Activatable and de-activatable fluorescent constructs as probes to determine cellular uptake

Objectives A tracer that is able to distinguish between intra- and extracellular localization by a change in fluorescence can help to provide insight in the uptake process of receptor-targeted tracers. In this study we explored this concept for the chemokine receptor 4 (CXCR4). Two different photophysical mechanisms were studied under the same conditions; using 1) a “traditional” activatable fluorescence tracer that displayed enhanced fluorescence upon internalization and 2) a novel de-activatable fluorescence tracer wherein the fluorescence of the extracellular portion of the tracer could be turned off via a secondary click chemistry step.

Methods The activatable tracer was synthesized by coupling the CXCR4-targeting peptide Ac-TZ14011 to an activatable dye construct which contains a Cy3 dye that was linked to a Cy5 dye through a reduction-cleavable disulfide bond. Reduction of Förster Resonance Energy Transfer (FRET)-based Cy3-signal quenching was used to assess disulfide cleavage in solution and after lysosomal internalization into cells. The de-activatable tracer was synthesized by conjugating of the Ac-TZ14011 peptide to a Cy5 dye that contained a chemoselective azide handle. FRET-based silencing of the Cy5 signal was evaluated after addition of a hydrophilic DBCO-containing Cy7 quencher. Binding of the tracers (1uM) to the CXCR4 receptor and subsequent internalization was evaluated in CXCR4 over-expressing MDAMB231 X4 cells using a Leica SP8 fluorescence confocal microscope. Membranous staining and internalization of the tracers was assessed over time (t=0 to 60 min), after addition of the tracers to the medium. The de-activatable tracer was evaluated via a two-step process wherein the Cy5 labeled peptide was added 1hr prior to addition of the quencher (2uM). Ac-TZ14011 functionalized with a Cy5 derivative that did not contain the azide handle was used as a control. Signal quantification of the Cy3 and Cy5 signals was performed using Leica LASX software.

Results The FRET efficiency of the activatable tracer was 55% and a limited 2.3-fold increase in fluorescence was seen upon disulfide reduction in solution. Fluorescence imaging in viable cells revealed a similar effect; only a weak membranous staining and subsequent increase in the Cy3 signal in the cytoplasm of the cells was seen which did not significantly improve over time. In contrast, addition of the Cy7-quencher to the Cy5-azide labeled peptide resulted in a fast (2-5 minutes) and >90% reduction of the Cy5 signal in solution. Membranous staining in cells was significantly more intense compared to staining achieved with the activatable tracer. Addition of the quencher resulted in an 80% decrease in the membranous staining within the first 10 minutes, while fluorescence of internalized tracer-component was preserved. Control experiments with a tracer lacking the azide handle did not show reduction of the Cy5 signal after addition of the quencher.

Conclusions In determining the cellular incorporation of receptor bound tracers, de-activatable fluorescence tracers provide a promising alternative for activatable tracers. Moreover, the click chemistry-based pretargeting-like approach proved to be efficient and can be utilized for other functionalization of extracellular bound tracers.