Fig. 3
Cell targeting and internalization of Atto488 carrying formulations. Representative histograms a and quantitative analysis b of MFI associated with MCF-7 and MDA-MB-231 cells upon incubation with ANANAS-Atto488 (Atto488 3.5 × 10−9 M), ANANAS-Atto488-Cetux10 (cetuximab 0.58 μg/mL, Atto488 3.5 × 10−9 M) and cetux-Atto488 at low and high concentration (Cetux-Atto488(1): same Cetux as in ANANAS-Atto488-Cetux10; Cetux-Atto488(2): same Atto488 as in ANANAS-Atto488-Cetux10; MFI values have been normalized for the Atto488 brilliance in each formulation (40% and 61% of that of the free dye when tethered to ANANAS or to cetuximab, respectively); c Ratio between cell-associated MFI in MDA-MB-231 when treated with ANANAS-Atto488-Cetux10 and Cetux-Atto488(1) at the same cetuximab concentration (0.58 μg/mL). The horizontal red bar indicates the ratio between FAR ANANAS-Atto488-Cetux10 and FAR Cetux-Atto488 and corresponds to the maximum enhancement factor expected for the ANANAS-based carrier in case of a pure FAR improvement mechanism. A similar trend was observed with the MCF-7 cells. However, owing to the low fluorescence registered in the cetux-Atto488(1)-treated samples, the actual MFI ratio—which can only be estimated—was not reported in the figure. (The original data set is reported in Supplementary Table 6). Data presented as mean of n = 3 experiments (each in triplicate) ± SEM. Paired t test of treated samples respect to time 0: *p < 0.05, **p < 0.01, ***p < 0.001. d Fluorescence microscopy imaging of MCF-7 and MDA-MB-231 after 6 h treatment with ANANAS-Atto488-Cetux10. Green: Atto488, red; Lysotracker, blue channel nuclei staining with DAPI. × 60 magnification, size bar = 20 µm. e Effect of the endocytic inhibitor chlorpromazine (CPZ) on ANANAS internalization: MFI associated with MCF-7 and MDA-MB-231 cells pre-treated with CPZ 30 µM, and then treated for 6 h with the different Atto488 formulations. Data presented as mean of three independent experiments (each in triplicate) ± SEM (paired t test): *p < 0.05, **p < 0.01. For the concentrations of Atto488-loaded reagents, see above
