Interactions between DNA and Synthetic Cationic Liposomes

6-diamidino-2-phenylindole (DAPI), a water-soluble fluorescent marker with affinity for the DNA minor groove, which is useful to directly observe the ...
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J. Phys. Chem. B 2000, 104, 2829-2835

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Interactions between DNA and Synthetic Cationic Liposomes I. S. Kikuchi and A. M. Carmona-Ribeiro* Departamento de Bioquı´mica, Instituto de Quı´mica, UniVersidade de Sao Paulo, Caixa Postal 26077, S. Paulo-SP, Brazil ReceiVed: October 7, 1999; In Final Form: January 18, 2000

The interaction between T2, T4, T5, T7, and λ bacteriophages double stranded DNA and cationic liposomes made up of one single synthetic cationic lipid, dioctadecyldimethylammonium bromide (DODAB), is quantitatively evaluated from a physicochemical point of view. The first step of the interaction is driven by the electrostatic attraction between DNA and bilayer; with probes being displaced from their DNA or bilayer sites. Under conditions of DODAB excess, at maximal DODAB adsorption on DNA, there are ca. 70 DODAB molecules adsorbed per nucleotide on DNA, a molar proportion (MP) that does not depend on DNA type. Above charge neutralization, there is DNA -induced liposomal rupture, as evaluated from dialysis of DNA/ liposome mixtures where liposomes contain [14C]-sucrose in their internal compartment. In water, this DNAinduced leakage of radioactive liposomal contents suggests that the interaction DNA/cationic bilayer is not superficial. The DODAB/DNA interaction led to formation of globules as visualized from dark-field optical microscopy and to occurrence of a linear dependence between turbidity for the mixture and 1/λ2. At maximal DODAB adsorption, the formation of DODAB/DNA globular complexes causes loss of double-stranded DNA hypochromism as detected from temperature effects on DNA absorbance at 260 nm in the presence or absence of DODAB. In summary, the DODAB/DNA interaction is not at all superficial as expected merely from the electrostatic attraction between oppositely charged molecules: liposome loses its integrity and DNA loses its double helix becoming single-stranded. The hydrophobic attraction between nitrogenous bases on DNA and hydrocarbon chains on liposome bilayers plays an important role in determining the new physicochemical properties of the complex.

Introduction The entrance of exogenous DNA in the nucleus of transfected cells is not well understood.1-8 Several cloned genes linked to their own or heterologous promoter-enhancer sequences are now available. Their deliverance to cells by processes such as calcium phosphate precipitation, electroporation and lipofection has allowed their expression in a large number of cell types.9-17 These methods somehow mediate cytoplasmic delivery of a small portion of DNA with its subsequent incorporation into the nucleus. Among the variety of cellular transfection techniques commonly used, only lipofection using cationic liposomes sucessfully introduced a glucocorticoid receptor derivative, overexpressed and purified from Escherichia coli, into mammalian cells.18 Despite its importance, the establishment of more general physicochemical principles driving the interaction between cationic liposomes and DNA is still in its infancy. In the DNA-cationic liposome complex, the nucleic acids or short, single-strand antisense oligonucleotides are believed to be simply complexed (instead of encapsulated) with cationic unilamellar vesicles by electrostatic interactions. Intricate topological rearrangements may occur, including DNA condensation, liposome aggregation, and fusion.19-21 Recently, an unexpected topological transition from liposomes to optically birefringent liquid-crystalline condensed globules was revealed by X-ray diffraction and optical microscopy: a novel multilamellar structure with alternating lipid bilayer and DNA monolayers.22 * Author to whom correspondence should be addressed. Phone: 55-118182164. Fax: 55-11-8155579.

