Figure 6
From: Cytosolic protein delivery using pH-responsive, charge-reversible lipid nanoparticles
Endocytic uptake of NLS-(− 30)GFP-LNPs and the importance of endosome acidification in the cytosolic release of NLS-(− 30)GFP. (A) CLSM observation of the cytosolic appearance of NLS-(− 30)GFP after treatment with NLS-(− 30)GFP-LNPs (1:10) (equivalent to 2.5 μM NLS-(− 30)GFP) for 6 h at 37 °C or 4 °C. Scale bar, 50 μm. (B) Percentages of cells bearing cytosolic NLS-(− 30)GFP signals in (A). (C) Total cellular uptake of NLS-(− 30)GFP after treatment with endocytosis inhibitors: 30 μM of pitstop2 (a clathrin-mediated endocytosis inhibitor), 80 μM of EIPA (a macropinocytosis and clathrin-mediated endocytosis inhibitor) and 0.5 μM of wortmannin (a macropinocytosis related PI3K inhibitor). Cells were incubated with NLS-(− 30)GFP-LNP (1:10) (equivalent to 2.5 μM NLS-(− 30)GFP) for 6 h. (D) CLSM observation of the cytosolic appearance of NLS-(− 30)GFP after treatment with NLS-(− 30)GFP-LNP (1:10) (equivalent to 2.5 μM NLS-(− 30)GFP) for 6 h in the presence and absence of NH4Cl, an inhibitor of endosomal acidification. Scale bar, 50 μm. (E) Percentages of cells bearing cytosolic NLS-(− 30)GFP signals in (D). Results are presented as mean ± SD (n = 3). **P < 0.01; ***P < 0.001 (ANOVA followed by unpaired t-test for (B, E) and by Dunnett’s post hoc test (C) vs. non-inhibitor).
