Investigation into the uptake mechanism. (A) Effect of low temperature
incubation of HeLa cells on liposomal delivery of TOPRO3 and endosomal
uptake of pHrodo. Cells were preincubated on ice with 5 μM CPK
(2 h), followed by 15 min incubation with 0.25 mM CPE-decorated liposomes
containing TOPRO3. After three washes confocal images were taken immediately
(0 min) and after 60, 120, and 180 min. Top row: TOPRO3 (red), bottom
row: pHrodo (blue). (B) Graphical representation of the percentage
of TOPRO dye uptake by HeLa cells on ice. Fluorescence intensities
were calculated by ImageJ and plotted as a percentage relative to
the fluorescence of TOPRO3 delivery at 37 °C (100%). Scale bar
is 25 μm. (C) Effect of endocytosis and macropinocytosis inhibitors
on delivery of PI by liposomes to HeLa cells. Cells were incubated
with medium (Ctrl+), or medium containing 0.25 μM wortmannin
(Wor), 40 μM chlorpromazine (Chl), 200 μM genistein (Gen),
40 μM nocodazole (Noc) for 1 h, 0.01% w/v sodium azide (NaN3),
followed by 2 h incubation with 5 μM CPK in the presence of
inhibitors, and then treated for 15 min with CPE-liposomes containing
PI. Final concentration of lipids (liposomes) was 0.25 mM. Cellular
uptake was measured by flow cytometry. Positive control (100%): fluorescence
of PI dye in the absence of inhibitors.