Recently, we used a monomer of the DNA biopolymer to further probe the interaction between a nucleotide and dioctadecyldimethylammonium bromide large liposomes23,24 from a physicochemical point of view.25 At maximal nucleotide adsorption onto the cationic liposomes, the positive sign of the liposome charge was kept and the hydrophobic attraction between the nucleotide and the bilayer core led to nucleotide insertion in the cationic bilayer with formation of a 2:1(molar ratio) lipid/adsorbed nucleotide complex accompanied by leakage of liposome internal contents.25 Energy minimization by molecular modeling also validated the hypothesis of nucleotide insertion in the cationic bilayer, suggesting absence of physical restrictions to the proposed structure for the complex.25 In this work, the results previously obtained for the nucleotide/DODAB interaction are extended and enriched to obtain a quantitative physicochemical description of the polynucleotide/DODAB interaction. The results shed new light into the major role played by the hydrophobic attraction in determining formation of the DODAB/DNA globular complex where cationic liposomes have lost their integrity and double-stranded DNA has lost its characteristic double helical structure. Material and Methods Chemicals. Dioctadecyldimethylammonium bromide 99.9% pure (DODAB) was obtained from Fluka Chemie AG (Switzerland) and used as such without further purification. Lyophilized T2, T4, T5, T7, and λ bacteriophage double-stranded DNA isolated from E. coli as host strain were purchased from Sigma and had their concentration in nucleotides determined from inorganic phosphorus analysis.26 Table 1 shows DNA properties

10.1021/jp9935891 CCC: $19.00 © 2000 American Chemical Society Published on Web 03/14/2000

2830 J. Phys. Chem. B, Vol. 104, No. 13, 2000

Kikuchi and Carmona-Ribeiro

TABLE 1: Properties from Bacteriophage DNA from Sigma and Analytical DNA Concentrations in Nucleotides for Double-Stranded Nucleic Acids Used in This Work [DNA] (in mM of nucleotides) DNA type

molecular weight (Da)

DNA size (in base pairs)

nominal concentration

analytical concentrationa

T2 T7 λ T4 T5

110 × 106 25 × 106 31.5 × 106 110 × 106 68 × 106

∼164 000 ∼37 900 48 502 ∼167 000 ∼103 000

1.49 1.50 0.16 0.46 1.50

1.80 1.70 0.19 0.52 1.40

a

Determined by inorganic phosphorus analysis.

and analytical determination for stock DNA solutions after adding 1 or 0.5 mL sterile water in the lyophilized DNA samples coming from Sigma. All other reagents were analytical grade and were used without further purification. Water was Milli-Q quality. Liposomes Preparation. DODAB vesicle dispersions were prepared by 3 different methods: (1) by heating the DODAB powder in water at 56 °C;24,27 (2) by ultrasonic dispersion of the DODAB powder in water solution kept at temperatures of ca. 50-70 °C,23,28 i.e., above the phase transition temperature of the DODAB bilayer, which was recently determined to be 43.5 °C;32 (3) by chloroform vaporization of a DODAB chloroformic solution in water.28 The second method yields small unilamellar vesicles (SUV) and/or bilayer fragments with a mean zeta-average diameter of 86 nm.29 DODAB concentration was analytically determined by microtitration.30 The first and the third methods yield large unilamellar vesicles (LUV) with a mean diameter of ca. 400 nm from measurements of entrapment efficiency24,31 and ca. 315 up to 711 nm from determination of mean ζ-average diameters using dynamic light scattered over a range of ionic strengths (0-5 mM monovalent salt).32 Determination of DODAB Adsorption onto Bacteriophage DNA from Small DODAB Vesicles. The interaction DNA/ small vesicles under conditions of DODAB excess was promoted by mixing 0.5 mL of a DODAB dispersion (ca. 1.1 mM) with 0.05 mL of a DNA solution (0.050-0.150 mM as the nucleotide concentration) to yield final concentrations of ca. 1.0 mM DODAB and 0.005-0.015 mM DNA nucleotides. Thereafter, the mixture was incubated (30 min/37 °C), centrifuged (15000 rpm/1 h/15 °C), and the supernatant filtered through polycarbonate membranes (0.2 µm cutoff) in order to separate DODAB adsorbed from free DODAB. Nonadsorbed DODAB concentration in the mixture was analytically determined in the filtered solution by solubilization of a dyeDODAB complex in nonionic micelles.33 Routinely, the large DNA macromolecule plus adsorbed DODAB were retained whereas free DODAB SUV freely passed through the filter. Adsorbed DODAB concentration was measured at least in quadruplicate for each mixture and expressed as adsorbed DODAB concentration ( mean standard deviation. Microelectrophoresis of the DNA/DODAB Complex. Because DODAB SUV are too small to be visualized under darkfield optical microscopy as usual for particle microelectrophoresis using the Rank Brothers apparatus, any particle scattering light under the dark field could only correspond to DNA/ DODAB particles coming from DODAB-induced transition bobine globule for DNA.34 Mixtures DNA/DODAB were prepared to yield DNA at a final fixed concentration (fixed at a value between ca. 0.0003-0.002 mM in nucleotides) and increasing DODAB SUV amounts in each mixture (DODAB

