Alignment of Bicelles Studied with High-Field Magnetic Birefringence

Article pubs.acs.org/Langmuir

Alignment of Bicelles Studied with High-Field Magnetic Birefringence and Small-Angle Neutron Scattering Measurements Marianne Liebi,† Peter G. van Rhee,§ Peter C. M. Christianen,§ Joachim Kohlbrecher,‡ 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 § High Field Magnet Laboratory, Institute for Molecules and Materials, Radboud University Nijmegen, Toernooiveld 7, 6525 ED Nijmegen, The Netherlands ∥ Department of Materials, ETH Zurich, Wolfgang-Pauli-Strasse 10, 8093 Zurich, Switzerland ‡

ABSTRACT: Birefringence measurements at high magnetic field strength of up to 33 T were used to detect magnetically induced alignment of bicelles composed of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), cholesterol, and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-diethylenetriaminepentaacetate (DMPE-DTPA) with complexed lanthanide ions. These birefringence measurements together with a small-angle neutron scattering (SANS) analysis in a magnetic field showed parallel alignment of the bicelles if the lanthanide was thulium (Tm3+), and perpendicular alignment with dysprosium (Dy3+). With the birefringence measurements, the order parameter S can be determined as a function of the magnetic field strength, if the magnetic alignment reaches saturation. Additional structural information can be obtained if the maximum induced birefringence is considered. The degree of alignment of the studied bicelles increased with decreasing temperature from 40 to 5 °C and showed a new bicellar structure comprising a transient hole formation at intermediate temperatures (20 °C) during heating from 5 to 40 °C.

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Magnetically induced birefringence Δn′ has been used to study various types of phospholipid systems31 including purple membrane suspensions, 32 mechanically oriented multilayers,33,34 vesicles,33 and tubular aggregates.35−37 Nevertheless, the potential for investigations of bicellar systems with birefringence in high magnetic field has not yet been exploited. Measurements of Δn′ can be carried out up to the highest fields possible to reach full saturation of the alignment, therewith permitting the determination of the order parameter S and quantitative analysis of the data. The detected Δn′ consists of an intrinsic component, which depends on the species and the molecular organization within the aggregate. For anisotropic shape, there is in addition a form component, which exists due to depolarization and has in general a negative value.36,38,39 At low magnetic fields (or small aggregates), birefringence depends quadratically on the magnetic field.31 For increasing magnetic field strength (or increasing aggregate size), the birefringence converges to the maximum induced birefringence Δn′max, that is, full alignment of the aggregates. As soon as the inflection point of the measured birefringence curve is reached, the order parameter S can be determined. The detected magnetically induced birefringence Δn′ is thereby proportional

INTRODUCTION

Bicelles are disk-like aggregates of amphiphilic molecules (mainly phospholipids) and have gained increasing interest to study membrane associated proteins and their interaction with phospholipids.1,2 Beside their use as biomembrane mimetic material, such nanometer-scaled disks, tunable both in size (e.g., molar ratio, cholesterol content3) and in magnetic orientability (doping with lanthanide ions4), have in addition potential as building blocks for new smart materials, whose properties can be triggered magnetically. So far, the structure of bicelles of different composition was studied intensively with various techniques, such as NMR spectroscopy,5−13 small-angle neutron and X-ray scattering, SANS14−18 and SAXS,19−21 atomic force microscopy AFM 22 electron microscopy EM,11,15,23−25 dynamic light scattering DLS,24,25 differential scanning calorimetry DSC,21 polarized optical microscopy, 17,21,26 Fourier-transformed infrared spectroscopy FTIR,20,21 rheometry,27 and EPR spectroscopy.28,29 Magnetic alignment of bicelles on the other hand has so far mainly been studied using only NMR spectroscopy or SANS in magnetic fields.3,4,13,18 As lanthanides can shift and broaden NMR absorption lines30 and therefore cause substantial problems in applying NMR methods, alternative tools such as birefringence to detect magnetically induced alignment of aggregates in solution are beneficial. © 2013 American Chemical Society

Received: December 21, 2012 Revised: February 13, 2013 Published: February 13, 2013 3467

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to the order parameter S and the maximum birefringence Δn′max: Δn′ = Δn′max ·S

(1)

2

∬ 3 cos 2θ − 1 f (θ) sin θ dθ dϕ

(2)

where θ and ϕ are the polar and azimuthal angles in spherical coordinates, describing the orientation of the long molecular axis with respect to the magnetic field direction, and f(θ) is the Maxwell−Boltzmann distribution function with the assumption of molecules with a cylindrical symmetry axis (orientation energy independent from ϕ):

