Dynamic Lipid Lateral Segregation Driven by Lauryl Cyclodextrin

Feb 14, 2013 - Dynamic Lipid Lateral Segregation Driven by Lauryl Cyclodextrin. Interactions at the Membrane Surface. Michel Roux,*. ,†. Edward Ster...
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Dynamic Lipid Lateral Segregation Driven by Lauryl Cyclodextrin Interactions at the Membrane Surface Michel Roux,*,† Edward Sternin,‡ Véronique Bonnet,§ Christophe Fajolles,∥ and Florence Djedaíni-Pilard§ †

CEA/DSV/iBiTec-S, UMR CNRS 8221, SB2SM, F-91191 Gif sur Yvette Cedex, France Department of Physics, Brock University, St. Catharines, Ontario, L2S 3A1 Canada § Laboratoire des Glucides, UMR 6219, Université de Picardie Jules Verne, F-80039 Amiens, France ∥ CEA/IRAMIS/UMR3299 SIS2M/LIONS, F-91191 Gif sur Yvette Cedex, France ‡

ABSTRACT: Amphiphilic cyclodextrins, with a cholesterol anchor (βChol) or an aspartic acid moiety esterified by two lauryl acyl chains (βDLC), were designed to combine the inclusion ability of the cyclodextrin cavity with the carrier properties of model membranes. Their insertion in phosphatidylcholine bilayers induces a marked lateral phase separation into a pure lipid phase and a cyclodextrin-rich phase (LCD), organized as a 2D cyclodextrin network stabilized by intermolecular hydrogen bonds between the saccharide ̈ headgroups at the membrane surface (Roux, M.; Perly, B.; Djedaini-Pilard, F. Self-Assemblies of Amphiphilic Cyclodextrins. Eur. Biophys. J. 2007, 36, 861−867). We have replaced the dilauryl anchor by a single lauryl chain grafted onto a leucine residue, giving monolauryl-β-cyclodextrin (βMLC), which readily inserts into bilayers of chain-deuterated DMPC-d27. The removal of one lauryl acyl chain leads to a dynamic membrane insertion of this new cyclodextrin derivative, with significant lipid exchange on the deuterium NMR time scale between a loosely packed cyclodextrin-enriched phase (L′CD) and free lipid regions, yielding broadened two-component NMR spectra. Like the LCD phases, the cyclodextrin-enriched L′CD regions remain (partially) fluid below the DMPC-d27 main fluid-to-gel transition but do not undergo a clear transition toward a gel state, as observed at 14.5 °C in the LCD phase induced by the dilauryl derivative. Partially fluid lipids of the βMLC-induced L′CD phase coexist with pure lipids in the Pβ′ gel phase with possible exchange between them until all of the lipids undergo a transition toward an Lβ′ gel state at around 7 °C. Trimethylated monolauryl-β-cyclodextrins induce only an ordering of the lipid acyl chains just above the main transition, without any lateral phase separation. Similar chain ordering is also observed within the βMLC-induced L′CD phase as a consequence of the deep membrane insertion of the monolauryl nonmethylated cyclodextrin derivative.

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INTRODUCTION

Cyclodextrin-containing organized supramolecular structures can also be obtained through self-assemblies of amphiphilic cyclodextrins or by their insertion into integrated systems such as lipid membranes.12−14 Polysubstitution of cyclodextrin hydroxyl groups with alkyl chains on the primary alcohol side, the secondary side, or both can lead to medusa-, skirt-, or bouquet-shaped derivatives, respectively. Such amphiphilic derivatives can lead to the formation of nanoparticles capable of encapsulating drugs.12,15 Monosubstituted cyclodextrins obtained by grafting a hydrophobic anchor, such as a cholesterol nucleus,16 a diacylated aspartic acid,17 or a phosphoramidyl moiety,18 onto a single hydroxyl group of the oligosaccharide cavity were designed to insert into lipid membranes and improve their vectorization through their liposome transportation in the organism. These cyclodextrin derivatives readily insert themselves into lipid membranes, and

Cyclodextrins are water-soluble molecular cages with a hydrophobic interior that allows the inclusion and hence solubilization of various water-insoluble molecular species.1,2 In addition to commercial applications in the food, cosmetics, and textile industries,3−6 these biocompatible cyclic oligosaccharides, with low toxicities and no immune response, have also been used extensively in pharmaceutical applications as hydrophobic drug carriers with a large number of approved and marketed formulations.7,8 Improved inclusion capacity and controlled drug release can be experimentally achieved through cyclodextrin-containing macromolecular structures, obtained by direct covalent cross-linking of cyclodextrin units or by their covalent attachment as side chains of a synthetic polymer backbone.9 There is growing interest in cyclodextrin-containing polycationic polymers especially designed for the gene delivery of oligonucleotides and plasmids.10 Noncovalent supramolecular structures made up of cyclodextrin units or more recently of cyclodextrin-containing polymers combined with other polymers also hold promise as tools for drug delivery.11 © XXXX American Chemical Society

Received: November 13, 2012 Revised: February 3, 2013

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some lead to a strong lateral segregation of the membrane lipids into a pure lipid domain and a cyclodextrin-enriched phase (LCD). Although more disordered than the pure lipids, the LCD phase is very stable and remains in the fluid state below the chain-melting transition of DMPC to coexist with pure lipids in the gel state at cyclodextrin concentrations as low as 5%.17 This remarkable stability is due to particular intermolecular interactions at the membrane surface of the β-cyclodextrin headgroups, known to aggregate as monomers in aqueous solution.19,20 Phase separation is indeed not observed when the hydroxyl groups are methylated, preventing hydrogen bonding between adjacent cyclodextrins. Monosubstituted β-cyclodextrins provide a straightforward case of microdomain formation within a lipid bilayer through finely tuned molecular interactions at the membrane−water interface.21 The amount of lipids trapped in the LCD phase depends critically on the hydrophobic anchor. The LCD phase induced by dilauryl-β-cyclodextrin contains 3 times more lipids (4 to 5 lipids per cyclodextrin) than that obtained with cholesteryl-βcyclodextrin (1.5 lipid per cyclodextrin). At a lipid molar concentration close to that of the LCD phase (∼20%), dilaurylβ-cyclodextrins are able to sequester most of the lipid molecules, leaving a single well-defined, stable LCD phase with only trace amounts of free lipids. As expected from the difference in their hydrophobic anchors, the two amphiphilic cyclodextrin derivatives are also different in their effects on the thermal behavior of the lipid phases. Although both LCD phases remain fluid below the DMPC main transition, the one obtained with dilauryl-β-cyclodextrin displays a sharp transition to a gel state with frozen acyl chains at 12.5 °C. The one observed with the cholesteryl derivative remains partially fluid below this temperature, possibly because of the well-known effect of “fluidization” of lipids in the gel phase by cholesterol.22 In this Article, we deal with a third class of monosubstituted amphiphilic cyclodextrins containing a single acyl chain as a hydrophobic anchor,23 prepared by grafting a leucine residue amidified by one lauryl chain via a succinyl spacer (Scheme 1). The results from the present study of monolauryl-β-cyclodextrin (βMLC) interactions with DMPC membranes are fully consistent with the model of a β-cyclodextrin network mediated through hydrogen bonds at the membrane surface. The formation of a βMLC-induced laterally segregated phase, found to be different from the LCD phases described previously, was monitored by deuterium NMR of chain-deuterated DMPC membranes. NMR spectra were also recorded from membranes containing the trimethylated derivative of monolauryl-β-cyclodextrin, where no lateral segregation was detected.

