DNA–cationic amphiphile interactions

Colloids and Surfaces A: Physicochem. Eng. Aspects 228 (2003) 43–55

DNA–cationic amphiphile interactions Maria G. Miguel a,b,∗ , Alberto A.C.C. Pais a,b , Rita S. Dias a,b , Cec´ılia Leal a,b , Mónica Rosa a,b , Björn Lindman a,b b

a Chemistry Department, Coimbra University, 3004-535 Coimbra, Portugal Physical Chemistry 1, Center for Chemistry and Chemical Engineering, P.O. Box 124, 22100 Lund, Sweden

Abstract DNA shows strong interactions with cationic cosolutes and these have both biological and technological significance. We outline our research on various mixed systems of DNA and cationic amphiphiles including the interaction of DNA with simple cationic surfactants as well as the interaction with catanionic mixtures and positively charged catanionic vesicles. An overview from phase behavior to microstructure will be presented. We will also address DNA compaction and decompaction phenomena in different systems. Finally, simulations on DNA confinement and interaction with cationic polyions are considered. © 2003 Published by Elsevier B.V. Keywords: DNA; Cationic amphiphiles/surfactants; Amino acid-based surfactants; Catanionic mixtures; Catanionic vesicles; DNA–amphiphile complexes; Polyions; Phase behavior; DNA hydration; DNA compaction/decompaction; Fluorescence microscopy; Cryo-TEM; Small angle scattering; Sorption microcalorimetry; Molecular simulations

1. Introduction The interactions in mixed solutions of DNA with cationic cosolutes—multivalent ions, cationic amphiphiles, and cationic macromolecules—have attracted a great interest from the biomedical sciences, not only because of its direct biological implications, but also for a number of applications concerning separation, purification and transfection of DNA. Given their fundamental relevance, it is time for physical chemists to devote particular attention to these systems, aiming to a better understanding of the driving

∗

Corresponding author. Fax: +351-239-82-7703. E-mail address: [email protected] (M.G. Miguel).

0927-7757/$ – see front matter © 2003 Published by Elsevier B.V. doi:10.1016/S0927-7757(03)00334-0

forces behind the molecular interactions; this is also expected to increase their efficiency and applicability. The introduction of foreign DNA to cells (transfection) is widely used in biotechnology and has received, over the recent years, considerable attention in medicine for curing genetic diseases (gene therapy). Gene “packaging”—the formulation of the genetic material in order to make it suitable for administration and delivery—is a physical pharmaceutics problem with significant aspects of fundamental physical chemistry involved. The basic requirements for effective transfection vectors is the ability to compact DNA, to protect it against degradation and to deliver it to the cell membrane with efficiency and specificity, and finally to facilitate the DNA transport through the cell membrane [1]. The most efficient transfection

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vectors currently used are based on viruses, but they suffer from severe limitations [1]. Much effort is aimed today to the development of “artificial” vectors formed by the complexation of DNA with cationic lipids or polymers [2]. Such self-assembling vectors are safer than viruses, have large carrier capacity, are applicable to all cell types, and easier to produce. However, a major drawback has been their low transfection efficiency. Cationic lipids promote the condensation of the negatively charged DNA into a more compact structure, capable of crossing biological membranes. This complexation also protects DNA from degradation. A number of cationic lipids have been used in transfection protocols [2,3]. However, “although cationic lipid-based gene delivery systems are being intensively investigated and novel cationic lipid molecules are synthesized routinely, no definite structure–activity relationship has clearly emerged so far” [3]. A pressing need emerges for a systematic characterization of the DNA–lipid complexes as “packaging” systems for transfection. Mixed systems with surfactants/lipids can be used as packaging agents for delivery of nucleic acids to cells. We are interested in understanding interactions and studying and controlling microstructures formed, both in bulk and at interfaces. The condensation and protection of double- and single-stranded DNA by complexation with cationic amphiphiles is currently studied by our group using a wide range of systems and experimental techniques. Topics investigated include phase behavior, microstructure characterization and compaction of DNA with simple quaternary ammonium surfactants, amino acid-based surfactants as well as mixtures of cationic and anionic surfactants, and thermodynamically stable catanionic vesicles [4–7]. We are using for this purpose a broad range of techniques as nuclear magnetic resonance (NMR), small-angle X-ray scattering (SAXS), small-angle neutron scattering (SANS), fluorescence, optical and cryogenic transmission electron microscopies, microcalorimetry, and ellipsometry. Our research program, started quite recently in this area, was initiated with studies in model systems. We plan to extend our investigations to more complex systems, and perform toxicity tests and in vitro and in vivo tranfections; a promising lead emerges from the correlation found between DNA compaction and

