Multi-cationic Lipid Complexes Driven by

J. Phys. Chem. B 2001, 105, 5291-5297

5291

Polymorphism of DNA/Multi-cationic Lipid Complexes Driven by Temperature and Salts Eric Raspaud,† Bruno Pitard,‡,⊥ Dominique Durand,§ Olivier Aguerre-Chariol,# Juan Pelta,†,! Gerardo Byk,| Daniel Scherman,‡ and Franc¸ oise Livolant*,† Laboratoire de Physique des Solides CNRS UMR 8502, UniVersite´ Paris-Sud, 91405 Orsay Cedex, France, UMR 7001 CNRS/ENSCP/AVentis, Centre de Recherche de Vitry-AlfortVille, 13, Quai Jules Guesdes, BP 14 94403 Vitry-sur-Seine, France, LURE, CNRS UMR 130, UniVersite´ Paris-Sud, BP 34, 91898 Orsay Cedex, France, Laboratory of Peptidomimetics and Genetic Chemistry, Department of Chemistry, Bar-Ilan UniVersity, 52900 Ramat Gan, Israel, and Rhodia, 52 rue de la Haie Coq, 93309 AuberVilliers, France ReceiVed: NoVember 15, 2000

The structural polymorphism of DNA/lipopolyamine (multi-cationic lipid) complexes has been studied by X-ray diffraction and cryo-electron microscopy. Monovalent salt and temperature effects have been analyzed. Depending on the treatment applied to the lipidic solution prior to DNA addition, two types of structure can be obtained for identical final conditions: a classical lamellar structure and a more complex structure. The latter was shown to appear when the lipidic solution prepared in high salt condition is vortexed and quenched below the hydrocarbon chain melting temperature, before addition to the DNA solution. It corresponds to some small facetted vesicles. Similar behavior is observed for the poly(styrene-sulfonate)/lipid system.

Introduction Synthetic gene delivery systems formed by the association of DNA and cationic lipids have been widely studied these last years for their potential therapeutic applications.1 Nevertheless, their in vivo efficiency remains low, revealing our poor understanding of the different mechanisms involved. Being able to predict and control the structure of the DNA-lipid complexes is a challenging task since we expect the structural polymorphism of the complexes to be correlated with their transfection efficiency. For instance, Koltover et al.2 reported that the fusogenic properties were improved while using inverted hexagonal complexes instead of lamellar complexes. In an earlier study,3 Mok and Cullis have also suggested that complexes exhibiting higher transfection potencies present nonbilayer lipid structures. In most cases, charged lipids or a mixture of charged and neutral lipids have been used and it has already been established that the global charge of the complexes (negative, neutral, or positive) can be adjusted by varying the charge concentration ratio between lipids and DNA.4,5 It has been shown that maximum transfection is observed in this neutral zone, at a charge ratio of the order of one.6,7 The size of the aggregates depends on this ratio. It is maximum in the neutral zone and may become larger than the micron forming macroscopic aggregates for high DNA and lipid concentrations. In * Author to whom correspondence should be addressed. E-mail: [email protected] or [email protected]. † Laboratoire de Physique des Solides CNRS UMR 8502, Universite ´ Paris-Sud. ‡ UMR 7001 CNRS/ENSCP/Aventis, Centre de Recherche de VitryAlfortville. § LURE, CNRS UMR 130, Universite ´ Paris-Sud. | Laboratory of Peptidomimetics and Genetic Chemistry, Department of Chemistry, Bar-Ilan University. # Rhodia. ⊥ Present address: INSERM U533, Faculte ´ de me´decine, 1 rue Gaston Veil, BP 53508, 44035 Nantes, France. ! Present address: ERRMECE, Universite ´ Cergy-Pontoise, 2, av. Adolphe Chauvin, BP 222, 95032 Cergy-Pontoise, France.

