Ethidium Bromide as a Promising Probe for Studying DNA Interaction

cationic dye ethidium bromide (EB) from DNA double helix to the solution which is ... increase of fluorescence intensity of EB intercalated in free si...
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Langmuir 2002, 18, 10348-10356

Ethidium Bromide as a Promising Probe for Studying DNA Interaction with Cationic Amphiphiles and Stability of the Resulting Complexes Vladimir A. Izumrudov,* Marina V. Zhiryakova, and Alevtina A. Goulko Department of Chemistry, Moscow State University, Leninskie gory, Moscow 119992, Russia Received June 26, 2002. In Final Form: September 20, 2002

The electrostatic binding of polycations with DNA‚EB complex results in displacement of intercalated cationic dye ethidium bromide (EB) from DNA double helix to the solution which is accompanied by a quenching of EB fluorescence. On the basis of this phenomenon, the fluorescence assay of DNA-containing polyelectrolyte complexes was recently developed. Data obtained in the current work demonstrate the applicability and advantages of this approach for monitoring both an interaction of DNA with cationic surfactants (CS) and stability of DNA-CS complexes. The comprehensive study was carried out with cationic detergents having different C12-C16 “tails” and “heads” with pyridinium or amino groups. In parallel, the similar experiments were performed with pyrenyl-tagged poly(methacrylate) anion (PMA*), in which the complex formation was monitored by quenching of PMA* fluorescence with pyridinium or nonquaternary amino groups of the detergents. The fluorescence titration curves of DNA‚EB or PMA* with CS consisted of two parts, with negligible quenching on the initial stage followed by the pronounced quenching. The critical aggregation concentration (CAC) determined from the intersection points of the curves decreased substantially with the length of “tail”. CAC values measured in DNA-CS mixtures proved to be noticeably higher than those from PMA*-CS mixtures. This finding suggests that DNAinduced self-assembly of CS molecules in the intramacromolecular aggregates is hindered due to rigidity of the double helix. Dissociation of DNA-CS complexes in salt (NaCl) solutions was monitored by the increase of fluorescence intensity of EB intercalated in free sites of DNA. Inasmuch as the addition of salt resulted in increase of CAC and decrease of critical micelle concentration (CMC), two regimes of destruction of DNA-CS complexes dependent on CS concentration were revealed. The regime of noncooperative destruction was realized if CS concentration was lower than CMC at the ionic strength of the complex dissociation, otherwise the second regime of the cooperative destruction took place. In the latter case, the salt concentration corresponding to the destruction virtually did not depend on the length of “tail” but markedly decreased with increase of a number of N-methyl groups in the “head” in the series C12NH2 > C12NHMe > C12NMe2 > C12N+Me3Br-. It implies that distance between charges in the ion pairs is the dominant factor determining the stability of the complexes. In the case of a DNA-C12NMe2 complex, the destruction was rather pH sensitive and occurred at pH and ionic strength that were close to physiological conditions. The results might create the basis for design of DNA-CS complexes with controlled stability which could be of particular promise for DNA delivery to the target cell.

* To whom correspondence should be addressed. E-mail: [email protected]. Telephone: 007-(095)-9393117. Fax: 007-(095)-9390174.

given and controlled stability. The assay was based on the measuring a fluorescence intensity of cationic dye ethidium bromide (EB). The electrostatic binding of polycation with DNA‚EB complex resulted in a competitive displacement of the intercalated dye from the double helix to the solution accompanied by a quenching of EB fluorescence. Dissociation of the polyplex in response to the addition of salt or the pH change made possible the intercalation of EB into free sites of DNA monitored by the ignition of EB fluorescence. Stability of the polyplexes in water-salt solutions determined by this approach dropped noticeably when the length and/or charge density of polycations chains decreased or longer N-alkyl substituents were introduced into polycations.2 It was demonstrated3 that the design of stimuli-response polyplexes might be accomplished not only by proper choice of polyamine of one or another type but also by using tailormade polycations with amino groups of different structure that provide different degrees of steric hindrances. Polyamines with tertiary amino groups endowed the polyplexes with pH sensitivity and resistance to added

(1) Smedt, S. De; Demeester, J.; Hennik, W. Pharm. Res. 2000, 5, 1425-1433. (2) Izumrudov, V. A.; Zhiryakova, M. V. Macromol. Chem. Phys. 1999, 200, 2533-2540.

(3) Izumrudov, V. A.; Zhiryakova, M. V.; Kudaibergenov S. E. Biopolymers 2000, 52, 94-108.

Introduction The development of nonviral gene therapy has motivated the extensive studies of complexes between DNA and molecules capable of compacting DNA and transferring it into cells. One of the general requirements for these complexes (so-called “polyplexes” and “lypoplexes”) as the ideal gene vehicles is that they should be able to combine high stability to environmental changes with the ability to dissociate within narrow and given intervals of pH, ionic strength, or temperature. The first major approach in nonviral gene therapy is based on “polyplexes”, complexes formed by mixing of DNA with synthetic polycations. A variety of polycation molecules with different chemical composition, number of repeating units, and architecture of the polymer backbone have been proposed for polyplex formation.1 Data obtained by the developed fluorescence assay2,3 appear to be the guideline in design of polyplexes with

10.1021/la020592u CCC: $22.00 © 2002 American Chemical Society Published on Web 12/02/2002

