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Electrochemical and Spectroscopic Study of Octadecyltrimethylammonium Bromide/DNA Surfoplexes Alberto Rodrıguez-Pulido, Emilio Aicart, and Elena Junquera* Departamento de Quımica Fısica I, Facultad de Ciencias Quımicas, Universidad Complutense de Madrid, 28040-Madrid, Spain Received October 14, 2008. Revised Manuscript Received January 15, 2009 The use of cationic micelles consisting of octadecyltrimethylammonium bromide (C18TAB) to compact calf thymus DNA has been investigated in aqueous buffered solution at 310.15 K by means of conductometry, electrophoretic mobility, and several fluorescence spectroscopy methods. The results indicate that C18TAB micelles, consisting of 44 monomers on average, may compact DNA molecule by an electrostatic interaction that takes place at the cationic spherical micelle surface. The surfoplexes thus formed show a surface density charge that goes from negative to positive values at a Lmic/D mass ratio of around 1.0 (where Lmic and D are the masses of micellized cationic surfactant and DNA), called the isoneutrality ratio (Lmic/D)φ.Values of this characteristic parameter, determined in this work not only from the electrochemical experimental data but also from spectroscopic measurements, are in very good agreement with those ones calculated from molecular parameters and some other properties also obtained in this work. The electrostatic character of the DNA-micelle interaction has been confirmed by analyzing the decrease in fluorescence emission of the fluorophore ethidium bromide, EtBr, initially intercalated between DNA base pairs, as long as the surfoplexes are formed. Fluorescence anisotropy experiments have revealed that micelle packing becomes more rigid in the presence of DNA, but once the surfoplex is formed, the fluidity increases with the Lmic/D mass ratio, attaining its maximum when the isoneutrality ratio is exceeded. This fact, together with the net positive charge of the surfoplexes with the Lmic/D mass ratio over the isoneutrality ratio, makes this regimen of lipid and DNA content the optimum for efficiency in the transfection process.
Introduction During the last few decades, much attention has been paid to the interaction between the DNA polyelectrolyte and oppositely charged cationic surfactants.1-8 This interaction is of great importance from biological and medical perspectives because cationic colloidal systems are known not only as DNA purifiers by condensation and precipitation but also as nonviral vehicles for gene transfer processes, capable of delivering genetic material to a patient’s cells.4 Cationic liposomes, cationic and/or cat-anionic vesicles, and *To whom correspondence should be addressed. E-mail: junquera@ quim.ucm.es. Tel: +34-91-394-4131. Fax: +34-91-394-4135. http:// www.ucm.es/info/coloidal/index.html. (1) Felgner, J. H.; Gadek, T. R.; Holm, M.; Roman, R.; Chan, H. W.; Wenz, M.; Northrop, J. P.; Ringold, G. M.; Danielsen, M. Proc. Nat. Acad. Sci. U.S.A. 1987, 84, 7413. (2) Felgner, P. L.; Heller, M. J.; Lehn, P.; Behr, J.-P.; Szoka, F. C. Artificial Self-Assembling Systems for Gene Delivery; American Chemical Society: Washington, DC, 1996. (3) Mahato, R. I.; Kim, S. W. Pharmaceutical Perspectives of Nucleic AcidBased Therapeutics; Taylor and Francis: London, 2002. (4) Lasic, D. D. Liposomes in Gene Delivery; CRC Press:Boca Raton, FL,1997. (5) Ewert, K.; Slack, N. L.; Ahmad, A.; Evans, H. M.; Lin, A. J.; Samuel, C. E.; Safinya, C. R. Curr. Med. Chem. 2004, 11, 133. (6) Lonez, C.; Vandenbranden, M.; Ruysschaert, J. M. Prog. Lipid Res. 2008, 47, 340. (7) Ma, B. C.; Zhang, S. B.; Jiang, H. M.; Zhao, B. D.; Lv, H. T. J. Controlled Release 2007, 123, 184. (8) Safinya, C. R.; Ewert, K.; Ahmad, A.; Evans, H. M.; Raviv, U.; Needleman, D. J.; Lin, A. J.; Slack, N. L.; George, C.; Samuel, C. E. Philos. Trans. R. Soc. London, Ser. A 2006, 364, 2573. (9) Kennedy, M. T.; Pozharski, E. V.; Rakhmanova, V. A.; MacDonald, R. C. Biophys. J. 2000, 78, 1620. (10) Pozharski, E.; MacDonald, R. C. Biophys. J. 2003, 85, 3969. :: (11) Radler, J. O.; Koltover, I.; Salditt, T.; Safinya, C. R. Science 1997, 275, 810.
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cationic micelles, forming what is known as lipoplexes (liposome or vesicle/DNA complexes)4,5,9-12 and surfoplexes (micelle/DNA complexes),13-23 have widely demonstrated their capability to compact the negatively charged polyelectrolyte by means of a strong surface electrostatic interaction, with the release of the counterions playing an important entropic role. Among them, lipoplexes have been widely studied from biochemical and physicochemical standpoints because of the biocompatibility, biodegradability, and nonimmunogenity of liposomes.4 However, surfoplexes are attracting interest given the their low cost and ease of preparation and handling of micelles makes them advantageous with respect to liposomes made with natural or synthetic lipids. Alkyl quaternary ammonium bromides are cationic surfactants that, either in aqueous binary systems or as part of (12) Caracciolo, G.; Pozzi, D.; Caminiti, R.; Marchini, C.; Montani, M.; Amici, A.; Amenitsch, H. J. Phys. Chem. B 2008, 112, 11298. (13) Bonincontro, A.; La Mesa, C.; Proietti, C.; Risuleo, G. Biomacromolecules 2007, 8, 1824. (14) Kawashima, T.; Sasaki, A.; Sasaki, S. Biomacromolecules 2006, 7, 1942. (15) Marchetti, S.; Onori, G.; Cametti, C. J. Phys. Chem. B 2006, 110, 24761. (16) Mel’nikov, S. M.; Lindman, B. Langmuir 1999, 15, 1923. (17) Nakanishi, H.; Tsuchiya, K.; Okubo, T.; Sakai, H.; Abe, M. Langmuir 2007, 23, 345. (18) Spink, C. H.; Chaires, J. B. J. Am. Chem. Soc. 1997, 119, 10920. (19) Zhu, D. M.; Evans, R. K. Langmuir 2006, 22, 3735. (20) Rudiuk, S.; Franceschi-Messant, S.; Chouini-Lalanne, N.; Perez, E.; Rico-Lattes, I. Langmuir 2008, 24, 8452. (21) Dias, R. S.; Mel’nikov, S. M.; Lindman, B.; Miguel, M. G. Langmuir 2000, 16, 9577. (22) Miguel, M. G.; Pais, A.; Dias, R. S.; Leal, C.; Rosa, M.; Lindman, B. Colloids Surf., A 2003, 228, 43. (23) Bhattacharya, S.; Mandal, S. S. Biochemistry 1998, 1998, 7764.