(

exp f (θ ) =

∬ exp

(

N ΔχB2 cos2 θ 2μ0 NAkT 2

2

N ΔχB cos θ 2μ0 NAkT

)

) sin θ dθ dϕ

MATERIALS AND METHODS

The bicelles studied here are composed of a mixture of DMPC (1,2dimyristoyl-sn-glycero-3-phosphocholine), cholesterol, and DMPEDTPA (1,2-dimyristoyl-sn-glycero-3-phosphoethanolaminediethylenetriaminepentaacetate) with complexed lanthanide ions and were produced as described previously.3,15 Briefly, the compounds were weighed as chloroform and methanol solutions in a round-bottom flask to yield a molar ratio of DMPC:cholesterol:DMPE-DTPA:lanthanide of 16:4:5:5 and a total lipid concentration of 15 mM. After evaporation of the solvents, the resulting dry lipid film was hydrated with a sodium phosphate buffer (50 mM, pH 7.5) and vortexed until a homogeneous suspenstion was obtained. After five freeze−thaw cycles, the samples were extruded 10 times with “The Extruder” from Lipex Biomembranes (Vancouver, Canada) through Nucleopore polycarbonate membranes from Sterico (Dietikon, Switzerland) with a pore size of 200 nm, followed by 10 times extrusion through membranes with 100 nm pores. Birefringence measurements based on phase modulation techniques were carried out as described previously.3,41,42 Briefly, the sample was placed in the temperature-controlled bore of a 33 T Bitter magnet at the HFML, Nijmegen, The Netherlands. The laser light (HeNe laser, 632.8 nm) was guided through a polarizer (set at 45° to the magnetic field direction), followed by a photoelastic modulator (PEM-90, Hinds Instruments, Hillsboro, USA) before passing the sample in horizontal direction to the magnetic field direction. After passing the second polarizer (set at −45°), the light was detected by a Si photodiode. Two lock-in amplifiers (SR830, SRS, Sunnyvale, USA) were used to detect the first and second harmonic of the ac signal, from which the birefringence can be calculated.3 For the SANS experiments performed at the SANS-I beamline at PSI, Villigen, Switzerland, the samples were produced in D2O buffer. The neutron wavelength was fixed at 8 Å and measured at detector distances of 2, 6, and 18 m covering a momentum transfer of 0.003 ≤ q ≤ 0.15 Å−1. Measurements were carried out in a super conductive magnet with horizontal field of 8 T perpendicular to the neutron beam. Data were corrected for background radiation, empty cell scattering, and detector efficiency. Radial averaged curves were fitted using the software package SASfit. A Porod’s approximation was used in the case of full cylinders (i.e., disks). Hollow cylinders (i.e., disks with a hole) were described by a cylindrical shell.

The order parameter S of the bicellar aggregate in reference to the magnetic field direction can be described with the help of the second Legendre polynomial: S=

Article

(3)

where Δχ is the molar magnetic susceptibility anisotropy (in SI units, m3 mol−1), N is the number of molecules in one bicelle, B is the magnetic field strength, NA is the Avogadro constant, k is the Boltzmann constant, and μ0 is the magnetic constant. In the case of a parallel orientation of the phospholipids to the magnetic field (Δχ > 0), the order parameter S is equal to 1 in the case of full alignment (cos θ = ±1). For perpendicular orientation (Δχ < 0), the phospholipids have one degree of freedom more (rotation around the magnetic field axis), and S is equal to −0.5 at full alignment (cos θ = 0). The bicellar system investigated here is based on a mixture of DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine), cholesterol, and the chelator lipid DMPE-DTPA (1,2-dimyristoyl-snglycero-3-phosphoethanolaminediethylenetriaminepentaacetate) with complexed thulium ions (Tm3+) as studied previously.3,15 Different morphologies were found as a function of temperature. At temperatures above 40 °C, the different components seem to be rather well mixed, and mainly vesicles were found, that is, self-closed bilayer. Below 40 °C, cryo-TEM as well as SANS measurements revealed the presence of disklike aggregates, that is, bicelles. Lipid segregation was proposed as the driving force for this structural change, with DMPEDTPA with complexed Tm3+ covering the highly curved edge of the bicelle, due to its large headgroup. At low temperatures, cryo-TEM micrographs indicated the presence of bicelles with an elongated shape. Increased lipid segregation with decreased temperatures might thereby be responsible for the augmented edge formation. In contrast to the more common bicellar mixtures composed of DMPC and DHPC (see, for example, the review by Katsaras et al.40 and references therein), the magnetic alignability was found to be most pronounced at low temperatures (5 °C). In the previous study, several indications for a transient appearance of holes in the disks at intermediate temperatures were found;3 however, further investigations are needed to gain a better understanding. Here, magnetically induced birefringence measurements in combination with SANS measurements were used to investigate the magnetic alignment and structural changes of the DMPC/Chol/DMPEDTPA/Tm3+ mixture in more detail. In addition, the effect of using two different lanthanide ions, thulium and dysprosium, on the magnetic alignment was studied.

