Perfluorooctanoic acid rigidifies a model lipid membrane

B. Bruning and B. Farago(Dated: May 6, 2014)

We report a combined dynamic light scattering (DLS) and neutron spin-echo (NSE) study on vesi-cles composed of the phospholipid 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC) underthe influence of varying amounts of perfluorooctanoic acid (PFOA). We study local lipid bilayerundulations using NSE on time scales up to 200 ns. Similar to the effect evoked by cholesterol, weattribute the observed lipid bilayer stiffening to a condensing effect of the perfluorinated compoundon the membrane.

PACS numbers:

Perfluorinated compounds (PFCs) are fully fluorinatedfatty acid analogues commonly used in a wide range ofapplications, such as the production of fire-extinguishingfoams, anticorrosion agents, lubricants or cosmetics [1, 2].As a consequence of their chemical stability, these com-pounds exhibit an environmental stability and are trans-mitted into the mammal food chain [3]. Due to their ten-dency to bioaccumulate, the compounds affect propertiesof cell membranes, causing developmental and reproduc-tive disorders [2, 4]. It is of particular interest to under-stand the effect of perfluorinated compounds not only oncellular membranes [5, 6], but also on their biomimeticcounterparts [7]. In mammal organisms, vesicular mem-branes often serve as natural carriers. It is assumed thatfunctional properties of a membrane depend likewise onits composition-dependent structure and dynamics [8, 9].In order to gain insight into membrane function, spe-cific material properties, such as e. g. the bilayer bend-ing rigidity κ can be aimfully influenced. Several studieshave addressed the insertion of perfluorinated compoundsinto binary model membranes. Oriented mono- and bi-layers were investigated by Matyszewska et al. usingmethods including surface pressure and potential mea-surements, infrared spectroscopy (IR), nuclear magneticresonance (NMR) techniques and molecular dynamics(MD) simulations [4, 10, 11]. While the phospholipidheadgroup tilt against the bilayer normal decreases withrising amounts of inserted PFOA, the lipid acyl chainorder increases. Lateral molecule diffusion in the mem-brane plane changes non-uniformly as the ratio of thecomponents in the binary mixture is varied [10]. Lehm-ler et al. study liposomes containing binary mixturesof varying phospholipids and perfluorinated surfactants[12–15]. They use fluorescence spectroscopy and differ-ential scanning calorimetry measurements to study thepartitioning of surfactants into the phospholipid bilayerand find that it is independ of the lipid acyl chain length.While the phase behaviour is largely independent of thetype of phospholipid, PFOA itself is found to partitionmore readily into lipid bilayers in their fluid phase [13].Several studies have taken advantage of a combinationof DLS and long-wavelength NSE for the investigationof the local bilayer undulation dynamics in unilamellarlipid vesicles (ULV’s) [16–21]. Here, we cover a windowof more than 200 ns. The membrane dynamics was in-

FIG. 1: (color online). Relaxation rate Γd vs. q2, for vesiclescomposed of DMPC and DMPC/PFOA (5mol%). Linear fitsyield the vesicle center-of-mass diffusion constant D.

vestigated by NSE using wavelengths of λ = 10 A and17 A. We highlight complementary dynamic regimes byadapting the time range of the correlation function decay.We use our DLS-results for a quantitative separation oftwo dynamical processes in the 17 A data.

The phospholipid DMPC was purchased from Avanti(Alabaster, AL), the perfluorinated surfactant PFOAfrom Sigma-Aldrich (Steinheim, Germany). Componentswere dissolved in chloroform in the desired molar propor-tions. The solvent was evaporated and the dry lipids werehydrated with heavy water (D2O) at 10 mg/ml. D2O wasused both for dynamic light scattering (DLS) and neu-tron spin-echo (NSE). For each specific composition, thePH was compensated through the addition of Na2CO3-base. To obtain unilamellar vesicles (ULV’s), the sus-pension was passed ten times through a polycarbonatefilter with 500 A pore diameter using a LiposoFast BasicExtruder (Avestin, Canada). For the NSE experiment,samples were poured into 1 mm thick quarz cuvettes witha quadratic cross section of 35 mm by 35 mm (Hellma,Mullheim, Germany).

