J. Phys. Chem. B 2008, 112, 11305–11309
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Enhanced Hydrophobicity of Fluorinated Lipid Bilayer: A Molecular Dynamics Study Hiroaki Saito,*,†,‡,§ Wataru Shinoda,*,†,‡ and Masuhiro Mikami†,‡ Research Institute for Computational Sciences (RICS), Research Institute of AdVanced Industrial Science and Technology (AIST), Central 2, 1-1-1 Umezono, Tsukuba 305-8568, Japan, and CREST, Japan Science and Technology Agency (JST), 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan ReceiVed: February 05, 2008; ReVised Manuscript ReceiVed: June 15, 2008
A molecular dynamics simulation of a partially fluorinated phospholipid bilayer has been carried out to understand the effects of fluorination of the hydrophobic chains on the structure and water permeability across the membrane. Fluorocarbon chains typically have an all-trans conformation, showing a highly ordered structure in the membrane core compared to ordinary hydrocarbon chains. The free energy profiles of water across the bilayers were successfully estimated by a revised cavity insertion Widom method. The fluorinated bilayer showed a higher free energy barrier than an ordinary nonfluorinated lipid bilayer by about 1.2 kcal/mol, suggesting a lower water permeability of the fluorinated bilayer membrane. A cavity distribution analysis elucidated the reduced free volume in the fluorinated membrane due to the neatly packed chains, which should account for the higher free energy barrier. Introduction Because of their extreme hydrophobicity and thermal stability, fluorinated molecules have been widely utilized in the chemical industry and in medical applications.1-9 For example, fluorinated amphiphiles are known as useful surfactants showing higher efficiencies and stronger tendencies toward self-assembly than the corresponding hydrogenated molecules.1-7 In medical applications, for example, a partially fluorinated liposome is expected to be a good material for a drug delivery system (DDS) because of its improved circulating times in the bloodstream.7,8 Fluorinated liposomal membranes display lower permeability coefficients than any of their hydrogenated counterparts, efficiently retaining the entrapped contents in the liposome.8 In such a fluorinated lipid bilayer, the water permeability is also expected to be reduced because of the high hydrophobicity of the fluorinated segment. However, it would not be simple to explain the high hydrophobicity of the fluorinated membrane. Compared with hydrogen, a fluorine atom has lower polarizability and a larger van der Waals radius,3 which implies weaker van der Waals interactions between fluorinated chains and lower cohesive energy densities in liquid fluorocarbons.4,5 The larger molecular surface presented by fluorination is partly responsible for the higher hydrophobicity of fluorocarbon chains in water because the hydrophobic effect of the chain is roughly proportional to the area exposed to water.6 This is explained by the large entropy loss due to the formation of a hydrogen-bond network among water molecules around the solute.10 However, the situation is quite different for a single water molecule in the membrane. In this case, the hydrogen-bond network obviously does not account for the lower permeability of water across the fluorinated membrane. As for transport of a small molecule through a dense polymeric membrane, several theoretical models based on free * To whom correspondence should be addressed. E-mail:
[email protected] (H.S.);
[email protected] (W.S.). † RICS-AIST. ‡ CREST-JST. § Present address: School of Mathematics and Physics, College of Science and Engineering, Kanazawa University.
