Fluorinated Vesicles Made from Combinations ofPhospholipids and Semifluorinated Alkanes. Direct

Experimental Evidence of the Location of theSemifluorinated Alkane within the Bilayer

Marc Schmutz,† Bernard Michels,‡ Pascal Marie,† and Marie Pierre Krafft*,†

Chimie des Systemes Associatifs, Institut Charles Sadron (UPR CNRS 22),6, rue Boussingault, 67 083 Strasbourg Cedex, France, and Laboratoire des Fluides Complexes(UMR CNRS), Universite Louis Pasteur, 4, rue Blaise Pascal, 67 070 Strasbourg Cedex, France

Received November 12, 2002. In Final Form: March 19, 2003

Previous work has demonstrated that codispersion of semifluorinated alkanes CnF2n+1CmH2m+1 (FnHmdiblocks) with phospholipids strongly modifies the properties of the resulting vesicles. The hypotheticalsegregation and ordered self-organization of FnHm diblocks as a continuous film within the bilayer weresuggested to account for the observed changes of bilayer behavior; however, no proof of the diblock locationand organization was provided. We now report direct experimental evidence of the diblock’s presence andorganization within the bilayer using cryogenic transmission electron microscopy (cryo-TEM), high-sensitivity differential scanning calorimetry (micro-DSC), and small-angle X-ray scattering (SAXS). Cryo-TEM demonstrated that a sonicated codispersion of dioleoylphosphatidylcholine (DOPC) and C6F13C10H21(F6H10) (1:1) led to an homogeneous population of small unilamellar vesicles (SUVs) with no F6H10emulsion droplets present. The bilayer of these SUVs appeared as a single, thick and dark ring, characteristicof the bilayer of vesicles made from fluorinated phospholipids. This aspect, very different from that of thebilayer of DOPC vesicles, which consists of two concentric rings, demonstrated unambiguously the presenceof a fluorinated core within the bilayer. The micro-DSC study on dimyristoylphosphatidylcholine (DMPC)/F6H10 SUVs showed a shift of the DMPC main transition phase temperature to lower values, indicatinginterdigitation of the H10 hydrogenated segments of the diblock with the fatty acid chains of the DMPC.In addition, F6H10 was observed to undergo a gel-to-fluid transition at ∼25 °C when incorporated in theDMPC bilayer, giving further evidence for an organized fluorinated film within the bilayer (F6H10 is liquidin the bulk). Finally, SAXS experiments were in agreement with a hollow spherelike particle modelconstituted by a∼3 nm thick fluorinated shell, indicating that the fluorinated segments are not interdigitated.

Introduction

Fluorinated vesicles, that is, vesicles formed fromperfluoroalkylated amphiphiles, have raised interestbecause of new properties, different from those of con-ventional vesicles made from hydrocarbon lipids, andbecause of their potential as drug delivery systems.1-5

Fluorinated chains possess the unique feature of beingstrongly hydrophobic and lipophobic at the same time.Fluorinated amphiphiles display a strong tendency to formhighly stable self-assemblies, as illustrated by the factthat even certain short single-chain surfactants can forma variety of bilayer-based structures, including vesicles6

and microtubules,7,8 while their hydrocarbon analoguesonly form micelles. Fluorinated vesicles made of perfluo-roalkylated phosphatidylcholines are less permeant withrespect to the release of both hydrophilic and lipophilic

encapsulated species9 and display increased in vivopersistence.10

An alternative approach to obtaining fluorinated vesiclesconsists of using a combination of standard hydrocarbonlipids and semifluorinated alkanes CnF2n+1CmH2m+1 (FnHmdiblocks).1,2,11 FnHm diblocks, because they appear to bewell tolerated biologically and are easy to synthesize,have potential as versatile membrane components.12

FnHm diblocks were shown to be efficient stabilizers ofphospholipid-based fluorocarbon emulsions and allowedprecise particle size control.13,14 It was recently found thatF8H16 substantially decreased the interfacial tensionbetween a phospholipid dispersion and a fluorocarbonpendant drop, revealing the presence of the diblocks atthe interface.15 Dipalmitoylphosphatidylcholine (DPPC)/CnF2n+1CHdCHCmH2m+1 vesicles are considerably morestable and less permeant to 5,6-carboxyfluorescein releasethan vesicles made from DPPC alone.16 FnHm diblocks* Corresponding author. Tel: (33) 3 88 41 40 60. Fax: (33) 3 88

