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Understanding the Enhanced Magnetic Response of Aminocholesterol Doped Lanthanide-Ion Chelating Phospholipid Bicelles Stéphane Isabettini, Sarah Massabni, Joachim Kohlbrecher, Lukas D Schuler, Peter Walde, Marina Sturm, Erich Josef Windhab, Peter Fischer, and Simon Kuster Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b01370 • Publication Date (Web): 31 Jul 2017 Downloaded from http://pubs.acs.org on August 7, 2017

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Understanding the Enhanced Magnetic Response of Aminocholesterol Doped Lanthanide-Ion Chelating Phospholipid Bicelles Stéphane Isabettini*,a, Sarah Massabnia, Joachim Kohlbrecherb, Lukas D. Schulerc, Peter Walded, Marina Sturme, Erich J. Windhaba, Peter Fischera, and Simon Kustera a

Laboratory of Food Process Engineering, ETH Zurich, Schmelzbergstrasse 9, 8092 Zurich,

Switzerland. b

Laboratory for Neutron Scattering and Imaging, Paul Scherrer Institute, 5232 Villigen PSI,

Switzerland. c

xirrus GmbH, Buchzelgstrasse 36, 8053 Zürich, Switzerland.

d

Department of Materials, ETH Zurich, Vladimir-Prelog-Weg 5, 8093 Zurich, Switzerland.

e

Max Planck Institute of Colloids and Interfaces, Am Mühlenberg 1, 14476 Potsdam, Germany.

ABSTRACT Cholesterol (Chol-OH) and its conjugates are powerful molecules for engineering the physicochemical and magnetic properties of phospholipid bilayers in bicelles. Introduction of aminocholesterol (3β-amino-5-cholestene, Chol-NH2) in bicelles composed of 1,2-dimyristoyl-snglycero-3-phosphocholine (DMPC) and the thulium-ion chelating phospholipid 1,2-dimyristoylsn-glycero-3-phospho-ethanolamine-diethylene

triaminepentaacetate

(DMPE-DTPA/Tm3+)

results in unprecedented high magnetic alignments by selectively tuning the magnetic susceptibility Δχ of the bilayer. However, little is known on the underlying mechanisms behind the magnetic response and, more generally, on the physico-chemical forces governing a Chol-

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NH2 doped DMPC bilayer. We tackled this shortcoming with a multiscale bottom-up comparative investigation of Chol-OH and Chol-NH2 mixed with DMPC. First, simplified monolayer models on a Langmuir trough were employed to compare the two steroid molecules at various contents in DMPC.

In a second step, a molecular dynamics (MD) simulation allowed for a more

representative model of the bicelle bilayer, while monitoring the amphiphiles and their interactions on the molecular level. In a final step, we moved away from the models and investigated the effect of temperature on the structure and magnetic alignment of Chol-NH2 doped bicelles by SANS. The DMPC/steroid monolayer studies showed that Chol-OH induces a larger condensation effect than Chol-NH2 at steroid contents of 16 and 20 mol%. However, this tendency was inversed at steroid contents of 10, 30 and 40 mol%. Although the MD simulation with 16 mol% steroid revealed that both compounds induce a liquid-ordered state in DMPC, the bilayer containing Chol-NH2 was much less ordered than the analogous system containing CholOH. Chol-NH2 underwent significantly more hydrogen bonding interactions with neighboring DMPC lipids than Chol-OH. It seems that by altering the dynamics of the hydrophilic environment of the bicelle, Chol-NH2 likely changes the crystal field and angle of the phospholipid-lanthanide DMPE-DPTA/Tm3+ complex. These parameters largely determine the magnetic susceptibility Δχ of the complex, explaining the SANS results, which show significant differences in magnetic alignment of the steroid doped bicelles. Highly magnetically alignable DMPC/Chol-NH2/DMPE-DTPA/Tm3+ (molar ratio 16:4:5:5) bicelles were achieved up to temperatures of 35 °C before a thermoreversible rearrangement into non-alignable vesicles occurred. The results confirm the potential of Chol-NH2 doped bicelles to act as building blocks for the development of the magnetically responsive soft materials of tomorrow. INTRODUCTION Bicelles play an integral role in soft matter research and have been extensively studied since early descriptions in the 1980s.1,2 They are invaluable membrane model systems for understanding the complex interactions and mechanisms occurring in lipid bilayers.3-5


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Moreover, their versatility in architecture and biocompatibility has high potential for diverse pharmaceutical technologies including dermal applications and drug delivery.6,7 Bicelles are commonly formed from a mixture of two phospholipids: 1,2-dimyristoyl-sn-glycero-3phosphocholine

(DMPC)

and

phosphocholine

(DHPC).8

A

the

comparatively

multitude

of

shorter

different

1,2-dihexanoyl-sn-glycero-3-

lipids,

including

glycerolipids,9

sphingolipids,10 and cholesterol (Chol-OH),11 are also used to construct and tailor the properties of these polymolecular disk-like assemblies. In addition to compositional adjustability, a high degree of tuneability is achievable through the optimization of physico-chemical properties such as temperature, pH, net charge and bilayer rigidity. Chol-OH is a weakly amphiphilic biomolecule of high importance in mammalian plasma membranes. It consists of a polar hydroxyl group and a bulky hydrophobic part composed of a rigid four ring system and a flexible tail. Chol-OH is employed in bicelle technology to harness the physico-chemical properties it delivers to the lipid bilayer.12 The clear transition between solid-ordered and liquid-disordered phospholipid states vanishes upon incorporating Chol-OH in the bilayer. Instead, an intermediate liquid-ordered state occurs.13 The induced increase in order is accompanied by lower membrane permeability, reduced lipid flip-flop and improved mechanical stability of the membrane. Chemical modifications of Chol-OH are of profound interest to harness the full potential of the molecule and tailor lipid-membrane properties. Synthetic derivatives of Chol-OH are exceptional biotechnological tools.14 The polar headgroup of Chol-OH provides control over the bilayer charge. For example, insertion of cholesterol sulphate in DMPC/DHPC bicelles prevents precipitation at lower temperatures and higher lipid concentrations allowing stabilisation of the aligned phase.15 Replacing the polar hydroxyl headgroup of Chol-OH with a primary amine in Chol-NH2 allows for the presence of a pH value dependant positive charge in the lipid bilayer.16 Furthermore, cholesteryl chloroformate and Chol-NH2 are common headgroup modified derivatives used as starting blocks for the synthesis of more complex compounds.14,17


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In addition to their versatility in design, the ability bicelles have to align in the presence of an external magnetic field has greatly contributed to their scientific success. Bicelles composed of DMPC and the lanthanide-ion (Ln3+) chelating DMPE-DTPA phospholipid are admirable candidates for the generation of highly tunable magnetically responsive soft materials.18,19 The magnetic alignment of bicelles is commonly controlled by changing their size through optimization of the total lipid content or ratio.20 Although this may be done with DMPC/DMPEDTPA/Ln3+ bicelle systems, a more viable approach consists of introducing Chol-OH within the bilayer. Chol-OH counteracts the curvature-inducing effect of the DMPE-DTPA/Ln3+ species, resulting in larger bicelle species.21 A two-fold increase in magnetic alignment could be achieved with DMPC/Chol-OH/DMPE-DTPA/Tm3+ (molar ratio 16:4:5:5) bicelles when compared to the Chol-OH free DMPC/DMPE-DTPA/Tm3+ (molar ratio 4:1:1) bicelles at 5 °C and an 8 T magnetic field.

