Methods: 14 Sprague Dawley rats undergoing LAD occlusion for 45 min were pretreated with i.v. injection of anti-thrombin PPACK (D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone)-conjugated PFC NP (~ 13,000 PPACK per PFC NP), or saline (control) followed by 2 hours of reperfusion. Sham controls (no LAD ligation) were compared. Simultaneous to treatments, a different and unique nontargeted CE PFC NP (CE: Crown Ether core) was injected for subsequent tracking and spectroscopic quantification of the relative distributions and amounts of PFC NP trapped in non-reperfused myocardium. Hearts were perfused with saline in situ, then extracted for ex vivo 19F (fluorine) MRI and spectroscopy at 7T (Bruker). T2* images were acquired for hemorrhage/edema in the area at risk. 19F MRI was acquired for PFC NP trapping (spin echo: TR 2000ms, TE 4.51 ms, FOV 20x20, 64x64 slice 1mm), along with whole heart 19PFC CE spectroscopy.
Results: Fluorine MRI of an intact heart illustrates the trapping of PFC NP in areas of nonreperfusion (Figure) amidst infarct scar region. Quantification of total heart CE PFC NP signal emanating from trapped NP revealed reduced accumulation (¯49%) in the hearts of treated rats (0.47±0.09 vs 0.92±0.19, treated vs control; p < 0.05). For sham occlusions, minimal deposition of CE PFC NP was observed irrespective of PPACK PFC NP treatment (n=10 per group). Areas of T2* signal indicative of vascular leakage extended beyond that of the 19F signals (Figure T2*), indicating more severe damage and trapping in central infarct zones than in more peripheral border zones after 2 hours.
Discussion: Continued inhibition of thrombin in cardiac IRI with locally-acting PPACK PFC NP preserved vascular integrity, and limited hemorrhage and No Reflow (less trapping). Because these PPACK PFC NP do not prolong bleeding times or coagulation parameters beyond ~30-60 min after injection, yet maintain prolonged local surveillance against activated thrombin, they represent a potentially useful therapeutic strategy for cardiac IRI.