DNA and Cationic Surfactant Complexes at Hydrophilic Surfaces. An

Marité Cárdenas,* José Campos-Terán, Tommy Nylander, and Björn Lindman. Center for Chemistry and Chemical Engineering, Physical Chemistry 1, ...
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Langmuir 2004, 20, 8597-8603

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DNA and Cationic Surfactant Complexes at Hydrophilic Surfaces. An Ellipsometry and Surface Force Study Marite´ Ca´rdenas,* Jose´ Campos-Tera´n, Tommy Nylander, and Bjo¨rn Lindman Center for Chemistry and Chemical Engineering, Physical Chemistry 1, University of Lund, P.O. Box 124, SE-221 00 Lund, Sweden Received December 15, 2003. In Final Form: July 15, 2004 The adsorption and formation of DNA and cationic surfactant complexes at the silica-aqueous interface have been studied by ellipsometry. The interaction between the DNA-surfactant complexes at the micaaqueous interface has been determined by the interferometric surface force apparatus. Adsorption was as expected not observed on negatively charged hydrophilic surfaces for DNA and when DNA-cationic surfactant complexes were negatively charged. However, adsorption was observed when there is an excess of cationic surfactant, just below the point of phase separation. The adsorption process requires hours to reach steady state. The adsorbed layer thickness is large at low surface coverage but becomes more compact and thinner at high coverage. A long-range repulsive force was observed between adsorbed layers of DNA-cationic surfactant complexes, which was suggested to be of both electrostatic and steric origin. The forces were found to be dependent on the equilibration time and the experimental pathway.

Introduction The development of methods for DNA extraction and purification1 and the potential use of DNA-cationic surfactant systems as vehicles in gene delivery and gene transfection2-4 have promoted the study of the bulk behavior of DNA and complexes formed of DNA and cationic surfactants during the last two decades,5-12 although the first reports appeared in 1979.5 Even though interactions with cell membranes and colloidal particles existing in the blood might influence how efficiently DNAsurfactant complexes are delivered to the target cells, their interfacial behavior has received little attention. The effect of pH and ionic strength on the adsorption of DNA to negatively and positively charged and neutral particles has been extensively studied.13-15 Recently, we have studied the adsorption of DNA, a cationic surfactant, cetyltrimethylammonium bromide (C16TAB), and their mixtures at hydrophobized silica surfaces by means of ellipsometry16 and dynamic light scattering (DLS).17 For * Corresponding author. E-mail: [email protected]. (1) Trewavas, A. Anal. Biochem. 1967, 21, 324. (2) Lasic, D. D.; Templeton, N. S. Adv. Drug Delivery Rev. 1996, 20, 221. (3) Verma, I. M.; Somia, N. Nature 1997, 389, 239. (4) Templeton, N. S.; Lasic, D. D. Mol. Biotechnol. 1999, 11, 175. (5) Chatterjee, R.; Chattoraj, D. K. Biopolymers 1979, 18, 147. (6) Hayakawa, K.; Santerre, J. P.; Kwak, J. C. T. Biophys. Chem. 1983, 17, 175. (7) Shirahama, K.; Takashima, K.; Takisawa, N. Bull. Chem. Soc. Jpn. 1987, 60, 43. (8) Gorelov, A. V.; McLoughlin, D. M.; Jacquier, J. C.; Dawson, K. A. Il Nuovo Cimento 1998, 20, 2553. (9) Koltover, I.; Salditt, T.; Ra¨dler, J. O.; Safinya, C. R. Science 1998, 281, 78. (10) Gani, S. A.; Chattoraj, D. K.; Mukherjee, D. C. Ind. J. Biochem. Biophys. 1999, 36, 165. (11) Dias, R.; Mel’nikov, S.; Lindman, B.; Miguel, M. G. Langmuir 2000, 16, 9577. (12) Smith, P.; Lynden-Bell, R. M.; Smith, W. Phys. Chem. Chem. Phys. 2000, 2, 1305. (13) Chattoraj, D. K.; Chowrashi, P.; Chakravarti, K. Biopolymers 1967, 5, 173. (14) Gani, S. A.; Mukherjee, D. C.; Chattoraj, D. K. Langmuir 1999, 15, 7130. (15) Mitra, A.; Chakraborty, P.; Chattoraj, D. J. Ind. Chem. Soc. 2001, 78, 689. (16) Ca´rdenas, M.; Braem, A.; Nylander, T.; Lindman, B. Langmuir 2003, 19, 7712.

