- Email: [email protected]
sodium dodecyl sulfate mixtures in liposomes modeling the stratum corneum lipid composition
COLLOIDS
AND ELSEVIER
Colloids and Surfaces A: Physicochemicaland EngineeringAspects 133 (1998)253 260
A
SURFACES
Sublytic effects caused by C14-alkyl betaine/sodium dodecyl sulfate mixtures in liposomes modeling the stratum corneum lipid composition A. de la Maza a,,, j. Baucells
b, p.
Gonzalez c, J.L. Parra a
a Consejo Superior de Investigaciones Cientificas (C. S. I. C.), Centro de Investigacidn y Desarrollo (C. I. D. ), Departamento de Tensioactivos, Calle Jorge Girona 18-26, 08034 Barcelona, Spain b Universidad Autdnoma de Barcelona (U.A.B.), Facultad de Veterinaria, Bellaterra, 08193, Barcelona, Spain Transtechnics. S.L., Sant Guillem, 21, 08006 Barcelona, Spain Received 18 January 1997; accepted 14 July 1997
Abstract It is known that a reduction in the skin irritation caused by anionic surfactants occurs in the presence of amphoteric ones. However, the mechanisms of this reduction are far from understood, since a detailed description of the process has yet to be given. The interaction of tetradecyl betaine (C14-Bet)/sodium dodecyl sulfate (SDS) mixtures with a simplified skin membrane model were investigated. To this end, liposomes modeling the stratum corneum (SC) lipid composition (40% ceramides, 25% cholesterol, 25% palmitic acid and 10% cholesteryl sulfate) were used. The surfactant/lipid molar ratios Re and the bilayer/aqueous phase partition coefficients K were determined by monitoring the increase in the fluorescence intensity of liposomes due to the carboxyfluorescein (CF) released from the interior of vesicles. These surfactant mixtures showed at the two interaction levels investigated (50 and 100% CF release) higher ability to alter the release of the CF trapped into SC bilayers (lower Re values) than that exhibited by the anionic component (SDS), although always showing a lower degree of partitioning into these structures (lower K values). Comparison of these findings with those reported for the interaction of these mixtures with phosphatidylcholine (PC) liposomes shows that SC liposomes were more resistant to the action of these surfactant mixtures, their degrees of partitioning into SC bilayers also being clearly lower than those reported for PC ones. © 1998 Elsevier Science B.V. Keywords." Tetradecyl betaine/sodium dodecyl sulfate mixtures; Stratum corneum liposomes; Stratum corneum liposomes/surfactant interactions; Carboxyfluorescein release; Surfactant/stratum corneum lipids molar ratios; Surfactant partition coefficients
1. Introduction Liposomes are aqueous lipid dispersions organized as bilayers which are widely used as simplified membrane models [1-3]. A number of studies have been devoted to the understanding of the * Corresponding author. Tel: (+ 34) 3 400 61 61; Fax: (+ 34) 3 204 59 04. 0927-7757/98/$19.00 © 1998Elsevier ScienceB.V. All rights reserved. PH S0927-7757 (97) 00206-9
principles governing the interaction of surfactants with these structures [4-9]. This interaction leads to the breakdown of lamellar structures and the formation of lipid-surfactant mixed micelles. A significant contribution has been made by Lichtenberg et al. [10], who postulated that the surfactant/lipid molar ratio Re producing solubilization of liposomes depends on the surfactant critical micelle concentration (CMC) and on the
254
A. de la Maza et al. / Colloids SurJaces A: Physicochem. Eng. Aspects 133 (1998) 253 260
bilayer/aqueous medium distribution coefficients K, which depends on the combination of amphiphilic properties of the lipid and the surfactant. Zwitterionic surfactants have strong interaction with anionic surfactants in water [ 11]. The effect of the micellar solution phase of these mixtures in avoiding, or at least reducing, the level of anionic/protein interaction has been suggested by several workers as a way of reducing the potential irritation of anionic surfactants [12,13]. Thus, a reduction in skin irritation by anionics has been reported by Rhein and Simion in the presence of amphoteric surfactants [14]. These authors attributed this reduction at two possible mechanisms. The formation of mixed micelles that sequester the irritating surfactant, preventing its interaction with the skin or, alternatively, the fact that the mild surfactant may compete with the irritating surfactant for binding sites on the skin's surface. In order to find out whether the lipids building the intercellular matrix of the stratum corneum (SC) could form bilayers, Wertz and coworkers prepared liposomes from lipid mixtures approximating the SC composition and studied the interaction of these liposomes with sodium dodecyl sulfate (SDS) to study its deleterious effect on human skin [15-17]. We previously studied the sublytic interaction of tetradecyl betaine (C14-Bet) and SDS when interacted individually or in mixtures with phosphatidylcholine (PC) liposomes [ 18 21 ]. We also investigated the formation and characterization of liposomes formed with mixtures of four commercially available synthetic lipids approximating the composition of the SC [22]. In the present work we extend these investigations by characterizing the Re and K parameters of C14-Bet/SDS mixtures when interacted with SC liposomes. This information could be useful in establishing a criterion for the evaluation of the skin irritation capacity of these surfactant mixtures at submicellar concentrations.
2. Materials and methods N-Tetradecyl-N,N-dimethylbetaine (C14-Bet) was specially prepared by Albright and Wilson,
Ltd. (Warley, West Midlands, UK); the active matter was 30% in water and the amino-free content was 0.50%. SDS was obtained from Merck and further purified by a column chromatographic method [23]. Piperazine-l,4-bis(2-ethanesulfonic acid) (PIPES buffer) obtained from Merck (Darmstadt, Germany) was prepared as 20mM PIPES buffer adjusted to pH 7.20 with NaOH, containing 110 mM Na2SO 4. The starting material 5(6)-carboxyfluorescein (CF), was obtained from Eastman Kodak (Rochester, NY) and further purified by a column chromatographic method [24]. Polycarbonate membranes and membrane holders were purchased from Nucleopore (Pleasanton, CA). Reagent-grade organic solvents, ceramides type III (Cer), cholesterol (Chol) and palmitic acid (PA) were supplied by Sigma Chemical Co. (St Louis, MO). Cholesteryl sulfate (Chol-sulf) was prepared by reaction of cholesterol with excess chlorosulfonic acid in pyridine and purified chromatographically. The molecular weight of ceramide type III used in the lipid mixture was determined by low-resolution fast atom bombardment mass spectrometry using a Fisons VG Auto Spec Q (Manchester, UK) with a cesium gun operating at 20 kV. From this analysis a molecular weight of 671 g was obtained for the majority compound of the Cer used (Sigma), this value being used to calculate the molarity of the lipid mixture investigated [22]. The lipids of the highest purity grade available were stored in chloroform/methanol 2:1 under nitrogen at - 2 0 ° C until use. 2.1. Liposome preparation and characterization
We previously reported the formation of liposomes formed by a mixture of lipids modeling the SC composition (40% Cer, 25% Chol, 25% PA and 10% Chol-sulf) [22] following the method described by Wertz et al. [15]. Liposomes (lipid conc. 0.5-5.0 mM) were prepared in PIPES buffer containing 100mM CF. Vesicles were freed of unencapsulated dye by passage through Sephadex G-50 medium resin (Pharmacia, Uppsala, Sweden) by column chromatography to study the changes in the release of the CF trapped in liposomes due to the action of surfactant mixtures.
