Synthesis and Gene Transfer Activities of Novel ... - ACS Publications

Bioconjugate Chem. 2007, 18, 1537−1546

1537

Synthesis and Gene Transfer Activities of Novel Serum Compatible Cholesterol-Based Gemini Lipids Possessing Oxyethylene-Type Spacers Avinash Bajaj,† Paturu Kondaiah,‡ and Santanu Bhattacharya*,†.§ Department of Organic Chemistry and Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science, Bangalore 560 012, and Chemical Biology Unit of JNCASR, Bangalore 560 064, India. Received January 11, 2007; Revised Manuscript Received March 26, 2007

Four novel cholesterol-based gemini cationic lipids differing in the length of oxyethylene-type spacers [-CH2(CH2-O-CH2)n-CH2-] between each ammonium headgroup have been synthesized. These formed stable suspensions in aqueous media. Cationic liposomes were prepared from each of these lipids individually and as mixtures of cationic lipid and DOPE. These were used as nonviral gene delivery agents. All the cholesterol-based gemini lipids induced better transfection activity than their monomeric counterpart. Inclusion of DOPE in coliposomal formulation of the cationic gemini lipid potentiates their gene transfer activity significantly. A major characteristic feature of these oxyethylene spacer based cholesterol gemini lipids was that serum does not inhibit the transfection activity of these gemini lipids, whereas the transfection activity of their monomeric counterpart decreased drastically in the presence of serum. One of the cholesterol-based gemini lipids 2a possessing a -CH2CH2-O-CH2-CH2- spacer showed the highest transfection activity.

INTRODUCTION Viral vectors, although capable of ensuring high levels of transgene expression, suffer from many disadvantages like generation of toxic inflammatory responses, possibility of random integration into host chromosome, limited insert size of virally packaged therapeutic gene, possibility of generating replication-competent virus through recombination with host genome, and so forth (1-4). Therefore, the development of effective and safe genetic carriers is the key to achieving clinical success for gene therapy. Major nonviral gene delivery reagents mainly comprises two types: polymers and cationic lipids (5). Cationic polymers like polyethyleneimines (PEI) (6), polylysines (7, 8), have already been studied in detail for gene transfection. Cationic liposomes are promising tools for delivery of genes, because of their lower toxicity, very low immunogenicity, convenient preparation, and so forth, as compared to their viral counterparts. Among cationic lipids, various structure-activity investigations have been reported using different types of cationic lipids with variation of hydrophobic parts, linker region, headgroups, and others (917). We have been investigating the role of various molecular modifications in different synthetic lipids on their membrane level properties and their further influence on gene delivery (1820). The functional group that links the polar head group and the hydrocarbon chains of such lipid molecules plays a crucial role in their utilization in gene transfer events. Thus, N-[1-(2,3dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA1), which contains an ether linkage between the headgroup and the long alkyl chains shows greater in ViVo transfection efficiency than the corresponding ester analogue N-[1-(2,3dioleyl)]-N,N,N-trimethylammonium chloride (DOTAP) (21, 22). We have shown the effect of this linkage functionality on * Corresponding author. E-mail: [email protected]. Phone: (91)-80-2293-2664. Fax: (91)-80-2360-0529. † Department of Organic Chemistry, Indian Institute of Science. ‡ Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science. § Chemical Biology Unit of JNCASR.

the gene transfection efficiencies of cholesterol-based amphiphiles. It was observed that cholesterol-based cationic lipids bearing ether linkages between cholesterol backbone and headgroup possessed greater transfection capability as compared to other linkages (23, 24). Gemini (dimeric) surfactants have also been found to possess transfecting abilities. The effect of the nature of the spacer, hydrocarbon chains, and headgroups on the aggregation and transfection properties has been studied in detail in the case of gemini surfactants (16, 25-30). The gemini analogue of the lipid is also known to exist in natural cell membranes. For instance, cardiolipin, a glycerol-bridged phosphatidic acid, is a dimeric lipid that occurs in the heart and skeletal muscles (31). Taking inspiration from the cardiolipins, we reported for the first time the synthesis and membrane-forming properties of pseudoglyceryl gemini lipids with polymethylene spacers (32). The aggregation properties of these gemini lipids were found to depend on the length of the spacer (33). Later on, Ahmad et al. showed the transfection activities of cardiolipin-based gemini lipids (34, 35). Recently, we reported that the properties of cationic gemini lipids were quite different when oxyethylenebased spacers were employed in place of polymethylene chains (36). We also described the gene transfection properties of gemini lipids where the monomeric cholesterol based units are connected by polymethylene spacer (37). Herein, we present the synthesis and transfection properties of cholesterol-based biscationic (2a-2d) gemini lipids, where each lipid monomer is connected at the level of headgroup via oxyethylene-based spacer chains of variable length (Figure 1). In this paper, we describe the detailed, optimized synthetic procedures and transfection activities of these new biologically active compounds. To put our results into appropriate perspective, we compared the transfection activities of these new gemini cationic lipids with that of monomeric lipid 1. We have observed 1

Abbreviations: DOPE, 1,2-dioleoyl-L-R-glycero-3-phosphatidylethanolamine; DOTMA, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride; DOTAP, N-[1-(2,3-dioleyl)]-N,N,N-trimethylammonium chloride; MFI, mean fluorescence intensity; FACS, fluorescence activated cell sorting.

10.1021/bc070010q CCC: $37.00 © 2007 American Chemical Society Published on Web 07/28/2007

1538 Bioconjugate Chem., Vol. 18, No. 5, 2007

Figure 1. Molecular structures of cholesterol-based cationic monomeric lipid 1 and gemini lipids (2a-2d) synthesized and used for transfection studies.

significantly enhanced gene transfection activities of these gemini lipids as compared to the monomeric counterpart. Serum is known to inhibit the transfection efficiency of cationic liposomes, whereas the major distinguishing feature of these gemini lipids is that they are able to transfect the mammalian cell lines even in the presence of serum.

