Lipid Bilayer Elasticity Measurements in Giant Liposomes in Contact

Mar 26, 2008 - Laboratoire des Liquides Ioniques et Interfaces Chargées, UMR 7612, Université Pierre et Marie Curie, ESPCI, CNRS, 4 place Jussieu, c...
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Lipid Bilayer Elasticity Measurements in Giant Liposomes in Contact with a Solubilizing Surfactant Christine Me´nager,*,† Dihya Guemghar,† Re´gine Perzynski,† Sylviane Lesieur,‡ and Vale´rie Cabuil† Laboratoire des Liquides Ioniques et Interfaces Charge´ es, UMR 7612, UniVersite´ Pierre et Marie Curie, ESPCI, CNRS, 4 place Jussieu, case 51, 75005 Paris, France, and Laboratoire de Physico-Chimie des Syste` mes Polyphase´ s, UMR CNRS 8612, UniVersite´ Paris-Sud, Faculte´ de Pharmacie, 5 rue Jean-Baptiste Cle´ ment F-92296 Chaˆ tenay-Malabry Cedex, France ReceiVed December 5, 2007. In Final Form: February 18, 2008 A new method to probe the modification of the elasticity of phospholipid bilayers is presented. The purpose here concerns the action of a solubilizing surfactant on a vesicle bilayer. This method is based on the measure of the under-field elongation of giant magnetic-fluid-loaded liposomes. The addition of the nonionic surfactant octyl-βD-glucopyranoside (OG) to vesicles at sublytic levels increases the elasticity of the membrane, as shown by the value of the bending modulus Kb, which decreases. Kb measured around 20 kT for a pure 1,2-dioleoyl-sn-glycero-3phosphocholine (DOPC) bilayer indeed reaches a few kT in the case of the mixed OG-DOPC bilayer. The purpose and interest of this study are to allow the determination of the membrane bending modulus before and after the addition of OG on the same magnetic liposome. Moreover, the experimental conditions used in this work allow the control of lipid and surfactant molar fractions in the mixed aggregates. Then, optical microscopy observation can be performed on samples in well-defined regions of the OG-phospholipid state diagram.

Introduction The action of surface-active agents on lipid bilayer assemblies is among the major concerns in biological membrane research and liposome technology. This arises from the fact that many metabolites and therapeutics are amphipathic molecules able to partition between water and natural or artificial membranes and then change lipid bilayer properties such as lateral organization, permeability, cohesion, or resistance to rupture. Moreover, understanding of the physical behavior of lipid-surfactant mixed systems in excess water is required for the control of biological membrane component solubilization and reconstitution processes by surfactants.1-4 Vesicle structures are a suitable model for investigating interactions between surfactants and membranes as they well mimic the compartmentalization of cell membranes. It is widely admitted that the progressive addition of a solubilizing surfactant, also called a detergent, to lipid vesicles finally yields mixedsurfactant-lipidmicellesthroughathree-stagemechanism.4-10 This work deals with the first stage of the vesicle-to-micelle transition at sublytic surfactant concentrations. At this stage, the surfactant molecules insert into the liposome membrane without disruption. The aim here is to study the changes in the physical properties of the membrane as a function of its composition. Our model is constituted by giant phospholipid vesicles (1,2-dioleoyl* Corresponding author. E-mail: [email protected]. † Universite ´ Pierre et Marie Curie. ‡ Universite ´ Paris-Sud. (1) Eytan, G. D. Biochim. Biophys. Acta 1982, 694, 185. (2) Racker, E. Reconstitution of Transporters, Receptors and Pathological States; Academic Press: New York, 1985. (3) Rigaud, J.; Pitard, B.; Levy, D. Biochim. Biophys. Acta 1995, 1231, 223. (4) Helenius, A.; Simons, K. Biochim. Biophys. Acta 1975, 415, 29. (5) Lichtenberg, D. Handbook of Nonmedical Application of Liposomes; Lasic, D., Barenholz, Y., Eds.; CRC Press: Boca Raton, FL, 1996. (6) Walter, A. Biomembrane Structure and Function: The State of the Art; Gaber, B. P., Easwaran, K. R. K., Eds.; Academic Press: 1992. (7) Lichtenberg, D. Biomembranes: Physical Aspects; Shinitzky, M., Ed.; VCH: Weinheim, Germany, 1993. (8) Lichtenberg, D. Biochim. Biophys. Acta 1985, 821, 470. (9) Inoue, T. Vesicles; Rosoff, M., Ed.; Marcel Dekker, Inc.: New York, 1996. (10) Dennis, E. A. Arch. Biochem. Biophys. 1974, 165, 764.

