Effect of the Hydrocarbon Chain and Polymethylene Spacer Lengths

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Bioconjugate Chem. 2007, 18, 2144–2158

Effect of the Hydrocarbon Chain and Polymethylene Spacer Lengths on Gene Transfection Efficacies of Gemini Lipids Based on Aromatic Backbone Avinash Bajaj,† Bishwajit Paul,† S. S. Indi,‡ Paturu Kondaiah,§ and Santanu Bhattacharya*,†,| Department of Organic Chemistry, Department of Microbiology and Cell Biology, and Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science, Bangalore 560 012, India, and Chemical Biology Unit of JNCASR, Bangalore 560 064, India. Received May 18, 2007; Revised Manuscript Received August 27, 2007

Design, syntheses, and gene delivery efficacies of fifteen novel gemini (dimeric) and three monomeric cationic lipids anchored on an aromatic backbone have been described. Each new lipid has been used for liposome formation, and optimal formulations were used to determine the structure–activity correlation of the gene transfection efficacies of these lipids in HeLa and HT1080 cells. The results of the present investigation bring out the effect of hydrocarbon chain lengths and the length of the spacer between the headgroups on gene transfection efficiencies of the cationic gemini lipids based on aromatic backbone. The lipids bearing n-C14H29 hydrocarbon chain lengths have been found to be the best transfecting agents compared to their counterparts with n-C16H33 and n-C12H25 chains in HeLa cells. On the other hand, in HT1080 cells, the lipids based on n-C12H25 and n-C14H29 chains were found to be more potent transfecting agents than lipids possessing n-C16H33 chains. Transmission electron microscopy examination revealed the existence of spherical lipid–DNA complexes.

INTRODUCTION Over the years, the definition of a “drug molecule” has changed considerably. Drugs have emerged from active components of certain natural products, or via the design of specific enzyme inhibitors. The recent realization of using the genetic material, DNA as a potential drug has led to the development of gene therapy (1). Gene therapy can be envisaged as an attempt to cure a disease by supplementing the aberrant gene with a functional one or by the delivery of a suicide gene (2) or by transfer of a gene for the production of a therapeutic protein (3, 4). In recent years, many cationic lipid based formulations have been developed to aid the delivery of DNA, mRNA, siRNA, antisense oligonucleotide, proteins and even drugs into eukaryotic cells. Because of low levels of toxicity and very low immunogenicity, their ability to deliver large pieces of DNA, and ease of preparation, cationic lipids are increasingly used as the nonviral transfection vectors as compared to viral vectors, which have several limitations (5–13). In addition, viral vectors add complications during gene transfer by generating adverse immunogenic responses through recombination events with the host genome (14). Cationic lipid and polymeric suspensions readily form complexes with the negatively charged DNA (gene) under ambient conditions (15). The molecular structure of cationic lipids is an important parameter that controls their DNA complexation and gene transfection activity (16). Accordingly, we have investigated the role of various molecular-level modifications in different synthetic lipids on their membraneforming properties (17, 18) and further influence on gene delivery events (19–22). For instance, the functional group that * Corresponding author. Phone: (91)-80-2293-2664. Fax: (91)-802360-0529. Email: [email protected]. † Department of Organic Chemistry, Indian Institute of Science. ‡ Department of Microbiology and Cell Biology, Indian Institute of Science. § Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science. | Chemical Biology Unit of JNCASR.

links the polar headgroup and the hydrocarbon chains of such lipid molecules plays a crucial role in their utilization in gene transferevents.Thus,N-[1-(2,3-dioleyloxy)propyl)]-N,N,N-trimethylammonium chloride (DOTMA), which contains an ether linkage between the headgroup and the long oleyl chains shows greater in ViVo transfection efficiency than the corresponding ester analogue N-[1-(2,3-dioleoyloxy)propyl)]-N,N,N-trimethylammonium chloride (DOTAP) (23). Similarly, cholesterol-based lipids (24) and lipopolymers (25, 26) are also known as potent transfection agents. Here also, the role of functional group that connects cationic headgroup with a cholesteryl backbone assumes importance (19). Gemini surfactants, i.e. amphiphiles molecules containing two head groups and two aliphatic chains, linked by a rigid or flexible spacer, show more interesting surfactant properties in solution compared to their monomeric counterparts. Recently, a number of reports appeared in the literature which describe the aggregation and transfection properties of gemini surfactants (27–30). We reported, for the first time, the synthesis and aggregation behavior of gemini pseudoglyceryl lipids (31, 32). It was shown that the aggregation properties of the gemini lipids could be modulated by the length of the spacer (33). Recently, the role of the oxyethylene-type spacer between the cationic headgroups on membrane aggregation and hydration properties was also investigated (34). Ahmad and co-workers showed the transfection properties of the cardiolipin-based cationic lipids (35). Cholesterol-based gemini lipids have been shown to deliver the DNA into the mammalian cells very efficiently and even in the presence of serum (36, 37). The gene delivery efficiencies strongly depend on the length of the spacer between the headgroups. Although there is no systematic study on the use of gemini lipids for gene transfer that are based on aromatic units, there are a few reports in the literature that describe the transfection activities of cationic liposomes based on aromatic headgroups or backbones. Hoekstra et al. showed the gene delivery properties of certain pyridinium surfactants (38). Balaban and co-workers reported the transfection properties of various pyridinium based cationic Monomeric, gemini, and oligomeric

10.1021/bc700181k CCC: $37.00  2007 American Chemical Society Published on Web 11/21/2007

Aromatic Backbone Based Lipids

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Figure 1. Molecular structures of the monomeric lipids 1–3, gemini lipids 4–6 synthesized and studied in this work, and structures of DOTMA, DOTAP, and DOPE.

surfactants having structural variations at the level of hydrophobic segment, linker, and counter ions (39). Safinya and coworkers have shown that multivalent cationic lipids with an aromatic backbone hold promise for superior gene transfection (40). Aromatic cationic lipids (TRX) possessing amidine headgroup have been developed by Sakurai and co-workers (41). Recently, Phanstial and co-workers reported the transfection properties of aromatic lipophilic polyamines (42). However, there is no systematic study that examines the transfection activities of a series of gemini lipids based on an aromatic backbone. In addition, none of above reports addressed the important issue of serum compatibility of the liposomal formulations. We previously reported the aromatic backbone based gemini lipids (43). Subsequently, we described the synthesis of aromatic lipids bearing n-C16H33 hydrocarbon chains that differ from each other in the length of the polymethylene spacer (44). Herein, we present the synthesis of two analogous series of gemini lipids based on aromatic backbone bearing n-C12H25 and n-C14H29 hydrocarbon chains (Figure 1). We also report the transfection properties of these new gemini and three monomeric cationic lipids based on an aromatic backbone that differ in the hydrocarbon chain lengths and the length of the spacer between the headgroups. We investigated their transfection properties in detail in HeLa and HT1080 cells in the absence and presence of serum conditions. Gemini lipids showed enhanced transfection as compared to Lipofectin, which is a monomeric lipid, structurally related to the present set of gemini lipids, and commercially available reagent based on a 1:1 (w/w) ratio of DOTMA/DOPE1 formulation. The gemini lipids bearing n-C14H29 chain lengths have been found to be more effective transfecting agents than their analogues with n-C16H33 and n-C12H25 chains in HeLa cells, whereas in HT1080 cells, we 1 Abbreviations: DOPE, 1,2-dioleoyl-L-R-glycero-3-phosphatidylethanolamine; DOTMA, N-[1-(2,3-dioleoyloxypropyl)]-N,N,N-trimethylammonium chloride; DOTAP, N-[1-(2,3-dioleoyloxy)propyl]-N,N,Ntrimethylammonium chloride; MFI, mean fluorescence intensity; FACS, fluorescence-activated cell sorting.

observed that the lipids based on n-C12H25 and n-C14H29 chains were more effective than lipids bearing n-C16H33 chains.

