pubs.acs.org/Langmuir © 2010 American Chemical Society
DNA Lipoplexes: Formation of the Inverse Hexagonal Phase Observed by Coarse-Grained Molecular Dynamics Simulation Josephine Corsi,† Robert W. Hawtin,† Oscar Ces,‡ George S. Attard,† and Syma Khalid*,† † ‡
School of Chemistry, University of Southampton, Highfield, Southampton, SO17 1 BJ, United Kingdom, and Membrane Biophysics Platform, Department of Chemistry, Imperial College, London, SW7 2AZ United Kingdom Received April 12, 2010. Revised Manuscript Received June 10, 2010
Mixtures of dsDNA and lipids, so-called lipoplexes, are widely used as less toxic alternatives to viral vectors in transfection studies. However, the transfection efficiency achieved by lipoplexes is significantly lower than that of viral vectors and is a barrier to their use in the clinic. There is now significant evidence suggesting that the molecular organization and structure (nanoarchitecture) of lipoplexes might correlate with biological activity. As a consequence, the ability to predict quantitatively the nanoarchitecture of new systems, and how these might change intracellularly, would be a major tool in the development of rational discovery strategies for more efficient lipoplex formulations. Here we report the use of a coarse-grain molecular dynamics simulation to predict the phases formed by two lipoplex systems: dsDNA-DOPE and dsDNA-DOPE-DOTAP. The predictions of the simulations show excellent agreement with experimental data from polarized light microscopy and small-angle X-ray diffraction (SAXS); the simulations predicted the formation of phases with d-spacings that were comparable to those measured by SAXS. More significantly, the simulations were able to reproduce for the first time the experimentally observed change from a fluid lamellar to an inverse hexagonal phase in the dsDNA-DOPE-DOTAP system as a function of changes in lipid composition. Our studies indicate that coarse-grain MD simulations could provide a powerful tool to understand, and hence design, new lipoplex systems.
Introduction The complexes (lipoplexes) that are formed when double stranded (ds) DNA is mixed with lipids, and with cationic lipids (CL) in particular, are widely used as transfection agents for intracellular gene delivery.1-3 The key attraction of lipoplexes as transfection agents is that they have a lower toxicity than viral gene vectors. While CL-DNA complexes are considered to be safer than viral methods of intracellular DNA delivery, they are often associated with poorer transfection efficiencies, thus limiting their use for gene delivery in somatic gene therapy. Consequently, improving the transfection efficiency of nonviral vectors is considered to be critical to allow the realization of gene therapy. For reviews on transfection using cationic lipids, see refs 2-5. CL-DNA lipoplexes are usually mixtures of cationic lipids, for example, dioleoyl trimethylammonium propane (DOTAP) and neutral “helper” lipids (HL), such as dioleoyl phosphatidylethanolamine (DOPE) or dioleoyl phosphatidylcholine (DOPC). A variety of CL-HL-DNA lipoplexes have been reported that are characterized by self-assembled ordered nanoarchitectures that have either lamellar or hexagonal periodicities. A typical example of lamellar (LR) nanoarchitecture in a lipoplex occurs in the case of DOPC-DOTAP systems; in this phase, dsDNA helices are sandwiched between lipid bilayers.4 In contrast, DOPE forms complexes with DNA that have an inverse hexagonal (HII) nanoarchitecture, in which the DNA is located within the aqueous pores of the phase.5 *To whom correspondence should be addressed. Mailing address: School of Chemistry, University of Southampton, Highfield, Southampton, SO17 1 BJ, U.K. Telephone: þ442380591476. E-mail:
[email protected]. (1) (2) (3) (4) 814. (5)
Patil, S. D.; Rhodes, D. G.; Burgess, D. J. AAPS J. 2005, 7, E61–77. Safinya, C. R. Curr. Opin. Struct. Biol. 2001, 11, 440–8. Chesnoy, S.; Huang, L. Annu. Rev. Biophys. Biomol. Struct. 2000, 29, 27–47. Radler, J. O.; Koltover, I.; Salditt, T.; Safinya, C. R. Science 1997, 275, 810– Koltover, I. E. A. Science 1998, 281, 78–81.
