Chemical Details on Nucleolipid Supramolecular Architecture

Apr 6, 2012 - Aditya G. Kohli , Paul H. Kierstead , Vincent J. Venditto , Colin L. Walsh , Francis C. Szoka. Journal of Controlled Release 2014 190, 2...
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Chemical Details on Nucleolipid Supramolecular Architecture: Molecular Modeling and Physicochemical Studies Nada Taib,†,‡ Ahissan Aimé,†,‡ Said Houmadi,§ Sabine Castano,§ Philippe Barthélémy,*,†,‡ Michel Laguerre,§ and Isabelle Bestel*,†,‡ †

Université Bordeaux Segalen, Bordeaux, F-33076, France INSERM U869, Bordeaux, F-33076, France § Université de Bordeaux, IECB - CBMN UMR 5248 CNRS, 2 rue Robert Escarpit, F-33607 Pessac Cedex, France ‡

S Supporting Information *

ABSTRACT: Nucleolipids are currently under investigation as vectors for oligonucleotides (ON) delivery thanks to their supramolecular organization properties and their ability to develop specific interactions (i.e., stacking and potential Watson and Crick hydrogen bonds) for lipoplexes formation. To investigate the factors that govern the interaction events at a molecular level and optimize nucleolipid chemical structures, physicochemical experiments (tensiometry, AFM, BAM, and ellipsometry) combined with molecular dynamics simulation were performed on a series of zwitterionic nucleolipids (PUPC, DPUPC, PAPC) featuring a phosphocholine chain (PC). After construction and initial equilibration, simulations of pure nucleolipid bilayers were run for 100 ns at constant temperature and pressure, and their properties were compared to experimental data and to natural dipalmitoylphosphatidylcholine (DPPC) bilayers. Nucleolipid-based membranes are significantly more ordered and compact than DPPC bilayers mainly due to the presence of many intermolecular interactions between nucleoside polar heads. The hydrophilic phosphocholine moieties connected to the 5′ hydroxyls are located above the bilayers, penalizing nucleic bases accessibility for further interactions with ON. Hence, a neutral nucleolipid (PUOH) without hydrophilic phosphocholine was inserted in the membranes. Simulations and experimental analysis of nucleolipid membranes in interaction with a single strand RNA structure indicate that PUOH interacts with ON in the subphase. This study demonstrates that molecular modeling can be used to determine the interactions between oligonucleotide and nucleolipids.

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INTRODUCTION A body of evidence demonstrates that oligonucleotides (ON) including antisense and RNA interference (RNAi) hold great promise as drugs to complete the therapeutic arsenal against miscellaneous severe pathologies.1−6 However, overcoming critical issues concerning, for instance, the poor stability and the limited cell-delivery of such ON-based therapeutics is a prerequisite to large-scale clinical applications. In the past decade, many efforts have been devoted to the implementation of ON effective delivery systems based on viral7,8 or on more safe synthetic vectors.9−13 However, despite important progress, several impediments still have to be circumvent, spurring the continuing interest in the development of delivery strategies. To date, most of the synthetic vectors overwhelmingly capitalize on either electrostatic interactions or electrostatic and hydrophobic interactions to assemble the negative ON to the © 2012 American Chemical Society

cationic vectors and to form supramolecular assemblies required for ON delivery. One caveat with this approach is the binding of these positively charged vectors to serum proteins, mostly negatively charged at physiological pH. To date, many cationic molecule-based systems have failed or been unimpressive in clinical trials mainly due to toxicity.14,15 In this context, new strategies of ON delivery based on reinforced interactions such as hydrogen bonds and π−π stacking to further modulate the interactions between ON and the synthetic vector are emerging. Among them, the development of hybrid molecules bearing both nucleic acid units (i.e., nucleoside) and amphiphilic moieties, also known as nucleolipids, is of major interest.16−20 The peculiar behavior Received: February 21, 2012 Revised: April 3, 2012 Published: April 6, 2012 7452

