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Molecular Order Parameter Profiles and Diffusion Coefficients of Cationic Lipid Bilayers on a Solid Support† Michael Junglas, Birgit Danner, and Thomas M. Bayerl* Universita¨ t Wu¨ rzburg, Physikalisches Institut EP-5, 97074 Wu¨ rzburg, Germany Received August 25, 2002. In Final Form: November 12, 2002 The molecular order and lateral diffusion in fluid cationic bilayers of chain perdeuterated DPTAP (1,2dipalmitoyl-d62-trimethylammonium-propane) on a spherical solid support were studied by deuterium NMR and microcalorimetry. In comparison to the zwitterionic perdeuterated DPPC (1,2-dipalmitoyl-d62sn-glycero-3-phosphocholine), the molecular order parameter profile of the palmitoyl chains of DPTAP shows significantly higher molecular order toward the interface while similar values were obtained in the vicinity of the bilayer center. The lateral diffusion coefficient D of DPTAP is D ) (13.2 ( 1.5) × 10-12 m2/s at 60 °C, slightly below that of DPPC at this temperature. Increasing the ionic strength caused the value of D to decrease further. The results suggest that despite the cationic charge the supported DPTAP bilayer features tighter molecular packing and a reduced lateral diffusivity of its constituents than for the zwitterionic DPPC on a solid support.
Introduction Cationic amphiphiles with close structural resemblance to typical membrane-forming lipids, so-called cationic lipids, are used for the functionalization of surfaces1 and for wetting purposes2 and are crucial constituents of aggregates used for shuttling genes into cells.3 Especially the mixing of DNA with lipids containing cationic amphiphiles, giving rise to well-ordered nanostructures in which the DNA condenses between the lipid bilayers, has been the subject of recent X-ray studies.4 The salient physiological feature of cationic lipids, which in general do not occur in nature, is their positive charge at neutral pH. This allows for a strong Coulomb interaction with many of the mostly negatively charged components of a biological cell. Since this interaction is long range at the length scale of intermolecular forces, cationic lipids embedded in a membrane environment may be considered as molecular sensing devices for the coupling of biomolecules. A very useful dynamical parameter for the assessment of molecular interactions is the (molecular) order parameter5,6 that can be detected by solid-state NMR and can be calculated out of molecular dynamics (MD) simulations of membranes.7 Order parameter studies may further our knowledge on the complex interaction pattern * Corresponding author. Phone: 49-931-888-5863. Fax: 49-931888-5851. E-mail:
[email protected]. † Part of the Langmuir special issue entitled The Biomolecular Interface. (1) Ra¨dler, J.; Sackmann, E. On the measurement of weak repulsive and frictional colloidal forces by reflection interference contrast microscopy. Langmuir 1992, 8, 848-853. (2) Ra¨dler, J.; Strey, H.; Sackmann, E. Phenomenology and kinetics of Lipid Bilayer Spreading on Hydrophilic Surfaces. Langmuir 1995, 11, 4539-4548. (3) Monk, K. W. C.; Cullis, P. R. Structural and Fusogenic Properties Of Cationic Liposomes In the Presence Of Plasmid DNA. Biophys. J. 1997, 73 (5), 2534-2545. (4) Ra¨dler, J. O., et al. Structure of DNA-cationic liposome complexes: DNA intercalation in multilamellar membranes in distinct interhelical packing regimes. Science 1997, 275, 810-814. (5) Seelig, J.; Seelig, A. Lipid conformation in model membranes and biological membranes. Q. Rev. Biophys. 1980, 13, 19-61. (6) Lafleur, M., et al. Smoothed Orientational Order Profile of Lipid Bilayers by 2H NMR. Biophys. J. 1989, 56, 1037-10ff. (7) Tu, K.; Tobias, D. J.; Klein, M. L. Constant pressure and temperature molecular dynamics simulation of a fully hydrated liquid crystal phase dipalmitoylphosphatidylcholine bilayer. Biophys. J. 1995, 69, 2558-2562.
