Cholesterol Increases the Magnetic Aligning of Bicellar Disks from an

Jun 23, 2012 - mol % cholesterol the disk size and the magnetic alignability were .... at PSI, Villigen, Switzerland, using a superconductive magnet w...
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Cholesterol Increases the Magnetic Aligning of Bicellar Disks from an Aqueous Mixture of DMPC and DMPE−DTPA with Complexed Thulium Ions Marianne Liebi,† Joachim Kohlbrecher,‡ Takashi Ishikawa,§ Peter Fischer,*,† Peter Walde,∥ and Erich J. Windhab† †

Laboratory of Food Process Engineering, ETH Zurich, Schmelzbergstrasse 9, 8092 Zurich, Switzerland Laboratory for Neutron Scattering, Paul Scherrer Institute, 5232 Villigen PSI, Switzerland § Biomolecular Research Laboratory, Paul Scherrer Institute, 5232 Villigen PSI, Switzerland ∥ Department of Materials, ETH Zurich, Wolfgang-Pauli-Strasse 10, 8093 Zurich, Switzerland ‡

ABSTRACT: Aqueous mixtures of 1,2-dimyristoyl-sn-glycero-3phosphocholine (DMPC), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine−diethylenetriamine pentaacetate (DMPE−DTPA) with complexed thulium ions (Tm3+), and cholesterol with varying molar ratio were studied at different temperatures in the presence and absence of a magnetic field. For mixtures without cholesterol weakly magnetically alignable small disks, so-called bicelles, are formed at temperatures below the phase transition temperature (5− 22 °C), as shown by cryo-transmission electron microscopy (cryoTEM) and small-angle neutron scattering (SANS). In presence of 16 mol % cholesterol the disk size and the magnetic alignability were larger within the entire temperature range studied (5−40 °C). Cholesterol acts as a spacer between DMPE−DTPA with complexed Tm3+, allowing these molecules to integrate more frequently into the planar part of the bicelles. Replacing DMPC partially by cholesterol thus lead to an increase in magnetic aligning by a higher amount of the magnetic handles (Tm3+ complexed to DMPE-DTPA) in the plane and by an increased number of phospholipids in the enlarged bicelles. The magnetic aligning was most pronounced at 5 °C. The temperature-dependent structural changes of the DMPC/cholesterol/DMPE− DTPA/Tm3+ aqueous mixtures are complex, including the transient appearance of holes in the disks at intermediate temperatures.

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INTRODUCTION Bicelles, bilayered micelles, are extensively studied because of their use as biomembrane mimetic material.1−3 They provide a fully hydrated phospholipid bilayer fragment, allowing the study of membrane-associated biomolecules, such as integral membrane proteins, for keeping the protein’s native conformation.4,5 Under the right conditions bicelles align in a magnetic field. The diamagnetic susceptibility anisotropy Δχ of a single phospholipid molecule, determining its preferred orientation in a magnetic field, is summed up in a bicellar aggregate with the aggregation number n. Thus, for a large enough bicelle the thermal fluctuation energy kT can be overpowered by the potential energy in an external magnetic field.6,7 The magnetic orientation energy Emag in a magnetic field H is calculated as follows 1 Emag = − μ0 nΔχH2 2 where μ0 is the magnetic constant. Emag can be increased by doping the membrane with lanthanide ions (such as Tm3+) conferring a large paramagnetic susceptibility Δχ.8−10 Especially structural determinations of membrane proteins using © 2012 American Chemical Society

NMR spectroscopy profit from the possibility of the aggregates to be aligned in magnetic fields.2,5,11,12 The alignment causes dipolar coupling, thereby increasing the accuracy of the protein structure determination.3,13,14 Besides their use in NMR spectroscopy, bicelles have recently been described as advantageous model in drug/ membrane partitioning studies, as more surface area is accessible as compared to closed vesicles.15−17 Well-known bicellar mixtures are composed of a long chain phospholipid (mostly DMPC, 1,2-dimyristoyl-sn-glycero-3phosphocholine) building the planar part and a short chain phospholipid (DHPC, 1,2-dihexanoyl-sn-glycero-3- phosphocholine) covering the highly curved edges.18−22 Bicelles also form in mixtures of long chain phospholipids with a detergent,23−25 with poly(ethylene glycol)-conjugated lipids (PEG-lipids)15,26 or with the T-shaped amphiphile A6/6.27 We investigated a mixture of DMPC and DMPE−DTPA (1,2dimyristoyl-sn-glycero-3-phosphoethanolamine−diethylenetriReceived: May 11, 2012 Revised: June 20, 2012 Published: June 23, 2012 10905

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Figure 1. Schematic illustration on how cholesterol is supposed to increase the magnetic alignability of bicelles from an aqueous mixture of DMPC and DMPE−DTPA with complexed thulium ions Tm3+. Cholesterol acts as a spacer molecule to drive the magnetic handles (DMPE−DTPA·Tm3+) from the bicelle edges toward the bicelle plane. Replacing DMPC partially with cholesterol thus led to an increase in disk size, increasing the magnetic orientation energy Emag through the aggregate number n. In addition, DMPE−DTPA·Tm3+ integrated in the plane can now contribute to Emag with its large value of magnetic susceptibility anisotropy Δχ.

