachea stent loaded with AL drug; and PAGL is the nanofiber film-covered trachea stent loaded with AgNPs and AL drugs, *
P <0.05, ** P <0.01 and * * * P <0.001.
FIG. 5 shows the results of in vivo antiangiogenic and antibacterial performance test pfj600291 drug-loaded nanofiber film-covered trachea stent. Where a: 4 weeks after implantation of airway stents, dual immunofluorescence staining of Ki67 and Caspase-3 in tracheal tissues and CD31 in tracheal tissues are performed, and DAPI is used to label nucleus; b-c: statistical analysis results of mean fluorescence intensity (MFI) of Ki67 and Caspase- 3; d: MFI statistical analysis results of CD31; e-f: the drug-loaded nanofiber film-covered airway stent implanted in the trachea of New Zealand rabbits is removed, the covered nanofiber film is removed, and cultured in plate medium. The figure shows the representative picture of plate cloning of microorganisms and the results of statistical analysis of microbial content; and g: analysis results of correlation between microbial content and granulation tissue formation in the control group and PAG nanofiber film- covered airway stents. In the figure: Ctrl is a commercial trachea stent; PAG is the nanofiber film-covered trachea stent loaded with AgNPs drug; PAL is the nanofiber film- covered trachea stent loaded with AL drug; and PAGL is the nanofiber film-covered trachea stent loaded with AgNPs and AL drugs, * P <0.05, * * P <0.01 and * * * P <0.001.
DETAILED DESCRIPTIONTo make the objectives, technical solutions and advantages of the present invention more apparent, the present invention will be further described in detail with examples and drawings. It is to be understood that the specific examples described herein are only for explaining the present invention and are not used to limit the present invention.
Example 1 nanofiber film-covered trachea stent construction
In the present invention, AgNPs and AL are loaded in PLA using an electrospinning technique to prepare a nanofiber film coated on SEMS, thereby obtaining a drug-loaded nanofiber film-covered trachea stent.
Example (1) Preparation of PLA solution
Four portions of the same PLA solution are prepared for use, each portion being prepared as follows: 1.25 g PLA is dissolved in 10 ml of hexafluoroisopropyl alcohol.
Example (2) Preparation of PLA/AgNPs/AL solution
S1: 1 portion of the PLA solution prepared in Example (1) is taken, 0.05 g AgNPs is dissolved in a part of the PLA solution to disperse the AgNPs sufficiently, and to prepar@;600291 a solution A for use;
S2: 0.01g AL is dissolved in part of PLA solution to prepare a solution B for later use; and
S3: the solution A, solution B and the remaining PLA solution are mixed evenly to prepare a PLA/AgNPs/AL solution, a mass of AL in the solution accounts for 1% of a mass of PLA, and a mass of AgNPs accounts for 4% of a mass of PLA.
Example (3) Preparation of PLA/AgNPs solution 1 portion of PLA solution prepared in Example (1) is taken, 0.05 g AgNPs is dissolved in a part of PLA solution to prepare a PLA/AgNPs solution with a mass of AgNPs accounting for 4% of a mass of PLA.
Example (4) Preparation of PLA/AL solution 1 portion of the PLA solution prepared in Example (1) is taken, and 0.01 g of AL is dissolved in a part of the PLA solution to prepare a PLA/AL solution with a mass of AL accounting for 1% of a mass of PLA.
The PLA solution, PLA/AgNPs solution, PLA/AL solution and PLA/AgNPs/AL solution prepared in Examples (1)-(4) are used as electrospinning solutions to perform electrospinning to obtain four different nanofibers, namely PLA nanofibers, PLA/AgNPs nanofibers (PAG nanofiber), PLA/AL nanofibers (PAL nanofiber) and PLA/AgNPs nanofibers (PAGL nanofiber). The above-mentioned nanofibers are coated on a surface of SEMS respectively, and four different nanofiber-coated trachea stents are obtained, namely, a PLA nanofiber-coated trachea stent, a PAG nanofiber-coated trachea stent, a
PAL nanofiber-coated trachea stent and a PAGL nanofiber-coated trachea stent.
