Effect of Helper Lipids on the Interaction of DNA with Cationic Lipid

Jun 22, 2012 - dimethyldioctadecylammonium bromide, with/without 50 mol % of a ... composed of dimethyldioctadecylammonium bromide binding the most...
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Effect of Helper Lipids on the Interaction of DNA with Cationic Lipid Monolayers Studied by Specular Neutron Reflection A. P. Dabkowska,† D. J. Barlow,† R. A. Campbell,‡ A. V. Hughes,§ P. J. Quinn,† and M. J. Lawrence*,† †

Institute of Pharmaceutical Science, School of Biomedical Sciences, King’s College London, 150 Stamford Street, London, SE1 9NH, U.K. ‡ Institut Laue-Langevin, B.P. 156, 38042 Grenoble Cedex, France § ISIS, Rutherford Appleton Laboratory, Chilton, Didcot, OX11 OQX, U.K. S Supporting Information *

ABSTRACT: The interaction of DNA with monolayers of the cationic lipid dimethyldioctadecylammonium bromide, with/without 50 mol % of a neutral “helper” lipid, either dioleoylphosphatidylethanolamine or cholesterol, has been studied using specular neutron reflection, surface pressure−area isotherms, and Brewster angle microscopy. The amount of DNA bound to the lipid head groups has been comprehensively quantified in the range of 8−39 vol% of DNA with respect to the monolayer composition (monolayers composed of dimethyldioctadecylammonium bromide binding the most DNA and monolayers containing dioleoylphosphatidylethanolamine binding the least) and surface pressure (DNA binding being greatest at highest surface pressures). Surprisingly, regardless of these variables, the thickness of the DNA-containing layer remained approximately constant between 18 and 25 Å. This systematic study is the first direct quantification of the binding of DNA with two different helper-lipid-containing multicomponent monolayers, an important step toward understanding interaction parameters in more realistic models of gene delivery systems.

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and total reflection X-ray fluorescence.13 Monolayers at the air−water interface have been shown to be particularly effective for studying the interaction between DNA and lipid, as the density of lipid molecules can be controlled and the binding of DNA to the lipid monolayer surface can be detected and, when used in conjunction with a technique such as GIXD or SNR, quantified. Sun et al.9 and Cárdenas et al.8 both used BAM to observe changes in the lateral phase behavior on the micrometer-scale of dimethyldioctadecylammonium bromide (DODAB) monolayers upon addition of DNA. GIXD experiments11 showed that DNA adsorbed under a doublechain cationic lipid and condensed the monolayer. More recently, work has been undertaken using SNR to study the interaction of entire lipoplexes with lipid bilayers fabricated as model cell membranes.14,15 The success of lipoplexes as gene delivery vehicles both in vitro and in vivo has been improved by the incorporation of neutral lipids, such as dioleoylphosphatidylethamolamine (DOPE) or cholesterol into the formulation. These neutral lipids are known as “helper” lipids because of their ability to increase transfection5,16 and decrease the toxicity17 that is generally associated with cationic lipids. Indeed, the majority of cationic lipid delivery systems currently being investigated

INTRODUCTION Complexes prepared from cationic lipid vesicles and DNA commonly known as lipoplexeshold much promise as gene delivery vectors: they have been shown to stabilize DNA against enzymatic degradation and also to facilitate the cellular uptake and subsequent translation of DNA.1 The use of lipoplexes for DNA delivery offers a very realistic alternative to the use of viruses as vectors because lipoplexes exhibit much lower immunogenicity and infectability, they are easy to manufacture, and have the ability to carry DNA of any size.2 To date, a range of techniques have been used to determine the molecular architecture of the lipoplexes formed between DNA and cationic vesicles. For example, small-angle X-ray scattering (SAXS) experiments have shown that lipoplexes prepared from cationic dioleoyl trimethylammonium propane are organized such that strands of DNA are arranged in a periodic fashion in the aqueous space between cationic lipid bilayers.3 However, the picture emerging is one where the lipids can exist with DNA in a wide range of structures, including lamellar3,4 and hexagonal5,6 depending on the type of lipid, the DNA-to-lipid ratio, and the method of lipoplex preparation. The complexation of cationic lipids with DNA at interfaces, such as monolayers and bilayers, has recently been studied using a wide range of techniques including specular neutron reflectivity (SNR),7 Brewster angle microscopy (BAM),8−10 grazing incident X-ray diffraction (GIXD) and scanning force microscopy,11 ellipsometry,7,10 cryo-electron tomography,12 © 2012 American Chemical Society

Received: April 24, 2012 Revised: June 18, 2012 Published: June 22, 2012 2391

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contain helper lipids.18 Although the detailed molecular structures of the lipoplexes formed from cationic lipids have been often investigated, relatively few studies have been conducted to determine the effect on their internal structure of the neutral helper lipids, such as DOPE or cholesterol. Due to the paucity of research in this area, there is a need for detailed studies establishing the role of neutral helper lipids in improving the effectiveness of lipoplexes in order to understand the relationship between their physicochemical properties and their transfection behavior and ultimately to aid in the rational design of improved gene therapy vectors. In the present work, DODAB monolayers, with and without 50 mol % of a neutral helper lipid, namely either DOPE or cholesterol, were used to model half of the lipid bilayer present in a lipoplex and SNR using isotopic contrast variation used to study the interaction of DNA with the monolayers. DODAB was selected as the cationic lipid for study because it has been shown to be effective in transfection of DNA in vivo.19 It was also shown that the presence either of DOPE or cholesterol in the formulation gave rise to an increased transfection efficiency.20