amine pentaacetate) with complexed lanthanides as “magnetic handle”.28 In this system the large headgroup of the chelator lipid with the complexed thulium ion (denoted as DMPE− DTPA·Tm3+) is localized preferentially at the edges of the bicelles.28 One drawback of the location of the chelator lipid with Tm3+ in the rim of the bicelles is the fact that they do not contribute to Emag, resulting only in a weak alignment of the bicelles. Implementing more chelator lipid in the plane of the bicelles requires a better mixing with the plane-forming lipid DMPC, which can be achieved by addition of a spacer molecule to counteract curvature introduced by the large headgroup of DMPE−DTPA·Tm3+. In this work, we investigated cholesterol as possible spacer molecule to drive DMPE−DTPA·Tm3+ from the bicelle edges toward the bicelle plane as illustrated in Figure 1. From literature it is well-known that cholesterol has the capacity to modulate the physical properties and lateral organization of lipid bilayers.29,30 According to the umbrella model, the mainly hydrophobic cholesterol must be covered by neighboring polar head groups of the phospholipids to be protected from interactions with water.31,32 With this model derived from Monte Carlo simulations, the following effects of cholesterol can be explained. With increasing cholesterol content the phosphocholine surface area per molecule is decreased, as the acyl chains and cholesterol pack more tightly in the limited space under the headgroup (condensing effect); because of this denser packing, the permeability of the bilayer is reduced. The acyl chains have less conformational freedom (ordering effect), and the bilayer thickness increases. Because of this ordering effect, the phase transition between the solid-ordered, so, and the liquid-disordered, ld, phases is broadened by the addition of cholesterol, introducing an intermediate liquid-ordered phase, lo.29,30,33,34 Furthermore, it has been suggested that the incorporation of cholesterol increases the lateral distance between the head groups in the bilayer29 and decreases spontaneous curvature in a monolayer.35 This property can be used to form planar bilayers, if cholesterol is mixed with a surfactant, building micelles on its own.36

In this study, bicellar mixtures of DMPC with DMPE− DTPA·Tm3+ were investigated regarding different mixing ratios and the influence of the temperature on structure. The main focus was on the incorporation of cholesterol into the mixtures in order to increase the magnetic alignability of the aggregates (Figure 1). The main characterization methods were cryotransmission electron microscopy (cryo-TEM) for direct visualization of the structures and small-angle neutron scattering (SANS) measurements in a magnetic field of up to 8 T in order to simultaneously characterize structure and alignment of the bicellar mixtures. As additional tool to detect magnetic aligning, birefringence measurements were carried out inside a superconducting magnet with maximum field strength of 5.5 T. Structural changes occurring were followed by turbidity measurements varying the temperature.

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MATERIALS AND METHODS

Materials. The phospholipids, 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine−diethylenetriamine pentaacetate (DMPE−DTPA), were purchased as chloroform solutions from Avanti Polar Lipids (Alabaster, AL) and used without further purification. TmCl3 (99.9%), cholesterol (99%), D2O (99.9 atom % D), and methanol (99.8%) were from Sigma-Aldrich (Buchs, Switzerland); chloroform (stabilized with EtOH) was from Ecsa (Balerna, Switzerland). Stock solutions of 10 mM TmCl3 in MeOH and of 10 mM cholesterol in chloroform were prepared. Bicelle Preparation. The bicelles were produced as described previously;28 unless otherwise indicated for a better pH control a sodium phosphate buffer (50 mM, pH 7.5) was used. For SANS measurements, the buffer salt was dissolved in D2O and HCl was added to give a pH meter reading of 7.0 (corresponding to a pD of 7.4).37 After weighing appropriate amounts of the lipid solutions and lanthanides into a round-bottom flask, chloroform and methanol were evaporated with a rotary evaporator followed by residual solvent removal under high vacuum overnight. Afterward, the dry lipid film was hydrated with buffer solution and vortexed until a homogeneous suspension was obtained. Five repeated freeze−thaw cycles were carried out by plunging the flask into liquid nitrogen followed by slow heating above the phase transition temperature (about 40−45 °C). The extrusion was performed under nitrogen at 40 °C with “The 10906

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and detected by a photodetector (Hinds Instruments). The first and second harmonic I1ω and I2ω of the ac signal were detected with two lock-in amplifiers (SR830, SRS, Sunnyvale, CA). The PEM amplitude A0 was chosen to be 2.405 rad, therewith making the dc component birefringence independent. The birefringence Δn is obtained using