The electrospinning conditions are: electrospinning is carried out using a 15 kV high voltage power supply, a flow rate of 0.5 mL/h, and a drum collector 20 cm from a needle tip; during the electrospinning, aligning the SEMS with a roller collector to collect spun fibers to obtain a nanofiber film having a thickness of 0.2-1000 um; and to ensure a relatively consistent nanofiber film thickness on the SEMS surface, performing electrospinning on each SEMS with 1.7 mL of the electrospinning solution.
The PLA nanofiber film-covered trachea stent is not loaded with a drug; the PAG nanofiber film-covered trachea stent is loaded with a AgNPs drug; the PAL nanofiber film-covered,500291 trachea stent is loaded with an AL drug; and the PAGL nanofiber film-covered trachea stent is loaded with the AgNPs and AL drugs. The above nanofiber film-covered trachea stent is dried and stored in a sealed container at -4°C for future use.
Example 2 Properties characterization of nanofiber film
In a process of preparing drug-loaded nanofiber film-covered trachea stent, the PLA nanofibers, PAG nanofibers, PAL nanofibers and PAGL nanofibers are coated on the surface of SEMS, and different types of nanofiber films are formed on the surface of
SEMS, namely, a PLA nanofiber film, a PAG nanofiber film, a PAL nanofiber film and a
PAGL nanofiber film. (1) AgNPs particle size and morphology detection
The smaller the particle size, the stronger the antibacterial property of AgNPs, and the inventors uses TEM (JEM-2100F; JEOL, Japan) to identify the particle size and morphology of AgNPs, and the results are shown in FIG. la.
It can be seen from FIG. 1a that AgNPs has a spherical shape with a diameter in the range of 10-15 nm, which lays a foundation for good antibacterial performance of drug- loaded nanofiber film-covered trachea stent in the later stage. (2) Chemical composition and chemical group detection of nanofiber film
The chemical compositions of PLA nanofiber film, PAG nanofiber film, PAL nanofiber film and PAGL nanofiber film are detected using FTIR (sample scanning range is 500-4000 cm”), and a FTIR-derived spectrum is shown in FIG. 1b. Further, Raman spectrum (DXR2;
Thermo Fisher Technology Co., LTD.) is used to analyze the chemical groups of the chemical components of the four nanofiber films, and the results are shown in FIG. lc.
It can be seen from FIG. 1a that typical peaks of the PLA nanofiber film appear at 1752 cm” for C = O stretching, 1453 cm”! for CH bending, 1182 cm”! for C-O-C stretching, 1129 cm”! and 1085 cm” for C-O stretching, and 1045 cm” for OH bending. The PAG nanofiber film, PAL nanofiber film and PAGL nanofiber film all have typical PAL characteristic peaks, but have slight shifts.
It can be seen from FIG. 1c that characteristic peaks of the PLA nanofiber film appear at
297 cm” for bending C-O-C, 399 cm” for C-COO stretching, 872 cm”! for C-CO groYR 600294 vibration, 1043 cm" for C-CH3 skeleton stretching, 1128 cm” for CH3 asymmetric rocking, 1454 cm”! for CH3 symmetric bending, 1772 em” for C = O asymmetric stretching and finally 2945 cm“ for CH3 symmetric stretching. Obviously, in the PAGL nanofiber film, main characteristic peaks of raw material components are shifted at 243 cm" and 873- 1593 cm”, in addition to the characteristic peaks of PLA. Taken together, these spectra confirm that AgNPs or/and AL are successfully introduced into the PAGL nanofibers. (3) Physico-chemical characterization of nanofiber films
An energy dispersive dpectroscopy (EDS) system-equipped SEM (JSM-7401 F; JEOL,
Japan) is used to observe the surface morphology and fiber diameter of PLA nanofiber film, PAG nanofiber film, PAL nanofiber film and PAGL nanofiber film, and the test results are shown in FIG. 1d. The fiber diameter distributions of PLA nanofibers, PAG nanofibers,
PAL nanofibers and PAGL nanofibers are analyzed by fitting curves, and the results are shown in FIG. If. Meanwhile, the elemental mapping in the PAGL nanofiber film is analyzed, and the results are shown in FIGS. 1e and 1g.
