The Effect of Postadded Ethylene Glycol Surfactants on DNA

Jul 1, 2009 - The addition of amphiphiles grafted with polyethylene glycol units to constructs of DNA-amphiphiles is of most relevance for application...
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J. Phys. Chem. B 2009, 113, 9909–9914

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The Effect of Postadded Ethylene Glycol Surfactants on DNA-Cationic Surfactant/Water Mesophases Cecilia Leal,* Azat Bilalov,§ and Bjo¨rn Lindman Physical Chemistry 1, Center of Chemistry and Chemical Engineering, UniVersity of Lund, POB 124, Lund 22100, Sweden ReceiVed: NoVember 19, 2008; ReVised Manuscript ReceiVed: April 10, 2009

The addition of amphiphiles grafted with polyethylene glycol units to constructs of DNA-amphiphiles is of most relevance for applications demanding colloidal stability. In this work, we study the self-assembly behavior of a true ternary mixture comprising (i) an electroneutral complex of DNA and a cationic surfactant (dodecyltrimethylammonium, DTA), (ii) water, and (iii) nonionic surfactant (dodecyl tetraethylene glycol, C12EO4; and dodecyl octaethylene glycol, C12EO8). The phase diagrams of the two systems: DNA-DTA/ C12EO4/water and DNA-DTA/C12EO8/water were carried out using 2H NMR, and small-angle X-ray scattering (SAXS). In both mixtures, the DNA-DTA complex incorporates the postadded nonionic surfactant and large liquid crystalline regions were found. The supramolecular assemblies evolve from a 2D hexagonal structure of the normal type to a lamellar phase as the content of nonionic surfactant is increased. The effect of ethylene glycol unit size in the phase behavior is discussed. We suggest that when longer ethylene glycol units (C12EO8 vs C12EO4) are used, the DNA-DTA aggregate gets saturated with the nonionic surfactant and there exists a coexistence of a fully swollen mesophase phase of C12EO8 alone presumably of the normal hexagonal type with the lamellar and hexagonal phases of DNA-DTA/C12EO8/water. 1. Introduction Mixtures of surfactants with water-soluble polymers are used in a vast number of fields including cosmetics, foodstuffs, and pharmaceutical formulations, among others. The particular case of charged polymers and oppositely charged surfactants in an aqueous medium is exceptionally interesting due to the interplay of forces of different nature: electrostatic and hydrophobic. In the cell, the same interactions govern unselective aggregation and organization between proteins and nucleic acids. Among water-soluble biopolymers, DNA is the most prominent. DNA is a highly negatively charged polymer that readily interacts with oppositely charged proteins to be compacted in the cell nucleus. Similar packing occurs in viruses and this has inspired the development of artificial vectors for gene delivery. Lipofection constitutes about 8% of the current clinical trials in gene therapy.1 The vectors used in this caseslipoplexessare constructs of DNA and oppositely charged amphiphiles such as lipids and/or surfactants.2 The basic constituents of a lipoplex are DNA and a cationic amphiphile, but normally a nonionic species is also added.3,4 The fabrication of a vector for exogenous DNA transfer (transfection) is rather intricate, and the basic understanding of the fundamental interactions taking place is of utmost importance. A number of different factors have been studied in order to correlate the physicochemical properties of the carrier to its transfection efficiency (TE). One interesting observation is that a universal behavior in TE is observed for lipoplexes of different structure and lipid species when normalized to the charge density of the complex.5 Surfactants and lipids have a very rich phase behavior,6 this is * To whom correspondence should be addressed. E-mail: cecilial@ mrl.ucsb.edu. Current address: Materials Department, University of California Santa Barbara, CA 93106. § On leave from Physical and Colloid Chemistry, Kazan State Technological University, Russia.

