Interactions of oxyethylenated nonylphenols with liposomes mimicking the stratum corneum lipid composition

Colloids and Surfaces A: Physicochemical and Engineering Aspects 145 (1998) 83–91

Interactions of oxyethylenated nonylphenols with liposomes mimicking the stratum corneum lipid composition A. de la Maza *, O. Lopez, L. Coderch, J.L. Parra Consejo Superior de Investigaciones Cientı´ficas (C.S.I.C.), Centro de Investigacio´n y Desarrollo (C.I.D.), Departamento de Tensioactivos, Calle Jorge Girona 18–26, 08034 Barcelona, Spain Received 13 December 1997; accepted 29 July 1998 Abstract The interactions of oxyethylenated nonylphenols (ethylene oxide units ( EO) average between 5 and 30) with liposomes modeling the stratum corneum lipid composition were investigated. Liposomes were formed by a lipid mixture containing 40% ceramides, 25% cholesterol, 25% palmitic acid and 10% of cholesteryl sulfate. The surfactant/lipid molar ratios (Re) and the bilayer/aqueous phase partition coefficients (K ) were determined at three interaction levels: 50% release of the 5(6) carboxyfluorescein (CF ) trapped into the vesicles, saturation, and complete liposome solubilization. The fact that the free surfactant concentrations for each surfactant tested were at sublytic and lytic levels lower than and similar to its critical micelle concentration (in the experimental working medium) indicated that these interactions were mainly governed by the action of surfactant monomers and the formation of mixed micelles respectively. The nonylphenols with 15 EO units always showed the highest activity on SC bilayers ( lowest Re values), whereas that with 5 EO units exhibited the highest degree of partitioning into liposomes or affinity with these bilayer structures (highest K values). Different trends in the interaction of nonylphenols with SC and phosphatidylcholine (PC ) liposomes were observed when comparing the present Re and K parameters with those reported for PC ones. Thus, whereas SC liposomes were more resistant to the action of nonylphenols, the degree of partitioning of these surfactants into SC bilayers was always greater than that reported for PC ones. © 1998 Elsevier Science B.V. All rights reserved. Keywords: Stratum corneum liposomes; Oxyethylenated nonylphenols; Permeability alterations and bilayer solubilization; Carboxyfluorescein release; Static light-scattering; Surfactant/stratum corneum lipids molar ratios; Surfactant partition coefficient

Abbreviations: SC=stratum corneum; TRIS=Tris-(hydroxymethyl )-aminomethane; NP( EO) =nonylphenol oxyethyX lenated with x average in ethylene oxide units (x=5, 10, 15, 20, 30); EO=ethylene oxide; CF=5(6)-carboxyfluorescein; Re= effective surfactant/lipid molar ratio; Re % =effective 50 CF surfactant/lipid molar ratio for 50%CF release; Re = SAT effective surfactant/lipid molar ratio for bilayer saturation; Re =effective surfactant/lipid molar ratio for bilayer SOL solubilization; S =surfactant concentration in the aqueous W =surfactant concentration in the aqueous medium; S W,50%CF * Corresponding author. Tel: +34 3 400 61 61; fax: +34 3 204 59 04.

medium for 50%CF release; S =surfactant concentration W,SAT in the aqueous medium for bilayer saturation; S =surfactant concentration in the aqueous medium for W,SOL bilayer solubilization; S =surfactant concentration in the B bilayers; K=bilayer/aqueous phase surfactant partition coefficient; K % =bilayer/aqueous phase surfactant partition 50 CF coefficient for 50% CF release; K =bilayer/aqueous phase SAT surfactant partition coefficient for bilayer saturation; K =bilayer/aqueous phase surfactant partition coefficient for SOL bilayer solubilization; PC=phosphatidylcholine; Cer=ceramides type III; Chol=cholesterol; PA=palmitic acid; Cholsulf=cholesteryl sulfate; PTT=phase transition temperature; PI=polydispersity index; CMC=critical micellar concentration; r2=regression coefficient; SLS=static light-scattering

0927-7757/98/$ – see front matter © 1998 Elsevier Science B.V. All rights reserved. PII S0 9 2 7- 7 7 5 7 ( 9 8 ) 0 06 9 5 - 5

