Unexpected Bilayer Formation in Langmuir Films of Nucleolipids

Article pubs.acs.org/Langmuir

Unexpected Bilayer Formation in Langmuir Films of Nucleolipids Bernard Desbat,†,‡ Nessim Arazam,§,∥ Salim Khiati,⊥,# Giovanni Tonelli,⊥,# Wilfrid Neri,§,∥ Philippe Barthélémy,⊥,# and Laurence Navailles*,§,∥ †

Univ. Bordeaux, CBMN, UMR 5248, F-33600 Pessac, France CNRS, CBMN, UMR 5248, F-33600 Pessac, France § CNRS, CRPP, UPR 8641, F-33600 Pessac, France ∥ Univ. Bordeaux, CRPP, UPR 8641, F-33600 Pessac, France ⊥ Université Bordeaux-2, F-33076 Bordeaux, France # ARNA lab INSERM U869, F-33076 Bordeaux, France ‡

S Supporting Information *

ABSTRACT: Langmuir monolayers have been extensively investigated by various experimental techniques. These studies allowed an in-depth understanding of the molecular conformation in the layer, phase transitions, and the structure of the multilayer. As the monolayer is compressed and the surface pressure is increased beyond a critical value, usually occurring in the minimal closely packed molecular area, the monolayer fractures and/or folds, forming multilayers in a process referred to as collapse. Various mechanisms for monolayer collapse and the resulting reorganization of the film have been proposed, and only a few studies have demonstrated the formation of a bilayer after collapse and with the use of a Ca2+ solution. In this work, Langmuir isotherms coupled with imaging ellipsometry and polarization modulation infrared reflection absorption spectroscopy were recorded to investigate the air−water interface properties of Langmuir films of anionic nucleolipids. We report for these new molecules the formation of a quasi-hexagonal packing of bilayer domains at a low compression rate, a singular behavior for lipids at the air−water interface that has not yet been documented.

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INTRODUCTION One of the main promising tools for medicine in the next few years will be gene therapy.1 It is based on the introduction of a therapeutic gene into the cell of a patient with a very high specificity of action.2−4 To enable this specific action, one necessary tool is a vector for delivering nucleic acids into targeted cells. This is what we call gene delivery or vectorization.5 Viruses are a very efficient vector, but they present a great risk in clinical application because of their immunogenic toxicity.6 To overcome this, various synthetic vector designs have been proposed in the past few years.4 The vector needs to form a complex with negatively charged DNA and have some transport properties. This is why the earliest ones were based on cationic lipids.7 They are still the most frequently studied type, but their cytotoxicity and inefficiency in vivo is a major problem in medicinal applications.8,9 Anionic liposomes are an alternative.3,9−11 To render these new vectors efficient, we have explored a new strategy using anionic nucleolipids where we can modulate the nature and the specificity of the interactions between the vector and the nucleic acid.12−15 Anionic nucleolipids are negatively charged synthetic molecules made of a polar nucleotide head and a hydrophobic lipid tail (Figure 1).16−19 This enables us to create © 2012 American Chemical Society

ionic interactions with a divalent cation, molecular recognition between nucleobases, and hydrogen bonding in base stacking. We have shown in a previous study the correlation between the transfection efficiency and the supramolecular structure of the vector.17 For a better understanding of the interactions at a molecular level between the nucleolipids and the nucleic acid, we need to perform experiments on a 2D model using Langmuir films with infrared spectroscopy techniques and optical imaging. Before studying the complete system (nucleolipid and nucleic acid), the first step in our approach is to understand the interfacial properties of these new amphiphilic molecules at the air/water interface. This approach has the advantage that the density of the molecules at the interface can be varied by lateral compression, film formation can be studied via surface tension measurements, and the molecular orientation can be probed via polarization modulation infrared reflection absorption spectroscopy (PMIRRAS).20−23 At the air/water interface, Langmuir monolayers have been extensively investigated by various experimental Received: December 2, 2011 Revised: March 7, 2012 Published: March 21, 2012 6816