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Over the past few years a number of studies have suggested a link between the transfection efficiency (TE) of lipoplexes and their nanoarchitecture.6,7 For example, it has been shown that TE in mammalian cells is significantly higher in CL-DNA complexes with a HII architecture compared to those with a LR architecture.8 Furthermore, it is thought that electrostatic interactions play a role in the initial binding of the lipoplex to the cell membrane,3 and this means that the charge ratio, which is set by the ratio of cationic lipid to DNA, is a second important determinant of transfection efficiency. However, despite recent intense interest in the relation between CL-HL-DNA lipoplex phase structure and biological activity, it is still not possible to predict quantitatively with any degree of accuracy the phase behavior of dsDNA, CL, and helper lipid mixtures, particularly in intracellular environments. The time scales encountered in lipoplex formation and interaction with cells are relatively long, and as a result the predictive capacity that is required to map the phase architecture of lipoplex mixtures cannot be achieved by established atomistic simulation methods.9 Coarse-grain (CG) molecular dynamics (MD) simulations in which ∼4 (or more) heavy atoms are mapped to a single particle enable the simulation of larger systems on longer time scales than is possible by atomistic simulation methods.10-12 (6) Koynova, R.; Wang, L.; Tarahovsky, Y.; MacDonald, R. C. Bioconjugate Chem. 2005, 16, 1335–1339. (7) Lin, A. J.; Slack, N. L.; Ahmad, A.; Koltover, I.; George, C. X.; Samuel, C. E.; Safinya, C. R. J. Drug Targeting 2000, 8, 13–27. (8) Safinya, C. R.; Ewert, K.; Ahmad, A.; Evans, H. M.; Raviv, U.; Needleman, D. J.; Lin, A. J.; Slack, N. L.; George, C.; Samuel, C. E. Philos. Trans. R. Soc., A 2006, 364, 2573–2596. (9) Khalid, S.; Rodger, P. M. Prog. React. Kinet. Mech. 2004, 29, 167–186. (10) Marrink, S. J.; Risselada, H. J.; Yefimov, S.; Tieleman, D. P.; de Vries, A. H. J. Phys. Chem. B 2007, 111, 7812–7824. (11) Bond, P. J.; Holyoake, J.; Ivetac, A.; Khalid, S.; Sansom, M. S. J. Struct. Biol. 2007, 157, 593–605. (12) Shih, A. Y.; Arkhipov, A.; Freddolino, P. L.; Schulten, K. J. Phys. Chem. B 2006, 110, 3674–3684.
Published on Web 06/28/2010
DOI: 10.1021/la101448m
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A number of theoretical studies have addressed the properties of lipoplex-forming materials, including the thermotropic phase behavior of phospholipids13 and the thermodynamics of lipid-DNA complexes;14 these have also been the subjects of recent reviews15,16 Here we report CG MD simulations of HII phase formation in a dsDNA-HL system as a function of hydration, and of a change from LR to HII phase stability in dsDNA-CL-HL mixtures at fixed hydration; this is the first such observation in lipoplexes by MD simulation. We have used a coarse-grain approach that is intermediate in detail between traditional atomistic simulations,17,18 and the implicit solvent coarse-grain approaches often used to study DNA-lipid systems.19-22 Our approach allows us to study much larger systems than atomistic methods, while the inclusion of explicit water, ions, and DNA details such as bases, backbone, sugar, as well as distinct major and minor grooves allows us to retain key features of the system. The structural predictions of our simulation were validated by comparison with small-angle X-ray scattering (SAXS) experimental data on the phase structures of the lipoplex mixtures. SAXS is a powerful non-invasive platform that enables the measurement of the morphology of the bulk phase as well as any modifications in the structural parameters of the system induced by the addition of DNA. SAXS has previously been used to study inverted hexagonal phases of cationic liposome-DNA complexes5 and phospholipid-DNA-metal complexes.23 In the present study, our simulations are able to predict the stabilization of lamellar-like phases with increasing DOTAP to DOPE ratios, and are in excellent agreement with structural data obtained by SAXS experiments and polarizing light microscopy observations of comparable mixtures.
Methods DNA Model. The DNA model used in this study is based on coarse-grain particle types developed by Marrink et al.24 In summary, there are four particle types: charged, polar, nonpolar, and apolar. Each CG particle replaces approximately four heavy (not hydrogen) atoms in an atomistic description. The DNA model was created by mapping appropriate particle types onto atomic positions in an atomistic representation of the canonical beta form of DNA. An elastic network of harmonic restraints connects particles within 0.7 nm with a force constant of 1500 kJ mol-1 nm-2. Full details of the model and validation are given by Khalid et al.25 Simulation System. The initial simulation system (lamellar) was created by placing three 36-base-pair DNA strands spaced 5 nm apart just above a preformed DOPE bilayer. Water molecules were added as required. The multilamellar system was then (13) Marrink, S. J.; Mark, A. E. Biophys. J. 2004, 87, 3894–3900. (14) May, S.; Harries, D.; Ben-Shaul, A. Biophys. J. 2000, 78, 1681–1697. (15) Marrink, S. J.; de Vries, A. H.; Tieleman, D. P. Biochim. Biophys. Acta 2009, 1788, 149–168. (16) Podgornik, R.; Harries, D.; Parsegian, V. A.; Strey, H. H. In Gene and Cell Therapy: Therapeutic Mechanisms and Strategies; Smyth Templeton, N., Ed.; CRC Press: Boca Raton, FL, 2003. (17) Khalid, S.; Hannon, M. J.; Rodger, A.; Rodger, P. M. Chemistry 2006, 12, 3493–3506. (18) Khalid, S.; Hannon, M. J.; Rodger, A.; Rodger, P. M. J. Mol. Graphics Modell. 2007, 25, 794–800. (19) Farago, O.; Gronbech-Jensen, N.; Pincus, P. Phys. Rev. Lett. 2006, 96, 018102. (20) Farago, O.; Gronbech-Jensen, N. Biophys. J. 2007, 92, 3228–3240. (21) Farago, O.; Ewert, K.; Ahmad, A.; Evans, H. M.; Gronbech-Jensen, N.; Safinya, C. R. Biophys. J. 2008, 95, 836–846. (22) Farago, O.; Gronbech-Jensen, N. J. Am. Chem. Soc. 2009, 131, 2875–2881. (23) Francescangeli, O.; Pisani, M.; V., S.; Bruni, P.; Weiss, T. M. Europhys. Lett. 2004, 67, 669–675. (24) Marrink, S. J.; de Vries, A. H.; Mark, A. E. J. Phys. Chem. B 2004, 108, 750– 760. (25) Khalid, S.; Bond, P. J.; Holyoake, J.; Hawtin, R. W.; Sansom, M. S. J. R. Soc. Interface 2008, 5, S241–50.
