DNA with Double-Chained Amphiphilic ... - ACS Publications

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DNA with Double-Chained Amphiphilic Counterions and Its Interaction with Lecithin Alexey Krivtsov,*,† Azat Bilalov,*,† Ulf Olsson, and Björn Lindman Physical Chemistry, Chemistry Department, Lund University, POB 124, 221 00 Lund, Sweden ABSTRACT: Complex salts of double-stranded DNA with amphiphilic counterions offer novel opportunities for studies of DNA−lipid interactions. Here the effect of the hydrophobicity of the amphiphilic counterion is in focus. For this purpose, double stranded DNA with didodecyldimethylammonium ions as counterions, DDADNA, is prepared and investigated with respect to microstructure. In particular, in order to monitor the interactions with phospholipids, the phase diagram of the DDADNA/lecithin/water system is determined and compared with the previously determined phase diagram with single alkyl chain counterions, dodecyltrimethylammonium, DTA. In both systems, there is a formation of lamellar and reverse hexagonal phases, where hydrated DNA is sandwiched between bilayers or forms the core of reverse cylindrical micelles, respectively. However, whereas the lecithin lamellar phase can incorporate large amounts of DDADNA, there is in the case of the single chain surfactant, DTADNA, a transition to a bicontinuous cubic phase at higher DTADNA concentrations. The general appearance of the phase diagrams, and in particular the role of counterion hydrophobicity, can be rationalized in a simple geometric model.

1. INTRODUCTION The interaction of DNA with amphiphilic compounds such as lipids and surfactants has both biological and biotechnological implications. The presence of significant amounts of lipid in the nucleus of eukaryotic cells has recently received much attention, and there are intense discussions about the possible biological role of the nuclear lipids.1 Combinations of DNA and lipids or lipid mixtures in lipoplexes are receiving increased attention in biotechnology as they constitute an efficient way of formulating DNA, for example, in transfection. Against this background, it is evident that a deeper understanding of the structures formed and the underlying interactions in mixed systems of DNA and lipids or surfactants is required. X-ray diffraction studies of DNA-lipid/surfactant systems have revealed the formation of organized structures, mainly liquid crystalline in nature.2,3 An important bridge between the studies of the simpler well-defined systems and the real biological systems is to investigate the effect of lipids on nucleosome core particles or arrays.4 Studies of phase structures and phase diagrams constitute a fundamental basis of understanding lipid and surfactant systems as well as mixed systems of those with macromolecules. In our group, we have previously reported phase diagrams for mixed systems of DNA and cationic surfactants.5−9 In order to avoid the complication that any mixture of two electrolytes without a common ion, from a thermodynamic point of view, must be considered as a four-component system, and thus requiring a complex representation of the phase diagram, we have eliminated the simple counterions. Thus, by forming the compound of DNA with the cationic surfactant, the “complex salt”,10 and mixing it with water and a cosolute we can obtain two-dimensional phase diagrams, straightforward to represent. From these phase diagrams, deductions about the interactions between DNA and lipids can be made. In a previous study we © 2012 American Chemical Society

described a systems composed of DNA, with a single-chain cationic surfactant as counterion, and lecithin. A quite complex phase behavior emerged, illustrating an intriguing packing of DNA rods and mixed lipid-surfactant aggregates. Whereas our previous work concerned single-chain surfactant ions, double-chain lipids and surfactants have a larger biological and biotechnological relevance. Therefore, we have extended our work to include also DNA with a cationic surfactant with two alkyl chains as counterion and investigated the phase diagram and phase structures in mixtures with lecithin; this mimics the amphiphilic components of efficient transfection systems.11 An investigation of this system also has significance for our understanding of mixed systems of polyelectrolytes and oppositely charged systems in general. These systems have broad applications in formulations (for example, personal care) and, therefore, have been extensively investigated. However, comparisons between singe- and double-chain surfactants are lacking. It is well established that the self-assembly of the surfactants themselves is entirely different. As is well understood on the basis of packing constraints, single-chain surfactants generally form discrete spherical or rodlike micelles, whereas double-chained ones pack into infinite bilayers in a lamellar liquid crystalline phase. The present study illustrates how this difference in packing affects also the more complex system with DNA and lecithin.

