Elsevier

Biomaterials

Volume 33, Issue 33, November 2012, Pages 8548-8556
Biomaterials

Suppression of post-angioplasty restenosis with an Akt1 siRNA-embedded coronary stent in a rabbit model

https://doi.org/10.1016/j.biomaterials.2012.07.045Get rights and content

Abstract

Restenosis is the formation of blockages occurring at the site of angioplasty or stent placement. In order to avoid such blockages, the suppression of smooth muscle cells near the implanted stent is required. The Akt1 protein is known to be responsible for cellular proliferation, and specific inhibition of Akt1 gene expression results in the retardation of cell growth. To take advantage of these benefits, we developed a new delivery technique for Akt1 siRNA nanoparticles from a hyaluronic acid (HA)-coated stent surface. For this purpose, the disulfide cross-linked low molecular polyethyleneimine (PEI) (ssPEI) was used as a gene delivery carrier because disulfide bonds are stable in an oxidative extracellular environment but degrade rapidly in reductive intracellular environments. In this study, Akt1 siRNA showed efficient ionic interaction with the ssPEI carrier, which was confirmed by polyacrylamide gel electrophoresis. Akt1 siRNA/ssPEI nanoparticles (ASNs) were immobilized on the HA-coated stent surface and exhibited stable binding and localization, followed by time-dependent sustained release for intracellular uptake. Cellular viability on the nanoparticle-immobilized surface was assessed using A10 vascular smooth muscle cells, and the results revealed that immobilized ASNs exhibited negligible cytotoxicity against the adhering A10 cells. Transfection efficiency was quantified using a luciferase assay; the transgene expression of Akt1 suppression through the delivered Akt1 siRNA was measured using RT-PCR and western blot, demonstrating higher gene silencing efficiency when compared to other carriers. ASN coated on HA stents were deployed in the balloon-injured external iliac artery in rabbits in vivo. It was shown that the Akt1 released from the stent suppressed the growth of the smooth muscle at the peri-stent implantation area, resulting in the prevention of restenosis in the post-implantation phase.

Introduction

Restenosis remains the most significant consequence of percutaneous transluminal angioplasty, despite efforts to address it both biologically and mechanically [1], [2]. Gene therapy has been attempted as a treatment option in terms of controlling gene expression in neighboring cells related to restenosis. Non-viral gene delivery has been considered safer than its viral counterpart. Strategies for enhancing non-viral gene delivery typically involve the complexation of plasmids with cationic polymers or lipids, which can self-assemble with DNA to form particles capable of being endocytosed by cells. Substrate-mediated delivery results in the immobilization of DNA complexed with the carrier onto the substrate. Immobilization on the target surface can enhance gene transfer by maintaining an elevated concentration of DNA within the cellular microenvironment via sustained release and by facilitating subsequent cellular internalization [3].

Polyethyleneimine (PEI) is a commonly used polymeric gene carrier possessing a high density of primary, secondary, and tertiary amino groups that can be protonated at different environmental pH levels. At physiological pH, polycation is very effective in binding to DNA and can mediate the transfection of eukaryotic cells. However, its relatively higher cytotoxicity hinders clinical application [4], [5], [6]. Disulfide cross-linked PEI (ssPEI) is preferred in the design and synthesis of biodegradable polymers for drug delivery. It was previously demonstrated that ssPEI has an efficient transfection property in the A10 vascular smooth muscle cell (VSMC) line derived from the thoracic aorta of embryonic rats. First, disulfide bonds are more hydrolytically stable than ester bonds in the extracellular environment, thus polycation with disulfide bonds can be used to prepare stable complexes with plasmid DNA. Second, disulfide bonds can be cleaved rapidly by glutathione and thioredoxin reductase in the cytoplasm [7], [8], [9]. As a result, DNA can be rapidly released from polyplexes in order to mediate efficient gene expression. In addition, cytotoxicity can be reduced by avoiding high charge density and long-term polymer accumulation.