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ternary mixed systems, have been widely studied.24-26 In particular, those with more than eight carbon atoms have specific pharmaceutical applications as a result of their antibacterial function.27 The surfoplexes consisting of DNA and several alkyltrimethylammonium bromides (CnTAB, where n = 12, 14 and 16) have been characterized using several experimental methods.13-17,20-23 Most of this work focuses on the interaction of DNA below the cmc; however, it has been reported19 that hydrophobic interaction-mediated surfactant aggregation plays a key role in the formation of the DNA-surfactant complex. In this work, we pretend to analyze the interaction between the cationic micelles (above the cmc) of a longer component of this family, octadecyltrimethylammonium bromide, C18TAB (Chart 1), and a doublestranded DNA of medium size. C18TAB may offer several additional advantages of the potential applications of alkyltrimethylammonium salts/DNA surfoplexes in gene therapy, compared with the shorter CnTAB surfactants and also with liposomes or vesicles. Thus, its critical micelle concentration, reported in the literature26,28 and measured in this work, is lower than that shown by the shorter homologous (n = 1216).29-32 This fact leads to a lower free monomeric surfactant concentration in the media, which also implies a lower toxicity. Furthermore, as will be demonstrated in this work, the affinity of DNA-micelle interaction is lower than that of liposomes/DNA or vesicles/DNA, which could be an interesting additional advantage because the surfoplex may more easily liberate DNA, after endocytosis, into the cell media. The characterization of the surfoplexes has been carried out using electrochemical and spectroscopic experimental techniques. Given that the DNA/micelle interaction process is dominated by electrostatic forces, a careful electrochemical study of the bulk solution by means of conductometry and of the micelle surface by means of the zeta potential is strongly recommended.4,33-38 Additionally, fluorescence spectroscopy (24) Holland, P. M.; Rubingh, D. N. Mixed Surfactant Systems; American Chemical Society: Washington, DC, 1992. (25) Ogino, K.; Abe, M. Mixed Surfactant Systems; Marcel Dekker: New York, 1993. (26) Maiti, K.; Chakraborty, I.; Bhattacharya, S. C.; Panda, A. K.; Moulik, S. P. J. Phys. Chem. B 2007, 111, 14175. (27) Arevalo, J. M.; Arribas, J. L.; Hernandez, M. J.; Lizan, M.; Herruzo, R. Med. Prevent. 2001, 7, 17. (28) Swanson-Vethamuthu, M.; Feitosa, E.; Brown, W. Langmuir 1998, 14, 1590. (29) Attwood, D.; Florence, A. T. Surfactant Systems: Their Chemistry, Pharmacy and Biology; Chapman and Hall: London, 1983. (30) Junquera, E.; Tardajos, G.; Aicart, E. J. Colloid Interface Sci. 1993, 158, 388. (31) Junquera, E.; Aicart, E. Langmuir 2002, 18, 9250. (32) Lainez, A.; del Burgo, P.; Junquera, E.; Aicart, E. Langmuir 2004, 20, 5745. (33) Janoff, A. S. Liposomes: Rational Design; Marcel Dekker: New York, 1999. (34) Rosoff, M. Vesicles; Marcel Dekker: New York, 1996. :: (35) Radler, J. O.; Koltover, I.; Jamieson, A.; Salditt, T.; Safinya, C. R. Langmuir 1998, 14, 4272. (36) Birchall, J. C.; Kellaway, I. W.; Mills, S. N. Int. J. Pharm. 1999, 183, 195. (37) Lobo, B. C.; Rogers, S. A.; Choosakoonkriang, S.; Smith, J. G.; Koe, G. S.; Middaugh, C. R. J. Pharm. Sci. 2001, 91, 454. (38) Xu, Y. H.; Hui, S. W.; Frederik, P.; Szoka, F. C. Biophys. J. 1999, 77, 341. (39) Lakowicz, J. R. Principles of Fluorescence Spectroscopy; Kluwer Academic/Plenum: New York, 1999. (40) Eastman, S. J.; Siegel, C.; Tousignant, J.; Smith, A. E.; Cheng, S. H.; Scheule, R. K. Biochim. Biophys. Acta 1997, 1325, 41. (41) MacDonald, R. C.; Ashley, G. W.; Shida, M. M.; Rakhmanova, V. A.; Tarahovsky, Y. S.; Pantazatos, D. P.; Kennedy, M. T.; Pozharski, E. V.; Baker, K. A.; Jones, R. D.; Rosenzweig, H. S.; Choi, K. L.; Qiu, R.; McIntosh, T. J. Biophys. J. 1999, 77, 2612.
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is a powerful tool for analyzing the structural aspects of both the colloidal vehicle, micelles in this case, and their complexes with DNA.23,36,38-48 Different types of fluorescence assays can be carried on depending on the choice of the fluorescence probe. In this work, we have used three different probes (Chart 1): (i) pyrene (Py) to determine the average aggregation number of C18TAB micelles as well as the micropolarity of the micelles inside by means of static quenching experiments; (ii) ethidium bromide (EtBr) to analyze the cationic micelleDNA binding by means of fluorescence intercalating assays; and (iii) diphenylhexatriene (DPH) to check the flexibility of the micelle packing in the absence and presence of DNA by means of fluorescence anisotropy measurements. Given that the properties of the complexes formed by colloidal aggregates and DNA are known to depend very much on their composition, all of the above-mentioned experiments have been carried out to cover a wide range of Lmic/D ratios, where Lmic and D stand for the masses of micellized cationic surfactant and DNA, respectively. Furthermore, because C18TAB is characterized by a Kraft temperature of around 308.15 K,49,50 all of the experiments herein reported are carried out at 310.15 K, which is approximately the human body temperature. The physiological medium has been mimicked by working at a physiological pH of around 7.4, conferred by an HEPES buffer that is widely used in biochemical assays.
