Novel Gemini Pyridinium Surfactants: Synthesis and Study of Their

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Novel Gemini Pyridinium Surfactants: Synthesis and Study of Their Surface Activity, DNA Binding, and Cytotoxicity Avinash Bhadani and Sukhprit Singh* Department of Chemistry, Guru Nanak Dev University, Amritsar, India Received May 8, 2009. Revised Manuscript Received August 12, 2009 New pyridinium gemini amphiphiles having ethane-1,2-dithiol spacer have been synthesized by regioselective electrophilic cobromination of R-olefins. Ethane-1,2-dithiol (1) and N-bromosuccinimide (6) on reaction with R-olefins (dodecene (2), tetradecene (3), hexadecene (4), and octadecene (5)) gave the respective 1,2-bis(2-bromoalkylthio)ethane (7-10). The bromoalkylthio ethers when reacted with pyridine (11) gave the respective gemini bispyridinium bromide (12-15). The surface properties of new geminis were evaluated by surface tension and conductivity measurements. These gemini surfactants have also been found to be having low cytotoxicity by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide) assay on C6 glioma cells. The DNA binding capabilities of these amphiphiles have been determined below as well as above critical micelle concentration. The preliminary studies by agarose gel electrophoresis indicated chain length dependent DNA binding abilities which has further been proved by ethidium bromide exclusion experiments and transmission electron microscopy (TEM).

Introduction With the development of new technology and close cooperation between several interdisciplinary fields molecular bases of many diseases have been properly recognized. This has eventually led to development of new line of treatment known as “gene therapy”. Gene therapy, a modern treatment protocol for compensating the defect occurring due to defective gene, involves sending the desired encoded information with the help of some agents (vectors).1 The vectors may be viral or nonviral. Although viral vectors have shown desired results in animal models, the clinical trials on human are still prohibited due to immunological responses associated with the virus.2 Gemini cationic amphiphiles are novel delivery agents having an excellent potential for use in gene therapy. This unique property of cationic gemini amphiphiles can be attributed to their greater ability to bind and condense DNA and relatively low cytotoxicity toward animal *Corresponding author: e-mail [email protected]; Tel +919855557324; Fax +911832258820. (1) Martin, B.; Sainlos, M.; Aissaoui, A.; Oudrhiri, N.; Hauchecorne, M.; Vigneron, J.-P.; Lehn, J.-M.; Lehn, P. Curr. Pharm. Des. 2005, 11, 375–394. (2) Lehn, P.; Fabrega, S.; Oudrhiri, N.; Navarro, J. Adv. Drug Delivery Rev. 1998, 30, 5–11. (3) Kirby, A. J.; Camilleri, P.; Engberts, J. B. F. N.; Feiters, M. C.; Nolte, R. J. M.; S€oderman, O.; Bergsma, M.; Bell, P. C.; Fielden, M. L.; Rodrı´ guez, C. L. G.; Guedat, P.; Kremer, A.; McGregor, C.; Perrin, C.; Ronsin, G.; van Eijk, M. C. P. Angew. Chem., Int. Ed. 2003, 13, 1448–1457. (4) Fisicaro, E.; Compari, C.; Duce, E.; Donofrio, G.; Rozycka-Roszak, B.; Wozniak, E. Biochim. Biophys. Acta 2005, 1722, 224–233. (5) Gaucheron, J.; Wong, T.; Wong, K. F.; Maurer, N.; Cullis, P. R. Bioconjugate Chem. 2002, 1, 671–675. (6) Zana, R. J. Colloid Interface Sci. 2002, 248, 203–220. (7) Ryhanen, S, J.; Saily, M, J.; Paukku, T.; Borocci, S.; Mancini, G.; Holopainen, J. M.; Kinnunen, P. K. J. Biophys. J. 2003, 84, 578–587. (8) Fielden, M. L.; Perrin, C.; Kremer, A.; Bergsma, M.; Stuart, M. C.; Camilleri, P.; Engberts, J. B. F. N. Eur. J. Biochem. 2001, 268, 1269–1279. (9) Bell, P. C.; Bergsma, M.; Dolbnya, I. P.; Bras, W.; Stuart, M. C. A.; Rowan, A. E.; Feiters, M. C.; Engberts, J. B. F. N. J. Am. Chem. Soc. 2003, 125, 1551–1558. (10) Camilleri, P.; Kremer, A.; Edwards, A. J.; Jennings, K. H.; Jenkins, O.; Marshall, I.; McGregor, C.; Neville, W.; Rice, S. Q.; Smith, R. J.; Wilkinson, M. J.; Kirby, A. J. Chem. Commun. 2000, 1253–1254. (11) McGregor, C.; Perrin, C.; Monck, M.; Camilleri, P.; Kirby, A. J. J. Am. Chem. Soc. 2001, 123, 6215–6220. (12) Karlsson, L.; van Eijk, M. C. P.; Soderman, O. J. Colloid Interface Sci. 2002, 252, 290–296.