sn-glycero-3-phosphocholine or DOPC) on which a nonionic surfactant, octyl-β-D-glucopyranoside (OG), is acting. Indeed, the mechanism of phospholipids solubilization by OG is one of the best documented (see ref 11 for review). We have already proved the existence of OG-catalyzed formation of pores by using small unilamellar magnetic-fluid-loaded liposomes (MFLs), the encapsulated iron oxide particles serving as a calibrated colloid for permeability measurements.12 Here, we examine the effect of OG on membrane properties of giant MFLs by applying a magnetic field, already shown as a noninvasive tool that flattens the thermal fluctuations of the membrane.13 Direct observation of the vesicle shape is performed by optical microscopy under a magnetic field of low intensity, typically several tenths of an oersted. Deformation of the same magnetoliposome is measured before and after the addition of adapted amounts of OG molecules. These are fixed on the basis on previous data found by fluorescence energy transfer experiments between two lipid probes inserted within the bilayer of phospholipid vesicles and giving the partitioning of OG molecules between the lipidic assemblies and the aqueous continuum.14 To our knowledge, the present work provides the first insight into the mechanical properties of the lipid membrane in the first stage of the vesicle-to-micelle transition by exactly controlling the surfactant bilayer composition of the vesicles. The shape of a giant liposome loaded with magnetic fluid submitted to a magnetic field can be described by an ellipsoid with axial symmetry around the field direction.13,15 The deformation and its amplitude are characterized by a parameter of ellipticity e2 ) 1 - b2/a2, where a is the semi-axis parallel (11) Ollivon, M.; Lesieur, S.; Grabielle-Madelmont, C.; Paternostre, M. Biochim. Biophys. Acta 2000, 1508, 34. (12) Lesieur, S.; Grabielle-Madelmont, C.; Me´nager, C.; Cabuil, V.; Dadhi, D.; Pierrot, P.; Edwards, K. J. Am. Chem. Soc. 2003, 125, 5266. (13) Bacri, J. C.; Cabuil, V.; Cebers, A.; Me´nager, C.; Perzynski, R. Europhys. Lett. 1996, 33, 235. (14) Paternostre, M.; Meyer, O.; Grabielle-Madelmont, C.; Lesieur, S.; Ghanam, M.; Ollivon, M. Biophys. J. 1995, 69, 2476. (15) Bacri, J. C.; Cabuil, V.; Cebers, A.; Me´nager, C.; Perzynski, R. Mater. Sci. Eng., C 1995, 5, 197.

10.1021/la703807t CCC: $40.75 © 2008 American Chemical Society Published on Web 03/26/2008

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Figure 1. Magnetic-field-induced deformation of a giant MFL without OG (a) and in the presence of 5 mM OG total concentration (b); H ) 64 Oe; bars ) 10 µm.