Experimental Section Materials. Cationic surfactant, octadecyltrimethylammonium bromide (C18TAB), sodium salt of calf thymus DNA with less than 5% protein, the fluorescent probes, ethidium bromide (EtBr), pyrene (Py), diphenylhexatriene (DPH), the quencher, hexadecylpyrydinium chloride (CePyCl), and the components of HEPES buffer (4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid and its sodium salt) were from Sigma-Aldrich. All of them, with the best available purities, were used without further purification. Distilled water was deionized using a Super Q Millipore system (with conductivity lower than 18 μS cm-1). Solutions were prepared by mass, and unless otherwise stated, all were buffered by using 40 mM HEPES with an ionic strength of 19.2 mM at 310.15 K. Preparation of Surfoplexes. A stock solution of DNA (1.0 mg/mL = 1.54 10-3 M base pair) was prepared by dissolving an appropriate amount of the solid in 40 mM HEPES, pH 7.4, 2 days before mixing with micelles. DNA concentrations (expressed in mM base pairs) were determined by absorbance at 260 nm (ε = 6600 M-1 cm).16,51 A A260/A280 ratio of 1.90 and a negligible absorbance at 320 nm (A320 = -0.003)16,51-53 reveal that the contamination of the DNA used in this work by the (42) Tarahovsky, Y. S.; Koynova, R.; MacDonald, R. C. Biophys. J. 2004, 87, 1054. (43) Geall, A. J.; Eaton, M. A. W.; Baker, T.; Catterall, C.; Blagbrough, I. S. FEBS Lett. 1999, 459, 337. (44) Borenstain, V.; Barenholz, Y. Chem. Phys. Lipids 1993, 64, 117. (45) Hirsch-Lemer, D.; Barenholz, Y. Biochim. Biophys. Acta 1998, 1370, 17. (46) Lentz, B. R.; Moore, B. M.; Barrow, D. A. Biophys. J. 1979, 25, 489. (47) Regelin, A. E.; Fankhaenel, S.; Gurtesch, L.; Prinz, C.; von Kiedrowski, G.; Massing, U. Biochim. Biophys. Acta 2000, 1464, 151. :: :: (48) Ryhanen, S. J.; Saily, M. J.; Paukku, T.; Borocci, S.; Mancini, G.; Holopainen, J. M.; Kinnunen, P. K. Biophys. J. 2003, 84, 578. (49) Davey, T. W.; Ducker, W. A.; Hayman, A. R.; Simpson, J. Langmuir 1998, 14, 3210. (50) Jaeger, D. A.; Li, G. W.; Subotkowski, W.; Carron, K. T.; Bench, M. W. Langmuir 1997, 13, 5563. (51) Barreleiro, P. C. A.; Lindman, B. J. Phys. Chem. B 2003, 107, 6208. (52) Gonc-alves, E.; Debs, R. J.; Heath, T. D. Biophys. J. 2004, 86, 1554. (53) Hirsch-Lerner, D.; Zhang, M.; Eliyahu, H.; Ferrari, M. E.; Wheeler, C. J.; Barenholz, Y. Biochim. Biophys. Acta 2005, 1714, 71.
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Chart 1. (A) Cationic Surfactant Octadecyltrimethylammonium Bromide, C18TAB, and (B) Fluorescence Probes(B1) Pyrene, (B2) EtBr, and (B3) DPH
presence of a certain percentage of proteins is negligible. Agarose gel electrophoresis experiments reveal that the DNA used herein consist of around 2700 bp fragments on average (i.e., 1.75 106 g mol-1). Equal volumes of DNA thus prepared and C18TAB micelle solutions were mixed by adding DNA over micelles, as usually done in these studies.38,54 The concentrations of both solutions were controlled to fit the final desired Lmic/D mass ratio, defined as Lmic L -cmcV ¼ D D
ð1Þ
where L and Lmic are the total and micellized mass of cationic surfactant, respectively, D is DNA mass, and cmc and V are the critical micelle concentration, expressed in mass per unit volume, and the solution volume, respectively. The mixing process, after optimization, was carried out at an adding speed of 0.2 mL/ min, with continuous, constant, and vigorous magnetic stirring. Once the addition was concluded, the solution was maintained under agitation for 10 min to favor the formation of surfoplexes.
Conductometric Measurements. Conductivity data were collected at 310.15 K ((1 mK) with a HewlettPackard 4263A LCR meter. The equipment, preparation of mixtures, and fully computerized procedure were described previously.55,56 The reproducibility of the specific conductivity, κ, obtained as an average of 2400 measurements for each concentration point, is better than 0.03%. The conductivity measurements were made as a function of total surfactant concentration at a constant DNA concentration of 0.182 mg/mL, covering Lmic/D = 0-4.0. Electrophoretic Mobility Measurements. A laser Doppler electrophoresis (LDE) technique (Zetamaster 2000, Malvern Instruments Ltd.), described previously,57 was used to measure electrophoretic mobilities. The cell used is a Zetasizer (54) Salvati, A.; Ciani, L.; Ristori, S.; Martini, G.; Masi, A.; Arcangeli, A. Biophys. Chem. 2006, 121, 21. (55) Junquera, E.; Aicart, E. Rev. Sci. Instrum. 1994, 65, 2672. (56) Junquera, E.; Aicart, E. Int. J. Pharm. 1999, 176, 169. (57) Junquera, E.; Arranz, R.; Aicart, E. Langmuir 2004, 20, 6619.
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2000 standard quartz rectangular capillary electrophoresis cell with dimensions of 5 2 50 mm3, which is calibrated with a zeta potential latex standard of ζ = -50 ( 5 mV. The temperature was controlled at 310.15 ( 0.01 K. Each electrophoretic mobility datum point is taken as an average over 10 independent measurements. The DNA concentration was kept constant at 0.495 mg/mL, and the total surfactant concentration ranged from 0.20 to 2.96 mg/mL (i.e., Lmic/D was varied from 0.3 to 5.9). Fluorescence Spectroscopy. Steady-state fluorescence experiments were carried out with a Perkin-Elmer LS-50B luminiscence spectrometer.58,59 A 10 mm stoppered rectangular silica cell was placed in a stirred cuvette holder whose temperature was kept constant at 310.15 ( 0.01 K with a recirculating water circuit. Three different types of experiments were done: (i) static quenching experiments of pyrene emission to determine the aggregation number of C18TAB micelles; (ii) ethidium bromide intercalation assays to analyze the type of interaction that takes place between the cationic micelles and the polyelectrolyte; and (iii) anisotropy measurements with the DPH probe to evaluate the flexibility of the micelle packing in the absence and presence of DNA. Quenching of Pyrene Emission. Both excitation and emission band slits were fixed at 2.5 nm, and the scan rate was selected at 240 nm/min. Pyrene was used as a luminescence probe, and CePyCl was chosen as a static quencher. The excitation wavelength was selected at 340 nm, and the emission spectra were collected from 360 to 500 nm. The first and third vibronic peaks of the pyrene appear at around 373 and 385 nm, respectively. The pair pyrene/CePyCl assures that the residence time of the quencher into the micelle is much longer than the fluorescence lifetime of the (58) Junquera, E.; Pe~ na, L.; Aicart, E. Langmuir 1997, 13, 219. (59) Rodriguez-Pulido, A.; Aicart, E.; Llorca, O.; Junquera, E. J. Phys. Chem. B 2008, 112, 2187.
