Fig. 2.

Evaluating nortriptyline (NT)-DOPE CADosomes for cytosolic delivery of eGFP-encoding mRNA in a HeLa cell line. (A–B) Evaluation of cellular uptake of NT-DOPE CADosomes with different N/P ratios, loaded with Cy5-labeled mRNA, in HeLa cells as analyzed via flow cytometry and expressed as Cy5+ cells and Cy5 relative mean fluorescence intensity (rMFI Cy5; normalized to non-treated control (NTC)), respectively. (C) Representative confocal images of HeLa cells after transfection with Cy5-mRNA NT-DOPE CADosomes N/P 9, with nuclei (blue) and intracellular Cy5-mRNA (magenta). Scale bars correspond to 100 μm. (D–E) eGFP expression 24 h after transfection with NT-DOPE CADosomes N/P 3, 6, 9 and 12, DOTAP-DOPE N/P 2 and negative controls (CTRL) complexing luciferase-encoding mRNA, expressed as percentage eGFP+ HeLa cells and eGFP rMFI, respectively. (F) Transfection yield (i.e. eGFP expression normalized to intracellular mRNA dose) of NT-DOPE CADosomes N/P 9 compared to DOTAP-DOPE lipoplexes. (G) Representative confocal images of HeLa cells 24 h after transfection with eGFP-mRNA NT-DOPE CADosomes N/P 9, with nuclei (blue) and enhanced green fluorescent protein (eGFP) expression (green). Scale bars correspond to 100 μm. Data are represented as mean ± the standard error of the mean (SEM) for three independent repeats (n = 3). Statistical analysis was performed using One Way Anova with Tukey Correction (ns p > 0.05, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)