pubs.acs.org/Langmuir © 2009 American Chemical Society
DNA Binding to Zwitterionic Model Membranes Marie-Louise Ainalem,*,† Nora Kristen,†,^ Karen J. Edler,‡ Fredrik H€oo€k,§ Emma Sparr,† and Tommy Nylander† †
Physical Chemistry, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden, ‡University of Bath, Claverton Down, Bath BA2 7AY, United Kingdom, and §Chemistry Department, Department of Applied Physics, Chalmers University of Technology, 41296 Gothenburg, Sweden. ^ Present affiliation: Stranski-Laboratorium, Institut f€ ur Chemie, Technische Universit€ at Berlin, D-10623 Berlin, Germany. Received September 25, 2009. Revised Manuscript Received November 9, 2009
This study shows that DNA (linearized plasmid, 4331 base pairs and salmon sperm, 2000 base pairs, respectively) adsorbs to model membranes of zwitterionic liquid crystalline phospholipid bilayers in solutions containing divalent Ca2þ cations, and also in solutions containing monovalent Naþ. The interaction between DNA and surface-supported model membranes was followed in situ using null ellipsometry, quartz crystal microbalance with dissipation, as well as neutron reflectometry. In the presence of Naþ (in the absence of multivalent ions), DNA adopts an extended coil conformation upon adsorption. The solvent content in the adsorbed layer is high, and DNA is positioned on top of the membrane. In the presence of divalent Ca2þ, the driving force for the adsorption of DNA is electrostatic, and the adsorbed DNA film is not as dilute as in a solution containing Naþ. Cryo-TEM and SANS were further used to investigate the interaction in bulk solution using vesicles as model membrane systems. DNA adsorption could not be identified in the presence of Naþ using SANS, but cryo-TEM indicates the presence of DNA between neighboring unilamellar vesicles. In the presence of Ca2þ, DNA induces the formation of multilamellar vesicles in which DNA intercalates the lamellae. Possible electrostatic and hydrophobic mechanisms for the adsorption of DNA in solutions containing monovalent salt are discussed and compared to the observations in divalent salt.
Introduction The use of cationic polymers or lipids to deliver DNA across lipid membranes is an important research area within nonviral gene delivery.1-3 Physicochemical studies have shown that DNA molecules condense upon interaction with cationic specimens and reduce their physical extension dramatically, which can allow for membrane crossing and protection against degradation.4-8 One of the drawbacks with using cationic species for DNA condensation is, however, their toxic effect on the cells.9,10 In pursuit of a noncytotoxic alternative, the interaction of DNA with zwitterionic phospholipids, which are a dominating constituent of cellular membranes, is therefore of great interest. This motivates the characterization of interactions in such systems, which is also the basis of the present study. Another important aspect of DNA-phospholipid interactions is the possibility of DNA interacting with phospholipids within the cell nucleus. In fact, the intranuclear space is known to contain lipids that are not connected with the double lipid bilayer
membrane that comprises the nuclear envelope.11-13 These endonuclear lipids have been related to structural function, signal transduction, and stimulating DNA and RNA synthesis,14-16 although their precise function is still not fully understood. One possibility is that endonuclear lipids also serve as components of chromatin, possibly for structural reasons.17-19 Even though the DNA-phospholipid interactions may have important implications for biological function, as well as gene therapy and other applications in biotechnology and medicine, the basic mechanism for the interaction between DNA and zwitterionic phospholipids is still not fully understood. There is ample evidence of attractive interactions between DNA and zwitterionic phospholipid bilayers in the presence of multivalent ions. For example, ordered structures of DNA and phospholipids have been observed using X-ray scattering, and DNA has been proposed to embed between bilayers in lamellar structures of varying morphology.20-25 The same system in the presence of
€ *Corresponding author. Marie-Louise Ainalem nee Orberg. E-mail:
[email protected]. Fax: þ 46 46 222 4413.
(12) Hunt, A. N.; Clark, G. T.; Attard, G. S.; Postle, A. D. J. Biol. Chem. 2001, 276, 8492–8499. (13) Irvine, R. F. Nat. Rev. Mol. Cell Biol. 2003, 4, 349–360. (14) Alessenko, A.; Burlakova, E. B. Bioelectrochemistry 2002, 58, 13–21. (15) Divecha, N.; Irvine, R. F. Cell 1995, 80, 269–278. (16) Martelli, A. M.; Fala, F.; Faenza, I.; Billi, A. M.; Cappellini, A.; Manzoli, L.; Cocco, L. Cell. Mol. Life Sci. 2004, 61, 1143–1156. (17) Albi, E.; Lazzarini, R.; Magni, M. V. Biochem. J. 2008, 410, 381–389. (18) Albi, E.; Magni, M. P. V. Biol. Cell 2004, 96, 657–667. (19) Martelli, A. M.; Tabellini, G.; Borgatti, P.; Bortul, R.; Capitani, S.; Neri, L. M. J. Cell. Biochem. 2003, 88, 455–461. (20) Bruni, P.; Pisani, M.; Amici, A.; Marchini, C.; Montani, M.; Francescangeli, O. Appl. Phys. Lett. 2006, 88. (21) Kharakoz, D. P.; Khusainova, R. S.; Gorelova, A. V.; Dawson, K. A. Febs Lett. 1999, 446, 27–29. (22) McManus, J. J.; Radler, J. O.; Dawson, K. A. Langmuir 2003, 19, 9630– 9637. (23) McManus, J. J.; Radler, J. O.; Dawson, K. A. J. Am. Chem. Soc. 2004, 126, 15966–15967. (24) Uhrikova, D.; Hanulova, M.; Funari, S. S.; Khusainova, R. S.; Sersen, F.; Balgavy, P. Biochim. Biophys. Acta, Biomembr. 2005, 1713, 15–28. (25) Uhrikova, D.; Lengyel, A.; Hanulova, M.; Funari, S. S.; Balgavy, P. Eur. Biophys. J. 2007, 36, 363–375.
(1) De Smedt, S. C.; Demeester, J.; Hennink, W. E. Pharm. Res. 2000, 17, 113– 126. (2) Felgner, P. L.; Gadek, T. R.; Holm, M.; Roman, R.; Chan, H. W.; Wenz, M.; Northrop, J. P.; Ringold, G. M.; Danielsen, M. Proc. Natl. Acad. Sci. U.S.A. 1987, 84, 7413–7417. (3) Mahato, R. I.; Rolland, A.; Tomlinson, E. Pharm. Res. 1997, 14, 853–859. (4) Dias, R.; Mel’nikov, S.; Lindman, B.; Miguel, M. G. Langmuir 2000, 16, 9577–9583. (5) Koltover, I.; Salditt, T.; Radler, J. O.; Safinya, C. R. Science 1998, 281, 78– 81. (6) Lasic, D. D.; Strey, H.; Stuart, M. C. A.; Podgornik, R.; Frederik, P. M. J. Am. Chem. Soc. 1997, 119, 832–833. (7) Melnikov, S. M.; Sergeyev, V. G.; Yoshikawa, K. J. Am. Chem. Soc. 1995, 117, 9951–9956. € (8) Orberg, M. L.; Schillen, K.; Nylander, T. Biomacromolecules 2007, 8, 1557– 1563. (9) Fischer, D.; Li, Y. X.; Ahlemeyer, B.; Krieglstein, J.; Kissel, T. Biomaterials 2003, 24, 1121–1131. (10) Lee, R. J.; Huang, L. Crit. Rev. Ther. Drug Carrier Syst. 1997, 14, 173–206. (11) DeLong, C. J.; Qin, L. Y.; Cui, Z. J. Biol. Chem. 2000, 275, 32325–32330.
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monovalent ions is, however, far less studied. There are a few studies that report measurable effects of DNA on zwitterionic lipid bilayers in the absence of multivalent ions. Pott et al. have proposed DNA to be confined between lamellar phosphatidylcholine (PC) bilayers,26,27 and Wu et al. have shown that aggregation of oligolamellar 1,2-dioleoyl-sn-glycero-phosphatidylcholine (DOPC) LR phase vesicles occurs in the presence of DNA.28 DNA has also been shown to adsorb to 1,2-dipalmitoylsn-glycero-3-phosphatidylcholine (DPPC) gel (Lβ) phase membranes,29 and Lu et al. propose that the adsorption of short oligonucleotides to Lβ phase zwitterionic bilayers is preferred over LR phase bilayers.30 In this study, we used LR phase DOPC or 1,2-dimyristoyl-snglycero-3-phosphatidylcholine (DMPC) as model systems for biological membranes. We first addressed the interaction of DNA with lipid bilayers deposited on solid surfaces in aqueous solutions containing Naþ or Ca2þ cations using the following techniques: (1) in situ null ellipsometry, which was used to characterize the adsorption process in terms of the adsorbed amount and layer thickness, by recording the change in state of the polarized light upon reflection at a surface,32 with a time resolution of a few seconds; (2) quartz crystal microbalance with dissipation monitoring (QCM-D),33 which operates as an ultrasensitive weighing device, and was used to characterize the formation of thin films on surfaces by measuring the energy dissipation, D (c.f. film rigidity), and the resonance frequency, f (c.f. coupled mass), of a quartz crystal; (3) neutron reflectometry (NR) by which the specular reflection of neutrons at an interface is recorded, and data can be evaluated to reveal the distribution of lipid and DNA across an adsorbed film.34 We have also addressed the interaction of DNA with model membranes in the presence of monovalent Naþ or divalent Ca2þ cations using extruded lipid vesicles, applying two experimental techniques: cryo-transmission electron microscopy (cryo-TEM), which allows for high-resolution observations of objects as small as a few nanometers at high water content;35 and small-angle neutron scattering (SANS), which frequently is applied for structural investigations of nanometer-sized objects and measures the intensity of the scattered neutrons, I, as a function of momentum transfer, Q.
