Local sustained delivery of acetylsalicylic acid via hybrid stent with biodegradable nanofibers reduces adhesion of blood cells and promotes reendothelialization of the denuded artery

Abstract

Incomplete endothelialization, blood cell adhesion to vascular stents, and inflammation of arteries can result in acute stent thromboses. The systemic administration of acetylsalicylic acid decreases endothelial dysfunction, potentially reducing thrombus, enhancing vasodilatation, and inhibiting the progression of atherosclerosis; but, this is weakened by upper gastrointestinal bleeding. This study proposes a hybrid stent with biodegradable nanofibers, for the local, sustained delivery of acetylsalicylic acid to injured artery walls. Biodegradable nanofibers are prepared by first dissolving poly(D,L)-lactide-co-glycolide and acetylsalicylic acid in 1,1,1,3,3,3-hexafluoro-2-propanol. The solution is then electrospun into nanofibrous tubes, which are then mounted onto commercially available bare-metal stents. In vitro release rates of pharmaceuticals from nanofibers are characterized using an elution method, and a highperformance liquid chromatography assay. The experimental results suggest that biodegradable nanofibers release high concentrations of acetylsalicylic acid for three weeks. The in vivo efficacy of local delivery of acetylsalicylic acid in reducing platelet and monocyte adhesion, and the minimum tissue inflammatory reaction caused by the hybrid stents in treating denuded rabbit arteries, are documented. The proposed hybrid stent, with biodegradable acetylsalicylic acid-loaded nanofibers, substantially contributed to local, sustained delivery of drugs to promote re-endothelialization and reduce thrombogenicity in the injured artery. The stents may have potential applications in the local delivery of cardiovascular drugs. Furthermore, the use of hybrid stents with acetylsalicylic acid-loaded nanofibers that have high drug loadings may provide insight into the treatment of patients with high risk of acute stent thromboses.

Keywords: acetylsalicylic acid; biodegradable drug-eluting nanofibers; cell adhesion to vascular stents; release characteristics.

Figure 1

Setup for electrospinning. Notes: (A) Electrospinning setup consists of a power supply (35 kV, 4.16 mA/125 W), a syringe pump, and a metallic pin (with a diameter of 0.95 mm) that is mounted on a motor with a rotational speed of 300 rpm. (B) Electrospun nanofibrous tube and bare-metal stent (BMS) (3.5 × 20 mm). (C) The tube was mounted onto a commercially available BMS.

Figure 2

Morphology of nanofibrous membrane elucidated by scanning electron microscopy. Notes: Images of acetylsalicylic acid-loaded nanofibers within the diameter range 50–8,720 nm, (A) before expansion (the pore size is around 5 μm), and (B) after expansion by a balloon (the pore size is around 10 μm). Scale bar: 10 μm.

Figure 3

Platelet adhesion test in vitro. Notes: After immersion of acetylsalicylic acid-loaded nanofibrous membranes, (A) (25 μg/mm²) and (B) (5 μg/mm²) in platelet-rich plasma. No significant difference existed between platelet adhesion at 1 hour (A1 and B1). At 3 hours, significantly fewer platelets adhered to the nanofibers with 25 μg/mm² than with 5 μg/mm² acetylsalicylic acid loading (A2 and B2). Significantly more platelets adhered on the nanofibrous membranes with the lowest acetylsalicylic acid loading (1 μg/mm²) at both 1 and 3 hours (C1 and C2). Scale bar: 10 μm.

Figure 4

In vitro release of acetylsalicylic acid. Notes: In vitro (A) daily and (B) accumulated release behaviors of acetylsalicylic acid from nanofibrous membranes. A biphasic release pattern was found, with an initial burst of release for the first few days, followed by a rather stable release for up to 3 weeks.

Figure 5

Stent implantation. Notes: (A) Schematic locations of implantation of two stents in rabbit descending abdominal aorta (arrow). (B) Angiograms of rabbit vasculature following injection of contrast dye, showing location of stented area (arrow). Scale bar: 20 mm.

Figure 6

Angiograms of rabbit vasculature. Notes: (A) Following injection of contrast dye. (B) stented descending abdominal aorta. (C) showing patent branch vessels following implantation. Asterisks indicate abdominal aorta branch. Arrows indicate implantation part of stent. Scale bar: 10 mm.

Figure 7

Quantitative analysis of endothelial coverage. Notes: Endothelial coverage above struts at 2 weeks (A1, B1 and C1) and 4 weeks (A2, B2 and C2) in Groups A, B, and C. Group A exhibited the most complete coverage at both 2 and 4 weeks.

Figure 8

Percentage of endothelial coverage on stent struts. Notes: Acetylsalicylic acid-loaded (Groups A and B), and non-acetylsalicylic acid-loaded (Group C) nanofibrous membrane stent. *P<0.05 Group A versus Group C in post hoc analysis; ^#P<0.05 Group B versus Group C in post hoc analysis.

Figure 9

Adherent platelets and monocytes at various times. Notes: Adhesion of platelets and monocytes on endothelial surface of stented arterial vessels in Groups A, B, and C at 1, 2, 3, and 4 weeks by scanning electron microscopy. Acetylsalicylic acid-loaded hybrid stents can inhibit the adhesion of platelets and monocytes on the endothelial surface of blood vessels. Scale bar: 50 μm.

Figure 10

Number of adherent blood cells on stent struts. Notes: (A) Number of adherent platelets on stent struts. Acetylsalicylic acid-loaded (Groups A and B) and non-acetylsalicylic acid-loaded (Group C) nanofibrous membrane stent. (B) Number of monocytes that adhered on stent struts. *P<0.05 Group A versus Group C in post hoc analysis. ^#P<0.05 Group B versus Group C in post hoc analysis; ^!P<0.05 Group A versus Group B in post hoc analysis.

Figure 11

Pathology of Groups A, B, and C at 2 and 4 weeks. Notes: Two-week (A1, B1 and C1) and 4-week (A2, B2 and C2) high-power photomicrographs (H&E staining; 200 × magnification) of pathology of Groups A, B, and C. The inflammation response was significant lower in Groups A and B than in Group C.

Figure 12

Vascular inflammation and injury scores. Notes: (A) Vascular inflammation score. (B) Vascular injury score. *P<0.05 Group A versus Group C in post hoc analysis; ^#P<0.05 Group B versus Group C in post hoc analysis.

Figure 13

Immunofluorescence of collagen I close to stented arteries. Notes: Collagen type I immunostaining (orange) of acetylsalicylic acid-loaded (Groups A and B) and non-acetylsalicylic acid-loaded (Group C) nanofibrous membrane stent. (Group D) normal vessels. Autofluorescence on tunica media (green), and DAPI-stained nuclei are also shown. Less collagen type I-positive labeling was observed close to the acetylsalicylic acid-loaded stent vessels. scale bar: 75 μm. Abbreviations: A, tunica adventitia; M, tunica media; D, control group.