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created by simply replicating this system four times in the z-dimension (normal to the bilayer plane). This resulted in a system comprising 12 DNA molecules and 5660 DOPE molecules. The number of water and DOTAP molecules was adjusted according to the requirements of the system being simulated; full details of the simulated systems are given in the Supporting Information, Table 1. For the study of the phase structure of lipoplexes as a function of DOPE/DOTAP mole ratios, we started with a pure DOPE system and progressively converted DOPE molecules to DOTAP molecules at random. This was done by converting lipid molecules in one bilayer and then replicating this to create the four-bilayer starting configuration. The topology for DOTAP was created from the DOPE model (included in the MARTINI forcefield),24 by removing the coarse-grain particle that represents the phosphate group (see the Supporting Information for structure of CG lipid molecules). Counterions were added to give an overall neutral system by balancing the residual charges of the DNA or the DOTAP lipids. Details of the simulations are summarized in Table 1 in the Supporting Information. Each system was energy minimized prior to a g300 ns MD production run. Simulation Protocols. All simulations were performed using the Gromacs 3.3.3 simulation package.26,27 Production simulations were performed in the NPT ensemble. The temperatures of the DNA and lipids were coupled separately to the water using the Berendsen algorithm at 323 K, with a coupling constant of 0.5 ps. The simulations were performed at 323 K as, while it is known that the inverse hexagonal phase of pure DOPE will form at temperatures above 295 K, on a simulation time scale this is best captured at elevated temperatures and low hydration levels.13 The system pressure was coupled anisotropically using the Berendsen algorithm at 1 bar, with a compressibility of 1 10-5 bar-1. Orthorhombic periodic boundary conditions were applied in all three dimensions. Lennard-Jones interactions were smoothly shifted to zero between 0.9 and 1.2 nm to reduce the cutoff noise, and electrostatics were smoothly shifted to zero between 0 and 1.2 nm. Analyses were performed using GROMACS tools, HOLE,28 and locally written scripts. VMD was used for visualization. d-Spacings were calculated by measuring the distances between the centers of the channel cross sections using standard trigonometric methods. Small-Angle X-ray Scattering Experiments. Lipids were purchased from Avanti Polar Lipids and used without further purification. Samples were prepared from chloroform solutions of the required lipid mixtures, from which the solvent was removed under vacuum. DNA was added to the dried lipids from a 10 mg mL-1 (in water) solution of salmon sperm DNA (Fluka). The samples were lyophilized before being rehydrated and transferred to Lindemann glass capillary tubes, 1.5 mm internal diameter (Agfa NDT Limited). These glass capillaries were flame-sealed to prevent loss of water. After overnight equilibration at room temperature, the structure and dimensions of the mesophases formed by the dsDNA-DOPE and dsDNA-DOTAP-DOPE mixtures were investigated using small-angle X-ray diffraction (SAXS). SAXS measurements were conducted using a specialized inhouse custom-built SAXS/WAXS beamline coupled to a copper target Bede Microsource (Durham, U.K.) X-ray generator with integrated glass polycapillary X-ray focusing optics. The Ni-filtered Cu KR radiation (λ = 1.54 A˚) was cut down with 300-μm pinholes. X-ray diffraction images were acquired on an X-ray intensified charge-coupled device (Gemstar Detector, Photonic Science, East Sussex, U.K.). The sample holder was equipped with a computer-driven Peltier-based unit capable of (26) Berendsen, H. J. C.; Vanderspoel, D.; Vandrunen, R. Comput. Phys. Commun. 1995, 91, 43–56. (27) Lindahl, E.; Hess, B.; Van Der Spoel, D. J. Mol. Model. 2001, 7, 306–317. (28) Smart, O. S.; Neduvelil, J. G.; Wang, X.; Wallace, B. A.; Sansom, M. S. J. Mol. Graphics 1996, 14, 354-360; 376.