transfection efficiency in previous studies of DNA interactions with polycations, chitosans with different chain lengths [8]. Support from theoretical and simulation studies will improve our knowledge on molecular interactions and on the general physical chemistry of these complex systems. Promising results have been obtained already in the several aspects of association [9,10], confinement [11], and compaction [12,13]. With our long experience on fluorescence methods we are developing techniques suitable to investigate these systems, correlating experimental and theoretical data [9], and studying kinetics, an important area, still in its infancy [14,15]. This background will allow us to better design systems for DNA compaction/decompaction and establish compaction/transfection relations, as a basis for more biological and medical needs.

2. DNA interaction with cationic surfactants A polyion interacts associatively with oppositely charged cosolutes, in particular if they are highly charged as cationic surfactant self-assemblies and polycations [16,17]. For surfactants, this can be described in terms of a binding isotherm. Such cooperative surfactant binding is found for many polymer–surfactant systems and is best described in terms of a polymer-induced surfactant self-assembly; in line with this the binding isotherm is shifted to lower surfactant concentrations with increasing surfactant alkyl chain length. Different types of surfactant self-assemblies are possible; the most studied case is where the surfactants form discrete, roughly spherical micelles; then a polymer-induced micellization, and a CMC, or a critical association concentration, CAC, can be considered. While the CMC/CAC is only weakly dependent on polymer molecular weight, for polyion–surfactant systems it decreases strongly with increasing linear charge density of the polymer, as shown by experimental [18,19] and theoretical work [20,21]. The role of polyion flexibility has also been investigated and it was found that as the polyion becomes more flexible there is a stronger association [22,23]. Due to hydrophobic interactions, surfactants, like other amphiphiles, self-assemble in aqueous systems. Depending on the balance between the polar and

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the non-polar parts, a large number of self-assembly structures are possible [24–26]. Other factors may also influence the structure formed, such as added cosolutes [27,28]. In the case of DNA–surfactant systems, micelles of different shapes, (normal or inverse) hexagonal and lamellar phases are the cases described so far [29–34]. Other structures such as cubic liquid crystalline phases, are also expected but remain to be studied. 2.1. General phase behavior Double helix DNA is a high molecular weight rigid polyanion, of high linear charge density, compared with other studied polyion–oppositely charged surfactant systems, with one charge per 0.17 nm (for the double-helix B-form DNA) [35]. For an oppositely charged polyelectrolyte–surfactant pair, an associative phase separation is in general observed over wide concentration regions, i.e. there is a separation into one phase rich in both polymer and surfactant, and one dilute solution phase. This phase separation is entropically driven, determined by the translational entropy of the counterions. The surfactant molecules are in an aggregated form in the concentrated phase, but different types of aggregates are formed in different systems [29–32,36–39]. At higher surfactant concentrations, “redissolution” usually takes place. If the electrostatic driving force for association is eliminated or screened, a segregative phase separation, characteristic of systems with a low entropic driving force of mixing, can be expected, i.e. there is a formation of two solution phases, one enriched in polymer and the other in surfactant, as large amounts of electrolyte have been added [39]. At intermediate salt concentrations, there is no phase separation [28]. DNA–cationic surfactant systems only partially appear to display this behavior. Our phase diagrams studies [4] show a strongly associative behavior. The associative phase separation is enhanced as the alkyl chain length of the surfactant increases, which is also observed for other polymer–surfactant systems. This is in line with a lower CMC/CAC and a larger surfactant aggregate for longer alkyl chains [40,41]. However, some features are different from previous studies of polyelectrolyte–surfactant interactions. Associative phase separation starts at very low