Interaction of DNA with Cationic Surfactants

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Table 1. Properties of Studied Surfactants surfactant

gross formula

C12NH2

C12H25NH2

C12NHMe C12NMe2 C12N+Me3Br-

C12H25NH(CH3) C12H25N(CH3)2 C12H25N+(C5H5)Cl-

C14N+Me3BrC16N+Me3Br-

C14H29N+(CH3)3BrC16H33N+(CH3)3Br-

C12Py+Cl-

C12H25N+(CH3)3Br-

C16Py+Br-

C16H33N+(C5H5)Br-

CMC, mol‚L-1 28 1.47 × 10-2 (25 °C) 1.48 × 10-2 (30 °C) 1.46 × 10-2 (30 °C) 1.61 × 10-2 (30 °C) 1.40 × 10-2 (25 °C) 1.42 × 10-2 (30 °C) 3.6 × 10-3 (30 °C) 9.0 × 10-4 (25 °C) 9.2 × 10-4 (30 °C) 1.43 × 10-2 (25 °C) 1.47 × 10-2 (30 °C) 9.0 × 10-4 (25 °C)

salt that are the most promising for addressing DNA packed in the polyplex to the target cell. The second major approach in nonviral gene therapy is based on “lypoplexes”. According to the nomenclature,4 “lypoplex” replaced all of the terms previously used for cationic lipid-nucleic acid complexes. For this definition, cationic lipid refers to all cationic amphiphiles, including different micelle-forming cationic detergents such as hexadecyltrimethylammonium bromide. Cationic lipids are currently used for cell transfection in vitro. Cationic detergents are able to condense DNA into discrete particles as well.5 However, for the same number of methylene groups in the “tail”, detergents have evidently much higher water solubility than lipids with two ”tails” in the molecule. Accordingly, upon addition to cells, fast release of detergents into the environment induces DNA recondensation and detergent-related citotoxicity. As a consequence, this class of amphiphiles is unable to transfect cells per se.5,6 Nevertheless, simple structure of detergents and availability of a numerous cationic detergents with different hydrophobic “tails” and hydrophilic “heads” make them attractive as model compounds for revealing the factors determining DNA interaction with cationic amphiphiles and for elucidation of mechanism of this interaction.7,8 The results of these experiments could be important in design of lypoplexes with high sensitivity to the environmental changes that is evidently a necessary step in the development of synthetic gene delivery systems. In addition, the approach developed by Blessing et al.9 and based on transformation of cystein-detergent into a cystine-lipid on the template DNA via simple air-induced dimerization shows that amphiphiles of this class are candidates for gene delivery as well. So, interaction of DNA with cationic detergents merits consideration. The data obtained in this work demonstrate the applicability and advantages of the assay based on measuring of EB fluorescence for monitoring an interaction of DNA with cationic surfactants (CS) in aqueous and water-salt solutions at different pH values. Since the method does not confine the CS to amphiphiles of special structure and properties, a comprehensive study has been carried out with various cationic detergents. Using C12-C16 detergents with a pyridinium group or (4) Felgner P. L.; Barenholz, Y.; Behr, J. P.; Cheng, S. H.; Cullis, P.; Huang, L.; Jessee, J. A.; Seymour, L.; Szoka, F.; Thierry, A. R.; Wagner, E.; Wu, G. Hum. Gene Ther. 1997, 8, 511-512. (5) Behr, J. P. Tetrahedron Lett. 1986, 27, 5861-5864. (6) Behr, J. P. Acc. Chem. Res. 1993, 26, 274-278. (7) Wang, J.; Dubin, P. L.; Zhang, H. Langmuir 2001, 17, 16701673. (8) Eskilsson, K.; Leal, C.; Lindman, B.; Miquel, M.; Nylander, T. Langmuir 2001, 17, 1666-1669. (9) Blessing, T.; Remy, J.-S.; Behr, J.-P. Biochemistry 1998, 95, 14271431.