Experimental Section Materials. Luciferase plasmid DNA (pET9, Promega), 4331 base pairs (bp), was amplified, linearized, and purified as described by Ainalem et al.36 As an additional source of DNA, salmon sperm DNA of 2000 ( 500 bp (Invitrogen) was used. The final concentration of salmon sperm DNA and linearized plasmid DNA used in each experiment was 0.15 mg mL-1 and 0.1 mg mL-1, respectively. These were chosen so that the DNA concentration in all experiments was below the overlap concentration c*. DOPC (Avanti Polar Lipids), DMPC (Avanti Polar Lipids), DMPC with perdeuterated acyl chains, d54-DMPC (Larodan), as (26) Pott, T.; Colin, A.; Navailles, L.; Roux, D. Interface Sci. 2003, 11, 249–257. (27) Pott, T.; Roux, D. Febs Lett. 2002, 511, 150–154. (28) Wu, C. M.; Chen, H. L.; Liou, W.; Lin, T. L.; Jeng, U. S. Biomacromolecules 2004, 5, 2324–2328. (29) Malghani, M. S.; Yang, J. J. Phys. Chem. B 1998, 102, 8930–8933. (30) Lu, D. M.; Rhodes, D. G. Biochim. Biophys. Acta, Biomembr. 2002, 1563, 45–52. (31) Landgren, M.; Jonsson, B. J. Phys. Chem. 1993, 97, 1656–1660. (32) Tiberg, F.; Landgren, M. Langmuir 1993, 9, 927–932. (33) Rodahl, M.; Hook, F.; Krozer, A.; Brzezinski, P.; Kasemo, B. Rev. Sci. Instrum. 1995, 66, 3924–3930. (34) Majkrzak, C. F.; Berk, N. F.; Krueger, S. K.; Borchers, J. A.; Dura, J. A.; Ivkov, R.; O0 Donovan, K. Neutron News 2001, 12, 25–29. (35) Vinson, P. K.; Bellare, J. R.; Davis, H. T.; Miller, W. G.; Scriven, L. E. J. Colloid Interface Sci. 1991, 142, 74–91. (36) Ainalem, M. L.; Carnerup, A. M.; Janiak, J.; Alfredsson, V.; Nylander, T.; Schillen, K. Soft Matter 2009, 5, 2310–2320.
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well as the surfactant n-doceyl-β-D-maltopyranoside, DDM (Anatrace), were used as received. DOPC containing perdeuterated acyl chains, d-DOPC, was synthesized in the group of Prof. Eugenijus Butkus at the department of Organic Chemistry, Vilnius University, Lithuania. This was done by esterification of the glycerophosphatidylcholine cadmium salt with fully deuterated oleic acid, which in turn was synthesized at the Physical and Theoretical Chemistry Laboratory, University of Oxford, United Kingdom. All experiments were performed in solutions containing either 10 mM NaBr (Aldrich) or 2.5 mM CaCl2 (Sigma Aldrich) and 5 mM NaBr. Ellipsometry, QCM-D, and cryo-TEM were also performed using a 1 mM CaCl2 and 8 mM NaBr solution. Ultrapure water with a specific resistivity of 18.2 MΩ cm was obtained through a Millipore Milli-Q purification system, and all solutions were prepared in either Milli-Q purified water and/or D2O (ARMAR Chemicals, D€ ottingen, Switzerland). Ellipsometry. In situ null ellipsometry was performed using an automated Rudolph Research thin-film ellipsometer, type 43603-200E, equipped with high-precision stepper motors and operating at a wavelength of 4015 A˚ with an angle of incidence of 68.04.31,32 A 5 mL trapezoidal sample cell of optical glass was used, which was magnetically stirred at about 300 rpm and had a temperature of 25.0 ( 0.1 C and Teflon tubes connected to a peristaltic pump (Ole Dich Instrument makers ApS, Hvidovre, Denmark) to allow a 5 mL min-1 flow of the desired electrolyte solution. The silicon (Si) wafers with a thermally grown oxide (SiO2) layer of ∼300 A˚ (provided by Bo Thuner, Department of Chemistry, IFM, Link€ oping University, Sweden) were used as substrates and cleaned as described by Vandoolaeghe et al.37 Prior to starting an experiment, the wafers (stored in ethanol) were dried under reduced pressure (0.02 mbar) and treated in an air plasma cleaner (Harrick Scientific Corp., model PDC-3XG) for 5 min. The optical properties of the substrate were determined at the start of each experiment in order to obtain reliable measures of the refractive index and thickness of the SiO2 layer as well as the Si refractive index. This was accomplished by measuring the angles Δ and Ψ of the bare substrate in two ambient media with different refractive indices, air and water, as described in detail by Tiberg et al.32 Four-zone measurements were performed to correct for imperfections in the optical components.38 Experimental Procedure. Bilayers of high coverage and DOPC purity were formed on the Si surfaces in 0.1 mM HCl by the well-established three-step method of adsorption and deposition from a mixed micellar solution containing DOPC and DDM (1:6 by weight).37,39-41 See available Supporting Information (Figure S1a) where the bilayer thickness of 42 ( 2 A˚ and the mass uptake of ∼4.2 mg m-2 corresponds to a complete bilayer surface coverage and a resulting area per DOPC molecule of about 62 A˚2 according to Tiberg et al.42 This headgroup area for a surface-deposited lipid bilayer is lower than that usually reported for DOPC LR phase in excess water, 72 A˚2 (Nagle and coworkers).43,44 We note, however, that Lis et al. report that DOPC bilayers are highly compressible and that areas between 60 A˚2 and 80 A˚2 can be obtained depending on water content.45 Following (37) Vandoolaeghe, P.; Rennie, A. R.; Campbell, R. A.; Thomas, R. K.; Hook, F.; Fragneto, G.; Tiberg, F.; Nylander, T. Soft Matter 2008, 4, 2267–2277. (38) Azzam, R. M. A.; Bashara, N. M. Ellipsometry and Polarized Light; Elsevier: Amsterdam, 1989. (39) Grant, L. M.; Tiberg, F. Biophys. J. 2002, 82, 1373–1385. (40) Vacklin, H. P.; Tiberg, F.; Fragneto, G.; Thomas, R. K. Langmuir 2005, 21, 2827–2837. (41) Vacklin, H. P.; Tiberg, F.; Thomas, R. K. Biochim. Biophys. Acta, Biomembr. 2005, 1668, 17–24. (42) Tiberg, F.; Harwigsson, I.; Malmsten, M. Eur. Biophys. J. 2000, 29, 196– 203. (43) Nagle, J. F.; Tristram-Nagle, S. Biochim. Biophys. Acta Rev. Biomembr. 2000, 1469, 159–195. (44) Tristram-Nagle, S.; Petrache, H. I.; Nagle, J. F. Biophys. J. 1998, 75, 917– 925. (45) Lis, L. J.; McAlister, M.; Fuller, N.; Rand, R. P.; Parsegian, V. A. Biophys. J. 1982, 37, 667–672.
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the formation of the bilayer, the solution in the cell was changed to the desired salt solution, and linearized plasmid DNA (4331 bp) was added. All additions of lipids and DNA were accomplished by injections of small volumes of concentrated stock solution into the bulk of the sample cell. Data Evaluation. The recorded ellipsometric angles were modeled on the basis of the assumption of isotropic media and planar interfaces within the framework of an optical fourlayer model (Si-SiO2-DOPC/DNA-solution).31 The adsorbed amount Γ was determined using the de Feijters formula38 for which the mean refractive index, nf, and the mean ellipsometric thickness, df, were calculated Γ ¼
ðnf - n0 Þdf dn=dc
ð1Þ
where n0 is the refractive index of the bulk (n0 = 1.3423) and dn/dc is the refractive index increment of the adsorbed material as a function of its bulk concentration (dn/dc = 0.148 g cm-3 for DOPC38).
Quartz Crystal Microbalance with Dissipation Monitoring (QCM-D). For the QCM-D experiments, we used a Q-Sense E4 (Q Sense), which was equipped with four thermally insulated liquid cells for measuring real-time kinetics with each cell containing a horizontally mounted Si crystal. The sensor crystals (Q-Sense, Gothenburg, Sweden) with a surface layer of SiO2 and a 5 MHz fundamental resonance frequency (QSX-303) were thoroughly cleaned before use as described by Vandoolaeghe et al.37 The crystals were stored in ethanol and dried under reduced pressure (0.02 mbar) followed by air plasma cleaning for 5 min prior to performing the experiments. During an experiment, the piezoelectric crystal is excited at its fundamental resonance frequency, and the difference in resonance frequency, Δf, and energy dissipation, ΔD, of a coated Si surface compared to that of a bare surface is measured at several harmonics simultaneously as a function of time.46 Continuous exchange of the bulk solution is achieved perpendicularly to the Si surface by means of a peristaltic pump (Ismatech, Z€ urich, Switzerland). All experiments were done at 22 C, and the temperature was controlled by a Peltier element. Experimental Procedure. The same experimental procedure as for ellipsometry was applied regarding the formation of a DOPC bilayer; see Supporting Information (Figure S1b). The average surface coverage reached was 85%, which corresponds to bilayers containing small defects. Salmon sperm DNA (2000 bp) was used as the DNA source and was circulated at a rate of 10 μL min-1. All steps were run for at least 20 min to ensure equilibrium conditions. Data Evaluation. The Sauerbrey equation gives a linear relation between the change in mass (Δm) and the shift in resonant frequency (Δf ) Δm ¼
C Δf on
ð2Þ
where C is 17.7 ng cm-2 Hz1- for a 5 MHz sensor crystal and depends on the thickness and intrinsic properties of the crystal.47 The overtone number is represented by on (3, 5, 7, and 13). Equation 2 is only valid when the adsorbed film is thin, rigid, €k et al.48 However, an and evenly distributed as discussed by H€ oo acoustically rigid film may also trap solvent, which becomes sensed as an additional mass through Δf. Hence, only in cases when the amount of coupled water is low, the “Sauerbrey mass” corresponds to the adsorbed molecular weight. For planar supported lipid bilayers, this is the case, as verified with other (46) Rodahl, M.; Hook, F.; Fredriksson, C.; Keller, C. A.; Krozer, A.; Brzezinski, P.; Voinova, M.; Kasemo, B. Faraday Discuss. 1997, 107, 229–246. (47) Sauerbrey, G. Angew. Phys. 1959, 155, 206–222. (48) Hook, F.; Kasemo, B.; Nylander, T.; Fant, C.; Sott, K.; Elwing, H. Anal. Chem. 2001, 73, 5796–5804.