dx.doi.org/10.1021/la2047596 | Langmuir 2012, 28, 6816−6825

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structure of fatty acids. For monolayers spread on Ca2+ solutions, the collapsed film consists of a bi- and trilayer mixture with a ratio that changes by the collapse protocol. Their analysis suggests that the bilayer structure is inverted (i.e., with the hydrophobic tails in contact with the water and the calcium ions bridging the polar heads). The authors provide theoretical arguments rationalizing that the observed structures have lower free energies compared to those of other possible structures and contend that the collapsed structures may, under certain circumstances, form spontaneously.41 The same behavior was observed for behenic acid Langmuir films before and after collapse on pure water and on Ca2+ solutions and was confirmed with in situ imaging ellipsometry.42,43 Indeed, the three-layer formation has been reported for various compounds (cholesterol, DOPS, and liquid crystals), but bilayer stabilization was reported only for DMPC at very high compression (45 mN/m) after the collapse point and in the work previously cited.39−43 To our knowledge, very few studies have been done with anionic nucleolipids at the air−water interface.44,45 In the present work, Langmuir isotherms coupled with imaging ellipsometry and PM-IRRAS were recorded to investigate the air−water interface properties for Langmuir films of anionic nucleolipids. We report for these new molecules the formation of a quasi-hexagonal distribution of bilayer domains at a low compression rate, a singular behavior for lipids at the air/water interface that has not yet been documented.

Figure 1. Chemical structures and nomenclature of the two nucleotide lipids used in this study: (a) thymidine-3′-(1,2-dipalmitoyl-sn-glycero3-phosphate) diC16-3′-dT and (b) adenosine-3′-(1,2-dipalmitoyl-snglycero-3-phosphate) diC16-3′-dA. The counterion is triethylammonium, (C2H5)3NH+.

techniques.24,25 These studies allowed an in-depth understanding of the molecular conformation in the layer, phase transitions, and the structure of the multilayer. Under controlled compression and temperature conditions, insoluble monolayers at gas/water interfaces (Langmuir monolayers) exhibit a significant number of phases, including quasi-2D gaseous, liquid, and several solid phases.26−30 As the monolayer is compressed and the surface pressure (π) is increased beyond a critical value, usually occurring over the minimal closely packed molecular area (i.e., the average cross section of the molecule, A0), the monolayer fractures and/or folds, forming multilayers in a process referred to as collapse.31−33 Spontaneous collapse, where the breakage and/ or folding occurs over molecular areas of A > A0 and the monolayer coexists with its bulk phase, has also been reported.32,34−37 Various mechanisms for monolayer collapse and the resulting reorganization of the film have been proposed. Recently, molecular dynamics simulations have been applied to the study of the monolayer collapse processes and vesicle conformations.38,39 On the experimental side, Saccani et al. have demonstrated that stable phospholipid multilayers can be formed at the air−water interface by the compression of monolayers beyond the collapse.40 Brewster angle microscopy (BAM) and polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS) results showed the formation of a fluid trilayer composed of a monolayer on top of a bilayer in the case of 1,2-di[cis-9-octadecenoyl]-snglycero-3-[phospho-L-serine] (DOPS) and a partial fluid bilayer in the case of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC). Thermodynamic considerations and the molecular dynamics simulations suggest a tail-to-tail contact organization for the latter system. More recently, neutron and synchrotron X-ray studies of arachidic acid monolayers compressed to the collapse region, beyond their densely packed molecular area, have revealed that the resulting structures show a surprising degree of reproducibility and order. The structure of the collapsed monolayers differs for films that are spread on pure water or on CaCl2 solutions. On pure water, the collapsed monolayer forms a stable crystalline trilayer structure, with acyl chain in-plane packing practically identical to the 3D crystal