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concentrations of DNA or surfactant, possibly due to a “double cooperativity” on the binding of surfactant to DNA, i.e. there is not only surfactant self-assembly but also binding of surfactant aggregates to one DNA molecule that facilitates further binding. No redissolution at high surfactant concentrations is observed, which may correspond to a quantitative rather than qualitative difference from other polymers and might then be possible to rationalize in terms of the very high linear charge density [18–21]. The salt effect was shown to be complex [4] since the associative phase separation could be either enhanced or decreased depending on the conditions. When working with DNA some electrolyte is necessary to keep the molecule in its native state (double helix). Without such additives the DNA strands drift apart due to electrostatic repulsions [35]. This indeed explains the unexpected results obtained: we have a different phase behavior for DNA as a double-helix and as denaturated single stranded. The fact that DNA–surfactant systems show a different behavior if the DNA molecule is in its native (ds-DNA) or denaturated (ss-DNA) state is relevant and has been the subject of several studies. We performed phase map studies relating the dependence of the phase behavior of DNA–cationic surfactant systems on temperature and the amount of salt. The characterization of the primary structure of the DNA molecule was carried out by circular dichroism (CD) and melting temperature curve determinations [42]. DNA structure will be determined in the DNA–cationic surfactant precipitate by infrared spectroscopy (IR). Theoretical phase maps will be built for comparison with experimental data. Many questions remain to be answered. What is the concentrated phase? What do we know about its structure, solubility and water uptake? How do polymeric counterions affect the interactions, shape and size of ionic surfactant aggregates? We are developing several approaches aiming to a better understanding of these issues. For this, we are re-investigating the phase behavior on these mixtures, determining the microstructure of the concentrated phase for different systems, and studying the hydration of DNA–amphiphile complexes by a new calorimetric method. All these studies are in progress in our laboratories. Some results will be reported below.

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2.2. A new approach to phase behavior The description of phase diagrams for these systems in two-dimensional plots is incomplete and can be misleading. A system of two electrolytes, without common counterions, and a solvent cannot in general be treated as a ternary system. One consequence is that the composition of separated phases does not in general lie in the “conventional” mixing plane used. The mixture of a simple surfactant (surfactant ion+counterion) and a polyelectrolyte (polyion+counterion) in water is a complicated four-component system, and therefore, a full description of its phase behavior requires a three-dimensional representation [24,25]. Due to the fact that even a sketchy three-dimensional phase diagram determination is a considerable undertaking, there are very few of these maps available in the literature [20]. In order to proceed we will introduce a simplification previously used for other polymer–surfactant mixtures [36], as well as for mixed surfactant systems [43]. In these ongoing investigations we start from the “pure complex salt” (synthesized from the surfactant ion and the polymeric ion), thus eliminating one of the inorganic counterions in the mixture. Then, the aqueous mixture of this “complex salt”, DNA with the surfactant as counterion, can be studied with either the simple surfactant or the polyelectrolyte. These aqueous mixtures give a truly ternary system, accessible for detailed study. Our most recent results for the system DTADNA/ DTAB/WATER show that all the samples studied so far are birefringent; the complex is extremely insoluble at high water content (75–100% water) and is less insoluble in the micellar (60–75% water) and hexagonal phase (55–35% water) the binary phase diagram of dodecyltrimethylammonium bromide (DTAB). 2.3. Hydration of DNA–surfactant complexes Water is ubiquitous in biological and surfactant systems, which makes sorption/drying processes of paramount importance. The sorption of water by a condensed phase can strongly influence its physical properties, such as phase state, molecular structure, solubility and reactivity [44–46]. Most of the molecular processes of life depend on intermolecular and interaggregate interactions in an aqueous environment

