Cholesterol-Diethylenetriaminepentaacetate Complexed with Thulium

First, we analyzed whether cholesterol-DTPA·Tm3+ can be integrated into a DMPC .... Foundation is acknowledged for funding (Project number 200021_132...
1 downloads 0 Views 4MB Size
Download PDF
Article pubs.acs.org/JPCB

Cholesterol-Diethylenetriaminepentaacetate Complexed with Thulium Ions Integrated into Bicelles To Increase Their Magnetic Alignability Marianne Liebi,† Simon Kuster,† 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 and §Biomolecular Research Laboratory, Paul Scherrer Institute, 5232 Villigen PSI, Switzerland ∥ Department of Materials, ETH Zurich, Wolfgang-Pauli-Strasse 10, 8093 Zurich, Switzerland ‡

S Supporting Information *

ABSTRACT: Lanthanides have been used for several decades to increase the magnetic alignability of bicelles. DMPE-DTPA (1,2-dimyristoyl-sn-glycero-3-phospho-ethanolamine-diethylenetriaminepentaacetate) is commonly applied to anchor the lanthanides into the bicelles. However, because DMPE-DTPA has the tendency to accumulate at the highly curved edge region of the bicelles and if located there does not contribute to the magnetic orientation energy, we have tested cholesterol-DTPA complexed with thulium ions (Tm3+) as an alternative chelator to increase the magnetic alignability. Differential scanning calorimetric (DSC) measurements indicate the successful integration of cholesterol-DTPA into a DMPC (1,2-dimyristoyl-sn-glycero3-phosphocholine) bilayer. Cryo transmission electron microscopy and small-angle neutron scattering (SANS) measurements show that the disklike structure, that is, bicelles, is maintained if cholesterol-DTPA·Tm3+ is integrated into a mixture of DMPC, cholesterol, and DMPE-DTPA·Tm3+. The size of the bicelles is increased compared to the size of the bicelles obtained from mixtures without cholesterol-DTPA·Tm3+. Magnetic-field-induced birefringence and SANS measurements in a magnetic field show that with addition of cholesterol-DTPA·Tm3+ the magnetic alignability of these bicelles is significantly increased compared to bicelles composed of DMPC, cholesterol, and DMPE-DTPA· Tm3+ only.

■

INTRODUCTION Because of their amphiphilic nature, phospholipids form different aggregates in aqueous solution by spontaneous selfassembly. The type of aggregate formed depends on various factors in particular on the chemical structure of the phospholipids on their concentration and on temperature. The main forces are thereby hydrophobic interactions inducing association of the molecules and the hydrophilic, ionic, or steric repulsion of the head-groups, demanding the contact with water.1 The geometry of the aggregates formed mainly depends on the molecular shape of the phospholipidic building blocks. One possible structure is a bicelle, which is a disklike object composed of bilayer forming molecules, here DMPC (1,2dimyristoyl-sn-glycero-3-phosphocholine) building the planar part and molecules with the preference for high curvature, here DMPE-DTPA (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-diethylenetriaminepentaacetate, Scheme 1) with complexed lanthanide ions, covering the highly curved edge region.2 An interesting property of phospholipid bicelles is their orientability in magnetic field caused by the magnetic susceptibility anisotropy Δχ. Phospholipid molecules experience an orientational force in a magnetic field toward a minimal magnetic energy Emag, which for a large enough aggregate can overcome the thermal fluctuation energy kT. Besides an increased size of the aggregate, the orientational force can be © 2013 American Chemical Society

