Article pubs.acs.org/Langmuir
Methods for Generating Highly Magnetically Responsive LanthanideChelating Phospholipid Polymolecular Assemblies Stéphane Isabettini,*,† Mirjam E. Baumgartner,† Pernille Q. Reckey,† Joachim Kohlbrecher,‡ Takashi Ishikawa,§ Peter Fischer,† Erich J. Windhab,† and Simon Kuster† †
Laboratory of Food Process Engineering, ETH Zurich, Schmelzbergstrasse 9, 8092 Zurich, Switzerland Laboratory for Neutron Scattering and Imaging and §Biomolecular Research Laboratory, Paul Scherrer Institute, 5232 Villigen PSI, Switzerland
‡
ABSTRACT: Mixtures of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and its lanthanide ion (Ln3+) chelating phospholipid conjugate, 1,2-dimyristoyl-sn-glycero3-phospho-ethanolamine-diethylene triaminepentaacetate (DMPE-DTPA), assemble into highly magnetically responsive polymolecular assemblies such as DMPC/DMPEDTPA/Ln3+ (molar ratio 4:1:1) bicelles. Their geometry and magnetic alignability is enhanced by introducing cholesterol into the bilayer in DMPC/Cholesterol/DMPEDTPA/Ln3+ (molar ratio 16:4:5:5). However, the reported fabrication procedures remain tedious and limit the generation of highly magnetically alignable species. Herein, a simplified procedure where freeze thawing cycles and extrusion are replaced by gentle heating and cooling cycles for the hydration of the dry lipid film was developed. Heating above the phase transition temperature Tm of the lipids composing the bilayer before cooling back below the Tm was essential to guarantee successful formation of the polymolecular assemblies composed of DMPC/DMPE-DTPA/Ln3+ (molar ratio 4:1:1). Planar polymolecular assemblies in the size range of hundreds of nanometers are achieved and deliver unprecedented gains in magnetic response. The proposed heating and cooling procedure further allowed to regenerate the highly magnetically alignable DMPC/Cholesterol/DMPE-DTPA/Ln3+ (molar ratio 16:4:5:5) species after storage for one month frozen at −18 °C. The simplicity and viability of the proposed fabrication procedure offers a new set of highly magnetically responsive lanthanide ion chelating phospholipid polymolecular assemblies as building blocks for the smart soft materials of tomorrow.
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valuable tool for bicelle engineering.13,14 For example, DMPC has a packing parameter close to unity, giving it a cylindrical molecular geometry when compared to DHPC that is more cone-like with a packing parameter of about 1/3. The bicellar size is readily tailored by both the ratio of the composing lipids and the overall concentration.12,15−17 Further doping DMPC/ DHPC bilayers with either anionic 1,2-dimyristoyl-sn-3phosphoglycerol (DMPG) or cationic 1,2-dimyristoyl-3-trimethylammoniumpropane (DMTAP) allows for tailoring of the bilayer’s charge.18 DMPE-DTPA is another common dopant that may be introduced in concentrations lower than 1% of the total lipid composition to chelate lanthanide ions on the bilayer’s surface. This enhances the magnetic alignability and permits control over the direction of alignment with respect to the field direction. In biomolecular membrane structural studies by NMR, DMPE-DTPA allows to benefit from the high magnetic susceptibility offered by the chelation of Ln3+, while reducing the paramagnetic shifts induced by the free ions.19−23 DMPE-DTPA may also be employed to chelate copper ions to speed up the T1 relaxation of the embedded membrane proteins and considerably shorten NMR measurement time.24
INTRODUCTION Bicelles are invaluable soft materials commonly employed as membrane mimicking models for the study of bound or associated membrane proteins by NMR spectroscopy.1 Furthermore, their viability for pharmaceutical applications in the field of dermal treatments or drug delivery has recently been revealed.2 We have also demonstrated the possibility of using bicelles for the design of optically active gels.3 Their tunability in terms of size, charge, and magnetic alignability is at the source of their success. The term “bicelle” is derived from binary, bilayered mixed micelles introduced in the 1990s by Sanders and co-workers.4−6 However, the first bicelle systems appeared a decade earlier and were formed with a mixture of bile salt and phospholipid.7 Since then, numerous systems made from various lipid families have been reported. The diversity of lipidic systems capable of forming bicelles has largely contributed to confirming their scientific importance.8,9 The most studied bicelle system is composed of a mixture of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), constituting the planar part of the bicelle, and the shorter 1,2dihexanoyl-sn-glycero-3-phosphocholine (DHPC) phospholipid covering the edge.10−12 The molecular geometry of the phospholipids composing the bilayer is proposed to be the underlying cause behind the spontaneous self-assembly into bicelles. Consequently, the packing parameter of lipids is a © XXXX American Chemical Society
Received: March 3, 2017 Revised: June 5, 2017 Published: June 8, 2017 A
DOI: 10.1021/acs.langmuir.7b00725 Langmuir XXXX, XXX, XXX−XXX
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Figure 1. Schematic representation of the current fabrication procedure of DMPC/DMPE-DTPA/Tm3+ (molar ratio 4:1:1) or DMPC/Cholesterol/ DMPE-DTPA/Tm3+ (molar ratio 16:4:5:5) bicelles.24,28 The dry lipid film is hydrated in a 50 mM phosphate buffer at a pH value of 7.5 before undergoing five freeze thaw cycles (FT) in liquid nitrogen (LN2). The resulting suspension is extruded 10 times through a polycarbonate membrane with a pore size of 200 nm and another 10 times through a membrane with a pore size of 100 nm. The extrusions are carried out at 40 and 60 °C for the cholesterol-free DMPC/DMPE-DTPA/Tm3+ and the DMPC/Cholesterol/DMPE-DTPA/Tm3+ bicelle systems, respectively. Herein, we simplify this procedure by replacing the freeze thawing and extrusion steps with a heating and cooling procedure (H&C) to generate highly magnetically responsive polymolecular assemblies.