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controlling the sample temperature over a range of 10-120 °C with a precision of (0.05 °C. SAXS Analysis. Diffraction patterns were analyzed with the AXcess software package developed by Dr. Andrew Heron at Imperial College London.29 The lamellar phase gives Bragg peaks whose intensities are in the ratio of 1:2:3:4, while√ for the √HII phase the diffraction peak intensities are in the ratio 1: 3:2: 7.30 In all samples, a minimum of three peaks were used to determine the relevant d-spacing. The estimated accuracy of the layer spacings is (0.15 A˚. Images were calibrated with silver behenate, which has a lamellar lattice spacing of 58.38 A˚.31 Polarized Light Microscopy Experiments. Samples for polarized light microscopy were prepared alongside those for X-ray study. To ensure binary mixtures were well mixed, they were incubated at 37 °C for several days before being sandwiched between a glass slide and coverslip to give samples approximately 50 μm thick. Samples were investigated using an Olympus BH-2 polarized light microscope equipped with a Linkam YMS90 heating stage and temperature control unit. Inverse hexagonal phases were identified by their birefringent optical textures when viewed through crossed polarizers. Biphasic samples with coexisting lamellar and hexagonal phases were characterized by regions with typical hexagonal optical textures and regions with greater fluidity and less well-defined optical textures.
Results For clarity, the results of our studies are presented in two parts. The first describes the effect of hydration on the structure and dynamics of dsDNA-DOPE mixtures, while the second describes the effect of the change from LR to HII phase on the structure and dynamics of dsDNA-DOPE-DOTAP mixtures. In the latter system, the change in phase stability is achieved by altering the molar ratio of DOTAP to DOPE at a fixed water content. Each simulation system has a DNA/lipid content of 12 DNA molecules (36 base pairs) and ∼5600 lipid molecules. Table 1 in the Supporting Information provides a summary of the simulations performed for both studies and full details of the simulation systems. The dsDNA-DOPE System. Formation of the HII Phase. Transitions between lipid/water phases arise as a result of changes in the balance between attractive forces in the headgroup region and repulsive forces in the hydrocarbon tails.30 This balance between attractive and repulsive forces is known to be affected by the amount of water in the system; indeed, for DOPE/ water phases, the effect of water on the formation of different phases has been shown both experimentally and via CG simulation.13,24 To determine the extent to which water may play a similar role in DNA-containing lipid phases, we undertook a hydration study in which the phase behavior of DNA-DOPEwater systems was mapped as a function of the water concentration. For reference, we first performed a simulation of the DNAfree DOPE-water system at a hydration level of ∼10 water molecules per lipid (Note that these are “real” water molecules, as one CG particle represents 4 real water molecules. The water content of this, and all of the other systems simulated, is within the range that corresponds to the range of limiting hydration values of the inverse hexagonal phase of this lipid as reported by Tate and Gruner.32 From herein, references to the number of water molecules will be in terms of real water molecules and not CG particles). This water to lipid ratio was chosen as a reference point, (29) Seddon, J. M.; Squires, A. M.; Conn, C. E.; Ces, O.; Heron, A. J.; Mulet, X.; Shearman, G. C.; Templer, R. H. Philos. Trans. R. Soc., A 2006, 364, 2635–2655. (30) Seddon, J. M. Biochim. Biophys. Acta 1990, 1031, 1–69. (31) Huang, T. C.; Toraya, H.; Blanton, T. N.; Wu, Y. J. Appl. Crystallogr. 1993, 26, 180–184. (32) Tate, M. W.; Gruner, S. M. Biochemistry 1989, 28, 4245–4253.
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Figure 1. Lameller to inverse hexagonal phase transition as a function of hydration level. The color scheme is as follows: DNA backbone is blue, DNA bases and sugars are shown as a purple surface, the DOPE lipid tails are orange, and the DOPE lipid headgroups are cyan. Water and ions are omitted for clarity. In some of the snapshots, lipids that obscure the DNA have also been removed for clarity.