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variation in the surface pressure with respect to the area over which the film was spread was continuously recorded. No differences were found for the isotherms measured by continuous compression using velocities in the range 10−50 cm2 min−1; differences in the isotherms were, however, observed at compression rates greater than 50 cm2 min−1. Note that the area per molecule reported here for the DODAB monolayers containing the helper lipids is the mean molecular area of DODAB with cholesterol or DODAB with DOPE. Each experiment was repeated using a fresh lipid film at least three times to ensure the reproducibility of the data. The lipid films were checked for stability by maintaining the surface pressure at a predetermined value and monitoring any change in surface area over time. Brewster Angle Microscopy. The lipid films, spread on an aqueous phase containing 10 mM NaCl in the presence and absence of 0.067 mg mL−1 of ctDNA, were imaged at 22 ± 2 °C using a Brewster angle microscope with a 10× objective (BAM2, NDF, Göttingen, Germany) mounted above a Nima 601 Langmuir trough (Coventry, UK) with a Wilhemy plate partially immersed in the subphase and attached to a pressure sensor to measure surface pressure. The angle of incidence for all measurements was the Brewster angle for water, at approximately 53° relative to the normal to the water surface at a wavelength of 532 nm. The BAM images were recorded on a chargecoupled device (CCD) camera with the background subtracted, and the images were corrected geometrically. The size of each image presented is 400 μm × 400 μm. As the surface structures of DODABcontaining monolayers have been previously reported to exhibit a dependence on the history of the film reported,21 the BAM images reported here were obtained from freshly prepared and compressed monolayers. Specular Neutron Reflectivity. The SNR monolayer experiments were carried out at 22 ± 2 °C on two neutron reflectometers: (1) the SURF beamline at the ISIS Facility, Rutherford Appleton Laboratory (Chilton, Oxfordshire, UK) where data were collected at one incident angle, namely, 1.5°, relative to the plane of the surface to give a scattering vector, Q, from 0.05 to 0.6 Å−1 with a resolution in Q of 4% and (2) the FIGARO beamline at the Institut Laue-Langevin (Grenoble, France) where data were collected at two incident angles (0.62° and 3.8°) to give a range of Q from 0.005 to 0.4 Å−1 with a resolution in Q of 8%. In particular, a number of the DOPE-containing systems were measured on both instruments with comparable results being obtained. Prior to analysis, a flat background was determined for the data obtained on SURF by extrapolation to high momentum transfer, and subtracted from all reflectivity profiles. The backgrounds subtracted from the data obtained on FIGARO were determined from the simultaneous acquisition of off-specular data for each measurement on the area detector. Regardless of the instrument used, standard calibration of the instrument was performed using a pure D2O subphase. Isotopic substitution between hydrogen and deuterium was used to provide contrast between the subphase, ctDNA, and the lipid monolayer. Three different isotopic contrasts were measured in the present study: (1) chain-deuterated DODAB (d74-DODAB) and, where appropriate, protiated helper lipid on air-contrast matched water (acmw), composed of 0.92 H2O:0.08 D2O to obtain a zero scattering length density (SLD); (2) d74-DODAB and, where appropriate, protiated helper lipid on a D2O subphase; (3) hydrogenated DODAB and, where appropriate, protiated helper lipid also on a D2O subphase. The aqueous subphases containing 10 mM NaCl are denoted as D2O(s) and acmw(s), while those phases containing 10 mM NaCl with 0.067 mg mL−1 ctDNA are denoted as D2O(s,DNA) and amcw(s,DNA). Note that neither the presence of NaCl nor that of the ctDNA in the bulk phase at the levels used in the study made a significant difference to the SLD calculated for the subphase. The SLD values are shown in the Supporting Information, Table 1. The SLD of D2O used on FIGARO was found to have an SLD of 5.90 × 106 Å−2 from the critical edge. The SLD of the ctDNA was taken to be 3.67 × 106 Å−2 in H2O and 4.46 × 106 Å−2 in D2O, as calculated assuming 41.9% G−C and 58.1% A−T content, as reported by the manufacturer, and the H-exchange within the bases was calculated as reported by Jacrot.22 For all SNR studies, lipid monolayers were prepared as described above on the

EXPERIMENTAL SECTION

Materials. DODAB (MW 630.95 g mol−1; >99.0% purity; C38H80NBr) was purchased from Sigma-Aldrich (Poole, Dorset, UK), while its chain-deuterated analogue, dimethyldi(octadecyld37)ammonium bromide (d74-DODAB; MW 705.41 g mol−1; 98% deuteration; C38H6D74NBr) was purchased from CDN Isotopes (Quebec, Canada). The purity of both forms of DODAB was verified using electrospray mass spectroscopy (EPSRC National Mass Spectrometry Service Centre, Swansea). Cholesterol (MW 386.66 g mol−1; >98% purity; C27H45OH) and 1,2-dioleyl-sn-glycero-3phosphoethamolamine (DOPE; MW 744.05 g mol−1; 100% purity; C41H78NO8P) were purchased from Avanti Polar Lipids (Alabaster, AL, USA) and used without further purification. The surface behavior of all lipids used was checked prior to their use in the study. Genomic calf thymus DNA (ctDNA) from Sigma-Aldrich (Poole, Dorset, UK) was used without further purification. The molecular weight of the ctDNA was reported to be 10−15 million daltons by the manufacturer. Ethanol (AnalaR) and chloroform (spectroscopic grade) were purchased from BDH Chemical Ltd. (Poole, Dorset, UK). Sodium chloride (NaCl; AnalaR, BDH Chemical Ltd., Poole, Dorset, UK) was heated to 530 °C for 26 h in a muffle furnace to remove any surfaceactive contaminants. The aqueous subphase used in all experiments contained 10 mM NaCl in an attempt to avoid DNA denaturation into single chains. Water was either deionized water that was double distilled in a well-seasoned still where the purity was regularly checked by surface tensiometry (a surface tension of 72.8 ± 1 mN m−1 at 20 °C was deemed acceptable) and spectroscopically, or purified with a Millipore Milli-Q system to a resistivity of 18 MΩ cm. Deuterated water (D2O; 99.9% deuteration) was obtained from Sigma-Aldrich (Gillinham, UK) or Euriso-top (Paris, France). Surface-Pressure Area Isotherms. Surface-pressure area isotherms were recorded using a Nima 601 Langmuir trough (Coventry, UK) cleaned using chloroform, ethanol and ultrapure water. The Langmuir trough was filled with 300 mL of the requisite aqueous subphase (either a 10 mM NaCl solution or a 10 mM NaCl solution containing ctDNA (0.067 mg mL−1, corresponding to 1.29 × 1017 DNA phosphate groups per mL)), and the cleanliness of the aqueous phase was confirmed by ensuring that surface pressure, measured using a Wilhelmy plate (10 mm × 50 mm filter paper; Whatman International, Maidstone, UK) partially immersed in the subphase and attached to a pressure sensor, did not increase upon closing of the barriers. Lipid films were spread from a chloroform solution (typically 20 μL of a ∼2 mg mL−1 lipid solution) using a Hamilton syringe (Bonaduz, Switzerland) onto the appropriate aqueous subphase. After evaporation of the chloroform solvent (approximately 10 min), the lipid film was slowly compressed at a rate of 30 cm2 min−1, and the 2392