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1. Introduction The permeability barrier of the skin, which prevents penetration of substances from the environment is located in the horny layer (stratum corneum, SC ), which is a compact mass of metabolically inactive cells, embedded in an extracellular matrix of non-polar continuous lamellar lipid layers [1,2]. In all cellular and intercellular membranes, such bilayer-forming lipids consist predominantly of phospholipids. However, SC has been shown to be virtually devoid of phospholipids, as a result of which its ability to form bilayers has proved to be somewhat surprising [3–6 ]. In order to find out whether SC lipids could form bilayers, Wertz and Abraham [7–9] prepared liposomes from lipid mixtures approximating the composition of SC lipids. These authors also investigated the action of sodium dodecyl sulfate on these liposomes to study the deleterious effect of this surfactant on human skin [10]. Nonionic surfactants are considered to be suitable agents for solubilization and reconstitution of membrane proteins [11–16 ]. However, despite the characteristic surface adsorption properties in aqueous solutions exhibited by the oxyethylenated nonylphenols [17] and the steric stabilizing effect caused in liposome suspensions [18], these surfactants have been not used up to now in studies of biological membranes. A number of studies have been devoted to the understanding of the principles governing the interaction of surfactants with simplified membrane models such as phospholipid or stratum corneum liposomes [19–21]. This interaction leads to the breakdown of lamellar structures and to the formation of lipid–surfactant mixed micelles. A significant contribution in this area has been made by Lichtenberg [22], who postulated that the effective surfactant-to-lipid molar ratio (Re) producing solubilization of liposomes depends on the surfactant critical micelle concentration (CMC ) and on the bilayer/aqueous medium distribution coefficients (K ). We previously studied the interaction of nonylphenols with PC liposomes [23]. We also investigated the formation of liposomes using different mixtures of four commercially available synthetic

lipids approximating the composition of the stratum corneum and their sublytic interaction with different surfactants [24–26 ]. In the present work we seek to extend these investigations by characterizing the Re and K parameters of a series of nonylphenols when interacted with SC liposomes in order to establish a criterion for the evaluation of their activity on biological membranes.

2. Materials and methods Polydisperse nonylphenol polyethoxylated surfactants having an average of 5, 10, 15, 20 and 30 ethylene oxide groups per molecule and an active matter of 100% were prepared by Tenneco Espan˜a S.A. According to the manufacturer’s specifications nonylphenol was oxyethylated in an autoclave using a batch process (temperature of 170°C, ethylene oxide pressure of 50 psig) using 0.5% NaOH as catalyst. When the reaction had been completed, neutralization was performed with phosphoric acid followed by a final filtration process. These products are hereafter referred to as NP( EO) , NP( EO) , NP( EO) , NP( EO) and 5 10 15 20 NP( EO) . The homogeneity of these nonionic 30 compounds was verified by the graphs of surface tensions versus logarithm of surfactant concentration; only samples exhibiting sharp breaks were used. The iodometric titration of the EO groups was carried out [27] and the average of a few determinations gave the same results: NP(EO) 5 53.89%, NP( EO) 70.93%, NP( EO) 76.46%, 10 15 NP( EO) 85.39%, and NP( EO) 93.2%. Triton 20 30 X-100 was purchased from Rohm and Haas (Lyon, France). 5(6) carboxyfluorescein (CF ) was obtained from Eastman Kodak (Rochester, NY ) and further purified by a column chromatography [28]. Tris-(hydroxymethyl )-aminomethane ( TRIS) was obtained from Merck. TRIS buffer was prepared as 5 mM TRIS adjusted to pH 7.20 with HCl, containing 100 mM NaCl and supplemented with 110 mM CF when studying permeabilities. Polycarbonate membranes and membrane holders were purchased from Nucleopore (Pleasanton, CA). Reagent grade organic solvents, ceramides type III (Cer) and cholesterol (Chol ) were supplied

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by Sigma Chemical Co. (St Louis, MO) and palmitic acid (PA) (reagent grade) was purchased from Merck. Cholesteryl sulfate (Chol-sulf ) was prepared by reaction of cholesterol with excess chlorosulphonic acid in pyridine and purified chromatographically. The molecular weight of ceremide type III used in the lipid mixture was determined by low resolution fast atom bombardment mass spectrometry (FAB-MS ) using a Fisons VG Auto Spec Q (Manchester UK ) with a cesium gun operating at 20 kV. A molecular weight of 671 g mol−1 was obtained for most compounds of the ceremide type III used (Sigma). This value was used to calculate the molarity of the lipid mixture investigated.

determined by proton magnetic resonance (1H NMR) showed a value of 55–56°C [24]. The vesicle size distribution and the polydispersity index (PI ) of liposomes after preparation ( lipid concentration ranging from 0.5 to 10.0 mM ) was determined with dynamic light-scattering measurements using a photon correlator spectrometer (Malvern Autosizer 4700c PS/MV ). The studies were made by particle number measurement. The sample was adjusted to the appropriate concentration range with TRIS buffer and the measurements were taken at 25°C at a scattering angle of 90°.