within cationic lipid-biomolecule complexes and can assist in the design of such structures optimized for drug delivery or gene shuttling. However, so far no order parameter profiles of cationic lipid bilayers have been published. Besides the need of specific deuterium labeling of the cationic lipids, there is a morphological problem. Owing to their charge, cationic lipids spontaneously form very small (e50 nm) vesicles in water. Due to the rapid thermally driven rotation rate (tumbling) of these vesicles which scales with the cube of their radius, the second rank tensor interactions of a 2H NMR spectrum become averaged to zero, resulting in a collapse of the spectrum to a single isotropic line.8 Unfortunately, these tensor interactions contain all the dynamic information of the system (including the order parameter). Using oriented multilayers of pure cationic lipids to overcome this obstacle is not feasible since the strong Coulomb repulsion between the stacked bilayers will prevent the formation of such structures. We have tackled this problem by using spherical silica beads of submicrometer dimensions coated with a single bilayer of cationic lipids (so-called spherical supported vesicles, SSVs).9 This causes the bilayer morphology to adopt the (well-defined) shape of the spherical support and eliminates by the size of the latter the tumbling problem as well. In aqueous solution, this system can mimic to the bulk a cell wall, which can (Coulomb) interact with dissolved biomolecules and be studied by solid-state NMR. This approach opens up another interesting feature, namely, the study of the molecular diffusion of the cationic lipids along the bilayer plane (lateral diffusion) and its modulation by the coupling of biomolecules such as DNA strands using the well-established Carr-Purcell-Meiboom-Gill (CPMG) pulse sequence.10 In this work, we concentrate on the basic system, single bilayers of pure cationic lipids coated on silica beads of 400 nm diameter. We used a chain perdeuterated 1,2(8) Abragam, A. The Principles of Nuclear Magnetism; Oxford University Press: Oxford, 1961; pp 1-599. (9) Bayerl, T. M.; Bloom, M. Physical properties of single phospholipid bilayers adsorbed to micro glass beads. Biophys. J. 1990, 58, 357-362. (10) Meiboom, S.; Gill, D. Modified Spin-Echo Method for Measuring Nuclear Relaxation Times. Rev. Sci. Instrum. 1958, 29 (8), 688-691.
10.1021/la026468s CCC: $25.00 © 2003 American Chemical Society Published on Web 01/01/2003
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Figure 1. Structure of DPTAP-d62.
dipalmitoyl-d62-trimethylammonium-propane (DPTAPd62) and evaluated the order parameter profile and its lateral diffusion coefficient as well as the dependence of these parameters on the ionic strength of the bulk solution. We consider this as a first step in the design of model systems, which allow for detailed dynamic studies of cationic lipid-biomolecule interactions at a molecular scale.
Figure 2. DSC of DPTAP-d62 SSVs. The full line is the heating cycle and the dashed line is the cooling cycle.