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cell line.3-13 The condensed DNA and cationic lipid complex often termed as lipoplexes are readily taken up by the cell by the phenomenon of endocytosis and are stable enough to escape endosome/lysosome compartment to pass to cytoplasm and finally to nucleus of the cell.14,15 Pyridinium amphiphiles, among the cationics, have proved to be more proficient in gene delivery in both in vitro and in vivo tests and have potential to deliver gene in wide variety of cells.16-19 Although the traditional gemini surfactant such as quaternary ammonium gemini surfactants having structural formula (m)a(s)b(m)a where “m” is alkyl chain, “s” is spacer, and “a” and “b” denote chain length of tail and spacer, respectively, have been widely studied3-7,20-23 and frequently utilized for biomedical applications, yet the properties, such as DNA binding affinity, cytotoxicity toward animal cell line, etc., of the corresponding pyridinium congener have so far been unexploited. In fact, only few reports24-26 are available regarding the synthesis and (13) Buijnsters, P. J. J. A.; Rodriguez, C. L. G.; Willighagen, E. L.; Sommerdijk, N. A. J. M.; Kremer, A.; Camilleri, P.; Feiters, M. C.; Nolte, R. J. M.; Zwanenburg, B. Eur. J. Org. Chem. 2002, 1397–1406. (14) Luo, D.; Saltzman, W. M. Nat. Biotechnol. 2000, 18, 33–37. (15) Gao, X.; Kim, K.; Liu, D. AAPS J. 2007, 9, E92–E104. (16) Ilies, M. A.; Seitz, W. A.; Ghiviriga, I.; Johnson, B. H.; Miller, A.; Thompson, E. B.; Balaban, A. T. J. Med. Chem. 2004, 47, 3744–3754. (17) Pijper, D.; Bulten, E.; Smisterova, J.; Wagenaar, A.; Hoekstra, D.; Engberts, J. B. F. N.; Hulst, R. Eur. J. Org. Chem. 2003, 4406–4412. (18) van der Woude, I.; Wagenaar, A.; Meekel, A. A. P.; ter Beest, M. B. A.; Rutters, M. H. J.; Engberts, J. B. F. N.; Hoekstra, D. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 1160–1165. (19) Ilies, M. A.; Seitz, W. A.; Johnson, B. H.; Ezell, E. L.; Miller, A. L.; Thompson, E. B.; Balaban, A. T. J. Med. Chem. 2006, 49, 3872–3887. (20) Uhrikova, D.; Zajac, I.; Dubnickova, M.; Pisarcik, M.; Funari, S. S.; Rapp, G.; Balgavy, P. Colloids Surf., B 2005, 42, 59–68. (21) Badea, I.; Verrall, R.; Baca-Estrada, M.; Tikoo, S.; Rosenberg, A.; Kumar, P.; Foldvari, M. J. Gene Med. 2005, 7, 1200–1214. (22) Alami, E.; Beinert, G.; Marie, P.; Zana, R. Langmuir 1993, 9, 1465–1467. (23) Barbero, N.; Quagliotto, P.; Barolo, C.; Artuso, E.; Buscaino, R.; Viscardi, G. Dyes Pigm. 2009, 82, 124–129. (24) Quagliotto, P.; Viscardi, G.; Barolo, C.; Barni, E.; Bellinvia, S.; Fisicaro, E.; Compari, C. J. Org. Chem. 2003, 68, 7651–7660. (25) Zhou, L.; Jiang, X.; Li, Y.; Chen, Z.; Hu, X. Langmuir 2007, 23, 11404– 11408. (26) Quagliotto, P.; Barolo, C.; Barbero, N.; Barni, E.; Compari, C.; Fisicaro, E.; Viscardi, G. Eur. J. Org. Chem. 2009, 19, 3167–3177.

Published on Web 09/04/2009

DOI: 10.1021/la901641f

11703

Article Scheme 1. Synthetic Route for Synthesis of Gemini Pyridinium Surfactants 12-15

Bhadani and Singh

and agarose gel electrophoresis. An interesting observation about these surfactants have been assessed by analyzing cationic amphiphile and DNA complexation at constant charge ratio with transmission electron microscopy(TEM) which further support the chain length dependent behavior observed by gel electrophoresis and EB exclusion experiments. The aggregation of negatively charged DNA and positively charged cationic amphiphile eventually resulted into nanometric particles showing different type of shapes. The MTT-based cytotoxicity assay of these gemini surfactants depicted that these compounds have low cytotoxicity toward C6 glioma cell (cancerous brain cell line) as compared to quaternary ammonium surfactant 16. It has also been observed that the cytotoxicity of new gemini pyridinium surfactants 12-15 decreases with increase in alkyl chain length.

Experimental Section Materials. 1-Dodecene, 1-tetradecene, 1-hexadecene, 1-octaproperties of such surfactants. This has kept these novel gemini pyridinium cationics away from biomedical applications. Further, no report could be found regarding DNA binding studies and cytotoxicity of gemini bispyridinium surfactants. Most of the biophysical and mechanistic studies of DNA interaction have been carried out on quaternary ammonium systems above or below cmc value of the surfactant under investigation.27-33 Further, the majority of these studies utilize single tail surfactants which are seldom used as gene delivery system. There is no report of the study of DNA-gemini pyridinium surfactant interaction, although pyridinium amphiphiles have been successfully used in gene delivery. A recent report34 suggests very different behavior of gemini pyridinium surfactants in aqueous system. In the present work new gemini pyridinium surfactants have been prepared by cobromination protocol (Scheme 1). The reaction involves direct synthesis of 1,2-bis(2-bromoalkylthio)ethanes where alkyl is dodecyl, tetradecyl, hexadecyl, and octadecyl by reacting the respective R-olefins (1-dodecene (2), 1-tetradecene (3), 1-hexadecene (4), and 1-octadecene (5)) with N-bromosuccinimide (6) and ethane-1,2-dithiol (1). The compounds 1,2-bis(2bromoalkylthio)ethanes (7-10) on reaction with pyridine (11) at 80 °C for 3 h give the corresponding 1,10 -(1,10 -(ethane-1,2-diylbis(sulfanediyl))bis(alkane-2,1-diyl))dipyridinium bromide (12-15). These pyridinium gemini surfactants exhibit better DNA binding capability and low cytotoxicity in comparison to the quaternary ammonium gemini surfactant 1,6-hexanediyl bis(dimethyldodecylammonium)bromide (16), the reference compound. It has also been found that the DNA binding capability of these surfactants 12-15 increase with increase in alkyl chain length. This behavior of the new surfactants 12-15 was supported by both EB exclusion experiments by fluorescence spectroscopy (27) Marchetti, S.; Onori, G.; Cametti, C. J. Phys. Chem. B 2005, 109, 3676– 3680. (28) Chen, X.; Wang, J.; Shen, N.; Luo, Y.; Li, L.; Liu, M.; Thomas, R, K. Langmuir 2002, 18, 6222–6228. (29) Cardenas, M.; Braem, A.; Nylander, T.; Lindman, B. Langmuir 2003, 19, 7712–7718. (30) Vongsetskul, T.; Taylor, D. J. F.; Zhang, J.; Li, P. X.; Thomas, R. K.; Penfold, J. Langmuir 2009, 25, 4027–4035. (31) Jadhav, V. M.; Valaske, R.; Maiti, S. J. Phys. Chem. B 2008, 112, 8824– 8831. (32) Bombelli, C.; Borocci, S.; Diociaiuti, M.; Faggioli, F.; Galantini, L.; Luciani, P.; Mancini, G.; Sacco, M. G. Langmuir 2005, 21, 10271–10274. (33) Leal, C.; Moniri, E.; Pegado, L.; Wennerstrom, H. J. Phys. Chem. B 2007, 111, 5999–6005. (34) Fisicaro, E.; Compari, C.; Biemmi, M.; Duce, E.; Peroni, M.; Barbero, N.; Viscardi, G.; Quagliotto J. Phys. Chem. B 2008, 112, 12312–12317.