Means of controlling the magnetic alignment of bicelles by selectively altering the

magnetic susceptibility Δχ without changing their dimensions remain scarce. We have recently addressed this shortcoming by replacing Chol-OH with Chol-NH2 to make DMPC/CholNH2/DMPE-DTPA/Tm3+ (molar ratio 16:4:5:5) bicelles.22 The magnetic alignment was increased two-fold and six-fold when comparing to DMPC/Chol-OH/DMPE-DTPA/Tm3+ (molar ratio 16:4:5:5) and DMPC/DMPE-DTPA/Tm3+ (molar ratio 4:1:1) bicelles at 5 °C and an 8 T magnetic field, respectively. This unprecedented magnetic response was achieved without changing the size of the bicelles, implying a selectively enhanced magnetic energy of the bicelles through a change in the magnetic susceptibility Δχ of the bilayer. However, the reasons behind this enhanced magnetic susceptibility Δχ remain unclear and calls for further characterization of the physico-chemical properties of Chol-NH2 doped lipid bilayer. According to the general theory of the magnetic anisotropy of lanthanide-containing liquid crystals proposed by Mironov et al, the magnetic susceptibility Δχ is influenced by various effects at the molecular level.23 The DMPE-DTPA phospholipid chelates a lanthanide ion in a distorted tricapped trigonal prismatic molecular geometry (or distorted mono capped square antiprismatic). The lanthanide ion coordinates eight out of nine ligands with DTPA and one with


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water. The largest contribution to the magnetic susceptibility comes from the molecular geometry of the DMPE-DTPA/Ln3+ complex, defining the crystal field of the chelated lanthanide ion. A second contribution of equal importance comes from the spatial orientation of the crystal field-induced magnetic axis with respect to the long molecular axis defined by the myristoyl tails of the phospholipid. The angle between these two axes define the sign and maximal magnitude of the magnetic susceptibility Δχ. Another two contributions include microscopic disorder, originating from the degrees of freedom for molecular motion in the bilayer, and macroscopic disorder, resulting from changes in assembly architecture. Fluctuations in the molecular geometry of the lanthanide-phospholipid complex diminish the magnetic susceptibility Δχ. Moreover, certain polymolecular assembly architectures will reduce the cumulative magnetic energy of the bilayer. The present work is directed towards understanding the reasons behind the enhanced magnetic susceptibility Δχ of DMPC/Chol-NH2/DMPE-DTPA/Tm3+ (molar ratio 16:4:5:5) bicelles. In order to characterise the physico-chemical phenomena governing the Chol-NH2 doped bicelle system and understand the resulting high magnetic alignments, we propose a systematic comparison with the well characterized Chol-OH doped systems.21 A multiscale bottom-up investigation was undertaken starting from simplified models of the lipid monolayer and bilayer before building up the complexity to best imitate the bicelle system as outlined in Figure 1. The physicochemical behavior of Chol-NH2 in DMPC bilayers has never been characterized. However, Lönnfors et al. provide a detailed account of the interaction of Chol-NH2 with phospholipids in other binary and ternary bilayer membranes.16 The characterization of simplified DMPC/CholNH2 systems offers a first glimpse into the physico-chemical properties of DMPC phospholipid bilayers doped with Chol-NH2 before dealing with the more complex bicelle systems.

In a first

step, we worked with DMPC/Chol-NH2 and DMPC/Chol-OH monolayers at the air/water interface and studied their surface pressure/molecular area isotherms on a Langmuir trough (Figure 1A). The sub-phase was composed of a 50 mM phosphate buffer at a pH value of 7 and at 5 °C to imitate the hydrophilic environment of the bicelles when they display an optimal


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magnetic response. In a next step, we proceed to MD simulations to move away from the monolayer and study a bilayer (Figure 1B), which was a more realistic representation of the bicelles. The introduction of the lanthanide-chelating phospholipid DMPE-DTPA/Tm3+ complex was made possible in the MD simulations. The lanthanide-phospholipid complexes present in the planar part of the bicelle hold the key to understanding the magnetic response of the bicelles due to the large magnetic susceptibility Δχ conveyed by the paramagnetic lanthanide ions. Therefore, a MD simulation of both the Chol-OH and Chol-NH2 doped bilayers was employed to observe the parameters defining the magnetic susceptibility Δχ (Figure 1C). These include the lipid order, the crystal field of the chelated lanthanide ion, and the orientation of the long molecular axis of the DMPE-DTPA/Tm3+ complex with respect to its magnetic axis. We conclude our investigations by monitoring the thermal behavior of DMPC/Chol-NH2/DMPE-DTPA/Tm3+ (molar ratio 16:4:5:5) bicelles by small angle neutron scattering (SANS). We characterise the structure of the bicelles at different temperatures and quantify their magnetic alignment. By again comparing to the well characterized DMPC/Chol-OH/DMPE-DTPA/Tm3+ (molar ratio 16:4:5:5) bicelles, we were able to comment on the different physico-chemical forces governing the bicelle properties (Figure 1D). These temperature studies expand the versatility of the CholNH2 doped bicelles by demonstrating their ability to remain in a highly-aligned state at higher temperatures. Understanding the thermal behavior of these polymolecular assemblies is of capital importance to envisage their application as building blocks for the magnetically responsive soft materials of tomorrow. EXPERIMENTAL Materials 1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and the hexa-ammonium salt of 1,2dimyristoyl-sn-glycero-3-phosphoethanolamine-N-diethylenetriaminepentaacetic

(DMPE-

DTPA) phospholipids were purchased from Avanti Polar Lipids (Alabaster, AL) in powdered form (99%). Ethanol stabilized Chloroform, anhydrous thulium(III) chloride (99.9%), and


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methanol (99.8%) were purchased from Sigma-Aldrich (Buchs, Switzerland). D2O (99.9 atom %D) and MeOD-d4 (99.9% atom %D) were purchased from ARMAR Chemicals (Döttingen, Switzerland). Chol-OH was purchased from AMRESCO (Solon, OH). Chol-NH2 was synthesized and characterized as described elsewhere.22,24 HR-MS(+): calculated for C27H48N [M+H]+: 386.3787. Found: 386.3781. Δm/z: 1.5 ppm. 1H NMR (400 MHz, CDCl3) δ: 5.32 (d, 1H), 2.61 (m, 1H), 0.85-2.16 (m, 43H), 0.67 (s, 3H) ppm.

13C

NMR (100 MHz, CDCl3/MeOD 4:1) δ: 140.69,

120.97, 56.53, 55.93, 51.25, 49.98, 42.03, 41.67, 39.54, 39.25 37.74, 36.21, 35.93, 35.56, 31.62, 31.57, 31.15, 27.96, 27.71, 23.98, 23.57, 22.39, 22.13, 20.73, 18.96, 18.34, 11.48 ppm.