Akt1 siRNA was delivered to the cells with enhanced proliferation for knocking down the Akt1 protein responsible for cellular proliferation at the mRNA level. It has been reported that the inhibition of Akt1 decreases the expression of caspase-8 in endothelial cells. Suppression of downstream Akt1 signaling proteins such as Fas ligand results in the stimulation of caspase activity and apoptosis in VSMCs. It has also been demonstrated that the specific inhibition of Akt1 protein expression results in the retardation of cell growth [10], [11], [12], [13], [14].

Hyaluronic acid (HA) is highly compatible with cells and the cellular matrix. It can be efficiently degraded using specific enzymes, such as hyaluronidase; furthermore, its degradation products could induce extracellular matrix production and the neo-formation of blood capillaries [15], [16], [17]. HA enhances proliferation and migration of endothelial cells at a later stage, whereas Akt1 siRNA delivered from nanoparticles can knock down the initial over-growth of VSMCs near the implanted stent. Here, Akt1 siRNA delivery was evaluated on an HA-coated surface, and the subsequent inhibition of Akt1 protein expression was also determined.

In this study, ssPEI was complexed with Akt1 siRNA (Akt1 siRNA/ssPEI nanoparticles [ASNs]) and immobilized on an HA-coated surface. The binding ability of the ASNs and the release of siRNA from the HA-coated surface were studied. The potential efficacy of ASNs was evaluated with the rat VSMC line. The suppression of the Akt1 protein and its downstream signaling proteins regulating the cellular proliferation after the treatment with ASNs was examined. ASNs were coated on an HA stent deployed in the balloon-injured external iliac artery in a rabbit in vivo. Finally, post-angioplasty restenosis was measured by micro-computed tomography (micro-CT) imaging.

Section snippets

Materials

Branched PEI (bPEI) 25K and linear PEI (lPEI) 25K were purchased from Sigma–Aldrich (St. Louis, USA). Plasmid gWiz-luc (Aldevron, USA) was transformed into the competent cells, Escherichia coli DH5α, using the heat shock method. gWiz-luc was then propagated in bacterial cultures grown in Luria-Bertani (LB) media (Becton Dickinson, USA) containing 100 μg/ml of kanamycin (Biosesang Inc., Korea), and extracted and purified using a mini DNA-spin kit (Intron Biotechnol Co., Korea). Akt1 siRNA and

Optimization of transfection efficiency with ssPEI

The ssPEI with different N/P ratios was complexed with luciferase pDNA and its transfection efficiency was compared with two positive controls of bPEI 25 kDa and lPEI 25 kDa. After 24 h of transfection, transgene expression was measured by luciferase assay (Fig. 1). Compared to other carriers, ssPEI exhibited higher gene transfection efficiency as well as increased gene expression with an increase in DNA concentration.

Cell attachment studies on HA-coated stents

A10 VSMC lines were used to study cell attachment on HA-coated stents. It was

Discussion

Substrate-mediated delivery enhances gene transfer by increasing the concentration of DNA in the cellular microenvironment. Immobilized complexes in a substrate-mediated fashion deliver therapeutic genes to primary cells with improved cellular viability and transfection efficiency, in comparison with bolus delivery. Maximal transfection from this approach requires the affinity of the DNA complex to the substrate that is sufficient for immobilization, but not so excessive that it limits cellular

Conclusion

VSMC proliferation over the stent was suppressed by the delivery of Akt1 siRNA complexed with an ssPEI carrier on an HA-coated stent surface. The surface-mediated delivery resulted in higher protein expression compared to the bolus-mediated delivery, which is a benefit of stent-mediated delivery. The ssPEI showed reduced toxicity and higher gene expression compared to its non-degradable counterparts. The Akt1 siRNA was successfully delivered, and Akt1 suppression at the mRNA and protein level

Acknowledgments

This work was financially supported by the Bio Imaging Research Center at GIST. IKP also acknowledges the financial support of the Regional Technology Innovation Program of the Ministry of Commerce, Industry, and Energy (Grant RT104-01-01); the Basic Science Research Program through the National Research Foundation of Korea, funded by the Ministry of Education, Science, and Technology (2010-0002940); the Korea Healthcare Technology R&D Project, Ministry for Health, Welfare, and Family Affairs,

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