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. 2016 Aug 22;2(9):621–630. doi: 10.1021/acscentsci.6b00172

Figure 5.

Figure 5

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.