Extruder” from Lipex Biomembranes (Vancouver, Canada) and Nucleopore polycarbonate membranes from Sterico (Dietikon, Switzerland). The suspension was first passed 10 times through a membrane with 200 nm pores, followed by 10 times through a membrane with 100 nm pores. Samples were stored before measurements at room temperature and were stable over at least 1 week. The method of preparation is crucial to obtain bicelles as shown and discussed previously.28 SANS. SANS experiments were performed on the SANS-I beamline at PSI, Villigen, Switzerland, using a superconductive magnet with horizontal field of 8 T perpendicular to the neutron beam. The neutron wavelength was fixed at 8 Å. Data were collected on a twodimensional 3He detector at distances of 2, 6, and 18 m covering a momentum transfer of 0.003 ≤ q ≤ 0.15 Å−1. All data were corrected for background signal from the empty setup, transmission, and detector efficiency. A part of the samples (Figure 3) were also studied without a superconductive magnet in standard temperature controlled holders for cuvettes. For fitting the software package SASfit was used.38 For the disk-shaped bicelles a Porod’s approximations for cylinder was used as a form factor.39 Vesicles were fitted using a form factor for spherical shells with an inner radius R and a thickness of the shell ΔR.38 To implement the appearance of holes in the disks, a cylindrical shell with circular cross section was used. The inner radius describes the hole within the bilayered disk, which is represented by the shell thickness. The length of the cylindrical shell depicts the thickness of the disk. Polydispersed samples were fitted with a log− normal distribution

Δn = − with

⎛ I1ωJ (A 0) ⎞ ⎟⎟ δ = arctan⎜⎜ 2 ⎝ I2ωJ1(A 0) ⎠ where d is the thickness of the sample (d = 10 mm), λ is the wavelength of the laser (λ = 635 nm), and J1 and J2 are Bessel functions of the first kind, with J1(2.405) = 0.5191 and J2(2.405) = 0.4317. The measured values of the birefringence were normalized with the values measured at 0 T. Turbidity Measurements. Turbidity measurements were carried out with a temperature-controlled double-beam spectrophotometer (V-670 spectrophotometer, Jasco, Hachioji, Japan). The wavelength was set to 400 nm, where no absorption peak of the sample itself was found in a spectrum taken at room temperature from 190 to 850 nm. Changes in transmitted intensity are expressed as optical density (l = 1 cm). The temperature was decreased from the starting temperature of 40 to 5 °C with a temperature rate of 1 °C/min. The temperature was hold at 5 °C for 5 min prior to the heating with the same temperature rate.

p(R , σ , μ) = (R 2πσ 2 )−1 exp(− (ln R − ln μ)2 /2σ 2)

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with the location parameter μ and the width parameter σ. To extract a measure of orientation from the scattering pattern, an alignment factor Af was calculated (adapted from ref 40): A f (q) =

δλ 2πd

RESULTS AND DISCUSSION In the following, data on the structure of the cholesterol-free system DMPC:DMPE-DTPA·Tm3+ are presented first. Then, the changes induced in structure and magnetic aligning by adding cholesterol to this system are described and discussed. DMPC/DMPE−DTPA·Tm3+. Previously, we have shown that bicelles form after appropriate preparation steps in aqueous solution from DMPC, DMPE−DTPA, and Tm3+ at certain lipid and Tm3+ concentrations and at certain molar ratio of the two lipids.28 The standard sample with a molar ratio of DMPC:DMPE−DTPA:Tm3+ of 4:1:1 is composed of bicelles with an average diameter of 36 ± 6 nm, as determined by cryoTEM (x = 54) (see Figure 2B).28 If the chelator lipid content is increased to a molar ratio of 3:2:2, the bicelles decrease in size to an average diameter of 22 ± 4 nm (x = 38), as shown in Figure 2A. If the content of chelator lipid DMPE−DTPA is decreased to 10 mol % (DMPC:DMPE−DTPA:Tm3+ = 9:1:1), mainly vesicles are found as shown in Figure 2C. These results were confirmed by SANS measurements at 25 °C (Figure 3). For a low chelator lipid content (DMPC:DMPE−DTPA:Tm3+ = 9:1:1) the scattering is dominated by vesicles, represented in the fit by a form factor for spherical shells with a membrane thickness of 4.2 nm, which is in good agreement with literature values.45 The vesicle radius was fitted with a log−normal distribution with μ = 37 nm and σ = 0.27. At a higher content of DMPE−DTPA·Tm3+ (molar ratio DMPC:DMPE−DTPA:Tm3+ = 4:1:1), the scattering intensity decays with q−2, which is typical for disklike structures,46 as found in cryo-TEM images (Figure 2B). The best fit resulted in disks with a thickness of 4 nm; the radius is polydisperse with μ = 9.1 nm and σ = 0.674. The reason for the large polydispersity might be the occurrence of larger ripple phase structures, as also found in cryo-TEM (data not shown). This is in agreement with observations made with other types of bicellar systems based on mixtures of DMPC and DHPC lipids.47 At high chelator lipid concentration the scattering

∑ I(ϕ) cos(2ϕ) ∑ I(ϕ)