volume concepts11 have been proposed so far.12-14 For example, in the solubility-diffusion model,15 the permeability coefficient can be expressed as the product of the solubility and diffusion coefficients of the penetrant in the membrane. These models commonly relate the mutual diffusion coefficient for a penetrant/ polymer system to the free volume of the system. In fact, several molecular dynamics (MD) simulations have shown that the calculated diffusion coefficient of a solute molecule correlates with the free volume distribution16-21 and polymer mobility.16,19 An advanced theoretical model for permeation of a small solute through lipid bilayers has been proposed by Marrink and Berendsen to take into account an inhomogeneous environment in the bilayer interior.22 Based on this theoretical model, several molecular simulations have been performed to estimate the permeability or free energy profile of small molecules through the lipid bilayer membranes,23-31 although no study has been reported on fluorinated bilayer membranes. In this study, we attempted to understand the highly hydrophobic nature of a fluorinated lipid bilayer membrane from the molecular viewpoint using MD simulations. A comparison of partially fluorinated and nonfluorinated phospholipid bilayers elucidates the effects of fluorination on the free energy profile of water across bilayers, as well as the structural properties such as the molecular packing in the bilayer core. Material and Methods Figure 1 shows molecular models of the partially fluorinated phospholipid 1,2-di(F8CCH8)PC (FPC) and the corresponding nonfluorinated counterpart 1,2-di(H8CCH8)PC (HPC) used in the simulations. Both HPC and FPC molecules have two etherlinked hydrophobic chains, both of which have a monoacetylene group (triple bond) in the middle. In the FPC lipid, the last eight hydrocarbon atoms in each chain are replaced by fluorocarbon atoms.9 In Figure 1, the lower hydrophobic chain (C1–C18) in both lipids is defined as the Sn–1 chain, and the other side of the chain is defined as the Sn–2 chain. We modeled the lipid molecules based on the united-atom model developed by Smondyrev et al.32 For the missing segments including the ether linkage, the triple bond, and the
10.1021/jp801057k CCC: $40.75 2008 American Chemical Society Published on Web 08/19/2008
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Saito et al.
Figure 1. Chemical structures of 1,2-di(H8CCH8)PC and 1,2-di(F8CCH8)PC lipid molecules. Atom types for the intra- and intermolecular potentials are shown in the each atom. The atom types of hydrocarbon atoms for the alkyl chain are denoted as CH2 and CH3 to distinguish them from the atoms in the headgroup (C2 and C3). Partial charges of atoms are located next to the atoms. The carbon numbers (C1-C17) of the hydrophobic chain are provided above some atoms.
fluorocarbon chain, several other potential parameter sets were utilized.33-35 For the fluorocarbon chain of the FPC lipid, the modified Ryckaert-Bellemans-type dihedral potential33 was employed to introduce a high trans-gauche barrier. AMBER potential sets34 were used for the dihedral angle of the etherlinkage segment. For the triple-bond segment, we chose the sp carbon (CYE) for the Lennard-Jones (LJ) potential parameter and applied the bending potentials for the alkyne group in the MM3 potential set35 to keep the straight conformation. The equilibrium bond lengths, r0, for the triple-bond segment were obtained from the optimized geometry of the acetylene-series molecules C8H14 and C8H7F7 with the Gaussian 03 program36 at the MP2/6-311G** level. We adopted the partial charges used for DPhPC lipid27 for the polar and ether-linkage regions. The partial charges for the triple-bond region were determined by MO calculations at the MP2/6-311G** level with the MerzSingh-Kollman scheme.37 The partial charges of united atoms were obtained by summing hydrogen charges into heavy atoms. The potential parameters used for these segments are summarized in Table 1, and the partial charges are given in Figure 1. The initial lipid conformation was built based on the crystal DMPC conformation obtained from X-ray diffraction data.38 The HPC and FPC bilayers consisted of 72 (6 × 6 × 2) lipid molecules and 2160 and 3240 water molecules, respectively, to simulate the fully hydrated conditions.9 Initial bilayer structures were generated by packing the lipid molecules into a hexagonally close-packed (hcp) lattice in the x-y plane to reproduce the experimental molecular area per lipid (HPC, 71.18 Å2; FPC, 87.9 Å2).9 The TIP3P water molecules39 were disposed outside the bilayer. MD calculations were performed in the NPnAT (constant normal pressure, area, and temperature) ensemble using the MPDyn program developed in our research group.40 The MD calculations for each system were performed at Pn ) 0.1 MPa and T ) 298 K. The Nose´-Hoover chain41 scheme (length ) 5) and Andersen barostat41 were used to yield