triggered with the addition of lanthanide ions with a large magnetic susceptibility anisotropy.3,4 Lanthanide ions can be divided into two groups, depending on the sign of their magnetic susceptibility. Here, we used Tm3+, which has a large positive magnetic susceptibility anisotropy and is known to lead to an alignment of the bicellar normal parallel to the magnetic field direction.5−8 Magnetic aligning of bicelles composed of DMPC and the short chain lipid DHPC sometimes doped with small amounts of lanthanide ions complexed to DMPE-DTPA is used in NMR studies of membrane-associated proteins to increase the accuracy of structure determination by dipolar coupling.9−11 The alignable phase of such bicellar systems however are rather extended lamellar sheets or a chiral nematic phase of wormlike micelles5,12,13 than separated disks as studied here. Recently, we reported about the potential of magnetic alignable disks as functional ingredient in a smart hydrogel with magnetically and thermally switchable optical properties.14 Optimizing the bicellar mixtures in terms of magnetic alignability is crucial for such applications. The main drawback of the mixtures studied so far is the partial location of the magnetic handles (DMPE-DTPA with complexed lanthanides) Received: July 4, 2013 Revised: November 6, 2013 Published: November 7, 2013 14743

dx.doi.org/10.1021/jp406599c | J. Phys. Chem. B 2013, 117, 14743−14748

The Journal of Physical Chemistry B

Article

Scheme 1. Chemical Structures of Cholesterol-DTPA (Top) and DMPE-DTPA (Bottom)

in the curved edge region of the bicelle.2 Thus to increase the magnetic orientation, lanthanide ions should be preferentially integrated into the plane of the bicelles. We also found that the inclusion of cholesterol into the bicelles increases the magnetic alignability of bicelles significantly.15 Cholesterol acted thereby as a spacer between the large head-groups of the chelator lipids (DMPE-DTPA with complexed lanthanides) allowing their integration into the plane of the bicelles to a higher extent than without cholesterol. On the basis of these previous findings, cholesterol was directly used as hydrophobic anchor for DTPA in order to increase the magnetic alignability of the bicelles even more. Rational in doing so is given by the work on lanthanide complexes with either DTPA or tetraazacyclododecanetetraacetate (DOTA) covalently bound to cholesterol, which have been described as possible contrast agents for magnetic resonance imaging studies.16−19 The synthesis of cholesterolDTPA (Scheme 1) used in this work was first described by Lattuada and Lux with complexed Gd3+ and an improved synthesis was published by Davies and Duhme-Klair.20 Such cholesterol-DTPA chelator complexed with Eu3+ was successfully integrated into liposomes and used as a marker to follow their biodistribution using time-resolved fluorescence spectroscopy.21 The bicelles studied here are improvements of a previously studied mixture of DMPC, cholesterol, and DMPE-DTPA with complexed Tm3+ (DMPE-DTPA·Tm3+).15 In the present work, cholesterol was replaced to 50 or 100% by cholesterol-DTPA· Tm3+. Differential scanning calorimetry (DSC) was used to test for a successful integration into DMPC bilayers. Cryo transmission electron microscopy (cryo-TEM), dynamic light scattering (DLS) and small-angle neutron scattering (SANS) were used to identify the bicellar self-assembly structure. SANS in magnetic field as well as magnetic-field-induced birefringence were conducted in order to determine the magnetic alignability of the bicelles.