It can be seen from FIG. 1d that the PLA nanofiber film, the PAG nanofiber film, the PAL nanofiber film and the PAGL nanofiber film all show a unique random interconnected fiber network structure. It can be seen from FIG. 1f that average diameters of PLA nanofibers,
PAG nanofibers, PAL nanofibers and PAGL nanofibers are 275.77 + 98.44 nm, 299.11 + 105.03 nm, 347.38 + 112.34 nm and 419.85 + 105.75 nm. The mapping of some elements in the PAGL nanofiber film is shown in FIG. 1e. It can be seen from FIGS. 1e and 1g that carbon (C), oxygen (O), nitrogen (N), sodium (Na), chlorine (CI), fluorine (F), and silver (Ag) are uniformly distributed throughout the PAGL nanofiber film, further confirming that
AL and AgNPs are successfully loaded onto the PAGL nanofibers. (4) Mechanical properties testing of nanofiber films
The hydrophilicity of the PLA nanofiber film, the PAG nanofiber film, the PAL nanofiber film, and the PAGL nanofiber film is evaluated by measuring water contact angles using a contact angle measuring device (Portsmouth, USA) in combination with the Laplace-
Young method, and the test results are shown in FIG. 1h. In addition, a universal testing machine (CMT6503;, Shenzhen SANS testing machine, China) is used to test the tensil& 590291 strength of the four nanofiber films at a tensile rate of 10 mm/min, and the test results are shown in FIG. 1i.
It can be seen from FIG. 1h that the droplet morphologies of PLA nanofiber film, PAG nanofiber film, PAL nanofiber film and PAGL nanofiber film remains basically unchanged compared with fresh contact after 15 s of stabilization, and the water contact angles thereof are 133.7 + 2.1°, 127.3 + 4.2°, 126.7 + 1.5° and 129.3 + 2.5°, and compared with an initial (0 s) water contact angle, the difference is not statistically significant. The results show that the doping content of AgNPs and AL in the present invention has no effect on an overall hydrophobicity of the PAGL nanofiber film, which is beneficial for preventing sputum adhesion and improving trachea cleanliness.
The nanofiber film has good mechanical properties, which is the necessary condition for its coating on a surface of trachea stent to achieve in vivo application. It can be seen from the stress-strain curve analysis results of the tensile test in FIG. 1i that, compared with the PLA nanofiber film, although the ductility of the nanofiber film is slightly decreased due to the introduction of AL in the PAL nanofiber film, the introduction of AgNPs in the
PAG nanofiber film and the introduction of AgNPs and AL in the PAGL nanofiber film enhance the mechanical strength of the nanofiber film, and the mechanical strength of the PAGL nanofiber film is the highest. (5) Drug release behavior detection of nanofiber films
In order to examine the drug release behavior of the nanofiber films, the nanofiber films are cut into small cubes of about 10 mg; the cubes are soaked in 5 ml PBS, 50 rpm, and stirred at 37°C to obtain a sustained-release solution; from day 0 to day 21, 1 ml of sustained-release solution is collected for test at specific time interval, a UV-VIS spectrophotometer (model: 3700, Shimadzu, Japan) is used to determine absorbance values of sustained-release solution at 411 nm and 222 nm to determine the release amount of AgNPs and AL in the release medium (namely, PBS with pH of 7.4); and finally, the percent release of AgNPs and AL is calculated based on the initial weight of AgNPs and AL in the nanofiber film, and the results are shown in FIGS. 1J and 1k.