Results Materials and Methods Chain deuterated DPTAP-d62 (Figure 1), a special synthesis, and chain deuterated 1,2-dipalmitoyl-d62-sn-glycero-3-phosphocholine (DPPC-d62) were purchased from Avanti Polar Lipids (Alabaster, AL) and used without further purification. 2-[4-(2Hydroxyethyl)-1-piperazinyl]-ethane-sulfonic acid (HEPES) and sodium chloride p.a. were obtained from Fluka (Mu¨hlheim, Germany). Monodisperse nonporous silica beads are a special synthesis by Degussa AG, Department of Anorganic Chemistry (Hanau, Germany), with a diameter of 400 ( 20 nm. The buffer was prepared with ultrapure water (Millipore Corp., Bedford, MA) using 20 mM HEPES, pH ) 7.0 (adjusted with NaOH), and NaCl (0, 150 mM, respectively). For the NMR experiments, the ultrapure water (Millipore) was replaced by deuterium-depleted water (DDW) purchased from Isotec (Miamisburg, OH). Samples were prepared by dispersing 10-30 mg of dry lipid in the buffer at a temperature of 60 °C under gentle vortexing. The dispersion was rod sonicated at 60 °C until the sample appeared opalescent in transmitted light and yellowish under a 90° incident angle, indicating that small unilamellar vesicles (SUVs) of less than 100 nm diameter were formed. Then the dry silica beads were added to the solution, and the mixture was rotated at 60 °C for 2 h to promote SUV fusion at the bead surface. After this, the sample was cooled to room temperature and then washed five times in excess buffer using a tabletop centrifuge. The last washing step was done with deuterium-depleted buffer. After this, the sample was transferred to the NMR tube (pressure plastic caps, 10 mm, Wilmad Glass, Buena, NJ); the final lipid concentration in the tube was 25 ( 4 mg. For the differential scanning calorimetry (DSC), the microcalorimeter MCII from Microcal (Northhampton, MA) operating at a scan rate of 20 °C/h was used. The lipid concentration of the SSV samples was 2.5 mg/mL. Endotherms and exotherms were recorded at temperatures between 20 and 70 °C. All NMR experiments were performed with an AMX 500 MHz Bruker spectrometer operating at 76.7 MHz for 2H using a 10 mm multinuclear solid-state probe. The experiments were done at temperatures of 50 and 60 °C that were controlled by a Bruker VT 100. The quadrupolar echo sequence was taken with an appropriate cyclops phase cycling11 and a pulse length of 6.7 µs. The dwell time was 0.4 µs for the CPMG experiments, 1 µs for the T2 measurements, and 2 µs for the spin-echo spectra. For the acquisition of the spectra, we used a delay time of 20 µs, except for the CMPG which were done with 16, 20, 25, 30, and 35 µs, respectively. The repetition time was 300 ms for all experiments. All other details of the NMR experiments were described previously.12 (11) Rance, M.; Byrd, A. Obtaining high-fidelity spin-1/2 powder spectra in anisotropic media: phase-cycled Hahn echo Spectroscopy. J. Magn. Reson. 1983, 52, 221-240. (12) Ko¨chy, T.; Bayerl, T. M. Lateral diffusion coefficients of phospholipids in spherical bilayers on a solid support measured by 2Hnuclear-magnetic-resonance relaxation. Phys. Rev. E 1993, 47, 21092116.
In a first series of experiments, the phase transition of DPTAP-d62 bilayers on a spherical support (SSVs) was studied by DSC. The heating and cooling scans of DPTAPd62 SSVs in an aqueous dispersion containing 150 mM NaCl are shown in Figure 2. The salt was added to prevent effects of spurious ions in the buffer on the phase transition. Without any salt in the buffer, the phase transition of the DPTAP-d62 SSVs was not reproducible, while any NaCl addition above 15 mM and up to 200 mM gave similar and highly reproducible DSC endotherms. Both endotherm and exotherm in Figure 2 show a rather broad feature that can be separated into two peaks of similar enthalpy at 52.5 and 56.0 °C (heating scan) and at 49.5 and 54.0 °C (cooling scan). The hysteresis observed of 2-3 °C is quite typical for bilayers on a solid support and is a result of the lateral tension the system