tate (DMPE-DTPA) in powder form was purchased from Avanti Polar Lipids (Alabaster, AL) and used without further purification. Chloroform (stabilized with ethanol) was from Ecsa (Balerna, Switzerland); cholesterol (99%), anhydrous thulium(III) chloride (99.9%), D2O (99.9 atom % D), and methanol (99.8%) were from Sigma-Aldrich (Buchs, Switzerland). Synthesis of Cholesterol-DTPA. Cholesterol-DTPA was prepared in a multistep synthesis from tert-butyl 2-bromoacetate. According to Micklitsch et al.22 the double nucleophilic substitution reaction of tert-butyl 2-bromoacetate and 2aminoethanol followed by the bromination of the alcohol function generated di-tert-butyl 2,2′-((2-bromoethyl)azanediyl)diacetate. This compound was then connected to a lysine derivative in a double nucleophilic substitution reaction according to Anelli et al.23 After modification of the protecting groups, the cholesterol unit was attached to the chelator precursor according to Lattuada and Lux.17 Following cleavage of the protecting functional groups the cholesterol-DTPA chelator was obtained.17 A detailed description of the synthesis including a scheme with the synthetic route as well as all analytical data can be found in the Supporting Information. Bicelle Preparation. The bicelles were produced with a procedure described previously.2,15,24 DMPC, cholesterol, cholesterol-DTPA, DMPE-DTPA, and TmCl3 (in methanol) were weighed into a round-bottom flask and chloroform was added until the powder was dissolved completely. The solvents were removed by evaporation in a rotatory evaporator resulting in a thin dry lipid film, which was hydrated with a phosphate buffer (50 mM, pH 7.5) yielding a total lipid concentration of 15 mM. For SANS measurements, a D2O phosphate buffer was used (50 mM, pH meter reading 7.0 corresponding to a PD of 7.4).25 Five freeze−thaw cycles were performed by plunging into liquid nitrogen followed by warming up to about 45 °C. The samples were extruded 10 times through polycarbonate membranes (Sterico, Dietikon, Switzerland) with a pore size of 200 nm followed by 10 times extrusion through membranes with a pore size of 100 nm by using “the Extruder” from Lipex Biomembranes (Vancouver, Canada). Samples for DSC measurements were produced similarly, though the lipid concentration was 30 mM in order to optimize the DSC signal.

■

MATERIAL AND METHODS Materials. A choloroform solution of 1,2-dimyristoyl-snglycero-3-phosphocholine (DMPC) and 1,2-dimyristoyl-snglycero-3-phospho-ethanolamine-diethylenetriaminepentaace14744

dx.doi.org/10.1021/jp406599c | J. Phys. Chem. B 2013, 117, 14743−14748

The Journal of Physical Chemistry B

Article

Differential scanning calorimetry (DSC). DSC measurements were performed under atmospheric conditions on a DSC-7 calorimeter (Perkin-Elmer, Waltham) and analyzed with the Pyris version 5 software (Perkin-Elmer). Twenty microliters of samples were put into an aluminum pan and sealed to avoid evaporation of the solution during measurements. Samples were scanned between 5 and 60 °C. The scanning rate was 10 °C/min. Cryo-TEM. Cryo-TEM was carried out as described previously.26 A VitrobotTM apparatus (FEI, Eindhoven, The Netherlands) was used to make thin aqueous films on holey carbon grids (Quantifoil, Jena, Germany) and frozen by plunging into liquid ethane at the temperature of liquid nitrogen. The samples were analyzed in a Tecnai G2 F20 microscope (FEI) with a field emission gun and Tridem energy filter (Gatan) operated at an accelerating voltage of 200 kV. Samples were kept at the temperature of liquid nitrogen during analysis in a cryo-holder (model 626, Gatan). The data were recorded using a 2048 × 2048 CCD camera (Gatan) with 5−10 μm underfocus in order to increase the contrast. SANS. SANS measurements were carried out at the SANS-I beamline at PSI, Villigen Switzerland. The sample was placed in a superconducting magnet with maximum field strength of 8 T and field direction perpendicular to the neutron beam. A twodimensional 3He detector positioned at a distance of 18, 6, and 2 m to the sample was used for data collection, which corresponds to a momentum transfer of 0.03 ≤ q ≤ 1.5 nm−1 at the fixed neutron wavelength of 8 Å. Data were corrected for background radiation, empty cell scattering, and detector efficiency. Radial-averaged scattering curves were fitted using the software package SASfit using a Porod’s approximation for cylinder as a form factor.27 The anisotropy of the scattering pattern representing the degree of orientation of the bicelles was quantified with an alignment factor Af as described previously.15 DLS. DLS measurements were performed on a Malvern Zetasizer Nano ZS with a He−Ne laser fixed at 90°. Analysis was conducted at undiluted samples using the non-invasive backscatter technology (NIBS). Birefringence Measurements. Birefringence measurements in high magnetic fields were performed at the HFML, Nijmegen, The Netherlands using phase modulation techniques following the procedure described previously.24,28,29 Thereby the laser light (He−Ne laser) was guided through the sample placed in a Bitter magnet with maximum field strength of 33 T after passing a polarizer (set at 45° to the magnetic field direction) and a photoelastic modulator (PEM-90, Hinds Instruments Hillsboro, U.S.A., set at 0°). A second polarizer set at −45° was placed in front of the Si photodiode detector. The first and second harmonic of the ac signal was detected by two lock-in amplifiers (SR830, SRS, Sunnyvale, U.S.A.) and the birefringence was calculated as described elsewhere.15