40 41 99. E-mail: krafft@ics.u-strasbg.fr.† Institut Charles Sadron.‡ Universite Louis Pasteur.(1) Riess, J. G. J. Drug Targeting 1994, 2, 455.(2) Riess, J. G. J. Liposome Res. 1995, 5, 413.(3) Riess, J. G.; Frezard, F.; Greiner, J.; Krafft, M. P.; Santaella, C.;

Wierling, P.; Zarif, L. In Handbook of Nonmedical Applications ofLiposomes. From Design to Microreactors, Vol. III; Barenholz, Y., Lasic,D. D., Eds.; CRC Press: Boca Raton, FL, 1996; Chapter 8, p 97.

(4) Krafft, M. P.; Riess, J. G. Biochimie 1998, 80, 489.(5) Krafft, M. P. Adv. Drug Delivery Rev. 2001, 47, 209.(6) Krafft, M. P.; Giulieri, F.; Riess, J. G. Angew. Chem., Int. Ed.

Engl. 1993, 32, 741.(7) Giulieri, F.; Krafft, M. P.; Riess, J. G. Angew. Chem., Int. Ed.

Engl. 1994, 34, 1514.(8) Giulieri, F.; Krafft, M. P. J. Colloid Interface Sci. 2003, 258, 335.

(9) Frezard, F.; Santaella, C.; Vierling, P.; Riess, J. G. Biochim.Biophys. Acta 1994, 1192, 61.

(10) Santaella, C.; Frezard, F.; Vierling, P.; Riess, J. G. FEBS Lett.1993, 336, 481.

(11) Trevino, L.; Frezard, F.; Postel, M.; Riess, J. G. J. Liposome Res.1994, 4, 1017.

(12) Riess, J. G.; Cornelus, C.; Follana, R.; Krafft, M. P.; Mahe, A.M.; Postel, M.; Zarif, L. Adv. Exp. Biol. Med. 1994, 345, 227.

(13) Cornelus, C.; Krafft, M. P.; Riess, J. G. Artif. Cells, Blood Subst.,Immob. Biotech. 1994, 22, 1183.

(14) Krafft, M. P.; Riess, J. G.; Weers, J. G. In Submicronic Emulsionsin Drug Targeting and Delivery; Benita, S., Ed.; Harwood Academic:Amsterdam, 1998; p 235.

(15) Bertilla, S. M.; Thomas, J. L.; Marie, P.; Krafft, M. P. Manuscriptin preparation.

4889Langmuir 2003, 19, 4889-4894

10.1021/la020906d CCC: $25.00 © 2003 American Chemical SocietyPublished on Web 05/08/2003

have demonstrated an inhibiting effect on the hydrolysisof lipids by phospholipase A2 when incorporated intovesicles made of DPPC or dimyristoylphosphatidylcholine(DMPC).17 The incorporation of FnH10 diblocks (n ) 4, 6,8) in the bilayer of small unilamellar vesicles (SUVs) madefrom bovine brain phosphatidylserine had a strong impacton the fusion kinetics of these SUVs.18 Both the Ca2+-induced rates of fusion and the release of the internalvesicle content were much lower for the fluorinated SUVsthan for SUVs made of phosphatidylserine alone. Thestrongest effect was obtained for F6H10.

In the above studies, the improvement in vesicleproperties, including shelf stability enhancement, resis-tance to enzymatic hydrolysis, fusion and release hinder-ing, observed when FnHm diblocks were present in thevesicle formulation were attributed to self-organizationof the diblocks within the phospholipid bilayer. The fluo-rinated segments were hypothesized to segregate and forma continuous, ordered hydrophobic and lipophobic centralfluorinated film, while the hydrogenated segments wouldpoint toward the phospholipid chains, likely inter-digitating with them (Scheme 1). This hypothesis isreasonable because FnHm diblocks have little tendencyto be molecularly dispersed or to form micelles in water.However, this hypothesis has never been confirmedexperimentally. Moreover, the formation of alternativecolloidal systems, such as phospholipid-coated FnHmemulsion droplets that would coexist with phospholipid/FnHm mixed vesicles, has never been ruled out. We havetherefore investigated vesicles made from combinationsof phospholipids (DOPC, DMPC) and FnHm diblocks(F6H10, F8H16) by cryogenic transmission electronmicroscopy (cryo-TEM), high-sensitivity differential scan-ning calorimetry (micro-DSC), and small-angle X-rayscattering (SAXS). Vesicles made from a perfluoroalkyl-ated phosphatidylcholine (F-PC) were also investigatedfor reference and comparison.