undergoes upon temperature changes.13 The two peaks may suggest a partial decoupling of the phase transition between inner and outer monolayer leaflets owing to the close proximity of the inner leaflet to the solid surface. Such behavior was observed previously for DPPC bilayers on a solid support.14 Since the silica bead surface exhibits negative charge, its interaction with the cationic DPTAP of the inner leaflet is likely to be even stronger than for the zwitterionic DPPC. After the initial DSC measurements, deuterium NMR studies were performed at a temperature of 60 °C corresponding to the fluid state of the DPTAP-d62 SSVs according to Figure 2. Deuterium (2H) NMR spectra of the SSVs with and without NaCl in the buffer solution are shown in Figure 3. For comparison, the spectrum of a zwitterionic DPPC-d62 SSV at 50 °C is also shown in Figure 3. In both cases (DPTAP and DPPC), the NMR line shape is free from any distortion due to diamagnetic susceptibility anisotropy effects as the solid support ensures a spherical distribution of the molecular directors of the sample. This allows a direct comparison of the line shapes and of the order parameter profiles of zwitterionic and cationic lipids on a solid support, provided that the transverse relaxation time T2e is similar for both lipid species. Our measurements of T2e at 50 °C (DPPC) and 60 °C (DPTAP, no NaCl added) gave 180 and 220 µs for DPTAP-d62 SSVs and DPPC-d62 SSVs, respectively. For the case of DPTAP-d62 SSVs in 150 mM NaCl, we obtained T2e ) 240 µs while the salt addition had no measurable effect on the DPPC-d62 SSVs. (13) Naumann, C.; Brumm, T.; Bayerl, T. M. Phase transition behavior of single phosphatidylcholine bilayers on a solid spherical support studied by DSC, NMR and FT-IR. Biophys. J. 1992, 63, 13141319. (14) Ka¨sbauer, M.; Junglas, M.; Bayerl, T. M. Effect of cationic lipids in the formation of asymmetries in supported bilayers. Biophys. J. 1999, 76, 2600-2605.
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Figure 3. Deuterium NMR spectra of SSVs of DPTAP-d62 without NaCl (bottom), of DPTAP-d62 with 150 mM NaCl (middle), and of DPPC-d62 without NaCl (top) at a temperature of 50 °C for DPPC-d62 and 60 °C for DPTAP-d62.
Figure 4. Molecular order parameter vs carbon chain position of DPTAP-d62 SSVs (T ) 60 °C) and of DPPC-d62 SSVs (T ) 50 °C).
As can be inferred from Figure 3, the NMR spectra for DPTAP-d62 SSVs and DPPC-d62 SSVs show similar Pake doublets with quadrupolar splittings of ∆νQDPTAP ) 22.0 kHz and ∆νQDPPC ) 21.4 kHz. However, the DPPC shows near the 90° edges a slightly higher intensity, most likely due to a different distribution of the molecular order parameters of the individual methylene groups along the acyl chain. To analyze this further, we calculated the order parameter profiles from the spectra in Figure 3 by the well-established de-Pake-ing procedure.15 The profiles shown in Figure 4 demonstrate clearly a significant increase of the molecular order of DPTAP in the methylene region close to the headgroup of the lipids, often termed the “plateau region”. In contrast, the region close to the bilayer center exhibits a similar order for both lipids, which decreases with increasing carbon number (Figure 4). As a consequence, the decrease of molecular order from the carbon positions near the interface down to the bilayer center is steeper for DPTAP compared to DPPC. In a further series of NMR relaxation measurements, we addressed the question of differences in lateral diffusion of DPTAP and DPPC bilayers on a solid support. We were particularly interested in possible alterations of the diffusive behavior due to the strong electrostatic coupling (15) Sternin, E.; Bloom, M.; MacKay, A. L. De-pake-ing of NMR spectra. J. Magn. Reson. 1983, 55, 274-282.
Junglas et al.
Figure 5. Semilogarithmic plot of the CPMG signal intensity of DPTAP-d62 SSVs at 60 °C versus time t ) 2nτ for τ ) 16, 20, 25, 30, and 35 µs as indicated. The slope of the linear fits gave 1/T2CPMG.