Surface pressure/molecular area isotherms The surface pressure/molecular area isotherms of DMPC/Chol-NH2 and DMPC/Chol-OH monolayers were measured at the air/water interface on a KSV-NIMA ISR Langmuir trough of 533 cm2 equipped with two moving barriers, a surface pressure microbalance and a platinum plate. A 50 mM phosphate buffer at a pH value 7 was prepared at room temperature from MilliQ water and used as a sub-phase. The temperature was controlled with the integrated water cooling system of the trough connected to a water bath. 1 mg/ml stock solutions of DMPC/steroid in chloroform were prepared with steroid contents of either 0, 10, 16, 20, 30, 40 or 100 mol%. 50 µL were spread dropwise on the surface of the sub-phase when the temperature reached 5 °C using a 100 µL glass Hamilton syringe equipped with a metal needle with a diameter of 0.5 mm. The lipid-containing chloroform droplets were deposited on the subphase and never dropped from above to avoid lipidic material entering the sub-phase. For the same reason, the metal tip of the needle never touched the surface of the sub-phase. The monolayer was incubated for 15 min at a constant area of approximately 150 Å2/ molecule to ensure the solvent fully evaporated and a uniform lipid distribution remained. The monolayer was then compressed at a rate of 100 mm/min until a clear collapse of the monolayer was observed. All experiments were performed at least three times from fresh solutions and the


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average isotherms were calculated. The trough and the platinum plate were fully washed and dried with isopropanol, MilliQ water and phosphate buffer after each experiment.

Molecular dynamics (MD) simulation A MD simulation was conducted in silico for the DMPC/steroid and the DMPC/steroid/DMPEDTPA/Tm3+ bilayers. The simulation conditions were chosen to best imitate the bicelle environment with a 50 mM phosphate buffer at a pH value of 7, see Table 1. GROMOS force-field 54a7 parameters for DPPC headgroups were employed.25-30 Convenient charge group derivations for the phosphate groups were applied according to Junker et al.31 The Gromacs software package (version 4.5.x) was used on an Intel Xeon multicore machine in parallel.32 Typical conventions of GROMOS were followed including: leap-frog integration schemes with a time-step of 2 fs, Berendsen thermostat ( = 0.1 ps) and pressure coupling ( = 0.5 ps). The long-range electrostatics PME was applied with the split between real and reciprocal Fourier space set to 1.0 nm.33 Molecular structures were sketched in Avogadro,34 minimized and exported to MDL mol format. The topology was generated for all bonds, angles, exclusions, improper dihedrals and proper dihedral angles according to the force-field. The employed DMPC structure was proposed by Tieleman et al.35,36 The introduction of Chol-OH within the GROMOS 87 force-field by M. Höltje et al. was used as a reference.37 The original parameters were modified to account for different partial charges of the hydroxyl group introduced in GROMOS 53A638 and special -CH2 ring aliphatic hydrocarbons introduced in GROMOS 45A3.39

These

modifications were applied analogously to the Chol-NH2 force-field. In a first step, the pure DMPC bilayer was equilibrated for 1 ns at 30 °C with both water and buffer molecules in the surroundings. The 20 steroid molecules were then introduced in the bilayer with one perturbation by slow growth from dummy atoms to the correct atom types. The primary amine headgroup of Chol-NH2 is expected to be mainly protonated at the studied pH.16,40,41 Protonated Lysine was employed as a basis for the simulation, which was also shown


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to be a robust basis for different anilines.31,42 20 water molecules were replaced with Cl- ions to act as counter-ions. The exact molecular composition of the simulated DMPC/steroid bilayer is presented in Table 1. Table 1. Nature and number of the investigated molecules and ions in the DMPC/Chol-OH and DMPC/Chol-NH2 MD simulations. Number of simulated molecules and ions DMPC Chol-OH Chol-NH2 H2PO4HPO42Na+ H2O Cl-

Steroid in the DMPC/steroid bilayer Chol-OH Chol-NH2 128 20 6 4 14 10415 -

128 20 6 4 14 10395 20

The simulation was performed for a total of 142 ns at 5 °C and 82 ns at 40 °C. The difference in simulation time between the two studied temperatures originates from the different speeds at which the modelled bilayers reach a steady state. The simulation was performed for another 30 ns at both temperatures to guaranty sufficient statistics. These last 30 ns of simulation were employed for computing the results and comparing the two steroid doped bilayers. The DMPE-DTPA/Tm3+ phospholipid-lanthanide complex was first modelled alone before incorporating it into the bilayer. Although the chemical structure of the phospholipid was readily constructed, the thulium ion was never applied in molecular simulation within the GROMOS force-field. Therefore, the Lennard-Jones parameters of the thulium ion were estimated. Thulium has been simulated in its ionic form Tm3+ + 3 Cl- in a cubic box containing 3005 simple point charge (SPC) water molecules. The salt was included in a system imitating the final complex with DTPA5- and 5 NH4+ in a cubic box containing 2984 SPC-water molecules. The final lipid was then introduced as DMPE-DTPA5-/Tm3+ and its 5 NH4+ and 3 Cl-counter-ions with 2949 SPC-water molecules. The radial distribution and the coordination numbers were cross-checked


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to guarantee reasonable characteristics in the simulation. Although a convincing model was obtained with the salt and the simplified DTPA chelator, the high frequency of reorientation of the amide bond in the DMPE-DTPA molecule caused regular loss of contact with the thulium ion. For the purpose of this simulation, the stability of the DMPE-DTPA/Tm3+ complex was artificially enhanced by introducing additional bonds between the ion and the DTPA ligands. These bonds were set to a length of 0.26 nm based on the energy minimization with Avogadro applying a universal force field and a force-constant of 6.0·106 kJ·mol−1nm−4.43 The chelator geometry was determined based on a simulated DTPA/Tm3+ complex at an energy minimum. The coordination of the resulting DMPE-DTPA/Tm3+ chelator was evaluated in aqueous solution before introducing it into the DMPC/steroid bilayers. The DMPC/steroid/DMPE-DTPA/Tm3+ bilayer simulations were conducted analogously to DMPC/steroid. A molar ratio of components of 16:4:5:5 was required to imitate the bicelle bilayer. However, DMPE-DTPA/Tm3+ species are believed to accumulate at the edges of the bicelles.18,21 Since we aim to simulate the planar part of the bicelle, only six of the modelled DMPE-DTPA/Tm3+ molecules were introduced into the bilayer. An additional 12 steroid molecules were introduced to guarantee a comparable phospholipid to steroid ratio. The exact molecular composition of the simulated DMPC/steroid/DMPE-DTPA/Tm3+ bilayer is presented in Table 2.


