Gene Transfection Efficacies of Novel Cationic Gemini Lipids

Biomacromolecules 2008, 9, 991–999

991

Gene Transfection Efficacies of Novel Cationic Gemini Lipids Possessing Aromatic Backbone and Oxyethylene Spacers Avinash Bajaj,† Paturu Kondaiah,‡ and Santanu Bhattacharya*,†,§ Department of Organic Chemistry, Indian Institute of Science, Bangalore 560 012, India, 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 August 22, 2007; Revised Manuscript Received October 17, 2007

Six novel gemini cationic lipids based on aromatic backbone, bearing n-C14H29 or n-C16H33 hydrocarbon chains, differing in the length of oxyethylene type spacers -CH2-(CH2-O-CH2)m-CH2- between each ammonium headgroups have been synthesized, where m varies from 1 to 3. Each of these lipids formed stable suspensions in aqueous media. Cationic liposomes were prepared from each of these lipids individually and as mixtures of each cationic lipid and DOPE. These were used as nonviral gene delivery agents. Transfection studies showed that among lipids bearing n-C14H29 chains, the transfection efficacies decreased with the increase in the length of the spacer, whereas in case of lipids bearing n-C16H33 chains, the transfection efficacies increased with the increase in the length of the spacer. Lipid bearing n-C16H33 hydrocarbon chains with a [-(CH2-CH2-O-CH2-CH2-O-CH2CH2-O-CH2-CH2)-] spacer was found to be a potent gene transfer agent and its transfection was highly serum compatible even in the presence of 50% serum conditions.

Introduction Completion of the Human Genome Project has generated a wealth of information that helps in finding out new therapeutic targets. Nucleic acids play the role of new drugs for the diseases, of which a genetic basis is known. Thus gene delivery could be exploited to correct missing genes, replace defective genes, or down-regulate aberrant gene expressions. Efficient delivery of nucleic acids into target cells however, continues to be a major impediment for the success of gene therapy.1 There are mainly two classes of vehicles for gene delivery: (i) viral and (ii) nonviral vectors. Viral vectors, however, suffer from major disadvantages like adverse immunogenic reactions, insertional mutagenesis, and sometimes fatal toxicity that limit their use in clinical trials.2–5 Nonviral vectors, on the other hand, consist of positively charged polymers,6,7 peptides, or lipids that form self-assemblies with DNA. While these nonviral vectors have the advantages of high DNA carrying capacity, ease of preparation, and lower immunogenicity and cytotoxicity, many of them are not nearly as efficient as viral vectors. Since the discovery by Felgner,8 many liposomal gene delivery reagents have been developed.9 Various modifications at the level of polar headgroup,10,11 chain-backbone linkage region, and the hydrocarbon chain or hydrophobic parts have been shown to significantly influence the efficacies of liposomal gene delivery systems.12–14 We have demonstrated the advantage of the ether linkage on the modulation of the gene transfection efficiencies of cholesterol based cationic lipids.15,16 Gemini lipids typically possess two polar head groups and four long aliphatic chains, linked by either a rigid or flexible spacer.17,18 Aggregation properties of the membranes strongly depend on the nature and length of the spacer between the headgroups.19 * Corresponding author. E-mail: [email protected]. Telephone: (91)-80-2293-2664. Fax: (91)-80-2360-0529. † Department of Organic Chemistry, Indian Institute of Science. ‡ Department of Molecular Reproduction, Development and Genetics. § Chemical Biology Unit of JNCASR.