CAC on PMA*-matrix, mol‚L-1

CAC on DNA-matrix, mol‚L-1

4.0 × 10-4 2.3 × 10-4 1.1 × 10-4 1.0 × 10-4

5.4 × 10-4 7.4 × 10-4 8.0 × 10-5 1.0 × 10-5

1.1 × 10-4

7.3 × 10-4

primary, secondary, tertiary, and quaternary amino groups allowed us to ascertain the influence of the hydrophobic “tail” and hydrophilic “head” on their interaction with DNA. In parallel, the experiments were performed with mixtures of pyrenyl-tagged poly(methacrylate) anion (PMA*) and detergent quenchers by fluorescence quenching technique. The data obtained might be a guide line in preparing DNA-amphiphile complexes of controlled stability that are particularly promising for development of gene delivery systems. Experimental Section Materials. NaCl, CHES, and HEPES buffers were purchased from Sigma (USA). Ethidium bromide (EB) was purchased from Sigma (USA). Concentration of EB in solution was determined spectrophotometrically assuming molar extinction coefficient 5600 L‚mol-1‚ cm-1 at 480 nm.10 Calf Thymus DNA. Na salt of highly polymerized calf thymus DNA (∼10000 base pairs) was purchased from Sigma (USA) and used without further purification. Concentration of DNA phosphate groups in the solutions was determined by UV absorbance measurements at 260 nm assuming a molar extinction coefficient of 6500 L‚mol-1‚cm-1.11 Poly(methacrylic) acid (PMAA) was synthesized by radical polymerization and fractionated by partial precipitation in methanol/ethyl acetate mixture.12 PMAA fraction with weightaverage degree of polymerization PW ) 3500 was used. PMAA tagged by fluorescence pyrenyl groups (PMAA*) was synthesized by interaction of the PMAA fraction with pyrenyldiazomethane as described elsewhere.12 PMAA* sample contained 1 fluorescence label per 1250 monomer units as determined from a UV spectrum of PMAA* solution assuming molar extinction coefficient 5 × 104 L‚mol-1‚cm-1 at 342 nm.12 Solutions of tagged sodium poly(methacrylate) (PMA*) were prepared by neutralization of PMAA* aqueous solution by adding of 1 equiv of NaOH with respect to carboxylic groups of the polyacid. Surfactants. 1-Dodecylpyridinium chloride (C12Py+Cl-), 1-hexadecylpyridinium bromide (C16Py+Br-), dodecyltrimethylammonium bromide (C12N+Me3Br-), tetradecyltrimethylammonium bromide (C14N+Me3Br-), and hexadecyltrimethylammonium bromide (C16N+Me3Br-) were purchased from Tokio Casei Inc. (Japan). Dodecylamine (C12NH2), N-methyldodecylamine (C12NHMe), and N,N-dimethyldodecylamine (C12NMe2) were purchased from Sigma (USA). All surfactants were used without further purification. Equivalent amounts of HCl were added to aqueous solutions of C12NH2, C12NHMe, and C12NMe2 to charge the amino groups of the surfactants. The characteristics of CS used are listed in Table 1. The obtained aqueous solutions of DNA, PMA*, and surfactants were diluted with CHES buffer so that the prepared solutions (10) Waring, M. J. J. Mol. Biol. 1965, 13, 269-282. (11) Olins, D. E.; Olins, A. L.; von Hippel, P. H. J. Mol. Biol. 1967, 24, 157-176. (12) Izumrudov, V. A.; Bronich, T. K.; Saburova, O. S.; Zezin, A. B.; Kabanov, V. A. Macromol. Chem., Rapid Commun. 1988, 9, 7-12.

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of given concentrations contained 0.02 mol‚L-1 CHES, pH 9.0. The solutions with lower pH were prepared using HEPES buffer of the same concentration. Methods. Fluorescence Measurements. Fluorescence intensity of the solutions was measured using Jobin-Yvon-3CS spectrofluorimeter (France) with water-thermostatic stirred cell holder. The measurements were made in a capped quartz fluorescence cell upon permanent stirring at 25 °C. The excitation and emission wavelengths in experiments with EB were set at 535 and 595 nm, respectively, whereas in experiments with PMA* the wavelengths were 342 and 395 nm, respectively. The DNA solution was directly mixed with EB in the fluorescence cell. The composition of the obtained complex DNA‚ EB was [EB]/[P] ) 0.25, where [P] is molar concentration of DNA phosphate groups. At this ratio, corresponding to one molecule of intercalated EB per two pairs of DNA bases (four nucleotides), the maximum of EB fluorescence intensity was observed.13 Complexes of DNA with CS were prepared in the same cells by step-by-step addition of a surfactant stock solution. The titration was finished when the pronounced quenching of the fluorescence was achieved. Fluorimetric titration of the prepared complex solution with salt was carried out in the same cell by successive addition of 4 mol‚L-1 NaCl and measuring fluorescence intensity I. Time interval between the addition of the salt portions was 5 min. In parallel, the titration of DNA‚EB complex of the same concentration by the same manner was done and the values of fluorescence intensity I0 were determined. Data obtained were presented mainly as the dependence of relative fluorescence intensity I/I0 on the salt concentration, CNaCl. In the experiments with PMA* the cationic detergents capable to quench PMA* fluorescence were used. PMA*-CS complex was prepared in the same way as the DNA-CS complex. The fluorimetric titration of the PMA*-CS complex with salt was performed analogously, and the fluorescence intensity I was determined. In parallel, the titration of PMA* solution of the same concentration was conducted and the values of fluorescence intensity I0 were determined. Data obtained were presented as the dependence of relative fluorescence intensity I/I0 on CNaCl.

Results and Discussion Ethidium bromide is a cationic dye that is widely used as a probe for native DNA. The ethidium ion displays a dramatic increase in fluorescence efficiency when it intercalates into DNA. The mechanism of this enhancement is that in free solution the excited state follows a nonradiative decay pathway that involves donation of an amino group proton to the solvent. When intercalated into the DNA helix, the ethidium cation is isolated from the solvent and the proton transfer pathway is virtually eliminated. This leads to an increase in the excited state lifetime from 1.8 ns in water to 23 ns and consequently an increase in the molar fluorescence intensity by about 20-fold.14 The ignition of EB fluorescence upon intercalation of the dye between base pairs of the double helix indicates the presence of native DNA in solution and lies in the basis of the DNA staining in gel electrophoresis. Competitive displacement of the dye from DNA to solution by positively charged species evidently leads to quenching of EB fluorescence. The role of these competitors can be played by basic polypeptides,15-17 polyamines of linear or branched structure,2,3,18,19 cationic dendrimers,19,20 or histones.18 (13) Le-Pecq, J.-B.; Paoletti, C. J. Mol. Biol. 1967, 27, 87-106. (14) Pasternack, R. F.; Caccam, M.; Keogh, B.; Stephenson, T. A.; Williams, A. P.; Gibbs, E. J. J. Am. Chem. Soc. 1991, 113, 6835-6840. (15) Plank, C.; Tang, M. X.; Wolfe, A. R.; Szoka, F. C. Hum. Gene Ther. 1999, 10, 319-332. (16) Dufourcq, J.; Neri, W.; Henry-Toulme, N. FEBS Lett. 1998, 421, 7-11. (17) Wyman, T. B.; Nicol, F.; Zelphati, O.; Scaria, P. V.; Plank, Ch.; Szoka, F. C. Biochemistry 1997, 36, 3008-3017.