the present study, we studied how the structural characteristics of the complexes formed in the neutral zone depend on their preparation method. Among all parameters that we have tested, two appear to be of importance: salt concentration and temperature. Combining X-ray diffraction with electron microscopy observations, we show that in our system, the polymorphism of the complexes depends on the treatment applied to the lipidic solution, prior to DNA addition and complex formation. Material and Methods Materials. Supercoiled plasmid DNA (pXL2774, 4500 bp) was dialyzed against 10 mM Tris-HCl buffer, 1mM EDTA, pH 7.4. Polydisperse poly(sodium styrene-sulfonate) (PSS) was purchased from Aldrich (average Mw ∼ 106 g/mol). Its degree of sulfonation is unknown. The lipopolyamine RPR120535 is composed of a sperminederived amino acid linked via a glycine to a dioctadecyl hydrophobic entity.8 At neutral pH, three amines are ionized and electroneutralized by acetate counterions. The melting temperature Tm of the hydrocarbon chain was found equal to 42 °C in pure water and equal to 47.1 °C when the lipid is dried in the powder form.9 Lipids were diluted in pure water or in saline NaCl solution at room temperature, heated to about 60 °C, well above the chain melting temperature, vortexed, and heated again. The lipids were removed from the oven, allowed to cool and to stabilize at room temperature from a few minutes up to 4 h before mixing with the DNA solution. In the samples studied by microscopy, large vesicles (>1µm) were observed in coexistence with the structures reported here. In the following, we will refer the initial lipids state to the lipid state in solution just prior to DNA addition. Lipid/DNA complexes were always formed by addition of the lipids to the DNA solution. Initial concentrations of lipids and DNA ranged from 0.5 to 20 g/L and from 0.17 to 2 g/L, respectively. For X-ray experiments, DNA/lipid concentrations were set to obtain macroscopic pellets (charge ratio ) 3 and a

10.1021/jp004214e CCC: $20.00 © 2001 American Chemical Society Published on Web 05/12/2001

5292 J. Phys. Chem. B, Vol. 105, No. 22, 2001

Figure 1. Two typical diffraction profiles of lipopolyamine/DNA macroscopic aggregates: the one peak profile (a) associated to a lamellar structure and the other one (b) which is denoted “X”. For both samples, the final concentrations (in NaCl, DNA, and lipids) are identical. Only the initial NaCl concentration of the lipid solution, prior to DNA addition, is different (no salt (a), 1 M NaCl (b)).

final DNA concentration ) 0.16 g/L). For electron microscopy, small complexes are required and observations were made in the presence of a large excess of lipids (charge ratio ) 15); the final DNA concentration was set to 0.4 g/L. The NaCl concentration ranged from 10 mM to 0.5 M in the DNA solution, and from 0 to 2 M in the lipid solution. Varying the volume ratio of initial DNA and lipid solutions from roughly 0.05 to 15 allowed us to explore a wide range of final NaCl concentrations (from 30 mM to 0.95 M when starting from saline lipid solutions and from 3 mM to 0.45 M when starting from saline DNA solutions) while keeping constant the lipid/ DNA ratio. Electron Microscopy. CryoTEM observations were performed to visualize the structure of the complexes in their aqueous environment. Complexes are confined in a liquid film of thickness of the order of 100 nm which is vitrified to an amorphous state by plunging into liquid ethane before structural changes may occur.10 Uranyl acetate or Na phosphotungstate was added sometimes to the sample before freezing. A few samples were also deposited onto a carbon film and negatively stained, air-dried, and observed in classical TEM. All observations were done with an accelerating voltage of 120 kV, in a Philips CM12 EM. X-ray Diffraction. Capillaries were filled with the aggregated samples and centrifuged to 150 g to collect the precipitates. X-ray diffraction experiments were performed using a synchrotron source (station D 24 and D 43) at LURE (Orsay, France) with selected wavelength λ ) 1.44 Å (D 43) and λ )1.488 Å (D 24). The detection system consists of phosphor image plates, scanned with a Molecular Dynamics PhosphoreImager. The explored range of the transfert vector q ) (4π/λ) sin θ (where 2θ is the scattering angle) was comprised between 0.01 and 2.2 Å-1. For heating experiments, the samples were equilibrated for 15 min at each temperature and the diffraction signals were integrated during the following 30 min. Results Increasing the salt concentration was already known to widen the range of lipids/DNA concentration ratios for which the complexes formation leads to macroscopic aggregates (neutral zone).5 We show how the salt may also modify the local structure of the lipid/DNA aggregates, as illustrated in Figure 1. The two X-ray diffraction spectra correspond to samples with identical final DNA, lipid, and salt concentrations but prepared

Raspaud et al.