concentrations over the 0-0.016 mM range). Mixtures were incubated (30 min/37 °C) and electrophoretic mobilities were measured and plotted as a function of DODAB concentration at two to three different DNA concentrations. Mobilities were measured using a Rank Brothers microelectrophoresis apparatus with a flat cell at 25 °C. The sample to be measured was placed into the electrophoresis cell, electrodes were connected, and a voltage of (60 V was applied across the cell. Velocities of individual particles over a given tracking distance were recorded, as was direction of particle movement. Average velocities were calculated from data on, at least, 20 individual particles. EM was calculated according to the equation EM ) cm(u/V)(1/t), where u is the distance over which the particle is tracked (micrometers), cm is the interelectrode distance (7.27 cm), V is the voltage applied ((60 V), and t is the average time in seconds required to track one particle a given distance u. At a given DNA concentration, EM was determined as a function of DODAB concentration. Determination of Probe Displacement Due to the DODAB/ DNA Interaction. Two probes were separately used to monitor occurrence of probe displacement due to the DODAB/DNA interaction: merocyanine 540 (MERO), a fluorescent dye for marking hydrophobic sites at the DODAB bilayer,35 and 4′, 6-diamidino-2-phenylindole (DAPI), a water-soluble fluorescent marker with affinity for the DNA minor groove, which is useful to directly observe the coil-globule transition in DNA molecules.34 Concentrations for DAPI and MERO stock solutions prepared in water were 0.012 and 1.0 mM. Final DAPI and MERO concentrations in the DODAB/DNA or in DODAB/ nucleotide (with 2′-deoxyadenosine 5′-monophosphate, DMP, as the nucleotide tested) mixtures were 0.0006 and 0.010 mM, respectively. Optical (for MERO) or fluorescence spectra (for DAPI) were recorded at three different experimental conditions: (1) the dye alone in water, (2) the dye in DNA or in DODAB, (3) the dye in the DNA/DODAB or in the DMP/ DODAB mixtures. Appropriate controls were performed to account for vesicles and/or DNA turbidity by using the same ingredients without dye as a blank. DAPI excitation spectra were obtained at 450 nm as emission wavelength whereas emission spectra were obtained at 365 nm as excitation wavelength. Determination of Liposome Rupture upon Interaction with DNA. Entrapment efficiency for DODAB liposomes prepared by method (1) was obtained from dialysis and radioactive labeling of the intraliposomal aqueous compartment.36 Samples in the amount of 8-10 mL of liposomes prepared in water (ca. 1 mM DODAB) containing [14C]-sucrose (LS) and 2 mL of a dialysis control of water containing [14C]sucrose (S) were dialyzed in two separate bags against 1.5 L of water (changed five times), respectively, over 24 h with vigorous stirring. Before dialysis, aliquots of LS and S were reserved for the determination of [14C]-sucrose entrapment efficiency of the liposomes (ENT). After dialysis, the radioactivity, in counts per minute (cpm), was determined for the dialyzates and for the reserved aliquots. Entrapment can be taken as36

