Compaction Process of Calf Thymus DNA by Mixed Cationic

J. Phys. Chem. B 2008, 112, 2187-2197

2187

Compaction Process of Calf Thymus DNA by Mixed Cationic-Zwitterionic Liposomes: A Physicochemical Study Alberto Rodrı´guez-Pulido,† Emilio Aicart,† Oscar Llorca,‡ and Elena Junquera*,† Departamento de Quı´mica Fı´sica I, Facultad de Ciencias Quı´micas, UniVersidad Complutense de Madrid, 28040-Madrid, Spain, and Centro de InVestigaciones Biolo´ gicas, CSIC, Ramiro de Maeztu 9, 28040-Madrid, Spain ReceiVed: October 1, 2007; In Final Form: NoVember 27, 2007

The compaction of calf thymus DNA (CT-DNA) by cationic liposomes constituted by a 1:1 mixture of a cationic lipid, 1,2-distearoyl-3-(trimethylammonio)propane chloride (DSTAP), and a zwitterionic lipid, 1,2dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE, null net charge at pH ) 7.4), has been evaluated in aqueous buffered solution at 298.15 K by means of conductometry, electrophoretic mobility, cryo-TEM, and fluorescence spectroscopy techniques. The results reveal that DSTAP/DOPE liposomes are mostly spherical and unilamelar, with a mean diameter of around 77 ( 20 nm and a positively charged surface with a charge density of σζ ) (21 ( 1) × 10-3 C m-2. When CT-DNA is present, the genosomes DSTAP/DOPE/CT-DNA, formed by means of a surface electrostatic interaction, are generally smaller than the liposomes. Furthermore, they show a tendency to fuse forming cluster-type structures when approaching isoneutrality, which has been determined by the electrochemical methods at around (L/D)φ ) 5.6. The analysis of the decrease on the fluorescence emission of the fluorophore ethidium bromide, EtBr, initially intercalated between DNA base pairs, as long as the genosomes are formed has permitted us to confirm the electrostatic character of the DNA-liposome interaction.

I. Introduction Nowadays, cationic lipid-DNA complexes, named genosomes or lipoplexes, are one of the most promising nonviral DNA-delivery systems, particularly in gene transfer therapy.1-21 Contrary to the virus-based methods, cationic liposomes offer biocompatibility, biodegradability, and nonimmunogenity in carrying DNA.2 Understanding the mechanism of interaction of DNA and the cationic surfactant is of great interest for its biological and clinic applications.1-3 However, in spite of the advances in the last years, there still remain many doubts related to the nature and mechanisms of the complex formation, the structure of the genosome, and in particular why genosomes act quite well in vitro but transfection efficiency decays in vivo.2,22,23 The driving force for the formation of lipoplexes is mostly electrostatic, between the negatively charged DNA and the positively charged cationic liposomes. In addition, it is an entropic driven process that comes from the release of counterions to the solution.12,17,18 Neutralization of the negative charges of DNA by cationic liposomes results not only in the complexation of DNA but the formation of different more compact and condensed structures.1,2,6,8,12-14,16-21,24 This compaction occurs on the surface of the cationic liposome. Additionally, the optimum morphology of the genosomes is much depending on the elasticity and the surface charge density, which are related to the nature, concentration, and composition of the cationic-zwitterionic lipid mixture, pH, temperature, and ionic strength.1,2,9,25,26 * To whom correspondence should be addressed. Tel.: +34-91-3944131. Fax: +34-91-394-4135. E-mail: [email protected]. Web: http://www.ucm.es/info/coloidal/index.html. † Universidad Complutense de Madrid. ‡ CSIC.

The purpose of this work is to investigate the formation in aqueous solution of genosomes formed by a double-stranded DNA with a mixed liposome composed by a cationic lipid, 1,2distearoyl-3-(trimethylammonio)propane chloride (DSTAP), and a zwitterionic lipid, 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE). DSTAP is an 18C double chain surfactant of the diacyltrimethylammoniopropane family that has not been studied yet as a nonviral vector, while DOPE, which has neutral charge at pH ) 7.4, is a helper lipid generally added to the cationic liposome for various reasons:2,9,14,22,26-33 (a) It increases the transfection efficiency. (b) It reduces the toxicity of cationic surfactants. (c) It increases the elasticity of the liposome bilayer. (d) It promotes the fusion of lipoplexes with the cell membrane and improves intracellular delivery. (e) Due to its low transition temperature, it decreases the transition temperature of the mixed cationic-zwitterionic liposome to around room temperature. The characterization of the lipoplexes has been done using a wide variety of experimental methods: electrochemical techniques, as ζ potential and conductometry; spectroscopic techniques, as fluorescence spectroscopy; electron microscopic techniques, as cryogenic transmission electron microscopy (cryo-TEM). To get a better comprehension of the properties of the lipoplex, all the above-mentioned experiments have been done covering a wide range of L/D ratios, where L stands for the sum of cationic and neutral lipids masses and D is the DNA mass, at 298.15 K and biological pH ()7.4) due to the action of HEPES buffer, widely used in biochemical assays. Being the lipoplex formation is a process dominated by electrostatic interactions, it is obvious that a careful electrochemical study of the bulk solution, by means of conductometry, and of the lipoplexes surface, from ζ potential, is strongly recommended to understand better the genosome formation.1-4,19,26,34 On the other hand, it is believed that when

10.1021/jp7095828 CCC: $40.75 © 2008 American Chemical Society Published on Web 01/26/2008