reflected also when DNA is present and a number of fascinating supramolecular structures can be obtained by mixing DNA and amphiphiles. Lamellar assemblies of lipid bilayers intercalated with DNA were the very first structure reported for lipoplexes and are still the most common phase obtained.7 However, depending on the lipid selection, other phases like reversed8-10 and normal11-13 2D hexagonal phases have been stabilized. Analogously to classic polyelectrolyte - oppositely charged surfactant aggregates, a system of DNA, a cationic amphiphile (lipid or surfactant) and water, should be treated as a fourcomponent system.14 The number of components can be reduced by one if an electroneutral complex salt is constructed with two of the components, normally the anionic polyelectrolyte and the oppositely charged amphiphile.15 A complex salt is a stoichiometric aggregate where every charge in the polyelectrolyte is compensated by the surfactant. In the DNA case, it has been demonstrated that an electroneutral DNA-amphiphile can be prepared with nearly complete counterion release.16 This approach is used to study a number of different polyelectrolyte mixtures17-19 because one obtains true ternary mixtures, the phase behavior of which can be more easily investigated and can be represented in two dimensions (at constant temperature and pressure). While the proper thermodynamic description requires simplification of the system this still needs to be experimentally relevant. One arising issue with the preparation of lipid vectors for gene and drug delivery is that the inherent stability of the carrier needs to be balanced to make them function in vivo. One important example is the requirement of long blood circulation times which has motivated the construction of liposomes sterically stabilized by polymeric lipids.20,21 Even if the mechanisms involved in extended circulation times are somewhat unclear22 most formulations nowadays include phospholipids grafted with polyethylene glycol (PEG). The amounts of PEG grafted phospholipids and the molecular weight of the PEG unit can be modulated to optimize the circulation

10.1021/jp810185k CCC: $40.75  2009 American Chemical Society Published on Web 07/01/2009

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time.23 In addition, the size of the PEG unit determines how much can be incorporated within the liposome lipid membrane.24,25 For gene delivery vectors the same concerns apply and “PEGylated” lipids are normally added to DNA-lipid complexes for transfection in vivo.26 The initial DNA-lipid construct is normally postgrafted with PEGylated lipid aiming to stabilize the outer-surface of the lipoplex without major rearrangements in the original supramolecular structure.27-29 In this paper we used the three-component simplification protocol to study the phase behavior of a stoichiometric DNA-cationic surfactant complex in the presence of nonionic surfactants comprising a PEG moiety (alkylethylene glycols) of two different sizes. Specifically, the ternary phase diagram of DNA-Dodecyltrimethylammonium (DTA), water, and dodecyl tetraethylene glycol (C12EO4) was investigated. For comparison, the ternary mixture using an equivalent alkylethylene glycol with a larger PEG moietysdodecyl octaethylene glycol (C12EO8) was studied as well. We are particularly interested in investigating to what extent a PEGylated amphiphile can be postadded into an existing DNAamphiphile complex and what implications that has on the phase behavior. 2. Materials and Methods Materials. Herring testes Na-DNA sodium salt (Sigma) was used as received. This DNA is highly polydisperse with an average molar mass of 700 bp, determined by electrophoresis. The concentration of DNA was determined by UV methods. The A260/A280 ratio of DNA solutions was determined to be 1.8 suggesting that DNA was free of proteins.30 Sodium bromide (Riedel-deHaen extra pure quality) was used as received. The cationic surfactant, dodecyltrimethylammonium bromide (DTAB) and the nonionic surfactants dodecyl tetraethylene glycol (C12EO4) and dodecyl octaethylene glycol (C12EO8) were used as received and obtained from Tokyo Kasei Kogyo Co., Ltd. and Nikko Chemical Co., (Tokyo, Japan), respectively. The water used was from a Milli-Q filtration system (Millipore). D2O was obtained from Dr. Glaser AG, Basel. Preparation of the Complex Salt DNA-DTA. DNA solutions were prepared by weighing the desired amount and dissolving it in 10 mM NaBr. The pH of all solutions was 7 ( 0.2. DNA-surfactant aggregates were prepared by mixing equal amounts of moles of negative charges of DNA and positive charges of DTA (200 mL of 5 mM solutions) under stirring. Under these conditions the counterion release should be maximal and nearly complete.16 The precipitate was equilibrated in solution for 48 h. It was then separated from the aqueous phase by filtration and washed extensively with Millipore water. The macromolecular complex salt (DNA-DTA) was dried for 3 days in a DW6-85 freeze-dryer. Sample Preparation. Appropriate amounts of the DNA-DTA complex dry precipitate, nonionic surfactant (powder form), and water (for the NMR experiments, heavy water was used) were loaded in 8 mm (i.d.) glass tubes, which were flame-sealed immediately. First the components were mixed with a Vortex vibrator; the mixing was continued in a centrifuge over a few days at 4000 rpm and 40 °C. The tubes were turned end over end every 15 min. The samples were left to equilibrate in a temperature-controlled room at 25 ( 0.5 °C for 2 to 6 months. The samples are expected to be at thermodynamic equilibrium at the time of the measurements. The order of addition of the different components seems to be irrelevant and reversibility is observed during dehydration/rehydration cycles.

