US EPA

7450634 

Journal Article 

Formation of plasmid-based transfection complexes with an acid-labile cationic lipid: characterization of in vitro and in vivo gene transfer 

Boomer, JA; Thompson, DH; Sullivan, SM 

2002 

1 

Pharmaceutical Research
ISSN: 0724-8741
EISSN: 1573-904X 

19 

9 

1292-1301 

English 

12403065 

10.1023/A:1020342523694 

WOS:000178212900007 

PURPOSE: This study tests the hypothesis that gene transfer efficiency may be improved through the use of transiently stable transfection complexes that degrade within endosomal compartments and promote plasmid escape into the cytosol.

METHOD: An acid labile cationic lipid, O-(2R-1,2-di-O-(1'Z, 9'Z-octadecadienyl)-glycerol)-3-N-(bis-2-aminoethyl)-carbamate (BCAT), was designed, synthesized, and tested for enhanced gene transfer activity relative to non-labile controls.

RESULTS: The O-alkenyl chains of BCAT were completely hydrolyzed after 4 h incubation in pH 4.5 buffer at 25 degrees C. Addition of BCAT to plasmid DNA in 40% ethanol followed by ethanol evaporation yielded transfection complexes that transfected several cell types in the presence of fetal calf serum and without the need of a helper lipid. Transfection complexes prepared from BCAT displayed higher luciferase expression than the corresponding DCAT complexes (an acid-insensitive derivative of BCAT) for all cell types tested. Uptake studies showed that this increase was not due to a difference in the amount of DNA being delivered. FAGS analysis for GFP expression showed that BCAT transfection complexes yielded 1.6 more transfected cells and 20% higher log mean fluorescence than DCAT transfection complexes. In vivo gene transfer was demonstrated in subcutaneous tumor-bearing mice by systemic administration of a 60 microg plasmid dose. Expression was observed in the lungs and in the tumor, with the highest activity being observed in the lungs.

CONCLUSIONS: Our results show that increased transfection can be obtained by coupling the cationic headgroup to the hydrophobic amphiphilic tails via acid-labile bonds. Acid-catalyzed release of the alkyl chains should facilitate dissociation of the cationic lipid headgroup from the plasmid, thus accelerating one of the rate-limiting steps in cationic lipid mediated transfection. 

Acid-sensitive lipid; Cytoplasmic delivery; Endosomal escape; Gene transfer; Transfection; Vinyl ether hydrolysis; alcohol; chloramphenicol acetyltransferase; drug carrier; lipid; o (1,2 di o (9' octadecenyl) glycerol) 3 n (bis 2 aminoethyl) carbamate; o [1,2 di o (1', 9' octadecadienyl) glycerol] 3 n (bis 2 aminoethyl) carbamate; plasmid DNA; plasmid vector; unclassified drug; animal cell; animal experiment; animal model; article; catalysis; cell type; complex formation; controlled study; degradation; dissociation; drug design; drug formulation; drug synthesis; endosome; evaporation; fetal calf serum; fluorescence; gene expression; gene targeting; gene transfer; genetic transfection; human; human cell; hydrolysis; hydrophobicity; in vitro study; in vivo study; lung; mouse; nonhuman; nonviral gene delivery system; pH; priority journal; 3T3 Cells; Acids; Animals; Cations; Cell Line; Chemistry, Pharmaceutical; Gene Transfer Techniques; Humans; Lipids; Mice; Mice, Inbred C3H; Plasmids; Transfection