11704 DOI: 10.1021/la901641f

decene, dithiol, and ethidium bromide were purchased from Sigma-Aldrich and were used without any purification. N-Bromosuccinimide (NBS) was purchased from Central Drug House, New Delhi, India, and pyridine from Qualigens Fine Chemicals, Mumbai, India. Agarose and Tris buffer were purchased from Sisco Research Laboratory Pvt Ltd., Mumbai, India. Plasmid DNA pUC 18 was purchased from Bangalore GeNei, Bangalore, India. Quaternary ammonium gemini surfactant 1,6-hexanediyl bis(dimethyldodecylammonium)bromide (16) was synthesized by previously reported procedure.35 Triply distilled autoclaved water was used in all experiments. Synthesis. Ethane-1,2-dithiol (1; 1.88 g, 20 mmol) was slowly added to the stirred suspension of 1-dodecene (2; 6.72 g, 40 mmol)/1-tetradecene (3; 7.84 g, 40 mmol)/1-hexadecene (4; 8.96 g, 40 mmol)/1-octadecene (5; 10.08 g, 40 mmol), and N-bromosuccinimide (6; 7.12 g, 40 mmol) in chloroform (150 mL) at 10-15 °C under inert conditions (nitrogen atmosphere). After addition, the reaction mixture was stirred for 30 min at room temperature. The progress of the reaction was monitored by thin layer chromatography (Silica gel G coated (0.25 mm thick) glass plates, using hexane:ethyl acetate (98:2) as mobile phase; the spots were visualized by iodine). The reaction was complete in 30 min in all the cases. Chloroform was then removed from the crude reaction mixture under reduced pressure in a rotary flash evaporator (Heidolph Labrota 4000-efficient, Germany) at 40 °C. It was then allowed to cool. The crude reaction mixture was then stirred with 60 mL of hexane and filtered to remove the precipitated succinimide. The filtrate was collected in a separating funnel and washed with 50 mL of water followed by drying over anhydrous sodium sulfate. Hexane was removed at 45 °C using a vacuum rotary flash evaporator. Hexane (30 mL) was added, the crude reaction mixture was stirred at -5 to -10 °C for 15 min, and the separated precipitate was filtered using a Buckner funnel under vacuum and washed with cold ethyl acetate. In the case of 1,2-bis(2-bromododecylthio)ethane purification was done using column chromatography on basic alumina (by using hexane to hexane:ethyl acetate (95:5), a stepwise increasing polarity elution method) to avoid loss in yield. Alternately, it can be purified by stirring at -30 °C followed by filtration as above-mentioned. The filtered material was vacuum-dried at 25 °C in a rotary evaporator for 2 h. The resulting compounds 1,2-bis(2-bromododecylthio)ethane (7; 5.88 g, 10 mmol)/1,2-bis(2-bromotetradecylthio)ethane (8; 6.42 g, 10 mmol)/1,2-bis(2-bromohexadecylthio)ethane (9; 7.00 g, 10 mmol)/1,2-bis(2-bromooctadecylthio)ethane (10; 7.57 g, 10 mmol) were heated with pyridine (11; 1.97 25 mmol) at 80 °C for 3 h. The resulting crude mixture was cooled to 20 °C. Cold acetone (30-40 mL) was added, and the reaction mixture was stirred at 0 °C for 30 min and filtered using a Buckner funnel under vacuum and washed with acetone. The step was repeated to (35) Zana, R.; Benrraou, M.; Rueff, R. Langmuir 1991, 7, 1072–1075.

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Bhadani and Singh remove the excess pyridine and vacuum-dried at 30 °C in rotary evaporator to get the final compounds 1,10 -(1,10 -(ethane-1,2diylbis(sulfanediyl))bis(dodecane-2,1-diyl))dipyridinium bromide (12)/1,10 -(1,10 -(ethane-1,2-diylbis(sulfanediyl))bis(tetradecane-2, 1-diyl))dipyridinium bromide (13)/1,10 -(1,10 -(ethane-1,2-diylbis(sulfanediyl))bis(hexadecane-2,1-diyl))dipyridinium bromide (14)/1,10 -(1,10 -(ethane-1,2-diylbis(sulfanediyl))bis(octadecane-2, 1-diyl))dipyridinium bromide (15). The structures of all the products were confirmed by IR, NMR, mass spectra, and elemental analysis. IR spectra were recorded as a thin neat film on a Shimadzu 8400s FT-IR (Kyoto, Japan) instrument. Mass spectra of intermediates 7-10 and surfactants 12-15 were recorded on Jeol SX 102/Da-600 using FAB as ion source and Waters Q-Tof Micromass using ESI as ion source, respectively. 1 H and 13C NMR spectra were recorded on a JEOL AL-300 (JEOL Japan), FT-NMR 300 MHz system and a BRUKER AVANCE II (Switzerland), FT NMR 400 MHz system as a solution in CDCl3, using tetramethylsilane (TMS) as an internal standard. Elemental analyses were recorded on a Thermo Electron (U.K.) made Flash EA 1112 Series CHNSO analyzer. 1,2-Bis(2-bromododecylthio)ethane (7): A white solid was obtained according to the general procedure. Yield 66.36%; mp 62-64 °C. 300 MHz 1H NMR (CDCl3, TMS): δ 0.88 (t, 6 H), 1.26 (br s, 32 H), 1.76 (m, 2 H), 2.00 (m, 2 H), 2.79 (s, 4H), 2.94-3.14 (ab-q, 4H) 4.04-4.11 (m, 2 H). 75 MHz 13C NMR (CDCl3): δ 55.17, 40.94, 37.08, 33.12, 31.82, 29.51, 29.48, 29.37, 29.24, 28.85, 27.62, 22.61, 14.05. IR (CCl4): 2953, 2916, 2843, 1465, 1375, 721, 669 cm-1. FAB MS m/z: 586 M+, 587 M+ + 1, 588 M+ + 2, 589 M+ + 3, 590 M+ + 4 (507/509, 339/341, 307/309 (base peak). Similarly, 1,2-bis(2-bromotetradecylthio)ethane (8), 1,2-bis(2bromohexadecylthio)ethane (9), and 1,2-bis(2-bromooctadecylthio)ethane (10) have also been obtained as white solids in 67.62%, 71.62%, and 73.18% yield. 1,10 -(1,10 -(Ethane-1,2-diylbis(sulfanediyl))bis(dodecane-2,1diyl))dipyridinium bromide (12): A white solid was obtained according to general procedure. Yield, 70.43% (50.35% with respect to starting 1-dodecene); mp 96-102 °C. 300 MHz 1H NMR (CDCl3, TMS): δ 0.87(t, 6 H), 1.24 (br s, 28 H), 1.40 (m, 4 H), 1.60 (m, 4 H), 2.71-2.80 (m, 4H), 3.49 (br s, 2 H), 5.03-5.05 (m, 2 H), 5.17 (m, 2 H), 8.17-8.21 (t, 4H), 8.60-8.65 (m, 2 H), 9.57 (s, 4H). 75 MHz 13C NMR (CDCl3): δ 145.75, 127.81, 64.70, 46.98, 32.03, 31.72, 30.76, 29.44, 29.40, 29.29, 29.16, 26.61, 22.49, 13.96. IR (CCl4): 3410, 3058, 2956, 2923, 2852, 1631, 1488, 1458, 1213, 1182, 684 cm-1. ESI-MS positive ions m/z (relative intensity %): 665.3 (84%) (M+ - Br), 667.3 (86.9%) (M+ - Br + 2), 668.4 (42.6%), 669.4 (13.9%), 293.2 (100%) (base peak, C18H31NS•+). Elemental analysis % calculated (found): C, 57.90; H, 8.37; N, 3.75 (C, 57.65; H, 8.46; N, 3.69). Similarly, 1,10 -(1,10 -(ethane-1,2-diylbis(sulfanediyl))bis(tetradecane-2,1-diyl))dipyridinium bromide (13), 1,10 -(1,10 -(ethane-1, 2-diylbis(sulfanediyl))bis(hexadecane-2,1-diyl))dipyridinium bromide (14), and 1,10 -(1,10 -(ethane-1,2-diylbis(sulfanediyl))bis(octadecane-2,1-diyl))dipyridinium bromide (15) have also been obtained in 77.34%, 77.87%, and 84.02% yield, respectively. Conductivity Measurements.36,37 Conductivity was measured on Equip-Tronics auto temperature conductivity meter model EQ.661 equipped with a conductivity cell. The solutions were thermostated in the cell at 25 ( 0.1 °C. For the determination of cmc adequate quantities of a concentrated surfactant solution (i.e., 2, 1, and 0.5 mM stock solution for surfactants 12, 13, and 14, respectively) were added in order to change the surfactant concentration from concentrations well below the critical micelle concentration (cmc) to up to at least 1-2 times the cmc. The degree of counterion binding (β) is calculated as (1 - R), where R = Smicellar/Spremicellar, i.e., ratio of the slope after and before cmc. (36) Rodriguez, A.; Graciani, M. M.; Moya, M. L. Langmuir 2008, 24, 12785– 12792. (37) Viscardi, G.; Quagliotto, P.; Barolo, C.; Savarino, P.; Barni, E.; Fisicaro, E. J. Org. Chem. 2000, 65, 8197–8203.