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EXPERIMENTAL SECTION

Materials. The nucleolipid thymidine-3′-(1,2-dipalmitoyl-sn-glycero-3-phosphate) diC16-3′-dT was designed and synthesized a few years ago by Barthélémy and co-workers.17 To compare the effect of the base on the interaction between nucleolipids, the analogue adenosine-3′-(1,2-dipalmitoyl-sn-glycero-3-phosphate) diC16-3′-dA was synthesized. Step 1. To a solution of 2 M MeNH2 in THF (20 mL) was added N6-benzoyl-5′-O-(4,4′-dimethoxytrityl)-2′-deoxyadenosine-3′-O-[O-(2cyanoethyl)-N,N′-diisopropylphosphoramidite] (1.5 g, 1.75 mmol) at room temperature. After being stirred for 2 h, the mixture was evaporated under reduced pressure. The residue underwent chromatographic characterization on a column of silica gel with 1% MeOH (methanol)/DCM (dichloromethane) containing 1% TEA and then 3% MeOH/DCM containing 1% TEA (triethylamine) to give the fractions containing the target 5′-O-(4,4′-dimethoxytrityl)-2′-deoxyadenosine-3′-O-[O-(2-cyanoethyl)-N,N′-diisopropylphosphoramidite]. Yield: 1.18 g (89.8%). Step 2. 5′-O-(4,4′-Dimethoxytrityl)-2′-deoxyadenosine-3′-O-[O-(2cyanoethyl)-N,N′-diisopropylphosphoramidite] (the product of step 1) (1.0 g, 1 equiv, 1.33 mmol), 1,2-dipalmitoyl-sn-glycerol (0.984 g, 1.3 equiv, 1.73 mmol/dissolved in 6 mL of THF), and a tetrazole solution in acetonitrile (0.45 M, 4 mL, 1.3 equiv, 1.73 mmol) were dissolved in 6 mL of dry acetonitrile under argon. The reaction mixture was stirred for 7 h at room temperature, followed by oxidation with 80 mL of a solution of I2 (0.02 M in THF/pyridine/H2O). After 12 h at room tempereature, the solvent was evaporated under high vacuum to yield intermediate products. The contents of the reaction flask were dissolved in 20 mL of methylene chloride and then washed sequentially with 3 × 20 mL of HCl (0.1 N) and with 3 × 20 mL of saturated Na2S2O3. The product was isolated after purification on silica gel (DCM/MeOH/TEA from 98:2:1 to 50:49:1). Yield: 481 mg (41%). 1 H NMR (300 MHz, CDCl3): δ 0.77 (t, 6H, J = 6.9 Hz, 2 CH3), 1.15 (m, 48H, 24 CH2), 1.48 (dd, 4H, J1 = 8.4 Hz, J2 = 15.6 Hz, 2CH2), 2.18 (m, 4H, 2CH2), 2.92 (t, 2H, J = 5.6 Hz, H2′), 3.6−4.4 (m, 7H, 2CH2(glycerol), H4′, H5′), 5.21 (s, 1H, CH(glycerol)), 6.28 (t, 1H, J = 6.7 Hz, H1′), 7.91 (s,1H, Hbase), 8.16 (s, 1H, Hbase). 6817


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equipped with a HgCdTe photovoltaic detector (SAT, Poitiers) cooled to 77 K. The spectra are composed of 600 scans coadded at a resolution of 8 cm−1. To remove the contribution of liquid water absorption and the dependence on Bessel functions, the film spectra are divided by the spectra of the subphase. The infrared beam is reflected to the interface at a 75° angle of incidence. Under this condition, a negative band indicates a transition dipole moment oriented preferentially perpendicular to the interface, and a positive band indicates a transition dipole moment oriented preferentially within the plane of the interface. The absorption band vanishes when the transition dipole moment has a 38° tilt from the interface.47