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probe. Pyrene,60 quencher, and micelle concentrations were :: prepared via the Infelta and Gratzel procedure,60 assuring a Poisson distribution for the equilibrium of solubilizates between the aqueous and micellar phases.61-64 Although the quencher is a surfactant itself and it is expected to mix with the C18TAB micelles, its concentration is so low, compared with that of the surfactants, that no interaction effect with the micelle is assumed.65 Ethidium Bromide Intercalation Assays. Fluorescence emission spectra of ethidium bromide in the 530-700 nm region were recorded with excitation at 520 nm (the molar extinction coefficient is the same at 520 nm for free and DNA-associated EtBr).59 The probe concentration was kept constant at [EtBr] = 0.14 mM in all cases. The emission of an EtBr/DNA solution (DNA/EtBr molar ratio is larger than 6:1 and [DNA] = 0.496 mg/mL) was registered at increasing [L] by adding an EtBr/DNA/L solution, thus covering the Lmic/D region from 0 to 5.9. In all cases, the excitation and emission band slits were fixed at 2.5 and 4 nm, respectively, and the scan rate was selected at 240 nm/min. Fluorescence spectra were corrected for the background intensities of the buffer solution. Fluorescence Anisotropy. Diphenylhexatriene (DPH) was included in C18TAB micelles to yield a molar ratio of probe to surfactant of 1:200. For that purpose, a small volume of DPH/ethanol solution (1.91 10-4 M) was added to a vial, and the solvent was evaporated by using an air current; the required amounts of C18TAB and HEPES buffer were then added to the dried DPH, and the resulting micelle-doped DPH solution was sonicated for a few minutes to favor its homogeneity. DNA (0.501 mg/mL) was added to the labeled micelles to yield the desired Lmic/D ratios of micellized surfactant and DNA (covering the Lmic/D region from 0 to around 3) using the experimental procedure widely explained in a previous section. The fluorescence intensities of the emitted light with the excitation and emission polarized following the modes vertical-vertical (IVV), vertical-horizontal (IVH), horizontal-horizontal (IHH), and horizontalvertical (IHV) were measured by exciting DPH at 360 nm and recording its fluorescence emission at 430 nm. The slit widths were 2.5 nm for both the excitation and the emission. Fluorescence anisotropy, r, was calculated by using the well-known equation39 r ¼
IVV -GIVH IVV þ 2GIVH
ð2Þ
where G (= IHV/IHH) is the instrument grating factor, estimated to be the average of 10 measurements for each solution at the chosen excitation and emission wavelengths, thus correcting optical and electronic differences in the parallel and perpendicular channels. The influence of the light scattering of samples on anisotropy values was also evaluated and considered with the corresponding blank solutions. Each anisotropy value is an average over 36 independent experimental measurements. (60) Malliaris, A.; Lang, J.; Zana, R. J. Chem. Soc., Faraday Trans. 1 1986, 82–109. (61) Infelta, P. P. Chem. Phys. Lett. 1979, 61, 88. :: (62) Infelta, P.; Gratzel, M. J. Chem. Phys. 1979, 70, 179. (63) Turro, N. J.; Yekta, A. J. Am. Chem. Soc. 1978, 100, 5951. (64) Zana, R. Surfactant Solutions: New Methods of Investigation; Marcel Dekker:New York, 1987. (65) Hansson, P.; Jonsson, B.; Strom, C.; Soderman, O. J. Phys. Chem. B 2000, 104, 3496.
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Results and Discussion C18TAB Micelles. Micelles consisting of C18TAB monomers have been characterized through conductivity and static fluorescence measurements. Experimental values of specific conductivity, κ, are plotted in Figure 1 as a function of surfactant concentration at 310.15 K (the Kraft point of C18TAB in water is around 308.15 K)49,50 in aqueous 40 mM HEPES medium, pH 7.4. The concentration at which there is a break in the property, determined by using the Phillips method66 is assigned to the critical micelle concentration, cmc (= 0.169 mM). The aggregation number of the micelles, N, has been determined from the steady-state fluorescence data, assuming a Poisson distribution for the equilibrium of solubilizates between the aqueous and micellar phases. The equation to be applied is63,64 ln II ¼ ln II, 0 -
N½Q N½Q ¼ ln II, 0 ½Lmic ½L -cmc
ð3Þ
where [Q], [L], and [L]mic (= [L] - cmc) are the concentrations of quencher, total surfactant, and micellized surfactant, respectively, and II,0 and II are the fluorescence intensities in the absence and in the presence of quencher for the first peak in the pyrene emission spectra. Figure 2 reports the pyrene emission spectra for C18TAB micellar solutions, well above the cmc, in the absence and presence of several quencher concentrations; the inset shows a plot of ln II versus [Q] (eq 3). The slope of this straight line reveals that C18TAB micelles consist of 44 monomers on average at 310.15 K in aqueous 40 mM HEPES medium, pH 7.4. It is well known that cmc and N are strongly dependent on the conditions of the experiment (water or buffer solution, pH, ionic strength, and temperature); literature values26,28,50,67-70 have been determined over a wide range of those conditions, making the comparison with the results reported herein somehow difficult. In spite of this, taking into account all previous considerations, the cmc and N of C18TAB determined in this work are consistent with literature values. Additionally, regarding the shape of the micelles, a sphere-to-rod transition is reported in the literature for C16TAB micelles in water at 298.15 K at a molality of 0.28,71 a surfactant concentration around 300 times its cmc.30 Accordingly, it could be deduced that C18TAB micelles studied in this work, consisting of only 44 monomers on average at a concentration of [L] = 14.87 mM, which is around 100 times the cmc, are estimated to be spherical, mostly considering that a value of N of around 115 has been reported29 as the maximum value for the aggregation number of micelles of C18TAB consistent with a spherical shape. Furthermore, the formation of spherical or rodlike micelles is governed by the packing parameter P (= v/a0lc), proposed by Israelachvili,72 that relates the volume v, the critical chain length lc of the surfactant (66) Phillips, J. N. Trans. Faraday Soc. 1955, 51, 561. (67) Das, M. L.; Bhattacharya, P. K.; Moulik, S. P. Langmuir 1990, 6, 1591. (68) Kodama, M.; Tsujii, K.; Seki, S. J. Phys. Chem. B 1990, 94, 815. (69) Lu, J. T.; Simister, E. A.; Thomas, R. K.; Penfold, J. J. Phys. Chem. B 1993, 97, 6024. (70) Swanson-Vethamuthu, M.; Almgren, M.; Karlsson, G.; Bahadur, P. Langmuir 1996, 12, 2173. (71) Alauddin, M.; Verrall, R. E. J. Phys. Chem. 1989, 93, 3724. (72) Israelachvili, J. Intermolecular and Surfaces Forces; Academic Press: London, 1992.
