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.

Selection of DNA aptamers that prevent the fibrillization of α-synuclein protein in cellular and mouse models.

A neuropathological hallmark of Parkinson's disease (PD) is the aggregation and spreading of misfolded α-synuclein (αSyn) protein. In this study, a selection method was developed to identify aptamers that showed affinity for monomeric αSyn and inhibition of αSyn aggregation. Aptamer a-syn-1 exhibited strong inhibition of αSyn aggregation in vitro by transmission electron microscopy and Thioflavin T fluorescence. A-syn-1-treated SH-SY5Y cells incubated with pre-formed fibrils (PFFs) showed less intracellular aggregation of αSyn in comparison with a scrambled oligonucleotide control, as observed with fluorescent microscopy. Systemic delivery of a-syn-1 to the brain was achieved using a liposome vehicle and confirmed with fluorescence microscopy and qPCR. Transgenic mice overexpressing the human A53T variant of αSyn protein were injected with a-syn-1 loaded liposomes at 5 months of age both acutely (single intraperitoneal [i.p.] injection) and repeatedly (5 i.p. injections over 5 days). Western blot protein quantification revealed that both acute and repeated injections of a-syn-1 decreased levels of the aggregated form of αSyn in the transgenic mice in the prefrontal cortex, caudate, and substania nigra (SNc). These results provide in vitro and in vivo evidence that a-syn-1 can inhibit pathological αSyn aggregation and may have implications in treatment strategies to target dysregulation in PD.

Adsorption-desorption nano-aptasensors: fluorescent screening assays for ochratoxin A.

In this study, a FRET-based fluorescent aptasensor for the detection of ochratoxin A (OTA) was optimized based on the quenching efficiency of single-walled carbon nanotubes (SWCNTs) and the binding affinity of aptamers. OTA aptamers were conjugated with quantum dots and adsorbed to the surface of both acid-modified and unmodified SWCNTs. The maximum fluorescence quenching efficiency of the SWCNTs were compared. Acid-modified SWCNTs (amSWCNTs) have moderate quenching efficiency, providing an optimal sensitivity for qualitative fluorescence-enhancement biosensor assays. The binding parameters of the QD-modified OTA aptamers (1.12.2 and A08min) on the surface of amSWCNTs were compared. Based on our results, the A08min aptamer is a better candidate for OTA detection. Using the A08min aptamer, the SWCNT method had a limit of detection (LOD) of 40 nM. The amSWCNT method had a significantly lower LOD of 14 nM. Turn-on fluorescent nano-aptasensors are emerging as an effective diagnostic tool for simple detection of mycotoxins. Nanocomplexes designed for the detection of mycotoxins in solution and paper-based tests have proven to be useful.