2.1. Liposome preparation and characterization

The distribution of surfactant between lipid bilayers and aqueous media for a mixing of lipids (mM ) and surfactant (mM ) in dilute aqueous media obeys a partition coefficient K, given (in mM−1) by the equation [23]:

We have previously reported the formation of liposomes using a mixture of lipids modeling the SC composition (40% Cer, 25% Chol, 25% PA and 10% Chol-sulf ) [24], which were prepared following the method described by Wertz et al. [7]. After preparation vesicles were annealed at 60°C for 30 min and incubated at 25°C under N 2 atmosphere. To study the changes in the release of the CF trapped into liposomes, vesicles containing CF were freed of the unencapsulated dye by passage through Sephadex G-50 medium resin (Pharmacia, Uppsala, Sweden) by column chromatography [26 ]. The final volumes of liposomes were adjusted with TRIS buffer to provide a final lipid concentration ranging from 0.5 to 10.0 mM. The lipid composition and concentration of SC liposomes after preparation were determined using thin-layer chromatography coupled to an automated flame ionization detection system ( TLC–FID) (Iatroscan MK-5, Iatron Lab. Inc. Tokyo, Japan) [29]. In order to determine whether all the components of the lipid mixture formed liposomes, vesicular dispersions were analyzed for these lipids [29]. The dispersions were then spun at 140 000g at 25°C for 4 h to remove the vesicles [30]. The supernatants were tested again for these components. No lipids were detected in any of the supernatants. The phase transition temperature (PTT ) of SC lipid mixture forming liposomes was

2.2. Parameters involved in the interaction of surfactants with SC liposomes

K=Re/[S (1+Re)] (1) W where Re is the surfactant-to-lipid molar ratio in the bilayers (Re=S /L, S being the surfactant B B concentration in the bilayers (mM ) and L the lipid concentration (mM )), and S is the surfactant W concentration in the aqueous medium (mM ). Changes in the release of the CF trapped in SC lipid vesicles due to the action of surfactants were determined quantitatively by monitoring the increase in the fluorescence intensity of liposomes due to the CF released [23]. Fluorescence measurements were made with a Shimadzu RF-540 spectrofluorophotometer. The fluorescence intensity measurements were taken 60 min after adding the surfactant to liposomes at 25°C. This interval was chosen as the minimum period needed to achieve a constant level of CF release ( lipid concentration range used 0.5–5.0 mM ). The experimental determination of this interval is given in Section 3. Solubilization of SC liposomes ( lipid concentration 0.5–10.0 mM ) was determined by monitoring the SLS variations of the systems [23,31]. The solubilization process was characterized by two parameters termed Re and Re that corresSAT SOL

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ponded to the surfactant/lipid molar ratios at which the surfactant: (i) saturated liposomes and (ii) led to a complete solubilization of liposomes [23]. SLS measurements were made 24 h after adding the surfactant to liposomes at 25°C [31,32] using the spectrofluorophotometer Shimadzu RF-540 at 25°C with both monochromators adjusted to 500 nm. This time was chosen as the optimum period at which a complete equilibrium between the surfactants and liposomes was reached. The assays were carried out in triplicate and the results given are the average of those obtained. The determination of Re (Re % , 50 CF Re and Re ) and S (S ,S and SAT SOL W W,50%CF W,SAT S ) was carried out on the basis of the linear W,SOL dependence between the surfactant concentrations needed to achieve 50% CF release, to saturate or to solubilize liposomes and the lipid concentration in liposomes (L) which can be described by the equation: S =S +ReL (2) T W where S is the total surfactant concentrations, Re T and the aqueous surfactant concentration S are W the slope and the ordinate at the origin (zero lipid concentration).