Af are the second cosine Fourier coefficients of the normalized azimuthally intensity I(ϕ). The values of Af range between 1 (scattering in direction of ϕ = 0, perpendicular to the magnetic field direction) and −1 (scattering in direction of ϕ = 90°, parallel to the magnetic field), with Af = 0 for isotropic scattering. The q range for the evaluation of Af was set to 0.03−0.04 Å−1. Cryo-TEM. The samples were analyzed by cryo-transmission electron microscopy (cryo-TEM) following the procedure described previously.41 A volume of 3.5 μL of the bicellar suspension was mounted onto holey carbon grids (Quantifoil, Jena, Germany), blotted to make thin aqueous films under controlled temperature and humidity, followed by plunging into liquid ethane at the temperature of liquid nitrogen with a VitrobotTM apparatus (FEI, Eindhoven, The Netherlands). The grids were examined at the temperature of liquid nitrogen using a cryo-holder (model 626, Gatan) and a Tecnai G2 F20 microscope (FEI) equipped with a field emission gun and Tridem energy filter (Gatan) operated at an accelerating voltage of 200 kV. The data were recorded with 5−10 μm underfocus by using a 2048 × 2048 CCD camera (Gatan). The size of the bicelles was determined by analyzing various micrographs. The number of bicelles counted, x, is given in the text. The small very contrast rich particles seen in the micrographs are ice crystal contaminations from the freezing procedure. Birefringence Measurements. Birefringence measurements based on the phase modulation technique in a magnetic field were used as a method to detect orientation of the bicellar disks.42−44 A photoelastic modulator (PEM-90, Hinds Instruments) set to operation frequency of 50 kHz was placed between two crossed linear polarizers (Newport, Irvine, CA); a diode laser (Newport, Irvine, CA) with a wavelength of 635 nm was used as light source. The sample was placed in a temperature-controlled cuvette (Hellma, Germany) inside a superconducting magnet (Cryogenic, UK) with maximum field strength of 5.5 T. The laser light propagates horizontally through the sample, deflected by nonpolarizing mirrors (Newport, Irvine, CA) 10907

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Figure 3. Radial averaged SANS curves and corresponding fit of DMPC:DMPE−DTPA:Tm3+ for molar ratio of 3:2:2, 4:1:1, and 9:1:1, respectively. Total lipid concentration was 15 mM, and measurements were carried out at 25 °C. The curves have been shifted vertically for better clarity.

potential, taking into account the interaction of the aggregates in the unbuffered solution. In addition fits of the form factors only are shown (dashed lines). The scattering curves taken at 5 and 22 °C are in agreement with small bicellar disks as described previously.28 The curves can be fitted well with a form factor for flat cylinders39 with a thickness of 4.5 nm and a log−normal distributed radius with μ = 13.5 nm and σ = 0.156. At 35 °C, the size of the disks as well as its polydispersity increases. The best fit was composed of two populations, both with a thickness of 3.7 nm and a log−normal distributed radius with μ = 16.5 nm and σ = 0.245 and μ = 39.1 nm and σ = 0.021, respectively. At 45 and 60 °C the scattering curve can be described best with a form factor for spherical shells (45 °C: d = 3.7 nm, μ = 16.9 nm, σ = 0.303; 60 °C: d = 3.5 nm, μ = 14.8 nm, σ = 0.359), indicating that the bilayers closed into vesicles at those temperatures. This was confirmed by cryo-TEM micrographs taken from samples which were heated to 60 °C prior to quick-freezing, showing mainly vesicles (data not shown). We assume that lipid segregation between the edge-forming DMPE-DTPA·Tm3+ molecules and the bilayer-forming phospholipid DMPC below the phase transition Tm is responsible for the formation of the disklike structures. At temperatures above Tm, where the lipids are in the liquid-disordered, ld, phase, the lipids are mixed and lead to larger bilayers, which are self-closing into vesicles above a certain size. The membrane thickness decreases with increasing temperature, in accordance with the literature.45 If the bilayer-forming phospholipid DMPC is replaced by POPC, which has a much lower phase transition temperature (Tm = −2.5 ± 2.4 °C48), only vesicles were observed in the temperature regime of 5−30 °C. In this case, lipid segregation caused by the phase transition of DMPE-

Figure 2. Cryo-TEM micrographs of lipid mixtures of DMPC and DMPE−DTPA with complexed Tm3+, total lipid concentration 15 mM, frozen from room temperature. Molar ratio of DMPC:DMPE− DTPA:Tm3+ was 3:2:2 (A), 4:1:1 (B), and 9:1:1 (C); scale bar = 200 nm. The small particles with high contrast are ice crystals.

curve cannot be explained by the presence of small disks alone; the best fit was obtained by inclusion of a population of rods with a diameter of 6.5 nm and a length of 22.7 nm together with a smaller population of disks with similar sizes. In the obtained cryo-TEM (Figure 2A) the distinction between rods and disks in edge-on view is not possible. This observation is consistent with the assumption of partial lipid segregation in such lipid aggregates, where the chelator lipid DMPE−DTPA with the complexed lanthanides is located preferably in the highly curved edge region of the bicelle, due to the large headgroup. DMPE−DTPA with complexed Tm3+ alone forms micelles in aqueous solution,28 confirming the preference of the complex for highly curved assemblies. Figure 4 shows SANS measurements of DMPC:DMPE− DTPA:Tm3+ at 15 mM in the intermediate molar ratio of 4:1:1 at 5, 22, 35, 45, and 60 °C. Fit curves shown in Figure 4 as solid lines include a structure factor of a repulsive interaction 10908

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Without cholesterol the size of the disks was 36 ± 6 nm (Figure 2B).28 Addition of cholesterol led to an increase in disk size, as obvious from cryo-TEM images (Figure 5). In the

Figure 4. Radial averaged SANS curves and corresponding fits of DMPC:DMPE−DTPA:Tm3+ in a molar ratio of 4:1:1 and a total lipid concentration of 15 mM at temperatures from 5 to 60 °C. Samples were prepared at pH 7 (at 25 °C) without buffer. The curves have been shifted vertically for better clarity.