Furthermore, the DMPE-DTPA/Gd3+ complex is a useful contrast agent in magnetic resonance imaging (MRI).25 We have recently developed highly magnetically responsive and tunable bicelle systems by replacing DHPC with DMPEDTPA.26 The resulting DMPC/DMPE-DTPA/Ln3+ bicelles allows for the association of many more paramagnetic lanthanide ions on the bilayer’s surface, resulting in an enhanced magnetic response of the bicelles. Moreover, moving away from the water-soluble DHPC molecule allows for the formation of dilution-resistant assemblies. The magnetic response is dictated by the bicelle’s overall magnetic energy 1 Emag described by Emag = − 2 μ0 nΔχB2 , where B is the magnetic field strength, μ0 is the magnetic constant, n is the aggregation number, and Δχ is the molecular diamagnetic susceptibility anisotropy of the lipids composing the bilayer.27 The magnitude and sign of Δχ may be controlled by complexing lanthanide ions on the surface of the bicelle.19 Nevertheless, the magnetic response is more commonly controlled by altering the aggregation number n. Larger bicelles have more phospholipids capable of contributing their individual Δχ to the final polymolecular assembly. Therefore, n is directly correlated to the readily engineered bicelle size.4−6,8,21,28,29 The response of DMPC/DMPE-DTPA/Ln3+ bicelles to magnetic fields is tailored by both, their size (aggregate number n) and the molecular diamagnetic susceptibility anisotropy Δχ. The latter is readily achieved by changing the nature of the chelated lanthanide ion. Introducing cholesterol results in the formation of DMPC/Cholesterol/DMPE-DTPA/Ln3+ bicelles. Controlling the aggregation number n (i.e., the bicellar size) and magnetic response with cholesterol is a viable approach in addition to the more conventional optimization of the composing lipid ratio or total concentration.30 Moreover, cholesterol alters the physicochemical forces governing the lipid bilayer resulting in the appearance of an intermediate liquid ordered phase.31,32 Cholesterol’s hydroxyl group offers a fertile playground for chemical modifications that open additional property-tailoring for bicelles.33 Cholesteryl sulfate has been implemented in DMPC/DHPC bicelles to broaden the temperature range over which stable alignment occurs. This
enabled the formation of aligned phases at lower temperatures and high lipid concentrations.34 In DMPC/DMPE-DTPA/Ln3+ bicelle systems, Cholesterol-diethylenetriaminepentaacetate conjugates were incorporated into the bilayer to enhance their magnetic response by chelation of additional lanthanide ions on the bicelle’s surface.35 The reported fabrication procedure of DMPC/DMPEDTPA/Tm3+ (molar ratio 4:1:1) and DMPC/Cholesterol/ DMPE-DTPA/Tm3+ (molar ratio 16:4:5:5) bicelles is similar to existing protocols for the formation of large unilamellar vesicles.36−38 This process involves freeze thawing cycles (FT) followed by multiple extrusion steps (Ext) once the dry lipid film has been hydrated as explained in Figure 1. These procedures are far from optimal for the generation of maximally magnetically responsive polymolecular assemblies. Furthermore, they contradict the reported fabrication protocols of DMPC/DHPC bicelles, developed to favor the self-assembly of disk-like polymolecular assemblies.39,40 Herein, we explored the possibility of forming large self-assembled polymolecular structures through removal of the extrusion step. We replaced the freeze thawing cycles and hydrated the dry lipid film with a more gentle procedure involving heating and cooling cycles (H&C). We investigated two lipid systems: DMPC/DMPEDTPA/Tm3+ (molar ratio 4:1:1) and DMPC/Cholesterol/ DMPE-DTPA/Tm3+ (molar ratio 16:4:5:5) by characterizing the resulting polymolecular assemblies and quantifying their magnetic alignability. In a final step, the possibility of regenerating the magnetic response of a one month old frozen DMPC/Cholesterol/DMPE-DTPA/Tm 3+ (molar ratio 16:4:5:5) sample was evaluated using the same heating and cooling procedure. This comparatively reduced shelf life of days to a few weeks impinges on the versatility of these magnetically responsive polymolecular assemblies.26,30,41 We address this frailty by studying the system’s magnetic alignment after prolonged storage before evaluating simple means of regenerating the sample. With the proposed optimized fabrication procedures, we aim to considerably enhance the magnetic response of the polymolecular assemblies without altering their lipid composition. The resulting systems are viable building blocks for the development of future soft B
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with the values obtained at 0 T, and the temperature cycles were conducted using a gradient of 1 °C/min. Small Angle Neutron Scattering. Small angle neutron scattering SANS measurements were conducted on the SANS-I beamline at the Paul Scherrer Institut (Villigen, Switzerland). Samples were prepared in a 50 mM D2O phosphate buffer with a pH value of 7.0 (pD of 7.4) following the aforementioned fabrication procedures. The neutron wavelength was fixed at 0.8 nm and a 2D 3He detector was placed at 6 m from the sample. A superconducting magnet was employed to supply an 8 T magnetic field perpendicular to the neutron beam. The alignment factor Af was computed from the 2D scattering pattern of the sample in the q-range of 0.3−0.4 nm−1 to quantify the magnetic alignment of the samples as described previously.30,41 The Af is a means of transforming the obtained anisotropic SANS 2D scattering patterns into a numerical value that is more readily presented and compared. The Af is the second cosine Fourier coefficient of the normalized azimuthal intensity I(φ) adapted from Hongladarom et al.45 It is computed with
