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Bioconjugate Chem. 2001, 12, 258−263

Diquaternary Ammonium Compounds as Transfection Agents Howard S. Rosenzweig, Vera A. Rakhmanova, and Robert C. MacDonald* From the Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, Illinois 60208. Received August 10, 2000; Revised Manuscript Received January 17, 2001

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-1,6hexanediamine (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 °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.

INTRODUCTION

The in vitro introduction of plasmid DNA into mammalian cells (transfection) for transient expression of a variety of desired proteins has become a standard technique in molecular biology. Earlier methods involved the use of DNA coprecipitation with calcium phosphate, soluble DEAE or Polybrene resins (1), as well as electroporation (2). More recently, the use of cationic amphiphiles or more simply “cationic lipids” for mediating DNA transfection has become widespread. This trend was initiated by the demonstration of Felgner et. al. that the cationic lipid, DOTMA (dioleoloxypropyl-trimethylammonium bromide), was much faster, simpler, cheaper, and generally more effective than alternative methods in transfecting DNA into cultured cells (3). Since that time, there have been additional reports of different cationic lipids with transfection activity (e.g., refs 4-11). One of the potentially most important applications of cationic lipids is as DNA delivery agents for gene therapy (12). Another potential clinical application is delivery of antisense oligonucleotides (13). Other molecules can be delivered to cells as cationic complexes, such as a transcription regulator protein (14), a viral trans-activator protein (15), and a mushroom toxin (16) and it is likely that additional applications will be developed. Our investigation was prompted by the fact that dimethyldioctadecylammonium bromide, when formulated with an equimolar amount of dioleoylphosphatidylethanolamine (DOPE), was shown to be quite an * To whom correspondence should be addressed. Phone: (847) 491-5062. Fax: (847) 467-1380. E-mail: [email protected].

effective DNA transfection agent (6). (This mixture is marketed by Life Technologies under the trade name Lipofectace.) It appeared possible that introduction of a second quaternary ammonium group could increase the strength of interaction with DNA and perhaps generate an improved transfection agent. We thus sought a synthetic route to generate a molecule with two dimethylammonium groups, and two long-chain alkyl groups. The route we chose involved alkylating tetramethyldiamines with long-chain alkyl halides; it was found to provide convenient and efficient access to dialkyltetramethyldiquaternary ammonium transfection agents. EXPERIMENTAL PROCEDURES

Materials. Chemical reagents and solvents were normally purchased either from Aldrich Chemical Co. (Milwaukee, WI) or Sigma Chemical Co. (St. Louis, MO) and were used without further purification. BHK-21 cells (Syrian or Golden Hamster) were obtained from American Type Culture Collection (Rockville, MD). Tissue culture reagents were purchased from Life Technologies (Grand Island, NY). The DNA plasmid with the β-galactosidase gene driven by the cytomegalovirus promoter (pCMV-β-gal) was also purchased from Life Technologies. It was propagated in DH5 cells and purified with a Plasmid Mega Kit from Qiagen (Valencia, CA). Synthetic Methods for Diquaternary Ammonium Salts. To an oven-dried and desicated 1.2 × 2.5 cm screw cap vial was added anhydrous acetonitrile (1 mL), followed by either tetramethylethylenediamine, tetramethyl-1,3-propanediamine, or tetramethyl-1,6-hexanediamine (300 µmol), and finally by the long-chain alkyl halide (cetyl iodide or oleyl bromide, 665 µmol), and the

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DNA Transfection with Diquaternary Ammonium Salts