Figure 5. Cryo-TEM micrographs of lipid mixtures of DMPC and DMPE−DTPA·Tm3+ containing 16 (A) and 40 mol % cholesterol (B) frozen from room temperature. The bicelles get larger with increasing cholesterol until they are self-closing into vesicles. Total lipid concentration 15 mM, molar ratio of (DMPC + cholesterol):DMPE−DTPA:Tm3+ was 4:1:1. Scale bar = 200 nm. The small particles with high contrast are ice crystals.

DTPA·Tm3+ below 25 °C lead to the formation of solid domains, surrounded by POPC in the ld phase.49 As discussed previously, the alignability of bicelles from a mixture of DMPC and DMPE−DTPA·Tm3+ is only weak.28 This is mainly due to the fact that the disks are relatively small and that the magnetic handles (large Δχ of DMPE− DTPA·Tm3+) are located preferentially in the curved edge region of the bicelle and thus do not contribute to the magnetic aligning. DMPC/Cholesterol/DMPE−DTPA/Tm3+. To investigate whether the presence of cholesterol increases the magnetic alignability of the disks by increasing the amount of DMPE− DTPA·Tm3+ in the plane of the bicelles (as illustrated in Figure 1), mixtures of DMPC, cholesterol, and DMPE−DTPA with complexed Tm3+ were analyzed. Cholesterol is well-known from the literature30,32,50 to integrate in an upright position into the membrane and therefore might act as a spacer between the large headgroup of the chelator lipid. This may lead to larger bicelles and thus to an increase in the number of associated magnetic field sensitive centers, n, and more chelator lipid with large Δχ contributing to Emag. Different mixtures of DMPC and DMPE−DTPA·Tm3+ with cholesterol were investigated. The total lipid concentration (including cholesterol) was kept constant at 15 mM, and the chelator lipid·thulium complex, DMPE−DTPA·Tm3+, accounted for 20 mol %. The bilayer forming phospholipid DMPC was gradually replaced by cholesterol to a cholesterol content of 12.5, 16, 19.2, and 40 mol %.

presence of 16 mol % cholesterol the disk diameter was 110 ± 23 nm (x = 146, see Figure 5A); cryo-TEM micrographs indicated the presence of a few vesicles. With addition of 40 mol % cholesterol almost exclusively vesicles were found (Figure 5B). SANS measurements are in agreement with an increase of the bicellar disk size with the addition of cholesterol (Figure 6). For cholesterol contents of 12.5, 16, and 19.2 mol % the scattering intensity again decays with q−2 typical for disks (indicated by the slope of −2). The deviations indicated with arrows in Figure 6 are most likely caused by scattering contributions from the ripple phase. For the sample without cholesterol the peak is quite broad between q = 0.24 nm−1 and q = 0.45 nm−1, corresponding to a size range from 14 to 26 nm. These values correspond well with ripple repeated distances reported in the literature, which show two dominant values at about 13 and 26 nm.51,52 According to the literature, the addition of cholesterol eliminates the bimodal distribution. With increasing cholesterol content the ripple repeat distance increases, whereas the amplitude decreases and above 15 mol % cholesterol no ripples could be detected.51 The same trend was observed in the scattering curves depicted in Figure 6, 16 mol % being the highest cholesterol concentration where a peak could be detected. As the ripple phase was not the main focus 10909

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Tm3+ complexes to be part of a flat bilayer to a larger extent than in the absence of cholesterol (see Figure 1). This is somewhat in apparent contrast with the structural behavior reported for the system composed of unsaturated phospholipids and PEG lipids, where the addition of cholesterol reduced the amount and size of liposomes in a mixture of different aggregate morphologies.53 However, in the same mixture and in mixtures with saturated phospholipids, cholesterol induces the transition from long threadlike micelles to discoidal structures, in which the edge area is minimized. This is similar to the curvature-reducing effect of cholesterol found in our system with saturated phospholipid and the chelator lipids. For the eggPC/PEG lipid system, Sandtröm et al.53 explained the vanishing of liposomes with inclusion of cholesterol with the increased bending modulus of the lipid membrane in the lo phase compared to the modulus in the ld phase. Differences in the physicochemical properties of PEG and the chelator headgroup are obvious since DMPE−PEG2000 above 10 mol % cannot be integrated into a planar bilayer even in the ld phase.54 On the other hand, DMPE−DTPA·Tm3+ can be integrated into a planar bilayer up to 20 mol % in the ld phase (Figure 4) or at a high cholesterol content (Figure 5). As a result of the increased disk size and the increased thulium content in the plane, the magnetic alignability of the disks was enhanced significantly with the addition of cholesterol as shown in Figure 7. In a mixture of DMPC:DMPE−

Figure 6. Radial averaged SANS curves and corresponding fit of lipid mixtures of DMPC and DMPE−DTPA·Tm 3+ with different cholesterol contents. Total lipid concentration was 15 mM, and measurements were carried out at 20 °C (cooled from room temperature) for 12.5, 16, and 19.2 mol % cholesterol content and at 25 °C for 0 and 40 mol % cholesterol content. Arrows indicate Bragg peaks originated by ripple phase structures. The curves have been shifted vertically for better clarity.

