Dicationic Lipophosphoramidates as DNA Carriers - American

Bioconjugate Chem. 2007, 18, 1604−1611

1604

Dicationic Lipophosphoramidates as DNA Carriers Mathieu Me´vel,† Tristan Montier,‡ Franç ois Lamarche,† Pascal Dele´pine,‡ Tony Le Gall,‡ Jean-Jacques Yaouanc,† Paul-Alain Jaffre`s,† Dominique Cartier,† Pierre Lehn,‡ and Jean-Claude Cle´ment*,† UMR CNRS 6521, Universite´ de Bretagne Occidentale, UFR Sciences, 6, Avenue Le Gorgeu, C.S. 93837, F-29238 Brest cedex 3, France, and INSERM U 613, Universite´ de Bretagne Occidentale, C.S. 2653, F-29275 Brest cedex, France. March 14, 2007; Revised Manuscript Received May 30, 2007

Lipophosphoramidates with two different permanent cations as polar heads were synthesized and evaluated for their gene transfer activity. Physicochemical measurements (particle size, zeta potentials) and gel retardation assays were also performed. In Vitro biological evaluation was conducted with A542 and HeLa cell lines, and cytotoxicity determined by a chemiluminescent assay. The set of results indicates that, on the whole, dicationic lipophosphoramidates constitute an interesting alternative to their monocationic analogues.

INTRODUCTION The use of cationic lipids for mediating DNA transfection has become widespread. Since the pioneering work of Felgner et al. (1), this trend has grown rapidly, so that now a variety of such compounds have been described and asserted as DNA carriers for future applications to gene therapy (2, 3). Mixing an anionic plasmid DNA with variable amounts of cationic lipids (in terms of ( charge ratio) results in a “lipoplex”, whose transfection efficiency will generally vary according to that ratio. In our previous in Vitro experiments with phosphonolipids (4, 5) or lipophosphoramidates (6), optimal ( ratios were in the range 2-4. Lower ratios exhibited lower efficiency and higher, generally, an increased cytotoxicity. A possible way to lower the proportion of cationic lipid to DNA is to increase twofold (or more) the number of cations of the lipid. Moreover, additional cations could increase the strength of the interaction with DNA. Indeed, many examples of polycationic lipids are reported, notably lipidic polyamines. Their different amino groups are protonated at variable pH, leading to multicationic species, especially once internalized into cells (2). Permanently polycationic compounds are less numerous. Apart from polymers or bis-guanidinium derivatives (7), most of them are recorded as “gemini” (8). Some examples of gemini compounds used as DNA carriers are described, and in all quoted compounds, the cations are quaternary ammoniums (9-15). We have previously shown that monocationic “phosphonolipids” (4, 5) and “lipophosphoramidates” (6) had good transfection efficacy, together with moderate cytotoxicity. Moreover, the replacement of the ammonium by a phosphonium or arsonium head group was generally beneficial in terms of efficiency and cytotoxicity. So, we wondered about the behavior of such phosphorylated derivatives including two cations, identical or different, e.g., an ammonium plus a phosphonium, or an ammonium plus an arsonium, for instance. Furthermore, one can suppose that the arrangement of the two cations and the lipidic part (e.g., in single file or T-shaped) would lead to different interactions with DNA, hence a different efficacy. That is the case for cholesteryl polyamines, for instance, where the * To whom correspondence should be addressed. E-mail: [email protected]. † UMR CNRS 6521. ‡ INSERM U 613.

T-shaped arrangement was found to be more efficient than the linear one (16).

EXPERIMENTAL PROCEDURES 1. Materials and Reagents. Diisopropylethylamine (DIPEA) was dried and distilled over KOH. Fatty phosphites were synthesized by heating diphenyl phosphite (1 equiv) with tetradecanol or oleoyl alcohol (2 equiv) for 3 h at 120 °C and elimination of the produced phenol by distillation under vacuum. Bromopropyltrimethylammonium bromide 2 was purchased from Aldrich. Solvents were freshly distilled on appropriate driers and reactions run under nitrogen atmosphere. All compounds were fully characterized by 1H (400 MHz), 13C (100 MHz) and 31P (121.49 MHz) NMR spectroscopy (Bruker AC 300 and Advance DRX 400 spectrometers). Coupling constants J are given in hertz. The following abbreviations were used: s for singulet, d doublet, t triplet, q quadruplet, qt quintuplet, and m for multiplet. High-resolution mass spectroscopy (HRMS) (ESI) was performed at the “Centre Re´gional des Mesures Physiques de l’Ouest”, Rennes, France. O,O-Ditetradecyl-N-(dimethylaminopropyl)phosphoramidate 1a and O,O-dioleoyl-N-(dimethylaminopropyl)phosphoramidate 1b Ditetradecyl phosphite (2.37 g, 5 mmol) or dioleoyl phosphite (2.91 g, 5 mmol), dimethylaminopropylamine (0.63 mL, 5 mmol), CH2Cl2 (15 mL), and CBrCl3 (0.55 mL, 5.5 mmol) were mixed and cooled at 5 °C. DIPEA (0.96 mL, 5.5 mmol) was then added and the temperature kept 1 h at 5 °C, then 1 h at room temperature (RT). Solvent was discarded under reduced pressure, then DIPEA halides were precipitated in diethyl ether and filtered off. Purification of crude products was accomplished by column chromatography (silica gel), eluting with a chloroform/methanol (9/1) mixture. Compound 1a (C14:0) is a white solid (80% yield), and 1b (C18:1) a pale yellow oil (80% yield). NMR data for 1a: 1H (CDCl3) 0.86 (t, 3JH-H ) 6.6, 6H), 1.26 (m, 44H), 1.65 (m, 4H), 1.66 (q, 3JH-H ) 6.7, 2H), 2.22 (s, 6H), 2.35 (t, 3JH-H ) 6.9, 2H), 2.98 (m, 2H), 3.47 (m, 1H), 3.98 (m, 3JH-H ) 3JP-H ) 6.4, 4H). 13C (CDCl3) 14.1, 22.632.0, 25.5, 30.3 (d, 3JP-C ) 6.2), 40.3, 45.2, 57.2, 66.1 (d, 2JP-C ) 6.6). 31P (CDCl3) 9.76. NMR data for 1b: 1H (CDCl3) 0.86 (t, 3JH-H ) 6.6, 6H), 1.26 (m, 44H), 1.65 (m, 4H), 1.68 (m, 2H), 1.99 (m, 8H), 2.21 (s, 6H), 2.34 (m, 2H), 3.01 (m, 2H), 3.52 (m, 1H), 3.98 (m, 3J 3 13C (CDCl ) 14.1, H-H ) JP-H ) 6.4, 4H), 5.32 (m, 4H). 3