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Table 2. Nature and number of the investigated molecules and ions in the DMPC/CholOH/DMPE-DTPA/Tm3+ and DMPC/Chol-NH2/DMPE-DTPA/Tm3+ MD simulations. Number of simulated molecules and ions

Steroid in the DMPC/steroid/DMPE-DTPA/Tm3+ bilayer Chol-OH Chol-NH2 128 32 6 4 14 10397 18 30 6

DMPC Chol-OH Chol-NH2 H2PO4HPO42Na+ H2O ClNH4DMPE-DTPA5-/Tm3+

128 32 6 4 14 10365 50 30 6

Bicelle preparation The bicelles were prepared as described previously.17-19,21 The mixture of compounds DMPC/Chol-OH/DMPE-DTPA/Tm3+

(molar

ratio

16:4:5:5)

or

DMPC/Chol-NH2/DMPE-

DTPA/Tm3+ (molar ratio 16:4:5:5) were prepared from 10 mg/ml chloroform (lipids) or 10 mM methanol (thulium) stock solutions. A dry lipid film was obtained by removal of the solvents under vacuum. The dry lipid film was hydrated with a 50 mM phosphate buffer at a pH value of 7 at room temperature to obtain solutions with 15 mM total lipid concentration. An equivalent phosphate buffer was prepared in D2O for SANS experiments. The samples were subject to five consecutive freeze-thawing cycles in liquid nitrogen before being extruded (Lipex Biomembranes, Vancouver, Canada) 10 times through 200 nm pores and another 10 times through 100 nm pores using polycarbonate membranes (Sterico, Dietikon, Switzerland). The bicelle samples were stored in the fridge and regenerated with two heating and cooling cycles from 5 to 60 °C and back at 1 °C/min before any measurement.


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Small Angle Neutron Scattering (SANS) SANS measurements were conducted on the SANS-I beamline at PSI, Villigen, Switzerland. The detector distance was set at 2, 6, or 18 m with a neutron wavelength of 0.8 nm. A 2D 3He detector covered the q-range from 0.03 to 1.3 nm-1. Radially averaged scattering curves were constructed from 2D neutron scattering patterns of samples measured at 0 T. The scattering curves were fitted with the SASfit software package.44 The disk-like bicelles were fitted with a form factor for Porod cylinders with a log-normal size distribution. Vesicles were fitted with a form factor for spherical shells with a log-normal size distribution. The scattering curves of a DMPC/Chol-NH2/DMPE-DTPA/Tm3+ (molar ratio 16:4:5:5) bicelle sample were obtained at 5, 10, 20, 30, and 40 °C on heating and at 20 °C on cooling at a rate of 1 °C/min. Furthermore, small angle neutron scattering patterns were obtained under a 8 T magnetic field at a detector distance of 6 m to evaluate the degree of alignment of DMPC/Chol-NH2/DMPE-DTPA/Tm3+ (molar ratio 16:4:5:5) bicelles undergoing a heating and cooling cycle covering the temperature range from 4 to 42 °C at either 0.1 or 1 °C/min. The degree of alignment of the bicelles was quantified by computing alignment factors Af from the 2D scattering patterns. Af are the second cosine Fourier coefficients of the normalised azimuthally intensity in the q-range of 0.3 to 0.4 nm-1 as described elsewhere.21 An isotropic scattering pattern will yield and Af of 0 (no alignment of the bicelles). Moreover, for the studied thulium-chelating Chol-NH2 doped bicelles, the Af become increasingly negative (with a theoretical limit at -1) with bicelles alignment. RESULTS AND DISCUSSION DMPC/steroid monolayer DMPC/Chol-NH2 and DMPC/Chol-OH monolayers isotherms were investigated with varying steroid contents at the air/water interface in Figure 2A and 2B, respectively. This model system allowed for a direct comparison between the two steroid molecules and their respective interactions with DMPC.45 Chol-OH readily mixes with phospholipids with an aliphatic tail length between 14 and 17 carbons.46 The Langmuir trough technique was employed and the surface


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pressure/molecular area isotherms of the DMPC/steroid monolayers were recorded at 5 °C with a 50 mM phosphate buffer sub-phase at a pH value of 7. Since we aim at understanding the forces governing Chol-NH2 doped bicelles, these conditions were chosen to best imitate the bicelle environment when displaying an optimal magnetic response. The monolayers were further studied by coupling Fourier Transform Infrared Reflection-Absorption Spectroscopy (FT-IRRAS) to the Langmuir trough experiment. Infrared measurements allowed monitoring of the chemical interactions occurring in the monolayer and supported our findings as detailed in the supplementary information.47 The appearance of a plateau at 71 Å2 for the pure DMPC monolayer was induced by the transition from a liquid-expanded to a liquid-condensed phase when working below the phase transition temperature of the lipid. The isotherms of the steroid compounds displayed a very different behavior. Condensed steroid domains grow until a critical area was reached and a steep lift-off into the condensed phase occurs. Both Chol-NH2 and Chol-OH resulted in similar isotherms with a lift-off at about 43 Å2/molecule, in line with previous findings.45,47 The effect of adding 10 mol% steroid in the DMPC monolayer was immediately visible as the liftoff point of the isotherms was shifted towards lower areas per molecule. When Chol-OH was employed, the plateau occurring in the pure DMPC monolayer disappeared. This finding is consistent with the ordering effect induced by Chol-OH in phospholipid systems.13,45,48,49 The disappearance of the phase transition was not as clear for the DMPC/Chol-NH2 system where a kink in the isotherm remained visible at about 65 Å2/molecule. At higher steroid contents of 30 and 40 mol%, the isotherms look very similar to those of the pure steroid molecules. The monolayer properties seem dominated by the presence of the steroid lipids. Although these steroid contents are not comparable to those of the bicelle systems, the isotherms emphasize the differences induced by the presence of primary amine in Chol-NH2 instead of a hydroxyl group in Chol-OH.


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The appearance of a more ordered lipid phase with Chol-NH2 was confirmed from the similar evolution of the isotherms compared to the Chol-OH doped systems with increasing steroid contents. A similar behavior was expected for Chol-NH2 that possesses the same steroid backbone as Chol-OH. Moreover, these findings go in line with the observations of Lönnfors et al.16 where Chol-NH2 was shown to form liquid-ordered phases in bilayers composed of other saturated

lipids,

such

as

palmitoyl

sphingomyelin

and

1,2-dipalmitoyl-sn-glycero-3-

phosphocholine (DPPC). However, the isotherms remained markedly different regardless of the similar trend upon increasing steroid contents, implying that different physico-chemical forces govern the properties of the Chol-OH and Chol-NH2 doped systems. To facilitate comparison between the two systems, the excess area per molecule was computed with = / − ( + ), where / is the mean measured area of the molecules in the DMPC/steroid monolayer at a fixed surface pressure.47 and are the areas of the individual molecules as evaluated from the single-component monolayers. and are the molar fractions of the respective components. Consequently, = 0 in the scenario where the lipids mix ideally and no steroidinduced condensation occurs. The excess area per molecule was plotted as a function of steroid content at different surface pressures in Figure 3. Both steroid compounds condensed the monolayer as evidenced by the negative values in Figure 3. This condensation effect was marked at lower surface pressures where the lipid tails are more prone to organise in the presence of Chol-OH.47 At 10 mol% steroid contents, Chol-NH2 had a stronger condensation effect than Chol-OH on the lipid monolayer as evidenced by the larger excess area per molecule obtained with the former. The peak shifts in the C-H bands observed in the FT-IRRA spectra of the monolayers supported this finding (see supporting information). However, the simultaneous unclear disappearance of the phase transition observed in the isotherm (see Figure 2B) suggests complex phase behaviours, which may not be fully appreciated only based on these findings. Contrastingly, Chol-OH had a larger impact on the