For dynamic light scattering (DLS), an ALV goniome-ter with a 35 mW He-Ne laser operating at a wavelengthof 632.8 nm was used with an ALV/High QE APD detec-

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tor and an ALV-6010/ 160 external multiple τ digital cor-relator unit. The vesicle center-of-mass diffusion can bedescribed by a correlation function g1(t) = exp(−D ·q2t),which is derived from the measured intensity correla-tion function g2(t) through the Siegert relation g1(t) =√g2(t)− 1. The center-of-mass diffusion coefficient D

and the hydrodynamic vesicle radius RH are linked ac-cording to the Stokes-Einstein equation: RH = kBT

6π·η(T )·D .

kB denotes the Boltzmann constant, T the absolute tem-perature and η(T ) the temperature-dependent solventviscosity (for D2O at 30◦ C, η(T ) = 0.973 · 10−3 Pa s).Long term measurements indicate that the averaged vesi-cle radii RH remained constant for at least a week. Thevesicles for the combined DLS and NSE experiment werefreshly prepared and measured within one day. Angle-dependent measurements were taken between 20◦ and150◦ in steps of 10◦, the obtained relaxation rates Γdvs. q2 are shown in fig. 1. Linear fits indicate a purelyFickian diffusion. The linear slope of these curves cor-responds to the vesicle center-of-mass diffusion constantD.

The neutron spin-echo (NSE) experiment was per-formed at the cold spectrometer IN15 at the InstitutLaue Langevin (ILL, Grenoble, France). Due to its fineangular resolution in the small-angle regime (small q),the instrument is well suited to probe mesoscopic lengthscales. An introduction into the NSE method is givenin [22]. A measurement yields a momentum transfer andtime-resolved intermediate scattering function S(q, t), inwhich the Fourier time t changes proportionally to thewavelength λ3 and the applied magnetic field integral,following t ∝ λ3

∫|B|dl. Error bars in the measured

data correspond to ±σ statistical error calculated fromthe counting statistics and are transferred to our leastsquares fits. The q-range probed by NSE lies in the rangeof the inverse length scales of local bilayer interface undu-lations. This allows a data interpretation on the basis ofmodels including a unique q-dependence of the measuredrelaxation rates Γ(q). On IN15, incident neutron wave-lengths between 6 and 25 A with a wavelength spread of15% are available. At a distance of 4.6 m from the sam-ple, a He-3/CF4 multidetector is located with 32·32 pixelsof 1 cm2 each. The instrumental resolution is determinedusing graphoil as a purely coherent elastic scatterer andaccounted for by division of sample scattering throughresolution signal [23].

Merely simple diffusion mechanisms were assumed forthe short wavelength data S(q, t) ∝ exp(−Γf t), with arelaxation rate Γf = Df · q2. An example is shownin fig. 2 (a) for DMPC at 30◦C, within the q-rangein which the most pronounced changes in the dynam-ics occur. Fig. 2 (b) then shows an effective diffusionconstant Dfeff(q) derived as Γf (q)/q2 for DMPC andDMPC/PFOA (10mol%). In both cases, this plot is notq-independent, as would be the case if only Fickian dif-fusion occured in the probed time range. Unfortunately,the different dynamic contributions cannot be unambi-giously separated, due to a lack of a full polarization

(a)

(b)

FIG. 2: (color online). (a) Normalized intermediate scat-tering function S(q, t)/S(q) for DMPC (30◦C) at λ = 10 A.Single-exponential fits yield the relaxation rates Γf (q); (b)the effective diffusion constant Dfeff(q) = Γf (q)/q2 exhibitsa q-dependence, indicating further dynamics.

decay at the largest measured q shown in (a). The dy-namics are not fully covered within the probed window,necessitating our choice of a higher wavelength to extendthe Fourier-time range. Here, a smaller q-range is coveredwith higher resolution, thus two complementary data setsare obtained. Regarding the origin of the quadratic modeΓf (q2), several possibilities exist: In their seminal the-oretical works, Evans and Yeung, as well as Seifert andLanger have already predicted modes, which relate bend-ing and local density changes between the two monolayersand taken into account resulting intermonolayer friction[24, 25]. The modes follow quadratic relations Γ(q2), andhave been discussed in recent experimental works by Ar-riaga et al. [18]. On the other hand, the contributionlies close to the one of the vesicle center-of-mass diffu-sion found by dynamic light scattering. Following, wediscuss vesicle center-of-mass translations and bilayer un-dulations as separable dynamic contributions in the longwavelength spin-echo data.