Leal et al. Methods. To classify anisotropic and isotropic phases, the samples were visualized under cross-polarized light. Birefringent (anisotropic) and nonbirefringent (isotropic) phases can be distinguished as well the presence of one (transparent) or two (nontransparent) macrophases. The more detailed structural investigation used 2H NMR and small angle X-ray scattering (SAXS). 2 H NMR (D2O). The 2H NMR spectrum of deuterated water is dominated by the interaction of the deuteron electric quadrupole moment with the electric field gradients at the nucleus. Both the magnetic dipole and the electric quadrupole moments are fixed within the nucleus and share the same orientation with respect to an external magnetic field. The anisotropy of liquid crystalline (LC) samples enforces a residual orientational order on interfacial water molecules. This results in a nonzero averaged quadrupole interaction giving rise to a NMR spectrum with two peaks of equal intensity (quadrupole splitting). For an isotropic LC phase, or a solution, a single sharp peak is obtained because of isotropic molecular motions which average the interactions to zero.31 The magnitude of the splitting obtained from 2H NMR depends, among other things, on the angle θ between the axis of cylindrical symmetry (director) and the magnetic field as well as the fraction of confined 2H2O in one or more anisotropic sites.32 For a system containing more than one phase, the 2H NMR spectra are superimposed unless there is fast 2H exchange between the phases. For a mixture of hexagonal and lamellar liquid crystalline phases, two splittings can then be observed with the one originating from the lamellar phase being ideally twice the magnitude of that obtained for the hexagonal phase.33 The experiments were performed at a frequency of 15.371 MHz on a Bruker DMX 100 spectrometer equipped with a 100 MHz (2.3 T) wide-bore superconducting magnet. The temperature was controlled with an air flow through the sample holder. Small Angle X-ray Scattering (SAXS). The measurements were performed on a Kratky compact small-angle system equipped with a position sensitive detector (OED 50 M from M Braun, Graz, Austria) containing 1024 channels with 53.0µm width. Cu KR radiation of wavelength 1.542 Å was provided by a Seifert ID300 X-ray generator operating at 50 kV and 40 mA. A 10 µm thick nickel filter was used to remove the Kβ radiation, and a 1.55-mm tungsten filter was used to protect the detector from the primary beam. The sample-to-detector distance was 277 mm. The volume between the sample and the detector was kept under vacuum during data collection in order to minimize the background scattering. The temperature was kept constant at 25 °C ((0.1 °C) with a Peltier element. 3. Results and Discussion General Outline of Phase Diagrams. It was our interest to investigate to what extent the phase behavior of a binary assembly containing DNA and a cationic amphiphile can be modulated by postaddition of a noncharged amphiphile. It is well-known that the structural (and even dynamic) features of a binary DNA-amphiphile aggregate can be altered by the addition of salt,13,34 alcohols,8-10 and also of a third lipid component.7,35-37 Specifically for DNA-cationic mixtures we recently found out that the supramolecular structure evolves from a lamellar phase to a reversed hexagonal phase by the addition of increasing amounts of long-chain primary alcohols.9 The effect of the alcohol on the phase behavior is controlled by the extent of incorporation within the DNA-surfactant aggregate.10 An existent DNA-amphiphile aggregate is able to accommodate a third component and reassemble into another