hydrophobic surfaces, hydrophobic interactions lead to adsorption of both surfactant-free DNA and DNA-cationic surfactant mixtures. In this work, interesting effects could be seen upon the addition of cationic surfactant to a preadsorbed DNA layer: a significant increase in adsorbed amount that is accompanied by a dramatic compaction of the adsorbed layer. Such an observation is correlated to the formation of DNA globules in the bulk.16 Interestingly, similar behavior was observed for the interaction of DNA/ cationic surfactant complexes onto hydrophobic latex particles by DLS.17 In the case of negatively charged hydrophilic surfaces, no adsorption is expected for DNA because both the surface and DNA are similarly charged. However, DNA-cationic surfactant complexes might be able to adsorb at a negatively charged surface because one of its components has affinity for negatively charged surfaces. Here, we study how the presence of dodecyltrimethylammonium bromide (C12TAB) affected the interfacial behavior of DNA on negatively charged surfaces. For this purpose, different DNA samples have been used, which include (i) large double-stranded (ds) DNA (2 kbp), (ii) large single-stranded (ss) DNA (heat-denatured 2kb), (iii) short ds-DNA (146 bp), and (iv) short ss-DNA (100b). The adsorption and formation of DNA-cationic surfactant complexes at the silica-aqueous interface have been studied by means of ellipsometry. The surface force apparatus (SFA) has been used to directly measure the forces between DNA-C12TAB complexes formed at the mica-aqueous interface. Experimental Section Materials. C16TAB and C12TAB (Merck pa quality), sodium dodecyl sulfate (SDS; Sigma), and sodium bromide (Aldrich, extra pure quality) were used as received. Salmon sperm DNA was purchased from Gibco, BRL, and used as received. As stated by the manufacturer, this DNA is double-stranded and 2000 ( 500 base pairs (bp) in length as determined by 1% TAE agarose gel analysis and free from Dnase and Rnase. Short mononuclesomal DNA, 146 bp as determined by 7.5% acrylamide gel analysis, was kindly provided by D. McLoughlin and was dialyzed against 10 mM NaBr solution prior to use. The 100 bases (b) ss-DNA (17) Ca´rdenas, M.; Schille´n, K.; Nylander, T.; Jansson, J.; Lindman, B. Phys. Chem. Chem. Phys. 2004, 6, 1603.

10.1021/la0363581 CCC: $27.50 © 2004 American Chemical Society Published on Web 08/28/2004

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(MWG-Biotech AG) was used as received. The DNA concentration was measured spectrophotometrically by its absorbance at 260 nm, using an extinction coefficient of 33 µg/mL for ss-DNA and 50 µg/mL for ds-DNA.18 The ratio of absorbance at 260 and 280 nm was about 1.8-1.9, and the absorbance at 320 nm was negligible, indicating the absence of protein contamination.18 Salmon sperm DNA was thermally denatured to produce ss-DNA by heating to 85 °C for 10 min and then cooling rapidly by injecting the sample into a mixture of cold ice and ethanol.19 The conformation of DNA in aqueous solution, that is, whether the chains are single- or double-stranded, was verified by circular dichroism measurements.20 Water purified by a Milli-Q system (Millipore Corp., Bedford, MA) was used in all measurements. All DNA and surfactant solutions were prepared in 10 mM NaBr solution. Surface Preparation. The silica surfaces (p-type, borondoped, resistivity 1-20 Ω‚cm) were thermally oxidized in an oxygen atmosphere at 920 °C for ∼1 h, followed by annealing and cooling in an argon flow. This procedure yields a SiO2 layer of ∼300-Å thickness. The oxidized wafers were cut into slides with a width of 12.5 mm and cleaned in a mixture of (1) NH4OHH2O2 and (2) HCl-H2O2 as described earlier.21 Finally, the cleaned oxidized wafers were stored in ethanol. The surfaces were dried under vacuum (0.001 mbar) and then treated in a plasma cleaner (Harrick Scientific Corp., model PDC-3XG) during 5 min prior to the start of the ellipsometric experiments. Ellipsometry. An automated Rudolph Research thin-film null ellipsometer, type 43603-200E, is used as described by Tiberg and Landgren21 to measure adsorbed amount and thickness of adsorbed layers in situ. The optical properties of the silica surfaces, that is, the refractive index of the silicon and the oxide layer as well as the oxide layer thickness, were characterized at the beginning of each experiment by measuring the ellipsometric angles, Ψ and ∆, in two different ambient media, air and 10 mM NaBr solution, as described earlier.22 All measurements were performed with a light-source wavelength of 4015 Å and with an angle of incidence of ∼68.5°. A 5-mL cuvette was thermostated to 25.0 ( 0.1 °C and agitated with a magnetic stirrer at about 300 rpm. The recorded ellipsometric angles were evaluated using a four-layer optical model, assuming isotropic media and planar interfaces. The mean refractive index, nf, and the ellipsometric thickness, df, of the adsorbed layer were calculated numerically as described elsewhere.23,24 The adsorbed amount, Γ, was calculated from nf and df using the formula

Γ)