materials and widen the frame of possibilities in, for example, the design of magnetically switchable optical gels.3
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EXPERIMENTAL SECTION
Materials. 1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dimyristoyl-sn-glycero- 3-phospho-ethanolamine-diethylene triaminepentaacetate acid hexa-ammonium salt (DMPE-DTPA) phospholipids were purchased from Avanti Polar Lipids (Alabaster, AL) in powdered form (99%) and were used without further purification. Cholesterol was purchased from Amresco (Solon, OH). Anhydrous thulium(III) chloride (99.9%), chloroform (stabilized by ethanol), and methanol (99.8%) were purchased from Sigma-Aldrich (Buchs, Switzerland). D2O (99.9 atom % D) was purchased from ARMAR Chemicals (Döttingen, Switzerland). Sample Preparation. The phospholipids and cholesterol were dissolved in a chloroform stock solutions of 10 mg/mL and a 10 mM stock solution of thulium chloride in methanol was prepared. All samples consisted of a total lipid concentration of 15 mM. The molar ratios of the bicelle constituents were: 4:1:1 for DMPC/DMPEDTPA/Tm3+ and 16:4:5:5 for DMPC/Cholesterol/DMPE-DTPA/ Tm3+. The lipids were weighed into a round-bottom flask together with the TmCl3 and optional cholesterol. After gentle evaporation of the organic solvents, the resulting lipid film was dried for a minimum of 2 h at 30 °C under high vacuum. The resulting dry lipid film was subsequently rehydrated with 50 mM sodium phosphate buffer at a pH value of 7.5 following the heating and cooling procedure. The heating and cooling procedure involved cooling the sample down to 5 °C after the phosphate buffer was added. The DMPC/DMPE-DTPA/Tm3+ (molar ratio 4:1:1) sample was then heated to 40 °C and cooled back to 5 °C with a temperature gradient of 1 °C/min. The DMPC/ Cholesterol/DMPE-DTPA/Tm3+ (molar ratio 16:4:5:5) sample was then heated to 60 °C and cooled back to 5 °C with a temperature gradient of 1 °C/min. This process was repeated a second time and the samples were stored at room temperature before proceeding to further measurements. Two minutes of vortexing was applied when the sample reached the maximum and minimum temperature of the heating and cooling cycle, respectively. Birefringence. Birefringence measurements of the samples exposed to a 5.5 T field were undertaken to quantify magnetic orientation and monitor changes in the architecture of the polymolecular assemblies.42−44 The method and experimental setup was analogous to that described in our previous works.3,30,41 The sample was placed in a temperature-controlled quartz cuvette (Hellma, Germany). Light from a diode laser with a wavelength of 635 nm was polarized by two crossed linear polarizers (Newport, Irvine, CA). A photoelastic modulator PEM-90 (Hinds Instruments, Hillsboro, OR) placed between the two polarizers was set to operate at 50 kHz. The PEM amplitude A0 was set to 2.405 rad, making the DC component independent of birefringence. The sample cuvette was placed between the PEM and the second polarizer in the magnet operating at 5.5 T. A set of nonpolarizing mirrors (Newport, Irvine, CA) were employed to guide the laser light through the different elements and was finally directed onto a photo detector (Hinds Instruments, Hillboro, OR). Two lock-in amplifiers SR830 (SRS, Sunnyvale, CA) were employed for recording of the first Iω1 and second Iω2 harmonic of the AC signal used to evaluate the degree of retardation δ of the polarized light with
A f (q) =
The Af ranges between +1, when the scattering direction φ = 0 (alignment parallel to the magnetic field direction) and −1 when the scattering direction φ = 90° (alignment perpendicular to the magnetic field direction). An isotropic scattering pattern yields an Af of 0 (no alignment). Cryo-Transmission Electron Microscopy (Cryo-TEM). Samples for cryo-TEM were prepared as described previously.30,46 The sample was suspended as a thin aqueous film on holey carbon grids (Quantifoil, Jena, Germany) before being flash-frozen in liquid ethane at 77 K using a cryoplunge 3 system (Gatan, Pleasanton, CA). Samples were measured with JEM2200FS (JEOL, Japan) equipped with a field emission gun and an in-column energy filter (JEOL) operated at 200 kV. A 4096 × 4096 CMOS camera (F416, TVIPS, Germany) with 5− 10 μm under focus was used to increase the contrast. High contrast particles on the micrographs are ice crystals resulting from the freezing procedure. Dynamic Light Scattering. Dynamic light scattering (DLS) measurements were conducted at 5 °C on a Malvern Zetasizer Nano ZS (Malvern, UK) equipped with a He−Ne laser fixed at 90° with noninvasive backscatter technology (NIBS). Spectrophotometry. Spectrophotometry measurements were conducted at 400 nm on a Cary 100 UV−vis spectrophotometer (Agilent Tech., Santa Clara, CA), equipped with an extended sample compartment containing a six by six multicell block Peltier element. Samples were kept at 5 °C for 5 min prior to starting the heat cycle experiment. A heat cycle consisted of ramping to 50 °C and back to 5 °C with a temperature rate of 1 °C/min. Changes in the transmitted intensity were expressed in terms of optical density with a cell path length of 1 cm. The 50 mM phosphate buffer was employed to correct the background.