Figure 2. The two diffraction profiles of lipopolyamine/PSS macroscopic aggregates associated with the lamellar structure (a) and with the “X” structure (b). The two structures are obtained in the same conditions as for DNA.

in two different ways: (in both samples, prior to DNA addition, lipid solutions have been heated and vortexed as described in the Material part). (a) Lipids are diluted in water and added to the saline DNA solution. A large single peak is observed at q ) 0.075 Å-1. This peak, which was attributed to the lamellar structure of DNA-lipopolyamine complexes,5 corresponds to the lamellar periodicity p ) 2π/q, with p ) 84 Å. The lamellar stacking does not extend over large distances (over ∼ five periods) which prevents the observation of the second-order reflection. (b) lipids are diluted in a saline solution (CNaCl g 0.5 M) before mixing with DNA. Two intense peaks and a shoulder can be detected. They are located at approximately 0.04, 0.07, and 0.10 Å-1. No other peak was observed at lower q values (until 0.01 Å-1). From one sample to another, the position of the first peak ranges from 0.039 to 0.047 Å-1 and the position of the second one from 0.07 to 0.083 Å-1, both positions being related by a factor 1.6-1.8. Dispersion of the values is not related to a variation of the charge ratio or to the different concentrations. They are observed even for samples prepared with the same initial and final concentrations. In all cases, because of the relative peak positions, this kind of profile cannot be associated with a lamellar structure. These peaks could be rather attributed to a unique structure, as predicted for dense packed spheres clusters11 or to different local structures in coexistence. In particular, the second peak is asymmetric and may hide another peak located at 0.075 Å-1, which may correspond to the lamellar peak. When lipids are diluted in a saline solution with a NaCl concentration CNaCl e 0.35 M prior to DNA addition, the profile of diffracted intensity by the macroscopic pellets presents only a large peak as observed in (a) indicating a similar lamellar structure. The range of 0.35 to 0.5 M NaCl was not explored. Similar X-ray diffraction spectra were also recorded for lipid/ PSS macroscopic aggregates (Figure 2). Aggregates, prepared with lipids diluted in water (Figure 2a), are characterized by a first peak and a second-order reflection (not shown in Figure 2a) located at 0.10 Å-1 and around 0.2 Å-1. This indicates a lamellar stacking of periodicity p ) 2π/q ) 62.8 Å which, according to the full width half-maximum value of the firstorder peak, extends over ∼ 9 periods. This small p value compared to the DNA case (p ∼ 84 Å) could be due to the PSS hydrophobic backbone leading to a PSS confinement inside the lipid bilayers as suggested in ref 12. When aggregates are prepared with lipids diluted in 1 M NaCl (Figure 2b), two other peaks located at smaller q values (0.0455 Å-1 and 0.082 Å-1)

Polymorphism of DNA/Multi-cationic Lipid Complexes

J. Phys. Chem. B, Vol. 105, No. 22, 2001 5293

Figure 3. The position of the first peak in the intensity profile (2π/q) is plotted as a function of the temperature to which lipids were heated prior to DNA addition at room temperature. Lipids are diluted in 1 M NaCl. A jump occurs between 40 and 50 °C at the gel-to-liquidcrystalline transition of the lipidic hydrocarbon chains.