of the cationic bilayer to the support (silica exhibits a negative surface charge at neutral pH) and due to the Coulomb repulsion between adjacent cationic lipids. The measurements were done using the well-established CPMG pulse sequence. This sequence can act as a low pass filter for diffusive motions and thus allows the determination of the diffusion constant, provided that the diameter of the spherical support is known.12,16 The results of a CPMG experiment on DPTAP-d62 SSVs (T ) 60 °C) for increasing time t ) 2nτ, where n is the number of echoes and τ is the time between the 90° refocusing pulses, are shown in Figure 5. Within the limit of short t (t < 500 µs), the plot of the CPMG intensities versus t for different values of τ (Figure 5) is exponential; the slope gave 1/T2CPMG(τ). According to the theory of transverse relaxation with lateral diffusion being the dominant slow motion mechanism, this relaxation can be described by (T2CPMG)-1 ) [2M2rD/R2]τ2 + (T′2)-1 in the limit of τD . τM. Here M2r is the residual second moment of the 2H NMR line shape, D is the lateral diffusion coefficient of the lipids, R is the radius of the bead, τM is the NMR time scale (≈10-5 s for 2H NMR), and τD ) R2/6D is the diffusion correlation time on a sphere of radius R. (T′2)-1 is the relaxation rate due to processes having correlation times (τ′2) which are fast on the NMR time scale (τ′2 , τM). Hence, a plot of 1/T2CPMG versus τ2 should give a straight line with the slope b ) 2M2rD/R2 (Figure 6). We obtained b ) 2.9 × 1012 s-3 and thus with R ) 200 ( 20 nm and the second moment in the limit M2r ≈ M2 ) (2p∆nQ)2/5 ) 4.4 × 109 s-2, a lateral diffusion constant D is obtained according to D ) bR2/(2Mr) as DDPTAP ) (13.2 ( 1.5) × 10-12 m2/s at 60 °C for the sample without NaCl and DDPTAP ) (10.6 ( 1.0) × 10-12 m2/s for the sample with 150 mM NaCl (b ) 1.9 × 1012 s-3 and M2 ) 3.6 × 109 s-2). For DPPC-d62, we obtained a value of DDPPC ) (14.2 ( 1.2) × 10-12 m2/s at 60 °C. The results of the diffusion measurements are summarized in Table 1. Discussion In this work, we compared the molecular order and diffusion in DPTAP bilayers with that of zwitterionic DPPC, both on a solid support. We have previously shown for DPPC that the presence of a solid support does not significantly alter the order parameter profile and the (16) Bloom, M.; Sternin, E. Transverse Nuclear Spin Relaxation in Phospholipid Bilayer Membranes. Biochemistry 1987, 26, 2101-2105.
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ated DPTAP would be required to establish the asymmetry in molecular order.
Figure 6. Plot of 1/T2CPMG(τ) obtained from Figure 5 versus τ2. The slope of the linear fit (full line) gave b, from which the diffusion coefficient D was calculated. Table 1 sample
diffusion coefficient D/10-12 m2/s
DPPC-d62 DPTAP-d62 without NaCl DPTAP-d62, 150 mM NaCl
14.2 ( 1.2 13.2 ( 1.5 10.6 ( 1.0
diffusion (compared to multilamellar vesicles) when chain perdeuterated lipids are used as probe molecules.9,12 However, owing to the intrinsic problems with pure DPTAP bilayer formation as mentioned in the Introduction, a direct comparison between the supported and the nonsupported states of DPTAP with respect to order and diffusion cannot be made. DPTAP-d62 bilayers are more highly ordered than those of the zwitterionic DPPC-d62. The region close to the headgroup shows significantly higher values of the order parameter, indicating reduced fluctuations of these chain segments with respect to the molecular director axis. However, in the vicinity of the bilayer center both DPPC and DPTAP exhibit similar order. As a result, the decrease of molecular order with the chain position is steeper for the cationic lipid. This may suggest that the positively charged headgroup of DPTAP has a significant influence on the molecular packing of the bilayer. However, the addition of 150 mM NaCl did not alter the order parameter profile, even though the Debye screening length is less than 0.8 nm at this ionic strength. This lets us conclude that not the charge but sterical reasons arising from the headgroup structure may account for the steeper gradient of molecular order. Indeed, the DPTAP headgroup is significantly less space filling than the phosphocholine group of DPPC, which will result in tighter packing and would explain the difference in the order parameter profile. Asymmetries in molecular order between the inner and outer monolayers of the bilayer (as suggested by the results of the DSC scans in Figure 2) owing to a stronger interaction of the inner leaflet with the oppositely charged silica surface are not obvious from the NMR spectra. Nevertheless, such asymmetry was clearly demonstrated previously for the case of DPPC.17 However, it became detectable by deuterium NMR only for selectively chain deuterated DPPC while for perdeuterated chains the effect became obscured by superposition of many individual Pake doublets. Further studies using selectively chain deuter(17) Hetzer, M., et al. Asymmetric molecular friction in supported phospholipid bilayers revealed by NMR measurements of lipid diffusion. Langmuir 1998, 14, 982-984.
