Cationic lipids derived from glycine betaine promote efficient and non-toxic gene transfection in cultured hepatocytes.
BACKGROUND
The low efficiency and toxicity of transfection in a primary culture of hepatocytes using cationic lipids remains a limiting step to the study of gene function and the setting up of non-viral gene therapy.


METHODS
A novel class of cationic lipids (GBs) derived from natural glycine betaine compounds covalently linked to acyl chains by enzymatically hydrolysable peptide and ester bonds, a structure designed to reduce cytotoxicity, was used to improve transfection efficiency in a primary culture of rat hepatocytes. The relationship between lipid structure, lipoplex formulation and transfection efficiency was studied using six GBs (12-14-16, 22-24-26) varying in their spacer and acyl chains.


RESULTS
GB12, characterized by short [(CH(2))(10)] acyl chains and spacer, allowed plasmid uptake in all cells and reporter gene expression in up to 40% of hepatocytes with a low cytotoxicity, a much higher efficiency compared with transfections using other reagents including Fugene6 and Lipofectin. We also showed that numerous cells accumulated high amounts of plasmids demonstrating that GB12 promoted a very efficient DNA transfer through plasma membrane leading to an increase in nuclear plasmid translocation, allowing a much higher gene expression. Moreover, GB12-transfected hepatocytes survived to injection in normal livers and were found to express the LacZ reporter gene.


CONCLUSIONS
The non-toxic GB12 formulation is a powerful vehicle for plasmid delivery in cultured hepatocytes with relevance in liver gene therapy.
Gilot
David
D
Hôpital Pontchaillou, Régulation des Equilibres fonctionnels du foie Normal et Pathologique, INSERM U522, Avenue de la Bataille Flandre/Dunkerque, 35033 Rennes, France. david.gilot@rennes.inserm.fr
Miramon
Marie-Laure
ML
Benvegnu
Thierry
T
Ferrieres
Vincent
V
Loreal
Olivier
O
Guguen-Guillouzo
Christiane
C
Plusquellec
Daniel
D
Loyer
Pascal
P
D000818:Animals; D001622:Betaine; D002478:Cells, Cultured; D022781:Hepatocytes / Q000502:physiology*; D008055:Lipids; D008297:Male; D010714:Phosphatidylethanolamines; D051381:Rats; D011917:Rats, Inbred Lew; D014162:Transfection / Q000379:methods*
C410262:1,2-dioleoyl-glycero-3-phosphatidyl ethanolamine; D008055:Lipids; D010714:Phosphatidylethanolamines; D001622:Betaine