Effect of cholesterol on the transfection efficiency of DOTAP-containing lipoplexes

D. Pozzi, F. Cardarelli, F. Salomone, C. Marchini, H. Amenitsch, G. Caracciolo
Sapienza University of Rome, IT

Keywords: lipoplexes, cholesterol, transfection efficiency, laser scanning confocal microscopy

Summary:

Here, we have studied the effect of cholesterol on the transfection efficiency (TE) of cationic liposomes made of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and dioleoylphosphocholine (DOPC) in Chinese hamster ovary (CHO) cells. We held the charge of the system constant by keeping the DOTAP content of complexes unchanged (XDOTAP=0.5), and we progressively replaced DOPC molecules by cholesterol molecules. We observed an increase in TE of cholesterol-containing lipoplexes. We asked whether differences in TE could be due differences in the uptake mechanism and/or in the final fate of lipoplexes. To address this issue, laser scanning confocal microscopy (LSCM) experiments were performed on DOTAP–DOPC/DNA and DOTAP–cholesterol/DNA lipoplexes. While DOTAP-DOPC/DNA lipoplexes enter CHO cells exclusively through fluid-phase macropinocytosis, cholesterol-containing lipoplexes use different endocytosis pathways. This finding may suggest that the higher TE of cholesterol-containing lipoplexes is due to the activation of multiple endocytic pathways each of them characterized by their relative cascade of intracellular processes.