Figure 1.

Targeted fib-GC-AuNPs are superior to non-targeted GC-AuNPs in fibrin-binding and mCT imaging of cerebral thromboemboli. A, Schematic diagram of fibrin-targeted gold nanoparticles. B, In vitro experiments to show a higher fibrin-binding capacity of fib-GC-AuNPs vs. GC-AuNPs. When either fibrinogen or thrombin was added to GC-AuNP colloid (upper row) or fib-GC-AuNP colloid (middle row), the red coloring caused by dissolute colloids did not change. In contrast, when either preformed fibrin clot was immersed in the fib-GC-AuNP colloid (upper row) or if fibrin clot was formed in situ in the fib-GC-AuNP colloid (middle row), the redness (upper and middle rows) and UV absorbance (lower row) caused by the nanoparticle colloids was decreased and the clots formed were stained a red color. These changes suggest that both non-targeted GC-AuNPs and fibrin-targeted fib-GC-AuNPs have bound to the clot, reducing the amount of colloid in free solution. Considering the relatively weak changes in the color and UV absorbance of the non-targeted GC-AuNP colloid as well as in the color of the preformed and in situ clots, the fibrin-binding capacity of GC-AuNPs is substantially lower than that of fib-GC-AuNPs. TBS denotes Tris-buffered saline. C and D, representative mCT thrombus images / Cy5.5 near-infrared fluorescent (NIRF) thrombus images of C57Bl/6 mice with embolic stroke (C) and grouped quantification data for the areas of embolic clots in the black and red dotted squares (D). Embolic stroke was induced by injecting Cy5.5-fluorescently labeled clot into the bifurcation area of the left distal internal carotid artery of mice (n = 56). One hour later, mCT thrombus images (upper row in C) were acquired 5 minutes after intravenous injection of 300 μL GC-AuNPs or fib-GC-AuNPs of either a low (12 mg/kg; n = 5 / group) or high concentration (120 mg/kg; n = 23 / group). After each animal was sacrificed and the brain was collected, ex vivo optical imaging was performed to obtain a Cy5.5 NIRF thrombus image (lower row in C), which served as a exogenously labeled standard for the purposes of comparing to the corresponding mCT image. At both the high and low concentrations, targeted fib-GC-AuNPs visualize the Y-shape cerebral thrombi better than non-targeted GC-AuNPs (black dotted squares in C). Note how the fluorescent embolic thrombus (red dotted squares in C) is similar for all animals, but how the same thrombi are much better visualized with targeted vs. non-targeted nanoparticles by CT (black dotted squares in C). Regardless of the type of imaging agent, the high concentration is superior to the low concentration in the CT visualization of cerebral thrombi. The above imaging findings from the representative animals (C) are corroborated by the quantification data for all 56 animals (D); the visualized thrombus area ratio (mCT/NIRF) is the highest in the high concentration fib-GC-AuNP group, followed by the high concentration GC-AuNP group, the low concentration fib-GC-AuNP group, and last the lowest in the low concentration GC-AuNP group. E and F, mCT visualization (E) and quantification (F) of cerebral thromboemboli after sequential administrations (blue colored +→) of first non-targeted GC-AuNP and then targeted fib-GC-AuNP, and vice versa. After intravenous (i.v.) injection of GC-AuNPs (120 mg/kg, 300 μL) 50 minutes after embolic stroke, there is very weak parenchymal mCT hyperdensity in the circle of Willis (black dotted square in the 1^st column of the upper row). A subsequent mCT image at 60 minutes, obtained after administering fib-GC-AuNPs (120 mg/kg, 300 μL), clearly visualizes cerebral thrombus (black dotted square in the 2^nd column of the upper row), which co-localizes to thrombus-marking Cy5.5 signal on the ex vivo NIRF image (red dotted square in the second column of the lower row). When the order of injecting the two types of imaging agent was reversed, mCT visualization of cerebral thrombus by using fib-GC-AuNPs is not further improved by additionally using GC-AuNPs (black dotted squares in the 3^rd and 4^th columns of the upper row), despite the presence of thrombus-marking Cy5.5 signal on the ex vivo NIRF image (red dotted square in the 4^th column of the lower row). These findings from the representative animals are corroborated by quantified data (the areas of embolic clots in the black and red dotted squares) for all 14 animals (F; n = 7 / group). Graphs show mean ± SEM. ^*P < 0.01 from paired t-tests between mCT vs. corresponding NIRF lesion areas; ^#P < 0.01 (vs. 120 mg/kg GC-AuNP and both 12 mg/kg and 120 mg/kg fib-GC-AuNP groups) from Student's t-tests; ^§P < 0.01 from paired t-tests. Concentrations of nanoparticles are reported as 'mg Au / kg of animal'. Scale Bars = 2 mm.