EXPERIMENTAL PROCEDURES 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 Jeol JNM λ-300 (300 MHz for 1H and 75 Hz for 13C) spectrometer. The chemical shifts (δ) are reported in ppm downfield from the internal standard: TMS, for 1H NMR and 13C 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 table-top GCMS or ESI-MS (HP1100LC-MSD). Infrared (IR) spectra were recorded on a Jasco FT-IR 410 spectrometer either using KBr pellets or neat. Gemini lipids were synthesized as described below and were characterized fully by their 1H NMR, 13C NMR, mass spectra, and elemental analysis. General Method for Synthesis of Gemini Lipids (2a-2e). A mixture of cholest-5-en-3β-oxyethan-N,N-dimethyl amine (24) (0.2 mmol) and an appropriate R,ω-dibromoalkoxyalkanes (0.07 mmol) in dry MeOH-EtOAc (4 mL, v/v 1/1) was refluxed over a period of 48-72 h in a screw-top pressure tube, after which TLC indicated complete disappearance of the starting dibromide. After that, the reaction mixture was cooled and solvent was evaporated to furnish a crude solid. It was repeatedly washed with ethyl acetate to remove any cholest-5-en-3β-oxyethan-N,Ndimethyl amine, and the residue was finally subjected to repeated crystallization from a mixture of MeOH and EtOAc. The product yields ranged from 50% to 60%. The purities of the individual lipids were ascertained from TLC; the Rf ranged from 0.2 to 0.3 in 10:1 CHCl3/MeOH. All the new gemini lipids were fully characterized by 1H NMR, 13C NMR, mass spectrometry, and C, H, N analysis. Pertinent spectroscopic and analytical data are given below. Lipid 2a. 1H NMR (300 MHz, CDCl3): δ 0.67 (s, 6H), 0.852.29 (m, 82H), 3.19 (m, 2H), 3.49 (s, 12H), 3.93 (br m, 8H),

Bajaj et al.

4.01 (br s, 4H), 4.29 (br s, 4H), 5.35 (d, J ) 4.5 Hz, 2H). 13C NMR (CDCl3, 75 MHz): δ 11.65, 18.50, 19.13, 20.87, 22.34, 22.58, 23.65, 24.08, 27.73, 27.80, 27.93, 28.01,31.65, 31.70, 35.60, 35.99, 36.57, 36.75, 38.54, 39.31, 39.53, 42.11, 49.89, 52.59, 55.98, 56.50, 61.57, 64.60, 65.25, 79.80, 121.17, 139.64. ESI-MS: 1067.4 [M+2 + Br-], 493.4 [M+2/2]. Anal. (C66H120O3N2Br2·H2O) C, H, N. Lipid 2b. 1H NMR (300 MHz, CDCl3): δ 0.67 (s, 6H), 0.852.37 (m, 82H), 3.19 (m, 2H), 3.47 (s, 12H), 3.78 (br s, 4H), 3.93 (br s, 12 H), 4.09 (br s, 4H), 5.35 (d, J ) 4.5 Hz, 2H). 13C NMR (CDCl3, 75 MHz): δ 11.76, 18.62, 19.28, 20.96, 22.45, 22.70, 23.79, 24.20, 27.90, 28.05, 28.11, 30.83, 31.75 31.81, 35.69, 36.09, 36.65, 36.83, 38.67, 39.40, 39.63, 42.19, 49.94, 52.85, 56.08, 56.59, 61.92, 64.70, 64.88, 70.22, 79.73, 122.22, 139.79. ESI-MS: 1110.2 [M+2 + Br-]. Anal. (C68H122O4N2Br2·H2O) C, H, N. Lipid 2c. 1H NMR (300 MHz, CDCl3): δ 0.67 (s, 6H), 0.852.35 (m, 82H), 3.19 (m, 2H), 3.49 (s, 12H), 3.65 (br s, 4H), 3.72 (br s, 4H), 3.94 (br s, 12 H), 4.06 (br s, 4H), 5.35 (d, J ) 4.5 Hz, 2H). 13C NMR (CDCl3, 75 MHz): δ 11.68, 18.52, 19.21, 20.89, 22.39, 22.63, 23.70, 24.11, 27.82, 27.98, 28.05, 31.66, 31.73, 35.61, 36.01, 36.58, 36.76, 38.59, 39.31, 39.54, 42.13, 49.86, 52.69, 55.99, 56.50, 61.88, 64.65, 64.88, 70.06, 70.22, 79.62, 122.09, 139.76. ESI-MS: 1155.5 [M+2 + Br-], 537.5 [M+2/2] Anal. (C70H128O5N2Br2·3H2O) C, H, N. Lipid 2d. 1H NMR (300 MHz, CDCl3): δ 0.67 (s, 6H), 0.852.35 (m, 82H), 3.19 (m, 2H), 3.49 (s, 12H), 3.63 (br s, 12H), 3.73 (br s, 4H), 3.95 (br s, 12 H), 4.06 (br s, 4H), 5.35 (d, J ) 4.5 Hz, 2H). 13C NMR (CDCl3, 75 MHz): δ 11.83, 18.69, 19.34, 21.04, 22.53, 22.78, 23.82, 24.26, 27.98, 28.14, 28.19, 31.83, 31.88, 35.76, 36.16, 36.75, 36.90, 38.75, 39.48, 39.69, 42.29, 49.86, 52.69, 55.99, 56.50, 61.88, 64.74, 65.06, 70.14, 70.35, 70.45, 70.54, 79.88, 122.35, 139.87. ESI-MS: 1206.1 [M+2 + Br-]. Anal. (C64H134O7N2Br2·3H2O) C, H, N. Liposome Preparation. Each lipid or its mixture with DOPE in the desired mole ratio was dissolved in dry 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 high vacuum overnight. Freshly autoclaved Milli-Q water was added to individual film such that the final concentration of the cationic lipid was maintained at 0.5 mM. The mixtures were kept for hydration at 4 °C for 1012 h and were subjected to repeated freeze-thaw cycles (icecold water to 60 °C) with intermittent vortexing to ensure hydration. Sonication of these suspensions for 15 min in a bath sonicator at 60 °C afforded closed cationic liposomes, as evidenced from transmission electron microscopy. Liposomes were prepared and kept under sterile conditions. Formulations were stable, and no precipitation was observed within 3 months if stored at 4 °C. Transmission Electron Microscopy. Freshly prepared aqueous suspensions of lipid-DNA complexes 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 200CX) 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