Experimental SectionMaterials. DOPC and DMPC were purchased from Sigma.

F6H10 and F8H16 diblocks were synthesized according to Brace19

(purity 99%, gas chromatography). HEPES was obtained fromFluka. The perfluoroalkylated phosphatidylcholine was a giftfrom Prof. J. G. Riess (MRI Institute, Medical Center, Universityof California at San Diego). The chemical structures of the vesiclecomponents investigated are shown in Chart 1.

Vesicle Preparation. One-Component Vesicles (DOPC,DMPC, or F-PC). A solution of the lipid (DMPC, DOPC, or F-PC)in CHCl3 was evaporated in a rotary evaporator to obtain a thinfilm. This film was hydrated at room temperature with HEPESbuffer and homogenized by vigorous stirring to yield 1 mMconcentrated dispersions of vesicles. Typically, 2 mL sizedsamples were prepared. The resulting coarse dispersion wassubmitted to sonication (Branson B-30 sonifier, 15 mm titaniumprobe, 20 min, power 5, 50% pulse) in a cell thermoregulated at25 °C. The resulting transparent dispersion was heated at 40 °Cfor 1 h in order to anneal defects that could have been producedby sonication. The vesicle dispersion was filtered on a 0.22 µmpoly(ether sulfone) (PES) membrane.

Two-Component Vesicles (DOPC/F6H10, DMPC/F6H10, andDMPC/F8H16). DOPC or DMPC was solubilized in CHCl3 withthe appropriate amount of F6H10 in order to yield DMPC/F6H10molar ratios of 1:1, 1:2, and 1:3. The solvent was evaporatedusing a rotary evaporator. The resulting thin film was thenhydrated with HEPES buffer and homogenized to yield 1 mMlipid concentrated dispersions of vesicles. Samples were sonicatedas described above. The annealing step was essential for obtainingDMPC/FnHm vesicles with reproducible characteristics. TheDMPC concentration of the DMPC/F8H16 (1:1) vesicle suspensionprepared for SAXS experiments was 1.087 mM. The thin filmwas hydrated at 60 °C, that is, above the melting point, 52 °C,of F8H16. The sample was then sonicated at 60 °C and heatedat 60 °C for 30 min for annealing.

Differential Scanning Calorimetry. High-sensitivity cal-orimetry experiments were performed on a DASM-4 microcalo-rimeter interfaced to a Bull Micral computer.20 Thermogramswere plotted as the variation of power as a function of temper-ature. Temperature was programmed only on heating. At leasttwo repetitive scans were carried out for each sample. The meltingpoint of the F8H16 diblock was determined with a Mettler-ToledoDSC TC 15.

Quasi-Elastic Light Scattering (QELS). A Malvern ZetaSizer 3000 HS was used for dynamic light scattering at ascattering angle of 90°. The temperature was 25 °C. Thez-averaged hydrodynamic mean diameters (Dh ) of the vesicleswere determined using the software provided by Malvern.

Cryogenic Transmission Electron Microscopy. A 400mesh lacey carbon film copper grid was rendered hydrophilic bya mild glow discharge. Five microliters of the vesicle solutionwas deposited and adsorbed for 2 min. Excess of sample solutionwas removed with filter paper (Whatman 2 or 5) to leave a thinfilm. The grid was rapidly plunged into liquid-nitrogen-cooledethane and stored in liquid nitrogen until observation. The gridwas transferred in a Gatan 626 cryo holder and observed in aCM120 Philips microscope. Low-dose images were recorded onSO163 films and developed under standard conditions.(16) Trevino, L.; Frezard, F.; Rolland, J. P.; Postel, M.; Riess, J. G.

Colloids Surf. 1994, 88, 223.(17) Privitera, N.; Naon, R.; Riess, J. G. Biochim. Biophys. Acta 1995,

1254, 1.(18) Ferro, Y.; Krafft, M. P. Biochim. Biophys. Acta 2002, 1581, 11.

(19) Brace, N. O. J. Org. Chem. 1973, 38, 3167.(20) Privalov, P. L. Pure Appl. Chem. 1980, 52, 479.