10.1021/bc070089z CCC: $37.00 © 2007 American Chemical Society Published on Web 08/03/2007

Dicationic Lipophosphoramidates as DNA Carriers

22.6-32.0, 25.4, 30.3 (d, 3JP-C ) 6.2), 40.1, 45.4, 57.1, 66.1 (d, 2JP-C ) 6.6), 129.6, 129.7. 31P (CDCl3) 9.77. 3-Iodopropyltrimethylphosphonium Iodide 3. Into 50 mL of degassed toluene were added under nitrogen atmosphere diiodopropane (1.32 mL, 12 mmol) and 10 mL of a 1 M solution of trimethylphosphine in THF (Aldrich). The mixture was then heated for 3 days at 40 °C. After cooling at RT, the white precipitate was washed twice with 10 mL of pentane and dried under vacuum (82% yield). NMR data: 1H (DMSO-d6) 1.85 (d, 9H), 2.03 (q, 2H), 2.24 (m, 2H), 3.33 (t, 2H). 13C (DMSO-d6) 7.3, 7.4, 23.8, 24.9. 31P (DMSO-d6) 28.4. HRMS (ESI): m/z calcd (z ) 1) 244.9956; found 244.9953 (an ionization cluster [2C+, I-] was also observed at m/z 617). 3-Iodopropyltrimethylarsonium Iodide 4. Into 50 mL of degassed toluene were added under nitrogen atmosphere diiodopropane (2.27 mL, 20.7 mmol) and trimethylarsine (1.95 mL, 18.4 mmol). After heating for 6 days at 70 °C and cooling at RT, the white precipitate was washed twice with 10 mL of pentane and dried under vacuum (86% yield). NMR data: 1H (CD3CN) 1.86 (s, 9H), 2.08 (q, 3JH-H ) 6.9, 2H), 2.42 (m, 2H), 3.28 (t, 3JH-H ) 6.8, 2H). 13C (CD3CN) 5.7, 7.7, 26.0, 27.1. HRMS (ESI): m/z calcd (z ) 1) 288.9434; found 288.9441 (an ionization cluster [2C+, I-]+ was also observed at m/z ) 705. Dicationic Lipophosphoramidates 5, 6, And 7. Into 2 mL of CH3CN were added 1a (632 mg, 1.1 mmol) or 1b (751 mg, 1.1 mmol) and 1 mmol of the “oniums” 2 (261 mg), 3 (372 mg), or 4 (416 mg). The mixture was heated at 70 °C for 3 days, then the solvent discarded under reduced pressure. The residues were precipitated and washed in ethyl acetate, then dried under vacuum. Ditetradecyl dicationic compounds 5a, 6a, and 7a are off-white powders. Dioleoyl 5b, 6b, and 7b are yellow to orange waxes. All yields were in the range 90-92%. NMR data for 5a: 1H (DMSO-d6) 0.85 (t, 3JH-H ) 6.6, 6H), 1.24 (m, 44H), 1.57 (m, 4H), 1.80 (m, 2H), 2.19 (m, 2H), 2.82 (m, 2H), 3.07 (s, 6H), 3.12 (s, 9H), 3.27 (m, 2H), 3.34 (m, 4H), 3.95 (m, 3JH-H ) 3JP-H ) 6.4, 4H), 5.01 (m). 13C (DMSO-d6) 13.9, 16.5, 20.1-31.3, 24.1, 29.0 (d, 3JP-C ) 6.2), 37.7, 50.0, 52.4, 59.6, 61.5, 61.9, 66.1 (d, 2JP-C ) 6.6). 31P (DMSO-d6) 9.34. NMR data for 5b: 1H (DMSO-d6) 0.85 (t, 3JH-H ) 6.6, 6H), 1.24 (m, 44H), 1.57 (m, 4H), 1.80 (m, 2H), 1.97 (m, 8H), 2.19 (m, 2H), 2.84 (m, 2H), 3.06 (s, 9H), 3.11 (s, 9H), 3.27 (m, 2H), 3.34 (m, 4H), 3.95 (m, 3JH-H ) 3JP-H ) 6.4, 4H), 5.01 (m, 1H), 5.31 (m, 4H). 13C (DMSO-d6) 13.9, 16.8, 20.1-31.3, 25.1, 29.0 (d, 3JP-C ) 6.2), 36.3, 51.0, 53.4, 59.6, 61.8, 62.1, 66.1 (d, 2JP-C ) 6.6). 31P (DMSO-d6) 9.40. NMR data for 6a: 1H (DMSO-d6) 0.87 (t, 3JH-H ) 6.6, 6H), 1.27 (m, 44H), 1.57 (m, 4H), 2.10 (m, 2H), 2.27 (d, 9H), 2.48 (m, 2H), 2.80 (m, 2H), 3.08 (m, 2H), 3.39 (s, 6H), 3.70 (m, 2H), 3.77 (m, 2H), 3.84 (m, 3JH-H ) 3JP-H ) 6.4, 4H), 4.17 (m, 1H). 13C (DMSO-d6) 10.0 (d), 14.1, 17.7, 20.5, 22.7-32.1, 25.7, 30.5 (d, 3JP-C ) 6.2), 37.9, 52.3, 62.8, 63.2, 67.1 (d, 2JP-C ) 6.6). 31P (DMSO-d6) 9.01, 27.9. NMR data for 6b: 1H (DMSO-d6) 0.87 (t, 3JH-H ) 6.6, 6H), 1.27 (m, 44H), 1.57 (m, 4H), 1.99 (m, 2H), 2.11 (m, 2H), 2.27 (d, 9H), 2.46 (m, 2H), 2.80 (m, 2H), 3.07 (m, 2H), 3.38 (s, 6H), 3.70 (m, 2H), 3.78 (m, 2H), 3.95 (m, 3JH-H ) 3JP-H ) 6.4, 4H), 4.18 (m, 1H), 5.45 (m, 4H). 13C (DMSO-d6) 10.0 (d), 14.1, 17.0, 20.5, 22.7-32.1, 25.7, 30.5 (d, 3JP-C ) 6.2), 38.0, 52.4, 62.9, 63.3, 67.0 (d, 2JP-C ) 6.6), 129.7, 130.0. 31P (DMSOd6) 9.1, 28.1. HRMS (ESI): m/z calcd (with z ) 2) 400.3526; found 400.3533 (an ionization cluster [2C+, I-]+ was also observed at m/z 927).

