Interaction of DNA with Cationic Vesicles: A Calorimetric Study

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J. Phys. Chem. B 2000, 104, 7795-7802

7795

Interaction of DNA with Cationic Vesicles: A Calorimetric Study Paula C. A. Barreleiro,*,† Gerd Olofsson,† and Paschalis Alexandridis‡ Center for Chemistry and Chemical Engineering, Physical Chemistry 1, Lund UniVersity, P.O. Box 124, S-22100 Lund, Sweden, and Department of Chemical Engineering, State UniVersity of New York at Buffalo, Buffalo, New York 14260-4200 ReceiVed: February 18, 2000; In Final Form: May 1, 2000

The interaction of DNA with vesicles of cationic lipids mixed with varying amounts of a zwitterionic lipid in dilute solutions was studied by isothermal titration microcalorimetry, differential scanning calorimetry (DSC), and turbidity measurements. Extruded pure dimethyldioctadecylammonium bromide (DODAB) and chloride (DODAC) vesicles and mixed vesicles with various amounts of dioleoylphosphatidylethanolamine (DOPE) were investigated at 25 °C. The interaction of DNA with cationic vesicles is fast and endothermic. The enthalpy of reaction per mole of lipid added is constant with varying charge ratio between cationic lipid and DNA but decreases with increasing amounts of the zwitterionic lipid in the vesicle. The enthalpy change is constant up to a critical charge ratio F+/- of about 0.8 for the addition of lipid vesicles to DNA and 1.2 for the addition of DNA to lipid vesicles. The enthalpy of reaction decreases with the replacement of Br- by Clion and is independent of cationic vesicle size in the range 50-200 nm. The effect of DOPE on the thermotropic behavior of DODAB in aqueous dispersions with and without DNA was investigated using DSC. Inclusion of DOPE in the DODAB cationic vesicle increases the bilayer fluidity, e.g., decreases the melting temperature, Tm. The status of the hydrocarbon chains, solid-like or fluid, has a pronounced effect on the measured enthalpy changes and most strongly for the formation of positively charged, lipid-enriched aggregates.

1. Introduction Felgner et al.1 showed for the first time that cationic vesicles made of a cationic lipid and a neutral lipid could be used to mediate intracellular delivery of DNA into cells. The key role of the cationic lipid is to provide an electrostatic attraction between the vesicle and the DNA molecule. It was shown that variations in the length and degree of unsaturation of the lipid chain, nature of the groups bound to the quaternary nitrogen and even the counterion can have large effects on the transfection efficiency of these complexes.2 The introduction of neutral or zwitterionic lipids in the cationic vesicle has been shown to increase the transfection efficiency of the complexes in different cell lines in vitro as well as in vivo.3 However, its role is not well understood. The most efficient neutral or zwitterionic lipid is the unsaturated dioleoylphosphatidylethanolamine (DOPE) with the exception of cholesterol for in vivo transfer.4 It is thought that DOPE increases transfection efficiency by promoting membrane fusion.1,5 However, for some cell types DOPE is not required for fusion and the transfection efficiency of some cationic lipids is not enhanced. Pure DOPE does not form bilayers and liposomes at normal conditions due to its packing parameter but participates in a bilayer structure if stabilized by a bilayer-forming lipid such as dimethyldioctadecylammonium bromide (DODAB). A significant number of studies dealing with the structure and morphology of DNA-cationic vesicle complexes has been reported. Cryo-TEM,6-8 freeze-fracture electron microscopy,9,10 synchrotron X-ray scattering,11-13 optical and fluorescence microscopy,14,15 and atomic force microscopy16 have given a fairly good picture of the structure of these complexes as a * Corresponding author. E-mail: [email protected]. † Lund University. ‡ State University of New York at Buffalo.

function of the two most important parameters: vesicle composition, e.g., type and quantity of the helper lipid, presence of cosurfactants, and charge ratio between cationic lipid and DNA. The use of synchrotron radiation allowed not only observation of the structures at higher concentrations (precipitates) but also in the extremely dilute solutions (99% of water) used in gene therapy.11,12 The structures observed are not specific for a certain DNA/lipid system but have been observed for different types of DNA and different cationic and neutral lipids.11-13 Despite the increasing number of publications describing the structure and morphology, little is know about the thermodynamics and kinetics of the formation of DNA-cationic lipid vesicle complexes. Isothermal titration calorimetry is an accurate technique to characterize the energetics of binding interactions of biological macromolecules. One of its strengths is that it does not require the immobilization and/or modification of the reactants as in fluorescence microscopy, where the modification of one of the binding partners with a fluorescent probe may alter the interaction that one wants to study. Differential scanning calorimetry has long been used to study a wide range of thermal transitions in biological systems: thermotropic properties of lipid membranes composed of pure and mixed lipids, and nature of lipid-protein interaction.17 In this paper, we report a study of the interaction of doublestranded DNA with cationic lipid vesicles in aqueous solution using isothermal titration calorimetry, differential scanning calorimetry and turbidity measurements. The enthalpy of binding of a cationic micelle forming surfactant CTAB (cetyltrimethylammonium bromide) to DNA, measured by titration microcalorimetry, has been reported18 but not the association of mixed cationic vesicles with DNA. Thus, we studied unilamellar DODAB and dimethyldioctadecylammonium chloride (DODAC) vesicles and DODAB vesicles containing varying amounts of

