Cationic Oxyethylene Lipids. Synthesis, Aggregation, and Transfection

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Bioconjugate Chem. 2004, 15, 508−519

Cationic Oxyethylene Lipids. Synthesis, Aggregation, and Transfection Properties Santanu Bhattacharya* and Padinjarae Vangasseri Dileep Department of Organic Chemistry, Indian Institute of Science, Bangalore, India - 560 012. Received February 13, 2003; Revised Manuscript Received December 30, 2003

Four cationic lipids (1-4) with oligo-oxyethylene units at the linkage region between the pseudoglyceryl backbone and the hydrocarbon chains have been synthesized. Two of these lipids (1 and 2) have an equal number of (CH2CH2O)n units attached to both C-1 and C-2 positions of the pseudoglyceryl backbone, making their linkage regions similar, while the other two (3 and 4) are unsymmetrical in terms of the number of oxyethylene units in the linkage. Synthesis of lipids 1 and 2 involved the coupling of benzyl glycerol with the corresponding tosylates as a key step. Each of these lipids formed membranous aggregates when dispersed in water and exhibited clear thermotropic phase transitions typical of vesicular assemblies. The lipids 1-4 exhibited enhanced biological activities as gene transfer agents compared to their non-oxyethylene diether analogue, DHTMA. Transfection experiments using aqueous suspensions of these lipids and also their mixtures with cholesterol or dioleoyl phosphatidyl ethanolamine (DOPE) were performed on HeLa cells. The best transfection activity was demonstrated by unsymmetrical lipid 3, which had two oxyethylene units only at the C-1 position of the pseudoglycerylbackbone.

INTRODUCTION

The recent concept of “the genetic material itself as the drug” identifies the need for efficient DNA delivery vectors (1, 2). This has led to the development of many synthetic gene transfer vectors; among them are the formulations based on cationic lipids (3-8). Due to the ease of preparation and low levels of toxicity and immunogenic responses, cationic lipid-mediated transfection offers wide scope, especially in in vivo experiments (9) and clinical trials (10). However, the transfection efficiency of cationic lipid formulations has not yet been optimized to its maximum level (11) Due to this, the design and synthesis of novel cationic lipid molecules capable of efficient gene delivery is attracting much attention (12-14). We have been investigating the role of various molecular level modifications in different synthetic lipids on their membrane properties and further influence on gene delivery events. We have observed that the linkage functionality between the hydrocarbon chain and the polar headgroup of a lipid molecule has significant influence on its aggregation behavior in water (15). Since this linkage is in a dynamic interfacial region in an aqueous suspension, even small changes at the linkage modulate the aggregation properties significantly. Such small modulations at the linkage regions of a cationic lipid is also known to have pronounced effects on its transfection efficiencies (4, 16, 17). Along this direction, recently we have designed four cationic lipids 1-4 (Figure 1) having hydrophilic oxyethylene ((CH2CH2O)n) groups at the linkage region (18). We envisaged the design of two types of oxyethylene-bearing cationic lipids. In one case we introduced an equal number of (CH2CH2O)n units to both C-1 and C-2 posi* To whom correspondence should be addressed. E-mail: [email protected]; also at Chemical Biology Unit, JNCASR, Bangalore.

Figure 1. Molecular structures of the cationic lipids mentioned in the present study.

tions of the pseudoglyceryl backbone. In the other case, only one (CH2CH2O)2 unit was introduced either at the C-1 or C-2 positions of the backbone, to result in an unsymmetrical system. Though synthetically more demanding, an ether linkage functionality was chosen between the pseudoglycerol backbone and the hydrophobic chains mainly due to the enhanced biological activities of cationic lipids having ether linkages compared to their ester analogues in gene delivery events (16, 17), and also due to the enhanced chemical stability of ether bonds. Though unnatural, such synthetic lipid molecules form an important class. The incorporation of (CH2CH2O)n groups at the linkage regions is expected to widen the membrane-bound water layer in such aggregates. Such variations at the interfacial region should in turn influence the association of such assemblies with various biomolecules. Ethyleneoxy units were chosen to be the appropriate hydrophilic linkage functionality in these lipid molecules by considering its literature precedence (19, 20). Incorporation of oxyethylene units in lipid molecules have been attempted before, also for achieving some specific biological functions (21, 22). We observed a pronounced enhancement in the gene delivery capabilities of cationic lipids upon incorporation of oxyethylene units at the linkage region (23). In this paper, we describe the detailed, optimized synthetic procedures of these biologically important compounds. We also present their aggregation properties and biological activities. To put

10.1021/bc0340215 CCC: $27.50 © 2004 American Chemical Society Published on Web 03/12/2004

Cationic Oxyethylene Lipids

our results in appropriate perspective, we compare the physicochemical and biological properties of the new lipids 1-4 with that of DHTMA. The molecular structure of DHTMA closely resembles that of N-(1-[2,3-dioleyloxy]propyl)-N,N,N-trimethylammonium chloride (DOTMA), an ingredient which has been used in several commercially available formulations for gene transfection. MATERIALS AND METHODS

General. All the reagents and chemicals used in this study were of the highest purity available. Cationic lipids were synthesized and characterized fully by their IR, 1H NMR, mass spectra, and elemental analysis. 1,2-Dioleoylsn-glycero-3-phosphatidylethanolamine (DOPE) was purchased from Avanti Polar Lipids. 1H NMR was recorded on JEOL JNM λ-300 (300 MHz for 1H) or Bruker DRX500 (500 MHz for 1H) spectrometers. Chemical shifts (δ) are reported in ppm downfield from the internal standard; TMS, in the case of proton NMR. IR spectra were recorded on a Perkin-Elmer-781 spectrometer or on a Jasco FT-IR 410 spectrometer. Mass spectra were recorded on a Kratos PCKompact SEQ V1.2.2 MALDI-TOF spectrometer. Eluents for column chromatography, MeOH, EtOAc, CHCl3 and petroleum ether (60-80 fraction), were distilled before use. Solvents used for reactions were dried before use. DMF, CH2Cl2, CH3CN, and Et3N were dried over P2O5. Et3N and pyridine were stored over KOH. THF, benzene, and toluene were dried over sodium benzophenone ketyl prior to use. DMSO was dried over CaH2, vacuum distilled, and was stored over molecular sieves. The purity of all the final lipids was checked by thin-layer chromatography (TLC) on silica gel G-60 plates (Merck) prior to vesicle preparation. TLC employed chloroform/methanol mixture as the eluent as indicated in the synthesis, and spots were visualized by staining the plates with iodine vapor. All the lipids used in this study exhibited single spots on TLC plates. O-Hexadecyl Monoethylene Glycol Tosylate (6a) and O-Hexadecyl Diethylene Glycol Tosylate (6a). Either of the alcohols (ethylene glycol monohexadecyl ether or diethylene glycol monohexadecyl ether) was dissolved in CH2Cl2 and cooled in an ice-water bath. To this was added dry pyridine (5 equiv), and the mixture was stirred for 10 min. p-TsCl (2.5 equiv) was added, and the mixture was stirred at 0 °C for 15 min and then for 25-50 h at rt. At the end of the reaction, the reaction mixture was dissolved in CHCl3 and washed with aq 1.2 N HCl followed by 25% NaHCO3, water, and brine solution. The organic layer was dried over anhyd Na2SO4. The desired product was isolated by silica gel column chromatography using an EtOAc-hexane solvent mixture. The isolated yields were 82% for 6a (Rf ∼ 0.5 in EtOAc-hexane (8:92) solvent mixture) and 68% for 6b (Rf ∼ 0.5 at EtOAc-hexane (10:90) solvent mixture). 1H NMR (CDCl3, 300 MHz): δ: (6a) 0.88 (t, terminal CH3, 3H), 1.25 (br. m, CH2 × 13, 26H), 1.54 (p, C14H29CH2CH2O, 2H), 2.45 (s, CH3Ar, 3H), 3.39 (t, C15H31CH2O, 2H), 3.46-3.5 (m, C15H31CH2OCH2, 2H), 3.54-3.57 (m,C15H31CH2OCH2CH2O, 2H), 3.69 (t, CH2CH2OTs, 2H), 4.14 (t, CH2C H2OTs, 2H), 7.33 (d, meta H, 2H), 7.79 (d, ortho H, 2H); MS (MALDI-TOF): Calcd for C25H44O4S: 440.7 Found: 463.9 (M + Na+); Anal. Calcd for C25H44O4S‚ 0.5H2O: C 66.77, H 10.09 Found: C 66.73, H 9.54. (6b) 0.88 (t, terminal CH3, 3H), 1.26 (br. m, CH 2 × 13, 26H), 1.48 (p, C14H29C H2CH2O, 2H), 2.45 (s, CH3Ar, 3H), 3.36 (t, C15H31CH2O, 2H), 3.59 (t, C H2CH2OTs, 2H), 4.13 (t, CH2CH2OTs, 7.32 (d, meta H, 2H), 7.79 (d, ortho H, 2H); IR (cm-1): 1590 (w), 1100-1150 (br. s, O-C-O str.); MS (MALDI-TOF): Calcd for C27H48O5S: 484.7. Found: 507.9