Bioconjugate Chem., Vol. 18, No. 5, 2007 1605

NMR data for 7a: 1H (DMSO-d6) 0.87 (t, 3JH-H ) 6.6, 6H), 1.27 (m, 44H), 1.57 (m, 4H), 2.11 (m, 2H), 2.30 (s, 9H), 2.60 (m, 2H), 2.90 (m, 2H), 3.08 (m, 2H), 3.35 (s, 6H), 3.66 (m, 2H), 3.72 (m, 2H), 3.84 (m, 3JH-H ) 3JP-H ) 6.4, 4H), 4.24 (m, 1H). 13C (DMSO-d6) 9.0, 13.6, 14.1, 22.7-32.1, 29.5, 25.4, 30.5 (d, 3JP-C ) 6.2), 37.8, 51.1, 63.0, 67.1 (d, 2JP-C ) 6.6). 31P (DMSO-d ) 9.11. 6 NMR data for 7b: 1H (DMSO-d6) 0.87 (t, 3JH-H ) 6.6, 6H), 1.27 (m, 44H), 1.57 (m, 4H), 1.99 (m, 2H), 2.06 (m, 2H), 2.32 (s, 9H), 2.55 (m, 2H), 2.86 (m, 2H), 3.08 (m, 2H), 3.37 (s, 6H), 3.68 (m, 2H), 3.74 (m, 2H), 3.95 (m, 3JH-H ) 3JP-H ) 6.4, 4H), 4.11 (m, 1H), 5.45 (m, 4H). 13C (DMSO-d6) 9.7, 14.1, 14.8, 19.3, 22.7-32.1, 25.8, 30.5 (d, 3JP-C ) 6.2), 38.1, 52.3, 63.8, 67.0 (d, 2JP-C ) 6.6), 129.7, 130.0. 31P (DMSO-d6) 9.05. O,O-Dioleoyl-N-(3-(N-imidazolyl)propyl)phosphoramidate 10. Into 15 mL of CH2Cl2 were added CBrCl3 (0.55 mL, 5.5 mmol), 3-aminopropylimidazole (0.59 mL, 5 mmol), and dioleoyl phosphite (2.91 g, 5 mmol). After cooling at 5 °C, DIPEA (0.96 mL, 5.5 mmol) was added and the mixture kept at this temperature for 1 h, then 1 h at RT. Solvent was evaporated and DIPEA halides filtered off, after precipitation from diethylether. Purification of crude product was accomplished by column chromatography (silica gel), eluting with a chloroform/ methanol (9/1) mixture. Dioleoyl phosphoramidate 10 was isolated as a yellow wax (80% yield). NMR data for 10: 1H (CDCl3) 0.86 (t, 3JH-H ) 6.6, 6H), 1.26 (m, 44H), 1.65 (m, 4H), 1.94 (q, 3JH-H ) 6.7, 2H), 1.99 (m, 8H), 2.89 (m, 2H), 3.61 (m, 1H), 3.98 (m, 3JH-H ) 3JP-H ) 6.4, 4H), 4.03 (t, 3JH-H ) 6.9, 2H), 5.32 (m, 4H), 6.92 (s, 1H), 7.04 (s, 1H), 7.49 (s, 1H). 13C (CDCl3) 13.2, 21.8-31.0, 24.7, 29.7 (d, 3JP-C ) 6.2), 37.1, 43.0, 65.4 (d, 2JP-C ) 6.6), 117.9, 128.4, 128.8, 129.1, 136.4. 