are detected instead. Their positions are comparable to those determined in the DNA case. Since the lamellar first-order peak (Figure 2a) is significantly shifted from the two peaks of Figure 2b, one may conclude in the PSS case, that the second peak of Figure 2b located at 0.082 Å-1 is not associated with the simple lamellar structure but rather with the structure giving the first peak at 0.0455 Å-1. Similar conclusions could be applied to the lipid/DNA system. From the comparison between the two profiles in Figure 2, one may also exclude in the PSS case, the presence of a significant amount of lamellae in this new structure. On condition that pellets or macroscopic aggregates are formed, the same (a) and (b) diffraction profiles are observed for different concentrations of DNA (PSS), and lipids and for different charge ratios. Also the peaks positions and the associated structures do not depend on the final NaCl concentration (in the range of 30 mM to 0.5 M). This means that under physiological conditions (about 150 mM NaCl), both types of structure may be formed. The local structure depends on the NaCl concentration in the initial lipid solution. In the next step, we just consider how experimental conditions may determine the formation of either the lamellar structure (“L”) or this new structure, denoted “X”. We focus on lipid/DNA system. Further work will be necessary to precise the structure of the complexes. A second series of experiments was performed using salted lipids (1 M NaCl in the initial solution), and varying the temperature to which the lipid solution was heated and vortexed before mixing with DNA. Mixing was always done at room temperature. Either “L” or “X” structures were obtained, as summarized in Figure 3, where the position of the first peak (2π/q) of the X-ray profiles is plotted as a function of the heating temperature. It clearly appears that the lamellae form when the lipid solution is heated to temperatures T < Tm (with Tm ∼ 42 °C) and that the “X” structure forms when the heating temperature T is above Tm. In a last experiment, the lipid solution was heated above Tm and mixed to DNA at this temperature (T ) 50 °C). The structure was then analyzed at room temperature. Instead of the “X” structure, the lamellae form. Thus, the structure of the aggregate does not only depend on the salt concentration of the cationic lipid solution but also on its temperature treatment, both effects acting on the initial lipids state. To summarize, two conditions are needed in order to form the “X” local structure of the lipid/DNA macroscopic aggregates: the initial lipid solution has to be salted with CNaCl g 0.5 M, heated above Tm and vortexed before mixing with DNA at room temperature. On the contrary, the lamellae can

Figure 4. X-ray scattering of lipid/DNA systems as a function of the temperature (data log-stacked for clarity): lamellar structure (Figure 4a) and “X” structure (Figure 4b). The inter-lamellar distance decreases at the melting temperature while the “X” structure melts at higher temperature.

be formed under multiple conditions: (i) no salt or low amounts of salt in the initial lipid solution (CNaCl e 0.35 M) whatever the temperature conditions, (ii) large amounts of salt in the initial lipid solution (CNaCl g 0.5 M) and no heating above Tm, (iii) large amounts of salt in the lipid solution (CNaCl g 0.5 M) and mixing with DNA at a temperature above Tm. A third series of X-ray diffraction experiments was performed on the two types of lipid/DNA macroscopic aggregates. The samples were progressively heated from room temperature to T > Tm. The diffracted intensities recorded at different temperatures are plotted in Figure 4a for the lamellar structure and in Figure 4b for the “X” structure. For the lamellar structure (Figure 4a), the peak located at q ) 0.076 Å-1 recorded for T < Tm is shifted to higher q values (q ≈ 0.087 Å-1) for T > Tm. The associated lamellar periodicity p ) 2π/q is then found to jump from 82.5 to 72 Å. Such a decrease of the lamellar repeat distance was previously observed at the gel-to-liquid-crystalline transition, on dimyristoylphosphatidyl-choline/dimyristoyltrimethylammonium propane (DMPC/DMTAP) mixed with DNA.13 It was associated to a decrease of the lipidic bilayer thickness. For the sample of type “X”, the thermotropic behavior of lipid/DNA system is illustrated in Figure 4b. For T ) 21 °C, the two intense peaks and the shoulder characteristic of the “X” structure may be seen whereas for T ) 83.5 °C, the scattering curve is characteristic of a lamellar structure with two Bragg reflections located in q1 ) 0.089 Å-1 and q2 ≈ 2q1. They are attributed to the fluid lamellar stacking of periodicity p ) 71 ( 1 Å, the same as described in Figure 4a, at high temperature. As shown in Figure 4b, the transition from the “X” structure to the fluid lamellar stacking occurs in two steps. In the first step, from T ) 35 to 55.5 °C, i.e., at the chain melting transition, the

5294 J. Phys. Chem. B, Vol. 105, No. 22, 2001

Raspaud et al.

