title: Cholesterol-dependent macropinocytosis and endosomal escape control the transfection efficiency of lipoplexes in CHO living cells.
abstract: Here we investigate the cellular uptake mechanism and final intracellular fate of two cationic liposome formulations characterized by similar physicochemical properties but very different lipid composition and efficiency for intracellular delivery of DNA. The first formulation is made of cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and the zwitterionic helper dioleoylphosphocholine (DOPC), while the second one is made of the cationic 3β-[N-(N,N-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol) and the zwitterionic lipid dioleoylphosphatidylethanolamine (DOPE). Combining pharmacological and imaging approaches we show that both DOTAP-DOPC/DNA and DC-Chol-DOPE/DNA lipoplexes are taken up in Chinese hamster ovary (CHO) living cells mainly through fluid-phase macropinocytosis. Our results also indicate that lipoplex macropinocytosis is a cholesterol-sensitive uptake mechanism. On the other side, both clathrin-mediated and caveolae-mediated endocytosis play a minor role, if any, in the cell uptake. Colocalization of fluorescently tagged lipoplexes and Lysosensor, a primary lysosome marker, reveals that poorly efficient DOTAP-DOPC/DNA lipoplexes are largely degraded in the lysosomes, while efficient DC-Chol-DOPE/DNA systems can efficiently escape from endosomal compartments.
authors.affiliation: Center for Nanotechnology Innovation @NEST, Istituto Italiano di Tecnologia, Piazza San Silvestro 12, 56127 Pisa, Italy.
authors.forename: Francesco
authors.initials: F
authors.lastname: Cardarelli
authors.forename: Daniela
authors.initials: D
authors.lastname: Pozzi
authors.forename: Angelo
authors.initials: A
authors.lastname: Bifone
authors.forename: Cristina
authors.initials: C
authors.lastname: Marchini
authors.forename: Giulio
authors.initials: G
authors.lastname: Caracciolo
mesh_terms: D000818:Animals; D016466:CHO Cells; D002784:Cholesterol / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism*; D006224:Cricetinae; D003412:Cricetulus; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D011992:Endosomes / Q000737:chemistry / Q000378:metabolism*; D005229:Fatty Acids, Monounsaturated / Q000737:chemistry / Q000378:metabolism; D005456:Fluorescent Dyes; D008081:Liposomes / Q000737:chemistry / Q000378:metabolism*; D018613:Microscopy, Confocal; D010713:Phosphatidylcholines / Q000737:chemistry / Q000378:metabolism; D010714:Phosphatidylethanolamines / Q000737:chemistry / Q000378:metabolism*; D010873:Pinocytosis; D000644:Quaternary Ammonium Compounds / Q000737:chemistry / Q000378:metabolism; D014162:Transfection
chemical_list: D005229:Fatty Acids, Monounsaturated; D005456:Fluorescent Dyes; D008081:Liposomes; D010713:Phosphatidylcholines; D010714:Phosphatidylethanolamines; D000644:Quaternary Ammonium Compounds; C070648:3-(N-(N',N'-dimethylaminoethane)carbamoyl)cholesterol; C094877:dioleoyl phosphatidylethanolamine; D004247:DNA; D002784:Cholesterol; C017251:1,2-oleoylphosphatidylcholine; C070046:1,2-dioleoyloxy-3-(trimethylammonium)propane