31P (CDCl3) 9.57. Dicationic Lipophosphoramidates 11 And 12. To 2 mL of CH3CN were added 0.781 mg (1.1 mmol) of phosphoramidate 10 and 372 mg (1 mmol) of onium 3 or 416 mg (1 mmol) of onium 4. The mixture was heated at 70 °C for 3 days, then the solvent evaporated. After precipitation from ethyl acetate, dicationic phosphoramidates 11 (P+) and 12 (As+) were isolated as yellow to orange waxes (90-95% yield). NMR data for 11: 1H (CDCl3) 0.87 (t, 3JH-H ) 6.6, 6H), 1.27 (m, 44H), 1.57 (m, 4H), 1.99 (m, 8H), 2.15 (d, 9H), 2.18 (m, 2H), 2.62 (m, 2H), 2.99 (m, 2H), 3.05 (m, 2H), 3.93 (m, 1H), 3.95 (m, 3JH-H ) 3JP-H ) 6.4, 4H), 4.41 (m, 2H), 4.66 (m, 2H), 5.45 (m, 4H), 7.37 (s, 1H), 7.97 (s, 1H), 9.89 (s, 1H). 13C (CDCl ) 10.0 (d), 14.1, 20.5, 22.7-32.1, 24.0, 30.5 (d, 3J 3 P-C ) 6.2), 30.7, 38.0, 47.8, 49.0, 67.0 (d, 2JP-C ) 6.6), 129.7, 130.0, 122.0, 123.4, 136.7. 31P (CDCl3) 9.52, 27.89. 15N (CDCl3) 201.7, 203.4, 341.5. NMR data for 12: 1H (CDCl3) 0.87 (t, 3JH-H ) 6.6, 6H), 1.27 (m, 44H), 1.57 (m, 4H), 1.99 (m, 8H), 2.19 (m, 2H), 2.25 (d, 9H), 2.66 (m, 2H), 2.99 (m, 2H), 3.03 (m, 2H), 3.86 (m, 1H), 3.95 (m, 3JH-H ) 3JP-H ) 6.4, 4H), 4.42 (m, 2H), 4.60 (m, 2H), 5.45 (m, 4H), 7.53 (s, 1H), 7.94 (s, 1H), 9.85 (s, 1H). 13C (CDCl ) 9.2, 14.1, 21.8, 22.7-32.1, 24.1, 30.5 (d, 3J 3 P-C ) 6.2), 31.2, 37.7, 47.5, 49.4, 67.0 (d, 2JP-C ) 6.6), 129.7, 130.0, 121.7, 123.1, 137.0. 31P (CDCl3) 9.52. 15N (CDCl3) 201.7, 203.4, 341.5. O,O-Dioleoyl-N-(N,N-bis-(3-(dimethylamino)propyl)))phosphoramidate 14. Dioleoylphosphite (2.91 g, 5 mmol), 3,3′iminobis-(N,N-dimethylpropylamine) 13, CH2Cl2 (15 mL), and CCl4 (2 mL, 10 mmol) were mixed at 5 °C. DIPEA (0.96 mL, 5.5 mmol) was then added and the mixture kept for 1 h at 5 °C, then 1 h at RT. Solvents were discarded and DIPEA halides precipitated and filtered from diethyl ether. Dioleoyl phosphoramidate 14 was isolated as a yellowish oil (88% yield). NMR data for 14: 1H (CDCl3) 0.87 (t, 3JH-H ) 6.6, 6H), 1.25 (m, 44H), 1.63 (m, 4H), 1.70 (m, 4H), 1.99 (m, 8H), 2.25

1606 Bioconjugate Chem., Vol. 18, No. 5, 2007

Me´vel et al.

Scheme 1. Synthesis of “in Single File” Dicationic Lipophosphoramidates

Scheme 2. Synthesis of Mixed Imidazolium-Onium Dicationic Lipophosphoramidatesa

a

(a) CBrCl3 (1.1 equiv), DIPEA (1.1 equiv), 5 °C, 1 h, then RT, 1 h. (b) I(CH2)3Z+(CH3)3, I- (0.9 equiv), CH3CN, 70 °C, 3 days.

Scheme 3. Synthesis of a T-shaped Dicationic Phosphoramidatea

a

(a) CCl4 (2 equiv), DIPEA (2.2 equiv), 5 °C, 1 h, then RT, 1 h. (b) CH3I (6 equiv), RT, 12 h.