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solvated starting structure with no water molecules in the hydrophobic section. All simulations were carried out using the GROMACS 4.0.3 software package.33 In all simulations, a leapfrog integrator was used with a 1 fs time step. A cutoff of 1.4 nm was used for calculating the Lennard-Jones interactions. Long-range electrostatics are computed using the particle mesh Ewald (PME) method.34,35 The real space interactions were evaluated using a 1.2 nm cutoff, and the reciprocal space interactions were evaluated on a 0.13 nm grid with eighth-order spline interpolation. The simulations were performed with semiisotropic pressure coupling to 1 bar, which allowed the area per nucleolipid to fluctuate during the simulation. Each component of the system (i.e., nucleolipid and water) was coupled separately to a temperature bath at 310 K for PUPC, DPUPC, PUPOH and at 317 K for PAPC, that is, above the gel to liquid phase transition temperature for all nucleolipids, using the V-rescale thermostat with a coupling constant of 0.1 ps.36 All of the molecular dynamics simulations were carried out using periodic boundary conditions. The SPC water model was used in all simulations, and water geometry is constrained using the SETTLE algorithm.37 Center of mass motion is removed separately for each component to avoid block shifting of layers due to rounding error accumulation.38 Four simulations were carried out: (1) the PUPC bilayer (PUPC240) consisting of 240 nucleolipids, 12 401 water molecules, (2) the DPUPC (DPUPC240) bilayer consisting of 240 nucleolipids and 21 330 water molecules, (3) the PAPC bilayer (PAPC240) consisting of 240 nucleolipids, 22 797 water molecules, and (4) the PUOH bilayer (PUOH240) consisting of 240 nucleolipids and 17 061 water molecules. All of the starting structures were energy minimized, before the MD simulations, to avoid unfavorable contacts between molecules. All bilayers were simulated during 100 ns. Next, to investigate the potential reorganization of nucleolipid bilayers (i.e., nucleic base orientation) triggered by interactions with ON, we included an ON during the simulations. For that purpose, we manually inserted a nonmodified single-strand RNA Poly-A (consisting of a short 15 adenosine oligonucleotide to simplify the system) 10 Å over PUPC and PUOH nucleolipid bilayers. The Poly-A coordinates and topology were generated with PRODRG topology generation server using the GROMOS force field 96. The starting structure was energy minimized, before a series of molecular dynamic simulations. Two simulations were carried out: (1) the (PUPC240)/Poly-A system consisting of equilibrated PUPC bilayer, Poly-A oligonucleotide, and 12 401 water molecules; and (2) the (PUOH240)/Poly-A system consisting of equilibrated PUOH bilayer, Poly-A oligonucleotide, and 17 058 water molecules. Langmuir−Blodgett Deposition, Isotherms. The Langmuir film experiments were performed on a 300 cm2 rectangular Langmuir trough made from Teflon (Nima Technology, Coventry, UK) using ultrapure water (doubly distillated with a resistance of 18.2 mΩ). PAPC was dissolved in HPLC-grade chloroform to get a concentration of 0.67 mg/mL. A volume of 30 μL of the solution of PAPC was spread onto water (21 ± 1 °C) using a calibrated Hamilton microsyringe; small droplets were deposited at different places distributed at the surface of water to get a uniform distribution on the subphase. Surface pressure was measured using a Wilhelmy plate attached to a sensitive balance with an accuracy of ±0.1 mN/m. Compression of the monolayer was started after 15 min relaxation by moving the barrier with a constant speed of 5 cm2/min. The monolayer was compressed to the deposition pressure of 25 mN/m and controlled by a feedback control loop to maintain a monolayer pressure constant. The films were then transferred at constant pressure onto freshly cleaved mica substrates by the vertical lifting method at a rate of 10 mm/min. The mica substrates were dipped vertically from the water to the air for the initial monolayer transfer, and then from the air into the water for the second monolayer transfer according to the procedure reported by Osborn et al.39 Tensiometry Experiments. Monolayer experiments were performed on a circular glass trough (Ø 6.5 cm) using 50 mL of ultrapure water (doubly distillated with a resistance of 18.2 mΩ). PAPC or

of these amphiphiles in self-assemblies relies on the diversity of functional groups capable of cooperative noncovalent interactions combined with specific base−base recognition. These molecules offer several advantages as compared to other commonly used synthetic vectors, including efficiency, stability, and absence of toxicity.21,22 As a consequence, scientists borrow nowadays nucleolipid features to build artificial molecular devices and develop novel therapeutic strategies for ON delivery.21−26 Conceptually, nucleolipids offer a versatile chemical platform allowing a fine-tuning of numerous parameters, including (i) base−base interactions (nucleoside moiety of the nucleolipids can stabilize self-assemblies), (ii) nature of the hydrophobic parts, and (iii) supramolecular structures of the ON− nucleolipid complexes (nucleolipoplexes, fibers, etc.), transfecting format27 (colloidal phases, hydrogel, etc). It is clear that ON can take advantage of nucleolipid molecules to be delivered in cells. Nevertheless, according to their chemical structure, interactions with ON may differ. In this context, combining molecular modeling and experiments presents a tremendous advantage: (i) to dissect at an atomic level ON−nucleolipid interactions and (ii) to reinforced these interactions by proposing nucleolipid chemical modification. Despite the documented importance of nucleolipids as vectors, far less is known about their atomic level structure in bilayers than is known for natural phospholipids.28,29 In this Article, we describe an investigation of ON− nucleolipid interactions in the 2′-3′ dipalmityl (C16)-based nucleolipid vector family.30,31 For that purpose, nucleolipids of different chemical structures were assembled in bilayers, leading to the creation of first models of nucleolipid-based membranes. Aiming at inferring how the chemical structure can influence the organization within bilayers, the orientation and accessibility of nucleic functionalities were investigated in details. Next, interactions between ON and built nucleolipid bilayers were simulated. In parallel, molecular modeling results were validated by several Langmuir film-based experiments including tensiometry, AFM, Brewster angle microscopy (BAM), and ellipsometry.