TABLE 1: Force-Field Parameters for the United-Atom Model bond
r0 (Å)
CH2-CYE CF2-CYE CYE-CYE
1.47 1.45 1.22
angle
kθ (kcal/mol)
θ0 (deg)
C2-OS-CH2 OS-CH2-CH2 CH-OS-CH2 CH2-CH2-CYE CF2-CF2-CYE CYE-CYE-CH2 CYE-CYE-CF2
100.0 80.0 100.0 100.0 100.0 22.661 22.661
111.8 109.5 111.8 111.8 111.8 180.0 180.0
dihedral angle
N
Vn (kcal/mol)
F1 (deg)
C2-CH-OS-CH2 C2-CH-OS-CH2 CH-OS-CH2-CH2 CH-C2-OS-CH2 C2-OS-CH2-CH2 CH2-CH2-CYE-CYE CH2-CYE-CYE-CH2 CYE-CYE-CF2-CF2
2 3 3 3 3 3 3 3
0.2 1.45 2.9 2.9 2.9 0.0 0.0 0.0
0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
nonbonded
σ (Å)
ε (kcal/mol)
CYE CH2 CH3 CF2 CF3
3.75 3.96 3.96 5.078 5.078
0.105 0.091 0.136 0.080 0.114
the NPnAT ensemble with response times of 0.5 and 2 ps, respectively. The equations of motion were integrated by the rRESPA42 algorithm with two different time steps: The time step size used for updating the force in the Ewald reciprocal space was 8 fs; all the rest of the forces were updated every 2 fs. All bond lengths were constrained using the SHAKE/
Enhanced Hydrophobicity of Fluorinated Lipid Bilayer
J. Phys. Chem. B, Vol. 112, No. 36, 2008 11307
TABLE 2: Comparison of the Structural Data with Experimental Measurementsa 1,2-di(H8CCH8)PC Dl (Å) dP-P (Å)
1,2-di(F8CCH8)PC
MD
expt
60.71 ( 0.23 34.57 ( 0.35
61.1 ( 1 34.9 ( 0.6b b
MD
expt
66.28 ( 0.24 35.56 ( 0.29
67.6 ( 0.8b 36.7 ( 0.4b
a Dl, lamella repeat distance (Å); dP-P, peak-to-peak distance of the area (Å). b Experimental data (at 298 K) are taken from ref 9.
RATTLE/ROLL41 algorithm with the tolerance ∆r/r ) 10-8. The Ewald method43 was used for the electrostatic interactions. In the first relaxation process of the system, 1-ns MD calculations with the large response time of the barostat (τb ) 40 ps) were carried out to relax the MD cell size. Also, the system temperature was set to 348 K to enhance chain melting in the first 500 ps. Both simulation systems were well equilibrated within 4 ns of NPnAT-MD calculations. The average P-P atom distance, dP-P, and the lamellar spacing, Dl, over a 20-ns MD trajectory show good agreement with the values from experiment (see Table 2). The total MD simulation time was 24 ns for each system, and the last 20 ns of the MD trajectory was used for the analyses. To check the quality of the potential force field, we calculated structural and dynamical properties. Although the details will be reported in a forthcoming article,44 we briefly mention them here. The calculated electron density profile for the fluorinated membrane shows characteristic peaks in the inner membrane, showing good agreement with the results obtained by X-ray diffraction measurements of 1,2-di(F4C11)PC bilayer.8 The calculated lateral diffusion coefficients also agree with the experimental measurements.44 However, the surface tension measured during the NPnAT-MD simulations deviates from zero, showing large positive values (HPC, 48.8 dyn/cm; FPC, 58.0 dyn/cm). This indicates that our model lipid would underestimate the molecular area when the NPT ensemble is used. The issue is rather well-known in this community that even the most widely used force fields such as CHARMM and AMBER underestimate the molecular area for straight-chain lipids such as DPPC (dipalmitoylphosphatidylcholine) and POPC (palmitoyloleoylphosphatidylcholine). However, these force fields produce reasonable membrane structure using the NPnAT and NPγT ensembles, where the molecular area should be known.45 The situation is similar to the current study, and as mentioned above, the agreement of structural and dynamical properties with experimental data encourages us to investigate the further details with the obtained NPnAT-MD trajectories. The free energy profile of water across each membrane was calculated from the difference between the excess chemical potentials, µex(z), along the bilayer normal, ∆G(z) ) µex(z) µex(z0), where the reference position, z0, is taken at the center of the water layer. The cavity insertion Widom method (CIW)28 was used to estimate the excess chemical potential at each z position, which was calculated by the equation
〈 (NPV+ 1) exp[-β∆U(z)]〉 -
µex(z) ) -kBT ln β
kBT ln〈Pcav(z)〉(1) where P, V, and N denote the pressure, volume of the MD cell, and number of particle in the system, respectively; β denotes the inverse temperature, kBT; and kB is the Boltzmann constant. ∆U(z) denotes the intermolecular interaction between an inserted water molecule at z position and the surrounding molecules, and Pcav(z) denotes the probability density of a cavity at z. The
Figure 2. Free energy profiles for bilayers of 1,2-di(F8CCH8)PC (FPC, solid line) and 1,2-di(H8CCH8)PC (HPC, dashed line) along the bilayer normal.
