Diquaternary Ammonium Compounds as Transfection Agents

Abstract

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'-tetramethyl-1,2-ethanediamine (TmedEce), N,N'-dioleyl-N,N,N',N'-tetramethyl-1,3-propanediamine (PropEce), N,N'-dioleyl-N,N,N',N'-tetramethyl-1,6-hexanediamine (HexEce), and their corresponding N,N'-dicetyl saturated analogues (TmedAce, PropAce and HexAce) have all been synthesized and characterized. They were prepared via a bis-Menshutkin reaction of the corresponding tetramethyldiamine with 2.2 M equiv of a long-chain alkyl halide (saturated or unsaturated). The reaction was run in anhydrous acetonitrile for ca. 3 days at 60 degrees C, which produced the diquaternary ammonium halides in good to nearly quantitative yields for most derivatives. DNA transfection comparable to commercially available reagents such as Lipofectin, Lipofectace, Lipofectamine, and O-ethyldioleoylphosphatidylcholinium triflate has been achieved in vitro with these new reagents. There was no need to use a colipid for effective transfection, but serum did significantly inhibit transfection. The saturated and the unsaturated derivatives differed with respect to hydration behavior. The saturated derivatives appeared to retain a lamellar-type crystalline array structure upon hydration, whereas the unsaturated versions formed micelles and/or liposomes, depending on the ionic strength: HexEce was micellar in both water and saline; PropEce was micellar in water but lamellar 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 quaternary sites affected transfection efficacy in the order HexAce > TmedAce = PropAce for the saturated derivatives and in the order PropEce = HexEce > TmedEce, with a smaller spread, for the unsaturated derivatives.

In vivo cutaneous interferon‐γ gene delivery using novel dicationic (gemini) surfactant–plasmid complexes

“…Rosenzweig et al [40] found that certain 16-n-16 series of diquaternary ammonium compounds were capable of transfecting BHK-21 hamster kidney cells in vitro in the absence of DOPE. Transfection efficiency was highest for an intermediate spacer length 16-6-16; shorter spacers (2 or 3) were 5-10-fold less effective.…”

“…DOPE inhibited transfection by the 16-2-16 gemini surfactant even at low amounts (2 : 1 gemini/DOPE ratio). The presence of serum also inhibited transfection efficiency by these gemini surfactants [40] leading to concerns about their utility in systemic gene therapy. However, our results in vivo indicate that this should not be a limiting factor for topical application.…”

Review on chemically bonded geminis with cationic heads: second-generation interfactants

“…the transport of genetic material through biological membranes into eukaryotic cells, are characteristic applications of green gemini surfactants [67,68]. Certain cationic gemini surfactants with low toxicity have a superior ability to introduce genes into cells [69][70][71][72]. pH regulation has profound effects on surface adsorption and aggregation behavior of gemini surfactants [73].…”

Structural, biocomplexation and gene delivery properties of hydroxyethylated gemini surfactants with varied spacer length

“…The use of the so-called nonviral vectors [4] makes it possible to solve problems connected with the condensation and compaction of giant DNA polyanions, with such limitations of viral vectors as immune response avoided. Although the application of synthetic vectors including those based on cationic surfactants and lipids is well documented [5][6][7], it is yet a challenging problem due to their relatively lower transfection efficacy compared to viral carriers. One of the main trends in the development of current protocols is the design of carriers with multicentered interactions with phosphate anions of DNA, including those based on gemini surfactants [8][9][10][11].…”