Author's School

School of Engineering & Applied Science

Author's Department/Program

Biomedical Engineering

Language

English (en)

Date of Award

Spring 3-24-2014

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Chair and Committee

Samuel A Wickline

Abstract

Optimization of the mediation of acute thrombi remains a significant research challenge in the treatment of emergency conditions including heart attack and ischemic stroke. We have demonstrated that a nanoparticle carrying potent direct thrombin inhibitors can advance the treatment of acute thrombosis arising from various cardiovascular pathologies. The thrombin-inhibiting nanoparticles presented herein are designed to focus the antithrombotic impact of direct thrombin inhibitors at the site of thrombus formation and to provide imaging contrast to highlight the formation or the abatement and eventual disintegration of the thrombus.

Perfluorocarbon nanoparticles were functionalized by covalent addition of PPACK or bivalirudin to carboxy-PEG capped lipid components to the stabilizing lipids. Over 10000 inhibitors were added per particle. In vitro experiments evaluated inhibition of thrombin cleavage of the chromogenic substrate Chromozym TH and defined the kinetics of the particle-thrombin interaction. Thrombin-inhibiting activity of the component inhibitors was undiminished on the nanoparticles and, as explored in appended work, the nanoparticles had a significant kinetic advantage over the lone inhibitors.

To demonstrate efficacy of the particles as inhibitors of clot-bound thrombin, fibrinopeptide A ELISAs assayed the production of fibrin in plasma exposed to the surface of forming clots treated with PPACK, bivalirudin, PPACK nanoparticles, or bivalirudin nanoparticles. Similarly treated clots were monitored for growth in plasma via magnetic resonance imaging. Nanoparticles exceeded the activity of the component inhibitors in blocking FPA production by bound thrombin and formed an inhibitory layer that stopped further growth of clots in plasma.

In vivo testing of thrombosis inhibition was performed in C57BL/6 mice and NZW rabbits using the Rose Bengal laser-induced thrombosis model (with ultrasonic flow probes tracking progress to occlusive thrombus). Thrombin-inhibiting particles were compared to Heparin, PPACK, bivalirudin, saline, and analogous non-functionalized particles as inhibitors of acute thrombosis in mice. Thrombin-inhibiting nanoparticles significantly delayed occlusion time in mice, outperforming heparin and the component inhibitors. Ultrasound and magnetic resonance imaging were employed to evaluate deposition of nanoparticles in mouse or rabbit thrombi. Thrombi were analyzed following in vivo experiments, using imaging and histochemical methods. Magnetic resonance imaging and spectroscopy revealed the specific deposition of thrombin-inhibiting nanoparticles in thrombi. For evaluation of fine clot morphology, mouse thrombi were examined with transmission electron microscopy. For evaluation of fibrin and platelet content in the thrombus Carstair's staining was employed. Clots formed following nanoparticle administration exhibited lesser platelet content.

In additional experiments, bleeding times and APTT measurements determined the systemic effects of the nanoparticles. Though the thrombin-inhibiting nanoparticles delayed thrombotic occlusion at a site of arterial injury to 1.5-2 hours, significant effects on blood pool coagulation parameters were observed for less than 20 minutes.

We have demonstrated that the thrombin-inhibiting nanoparticle is kinetically superior to conventional direct inhibitors. In vivo, the potent inhibition kinetics, combined with the pharmacokinetic and pharmacodynamic properties of perfluorocarbon nanoparticles enabled superior inhibition of thrombosis while maintaining an excellent safety profile with short-lived systemic effects. Imaging data indicated the formation of layers of nanoparticles at sites of arterial injury. Thrombin-inhibiting nanoparticles are thus derived here as a new tool for treatment of acute thrombosis. The particles open a new avenue in this field of medical research as the first therapeutic to form a detectable, site-specific, and quantifiable anticoagulant layer that seals against the progress of acute thrombosis.

Comments

Permanent URL: http://dx.doi.org/10.7936/K7T43R26

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