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Article

Surface Tension Measurements. Critical micelle concentration (cmc) and surface tension attained at cmc were determined using a CSC Du Nouy interfacial tensiometer (Central scientific Co., Inc.) using platinum-iridium ring (circumference 5.992 cm) at 25 ( 0.1 °C. The tensiometer was calibrated using triple distilled water. The surfactant solution was aged for 12 h prior to determination of surface activity. Agarose Gel Electrophoresis.38 170 ng of pDNA and 10 μL of 12.5, 25, and 50 μM gemini surfactants 12-16 solution was loaded with 5 μL glycerol into 1% agarose gel containing 2 μL of ethidium bromide (0.5 mg/mL). Electrophoresis was carried out at 100 V in Tris buffer for 30 min. The DNA band was visualized under UV transillumination Alpha Imager HP (Alpha Innotech Corp.). Photographs were taken using an Alpha Imager. Ethidium Bromide Exclusion.39,40 The fluorescence intensity of each solution was measured using a Shimadzu RF 5301 PC fluorescence spectrometer (Shimadzu Corp., Japan). The measurements were made in a quartz fluorescence cell. The excitation and emission wavelengths in experiments with EB were set at 320 and 599 nm, respectively. 1 μg of pDNA was used for each experiment, and DNA solution was directly mixed with 1 μL of EB (0.5 mg/mL) in the fluorescence cell; triply distilled autoclaved water was added to make total volume 2 mL. Titrations with surfactants were done by adding 100 μL of 10 mM surfactant solution in each step. Ten such additions gave 10 observations to generate intensity vs wavelength plots. (Figure 5). Transmission Electron Microscopy.41,42 A chloroform solution of the gemini surfactants 12-15 (1 mg/mL) was dried by rotary evaporation to form a thin film. The film was kept under vacuum for 4 h to ensure the complete removal of the residual solvent. The dried lipid was hydrated at 30 °C under a N2 stream with the appropriate amount of deionized water to make final concentration 2 mM. The resulting solution was stored at 4 °C. 1 μg of pDNA was added to 5 μL of the above-prepared solution. Freshly prepared aqueous suspensions of lipoplexes were examined under transmission electron microscope by negative staining using 2% PTA (phosphotungstic acid). A 5 μL sample of the suspension was loaded onto collodion-coated copper grids and allowed to remain for 1 min. Excess fluid was wicked off the grids by touching their edges to filter paper, and 2 μL of 2% PTA was applied on the same grid, after which the excess stain was similarly wicked off. The grid was air-dried, and the specimens were observed under TEM (Morgagni 268 D, Philips, Holland) operating at an acceleration voltage of 70 kV. Cytoxicity Assay.43 The MTT (3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide)-based cytotoxicity test was used to evaluate all the Gemini surfactants, and the tests were carried out on C6 glioma (cancerous brain cell line, passage number 65). Cells were seeded in 96 well flat bottom microplates at the density (2.5-3.0)  104 per mL, 100 μL per well, and were allowed to grow for 24 h. The compounds dissolved in double distilled water were sterilized using Millipore filter (pore size 0.22 μm) and were added to the culture media over a concentration range of 1-100 μM. The cytotoxicity of the compounds was assessed after 24 h of exposure. Absorbance was read at 550 nm using a Muliskan PLUS plate reader (Labsystem, Finland). The statistical analysis was performed using Sigma Stat 3.5.1 and Sigma Plot 11.0 (38) Wigglesworth, T. J.; Teixeira, F., Jr.; Axthelm, F.; Eisler, S.; Csaba, N. S.; Merkle, H. P.; Meier, W.; Diederich, F. Org. Biomol. Chem. 2008, 6, 1905–1911. (39) Izumrudov, V. A.; Zhiryakova, M. V.; Goulko, A. A. Langmuir 2002, 18, 10348–10356. (40) Zhao, X.; Shang, Y.; Liu, H.; Hu, Y. J. Colloid Interface Sci. 2007, 314, 478– 483. (41) Bajaj, A.; Paul, B.; Kondaiah, P.; Bhattacharya, S. Bioconjugate Chem. 2008, 19, 1283–1300. (42) Bajaj, A.; Paul, B.; Indi, S. S.; Kondaiah, P.; Bhattacharya, S. Bioconjugate Chem. 2007, 18, 2156. (43) Vyas, S. M.; Turanek, J.; Kn€otigova, P.; Ka^sna, A.; Kvardova, V.; Koganti, V.; Rankin, S. E.; Knutsonc, B. L.; Lehmler, H.-J. New J. Chem. 2006, 30, 944–951.

