Near-infrared fluorescence imaging of lymph nodes using a new enzyme sensing activatable macromolecular optical probe

The aim of this study was to validate the use of near infrared fluorescence imaging (NIRF) using enzyme-sensitive optical probes for lymph node detection. An optical contrast probe that is activated by cystein proteases, such as cathepsin B, was used to visualize lymph nodes by NIRF reflectance imaging. In order to quantitate the uptake of the optical probe in lymphatic tissue, the biodistribution was assessed using the Indium-111 labeled optical probe. Sixteen Balb-c mice were injected either intravenously (i.v.) or subcutaneously (s.c.) with the NIRF-probe (2 mol cyanine (Cy)/animal; i.v., n=10; s.c., n=6) and imaged 24 h after injection. Signal intensities and target-to-background ratios of various lymph nodes were measured by manual regions of interest (ROIs). Additional signal intensity measurements were performed of excised lymph nodes (n=21) from i.v. injected mice (24 h after injection) and compared with excised lymph nodes (n=8) of non-injected mice. The probe employed in this study was lymphotropic with approximately 3–4% accumulation in lymph nodes (3.4±0.8% ID/g). Measurements of the excised lymph nodes (after i.v. injection) confirmed a significant increase in lymph node fluorescence signal from baseline 26±7.6 arbitary units (AU) to 146±10.9 AU (p<0.0001). A significant increase in lymph node fluorescence signal was also seen in vivo throughout the body after i.v. injection (96±7.8 AU) and/or regionally after s.c. injection (141±11.5 AU) in comparison with baseline autofluorescence (26±7.6 AU). Target-to-background ratio was significantly higher after s.c. injection (6.6%±0.81) compared with i.v. injection (4.8±0.67%). Detection and visualization of lymph nodes is feasible by NIRF imaging using a cystein-protease sensitive optical probe.