10.1021/jp000636c CCC: $19.00 © 2000 American Chemical Society Published on Web 07/25/2000

7796 J. Phys. Chem. B, Vol. 104, No. 32, 2000 DOPE, all prepared by extrusion. The results show that the state of the bilayer (gel versus fluid) has a marked effect on the energetics of interaction between DNA and mixed cationic lipid vesicles. 2. Experimental Section 2.1. Materials. The zwitterionic lipid dioleoylphosphatidylethanolamine (DOPE) and the cationic lipid dimethyldioctadecylammonium bromide (DODAB) with stated purity better than 99%, as determined by HPLC, were purchased from Avanti Polar Lipids (Alabaster, AL) and used without further purification. Recrystallized dimethyldioctadecylammonium chloride (DODAC), obtained by counterion exchange from DODAB as described elsewhere,19 was kindly supplied by Dr. I. M. Cuccovia, to whom we are indebted. Salmon sperm DNA (2000 ( 500 bp as determined by 1% TAE Agarose gel analysis and free from Dnase and Rnase as stated by the manufacturer) prepared from highly pure, phenol extracted DNA was purchased from Gibco, BRL. The DNA concentration was measured by its absorbance at 260 nm. The ratio of the absorbance at 260 nm vs 280 nm was about 1.8-1.9 and the absorbance at 320 nm was negligible, so no contamination of protein was observed. The conformation of DNA in aqueous solution was confirmed by circular dichroism (CD). A melting temperature of 49 °C was measured by DSC. 2.2. Sample Preparation. The vesicles were prepared by mixing the cationic and zwitterionic lipids at the desired mole ratio in chloroform. A film of the lipid mixture was made by evaporation of the chloroform under a stream of N2, and it was dried under vacuum overnight to remove residual organic solvent. The film was then hydrated in water and sonicated for 10 min (using a ultrasound bath Starsonic 90, Liarre) above the gel-to-liquid-crystalline phase transition temperature (Tm) of the lipid mixture to suspend the lipids. The Tm for DODAB is 44.8 °C in aqueous solution20 and for DOPE is -16 °C in 20 mM HEPES buffer and 10 mM NaCl.21 An extrusion system (Avanti Polar Lipids, Alabaster, AL) was used to prepare unilamellar vesicles of the desired size. Extrusions were performed manually through two stacked 13 mm polycarbonate filters with nominal pore diameters of 50, 100, and 200 nm and repeated 25-29 times. An odd number of passages was used to avoid the presence of multilamellar vesicles that might not have crossed the filter. The extrusion temperature was 55 °C. After extrusion, the vesicle dispersions were cooled to room temperature at which they were stored for 2 h before used. Sizes were determined by dynamic light scattering, which revealed a very narrow size distribution. No multilamellar vesicles were observed by cryoelectron microscopy. The DNA-lipid complexes, or lipoplexes,22 for DSC measurements were prepared at 25 °C from aqueous stock solutions of DNA and cationic lipid vesicles and diluted in water. The vesicle solution was added to the DNA solution and mixed to prepare anionic (or cationic) complexes of the desired charge ratio between lipids and DNA. 2.3. Isothermal Titration Microcalorimetry. The enthalpies of reaction between DNA and the cationic vesicles were measured using a stainless steel titration vessel of 1 mL in the prototype of the TAM four-channel microcalorimetric system23 at 25 °C. Values of the calibration constant, c, and time constant, τ, were determined from electrical heating experiments performed using insertion heaters immersed in water inside the vessel. The calibration constant was also obtained by dissolution of propan-1-ol in water,24 and the two methods agreed within 0.5%. In the experiments, small aliquots of a cationic vesicle