ENT ) (1/C)(cpm2/cpm1 - cpm2c/cpm1c) where 1 and 2 subscripts refer to counts of LS before and after dialysis, respectively; 1c and 2c subscripts are counts of S before and after dialysis, respectively; and C is the molar DODAB concentration. Thus, ENT is expressed in M-1. ENT determined was similar to figures previously reported for this preparation method,24 15.5 ( 5.7, and in agreement with ENT previously reported for DODAB vesicles obtained by chloroform vaporization.24,28

DNA Interactions with Synthetic Cationic Liposomes DNA-induced vesicle disruption was evaluated for λ and T4 DNA from equilibrium dialysis assays. The liposomes prepared in a [14C-sucrose] solution were previously dialyzed (LS dialyzate). The LS dialyzate (0.8 mL DODAB 1.25 mM) containing a total radioactivity equal to cpmtotal was previously added of the DNA solution (0.2 mL λ or T4 DNA at 0.154 or 0.456 mM in nucleotides, respectively). The mixture was incubated (0.5 h/37 °C). Thereafter, it was placed inside the hemichamber a of a equilibrium dialysis chamber. Leakage was inferred from total radioactivity in hemichamber b (cpmb) once attained the equilibrium between a and b compartments which were separated by a cellulose dialysis membrane. The percentiles of liposome rupture from the equilibrium dialysis procedure described above was calculated as

%Re ) 100 [cpmb/(cpmtotal/2)] Final DODAB and DNA concentration in the mixtures submitted to equilibrium dialysis were 1.0 and 0.031 (λ DNA) or 0.091 (T4 DNA) mM. Detection of Microscopic Globules from Determination of Turbidity Spectra and Application of the Joebst Law for Light Scattering from the DNA/DODAB Mixtures. DODAB/ DNA mixtures were prepared at a molar proportion (MP) above 2:1, i.e., above charge neutralization for the complex (see section on microelectrophoresis). For this set of experiments only DODAB SUV were used. This criterion was empirically established from visualization of particles scattering light in the dark field of the microelectrophoresis Rank Brothers apparatus which was definitely possible from charge neutralization for the complex. Final concentrations for DODAB and DNA (in nucleotides) in the mixtures were 0.5 and ca. 0.1 mM. The mixture was incubated (0.5 h/37 °C) and turbidity spectra recorded (400-600 nm) using as a blank DODAB SUV at the same concentration as used in the DODAB/DNA mixture being tested. Turbidity was plotted as a function of 1/λ2 in order to ascertain the globular character of the light-scattering species in the dispersion.37-39 To control any eventual globule aggregation contributing to turbidity, turbidity spectra were registered at two different times: (1) immediately after incubation; (2) 8 h thereafter. No difference was obtained between the two turbidity spectra. Determination of DODAB Effects on DNA Melting Properties. Temperature affects double-stranded DNA causing separation (melting) of complementary strands of the double strand and hyperchromism at 260 nm. To determine any effect DODAB SUV might have on the DNA double helix, DNA melting curves for several types of bacteriophage DNA (λ, T2, T5, and T7) were obtained in the presence or absence of DODAB SUV. Absorbance at 260 nm was recorded as a function of temperature inside a quartz cuvette containing DODAB SUV/DNA mixtures at final concentrations of 1.0 and 0.014 mM DODAB and DNA, respectively. The temperature of the sample inside the cuvette was increased using a circulating water bath connected to the cuvette holder and measured from a thermocouple in direct contact with the sample, which was connected to a Jencons digital thermometer, model 2003. The temperature variation rate was constant and equal to ca. 1.0 °C/ min. In a second set of experiments using similar experimental conditions, absorbance at 260 nm was measured for each sample mixture at 35 or 85 °C and compared with absorbance for DNA alone. Hyperchromism can be taken as the difference between absorbancies at 85 and 35 °C (∆A). As a blank for DODAB/ DNA mixtures was used a DODAB dispersion containing the same final DODAB concentration as the one in the sample

J. Phys. Chem. B, Vol. 104, No. 13, 2000 2831

Figure 1. Electrophoretic mobility (EM) for particles in DNA/DODAB SUV mixtures as a function of DODAB concentration at three different T4 DNA concentrations.

