Promoting endothelial recovery and reducing neointimal hyperplasia using sequential-like release of acetylsalicylic acid and paclitaxel-loaded biodegradable stents

Abstract

Introduction: This work reports on the development of a biodegradable dual-drug-eluting stent with sequential-like and sustainable drug-release of anti-platelet acetylsalicylic acid and anti-smooth muscle cell (SMC) proliferative paclitaxel.

Methods: To fabricate the biodegradable stents, poly-L-lactide strips are first cut from a solvent-casted film. They are rolled onto the surface of a metal pin to form spiral stents. The stents are then consecutively covered by acetylsalicylic acid and paclitaxel-loaded polylactide-polyglycolide nanofibers via electrospinning.

Results: Biodegradable stents exhibit mechanical properties that are superior to those of metallic stents. Biodegradable stents sequentially release high concentrations of acetylsalicylic acid and paclitaxel for more than 30 and 60 days, respectively. In vitro, the eluted drugs promote endothelial cell numbers on days 3 and 7, and reduce the proliferation of SMCs in weeks 2, 4, and 8. The stents markedly inhibit the adhesion of platelets on days 3, 7, and 14 relative to a non-drug-eluting stent. In vivo, the implanted stent is intact, and no stent thrombosis is observed in the stent-implanted vessels without the administration of daily oral acetylsalicylic acid. Promotion of endothelial recovery and inhibition of neointimal hyperplasia are also observed on the stented vessels.

Conclusion: The work demonstrates the efficiency and safety of the biodegradable dual-drug-eluting stents with sequential and sustainable drug release to diseased arteries.

Keywords: biodegradable drug-eluting stents; mechanical properties; poly-L-lactide; polylactide-polyglycolide; sequential-like and sustainable release.

Figure 1

Steps for manufacturing newly developed stents. Notes: (A) A poly-L-lactide strip was first cut from a solvent-cast film and rolled onto the surface of a metal pin that was fixed at the two ends with tape. (B) The metal pin, wrapped with the strip, was placed in an isothermal water bath at 70°C, and then in a water bath at 0°C for quenching. (C) The electrospinning of acetylsalicylic acid-loaded poly-D-L-lactide-glycolide nanofibers onto the biodegradable stent. (D) The spinning of paclitaxel-loaded nanofibers onto the stent.

Figure 2

Scanning electron microscopy images of electrospun nanofibers. Notes: (A) Acetylsalicylic acid nanofibers with a diameter of 530–710 nm and 75.3% porosity. (B) Paclitaxel-loaded nanofibers with a diameter of 690–1,210 nm and 70.5% porosity (scale bar: 1 μm).

Figure 3

Balloon expansion of a biodegradable stent inside a plastic tube. Notes: (A) The electrospun nanofibers mounted on a spiral poly-L-lactide stent (3.5×20 mm). (B) During inflation of the balloon, the stents with the nanofibers expanded inside a plastic tube. The balloon was removed, leaving the stent and the nanofibers in situ (scale bar: 10 mm).

Figure 4

Comparison of biodegradable drug-eluting stents and metallic stents under compression. Biodegradable spiral stents had higher mechanical strengths than metallic stents. Abbreviation: PLLA, poly-L-lactide.

Figure 5

Effect of drug loading on rate of release by biodegradable stents. Notes: The combined release curves of the drug-eluting stent/nanofibers exhibited ‘sequential-like’ release behavior: a high concentration of acetylsalicylic acid was released in the first few days, and then a high concentration of paclitaxel was released from day 10.

Figure 6

Platelet adhesion test in vitro. Notes: Following immersion of the drug-loaded and non-loaded nanofibrous membranes in platelet-rich plasma, significantly fewer platelets adhered to the drug-loaded nanofibers at 3 (A1), 7 (A2), and 14 (A3) days than to the non-loaded nanofibers (B1–3) on days 3, 7, and 14, respectively. Red arrows indicate platelet adhesion. B2 exhibits larger platelet aggregates and more extensive platelet pseudopod formation than B3 (scale bar: 50 μm).

Figure 7

Number of adherent platelets. Notes: Following immersion of the two groups of stents in platelet-rich plasma for 3 hours, significantly fewer platelets adhered to the drug-loaded stents than to the non-drug-loaded stents on days 3, 7, and 14 (^#P<0.01).