Barreleiro et al. (or DNA) solution were added to DNA (or cationic vesicle) solution in the vessel. The calorimetric vessel was charged with 0.9 mL of liquid to be titrated, and typically 12 µL aliquots were added over 180 s with at least 30 min between each injection. The additions were made using a gastight Hamilton syringe connected with a computer-operated syringe drive. Thus injection volume and injection rates could be controlled with great precision. The system was stirred at 100 rpm with a gold propeller. Each titration series was repeated two or three times, and good reproducibility was observed. Null experiments, in which vesicle (or DNA) solution was injected into water and water was injected into vesicle (and DNA) solution in the vessel, were also made. 2.4. Turbidity. The turbidity was measured at λ ) 410 nm using a Perkin-Elmer Lambda 14 UV/visible spectrophotometer, equipped with a temperature-controlled sample cell holder. Consecutive portions of DNA solution were added to the vesicle solution in a 1 mL quartz cuvette cell. The sample was kept in situ and the turbidity was measured continuously with time. The temperature was controlled to within (0.1 °C by a Haake circulating water bath. The temperature in the cell was measured using a thermocouple. Reproducibility was evaluated from at least three runs. 2.5. Differential Scanning Calorimetry. The high-sensitivity differential scanning calorimeter MicroCal MC-2 (Microcal Inc. Northampton, MA) was used to make the DSC measurements. It is equipped with twin total-fill cells of 1.2 mL for the reference and sample solutions. The solutions were degassed (Nueva II stirrer, Thermolyne) before being transferred to the cells using a Hamilton syringe. The instrument measures the power required to keep the temperature of the sample and reference cells equal while the temperature is raised (upscan) or lowered (downscan) at a constant rate, 1 °C/min in this work. Lower scanning rates were also used to verify transition kinetics. The reference thermogram for both cells filled with the same solvent was recorded under the same conditions. The difference between the sample and reference thermograms is proportional to the excess heat capacity, ∆Cp. The experiments were repeated several times and a good reproducibility was observed. 3. Results We investigated the association of double-stranded DNA with cationic lipid vesicles in solution using isothermal titration calorimetry, differential scanning calorimetry, and turbidity measurements as a function of the charge ratio between lipid and DNA, F+/-, and lipid composition of cationic vesicles. The charge ratio, F+/-, is defined as the ratio of positive charge equivalents of the cationic component to negative charge equivalents of the nucleic acid,22 and XDODAB is defined as the mole fraction of DODAB in the cationic vesicle. These studies were performed in distilled water (pH 5.5-6.0) at very low ionic strength (electrolyte introduced from the dilute DNA solution), so the electrostatic interactions to a large extent were unscreened. The addition of cationic vesicles to DNA (and Vice Versa) did not cause any significant change of pH (results not shown). In solutions with F+/- below 1, excess DNA interacts with the complexes to give negatively charged aggregates that are stable in solution. Likewise, positively charged aggregates are formed by lipids bound to the complex in excess of the isoelectric amount. In solutions where the amount of positive charges from lipids balances the negative charges from DNA (F+/- ) 1), the particles agglomerate and the complex precipitates. The arrangement of lipids and DNA is the same in the positively charged and negatively charged aggregates as in the isoelectric complexes.25

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(a)

(b)

Figure 1. Typical calorimetric record from a titration experiment. (a) Eighteen injections of 12 µL cationic vesicle dispersion to 0.90 mL DNA solution (Ci ) 0.30 mg/mL). (b) Peak number 3 after dynamic correction of the potential-time curve.

3.1. Isothermal Titration Calorimetry. In solutions containing DNA and lipids, precipitation occurs close to charge neutrality. The presence of electrolytes increases the precipitation (since it decreases the Debye-Hu¨ckel screening length, and the repulsive barrier between particles) and, therefore, we have chosen to work at low ionic strength. Two different approaches to prepare the DNA-lipid complexes (lipoplexes) were used to avoid crossing the solubility gap. Anionic complexes (F+/< 1) were prepared by injecting cationic vesicles into DNA solution and cationic complexes (F+/- > 1) were prepared by injecting DNA into cationic vesicle solution. A typical calorimetric record for the titration of DNA solution with a dispersion of cationic vesicles is presented in Figure 1a, which shows the thermal power, P, as a function of time, t. The time between each injection, 30 min, is sufficiently long to allow the equilibrium state to be reached, as shown by a small length of baseline between the peaks. The experimental potential (thermopile voltages)-time curve was deconvoluted by adding to the original signal its time derivative multiplied by the time constant as described by the simple Tian equation,26 P ) c(V + τdV/dt), where P is the thermal power (dq/dt), τ is the time constant of the instrument, c is the calibration constant, and dV/dt is the time derivative of the voltage signal. The calori-

Figure 2. Enthalpies of interaction ∆Hr calculated per mole of injected lipid, as a function of the charge ratio F+/- for the titration of DNA (Ci ) 0.30 mg/mL) with (b) DODAB and (O) DODAC vesicles at 25 °C.