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evolutionary algorithm30 in order to minimize the χ2 values obtained for the fitting. In order to minimize the probability of correlated parameters, the “known” parameters (such as the SLDs of the layers, solvent and air) were fixed to the calculated value, while those parameters that were common to all contrasts (such as the thickness of the layers and the solvent penetration) were constrained between the three contrasts. The roughness between the various layers in the model was fixed at 4 Å, deemed to be physically reasonable as the roughness of the lipid interface. Note that although it was expected that the DNA/water interface would be rougher, the effect of the roughness on the fitted parameters was found to be minor. There is not sufficient resolution in the measurements to determine this roughness unambiguously, but in testing physically plausible values of 4 Å and 6 Å for the DODAB monolayer on a DNA-containing subphase there was only a mean 4% effect on the data, so the average thickness values were taken from the fitting using the two different roughness values to take this uncertainty into consideration (Supporting Information, Table 4). During fitting, each system was modeled using the minimum number of sublayers required to give an adequate fit to the reflectivity profiles recorded under each of the three measured contrasts, with constraints applied to limit the fitted parameters to physically reasonable values. All of the data were also fitted using AFit software25 in which a model of the interface as described by the optical matrix model is calculated using user-specified parameter values and overlaid over the data points. All three of the analysis methods provided optimal values for the fitted parameters that were within a few percent of each other. The errors on the RasCAL fitted parameters were calculated using the Bootstrap method31 and are provided in the tables (±value). It should be noted, however, that while each contrast is differentially sensitive to each of the parameters, the fitting algorithms in RasCAL weight all contrasts equally. In the case of the DODAB:DOPE monolayers, the errors were determined by visual inspection of the fits achieved for each of the contrasts in AFit; however, the fitting was carried out independently using RasCAL.

NIMA Langmuir trough placed on an antivibration table. The resulting surface films were compressed (at a speed of 30 cm2 min−1) to 5, 20, 30, or 40 mN m−1, and held at each of these surface pressures using the automated controls (for approximately 1 h, depending upon the contrast of the subphase used), while the SNR measurements were performed on the surface films. The films were stable under the conditions of the experiment. Specular Neutron Reflectivity Data Analysis Using the Optical Matrix Method. SNR data can be analyzed by determining the variation in the SLD profile normal to the plane of the interface. The specular reflection of neutrons from a monolayer is measured using a collimated neutron beam as a function of the momentum transfer perpendicular to the water surface (Qz): Qz =

4π sin θ λ

(1)

where Qz is related to the angle of incidence of the neutron beam (θ) and the wavelength (λ) of the neutrons. The SNR profiles presented in this paper show the ratio between the intensity of the specular reflection of neutrons, corrected for background scattering, to that of the incident beam (determined for each incident angle) versus Qz. The SNR profiles are shown including the values of Qz when background or residual scattering levels were reached. The SNR profiles were analyzed using RasCAL software,23 which uses the optical matrix method simultaneously to fit data of multiple isotopic contrasts. It was ensured that SNR measurements were taken in regions of the isotherm where the deuteration had negligible effect on the isotherm (i.e., not in the region of a phase transition), and it was assumed that the structure was not altered by deuterium labeling in these regions.24 Analysis of the SNR profiles was repeated independently using two other software programs, namely, AFit25 and MOTOFIT.26 All of these programs use the Abeles matrix method to calculate the reflectivity profiles, approximating the interfacial monolayer of interest as a user-defined number of stratified layers oriented parallel to the plane of the surface, with RasCAL and MOTOFIT performing a simultaneous fitting of the data sets obtained using different isotopic contrasts on the assumption that they are chemically equivalent. The composition of each layer may consist of a whole molecule (such as a lipid) or part of a molecule (such as the headgroup of a lipid) together with solvent. In the present study, the models used to describe the monolayer comprised either one or two layers. One-layer models were successfully applied to all monolayers. The single layer represented the hydrophobic chain region of the lipid molecule, as the headgroup region is very small and was available only in one, protiated, contrast. Owing to the presence of the larger phosphoethamolamine headgroup in DOPE, however, the headgroup was included for the DODAB:DOPE monolayers. Regardless of the number of layers, each layer was described by four parameters, namely, a thickness, an SLD, the penetration of solvent into the layer (% by volume), and a roughness. The SLD of each layer was calculated as

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RESULTS AND DISCUSSION Surface-Pressure Area Isotherms and BAM. DODAB Monolayers. The interactions of ctDNA with monolayers of DODAB alone or DODAB mixed with a 1:1 molar ratio of helper lipid were investigated by examining the isotherms produced by the compression of the DODAB-containing layers spread on an aqueous subphase containing 10 mM NaCl in the absence or presence of 0.067 mg mL−1 ctDNA. BAM images were collected at several points during the production of the isotherm, namely 5, 20, 30, and 40 mN m−1. Figure 1A shows the surface pressure−area isotherm obtained for DODAB spread on an aqueous phase containing 10 mM NaCl in the absence and presence of ctDNA. The deviation between repeat isotherms at each condition was less than a few percent. The isotherms of the DODAB and DODAB:helper lipid mixtures in the absence of ctDNA have been published previously.24 The presence of ctDNA in the subphase at a concentration of 0.067 mg mL−1 caused pronounced changes in the appearance of the surface pressure isotherm obtained upon compression of the DODAB monolayer (Figure 1A). These changes in the isotherm are not considered to be the result of any intrinsic surface activity in the DNA, since surface balance measurements (not shown) indicate that, at the concentrations used in the present study, ctDNA does not cause any reduction in the surface tension of a 10 mM NaCl solutiona result that, although at odds with the findings reported by others,12 nevertheless accords with the findings reported by Cárdenas et al.8

n

SLD =

∑i = 1 bi vcomponent

(2)

where bi is the scattering length of the ith of n atoms and Vcomponent is the volume of each component of the molecule (Supporting Informnation, Table 1). The volumes of the alkyl chains were calculated from the volume increments for methyl and methylene groups reported by Armen et al.27 The volume of the DODAB headgroup was calculated from the summed volumes of two methyl groups,27 nitrogen and bromide,28 while the volume of cholesterol was taken as 630 Å3,29 and that for the ethanolamine headgroup was calculated from the summed volumes of phosphate, glycerol, and carbonyl moieties.27 In modeling the DODAB:cholesterol monolayers, the cholesterol molecule was assumed to be located predominately in the alkyl chain region of the lipid monolayer, with only its terminal hydroxyl group being present in the headgroup region. To find the parameters that gave the most realistic model, the SNR data obtained for three contrasts, measured at each experimental condition, were simultaneously corefined in RasCAL using an 2393