Cholesterol-Based Gemini Lipids

(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) 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 toward HeLa cells was determined using 3-(4,5-dimethyl thiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay following literature procedures (38, 39). 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 5-30 µM were investigated for toxicity studies. Cytotoxicity of the lipid formulations optimal for transfection experiments was determined under conditions exactly the same as transfection conditions. Nearly 12 000 cells/well were plated in 96 well plates. After 24 h, optimized lipid/DOPE formulations were complexed with 0.2 µg of the DNA at various N/P ratios for 30 min. DNA-lipid complexes were added to the cells in the absence of serum. After 6 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 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 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, 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 0.8 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 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 6 h at 37 °C in a humidified atmosphere containing 5% CO2. 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. Plates were further incubated for a period of 42 h before checking for the reporter gene expression. Green fluorescent protein (GFP) expression was examined by fluorescence microscopy and was quantified by flow cytometry analysis. All the experiments were done in duplicate, and results presented are the average of at least two such independent

Bioconjugate Chem., Vol. 18, No. 5, 2007 1539

experiments done on two different days. For comparison, transfections using lipid 1 were performed using optimized lipid/ DOPE ratio (24) of 1:1 at N/P ratio of 1.0, using 0.8 µg of plasmid DNA. 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 this complex for 6 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 to 400 µL 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 transfection experiments using 0.2 µg of the plasmid DNA. After 30 min of incubation, these complexes were electrophoretically run on a 1.0% agarose gel. The uncomplexed DNA moved out of the well, but the DNA that was complexed with lipid remained inside the well (40). 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 percentages of transfected cells were 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 obtain the statistical data, which have been presented as the average of at least two independent measurements. For flow cytometry analysis, ∼48 h posttransfection, 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 forwardscatter). 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. 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. Four cholesterol-based gemini lipids possessing oxyethylene-based spacer chains have been synthesized. Each lipid differed from the others based on the spacer lengths [-CH2-(CH2-O-CH2)n-CH2-]. These gemini lipids have been synthesized from the precursor cholest-5-en-3β-oxyethanN,N-dimethyl amine by Menshutkin reaction with the corresponding R,ω-dibromoalkoxyalkanes. The synthesis of precursor cholest-5-en-3β-oxyethan-N,N-dimethyl amine and monomeric lipid 1 has already been reported (24). All the gemini lipids have been purified by repeated precipitation with MeOH, EtOAc, and acetone. All the gemini lipids were fully characterized by 1H NMR, 13C NMR, ESI-MS, and C, H, N analysis. Aggregate Formation from Cationic Gemini Cholesterol Lipids. Upon hydration, each of the gemini cholesterol lipids


Bajaj et al.

Figure 2. Electrophoretic gel patterns for lipoplex-associated DNA in gel retardation for gemini cholesterol lipids: (a) 2a; (b) 2b; (c) 2c; (d) 2d. The N/P ratios are indicated at the top of each lane. 0.2 µg of the DNA was complexed with liposomes at various N/P ratios for 30 min, and lipoplexes were run electrophoretically on 1% agarose gel.

Figure 3. Transfection efficiencies of cholesterol-based gemini lipids with various mole ratios of DOPE: (a) 2a; (b) 2b; (c) 2c; (d) 2d. Concentration of the DNA ) 0.8 µg/well, and lipids were used at N/P ratio of 0.5. Lipoplexes were incubated with cells for 6 h. Data are expressed as the number of transfected cells and MFI as obtained from flow cytometry analysis after 48 h of transfection.

was found to disperse easily in water. All the lipid molecules mentioned here formed stable suspensions in water. The suspensions formed from lipids were found to be translucent. Generally, TEM examination of air-dried, aqueous suspensions revealed the existence of closed vesicle-like aggregate structures for all the gemini lipids (not shown). The aggregates formed from all the gemini lipids ranged from 30 to 80 nm in size. When compared, all gemini lipid aggregates were generally found to be smaller in size as compared to the aggregates of monomeric lipid 1, which ranged ∼100 ( 30 nm in size (not shown). Mixed Liposome Formation with 1,2-Dioleoyl-L-r-glycero3-phosphatidyl Ethanolamine (DOPE). Liposomes could be conveniently prepared from a mixture of gemini lipid with helper lipid 1,2-dioleoyl-L-R-glycero-3-phosphatidyl ethanolamine (DOPE) by first subjecting the films of lipid mixtures to hydration, repeated freeze-thaw cycles, and followed by sonication at 60 °C for 15 min. All the gemini lipid/DOPE formulations formed optically transparent suspensions. Liposomes were prepared under sterile conditions and were resonicated for 5 min at room temperature before transfection experiments. The vesicular suspensions were sufficiently stable, and no precipitation was observed within 3 months if stored at 4 °C. Gel Electrophoresis. To characterize the electrostatic binding interactions between the plasmid DNA and the mixed cationic liposomes as a function of different N/P charge ratios (or lipid/ DNA mole ratios), we performed conventional electrophoretic gel retardation assays (Figure 2). All gemini lipid suspensions