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RESULTS DMPC/DMPE-DTPA/Tm3+ (Molar Ratio 4:1:1). Removal of the extrusion steps in the fabrication procedure of DMPC/ DMPE-DTPA/Tm3+ (molar ratio 4:1:1) bicelles offered the possibility of generating larger and more alignable polymolecular assembly structures. We optimized the fabrication procedure by replacing the freeze thawing cycles with gentle heating and cooling cycles (H&C). This procedure was directly inspired from commonly reported fabrication protocols of DMPC/DHPC bicelles and is expected to favor the selfassembly procedure.39,40 In a first stage, the attainable architectures were investigated by cryo-TEM and DLS. Their magnetic alignment was quantified and compared with the first generation of extruded bicelle systems.26 In order to comprehend the origin of the polymolecular rearrangements
⎛ Iω2J (A 0) ⎞ 2 ⎟⎟ δ = arctan⎜⎜ ⎝ Iω1J1(A 0) ⎠ where J1 and J2 are Bessel functions of the first kind, with J1(2.405) = 0.5191 and J2(2.405) = 0.4317. The retardation δ was then converted into a birefringence signal Δn′ to quantify the degree of anisotropy in the material using
Δn′ = −
∑ I(φ) cos(2φ) ∑ I(φ)
δλ 2πd
where λ is the wavelength of the laser at 635 nm and d is the thickness of the sample (10 mm). The birefringence signal Δn′ was normalized C
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(i.e. perpendicular alignment of the assemblies). The DMPC/ DMPE-DTPA/Tm3+ (molar ratio 4:1:1) sample revealed an Af of −0.30, which is 2-fold greater than the first generation of extruded bicelles.41 Nevertheless, the nonplanar nature of the larger assembly structures hinders their contribution to the sample’s magnetic alignment. The twists and folds present in their structure results in differing orientations of the composing lipid’s molecular axis. The cumulative magnetic energy of the bilayer is reduced by the opposing directional components of the magnetic force acting on the individual lipids. Consequently, the magnetic alignment of the associated polymolecular assembly will be lower than that of a bicelle architecture with equivalent planar surface area. Regardless of the higher complexity of the nonextruded DMPC/DMPE-DTPA/Tm3+ (molar ratio 4:1:1) systems, the change in Af as a function of temperature under a 8 T magnetic field revealed a similar trend to that observed in the previously reported extruded systems.41 The results presented in Figure 3A show an enhanced Af between 10 and 20 °C before a thermoreversible collapse of the magnetically alignable polymolecular assemblies into nonalignable vesicles occurred between 22 and 24 °C. This temperature range corresponds to the phase transition temperature Tm of DMPC from the solid ordered to the liquid disordered phase. The characteristic evolution of the DMPC/DMPE-DTPA/Tm3+ (molar ratio 4:1:1) system’s Af with temperature is dictated by the nature of the phospholipids composing the bilayer. Solely the magnitude of the alignment is influenced by the fabrication procedure due to the resulting different polymolecular assembly architectures. We further investigated the phenomena occurring in the polymolecular assembly’s bilayer upon changes in temperature with birefringence and spectrophotometry measurements in Figure 3B. The sample’s birefringence signal under a 5.5 T magnetic field was employed as a complementary method to monitor magnetic alignment during a heating and cooling cycle from 5 to 40 °C and back at a rate of 1 °C/min.30,43 Unlike for the alignment factor computed from SANS scattering patterns, birefringence is also sensitive to polymolecular rearrangements occurring in the bilayer.42 Therefore, the birefringence signal originating from changes in the sample’s magnetic alignment may be decoupled from the signal caused by molecular rearrangements in the bilayer. This was done by comparing how the Af and birefringence signals evolve with temperature in Figure 3A and B, respectively. The birefringence results confirmed the higher magnetic alignment at 5 °C with a value of 1.17 × 10−5, which is almost twice as strong as for the reported extruded systems.41 The zeroing of the birefringence signal above 24 °C supports the formation of nonalignable vesicles. This temperature corresponds to the Tm of DMPC, which acts as a trigger point for major rearrangements in the polymolecular assemblies. These transformations are thermoreversible as alignable species were regenerated upon cooling below Tm, where the birefringence signal followed the same trend as on heating. The distinct peaks occurring around Tm are characteristic of the DMPC/DMPE-DTPA/Tm3+ (molar ratio 4:1:1) assemblies. These peaks cannot be caused by a sudden enhancement of the sample’s magnetic alignability as the Af collapsed in this temperature range. Instead, the peaks suggest the presence of a transition structure from the planar alignable polymolecular assemblies below Tm to the nonalignable vesicles present above Tm. The slow kinetics of the molecular rearrangements with respect to the applied heating and cooling
occurring upon changing temperature and their impact on the sample’s magnetic alignment, complementary analytical methods were employed including SANS, birefringence, cryo-TEM, and spectrophotometry. The cryo-TEM micrographs of a DMPC/DMPE-DTPA/ Tm3+ (molar ratio 4:1:1) sample at 5 °C after the heating and cooling cycles in Figure 2A reveal a multitude of bicelles (red
Figure 2. (A) Cryo-TEM micrographs of DMPC/DMPE-DTPA/ Tm3+ (molar ratio 4:1:1) at 5 °C fabricated by heating and cooling cycles. Large sheetlike structures with folds and twists are indicated with blue arrows. They were surrounded by numerous smaller bicelles shown with red arrows. The scale bars represent 200 nm. Note that the micrograph surrounded by a red box was obtained from another micrograph of the same sample. (B) Intensity (red) and number (black) distribution of the sample obtained from DLS at 5 °C.