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mN m−1) except for the presence of very occasional small, rather sparse, circular domains. Although not shown here, “flower-like” features were observed in the plateau (coexistence) region of the DODAB monolayers, occurring around a surface pressure of 10 mN m−1, as previously reported by Sun et al.9 and McLoughlin et al.34 for DODAB monolayers prepared under similar conditions. In contrast, when ctDNA was present in the subphase, no “flower-like” structures, typical of those seen in the coexistence (plateau) region in its absence, were observed. Instead the film appeared to be homogeneous at all surface pressures supporting the surface pressure isotherm results, and indicating that the presence ctDNA in the subphase suppressed the phase transition from expanded to condensed phase. Although Sun et al.9 also reported an expansion of the DODAB monolayer when ctDNA was present in the subphase, in contrast to the results reported here, they found that the plateau region seen in the absence of ctDNA was retained, albeit shifted to higher surface pressures: from 20 mN m−1 in the absence of ctDNA to 30 mN m−1 in its presence. In addition, these workers using BAM detected, regardless of surface pressure, the presence of DNA induced “dot-like” domains, which were found only at higher pressures in the absence of DNA, and were attributed to a condensation of DNA. We believe that the differences observed in surface morphology between the present study and that reported by Sun et al.9 are the result of differences in experimental conditions including subphase (i.e., water as opposed to the water containing 10 mM NaCl used in the present study). Indeed it is widely reported that slight differences in experimental factors can cause significant changes in the DODAB isotherm.35 DODAB:Cholesterol Monolayers. The addition of cholesterol at a 1:1 molar ratio to the DODAB in the absence of ctDNA abolished the phase transition from an expanded to condensed phase (Figure 2A) in agreement with previous reports.36 In addition, the curve was shifted toward much smaller molecular areas such that the isotherm was in a condensed phase at all areas per molecule − the lift off for the mixed DODAB:cholesterol monolayer occurred at a mean area per molecule of approximately 55 Å2. When ctDNA was present in the subphase, the surface pressure of the mixed DODAB:cholesterol film increased significantly at large areas per molecule (Figure 2A), i.e., the presence of ctDNA caused an expansion in the DODAB:cholesterol monolayer, just as it did for the DODAB monolayer. At smaller areas per molecule, where the monolayer was more condensed, the isotherm for DODAB:cholesterol in the presence of ctDNA became much more comparable to that recorded in its absence, although the presence of ctDNA did tend very slightly to increase the area per molecule. BAM images of the mixed DODAB:cholesterol film in the presence of ctDNA revealed that, at low surface pressures (i.e., less than or equal to 5 mN m−1) the “bubble-like” features seen in the DODAB:cholesterol monolayer were absent (Supporting Information Figure 1), suggesting that even at very low surface pressures ctDNA interacts with the monolayer to cause slight changes in its structure, such as increasing the lateral homogeneity of the film. At the higher surface pressures, no notable differences were observed between the DODAB:cholesterol monolayers in the presence and absence of ctDNA (Figure 2B and Supporting Information Figure 1).

Figure 1. (A) Surface pressure−area isotherms of DODAB on a 10 mM NaCl subphase without [] and with [---] ctDNA (0.067 mg mL−1) at 22 °C. Number of repeats (n) = 3. Note that the area per molecule reported in Figure 1A is that calculated from the knowledge of the amount of lipid added to the surface. (B) BAM images of a pure DODAB monolayer at 30 mN m−1 on a 10 mM NaCl subphase without and with DNA as indicated. BAM images at surfaces pressures of 5, 20, and 40 mN m−1 are given in Supporting Information Figure 1. The size represented by each image is 400 × 400 μm.

As can be seen in Figure 1A, the presence of ctDNA causes the film to become more expanded at all surface pressures, with the lift-off area increasing from approximately 130 Å 2 molecule−1 to approximately 140 Å2 molecule−1 in the presence of ctDNA. In addition, the presence of ctDNA in the subphase abolished the plateau/condensation region seen in its absence. The increase in the surface pressure isotherm in the presence of ctDNA suggests that ctDNA interacts with the surface monolayer of DODAB, probably via electrostatic interaction, to form an interfacial complex. The expansion in the cationic monolayer at the air−water interface upon the addition of DNA into the aqueous subphase has been explained on the basis of the linearized Poisson−Boltzmann equation as being due to differences in the charge density between DNA and the cationic monolayer.8 Similar expansions in the monolayers of DODAB spread on water have been reported in the presence of DNA,8 the negatively charged polymer, sodium poly(styrenesulfonate)32 and acidic polysaccharides.33 By way of contrast, the neutral polysaccharide dextran was not seen to interact with a DODAB monolayer spread on water.33 During compression, corresponding changes in the in-plane morphology of the DODAB monolayers, both in the absence and presence of ctDNA, were observed using BAM. Figure 1B shows the BAM images obtained for the DODAB systems at a surface pressure of 30 mN m−1; BAM images at surfaces pressures of 5, 20, and 40 mN m−1 are given in the Supporting Information, Figure 1. The BAM images for the DODAB monolayers in the absence of ctDNA have been previously reported by us,24 and are given here for the purposes of comparison. In the absence of ctDNA, the DODAB monolayer was largely homogeneous in the condensed region (20 −40 2394

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Figure 2. (A) Surface pressure−area isotherms of DODAB:cholesterol (1:1 molar ratio) on a 10 mM NaCl subphase without [] and with [---] ctDNA (0.067 mg mL−1) at 22 °C. Number of repeats (n) = 3. Note that the area per molecule reported here for the DODAB monolayers containing cholesterol is the mean molecular area of DODAB with cholesterol. (B) BAM images of a DODAB:cholesterol monolayer at 30 mN m−1 on a 10 mM NaCl subphase without and with DNA as indicated. BAM images at surfaces pressures of 5, 20, and 40 mN m−1 are given in Supporting Information Figure 1. The size represented by each image is 400 × 400 μm.