2. EXPERIMENTAL SECTION Materials. Herring testes Na-deoxyribonucleic acid sodium salt (Sigma) was used as received. This DNA is highly polydisperse with an Received: July 6, 2012 Revised: August 25, 2012 Published: September 1, 2012 13698

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average molar mass of 700 bp, determined by electrophoresis. The concentration of DNA was determined by UV methods. The nitrogenous bases in nucleotides have an absorption maximum at about 260 nm. In contrast to nucleic acids, proteins have a UV absorption maximum of 280 nm, due mostly to the tryptophan residues. The absorbance of a DNA sample at 280 nm gives an estimate of the protein contamination of the sample. The ratio of the absorbance at 260 nm/absorbance at 280 nm (A260/A280) is a measure of the purity of a DNA sample; it should be between 1.65 and 1.85.12,13 The A260/A280 ratio of DNA solutions was determined to be 1.8 suggesting that DNA was free of proteins. Sodium bromide (Riedel-deHaen extra pure quality) was used as received. The cationic surfactant, didodecyldimethylammonium bromide (DDAB), was used as received and obtained from Tokyo Kasei Kogyo Co., Ltd. Soybean lecithin (1,2-diacyl-sn-3-phosphatdicholine), with the trade name Epikuron 200, was obtained from Lucas Meyer (Hamburg, Germany). Its density is 1.02 g mL−1, and the main fatty acid component is the C18 acid with double bonds. The high content of unsaturated fatty acid chains (>78%) gives a chain melting point well below 0 °C. Epikuron 200 contains about 2.5% water, with the molecular weight of lecithin being 773. Lecithin contains β-carotene as an added antioxidant. βCarotene is the lipophilic pigment responsible for the orange-yellow color of lecithin. Content of β-carotene in Epikuron 200 is negligible ( RDNA ≈ 10 Å. At the same time, Rw can only be a few Ångströms larger than RDNA. The cationic amphiphilic counterions of DNA are anchored at the interface, and there is a significant attraction between the cylindrical interface and DNA that gives an upper limit to Rw. From eq 6, we see that constraining Rw to a narrow range around, say, 15 Å, sets a corresponding constraint on ϕs and, further, by eq 8 on ϕDNA. Hence, the reverse hexagonal phase should be limited to a narrow “island” in the phase diagram, the location of which can be calculated from eqs 6 and 8 and the constraint on Rw. For DDADNA we have ϕDNA = 0.40ϕDDADNA and the total surfactant volume fraction ϕs = ϕlecithin + 0.60ϕDDADNA. Setting Rw ≈ 15 Å and using ls = 20 Å, which is a reasonable value for lecithin, that is the major component, we obtain with eqs 4 and 6, ϕlecithin ≈ 0.55 and ϕDDADNA ≈ 0.25 as the location of the reverse hexagonal phase, in good agreement with the experiment. In Figure 5, we show a model phase diagram of the DDADNA/lecithin/water system with the calculated location of the reverse hexagonal phase shown as a filled circle.

(3)

Here, ADNA = πRDNA2 is the effective cross section area of DNA, with radius RDNA ≈ 1.0 nm, and ϕDNA is the DNA volume fraction. The total volume fraction of the aqueous micellar core, ϕDNA + ϕw = 1 − ϕs, can be written as

1 − ϕs =

πR w 2Lcyl V

(4)

where Rw is the radius of the aqueous cylinder core and Lcyl/V is the length per unit volume of the reversed cylindrical micelles. Assuming that all surfactant occupies the interface of the aqueous domains, the total interfacial area per unit volume is given by ϕs/ls. Here, the effective surfactant length, ls, is strictly the surfactant volume-to-area ratio ls = vs/As, where vs is the surfactant molecular volume and As is the average area that each surfactant molecule occupies at the interface (Figure 2), here defined by Rw. In analogy with eq 4, this area density can be written as ϕs ls

=

Figure 5. Calculated partial phase diagram of the DDADNA/lecithin/ water system.