as this is just below the water limit at which the lamellar to inverse hexagonal phase transition was observed in earlier simulation studies of DNA-free lipid systems.13 This level of hydration, which corresponds to a water volume fraction of ∼0.2, is near but below the limiting hydration for DOPE (volume fraction 0.21-0.3) that has been reported experimentally. We observed a transformation from the initial lamellar phase to an ordered inverse hexagonal phase with a d-spacing of ∼6.2 nm. The transformation proceeded via stalk formation between the layers of the lamellar phase, as described in previous studies.13 The formation of an ordered inverse hexagonal phase in the dsDNA-DOPE mixtures was observed at lower hydration levels than the pure DOPE system. At hydration levels corresponding to g18 waters/lipid, the DNA-containing system formed a lamellar phase with an “undulating” appearance; that is, the lipids had a (static) wavelike appearance. At this level of hydration, the lamellar phase was stable and the undulation induced by the DNA did not appear to induce the formation of lipid stalks. This is in agreement with our own simulations of the pure DOPE system and an earlier study of DNA-free DOPE phases in which Marrink and Mark reported that spontaneous stalk formation was not observed at hydration levels of >12 waters/lipid.13 In our simulations, removal of water particles from the system resulted in some hexagonal character developing; a mixed phase was observed at ∼11 waters/lipid (Figure 1). Lipid stalk formation occurred within the first 50 ns of simulation, followed by formation of porelike structures around the DNA molecules. This mixed phase (or biphasic system) was stable over the next 350 ns of simulation. Further reduction of the water content resulted in a imperfect inverse hexagonal phase at ∼7 waters/lipid; channels of different diameters were arranged in a loose but identifiably hexagonal geometry with DNA surrounded by water, then lipid (Figure 1). At least two of the channels have two DNA molecules located within them. In contrast, just a small reduction in the water content to ∼10 waters per lipid resulted in an ordered inverse hexagonal phase in the pure DOPE system. At a hydration level of ∼2 waters/lipid, an ordered inverse hexagonal phase was observed, with DNA molecules located within channels formed by the lipid headgroups, with the channels being arranged in a DOI: 10.1021/la101448m
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Figure 3. Lamellar to inverse hexagonal phase transition as a function of lipid composition. Color scheme is as in Figure 1.
Figure 2. Radius profiles of the channels formed in the inverse hexagonal phase (∼2 waters per lipid). The top diagram shows an isolated channel, and the lipid phosphate particles are displayed as gray spheres. The surface of the channel is shown in blue. The plot shows the average (calculated over the last 20 ns) radius and standard deviation along the principal (z) axis of the channel. The reference system (no DNA) is in black, while the system containing DNA is shown in red.
hexagonal pattern (Figure 1). The d-spacing for this phase was 6.2 ( 0.5 nm. The evolution of the inverse hexagonal phase proceeds via movement of the DNA molecules from an originally stacked arrangement in the lamellar phase to a hexagonal arrangement. This movement is accompanied by stalk formation near the DNA molecules. The stalks subsequently elongate and fuse to form water-containing channels around the DNA molecules. An ordered inverse hexagonal phase was observed at a ratio of 10 waters/lipid in the absence of DNA. However, in the presence of DNA, the water content was reduced to ∼2 waters/ lipid before an ordered HII phase was observed. This observation suggests that the DNA is replacing the water in the pores, thus appearing to stabilize the phase at lower levels of hydration. Channel Geometry. The geometry and flexibility of the water/ DNA channels are both likely to play a key role in stabilizing the inverse hexagonal phase. To characterize the dynamical behavior of the channels in the present simulations, we calculated the average channel radius profile (the circumference of the channel is defined by the phosphate particles of the DOPE headgroups); this analysis was performed for all of the channels in dsDNA-DOPE simulations. As the resulting geometries were very similar in all of the channels, here we focus our description on two different channels from the same dsDNA-DOPE system (Figure 2). The radius profiles of both channels were similar, with average radii of ∼1 nm. The channels did not exhibit uniform radii along the principal axis, with radii ranging from 0.93 to 1.08 nm ( 0.5. The nonuniform radii along the pore together with the deviations from the average radius at each point along the principal axis over the last 50 ns are indicative of a flexible, undulating channel. Analysis of the movement of the water within the channels revealed that the individual water particles are not “bound” to specific phosphate groups of the backbone, but move around quite freely within the channels with lateral and longitudinal motion of up to ∼0.6 nm within just the last 5 ns of the simulation 12122 DOI: 10.1021/la101448m