Enhanced gene transfection properties of cholesterol based gemini lipids had also been reported recently.20,21 Development of alternative molecular structures22–25 for gene transfer holds key to the discovery of new potent nonviral gene delivery vehicles.26 For instance, the transfection properties of a few cationic lipids based on aromatic backbone have been reported.27–30 Hoekstra et al. described the gene delivery properties of certain pyridinium surfactants.31 Balaban and coworkers investigated the transfection properties of various cationic lipids based on pyridinium, dimeric, and oligomeric surfactants having structural variations at the level of hydrophobic segment, linker, and counterions.32–34 Safinya and coworkers have shown that multivalent cationic lipids with an aromatic backbone hold a promise for superior gene transfections activities.28,35,36 Cationic lipid (TRX) having an amidine group with an aromatic backbone coupled with dodecyl hydrocarbon chains have been developed by Sakurai and coworkers.37 Recently, Phanstiel and co-workers disclosed the transfection properties of certain lipophilic polyamines.38 In our search for promising lipid molecules for efficient gene transfection, we have described certain cationic lipids where hydrocarbon chains are anchored via aromatic backbone.26,39 The nature of the spacer and their hydratability influence the aggregation and transfection properties significantly.19–21,40 Gene transfection involves DNA-lipid complexation (lipoplex formation), which is governed by electrostatic interactions. These interactions are in turn influenced by both the hydration of the lipids during lipoplex formation as well as the spine of hydration at the DNA backbone. Therefore it would be interesting to examine transfection properties of aromatic gemini lipids possessing oxyethylene type spacer. We synthesized six new cationic gemini lipids based on an aromatic backbone bearing n-C14H29 or n-C16H33 hydrocarbon chains, where the two lipid units are connected via oxyethylene type spacers of variable lengths at the level of the cationic ammonium headgroups (Figure 1). Herein we present the transfection properties of this class of lipid based formulations

10.1021/bm700930y CCC: $40.75  2008 American Chemical Society Published on Web 02/14/2008

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Figure 1. Molecular structures of the monomeric lipids 1,2, gemini lipids 3a-3c,4a-4c (synthesized and studied in this work), and that of naturally occurring gemini lipid, cardiolipin, DOPE, and cationic lipids DOTMA and DOTAP.

and discuss how the lipid chain length and spacer determine the gene delivery activities.

Experimental Details 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 an individual film such that final concentration of the cationic lipid was 0.5 mM. The mixtures were kept for hydration at 4 °C for 10–12 h and were repeatedly freeze–thawed (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. Plasmid DNA. pEGFP-c3 (Clontech), 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) 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-dimethylthiazole-2-yl)-2,5diphenyltetrazolium bromide reduction assay following literature procedures.41,42 Cytotoxicity of the lipid formulations optimal for transfection experiments were found out under conditions exactly similar for

transfection conditions. Nearly 12000 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 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 at a well when checked under a microscope. Media was removed and 200 µL of DMSO was added per well. The absorbance was measured using a microtiter plate reader. The percent 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 45000 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 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, the medium was removed and cells were washed with DMEM, and 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

Aromatic Backbone Based Lipids

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Scheme 1a

a Reagents, Conditions, and Yields: (i) HCl, MeOH, 80 °C, 12 h, 90%; (ii) RBr, K2CO3, dry acetone, 65 °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) dibromoalkoxy alkanes, MeOH-EtOAc (2:1), 80 °C, screw-top pressure tube, 2–4 days, 40–50%.

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 this complex for 4 h. At the end of the incubation period, medium was removed and cells were washed with DMEM, and 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 a well, but the DNA that was complexed with lipid remained inside the well.43 Transmission Electron Microscopy. Freshly prepared aqueous suspensions of liposomes were examined under transuspensions of lipoplexessmission electron microscopy by negative staining. A 10 µL sample of the suspension was loaded on to 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 5000–20000×. Flow Cytometry. The reporter gene expression was examined by fluorescence microscopy at regular intervals and was quantified 48 h post transfection by flow cytometry. Percentage 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, 10000 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 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 side-scatter than living cells. The FACS scans have 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 were eliminated by gating out brightly fluorescent cells.