metric record obtained from the dynamic correction is presented in Figure 1b. The corrected curve has a higher noise level since the time derivative of the potential signal is used to correct the voltage output. This noise is random and does not affect the calculated integrals. The length of each peak is equal to the injection time (3 min), thus it seems that the reaction occurs instantaneously. However, the possibility of the occurrence of athermal processes of longer duration cannot be ruled out. The shape of the time-corrected peak is the same for all the peaks in the titration series that give a measurable enthalpy change. The observed reaction enthalpies were obtained by integration of the area under the calorimetric peaks. They were fairly small, for instance, the peak in Figure 1b corresponds to 670 µJ. The injection of the cationic vesicle solution into pure water without DNA and the injection of water into the DNA solution gave rise to negligible enthalpy changes (results not shown). In Figure 2, the observed enthalpy of reaction expressed per mole of added lipid in each injection is plotted as a function of charge ratio, F+/-, for the titration of DNA solution with dispersions of pure vesicles of DODAB and DODAC. The diameter of the vesicles was 230 ( 10 nm for DODAB and 210 ( 10 nm for DODAC as measured by dynamic light scattering. We observe that as the vesicle dispersion is added to the DNA solution, the enthalpy of reaction is constant and endothermic (heat absorbed) up to a charge ratio around 0.8 where an abrupt decrease in the enthalpy is observed. At the same time the abrupt decrease in enthalpy was observed, precipitation occurred (see below). Further injections to give charge ratios above 0.8 gave very small and not reproducible enthalpy changes. The enthalpies of interaction, ∆Hr, between DNA and cationic vesicles were calculated as the average of all the injections up to precipitation. The enthalpy was 9.8 ( 0.5 kJ mol-1 for DODAB and 5.3 ( 0.4 kJ mol-1 for DODAC, expressed per mole of added lipid in each injection. The difference of 4.5 kJ mol-1 is surprisingly large considering that the only difference between DODAB and DODAC is the replacement of Br- counterions by Cl- ions. Strong counterion effects on the thermotropic behavior of cationic vesicles have recently been observed.20 Titration experiments with DODAC vesicles with three different sizes, 95 ( 5 nm, 130 ( 6 nm, and 210 ( 10 nm, gave the same calorimetric results when added to DNA solution (results not shown).

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Barreleiro et al.

(a)

(b)

Figure 3. Titration of DNA (Ci ) 0.30 mg/mL) with DODAB/DOPE vesicles with varying mole fractions of DODAB in the mixed cationic vesicle: XDODAB (2) 1; (]) 0.75; (b) 0.67; (9) 0.5; (O) 0.33 and ([) 0.25 at 25 °C. Enthalpies of interaction ∆Hr (a) calculated per total amount of injected lipid and (b) calculated per mole of cationic lipid as a function of charge ratio F+/-.

To evaluate how the addition of DOPE influences the energetics of interaction between DNA and cationic vesicles, we measured the enthalpies of reaction between DNA and mixed cationic vesicles with varying mole fractions of DOPE. Figure 3a shows the ∆Hr expressed as kJ per total amount of lipid added in each injection as a function of charge ratio, F+/-, for the various mixed cationic vesicles studied. The enthalpy is constant and endothermic up to a charge ratio about 0.8, as for the pure cationic vesicles. The observed enthalpies decrease with increasing amounts of DOPE. The enthalpy changes calculated per mole of added cationic lipid are plotted against charge ratio, F+/-, in Figure 3b. It is noteworthy that in this representation, ∆Hr is the same for the three vesicle dispersions containing 0.5, 0.33, and 0.25 mole fraction of DODAB. Obviously the amount of charged lipid in these cationic vesicles determines the reaction enthalpy. The main transition temperature, Tm, is below 25 °C, and the lipids in these vesicle dispersions are in the fluid state (see below DSC measurements). The vesicles with pure DODAB or with high DODAB content are in the gel state at the temperature of the measurements, which leads to a signifi-

Figure 4. Titration of DODAB/DOPE vesicle dispersions containing varying mole fractions of DODAB with DNA: XDODAB (b) 1; (O) 0.67; (]) 0.5. The initial concentration of cationic lipid was 0.3 mM.

cantly more endothermic interaction. Further, the mixed vesicles with high DODAB content give significantly higher interaction enthalpies than do pure DODAB when the enthalpy is calculated per mole of added cationic lipid. When preparing positively charged complexes, the cationic vesicles were kept in the cell and the DNA solution was injected. Results from the addition of DNA solution to a dispersion of cationic vesicles are shown in Figure 4. The enthalpy of reaction per injection calculated per mole of charge unit in DNA is constant and endothermic up to a charge ratio about 1.2, where an abrupt decrease is observed. Also, in this type of experiment the decrease is due to the start of precipitation, as verified by turbidimetric titrations. The results of the calorimetric titrations are consistent with the picture that for a charge ratio smaller than 1, free DNA coexists with lipoplexes and for charge ratio higher than 1, free vesicles coexist with the lipoplexes as recently reported25,27,28 using optical microscopy, cryo-TEM, and synchrotron X-ray diffraction. The enthalpy of interaction was found to be 5.1 ( 0.3 kJ mol-1 for the addition of DNA to pure DODAB dispersion and 9.1 ( 0.5 kJ mol-1 for mixed DODAB/DOPE (2:1) (expressed per mole of charge unit in DNA) when the initial concentration of cationic lipid was of 0.3 mM. When the initial concentration of cationic lipid in the calorimetric vessel was increased to 0.6 mM, ∆Hr was found to be 17.5 ( 0.3 kJ mol-1 for pure DODAB vesicles, 37.0 ( 0.8 kJ mol-1 for DODAB/DOPE (2:1), and 1.7 ( 0.3 kJ mol-1 for DODAB/DOPE (1:1) vesicles (calculated per mole of charge unit in DNA). Thus, the enthalpy of reaction was dependent on the initial concentration of vesicles in the calorimetric vessel. 3.2. Turbidity. Turbidity measurements at 410 nm were performed under the same conditions as the calorimetric titration experiments. Figure 5 shows the turbidity after each injection, τ, versus charge ratio, F+/-, for the addition of vesicles containing equimolar amounts of DODAB and DOPE to DNA solution, corrected for the turbidity of the cationic vesicles in the absence of DNA. The conditions were kept as similar as possible to the calorimetric titration, with 30 min intervals between injections. Addition of vesicles to DNA (or DNA to vesicles) caused an instantaneous increase of turbidity. Fast binding of DNA to the cationic vesicles was also observed in the isothermal titration calorimetry experiments. The increase of turbidity indicate the formation of larger particles as observed