vial was sealed tightly. The reaction vial was then placed in a dry heating block at 60 °C, covered with aluminum foil, and allowed to react for ca. 3 days. In the case of the saturated analogues TmedAce and HexAce, significant precipitation of product was observed when the reaction mixture was heated, while PropAce only precipited after it cooled. After cooling, the solid was triturated with absolute ethanol and then suction filtered. The solid was dried under high vacuum and weighed. Yields were 60-84% for TmedAce and HexAce and 17% for PropAce. The unsaturated analogues did not precipitate in the reaction mixture until they cooled to RT. Their workup was modified due to their different solubility characteristics (soluble in absolute ethanol and chloroform). The acetonitrile above the solid was first decanted away, and then the residue dissolved in 1-2 mL of chloroform. The latter solution was transferred to a culture tube, and the bulk of the volatiles were removed with a stream of argon under warming. The solid residue was redissolved in 3-6 mL of chloroform and subjected to silica gel chromatography. Typically, the CHCl3 solution containing the crude product was applied to a dry silica gel column of g2.5 mL. The column was washed with ca. 5 vol of CHCl3, followed by 9:1 CHCl3:CH3OH, at which point the bulk of the product eluted from the column. After collection of six 6-mL fractions, the remaining product was eluted with 5:1 and then 4:1 CHCl3: CH3OH (three fractions each). The desired product has an Rf value of ca. 0.57 when run on an analytical silica gel TLC plate (in CHCl3:CH3OH:acetic acid, 65:25:4). The yield for TmedEce was 19%, while the yields for PropEce and HexEce were essentially quantitative. Synthesis of Dimethyl-2-dimethylaminoethylcetylammonium Iodide (Quatamine-1). To an oven-dried and desicated 1.2 × 2.5 cm screw cap vial was added anhydrous acetonitrile (1 mL) followed by tetramethylethylenediamine (35 mg, 302 µmol) and finally cetyl iodide (106 mg, 301 µmol), and the vial was sealed tightly. The vial was then placed in a dry heating block at 60 °C, covered with aluminum foil, and then allowed to react overnight. After this time, the sample was cooled to room temperature, and a small precipitate was removed by gravity-filtration through Celite (ca. 2 cm). The bulk of the volatiles were evaporated away with a gentle stream of argon (while warming in a water bath), yielding a light orange-yellow residue. Unreacted tetramethylethylenediamine was removed by extraction as follows. The mixture was dissolved in chloroform (5 mL), which solution was then transferred to a 50 mL graduated cylinder. Methanol (2.5 mL) was added, followed by 10% HCl (3 mL). After mixing, the lower organic layer was removed and gravity-filtered through granular sodium sulfate. It was then transferred to a tared vial, and the bulk of the volatiles removed as described above. The sample was then placed under high vacuum for 35 min and weighed. Fifty milligrams (35% Yield) of very pure monoquaternerized salt (as determined by 1H NMR) was obtained. Product Characterization. Liquid secondary ion mass spectra were obtained on a VG70 mass spectrometer (VG Analytical) with Cs ion as a primary source. Proton NMR spectra were obtained on a Varian Gemini 300 MHz spectrometer, using tetramethylsilane as an internal reference, and chemical shifts are given in δ (ppm). By TLC and proton NMR, all of the diquaternary ammonium compounds were found to be pure. No evidence of starting materials or monoquaternary interme-