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methods such as ellipsometry and neutron reflectometry.37 In the case of polynucleotide coupling to supported lipid bilayers, the situation is more complicated.49 The combined effect of high water content and the nonrigid character of DNA induce frictional loss and thus damping of the crystal oscillation. ΔD is for that reason simultaneously measured, since it provides structural information in terms of the viscoelastic properties of adsorbed films in real time. It should be noted, though, that as long as the damping is low, relative differences between different systems, e.g., DNA in the absence or presence of Ca2þ, can still be trusted with high accuracy. Changes in reference frequency from higher harmonics (n = 3, 5, ...) were normalized to the fundamental frequency (n = 1; 5 MHz). Neutron Reflectometry (NR). NR was performed at the National Institute for Standards and Technology (NIST) Center for Neutron Research, Gaithersburg, Maryland, USA, using the NG7 reflectometer operated at a fixed wavelength of λ = 4.75 A˚.34 The specular reflectivity from a flat surface is measured using a collimated neutron beam as a function of momentum transfer (Q) Q ¼ ð4π sin θÞ=λ
ð3Þ
where θ is the glancing angle of incidence and λ is the wavelength.50 When the angle of incidence of the incoming beam is changed (typically between 0 and 3), the magnitude of Q is varied. In order to correct for background scattering, i.e., small-angle scattering from the bulk, the off-specular scattering is also measured. In the resulting NR profiles, it is the ratio of the specular reflection of a neutron beam to that of the incident beam, obtained through a slit scan (reflectivity of the direct beam at varying slit width), that is displayed. The measurements were performed using a liquid cell holding 10 mL in which the Si block is horizontally arranged on top of a Teflon trough that contains the solution.51 The cell was connected to a syringe pump for efficient solution exchange, set to a constant temperature of 25.0 ( 0.1 C, and equipped with magnetic stirring. The Si substrates (5 cm 5 cm 1 cm) were single crystals of (111) crystal face that prior to the experiments had been polished (Siltronix, France) and cleaned as described by Vandoolaeghe et al.37 As a result of the interaction between a neutron and the nuclei of atoms, the scattering from hydrogen will be different from that of deuterium. This allows for contrast variation, and the surfaces were characterized in D2O and H2O, as well the water contrast corresponding to the scattering length density (F) of the Si block (cmSi), containing 61.9% H2O (by volume). The scattering length density of a specific material is expressed as F ¼
X
ni bi
ð4Þ
where n is the number of nuclei found in a given volume and b is the coherent scattering length.52 The F values of D2O, H2O, and cmSi are 6.34 10-6 A˚-2, -0.56 10-6 A˚-2, and 2.07 10-6 A˚-2, respectively. Experimental Procedure. The d-DOPC bilayers were deposited using the same experimental procedure as for QCM-D (Supporting Information). Bilayers were formed in D2O and further characterized in cmSi and H2O. All solutions were injected by means of disposable polypropylene syringes and 40 mL (4 cell volume) ensured exchange of the bulk solution during rinsing. When studying the interaction of DNA with the bilayer, linearized plasmid DNA (4331 bp) was injected in D2O and electrolyte. When changing the contrast, only a salt solution was used due to limited DNA availability. (49) Larsson, C.; Rodahl, M.; Hook, F. Anal. Chem. 2003, 75, 5080–5087. (50) Lu, J. R.; Thomas, R. K. J. Chem. Soc., Faraday Trans. 1998, 94, 995–1018. (51) Vandoolaeghe, P.; Rennie, A. R.; Campbell, R. A.; Nylander, T. Langmuir 2009, 25, 4009–4020. (52) Schurtenberger, P. In Neutrons, X-rays and Light: Scattering Methods Applied to Soft Condensed Matter, 1st ed.; Lindner, P., Zemb, T., Eds.; Elsevier: Amsterdam, 2002; pp 145-170.
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Data Evaluation. All NR profiles obtained in this study were simulated using the Motofit package,53 which uses the Abeles matrix method to calculate the reflectivity of thin layers and enables simultaneous least-squares fitting of the data sets (from measurements of different isotopic compositions). The fitting parameters for each layer are the thickness (d), the interfacial roughness that is related to the preceding layer in the model (ξ), and either F or the solvent volume fraction (φ) when the layer is composed of a single component and solvent. The bare surfaces were modeled according to a three-layer model (Si-SiO2solution) in which F of SiO2 is 3.41 10-6 A˚-2. Bilayers were modeled as three-layer structures consisting of the interior acyl chains (ACs) as well as the inner and outer headgroups (HGs); see Supporting Information (Figure S2 and Table S1).40,41 The scattering length densities of HGs and ACs agree with those previously reported for surface-deposited DOPC bilayers, 1.80 10-6 A˚-2 for HGs and 6.00 10-6 A˚-2 for ACs.51 The initial thickness and roughness values used for fitting were obtained from the same study for which the resulting total bilayer thickness also agrees well with the in situ null ellipsometry data; d = 7 A˚ and ξ = 2 A˚ for HGs, and d = 29 A˚ and ξ = 4 A˚ for ACs. We also note that the obtained thickness for a surface-deposited DOPC bilayer agree with DOPC bilayers in bulk solution for which TristramNagle et al. have reported that d = 27 A˚ for ACs and d = 9 A˚ for HGs.44 During fitting, the water distribution in each lipid monolayer was restricted to maintain identical mean molecular areas (A) for the HG and AC regions in order to obtain a physically realistic bilayer fit. This was achieved to within 2 A˚2 (the minimum value that allowed for global fitting of all data sets).40 The volumes of HGs and ACs were taken as 319 A˚3 and 984 A˚3 (per DOPC lipid), respectively, in accordance with Nagle et al.43 The resulting adsorbed amount was ∼2.2 mg m-2 and equals a surface coverage of ∼50%.41 A solvent layer of 3 A˚ was also found to be located between the bare surface and the inner lipid HGs. Cryo-Transmission Electron Microscopy (Cryo-TEM). Transmission electron micrographs were digitally recorded using a Philips CM120 Bio TWIN electron microscope, operated at 120 kV, equipped with a Gatan MSC791 cooled-CCD camera system. Since beam damage is resolution-limiting, all samples were imaged under minimal electron dose conditions. The cryo-TEM samples were prepared using a controlled environment vitrification system (CEVS),54 where relative humidity and temperature can be regulated. A 5 μL drop was placed on a lacey carboncoated copper grid, made hydrophilic using a Emitech glow discharge unit, and excess fluid was gently blotted away leaving a thin film of aqueous sample covering the grid. The grid was plunged into liquid ethane at -180 C to allow rapid vitrification of the specimen. All prepared grids were stored in liquid nitrogen until transfer to the EM. Experimental Procedure. Dry DOPC powder was hydrated using the salt solution of interest to a total concentration of 15 mM followed by vortexing. The lipid dispersion was repeatedly frozen using dry ice in ethanol and thawed at 30 C for ten cycles in order to produce multilamellar vesicles (MLVs). The lipid dispersion was then extruded ten times at room temperature through a 0.100 μm pore size filter (Minisart, Sartorius). Extruded vesicle dispersions were used for measurements within 12 h of preparation. The samples with DNA were prepared by adding the vesicles into equal volumes of linearized plasmid DNA (4331 bp) to a final lipid concentration of 7.5 mM. All samples were equilibrated in polypropylene containers at 25 C for at least 3 h.
(53) Nelson, A. J. Appl. Crystallogr. 2006, 39, 273–276. (54) Bellare, J. R.; Davis, H. T.; Scriven, L. E.; Talmon, Y. J. Electron Microsc. Technol. 1988, 10, 87–111. (55) Buttner, H. G.; Lelievre-Berna, W.; Pinet, F. The Yellow Book, Guide to Neutron Research Facilities at the ILL; Institut Laue-Langevin: Grenoble, 1997; pp 229-246.