were able to retard the plasmid DNA from the well at N/P ratio of 1.0. It should be noted that every gemini molecule possesses two hard charges, which indicates that the whole plasmid DNA gets retarded at a lipid/DNA mol ratio of 0.5. Significant retardation of DNA was also observed at an N/P ratio of 0.5 (or lipid/DNA mole ratio of 0.25) as well. Transfection Biology. To find out the optimized transfection efficiency, both the number of transfected cells and the mean fluorescence intensity (MFI) have been considered. The MFI defined for GFP-positive cells reveal that the level of GFP expression with a higher MFI value correlates positively with high GFP expression (41). These data were obtained from flow cytometric analysis. Figure 3 clearly indicates that individual lipid formulations were able to transfect the high number of cells, but the MFI observed was very low. When cells transfected with individual lipids were observed under the fluorescence microscope (not shown), very few green fluorescent cells were visible. This is confirmed by FACS analysis, which showed very low MFI. Optimization of Lipid/DOPE Ratio. Naturally occurring lipid such as DOPE has been known to increase the efficiency of transfection in lipid-mediated gene transfer applications. In order to see the most effective formulations, transfections with identical lipid/DNA mole ratios (or N/P ratio), varying the mole ratio of gemini lipids (2a-2d) in DOPE, were performed (Figure 3). FACS analysis (Supporting Information) indicates that incorporation of DOPE enhances the transfection efficacy of gemini lipids dramatically, especially in terms of MFI. When cells transfected with lipid-DOPE co-liposomes were observed



Figure 4. Transfection efficiencies of gemini lipids using optimized lipid/DOPE formulations at various N/P ratios in the absence of serum (-FBSFBS). (a) 2a; (b) 2b; (c) 2c; (d) 2d. Concentration of the DNA ) 0.8 µg/well. Lipoplexes were incubated with cells for 6 h. Data are expressed as the number of transfected cells and MFI as obtained from flow cytometry analysis after 48 h of transfection.

under the fluorescence microscope, we observed highly fluorescent cells which were confirmed by the increase in MFI observed from FACS analysis. In our studies, we had considered both the number of transfected cells and the MFI for optimizing the transfection efficacies of gemini lipids, both as the number of transfected cells and the amount of gene expression matter for successful gene therapy. The optimized lipid/DOPE ratio was found to vary with the length of the oxyethylene spacer of gemini lipids. Lipid 2a showed maximum gene transfection activity at a lipid/DOPE ratio of 1:4, whereas 2b showed maximum activity at a lipid/ DOPE ratio of 1:2, and with further increase in DOPE mole ratio, transfection activity started to decrease. Gemini lipid 2c showed maximum activity at a lipid/DOPE mol ratio of 1:3. Lipid 2a at a lipid/DOPE ratio of 1:4 was able to transfect nearly 50% of the cells with MFI of 100, whereas 2b was able to show the same activity at a lipid/DOPE mole ratio of 1:2. Lipid 2c at a lipid/DOPE mole ratio of 1:3 was found to be most effective, transfecting nearly 60% of cells with MFI greater than 100, and with further increase in the DOPE mole ratio, transfection efficiency decreased. Lipid 2d was not found to be highly effective at any of the lipid/DOPE ratios, although maximum transfection activity was observed at a lipid/DOPE mol ratio of 1:5. While comparing all the gemini lipids, lipid 2c was found to be the most effective gemini lipid and even required a lower mole percentage of DOPE as compared to other gemini lipids. Each gemini lipid required different amounts of DOPE for optimized transfection efficiency; this may be because of the change in the balance between hydrophilicity and hydrophobicity of the gemini lipids. Optimization of N/P Ratio. After optimization of the lipid/ DOPE mole ratio for each gemini lipid, all the gemini lipids were tested by taking the identical amount of DNA (0.8 µg) and varying the amount of lipid using the respective optimized lipid/DOPE ratio. Lipid 2a was able to transfect a maximum of 50% of the cells with an MFI of 100 at N/P ratio of 0.25, whereas lipid 2b formulations could transfect approximately the

same number of cells but with low MFI at N/P ratio of 0.5 (Figure 4). Transfection efficiency of the lipid 2a formulation decreased at higher N/P ratios, especially in terms of MFI. Gemini lipid 2c was able to transfect more than 60% of the cells at N/P ratio of 0.5 with MFI of ∼100. While comparing the transfection efficiencies, the lipid 2b formulation induced a lower transfection efficiency than lipid 2a, whereas the transfection efficiency of the lipid 2c formulation was greater than that of lipid 2b and was comparable to that of 2a. The lipid 2d formulation was found to be the least effective gene delivery agent in this series of gemini cholesterol lipids. All the gemini lipids followed a bell-shaped graph when transfection efficiencies were plotted against N/P ratio. When all the gemini lipids were compared at N/P ratio of 0.5, lipid 2a and 2c formulations were found to be the best transfecting agents. Effect of Serum. One of the serious shortcomings 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. Serum proteins that are negatively charged are known to interact with cationic lipids, thereby competing with DNA for cationic lipids, leading to inhibition of transfection. To have better potential especially in gene therapy, it is important to have reagents that are capable of delivering and expressing an external gene inside a cell in the presence of serum. There are very few reports of cationic lipids, including our own, which induce transfection activity even in the presence of serum (19, 42, 43). Therefore, we planned to study the efficiencies of these cholesterol-based gemini lipids in the presence of serum as well and compared the transfection activities of these lipid formulations against a commercially available reagent, Lipofectin. To our pleasant surprise, serum did not inhibit the transfection efficiency of any of the gemini lipids; an enhancement in transfection efficiency was even observed in the presence of 10% serum conditions (Figure 5). Gemini lipid 2a showed better transfection efficiency in the presence of serum. Nearly 60-70% of the cells were found to be transfected with MFI of 100-135 using the lipid


Bajaj et al.