A. de la Maza et al. / Colloids Surfaces A: Physicochem. Eng. Aspects 133 (1998) 253~60
The bilayer lipid composition after liposome preparation was determined using thin-layer chromatography coupled to an automated flame ionization detection system [22,25]. In order to find out whether all the components of the lipid mixture formed liposomes, vesicular dispersions were analyzed for these lipids [25]. The dispersions were then spun at 140 000g at 25°C for 4 h to remove the vesicles [26]. The supernatants were tested again for these components. No lipids were detected in any of the supernatants. Analyses of proton magnetic resonance (1H NMR) were carried out at temperatures ranging from 25°C to 90°C to determine the phase transition temperature of the lipid mixture forming liposomes, which showed a value of 55-56°C [22]. The vesicle size distribution and the polydispersity index (PI) of liposome suspensions after preparation were determined with dynamic lightscattering measurements using a photon correlator spectrometer (Malvern Autosizer 4700c PS/MV) [27]. After preparation, the vesicle size distribution varied very little, always showing a similar value of about 200nm (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 PIPES buffer and equilibration for 60 min showed in all cases values similar to those obtained after preparation, with a slight increase in the PI (between 0.10 and 0.12). Hence, liposome preparations appeared to be reasonably stable in the absence of surfactant mixtures under the experimental conditions used. 2.2. Parameters involved in the interaction o f surfactant mbctures with S C liposomes
In the analysis of the equilibrium partition model proposed by Schurtenberger et al. [28] for bile salt/lecithin systems, Lichtenberg et al. [10] and Almog et al. [26] have shown that for a mixing of lipids in dilute aqueous media, the distribution of surfactant between lipid phase and aqueous media obeys a partition coefficient K ( m M - 1), given by [20] K = Re/[Sw ( 1 + Re)]
( 1)
where Re is the effective molar ratio of surfactant
255
to lipid in the bilayers (Re = SB/L, SB(mM) being the concentration of surfactant in the bilayers ) and Sw(mM) is the surfactant concentration in the aqueous medium. Given that the range of lipid concentrations used in the mixture is similar to that used by Almog et al. to test their equilibrium partition model, the K parameter has been determined using this equation. The determination of these parameters can be carried out on the basis of the linear dependence existing between the surfactant concentrations required to achieve 50% and 100% CF release and the SC lipid concentration [L], which can be described by the equations ST,5Oo/oCF = SW,SOO/oCF -'~ Resoo/ocF[L ]
(2)
ST, IO0%CF : Sw, IO0°/oCF "t- Reloo%cv[L]
(3)
where ST,5OnCF and Sv,~o0o/ocvare the total surfactant concentrations. The surfactant/lipid molar ratios Reso~cF and Relooo/ocv and the aqueous surfactant concentrations Sw.5o%cF and Sw,~oOo/ocv are in each curve respectively the slope and the ordinate at the origin (zero lipid concentration) [21]. Changes in the release of the CF trapped in SC vesicles were determined quantitatively by monitoring the increase in the fluorescence intensity of liposomes due to the CF liberated [21,24]. The fluorescence intensity measurements were taken 60 rain after adding the surfactant solution to liposomes at 25°C using a Shimadzu RF-540 spectrofluorophotometer (Kyoto, Japan). This interval was chosen as the minimum period of time needed to achieve a constant level of CF release for the lipid concentration range used. The experimental determination of this interval is given in the Section 3. The CMC of C~4-Bet/SDS mixtures was determined from the abrupt change in the slope of the surface tension values vs. surfactant concentration [21]. The values obtained for each mole fraction of the zwitterionic component Xzwitte r a r e given in Table 1. 3. Results and discussion
In preliminary experiments we determined the suitable sonication temperature of the lipid mixture
256
A. de la Maza et al. / Colloids Surfaces A." Physicochem. Eng. Aspects 133 (1998) 253 260
Table 1 Surfactant to lipid molar ratios Re, partition coefficients K and surfactant concentrations in the aqueous medium Sw resulting in the interaction (50% and 100% CF release) of C14-Bet/SDS mixtures for different mole fractions of the zwitterionic surfactant Xzwitto,. The CMCs of each surfactant mixture tested are also included, together with the regression coefficients of the straight lines obtained Xzwitte r
0 0.2 0.4 0.6 0.8 1.0
CMC (mM)
Sw.soo/,cv SW,lOOO/oCF Reso*/oCV (mM) (raM) (mol tool -1)
Relooo/,cv (mol tool -1)
0.500 0.175 0.090 0.080 0.115 0.190
0.083 0.048 0.029 0.028 0.051 0.102
1.0 0.224 0.108 0.100 0.172 0.349
0.289 0.110 0.061 0.058 0.089 0.160
0.350 0.125 0.063 0.057 0.115 0.291
investigated b y p r e p a r i n g l i p o s o m e s at t e m p e r atures a p p r o x i m a t i n g its phase t r a n s i t i o n tempera t u r e ( 5 5 - 5 6 ° C ) . It was f o u n d t h a t t e m p e r a t u r e s exceeding this t e m p e r a t u r e b y m o r e t h a n 10°C caused n o t i c e a b l e a l t e r a t i o n s in Cer a n d Chol-sulf. A s a consequence, the lipid m i x t u r e was s o n i c a t e d at 60°C. To d e t e r m i n e the time n e e d e d to o b t a i n a constant level o f C F release o f l i p o s o m e s in the lipid c o n c e n t r a t i o n range investigated, a kinetic s t u d y o f the i n t e r a c t i o n o f C14-Bet/SDS m i x t u r e s with SC l i p o s o m e s was carried o u t at different Xzwitte r. L i p o s o m e s at lipid c o n c e n t r a t i o n s o f 1.0 m M a n d 5.0 m M were t r e a t e d with s u r f a c t a n t m i x t u r e s at c o n c e n t r a t i o n o f 0.1 m M a n d 0.6 m M respectively. The s u b s e q u e n t changes in C F release for each Xzwitte r were studied as a function o f time. The results o b t a i n e d are i n d i c a t e d in Figs. 1 (a) a n d ( b ) respectively. A b o u t 60 m i n was n e e d e d to achieve a c o n s t a n t level o f C F release in all cases. This finding c o n t r a s t s with t h a t r e p o r t e d for the interaction o f these s u r f a c t a n t mixtures with P C liposomes [21], where the time n e e d e d to o b t a i n a c o n s t a n t C F release level was always lower (40 rain). T h e fact t h a t C F release curves vs. time exhibited p l a t e a u x for different Xzwitte r (in accordance with the results r e p o r t e d for P C liposomes) c o u l d be a t t r i b u t a b l e to the release o f the fluorescent dye t h r o u g h holes, o r channels, created in the m e m b r a n e . The i n c o r p o r a t i o n o f s u r f a c t a n t m o n o m e r s to m e m b r a n e s m a y directly induce the f o r m a t i o n o f h y d r o p h i l i c p o r e s in these structures o r merely stabilize transient holes, in a g r e e m e n t with the c o n c e p t o f transient channels suggested
100
KSOO/,CF
Klooo/ocv
r2
(mM -1)
(mM 1)
(50% CF)
r2 (100%CF)
3.12 2.31 2.04 1.93 2.02 2.21
1.73 1.66 1.60 1.56 1.65 1.617
0.994 0.998 0.994 0.991 0.995 0.997
0.996 0.993 0.997 0.998 0.997 0.994
• x = 1.0, o x = 0.4,
o x = 0.8, • x = 0.2,
• x = 0.6, ~ x = 0
80,
0
0
40 V
[3
t3
60
70
80
60
70
80
20
10
20
30
a
40 Time
50 [mini
100
80
60
40
20
10 b
20
30
40
50
Time [min]
Fig. 1. Time curves of the release of CF trapped in SC liposomes (lipid concentration: (a) 1.0 raM; (b) 5.0 raM) caused by the addition of a constant concentration of C14-Bet/SDS surfactant mixtures ((a)0.1 raM; (b)0.6 mM) at different mole fractions of the zwitterionic surfactant (Xzwitt~r).X = 1.0 ( I ) , X = 0 . 8 (D), X = 0 . 6 ( e ) , X = 0 . 4 (O), X=0.2 (I?), X=0 (V).