2188 J. Phys. Chem. B, Vol. 112, No. 7, 2008 the charge ratio (CR) between cationic liposome and DNA approaches 1, the membranes of the liposomes go on to fuse by a cooperative process to lead DNA to collapse onto condensed structures together with the liposome.1,2,19 There is a great controversy with respect to the possible structure of these compact aggregates, and several packing structures have been proposed: spaghetti and meat-ball models, lamellar structures with DNA intercalated between the layers, inverse micelles hexagonal phases HIIC, etc.9,11,13,14,26,35-37 In this sense, cryoTEM is a powerful microscopic technique that can shed light about the size, shape, and morphology of the lipoplexes.4,6,9,11,14,15,26,38-42 Additionally, DNA and the lipids used in this work do not contain fluorescent groups, but it is wellknown that ethidium bromide (EtBr) is a fluorescent probe widely used in intercalating assays to analyze the cationic liposome-DNA bind-ing.4,7,26,43-46 This probe scarcely fluoresces in water, but its intensity increases up to 30 times when it inserts between the DNA base pairs. Thus, the variation of this emission as long as cationic liposome is added to a DNA-EtBr solution will inform about the type of interactions that take place between DNA and liposomes to yield lipoplexes formation. II. Experimental Section A. Materials. Cationic lipid, 1,2-distearoyl-3-(trimethylammonio)propane chloride (DSTAP), and zwitterionic lipid, 1,2dioleoyl-sn-glycero-3-phosphatidyletanolamine (DOPE), with purities greater than 99% mass, were from Avanti Polar Lipids. Both lipids contain 18 carbons in any of their double tails, and in addition, DOPE contains an unsaturation on the cis configuration at the 9 position of both hydrocarbon chains. The sodium salt of calf thymus DNA (CT-DNA), with less than 5% protein, the fluorescent probe, ethidium bromide (EtBr), and the components of HEPES buffer (4-(2-hydroxyethyl)piperazine1-ethanesulfonic acid and its sodium salt) were from SigmaAldrich. All of them were used without further purification. Distilled water was deionized using a Super Q Millipore system (with a conductivity lower than 18 µS cm-1) and finally was also degassed with a vacuum pump prior to the preparation of the solutions. Solutions were prepared by mass, and unless otherwise stated, all were buffered by using 40 mM HEPES, ionic strength ) 15.3 mM, at 298.15 K. B. Preparation of Mixed Liposomes. Appropriate amounts of DSTAP (L+) and DOPE (L0) were dissolved in chloroform to obtain the desired 1:1 (L+/L0) ratio. After this organic solution was vortexed, chloroform was removed by evaporation under high vacuum during 2-3 h to yield a dry lipid film. For removal of residual amounts of organic solvent, the films were further maintained under high vacuum for an additional 2-3 h. The resulting dry lipid films were then hydrated with 40 mM HEPES, pH ) 7.4. Alternating cycles of vigorous vortexing and sonication were used to help with homogeneization of solutions; in any case, the total time of sonication did not last more than 20 min to avoid the formation of small unilamellar vesicles, SUV.47 Also with the aim of favoring the hydration process, these solutions were heated to a temperature not exceeding 333 K but above the temperatures of gel transition of the lipid components and their mixtures.28 This combination of vortexing, sonication, and heat yields a polydisperse population of multilamellar vesicles, MLV,5,8,48 which were transformed into the desired large unilamelar vesicles, LUV (with a reduction on the polydispersity), by a sequential extrusion procedure.21,30,49-51 This process consisted on subjecting the hydrated solution through polycarbonate membranes (Nucleopore, Whatman International Ltd., Kent, U.K.): 5 passes

Rodrı´guez-Pulido et al. through 400 nm pore size; 5 passes through 200 nm pore size; at least 10 passes through 100 nm pore size. This extrusion was carried on by using a Thermobarrel Lipex Extruder (Northern Lipids, Inc., Vancouver, Canada), with 10 mL of capacity, that uses a N2 current under pressure, which forces the solution to pass through the membrane. In most extrusion cycles, pressures of around 15 atm were enough to extrude the solutions, with the exception of the higher lipid concentrations where pressures up to 30 atm were necessary. The liposomes thus prepared were left to rest during 24 h before their use or their mixing with DNA to form the lipoplexes. C. DNA Preparation. A stock solution of CT-DNA (0.100 mg/mL ) 1.18 × 10-4 M base pair) was prepared by dissolving an appropriate amount of the solid in 40 mM HEPES, pH ) 7.4, 2 days before the mixing with liposomes. DNA concentrations (expressed in mM base pairs) were determined by absorbance at 260 nm ( ) 6600 M-1 cm-1).17,52,53 An A260/ A280 ratio of 1.90 and a negligible absorbance at 320 nm (A320 ) -0.003)8,21,31,52,53 reveal that the contamination of the DNA used in this work by the presence of a certain percentage of proteins is negligible. D. Preparation of Lipoplexes. Equal volumes of DNA and DSTAP/DOPE liposome extruded solutions were mixed by adding DNA over liposome, as usually done in these studies.26,29 Concentrations of both solutions were controlled to fit the final desired L/D ratio, defined as

L/D )

