Polycation liposomes as a vector for potential intracellular delivery of microRNA.

BACKGROUND: We previously developed a microRNA (miRNA) delivery system by using dicetyl phosphate-tetraethylenepentamine-based polycation liposomes (TEPA-PCL), applied it to miR-92a delivery, and demonstrated its gene-silencing potential and effective anti-angiogenic effects. In the present study, we investigated the mechanism of intracellular delivery of cholesterol-grafted miR-92a (miR-92a-C) into cells.

METHODS: To investigate the intracellular distribution of miR-92a-C/TEPA-PCL complex, we used human umbilical vein endothelial cells and examined certain points after transfection: (i) the time-course of miR-92a-uptake into the cells; (ii) the endocytosis pathway induced by miR-92a-C/TEPA-PCL; (iii) the capability of miR-92a-C/TEPA-PCL to escape from the endosomes; and (iv) the release of miR-92a-C from TEPA-PCL in the cytoplasm.

RESULTS: Our data indicated that miR-92a-C formulated in TEPA-PCL accumulated in and was spread throughout the cytoplasm in a time-dependent manner, and was taken up into the cells by macropinosome-mediated endocytosis. In addition, the surface charge of miR-92a-C/TEPA-PCL was neutral at pH 7.4 and was charged positively at around pH 5.5, which is the inner pH of endosomes. When the late endosomes/lysosomes were stained with Lysotracker, miR-92a-C/TEPA-PCL efficiently escaped from the endosomes into the cytoplasm, possibly through the proton-sponge effect. Furthermore, miR-92a-C spread throughout whole cytoplasm and did not co-localize completely with TEPA-PCL, indicating that some of the miR-92a-C was present in free form in the cytoplasm.

CONCLUSIONS: The results of the present study suggest that TEPA-PCL-based lipoplexes have an excellent potential to deliver microRNAs into the cytoplasm of cells and to induce RNA silencing action mediated by microRNAs and other small RNAs.