cavity density, Pcav(z), was calculated using a uniform grid. Each bin was examined whether atoms existed or not. In these calculations, the selected regions for cavity searches were 50 × 50 × 60 Å in both systems. The solute molecules were directly inserted into the center of cavity sites at position z so that the configurations contributing the value of exp[-β∆U(z)] could be sampled efficiently. The sampling to the whole system was estimated by multiplying the probability of the cavity distribution, Pcav(z), by exp[-β∆U(z)]. Although the insertion method is suitable for estimation of µex(z) in the bilayer core region, the method works less efficiently when it is applied to a high-density or polar region.28 To improve the efficiency, we made a modification in searching the cavity of the system by taking into account each particle size of the molecules making up the system. In this calculation, the van der Waals (vdW) radius for each atom was assumed to be 1/2σ, where σ is one of the LJ nonbonded interaction parameters. Thus, a more accurate cavity profile for the trial insertion would be obtained. Also, we used a smaller grid size, Rcav, of 0.5 Å because the previous grid size (Rcav ) 2.6 Å)28 used in the cavity search was so large that the cavity sites could hardly be found when the vdW radius of the atom was taken into account. Also it should be smaller than the vdW radius of hydrogen atom used for the water molecule to search the cavity space in water/membrane region, where the density is high. A similar cavity-biased insertion method with the van der Waals radius of atoms was implemented by Pohorille et al. and showed good convergence of the chemical potential.46 For the calculation of the interaction energy, ∆U(z), between the inserted molecule and the surroundings, the van der Waals interaction was truncated at 12 Å, and the Ewald method was used for the electrostatic interaction. The Ewald method should be used in the CIW computation because a truncation of the electrostatic interaction give a systematic error in the calculated chemical potential.31 For each configuration from the MD trajectories, 360 trial insertions of randomly oriented water molecules were performed. The standard deviation of the excess chemical potential was estimated from 10 block averages of µcav(z), each of which was calculated from 2-ns MD trajectories. Results and Discussion The calculated free energy profiles of water along the bilayer normal for FPC and HPC bilayers are shown in Figure 2. The excess chemical potential energies µex(z) were calculated at every 2.5 Å along the bilayer normal. The insertion technique works less efficiently when it is used in a high-density or polar region.28 However, as shown in Figure 2, the calculated µex(z)
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Figure 3. Probabilities of gauche conformer as a function of carbon number in Sn-1 and Sn-2 chains of 1,2-di(F8CCH8)PC (FPC, solid line) and 1,2-di(H8CCH8)PC (HPC, dashed line) lipids.