It can be seen from FIG. 1j that the release of AgNPs in the PAG nanofiber film and th&,500291
PAGL nanofiber film can be divided into burst release period (0-7 days) and constant release period (8-21 days). During the burst release, the PAG nanofiber film and PAGL nanofiber film can rapidly release the drug in a short time to exert drug effect, and achieve an objective of anti-inflammation and anti-proliferation; and the PAG nanofiber film and
PAGL nanofiber film release drug slowly and steadily in a constant release period, which can maintain a certain drug concentration in the local area and exert the drug effect continuously. The release rates of AgNPs from the PAG nanofiber film and PAGL nanofiber film in the PBS solution are 57.03 + 4.89% and 59.38 + 5.64% in the first 7 days.
After that, AgNPs in the PAG nanofiber film and PAGL nanofiber film is slowly and uniformly released for 21 days, and the release rates are 61.7 + 6.16% and 65.23 + 7.12%, without significant difference.
It can be seen from FIG.1k that in the PAL nanofiber film and the PAGL nanofiber film, the release of AL Is also divided into burst release period (0-7 days) and constant release period (8-21 days). During the burst release, the PAL nanofiber film and PAGL nanofiber film can rapidly release the drug in a short time to exert drug effect, and achieve an objective of anti-inflammation and anti-proliferation; and the PAL nanofiber film and PAGL nanofiber film release drug slowly and steadily in a constant release period, which can maintain a certain drug concentration in the local area and exert the drug effect continuously. The burst release of AL is 64.83 + 4.84% in the PAL nanofiber film and 68.38 + 4.65% in PAGL nanofiber film in the first 7 days. AL is slowly released over the next 8- 21 days.
Although there is a sudden release process of AgNPs and/or AL in the nanofiber film, an almost closed environment is formed between the drug-loaded nanofiber film-covered trachea stent and the trachea, which is not eroded by the flowing liquid and air stream, and the released drug is still locally concentrated in the trachea, thereby maintaining an effective high drug concentration.
Example 3 Testing of the antibacterial properties of nanofiber films
With methicillin-resistant Staphylococcus aureus MRSA (ATCC 33592) as an experimental subject, the inventor respectively tests the inhibitory effects of PAG;500291 nanofiber film, PAL nanofiber film and PAGL nanofiber film on drug-resistant bacteria. The mechanism of inhibiting MRSA growth by PAG nanofiber film loaded with the AgNPs drug is shown in FIG. 2a. During the experiment, the nanofiber film is cut into a square of 1 x 1 cm”, irradiated under ultraviolet rays for 30 minutes, and sterilized. Subsequently, the sheared nanofiber films are co-incubated with MRSA, and an optical density (OD) value at 600 nm is measured as shown in FIG. 2b. Meanwhile, MRSA is inoculated on a solid medium for cultivation, and the sheared nanofiber films are added to the medium to observe the formation of MRSA colonies, and the test results are shown in FIG. 2c.
It can be seen from FIG. 2b that after th PAL nanofiber film loaded with AgNPs is incubated with the MRSA, the OD value at 600 nm is not significantly different from that of a blank control (BC) group, suggesting that AL does not exert antibacterial function. The OD value at 600 nm of PAG nanofiber film loaded with AgNPs and PAGL nanofiber film loaded with
AgNPs and AL are significantly lower than that of PAL nanofiber film group after co- incubation with the MRSA. Compared with positive control group (added with antibiotics,
PC group), there is no significant difference in OD value at 600 nm between PAG nanofiber film and PAGL nanofiber film co-incubated with MRSA group. The results show that AgNPs could significantly inhibit the proliferation of MRSA.
It can be seen from FIG. 2c that, compared with BC group, co-incubation of PAL nanofiber film with MRSA does not affect the clonal proliferation of MRSA. Compared with PAL nanofiber film, the co-incubation of PAG nanofiber film and PAGL nanofiber film with
MRSA significantly have inhibited the formation of MRSA colonies, and the inhibition rates of PAG nanofiber film and PAGL nanofiber film on MRSA growth are 80.56 + 6.50% and 83.81 + 8.32%.
