H. A. Kassam1, C. Gillis1, N. Tsihlis1, S. Stupp2, M. Kibbe1 1University Of North Carolina At Chapel Hill,Surgery,Chapel Hill, NC, USA 2Northwestern University,Chicago, IL, USA
Introduction: The aim of this study is to develop and evaluate a novel, systemically delivered, targeted therapy that will prevent restenosis following all cardiovascular interventions. Increased fractalkine (CX3CL1) levels have been seen in atherosclerotic arteries; however, limited studies have demonstrated presence in injured vessels. Recently, our lab discovered that fractalkine levels are increased after arterial injury. Thus, for this study we identified a unique peptide sequence that binds to fractalkine with the goal of synthesizing a self-assembled peptide amphiphile (PA) nanofiber targeted to fractalkine after arterial injury. In addition, our goal was to determine the optimal dose of the fractalkine-targeted nanofiber to bind to the site of injury, along with binding duration of the targeted nanofiber to the site of arterial injury.
Methods: The fractalkine-targeted PA (C16-VVAASFPELDLENFEYDDSAEA) nanofiber containing a fluorescent TAMRA tag was synthesized using solid-phase peptide synthesis and purified by reversed-phase high-pressure liquid chromatography (HPLC). Nanofiber formation was assessed via transmission electron microscopy (TEM). Controls included a PA without the binding sequence (backbone PA, C16-VVAAK), a lower charged fractalkine PA (C16-VVAASFPELDLQNFQYNNSAEA), and injury alone. Male Sprague Dawley rats (250-300g) underwent the carotid artery balloon injury model followed by intravenous injection of the PA (0.5-5.0 mg) 24 hours after injury. Arteries were harvested after 5 hours of circulation time (n=3/group). To assess binding duration, balloon-injured rats received intravenous injections of fractalkine PA (5 mg) 24 hours after injury and arteries were harvested after 5, 24, 48, and 72 hours of circulation time (n=3/group). Harvested carotid arteries were frozen, cross-sectioned at 5 microns thick, and imaged for fluorescence.
Results: HPLC confirmed PAs were >95% pure, and TEM showed PAs formed nanofibers. Animals whose carotid arteries were injured and underwent injection of the fractalkine-targeted PA showed fluorescence at the site of injury in the area of the arterial media. In contrast, no binding was observed with the backbone PA or lower charged fractalkine PA. No significant fluorescent signal was detected in the uninjured arteries. We observed binding of fractalkine-targeted PA at doses as low as 0.5 mg. Furthermore, PA binding was seen at the injured site for up to 48 hours after injection.
Conclusion: We have demonstrated specific binding of our fractalkine-targeted PA nanofiber to the site of arterial injury compared to control PA nanofibers, with binding duration observed up to 48 hours after injection and at doses as low as 0.5 mg. This research serves as the foundation upon which a targeted, drug-eluting therapy to prevent restenosis, and possibly prevent atherosclerosis, will be evaluated.