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(M + Na+), 523.9 (M + K+); Anal. Calcd for C27H48O5S‚ 0.7H2O: C 65.21, H 10.01. Found: C 65.18, H 9.44. O-Benzyl Glycerol (8). 1,2-O-Isopropylidene glycerol (10 g, 75.6 mmol) was mixed with 8.8 g (156.6 mmol, 2 equiv) of powdered KOH in 45 mL of dry benzene. The solution turned yellow and then rose red within 5 min. To that was added 18 mL (19.8 g, 156.6 mmol, 2 equiv) of benzyl chloride. The mixture was refluxed at 100 °C for 23 h. The water formed during the reaction was removed by means of a phase-separating head (DeansStark apparatus). Then the cooled mixture was diluted with benzene (100 mL) and washed successively with water (50 mL), 0.1 N aq HCl (50 mL), 2.5% aq NaHCO3 (50 mL), and again with water. The benzene phase was dried over anhyd Na2SO4, and the solvent was removed under reduced pressure. The crude product, 1,2-O-isopropylidene glycerol benzyl ether was homogenized in 100 mL of MeOH-H2O (2:1) and to that was added 5.75 g (30.3 mmol) of p-TsOH. The mixture was stirred at room temperature for 1 h. The reaction mixture was neutralized with NaHCO3 powder. The mixture was filtered and washed with CHCl3, and then the organic layer was evaporated under reduced pressure. Traces of the remaining acid were removed by a subsequent base workup. The concentrated organic layer was diluted with CHCl3 and washed with saturated aq NaHCO3 (100 mL), water (50 mL), and saturated brine (50 mL). The organic layer was dried over anhyd Na2SO4. A 53% yield (7.3 g) of the pure compound was obtained by column chromatography on silica gel (24). Compound was eluted in 5% MeOH-CHCl3 (Rf ∼ 0.8 at MeOH-CHCl3 (2:98)). 1H NMR (CDCl3, 300 MHz): δ: 2.65 (br. s, OH, 2H), 3.453.7 (m, 2 × CH2O, 4H), 3.75-4 (q, OCH2CHOCH2O), 4.05 (s, OCH2Ar, 2H), 7.35 (s, Ar, 5H); IR (cm-1): 3100-3650 (br., OH). 1,2-Bis(2′-hexadecyloxyethoxy)-3-benzyloxypropane (9a). O-Benzyl glycerol (300 mg, 1.65 mmol) in 5 mL of dry DMSO was added to a precooled DMSO suspension (5 mL) of NaH (55% in oil) (237 mg, 3.3 equiv; prewashed with dry hexane) in a two-necked flask equipped with a septum and a guard tube. The mixture was stirred for 2 h at room temperature. To that were added 1.6 g of O-hexadecyl monoethylene glycol tosylate (11a) (3.6 mmol, 2.2 equiv) and 150 mg of Bu4N+I- (0.4 mmol, 0.25 equiv) in 2 mL of DMSO through a syringe. The reaction mixture was further stirred at room temperature for 24-30 h (till the alkyl tosylate was consumed, as indicated by TLC). Once the reaction was over, the reaction mixture was diluted with EtOAc and washed with 1 N aq HCl followed by 2.5% aq NaHCO3, water, and brine. The aqueous layer was extracted again with EtOAc. The organic phase was dried over anhyd Na2SO4. Pure compound was isolated as a pale yellowish liquid by column chromatography over silica gel using 5% EtOAc-hexane as the solvent system. The isolated yield was 880 mg (74%) (Rf ∼ 0.4 at EtOAc-hexane (5:95)). 1 H NMR (CDCl3, 300 MHz): δ: 0.88 (t, 2 × terminal CH3, 6H), 1.25 (br. m, 26 × CH2, 52H), 1.55 (p, 2 × C14H29CH2CH2O, 4H), 3.41 (t, 2 × C15H31CH2O, 4H), 3.49-3.75 (m, [2 × OCH2CH2O] + [2 × OCH2CHO] + OCH2CHOCH2O, 13H), 4.53 (s, OCH2Ar, 2H), 7.23-7.37 (m, Ar, 5H); IR (cm-1): 1100 (br. strong, C-O-C str.); MS (MALDITOF): Calcd for C46H86O5: 719.2. Found: 742.5 (M + Na+). 1,2-Bis(2′-[2′′-hexadecyloxyethoxy]ethoxy)-3-benzyloxypropane (9b). 9b was synthesized following the same procedure for that of 9a. Pure compound was isolated in 67% yield as a colorless liquid (which forms a wax on standing) by column chromatography over silica

510 Bioconjugate Chem., Vol. 15, No. 3, 2004

gel using EtOAc-hexane (15:85) as the solvent system (Rf ∼ 0.4 at EtOAc-hexane (15:85)). 1H NMR (CDCl3, 300 MHz): δ: 0.88 (t, 2 × terminal CH3, 6H), 1.20 (br. m, 26 × CH2, 52H), 1.5 (p, 2 × C14H29CH2CH2O, 4H), 3.6 (t, 2 × C15H31CH2O, 4H), 3.44-3.71 (m, [4 × OCH2CH2O] + [2 × OCH2CHO] + OCH2CHOCH2O, 21H), 4.47 (s, OCH2Ar, 2H), 7.17-7.28 (m, Ar, 5H); IR (cm-1): 1110 (br. strong, C-O-C str.); MS (MALDI-TOF): Calcd for C50H94O7: 806.7. Found: 829.7 (M + Na+). 2,3-Bis(2′-hexadecyloxyethoxy)-1-propanol (10a). 9a (500 mg) was dissolved in 10 mL of EtOAc-MeOH mixture in a hydrogenation setup, and to that was added 240 mg of 10% Pd-charcoal. Hydrogenation was performed for 12 h at atmospheric pressure. Then Pdcharcoal was filtered off from the reaction mixture with a sintered funnel and was washed with CHCl3 and EtOAc. Solvent was evaporated under reduced pressure, and the pure compound was isolated as a slight yellow gummy solid by column chromatography over silica gel with EtOAc-hexane (1:4) as the eluting solvent (Rf ∼ 0.7 at EtOAc-hexane (1:4) solvent mixture). The isolated yield was 380 mg (87%). 1H NMR (CDCl3, 300 MHz): δ: 0.88 (t, 2 × terminal CH3, 6H), 1.25 (br. m, 26 × CH2, 52H), 1.55 (p, 2 × C14H29CH2CH2O, 4H), 3.4-3.71 (m, [2 × C15H31CH2O] + [2 × OC H2CH2O] + [2 × OCH2CHO], 16H), 3.82-3.88 (m, OCH2CHOCH2O, 1H); MS (MALDITOF): Calcd for C39H80O5: 628. Found: 651 (M + Na+), 667 (M + K+); Anal. Calcd for C39H80O5: C 74.46, H 12.82. Found: C 74.46, H 12.61. 2,3-Bis(2′-[2′′-hexadecyloxyethoxy]ethoxy)-1-propanol (10b). 9b (400 mg) was dissolved in 8 mL of EtOAc-MeOH mixture in a hydrogenation setup, and to that was added 135 mg of 10% Pd-charcoal. Hydrogenation was performed for 12 h at atmospheric pressure. Then Pd-charcoal was filtered off from the reaction mixture with a sintered funnel and was washed with CHCl3 and EtOAc. Solvent was evaporated under reduced pressure and the pure compound was isolated as a yellow wax by column chromatography over silica with EtOAc-hexane (1:1) as the eluting solvent (Rf ∼ 0.7 at EtOAc-hexane solvent (1:1) mixture). The isolated yield was 350 mg (80%). 1H NMR (CDCl3, 300 MHz): δ: 0.88 (t, 2 × terminal CH3, 6H), 1.25 (br. m, 26 × CH2, 52H), 1.56 (p, 2 × C14H29CH2CH2O, 4H), 3.42 (t, 2 × C15H31CH2O, 4H), 3.47-3.73 (m, [4 × OCH2CH2O] + [2 × OCH2CHO], 20H), 3.8-3.87 (m, OCH2CHOCH2O, 1H); IR (cm-1): 1110 (br. strong, C-O-C str.), 3400-3480 (br., OH); Anal. Calcd for C43H88O7‚0.6H2O: C 70.95, H 12.35. Found: C 71.04, H 12.25. 2,3-Bis(2′-hexadecyloxyethoxy)-1-propyl Tosylate (11a). 2,3-Bis(2′-[2′′-hexadecyloxyethoxy]ethoxy)-1-propanol (10a) (130 mg, 0.2 mmol) was dissolved in 1 mL of dry CH2Cl2 and was cooled in an ice-water bath. To that were added 2 mL of dry pyridine and 78.8 mg of freshly recrystallized p-TsCl (2 equiv). The reaction mixture was stirred at 0 °C for 30 min and then for 24 h (till the starting alcohol disappeared as indicated from TLC) at room temperature. The reaction mixture was then diluted with CHCl3 and was washed with water followed by 2 N aq HCl and again with water. The organic phase was washed with brine and dried over anhyd Na2SO4. Solvent was evaporated under reduced pressure, and pure compound was isolated (140 mg, 85%) as a white wax by column chromatography over silica gel using EtOAchexane solvent mixture (Rf ∼ 0.5 at EtOAc-hexane (15: 85) solvent mixture). 1H NMR (CDCl3, 300 MHz): δ: 0.88 (t, 2 × terminal CH3, 6H), 1.26 (br. m, 26 × CH2, 52H), 1.54 (p, 2 × C14H29CH2CH2O, 4H), 2.45 (s, CH3Ar, 3H), 3.38-3.67 (m, [2 × C15H31C H2O] + [2 × OCH2CH2O] +