In conclusion, the PAL nanofiber film loaded with AL alone do not have, but the PAG nanofiber film loaded with AgNPs and the PAGL nanofiber film loaded with AgNPs and AL have excellent antibacterial properties.
Example 4 Testing of anti-angiogenesis ability of nanofiber films
HUVECs are selected for in vitro anti-angiogenesis experiments. The PAG nanofiber film,
the PAL nanofiber film, and the PAGL nanofiber film are cut into squares of 1 x 1 cm? 600291 respectively, and irradiated under ultraviolet rays for 30 minutes. (1) Detection of HUVECS proliferation activity by cell counting kit (CCK-8) experiment
CCK-8 kit is used in cell proliferation experiment. Cells (1 x 103/mL) are inoculated in 96- well plates overnight. To each well, PBS or each experimental group of fiber film extract solution is added. On days 1, 2, and 3, CCK-8 reagent is added to each well, and incubated in an incubator at 37°C, 5% CO» for 3 hours to develop color. The OD value at 450 nm is measured with a microplate reader. The relative cell proliferation rate is calculated according to the following formula: relative cell proliferation rate = [(ODc-
ODs)/(ODc-ODb)] x 100%. ODb is an OD value of blank well; ODc is an OD value of the control group on the first day; and ODs is an OD value of the sample group per day. The test results are shown in FIGS. 3a and 3b.
It can be seen from FIG. 3a that, compared with BC group, co-incubation of PAG nanofiber film, PAL nanofiber film and PAGL nanofiber film with HUVECs can significantly inhibit the proliferation activity of HUVECs, and the PAGL nanofiber film has the strongest inhibitory effect on the proliferation activity of HUVECs. In order to characterize the effect of nanofiber films on the proliferation of HUVECs, CCK-8 measurements are performed for 3 consecutive days and a curve of OD values of HUVECs at 450 nm are plotted. It can be seen from FIG. 3b that the PAG nanofiber film, PAL nanofiber film and PAGL nanofiber film significantly inhibit the proliferation of HUVECs, and the PAGL nanofiber film has the most significant inhibitory effect on the proliferation of HUVECs. (2) Detection of HUVECs migration ability by scratch test and Transwell test
HUVECs are inoculated into a 6-well plate at a density of 1 x 10° cells/mL, and after a cell monolayer is formed, a sterile P200 pipette tip is used to scratch, and PBS is used to rinse free cells; subsequently, different nanofiber films are added to the cell culture plates.
Photographs of the HUVECs are taken under a fluorescent microscope (1X53, Olympus,
Japan) at 0 and 24 h to calculate wound healing rates, and the results are shown in FIG. 3c and FIG. 3d. Wound healing rate = (WO0-W24)/\WO0 x 100%, the WO is a wound area scratched at 0 h, and the W24 is a wound area scratched at 24 h.
HUVECs are inoculated into an upper chamber of Transwell at a density of 5 x 100600291 cells/mL, different nanofiber films are added into the upper chamber of Transwell, and after a total incubation time of 36 h, migrated cells remaining on a lower surface of a filter film of the upper chamber of Transwell are removed with cotton swabs; and the migrated cells on the lower surface of the filter film are stained with 0.5% crystal violet solution and photographed with a light microscope after fixation with 4% paraformaldehyde. The number of cells migrating is statistically analyzed using the Image J software (National
Institutes of Health, Bethesda, USA) and the results are shown in FIGS. 3C and 3e.
