Progr Colloid Polym Sci (2001) 118: 163167 Springer-Verlag 2001

COLLOIDS IN PHARMACEUTICAL AND BIOLOGICAL APPLICATIONS

R. S. Dias B. Lindman M. G. Miguel

Interactions between DNA and surfactants

R. S. Dias B. Lindman M. G. Miguel (&) Chemistry Department, Coimbra University, 3004-535 Coimbra, Portugal e-mail: mgmiguel@ci.uc.pt R. S. Dias B. Lindman Physical Chemistry 1, Center for Chemistry and Chemical Engineering P.O. Box 124, 221 00 Lund, Sweden Invited lecture, 14th Conference of the European Colloid and Interface Science Patras, Greece, 2000

Abstract The interaction between DNA and alkyltrimethylammonium bromides of dierent chain lengths is studied, both on a macroscopic and on a single-molecule level. The phase maps for aqueous DNAsurfactant systems as well as some interesting salt eects are presented. Some preliminary results on the structure of DNAsurfactant complexes are also given. Studies involving DNA and surfactant mixtures are also conducted.

Key words DNAsurfactant systems Catanionic mixtures DNAamphiphiles complexe Phase behavior

Introduction
Mixtures of water-soluble polymers and surfactants are present in a large number of systems in nature and industrial applications, such as in foods, pharmaceutical formulations, cosmetics, detergents, paints, etc., and these systems have been the subject of a large number of studies [1, 2]. The interactions between amphiphilic molecules and biologically active polyelectrolytes have received particular attention not only from the physical chemistry viewpoint, but also were specially investigated in biomedical studies. Within this group we consider DNAcationic surfactants systems. These systems have a number of applications, such as the purication of DNA by condensation and precipitation [3], DNA renaturation and ligation [4], and selective separation of DNA in the presence of RNA [5]. Surfactants can also be used for positive charging of neutral liposomes for gene delivery purposes [6]. Owing to the growing interest in this eld and numerous applications of these systems several studies have been presented in the literature concerning DNA interaction with cationic amphiphiles [713]. In spite of this, a number of facts are still not quite clear in these systems. The mechanism of surfactant binding and

the structure of the complex are some examples. In our work an attempt is made to understand these and other points. The surfactants used are cetyltrimethylammonium bromide (CTAB), tetradecyltrimethylammonium bromide (TTAB), and dodecyltrimethylammonium bromide (DTAB).

DNA association with cationic surfactants

Some applications of the DNAcationic surfactant systems are based in the fact that DNA phase-separates associatively with the amphiphiles. The formation of a precipitate, or phase concentrated in both components, has been reported for several systems of polyelectrolytes and oppositely charged surfactants [1416]. The electrostatic interactions between them are obviously strong and lead to strong association. Surfactant aggregates induced by the polymer will act as counterions, thereby reducing the charge of the complex and the entropic driving force for mixing and the interpolymer repulsions [2]. However, in contrast to other polyelectrolyte surfactant systems reported [15, 16] the precipitate does not redissolve with an excess of surfactant. Other

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information obtained is that the precipitate is formed at very low amounts of DNA and minor surfactant concentrations, far below the surfactant critical micelle concentration (cmc). Polyelectrolyteoppositely charged surfactant systems are known to have a critical aggregation concentration (cac) lower than the cmc of free surfactants, often by orders of magnitude. The fact that the cationic surfactant binding occurs preferentially to anionic polyelectrolytes of high charge density further enhances this behavior. These results are presented schematically in Fig. 1. These phase maps, studied by turbidimetry, are presented in a simplied two-dimensional representation. Since the amount of water in these systems is extremely high, this type of representation provides a better visualization.

The salt effect

The same systems were studied with the addition of salt [17]. The results are presented in Fig. 2. It is a commonly accepted viewpoint that the cac of the polyelectrolyte oppositely charged surfactant systems increases on addition of salt [18]. This is due to a weakened interaction between the polymer and the surfactant induced by the stabilization of free micelles and a screening of the electrostatic interactions. With this we would expect a decrease in the two-phase region and not the observed broadening of it. By looking at the DNA TTAB phase map, for example, we observe that the lines of precipitation of the system with and without salt cross instead of being dislocated one in relation to the other (Fig. 3); this is unexpected behavior. We believe that the knowledge of the DNAsurfactant complex structure can help us in understanding this trend.

