Chemistry and Physics of Lipids 154 (2008) 79–86

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Chemistry and Physics of Lipids

journa l homepage: www.e lsev ier .com/ locate /chemphys l ip

Partitioning of 1-pyrenesulfonate into zwitterionic and mixed

zwitterionic/anionic fluid phospholipid bilayers

Miguel Manuel, Jorge Martins ∗

ro, Por

to bioajor

phatrionic1-pyrative

osphoanionxed zpera×104

rfacia

IBB-CBME and DQBF-FCT, Universidade do Algarve, Campus de Gambelas, 8005-139 Fa

a r t i c l e i n f o

Article history:Received 28 November 2007Received in revised form 23 April 2008Accepted 25 April 2008Available online 4 May 2008

Keywords:Fluorescent probesLiposomesMixed zwitterionic/anionic bilayersPartition equilibriumSecond derivative spectrumUV–vis absorption

a b s t r a c t

Molecular partitioning incesses and reactions. The mis obtained with pure phosbilayers containing zwittesolvatochromic effects ofmonitored by second derivconstants into defined phtions, the partition of theat 25 ◦C), and into fluid mipholipids. At the same temmixed with POPC (Kp = 3.4tation is based on the intebilayers.

1. Introduction

Biological membranes come in contact with a variety ofmolecules, whether endogenous or xenobiotics, interactingdiversely as a function of specificity of the biomembrane-associatedprocesses, e.g. permeability or transport, chemoreception andmembrane enzymology (Gennis, 1989). The kinetics of manymembrane-based processes are influenced by molecular partition-ing between aqueous and membrane environments, because itis not the aqueous concentrations of substrates (Heirwegh et al.,1989; Mason et al., 1991) that determines the activity of target pro-teins, but their concentrations within the membrane. Furthermore,since these volume-surface reactions occur involving substratesinserted in lipid bilayers, their rates involve a so-called reductionin dimensionality (Adam and Delbruck, 1968), e.g. involvement oftwo-dimensional reactions kinetics (depending on the concentra-tion of reactants per unit area), preceded by a three-dimensionalstep, relaying in volume concentration (Martins et al., 2004; Meloand Martins, 2006).

When studying the interaction of chemicals with model mem-branes the analysis of partition process must come first (Santos et

∗ Corresponding author. Tel.: +351 289800900; fax: +351 289800066.E-mail address: jmartin@ualg.pt (J. Martins).

0009-3084/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved.doi:10.1016/j.chemphyslip.2008.04.007

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membranes is of fundamental importance in diverse biochemical pro-ity of aqueous/membrane partition data using model membrane systems,idylcholine bilayers, being lipid mixtures less used, while studies involving/anionic mixtures of phospholipids are even more scarce. In this study, theenesulfonate observed at 375 nm in aqueous liposome suspensions, andabsorption spectrophotometry, enabled the determination of its partitionlipid bilayers. We compare, under cautiously settled experimental condi-ic amphiphile PSA into fluid zwitterionic bilayers of POPC (Kp = 6.7×103,

witterionic/anionic bilayers containing small proportions of anionic phos-ture, we found increasing Kp values in parallel with the proportion of POPSand Kp = 7.3×104, with 5 and 10 mol% of POPS, respectively). Our interpre-l properties of fluid and flexible mixed zwitterionic/anionic phospholipid

© 2008 Elsevier Ireland Ltd. All rights reserved.

al., 2003). The mainstream of data is obtained using zwitterionicphosphatidylcholine vesicles (Ottiger and Wunderli-Allenspach,1997), since it is the major phospholipid in biological membranes.However, the diversity of biomembranes requires models reflect-

ing some basic features of the various lipid compositions (Gennis,1989). Nevertheless, lipidic mixtures are used at a lesser extent, andfewer attention is dedicated to mixed zwitterionic/anionic phos-pholipid bilayers. These mixtures mimic some characteristics of theplasma membranes’ lipid composition, since negatively chargedcomponents are present at around 10–20% (Gennis, 1989). Partitionto zwitterionic bilayers generally decreases in the sequence apolar,polar, anionic and cationic solutes. In comparison, partition to nega-tively charged bilayers is increased to cationic species (Takegami etal., 2005), decreased to anionic (Waczulikova et al., 2002; Thomaeet al., 2007), and unaltered for apolar and polar molecules. How-ever, using small unilamellar vesicles (SUV) as model membranes,the interaction of polar amphiphiles with mixed negatively chargedvesicles is found augmented when compared with neutral lipo-somes (Omran et al., 2002), and it is increased for anionic bile salts,from pure zwitterionic to pure anionic vesicles (Hildebrand et al.,2004). Distinctive findings were also reported on the interaction ofSDS (sodium dodecyl sulfate) with zwitterionic and mixed zwitte-rionic/anionic SUV (Tan et al., 2002), and large unilamellar vesicles(LUV) (Cocera et al., 2004). Still, no interpretations about the molec-ular basis of these phenomena were presented. Partition of small

and P

80 M. Manuel, J. Martins / Chemistry

molecules from aqueous media into model membranes generallydepends on the physical–chemical properties of the partitioningspecies, on their differential interactions between polar and apolarmedia, as well as on the different lipid bilayer interfacial character-istics such as lipid packing, entropic costs for particular locations,surface charge density, internal dipole potential, as well as bilayerpolarity and/or water penetration (De Young and Dill, 1988; Gennis,1989; Xiang and Anderson, 1995; Langner and Kubica, 1999; Arraisand Martins, 2007).