Bhattacharya and Dileep

[2 × OCH2CHO], 14H), 3.72 (q, OCH2CHOCH2O, 1H), 4.06 (dd [J ) 9 Hz, 6 Hz], HCHOTs, 1H), 4.17 (dd, [J ) 9 Hz, 3 Hz], HCHOTs, 1H), 7.34 (d, meta H, 2H), 7.79 (d, ortho H, 2H); IR (cm-1): 1110 (br. strong, C-O-C str.) 2,3-Bis(2′-[2′′-hexadecyloxyethoxy]ethoxy)-1-Propyl Tosylate (11b). 2,3-Bis(2′-[2′′-hexadecyloxyethoxy]ethoxy)-1-propanol (10b) (150 mg, 0.2 mmol) was dissolved in 5 mL of dry CH2Cl2 and was cooled in an icewater bath. To that were added 0.09 mL (1 mmol, 5 equiv) of dry pyridine and 79.7 mg of recrystallized p-TsCl (2 equiv). The reaction mixture was stirred for 27 h (till the starting alcohol disappeared as indicated from TLC) at room temperature. The reaction mixture was then diluted with CHCl3 and washed with water followed by 2 N aq HCl and again with water. The organic phase was washed with brine and dried over anhyd Na2SO4. Solvent was evaporated under reduced pressure, and pure compound was isolated as a yellow liquid by column chromatography over silica gel. Yield was practically quantitative (Rf ∼ 0.5 at EtOAc-hexane (30:70) solvent mixture). 1H NMR (CDCl3, 300 MHz): δ: 0.88 (t, 2 × terminal CH3, 6H), 1.25 (br. m, 26 × CH2, 52H), 1.57 (p, 2 × C14H29CH2CH2O, 4H), 2.45 (s, CH3Ar, 3H), 3.43 (t, 2 × C15H31CH2O, 4H), 3.48-3.76 (m, [4 × OCH2CH2O] + [2 × OCH2CHO] + [OCH2CHOCH2O], 19H), 4.06 (dd [J ) 9 Hz, 6 Hz], HCHOTs, 1H), 4.17 (dd, [J ) 9 Hz, 3 Hz], HCHOTs, 1H), 7.34 (d, meta H, 2H), 7.80 (d, ortho H, 2H). 1-Bromo-2,3-bis(2′-hexadecyloxyethoxy)propane (12a). 2,3-Bis(2′-hexadecyloxy ethoxy)-1-propyl tosylate (11a) (140 mg, 0.17 mmol) and LiBr (46.56 mg, 0.53 mmol, 3 equiv) were dissolved in 2 mL of dry DMF and refluxed at 70 °C for 24 h. Then the reaction mixture was diluted with 125 mL of EtOAc and was washed with 100 mL of water followed by brine. The organic phase was dried over anhyd Na2SO4. Pure compound was isolated in 81% yield (100 mg) by column chromatography over silica gel (Rf ∼ 0.8 at EtOAc-hexane (15:85)). 1H NMR (CDCl3, 300 MHz): δ: 0.88 (t, 2 × terminal CH3, 6H), 1.25 (br. m, 26 × CH2, 52H), 1.56 (p, 2 × C14H29CH2CH2O, 4H), 3.4-3.74 (m, [7 × CH2O] + CH2Br + OCH2CHOCH2O, 17H); IR (cm-1): 1100-1130 (broad, C-O-C str.) 1-Bromo-2,3-bis(2′-[2′′-hexadecyloxyethoxy]ethoxy)propane (12b). 2,3-Bis(2′-[2′′-hexadecyloxy ethoxy]ethoxy)-1-propyl tosylate (11b) (200 mg, 0.23 mmol) and LiBr (59.80 mg, 0.7 mmol, 3 equiv) were dissolved in 3 mL of dry DMF and refluxed at 70 °C for 8 h. Then the reaction mixture was dissolved in 100 mL of EtOAc and washed twice with water (2 × 20 mL) followed by brine. The organic phase was dried over anhyd Na2SO4. EtOAc was evaporated under reduced pressure, and pure compound was isolated in 84% yield (150 mg) by column chromatography over silica gel (Rf ∼ 0.7 at EtOAchexane (3:7)). 1H NMR (CDCl3, 300 MHz): δ: 0.88 (t, 2 × terminal CH3, 6H), 1.25 (br. m, 26 × CH2, 52H), 1.56 (p, 2 × C14H29CH2CH2O, 4H), 3.42 (t, 2 × C15H31CH2O, 4H), 3.46-3.75 (m, [9 × CH2O] + CH2Br + OCH2CHOCH2O, 21H); IR (cm-1): 1140, 1200-1240 (broad, C-O-C str.) N-(2,3-Bis[2′-hexadecyloxyethoxy]propyl)N,N,Ntrimethylammonium Bromide (1). Trimethylamine was introduced into dry CH3CN (1 mL) in a pressure tube in an ice-acetone bath. This resulted in a 3-fold expansion of the initial volume of acetonitrile. 1-Bromo-2,3bis(2′-hexadecyloxyethoxy)propane (12a) (100 mg, 0.15 mmol) was dissolved in CH3CN-toluene mixture by slight warming and was transferred to the pressure tube.


The reaction mixture was heated at 80 °C for 24 h. The reaction mixture was cooled, and the excess trimethylamine was evaporated by warming in a water bath. Solvent was then removed under reduced pressure. Pure compound was isolated as a white solid by column chromatography over silica using MeOH-CHCl3 as the solvent mixture. Yield of the reaction was 74% (80 mg). 1H NMR (500 MHz, CDCl ): δ: 0.88 (t, 2 × terminal CH , 3 3 6H), 1.26 (br. m, 26 × CH2, 52H), 1.52-1.57 (m, 2 × C14H29CH2CH2O, 4H), 3.41 (s, 3 × N+CH3, 9H), 3.433.71 (m, 7 × CH2O, 14H), 3.90-3.92 (m, OCH2-CHOCH2N+, 1H), 4.11-4.18 (m, CH2N+, 2H); IR (cm-1): 1060-1150 (broad, C-O-C str.); MS (MALDI-TOF): Calcd for C42H88O4N: 671.2. Found: 672.2 (M + H+); Anal. Calcd for C42H88O4NBr‚1.5H2O: C 64.83, H 11.79, N 1.8. Found: C 64.93, H 12.03, N 1.65. N-(2,3-Bis[2′-{2′′-hexadecyloxyethoxy}ethoxy]propyl)-N,N,N-trimethylammonium Bromide (2). Trimethylamine was introduced into dry CH3CN (1 mL) in a pressure tube in an ice-acetone bath which nearly resulted in a 3-fold expansion of the volume of CH3CN from its initial value. 1-Bromo-2,3-bis(2′-[2′′-hexadecyloxyethoxy]ethoxy)propane (12b) (150 mg, 0.19 mmol) was dissolved in CH3CN-toluene mixture by light heating and was transferred to the pressure tube. The reaction mixture was heated at 80 °C for 24 h. The reaction mixture was cooled, and the excess Me3N was evaporated by warming. Solvent was then removed under reduced pressure. Pure compound was isolated as a white wax (110 mg, 68%) by column chromatography over silica gel (100-200 mesh) using MeOH-CHCl3 (1:9) as the solvent mixture. After the chromatography separation, the compound was dissolved in chloroform and was passed through a sintered funnel to remove any silica gel impurity in the eluant (Rf ∼ 0.5 at MeOH-CHCl3 (1:9) solvent mixture). 1H NMR (500 MHz, CDCl3): δ: 0.88 (t, 2 × terminal CH3, 6H), 1.26 (br. m, 26 × CH2, 52H); 1.55 (p, 2 × C14H29CH2CH2O, 4H), 3.38 (s, 3 × N+CH3, 9H), 3.39-3.67 (m, 11 × C H2O, 22H), 3.89-3.91 (m, OCH2-CHO-CH2N+, 1H), 4.04 (d, HCH-N+, 1H), 4.16 (m, HC H-N+, 1H); IR (cm-1): 1100 (broad, C-O-C str.); MS (MALDI-TOF): Calcd for C46H96O6N: 759.3. Found: 760.1 (M + H+); Anal. Calcd for C46H96O6NBr‚1.9H2O: C 63.26, H 11.52, N 1.6. Found: C 63.25, H 11.77, N 1.43. 2-(2′-Chloroethoxy)ethyl-2′′-tetrahydropyranyl Ether (13). To 2 g of 2-(2′-chloroethoxy)ethanol (16.1 mmol) were added 2.03 g of dihydropyran (24.1 mmol, 1.5 equiv) and 1 drop of concentrated HCl. It was stirred at rt for 2.5 h. The reaction mixture was initially neutralized by the addition of solid NaHCO3. The reaction mixture was then diluted with EtOAc and washed with saturated aq NaHCO3 solution followed by brine. The organic phase was dried over anhyd Na2SO4. Pure compound was isolated as a colorless liquid (quantitative conversion) chromatographically by passing through a short column of silica gel and eluting with EtOAchexane solvent (2:98) mixture (25). 1H NMR (90 MHz, CDCl3): δ: 1.4-2 (m, 3 × CH2, 6H), 3.3-4 (m, [4 × C H2O] + CH2Cl, 10H), 4.65 (t, O-CH2C HCH2-O, 1H); IR (cm-1): 1120 (broad, C-O-C str.) 3-Benzyloxy-2-hydroxy-1-(2′-[2′′-hydroxyethoxy]ethoxy)propane (14). To a solution containing O-benzyl glycerol (8) (0.5 g, 2.75 mmol) were added 2-(2′-chloroethoxy)ethyl 2′′-tetrahydropyranyl ether (13) (1.72 g, 8.24 mmol, 3 equiv) and 82.1 mg of Bu4N+HSO4- (0.242 mmol, 0.09 equiv) dropwise in 2 mL of 50% aq NaOH (2 g, 55 mmol, 20 equiv). The mixture was stirred and heated at 65 °C for 5 h. Then the reaction mixture was diluted with CH2Cl2 and washed with water followed by brine. The