EXPERIMENTAL DETAILS Materials and Methods. All reagents, solvents, and chemicals used in this study were of the highest purity available. The solvents were dried prior to use. Column chromatography was performed using 60–120 mesh silica gel. NMR spectra were recorded using a Jeol JNM λ-300 (300 MHz for 1H) spectrometer. The chemical shifts (δ) are reported in ppm downfield from the internal standard, TMS, for 1H NMR. Mass spectra were recorded on a Kratos PCKompact SEQ V1.2.2 MALDI-TOF spectrometer or on a MicroMass ESI-TOF spectrometer or Shimadzu tabletop GC-MS or ESI-MS (HP1100LC-MSD). Infrared (IR) spectra were recorded on a Jasco FT-IR 410 spectrometer using KBr pellets or neat. Lipids were synthesized as described below and were characterized fully by their 1H NMR, mass spectra, and elemental analysis. Synthesis. Methyl-3,4-dihydroxybenzoate (8) (45). To a solution of 2 g (12.98 mol) of 3,4-dihydroxy benzoic acid (7) in dry MeOH (8 mL) was added conc HCl dropwise in ice–cold conditions with constant stirring. The resultant mixture was then refluxed at 70 °C for 12 h. After that, the solution was concentrated and extracted using EtOAc, and the pure product was isolated as a brown solid in 90% yield upon rotary evaporation. IR (neat, cm-1): 3351, 1696, 1603, 1532, 1435. 1 H NMR (CDCl3, 300 MHz): δ 3.87 (s, 3H, -O-CH3), 5.52 (s, 1H, -OH), 5.77 (s, 1H, -OH), 6.89–6.92 (d, 1H, J ) 9.0 Hz, ArH), 7.57–7.59 (m, 2H, ArH). Methyl-3,4-dialkyloxybenzoate (9a–9c). A mixture of 1.84 g (11.2 mmol) of 8, 28 mmol of appropriate alkyl bromide, and 33.6 mmol of K2CO3 in acetone was refluxed at 70 °C for 3 days. After that, the solution was concentrated, and the reaction mixture was taken in EtOAc. The organic layer was washed with water, brine, and then dried over anhydrous Na2SO4. The resulting solution was concentrated, and the residue was purified by chromatography over silica gel column using n-hexane/ EtOAc as eluent. The pure product was isolated as a white solid in 70–75% yield.

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Methyl-3,4-didodecyloxybenzoate (9a) (46). IR (neat, cm-1): 2910, 2848, 1712, 1590, 1517, 1467. 1H NMR (CDCl3, 300 MHz): δ 0.86–0.90 (t, 6H, J ) 6Hz, 2× -CH3), 1.26–1.46 (br m, 36H, 2× -(CH2)9), 1.78–1.86 (m, 4H, 2× -O-CH2-CH2), 3.88 (s, 3H, -O-CH3), 4.01–4.06 (m, 4H, 2× -O-CH2), 6.84–6.87 (d, 1H, J ) 9.0 Hz, ArH), 7.53 (s, 1H, ArH), 7.61–7.65 (m, 1H, ArH). LRMS: 505 (M + H+). HRMS: Calcd 505.4257 (M + H+); found 505.4253. Methyl-3,4-ditetradecyloxybenzoate (9b) (46). IR (neat, cm-1): 2917, 2850, 1712, 1598, 1517, 1467. 1H NMR (CDCl3, 300 MHz): δ 0.86–0.90 (t, 6H, J ) 6Hz, 2× -CH3), 1.26–1.46 (br m, 44H, 2× -(CH2)11), 1.82 (m, 4H, 2× -O-CH2-CH2), 3.88 (s, 3H, -O-CH3), 4.04 (m, 4H, 2× -O-CH2), 6.84–6.88 (d, 1H, J ) 9.0 Hz, ArH), 7.54 (s, 1H, ArH), 7.64 (m, 1H, ArH). LRMS: 561 (M + H+). HRMS: Calcd 561.4883 (M + H+); found 561.4871. Methyl-3,4-dihexadecyloxybenzoate (9c) (46). IR (neat, cm-1): 2910, 2848, 1712, 1590, 1517, 1467. 1H NMR (CDCl3, 300 MHz): δ 0.86–0.90 (t, 6H, J ) 6Hz, 2× -CH3), 1.26–1.46 (br m, 52H, 2× -(CH2)13), 1.78–1.86 (m, 4H, 2× -O-CH2-CH2), 3.88 (s, 3H, -O-CH3), 4.01–4.06 (m, 4H, 2× -O-CH2), 6.84–6.87 (d, 1H, J ) 9.0 Hz, ArH), 7.53 (s, 1H, ArH), 7.61–7.65 (1H, m, ArH), LRMS: 656 (M + K+). 3,4-Dialkyloxybenzyl aAlcohol (10a–10c). To a suspension 0.6 g (5 mmol) of LiAlH4 in dry THF (5 mL) was added a solution of 3.24 mmol of 9a–9c dropwise in ice–cold conditions with constant stirring under a nitrogen atmosphere. The ice bath was removed, and the resultant was refluxed at 70 °C overnight. Then, moist EtOAc (20 mL) was added slowly and stirred for 10 min at room temperature. The solvent was removed under vacuum, and then the reaction mixture was taken in CHCl3 and washed with saturated Na, K-tartrate solution (50 mL). The aqueous layer was extracted twice with CHCl3. All organic layers were collected together and dried over anhydrous Na2SO4, and the solvent was evaporated to leave a solid, which was purified by column chromatography using n-hexane/EtOAc as eluent. The pure product was isolated as a white solid in 90–95% yield. 3,4-Didodecyloxybenzyl Alcohol (10a). IR (neat, cm-1): 3332, 2956, 2917, 2850, 1590, 1517, 1467. 1H NMR (CDCl3, 300 MHz): δ 0.86–0.90 (t, 6H, J ) 6Hz, 2× -CH3), 1.26–1.46 (br m, 36H, 2× -(CH2)9) 1.79 (m, 4H, 2× -O-CH2-CH2), 3.96–4.02 (m, 4H, 2× -O-CH2), 4.60 (s, 2H, -CH2-Ar), 6.85 (s, 2H, ArH), 6.92 (s, 1H, ArH). LRMS: 499 (M + Na+). HRMS: Calcd 499.4127 (M + Na+); found 499.4231. 3,4-Ditetradecyloxybenzyl Alcohol (10b). IR (neat, cm-1): 3342, 2917, 2850, 1590, 1517, 1467. 1H NMR (CDCl3, 300 MHz): δ 0.86–0.90 (6H, t, J ) 6Hz, 2× -CH3), 1.26–1.46 (br m, 44H, 2× -(CH2)11), 1.79 (m, 4H, 2× -O-CH2-CH2), 3.96–4.02 (m, 4H, 2× -O-CH2), 4.60 (s, 2H, -CH2-Ar), 6.85 (s, 2H, ArH), 6.92 (s, 1H, ArH). LRMS: 555 (M + Na+). HRMS: Calcd 555.4753 (M + Na+); found 555.4743. 3,4-Dihexadecyloxybenzyl Alcohol (10c). IR (neat, cm-1): 3348, 2954, 2916, 2849, 1518, 1466. 1H NMR (CDCl3, 300 MHz): δ 0.86–0.90 (t, 6H, J ) 6Hz, 2× -CH3), 1.26–1.46 (br m, 52H, 2× -(CH2)13), 1.80 (m, 4H, 2× -O-CH2-CH2), 3.96–4.02 (m, 4H, 2× -O-CH2), 4.6 (s, 2H, -CH2-Ar), 6.86 (s, 2H, ArH), 6.92 (s, 1H, ArH). LRMS: 612 (M + Na+). HRMS: Calcd 611.5379 (M + Na+); found 611.5370. 1-(3,4-Dialkanoyloxyphenylmethyl) Dimethylamine (11a– 11c). To a stirring solution of 0.76 mmol of the benzyl alcohol (10a–10c), 0.620 g (3 mmol) of PBr3 in dry DCM was added dropwise in ice–cold conditions. Slowly, the resultant was allowed to rise to room temperature, and the mixture continued to stir for 24 h. Since the bromo compound was unstable, no isolation was attempted. The subsequent step was done in situ by adding excess Me2NH in MeOH and again kept for 12 h.

Bajaj et al.