lipid organization can conveniently be measured by DSC. As shown in Figure 1, for pure DMPC a distinct peak at 22 °C is

Figure 1. DSC curves of pure DMPC, DMPC/cholesterol (molar ratio 4:1) and DMPC/cholesterol-DTPA·Tm3+ (molar ratio 4:1) measured at a heating rate of 10 °C/min and a total lipid concentration of 30 mM.

visible that reflects the solid-ordered to liquid-disordered phase transition. This transition disappears after inclusion of 20 mol % cholesterol. The same is observed if cholesterol-DTPA·Tm3+ is mixed with DMPC, indicating the successful integration of cholesterol-DTPA·Tm3+ into the DMPC bilayer. In a next step, cholesterol-DTPA·Tm3+ was integrated into the bicellar formulation composed of DMPC, DMPE-DTPA· Tm3+ and cholesterol, whereby cholesterol was either completely replaced by cholesterol-DTPA·Tm3+ or partially. The resulting compositions were DMPC/cholesterol-DTPA/ DMPE-DTPA/Tm3+ (molar ratio 16:4:5:9) and DMPC/ cholesterol/cholesterol-DTPA/DMPE-DTPA/Tm3+ (molar ratio 16:2:2:5:7) with a total lipid concentration of 15 mM. In order to investigate the influence of cholesterol-DTPA·Tm3+ on the self-assembly structure of the bicellar mixture, electron microscopy, and SANS measurements were performed. For both mixtures, cryo-TEM micrographs revealed the presence of large bicelles as shown in Figure 2. SANS measurements of DMPC/cholesterol/cholesterolDTPA/DMPE-DTPA/Tm3+ (molar ratio 16:2:2:5:7) at 5 °C are also in agreement with large bicelles, as the radial averaged curve could be well fitted with a form factor for flat cylinders (disks), with R = 86.3 nm and a height of 4.9 nm (see Figure 3). From DLS measurements the disk radius was extracted from the measured hydrodynamic radius according to Glover et al.,31 being Rdisk = 91.1 nm at 5 °C. This is in good agreement with the SANS measurements. For the mixture where cholesterol was completely replaced by cholesterol-DTPA·Tm3+ (DMPC/ cholesterol-DTPA/DMPE-DTPA/Tm3+, molar ratio 16:4:5:9) a disk radius of Rdisk = 83.0 nm was obtained. As the bicelles containing cholesterol-DTPA·Tm3+ are larger than the reference bicelles with cholesterol (R = 55 nm determined from cryo-TEM micrographs)15 the cholesterol-chelator complex seems to integrate well into the planar part of the bicelles. Mixtures containing both cholesterol and cholesterolDTPA·Tm3+ yielded the largest bicelles thus this combination reduced the highly curved edge-area most efficiently. The main goal of this study was to increase the magnetic alignability of the previously studied bicelles based on DMPC, cholesterol, and DMPE-DTPA/Tm3+. The alignment of the