A. de la Maza et al. / Colloids Surfaces A: Physicochem. Eng. Aspects 133 (1998) 253-260
by Edwards and Almgren in the surfactant-mediated increase in PC membrane permeability [29,30]. The differences in the surfactant-induced CF release kinetics in both PC and SC liposomes could be correlated with the different gel-liquid crystal phase transition temperature of lipids building these two bilayer structures, which affects both the positional organization of lipid molecules and their polar heads as well as their mobility, and also affects the formation and stabilization of the aforementioned membrane holes. The spontaneous release of the CF trapped in liposomes in the absence of surfactant in this period of time was negligible. To determine the Re and Sw parameters a systematic investigation of CF release alteration caused by the addition of C14-Bet/SDS mixtures was carried out for various SC lipid concentrations (from 0.5 to 5 . 0 m M ) . To this end, liposome aliquots (2.0 ml, lipid concentration from 1.0 to 10.0mM) were mixed with equal volumes of buffered surfactant solutions at different Xzwitte r. 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 Xzwitter = 0.6 are given in Fig. 2. The C14-Bet/SDS concentrations resulting in 50% and
100% of CF release for each Xzwitte r tested were obtained graphically and plotted vs. lipid concentration. An acceptable linear relationship was established in each case. These results are plotted in Fig. 3(a) and (b) (50% and 100% CF release respectively). The error bars of these figures are the SD and represent the error of three replicates. The straight lines obtained corresponded to the Eqs. (2) and (3) from which Re and Sw were determined. These parameters, including the regression coefficients r 2 of the straight lines, are given in Table 1. The Sw values increased as the CF release rose, although showing smaller values than those for the surfactant CMCs in all cases. This finding
2"5r
• o
X = 1,0, X = 0,4,
[] X = 0.8, • X = 0.2,
f1
I] J[
•
[
o [L]= 3.0 mM,
'°°I40
I L l = 0. 5 I I ] ~
[] ILl = L 0 lnM~
• [L] = 2.0 I n M
• [LI= 4.0 raM,
v [L]= 5.0 mM
• v
X = 0.6,
X=0
I ]
N
~'--~2.0 ~1.5 ra0 1.0 0.5.
1
2 Lipid
/
257
3
4
[mM]
ii [[
I 5.0
..............
I
"Tr'r
.......
--
~
4.0
~
3.0 2.0
2O
0.01
1.0
0.1 Surfactant
1,0
1
[raM]
Fig. 2. Percentage changes in CF release of SC liposomes, (lipid concentration ranging from 0.5 to 5.0mM), induced by the presence of increasing concentrations of C14-Bet/SDS surfactant mixtures at the mole fraction of the zwitterionic surfactant of 0.6. Lipid concentrations: 0.5 mM ( I ) , 1.0 mM ([3), 2.0 mM (O), Y 0 m M (©), 4 . 0 m M ( T ) , 5.0 m M ( V ) .
b
2
3
4
Lipid [mM]
Fig. 3. Snrfactant concentrationsfor differentmole fractions of the zwitterionicsurfactant ( g z w i t t e r ) resultingin (a) 50% and (b) 100% CF release vs. lipid concentration of liposomes. X= 1.0 (I), X=0.8 ([1), X=0.6 (O), X=0.4 (©), X=0.2 (T), X= 0 (v).
258
A. de la Maza et al. / Colloids Surfaces A." Physicochem. Eng. Aspects 133 (1998) 253-260
suggests that the surfactant-liposome interaction must be ruled mainly by the action of surfactant monomers. These findings are in agreement with those reported for sublytic interactions of these surfactant mixtures with PC liposomes [21]. Furthermore, Sw showed the lowest values at the same mole fraction (Xzwitte r = 0.6), at which a minimum in the CMC of these mixtures took place (see Table 1). Thus, the lower the surfactant mixture CMC the lower the Sw concentration at which 50% and 100% CF release of SC liposomes occurred. As for the R e parameter, this value also increased as the CF release (percent) rose, regardless of the Xzwitte r. Given that the surfactant capacity to alter the release of the CF trapped in the bilayers is inversely related to the R e parameter, the activity of these mixtures was always higher than that for the anionic component (SDS) and the maximum activity (at both 50% and 100% CF release) corresponded to the Xzwitter=0.6. These findings contrast sharply with the reduced skin irritation caused by the micellar solutions of SDS in the presence of alkyl betaines [12-14]. Comparison of the R e values with those reported for the interaction of these surfactant mixtures with PC liposomes [21] shows that the ability of these mixtures to alter the release of the dye trapped into SC bilayers (at 50% CF release) was always slightly less (higher R e values) than that reported for PC bilayers, although showing similar tendencies with respect to the influence of the Xzwitte r. Thus, SC liposomes exhibited more resistance to the surfactant perturbations than PC ones at the sublytic interaction level investigated. This different characteristic could be explained by bearing in mind the more hydrophilic nature of PC; this could facilitate the permeation of water and some other molecules (as surfactants) in PC liposomes, either through the hydrophilic holes created by the surfactants on the PC polar heads or via formation short-lived complexes surfactants-PC polar heads, and subsequent transfer of the bilayers via flip-flop [31]. The K parameter for SC liposomes at both 50% and 100% CF release show that the surfactant molecules for Xzwitter=0.6 had the lowest degree of partitioning into bilayers (minimum K values).
Given that this Xzwitte r also showed a minimum in the surfactant CMC (see Table 1), a direct correlation could be established between the partitioning of these mixtures into SC bilayers and their CMCs. Thus, the lower the CMC of the surfactant mixtures, the higher their ability to alter the permeability of SC liposomes and the lower their degree of partitioning into these bilayer structures. The fact that these surfactant mixtures showed at 100% CF release lower K values than those for 50% may be explained by assuming that at low R e possibly only the outer vesicle leaflet was available for interaction with surfactant molecules, the binding of additional molecules to bilayers being hampered at slightly higher R e values. These findings are in agreement with those reported by Schubert et al. [32] for sodium cholate/PC liposomes and with our investigations involving the interaction of different anionic and non-ionic surfactants also with PC liposomes [20, 33]. An opposite tendency in the variation of K vs. the Xzwitte r w a s observed when comparing the present K values with those reported for the interaction of the same surfactant mixtures with PC liposomes [21]. Thus, whereas the degree of partitioning of these mixtures into SC bilayers (or bilayer affinity) showed a minimum for Xzwitte r = 0 . 6 , that for PC liposomes showed a maximum at the same mole fraction. Furthermore, the K values for SC liposomes were always smaller than those for PC ones. This opposite tendency may be explained by bearing in mind the different chain length of the lipid building these two bilayer structures, the degree of saturation and the nature of the polar heads. If the R e and K values obtained are plotted as a function of the Jf'zwitter, then the graphs shown in Fig. 4(a) and (b) are obtained. The curves of both parameters showed a minimum for X'zwitter=0.6, although K for 100% CF release showed almost a constant value in all cases. Thus, the Xzwitter=0.6 was a critical mole fraction at which the maximum activity of these surfactant mixtures and the minimum degree of partitioning into SC liposomes occurred simultaneously. The strong interaction of C14-Bet and SDS in water, which results in a greater surface activity of these systems than that attainable with any of the indivi-
A. de la Maza et al. / Colloids Surfaces A." Physicochem. Eng. Aspects 133 (1998) 253-260
1.0
l
I
50% CF Release o 100% CF Release •
259
with an opposite tendency with respect to the Yzwitter, than that reported for PC liposomes.