TABLE 2: Molar Proportion (MP′) DODAB/DNA at Charge Neutralization Depicted from Curves of Electrophoretic Mobility as a Function of DODAB Final Concentration in the Mixtures as Those Shown in Figure 1a final [DNA] (µM nucleotides)

final [DODAB] (µM)

DODAB/DNA (MP′ at EM ) 0)

0.456 0.912 1.824

1.0 1.5 5.0

2.2:1 1.6:1 2.7:1

a

Mixtures contain T4 DNA and DODAB SUV.

mixture. For DNA samples, the blank was the same solution in which DNA was, i.e., pure water. Results and Discussion 1. Charge Neutralization for the DODAB/DNA Complex Occurs upon Mixing a Molar Proportion of 2:1 (DODAB: DNA nucleotides). Figure 1 shows electrophoretic mobility (EM) for particles in DODAB/DNA mixtures as a function of final DODAB concentration for a fixed concentration of T4 DNA at three different T4 DNA concentrations. Particle visualization was difficult below, and rather easy above charge neutralization (EM ) 0). Under the dark field of the optical microscope, visualization itself ascribes to the particle a diameter larger than 0.15 µm. In fact, sizes of ca. 1.0 µm mean diameter were reported for globular T4 DNA formed upon addition of a final concentration of 0.1 mM of cetyltrimethylammonium bromide.40 The internal consistency of the results in Figure 1 can also be depicted from the increasing amounts of DODAB required to neutralize increasing amounts of DNA. In Table 2 are presented the DODAB concentrations required for EM ) 0 (charge neutralization for the complex) at 3 different T4 DNA concentrations. Although the DODAB amount required increases, the molar proportion remains approximately constant and equal to 2:1 DODAB:DNA. This figure is interesting because it allows a straightforward interpretation: it is necessary to add at least two DODAB bilayers to neutralize both strands of one single molecule of double-stranded DNA. Why is this so? Simply because, initially, the electrostatic attraction directs coverage of one DNA strand with one DODAB bilayer so that the first step of the interaction will lead to an intermediate equal to a double-stranded DNA electrostatically bound to a DODAB bilayer on each strand. However, this first arrangement is not stable, the bilayer is destroyed, and the helicoidal DNA attached to two continuous DODAB bilayers becomes a globule where the total charge is zero (charge neutralization), i.e., a DODAB amount equivalent to one of the two DODAB bilayers must be expelled from the complex in order to obtain EM ) 0 and the

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Figure 2. Optical spectra of merocyanine 540 in pure water (a), in DODAB LUV (b) and in λDNA/DODAB LUV mixtures (c in A and B), in DMP/DODAB LUV mixtures (c in C and D). The typical spectra for merocyanine (0.010 mM) in water acquires a different shape in the presence of nucleic acid (0.15 mM λDNA) or DMP nucleotide (2.0 mM) which closely resembles the spectra for merocyanine in pure water illustrating the dye displacement from its site in the DODAB bilayer. Vesicles were obtained by heating (A, C) or by chloroform vaporization (B, D).