to the magnetic field H B , and b is the value of the two other semi-axes perpendicular to H B . As a proof, the principle of the undertaken experiments is given in Figure 1, where the underfield deformation of a pure DOPC liposome is compared to that of a DOPC-OG mixed one. It can be noticed that, at the same value of the magnetic field (H ) 64 Oe), the elongation of the liposome is stronger in the case of the mixed DOPC-OG liposome (e ) 0.9, Figure 1b) than for the surfactant-free DOPC liposome (e ) 0.5, Figure 1a). Materials and Methods Materials. The phospholipid constituting the bilayer is cis-1,2dioleoyl-sn-glycero-3-phosphocholine (DOPC, Sigma). Its phase transition temperature is -22 °C so that the phospholipid molecules are always in a fluid-like state at room temperature. The magnetic colloid used is a dispersion of citrate-coated maghemite nanoparticles dispersed in water at pH 7 and synthesized as previously described.16,17 Briefly, superparamagnetic Fe3O4 (magnetite) nanocrystals are prepared by alkaline coprecipitation of FeCl2 and FeCl3 salts and then oxidized into maghemite γ-Fe2O3 particles in an acidic nitrate ferric solution. The negative surface charges of the grains are ensured by anionic citrate ligands provided by adding sodium citrate to the preparation. A residual ionic strength due to nonadsorbed citrate species in equilibrium with the adsorbed ones is always present. Final adjustment of both aqueous medium and maghemite concentrations is performed by ultrafiltration. A 10 mL aliquot of the colloidal particle suspension is centrifuged in a MACROSEP filter, cutoff 50 kD (Fisher ScientificLabosi, France) at 5000g for 30 min. The particles are redispersed in a 43 mM sodium citrate solution. Final volume fraction of particles (between 5 and 8%) is checked by flame spectrometry, and ionic strength is determined by conductivity measurements (1.3 mS, Tacussel type CD 810). Preparation of Giant MFLs. The preparation of giant liposomes encapsulating magnetic nanoparticles has already been described.18,19 As usual, it is based on the spontaneous swelling of a phospholipidic film. But instead of starting the procedure with a dried lipid film, the lipid is prehydrated with the magnetic colloid. Thus an oily film containing the particles is obtained, which is swollen thereafter with triply distilled water. The procedure is as follows: 1 mg of dry DOPC powder is mixed with 10 µL of the aqueous dispersion of magnetic particles and shearing with a glove finger on a glass support (Petri dish) to obtain an oily orange film. Immediately after the shear, 1 mL of triply distilled water is poured onto the film to start the spontaneous swelling. The Petri dish is then placed in a water bath at 45 °C for 20 min. Preparation of Magnetic-Fluid-Free Large Unilamellar Vesicles (LUVs). Magnetic-fluid-free LUVs are prepared by hydration of a DOPC film with a 43 mM sodium citrate aqueous solution followed by sequential extrusion down through 0.8 µm/0.4 µm/0.2 µm (two (16) Massart, R. IEEE Trans. Magn. 1981, 17, 131. (17) Lefe´bure, S.; Dubois, E.; Cabuil, V.; Neveu, S.; Massart, R. J. Mater. Res. 1998, 13, 2975. (18) Sandre, O.; Me´nager, C.; Prost, J.; Cabuil, V.; Bacri J. C.; Cebers, A. Phys. ReV. E 2000, 62 (3), 3865. (19) Me´nager, C.; Cabuil, V. J. Phys. Chem. B 2002, 106, 7913.

