Date of Award

August 2020

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Biological Sciences

First Advisor

Julie A Oliver

Committee Members

Heather A Owen, Douglas A Steeber, Charles F Wimpee, Marija Gajdardziska-Josifovska

Keywords

Blood plasma, Coated nanoparticle, Hyperthermia, Integrin receptors, Ischemic stroke, Scanning electron microscopy

Abstract

Activated platelets are a key component of the arterial thrombi responsible for heart attack and stroke. Tissue plasminogen activator (tPA) is currently the only FDA-approved drug for ischemic stroke. It works by dissolving fibrin, thus breaking down the clot and restoring blood flow to the brain. However, it must be administered within 4.5 hours of the onset of stroke. This leaves a significant number of patients who are ineligible for intervention and who have poor clinical outcomes. Additionally, tPA is not injury site-specific; consequently, the side effects range from mild to severe. We are investigating targeting activated platelets in occlusive thrombi for magnetically-induced hyperthermia as an alternative therapy for ischemic stroke.

The binding of plasma fibrinogen to its platelet surface receptor is dependent on cell activation and could therefore function in selective targeting of activated platelets. When fibrinogen receptors are cross-linked by ligand, they engage the actin cytoskeleton and clear from the edges of platelets or groups of platelets, thereby exposing additional unoccupied receptors. We propose that fibrinogen-conjugated nanoparticles can be used to target activated platelets rather than fibrin within an existing occlusive clot as a means to restore blood flow in the vessel. In our model, selection for activated platelets over quiescent circulating platelets is critical in order to minimize bleeding complications that are especially dangerous in ischemic brain. We hypothesize that fibrinogen-conjugated, gold-coated magnetite nanoparticles can specifically target activated platelets and disrupt occlusive thrombi when exposed to an oscillating magnetic field. We tested this using an in vitro model system of thrombosis that allows platelet activation and fibrin formation in platelet-rich plasma or whole blood. Exposing labeled samples to an oscillating magnetic field will cause hyperthermia and disrupt clots. Dense fibrin networks make nanoparticle penetration into the clots very difficult, so we have described clot structure and quantified nanoparticle access to the clot interior using the robust gold nanoparticle system. Our experiments found nanoparticles interact with both activated platelets, and fibrin, giving us multiple targets; clot structure influences how well the nanoparticles are incorporated; and multiple applications of nanoparticles may increase the power of clot busting. These results show our specific cell targeting with hyperthermia can be developed into a viable therapy for ischemic stroke.

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