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RESULTS AND DISCUSSION Lanthanide Ion Dependency of Bicellar Alignment in Magnetic Fields. In accordance with literature,4,43 it was found that the orientation direction is dependent on the lanthanide ion used. These ions confer a large Δχ to the bicelle, which originates from the crystal field coupling terms.43−46 The lanthanide ions can be classified into two groups, having opposite sign of Δχ. Dysprosium (Dy3+) enhances the negative magnetic susceptibility anisotropy Δχ of the phospholipids, resulting in a preferred orientation of the phospholipids, and with it the disk normal (see sketch in Figure 1), perpendicular to the magnetic field. The measured magnetically induced birefringence shows a negative sign, and the order parameter converges to −0.5 at high field strength, as shown in Figure 1. Thulium (Tm3+) changes the orientation of the phospholipids and the disk normal to parallel, as the sign of Δχ is switched to positive. The order parameter thereby converges toward +1 with increasing field strength (see Figure 1). The curves have been fitted using eqs 1−3, yielding NΔχ and Δn′max as fitted values. The magnetic field strength B at which the order parameter S reaches one-half its maximum value (S1/2 = 0.5 for Tm3+, and S1/2 = −0.25 for Dy3+, respectively) is directly related to NΔχ and is a measure for the magnetic alignability of the bicelles. 3468

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Figure 3. Magnetic field induced birefringence Δn′ of DMPC/Chol/ DMPE-DTPA/Tm3+ bicelles as a function of the magnetic field B at different temperatures and cooling cycles. Closed symbols represent the cooling, open symbols represent the heating cycle, and solid line represents water measured at 40 °C.

Figure 1. Magnetic induced birefringence Δn′ (○) and corresponding fit of the order parameter S (solid line) as a function of magnetic field for DMPC/Chol/DMPE-DTPA bicelles with different lanthanides measured at 5 °C. Notice that the scale of S for positive and negative values is different, to account for the difference in Δn′max between Dy3+ and Tm3+ samples.

Data in Figure 3 originate from various effects acting simultaneously: (i) enhanced alignment with increasing field strength and decrease in temperature, (ii) contributions from intrinsic (molecular organization within the aggregate) and (iii) form birefringence (shape of the aggregates), as discussed in the following. All curves in Figure 3 have the same general shape but differ in Δn′max and B(S1/2). Figure 4 shows B(S1/2) of DMPC/Chol/ DMPE-DTPA bicelles with Tm3+ and Dy3+ at the measured temperatures during cooling and heating.

The opposing orientation of the bicelle doped with the two different lanthanides was confirmed with SANS in a magnetic field of 8 T. The larger is the scattering angle, the smaller is the scattering object; thus the direction of pronounced scattering at larger scattering angles is the direction of the disk normal. In accordance with birefringence measurements, the sample with complexed Dy 3+ oriented with its disk normal perpendicular to the magnetic field (Figure 2a), whereas the sample with complexed Tm3+ oriented parallel to the magnetic field (Figure 2b).

Figure 2. SANS 2D scattering pattern of DMPC/Chol/DMPE-DTPA bicelles with complexed Dy3+ (a) and Tm3+ (b) at 5 °C and 8 T. Magnetic field direction is vertical in the plane.

Figure 4. Magnetic field strength at which the order parameter S reaches one-half of its maximum value B(S1/2) for DMPC/Chol/ DMPE-DTPA bicelles with complexed Tm3+ (black) and Dy3+ (gray) as a function of temperature. Closed symbols represent the cooling, and open symbols represent the heating cycle.

Temperature Dependency of the Bicellar Alignment in Magnetic Fields. DMPC/Chol/DMPE-DTPA bicelles with either Tm3+ or Dy3+ in a molar ratio of 16:4:5:5 showed structural changes with temperature. The Δn′ from 0 to 33 T showed a pronounced temperature dependence; see Figure 3. The experiment reported in Figure 3 started at 40 °C, where a first magnetic field sweep was performed. Upon cooling, the magnetic field induced birefringence increased up to 10 °C, after which it slightly decreased till 5 °C. Upon heating, the 10, 20, and 30 °C curves did not coincide with those at the same temperature during cooling, evidencing thermal hysteresis, up to 40 °C where the birefringence again coincided with the signal at the start.