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Small-Angle Neutron Scattering (SANS). SANS was carried out at the Institute Laue-Langevin, France, on the smallangle diffractometer D22.55 The D22 instrument has a 2-D area detector consisting of 128 128 cells, each measuring 8 8 mm2. In order to collect data over a wide Q-range (∼0.001-0.1 A˚-1), two detector distances were used, 3.5 and 17.5 m, and λ was 16 A˚. Experimental Procedure. All samples were prepared according to the previously stated cryo-TEM protocol with the exception that the sample preparation and measurement were done at 30 C in order to be above the chain melting temperature of the phospholipids used, DMPC and d54-DMPC. Measurements were performed using banjo-shaped quartz cells (Hellma, M€ ullheim, Germany) and the instrument aperture was 14 mm. The optical path length was 2 mm for samples containing 83% or 100% D2O and 1 mm for samples containing 100% H2O (by volume). Data Evaluation. The total intensity of a measurement is the product of a structure factor, S(Q), which contains information regarding interactions or correlations and a form factor, P(Q), which is related to the shape of the scattering objects IðQÞ ¼ NV 2 ðΔFÞ2 PðQÞSðQÞþB
ð5Þ
where N is the number concentration, V is their volume, and B is the incoherent background.52 The term ΔF is the difference in scattering length density between the scattering body and the surrounding media. The contrast match point of d54-DMPC was determined experimentally (data not shown) to be 83% D2O (by volume). This was achieved by calculating the D2O concentration at which the intensity of neutrons scattered in the forward direction goes to zero (I(0) = 0), in accordance with the approximation by Guinier et al.56 The obtained F of 5.17 10-6 A˚-2 agrees well with that calculated, 5.40 10-6 A˚-2.57 The scattering curves were corrected for nonuniform detector response by dividing the measurements with that of pure water displaying high incoherent scattering. The collected raw intensity data were then placed on an absolute scale (cm-1) with the knowledge of the solid angle and the primary beam intensity. The background noise was measured using the neutron absorber boron carbide (B4C) and subtracted from all data sets, and sample transmissions verified the used content of H2O/D2O. The scattering data were analyzed using Igor Macros made available by the SANS group at the NIST Center for Neutron Research, and the data sets (different isotopic composition) were simultaneously modeled as for NR.58
Results Surface-Deposited Lipid Bilayers. Adsorption of DNA from Solutions Containing Monovalent Naþ Cations. Figure 1a shows the adsorption of linearized plasmid DNA (4331 bp) to a deposited DOPC bilayer using in situ null ellipsometry (see Experimental Section) for a 10 mM NaBr solution. The surface excess (plateau value), Γ, of DNA was 0.8 mg m-2, and the recorded thickness, d, did not increase upon DNA addition. It is important here to note that data were evaluated using an optical model assuming a single homogeneous layer, and a adsorbed film of low density might therefore not be resolved. The adsorbed amount decreased upon rinsing using a solution containing 10 mM NaBr, indicating DNA desorption. Figure 1b shows the analogous results for salmon sperm DNA (2000 bp) interacting with a deposited DOPC bilayer obtained from QCM-D (see Experimental Section) for a 10 mM NaBr solution. The (56) Guinier, A.; Fournet, G.; Walker, C. B.; Yudowitch, K. L. Small-Angle Scattering of X-rays; John Wiley: New York, 1955. (57) Nakano, T. M.; Kudo, M.; Fukuda, T. H. Chem. Phys. Lipids 2008, 154, S27–S27. (58) Kline, S. R. J. Appl. Crystallogr. 2006, 39, 895–900.
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Figure 1. Effect of adding DNA to supported DOPC bilayers on silica surfaces from aqueous solutions containing 10 mM NaBr. DNA addition is indicated using solid lines, and dashed lines correspond to the start of the rinsing cycles. At t = 0, the bilayer formation is completed. (a) shows the adsorbed amount, Γ, (O) and film thickness, d, (b) as a function of time, t, obtained from ellipsometry. (b) shows the adsorbed amount, Δm, (O) and the difference in dissipation, ΔD, (b, for on = 13) as a function of time, t, obtained from QCM-D.
DNA surface excess was 0.7 mg m-2, which is in good agreement with the ellipsometry results for linearized plasmid DNA (4331 bp). The difference in energy dissipation, ΔD (on = 13), increased upon DNA adsorption, which indicates the formation of a structure that extends into solution. It was also noted that the kinetic evolution of ΔD was independent of harmonics used. Rinsing using an aqueous solution of 10 mM NaBr induced Γ to decrease, indicating removal of adsorbed DNA. Comparison among the different DNA species showed that the maximal Γ was not affected by doubling the DNA size, as complementary ellipsometry data using the shorter DNA molecule (2000 bp) showed identical plateau values (not shown). In Figure 2, the NR profile of DNA (4331 bp) interacting with a d-DOPC bilayer in D2O (corresponding to steady state in Figure 1) is compared to that of the corresponding bilayer profile. The critical edge shifts toward lower Q upon DNA addition and the reflectivity is higher at higher Q. The NR profiles in H2O and cmSi (not shown) were further identical to those of the bilayer itself (Supporting Information, Figure S2), which is explained by the fact that the exchange of bulk solution to a new contrast during rinsing, without DNA, causes desorption of DNA as observed using ellipsometry and QCM-D (Figure 1). The adsorbed DNA was modeled as a layer both on top of the bilayer (model 1) and located within the outer HGs of the bilayer (model 2). The starting values used for modeling the DNA-containing layer for both models 1 and 2 were d = 25 A˚,59 the known hydrated cross section of DNA, and ξ = 3 A˚. The previously reported DNA scattering length density in D2O was 4.1 10-6 A˚-2.60 The best fit, which is included in Figure 2, was obtained using model 2 for a three-layer structure separated into an inner layer of HGs, an intermediate region of ACs and an outer mixed layer consisting of HGs and adsorbed DNA. The D2O, H2O, and cmSi contrasts were simultaneously modeled, and the resulting fitting parameters are summarized in Table 1. To model the shift of the critical edge, F of the subphase was fitted to 5.17 10-6 A˚-2, which should be compared to 6.34 10-6 A˚-2 for pure D2O. This could indicate the presence of remaining hydrogen in the sample cell. Still, the shift of the critical edge was found to increase over time, which is not consistent with incomplete rinsing. It is interesting that the critical edge is known to be rounded for rough surfaces, and if the shift observed in this study is interpreted in the (59) Podgornik, R.; Rau, D. C.; Parsegian, V. A. Macromolecules 1989, 22, 1780–1786. (60) May, R. P. In Neutrons, X-rays and Light: Scattering Methods Applied to Soft Condensed Matter, 1st ed.; Lindner, P., Zemb, T., Eds.; Elsevier: Amsterdam, 2002; pp 463-480.
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Figure 2. NR (R) as a function of momentum transfer (Q) in a 10 mM NaBr solution of D2O for a d-DOPC bilayer (O) and a d-DOPC bilayer in the presence of DNA (b). Error bars were omitted due to clarity but were all smaller or equal to the marker size. Lines corresponds to the fits; see Table 1. Table 1. Parameters Obtained for Fits to NR Profiles of the DNA Adsorption to a d-DOPC Bilayer in 10 mM NaBr Solutions (D2O, H2O, and cmSi)a layer
d (A˚)
ξ (A˚)
F 10-6 (A˚-2)
φ (%)
A (A˚2)
HG 6 1 1.80 58 127 AC 29 1 6.10 46 125 HG/DNA 23 15 4.10 33 ---HG* 6 1 1.90 58 127 AC* 31 1 6.20 49 125 a A three-layer structure, separated into an inner layer of HGs, an intermediate region of ACs, and an outer layer consisting of HGs and adsorbed DNA, was used. The subphase F in the D2O contrast was fitted to 5.17 10-6 A˚-2 .The layer thickness is d, and the interfacial roughness is described by ξ. F is the scattering length density of the component, and the solvent content in the layer equals φ. A is the resulting mean molecular area including solvent. The asterisk, *, denotes the bilayer fit in the absence of DNA (taken from Supporting Information Table S1).
same way, this indicates a change of the film structure upon DNA addition to a model membrane.61 The fact that DNA is hydrogenated could also affect the subphase F, provided that enough DNA is close to the interface. The mean molecular area of the inner HGs and ACs are identical with that of the bilayer in the absence of DNA. The DNA surface excess was obtained by subtracting the adsorbed amount that corresponds to HGs in the (61) Harris, P. J.; Bayliss, S. C.; Bardrick, T.; Hillman, R.; Cubitt, R. J. Porous Mater. 2000, 7, 47–49.
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Figure 3. Effect of adding DNA to supported DOPC bilayers on silica surfaces from aqueous solutions containing 2.5 mM CaCl2. DNA addition is indicated using solid lines, and dashed lines correspond to the start of the rinsing cycles using solutions containing Naþ. At t = 0, the bilayer formation is completed. (a) shows the adsorbed amount, Γ, (O) and film thickness, d, (b) as a function of time, t, obtained from ellipsometry. (b) shows the adsorbed amount, Δm, (O) and the difference in dissipation, ΔD, (b, for on = 13) as a function of time, t, obtained from QCM-D.