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MATERIALS AND METHODS

Molecular Dynamics Simulations. The single PUPC, DPUPC, PAPC, and PUOH coordinates and topologies were generated using the PRODRG topology generation server with the GROMOS force field 96 (43a1).32 The initial structure of each bilayer (PUPC, DPUPC, PAPC, and PUOH) was obtained by setting a 12 × 10 array of nucleolipid molecules in the x, y plane with random rotation around the z axis and random translations along the z axis with a maximum of 0.5 nm, which constituted the first layer. To generate the second layer, the starting layer is copied and then flipped by rotating it 180° around either the x or the y axis. The two leaflets are translated to provide approximately the correct bilayer thickness and then centered in a new box of appropriate size. The x and y dimensions of this box can be exactly calculated from the lattice spacing used during generation of the first leaflet. Next, bilayer and box size are compressed by a series of scaling steps alternating with energy minimization. The x and y coordinates of the atoms, and the x and y box dimensions, are scaled using a scaling factor less than 1 (0.95 is appropriate), which laterally compresses the entire system. Once the bilayer was minimized, a water slab was added to solvate the headgroup region of the nucleolipid bilayer. As the partitioning of water into a bilayer is unfavorable enough to see no water inside the hydrophobic part of a bilayer, the goal is therefore a sufficiently 7453

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PUPC or DPUPC or a mixture of PUPC/PUOH (25/75) was dissolved in HPLC-grade chloroform (400 μM) at a temperature of 22 ± 2 °C. The solutions of lipid were gradually spread onto water (22 ± 2 °C) at the air−water interface using a calibrated Hamilton microsyringe; small droplets were deposited at different places distributed at the surface of water to get a uniform distribution on the subphase, until the surface pressure of 45 mN/m was reached (nucleolipid monolayer collapse pressure). The surface pressure (π) was measured using a Wilhelmy plate attached to a sensitive balance with an accuracy of ±0.1 mN/m. Polar head areas were calculated from the relation:

that in contrast with diacyl nucleoside amphiphiles, the ketal functionality locks this bicyclic ribonucleoside in the southern conformation (C′2 endo), restricting the conformations to probably favor recognition. On the basis of these results, we started our approach by focusing on ketal derived nucleolipids. To compare the gain brought by ketal versus diacyl functions in term of recognition properties, a diacyl analogue was also considered. Three amphiphiles were then selected differing in the structure of the base (U or A) and in the structure of the lipid attachment (constrained ketal group or diacyl groups) on nucleoside (Figure 1) (PUPC, dipalmityl uridine phosphocho-

trough area nnucleolipid × Na nnucleolipid = mol number of nucleolipid Na = Avogadro number AFM Experiments. The film was transferred from the water subphase to mica substrates. The different areas of the sample were imaged by AFM operating in the tapping mode using oxidized silicon cantilevers. The AFM experiment was performed in air at atmospheric pressure using an MFP-3D atomic force microscope from Asylum Research. Brewster Angle Microscopy and Ellipsometry Experiments. Nucleolipid monolayers were formed at the surface of a small homemade of Teflon circular Langmuir trough (20 cm2, 8 cm3). The surface pressure (π) was measured by the Wilhelmy method using a filter paper plate. The trough was filled with buffer (Tris 20 mM, NaCl 150 mM, pH 7.4), and experiments were carried out at 22 °C. Pure PUPC or mixed PUPC, PUOH (75/25) films were obtained by spreading a few microliters of nucleolipid in HPLC-grade chloroform (400 μM) at the air/water interface. For RNA interaction experiments, Poly-A or Poly-U dispersion (100 mers, 1 mg/mL) in ultrapure water (200 μL) was injected underneath the lipid monolayer, which represents a stoichiometry of approximately 1 nucleolipid for 30 poly-U or poly-A bases. The morphology of PUPC or mixed PUOH/PUPC layers before and after RNA interaction at the air/water interface was observed using a Brewster angle microscope (NFT BAM2plus, Göttingen, Germany). The microscope was equipped with a frequency doubled Nd:Yag laser (532 nm, 50 mW), a polarizer, an analyzer, and a CCD camera. The exposure time (ET), depending on the image luminosity, was adjusted to avoid saturation of the camera. The spatial resolution of the Brewster angle microscope was about 2 μm, and the image size was 600 × 450 μm with a 10× magnification lens. Thickness estimations were obtained by ellipsometric measurements using the same setup as that for BAM with an imaging ellipsometer at an incidence angle of 54.6°. It operates on the principle of classical null ellipsometry.40 The angles of the polarizer, compensator, and analyzer that obtained the null condition allow one to get the (Δ, Ψ) angles that are related to the optical properties of the sample. In ultrathin film conditions, Δ is proportional to the film thickness. Comparison of the measured data with computerized optical modeling included in the BAM software leads to a deduction of film thickness when an estimation of the refractive index can be obtained.

