Polyethylenimine but not cationic lipid improves antisense activity of 3'-capped phosphodiester oligonucleolides

Lipofectin, which is a mixture of neutral lipid with a cationic lipid, has been widely used to enhance cellular delivery of phosphorothioate, 2'-sugar -modified, and chimeric antisense oligonucleotides, Phosphodiester oligonuc leotides delivered with Lipofectin usually do not elicit antisense activity probably because cationic lipid formulations do not sufficiently protect u nmodified oligonucleotides from nuclease degradation. We show that a cation ic polymer, polyethylenimine (PEI), improves the uptake and antisense activ ity of 3'-capped 20-mer and 12-mer antisense phosphodiester oligonucleotide s (PO-ODN) targeted to different regions of Ha-ras mRNA and to the 3'-untra nslated region (3'-UTR) of C-raf kinase, In contrast, PEI, which forms a ve ry stable complex with the 20-mer phosphorothioate oligonucleotide (PS-ODN) , does not enhance its antisense activity. Using fluorescently labeled carr iers and ODN, we show that PEI-PS-ODN particles are very efficiently taken up by cells but PS-ODN is not dissociated from the carrier. Our results ind icate that carrier-ODN particle size and stability and ODN release kinetics vary with the chemical nature of the ODN and the carrier being transfected into the cells. The very low cost of PEI compared with cytofectins and the increased affinity for target mRNA and decreased affinity for proteins of PO-ODN compared with PS-ODN make the use of PEI-PO-ODN very attractive.