Scheme 4. Monocationic Lipophosphoramidates Used for Comparison Tests

(s, 12H), 3.06 (m, 4H), 3.83 (m, 4H), 3.95 (m, 3JH-H ) 3JP-H ) 6.4, 4H), 5.34 (m, 4H). 13C (CDCl3) 14.0, 22.6-32.0, 25.6, 30.2 (d, 3JP-C ) 6.5), 44.0, 45.3, 56.8, 66.4 (d, 2JP-C ) 5.9), 129.7, 129.9. 31P (CDCl3) 10.6. O,O-Dioleoyl-N-(N,N-bis-(3-(trimethylamonium)propyl)))phosphoramidate, diiodide 15. Compound 14 was shaken with an excess of CH3I for 24 h at RT. Excess of CH3I was evaporated and the diammonium 15 isolated as a yellow wax (94% yield). NMR data for 15: 1H (CDCl3) 0.86 (t, 3JH-H ) 6.6, 6H), 1.25 (m, 44H), 1.66 (m, 4H), 1.99 (m, 8H), 2.21 (m, 4H), 3.23

(m, 4H), 3.46 (s, 18H), 3.70 (m, 4H), 3.96 (m, 3JH-H ) 3JP-H ) 6.4, 4H), 5.34 (m, 4H). 13C (CDCl3) 14.0, 22.8-32.0, 24.0, 29.8 (d, 3JP-C ) 6.6), 42.6, 53.6, 64.0, 66.6 (d, 2JP-C ) 5.9), 129.1, 129.3. 31P (CDCl3) 10.0. 2. In Vitro Experiments. Cell Culture. Two different cell lines (A549 and HeLa) were used. The alveolar type II epithelial cell line A549 and the human adenocarcinoma epithelial cell line HeLa were obtained from the American Type Culture Collection (respectively, cat. nos .CCL-185 and ccl-2, ATCC, Rockville, MD). These cells were grown in D-MEM (GibcoBRL, UK) and supplemented with 10% fetal calf serum (FCS) (Gibco-BRL, UK) and 100 U/mL of penicillin/streptomycin. All the cells were maintained in a humidified incubator with 5% CO2 at 37 °C. Complex Formulation. Each cationic lipid was prepared alone or in combination with the neutral co-lipid DOPE (w/w ) 1:1). Lipids were kept and formulated in chloroform solutions; then, chloroform was removed by rotary evaporation to produce dried lipid films. A total of 1 mL of sterile pyrogen-free DI



Chart 1. In Vitro Transfection Efficiency of Dicationic 6b with A549 cells, in Comparison with Monocationic Compounds 17 and 18 and Lipofectaminea

a Numbers in brackets correspond to the charge ratio (. The letter D indicates a formulation with DOPE (vector/ DOPE ratio: 1/1). The letter C indicates a formulation with cholesterol (vector/ DOPE ratio: 1/1).

Chart 2. In Vitro Transfection Efficiency of Dicationic 6b with HeLa cells, in Comparison with Monocationic Compounds 17 and 18 and Lipofectaminea

a Numbers in brackets correspond to the charge ratio (. The letter D indicates a formulation with DOPE (vector/ DOPE ratio: 1/1). The letter C indicates a formulation with cholesterol (vector/ DOPE ratio: 1/1).

water per milligram of lipid was added, and the vials were sealed and stored at 4 °C overnight. Vesicles were then prepared by sonicating the aqueous suspension for 10 min in a bath sonicator. Cationic lipid/DNA complexes were formed as follows: plasmid DNA (1 µg) was diluted with sterile pyrogen-free deionized water and added to the lipid solution in a polystyrene tube. Lipoplexes were kept at room temperature for 30 min before being used for transfection. Determination of the Lipoplex Charge Ratio. The charge ratio was calculated theoretically as the molar ratio of the cationic lipids to DNA. In order to study the impact of the formulation on gene expression, various charge ratios (() were prepared with a constant amount of pDNA (1 µg). As a control, 2 µL of Lipofectamine (Invitrogen) was used, accordingly to manufacturer’s instructions, and in these conditions, for 1 µg of delivered DNA, the N/P ratio is equal to 2.

Transfection Protocol. Cells were seeded 24 h before transfection onto a 24-well plate at a density of 100 000 cells per well and incubated overnight in a humidified 5% CO2 atmosphere at 37 °C. Transfection was performed as described by Felgner et al. (1), with the following modifications. Appropriate amounts of the cationic lipids and the plasmid vector in OptiMem were complexed, and about 200 µL was added to each well. After 2 h 30 min incubation at 37 °C, the medium was removed, and fresh medium was added. Following a further 48 h at 37 °C, the cells were assayed for luciferase expression using a chemiluminescent assay (Promega). Assays were carried out as described by the manufacturer. The total protein concentration of the cell culture was determined using the BC assay kit (Uptima). Luciferase activity of each condition was expressed to total relative light units (Total RLU) per milligram of total protein. Results are reported as means ( SEM.


Me´vel et al.

Table 1. Luciferase Activity of Dicationic Lipids and Comparison with Monocationic Lipids dicationic lipid no./lipid alone/etc.

cells

DOPEa

(b ratio

TRLU ×106

5b

HeLa

6b

HeLa

+ +

2 2 4 1

1.1 0.8 2.7 1.6

A549

+

2 4

2.3 1.45

HeLa

+ + + + + + + + + + + + +

4 2 4 4 4 1 8 1 8 2 8 4 2 8 8 4 4 8 4 2 2 2 2 2 2 2 4 4

2.0 0.55 7.5 1.6 0.10 0.12 0.08 0.13 0.17 0.01 0.5 0.07 1.1 0.3 1.3 0.3 0.9 0.4 0.55 0.6 1.4 1.55 0.3 1.55 1.2 0.9 2.1 2.1

7b

A549 11bc

HeLa A549

12bc

HeLa A549

15

HeLa A549

16 17 18 17 18 19 19

a Without (-) or with (+) DOPE (1:1) added. b The better ( ratio in the range 1-8 is quoted. c Because of values about a decade lower, no comparison was made with monocationic lipids.