Chart 1. Chemical Structures of the Semifluorinated Alkanes F6H10 (a) and F8H16 (b) and of theVesicle-Forming Perfluoroalkylated Phosphatidylcholine (F-PC) (c), Dimyristoylphosphatidylcholine (DMPC) (d),

and Dioleoylphosphatidylcholine (DOPC) (e)

Scheme 1. Hypothetical Representation of aFluorinated Bilayer Made from a Combination of a

Hydrogenated Phospholipid (PL) and aSemifluorinated Alkane (FnHm)

4890 Langmuir, Vol. 19, No. 12, 2003 Schmutz et al.

Small-Angle X-ray Scattering. Data Collection. X-rayscattering experiments were carried out with a Brucker Nanostarspectometer operating at 40 kV and 40 mA. The scattering vectoris defined by q ) (4π/λ) sin(θ/2) where λ is the wavelength of theincident X-ray beam and θ is the scattering angle. The sample-to-detector length was 1050 mm, and the range of scatteringangles corresponded to wave vectors between 0.0014 and 0.15Å-1 for the Cu KR1 radiation (λ ) 1.514 Å). The samples wereplaced in glass capillaries 1 mm in diameter. The data weretreated according to standard procedures for small-angle isotropicscattering.21,22 The radially averaged data were corrected fromsample quartz capillary scattering, sample thickness, andtransmission. Sample transmission was checked before and aftereach measurement to detect possible alteration of material(solvent evaporation, bubble formation, etc.) and to correct forself-absorption of the sample. The intensity data were normalizedto the scattering intensity of water (a 1 mm thick sample) ratherthan that of Lupolen (a solid polymer film) used classically tocorrect fluctuations in the X-ray source intensity.23 Data werenot plotted on an absolute scale. The differential scattering crosssection per volume unit, denoted I(q), was obtained by subtractingan appropriate background, I(q) ) (IS - ΦVISB)/IH2O, where IS,ISB, and IH2O are the scattering intensities of the sample, samplebackground, and water, respectively, and ΦV is the sample volumefraction.

Data Analysis and Model of the Particle.The general expressionof the intensity scattered by a system composed of n correlated,identical, and homogeneous globular particles dispersed in acontinuous and uniform medium is given by I(q) ) A{nI1(q) +n2I2(q)}, where A is the instrument constant, I1 is the singleparticle intensity (intraparticle term), averaged over particleorientations, and I2 is the structure factor for the correlationsbetween particles (interparticle term), averaged over orientationsand positions of the particles.

Assuming that the particles are spherically symmetric andthat their positions are correlated with neither their orientations(randomly oriented) nor their size, I(q) ) ANpP(q)S(q), whereNp is the number of particles per volume unit and P(q) is themean squared particle shape factor. For a dilute system ofnoninteracting particles, S(q) ∼ 1. The equation is then reducedto I(q) ) AΦpVpP(q), Vp and Φp being the particle volume andvolume fraction, respectively (Φp ) NpVp). The shape factorP(q) of a particle constituted of n concentric spherical shells (j)of homogeneous scattering length density Fj, defined betweenradii Rj and Rj+1 (R0 ) 0 ) particle center), is given by P(q) )[∑j)1

n Vj(bj - bj+1)f(qRj)]2, with Vj ) 4π/3Rj3 and f(qRj) is a form

factor,

In our model, the particle is composed by a water coresurrounded by a shell formed by phospholipids and semifluori-nated alkanes (Scheme 2). The core radius is Rc, the shellthickness is d, and the overall particle radius is RT ) Rc + d.The coherent scattering lengths of the shell, core, and mediumare bs, bc, and bm, respectively. I(q) ) ANp[VT(bs - bm)f(qRT) +Vc(bc - bs)f(qRc)]2, with VT ) 4π(Rc + d)3/3 and Vc ) 4π(Rc)3/3.In the case of vesicles, the core has the same electron density asthe continuous medium (bc ) bm ) bH2O) and the interface hasa high electron density. The scattered intensity is expressed by

where ∆b ) bs - bm is the contrast factor between the hollowparticles and the continuous medium. The volume of the coreparticle, Vc, was calculated using the mean radius Rh , and thevolume of the whole particle, VT, by taking Rh plus the thickness

of the shell d. The values of f(qRT) and f(qRc) were, however,averaged over the whole particle size distribution range.

