Self-Reporting Theranostic: Nano Tool for Arterial Thrombosis

Abstract

Arterial thrombosis (AT) originates through platelet-mediated thrombus formation in the blood vessel and can lead to heart attack, stroke, and peripheral vascular diseases. Restricting the thrombus growth and its simultaneous monitoring by visualisation is an unmet clinical need for a better AT prognosis. As a proof-of-concept, we have engineered a nanoparticle-based theranostic (combined therapy and monitoring) platform that has the potential to monitor and restrain the growth of a thrombus concurrently. The theranostic nanotool is fabricated using biocompatible super-paramagnetic iron oxide nanoparticles (SPIONs) as a core module tethered with the anti-platelet agent Abciximab (ReoPro) on its surface. Our in vitro feasibility results indicate that ReoPro-conjugated SPIONS (Tx@ReoPro) can effectively prevent thrombus growth by inhibiting fibrinogen receptors (GPIIbIIIa) on the platelet surface, and simultaneously, it can also be visible through non-invasive magnetic resonance imaging (MRI) for potential reporting of the real-time thrombus status.

Keywords: MRI; ReoPro; arterial thrombosis; platelet aggregations; theranostics.

Figure 1

Chemical fabrication of Tx@ReoPro nanotool and its physicochemical characterisation: (A) SPION conjugated with oleylamine; (B) formation of aqueous phase SPION by substituting oleylamine with dopamine through ligand exchange method; (C) PEG functionalisation of the dopamine-conjugated SPION (SPION-PEG) is ~5 nm diameter in TEM micrograph image (scale bar 10 nm); (D) Abciximab (ReoPro) drug amalgamation to the chemically modified SPION through amide bond by EDC/NHS reaction resulted in Tx@ReoPro. TEM micrographs of Tx@ReoPro is showing a diameter of around 7 nm (scale bar 5 nm); (E) Hydrodynamic diameter of SPION-PEG (21.04 nm) is represented by blue line, and hydrodynamic diameter of Tx@ReoPro (28.21 nm) is represented by red line. (F) Shows absorption spectra of the SPION nanocomposite Tx@ReoPro.

Figure 2

Inhibition of platelet aggregation by Tx@ReoPro: (A) Schematic representation of whole blood impedance-based platelet aggregometry method; (B) ADP (6.5 μM)-induced platelet aggregation inhibition in presence of increasing concentration of Tx@ReoPro; (C) Effect of ReoPro and Tx@ReoPro on 6.5 μM ADP induced activated platelets where drug concentration is same (i.e., 3.22 μg/mL); (D) Effect of SPION on platelet aggregation with ADP (6.5 μM); (E) One-way ANOVA analysis of Aggregation Unit (final aggregation) showed a significant change in platelet aggregation in presence of Tx@ReoPro (16.12 μg/mL and 32.25 μg/mL) when activated with ADP 6.5 μM, where * = p value < 0.05, and n = 3. Blue and red indicate duplicate test for each electrode pair (B–D).

Figure 3

Rheometric analysis of thrombus formation in the presence of Tx@ReoPro (80 μg/mL) using TEG and FOR: (A) Thromboelastography (TEG)-based real-time thrombus monitoring showed reduced maximum amplitude (MA) (solid black line), i.e., 42.6 mm in presence of Tx@ReoPro compared to control set (dotted purple line); (B) Free Oscillation Rheometry (FOR) showed longer clot onset times (COT1 representing blue line and COT2 representing red line) with reduced clot strength (G’max), i.e., 105.5 pa in presence of Tx@ReoPro compared to control set. Each of these experiments was performed in duplicate set and, in total, 4 times. One set of characteristic TEG and FOR data is presented here.

Figure 4

Inhibition of the fibrinogen receptor GPIIbIIIa by Tx@ReoPro along with its real-time monitoring: (A) Fibrinogen binding experiments were performed using flow cytometry, where FITC-conjugated fibrinogen was used to monitor Tx@ReoPro-mediated inhibition of GPIIbIIIa when activated with ADP (10 μM); F is representing percentage of population; (B) MFM images showed prominent magnetic force lines, indicating the active magnetic property of both SPION and Tx@ReoPro; (C) MRI shows increased R2* intensity with increasing dose of Tx@ReoPro; (D) Represents bright-field microscopic image of the same samples (resting platelets, platelets activated with ADP 10 μM with and without Tx@ReoPro) from MRI experiment. The flow cytometry was performed twice, and PoC MRI experiment was performed once.