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Figure 1. Phase diagrams of the (a) DNA-DTA/C12EO4/water (b) DNA-DTA/C12EO8/water: (1) One-phase region, lamellar liquid crystalline phase (L); (2) one-phase region, hexagonal liquid crystalline phase (H); (3) two-phase region, isotropic aqueous solution (I) and H; (4) three-phase region, I + H + L; and (5) two-phase region, I + L. Illustration of the 2D hexagonal phase (left) and lamellar (right) obtained for this mixture containing cationic and nonionic surfactants (red) and DNA (blue).

mesophase. This a characteristic of materials formed by noncovalent self-assembly and has a number of important applications. One example is the therapeutic usage of DNA-amphiphile assemblies where the cationic lipid is the only required entity to compact DNA but other components are necessarily included in order to achieve the desired cell toxicity, blood circulation time, and targeting. The partial phase diagrams for the ternary DNA-DTA/ C12EO4/water and DNA-DTA/C12EO8/water mixtures are shown in Figure 1. Sample compositions are given in weight percent of the components. First the samples were visualized under polarized light to detect anisotropic phases. The samples were then studied by 2H NMR and it was established whether a certain sample consisted of a single homogeneous phase, two phases, or three phases. The structure of the various phases was investigated by 2H NMR and SAXS. There are basically five significant phase regions in the DNA-DTA/C12EO4/water phase diagram (Figure 1a): 1) Onephase region-lamellar liquid crystalline phase (L); 2) One-phase region-hexagonal liquid crystalline phase (H); 3) Two-phase region-isotropic aqueous solution (I) and H; 4) Threephase region- I + H + L; and 5) Two-phase region-I + L. The DNA-DTA/C12EO8/water phase diagram (Figure 1b) is studied in less detail but nevertheless a similar phase behavior could be observed: 1) One-phase region-lamellar liquid crystalline phase (L) and 2) One-phase region-hexagonal liquid crystalline (H). The Lamellar Liquid Crystalline Region. The samples in this area have a transparent gel-like morphology and show strong optical birefringence. Figure 2 shows the SAXS data for the mixtures (a) DNA-DTA/C12EO4/water (6:19:75 wt %) and (b) DNA-DTA/C12EO8/water (40:20:40 wt %). The relative positions of the Bragg peaks at 1:2 indicate the presence of a lamellar phase. This is most likely arranged as first suggested by the

Safinya lab:7 mixed bilayers containing cationic and nonionic surfactants intercalated by DNA molecules. One interesting feature in DNA-amphiphile lamellar phases is that, normally, a peak corresponding to the correlation between aligned DNA molecules can also be observed in the SAXS data. Here we could not detect that Bragg reflection. This could suggest that in the DNA-DTA:C12EO4/C12EO8 aggregate DNA is not aligned in a liquid crystalline fashion. This feature has been observed for other amphiphilic aggregates containing polypeptides38 and short nucleic acids.39 Another possible explanation is that the DNA correlation peak could be convoluted with the second Bragg peak of the amphiphile lamellar phase. It could be argued that the DNA correlation peak is absent due to equilibration times; however, in similar systems made of concentrated DNA-lipid aggregates, very long DNA molecules were observed to equilibrate in their interhelical distance in an average time of 3 days,7 here the time scale for sample equilibration is months. The position of the first Bragg peak of the lamellar structure can be used to calculate the lamellar spacing. Here we can observe that the first peak for the DNA-DTA/C12EO4/water system is located at smaller q values compared to the DNA-DTA/C12EO8/water case. In real space this corresponds to a lamellar spacing of d ) 2π/q of 57 Å for the C12EO4 containing aggregate and d ) 46 Å for C12EO8. The water content of the C12EO4 system is almost twice that for the C12EO8 containing complex so naturally the more swollen system gives rise to a larger lamellar spacing. While the hydrophobic tails of both nonionic surfactants are of the same size, the hydrophilic chain of C12EO8 is longer than that of C12EO4 (8 ethylene oxide units vs 4). In fact, if the C-C bonds are all-trans the stretched length of the hydrophilic tail is twice as long as the hydrophobic tail for C12EO8. For C12EO4 the length is equivalent to a 12-carbon hydrophilic chain equaling the 12-carbon hydrophobic chain. In the inset of Figure 2 is