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The decay of EB fluorescence signal was also observed upon addition of cationic surfactants (detergents and/or lipids)21-24 or cationic liposomes25 to DNA‚EB complex. The results obtained revealed much more effective binding of CS with DNA than that of the small organic cations or salt.21,22 It was noted23 that the mechanism of exclusion of the intercalated dye from DNA is not clear and suggested that the EB removal can be promoted by hydrophobic interactions between the planar aromatic ethidium ring and the surfactant tail groups. Model cationic lipids with +1, +2, +3, and +5 charges in the “head” were found to interact in similar ways with DNA if this interaction was compared in terms of the apparent molar charge ratio of lipid to DNA. In particular, for this lipid:DNA charge ratio from 1.25:1 to 1.5:1, all the DNA became inaccessible to EB in all mixtures.24 The specific liposome-to-DNA ratio at which the EB fluorescence quenching occurred was also corresponded to a positive to negative charge ratio close to 1:1 independently on DNA size in the range 100-23000 base pairs.25 The fact that EB dissociation from the DNA‚ EB complex has a critical dependence on the charge neutralization was attributed to a well-known collapse of DNA molecules into packed forms proceeding as a highly cooperative process.26,27 Finally, the effect of ionic strength on the ability of a cationic lipid to prevent the EB intercalation into DNA has been established. The intercalation was enhanced at high ionic strength, and in concentrated salt solution the DNA was totally accessible to EB.24 Being encouraged by the above findings, we aimed to use the EB probe to elucidate the factors affecting both an interaction of DNA with cationic detergents in aqueous solutions and stability of the complexes formed in watersalt media. Of special interest was a question whether DNA-induced self-assembly of the detergent molecules or the misellization in the bulk solution endows the cationic amphiphiles with the ability to bind DNA so strongly that stability of these complexes and polyplexes could be comparable. Interaction of DNA and PMA* with Cationic Detergents in Salt-Free Aqueous Solutions. Typical curves of fluorimetric titration of solution of the DNA‚EB complex with detergents having quaternary amino groups in the “head” are shown in Figure 1 for alkyltrimethylammonium bromides with C12 (curve 1), C14 (curve 2), and C16 (curve 3) “tails”. Similar curves were obtained with pyridinium detergents (Figure 2). The data are presented as dependencies of fluorescence intensity I on the ratio of molar concentrations of the amino groups and phosphate groups of DNA, Z ) [amino groups]/[P] ≡ [+]/ [-]. It is seen that the addition of CS results in the fluorescence quenching. As expected, efficiency of the quenching decreases with the shortening of the “tail”. It is well-known that electrostatic interaction of CS with (18) Izumrudov, V. A.; Zezin, A. B.; Kargov, S. I.; Zhiryakova, M. V.; Kabanov, V. A. Dokl. Phys. Chem. 1995, 342, 150-153. (19) Tang, M. X.; Szoka, F. C. Gene Ther. 1997, 4, 823-832. (20) Chen, W.; Turro, N. J.; Tomalia, D. A. Langmuir 2000, 16, 1519. (21) Bhattacharaya, S.; Mandal, S. S. Indian J. Biochem. Biophys. 1997, 34, 11-17. (22) Bhattacharaya, S.; Mandal, S. S. Biochim. Biophys. Acta 1997, 1323, 29-44. (23) McLoughlin, D. M.; O’Brien, J.; McManus, J. J.; Gorelov, A. V.; Dawson, K. A. Bioseparation 2001, 9, 307-313. (24) Eastman, S. J.; Siegel, C.; Tousignant, J.; Smith, A. E.; Cheng, S. H.; Scheule, R. K. Biochim. Biophys. Acta 1997, 1325, 41-62. (25) Gershon, H.; Ghirlando, R.; Guttman, S. B.; Minsky, A. Biochemistry 1993, 32, 7143-7151. (26) Manning, G. S. Biopolymers 1980, 19, 37-59. (27) Manning, G. S. Biopolymers 1981, 20, 1261-1270.

Interaction of DNA with Cationic Surfactants

Figure 1. Dependencies of fluorescence intensity I of mixtures of DNA‚EB solution with different alkyltrimethylammonium bromides, C12N+Me3Br- (1), C14N+Me3Br- (2), and C16N+Me3Br(3), on the ratio Z ) [+]/[-]. [P] ) 4 × 10-5 mol‚L-1, [EB]/[P] ) 0.25; 0.02 mol‚L-1 CHES, pH 9.0, 25 °C.

