The carboxylic acid starting materials for the amide couplings to make compound 1-3 were purchased from Steraloids Inc. (Newport, RI). All other reagents were obtained from Sigma-Aldrich (St. Louis, MO) and used as received. Solvents were of HLPC grade.
Compounds 1-4 were synthesized in a similar manner, starting with the carboxylic acid sterol derivatives. The carboxylic acid was charged to a clean, dry 3-neck round bottom flask equipped with magnetic stirring. Dicyclohexylcarbodiimide was added in 1.1 equivalents along with enough anhydrous tetrahydrofuran to afford a 2.7mM solution. 0.2 equivalents of 1-hydroxybenzotriazole hydrate and 1.1 equivalents of spermine were added to the reaction mixture and stirred overnight at room temperature. Solvents were removed by rotary evaporation at 40°C and 0mbar to produce an oily residue. The residue was dissolved up in a 1:1 v/v mixture of dimethylsulfoxide and acetonitrile (0.6mM with respect to starting material). The resulting clear solution was filtered and purified on a semi-preparative C18 column (Shimadzu Premier 10mm × 150mm) equipped with LC/MS (Shimadzu LC2010) positive mode electrospray ionization and an acetonitrile and water solvent system buffered with 0.05% formic acid. Mass-selected fractions were concentrated to a residue as the formic acid salt and then dissolved in methanol for storage at −20°C. Final product yields of 30–40% were typical.
Compound 5 was prepared in the same manner except with 0.5 equivalents of spermine.
Reactions were also monitored by LC/MS with the Shimadzu C18 Premier 4.6mm × 0mm column. 1H NMR was conducted on a Bruker DMX300 spectrometer.
5.1.1 5β-cholanyl-spermide-3α-ol 1
MS m/z 561 [M+H]+. 1H NMR (CD3OD, 300MHz) δ(ppm) = 4.96 (t, 1 H, NH), 4.76 (m, 4 H, 2x NH 1x NH2), 3.37 (m, 1 H, CH), 3.37 (d, J = 0.66 Hz, 2 H, CH2-N), 3.24 (d, J = 1.45 Hz, 8 H, 4x CH2-N), 3.23 (t, 2 H, CH2-N), 3.17 (s, 2 H, CH2-CO), 3.08 (s, 4 H, 2x CH2), 3.07 (s, 2 H, CH2), 3.06 (d, J = 2.01 Hz, 1 H, OH), 2.66 (s, 2 H, CH2), 2.65 (s, 2 H, CH2), 1.34 (t, 2 H, CH2), 1.33 (d, J = 0.57 Hz, 2 H, CH2), 1.30 (m, 14 H, 7x CH2), 0.94 (m, 6 H, 6x CH), 0.20 (s, 9 H, 3x CH3).
5.1.2 5-cholenyl-spermide-3β-ol 2
MS m/z 560 [M+H]+. 1H NMR (CD3OD, 300MHz) δ(ppm) = 4.96 (t, 1 H, NH-CO), 4.90 (t, 1 H, CH), 4.75 (m, 4 H, 2x NH, NH2), 3.37 (m, 1 H, CH), 3.37 (d, J = 1.07 Hz, 2 H, CH2-N), 3.24 (d, J = 1.21 Hz, 8 H, 4x CH2-N), 3.23 (t, 2 H, CH2-N), 3.17 (s, 2 H, CH2-CO), 3.07 (s, 4 H, 2x CH2), 3.06 (s, 2 H, CH2), 3.05 (d, J = 1.53 Hz, 1 H, OH), 2.65 (s, 2 H, CH2), 2.64 (s, 2 H, CH2), 2.08 (d, J = 0.89 Hz, 2 H, CH2-CO), 1.54 (t, 2 H, CH2), 1.43 (d, J = 0.57 Hz, 2 H, CH2), 1.28 (m, 10 H, 5x CH2), 1.02 (m, 2 H, 2x CH), 0.72 (m, 3 H, 3x CH), 0.07 (d, J = 0.3 Hz, 3 H, CH3), 0.06 (s, 6 H, 2x CH3).
