Phosphate-enhanced transfection of cationic lipid-complexed mRNA and plasmid DNA

Cationic lipid-mediated gene transfer has been shown to be a competent albeit inefficient mechanism of promoting cellular gene transfer. One way to improve the efficacy of cationic lipid-mediated transgene expression is to optimize conditions for complex formation between the lipids and nucleic acids. In this report we describe the beneficial effects of using phosphate buffer to precondition lipofectin (a 1:1 (w/w) mixture of N-[1-(2,3-dioleyloxy)propyl]-n,n, n-trimethylammonium chloride (DOTMA), and dioleoyl phosphatidylethanolamine (DOPE)) prior to complexing with plasmid DNA or mRNA. Under such optimized conditions we studied the kinetics of DNA- and RNA-mediated transgene expression in a human osteosarcoma cell line (HOS). Preincubation of lipofectin in phosphate buffer resulted in up to 26- and 56-fold increases in luciferase expression from plasmid DNA and mRNA, respectively. Addition of chloroquine (50 microM), which enhanced plasmid-mediated gene delivery 3-fold, was synergistic with phosphate resulting in an additional 46-fold increase in luciferase expression. The preincubation with phosphate shortened both the time required for cellular uptake and the time to achieve maximal transgene expression. Optimal transfection was achieved in the presence of 30-80 mM phosphate, at pH 5.6-6.8 under which the phosphate anion is divalent. The effect of phosphate anion was specific in that monovalent Cl- and acetate anions were not stimulatory. These results demonstrate that divalent phosphate anion plays a stimulatory role during complex formation and transfection when cationic lipids come in contact with negatively charged nucleic acids and cell membranes. These findings delineate specific conditions which dramatically enhance transfection efficiency for both DNA and mRNA, and provide an effective procedure for gene transfection studies.