3. Results and discussion After preparation the vesicle size distribution varied very little, showing in all cases a similar value of about 200 nm (PI lower than 0.1), thereby indicating that the size distribution was very homogeneous. The size of vesicles after the addition of equal volumes of TRIS buffer and equilibration for 24 h showed in all cases values similar to those obtained after preparation, with a slight increase in the PI (between 0.12 and 0.14). Hence, the liposome preparations were stable in the absence of surfactant under the experimental conditions used. 3.1. Parameters involved in the surfactant–SC liposomes interaction We have previously determined the suitable sonication temperature of the lipid mixture studied by

preparing liposomes at temperatures approximating its PTT (55–56°C ). It was found that temperatures exceeding this value by more than 10°C caused alterations in Cer and Chol-sulf. Hence, lipid mixture was sonicated at 60°C. It is known that, in surfactant/lipid systems, complete equilibrium may take several hours [22,31]. However, in sublytic interactions a substantial part of the surfactant effect takes place about 30 min after its addition to the liposomes [33]. To determine the time needed to obtain a constant level of CF release of liposomes, a kinetic study of the interaction of the nonylphenols investigated with SC liposomes was carried out. Liposomes were treated with a constant sublytic surfactant concentration and subsequent changes in CF release were studied as a function of time. The curves obtained for each surfactant tested at two concentrations (0.1, and 0.8 mM ) and using liposomes at two lipid concentrations (0.5 and 5.0 mM ) are shown in Fig. 1, parts (a) and (b), respectively. It may be seen that about 60 min was needed to achieve a constant level of CF release in all cases. Hence, CF release changes were studied 60 min after addition of surfactants to liposomes at 25°C. This finding contrasts with that reported for the interaction of these surfactants with PC liposomes, where the time needed to obtain a constant CF release level was clearly lower (about 40 min) [23]. The fact that CF release curves versus time exhibited plateaux at different levels for the different lipid concentrations investigated (in accordance with the results reported for PC liposomes) could be attributable to the release of CF through holes, or channels, created in the membrane. The incorporation of surfactant into membranes may directly induce the formation of hydrophilic pores or merely stabilize transient holes, in agreement with the concept of transient channels suggested by Edwards et al. [11,34]. The differences in the surfactant-induced CF release kinetics in PC and SC liposomes could be related to the different PTT of lipids building these two liposomes, which affects the positional organization of lipid molecules and their polar heads as well as their mobility. The more hydrophilic nature of PC could also facilitate the formation of hydro-

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volumes of buffered surfactant solutions at various concentrations. The final surfactant concentration (mM ) was calculated from each mixture. Changes in the CF release were determined 60 min after surfactant addition at 25°C. The assays were also carried out in triplicate and the results given are the average of those obtained. The curves obtained for NP( EO) are given in Fig. 2. The surfactant 15 concentrations resulting in 50% CF release (S % ) for each surfactant tested were obtained 50 CF graphically and plotted versus lipid concentration ( Fig. 3). An acceptable linear relationship was (a)

(b) Fig. 1. (a) Time curves of the release of CF trapped in SC liposomes caused by the addition of a constant surfactant concentration (0.1 mM ), the lipid concentration also remaining constant at 0.5 mM: %, NP( EO) ; $, NP( EO) ; #, 5 10 NP(EO) ; &, NP( EO) ; (, NP( EO) . (b) Time curves of 15 20 30 the release of CF trapped into SC liposomes caused by the addition of a constant concentration (0.8 mM ), the lipid concentration also remaining constant at 5.0 mM: %, NP( EO) ; 5 $, NP( EO) ; #, NP( EO) ; &, NP( EO) ; (, NP(EO) 10 15 20 30

philic pores due to the action of surfactants on the PC polar heads and the subsequent permeation of CF through these created holes [35]. The spontaneous release of CF trapped into SC liposomes in the absence of surfactant in this period of time was negligible. To determine the Re and S parameters at W a sublytic level, a systematic investigation of permeability alterations (50% CF release) of CF-containing liposomes was carried out for various SC lipid concentrations (0.5–5.0 mM ). Liposome aliquots (2.0 ml ) were mixed with equal

Fig. 2. Percentage changes in CF release of SC lipid liposomes ( lipid concentration ranging from 0.5 to 5.0 mM ) induced by the presence of increasing amounts of NP(EO) . Lipid con15 centratio: (, [L]=0.5 mM; ,, [L]=1.0 mM; %, [L]=2.0 mM; &, [L]=3.0 mM; #, [L]=4.0 mM; $, [PL]=5.0 mM.

