Near infrared probes for biochemical, cellular, and whole animal analysis of disease processes

Abstract

The study of disease processes requires a number of tools for detection of proteins and biomarkers in cell and animal based assays. Near infrared (NIR) technologies offer the advantage of high signal without interference from background producing factors such as tissues, blood, or plastics. NIR fluorescence quenching biochemical assays employing a novel NIR quencher are homogeneous and sensitive. NIR-based immunocytochemical assays offer a means of quantitatively evaluating cell signaling pathways. The technology can be extended to the development of targeted molecular imaging agents for disease analysis in animal models. We describe here model assays for each of these categories. A fluorescence quenching caspase-3 assay was developed employing a novel, broadly applicable quencher dye suitable for use with both visible and NIR dye chemistries. An NIR cell based assay is described for assessment of phosphorylation of p53 in response to a cellular stimulus. Finally, we describe the development and application of a targeted NIR optical imaging agent for monitoring tumor growth in whole animals. The NIR biochemical and cell based assays are robust with Z' factors greater than 0.7. The use of an IRDye ® 800CW-labeled cyclic RGD peptide is presented as a model for development and application of targeted imaging agents. NIR technologies are compatible with the complete spectrum of assay needs for disease analysis and therapeutic development.

‘In vivo’ optical approaches to angiogenesis imaging

“…Based on the binding of RGD to α v β 3 integrin, Haubner et al [ 74 ] developed 125 I and later 18 F [ 75 ] labeled tracers to image α v β 3 integrin expression and tumor angiogenesis. In recent years, many fluorescently labeled cyclic RGD peptides have been developed for near infrared fluorescence imaging like RGD-Cy7, RGD-Cy5.5, RGD-QD705 [ 76 – 78 ] and cyclic RGD coupled to IRDye 800CW-labeled peptide which is even more shifted towards the infrared parts of the spectrum resulting in ideal fluorescent characteristics for in vivo use [ 79 , 80 ]. Mulder et al [ 81 , 82 ] recently described paramagnetic lipid-encapsulated quantum dots with RGD presented at the outside and a green fluorescent quantum dot at the inside of the micellar shell.…”