[47]. Water activity is the main factor affecting the conformation of nucleic acid [48]. Some intercellular environments in which DNA functions in vivo resemble hydrated powders and films more than dilute solutions. For these low water content states, variations in the amount of internal and surface water can have pronounced effects on DNA structure and function. In spite of its relevance, and although several methods have been used to picture the hydration of DNA and DNA–amphiphile complexes [49–51], a clear characterization has not been achieved for these systems. We are investigating the isothermal DNA and DNA– amphiphile (1:1) hydration by sorption microcalorimetry, using a double-twin isothermal microcalorimeter. This novel method provides simultaneous measurement of partial molar enthalpy of water in the sorption process (differential sorption enthalpies) and the amount of water uptake (sorption isotherms) by DNA and the complex [52]. The versatility of the method has recently been demonstrated for studies of phospholipids hydration [53]. Our results allow some interesting conclusions: DNA hydration is exothermic; stabilization of A form is observed for approximately ten water molecules per base pair and 0.62 relative water activity; and the partial molar enthalpy of water is −3.2±0.2 kJ mol−1 (H2 O). Stabilization of DNA B form appears at 0.80 water activity for ∼20 water molecules; and the partial molar enthalpy of water is 0. At this point the hydration is complete. DNA additionally takes up to 35 water molecules per base pair, reaching 0.92 water activity, with no further changes in the partial molar enthalpy of water, which is consistent with the growth in water layers in the sample [54]. In respect to DNA–amphiphile complexes, reported below for the CTADNA complex (DNA:CTAB (cetyltrimethylammonium bromide), 1:1), the complex takes a considerable amount of water and no DNA conformational changes were observed during hydration. Hydration is exothermic for the incorporation of the first seven water molecules, at 0.68 water activity. Further incorporation of water up to ∼22 water molecules, at 0.96 water activity is endothermic (Fig. 1). The process is then entropically driven, which seems consistent with an order–disorder transition of the hydrocarbon surfactant chains [54]. We believe that this ongoing project, based on the simultaneous study of the partial molar enthalpy of

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Fig. 1. Differential enthalpy of sorption (−sorp H) vs. water content for DNA (—, 25.0 mg) and DNACTA (· · · , 21.3 mg) (from [54]).

water and the chemical potential (partial molar free energy), provides unique thermodynamic data for the water sorption on the DNA–amphiphile systems currently under investigation and will bring valuable understanding to more complex systems. 2.4. Microstructure of DNA–surfactant complexes The surfactant molecules are in an aggregated form in the concentrated phase; spherical and rod-like micelles (normal or inverse) hexagonal phase and lamellar phase have been discussed [27–32,36–38]. The factors determining the type of aggregate formed can be assumed to be the same as for simple surfactant systems, a statement supported by some recent work [36]. For studies of the “precipitate”, we determine the average and “local” microstructure of the complexes, after removing excess water, by SAXS and optical and cryogenic transmission electron microscopy (cryo-TEM). Some amphiphiles are well known to self-assembly into lamellar, cubic, and hexagonal structures. Also, several structures have been proposed for DNA–lipid complexes [29–34]. Based on SAXS studies (Fig. 2) we conclude that DNA–cationic surfactant complexes present a hexagonal structure; although this is less clear for shorter chain surfactants.

An inverted hexagonal structure has been claimed for some DNA–lipid complexes [55]. In our studies, however, we do not have any evidence of an inverted structure, a topic that deserves further investigation. In view of the self-assembly of these cationic surfactants we would, however, expect the hexagonal phase to be of the “normal” type. SAXS is powerful in assessing the lyotropic liquid crystalline organization in the complexes [56], but it

Fig. 2. Small-angle X-ray diffractograms of DNA–cationic surfactant precipitated complexes. T = 25 ◦ C (redrawn from [95]).

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averages over different microdomains in the sample volume. This is where cryo-TEM is very useful; EM has a limited spatial range, but great spatial resolution, and easily discriminates between microdomains of different structures [57]. In some of our studies on microstructure characterization we have then used more than one technique, as shown below for the interaction of DNA with catanionic vesicles [7].