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Scheme 1. Chemical Structures of the Monolauryl (βMLC), Dilauryl (βDLC), and Cholesteryl (βChol) Derivatives of βCyclodextrin with Their Succinyl Spacera

a

The trimethylated derivatives were obtained by permethylation of the primary (6) and secondary (2, 3) hydroxyl groups.

(Eurisotop, St Aubin, France) equilibrated at pH 7.5, giving ∼200 mM lipid dispersions. The samples were then subjected to three freeze (in liquid nitrogen) and thaw (up to 45 °C) cycles. 2 H NMR Experiments. 2H NMR spectra were recorded at 46 MHz on a Bruker DMX 300 spectrometer equipped with a probe specifically designed for solid-state deuterium NMR experiments (Morris Instruments Inc., Gloucester, Ontario, Canada). Membrane samples were cooled from 37 to −15 °C, and NMR spectra were acquired with a dwell time of 2 μs, 4K data points, and a repeat time of 200 ms. A quadrupolar echo pulse sequence was employed with a pulse length of 4 μs and a pulse separation, τ, of 40 μs.24 A zeroth-order phase correction was applied to obtain no signal in the imaginary channel. When necessary, the free induction decay was shifted by a fraction of the dwell time using an orthogonal polynomial interpolation routine so that the Fourier transform could start at the top of the echo.25 Oriented 2H NMR spectra (0°) were obtained numerically using dePakeing.26,27 Order parameters SCD of the methyl and methylene groups of fluid acyl chains were obtained from their dePaked quadrupolar splittings ΔνQ according to SCD =

MATERIALS AND METHODS

Synthesis of Amphiphilic Cyclodextrins. The synthesis of monolauryl-β-cyclodextrin and its trimethylated derivative was performed as described previously.23 The chemical structures of monolauryl (βMLC), dilauryl (βDLC), and cholesteryl (βChol) βcyclodextrins discussed in this Article are shown in Scheme 1. Sample Preparations. Chain-deuterated DMPC-d27 and DMPCd54 were from Avanti Polar Lipids (Alabaster, AL, USA), and the cholesterol was from Sigma-Aldrich (St Louis, MO, USA). Multilamellar liposomes were prepared by mixing chloroform solutions of the lipid and the appropriate cyclodextrin derivative, removing the solvent by evaporation under reduced pressure, and resuspending the solid residues in 1 to 2 mL of water equilibrated at pH 7 with vortex mixing. The resulting mixture was lyophilized, and the obtained powder was dispersed by continuous vortex mixing in 100−300 μL of buffer (50 mM Tris, 40 mM NaCl) in deuterium-depleted water

⎛ 4 ⎞⎛ h ⎞ ⎜ ⎟⎜ ⎟Δν ⎝ 3 ⎠⎝ e 2qQ ⎠ Q

(1)

2

where e qQ/h is the quadrupolar coupling constant, 167 kHz for a static C−D bond.25 The first moments M1 of the deuterium powder spectra were determined,25,28 and the average order parameters ⟨SCD⟩ of the DMPC-d27 acyl chains calculated according to ⟨SCD⟩ =

⎛ 4 ⎞⎛ h ⎞⎛ 3 3 ⎞ ⎜ ⎟⎜ ⎟⎜ ⎟M1 ⎝ 3 ⎠⎝ e 2qQ ⎠⎝ 4π ⎠

(2)

For some motionally averaged quadrupolar splittings ΔνQ that were individually resolved in the dePaked spectra from the sn2 acyl chain of DMPC-d27, moments M1 and M2 were calculated as

⎛ 4π ⎞ M1 = ⎜ ⎟⟨Δν ⟩ ⎝3 3 ⎠ Q B

(3)

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Figure 1. 2H NMR powder (b, j) and dePaked spectra (a, c−h, i, k−p) of DMPC-d27 membranes recorded in the fluid phase at 23 °C (a−h) and at 20 °C (i−p) below the main transition, either pure (b, c, j, k) or with 5, 10, 20, 30, and 40% monolauryl-β-cyclodextrin (d−h, l−p) or 7.5% dilaurylβ-cyclodextrin (a, i) expressed in mol %. The methyl doublet of spectrum a was simulated with Gaussian functions (light trace). The digits and Greek letters on the pure DMPC dePaked spectrum (c) point to the quadrupolar splittings attributed to the C14 methyl deuterons (1), the C13 (2), C12 (3) and C2 (α, β) methylene deuterons, and those (C3−C8) of the plateau region (4). Components Iα, Igel, Ipf, I, and II are described in the text. The dePaked spectra were scaled with normalization factors obtained by the area normalization of their related FT spectra (b). Superimposed light trace j is for data recorded at 21.7 °C, showing the coexistence of fluid lipids in the Lα phase (Iα) with pseudofluid (Ipf) and gel (Igel) lipids of the Pβ′ phase.