organic phase was dried over anhyd Na2SO4. However, mono- and diether derivatives could not be isolated upon column chromatography. The mixture was isolated by silica gel column chromatography using EtOAc-hexane (1:1) as the solvent system (Rf of the mixture being 0.5 at this solvent mixture). This mixture was then subjected to the acid-catalyzed deprotection of THP by stirring the compound with 10 drops of concentrated HCl in 10 mL of 1:1 MeOH-CH2Cl2 solvent mixture for 2 h at room temperature. The reaction mixture was then carefully neutralized with solid NaHCO3 and was extracted in EtOAc and finally with CHCl3. The organic solvent from the organic extracts was evaporated under reduced pressure and the required mono derivative was isolated in the pure form, as a yellow liquid, by column chromatography over silica using MeOH-EtOAc solvent system (Rf of the required mono derivative ∼ 0.6 at EtOAc). The isolated yield was 250 mg (34% overall). 1H NMR (300 MHz, CDCl3): δ: 3.41-3.83 (m, 6 × CH2, 12H), 3.94 (q, OCH2CHOHCH2O, 1H), 4.48 (s, OCH2Ar, 2H), 7.19-7.31 (m, Ar, 5H); LRMS: Calcd for C14H22O5: 270. Found: 271 (MH+) 1-(2′-[2′′-Hexadecyloxyethoxy]ethoxy)-2-hexadecyloxy-3-benzyloxypropane (15). To a mixture containing 3-benzyloxy-2-hydroxy-1-(2′-[2′′-hydroxyethoxy]ethoxy)propane (14) (320 mg, 1.2 mmol), Bu4N+HSO4(26.7 mg, 0.79 mmol, 0.09 equiv), and hexadecyl chloride (699 mg, 2.68 mmol, 2.2 equiv) was added dropwise 1.4 mL of 50% NaOH. The mixture was then stirred vigorously at 65 °C for 5 days. After the reaction, CH2Cl2 was added to it and the organic layer was washed with water followed by brine. The organic phase was dried over anhyd Na2SO4, and the solvent was evaporated under reduced pressure. Pure compound was isolated as a slight yellow liquid at 53% yield (450 mg) by silica gel column chromatography using EtOAc-hexane (15:85) as the solvent system (Rf ∼ 0.8 at this solvent system). 1H NMR (300 MHz, CDCl3): δ: 0.86 (t, 2 × terminal CH3, 6H), 1.23 (br. m, 26 × CH2, 52H), 1.54 (p, 2 × C14H29CH2CH2O, 4H), 3.42 (t, 2 × C15H31CH2O, 4H), 3.48-3.67 (m, 6 × CH2O, 12H), 3.76 (q, OCH2CHOCH2O, 1H), 4.53 (s, OCH2Ar, 2H), 7.26-7.32 (m, Ar, 5H); IR (cm-1): 1100 (broad, C-O-C str.) 2-Hexadecyloxy-3-(2′-[2′′-hexadecyloxyethoxy]ethoxy)-1-propanol (16). 1-(2′-[2′′-Hexadecyloxyethoxy]ethoxy)-2-hexadecyloxy-3-benzyloxypropane (15) (450 mg, 0.6 mmol) was dissolved in EtOAc-MeOH solvent mixture. To that was added activated 10% Pd-charcoal (100 mg). Hydrogen gas was introduced into the flask, and hydrogenation was carried out under atmospheric pressure, at room temperature for 12 h. Pd-charcoal was filtered off from the reaction mixture, and the pure compound was isolated as a low melting white solid (340 mg, 85% yield) by column chromatography over silica gel (Rf ∼ 0.4 at EtOAc-hexane (1:4) solvent mixture). 1H NMR (300 MHz, CDCl3): δ: 0.86 (t, 2 × terminal CH3, 6H), 1.23 (br. m, 26 × CH2, 52H), 1.55 (m, 2 × C14H29CH2CH2O, 4H), 3.42 (t, 2 × C15H31CH2O, 4H), 3.47-3.7 (m, 6 × CH2O, 12H), 3.83 (m, OCH2CHOCH2O, 1H); IR (cm-1): 1100-1130 (broad, C-O-C str.), 3400 (br., OH); LRMS Calcd for C39H80O5: 628. Found: 629 (MH+); Anal. Calcd for C39H80O5‚0.4H2O: C 73.62, H 12.8. Found: C 73.67, H 12.84. 2-Hexadecyloxy-3-(2′-[2′′-hexadecyloxyethoxy]ethoxy)-1-propyl Tosylate (17). 2-Hexadecyloxy-3-(2′[2′′-hexadecyloxyethoxy]ethoxy)-1-propanol (16) (140 mg, 0.2 mmol) was dissolved in 1 mL of dry CH2Cl2 and cooled in an ice-water bath. To that were added 2 mL of dry pyridine and 106 mg of freshly recrystallized p-TsCl (0.5