Figure 5. Type “X” lipopolyamines/DNA complexes (b,c) and lipids in excess (a) observed in cryoTEM after staining with uranyl acetate. Bar ) 100 nm. a: Lipids in excess form isolated polygonal vesicles (arrows). Some of them appear as thin platelets when seen in side view (arrowheads). b,c: 3D lipopolyamines/DNA complexes formed by aggregation of lamellae and polygonal vesicles. The lamellar stacking, which does not extend over more than 3-4 layers, can be also seen in some places (white arrows). Lipids vesicles are also glued to the aggregates (arrows). d: upon drying of the sample, polygonal lipid vesicles close pack as seen in the background (arrows). Dense lipids/DNA aggregates are seen as well (white arrows). Their 3D structure seems to be preserved. Classical TEM observation after uranyl acetate staining.

first Bragg reflection due to the fluid lamellar stacking appears and the second-order reflection also emerges from the scattered intensity (indicated by arrows). This first Bragg reflection enlarges the second peak associated with the “X” structure. In the second step, from T ) 68.5 to 83.5 °C, the first and second Bragg reflection due to the fluid lamellae become more intense and the two peaks associated with the “X” structure disappear. Although the two peaks disappear when the temperature is at least 20 °C above Tm, we do not suspect a structural reorganization independent of the Tm transition. More probably a slow process occurs and the stabilization times at each temperature were not long enough compared to the structural reorganization time. It can be also noted that the q-values of the peak(s) sligthly increase with the temperature for the lamellar structure while

they decrease for the “X” structure. This two-step process also confirms that the second peak observed in Figure 1b is not related to the lamellar stacking but is truly associated with the “X” structure. To check that this “X” structure is well-induced by the presence of DNA, a lipidic solution was prepared in the conditions required to form this “X” structure. This lipidic solution, without DNA, was centrifuged (up to 400 000 g) and no precipitate was detected, meaning that lipids alone do not aggregate. We also checked that DNA is well-condensed by the lipids since DNA is not accessible anymore to fluorescent probes. Finally, electron microscopy experiments were performed to analyze the structure of lipid/DNA complexes of type “X” and

Polymorphism of DNA/Multi-cationic Lipid Complexes the structure of pure lipids. Because of the limiting thickness of the vitrified liquid film studied by cryo-TEM, small complexes have been prepared in excess of lipids (at a lipid/ DNA charge ratio ) 15 and at a DNA concentration ) 0.4 g/L). The structure of the complexes and of the pure lipids can be observed simultaneously in the same sample (Figure 5). Isolated lipids are organized into faceted vesicles, 15 to 20 nm in diameter on average (Figure 5a). Polyhedral vesicles were already described in dimyristoylphosphatidylcholine (DMPC)14 and in dioctadecylamidoglycylspermine (DOGS)],9 below the chain melting temperature. They are due to the fluid-to-solid transition of the chains, and the polyhedral shape comes from the impossibility for solid bilayers to pack into a regular sphere.15,16 Along with this transition, part of the vesicles are broken, and a lot of platelets 15 to 20 nm large and 6 nm thick may be seen around (arrowheads in Figure 5a). DNA/lipids complexes are formed by lamellae stackings and by vesicles and platelets (Figure 5b,c). When stained and dried onto a grid, these DNA/lipid aggregates and faceted lipid vesicles in excess agglomerate. We observe a dense packing of the polyhedral vesicles and from place to place the stacking of a few lamellae (Figure 5d). X-ray diffraction has been done in parallel on the sample analyzed by cryo-TEM (Figure 5 a-c). In the absence of any pellet, a weak signal is recorded (not shown) and instead of the expected peaks associated with the “X” structure, a single large peak is observed at a q value equal to 0.075 Å-1. This peak is characteristic of the lamellar stacking seen on the micrograph (Figure 5b,c). No periodic organization of characteristic size (2π/0.04) ∼ 150 Å is detected, which is in fact consistent with the observation made by cryo-TEM. This means that the “X” structure can be observed only in the case of macroscopic aggregates. Discussion The series of experiments reported here show that the polymorphism of the DNA/lipid complexes is driven by the initial state of the cationic lipid. Lipopolyamine RPR120535 is known to form micelles or other shapes of aggregates, depending on the pH of the solution and temperature,5,9 as classically reported with other lipids, but its complete phase diagram is not known. Moreover, in our systems, the “temperature history” of the lipid sample is of importance since experiments are performed in a range of temperature including Tm. Therefore, different lipid phases might be found, the nature of which depends on the combination of three parameters: salt concentration, temperature, and “history” of the sample. In addition to these three parameters, one may include the fact that our lipidic samples have been also vortexed. The vortex effect, which is comparable to sonication effect, is well-known to modify significantly the shape of lipid aggregates (cf for instance ref 17). From our observations, we propose the tentative diagram presented on Figure 6. We suspect the micellar phase to exist at low salt (CNaCl < 0.35 M) and to be temperature insensitive for lipid concentrations below 20 g/L. When micelles are diluted above 0.5 M NaCl, lipids may organize in three different ways: (i) for T < Tm, bilayers probably arrange into large platelets, as suspected from the observation of the solutions (not vortexed) in phase contrast microscopy (data not shown), (ii) small fluid quasi spherical vesicles at T > Tm, and (iii) faceted vesicles, some of them broken, when these fluid vesicles are cooled below Tm too rapidly to let them reach the equilibrium state. We suspect the fluidity of the aliphatic chains of the lipids be restricted and this quenched state be metastable. These deduc-