■

RESULTS AND DISCUSSION First, we analyzed whether cholesterol-DTPA·Tm3+ can be integrated into a DMPC bilayer or whether this hydrophobic complex would rather form self-assembly structures on their own. Lattuada and Lux17 have found indirect evidence by MRI that micelles can form from cholesterol-DTPA·Tm3+ in contrast to the molecule containing DOTA.16,17,19 Cholesterol inclusion into a membrane leads to broadening and elimination of the phase transition, as a liquid-ordered phase with intermediate degree of organization forms.30 This change in 14745

dx.doi.org/10.1021/jp406599c | J. Phys. Chem. B 2013, 117, 14743−14748

The Journal of Physical Chemistry B

Article

Figure 4. Magnetic field strength at which the order parameter S reaches half of its maximum value B(S1/2) as function of temperature extracted from birefringence measurements in high magnetic fields for DMPC/cholesterol/DMPE-DTPA/Tm3+ (16:4:5:5, stars), DMPC/ cholesterol-DTPA/DMPE-DTPA/Tm3+ (16:4:5:9, open circles), and DMPC/cholesterol/cholesterol-DTPA/DMPE-DTPA/Tm 3 + (16:2:2:5:7, closed circles). Total lipid concentration was 15 mM.

saturation (S1/2). This indicates a better magnetic alignability of the cholesterol-DTPA·Tm3+ containing bicelles as compared to bicelles containing DMPE-DTPA·Tm3+ only. The alignability of the bicelles containing cholesterol and cholesterol-DTPA· Tm3+ is thereby slightly higher if compared to the sample with cholesterol-DTPA·Tm3+ only. This can be explained by the increased aggregation number (as found by DLS measurements) increasing the magnetic orientation energy of the larger bicellar aggregates. SANS measurements in a magnetic field of up to 8 T confirmed the better alignability of mixtures containing cholesterol-DTPA·Tm3+, as found with the birefringence measurements. The alignment factor Af characterizing the anisotropy of the measured scattering pattern for DMPC/ cholesterol/cholesterol-DTPA/DMPE-DTPA/Tm3+ (molar ratio 16:2:2:5:7) is shown as a function of the magnetic field in Figure 5. The lower the value, the higher is the alignability in the direction perpendicular to the magnetic field.15 At 8 T, the alignment factor reaches a value of −0.60, which is significantly lower than -0.36, which was measured for the reference sample without cholesterol-DTPA·Tm3+.15 From the results presented, two conclusions can be drawn. On the one hand, the increased alignability of the samples

Figure 2. Cryo-TEM micrographs of mixtures of DMPC/cholesterolDTPA/DMPE-DTPA/Tm3+, molar ratio 16:4:5:9 (A) and DMPC/ cholesterol/cholesterol-DTPA/DMPE-DTPA/Tm3+, molar ratio 16:2:2:5:7 (B), samples were kept at 5 °C prior to quick-freezing, total lipid concentration was 15 mM. Scale bar = 200 nm. The dark spots in the micrograph are most likely ice crystals.

Figure 3. Radial-averaged SANS curve and corresponding fit with a form factor of flat cylinders with a radius of 86.3 nm and a height of 4.9 nm of DMPC/cholesterol/cholesterol-DTPA/DMPE-DTPA/ Tm3+ (molar ratio 16:2:2:5:7, total lipid concentration 15 mM) measured at 5 °C without magnetic field applied.

bicelles in a magnetic field of various field strengths was studied with birefringence measurements. Field strengths of up to 33 T were used. Such high magnetic fields allow reaching full saturation of the alignment, therewith permitting the determination of the order parameter S.24 The magnetic field strength B at which the order parameter S reaches half its maximum value is taken as a measure for the capability of the aggregates to orient in a magnetic field. As shown in Figure 4 the mixtures containing cholesterol-DTPA·Tm3+ require a significantly lower B for reaching half the value of S at