0.8
~a 0 . 6
Acknowledgment
0.4
We are grateful to Mr. G. von Knorring for expert technical assistance. This work was supported by funds from D.G.I.C.Y.T. (Direcci6n General de Investigaci6n Cientifica y T6cnica) (Prog. n ° PB94-0043), Spain.
0.2
Mole Fraction X I e 50% CF Release o 100% CF Release
5.0
References
4.0 "3
2
3.0 2.0
0
o
~ 1.o
0
b
0.2
0.4
0.6
0.8
1.0
Mole Fraction X
Fig. 4. (a) Surfactant to lipid molar ratios Re and (b) partition coefficients K for C14-Bet/SDS mixtures at 50% and 100% CF release vs. the mole fraction of the zwitterionic surfactant (Xzwitter). (O) 50% CF release and (©) 100% CF release.
dual surfactants of the mixture [11] seems to be responsible for this behavior. As mentioned above, these findings contrast with the reduced skin irritation caused by the micellar alkyl betaines/SDS solutions [12-14]. The fact that the CF release alterations were caused mainly by surfactant monomers (sublytic interactions) and that skin irritation appears to be closely connected with the surfactant/proteins interaction (corneocytes and corneocyte envelopes building the SC) could explain in part these differences. In general terms, different trends in the interaction of these surfactant mixtures with SC and PC liposomes may be observed when comparing the corresponding Re and K parameters [21]. Thus, SC liposomes were more resistant to the action of surfactant monomers and the partitioning of these mixtures into SC structures was always lower, and
[1] B. Sternberg, Liposomes as a model for membrame structures and structural transformations: a liposome album, in: D.D. Lasic, Y. Barenholz (Eds.) Nonmedical Applications of Liposomes, From Gene Delivery and Diagnostics to Ecology, vol. IV, CRC Press, Boca Raton, FL, 1995, pp. 271-297. [2] D.D. Lasic (Ed.) Liposomes: From Physics to Applications, Elsevier, Amsterdam, 1993, pp. 219-241. [3] R.C. Scott, R.H. Guy, J. Hadgraft (Eds.), Factors Affecting the Formation Morfology and Permeability of Stratum Corneum Lipid Bilayers "in vitro", IBC Technical Services Ltd., UK, 1990, pp. 110 180. [4] U. Kragh-Hansen, M. le Marie, J.P. N6el, T. GulikKrzywicki, J.V. Moiler, Biochemistry 32 (1993) 1648. [5] M.B. Ruiz, A. Prado, F.M. Gofii, A. Alonso, Biochim. Biophys. Acta 1193 (1994) 301. [6] A.I. Polozava, G.E. Dubachev, T.N. Simonova, L.I. Barsukov, FEBS Lett. 358 (1995) 17. [7] M. Silvander, G. Karlsson, K. Edwards, J. Colloid Interface Sci. 179 (1996) 104. [8] T. Inoue, Interaction of surfactants with phospholipid vesicles, in: M. Rosoff (Ed.), Vesicles, Marcel Dekker, New York, 1996, pp. 151 195. [9] M.A. Partearroyo, A. Alonso, F.M. Gofii, M. Tribout, S. Paredes, J. Colloid Interface Sci. 178 (1996) 156. [10]D. Lichtenberg, J. Robson, E.A. Dennis, Biochim. Biophys. Acta 821 (1985) 470. [11] T. Iwasaki, M. Ogawa, K. Esumi, K. Meguro, Langmuir 7 (1991) 30. [12] E.R. Cooper, B. Berner, in: M.M. Rieger (Ed.) Surfactant in Cosmetics, Surfactant Science Series, vol. 16, Marcel Dekker, New York, 1985, pp. 195 211. [13] J. Garcia Dominguez, F. Balaguer, J.L. Parra, C.M. Pelegero, Int. J. Cosmet. Sci. 3 (1981) 57. [14] L.D. Rhein, F.A. Simion, in: M. Bender (Ed.) Interfacial Phenomena in Biological Systems, Surfactant Science Series, vol. 39, Marcel Dekker, New York, 1991, pp. 33-50.
260
A. de la Maza et at / Colloids Surfaces A: Physicochem. Eng. Aspects 133 (1998) 253-260
[15] P.W. Wertz, W. Abraham, L. Landmann, D.T. Downing, J. Invest. Dermatol. 87 (1986) 582. [16]P.W. Wertz, in: O. Braun-Falco, H.C. Korting, H. Maibach (Eds.) Liposome Dermatics (Griesbach Conference), Springer, Heidelberg, 1992, pp. 38 43. [17] W. Abraham, P.W. Wertz, L. Landman, D.T. Downing, J. Invest. Dermatol. 88 (1987) 212. [18] A. de la Maza, J.L. Parra, M.T. Garcia, I. Ribosa, J. Sanchez Leal, Colloids Surf. 61 ( 1991 ) 281. [19] A. de la Maza, J.L. Parra, J. Sanchez Leal, Langmuir 8 (1992) 2422. [20] A. de la Maza, J.L. Parra, Langmuir 11 (1995) 2435. [21] A. de la Maza, J.L. Parra, Colloids Surf. A: 90 (1994) 79. [22] A. de la Maza, A.M. Manich, L. Coderch, P. Bosch, J.L. Parra, Colloids Surf. A: 101 (1995) 9. [23] M.J. Rosen, J. Colloid Interface Sci. 79 (1981) 587. [24]J.N. Weinstein, E. Ralston, L.D. Leserman, R.D. Klausner, P. Dragsten, P. Enkart, R. Blumenthal, in: G.
[25] [26]
[27] [28] [29] [30] [31] [32] [33]
Gregoriadis (Ed.) Liposome Technology, vol. III, CRC Press, Boca Raton, FL, 1986, pp. 183-204. R.G. Ackman, C.A. McLeod, A.K. Banerjee, J. Planar Chromatogr. 3 (1990) 450. S. Almog, B.J. Litman, W. Wimley, J. Cohen, E.J. Wachtel, Y. Barenholz, A. Ben-Shaul, D. Lichtenberg, Biochemistry 29 (1990) 4582. A. de la Maza, J.L. Parra, Langmuir 12 (1996) 3393. P. Schurtenberger, N. Mazer, W. Kanzig, J. Phys. Chem. 89 (1985) 1042. K. Edwards, M. Almgren, Prog. Colloid Polym. Sci. 82 (1990) 190. K. Edwards, M. Almgren, Langmuir 8 (1992) 824. D.D. Lasic (Ed.) Liposomes: From Physics to Applications, Elsevier, Amsterdam, 1993, pp. 43-62. R. Shubert, K. Beyer, H. Wolburg, K.H. Schmidt, Biochemistry 25 (1986) 5263. A. de la Maza, J.L. Parra, Biophys. J. 72 (1997) 1668.
