Figure 2. Confocal microscopic images of live cells dual-labeled with AFDye532- and NF-penetratin. Cells were incubated with AFDye532-penetratin (A) and NF-penetratin (B) at 37°C for 20 min and images were captured by confocal microscopy. Panel C displays a differential interference contrast image of the cells. The arrows in the fluorescence images point at areas showing the anti-correlation between the pH-insensitive fluorescence of AFDye532 and the fluorescence of NF quenched at acidic pH.
Reduction of the membrane dipole potential enhances uptake and endo-lysosomal release of penetratin
Since the strong intramembrane dipole potential is expected to affect the uptake and/or endo-lysosomal release of positively-charged penetratin, we treated cells with 6-ketocholestanol and phloretin, agents known to increase and decrease, respectively, the positive, intramembrane dipole potential (Gross, Bedlack & Loew, 1994; Kovács et al., 2016). Flow cytometric analysis of the fluorescence intensity ratio of the dipole potential sensitive dye, di-8-ANEPPS, confirmed that 6-ketocholestanol significantly increased the dipole potential. On the other hand, phloretin decreased the dipole potential although its effect did not reach statistical significance (Fig. 3A). These observations are in accordance with our previous results (Kovács et al., 2016).
Having established that the treatments modify the dipole potential according to expectations, we set out to characterize the effect of an altered dipole potential on the uptake of penetratin. A diminished dipole potential significantly enhanced both the total cellular uptake of penetratin and its concentration in non-acidic compartments in both SKBR-3 and MDA-MB-231 cells (Fig. 4). Since both of these processes were enhanced at the lower dipole potential achieved by phloretin treatment, the ratio of the NF and AFDye532 intensities, characterizing the fractional release from acidic compartments, remained unchanged. On the other hand, 6-ketocholestanol, which significantly increased the dipole potential (Fig. 3A), had a statistically non-significant effect on both the total cellular uptake and endo-lysosomal release of penetratin (Fig. 4). In conclusion, we have established that the physiological level of the intramembrane, positive dipole potential significantly inhibits the uptake of penetratin.