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Figure 1. Specific conductivity, κ, as a function of total surfactant concentration at 310.15 K in 40 mM aqueous medium HEPES, pH 7.4.
hydrophobic tail, obtained according to the Tanford’s model,73,74 and the optimal head-group area a0. If P < 1/3, then the optimum aggregates with a minimum free Gibbs energy will be the spherical micelles, whereas if 1/3 < P < 1/2 then globular or rodlike micelles are preferred. In the present case, values29,32 of v = 0.51 nm3, lc = 2.43 nm, and a0 = 0.74 nm2 yield an estimated packing parameter P of around 0.28 for C18TAB micelles, characteristic of spherical aggregates and consistent with our reasoning on the basis of the aggregation number. Finally, the relative intensities of the first and third vibronic peaks of the pyrene spectra (Figure 2) are directly related to the apparent dielectric constant of the medium where the probe is housed. Characteristic values of the ratio II/IIII in toluene, methanol, and water are 1.04, 1.33, and 1.84, respectively.75 The average value obtained for the ratio II/IIII among all of the curves in Figure 2 is 1.26, similar to that of methanol, indicating that the microenvironment where the probe is housed, either close to the micellar surface76,77 or deeper inside the micelle core, is rather polar (i.e., having a polarity characteristic of a methanolic medium). It seems that 44 monomers of C18TAB yield a small packed micelle, allowing bulk water molecules to penetrate the so-called palisade layer of the micelle. C18TAB/DNA Surfoplexes. Some studies reported in the literature reveal the ability of cationic micellar aggregates to compact DNA molecules, thus forming surfoplexes.13-17,19,20,22,23 With the aim of improving the knowledge of this kind of interaction, we report here an electrochemical and spectroscopic study of the surfoplexes consisting of C18TAB micelles and DNA. Electrochemical Study. The analysis has been done at two levels: the surface of the charged micelles and surfoplexes, by the LDE technique (electrophoretic mobility), and the bulk, by conductometry experiments. Electrophoretic mobility, (73) Tanford, C. J. Phys. Chem. 1974, 78, 2469. (74) Tanford, C. The Hydrophobic Effect: Formation of Micelles and Biological Membranes; Wiley & Sons: New York, 1980. (75) Kalyanasundaram, K.; Thomas, J. K. J. Am. Chem. Soc. 1977, 99, 2039. (76) Zana, R.; Yiv, S.; Strazielle, C.; Lianos, P. J. Colloid Interface Sci. 1981, 80, 208. (77) Lianos, P.; Zana, R. J. Colloid Interface Sci. 1981, 84, 100.
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Figure 2. Emission fluorescence spectra of a 1.6 10-6 M solution of pyrene in 40 mM aqueous HEPES medium (pH 7.4) at 310.15 K in the presence of C18TAB micelles ([L] = 14.87 mM): curve 0, in the absence of CePyCl; curves 1-14, in the presence of CePyCl, with concentrations ranging from 0.30 10-5 to 24.6 10-5 M. The inset is a plot of the logarithm of the fluorescence intensity of the first peak in the pyrene emission spectra, ln II, as a function of [CePyCl].
μe, zeta potential, ζ, and surface charge density enclosed by the shear plane, σζ, are electrochemical properties that can give interesting information about the distribution of charges on the surface of surfoplexes. They are related through the well-known equations78-80 ζ ¼ and
3η μ 2ε0 εr f ðKD aÞ e
2ε0 εr KD kB T zeζ sinh σζ ¼ ze 2kB T
ð4Þ
ð5Þ
where η is the viscosity of water (6.824 10-4 N m-2 s at 310.15 K); ε0 and εr are the vacuum and relative permittivities (8.854 10-12 J-1 C2 m-1 and 74.2, respectively); f (kDa) is the Henry function, which depends on the reciprocal Debye length, κD, and the particle radius, a; e is the elemental charge; z is the valence of the ion; kB is the Boltzmann constant; and T is the absolute temperature. The Smoluchowski limit ( f (kDa) = 1.5) has been applied to estimate the Henry function,78-80 as usually done for medium to large particles in a medium of moderate ionic strength (a . κ-1 D ). Table 1 shows the values of the electrophoretic mobility, μe, zeta potential, ζ, and surface charge density, σζ, obtained at all Lmic/D ratios. Figure 3 shows the plot of ζ versus Lmic/D for C18TAB/DNA surfoplexes. The variation of zeta potential (and also of surface charge density, not plotted) follows a sigmoidal habit, as previously found for other lipoplexes and surfoplexes,4,36,38,81,82 with (78) Delgado, A. V. Interfacial Electrokinetics and Electrophoresis; Marcel Dekker: New York, 2002; Vol. 106. (79) Hunter, R. J. Zeta Potential in Colloids Science: Principles and Applications; Academic Press:London, 1981. (80) Ohshima, H.; Furusawa, K. Electrical Phenomena at Interfaces: Fundamentals, Measurements, and Applications; Marcel Dekker: New York, 1998. (81) Ciani, L.; Ristori, S.; Calamai, L.; Martini, G. Biochim. Biophys. Acta 2004, 1664, 70. (82) Lobo, B. C.; Davis, A.; Koe, G.; Smith, J. G.; Middaugh, C. R. Arch. Biochem. Biophys. 2001, 386, 95.
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Table 1. Values of Electrophoretic Mobility, μe, Zeta Potential, ζ, and Surface Density Charge (at the Shear Plane), σζ, at Different Values of the Lmic/D Mass Ratio for C18TAB/DNA Surfoplexesa Lmic/D
108μe (m2 V-1 s-1)
ζ (mV)
103σζ (C m-2)
0.3 0.6 0.7 0.9 1.2 2.9 4.4 5.9
-4.48 -3.70 -1.62 0.73 3.58 4.23 3.87 3.94
-46 -38 -17 8 37 44 40 41
-16 -13 -5 2 13 16 15 16
a The DNA concentration was kept constant at 0.495 mg/mL. Errors are estimated to be around 2% in electrophoretic mobility, 3% in zeta potential, and 6% in surface density charge.
Figure 4. Specific conductivity, κ, of C18TAB/DNA surfoplexes at 310.15 K as a function of Lmic/D at a constant DNA concentration of 0.182 mg/mL in 40 mM HEPES aqueous medium, pH 7.4.