DNA/HG layer and using the DNA molecular volume that was estimated from the contour length, 1.47 μm, and cross section, 25 A˚.59 The calculated DNA surface excess of ∼0.7 mg m-2 agrees with that from ellipsometry and QCM-D. The thickness value for the DNA-containing layer, obtained from the NR data, is lower than the value for the known DNA molecular cross section. We ascribe this underestimation of the DNA film thickness to the slab model used for analysis, which assumes a homogeneous layer and does not take into account the conformation and distribution of the adsorbed DNA segments. This is in agreement with the fact that ellipsometry did not resolve an increased film thickness upon DNA addition. Adsorption of DNA from Solutions Containing Divalent Ca2þ Cations. Figure 3 displays the results from both ellipsometry (Figure 3a) and QCM-D (Figure 3b) for the adsorption of DNA to model membranes in the presence of Ca2þ. The increase in Γ upon DNA addition varies depending on technique and was measured to be 0.5 mg m-2 with ellipsometry and 1.8 mg m-2 with QCM-D. The fact that QCM-D displays a higher value than ellipsometry is a consequence of the fact that the ellipsometer measures the “dry” mass while QCM-D measures the adsorbed component including coupled water (wet mass). DNA adsorption is also accompanied by an increase of the film thickness, d (from ellipsometry measurements), by 10 A˚ and ΔD (from QCM-D measurements, on=13). The kinetic evolution of ΔD was independent of harmonics used. The adsorbed amount decreased upon rinsing using Naþ, and ellipsometry indicates that all DNA is removed, while QCM-D suggests that a fraction still remain on the bilayer surface. We note that the results do not depend on whether the CaCl2 concentration is 1 mM or 2.5 mM. The data in the presence of Ca2þ should also be compared with the observations for Naþ solutions. Similar values for ΔD, indicating extending DNA conformations, and Γ were obtained, but DNA adsorption from solutions containing Naþ resulted in an apparent zero increase in d and lower Δm. In Figure 4, the NR profile in D2O of DNA interacting with a d-DOPC bilayer in a solution containing 2.5 mM CaCl2 is compared to that of the corresponding bilayer profile. DNA adsorption was followed over time, and the NR profile displayed is the one at steady state. The critical edge shifts toward lower Q upon DNA addition, but the effect is not as pronounced as when DNA is added from 10 mM NaBr solutions. The observed reflectivity at higher Q is also higher compared to that of the bare bilayer. After rinsing to change the contrast, the NR profiles were identical to those of the bilayer in the absence of DNA. This 4970 DOI: 10.1021/la9036327
Figure 4. NR profiles in D2O and 2.5 mM CaCl2 for a d-DOPC bilayer (O) and a d-DOPC bilayer in the presence of DNA (b). Error bars were omitted due to clarity but were all smaller or equal to the marker size. Lines correspond to the fits; see Table 2.
is in accordance with the removal of DNA upon rinsing that was observed for the 10 mM NaBr solution as well as for both Naþ and Ca2þ using ellipsometry and QCM-D. The NR profiles were modeled using the same approach as for the adsorption of DNA from an aqueous solution of 10 mM NaBr. The best fit, which is included in Figure 4, corresponds to model 2 for a three-layer structure separated into an inner layer of HGs, an intermediate region of ACs, and an outer mixed layer consisting of HGs and adsorbed DNA. The fitting parameters for the bilayer and DNA are summarized in Table 2, and the resulting thickness for the AC and the inner HG regions agree with the bilayer fit in the absence of DNA (Supporting Information, Table S1). The DNA in the DNA/HG layer corresponds to a surface excess of ∼0.5 mg m-2, and QCM-D, which includes bound solvent into its readings, is concluded to display a higher surface excess compared to both NR and ellipsometry. The thickness of the DNA-containing film agrees with ellipsometry and suggests that a thin film of DNA is located on top of the membrane. In accordance with the measurements in Naþ solutions, the shift of the critical edge was modeled by fitting F of the D2O subphase to 5.82 10-6 A˚-2. The critical edge was only observed to shift in the presence of DNA, which agrees with the Naþ solutions and suggests that the shift indicates the presence of DNA also further from the bilayer surface. Effect of Calcium on the Lipid Bilayer. To monitor the properties of zwitterionic lipid bilayers in the presence of monovalent or divalent salt, we recorded the corresponding change in Δm (QCM-D) and Γ (ellipsometry). An uptake in mass in the Langmuir 2010, 26(7), 4965–4976
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Table 2. NR Fit Parameters of the DNA Adsorption to a d-DOPC Bilayer in Solutions Containing 2.5 mM CaCl2 (D2O, H2O, and cmSi)a layer
d (A˚)
ξ (A˚)
F 10-6 (A˚-2)
φ (%)
A (A˚2)
HG 6 1 1.80 58 127 AC 29 1 6.20 46 125 HG/DNA 16 16 3.60 25 ---a The same three-layer structure as for bilayers in Naþ solutions was used. In addition, F of the subphase was fitted to 5.82 10-6 A˚-2 in the D2O contrast. The parameters included in the model are defined in the footnote of Table 1.
presence of CaCl2 was observed using QCM-D; see Supporting Information (Figure S3). The surface excess corresponding to associated divalent cations equals the amount of one Ca2þ ion per two lipid molecules. Upon rinsing using solutions of 10 mM NaBr, the divalent ions were removed (decreased Δm). The effect on Γ by addition of salt was below the detection limit for ellipsometry (data not shown). Lipid Vesicles. Vesicle Characterization. Figure 5 displays cryo-TEM images of DOPC vesicles in solutions containing 10 mM NaBr (a,b) and 2.5 mM CaCl2 (c,d). The mean vesicle radius was 650 ( 50 A˚, independent of salt concentration and in agreement with using 0.1 μm pore size filters for extrusion. Unilamellar vesicles dominate the samples, but MLVs were detected as well, and most of them were observed in the 10 mM NaBr solution. The largest vesicles were the ones with two or more lamellae (Figure 5b). For the MLVs in 10 mM NaBr solutions, the interlamellar repeat distance was measured to be 66 ( 5 A˚ from the images, which agrees well with the reported interlamellar repeat distance of 63 A˚ at 30 C for DOPC.44 Too few MLVs were observed in the presence of divalent Ca2þ cations to quantitatively state a repeat distance. Figure 5d displays, however, vesicles (dark circles) that cover the carbon support film. Due to the low contrast on the carbon film, it is difficult to determine the number of lamellae and the size of the vesicles located on the support film, and only the vesicles found in the vitrified film in the holes of the grid (Figure 5c) were measured when determining the average radius. Vesicles on the carbon support film were only observed in the presence of CaCl2, and are ascribed to the use of glowdischarged cryo-TEM grids which are hydrophilic and negatively charged to ease the spreading from aqueous suspensions. Electrostatic attractive interactions between the vesicles and the negatively charged carbon support film, which can only occur if the vesicle appears cationic, induces adsorption of the lipids film.62 We suggest that it is the introduction of the charge that decreases the probability for MLVs. The charge may further vary over the grid, as observed in Figure 5c,d. We note that MLVs can form after extrusion as a consequence of the fact that a vesicle is metastable and not in thermodynamic equilibrium and the shearing forces involved in the blotting procedure when preparing samples for cryo-TEM can cause distortions in the nanostructures (Danino et al.63). Figure 6 shows SANS data obtained for DMPC and d54DMPC vesicles at 30 C in aqueous solutions containing 10 mM NaBr (a) and 2.5 mM CaCl2 (b). No good fit of the experimental data recorded in 10 mM NaBr solutions was obtained using a size-averaged scattering function according to Bartlett et al.64 (62) Lis, L. J.; Lis, W. T.; Parsegian, V. A.; Rand, R. P. Biochemistry 1981, 20, 1771–1777. (63) Danino, D.; Talmon, Y. In Molecular Gels - Materials with Self-Assembled Fibrillar Networks; Weiss, R. G., Terech, P., Eds.; Springer: Dordrecht, 2006; pp 253-274. (64) Bartlett, P.; Ottewill, R. H. J. Chem. Phys. 1992, 96, 3306–3318.
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Figure 5. Cryo-TEM images of extruded DOPC vesicles, the model membranes applied in bulk solutions, containing 10 mM NaBr (a,b) and 2.5 mM CaCl2 (c,d). Scale bars are 100 nm.
However, an excellent fit was obtained when applying the same model to the vesicles in Ca2þ solutions: ( 2 16π2 2 Zþ2 2 F ðxÞ ¼ 6 Fshell -Fcore c1 þ c2 x þ c3 x Zþ1 Q þ BðxÞðxþ1Þ=2 ðc4 cos½ðZ þ 1ÞDðxÞ þ c7 sin½ðZ þ 1ÞDðxÞÞ þ xBðxÞðxþ2Þ=2 ðc5 cos½ðZ þ 2ÞDðxÞ þ c8 sin½ðZ þ 2ÞDðxÞÞ Zþ2 2 þ x BðxÞðxþ3Þ=2 ðc6 cos½ðZ þ 3ÞDðxÞ Zþ1 ) þ c9 sin½ðZ þ 3ÞDðxÞÞ
ð6Þ
which corresponds to a form factor for spherical core-shell particles of constant shell thickness and polydisperse core radius with a Schulz distribution. Fshell and Fcore describe the scattering length density of the shell and the core, respectively, Z is related to the normalized second moment (or polydispersity) of σc, the particle core radius distribution, and rcore is the mean core radius ! r2 core 1 2 x ¼ Qrcore , σ core ¼ 2 -1 ¼ ð7Þ Zþ1 r core The functions B(x) and D(x) are given by BðxÞ ¼
ðZ þ 1Þ2 2
ðZ þ 1Þ þ 4x
2
,
DðxÞ ¼ tan -1
2x ðZ þ 1Þ
ð8Þ
and the expressions for the coefficients c1-c9 can be obtained from Bartlett et al.64 Four out of eight variables were considered independent of contrast; the volume fraction of the scattering objects (φULV), rcore, σcore, and the shell thickness, i.e., the bilayer thickness (dbil). The volume fraction was estimated to 0.015 when the vesicle radius is 650 A˚ (cryo-TEM) and the lipid loss during extrusion corresponds to 50% (final lipid concentration of 3.75 mM). The remaining four variables were not independent of the contrast: the incoherent background B, Fcore, Fshell, as well as DOI: 10.1021/la9036327
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Figure 6. SANS spectra showing the intensity as a function of momentum transfer (Q) for extruded DMPC and d54-DMPC vesicles in solutions containing 10 mM NaBr (a) and 2.5 mM CaCl2 (b). The three contrasts displayed are DMPC in D2O (O), DMPC in 83% D2O (b), and d54-DMPC in D2O ()). The best fit in solutions containing Ca2þ was obtained using the spherical core-shell model resembling ULVs, and the best fit in solutions containing Naþ was obtained by combining the ULV model with a MLV model. Error bars were omitted due to clarity but were all smaller or equal to the marker size, and the parameter values for the best fits are shown in Table 3. Table 3. Parameters Obtained for Fits to SANS Data of DMPC and d54-DMPC Vesicles in 10 mM NaBr (ULVs and MLVs) and 2.5 mM CaCl2 (ULVs) Solutions, Respectivelya ULVs φULV
rcore (A˚)
MLVs σcore
dbil (A˚)
φMLV
rcore (A˚)
σcore
dbil (A˚)
dwater.(A˚)
N
0.012 505 0.36 40 0.002 360 0.37 40 23 4 a The data from vesicles in Ca2þ solutions were modeled using a form factor for a ULV, i.e., a spherical core-shell particle with constant shell thickness and polydisperse core radius having a Schulz distribution. The data from vesicles in Naþ solutions were fitted using a combined model consisting of the ULV form factor and a similar MLV model. The volume fraction of ULVs is given by φULV, and φMLV states the volume fraction of MLVs. The core radius and polydispersity index is given by rcore and σcore, respectively. The thickness of the shell (the bilayer) is dbil. dwater is the thickness of the water film, and N is the number of shells surrounding the core.