Figure 1. Molecular structure of studied nucleolipids.

line; PAPC, dipalmityl adenosine phosphocholine; DPUPC, dipalmitoyl uridine phosphocholine). They possess zwitterionic phosphocholine (PC) polar side chains in the 5′ position and palmityl chains in the 2′−3′ position of the nucleic ribose. The main aim was to investigate the influence of nucleic heads (pyrimidine or purine) and of sugar conformational restrictions on the molecular organization of bilayers that can further favor specific interactions with ON to be delivered. Note that the phase transition temperature of the selected nucleolipids was previously determined by modulated differential scanning calorimetry.41 Data revealed that the nucleoside moiety does not seem to affect the Tm values, PUPC (14.1 °C) being similar to PAPC (14.5 °C), whereas the sugar conformational restriction of ketal derivatives increases the Tm largely to 41.8 °C.

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RESULTS AND DISCUSSION 1. Optimization of Nucleolipids. Recently, we investigated the formation of self-assemblies resulting from the interactions of either complementary ketal-based adenosine and uridine nucleolipids or their diacyl analogues. We showed that the dynamics of the intersystem recognition and supramolecular assembly formation was dependent on the nucleobase complementary recognition and the hydrophobic chains, the ketal derivatives only allowing a potential base−base pair recognition.30,31 A conformational analysis of the ketal and diacyl nucleolipids using Monte Carlo simulations suggested 7454

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Significantly, they all were shown to assemble into lamellar phases above their Tm. Thanks to molecular modeling results (described in the next sections), a new nucleolipid vector was also proposed (PUOH: dipalmityl uridine hydroxyl), resulting from the variation of a supplementary parameter: the structural composition of the 5′ side chain (phosphocholine chains or hydroxyl). For experimental validation, these molecules were synthesized as previously described.30,41 2. Equilibration and Structural Properties of the Nucleolipid Membranes. We have carried out molecular dynamics simulations of fully hydrated nucleolipid bilayers. After construction and initial equilibration, simulations were run for 100 ns at a constant temperature of 310K for PUPC, PAPC, and PUOH or 317 K for DPUPC and at a constant pressure of 1 atm. As the main phase transition temperature is 287 K for ketal-based nucleolipids and 315 K for DPUPC, bilayers should be in a biologically relevant fluid phase (Lα). To validate the quality of our simulations and to characterize the effect of the nucleolipid chemical structure, we describe in detail key macroscopic properties of the systems. Nucleolipid membranes clearly equilibrate during the simulation based on both the energy of the system and the convergence of lipid membrane structural properties. In general, these and other properties reached equilibration during the first half of the trajectory, and average properties calculated over the last 50 ns and the last 2 ns were essentially identical. Throughout the rest of this Article, all nucleolipid properties reported are averaged over the final 2 ns of each trajectory. Phosphocholine Nucleolipids (PUPC, PAPC, DPUPC) versus Natural Phospholipid DPPC. Area per Lipid. The area per lipid was computed as the lateral area of the simulation box divided by the number of lipids per leaflet. Although area per lipid was determined from bilayers of nucleolipid in simulations, the calculated values (Figure 2b) can be directly compared to those obtained from the area of lipid systems (monolayers) measured by tensiometry experiments (Figure 2a). Figure 2 and Table 1 illustrate the good agreement of the lipidic area between simulation data and tensiometry for PAPC, PUPC, and DPUPC bilayers. Experimental surfaces area/lipid were determinate at a pressure of 25 mN/m, which is close to that of biological lipid bilayer. Surprisingly, the presence of the nucleobase on the amphiphile structure leads to a decrease of the polar head area (41.5 Å2 for PUPC, 44.6 Å2 for DPUPC, and 46.3 Å2 for PAPC (Table 1)) as compared to homologue conventional lipids such as DPPC (64 Å2)42,43 even though the incorporation of the nucleobase in the amphiphile increases the effective size of the headgroup. In details, PAPC nucleolipids that possess a purine nucleobase display the largest area/lipid as compared to PUPC: the surface is there a direct consequence of the nucleobase size. This result differs from that of Berti et al., who observed a smaller area/lipid for purine bases argued by a dominant stacking event.44 This difference can result from nucleolipid structure, which largely differs from that studied by Berti et al.44 For a similar uridine nucleobase, side chain (phosphocholine), and lipidic part (dipalmityl tail), the chemistry of the lipidic attachment impacts obviously on the area per lipid: DPUPC that displays a diester linker has an area/ lipid of 46.3 Å2, whereas PUPC that possesses a constrained structure displays only 43.5 Å2. The relatively low area per molecule above its phase transition temperature as compared to phospholipids indicates that the fluid phase of nucleolipids is dramatically more ordered. This result could be related to the