DOI: 10.1021/la901641f

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Article

Bhadani and Singh

Results and Discussion The preliminary structure of new gemini surfactants 12-15 have been established by 1H, 13C, DEPT (distortionless enhanced polarization transfer), 2D HETCOR (heteronuclear chemical shift correlation), and 2D COSY (correlation spectroscopy) experiments. Two multiplets at δ 4.96-5.08 ppm and δ 5.13-5.18 ppm correspond to four protons adjacent to thiol group. Each signal represent two protons. A broad singlet at δ 3.49-3.59 ppm was assigned to the proton directly attached to second carbon of alkyl chain which is attached to pyridinium nitrogen. The four protons attached to spacer carbon (i.e., -S-CH2-CH2-S-) appeared as multiplet at δ 2.60-2.90 ppm. The signal at δ 0.87 ppm represents six protons of the terminal CH3. The ring protons of pyridine were observed at δ 8.12-8.21, 8.51-8.65, and 9.51-9.59 ppm. 13C NMR spectra depicted sp3 carbon for terminal methyl at δ 13.96-14.12 ppm. The sp3 carbons for -CH2- were observed in the range of δ 22.49-32.20 ppm, including the carbons of the spacer that were observed at δ 30.76-31.00 ppm. The first carbon directly attached to thiol group was observed at δ 46.81-47.00 ppm, and the second carbon of alkyl chain directly attached to heteroatom nitrogen was observed at δ 64.68-64.92 ppm. All carbons of pyridinium group were observed at δ 127.81-127.97 ppm and δ 145.75-145.92 ppm. The structure of these gemini pyridinium surfactants 12-15 have further been established by ESI-MS (positive ion) mass spectroscopy. The parent ion peak for gemini surfactants 12-15 have been observed at m/z 665.3/667.3, 721.5/723.5, 775.5/779.5, and 833.6/ 835.6 for monopositive ion C36H62BrN2S2+ (12), C40H70BrN2S2+ (13), C44H78BrN2S2+ (14), and C48H86BrN2S2+ (15), respectively, where loss of one bromide ion from each molecule led to the formation of positively charged parent ion (M+ - Br). The (M+ - Br) + 1 and (M+ - Br) + 2 peaks were also observed in each case. The base ion peak for gemini surfactants 12-15 were observed at 293.2 (C18H31NS•+), 321.3 (C20H35NS•+), 349.3(C22H39NS•+), and 377.3 (C24H43NS•+), respectively. These peak correspond to dipositive molecular ions, lacking both bromides, observed by the ratio mass/charge (charge = 2). Critical Micellization Concentration. Geminis have astonishingly low cmc value than the corresponding single tail surfactants. Only few reports are available regarding synthesis and cmc value of gemini pyridinium surfactants.24,25 For better understanding we have divided gemini pyridinium surfactant into three types (Figure 1): Type 1: where two pyridinium ring are attached with -CH2- spacer to R position of the pyridinium nitrogen and alkyl chain is directly attached to pyridinium nitrogen. Type 2: where two pyridinium nitrogen are attached with -CH2- spacer and alkyl chain is attached to R position to the pyridinium ring. Type 3: series of new geminis, the two alkyl chains separated by a spacer (-S-CH2-CH2-S-) are attached to a pyridinium nitrogen through the β-carbon atom (β to the carbon directly attached to spacer heteroatom) of the alkyl chain (12-15). The new pyridinium gemini surfactants 12-14 (type 3) synthesized in the present work have lower cmc value as compared to other pyridinium geminis (types 1 and 2). Table 1 shows CMC (mM) and degree of counterion binding, β (%), values of pyridinium geminis. A general trend has been observed: the cmc decreases with increasing chain length. The CMC of 1,10 -(1,10 -(ethane-1,2-diylbis(sulfanediyl))bis(dodecane-2,1-diyl))dipyridinium bromide (12) is 0.43 mM while that of 1,10 -(1,10 -(ethane-1,2-diylbis(sulfanediyl))bis(tetradecane-2, 1-diyl))dipyridinium bromide (13) is 0.10 mM and 1,10 (1,10 -(ethane-1,2-diylbis(sulfanediyl))bis(hexadecane-2,1-diyl))dipyridinium bromide (14) has been observed to have a cmc 11706 DOI: 10.1021/la901641f

Figure 1. Types of gemini pyridinium surfactant.

of 0.03 mM. Surface properties of surfactant 15 cannot be evaluated because of its solubility limit in water. Conductivity data of surfactants 12-14 may also be presented as molar conductivity vs square root of concentration plot (Figure 2d-f). The plot gives clear idea of existence of premicellar aggregates. Surfactants 12 and 13 show onset of premicellar aggregate formation at concentration of 7 10-5 and 3.8  10-5 M, respectively. The second arrow (Figure 2d) indicates the point where there is existence of sufficient premicellar aggregates in solution. However, surfactant 14 is able to form true micelle at very low concentration, i.e., 3  10-5 M. Thus, in the case of surfactant 14 the plot of Λ vs C0.5 does not show formation of premicellar aggregates as prominently as that has been observed for surfactants 12 and 13. The Degree of Counterion Binding (β). The ratio of the slopes of the conductivity vs concentration curve above and below cmc gives degree of counterion dissociation R (i.e., R = Smicellar/ Spremicellar) and (1 - R) gives the degree of counterion binding, β. It is an important parameter because it manifests the counterions that are contained in the Stern layer to counterbalance the electrostatic force that opposes micelle formation. Quagliotto et al.24 determined the β value for a series of gemini bispyridinium bromides having different spacers where they had shown that different spacer is responsible for different β value. We in our study on new series of gemini bispyridinium surfactants 12-14 have found that β value decreases with increase in chain length (Table 1). The β value signifies the ability of counterion to bind micelles. It has been found that keeping the counterion (bromide ion), spacer (ethane dithiol), and cationic center (pyridinium group) the same and just varying the chain length resulted in decrease in β value with increase in binding ability of surfactants toward DNA. Surface Tension Measurements. The surface activity of gemini pyridinium surfactants 12 and 13 was also evaluated by measurement of surface tension. The critical micellar concentration (cmc) values determined were 0.35 and 0.082 mM for surfactants 12 and 13, respectively. Surface tension attained at the cmc of the surfactants was 40.67 and 42.90 dyn/cm for surfactants 12 and 13, respectively. However, it was not possible to determine the cmc values of surfactants 14 and 15 by this technique due to transition of surfactant behavior to cationic lipid behavior with increase in hydrophobic chain length. The values of cmc from surface tension are lower than those of conductivity. Similar results have been obtained by Fisicaro et al.34 for type 1 (Figure 1)gemini pyridinium surfactants. Similar results were also obtained by Pinazo et al.44 for arginine-based gemini surfactants and Esumi et al.45 for trimeric surfactants. This behavior has (44) Pinazo, A.; Wen, X.; Perez, L.; Infante, M. R.; Franses, E. I. Langmuir 1999, 15, 3134. (45) Yoshimura, T.; Yoshida, H.; Ohno, A.; Esumi, K. J. Colloid Interface Sci. 2003, 267, 167.