mmol, 2.5 equiv). The reaction mixture was stirred at 0 °C for 15 min and then at room temperature for 24 h (till the starting alcohol disappeared as indicated from TLC). The reaction mixture was then diluted with CH2Cl2 and was washed with water followed by 2 N aq HCl and again with water. The organic phase was washed with brine and dried over anhyd Na2SO4. Solvent was evaporated under reduced pressure, and pure compound was isolated as a colorless liquid (140 mg, 80% yield) by column chromatography over silica (Rf ∼ 0.3 at EtOAchexane (15:85) solvent mixture). The isolated product was found to be very hygroscopic. 1H NMR (CDCl3, 300 MHz): δ: 0.88 (t, 2 × terminal CH3, 6H), 1.25 (br. m, 26 × CH2, 52H), 1.46 (m, C14H29CH2CH2O, 2H), 1.55 (m, C14H29CH2CH2O, 2H), 2.46 (s, C H3Ar, 3H), 3.32-3.7 (m, [7 × CH2O] + [OCH2CHOCH2O], 15H), 3.96-4.01 (m, HC HOTs, 1H), 4.13 (dd [9 Hz, 3 Hz], HCHOTs, 1H), 7.32 (d, meta H, 2H), 7.78 (d, ortho H, 2H); IR (cm-1): 10801120 (br., C-O-C str.) 1-Bromo-2-hexadecyloxy-3-(2′-[2′′-hexadecyloxyethoxy]ethoxy)propane. 2-Hexadecyloxy-3-(2′-[2′′-hexadecyloxyethoxy] ethoxy)-1-propyl tosylate (140 mg, 0.2 mmol) and LiBr (46.6 mg, 0.5 mmol, 3 equiv) were dissolved in 2 mL of dry DMF, and the mixture was refluxed at 70 °C for 8 h. Then the reaction mixture was diluted with 100 mL of EtOAc and was washed twice with 30 mL portions of water. The water layer was extracted with EtOAc. The organic phase was then washed with brine and was dried over anhyd Na2SO4. EtOAc was evaporated under reduced pressure, and the pure compound was isolated in 73% yield (90 mg) by column chromatography over silica gel (Rf ∼ 0.8 at EtOAchexane (1:9)). The bromo compound was used for the next step immediately without further characterization. N-(2-Hexadecyloxy-3-[2′-{2′′-hexadecyloxyethoxy}ethoxy] propyl)-N,N,N-trimethylammonium Bromide (3). Dry NMe3 gas was passed into dry CH3CN (1 mL) in a pressure tube which was precooled cooled using an ice-acetone bath. 1-Bromo-2-hexadecyloxy-3-(2′-[2′′hexadecyloxyethoxy]ethoxy)propane (90 mg, 0.13 mmol) was dissolved in a mixture of CH3CN (2 mL)-toluene (0.5 mL) by slight warming and was transferred to the pressure tube. (It may be mentioned herein that the bromide was not soluble in CH3CN while it was very soluble in toluene. Thus, good yields were obtained when a minimum amount of toluene was used for the reaction.) The reaction mixture was then heated at 80 °C for 24 h. The resulting material was then cooled, and the excess trimethylamine was evaporated. This was then dissolved in CHCl3, and the solvent was removed under reduced pressure. Pure compound was isolated as a colorless waxy solid (100 mg, quantitative conversion) by column chromatography over silica gel (100-200 mesh) using 10% MeOH-CHCl3 as the eluting solvent. 1H NMR (500 MHz, CDCl3): δ: 0.90 (t, 2 × terminal CH3, 6H), 1.27 (br. m, 26 × CH2, 52H), 1.55 (br. m, 2 × C14H29CH2CH2O, 4H), 3.41-3.72 (m + s, singlet at 3.49, [7 × CH2O] + [3 × CH3N+], 14H + 9H), 3.97-4.14 (m, [OCH2CHOCH2N+] + [CH2N+], 1H + 2H); IR (cm-1): 1100 (broad, C-O-C str.); MS (MALDI-TOF): Calcd for C42H88O4N: 671.2. Found: 672.0 (M + H+); Anal. Calcd for C42H88O4NBr‚ 0.3H2O: C 66.69, H 11.81, N 1.85. Found: C 66.73, H 12.1, N 1.56. 3-Benzyloxy-2-hydroxy-1-hexadecyloxypropane (18). O-Benzyl glycerol (8) (299 mg, 1.6 mmol) was dissolved in dry THF and was cooled in an ice-water bath. NaH (55% in oil) (144 mg, 3.2 mmol), washed with dry hexane (to remove oil), was added to it and stirred at 0 °C for 15 min. To this was added 0.5 g (1.3 mmol) of


hexadecyl tosylate, and the reaction mixture was refluxed for 24 h. CH2Cl2 was added to the reaction mixture, and the resulting mass was acidified with 1.2 N aq HCl. The organic phase was then washed with water, followed by brine. The organic layer was isolated and dried over anhyd Na2SO4. Solvent was evaporated under reduced pressure and the pure compound was separated (200 mg, 40%) as a colorless liquid by column chromatography over silica gel (Rf ∼ 0.5 at EtOAc-hexane(1:4)). The spectroscopic properties match those of the reported compound. 1 H NMR (CDCl3, 300 MHz): δ: 0.88 (t, terminal CH3, 3H), 1.25 (br. m, 13 × CH2, 26H), 1.55 (p, C14H29C H2CH2O, 2H), 3.4-3.71 (m, 3 × CH2O, 6H), 3.93 (q, OCH2CHOCH2O, 1H), 4.54 (s, OC H2Ar, 2H), 7.25-7.34 (m, Ar, 5H); IR (cm-1): 1120 (br., C-O-C str.), 3400 (br., OH). 1-Hexadecyloxy-2-(2′-[2′′-hexadecyloxyethoxy]ethoxy)-3-benzyloxypropane (19). The alcohol 18 was taken (200 mg) in dry THF and was cooled in an icewater bath. NaH (55% in oil) (1.5 equiv) was washed with dry hexane to remove oil and was added to the solution of alcohol in THF. The mixture was stirred at room temperature for 15 min. A white precipitate was formed in the reaction mixture. To that was added 1.5 equiv of O-hexadecyl diethylene glycol tosylate, and the mixture was refluxed at 89 °C. After the reaction, THF was removed by evaporation under reduced pressure. Crude mixture was then dissolved in CH2Cl2 and washed with 1 N aq HCl, followed by water and brine. The organic phase was dried over anhyd Na2SO4. The required compound was isolated in 74% isolated yielding pure form as a slight yellow colored liquid by silica gel column chromatography using 20 EtOAc-hexane (Rf ∼ 0.4) as the elution mixture. 1H NMR (CDCl3, 300 MHz): δ: 0.88 (t, 2 × terminal CH3, 6H), 1.25 (br. m, 26 × CH2, 52H), 1.55 (p, 2 × C14H29CH2CH2O, 4H), 3.38-3.76 (m, [8 × CH2O] + [OCH2CHOCH2O], 17H), 4.53 (s, OCH2Ar, 2H), 7.25-7.31 (m, Ar, 5H); IR (cm-1): 1110 (br., C-O-C str.). 3-Hexadecyloxy-2-(2′-[2′′-hexadecyloxyethoxy]ethoxy)-1-propanol (20). 1-Hexadecyloxy-2-(2′-[2′′hexadecyloxyethoxy]ethoxy)-3-benzyloxypropane (19) (260 mg, 0.36 mmol) was dissolved in 5 mL of a mixture of dry EtOAc-MeOH (1:1). To that was added activated 10% Pd-charcoal (340 mg). Hydrogen gas was introduced to the flask. Hydrogenation was allowed to continue at atmospheric pressure, at room temperature for 15 h. Pdcharcoal was filtered off from the reaction mixture, and the pure compound was isolated as a low melting white solid (180 mg, 79% yield) by column chromatography over silica gel (Rf ∼ 0.5 at EtOAc-hexane (1:1)). 1H NMR (CDCl3, 300 MHz): δ: 0.88 (t, 2 × terminal CH3, 6H), 1.25 (br. m, 26 × CH2, 52H), 1.54-1.56 (m, 2 × C14H29CH2CH2O, 4H), 3.36-3.72 (m, 8 × CH2O, 16H), 3.83-3.87 (m, OCH2CHOCH2O, 1H); IR (cm-1): 1100 (br., C-O-C str.), 3400 (br., OH). 3-Hexadecyloxy-2-(2′-[2′′-hexadecyloxyethoxy]ethoxy)-1-propyl Tosylate (21). 3-Hexadecyloxy-2-(2′[2′′-hexadecyloxyethoxy]ethoxy)-1-propanol (180 mg, 0.29 mmol) was dissolved in 5 mL of dry CH2Cl2 and was cooled in an ice-water bath. To that were added 0.2 mL of dry pyridine and 138 mg of freshly recrystallized p-TsCl (0.725 mmol, 2.5 equiv). The reaction mixture was stirred at 0 °C for 15 min and then at room temperature for 24 h (till the starting alcohol disappeared as indicated from TLC). The reaction mixture was then diluted with CH2Cl2 and was washed with 2 N aq HCl followed by water and brine. The organic layer was separated and dried over anhyd Na2SO4. Solvent was evaporated under reduced pressure, and the pure compound was isolated


