A Physicochemical Characterization of the Interaction between DC

J. Phys. Chem. B 2008, 112, 12555–12565

12555

A Physicochemical Characterization of the Interaction between DC-Chol/DOPE Cationic Liposomes and DNA Alberto Rodrı´guez-Pulido,† Francisco Ortega,† Oscar Llorca,‡ Emilio Aicart,† 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: May 8, 2008; ReVised Manuscript ReceiVed: July 3, 2008

A 1:1 mixture of the cationic lipid 3β-[N-(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-Chol) and the zwitterionic lipid, 1,2-dioleoyl-sn-glycero-3-phosphoetanolamine (DOPE), has been used to compact calf-thymus DNA (CT-DNA) in aqueous buffered solution at 298.15 K. The formation process of this lipoplex has been analyzed by means of electrophoretic mobility, cryo-TEM, dynamic light scattering, and fluorescence spectroscopy techniques. The experimental results indicate that DC-Chol/DOPE liposomes are mostly spherical and unilamellar, with a mean diameter of around 99 ( 10 nm and a bilayer with a thickness of 4.5 ( 0.5 nm. In the presence of CT-DNA, DC-Chol/DOPE/CT-DNA lipoplexes are formed by means of a strong entropically driven surface electrostatic interaction, as confirmed by zeta potential and fluorescence results, as a consequence of which DNA is compacted and condensed at the surface of the cationic liposomes. The negative charges of DNA phosphate groups are neutralized by the positive charges of cationic liposomes at the isoneutrality L/D ratio, (L/D)φ around 4, obtained from electrophoretic, fluorescence, and DLS measurements. The decrease in the fluorescence emission intensity of ethidium bromide, EtBr, initially intercalated between DNA base pairs, as long as the association between the biopolymer and the cationic liposomes takes place has permitted one to confirm its electrostatic character as well as to evaluate the different microenvironments of varying polarity of DNA-double helix, liposomes, and/or lipoplexes. Electronic microscopy reveals a rich scenario of possible nanostructures and morphologies for the lipoplexes, from unilamellar DNA-coated liposomes to multilamellar lipoplexes passing through cluster-like structures and several intermediate morphologies. I. Introduction Cationic liposomes are self-assembled and self-organized nanosystems currently investigated as possible nonviral carriers of nucleic acids in gene delivery.1-15 Lipofection, that is, transfection by cationic liposomes, offers a series of advantages over other methods of transfection; cationic liposomes are known to be efficient in gene delivery in vitro because they do not disrupt the cell membrane, are metabolizable, are capable of transfecting many cell types, and have the potential to target specific tissues in vivo.16 Although in the last decades lipoplexes (cationic liposome/DNA complexes) have been used for in vivo and in vitro experiments, the differences between in vitro and in vivo transfection are still unclear. Thus, the results of in vitro experiments cannot be directly translated and/or extrapolated to the in vivo process. Consequently, physicochemical and biological studies are still necessary to improve our understanding of lipoplex structure and lipofection process, both aspects of great interest for its biological and clinic applications.1,2 It is well-known that electrostatics plays a major role in lipoplex formation and in various steps of the transfection process, which is entropically driven due to the release of counterions (cations from the DNA surface and anions from the cationic liposome surface) to the solution.9,14 Neutralization * Corresponding author. Phone: +34-91-3944131. Fax: +34-91-3944135. E-mail: [email protected]. † Universidad Complutense de Madrid. ‡ CSIC.

of the negative charges of DNA by cationic liposomes results in the formation of different structures where DNA is compacted and condensed.1-3,5,9-11,13-15,17 This compaction, which occurs on the surface of the cationic liposome, leads to several packing structures: spaghetti and meatball models, lamellar structures with DNA intercalated between the layers, inverse micelles hexagonal phases, etc.6,8,10,11,18,19 The optimum morphology of the resulting lipoplex depends very much on the elasticity and the surface charge density of the liposome membrane, which are related to the nature, concentration, and composition of the cationic-zwitterionic lipid mixture, pH, temperature, and ionic strength.1,2,6,19 We have recently reported20 an extensive study of the compaction of DNA by cationic liposomes constituted by a 1:1 mixture of a cationic lipid, 1,2-distearoyl-3-trimethylammoniumpropane chloride (DSTAP), a 18 C-double chain surfactant of the diacyltrimethylammoniumpropane family that was not studied yet as nonviral vector, and a zwitterionic lipid, 1,2dioleoyl-sn-glycero-3-phosphoetanolamine (DOPE). The results revealed that DSTAP/DOPE/DNA lipoplexes were formed by means of a strong surface electrostatic interaction. The size of the spherical unilamelar DSTAP/DOPE liposomes decreased in the presence of DNA, while in the proximity of isoneutrality, where there is no electrical barrier to avoid aggregation, liposomes showed a clear tendency to form cluster-type structures, DNA-mediated. Now, we present in this work the interaction in aqueous solution between a double-stranded DNA

10.1021/jp804066t CCC: $40.75  2008 American Chemical Society Published on Web 08/27/2008

12556 J. Phys. Chem. B, Vol. 112, No. 39, 2008 CHART 1: (a) Cationic Lipid Molecule, DC-CHOL; and (b) Zwitterionic Lipid Molecule, DOPE