Figure 2. Dependencies of fluorescence intensity I of mixtures of DNA‚EB solution with pyridinium surfactants C12Py+Cl(1) and C16Py+Br- (2) on the ratio Z ) [+]/[-]. The other conditions are the same as in Figure 1.

polyanion weakens repulsion between amphiphile molecules and facilitates the aggregation. The onset of this polyanion-induced self-assembly of the detergent molecules occurs upon achievement of critical aggregation concentration (CAC), which is much lower than critical concentration of bulk micellization (CMC) of the cationic detergent. CAC of hexadecyltrimethylammonium bromide with the longest “tail” and the lowest CMC (9 × 10-4 mol‚L-1) is evidently smaller as compared with CAC of C14N+Me3Br- and C12N+Me3Br-, which have CMC values of 3.6 × 10-3 and 1.4 × 10-2 mol‚L-1, respectively.28 Accordingly, the quenching by C16N+Me3Br- is the most efficient (Figure 1, curve 3). (28) Abramzon, A. A. Poverkhnostno-aktivniye veschestva (Surfactants); Khimiya: Leningrad, 1979 (in Russian).

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Figure 3. Dependencies of fluorescence intensity I of (DNA‚ EB + C12N+Me3Br-) mixtures containing different concentrations of DNA on the ratio Z ) [+]/[-]: [P] ) 2 × 10-5 mol‚L-1 (1), 4 × 10-5 mol‚L-1 (2), and 1 × 10-4 mol‚L-1 (3). The other conditions are the same as those given in Figure 1.

Significant and monotonic decrease of the fluorescence intensity reflected by curve 3 of Figure 1 suggests that the detergent concentration in the mixture established on addition of a first portion of C16N+Me3Br- solution to DNA‚EB complex is equal to, or higher than the CAC. The results are drastically different when the weakest competitor, C12N+Me3Br-, is used (curve 1). The titration curve consists of two parts, with negligible quenching at the initial stage followed by the pronounced decrease in the fluorescence intensity. It is reasonable to assume that the transition region at the critical ratio Z* ≈ 20 corresponds to onset of DNA-induced self-assembly of the detergent molecules. This assumption is supported by the experiment, in which DNA‚EB complex of different concentrations was titrated with C12N+Me3Br-. As seen in Figure 3, all the curves are S-shaped, and no quenching is observed until a certain Z* value is achieved. It is also seen that Z* decreases with the increase in concentration of DNA‚EB complex. Note that concentrations of C12N+Me3Br- at the transition point calculated from the obtained Z* proved to be virtually the same, (7.4-7.5) × 10-4 (7.4 × 10-4, 7.4 × 10-4, and 7.5 × 10-4 mol‚L-1 for curves 1, 2, and 3, respectively). Thus, the value 7.4 × 10-4 mol‚L-1 can be taken as DNA-induced critical aggregation concentration of dodecyltrimethylammonium bromide. Similar inflection points were also observed in the titration curves when other cationic detergents were employed and the proper concentration range for the DNA‚ EB complex was chosen. Note that the smaller the CMC of the detergent, the larger the dilution of the DNA‚EB had to be in order to perform measurements. It is important that similar S-shaped curves with the inflection points were obtained on the fluorimetric titration of PMA* with CS that were capable to quench PMA* fluorescence, such as pyridinium detergents. The data are shown in Figure 4 and Figure 5 where fluorescence intensity is plotted as a function of Z ) [amino groups]/[COO-] ≡ [+]/[-]. Z* values were found in a similar way, and the corresponding concentrations of the detergents were calculated. Similarly to DNA-CS mixtures (Figure 3), Z*

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Figure 4. Dependencies of fluorescence intensity I of PMA* mixtures with pyridinium surfactants C12Py+Cl- (1) and C16Py+Br- (2) on the ratio Z ) [+]/[-]. [PMA*] ) 4 × 10-5 mol‚L-1, 0.02 mol‚L-1 CHES, pH 9.0, 25 °C.

Figure 5. Dependencies of fluorescence intensity I of (PMA* + C12Py+Cl-) mixtures containing different PMA* concentrations on the ratio Z ) [+]/[-]: [PMA*] ) 2 × 10-5 mol‚L-1 (1), 4 × 10-5 mol‚L-1 (2), and 1 × 10-4 mol‚L-1 (3). The conditions are the same as those given in Figure 4.

decreased systematically with increase in PMA* concentration, and at high concentrations of PMA*, the complete quenching at the charge ratio 1:1 was achieved (Figure 5, curve 3). At the same time, concentrations of dodecylpyridinium chloride calculated from Z* values of curves 1-3 of Figure 5 were 1.1 × 10-4 mol‚L-1 for all PMA* concentrations. Thus, the transition regions are determined not by the charge ratio Z of the components but by the detergent concentration in solution. This finding strongly suggests that the inflection point on the fluorimetric titration curves indicates the onset of polyanion-induced CS aggregation. The CAC values, calculated from Z* values of the titration curves are listed in Table 1. CMC values of the detergents were taken from the literature and are also

Izumrudov et al.