In the lamellar phase, the schematic drawing of which is shown in Figure 6, the DNA molecules are confined to a thin water layer of thickness d w = 2ls

2πR wLcyl V

2ls(1 − ϕs) ϕs

ϕs

(9)

where 2ls is the effective bilayer thickness, ds. For the reversed hexagonal phase, we noted that the DNA−interface attraction constrains Rw ≥ RDNA. In the lamellar phase, for the same reason, we expect dw to be constrained to values similar to, but slightly larger than, 2RDNA. This implies that the lamellar phase has essentially the shape of a line in the phase diagram (Figure 3a). Assuming, for simplicity, a constant bilayer thickness 2ls = 40 Å and dw = 30 Å, the line begins at ϕlecithin = 2ls/(2ls + dw) =

(5)

Dividing eq 4 by eq 5, we obtain an expression for Rw

Rw =

1 − ϕs

(6)

which incorporated into eq 4 gives 13701

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example, to the packing in the binary DDAB/water lamellar phase,24 but it results from the matching of the DNA charge distribution. In the lecithin rich reverse hexagonal phase, the DDA layer is “diluted” with lecithin and the molecules can adopt a packing with a smaller area per headgroup. A similar effect can be obtained by adding salt. This is a very interesting issue for future exploration, especially in view of speculations on the interaction between lecithin and calcium and magnesium ions in the literature.25−27 DNA with amphiphilic counterions, cationic surfactant− DNA complex (CSDNA), is stable due to strong electrostatic attraction between highly charged polymer and oppositely highly charged surfactant aggregates. Salt effects will be different depending on osmotic pressure and thus charge number of the small counterions. Previously, we showed that adding 1:1 electrolyte to the ternary DTADNA/lecithin/water mixtures causes a slight increase in the unit cell dimension of the LC phases.6 Very high ionic strength may lead to DNA release from liquid crystals and the formation of DNA free liquid crystalline phases. In particular, we observe a coexistence of two lamellar phases. One contains DNA, and the other is essentially free of DNA. We expect similar effect of adding salt in the case of the DDADNA/lecithin/water system. Divalent cations have been shown to condense DNA in the lamellar cationic surfactant−DNA complexes.25 Adding divalent ions would screen the negatively charged DNA and reduce the repulsion between the DNA molecules confined between the surfactant bilayers. Lecithin as a zwitterionic lipid can also form a multilamellar complex with DNA in the presence of divalent cations of calcium, in which DNA is embedded between the lipid layers.26,27 We expect at relatively high salt contents coexistence of two lamellar phases: one lamellar phase of DDADNA/lecithin/ water and a second lamellar phase formed by DNA with divalent counterions and lecithin. We will address this further in a forthcoming publication.

Figure 6. Structural model of the lamellar phase of the system DDADNA/lecithin/water: dpolar is the thickness of the polar layer, including water, DNA, polar groups of lecithin, and DDA; dapolar is the thickness of the apolar layer, including the hydrocarbon tails of the surfactants; dS is the bilayer thickness of a surfactant; d is the repeat distance between bilayers; dDNA is the distance between DNA molecules; dW is the thickness of the water layer, including water and DNA; ls is the average surfactant molecule length (ls ≈ 0.5 ds); ADNA is the cross section of the DNA cylinder; As is the interface section per surfactant molecule. The repeat distance between bilayers can be obtained from the SAXS patterns (this corresponds to a lamellar spacing of d = 2πh/qm, at Miller indices [h] = [1], [2], [3], etc.). The bilayer thickness of a surfactant, ds, can be estimated on the basis of SAXS data as a slope in the coordinates of the equation d = ds/ ϕs.

0.57 on the binary lecithin/water axis. As DDADNA is added, the water content in the lamellar phase is slightly decreased as DNA partly replaces water at constant dw. When DNA gets added to this lamellar phase, confined to two dimensions, we first expect at low DNA concentrations an isotropic phase where the DNA rods are free to rotate in the plane. At higher DNA concentrations, we expect the rods to order into a nematic phase, with an order parameter that increases with increasing DNA concentration. This two-dimensional ordering of the DNA rods in the lamellar phase often results in a (broad) diffraction peak,20−22 from which the average separation, dDNA, between approximately parallel DNA rods can be obtained (Figure 6).6 We expect the lamellar line to terminate approximately when the DNA rods get close packed, that is, when dDNA≈ dw. Setting this termination to dDNA= dw = 30 Å corresponds to ϕDNA/ϕw = 0.54 and ϕDDADNA/ϕw = 1.3. In Figure 5, the predicted location of the lamellar phase is shown as a line defined by dDNA ≥ dw = 30 Å. At higher water contents, this lamellar phase coexists with pure water, and so does the fully hydrated DDADNA complex. For excess DDADNA, beyond DNA close packing in the lamellar phase, the hydrated complex forms and we thus have the three phase triangle: hydrated DDADNA (hexagonal) + water + lamellar phase, which is also shown in Figure 5. The calculated phase diagram, Figure 5, clearly catches the salient features of the DDADNA/lecithin/water phase diagram (Figure 3a). With the corresponding single chain surfactant counterion, DTA (Figure 3b), the situation is slightly different. At higher DTADNA concentrations, the lamellar phase becomes unstable because the single chained DTA, unlike the double-chained DDA, is not a lamellae forming surfactant. Instead a cubic phase23 having a weakly curved surfactant interface is formed. The location of the reverse hexagonal phase is however similar in the two systems. In the DDADNA/lecithin/water system, there are in fact two reverse hexagonal phases, located at very different positions in the phase diagram, one being hydrated DDADNA, whereas the other is rich in lecithin. In hydrated DDADNA, DDA occupies a larger area per molecule (130 Å2) at the polar−apolar interface. This packing is significantly different compared, for