We analyze here the partition of the anionic amphiphile1-pyrenesulfonate (PSA) into multilamellar vesicles (MLV) andLUV (produced by the extrusion technique), composed bypure 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC),and mixtures of POPC with negatively charged 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (POPS), or with 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (POPG). The modelanionic amphiphile PSA mimics several xenobiotics (e.g. pharma-ceuticals, pesticides) and endogenous substrates that interact withbiological membranes. Additionally, PSA has advantageous pho-tophysical (Bohne et al., 1990; Hara et al., 2004) and chemicalproperties, being as well reasonably soluble in water and in diverseorganic solvents. PSA exhibits solvatochromic effects required forsecond derivative UV–vis spectrophotometry (Perkampus, 1992),and for instance, its quantification in aqueous liposome suspen-sions is based on the simultaneous detection of the “aqueous” and“bilayer” fractions, being their relative weight dependent on theequilibrium partition constant (Kp) (Custodio et al., 1991; Kitamuraet al., 1995). At 25 ◦C, we find increasing Kp values in parallelwith relatively low molar proportions of POPS in mixed zwitteri-onic/anionic fluid bilayers, comparing with fluid zwitterionic POPCbilayers. A suitable interpretation, based on the interfacial proper-ties of these negatively charged bilayers, is altogether presented.

2. Material and methods

2.1. Chemicals and solvents

POPC, POPS (sodium salt), POPG (sodium salt), and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) (purity > 99.9%)were purchased from Avanti Polar Lipids (Alabaster, AL, USA). PSA(1-pyrenesulfonic acid sodium salt, for fluorescence grade) wasfrom Fluka (Buchs, Switzerland). MilliQ water is obtained usinga Millipore Simplicity 185 system (electrical conductivity and pH

−6 −1

are 5.4×10 S m and around 6.6–7, respectively, at room tem-perature). All organic solvents (highest purity) and NaCl (analyticalgrade) were from Merck (Darmstadt, Germany).

2.2. Aqueous liposome suspensions

MLV were prepared according to a modified protocol of a firmlyestablished method (Szoka and Papahadjopoulos, 1980). Phospho-lipid stock solutions, dissolved in chloroform:methanol 2:1 (v/v),were mixed in settled proportions and dried using a rotary evapo-rator Heidolph VV-micro (vacuum pump Buchi V-500, coupled toa Buchi V-800 digital control), 15 min at 150 mbar, followed by 1 hat 5 mbar, to remove traces of organic solvents. The lipidic film washydrated with MilliQ water during at least 1 h at 30 ◦C, or well abovethe phase transition temperature (Tm) of POPC (Tm =−2.6 ◦C), POPS(Tm = 14 ◦C), POPG (Tm =−2 ◦C), and DPPC (Tm = 41.5 ◦C) (Marsh,1990), vortexing regularly. By using only freshly deionised MilliQwater, possible spurious and cumulative effects arising from theinorganic and/or organic ionic species ubiquitous to buffer solu-tions are discarded, and thereby minimizing the electrostaticscreening of the surface potentials in zwitterionic/anionic bilayers.

hysics of Lipids 154 (2008) 79–86

Even so, NaCl solutions were used in hydration to evaluate puta-tive effects of ionic strength. The pH of MilliQ water was measuredbefore and after the partition assays, and was around 6.8 (aver-aged standard deviation of 0.2 pH units). Therefore, PSA (pKa < 1for aromatic sulfonic acids (IUPAC Chemical Data Series no 23,1979)), POPS and POPG (Egorova, 1998) are all completely disso-ciated in their anionic forms, excluding interferences arising fromacid-base multiequilibria. Even considering the lowering of pH nearthe surface of the bilayers due to the negative surface potential(Gennis, 1989), for the proportion of 10 mol% (molar proportion)of the anionic component, this would result at most in a lower-ing of roughly 0.5 pH units, which is not sufficient to influence anyof the anionic species involved. Several MLV compositions wereused, including pure POPC and DPPC vesicles, as well as mixturesof POPC with 5, 10 and 20 mol% of POPS, and with 5 and 10 mol%of POPG. No calcium quelating agents were used, since calcium inthe mM range (threshold to induce phase separation; Silvius andGagne, 1984) cannot be attained by ions evolving from the glassvessels, and counterfeit effects could occur from an unnecessaryuse. The calcium concentration in MilliQ water was ascertained byatomic absorption spectroscopy GBC Avant (GBC Scientific Equip-ment, Australia), obtaining values below the nM range (e.g. belowppb).