arrows) in the range of 20−70 nm in diameter along with larger species with folded and twisted sheet-like structures (blue arrows). The polymolecular assemblies were mainly composed of smaller structures with a mean hydrodynamic diameter DH of 68 nm as revealed by the number distribution obtained from DLS measurements of the sample at 5 °C in Figure 2B. The intensity distribution confirmed the presence of larger entities with a mean hydrodynamic diameter DH of 500 nm. In comparison, previously reported DMPC/DMPE-DTPA/Tm3+ (molar ratio 4:1:1) bicelles displayed a DH of 34 nm.26 Removal of the extrusion step allowed for the successful spontaneous formation of larger polymolecular assemblies that have the potential to deliver enhanced magnetic alignment. SANS measurements at 5 °C and under a 8 T magnetic field were employed to compute the alignment factor Af.30 The alignment factor Af allowed us to quantify the sample’s alignment as it becomes more negative as the bicelles move from a disordered state (Af = 0) to an aligned state, resulting in anisotropic scattering parallel to the magnetic field direction D
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Figure 3. (A) Alignment factor Af as a function of temperature on heating for DMPC/DMPE-DTPA/Tm3+ (molar ratio 4:1:1) under a 8 T field. (B) Birefringence signal as a function of temperature for the same sample under a 5.5 T field (filled lines) and absorbance as a function of temperature in the absence of a magnetic field (dashed lines). The data recorded on heating is shown in red and on cooling in blue. Tm corresponds to the phase transition temperature of DMPC.
rate of 1 °C/min could explain why the peaks were not overlapping. Nevertheless, both peaks started at the Tm of DMPC, suggesting that the bilayer lipids must have a certain degree of order to favor the formation of alignable polymolecular assemblies. The flattening of the birefringence curve at around 10 °C in Figure 3B is correlated to the sudden enhanced sample alignment observed in SANS at the equivalent temperature in Figure 3A. Intermediate ripple phases occurring within the bilayer are proposed to be at the origin of this phenomenon.41,47,48 Spectrophotometry experiments were conducted in the absence of a magnetic field. A heating and cooling gradient of 1 °C/min was applied and the change in sample absorbance was monitored from 5 to 50 °C and back for a DMPC/DMPEDTPA/Tm3+ (molar ratio 4:1:1) sample. To facilitate comparison with the birefringence results, the absorbance curves are also presented in Figure 3B with dashed lines. The large changes in absorbance occurring around the Tm of DMPC supports the presence of major polymolecular rearrangements in the sample. A variation in the composing lipid’s miscibility may be the driving force behind these structural changes. Such phenomena have been reported in DMPC/DHPC bicelles subject to higher temperatures where perforated and unperforated vesicles exist at 45 and 55 °C, respectively.49 The sudden collapse in absorbance upon heating the DMPC/ DMPE-DTPA/Tm3+ (molar ratio 4:1:1) bicelles could suggest an analogous process with the appearance of numerous perforations in the bilayer. Stromatosomes have been reported in numerous systems composed of self-assembling amphiphilic molecules and induce similar drops in light absorbance.50−53 Therefore, stromatosomes could be possible transition structures around the Tm of DMPC. The simultaneous sudden increase in birefringence signal goes in line with the presence of these perforated structures that could enhance the formbirefringence as proposed by Liebi et al.42 DMPC/Cholesterol/DMPE-DTPA/Tm3+ (Molar Ratio 16:4:5:5). Building from our success in generating more alignable polymolecular assemblies with DMPC/DMPEDTPA/Tm3+ (molar ratio 4:1:1), we introduced cholesterol in the phospholipid bilayer to create larger and more magnetically responsive temperature-resistant assembly structures. Cholesterol, with its inverse cone-like molecular geometry, acts to reduce curvature in the bilayer. This results
in larger polymolecular species with enhanced magnetic alignability.30 The replacement of a clear transition temperature from the solid ordered to the liquid disordered phase allows magnetically alignable DMPC/Cholesterol/DMPE-DTPA/ Tm3+ (molar ratio 16:4:5:5) systems to exist at temperatures well above the Tm of DMPC up to 40 °C. In a first stage, we evaluated the polymolecular assemblies formed by the DMPC/ Cholesterol/DMPE-DTPA/Tm3+ (molar ratio 16:4:5:5) system after hydration of the dry lipid film following the simplified heating and cooling procedure. The magnetic alignability of the resulting assemblies when exposed to an external field was quantified by computing the Af from SANS results. The system’s response to temperature was evaluated by monitoring the birefringence signal under a 5.5 T magnetic field to identify the driving forces behind spontaneous formation of the polymolecular assemblies. In a final step, we exploited the heating and cooling procedure to regenerate the highly magnetically responsive samples stored frozen for one month at −18 °C. The intensity and number distributions of the DMPC/ Cholesterol/DMPE-DTPA/Tm3+ (molar ratio 16:4:5:5) system produced by the heating and cooling procedure were determined by DLS measurements at 5 °C; see Figure 4A. The intensity distribution revealed an average hydrodynamic diameter DH of 712 nm, while the number distribution showed a population dominated by species in the size range of 220 nm. However, the highly polydisperse nature of the sample must be stressed. The sample was further characterized by cryo-TEM in Figure 4B. The micrographs confirmed the existence of a polydisperse population of bicelles in the size range of 200 nm in diameter. The presence of such large planar structures foresees large gains in magnetic alignment through an enhanced aggregate number n. The DMPC/Cholesterol/DMPE-DTPA/Tm3+ (molar ratio 16:4:5:5) system’s alignment factor Af was evaluated as a function of the magnetic field strength in Figure 5A (black circles). The sample produced following the simplified heating and cooling procedure revealed an Af of −0.85 at 8 T as calculated from the 2D SANS pattern in Figure 5B. In comparison, the reported extruded system has an Af of −0.35 at equivalent conditions.41 The same Af was achieved at magnetic field strengths below 2 T at 5 °C with the newly achieved bicelles. Furthermore, the Af curve tended toward a plateau E