Figure 2. (a) Elongation along uniform magnetic field H B . (b) Magnetophoresis setup (field gradient ∇H B superimposed to H B ). passages) polycarbonate filters (poretics, Osmonics, U.S.A.) according to a procedure already described.12,20 Final DOPC concentration is adjusted by weight to 20 mM total phospholipids, and the mean hydrodynamic diameter is measured by quasi-elastic light scattering (ZetaSizer Nano ZS 90, Malvern Instruments, France) and found to be equal to 180 ( 35 nm. Calculation of the Lipid and OG Concentrations. Experimental conditions are optimized and concentrations of DOPC and OG adjusted to get precise OG/DOPC ratios in the vesicle bilayer. In this respect, to reduce the error mainly due to the very low DOPC concentration ([DOPC]GUV) in the giant MFL preparation, DOPC LUVs empty of magnetic fluid are added to the vesicle preparation at known concentrations ([DOPC]LUV) to fix the total DOPC concentration [DOPC]tot at 1.5 mM. The lipid concentration of giant unilamellar vesicles (GUVs) is determined by weighting the Petri dish at each step of the synthesis. Volumes of GUV and LUV dispersions to be mixed are calculated from the relation [DOPC]tot ) ([DOPC]GUVVGUV + [DOPC]LUVVLUV)/Vtot, where Vtot is the volume of the final sample containing OG (Vtot ) VGUV + VLUV + VOG). The volume of surfactant solution VOG is chosen as being 4 times the volume of the giant vesicles (VOG ) 4VGUV). The value of the total sample volume Vtot is fixed as ranging from 170 to 180 µL, and the volume of the GUV dispersion could be then deduced according to VGUV ) [(1.5 - [DOPC]LUV)/([DOPC]GUV - 5[DOPC]LUV)]Vtot. The amount of OG to be added is determined by the final concentration desired: [OG]final ) [OG]iVOG/Vtot, where [OG]i is the concentration of the initial surfactant solution added to the vesicles. This is chosen lower than the critical micelle concentration of the surfactant (17.5 mM ( 0.5 mM, from surface tension measurements at 25 °C by the Wilhelmy plate method using a thermostated automatic digital tensiometer Kru¨ss K10T, Germany), to make sure that OG is added as monomers. OG Addition Experiments. Aliquots of LUVs and giant MFLs (GUVs) are mixed into a 2.5 mL flask placed in a water bath at 25 °C before being deposited in a customized cell: the bottom plate is a cover-glass, and the top one is a porous membrane Anodisc (Whatman) with 0.02 µm diameter pores. This setup enables separation of the sample from the reservoir made of Teflon and allows continuous observation in situ of the selected liposome before, during and after surfactant addition. The reservoir is sealed to the membrane with Parafilm (laboratory film, Pechiney, Chicago) (see observation device in Supporting Information). Magnetic Field Experiments. The magnetic field intensity (0 < H < 300 Oe) is measured with a Hall effect probe in the center of the two coils where the sample is placed (Figure 2a). Before studying the liposome deformation by the surfactant, a first experiment without (20) Martina, M. S.; Fortin, J. P.; Me´nager, C.; Cle´ment, O.; Barrat, G.; GrabielleMadelmont, C.; Gazeau, F.; Cabuil, V.; Lesieur, S. J. Am. Chem. Soc. 2005, 127, 10676.

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OG has been made, gradually increasing the uniform magnetic field intensity. Then, the magnetic field is cut off, and the required volume of the OG solution is added in the reservoir. Equilibrium is reached about 20 min after addition. Thereafter, a second magnetic field exposure is performed on the same liposome. Optical Microscopy. Dimensions of the giant MFLs are measured by optical bright-field microscopy (Leica DMIL 40×, NA O.65) using image-analysis software (Scion 4.0.2). Pictures from a chargecoupled device camera are digitized with a frame grabber (LG-3, Scion Corporation, Frederick, MD). Magnetophoresis Measurements. Magnetic field gradient is super-imposed perpendicularly to the uniform magnetic field as described in the setup of Figure 2b. The liposomes are placed in a capillary (0.2 × 2 mm i.d., Vitro Com, Inc.) in the middle region. The gradient ∇H ranges between 500 and 1000 Oe/cm (measured with a Hall effect probe) with a field H varying between 300 and 600 Oe.

Figure 3. Distribution of the giant MFLs in number per susceptibility value of the magnetic fluid contained in their aqueous core.

Results and Discussion Determination of the Magnetic Content within an Individual Giant MFL. An in situ measurement of the magnetic susceptibility χ is provided by a magnetophoresis experiment that consists of the study of the migration of a liposome submitted to a controlled gradient of magnetic field. Because magnetoliposomes have a higher magnetic susceptibility than the surrounding liquid, they move toward increasing field intensity. Their velocity is constant and corresponds to the balance of the magnetic force and the drag force exerted by the outer fluid. The magnetic force that causes the migration is Fm ) µ0χvesHy(∇xHy)V, with V being the volume of the liposome. For the calculation of the drag force, two cases are under consideration. In fact, the magnetic field for the magnetophoresis experiments is not very intense, and some magnetoliposomes remain spherical. The drag force corresponds to the Stockes law: Ff ) 6πηRV, where R is the radius of the vesicle, V is its velocity and η is water viscosity. Thus the magnetic susceptibility is χves ) 9πη/2µ0Hy(∇xHy)R2. In the other cases, when the liposome deforms into an ellipsoid, the Perrin law gives [32πη(a2 - b2)ν]/ {[(2(2a2 - 3b2))/(xa2-b2)] ln[(a + xa2-b2)/b] + 2a} for the drag force, where a is the semi-axis parallel to the magnetic field, and b is the value of the two other semi-axes perpendicular to H. The magnetic susceptibility is then related to the velocity by the following analytical expression:

