Optimization and characterization of anionic lipoplexes for gene delivery.
Anionic lipoplexes, comprising divalent cations, DNA and anionic liposomes, were optimized for high transfection efficiency and low cytotoxicity. Different molar ratios of anionic to zwitterionic lipid, 1:9 to 1:1 (1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DOPG): 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), various cations (Ca2+, Mg2+ and Na+) and different anionic lipid/DNA ratios were investigated. The optimized formulation was composed of: anionic/zwitterionic lipid molar ratio 1:4 (DOPG:DOPE); 15-20 mM Ca2+; and 15-20 microg lipid for complexation with 0.8 microg plasmid DNA. Biophysical studies (particle size analysis, gel electrophoresis, transmission electron microscopy (TEM), and confocal microscopy) were conducted to characterize the different formulations. TEM revealed structural differences between the complexed and uncomplexed lipoplexes. Gel electrophoresis confirmed the formation of anionic lipoplexes with the amount of free DNA minimized for the optimized formulation. Confocal imaging showed cellular uptake of the anionic lipoplexes. Most significantly the anionic lipoplex formulation, optimized for the highest transfection efficiency (approximately 78% in the presence of serum) exhibited the highest cell viability (approximately 93%, (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide) MTT assay). This was compared to Lipofectamine 2000 which had a transfection efficiency and cell viability of approximately 68% and 35%, respectively. The anionic lipoplex formulation developed here shows promise as a non-viral vector with high transfection efficiency and low cytotoxicity.
Srinivasan
Charudharshini
C
Department of Pharmaceutical Sciences, University of Connecticut, 69 North Eagleville Road, Unit 3092, Storrs, CT 06269, USA. csrinivasan@idi.harvard.edu
Burgess
Diane J
DJ