Figure 3. (A) Surface pressure−area isotherms of DODAB:DOPE (1:1 molar ratio) on a 10 mM NaCl subphase without [] and with [---] calf-thymus DNA (0.067 mg mL−1) at 22 °C. Number of repeats (n) = 3. Note that the area per molecule reported here for the DODAB monolayers containing DOPE is the mean molecular area of DODAB with DOPE. (B) BAM images of a DODAB:DOPE monolayer at 30 mN m−1 on a 10 mM NaCl subphase without and with DNA as indicated. BAM images at surfaces pressures of 5, 20, and 40 mN m−1 are given in Supporting Information Figure 1. The size represented by each image is 400 × 400 μm.

DODAB:DOPE Monolayers. The isotherm of a unimolar mixture of DODAB and DOPE on the aqueous 10 mM NaCl subphase (Figure 3A) was, regardless of surface pressure, in an expanded state, and, just like the cholesterol-containing monolayer, the presence of DOPE abolished the plateau region seen for the DODAB monolayer. The BAM studies revealed the presence of small (less than 8 μm diameter), irregular “bean-shaped” domains that retained their integrity and approached one another more closely upon compression (Figure 3B and Supporting Information Figure 1). When ctDNA was present in the subphase, the mixed DODAB:DOPE monolayer was, at all surface pressures, in an even more expanded state (Figure 3A). In contrast to the case when cholesterol was used as a helper lipid, at high surface pressures, the DOPE-containing monolayer was in a more expanded state in the presence of ctDNA than in its absence. The BAM images of the DODAB:DOPE monolayers show that the “bean-shaped” domains persisted at all surface pressures in the absence and presence of ctDNA (Figure 3A and Supporting Information Figure 1). This observation is consistent with the results reported by Cárdenas et al. (2005) who observed no dramatic changes in the BAM images of the interaction of salmon sperm DNA (2000 ± 500 base pairs) with monolayers of DODAB and the zwitterionic lipid, distearylphosphatidylcholine (DSPC) in either a 1:1 or 1:3 molar ratio. Specular Neutron Reflectivity: The Effect of Helper Lipids. The optical matrix analysis was performed on the SNR profiles obtained from monolayers of DODAB, DODAB:cholesterol, and DODAB:DOPE on an aqueous 10 mM NaCl subphase (Figure 4). In modeling the data using the optical matrix

method, the three contrasts measured for each experimental condition were fitted using one set of structural parameters. It was possible to simultaneously fit all three contrasts obtained for all three lipid monolayers assuming a single layer (Supporting Information Table 2). In all cases, the best fit was obtained without the presence of any solvent in the lipid layer, although no difference was found in the quality of the fit with up to ∼3% solvent included in the lipid layer. This result was not unexpected as the DODAB headgroup is protiated, and the thickness of the region it occupies is relatively small (at approximately 2−3 Å, as estimated from bond lengths) and consequently would be expected to contribute little to the SNR profile. The OH group in the cholesterol is also protiated and small. Note, therefore, that although the SNR data for the monolayers of DODAB, DODAB:cholesterol, and DODAB:DOPE could also be fitted with a two-layer model, there is insufficient information content of the profiles to permit the hydration level and thickness of the headgroup region to be fitted using unique and physically reasonable values, and they were successfully fitted with a simpler, one layer model. Note here too, that although the DODAB:DOPE monolayer is inhomogeneous, the domain sizes, as revealed through BAM, are sufficiently small that the reflectivity measurements furnish the average structure of the monolayer.24 DODAB monolayers. For monolayers composed only of the cationic DODAB lipid, the thickness of the monolayer was only 9.9 Å at 5 mN m−1 but increased with increasing surface pressure to 19.1 Å at 40 mN m−1 (Supporting Information Table 2). Between 5 and 40 mN m−1, the area per molecule calculated from the SNR thickness decreased from 107 Å2 to 56 2395

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Figure 4. SNR profiles for (A) DODAB, (C) 1:1 DODAB:cholesterol, and (E) 1:1 DODAB:DOPE monolayers on a 10 mM NaCl subphase at the surface pressure of 30 mN m−1. The contrasts shown are deuterated lipid on acmw (△), deuterated lipid on D2O (■) and protiated lipid on D2O (○). The data for two contrasts of the DODAB:DOPE system (△ and ■) extends lower into Q as it was measured on FIGARO, while all other contrasts for all systems were measured on SURF. The simultaneous fit for each data set is shown as solid lines on each contrast. The profiles of the SLD calculated from the fit as a function of the interface in the z-direction are shown for (B) DODAB, (D) 1:1 DODAB:cholesterol, and (F) 1:1 DODAB:DOPE monolayers. Error bars that are not visible are smaller than or equal to the data markers.

Å2 per molecule, consistent with a decrease from 105 Å2 to 58 Å2 per molecule obtained from the surface pressure−area isotherm. The largest increase in thickness of the DODAB monolayer was seen between 5 and 20 mN m−1, in the range where the monolayer underwent a transition from an expanded to a condensed phase (Figure 1A). The thickness of the DODAB monolayer at the higher pressure of 40 mN m−1 was found to be 27% lower than the ∼26 Å thickness reported from

ellipsometric studies of condensed DODAB monolayers on a 10 mM NaCl aqueous subphase.37 The increased thickness obtained from the ellipsometric studies may be due to the SNR data being most sensitive to the chains with the head groups having little effect on the fitted thickness. The length of the fully extended chains was calculated to be ∼23 Å,24 which indicates that the lipids in this study are tilted in the monolayer even at the highest surface pressure. In the case of a monolayer 2396

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Figure 5. SNR profiles for (A) DODAB, (C) 1:1 DODAB:cholesterol, and (E) 1:1 DODAB:DOPE monolayers on a 10 mM NaCl subphase containing ctDNA (0.067 mg mL−1) at the surface pressure of 30 mN m−1. The contrasts shown are deuterated lipid on acmw (Δ), deuterated lipid on D2O (■) and protiated lipid on D2O (○). The data for two contrasts of the DODAB:DOPE system (△ and ■) extends lower into Q as it was measured on FIGARO while all other contrasts for all systems were measured on SURF. The simultaneous fit for each data set is shown as solid lines on each contrast. The profiles of the SLD calculated from the fit as a function of the interface in the z-direction are shown for (B) DODAB, (D) 1:1 DODAB:cholesterol, and (F) 1:1 DODAB:DOPE monolayers. Error bars that are not visible are smaller or equal to the data markers.