Results and Discussion Chemistry. Six new gemini cationic lipids (3,4) bearing different lengths of hydrocarbon chains have been synthesized. For comparison of their transfection properties with that of their corresponding monomeric counterparts, lipids 1,2 were also synthesized (Figure 1). These lipids possess an aromatic backbone that anchors the linkage between the hydrocarbon chains and the charged headgroup. Two cationic ammonium headgroups are joined via oxyethylene type spacers, which differ in the length in the gemini lipids. For the synthesis, first the 3,4-dihydroxy benzoic acid (5) was esterified in the presence of conc HCl and dry MeOH under reflux conditions to afford corresponding ester (6) in 90% yield


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(Scheme 1). The ester (6) 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 (7a-7b) in 70–75% yields. The ester group of the product (7a-7b) was then reduced with LiAlH4 in dry THF under reflux conditions over a period of 12 h, to afford 8a-8b in 90–95% isolated yields. Next, the conversion of 8a-8b 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 ensuring 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 workup, compounds 9a-9b were obtained in 80–85% yields. The lipids 1,2 were prepared upon refluxing the tertiary amine 9a,9b with an excess of MeI in EtOH in quantitative yields (Scheme 1). The gemini cationic lipids with oxyethylene type spacers, 3,4 were synthesized by heating 9a-9b with the appropriate R,ω-dibromoalkoxy alkanes 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 (see Supporting Information). Aggregate Formation from Cationic Gemini Lipids. Upon hydration, all the cationic lipids were found to be dispersed in water. The suspensions formed from lipids were found to be translucent. Generally TEM examination of air-dried, aqueous suspensions revealed the existence of vesicular aggregate structures for all the gemini lipids (not shown). Mixed Liposome Formation with 1,2-Dioleoyl-L-r-glycero3-phosphatidyl ethanolamine (DOPE). Liposomes could be conveniently prepared from each of gemini lipid with naturally occurring helper lipid (1,2-dioleoyl-L-R-glycero-3-phosphatidyl ethanolamine (DOPE)) by first subjecting the films of lipid mixtures to hydration then repeated freeze–thaw cycles followed by sonication at 80 °C for 15 min. The liposomal suspensions were sufficiently stable, and no precipitation was observed within three 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). Gemini lipid suspensions bearing n-C16H33 hydrocarbon chains were able to retard the plasmid DNA from the well at N/P ratio of 1.0 and more than 90% plasmid DNA got retarded at N/P ratio of 0.75. It should be noted that every gemini molecule possesses two hard charges, which indicates that the whole plasmid DNA gets retarded at lipid/DNA mole ratio of 0.5. For gemini lipid suspensions bearing n-C14H29 hydrocarbon chains, nearly 90% of the DNA gets retarded at N/P ratio of 0.75. We observed maximum transfection efficiencies at N/P ratio of 0.75–1.0 in the absence of serum, where a maximum amount of the DNA was bound to liposomes.

Figure 2. 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. Table 1. Optimized Lipid:DOPE Mole Ratios for Cationic Lipids for Transfection Studies. lipid

lipid:DOPE

lipid

lipid:DOPE

1 3a 3b 3c

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

2 4a 4b 5c

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

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.44 Therefore, we first decided to investigate the optimized lipid:DOPE mole ratio for our newly cationic lipids as their neat lipid suspensions were found to show very low transfection efficacies (not shown). To obtain the most effective formulations, transfections with identical lipid/DNA mole ratio (or N/P ratio) with variations in the mole ratio of lipids in DOPE were performed. To find out the optimized transfection efficiency, both the number of transfected cells and mean fluorescence intensity (MFI) were considered. The mean fluorescence intensities (MFI) defined for GFP positive cells reveal that the level of GFP expression with a higher MFI value correlates positively with a high GFP expression.45 These data were obtained from flow cytometric analysis. Table 1 shows the optimized lipid:DOPE ratios for these gemini lipids. Monomeric lipids 1 and 2 showed maximum transfection efficacy at lipid:DOPE mole ratio of 1:2, whereas for the gemini lipids, the optimized lipid:DOPE mole ratios were found to be high. For lipids possessing [-(CH2-CH2-O-CH2-CH2)-] spacer, optimized lipid:DOPE ratio was 1:8, whereas for other gemini lipids possessing longer spacers, the optimized mole ratio was 1:6. These optimized lipid:DOPE formulations were used for further studies. Optimization of Lipid/DNA (N/P) Charge Ratio. After optimization of the lipid:DOPE mole ratio for each gemini lipid, each gemini lipid was tested by taking the identical amount of DNA (0.8 µg) and varying the amount of lipid using the