Bioconjugate Chem., Vol. 12, No. 2, 2001 259

diate was observed in the proton NMR spectra or analytical TLC of the purified products. Spectral Data (all 1H NMR spectra were obtained at 300 MHz). TmedAce, in deuteriochloroform:methanold4 [1:1]: δ 4.21 (singlet, 4H, internal methylenes adjacent to quaternary nitrogens), 3.57 (broad triplet, J ) 8 Hz, 4H, external methylenes adjacent to quaternary nitrogens), 3.32 (singlet, 12H, quaternary methyl groups), 1.84 (broad singlet, 4H), 1.5-1.2 (multiplet, 52H), 0.89 (triplet, J ) 7 Hz, 6H, terminal methyl groups). MS: 552 (M+ methyl). PropAce, in deuteriochloroform:methanol-d4: δ 3.57 (broad triplet, J ) 8 Hz, 4H, internal methylenes adjacent to quaternary nitrogens), 3.46 (broad triplet, J ) 8 Hz, 4H, external methylenes adjacent to quaternary nitrogens), 3.23 (singlet, 12H, quaternary methyl groups), 2.41 (broad singlet, 2H, central internal methylene), 1.83 (broad singlet, 4H), 1.5-1.2 (multiplet, 52H), 0.89 (triplet, J ) 7 Hz, 6H, terminal methyl groups). MS: 708 (M+ + I-), 566 (M+ - methyl). HexAce, in deuteriochloroform:methanol-d4: δ 3.44 (broad triplet, J ) 8 Hz, 4H, internal methylenes adjacent to quaternary nitrogens), 3.36 (broad triplet, J ) 8 Hz, 4H, external methylenes adjacent to quaternary nitrogens), 3.14 (singlet, 12H, quaternary methyl groups), 1.95-1.7 (multiplet, 8H), 1.55 (broad singlet, 4H), 1.481.2 (multiplet, 52H), 0.89 (triplet, J ) 7 Hz, 6H, terminal methyl groups). MS: 750 (M+), 608 (M+ - methyl). TmedEce, in deuteriochloroform: δ 5.38-5.3 (overlapping triplets, J ) 6 Hz, 4H, vinyl), 4.77 (singlet, 4H, internal methylenes adjacent to quaternary nitrogens), 3.71 (broad triplet, J ) 8 Hz, 4H, external methylenes adjacent to quaternary nitrogens), 3.50 (singlet, 12H, quaternary methyl groups), 2.06-1.92 (multiplet, 8H, allylic), 1.92-1.70 (multiplet, 12H), 1.5-1.2 (multiplet, 36H), 0.88 (triplet, J ) 7 Hz, 6H, terminal methyl groups). MS: 697/699 (M+ + Br-), 604 (M+ - methyl). PropEce, deuteriochloroform: δ 5.38-5.3 (overlapping triplets, J ) 6 Hz, 4H, vinyl), 3.92 (broad triplet, J ) 8 Hz, 4H, internal methylenes adjacent to quaternary nitrogens), 3.48 (broad triplet, J ) 8 Hz, 4H, external methylenes adjacent to quaternary nitrogens), 3.36 (singlet, 12H, quaternary methyl groups), 2.83-2.7 (multiplet, 2H, central internal methylene), 2.06-1.92 (multiplet, 8H, allylic), 1.92-1.70 (multiplet, 12H), 1.46-1.2 (multiplet, 36H), 0.88 (triplet, J ) 7 Hz, 6H, terminal methyl groups). MS: 711/713 (M+ + Br-), 618 (M+ methyl). HexEce, deuteriochloroform: δ 5.38-5.3 (overlapping triplets, J ) 6 Hz, 4H, vinyl), 3.80-3.65 (multiplet, 4H, internal methylenes adjacent to quaternary nitrogens), 3.50-3.29 (overlapping broad triplet and singlet, 16H, external methylenes adjacent to quaternary nitrogens and quaternary methyl groups), 2.06-1.92 (multiplet, 8H, allylic), 1.81-1.55 (multiplet, 12H), 1.42-1.20 (multiplet, 44H), 0.88 (triplet, J ) 7 Hz, 6H, terminal methyl groups). MS: 753/755 (M+ + Br-), 660 (M+ - methyl). Quatamine-1, deuteriochloroform: δ 4.40-4.30 (broad singlet, 2H, internal methylene adjacent to quaternary nitrogen), 4.20-4.10 (broad singlet, 2H, external methylene adjacent to quaternary nitrogen), 3.60-3.50 (multiplet, 2H, methylene adjacent to dimethylamino-group), 3.45 (broad singlet, 6H, quaternary methyl groups), 3.13 (broad singlet, 6H, dimethylamino-group), 1.88-1.74 (broad singlet, 2H), 1.40-1.16 (multiplet, 26H), 0.88 (triplet, J ) 7 Hz, 3H, terminal methyl group). MS: 341 (M+). Storage of Diquaternary Ammonium Salts and their Dispersal. Diquaternary ammonium salts were

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Figure 1. Reactions used for the synthesis of the diquaternary ammonium compounds. The upper scheme describes the synthesis of the saturated series and the lower scheme describes that of the unsaturated derivatives. In each series, compounds with 2, 3, and 6 carbons separating the nitrogen atoms were prepared.