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physico-chemical forces governing the monolayer at higher contents of 16 and 20 mol%. The larger of Chol-NH2 with respect to Chol-OH suggests a smaller induced condensation effect of the former. The appearance of a minimum in at steroid contents between 30 and 40 mol% has been associated to the umbrella effect where phospholipid headgroups shield Chol-OH from the bulk water phase. Nevertheless, numerous interaction models exist and the complexity of phospholipid-steroid interactions may not be solely understood in this manner.50 At these larger steroid contents, Chol-NH2 had a larger condensation effect than Chol-OH as evidenced by the more negative Aex values of the former in Figure 3 and the larger maximum intensity of the C-H bands in FT-IRRAS, see Figure S2. The opposite effect was observed at lower steroid contents of 16 and 20 mol%. It is possible that the physical interactions resulting from headgroup interactions between Chol-NH2 and neighbouring lipids only comes into play at higher steroid contents where it becomes a more dominant component. The replacement of the hydroxyl headgroup of Chol-OH with a positively charged primary amine certainly induces large changes in the already complex phase-diagram of Chol-OH doped phospholipid bilayers.12,13,48,49 Molecular dynamics (MD) of the DMPC/steroid bilayer A MD simulation of the DMPC/Chol-NH2 and the DMPC/Chol-OH bilayer was undertaken to complement the findings of the monolayer study and serve as another simplified model of the bicelle systems. The simulation conditions were chosen to best imitate the bicelle environment. The bilayer consisted of 128 DMPC phospholipids with 16 mol% steroid (20 molecules). The hydrophilic surroundings of the bicelle was composed of a 50 mM sodium phosphate buffer at a pH value of 7. The average number of hydrogen bonds per time frame to surrounding water molecules and DMPC are presented for both steroids at 30 and 5 °C in Table 3. Regardless of the temperature, Chol-NH2 showed double the amount of hydrogen bonds to DMPC and one third more with water molecules when compared to Chol-OH. At a pH value of 7, the primary amine of Chol-NH2 is mainly protonated.16,40,41 The increased extent of hydrogen bonding with Chol-NH2


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was expected when compared to Chol-OH that only contains one hydrogen atom in its hydroxyl group. Table 3. Computed hydrogen bonds per time frame of the steroid headgroup to surrounding molecules for the simulated DMPC/steroid bilayers at 5 and 30 °C. Steroid

Temperature [°C]

Average hydrogen bonds per time frame to H2O

Chol-OH Chol-NH2

5 30 5 30

19.5 20.3 32.8 32.5

DMPC 9.5 9.9 17.8 18.3

The simulation revealed a considerably larger diffusion coefficient for Chol-NH2 than Chol-OH at 5 °C. Moreover, the snapshot of the DMPC/Chol-NH2 bilayer taken at the end of the simulation at 5 °C in Figure 4A revealed that three out of the twenty Chol-NH2 molecules do not seek further contact with the DMPC bilayer. They remain in solution at the surface of the bilayer. This was not the case for Chol-OH that fully integrated into the bilayer, see Figure 4B. A lower ordering effect could explain the larger condensation effect of Chol-OH over Chol-NH2 at 16 mol% steroid contents observed in the monolayer study. The deuterated chain order parameters -SCD were evaluated from the simulated trajectories and are presented for both steroid doped bilayers in Figure 5. In the case of Chol-OH, a clear increase in order was observed when cooling from 30 to 5 °C as the curves shift upwards towards larger -SCD values. These results are consistent with the simulations of De Meyer et al.13 where DMPC bilayers containing 16 mol% Chol-OH move from semi-ordered phases to a complete liquid-disordered phase at temperatures above 28 °C. In the case of Chol-NH2, no change in order or in phase behavior occurred from 5 to 30 °C. The number of molecules per unit area was computed for the two bilayers at 5 °C. The DMPC/Chol-NH2 bilayer revealed a value of 50 Å2/molecule compared to 48 Å2/molecule for the DMPC/Chol-OH bilayer. The larger condensation effect of Chol-OH at 5 °C goes in line with the computed larger order in the bilayer. Furthermore, these simulation results support our findings where Chol-OH


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induced a larger condensation effect than Chol-NH2 in the 16 mol% DMPC/steroid monolayer at the air/water interface, see Figure 3. Molecular dynamics (MD) of the DMPC/steroid/DMPE-DTPA/Tm3+ bilayer Six DMPE-DTPA/Tm3+ molecules were incorporated into the DMPC/steroid bilayer to truly simulate a portion of the bicelle. Herein, we aimed to understand the reported large gains in magnetic response upon replacement of Chol-OH with Chol-NH2 in DMPC/steroid/DMPEDTPA/Tm3+ (molar ratio 16:4:5:5) bicelles. We propose a three-step analysis of the bilayer focusing on the factors capable of altering magnetic susceptibility Δχ as described in the general theory by Mironov et al.23 In the first step (i), we investigated if an increase in order could explain the enhanced magnetic response of the Chol-NH2 doped bilayer. In a second step (ii), we focused on the crystal field of the chelated lanthanide ion. In a third and final step (iii), we evaluated the possible change in orientation of the long molecular axis with respect to its magnetic axis in the DMPE-DTPA/Tm3+ complex. (i) We compared the degree of order in the two steroid doped systems. Bilayers with a higher degree of order were expected to enhance the total magnetic energy of the bicelle. More order coincides with reduced fluctuations arising from the nonrigidity of phospholipid-lanthanide species within the bilayer. Consequently, the microscopic disorder contribution was reduced, as proposed in the general theory of magnetic anisotropy of lanthanide-containing liquid crystals by Mironov et al.23 The deuterated chain order parameter -SCD was computed for the myristoyl tails of the lipids in the respective bilayer systems. For DMPC, the values were analogous to those obtained in the DMPC/steroid bilayer. The -SCD parameters of the DMPE-DTPA/Tm3+ phospholipids revealed a higher degree of order with Chol-OH in the bilayer than with Chol-NH2. To further evaluate the order in the two systems, a cluster analysis was undertaken according to Daura et al.51 The results did not allow for any noticeable difference between the two systems. Therefore, the larger degree of magnetic alignment in DMPC/Chol-NH2/DMPE-DTPA/Tm3+ (molar ratio 16:4:5:5) bicelles may not be attributed to a difference in order within the bilayer.