L+ + L0 D

(1)

where L is the sum of cationic (L+) and neutral (L0) lipid masses and D is DNA mass. The ratio L+/L0 is kept constant at around 1:1. The mixing process, after optimization, was done at an adding speed of 0.2 mL/min, with continuous, constant, and vigorous magnetic stirring. Once the addition was concluded, the solution was maintained under agitation during 10 min to favor the formation of lipoplexes. Finally, the lipoplexes thus prepared were kept at room temperature and resting for at least 20 min before their use. E. Electrophoretic Mobility Measurements. A laser Doppler electrophoresis (LDE) technique (Zetamaster 2000, Malvern Instruments Ltd.), previously described,54 was used to measure electrophoretic mobilities. The cell used is a Zetasizer 2000 standard quartz rectangular capillary electrophoresis cell of 5 × 2 × 50 mm, which is calibrated with a ζ potential latex standard of ζ ) -50 ( 5 mV. Temperature was controlled at 298.15 ( 0.01 K. Each electrophoretic mobility data point is taken as an average over 10 independent measurements made at different L/D mass ratios. DNA concentration was kept constant at around 0.05 mg/mL, and the total lipid concentration ranged from 0.05 to 0.75 mg/mL; i.e., L/D ratio was varied from 1 to 15. Electrophoretic mobility for liposome solution in the absence of DNA (L/D ) ∞) was also measured. F. Conductometric Measurements. Conductivity data were collected at 298.15 K ((1 mK) with a Hewlett-Packard 4263A LCR Meter. The whole equipment, the preparation of mixtures, and the fully computerized procedure were widely described previously.55,56 The reproducibility on the specific conductivity, κ, obtained as an average of 2400 measurements for each concentration point, is better than 0.03%. The conductivity measurements were made as a function of total lipid concentration at constant DNA concentration of 0.050 mg/mL, thus varying L/D ratio within the same range as that one used for electrophoretic mobility measurements. A delay time of around

Compaction Process of Calf Thymus DNA

J. Phys. Chem. B, Vol. 112, No. 7, 2008 2189

12 min has been employed prior to measurement after addition of concentrated liposome/DNA solution to ensure the stability of the data. G. Cryo-TEM Measurements. Transmission electron microscopy experiments under liquid-nitrogen temperatures (cryoTEM) were run on samples of DSTAP/DOPE liposomes in the absence and presence of DNA at different L/D ratios below, around, and above the value determined by electrochemical studies for the isoneutrality (L/D)φ. All the samples were prepared and vitrified according to the method devised by Dubochet et al.57 and also described in Llorca et al.58 Aliquots of 5 µL of the different samples were applied to glow-discharged 300 mesh copper holey carbon coated grids (Agar Scientific Ltd.) for 1 min, blotted, and frozen rapidly in liquid ethane at 93 K and kept at this temperature throughout the whole procedure using a GATAN cryoholder and an anticontaminator. Observations were conducted at a JEOL 1230 microscope operated at 100 kV. Micrographs were recorded on Kodak 4489 film at 0˚ tilt and a nominal magnification of 40 000×. Selected micrographs were digitalized in a Dimage Scan Multi Pro scanner (Minolta) at 2400 dpi with a final sampling window of 2.6 Å/pixel at the specimen. H. Fluorescence Emission Spectra: Ethidium Bromide Intercalation Assays. Fluorescence emission spectra of ethidium bromide in the 530-700 nm region were recorded with excitation at 520 nm (the molar extinction coefficient is the same at 520 nm for free and DNA-associated EtBr) by using a PerkinElmer LS-50B luminescence spectrometer.56,59-61 A 10 mm stoppered rectangular silica cell was placed in a stirred cuvette holder whose temperature was kept constant at 298.15 ( 0.01 K. Probe concentration was kept constant at [EtBr] ) 62.9 µM in all cases. Two set of experiments were done: (i) The emission of a EtBr/DNA solution was registered at increasing L concentrations by adding a EtBr/DNA/L solution, thus covering from L/D ) 0 to L/D ) 10 (in both solutions, DNA:EtBr molar ratio is 6:1 and [DNA] ) 0.025 mg/mL. (ii) EtBr emission is measured as long as L concentration increases by adding an EtBr/L solution (blank tests). In all cases, excitation and emission band slits were fixed at 2.5 and 5 nm, respectively, scan rate was selected at 240 nm/min, and emission spectra were recorded at least 10 min after addition of genosomes (study i) and/or liposomes (study ii, blanks).

(CR)φ ) 198

(

)

n+ L+/ML+ L+ M h bp h bp L - L0 M CR ) ) ) ) (2) n- 2D/M D 2ML+ h bp D 2ML+ where n+ and n- are the number of moles of positive and negative charges, coming from cationic lipid and DNA molecule, respectively, ML+ is the molar mass of cationic lipid, and M h bp is the base pairs averaged molar mass ()649.9 g mol-1). Among all L/D or CR ratios, there is one with special and important significance; it is called the isoneutrality ratio, (L/D)φ, defined as the L/D at which the positive charges of the liposome are stochiometrically equal to the negative charges of DNA, i.e., CR ) 1. This parameter is characteristic of each genosome and is expected to appear at a value which can be calculated from eq 2 as

L+ D

φ

)

2ML+ M h bp

(3)

In the present case, ML+ for DSTAP is 702.59 g mol-1, which implies, from eqs 2 and 3, that for the DSTAP/DOPE/CT-DNA genosome studied herein L/D ) 4.5 CR and, thus, the isoneutrality would be reach at (L/D)φ ) 4.5. The electrochemical analysis has been done at two levels: the bulk, by conductometry experiments; the surface of the charged liposomes and genosomes, by the LDE technique (electrophoretic mobility). Figure 1 shows, as an example, a plot of 10 independent experiments of light scattered intensity, ILS, as a function the experimental electrophoretic mobility, µe, for DSTAP/DOPE/CT-DNA genosomes at L/D ) 10.5 (DNA concentration was kept constant at 0.050 mg/mL, in aqueous HEPES medium at pH ) 7.4 and 298.15 K) that yield an averaged electrophoretic mobility of µ j e ) (3.66 ( 0.12) × 10-8 m2 V-1 s-1. Table 1 resumes the values obtained at all L/D ratios studied, including the value of L/D ) ∞, i.e. only liposome. As can be seen in the table, there is a sign inversion between L/D ) 5.1 and L/D ) 6.1; i.e., the DSTAP/DOPE/ CT-DNA genosomes change the electrophoretic mobility from negative to positive when the mass of lipid is between five times and six times that of DNA. The well-known Henry equation has been used to relate electrophoretic mobility and ζ potential:62-64