(nf - n0)df dn/dc

where n0 is the refractive index of the bulk solution and dn/dc is the refractive index increment as a function of the bulk concentration. For DNA solutions,25 dn/dc ) 0.134 g/cm3, and for surfactant solutions, dn/dc ) 0.15 g/cm3. In the data presented below for the mixed systems, surface excess concentrations are calculated using the dn/dc of DNA. The highest relative error in the adsorbed amount due to this simplification was ∼10% (calculated by assuming that the layer was purely surfactant). For typical experimental conditions, the errors in Ψ and ∆ are normally distributed with standard deviations of 0.001 and 0.002°, respectively. The relative errors in refractive index and thickness are rather high for small surface excess concentrations, Γ < 0.5 mg/m2, but decrease rapidly to values around 5-10% for Γ > 1 mg/m2. However, the relative error in the surface excess concentration is much smaller: 15% at ∼0.1 mg/m2 and less than 1% for Γ ) 2 mg/m2.26 (18) Cantor, C. R.; Schimmel, P. R. Biophysical Chemistry; W. H. Freeman and Co.: San Francisco, 1980; p 1371. (19) Krasna, A.-I. Biopolymers 1970, 9, 1029. (20) Gray, D. M.; Ratliff, R. L.; Vaughan, M. R. Methods Enzymol. 1992, 211, 389. (21) Tiberg, F.; Landgren, M. Langmuir 1993, 9, 927. (22) Landgren, M.; Jo¨nsson, B. J. Phys. Chem. 1993, 97, 1656. (23) Jenkins, T. E. J. Phys.: Appl. Phys. 1999, 32, R45. (24) Azzam, R. M. A.; Bashara, N. M. Ellipsometry and polarized light; North-Holland Publishing Co.: Amsterdam, 1977. (25) Bhattacharya, S.; Mandal, S. S. Biochim. Biophys. Acta 1997, 1323, 29. (26) Tiberg, F. J. Chem. Soc., Faraday Trans. 1996, 92, 531.

Ca´ rdenas et al. The compaction studies were performed as follows. After characterizing the optical properties of the silica plate, the DNA sample was injected by a small aliquot of a concentrated stock, typically 10 µL, into 5 mL of NaBr solution in the ellipsometric cuvette. Successive aliquots of cationic surfactant stock solution, typically ranging from 10 to 100 µL, were added to the DNA solution until either adsorption or turbidity was observed. In all experiments, a fixed DNA concentration of 0.06 mg/mL (1.85 × 10-4 M of negative charges) was used. SFA. The interferometric SFA was used to measure the interaction between two mica surfaces across an aqueous 10 mM NaBr solution containing DNA, C12TAB, or DNA-C12TAB mixtures. The technique27 as well as the particular version of the apparatus (Mark IV) used in this study28 is described in more detail elsewhere. The force is measured between two mica surfaces (mean radius curvature, R, of about 1-2 cm) in a crossed cylinder configuration. The two mica sheets with silver covering their backsides and supported on half-cylindrical silica disks are mounted on a double cantilever spring (with a spring constant K) and on a piezoelectric crystal, respectively. The surface separation, D, between the two surfaces is controlled by the piezoelectric crystal and measured by an interferometric technique with an accuracy of 2 Å. The magnitude of the force, F, can be determined from the measured spring deflection down to about 10-7 N and is given normalized with the mean radius of curvature. The SFA, equipped with a small volume chamber (∼40 mL), was dismantled, and all inner parts were rinsed with water and ethanol and finally dried with ultrapure nitrogen flow before it was assembled again. Green muscovite mica (S & J Trading Inc., New York) was cleaved into thin molecularly smooth sheets, cut in about 1 × 1 cm pieces, and put down on a freshly cleaved mica baking sheet; then a 520 Å thick silver layer was evaporated onto this mica sheet. The mica pieces were then glued with the silver side down onto optically polished half-cylindrical silica disks, which were then mounted in the SFA. The assembly of the instrument and surface preparation were performed in a clean room under essentially dust-free conditions. At the beginning of each experiment, the mica-mica contact position was measured in air. After that the contact position as well as a control force curve across 10 mM NaBr solution was measured to verify that the system was clean. Aqueous solutions were degassed under a vacuum during at least 15 min, before they were filled into the SFA. If the force curve measured agreed with what was expected theoretically, the salt solution was then extracted from the SFA chamber, leaving a drop of solution trapped between the mica surfaces, and replaced either by 0.06 mg/mL DNA (1.8 × 10-4 M DNA charges), 6 × 10-4 M C12TAB, or the mixture of them at these concentrations in 10 mM NaBr solution. After each change of the reservoir solution, the system was allowed to equilibrate for at least 1 h before making the first measurement. All force measurements were performed at 19 °C.

Results and Discussions Ellipsometry. None of the studied surfactant-free DNA samples adsorb at the hydrophilic silica surface. This is a consequence of the electrostatic repulsions between DNA and the negatively charged surface at the low ionic strength used here (10 mM NaBr solution). Adsorption of a polyelectrolyte to a similarly charged surface can only occur at a sufficiently high salt concentration, where electrostatic contributions are highly screened and the interaction between the polymer-surface is similar or larger than that of the polymer-solvent.29 DNA has been found to adsorb to negatively charged surfaces at a high ionic strength where electrostatic forces are sufficiently screened and the solubility of DNA is decreased.13-15 In these studies, adsorption of DNA macromolecules to (27) Israelachvili, J. N.; Adams, G. E. J. Chem. Soc., Faraday Trans. 1978, 174, 975. (28) Parker, J. L.; Christenson, H. K.; Ninham, B. W. Rev. Sci. Instrum. 1989, 60, 3135. (29) Fleer, G. J.; Cohen Stuart, M. A.; Scheutjens, J. M. H. M.; Cosgrove, T.; Vincent, B. Polymer at Interfaces, 1st ed.; Chapman & Hall: London, 1993.