Figure 8

Incorporation of BrdU into HUVACs was assayed by BrdU cell proliferation ELISA, as described in the Methods section. Notes: Data from BrdU assays revealed that elution on day 3 promoted the proliferation of HUVACs over that of the control group. The reduction of cell proliferation on days 14 and 21 relative to the control group and day 7 was also significant. Each value (mean ± standard error [n=4]) is expressed as a multiplier of the amount of BrdU incorporated into control cells (*P<0.05; ^#P<0.01). Abbreviations: BrdU, 5-bromo-2-deoxyuridine; ELISA, enzyme-linked immunosorbent assay; HUVACs, human umbilical cord veins.

Figure 9

Incorporation of BrdU into vascular SMCs was assayed by BrdU cell proliferation ELISA, as described in the Methods section. Notes: These BrdU assays revealed that elution reduced the proliferation of cells below that of the control group as detected on different days. Each value (mean ± standard error [n=4]) is expressed as a multiplier of the amount of BrdU incorporated into control cells (*P<0.05). Abbreviations: BrdU, 5-bromo-2-deoxyuridine; ELISA, enzyme-linked immunosorbent assay; SMCs, smooth muscle cells.

Figure 10

The delivery of the developed stent in the descending abdominal aorta of the rabbit. Notes: (A) Photographs of stent on X-ray film. (B and C) A balloon was used to deliver biodegradable stents into the rabbit abdominal aorta. (D) No migration of the implanted stent was observed after deployment and dye injection (scale bar: 10 mm). Arrows indicate stent site.

Figure 11

No migration of the implanted stent (arrows) was observed at 3 days and 4 weeks following the procedure, and the stented abdominal aorta was patent for all test animals based on the peripheral vascular ultrasound study (see supplemental video).

Figure 12

In vivo assessment of endothelial function in rabbits. Notes: Change in diameter of abdominal aorta in response to acetylcholine infusions. Endothelial function was evaluated after 4 weeks of stenting treatment. Endothelial-dependent vasodilatory response to Ach was maintained in drug-loaded stenting for a significantly greater period than in balloon-injury aorta (*P<0.01). Abbreviation: Ach, acetylcholine.

Figure 13

Photomicrographs of cross-sections showing the condition of the stent on the vessel lumen three days following balloon angioplasty and stent deployment. Notes: Segment of vessel of a treated animal shows a characteristic fragment of a spiral stent, wide vessel lumen without thrombus formation and non-significant stenosis without a lumen reduction of greater than 50% (A1, and B1, 40×) (scale bar: 250 mm). Additionally, segment of vessel of a treated animal has greater endothelial injury (asterisk) comparing with other vessel area (single arrow) (A2, and B2, 100×) (scale bar: 100 mm).

Figure 14

Photomicrographs of representative rabbit vessel sections 28 days after intervention. Notes: Four-week high-power photomicrographs (hematoxylin-eosin staining, ×200) of pathology of groups A and B were shown. Inflammation response was low in stent groups (A). By day 28, an anatomically intact endothelium had been re-constituted in group A (almost no intimal hyperplasia), and groups (B) exhibited consistent intimal hyperplasia with a thickness of approximately 100 μm. A single arrow represents the endothelial cells, and asterisk indicates the inflammatory cells surrounding the stent struts. Scale bar: 50 mm. # indicates the stent strut area.

Figure 15

Immunofluorescence of calponin on stented arteries after 4 weeks. Notes: Calponin immunostaining (red) of (A) a drug-eluting spiral stent and (B) balloon-injury artery. Autofluorescence on tunica media (green) and DAPI-stained nuclei are also shown. A high degree of positive labeling with calponin was observed on the drug-loaded spiral stent vessels. The double arrow depicts tunica media. Group B showed that marked neointimal hyperplasia resulted from proliferation of smooth muscle cells. Abbreviation: DAPI, diamidine-2-phenylindole.

Figure 16

Quantitative analysis of calponin content of arteries on day 28. Notes: Drug-eluting spiral stent and control groups had significantly higher calponin levels than did the balloon-injury group (*post hoc P<0.05).