Figure 5. Transfection efficiencies of gemini lipids using optimized lipid/DOPE formulations at various N/P ratios in the presence of 10% FBS (-FBS+FBS). (a) 2a; (b) 2b; (c) 2c; (d) 2d. Concentration of the DNA ) 0.8 µg/well. Lipoplexes were incubated with cells for 6 h. Data are expressed as the number of transfected cells and MFI as obtained from flow cytometry analysis after 48 h of transfection.

2a formulation at N/P ratio of 0.25 in the presence of 10% serum conditions. Serum also did not affect the transfection efficiency of gemini lipid 2b, although efficiency was lower than that of the lipid 2a formulation. The transfection efficiency of lipid 2c was nearly 60-70% of cells with MFI of 100-110 at N/P ratio of 0.5. Gemini lipid 2d was much less efficient in the presence of 10% serum as compared to other gemini lipids. Therefore, all the gemini lipids (except 2d) were found to show high transfection activity in the presence of serum. Gemini Lipids vs Monomeric Lipid. To put our results into proper perspective, we compared the transfection activities of the cholesterol gemini lipid formulations with those of their corresponding monomer. Figure 6 shows the transfection efficiencies of the gemini lipids (2a-2d) in comparison with that of monomeric lipid 1 formulation. Although the monomeric lipid 1 showed transfection efficiency of ∼60%, but the mean fluorescence intensity in the case of monomeric lipid 1 was much lower as compared to any of the gemini lipids tested (Figure 6). MFI indirectly tells the amount of the protein expressed in the cells, which gives the number of copies of the plasmids delivered inside the cell. It is clear that each gemini lipid shows enhanced mean fluorescence intensity as compared to the monomeric lipid counterpart 1. Figure 6b shows that there is a dramatic decrease in the transfection efficiency of the monomeric lipid 1 in the presence of 10% serum conditions, whereas in the case of gemini lipids, there is hardly any effect on the transfection activity in the presence of serum. The lipid 2a formulation was found to transfect nearly 70% of the cells with MFI of ∼100, whereas formulations based on monomeric lipid 1 transfected only nearly 20% of cells with very low MFI of ∼40. Therefore, in conclusion, all gemini lipids were much better transfecting agents as compared to the monomeric lipid 1. Among gemini lipids, the lipid 2a formulation was found to be the best transfecting agent in the presence of serum, whereas the lipid 2d formulation was least effective.

Figure 6. Comparative transfection efficiencies of gemini lipids (2a2d) and monomeric lipid 1 in the absence (-FBS-FBS) and presence of 10% serum (-FBS+FBS). Concentration of the DNA ) 0.8 µg/ well. Lipoplexes were incubated with cells for 6 h. Data are expressed as the number of transfected cells and MFI as obtained from flow cytometry analysis after 48 h of transfection.

GFP Assay at High Serum Concentrations. To see the effect of high concentrations of serum on the gene transfection efficiencies, we performed the gene transfection efficiencies using optimized lipid/DOPE formulations at 30% and 50% serum concentrations (Figures 7 and 8). The lipid 2a/DOPE formulation was able to transfect ∼50% of cells at 30% and



Figure 7. Transfection efficiencies of gemini lipids using optimized lipid/DOPE formulations at various N/P ratios in the presence of 30% FBS. (a) 2a; (b) 2b; (c) 2c; (d) 2d. Concentration of the DNA ) 0.8 µg/well. Lipoplexes were incubated with cells for 6 h. Data are expressed as the number of transfected cells and MFI as obtained from flow cytometry analysis after 48 h of transfection.

Figure 8. Transfection efficiencies of gemini lipids using optimized lipid/DOPE formulations at various N/P ratios in the presence of 50% FBS. (a) 2a; (b) 2b; (c) 2c; (d) 2d. Concentration of the DNA ) 0.8 µg/well. Lipoplexes were incubated with cells for 6 h. Data are expressed as the number of transfected cells and MFI as obtained from flow cytometry analysis after 48 h of transfection.

50% serum concentrations, although there was a decrease in the MFI at higher serum conditions and maximum transfections were observed at N/P ratio of 2.0. This is expected, because at such higher serum conditions, negatively charged serum proteins start to compete with DNA for complexation with cationic lipids. One major characteristic feature of the lipid 2a formulation is that there is no effect on the number of cells getting transfected even in the presence of high concentrations of serum. Therefore, it indicates that serum does not inhibit the number of transfected cells but reduces the amount of the DNA delivered inside the cells as indicated by FACS analysis. Lipid 2b is able to transfect nearly 40% of the cells at N/P ratio of 3.0 at 30% and 50% serum conditions. The MFI observed in the case of lipid 2b formulations was nearly twice that of the lipid 2a formulation, which indicates that lipid 2b was able to deliver double the amount of DNA into the cells as compared to the lipid 2a

formulation, whereas the transfection efficacy of commercially available Lipofectin diminished completely at higher serum concentrations. Lipid 2c was able transfect 30% of cells with low MFI at N/P ratios 2.0-3.0. Lipid 2d was not able to transfect at high serum concentrations. Effect of Variation of the Amount of DNA. To see whether the varying the amount of DNA affects the transfection efficiency of gemini lipids, we have performed transfection experiments with all gemini lipids at N/P ratios of 0.25 and 0.5, varying the amount of DNA from 0.4 to 2.0 µg. We did not observe any increase in gene transfection activity by varying the amount of DNA in all cases of gemini lipids, except gemini lipid 2a, which showed an enhancement in gene delivery when 0.4 µg of DNA was used (Figure 9). This indicates that lipid 2a is more efficient at low DNA concentrations. Lipid 2a was able to transfect nearly 70% of cells with MFI of ∼170 at an


Bajaj et al.