AND ELSEVIER
Colloids and Surfaces A: Physicochemicaland EngineeringAspects 133 (1998)253 260
A
SURFACES
Sublytic effects caused by C14-alkyl betaine/sodium dodecyl sulfate mixtures in liposomes modeling the stratum corneum lipid composition A. de la Maza a,,, j. Baucells
b, p.
Gonzalez c, J.L. Parra a
a Consejo Superior de Investigaciones Cientificas (C. S. I. C.), Centro de Investigacidn y Desarrollo (C. I. D. ), Departamento de Tensioactivos, Calle Jorge Girona 18-26, 08034 Barcelona, Spain b Universidad Autdnoma de Barcelona (U.A.B.), Facultad de Veterinaria, Bellaterra, 08193, Barcelona, Spain Transtechnics. S.L., Sant Guillem, 21, 08006 Barcelona, Spain Received 18 January 1997; accepted 14 July 1997
Abstract It is known that a reduction in the skin irritation caused by anionic surfactants occurs in the presence of amphoteric ones. However, the mechanisms of this reduction are far from understood, since a detailed description of the process has yet to be given. The interaction of tetradecyl betaine (C14-Bet)/sodium dodecyl sulfate (SDS) mixtures with a simplified skin membrane model were investigated. To this end, liposomes modeling the stratum corneum (SC) lipid composition (40% ceramides, 25% cholesterol, 25% palmitic acid and 10% cholesteryl sulfate) were used. The surfactant/lipid molar ratios Re and the bilayer/aqueous phase partition coefficients K were determined by monitoring the increase in the fluorescence intensity of liposomes due to the carboxyfluorescein (CF) released from the interior of vesicles. These surfactant mixtures showed at the two interaction levels investigated (50 and 100% CF release) higher ability to alter the release of the CF trapped into SC bilayers (lower Re values) than that exhibited by the anionic component (SDS), although always showing a lower degree of partitioning into these structures (lower K values). Comparison of these findings with those reported for the interaction of these mixtures with phosphatidylcholine (PC) liposomes shows that SC liposomes were more resistant to the action of these surfactant mixtures, their degrees of partitioning into SC bilayers also being clearly lower than those reported for PC ones. © 1998 Elsevier Science B.V. Keywords." Tetradecyl betaine/sodium dodecyl sulfate mixtures; Stratum corneum liposomes; Stratum corneum liposomes/surfactant interactions; Carboxyfluorescein release; Surfactant/stratum corneum lipids molar ratios; Surfactant partition coefficients
1. Introduction Liposomes are aqueous lipid dispersions organized as bilayers which are widely used as simplified membrane models [1-3]. A number of studies have been devoted to the understanding of the * Corresponding author. Tel: (+ 34) 3 400 61 61; Fax: (+ 34) 3 204 59 04. 0927-7757/98/$19.00 © 1998Elsevier ScienceB.V. All rights reserved. PH S0927-7757 (97) 00206-9
principles governing the interaction of surfactants with these structures [4-9]. This interaction leads to the breakdown of lamellar structures and the formation of lipid-surfactant mixed micelles. A significant contribution has been made by Lichtenberg et al. [10], who postulated that the surfactant/lipid molar ratio Re producing solubilization of liposomes depends on the surfactant critical micelle concentration (CMC) and on the
254
A. de la Maza et al. / Colloids SurJaces A: Physicochem. Eng. Aspects 133 (1998) 253 260
bilayer/aqueous medium distribution coefficients K, which depends on the combination of amphiphilic properties of the lipid and the surfactant. Zwitterionic surfactants have strong interaction with anionic surfactants in water [ 11]. The effect of the micellar solution phase of these mixtures in avoiding, or at least reducing, the level of anionic/protein interaction has been suggested by several workers as a way of reducing the potential irritation of anionic surfactants [12,13]. Thus, a reduction in skin irritation by anionics has been reported by Rhein and Simion in the presence of amphoteric surfactants [14]. These authors attributed this reduction at two possible mechanisms. The formation of mixed micelles that sequester the irritating surfactant, preventing its interaction with the skin or, alternatively, the fact that the mild surfactant may compete with the irritating surfactant for binding sites on the skin's surface. In order to find out whether the lipids building the intercellular matrix of the stratum corneum (SC) could form bilayers, Wertz and coworkers prepared liposomes from lipid mixtures approximating the SC composition and studied the interaction of these liposomes with sodium dodecyl sulfate (SDS) to study its deleterious effect on human skin [15-17]. We previously studied the sublytic interaction of tetradecyl betaine (C14-Bet) and SDS when interacted individually or in mixtures with phosphatidylcholine (PC) liposomes [ 18 21 ]. We also investigated the formation and characterization of liposomes formed with mixtures of four commercially available synthetic lipids approximating the composition of the SC [22]. In the present work we extend these investigations by characterizing the Re and K parameters of C14-Bet/SDS mixtures when interacted with SC liposomes. This information could be useful in establishing a criterion for the evaluation of the skin irritation capacity of these surfactant mixtures at submicellar concentrations.
2. Materials and methods N-Tetradecyl-N,N-dimethylbetaine (C14-Bet) was specially prepared by Albright and Wilson,
Ltd. (Warley, West Midlands, UK); the active matter was 30% in water and the amino-free content was 0.50%. SDS was obtained from Merck and further purified by a column chromatographic method [23]. Piperazine-l,4-bis(2-ethanesulfonic acid) (PIPES buffer) obtained from Merck (Darmstadt, Germany) was prepared as 20mM PIPES buffer adjusted to pH 7.20 with NaOH, containing 110 mM Na2SO 4. The starting material 5(6)-carboxyfluorescein (CF), was obtained from Eastman Kodak (Rochester, NY) and further purified by a column chromatographic method [24]. Polycarbonate membranes and membrane holders were purchased from Nucleopore (Pleasanton, CA). Reagent-grade organic solvents, ceramides type III (Cer), cholesterol (Chol) and palmitic acid (PA) were supplied by Sigma Chemical Co. (St Louis, MO). Cholesteryl sulfate (Chol-sulf) was prepared by reaction of cholesterol with excess chlorosulfonic acid in pyridine and purified chromatographically. The molecular weight of ceramide type III used in the lipid mixture was determined by low-resolution fast atom bombardment mass spectrometry using a Fisons VG Auto Spec Q (Manchester, UK) with a cesium gun operating at 20 kV. From this analysis a molecular weight of 671 g was obtained for the majority compound of the Cer used (Sigma), this value being used to calculate the molarity of the lipid mixture investigated [22]. The lipids of the highest purity grade available were stored in chloroform/methanol 2:1 under nitrogen at - 2 0 ° C until use. 2.1. Liposome preparation and characterization
We previously reported the formation of liposomes formed by a mixture of lipids modeling the SC composition (40% Cer, 25% Chol, 25% PA and 10% Chol-sulf) [22] following the method described by Wertz et al. [15]. Liposomes (lipid conc. 0.5-5.0 mM) were prepared in PIPES buffer containing 100mM CF. Vesicles were freed of unencapsulated dye by passage through Sephadex G-50 medium resin (Pharmacia, Uppsala, Sweden) by column chromatography to study the changes in the release of the CF trapped in liposomes due to the action of surfactant mixtures.