Figure 3. Values of zeta potential, ζ, of C18TAB/DNA surfoplexes at different Lmic/D ratios (bottom) or charge ratios CR (top) in an aqueous buffered medium (HEPES 40 mM) at pH 7.4. [DNA] = (0.495 ( 0.002) mg/mL. The solid line is a sigmoidal fit to the experimental values. Errors are estimated to be around 3%. three clear zones in the Figure: (i) the zone where the zeta potential of the surfoplexes is negative, ζ = (-45 ( 2 mV); (ii) the zone where ζ sharply inverts the sign; and (iii) the zone where ζ of the surfoplexes is positive and constant at ζ = (42 ( 2 mV), similar to that determined for other lipoplexes in our laboratory.59,83 The value of Lmic/D at which μe, zeta potential, ζ, and/or the surface charge density, σζ, is null is known as the isoneutrality ratio, Lmic/Dφ, which in this case corresponds to a value of (Lmic/D)φ,exp,ζ = 0.83. Figure 4 reports a plot of the specific conductivity, κ, as a function of [L] in aqueous HEPES medium at constant DNA concentration (= 0.182 mg/mL). As can be seen in the Figure, in the presence of DNA, the conductivity shows a clear change in the positive slope at [L] = 0.62 mM. At [L] below the break, electrostatic interactions between the positive charges facing outward from the micellar surface and the negative charges of DNA provoke a release of counterions (Na+ from DNA and Br- from the micelles), thus justifying the increase observed in conductivity. Once the break occurs, a lower slope is seen given in which only the ionic free micelles and counterions coming from micelle dissociation contribute to the conductivity. The value of [L] = 0.62 mM, which corresponds to Lmic/D = 0.97, has been attributed to the (83) Rodriguez-Pulido, A.; Ortega, F.; Llorca, O.; Aicart, E.; Junquera, E. J. Phys. Chem. B 2008, 112, 12555.
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isoneutrality ratio (i.e., (Lmic/D)φ,exp,κ) of the C18TAB/DNA surfoplex. Fluorescence Spectroscopic Study. The spectroscopic analysis of the surfoplexes has been carried out with two kinds of fluorescence studies: (i) ethidium bromide (EtBr) intercalation assays and (ii) anisotropy measurements. The former permits us to confirm and characterize the DNA-micelle surface electrostatic interaction, whereas anisotropy values give interesting information about the changes on micelle packing flexibility when DNA is present. Both type of experiments also allow for the determination of (Lmic/D)φ. It is known that the fluorescence intensity of ethidium bromide, an aromatic planar cationic fluorophore (Chart 1) increases around 30-fold upon its intercalation between base pairs of double-stranded DNA;38-42 the hydrophobic environment inside the base pairs protects the probe from water molecules and molecular oxygen that may quench its fluorescence emission. When DNA is condensed or compacted by cationic aggregates, EtBr intercalation is prevented and, as a consequence, fluorescence intensity will be quenched because the probe will remain fully accessible to the bulk solvent. Thus, the decrease on probe emission intensity, as long as a solution with C18TAB micelles is added at constant DNA concentration, can be used as a means to asses DNA accessibility and, accordingly, DNA-micelle compaction. Figure 5 shows the emission fluorescence spectra of EtBr at a constant DNA concentration of 0.496 mg/mL as long as the surfactant concentration, and thus the Lmic/D ratio, increases. Also included in the Figure are the emission spectra of EtBr in the absence of DNA (dashed line, Lmic/D = ¥, [L] = 0.586 mg/mL) and in the absence of micelles (i.e. just in the HEPES-buffered medium (dotted line)). Several features can be observed in the Figure: (i) EtBr does not interact with cationic micelles, as would be expected for a cationic probe, because the emission in the presence of micelles is comparable to that in the bulk and in both cases very low (i.e., around 45 au, dashed and dotted lines in the Figure); (ii) the emission of EtBr experiments clearly changes when DNA is present, decreasing from around 750 au at Lmic/D = 0 (only DNA and EtBr are present, curve 0 in the Figure) to around 150 au at Lmic/D = 1.0 (curve 5 in the Figure), from which it remains almost constant with Lmic/D increasing up to a value DOI: 10.1021/la8034038
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Figure 5. Emission fluorescence spectra of EtBr in the presence of C18TAB/DNA surfoplexes at different Lmic/D ratios for curves 0-9: (0) 0, (1) 0.1, (2) 0.4, (3) 0.6, (4) 0.8, (5) 1.1, (6) 1.9, (7) 2.9, (8) 4.4, and (9) 5.9. Medium: 40 mM aqueous HEPES, pH 7.4. 6:1 DNA/EtBr. [DNA] = 0.496 mg/mL. The dotted line shows the emission fluorescence spectra of EtBr in the absence of micelles and surfoplexes. The dashed line shows an example of the emission fluorescence spectra of EtBr only in the presence of micelles at [L] = 0.586 mg/ mL. Both spectra (dotted and dashed lines) are very similar and overlap in most parts of the studied λ region. of around 6 times the isoneutrality ratio ((Lmic/D)φ). Figure 6 shows that the maximum intensity at λ = 588 nm as a function of Lmic/D allows for the determination of this isoneutrality ratio, (Lmic/D)φ,exp,fl = 1.09, as calculated by the Phillips method,66 in good agreement with the zeta potential and conductivity results. The trend observed in both Figures reveals that EtBr, initially within the DNA helix, is displaced toward the aqueous bulk when the surfoplex is formed in the presence of C18TAB micelles, confirming that the EtBr-DNA interaction is weaker than the electrostatic micelle-DNA interaction. However, and in contrast with what we found in the presence of lipoplexes,59,83 the emission does not fall until that measured for the probe in the bulk is IF ≈ 45 au, indicating that some probe molecules may remain inside the DNA helix when it is compacted by the micelles. It seems that C18TAB micelles do not displace all EtBr probe molecules from inside the DNA helix, which leaves some parts still accessible to EtBr, justifying why its emission intensity drops when surfoplexes are formed but does not reach bulk levels, where it would show negligible values typical of a fluorescence probe in a polar environment. Furthermore, to reinforce this argument, the experimental π f π* bands of Figure 5 have been interpreted84 as consisting of several bands at different wavelengths, each of which is attributable to the π f π* emission of the probe immersed in different microenvironments, characterized by its hydrophobicity, microviscosity, rigidity, and/or solvation features. Accordingly, all spectra were deconvoluted into the optimum number of overlapping Gaussian curves following a procedure widely explained elsewhere.85 Table 2 reports the results of these deconvolutions (in terms of the wavelengths, λi, areas, Ai, and widths, (84) Karukstis, K. K.; Zieleniuk, C. A.; Fox, M. J. Langmuir 2003, 19, 10054. (85) Junquera, E.; del Burgo, P.; Boskovic, J.; Aicart, E. Langmuir 2005, 21, 7143.