P NMR (121 MHz, CDCl3): 2.00 ppm High-resolution ESI MS [M−H]−: theoretical m/z, 880.5570; observed m/z, 880.5574. These anionic nucleolipids are amphiphilic molecules with a polar head composed of a DNA nucleobase (adenine or thymidine) and a hydrophobic part composed of two alkyl chains. The triethylammonium counterion was provided during the nucleolipid synthesis. All of the buffers were made of trishydroxymethylaminomethane (Tris), calcium chloride (CaCl2), sodium chloride (NaCl), and hydrochloric acid (HCl) dissolved in Milli-Q ultrapure water with a resistivity of 18.2 MΩ.cm (pH ∼5.6). The Tris buffer is 20 mM Tris, 150 mM NaCl (pH 7.4). The Tris−calcium buffer is 20 mM Tris, 150 mM NaCl, and 15 mM CaCl2 (pH 7.4). HPLC-grade ethanol-stabilized chloroform (CHCl3) and HPLC-grade methanol (CH3OH) were purchased from Sigma-Aldrich. Heavy water (D2O) was purchased from CEA (Saclay, France). Formation of Langmuir Films. We used two Teflon Langmuir troughs (Nima Co., Birmingham) filled with an aqueous subphase of buffer. One trough has an area of 100 cm2, and the other has an area of 45 cm2. The Langmuir film was formed by spreading a few microliters of nucleolipid solution (nucleolipid concentration of 0.5 mg/mL) in chloroform/methanol (50/50, v/v) on the subphase, and we waited 15 min until the solvents had evaporated. The experiments were carried out at room temperature (T = 22 ± 1 °C). Isotherm Measurements. The films were compressed by a barrier at the rate of 5 cm2/min (3 Å2/mol/min), and the surface pressure, Π (mN/m), was measured according to the Wilhelmy method using a computer-controlled Nima surface tensiometer. The Wilhelmy plate was made of a Whatman paper filter. The experiments were carried out at 22 °C. The water surface tension was 72.8 mN/m. Imaging Ellipsometry. We used an IElli2000 ellipsometer (NFTNanofilm Technologie GmbH, Göttingen, Germany) mounted on the Langmuir trough. The microscope is composed of a doubled Nd:YAG laser (50 mW, 532 nm), a polarizer, a compensating plate, an analyzer, and a 10× objective coupled to a CCD camera. The exposure time of the camera is adjusted in order to avoid saturation. The images size is 670 μm × 450 μm with a lateral resolution of 2 μm. The setup was used as an imaging ellipsometer at an angle of incidence of 54.58° close to the Brewster angle (53.12°) for the air/water interface at 532 nm. It operates on the principle of classical null ellipsometry.46 The angles of the polarizer, compensator, and analyzer that give the null condition allow us to obtain the (Δ, Ψ) angles that are related to the optical properties of the sample. Δ = (δp − δs) is the phase retardation parameter (with δp being the phase of the p polarization and δs being the phase of the s polarization), and Ψ is the amplitude parameter (tg2Ψ = Rp/Rs, with Rp being the p-polarized reflectance and Rs being the s-polarized reflectance). Under ultrathin film conditions, Δ is proportional to the film thickness, and it is convenient to define the variable δΔ = Δ0 − Δ, with Δ0 being the buffer delta value and Δ being the sample delta value. When the sample contains different domains, the imaging ellipsometer allows us to obtain, successively, the null condition and the δΔ for each specific domain on the surface. A comparison of the measured data with computerized optical modeling included in the ellipsometer software leads to a deduction of the domain thickness when an estimation of the refractive index (n) can be obtained. The Ψ parameter cannot be used to determine the n value because its value is almost constant for ultrathin films samples. Fourier Transform Infrared Spectroscopy. ATR-FTIR infrared spectroscopy was used to obtain absorbance spectra of the bulk of the different molecules studied. The spectrometer was a Nicolet Nexus 6700 equipped with a HgCdTe detector cooled to 77 K, a diamond ATR crystal, and a BaF2 polarizer in order to perform polarized ATRFTIR spectroscopy. The spectra are composed of 600 scans coadded at a resolution of 4 cm−1 taken between 4500 and 600 cm−1. From the polarized ATR-FTIR spectra, one can obtain for each absorption band the dichroic ratio (R = Ap/As), which contains the orientation information. The PM-IRRAS setup was mounted on the Langmuir trough to obtain spectra of the films at the interface as previously described.34 The spectra were recorded on a Nicolet Nexus 870 spectrometer

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RESULTS Langmuir Films: Compression Isotherm and Microscopic Imaging Ellipsometry. Nucleolipid Films on Water. DiC16-3′-dA and DiC16-3′-dT were spread on pure water, and the surface pressure−area isotherm was recorded (Figure 2). The surface-pressure isotherm shows a liquid-

Figure 2. Surface pressure isotherms for (a) diC16-3′-dA and (b) diC16-3′-dT for three different subphases: H2O, Tris (20 mM)−NaCl (150 mM), and Tris (20 mM)−NaCl (150 mM)−CaCl2 (15 mM).

expanded (LE) phase at low pressure with a homogeneous layer as seen on ellipsometric images (Figure 3a). At 18 mN/m, a transition to a liquid-condensed (LC) phase occurs with a most visible plateau on the DiC16-3′-dT′s isotherm: this transition corresponds to the formation of domains (Figure 3b). These domains are not fluid but crystalline because they do not have a circular or curved shape but sharp edges. After the transition, these domains fold compactly and form quasihexagonal packing with increasing compression (Figure 3c). The DiC16-3′-dT and DiC16-3′-dA isotherm curves on pure water are superimposed for large molecular area values (>50 Å2), and then they become distinct when the surface pressure reaches a value of close to 18 mN/m. At a surface pressure greater that 18 mN/m, as we see from the images (Figures 3c 6818


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Figure 3. Representative ellipsometric images for a film (formed on pure water) of the DiC16-3′-dT nucleolipid under compression. Each image size is 670 μm × 450 μm with a lateral resolution of 2 μm.

Figure 4. δΔ ellipsometric variation versus surface pressure for (a) DiC16-3′-dA and (b) DiC16-3′-dT nucleolipids with three different subphases: H2O (), Tris (20 mM)−NaCl (150 mM)−CaCl2 (15 mM) (···), and Tris (20 mM)−NaCl (150 mM) (•••). The values plotted on the graph correspond to measurements in the dark areas (bilayer domains) of the images.