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Figure 2. SAXS data for the mixtures (a) DNA-DTA/C12EO4/water (6:19:75 wt %) and (b) DNA-DTA/C12EO8/water (40:20:40 wt %) in the lamellar phase region (area 1) of the phase diagrams. The arrows indicate the Bragg peaks at the relative positions 1:2. The cross arrow in panel b corresponds to a Bragg peak presumably arising from a liquid crystalline phase of C12EO8 alone. In the top-right is a model (POVRAY) of the DNA(blue)-DTA(red headgroup)/C12EO4 (green headgroup)/water lamellar phase (DNA-DTA:C12EO4 6:19 wt %) showing the penetration of the ethylene glycol units of the nonionic surfactant (green) in the DNA-water layer. In the bottom-right of the figure is a model of a crowded DNA(blue)-DTA(red headgroup)/C12EO8 (green headgroup)/water lamellar phase (DNA-DTA: C12EO8 40:20 wt %) assuming that all C12EO8 is incorporated in the DNA-DTA complex. This scenario is difficult to realize physically given that there is hardly any space to fill water in the DNA layer.

illustrated a model of the lamellar phase where the hydrophilic part of the nonionic surfactant penetrates the DNA water layer. This model assumes that the cationic surfactant (headgroup in red) is preferentially located in the vicinity of DNA (blue) and the nonionic surfactant (headgroup in green) fills the gap between DNA molecules.35 As the length of the hydrophilic chain of the surfactant increases (C12EO8 vs C12EO4), it is difficult to realize that it can be incorporated to a large extent into the DNA water layer of the DNA-cationic surfactant complex.21,24,25,40 The schematic representation in Figure 2b models the lamellar DNA-DTA:C12EO8 (40:20 wt %) complex. It is clearly a rather crowded system where there would be essentially no space to incorporate water. Most likely not all the C12EO8 is included in the DNA-DTA aggregate. In fact, the X-ray pattern displays a Bragg peak at lower q, corresponding to a d ) 90 Å, this indicates the presence of liquid crystalline phases with larger lattice spacing. The nature of this phase is rather difficult to identify by the X-ray data because only one peak can be detected. Furthermore, should there be a Bragg peak characteristic for a lamellar phase, this would be located at q ) 0.14 Å-1 or at q ) 0.12 Å-1 for a hexagonal phase; both of which would be totally submerged by the first order peak of the lamellar DNA-DTA:C12EO8 aggregate. Taking into consideration the phase diagram by Mitchell et al. for the C12EO8/ water binary mixture,41 where the region for the 2D normal hexagonal phase is far wider than the lamellar phase region, it is reasonable to assume that the mesophase of C12EO8 alone coexisting with the lamellar phase of the DNA-DTA:C12EO8

aggregate is most likely a 2D hexagonal phase of the normal type. For conventional mixtures of nonionic and cationic surfactants the departure of the nonionic surfactant from the ionic surfactant-water interface is not a common observation, as exemplified by the micelle stabilization of mixed solutions.42,43 The negative effective interaction parameter can mainly be referred to the entropic penalty arising from the counterion distribution in forming ionic surfactant micelles. However, this mechanism is not operating for a surfactant with a highly polyvalent counterion such as DNA. Therefore, the general strong driving force for forming mixed aggregates is absent for the system considered here. Another issue to consider is that the ethylene glycol chains experience van der Walls attraction forces inducing a considerable lateral pressure at the DNA-cationic surfactant interface. In addition, the DNAcationic surfactant film is expected to be rather rigid.9 Hence, the incorporation of surfactants of long polyethylene glycol units (e.g., C12EO8) might not be optimal for high concentration regimes.21,40 The Hexagonal Liquid Crystalline Region. In Figure 3 are shown the SAXS spectra obtained for mixtures (a) DNA-DTA/ C12EO4/water (28.5:28.5:43 wt %) and (b) DNA-DTA/C12EO8/ water (50:25:25 wt %). The relative positions of the Bragg peaks 1:3:2 indicate the presence of a 2D hexagonal phase. This can be of the reverse type with the surfactant molecules decorating the DNA molecules8 typically obtained with long chain phosphoethanolamine lipids or surfactants mixed with long-chain alcohols.9 Alternatively the 2D hexagonal arrange-