Figure 2. (a) Surface pressure−molecular area (π−A) isotherms of PAPC (purple), DPUPC (green), PUPC (blue), and PUOH/PUPC (75/25) (red) nucleolipid membranes. Values are the means of a least three independent experiments. (b) Plot of area per lipid during the course of molecular dynamics simulations. Areas for PAPC, DPUPC, PUPC, and PUOH are shown in purple, green, blue, and red, respectively.

Table 1. Surface Area/Lipid from Molecular Modeling and Tensiometry Experiments,a and Thickness from Molecular Modeling surf. area/lipid (Å2)b exp. surf. area/ lipid (Å2)c,d thickness (A) d(P−P)

PUPC

DPUPC

PAPC

PUOH

43.7 ± 0.1

46.0 ± 0.7

48.4 ± 0.9

40.7 ± 0.9

41.5 ± 0.9

44.6 ± 0.7

46.3 ± 1.1

38.2 ± 1

45.8 ± 0.2

48.5 ± 0.4

48.9 ± 0.6

44.39 ± 0.9e

a

Values are the means and standard deviations of at least three independent experiments. bFrom bilayers of nucleolipids. cFrom monolayers of nucleolipids. dExperiments done at a temperature of 22 °C. ed(O−O).

presence of interactions that may occur between the nucleoside part of nucleolipids. To get more insight into the likely arrangement, the interaction partners of nucleic bases within membranes were calculated by the radial distribution function (RDF) between bases and between bases and phosphorus (P) or nitrogen (N) of PC chains within 4.5 Å. Under this distance, the partner proximity is reminiscent of potential interactions such as hydrogen bonds or stacking events. Results of the cumulative 7455

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RDF numbers (i.e., average number of atoms within a distance of 4.5 Å) are presented in Table 2. Globally, each base is in the Table 2. Intramembrane Interactions: RDF and HydrogenBond Calculations intramembrane interactions RDF < 4.5 Å

hydrogen bonds

base−base base−P base−N base−OH

PUPC 2 1 1 3 1.2

DPUPC

PAPC

3 2 0

2 1 2

1.9

0.7

PUOH

1 3.4

neighboring of two (PUPC, PAPC) or three (DPUPC) other bases within a distance of 4.5 Å. Moreover, a proximity is also observed between bases and P or N of the PC chain. Altogether these results confirm that many interactions can occur between bases themselves and between bases and PC chains. The number of intramembrane hydrogen bonds (H-bonds) was also calculated (Table 2). By considering a general form of the hydrogen bond X−H···Y, the criteria of the H-bonds formation we adopted in the work are: the X−Y distance is less than 3 Å, and the H−X−Y angle is less than 20°. We used the H-bonds plugin implemented in Analysis module of VMD (visual molecular dynamic) version 1.8.7. As compared to conventional phospholipids (DPPC), the presence of H-bonds between nucleolipid polar heads can be emphasized. As a consequence, the driving force behind the nucleolipid specific surface is more the nucleobase polar head interactions than the dynamics of fatty chains as in conventional lipids such as DPPC. Another consequence of these strong lateral interactions and the low specific surface of lipids is the large increase of the angle between the PC chains (P···N vector) and the layer plane. The vector connecting P to N atoms of PC chains tended to point clearly into the water for nucleolipids (Figure 3a). Figure 3b illustrates the relative location of nucleic bases as compared to the P and N atoms in PUPC bilayer. Similar data were obtained for DPUPC and PAPC (data not shown). A measure of distances between center of mass of bases, P, and N in the upper and lower bilayer leaflet plotted along the simulation time reveals that all along the simulation, nucleic bases remained located under PC chains. To evaluate, at a molecular level, the positioning of PC chains, the tilt angle of PN vectors with respect to the plan of each bilayer was calculated. Although tilt angles are similar whatever the nucleolipid chemical structure (67 ± 1.7° for PAPC, 71.2 ± 2.2° for DPUPC, and 72.6 ± 4.6° for PUPC), they sharply differ from that obtained for more classical bilayers like DMPC or DPPC (10 ± 1°).45−48 These results again indicate that the compact structuration triggered by nucleoside heads has several consequences on nucleolipid global organization: an ordering of lipid tails and a main covering of nucleic bases by PC chains. The top view of simulated nucleolipid bilayers also confirmed the shielding by PC chains and emphasized the poor accessibility of nucleic bases, which are buried within the membrane (Figure 4a−c). Moreover, a precise analysis of polar heads organization within the membrane reveals that no preferred orientation of nucleic bases can be observed. Surprisingly, the ketal induced conformational restrictions do not favor a particular orientation as compared to the more flexible diacyl analogues.