It can be seen from FIGS. 3c and 3d that an intercellular space closure rate in BC group is as high as 80.13 + 7.58%. However, after co-incubation with the PAG nanofiber film,
PAGL nanofiber film and PAGL nanofiber film, the gap closure of HUVECs cells is hindered. The HUVECs treated with the PAGL nanofiber film have the largest intercellular space (8.93 + 2.22%) after 24 h. It can be seen from FIGS. 3c and 3e that the HUVECs in BC group can pass through the filter film and cover almost the whole lower surface of the upper and lower chamber of the Transwell, and the migration number of HUVECs in
BC group is as high as 1038.00 + 67.51, indicating that HUVECs have a strong migration ability. However, the number of HUVECs migrated cells incubated with the PAG nanofiber film, PAL nanofiber film, and PAGL nanofiber film decreased significantly to 568.33 + 51.93, 288.00 + 20.07, and 56.67 + 5.69, respectively. In conclusion, the PAG nanofiber film, PAL nanofiber film and PAGL nanofiber film can effectively inhibit the migration ability of HUVECs, and the PAGL nanofiber film loaded with AgNPs and AL has the highest inhibition rate on HUVECs migration. The AgNPs and AL can synergically enhance the anti-angiogenesis ability of nanofiber films.
Example 5 Experimental verification of nanofiber film-covered trachea stent against TISR in vivo (1) Experimental animals and grouping treatment
The anti-TISR effect of nanofiber film-covered trachea stent in vivo is validated using male
New Zealand rabbits. The experimental animals are from the Laboratory Animal Center of Henan Hualan Biological Co. Ltd. China. All implantation procedures are approved by the Animal Care Committee of the First Affiliated Hospital of Zhengzhou University and;goo291 are conducted in accordance with the guidelines of the National Institutes of Health for the care and use of laboratory animals.
New Zealand rabbits are randomly divided into 4 groups (N = 5): Ctrl group, PAG group,
PAL group, and PAGL group. Ctrl group: s commercial trachea stent is implanted in the trachea; PAG group: a PAG nanofiber film-covered trachea stent loaded with AgNPs drugs is implanted in the trachea; PAL group: a PAL nanofiber film-covered trachea stent loaded with AL drugs is implanted in the trachea; and PAGL group: a PAGL nanofiber film- covered trachea stent loaded with AgNPs and AL drugs is implanted in the trachea. (2) In vivo trachea stent implantation
In order to verify the potential of the nanofiber film-covered trachea stent of the present invention against TISR, the nanofiber film-covered trachea stents prepared in Example 1 is placed in the New Zealand rabbits. The schematic diagram of time axis of animal experiment is shown in FIG. 4a. The specific operation of in vivo trachea stent implantation is as follows.
Prior to surgery, New Zealand rabbits are anesthetized by intramuscular injection with the animal in supine position and hyperextension of the neck. Portal dilation is performed using a 12-Fr dilator (Cook Medical). Subsequently, a 0.035-inch guidewire (Terumo
Corporation; Tokyo, Japan) and a 5-Fr catheter (Terumo Corporation) are used to enter the trachea, under fluoroscopic guidance from the Artis zee DSA system (Siemens,
Germany), the 5-Fr stent delivery system is advanced over the guidewire along with the trachea stent, which is placed at least 1.5-2 cm from the carina. Following trachea stent implantation, the animals are closely monitored and euthanized 4 weeks later. The operation time for trachea stent implantation is shown in FIG. 4e. A fluoroscopic monitoring image of the DSA system is shown in FIG. 4b.
It can be seen from FIG. 4b that the nanofiber film-covered trachea stent is successfully implanted into the trachea of New Zealand rabbit. It can be seen from FIG. 4e that there is no difference in the operation time for trachea stent implantation between the groups. (3) Computed tomography of trachea
After 4 weeks of nanofiber film-covered trachea stent implantation, cervical and thoraci€,600291
CT (Sensation 64, Siemens, Germany) scans are performed in New Zealand rabbits, and
VRT and maximum intensity projection (MIP) are used to observe trachea anatomy, trachea condition, morphology of nanofiber film-covered trachea stent and granulation tissue hyperplasia. Before scanning, New Zealand rabbits are injected intramuscularly with 0.02 ml/kg thiazine hydrochloride to anesthetize experimental animals. After the anesthesia is effective, the test animals are fixed on a CT table to complete the scanning.
Multiplanar reconstructions are performed using 1 mm thick sections spaced 0.5 mm apart in this study. Siemens image post-processing software is used to analyze the data and images. The results of the scan are shown in FIG. 4c, and the tracheal ventilation rate is shown in FIG. 4f.