The results of our diffusion measurements (Table 1) suggest a close resemblance of DPTAP and DPPC diffusion at 60 °C. This is rather unexpected since the positive charge of the DPTAP and the resulting repulsion between adjacent molecules in the bilayer plane may create a free volume different from that for the case of the (neutral) DPPC. In terms of the free volume model of lateral diffusion,18,19 this will result in a different critical free area and may also affect the frictional coefficient. Since the increase of the ionic strength only slightly reduced D (cf. Table 1) although this NaCl addition decreased the Debye screening length below 0.8 nm, we conclude that the headgroup charge does not provide the dominant contribution to D. Assuming that an asymmetry in molecular order exists for DPTAP SSVs similar to that observed previously for DPPC SSVs, we can expect differences in the lateral diffusion between the two monolayers with the inner one (i.e., the monolayer facing the silica) exhibiting significantly slower diffusional motion. Hence, the value of DDPTAP as given in Table 1 should be considered as an average over the two monolayers. Unfortunately, we could not find in the literature any D values for a bilayer consisting solely of DPTAP or of other cationic lipids for comparison, obtained by fluorescence methods such as fluorescence recovery after photobleaching (FRAP) or fluorescence correlation spectroscopy (FCS). For DPPC, such values are available (D ) 1.7 × 10-11 m2/s) at 60 °C measured by FRAP18 and compare well with our results, taking into account that the length scales of the NMR and FRAP techniques are different.20 The two-dimensional diffusion of DNA oligonucleotides on the surface of cationic bilayers as measured by fluorescence techniques shows D values which are only 1 magnitude less than our D values for the lipids.21,22 Extrapolating these DNA diffusion coefficients to molecular sizes comparable to those of a lipid provides D values that are close to those obtained for the DPTAP. This raises the question about a possible diffusive coupling between the negatively charged DNA and the cationic lipids. After all, the DNA does not diffuse inside but rather on top of the bilayer where geometrical constraints for diffusive motion comparable to that within the bilayer plane do not exist. Further NMR studies presently under way in our lab will concentrate on the interaction of DNA with the cationic lipids in the supported bilayer in order to obtain insight into the dynamical consequences of this interaction. LA026468S (18) Vaz, W. L. C.; Clegg, R. M.; Hallman, D. Translational diffusion of lipids in liquid crystalline phases phosphatidylcholine multibilayers. A comparison of experiment with theory. Biochemistry 1985, 24, 781786. (19) Galla, H. J., et al. On two-dimensional passive random walk in lipid bilayers and fluid pathways in biomembranes. J. Membr. Biol. 1979, 48, 215-236. (20) Karakatsanis, P.; Bayerl, T. M. Diffusion measurements in oriented phospholipid bilayers by 1H NMR in a static fringe field gradient. Phys. Rev. E 1996, 54, 1785-1790. (21) Ra¨dler, J. O.; Maier, B. Conformation and Self-Diffusion of Single DNA Molecules Confined to Two Dimensions. Phys. Rev. Lett. 1999, 82 (9), 1911-1914. (22) Maier, B.; Ra¨dler, J. O. DNA on fluid membranes: A model polymer in two dimensions. Macromolecules 2000, 33 (19), 7185-7194.
