Acid-induced degradation of widely used NIR dye DiR causes hypsochromic shift in fluorescence properties

DiR (1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine iodide) is one of the most widely used near-infrared dyes for in-vivo imaging due to its favorable optical properties. So far, chemical stability has been taken for granted by most investigators. However, in a recently published imaging study, we found that DiR can exert a hypsochromic shift in fluorescence in-vivo, potentially induced by low pH. This behavior may disturb kinetic measurements and the readout of additional markers fluorescing at lower wavelengths. The present in-vitro experiments were conducted to verify the findings from our in-vivo study and to elucidate the changes of the optical properties of DiR. For this purpose, DiR was incubated in a pH range from pH 2 to neutral pH over 42 days. Fluorescence and absorption measurements as well as mass spectrometry analytics (MS) were conducted to monitor the degradation process of DiR. The protonation effect on DiR optical behavior was estimated using in-silico modelling. For the most acidic sample, a distinct decrease in DIR-fluorescence was noted and several degradation products could be analyzed via MS, confirming the initial hypothesis. Ultimately, scientists should be aware of the possibility of acid-induced DiR degradation, especially when adding a second fluorescence label for dual dye imaging or performing quantitative data analysis.