Figure 5. Alignment factor as a function of the magnetic field strength of DMPC/cholesterol/cholesterol-DTPA/DMPE-DTPA/Tm3+ at 5 °C, molar ratio 16:2:2:5:7, total lipid concentration 15 mM. 14746

dx.doi.org/10.1021/jp406599c | J. Phys. Chem. B 2013, 117, 14743−14748

The Journal of Physical Chemistry B

■

containing cholesterol-DTPA·Tm3+ is caused by the enlarged self-assembly structures build, because the magnetic orientation energy Emag is proportional to the aggregation number N.4 On the other hand, Δχ conferred by the individual molecules is equally important, thus Δχ from cholesterol-DTPA·Tm3+ is adding to the increased alignability. However, no predictions for the value of Δχ of cholesterol-DTPA·Tm3+ can be made at this point. Δχ highly depends on the orientation of the ligand field as well as on the mobility of the ligand complex and cannot easily be predicted.32−34

■

CONCLUSION

■

ASSOCIATED CONTENT

REFERENCES

(1) Israelachvili, J. N. Intermolecular and Surface Forces; Academic Press: New York, 2011. (2) Beck, P.; Liebi, M.; Kohlbrecher, J.; Ishikawa, T.; Rüegger, H.; Fischer, P.; Walde, P.; Windhab, E. J. Novel Type of Bicellar Disks from a Mixture of DMPC and DMPE-DTPA with Complexed Lanthanides. Langmuir 2010, 26, 5382−5387. (3) Mironov, V. S.; Galyametdinov, Y. G.; Ceulemans, A.; GorllerWalrand, C.; Binnemans, K. Room-temperature magnetic anisotropy of lanthanide complexes: A model study for various coordination polyhedra. J. Chem. Phys. 2002, 116, 4673−4685. (4) Qiu, X. X.; Mirau, P. A.; Pidgeon, C. Magnetically Induced Orientation of Phosphatidylcholine Membranes. Biochim. Biophys. Acta 1993, 1147, 59−72. (5) Katsaras, J.; Harroun, T. A.; Pencer, J.; Nieh, M. P. “Bicellar” lipid mixtures as used in biochemical and biophysical studies. Naturwissenschaften 2005, 92, 355−366. (6) Prosser, R. S.; Hunt, S. A.; DiNatale, J. A.; Vold, R. R. Magnetically Aligned Membrane Model Systems with Positive Order Parameter: Switching the Sign of Szz with Paramagnetic Ions. J. Am. Chem. Soc. 1996, 118, 269−270. (7) Prosser, R. S.; Shiyanovskaya, I. V. Lanthanide ion assisted magnetic alignment of model membranes and macromolecules. Concepts Magn. Reson. 2001, 13, 19−31. (8) Prosser, R. S.; Volkov, V. B.; Shiyanovskaya, I. V. Novel chelateinduced magnetic alignment of biological membranes. Biophys. J. 1998, 75, 2163−2169. (9) Bax, A.; Kontaxis, G.; Tjandra, N. Dipolar couplings in macromolecular structure determination. Methods Enzymol. 2001, 339, 127−174. (10) Sanders, C. R.; Prosser, R. S. Bicelles: a model membrane system for all seasons? Structure 1998, 6, 1227−1234. (11) Tjandra, N.; Bax, A. Direct measurement of distances and angles in biomolecules by NMR in a dilute liquid crystalline medium. Science 1997, 278, 1111−1114. (12) Nieh, M.-P.; Raghunathan, V. A.; Glinka, C. J.; Harroun, T. A.; Pabst, G.; Katsaras, J. Magnetically Alignable Phase of Phospholipid “Bicelle” Mixtures Is a Chiral Nematic Made Up of Wormlike Micelles. Langmuir 2004, 20, 7893−7897. (13) van Dam, L.; Karlsson, G.; Edwards, K. Direct observation and characterization of DMPC/DHPC aggregates under conditions relevant for biological solution NMR. Biochim. Biophys. Acta 2004, 1664, 241−256. (14) Liebi, M.; Kuster, S.; Kohlbrecher, J.; Ishikawa, T.; Fischer, P.; Walde, P.; Windhab, E. J. Magnetically Enhanced Bicelles Deliver Switchable Anisotropy in Optical Gels, submitted for publication, 2013. (15) Liebi, M.; Kohlbrecher, J.; Ishikawa, T.; Fischer, P.; Walde, P.; Windhab, E. J. Cholesterol Increases the Magnetic Aligning of Bicellar Disks from an Aqueous Mixture of DMPC and DMPE−DTPA with Complexed Thulium Ions. Langmuir 2012, 28, 10905−10915. (16) Accardo, A.; Tesauro, D.; Aloj, L.; Pedone, C.; Morelli, G. Supramolecular aggregates containing lipophilic Gd(III) complexes as contrast agents in MRI. Coord. Chem. Rev. 2009, 253, 2193−2213. (17) Lattuada, L.; Lux, G. Synthesis of Gd-DTPA-cholesterol: a new lipophilic gadolinium complex as a potential MRI contrast agent. Tetrahedron Lett. 2003, 44, 3893−3895. (18) Laurent, S.; Henoumont, C.; Vander Elst, L.; Muller, R. N. Synthesis and Physicochemical Characterisation of Gd-DTPA Derivatives as Contrast Agents for MRI. Eur. J. Inorg. Chem. 2012, 2012, 1889−1915. (19) Oliver, M.; Ahmad, A.; Kamaly, N.; Perouzel, E.; Caussin, A.; Keller, M.; Herlihy, A.; Bell, J.; Miller, A. D.; Jorgensen, M. R. MAGfect: a novel liposome formulation for MRI labelling and visualization of cells. Org. Biomol. Chem. 2006, 4, 3489−3497. (20) Davies, C. L.; Duhme-Klair, A.-K. Synthesis of a cholesterolappended Tb−DTPA complex by combined removal of tert-Butyl protecting groups and complexation of terbium(III). Tetrahedron Lett. 2011, 52, 4515−4517.

