Detection and monitoring of the multiple inflammatory responses by photoacoustic molecular imaging using selectively targeted gold nanorods

In vitro cell experiments have been performed to detect and monitor the upregulation of intercellular adhesion molecule-1 (ICAM-1) and E-selectin simultaneously by photoacoustic molecular imaging (PMI). Human umbilical vein endothelial cells (HUVECs) were grown on gelatin-coated glass slides and stimulated with inflammatory cytokines to induce the expression of the inflammatory biomarkers, ICAM-1 and E-selectin. Gold nanorods (GNRs) of aspect ratio (AR) 1:3 with absorption centered at 715 nm conjugated to anti-ICAM-1 antibody and GNRs of AR 1:3.5 with absorption centered at 800 nm conjugated to anti-E-selectin were exposed to HUVECs with different stimulation conditions. A focused high frequency ultrasonic transducer (60 MHz, f/1.5) was used to scan the photoacoustic (PA) signal over the top surface of the cell containing slides. Averaged PA signal intensity from the stimulated cells was about 3 folds higher (~10 dB) compared to the un-stimulated cells for both ICAM-1 and E-selectin. The strong binding of GNRs to the stimulated HUVEC cells was evidenced by fluorescence imaging. Exposure of HUVEC cells to GNRs conjugated to isotype control antibodies confirms a low level non-specific binding. Also, at 0, 2, 6, and 24 hours after inflammatory stimulation, the HUVECs were exposed to GNRs conjugated anti-ICAM-1 antibody and anti-E-selectin antibody. PA intensity at each stage of inflammation compares well with fluorescence imaging and rt-PCR quantification.