⎛ 4π 2 ⎞ 2 M2 = ⎜ ⎟⟨ΔνQ ⟩ ⎝ 5 ⎠

compare with those obtained previously in DMPC-d 54 membranes at various concentrations up to 30% βDLC.17 DMPC-d27 Membranes. Deuterium NMR spectra of membranes recorded above the gel-to-fluid transition are characterized by a well-defined distribution of quadrupolar splittings typical of the liquid-crystalline Lα phase (Figure 1b), which can be partially resolved by the deconvolution of the powder pattern with the standard iterative dePakeing procedure (Figure 1c). The ordered methylene groups located near the membrane surface appear together as a large composite quadrupolar splitting found at the edge of the dePaked spectrum (feature 4), in a plateau region that is the signature of a lipid bilayer. The narrow doublet at ±4 kHz (feature 1) is attributed to the methyl groups of the disordered end of the acyl chains (C14) and has proven to be very useful to distinguish between lipid phases.17,30−32 The use of DMPC-d27 selectively deuterated on the sn2 chain allows for an unambiguous assignment of the other resolved quadrupolar splittings: the C12 (feature 3) and C13 (feature 2) deuterons

(4)

Unresolved quadrupolar splittings overlapping in the plateau region were estimated according to the smoothed average order profile method.29

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RESULTS H NMR of deuterated phospholipids allows a precise analysis of the lipid bilayer organization through measurement of the order parameters of the lipid acyl chains. Figure 1 displays deuterium NMR spectra obtained with DMPC-d27 membranes at 23 °C (left) and 20 °C (right), above and below the main gel-to-fluid transition. NMR spectra were obtained with pure DMPC membranes (traces b, c, j, and k) and in the presence of various concentrations of monolauryl-β-cyclodextrin (traces d− h and l−p). Data obtained at a single representative concentration of the dilauryl derivative are also included at the top of the figure (traces a and i) as a control in order to 2

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Figure 2. Methyl region of the dePaked 2H NMR spectra of DMPC-d27 membranes recorded without (A) and with 30% (B) or 40% (C) monolauryl-β-cyclodextrin expressed in mol %. The NMR spectra were obtained by cooling the sample in 1° steps, with 0.1° steps between 23 and 20 °C. Selected spectra are shown between 30 and 12 °C. The light dePaked traces superimposed with the 12 °C spectra are those recorded at 5 °C. Note the increased scaling applied for the data obtained between 19 and 13 °C (× 2) and at 12 and 5 °C (× 4). The continuous line connecting the peaks of the NMR data recorded at 30% βMLC (B) was drawn manually through the peak maxima and then translated as a dotted line to the corresponding data at 40% (C). The components (I, II, Igel, and Ipf) indicated by the arrows are described in the text.

and the α and β deuterons of the C2 carbon next to the glycerol backbone.33 DMPC-d54 deuterated on both acyl chains has also been used to differentiate the sn1 and sn2 methyl groups. Unless otherwise stated, all results discussed with DMPC-d54 were obtained in previous studies.16,17 DMPC-d27 deuterated on a single chain allows also a more precise analysis of the NMR spectra recorded in the Pβ′ ripple phase found between the pretransition and the main transition that occur at 7 and 21.5 °C, respectively, as determined by DSC (cooling scan of Figure 3). At these temperatures, the sn2 acyl chain methyl signal of pure DMPC molecules contains two quadrupolar splittings characteristic of the lipids found in this intermediate gel phase observed with saturated phosphatidylcholines.34−38 The Pβ′ ripples are generally asymmetric with a sawtooth profile resulting from the alternation of major (M) and minor (m) bilayer sections of different thicknesses, connected by a kink and leading to a nonplanar corrugated membrane geometry. The major sections have the thickness of a bilayer in the gel phase, whereas the thickness of the minor sections is smaller, consistent with that found in the fluid Lα phase. The coexistence of gel and fluid-like lipids in the Pβ′ phase has

been verified by various methods,39−41 including 13C NMR studies.42,43 According to previous 2H NMR work,22,44,45 the large and the small methyl quadrupolar splittings detected in the Pβ′ phase are very likely due to these gel (Igel) and “pseudofluid” (Ipf) lipids coexisting within the ripple phase. At a temperature very close to the main transition temperature (21.7 °C), a third quadrupolar splitting attributed to the DMPC in the Lα phase (Iα) is clearly detected, indicating that (i) the Lα and Pβ′ phases coexist at this temperature and (ii) the fluid components of the Lα and Pβ′ NMR spectra reflect two distinct order parameters and are not related.45 Dilauryl-β-cyclodextrin. As reported previously for DMPC-d54 membranes containing various molar concentrations (up to 30%) of dilauryl-β-cyclodextrin,17 the insertion of this derivative into DMPC-d27 membranes is also coupled with the appearance of a second spectral component (II) attributed to the lipids of a cyclodextrin-enriched phase (LCD) in slow exchange with the free lipids (I) on the NMR time scale (Figure 1a). Simulation with Gaussian functions of the two methyl components separated by 1.7 kHz indicates that after the membrane insertion of 7.5% βDLC, ∼35% of the total D

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lipids are trapped in the LCD phase, yielding a lipid-tocyclodextrin ratio similar to that obtained with DMPC-d54 membranes (∼4:1 mol/mol) at similar and other βDLC concentrations.17 Below the main transition (Figure 1i), the free lipids are in the gel phase, although those of the LCD phase remain fluid, producing well-resolved dePaked NMR spectra down to 14.5 °C and gel phase spectra below this temperature (data not shown). Monolauryl-β-cyclodextrin. Membrane insertion of increasing amounts of the monolauryl cyclodextrin derivative βMLC is also associated with the progressive appearance of a second spectral component (II) close to that of the pure lipids (I). In the fluid phase above the main transition, two methyl group signals are clearly visible at and above 20% of βMLC (Figure 1f−h) in spite of an increase in line broadening not observed with 7.5% of the dilauryl derivative nor at higher concentrations in the DMPC-d54 membranes studied previously.17 For instance, the free lipid signal is broadened with a line width increase of about 50% in the presence of 30% of the cyclodextrin derivative. This important line broadening is probably related to exchanges between the environments giving rise to the two NMR components (I, II). This will be developed further in the Discussion in relation to lipid exchanges between βMLC-depleted and βMLC-enriched phases. The quadrupolar splitting of the additional component observed with the monolauryl cyclodextrin is greater than that of the free lipids, whereas it was smaller in the presence of the dilauryl derivative. The βMLC-induced second component is also clearly detected below the fluid-to-gel transition, with a methyl quadrupolar splitting very close to that of the pseudofluid component (Ipf) of the ripple phase. The intensity of the second methyl signal (II) grows with the increasing amounts of the monoacyl cyclodextrin derivative, whereas that of the free lipids in the gel state (Igel) decreases (Figure 1k−p). For a concentration of 40%, the gel component of the methyl spectrum almost vanishes (Figure 1p). The emergence of the second methyl spectral component (II) is illustrated in more detail in Figure 2 where temperaturedependent changes in the dePaked 2H NMR spectra at high concentrations (30 and 40%) of βMLC are shown (Figure 2B,C). Figure 2A presents the pure DMPC spectra for comparison, and here the transition is seen to occur at around 21.5 °C, in good agreement with our calorimetric data (Figure 3). Below this temperature, the two methyl signals characteristic of the Pβ′ phase (Igel and Ipf) are well-resolved. There is a gradual decrease of the pseudofluid component (Ipf), which is barely visible at 12 °C and disappears completely at 5 °C, below the DMPC-d27 pretransition found at 7 °C by calorimetry. As shown in Figure 2B, for 30% βMLC the second component (II) appears at around 26 °C and is partially resolved at 24 °C. At higher temperatures, the spectra of the βMLC-induced component (II) coincide with that of the free lipids (I) and the resulting spectral lines are considerably broadened. The temperature dependence of the smaller quadrupolar splitting (I) associated with the free lipids in the fluid phase above 24 °C is similar to that observed with the pure DMPC-d27 membranes shown in Figure 2A but differs below this temperature. The quadrupolar splitting of the free lipids (I) shows a smooth sigmoidal change without a sharp transition and gradually approaches the quadrupolar splitting (II) of the lipids trapped in the βMLC-enriched phase. Around 21 °C, there is a region where the two components are again superimposed. At this temperature, the NMR spectra recorded