The field strength needed to reach S1/2 decreased with decreasing temperatures. This is in accordance with SANS data reported previously, showing an increasing degree of alignment of DMPC/Chol/DMPE-DTPA bicelles with complexed Tm3+ with decreasing temperatures.3 Samples with Dy3+ showed the same temperature dependency, but are better aligned over the entire temperature range. The reason for this is the increased Δχ value in bicelles containing Dy3+ as compared to Tm3+-bicelles, as the bicellar size was found with DLS and SANS measurements to be 3469

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the same at 20 °C in the cooling and heating cycle. The molecular polarizability Δα is a property of a single molecule and is not dependent whether the sample is cooled or heated prior to the measurements. Thus, the difference in Δn′max is most probably caused by a difference in the form birefringence. The depolarization factors determining the contribution of the form birefringence and corresponding references for different aggregate shapes are listed in Table 1.

independent of the lanthanide ion used (data not shown). According to the Bleany theory, Dy3+ shows the largest absolute magnetic anisotropy of the lanthanides, with a ratio between the magnetic anisotropies of Δχ(Tm3+)/Δχ(Dy3+) = −0.53.30 However, Δχ conferred by lanthanides highly depends on the metal complex, and thus on the orientation of the ligand fields and the mobility of the ligand complex.45,47 Mironov et al.48 have shown with numerical calculations that the Bleany theory is only limited valid, and the ratio of Δχ(Tm3+)/Δχ(Dy3+) can vary between −0.65 and −1.28 with large differences in absolute values in different types of complexes. As no precise values are available of DMPE−DTPA complexes, no further quantitative analysis is possible. In addition to the expected larger Δχ value of the DMPE−DTPA·Dy3+ complex, the phospholipid molecules composing the bicelles have a small negative Δχ value, acting toward a perpendicular orientation, enhancing the large negative Δχ value of the bicelles even further. No temperature hysteresis was visible for B(S1/2), meaning that NΔχ only depends on temperature, not on the temperature history. This temperature dependency can be explained by the increased Δχ value of the phospholipids at low temperatures caused by the higher molecular order below the phase transition temperature.49 Literature values of Δχ for DPPC in the crystalline Lβ′ phase are about an order of magnitude higher than for DMPC in the liquid-analogue Lα phase, which is attributed to the well-ordered acyl chains in Lβ′.50 The absence of a sharp increase at the phase transition of DMPC (Tm = 23.6 °C51) is most likely caused by cholesterol, which leads to a broadening and elimination of the phase transition, as a phase of intermediate degree of organization, Lo, is formed.52 Beside the B(S1/2) value, the measured birefringence curves (Figure 3) are defined by Δn′max, the value of the maximum induced birefringence; see Figure 5. Δn′max shows a temperature hysteresis in the cooling and heating cycle, which is most pronounced at 20 °C. Detection of Structural Changes with Temperature. Δn′max depends on the number of oriented molecules in the sample, on their molecular polarizability Δα, and on the form birefringence. As no hysteresis was found in the B(S1/2) values, the number of oriented molecules in the sample is most likely

Table 1. Depolarization Factors Determining Form Birefringence for Different Aggregate Shapes Parallel (D∥) and Perpendicular (D⊥) to Ba sphere53 infinite plane53 full cylinder54 hollow cylinder55

D∥

D⊥

1/3 0 0.11 0.2

1/3 1 0.78 0.6

a

Full cylinder with a length to diameter ratio of L/d = 0.1, and hollow cylinder with L/d = 0.1 and ratio of inner radius to outer radius R1/R2 = 0.4.

