Fig. 7

Investigation of the gene delivery process in HEK293T cells. A + B Cellular uptake of polyplexes in HEK293T cells. Cells were incubated with (layered) polyplexes of polymers and YOYO-1-labeled pDNA at N*/P 30 for different time periods and analyzed via flow cytometry. Cells incubated with labeled pDNA served as control (rMFI = 1). Values represent mean ± SD of A viable, single YOYO-1 positive cells and B rFMI of all viable, single cells relative to cells treated with YOYO-1 labeled pDNA only. ^#/##/###Significant difference to LPEI at respective time point (p < 0.05/0.01/0.001), */**/***significant difference to same polymer after 24 h (p < 0.05/0.01/0.001). C + D Endosomal escape was analyzed via CLSM following simultaneous incubation with the non-permeable dye calcein (green) and (layered) polyplexes of polymers and pDNA at N*/P 30 (not stained) for 1 h. The cell nuclei were stained with Hoechst 33342 (blue). Values in (C) were obtained by image analysis of all acquired images using ImageJ and represent mean ± SD (n = 3) of the number of cells with extensive green fluorescence relative to the total amount of cells (Hoechst-stained nuclei). **/***Significant difference to HC-mic (p < 0.01/0.001). Shaded columns represent the proportion of cells with calcein release divided by the proportion of YOYO-1 positive cells at the same time point (in percent). Green dots in (D) indicate calcein within cellular compartments, whereas a diffuse green fluorescence pattern indicates calcein released to the cytosol