Diquaternary ammonium compounds as transfection agents

Diquaternary ammonium salts constitute a new class of reagent for mediating transfection of DNA in mammalian cell lines. N,N'-dioleyl-N,N,N',N'-tetram ethyl-1,2-ethanediamine (TmedEce), N,N' dioleyl-N,N,N',N'-tetramethyl-1,3-p ropanediamine (PropEce), N,N'-dioleyl-N,N,N',N'-tetramethyl-1,6-hexanediami ne (HexEce), and their corresponding N,N'-dicetyl saturated analogues (Tmed Ace, PropAce and HexAce) have all been synthesized and characterized. They were prepared via a bis-Menshutkin reaction of the corresponding tetramethy ldiamine with 2.2 M equiv of a long-chain alkyl halide (saturated or unsatu rated). The reaction was run in anhydrous acetonitrile for ca. 3 days at 60 degreesC, which produced the diquaternary ammonium halides in good to near ly quantitative yields for most derivatives. DNA transfection comparable to commercially available reagents such as Lipofectin, Lipofectace, Lipofecta mine, and O-ethyldioleoylphosphatidylcholinium triflate has been achieved i n vitro with these new reagents. There was no need to use a colipid for eff ective transfection, but serum did significantly inhibit transfection. The saturated and the unsaturated derivatives differed with respect to hydratio n behavior. The saturated derivatives appeared to retain a lamellar-type cr ystalline array structure upon hydration, whereas the unsaturated versions formed micelles and/or liposomes, depending on the ionic strength: HexEce w as micellar in both water and saline; PropEce was micellar in water but lam ellar in saline; and TmedEce was lamellar in both. Despite these different hydration patterns, all of these unsaturated derivatives formed productive transfection complexes with DNA. Varying the distance between the quaternar y sites affected transfection efficacy in the order HexAce > TmedAce = Prop Ace for the saturated derivatives and in the order PropEce = HexEce > TmedE ce, with a smaller spread, for the unsaturated derivatives.