(calf-thymus DNA, CT-DNA) and a mixed liposome composed by a completely different cationic lipid belonging to the sterols family, 3β-[N-(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-Chol), and the same zwitterionic lipid, DOPE (see Chart 1). DOPE, which has neutral charge at pH ) 7.4, is frequently used as a helper lipid,2,6,11,19,21-25 for in vitro and in vivo transfection, because it increases the transfection efficiency, reduces the toxicity of cationic surfactants, increases the elasticity of the liposome bilayer, promotes the fusion of lipoplexes with the cell membrane improving intracellular delivery, and decreases the transition temperature of the mixed cationic-zwitterionic liposome to around room temperature. Lipoplexes formed by DC-Chol/DOPE liposomes and different DNA structures have been evaluated in the literature not only from a biological point of view, that is, the transfection process, its toxicity, efficiency, activity, and/or the delivery of the chloride transporter gene to the lungs on the treatment of cystic fibrosis disease,26-33 but also from a physicochemical standpoint, through SAXS and/or circular dichroism studies as well as experiments by means of freeze-fracture EM, atomic force microscopy AFM, dynamic light scattering DLS, isothermal titration calorimetry ITC, differential scaning calorimetry DSC, zeta potential, etc.24-26,31,33-40 However, many aspects of the compaction phenomena, mainly related to the structure and morphology of this lipoplex, still remain unclear. The lipoplexes studied in this work have been characterized by means of a wide variety of experimental methods, such as zeta potential, fluorescence spectroscopy, dynamic light scattering (DLS), and cryogenic transmission electron microscopy (cryo-TEM). The analysis of the experimental results is carried out with great accuracy and in depth details. Because the composition of the lipoplex is known to have a marked influence on its properties, all of 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 () L+ + L0) 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. With the electrostatic interactions at the liposome surface as the driving force of the DNA-liposome compaction process, electrophoretic mobility and/or zeta potential measurements seem to be of clear interest to understand better the lipoplex formation.1,2,19,41 Moreover, cryo-TEM is a powerful microscopic technique that can shed light on the size, shape, and morphology of the lipoplexes.3,6,8,11,12,19,42-45 DLS experiments also assist in analyzing the changes on the size of liposomes and/or lipoplexes during the compaction process. Additionally, ethidium bromide intercalating assays (EtBr, see Chart 2) will inform about the type of interactions that occur among DNA and liposomes to yield lipoplexes. In this study, we will show how DC-Chol/DOPE/CT-DNA lipoplexes present a broad distribution of morphologies, that is, DNA-coated unilamellar lipoplexes, clusters of liposomes-DNA mediated, nanostruc-

Rodrı´guez-Pulido et al. CHART 2: Fluorescence Probe Molecule, EtBr

tures with thickened, flattened, and deformed walls, and also multilamellar lipoplexes with or without open bilayers rolled over the lipoplex. The periodicity in the multilamellar structures was determined by digitizing and image processing techniques and indicates that DNA helixes are sandwiched and aligned between cationic lipidid bilayers. Furthermore, among the wide variety of detected structures, DC-Chol/DOPE liposomes do not decrease their size when compacting DNA, unlike the DSTAP/ DOPE/CT-DNA system, previously reported by our group.20 II. Experimental Section A. Materials. Cationic lipid, 3β-[N-(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-Chol), and zwitterionic lipid 1,2-dioleoyl-sn-glycero-3-phosphoetanolamine (DOPE) were from Avanti Polar Lipids. Cationic lipid belongs to the sterols family, and DOPE contains an unsaturation on the cis configuration at the 9 position of both hydrocarbon chains. Sodium salt of calf thymus DNA (CT-DNA), the fluorescent probe, ethidium bromide (EtBr), and the components of HEPES buffer (4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid and its sodium salt) were from Sigma-Aldrich. All of them, with the best purities, were used without further purification. Solutions were prepared by mass with distilled and deionized water (Super Q Millipore system, conductivity lower than 18 µS cm-1), and all were buffered using HEPES 40 mM, ionic strength ) 15.3 mM, at 298.15 K. B. Preparation of DNA Solutions. A stock solution of CTDNA (0.100 mg/mL ) 1.18 × 10-4 M base pair) was prepared by dissolving an appropriate amount of the solid in HEPES 40 mM, pH ) 7.4, two days before the mixing with liposomes. DNA concentrations (expressed in mM base pairs) were determined by absorbance at 260 nm (ε ) 6600 M-1 cm).46,47 A A260/A280 ratio of 1.90 and a negligible absorbance at 320 nm (A320 ) -0.003) reveal5,24,46,47 that the contamination of the DNA used in this work by the presence of a certain percentage of proteins is negligible. C. Preparation of Cationic Liposome Solutions. Appropriate amounts of DC-Chol (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 2-3 additional hours. The resulting dry lipid films were then hydrated with 40 mM HEPES, pH ) 7.4, and homogenized with the help of alternating cycles of vigorous vortexing and sonication; in any case, the total time of sonication did not last more than 20 min to avoid the formation of SUV.48 Also with the aim of favoring the hydration process, these solutions were moderately heated to a temperature not exceeding 313 K. This combination of vortexing, sonication, and heat yields a polydisperse population of MLVs,5,49,50 which were transformed into the desired unilamelar vesicles, LUVs (with a reduction on the polydispersity), by a sequential extrusion procedure, widely explained

