Formation of Novel Hydrophobic Complexes between Cationic Lipids and Plasmid DNA

Abstract

An ability to generate a well defined lipid-based carrier system for the delivery of plasmid DNA in vivo requires the characterization of factors governing DNA/lipid interactions and carrier formation. We report that a hydrophobic DNA/lipid complex can be formed following addition of cationic lipids to DNA in a Bligh and Dyer monophase consisting of chloroform/methanol/water (1:2.1:1). Subsequent partitioning of the monophase into a two-phase system allows for the extraction of DNA into the organic phase. When using monovalent cationic lipids, such as dimethyldioctadecylammonium bromide, dioleyldimethylammonium chloride, and 1,2-dioleyl-3-N,N,N-trimethylaminopropane chloride, greater than 95% of the DNA present can be recovered in the organic phase when the lipid is added at concentrations sufficient to neutralize DNA phosphate charge. When the polyvalent cationic lipids 2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl- 1- propanaminium trifluoroacetate and diheptadecylamidoglycyl spermidine are used, efficient extraction of the DNA into the organic phase is also achieved when the charge ratio between lipid and DNA is approximately equal. Formation of the hydrophobic DNA complex can only be achieved with cationic lipids. In the absence of added cations or in the presence of excess Ca2+, L-lysine, or poly(L-lysine), 100% of the DNA is recovered in the aqueous fraction. The monovalent cationic lipid/DNA complexes can also be prepared in the presence of detergent; however, low concentrations of NaCl (< 1 mM) lead to dissociation of the complex. Importantly, these results clearly demonstrate that cationic lipid binding does not lead to DNA condensation. The methods described, therefore, enable DNA/lipid complexes to be characterized in the absence of DNA condensation.(ABSTRACT TRUNCATED AT 250 WORDS)

Stabilized plasmid-lipid particles: construction and characterization

“…11 It is of interest to determine whether this hydrophobic particle can be surrounded by an outer monolayer of lipid, which would then result in small, plasmid-containing particles stabilized in an aqueous medium. Detergent dialysis is a logical technique for achieving this, as the detergent may be expected to solubilize the hydrophobic plasmid DNAcationic lipid particles.…”

A novel, long-circulating, and functional liposomal formulation of antisense oligodeoxynucleotides targeted against MDR1

“…The use of a Bligh-Dyer monophase to extract plasmid DNA into an organic phase using cationic lipid has been reported previously. 19,27 PC40, Chol, and PEG-DSPE, all in CHCl 3 , were added to the organic phase to give a molar ratio of PC40 to Chol to DOTAP to PEG-DSPE of 3:2:1:0.2. ddH 2 O was added to give a PL concentration of 20 -30 mM (in ddH 2 O), and this mixture was vortexed and then sonicated for ϳ1 minute to form an emulsion. The organic phase was then evaporated by rotary evaporation at ϳ500 mm Hg.…”

“…administration. A Bligh and Dyer 18 extraction procedure was used by Reimer et al 19 to evaluate the formation of hydrophobic complexes between cationic lipid and plasmid DNA. These complexes exist in an organic solvent (CHCl 3 ), and therefore will not be suitable for transfection; however, they may serve as useful intermediates in the formation of liposomal DNA carriers.…”

“…These complexes exist in an organic solvent (CHCl 3 ), and therefore will not be suitable for transfection; however, they may serve as useful intermediates in the formation of liposomal DNA carriers. 19 Because the complexes form as a result of the electrostatic interaction between the cationic lipid and the DNA, this procedure may also be used for the extraction of phosphorothioate asODN. We hypothesized that the hydrophobic cationic lipid-asODN particles could be coated with neutral phospholipids (PLs) and lipid derivatives of polyethylene glycol (PEG) through a reverse phase evaporation procedure similar to that described by Szoka and Papahadjopoulos.…”

Protamine sulfate enhances lipid-mediated gene transfer

“…The most critical obstacles to transgene expression. [5][6][7][8][9][10][11][12][13][14][15] step, which appears to be rate limiting and needs to be Recently, much work has focused on efforts to increase addressed, is the efficient delivery of the gene from the the level of transfection efficiency. New lipid formucytoplasm into the nucleus.…”