To account for particle size polydispersity, a zero-order normallog distribution was used:24

X(R) gives the percentage of particles of radius R, Rh is thearithmetic mean particle radius, and σ is the standard deviation.Polydispersity is expressed by the term σ/Rh . The computedscattered intensity was approximated by

where Vh p ) 4π/3(Rh )3(1 + σ2/Rh 2)3 is the mean particle volume. Thevalues giving the best fit with experimental data were obtainedusing a nonlinear least squares procedure.

Results and DiscussionCharacterizationof theVesiclesbyCryo-TEM.The

morphology of DOPC and DOPC/F6H10 vesicles wasdetermined by cryo-TEM, which allows direct observationof vesicles without addition of staining agents. Themicrographs show that the DOPC/F6H10 codispersion ledexclusively to SUVs (Figure 1a), as observed for DOPCvesicles. No droplets of emulsion of F6H10 coated by DOPCwere detected. Emulsion droplets would scatter electrons

(21) Guinier, A.; Fournet, G. Small angle scattering of X-rays; Wiley:New York, 1955.

(22) Lindner, P.; Zemb, T. Neutron, X-Ray and Light Scattering:Introduction to an Investigative Tool for Colloidal and PolymericSystems; North-Holland: Amsterdam, 1991.

(23) Zemb, T.; Charpin, P. J. Phys. 1985, 46, 249.(24) Epenscheid, W. F.; Kerker, M.; Matijevic, E. J. Phys. Chem.

1964, 68, 3093.

f(qRj) ) 3sin(qRj) - qRj cos(qRj)

(qRj)3

I(q) ) ANp(∆b)2[VT f(qRT) - Vc f(qRc)]2 (1)

Scheme 2. Concentric Shell Model for a Particlewith Its Radial Scattering Length Density Profilea

a HC and FC represent the hydrogenated and fluorinatedchain zone thicknesses. Rc is the particle core radius, d is theshell thickness, and RT is the overall particle radius.

X(R) ) 1

x2π Ln(σ2

Rh+ 1)

1R

exp[-(Ln(R

Rhxσ2

Rh 2+ 1))2

2 Ln(σ2

Rh 2+ 1) ]

I(q) ) ∫0

∞I(q)X(R) dR ) A(∆b)2Φp(1/Vh p) ∑Vp

2 P(q)X(R) (2)

Vesicles of Phospholipids and Semifluorinated Alkanes Langmuir, Vol. 19, No. 12, 2003 4891

more effectively and hence would be much darker thanthe water contained in SUVs. However, a significantdifference can be noted in the aspect of the bilayers of thetwo types of vesicles. The bilayer of DOPC vesicles is seenas two concentric dark rings (Figure 1b), as previouslydescribed.25 On the other hand, the DOPC/F6H10 vesiclebilayer appears as a single, thicker and darker ring (Figure1a). This pattern is very similar to that observed for thefluorinated vesicles made from perfluoroalkylated phos-phatidylcholine (Figure 1c).26 In these vesicles, the centralfluorinated core constituted by the fluorinated tailsstrongly scatters electrons, forming a new zone of intensecontrast in addition to that of the polar heads. As aconsequence, under optimum conditions, one could haveexpected the fluorinated bilayer to be visualized as threeconcentric rings, a thick dark central one flanked by twolighter ones. The fact that only one ring can be observedon the cryo-TEM pictures of F-PC vesicles is likely due tothe fact that the low-contrast regions corresponding tothe hydrogenated segments (only six carbons) are toonarrow to be distinguishable from the thick central ringdue to the fluorinated tails. The three concentric ringsexpected for DOPC/F6H10 vesicles were not observed

either. The fact that the aspect of the bilayer of DOPC/F6H10 vesicles does not present any distinguishabledifference to that of F-PC vesicles demonstrates that theF6H10 diblocks are located within the bilayer and forma continuous central fluorinated core like for F-PC, forwhich the covalently attached fluorinated chains are forcedto pile up in the center of the bilayer.27 The fluorinatedchains of the diblocks are segregated in the center of thebilayer of the DOPC/F6H10 vesicles rather than homo-geneously and randomly dispersed along with the hy-drogenated chains.