Interaction between DC-Chol/DOPE Liposomes and DNA elsewhere.20 Liposomes thus prepared were left to rest during 24 h before their use or their mixing with DNA to form the lipoplexes. D. Preparation of Lipoplex Solutions. Equal volumes of DNA and DC-Chol/DOPE liposome extruded solutions were mixed by adding DNA over liposome, as usually done in these studies.19,23 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, with the exception of DLS measurements where we waited for 24 h. E. Electrophoretic Mobility Measurements. A laser Doppler electrophoresis (LDE) technique (Zetamaster 2000, Malvern Instruments Ltd.), fully described,51 was used to measure electrophoretic mobilities. Temperature was controlled at (298.15 ( 0.01 K). Each electrophoretic mobility datum 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 0.51 mg/mL; that is, L/D ratio was varied from 1 to 10. Electrophoretic mobility for liposome solution in the absence of DNA (L/D ) ∞) was also measured. F. Cryo-TEM Measurements. Transmission electron microscopy experiments under liquid nitrogen temperatures (cryoTEM) were run on samples of DC-Chol/DOPE liposomes in the absence and presence of DNA. All of the samples were prepared and vitrified according to the method devised by Dubochet et al.,52 and also described in Llorca et al.,53 with minor modifications. A few microliters of the samples was applied on QUANTAFOIL R2/4 holey carbon film, blotted, and the grids were plunged into liquid ethane. Observations were conducted at a JEOL 1230 microscope operated at 100 kV and equipped with a Gatan liquid nitrogen specimen holder. Images were recorded at a nominal magnification of 30 000 on Kodak SO-163 film and digitized using a Minolta Dimage Scan Multi Pro scanner at 4800 dpi, which corresponded to 1.75 Å/pixel at the specimen. G. Ethidium Bromide Intercalation Assays. Fluorescence emission spectra of ethidium bromide in the 535-700 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 Perkin-Elmer LS-50B luminescence spectrometer.54-56 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] ) 6.34 µM in all cases. The emission of an EtBr/DNA solution (DNA:EtBr molar ratio is 6:1 and [DNA] ) 0.025 mg/mL) was registered at increasing L concentrations by adding a EtBr/DNA/L solution, thus covering from L/D ) 0 to 6.9. 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 lipoplexes. Fluorescence spectra were corrected for the background intensities of the buffer solution. H. Dynamic Light Scattering Experiments. Dynamic light scattering was performed with an ALV (CGS-8) working in pseudocross-correlation mode, using the green line (µ ) 514.5 nm) of an argon ion laser (Coherent I300). The intensity correlation functions were obtained at a fixed temperature of 298.15 K and as a function of the scattering angle, µ, between 30° and 150°, corresponding to wavevectors, q, from 8.42 ×

J. Phys. Chem. B, Vol. 112, No. 39, 2008 12557 104 to 3.14 × 105 cm-1 defined as q ) (4πn/µ) sin(µ/2), where n is the solution refractive index. The normalized second-order correlation functions, g(2)(t), were analyzed using both GENDIST57 and CONTIN58,59 inverse Laplace algorithms, giving both similar relaxation time distributions. From the average relaxation times, τ, the apparent diffusion coefficients, Dapp, were obtained, and using the Stokes-Einstein relation,60 the apparent hydrodynamic radii were calculated.

kBT 1 q2 ) Dappq2 ) τ 6πηRh,app

(1)

The intensity correlation functions of liposomes, DNA, and lipoplexes at different L/D ratios were measured at least twice. Lipoplexes were formed at [DNA] ) 0.100 mg/mL. III. Results and Discussion The isoneutrality L/D ratio, (L/D)φ, is known to be one of the most characteristic lipoplex parameters, because many properties of the nanoaggregate change at (L/D)φ. It is defined as the L/D ratio at which the charge ratio of the lipoplex, CR, equals 1, that is, the L/D ratio at which the negative charges of DNA are stoichiometrically neutralized by the positive charges of the liposome. If n+ and n- stand for 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 jMbp is the base pairs averaged molar mass ()649.9 g mol-1), the charge ratio is given by:

CR )

(

)

j n+ L+/ML+ L+ j Mbp L - L0 Mbp ) ) ) n- 2D/M j D 2ML+ D 2ML+ bp

and the isoneutrality ratio by:

(CR)φ ) 1f

( ) L+ D

) φ

2ML+ j M

(2)

(3)

bp

(L/D)φ can be determined from different physicochemical properties, the electrochemical ones being the most appropriate because they reflect the charge balance that takes place when the lipoplex is formed. Among all, the electrophoretic mobility and those magnitudes related to it, such as the zeta potential and the surface density charge, are very often used to obtain (L/D)φ because all of them show an inversion of sign at this particular L/D. Figure 1 shows a plot of ILS versus the experimental electrophoretic mobility, µe, for DC-Chol/DOPE/ CT-DNA lipoplexes at L/D ) 3.4, as an example, in 10 independent experiments carried on in aqueous HEPES medium at pH ) 7.4, (DC-Chol:DOPE ) 1:1, DNA concentration was kept constant at 0.050 mg/mL), that yield an averaged electrophoretic mobility of jµe ) -(3.54 ( 0.14) × 10-8 m2 V-1 s-1. From this averaged electrophoretic mobility, zeta potential, ζ, and surface charge density enclosed by the shear plane, σζ, are determined at each L/D value by using the well-known equations:61,62

ζ)

3η µ 2ε0εrf(κDa) e

(4)

and

σζ )

( )

2ε0εrκDkBT zeζ sinh ze 2kBT

(5)

where η is the viscosity of water (8.904 × 10-4 N m-2 s at 298.15 K); ε0 and εr are the vacuum and relative permittivity

12558 J. Phys. Chem. B, Vol. 112, No. 39, 2008

Rodrı´guez-Pulido et al.