normally stored at -20 °C as 10-40 mg/mL CHCl3:CH3OH [1:1] or CHCl3 solutions for the dicetyl and dioleyl derivatives, respectively. An age-dependent reduction of transfection activity was observed with the unsaturated derivatives, and therefore, we recommend that they be used within 1-2 months after final purification and solution preparation. Formation of Aqueous Dispersions of Diquaternary Ammonium Salts. Aliquots of stock were transferred to vials where the bulk of the solvent was removed under a stream of argon. The vial was then placed under an oil pump vacuum for at least 30 min/mg to remove residual solvent. Next, the appropriate amount of phosphate-buffered saline (PBS) was added in order to obtain a 1 mg/mL solution, and the tube was vortexed or bathsonicated briefly. Transfection Protocol. BHK cells were transfected with pCMV-β gal plasmid in 96 well microplates. Complete medium was GMEM containing 5 or 10% fetal bovine serum, 2 mM glutamine, 2% tryptose phosphate, and 1/100 PenStrep. The cells were incubated at 37 °C in 5% CO2. Cells were seeded on the day before transfection at densities such that they would be approximately 70% confluent when transfected. Cells were prepared for transfection by removal of complete medium and replacing it with medium lacking serum, tryptose, and antibiotics. Amphiphiles, suspended in PBS at 1 mg/ mL, were added to the plasmid DNA in PBS at 0.1 mg/ mL with gentle mixing and allowed to incubate for 2030 min. The DNA-amphiphile complex was then added to the cells and incubated 4-6 h at 37 °C. After this time, the cells were returned to complete medium, either following washing with PBS or simply upon addition of 1/10 or 1/20 volume of fetal bovine serum. Twenty hours after treatment with the DNA-amphiphile complex, the cells were assayed for β-galactosidase activity using a microplate fluorometric assay (17). Briefly, the cells were washed with PBS and permeabilized by adding 100 µL of PBS containing 0.03% Triton X-100. Ten microliters of 0.1 mM fluorescein digalactoside was added to the wells (final volume was 100 µL); 30 and 60 min after addition of substrate, the plate was read in a microplate fluorometer (Packard Instruments). The rate of reaction was linear in this time range and the fluorescence intensity difference between the two times was propor-

tional to β-galactosidase activity. Standards of purified β-galactosidase (Sigma) were run on each plate to validate the assay reaction. We did not routinely quantify the number of cells or protein in transfected cultures, so we normally express the galactosidase activity in terms of light units. To date, optimum conditions yield approximately 10 milliunits of enzyme activity/well of a 96 well plate. RESULTS

All of the diquaternary ammonium salts were prepared via a bis-Menshutkin reaction of their corresponding tetramethyldiamines with 2.2 M equiv of a long-chain alkyl halide (saturated or unsaturated). The reaction was run in anhydrous acetonitrile for 3 days at 60 °C in the dark, which provides the diquaternary ammonium halides in good to nearly quantitative yields for most derivatives (see Figure 1). An example of a monoquaternarized version of TmedAce, Quatamine-1, was produced through an analogous procedure, with the exception that only 1 M equiv of the cetyl iodide was used, and the reaction was only run overnight. For more details, see Materials and Methods. The diquaternary ammonium halides were characterized by both 1H NMR and mass spectral analysis. As can be seen in the Spectral Data section of Materials and Methods, the NMR resonances are consistent with the diquaternary structure assigned for each derivative. The mass spectral data consistently show both a parent ion minus one halide, as well as a parent ion minus one methyl group, for each derivative. Most cationic amphiphiles described in the literature as transfection reagents are more effective in the presence of DOPE (4, 11, 15), although there are exceptions (7, 18). In the case of the diquaternary ammonium salts, transfection efficiency was actually reduced by DOPE (see Figure 2 for a representative example), therefore, most of our studies were done without colipid. The monoquaternary Quatamine-1 was found to be highly toxic, even at low doses (data not shown) and underscores the need to isolate pure diquaternary ammonium salt for transfection purposes. Generally, investigators have not examined the effects of small amounts

DNA Transfection with Diquaternary Ammonium Salts

Figure 2. Transfection activity of TmedAce/DOPE at different ratios of TmedAce to DOPE. Transfection complexes were prepared in DPBS according to the standard protocol. Then lipid/ DNA complexes were applied to BHK cells in the amounts: 0.2/ 0.2, 0.5/0.5, and 1.0/1.0 µg (first, second, and third bars, of each set, respectively). Error bars indicate high and low values of replicates.