The mixture was concentrated, and the residue was dissolved in CHCl3. The organic mixture was washed with 1 N HCl (2 × 50 mL) followed by water, saturated NaHCO3, and brine, and then dried over anhydrous Na2SO4. The organic solvent was removed and then subjected to column chromatography over silica gel using MeOH/CHCl3as an eluent. The pure compound was isolated as a white solid in 80–85% yield. 1-(3,4-Didodecyloxyphenylmethyl) Dimethylamine (11a). IR (neat, cm-1): 2921, 2852, 1589, 1513, 1467. 1H NMR (CDCl3, 300 MHz): δ 0.86–0.90 (t, J ) 6Hz, 6H, 2× -CH3), 1.26–1.46 (br m, 36H, 2× -(CH2)9), 1.79–1.82 (m, 4H, 2× -O-CH2-CH2), 2.23 (s, 6H, 2× -N-CH3), 3.78 (s, 2H, -CH2-Ar), 3.95–4.01 (m, 4H, 2× -O-CH2), 6.78–6.87 (m, 3H, ArH). LRMS: 504 (M + H+). HRMS: Calcd 504.4780 (M + H+); found 504.47. 1-(3,4-Ditetradecyloxyphenylmethyl) Dimethylamine (11b). IR (neat, cm-1): 2923, 2854, 1590, 1511, 1467. 1H NMR (CDCl3, 300 MHz): δ 0.86–0.90 (t, 6H, J ) 6Hz, 2× -CH3), 1.26–1.46 (br m, 44H, 2× -(CH2)11), 1.80 (m, 4H, 2× -O-CH2-CH2), 2.21 (s, 6H, 2× -N-CH3), 3.33 (s, 2H, -CH2-Ar), 3.97 (m, 4H, 2× -O-CH2), 6.79 (m, 3H, ArH). LRMS: 561 (M + H+). HRMS: Calcd 560.5406 (M + H+); found 560.5416. 1-(3,4-Dihexadecyloxyphenylmethyl) Dimethylamine (11c). IR (neat, cm-1): 2917, 2850, 1590, 1517, 1467. 1H NMR (CDCl3, 300 MHz): δ 0.86–0.90 (t, 6H, J ) 6Hz, 2× -CH3), 1.26–1.46 (br m, 52H, 2× -(CH2)13), 1.79 (m, 4H, 2× -O-CH2-CH2), 2.56 (s, 6H, 2× -N-CH3), 3.78 (s, 2H, -CH2-Ar), 3.92–3.98 (m, 4H, 2× -O-CH2), 6.80 (s, 2H, ArH), 6.93 (s, 1H, ArH). LRMS: 617 (M + H+). HRMS: Calcd 616.6032 (M + H+); found 616.5940. 1-(3,4-Dialkyloxyphenylmethyl)- N,N,N-trimethyl Ammonium Iodide (1, 2, 3). To a solution of 11a–c (0.11 mmol) in dry EtOH, MeI (excess) was added in a screw-top pressure tube. The resulting mixture was heated to reflux at 80 °C for 24 h. The removal of solvent and subsequent washing with EtOAc– MeOH (9:1) produced a yellowish–white compound. The compound was isolated in quantitative yield. Lipid 1. 1H NMR (300 MHz, CDCl3): δ 0.86–0.90 (t, 6H, J ) 6.0 Hz, 2× -CH3), 1.26–1.46 (m, 36H, 2× -(CH2)9), 1.75–1.87 (m, 4H, 2× -O-CH2-CH2-), 3.35 (s, 9H, 3× -N+CH3), 3.98–4.06 (m, 4H, 2× -O-CH2-CH2-), 4.87 (s, 2H, -N+CH2-Ar), 6.88 (d, 1H, J ) 9.0 Hz, ArH), 7.11 (d, 1H, J ) 9.0 Hz, ArH), 7.20 (s, 1H, ArH). ESI-MS: 519.2 (M+). Anal. (C34H64NO2I · 2H2O) C, H, N. Lipid 2. 1H NMR (300 MHz, CDCl3): δ 0.86–0.90 (t, 6H, J ) 6.0 Hz, 2× -CH3), 1.26–1.46 (m, 44H, 2× -(CH2)11), 1.80 (m, 4H, 2× -O-CH2-CH2-), 3.36 (s, 9H, 3× -N+-CH3), 3.98–4.06 (m, 4H, 2× -O-CH2-CH2-), 4.92 (s, 2H, -N+-CH2Ar), 6.86–6.89 (d, 1H, J ) 9.0 Hz, ArH), 7.10–7.13 (d, 1H, J ) 9.0 Hz, ArH), 7.23 (m, 1H, ArH). ESI-MS: 574.5 (M+). Anal. (C38H72NO2I) C, H, N. Lipid 3. 1H NMR (300 MHz, CDCl3): δ 0.85 (t, 6H, J ) 7.0 Hz, 2× -CH3), 1.25–1.46 (m, 52H, 2× -(CH2)13), 1.78–1.80 (m, 4H, 2× -O-CH2-CH2-), 3.35 (s, 9H, 3× -N+-CH3), 3.97–4.05 (m, 4H, -O-CH2-CH2-), 4.91 (s, 2H, -N+-CH2-Ar), 6.87 (d, 1H, J ) 8.1 Hz, ArH), 7.11 (d, 1H, J ) 8.1 Hz, ArH), 7.21 (s, 1H, ArH). ESI-MS: 631 (M+). Anal. (C42H80 NO2I · 2H2O) C, H, N. General Method for the Synthesis of Gemini Lipids (4, 5, 6). A solution of 11a–c (0.24 mmol) and appropriate R,ωdibromoalkane (0.080 mmol) in dry MeOH/EtOAc mixture was refluxed over a period of 48–96 h. It was cooled, and solvent was evaporated to produce a crude solid. It was repeatedly washed with dry EtOAc to remove unreacted starting material and finally subjected to repeated crystallization from a mixture of EtOAc and MeOH (9:1), which gives white solid. Yield 40–50%. Lipid 4a. 1H NMR (300 MHz, CDCl3): δ 0.86–0.90 (t, J ) 6.0 Hz, 12H, 4× -CH3), 1.26–1.46 (m, 72H, 4× -(CH2)9), 1.80