Figure 1. Intensity vs electrophoretic mobility, µe, for DC-Chol/DOPE/ CT-DNA lipoplexes at L/D ) 3.4, in aqueous buffered medium (HEPES 40 mM) at pH ) 7.4. DC-Chol:DOPE ) 1:1. µe ) -(3.54 ( 0.14) × 10-8 m2 V-1 s-1.

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 DC-Chol/DOPE/CT-DNA Lipoplexes (DNA Concentration Was Kept Constant at 0.050 mg/mL) L/D

108 µe (m2 V-1 s-1)

ζg (mV)

103σζg (C m-2)

1.0 1.2 2.6 3.4 4.5 6.2 6.3a 6.3b 6.4c 6.4d 6.4e 8.1 10.3 ∞f

-4.51 -4.08 -4.72 -3.54 1.82 3.41 3.13 3.31 3.20 3.23 3.16 3.38 3.57 3.87

-57 -52 -60 -45 23 43 40 42 41 35 33 43 46 49

-20 -17 -21 -15 7 14 18 23 18 11 10 14 15 16

a Ionic strength modified with NaCl, I ) 30.7 mM. b Ionic strength modified with NaCl, I ) 45.7 mM. c Ionic strength modified with CaCl2, I ) 30.7 mM. d Temperature modified at T ) 308.15 K. e Temperature modified at T ) 313.15 K. f Liposome in the absence of DNA. g Errors are estimated to be around 3% in zeta potential and around 6% in surface density charge.

(8.854 × 10-12 J-1 C2 m-1 and 78.5, respectively); e is the elemental charge; z is the valence of the ion; kB is the Boltzmann constant; T the absolute temperature; and f(κDa) is the Henry function, which depends on the reciprocal Debye length, κD, and the particle radius, a. For medium-to-large particles in a medium of moderate ionic strength (a > κD-1), Smoluchowski limit is usually applied (f(κDa) ) 1.5) to estimate the Henry function.61,62 Table 1 resumes the values of the above-defined electrochemical properties obtained at all L/D ratios, including the value of L/D ) ∞, that is, only liposome, and Figure 2 shows the plot of ζ versus L/D for the DC-Chol/DOPE/CT-DNA lipoplexes. The dotted line corresponds to the value for DCChol/DOPE (1:1) liposome in the absence of DNA (L/D ) ∞, ζ ) (49 ( 2) mV). A sigmoidal curve, similar to that previously found for other lipoplexes,2,19,49,63 can be observed. This trend shows three different zeta potential regions: (i) the region where the net charge of lipoplexes is negative and almost constant at

Figure 2. Values of zeta potential, ζ, of DC-Chol/DOPE/CT-DNA lipoplexes at different L/D ratios in aqueous buffered medium (HEPES 40 mM) at pH ) 7.4. DC-Chol:DOPE ratio is 1:1; [DNA] ) (0.050 ( 0.001) mg/mL. Solid line: sigmoidal fit of experimental values. Dotted line corresponds to the value for DC-Chol:DOPE (1:1) liposome in the absence of DNA (L/D ) ∞). The inset at the bottom shows a detail of the zeta potential values of DC-Chol/DOPE/CT-DNA lipoplexes (L/D ) 6.4) at different temperatures (!, T ) 308.15 K and 9, T ) 313.15 K) and at different ionic strengths (4, I ) 30.7 mM and 2, I ) 45.7 mM either with NaCl added and b, I ) 30.7 mM with CaCl2 added). Errors are estimated to be around 3%.

(ζ ) (-56 ( 3) mV); (ii) the region where the inversion of zeta potential sign takes place; and (iii) the region where the net charge of the lipoplexes is positive and tends to the value for the DC-Chol/DOPE liposome. Thus, (L/D)φ has been unambiguously calculated from the L/D value at which zeta potential is zero, that is, (L/D)φ,ζ ) 4.1. This behavior points to a typical surface liposome-DNA interaction, mainly electrostatically driven. The inset at the bottom of the figure shows a detail of the zeta potential values of DC-Chol/DOPE/CT-DNA lipoplexes (L/D ) 6.4) at different temperatures (T ) 308.15 and T ) 313.15 K) and at different ionic strengths (I ) 30.7 mM and I ) 45.7 mM with NaCl added, and I ) 30.7 mM with CaCl2 added). It can be observed that the presence of an electrolyte does not affect very much the zeta potential of the lipoplex, irrespective of the type and/or concentration of the added electrolyte. However, an increase in temperature seems to provoke a clear decrease on the zeta potential. Because electrophoretic mobility (see Table 1), viscosity, and permittivity60 decrease with temperature, the combination of the three properties in eq 2 results in a decrease on zeta potential with temperature, a behavior that has been also found in other charged interfaces.64 The (L/D)φ has been also determined from ethidium bromide (EtBr) intercalation assays, which are very often used to confirm and characterize this surface electrostatic interaction. For that purpose, the variation on the emission of aqueous buffered solutions of EtBr, an aromatic planar cationic fluorophore (see Chart 2), is followed at constant CT-DNA concentration as long as a solution with DC-Chol/DOPE liposomes is added, and, thus, the lipoplex is formed. Figure 3 shows the emission fluorescence spectra of EtBr at a constant DNA concentration of 0.025 mg/ mL as long as lipid concentration, and thus L/D ratio, increases. The figure also includes the emission spectra of EtBr in the absence of DNA (L/D ) ∞, [L] ) 0.098 mg/mL) and also in the absence of liposomes, that is, just in the buffered medium. The spectra reported in the figure evidence that EtBr does not