given in Table 1. It is seen that the CAC determined in PMA* solutions are about 2 orders of magnitude smaller than the CMC of the detergents. Notice that in the case of DNA this difference is much smaller, while still significant. Thus, the CAC values of the detergents determined in the presence of DNA proved to be noticeably higher than those obtained in their mixtures with PMA*, as is seen in Table 1. The reason for the different effects of PMA* and DNA on the polyanion-induced self-assembly of the detergent molecules is probably rooted in different flexibility of PMA* and DNA molecules as well as their conformational changes upon the titration. It can be envisioned that neutralization of mutual repulsion of negative charges on the PMA* chain after binding of the detergent molecules results in collapse of this flexible vinyl polymer. The latter should facilitate the aggregation of CS molecules and, hence, the formation of CS micelle on the PMA* template. Molecules of native DNA are evidently much more rigid than PMA* chains. Most likely, the titration of DNA‚EB solution by CS results in formation of micelles or hairlike structures29 immobilized on the DNA molecule, since compact particles of DNA-CS complex are discrete and consist of a single nucleic acid molecule.30 In this particular case, high rigidity of the double helix should hinder the DNA-induced self-assembly of CS molecules that is reflected by the rise in CAC. The fact that the charge ratio Z* approaches unity with the increase in DNA concentration (Figure 3) seems to be important. It probably indicates that at relatively high concentration of the detergents their binding to DNA is as efficient as the binding of cationic lipids24 or liposomes.25 In both cases mentioned above the dye dissociation from the DNA‚EB complex occurs cooperatively and proceeds to completeness when the charge ratio of 1:1 is achieved. To further explore the demonstrated cooperativity of the DNA-CS interaction, we studied dissociation of complexes in solutions with increasing salt concentration. Stability of DNA-CS and PMA*-CS Complexes in Water-Salt Solutions. The results of fluorimetric titration of a DNA‚EB complex and (DNA‚EB + C16N+Me3Br-) mixture with the salt are shown in Figure 6. The titrations were conducted as described in the Experimental Section. The observed decrease of fluorescence intensity I0 of the DNA‚EB complex (curve 1) is caused by the change of microenvironment of intercalated EB since DNA‚EB complex is stable at high ionic strength.13 The small fluorescence intensity I of the initial (DNA‚EB + C16N+Me3Br-) mixture at low ionic strength suggests that almost all EB molecules are dissociated from the DNA in the presence of the detergent. It is seen that EB remains bound with DNA at CNaCl < 0.5 mol‚L-1. A pronounced kink in curve 2, Figure 6, reflects dissociation of DNAC16N+Me3Br- complex. The fact that curves 1 and 2 overlap at CNaCl > 0.6 mol‚L-1 suggests that detergent is completely dissociated from the DNA and EB is completely intercalated into the DNA double helix at high salt concentration. The cooperativity of the destruction of DNA-C16N+Me3Br- complex can be more clearly seen when data in Figure 6 are replotted as a dependence of relative fluorescence intensity I/I0 on the salt concentration (Figure 7). The steep transition at 0.6 M NaCl implies that the dissociation is highly cooperative. The fluorimetric titrations of DNA-CS complexes with the salt solution were also conducted using different (29) Smith, P.; Lynden-Bell, R. M.; Smith, W. Phys. Chem. Chem. Phys. 2000, 2, 1305-1310. (30) Melnikov, S. M.; Sergeyev, V. G.; Yoshikawa, K. J. Am. Chem. Soc. 1995, 117, 2401-2408.

Interaction of DNA with Cationic Surfactants

Figure 6. Dependencies of fluorescence intensity I0 of DNA‚ EB solution (1) and fluorescence intensity I of (DNA‚EB + C16N+Me3Br-) mixture (2) on NaCl concentration. Z )10, [P] ) 2 × 10-5 mol‚L-1. The other conditions are the same as those given in Figure 1.

Figure 7. Dependencies of relative fluorescence intensity I/I0 of solution of (DNA‚EB + C16N+Me3Br-) mixture on NaCl concentration. The conditions are the same as those given in Figure 6.

alkyltrimethylammonium bromides. In these experiments the concentration of DNA was the same as above, 2 × 10-5 mol‚L-1, while values of the Z ratio in the mixtures increased with the decrease in a number of methylene groups in the “tail”. The latter was dictated by the evident requirement that the quenching of EB fluorescence in the initial mixtures should be achieved. Figure 8 shows the results of the titrations when C12N+Me3Br- (curve 1), C14N+Me3Br- (curve 2), and C16N+Me3Br- (curve 3) were used. It is seen that dissociation of the DNA-CS complex shifts toward high ionic strength as the number of methylene groups in the “tail” increases, but the shift is not proportional to the growth of this parameter. This finding suggests that the specific mechanism of CS dissociation from DNA depends on “tail” length. When

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Figure 8. Dependencies of relative fluorescence intensity I/I0 of DNA‚EB mixtures with different alkyltrimethylammonium bromides C12N+Me3Br- (1), C14N+Me3Br- (2), and C16N+Me3Br(3) on NaCl concentration. [P] ) 2 × 10-5 mol‚L-1, Z ) 100 (1), 20 (2), and 10 (3). The other conditions are the same as those given in Figure 1.

Figure 9. Dependencies of relative fluorescence intensity I/I0 of (DNA‚EB + C14N+Me3Br-) mixtures of different composition Z on NaCl concentration: Z ) 5 (1), 10 (2), 12 (3), and 15 (4). [P] ) 2 × 10-5 mol‚L-1. The other conditions are the same as those given in Figure 1.

detergents with long C14 and C16 “tails” are used, the destruction of the DNA-CS complexes is cooperative (curves 2 and 3), which means that CS aggregates move into the solution without being dissociated into molecules of detergent. The noncooperative destruction of the DNAC12N+Me3Br- complex (curve 1) suggests that dissociation of individual CS molecules from the complex is involved. The switch of the mechanism of CS dissociation can be also achieved by decrease of the detergent concentration (Figure 9). In the mixtures of DNA‚EB and C14N+Me3Brwith the same DNA concentration but different composition Z, one can realize either the cooperative destruction