and 4a,b, images 2 and 3), the surface is covered with the coexistence of two phases (dark and bright domains). The proportion of each phase depends on the surface pressure. At high surface pressure, the value of the molecular area (A0) corresponds to an average value. We observe that this value is significantly lower (27 or 21 Å2 for the dT or dA compounds, respectively) than the molecular area expected for an amphiphilic molecule with two hydrophobic chains (around 36 Å2 for phospholipids as DPPC). This observation suggests the existence of multilayer domains because the density of molecules is greater per unit area. The difference between the two compounds is related to the distinct proportion between monolayer and multilayer areas and the difference in the chemical nature of the polar headgroup (adenine versus thymine). At the same pressure, the comparison of the ellipsometric δΔ (Figure 4a,b) inside the domains (dark areas) shows a 2-fold increase in this parameter (6−13° for DiC16-3′-dA and 7−14° for DiC16-3′-dT). The values plotted on the graph (Figure 4) correspond to measurement in the dark areas (inside the domains) of the images presented in Figure 4. The rotation of the polarizer allows for the measurement of the δΔ value outside the domains (bright

areas).The corresponding values are not transferred to the graph because it was a simple value control to confirm the existence of the monolayer in these areas. Nucleolipid Films on a Tris Buffer. The two nucleolipids DiC16-3′-dA and DiC16-3′-dT show the same behavior. The increase in the surface pressure begins over a larger molecular area (140 Å2) than on the water subphase (100 Å2) because of the strong repulsive ionic interactions between the negatively charged phosphate groups at the interface. Compared to conventional phospholipids, this area is much larger (for example, the DMPC surface pressure starts to increase over a 100 Å2 molecular area), suggesting that the polar head was spread out and parallel to the interface. The film remains stable and homogeneous until the collapse at high pressure (60 mN/ m) (Figure 2). The surface-pressure isotherms show an LE phase at low pressure and a transition at 35 mN/m to an LC phase. However, this phase transition is not associated with the formation of domains on the ellipsometric images. The images are homogeneous at low and high surface pressure as shown in Figure 4a,b (images 1 and 4), and the δΔ measurement shows, over a large surface pressure range, a continuous variation of this parameter from 5 to 8° (DiC16-3′-dA and DiC16-3′-dT) 6819


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(Figure 4a,b). It is indeed in this case an average value of the illuminated area between the two domains formed by deoriented chains at low pressure (LE) and well-oriented chains at high pressure (LC). Unlike the example of DPPC for which the domains are large enough to be observed, we are limited here by the instrumental resolution indicating that the two domains are smaller than 1 to 2 μm. At surface pressure equal to 40 mN/m, the surface is covered with a majority of well-oriented chains (LC phase) in a 2D monolayer system. The molecular area is close to 40 Å2 as expected for an amphiphilic molecule with two hydrophobic chains. Nucleolipid Films on a Tris−Calcium Buffer. For both nucleolipids, the pressure−area isotherms show a phase transition with a plateau at low surface pressure (around 15 mN/m). The increase in the surface pressure begins over a molecular area similar to that on the Tris buffer (140 Å2/mol). However, like on water, the Langmuir monolayer remains stable and homogeneous and the parameters present an abrupt transition at around 15 mN/m. At this pressure, a large number of domains, with an increased gray level, appear with the same morphology as on water. The domains show a 2-fold increase in δΔ (7−14° for DiC16-3′-dA and 6−13° for DiC16-3′-dT). As described for the water system, the surface is covered with the coexistence of two phases (dark and bright domains). The proportion of each phase depends on the surface pressure. At high surface pressure, the value of the molecular area (A0) corresponds to an average value in a 3D system and cannot be compared to expected values for 2D monolayer systems. For very high surface pressures, one should be aware, however, of a possibly non-negligible systematic error due to area increments resulting from technical peculiarities of the experimental setup used.48,49 This particularly concerns our case where a Wilhelmy plate made of filter paper is used in order to measure the surface pressure. This is even more possible when considering our systems, not with fluid but with crystalline domains. The limited molecular areas obtained for these rigid 3D systems must be used carefully. PM-IRRAS Experiments. The PM-IRRAS spectra of DiC16-3′-dT and DiC16-3′-dA at the air−water interface were recorded. To ease the IR band assignment (Table 1), the ATR spectra of the dry nucleolipids were also recorded (Figure 5). The orientation of the dipolar transition moment of the principal base vibrations are reported in the same figure using the assignments reported in the literature.50 The PM-IRRAS spectra corresponding to the CH2 transition moments from the alkyl chains are similar for DIC16-3′-dA and DiC16-3′-dT on water and on the tris−calcium buffer (Figure 6). The strong positive νas CH2 and νs CH2 bands with maxima at 2918 and 2850 cm−1 indicate that the alkyl chains are mainly in the trans conformation. When the film is compressed from 10 to 50 mN/m, the band widths decrease slightly and their absolute intensities increase. The PM-IRRAS signal (SPM‑IRRAS) depends on the orientation of the transition moment of the vibration with respect to the surface plane and on the concentration on the surface. In previous papers, we demonstrated that the PM-IRRAS signal is proportional to the surface concentration of the molecule and to the orientation function of each vibration (expression 1).51,52 SPM ‐ IRRAS ∝

