Responsive Contrast Agents: Synthesis and Characterization of a Tunable Series of pH-Sensitive Near-Infrared Pentamethines

Abstract

The demand for responsive dyes in optical imaging is high to achieve a better signal-to-noise ratio and, more specifically, to visualize acidic compartments of the endocytic pathway. Herein, we present a new synthetic route, with a step-by-step synthesis of water-soluble pH-sensitive cyanine dyes exhibiting p<i>K</i>_a values in the region of physiological pH, as confirmed by absorption and fluorescence spectra. Moreover, modification of p<i>K</i>_a values was achieved by two different substitution patterns, creating tunable pH-sensitive dyes. We demonstrated the functionality of the pH-sensitive dyes and their suitability as contrast agents for cellular uptake studies by preparing dye-labeled cetuximab and transferrin conjugates. Sulfonated head chains increased water solubility and prevented the formation of dimers, even in the context of dye-labeled bioconjugates. Confocal microscopy images of living cells revealed their pH-responsiveness, as specific fluorescence signal enhancements were observed in acidic compartments of the endocytic pathway (endosomes and lysosomes), although the background signal was low in a pH-neutral environment. Using mixtures of conjugates labeled with either a pH-sensitive or non-pH-sensitive dye for the uptake studies, we could follow the receptor binding and distinguish it from the endocytic uptake process of the conjugates in a simultaneous manner. Moreover, we used flow cytometry to quantify the fluorescence and observed a 3-fold signal enhancement for the pH-sensitive dye conjugates over a period of 3 h.