Interaction between DC-Chol/DOPE Liposomes and DNA

Figure 3. Emission fluorescence spectra of EtBr in the presence of DC-Chol/DOPE/CT-DNA lipoplexes at different L/D ratios: 0, L/D ) 0; 1, L/D ) 1.0; 2, L/D ) 2.0; 3, L/D ) 3.0; 4, L/D ) 4.0; 5, L/D ) 5.0; 6, L/D ) 5.9; and 7, L/D ) 6.9. Medium: aqueous HEPES 40 mM, pH ) 7.4. DC-Chol:DOPE ratio is 1:1; DNA:EtBr ratio is 6:1; [DNA] ) 0.025 mg/mL. Dotted line shows the emission fluorescence spectra of EtBr in the absence of liposomes and lipoplexes. Dashed line shows an example of the emission fluorescence spectra of EtBr only in the presence of liposomes at [L] ) 0.098 mg/mL.

interact with cationic DC-Chol/DOPE liposome, as would be expected for a cationic probe, because the emission in the presence of liposome is comparable to that in the bulk. However, it is also obvious in the figure that the emission of EtBr clear changes when DNA is present. It is known that the fluorescence intensity of this probe increases around 30 times upon its intercalation between base pairs of double-stranded DNA,4,19,65-67 because the hydrophobic environment found between the base pairs protects the probe from water molecules and molecular oxygen that may quench its fluorescence emission. When DNA is condensed or compacted by cationic liposome, EtBr intercalation is prevented and, as a consequence, fluorescence intensity will be quenched, as the probe will remain fully accessible to the bulk solvent. Thus, the decrease in probe emission intensity observed in the figure can be used as a way to asses DNA accessibility and, accordingly, DNA-liposome compaction. Figure 4 reports the maximum intensity (at λ ) 588 nm) as a function of L/D. It can be observed that the emission intensity gradually decreases from around 180 au at L/D ) 0 to around 80 au at L/D ) 4.0, while it remains almost constant at very low values from L/D ) 5.0 to 6.9, comparable to those found in the absence of DNA and even in the absence of liposomes (i.e., in the bulk). This trend confirms that DNA-liposome interaction is stronger than that between EtBr and DNA, the probe being displaced by the addition of lipid from the DNA base pair microenvironment toward the aqueous bulk, where its quantum fluorescence yield falls dramatically down. Additionally, this figure allows the determination of the isoneutrality ratio, (L/D)φ,fl ) 4.1, calculated by Phillip’s method,68 in total agreement with zeta potential results. Furthermore, both values are in very good agreement with that one calculated from eqs 2 and 3 (ML+ for DC-Chol is 537.27 g mol-1, which implies that (L/D)φ,calc ) 3.95 for DC-Chol/DOPE/CT-DNA lipoplexes). Although structurally very different (no double chain, see Chart 1) and with a molecular mass around 30% lower, it can be observed that the (L/D)φ for DC-Chol/DOPE/CT-DNA lipoplexes is comparable to those ones reported for other

J. Phys. Chem. B, Vol. 112, No. 39, 2008 12559

Figure 4. Emission fluorescence intensity of EtBr at 588 nm in the presence of DC-Chol/DOPE/CT-DNA lipoplexes as a function of L/D ratio. Medium: aqueous HEPES 40 mM, pH ) 7.4. DC-Chol:DOPE ratio is 1:1; DNA:EtBr ratio is 6:1; [DNA] ) 0.025 mg/mL. The emission fluorescence intensities of EtBr in the absence of liposomes and lipoplexes and only in the presence of liposomes ([L] ) 0.098 mg/mL) are shown as dotted and dashed lines, respectively.

lipoplexes, formed by double-chain cationic lipids (with or without helper lipids) and DNA, obtained by different experimental methods.5,9,15,19,20,25,41,69-71 With the aim of justifying all of the above-mentioned evidence, the experimental π f π* bands of Figure 3 have been interpreted72 as consisting of several bands at different wavelengths, each of which is 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, all of the spectra were deconvoluted 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.55 Figure 5 shows a resume of the deconvolution process into one and two 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 5 and Table 2 deserves some remarks. At L/D ) 0, the best fit corresponds to the two-gaussians option, indicating that in the absence of liposome but in the presence of DNA, EtBr is partitioned between two possible microenvironments, which correspond to λ1 ) (580 ( 2) nm and λ2 ) (598 ( 5) nm. On the other hand, the one-Gaussian fit is the correct choice at L/D ) ∞ (in the absence of DNA) and just in the medium (absence of DNA and liposomes) because only one microenvironment, the bulk, is expected in these cases (Gaussian centered at λ ) 598 and 597 nm, respectively). Although these deconvolutions are omitted in this Article for the sake of conciseness, Table 2 reports the corresponding fit parameters (last two lines). Accordingly, the two bands found in the presence of DNA (L/D ) 0) are assigned to the π f π* emission of EtBr intercalated between the base pairs of the DNA double helix (λ1) and immersed in the aqueous bulk (λ2), respectively, confirming that the more hydrophobic is the microenvironment, the more blue-shifted is the emission band. As can be seen in the table, this assignment can be also applied in the presence of liposomes, as L/D increases up to L/D )



Figure 5. Emission fluorescence spectra of EtBr in the presence of DC-Chol/DOPE liposomes at different L/D ratios, together with their deconvolutions into 1 or 2 Gaussian components. Solid line: experimental spectra. Dashed line: Gaussian components. Dot line in 2 Gaussian cases: total sum of Gaussian components. Medium: aqueous HEPES 40 mM, pH ) 7.4. DC-Chol:DOPE ratio is 1:1.