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(a)

Figure 5. Turbidity τ at 410 nm as a function of the charge ratio F+/for DNA (Ci ) 0.30 mg/mL) titrated with DODAB/DOPE (1:1).

by dynamic light scattering.27,29 The turbidity increases in a linear way (the turbidity increment is the same for each injection) up to a charge ratio, F+/-, roughly equal to 0.8 where it decreases. At charge ratios lower than 0.8 the solutions were bluish, but at F+/- roughly equal to 0.8, they became cloudy and further addition of cationic vesicles (or DNA) led to precipitation or coacervation as observed by visual inspection. The same results were obtained in a calorimeter30 that allows simultaneous detection of heat and turbidity.31 3.3. Differential Scanning Calorimetry. Figure 6a and b shows the differential scanning calorimeter (DSC) traces for DODAB/DOPE mixed vesicles at different mole fractions of DOPE in the absence of DNA. The vertical dashed line in Figure 6a-c at 25 °C indicates the temperature of the titration measurements. The concentration of DODAB was 1 mM in samples shown in Figure 6a and 0.225 mM in Figure 6b. The pure cationic lipid (XDODAB ) 1) shows a sharp peak from the main transition, that is, the gel (Lβ)-to-liquid crystalline (LR) transition at 44.8 °C, as previously reported.20 When DOPE was added, the peak became broader and the temperature of the main transition decreased and exhibited a low-temperature shoulder. At XDODAB ) 0.6 the DSC trace showed two broad peaks, see Figure 6a, which indicates phase separation resulting in domains with a heterogeneous distribution of DOPE and DODAB. At XDODAB ) 0.50 a very broad peak located at around 19.6 °C appeared. The melting temperature for the sample containing XDODAB ) 0.33 was even lower, and for XDODAB < 0.33 no phase transition was observed within the detection range of the instrument. Dilution to 0.225 mM DODAB did not significantly change the DSC traces. The pre-transition observed in Figure 6b for dispersions of pure DODAB may be due to a different batch of DODAB used. Till now the nature of this pre-transition has not been explained satisfactorily, but it seems to be dependent on the purity of the sample, preparation method, etc.20 The transition temperature Tm for lipid mixtures containing equimolar amounts of DODAB and DOPE or DOPE in excess is below 25 °C and, thus, these samples were in the fluid phase in the titration calorimetric measurements. Vesicles of pure DODAB and DODAC (Tm ) 47.1 °C)20 were in the gel state as also were mixtures of DODAB and DOPE with mole fractions of DODAB higher than 0.67. DSC thermograms of vesicle dispersions to which DNA had been added are shown in Figure 6c. The vesicle dispersions are

(b)

(c)

Figure 6. DSC traces for aqueous dispersions of DODAB/DOPE vesicles of varying composition. Mixed vesicle dispersions containing (a) 1 mM DODAB, (b) 0.225 mM DODAB (without DNA), and (c) 0.037 mg/mL DNA and 0.225 mM DODAB, giving a charge ratio ratio F+/- ) 0.5. The mole fraction of DODAB in the mixed cationic vesicles XDODAB is indicated in the figures. Curves have been offset from each other to avoid overlap.

