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Polycation liposomes as a vector for potential intracellular delivery of microRNA
Author(s) -
Ando Hidenori,
Okamoto Ayaka,
Yokota Masafumi,
Asai Tomohiro,
Dewa Takehisa,
Oku Naoto
Publication year - 2013
Publication title -
the journal of gene medicine
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.689
H-Index - 91
eISSN - 1521-2254
pISSN - 1099-498X
DOI - 10.1002/jgm.2744
Subject(s) - endosome , endocytosis , cytoplasm , intracellular , microbiology and biotechnology , transfection , liposome , gene silencing , chemistry , biophysics , biology , cell , biochemistry , gene
Abstract 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. Copyright © 2013 John Wiley & Sons, Ltd.