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Figure 1. Adsorption isotherm for C12TAB (filled circles) and DNA-C12TAB mixtures (open circles) as measured by ellipsometry. The 0.06 mg/mL DNA was used for the mixed system. Lines are included as guides for the eye.

various types of particles including silica particles was believed to be an entropically controlled process, in which DNA competes with water for the active sites at the surface. The adsorption isotherm for C12TAB at the silica surface is given in Figure 1. At low concentrations, adsorption occurs through ion exchange and the adsorbed amount is low because of the low surface charge density of silica under the used conditions (∼0.01 OH-/nm at pH 6).30 At higher concentrations, the adsorption increases in a cooperative way with the formation of surface aggregates, to values well above the ion exchange capability. At even higher concentrations, the adsorbed amount finally reaches a plateau value that is independent of surfactant concentration. This isotherm is in good agreement with that of C12TAB adsorption on silica particles reported earlier.31 Figure 1 also shows the adsorption from 0.06 mg/mL DNA solution (1.85 × 10-4 M of negative charges) as a function of C12TAB concentration. Measurable adsorption is observed once the surfactant concentration reaches 6 × 10-4 M C12TAB. This corresponds to a DNA/C12TAB molar charge ratio, F-/+, of 0.31; that is, there are about three cationic surfactants per phosphate group. At these low C12TAB concentrations, no significant adsorption for the single-component system is observed (Γ < 0.05 mg/ m2). The volume fraction of DNA-surfactant complex in the adsorbed layer is ∼25% as calculated from the adsorbed amount and layer at steady-state conditions (1.2 mg/m2 and 50 Å, respectively) if we assume a density of the complex of 1.1 g/mL. This value is in agreement with recent neutron reflectivity measurements performed on the same system by our group (unpublished data). Similar results were obtained by Gani el at.,14 who studied the adsorption of DNA to charged particles. They argued that DNA had to compete with water for adsorption on hydrophilic solid surfaces. Figure 2a shows the adsorbed amount (open circles) and adsorbed layer thickness (crosses) as a function of time for 2 kbp ds-DNA and C12TAB at F-/+ ) 0.31. Slow adsorption kinetics is observed, and it takes at least 15 h to reach steady-state conditions. If more C12TAB is added, further adsorption occurs (Figure 1). However, turbidity is observed already at 7 × 10-4 M C12TAB (F-/+ ) 0.26) indicating the beginning of the phase separation (30) Iler, R. K. The Chemistry of silica, 1st ed.; John Wiley & Sons: New York, 1979; p 866. (31) Bijsterbosch, B. H. J. Colloid Interface Sci. 1974, 47, 186.

Figure 2. (a) Adsorbed amount (open circles) and adsorbed layer thickness (crosses) as a function of time for 2 kbp ds-DNA and C12TAB at F-/+ ) 0.31 as measured by ellipsometry. (b) Adsorbed amount (open circles) and adsorbed layer thickness (crosses) as a function of time for 2 kbp ds-DNA and C12TAB at F-/+ ) 0.26 as measured by ellipsometry. The arrow indicates the point at which continuous rinsing with 10 mM NaBr solution was initiated. The time scale was increased by a factor of 20 after rinsing was started. Lines are drawn as a guide for the eye.

process. Once phase separation occurred, macroscopic precipitation was observed at both the surface and the glass walls of the cuvette. No ellipsometric data were recorded after this point because of the large scatter of light. The adsorbed amount and layer thickness versus time when C12TAB concentration reaches 7 × 10-4 M (F-/+ ) 0.26) is given in Figure 2b. Note that the adsorption kinetics becomes faster and the adsorbed amount increases with increasing the surfactant concentration, that is, by getting closer to the phase separation boundary. At both charge ratios, there is a decrease in layer thickness from >100 to ∼50 Å once the surface coverage reached ∼0.5 mg/m2. Clearly, above this surface coverage the DNA-C12TAB layer acquires a quite compact structure at the solid-liquid interface, as has previously been shown in our group.16,17 It must be pointed out that if the adsorption process was limited by the diffusion of the compacted DNA only (4-5 × 10-12 m2/s as determined by DLS),17 a higher adsorption rate should be observed. This suggests that the time needed to reach steady state