χves )

(

2(2a2 - 3b2)

xa

2

-b

2

(

32πη(a2 - b2)ν

)

)

Figure 4. Schematic drawing of a typical deformation of a flattening magnetoliposome from sphere to prolate ellipsoid as induced by applying a uniform magnetic field.

of any elongation.21 The eccentricity of the liposomes is linked to the applied magnetic field through the following expression:

(

( )

(1)

where Kb is the bending modulus, H* is a reduced parameter homogeneous to a magnetic field and constant for a given liposome: H*2 ) (x45kT/32πKb)[4/(µ0(µ - 1)2)](τ0/R), with R being the initial radius of the liposome, τ0 its initial tension, and µ ) 1 + χves. f(e) and g(e) are the following functions of eccentricity e:

(x

1 - e2 +

f(e) )

a + xa2 - b2 ln + 2a µ0Hy(∇xHy)V b

Figure 3 shows the distribution of the giant MFL vesicles as a function of the magnetic susceptibility of their iron oxide content. The distribution is rather large. An average susceptibility value χves taken equal to 3 × 10-4 and corresponding to a volume fraction of maghemite nanoparticles close to 4 × 10-5 is used in the following. MFL Deformation under a Uniform Magnetic Field: Theoretical Background. It is well-known that the projected area of giant liposomes is smaller than the true surface of the membrane, the difference being adsorbed by thermal undulations. Initially, quasi-spherical vesicles can change their shape at constant volume by using the excess area hidden in the fluctuating membrane (Figure 4). The basis of the model describing the deformations of isolated magnetic liposomes has been previously established in the case of weak magnetic field and extended later in the case of ellipsoids

)

16πKb 8πKb H (f(e) - 1) + ln g(e)2 ) 4 ln kT 45kT H*

and

g(e) )

(

)

arcsin(e) 1 e 2(1 - e2)1/6

arcsin(e) 3 - 2e2 - (3 - 4e2) 3 2 e e (1 - e2)1/2

(1 - e )

2 2/3

(( ) (

6 3 - e2 1 + e ln - 4 5 1 e e e

)

))

(2)

Equation 1 links e and H, two quantities that can be experimentally determined. Figure 6 sketches eq 1 as solid lines in a semilog plot for three values of Kb: 20 and 1 kT. MFL Deformation under a Uniform Magnetic Field without and after OG Addition. The total lipid concentration is fixed at 1.5 mM for all the experiments, while the total OG concentration is gradually increased according to the procedure described in (21) Me´nager, C.; Meyer, M.; Cabuil, V.; Cebers, A.; Bacri, J. C.; Perzynski, R. Eur. Phys. J. E 2002, 7, 325.

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Figure 5. Magnetic-field-induced deformations of the same MFL without OG (a) and in the presence of OG (b). Total DOPC and OG concentrations are 1.5 mM and 5 mM, respectively.

Figure 6. Deviation from spherical shape of giant MFLs as the applied uniform magnetic field is increased for different bending modulus values Kb. Symbols indicate the experimental values of e4 at surfactant concentrations of 0 mM (open symbols, 10 vesicles) and 5 mM (filled symbols, 5 vesicles) versus the natural logarithm of the square of the reduced magnetic field ln(H/H*)2. Solid lines report the theoretical curves calculated from eq 1 for three values of the bending modulus (Kb ) 20, 5, and 1 kT).