Figure 9. Effect of the variation of the amount of the DNA on gene transfection efficiency of gemini lipid 2a/DOPE (1:4 mol ratio) formulation at N/P ratio of 0.5. Lipoplexes were incubated with cells for 6 h. Data are expressed as the number of transfected cells and MFI as obtained from flow cytometry analysis after 48 h of transfection.

N/P ratio of 0.5. Therefore, among all gemini lipids, the lipid 2a formulation was found to be the best, transfecting nearly 70% of cells using 0.4 µg of DNA at an N/P ratio of 0.5. Cytotoxicity. The cytotoxic effects of the cationic lipids toward HeLa cells had been examined in detail, as shown in Figure 10. First, all the liposome formulations of lipids had been tested at different concentrations of 5, 10, 15, 20, and 30 µM, as the maximum amount of lipid used in all transfection is 18.8 µM. All the lipid formulations were found to be nontoxic at all lipid concentrations, except lipid 2d formulation, where 80% cell viability was found at 20 µM concentration and 70% cell viability at 30 µM concentration (Figure 10a). To see the effect of the DOPE on cell viability, we also investigated the cell viability assays on HeLa cells using optimized lipid/DOPE formulations at different concentrations of 5, 10, 15, 20, and 30 µM (Figure 10b). Lipid/DOPE formulations were also found be nontoxic at all concentrations, except lipid 2d where nearly 80% cell viability was found. The lipid 2d formulation was also found to be the least efficient transfecting agent among the series of lipids studied herein. Lipid-DNA lipoplexes using optimized lipid/DOPE formulations were studied for cell viability assays. More than 80% cell viability was observed with all formulations at all N/P ratios (Figure 10c). Characterization of Lipid-DNA Complexes. Morphology of lipid/plasmid complexes was visualized under a transmission electron microscope after negative staining using uranyl acetate. The complexes formed with the best gemini lipids (2a and 2c) at their optimized transfection ratios with plasmid DNA revealed some distinct morphological changes depending on the length of the spacer in between the cationic ammonium headgroups of the gemini cholesterol lipid molecules (Figure 11). The lipoplexes formed from gemini lipid 2a/DNA showed a tendency to form “elongated” structures (Figure 11a). It appears the lipid vesicles are organized on the templates of plasmid DNA surface. In contrast, the lipid 2c/DNA complexes showed aggregation during such complexation, leading to the formation of irregular morphologies.

CONCLUSIONS We have presented here the syntheses of four cholesterolbased gemini cationic lipids, which bear oxyethylene-based spacers in between the headgroups. These gemini lipids are dimers of the monomeric lipid 1, which also possess a nonhydrolyzable ether-type linkage between the cationic headgroup and cholesterol backbone. The gemini lipids differ from each other in the number of oxyethylene groups present between the headgroups. The gemini lipids have been synthesized in a single step from cholest-5-en-3β-oxyethan-N,N-dimethylamine, by reacting with appropriate R,ω-dibromoalkoxyalkanes. Electron micrographs showed the presence of vesicle-like aggregates formed from the aqueous suspensions of these gemini

Figure 10. (a) Cytotoxicity exhibited toward HeLa cells by liposomes. (b) Cytotoxicity exhibited toward HeLa cells by optimized lipid/DOPE co-liposomes. (c) Cytotoxicity toward HeLa cells by lipoplexes at different N/P ratios using optimized lipid/DOPE formulations. All the experiments were performed in triplicate in 96-well plates. The % viability was then calculated as [{A590(treated cells) - background]/ [A590(untreated cells) - background}] × 100.

lipids. All gemini cholesterol lipids were able to transfect the mammalian cells. Incorporation of an oxyethylene spacer between the cationic ammonium headgroups dramatically increased the transfection activities of the gemini cholesterol lipids as compared to their monomeric counterparts. Serum is known to be a major impediment for in ViVo applications of cationic liposomes. All the gemini lipid formulations were, however, able to transfect the mammalian cells even in the presence of serum, in contrast to their monomeric lipid counterparts, where a dramatic decrease in transfection efficacy



ACKNOWLEDGMENT This work was supported by Department of Biotechnology, Government of India, New Delhi, India. Avinash Bajaj thanks CSIR for a senior research fellowship. Supporting Information Available: Elemental analysis and FACS details. This material is available free of charge via the Internet at http://pubs.acs.org.

LITERATURE CITED

Figure 11. Transmission electron micrographs for the lipoplexes prepared from most effective gemini lipid/DOPE liposome/DNA complexes at the optimized N/P ratio. (a) 2a; (b) 2c.

was observed. The transfection efficiency of gemini lipid 2a was in fact enhanced in the presence of 10% serum conditions. The lipid 2a formulation was able to transfect nearly 70% of the cells with high MFI. A characteristic feature of the lipid 2a formulation is that highest transfection efficiency was achieved using only 0.4 µg of the DNA at an N/P ratio of 0.5. The lipid 2c formulation was found to be effective at a lipid/DOPE ratio of 1:2 and also compatible in the presence of serum. The lipid 2d formulation was found to be the least effective. Maximum transfection efficacies of gemini lipids were observed at N/P ratios of 0.5-0.75 in the absence of serum, whereas we observed the complete complexation of DNA at N/P ratios higher than 1.0. Similar observations have also been reported by others. Complete retardation of the plasmid DNA is not essential for maximum gene transfection efficacy. There are many interactions including hydrophobic interactions between liposomes and DNA and between lipoplexes and cell membranes, other than electrostatic interactions, that may be responsible for enhanced gene transfection activities at low N/P ratios. Although the precise molecular mechanism for their compatibility in the presence of serum is beyond the scope of this presentation, these results indicate that gemini cholesterol lipids with proper design may afford new carriers which may be useful in the field of gene therapy. Cytotoxicity assay shows nearly 80% of the cell viability at the optimum lipid/DNA concentrations. Since the resulting gemini lipids and their liposomes with DOPE are stable both chemically and as colloidal suspensions, these could be used for more practical applications.