(see Supporting Information Figure 3). It is interesting to compare the geometry and flexibility of these channels with those formed in our reference simulation of a DOPE-water system (10 waters/lipid). In the DNA-free system, the channels had a mean radius of 1.08 nm that varied within the range ∼1.02-1.18 nm ( 0.5. Thus, in the absence of DNA, the channels formed by the lipid headgroups are slightly wider but are just as irregular and flexible as in the presence of DNA. The slightly narrower channels in the presence of DNA presumably arise from the tight association of the lipid headgroups with the DNA backbone.25,33 The dsDNA-DOPE-DOTAP System. Phase Nanoarchitectures. The effect of CL on the structure of lipoplexes was investigated using mixtures in which increasing amounts of DOTAP, which in its pure state hydrates to form a lamellar phase, is added to DOPE, which in its pure state hydrates to form an inverse hexagonal phase (Figure 3). The simulations were performed at a hydration level of ∼2 waters/lipid, since this composition yields the most clearly defined inverse hexagonal phases in the simulation of the dsDNA-DOPE system. Our simulations revealed that as the ratio of DOTAP to DOPE is increased, there is a greater propensity to retain lamellar-like character. Visual inspection of the molecular arrangement in the simulated phases formed at ∼2 waters/lipid revealed that, as the concentration of DOTAP increases, the HII phase becomes more disordered. The structure appeared to swell slightly as the DOTAP content was increased to 20% DOTAP, with the d-spacing increasing to 6.4 ( 0.5 nm; this is consistent with our experimental observations by SAXS. In our simulations, at 40% DOTAP, the DNA molecules and the channels surrounding them were not arranged in a hexagonal lattice; the morphology was substantially distorted and the d-spacing could not be easily defined (estimating the center of the distorted channels gives an approximate d-spacing of ∼6.9 ( 1.2 nm). As the concentration of DOTAP was increased, the formation of pores was less evident than that in the dsDNA-DOPE systems, such that, at 40% and 50% DOTAP, large areas of lamellar-type phases with little stalk formation were observed. d-Spacings could not be defined in these systems. A qualitative phase diagram of DOPE and DOTAP mixtures was determined using polarized light microscopy. This study shows a change from a HII to LR phase with increasing DOTAP content, with the transition occurring via a biphasic system from ∼30 wt % DOTAP (see Supporting Information Figure 4). The experimental phase diagram shows that pure DOTAP is lamellar in character, with no biphasic region. Similarly SAXS measurements show a phase change at this range of compositions. These observations are in excellent agreement with the predictions from our simulations, both in terms of the architecture formed and the dimensions of the phase. Lipid Arrangement. We recently reported that, perhaps contrary to chemical intuition, the equilibrium distance between the (33) Bandyopadhyay, S.; Tarek, M.; Klein, M. L. J. Phys. Chem. B 1999, 103, 10075–10080.
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Figure 4. Top left: RDF between DNA backbone and DOPE headgroups. Top right: RDF between DNA backbone and DOPE ethanolamine groups (with differing amounts of DOTAP present in the simulation system). Bottom left: RDF between DOTAP ethanolamine groups and DNA. The DOPE ethanolamine-DNA backbone curve RDF is also shown for reference. Bottom right: RDF of DOPE phosphate groups with DOPE ethanolamine groups and DOTAP ethanolamine groups. All systems contain ∼2 waters per lipid.
zwitterionic or cationic lipids and the DNA backbone in lipidDNA mixtures tends to be roughly equal.25,34,35 Molecular simulation studies in which the lipid arrangement around DNA has been calculated have thus far mostly been reported for the lamellar phase.25,36 To characterize the arrangement of lipids in the inverse hexagonal phase, we have calculated lipid-DNA (lipid headgroup-DNA phosphate particles) and lipid (headgroup)-lipid (headgroup) radial distribution functions (RDFs) from our simulations (Figure 4). Two aspects of the lipid arrangement are particularly interesting: first, the arrangement of the lipid headgroups relative to the DNA and, second, the arrangement of the DOPE and DOTAP lipids with respect to each other. The proximity of DOPE lipids to the dsDNA, in the absence of any DOTAP lipids, is shown in Figure 4, top left. As expected, the highest peak corresponds to the probability of finding the ethanolamine group of DOPE within the first hydration shell of the DNA phosphate groups (∼0.52 nm). The peak corresponding to the second hydration shell is much lower and located at a distance of ∼0.92 nm. In contrast, the phosphate group of DOPE has a higher propensity to be located within the second solvation shell of the DNA phosphate groups. This arrangement is presumably driven by the electrostatic repulsion between DNA and DOPE phosphate groups and the attraction of the DNA phosphate group to the DOPE ethanolamine group. Interestingly, the equilibrium distance of the DOPE ethanolamine group to the DNA backbone is not altered by addition of the cationic DOTAP (34) Gurtovenko, A. A.; Vattulainen, I. J. Am. Chem. Soc. 2005, 127, 17570– 17571. (35) Zantl, R.; Baicu, L.; Artzner, F.; Sprenger, I.; Rapp, G.; Radler, J. O. J. Phys. Chem. B 1999, 103, 10300–10310. (36) Nielsen, S. O.; Lopez, C. F.; Ivanov, I.; Moore, P. B.; Shelley, J. C.; Klein, M. L. Biophys. J. 2004, 87, 2107–2115.