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(ii) It was necessary to take a closer look at the phospholipid-lanthanide complex and consider other contributions that define the magnetic susceptibility Δχ to explain the observed large differences in magnetic response of the steroid doped bicelles. The analysis revealed that CholNH2 underwent significantly more hydrogen bonding interactions with its surroundings than Chol-OH. It interacted to a greater extent with DMPC, DMPE-DTPA/Tm3+, and the phosphate buffer. These results confirm those observed in the DMPC/steroid simulation in Table 2. CholNH2 has the ability to influence the hydrophilic environment of the bilayer to a larger extent than Chol-OH. Surrounding water molecules act as the 9th coordination site of thulium in the DTPA complex.52 This will define the crystal field of the chelated lanthanide, which in turn determines the magnetic susceptibility Δχ of the molecule. Therefore, we analyzed the cumulative occupation time of the 9th coordination site for the two DMPC/steroid/DMPE-DTPA/Tm3+ systems in Table 4. As expected, water molecules account for most of the occupation time. Nevertheless, a clear difference was observed as the coordination to oxygen from neighbouring DMPC phospholipids was preferred in the Chol-NH2 doped bilayer. The oxygen from the carboxylic group in the ester bond of the phospholipids acted as a ligand. Consequently, CholNH2 influences the pool of molecules available to act as a 9th coordination site in the DTPA/Tm3+ complex by altering the dynamics of the hydrophilic environment of the bilayer. The resulting differences in the crystal field and magnetic susceptibility Δχ offers an element of explanation to the contrasting magnetic properties of the steroid doped bicelles. Table 4. Cumulative occupation times of the 9th coordination site in the DTPA/Tm3+ complex. The occupation times were evaluated over the entire 30 ns trajectory and averaged over all six DMPE-DTPA/Tm3+ molecules in either the Chol-OH or Chol-NH2 bilayers. Oxygen atoms from water or from the carboxylic group in the ester bond of neighbouring phospholipids may act as a 9th ligand. In the case of the Chol-NH2 doped bilayer, one of the six DMPE-DTPA/Tm3+ molecule’s headgroup adopted a closed conformation resulting in an intramolecular 9th ligand (also on the oxygen from the carboxylic group in the chelator head). Chol-OH DMPC DMPE-DTPA phosphate buffer H2O

15.6% 16.7% 67.7%

Chol-NH2 37.1% 0.3% 62.6%


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(iii) The orientation of the myristoyl tails with respect to the chelator headgroup was analyzed for all the DMPE-DTPA/Tm3+ molecules in the bilayer. The myristoyl tails define the long molecular axis. The crystal field of the DTPA/Tm3+ complex defines the principal magnetic axis. The orientation of the long molecular axis with respect to the principal magnetic axis determines the sign and magnitude of the magnetic susceptibility Δχ. This corresponds to the second contribution in the general theory of magnetic anisotropy of lanthanide-containing liquid crystals by Mironov et al.23 For example, rotations of rod-like molecules around their long molecular axis reduces the magnetic susceptibility Δχ of the molecule.23 Although the exact position of the principal magnetic axis was not computed, we propose a simplified interpretation using various identified angles in the DMPE-DTPA/Tm3+ molecule. Phosphorus or thulium atoms were employed as a hinge for computing the angles shown in Figure 6A and 6B, respectively. The Tm3+-P-CH3 angles shown in Figure 6A were measured for both lipid tails and averaged for each molecule. Analogously, the two P-Tm3+-O angles shown in Figure 6B were averaged for each molecule. Both angles were averaged over all six DMPE-DTPA/Tm3+ molecules and the resulting difference between the two DMPC/steroid/DMPE-DTPA/Tm3+ (molar ratio 16:4:5:5) systems are presented in Table 5. Table 5. Average angles of the DMPE-DTPA/Tm3+ molecules incorporated in either the Chol-OH or Chol-NH2 doped DMPC/steroid/DMPE-DTPA/Tm3+ (molar ratio 16:4:5:5) bilayers. Two different angles were calculated employing either phosphorus (P) or the complexed thulium ion (Tm3+) as a hinge. The angles are schematically represented in Figure 6 and were averaged over all six of the DMPE-DTPA/Tm3+ molecules in the respective steroid doped bilayers. CH3 corresponds to the terminal carbon in the myristoyl tails and O to the deprotonated carboxylic acid oxygen atoms acting as ligands in the DTPA/Tm3+ complex. Steroid in the DMPC/steroid/DMPE-DTPA/Tm3+ (molar ratio 16:4:5:5) bilayer Chol-OH Chol-NH2

Average angle Tm3+-P-CH3 132° 108°

P-Tm3+-O

96° 105°

The results from the computed angles in Table 5 revealed a contrasting orientation of the phospholipid tails with respect to the chelator headgroup in the two steroid doped bilayer systems. Consequently, the different hydrophilic environment around the bilayer resulting from


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the replacement of Chol-OH with Chol-NH2 induced a change in the molecular geometry of the lanthanide-chelating phospholipids. The magnetic susceptibility Δχ of the DMPE-DTPA/Tm3+ molecule was altered through a change in orientation of the long molecular axis with respect to the principal magnetic axis. These results offer another element of explanation to the contrasting magnetic properties of the steroid doped bicelles.22 Thermal behavior of DMPC/Chol-NH2/DMPE-DTPA/Tm3+ (molar ratio 16:4:5:5) bicelles Chol-OH increases the thermal resistance of DMPC/DMPE-DTPA/Tm3+ bicelle systems by inducing a liquid-ordered phase in the bilayer.21 In the absence of Chol-OH, the thermoreversible collapse of the bicelles into vesicles occurs at the phase-transition temperature of DMPC at 24 °C.18,21 A similar behavior was expected for Chol-NH2 that removed the phase-transition temperature of pure DMPC bilayers and induced a type of liquid-ordered state of its own. However, the lack of a clear phase transition revealed from the molecular dynamic simulation makes it difficult to predict the behavior of DMPC/Chol-NH2/DMPE-DTPA/Tm3+ (molar ratio 16:4:5:5) bicelles with changing temperature. Therefore, the structure and alignment of DMPC/Chol-NH2/DMPE-DTPA/Tm3+ (molar ratio 16:4:5:5) bicelles was monitored upon heating and cooling in Figure 7A and 7B, respectively. The radially averaged SANS curves in Figure 7A were recorded in the absence of a magnetic field to obtain structural information on the DMPC/Chol-NH2/DMPE-DTPA/Tm3+ (molar ratio 16:4:5:5) sample at various temperatures. The data obtained at 5 °C was fitted with a Porod cylinder model with a log-normal size distribution ( = 0.5). The bilayer thickness was 4.6 nm and the disk radius 61 nm, in line with our previous findings.21,22 Moreover, the scattering intensity decays with q−2, which is characteristic of planar disk-like structures.31,32 Upon heating, the dimension of the bicelle remained unchanged as the SANS curves at 10, 20, and 30 °C could be fitted with the same model. A clear difference was observed at 40 °C with the appearance of a kink at a q-value of 0.08 nm-1. This phenomenon is consistent with the appearance of vesicles.21 The data was fitted with a form factor for spherical shells with a thickness of 4.6 nm and a log-