Figure 3. Transfection efficiencies of cationic lipids using optimized lipid:DOPE formulations at various N/P ratios in the absence of serum (-FBS-FBS). (a): lipid 1 (SE ) 3.415 (GFP), 10.23 (MFI); SD ) 7.637 (GFP), 22.87 (MFI)); (b): lipid 3a (SE ) 4.201 (GFP), 5.545 (MFI); SD ) 9.393 (GFP), 12.40 (MFI)); (c): lipid 3b (SE ) 5.504 (GFP), 2.782 (MFI); SD ) 12.31 (GFP), 6.221 (MFI)); (d): lipid 3c (SE ) 5.607 (GFP), 1.265 (MFI); SD ) 12.54 (GFP), 2.829 (MFI)). Concentration of the DNA ) 0.8 µg/well. Data are expressed as number of transfected cells and MFI as obtained from flow cytometry analysis.

respective optimized lipid:DOPE ratio. Monomeric lipid 1 based formulation was able to transfect nearly 40% of cells with MFI ∼ 75 (Figure 3). With the incorporation of -CH2-CH2-O-CH2CH2-, the spacer between the headgroups did not influence the transfection activity of lipid 3a formulation, which transfects nearly 40% of cells with MFI of ∼70 at N/P ratio of 1.0. At higher N/P ratios, although there was no change in the number of transfected cells, there was a decrease in MFI observed. It means at higher N/P ratios, although the number of transfected cells were the same, the amount of the DNA delivered decreased. FACS analysis showed nearly 30% GFP positive cells at N/P ratio of 1.0 in the case of lipid 3b formulation, and at N/P ratio of 1.5, nearly 40% cells got transfected with MFI ∼ 50. Similarly, for the formulation from lipid 3c, a maximum of 30% cells got transfected at N/P ratio of 1.5. Overall, among the lipids 3a,3b,3c bearing n-C14H29 hydrocarbon chains, the transfection efficacy decreased with the increase in length of the spacer, having the maximum transfection observed in the case of a lipid 3a based formulation with nearly 40% GFP positive cells with MFI of 75 and dimerization of monomeric lipid 1 did not enhance the transfection efficacy in this series of lipids. Among the lipids bearing n-C16H33 hydrocarbon chains, MFI of ∼40 with nearly 30% GFP positive cells was observed in case of monomeric lipid 2 formulation. Among gemini lipids, lipid 4a formulation with a -CH2-CH2-O-CH2-CH2- spacer was found to be least effective, showing only ∼10% transfection efficacy with very low MFI (Figure 4). With increase in the length of the spacer, transfection efficacy increased. Lipid 4b bearing a [-(CH2-CH2-O-CH2-CH2-OCH2-CH2)-] spacer showed ∼20% transfection efficacy, whereas in the case of lipid 4c, there was a dramatic enhancement in the transfection efficacy. Lipid 4c formulation was able to transfect nearly 60% of cells with MFI ∼ 100, which is even greater than that observed in the case of lipid 3a formulation and nearly two times higher than monomeric lipid 2 formulation.