LUV of pure POPC were further analyzed, in addition to LUVcontaining 5 and 10 mol% of POPS, and procedures were homolo-gous to those described for MLV suspensions. LUV were preparedby the extrusion method (MacDonald et al., 1993), using an AvantiMini-Extruder (Alabaster, AL, USA), forcing the MLV to pass 21times through a pair of Nucleopore polycarbonate filters (Wath-man, USA), with pore size of 0.6 �m. This approach produces LUVwithout introducing contaminants, but one must be aware of thepolydispersity of vesicles (populations of vesicles with 1, 2 or 3lamellae), even for pores lower than 0.2 �m, given the moderateand irregular values of pressure (6.9–10.3 bar) obtained by handwith syringes of 1 ml (Patty and Frisken, 2003). On the other hand,the presence of external solutes in liposome suspensions, as whenincubating with PSA, must be also taken into account. LUV displayosmotic properties, which may lead to alterations in bilayer hydra-tion and/or in lipid packing density (White et al., 1996). To minimizeartefacts from the osmotic stress in the incubation step, we uselarger LUV to maintain a phospholipid packing density, and its vari-ation, the closest possible to the much bigger MLV. Even though, theLUV used present sufficiently different lamellarity, when compar-ing with MLV, while the exposed bilayer surface available to solute

partition was maintained equal for both systems. For calculationsof the partition constants, we assumed that 15% of the total phos-pholipid concentration is on the outer bilayer of MLV (Szoka andPapahadjopoulos, 1980), and for this type of LUV (mean lamellarityof about 1.3), it is estimated that 75% (Berger et al., 2001; Patty andFrisken, 2003) of the total phospholipid concentration is accessible(instead of 100%, when precise unilamellarity is verified in LUV).

2.3. UV–vis absorption and second derivative spectrophotometry

Liposome suspensions (with varying phospholipid composi-tions) were incubated with PSA 5 �M (ε = 8.45 × 103 M−1 cm−1,at 346 nm; Hara et al., 2004), for total phospholipid concentra-tions varying between 0.065 and 1.0 mM, during 15–30 min, at25 ◦C. Aqueous PSA is strictly in the monomer form, since thethreshold value for aggregation in water is 0.4 mM (Menger andWhitesell, 1987). Even for the referred proportion of exposed bilay-ers, it is ensured that the PSA/phospholipid molar ratio is wellbelow the critical values for saturation of bilayers (Lasch, 1995).Higher (10 �M) and lower (2.5 �M) concentrations of PSA havebeen examined, and no variations in the final equilibrium values

and P

M. Manuel, J. Martins / Chemistry

of partition were found. At room temperature, the translocation ofcharged amphiphiles analogous to PSA across fluid bilayers occurswithin a characteristic half-lifetime of several hours (Lasch, 1995;Tan et al., 2002; Keller et al., 2006), and even for overnight incu-bations (18–20 h), no influences from the flip-flop process in thediverse partition equilibrium states were detected in the underly-ing spectroscopic measurements, either for zwitterionic and mixedzwitterionic/anionic bilayers. Accordingly, it is fair to conclude thatthe potential impact of flip-flop is negligibly small, within the timeinterval (30 min) to complete each partitioning study.

Absorption spectra were recorded on a Shimadzu UV-2401PC spectrophotometer (Shimadzu, Japan), equipped with a ther-mostated cell-holder coupled to a circulator Savant RWC825(Savant Instruments, USA), at 25±0.2 ◦C, in 1 cm quartz cuvettes,slit width of 1 nm, and wavelength sampling interval of 0.1 nm.Either MilliQ water or suspension of liposomes were used as ref-erence in UV–vis measurements producing equal results in doublederivative spectra, so the economical procedure was chosen. Thesecond derivative spectra were obtained using the software UVProbe 2.0SU1 (Shimadzu, Japan), whose smoothing and differen-tiation calculation is based on a convolution function using 17 datapoints. The wavelength sampling interval of 0.1 nm was evaluatedas adequate to ensure both background elimination and spectralresolution.

2.4. Determination of partition constants

Second derivative absorption spectrum method has been sel-dom used to characterize partition into model and biologicalmembranes (Omran et al., 2002; Santos et al., 2003; Pola et al.,2004; Takegami et al., 2005; Ferreira et al., 2005). Custodio etal. (1991) and Kitamura et al. (1995) presented a second deriva-tive spectrophotometry procedure to obtain partition coefficientsof chromophores that display solvatochromic effects betweenaqueous and bilayer media. In brief, the complete mathematicalformulation lying behind is as follows. Defining the partition coef-ficient (K ′p) of solute (S) between the lipidic and aqueous phases,as:

K ′p =fraction of S in lipidic phase/[L]fraction of S in water phase/[W]

(1)

where [L] is the molar lipidic concentration (proportion of lipidbilayers available for partition) and [W] is the concentration ofwater ([W] = 55.6 M, at 25 ◦C).