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depends on the internal rearrangement in the layer. The initially adsorbed DNA-C12TAB complex, which in bulk has a hydrodynamic radius of about 55 nm,17 is disrupted due to the interaction with the surface as well as with other adsorbed complexes to reach an adsorbed layer thickness of just about 5 nm. Consequently, the decrease of layer thickness to 5 nm occurred only after a certain concentration is reached. Similar kinetically controlled adsorption processes have been observed earlier for many polymeric systems.32 If displacement of the DNA/C12TAB solution by continuous rinsing with 10 mM NaBr solution was performed (arrow shown in Figure 2b), total desorption occurred within minutes. The same effect was observed upon addition of the anionic surfactant SDS. SDS is known to induce DNA decompaction in the bulk as a result of a competitive interaction with the cationic surfactant.33 Because there is no strong affinity of any of the individual components for the interface, total desorption is observed once the complex is disrupted by the action of SDS. The same interfacial behavior was observed for all DNA samples regardless of the molecular weight or conformation (double- or single-stranded). If, on the other hand, hexadecyltrimethylammonium bromide (C16TAB) was used as the compacting agent, no adsorption was observed even when the surfactant concentrations were so high that the phase separation region was reached. This is in agreement with results presented by Thomas’ group; they found that there is adsorption of DNA-C12TAB but not of DNA-C16TAB complexes at the air-water interface even well beyond phase separation.34 Chatterjee and Chattoraj5 showed that the free energy of alkyltrimethylammonium bromide binding to DNA in the bulk decreases as the surfactant’s chain length increases. Thus, DNAC16TAB complexes are considerably more stable in the bulk than those formed with C12TAB, and the integrity of the structure is, therefore, not perturbed by an interaction with the surface. Surface Force Measurements. Figure 3 shows the surface forces measured between two mica surfaces across an aqueous 10 mM NaBr solution containing 0.06 mg/mL DNA. As mentioned above, ellipsometry measurements do not indicate any adsorption for surfactant-free DNA samples and because both the surface and the DNA are highly negatively charged, no change in the force curve is expected as compared to the pure salt background solution.35 However, when a surfactant-free DNA solution is injected in the SFA chamber, a displacement of the force curve is observed when measured at the same speed of approach as the one for the pure salt solution. This behavior could be explained by an entrapment of DNA molecules between the mica surfaces. If sufficient force is applied, mica-mica contact can eventually be reached at a separation distance of ∼3 Å, as expected from one layer of strongly associated sodium counterions on each surface. The force curve measured across 0.06 mg/mL DNA (1.8 × 10-4 M) in 10 mM NaBr solutions can be fitted to the Derjaguin-Landau-Verwey-Overbeek (DLVO) theory, with a Debye length of 30 Å, as expected for a 0.01 M 1:1 electrolyte. If we place the onset of van der Waals and electrostatic forces 60 Å out from the mica surface, we obtain the fit shown in Figure 3 for a surface potential of (32) Van de Ven, T. G. M. Adv. Colloid Interface Sci. 1994, 48, 121. (33) Dias, R. S.; Lindman, B.; Miguel, M. G. J. Phys. Chem. B 2002, 106, 12608. (34) Thomas, R. K. Department of Physical Chemistry, Oxford University, U.K. Private communication. (35) Tadmor, R.; Herna´ndez-Zapata, E.; Chen, N.; Pincus, P.; Israelachvili, J. N. Macromolecules 2002, 35, 2380.

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Figure 3. Force normalized by the radius of curvature, F/R, as a function of surface separation between mica surfaces in 0.06 mg/mL (1.8 × 10-4 M negative charges) DNA solution (closed circles). As a reference, the force curve across the 10 mM NaBr solution is included (crosses). Arrows indicate that the surfaces can be taken to mica-mica contact upon exerting enough force. Fitting to DLVO theory is shown by a solid line. Chart 1. Schematics of the SFA Experimental Procedure Followed in This Worka

a Between all the steps, a drop of solution was left between the surfaces when replacing the reservoir solution.

100 mV at constant charge. No adhesion was observed upon retraction from surface separations of 120 Å or larger. For mixed DNA-C12TAB mixtures, surface force curves were measured as follows. First, after recording the force curve for surfactant-free DNA solutions, the SFA chamber was emptied leaving a droplet of DNA solution trapped between the mica surfaces. Then, a solution containing DNA-C12TAB complexes (F-/+ ) 0.31) in 10 mM NaBr was filled in the SFA chamber. A schematic representation of the SFA experimental procedure is given in Chart 1. Figure 4 shows the surface forces measured between two mica surfaces across DNA-C12TAB solutions (F-/+ ) 0.31) after 1 and 12 h of equilibration time. For DNA-C12TAB mixtures, a different behavior is observed as compared to the single-component systems, which is in agreement with the adsorption of DNA-C12TAB complexes to the mica surfaces. After 1 h of equilibration time, a long-range repulsive force is observed with an onset at 400 Å. This force curve seems to be a result of a combination of