values were sufficiently converged even in the interfacial regions. A comparison with previous CIW studies shows that the refined cavity search does help improve the convergence of the chemical potential sampling significantly. The free energy barriers of FPC and HPC bilayers for water were estimated to be 6.4 and 5.2 kcal/mol, respectively, suggesting a lower water permeability for the FPC bilayer. This observation is consistent with the experimental observation that the fluorinated core with a liposomal membrane acts as an efficient barrier to the permeation of the small solvent molecules.8 The higher free energy barrier in the FPC membrane is not straightforwardly understood from the intermolecular interactions, because once a water molecule enters the membrane core, the net interaction energy of the water molecule in the FPC membrane is assumed to be similar to that in the HPC membrane. Thus, we should find an alternative explanation for the difference in free energy barrier, e.g., the accessibility of water molecules in the membrane core. To understand this, we turn our attention to the molecular packing in the membrane core. Figure 3 shows a plot of the probability of a gauche conformer in the hydrophobic chains for each lipid, Pgauche, as a function of the carbon number. Here, the carbon number is indicated in Figure 1. The hydrocarbon region contains about 25% gauche conformers, whereas the fluorinated segment (10-15) contains almost none; i.e., most of the fluorocarbon chains are in the all-trans conformation. This is due to the high trans-gauche energy barrier in the fluorocarbon chain of 4.6 kJ/mol, compared to 2 kJ/mol in the hydrocarbon chain.2 This high trans-gauche energy barrier prevents configurational isomerization of the fluorinated segment, making the chain stiff. A similar observation was made experimentally that fluorinated amphiphiles tend to be rod-shaped.47 The rigidity of the fluorinated segment results in a very different arrangement or packing of the chains in the bilayer core. The difference is characterized by the molecular order parameters, Smol, as shown in Figure 4. The Smol values of the fluorocarbon segment (C10-C18) in the FPC lipid are higher than those of any other hydrocarbon segments. Such high order parameters of the fluorinated segment were also reported by experimental8 and computational32 studies of a fluorinated bilayer. According to the experimental observation using a fluorescence probe, a significant rigidity of the fluorinated chain tail increases the membrane order, and this higher order of the fluorinated membrane is correlated with the higher phasetransition temperature of the fluorinated membrane.8 In addition to the highly ordered chain, because most of the fluorocarbon chains are in the all-trans conformations, the effect of steric hindrance due to the gauche conformer is largely weakened.
Saito et al.
Figure 4. Order parameter profiles as a function of carbon number in Sn-1 and Sn-2 chains of 1,2-di(F8CCH8)PC (FPC, solid line) and 1,2di(H8CCH8)PC (HPC, dashed line) lipids.
Figure 5. Cavity distributions for bilayers of 1,2-di(F8CCH8)PC (FPC, solid line) and 1,2-di(H8CCH8)PC (HPC, dashed line) along the bilayer normal.
Consequently, these properties lead to the neat packing of the fluorocarbon chains in the bilayer core and change the density of atoms in this region. Figure 5 shows a plot of the probability distributions of a cavity, Pcav, along the bilayer normal for both bilayers. The FPC bilayer considerably reduces Pcav in the fluorinated core region (|z| < 14 Å), showing a denser packing than in the HPC bilayer. Thus, we conclude that the high hydrophobicity of FPC bilayer mainly arises from the neat packing of the fluorinated chains. From the molecular viewpoint, the dense packing in the fluorinated segment leads to the restricted translational and rotational motions of water molecules in the membrane; the entropy loss could be responsible for the increase of the free energy of water in the membrane core. This was