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of the complex at high concentration of the detergent (at high Z, curve 4) or the noncooperative destruction if solution with a low concentration of the detergent is used (curve 1). A DNA-CS complex is evidently formed at [CS] > CAC; i.e., when concentration of CS in the mixture exceeds CAC. The initial solutions of the mixtures used for the titration (Figure 8 and Figure 9) met this requirement. However, upon titration with the salt, the electrostatic interactions in the complex are weakened due to electrostatic shielding of the charges in the ion pairs by added low molecular weight ions. Subsequently, CAC increases in the course of the titration, and at CAC ≈ [CS], the complex dissociates. So, the higher the concentration of the detergent in the mixture, the more salt has to be added to dissociate the complex. This is supported by the shift of the fluorescence ignition in the curves of Figure 9 to higher salt concentration with the increase of Z. It is important that in contrast to polyanion-induced aggregation, bulk micellization of CS is enhanced by the added salt. The electrostatic shielding of the charged “heads” weakens mutual repulsion of the detergent molecules and, hence, favors their assembly in the micelle. It has been reported that CMC values of the ionic detergents determined in water are more than an order of magnitude higher than those obtained in a 1 mol‚L-1 solution of 1,1-salt.28 So, though the concentrations of C14N+Me3Br- used in the experiments with DNAC14N+Me3Br- complex were much smaller than its CMC ()3.6 × 10-3) in water (Table 1), in the course of the titration, this parameter could significantly decrease. We estimated the CMC of water-salt solutions of C14N+Me3Br- by the I3/I1 ratio in the fluorescence spectrum of pyrene31 and revealed that at [NaCl] ≈ 0.3 mol‚L-1, the CMC value approaches the detergent concentration corresponding to the composition Z ≈ 8 of DNA-C14N+Me3Br- mixture used in the experiments (Figure 9) (data not shown). In other words, at [NaCl] g 0.3 mol‚L-1, concentration of the detergent in DNA-C14N+Me3Br- mixtures with Z > 8 (Figure 9, curves 2-4) is larger than CMC. By this means, the external salt produces a dual effect on the stability of the DNA-CS complex, with two trends acting in opposite directions. CAC increases on addition of salt, whereas CMC decreases. Accordingly, there are two regimes of the complex destruction. The first regime of the noncooperative dissociation is realized if [CS] is smaller than CMC at the ionic strength corresponding to the dissociation (Figure 9, curves 1-3). In the second regime, [CS] is larger than CMC in the whole range of the titration; the detergent molecules remain in the aggregated state both in DNA-CS complex and in bulk solution after the complex dissociation. It is apparent that in the latter case the DNA interaction with CS becomes similar to the interaction of DNA with polycation. In both systems, a driving force of the complex formation is the gain in entropy due to release of a large amount of small counterions that were localized in the vicinity of both DNA and the partner (polycation or CS aggregate) before the interaction. Consequently, to destruct the complex one should introduce the salt in an amount sufficient to compensate for this effect. There are three arguments supporting this view. First, curves 2 and 3, Figure 9, of the salt titration of DNA-C14N+Me3Br- mixtures with intermediate values of Z have a pronounced two-step appearance. After the ignition of fluorescence at the first step of the titration, (31) Chandar, P.; Samasandaran, P.; Turro, N. J. Macromolecules 1988, 21, 950-953.

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a clearly defined maximum on the curves is observed. Further addition of salt results in decrease in the fluorescence intensity that indicates stabilization of the complex on the second step of the titration. It is important that stabilization of DNA-CS complexes occurs at [NaCl] g 0.3 mol‚L-1 and Z > 8, i.e., under conditions corresponding to aggregation of C14N+Me3Br- molecules in bulk solution. In other words, the switch to another mechanism of the complex dissociation results in significant increase in tolerance of DNA-CS complex to destructive action of salt. Second, in the mixtures with Z g 10 (Figure 9, curves 2-4), regardless the detergent concentration, a pronounced fluorescence ignition on the final stage of the titrations occurs at the same ionic strength. The range of the ignition did not change with the further increase of the detergent concentration: the cooperative destruction of DNA-C14N+Me3Br- complexes in the mixtures with Z ) 20 and Z ) 25 proceeded at the same critical salt concentration, [NaCl]* (data not shown). This agrees well with the above mechanism of the complex destruction since the dissociation via the aggregates transfer to the solution suggests insignificant, if any, dependence of [NaCl]* on the detergent concentration. For polyplexes, an increase of a number of the charged units in the polycation chain above 50 resulted in relatively insignificant increase in [NaCl]* of the destruction of the DNA-polycation complex.2 Analogously, inasmuch as the aggregation number of the micelles in bulk solutions is relatively high (at least more than 50), even the pronounced change of this parameter upon variation of the ionic strength and the detergent concentration should not result in noticeable change of [NaCl]*. Finally, as is seen in Figure 8 (curves 2 and 3), [NaCl]* of the cooperative dissociation of the complexes is almost independent of a number of methylene groups in the “tail”. In other words, the possible variation in the aggregation number of the micelles with C14 and C16 detergents does not significantly affect the stability of a DNA-CS complex. This is consistent with our earlier findings on studying DNA-polycation systems in which the cooperative dissociation of DNA complexes with quaternized polyamines occurs at the same range of NaCl concentrations.2 The revealed similarity in the cooperative dissociation of DNA-CS complexes and polyplexes suggests that the tolerance of both complexes to the salt is controlled by the same factors. Results obtained on the titration of DNA complexes with detergents having different structure of amino group in the “head” give proof to this assumption. Figure 10 shows the salt titration of mixtures of DNA‚ EB and dodecylamines in which the “head” contained primary (curve 1), secondary (curve 2), tertiary (curve 3), or quaternized (curve 4) amino groups. Note that these detergents were reported to have the same CMC of (1.5 ( 0.1) × 10-4 mol‚L-1 in salt-free aqueous solutions.28 The S-shape of the curves 1-3 is indicative of the cooperative dissociation of the complexes. It is seen that the stability of the complexes in salt solutions systematically weakened in the series C12NH2 > C12NHMe > C12NMe2 > C12N+Me3Br-. The result suggests that the increase of a number of methyl groups at the nitrogen atom in above series results in increase of the steric hindrance that makes difficult a close approach of the positively charged “head” to negatively charged phosphate group of DNA. It implies that the structure of amino group in the “head” determines the distance between charges in the ion pairs and, hence, is a dominant factor of the