of side products that may be carried over in the final product as impurities. Given the high toxicity of some monoalkylamines, small variations in the purity of even highly pure materials could generate errors in evaluation of transfection agents and perhaps also account for some of the unexplained variations in the response of different cells to different compounds. The diquaternary compounds exhibited a variety of hydration behavior. The saturated derivatives, according to freeze-fracture electron microscopy, formed stacked, planar lamellae. The unsaturated compounds were observed by light microscopy to form liposomes at high ionic strength. At low ionic strength, two of the unsaturated compounds generated clear solutions, implying the formation of a micellar phase. Specifically, HexEce was micellar in both water and saline, PropEce was micellar in water but lamellar in saline, and TmedEce was lamellar in both aqueous phases. All of the diquaternary ammonium salts exhibited good to excellent transfection activity in BHK cells. Many transfection lipids are more active when formulated with equimolar DOPE, although this is not the case with the diquaternary compounds. Transfection activity of TmedAce formulated with DOPE at several ratios is shown in Figure 2. The TmedAce without colipid was most active and a similar pattern was found with the other diquaternary compounds (data not shown). The most efficient reagent of the saturated derivatives was HexAce (Figure 3). TmedAce and PropAce were 3-4 times less effective. This was observed under a variety of doses and lipid to DNA ratios (Figure 4). In the case of the unsaturated derivatives, PropEce and HexEce were both found to be highly efficient and essentially equivalent (Figures 5 and 6, respectively), with TmedEce only slightly less effective (Figure 7). In all cases, the presence of 10% serum was found to significantly reduce if not completely inhibit the transfection efficacy of these reagents. The diquaternary ammonium salts were, at optimal lipid:DNA ratio and dose, at least as effective transfection agents as O-ethyl-dioleoylphosphatidylcholine (EDOPC), the cationic lipid with which we have had the most experience. EDOPC, a phospholipid derivative, was previously shown to be as effective as one of the current

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Figure 3. Comparison of TmedAce, PropAce and HexAce in transfection of BHK cells. Transfection complexes were formed in DPBS and applied to cells in the amounts: 0.5/0.5, 1.0/0.5, and 1.0/1.0 µg (first, second, and third bars, of each set, respectively). Error bars indicate high and low values of replicates.

Figure 4. Comparison of TmedAce and HexAce transfection activities for different lipid/DNA ratios and doses. BHK cells were treated with plasmid DNA complexed with either TmedAce (first bar of each pair) or HexAce (second bar of each pair) in the amounts (µg/well) shown. Bars indicate high and low values of replicates.

Figure 5. Transfection activity of PropEce as a function of the lipid/DNA ratio and dose. BHK cells were treated with plasmid DNA complexed in DPBS with PropEce in the amounts (µg/well) shown. The data of transfection with EDOPC are presented for comparison. Bars indicate high and low values of replicates.

benchmarks of transfection reagents, Lipofectamine, in mediating transfection of BHK cells (19, 20). Its activity was also similar to that of Lipofectace and considerably superior to that of Lipofectin (unpublished).

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Figure 6. Transfection efficiency of HexEce with and without serum. BHK cells were transfected with HexEce/DNA complexes prepared in DPBS at different lipid/DNA ratios. Transfection was performed in medium without supplements (first and open bar of each group) and in medium containing 10% FBS (second and filled bar of each group) in the amounts (µg/well) shown. Bars indicate high and low values of replicates.

Figure 7. Transfection efficiency of TmedEce with and without serum. BHK cells were transfected with TmedEce/DNA complexes prepared in DPBS at different lipid/DNA ratios. Transfection was performed in medium without supplements (first bar of each group) and in medium containing 10% FBS (second bar of each group) in the amounts (µg/well) shown. Bars indicate high and low values of replicates. DISCUSSION

The diquaternary ammonium salts are conveniently synthesized in a single step, via a bis-Menshutkin reaction of their corresponding tetramethyldiamines with long-chain alkyl halides (either saturated or unsaturated). Yields ranged from good to nearly quantitative. Such straightfoward chemistry as well as the commercial availability of a range of various tetramethyldiamines as well as long-chain alkyl halides, provides the opportunity to explore many permutations through solution phase combinatorial chemistry. The observation that DOPE is not necessary for highly efficient transfection contrasts with the requirement of LipofectAce for DOPE. A number of other transfection lipid mixtures also contain DOPE as an integral component. Therefore, it appears that the addition of a second permanent positive charge in the diquaternary compounds somehow obviates the requirement for DOPE that is exhibited by the monoquaternary dimethyldioctadecylammonium bromide component of LipofectAce. The low transfection efficacy observed in the presence of 10% serum may preclude the in vivo usage of the