For isotropic objects, such as a sphere, the depolarization factor is equal in all three spatial directions, and the measured birefringence will only depend on the intrinsic birefringence. In case of cylinders, the depolarization factor is larger perpendicular to the plane than parallel to it, and thus the detected birefringence is diminished by the contribution of the form birefringence.56 As shown in Table 1, the difference between D∥ and D⊥ is smaller for hollow cylinders as compared to full cylinders, providing a possible explanation for the differences in Δn′max at intermediate temperature. The theoretical birefringence was calculated according to Gielen et al.56 using the depolarization factor for a full cylinder and for a hollow cylinder (Table 1) while keeping all other parameters constant as follows: molecular polarizability αx″x″ = αy″y″ = 304 au, αz″z″ = 404 au,57 concentration C = 15 mM, volume fraction f = 9.36 × 10−6, refractive index of water n1 = 1.33,58 refractive index of phospholipids with cholesterol nf = 1.4372,59 T = 20 °C, and NΔχ = 4.32 × 10−5 m3 mol−1 (as extracted from fitting). Comparison of the calculated birefringence curve and the measured data at 20 °C (Figure 6) indicates that the temperature hysteresis in the birefringence measurement can indeed be explained by the transient appearance of holes in the bicelles. Thereby, the bicelles have a length to diameter ratio of L/d = 0.1, and a ratio of the radius of the hole to the radius of the bicelle of R1/R2 = 0.4. This finding was cross-checked with SANS measurements without magnetic field applied at 20 °C taken during the heating and cooling cycle.3 Figure 7 shows radial averaged SANS curves and the corresponding fit with a form factor for a flat cylinder during cooling, and a form factor for a hollow flat cylinder during heating. The characteristics of hollow cylinders of the best fit (L/d = 0.13, R1/R2 = 0.45) are in good agreement with the values found in the birefringence measurements. The holes were observed in a temperature range, in which the main phase transition temperatures Tm of DMPC (Tm = 23.6 °C)51 lay, and thus it is supposed that molecular rearrangements occurring at Tm are involved in this process. The opening of small transient pores in lipid bilayer has been observed at the phase transition temperature.60,61 For the charged phospholipid DMPG (1,2-dimyristoyl-sn-glycero-3phosphoglycerol) at low ionic strength, considerably larger

Figure 5. Maximum induced birefringence Δn′max for DMPC/Chol/ DMPE-DTPA bicelles with complexed Tm3+ (black) or Dy3+ (gray) as a function of temperature. Closed symbols represent the cooling, and open symbols represent the heating cycle. 3470

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High magnetic fields of up to 33 T allow the determination of the order parameter S as a function of the magnetic field strength and allow therewith the analysis of magnetic alignment without the impact of structural changes on the birefringence signal. On the other hand, the maximum induced birefringence at full alignment Δnmax comprises information about the aggregate structure. The studied mixture, DMPC/Chol/ DMPE-DTPA, exhibits parallel orientation of the bicellar normal to the magnetic field if Tm3+ is attached, and perpendicular orientation with Dy3+. The order parameter S increases with decreasing temperature. The maximum induced birefringence shows a complex temperature dependency due to structural changes influencing both form and intrinsic birefringence. Comparison of the theoretically calculated birefringence with the measured Δn′ values reveals a new structure with the transient hole formation in the bicelles during the heating cycle, which was verified with SANS measurements.

Figure 6. Magnetic field induced birefringence Δn′ of DMPC/Chol/ DMPE-DTPA/Tm3+ bicelles at 20 °C during cooling (gray △) and heating (○). Solid lines represent calculations of the theoretical birefringence according to Gielen et al.56 with a depolarization factor for a full cylinder (gray) and a hollow cylinder (black) while keeping all other parameters constant.

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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 no. 200021_132132). This work is based on experiments performed at the Swiss spallation neutron source SINQ, Paul Scherrer Institute, Villigen, Switzerland and at the high field magnetic laboratory HFML, Nijmegen, The Netherlands. Part of this work has been supported by EuroMagNET under the EU contract no. 228043.

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Figure 7. Radial averaged SANS curves and corresponding fit for DMPC/Chol/DMPE-DTPA/Tm3+ bicelles at 20 °C during cooling (gray △) and heating (○) at 0 T. Values of the best fit for full cylinder (gray) resulted in a log-normal distributed radius R with the location parameter μ = 30 nm and the width parameter σ = 0.5. For the hollow cylinder (black), a log-normal distribution was assumed for the radius of the hole R1, with best fit values μ = 19 nm and σ = 0.5, surrounded by a rim of 23 nm. Thickness was in both cases 4.2 nm.

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pores were found in the phase transition region, probably caused by the electrostatic repulsion between the headgroup.62,63 Similar electrostatic repulsion may also play a role in the DMPC/Chol/DMPE-DTPA/Tm3+ described here. Both birefringence and SANS measurements could be fitted well with a disk containing one single large hole; however, there is no argument against a model containing many small pores (with increased number of fit parameters). In an earlier cryoTEM study, there has been indication for both bicelles containing one single hole as well as bicelles with a few smaller pores.3 In accordance with the intermediate phase found for DMPG, we assume that the charges present in the DMPEDTPA/Tm3+ headgroup hereby cause the enlargement of small cavities occurring at the main phase transition.

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CONCLUSION We have shown that magnetically induced birefringence is a useful tool to study bicellar systems. Magnetic alignment can be determined even in the presence of high lanthanide contents. 3471

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