TABLE 2: Parameters of the Deconvoluted Gaussian Bands of EtBr Fluorescence Emission Spectra in the Presence of DC-Chol/DOPE/CT-DNA Lipoplexes at Different L/D Ratios: Wavelength λi, Width Wi, and Area Ai in Terms of % Contribution to the Overall Fluorescence Emission Area (within Parentheses)a 2 gaussians

1 gaussian 2

L/D

I588

λ1 (nm)

W1

A1 (%)

λ2 (nm)

W2

A2 (%)

r

0 1.0 2.0 3.0 4.0 5.0 5.9 6.9 ∞b buffer

181 174 144 114 86 20 21 22 11 11

582 582 581 582 582

36 36 33 31 25

4575(41) 3441(33) 1790(21) 1063(15) 438(9)

603 600 597 595 594

49 50 51 52 53

6543(59) 7049(67) 6728(79) 5850(85) 4687(91)

0.999 0.999 0.999 0.999 0.999

λ1 (nm)

W1

A1

r2

594 593 591 597 598

52 54 52 57 59

1307 1295 1353 699 883

0.997 0.996 0.995 0.998 0.997

a Medium: aqueous HEPES 40 mM, pH ) 7.4. DC-Chol:DOPE ratio is 1:1; DNA:EtBr ratio is 6:1; [DNA] ) 0.025 mg/mL. b [L] ) 0.098 mg/mL.

Figure 7. Selected cryo-TEM micrographs of DC-Chol:DOPE (1:1) liposomes at L/D ) ∞, that is, liposomes in the absence of CT-DNA. Scale bar: 100 nm.

Figure 6. Plot of the areas (in terms of % of the total area) of Gaussian bands as a function of L/D ratios.

4.0, that is, around (L/D)φ. However, as long as L/D increases (Figure 5), the two-gaussians fit is gradually losing physical and mathematical basis in favor of the one-gaussian fit. In fact, once the isoneutrality is passed, for example, at L/D ) 5.0, the correct choice is the one-gaussian fit, with λ1 ) (594 ( 5) nm, assigned to the probe in the bulk. This evidence corroborates that there is a competition between the probe and the liposome for DNA; EtBr, initially within the DNA helix, is displaced to the aqueous bulk when the lipoplex is formed, confirming that EtBr-DNA interaction is weaker than the electrostatic liposomeDNA interaction. This fact explains that EtBr can be within two microenvironments (bulk and DNA helix) at L/D < (L/ D)φ, but when the probe is totally displaced from inside of DNA helix at L/D > (L/D)φ, it can only be in the bulk. Consistent with this reasoning is the trend shown in Figure 6 by the areas of the gaussians above commented (in terms of % of the total area) as a function of the L/D ratio. As can be observed, Α1 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 lipoplex is formed. The plot of the total area is omitted because it is similar to that shown in Figure 4 by the intensity, as expected.

The structure and morphology of the liposomes and lipoplexes have been evaluated by using transmission electron microscopy techniques. For that purpose, cryo-TEM experiments were run for samples of DC-Chol/DOPE liposomes and DC-Chol/DOPE/ CT-DNA lipoplexes at L/D ) 8.1, that is, above (L/D)φ. Figure 7 shows four micrographs selected among those taken on cryoTEM experiments of liposome samples. These micrographs reveal the presence of a homogeneous population of unilamellar spherical liposomes, characterized by an average diameter of around (99 ( 10) nm and a bilayer with a thickness of (4.5 ( 0.5) nm, both parameters averaged over the structures found in all of the micrographs. The formation of lipoplexes induces a clear change in liposome morphology. Figure 8 is a micrograph showing a general view of the nanostructures obtained when cationic DCChol/DOPE liposomes are mixed with CT-DNA at L/D ) 8.1, that is, in excess of cationic lipid. This micrograph and many others, omitted for the sake of conciseness, reveal a rich distribution of complex morphologies, from unilamellar DNAcoated liposomes to multilamellar lipoplexes passing through cluster-like structures and including several intermediate morphologies. In all of the cases, the unilamellar DNA-coated liposomes show diameters of around 100 nm that roughly correspond to the size of liposomes in the absence of DNA. It means that the addition of the biopolymer mainly affects the structure and morphology of the liposome but not its size. Another important feature that can be observed in all of the above-mentioned micrographs is that, as compared to the structures seen in the micrographs of liposomes, lipoplex



Figure 8. A selected cryo-TEM micrograph showing a general view of DC-Chol/DOPE/CT-DNA lipoplexes at L/D ) 8.1 (DC-Chol:DOPE ) 1:1). Scale bar: 200 nm.