Figure 7. SANS 2D scattering pattern of DMPC:DMPE−DTPA:Tm3+ 4:1:1 (A) and DMPC:Chol:DMPE−DTPA:Tm3+ 16:4:5:5 (16 mol % cholesterol) (B) at 5 °C and 8 T; magnetic field direction is horizontal in the plane, detector distance 18 m.

of this study, no detailed fit was implemented. Nevertheless, it is obvious that the scattering at low q values (0.03 nm−1 < q < 0.8 nm−1) increases with increasing cholesterol content, indicating an increase in the size of the bicelles. The scattering curve for a cholesterol content of 40 mol % could be fitted with a form factor for spherical shells, with a thickness of 3.7 nm and a broad log−normal distribution with μ = 36.4 nm and σ = 0.25. The missing forward scattering in the fit can be explained by the presence of elongated vesicles in the sample (as seen by cryo-TEM in Figure 5B). The short elliptical axis being in the size range of the spherical vesicles and the long axis being responsible for the increased forward scattering compared to the fit, which accounted for spherical vesicles only. In conclusion, SANS and cryo-TEM are in agreement with an increased disk size with increasing cholesterol content; the bilayer self-closes into vesicles upon a certain size, i.e., cholesterol content. It seems that cholesterol indeed acts as a spacer between the large headgroup of the DMPE− DTPA·Tm3+ molecules, thus allowing these chelator lipid

DTPA:Tm3+ in a molar ratio of 4:1:1 the disks were only weakly aligned (Figure 7A) at 5 °C and a magnetic field of 8 T.28 Replacing 16 mol % DMPC by cholesterol resulted in an highly anisotropic scattering pattern, indicating a higher degree of orientation (Figure 7B). The disks are oriented with their normal parallel to the magnetic field, as can be expected for phospholipid bilayers doped with Tm3+.8,10,55 The anisotropy of the scattering patterns taken at different temperatures was characterized by the alignment factor Af. The larger the modulus of this value gets, the more anisotropic is the scattering pattern indicating a higher degree of orientation in a magnetic field of 8 T. A negative value of Af means an orientation of the normal of the disk surface toward the magnetic field direction and a positive value perpendicular to it. As shown in Figure 8, the alignment factor at 5 °C was reduced from −0.08 for DMPC:DMPE−DTPA:Tm3+ (4:1:1) to −0.36 if 16 mol % of DMPC was replaced by cholesterol (DMPC:cholesterol:DMPE−DTPA:Tm3+, 16:4:5:5). For the bicelles containing cholesterol, the orientation decreased with increasing temperature in the temperature range from 5 to 40 10910

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Figure 8. Alignment factor calculated from SANS measurements at 8 and 0 T from 5 to 40 °C with a heating rate of about 0.5 °C/min of DMPC:Chol:DMPE−DTPA:Tm3+, molar ratio 16:4:5:5 (16 mol % cholesterol) and DMPC:DMPE−DTPA:Tm3+, molar ratio 4:1:1 at 5 and 30 °C, total lipid concentration 15 mM.

Figure 9. Change of birefringence in a DMPC/Chol/DMPE−DTPA/ Tm3+ aqueous mixture between 5 and 50 °C, measured at 0 T (gray) and 5.5 T (black, solid line: cooling down; dashed line: heating up). Molar ratio 16:4:5:5 (cholesterol content 16 mol %), total lipid concentration 15 mM. Heating and cooling rate was 0.3 °C/min.

°C, but was at 40 °C with Af = 0.12 still significantly higher than any alignment observed in the sample without cholesterol. It is worth noting that the observed higher alignability at lower temperatures is different from the behavior of other bicellar systems composed of DMPC and DHPC, sometimes doped with Tm3+, which are extensively discussed in the literature.19,20,22,23 For such mixture the magnetic alignable phase is found above the phase transition temperature of DMPC and is not composed of disks but of long, wormlike micelles47,56 which transform into extended perforated lamellae with the addition of charged lipids or ions (e.g., Tm3+).19,57 Below the phase transition of DMPC, a mixture of DMPC and DHPC forms small bicelles, which cannot be aligned in magnetic field. This is in contrast to the system described here, which shows most pronounced orientation at temperatures below the phase transition temperature. The increased alignability at lower temperatures can be partly explained by the increased Δχ value of the phospholipids below Tm caused by the higher molecular order in the so phase.58 In addition, the thermal fluctuation energy kT opposing Emag is decreased with decreasing temperature. Furthermore, a structural change in the sample during cooling could also add to the increased alignability. To clarify this point, additional measurements at different temperatures were carried out. The change in birefringence of a sample of DMPC/ cholesterol/DMPE−DTPA·Tm3+ as a function of temperature was measured in the absence and presence of a magnetic field of 5.5 T, as shown in Figure 9. Birefringence is a sensitive tool to measure orientation of optically anisotropic structures. Without applied magnetic field no change in birefringence was detected over the whole investigated temperature range of 5− 50 °C, as expected. At a magnetic field strength of 5.5 T the birefringence signal increased with decreasing temperature, indicating an enhanced orientation of the molecules, supporting the findings of the SANS measurements. A temperature hysteresis was found, where the birefringence increased to higher values if the sample was heated up again. This process was reversible after reaching 50 °C. It is worth mentioning that the temperature hysteresis in the birefringence measurement was not observed for the alignment factor extracted from SANS measurements. Therefore, the hysteresis might not be caused by a better aligning of the