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Figure 2. Specific conductivity vs concentration plot where (a), (b), and (c) represent gemini bispyridinium surfactants 12, 13, and 14, respectively. (d), (e), and (f) represent molar conductivity vs C0.5 plot of gemini bispyridinium surfactants 12, 13, and 14, respectively.

already been discussed by Fisicaro et al.34 and is due the formation of non-surface-active premicellar aggregates by surfactants. Further the plot of Λ vs C0.5 (Figure 2d,e) also indicates the existence of such premicellar aggregates. Finally, Rosen et al.46 have also reported the existence of premicellar aggregates for (46) Tsubone, K.; Arakawa, Y.; Rosen, M. J. J. Colloid Interface Sci. 2003, 26, 2516.

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short spacer gemini surfactants due to which there is a significant difference in determination of cmc values by surface tension and conductivity. Figure 3 shows surface tension vs log concentration plot for surfactants 12 and 13. Agarose Gel Electrophoresis. All the new gemini pyridinium surfactants 12-15 and quaternary ammonium gemini surfactant 1,6-hexanediyl bis(dimethyldodecylammonium)bromide (16) were examined for their DNA binding capability with agarose gel DOI: 10.1021/la901641f

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Table 1. CMC (mM) and β (%) Values of Gemini Pyridinium Surfactants entry

typea

Ra

na

Xa

β (%)

CMC (mM)

Br 75 0.43 3 C10H23 Br 63 0.10 3 C12H25 Br 55 0.03 3 C14H27 3 Br 82 0.83b 1 C12H25 1 C12H25 3 Cl 69 1.51 4 Cl 67 1.28 1 C12H25 8 Cl 44 1.11 1 C12H25 12 Cl 48 0.22 1 C12H25 1 C12H25 2 CH3SO3 74 2.07 3 CH3SO3 72 2.09 1 C12H25 4 CH3SO3 40 1.93 1 C12H25 8 CH3SO3 57 1.32 1 C12H25 12 CH3SO3 57 0.75 1 C12H25 4 Br 2.69 2 C10H21 6 Br 2.00 2 C10H21 4 Br 0.56 2 C12H25 6 Br 0.54 2 C12H25 a Type, R, n, and X are defined in Figure 1. b At 30 °C.; entries 4-13 determined by conductivity (classical method) [ref 24] and entries 14-17 determined by a tensiometer [ref 25]. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

electrophoresis. The results of the experiments demonstrated better DNA binding capability of the pyridinium gemini surfactants than 1,6 hexanediyl bis(dimethyldodecylammonium)bromide (16) (Figure 4). The results also established alkyl chain length dependent binding capability of the surfactants irrespective of charge ratio. The better binding capability of the pyridinium geminis have been indicated by the fact that no retardation of pDNA was observed when 25 μM solution of 1,6-hexanediyl bis(dimethyldodecylammonium)bromide was used, whereas the new pyridinium gemini surfactant 12 was able to retard the migration of the DNA at the same concentration to some extent and all other geminis 13-15 were able to retard the migration of pDNA at a concentration of 25 μM . Compound 13 was even able to cause retardation in the migration of pDNA at 12.5 μM concentration. Gemini surfactants 14 and 15 were able to cause retardation of negatively charged pDNA toward positive electrode to greater extent at the same concentration. All the surfactants were able to bind the DNA at 50 μM concentration of surfactant solution. EB Exclusion Experiment. It is a reported fact that the fluorescence emission of ethidium bromide (EB, 3,8-diamino-5ethyl-6-phenylphenanthridinium bromide) is enhanced as a result of intercalation between the DNA base pairs relative to that in water.47,48 The extent of binding of a particular surfactant can be determined by the ability of a surfactant to displace EB from this intercalation complex causing a quenching of the fluorescence signal due to formation of surfactant-DNA complex.49 Thus, titration of EB-DNA complex gives clear idea about the nature of binding. Keeping the charge ratio constant it has been observed by agarose gel electrophoresis that binding is greatly influenced by alkyl chain length of surfactant. To prove the chain length dependent binding capability, the fluorescence titration was performed with the gemini pyridinium surfactants 12-15. It has been observed that 50% fluorescence is quenched in between 4 and 5 additions of gemini surfactant 12. Gemini 13 and 14 cause 50% quenching after 4 and 3 additions of respective surfactant solution. However, gemini 15 was able to cause 50% quenching of fluorescence signal in between 2 and 3 additions (Figure 5). This relation explains greater ability of long chain gemini to replace (47) Llres, D.; Clamme, J. -P.; Dauty, E.; Blessing, T.; Krishnamoorthy, G.; Duportail, G.; Mly, Y. Langmuir 2002, 18, 10340–10347. (48) Rodriguez-Pulido, A.; Aicart, E.; Junquera, E. Langmuir 2009, 25, 4402–4411. (49) Moreau, L.; Barthelemy, P.; Li, Y.; Luo, D.; Carla, A. H.; Prata, C. A. H.; Grinstaff, M. W. Mol. Biosyst. 2005, 1, 260–264.

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Figure 3. Surface tension vs log C plot for the surfactants 12 (9) and 13 (b).