Figure 3. (a) Representation of PN vector and tilt angle. (b) Plot of the distance along the membrane normal between the center of mass of phosphate atoms (green), nitrogen atoms (blue), and of uridine groups (magenta) for PUPC membrane in the two leaflets. (c) Plot of the distance along the membrane normal between the center of mass of hydroxyl oxygen atoms (violet) and of uridine groups (magenta) for PUOH membrane in the two leaflets. (d) AFM on Langmuir− Blodgett film of PAPC. The image size is 1 × 1 μm2.

Thickness. The thickness of nucleolipid bilayers was estimated as the average distance between center of mass of the P atoms in each leaflet (Pcom−Pcom). As compared to natural phospholipids such as DPPC for which thickness bilayer experimentally ranges between 36.4 and 39.6 Å,49 the width of nucleolipid bilayers obtained after 100 ns of simulation is strikingly larger: 45.8, 48.5, and 48.9 Å for PUPC, DPUPC, and PAPC, respectively (Table 1). The large thickness observed in molecular modeling is confirmed by AFM measurements on the depth of a defect on the Langmuir−Blodgett film of PAPC (Figure 3d, around 5.5 nm in a hole present on the bilayer) and by previous SAXS analyses in the fluid state (lamellar repeat period of 4.6 nm) of DPUPC.41 Once again, we can interpret 7456

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Figure 4. Snapshots of the top view of PUPC (a), DPUPC (b), PAPC (c), and PUOH (d) simulated nucleolipid bilayers. Bases available are shown in orange, and other lipid groups are shown in blue.

the data as evidence for numerous lateral interactions between nucleic polar functionalities within bilayers. These features trigger the rigidification of lipidic chains, increasing ordering within the membrane and impairing their tilting with respect to the bilayer plan as is observed in a more fluid bilayer (DPPC). The membrane thickness is increased as the lipid chain order increases. Water Accessibility. We observed that nucleic bases are not easily accessible, being in fact buried under PC moieties that are exposed on bilayer surface. To evaluate nucleic bases accessibility, we calculated RDF between pairs of atoms likely to participate in intermolecular H-bonding between nucleolipids (base, P, or N) and surrounding water (oxygen atom). Results (Table 3) clearly indicate the formation of hydratation shells around the ammonium part of PC chains

Figure 5. Number density profiles of the nucleolipid phosphorus (green), choline (blue), and base (magenta) groups along the membrane normal for PUPC (a), DPUPC (b), PAPC (c), and PUOH (d) bilayers.

is not surprising that most hydrogen bonds occurred with PAPC and in less extent DPUPC systems as compared to PUPC. In the latter case, the poor hydratation of bilayers can be directly correlated to the very small polar area/lipid reminiscent of a high compacting. Altogether, these data demonstrated that in our set of nucleolipids, PC chains clearly hamper the development of potential interactions with ON to vehiculate. Phosphocholine Nucleolipid PUPC versus Hydroxyl Nucleolipid PUOH. Thanks to molecular modeling simulation analyses, which indicate that PC moieties are an obstacle to ON interactions, a nucleolipid structure consisting of the replacement of the PC chain of PUPC by an hydroxyl group (PUOH) was inserted in bilayers (Figure 1). The area/lipid is slightly smaller for PUOH (40.7 Å2) than that obtained for the corresponding PUPC (43.7 Å2) (Figure 2b). Tensiometry experiments reveal that pure PUOH Langmuir film at 25 mN/ m cannot be formed certainly due to nonsufficient hydrophilic properties. This behavior can be related to that of nonionic thymidine nucleolipids developed by Mulet et al.,20 which displayed a monolayer collapse at a surface pressure of only 14 mN/m. BAM experiments on PUOH show the formation of spheric supramolecular assemblies at the air/water interface instead of a monolayer (Figure S1a). Nevertheless, PUOH area/lipid was estimated from a mixture of PUOH and PUPC nucleolipids (75/25). BAM images show a homogeneous (without any domains) and relatively viscous phase because very few lateral movements are observed at the interface (Figure S1b). For this calculation, we made the assumption that PUPC polar head in the mixture does not differ from pure film and find an area of 38.2 Å2 (Figure 2a). The RDF calculations