Rosenzweig et al.

diquaternary ammonium salts as vectors for systemic gene therapy, although procedures to reduce or eliminate the serum sensitivity of other cationic lipid formulations (18, 21) may well be applicable to the diquaternary compounds. The hydration behavior of the unsaturated derivatives follows a logical pattern, given the length of the alkyl chain that separates the two quaternary sites and the known effects of molecular shape on polymorphic phase preferences (22). TmedEce remains lamellar in both water and saline since the ethyl bridge between the two quaternary sites geometrically precludes anything but a parallel orientation of the two oleyl chains, which thus leads to a lamellar phase in both media. PropEce, possessing an extra carbon in its tether group, has enough space to allow ionic strength to affect its phase stability; it assumes a lamellar configuration in saline, wherein chloride anions screen the repulsion between the two positive sites and allow them to come close enough that the oleyl chains may be parallel, whereas in water, the two quaternary sites must move further away due to electrostatic repulsion, which consequently causes the oleyl chains to assume a wedge shape, favoring a micellar phase. Finally, HexEce was micellar in both water and saline since the two quaternary sites are held apart by the hexyl chain tether, the steric effect of the oleyl chain is independent of ionic strength, so the molecule is wedgeshaped and hence micellar in both media. The fact that efficient transfection was obtained with both HexEce and TmedEce shows that their different initial hydration behavior does not preclude either from forming a productive complex with DNA [in analogy to what has been observed with O-alkyl-dioleoylphosphatidylcholinium compounds (23)]. In conclusion, diquaternary ammonium salts constitute a new class of reagent for mediating transfection of DNA. Some of the derivatives we studied do so with high efficiency without a requirement for colipid. At least in the absence of serum, diquaternary compounds exhibit transfection activity that is at least as effective, if not superior to that of a number of popular, commercially available agents. Their low cost, extreme ease of synthesis and suitability to varying their chemical (and hence also physical) properties in a very straightfoward manner allows for optimizing activity for particular cell and transfection conditions. ACKNOWLEDGMENT

This work was presented in part at the 214th American Chemical Society National Meeting, Las Vegas, NV, in September 1997. H.S.R. wishes to thank Dr. Maria Papadopoulou for carefully reviewing the manuscript, as well as for her constant encouragement and inspiration. We thank Yury Tarahovsky for electron microscopy. This research was supported by the NIH (Grant GM52329). LITERATURE CITED (1) Keown, W. A., Campbell, C. R., and Kucherlapati, R. S. (1990) Methods for introducing DNA into mammalian cells. Methods Enzymol. 185, 527-537. (2) Forster, W., and Neumann, E. (1989) Gene Transfer by Electroporation- -A Practical Guide. In Electroporation and Electrofusion in Cell Biology (E. Neumann, A. E. Sowers, and C. A. Jordan, Eds.) pp 299-318. Plenum Press, New York. (3) Felgner, P. L., Gadek, T. R., Holm, M., Roman, R., Chan, H. W., Wenz, M., Northrop, J. P., Ringold, G. M., and Danielsen, M. (1987) Lipofection: a highly efficient, lipidmediated DNA-transfection procedure. Proc. Natl. Acad. Sci. U.S.A. 84, 7413-7417.