fair visualization that corresponds to formation of globules. One should notice that DODAB SUV are too small (0.086 nm mean diameter) to be visualized under dark field optical microscopy.41 2. The First Step of the DODAB/DNA Interaction Involves Ion Pairing with Displacement of Probes from DNA or from the Cationic Bilayer. The first step of the DODAB/DNA interaction indeed involves electrostatic attraction between the DODAB vesicle and DNA, possibly with formation of ion pairs as further evidenced from displacement of probes located either at the DNA surface or at the DODAB bilayer surface. Figure 2 shows that the interaction leads to merocyanine spectra indicative of merocyanine displacement from its site on the DODAB bilayer surface35 into water. The nucleotide DMP generates a similar spectral change for MERO in the DODAB/DMP mixtures (Figure 2C,D). The resultant MERO spectra are a composition of the spectrum in pure water (corresponding to the displaced MERO from its site in the DODAB bilayer) and the spectrum in the DODAB bilayer (corresponding to the MERO molecules that still remain at the DODAB bilayer surface). For a probe located at the DNA surface, analogous results were obtained (Figure 3). DAPI, a fluorescent probe typically located at the DNA minor groove, also is displaced from its usual location due to the DODAB/DNA interaction. DAPI fluorescence spectra in the DODAB/DNA mixture (Figure 3F) are similar to DAPI spectra in pure water (compare spectra in Figure 3B,F). 3. Cationic Liposome Disruption and Separation of Single Strands in the DNA Double Helix Causes Formation of a Cationic Globule, a Pill to be Swallowed by Cells To Be Transfected. Under conditions of DODAB excess, i.e., at limiting DODAB adsorption onto DNA, the DODAB/DNA complex accommodates a molar proportion of ca. 70 adsorbed DODAB molecules per DNA nucleotide (Table 3). This indicates the positively charged character of the complex as also obtained at final DODAB concentrations above charge neutralization for the complex in Figure 1. Furthermore, this proportion is not affected by DNA type as expected from formation of ion pairs driving the complexation. Conformational changes on

Kikuchi and Carmona-Ribeiro

Figure 3. Excitation and emission fluorescence spectra for DAPI in pure water (A, B), incorporated in λ DNA (C, D), in λ DNA/DODAB LUV mixtures (E, F). Final DAPI concentrations are 0.6 µM (C, D, E, F) at three different DNA concentrations (b, c, d), without DODAB in C and D, and at 2 different DODAB concentrations for the DODAB/ DNA mixtures (E, F). DAPI spectra in the DODAB/DNA mixtures become similar to those obtained for DAPI in pure water showing its displacement from its former site in DNA.

TABLE 3: DODAB Adsorption from DODAB SUV onto Bacteriophage DNA from DNA/DODAB Mixtures and Mean Molar Proportions (MP) Calculated As Adsorbed DODAB/DNA adsorbed [DODAB] DODAB concenin the [DNA] tration filtrate [DODAB] (in mM (mM) MP (mM) nucleotide) DNA (mM) T4 λ T2

T5

T7

0.96 0.96 1.01 1.01 0.96 0.96 0.87 0.87 0.96 0.96 0.87 0.87 0.96 0.96 0.87 0.87

0.040 0.020 0.015 0.007 0.007 0.014 0.007 0.014 0.007 0.014 0.007 0.014 0.007 0.014 0.007 0.014

0 0 0.12 0.16 0.18 0.31 0.52 0.28 0.35 0.12 0.19 0.03 0.43 0.06 0.26 0.17

0.96 0.96 0.89 0.85 0.78 0.65 0.35 0.59 0.61 0.83 0.68 0.84 0.53 0.90 0.60 0.69

24a 48a 61 115 112 46 49 42 90 61 100 62 78 66 88 51

mean MP

88 ( 38 62 ( 33

71 ( 16

71 ( 16

a No DODAB excess occurs under these experimental conditions, i.e., adsorption is not maximal.