4. SUMMARY Here we have demonstrated that depending on the cationic surfactant, the preferred packing and phase structure of the DNA with cationic surfactant as counterion in the ternary mixtures with lecithin and water will be different. When comparing the phase diagrams of the single- and double-chain surfactant systems, the following two differences appear. (i) DTA with lecithin forms a cubic liquid crystalline phase (gyroid symmetry, normal type), where the DNA duplexes are incorporated within the water domains. This phase is not present in the case of DDA, obviously because this doublechained surfactant does not form wormlike surfactant micelles in mixtures with lecithin. (ii) The aqueous lecithin lamellar phase can solubilize up to 55 wt % DDADNA, where DDA and lecithin form mixed bilayers. On the other hand, only 25 wt % DTADNA can be incorporated into the lamellar phase of aqueous lecithin. Double-chained DDA counterions cannot form normal micelles in water. Instead, bilayer or reverse structures are preferred. The phase behavior of surfactant can be shifted. When the volume of the hydrophobic part of surfactant is increased, by changing of single-chained amphiphile with a double-chained one, the complexes will adopt a lamellar structure. A further increase of the hydrophobic part leads to the formation of the inverted structures, such as inverted 13702

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(3) Koltover, I.; Salditt, T.; Rädler, J. O.; Safinya, C. R. An Inverted Hexagonal Phase of Cationic Liposome-DNA Complexes Related to DNA Release and Delivery. Science 1998, 281, 78−81. (4) Lundberg, D.; Berezhnoy, N. V.; Lu, C.; Korolev, N.; Su, C.-J.; Alfredsson, V.; Miguel, M.; Lindman, B.; Nordenskiöld, L. Interactions Between Cationic Lipid Bilayers and Model Chromatin. Langmuir 2010, 26, 12488−12492. (5) Bilalov, A.; Carlstedt, J.; Krivtsova, E.; Lindman, B.; Olsson, U. DNA with Amphiphilic Counterions: Tuning Colloidal DNA with Cyclodextrin. Soft Matter 2012, 8, 4988−4994. (6) Bilalov, A.; Olsson, U.; Lindman, B. DNA-Lipid Self-Assembly: Phase Behavior and Phase Structures of a DNA-Surfactant Complex Mixed with Lecithin and Water. Soft Matter 2011, 7, 730−742. (7) Leal, C.; Bilalov, A.; Lindman, B. The Effect of Postadded Ethylene Glycol Surfactants on DNA-Cationic Surfactant/Water Mesophases. J. Phys. Chem. B 2009, 113, 9909−9914. (8) Leal, C.; Bilalov, A.; Lindman, B. Phase Behavior of a DNA-Based Surfactant Mixed with Water and n-Alcohols. J. Phys. Chem. B 2006, 110, 17221−17229. (9) Bilalov, A.; Leal, C.; Lindman, B. Mixing Oil and Water by a DNA-Based Surfactant. J. Phys. Chem. B 2004, 108, 15408−15414. (10) Svensson, A.; Piculell, L.; Cabane, B.; Ilekti, P. A New Approach to the Phase Behavior of Oppositely Charged Polymers and Surfactants. J. Phys. Chem. B 2002, 106, 1013−1018. (11) Lundberg, D.; Faneca, H.; Moran, M.; Lima, M.; Miguel, M.; Lindman, B. Inclusion of a Single-Tail Amino Acid-Based Amphiphile in a Lipoplex Formulation: Effects on Transfection Efficiency and Physicochemical Properties. Mol. Membr. Biol. 2011, 28, 42−53. (12) Glasel, J. A. Validity of Nucleic Acid Purities Monitored by 260/ 280 Absorbance Ratio. Biotechniques 1995, 18, 62−63. (13) Saenger, W. Principles of Nuclei Structure; Springer-Verlag: NewYork, 1984. (14) Ven, M.; Kattenberg, M.; Ginkel, G.; Levine, Y. K. Study of the Orientational Ordering of Carotenoids in Lipid Bilayers by Resonance Raman Spectroscopy. Biophys. J. 1984, 45, 1203−1210. (15) Lee, S. B.; Clabaugh, K. C.; Silva, B.; Odigie, K. O.; Coble, M. D.; Loreille, O.; Scheible, M.; Fourney, R. M.; Stevens, J.; Carmody, G. R.; Parsons, T. J.; Pozder, A.; Eisenberg, A. J.; Budowle, B.; Ahmad, T.; Miller, R. W.; Crouse, C. A. Assessing a Novel Room Temperature DNA Storage Medium for Forensic Biological Samples. Forensic Sci. Int.: Genet. 