as a colorless liquid in 85% yield by column chromatography (silica) (Rf ∼ 0.4 at EtOAc-hexane (1:9) solvent mixture). 1H NMR (CDCl3, 300 MHz): δ: 0.88 (t, 2 × terminal CH3, 6H), 1.25 (br. m, 26 × CH2, 52H), 1.471.55 (m, 2 × C14H29CH2CH2O, 4H), 2.45 (s, CH3Ar, 3H), 3.32-3.71 (m, [7 × CH2O] + [OCH2CHOCH2O], 15H), 3.98-4.15 (m, C H2OTs, 2H), 7.32 (d, meta H, 2H), 7.79 (d, ortho H, 2H); IR (cm-1): 1080-1110 (br., C-O-C str.) 1-Bromo-2-(2′-[2′′-hexadecyloxyethoxy]ethoxy)-3hexadecyloxypropane. 3-Hexadecyloxy-2-(2′-[2′′-hexadecyloxyethoxy]ethoxy)-1-propyl tosylate (190 mg, 0.24 mmol) and LiBr (63.2 mg, 0.7 mmol, 3 equiv) were dissolved in 3 mL of dry DMF and refluxed at 70 °C for 8 h. The reaction mixture was then diluted with 150 mL of EtOAc and washed twice with 30 mL portions of water. The water layer was extracted with EtOAc. The organic layers were collected together, washed with brine, and dried over anhyd Na2SO4. EtOAc was evaporated under reduced pressure, and pure compound was isolated in 59% yield (90 mg) by column chromatography over silica gel (Rf ∼ 0.5 at EtOAc-hexane(5:95)). 1H NMR (CDCl3, 300 MHz): δ: 0.88 (t, 2 × terminal CH3, 6H), 1.25 (br. m, 26 × CH2, 52H), 1.54-1.56 (m, 2 × C14H29CH2CH2O, 4H), 3.42 (t, 2 × C15H31CH2O, 4H), 3.46-3.75 (m, [5 × CH2O] + [OCH2CHOCH2Br] + [CH2Br], 13H); IR (cm-1): 1100-1120 (br., C-O-C str.) N-(3-Hexadecyloxy-2-[2′-{2′′-hexadecyloxyethoxy}ethoxy]propyl)-N,N,N-trimethylammonium Bromide (4). Dry trimethylamine gas was passed into dry CH3CN (1 mL) in a pressure tube. 1-Bromo-2-hexadecyloxy-3-(2′-[2′′-hexadecyloxyethoxy]ethoxy)propane (90 mg) was dissolved in CH3CN-toluene (1:1) mixture by warming and was transferred to the pressure tube. The reaction mixture was heated at 80 °C for 24 h. The reaction mixture was then cooled, and the excess trimethylamine was evaporated by warming the mixture on a water bath. The crude mixture was then diluted with CHCl3, and the solvent was removed under reduced pressure. Pure compound was isolated as a colorless waxy solid (70 mg, 72%) by column chromatography over silica gel (100-200 mesh) using MeOH-CHCl3 (1:9) as eluent. 1H NMR (500 MHz, CDCl ): 0.88 (t, 2 × terminal CH , 3 3 6H), 1.25 (br. m, 26 × CH2, 52H), 1.54 (p, 2 × C14H29CH2CH2O, 4H), 3.35 (s, 3 × CH3N+, 9H), 3.39-3.66 (m, 7 × CH2O, 14H), 3.89-3.91, 4.13 (m, [CH2N+] + [OCH2CHOCH2N+], 2H + 1H); IR (cm-1): 1070-1150 (broad, C-O-C str.); MS (MALDI-TOF): Calcd for C42H88O4N: 671.2. Found: 671.6; Anal. Calcd for C42H88O4NBr‚ 0.2H2O: C 66.84, H 11.81, N 1.86. Found: C 66.8, H 11.93, N 1.51. Vesicle Preparation. Thin lipid films from individual lipids 1-4 were made in Wheaton glass vials by dissolving individual lipids in chloroform and evaporating the organic solvent under a steady stream of dry nitrogen. Last traces of organic solvent were removed by keeping these films under vacuum overnight. Water was added to each individual film and kept for hydration at 4 °C for 6-10 h. Then these samples were repeatedly freezethawed (ice-cold water to 50 °C) with intermittent vortexing to afford multilamellar vesicular aggregates (MLVs). Small unilamellar vesicles (SUV) were prepared by sonicating these MLVs in a bath sonicator above the phase transition temperature of the individual lipids for 10-15 min. Transmission Electron Microscopy. Lipid suspensions were examined under transmission electron microscopy by negative staining with 1% uranyl acetate. Vesicular suspensions were made in 1% uranyl acetate solution at a concentration of 1 mM. Sonicated suspen-


sions were spreaded onto carbon-Formvar coated copper grids (400 mesh), and the vesicles were allowed to settle onto the grid. Excess solution was wicked off using a filter paper, and the grid was air-dried for 30 min before examining under TEM. TEM was performed on a JEOL200 CX electron microscope typically with an accelerating voltage of 100 keV. Micrographs were recorded at a magnification of 80 000. Differential Scanning Calorimetry. Multilamellar vesicles were prepared in degassed water as mentioned above (concentration ) 1 mM), and their thermotropic behavior was investigated by differential scanning calorimetry using a CSC-4100 model multicell Differential Scanning Calorimeter (Calorimetric Sciences Corporation, Utah). Baseline thermogram was obtained by using degassed water in all the ampules including the reference cell to normalize cell to cell differences. Water (0.5 mL) was used for the baseline thermogram. The measurements were carried out in the temperature range of 25 °C to 65 °C at a scan rate of 20 °C/h. Prior to the experimental thermogram, samples were subjected to repeated heating and cooling scans in DSC. The thermograms for the vesicular suspensions were obtained by subtracting the respective baseline thermogram from the sample thermogram using the software ‘CpCalc’ supplied by the manufacturer. Peak position in the plot of excess heat capacity vs temperature was taken as the gel to liquid crystalline phase transition temperature for each vesicular suspension. The molar heat capacities, calorimetric enthalpies (∆Hc) and entropies (∆S) were also computed using the same software. The size of the cooperativity unit (CU) for the phase transition of each lipid was determined using the formula

CU )

∆HVH ∆Hc

where ∆HVH is the vant Hoff enthalpy and ∆Hc is the calorimetric enthalpy. vant Hoff enthalpy was calculated from the equation

∆HVH )

6.9T2m ∆T1/2

where Tm is the phase transition temperature (in Kelvin) and ∆T1/2 is the full width at half-maximum of the thermogram (26-28). Cell Culture, Plasmid DNA. HeLa cells were cultured in DMEM (Gibco BRL) supplemented with 10 fetal bovine serum (FBS) in T-25 culture flasks (Nunc, Denmark) and were incubated at 37 °C in a humidified atmosphere containing 5% CO2. Cells were regularly passaged by trypsinization using 0.1 trypsin (EDTA 0.02%, dextrose 0.05%, and trypsin 0.1%) in PBS (pH 7.2). Plasmid pEGFP-N1 (Clontech), which encodes for an enhanced green fluorescence protein (GFP) under a CMV promoter, was amplified in Escherichia coli (DH5R) and purified using Qiagen Maxi Prep Plasmid Purification protocol (Qiagen, Germany). Purity of the plasmid was checked by electrophoresis on 0.8% agarose gel and was found to be more than 80% supercoiled. Concentration of the DNA was estimated spectroscopically by measuring the absorption at 260 nm. Cytotoxicity. Toxicity of each cationic lipid formulation toward HeLa cells in the presence of 10% FBS was determined using 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay following literature procedures (29, 30). MTT gets converted


to an insoluble blue dye, formazan, in actively respiring cells. The assay estimates the concentration of the formazan formed, which, in turn, gives a measure of the population of cells that are metabolically active. A range of liposomal concentrations between 200 nM to 500 µM were investigated for toxicity studies. Cytotoxicity of the lipid formulations optimal for transfection experiments were found out under conditions exactly similar to transfection experiments. The lipid formulations (lipid + DOPE) were complexed with DNA (0.1 µg) at various charge ratios in a range that was found to be optimal for transfections (lipid to DNA charge ratio 1-4). DNA-lipid complexes were added to the cells in the absence of serum. After 5 h of incubation, enough serum containing medium was added so as to have a final serum concentration of 10%. After 44 h, 20 µL media was replaced with 20 µL of MTT solution and was incubated further for 5 h. Following 5 h of incubation, blue formazan microcrystals were seen at the wells when checked under the microscope. One hundred microliters of 10% SDS in 0.01 N HCl was then added to each well and incubated at 37 °C for 10 h for the lysis of the cells and the solubilzation of the formazan formed. The absorbance was measured using a microtiter plate reader. The % viability was then calculated as (OD570 - OD690)sample/ (OD570 - OD690)control × 100, where the untreated cells (where no lipid suspension was added) were used as control. The assay was carried out in triplicates. Transfection. All transfection experiments were carried out in antibiotic-free media. In a typical experiment, 96-well plates were seeded with 9000 cells/well in antibiotic-free DMEM 24 h before transfection such that they were ∼70% confluent at the time of transfection. Lipid formulation and DNA were serially diluted separately in DMEM containing no serum to have the required working stocks. DNA was used at a concentration of 0.1 µg/ well. The lipid and DNA were complexed in a volume of 20 µL by incubating them together at room temperature for about 30 min. The lipid concentrations were varied so as to obtain the required lipid/DNA charge ratios of 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8, and 2.0. Charge ratios here represent the ratio of charge on cationic lipid (in moles) to nucleotide base molarity and were calculated by considering the average nucleotide mass of 330. After 30 min of complexation, 50 µL of medium was added to the complexes (final DNA concentration ) 4.33 µM). Old medium was removed from the wells, and lipid-DNA complexes in 70 µL media were added to the cells. The plates were then incubated for 5 h at 37 °C in a humidified atmosphere containing 5% CO2. At the end of the incubation period, 130 µL of DMEM containing 15.4% FBS was added per well such that final serum concentration in the medium is 10%. Plates were further incubated for a period of 43 h before checking for the reporter gene expression. GFP expression was estimated by fluorescent microscopy and was quantified by flow cytometry analysis. All the experiments were performed in duplicate, and the results presented are the average of at least four such independent experiments. RESULTS AND DISCUSSION