J. Phys. Chem. B, Vol. 105, No. 22, 2001 5295

Figure 6. Proposed diagram of pure lipopolyamines as a function of temperature (T) and NaCl concentration. Depending on the initial state of the lipids, either a lamellar structure “L” or aggregates “X” are formed when DNA is added. t1, t2, t3, and t4 correspond to the four transitions induced by the addition of DNA to the different states of pure lipids solutions.

tions are also supported by preliminary light scattering experiments (data not shown) showing that vesicles formed in high salt above and below Tm have a comparable size (typically 1525 nm). Multiple types of lipid/DNA complexes are obtained accordingly, as summarized in Figure 6. They may originate from the association of DNA with lipid micelles (transition 1, t1), unknown structures (t2), spherical vesicles (t3), or intact plus broken facetted vesicles (t4). As regards the lamellar complexes, all the characteristics of the stacking are identical whatever the transition t1, t2 and t3. As shown in a previous study,5 the extension of the stacking to approximately 5 lamellae is also preserved whatever the final size of the lipid/DNA aggregates. In excess of lipids, these lamellar stackings are isolated and coexist with micelles while for charge ratios of the order of unity, they agglomerate and form a macroscopic aggregate. The formation of such aggregates suggests a two-step process: in a first step, a fast association kinetic of lipids with DNA to form the lamellar stacking (cooperative process), acting as a kinetic trap and in a second step, a slower agglutination kinetic of the lamellae among themselves. This second step should depend on the DNA and lipid concentrations and on the surface potential of the complexes. Since the lamellar stackings are clumped into large aggregates, one might expect a third step corresponding to a fusion of the lamellae. In our case, this third step is not seen experimentally at room temperature probably because of the slow lipids motion. Moreover, this motion is frozen because lipids are below their chain melting temperature Tm. This is