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Figure 5. (A) Alignment factor Af as a function of magnetic field strength for DMPC/Cholesterol/DMPEDTPA/Tm3+ (molar ratio 16:4:5:5) produced by the heating and cooling procedure at 5 °C (black circles) and 40 °C (red triangles). The 2D SANS patterns from which the Af were calculated at 8 T are shown at (B) 5 °C and (C) 40 °C. The magnetic field direction is shown with a white arrow labeled B. Figure 4. (A) Intensity (red) and number (black) distribution obtained from DLS at 5 °C and (B) cryo-TEM micrographs at 5 °C of DMPC/Cholesterol/DMPE-DTPA/Tm3+ (molar ratio 16:4:5:5) produced by the heating and cooling procedure. Bicelle structures are observed in the cryo-TEM micrographs. The scale bar represents 200 nm. Dark patches are water crystals resulting from the flashfreezing procedure.
tems, we evaluated the possibility of regenerating the samples after one month of storage frozen at −18 °C. A one month old frozen DMPC/Cholesterol/DMPEDTPA/Tm3+ (molar ratio 16:4:5:5) sample was melted by heating to 5 °C before being characterized by cryo-TEM and DLS in Figure 6A and B, respectively. Long ribbonlike structures were observed in the cryo-TEM micrographs. The DLS results revealed a bimodal distribution with a large population of smaller species in the 100 nm range. The large bicelle species observed in the freshly fabricated sample (Figure 4B) were no longer visible. We evaluated the possibility of regenerating these highly magnetically responsive polymolecular assemblies with the heating and cooling procedure. The magnetic alignment of the sample was monitored by birefringence measurements under a 5.5 T field as it underwent two consecutive heating and cooling cycles from 5 to 60 °C and back. The results from the first and second cycles are presented in red and black in Figure 6C, respectively. Studying the evolution of the sample’s birefringence signal over the two heating and cooling cycles allowed for monitoring of the changes occurring in the polymolecular assemblies throughout the process. Furthermore, the intensity of the birefringence signal is directly related to the sample’s magnetic alignability. If the birefringence signal increases after the heating and cooling cycles, a higher degree of alignment is achieved that must result from the regeneration of the magnetically responsive species. By further monitoring the intensity of the signal as a function of temperature, it is possible to identify important temperatures acting as trigger points in the regeneration process. Analogously
when reaching 8 T, indicating that the system was reaching full alignment. This saturation effect has only been observed for the former extruded sample by birefringence measurements at 35 T.42 When increasing the temperature to 40 °C, the DMPC/ Cholesterol/DMPE-DTPA/Tm3+ (molar ratio 16:4:5:5) sample produced by the heating and cooling procedure remained alignable as revealed by the red triangles in Figure 5A. A maximum Af of −0.35 was achieved at 8 T and 40 °C as calculated from the 2D SANS pattern in Figure 5C. Such a degree of alignment is equivalent to what could be achieved with the former extruded system at 5 °C. The overall magnetic energy of the large polymolecular assemblies formed after the heating and cooling procedure were able to withstand the high thermal energy acting against them at 40 °C and maintain a respectable degree of alignment. Aging and Regeneration of DMPC/Cholesterol/DMPEDTPA/Tm3+ (Molar Ratio 16:4:5:5). The unprecedented magnetic alignments and the simplicity of the heating and cooling procedure highlights the improvements made over the previously reported methods. In order to ascertain the value of the procedure and increase the versatility of DMPC/ Cholesterol/DMPE-DTPA/Tm3+ (molar ratio 16:4:5:5) sysF
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heating, which marks a change in lipid arrangement in the bilayer. This peak is similar to that observed for the cholesterolfree DMPC/DMPE-DTPA/Tm3+ (molar ratio 4:1:1) system in Figure 3B as the bilayer moves into a liquid disordered state above Tm. A similar mechanism could be responsible for the peak at 28 °C in the DMPC/Cholesterol/DMPE-DTPA/Tm3+ (molar ratio 16:4:5:5) system. This temperature corresponds to the phase boundary between the semiordered state and fully disordered state of a DMPC bilayers with 16 mol % cholesterol.54 Upon further heating, the birefringence signal gradually decreased as all the alignable polymolecular assemblies rearranged into vesicles at a temperature of 50 °C. In the second cycle, a clear hysteresis occurred in the birefringence signal upon heating and cooling. Liebi and coworkers reported this phenomenon for DMPC/Cholesterol/ DMPE-DTPA/Tm3+ (molar ratio 16:4:5:5) bicelles.30,41,42 The appearance of the hysteresis originates from large holes in the bicelle’s bilayer that form only during heating. The peak in the birefringence signal at 28 °C remained visible in the hysteresis envelope. The birefringence signal evolved in the same way with temperature for both cooling cycles. This suggests that the key parameter responsible for regenerating the highly magnetically alignable structures already occurred on heating. Nevertheless, the movement of the lipids from a fully disordered state into an ordered state upon cooling back to 5 °C was necessary to regenerate the large bicelles from the vesicles existing at 50 °C. Consequently, the proposed heating and cooling procedure was sufficient to guaranty the successful formation and regeneration of the highly magnetically responsive species.