3. DNA interaction with catanionic mixtures The relevance of investigating DNA compaction in mixed systems of a cationic surfactant and an anionic surfactant or polymer relates not only to the control of DNA compaction and to the possibility of reversing compaction, but also to the fate of compacted DNA in a biological system, where it will be interacting both with anionic lipids and with anionic polysaccharides. We will present some of our results on the interaction between DNA and catanionic mixtures, i.e. mixtures of cationic and anionic surfactants. 3.1. DNA compaction and decompaction The compaction of DNA can be conveniently monitored by fluorescence microscopy [58,59]. The general aspects of DNA compaction by cationic

surfactants have been described by Yoshikawa’s group [31,60–64] and others and by our group recently [4,65]. Some illustrative fluorescence micrographs are presented in Fig. 3. A number of features can be observed. On addition of cationic surfactant to a DNA solution there is a compaction of DNA molecules from an elongated “coil” conformation to a compacted one, “globule”. For intermediate concentrations a coexistence of “coils” and “globules” is found. Additionally, as the surfactant alkyl chain length increases, compaction occurs at lower surfactant concentrations. A number of other features can be observed in microscopy as surfactants are added to DNA solutions, such as different topologies, like toroids, DNA precipitation and DNA aggregation. Compaction is a general phenomenon in the presence of multivalent ions and positively charged surfaces [66–68] and can be reversed on addition of electrolyte and anionic surfactants [5,6]. It seems that in view of the high molecular weight of DNA we can to some extent regard this compaction as an associative phase separation on the single molecular level. We see indeed that this “microscopic phase behavior” parallels the macroscopic one reviewed above. A striking observation appears to be that of coexistence of coils and globules in a sizeable concentration range, also found with other cosolutes like flexible

Fig. 3. Schematic diagram of the compaction and decompaction of DNA by cationic and anionic surfactants. Fluorescence microscopy allows the visualization of DNA molecules in compacted conformation after the addition of a positively charged surfactant. With addition of an anionic surfactant, the DNA molecule is released into solution as a coil. For intermediate concentrations of surfactants both DNA conformations coexist in solution.

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polymers [69] and multivalent ions [66,67,70]. Surfactant molecules are unevenly distributed between DNA molecules [31] and binding shows a “double cooperativity”. The mechanism for this is not clear and deserves further investigation, but in our opinion it has to be analyzed in terms of extended (“infinite”) self-assemblies formed and the polyion compaction due to strong attractive electrostatic correlation effects [71–75]. It should be observed that the coexistence is in agreement with the phase diagram work giving a two-phase behavior at very low surfactant contents. DNA compaction can be reversed in mixed surfactant systems. Fig. 3 illustrates how mixed surfactant systems can be used for control of compaction. The unfolding of DNA molecules previously compacted with cationic surfactant was shown to be dependent on the anionic surfactant chain length; lower amounts of a longer chain surfactant were needed to release DNA into solution. This is in line with the chain length dependence of surfactant self-assembly. When adding the anionic surfactant to the solution of DNA and cationic surfactant, above a certain concentration, it will associate and form mixed self-assemblies with the oppositely charged amphiphile and release DNA back into the solution as a coil. The onset of this association will be determined by a critical micellar concentration for the mixture of the two surfactants (CMCmixt ). As an example, sodium dodecylsulfate (SDS) is much more efficient in unfolding DNA than the shorter chained surfactant, sodium octylsufate (SOS) [6]. On the other hand, our results show no dependence of decompaction on the hydrophobicity of the compacting amphiphile (CTAB, tetradecyltrimethylammonium bromide (TTAB), DTAB). Above a certain CMCmix two types of structures can be formed in solution, DNA–cationic surfactants complexes (“globules”) and cationic–anionic surfactant aggregates. Contrary to the process described above, complexes dissociation imply a “transfer” of molecules between two surfactant self-assemblies, which will not depend on the surfactant chain length [6]. 3.2. Reversible amphiphile self-assembly An important conclusion is that surfactant–surfactant interactions are stronger than polymer–surfactant interactions. This leads to the formation of surfactant aggregates in solution on the sequence of DNA