ζ)

3η µ 20rf(κDa) e

(4)

Here η is the viscosity of water (8.904 × 10-4 N m-2 s at 298.15 K), 0 and r are the vacuum and relative permittivity (8.854 × 10-12 J-1 C2 m-1 and 78.5, respectively), and f(κDa) is the Henry function that depends on the reciprocal Debye length, κ, and the particle radius, a. For medium-to-large particles in a medium of moderate ionic strength (a . κD-1), the Smoluchowski limit is usually applied (f(κDa) ) 1.5) to estimate the Henry function.62-64 Another property that can give an interesting information about the distribution of charges on the surface of liposomes and genosomes is the surface charge density enclosed by the shear plane, σζ; it can be calculated from ζ potential, assuming a Gouy-Chapman double layer, by using the equation62-64

III. Results and Discussion The interaction of DSTAP/DOPE mixed liposome with CTDNA has been studied in this work by covering a wide range of L/D mass ratios, including the values of L/D ) 0 (only DNA is present) and L/D ) ∞ (only liposome is present). This L/D mass ratio is related with charge ratio, CR, by the expression

( )

σζ )

( )

20rκDkBT zeζ sinh ze 2kBT

(5)

where e is the elemental charge, z is the valence of the ion, kB is the Boltzmann constant, and T is the absolute temperature. Table 1 also reports the values of ζ potential and surface charge density at different L/D ratios, and Figure 2 shows the plot of σζ vs L/D for the DSTAP/DOPE/CT-DNA genosomes. The dotted line corresponds to the value for DSTAP/DOPE (1:1) liposome in the absence of DNA (L/D ) ∞, σζ ) (21 ( 1) × 10-3 C m-2). As can be seen in the figure, the variation of surface charge density follows a sigmoidal habit, as previously found for other genosomic systems.2,4,5,26,65 It means that three clear zones can be distinguished in the figure: (i) the zone where the net charge of genosomes is negative and almost constant at σζ ) (-19 ( 1) × 10-3 C m-2; (ii) the zone where the inversion of sign takes place at (L/D)φ ) 5.6; (iii) the zone where the net charge of the genosomes is positive and tends to the value for the DSTAP/DOPE liposome (σζ ) (21 ( 1) × 10-3 C m-2). Since the buffered medium used in the ζ potential, cryo-TEM, and fluorescence studies (40 mM HEPES, ionic strength I )

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Rodrı´guez-Pulido et al.

Figure 1. Scattered light intensity vs electrophoretic mobility, µe (10 independent experiments), for DSTAP/DOPE/CT-DNA genosomes at L/D ) 10.5, in aqueous HEPES medium at pH ) 7.4. DSTAP:DOPE ) 1:1. µ j e ) (3.66 ( 0.12) × 10-8 m2 V-1 s-1.

Figure 2. Values of surface density charge, σζ, of DSTAP/DOPE/ CT-DNA genosomes at different L/D ratios in aqueous 40 mM HEPES medium at pH ) 7.4. DSTAP:DOPE ratio is 1:1. [DNA] ) 0.050 ( 0.001 mg/mL. Solid line: sigmoidal fit of experimental values. Dot line corresponds to the value for DSTAP:DOPE (1:1) liposome in the absence of DNA (L/D ) ∞) (σζ ) (21 ( 1) × 10-3 C m-2).

TABLE 1: Values of Electrophoretic Mobility, µe, Zeta Potential, ζ, and Surface Density Charge (at the Shear Plane), σζ, at Different Values of L/D Mass Ratios for DSTAP/DOPE/CT-DNA Genosomesa L/D

108µe/m2 V-1 s-1

ζc/mV

103σζc/C m-2

1.8 3.1 3.1 3.8 4.8 5.1 5.1 6.1 6.9 8.0 10.5 12.2 15.2 ∞b

-4.12 -4.15 -4.55 -4.15 -4.40 -3.83 -4.12 3.17 3.88 3.08 3.66 3.99 4.35 4.62

-52 -53 -58 -53 -56 -49 -52 40 49 39 47 51 55 59

-18 -18 -20 -18 -19 -18 -16 13 16 12 15 17 19 21

a DNA concentration was kept constant at 0.050 ( 0.001 mg/mL. Liposome in the absence of DNA. c Errors are estimated to be around 3% in ζ potential and around 6% in surface density charge.

b

15.3 mM, pH ) 7.4) yields such a high conductivity (∼10-3 S cm -1) that masks all the changes assignable to genosomes formation, the conductivity study was done in aqueous medium at constant DNA concentration ()0.050 mg/mL), as a function of lipid concentration [L]. As can be seen in Figure 3, the conductivity increases linearly with [L] for DSTAP/DOPE liposomes; it is worth noting that the critical vesicle concentration, CVC, for this mixed liposome is too low to be detected within this concentration range. However, in the presence of DNA, conductivity shows a clear change on the positive slope at [L] ) 0.25 mM, which corresponds to an L/D ratio of 3.6. At [L] below the break, electrostatic interactions between the positive charges facing outward from the liposome surface and the negative charges of DNA provoke a release of counterions, Na+ from DNA and Cl- from liposomes, thus justifying the increase observed in conductivity. Once the break occurs, a lower slope is seen given that only the counterions coming from liposome dissociation contribute to the conductivity. Similar behavior has been recently found for cat-anionic vesicles binding DNA.66 The (L/D)φ value thus obtained is lower than

Figure 3. Specific conductivity, κ, as a function of total DSTAP/DOPE liposome concentration, in the absence (open symbols) and presence (solid symbols) of DNA at constant concentration ()0.050 mg/mL) in aqueous medium.