DNA and Cationic Surfactant Complexes

Figure 4. Force normalized by the radius of curvature, F/R, as a function of surface separation between mica surfaces in DNA-C12TAB solution at F-/+ ) 0.31. Here, the mixed DNACTAB solution was filled in after equilibration of the mica surfaces with a 1.8 × 10-4 M DNA solutions. Closed and open circles indicate the first and second approaches, respectively. The arrow indicates the observed attractive inward jump into contact. Scheme 1. Schematic Illustration Showing the Suggested Structure of the Adsorbed DNA-C12TAB Complexes between Two Mica Surfaces

electrostatic and steric repulsion because it cannot be fitted to the DLVO theory. The surfaces can be approached until a surface separation of ∼50 Å. Adhesion was measured upon retraction with a pull-off force of 30.5 mN/m. In bulk solution, complexes formed with 2 kbp ds-DNA and C16TAB form globular species with a hydrodynamic radius of ∼50 nm.17 Initially the DNA-surfactant complexes acquire a quite bulky conformation at the surface that gives rise to the large repulsion measured. This behavior is also in agreement with that found with ellipsometry where, at low surface coverage, a large thickness is found (Figure 2a,b). This is schematically represented in Scheme 1. A dramatically different force behavior is found after 12 h of equilibration time. Now the range of the repulsive interactions is reduced, with an onset at about 270 Å instead of 400 Å. Additionally, there is an attractive force that pulls the surfaces from about 117 Å to a surface separation of 60 Å. If more force is applied, this layer can

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be compressed to about 55 Å. The adhesion force increased slightly with time up to 37 mN/m, as compared to the first approach. The long-range force can be fitted to the DLVO theory with a hard wall of ∼110 Å and a decay length of 30 Å, yielding a surface potential of ∼35 mV, indicating that the surface charge is drastically diminished by the adsorption of DNA-C12TAB complexes. After long times of equilibration, the hydrophilic surface seems to change the structure of the initially adsorbed DNA-C12TAB complexes, resulting in a more homogeneous compact adsorbed layer. The measured inward jump distance is about 57 Å, which is close to the separation distance found at the wall (60 Å). A plausible structure of the adsorbed layer of the DNA-C12TAB complex is given in Scheme 1 where the DNA strands are sandwiched between two C12TAB aggregates. The cross-sectional diameter of such an aggregate (∼62 Å) is comparable with both the ellipsometer thickness and the SFA contact distance. This fact suggests that one adsorbed complex layer is removed from the gap upon reaching contact. Indeed, an attractive force is predicted for adsorbed neutral polymers that are in full equilibrium with a polymer solution of constant concentration regardless of the solvent conditions.36,37 In this context, full equilibrium means that adsorbed polymers can flow out of the gap upon approach of the surfaces and thus requires a weak interaction between the polymer and the surface. The onset of the interaction occurs at surface separations that are comparable with the diameter of the polymers. The lattice model predicts that the ultimate equilibrium situation will be a sandwich structure of two plates with one layer of polymer segments in between.37 Although the system studied here does not consist of a neutral polymer, it is a weakly charged complex (PB fittings give low surface potentials) that do not have strong affinity for the surface (rinsing with salt solution or SDS addition induces total desorption, as observed in ellipsometry, and small pull-off forces are measured for the adsorbed DNA-C12TAB layers by SFA). Moreover, the attractive jump occurs at a separation distance that is comparable with the cross-sectional diameter of the complexes as discussed above. It must be pointed out that limited recharging occurs in the DNA-cationic surfactant systems and that the surfactant binding ceases close to the charge neutralization condition.38 Therefore, no resolubilization of the precipitate is observed upon increasing the surfactant concentration.6 Similar behavior has been observed for chitosan-SDS mixtures in equilibrium with mica surfaces as measured by the SFA.39 This system also shows limited resolubilization upon increasing SDS concentration. In this case, force measurements show that on approach an inward jump from ∼8 to ∼4 nm is observed. This ∼4-nm jump is comparable to the cross-sectional area of the SDSchitosan aggregates. These authors propose that bridging occurs as a result of the SDS-mediated intersurface links once the repulsive electrostatic double layer is balanced by the attraction due to hydrophobic interactions. Figure 5 shows the force curve after the solution in the SFA reservoir was exchanged from a mixed DNA-C12TAB solution to a 10 mM NaBr solution. Here, the force range is similar to the one found before dilution but the inward jump is lost. Additionally, no adhesion is found unless the surfaces are taken to mica-mica contact. Although rinsing induces total desorption of DNA-C12TAB mixtures from (36) de Gennes, P. G. Macromolecules 1982, 15, 492. (37) Scheutjens, J. M. H. M.; Fleer, G. J. Macromolecules 1985, 18, 1882. (38) Bruinsma, R. Eur. Phys. J. B 1998, 4, 75. (39) De`dinaite´, A.; Ernstsson, M. J. Phys. Chem. B 2003, 107, 8181.