2011, DOI: 10.1016/j.fsigen.2011.01.008. (16) Knaapila, M.; Svensson, C.; Barauskas, J.; Zackrisson, M.; Nielsen, S. S.; Toft, K. N.; Vestergaard, B.; Arleth, L.; Olsson, U.; Pedersen, J. S.; Cerenius, Y. A New Small-Angle X-Ray Scattering SetUp on the Crystallography Beamline I711 at MAX-Lab. J. Synchroton Radiat. 2009, 16, 498−504. (17) Leal, C.; Wadsö, L.; Olofsson, G.; Miguel, M.; Wennerström, H. The Hydration of a DNA-Amphiphile Complex. J. Phys. Chem. B 2004, 108, 3044−3050. (18) Thalberg, K; Lindman, B; Karlstrom, G. Phase-Behavior of Systems of Cationic Surfactant and Anionic Polyelectrolyte - Influence of Surfactant Chain-Length and Polyelectrolyte Molecular-Weight. J. Phys. Chem. 1991, 95, 3370−3376. (19) Montalvo, G.; Khan, A. Self-assembly of a Mixed Ionic and Zwitterionic Zmphiphiles: Associative and Dissociative Interactions between Lamellar Phases. Langmuir 2002, 18, 8330−8339. (20) Rädler, J. O.; Koltover, I.; Jamieson, A.; Salditt, T.; Safinya, C. R. Structure and Interfacial Aspects of Self-Assembled Cationic LipidDNA Gene Carrier Complexes. Langmuir 1998, 14, 4272−4283. (21) Lasic, D. D.; Strey, H.; Stuart, M. C. A.; Podgornik, R.; Frederik, P. M. DNA-Cationic Liposome Complexes: Structure and StructureActivity Relationships. J. Am. Chem. Soc. 1997, 119, 832−833. (22) Dias, R. S.; Lindman, B.; Miguel, M. G. DNA interaction with catanionic vesicles. J. Phys. Chem. B 2002, 106, 1260−12607. (23) Bilalov, A.; Olsson, U.; Lindman, B. A Cubic DNA-Lipid Complex. Soft Matter 2009, 5, 3827−3830. (24) Fontell, K.; Ceglie, A.; Lindman, B.; Ninham, B. Some Observations on Phase Diagrams and Structure in Binary and Ternary

hexagonal; therefore, the nature of the hexagonal phase obtained for the hydrated DDADNA complex has to be the inverted type, found for double-chained lipids with small headgroups. We summarize our conclusions in the scheme shown in Figure 7.

Figure 7. Schematic illustration of the transformation of the LC structures found in the DDADNA/lecithin/water and DTADNA/ lecithin/water systems. When we increase the spontaneous curvature of the interface, by changing of a double-chained amphiphile with a single-chained one, the complexes will transfer from reversed to a normal (direct) structure.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (A.K.); [email protected] (A.B.). Notes

The authors declare no competing financial interest. † On leave from Physical and Colloid Chemistry, Kazan National Research Technological University, Russia.

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ACKNOWLEDGMENTS We thank Elena Krivtsova for technical assistance and acknowledge help rendered by Mo Segad for running SAXS experiments. Financial support by the Swedish Research Council (VR), partly through the Linnaeus grant Organizing Molecular Matter (OMM) Center of Excellence (239-20096794) is gratefully acknowledged.

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REFERENCES

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