In absorption spectrum of S, the scatter due to liposomesuspensions is eliminated in the second derivative of spectrum(D = d2(Abs)/d�2), along the wavelength range of measurements,i.e. background scatter should be nearly described by a linearfunction of �, whose second derivative is zero. Since aqueousliposome suspensions can contain S molecules distributed inwater (subscript W) and in lipid bilayer (subscript L) phases, Dis equal to the cumulative contributions from each phase, e.g.D = d2εL/d�2[S]L + d2εW/d�2[S]W, where [S]L and [S]W are the molarconcentrations of S in the lipidic and aqueous phases, respectively;εL and εW are the molar absorption coefficients of chromophoreS, in each medium; the optical length is 1 cm. As [S]L and [S]Ware unknown, on experimental condition that the total concen-tration of S is fixed ([S]T = [S]L + [S]W), by introducing �D = D−D0,where D0 is the second derivative of absorbance of S in water(without liposomes), �D is proportional to the concentration of Spartitioned into the lipid bilayer (�D = (d2εL/d�2−d2εW/d�2)[S]L).The subtraction course of action results from a foregoing approachusing absorption difference spectroscopy (Welti et al., 1984). Itfollows that the fraction of S in the lipidic phase is defined as�D/�Dmax, where �Dmax = (d2εL/d�2)[S]T, is the maximum value

hysics of Lipids 154 (2008) 79–86 81

of �D (assuming all S molecules partitioned within the bilayer,or D0→0). Taking into account Eq. (1), one obtains the followingrelationship between �D and [L]:

�D = �Dmax[L]([W]/K ′p)+ [L]

(2)

Given that Eq. (2) is analogous to the Henri–Michaelis–Mentenequation of classical enzyme kinetics (representing graphicallya rectangular hyperbolae) (Cornish-Bowden, 1995), the retrievedparameters ([W]/K ′p) and �Dmax are determined from a straight-forward graphical non-parametric method (the direct linear plot)(Cornish-Bowden, 1995), instead of using the usual lineariza-tion procedure (Hanes-Woolf type plot: [L]/�D = (1/�Dmax)[L]+([W]/K ′p�Dmax)) (Pola et al., 2004), that although enabling satis-factory accuracy, imposes the independent variable [L], both in theordinate and in the abscissa. The direct linear plot, represents eachexperimental result (�D and respective [L]) as a straight line, havingintercepts −[L] and �D on the abscissa and ordinate, respectively.Ideally, the point of intersection of the lines would give the coor-dinates of the best-fit values of ([W]/K ′p) (on abscissa) and �Dmax

(on ordinate), however, due to experimental uncertainty, they areobtained in practice from the median of the sets of coordinates(Cornish-Bowden, 1995). This method provides clear and accurateinformation about the quality of the data, and unbiased estimatesof the fitted parameters, as those from non-linear fitting computerprograms. The fitting parameters can be retrieved as well fromnon-linear least-squares fitting methods to Eq. (2), using the Solverfeature of Microsoft Excel®, which has enough accuracy for mostpractical purposes. Still, it must be emphasized that the non-linearoptimization methods require good initial guesses for fitted param-eters, careful balancing of intrinsic factors of iterative minimizationroutines and even so, there is no guarantee of convergence to theglobal minimum (Cornish-Bowden, 1995).

In literature, the characterization of partition process is com-monly expressed by the equilibrium partition constant (Santos etal., 2003), as defined by Nernst (Lasch, 1995):

Kp = [S]L

[S]W(3)

The relationship between the two formulations for equilibriumpartition (Eqs. (1) and (3)) is simply Kp = K ′p(VW

m /VLm) (Santos et al.,

2003), where VLm and VW

m are the molar volumes of POPC and ofwater at 25 ◦C, e.g. 752.75 and 18.07 cm3 mol−1, respectively. Weassumed that the phospholipid molecular area and hydrophobic

length in the zwitterionic/anionic mixtures, are equal to pure POPCbilayers in the fluid phase (Kucerka et al., 2005).

Evaluation of partition using the separation of aqueous and gelDPPC MLV phases, was achieved using a Beckman-Coulter AvantiJ-251 centrifuge (Beckman-Coulter, USA), at 27,200× g, 45 min, at25 ◦C. Although this procedure is not trouble-free and could resultin equilibria perturbations, it certainly provides additional infor-mation. Non-partitioned PSA was quantified from the aqueoussupernatant (effectively separated from the MLV pellet) by monitor-ing the fluorescence emission at 374 nm, using a spectrofluorimeterSpex Fluoromax-3 (Jobin Yvon–Horiba, France).

3. Results and discussion

3.1. Absorption and second derivative spectra

Additive effects, caused by light scattering from aqueous lipo-some suspensions, are observed in the absorption spectra of PSAin MilliQ water, and in aqueous suspensions samples containingincreasing amounts of POPC MLV (Fig. 1). The absorption maximumof PSA at 346 nm in liposomes exhibited a slight red-shift of about

82 M. Manuel, J. Martins / Chemistry and P

Fig. 1. Absorption spectra of PSA 5 �M, at 25 ◦C, in (a) MilliQ water and in POPCMLV aqueous suspensions, whose phospholipid concentration is (b) 0.065 mM, (c)0.125 mM, (d) 0.25 mM, (e) 0.5 mM and (f) 1.0 mM.