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Figure 6. (A) Cryo-TEM micrographs of a one month old frozen DMPC/Cholesterol/DMPE-DTPA/Tm3+ (molar ratio 16:4:5:5) sample as it was melted by heating to 5 °C before being flash-frozen. The scale bars represent 200 nm. Dark patches are water crystals resulting from the flash-freezing procedure. Note that the micrograph surrounded by a red box was obtained from another micrograph of the same sample. (B) Intensity (red) and number (black) distribution of the same sample obtained from DLS at 5 °C. C) Birefringence signal as a function of temperature at 5.5 T of the same sample as it was regenerated by two consecutive heating and cooling cycles at 1 °C/ min. Heating is represented with full lines and cooling with dashed lines. In the first cycle (red lines), the sample was melted out of its frozen state. Arrows indicate the direction of temperature change. The schematic representations of the polymolecular assembly geometries are shown where appropriate. The ribbons rearranged into vesicles upon heating and bicelles were regenerated when cooling back to 5 °C (first cycle, red lines) as evidenced by the larger birefringence signal. Bicelles with holes are a likely cause of the hysteresis on heating observed in the second cycle (black lines).42
CONCLUSION Polymolecular assemblies displaying an unprecedented magnetic response were successfully generated from both DMPC/ DMPE-DTPA/Tm3+ (molar ratio 4:1:1) and DMPC/Cholesterol/DMPE-DTPA/Tm3+ (molar ratio 16:4:5:5) lipid systems by following simple heating and cooling cycles from 5 to 60 °C to hydrate the dry lipid film as summarized in Figure 7A. The added value of this simplified fabrication procedure is evident when comparing the magnetic alignment of the resulting polymolecular assemblies with those formed following the previously reported fabrication procedure in Figure 7B. The heating and cooling procedure allowed for a 2-fold increase in alignment factor for the cholesterol-free DMPC/DMPEDTPA/Tm3+ (molar ratio 4:1:1) system and a 2.5-fold increase for the DMPC/Cholesterol/DMPE-DTPA/Tm3+ (molar ratio 16:4:5:5) at 5 °C and 8 T. The movement of the lipids to the liquid disordered and back to the solid ordered state induced major rearrangements of the polymolecular assemblies. Consequently, going above and below the phase transition temperature Tm was the key parameter responsible for the successful formation of the highly magnetically responsive DMPC/DMPE-DTPA/Tm3+ (molar ratio 4:1:1) species. The DMPC/Cholesterol/DMPE-DTPA/Tm 3+ (molar ratio 16:4:5:5) samples must be heated up to 60 °C to move out of the broad liquid-ordered state into a fully disordered lipid bilayer before cooling back down. The proposed heating and cooling procedure is ideally suited for these purposes. DMPC/ Cholesterol/DMPE-DTPA/Tm3+ (molar ratio 16:4:5:5) samples may be stored for prolonged periods of time in a frozen state, similarly to known bicelle systems.39,40 They were readily regenerated by following the same heating and cooling procedure initially employed to create them from a dried lipid film.