also found for a fluorinated polymer membrane where the diffusivity and permeability of the penetrants were largely reduced in comparison to the nonfluorinated counterpart.48,49 Conclusion The effects of fluorination of the lipid hydrophobic tails on bilayer properties were investigated by MD simulations. It was found that the fluorinated bilayer produces a higher free energy barrier for water than the nonfluorinated bilayer, demonstrating an enhanced hydrophobicity due to the fluorinated chain. Because the fluorinated chains are mostly in the all-trans conformation, they are neatly packed in the fluorinated membrane, which leads to a large reduction of the accessible volume for a water molecule in the fluorinated bilayer. This observation suggests that the higher free energy barrier of water across the fluorinated membrane is attributable to the reduced accessible
Enhanced Hydrophobicity of Fluorinated Lipid Bilayer volume for water in the membrane core rather than the interaction energy. Acknowledgment. The authors thank Dr. Baba for providing experimental data before publication. This research was supported by CREST (Core Research for Evolutional Science and Technology), Japan Science and Technology Agency (JST). References and Notes (1) Ravey, J. C.; Ste´be´, M. J. Colloids Surf. A: Physicochem. Eng. Aspects 1994, 84, 11. (2) (a) Gonza´lez-Pe´rez, A.; Prieto, G.; Ruso, J. M.; Sarmiento, F. Mol. Phys. 2003, 101, 3185. (b) Blanco, E.; Gonza´lez-Pe´rez, A.; Ruso, J. M.; Pedrido, R.; Prieto, G.; Sarmiento, F. J. Colloid Interface Sci. 2005, 288, 247. (3) Kissa, E. Fluorinated Surfactants: Synthesis, Properties, Applications; Surfactant Science Series; Marcel Dekker: New York, 1994, Vol. 50. (4) Reed, T. M. Fluorine Chemistry; Academic Press: New York, 1964; Vol. 5, pp 133-221. (5) Riess, J. G. Colloids Surf. 1994, 84, 33. (6) Tanford, C; Hydrophobic Effect: Formation of Micelles and Biological Membrane; John Wiley and Sons: New York, 1980. (7) Krafft, M. P. AdV. Drug DeliV. ReV. 2001, 47, 209. (8) (a) Santaella, C.; Fre´zard, F.; Vierling, P.; Riess, J. G. FEBS Lett. 1993, 336, 481. (b) Fre´zard, F.; Santaella, C.; Vierling, P.; Riess, J. G. Biochim. Biophys. Acta 1994, 1192, 61. (c) Fre´zard, F.; Santaella, C.; Montisci, M. -J.; Vierling, P.; Riess, J. G. Biochim. Biophys. Acta 1994, 1194, 61. (d) Trevino, L.; Fre´zard, F.; Rolland, J. P.; Postel, M.; Riess, J. G. Colloids Surf. A: Physicochem. Eng. Aspects 1994, 88, 223. (e) Rolland, J. P.; Santaella, C.; Vierling, P. Chem. Phys. Lipids 1996, 79, 71. (f) McIntosh, T. J.; Simon, S. A.; Vierling, P.; Santaella, C.; Ravily, V. Biophys. J. 1996, 71, 1853. (g) Ravily, V.; Clary, L.; Santaella, C.; Vierling, P. Chem. Phys. Lipids 1997, 90, 75. (9) (a) Takagi, T.; Takai, K.; Baba, T.; Kanamori, T. J. Fluorine Chem. 2007, 128, 133. (b) Baba, T.; Takagi, T.; Takai, K.; Kanamori, T.,in preparation. (10) Frank, H.; Evans, M. J. Chem. Phys. 1945, 13, 507. (11) Frisch, H. L.; Klempner, D.; Kwei, T. K. Macromolecules 1971, 4, 237. (12) Fujita, H. Fortschr. Hochpolym. Forsch. 1964, 3, 1. (13) Ganesh, K; Nagrajan, R; Duda, J. L. Ind. Eng. Chem. Res. 1992, 31, 746. (14) Lee, W. H. Polym. Eng. Sci. 1980, 20, 65. (15) Graham, T. Philos. Mag. 1866, 32, 401. (16) Krishna Pant, P. V.; Boyd, R. H. Macromolecules 1993, 26, 679. (17) Tamai, Y.; Nakanishi, K. Macromolecules 1994, 27, 4498. (18) Tamai, Y.; Tanaka, H.; Nakanishi, K. Macromolecules 1995, 28, 2544. (19) Hofmann, D.; Fritz, L.; Ulbrich, J.; Schepers, C.; Bo¨hning, M. Macromol. Theory Simul. 2000, 9, 293. (20) Gee, R. H; Boyd, R. H. Polymer 1995, 36, 1435. (21) Pavel, D.; Shanks, R. Polymer 2005, 46, 6135. (22) Marrink, S. J.; Berendsen, H. J. C. J. Phys. Chem. 1994, 98, 4155. (23) Marrink, S. J.; Berendsen, H. J. C. J. Phys. Chem. 1996, 100, 16729. (24) Jedlovszky, P.; Mezei, M. J. Am. Chem. Soc. 2000, 122, 5125. (25) Carl, D. R.; Feller, S. E. Langmuir 2003, 19, 8560. (26) Bemporad, D.; Essex, J. W. J. Phys. Chem. B 2004, 108, 4875.
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