Importance of the physicochemical properties of fluorescent dyes for obtaining target-specific in vivo images by membrane-permeable macromolecular imaging probes

Abstract

Background: Membrane-permeable macromolecular (MPM) probes are designed to deliver functional proteins to disease sites and into cells by using protein transduction domain (PTD). To visualize intracellular molecular events in tumor sites, we previously developed MPM imaging probes conjugated with near-infrared fluorescence (NIRF) dyes. However, the factors which influence target-specific in vivo fluorescence images of MPM imaging probes are still unclear. We studied whether the physicochemical properties of NIRF dye significantly affect the in vivo images of MPM imaging probes. Methods: We constructed three MPM imaging probes conjugated with NIRF dyes including IRDye800, AlexaFluor750 and the newly synthesized PBI3921 and compared their biodistribution in tumor-bearing mice using in vivo optical imaging. In addition, we addressed to reveal relationships between biodistribution of MPM imaging probes and conjugated NIRF properties. Results: In vivo images obtained by three MPM probes were spatiotemporally different each other. These probes (~46 kDa) are different only in their NIRF dyes (~2 kDa), which have different physicochemical properties including surface charge and hydrophobicity based on chromatograph analyses. Moreover, the results of clearance efficiency obtained by using cancer cells were not necessarily correlated with the ones obtained by using mouse models. Conclusions: We concluded that physicochemical properties of NIRF dyes do affect the in vivo off-target images after injection of MPM imaging probes and that clearance of non-specific image may be predicted by the analysis of the property of a fluorescent dye conjugated to MPMs. These results would provide useful information to design MPM imaging probes with high target specificity in vivo.