Figure 3. Average order parameter ⟨SCD⟩ of the DMPC sn2 chain, calculated from the first moment M1 of the 2H NMR powder spectra of DMPC-d27 membranes (A) and D2 function (see text) (B) as a function of temperature: pure (∗, ◊) and with 10% (●), 30% (▲), and 40% (+) monolauryl-β-cyclodextrin and 7.5% (□) dilauryl-β-cyclodextrin, expressed in mol %. The DSC cooling scan (arbitrary units) of pure DMPC-d27 membranes is plotted in panel A and rescaled vertically (×10) in the pretransition region. Partial magnetic orientation of the pure DMPC-d27 membranes leads to a slightly distorted powder pattern with an artificially low D2 value (∗), which can be corrected (◊) by calculating M1 and M2 from the order parameter distribution. E

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with 30% βMLC remain fluidlike, with barely a trace of the gel signal (Igel) like that observed at about ±15 kHz for the pure DMPC membrane sample (Figure 2A). In the 30% βMLC sample, such a gel signal (Igel) emerges only at around 20 °C. It coexists with the remaining fluidlike methyl quadrupolar splitting containing contributions from both the free lipid pseudofluid component (Ipf) and the second component associated with the βMLC-enriched phase (II). This composite splitting begins to decrease slowly below 20 °C, is just detected at 12 °C, and vanishes completely between 5 and 0 °C. Similar data are obtained for 40% βMLC (Figure 2C) except that the intensity of the second component (II) is increased by a greater fraction of the lipids in the βMLC-enriched phase. The sigmoidal temperature dependence of the remaining free lipid quadrupolar splitting (I) is still apparent, although its appreciation is hindered by the overlap of NMR lines. Because of the increased βMLC concentration, the intensity of the signal associated with free lipids in the gel state (I) has considerably decreased and appears only at around 17 °C. At 12 °C, there is still roughly 50% of the βMLC-enriched phase signal (II), some signal is still detected at 5 °C and disappears between 5 and 0 °C (spectra not shown). Internal changes in the DMPC-d27 membranes associated with the emergence of the βMLC-induced phase can be further illustrated through the temperature dependence of the average order parameter ⟨SCD⟩ determined from the first moment M1 of the powder spectra. In Figure 3, ⟨SCD⟩ is seen to increase upon cooling, with a sharp change at around 21.5 °C associated with the main fluid-to-gel transition of the pure lipids, as discussed above, and in good agreement with the DSC cooling scan. It is known that the fluid-to-gel transition temperature of saturated phospholipids such as DMPC is decreased by the deuteration of their acyl chains.46 On the basis of similar DSC cooling scans, the fluid-to-gel transitions of unlabeled DMPC and DMPC-d54 deuterated on both sn1 and sn2 chains were observed at 23.5 and 20.5 °C, respectively. Another sharp singularity at −3.5 °C, is associated with the subtransition of the phospholipids from the Lβ′ gel phase to the Lc lamellar gel phase.47 The pretransition detected at around 7 °C in the DSC cooling scan does not affect the averaged bilayer order. Both the main transition and the subtransition are still detected in the presence of 10% βMLC, but at 30% the subtransition has vanished. The curve is considerably smoothed at 40% with an almost linear increase in the average order parameter with temperature, without any apparent transition. Below the main transition, there is a significant and gradual decrease in the average membrane order with increasing βMLC concentrations. The effect of βMLC on the Lα → Pβ′ → Lβ′ → Lc transitions can be seen through the temperature dependence of the function D2, defined as D2 =