aggregates, but rather by a significant change of the aggregate structure. This was indeed the case (see below). The measured birefringence is composed of a form component and an intrinsic component.59 The intrinsic birefringence is normally much stronger and depends on the species and the orientation of the individual molecules within the aggregate. The form birefringence depends on differences between the refractive indices of the aqueous solution and the bilayer and varies e.g. with the thickness of the lipid bilayer or with stacking of lamellae, if such stacking would exist. For optically anisotropic samples, aligned by the magnetic field, the measured birefringence hence depends on the internal structure (molecular organization within the aggregates) and the shape of the aggregates, as well as on the order parameter (degree of aligning of the aggregates).60 The magnetic field strength available for these measurements allowed only measurements in the Cotton−Mouton regime, where the induced birefringence is proportional to the squared magnetic field strength H2.43 No full alignment was reached; thus, a determination of the order parameter was not possible. Turbidity measurements in a temperature-controlled double beam spectrophotometer were carried out at a wavelength of 400 nm in order to observe whether the bicellar systems show significant temperature dependency of the light scattering without applied magnetic field. As also observed visually, the optical density of the sample increased if the sample was cooled down (Figure 10). The increased optical density at low temperatures corresponds to findings on DMPG assemblies, showing that the dispersion turbidity in the gel phase is higher than in the fluid phase.61 Similar to birefringence measurements, there was a temperature hysteresis. The sample got very transparent at about 20−25 °C during heating up from 5 to 40 °C. The hysteresis was reversible after cycling through 40 °C. The position of the transparent region was slightly shifted, if the temperature rate was changed. If the temperature ramp was stopped at 20 °C, the low optical density (A ≈ 0.4, see Figure 10) stayed constant for at least 1 h. If the sample was heated up to 25 °C, the value of optical density increased immediately to 10911

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Figure 10. Absorbance of a sample of DMPC:Chol:DMPE− DTPA:Tm3+ (molar ratio 16:4:5:5, 16 mol % cholesterol, total lipid concentration 15 mM) at a wavelength of 400 nm between 5 and 40 °C with a temperature rate of 1 °C/min. Solid line: cooling down; dashed line: heating up.

0.7. The similar appearance of the temperature hysteresis in the magnetic birefringence measurements and the optical density measurements without magnetic field support the assumption that significant structural changes occur in the sample upon varying the temperature. These changes in structure are not caused by the applied magnetic field. SANS measurements without applied magnetic field were carried out at 40, 20, 10, and 5 °C in order to obtain structural information for various temperatures (Figure 11). The membrane thickness, obtained from a Porod Cylinder fit, increased with decreasing temperature as expected, from 3.8 nm at 40 °C to 4.2 nm at 10 °C and 4.4 nm at 5 °C. The scattering curves showed a temperature hysteresis for cooling down and heating up, respectively, supporting findings from birefringence and turbidity measurements. Whereas the curve taken at 20 °C during cooling is consistent with the presence of bicellar disks (slope −2, deviation originated from ripple phase as described above), the scattering curve at 20 °C during heating cycle could be best described with a form factor for a flat, hollow, cylindrical shell, describing a disk with a hole. For the radius of the hole a log−normal distribution was assumed, best fit values resulted in μ = 19 nm and σ = 0.50, surrounded by a rim of 23 nm. Cryo-TEM micrographs were taken from samples of DMPC:Chol:DMPE−DTPA:Tm3+ (molar ratio 16:4:5:5, total lipid concentration 15 mM) subjected to different temperature histories. Figure 12A shows a sample, which was cooled from 40 to 20 °C before quick-freezing; mostly spherical disks can be seen from different angles. The sample was cooled down to 5 °C and heated up again to 20 °C in order to capture the transparent phase found with the absorbance measurements. SANS measurements indicate that the bicelles appear as perforated disks. We also see white spots in the cryomicrographs (Figure 12B, arrows) supporting our interpretation of the SANS data. Furthermore, some of the bilayered aggregates have a more elongated shape. Even more of such elongated structures beside circular disks were found in micrographs from a sample, which was kept in an ice bath before quick-freezing (Figure 12C). The perforated disks seem to be responsible for the appearance of a transparent intermediate phase, comparable with the transparent intermediate phase of the charged