Determination of Cell Toxicity. The toxicity of the different lipid/DNA complexes was determined by using a chemiluminescent assay (TOXILIGHT - Cambrex, Lie`ge, Belgium). Twenty-four hours before the assay, the cells were plated in a 24-well plate (100 000 cells per well). Transfections were performed as described above, and the cells were incubated for

48 h at 37 °C. The toxicity assay was then carried out as specified by the manufacturer. This kit quantitatively measures the release of adenylate kinase (AK) from damaged cells into the surrounding medium. The reaction involves two steps. The first one involves the addition of ADP as a substrate for AK. In the presence of AK, the ADP is converted to ATP for assay by bioluminescence. Then, the bioluminescence method utilizes an enzyme Luciferase, which catalyzes the emission of photons from ATP and luciferin. By combining both reactions, the emitted light intensity is directly related to the AK concentration. The relative light units (RLU) were conversely proportional to the number of living cells. Untransfected cells were used as a reference. 3. DNA Binding Ability. To 1 µg of plasmid DNA in Optimem (Gibco) were added cationic lipids at concentrations corresponding to an N/P charge ratio ranging from 0.5 to 8. The mixture was incubated for 30 min at room temperature. The complexes were subjected to electrophoresis in 1% agarose gel at 100 V, 90 mA. The gel was stained with ethidium bromide (10 mg/mL) and visualized on an UV illuminator (Fischer Bioblock). 4. Sizes and ζ Potential Determination. Particle size and ζ potentials were determined using a Malvern Zetasizer 3000 HAS. For lipids alone, 2.7 mM (about 3 mg/mL) solutions in 10 mM Hepes buffer were prepared. Then, 0.2 mL of the solution was put into the cuvette and diluted up to 4 mL with Hepes. For lipoplexes, plasmidic DNA (2.5 µg) in Optimem (20 µL) was added to 5.5 µL (( ratio ) 4) or 11 µL (( ratio ) 8) of the above lipid 2.7 mmol solution diluted in 0.2 mL of Optimem, and left at RT for 30 min. The solution was then diluted to 4 mL with 10 mM Hepes buffer into the cuvette for measurement.

RESULTS AND DISCUSSION 1. Synthesis of Dicationic Lipophosphoramidates. We thus sought a synthetic route to “dicationic lipophosphoramidates”. For the “in single file” ones, since the phosphoramidate 1 is easily accessible by a Todd-Atherton reaction (17), a subsequent quaternarization by haloalkyl “onium” salts 2, 3, or 4 would lead directly to dicationic phosphoramidates 5, 6, or 7, respectively. Bromopropyltrimetylammonium 2 is commercially available, whereas iodopropyltrimethylphosphonium 3 and arsonium 4 were synthesized by adapting a procedure described for bromopropyltrimethylphosphonium (18). Indeed, heating

Chart 3. Cell Toxicity of Monocationic 18 and Dicationic 5b, 6b, and 7b



Figure 1. DNA binding ability of monocationic lipid 18 and dicationic 7b. Figure 2. DNA binding ability of dicationic lipids 5b and 6b.

salts 2, 3, and 4 with an equivalent quantity of dimethylamino phosphoramidate 1 in acetonitrile at 70 °C for 3 days led to the wanted dicationic compounds 5, 6, and 7 in a 90-92% yield range (Scheme 1). Then, we considered synthesizing imidazolium cations too, since some DNA carriers with such polar heads have been described (19). Propylimidazole phosphoramidate 10 was easily accessible from aminopropylimidazole 8 and fatty phosphite 9. Quaternarization by the same onium salts 3 or 4 led to dicationic compounds 11 and 12 with excellent yields (90-95%) (Scheme 2). A T-shaped dicationic phosphoramidate 15 was also synthesized from triamine 13 and fatty phosphite 9, followed by a quaternarization of the resulting diamino compound 14 with methyl iodide (Scheme 3). However, its in Vitro efficiency was lower than the corresponding “in single file” diammonium 5 (see below), and that way was not continued further. In comparison with the already described (6) very efficient monocationic lipophosphoramidates 16-19 (Scheme 4), these dicationic compounds were then evaluated for their transfection efficiency, their cytotoxicity, and their DNA binding ability. Some physicochemical parameters (lipoplexes size, zeta potentials) were also established.

2. Transfection Efficiency and Cytotoxicity. Transfection efficiency (expressed as luciferase activity) of dicationic compouds was performed on two different cell lines (A549 and HeLa), in comparison with monocationic phosphoramidates 1619, according to routinely used and previously described procedures (5, 20). Each lipid was tested at increasing ( charge ratios (1-8). Charts 1 and 2 show an example of the superiority of dicationic 6b (ammonium/phosphonium) vs monocationic 17 (phosphonium) or 18 (arsonium). Table 1 recapitulates the observed efficiencies for each dicationic lipid described above. As a general rule, dicationic lipids 5, 6, and 7 exhibit slightly greater values than the corresponding monocationic ones, at the same ( charge ratio, and are more efficient without DOPE added. On the other hand, dicationic lipids 11b and 12b with a central imidazolium core exhibit rather disappointing lower values. T-shaped diammonium 15 shows less efficiency than the corresponding “in single file” 5b. Insofar as at the same ( charge ratio dicationic lipids require half the material as the monocationic ones, a lower cytotoxicity was expected. To that effect, the toxicity of the different lipoplexes was determined by using a chemiluminescent assay (see the Experimental Procedures for details).