the experimental section. First, in zero magnetic field, an increase in the apparent surface area of the initially liposome can be noticed due to the modification of both the osmotic and mechanic equilibrium of the DOPC bilayer, which incorporated a part of the added OG molecules. Qualitatively, as already said in the introduction, by applying a given magnetic field, the magnetic elongation observed by optical microscopy (after 20-min equilibrium setting), clearly shows higher eccentricity of the giant MFL in the presence of 5 mM surfactant than that seen in OG-free aqueous medium. The values of the eccentricity appear to increase with increasing magnetic field intensity (see Figure 5). These are indicated and compared to the theory in Figure 6. The experiment is performed on 5 to 10 vesicles with R ranging between 12 and 23 µm. In Figure 6, the experimental variations of e4 recorded for the various magnetic vesicles are plotted as a function of ln(H/H*)2, i.e., a reduced expression of ln H2. An adjustment along the logarithmic field axis determines the value of the parameter H*. Without OG, the experimental data fit well the theoretical curve obtained for a Kb value of 20 ( 2 kT with 10 < H* < 30 Oe. It means τ0 ≈ 5 × 10-12 N‚m-1. This value of Kb is in agreement with our previous reports13,21 and also in agreement with the

Figure 7. Deviation from the spherical shape of giant MFLs as induced by a uniform magnetic field of variable intensity for a bending modulus Kb ) 0.5 kT. Symbols indicate e4 experimental values versus the natural logarithm of the square of the magnetic field ln(H/H*)2 at a surfactant concentration of 10 mM OG. The solid line reports the theoretical curve calculated from eq 1. (a). Optical microscopy picture of a magnetoliposome after 53 min of incubation time with OG 10 mM. In zero magnetic field, the shape is very distorted (b). Bar ) 15 µm.

results already provided by other methods.22-24 When OG ) 5 mM is added to the magnetic vesicles, the adjustment to the theoretical data shows a dramatic decrease of the bending modulus Kb; a value of 1 kT ( 0.5 kT with 13 < H* < 29 Oe is then found, corresponding to τ0 ) 10-12 N‚m-1. For 10 mM OG addition, the enrichment of the membrane in OG molecules makes the liposomes very soft and the lipid bilayer brittle. Despite the difficulty to follow the liposome deformations before some pieces of membrane are expelled, some measure(22) Kummrow, M.; Helfrich, W. Phys. ReV. A 1991, 44 (12), 8356. (23) Kwok, R.; Evans, E. Biophys. J. 1981, 35, 637. (24) Evans, E.; Rawicz, W. Phys. ReV. Lett. 1990, 64 (17), 2094.

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Figure 8. Microscopy images visualizing giant MFL deformation as a function of time at H ) 100 Oe upon the addition of 12 mM total OG; bar ) 10 µm.

ments are performed. They are plotted in Figure 7, and an estimate of the bending modulus on the order of 0.5 kT with 13 < H* < 70 Oe can be obtained (Figure 7a). This increment of the membrane elasticity is indeed correlated to the highly fluctuating membranes and frequently distorted shapes of the liposomes observed in zero magnetic field, as it is illustrated by Figure 7b, 53 min after OG addition. A stationary shape is sometimes obtained after a long sequence of instable distorted shape. From literature data giving the molar fraction of OG in the lipid bilayer as a function of the total phospholipid and surfactant concentrations, the membrane composition of the giant MFL ([DOPC] ) 1.5 mM) corresponds to OG/DOPC molar ratios close to one molecule of OG per molecule of DOPC at 5 mM total OG and not exceeding 1.5 molecule of OG per lipid at 10 mM total OG.14 These liposomes are still in the vesicle domain of their phase diagram.25 The experiments of MFL deformations under magnetic field in the presence of and without surfactant allow the determination of the bending modulus of the phospholipid bilayer as a function of OG insertion. The role of the surfactant on the membrane elasticity is clearly shown. OG insertion causes an increase in the elasticity and thus a dramatic decrease of the bending modulus. In order to explain the very low measured value of the bending modulus associated with the picture of Figure 7b, a twofold theory is possible. It can be related to spatial heterogeneities of OG along the membrane, OG being accumulated in the high curvature domain of the membrane. Taking into account the packing parameter of the OG molecule as calculated close to p ) V/al ) 0.52,26 it is realistic to think that the OG molecules modify the local curvature of the bilayer by preferably accumulating in local sites prior to diffusion in the bilayer.27-31 The insertion of OG molecules induces an increment of the elasticity until it is near the breakdown threshold of the vesicle. Compared to the experiments performed with cholesterol-based membranes, in which cholesterol molecules make the membrane stiffer,32 here the opposite effect is evidenced: OG makes the membrane softer. The value obtained of the order of kT is of the (25) Guemghar, D. Ph.D. Thesis, University of Paris 6, France, 2006. (26) Israelachvili, J. Intermolecular and Surface Forces, 2nd ed.; Academic Press: New York, 1991. (27) Beugin-Deroo, S. Ph.D. Thesis, University Paris-Sud, France, 1997. (28) Lesieur, S.; Ollivon, M. In Synthetic Surfactant Vesicles; Uchegbu, I. F., Ed.; Harwood Academic Publishers: Amsterdam, 2000; Vol. 11, pp 49-79. (29) Seras, M.; Grabielle-Madelmont, C.; Paternostre, M. T.; Ollivon, M.; Handjani-Villa, R.M.; Lesieur, S. Prog. Colloid Polym. Sci. 1991, 84, 502. (30) Seras, M.; Gallay, J.; Vincent, M.; Ollivon, M.; Lesieur, S. J. Colloid Interface Sci. 1994, 167 (1), 159. (31) Seras, M.; Edwards, K.; Almgren, M.; Carlson, G.; Ollivon, M.; Lesieur, S. Langmuir 1996, 12, 330. (32) Needham, D.; Rashmi, S.N. Biophys. J. 1990, 58, 997.