A. de la Maza et al. / Colloids Surfaces A: Physicochem. Eng. Aspects 133 (1998) 253~60
The bilayer lipid composition after liposome preparation was determined using thin-layer chromatography coupled to an automated flame ionization detection system [22,25]. In order to find out whether all the components of the lipid mixture formed liposomes, vesicular dispersions were analyzed for these lipids [25]. The dispersions were then spun at 140 000g at 25°C for 4 h to remove the vesicles [26]. The supernatants were tested again for these components. No lipids were detected in any of the supernatants. Analyses of proton magnetic resonance (1H NMR) were carried out at temperatures ranging from 25°C to 90°C to determine the phase transition temperature of the lipid mixture forming liposomes, which showed a value of 55-56°C [22]. The vesicle size distribution and the polydispersity index (PI) of liposome suspensions after preparation were determined with dynamic lightscattering measurements using a photon correlator spectrometer (Malvern Autosizer 4700c PS/MV) [27]. After preparation, the vesicle size distribution varied very little, always showing a similar value of about 200nm (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 PIPES buffer and equilibration for 60 min showed in all cases values similar to those obtained after preparation, with a slight increase in the PI (between 0.10 and 0.12). Hence, liposome preparations appeared to be reasonably stable in the absence of surfactant mixtures under the experimental conditions used. 2.2. Parameters involved in the interaction o f surfactant mbctures with S C liposomes
In the analysis of the equilibrium partition model proposed by Schurtenberger et al. [28] for bile salt/lecithin systems, Lichtenberg et al. [10] and Almog et al. [26] have shown that for a mixing of lipids in dilute aqueous media, the distribution of surfactant between lipid phase and aqueous media obeys a partition coefficient K ( m M - 1), given by [20] K = Re/[Sw ( 1 + Re)]
( 1)
where Re is the effective molar ratio of surfactant
255
to lipid in the bilayers (Re = SB/L, SB(mM) being the concentration of surfactant in the bilayers ) and Sw(mM) is the surfactant concentration in the aqueous medium. Given that the range of lipid concentrations used in the mixture is similar to that used by Almog et al. to test their equilibrium partition model, the K parameter has been determined using this equation. The determination of these parameters can be carried out on the basis of the linear dependence existing between the surfactant concentrations required to achieve 50% and 100% CF release and the SC lipid concentration [L], which can be described by the equations ST,5Oo/oCF = SW,SOO/oCF -'~ Resoo/ocF[L ]
(2)
ST, IO0%CF : Sw, IO0°/oCF "t- Reloo%cv[L]
(3)
where ST,5OnCF and Sv,~o0o/ocvare the total surfactant concentrations. The surfactant/lipid molar ratios Reso~cF and Relooo/ocv and the aqueous surfactant concentrations Sw.5o%cF and Sw,~oOo/ocv are in each curve respectively the slope and the ordinate at the origin (zero lipid concentration) [21]. Changes in the release of the CF trapped in SC vesicles were determined quantitatively by monitoring the increase in the fluorescence intensity of liposomes due to the CF liberated [21,24]. The fluorescence intensity measurements were taken 60 rain after adding the surfactant solution to liposomes at 25°C using a Shimadzu RF-540 spectrofluorophotometer (Kyoto, Japan). This interval was chosen as the minimum period of time needed to achieve a constant level of CF release for the lipid concentration range used. The experimental determination of this interval is given in the Section 3. The CMC of C~4-Bet/SDS mixtures was determined from the abrupt change in the slope of the surface tension values vs. surfactant concentration [21]. The values obtained for each mole fraction of the zwitterionic component Xzwitte r a r e given in Table 1. 3. Results and discussion
In preliminary experiments we determined the suitable sonication temperature of the lipid mixture
256
A. de la Maza et al. / Colloids Surfaces A." Physicochem. Eng. Aspects 133 (1998) 253 260
Table 1 Surfactant to lipid molar ratios Re, partition coefficients K and surfactant concentrations in the aqueous medium Sw resulting in the interaction (50% and 100% CF release) of C14-Bet/SDS mixtures for different mole fractions of the zwitterionic surfactant Xzwitto,. The CMCs of each surfactant mixture tested are also included, together with the regression coefficients of the straight lines obtained Xzwitte r
0 0.2 0.4 0.6 0.8 1.0
CMC (mM)
Sw.soo/,cv SW,lOOO/oCF Reso*/oCV (mM) (raM) (mol tool -1)
Relooo/,cv (mol tool -1)
0.500 0.175 0.090 0.080 0.115 0.190
0.083 0.048 0.029 0.028 0.051 0.102
1.0 0.224 0.108 0.100 0.172 0.349
0.289 0.110 0.061 0.058 0.089 0.160
0.350 0.125 0.063 0.057 0.115 0.291
investigated b y p r e p a r i n g l i p o s o m e s at t e m p e r atures a p p r o x i m a t i n g its phase t r a n s i t i o n tempera t u r e ( 5 5 - 5 6 ° C ) . It was f o u n d t h a t t e m p e r a t u r e s exceeding this t e m p e r a t u r e b y m o r e t h a n 10°C caused n o t i c e a b l e a l t e r a t i o n s in Cer a n d Chol-sulf. A s a consequence, the lipid m i x t u r e was s o n i c a t e d at 60°C. To d e t e r m i n e the time n e e d e d to o b t a i n a constant level o f C F release o f l i p o s o m e s in the lipid c o n c e n t r a t i o n range investigated, a kinetic s t u d y o f the i n t e r a c t i o n o f C14-Bet/SDS m i x t u r e s with SC l i p o s o m e s was carried o u t at different Xzwitte r. L i p o s o m e s at lipid c o n c e n t r a t i o n s o f 1.0 m M a n d 5.0 m M were t r e a t e d with s u r f a c t a n t m i x t u r e s at c o n c e n t r a t i o n o f 0.1 m M a n d 0.6 m M respectively. The s u b s e q u e n t changes in C F release for each Xzwitte r were studied as a function o f time. The results o b t a i n e d are i n d i c a t e d in Figs. 1 (a) a n d ( b ) respectively. A b o u t 60 m i n was n e e d e d to achieve a c o n s t a n t level o f C F release in all cases. This finding c o n t r a s t s with t h a t r e p o r t e d for the interaction o f these s u r f a c t a n t mixtures with P C liposomes [21], where the time n e e d e d to o b t a i n a c o n s t a n t C F release level was always lower (40 rain). T h e fact t h a t C F release curves vs. time exhibited p l a t e a u x for different Xzwitte r (in accordance with the results r e p o r t e d for P C liposomes) c o u l d be a t t r i b u t a b l e to the release o f the fluorescent dye t h r o u g h holes, o r channels, created in the m e m b r a n e . The i n c o r p o r a t i o n o f s u r f a c t a n t m o n o m e r s to m e m b r a n e s m a y directly induce the f o r m a t i o n o f h y d r o p h i l i c p o r e s in these structures o r merely stabilize transient holes, in a g r e e m e n t with the c o n c e p t o f transient channels suggested
100
KSOO/,CF
Klooo/ocv
r2
(mM -1)
(mM 1)
(50% CF)
r2 (100%CF)
3.12 2.31 2.04 1.93 2.02 2.21
1.73 1.66 1.60 1.56 1.65 1.617
0.994 0.998 0.994 0.991 0.995 0.997
0.996 0.993 0.997 0.998 0.997 0.994
• x = 1.0, o x = 0.4,
o x = 0.8, • x = 0.2,
• x = 0.6, ~ x = 0
80,
0
0
40 V
[3
t3
60
70
80
60
70
80
20
10
20
30
a
40 Time
50 [mini
100
80
60
40
20
10 b
20
30
40
50
Time [min]
Fig. 1. Time curves of the release of CF trapped in SC liposomes (lipid concentration: (a) 1.0 raM; (b) 5.0 raM) caused by the addition of a constant concentration of C14-Bet/SDS surfactant mixtures ((a)0.1 raM; (b)0.6 mM) at different mole fractions of the zwitterionic surfactant (Xzwitt~r).X = 1.0 ( I ) , X = 0 . 8 (D), X = 0 . 6 ( e ) , X = 0 . 4 (O), X=0.2 (I?), X=0 (V).