Me´vel et al.

Figure 3. DNA binding ability of dicationic lipids 11b and 12b.

Chart 3 presents a comparison among dicationic lipids 5b, 6b, and 7b and monocationic 18 (more RLUs are detected, more the cytoxicity increases correlated to the toxicity of the tested cationic lipid formulations). For each ( charge ratio (2, 4, or 8), the cytotoxicity of dicationic compounds is about one-third lower than that of 18. 3. DNA Binding Ability. Electrophoretic mobility of plasmid DNA was examined with increasing N/P ratios (2-8) using 1% agarose gel (DNA was visualized by ethidium bromide staining). Complete binding of DNA was observed at an N/P ratio lower than 4 for phosphoramidates 5b, 6b, 7b, and 18, whereas binding was not complete at an N/P ratio of 4 for imidazolium compound 12b or 8 for 11b (Figures 1-3). This difference may explain the lower values quoted above. 4. Lipoplex Sizes and ζ Potentials. On the basis of purely electrostatic interactions, dicationic lipids were anticipated to be better DNA-condensing amphiphiles than their monocationic analogues. In other words, the hydrodynamic diameters for lipoplexes prepared from lipids 5-7 and 11-12 were expected to be smaller than those prepared from monocationic lipids.

Table 2. Sizes and ζ Potentials of Lipids and Their Lipoplexes (formulation without neutral co-lipid) ζ potential (mV)

size (µm) lipoplex

lipoplex

lipid no

lipid alone

r)4

r)8

lipid alone

r)4

r)8

5b 6b 7b 11b 12b 15 18

175 176 146 100 140 259 210

240 268 187 328 332 247

235 249 175 344 328 229

69 80 79 90 85 89 62

26 19 30 20 20 26 44

33 46 43 24 26 39 60

Table 2 recapitulates sizes and ζ potentials for lipids alone and their lipoplexes (at a ( charge ratio of 4 and 8) (see Experimental Procedures for details). Indeed, average diameters of dicationic lipids are in the 100175 range, compared to 210 for monocationic 18, for lipids alone, but the lipoplex of 18 appears in the same range as dicationics. Changing lipid for lipoplex is accompanied by a 35-50 mV downshift of the ζ potentials for dicationic lipids,



whereas the same change keeps the values basically unchanged for 18, at the same ( charge ratio. From these data, a correlation between size, ζ potentials, and transfection efficacies of lipoplexes is not obvious. The same conclusions were made by Chaudhuri et al., from a series of cationic amphiphiles containing mono-, di-, and trilysine headgroups (21).

CONCLUSION The synthesis of permanently charged dicationic phosphoramidates was reported. The new vectors were readily obtained in good yield by using as a key reaction a Todd-Atherton coupling followed by a quaternarization step involving a halogeno-alkane ω-functionalized by either an ammonium, a phosphorinum, or an arsonium. The central cationic charge is either an ammonium or an imidazolium. Moreover, the synthesis of a T-shaped diammonium phosphoramidate was reported in high yield. These dicationic lipophosphoramidates are slightly more efficient than their monocationic analogues for in Vitro gene delivery. The best vector for the transfection of the two cell lines studied (A549 and Hela) is compound 6b, which is characterized, on the polar head, by the presence of a central ammonium and a terminal phosphonium. Furthermore, its cytotoxicity is about one-third lower than that of the monocationic vector 18. In another work (22), we have examined the transfection efficacies of lipophosphoramidates bearing a spermine as the polar head. Biological evaluation was performed on the same cell lines as above and in the same experimental conditions. Spermine derivatives expressed luciferase levels onehalf the values of permanently dicationic compounds described here. Since monocationic phosphoramidates exhibit interesting in ViVo activity (6), their dicationic analogues are currently under investigation to this end.

ACKNOWLEDGMENT “Brest Me´tropole Oceáne” is gratefully acknowledged for a grant to M.M. The authors thank also the “Conseil Re´gional de Bretagne” and “Vaincre La Mucoviscidose” for financial support and the CNRS for a postdoctoral grant to F.L.

LITERATURE CITED (1) 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, lipid-mediated DNA-transfection procedure. Proc. Natl. Acad. Sci. U.S.A. 84, 7413-7417. (2) Miller, A .D. (1998) Cationic liposomes for gene therapy. Angew. Chem., Int. Ed. 37, 1768-1785. (3) Martin, B., Sainlos, M., Aissaoui, A., Oudrhiri, N., Hauchecorne, M., Vigneron, J. P., Lehn, J. M., and Lehn, P. (2005) The design of cationic lipids for gene delivery. Curr. Pharm. Des. 11, 375394. (4) Gueńin, E., Herve´, A. C., Floch, V., Loisel, S., Yaouanc, J. J., Cle´ment, J. C., Fe´rec, C., and Des Abbayes, H. (2000) Cationic phosphonolipids containing quaternary phosphonium and arsonium groups for DNA transfection with good efficiency and low cellular toxicity. Angew. Chem., Int. Ed. 39, 629-631. (5) Floch, V., Loisel, S., Gueńin, E., Herve´, A. C., Yaouanc, J. J., Cle´ment, J. C., Fe´rec, C., and Des Abbayes, H. (2000) Cation substitution in cationic phosphonolipids: A new concept to improve transfection activity and decrease cellular toxicity. J. Med. Chem. 43, 4617-4628. (6) Picquet, E., Le Ny, K., Dele´pine, P., Montier, T., Yaouanc, J. J., Cartier, D., Des Abbayes, H., Fe´rec, C. and Cle´ment, J. C. (2005)