Figure 11. Radial averaged SANS curves from DMPC:Chol:DMPE− DTPA:Tm3+, molar ratio 16:4:5:5 (16 mol % cholesterol), total lipid concentration 15 mM, measured without magnetic field applied at 40, 20 (cooling −1 and heating −2), 10, and 5 °C. The curves have been shifted vertically for better clarity.

phospholipid DMPG, which is built up by perforated vesicles.62−65 It is known that small pores (1.0−1.7 nm) open in lipid bilayers at the phase transition temperature.66 However, extensive perforation at the phase transition leading to decreased bilayer optical contrast and therefore to a decreased sample turbidity was so far only found for short chain PG lipids at low ionic strength.61,67 DSC data of DMPG show that the phase transition is extended over more than 10 °C, corresponding to the transparent intermediate phase mentioned. In contrast to our system, however, no temperature hysteresis has been reported in those cases. In addition, we found that the transient structure is also influenced by the cholesterol content. In a sample with a molar ratio of DMPC:Chol:DMPE−DTPA:Tm3+ of 16:3:5:5 (12.5 mol % cholesterol) the holes seemed to be much more pronounced (Figure 13A) and are less frequently seen in samples with molar ratio of 16:5:5:5 (19.2 mol % cholesterol, Figure 13B). Cholesterol is again acting as a spacer molecule between the edge forming chelator lipids, which tend to build highly curved edge regions, leading to such an intermediate phase. With its small headgroup cholesterol decreases the spontaneous curvature and therefore destabilizes the intermediate phase as was also described for the perforated vesicles consisting of DMPG.35 In samples without cholesterol no such hole formation could be observed neither in SANS nor in cryo10912

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Figure 13. Cryo-TEM micrographs of DMPC:Chol:DMPE− DTPA:Tm3+ which were subjected to 40 °C → 5 °C → 20 °C. Molar ratio was 16:3:5:5 (12.5 mol % cholesterol) (A) and 16:5:5:5 (19.2 mol % cholesterol) (B), respectively. Holes in the disks are more pronounced for lower cholesterol contents. Total lipid concentration 15 mM; scale bar = 200 nm. The small particles with high contrast are ice crystals.

Figure 12. Cryo-TEM micrographs of DMPC:Chol:DMPE− DTPA:Tm3+ (molar ratio 16:4:5:5, 16 mol % cholesterol, total lipid concentration 15 mM), which were subjected to different temperature histories prior to rapid freezing. (A) 40 °C → 20 °C, (B) 40 °C → 5 °C → 20 °C, and (C) was stored in an ice bath before freezing. Scale bar = 200 nm. The small particles with high contrast are ice crystals.

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CONCLUSION We have shown that bicellar disks consisting of DMPC, the chelator lipid DMPE−DTPA with complexed Tm3+, and cholesterol can be aligned in external magnetic fields more efficiently than in absence of cholesterol. The lower the temperature, the better is the aligning. The structural changes occurring with a change of temperature are complex and most likely driven by lipid segregation of the different molecules if the temperature is lowered below the phase transition of DMPC (Tm = 25 °C). The large headgroup of DMPE− DTPA·Tm3+ is thereby responsible for covering the highly curved edge region of the aggregates. The size of the disks is dependent on the molar ratio of DMPC:DMPE−DTPA·Tm3+ and the amount of cholesterol. Cholesterol acts as a spacer between the lipids bearing the large DTPA·Tm3+ headgroup, allowing more of the chelator lipid with complexed Tm3+ to integrate into the plane of the disk. As a consequence of the

TEM, probably due to the significant smaller size of the disks without cholesterol (see Figure 2B). We propose that again lipid segregation is the driving force for the structural change. In phase diagrams of DMPC and cholesterol, there appears a phase transition at about 25 °C. At cholesterol contents from about 5 to 20 mol % the liquidordered phase, lo induced by cholesterol coexists with either the ld (above 25 °C) or the so phase (below 25 °C).34 At temperatures below this phase transition, lipid segregation leads to structures with an increased amount of edge regions; thus, holes appear in the disk or elongated planar aggregates form. The presence of charges might also be necessary for the opening of the holes, as discussed for DMPG61 as well as for the opening of pores caused by the addition of ionic surfactants to lipid bilayers.67 The mechanism of this hole formation needs to be investigated in future studies. 10913

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increased disk size and the increased amount of Tm3+ on the surface of the plane of the disk, the addition of cholesterol leads to a significantly increased magnetic alignability compared to the cholesterol-free system.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: peter.fi[email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The Swiss National Foundation is acknowledged for funding (Project 200021_132132). The authors acknowledge EMEZ for technical support. This work is based on experiments performed at the Swiss Spallation Neutron Source SINQ, Paul Scherrer Institute, Villigen, Switzerland.

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ABBREVIATIONS Chol, cholesterol; DMPC, 1,2-dimyristoyl-sn-glycero-3-phosphocholine; DMPE−DTPA, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine−diethylenetriamine pentaacetate; Emag, magnetic orientation energy; ld, liquid-disordered phase of phospholipids; lo, liquid-ordered phase of phospholipids; SANS, small-angle neutron scattering; so, solid-ordered phase of phospholipids; TEM, transmission electron microscopy; Tm, main phase transition temperature of phospholipids.

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