⎛ M2 ⎞ ⎜ ⎟ − 1 ⎝ 1.35M12 ⎠

D2 at the main transition leading to the Pβ′ ripple phase, with a maximum value of about 0.2 at 21 °C for the pure lipids, followed immediately by (i) a smooth decrease upon further cooling and (ii) a quasi-plateau (D2 ≈ 0.15) starting at around 7 °C at the Pβ′ → Lβ′ pretransition of DMPC-d27. In the presence of βMLC, the D2 maximum observed at the main transition is increased and shifted to lower temperatures, giving 0.24 and 0.26 for 10 and 30%, respectively, of the cyclodextrin derivative at 19 °C and 0.3 for 40% at 14 °C. A plateau is also observed at the pretransition after a D2 decrease in the presence of 10% (0.19) and 30% (0.22) βMLC. For a concentration of 40%, there is a continuous D2 decrease, without either a plateau or a significant change in slope. Finally, below the plateau in the pure lipid data, there is a sharp D2 decrease at the subtransition leading to the Lc lamellar gel phase with D2 values at around 0.1 indicating a narrower dispersion of order parameters. A sharp subtransition is also detected for 10% βMLC but not with 30 and 40% of the cyclodextrin derivative where there is only a smooth decrease in the D2 values. Data recorded with membranes containing 5 and 20% βMLC (data not shown) were found to be similar to those obtained at 10%. Measurements of ⟨SCD⟩ and D2 were also carried out in DMPC-d27 membranes containing 7.5% of dilauryl derivative βDLC. The resulting D2 curve shown in Figure 3B is similar to those obtained with intermediate concentrations of the monolauryl-β-cyclodextrin. Quasi-constant D2 is obtained in the fluid Lα phase, with slightly larger values (0.14). Similarly, the maximum value reached after a sharp increase in D2 at the transition is higher (0.32). It is also followed by a decrease leading to a plateau (0.17) between the DMPC-d 27 pretransition and subtransition. The sharp first-order fluid-togel transition of the lipids trapped in the LCD cyclodextrinenriched phase, which occurs at 14.5 °C, between the main transition and the pretransition, is apparent on the ⟨SCD⟩ and D2 curves shown in Figure 3 (spectra not shown). Below this temperature, ⟨SCD⟩ is not decreased, as observed with the monolauryl cyclodextrin derivative, but it is similar to that of pure DMPC-d27 membranes (Figure 3A). Trimethylated Lauryl-β-cyclodextrin. Deuterium NMR spectra obtained in the presence of 10% trimethylated derivatives of monolauryl and dilauryl-β-cyclodextrin in DMPC-d27 membranes, just above the main transition at 23 °C (Figure 4), are essentially identical to those obtained previously for the dilauryl analog inserted into DMPC-d54 membranes.17 We observe (i) well-resolved and narrow single-component NMR spectra indicating that the free lipids and those interacting with the methylated derivatives exchange rapidly on the NMR time scale and (ii) larger quadrupolar splittings, with 25 and 29% increases for the methylenes of the plateau and the terminal methyl groups, respectively. In addition, when inserted into DMPC-d54 membranes, the trimethylated monolauryl derivative, like the dilauryl derivative studied earlier,17 induces two methyl doublets with equal intensities (data not shown). As pointed out previously,17 such a perturbation is similar to that observed in the presence of the same amount (10%) of cholesterol (Figure 4e). The line shape of the powder spectrum obtained with 10% TrimβMLC (Figure 4b) is typical of a nonrandom distribution of magnetically oriented lipid membranes with the bilayer normal mostly perpendicular to the magnetic field, yielding intense narrow 90° doublets and only a trace of 0° shoulders.48 Applying the standard iterative dePakeing method leads to

(5)

For axially symmetric 2H NMR spectra, D2 is the relative mean square width of the distribution of quadrupolar splittings and is highly sensitive to changes found in heterogeneous twophase coexistence regions.22,28 Each of the curves in Figure 3B can be divided into four parts. In the liquid-crystalline Lα phase, the D2 values are relatively small (∼0.1) and quasi-constant, indicative of a well-defined order profile with a low dispersion of the lipid chain order parameters. There is a sharp increase in F

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Figure 4. 2H NMR powder (b) and dePaked (a, c−e) spectra of DMPC-d27 membranes recorded at 23 °C, either pure (a) or with a 10% mol concentration of TrimβMLC (b, c), TrimβDLC (d), or cholesterol (e). The powder spectrum (b) was deconvoluted (c) with either standard iterative dePakeing (light trace) or with a Tikhonov regularization inverse algorithm.

distorted data with nonphysical negative intensities (Figure 4c, light trace). Depakeing such a spectrum requires the use of a Tikhonov-regularization inverse algorithm capable of extracting both the order parameter distribution (Figure 4c) and the (unknown) orientation distribution function p(θ) of the sample.27 This analysis indicates that at least 95% of the sample is in a highly aligned state. If this alignment distribution is described as an ellipsoidal deformation of the liposomes, then the square of the ratio κE of the long and small axes of the ellipsoid would be at least 18. The effects of trimethylated amphiphilic cyclodextrins and cholesterol on DMPC-d27 membranes are summarized in Figure 5. Panel A displays the temperature dependence above Tc of the order parameters of the methylene deuterons located at each end of the lipid acyl chains, namely, those of the plateau region and those of the C13 carbon next to the methyl group of the sn2 acyl chain. The order parameter curves obtained in the presence of the mono and dilauryl trimethylated β-cyclodextrins are superimposable. At high temperature (45 °C), their order parameters are close to those obtained with pure DMPC membranes and much smaller than those measured in the presence of 10% cholesterol. For all membranes, the order parameters increase with decreasing temperature, with more pronounced variations below the main transition. Order

Figure 5. Interaction of DMPC-d27 membranes with amphiphilic derivatives of trimethyl and hydroxylated β-cyclodextrin. Temperature dependence of the sn2 acyl chain order parameters SCD of the plateau region and C13 methylene (A) and end-terminal methyl group (B) resonances measured from the deuterium dePaked spectra. The membranes were either pure (●) or codispersed with 10% mol TrimβMLC (*), TrimβDLC (○), or cholesterol (+), 30% βMLC (□ containing ×, □), or 7.5% βDLC ( × ).

parameters increase more sharply for the TrimβDLC- and TrimβMLC-containing membranes, and these curves shift from that of the pure lipids to those of the cholesterol-containing membranes with decreasing temperatures. G

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the cholesteryl and dilauryl derivatives. To mark this difference, we will hence refer to the βMLC-enriched phase as the L′CD phase. L′CD Phase above the Main Transition. The NMR data (Figures 1 and 2) indicate that there are two striking differences between the LCD and L′CD phases. First, there is considerable line broadening in the fluid phase of the NMR resonances of the two components (I and II) associated with either pure lipids or with those sequestered in the cyclodextrin-enriched L′CD phase, quite unlike the resonances of the LCD phase, which are well-defined and very narrow. This suggests that there is some exchange between the pure lipids and those in the L′CD phase. This lipid exchange is also responsible for the smoothed fluid-to-gel transition of the free lipids observed in the presence of βMLC. One should then envision such a model of a dynamic lipid exchange between enriched and depleted regions rather than that of the coexistence of a pure lipid phase and a separate LCD phase, as observed with dilauryl derivative βDLC. The second difference concerns the temperature dependence of the acyl chain order parameters of the lipids sequestered in the LCD and L′CD phases (Figure 5B). The DMPC order parameters show a slight monotonic change in the LCD phase but a much stronger variation with a sigmoidal shape in the L′CD phase. The L′CD curve obtained with βMLC actually closely matches the averaged methyl quadrupolar splitting measured in the presence of trimethylated derivative TrimβMLC, which does not induce phase separation. Both curves have the same dramatic order parameter increase in the vicinity of the main fluid-to-gel transition (Figure 5B). This behavior has been observed and discussed previously with DMPC-d54 membranes containing the trimethylated derivative of dilauryl-β-cyclodextrin and is reviewed in the following paragraph. In that work,17 the TrimβDLC-induced ordering of the lipid acyl chains was compared with the well-known ordering effect of cholesterol on fluid lipid bilayers. The chain ordering occurred only below 30 °C in the vicinity of the main transition, as observed here for both hydroxylated and trimethylated monolauryl cyclodextrin derivatives. This temperature range is associated with enhanced density fluctuations due to a softening of the bilayer bending modulus, leading to the well-known anomalous swelling of saturated phosphatidylcholine bilayers.50,51 This effect can be observed through various physical characteristics of the bilayer, most commonly through a nonlinear increase in the lamellar repeat distance D of multilamellar liposomes when approaching the main transition temperature. We have proposed that the increased order observed with the addition of amphiphilic derivatives of trimethyl β-cyclodextrin is related to this anomalous pretransitional behavior of DMPC multibilayers.17 This hypothesis was supported later by the observation that methylated amphiphilic cyclodextrins do not induce such order increase with membranes of POPC with an unsaturated sn2 oleoyl chain, which are not subject to anomalous swelling (Roux, unpublished results). The proposed mechanism for the observed straightening of the DMPC acyl chains was based on the idea that the pretransitional softening of the membrane bending modulus could favor a deeper membrane penetration of the amphiphilic methylated cyclodextrin derivatives, leading to a cholesterol-like bilayer perturbation with a corresponding increase in the acyl chain order parameters. The DMPC-d27 order parameter curves of Figure 5A obtained in the present work with the two trimethylated