that one obtained from surface density charge sigmoidal plots (L/D)φ ) 5.6, this difference being attributable to the different ionic strength of the media used in both experiments. But in any case both are in reasonable good agreement with that one calculated from eqs 2 and 3 and with that reported for other genosomes, formed by DOTAP, DOTIM, DDAB, DOPC, or EDOPC cationic lipids (with or without helper lipids) and DNA, using different experimental methods as ζ potential,4,19,26,34 isothermal titration calorimetry,8,12 dynamic light scattering,19,20,32-34,46,67,68 or fluorescence spectroscopy.4,20,21,26,33,46 The shape, size, and morphology of the liposomes and genosomes have been evaluated by using transmission electron microscopy techniques. For that purpose, cryo-TEM experiments were run on one sample of DSTAP/DOPE liposome solution and three samples of DSTAP/DOPE/CT-DNA genosomes at L/D below, around, and above (L/D)φ. Figure 4 shows a selection



Figure 4. Gallery of selected cryo-TEM micrographs of DSTAP:DOPE (1:1) liposomes (L/D ) ∞, liposomes in the absence of CT-DNA). Scale bar: 100 nm.

Figure 5. Gallery of selected micrographs of DSTAP/DOPE/CT-DNA genosomes. (DSTAP:DOPE ) 1:1) at different L/D ratios: (a, b) (L/D) < (L/D)φ; (c-f) (L/D) > (L/D)φ. Scale bar: 50 nm.

of the micrographs taken on cryo-TEM experiments of DSTAP/ DOPE liposomes. These photographs reveal the presence of unilamellar spherical liposomes, with an average diameter of around 77 ( 20 nm, in agreement with other cationic mixed liposomes reported in the literature.60,69,70 Figures 5 and 6 present a gallery of micrographs selected among those taken on cryoTEM experiments of DSTAP/DOPE/CT-DNA genosomes at L/D below (Figure 5a,b), above (Figure 5c-f), and around (L/ D)φ (Figure 6). Several features can be observed on these micrographs: (i) The main one is that, compared with the structures seen on the micrographs of liposomes, genosomic structures are more condensed, with a clear accumulation of electronic density on the surface, irrespectively of L/D ratio. The neat line representing the lipidic bilayer, characteristic of liposomes and clearly observed on micrographs of Figure 4, has been lost. (ii) However, the size and shape of genosomes do depend on L/D value. Thus, at L/D below and above (L/ D)φ, the structures are mostly roughly spherical, as liposomes, but with smaller diameters (around 20 nm), while, at L/D close

to isoneutrality, a certain percentage of cluster-type structures appear together with those ones also found at the other L/Ds. A possible explanation to all these observations is as follows: when the liposomes are in the presence of DNA, the genosomes are formed, the driving force being a surface electrostatic interaction. It means that DNA is compacted and condensed at the surface of liposomes by action of a strong interaction between the positive charges of liposome surface and the negative charges of phosphodiester DNA groups. This would explain the increase of electronic density on the surface of the spherical structures seen in Figures 5 and 6. Furthermore, this interaction may have two consequences: (i) Liposomes are disrupted to form smaller structures, irrespectively of L/D ratio. (ii) When the positive charge of liposomes are neutralized by the negative charges of DNA, i.e., L/D ∼ (L/D)φ or CR ∼ 1, liposomes may go on to fuse mediated by condensed DNA, as also found in the literature for other genosomes.4,8,9,18,19,26,40,42,71 This behavior may be explained by considering that, at CR ) 1, the net charge on the surface of the genosomes would be



Figure 6. Gallery of selected micrographs of DSTAP/DOPE/CT-DNA genosomes. (DSTAP:DOPE ) 1:1) at L/D ∼ (L/D)φ. Scale bar: 50 nm.

CHART 1: Drawing of the DNA Compaction Process, Accompanied by a Reduction on Liposome Size and by the Formation of DNA-Mediated Cluster Type Structures

neutral or “isoelectric” and, therefore, there would be no electrical barrier to aggregation between complexes. With the information yielded by ζ potential and cryo-TEM results, and considering the molecular data for DSTAP and DOPE,72-75 a model based on the geometry of the liposome can be designed to explain the experimental evidence found on the genosome formation process. Thus, in the absence of DNA, DSTAP/DOPE liposomes, with an averaged diameter of 77 nm, would contain around 48 400 lipids, i.e., 24 200 cationic lipids/ liposome. For this calculation, we have assumed72-75 a lipidic bilayer of approximately 5 nm width and a polar head surface of around 0.68 nm2; the surface area of the spherical liposomes ()8π(0.25d2 - 2d + 10), d is the diameter) is calculated by using the diameter obtained on cryo-TEM experiments. However, in the presence of DNA, the liposomes reduce their size (SUV, with averaged diameters of around 21 nm) and, accordingly, they contain fewer lipid molecules, only around 2600, 1300 of which would be cationic lipids. Considering that the CT-DNA used in these experiments consists of 9200 base pairs/ double-stranded segment,76 it would be necessary for 14 liposome aggregates in close proximity/DNA segment to reach isoneutrality and, thus, DNA compaction. This estimation is in good agreement with what cryo-TEM micrographs have shown.