⎛N⎞ ⎜ ⎟ × f θ ⎝A⎠

Table 1. Spectral Assignments Reported for the Polarized ATR Spectra of DiC16-3′-dT and DiC16-3′-dA in the 1800− 900 cm−1 Region position

assignment

970−950 1010−1000 1070−1060 1015−1090 1175−1165 1240−1220 1275 1475−1467 1510−1500 1576−1570 1605−1600 1611 1642−1638 1655−1649 1670−1660 1695−1685 1745−1730 2852−2848 2925−2915 2956−2952

δ(C−C−H) δ(O4′−C1′−H) δ(O4′−C1′−H) νs(PO2−) ? νas(PO2−) ν(C1′−N1) δ(CH2) ν(CN) + δ(NH2) ν(CN) ν(CN) + δ(NH2) ν(CN) ν(CC) ν(CN) + δ(NH2) ν(CO) ν(CO) ν(CO) νs(CH2) νas(CH2) νas(CH3)

sugar sugar sugar phosphate phosphate base A lipid base A base A base A base A in D2O base T base A carbonyl base T carbonyl base T lipid lipid lipid lipid

orientation function of the vibration (with θ being the average angle of the transition moment with respect to the normal at the interface); fθ can be positive, zero, or negative depending on the orientation of the transition moment. From this expression, it follows that the multiplication of the PM-IRRAS intensity by

Figure 5. ATR spectra of (a) DiC16-3′-dA and (b) DiC16-3′-dT in powder. The solid line corresponds to the experimental spectra. The inset presents the direction of the main transition moment with respect to the nuclear base.

(1)

N is the number of molecules at the interface, A is the area of the trough when the PM-IRRAS spectrum is recorded, fθ is the 6820


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chains. The doubling of the δΔ parameter on the domains can be associated only with the formation of a bilayer because the formation of a trilayer on the domains requires much more tilted chains to obtain a thickness that increases only 2-fold with respect to the monolayer thickness. The same behavior has been obtained for diC16-3′-dA with the same relation between the organization and buffer solution (data not presented). One question remains: what is the organization of the bilayer domains? Three hypotheses can be proposed (Scheme 1). Scheme 1

Figure 6. PM-IRRAS spectra for CH2 domain normalized by the area for DiC16-3′-dT: (a) a pure water subphase, (b) a Tris−NaCl−CaCl2 subphase, and (c) a Tris−NaCl subphase.

In hypothesis a, the molecules adopt an organization similar to the organization of lipids in membrane models, but the headgroup of the external layer is in contact with air. Such organization seems difficult to stabilize because until now such organization has been observed only on DMPC, at high surface pressure, and in a very small domain of temperature. Hypothesis b has never been observed and requires the interaction of the external headgroup with a methyl group of the internal layer, which is not very favorable in terms of energy. Hypothesis c has never been observed without the use of divalent cations and requires the interaction of a methyl group with the water surface and a gain in energy by the mutual interaction between the headgroups of the two layers. This type of interaction should be considered with the nucleolipids here because their headgroups contain donor/acceptor moieties that are able to form hydrogen bonds and because the water surface is a dynamic interface where the water molecules may adapt their orientation to produce a quasi “hydrophobic” interface; this is what happens when oil or a fluorinated fluid spreads on water.53,54 To choose among the three hypotheses, we have analyzed the low-frequency part of the PM-IRRAS spectra where we can find the main vibrations of the polar headgroup (Figures 7 and 8). On water (Figure 7a), diC16-3′-dT presents, at low and high surface pressure, one broad absorption near 1720 cm−1 resulting from the superposition of the ester carbonyl vibration and the carbonyl vibration of thymidine. The relative intensity of the two bands changes only a little on the phase transition. More significant modifications appear at the phosphate vibrations. The relative intensity change indicates the reorientation of this group, and the large shift at high frequency of the antisymetric phosphate vibration (1221 to 1255 cm−1) is clear evidence of phosphate dehydration with the phase transition.55 In the case of diC16-3′-dT on the Tris−calcium buffer (Figure 7b), a band at low surface pressure appears near 1620 cm−1 and should be assigned to the Tris molecule. This

the area gives access to one variable that is sensitive only to the orientation function if the number of molecules is constant all along the compression (expression 2). SPM‐IRRAS × A = N × fθ