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(Figure 4). If we consider the system containing ∼2 waters/lipid, it can be seen in Figure 4 that the shapes of the RDFs at 0%, 20%, and 40% TAP are almost identical. There is a slight shift in the main peak from 0.52 to 0.53 nm, but it is not possible to determine from these simulations if this is a significant difference. A similar trend is seen in the DOTAP ethanolamine-DNA phosphate distances. At 20% DOTAP, the peak corresponding to the first hydration shell occurs at ∼0.51 nm, while at 40% DOTAP a rather broader peak that spans ∼0.50-0.52 nm is observed. These differences suggest a possible preference for closer proximity to the DNA backbone of the TAP headgroups compared with the PE headgroups. However, the small differences in the equilibrium distances argue for further investigation with a more detailed (atomistic) force field. The lipid-lipid RDFs indicate tight headgroup packing of the two lipid types; there is slightly higher probability of finding a DOTAP ethanolamine group within the first solvation shell of a DOPE phosphate group, than a DOPE ethanolamine group (Figure 4). This is in agreement with our previously reported arrangement of TAP and PC headgroups from simulations of the LR phase of this binary mixture.25 Channel Geometry. Analysis of the geometry of the channels formed at ∼2 waters/lipid and 20% DOTAP (i.e., the conditions under which the most regular, DOTAP-containing inverse hexagonal phases were formed) revealed dynamic channels with a radius of ∼0.99 nm ( 0.5 (Figure 5). Thus, these channels were comparable to those formed in the dsDNA-DOPE systems at a similar hydration level, but slightly narrower than the channels formed in the absence of DNA. The channels formed at 40% DOTAP were irregular in shape and rather short, as the change to a complete inverse hexagonal phase did not occur on the time scale of the simulations. Thus, it was not possible to calculate the radii of these channels. The DOI: 10.1021/la101448m
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Figure 5. Average radius profiles of inverse hexagonal phase channels of the 0% TAP system (black) and the 20% TAP system (red). Both systems contain ∼2 waters per lipid.
irregular geometries of the simulated channels at 40% DOTAP are in qualitative agreement with observations from SAXS experiments (see below). Ion Location and Dynamics. A key difference between our treatment of DNA and that in more theoretical models14 is that the level of detail in our model enables us to study the DNA-ion interactions in rather more detail. In particular, we have shown previously that the model is able to reproduce qualitatively the pattern of ion clouds around DNA observed in atomistic simulations.25 In the present study, we have calculated RDFs of Clions with respect to DNA and lipid headgroups to determine their preferred location (Figure 6). This analysis has been performed for the system containing 20% DOTAP and ∼2 waters per lipid, as this system contained both lipid types and also produced a welldefined inverse hexagonal phase. The highest peaks corresponding to the first hydration shell of Cl- ions are those of the DOTAP and DOPE ethanolamine groups. Interestingly, the equilibrium distance of both the DOTAP and DOPE ethanolamine particles from the Cl- ions is almost identical at ∼0.5 nm. Visual inspection reveals interdigitation of the ions between the lipid headgroups of both lipid types (see Supporting Information Figure 5). This has also been observed in simulation and experimental studies of lamellar phases of cationic and zwitterionic lipid mixtures.25 As expected, the peak for the DNA backbone (phosphate particles) corresponds to the second hydration shell of the Cl- ions, at about ∼1 nm, due to electrostatic repulsion effects. To gain a better understanding of the role of ions in these systems, it is useful to consider their mobility within the channels of the inverse hexagonal phase. In particular, are they “bound” to the lipid headgroups or are they mobile within the channels? To address this, we have calculated the movement of Cl- ions along the channels during the last 100 ns of the 20% TAP, ∼ 2 waters per lipid simulation (Figure 7). We observe two types of general behavior; the vast majority of ions remain interdigitated between lipid headgroups for long periods (∼ 50 ns) during which they only experience slight fluctuations in their motion (thermal motion) and then they “escape” from in between the headgroups to move into the channel and then occupy a position between neighboring headgroups. However, for the majority of the simulation, at any one time there is a proportion of ions that do not remain in between specific headgroups for any significant length of time. These ions are able to move more freely (up to ∼1.5 nm). 12124 DOI: 10.1021/la101448m
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Figure 6. RDF of Cl- ions with respect to lipid headgroups and DNA phosphate groups.
Figure 7. Movement of two selected Cl- ions along (z dimension) the same channel in the inverse hexagonal phase. The black line corresponds to an ion that remains interdigitated between lipids headgroups from 200 to 240 ns and then moves more freely thereafter. The red line corresponds to an ion that is not interdigitated between any particular headgroups for the time frame of the calculation.
Small-Angle Scattering Experiments. Samples of lipid and salmon sperm dsDNA with an average length of 200 base pairs, but with a high degree of polydispersity, were prepared such that DNA/lipid ratios were comparable to the systems studied in our coarse-grain MD simulations. The DNA content was calculated such that the ratio of DNA base pairs to lipid molecules was conserved between simulations and experimental samples. Using an estimate of (a) the area of the headgroup of DOPE (48-65 A˚2),37 (b) the diameter, and therefore circumference of the pores (∼40 and ∼126 A˚, respectively), (c) DNA length (∼200 base pairs 3.4 A˚ per base pair), and (d) the diameter of the DNA duplex (20 A˚) and comparing the total length of channel to the total length of dsDNA, it was calculated that in these mixtures 1 in 1.7 to 1 in 2.4 channels were occupied by a salmon sperm dsDNA molecule. This is a slightly lower, but comparable rate of DNA channel occupation than that in our simulations (1 in 1.4 channels occupied by DNA), as the bulk inverse hexagonal phase is formed (37) Corsi, J.; Dymond, M. K.; Ces, O.; Muck, J.; Zink, D.; Attard, G. S. Chem. Commun. (Cambridge, U.K.) 2008, 2307–2309.