1 nm, as previously observed for other chromophores (Kitamuraet al., 1995; Omran et al., 2002). This deviation is attributed to adecrease in the polarity of the environment, indicating an incorpo-ration of chromophores in the hydrophobic region of zwitterionicbilayers. These findings are in accordance with the determinedlocation and orientation of PSA within lipid bilayers (Kachel et al.,1998).

Fig. 2 displays the second derivative spectra (D = d2(Abs)/d�2 vs.�), computed from the absorption spectra in Fig. 1. The scatter-ing effects are completely eliminated in the 380–400 nm region,where the absorption of PSA is null. Two points (roughly equiva-lent to isosbestic points in absorbance) are observed in the second

Fig. 2. Second derivative spectra calculated from the absorption spectra of PSA 5 �Min Fig. 1 in (a) MilliQ water and in POPC MLV suspensions (b) 0.065 mM, (c) 0.125 mM,(d) 0.25 mM, (e) 0.5 mM and (f) 1.0 mM. Insert: Expansion in the region 372–378 nm.

hysics of Lipids 154 (2008) 79–86

derivative spectra at 373 and 376 nm (see insert in Fig. 2), demon-strating that the background scatter is effectively eliminated, andthat PSA do exists in two different environments at thermal equi-librium. For zwitterionic/anionic vesicles containing POPC/POPSand POPC/POPG mixtures, similar findings were observed; the onlydifference was the occurrence of a blue-shift of the absorption max-imum, suggesting that the pyrenyl moiety is exposed to a morepolarizable environment.

The �D values were averaged over at least five independentassays for each partitioning assay, using either MLV or LUV. It isrecommended that the �D values should be measured aroundthe absorption maximum (�max) (Perkampus, 1992), however, theywere only found reproducible at 375 nm, and not in the mostintense peaks at 330 and 346 nm (Fig. 1). At 330 and 346 nm, wewere not able to find any type of correlation between �D and [L].This can be understood based on the solvent dependent photo-physical properties of the pyrene moiety (Karpovich and Blanchard,1995). While at 330 and 346 nm, the various bands corresponds tovibronic transitions of the allowed electronic transition S2← S0, thepeak at 375 nm includes only the 0–0 vibronic transition of S1← S0.In pyrene (Karpovich and Blanchard, 1995), this electronic tran-sition is symmetry forbidden, and therefore the 0–0 transition ispurely electronic in nature and formally forbidden in the totallysymmetric ground state geometry. Interactions with the mediumrelax this symmetry, augmenting the intensity of this transition.In PSA, although the sulfonate group lowers the molecular sym-metry, the low down intensity at 375 nm indicates that much ofthe solvent effects in this peak are still retained and is thereforesensitive to solvent effects, providing in this way a suitable signalfor double derivative UV–vis spectrophotometry (Fig. 2). This effecthas been noticed for other 1-pyrenyl derivatives, with methylether(Winnik et al., 1991), methylamide (Morishima et al., 1991) and sul-fonyl (Gonzalez-Benito et al., 2000) linkages. In relation with this,fluorescence would be far more sensitive to characterize the par-titioning process of PSA, nevertheless, we found that the spectralalterations of PSA emission in alcoholic solvents of varying polarity(methanol, ethanol, 1-propanol, and 2-butanol) are not sufficientlyresponsive to this end.

Concerning the somewhat sizeable standard deviationsobserved in some few cases for the �D values obtained (coefficientof variation of the mean values until the maximum of 15%) eitherfor MLV and LUV, the following comments should be made. Sincethe numerical double differentiation calculations are extremelysensitive to changes of the slope in absorption spectra, using the

375 nm band of relatively low intensity and width, produces largerdeviations (even with an algorithm using 17 data points). Further-more, there must be an influence in �D of the random fluctuationsin the [L] values, due to the polidispersion in the shape of the MLVand consequently in the exposed proportion of phospholipids. Aswell, LUV are more precise in the exposed fraction of phospho-lipids, but present some disadvantages due to its oligolamellarityand from likely lipid losses during the preparation. Nevertheless,the overall results described afterwards are self-consistent, andare in accordance with the partitioning outcomes obtained viaother independent experimental approaches.

3.2. Determination of Kp

Although being extensively used in enzyme kinetics analysis(Cornish-Bowden, 1995), to the best of our knowledge, it is thefirst time that the direct linear plot is applied in the analysis ofan analogous detailed formalism to evaluate partition into lipidbilayers. The retrieved values from the direct linear plot and fromthe non-linear fitting using the Solver feature of Microsoft Excel,are indistinguishable in statistical terms. In direct linear plots, the

M. Manuel, J. Martins / Chemistry and P

fitted values are read right away: (55.6/K ′p) (indicated in the posi-tive values in the abscissa, Fig. 3A) and �Dmax (in the correspondingordinate). In Fig. 3, [L] corresponds to 15% of the total phospho-lipid concentration in MLV suspensions. Very good non-parametric

Fig. 3. Direct linear plots determining promptly the fitted values ([W]/K ′p) and�Dmax for the equilibrium partition at 25 ◦C, of PSA into MLV containing the follow-ing compositions: (A) POPC, (B) POPC-POPS (90-10, mol%), (C) POPC-POPS (80-20).The concentrations corresponding to the exposed proportion of lipid bilayers arethe following: (�) 9.75 �M, (�) 18.75 �M, (�) 37.5 �M, (�) 75 �M, and (�) 150 �M.