Figure 9. Temporal evolution of the eccentricity for an MFL in the presence of 12 mM total OG and submitted to H ) 100 Oe.

same order of magnitude as the value found in the case of a bicontinuous phase, for example, AOT/octanol.33 For OG concentrations higher than 10 mM, it is impossible to perform measurements in conditions of variable magnetic field, so the experiments are carried out at constant magnetic field. MFL Deformation at High OG Concentrations under a Constant and Uniform Magnetic Field. Figure 8 illustrates the MFL deformation at H ) 100 Oe as a function of time at constant magnetic field after OG addition to a 12 mM final concentration (1.5 mM DOPC concentration). The magnetic vesicle exhibits significant deformations, and the eccentricity evolution is presented in Figure 9. It increases during the first 10 min, reaches a plateau (e ) 1), and suddenly decreases 40 min after OG addition. The shape then relaxes for 8 min toward a spherical shape, as if releasing materials, before again becoming ellipsoidal in a 49 min period of time (see Supporting Information for an accelerated movie of such experiment). After 1 h, the liposome remains prolate with an eccentricity of 0.9, and no leakage of the nanoparticles is observable. Some small magnetic vesicles accumulate at each pole of the giant MFL observed (Figure 8). In the whole preparation, magnetic vesicles form chains when the magnetic field is applied for a few minutes. The same kind of experiment has been done at a 15 mM total OG concentration and is presented in Figure 10. The eccentricity follows the same kind of profile, but a definitive decrease begins around t ) 38 min (see Figure 11). The images in Figure 10 show that the MFL is progressively elongating until it reaches a “cigar” shape. Two pores open, the first one around t ) 28 min because it causes the decrease of the elongation and the second one at t ) 38 min just before the disappearance of the liposome. The liposome is likely turned into submicronic structures. The two experiments made as a function of time at constant and uniform magnetic field prove the formation of a pore in the (33) Bisceglia, M.; Acosta, E. Colloids Surf., B 2003, 213, 1.

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Figure 10. MFL elongation as a function of time under H ) 96 Oe in the presence of 15 mM total OG. Arrow indicates pore opening. Bar is 10 µm.

Figure 11. Temporal evolution of the eccentricity versus time for an MFL in the presence of 15 mM total OG and submitted to H ) 96 Oe.