Table 3. Membrane/Water Interactions: RDF and Hydrogen-Bond Calculations memb./water interactions RDF < 4.5 Å

hydrogen bonds

base−water P−water N−water OH−water

PUPC

DPUPC

PAPC

PUOH 11

23 7.5

3 26

2 25

18.7

21.9

16 27

and confirm the very poor water accessibility of nucleic bases. To further examine and visualize the intimate structure of the bilayers, density number profiles of individual components (bases, P, N, and water) in each system were calculated for PUPC, DPUPC, and PAPC (Figure 5a−c). In PUPC bilayer, the density number profile of water stops at the average density number profile of P. It means that water passes through the choline level, with access to P, but does not reach nucleic bases. Comparatively, in DPUPC and to a more extent in PAPC bilayer, water can penetrate more deeply. Altogether, the density profiles confirm that water reaches easily the PC ammonium group but hardly the nucleic bases embedded within membranes. The number of hydrogen bonds between nucleolipid polar heads and surrounding water was also calculated (Table 3). According to density number profiles, it 7457

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between bases and between bases and OH confirm the presence of many partners able to interact within the membrane (Table 2). For instance, the OH side chain is pointing into water as did PUPC PC chain, and lipidic tails adopt an ordered organization. Thanks to the compact structure of the membrane, it is thus not surprising that the replacement of the bulky PC chain by a hydroxyl has only a slight impact on polar head areas (minoration of only 3 Å). On the contrary, sharp consequences on nucleic base accessibility are observed. RDF estimations between PUOH (nucleic base and OH) and water indicate the presence of hydratation shells around both components (Table 3). This is in agreement with the top view of PUOH bilayer (Figure 4d) as compared to PUPC (Figure 4a), with the relative location of nucleic bases and side chains plotted along the simulation time (Figure 3c) and with H-bond calculations between nucleolipids and water (Table 3). Further analyses based on density profiles again confirm the great accessibility of nucleic bases to water and to potential interactions with ON: as compared to PUPC in which water molecules hardly reach the uridine, in PUOH one-half of the atoms of the base are within water, and the OH group is freely accessible from outside (Figure 5d). 3. Interactions between Nucleolipid Membrane and ON. In an attempt to anticipate the potential development of specific interactions between nucleolipid bilayers and ON to be delivered, molecular dynamics simulations of these two component systems were carried out. Our aim is to examine at the molecular level a potential reorganization of nucleolipid membranes (i.e., orientation of nucleic bases) triggered by the presence of an interacting ON. Furthermore, these investigations will highlight the relevance of using a molecular modeling approach in this study. For that purpose, PUPC versus PUOH bilayers were used, and a short 15 mer single strand RNA Poly-A oligomer was constructed to both simplify the system and induce most favorable interactions. The Poly-A was manually inserted, initially located 10 Å above the nucleolipid bilayer surfaces (PUPC or PUOH) and in a parallel orientation to the bilayer plane. The started structures were energy minimized, before a series of molecular dynamics simulations. After only 1 ns of simulation, Poly-A quickly approaches the bilayer surface for both nucleolipids, and the systems were found to be equilibrated after 50 ns (Figure 6). To further quantify interactions between bilayers and ON, the number of intermolecular H-bonds was calculated over the last 2 ns of simulations. The results are listed in Table 4. Although the choline groups, which occupy the outermost layer of the membrane, are positively charged, and therefore should attract the negatively charged ON, the number of H-bonds is far more important for the PUOH system (25) than for the PUPC one (12). Moreover, in the PUPC system, the Poly-A oligomer mainly plays the role of H-bond donor (11/12 Hbonds). PUPC nucleolipids act as H-bond acceptor, and 36% of H-bonds result from nucleic base interactions. This result confirms the low accessibility of PUPC N3 group, which is deeply located within the bilayer and cannot easily go through the PC shield. At the opposite, as established by dynamics simulations, the N3 position of PUOH nucleic base is much more accessible. It is thus not surprising that among the 25 H-bonds, 10 resulted from PUOH as donor among which 60% derived from nucleic bases. As an acceptor, PUOH mainly participates through nucleic bases (74%). To further investigate poly-A/

Figure 6. Snapshots of the side view of the model system with PUPC membrane/Poly_A oligonucleotide before (a) and after 50 ns of molecular dynamic simulations (b). Water molecules are omitted for clarity reasons.

