Development and characterization of aptamer-conjugated imaging tools for diagnostic applications

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Creator: 

Koudrina, Anna

Date: 

2020

Abstract: 

Magnetic resonance imaging (MRI) and computed tomography (CT) are imaging modalities commonly used to assess blood flow; however, neither can label a blood clot that may either disrupt flow, causing an ischemic stroke, or have the ability to demonstrate where a cerebral hemorrhage has occurred (hemorrhagic stroke). To improve the diagnosis and treatment of blood vessel diseases, such as stroke and aneurysms, we developed fibrinogen aptamer (FA)-functionalized contrast agents, enabling the identification and labelling of blood clots. Fibrin was chosen as the target of interest as it a major constituent of aforementioned conditions. Since FA was originally selected to bind fibrinogen, fibrin-binding validation was required. It was hypothesized that FA would retain some binding affinity towards the polymerized form, fibrin, given that most of the structural elements of fibrinogen remain unmodified in the final form. To assess the affinity and selectivity of FA towards non-solution-based fibrin, FA was tagged with a green emitting fluorophore and fluorescence co-localization was monitored. Solubilized fibrin was also used in a number of binding validation studies, including microscale thermophoresis, isothermal titration calorimetry, and circular dichroism. These techniques were used to calculate the apparent Kd, which was found to be within the acceptable range. Four different FA-targeted contrast materials were produced, including gadolinium conjugates (Gd(III)-DOTA/NOTA-FA) for MRI, iodinated-FA and FA-functionalized gold nanoparticles (FA-AuNPs) for CT, and FA-functionalized gold-coated iron-oxide nanoparticles (FA-Fe3O4-AuNPs) as dual-imaging agents for MRI and CT. All formulations were tested for their ability to produce significant contrast enhancement. In each case, when treated with aptamer-targeted formulations, contrast development was specific to the location of the clot. In MRI, T1- and T2-weighted scans revealed hyperintense contrast enhancement of fibrinogen in the blood pool and fibrin clots, respectively. In CT, both iodo-FA and FA-AuNPs showed high positive contrast enhancement, confirming that targeting minimizes dilution of the contrast material in the presence of the blood pool. FA-Fe3O4-AuNPs were successful in producing clot-specific hypointense contrast in MRI, allowing for delineation of clot parameters. However, CT imaging demonstrated that higher concentrations of the nanoparticles is required, and the thin gold coating is not sufficient to produce contrast enhancement.

Subject: 

Chemistry

Language: 

English

Publisher: 

Carleton University

Contributor: 

Collaborator and co-author: 
Eve Tsai
Co-author: 
Erin McConnell
Co-author: 
Joseph Zurakowski
Co-author: 
Greg Cron
Co-author: 
Suzan Chen
Co-author: 
Jon O'Brien
Co-author: 
Robert Garcia
Co-author: 
Spencer Boisjoli
Co-author: 
Peter Kan
Co-author: 
Celine Chartrand

Thesis Degree Name: 

Doctor of Philosophy: 
Ph.D.

Thesis Degree Level: 

Doctoral

Thesis Degree Discipline: 

FLAG

Parent Collection: 

Theses and Dissertations

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