Comparative gene transfer between cationic and thiourea lipoplexes.
BACKGROUND
We have previously developed lipopolythiourea lipids as neutral DNA condensing agents for systemic gene delivery. Optimization of the lipopolythiourea structure led to efficient transfecting agents. To further evaluate these lipids, we investigated the internalization process of the thiourea lipoplexes and their intracellular mechanism of transfection versus that of cationic lipoplexes.


METHODS
The MTT test was used for cytotoxicity assessment. Transfection efficiency was determined by luciferase read-out. Permeation to propidium iodide and enhanced green fluorescent protein was evaluated by flow cytometry. Kinetics of internalization and DNA release were monitored by confocal microscopy with labelled DNA. Endocytosis inhibitors were used to study the mechanisms of lipoplex internalization.


RESULTS
Although thiourea/DNA complexes exhibit an almost similar level of transfection compared to that of cationic complexes, the thiourea lipoplexes were shown to be six-fold less internalized. Complexes were able to permeabilize the cytoplasmic membrane to 30 kDa molecules. Finally, DNA was shown to be released in less than 10 min in the cellular cytoplasm versus 30 min for cationic lipoplexes.


CONCLUSIONS
Despite a weaker internalization compared to cationic lipids, the thiourea lipoplexes were able to transfect cells at a similar level as a result of its greater ability to destabilize the cytoplasmic membrane and release DNA.
Breton
Marie
M
Inserm, U640, CNRS, UMR8151, Unité de Pharmacologie Chimique et Génétique, Université Paris Descartes, Faculté de Pharmacie, Paris, France.
Leblond
Jeanne
J
Seguin
Johanne
J
Midoux
Patrick
P
Scherman
Daniel
D
Herscovici
Jean
J
Pichon
Chantal
C
Mignet
Nathalie
N
D000818:Animals; D001692:Biological Transport; D002412:Cations; D002460:Cell Line; D004705:Endocytosis; D005453:Fluorescence; D018014:Gene Transfer Techniques; D006801:Humans; D042541:Intracellular Space / Q000378:metabolism; D007700:Kinetics; D008055:Lipids / Q000737:chemistry*; D051379:Mice; D018613:Microscopy, Confocal; D010539:Permeability; D010957:Plasmids / Q000235:genetics; D013696:Temperature; D013890:Thiourea / Q000737:chemistry*; D014162:Transfection
D002412:Cations; D008055:Lipids; D013890:Thiourea