Assessment of Aptamer-Targeted Contrast Agents for Monitoring of Blood Clots in Computed Tomography and Fluoroscopy Imaging.

Objective: Random formation of thrombi is classified as a pathological process that may result in partial or complete obstruction of blood flow and limited perfusion. Further complications include pulmonary embolism, thrombosis-induced myocardial infraction, ischemic stroke, and others. Location and full delineation of the blood clot are considered to be two clinically relevant aspects that could streamline proper diagnosis and treatment follow-up. In this work, we prepared two types of X-ray attenuating contrast formulations, using fibrinogen aptamer as the clot-seeking moiety. Methods: Two novel aptamer-targeted formulations were designed. Iodine-modified bases were directly incorporated into a fibrinogen aptamer (iodo-FA). Isothermal titration calorimetry was used to confirm that these modifications did not negatively impact target binding. Iodo-FA was tested for its ability to produce concentration-dependent contrast enhancement in a phantom CT. It was subsequently tested in vitro with clotted human and swine blood. This allowed for translation into ex vivo testing, using fluoroscopy. FA was also used to functionalize gold nanoparticles (FA-AuNPs), and contrast capabilities were confirmed. This formulation was tested in vitro using clotted human blood in a CT scan. Results: Unmodified FA and iodo-FA demonstrated a nearly identical affinity toward fibrin, confirming that base modifications did not impact target binding. Iodo-FA and FA-AuNPs both demonstrated excellent concentration-dependent contrast enhancement capabilities (40.5 HU mM-1 and 563.6 HU µM-1, respectively), which were superior to the clinically available agent, iopamidol. In vitro CT testing revealed that iodo-FA is able to penetrate into the blood clots, producing contrast enhancement throughout, while FA-AuNPs only accumulated on the surface of the clot. Iodo-FA was thereby translated to ex vivo testing, confirming target-binding associated accumulation of the contrast material at the location of the clot within the dilation of the external carotid artery. This resulted in a 34% enhancement of the clot. Conclusions: Both iodo-FA and FA-AuNPs were confirmed to be effective contrast formulations in CT. Targeting of fibrin, a major structural constituent of thrombi, with these novel contrast agents would allow for higher contrast enhancement and better clot delineation in CT and fluoroscopy.

Screening and initial binding assessment of fumonisin b(1) aptamers.

Fumonisins are mycotoxins produced by Fusarium verticillioides and F. proliferatum, fungi that are ubiquitous in corn (maize). Insect damage and some other environmental conditions result in the accumulation of fumonisins in corn-based products worldwide. Current methods of fumonisin detection rely on the use of immunoaffinity columns and high-performance liquid chromatography (HPLC). The use of aptamers offers a good alternative to the use of antibodies in fumonisin cleanup and detection due to lower costs and improved stability. Aptamers are single-stranded oligonucleotides that are selected using Systematic Evolution of Ligands by EXponential enrichment (SELEX) for their ability to bind to targets with high affinity and specificity. Sequences obtained after 18 rounds of SELEX were screened for their ability to bind to fumonisin B(1). Six unique sequences were obtained, each showing improved binding to fumonisin B(1) compared to controls. Sequence FB(1) 39 binds to fumonisin with a dissociation constant of 100 ± 30 nM and shows potential for use in fumonisin biosensors and solid phase extraction columns.