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Figure 6. Emission fluorescence intensity of EtBr at 588 nm in the presence of C18TAB/DNA surfoplexes as a function of Lmic/D (bottom) or charge ratio CR (top). Medium: 40 mM aqueous HEPES, pH 7.4. 6:1 DNA/EtBr. [DNA] = 0.496 mg/mL. The emission fluorescence intensity of EtBr in the absence of micelles and surfoplexes and only in the presence of micelles ([L] = 0.586 mg/ mL) is shown as dotted and dashed lines, respectively. Table 2. Parameters of the Deconvoluted Gaussian Bands of EtBr Fluorescence Emission Spectra in the Presence of C18TAB/DNA Surfoplexes at Different Lmic/D Ratiosa two Gaussians Lmic/D I588 λ1 (nm) W1
A1
λ2 (nm) W2
one Gaussian A2
λ1 (nm) W1 A1
0 776 586 35 20 821 608 47 22 634 0.1 752 586 35 19 827 607 47 23 066 0.4 627 586 36 18 553 608 48 18 796 0.6 481 588 38 16 156 611 50 12 542 0.8 330 588 38 10 035 609 50 9561 1.1 155 576 22 601 598 48 9178 1.9 145 587 39 4808 610 53 4444 2.9 176 587 38 4912 606 52 5742 4.4 120 585 38 3103 606 52 4155 5.9 161 587 39 5258 609 52 4765 41 599 55 2867 ¥b buffer 44 599 56 3065 a Wavelength λi, width Wi, and area Ai. Medium: aqueous HEPES 40 mM, pH 7.4. The DNA/EtBr ratio is 6:1; [DNA] = 0.496 mg/mL. b [L] = 0.586 mg/mL
Wi, of the deconvoluted peaks), and Figure 7 shows an example for Lmic/D = 2.9. As can be seen in the Table, the one-Gaussian fit is the correct choice at Lmic/D = ¥ (in the absence of DNA) and/or for EtBr just in the medium (in the absence of DNA and micelles) because only one microenvironment, the bulk, is expected in these cases (Gaussian centered at λ = 599 nm, last two lines of the Table). However, in the absence of micelles but in the presence of DNA (i.e., at Lmic/D = 0, first line of Table 2), the best fit corresponds to the two-Gaussian option, indicating that EtBr is partitioned between two possible microenvironments located at λ1 = (586 ( 1) nm and λ2 = (608 ( 1) nm that are accordingly assigned to the π f π* emission of EtBr intercalated between the base pairs of the DNA double helix (λ1) and immersed in the aqueous bulk (λ2), respectively. It is thus confirmed that the more blue-shifted the emission band, the Langmuir 2009, 25(8), 4402–4411
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Article Chart 2. Schematic Drawing to Show the Displacement of EtBr Probe Molecules (in Red) from inside the DNA Helix as a Result of the Formation of (A) Surfoplexes and/or (B) Lipoplexes at (C) Different Lipid/DNA Mass Ratiosa
Figure 7. Emission fluorescence spectra of EtBr in the presence of C18TAB/DNA surfoplexes at Lmic/D = 2.9, together with their deconvolutions into two Gaussian components. Solid line: experimental spectra. Dashed line: Gaussian components. Dotted line: total sum of Gaussian components. Medium: 40 mM aqueous HEPES, pH 7.4. 6:1 DNA/EtBr. [DNA] = 0.496 mg/mL. more hydrophobic the microenvironment. As can be seen in the Table, this assignment (λ1 around 586 nm and λ2 around 608 nm) can also be applied on average in the presence of micelles as Lmic/D increases, even for Lmic/D > (Lmic/D)φ, in contrast with what we found previously for lipoplexes,59,83 where once the isoneutrality ratio was passed, only one microenvironment was found, indicating that all EtBr molecules were displaced from the helix inside to the bulk. Furthermore, both A1 and A2 in Table 2 (areas of the deconvoluted peaks) remain almost constant at around 50%, again in contrast with what we found in the case of lipoplexes where A1 decreased while A2 increased with L/D. This feature also point to the fact that a certain percentage of probe molecules remain inside the helix even when the surfoplex is formed, whereas in the case of lipoplexes all EtBr molecules are displaced toward the bulk. This clear difference among lipoplexes and surfoplexes can be due to (i) structure or geometrical reasons; micelles are smaller than liposomes (for a given number of C atoms in the hydrocarbon chain/s) and DNA can not bend itself on the compaction process around the spherical micelle surface more than its flexibility limit, leaving regions accessible to EtBr molecules and/or (ii) a different affinity on the DNA/colloidal vehicle interaction; it is clear that both DNA/liposome and DNA/micelle interactions are more favorable than that between DNA and EtBr, but the affinity of the former seems higher. For that reason, cationic liposomes are able to displace EtBr completely from inside the DNA helix, whereas cationic micelles provoke only a partial displacement. We do think that both reasons may explain the experimental evidence, although more studies to confirm it will be welcomed. Chart 2 shows a comparative schematic diagram. Fluidity is known to be an important biophysical parameter characterizing lipid layers and bilayers. Fluorescence anisotropy, r, serves as a measure of membrane fluidity. Thus, the higher the degree of rotation of an excited fluorophore in lipid packing (i.e., with increasing fluidity), the smaller the anisotropy. In this work, we have used nonpolar fluorophore DPH, which is completely buried in the Langmuir 2009, 25(8), 4402–4411
a (A) EtBr in the presence of DNA; (B) EtBr in the presence of DNA and C18TAB cationic micelles (B1) at (Lmic/D) < (Lmic/D)φ, and (B2) (Lmic/D) > (Lmic/D)φ; and (C) EtBr in the presence of DNA and cationic liposomes at (C1) (L/D) < (L/D)φ and (C2) (L/D) > (L/D)φ.
hydrophobic core of C18TAB micelles. Consequently, changes in the anisotropy can be directly related to changes in the fluidity (or rigidity) of the micelle packing. Figure 8 shows anisotropy values of C18TAB-micelle-doped DPH in the presence of DNA at several Lmic/D values. As can be observed in the Figure, anisotropy decreases almost linearly up to Lmic/D around 1 and remains almost constant at r = 0.07 at higher Lmic/D values. An anisotropy value of r = 0.051, in agreement with literature values,86 was also measured for C18TAB micelles in the absence of DNA (Lmic/ D = ¥). This Figure permits us to obtain the isoneutrality ratio (Lmic/D)φ,exp,r at 1.12, in very good agreement with the values also obtained in this work by using the other techniques. Figure 8 deserves some remarks in this respect: (i) Anisotropy increases when the micelles are in the presence of DNA, indicating that micelle packing becomes less flexible (more ordered) once the surfoplexes are formed. The interaction of the positively charged head groups with the polyanion DNA seems to reduce the lateral diffusion of the lipids in the micelle packing, as recently confirmed in lipoplexes by NMR studies.87 Also, the complexation of the polyelectrolyte by cationic micelles through electrostatic interactions is very cooperative in nature, which is consistent with the reported elongated coil-to-globular state transition for DNA upon binding cationic micelles.15,88,89 (ii) Surfoplex composition has a clear effect on the anisotropy and, accordingly, on the flexibility of the complex; the packing between the hydrocarbon surfactant chains forming the micelles (86) Alves, F. R.; Zaniquelli, M. E. D.; Loh, W.; Castanheira, E. M. S.; Oliveira, M.; Feitosa, E. J. Colloid Interface Sci. 2007, 316, 132. (87) Leal, C.; Sandstrom, D.; Nevsten, P.; Topgaard, D. Biochim. Biophys. Acta 2008, 1778, 214. (88) Mel’nikov, S. M.; Sergeyev, V. G.; Yoshikawa, K. J. Am. Chem. Soc. 1995, 117, 2401. (89) Mel’nikov, S. M.; Sergeyev, V. G.; Yoshikawa, K.; Takahashi, H.; Hatta, I. J. Chem. Phys. 1997, 107, 6917.