(m, 8H, 4× -O-CH2-CH2), 3.07 (br m, 2H, -N+-CH2-CH2-), 3.22 (s, 12H, 4× -N+-CH3), 3.98–4.01 (m, 12H, 4× -O-CH2CH2- and 2× -N+-CH2-CH2), 4.71 (s, 4H, 2× -N+-CH2-Ar), 6.86 (d, J ) 9.0 Hz, 2H, ArH), 7.08 (m, 4H, ArH). ESI-MS: 459.4 ((C12H25O)2C6H3CH2+), 589.5 ((C12H25O)2C6H3CH2N+ (CH3)2(CH2)3N(CH3)2). Anal. (C69H128N2O4Br2 · 2H2O) C, H, N. Lipid 4b. 1H NMR (300 MHz, CDCl3): δ 0.86–0.90 (t, J ) 6.0 Hz, 12H, 4× -CH3), 1.25–1.46 (m, 72H, 4× -(CH2)9), 1.77 (m, 12H, 4× -O-CH2-CH2- and 2× -N+-CH2-CH2-), 3.12 (s, 12H, 4× -N+-CH3), 3.96–4.02 (m, 12H, 4× -O-CH2-CH2- and -N+-CH2-CH2), 4.67 (s, 4H, 2× -N+-CH2-Ar), 6.88 (d, J ) 9.0 Hz, 2H, ArH), 7.11 (m, 4H, ArH). ESI-MS: 459.4 ((C12H25O)2C6H3CH2+), 603.5 ((C12H25O)2C6H3CH2N+(CH3)2(CH2)4N(CH3)2). Anal. (C70H130N2O4Br2 · 3H2O) C, H, N. Lipid 4c. 1H NMR (300 MHz, CDCl3): δ 0.86–0.90 (t, J ) 6.0 Hz, 12H, 4× -CH3), 1.25–1.47 (m, 74H, 4× -(CH2)9 and -N+-CH2-CH2-CH2-), 1.80 (m, 8H, 4× -O-CH2-CH2), 2.23 (m, 4H, 2× -N+-CH2-CH2-), 3.18 (s, 12H, 4× -N+-CH3), 3.99 (m, 12H, 4× -O-CH2-CH2- and 2× -N+-CH2-CH2), 4.75 (s, 4H, 2× -N+-CH2-Ar), 6.89 (d, 2H, J ) 9.0 Hz, ArH), 7.13 (m, 4H, ArH). ESI-MS: 459.4 ((C12H25O)2C6H3CH2+), 617.6 ((C12H25O)2C6H3CH2N+(CH3)2(CH2)5N(CH3)2). Anal. C, H, N, (C71H132N2O4Br2). Lipid 4d. 1H NMR (300 MHz, CDCl3): δ 0.86–0.90 (t, J ) 6.0 Hz, 12H, 4× -CH3), 1.26–1.46 (m, 76H, 4× -(CH2)9 and 2× -N+-CH2-CH2-CH2-), 1.79 (m, 12H, 4× -O-CH2-CH2- and 2× -N+-CH2-CH2-CH2-), 3.23 (s, 12H, 4× -N+-CH3), 3.82 (m, 4H, -N+-CH2-CH2), 3.96–4.03 (m, 8 H, 4× -O-CH2-CH2-), 4.78 (s, 4H, 2× -N+-CH2-Ar), 6.85–6.88 (d, J ) 9.0 Hz, 2H, ArH), 7.12–7.16 (m, 4H, ArH). ESI-MS: 547.5 (M+2/2). Anal. (C72H134N2O4Br2) C, H, N. Lipid 4e. 1H NMR (300 MHz, CDCl3): δ 0.86 (t, J ) 6.0 Hz, 12H, 4× -CH3), 1.26 (m, 92H, 4× -(CH2)9 and 2× -N+-CH2CH2-CH2-CH2-CH2-CH2-), 1.79 (m, 8H, 4× -O-CH2-CH2-), 3.05 (s, 12H, 4× -N+-CH3), 3.73 (m, 4H, 2× -N+-CH2-CH2), 3.98 (m, 8H, 4× -O-CH2-CH2-), 4.82 (s, 4H, 2× -N+-CH2-Ar), 6.84–6.87 (d, J ) 9.0 Hz, 2H, ArH), 7.14–7.18 (m, 4H, ArH). ESI-MS: 588 (M+2/2). Anal. (C78H146N2O4Br2) C, H, N. Lipid 5a. 1H NMR (300 MHz, CDCl3): δ 0.86–0.90 (t, 12H, J ) 6.0 Hz, 4× -CH3), 1.25–1.47 (m, 88H, 4× -(CH2)11), 1.83 (m, 8H, 4× -O-CH2-CH2-), 3.05 (m, 2H, -N+-CH2-CH2-), 3.22 (s, 12H, 4× -N+-CH3), 4.00 (m, 12H, 4× -O-CH2-CH2- and 2× -N+-CH2-CH2), 4.68 (s, 4H, 2× -N+-CH2-Ar), 6.88–6.91 (d, 2H, J ) 9.0 Hz, ArH),7.06 (m, 4H, ArH). ESI-MS: 515.5 ((C14H29O)2C6H3CH2+), 557.5 ((C14H29O)2C6H3CH2N+(CH3)2), 645.6 ((C14H29O)2C6H3CH2N+(CH3)2(CH2)3N(CH3)2), 1241.9 (M+2 + Br-). Anal. (C77H144N2O4Br2) C, H, N. Lipid 5b. 1H NMR (300 MHz, CDCl3): δ 0.86–0.90 (t, 12H, J ) 6.0 Hz, 4× -CH3), 1.25–1.46 (m, 88H, 4× -(CH2)11), 1.80 (m, 8H, 4× -O-CH2-CH2-), 2.31 (m, 4H, 2× -N+-CH2-CH2-), 3.13 (s, 12H, 4× -N+-CH3), 3.98 (t, 8H, J ) 6.0 Hz, 4× -OCH2-CH2-), 4.12 (m, 4H, -N+-CH2-CH2), 4.63 (s, 4H, 2× -N+CH2-Ar), 6.88–6.90 (d, 2H, J ) 6.0 Hz, ArH), 7.04 (m, 4H, ArH). ESI-MS: 515.5 ((C14H29O)2C6H3CH2+), 659.7 ((C14 H29O)2C6H3CH2N+(CH3)2(CH2)3N(CH3)2). Anal. (C78H146 N2O4Br2 · 2H2O) C, H, N. Lipid 5c. 1H NMR (300 MHz, CDCl3): δ 0.86–0.90 (t, 12H, J ) 6.0 Hz, 4× -CH3), 1.25–1.47 (m, 90H, 4× -(CH2)11 and -N+-CH2-CH2-CH2-), 1.80 (m, 8H, 4× -O-CH2-CH2), 2.23 (m, 4H, 2× -N+-CH2-CH2-), 3.18 (s, 12H, 4× -N+-CH3), 3.99 (m, 12H, 4× -O-CH2-CH2- and 2× -N+-CH2-CH2), 4.75 (s, 4H, 2× -N+-CH2-Ar), 6.87–6.89 (d, 2H, J ) 6.0 Hz, ArH), 7.13 (m, 4H, ArH). ESI-MS: 515.5 ((C14H29O)2C6H3CH2+), 595.0 (M+2/2), 673.7 ((C14H29O)2C6H3CH2N+(CH3)2(CH2)3N(CH3)2), 1270 (M+2 + Br-). Anal. (C79H148N2O4Br2) C, H, N.


Lipid 5d. 1H NMR (300 MHz, CDCl3): δ 0.86–0.90 (t, 12H, J ) 6.0 Hz, 4× -CH3), 1.26–1.46 (m, 92H, 4× -(CH2)11 and 2× -N+-CH2-CH2-CH2-), 1.79 (m, 8H, 4× -O-CH2-CH2-), 2.17 (m, 4H, 2× -N+-CH2-CH2-), 3.21 (s, 12H, 4× -N+-CH3), 3.83 (m, 4H, 2× -N+-CH2-CH2), 4.02 (m, 8H, 4× -O-CH2-CH2-), 4.72 (s, 4H, 2× -N+-CH2-Ar), 6.86–6.89 (d, 2H, J ) 8.1 Hz, ArH), 7.12 (m, 4H, ArH). ESI-MS: 602.0 (M+2/2). Anal. (C80H150N2O4Br2) C, H, N. Lipid 5e. 1H NMR (300 MHz, CDCl3): δ 0.86–0.90 (t, 12H, J ) 6.0 Hz, 4× -CH3), 1.26–1.40 (m, 108H, 4× -(CH2)11 and 2× -N+-CH2-CH2-CH2-CH2-CH2-CH2-), 1.82 (m, 8H, 4× -OCH2-CH2-), 3.21 (s, 12H, 4× -N+-CH3), 3.72 (m, 4H, 2× -N+CH2-CH2), 4.01 (m, 8H, 4× -O-CH2-CH2-), 4.13 (s, 4H, -N+CH2-Ar), 6.84–6.87 (d, 2H, J ) 8.1 Hz, ArH), 7.14–7.18 (m, 4H, ArH). ESI-MS: 644.0 (M+2/2). Anal. (C86H162N2O4Br2 · 2H2O) C, H, N. Lipid 6a. 1H NMR (300 MHz, CDCl3): δ 0.87 (t, 12H, J ) 7.0 Hz, 4× -CH3), 1.25–1.46 (m, 104H, 4× -(CH2)13), 1.82–1.86 (m, 8H, 4× -O-CH2-CH2-), 3.01–3.10 (br m, 2H, -N+CH2-CH2-), 3.21 (s, 12H, 4× -N+-CH3), 3.96–4.01 (m, 12H, 4× -O-CH2-CH2- and 2× -N+-CH2-CH2), 4.72 (s, 4H, 2× -N+CH2-Ar), 6.85–6.88 (d, 2H, J ) 8.1 Hz, ArH), 7.07–7.10 (m, 4H, ArH). ESI-MS: 637 (M2+/2). Anal. (C85H160N2O4Br2) C, H, N. Lipid 6b. 1H NMR (300 MHz, CDCl3): δ 0.87 (t, 12H, J ) 7.0 Hz, 4× -CH3), 1.25–1.46 (m, 104H, 4× -(CH2)13), 1.76–1.86 (m, 8H, 4× -O-CH2-CH2-), 2.29–3.10 (m, 4H, 2× -N+-CH2CH2-), 3.12 (s, 12H, 4× -N+-CH3), 3.99 (t, 8H, J ) 6.0 Hz, 4× -O-CH2-CH2-), 4.02–4.11 (m, 4H, 2× -N+-CH2-CH2), 4.62 (s, 4H, 2× -N+-CH2-Ar), 6.88 (d, 2H, J ) 8.1 Hz, ArH), 7.05–7.12 (m, 4H, ArH). ESI-MS: 644 (M2+/2). Anal. (C86H162N2O4Br2 · 3H2O) C, H, N. Lipid 6c. 1H NMR (300 MHz, CDCl3): δ 0.88 (t, 12H, J ) 7.0 Hz, 4× -CH3), 1.25–1.47 (m, 106H, 4× -(CH2)13 and -N+CH2-CH2-CH2-), 1.79–1.85 (m, 8H, 4× -O-CH2-CH2-), 2.10–2.24 (br m, 4H, 2× -N+-CH2-CH2-), 3.18 (s, 12H, 4× -N+-CH3), 3.99–4.01 (m, 12H, 4× -O-CH2-CH2- and 2× -N+-CH2-CH2), 4.75 (s, 4H, 2× -N+-CH2-Ar), 6.87 (d, 2H, J ) 8.1 Hz, ArH), 7.13 (m, 4H, ArH). ESI-MS: 650 (M2+/2). Anal. (C87H164N2O4Br2) C, H, N. Lipid 6d. 1H NMR (300 MHz, CDCl3): δ 0.88 (t, 12H, J ) 7.0 Hz, 4× -CH3), 1.25–1.46 (m, 108H, 4× -(CH2)13 and 2× N+-CH2-CH2-CH2-), 1.78–1.82 (m, 8H, 4× -O-CH2-CH2-), 2.24–2.40 (m, 4H, 2× -N+-CH2-CH2-), 3.21 (s, 12H, 4× -N+CH3), 3.84–3.95 (m, 4H, 2× -N+-CH2-CH2), 3.97–4.03 (m, 8H, 4× -O-CH2-CH2-), 4.62 (s, 4H, -N+-CH2-Ar), 6.87 (d, 2H, J ) 8.1 Hz, ArH), 7.13–7.19 (m, 4H, ArH). ESI-MS: 657 (M2+/2). Anal. (C88H166N2O4Br2) C, H, N. Lipid 6e. 1H NMR (300 MHz, CDCl3): δ 0.86 (t, 12H, J ) 7.0 Hz, 4× -CH3), 1.16–1.40 (m, 124H, 4× -(CH2)13 and 2× -N+-CH2-CH2-CH2-CH2-CH2-CH2-), 1.76–1.78 (m, 8H, 4× -OCH2-CH2-), 3.17 (s, 12H, 4× -N+-CH3), 3.62–3.70 (m, 4H, 2× -N+-CH2-CH2), 3.94–3.99 (m, 8H, 4× -O-CH2-CH2-), 4.79 (s, 4H, 2× -N+-CH2-Ar), 6.82 (d, 2H, J ) 8.1 Hz, ArH), 7.09 (d, 2H, J ) 8.1 Hz, ArH), 7.15–7.21 (m, 2H, ArH). ESI-MS: 700 (M2+/2). Anal. (C94H178N2O4Br2) C, H, N. Liposome Preparation. Individual lipid or its mixture with DOPE in the desired mole ratio was dissolved in chloroform in autoclaved Wheaton glass vials. Thin films were made by evaporation of the organic solvent under a steady stream of dry nitrogen. Last traces of organic solvent were removed by keeping these films under vacuum overnight. Freshly autoclaved water (Milli-Q) was added to individual film such that the final concentration of the cationic lipid was 0.5 mM. The mixtures were kept for hydration at 4 °C for 10–12 h and subjected to repeated freeze–thaw cycles (ice–cold water to 70 °C) with intermittent vortexing to ensure hydration. Sonication of these