It can be seen from the trachea VRT images in FIG. 4c, varying degrees of tracheal stenosis penetrated through the covered segment of trachea stent in Ctrl group, and significant stenosis at the end of trachea stent placement is observed in PAG group and
PAL group, while only mild trachea stenosis is observed in PAGL group. The position relationship between trachea stent and surrounding tissues is observed by using trachea
VRT image and MIP image. The results show that the trachea stent is in a safe position in the 4 groups of experimental animals, and no displacement or dislocation occurs in the trachea. It can be seen from the sagittal CT image in FIG. 4c that there is obvious granulation tissue formation in the trachea of experimental animals in Ctrl group, which is protruding into the tracheal cavity through the trachea stent. There are different degrees of granulation tissue proliferation at two ends of trachea stent in PAG group and PAL group, and the amount of granulation tissue formation is the least in PAGL group. It is noteworthy that the PAGL group shows the best fit between the trachea stent and the trachea compared to the other three groups.
It can be seen from FIG. 4f that the levels of tracheal ventilation are improved in New
Zealand rabbits in the PAG, PAL, and PAGL groups compared to the Ctrl group, with the improvement in the PAGL group being the most significant. In conclusion, the PAG nanofiber film-covered trachea stent, PAL nanofiber film-covered trachea stent and PAGL nanofiber film-covered trachea stent are effective in improving TISR, and the PAGL, 50091 nanofiber film-covered trachea stent has the best improvement effect. (4) Pathological damage detection of trachea tissue
After removal of the trachea stent, the trachea is dissected transversely near the proximal and distal ends of the trachea stent to obtain tracheal specimens. The above specimens are embedded in paraffin and sectioned to obtain tracheal tissue sections. Tracheal tissue sections are stained with hematoxylin and eosin (H&E) and Masson staining to evaluate tracheal tissue inflammation, degree of granulation tissue and collagen deposition. The results of the tests are shown in FIGS. 4d, 4g and 4h.
It can be seen from FIG. 4d that the trachea of New Zealand rabbits in Ctrl group shows structural injury with obvious granulation tissue hyperplasia and extensive collagen deposition, and the degree of trachea injury of New Zealand rabbits in PAG, PAL and
PAGL groups is reduced, and granulation tissue hyperplasia and collagen deposition are reduced; and New Zealand rabbits in the PAGL group have the most complete trachea structure with the least granulation tissue and collagen deposition. It can be seen from
FIG. 4g that the thickness of trachea epithelial cells of New Zealand rabbits in Ctrl group is 742.73 + 55.88 um, and that of New Zealand rabbits in PAGL group is 174.29 + 18.44 pm, which is significantly decreased. It can be seen from FIG. 4h, compared with Ctrl group, the collagen density in trachea of New Zealand rabbits in PAG, PAL and PAGL groups is gradually decreased, 42.30 + 5.98% in Ctrl group, 34.18 + 3.54% in PAG group, 25.53 + 4.44% in PAL group, and the lowest is 10.33 + 2.49% in PAGL group. In conclusion, the PAG nanofiber film-covered trachea stent, PAL nanofiber film-covered trachea stent and PAGL nanofiber film-covered trachea stent are effective in improving pathological injury of trachea tissue, inflammatory reaction and granulation tissue hyperplasia in TISR, among which the PAGL nanofiber film-covered trachea stent is the best. (5) Evaluation of antiangiogenic effect of nanofiber film-covered trachea stent in vivo
CD31, an important marker of vascular endothelial cells, plays a crucial role in early angiogenesis, and is commonly used to assess vascularization. After removal of the trachea stent, the trachea is dissected transversely near the proximal and distal ends 9f;500291 the trachea stent to obtain tracheal specimens. The above specimens are embedded in paraffin and sectioned to obtain tracheal tissue sections. Immunofluorescence staining of apoptosis-related protein Caspase-3, proliferation-related protein Ki67 and angiogenesis marker CD31 is performed on tracheal tissue sections, and the expression of these proteins are quantified. The detection results are shown in FIGS. 5a-5d.