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Figure 4. 2H NMR spectra for DNA-DTA/C12EO4/water samples in phase regions (from bottom to top): (3) H + I; (4) L + H +I; (5) L + I; and (1) L.

Figure 3. SAXS data for the mixtures (a) DNA-DTA/C12EO4/water (28.5:28.5:28.5 wt %) and (b) DNA-DTA/C12EO8/water (50:25:25 wt %) in the hexagonal phase region (area 2) of the phase diagrams. The arrows indicate the Bragg peaks at the relative positions 1:3:2. The cross arrow in panel b corresponds to a Bragg peak presumably arising from a liquid crystalline phase of C12EO8 alone.

ment can be of the normal type with rodlike micelles of surfactants arranged with DNA in a “honeycomb” fashion. This has been observed for DNA complexed with single chain cationic surfactants12,13,44,45 or alternatively with lipids holding a big and highly charged headgroup.11 The natural evolution of surfactant phase behavior as a function of surfactant concentration is from spherical to rodlike micelles that further arrange in a hexagonal array. Increasing further the surfactant concentration leads to a bilayer lamellar arrangement (in between these phases, cubic structures are also found).6,46 In the systems presented in this paper the nonionic surfactants C12EO4 and C12EO8 are added to the DNA-DTA complex and as their concentration is increased the evolution is from 2D hexagonal to a lamellar phase. It is reasonable to assume that the hexagonal phase is of the normal type. Analogously to the lamellar structure, the lattice spacing of the 2D hexagonal phase of the C12EO4 containing complex is larger than for the C12EO8 case (d ) 52 Å vs d ) 42 Å, respectively) due to the higher degree of swelling. For the C12EO8 case, the same peak at smaller q, presumably arising from a swollen liquid crystalline phase of C12EO8 alone, is present indicating that also in the hexagonal phase the bulky hydrophilic chain hinders a full incorporation of this nonionic surfactant in the DNA-cationic surfactant complex. Two and Three-Phase Regions. 2H NMR spectra obtained for DNA-DTA/C12EO4/water mixtures are shown in Figure 4. Representative samples of the five regions displayed in the phase diagram in Figure 1 are shown: two-phase region hexagonal + isotropic solution (area 3 in the phase diagram); three-phase

