Transmission electron microscopy.

<p>Negative-stain transmission electron micrographs of aqueous suspensions of the cholesterol-based cationic geminis (a) CholG-D, (b) CholHG-D, (c) CholHG-1ox, (d) CholHG-2ox, (e) CholHG-3ox and (f) CholHG-4ox.</p

Efficacious Gene Silencing in Serum and Significant Apoptotic Activity Induction by Survivin Downregulation Mediated by New Cationic Gemini Tocopheryl Lipids

Nonviral gene delivery offers cationic liposomes as promising instruments for the delivery of double-stranded RNA (ds RNA) molecules for successful sequence-specific gene silencing (RNA interference). The efficient delivery of siRNA (small interfering RNA) to cells while avoiding unexpected side effects is an important prerequisite for the exploitation of the power of this excellent tool. We present here six new tocopherol based cationic gemini lipids, which induce substantial gene knockdown without any obvious cytotoxicity. All the efficient coliposomal formulations derived from each of these geminis and a helper lipid, dioleoylphosphatidylethanolamine (DOPE), were well characterized using physical methods such as atomic force microscopy (AFM) and dynamic light scattering (DLS). Zeta potential measurements were conducted to estimate the surface charge of these formulations. Flow cytometric analysis showed that the optimized coliposomal formulations could transfect anti-GFP siRNA efficiently in three different GFP expressing cell lines, viz., HEK 293T, HeLa, and Caco-2, significantly better than a potent commercial standard Lipofectamine 2000 (L2K) both in the absence and in the presence of serum (FBS). Notably, the knockdown activity of coliposomes of gemini lipids was not affected even in the presence of serum (10% and 50% FBS) while it dropped down for L2K significantly. Observations under a fluorescence microscope, RT-PCR, and Western blot analysis substantiated the flow cytometry results. The efficient cellular entry of labeled siRNA in GFP expressing cells as evidenced from confocal microscopy put forward these gemini lipids among the potent lipidic carriers for siRNA. The efficient transfection capabilities were also profiled in a more relevant fashion while performing siRNA transfections against survivin (an anti-apoptotic protein) which induced substantial apoptosis. Furthermore, the survivin downregulation improved the therapeutic efficacy levels of an anticancer drug, doxorubicin, significantly. In short, the new tocopherol based gemini lipids appear to be highly promising for achieving siRNA mediated gene knockdown in various cell lines

Reaction conditions and yields: (i) <i>p</i>-TsCl, Py, CHCl₃, DMAP, 0°C, 6 h, 91.5%; (ii) Ethylene glycol, dry dioxane, 4 h, reflux, 80%; (iii) <i>p</i>-TsCl, Py, CHCl₃, 0°C, 6 h, 88%; (iv) Dimethylamine, MeOH, screw-top pressure tube, 80°C, 24 h, 99%; (v) 1,4-Dibromobutane-2,3-diol, MeOH-EtOAc (4 mL, v/v: 1/1), screw-top pressure tube, 80°C, 48–72 h, 48%; (vi) <i>N</i>-Methylethanolamine, CH₃CN, 24 h, reflux, 97%; (vii) 1,4-Dibromobutane-2,3-diol, MeOH-EtOAc (4 mL, v/v: 1/1), screw-top pressure tube, 80°C, 48–72 h, 41%; (viii) α, <i>ω</i>-dibromoalkoxyalkane, MeOH-EtOAc (4 mL, v/v: 1/1), screw-top pressure tube, 80°C, 48–72 h, 40–50%.

<p>Reaction conditions and yields: (i) <i>p</i>-TsCl, Py, CHCl₃, DMAP, 0°C, 6 h, 91.5%; (ii) Ethylene glycol, dry dioxane, 4 h, reflux, 80%; (iii) <i>p</i>-TsCl, Py, CHCl₃, 0°C, 6 h, 88%; (iv) Dimethylamine, MeOH, screw-top pressure tube, 80°C, 24 h, 99%; (v) 1,4-Dibromobutane-2,3-diol, MeOH-EtOAc (4 mL, v/v: 1/1), screw-top pressure tube, 80°C, 48–72 h, 48%; (vi) <i>N</i>-Methylethanolamine, CH₃CN, 24 h, reflux, 97%; (vii) 1,4-Dibromobutane-2,3-diol, MeOH-EtOAc (4 mL, v/v: 1/1), screw-top pressure tube, 80°C, 48–72 h, 41%; (viii) α, <i>ω</i>-dibromoalkoxyalkane, MeOH-EtOAc (4 mL, v/v: 1/1), screw-top pressure tube, 80°C, 48–72 h, 40–50%.</p

Optimized formulations of different cholesterol based lipids.