hysics of Lipids 154 (2008) 79–86 83

fittings were obtained for pure POPC (Fig. 3A), and for MLV con-taining 5 mol% (data not shown) and 10 mol% (Fig. 3B) of POPS.However, 20 mol% of POPS (Fig. 3C) yielded a plot displaying verypoor precision and even some intersections off-scale, obviating thecalculation of Kp. This observation is also supported by parallelanalysis through non-linear and linear least-squares fittings. Con-sequently, for 20 mol% of POPS there is no experimental supportof partition under the same conditions for lower POPS contents,probably because of altered miscibility of the phospholipid compo-nents (Sinn et al., 2006) and/or changes in lamellarity (Szoka andPapahadjopoulos, 1980) and/or variations in vesicles shape and size(Traıkia et al., 2002). Mixtures with 5 and 10 mol% of POPG (data notshown) also produced reasonable fittings, but the underlying �Ddata showed a significant increase in the dispersion of experimentalvalues. We attribute this larger scattered data to a less ideal char-acter of the PC/PG mixtures, as described previously (Lewis et al.,2005). Contrasting, DPPC MLV in the gel phase did not exhibit anytype of non-linear and non-parametric fittings (data not shown).Therefore, double derivative spectrophotometry does not provideexperimental evidences of PSA partitioning into bilayers of DPPC,at 25 ◦C. However, this methodology can not discriminate betweenadsorbed and aqueous PSA, because there are no solvatochromiceffects in this case. To ascertain this issue, we used phase sepa-ration methodology, through centrifuging liposome suspensionswith added PSA, to evaluate its partition into the gel phase DPPC.Although not unequivocal, the fact that the concentration of PSAin the supernatant remained unaltered (within the experimentalerror), supports the non-observation of partition into the gel phaseDPPC.

The Nernst partition constant of PSA into POPC MLV at 25 ◦C,Kp = 6.9×103, is consistent with comparable values obtained forother partitioning molecules (Kp≈5×103, at room temperature)using this method (Kitamura et al., 1995; Omran et al., 2002; Polaet al., 2004; Takegami et al., 2005; Ferreira et al., 2005), and for

similar anionic amphiphiles through diverse methodologies (Tanet al., 2002; Cocera et al., 2004; Hildebrand et al., 2004; Keller etal., 2006). What’s more, our findings are in accordance with thefree-volume model of membrane partitioning (De Young and Dill,1988; Gennis, 1989; Xiang and Anderson, 1995), since the parti-tion coefficient of SDS into POPC bilayers (Keller et al., 2006) ishigher than the value obtained here for PSA, as the moleculararea of SDS (30 A2) is lower than the calculated value (≈40 A2)for PSA (Edward, 1970). Surprisingly at a first glance, we foundincreasing Kp values in parallel with the proportion of POPS andPOPG in the mixtures (Table 1). For mixtures with 5 mol% of POPSthe partition increases to Kp = 3.3×104, and it is even higher for10 mol% of POPS, Kp = 7.4×104, while the corresponding valuesof �Dmax varied inversely (Table 1). Ionic strengths until 0.2 MNaCl, bestow statistically identical values with 5 and 10 mol% ofPOPS, as when using MilliQ water. Partition into POPC LUV andinto LUV containing from 5 until 10 mol% of POPS, endow valuesfor Kp, within experimental error, reproducing those obtained forMLV, e.g. Kp = 6.5×103 for pure POPC, until Kp = 7.1×104 for POPCmixed with 10 mol% of POPS. Taken together, the results obtained

Table 1Retrieved values for Kp of PSA, at 25 ◦C, into MLV vesicles of defined phospholipidcompositions, and respective �Dmax

MLV composition Kp �Dmax

POPC 6.9×103 4.7×10−3

POPC-POPS (95-5, mol%) 3.3×104 8.8×10−4

POPC-POPS (90-10, mol%) 7.4×104 5.5×10−4

POPC-POPG (95-5, mol%) 3.5×104 5.3×10−4

POPC-POPG (90-10, mol%) 7.1×104 3.0×10−4

84 M. Manuel, J. Martins / Chemistry and P

Fig. 4. Fraction (�D/�Dmax) of PSA within the phospholipid bilayers at 25 ◦C, as afunction of phospholipid concentration, [L], and for the following compositions ofMLV: (a, �) POPC, (b, �) POPC-POPS (95-5, mol%), (c, �) POPC-POPS (90-10, mol%).The trend lines are included only for purposes of eye guiding.

for the partition of PSA into the described model membrane sys-tems and for the carefully chosen physical–chemical conditions areon average: Kp = 6.7×103 for pure zwitterionic POPC, Kp = 3.4×104

for mixed zwitterionic/anionic with 5 mol% POPS, and Kp = 7.3×104

for 10 mol% POPS.