suspensions for 15 min in a bath sonicator at 70 °C afforded closed cationic liposomes as evidenced from transmission electron microscopy. Liposomes were prepared and kept under sterile conditions. Formulations were stable and, if stored frozen, possessed long shelf life. Transmission Electron Microscopy. Freshly prepared aqueous suspensions of lipoplexes were examined under transmission electron microscopy by negative staining using 1% uranyl acetate. A 10 µL sample of the suspension was loaded onto Formvar-coated, 400 mesh copper grids and allowed to remain for 1 min. Excess fluid was wicked off the grids by touching their edges to filter paper, and 10 µL of 1% uranyl acetate was applied on the same grid, after which the excess stain was similarly wicked off. The grid was air-dried for 30 min, and the specimens were observed under TEM (JEOL 200-CX) operating at an acceleration voltage of 120 keV. Micrographs were recorded at a magnification of 5000–20 000×. Plasmid DNA. pEGFP-c3 (Clontech, USA), which encodes for an enhanced green fluorescence protein (GFP) under a CMV promoter, was amplified in Escherichia coli (DH5R) and purified using Qiagen Midi Prep Plasmid Purification protocol (Qiagen, Germany). Purity of the plasmid was checked by electrophoresis on 1.0% agarose gel. Concentration of the DNA was estimated spectroscopically by measuring the absorption at 260 nm and confirmed by gel electrophoresis. The plasmid preparations showing a value of OD260/OD280 > 1.8 were used. Cell Culture. Cells (HeLa or HT1080) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Sigma) supplemented with 10% fetal bovine serum (FBS) in T25 culture flasks (Nunc, Denmark) and were incubated at 37 °C in a humidified atmosphere containing 5% CO2. Cells were regularly passaged by trypsinization with 0.1% trypsin (EDTA 0.02%, dextrose 0.05%, and trypsin 0.1%) in PBS (pH 7.2). Cytotoxicity. Toxicity of each cationic lipid formulation towards HeLa cells was determined using 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide reduction assay following the literature procedures (47). MTT is 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. Cytotoxicity of the lipid formulations optimal for transfection experiments were found out under conditions exactly similar for transfection conditions. Nearly 12 000 cells/well were plated in a 96 well plate. After 24 h, optimized lipid/DOPE formulations were complexed with 0.2 µg of the DNA at an N/P ratio of 0.75 for 30 min. DNA–lipid complexes were added to the cells in the absence of serum. After 4 h of incubation, lipoplexes were removed and 200 µL of media with 10% FBS was added. After 42 h, 20 µL of MTT solution was added and the cells were incubated further for 4 h. Blue formazan crystals were seen as well when checked under the microscope. Media was removed and 200 µL of DMSO was added per well. The absorbance was measured using a microtiter plate reader. The % viability was then calculated as [{A590(treated cells) – background]/[A590(untreated cells) – background}] × 100. Transfection Procedure. All transfection experiments were carried out in HeLa or HT1080 cells in antibiotic-free media unless specified otherwise. In a typical experiment, 24 well plates were seeded with 45 000 cells/well in antibiotic-free media 24 h before transfection such that they were at least ∼70% confluent at the time of transfection. For transfection, to have the required working stocks lipid formulation and DNA were serially diluted separately in DMEM containing no serum. DNA was used at a concentration of 0.8 µg/well unless specified otherwise. The lipid and DNA were complexed in a volume of 200 µL by incubating the desired amount of lipid formulation

Bajaj et al.

and DNA together at room temperature for about 30 min. The lipid concentrations were varied so as to obtain the required lipid/DNA (N/P) charge ratios. Charge ratios here represent the ratio of charge on cationic lipid (in mol) to nucleotide base molarity and were calculated by considering the average nucleotide mass of 330. After 30 min of complexation, 200 µL of media were added to the complexes (final DNA concentration ) 12.12 µM). Old medium was removed from the wells, cells were washed with DMEM, and lipid–DNA complexes in 200 µL media were added to the cells. The plates were then incubated for 4 h at 37 °C in a humidified atmosphere containing 5% CO2. At the end of incubation period, medium was removed and cells were washed with DMEM, 500 µL of DMEM containing 10% FBS was added per well. Plates were further incubated for a period of 44 h before checking the reporter gene expression. GFP expression was examined by fluorescence microscopy and was quantified by flow cytometry analysis. Control transfections were performed in each case using a commercially available transfection reagent Lipofectin based on the standard conditions specified by the manufacturer. All the experiments were done in duplicates, and results presented are the average of at least two such independent experiments done on two different days. For transfections in the presence of serum, lipid and DNA were separately diluted in serum-free media as already mentioned, and the complexation was done in serum-free media (200 µL) for 30 min. The complex was then diluted to 400 µL with DMEM containing 20% FBS so as to achieve a final serum concentration of 10%. The cells were then incubated with these complexes for 4 h. At the end of the incubation period, medium was removed and cells were washed with DMEM; 500 µL of DMEM containing 10 % FBS was added per well. For transfections at 30% and 50% of serum concentrations, complexes were diluted with DMEM containing 60% FBS or with neat FBS, respectively. Gel Electrophoresis. To examine the complexation of DNA with cationic lipid suspensions at different lipid–DNA ratios, we prepared lipid–DNA complexes at different lipid–DNA charge ratios in an identical manner as was done with the transfection experiments. After 30 min of incubation, these complexes were electrophoretically run on a 1.0% agarose gel. The uncomplexed DNA moved out of well, but the DNA that was complexed with lipid remained inside the well (7). Flow Cytometry. The reporter gene expression was examined by fluorescence microscopy at regular intervals and was quantified 48 h post-transfection by flow cytometry. The percentage of transfected cells was obtained by determining the statistics of cells fluorescing above the control level wherein nontransfected cells were used as the control. Approximately 10 000 cells were analyzed to achieve the statistical data, which have been presented as the average of at least two such independent measurements. For flow cytometry analysis, ∼48 h post-transfection, old medium was removed from the wells; cells were washed with PBS and trypsinized by adding 100 µL of 0.1% trypsin. To each well, 200 µL of PBS containing 20% FBS was added. Duplicate cultures were pooled and analyzed by flow cytometry immediately using a Becton and Dickinson flow cytometer equipped with a fixed laser source at 488 nm. FACS Analysis. FACS data were analyzed by public domain WinMDI software to eliminate data from cell debris (particles smaller than cells), dead cells, and clumps of two or more cells. Subcellular debris and clumps can be distinguished from single cells by size (estimated by the intensity of low angle forward scatter). Dead cells have lower forward-scatter and higher sidescatter than living cells. The FACS scans had been configured to display the fluorescence signals only from those particles with a specified set of scatter properties, namely, living single cells.