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compaction, unfolding and release of DNA into the solution. The structure of these self-assemblies was also investigated by cryo-TEM [6]. We detected very interesting phases, like spontaneously formed catanionic vesicles. We also found that it is possible to predict and control these structures, if the phase behavior of the mixed-amphiphile system involved is well established. This brings a new insight on the compaction/decompaction phenomena. Several interesting opportunities for applications may emerge, like DNA purification and controlled DNA delivery. Specifically for DNA delivery purposes, and taking into account toxicity factors, the phase behavior of mixtures with oppositely charged biocompatible lipids, with a special interest on possible vesicles regions, should be studied. 4. DNA interaction with catanionic vesicles Kinetically stable (i.e. non-equilibrium) vesicles have been extensively considered as DNA vehicles; in these cases, a two-phase dispersion lamellar phasedilute molecular solution is the equilibrium state. Vesicles of different charges have been investigated with a focus on positively charged ones [5]. Though not always essential, cationic lipids are typically employed in combination with neutral/zwitterionic lipids. These “helper” neutral lipids are incorporated in the liposomes in order to increase their stability [76], reduce the cytotoxicity of the cationic lipids [77], or increase the membrane fluidity during the transfection [78,79]. Our group has during some time investigated vesicles in mixed cationic+anionic surfactant systems which form spontaneously, have an apparently unlimited life-time and which are believed to be thermodynamically stable [25,80,81]. The vesicles are polydisperse with sizes in the approximate range of 0.05–0.5 ␮m although there are also a few larger ones; these last can be directly visualized by optical microscopy while the smaller ones were conveniently studied by cryo-TEM [80,82]. In our work we have introduced investigations on the interactions between these “catanionic” vesicles and DNA [5,7]. 4.1. Phase behavior Phase maps for mixed polymer–vesicle systems are rare in literature. The aqueous system of DNA and

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positively charged catanionic vesicles, composed of CTAB and SOS showed a strong associative phase behavior with the formation of a precipitate. A two-phase region was observed over all the studied concentrations. DNA–vesicle complexes are formed for very low amounts of DNA and coexist with undisturbed vesicles until a DNA-to-lipid charge ratio of 1.3. For higher DNA concentrations only DNA–vesicle complexes were observed in solution. A full structural characterization was performed by optical and electron (cryo-TEM) microscopy and by SAXS [7]. 4.2. Microstructure of DNA–catanionic vesicles complexes The interaction between DNA and positively charged catanionic vesicles led to the formation of complexes, characterized by a lamellar structure [7], that is illustrated in the Figs. 4 and 5 for cryo-TEM and SAXS studies, respectively. Based on these results we propose a multilayer lamellar structure with DNA intercalated in the bilayers; this kind of structure has been found in other similar DNA–lipid complexes [32,57,83–85]. There is, however, an interesting difference. For DNA–liposome systems, DNA molecules are enclosed within the complexes until the charge neutralization of the system is achieved. After this point, DNA in excess coexists with the lipoplexes. For DNA–catanionic vesicle systems, DNA molecules are incorporated into the complexes even above the overall charge neutralization of the aggregate and anionic micelles are instead expelled from the complex (Fig. 5). Considering that the degree of DNA compaction is an important parameter on the efficiency of gene delivery, we may suggest that the inclusion of a negative amphiphile in the vesicle preparation may induce a denser packing of DNA within the lipoplex. 4.3. DNA compaction In the dilute regime of a phase diagram of some aqueous catanionic mixtures, two regions of stable vesicles may exist; in one the vesicles have a net negative charge, in another a net positive charge [86]. We have investigated the interaction of DNA with catanionic vesicles for different surfactant ratios (water–CTAB–SOS).

Fig. 4. Cryo-TEM image of multilamellar structures formed by the interaction of DNA with catanionic vesicles (CTAB/SOS/water).

In solutions of negatively charged vesicles, no attraction is indicated; there is no compaction of DNA. This indicates that there is no extraction of cationic surfactant from the mixed vesicles, suggesting, once again, that the surfactant–surfactant interaction is stronger than the surfactant–DNA interaction. However, if DNA is mixed with cationic vesicles, on the other hand, a strong interaction is clearly demonstrated. One direct demonstration is the compaction of DNA. We observed that DNA compacts and adsorbs onto the surface of positively charged vesicles, and that the addition of anionic surfactant can release DNA back into solution after being compacted with cationic surfactant. Under conditions of higher DNA concentrations, the situation becomes different, including vesicle disruption and vesicle–vesicle flocculation