Chart 1 shows a schematic drawing of the DNA compaction process, accompanied by a clear reduction in liposomes size. To confirm and characterize this surface electrostatic liposome-DNA interaction, a complete fluorescence spectroscopic study has been carried out, by following the variation on the emission of ethidium bromide, in aqueous solutions at constant CT-DNA concentration as long as a DSTAP/DOPE solution is added, and thus, the genosome is formed. EtBr is an aromatic planar cationic fluorophore, whose fluorescence intensity increases around 30 times upon its intercalation between base pairs of double-stranded DNA.4,7,26,43-45 The reason for ethidium bromide’s intense fluorescence after binding with DNA is probably not due to rigid stabilization of the phenyl moiety, because the phenyl ring has been shown to project outside the intercalated bases. In fact, the phenyl group is found to be almost perpendicular to the plane of the ring system, as it rotates about its single bond to find a position where it will abut the ring system minimally. Instead, the hydrophobic environment found between the base pairs is believed to be responsible. By moving into this hydrophobic environment and away from the solvent, the ethidium cation is forced to shed any water molecules that were associated with it. As water is a highly efficient fluorescent quencher, the removal of these water molecules allows the


Figure 7. Emission fluorescence spectra of EtBr in the presence of DSTAP/DOPE/CT-DNA genosomes at different L/D ratios (corrected with the blank experiments, Figure 8): 0, L/D ) 0; 1, L/D ) 1.0; 2, L/D ) 2.1; 3, L/D ) 3.1; 4, L/D ) 4.1; 5, L/D ) 5.1; 6, L/D ) 6.1; 7, L/D ) 7.1; 8, L/D ) 8.1; 9, L/D ) 9.1; 10, L/D ) 10.1. Medium: aqueous 40 mM HEPES, pH ) 7.4. DSTAP:DOPE ratio is 1:1. DNA: EtBr ratio is 6:1. [DNA] ) 0.025 mg/mL.

ethidium to fluoresce. Furthermore, thanks to this intercalation, EtBr is protected as well from molecular oxygen thus avoiding other quenching processes. When EtBr intercalation is prevented by DNA condensation or compaction on cationic liposome surface, EtBr fluorescence intensity will be quenched, as the probe will remain fully accessible to the bulk water and molecular oxygen.43 Accordingly, the decrease on probe emission intensity can be used as a mean to asses DNA accessibility and, thus, DNA-liposome condensation. Figure 7 shows the emission fluorescence spectra of EtBr at constant DNA concentration ([DNA] ) 0.025 mg/mL) as long as lipid concentration, and thus L/D ratio, increases. The spectra shown in this figure are corrected with the blank experiments (see Figure 8), which consist of the measurement of EtBr emission, in the absence of DNA, at certain values of lipid concentration that had necessarily to be the same as those used in the experiments with DNA. In all the cases, the aqueous solution is buffered at pH ) 7.4 to ensure the neutral charge of DOPE by using 40 mM HEPES. It can be observed in these figures that EtBr emission decreases in the presence of DNA at increasing L/D ratios (Figure 7), but it remains basically unaffected and very low in the absence of DNA with the same increase in L/D (Figure 8). Also remarkable in Figure 7 is that there are two groups of spectra: from L/D ) 0 to L/D ) 5.1, the emission decreases gradually from around 200 au to around 100 au, and from L/D ) 6.1 to L/D ) 10.1, the emission intensity remains almost constant at negligible values. This evidence confirms that (i) EtBr does not interact with cationic DSTAP/DOPE liposome, as would be expected for a cationic probe, (ii) EtBr interacts with DNA, as described above, but the DNA-liposome interaction is stronger and, with the addition of lipid, the probe is displaced from the DNA base pair microenvironment toward the aqueous bulk, where the quantum fluorescence yield of the probe falls dramatically down, and (iii) the isoneutrality must occur at 5.1 < L/D < 6.1, in total agreement with ζ potential results. The ground state of the fluorescent probe used in this work yields, upon excitation, a locally excited state (LE) (via π f


Figure 8. Emission fluorescence spectra of EtBr in the presence of DSTAP/DOPE liposomes at different concentrations (blank experiments). Medium: 40 mM aqueous HEPES, pH ) 7.4. DSTAP:DOPE ratio is 1:1. Although these experiments are done in the absence of DNA, L/D notation has been kept to refer to the same ratios for the corresponding experiments in the presence of DNA: 0, L/D ) 0; 1, L/D ) 1.0; 2, L/D ) 2.1; 3, L/D ) 3.1; 4, L/D ) 4.1; 5, L/D ) 5.1; 6, L/D ) 6.1; 7, L/D ) 7.1; 8, L/D ) 8.1; 9, L/D ) 9.1; 10, L/D ) 10.1.

π* electronic transition). To interpret the mechanism by which this excited state is formed and deactivated, we have followed in this work the approach77 that considers the π f π* emission band of the fluorescent probe as consisting of different bands at different wavelengths, each of which attributable to the π f π* emission of the probe immersed within different microenvironments, characterized by its hydrophobicity, microviscosity, rigidity, and/or solvation features. Accordingly, experimental emission spectra of Figure 7 were analyzed by deconvolution into overlapping Gaussian curves with a commercial nonlinear least-squares multipeaks fitting procedure that uses an iterative Marquardt-Levenberg fitting algorithm. Both the procedure and the control of the goodness of the fits were widely explained elsewhere.60 Figure 9 shows a resume of the deconvolution process into 1 and 2 Gaussian components at a selection of the L/D ratios studied herein, and Table 2 reports the spectroscopic parameters of both fits. The information recorded in Figure 9 and Table 2 deserves some remarks. At L/D ) 0, the best fit corresponds to the 2-Gaussian option, indicating that, in the absence of liposome, EtBr is partitioned between two possible microenvironments, which correspond to λ1 ) 580 ( 5 nm and λ2 ) 600 ( 5 nm. These two bands are assigned to the π f π* emission of EtBr intercalated between the base pairs of the DNA double helix and immersed in the aqueous bulk, respectively. The more hydrophobic is the microenvironment, the more blueshifted the emission band appears. As can be seen in the table, this assignment can be also applied in the presence of liposomes, as L/D increases, up to the surroundings of isoneutrality (L/ D)φ. However, it can be also observed in the figure that as long as L/D increases, the two-Gaussian fit is gradually losing physical and mathematical basis in favor of the 1-Gaussian fit. In fact, once the isoneutrality is passed, for example, at L/D ) 6.1, the correct choice is the 1-Gaussian fit, with λ1 ) 590 ( 5 nm, assigned to the probe in the bulk. This evidence