A. de la Maza et al. / Colloids Surfaces A: Physicochem. Eng. Aspects 133 (1998) 253-260
by Edwards and Almgren in the surfactant-mediated increase in PC membrane permeability [29,30]. The differences in the surfactant-induced CF release kinetics in both PC and SC liposomes could be correlated with the different gel-liquid crystal phase transition temperature of lipids building these two bilayer structures, which affects both the positional organization of lipid molecules and their polar heads as well as their mobility, and also affects the formation and stabilization of the aforementioned membrane holes. The spontaneous release of the CF trapped in liposomes in the absence of surfactant in this period of time was negligible. To determine the Re and Sw parameters a systematic investigation of CF release alteration caused by the addition of C14-Bet/SDS mixtures was carried out for various SC lipid concentrations (from 0.5 to 5 . 0 m M ) . To this end, liposome aliquots (2.0 ml, lipid concentration from 1.0 to 10.0mM) were mixed with equal volumes of buffered surfactant solutions at different Xzwitte r. 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 Xzwitter = 0.6 are given in Fig. 2. The C14-Bet/SDS concentrations resulting in 50% and
100% of CF release for each Xzwitte r tested were obtained graphically and plotted vs. lipid concentration. An acceptable linear relationship was established in each case. These results are plotted in Fig. 3(a) and (b) (50% and 100% CF release respectively). The error bars of these figures are the SD and represent the error of three replicates. The straight lines obtained corresponded to the Eqs. (2) and (3) from which Re and Sw were determined. These parameters, including the regression coefficients r 2 of the straight lines, are given in Table 1. The Sw values increased as the CF release rose, although showing smaller values than those for the surfactant CMCs in all cases. This finding
2"5r
• o
X = 1,0, X = 0,4,
[] X = 0.8, • X = 0.2,
f1
I] J[
•
[
o [L]= 3.0 mM,
'°°I40
I L l = 0. 5 I I ] ~
[] ILl = L 0 lnM~
• [L] = 2.0 I n M
• [LI= 4.0 raM,
v [L]= 5.0 mM
• v
X = 0.6,
X=0
I ]
N
~'--~2.0 ~1.5 ra0 1.0 0.5.
1
2 Lipid
/
257
3
4
[mM]
ii [[
I 5.0
..............
I
"Tr'r
.......
--
~
4.0
~
3.0 2.0
2O
0.01
1.0
0.1 Surfactant
1,0
1
[raM]
Fig. 2. Percentage changes in CF release of SC liposomes, (lipid concentration ranging from 0.5 to 5.0mM), induced by the presence of increasing concentrations of C14-Bet/SDS surfactant mixtures at the mole fraction of the zwitterionic surfactant of 0.6. Lipid concentrations: 0.5 mM ( I ) , 1.0 mM ([3), 2.0 mM (O), Y 0 m M (©), 4 . 0 m M ( T ) , 5.0 m M ( V ) .
b
2
3
4
Lipid [mM]
Fig. 3. Snrfactant concentrationsfor differentmole fractions of the zwitterionicsurfactant ( g z w i t t e r ) resultingin (a) 50% and (b) 100% CF release vs. lipid concentration of liposomes. X= 1.0 (I), X=0.8 ([1), X=0.6 (O), X=0.4 (©), X=0.2 (T), X= 0 (v).
258
A. de la Maza et al. / Colloids Surfaces A." Physicochem. Eng. Aspects 133 (1998) 253-260
suggests that the surfactant-liposome interaction must be ruled mainly by the action of surfactant monomers. These findings are in agreement with those reported for sublytic interactions of these surfactant mixtures with PC liposomes [21]. Furthermore, Sw showed the lowest values at the same mole fraction (Xzwitte r = 0.6), at which a minimum in the CMC of these mixtures took place (see Table 1). Thus, the lower the surfactant mixture CMC the lower the Sw concentration at which 50% and 100% CF release of SC liposomes occurred. As for the R e parameter, this value also increased as the CF release (percent) rose, regardless of the Xzwitte r. Given that the surfactant capacity to alter the release of the CF trapped in the bilayers is inversely related to the R e parameter, the activity of these mixtures was always higher than that for the anionic component (SDS) and the maximum activity (at both 50% and 100% CF release) corresponded to the Xzwitter=0.6. These findings contrast sharply with the reduced skin irritation caused by the micellar solutions of SDS in the presence of alkyl betaines [12-14]. Comparison of the R e values with those reported for the interaction of these surfactant mixtures with PC liposomes [21] shows that the ability of these mixtures to alter the release of the dye trapped into SC bilayers (at 50% CF release) was always slightly less (higher R e values) than that reported for PC bilayers, although showing similar tendencies with respect to the influence of the Xzwitte r. Thus, SC liposomes exhibited more resistance to the surfactant perturbations than PC ones at the sublytic interaction level investigated. This different characteristic could be explained by bearing in mind the more hydrophilic nature of PC; this could facilitate the permeation of water and some other molecules (as surfactants) in PC liposomes, either through the hydrophilic holes created by the surfactants on the PC polar heads or via formation short-lived complexes surfactants-PC polar heads, and subsequent transfer of the bilayers via flip-flop [31]. The K parameter for SC liposomes at both 50% and 100% CF release show that the surfactant molecules for Xzwitter=0.6 had the lowest degree of partitioning into bilayers (minimum K values).
Given that this Xzwitte r also showed a minimum in the surfactant CMC (see Table 1), a direct correlation could be established between the partitioning of these mixtures into SC bilayers and their CMCs. Thus, the lower the CMC of the surfactant mixtures, the higher their ability to alter the permeability of SC liposomes and the lower their degree of partitioning into these bilayer structures. The fact that these surfactant mixtures showed at 100% CF release lower K values than those for 50% may be explained by assuming that at low R e possibly only the outer vesicle leaflet was available for interaction with surfactant molecules, the binding of additional molecules to bilayers being hampered at slightly higher R e values. These findings are in agreement with those reported by Schubert et al. [32] for sodium cholate/PC liposomes and with our investigations involving the interaction of different anionic and non-ionic surfactants also with PC liposomes [20, 33]. An opposite tendency in the variation of K vs. the Xzwitte r w a s observed when comparing the present K values with those reported for the interaction of the same surfactant mixtures with PC liposomes [21]. Thus, whereas the degree of partitioning of these mixtures into SC bilayers (or bilayer affinity) showed a minimum for Xzwitte r = 0 . 6 , that for PC liposomes showed a maximum at the same mole fraction. Furthermore, the K values for SC liposomes were always smaller than those for PC ones. This opposite tendency may be explained by bearing in mind the different chain length of the lipid building these two bilayer structures, the degree of saturation and the nature of the polar heads. If the R e and K values obtained are plotted as a function of the Jf'zwitter, then the graphs shown in Fig. 4(a) and (b) are obtained. The curves of both parameters showed a minimum for X'zwitter=0.6, although K for 100% CF release showed almost a constant value in all cases. Thus, the Xzwitter=0.6 was a critical mole fraction at which the maximum activity of these surfactant mixtures and the minimum degree of partitioning into SC liposomes occurred simultaneously. The strong interaction of C14-Bet and SDS in water, which results in a greater surface activity of these systems than that attainable with any of the indivi-
A. de la Maza et al. / Colloids Surfaces A." Physicochem. Eng. Aspects 133 (1998) 253-260
1.0
l
I
50% CF Release o 100% CF Release •
259
with an opposite tendency with respect to the Yzwitter, than that reported for PC liposomes.