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environmental and adverse biological effects of surfactants, involves designing new molecules that mimic natural amphiphilic structures [87]. In this regard, amino acid-based surfactants are of interest because, given their chemical structure, which mimics natural lipoaminoacids, they are expected to be highly biodegradable, milder, and less irritant than conventional surfactants. Arginine-based surfactants, like LAM (N␣ -acyl-arginine-methyl esther hydrochloride) and ALA (arginine-N-dodecyl amide dihydrochloride), are cationic surfactants reported to have satisfactory toxicity profile, high degradability, and a surface activity comparable to that of the conventional long-chain quaternary ammonium salts [88]. Phase diagram studies are being performed on mixed aqueous systems amino acid-based surfactant/anionic surfactant. Different phases are characterized by visual inspection between crossed polaroids, optical and electron microscopy, and SAXS. An important step in this work was taken recently when we were able to demonstrate a spontaneous vesicle formation in such mixed systems (see Fig. 6) [89]. New compounds are being synthesized and, similarly to cationic surfactants, we observed DNA compaction with some of these and other amino Fig. 5. (a) Small angle X-ray diffractogram of precipitated DNA–CTAB/SOS complexes. The amount of DNA on the samples increases from the upper to the bottom curves, varying the concentration ratios [DNA]/[S+ ], D/S. (䉬), D/S = 0.83; (䉱), D/S = 1.00; (䊊), D/S = 1.67; ( ), D/S = 2.00; ( ), D/S = 2.50. (b) Repeated distances, taken from (a), for DNA–DNA spacing, dDNA (filled symbols), and the interlayer distance, dmem (open), as a function of the concentration ratios, [DNA]/[S+ ], D/S. Different symbols correspond to different net surfactant charge concentrations: (䊏), [S+ ] = 1.2 mM; (䉬), [S+ ] = 2.3 mM; (䊉), [S+ ] = 3.5 mM (redrawn from [7]).

[5,7]. These results reveal some non-trivial points that we are investigating both experimentally and theoretically. 4.4. Amino acid-based vesicles Currently, with the basis of the knowledge acquired on the interaction between DNA and conventional surfactants, we are studying the complexation of DNA with amino acid-based surfactants. One reason for this is the biological effects of conventional cationic surfactants. An important strategy in general to minimize

Fig. 6. Cryo-TEM image of amino acid-based catanionic vesicles (ALA/SOS).

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acid-based surfactants, a topic currently under investigation. An important part of our ongoing work is the interaction between single- and double-helix DNA and amino acid-based catanionic vesicles. The observation of single DNA molecules in the presence of surfactants and vesicles are of importance for the understanding of molecular mechanisms of genosome formation, as well as for the formulation of liposome-based transfection delivery systems with optimal molar ratios between cationic and anionic lipid molecules. Further structural information on the DNA–lipid complexes can be obtained with cryo-TEM, X-ray and neutron scattering techniques. These studies would also allow complementary information on the properties of the lipid systems, e.g. the liposome shape, the existence of other liquid crystalline phases, etc. Also, for comparison, dynamic light scattering technique will be used to generate data describing the conformational changes of DNA chains in terms of a cooperative continuous binding. Further studies in this direction may shed light on the correlation between the microstructure of DNA–lipid complexes and their transfection efficiency.

5. Simulating DNA–polyion interactions Important theoretical and molecular simulation contributions have been made to the understanding of interactions on polyelectrolyte–surfactant solutions [20–23,90,91]. Based on our experimental results, and questions to be answered at a molecular level, a research program on molecular simulations of mixed polyelectrolyte–cosolute systems has recently been started in our group. We have used simple model descriptions of the systems in which chains are described as bead and spring assemblies. The solvent enters the model through its relative permittivity. All coulombic interactions are treated explicitly. Models are solved by Monte-Carlo [92]. Some developments and directions of our work will be presented below. 5.1. Confinement studies Studies on molecular confinement provide relevant information for practical systems of polyions confined