7800 J. Phys. Chem. B, Vol. 104, No. 32, 2000 the same as shown in Figure 6b and the charge ratio, F+/-, is 0.5. The system contains excess negative charges, and all lipid can be expected to be bound in the lipid-DNA complex. For the pure DODAB dispersion, Tm increased to 52 °C in the cationic lipid-DNA complex. The tendency to phase separation appears increased, see traces for XDODAB of 0.8 and 0.67, giving one hump around a temperature which is lower than Tm for the pure DODAB-DNA complex but higher than Tm in the mixed vesicles of the same composition. The low-temperature hump centered at 15 °C narrows as XDODAB decreases to XDODAB) 0.5 but stays at the same temperature. DSC measurements at other charge ratios below and above 1 were also performed. No significant differences between the DSC curves for various charge ratios below 1 were observed. For charge ratios smaller than 1, all of the lipids are associated with DNA and in solution there are “free” DNA and DNAlipid complexes. The DSC instrument was not sensitive enough to register the melting of DNA at this concentration. At charges ratios higher than 1, where DNA-lipid complexes and “free” vesicles coexist, the DSC curves consist of two sets of peaks which at least partially overlap. However, the main features of the melting curves for the complexes are the same. 4. Discussion The results presented in this paper are from measurements of the interaction between cationic vesicles and DNA to give soluble complexes, suitable for gene therapy. During the interaction, two processes have been shown to take place: the fusion of the vesicles induced by DNA and the collapse of DNA induced by the vesicles.11,13 The complex consists of alternating layers of lipids and semiflexible DNA double-helices with a periodicity d ) dm + dw, where dm is the thickness of the lipid bilayer and dw is the thickness of the DNA monolayer.11 The distance dm is determined by the length of the lipid. In these liquid crystalline lamellar aggregates, the DNA monolayers are ordered in a two-dimensional smectic order with a well-defined spacing between the DNA rods. The distance between DNA rods is shortest in negatively charged aggregates and longest in positive aggregates. The spacing increases with the fraction of uncharged (zwitterionic) lipid in the bilayer. The bilayer dominates the molecular ordering, and there were no indications of major changes in the distribution of lipids accompanying the complex formation.25 For charge ratios around 1, the aggregates precipitated into big flocs. These studies were performed with bilayers in the fluid state. However, the state of the hydrocarbon chains appears not to have a significant influence on the structure of the lipid-DNA complexes, as evidenced by the study of the DMPC/DMTAP (dimyristoylphosphatidylcholine/dimyristoyltrimethylammonium propane)-DNA system.32 Bilayer structures with intercalated lamellar order are formed with lipids in either the gel or fluid phase. The complexation with DNA had only a small effect on the overall phase behavior of the binary lipid system.33 The chain-melting transition in the lipid-DNA complexes is entirely dominated by the lipid phase behavior and the structural changes at the gel-to-fluid phase transition are governed by the volume change of the lipid bilayer. Smallangle X-ray scattering and cryo-TEM performed in our group indicate that lamellar liquid-crystalline aggregates are also formed with DODAB/DOPE for a mole fraction of DODAB above 0.25, under the conditions of our experiments.6,34 The turbidity of the solutions increases as the cationic vesicles are added to DNA solution (or vice versa), see Figure 5. This indicates a size change or a change of the particle concentration. A size change was observed by dynamic light scattering.25,29

Barreleiro et al. However, this aggregation is limited and restricted to the fast reaction between the components with what appears no secondary growth as the solutions are stable and no precipitation was observed for days at 25 °C. The requirement is that the ratio of positive charges from the cationic lipid to negative charge equivalents of DNA, F+/-, is below a certain charge ratio that is dependent on the conditions used (concentrations, temperature, ionic strength, etc). Spink and Chaires18 studied the binding of hexadecyltrimethylammonium bromide, CTAB, to DNA using titration microcalorimetry. CTAB is a micelle forming single-chain amphiphile, and the hexagonal structure of the resulting DNACTAB complex35 differs from the lamellar DNA-lipid complex in our study.6,34 Their titration series consisted of consecutive additions of micellar CTAB solution to DNA in buffer solution. They observed initially an exothermic process ascribed to the binding of individual CTAB molecules to DNA, followed by an endothermic process. This process was ascribed to the cooperative binding of CTAB monomers to give DNA-bound aggregates with an enthalpy change of 3.3 kJ (mol CTAB)-1. The enthalpy of micelle formation of CTAB is 1.8 kJ mol-1 under the condition of the experiments, so the second process can also be seen as the binding of CTAB micelles to DNA now with an enthalpy change of 1.5 kJ mol-1. Thus, the binding of surfactant aggregates is weakly endothermic. In this study, high local concentrations were avoided as the reactant solution was added slowly (12 µL during 3 min) to the well-stirred solution in the calorimeter vessel. Attempts were made to use the same conditions in the turbidity measurements. A striking feature of the titration calorimetric results is the constant enthalpy change when small amounts of cationic vesicles were added to DNA solution up to what appears a critical charge ratio, see Figure 3. The process(es) that give(s) the enthalpy changes are fast as seen from Figure 1b, which shows that the enthalpic effect stopped when the injection ended. The turbidity measurements show the same feature, namely, a linear increase in turbidity with increasing concentration of the cationic lipid up to F+/- equal to about 0.8, and then precipitation occurs. The results indicate complete binding of added lipid from the first injection and the complex formation proceeds in the same way until the critical charge ratio is reached when further added lipid gives rise to precipitation. Note that all added lipid is associated in bilayer form and the monomer concentration is negligible, less than 10-9 M-1.36 In our experiments the vesicles are large objects compared to the linear DNA used, which contains about 2 kbp. This means that about 100 DNA molecules are needed to fully neutralize one DODAB vesicle (200 nm). The enthalpy of formation of the DNA-cationic lipid complex was endothermic, which means that the driving force is of entropic origin (∆S > 0). DNA is a linear polyelectrolyte with a high charge density with negative charges located at 1.7 Å distance. Also, the surface of the lipid vesicles has a high charge density. Thus, both DNA and lipid bilayers are surrounded by clouds of oppositely charged small ions (“ion condensation”). A dominating contribution to the formation of the lipoplexes can be expected to be the release of counterions (free energy gain by ∼kT per released counterion) accompanying the binding of the two macroions.37 As seen in Figure 3b, the observed enthalpy changes are directly related to the amount of positively charged lipid, if the measurements are made above the main transition temperature, Tm, where the hydrocarbon chains are in the fluid state. The reaction enthalpies, ∆Hr, are moderately endothermic, 5.3 ( 0.3 kJ(mol DODAB)-1, when mixed vesicles with mole fraction