5.1.3 8-cholenyl-spermide-3α,12α-diol 3
MS m/z 576 [M+H]+. 1H NMR (DMSO-d6, 300MHz) δ(ppm) = 3.51 (s, 2 H, 2x OH), 3.32 (d, J = 39.89 Hz, 12 H, 6x CH2-N), 3.11 (m, 5 H, 3x NH, NH2), 1.27–2.30 (m, 20 H, 10x CH2), 1.24 (s, 10 H, 5x CH2), 1.15 (s, 6 H, 2x CH3), 0.82–1.09 (m, 5 H, 5x CH), 0.76 (d, J = 4.62, 3 H, CH3).
5.1.4 5β-cholanyl-spermide-3α,7α,12α-triol 4
MS m/z 594 [M+H]+. 1H NMR (DMSO-d6, 300mHz) δ(ppm) = 3.51 (s, 3 H, 3x OH), 3.34 (d, J = 39.81, 12 H, 6x CH2-N), 2.71–2.75 (m, 5 H, 3x NH, NH2), 1.56–1.90 (m, 20 H, 10x CH3), 1.24 (s, 10 H, 5x CH2), 1.15 (s, 6 H, 2x CH3), 0.93 (d, J = 6.57, 3 H, CH3), 0.74–0.91 (m, 8 H, 8x CH).
5.1.5 bis-cholanyl-spermide-triol 5
MS m/z 984 [M+H]+. 1H NMR (DMSO-d6, 300mHz) δ(ppm) = 3.51 (s, 6 H, 6x OH), 3.34 (d, J = 39.81, 12 H, 6x CH2-N), 2.71–2.75 (m, 4 H, 4x NH), 1.56–1.90 (m, 40 H, 20x CH3), 1.24 (s, 10H, 5x CH2), 1.15 (s, 12 H, 4x CH3), 0.93 (d, J = 6.57, 6 H, 2x CH3), 0.74–0.91 (m, 16 H, 16x CH).
Bovine aortic endothelial cells (BAEC) of passage 4–15 were seeded and maintained in T75 flasks prior to use in these experiments. A full growth media of 87% Dulbecco’s modified eagle media (D-MEM) (Gibco®), 10% fetal bovine serum(Gibco®), 2% penicillin streptomycin(Gibco®), and 1% L-glutamine(Gibco®) was changed out every 2–3 days and the cells were passaged every 5–10 days. 1–2 days prior to transfection, cells were seeded on 96-well plates for pGL4.75 transfections and 24-well plates for pEGFP-N3 transfections.
High throughput screening techniques were used to identify optimal liposome and lipoplex formulations for each compound based on the maximization of transfection efficiency and minimization of cytotoxicity. 96-well plates were used to formulate 12 different liposomal conditions per experimental set (including controls), each prepared in replicates of eight. Lipoplexes were generated in one 384-well plate for each 96-well liposome plate such that 4 charge ratios could be screened for each set of liposomes. The luciferase reporter plasmid, pGL4.75 (Promega) was complexed with each of the test liposomes, a positive control gene delivery vector Lipofectamine™ 2000 (Invitrogen™), and left as naked DNA. This vector provides a luminescent signal when the expressed Renilla luciferase reacts with coelenterazine-h which is generated by the digestion of EnduRen™ Live Cell substrate (Promega) by intracellular esterase. The reporter plasmid pEGFP-N3 (Promega) was also used to form lipoplexes in the same manner. Successful delivery of this plasmid results in a fluorescent signal on expression which can be imaged directly. All of the lipoplex formulations were prepared, pipette-mixed, and allowed to incubate for 30 minutes prior to delivery. Lipoplexes were transferred to well plates of confluent bovine aortic endothelial cells (BAEC) in OptiMem™ (Gibco®) serum-free media. After two hours of incubation (37°C, 5% CO2), the media in the cell well plates were replaced with full growth media and the cells were incubated overnight. 20 hours post-transfection, full growth media was aspirated from the cell well plates and EnduRen™ Live Cell substrate diluted in full growth media was added to each well. Following incubation for at least 1.5 hours, each well plate was analyzed for luminescent intensity in an Envision plate reader equipped with luminescent filter.