EB from DNA and proves the chain length dependent binding behavior of the series of new gemini surfactants. Thus, it may be concluded that hydrophobic interactions cause enhanced rate of exclusion of EB from DNA. Further, it has been observed that quaternary ammonium gemini 16 shows far less quenching as compared to the pyridinium analogue, which has also been witnessed by agarose gel electrophoresis. The better binding capability of pyridinium gemini over quaternary ammonium may be explained by restrictive conformational freedom of pyridinium group. The exact concentration of the surfactants 12-15 in mM required to displace ethidium bromide from the DNA which brings about 50% decrease in fluorescence intensity has been determined by plot of fluorescence intensity at 600 nm vs concentration of surfactant (in mM) shown as Figure 6. As expected, lesser concentration of long chain gemini (1.20, 1.38, 1.66, and 1.77 mM of surfactants 15, 14, 13, and 12, respectively) was able to cause 50% decrease in fluorescence intensity. Transmission Electron Microscopy. To look into the morphologies of the particles/vesicles formed by cationic amphiphiles with pDNA, the films formed on the collodion-coated copper grids were examined under a transmission electron microscope. The selected micrograms are shown in Figure 7. It has been observed that the chain length dependent binding behavior of new gemini pyridinium surfactants due to hydrophobic interactions resulted in the formation of different type of nanometric particle with pDNA in the aqueous solution at constant charge ratio. Gemini 12 formed spread-out and elongated clusters of width 100-150 nm. Such nanostructures had also been observed by Mahato et al.,50 3-(N-ethyl-2-methanesulfonyldimethylamino)1,2-dioleoylpropanediol, cholesterol, and DNA system, while the gemini 13 formed hexagonal phases of 200-400 nm. Recently Safinya et al.51 have shown the existence of a hexagonal phase for cationic dendrimer lipids, neutral lipid 1,2-dioleoyl-sn-glycerophosphatidylcholine, and DNA system. 1,10 -(1,10 -(Ethane-1,2diylbis(sulfanediyl))bis(hexadecane-2,1-diyl))dipyridinium bromide (14) formed encapsulated globular structure of 50-150 nm diameter. A similar related nanostructure had recently been reported by Sakurai et al.52 having diameter 50-200 nm for (50) Ajit, S. Narang; Thoma, L.; Miller, D. D.; Mahato, R. I. Bioconjugate Chem. 2005, 16, 156–168. (51) Zidovska, A.; Evans, H. M.; Ewert, K. K.; Quispe, J.; Carragher, B.; Potter, C. S.; Safinya, C. R. J. Phys. Chem. B 2009, 113, 3694–3703. (52) Koiwai, K.; Tokuhisa, K.; Karinaga, R.; Kudo, Y.; Kusuki, S.; Takeda, Y.; Sakurai, K. Bioconjugate Chem. 2005, 16, 1349–1351.

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Figure 4. Agarose gel electrophoresis of pDNA (170 ng per lane) and gemini surfactants 12-16 at different concentrations.

Figure 5. Fluorescence titration of pDNA-EB complex with gemini pyridinium surfactants. Fluorescence intensity decreases with subsequent addition of surfactant solution. (a), (b), (c), and (d) represent fluorescence quenching due to addition of gemini pyridinium surfactants 12, 13, 14, and 15, respectively.

cationic lipid having an amidine headgroup. The only difference is that it had shown existence of long network of globular structure Langmuir 2009, 25(19), 11703–11712

whereas gemini 14 formed closed end structures having length from 200 nm to 1 μm. Gemini 15 formed a different type of DOI: 10.1021/la901641f

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Figure 6. Intensity observed at 600 nm vs concentration (mM) plot of surfactants 12-16.

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Figure 8. Absorbance vs concentration (μM) plot of gemini surfactants 12-16 for determination of IC50 value. The values represent the mean of IC50 of three different experiments done in triplicate.

Figure 7. Transmission electron micrograph (TEM) of pDNA and gemini pyridinium surfactant complex. (a), (b), (c), (d) represent lipoplexes of pDNA and gemini pyridinium surfactants 12, 13, 14, and 15, respectively. 1 μg of pDNA and 5 μL of of 2 mM surfactant solution were used for each experiment.

independent structural identity ranging from 100 nm to 1 μM particle size. Similar structures were observed for the gemini lipid/ DOPE liposome/DNA system by Bhattacharya et al.41 Cytotoxicity Assay. Low toxicity of a molecule toward animal cell line is very crucial for noble applications such as gene delivery. Cytotoxicity of these gemini pyridinium surfactants 12-15 have been assessed on C6 glioma cells and compared with quaternary ammonium gemini surfactant 1,6-hexanediyl bis(dimethyldodecylammonium)bromide (16). The results of these assays for the new surfactants show that gemini pyridinium surfactants are less toxic than the quaternary ammonium gemini congener (16) and cytotoxicity of new gemini pyridinium surfactants decreased with increased alkyl chain length. Recently, we had reported glycerol-based pyridinium surfactants53 having two hydroxyl groups for which cytotoxicity increases with increasing alkyl chain length, but a reverse trend is observed for these new gemini pyridinium surfactants. IC50 values of the gemini surfactants 12-16 are given in Figure 8. These values denote the concentration of gemini surfactants (in μM) which cause the death of 50% of the living cells. Among all the surfactants that have been evaluated for cytoxicity toward the cell line under consideration, the most toxic was the reference 1,6-hexanediyl bis(dimethyldodecylammonium)bromide (16) having IC50 value 12.5 μM. The most toxic gemini pyridinium surfactant observed among the gemini pyridinium surfactants 12-15 is 1,2-1,10 (1,10 -(ethane-1,2-diylbis(sulfanediyl))bis(dodecane-2,1-diyl))dipyridinium bromide (12) with an IC50 value of 15.7 μM, while 1,10 -(1,10 -(ethane-1,2-diylbis(sulfanediyl))bis(octadecane-2,1-diyl))dipyridinium bromide (15) with an IC50 value 53.1 μM has been found to be the least toxic. (53) Singh, S.; Bhadani, A.; Kataria, H.; Kaur, G.; Kamboj, R. Ind. Eng. Chem. Res. 2009, 48, 1673–1677.

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The hydrophobic interactions54 and restrictive conformational freedom of pyridinium group may be playing an important role in strong binding with DNA below and above cmc value of the surfactant. The DNA molecule is a polymeric amphiphile consisting of bases, carbohydrate, and phosphate group. The bases constitute hydrophobic part whereas the sugars and phosphate constitute a hydrophilic part. Recently, Dies et al.55 established that increase in hydrophobicity of nonpolar part of surfactants influences association with DNA and hydrophilic ionic group oppose self-assembly. However, balance of both the forces is essential for the integrity of the double-helical structure of DNA. The fact that hydrophilic interactions are more prominent and hydrophobic interactions least in short chain gemini surfactant may have resulted in opposition of self-assembly between surfactant and DNA and the binding capability of gemini surfactant 12 having shortest chain length has been observed to be the least among the new series of surfactants synthesized. With the increase in alkyl chain length the influence of hydrophobic interactions comes into play and regulate self-assembly of DNA and surfactants; as a result, the binding capability increases with increasing alkyl chain length at a constant charge ratio. The better binding capability of pyridinium gemini surfactants than the quaternary ammonium congener may be explained on the basis of greater ability to form premicellar aggregates in the solution at very low concentration and restrictive conformational freedom of pyridinium group. Recently, Fisicaro et al.34 have proved greater ability of gemini pyridinium surfactants to form aggregates instead of adsorbing at the air water interface. The plot of molar conductivity versus concentration for surfactants 12 and (54) Zhu, D.-M.; Evans, R. K. Langmuir 2006, 22, 3735–3743. (55) Dias, R. S.; Magno, L. M.; Valente, A. J. M.; Das, D.; Das, P. K.; Maiti, S.; Miguel, M. G.; Lindman, B. J. Phys. Chem. B 2008, 112, 14446–14452.