perpendicular direction, thereby preventing their tilt.39 For example, the thickness of the cholesterol-containing monolayer at 5 mN m−1 was determined to be 18.9 Å (cf. 9.9 Å for the pure lipid monolayer). At higher surface pressures, the presence of cholesterol had less of an effect on the thickness of the monolayer with a fitted thickness of 21.4 Å at 40 mN m−1 (cf. 19.1 Å for the pure lipid monolayer), presumably because at this surface pressure the longer DODAB molecules, although still slightly tilted, are oriented more closely parallel to the interface normal.

of dihexadecyldimethylammonium bromide, a molecule that has two less carbons in the acyl chains than DODAB and a fully extended chain length of ∼21 Å, Ruggles et al. also obtained a small thickness for the chains of 15.7 Å at a surface pressure of 30 mN m−1.38 DODAB:Cholesterol Monolayers. At lower surface pressures, the presence of cholesterol in the monolayer resulted in a thicker layer when compared to the pure DODAB monolayer (Supporting Information Table 2). This result was as expected because cholesterol is known to reorient lipid chains into a 2397

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Table 1. Parameters from Modeling Neutron Reflectometry Data for Cationic Monolayers at 30 mN m−1 on a 10 mM NaCl Subphase Containing 0.067 mg mL−1 ctDNAa

a

lipid

area per molecule (Å2)

lipid thickness (Å)

DNA thickness (Å)

DNA amount (% by volume)

DODAB DODAB:cholesterol DODAB:DOPE

82 75 118

13.0 ± 0.2 22.7 ± 0.8 20.4 ± 0.1

22.5 ± 2.4 19.9 ± 2.8 19.1 ± 6.3

35 ± 2 25 ± 5 14 ± 3

For monolayers containing helper lipid, the area per molecule reported here is the mean molecular area of DODAB with the helper lipid.

DODAB:DOPE Monolayers. Despite the expanded nature of the monolayer, the lipid thickness of the DOPE-containing monolayers was, at the lower surface pressures of 5 mN m−1, thicker than the DODAB-only monolayer (viz., 14.7 Å, versus 9.9 Å, Supporting Information Table 2). The increased thickness of the DOPE-containing monolayer is undoubtedly partly due to the large ethanolamine headgroup, which contributes to the scattering of the monolayer to a greater extent than the small DODAB headgroup (Supporting Information Table 1). The DOPE-containing monolayer steadily increased in thickness over the compression range, as expected from the isotherm (Figure 3A). The thickness of the head region was included in the fitted thickness of the layer; however, in this case the headgroup thickness would be an average of the DOPE headgroup (previously reported to be ∼9 Å40) with the DODAB headgroup, which is much thinner, at ∼2−3 Å. The presence of the larger DOPE head would suggest that the thickness of the alkyl chains’ layer in the DODAB:DOPE monolayers was somewhat thinner than that recorded for the pure DODAB and the DODAB:cholesterol monolayers, which is exactly as would be expected from the more expanded nature of the DODAB:DOPE lipid film. The areas per molecule extrapolated from the isotherms for the DODAB and DODAB:cholesterol monolayers typically differ only by a few percent (ranging from 0 to 9%) from the areas per molecule obtained from the fitted data, calculated by dividing the molecular volumes by the fitted thicknesses (Supporting Information Table 2). However, for the DODAB:DOPE monolayers, the areas per molecule calculated from the SNR measurements were much smaller (ranging from 3 to 20%) than those extrapolated from the isotherm and here the differences may be explained by the nonideal mixing of the two lipids, as seen in the BAM (Figure 3B and Supporting Information Figure 1). Note that, in the case of the mixed lipid films, the areas per molecule extrapolated from the SNR thicknesses are those calculated for the combined areas of the two lipids. Thus, it is necessary to divide the area per molecule obtained from the SNR by a factor of 2, in order to compare it to the area per molecule obtained from the surface-pressure area isotherm. Specular Neutron Reflectivity: Presence of ctDNA in the Subphase. In order to investigate the effect of the presence of ctDNA on the structure of lipid monolayers at the air−water interface, monolayers of DODAB alone or mixed with a helper lipid (i.e., DOPE or cholesterol) were deposited on an aqueous subphase of 10 mM NaCl which contained 0.067 mg mL−1 ctDNA (Figure 5). The SNR profiles were measured at the same four surface pressures as those used to characterize the lipid monolayers deposited on an aqueous solution of 10 mM NaCl (SNR profiles of monolayers at surface pressures other than 30 mN m−1 are in the Supporting Information). It is clear from a comparison of Figures 4 and 5 that the presence of ctDNA in the subphase caused changes in the SNR profiles

measured for the various DODAB monolayers, although no Bragg peaks indicative of long-range repeat ordering were seen. Furthermore, it was no longer possible to fit the SNR in the presence of ctDNA using a single layer model, as was the case for the cationic monolayers in its absence. Instead, it was found necessary to use a two-layer model to adequately fit the SNR data in the presence of ctDNA, i.e., an upper lipid layer and a lower ctDNA-containing layer. Note that when modeling the SNR data, the SLD of the ctDNA was calculated, taking into account the differential exchange of its labile protons22 and was therefore dependent on whether the subphase was acmw or D2O. Regardless of the helper lipid used, DNA was not found to notably penetrate the lipid alkyl chains’ layer. Models that assumed penetration of different amounts of DNA into the lipid layer were tested, and it was found that it was possible to accommodate only up to 2−5% DNA by volume into the lipid region in the most sensitive contrast. Beyond this amount, it was not possible to adequately model the data within physically reasonable constraints. Since the (majority of the) DNA is thus confined at the level of the lipid head groups, we conclude that the DNA molecules are attracted to the air−water interface via an electrostatic interaction with the oppositely charged lipid head groups, rather than through a hydrophobic driving force. For the DODAB and DODAB:helper lipid monolayers, while it was feasible to model an intermediate layer between the lipid chains and the DNA, it was not possible to find a unique composition for this layer, and so the hydrated DNA layer in these monolayers will also contain a contribution from the lipid head groups. To make matters simple, however, the presence of the head groups in this layer was ignored in the subsequent calculation of the volume fractions of DNA and solvent in the layer. DODAB Monolayers. Table 1 and Supporting Information Table 3 show the parameters for layer thicknesses and percent solvent used to fit the DODAB monolayers deposited on the ctDNA-containing subphase. In the fitted two-layer model, the top layer represents the lipid region, and the lower layer represents a heavily hydrated ctDNA layer. As seen in Supporting Information Table 3, compared to the data obtained for the DODAB monolayer in the absence of ctDNA (Supporting Information Table 2), the thickness of the lipid region was reduced, indicating a greater tilt in the DODAB molecules. This result is in agreement with that reported by Kago et al.41 who determined using X-ray reflectivity that DODAB monolayers spread on pure water were thicker than those spread over ctDNA-containing subphases. From the areas per molecule calculated in the present study, it is clear that there are fewer lipid molecules required on the surface of the DNA-containing subphase in order to achieve the same surface pressure as on the 10 mM NaCl subphase. This observation is consistent with the isotherm data which showed that a more expanded monolayer was formed when ctDNA was present in the subphase (Figure 1). 2398