Curvature undulations of elastic membranes are com-monly described by the well-known Helfrich Hamiltonian[26]. Based on this continuum mechanical approach Mil-ner and Safran further describe the fluctuation dynamicsof microemulsion droplets and vesicles [27]. In their the-ory, the normal bending modes of the flexible interfaceare coupled to the viscous friction exerted by the sus-pending medium according to a single exponential decayexp(−ΓMSt) with a relaxation rate ΓMS = κ

4η q3, where η

3

is the effective viscosity of the solvent medium and κ thebilayer bending rigidity. Faster relaxations are assignedto stiffer membranes. While suited to describe soft inter-faces with bending rigidities on the order of kBT , such asmicro-emulsion droplets and sponge phases, the expres-sion fails to accurately account for dynamics of modellipid membranes with bending rigidities of several kBT .Zilman and Granek describe curvature shape fluctuationsof freely suspended phospholipid bilayers [28, 29]. Theirmodel takes into account a coupling of the bending modesand local diffusion processes: in a rigid membrane witha bilayer bending rigidity of (κ� kBT ), less free volumecan be explored by single molecules; this means that arelaxation rate for a coupled process of undulation andlocal curvature will increase, whereas the average ampli-tude of the modes will decrease. The anomalous subdif-fusive relaxation of the bending motions is described bya stretched exponential decay with a stretching exponentof β = 2/3 (eq. 1):

S(q, t) ∝ exp(−Γu(q) · t)β

with Γu(q) = 0.025γq

(kBT

κ

)1/2

·(kBT

η(T )

)q3(1)

The relaxation rate Γu(q) includes the temperature-de-pendent solvent viscosity η(T ). Further, γq is a weakmonotonous function of the bending rigidity κ accordingto γq = 1 − 3

4π

(kBTκ

)· ln(qh), where h is the membrane

thickness with q · h ≈ 1. When κ lies on the order ofseveral kBT , γq can be approximated to unity. Usingatomistic and coarse-grained molecular dynamics (MD)simulations, Brandt and Edholm describe the nanometerlength scale fluctuation decay in fluid biomembranes bya stretched exponential [30]. We discuss our experimen-tal data on the basis of this approach, augmented by theoccurrence of underlying vesicle center-of-mass diffusion(eq. 2). A is a normalization parameter close to one, themass diffusion relaxation rate Γd = D · q2 is fixed, therelaxation rate of bilayer undulations Γu is a free param-eter, and the stretched exponential is held to β = 0.66,following the Zilman-Granek approach. With respect toprevious works, such as [16, 17], this seems to be a moregeneral approach. Comparing the outcome to the resultsobtained on the basis of the previous description of S(q,t)/S(q), we find no significant differences within experi-mental precision.

S(q, t)/S(q, 0) = A · exp(−Γdt) · exp(−Γut)β (2)

The normalized intermediate scattering function of theDMPC standard S(q, t)/S(q) is shown in fig. 3 (a) forvarying q. Fourier times extend up to 200 ns, and the nor-malized polarization nearly fully decays. This means, thedynamics are captured within the spectrometer window,enabling more precise treatment than before. Over thewhole q-range, fits can be improved taking into account

(a)

(b)

FIG. 3: (color online). Normalized intermediate scatteringfunctions S(q, t)/S(q) for DMPC (30◦C): (a) q-range coveredat λ = 17 A; (b) combined fit according to eq. 2, single con-tributions are indicated by dotted lines.