Fig. 1 a Schematic representation of the isothermal pseudoternary phase diagram for the DNAdodecyltrimethylammonium bromide (DTAB)water system. There is a phase separation into two phases in almost the entire region considered. b Expanded view of the water-rich corner of the system (diamonds), including the DNAtetradecyltrimethylammonium bromide (TTAB)water (triangles), and DNA cetyltrimethylammonium bromide (CTAB)water (circles) systems for comparison. The open symbols refer to the clear one-phase solutions and the lled symbols to two-phase samples. The dashed line indicates the charge neutralization. T 25 C. From Ref. [17]

Fig. 2 a Same as Fig. 1b, but the phase map is represented on a logarithmic scale for better visualization of the eect of surfactant chain length on the phase transition. b Phase map for the three systems in the presence of 0.1 M NaBr. The circles represent samples without salt and the triangles samples in the presence of salt. The open symbols refer to the clear one-phase solution and the lled symbols to two-phase samples. T 25 C. From Ref. [17]

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Fig. 3 Eect of the addition of NaBr on the phase behavior of the DNATTAB aqueous system. The circles represent samples without salt and the triangles samples in the presence of salt. As before, the open symbols refer to the clear one-phase solution and the lled symbols to two-phase samples. The dashed line represents the expected direction of change for the system with salt. T 25 C. Redrawn from Ref. [17]

DNAsurfactant complexes
In spite of all studies made with these systems [813], the structure of the complex is not fully known. It is agreed [9, 19] that the complexes formed have a loosely packed hexagonal structure in which the DNA induces and stabilizes the formation of rodlike micelles. In fact, our preliminary results point in that way (Fig. 4a). We can also see that the structure is more ordered for the longerchain surfactant. The same experiments were performed with the addition of salt and the results are very similar (Fig. 4b). The only dierence appears to be that with salt the hexagonal lattice becomes less loose. This could be due to the salt osmotic pressure. Studies were also conducted within the two-phase region to investigate the dependence of the amount of the precipitate and water in the supernatant on the variation of the DTAB/DNA mixing concentration ratio [17]. We obtained some interesting results. The amount of precipitate reaches the maximum amount for a mixing ratio of approximately 1.0 and remains constant with further addition of surfactant, at least for the region studied. This indicates that the excess of surfactant added to the samples remains in the supernatant, probably as free micelles, and can give an explanation for the nonredissolution of the DNAsurfactant complexes. Since there is no binding of the surfactant to the complex after its neutralization, an inversion of the complex charge is not observed, as happens with many similar systems [15, 16]. The same studies were made with the addition of salt, but again there were no major dierences between the results. The precipitation starts for higher amounts of surfactant,

Fig. 4 Small-angle X-ray scattering diractograms of DNAsurfactant complexes a in the absence and b in the presence of NaBr. These preliminary results show a loosely packed hexagonal structure. The lled diamonds correspond to the DNACTAB complexes, the open circles to DNATTAB, and the lled triangles to the DNADTAB system. T 25 C

as expected, and there is a slight decrease in the amount of water in the samples, which corresponds to the salt contribution to the weight of the sample and means, probably, that the major fraction of salt is dissolved in the supernatant. Further studies are being conducted on this.

DNA compaction in the presence of cationic surfactants

Fluorescence microscopy is a technique that has recently been used in the study of DNA conformational behavior in the presence of several cosolutes, having as its main advantage the visualization of single molecules in solution. DNA molecules in aqueous solution present an extended conformation, owing in the solution and exhibiting a relatively slow wormlike motion, the designated unfolded coil conformation. With the

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addition of cationic surfactants, for example, the DNA molecules undergo compaction into a globular state. The interesting point in these systems is that the DNA molecules undergo a discrete, or a rst-order, phase transition [20, 21]. Associative phase behavior is well documented for mixed aqueous systems of a polyelectrolyte and oppositely charged surfactants [14, 15, 18] and so is the eect of surfactant on the polyion conformation [15, 16]. However the ndings for exible polyions of low charge density dier qualitatively from what we nd for DNA. In these cases it can be inferred that while surfactant binding is cooperative, in that it results in surfactant self-assembly aggregates and is characterized by a well-dened cac, there is an essentially uniform distribution of surfactant aggregates among the dierent polyions. Phase separation takes place when the net charge of the polyionsurfactant aggregates attains a low value and the surfactant distribution explains why rather high amounts of surfactant are needed for phase separation in these cases. For DNA, the very low values of surfactant concentration at which phase separation starts demonstrate a dierent binding situation and either complete or no binding: as binding to DNA starts, further binding is facilitated and one DNA double helix molecule is saturated before binding starts at another, i.e. there is double cooperativity. This is directly conrmed by the uorescence microscopy results, demonstrating the coexistence of original extended coils and globules compacted to the nal state. Our results for the DNA CTAB, DNATTAB, and DNADTAB systems show that this coexistence interval is narrower for DNACTAB aqueous mixtures and becomes wider for the shorter-chain surfactant. It is shown in Fig. 5 that a larger amount of DTAB is needed to induce the compaction of the DNA molecules, which is in agreement with our previous results.