Synthesis. The synthesis of the symmetric cationic lipids (1 and 2), which contain an equal number of oxyethylene units in both the chains at the C-1 and C-2 positions of the pseudoglyceryl backbone, is outlined in Scheme 1. The main step employed the coupling of the tosylates 6a and 6b with benzyl glycerol. The ether bond formation between benzyl glycerol and hexadecyl tosylate

Bhattacharya and Dileep Scheme 1a

a Reagents and conditions: (i) pTsCl, Py, CH Cl , rt, 25-50 2 2 h (82% for a and 68% for 6b); (ii) KOH, PhCH2Cl, benzene, reflux, 23 h; (iii) p-TsOH, MeOH,CH2Cl2, rt, 5 h (overall 53% for the steps ii and iii); (iv) 6, NaH, DMSO, Bu4N+I-, rt, 24-30 h (74 for n ) 0 and 67% for n ) 1); (v) H2, Pd/C, MeOH, EtOAc, 10 h (88% and 80%); (vi) p-TsCl, Py, CH2Cl2, rt, 27h (87% and 98%); (vii) LiBr, DMF, 70 °C, 8 h (81% and 84%); (viii) NMe3, MeCN-toluene, 80 °C, pressure tube, 24 h (74% ( 68%).

works reasonably well under classical etherification conditions involving KOH/benzene (24) or NaH/benzene under reflux. But, we observed that, in case of tosylates containing hydrophilic oxyethylene groups 6a and 6b, although such conditions (NaH/benzene, reflux, 24 h) yielded the required products 9a and 9b, the yields were only moderate (48% and 45%, respectively) (18). The reaction of benzyl glycerol with the corresponding chlorides under phase-transfer conditions (50% aq NaOH, Bu4N+HSO4-) (31) also yielded the required compounds, but again the yields were substantially lower. Moreover, in this case, isolation of the pure compounds from the reaction mixture was also cumbersome. The coupling under NaH/DMF (21) and KOH/DMSO reflux conditions were also not satisfactory. Interestingly, good yields were obtained when benzyl glycerol was treated with the corresponding tosylates 6a and 6b under NaH/DMSO in the presence of a phase-transfer catalyst, Bu4N+I-. The reaction could be carried out at room temperature, and the yields were much better (74% for 9a and 67% for 9b). More importantly, isolation of the pure products by column chromatography was comparatively easier under this condition. We found this method to be the most suitable one for etherification involving such hydrophilic tosylates. In general, the synthesis of lipids 1 and 2 was achieved as follows (Scheme 1). Ethylene glycol monohexadecyl ether 5a and diethylene glycol monohexadecyl ether 5b on reaction with tosyl chloride in the presence of pyridine in CH2Cl2 afforded the corresponding tosylates 6a and 6b. Benzyl glycerol 8 was synthesized by slight modification of a literature procedure from (()-1,2-O-isopropylidene glycerol (24). Benzyl glycerol 8 on reaction with either tosylate, 6a, or 6b in the presence of NaH/DMSO/ Bu4N+I- at room temperature afforded 9a or 9b, respectively, in good yields. Debenzylation of 9 to the glycerol derivatives 10 were achieved conveniently by hydrogenolysis. The direct bromination of 10 to the primary bromide 12 under Appel conditions (CBr4/PPh3 in CH2Cl2) was a very low-yielding reaction. So the alcohols were

Cationic Oxyethylene Lipids Scheme 2a

a Reagents and conditions: (i) DHP, concentrated HCl, rt, 1 h (98%); (ii) 13, aq NaOH (50%), Bu4N+HSO4-, 65 °C, 5 h; (iii) MeOH-CH2Cl2, concentrated HCl, rt, 2 h (overall 34% for the steps ii and iii); (iv) C16H33Cl, aq NaOH (50%), Bu4N+HSO4-, 65 °C, 5 days (53%); (v) H2, Pd/C, MeOH, EtOAc, 12 h (85%); (vi) p-TsCl, Py, CH2Cl2, rt, 24 h (80%); (vii) LiBr, DMF, 70 °C, 6 h (73%); (viii) NMe3, MeCN-toluene, 80 °C, pressure tube, 24 h (95%).

first converted to the corresponding tosylates 11a or 11b which on further reaction with LiBr in DMF under refluxing conditions yielded the corresponding bromides in good yields. The substitution of the tosylate residue in 11 with NaBr in acetone was, in contrast, very sluggish. The bromides 12a and 12b were finally converted in good yields to the corresponding cationic lipids 1 and 2, respectively, by reaction with trimethylamine in acetonitrile-toluene mixture in a screw-top pressure tube. Scheme 2 depicts the synthesis of the mixed-chain lipid, 3, in which one hydrophilic (CH2CH2O)2 linkage has been introduced only at the C-1 position of the pseudoglyceryl backbone. The alcohol 14 was synthesized by modification of a literature procedure (31). Briefly, chloroethoxyethanol was protected as a tetrahydropyranyl (THP) ether, 13 (25, 32). Benzyl glycerol 8 was alkylated at the primary position using 13 at the primary OH residue via a phase-transfer-catalyzed reaction, followed by the subsequent removal of the THP protecting group to yield the diol 14. The yield of this reaction was found to be strongly dependent upon the reaction time since the corresponding dialkylated derivative (31) was also formed if the reaction was allowed to continue beyond 4-5 h. The separation of the individual diol derivatives resulting from mono- and dialkylation of 8 was possible although difficult. The positional identity of the diol derivative 14, obtained by the monoalkylation, was established by converting the isolated diol 14 to a diester with palmitic acid. The downfield chemical shift of the methine proton (OCH2CHOCH2O) upon esterification was clearly observable. The diol 14 upon reaction with hexadecyl chloride under a similar phase-transfer etherification condition gave 15 in moderate yields. From 15, the cationic lipid 3 was then prepared via removal of the benzyl protecting group under hydrogenolytic conditions, conversion of the alcohol to bromide via tosylate, and quaternization with trimethylamine in a manner similar to the one described in Scheme 1. The synthesis of cationic lipid 4, in which the (CH2CH2O)2 unit remains at C-2 of the pseudoglyceryl backbone, has been achieved as summarized in the Scheme 3. Benzyl glycerol 8 was refluxed with 1 equiv of hexa-

Bioconjugate Chem., Vol. 15, No. 3, 2004 515 Scheme 3a

a Reagents and conditions: (i) C H OTs, NaH, THF, reflux, 16 33 24 h (40%); (ii) 6b, NaH, THF, reflux, 48 h (74%); (iii) H2, Pd/C, MeOH, EtOAc, 12 h (79%); (iv) p-TsCl, Py, CH2Cl2, rt, 24 h (85%); (v) LiBr, DMF, 70 °C, 20 h (59%); (vi) NMe3, MeCNtoluene, 80 °C, pressure tube, 24 h (72%).

Figure 2. Negatively stained transmission electron micrographs of aqueous suspensions of lipids 1 and 3 (scale: 90 nm for 1 cm).

decyl tosylate in the presence of NaH in THF to yield the monoalkylated derivative 18. It was then treated with 6b in the presence of NaH in dry THF to afford 19. This upon hydrogenolysis yielded the corresponding alcohol, which was finally converted to the desired lipid 4 following the same route as described in the previous cases. All the final compounds and those intermediates that are stable upon isolation were appropriately characterized by TLC, FT-IR, 1H NMR, mass spectrometry, and elemental analysis (cf. Experiment Section). Aggregation Properties. All the lipid molecules mentioned here formed stable suspensions in water. No precipitation was observed even after one month when these suspensions were stored at 4 °C. Upon hydration, the lipids having oxyethylene units at the linkage regions (1-4) were found to get dispersed in water with greater ease when compared to the lipid having just an ether linkage (DHTMA). Moreover, the suspensions formed from the lipids 1-4 were optically clear in contrast to DHTMA which yielded translucent suspension. Transmission Electron Microscopy. TEM examination of air-dried aqueous suspensions revealed the existence of membranous structures for all the four lipids 1-4. The suspensions formed from the lipids 1 and 2 having symmetrical chains appeared as distinct spherical aggregates in the micrographs. In contrast, the lipid 3 that has a di(oxyethylene) linkage only at the C-1 chain formed aggregates of irregular morphologies (See Figure 2). Lipid 4 formed vesicular aggregates which were considerably larger in comparison with the aggregates observed with the other lipids (not shown). The diameters of the aggregates formed from the symmetrical lipids 1 and 2 were in the range of 40-100 nm. Lipid 3 formed vesicles of diameters 90-150 nm whereas relatively larger aggregates (∼220 nm) were observed in the aqueous suspensions of lipid 4 (Table 1).