3.3. Extent of partitioning

The ratio between the various experimental values of �D, andthe retrieved values for the corresponding values of �Dmax, gives adirect estimate of the fraction of PSA partitioned into lipid bilayers(�D/�Dmax ∝ [S]L/[S]T). A higher fraction of PSA partitioned intobilayers is observed in parallel with the exposed fraction of lipidamount in MLV (as well as using LUV), and with the augmenting ofthe POPS content mixed with POPC (Fig. 4). The fact that the plot of�D/�Dmax vs. [L] displays a typical isotherm variation reinforcesthe consistency of the partition analysis undertaken. It must beemphasized that the exclusive purpose of this plot is to estimate theextent of partitioning. The lower extent of partition is observed forPOPC, in which the maximum fraction of PSA partitioned is around0.4, for 150 �M in the lipid concentration (PSA/phospholipid molarratio of 0.013). The partitioned fraction reaches values close to 1

with a proportion of 10 mol% of POPS (molar ratio of 0.033).

3.4. Lamellarity of liposomes and ionic strength

The lamellarity of liposomes can adversely influence the deter-minations and analysis of partition experiments (Ferreira et al.,2005), therefore the use of unilamellar vesicles should be preferred.For extruded LUV, even for fixed high pressures and filters withpore size of 0.05 �m, depending on the device, the mean lamel-larity can be higher than 1 (Berger et al., 2001). Only SUV wouldgrant an homogeneous population of strictly unilamellar vesicles;however, the small radius of curvature results in disadvantageousheterogeneous molecular packing and asymmetric transversal dis-tribution of lipid mixtures (Gennis, 1989). The measurement ofrepulsive forces in zwitterionic and mixed zwitterionic/anionicmultibilayers (Rand and Parsegian, 2005) established that, until10% of the anionic component, there are no evidences of alterationsin size and lamellarity between zwitterionic and mixed zwitteri-onic/anionic MLV. Therefore, MLV can be used accordingly, as longas the due attention is paid to the exposed fraction of bilayers(Szoka and Papahadjopoulos, 1980). In order to critically evalu-

hysics of Lipids 154 (2008) 79–86

ate the outcomes from MLV, we used extruded oligolamellar LUV,whose vesicle populations are well characterized (Berger et al.,2001; Patty and Frisken, 2003), and the coherence of the resultsobtained from the two actually different model membranes, renderthe partitioning analysis justly consistent.

Another factor that may influence the phase behaviour ofcharged lipid bilayers is the ionic strength in the aqueous phase,and since we choose to use MilliQ water, the correspondent analy-sis of influences that putatively arise from this parameter has beenundertaken. Fixing the ionic strength, for example, at the bench-mark value of 100 mM NaCl, we obtained, within experimentalerror, the same values for proportions of 5 and 10 mol% of POPS,as when using MilliQ water. In general, only ionic strengths of0.8–1 M NaCl would screen out electrostatic interactions (Rand andParsegian, 2005). This way, we were not able to scrutinize any effectarising from the usual values for ionic strength.

3.5. Interpretation of the partition processes

To understand the partition of anionic amphiphiles into fluidzwitterionic membranes, one must first recognize that this pro-cess simply reallocates the aqueous partitioning molecules. Theseare inserted in lipid bilayers due to the hydrophobic effect, thepyrenyl moiety residing aligned within the organized portion of themethylenic chain palisade, remaining the sulfonate group anchoredin the interface headgroup region exposed to water (Kachel et al.,1998). The outer sodium counterions do not exert any additionalscreening in the bilayer interface, since they are present at stoi-chiometric amounts, namely with PSA and anionic phospholipids.Additionally, given that the concentration of counterions is higheroutside of vesicles, it is likely that flip-flop of PSA into the innermonolayer is hindered, due to the resulting electrostatic imbalance,since the permeability of Na+ across lipid bilayers is insignificant toensure a symmetric distribution of charges in both sides.

In the last decades, there have been unequivocal evidences thatpartition of anionic species into negatively charged amphiphilicstructures does occur, from the PSA partitioning into non-ionicand anionic micelles (Turro and Kuo, 1987), until the sulfoneph-talein dyes partition into anionic micelles (Saikia et al., 2003).Furthermore, the partition of sodium bile salts cholate and deoxy-cholate is found increased when comparing from DPPC SUV,to SUV of DPPG (1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)]), the bilayers being in the fluid phase in both cases(Hildebrand et al., 2004). Some works (Cocera et al., 2004;

Hildebrand et al., 2004) established that the main effect involved inthe incorporation into anionic liposomes of amphiphilic molecules,either anionic or polar, is of hydrophobic nature. At least for the lowproportions of anionic phospholipids in mixed bilayers, used as wellin the present studies, electrostatic interactions play a minor role.