the solvent scattering length density, Fsolvent, where Fsolvent = Fcore. Table 3 displays the best ULV fit where the radius is equal to the sum of dbil and rcore. The difference in the radii obtained using cryo-TEM (650 A˚) and SANS (545 A˚) is most probably explained by the fact that different PC LR lipid systems were used. In addition, the shear associated with cryo-TEM sample preparation complicates a quantitative comparison. The φULV is slightly lower than the theoretical value as explained by lipid loss during extrusion, as well as the fact that the radius used for estimating the volume fraction corresponds to DOPC and was obtained from cryo-TEM. It should also be noted that the Guinier approximation applied when determining F for d54-DMPC is only valid for Q e Rg-1,65 which for the known radius obtained from cryo-TEM equals Q e 0.0015. However, the linear relationship indicates that the extrapolation procedure shows the correct functional dependence on the solvent contrast level even if only a few data points were recorded. To obtain good agreement with the experimental data for the NaBr solution, the ULV fit from solutions containing divalent Ca2þ had to be combined with the corresponding core-shell model extended to simulate MLVs through the addition of two variables (independent of contrast):58 the thickness of the water layer (dwater) and the number of shell layers surrounding the core (N). The addition of a fraction of MLVs to the SANS model is in agreement with the fact that more MLVs were observed using cryo-TEM in the presence of a monovalent salt compared to in a divalent salt. The volume fraction of MLVs was optimized by the scale factor (65) Feigin, L. A.; Svergun, D. I. Structure Analysis by Small-Angle X-ray and Neutron Scattering; Plenum Press: New York, 1987.
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φMLV (see Table 3). The scale factor obtained for 10 mM NaBr solutions (φULV þ φMLV) agrees well with the value found for solutions containing Ca2þ (φULV). The bilayer (dbil) and water (dwater) thickness of the MLVs obtained from SANS on the LR phases of DMPC and d54-DMPC was 40 A˚ and 23 A˚, respectively, and is in agreement with previously reported values.66,67 The number of lamellae (N) and the interlamellar spacing agrees further with the cryo-TEM images of the DOPC vesicles (Figure 5). σcore as well as dbil were independent of electrolyte. Adsorption of DNA from Solutions Containing Monovalent Naþ Cations. Figure 7a,b shows cryo-TEM images of DNA and DOPC vesicles in 10 mM NaBr solutions. The vesicles are confined in clusters where neighboring vesicles have flat contact areas. This was not observed in the absence of DNA. The average vesicle size could not be determined unambiguously due to vesicle deformation. Many of the vesicles observed are unilamellar, although the fraction of MLVs is higher compared to the situation in which no DNA is present. Some highly condensed vesicles are also observed (see white star in Figure 7b). The black arrows point to radiation-damaged areas in the cryo-TEM images, which could indicate the presence of DNA, as DNA is radiation-sensitive.68,69 As a result of that beam damage was only visible between (66) Kucerka, N.; Kiselev, M. A.; Balgavy, P. Eur. Biophys. J. 2004, 33, 328–334. (67) Kucerka, N.; Liu, Y. F.; Chu, N. J.; Petrache, H. I.; Tristram-Nagle, S. T.; Nagle, J. F. Biophys. J. 2005, 88, 2626–2637. (68) Rosa, M.; Miguel, M. D.; Lindman, B. J. Colloid Interface Sci. 2007, 312, 87–97. (69) Wasan, E. K.; Harvie, P.; Edwards, K.; Karlsson, G.; Bally, M. B. Biochim. Biophys. Acta, Biomembr. 1999, 1461, 27–46.
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Figure 7. Cryo-TEM images of extruded DOPC vesicles in the presence of DNA in 10 mM NaBr solutions. Black arrows in (a) and (b) shows areas of high brightness and indicate the presence of DNA located between lamellae. Inset in (a) displays a section of the image at higher magnification. In (b), the white arrow indicates crystalline ice, and the white star shows the presence of multilamellar vesicles in which DNA is not believed to be present. Scale bars are 100 nm.
Figure 8. Cryo-TEM images of extruded DOPC vesicles in the presence of DNA and 1 mM (a,b) as well as 2.5 mM (c,d) CaCl2. Inset in (a) shows a magnification where the black arrows points at a DNA coil. White arrow in (a) indicates an ice crystal, and the black arrow points at another DNA molecule. Scale bars are 500 nm in (c) and 100 nm in (a), (b), and (d).
neighboring vesicles, for which the average interlamellar repeat distance is 81 ( 5 A˚; DNA is indicated to be located between the lamellae. The repeat distance within the MLVs is identical to that of DOPC in the absence of DNA (∼65 A˚), and we suggest that DNA is not present in the highly condensed lamellar vesicles.24,44 A good fit of the DMPC and d54-DMPC vesicles in the presence of DNA and monovalent salt was obtained using the model for the vesicles alone in a solution of Naþ (Supporting Information, Figure S5). This implies that no association between DMPC and DNA can be detected using SANS under the conditions used. This can be due to experimental limitations, and it seems more appropriate to study the adsorption of DNA to zwitterionic lipids using surface-deposited bilayers. We note that SANS data of DNA alone also were acquired and fitted using a power-law model in which a flat background was included (Supporting Information Figure S4, Table S2).58 The scattering from free DNA coils in solution was subtracted from the scattering from DMPC and d54-DMPC vesicles in the presence of DNA and 10 mM NaBr. This did, however, not change the resulting scattering pattern, which indicates that the scattering contribution from free DNA can be neglected (for the conditions used). Adsorption of DNA from Solutions Containing Divalent Ca2þ Cations. Figure 8 displays cryo-TEM images of DOPC vesicles in the presence of DNA and 1 mM CaCl2 (a,b) and Langmuir 2010, 26(7), 4965–4976
2.5 mM CaCl2 (c,d). Large clusters are observed, and the largest are found in solutions containing 2.5 mM CaCl2, which sometimes cover the entire carbon grid holes (Figure 8c). As was also the case for the 10 mM NaBr solution above, it is impossible to quantitatively state the effect of DNA on the vesicle size. However, the comparison shows that more bilamellar and highly condensed MLVs are present after the addition of DNA for solutions containing Ca2þ (Figure 8d). The mean repeat distance of the bilayers, which was independent of CaCl2 concentration, was measured to 79 ( 7 A˚, which indicates that DNA, with a diameter of 25 A˚, is present between the lamellae. Compared to the data in 10 mM NaBr solutions, one population of lipid-DNA structures is further formed, and more DNA is suggested to interact with the lipids. Black arrows in the magnified section of Figure 8a show a DNA strand that is confined between two neighboring vesicles. The additional black arrow in Figure 8a indicates another DNA strand, and the white arrow points at an ice crystal. No free DNA segments are observed in 2.5 mM CaCl2 solutions. It should be noted that sample vitrification occurred far from equilibrium and the structures observed are still evolving. SANS data obtained for DMPC and d54-DMPC vesicles in the presence of DNA in 2.5 mM CaCl2 solutions are shown in Figure 9. The best fit, which is listed in Table 4, corresponds to a concentrated lyotropic LR phase where the form factor and DOI: 10.1021/la9036327
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Ainalem et al. Table 4. Parameters Obtained for Fits to SANS Data of DMPC and d54-DMPC Vesicles in the Presence of DNA and 2.5 mM Ca2þa φLam
d (A˚)
dbil (A˚)
σbil
η
N
0.011 520 83 0.34 0.75 3 a The model was a combined form and structure factor for a concentrated LR phase in which the lamellae had polydisperse thickness and uniform scattering length density.The scale factor φLam states the volume fraction of lamellae, d is the lamellar spacing, and dbil is the thickness of the bilayer in the presence of DNA. The polydispersity of the bilayer thickness is σbil, η is the Caille ordering parameter, and N is the number of lamellae.
for cryo-TEM and 545 A˚ for SANS) and with the cryo-TEM observation for DNA and vesicles in 2.5 mM Ca2þ solutions where the vesicles (of high size polydispersity) are shown to be tightly packed in large clusters.