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based on charge distribution only if the charged lipids were miscible with the spacing helper lipid, DOPE.24 However, the lateral heterogeneities observed in the DODAB:DOPE monolayer persist in the presence of ctDNA, as observed by BAM (Figure 3B and Supporting Information Figure 1), which suggests that the lipids do not undergo a rearrangement of the surface charges to induce a more favorable interaction with ctDNA. Therefore, the DODAB:DOPE monolayer is likely regionalized into areas of differential charge density where DNA may mostly adsorb to the positively charged areas. In terms of transfection efficiency in vitro, DODAB:DOPE is most successful, which suggests that it is lipid packing properties that determine the ability of a cationic lipid-containing system to act as a nucleotide vector, rather than relative DNA-binding affinity. Indeed, the transfection efficiency of lipoplexes is strongly correlated with their stability, which is known to be influenced by the membrane fluidity of the complexes.42 The fluid nature of vesicles prepared from a 1:1 molar ratio of DODAB:DOPE is demonstrated by their exhibiting a phase transition of less than 20 °C, even when bound to DNA,43 supporting the fact that lipid chains are in the fluid phase. In contrast, vesicles prepared solely from DODAB exhibit a phase transition of ∼45 °C43 and thus are present in the gel phase. It is reasonably expected that, in the presence of cholesterol, the lipid chains will also be in the gel phase at the experimental temperature. DOPE is believed to act as a helper lipid by enabling escape from the endosome by increasing the fusion of the vector lipids with those of the endosome; the overall DNA content of lipoplexes may matter less than its ability to release that DNA from the endosome into the cytoplasm. In contrast, DODAB:cholesterol was found to form tightly packed monolayers that bound less ctDNA, by volume, than DODAB alone (Supporting Information Table 3). At 5 mN m−1 approximately a quarter of the volume of the lower layers were filled with DNA for both DODAB and DODAB:cholesterol monolayers; however, as the monolayers were further compressed, progressively more ctDNA bound to the DODAB monolayer while a relatively constant amount of ctDNA bound to the DODAB:cholesterol throughout the compression. This result is consistent with the different natures of the isotherm, the DODAB isotherm showed that the surface positive charge density increased steadily whereas DODAB:cholesterol presented similar positive charge density throughout the compression above 5 mN m−1. At the highest surface pressure, the amount of ctDNA bound by DODAB and DODAB:cholesterol was 39 ± 2 and 31 ± 6 vol%, respectively. This observation was not surprising given that the addition of cholesterol causes only a small decrease in the charge per unit area at high surface pressures when compared against that for the monolayer containing DODAB alone. Indeed the average area per molecules are 74 and 76 Å2 molecule−1 for the DODAB and DODAB:cholesterol monolayers on the ctDNAcontaining subphase, respectively (Supporting Information Table 3). The DODAB:cholesterol monolayer at lower pressures is more densely packed than that of DODAB alone, and this condensed nature may be how the addition of cholesterol increases the circulation time and improves the transfection in vivo. In contrast, in vitro, this persistence of cholesterol in the layer may be a hindrance to endosomal escape, even though more ctDNA is bound to DODAB:cholesterol compared to DODAB:DOPE.

Underneath the DODAB monolayer, a ctDNA-containing layer was present, which was best fitted as a hydrated layer of ctDNA of approximately 21 Å thickness taking up ∼27−39% of this layer by volume, depending on the surface pressure (Supporting Information Table 3). Interestingly, the amount of ctDNA adsorbed increased as the monolayer was compressed. Thus, it appears that water and not ctDNA was squeezed out from below the layer of cationic lipid upon compression, as the charge ratio between the ctDNA and lipids must remain neutral, and removal of the ctDNA would be energetically unfavorable once binding and counterion release had occurred. It is important to emphasize here that there was no penetration of ctDNA into the lipid chains beyond 5% by volume, and we can be quite confident in this assertion because the relatively high SLD of the ctDNA affords that the analysis is very sensitive to any such change in the absorbed layer composition, the ctDNA penetration resulting in an increased SLD for the lipid layers in the air-contrast matched subphase and a decreased SLD when the subphase is D2O. These results are thus consistent with an electrostatic interaction between the ctDNA and the oppositely charged lipid headgroups, with the ctDNA content in the layer seemingly remaining constant over the range of surface pressures studied here. DODAB:Cholesterol Monolayers. Table 1 and Supporting Information Table 3 shows the parameters obtained for layer thicknesses, percent solvent, and interfacial roughness for a two-layer model of a mixed film of DODAB:cholesterol on the ctDNA-containing subphase. Compared to the larger effect of ctDNA on DODAB monolayer thickness, the presence of ctDNA caused very slight changes in the thickness of the mixed DODAB:cholesterol films. The largest difference in thickness was observed at the lower surface pressures, consistent with the isotherms that are most divergent at these pressures. This observation suggests that at the higher surface pressures there was very little rearrangement needed in the cholesterolcontaining mixed monolayer to allow its interaction with ctDNA. The ctDNA layer was found to be approximately 21 Å thick and contained 24−31 vol % ctDNA, almost invariant of surface pressure, which is similar to that found at the highest compression of the pure DODAB monolayer. Again, the compression of the monolayer does not lead to the “squeezing out” of the ctDNA from the interface. DODAB:DOPE Monolayers. The parameters obtained for layer thickness, percent solvent and interfacial roughness for a three-layer model of a mixed lipid films of DODAB:DOPE deposited on the DNA-containing subphase are given in Table 1 and Supporting Information Table 3. As would be expected from the Langmuir isotherm recorded for this system, which shows a more expanded behavior in the presence of ctDNA , the thickness of the lipid monolayer increases slowly with surface pressure. The thickness of the ctDNA-containing layer was found to be in the region of 22 Å, which is within error of the thickness of ctDNA obtained for the other lipid systems studied here. Interestingly, however, the ctDNA layer under the DODAB:DOPE monolayer was found to occupy only approximately 8− 18 vol %, depending on the compression. This amount of ctDNA is lower than the approximately 24−39 vol % ctDNA found for the other two lipid monolayers at high surface pressure. Comparison of the Effect of the Helper Lipids. We have previously predicted that the DODAB:DOPE mixed monolayer was expected to have the greatest ctDNA-binding capacity 2399