The order parameter data for the terminal methyl group of the DMPC sn2 acyl chain lead to the same observations (Figure 5B). Curves obtained above the main transition in the fluid phase with TrimβMLC and TrimβDLC (data not shown) are also superimposable. Because of the high symmetry of the rotational motion of the terminal methyl group, its quadrupolar splitting can still be measured in the gel phase. In the absence of amphiphilic cyclodextrins, the main transition of pure DMPC membranes is seen in a sudden increase in the methyl order parameter, giving two curves reflecting the order parameters of the gel and pseudofluid components, Igel and Ipf, of the Pβ′ phase shown in Figure 2. With 10% TrimβMLC, the methyl order parameter continues its steep increase below the main transition and reaches a small plateau at around 18 °C at the level of the Pβ′ pseudofluid component. It is followed by a sharp transition occurring at 14.5 °C, leading to values obtained for the Pβ′ gel component. A discussion of the latter data, shown here for reference, is beyond the scope of this Article and will be presented elsewhere with a comprehensive data set obtained at various concentrations of the mono and dilauryl trimethylated cyclodextrin derivatives. The two methyl order parameters measured from the NMR spectra obtained with 30% nonmethylated monolauryl-βcyclodextrin shown in Figure 2B are also plotted in Figure 5B. Interestingly, the curve obtained with the methyl signal (II) attributed to the lipids found in the monolauryl-β-cyclodextrinenriched phase is also superimposable with that obtained with its trimethylated derivative. As pointed out before, the temperature dependence of the first component (I) associated with the free lipids is similar to that obtained for pure DMPCd27 membranes, except in the vicinity of the main transition, below 24 °C, where the curve of the pure lipids coexisting with the βMLC-enriched phase shows a much more pronounced sigmoidal dependence (inset in Figure 5B). Finally, order parameters of the DMPC-d27 methyl groups measured with the nonmethylated dilauryl-β-cyclodextrin are also shown in Figure 5B. The signal associated with the lipids trapped in the βDLCenriched LCD phase shows a weak monotonic increase with a single slope over the entire temperature range, except for a steep first-order fluid-to-gel transition at 14.5 °C, as observed previously at 12.5 °C with DMPC-d54 membranes at various concentrations of βDLC.17 The curve obtained for the quadrupolar splittings of the pure lipids (I) that coexists with the LCD phase is superimposable with that obtained from NMR spectra of pure DMPC membranes and does not display any increased sigmoidal dependence below 24 °C (data not shown).

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DISCUSSION Cyclodextrins, especially the β form containing seven glucose units, tend to self-aggregate in aqueous solutions19,20 and can form rodlike structures held together through intermolecular hydrogen bonds.49 β-Cyclodextrins inserted at the surface of phospholipid bilayers via two lauryl chains are also able to selfaggregate, sequestering lipid molecules, to induce a lateral separation of a cyclodextrin-enriched LCD phase. The data obtained with βMLC show that altering the strength of the cyclodextrin membrane insertion by removing one of the two lauryl chains does not suppress the lateral separation of cyclodextrin-enriched regions. However, it does change the nature of the resulting segregated phase, which becomes highly dynamic, showing significant lipid exchanges with the βMLCdepleted regions, as opposed to the LCD phases observed with H

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contrast to the tightly packed fluid LCD phase obtained with the dilauryl derivative. On the contrary, partially fluid lipids remain in the L′CD phase below 14.5 °C where a significant fluid methyl signal is still detected (Figure 2B,C). The model of a “loose” cyclodextrin-enriched L′CD phase developed above is indeed consistent with the absence of a cooperative transition in βMLC-containing membranes. As discussed previously,17 the sharp fluid-to-gel transition observed with the dilauryl derivative is a consequence of efficient mixing in the cyclodextrin-enriched LCD phase of the structurally similar lauryl and myristoyl acyl chains, differing in length by only two methylene groups. It is conceivable that such cooperative freezing of the DMPC and cyclodextrin acyl chains might be reduced with the monolauryl derivative because one lauryl chain is missing, leaving “holes” below the bulky cyclodextrin headgroups, ultimately yielding a loosely packed arrangement of hydrocarbon chains in the L′CD regions. Interestingly, an important smoothing of the first-order LCD fluid-to-gel transition was observed in excess of dilauryl derivative βDLC. This smoothing was explained in a similar manner, with holes arising from the shortage of myristoyl chains in the oversized LCD cyclodextrin clusters partially filled with DMPC.17 Therefore, with a given mesh of the cyclodextrin network at the membrane surface, fixed by the size of the interacting saccharide headgroups, we can obtain holes in both cases either because of the loss of a lauryl chain below the cyclodextrin headgroup in the βMLC-induced L′CD phase or as a result of the missing DMPC molecules in the presence of excess βDLC in the LCD phase. Below the pretransition, D2 of both βMLC- and βDLCcontaining membranes reaches a plateau, just as in the case of pure DMPC, indicating that these composite membranes have now reached an equilibrium state, with a stable distribution of frozen lipids. In particular, any remaining fluid lipids coming from either cyclodextrin-enriched or -depleted regions of the βMLC-containing membranes are now in a frozen state. The loosely packed L′CD regions lead to a heterogeneous and disordered gel phase, with larger D2 plateau values and lower average membrane order parameters dependent on the βMLC concentration. Consistently, the gel phase obtained after the freezing of the compacted βDLC-induced LCD phase is more homogeneous, being apparently quite close to the Lβ′ gel phase of the pure lipids, with similar ⟨SCD⟩ and D2 plateau values. In any case, the observation that there is still a D2 plateau at the pretransition is indicative of a Pβ′ → Lβ′ phase change associated with the complete freezing of Pβ′ ripple structures within the cyclodextrin-depleted regions. The freezing of the partially fluid lipids trapped in the L′CD phase appears to be correlated with this Pβ′ → Lβ′ transition taking place in the cyclodextrin-depleted regions. Hence, there could be some lipid and βMLC monomers exchanging between the cyclodextrinenriched L′CD regions and the fluidlike segments of the cyclodextrin-depleted ripple phase, with both regions having lipids with similar order parameters. Such selective partitioning along the ripples of the Pβ′ phase has been described recently with DPPC membranes.52 With 40% βMLC, no membrane regions contain enough free lipid to undergo a cooperative transition to the gel state upon cooling (Figure 3A). Most of the lipids are in direct contact with βMLC in the L′CD phase, although the asymmetric shape of the dePaked methyl peaks obtained at around 23 °C indicates that there is still some exchange between free and bound lipid molecules at this concentration (Figure 2C). This