Overall, while among n-C14H29 hydrocarbon chain based gemini lipids, the transfection efficacy decreased with the increase in the length of the spacer, among aromatic-gemini lipids possessing n-C16H33 hydrocarbon chains, transfection efficacy increased with the increase in the length of the spacer. Thus lipid 4c bearing n-C16H33 hydrocarbon chains with a [-(CH2-CH2-O-CH2-CH2-O-CH2-CH2-O-CH2-CH2)-] spacer showed the maximum transfection activity, while among lipids bearing n-C14H29 hydrocarbon chains, lipid 3a with a [-(CH2CH2-O-CH2-CH2)-] spacer was the most effective. Effect of Serum. One of the serious shortcoming 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 the cationic liposomes, thereby competing with the DNA for cationic lipids, often 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. We therefore performed the transfection with all gemini lipid formulations in the presence of 10% FBS conditions (Figures 5, 6). In the case of monomeric 1 formulation, although there was not much change in number of transfected cells, there were 2-fold decreases in the MFI. Gemini lipid 3a-3c based formulations were able to transfect 20–30% cells without much change in the MFI. There were 2-fold decreases in the number of transfected cells in the case of monomeric lipid 2 formulation. In the case of n-C16H33 chain based lipid formulations, lipid 4c formulation was able to transfect nearly 30% of the cells at a N/P ratio of 0.75 with a MFI of ∼40, where only 5–10% transfection efficacy was observed in the case of other lipid formulations. Therefore, we decided to perform the transfection experiments using lipid 4c formulation at altered N/P ratios in the presence of high serum concentrations, as shown in Figure 7. In the presence of 10% serum conditions, lipid 4c formulation was able to transfect nearly 50% of the cells at N/P ratio of


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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). (a) lipid 2 (SE ) 3.243 (GFP), 1.134 (MFI); SD ) 7.251 (GFP), 2.535 (MFI)); (b): lipid 4a (SE ) 3.192 (GFP), 0.338 (MFI); SD ) 7.137 (GFP), 0.7564 (MFI)); (c): lipid 4b (SE ) 4.751 (GFP), 1.166 (MFI); SD ) 10.62 (GFP), 2.607 (MFI)); (d): lipid 4c (SE ) 5.886 (GFP), 8.975 (MFI); SD ) 13.16 (GFP), 20.07 (MFI)). 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 5. Transfection efficiencies of cationic lipids using optimized lipid:DOPE formulations at various N/P ratios in the presence of 10% serum concentrations (-FBS+FBS). (a): lipid 1 (SE ) 6.955 (GFP), 3.390 (MFI); SD ) 15.55 (GFP), 7.580 (MFI)); (b): lipid 3a (SE ) 3.963 (GFP), 2.371 (MFI); SD ) 8.862 (GFP), 5.302 (MFI)); (c): lipid 3b (SE ) 4.645 (GFP), 3.624 (MFI); SD ) 10.39 (GFP), 8.103 (MFI)); (d): lipid 3c (SE ) 2.619 (GFP), 0.7075 (MFI); SD ) 5.856 (GFP), 1.582 (MFI)). Concentration of the DNA ) 0.8 µg/well. Data are expressed as number of transfected cells and MFI as obtained from flow cytometry analysis.

4.5. In the presence of 30% serum conditions, there was no change in the number of transfected cells, whereas in the case of 50% serum, nearly 40% of the cells were found to be transfected. Therefore, lipid 4c formulation was able to transfect the cells efficiently even at high serum conditions where most of the commercially available formulations failed to transfect although the MFI intensity observed was low. This is expected, as at such high serum concentrations, negatively charged serum proteins compete with gemini lipid formulations for plasmid

DNA binding, which leads to the transfection of low amount of the DNA. Gemini Vs Monomeric Lipids. To put our results in proper perspective, we compared the transfection activities of the gemini lipid formulations with that of their corresponding monomer and commercially available reagent Lipofectin. Lipofectin is a liposomal formulation containing DOTMA and DOPE in 1:1 (w/w) ratio. Among n-C14H29 hydrocarbon chain based lipids, we could not get any advantage of the