In the absence of calcium ions and above Tm of the mixed bilayersystem, the PC/PS mixtures display an homogeneous fluid phaseat least until 10 mol% of the anionic component, as establishedby phase diagrams (Silvius and Gagne, 1984), NMR (Roux et al.,2000), atomic force microscopy (Spurlin and Gewirth, 2006), andcomputational simulations (Rodrıguez et al., 2007). Hence, beingensured that the mixed bilayers are in the fluid phase, electrostaticfactors, surface density and elastic properties must be consideredto comprehend the increased partition of PSA into mixed zwit-terionic/anionic bilayers. The partition of anionic solutes into PCbilayers is certainly increased by the positive internal dipole poten-tial (Gennis, 1989), but its effects in PC/PS mixtures should be nearlyequivalent to PC bilayers. Theoretical calculations on the distribu-tion of dipole potential and charge density along the bilayer normal(Polyansky et al., 2005) concluded that the presence of the cor-responding counterions render the overall electrostatic properties

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M. Manuel, J. Martins / Chemistry

of bilayers incorporating negative lipids, similar to those of zwit-terionic bilayers. Accordingly, the comprehension of our resultsmust be focused in the free-volume properties of mixed zwitte-rionic/anionic bilayers and asymmetric interfacial stress resultingfrom intercalation of amphiphiles (Traıkia et al., 2002). There is anelectrostatic repulsion between the phosphates of the anionic andzwitterionic phospholipids, and adding PS to PC bilayers tilts the PChead groups, because the negative charge pulls the P−–N+ dipoleof PC backward toward the bilayer interior (Scherer and Seelig,1989; Macdonald et al., 1991), and these stereochemical changesthus expand the free-volume in bilayers, in comparison with zwit-terionic bilayers. However, the magnitude of this effect does notseem sufficient to solely explain the increased partition observed.The increased occurrence of cavities of critical dimensions forsolute insertion (De Young and Dill, 1988; Gennis, 1989; Xiang andAnderson, 1995), should be understood in terms of morphologi-cal changes in bilayers (Mui et al., 1995), induced by asymmetricinsertion of PSA. The augmented bending of bilayers (extension ofthe outer monolayer and compression of the inner layer) results ina higher interfacial area in the outer monolayer, allowing a supe-rior amount of free-volume. Comparing with POPC, this effect islikely amplified in POPC/POPS bilayers, because of the extent ofelectrostatic repulsions at the level of the head-groups interfaceis now greater than in pure zwitterionic bilayers. In agreement,the retrieved �Dmax values decrease with increasing POPS con-tent (see Table 1). From the definition �Dmax = (d2εL/d�2)[PSA]T,where [PSA]T is the maximum concentration of PSA in the bilayers,and since changes in d2εL/d�2 should be numerically rather smalland the number of PSA molecules is kept constant, the increase ofthe free-volume in the bilayer should be controlling the decreasein �Dmax, e.g. an increase in the free-volume lowers the values of[PSA]T, and accordingly diminishes �Dmax. This can be reinforcedfrom the fact that the value of Kp increases around five times frompure POPC to mixed 5 mol% POPS, and for 10 mol% of POPS, is morethan twice the value observed for half the proportion of POPS.

4. Conclusions

The purpose of this study was to analyze the equilibrium parti-tion at 25 ◦C, of a model anionic amphiphile, the fluorescent probePSA, into bilayers composed by zwitterionic (fluid POPC and gelDPPC) and by defined fluid mixtures of zwitterionic/anionic phos-pholipids (POPC/POPS and POPC/POPG). We have taken advantageof the second derivative UV–vis spectrophotometry capabilities, to

determine partition constants as a function of the varied composi-tions of bilayers. We used concentrations of PSA in the �M range(evaluated as adequate for practical reasons and sensitivity for theUV absorption quantification), and the total phospholipid concen-tration varied from the �M until the mM range (to control theconstant background effects from light scattering, in the absorp-tion). We used MLV and LUV of several compositions, including purePOPC and pure DPPC vesicles, as well as mixtures of POPC with 5, 10and 20 mol% of POPS, and mixed POPC with 5 and 10 mol% POPG.The Nernst partition constant into pure POPC bilayers at 25 ◦C isKp = 6.9×103. In accordance with established findings, we foundincreasing Kp values in parallel with narrow POPS (and POPG, atsome extent) content in bilayers, proposing additionally a suit-able interpretation based on the interfacial properties of mixedzwitterionic/anionic phospholipid bilayers. This way, the analysisof partitioning of PSA into fluid neutral and negatively chargedlipid bilayers, allows the establishment of the experimental condi-tions for which the process occurs, together with its extension. Thisprovides the necessary and sufficient characterization of the parti-tion process of PSA into fluid phospholipid bilayers, to undertakefurther dynamical and/or structural studies in model membranes

hysics of Lipids 154 (2008) 79–86 85

using this fluorescent probe. Ensuing, it is also worthwhile toemphasize the necessity to carefully choose the experimental con-ditions to conduct partitioning studies of anionic amphiphiles intomixed zwitterionic/anionic bilayers, as well as the resulting elasticchanges in lipid bilayers.

Acknowledgements

We thank Professor Eurico Melo for valuable discussions andsuggestions, and Professor Manuel Aureliano for beneficial consid-erations and technical guidance.

This work was supported by the Fundacao para a Ciencia e aTecnologia (FCT), Portugal, through project POCTI/BCI/46174/2002,being M.M. recipient of a Ph.D. Grant (SFRH/BD/40671/2007).

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