5296 J. Phys. Chem. B, Vol. 105, No. 22, 2001 confirmed by an X-ray diffraction experiment performed at wide angle on macroscopic aggregates at room temperature (data not shown). A thin peak is detected at q ) 1.57 Å-1, corresponding to a distance of 4 Å between the ordered hydrocarbon chains. For T > Tm, the faster lipid motion yields possible the fusion of the lamellae. As shown in Figure 4, the width of the first lamellar reflection is reduced and the second-order Bragg reflection is detected, indicating an extension of the lamellar domains. The “X” structure, which remains to elucidate, is only observed via the transition t4. Moreover, the two intense peaks associated with this structure disappear when microscopic aggregates are formed in a large excess of lipids. It suggests either that the internal structure of the complexes change with the charge ratio or that this signal is due to a local structure induced by the agglutination of the complexes. In fact, we have tested the first hypothesis by performing X-ray scattering experiments on macroscopic aggregates formed at different charge ratios. For charge ratios comprised between 2 and 10, macroscopic pellets have been analyzed and no change was observed, meaning that the local organization of type “X” is independent of the charge ratio. As regards the second hypothesis, which becomes the most probable, all components present in the microscopic aggregates should be preserved in the macroscopic aggregates. As observed by cryo-TEM, they are composed of some lamellar stackings, connected vesicles, and platelets. As mentionned previously, the peak due to the lamellar stackings could be included in the second peak in Figure 1b. The two intense peaks observed at lower q-values should then be due to the connected vesicles. One may suggest that during the growth of the macroscopic aggregates, vesicles coming from different small lipid/DNA aggregates could be packed together and form a dense close-packing as in Figure 5d. One may not exclude that at the same time, some isolated vesicles could be also caught into this packing. This kind of organization could give the whole X-ray diffraction profile (refer to the sphere packing clusters diffraction diagram11). The characteristic distance of this packing given by the first peak position d ∼ 150 Å should then correspond to the vesicle size. This value is small considering that the bilayer thickness is of the order of 50 Å but it remains consistent with the size of the smallest vesicles observed by cryo-TEM. This interpretation explains why for DNA and PSS systems, one observes a characteristic distance of the same order of magnitude. This is also consistent with a characteristic distance varying from one sample to another because the preparation method of the lipidic samples, such as the vortex conditions (magnitude and period) which induce the formation of vesicles of a given size, cannot be rigorously identical for all the samples. Further work will be necessary to determine precisely the origin of this structure and especially to determine if the packed vesicles are distorted and in particular flattened as observed experimentally by Huebner et al. (see Figure 4 in ref 18) and Sternberg et al.19 We rather suspect that these packed vesicles remain faceted because of their surprising stability and because lipids are thought to be strongly trapped into the faceted bilayers. In the sample analyzed by cryo-TEM, where isolated faceted vesicles coexist with lipid/DNA aggregates, the NaCl concentration of the initial lipid solution is 1 M. It is lowered to 0.26 M in the final lipid/DNA solution. In such conditions, lipids alone should reorganize into micelles (cf Figure 6) but they remain trapped into faceted vesicles. In the same way, this is illustrated by the thermotropic behavior of the vesicles packing as described in Figure 4b, showing that

Raspaud et al. one must heat at higher temperature than Tm or longer to destroy such organization. The formation of clusters of small faceted vesicles and broken fragments stuck together by the polyelectrolyte was already reported with the cationic polyelectrolytes JR400 and LM200 and mixtures of anionic SDS and cationic DDAB surfactants.20,21 In the reported conditions, the adsorption of the polyelectrolyte onto the vesicles raised the Tm value and induced the faceting of the vesicles. Contrary to our system, the two effects could not be uncoupled. In our system, the same variety of structures was obtained by using linear DNA (150 to 48 000 bp) instead of supercoiled DNA, and KCl or MgCl2 instead of NaCl as added salts into the lipid solution (data not shown). On the contrary, changing the lipid counterion (fluoroacetate instead of acetate) or using another lipopolyamine prevents the formation of the “X” structure. Either the lipids are insoluble when they are cooled from the fluid to the solid state, or lipid/DNA complexes are always lamellar. This confirms the preponderant effect of the lipid with its counterions on the polymorphism of the complexes. In the course of the complex formation, both reactants (DNA and lipids) usually undergo coupled phase transitions.22 In all cases reported here, DNA condenses from an extended state to a confined state, and whatever the structure of the complexes, DNA is thought to be finally sandwiched between planar lipid bilayers. Under our range of experimental conditions, lipids also exhibit a large polymorphism: the apparent head diameter is significantly decreased when NaCl goes from 1 mM to 1 M and the aliphatic chains are fluid above Tm. Both parameters will change the head-to-chains volume ratio, and consequently promote reorganization of the lipids from micelles to bilayer structures. The pilot role played here by the cationic lipid is therefore easily understood. In the absence of NaCl, the neutralization of the charged heads of the lipid and consequently their reorganization into bilayers is due to the DNA molecule itself. In conclusion, we report here a polymorphism of cationic lipid/DNA complexes, which is dependent upon the temperature treatment applied to the cationic lipid solution, prior to DNA addition and complex formation. Acknowledgment. Preliminary X-ray diffraction and light scattering experiments were done at the LPS with the help of Olivier Pelletier, Marianne Clerc-Imperor, and Patrick Davidson and at LLB, CEA Saclay with the help of Didier Lairez, respectively. We also thank Jean-Franc¸ ois Sadoc, Pierre Wills, and Virginie Escriou for fruitful discussions, Marc Airiau for discussions on complex formation, and Marc Frederic for contributing to the synthesis of RPR120535. We thank Joe¨l Crouzet for his constant support and interest. This work was supported by a CNRS-Rhone Poulenc Rorer contract. References and Notes (1) Huang, L. Gene Ther. 2000, 7, 31. (2) Koltover, I.; Salditt, T.; Ra¨dler, J. O.; Safinya, C. R. Science 1998, 281, 78. (3) Mok, K. C.; Cullis, P. R. Biophys. J. 1997, 73, 2534. (4) Ra¨dler, J. O.; Koltover, I.; Salditt, T.; Safinya, C. R. Science 1997, 275, 810. (5) Pitard, B.; Aguerre, O.; Airiau, M.; Lachage`s, A.-M.; Boukhnikachvili, T.; Byk, G.; Dubertret, C.; Herviou, C.; Scherman, D.; Mayaux, J.-F.; Crouzet, J. Proc. Natl. Acad. Sci U.S.A. 1997, 94, 14412. (6) Gao, X.; Huang, L. Biochem. Biophys. Res. Commun. 1991, 179, 280. (7) Felgner, J. H.; Kumar, R.; Sridhar, C. N.; Wheeler, C. J.; Tsai, Y. J.; Border, R.; Ramsey, P.; Martin, M.; Felgner, P. L. J. Biol. Chem. 1994, 269, 2550.