Fig. 3. Surfactant concentrations resulting in 50% of CF release vs. lipid concentration of liposome suspensions: %, NP( EO) ; 5 $, NP(EO) ; #, NP(EO) ; &, NP( EO) ; (, NP( EO) . 10 15 20 30

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established in each case. The error bars are SD and represent the error of three replicates. The straight lines obtained corresponded to Eq. (2) were determined. from which Re % and S W,50%CF 50 CF These values including the regression coefficients (r2) of the straight lines and the surfactant CMC values previously reported in the experimental working medium [23] are given in Table 1. To determine the Re and S parameters at lytic W levels (saturation and solubilization of liposomes), SLS variations of the aggregates resulting in the interaction of the surfactants tested with SC liposomes were studied for various lipid concentrations. The curves for NP(EO) (SC lipid 15 concentration 1.0–10.0 mM ) are given in Fig. 4. The addition of surfactant led to an initial increase and a subsequent fall in the SLS intensity up to a constant value for complete bilayer solubilization was achieved. The curves obtained for the other nonylphenols investigated showed similar trends (results not shown). The SLS behavior is in similar to that reported for the interaction of these surfactant with PC liposomes [23], although showing in all cases a more pronounced initial SLS increase. The surfactant concentrations producing 100% (S ) and 0% (S ) of SLS in the system were SAT SOL obtained for each lipid concentration by graphical methods. The arrows A and B (curve for SC

Fig. 4. Percentage changes in static light-scattering of SC liposomes ( lipid concentration ranging between 1.0 and 10.0 mM ) induced by the presence of increasing amounts of NP( EO) . 15 Lipid concentration: (, [L]=1.0 mM; ,, [L]=3.0 mM; %, [L]=5.0 mM; &, [L]=6.0 mM; #, [L]=8.0 mM; $, [PL]= 10.0 mM.

concentration 10.0 mM, Fig. 4) correspond to these values. When plotting the surfactant concentrations thus obtained versus lipid concentration, curves were obtained in which an acceptable linear relationship was also established in each case. The results obtained for 100% and 0% of SLS are plotted in Fig. 5, parts (a) and (b), respectively. The error bars are also SD and also represent the error of three replicates. These straight lines corresponded to Eq. (2) from which Re , Re , and SAT SOL S ,S were determined. These values W,SAT W,SOL including the r2 of the straight lines are also given in Table 1. The free surfactant concentrations for each sur,S and S ) were factant tested (S W,SOL W,50%CF W,SAT at sublytic and lytic levels lower than and similar to its CMC in all cases. From these findings we may assume that the surfactant/liposomes sublytic and lytic interactions were mainly ruled by the action of surfactant monomers and by the formation of mixed micelles respectively, in agreement with our previous results involving the interaction of these surfactants with PC liposomes [23]. As for the Re parameters, these values increased from Re % to Re , regardless of the average 50 CF SOL in EO units of the surfactant tested. Given that the surfactant ability to interact with liposomes is inversely related to the Re parameter, the maximum activity at the three interaction levels studied corresponded to the NP( EO) ( lowest Re values). 15 Comparison of the Re values with those reported for the interaction of these surfactants with PC liposomes [23] shows that the surfactant’s ability to interact with SC liposomes was always less (higher Re values) than that reported for PC ones. However, a similar influence of the surfactant hydrophilic moiety was detected in both cases at lytic level. At sublytic level the activity of these surfactants depended on the liposome composition. Thus, whereas the NP(EO) exhibited the 5 highest ability to produce 50% CF release on PC liposomes, a higher average of EO units was needed (hydrophilic moiety EO ) to obtain the 15 highest surfactant activity on SC liposomes. The surfactant partition coefficients between SC bilayers and aqueous medium at the three interaction steps investigated show that the NP( EO) 5 molecules had the highest degree of partitioning

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(a)

(b) Fig. 5. (a) Surfactant concentrations resulting in 100% static light-scattering of the liposome/surfactant systems vs. lipid concentration of liposome suspensions: %, NP( EO) ; $, 5 NP(EO) ; #, NP( EO) ; &, NP( EO) ; (, NP(EO) . (b) 10 15 20 30 Surfactant concentrations resulting in 0% static light-scattering of the liposome/surfactant systems vs. lipid concentration of liposome suspensions: %, NP( EO) ; $, NP( EO) ; #, 5 10 NP(EO) ; &, NP(EO) ; (, NP(EO) . 15 20 30

