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Cholesterol-Dependent Macropinocytosis and Endosomal Escape Control the Transfection Efficiency of Lipoplexes in CHO Living Cells
journal contribution
posted on 2012-02-06, 00:00 authored by Francesco Cardarelli, Daniela Pozzi, Angelo Bifone, Cristina Marchini, Giulio CaraccioloHere 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.
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Keywords
lipid compositioncationic liposome formulationsimaging approachesDOTAPintracellular deliveryzwitterionic lipid dioleoylphosphatidylethanolaminezwitterionic helper dioleoylphosphocholineDNAintracellular fateuptake mechanismTransfection Efficiencyphysicochemical propertiesChinese hamster ovarycell uptakeendosomal compartmentsDOPEDOPClysosome markerlipoplexeEndosomal Escape ControlCHO Living CellsHerelipoplex macropinocytosis
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