to the birefringence peaks observed around the Tm for the DMPC/DMPE-DTPA/Tm3+ (molar ratio 4:1:1) assemblies in Figure 3B. These experiments aim at understanding the underlying mechanism responsible for the possible regeneration of the magnetically responsive species. The birefringence signal increased 2-fold at 5 °C after the first cycle. The corresponding increase in magnetic alignment was attributed to the reappearance of large bicelles starting from the ribbonlike structures observed after melting of the one month old sample. The enhanced magnetic response of a bicelle structure when compared to a ribbon of equivalent surface area comes from less edge-area in the former. Consequently, more phospholipids are located in the planar region of the polymolecular assembly, capable of contributing to the cumulative magnetic energy Emag of the bilayer.30,35,41 A distinct peak in the birefringence signal appeared at 28 °C on G
DOI: 10.1021/acs.langmuir.7b00725 Langmuir XXXX, XXX, XXX−XXX
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Langmuir
Figure 7. (A) Schematic representation of the polymolecular assemblies obtained by the heating and cooling (H&C) procedure for studied DMPC/ DMPE-DTPA/Tm3+ (molar ratio 4:1:1) and DMPC/Cholesterol/DMPE-DTPA/Tm3+ (molar ratio 16:4:5:5) lipid mixtures. The former cholesterol-free system was composed of sheet-like structures with folds and twists surrounded by numerous smaller bicelles. The later cholesterolcontaining system was composed of large polydisperse bicelles with diameters in the range of several hundreds of nanometers. The drawings are not to scale. (B) Alignment factor Af at 8 T and 5 °C of the samples prepared following the previously reported fabrication procedure involving freeze thawing cycles (FT) followed by extrusion (Ext) in red and following the new heating and cooling procedure (H&C) in black. dimyristoylphosphatidylcholine by solid-state NMR. Biochemistry 1992, 31, 8898−8905. (5) Sanders, C. R.; Landis, G. C. Reconstitution of membrane proteins into lipid-rich bilayered mixed micelles for NMR studies. Biochemistry 1995, 34, 4030−4040. (6) Sanders, C. R.; Prosser, R. S. Bicelles: a model membrane system for all seasons? Structure 1998, 6, 1227−1234. (7) Ram, P.; Prestegard, J. H. Magnetic field induced ordering of bile salt/phospholipid micelles: new media for NMR structural investigations. Biochim. Biophys. Acta, Biomembr. 1988, 940, 289−294. (8) Dürr, U. H. N.; Soong, R.; Ramamoorthy, A. When detergent meets bilayer: birth and coming of age of lipid bicelles. Prog. Nucl. Magn. Reson. Spectrosc. 2013, 69, 1−22. (9) Yamaguchi, T.; Suzuki, T.; Yasuda, T.; Oishi, T.; Matsumori, N.; Murata, M. NMR-based conformational analysis of sphingomyelin in bicelles. Bioorg. Med. Chem. 2012, 20, 270−278. (10) Sanders, C. R.; Hare, B. J.; Howard, K. P.; Prestegard, J. H. Magnetically-oriented phospholipid micelles as a tool for the study of membrane-associated molecules. Prog. Nucl. Magn. Reson. Spectrosc. 1994, 26, 421−444. (11) Glover, K. J.; Whiles, J. A.; Wu, G.; Yu, N.; Deems, R.; Struppe, J. O.; Stark, R. E.; Komives, E. A.; Vold, R. R. Structural evaluation of phospholipid bicelles for solution-state studies of membraneassociated biomolecules. Biophys. J. 2001, 81, 2163−2171. (12) 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. (13) Israelachvili, J. N.; Mitchell, D. J.; Ninham, B. W. Theory of selfassembly of hydrocarbon amphiphiles into micelles and bilayers. J. Chem. Soc., Faraday Trans. 2 1976, 72, 1525−1568. (14) Kumar, V. V. Complementary molecular shapes and additivity of the packing parameter of lipids. Proc. Natl. Acad. Sci. U. S. A. 1991, 88, 444−448. (15) McKibbin, C.; Farmer, N. A.; Jeans, C.; Reeves, P. J.; Khorana, H. G.; Wallace, B. A.; Edwards, P. C.; Villa, C.; Booth, P. J. Opsin stability and folding: modulation by phospholipid bicelles. J. Mol. Biol. 2007, 374, 1319−1332. (16) Yamamoto, K.; Soong, R.; Ramamoorthy, A. Comprehensive analysis of lipid dynamics variation with lipid composition and hydration of bicelles using nuclear magnetic resonance (NMR) spectroscopy. Langmuir 2009, 25, 7010−7018. (17) Son, W. S.; Park, S. H.; Nothnagel, H. J.; Lu, G. J.; Wang, Y.; Zhang, H.; Cook, G. A.; Howell, S. C.; Opella, S. J. ‘q-Titration’ of long-chain and short-chain lipids differentiates between structured and mobile residues of membrane proteins studied in bicelles by solution NMR spectroscopy. J. Magn. Reson. 2012, 214, 111−118. (18) Crowell, K. J.; Macdonald, P. M. Surface charge response of the phosphatidylcholine head group in bilayered micelles from phosphorus
The direction of magnetic alignment of the proposed polymolecular assemblies may further be tuned by switching the chelated lanthanide ion with, for example, dysprosium.19−23,26,41,42 The possibility of tailoring the direction of alignment, while simultaneously benefiting from the enhanced magnetic response, emphasizes the viability of the newly achieved DMPC/DMPE-DTPA/Ln3+ and DMPC/Cholesterol/DMPE-DTPA/Ln3+ polymolecular assemblies. The numerous advantages offered by these systems including their thermoreversible nature, their magnetic responses at field strengths as low as 1 T, their resistance to environmental changes, and the freedom in design offered by introducing cholesterol or altering the nature of the chelated lanthanide ion open doors for numerous applications in the field of smart soft materials. These may range from pharmaceutical applications to the fabrication of optically active gels.2,3
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AUTHOR INFORMATION
ORCID
Stéphane Isabettini: 0000-0003-3416-8103 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS 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.
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REFERENCES
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DOI: 10.1021/acs.langmuir.7b00725 Langmuir XXXX, XXX, XXX−XXX