(2)

Therefore, the comparison of the PM-IRRAS spectra normalized with respect to the area gives direct information on the reorientation of the molecular group during the compression. Taking into account this property, we present in the CH2 domain (Figure 6) the PM-IRRAS signal multiplied by the area for diC16-3′-dT on the three buffer solutions. The spectrum is shown at low pressure and high pressure for water and the Tris−calcium buffer to compare the chain orientation before and after the transition. Figure 6a,b shows that the phase transition observed for water and the Tris−calcium buffer cannot be associated with a large reorientation of the carbon chains because the PM-IRRAS CH2 intensities are almost the same after the area correction. We observe simply a sharpening of the bands at high pressure correlated with a shift of the CH2 maxima at lower frequency. This observation is coherent with a better organization of the carbon chains under increasing surface pressure. In contrast to this, in the case of the Tris buffer we observe a continuous change in the orientation function of the carbon chains. The clear increase in the corrected PM-IRRAS signal with surface pressure suggests a continuous reorientation of the CH2 vibrations in the interface plane due to the reorientation of the carbon chains that are more perpendicular to the interface. The PM-IRRAS data suggest that the continuous increase in the δΔ ellipsometer angle for diC16-3′-dT on the Tris buffer is simply due to the progressive reorientation of the carbon chains at the interface with a continuous increase in the monolayer thickness. On water and on Tris−calcium buffer, diC16-3′-dT does not show any significant reorientation of the carbon 6821


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Figure 7. PM-IRRAS spectra, normalized by the trough area, for the polar head domain (1000−1900 cm−1) for DiC16-3′-dT: (a) pure water subphase, (b) Tris−NaCl−CaCl2 subphase, and (c) Tris−NaCl subphase.

Figure 8. PM-IRRAS spectra, normalized by the trough area, for the polar head domain (1000−1900 cm−1) for DiC16-3′-dA: (a) pure water subphase, (b) Tris−NaCl−CaCl2 subphase, and (c) Tris−NaCl subphase.

band disappears at high surface pressure, and the band centered at 1710 cm−1 is mainly characteristic of the thymidine carbonyl bond. As in the previous water experiment, the phosphate groups show reorientation and dehydration behavior during the phase transition. Finally, on the Tris buffer (Figure 7c), the PM-IRRAS spectrum indicates that the thymidine group shows a large reorientation with the surface pressure because the carbonyl of this group is intense at low surface pressure and disappears almost completely at high surface pressure. From the PM-IRRAS selection rule, this suggests a thymidine carbonyl reorientation from the interface plane to the magic angle of 38° with respect to the normal. Some reorientation of the

phosphate group is also observed, but the dehydration of this group is less pronounced that in the previous case. In the case of diC16-3′-dA (Figure 8), the PM-IRRAS spectra show that the adenine group can adopt two forms depending of the buffer of the subphase. Indeed, at low surface pressure on the Tris buffer (Figure 8c), adenine is characterized by a vibration near 1650 cm−1,. which is typical of the neutral molecule, but on pure water (Figure 8a) and Tris−calcium buffer (Figure 8b), the spectra present one absorption at 1695 cm−1 that has been assigned to the protonated form of the adenine group.56 During the transition, the 1695 cm−1 band decreases in intensity on the water and Tris−calcium buffer, which seems to indicate a reorientation of the protonated 6822


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allows us to exclude the possibility of protonation of the phosphate group, which would make the molecule neutral and thus alter the balance between the electrostatic interactions (repulsive or attractive) and weak interactions such as hydrogen bonding and π stacking. Considering this pH/pKa argument, we can consider that the lipid is always negatively charged on the anionic phosphate group (PO2−). The hypothesis that we support is to consider the various counterions involved in the system with the different subphases. In the water subphase, the only counterion is triethylammonium [(C2H5)3NH+]. It makes a directed and localized hydrogen bond with the phosphate group and therefore promotes the formation of a quasi “zwitterionic” object and globally decreases the repulsive electrostatic interactions between the polar heads. At low surface pressure, the repulsive electrostatic interactions are weak as evidenced by the relatively small value of the molecular area. At high surface pressure, hydrogen bonds and π stacking between polar heads are attractive interactions sufficient to form the bilayer. In this case, the fine internal structure of the bilayer corresponds strictly to hypothesis c from Scheme 1. On the Tris−NaCl subphase, the sodium ions are added in a very high concentration ([NaCl] = 150 mM), much higher than the triethylammonium concentration that is present in solution. The highly solvated sodium ions will easily exchange with the triethylammonium ions and will gently associate with the phosphate group. The result is a strong negatively charged interface where repulsive electrostatic interactions dominate. It appears impossible to promote the formation of the bilayer because it is very difficult to form multilayers with anionic phospholipids such as dipalmitoylphosphatidylglycerol (DPPG) at the interface. After the addition of calcium (CaCl2) and at low surface pressure, the repulsive electrostatic interactions still dominate and the molecular area is very similar to the one on the Tris− NaCl subphase. For a sufficient surface pressure, the divalent cation will be inserted between the two phosphate groups to promote a strong attractive electrostatic interaction bridging the polar heads and will induce the formation of the inverted bilayer. This enables the formation of ionic interactions with a divalent cation, hydrogen bonding between nucleobases, and π stacking. In this case, the fine structure of the bilayer does not correspond to hypothesis c of Scheme 1 because Ca2+ is located between the polar heads. X-ray reflectivity experiments are expected to reconstruct the fine structure of the bilayer more accurately by taking into account various density profiles.