Cholesterol-DTPA·Tm3+ was tested regarding its ability to integrate into the bilayer plane of bicelles and increase the bicelles’ magnetic alignability. DSC measurements indicated that cholesterol-DTPA·Tm3+ was integrated into the DMPC bilayer, as the sharp phase transition of DMPC at 22 °C vanished. Cryo-TEM and SANS measurements were in accordance with an integration of cholesterol-DTPA·Tm3+ into the bilayer plane of the bicelles, whereby the bicelle size increased substantially if compared to the reference sample (DMPC/cholesterol/DMPE-DTPA/Tm3+). Both birefringence as well as SANS measurements in magnetic fields showed a significantly increased alignability of the bicellar mixtures including cholesterol-DTPA·Tm3+ compared to the reference bicelle. Overall, the increased alignment of the bicelles is caused by the increased aggregation number found for bicelles containing cholesterol-DTPA·Tm3+ and most likely by the magnetic susceptibility anisotropy Δχ conferred by the additional lanthanide ions complexed to cholesterol-DTPA.

S Supporting Information *

Prescription of the Synthesis of Cholesterol-DTPA, schema showing the synthetic path way, and analytical data. This material is available free of charge via the Internet at http:// pubs.acs.org.

■

Article

AUTHOR INFORMATION

Corresponding Author

*E-Mail: peter.fi[email protected]. Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS The authors acknowledge EMEZ for technical support and C. Holenstein for the performance of the DSC measurements and assistance during SANS measurements. The Swiss National Science Foundation is acknowledged for funding (Project number 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. 14747

