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

Seunghan Ha,Kang Kim,Andrew Carson,Nicholas A Kotov,Ashish Agarwal

doi:10.1364/boe.2.000645

Abstract

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.

Highlights

Recent reports have shown that inflammation plays a key role in a variety of systemic diseases such as atherosclerosis, coronary artery disease (CAD), rheumatoid arthritis (RA), and systemic lupus erythematosus [1]
The PA intensity from the stimulated Human umbilical vein endothelial cells (HUVECs) was on the average about 8-10 dB higher than the PA intensity from the un-stimulated cells for both intercellular adhesion molecule-1 (ICAM-1) and E-selectin
The sample slide was positioned in the sample holder for PA imaging (See Fig. 2) and by changing wavelengths PA images for intercellular adhesion molecule (ICAM)-1 at 715 nm and E-selectin at 800 nm were obtained

Summary

Introduction

Recent reports have shown that inflammation plays a key role in a variety of systemic diseases such as atherosclerosis, coronary artery disease (CAD), rheumatoid arthritis (RA), and systemic lupus erythematosus [1]. Prospective epidemiological studies have suggested that vascular risk is affected by increased basal levels of inflammatory cytokines such as interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α); cell adhesion molecules such as soluble or membrane-bound ICAM-1, E-selectin, and P-selectin; and acute-phase reactants such as C-reactive protein and fibrinogen [6]. Detection of these specific biomarkers, especially simultaneously, could show the bioactivities of inflammation and may provide prognostic information and further characterize systemic diseases such as atherosclerosis. ICAM-1 and E-selectin, were chosen as typical biomarker of inflammation and activation of endothelial cells (ECs)

Cell preparations and immunfluorescence analysis

Real-time quantitative PCR

GNRs synthesize and bio-conjugation

Results and discussion

Conclusion