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normal distribution with = 43 nm and = 0.26. The unchanged bilayer thickness, when compared to the sample at lower temperatures, was consistent with the results from MD simulation showing no marked difference in the phase behavior of the bilayer. Similarly to CholOH doped systems, the bicelles reform upon cooling as evidenced by the SANS curve recorded at 20 °C in Figure 7A. The data could be fitted using the same Porod cylinder model and log-normal distribution employed for the fitting at 20 °C on heating. The alignment factor Af of the DMPC/Chol-NH2/DMPE-DTPA/Tm3+ (molar ratio 16:4:5:5) sample was monitored as a function of temperature under a 8 T magnetic field in Figure 7B. The Af ranges from 0 (isotropic scattering, no alignment) to -1 (scattering is parallel, maximal alignment). The highest alignment was observed at 5 °C with an Af value of -0.78, in line with our previous findings.22 The alignment weakens with increasing temperature as the Af values become less negative. The lower alignment may be explained by the increasing thermal energy of the solution, opposing the magnetic energy of the bicelle. A sharp collapse in the Af was observed upon heating at 35 °C, corresponding to the collapse of bicelles into vesicles. However, the bicelles do not regenerate at the same temperature upon cooling. The Af values only increase again at temperatures below 28 °C. The alignment was fully retrieved when cooling back to 5 °C. DMPC/Chol-OH/DMPE-DTPA/Tm3+ (molar ratio 16:4:5:5) bicelles display a similar hysteresis effect where complex lipid rearrangements induce very different polymolecular assemblies upon heating from bicelles to vesicles than upon cooling from vesicles to bicelles.21,53 The different mechanisms of lipid segregation upon cooling and heating could also be responsible for the observed hysteresis in the Chol-NH2 doped bicelles. To ensure that the hysteresis was not caused by the slow kinetics of rearrangement of the polymolecular assemblies, the bicelle-to-vesicle collapse was interrupted upon heating at an Af value of -0.1 by introducing a gentler cooling cycle of 0.1 °C/min in Figure 7B (blue empty triangles). The fall in alignment immediately stopped and remained constant until the limiting temperature of 28 °C was reached and the bicelles start to regenerate. Here again the


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regeneration process could be immediately interrupted at an Af value of -0.3 by applying a gentler heating cycle of 0.1 °C/min (red filled triangles). These results show the highlyresponsive nature of the DMPC/Chol-NH2/DMPE-DTPA/Tm3+ (molar ratio 16:4:5:5) system, where the degree of alignment is readily tuned by thermal means within the hysteresis region. Previously reported DMPC/Chol-OH/DMPE-DTPA/Tm3+ (molar ratio 16:4:5:5) bicelles provided only weak alignments above 5 °C, emphasizing the viability of moving toward Chol-NH2 doped systems when working at higher temperatures. CONCLUSION Replacement of the steroid Chol-OH with the steroid Chol-NH2 in DMPC/steroid/DMPEDTPA/Tm3+ (molar ratio 16:4:5:5) bicelles enhances the magnetic energy of the lipid bilayer and results in unprecedented gains in magnetic alignability. Since the dimension of the bicelles remained unchanged, the increase in magnetic energy must be the result of an enhanced magnetic susceptibility Δχ of the Ln3+ chelating phospholipid DMPE-DTPA/Tm3+.22 We investigated the underlying mechanisms behind the magnetic response by comparing the physico-chemical forces governing a Chol-NH2 and a Chol-OH doped DMPC monolayer, bilayer and bicelle as outlined in Figure 8. The results from MD simulation were in good agreement and complementary to experimental data obtained from different methods including a model monolayer study and a SANS study of the bicelles. The combined monolayer study (Figure 8A) and MD bilayer simulation (Figure 8B) of the simplified Chol-NH2/DMPC and Chol-OH/DMPC systems revealed that Chol-NH2 underwent significantly more hydrogen bonding interactions with neighboring DMPC lipids. Furthermore, Chol-OH displayed a larger condensation effect and an enhanced degree of order in the lipid monolayers at steroid contents of 16 and 20 mol% and at 5 °C. This effect was inversed at higher steroid contents, where Chol-NH2 becomes more dominant and induced a larger condensation effect. The MD simulation of the DMPC/CholNH2/DMPE-DTPA/Tm3+ bilayer (Figure 8C) demonstrated how the hydrophilic environment was altered around the headgroup region of the DMPE-DTPA/Tm3+ lanthanide-phospholipid


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complex. The positively charged primary amine headgroup of Chol-NH2 influenced the pool of molecules available to act as a 9th coordination site in the DTPA/Tm3+ complex. This may result in alterations of the crystal field of the molecule. The enhanced magnetic susceptibility Δχ could also result from a change in the angle between the principal magnetic axis and the long molecular axis in DMPE-DTPA/Tm3+. The introduction of Chol-NH2 allowed for an enhanced thermal resistance of the DMPC/Chol-NH2/DMPE-DTPA/Tm3+ (molar ratio 16:4:5:5) bicelles (Figure 8D). Large alignment factors Af of -0.6 were achieved up to 35 °C upon heating before the bicelles collapsed into vesicles. The bicelles could be regenerated upon cooling below 28 °C. The thermoreversible nature of these systems combined with their high magnetic response makes them valuable candidates for the development of future smart soft-materials. For example, DPPC/Chol-OH/DPPE-DTPA/Tm3+ (molar ratio 16:4:5:5) bicelles have been employed to generate switchable anisotropy in optical gels.19 The additional thermal resistance offered by Chol-NH2 opens the possibility to work with the shorter DMPC lipid, whilst simultaneously benefiting from the enhanced magnetic response of the DMPC/Chol-NH2/DMPE-DTPA/Tm3+ (molar ratio 16:4:5:5) bicelles. Moreover, the existence of a temperature-alignment hysteresis, combined with the rapid response of the system to temperature change, allows for a fine-tuning of the degree of alignment of the colloidal system.

AKNOWLEDGEMENTS The authors acknowledge the Swiss National Science Foundation for funding (SMhardBi project number 200021_150088/1). The SANS experiments were performed at the Swiss spallation neutron source SINQ, Paul Scherrer Instute, Villigen, Switzerland. The FT-IRRAS experiments were conducted in collaboration with the Max Planck institute of colloids and interfaces in the lab of Prof. Dr. Gerald Brezesinski, Potsdam, Germany. We acknowledge P. Q. Reckey for her preliminary contributions to the Langmuir-trough monolayer experiments. Supporting Information Available


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Characterization of the DMPC/Chol-NH2 and DMPC/Chol-OH monolayers with Infrared Reflection-Absorption Spectroscopy (FT-IRRAS) is provided. This information is available free of charge via the Internet at http://pubs.acs.org/.

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(21) 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. (22) Isabettini, S.; Liebi, M.; Kohlbrecher, J.; Ishikawa, T.; Fischer, P.; Windhab, E. J.; Walde, P.; Kuster, S. Mastering the magnetic susceptibility of magnetically responsive bicelles with 3βamino-5-cholestene and complexed lanthanide ions. Phys. Chem. Chem. Phys. 2017, 19, 10820-10824. (23) 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. (24) You, Y.; Gong, S.; Sun, Q. A practical synthesis of 3β-Amino-5-Cholestene. Adv. Mater. Res. 2013, 830, 151–154. (25) Schmid, N.; Eichenberger, A. P.; Choutko, A.; Riniker, S.; Winger, M.; Mark, A. E.; van Gunsteren, W. F. V. Definition and testing of the GROMOS force-field Versions 54A7 and 54B7. Eur. Biophys. J. 2011, 40, 843–856. (26) Chiu, S. W.; Clark, M.; Balaji, V.; Subramaniam, S.; Scott, H. L.; Jakobsson, E. Incorporation of surface tension into molecular dynamics simulation of an interface: a fluid phase lipid bilayer membrane. Biophys. J. 1995, 69, 1230–1245. (27) Chandrasekhar, I.; Kastenholz, M.; Lins, R. D.; Oostenbrink, C.; Schuler, L. D.; Tieleman, D. P.; van Gunsteren, W. F. V. A consistent potential energy parameter set for lipids: Dipalmitoylphosphatidylcholine as a benchmark of the GROMOS96 45A3 force field. Eur. Biophys. J. 2003, 32, 67–77. (28) Poger, D.; van Gunstern, W. F.; Mark A. E. A new force field for simulating phosphatidylcholine bilayers. J. Comput. Chem. 2010, 31 , 1117-1125. (29) Poger, D.; Mark, A. E. On the validation of molecular dynamics simulations of saturated and cis -monounsaturated phosphatidylcholine lipid bilayers: a comparison with experiment. J. Chem. Theory. Comput. 2010, 6 , 325–336.