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Figure 5. Force normalized by the radius of curvature, F/R, as a function of surface separation between mica surfaces measured across the DNA-C12TAB solution at F-/+ ) 0.31 (closed circles) and after replacement with the 10 mM NaBr solution has been performed (open circles).

silica surfaces as determined by ellipsometry (see Figure 2b), some material seems to still be adsorbed within the mica surfaces. The lack of attractive interactions at separation distances lower than 120 Å suggests that most of the amphiphile has diffused out of the gap between the surfaces, leaving some partially compacted DNA in the contact position. Fitting to DLVO theory indicates that the surface potential considerably increases, to ∼70 mV for a hard wall at 55 Å. Indeed, the idea that the surfactant molecules desorbed from the complexes is supported by the finding that DNA-C12TAB globules dissolve upon dilution as evidenced for individual DNA-cationic surfactant complexes in the bulk solution measured by fluorescence microscopy.40 To investigate whether the adsorbed layer depends on the history of DNA-C12TAB complex formation, a different experiment was performed in which the mica surfaces are pre-equilibrated in 4.2 × 10-4 M C12TAB solution, and then the reservoir was changed to the mixed DNAC12TAB solution at F-/+ ) 0.31 (see Chart 1). In this way, it is possible to study the effect of the excess positive charge present under the conditions when adsorption occurred (F-/+ ) 0.31), corresponding to 4.2 × 10-4 M C12TAB. Figure 6 shows the surface forces measured between two mica surfaces across the 4.2 × 10-4 M C12TAB solution. The force curves obtained are clearly different to that in pure salt solution. In the first approach, the range and extension of the repulsive force was considerably decreased as compared with the pure salt system. This indicates that the surface charge has been reduced but not to the extent of charge neutralization. Once the surfaces come to a separation of ∼105 Å, they jump into adhesive contact at a separation distance of 16 Å. This value is similar to a fully extended C12 alkyl chain length (about 17 Å). Thus, at contact, the surfactant aggregates on the surfaces interact with each other giving rise to some sort of intercalated bilayer. At large separation distances, however, the surfactant molecules are expected to form micelles. A second approach does not show any repulsive force at all until an attractive force takes the surfaces to the same final contact thickness value as in the first approach. In both approaches, a pull-off adhesion force of (40) Mel’nikov, S. M.; Sergeyev, V. G.; Yoshikawa, K. J. Am. Chem. Soc. 1995, 117, 9951.

Ca´ rdenas et al.

Figure 6. Force normalized by the radius of curvature, F/R, as a function of surface separation between mica surfaces in the 4.2 × 10-4 M C12TAB solution. Closed and open circles indicate the first and second approaches, respectively. As a reference, the force curve across the 10 mM NaBr solution is included (crosses). Arrows indicate attractive jumps into contact.

about 106 mN/m was found. This clearly indicates that the surface properties are influenced by the presence of the cationic surfactant even at concentrations far below the critical micelle concentration (cmc). Similar results have been reported earlier for the interactions between glass surfaces immersed in C16TAB solutions as measured by a modified SFA.41 The authors argued that at low surfactant concentrations (10-6 M) a monolayer was formed and the surfaces became adhesive in contact, but this is unlikely at hydrophilic surfaces; instead, micellarlike aggregates build at the surface. At the 5 × 10-5 M C16TAB surface, charge neutralization occurred. Although both a different surface and a different surfactant chain length have been used in the present work, the same qualitative results are expected because both surfaces are highly negatively charged. The higher cmc for shorter surfactant chains (C12 as compared to C16) shifts the adsorption isotherm to higher concentrations. Moreover, Boschkova et al.42 measured the forces between two mica surfaces across 0.3 × cmc (2.7 × 10-4 M) C12TAB and found that an ∼8-Å-thick monolayer was built up on each surface at contact. Indeed, even though the concentration used by Boschkova et al.42 is higher than in the present work, the same general features for the force curve and the layer thickness are observed. The force curve measured after replacing the reservoir solution for DNA-C12TAB mixtures at F-/+ ) 0.31 is given in Figure 7. The magnitude of the long-range force lies between the forces recorded at low and high coverage for the mixed DNA-C12TAB system, where instead the mica surfaces had been equilibrated with surfactant-free DNA solution. In this case, no inward jumps are observed until the force barrier is reached at 65 Å. DLVO fitting also yields a low surface potential of 33 mV when the hard wall is placed 110 Å out from the mica-mica contact. A second approach, after 12 h of equilibration time, increased the range of the repulsive force as well as the force barrier. Adhesion was observed upon retraction. The extension of the adhesion depends on the extent to which the adsorbed layers had been compressed. An example of the adhesive (41) Parker, J. L.; Yaminsky, V. V.; Claesson, P. M. J. Phys. Chem. 1993, 97, 7706. (42) Boschkova, K.; Kronberg, B.; Stålgren, J. J. R.; Persson, K.; Salagean, M. R. Langmuir 2002, 18, 1680.