adenine in the multilayer domains. Moreover, with both buffers the ester carbonyl bond adopts almost the same orientation during the phase transition. The phosphate group still presents some reorientation but only a small dehydration effect. In contrast, with the Tris subphase, the adenine and the ester carbonyl groups show a large reorientation upon compression, such as for the thymidine analogue under the same conditions. We have observed this behavior previously with other nucleolipid monolayers; in correlation with the phase transition observed near 35 mN/m on the isotherm, this indicates that the nucleic base changes from a flat organization at the interface to a more tilted orientation. This large adenine and ester reorientation leads to a reorientation of the phosphate group with a slight dehydration of this group. Regarding the stability of the monolayer and bilayer domains, we can observe that the PM-IRRAS and ellipsometry measurements are performed over several hours (3 and 1.5 h, respectively). Over these periods, the monolayers and bilayers are stable. At constant surface pressure during the acquisition of spectra, no change in pressure was observed.

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DISCUSSION AND CONCLUSIONS From the ellipsometry data and the PM-IRRAS spectra in the CH domains, we demonstrated that nucleolipids diC16-3′-dA and diC16-3′-dT form bilayers at relatively low surface pressure on pure water and the Tris−calcium buffer and adopt a simple monolayer organization on the Tris buffer. The organization of the bilayer is not simple to determine. In the case of diC16-3′dT, the organization with layers joined by their apolar sides (a in Scheme 1) is not probable because such organization has been observed only at high surface pressure and we do not expect PM-IRRAS spectra to be significantly different between the monolayer and the bilayer even if the polar headgroup of the second layer was in the air. Above all, this organization cannot explain the strong dehydration of the phosphate group. The organization with the polar sides of both layers downward (b in Scheme 1) seems less probable and is also not in agreement with the complete dehydration of the phosphate. The organization with layers joined by their polar sides (c in Scheme 1) seems more in agreement with our observations because the formation of a hydrogen-bonded interface can stabilize the bilayer and generate the dehydration of the phosphate groups. The case of adenine is more complex because even if we observed the same behavior as with diC163′-dT with the surface pressure, ellipsometry measurements, and analysis of the CH orientation function we do not detect any large modification of the phosphate hydration during the phase transition. We can simply postulate that the protonated form of adenine interacts with the phosphate group in the bilayer, which maintains the antisymetric PO2 vibration in the same position as in a hydrated phosphate. The latter organization (inverted bilayer) is thus the most probable for the diC16-3′-dT and diC16-3′-dA bilayers stabilized on water and Tris−calcium buffer. The formation of this inverted bilayer shows a particularly unusual behavior for an amphiphilic molecule. The formation of the bilayer is of course strongly related to the polar head part of these original molecules, but the fact that the bilayer occurs on water and Tris−NaCl−CaCl2 subphases whereas the bilayer is not formed in the presence of a Tris NaCl subphase is surprising. The PM-IRRAS experiments (analysis of frequency and shape of the peaks) show unambiguously that the phosphate group is not protonated in all cases. This observation

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ASSOCIATED CONTENT

S Supporting Information *

NMR spectra (1H, 13C, and 31P) and mass spectrometry data for the DiC16-3′-dA compound. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +33 556 84 56 61. Fax: +33 556 84 56 00. Notes

The authors declare no competing financial interest. 6823


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ACKNOWLEDGMENTS We acknowledge financial support from the Conseil Regional d′Aquitaine and CNRS within grant AP-07- 20071102027.

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Unexpected Bilayer Formation in Langmuir Films of Nucleolipids

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