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was present directly beneath the lipid head groups, without extensive penetration of the ctDNA into the layer occupied by the lipid chains. Using SNR, it was also possible to characterize the ctDNA layer, which was found to be made up of a single hydrated layer of DNA of a similar thickness to the diameter of the molecule for all the lipid mixtures studied. The positive charge densities of the DODAB and DODAB:cholesterol monolayers were found to match the negative charge density of a packed layer of DNA, while this was not the case for the DODAB:DOPE monolayer, probably due to inhomogenous mixing of this helper lipid. The presence of cholesterol was found to result in stable, compressed monolayers with good ctDNA-binding ability. In contrast, DOPE-containing monolayers bound less ctDNA, by volume, than DODAB or DODAB:cholesterol. Via BAM it was observed that the immiscibility of DODAB in DOPE was found to persist in the presence of ctDNA showing that DOPE does not act to optimize the charge distribution of the DODAB monolayer for ctDNA-binding. The difference between cholesterol and DOPE raises important questions about the structure−function relationship as it pertains to the application of the lipoplexes because while in vitro transfection was found to be improved by the presence of fusion-inducing hexagonal mesophases, such as the ones encouraged by DOPE,49 it has been shown that high in vivo transfection efficiency requires small, stabilized lipoplexes.20 Thus, the condensed, protective nature of DODAB:cholesterol monolayers found in this study is more appropriate for in vivo DNA delivery. In this study, a direct comparison revealed striking differences in the amount of DNA bound to monolayers containing two different helper lipids. This marks an important step to understanding the DNA:lipid interactions in a more realistic, multicomponent monolayer models of gene delivery vectors.

A look at the surface charge density also reveals differences between the monolayers. As the form of DNA used in the present study, namely, the B form, has 10 base pairs per turn, there are 20 negative charges on a turn with a pitch of 34 Å. Assuming a spacing of 45 Å between adjacent strands of packed DNA,44 the charge density of ctDNA would be expected to be one negative charge for every 75 Å2. This calculated negative charge density corresponds closely to the positive charge density obtained from the area per molecule of the DODAB and DODAB:cholesterol monolayers at 30 mN m−1. In the case of DODAB:DOPE, the area per positive charge is much larger (110 Å2), which gives a lateral spacing of 65 Å for ctDNA, suggesting fewer or more disperse strands of ctDNA adsorb at the interface of this mixed monolayer. Note that in the case of the mixed lipid monolayers, it was assumed that there was one positive charge for every DODAB:helper lipid pair. However, one major limitation of this calculation is that it assumes ideal mixing between DODAB and helper lipids which has been shown not to be the case for DODAB:DOPE. Importantly, the presence of helper lipids did not appear to change the thickness of the adsorbed layer of ctDNA which was consistently in the range of 19 to 25 Å, regardless of surface pressure. Sun et al.9 reported the layer of fish testes DNA to be around 14 Å thick after compression of a cationic monolayer composed of DODAB, while Zhang et al. found that ctDNA adsorbs with the single-chain cationic surfactant, C 12 trimethylammonium bromide, as single layers of ctDNA with thicknesses between 19 and 26 Å.45 Other reports indicate thicker but also more disordered layers of DNA underneath cationic monolayers. For instance, using X-ray reflectometry Kago et al. investigated ctDNA layers formed underneath monolayers of DODAB, and derived a model where the ctDNA was arranged as a undulating strands, with the DNA closest to the monolayer having 70 vol% DNA (thickness of 25−28 Å) and the outer layer about 30 vol% DNA;41 however, salt was not included in the subphase, and thus the ctDNA may be adsorbed, in part at least, in single-stranded form. While the cross-sectional diameter of the B-form DNA helix was found from crystallography to be approximately 24 Å,46 the diameter of a cylinder of DNA has been calculated using bond length data as 19 Å.46 However, as noted by Gueron et al.,47 although the calculated diameter for the cylinder of B-form DNA includes the phosphates which jut out from the backbone, it does not take into account the hydration of the DNA which fills the space of the grooves of DNA in the B form. When this hydration and experimental error are taken into account, along with the concomitant roughness on both interfaces of the DNA, this would make the thickness of the DNA cylinder measured in the present study using SNR appear as expected. The consistency of the thickness of the DNA across all the lipid compositions is not surprising, as DNA has been shown to remain in B-form upon being condensed by DODAB, alone and in combination with both helper lipids.48

■

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information includes tables with the structural parameters obtained from the fitting. BAM images and fitted SNR profiles of the monolayers at other surface pressures are also included as Supporting Information. This material is available free of charge via the Internet at http://pubs.acs.org.

■

AUTHOR INFORMATION

Corresponding Author

*Tel: 0207 848 4808; fax: 0207 848 4800; e-mail: jayne. [email protected]. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS The authors thank the Science and Technology Facilities Council (STFC) for access to the facilities at ISIS Pulsed Neutron and Muon Source (Chilton, Oxfordshire, UK) and the Institut Laue-Langevin (Grenoble, France). We also thank Robert Barker for his assistance in setting up the Brewster angle microscope.

■

CONCLUSIONS A quantitative comparison of the binding of ctDNA monolayers of DODAB, alone or containing one of the helper lipids, cholesterol or DOPE, has been systematically carried out at different surface pressures. An interaction was demonstrated on the Langmuir trough by an increase in the effective area per molecule, as well as a change in the structure of the monolayers as observed by BAM. SNR showed that, regardless of surface pressure, an electrostatically attracted hydrated ctDNA layer

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REFERENCES

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