Langmuir 2010, 26(14), 12119–12125
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Article Table 1. Comparison of d-Spacings Obtained from SAXS Experiments and Simulations lattice parameter/A˚
visual inspection of phase mol % DOTAP
DNA
microscope
0
no yes
HII HII
HII HII
20
no yes
HII HII
no yes
HII HII
40
SAXS
SAXS
simulation
HII HII
62.19 62.24
62 ( 5 62 ( 5
HII HII
HII
68.58 60.59
64 ( 5
HII and LR HII
distorted HII
69.77 66.58
69 ( 12
of channels organized in many small domains oriented in different directions, meaning that some channels will not be accessible to dsDNA molecules in the experimental setup. SAXS measurements revealed that adding increasing amounts of DOTAP to DOPE caused an increase in the inverse hexagonal d-spacing from 6.2 to 6.8 nm. Indeed, when the DOTAP content was increased to 40%, coexisting biphasic inverse hexagonal and lamellar phases were observed; this is consistent with previous studies.5 The addition of DNA lessened the compositional extent of the biphasic domain such that, at 40 mol % DOTAP, a HII phase was observed, rather than a biphasic system (which is the case for DNA-DOPE mixtures). Estimates of the d-spacings of the inverse hexagonal phases obtained in CG MD simulations showed excellent correlation with the experimental results. Table 1 provides a comparison of the d-spacing values obtained by simulation and experimental methods.
Conclusions We have described a coarse-grain approach to the simulation of dsDNA-lipid mixtures that captures the changes from HII to LR phase stability that is observed experimentally and so has the potential to be applicable to the prediction of the phase behavior of a range of lipid-DNA mixtures. The behavior of dsDNA-DOPE mixtures was observed to be significantly different from that of pure DOPE. While in the absence of DNA an ordered inverse hexagonal phase was observed at a ratio of 10 waters/lipid, our simulations indicate that when DNA is present, there is incomplete formation of a hexagonal structure from the starting lamellar disposition of the components at levels of hydration that are above ∼2 water molecules per lipid. The simulations also suggest that channels formed in the inverse hexagonal phase are narrow and comparable to the DNA radius; that is, there is room only for a few waters around the DNA. The water residing within the channels is not bound to specific regions of the backbone but is quite mobile along the channel. The presence of the cationic lipid DOTAP does not make a significant difference to the channel geometry. Furthermore, the addition of DOTAP does not alter the equilibrium distance of DOPE headgroups from the DNA backbone. Our simulations provide some evidence to suggest that the DOTAP headgroups occupy positions closer to
Langmuir 2010, 26(14), 12119–12125
simulation
the DNA backbone than the DOPE headgroups, and that this effect is more pronounced when the ratio of DOTAP to DOPE headgroups is increased. However, we appreciate that the structural subtleties of the lipid headgroups may not be captured completely by the coarse-grain force field we have used, and thus, this effect should be investigated further and verified with an atomistic force field. In agreement with earlier coarse-grain studies of zwitterionic and cationic lipids in the lamellar phase, our simulations reveal that the DOTAP and DOPE headgroups are tightly packed together in the inverse hexagonal phase. Furthermore, we observed Cl- ions interdigitating equally between PE and TAP headgroups in the inverse hexagonal phase. The ions were able to remain between the same lipid headgroups for up to ∼50 ns before moving to a different location in the lipid headgroup region of the phase. This tight packing of ions between lipid headgroups has also been reported for lamellar phases.35 The close agreement between the nanoarchitectures resulting from the CG MD simulations and the experimental observations, particularly the ability to reproduce the HII-LR phase change, suggests that this simulation approach could provide a powerful new tool in the development of rational discovery strategies for more effective transfection agents. Acknowledgment. This work was supported by grants from the EPSRC (Platform Grant EP/G00465X/1). S.K. and J.C.C. acknowledge support from the University of Southampton, Life Sciences Interfaces Forum, G.S.A. and J.C.C. acknowledge support form a European Union FP6 award (NEONUCLEI, Project Number 12967). Supporting Information Available: One figure describing how the d-spacings were calculated from simulations; one plot showing movement of water molecules within the inverse hexagonal channels; one figure showing the structures of the CG lipid molecules; one figure showing interdigitation of ions between lipid headgroups; one experimental phase diagram; one table summarizing simulation systems. This material is available free of charge via the Internet at http://pubs.acs.org.
DOI: 10.1021/la101448m
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