DNA in the complex must have occurred since the light scattered by the dispersion follows the Joebst law: turbidity displays a linear behavior as a function of the inverse of the square wavelength of the incident light for all DODAB/DNA mixtures tested above charge neutralization for the complex (Figure 4A,B). The Joebst law typically describes light scattering by perfectly spherical particles with sizes comparable to those of the wavelength of the incident radiation.37-39 Therefore, it is

DNA Interactions with Synthetic Cationic Liposomes

J. Phys. Chem. B, Vol. 104, No. 13, 2000 2833 TABLE 4: Determination of Size for Globular DODAB/ DNA Complexes from Linear Turbidity vs 1/λ2 Plots in Figure 4Ba DNA type T4 T2 λ T7

Figure 4. Turbidity as a function of wavelength of the incident light λ (A) or as a function of 1/λ2 for DNA/DODAB mixtures at 0.5 mM DODAB SUV and 0.090 mM T2, 0.095 mM λ DNA or 0.085 mM T7 DNA. As a blank, a 0.5 mM DODAB SUV dispersion was used. The linear dependence is typical of the Joebst law for light scattering by spherical particles, as the DNA/DODAB complex is expected to be at this molar proportion (ca. 1:5 DNA:DODAB) above charge neutralization for the complex.

straigthforward, from results in Figure 4, to conclude that the DODAB/DNA complex has a spherical, globular shape and that the dispersion is formed by particles with sizes approximately equal to the wavelength of the incident radiation: 400-600 nm. In fact, sizes previously reported for DNA/cationic globular complexes do depend on DNA length being larger for the longer DNA strands. In this work we specially selected lengthy bacteriophage DNA so that globular complexes could be visualized for particle microelectrophoresis and the formation of DODAB/DNA globules could be detected very simply from turbidimetric measurements. In Figure 4, one should notice that the longest DNA chain which is the T2 DNA (164 000 bp) yields the largest slope for the turbidity vs λ-2 plot. The shortest DNA chain tested corresponds to T7 DNA (37 900 bp) which yielded the smallest slope among the three straight lines shown in Figure 4B. The DNA of intermediate size, the λ DNA (48 502 bp), consistently resulted in an intermediate slope (Figure 4B). For synthetic spherical vesicles of different sizes, we have previously reported a good correlation between particle size and slope of the turbidity vs 1/λ2 plot.39 From the present results, this correlation seems to hold also for DODAB/DNA globules of different sizes. Minagawa et al. previously obtained fluorescence images of T4 DNA molecules using a Nikon TMD microscope equipped with a 100× oil-immersed objective, recording the images on a videotape with a highly sensitive Hamamatsu SIT TV camera.34 Induction of the coil-globule transition for the T4 DNA was achieved via poly(ethylene glycol) (PEG) addition since the effect of immiscibility of stiff chains (DNA) and flexible coils (PEG) causes a poor solubility for the DNA biopolymer in PEG and conformational change from the extended coil to the more compact globule.42 Size for the T4 DNA globule was ca. 1 µm.34 Therefore, assuming 1 µm for the DODAB/T4 DNA globule, sizes for the other two globular complexes can be estimated from slopes of straight lines in Figure 4B (Table 4). These sizes correlate well with increasing molecular weights for the DNA used to form the DODAB/DNA complexes. From the cationic vesicle point of view, a simple DNA nucleotide as is DMP was shown to cause rupture of large DODAB vesicles.25 Table 5 shows that DNA also causes rupture of DODAB LUV. In fact, the DODAB/DNA interaction is more intimate than predicted from the initial interacting step merely driven by the opposite charges on both molecules. The hydrophobic interaction between nitrogenous bases in DNA and hydrocarbon chains in the DODAB bilayer may even cause separation of the two single strands that constitute the bacte-

molecular weight (Da)

DNA length (in base pairs)

110 × 106 ∼167 000 110 × 106 ∼164 000 31.5 × 106 48 502 6 25 × 10 ∼37 900

final final [DODAB] [DNA] (mM) (mM) 0.50 0.50 0.50

0.090 0.095 0.085

slope/ [DNA]

globule size (µm)

383 ( 1 320 ( 64 298 ( 66

1.00b 1.00 0.84 0.78

a Slopes were normalized to the final DNA concentration in the mixtures. b Size used as reference for calculation of the other sizes.