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(50) Björkbom, A.; Róg, T.; Kaszuba, K.; Kurita, M.; Yamaguchi, S.; Lönnfors, M.; Nyholm, T. K. M.; Vattulainen, I.; Katsumura, S.; Slotte, J. P. Effect of sphingomyelin headgroup size on molecular properties and interactions with cholesterol. Biophys. J. 2010, 99, 3300–3308. (51) Daura, X.; Gademann, K.; Jaun, B.; Seebach, D.; van Gunsteren, W. F. V.; Mark, A. E. Peptide folding: when simulation meets experiment. Angew. Chem. Int. Ed 1999, 38, 236–240. (52) Bottrill, M.; Kwok, L.; Long, N. J. Lanthanides in magnetic resonance imaging. Chem. Soc. Rev. 2006, 35, 557–571. (53) 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.


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Table of contents graphic:


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Figure 1. Schematic representation of the undertaken multiscale bottom-up comparative investigation of aminocholesterol (Chol-NH2) and cholesterol (Chol-OH) mixed with DMPC. The physico-chemical forces governing the two steroid doped systems were first identified and studied with simplified models involving monolayers at the air/water interface (A), followed by bilayers in a molecular dynamics (MD) simulation (B). A more representative model of the bicelle bilayer was achieved by introducing the DMPE-DTPA/Tm3+ phospholipid-lanthanide complex (C). In a final step, the thermal resistance and magnetic alignability of the DMPC/Chol-NH2/DMPE-DTPA/Tm3+ (molar ratio 16:4:5:5) bicelles was investigated and compared to the analogous Chol-OH doped bicelles (D). By characterizing the physico-chemical properties governing these amphiphilic systems, this study aims at understanding how Chol-NH2 alters the magnetic susceptibility ∆χ of Ln3+ chelating bicelles, resulting in a high magnetic response. 104x120mm (300 x 300 DPI)


Langmuir

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Figure 2. Surface pressure/molecular area isotherms of A) DMPC/Chol-OH and B) DMPC/Chol-NH2 monolayers on a 50 mM phosphate buffer sub-phase at 5 °C and with a pH value of 7. The steroid contents were either 0, 16, 20, 30, 40, or 100 mol%. 177x76mm (300 x 300 DPI)


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Figure 3. Excess area per molecule Aex as a function of steroid content evaluated at 5, 10, 15, and 25 mN/m. The monolayers were measured with a 50 mM phosphate buffer sub-phase at 5 °C and with a pH value of 7. Filled circles represent data points measured from Chol-OH doped monolayers and open squares from Chol-NH2 doped monolayers. A standard error of ± 0.8 Å2 was observed in the excess area and is not shown on the figure for clarity. These results were complemented with an FT-IRRAS study available in the supplementary information. 287x270mm (300 x 300 DPI)


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Figure 4. Snapshot of the simulated A) DMPC/Chol-NH2 and B) DMPC/Chol-OH bilayers at 5 °C. Atoms and bonds constituting the DMPC phospholipids are represented by a wireframe model and the steroids with a space-filling model at their van der Waals radii, water is omitted. Carbon atoms are shown in black, oxygen in red, nitrogen in blue, phosphorus in purple, sodium in teal and chlorine in green. The bilayer consists of 128 DMPC phospholipids with 20 steroid (16 mol%) molecules in a 50 mM sodium phosphate buffer at a pH value of 7. 177x76mm (300 x 300 DPI)


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Figure 5. The deuterated chain order parameters -SCD of the myristoyl tails of DMPC were evaluated for A) DMPC/Chol-OH and B) DMPC/Chol-NH2 bilayers with 16 mol% steroid at 5 °C (blue triangles) and 30 °C (red squares). Each myristoyl tail of DMPC resulted in a curve at a given temperature. The -SCD parameters were computed for the numbered atoms visible alongside the chemical structure of DMPC. 177x76mm (300 x 300 DPI)


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Figure 6. Ball and stick molecular representation of the DMPE-DTPA/Tm3+ molecules. Red atoms correspond to oxygen, orange phosphorus, grey carbon, blue nitrogen and green thulium. The hydrogen atoms are not represented for clarity. A) The angles formed by the positions of the thulium ion (Tm3+), the phosphorus atom (P) and the last carbon of each of the myristoyl tails (1CH3 and 2CH3) were evaluated. B) The angles formed by the position of the phosphorus atom (P), the thulium ion (Tm3+), and two of the furthest deprotonated carboxylic acid oxygen atoms acting as ligands in the DTPA/Tm3+ complex (O1 and O8) were evaluated. In both cases, the calculated angles are shown in green and yellow shades and where averaged for each DMPE-DTPA/Tm3+ molecule. The hydrogen atoms on the myristoyl tails are not represented in the united-atom model for clarity. 84x117mm (300 x 300 DPI)


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Figure 7. A) Radially averaged SANS curves of DMPC/Chol-NH2/DMPE-DTPA/Tm3+ (molar ratio 16:4:5:5) at 5, 10, 20, 30 and 40 °C on heating and 20 °C on cooling. The curves were shifted vertically for clarity. The q-2 decay of the scattering intensity is typical of disk-like structures and is shown with a red line. B) Alignment factor Af as a function of temperature of DMPC/Chol-NH2/DMPE-DTPA/Tm3+ (molar ratio 16:4:5:5) measured at 1.0 °C/min (circles) from 4 to 42 °C and at 0.1 °C/min (triangles) from 25 to 37 °C. The heating and cooling cycles are presented in red and blue, respectively. The highest degree of alignment of the bicelles was recorded at 4 °C with an Af of -0.77. 177x76mm (300 x 300 DPI)


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Figure 8. Schematic summary of the main conclusions of this work. The characterization techniques employed in the proposed multiscale bottom-up comparative investigation of aminocholesterol (Chol-NH2) and cholesterol (Chol-OH) mixed with DMPC are presented in the first column. They are labelled from A to D, analogously to the introductory Figure 1. The steroid content was 16 mol% to best imitate the composition of the bicelle. The identified and characterized key physico-chemical properties are presented in the second column. The impact of Chol-NH2 and Chol-OH on these properties is presented with a + (property is influenced) or – (property is not influenced) sign in the last two columns. The relative degree of influence induced by the respective steroids is stressed with multiple + signs. 104x73mm (300 x 300 DPI)


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Understanding the Enhanced Magnetic Response of

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