Figure 6. Transfection efficiencies of cationic lipids using optimized lipid:DOPE formulations at various N/P ratios in the presence of 10% serum concentrations (-FBS+FBS). (a) lipid 2 (SE ) 2.901 (GFP), 2.977 (MFI); SD ) 6.487 (GFP), 6.658 (MFI)); (b): lipid 4a (SE ) 2.540 (GFP), 0.5707 (MFI); SD ) 5.477 (GFP), 1.275 (MFI)); (c): lipid 4b (SE ) 4.022 (GFP), 1.684 (MFI); SD ) 8.995 (GFP), 3.764 (MFI)); (d): lipid 4c (SE ) 4.449 (GFP), 2.217 (MFI); SD ) 9.948 (GFP), 4.755 (MFI)). 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 7. Transfection efficiencies of cationic lipid 4c using optimized lipid:DOPE formulations at various N/P ratios in the presence of different serum concentrations. (a): 10% serum (SE ) 7.696 (GFP), 3.753 (MFI); SD ) 13.33 (GFP), 6.5 (MFI)); (b): 30% serum (SE ) 7.552 (GFP), 3.032 (MFI); SD ) 13.08 (GFP), 5.252 (MFI)); (c): 50% serum (SE ) 5.920 (GFP), 1.590 (MFI); SD ) 10.25 (GFP), 2.754 (MFI)) concentrations. Concentration of the DNA ) 0.8 µg/well. Data are expressed as number of transfected cells and MFI as obtained from flow cytometry analysis.

gemini lipids as compared to monomeric lipid 1. Whereas in case of n-C16H33 hydrocarbon chain based lipids, lipid 4c was found to be the most effective of other gemini lipids as well as monomeric lipids and lipofectin. Serum compatible efficacies of this gemini lipid formulation had already been shown previously, which shows that this lipid formulation is effective even in the presence of high serum concentrations,

where Lipofectin and most commercially available formulations fail to transfect at all. Cytotoxicity. We performed the MTT based cell viability assays for all the lipid-DNA complexes. (Figure 8) The cell viability results shown in Figure 8 clearly demonstrate that all the formulations show more than 75% cell viability. Monomeric lipid 2 and gemini lipid 4a were found to be least toxic, but


Figure 8. Cellular cytotoxicities of cationic lipid-DNA complexes at their optimized lipid/DNA ratios as determined from MTT assay. These cytotoxicity experiments were performed after 48 h of transfection and the data presented is the average of triplicates performed on the same day.

these are also found to show low transfection activities. Most effective formulation 4c showed ∼70% cell viability.

Conclusions A structure–activity relationship had been investigated for gene transfection activities of aromatic gemini lipids possessing oxyethylene spacer between headgroups. Six new cationic gemini lipids had been synthesized and used as liposomal formulations for gene transfer activities. Gene transfer activities of these gemini lipids were found to depend on the hydrocarbon chain lengths and the length of the oxyethylene spacer. Among n-C14H29 hydrocarbon chain based gemini lipids, lipids possessing a short spacer were found be most active, which is only comparable to its monomeric analogue, and transfection activity decreases with increase in spacer length. Among n-C16H33 chain based lipids, gene transfection activities increase with increase in spacer length and the lipid 4c was found to be the most active, which even possess two times higher activity than its monomeric analogue. Major characteristic feature of this formulation is its serum compatibility showing 30% transfection activity in presence of 50% serum conditions. More than 70% cell viabilities had been found in case of lipoplexes. The actual mechanism for transfection mediated by these lipid formulations is not obvious. However, the interesting results obtained with these novel aromatic gemini lipids should be of interest to researchers working in the field of designing nonviral vectors for gene transfer. 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 and Ms. Padmini for their help during the work. Supporting Information Available. Synthetic details and FACS graphs. This information is available free of charge via the Internet at http://pubs.acs.org.

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BM700930Y

Gene Transfection Efficacies of Novel Cationic Gemini Lipids

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