Cationic lipophosphoramidates and lipophosphoguanidines are very efficient for in vivo DNA delivery. Bioconjugate Chem. 16, 10511053. (7) Vigneron, J. P., Oudrhiri, N., Fauquet, M., Vergely, L., Bradley, J. C., Basseville, M., Lehn, P., and Lehn, J. M. (1996) Guanidiniumcholesterol cationic lipids: efficient vectors for the transfection of eukaryotic cells. Proc. Natl. Acad. Sci. U.S.A. 93, 9682-9686. (8) Menger, F. M., and Keiper, J. S. (2000) Gemini surfactants. Angew. Chem., Int. Ed. 39, 1907-1920. (9) Kirby, A. J., Camilleri, P., Engberts, J. B. F. N., Feiters, M. C., Nolte, R. J. M., So¨derman, O., Bergsma, M., Bell, P. C., Fielden, M. L., Garcia Rodriguez, C. L., Gue´dat, P., Kremer, A., Mc Gregor, C., Perrin, C., Ronsin, G., and Van Eijk, M. C. P. (2003) Gemini surfactants: new synthetic vectors for gene tranfection. Angew. Chem., Int. Ed. 42, 1448-1457. (10) Meekel, A. P., Wagenaar, A., Smisterova, J., Kroeze, J. E., Haadsma, P., Bosgraaf, B., Stuart, M. C. A., Brisson, A., Ruiters, M. H. J., Hoekstra, D. and Engberts, J. B. F. N. (2000) Synthesis of pyridinium amphiphiles used for transfection and some characteristics of amphiphile/DNA complex formation. Eur. J. Org. Chem. 665673. (11) Rosenzweig, H. S., Rakhmanova, V. A., and MacDonald, R. C. (2001) Diquaternary ammonium compounds as transfection reagents. Bioconjugate Chem. 12, 258-263. (12) Gaucheron, J., Wong, T., Wong, K. F., Maurer, N., and Cullis, P. R. (2002) Bioconjugate Chem. 13, 671-675. (13) Kasireddy, K., Ahmad, M. U., Ali, S. M. and Ahmad, I. (2004) Synthesis of novel cationic cardiolipin analogues for the optimal delivery of therapeutic agents. Tetrahedron Lett. 2743-2746. (14) Bombelli, C., Faggioli, F., Luciani, P., Mancini, G., and Sacco, M. G. (2005) Efficient transfection of DNA by liposomes formulated with cationic gemini amphiphiles. J. Med. Chem. 48, 5378-5382. (15) Paul, B., Bajaj, A., Indi, S. S., and Bhattacharya, S. (2006) Synthesis of novel dimeric cationic lipid based on an aromatic backbone between the hydrocarbon chains and headgroup. Tetrahedron Lett. 47, 8401-8405. (16) Lee, E. R., Marshall, J., Siegel, C. S., Jiang, C., Yew, N. S., Nichols, M. R., Nietupski, J. B., Ziegler, R. J., Lane, M., Wang, K. X., Scheule, R. K., Harris, D. J., Smith, A. E., and Cheng, S. H. (1996) Detailed analysis of structure and formulations of cationic lipids for efficient gene transfer to the lung. Human Gene Ther. 7, 1701-1717. (17) Atherton, F. R., Openshaw, H. T., and Todd, A. R. (1945) Studies on phosphorylation. Part II. The reaction of dialkyl phosphates with polyhalogen compounds in presence of bases. A new method for the phosphorylation of amines. J. Chem. Soc. 660. (18) Schmidbaur, H. and Scherm, H. P. (1976) 1-Methyl-1-methylen1λ5-phosphorinan und cyclopropyl-(dimethyl)methylenphosphoran: Zwei verschiedene Ylid-typen aus einer analogreaktion und ihre Komplexbildung. Chem. Ber. 1576-1585. (19) 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 imidazolium compounds for in vitro and in vivo gene delivery. Biochemistry 34, 13537-13544. (20) Montier, T., Dele´pine, P., Le Ny, K., Fichou, Y., Le Bris, M., Hardy, E., Picquet, E., Cle´ment, J. C., Yaouanc, J. J., and Fe´rec, C. (2004) KLN-5: a safe monocationic lipophosphoramidate to transfect efficiently haematopoietic cell lines and human CD34+ cells. Biochim. Biophys. Acta 1665, 118-133. (21) Karmali, P. P., Kumar, V. V., and Chaudhuri, A. (2004) Design, syntheses and in vitro gene delivery efficacies of novel mono-, diand trilysinated cationic lipids: a structure-activity investigation. J. Med. Chem. 47, 2123-2132. (22) Lamarche, F., Me´vel, M., Montier, T., Burel-Deschamps, L., Giamarchi, P., Tripier, R., Dele´pine, P., Le Gall, T., Cartier, D., Lehn, P., and Cle´ment, J. C. Lipophosphoramidates as lipidic part of lipospermines for gene delivery. Bioconjugate Chem., in press. BC070089Z

Dicationic Lipophosphoramidates as DNA Carriers - American

Recommend Documents