Discussion Figure 9. SANS spectra showing the intensity as a function of momentum transfer (Q) for extruded DMPC and d54-DMPC vesicles in the presence of DNA and 2.5 mM Ca2þ. The contrasts displayed are DMPC in D2O (O), DMPC in 83% D2O (b), and d54DMPC in D2O ()). Lines correspond to the best fit and are obtained by modeling a lamellar lyotropic phase; see Table 4. Error bars were omitted due to clarity but were all smaller or equal to the marker size.
structure factor are given by i 2ΔF2 h -q2 δ2 =2 1 -cosðQd Þ e SðQÞ bil Q2 N -1 X n Qdn cos 1¼ 1þ2 N 1 þ 2ΔQ2 d 2 RðnÞ 1 ! 2Q2 d 2 RðnÞ þ ΔQ2 d 2 n2 1 pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi exp 2 2 2ð1 þ 2ΔQ d RðnÞÞ 1 þ 2ΔQ2 d 2 RðnÞ PðQÞ ¼
ð9Þ
where N is the number of lamellae, d is the repeat spacing, dbil is the bilayer thickness, ΔQ is the instrumental resolution, and δ is the variation in bilayer thickness.70 The lamellae have a uniform scattering length density and a polydisperse thickness, and the Caille parameter, which describes the lamellar membrane rigidity, is η ¼
Q2 0 kB T η pffiffiffiffiffiffiffiffiffi RðnÞ ¼ 2 lnðπnÞ þ γE 4π 8π KM
ð10Þ
where kB is the Boltzmann constant, T is the temperature, K is the smectic bending elasticity, M is the compression modulus, and γE is Eulers constant. The fitted F of the bilayer is dependent on contrast; 5.20 10-6 A˚-2 for d54-DMPC in H2O, 1.73 10-6 A˚-2 for DMPC in D2O, and 1.50 10-6 A˚-2 for DMPC in 83% D2O. δ are within experimental error and N and η were set to agree with the cryo-TEM images and with the use of zwitterionic LR-phase phospholipids. The scale factor of the total intensity (φLam) is related to the concentration of lamellae and agrees with the previous estimation of the lipid volume fraction after extrusion. The scattering from free DNA is further negligible, in agreement with the observation in the 10 mM NaBr solution. The fitted bilayer spacing was 520 A˚ and is in fair agreement with the obtained radius of the vesicles in the absence of DNA (650 A˚ (70) Nallet, F.; Laversanne, R.; Roux, D. J. Phys. II 1993, 3, 487–502.
4974 DOI: 10.1021/la9036327
Zwitterionic Phospholipids Become Cationic in the Presence of Calcium. From the data presented in this paper, we can make comparisons of LR PC bilayers in the presence of monovalent and divalent cations. Our Cryo-TEM and SANS results show that the vesicle morphology is different in the presence of divalent compared to monovalent ions. The fact that the formation of MLVs appears more frequent for the Naþ solution is ascribed to the association between Ca2þ and HGs which increases the repulsion between neighboring lipid lamellae. Uhrikova et al. have also shown spontaneous formation of DPPC ULVs of LR phase in the presence of Ca2þ.71 We also observed that zwitterionic PC vesicles adsorb to the negatively charged lacey carboncoated copper grid used for cryo-TEM (in the presence of Ca2þ). Our observations agree with previous studies where the interbilayer separation has been shown to increase in the presence of divalent cations as a result of the fact that the ions turned zwitterionic lipid HGs cationic.62,72 In this study, we also reported, using QCM-D (Supporting Information Figure S3), that Ca2þ associates with a surface-deposited lipid bilayer. It is interesting to note that the observed PC:Ca2þ ratio of 2:1 is in perfect agreement with the model proposed by Brezesinski et al. and R€adler et al. that one Ca2þ cation associates with two lipid HGs.73,74 Their conclusion was based on an increased lipid ordering which was ascribed to a reorientation of the lipid HGs to expose their positive charges to the bulk solution. However, these observations by Brezesinski et al. and R€adler et al. were made in the presence of DNA, and it was proposed that divalent cations do not turn zwitterionic lipids cationic in the absence of DNA.74 On the basis of our data, it appears as the Ca2þ-lipid association is similar whether DNA is present or not. DNA Adsorbs to Phospholipid Bilayers Even in the Absence of Divalent Salt. We have with three different and complementary techniques (ellipsometry, QCM-D, and NR) clearly shown that DNA adsorbs to surface-deposited bilayers from aqueous solutions containing 10 mM NaBr and no multivalent ions. The combination of these techniques can further provide insight into the molecular conformation of DNA at the bilayer surface. The QCM-D data showed a marked increase in ΔD upon DNA adsorption, which implies extended conformations (71) Uhrikova, D.; Teixeira, J.; Lengyel, A.; Almasy, L.; Balgavy, P. Spectrosc. Int. J. 2007, 21, 43–52. (72) Lis, L. J.; Parsegian, V. A.; Rand, R. P. Biochemistry 1981, 20, 1761–1770. (73) Gromelski, S.; Brezesinski, G. Langmuir 2006, 22, 6293–6301. (74) McManus, J. J.; Radler, J. O.; Dawson, K. A. J. Phys. Chem. B 2003, 107, 9869–9875.
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of the adsorbed molecules. However, even if ellipsometry data gave a significant increase in mass, the technique could not resolve a corresponding increase of the film thickness upon DNA addition. This is commonly observed for highly hydrated films when the refractive index contrast is too low to independently resolve thickness and refractive index. Again, the most likely explanation for this is that the adsorbed DNA retains an extended coil conformation and only forms a few direct contacts with the bilayer. The NR data also suggest that the layer containing DNA is rougher than before adding the DNA. However, the layer measured by NR is very thin, and the solvent content in this thin film of DNA segments next to the surface of the bilayer is high. It should be noted here that a good fit to the NR data could not be obtained without changing the F of the bulk. This would account for the presence of DNA further from the surface, an extended DNA conformation, which the multilayer model we used cannot easily take into account. On the basis of the combination of these observations from all three complementary techniques, and the fact that the adsorbed amount agrees for all techniques (0.7-0.8 mg m-2), we conclude that the adsorbed DNA conformation is extended and that the DNAfilm is dilute. When studying the same system using vesicles, no adsorption of DNA was detected using SANS. The clusters of vesicles that were observed using cryo-TEM were large and would only scatter at very low Q and were therefore not detected using SANS. The fact that cryo-TEM observed a higher fraction of MLVs in the presence of DNA agrees with similar studies using cationic lipids where DNA has been shown to intercalate between the lamellae (see, for example, a review by Alfredsson et al.75). Here, we note, however, that the interlamellar spacing within an MLV is smaller (same as for DOPC alone) than between neighboring vesicles in clusters. In the discussion, we therefore need to distinguish between the interlamellar layers within the MLVs and the interlamellar layer in between neighboring vesicles within clusters, where the latter is believed to incorporate DNA. For the interlayer spacing of 81 A˚ between neighboring vesicles (as determined using cryo-TEM), the aqueous space is ∼39 A˚ if the bilayer is assumed to have a thickness of 42 A˚ (as for the surface-deposited DOPC bilayers). This is enough to incorporate a DNA monolayer of 25 A˚ (in the presence of a surrounding hydration shell).59 The aqueous space in the presence of DNA should be compared to the corresponding thickness of (63 - 42 =) 21 A˚ for the vesicles in the absence of DNA. These results are in good agreement with data by Wu et al. who proposed that a limited DNA fraction bind electrostatically to DOPC vesicles and unbound DNA induce aggregation into MLVs.28 Comparisons with previous X-ray data also agree with the cryo-TEM results, and Pott et al. state that the interlamellar repeat distance for a zwitterionic fully hydrated LR phase in the absence of divalent ions increases from 62.8 A˚ to 75.8 A˚ upon DNA addition.27 Another study shows the presence of two lamellar populations with repeat distances of 64.5 A˚ and 77.8 A˚ in mixtures of DNA and DOPC, where it was suggested that DNA was present in the larger spacing regions.24 When analyzing cryo-TEM data, it is important to know that the film of the sample is thickest close to the carbon grid.76 Large vesicles (globular structures) and vesicle clusters would therefore possibly only fit in areas of thicker films. Free DNA can, however, fit where films are thinner. (75) Alfredsson, V. Curr. Opin. Colloid Interface Sci. 2005, 10, 269–273. (76) Almgren, M.; Edwards, K.; Karlsson, G. Colloids Surf., A 2000, 174, 3–21.