Polymorphism of DNA/Multi-cationic Lipid Complexes (8) Byk, G.; Dubertret, C.; Escriou, V.; Frederic, M.; Jaslin, G.; Rangara, R.; Pitard, B.; Crouzet, J.; Wils, P.; Schwartz, B.; Scherman, D. J. Med. Chem. 1998, 41, 224. (9) Boukhnikachvili, T. Thesis, Universite´ Paris XI, France 1988. (10) Dubochet, J.; Adrian, M.; Chang, J.-J.; Homo, J.-C.; Lepault, J.; McDowald, A. W.; Schultz, P. Quarterly ReV. Biophys. 1988, 21, 129. (11) Sadoc, J. F. Thesis, Universite´ Paris-Sud, Orsay, France, 1976. (12) Radlinska, E. Z.; Gulik-Krzywicki, T.; Lafuma, F.; Langevin, D.; Urbach, W.; Williams, C. E. J. Phys. II (France) 1997, 7, 1393. (13) Zantl, R.; Artzner, F.; Rapp, G.; Ra¨dler, J. O. Europhys. Lett. 1998, 45 (1), 90. (14) Sackmann, E. FEBS Lett. 1994, 346, 3. (15) Lasic, D. D. Liposomes. In From Physics to Applications; Elsevier: Amsterdam, 1993.

J. Phys. Chem. B, Vol. 105, No. 22, 2001 5297 (16) Sackmann, E. Physical basis of self-organization and function of membranes: physics of vesicles. Handbook of Biological physics, Vol 1; Lipowsky, R., Sackmann, E., Eds.; Elsevier: Amsterdam, 1995. (17) Marques, E. F. Langmuir 2000, 16, 4798. (18) Huebner, S.; Battersby, B. J., Grimm, R.; Cevc, G. Biophys. J. 1999, 76, 3558. (19) Sternberg, B.; Sorgi, F. L.; Huang, L. FEBS Lett. 1994 356, 361. (20) Regev, O.; Marques, E.; Khan, A. Langmuir 1999, 15, 642. (21) Marques, E. F.; Regev, O.; Khan, A.; Miguel, M.; Lindman, B. Macromolecules 1999, 32, 6626. (22) Lasic, D. D.; Templeton, N. S. AdV. Gene Ther. 1996 20, 221.