TABLE 5: DNA Effect on Integrity of Large DODAB Vesicles (LUV) from Equilibrium Dialysisa sample

%Re

DODAB LUV 1.0 mM DODAB LUV 1.0 mM + λ DNA 0.031 mM DODAB LUV 1.0 mM + T4 DNA 0.091 mM

24.4 ( 9.1 46.1 ( 11.0 68.0

a Percentage of rupture (%R ) for the large vesicles was measured e for two different bacteriophage DNA under conditions of excess DODAB.

Figure 5. Effect of DODAB SUV on λ DNA melting curve. In A, absorbance at 260 nm (AT) is plotted as a function of temperature (T) for DNA (9) or for a DNA/DODAB mixture (0.014 mM DNA and 1.0 mM DODAB) (b). In B, absorbancies normalized to the absorbance at 35 °C as a function of temperature.

riophage double-stranded DNA. This is demonstrated from experiments for recording DNA absorbance at 260 nm as a function of temperature (Figure 5). There is a reduction of DNA hyperchromism in the presence of DODAB SUV that is typical of DODAB-induced double-strand separation. The result was generalized for several bacteriophage DNA in Figure 6. Hyperchromism was basically reduced to zero for all doublestranded DNA tested, a strong indication that the DODAB bilayer is capable of accomplishing the task previously accomplished by the increasing temperatures, i.e., melting of the double- stranded DNA. The explanation for this effect is simple: DODAB hydrocarbon chains are intimately mixing with

2834 J. Phys. Chem. B, Vol. 104, No. 13, 2000

Kikuchi and Carmona-Ribeiro with our major claim for the importance of the hydrophobic interaction: the thickness of the DNA single strand (0.9-1.3 nm) plus the thickness of an hydrophobically interacting DODAB monolayer (2.5 nm) plus the layer of strongly bound water in between phosphate moieties and DODAB polar heads (1.0 nm) would indeed yield 4.4-4.8 nm for the repeating distance. Figure 7 schematically illustrates this reasoning unifying our observations with those coming from ref 45 and ascribing the due importance to the hydrophobic interaction that keeps the complex integrity. Acknowledgment. I.S.K. gratefully acknowledges a FAPESP fellowship. A.M.C.R. thanks FAPESP and CNPq for research grants. References and Notes

Figure 6. DODAB SUV effect on DNA hyperchromism (∆A) for four different DNA types. There is a reduction of the hyperchromic effect expected for separation of strands from the double helix due to the hydrophobic interaction between the cationic DODAB SUV bilayers and the hydrophobic moiety of nitrogenous bases in DNA. In A and B, final DNA (in nucleotides) and DODAB concentrations are 0.014 and 1.0 mM, respectively. In C, final DNA and DODAB concentrations are 0.028 and 1.0 mM, respectively. Each error bar represents the mean standard deviation obtained from at least four independent measurements of (∆A).

Figure 7. An hypothetical model for the T4 DNA/DODAB complex that accounts for the X-ray repeating distance of 4.84 nm reported in ref 45 and for the hydrophobic interaction between nitrogenous bases and the cationic lipid hydrocarbon chains reported in this work and in ref 25.

the nitrogenous bases of the double-stranded DNA, bilayervesicles are being disrupted and double strands are being separated. As pointed out by a reviewer, although most of our results are consistent with the published information, the very high ratios of DODAB to DNA, in nucleotides, ca. 70:1, are not usually obtained for the complexes and might be related to a particular behavior for the sonicated DODAB dispersions, where bilayer fragments are present.43,44 The hydrophobic effect would then help to aggregate bilayer fragments onto the complex. Finally, the complex formed between T4 DNA and DODAB was recently evaluated from the point of view of X-ray diffraction analysis.45 The DNA-DODAB complex was reported to form a highly ordered multilamellar structure with a lamellar spacing of ca. 4.84 nm.45 These results are in agreement

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