Table 1. Characteristics of the Aggregates Formed by the Lipids 1-4 lipid

T m°a

∆H (kcal mol-1)

∆Sb (cal mol-1 K-1)

CUc

avg diamd (nm)

ID50e (µM)

alone

1 2 3 4 DHTMA

46.3 45.6 41.2 45.1 45.8

11.3 9.7 12.3 6.7 16.7

35.4 30.4 39.1 21.0 52.4

115 92 61 -g 42

40-80 55-100 90-150 220 -

190 75 175 195 >500

37 5 26 34 95

% viabilityf with 50 mol % chol 75 81 78 76 84

a The maximum deviation was (0.1 °C. b Enthalpy and entropy for the heating scan. The ∆H and ∆S values are average of three successive runs and the values are within (5%, c Size of cooperativity unit. d Average diameter as indicated from TEM. e Concentration that caused 50% cell death. f At [cationic lipid] ) 250 µM. g Could not be measured reliably due to baseline drifts.

Differential Scanning Calorimetry. With all the lipid suspensions examined, clear thermotropic solid gel to fluid liquid crystalline-like phase transitions typical of membranous aggregates were observed. The phase transitions of all the individual liposomal suspensions were found to be reversible with subsequent heating and cooling scans showing essentially the same histograms. The experimentally obtained phase transition temperature (Tm) values obtained are presented in Table 1. The phase-transition temperatures of lipids 1, 2, and 4 were comparable to that of DHTMA (45-46 °C). In contrast, the phase transition temperature of 3 was relatively lower (41 °C). These cationic lipids 1-4 and DHTMA have identical headgroup and hydrocarbon chain lengths. However, they differ at the linkage region in terms of the number of oxyethylene units. Clearly the DSC results suggest that insertion of oxyethylenes at the linkage region do not affect the phase-transition temperature of the resulting membranes. In a given series of lipids, the lengthening of the chain length is expected to enhance the phase-transition temperature (15). However, since the Tm values do not increase relative to that of DHTMA, it is clear that incorporated oxyethylene units do not significantly contribute to chain melting transition. It may be further noted that even the unsymmetrical lipid, 4 exhibited comparable Tm as that of DHTMA. Only the lipid 3 showed a lower Tm value. Therefore the effect on the thermal properties of these membranes are manifested only when the oxyethylenes are incorporated in the C-1 position alone. In general, there was a decrease in the enthalpy of phase transition upon incorporation of oxyethylene units at the linkage region of cationic lipids. Incorporation of oxyethylene units at the interfaces most likely enhances the hydration of the lipid assemblies. The effect of such interfacial hydration will be more predominant in the melted, disordered phase of the lipid assemblies. This may be responsible for lower phase-transition enthalpies observed with lipids 1-4. The nature of the thermal transitions of these oxyethylene-bearing cationic lipids were found to be more cooperative compared to their diether analogue DHTMA (see Table 1). Again enhanced hydration could be responsible for more cohesive interactions among such lipids leading to the manifestation of higher cooperativity. Biological Activity: Cytotoxicity and Gene Delivery. Having investigated the aggregation properties of these lipids 1-4 by electron microscopy and calorimetry, we were interested in examining their biological usefulness. Toward this end, we first systematically examined the cytotoxicity manifested by these lipids and then investigated their gene transfer activity in HeLa cells. The cytotoxic effects of the cationic lipids 1-4 and their diether counterpart DHTMA toward HeLa cells are shown in Figure 3 and are summarized in Table 1 along with their ID50 values. Incorporation of the oxyethylene

Figure 3. Cytotoxicity exhibited by the cationic lipids 1-4 toward HeLa cells. Toxicity exhibited by the diether lipid DHTMA is also shown. Shown are the data from one representative experiment. Standard deviations were within 5%.

units at the linkage region of a lipid molecule is found to increase its cytotoxicity. Among the lipids investigated, the lipid 2, containing two oxyethylene units in both of the chains, is found to be the most cytotoxic formulation. The morphology of dying cells in the case of these oxyethylene lipids were considerably different from that of DHTMA. The lipid DHTMA, which has just an ether functionality at the linkage region, was least cytotoxic. Its ID50 value was above the range of concentrations studied (>500 µM). Notably, all the ID50 values were much higher than the range of concentration at which a typical transfection experiment was performed (i.e., ∼5 µM). The transfection efficiencies (% of fluorescent cells due to the expressed green fluorescence protein (GFP)) of various cationic lipid formulations are shown in Figure 4. The untransfected control cells and the cells exposed only to the plasmid DNA did not show any fluorescent cells 48 h posttransfection. The cationic lipid, DHTMA which possess single ether links between the pseudoglyceryl skeleton and the hydrocarbon chains, showed only basal level transfection activity. A clear enhancement in transfection efficiencies were observed with the cationic lipids bearing oxyethylene linkage functionalities. The unsymmetrical lipids 3 and 4 were more efficient than the symmetrical ones 1 and 2. Lipid 3, bearing two oxyethylene units only at the C-1 position of the pseudoglyceryl backbone, was found to be the best cytofectin among the lipids studied. Effect of Helper Lipids. Incorporation of helper lipids such as cholesterol (33, 34) and dioleyoyl phosphatidyl ethanolamine (DOPE) (3, 4) is known to enhance the transfection efficiencies of many of the cationic lipidbased formulations. So, we decided to investigate how these helper lipids modify the activities of presently described oxyethylene bearing cationic lipids.


Figure 4. Potentiation of transfection abilities of cationic lipids toward HeLa cells upon incorporation of oxyethylene units at the linkage region. (control: untransfected cells; no lipid: cells exposed to DNA alone). Shown are the optimal transfection efficiencies of each liposomal formulation. Lipid to DNA charge ratio was 1-1.4. Cell viability was >80% for all the lipid formulations.


Figure 6. Reduction of cytotoxicity of cationic lipids 1-4 on incorporation of 50 mol % of cholesterol. Cationic lipid concentraion was 250 µM in all the formulations.

Figure 7. Enhancement in transfection efficiencies of cationic lipids 1-4 upon incorporation of 50 mol % of DOPE. Shown are the optimal transfection efficiencies of each lipid formulations.

Figure 5. Cytotoxicity exhibited by the cationic liposomes containing 50 mol of cholesterol toward HeLa cells. Shown are the data from one representative experiment. Standard deviations were within 5%.

Cholesterol-Containing Cationic Liposomes. The cytotoxicities of all the oxyethylene-bearing cationic lipids were significantly reduced when cholesterol was incorporated in their liposomal formulations (Figure 5). When cholesterol was present at a 1:1 molar ratio, the ID50 values of all the lipid formulations were much above the range of concentrations studied. This reduction in toxicity was as high as 94% in case of lipid 2 at a cationic lipid concentration of 250 µM (Table 1). For lipids 1, 3, and 4, the cell viability was increased by 50%, 66%, and 55%, respectively, upon incorporation of cholesterol under similar conditions. It is not clear whether the reduction in cytotoxicity upon inclusion of cholesterol in these liposomes is just due to the cholesterol inhibiting the cellular liposomal uptake in some fashion. The liposomal formulations containing cholesterol at molar ratios 20 or 50 was investigated for their transfection efficiencies. The formulations in the mol-to-mol ratios of cholesterol/cationic lipid (1:4) and (1:1) were prepared by freeze-thaw followed by sonication. The transfection efficiencies of both multilamellar and unilamellar aggregates against HeLa cell line for plasmid p EGFP-N1 were investigated. No significant improvement in the transfection efficiencies was observed upon incorporation of cholesterol at either of these molar ratios. The observed trasnsfection efficiencies of cholesterol containing formulations were comparable to that of neat lipid formulations. Effect of Inclusion of Dioleyoyl Phosphatidyl Ethanolamine. Though cholesterol did not influence gene trans-

fection of cationic lipids 1-4, DOPE, when used as a helper lipid, increased their transfection efficiencies significantly. There was ∼3-4 times enhancement in transfection efficiency when the liposomal formulations contained 50 mol of DOPE (see Figure 7). The lipid formulation involving the unsymmetrical lipid 3 (lipid 3/DOPE at molar ratio 1:1) was the most efficient formulation under this condition also. The addition of DOPE to DHTMA also improves the transfection efficiency, but the efficiency of formulation, 1:1 DHTMA/ DOPE, is orders of magnitude lower than the highly active formulations, e.g. 3/DOPE. It is known that lipoFECTACE (a recipe composed of 1:1 DOPE and cationic lipid) is better than the cationic lipid alone (35). The modulation of transfection efficiencies were only moderate when DOPE was present at 25 and 75 mol % in the lipid formulations. The transfection efficiencies showed a bell-shaped dependency on cationic lipid concentration, reaching an optimal value near lipid/DNA charge ratio 1.2 (see Figure 8). The active formulations (formulations containing 50 mol % of DOPE) exhibited very little cytotoxicity during transfection experiments. The % viability of HeLa cells under exact transfection conditions is presented in Figure 9. The toxicities of all the lipid formulations were

Cationic Oxyethylene Lipids. Synthesis, Aggregation, and Transfection

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