Mean Vesicle Diameters. Vesicle mean diameterswere measured by QELS (Figure 2a-c). Dh values were 33nm (polydispersity ) 0.27) for DOPC vesicles, 36 nm(polydispersity ) 0.25) for DOPC/F6H10 vesicles, 30 nm(polydispersity ) 0.27) for DMPC/F8H16 vesicles, and 40nm for F-PC vesicles (polydispersity ) 0.27). They werevery comparable, irrespective of the nature of the lipidsthat constituted the bilayer. The diameter measured byQELS cannot be directly compared to that measured oncryo-TEM pictures because the latter method gives onlya snapshot of a specimen, while QELS gives an averagemeasurement on the bulk sample.

Thermal Behavior of SUVs of DMPC/F6H10. InitialDSC Profiles. We have chosen DMPC instead of DOPCbecause the DOPC phase transition was too low (-21 °C)to allow a DSC study. The DSC heating curve of pureDMPC SUVs is plotted in Figure 3 (insert). It shows thetypical thermotropic behavior for such vesicles. The main-transition temperature Tc (P′â T LR) at 23.3 °C is inagreement with results in the literature.28,29 Tc is lowerfor SUVs than for multilamellar vesicles (MLVs). Thepretransition (L′â T P′â) that occurs for MLVs at ∼16 °Cis, in SUVs, part of the broad transition occurring overthe 14-24 °C temperature range (secondary-Tc peak30).

Figure 3 also shows that the DSC heating curve forSUVs made of the DMPC/F6H10 1:3 mixture is signifi-cantly different from those of DMPC SUVs. The thermo-grams are characterized by two peaks: a first peak, A, at∼23 °C, and a second one, B, at ∼25 °C. The same peaksappeared in the thermogram of the 1:1 mixture, peak Bbeing, however, less intense. From the shape and positionof peak A, it can be seen that there is both a broadeningof the DMPC main transition peak and a lowering of theDMPC Tc. This demonstrates that F6H10 diblocks havea disordering effect on the DMPC bilayer and henceconfirms their presence in the bilayer. Because thefluorinated segments are lipophobic, it is likely that it isthe hydrogenated segments H10 that interact with theDMPC chains. Peak B is characteristic of a gel-fluidtransition of F6H10. The intensity of this peak increasedwith the F6H10 molar fraction. This indicates thatalthough F6H10 is a liquid at room temperature, it is ina gel state when incorporated in the DMPC bilayer fortemperatures lower than 25 °C. This is in favor of anorganized arrangement of the fluorinated segments of theF6H10 molecules instead of a liquidlike behavior.

Cooling and Reheating Behavior. Peak B, althoughclearly visible during the first scan of DMPC/F6H10 (1:3)SUVs, disappeared when a second scan was run im-mediately after the first one. However, after 5 days atrest at 0 °C, the dispersion of vesicles being kept in thecalorimeter cell, peak B reappeared (Figure 4). This means

(25) Lepault, J.; Pattus, F.; Martin, N. Biochim. Biophys. Acta 1985,820, 315.

(26) Krafft, M. P.; Schieldknecht, L.; Marie, P.; Giulieri, F.; Schmutz,M.; Poulain, N.; Nakache, E. Langmuir 2001, 17, 2872.

(27) Riess, J. G. Tetrahedron 2002, 58, 4113.(28) van Dijck, P. W. M.; de Kruijff, B.; Aarts, A. M. M.; Verkleij, A.

J.; de Gier, J. Biochim. Biophys. Acta 1978, 506, 183.(29) Suurkuusk, M.; Singh, S. K. Chem. Phys. Lipids 1998, 94, 119.(30) Kodema, M.; Miyata, T.; Takaichi, Y. Biochim. Biophys. Acta

1993, 1169, 90.

Figure 1. Cryo-TEM micrographs of dispersions of (a) DOPC/F6H10 (1:1), (b) DOPC, and (c) F-PC. Part a shows that theDOPC/F6H10 sample is exclusively composed of SUVs withoutoccurrence of phospholipid-coated F6H10 emulsion droplets.DOPC/F6H10 and F-PC SUVs show identical, thick dark centralrings due to the strongly electron-scattering fluorinated chains.The two narrower and less intense concentric rings typical ofphospholipid bilayers (b) are not observed in the fluorinatedvesicles (a,c) because they are obscured by the much strongercentral rings due to the fluorinated chains.

4892 Langmuir, Vol. 19, No. 12, 2003 Schmutz et al.

that the gel-to-fluid transition of F6H10 within the bilayeris slow. This is confirmed by the fact that after 5 days atroom temperature, peak B is still smaller than at 0 °C.The same behavior was observed for 1:1 and 1:2 DMPC/F6H10 molar ratios, indicating that the DMPC/F6H10bilayer reached a thermodynamical equilibrium with slowkinetics.

