Figure 7.

L-5FA significantly increases the cellular exposure compared to untargeted 5FA by fluorescence microscopy. (A,B) BT474 cells were treated with 5 μM of unlabeled 5FA or L-5FA and fixed at 4 and 37 °C to visualize csGRP78-dependent internalization. Cells were visualized by three-dimensional super resolution laser scanning confocal microscopy. Targeted L-5FA was present on the membrane at a higher abundance at 4 °C as well as inside the cell at 37 °C, which are consistent with ligand-dependent enhancement of uptake compared to 5FA. (C) Degradation and/or export of the probe after internalization was characterized in live cells by epifluorescence microscopy, which was quantified by image analysis. Rhodamine-labeled 5FA and L-5FA were visualized in BT474 cells to calculate average fluorescence per cell over time. These data show consistently higher L-5FA cell association; furthermore, they suggest similar degradation kinetics between both constructs. (D) Although degradation kinetics appear to be similar, because L-5FA starts at such an elevated cell association, it yields a significantly higher exposure (AUC) to the rhodamine probe over a 240 h period (n = 3 per group, *p = 0.001). Mean ± SD.