cyclodextrin derivatives show that the membrane perturbation observed with the monolauryl derivative is identical to that induced by the dilauryl derivative reviewed above. Thus, both monolauryl and dilauryl methylated derivatives appear to be equally effective in pretransitional lipid chain ordering and bilayer penetration, independent of their number of lauryl chains. However, the number of acyl chains appears to determine the way in which the corresponding nonmethylated derivatives interact with the bilayers and segregate lipids through hydrogen bonds between cyclodextrin headgroups. With two lauryl acyl chains, βDLC leads to the formation of a well-separated LCD phase without an anomalous orderparameter increase near the main transition,17 whereas βMLC with a single acyl chain induces chain ordering above the main transition similar to that observed with the two methylated cyclodextrin derivatives TrimβMLC and TrimβDLC. Such ordering of DMPC acyl chains suggests that βMLC also penetrates the lipid bilayer deeply, similarly to the methylated cyclodextrin derivatives described above. Such bilayer penetration, resulting perhaps from a wobbly insertion at the membrane surface of the cyclodextrin through a single lauryl chain, is consistent with the formation of a loosely packed dynamic L′CD phase in exchange with the free lipid regions. L′CD Phase below the Main Transition. The βMLCinduced lateral separation of the cyclodextrin-enriched L′CD phase is also clearly visible below the DMPC-d27 main transition. The presence of a fluidlike methyl signal shows that partially fluid L′CD regions persist even at low temperatures (Figure 1 and 2). At these temperatures, pure DMPC membranes are now in the Pβ′ ripple phase, with lipids coexisting in the Lβ′ phase and in a pseudofluid state.39−45 Accordingly, the multicomponent NMR spectra obtained with the DMPC membranes in the Pβ′ phase at the main transition present a large order parameter dispersion with high D2 values (Figure 3). Upon further cooling, D2 is found to decrease, as a result of a gradual freezing out of the pseudofluid lipid fraction, leading at the pretransition to a stable and homogeneous Lβ′ gel phase characterized by a plateau in the temperature dependence of D2. In the presence of amphiphilic cyclodextrins, the frozen and pseudofluid lipids of the Pβ′ phase coexist with the lipids of the LCD or L′CD regions, leading to an even more heterogeneous lipid phase, with a superposition of several NMR spectra associated with higher D2 values (Figure 3). The highest D2 is obtained with the βDLC-containing membranes, and the NMR spectrum contains contributions from very disordered fluid lipids trapped in the LCD phase and much more ordered ones in the Pβ′ gel phase. Above 14.5 °C, the fluid LCD lipids show little variation with temperature, as indicated by their methyl order parameter curve (Figure 5B), and D2 decreases with cooling only as a result of the freezing of the free Pβ′ phase fluidlike lipids. After an instant decrease in D2 at 14.5 °C, associated with the first-order cooperative freezing of the fluid lipids trapped in the LCD phase, the freezing of the remaining free Pβ′ fluidlike lipids then leads to the Lβ′ phase at the pretransition. Because the ordered fluidlike lipids found in the βMLCinduced L′CD phase (II) and the Pβ′ phase (Ipf) are similar, they generate a narrower range of order parameters and comparatively lower D2 maxima. Their NMR signals decrease continuously below the main transition and disappear below the pretransition (Figure 2B,C), with both contributing to the monotonic D2 decrease observed with the βMLC-containing membranes (Figure 3). No sharp transition is observed, in I

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set an upper limit of the lipid-to-cyclodextrin ratio of ∼1.5 in the L′CD phase. Instead of a marked cooperative fluid-to-gel transition on cooling, the quantity of frozen lipids gradually increases over a temperature range of about 7 °C below the main transition, with the broadest distribution of order parameter values being seen with a maximum in D2 at 14 °C (Figure 3B). Further cooling toward a completely frozen lipid phase results in a continuous and quasi-monotonic decrease in D2 through the pretransition and the subtransition, supporting the idea of a uniform lipid phase quasi-saturated in monolaurylβ-cyclodextrin, with frozen lipids coexisting with fluid lipid regions that gradually disappear with decreasing temperature. At this point, there are apparently no more lipids left in the Pβ′ phase, although for 30% βMLC it remains observable between the pretransition and main transition. Assuming a lipid-tocyclodextrin ratio of 1, ∼43 and ∼66% of the total lipids should be trapped in the L′CD phase at 30 and 40% βMLC, respectively. Therefore, the minimum fraction of the sample that should be in cyclodextrin-depleted regions if DMPC membranes are to go through an Lα → Pβ′ → Lβ′ transition cycle, with a Pβ′ phase appearing between the transition and pretransition, occurs between 57 and 34%.