DNA Transfection with Diquaternary Ammonium Salts (4) Leventis, R., and Silvius, J. R. (1990) Interactions of mammalian cells with lipid dispersions containing novel metabolizable cationic amphiphiles. Biochim. Biophys. Acta 1023, 124-132. (5) Gao, X., and Huang, L. (1991) A novel cationic liposome reagent for efficient transfection of mammalian cells. Biochem. Biophys. Res. Commun. 179, 280-285. (6) Rose, J. K., Buonocore, L., and Whitt, M. A. (1991) A new cationic liposome reagent mediating nearly quantitative transfection of animal cells. Biotechniques 10, 520-525. (7) Barthel, F., Remy, J. S., Loeffler, J. P., and Behr, J. P. (1993) Gene transfer optimization with lipospermine-coated DNA. DNA Cell Biol. 12, 553-560. (8) Solodin, I., Brown, C. S., Bruno, M. S., Chow, C. Y., Jang, E. H., Debs, R. J., and Heath, T. D. (1995) A novel series of amphiphilic imidazolinium compounds for in vitro and in vivo gene delivery. Biochemistry 34, 13537-13544. (9) Akao, T., Nakayama, T., Takeshia, K., and Ito, A. (1994) Design of a new cationic amphiphile with efficient DNAtransfection ability. Biochem. Mol. Biol. Int. 34, 915-920. (10) Bichko, V., Netter, H. J., and Taylor, J. (1994) Introduction of hepatitis delta virus into animal cell lines via cationic liposomes. J. Virol. 68, 5247-5252. (11) Felgner, J. H., Kumar, R., Sridhar, C. N., Wheeler, C. J., Tsai, Y. J., Border, R., Ramsey, P., Martin, M., and Felgner, P. L. (1994) Enhanced gene delivery and mechanism studies with a novel series of cationic lipid formulations. J. Biol. Chem. 269, 2550-2561. (12) Morgan, R. A., and Anderson, W. F. (1993) Human gene therapy. Annu. Rev. Biochem. 62, 191-217. (13) Bennett, C. F., Chiang, M. Y., Chan, H., Shoemaker, J. E., and Mirabelli, C. K. (1992) Cationic lipids enhance cellular uptake and activity of phosphorothioate antisense oligonucleotides. Mol. Pharm. 41, 1023-1033. (14) Debs, R. J., Freedman, L. P., Edmunds, S., Gaensler, K. L., Du¨zgu¨nes, N., and Yamamoto, K. R. (1990) Regulation of gene expression in vivo by liposome-mediated delivery of a purified transcription factor. J. Biol. Chem. 265, 1018910192.

Bioconjugate Chem., Vol. 12, No. 2, 2001 263 (15) Farhood, H., Serbina, N., and Huang, L. (1995) The role of dioleoyl phosphatidylethanolamine in cationic liposome mediated gene transfer. Biochim. Biophys. Acta 1235, 289295. (16) Barber, K., Mala, R. R., Lambert, M. P., Qiu, R., MacDonald, R. C., and Klein W. L. (1996) Delivery of membraneimpermeant fluorescent probes into living neural cell populations by lipotransfer. Neurosci. Lett. 207, 17-20. (17) Rakhmanova, V. A., and MacDonald, R. C. (1998) A microplate fluorimetric assay for transfection of the bgalactosidase reporter gene. Anal. Biochem. 257, 234-237. (18) Felgner, P. L., Tsai, Y. J., Sukhu, L., Wheeler, C. J., Manthorpe, M., Marshall, J., and Cheng, S. H. (1995) Improved cationic lipid formulations for in vivo gene therapy. Ann. N. Y. Acad. Sci. 772, 126-139. (19) MacDonald, R. C., Ashley, G. W., Shida, M. M., Rakhmanova, V. A., Tarahovsky, Y. S., Pantazatos, D. P., Kennedy, M. T., Pozharski, E. V., Baker, K. A., Jones, R. D., Rosenzweig, H. S., Choi, K. L., Qiu, R., and McIntosh, T. J. (1999) Physical and biological properties of cationic triesters of phosphatidylcholine. Biophys. J. 77, 2612-2629. (20) MacDonald, R. C., Rakhmanova, V. A., Choi, K. L., Rosenzweig, H. S., and Lahiri, M. K. (1999) O-Ethylphosphatidylcholine: A metabolizable cationic phospholipid which is a serum-compatible DNA transfection Agent. J. Pharm. Sci. 88, 896-904. (21) Crook, K., Stevenson, B. J., Dubouchet, M., and Porteous, D. J. (1998) Inclusion of cholesterol in DOTAP transfection complexes increases the delivery of DNA to cells in vitro in the presence of serum. Gene Ther. 5, 137-143. (22) Israelachvili, J. N., Marcelja, S., and Horn, R. G. (1980) Physical principles of membrane organization. Q. Rev. Biophys. 13, 121-200. (23) Rosenzweig, H. S., Rakhmanova, V. A., McIntosh, T. J., and MacDonald, R. C. (2000) O-alkyl dioleoylphosphatidylcholinium compounds: The effect of varying alkyl chain length on their physical properties and in vitro DNA transfection activity. Bioconjugate Chem. 11, 306-313.

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