dynamic contributions just outside the instrumental win-dow in the µs-regime. Therefore, we add a vesicle center-of-mass diffusion contribution through the diffusion con-stant D obtained from DLS measurements (eq. 2). Infig. 3 (b), both vesicle center-of-mass diffusion, as wellas bilayer undulation contributions are shown for one ex-emplary q-value (q = 0.097 A). The double logarithmicscale shows that the decay curvature is well matched bythe combined fit. At momentum transfers of q = 0.071 Aand above, a deviation from the single-exponential be-haviour is observed, starting at Fourier times between 50and 100 ns, cf. (a). The undulation relaxation rate Γu(q)can now be further analyzed. For our DMPC standardin its fluid phase (30◦C), the result of κ = 17.68 ± 0.15kBT derived from linear regression of Γu(q3) is well inaccordance with literature values [31–33]. The inherentexperimental error for κ is estimated to lie on the orderof kBT . The undulation relaxation rate Γu(q3) obtainedfrom these fits are shown in fig. 4. Insertion of risingamounts of PFOA evokes a decrease in the linear slopeof Γu(q3). The resulting increase in the bilayer bendingrigidities κ is shown in (b).

Comparing unilamellar vesicles composed of DMPCand DMPC/PFOA mixtures, we have investigated theeffect of the perfluorinated compound on local bilayerundulations and on the bilayer bending rigidity κ. Ef-fects on vesicle self-diffusion were also investigated. TheNSE data can be meaningfully analyzed, assuming a com-bination of two separable contributions within the dy-

4

(a)

(b)

FIG. 4: (color online). Bilayer undulations: (a) Relaxationrate Γu vs. q3, for DMPC and DMPC/PFOA. Linear fitsyield the concentration dependent bilayer bending rigidity κshown in (b).

namic window up to 200 ns, namely vesicle center-of-mass diffusion and local bilayer undulations. The latterwas described on the basis of the well-known Zilman-Granek approach for free film fluctuations. Our datasupports a view that lipid bilayer undulation dynamicsand corresponding bending rigidities κ can be tuned bydirectly inducing changes at the lipid acyl chains: theperfluorinated compound PFOA, which partitions intothe membrane [12–15], reduces the free volume in themembrane plane, since the CF2-segments of the surfac-tant tail occupy more space than the neighbouring lipidchain CH2-segments. Consequently, an increase in thebilayer bending rigidity κ is observed. In a previouswork, we have also investigated binary lipid mixtures

containing DMPC and the monounsaturated phospho-lipid DOPC [19]. Here, a significant decrease in κ wasobserved, the higher the amount of the latter was. Wesuggested, this decrease might be explained by a meso-scopic lateral phase segregation leading to domain struc-ture fluctuations and a corresponding softening of themembrane [34, 35]. In the case of lipid/surfactant mix-tures on the other hand, a homogenous lateral distribu-tion of the two components is likely, similar to the one inbinary cholesterol mixtures. The stiffening of a DMPCbilayer through cholesterol insertion has long since beenassociated with the condensing effect of the sterol [36].Nakahara et al. have suggested similar effects might oc-cur for partially fluorinated alcohols in DPPC and DPPGmembranes [37]. In our case, the PFOA-induced stiffen-ing of the DMPC bilayer might be explained as follows:The ionic surfactant head is more hydrophillic and theperfluorinated tail more hydrophobic than the zwitteri-onic phospholipid headgroup and its acyl chains. Repul-sion between neighboring surfactant heads as well as thebulkiness of the surfactant CF2-segments in comparisonto their CH2 lipid acyl chain counterparts make an al-ternating in-plane molecule distribution of lipid and sur-factant likely. The rotation of lipid acyl chain segmentsaround the bilayer normal axis is then restricted. Thus,by PFOA-inclusion, the in-plane area density of lipidacyl chain and surfactant tail segments is then increased,causing lipid bilayer stiffening. Such a bilayer stiffeningmay well be the origin of the compounds harmful effectson cellular membranes, as it is likely to influence fusio-genic properties of lipid vesicles. Therefore, it would beinteresting to study how the compounds influence the for-mation of stalk intermediates as precursor states for thefusion of lipid vesicles. This could further elucidate, howreproductive and developmental processes are inhibitedin mammal organisms after perfluorinated compound in-gestion.

We are greatful to Ralf Kohler and Ralf Stehle for ex-perimental support. Roland Steitz is thanked for helpfulcomments on the manuscript. We thank HZB for finan-cial support and for the allocation of beam time on IN15,as well as ILL for technical support.

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