Figure 9. Emission fluorescence spectra of EtBr in the presence of DSTAP/DOPE/CT-DNA genosomes at different L/D ratios, together with their deconvolutions into 1 or 2 Gaussian components. Solid line: experimental spectra. Dashed line: Gaussian components. Dotted line in 2 Gaussian cases: total sum of Gaussian components. Medium: 40 mM aqueous HEPES, pH ) 7.4. DSTAP:DOPE ratio is 1:1. DNA:EtBr ratio is 6:1. [DNA] ) 0.025 mg/mL.



TABLE 2: Parameters of the Deconvoluted Gaussian Bands of EtBr Fluorescence Emission Spectra in the Presence of DSTAP/ DOPE/CT-DNA Genosomes at Different L/D Ratiosa 2 Gaussians

1 Gaussian

L/D

I588

λ1/nm

W1

A1 (%)

λ2/nm

W2

A2 (%)

r2

0 1.0 2.1 3.1 4.1 5.1 6.1

203 197 170 152 129 98 11

580 581 581 585 582 580

33 34 34 40 32 24

4109 (34) 3614 (32) 3004 (30) 4666 (52) 1728 (22) 505 (9)

601 599 599 604 596 593

48 49 50 51 52 50

7879 (66) 7743 (68) 6857 (70) 4224 (48) 6019 (78) 5207 (91)

0.999 0.998 0.999 0.998 0.998 0.997

λ1/nm

W1

A1

r2

592

51

1226

0.988

a

Wavelength, λi, width, Wi, and area, Ai, in terms of % contribution to the overall fluorescence emission area (within parentheses). Medium: aqueous 40 mM HEPES, pH ) 7.4. DSTAP:DOPE ratio is 1:1. DNA:EtBr ratio is 6:1. [DNA] ) 0.025 mg/mL.

TABLE 3: Parameters of the Fit of EtBr Fluorescence Emission Spectra to 1 Gaussian Band, in the Presence of DSTAP/DOPE (1:1) Liposomes at Different Concentrations (Blank Tests)a L/D

I588

λ1/nm

W1

A1

R2

0 1.0 2.1 3.1 4.1 5.1 6.1 7.1 8.1 9.1 10.1

12 10 11 11 10 10 9 10 11 11 10

597 597 597 597 597 597 595 595 595 594 595

57 56 55 56 55 58 56 65 57 67 67

834 722 680 673 655 657 611 821 630 883 881

0.998 0.982 0.998 0.996 0.998 0.995 0.995 0.994 0.996 0.994 0.994

a Wavelength, λi, width, Wi, and area, Ai. Medium: aqueous 40 mM HEPES, pH ) 7.4. DSTAP:DOPE ratio is 1:1. Although these experiments are done in the absence of DNA, L/D notation has been kept to refer to the same ratios for the corresponding experiments in the presence of DNA.

corroborates the type of interactions that are taking place: EtBr, initially within the DNA helix, is displaced to the aqueous bulk when the liposome is added, and the genosome is formed, given that the EtBr-DNA interaction is weaker than the electrostatic liposome-DNA interaction. In other words, the probe and the liposome compete for DNA. For that reason, at the beginning of the experiment EtBr can be within two microenvironments (bulk and DNA helix), but when the probe is totally displaced from inside of DNA helix, it can only be in the bulk. These conclusions and assignments are reinforced when the same deconvolution protocol is applied to the corresponding spectra in the absence of DNA (spectra of Figure 8, blank tests). A figure with the 1- and 2-Gaussian fits is given as Supporting Information, and Table 3 summarizes the fitting parameters of the best fit. As can be seen, only one Gaussian centered at 595 ( 5 nm is found irrespectively of L concentration, and in this case, it is unambiguously assigned to the emission of the probe in the bulk. Figure 10 shows a plot of the emission intensity at maximum wavelength (λmax ) 588 nm) vs L/D, together with some schematic drawings that try to sumarize all the abovedescribed evidence and microenvironments. With application of Phillips’s method to the data in the figure, an (L/D)φ is determined at 5.4, in very good agreement with that one obtained from ζ potential results. Accordingly, it can be concluded that EtBr emission decreases linearly with L/D up to (L/D)φ; above it, the emission is constant at negligible values and the probe has been totally displaced to the aqueous medium. A similar trend is followed by the total area, i.e., the sum of areas, reported in Table 2. However, in terms of % of the total area, it can be deduced from the data in Table 2 that, below (L/D)φ, Α1

Figure 10. Emission fluorescence intensity of EtBr at 588 nm in the presence of DSTAP/DOPE/CT-DNA genosomes as a function of L/D ratio. Medium: 40 mM aqueous HEPES, pH ) 7.4. DSTAP:DOPE ratio is 1:1. DNA:EtBr ratio is 6:1. [DNA] ) 0.025 mg/mL. The drawings show schemes of the microenvironments where the probe may be housed at different L/D ratios, and the numbers indicate the optimum number of Gaussian components, which is the number of possible microenvironments in each case.