into bilayers (maximum K values), whereas the NP( EO) showed the lowest (minimum K values) 30 ( Table 1). Thus, the rise in the number of EO units resulted during the transition steps from mixed vesicles (sublytic level ) to mixed micelles ( lytic level ) in a drastic decrease in the surfactant affinity with these bilayer structures. Comparison of the present K values with those reported for the interaction of these surfactants with PC liposomes shows that the degree of partitioning of these surfactants into the SC bilayers (or bilayer affinity) was always greater (higher K values) than that for PC ones. However, the influence of the surfactant hydrophilic moiety on this affinity was similar in both cases in spite of the different bilayer compositions and properties [23]. The different liposome composition, degree of saturation and nature of polar heads appears to be responsible for these differences. If the Re and K values obtained are plotted as a function of the surfactant CMC the graphs shown in Figs. 6 and 7 are obtained. The Re parameters for the three interaction levels investigated showed a minimum in the range of CMCs between 0.050 and 0.075 mM, which corresponded approximately to the NP( EO) . This minimum 15 was specially pronounced for the Re parameter SOL and very slight for Re % . 50 CF The rise in the surfactant CMC also resulted in a marked decrease in the K % , K and K 50 CF SAT SOL parameters, especially at low CMC values (Fig. 7). Thus, in this CMC range the partitioning of these surfactants into SC liposomes decreased markedly as the surfactant CMC increased. Thus, although

Table 1 Surfactant to lipid molar ratios, partition coefficients and surfactant concentrations in the aqueous medium for 50% CF release, saturation and complete solubilization of SC liposomes resulting in the interaction of the oxyethylenated nonylphenols ( EO units average 5, 10, 15, 20, 30) with SC liposomes. The surfactant CMCs and the regression coefficients of the straight lines obtained are also indicated

NP(EO) 5 NP(EO) 10 NP(EO) 15 NP(EO) 20 NP(EO) 30

CMC (mM )

S W,50%CF

S W,SAT

S W,SOL

Re % 50 CF

Re SAT

Re SOL

K

0.025 0.040 0.065 0.087 0.130

0.009 0.014 0.025 0.035 0.048

0.022 0.035 0.060 0.083 0.124

0.025 0.041 0.065 0.087 0.130

0.130 0.128 0.123 0.137 0.182

0.60 0.45 0.39 0.44 0.88

1.34 1.06 0.91 1.18 2.67

12.78 8.15 4.39 3.45 3.20

50%CF

K

SAT

17.04 8.87 4.67 3.68 3.77

K

SOL

22.90 12.61 7.33 6.24 5.59

r2 (50%CF )

r2 (SAT )

r2 (SOL)

0.996 0.993 0.995 0.992 0.997

0.994 0.996 0.994 0.998 0.994

0.997 0.991 0.998 0.995 0.996

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(#) and Re ($) vs. Fig. 6. Variation of Re % (%), Re SAT SOL 50 CF the surfactant CMCs for the oxyethylenated nonylphenols with different average in EO units (EO units=5, 10, 15, 20, 30).

tuberculous infection, and with long chains it was stimulated. In general terms, different trends in the interaction of these surfactant with SC and PC liposomes may be observed when comparing the corresponding Re and K parameters. Thus, whereas SC liposomes appeared to be more resistant to the action of surfactant monomers the partitioning of these surfactants into SC structures was always greater than that for PC ones. Thus, although a greater number of surfactant molecules was needed to produce alterations in SC bilayers, these molecules showed increased affinity with these structures. However, a similar influence of the hydrophilic–lipophilic balance of each surfactant tested in the Re and K parameters (except for Re % ) could be observed for both bilayered 50 CF structures, in spite of their different lipid compositions and physico-chemical characteristics.

Acknowledgment We are grateful to Mr. G. von Knorring for expert technical assistance. This work was supported by funds from DGICYT (Direccio´n General de Investigacio´n Cientı´fica y Te´cnica) (Prog. no. PB94-0043), Spain.

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(#) and K ($) vs. the Fig. 7. Variation of K % (%), K SAT SOL 50 CF surfactant CMCs for the oxyethylenated nonylphenols with different average in EO units (EO units=5, 10, 15, 20, 30).

the maximum ability to interact with SC liposomes corresponded to the NP( EO) , the maximum 15 affinity with these bilayer structures (maximum K ) always corresponded to the NP( EO) . This selec5 tive behavior may be correlated with the findings reported by Hart et al. [36 ], who demonstrated that polyoxyethylene alkylphenols with short chains (Macrocyclon) inhibited experimental

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