bilayer because the prolate ellipsoid relaxes toward a spherical shape without change of the external stress. Note that the total concentration of 12 mM OG at 1.5 mM DOPC corresponds to a range of surfactant/phospholipid molar ratio still in the vesicle domain of the vesicle-to-micelle transition process, while 15 mM total OG is located at the limit of the domain of coexistence between micelles and lamellar structures.14,34-36 At 12 mM OG, clearly the opening of a transient pore is observed, but the vesicle structure is preserved, whereas, at 15 mM OG, the pore formation precedes the liposome vanishing. It breaks as the solubilization starts. These observations are remarkably correlated to the diagrams of state already described.32,33 The proof of pore formation within a lipid bilayer due to its interaction with OG molecules has already been illustrated with egg-PC LUVs in which magnetic nanoparticles have been encapsulated.12 The study of the solubilization for this system showed that pores form in the first stage of the process when the surfactant equilibrates between the bilayer and the continuous aqueous phase without disappearance of the vesicle structure. These pores are big enough to let the nanoparticles leak from the inner core of the liposome. The observation of a pore by optical microscopy is possible when the event occurs in the focal plane of observation and when the lifetime of the pore is long enough. Here, it is the relaxation of the membrane that proves the existence of a pore. It is possible that the magnetic field facilitates the insertion of OG at the magnetic poles where the curvature imposed by the magnetic field is higher. The accumulation of OG then weakens the bilayer and enables the opening of a pore. When a bilayer is submitted to an external (34) Ueno, M. Biochemistry 1989, 28, 5631. (35) Ollivon, M.; Eidelman, O.; Blumenthal, R.; Walter, A. Biochemistry 1988, 27, 1695. (36) Beugin, S.; Grabielle-Madelmont, C.; Paternostre, M. T.; Ollivon, M.; Lesieur, S. Prog. Colloid Polym Sci. 1995, 98, 206.

stress such as a magnetic or electric field, the membrane tension increases and a pore opens. The line tension Τ of the membrane is the force that closes the pore. It has been demonstrated that a surfactant with a positive local curvature like OG localizes in a domain of strong curvature, such as the edges of the pore, and therefore stabilizes it.37 The consequence is a decrease of the line tension and an increase of the pore lifetime. Here, the magnetic field applied increases the membrane tension τ, whereas the surfactant decreases the initial tension τ0 by a factor of 4 in the case of OG addition. The critical radius of pore nucleation is rc ) Τ/τ, therefore a pore is opening if τ increases (rule of the magnetic field) and if the line tension Τ vanishes (rule of the surfactant). Then the mechanism is a molecular phenomenon of interaction between the membrane and the surfactant assisted (or not) by the magnetic field.

Conclusion We describe here a very promising method to probe the modification of the elasticity of phospholipid bilayers by way of the addition of a host molecule. This method is based on the measure of the under-field elongation of giant MFLs. The experiments presented here are related to the action of a solubilizing surfactant and to the vesicle-to-micelle transition process it generates. Clearly, the addition of the nonionic surfactant OG to vesicles at sublytic levels increases the elasticity of the membrane as shown by the value of the bending modulus Kb, which decreases. Kb measured around 20 kT for a pure DOPC bilayer indeed reaches a few kT in the case of the mixed OGDOPC bilayer. We demonstrate that the first stage of the transition from vesicle to mixed micelles, i.e., in the vesicular domain, does not correspond to drastic events but rather to successive transient states of the membrane. These states coincide with an evolving distribution of the surfactant within the lipid bilayer, which progressively becomes homogeneous, making the membrane very soft. The purpose and interest of the procedure developed in this study are to allow the determination of the membrane bending modulus before and after the addition of OG on the same magnetic liposome. The experimental conditions used in this work that allow the control of lipid and surfactant molar fractions in the mixed aggregates are pioneer compared with most reported studies about giant liposomes, which have never specified the effective composition of the vesicle bilayer. Then, optical microscopy observation can be performed on samples in well-defined regions of the OG-phospholipid state diagram. Providing the lipidwater partitioning law is established, this powerful method can be generalized to a lot of molecules that interact with membranes, (37) Puesh, P. H.; Borghi, N.; Karatekin, E.; Brochard-Wyart, F. Phys. ReV. Lett. 2003, 90, 128304.

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such as peptides, polymers, surfactants, or all molecules of interest for the biomedical domain. Acknowledgment. The authors thank Olivier Sandre and Andrejs Cebers for helpful discussions.

Me´ nager et al.

Supporting Information Available: Film of the MFL deformation at high OG concentration under a constant magnetic field and picture of the observation cell. This material is available free of charge via the Internet at http://pubs.acs.org. LA703807T