Date of Award

August 2013

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Biological Sciences

First Advisor

Julie A. Oliver

Committee Members

Ralph M. Albrecht, Marija Gajdardziska-Josifovka, Peter Geissinger, Heather A. Owen, Douglas A. Steeber

Keywords

Electron Microscopy, Hyperthermia, Magnetic Nanoparticles, Platelets, Stroke, Thrombus

Abstract

Ischemic stroke is the world's second leading cause of death and accounts for 2-4% of total worldwide healthcare costs. Ischemic stroke is caused by the occlusion of arteries responsible for supplying blood to the brain, which can result in disability or death. Arterial blood clots consist of aggregates of activated platelets wrapped in a mesh of fibrin. Tissue plasminogen activator, the only current FDA-approved treatment for ischemic stroke, functions by lysing fibrin in a blood clot. Unfortunately, tissue plasminogen activator significantly increases bleeding risks, which restricts its use. Alternatively, targeting and disrupting platelets within a clot could improve stroke outcome. To test this hypothesis, we have developed a targeting system utilizing fibrinogen to specifically target nanoparticles to activated platelets. Human fibrinogen was evaluated for targeting both human and murine platelets under conditions that are similar to an in vivo blood clot. Our results indicate that human fibrinogen conjugated to gold nanoparticles was capable of targeting activated human and murine platelets. Further, human fibrinogen conjugates bound to preformed platelet aggregates while in the presence of plasma levels of unconjugated fibrinogen. To disrupt platelets, we developed a system to cause localized hyperthermia to the platelet surface by utilizing inductively heated magnetic nanoparticles. Magnetic gold-coated magnetite nanoparticles were synthesized and characterized. The morphology of the gold-coated magnetite product differed substantially from the expected core-shell structure often reported for such nanoparticles. Despite the unexpected morphology, the nanoparticles could still be functionalized with protein and targeted to activated platelets. Localized hyperthermia was created when platelet-bound, fibrinogen-conjugated, gold-coated magnetite nanoparticles were exposed to an oscillating magnetic field. The effects of the hyperthermic treatment to surface-activated and aggregated platelets were evaluated by electron microscopy. The treated platelets demonstrated considerable structural damage, with the cell membrane showing significant disruption when compared to controls. A method to quantify platelet damage was developed and utilized to refine the length of exposure to the oscillating magnetic field and dose of nanoparticles. In the future, it may be feasible to use fibrinogen-conjugated, gold-coated magnetite to target and disrupt platelets in a thrombus in vivo, thereby restoring blood flow to ischemic brain.

Download

Share

COinS