dx.doi.org/10.1021/jp406599c | J. Phys. Chem. B 2013, 117, 14743−14748

The Journal of Physical Chemistry B

Article

(21) Mignet, N.; de Chermont, Q. L.; Randrianarivelo, T.; Seguin, J.; Richard, C.; Bessodes, M.; Scherman, D. Liposome biodistribution by time resolved fluorimetry of lipophilic europium complexes. Eur. Biophys. J. 2006, 35, 155−161. (22) Micklitsch, C. M.; Yu, Q.; Schneider, J. P. Unnatural multidentate metal ligating α-amino acids. Tetrahedron Lett. 2006, 47, 6277−6280. (23) Anelli, P. L.; Fedeli, F.; Gazzotti, O.; Lattuada, L.; Lux, G.; Rebasti, F. L-Glutamic Acid and L-Lysine as Useful Building Blocks for the Preparation of Bifunctional DTPA-like Ligands. Bioconjugate Chem. 1998, 10, 137−140. (24) Liebi, M.; van Rhee, P. G.; Christianen, P. C. M.; Kohlbrecher, J.; Fischer, P.; Walde, P.; Windhab, E. J. Alignment of Bicelles Studied with High-Field Magnetic Birefringence and Small-Angle Neutron Scattering Measurements. Langmuir 2013, 29, 3467−3473. (25) Covington, A. K.; Paabo, M.; Robinson, R. A.; Bates, R. G. Use of the Glass Electrode in Deuterium Oxide and the Relation between the Standardized pD (paD) Scale and the Operational pH in Heavy Water. Anal. Chem. 1968, 40, 700−706. (26) Ishikawa, T.; Sakakibara, H.; Oiwa, K. The architecture of outer dynein arms in situ. J. Mol. Biol. 2007, 368, 1249−1258. (27) Kohlbrecher, J. Software package SASfit for fitting small-angle scattering curves. http://kur.web.psi.ch/sans1/SANSSoft/sasfit.html (accessed February 11, 2013). (28) Christianen, P. C. M.; Shklyarevskiy, I. O.; Boamfa, M. I.; Maan, J. C. Alignment of molecular materials in high magnetic fields. Phys. B 2004, 346−347, 255−261. (29) Shklyarevskiy, I. O.; Jonkheijm, P.; Christianen, P. C. M.; Schenning, A. P. H. J.; Del Guerzo, A.; Desvergne, J.-P.; Meijer, E. W.; Maan, J. C. Magnetic Alignment of Self-Assembled Anthracene Organogel Fibers. Langmuir 2005, 21, 2108−2112. (30) Mannock, D. A.; Lewis, R. N. A. H.; McMullen, T. P. W.; McElhaney, R. N. The effect of variations in phospholipid and sterol structure on the nature of lipid-sterol interactions in lipid bilayer model membranes. Chem. Phys. Lipids 2010, 163, 403−448. (31) Glover, K. J.; Whiles, J. A.; Wu, G. H.; Yu, N. J.; Deems, R.; Struppe, J. O.; Stark, R. E.; Komives, E. A.; Vold, R. R. Structural evaluation of phospholipid bicelles for solution-state studies of membrane-associated biomolecules. Biophys. J. 2001, 81, 2163−2171. (32) Delli Castelli, D.; Terreno, E.; Carrera, C.; Giovenzana, G. B.; Mazzon, R.; Rollet, S.; Visigalli, M.; Aime, S. Lanthanide-Loaded Paramagnetic Liposomes as Switchable Magnetically Oriented Nanovesicles. Inorg. Chem. 2008, 47, 2928−2930. (33) Mironov, V. S.; Galyametdinov, Y. G.; Ceulemans, A.; Binnemans, K. On the magnetic anisotropy of lanthanide-containing metallomesogens. J. Chem. Phys. 2000, 113, 10293−10303. (34) Prosser, R. S.; Volkov, V. B.; Shiyanovskaya, I. V. Solid-state NMR studies of magnetically aligned phospholipid membranes: taming lanthanides for membrane protein studies. Biochem. Cell Biol. 1998, 76, 443−451.

14748

dx.doi.org/10.1021/jp406599c | J. Phys. Chem. B 2013, 117, 14743−14748