(1) Yang, Y., Nunes, F. A., Berencsi, K., Furth, E. E., Gonczol, E., and Wilson, J. M. (1994) Cellular immunity to viral antigens limits E1-deleted adenoviruses for gene therapy. Proc. Natl. Acad. Sci. U.S.A. 91, 4407-4411. (2) Knowles, M. R., Hohneker, K. W., Zhou, Z., Olsen, J. C., Noah, T. L., Hu, P. C., Leigh, M. W., Engelhardt, J. F., Edwards, L. J., and Jones, K. R. (1995) A controlled study of adenoviral-vectormediated gene transfer in the nasal epithelium of patients with cystic fibrosis. New Engl. J. Med. 333, 823-831. (3) Crystal, R. G., McElvaney, N. G., Rosenfeld, M. A., Chu, C.-S., Mastrangeli, A., Hay, J. G., Brody, S. L., Jaffe, H. A., Eissa, N. T., and Danel, C. (1994) Administration of an adenovirus containing the human CFTR cDNA to the respiratory tract of individuals with cystic fibrosis. Nat. Genet. 8, 42-51. (4) Yang, Y., Nunes, F. A., Berencsi, K., Goenczoel, E., Engelhardt, J. F., and Wilson, J. M. (1994) Inactivation of E2a in recombinant adenoviruses improves the prospect for gene therapy in cystic fibrosis. Nat. Genet. 7, 362-369. (5) Gao, X., and Huang, L. (1995) Cationic liposome-mediated gene transfer. Gene Ther. 2, 710-22. (6) Boussif, O., Lezoualc’h, F., Zanta, M. A., Mergny, M. D., Scherman, D., Demeneix, B., and Behr, J.-P. (1995) A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine. Proc. Natl. Acad. Sci. U.S.A. 92, 72977301. (7) Wagneer, E., Ogris, M., and Zauner, W. (1999) Polylysine-based transfection systems utilizing receptor mediated delivery. AdV. Drug DeliVery ReV. 30, 97-113. (8) Kabanov, A. V., and Kabanov, V. A. (1995) DNA Complexes with Polycations for the delivery of genetic material into cells. Bioconjugate Chem. 6, 7-20. (9) Miller, A. D. (2003) The problem with cationic liposome/micellebased non-viral vector systems for gene therapy. Curr. Med. Chem. 10, 1195-1211. (10) Nakanishi, M. (2003) New strategy in gene transfection by cationic transfection lipids with a cationic cholesterol. Curr. Med. Chem. 10, 1289-1296. (11) Nicolazzi, C., Garinot, M., Mignet, N., Scherman, D., and Bessodes, M. (2003) Cationic lipids for transfection. Curr. Med. Chem. 10, 1263-1277. (12) Niculescu-Duvaz, D., Heyes, J., and Springer, C. J. (2003) Structure-activity relationship in cationic lipid mediated gene transfection. Curr. Med. Chem. 10, 1233-1261. (13) Ilies, M. A., Seitz, W. A., Ghiviriga, I., Johnson, B. H., Miller, A., Thompson, E. B., and Balaban, A. T. (2004) Pyridinium cationic lipids in gene delivery: a structure-activity correlation study. J. Med. Chem. 47, 3744-3754. (14) Karmali, P. P., Kumar, V. V., and Chaudhuri, A. (2004) Design, syntheses and in vitro gene delivery efficacies of novel mono-, diand tri-lysinated cationic lipids: a structure-activity investigation. J. Med. Chem. 47, 2123-2132. (15) Majeti, B. K., Karmali, P. P., Reddy, B. S., and Chaudhuri, A. (2005) In vitro gene transfer efficacies of N,N-dialkylpyrrolidinium chlorides: a structure-activity investigation. J. Med. Chem. 48, 3784-3795. (16) Ilies, M. A., Seitz, W. A., Johnson, B. H., Ezell, E. L., Miller, A. L., Thompson, E. B., and Balaban, A. T. (2006) Lipophilic pyrylium salts in the synthesis of efficient pyridinium-based cationic lipids, gemini surfactants, and lipophilic oligomers for gene delivery. J. Med. Chem. 49, 3872-3887.