<p>(A) Optimized DOPE:lipid ratios; (B) optimized N/P ratios; (C) best transfection efficiency of lipids in absence of serum and (D) best transfection efficiency of lipids in serum. Concentration of DNA = 0.8 µg/well. Data are expressed as number of transfected cells and MFI as obtained from flow cytometry. Statistical differences from the controls (CholH-M) are labelled ** <i>P<</i>0.005 and *** <i>P<</i>0.0005.</p

Molecules of interest.

<p>Molecular structures of the cholesterol-based cationic gemini lipids used in the present investigation.</p

Gene Transfection in High Serum Levels: Case Studies with New Cholesterol Based Cationic Gemini Lipids

<div><p>Background</p><p>Six new cationic gemini lipids based on cholesterol possessing different positional combinations of hydroxyethyl (-CH₂CH₂OH) and oligo-oxyethylene -(CH₂CH₂O)_n- moieties were synthesized. For comparison the corresponding monomeric lipid was also prepared. Each new cationic lipid was found to form stable, clear suspensions in aqueous media.</p><p>Methodology/Principal Findings</p><p>To understand the nature of the individual lipid aggregates, we have studied the aggregation properties using transmission electron microscopy (TEM), dynamic light scattering (DLS), zeta potential measurements and X-ray diffraction (XRD). We studied the lipid/DNA complex (lipoplex) formation and the release of the DNA from such lipoplexes using ethidium bromide. These gemini lipids in presence of a helper lipid, 1, 2-dioleoyl phophatidyl ethanol amine (DOPE) showed significant enhancements in the gene transfection compared to several commercially available transfection agents. Cholesterol based gemini having -CH₂-CH₂-OH groups at the head and one oxyethylene spacer was found to be the most effective lipid, which showed transfection activity even in presence of high serum levels (50%) greater than Effectene, one of the potent commercially available transfecting agents. Most of these geminis protected plasmid DNA remarkably against DNase I in serum, although the degree of stability was found to vary with their structural features.</p><p>Conclusions/Significance</p><p>-OH groups present on the cationic headgroups in combination with oxyethylene linkers on cholesterol based geminis, gave an optimized combination of new genera of gemini lipids possessing high transfection efficiency even in presence of very high percentage of serum. This property makes them preferential transfection reagents for possible <i>in vivo</i> studies.</p></div

Bovine serum albumin (BSA) induced gel retardation.

<p>Gel electrophoretic patterns for the lipoplex-associated pEGFP-C3 plasmid DNA in the gel retardation assay for cationic lipid formulations where complexes were further treated with BSA. Experiment was performed using 0.2 µg of DNA per well at the N/P ratio of 5.</p

Alkaloid and Polyphenol fractions of Areca nut induce TGF-β signaling in HaCaT cells.

<p>Treatment of HaCaT cells with both the Alkaloid and Polyphenol fractions of areca nut water extract induced TGF-β signaling (p-SMAD2) and its down-stream target TGM2 as shown by the western blot (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051806#pone-0051806-g003" target="_blank">Figure 3A</a>). Expression of TGF-β down-stream targets were also studied by Real Time PCR (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051806#pone-0051806-g003" target="_blank">Figure 3B</a>) and semi quantitative PCR (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051806#pone-0051806-g003" target="_blank">Figure 3C</a>). Induction of genes by alkaloid and polyphenol fractions of areca nut was compromised in presence of TβRI inhibitor (ALK5 inhibitor). (UN- untreated, WS- water supernatant, POL- Polyphenol supernatant, DCM- Dichloromethane fraction).</p

Average hydrodynamic diameters and sizes of the lipid aggregates as obtained from the DLS measurements, and TEM studies respectively.

<p>The bilayer widths of the aggregates of the cationic gemini lipids as revealed from the x-ray diffractions.</p>a<p>Hydrodynamic diameters as obtained from DLS measurements; each value is shown as the mean ± S.D. (standard deviation) (<i>n = </i>3).</p>b<p>As evidenced from TEM.</p>c<p>Lipid bilayer width from the XRD experiments; the error in the measurements of width were within ±1%.</p

Gel electrophoresis to find out DNA binding and release efficiency.

<p>Electrophoretic gel patterns for the lipoplex-associated pEGFP-C3 plasmid DNA. (A) DNA binding efficiency of different gemini lipid based lipoplexes. The N/P ratios are indicated at the top of each lane. (B) SDS mediated DNA release from representative lipid based lipoplexes. The SDS/lipid ratios are indicated below each lane. Both experiments were performed using 0.2 µg of DNA per well.</p