The role of endosome destabilizing activity in the gene transfer process mediated by cationic lipids.
We used a 32P-labeled pCMV-CAT plasmid DNA to estimate the DNA uptake efficiency and unlabeled pCMV-CAT plasmid DNA to quantify the CAT activity after transfection of COS cells using each of the three following cationic compounds: [1] vectamidine (3-tetradecylamino-N-tert-butyl-N'-tetradecylpropionamidine, and previously described as diC14-amidine [1]), [2] lipofectin (a 1:1 mixture of N-(1-2,3-dioleyloxypropyl)-N,N,N-triethylammonium (DOTMA) and dioleylphosphatidylethanolamine (DOPE)), and [3] DMRIE-C (a 1:1 mixture of N-[1-(2,3-dimyristyloxy)propyl]-N,N-dimethyl-N-(2-hydroxyethyl) ammonium bromide (DMRIE) and cholesterol). Surprisingly, a high CAT activity was observed with vectamidine although the DNA uptake efficiency was lower as compared to lipofectin and DMRIE-C. Transmission electron microscopy (TEM) revealed endocytosis as the major pathway of DNA-cationic lipid complex entry into COS cells for the three cationic lipids. However, the endosomal membrane in contact with complexes containing vectamidine or DMRIE-C often exhibited a disrupted morphology. This disruption of endosomes was much less frequently observed with the DNA-lipofectin complexes. This comparison of the three compounds demonstrate that efficient transfection mediated by cationic lipids is not only correlated to their percentage of uptake but also to their ability to destabilize and escape from endosomes.
El Ouahabi
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A
Laboratoire de Chimie-Physique des Macromolécules aux Interfaces (LCPMI), Université Libre de Bruxelles, Brussels, Belgium. aelouaha@dbm.ulb.ac.be
Thiry
M
M
Pector
V
V
Fuks
R
R
Ruysschaert
J M
JM
Vandenbranden
M
M
D000578:Amidines; D000818:Animals; D019556:COS Cells; D002462:Cell Membrane / Q000502:physiology / Q000648:ultrastructure; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis; D003587:Cytomegalovirus / Q000235:genetics; D011992:Endosomes / Q000502:physiology* / Q000648:ultrastructure; D008055:Lipids; D008081:Liposomes; D010714:Phosphatidylethanolamines; D010957:Plasmids; D000644:Quaternary Ammonium Compounds; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D014162:Transfection / Q000379:methods*
D000578:Amidines; D008055:Lipids; D008081:Liposomes; C089665:N-t-butyl-n'-tetradecyl-3-tetradecylaminopropionamidine; D010714:Phosphatidylethanolamines; D000644:Quaternary Ammonium Compounds; D011993:Recombinant Fusion Proteins; C087276:(3-dimyristyloxypropyl)(dimethyl)(hydroxyethyl)ammonium; C020888:1,2-dielaidoylphosphatidylethanolamine; D015500:Chloramphenicol O-Acetyltransferase