1546 Bioconjugate Chem., Vol. 18, No. 5, 2007 (17) Gardner, R. A., Belting, M., Svensson, K., and Phanstiel, O., IV (2007) Synthesis and transfection efficiencies of new lipophilic polyamines. J. Med. Chem. 50, 308-318. (18) Bhattacharya, S., and Mandal, S. S. (1998) Evidence of interlipidic ion-pairing in anion-induced DNA release from cationic amphiphile-DNA complexes. mechanistic implications in transfection. Biochemistry 37, 7764-7777. (19) Dileep, P. V., Antony, A., and Bhattacharya, S. (2001) Incorporation of oxyethylene units between hydrocarbon chain and pseudoglyceryl backbone in cationic lipid potentiates gene transfection efficiency in the presence of serum. FEBS Lett. 509, 327-331. (20) Bhattacharya, S., and Dileep, P. V. (2004) Cationic oxyethylene lipids. Synthesis, aggregation, and transfection properties. Bioconjugate Chem. 15, 508-519. (21) Felgner, J. H., Kumar, R., Sridhar, C. J., Wheeler, Y. J., Tsai, R., Border, P., Ramsey, M., Martin, C. N., and Felgner, P. L. (1994) Enhanced gene delivery and mechanism studies with a novel series of cationic lipid formulations. J. Biol. Chem. 269, 2550-2561. (22) Song, Y. K., Liu, F., Chu, S., and Liu, D. (1997) Characterization of cationic liposome-mediated gene transfer in vivo by intravenous administration. Hum. Gene Ther. 8, 1585-1594. (23) Ghosh, Y. K., Visweswariah, S. S., and Bhattacharya, S. (2000) Nature of linkage between the cationic headgroup and cholesteryl skelton controls gene transfection efficiency. FEBS Lett. 473, 341344. (24) Ghosh, Y. K., Visweswariah, S. S., and Bhattacharya, S. (2002) Advantage of the ether linkage between the positive charge and the cholesteryl skeleton in cholesterol-based amphiphiles as vectors for gene delivery. Bioconjugate Chem. 13, 378-384. (25) McGregor, C., Perrin, C., Monck, M., Camilleri, P., and Kirby, A. J. (2001) Rational approaches to the design of cationic gemini surfactants for gene delivery. J. Am. Chem. Soc. 123, 6215-6220. (26) Bell, P. C., Bergsma, M., Dolbnya, I. P., Bras, W., Stuart, M. C. A., Rowan, A. E., Feiters, M. C., and Engberts, J. B. F. N. (2003) Transfection mediated by gemini surfactants: engineered escape from the endosomal compartment. J. Am. Chem. Soc. 125, 1551-1558. (27) Kirby, A. J., Camilleri, P., Engberts, J. B. F. N., Feiters, M. C., Nolte, R. J. M., Soderman, O., Bergsma, M., Bell, P. C., Fielden, M. L., Garcia, Rodriguez, C. L., Guedat, P., Kremer, A., McGregor, C., Perrin, C., Ronsin, G., and van Eijk, M. C. P. (2003) Gemini surfactants: New synthetic vectors for gene transfection. Angew. Chem., Int. Ed. 42, 1448-1457. (28) Prata, C. A. H., Zhao, Y., Barthelemy, P., Li, Y., Luo, D., McIntosh, T. J., Lee, S. J., and Grinstaff, M. W. (2004) Chargereversal amphiphiles for gene delivery. J. Am. Chem. Soc. 126, 12196-12197. (29) Chittimalla, C., Zammut-Italiano, L., Zuber, G., and Behr, J.-P. (2005) Monomolecular DNA Nanoparticles for Intravenous Delivery of Genes. J. Am. Chem. Soc. 127, 11436-11441. (30) Paul, B., Bajaj, A., Indi, S. S., and Bhattacharya, S. (2006) Synthesis of novel dimeric cationic lipids based on an aromatic backbone between the hydrocarbon chains and headgroup. Tetrahedron Lett. 47, 8401-8405.

Bajaj et al. (31) Hubner, W., Mantsch, H. H., and Kates, M. (1991) Intramolecular hydrogen bonding in cardiolipin. Biochim. Biophys. Acta 1066, 166174. (32) Bhattacharya, S., De, S., and George, S. K. (1997) Synthesis and vesicle formation from novel pseudoglyceryl dimeric lipids. Evidence of formation of widely different membrane organizations with exceptional thermotropic properties. Chem. Commun. 2287-2288. (33) Bhattacharya, S., and De, S. (1999) Synthesis and vesicle formation from dimeric pseudoglyceryl lipids with (CH2)m spacers: pronounced m-value dependence of thermal properties, vesicle fusion, and cholesterol complexation. Chem.sEur. J. 5, 2335-2347. (34) Kasireddy, K., Ali, S. M., Ahmad, M. U., Choudhury, S., Chien, P.-Y., Sheikh, S., and Ahmad, I. (2005) Synthesis of cationic cardiolipin analogs. Bioorg. Chem. 33, 345-362. (35) Chien, P.-Y., Wang, J., Carbonaro, D., Lei, S., Miller, B., Sheikh, S., Ali, S. M., Ahmad, M. U., and Ahmad, I. (2005) Novel cationic cardiolipin analog-based liposome for efficient DNA and small interfering RNA delivery in vitro and in vivo. Cancer Gene Ther. 12, 321-328. (36) Bhattacharya, S., and Bajaj, A. (2007) Membrane forming properties of pseudoglyceryl backbone based gemini lipids possessing oxyethylene spacers. J. Phys. Chem. B 111, 2463-2472. (37) Bajaj, A., Kondiah, P., and Bhattacharya, S. (2007) Design, synthesis, and in vitro gene delivery efficacies of novel cholesterolbased gemini cationic lipids and their serum compatibility: a structure-activity investigation. J. Med. Chem. 50, 2432-2442. (38) Mosmann, T. (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods 65, 55-63. (39) Hansen, M. B., Nielsen, S. E., and Berg, K. (1989) Re-examination and further development of a precise and rapid dye method for measuring cell growth/cell kill. J. Immunol. Methods 119, 203210. (40) Choi, J. S., Lee, E. J., Jang, H. S., and Park, J. S. (2001) New cationic liposomes for gene transfer into mammalian cells with high efficiency and low toxicity. Bioconjugate Chem. 12, 108-113. (41) Chabaud, P., Camplo, M., Payet, D., Serin, G., Molreau, L., Barthelemy, P., and Grinstaff, M. W. (2006) Cationic nucleoside lipids for gene delivery. Bioconjugate Chem. 17, 466-472. (42) Matsui, K., Sando, S., Sera, T., Aoyama, Y., Sasaki, Y., Komatsu, T., Terashima, T., and Kikuchi, J.-I. (2006) Cerasome as an infusible, cell-friendly, and serum-compatible transfection agent in a viral size. J. Am. Chem. Soc. 128, 3114-3115. (43) Karmali, P. P., Majeti, B. K., Sreedhar, B., and Chaudhuri, A. (2006) In vitro gene transfer efficacies and serum compatibility profiles of novel mono-, di-, and tri-histidinylated cationic transfection lipids: a structure-activity investigation. Bioconjugate Chem. 17, 159-171. BC070010Q

Synthesis and Gene Transfer Activities of Novel ... - ACS Publications

Recommend Documents