structures are more condensed, with a clear increase of electronic density on the surface of the spherical structures seen in Figure 7. It is known that DNA is compacted and condensed at the surface of the cationic liposomes by action of a strong electrostatic interaction, as confirmed by zeta potential and fluorescence results. With the aim of analyzing all of these changes, some details or zoom views have been extracted from the original micrographs and are reported all together in Figures 9 and 10. Figure 9A and B shows, as examples, how the presence of the biopolymer induces liposome aggregation to form cluster-like structures, where the liposomes are deformed at the surface of contact with adjacent liposomes, without rupture. A minority of the structures seen in the figure retains the unilamelar lipidic bilayer (see mainly Figure 9A) coated with a monolayer of DNA; most of the nanostructures in the figures, however, show a clear thickening of the walls, indicating the presence of multilamellar complexes that will be commented later on. These aspects, lipoplexes aggregation and wall thickening, flattening, and deformation, are evident in Figure 9B. This deformation indicates that either DC-Chol/DOPE liposomes are very flexible structures or the DNA-liposome interaction is quite asymmetric. When lipoplex is formed, positive charges are partially compensated on only one side of the bilayer because DNA is adsorbed and compacted only at the outer positive-charged surface of the liposome, reducing the effective headgroup of the cationic lipids and provoking a clear asymmetry in packing pressure.8 This stresses and destabilizes the membrane, thus promoting lipoplexes fusion and/or aggregation, as also postulated by similar systems.8,73 On the other hand, the flexibility of the lipidic bilayer + DNA is quite evident in the micrographs shown in Figure 9C (see a good example highlighted with white arrows in the third panel), where the structures reveal the existence of several loops and twisted bilayers. As mentioned above, one of the most striking characteristics that come to the eye in the micrographs is the thickness of the lipoplexes wall, clearly higher than that reported for the lipidic bilayer of the liposomes. This thickening has been attributed to the formation of multilamellar complexes constituted by a series of lipidic bilayer with DNA superficially compacted and adsorbed between bilayers (fingerprint pattern). It has been postulated8 that the growth of these multilamellar complexes starts with one DNA-coated liposome and follows two possible

Figure 9. (A-D) Details extracted from the original cryo-TEM micrographs of DC-Chol/DOPE/CT-DNA lipoplexes at L/D ) 8.1. Scale bar: 100 nm.

mechanisms: (i) adsorbed lipoplexes may rupture and roll over the lipoplex host, as can be seen in Figure 9D (see, for example, the asterisk in the first micrograph to observe the disruption of the bilayer previous to the envelop action and to the formation of the multilayer); all of these micrographs seem to depict a stage previous to the formation of the multilamellar structure; and/or (ii) flattened lipoplexes stack to a template lipoplex to form the DNA adsorbed bilayers, as can be seen for example in Figure 10A (stacking pointed with white arrows). Open bilayers (as that one shown in Figure 9D) appear if the energetic cost (in terms of free energy) associated with the formation of the edge is lower than that involved in the bilayer adsorption process; they are explained in the literature as due to the preference of surfactants for high curvature structures.8,74,75 In fact, DC-Chol does not form bilayers in the absence of helper lipid. The high cationic charge density at the edges results in an accumulation of negatively charged DNA near the edge, as seen in the micrographs. To analyze the regular arrangement of the multilamellae in the multilamellar lipoplexes above commented, 2000 × 2000 pixel square images were extracted from these micrographs where clear thickening of the walls is observed, using the boxer command found in the EMAN software for image processing,76 and these were low-pass filtered to remove high frequency noise. An example is shown in Figure 10B. The filtered images were then scaled down to 500 × 500 and 7 Å/pixel and saved as .png files. To analyze the presence of patterns, these images were opened with the program Image J (Image processing and analysis in Java). A collection of lines following the radius of the circular structures was drawn (see white dashed lines in Figure 10 B), and the gray levels along these lines were plotted



Figure 12. Variation of the frequencies of the relaxation modes as a function of q2 for DC-Chol/DOPE liposomes in the absence (top) and in the presence of DNA at ratio L/D ) 6.6 (bottom). In the mixtures, two relaxation modes were obtained: b and solid line correspond to DC-Chol/DOPE-DNA lipoplexes (slow relaxation mode), and O and dotted line, which is in accordance with the relaxation mode of free liposomes or DNA (fast relaxation mode). Medium: aqueous HEPES 40 mM, pH ) 7.4. DC-Chol:DOPE ratio is 1:1; [DC-Chol/DOPE]liposomes ) 0.366 mg/mL and [DC-Chol/DOPE]lipoplexes ) 0.324 mg/mL.

Figure 10. (A) Details extracted from the original cryo-TEM micrographs of DC-Chol/DOPE/CT-DNA lipoplexes at L/D ) 8.1. (B) 2000 × 2000 pixel square image extracted from one micrograph, then low-pass filtered to remove high frequency noise, and finally scaled down to 500 × 500 and 7 Å/pixel. (C). Plot of the gray level vs distance along a straight line across the 2D image shown in (B). The maximum densities are revealed as a peak within a distance of around 10 pixels on average (the most frequently observed value, corresponding to 7 nm (7 Å/pixel after the scanning of the micrographs)). Scale bar: 100 nm.

Figure 13. Hydrodynamic radius Rh of DC-Chol/DOPE-DNA lipoplexes at different L/D ratios, calculated by means of the Stokes-Einstein relationship from diffusion coefficients D (eq 1). The solid line is a guide for the eye obtained as a Gidding peak function, that is, the product of two Gaussian functions. The error bars show the upper and lower 95% confidence limit. Medium: aqueous HEPES 40 mM, pH ) 7.4. DC-Chol:DOPE ratio is 1:1; [DNA] ) (0.050 ( 0.001) mg/mL. Figure 11. Relaxation time distributions at a scattering angle θ ) 60° for DC-Chol/DOPE liposomes (dotted line), DNA (dashed line), and DC-Chol/DOPE-DNA lipoplexes at ratio L/D ) 6.6 (solid line). The inset shows the plot of (g(2)(t) - 1) vs τ. Medium: aqueous HEPES 40 mM, pH ) 7.4. DC-Chol:DOPE ratio is 1:1; [L]liposomes ) 0.366 mg/mL, [L]lipoplexes ) 0.324 mg/mL, and [DNA] ) 0.098 mg/mL.