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Figure 9. (a) Proposed mechanism for interaction of gemini pyridinium surfactants 12-15. (b) Structure showing interaction between dimer premicellar aggregate of gemini bispyridinium surfactant 15 and DNA segment in charge ratio 1:1. The energy-minimized structure show existence of strong interaction between positively charged pyridinium ring of surfactant and negatively charged phosphate of DNA. The energy minimization was performed with ChemBio3D Ultra 11.0 software (ChemOffice, CambridgeSoft Corp.) using MM2 molecular mechanics routine toward a 0.001 rms gradient.

13 clearly shows existence of premiceller region at very low concentration for surfactants 12 and 13 while in case of surfactant 14 such a distinction could not be observed from its molar conductivity versus square root of concentration plot. However, this does not rule out the existence of such premicellar aggregate in this case. It has been observed from the agarose gel electrophoresis that these gemini pyridinium surfactants 12 and 13 were able to retard migration of DNA at 2.5  10-5 and 1.25  10-5 M concentration, respectively, concentrations lower than the premicellar aggregation concentration. Gemini surfactants 14 and 15 are also able to retard migration of DNA toward positive electrode at extremely low concentration. This behavior can be explained on the basis of formation of dimer type premicellar aggregate at very low concentration. The existence of this type of premicellar aggregate was proposed by Rosen et al.56 In these premicellar aggregates the molecule of gemini are arranged with their hydrophilic groups at opposite ends of the structure and their hydrophobic groups oriented toward each other. Such aggregates are facilitated by increase in length of hydrophobic group and predominate in surfactant having long hydrophobic chain. Surfactants 12 and 13 initially form such types of micellar aggregates, with increase in concentration these surfactants form oligomers (trimers, tetramers, and so on) as observed by a Λ vs C0.5 plot (Figure 2d,e). However, surfactants 14 and 15 predominantly form dimer type of aggregate as plot of Λ vs C0.5 show no such deviation before CMC (Figure 2e). It must be noted that surfactant 14 is able to form micelle at 3  10-5 M concentration, much lower than premicellar concentration of surfactants 12 and 13 observed through a Λ vs C0.5 plot. The probable mechanism for binding of gemini pyridinium surfactants at concentration much lower than cmc value has been shown in Figure 9a. At concentration above CMC these surfactants form different types and shapes of lipoplexes as observed through transmission electron microscopy. Further, pronounced interaction of the pyridinium surfactant can be explained on the basis of restrictive conformational free(56) Song, L. D.; Rosen, M. J. Langmuir 1996, 12, 1149–1153.

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dom of pyridinium group. The pyridinium ring decreases the conformational freedom with respect to gemini ammonium surfactants as suggested by Quagliotto et al.24 Thus, conformational rigidity of pyridinium group may be responsible for strong interaction of gemini pyridinium surfactants with DNA. Pyridinium gemini surfactants are stabilized by keeping one of the two counterion between them in solution, thus minimizing the Coulombic repulsion. The addition of DNA to this system may have resulted in displacement of counterion with the negatively charged phosphate of the DNA segment. Figure 9b shows energy-minimized illustration of DNA segment consisting of two base pairs having four negatively charged phosphate and two gemini pyridinium surfactant 15 molecules (dimer premicellar aggregate) with a total of four pyridinium rings (charge ratio 1:1). Thus, conformational rigidity and stacking of pyridinium ring between adjacent phosphate groups and vice versa, of one side of DNA chain may be responsible for strong interaction between gemini pyridinium surfactants and DNA. In short, we can say that a single mechanism cannot explain the interaction of DNA with cationic surfactant/cationic lipids; it is a cooperative mechanism involving both the electrostatic interaction and the hydrophobic interaction which is perhaps resulting in the formation of mixed aggregates of DNA and the cationic surfactant. Conclusion. In the present study four new pyridinium gemini surfactants 12-15 have been synthesized by simple synthetic methodology using cobromination protocol in good yields. The CMC of these surfactants was determined by surface tension and conductivity measurements and compared with other pyridinium gemini surfactants. It has been found that these new geminis possess low CMC values. DNA binding capabilities of these surfactants were determined by agarose gel electrophoresis, fluorescence titration, and transmission electron microscopy. All new gemini surfactants possess good binding capability toward pDNA. The new geminis show chain length dependent binding capability as evidenced by agarose gel electrophoresis. The binding capability increases with increase in alkyl chain length. This trend of binding behavior has been further confirmed DOI: 10.1021/la901641f

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by titration using fluorescence spectroscopy. Transmission electron microscopy images have shown the formation of different type of nanostructure with pDNA due to different binding capability of each individual gemini bispyridinium surfactants. The cytotoxicity of these new surfactants decreases with increase in alkyl chain length. Thus, these new gemini bispyridinium surfactants have better DNA binding capability and low cytotoxicity than standard quaternary ammonium-based gemini surfactant 16. Acknowledgment. The authors are thankful to CSIR (Council of Scientific & Industrial Research) India for providing the research grant [01(2077)106/EMR-II] for this work, Sophisticated Analytical Instrumentation Facility (SAIF), Panjab University, Chandigarh for the 13C DEPT, 2D COSY, HETCOR, and mass spectra of the compounds and Sophisticated Analytical Instrumentation Facility (SAIF), AIIMS, New Delhi, for TEM evalua-

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tion of the samples. The authors are extremely grateful to Professor Gurcharan Kaur and Hardeep Kataria (Junior Research Fellow) of Department of Biotechnology, Guru Nanak Dev University, Amritsar, India, for the cytotoxicity assay of the gemini surfactants 12-16. To end the authors are highly indebted to the anonymous reviewers, who have suggested some important changes enabling the authors to improve the quality of this manuscript. Supporting Information Available: Characterization data of intermediates (8, 9, and 10) and gemini pyridinium surfactants (13, 14, and 15), 1H, 13C NMR spectra, 13C DEPT, 2D COSY, and 2D HETCOR spectra for gemini pyridinium surfactant 13, mass spectroscopic analysis of all the surfactants studied in this research, fluorescence titration spectra of surfactant 16, and phase contrast photographs of cytotoxicity evaluation. This material is available free of charge via the Internet at http://pubs.acs.org.

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