Dual-dye systems comprising activatable fluorescein dye and hydrophobic or hydrophilic Cy5 reference fluorophore for ratiometric drug delivery monitoring

[Display omitted] •Dual-dye systems Aza–Flu–Cy5h and Aza–Flu–Cy5s are developed.•Anticancer drug azatoxin (Aza) is bound to activatable fluorescein green-dye Flu.•Reference red-dye is either hydrophobic (Cy5h) or hydrophilic (Cy5s) indocyanine.•These systems enable ratiometric monitoring of drug release events.•Aza–Flu–Cy5s exhibits higher brightness in both red and green channels. Ratiometric fluorescence detection in biological samples in vitro and in vivo mitigates several issues in conventional single-signal measurements such as light absorption and scattering, dye photobleaching, and instrumental parameters. Herein, two new systems for ratiometric monitoring of drug delivery are synthesized and investigated. These systems comprise a non-fluorescent fluorescein-based dye, Flu, bound to the anticancer drug, azatoxin (Aza), through a hydrolytically cleavable acrylate ester linker and either hydrophobic (Cy5h) or hydrophilic (Cy5s) pentamethine cyanine dye providing a red-light reference signal. Upon drug release, the green Flu fluorescence notably increases enabling ratiometric tracking of drug release events. The spectral properties of the developed conjugates and fluorescently monitored drug release rates are investigated in biologically relevant media with respect of the Cy5 dye hydrophilicity.