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Notes Aggregation Properties of a Novel Class of Cationic Gemini Surfactants Correlate with Their Efficiency as Gene Transfection Agents Kevin H. Jennings,† Ian C. B. Marshall,*,† Michael J. Wilkinson,† Andreas Kremer,‡ Anthony J. Kirby,§ and Patrick Camilleri† Department of Analytical Chemistry, GlaxoSmithKline, New Frontiers Science Park, Third Avenue, Harlow, Essex, U.K. CM19 5AW, Department of Biopharmaceutical Research, GlaxoSmithKline, New Frontiers Science Park, Third Avenue, Harlow, Essex, U.K. CM19 5AW, and University Chemical Laboratory, Lensfield Road, Cambridge, U.K. CB2 1EW Received July 5, 2001. In Final Form: November 7, 2001
A number of cationic lipids that complex with DNA have been synthesized specifically for use as gene transfection agents, leading to the successful expression of several recombinant proteins in mammalian cells.1-3 These cationic lipids consist of one or two hydrophobic “tails” containing saturated or mono-unsaturated hydrocarbon chains 16-18 carbon atoms in length. In general, cationic lipids containing two hydrocarbon chains are found to be more efficient gene transfer agents than corresponding single-chain molecules.4 Gemini surfactants5 comprise two hydrocarbon chains linked together by two ionic groups and a spacer. Those with flexible spacers have different surface and bulk properties compared to conventional ionic surfactants, displaying greater propensity to form micelles and efficiently reduce surface tension.6 We recently synthesized a group of novel cationic gemini surfactants, GS1-GS5, comprising C12 saturated hydrocarbon “tails” and a short peptide “headgroup” with one or more basic amino acid residues (Figure 1).7 GS1-GS5 were all found to bind DNA, but they showed different efficiencies in mediating DNA transfection. GS1 and GS3 were ineffective in mediating transfection of a luciferase * To whom correspondence may be addressed. † Department of Analytical Chemistry, GlaxoSmithKline. ‡ Department of Biopharmaceutical Research, GlaxoSmithKline. § University Chemical Laboratory. (1) Felgner, P. L.; Gadek, T. R.; Holm, M.; Roman, R.; Chan, H. W.; Northrop, J. P.; Ringold, G. M.; Danielsen, M. Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure. Proc. Natl. Acad. Sci. U.S.A. 1987, 84, 7413-7417. (2) Wang, C. Y.; Huang, L. Highly efficient DNA delivery mediated by pH-sensitive immunoliposomes. Biochemistry 1989, 28, 9508-9514. (3) Felgner, J. H.; Kumar, R.; Sridhar, C. N.; Wheeler, C. J.; Tsai, Y. J.; Border, R.; Ramsey, P.; Martin, M.; Felgner, P. L. Enhanced gene delivery and mechanism studies with a novel series of cationic lipid formulations. J. Biol. Chem. 1994, 269, 2550-2561. (4) Lee, R. J.; Huang, L. Lipidic vector systems for gene transfer. Crit. Rev. Ther. Drug Carrier Syst. 1997, 14, 173-206. (5) Menger, F. M.; Littau, C. A. Gemini surfactants: synthesis and properties. J. Am. Chem. Soc. 1991, 113, 1451-1452 (6) Rosen, M. J. Geminis: A new generation of surfactants. CHEMTECH 1993, 30-34. (7) Camilleri, P.; Kremer, A.; Edwards, A.; Jennings, K.; Jenkins, O.; Marshall, I.; McGregor, C.; Neville, W.; Rice, S.; Smith, R.; Wilkinson, M.; Kirby, A. A novel class of cationic gemini surfactants with efficient in vitro gene transfection properties. J. Chem. Soc., Chem. Commun. 2000, 1253-1254.