region hexagonal + lamellar + isotropic solution (area 4); twophase region lamellar + isotropic solution (area 5); and the lamellar phase (area 1). Both lamellar and hexagonal liquid crystalline phases are anisotropic and give rise to quadrupolar splittings when water is substituted by D2O. The spectrum for lamellar and hexagonal phases can be distinguished because the 2H powder pattern for water confined to the hexagonally packed surfactant rods has a splitting half of that obtained for lamellar phases.47 An isotropic phase (e.g., solution or liquid crystalline cubic) gives rise to a single peak. This enables the detection of coexistence regions in the phase diagrams. The bottom of the spectrum corresponds to a coexistence of an isotropic solution (middle single peak) and a hexagonal phase (powder pattern). In area 4 of the phase diagram where both lamellar and hexagonal liquid crystalline phases are in equilibrium with a solution phase, the spectra consists of a splitting for the lamellar phase, another one for the hexagonal phase with roughly half the width, and a middle isotropic peak. In area 5 of the phase diagram the lamellar liquid crystalline phase coexists with an isotropic solution phase giving rise to a powder pattern and a middle peak. For the single phaselamellar (area 1) only the powder pattern is obtained. To evaluate further the hypothesis of a coexistence of a fully swollen liquid crystalline of C12EO8 alone with the lamellar and the hexagonal liquid crystalline phases of DNA-DTA/C12EO8/ water we selected the sample used for SAXS (see Figure 3) and performed 2H NMR. The spectra obtained for the sample DNA-DTA/C12EO8:water (50:25:25 wt %) is displayed in Figure 5. The splitting arising from the hexagonal phase is present and in addition another wider splitting is also observed. This could be assigned to the mesophase of C12EO8 alone. Alternatively this could also correspond to a coexistence of a two liquid crystalline phases of DNA-DTA/water both containing C12EO8. At this point more insight in the DNA-DTA/ C12EO8/water system is needed to clarify this issue. 4. Summary The primary objective of this work was to understand the self-assembly behavior of a fully hydrated DNA-cationic surfactant complex upon postaddition of a nonionic surfactant. Mixtures of DNA-DTA/C12EO4/water and DNA-DTA/C12EO8/ water were investigated. The main observation is that both

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Figure 5. 2H NMR spectrum for DNA-DTA/C12EO8/water (50:25: 25 wt %) displaying the coexistence of a 2D hexagonal phase of DNA-DTA/C12EO8/water (H) and a presumable mesophase of C12EO8 alone [H(C12EO8)].

nonionic surfactants penetrate the existent DNA-cationic surfactant aggregate giving rise to a rich phase behavior. In the case of C12EO4, increasing amounts of this nonionic surfactant results in an evolution from a normal 2D hexagonal phase into a lamellar phase. Because of the bulky nature of the hydrophilic part of the nonionic surfactant, they penetrate in the DNA/water layer of the DNA-DTA complex and are preferentially located in the gaps between DNA molecules. In the case of C12EO8 the hydrophilic chain is very long (equivalent to 24 C-C bonds) and the incorporation within the DNA-DTA/water aggregate is expected to be much less efficient. In this case, the SAXS spectra for both the liquid crystalline lamellar and hexagonal phases display a peak at lower q corresponding to 90 Å in real space. We attribute this to a swollen liquid crystalline phase of C12EO8 alone, presumable a 2D normal hexagonal phase. The same sample-DNA-DTA/C12EO8/water (50:25:25 wt %) giving rise to a SAXS spectrum showing a peak at higher lattice spacing (presumable mesophase of C12EO8 alone) in addition to the hexagonal pattern (DNA-DTA/C12EO8/water) was investigated by 2H NMR. Also in this case, the splitting due to the anisotropic hexagonal phase is present in coexistence with a wider one consistent with a coexisting phase of liquid crystalline nature. Acknowledgment. This work was supported by the Foundation for Strategic Research-SSF, The Swedish Institute, and The Swedish Research Council-VR. Part of this work was performed when B.L. was a visiting professor at University of Coimbra with support from FCT Grant No. PCTI/99/QUI/35415. We would like to acknowledge Daniel Topgaard for providing the POVRAY code for the conformational state of the ethylene oxide chains in the nonionic surfactants. Supporting Information Available: The data points used to construct the phase diagrams are available as Supporting Information. This information is available free of charge via the Internet at http://pubs.acs.org. References and Notes (1) Extensive and current information on clinical trials in the field of gene therapy can be found on the Internet at http://www.wiley.co.uk/ genetherapy/clinical. (2) Felgner, P. L.; Gadek, T. R.; Holm, M.; Roman, R.; Chan, H. W.; Wenz, M.; Northrop, J. P.; Ringold, G. M.; Danielsson, M. Proc. Natl. Acd. Sci. U.S.A. 1987, 84, 7413. (3) Zhou, X. H.; Huang, L. Biochim. Biophys. Acta 1994, 1189, 195. (4) Farhood, H.; Serbina, N.; Huang, L. Biochim. Biophys. Acta 1995, 1235, 289.

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