Interaction of DNA with Cationic Surfactants

Figure 10. Dependencies of relative fluorescence intensity I/I0 of DNA‚EB mixtures with surfactants C12NH2 (1), C12NHMe (2), C12NMe2 (3), and C12N+Me3Br- (4) on NaCl concentration. Z ) 50, [P] ) 4 × 10-5 mol‚L-1, [EB]/[P] ) 0.25. 0.02 mol‚L-1 HEPES, pH 7.0, 25 °C. Dotted line corresponds to (DNA‚EB + C12NMe2) mixture obtained at pH 7.5.

Figure 11. Dependencies of relative fluorescence intensity I/I0 of PMA* mixtures with different dodecylamines C12NH2 (1), C12NHMe (2), and C12NMe2 (3) on NaCl concentration. Z ) 3, [PMA*] ) 4.8 × 10-4 mol‚L-1; 0.02 mol‚L-1 HEPES, pH 7.0, 25 °C. Dotted line corresponds to (PMA* + C12NMe2 ) mixture obtained at pH 9.0.

stability of DNA-CS complexes. This is also true for complexes formed in the PMA*-dodecylamines mixtures, as illustrated in Figure 11. It is worth noting that for polyelectrolyte complexes of DNA with linear polycations, a similar correlation between the structure of amino group and the dissociation of polyplex in salt solutions has been established.3 All these findings demonstrate that by choice of amphiphile with amino group of proper structure, one can design lypoplex with given and controlled stability in water-salt media.

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Figure 12. Dependencies of fluorescence intensity I of (DNA‚ EB + C12NMe2) mixture on the ratio Z ) [+]/[-] obtained at different pH values: 7.0 (1), 7.5 (2), 8.0 (3), and 9.0 (4). Z ) 50, [P] ) 4 × 10-5 mol‚L-1, [EB]/[P] ) 0.25; 0.02 mol‚L-1 HEPES (1, 2, 3) and 0.02 mol‚L-1 CHES (4), 25 °C.

The stability of the DNA-CS complexes could be also modulated by changes in pH. As also seen in Figure 10, CS containing tertiary amino groups with the lower pK forms a complex whose stability is sensitive to pH changing in neutral media. At pH 7.5 (Figure 10, dotted line), the titration of (DNA‚EB + C12NMe2) mixture shows significant shift of the destruction profile to lower ionic strength as compared to the profile obtained at pH 7.0 (Figure 10, curve 3). The effect was specific for the detergent containing tertiary amino groups, the corresponding curves of the titration performed at pH 7.5 with the use of other dodecylamines virtually coincided with curves 1, 2, and 4 obtained at pH 7 (data not shown). The fluorescence quenching profiles obtained upon addition of C12NMe2 to DNA‚EB (Figure 12) show that the quenching becomes progressively less effective as pH is increased from 7.0 (curve 1) to 7.5 (curve 2) and 8.0 (curve 3); no quenching is observed at pH 9.0 (curve 4). Similar effects were observed for polyplexes. Thus, tertiary amino groups of polyamines which were protonated in neutral media endowed the DNA-polyamine complex with the most pronounced pH sensitivity as compared with polyplexes formed by DNA and polyamines with amino groups of another structure.3 Destabilization of PMA*-C12NMe2 complex with increase in pH was weaker as compared with DNA-C12NMe2 complex. Increase in pH from 7 to 9 shifted the destruction profile to lower ionic strength, cf. curve 3 and dotted line of Figure 11, but also did not change curve 1 and curve 2 corresponding to dodecylamines with primary and secondary amino groups (data not shown). In contrast to the mixture of DNA with C12NMe2 (Figure 12, curve 4), the PMA*-C12NMe2 complex remained quite stable at pH 9.0 as is seen in Figure 11 (dotted line). This fact might serve as further proof of above suggestion about relatively high flexibility of PMA* chain as a contributory factor for the polyanion-induced aggregation of the detergent molecules. In conclusion, destruction of the DNA-C12NMe2 complex can be controlled by pH and occurs at pH and ionic strengths which are close to the physiological conditions. It suggests that amphiphiles with tertiary amino groups

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can endow the lypoplexes with pH-controllable stability in water-salt solutions that is the most promising for delivery of gene material to the target cell. The revealed similarity in cooperative dissociation of lypoplexes and polyplexes controlled by the same factors can be considered a major step toward creating selfadjusted DNA-containing systems.

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Acknowledgment. We thank Professor Svetlana Sukhishvili (Stevens Institute of Technology, Hoboken, NJ) for many discussions. This work was supported by INTAS (Grant 00-113). LA020592U