Contents lists available at ScienceDirect European Journal of Pharmaceutical Sciences journal homepage: www.elsevier.com/locate/ejps Acid-induced degradation of widely used NIR dye DiR causes hypsochromic shift in fuorescence properties Markus Riehl a,b , Meike Harms b , Benedikt Göttel a , Holger Kubas b , Dirk Schiroky b , Karsten Mäder a, ⁎ a Institute of Pharmacy Faculty I of Natural Sciences, Martin Luther University Halle-Wittenberg, Wolfgang-Langenbeck-Strasse 4, 06120 Halle/Saale, Germany b Department of Pharmaceutical Technologies, Merck KGaA, Frankfurter Strasse 250, 64293 Darmstadt, Germany ARTICLEINFO Keywords: In-vivo imaging NIR Dye Hydrolysis Wavelength shift Fluorescence ABSTRACT DiR (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindotricarbocyanine iodide) is one of the most widely used near-in- frareddyesfor in-vivo imagingduetoitsfavorableopticalproperties.Sofar,chemicalstabilityhasbeentakenfor granted by most investigators. However, in a recently published imaging study, we found that DiR can exert a hypsochromic shift in fuorescence in-vivo, potentially induced by low pH. This behavior may disturb kinetic measurements and the readout of additional markers fuorescing at lower wavelengths. The present in-vitro experiments were conducted to verify the fndings 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 pH2 to neutral pH over 42 days. Fluorescence and absorption measurements as well as mass spectrometry analytics (MS) were con- ducted to monitor the degradation process of DiR. The protonation efect on DiR optical behavior was estimated using in-silico modelling. For the most acidic sample, a distinct decrease in DIR-fuorescence was noted and several degradation products could be analyzed via MS, confrming the initial hypothesis. Ultimately, scientists should be aware of the possibility of acid-induced DiR degradation, especially when adding a second fuorescence label for dual dye imaging or performing quantitative data analysis. 1. Introduction DiR (1,1′-Dioctadecyl-3,3,3′,3′-tetramethylindotricarbocyanine io- dide) is a commonly used near-infrared (NIR) dye. Its favorable prop- erties, e.g. low toxicity to living cells (Eisenblaetter et al., 2009; Granot et al., 2007) and low interference with intrinsic fuorescence signals from ingested food or hair, have made it a valuable tool for in-vivo imaging studies. Since the herein investigated label DiR is a tool in many studies in the context of biopharmaceutical research (Carlson et al., 2013; Etrych et al., 2016; Weiss et al., 2017), detailed knowledge about the properties of the dye in diferent environments (e.g. hydro- philic/lipophilic or acidic/alkaline media) is crucial for proper data interpretation. Recently, our group made the observation that DiR can exert a shift in fuorescence when embedded into poly(lactic-co-glycolic) acid (PLGA) microparticles (Riehl et al., 2018). It is well known that PLGA matrices cause a remarkable drop in microenvironment pH upon de- gradation (Anderson and Shive, 2012; Mäder et al., 1996), which is caused by the formation of acidic degradation products. In the men- tioned study, the fuorescence shift behavior of DiR to lower wavelengths caused false negative signals in a second fuorescence flter which was initially intended to monitor fuorescence emissions from a second label in the far-red spectrum. As a follow-up action, the herein presented in-vitro stability study of DiR was started with the aim to verify the in-vivo fndings and to further investigate the fate of DiR when being incubated at diferent pH-values over several weeks. For this purpose, ethanolic solutions of DiR were diluted with var- ious mixtures of lactic acid/water and incubated at 37 °C. Various parameters such as pH, fuorescence, absorption and mass spectra were assessed at predefned time points to monitor the hypothesized DiR degradation. In-silico modelling has been conducted to better under- stand the efect of protonation on the optical properties of DiR. Finally, the degradation of DiR especially in acidic media is dis- cussed in this work, elaborating implications for upcoming in-vivo imaging studies and the risk of signal interference. To our knowledge, these focused investigations on the behavior of DiR in acidic environ- ment were not conducted beforehand, although they reveal critical points to consider especially when adding a second fuorescence label for dual dye imaging or performing quantitative data analysis. https://doi.org/10.1016/j.ejps.2019.02.031 Received 13 June 2018; Received in revised form 11 February 2019; Accepted 19 February 2019 ⁎ Corresponding author. E-mail address: Maeder@pharmazie.uni-halle.de (K. Mäder). European Journal of Pharmaceutical Sciences 132 (2019) 27–33 Available online 21 February 2019 0928-0987/ © 2019 Elsevier B.V. All rights reserved. T