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.