Scheme 1a

a Reagents, conditions, and yields: (i) HCl, MeOH, 80 °C, 12 h, 90%; (ii) RBr, K2CO3, dry acetone, 70 °C, 72–86 h, 70–75%; (iii) LiAlH4, dry THF, 70 °C, 12 h, 90–95%; (iv) (a) PBr3, CH2Cl2, rt, 24 h; (b) Me2NH (excess), MeOH, rt, 12 h (overall yield ) 80–85% for steps (a) and (b)); (v) MeI, EtOH, 80 °C, 24 h, screw-top pressure tube, quantitative yields; (vi) Br(CH2)mBr, MeOH–EtOAc (2:1), 80 °C, screw-top pressure tube, 2–4 days, 40–50%.

This is called a scatter-gated fluorescence analysis. Therefore, the data from dead cells had been eliminated by gating out brightly fluorescent cells.

RESULTS AND DISCUSSION Chemistry. Fifteen new gemini cationic lipids (4–6) of different hydrocarbon chain lengths have been synthesized. For comparison of their transfection properties with those of their monomeric counterparts, lipids 1–3 were also synthesized (Figure 1). Each of these lipids possesses an aromatic backbone which anchors the linkage between the hydrocarbon chains and the charged headgroup. Two cationic ammonium headgroups are joined through flexible polymethylene spacers. For the synthesis, first the 3,4-dihydroxy benzoic acid (7) was esterified in the presence of conc HCl and dry MeOH under reflux conditions to protect the -COOH moiety (Scheme 1). The corresponding ester (8) was obtained in 90% yield. The ester (8) was then subjected to alkylation with appropriate n-alkyl bromide under reflux conditions in dry acetone in the presence of K2CO3 to furnish the dialkylated products (9a–9c) in 70–75% yields. The ester group of the product (9a–9c) was then reduced with LiAlH4 in dry THF, under reflux conditions over a period of 12 h, to afford 10a–10c in 90–95% isolated yields. Next, the conversion of 10a–10c to corresponding benzylic bromide was attempted. However, under Appel conditions (CBr4/PPh3 in CH2Cl2), although TLC indicated satisfactory conversion, during the isolation, a significant part of the product decomposed. The conversion of the alcohol to the corresponding bromide by PBr3 in CH2Cl2 appeared to be complete by TLC. However, again it was not possible to isolate the corresponding bromide presumably because of its high instability. Therefore,

upon confirming the formation of the bromide after reaction with PBr3 in CH2Cl2 for 24 h by TLC, a methanolic solution of dimethylamine was introduced directly into the reaction mixture, and the mixture was stirred at room temperature for 12 h. After the usual workup, compounds 11a–11c were obtained in 80–85% yields. Lipids 1–3 were prepared upon refluxing the tertiary amines 11a–11c with an excess of MeI in EtOH in quantitative yields (Scheme 1). The gemini cationic lipids with polymethylene spacers, 4–6, were synthesized by heating 11a–11c with the appropriate R,ω-dibromoalkanes to 80 °C in a mixture of MeOH–EtOAc (1:1) for 48–96 h in a screw-top pressure tube (Scheme 1). After repeated crystallizations from a mixture of MeOH-EtOAc, the isolated yields of the gemini lipids ranged from 40% to 50%. All the gemini lipids were adequately characterized by 1H NMR, mass spectra, and elemental analysis. Aggregate Formation from Cationic Gemini Lipids. Upon hydration, all the cationic lipids were dispersed in water at ∼70 °C. The suspensions formed from lipids were found to be translucent. Lipids bearing n-C14H29 and n-C16H33 chains were found to be dispersed with difficulty even at ∼80 °C. Therefore, we also did not observe their thermal phase transition temperature, whereas lipids bearing n-C12H25 chains were found to form suspensions easily at ∼70 °C and they afforded detectable phase transition temperatures. TEM examination of air-dried, aqueous suspensions of each lipid revealed the existence of vesicular aggregate structures (not shown). Mixed Liposome Formation with 1,2-dioleoyl-L-r-glycero3-phosphatidyl Ethanolamine (DOPE). Liposomes could be conveniently prepared from each lipid with naturally occurring lipid (1,2-dioleoyl-L-R-glycero-3-phosphatidyl ethanolamine


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Table 1. Optimized Lipid/DOPE Mole Ratios for Cationic Lipids for Transfection Studies lipid

lipid/DOPE

lipid

lipid/DOPE

lipid

lipid/DOPE

1 4a 4b 4c 4d 4e

1:1 1:2 1:2 1:2 1:2 1:2

2 5a 5b 5c 5d 5e

1:2 1:8 1:6 1:2 1:2 1:2

3 6a 6b 6c 6d 6e

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

(DOPE)) by first subjecting the films of lipid mixtures to hydration, repeated freeze–thaw cycles, followed by sonication at 70 °C for 15 min. All the DOPE doped lipids formed optically transparent suspensions. The vesicular suspensions were sufficiently stable and no precipitation was observed within three months if stored at 4 °C.

TRANSFECTION BIOLOGY Optimization of the Lipid/DOPE Ratio. Incorporation of the helper lipid DOPE is known to enhance the transfection efficiencies of many of the cationic lipid-based formulations (48). Therefore, we first decided to investigate the optimized lipid/DOPE mole ratio for our newly developed cationic lipids in HeLa cells, as their neat lipid suspensions were found to show very low transfection efficacies (not shown). To find out the optimized transfection efficiency, both the number of transfected cells and mean fluorescence intensity were considered. The mean fluorescence intensities (MFI) defined for GFP-positive cells reveal that the level of GFP expression with a higher MFI value correlate positively with a high GFP expression (49). These data were obtained from the flow cytometric analysis. In order to determine the most effective formulations, transfections were performed with identical amounts of the DNA (0.8 µg) and lipid (2.5 µg) upon variation of the mole ratios of cationic lipids in DOPE. Optimized lipid/DOPE ratios were found to vary with different cationic lipids depending upon the hydrocarbon chain lengths and the spacer segment between the cationic headgroups. The optimized lipid/DOPE ratios for all cationic lipids are given in Table 1. Monomeric lipids 2 and 3 showed the maximum transfection at optimized lipid/DOPE ratio of 1:2, whereas monomeric lipid 1 showed the optimum activity at a lipid/DOPE ratio of 1:1. Gemini lipids (4a–4d) bearing n-C12H25 hydrocarbon chains showed the maximum transfection efficiency at lipid/ DOPE ratio of 1:2. In the case of the other gemini lipids, optimized lipid/DOPE ratios differed depending on both the length of the spacer and the hydrocarbon chain lengths. Lipids 5a, 6d, and 6e showed transfections at the highest optimized lipid/DOPE ratios of 1:8, whereas lipids 5c and 5d showed the best activity at lipid/DOPE ratio of 1:2. These differences in the optimized lipid/DOPE ratios could be attributed to their different interaction behavior with DNA and the cell membranes, depending upon the lipid chain lengths and spacers between the headgroups. Optimization of Lipid/DNA Charge Ratio. After the optimization of the lipid/DOPE ratios for lipids, we performed transfection studies in HeLa cells at different lipid/DNA charge ratios using optimized lipid/DOPE ratios. Monomeric lipid 1 formulation showed 30–40 % GFP cells with MFI of ∼40 by FACS analysis. With the introduction of the spacer between the two monomeric lipid 1 molecules, transfection efficiency increased especially in the number of cells getting transfected (Figure 2). Gemini lipid formulations based on 4a–4e were able to transfect 60–80% of the cells, but the MFI values observed in all the formulations were very low. This suggests that these gemini lipid (4a–4e) formulations were able to transfect a large number of cells, but the amount of the DNA delivered was low. All these gemini lipid formulations (4a–4e) were found to be