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and Lmic L cmcV 2ML cmcV ¼ ¼ D φ D D D φ M bp
Figure 8. Fluorescence anisotropy at 430 nm, r430, of DPH in C18TAB/DNA surfoplex solutions at 310.15 K as a function of Lmic/D (bottom) or charge ratio CR (top). [DNA] = 0.501 mg/mL. Medium: 40 mM aqueous HEPES, pH 7.4. Errors in light scattering are less than 3%.
becomes looser as long as the micellized lipid content increases (i.e,. with increasing Lmic/D). Similar trends have been found for other surfoplexes23 and lipoplexes.23,44,45,47,48 Furthermore, it seems that once the isoneutrality ratio, (Lmic/D)φ,exp,r, is exceeded (i.e., surfoplexes are positively charged (see Figure 3, as already mentioned), the anisotropy reaches its lowest (and constant) value. Considering that fluidity plays a key role in the efficiency of the transfection process (the fluid behavior of surfoplex membranes determines their stability, which is a prerequisite for efficient lipofection) and that a net positive charge also favors the interaction between the surfoplex and the negatively charged cellular membrane, it can be concluded that C18TAB/DNA surfoplexes that are potentially efficient in transfecting cells are those with a mass of micellized surfactant of J1.12 times that of DNA (i.e., those with (Lmic/D) above(Lmic/D)φ). Calculation of the Isoneutrality Ratio. This parameter, defined as the ratio at which the positive charges of the micelles are stoichiometrically equal to the negative charges of DNA (i.e., CR = 1), is characteristic of the surfoplex and/ or lipoplex and can be not only experimentally determined by using different experimental methods, as done in this work, but also calculated. CR is given by the equation CR ¼
LM bp nþ L=ML ¼ ¼ n2DML 2D=M bp
ð6Þ
where n+ and n- are the numbers of moles of positive and negative charges coming from cationic surfactant and the DNA molecule, respectively; ML is the molar mass of the cationic surfactant (= 392.5 g mol-1); and M bp is the basepair-averaged molar mass (= 649.9 g mol-1), (L/D)φ. Lmic/Dφ can then be calculated via the equations L 2ML ðCRÞφ ¼ 1f ¼ D φ M bp 4410
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ð7Þ
ð8Þ
Equation 8 has been used in this work to calculate (Lmic/ D)φ,calc given the values of MLM bp, cmc (determined in a previous section), and D (that used in the experiments reported herein). Figures 3, 6, and 8 also show the corresponding properties as a function of CR (top x axis). Table 3 reports and resumes both experimental, (Lmic/D)φ,exp, and calculated, (Lmic/D)φ,calc, ratios for all methods used in the present work. As can be seen in the Table, there exists good agreement between the isoneutrality ratios determined by using both electrochemical (zeta potential and conductivity) and spectroscopic (EtBr fluorescence intercalation assays and DPH fluorescence anisotropy) techniques. Also, very good agreement has been found between the experimental and calculated values. From all of these values, it can be concluded that on average (Lmic/D)φ,exp and (Lmic/D)φ,calc (i.e., the isoneutrality ratio of C18TAB/DNA surfoplexes is reached when the mass of micellized C18TAB is close to the DNA mass). This value is around one-fourth that previously found in our laboratory for lipoplexes consisting of the same DNA and a 1:1 mixture of a zwitterionic helper lipid (DOPE) and a cationic lipid belonging either to the cholesterol derivative family (DC-Chol) or to the acylammoniumpropane family (DSTAP).59,83 This difference is due to the fact that (i) the molecular weights of these cationic lipids are almost double that of C18TAB and (ii) surfoplexes, unlike lipoplexes, are formed in the absence of a helper lipid (DOPE). Whatever the reason, the fact is that these positively charged C18TAB/DNA surfoplexes, with a low level of packing rigidity, can be formed with a lower content of cationic surfactant with respect to that of DNA, which may be quite advantageous regarding the toxicity problem associated with these lipids.
Conclusions It has been shown that around 44 monomers of the cationic surfactant C18TAB, with a packing parameter of P around 0.28, may form spherical micelles in HEPES medium at a critical micelle concentration of 0.169 mM at 310.15 K. The positively charged surfaces of these micelles are able to compact and condense double-stranded DNA segments by means of a strong, entropically driven surface electrostatic interaction that increases the order or rigidifies micelle packing. This DNA-micelle interaction seems to be weaker than that between liposomes/vesicles and DNA, a fact that could indicate that surfoplexes may liberate DNA more easily, after endocytosis, into the cell media. The isoneutrality of the surfoplex, experimentally determined by zeta potential, conductometry, EtBr intercalation essays, and DPH anisotropy measurements, and also calculated from molecular parameters and the cmc obtained in this work, is reached when the micellized lipid mass is approximately equal to that of DNA (i.e., (Lmic/D)φ). EtBr intercalating essays have revealed not only the electrostatic character of the micelle/DNA interaction but also that DNA could be less condensed than in the case of lipoplexes consisting of the same DNA and a 1:1 mixture of a zwitterionic helper lipid and a cationic lipid. Once the surfoplex is formed, the flexibility in micelle packing increases along with Lmic/D, reaching a constant, maximum value once the isoneutrality ratio is passed (i.e., when the mass Langmuir 2009, 25(8), 4402–4411
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Table 3. Experimental and Calculated Values of the Isoneutrality Ratio (Lmic/D)O,exp and (Lmic/D)O,calc, Obtained from All the Experimental Methods Reported in This Work and Equation 8, Respectively
[D] (mg/mL) ðLDmic Þφ, exp, i ðLDmic Þφ, calc, i
LDE i = ζ
conductivity i = κ
fluorescence EtBr intercalation i = fl
fluorescence anisotropy i = r
0.495 0.83 1.07
0.182 0.97 0.84
0.496 1.09 1.07
0.501 1.12 1.08
of micellized surfactant exceeds that of DNA). It can be concluded, then, that in this regime (i.e., (Lmic/D) > (Lmic/D)φ) C18TAB/DNA surfoplexes, with a surface density charge of σζ = (+15 ( 1) 10-3 C m-2, moderately flexible packing, and a low content of cationic surfactant are potentially adequate for transfecting purposes, although transfection assays would be necessary and welcomed.
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Acknowledgment. We thank the Spanish Ministry of Education (project no. CTQ2005-1106) and the Comunidad Aut onoma of Madrid (project no. S-SAL-0249-2006). We also thank C. Aicart for carrying on gel agarose electrophoresis experiments at the Biochemistry and Molecular Biology Department of the University Complutense of Madrid.
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