Table 4. Hydrogen Bonds (H-Bonds) between Poly-A and PUPC or PUOH Bilayers donors

acceptors

nucleolipid

PUPC/ Poly-A PUOH/ Poly-A

total Hbonds

poly-A

12

11

25

15

ribose and side chain

4

nucleolipid

base only

poly -A

ribose and side chain

1

1

7

4

6

10

4

11

nucleolipid bilayer association, RDF between center of mass of each bases of Poly-A and nucleolipid nucleic base or P or N atoms of the PC chain were calculated. Representative profiles of RDF are presented in Figure 7. Results clearly distinguish differences between PUPC and PUOH systems. They show that within a distance of 4 Å corresponding to π−π stacking or H-bonds, Poly-A base/nucleolipid base interactions mainly occurred in the PUOH association (Figure 7a and b), whereas Poly-A base/nucleolipid side-chain interactions are more 7458

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around 27.0 ± 0.5 Å taking a refractive index n = 1.46, compatible with a well-organized monolayer of the nucleolipids. The first injection of Poly-A into the subphase (70 μL) does not lead to modification of the morphology of the interface, which remains homogeneous, without thickness change. For further Poly-A injections (up to 220 μL), if there is no change in the morphology of the nucleolipid monolayer, fluctuations of gray levels are observed (Figure S3). Ellipsometric angle variations from D = 348.7° to D = 346.42° allow one to estimate thickness variations from 27.0 ± 0.5 to 33.2 ± 0.5 Å. In parallel, a weak increase of lateral pressure is observed, ΔP = +1 mN/m. Both of these observations are characteristic of weak and dynamic interactions of Poly-A with the nucleolipid PUOH/PUPC phase at the interface. In comparison, nothing is observed in the same conditions with a PUPC phase deposited at the air/water interface at 23 mN/m after 220 μL injection of Poly-A. The initial homogeneous nucleolipid phase remains homogeneous without thickness change (20.8 ± 0.5 Å). BAM and ellipsometry results confirm molecular modeling investigations to some extent, with many more H-bonds obtained after dynamics simulation between Poly-A and PUOH as compared to PUPC. The difference of interactions between Poly-A in the subphase and PUPC or PUOH/PUPC membranes reflects the difference of H-bonds observed in molecular modeling. To evaluate the specificity of interactions, BAM and ellipsometry experiments were conducted with PUPC/PUOH membrane and a non complementary RNA Poly-U. Weak and dynamic interactions were also detected, suggesting a lack of specificity as was previously observed by Mulet et al.20

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CONCLUSION This Article describes the creation and equilibration of pure nucleolipid membranes in 100 ns molecular dynamics simulations. The properties of these equilibrated membranes were consistent with experimental observations and provide molecular insights into nucleolipid organization. Thanks to lateral interactions between polar heads, the molecules adopt an ordered and close packing assembly. In addition to experimental findings, molecular dynamic simulations have been used to provide detailed structural information on ON− nucleolipid membrane systems. Thanks to these calculations, a neutral nucleolipid (PUOH) has been proposed and inserted into the bilayers. The data collected indicate that PUOH interacts favorably with ON in the subphase, whereas the uridine of the zwitterionic phosphocholine derivatives is not accessible for such interactions. A molecular modeling-based exploration of the structural elements that contribute to the formation of nucleolipid assemblies may open new avenues for the design of optimized structures for ON delivery.

Figure 7. Radial distribution functions (RDFs) for equilibrated PUPC/Poly_A and PUOH/Poly_A systems. RDFs of five bases of Poly-A from PUPC lipid bases (a), from PUOH lipid bases (b), from PUPC choline groups (c), and from PUOH hydroxyl (d).

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present in the case of PUPC bilayer (Figure 7c and d). Note that ON backbone (i.e., phosphodiester groups) interacts with PC side chains of PUPC, which is not observed for the PUOH side chain (Figure S2). These different behaviors were further confirmed by BAM and ellipsometry of both systems at the air/water interface of a Langmuir trough. Concerning PUOH, a monolayer of a mixture of PUOH/ PUPC (75/25) was deposited at the air/water interface of a Langmuir trough filled with buffer (Tris 20 mM, NaCl 150 mM, pH 7.4) at a lateral pressure around 23 mN/m. Ellipsometric angle measurements (D = 348.7°, Y = 2.37°) allow one to estimate the thickness of the nucleolipid mixture

ASSOCIATED CONTENT

S Supporting Information *

BAM images and additional molecular modeling data. 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] (I.B.), philippe.barthelemy@ inserm.fr (P.B.). 7459

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Notes

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The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the AFM (Association Française contre les Myopathies) and the Region Aquitaine for their financial support.

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ABBREVIATIONS

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REFERENCES

BAM, Brewster angle microscopy; PC, phosphocholine chain; ON, oligonucleotide; RDF, radial distribution function; P, phosphorus; N, azote; PUPC, dipalmityl uridine phosphocholine; PAPC, dipalmityl adenosine phosphocholine; DPUPC, dipalmitoyl uridine phosphocholine; PUOH, dipalmityl uridine hydroxyl

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