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DNA Adsorption from Solutions with Monovalent Naþ Ions Is Similar, but the DNA Conformation Is More Extended than from Solutions with Divalent Ca2þ. In the CaCl2 solution, the QCM-D measurements show higher adsorbed amounts of DNA to PC bilayers compared to the adsorption measured by ellipsometry and NR. The combination of these data therefore indicates that the bound water content, which is included in the recorded mass using QCM-D, is substantial and should not be neglected. Still, the increase in the layer thickness obtained using NR and ellipsometry, which corresponds to the presence of DNA, is lower than the value of the DNA cross section. This indicates that the thickness of the DNA film is underestimated due to the slab model, assuming a stack of homogeneous layers, used for evaluating the data. Compared to Naþ solutions, we observed a significant increase in thickness, as recorded by ellipsometry, and a higher fraction of bound solvent, as recorded by QCM-D, for the addition of DNA in the presence of Ca2þ. The change in dissipation using QCM-D and the loss of the critical edge using NR was, however, similar, which indicates extended DNA structures for both divalent and monovalent salt solutions. While DNA compaction has previously been reported in the presence of high amounts of divalent ions,77 we note that, already at concentrations of Ca2þ ions comparable to those used in this study, Zhang et al. has proposed that an attractive force arises within the DNA molecule.78 This force gives rise to a reduction of the DNA extension (compared to in monovalent salt) without resulting in DNA compaction. For the adsorbed DNA in this study, the plausible presence of smaller DNA loops therefore suggests that the DNA adopts a flatter conformation in solutions containing divalent ions compared to monovalent ions. In DNA-lipid samples containing Ca2þ, cryo-TEM displayed large vesicle clusters, which indicates that DNA associates with zwitterionic lipids extensively in the presence of divalent salt. The flat contact areas that were observed in between vesicles may be explained within the schematic model for the formation of lipoplexes as postulated by Weisman et al. for cationic lipids, where intervesicle adsorption is shown to be mediated by DNA.79 The lipoplex formation is reported to be a consequence of partial charge neutralization causing aggregation and eventually membrane rupturing. When Ca2þ associates with zwitterionic lipid HGs, the lipid ordering increases and the lipid is thought to behave like a cationic lipid.22,73,74,80 When considering the cryoTEM images, it is not surprising that the SANS model fails to perfectly describe the data in the presence of divalent salt. The deviation between the fit and the observed minima is explained by the polydispersity within the samples. The divergence at very low Q is explained by the scattering from the clusters or large MLVs, which are not taken into account in the model used for fitting the data. The data at higher Q, however, agree well with the cryoTEM observations of polydisperse lamellar phased structures in which DNA is intercalated between bilayers. We conclude that the vesicles can respond more readily to the presence of DNA compared to the deposited bilayers, and that this difference lies in the fact that vesicles are more dynamic structures. This is also in agreement with a study by Caracciolo et al. which showed that surface-deposited preformed aggregates of DNA, MLVs, and (77) Hackl, E. V.; Kornilova, S. V.; Blagoi, Y. P. Int. J. Biol. Macromol. 2005, 35, 175–191. (78) Zhang, C.; Zhang, F.; van Kan, J. A.; van der Maarel, J. R. C. J. Chem. Phys. 2008, 128. (79) Weisman, S.; Hirsch-Lerner, D.; Barenholz, Y.; Talmon, Y. Biophys. J. 2004, 87, 609–614. (80) Gromelski, S.; Brezesinski, G. Phys. Chem. Chem. Phys. 2004, 6, 5551– 5556.
DOI: 10.1021/la9036327
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divalent cations displayed similar multilayered structures as those observed in bulk solution where DNA was located between lipid bilayers.81 Our findings are also consistent with the reports that DNA promotes a higher fraction of MLVs in aqueous solutions containing CaCl2 as compared to NaBr. Hydrophobic and Electrostatic Interactions Are Two Possible Mechanisms for the Interaction Between DNA and PC Lipids in Solutions Containing Naþ. The observed attractive interaction between DNA and zwitterionic phospholipid bilayers in the absence of multivalent salt is concluded to be significant but weak, since, for example, the flow used for rinsing is enough to induce removal of the adsorbed DNA. Multiple characterization techniques have therefore been needed to demonstrate adsorption. It is possible that the driving force for the adsorption of DNA in monovalent salt solutions could be electrostatic as a result of the fact that the lipid HGs become polarized in the vicinity of a negatively charged DNA molecule. This agrees with what has previously been proposed in experimental studies27-29 and in a recent theoretical study by Mengistu et al. where the interaction between DNA and zwitterionic LR phase bilayers was modeled in the presence and absence of divalent cations.82 Mengistu et al. showed that, even though divalent ions enhanced the interaction by being redistributed from the DNA (before binding) to the HGs (after binding), thereby leading to reorientation of the HGs, the adsorption free energy was not zero in the absence of divalent ions. Favorable adsorption was observed for salt contents that reduced the lipid perturbation of the DNA’s counterion cloud. The extended coillike conformation of adsorbed DNA proposed in this study could then be the result of the inter-repulsive DNA interactions. The stronger electrostatic attraction between DNA and the bilayer in Ca2þ solutions implies a flatter DNA conformation at the bilayer surface as compared to Naþ solutions, which is also consistent with the present data. Still, DNA can adopt a conformation where loops are protruding also in solutions containing CaCl2, as was suggested from the cryo-TEM studies. When comparing the cryo-TEM images recorded in the presence of CaCl2 with those in 10 mM NaBr solutions, the interlamellar spacing is reduced in solutions containing Ca2þ (79 81 = -2 A˚). A similar result was reported by Uhrikova et al. who determined the repeat distance of DOPC in the presence of DNA and 12.75 mM CaCl2 to be 74.4 A˚, which should be compared to the value of 77.8 A˚ in the absence of divalent salt.24 Another source of attraction that might play a role for DNA adsorption to PC bilayers is hydrophobic interactions between the (interior) bases of the DNA double helix and the ACs of the bilayer. Indeed, the best fits obtained for the NR data could not resolve the individual layers of outer HGs and DNA. Cardenas et al. have reported that the adsorption of DNA to polystyrene particles of a net negative charge is hydrophobically driven.83,84 Interestingly, Peyrard et al. have further shown that thermal fluctuations cause the lifetime of a base pair, i.e., the time during which it stays closed, to be only on the order of a few milliseconds even at temperatures well below the melting transition.85,86 (81) Caracciolo, G.; Sadun, C.; Caminiti, R.; Pisani, M.; Bruni, P.; Francescangeli, O. Chem. Phys. Lett. 2004, 397, 138–143. (82) Mengistu, D. H.; Bohinc, K.; May, S. J. Phys. Chem. B 2009, 113, 12277– 12282. (83) Cardenas, M.; Braem, A.; Nylander, T.; Lindman, B. Langmuir 2003, 19, 7712–7718. (84) Cardenas, M.; Schillen, K.; Pebalk, D.; Nylander, T.; Lindman, B. Biomacromolecules 2005, 6, 832–837. (85) Peyrard, M.; Cuesta-Lopez, S.; Angelov, D. J. Phys.: Condens. Matter 2009, 21. (86) Peyrard, M.; Cuesta-Lopez, S.; James, G. J. Biol. Phys. 2009, 35, 73–89.
4976 DOI: 10.1021/la9036327
Ainalem et al.
Using the results by Peyrard et al., it is possible to envision how a minor fraction of the hydrophobic bases within the DNA double helix might be accessible to interactions with the hydrophobic AC regions in the bilayers. In relation to this, we could speculate that the presence of hydrophobic moieties in the vicinity of a DNA molecule could stabilize the opened base pair conformations. It is further interesting to note that the incorporation of Ca2þ in the DNA double helix, under the conditions used, will neutralize its charge to a higher extent compared to Naþ and thus increase its hydrophobicity, which should make the DNA even more likely to stick to the bilayers.77,87 This is, however, possibly in opposition to the studies showing that monomers of cationic surfactants do not to interact with DNA, and that the surfactant concentration needs to be above the critical aggregation concentration (CAC) for a DNA interaction to be detected.88 Compared to RNA and single-stranded DNA, double-stranded DNA, which exposes its hydrophobic bases to a lesser extent, should furthermore have a lower propensity for hydrophobic association. In support of this theory, recent QCM-D data from our group on the adsorption of single stranded DNA to surface-deposited DOPC bilayers (not yet published) shows lipids to be removed together with the DNA upon rinsing.
Conclusions This study shows association between DNA and zwitterionic phospholipids of LR phase even in monovalent salt solutions. Adsorbed DNA is situated on the surface of the bilayer in an extended coil-like conformation. Rinsing using a 10 mM NaBr solution removes the adsorbed DNA indicating that the interaction is weak. CaCl2 turns zwitterionic lipids cationic and mediates a stronger electrostatic interaction where DNA possibly adsorbs in a less extended conformation as a film which traps more solvent and is less dilute. Acknowledgment. The sixth EU framework program is acknowledged for work as being a part of a EU-STREP project with NEST program (NEONUCLEI, Contract 12967). The Linnaeus center of excellence on Organizing Molecular Matter through the Swedish Research Council is also thanked for financial support. E. S. acknowledges in addition financial support from the Swedish Foundation for Strategic Research (SSF). Travel grants for neutron experiments were received from the Swedish Research Council (VR). Dr Daiva Tauraite, Vilnius University, Lithuania, and Dr Chu Chuan Dong, University of Oxford, UK, are acknowledged for supplying perdeuterated DOPC for NR. NCNR, USA, and ILL, France, are recognized for allocations of beam time for NR and SANS. Also, Adrian Rennie, Roland May, and Sushil Satija are acknowledged for assistance during neutron experiments. Gunnel Karlsson, Biomicroscopy Unit, Lund University, is thanked for technical assistance during Cryo-TEM. Supporting Information Available: (1) Experimental Section: experimental data which shows the formation and characterization of surface-deposited model membranes using in situ null ellipsometry, QCM-D, and NR. (2) Results: experimental QCM-D data which shows the association between Ca2þ ions and a surface-deposited bilayer. (3) Results: experimental SANS data which shows the interaction between DNA and lipid vesicles in aqueous solutions containing 10 mM NaBr. This material is available free of charge via the Internet at http://pubs.acs.org. (87) Hartzell, B.; McCord, B. Electrophoresis 2005, 26, 1046–1056. (88) Zhu, D. M.; Evans, R. K. Langmuir 2006, 22, 3735–3743.
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