(4) Szente, L.; Szejtli, J. Cyclodextrins as Food Ingredients. Trends Food Sci. Technol. 2004, 15, 137−142. (5) Buschmann, H. J.; Schollmeyer, E. Applications of Cyclodextrins in Cosmetic Products: A Review. J. Cosmet. Sci. 2002, 53, 185−191. (6) Buschmann, H. J.; Knittel, D.; Schollmeyer, E. New Textile Applications of Cyclodextrins. J. Inclusion Phenom. Macrocyclic Chem. 2001, 40, 169−172. (7) Davis, M. E.; Brewster, M. E. Cyclodextrin-Based Pharmaceutics: Past, Present and Future. Nat. Rev. Drug Discovery 2004, 3, 1023− 1035. (8) Loftsson, T.; Duchêne, D. Cyclodextrins and Their Pharmaceutical Applications. Int. J. Pharm. 2007, 329, 1−11. (9) Van de Manakker, F.; Vermonden, T.; Van Nostrum, C. F.; Hennink, W. E. Cyclodextrin-Based Polymeric Materials: Synthesis, Properties, and Pharmaceutical/Biomedical Applications. Biomacromolecules 2009, 10, 3157−75. (10) Ortiz Mellet, C.; García Fernández, J. M.; Benito, J. M. Cyclodextrin-Based Gene Delivery Systems. Chem. Soc. Rev. 2011, 40, 1586−608. (11) Laza-Knoerr, A. L.; Gref, R.; Couvreur, P. Cyclodextrins for Drug Delivery. J. Drug Target. 2010, 18, 645−56. (12) Roux, M.; Perly, B.; Djedaïni-Pilard, F. Self-Assemblies of Amphiphilic Cyclodextrins. Eur. Biophys. J. 2007, 36, 861−867. (13) Bauer, M.; Charitat, T.; Fajolles, C.; Fragneto, G.; Daillant, J. Insertion Properties of Cholesteryl Cyclodextrins in Phospholipid Membranes: A Molecular Study. Soft Matter 2012, 8, 942−953. (14) Messner, M.; Kurkov, S. V.; Jansook, P.; Loftsson, T. SelfAssembled Cyclodextrin Aggregates and Nanoparticles. Int. J. Pharm. 2010, 387, 199−208. (15) Bilensoy, E. Nanoparticulate Delivery Systems Based on Amphiphilic Cyclodextrins. J. Biomed. Nanotechnol. 2008, 4, 293−303. (16) Roux, M.; Auzély-Velty, R.; Djedaïni-Pilard, F.; Perly, B. Cyclodextrin-Induced Lipid Lateral Separation in DMPC Membranes. A 2H-NMR Study. Biophys. J. 2002, 82, 813−822. (17) Roux, M.; Moutard, S.; Perly, B.; Djedaïni-Pilard, F. Lipid Lateral Segregation Driven by Diacyl Cyclodextrin Interactions at the Membrane Surface. Biophys. J. 2007, 93, 1620−1629. (18) Gervaise, C.; Bonnet, V.; Wattraint, O.; Aubry, F.; Sarazin, C.; Jaffrès, P. A.; Djedaïni-Pilard, F. Synthesis of LipophosphoramidylCyclodextrins and Their Supramolecular Properties. Biochimie 2012, 94, 66−74. (19) Coleman, A. W.; Nicolis, I.; Keller, N.; Dalbiez, J. P. Aggregation of Cyclodextrins: An Explanation of the Abnormal Solubility of βCyclodextrin. J. Inclusion Phenom. Macrocycle Chem. 1992, 13, 139− 143. (20) Rossi, S.; Bonini, M.; Lo Nostro, P.; Baglioni, P. Self-Assembly of β-Cyclodextrin in Water. 2. Electron Spin Resonance. Langmuir 2007, 23, 10959−10967. (21) Binder, W. H.; Barragan, V.; Menger, F. M. Domains and Rafts in Lipid Membranes. Angew. Chem., Int. Ed. 2003, 42, 5802−5827. (22) Vist, M. R.; Davis, J. H. Phase Equilibria of Cholesterol/ Dipalmitoyl Phosphatidylcholine Mixtures: 2H Nuclear Magnetic Resonance and Differential Scanning Calorimetry. Biochemistry 1990, 29, 451−464. (23) Angelova, A.; Fajolles, C.; Hocquelet, C.; Djedaïni-Pilard, F.; Lesieur, S.; Bonnet, V.; Perly, B.; Lebas, G.; Mauclaire, L. PhysicoChemical Investigation of Asymmetrical Peptidolipidyl-Cyclodextrins. J. Colloid Interface Sci. 2008, 322, 304−314. (24) Davis, J. H.; Jeffrey, K. R.; Bloom, M.; Valic, M. L.; Higgs, T. P. Quadrupolar Echo Resonance Spectroscopy in Ordered Hydrocarbon Chains. Chem. Phys. Lett. 1976, 42, 390−394. (25) Davis, J. H. The Description of Membrane Lipid Conformation, Order and Dynamics by 2H NMR. Biochim. Biophys. Acta 1983, 737, 117−171. (26) Sternin, E.; Bloom, M.; MacKay, A. L. dePake-Ing of NMR Spectra. J. Magn. Reson. 1983, 55, 274−282. (27) Sternin, E.; Schäfer, H.; Polozov, I.; Gawrisch, K. Simultaneous Determination of Orientational and Order Parameter Distributions

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CONCLUSIONS The cyclodextrin-enriched L′CD phase observed with monolauryl-β-cyclodextrin is very different from the LCD phase obtained with dilauryl derivative βDLC. The removal of one lauryl chain leads to dynamic lateral segregation because of the cyclodextrin wobbly insertion at the membrane surface. There is a significant exchange on the NMR time scale between free and segregated lipids in the Lα phase, leading to highly correlated cyclodextrin-enriched and -depleted phases, unlike those observed with the more firmly anchored dilauryl derivative, which are clearly independent, in the slow-exchange regime. As a result of its dynamic membrane insertion, the monolauryl derivative induces an ordering of the lipid acyl chains near the main transition, which is not observed with the dilauryl derivative. Below the main transition, the L′CD phase appears to remain coupled with the cyclodextrin-depleted regions in the Pβ′ phase through possible lipid exchange with the fluidlike regions of the ripple phase. NMR data obtained with DMPC-d27 membranes in the Pβ′ phase, with the permethylated derivatives of mono- and dilauryl β-cyclodextrin, suggesting also an interaction of these cyclodextrin derivatives with the lipid ripples will be described elsewhere.

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AUTHOR INFORMATION

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by grants from the Centre National de la Recherche Scientifique. We gratefully acknowledge Angelina Angelova for conducting the DSC experiments.

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REFERENCES

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K

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