Internalisation of mRNA-lipoplexes by dendritic cells

Lipoplexes, composed of Lipofectamine and mRNA encoding HIV Gag protein, were shown to be internalised by dendritic cells (DCs) and promote antigen presentation to stimulate HIV-specific T cell responses. Using confocal microscopy, we showed that one third of fluorescently labelled mRNA containing lipoplexes are co-localised with late endosomes. We further investigated the effect of inhibitors, blocking phagocytosis, macropinocytosis, clathrin- and caveolae- mediated endocytosis, on both the internalisation of the lipoplexes by DCs and the transfection efficiency. We observed that chloropromazine had no effect on the cellular uptake or transfection efficiency, excluding the involvement of clathrin-mediated endocytosis. Cytochalasine D, inhibiting macropinocytosis and phagocystosis, strongly reduced internalisation (50%) of the lipoplexes as well as protein expression (70%). Amiloride, which should specifically block macropinocytosis, induced only a modest reduction of uptake and transfection. Genistein and dynasore induced a strong reduction of on the level of protein expression (>70%), but not the overall uptake. Our results indicate that transfection-effective mRNA lipoplex internalisation by DCs, i.e. uptake that results in protein expression, preferentially proceeds by macropinocytosis and/or phagocytosis.