Interaction of DNA with Cationic Vesicles of DODAB between 0.25 and 0.5 were added to DNA. If the calorimetric measurements were made below Tm, ∆Hr became significantly more endothermic, 9.8 kJ mol-1 for pure DODAB and about 11 kJ(mol DODAB)-1 in mixed vesicles with XDODAB of 0.67 and 0.75. Thus, the state of the bilayer has a pronounced influence on ∆Hr. One could anticipate that a (partial) transition of the bilayer from the gel to the fluid state accompanying the binding to DNA could contribute significantly to the high ∆Hr observed for lipids below Tm. However, DSC measurements show that the Tm of DODAB increases upon complex formation, see Figure 6c. An increase in Tm was also observed for DMTAP when bound to DNA.33 Thus, the observed enthalpic effect does not stem from the chain-melting transition of the DODAB bilayer. When DNA interacts with mixed vesicles, the tendency of the lipids within the bilayer to demix becomes more pronounced already at XDODAB of 0.8, as indicated by the hump centered at 15 °C. At higher mole fractions of DOPE this hump becomes bigger but is centered at the same temperature. For bilayers in the fluid state Tm decreases upon addition of DNA (Figure 6c). Figure 4 shows the calorimetric results when DNA solution was added stepwise to vesicle dispersions containing 0.3 mM DODAB. Also in these titration series the observed enthalpies are constant up to a critical degree of charge neutralization where there is an abrupt drop in the enthalpy values. Under the conditions of our experiments the critical value corresponds to about 80% charge neutralization. The observed enthalpies vary strongly with the lipid composition increasing from 5.1 ( 0.3 kJ mol-1 for pure DODAB vesicles to 9.1 ( 0.5 kJ mol-1 in the mixed vesicle dispersion with XDODAB equal to 0.67. In both cases the lipids are in the gel state. In vesicles with an equimolar mixture of DODAB and DOPE, the lipids are in the fluid state at the temperature of the measurement, and somewhat unexpectedly we do not observe any measurable enthalpy change. There is also a surprising increase in the enthalpy changes when the concentration of lipid was increased to 0.6 mM DODAB with a more than 3-fold increase in the observed enthalpies in the pure DODAB and 0.67 mole fraction DODAB dispersions. In the DODAB/DOPE (1:1) dispersion, the reaction enthalpy was small but now measurable, 1.7 kJ mol-1. In this set of experiments, positively charged lipid-DNA aggregates were formed containing lipid in excess. Injections of additional DNA above 80% charge neutralization gave no observable enthalpy changes. Precipitation takes place as soon as the critical charge ratio is exceeded, as seen from the turbidity measurements and visual observations. But why do the enthalpy peaks vanish before the stoichiometric charge ratio is reached? There should be an appreciable amount of reactant left, but still no measurable amount of complex is formed. One possibility is that a significant amount of excess DNA (or lipid) is bound in the complex to give charged aggregates, as determined from zeta-potential measurements,25 and that the concentration of free DNA (or lipid) is very low. When the critical charge ratio is exceeded, the colloidal dispersion becomes unstable and the aggregates precipitate. Apparently, the formation of precipitate gave no measurable enthalpy change, which maybe is not surprising as the precipitate forms by clustering of the aggregates when the electrostatic repulsion that keeps the aggregates in solution diminishes. The constant enthalpy changes in the titration experiments are consistent with the view that the gain in free energy upon selfassembly of the complex is sufficiently large that lipid-DNA aggregates will form quantitatively until either the supply of lipid or DNA has run out.38 This limit appears to be reached at

