Korean Circ J.  2016 Jan;46(1):23-32. 10.4070/kcj.2016.46.1.23.

Novel Fabrication of MicroRNA Nanoparticle-Coated Coronary Stent for Prevention of Post-Angioplasty Restenosis

• Che HL
• Bae IH
• Lim KS
• Uthaman S
• Song IT
• Lee H
• Lee D
• Kim WJ
• Ahn Y
• Park IK
• Jeong MH

• KMID: 2223766
• DOI: http://doi.org/10.4070/kcj.2016.46.1.23

Abstract

BACKGROUND AND OBJECTIVES
MicroRNA 145 is known to be responsible for cellular proliferation, and its enhanced expression reportedly inhibits the retardation of vascular smooth muscle cell growth specifically. In this study, we developed a microRNA 145 nanoparticle immobilized, hyaluronic acid (HA)-coated stent.
MATERIALS AND METHODS
For the gene therapy, we used disulfide cross-linked low molecular polyethylenimine as the carrier. The microRNA 145 was labeled with YOYO-1 and the fluorescent microscopy images were obtained. The release of microRNA 145 from the stent was measured with an ultra violet spectrophotometer. The downstream targeting of the c-Myc protein and green fluorescent protein was determined by Western blotting. Finally, we deployed microRNA 145/ssPEI nanoparticles immobilized on HA-coated stents in the balloon-injured external iliac artery in a rabbit restenosis model.
RESULTS
Cellular viability of the nanoparticle-immobilized surface tested using A10 vascular smooth muscle cells showed that MSN exhibited negligible cytotoxicity. In addition, microRNA 145 and downstream signaling proteins were identified by western blots with smooth muscle cell (SMC) lysates from the transfected A10 cell, as the molecular mechanism for decreased SMC proliferation that results in the inhibition of in-stent restenosis. MicroRNA 145 released from the stent suppressed the growth of the smooth muscle at the peri-stent implantation area, resulting in the prevention of restenosis at the post-implantation. We investigated the qualitative analyses of in-stent restenosis in the rabbit model using micro-computed tomography imaging and histological staining.
CONCLUSION
MicroRNA 145-eluting stent mitigated in-stent restenosis efficiently with no side effects and can be considered a successful substitute to the current drug-eluting stent.

Keyword

Drug-eluting stents; Gene delivery; MicroRNA; Nanoparticle; Restenosis

MeSH Terms

Blotting, Western
Cell Proliferation
Drug-Eluting Stents
Genetic Therapy
Hyaluronic Acid
Iliac Artery
MicroRNAs*
Microscopy
Muscle, Smooth
Muscle, Smooth, Vascular
Myocytes, Smooth Muscle
Nanoparticles
Polyethyleneimine
Stents*
Viola
Hyaluronic Acid
MicroRNAs
Polyethyleneimine

Figure

• 

  Fig. 1 (A) Fluorescent microscopic image of YOYO1-labeled MSNs immobilized on the HA-coated stent surfaces. (B) The binding efficiency of YOYO1-labeled MSN on an HA-coated stent surface. YOYO1-labeled miR-145 was complexed with ssPEI at N/P ratios of 10 and 20 and was then immobilized on the HA-coated stent surface. The unbound miR-145 was measured by a UV spectrophotometer. HA: hyaluronic acid, MSN: microRNA 145/disulfide cross-linked low molecular polyethylenimine nanoparticles, ssPEI: disulfide cross-linked low molecular polyethylenimine, N/P: nitrogen to phosphate, miR: microribonucleic acid, UV: ultra violet.

• 

  Fig. 2 SEM images of VSMCs adhering on the MSN-immobilized HA stent surface after overnight culture. SEM: scanning electron microscopy, VSMCs: vascular smooth muscle cells, MSN: microRNA 145/disulfide cross-linked low molecular polyethylenimine nanoparticles, HA: hyaluronic acid.

• 

  Fig. 3 The uptake of YOYO1-labeled microRNA-145/ssPEI from the HA stent was measured by confocal microscopy from 1 to 12 hours. miR: microRNA, ssPEI: disulfide cross-linked low molecular polyethylenimine, HA: hyaluronic acid.

• 

  Fig. 4 The cell viability of VSMCs cultured on a luciferase/ssPEI nanoparticle-immobilized, HA stent surface. Different amounts of plasmid-luciferase (1-3 µg) were complexed with ssPEI at a fixed N/P ratio of 10 to 30 and immobilized on the HA-coated surface. The cellular viability of VSMCs on the surface was measured using MTS assay (Promega, Madison, WI, USA). VSMCs: vascular smooth muscle cells, ssPEI: disulfide cross-linked low molecular polyethylenimine, HA: hyaluronic acid, N/P: nitrogen to phosphate.

• 

  Fig. 5 Confirmation of miR-145 downstream pathway protein (c-Myc) and miR-145-GFP expression silencing after miR-145 treatments using ssPEI and bPEI carrier. Suppression of miR-145-GFP and downstream signaling proteins after the delivery of miR-145-GFP. The protein level was measured using western blotting. miR: microRNA, GFP: green fluorescent protein, ssPEI: disulfide cross-linked low molecular polyethylenimine, bPEI: branched polyethylenimine.

• 

  Fig. 6 Observation of post-angioplasty restenosis by micro-CT imaging. In MSN/HA-immobilized and HA-coated stents, a thin vascular overgrowth of cells was observed, and the stent linings were visible (n=3). BMS: bare metal stent, miR: microRNA, ISR: in-stent restenosis, CT: computed tomography, MSN: microRNA 145/disulfide cross-linked low molecular polyethylenimine nanoparticles HA: hyaluronic acid.

• 

  Fig. 7 Measurement of neointima by hematoxylin & eosin (H&E) staining. The iliac arteries were harvested 4 weeks after stent implantation, fixed with paraformaldehyde, embedded in Glycol methacrylate, and stained with hematoxylin & eosin (H&E) staining. The upper panel shows representative arteries from the BMS and HA-coated stent, and MSN/HA-immobilized stent groups. BMS: bare metal stent, HA: hyaluronic acid, miR: microRNA, MSN: microRNA 145/disulfide cross-linked low molecular polyethylenimine nanoparticles.

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