reporter gene above control levels in Chinese hamster ovary (CHO) cells7 whereas GS2, GS4, and GS5 were highly effective.7 Indeed, cells incubated overnight with GS4 produced expression levels in the same order of magnitude as those of cells incubated with LIPOFECTAMINE under the same conditions.7 The critical micellar concentrations measured for these surfactants were well above the concentrations used in DNA-binding and transfection experiments suggesting that micelle formation was not necessary for complexation to DNA.7 Therefore, major differences in transfection efficiency were presumed to derive from differences in the physicochemical properties (size, stability, net surface charge) of the cationic lipid-DNA complex. The size and stability of aggregates formed by cationic lipid vectors are thought to be crucial for both in vitro and in vivo gene delivery.8 Here, we report that GS1-GS5 show differences in their aggregation properties when dissolved in water (1 mg/mL) and adsorbed onto carbon/ Formvar coated grids or freshly cleaved mica for analysis by transmission electron microscopy (TEM) or atomic force microscopy (AFM) respectively (Figure 2 and Figure 3). GS3 formed closely packed aggregates after 24 h in solution (Figure 2A) which appeared to coalesce to form strings and longer fibrils >1 µm in length. GS2, GS4, and GS5 showed evidence of irregular shaped aggregates up to ∼500 nm diameter made up of smaller 4-7 nm diameter structures (Figure 2B-D) with very few or no fibrils apparent. For GS4, no evidence of fibrillisation was seen even after prolonged storage in water (over 1 month at 4 °C). GS1 showed the most structural order with fibrillar forms present immediately after dissolution (Figure 3). Fibrils imaged by TEM ranged in length from 5 to 930 nm (mean 229 ( 162 nm, n ) 102) and 3 to 20 nm in width (mean 11 ( 4 nm, n ) 77). In some instances a distinct 68 ( 16 nm periodicity (n ) 12) of thick and thin regions was seen, giving the appearance of “twistlike” features (Figure 3). These features were confirmed by AFM in which no stain was applied to samples (Figure 3). After being aged for 24 h, fibrils were found to exceed 4 µm in length (Figure 3). As is normal for TEM analysis by negative staining,9 sample preparation involved drying of surfactant solutions. This methodology could give rise to artifactual structures and we therefore checked the existence of fibrils using cryo-TEM without resort to drying.10 Fully hydrated samples of GS1 were analyzed by cryo-TEM, and the presence of fibrils with similar periodic features was confirmed (Figure 4). We also tried to image a sample preparation of GS4 by cryo-TEM, but this was unsuccessful. This suggests that preparations of this surfactant (8) Miller, A. D. Cationic liposomes for gene therapy. Angew. Chem., Int. Ed. 1998, 37, 1768-1785. (9) Blessing, T.; Remy, J. S.; Behr, J. P. Monomolecular collapse of plasmid DNA into stable virus-like particles. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 1427-1431. (10) Jennings, K.; Marshall, I.; Birrell, H.; Edwards, A.; Haskins, N.; Sodermann, O.; Kirby, A. J.; Camilleri, P. The synthesis and aggregation properties of a novel anionic gemini surfactant. J. Chem. Soc., Chem. Commun. 1998, 1951-1952.
10.1021/la0110242 CCC: $22.00 © 2002 American Chemical Society Published on Web 02/22/2002
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Figure 1. Molecular structure of gemini surfactants GS1-GS5. Gemini surfactants GS1 to GS5 were prepared under contract by Syncom (Groningen, The Netherlands) and used without further purification.
Figure 2. TEM negative stain images of surfactants 24 h after dissolution. Aliquots of surfactant in water (1 mg/mL) were adsorbed on carbon/Formvar-coated grids (Agar Scientific) and stained with 2.5% (w/v) ammonium molybdate containing 0.5% (w/v) trehalose, pH 7.0, for 15 s and then blotted dry. Samples were examined in a Hitachi H7100 TEM fitted with a Gatan MSC791 digital camera controlled by Gatan Digital micrograph V.2.5 software (Gatan). Panels show (A) GS3, (B) GS2, (C) GS4, and (D) GS5.
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Figure 3. Formation of GS1 fibrils over time. Surfactant GS1 was dissolved in water (1 mg/mL), incubated for either 0 (A, C) or 24 h (B, D) and adsorbed onto carbon/Formvar grids for TEM analysis (A, B) or onto freshly cleaved mica for AFM analysis (C, D). TEM analysis was conducted using a Hitachi H7100 TEM as above. AFM was conducted in air using a TopoMetrix Explorer AFM operating in contact mode (10:1 aspect ratio silicon nitride probes; nominal spring constant 0.032 N m-1; scanning frequency of 2-3 Hz). For AFM images, height of features in the z-plane are indicated by the grayscale.
Figure 4. Gemini surfactant GS1 imaged in fully hydrated state by cryo-TEM. Aliquots of surfactant in water (1 mg/mL) were placed on holey carbon grids (Agar Scientific), blotted with filter paper, and ultrarapidly plunge frozen in liquid ethane. Frozen samples were cryotransferred to an Oxford CT3500 cryo holder (Oxford Instruments) and examined in a Hitachi H7100 TEM as above. Panels A and B show different magnifications (as indicated) for the same sample.
are in a nonmicellar form and lack the necessary contrast for visualization by this technique.11 (11) Talmon, Y. Transmission electron microscopy of complex fluids - the state of the art. Ber. Bunsen-Ges. Phys. Chem. 1996, 100, 364-372.
A correlation was observed between surfactant morphology and transfection efficiency. Gemini surfactants that formed arrays or fibrils (GS1 and GS3) were ineffective as transfection agents in CHO-K1 cells, whereas those lacking these features (GS2, GS4, and GS5) were
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highly effective.7 Higher concentrations of surfactant are needed for imaging compared with transfection. This may in turn exaggerate the fibrillization properties of the surfactants which may be presumed from critical micelle concentration values to be operating at the submicellar level in transfection experiments. Nevertheless, identifying the propensity of surfactants to form close packed arrays and fibrils at these elevated concentrations appears to serve as a useful predictor of transfection efficiency. We have now embarked on an extensive program to design
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other peptide-based cationic gemini surfactants. Detailed relations between structure and transfection efficiency may help to provide valuable information concerning the features essential for the optimization of biological activity and the development of improved formulations.12 LA0110242 (12) Anchordoquy, T. J.; Girouard, L. G.; Carpenter, J. F.; Kroll, D. J. Stability of lipid/DNA complexes during agitation and freeze-thawing. J. Pharm. Sci. 1998, 87, 1046-1051.