SAXS Studies. SUVs made from equimolar combina-tions of DMPC and F8H16 were studied by SAXS. Thevariation of the diffracted intensity I(q) as a function ofthe wave vector is shown in Figure 5 for various concen-

trations (c ) 1.087×10-3 mol L-1, c/2, and c/4). The positionof the first maximum does not change with vesicleconcentration, meaning that the shape and size of thevesicles do not change with dilution and that the firstpeak is a peak of structure and not a peak of interaction.The positions of the following maxima and minima were,

Figure 2. Mean diameter of (a) DOPC/F6H10, (b) DOPC, and(c) F-PC SUVs, as determined by QELS.

Figure 3. Micro-DSC heating curves of DMPC/F6H10 1:1 (solidline) and 1:3 (dashed line) SUVs. The thermogram of DMPCSUVs is represented in the insert.

Figure 4. Thermograms of DMPC/F6H10 (1:3) SUV samplesobtained (a) immediately after a first heating-cooling cycleand after 5 days at 0 °C (b) or at room temperature (c).

Figure 5. Scattered X-ray intensity as a function of wavevector, q, for a suspension of DMPC/F8H16 (1:1) SUVs at 2.5%(circles, Φp ) 0.0433), 5% (diamonds, Φp ) 0.0867), and 10%(squares, Φp ) 0.1734). The solid line corresponds to the fitobtained using our hollow sphere model.

Vesicles of Phospholipids and Semifluorinated Alkanes Langmuir, Vol. 19, No. 12, 2003 4893

however, not easy to determine. This is due to weak signalintensity and to polydispersity.

Figure 5 also shows that the fit of the data accordingto eq 1 under the assumption of noninteracting, hollowspheres (polydispersity ) 0.3) provides a good descriptionof the data. The fit, ranging over 2 decades in intensityand over 1 decade in q, yields a core radius Rc of 9 nm anda shell thickness d of 3 nm. Deviations from the fit areseen at small q values (<0.01 Å-1), likely due to intra-vesicular contributions to the scattering. Our model maybe refined by taking into account two or more concentricshells. However, this would result in extra fitting pa-rameters, which was not thought to be worthwhile withinthe accuracy of the present experiments. In the presentwork, it was assumed that the density scattering of theshell would essentially be constant. Polydispersity of thecore particles, however, was taken into account by usinga zeroth order log-normal distribution.

SAXS experiments on similarly concentrated SUVsprepared with DMPC alone did not yield any detectablesignal. We can therefore assume that the scatteredintensity measured on the DMPC/F8H16 vesicles origi-nates from the fluorinated F8 segments. Knowing thatthe length of the F8 segments is ∼1.2 nm (total length ofF8H16 ) 3.3 nm), the SAXS results further confirm thatthe F8 segments form an internal shell and indicate thatthese segments are not interdigitated but rather adoptan extended configuration (Scheme 1).

Conclusions

Cryo-TEM, micro-DSC, and SAXS data establish thatcombinations of phospholipids and semifluorinated al-kanes FnHm lead to homogeneous populations of SUVswith a mixed bilayer membrane, without evidence offormation of phospholipid-coated FnHm emulsion drop-lets. The other results concern the structure of the mixedfluorinated bilayer. The fluorinated segments Fn form asegregated, organized continuous layer in the center ofthe vesicle bilayer. The thickness of this fluorinated layer,3 nm, indicates that the F8 segments are not interdigi-tated. On the other hand, the hydrogenated segments Hmare interdigitated with the fatty acid chains of thephospholipids. Semifluorinated alkanes provide a meansof obtaining fluorinated vesicles without using fluorinatedlipids, thus facilitating the access to and biologicalevaluation of such vesicles.

Acknowledgment. The authors thank ProfessorJ. G. Riess (University of California at San Diego) forhelpful discussions and Dr. D. Spehner (INSERM EPI99-08, EFS-Alsace) for kind access to the electron micro-scope. Thanks are also due to Dr. M. Rawiso for preciousadvice in SAXS experiments and to F. Schnell for his helpin SAXS measurements. The authors also acknowledgeAtoFina (Pierre Benite, France) for fluorinated precursors.

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