decreases while A2 increases with (L/D) confirming that the probe content decreases inside the helix (blue-shifted Gaussian, 1) and increases in the bulk (red-shifted Gaussian, 2) as long as the genosome is formed. Conclusions A set of electrochemical (conductometry and ζ potential), spectroscopic, and microscopic studies of CT-DNA in the presence of cationic DSTAP/DOPE liposomes has revealed that the polyelectrolyte is nicely condensed and compacted by the liposomes by means of a strong surface electrostatic interaction. The isoneutrality of the genosome thus formed is reached when the lipid mass is around 5.5 ( 0.1 times the DNA mass. The size of the spherical unilamelar DSTAP/DOPE liposomes of around 77 ( 20 nm of diameter decreases in the presence of DNA. In the proximity of isoneutrality, where there is no electrical barrier to avoid aggregation, it is observed that liposomes tend to form cluster-type structures, DNA-mediated. Calculations made with molecular parameters of both lipid and

2196 J. Phys. Chem. B, Vol. 112, No. 7, 2008 DNA molecules indicate that 14 SUV liposomes of around 21 nm of diameter are necessary to compact one DNA doublestranded segment, in agreement with experimental evidence. The experimental results herein reported allow one to conclude that DSTAP/DOPE liposomes efficiently compact and condense CTDNA, thus being potentially useful on developing DNA vectors, necessary in gene therapy protocols. It can be also concluded that experiments with different approaches to the studied interactions (electrochemical, spectroscopic, microscopic, etc.) are necessary to draw a complete global picture of the DNA compaction process. Acknowledgment. We thank the Spanish Ministry of Education, Project No. CTQ2005-1106, and the Comunidad Autońoma of Madrid, Project No. S-SAL-0249-2006. O.L. thanks the MEC of Spain (Project No. SAF2005-00775) for supporting cryo-TEM measurements. Supporting Information Available: Emission fluorescence spectra of EtBr in the presence of DSTAP/DOPE liposomes at different L/D ratios. This material is available free of charge via the Internet at http://pubs.acs.org. References and Notes (1) Janoff, A. S. Liposomes: Rational Design; Marcel Dekker, Inc.: New York, 1999. (2) Lasic, D. D. Liposomes in Gen DeliVery; CRC Press: Boca Raton, FL, 1997. (3) Rosoff, M. Vesicles; Marcel Dekker, Inc.: New York, 1996. (4) Birchall, J. C.; Kellaway, I. W.; Mills, S. N. Int. J. Pharm. 1999, 183, 195. (5) Ciani, L.; Ristori, S.; Calamai, L.; Martini, G. Biochim. Biophys. Acta 2004, 1664, 70. (6) Dias, R. S.; Lindman, B.; Miguel, M. G. J. Phys. Chem. B 2002, 106, 12600. (7) Eastman, S. J.; Siegel, C.; Tousignant, J.; Smith, A. E.; Cheng, S. H.; Scheule, R. K. Biochim. Biophys. Acta 1997, 1325, 41. (8) Gonç alves, E.; Debs, R. J.; Heath, T. D. Biophys. J. 2004, 86, 1554. (9) Gustafsson, J.; Arvidson, G.; Karlsson, G.; Almgren, M. Biochim. Biophys. Acta 1995, 1235, 305. (10) Harries, D.; May, S.; Gelbart, W. M.; Ben-Saul, A. Biophys. J. 1998, 75, 159. (11) Huebner, S.; Battersby, B. J.; Grimm, R.; Cevc, G. Biophys. J. 1999, 76, 3158. (12) Kennedy, M. T.; Pozharski, E. V.; Rakhmanova, V. A.; MacDonald, R. C. Biophys. J. 2000, 78, 1620. (13) Koltover, I.; Salditt, T.; Ra¨dler, J. O.; Safinya, C. R. Science 1998, 1998, 78. (14) Lasic, D. D.; H., S.; Stuart, M. A. C.; R., P.; Frederik, P. J. Am. Chem. Soc. 1997, 119, 832. (15) Mel’nikova, Y. S.; Mel’nikov, S. M.; Lofroth, J. E. Biophys. Chem. 1999, 81, 125. (16) Pozharski, E.; MacDonald, R. C. Biophys. J. 2002, 83, 556. (17) Pozharski, E.; MacDonald, R. C. Biophys. J. 2003, 85, 3969. (18) Ra¨dler, J. O.; Koltover, I.; Salditt, T.; Safinya, C. R. Science 1997, 275, 810. (19) Ra¨dler, J. O.; Koltover, I.; Jamieson, A.; Salditt, T.; Safinya, C. R. Langmuir 1998, 14, 4272. (20) Zuidam, N. J.; Barenholz, Y. Int. J. Pharm. 1999, 183, 43. (21) Zuidam, N. J.; Hirsch-Lemer, D.; Margulies, S.; Barenholz, Y. Biochim. Biophys. Acta 1999, 1419, 207. (22) de Lima, M. C. P.; Simoes, S.; Pires, P.; Faneca, H.; Duzgunes, N. AdV. Drug. DeliVery ReV. 2001, 47, 277. (23) Nicolazzi, C.; Garinot, M.; Mignet, N.; Scherman, D.; Bessodes, M. Curr. Med. Chem. 2003, 10, 1263. (24) Chesnoy, S.; Huang, L. Annu. ReV. Biophys. Biomol. Struct. 2000, 29, 27. (25) Jones, M. N.; Chapman, D. Micelles, Monolayers and Biomembranes; Wiley-Liss: New York, 1995. (26) Xu, Y. H.; Hui, S. W.; Frederik, P.; Szoka, F. C. Biophys. J. 1999, 77, 341. (27) Barreleiro, P. C. A.; Olofsson, G.; Brown, W.; Edwards, K.; Bonassi, N. M.; Feitosa, E. Langmuir 2002, 18, 1024. (28) Feitosa, E.; Alves, F. R.; Niemiec, A.; Oliveira, M.; Castanheira, E. M. S.; Baptista, A. L. F. Langmuir 2006, 22, 3579.

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Compaction Process of Calf Thymus DNA by Mixed Cationic

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