J. Phys. Chem. B, Vol. 104, No. 32, 2000 7801 about 80% charge neutralization under the condition of our experiments. With DNA in excess, the complex will contain more DNA than in the isoelectric precipitate and include sodium ions to maintain charge neutrality. In the second case, the amount of cationic lipid in the complex will exceed the amount required for isoelectricity and negative counterions will be present.38,39 The formation of the charged aggregates with lipids in the gel-state gives larger (endothermic) enthalpy changes than in the fluid state. The effect is pronounced for the formation of aggregates containing excess lipid. Partial segregation could be at least part of the reason for the large positive enthalpy changes observed when adding DNA solution to DODAB/DOPE (2:1) vesicles. Our DSC results indicate a tendency to phase separation in the DODAB/DOPE system, and this is increased upon addition of DNA. The enthalpic effect of formation of enriched regions can be expected to be significantly more unfavorable in the gel state than in the fluid. Contributions from such a change will arise only in systems with mixed vesicles, but pure DODAB vesicles also gave a significant enthalpy change, which indicates that the state of the bilayer is of importance. The difference between pure DODAB and mixed lipid vesicles is much less in the formation of negatively charged aggregates, Figure 3. In this situation the state of the lipid chains has a stronger influence on the enthalpy changes than the composition of the bilayers. The structural properties such as interlayer distance and DNA-DNA spacing differ between complexes containing fluid or gel-state lamellar phases,32 which may have enthalpic consequences for the formation of the charged aggregates. The reorganization of the lipid bilayers in the unilamellar vesicles to give the DNA complexes in the form of highly ordered lamellar globules may also contribute to the endothermic enthalpy of formation of the complexes, particularly in the gel state. The change in Tm accompanying complex formation indicates that the packing of the lipids has altered. However, the nature of the lipid counterion also has a significant influence on the formation of negatively charged, DNA-enriched aggregates where no halide ions should be present in the interior of the complex.38 As seen from Figure 2, the bromide counterion gave 4.5 kJ mol-1 more endothermic ∆Hr than chloride for the formation of pure DODAB-DNA and DODAC-DNA aggregates. Enthalpic contributions also may arise from changes in the DNA chains upon complex formation. The DNA keeps its helical conformation in the complexes, but circular dichroism (CD) measurements indicate transformations of the secondary and tertiary structure of DNA in the presence of cationic vesicles.40,41 The fluidity of the vesicle bilayer seems to be a primary requirement for the transfection of DNA, as shown by Akao et al.42 who observed that only amphiphiles that had a phase transition temperature lower than 37 °C could introduce DNA into the eukaryotic cells. Thus, it seems that the bilayers of the lipid mixtures need to be in the fluid state to improve the efficiency of the DNA-lipid complexes. The design of new gene delivery vehicles requires a deeper understanding of both the structures involved and the underlying energetics of binding. Further studies using calorimetric and other techniques are needed to better understand the factors contributing to the formation mechanism of DNA-cationic lipid complexes. 5. Conclusion We have used titration microcalorimetry to characterize the enthalpic changes associated with the formation of DNAcationic vesicle complexes in dilute aqueous solutions. The interaction is fast and endothermic, thus the formation of the

7802 J. Phys. Chem. B, Vol. 104, No. 32, 2000 complex is entropically driven at room temperature. The precise time scale, however, is difficult to measure due to the time resolution of the calorimeter. The enthalpy of complex formation is constant up to a critical charge ratio, F+/-,of about 0.8 when adding vesicles to DNA and up to 1.2 for the addition of DNA to lipid vesicles. Further addition of reactant gave no observable enthalpy changes, which indicates that above 80% charge neutralization no complex formation takes place. The charged DNA-lipid complexes contain excess DNA (or lipid), and the concentration of free DNA (or vesicles) in solution becomes too low for complexes to form. The same critical charge ratio was observed for precipitation in the turbidity measurements. When adding the zwitterionic DOPE to positively charged DODAB vesicles, the melting temperature decreases and, thus, the membrane fluidity increases. The status of the hydrocarbon chains, i.e., solid or fluid, has a pronounced effect on the enthalpy of interaction between DNA and cationic vesicles, especially in the formation of positively charged, lipid-enriched aggregates. We have also made measurements in other systems (e.g., 1,2dioleoyl-3-trimethylammonium propane/dioleoylphosphatidylethanolamine)34 and have found similar results, thus suggesting that the observed thermal behavior is a general feature occurring in the formation of lamellar liquid crystalline complexes made of double-chain lipids and DNA. Acknowledgment. We are grateful to P. Johansson for sharing and helping with the use of the calorimeter equipped with spectrophotometer. We would like to thank T. D. Le for helpful suggestions. P.C.A.B. acknowledges the PRAXIS XXI, JNICT for financial support, scholarship BD/13788/97. Support from The Swedish Research Council for Engineering Sciences (TFR) is acknowledged. References and Notes (1) Felgner, P. L.; Gadek, T. R.; Holm, M.; Roman, R.; Chan, H. W.; Wenz, M.; Northrop, J. P.; Ringold, G. M.; Danielsen, M. Proc. Natl. Acad. Sci. U.S.A. 1987, 84, 7413. (2) Malone, R. W.; Felgner, P. L.; Verma, I. M. Proc. Natl. Acad. Sci. U.S.A. 1989, 86, 6077. (3) Aksentijevich, I.; Pastan, I.; Lunardi-Iskandar, Y.; Gallo, R. C.; Gottesman, M. M. Human Gene Therapy 1996, 7, 1111. (4) Bennet, M. J.; Malone, R. W.; Nantz, M. H. Tetrahedron Lett. 1995, 36, 2207. (5) Farhood, H.; Serbina, N.; Huang, L. Biochim. Biophys. Acta 1995, 1235, 289. (6) Gustafsson, J.; Arvidson, G.; Karlsson, G.; Almgren, M. Biochim. Biophys. Acta 1995, 1235, 305. (7) Templeton, N. S.; Lasic, D. D.; Frederik, P. M.; Strey, H. H.; Roberts, D. D.; Pavlakis, G. N. Nature Biotechnology 1997, 15, 647. (8) Battersby, B. J.; Grimm R.; Huebner S.; Cevc G. Biochim. Biophys. Acta 1998, 1372, 379.

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