DNA and Cationic Surfactant Complexes

Figure 7. Force normalized by the radius of curvature, F/R, as a function of the surface separation between mica surfaces in the DNA-C12TAB solution at F-/+ ) 0.31. Here the mixed DNA-CTAB solution was filled in after equilibration of the mica surfaces with 4.2 × 10-4 M C12TAB solutions. Closed and open circles indicate the first and second approaches, respectively. The inset shows the adhesion observed when retrieving the surfaces.

force for the mixed DNA-C12TAB solutions is shown in the inset in Figure 7. It has been shown earlier that the order in which polyelectrolytes and oppositely charged surfactants43,44 are added to the solution determines the properties of adsorbed layers. Depending on the experimental pathway, the adsorbed layers can be trapped in quasi-equilibrium states, requiring inaccessible experimental times to reach true equilibrium. Moreover, a slightly higher surfactant concentration is present in the gap for the present experiment as compared to the experiment in which the complexes were formed after the surfaces were immersed in DNA solution. Thus, surfaceinduced phase separation could take place due to the close proximity to the phase separation border. Concluding Discussion DNA compacts as a result of ion-correlation effects, where its original sodium counterions are replaced by surfactant aggregates acting as multivalent counterions.45-48 In the binding of C12TAB to short DNA molecules (∼220 bp), a two-stage process has been observed45 in which the surfactant ions initially exchange with condensed sodium counterions without altering the charge of the complex so that the complex remains soluble. At this point, no adsorption to negatively charged surfaces is expected because the complex is still negatively charged. If more cationic surfactant is added, more surfactant molecules will bind due to DNA-induced surfactant self(43) De`dinaite´, A.; Claesson, P. M.; Bergstro¨m, M. Langmuir 2000, 16, 5257. (44) Braem, A.; Biggs, S.; Prieve, D. C.; Tilton, R. D. Langmuir 2003, 19, 2736. (45) Gorelov, A. V.; Kudryashov, E. D.; Jacquier, J. C.; McLoughlin, D. M.; Dawson, K. A. Physica A 1998, 249, 216. (46) Barreleiro, P. C. A.; Olofsson, G.; Alexandridis, P. J. Phys. Chem. B 2000, 104, 7795. (47) Wagner, K.; Harries, D.; May, S.; Kahl, V.; Ra¨dler, J. O.; BenShaul, A. Langmuir 2000, 16, 303. (48) Gelbart, W. M.; Bruinsma, R. F.; Pincus, P. A.; Parsegian, V. A. Phys. Today 2000, 53, 38.

Langmuir, Vol. 20, No. 20, 2004 8603

assembly, causing DNA charge neutralization, or close to that, and thereby phase separation.45 For the DNAC12TAB system, the bulk behavior correlates with the adsorption phenomena at similarly charged (negative) hydrophilic surfaces because no adsorption occurs for F-/+ > 1. However, adsorption occurred at an excess of C12TAB to DNA (F-/+ < 0.3). DNA is a large polyelectrolyte and remains soluble as a result of the large entropic penalty imposed for confining its counterions at the surface. However, adsorption is favored once the charge density of both the DNA macromolecule and the surface is sufficiently decreased, due here to the interaction with C12TA+ ions. Thus, the excess of cationic surfactant induces a shift in the adsorbed species from only surfactant aggregates (below F-/+ ) 0.31) to mixed surfactant-DNA complexes (above F-/+ ) 0.31). Indeed, DNA-C12TAB complexes adsorb in a cooperative manner to the surface (Figure 1), which indicates that certain aggregate formation is required for adsorption to occur. Additionally, the formation of these aggregates would also explain the slow kinetics observed for DNA-C12TAB complex adsorption to hydrophilic surfaces. Both ellipsometry and the SFA indicate that the structure of the adsorbed layer rearranged as the surface coverage increases. Shortly after the DNA-C12TAB complex solution has come into contact with the surface (the low-coverage region), both the thickness of the adsorbed layer is large (>100 Å) as determined by ellipsometry and surface force measurements. In addition, a long-range repulsive force was found. At longer equilibration times (the high coverage region), both the layer thickness and the range of the repulsive forces are considerably reduced. Moreover, the contact distance is 50-60 Å, which corresponds to one layer of complexes, as determined by ellipsometry (∼50-Å thick layers), trapped between the mica surfaces. In conclusion, the surface behavior correlates to that observed in the bulk and no adsorption occurs until there is an excess of surfactant with respect to negative charges of DNA. Once this excess is achieved, changes in the solvent quality induce a slow cooperative adsorption process onto both mica and silica surfaces. Interestingly, the DNA molecular weight and conformation (singleversus double-stranded chains) does not seem to influence the interfacial behavior of the mixed DNA-C12TAB system at negatively charged surfaces. It is rather the changes in the interaction parameters upon association with cationic surfactant that controls the adsorption process. These results are of relevance for the development of gene delivery vehicles in the sense that they provide an insight into the interactions of DNA-cationic amphiphile complexes with negatively charged surfaces that could be seen as a simple model for cell membranes. It also provides an insight into the type of structures that DNA might form with positively charged species inside the cell, for example, histone proteins. Acknowledgment. Thanks to D. McLoughlin for kindly providing the 146 bp ds-DNA sample. Thanks to Stefan Klintstro¨m for kindly supplying the silica plates. Special thanks are given to Andra De`dinaite´ for fruitful discussions. The Swedish Foundation for Strategic Research Program Colloid and Interface Technology and the Swedish research Council financially supported this work. LA0363581