(see Figure 10C) as a function of distance. The maximum and minimum levels of density of all of the images studied revealed a clear pattern, and the distance between repetitions was estimated by measuring the number of pixels between maximums. The analysis of a large number of images revealed that

these values fit within a narrow range of distances. The most frequently found value in the images analyzed was 10 pixels, which corresponds to 7 nm (after multiplying 10 pixels by the 7 Å/pixel). Thus, it means that interlamellar spacing is around 7 nm, consistent with the fact that DNA helix is effectively sandwiched and aligned between each two bilayers (around 4.5 nm/bilayer + around 2.5 nm/DNA helix). SAXS experiments reported in the literature yield similar results for other lipoplexes.2,14 Chart 3 sketches a resume of the broad distribution of different morphologies that have been found in the electronic



CHART 3: Schematic Drawings of the Different Structures of DC-Chol/DOPE-CT-DNA Lipoplexes

microscopy study: (i) DNA-coated unilamellar lipoplexes; (ii) aggregation of two or more (clusters) lipoplexes, with thickened, flattened, and deformed walls; (iii) structures with twisted and looped walls; and (iv) multilamellar lipoplexes with or without open bilayers rolled over the host lipoplex. It is worth mentioning that this scenario is not pretended to be exhaustive; it stands for only a fraction of the multitude of nanostructures found in lipoplexes systems that depend very much on its composition (i.e., cationic lipid, helper lipid, and DNA) and the relative amount of these components. With the aim of confirming and justifying the changes on the size of the nanostructures when DNA is present, and analyzing the influence that the composition of the lipoplex (L/D ratio) has on these changes, DLS experiments have been carried out on DNA, DC-Chol/DOPE liposome, and DC-Chol/DOPE/ DNA lipoplex samples at 298.15 K and different L/D ratios, below and above (L/D)φ. Figure 11 shows the relaxation time distributions and the second-order correlation functions g(2)(τ) (inset) at θ ) 60° for DC-Chol/DOPE liposomes, DNA, and DC-Chol/DOPE-DNA lipoplexes at L/D ) 6.6, as an example. It can be observed that lipoplex distribution shows as well a small peak assignable to liposomes without DNA superficially compacted (dotted line), in agreement with what has been seen in cryo-TEM micrographs, or to free DNA (dashed line). The variation of the frequencies of the relaxation modes (maxima in Figure 11) is plotted in Figure 12 as a function of q2 for DC-Chol/DOPE liposomes (top) and DC-Chol/DOPE-DNA lipoplexes at ratio L/D ) 6.6 (bottom). As noticed in the figure, when DNA is mixed with the liposomes (bottom), two different relaxation modes are observed: a fast mode (dotted line), which corresponds to free liposomes or free DNA, and a slow mode (solid line), which is related with the lipoplexes. From the slopes of the straight lines of Figure 12, applying eq 1, apparent diffusion coefficients can be determined, and from them and the Stokes-Einstein relation, the hydrodynamic radii, Rh, of the nanoparticles. Figure 13 reports these results as a function of L/D ratios; dashed and dotted lines correspond to free DNA and free liposome. It can be concluded that there is a good agreement with cryo-TEM results with respect to DC-Chol/ DOPE liposome size. Furthermore, the results reported in the figure confirm that the isoneutrality is reached around L/D ) 4.0, in very good consistency with the value found from zeta potential, EtBr intercalation essays, and the calculations (eqs 2 and 3). Additionally, it can be noticed in the figure a clear increase in the size of lipoplexes nanostructures in a wide L/D

range around isoneutrality (L/D)φ; this feature corroborate the cluster-like structures seen in cryo-TEM micrographs (Figures 8 and 9), where several liposomes are aggregated induced by the presence of the biopolymer, in some cases causing the deformation, the flattening, or even the disruption of the lipidic bilayer. IV. Conclusions A series of electrochemical, spectroscopic, light-scattering, and electronic microscopy techniques has evidenced that DCChol/DOPE cationic liposomes properly condense and compact CT-DNA by means of a strong entropically driven surface electrostatic interaction. The isoneutrality of the lipoplex thus formed, determined by zeta potential, EtBr intercalation essays, and DLS, is reached when the lipid mass is around (4.0 ( 0.1) times the DNA mass. DC-Chol/DOPE liposomes are mainly spherical and unilamellar with a diameter of around (99 ( 10) nm and a lipidic bilayer of around (4.5 ( 0.5) nm thick. DCChol/DOPE/CT-DNA lipoplexes present a broad distribution of different morphologies as revealed by the electronic microscopy study, from DNA-coated unilamellar lipoplexes, clusters of liposomes-DNA mediated, lipoplex nanostructures with thickened, flattened, and deformed walls, and also multilamellar lipoplexes with or without open bilayers rolled over the host lipoplex. The periodicity in these multilamellar structures has been determined by digitizing and image processing techniques as being around 7 nm, indicating that DNA helixes are effectively sandwiched and aligned between cationic lipidid bilayers. The experimental results reported in this work point to DC-Chol/DOPE liposomes as efficient DNA vectors, because they are capable of compacting and condensing CT-DNA, thus being of potential interest in developing new gene therapy protocols. Additionally, this work emphasizes the convenience of characterizing lipoplex systems from the point of view of different techniques and scanning a wide range of L/D ratios, from those with DNA in excess to those with cationic lipid in excess. 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 projects SAF2005-00775 from the Spanish Ministry of Education and Science (MEC), S-BIO-0214-2006 from the Autonomous Government of Madrid (CAM), and RD06/0020/1001 of

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A Physicochemical Characterization of the Interaction between DC

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