better than the monomeric lipid 1 formulation in terms of their ability to gene transfer. These gemini lipid formulations showed maximum transfection at an N/P ratio of 0.75–1.0 and followed a bell-shaped graph (Figure 2). The formulation based on monomeric lipid 2 was able to transfect nearly 40% of cells with MFI of ∼75 as compared to the monomeric lipid 1 formulation, which transfected 40% of cells with MFI of ∼40 (Figure 3). This shows that the tetradecyl chain based monomeric lipid 2 is nearly two times better than the monomeric lipid 1 with dodecyl chains especially in terms of the MFI. On dimerization of lipid 2 with -(CH2)3- propanediyl spacer (lipid 5a), the transfection efficiency decreased in terms of the number of transfected cells, while the MFI remained nearly the same. This indicates that propanediyl spacer based lipid (5a) aggregates were able to transfer nearly the same amount of the DNA but the efficiency reduced to nearly 30%. With an increase in the spacer length to -(CH2)5- pentanediyl, the transfection efficacy increased dramatically. Lipid 5c was able to transfect ∼60% of the cells with high MFI of ∼100 at an N/P ratio of 1.0 as compared to its monomeric lipid counterpart 2, which transfected only 40% of cells with MFI of ∼75. In the case of lipid 5d and 5e formulations, the transfection efficacies, however, decreased. The lipid 5d formulation transfected nearly 40% of the cells with an MFI of ∼75, and lipid 5e was able to transfect only 30% of the cells with MFI of ∼70. Therefore, among the n-C14H29 chain-based gemini lipids, lipid 5c bearing -(CH2)5- showed maximum transfection efficiency. Gemini lipid formulations based on hexadecyl chains were found to show the lowest transfection efficiency, in terms of both the number of transfected cells and the MFI. The monomeric lipid 3 formulation was able to transfect nearly 30% of the cells with an MFI of ∼40 (Figure 4). The maximum transfection was observed with the lipid 6d formulation, transfecting nearly 20% of the cells with an MFI of ∼60, whereas the lipid 6e formulation transfected ∼20% of the cells with a high MFI of ∼80. The overall analysis of the data clearly shows that cationic lipids based on n-C12H25 and n-C14H29 hydrocarbon chains are better transfecting agents than the lipids possessing n-C16H33 chains in HeLa cells (Figure 5). Lipids possessing n-C12H25 chains (1, 4a–4e) were able to transfect the highest number of cells with a low MFI, whereas n-C14H29 chain based lipids (2, 5a–5e) transfected cells with a high MFI. This indicates that lipids 2 and 5a–5e were able to deliver a larger amount of the DNA as indicated from MFI as compared to that delivered by lipid 1 and 4a–4e based formulations. Gemini lipids possessing n-C16H33 chains were found to be the least effective. Formulations based on lipid 5c possessing the n-C14H29 chain and the pentamethylene -(CH2)5- spacer were found to be the best recipe, which transfected nearly 60% of the cells with an MFI of ∼100, which was nearly three times more than a commercially available Lipofectin in terms of the number of cells and four times more efficient in terms of the MFI. Transfection Studies in HT 1080 Cells. To assess the general utility of these new lipid formulations, transfection studies with optimized lipid formulations were performed in HT1080 cells as well. Nearly 70% of the cells were transfected with the monomeric lipid 1 formulation with an MFI of ∼45 at an N/P ratio of 1.5 as observed by FACS analysis (Supporting Information Figure S-1). Gemini lipid formulations based on 4a and 4d were able to transfect 60–80% of the cells with MFI values of ∼40 and 50, respectively, whereas lipid 4b, 4c, and 4d formulations showed ∼40–50% transfection activity. Monomeric lipid 2 based formulations were able to transfect nearly 50% of cells with MFI ∼35 (Supporting Information Figure S-2). Lipid 5a and 5b formulations bearing -(CH2)3- and



Figure 2. Transfection efficiencies of cationic lipids using optimized lipid/DOPE formulations at various N/P ratios in the absence of serum (-FBS-FBS) in HeLa cells: (a) lipid 1; (b) lipid 4a; (c) lipid 4b; (d) lipid 4c; (e) lipid 4d; (f) lipid 4e. Concentration of the DNA ) 0.8 µg/well. Data are expressed as number of transfected cells and MFI as obtained from flow cytometry analysis.

Figure 3. Transfection efficiencies of cationic lipids using optimized lipid/DOPE formulations at various N/P ratios in the absence of serum (-FBS-FBS) in HeLa cells: (a) lipid 2; (b) lipid 5a; (c) lipid 5b; (d) lipid 5c; (e) lipid 5d; (f) lipid 5e. Concentration of the DNA ) 0.8 µg/well. Data are expressed as the number of transfected cells and MFI as obtained from flow cytometry analysis.

-(CH2)4- spacers are not very effective, transfecting ∼20% cells with MFI of ∼20, whereas the lipid 5c formulation with a -(CH2)5- spacer showed ∼50% transfection efficacy with MFI of ∼35. Nearly 40% of the cells with an MFI of ∼20 were transfected with lipid 5d formulations. The lipid 5e formulation was found to be highly effective with more than 80% transfection efficiency with an MFI of ∼40. Gemini lipid formulations based on n-C16H33 chains were found to show the least transfection activity, in terms of both the number of transfected cells and MFI even in HT1080 cells, as was seen with HeLa cells (Supporting Information Figure S-3).

Figure 6 presents the comparative transfection efficacies of all lipid formulations at N/P ratio of 0.75 in HT1080 cells. Lipid formulations 1, 4a, 4c, 4d, and 5c were found to be the best transfecting agents among all lipid formulations. Formulations of lipids possessing n-C16H33 chains were not found to be effective at all. Comparing the transfection efficacies of lipid formulations in HeLa and HT1080 cell lines showed interesting features. Overall, the MFI observed in HT1080 cell lines was lower compared to HeLa cells, which indicates that the release of the DNA from lipoplexes in HT1080 is not as easy as in HeLa cells where high MFI was observed.


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Figure 4. Transfection efficiencies of cationic lipids using optimized lipid/DOPE formulations at various N/P ratios in the absence of serum (-FBS-FBS) in HeLa cells: (a) lipid 3; (b) lipid 6a; (c) lipid 6b; (d) lipid 6c; (e) lipid 6d; (f) lipid 6e. Concentration of the DNA ) 0.8 µg/well. Data are expressed as the number of transfected cells and MFI as obtained from flow cytometry analysis.

Figure 5. A comparative study showing the effect of the spacer length and hydrocarbon chain lengths on gene transfection efficiencies of gemini lipids based on aromatic backbone in the absence of serum (-FBS-FBS) at an N/P ratio of 0.75 in HeLa cells.

Effect of the Serum. One of the serious limitations of the cationic lipid-mediated gene delivery, especially involving in ViVo trials, is that the transfection is inefficient particularly in the presence of serum proteins. Transfections in the absence of serum even for in Vitro experiments are also cumbersome due to the increased toxicity of cationic lipid formulations. To have better potential especially in gene therapy, it is important to develop lipids that are capable of delivering and expressing an external gene inside a cell in the presence of serum (13, 21, 50). To have a clear understanding of the effect of the serum in the present set of cationic lipid mediated gene delivery, we performed transfections with our optimized lipid formulations in the presence of serum in HeLa and HT1080 cells. Monomeric lipid 1/DOPE was able to transfect nearly 30% of the cells with a low MFI of ∼30 in the presence of 10% serum (Figure 7). With the introduction of the spacer, the transfection efficiency increased in the presence of serum especially in the number of cells. Lipid 4b–4d formulations were able to transfect nearly 60% of the cells in the presence of 10%

serum with low MFI, whereas the lipid 4e/DOPE formulation transfected only 20% of cells. A closer examination of these results suggests that lipid dimerization increases the transfection efficiency in the presence of 10% serum conditions, and this efficiency depends on the length of the spacer. The maximum transfection efficiency was achieved at N/P ratio of 0.75–1.5. The transfection efficiency observed in the presence of serum is comparable to that observed in the absence of serum, which clearly shows that serum does not inhibit the transfection efficacy of these formulations. The monomeric lipid 2/DOPE formulation in the presence of 10% serum was able to transfect nearly 40% of cells at N/P ratio of 1.0–1.5 (Figure 8). The lipid 5a/DOPE formulation was able to transfect nearly 30% of the cells with an MFI of ∼80 at N/P ratio of 1.5. Lipid 5c and 5d formulations in the presence of DOPE showed maximum transfection efficiency in the presence of serum, transfecting nearly 60% of cells with MFI which was nearly two times more than that observed in the case of 4b–4d/DOPE formulations. This indicates that the n-C14H29



Figure 6. A comparative study showing the effect of the spacer length and hydrocarbon chain lengths on gene transfection efficiencies of gemini lipids based on aromatic backbone in the absence of serum (-FBS-FBS) at N/P ratio of 0.75 in HT 1080 cells.