0.8
~a 0 . 6
Acknowledgment
0.4
We are grateful to Mr. G. von Knorring for expert technical assistance. This work was supported by funds from D.G.I.C.Y.T. (Direcci6n General de Investigaci6n Cientifica y T6cnica) (Prog. n ° PB94-0043), Spain.
0.2
Mole Fraction X I e 50% CF Release o 100% CF Release
5.0
References
4.0 "3
2
3.0 2.0
0
o
~ 1.o
0
b
0.2
0.4
0.6
0.8
1.0
Mole Fraction X
Fig. 4. (a) Surfactant to lipid molar ratios Re and (b) partition coefficients K for C14-Bet/SDS mixtures at 50% and 100% CF release vs. the mole fraction of the zwitterionic surfactant (Xzwitter). (O) 50% CF release and (©) 100% CF release.
dual surfactants of the mixture [11] seems to be responsible for this behavior. As mentioned above, these findings contrast with the reduced skin irritation caused by the micellar alkyl betaines/SDS solutions [12-14]. The fact that the CF release alterations were caused mainly by surfactant monomers (sublytic interactions) and that skin irritation appears to be closely connected with the surfactant/proteins interaction (corneocytes and corneocyte envelopes building the SC) could explain in part these differences. In general terms, different trends in the interaction of these surfactant mixtures with SC and PC liposomes may be observed when comparing the corresponding Re and K parameters [21]. Thus, SC liposomes were more resistant to the action of surfactant monomers and the partitioning of these mixtures into SC structures was always lower, and
[1] B. Sternberg, Liposomes as a model for membrame structures and structural transformations: a liposome album, in: D.D. Lasic, Y. Barenholz (Eds.) Nonmedical Applications of Liposomes, From Gene Delivery and Diagnostics to Ecology, vol. IV, CRC Press, Boca Raton, FL, 1995, pp. 271-297. [2] D.D. Lasic (Ed.) Liposomes: From Physics to Applications, Elsevier, Amsterdam, 1993, pp. 219-241. [3] R.C. Scott, R.H. Guy, J. Hadgraft (Eds.), Factors Affecting the Formation Morfology and Permeability of Stratum Corneum Lipid Bilayers "in vitro", IBC Technical Services Ltd., UK, 1990, pp. 110 180. [4] U. Kragh-Hansen, M. le Marie, J.P. N6el, T. GulikKrzywicki, J.V. Moiler, Biochemistry 32 (1993) 1648. [5] M.B. Ruiz, A. Prado, F.M. Gofii, A. Alonso, Biochim. Biophys. Acta 1193 (1994) 301. [6] A.I. Polozava, G.E. Dubachev, T.N. Simonova, L.I. Barsukov, FEBS Lett. 358 (1995) 17. [7] M. Silvander, G. Karlsson, K. Edwards, J. Colloid Interface Sci. 179 (1996) 104. [8] T. Inoue, Interaction of surfactants with phospholipid vesicles, in: M. Rosoff (Ed.), Vesicles, Marcel Dekker, New York, 1996, pp. 151 195. [9] M.A. Partearroyo, A. Alonso, F.M. Gofii, M. Tribout, S. Paredes, J. Colloid Interface Sci. 178 (1996) 156. [10]D. Lichtenberg, J. Robson, E.A. Dennis, Biochim. Biophys. Acta 821 (1985) 470. [11] T. Iwasaki, M. Ogawa, K. Esumi, K. Meguro, Langmuir 7 (1991) 30. [12] E.R. Cooper, B. Berner, in: M.M. Rieger (Ed.) Surfactant in Cosmetics, Surfactant Science Series, vol. 16, Marcel Dekker, New York, 1985, pp. 195 211. [13] J. Garcia Dominguez, F. Balaguer, J.L. Parra, C.M. Pelegero, Int. J. Cosmet. Sci. 3 (1981) 57. [14] L.D. Rhein, F.A. Simion, in: M. Bender (Ed.) Interfacial Phenomena in Biological Systems, Surfactant Science Series, vol. 39, Marcel Dekker, New York, 1991, pp. 33-50.
260
A. de la Maza et at / Colloids Surfaces A: Physicochem. Eng. Aspects 133 (1998) 253-260
[15] P.W. Wertz, W. Abraham, L. Landmann, D.T. Downing, J. Invest. Dermatol. 87 (1986) 582. [16]P.W. Wertz, in: O. Braun-Falco, H.C. Korting, H. Maibach (Eds.) Liposome Dermatics (Griesbach Conference), Springer, Heidelberg, 1992, pp. 38 43. [17] W. Abraham, P.W. Wertz, L. Landman, D.T. Downing, J. Invest. Dermatol. 88 (1987) 212. [18] A. de la Maza, J.L. Parra, M.T. Garcia, I. Ribosa, J. Sanchez Leal, Colloids Surf. 61 ( 1991 ) 281. [19] A. de la Maza, J.L. Parra, J. Sanchez Leal, Langmuir 8 (1992) 2422. [20] A. de la Maza, J.L. Parra, Langmuir 11 (1995) 2435. [21] A. de la Maza, J.L. Parra, Colloids Surf. A: 90 (1994) 79. [22] A. de la Maza, A.M. Manich, L. Coderch, P. Bosch, J.L. Parra, Colloids Surf. A: 101 (1995) 9. [23] M.J. Rosen, J. Colloid Interface Sci. 79 (1981) 587. [24]J.N. Weinstein, E. Ralston, L.D. Leserman, R.D. Klausner, P. Dragsten, P. Enkart, R. Blumenthal, in: G.
[25] [26]
[27] [28] [29] [30] [31] [32] [33]
Gregoriadis (Ed.) Liposome Technology, vol. III, CRC Press, Boca Raton, FL, 1986, pp. 183-204. R.G. Ackman, C.A. McLeod, A.K. Banerjee, J. Planar Chromatogr. 3 (1990) 450. S. Almog, B.J. Litman, W. Wimley, J. Cohen, E.J. Wachtel, Y. Barenholz, A. Ben-Shaul, D. Lichtenberg, Biochemistry 29 (1990) 4582. A. de la Maza, J.L. Parra, Langmuir 12 (1996) 3393. P. Schurtenberger, N. Mazer, W. Kanzig, J. Phys. Chem. 89 (1985) 1042. K. Edwards, M. Almgren, Prog. Colloid Polym. Sci. 82 (1990) 190. K. Edwards, M. Almgren, Langmuir 8 (1992) 824. D.D. Lasic (Ed.) Liposomes: From Physics to Applications, Elsevier, Amsterdam, 1993, pp. 43-62. R. Shubert, K. Beyer, H. Wolburg, K.H. Schmidt, Biochemistry 25 (1986) 5263. A. de la Maza, J.L. Parra, Biophys. J. 72 (1997) 1668.