It can be seen from FIGS. 5-5c that the tracheal tissues of New Zealand rabbits in the
Ctrl group shows obvious proliferation state without signs of apoptosis. Compared with
Ctrl group, the expression of Ki67 is significantly decreased in PAG and PAL groups, and further decreased in PAGL group. Compared with Ctrl group, the expression of Caspase- 3 is significantly increased in PAG and PAL groups, and the expression of Caspase-3 is further increased in PAGL group. Compared with Ctrl group, the PAGL group has the lowest Ki67 MFI of 0.32 + 0.08, and Caspase-3 has the highest MFI of 5.73 + 0.60. It can be seen from FIGS. 5a and 5d that the expression of CD31 is significantly decreased in
PAG and PAL groups compared with Ctrl group, and is further decreased in PAGL group.
The MFI of CD31 is reduced by about 77% in the PAGL group relative to the Ctrl group.
In conclusion, the PAG nanofiber film-covered trachea stent, PAL nanofiber film-covered trachea stent and PAGL nanofiber film-covered trachea stent are effective in promoting vascular endothelial cell apoptosis and inhibiting its proliferation, and the PAGL nanofiber film-covered trachea stent has the best effect. AQNPs synergized with AL to enhance the anti-angiogenesis effect of PAGL nanofiber film-covered trachea stent. (6) Evaluation of antibacterial effect of nanofiber film-covered trachea stent in vivo
After 4 weeks of endobronchial stent implantation, New Zealand rabbits are euthanized by pure carbon dioxide inhalation. The nanofiber film-covered trachea stent in the experimental animal is removed, and the nanofiber film on the trachea stent is carefully peeled off to evaluate antibacterial effect thereof in vivo. The PAG nanofiber film, PAL nanofiber film and PAGL nanofiber film are washed three times with sterile normal saline.
Subsequently, the nanofiber films are ultrasonic treated in 1 mL sterile saline solution for s, and the microorganisms on the nanofiber films are collected respectively. After proper dilution, the obtained microorganisms are inoculated on solid medium, and th&,600291 contents of microorganisms in trachea of different groups are compared. In addition, linear regression is used to analyze the relationship between microbial content and intratracheal granulation tissue growth. The results of the assay are shown in FIGS. 5e-
Sa.
It can be seen from FIGS. 5e and 5f that compared with Ctrl group, the PAL nanofiber- covered trachea stent has no inhibitory effect on the proliferation of microorganism; compared with the PAL nanofiber film-covered trachea stent, PAG nanofiber film-covered trachea stent and PAGL nanofiber film-covered trachea stent significantly reduce the content of microorganisms in trachea, and the PAGL nanofiber film-covered trachea stent has the strongest inhibitory effect on the proliferation of microorganisms, suggesting that nanofiber film-covered trachea stent loaded with AgNPs can effectively inhibit the proliferation of microorganisms in trachea, and has the ability to maintain trachea microecology. It can be seen from FIG. 5g that the linear regression analysis between the microbial content on the surface of trachea stent and the thickness of tracheal epithelial layer in Ctrl group and PAG group shows that the microbial content on the surface of trachea stent is positively correlated with the thickness of tracheal epithelial layer (R2 = 0.9066). The results show that the higher the microbial content on the surface of trachea stent, the more obvious the intratracheal granulation tissue hyperplasia. Therefore, inhibition of intratracheal microorganisms and improvement of the tracheal microenvironment can reduce the incidence of TISR and have a positive effect on improving the patency of trachea stents.
In view of the foregoing, the present invention effectively overcomes the deficiencies of the prior art and is highly valuable for industrial use. The above examples are intended to be illustrative of the substance of the present invention, but are not intended to limit the scope of the present invention. It is to be understood by those skilled in the art that the technical solutions of the present invention can be modified or replaced by equivalents without departing from the essence and protection scope of the technical solutions of the present invention.