Forthcoming work
Since many details in these systems are not well understood more experiments are being conducted in our groups. One of them is the study of the DNA interactions with alkyltrimethylammonium surfactants of even shorter chain length, such as decyltrimethylammonium bromide and octyltrimethylammonium bromide. Some preliminary phase diagrams are presented in Fig. 6. Hexyltrimethylammonium bromide was found not to precipitate DNA. As expected the area of the twophase region decreases with the decrease in the surfactant hydrophobicity. For the short-chain surfactant the association is no longer observed. One problem with these cationic systems is the toxicity of the surfactants, which reduces substantially their applications. An eort is being made to use more biocompatible amphiphiles. The use of surfactant mixtures is another way of dealing with this problem. By reducing the amount of cationic amphiphile we decrease the toxicity of the systems. Cationic liposomes, for example, made of neutral and cationic lipids, are well known as gene delivery vehicles. Owing to the undeniable importance of these systems, the interaction between DNA and surfactant mixtures is being carefully studied.

Interactions between DNA and surfactant mixtures

We studied the DNA conformational behavior in the presence of nonstoichiometric mixtures of two oppositely charged surfactants, CTAB and sodium octyl sulfate [22]. The aqueous mixtures of these surfactants exhibit a rich

Fig. 5 DNA conformational behavior in the presence of cationic surfactants CTAB, TTAB, and DTAB. The DNA charge concentration was maintained at 0.5 lM. The open circles correspond to the coil conformational state of DNA and the lled circles to the presence of globular molecules. The shaded circles represent the coexistence between elongated coils and compacted globules. T 25 C. From Ref. [17]

Fig. 6 Same as Fig. 2a with some preliminary results on the DNA decyltrimethylammonium bromidewater (squares) and DNAoctyltrimethylammonium bromidewater (crosses) systems. As before, the open symbols correspond to one-phase solution and the lled symbols to two-phase samples. T 25 C

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Fig. 7 The addition of a nonionic surfactant prevents the precipitation of DNAcationic surfactant. A simplied two-dimensional representation of the preliminary results is given for the aqueous system of DNAoctaethyleneglycol mono-n-dodecyl etherCTAB. The lled symbols refer to two-phase samples and the open symbols to clear one-phase solution. T 25 C

phase behavior, with the formation of surfactant aggregates with dierent charges and geometries [23, 24]. We were mainly interested in the phases of the vesicles. We observed that the presence of anionic-rich vesicles in aqueous DNA solutions does not lead to any associative interaction and does not aect the DNA conformational behavior; however, in the cationic-rich phase, DNA is in the globular state and it adsorbs onto the surface of the

vesicles [22]. Moreover, we observed that the change in the molar ratio between cationic and anionic surfactants in solution leads to DNA unfolding. Owing to the interesting results and promising use in several applications other studies are being conducted on this system, such as phase diagrams and the complex structure determinations. Another interesting part in the study of the DNA cationic surfactant interactions is the addition of a nonionic surfactant. We observed that the addition of octaethyleneglycol mono-n-dodecyl ether induces a weakening in the interaction between the polyion and the oppositely charged surfactant and is capable of preventing the precipitation of the systems at high enough concentrations. Some preliminary results are presented in Fig. 7. During this project other amphiphiles will be used.
Acknowledgements This work was supported by grants from JNICT and Praxis XXI (PRAXIS/BD/21227/99), the Caloust Gulbenkian Foundation, the Swedish Research Council for Engineering Sciences, and the Center for Amphiphilic Polymers in Lund. Sergey Mel'nikov is thanked for valuable participation in the initiation of this project and for useful discussions. We are grateful to Filipe Antunes and Stina Lindman for technical assistance.

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