into several environments as viruses, vesicles, and zeolite cavities. Model systems representing solutions of a single polyion and its counterions confined to a spherical cavity have allowed the determination of structure, energy, and free energy properties as a function of the cavity radius [11]. Both polyion linear charge densities and different counterion valences have been considered. Systems containing the polyion with the largest linear charge densities and monovalent counterions displayed the largest resistance to confinement in a small sphere. The segment distribution was radially inhomogeneous at all radii considered. For small cell radii, the structure factor displayed signatures of the spherical confinement, and the end-to-end separation and shape ratio were consistent with a spherical coil. The free energy cost in confining the polyelectrolyte was dominated by the ideal contribution of the counterions. At a reduction of the linear charge density, the overall chain flexibility is increased. The reduced extension of the polyion facilitated its adaptation to a smaller cell as concluded from the smaller variation of the single chain properties as the cell radius was reduced. However, for the smallest sphere the conformation of also the most flexible chain was affected. The confining free energy was reduced and became dominated by the ideal contribution of the counterions as the linear charge density was reduced. Finally, at increasing counterion valence, the polyion becomes more compact by the accumulation of the multivalent counterions nearby, and with trivalent counterions the polyion is more compact than a Gaussian chain. Naturally, polyions with multivalent counterions experience less constraints of being confined in a small cavity. The strong coupling between the polyion and its trivalent counterions made the ideal counterion contribution to the confinement free energy to be nearly fully compensated by the contribution from the electrostatic interactions. 5.2. Compaction by oppositely charged chains In work carried out by a significant number of groups, a large variety of compacting agents have been studied. Oppositely charged molecules have proved to induce DNA compaction at significantly lower concentrations than, e.g. non-ionic surfactants. This has prompted significantly work on the interactions

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between DNA and histones [93], chitosans [8] and polyamines [94], which have proven to be very efficient compacting agents. We have recently [12] studied the action of polycations as compacting agents of a medium size polyanion. The systems were characterized in terms of a conformational analysis in which shape, overall dimensions, structure factors and contact of the polyanion with the compaction agents were taken into consideration. Results have shown that the degree of compaction is determined by the size of the positive chains and their number. The role of electrostatic interactions is paramount in the compaction process and, an increase in the number of molecules of the compacting agent or in the number of charges on each molecule causes sudden collapse on the polyion molecule (see Fig. 7 for an illustration of the compacting process). Compaction is associated with polycations promoting bridging between different sites in the polyanion. Another interesting finding is that the interaction for polycation/polyanion charge ratios significantly below 1 produces only a small degree of intrachain segregation, allowing for significant translational freedom of the agent along the longer chain. An important conclusion is that counterions play only a minor role in the folding process. Analysis of the equilibration steps has shown that compaction is

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initiated at both ends of the polyanion, and is completed only when the available positively charged chains are bound to the longer chain. Complete charge neutralization is not necessary for obtaining an appreciable degree of compaction, in accordance with previous experimental studies [94].

Acknowledgements The work described here was supported by grants from JNICT and Praxis XXI (PRAXIS/BD/21227/99), the Fundação para a Ciˆencia e Tecnologia (FCT) (Project Sapiens PCTI/99/QUI/35414), Portugal, the Swedish Research Council for Engineering Sciences (TFR), and the Center for Amphiphilic Polymers (CAP), in Lund, Sweden. We acknowledge greatly the support from extensive knowledge of simple and complex systems and self-assembly, as well and experimental facilities, of Physical Chemistry 1 in Lund. The work reported on DNA hydration is done under the direction of Gerd Olofsson, Lars Wadsö and Håkan Wennerström. The simulation work is much stimulated by collaboration and support from Per Linse. The work on amino acid-based surfactant is made in collaboration with Maria Rosa Infante, Barcelona. We also acknowledge discussions in our network, including groups in Munich (Joachim Rädler), Dublin (Kenneth Dawson) and Paris (Dominique Langevin).

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Fig. 7. Conformational changes induced by the action of polycations (30 segments) upon a negatively charged chain (120 beads). Panel (a) corresponds to the polyanion shape in presence of the loosely bound counterions, while panel (b) illustrates the collapse in the presence of polycation for a polyanion/polycation unitary charge ratio.

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