Figure 7. Transfection efficiencies of cationic lipids using optimized lipid/DOPE formulations at various N/P ratios in the presence of serum (-FBS + FBS) in HeLa cells: (a) lipid 1; (b) lipid 4a; (c) lipid 4b; (d) lipid 4c; (e) lipid 4d; (f) lipid 4e. Concentration of the DNA ) 0.8 µg/well. Data are expressed as the number of transfected cells and MFI as obtained from flow cytometry analysis.

chain based lipid 5c and 5d formulations are two times better in transfection activity as compared to n-C12H25 chain based lipid 4c and 4d formulations in terms of MFI. This means that lipid 5c and 5d formulations are able to transfect a greater amount of the DNA into the cells in the presence of serum as well. As observed in the absence of serum conditions, n-C16H33 chain based lipids (3, 6a–6e) were again less efficient in the presence of serum as compared to other lipids (Figure 9). Effect of Serum on the Transfection in HT1080 Cells. We also performed transfection studies in the presence of 10% serum conditions in HT1080 cells. In the presence of serum, as expected, there is little decrease in the transfection activities of all the lipid formulations in terms of both the number of transfected cells and the MFI. Among the dodecyl chain based lipid formulations, monomeric lipid 1 and gemini lipid 4d formulations showed maximum activities transfecting ∼60% cells with MFI of ∼40 (Supporting Information Figure S-4). The gemini lipid 5e formulation was found to be the best among all the formulations in HT1080 cells (Supporting Information Figure S-5). Lipids (3, 6a-6e) with n-C16H33 were found to be

the least efficient even in the presence of serum as compared to other analogues in HT1080 cells. (Supporting Information Figure S-6). Effect of High Percentage of Serum. To approach the realistic in ViVo conditions, we performed transfections in the presence of high percentages of serum using the lipid 5c formulation at different N/P ratios in HeLa cells. We performed transfection in the presence of 10%, 30%, and 50% serum conditions using the optimized lipid/DOPE formulation of 5c. The transfection profiles are shown in Figure 10. In the presence of 10% serum, nearly 60% of the cells were found to get transfected at an N/P ratio of 4.5 with MFI of 40. The decrease in MFI in the presence of serum is because of the delivery of a low amount of the plasmid DNA. In the presence of serum, negatively charged serum proteins start competing with cationic lipids for negatively charged plasmid DNA, which leads to the delivery of less amount of the DNA inside the cells, because of which the MFI decreased. In the presence of 30% serum, the lipid 5c formulation was able to transfect nearly 50% of the cells with MFI of 40. To


Bajaj et al.

Figure 8. Transfection efficiencies of cationic lipids using optimized lipid/DOPE formulations at various N/P ratios in the presence of serum (-FBS + FBS) in HeLa cells: (a) lipid 2; (b) lipid 5a; (c) lipid 5b; (d) lipid 5c; (e) lipid 5d; (f) lipid 5e. Concentration of the DNA ) 0.8 µg/well. Data are expressed as the number of transfected cells and MFI as obtained from flow cytometry analysis.

Figure 9. Transfection efficiencies of cationic lipids using optimized lipid/DOPE formulations at various N/P ratios in the presence of serum (-FBS + FBS) in HeLa cells: (a) lipid 3; (b) lipid 6a; (c) lipid 6b; (d) lipid 6c; (e) lipid 6d; (f) lipid 6e. Concentration of the DNA ) 0.8 µg/well. Data are expressed as number of transfected cells and MFI as obtained from flow cytometry analysis.

our pleasant surprise, nearly 40% of the cells have been found to be GFP positive in the presence of 50% serum conditions, with very little decrease in MFI. These results showed the unusual behavior of the lipid 5c formulation, transfecting as many as 40 % of the cells in the presence of 50% serum conditions, where the commercially available formulation Lipofectin failed to transfect. Cytotoxicity. We performed the MTT-based cell viability assays for all the lipid–DNA complexes in HeLa cells. The cell viability results shown in Figure 11 clearly demonstrate that all the formulations showed more than 80% cell viability except the 4d, 5d, and 6d lipid (based on -(CH2)6- hexanediyl spacer

between the headgroups) based lipid–DNA complexes. The best lipid formulation 5c showed more than 90% cell viability.

CHARACTERIZATION OF LIPID–DNA COMPLEXES Gel Electrophoresis. Electrostatic interactions between the plasmid DNA and cationic liposomes as a function of lipid/ DNA charge (N/P) ratios were determined by electrophoretic gel retardation assay. Gel electrophoretic patterns for all lipid formulations have been shown in Figures 12 and 13. All lipid formulations showed more than 90% DNA retardation at an N/P ratio of 1.0. Nearly 50% retardation was observed at an N/P ratio of 0.75. The best lipid 5c formulation showed DNA



Figure 10. Transfection efficiencies using the optimized lipid 5c/DOPE formulation at various N/P ratios in the 10%, 30 %, and 50% serum conditions (-FBS + FBS) in HeLa cells: (a) 10% FBS; (b) 30% FBS; (c) 50% FBS. Concentration of the DNA ) 0.8 µg/well. Data are expressed as the number of transfected cells and MFI as obtained from flow cytometry analysis.

Figure 11. MTT-assay based cellular cytotoxicities of cationic lipid/DNA complexes at their optimized lipid/DNA ratios in HeLa cells: (a) lipids 1, 4a–4e; (b) lipids 2, 5a–5e; (c) lipids 3, 6a–6e.

Figure 12. Electrophoretic gel patterns for lipoplex-associated DNA in gel retardation for cationic lipid formulations. The N/P ratios are indicated at the top of each lane.

retardation at an N/P ratio of 1.0, and more than 50% DNA was retarded at an N/P ratio of 0.75. At an N/P ratio of 1.0, maximum transfection had been achieved with this formulation. Electron Microscopy. To get further insights into the lipid–DNA complexes, we performed electron microscopy of the lipid–DNA complexes using the optimized lipid/DOPE formulations. (51–56) Representive electron micrographs of lipid–DNA complexes have been shown in Figure 14. Most of

the lipid formulations formed spherical lipid–DNA complexes. Lipid–DNA complexes from lipid 3a formulations were very uniform and small in size with diameters of ∼150 nm, whereas other lipid–DNA complexes (4b, 4c, 4d, 4e) in this series were considerably larger in size ranging from 200 to 400 nm. Lipid 5a and 5c formulations showed irregular morphologies, whereas the 5d formulation showed spherical lipid–DNA complexes. The lipid 5e formulation formed thread-like lipid–DNA complexes.


Bajaj et al.

Figure 13. Electrophoretic gel patterns for lipoplex-associated DNA in gel retardation for cationic lipid formulations. The N/P ratios are indicated at the top of each lane.

Figure 14. Transmission electron micrographs for the lipoplexes prepared from most effective gemini lipid/DOPE liposome/DNA complexes at the optimized N/P ratio.

Similar observation were also noted by Mahato and co-workers. (57) Among n-C16H33 chain based lipid formulations, lipids 6a and 6e formed spherical structures, whereas lipid 6b formed condensed DNA structures and lipid 6d afforded small rodlike structures in their electron micrographs.

CONCLUSIONS Although diverse kinds of cationic lipids have been designed and tested as gene delivery vehicles, there remains a need for continuous improvement in the design of lipids for gene transfer. Keeping these targets in mind, we designed and synthesized a new kind of gemini lipid molecule based on aromatic backbone. Three series of gemini lipids differing in the length of the hydrocarbon chains have been synthesized. Polymethylene spacers of varying length were incorporated between the headgroups. In HeLa cells, gemini lipids bearing n-C12H25 chains

were found to transfect a greater number of cells with low MFI intensity, whereas n-C14H29 chain based lipids showed transfection with high MFI, and lipids based on n-C16H33chains were found to be the least effective. Lipid formulation 5c, possessing tetradecyl chains and pentamethylene spacers, showed the highest gene transfection efficacy in this series. A major characteristic feature of the lipid 5c formulation is its high serum compatibility, which is one of the major challenges in the lipidmediated gene delivery. In HT1080 cells, lipid formulations 1, 4a, 4c, 4d, and 5c were found to be the best transfecting agents among all lipid formulations. MFI observed in HT1080 cell lines was lower compared to that in HeLa cells, which indicates that the release of the DNA from lipoplexes in HT1080 is more difficult than in HeLa cells where a high MFI was observed. Transmission electron microscopy revealed spherical lipid–DNA complexes for most of the formulations. Regardless of the actual


mechanism (58–62) for transfection mediated by this class of gemini lipid formulations, the interesting and meaningful results obtained with these novel aromatic gemini lipids should be of interest to researchers working in the field of gene therapy using nonviral vectors.

ACKNOWLEDGMENT This work was supported by Department of Biotechnology, Government of India, New Delhi, India. Avinash Bajaj is thankful to the CSIR for senior research fellowship. We thank Dr. Omana Joy, Ms. Padmini, and Mr. Vamsi for their help during the work. Supporting Information Available: Elemental analysis, transfection studies in HT1080 cells, and FACS details. This material is available free of charge via the internet at http:// pubs.acs.org.

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