Sublytic Alterations Caused by the Nonionic Surfactant Dodecyl

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Notes Sublytic Alterations Caused by the Nonionic Surfactant Dodecyl Maltoside in Stratum Corneum Lipid Liposomes M. Co´cera, O. Lo´pez, L. Coderch, J. L. Parra, and A. de la Maza* Departamento de Tecnologı´as de Tensioactivos, Instituto de Investigaciones Quı´micas y Ambientales de Barcelona (I.I.Q.A.B.), Consejo Superior de Investigaciones Cientı´ficas (C.S.I.C.), Calle Jorge Girona 18-26, 08034 Barcelona, Spain Received May 17, 2001. In Final Form: September 26, 2001

Introduction The stratum corneum (SC) consists of corneocytes that are separated by an intercellular matrix mainly composed of lipids. These lipids are organized into bilayers that have been postulated both to account for the permeability properties and to ensure the cohesiveness between corneocytes.1-4 In all intracellular membranes, such bilayerforming 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. To find out whether SC lipids formed bilayers, Wertz et al.5 prepared and characterized liposomes from lipid mixtures modeling the SC composition. The surfactant dodecylmaltoside (DM) has been used for solubilization of cytochrome oxidase in active form6,7 and has been found to have good properties for solubilization of diverse membrane proteins.8-11 We first studied the formation of liposomes from lipid mixtures modeling the SC composition12 and the phase transitions involved in the interaction of DM with phosphatidylcholine (PC) * To whom correspondence should be addressed. Tel: (34-93) 400 61 61. Fax: (34-93) 204 59 04. (1) Wertz, P. W.; Downing, D. T. In Transdermal Drug Delivery. Developmental Issues and Research Initiatives; Hadgraft, J., Guy, R. H., Eds.; Marcel Dekker: New York, 1989; pp 1-22. (2) Friberg, S. E.; Goldsmith, L. B.; Kayali, I.; Suhaimi, H. In Interfacial Phenomena in Biological Systems; Bender, M., Ed.; Surfactant Science Series; Marcel Dekker: New York, 1991; Vol. 39, Chapter 1. (3) Imokawa, G.; Abe, A.; Jin, K.; Higaki, Y.; Kamashima, M.; Hidano, A. J. Invest. Dermatol. 1991, 96, 523-526. (4) Bouwstra, J. A.; Gooris, G. S.; Bras, W.; Downing, D. T. J. Lipid Res. 1995, 36, 685-695. (5) Wertz, P. W.; Abraham, W.; Landman L.; Downing, D. T. J. Invest. Dermatol. 1986, 87, 582-584. (6) Suarez, M. D.; Revzin, A.; Narlock, R.; Kempner, E. S.; Thompson, D. A.; Fergusom-Miller, S. J. Biol. Chem. 1984, 259, 13791-13799. (7) Bolli, R.; Nalecz, K. A.; Azzi, A. Arch. Biochem. Biophys. 1985, 240, 102-116. (8) Foresta, B.; Henao, F.; Champeil, P. Eur. J. Biochem. 1992, 209, 1023-1034. (9) le Marie, M.; Garrigos, M.; Møller, J. V. In Technologies on Protein Studies and Purification; Briand, Y., Doinel, C., Gagnon, J., Faure, A., Eds.; G.R.B.P.: Villebon sur Yvette, France, 1992; Vol. 5, pp 75-85. (10) Kragh-Hansen, U.; le Marie, M.; No¨el, J. P.; Gulik-Krzywicki, T.; Møller, J. V. Biochemistry 1993, 32, 1648-1656. (11) Lambert, O.; Levy, D.; Ranck, J. L.; Leblanc, G.; Rigaud, J. L. Biophys. J. 1998, 74, 918-930. (12) de la Maza, A.; Manich, A. M.; Coderch, L.; Bosch, P.; Parra, J. L. Colloids Surf., A 1995, 101, 9-19.

liposomes.13 Here, the Re and K parameters for the sublytic interaction of DM with SC lipid liposomes have been investigated to establish a criterion for the evaluation of its activity on a complex membrane such as the SC. Materials and Methods The nonionic surfactant dodecylmaltoside (n-dodecyl β-Dmaltoside, DM) was purchased from Sigma Chemicals Co. (St. Louis, MO). The starting material 5(6)-carboxyfluorescein (CF) was obtained from Eastman Kodak (Rochester, NY) and purified chromatographically.14 Piperazine-1,4 bis(2-ethanesulfonic acid) (PIPES) was obtained from Merck. PIPES buffer was prepared as 10 mM PIPES containing 110 mM Na2SO4 and 110 mM CF and adjusted to pH 7.2.12 Reagent grade organic solvents, ceramides type III (Cer), and cholesterol (Chol) were supplied 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 chlorosulfonic acid in pyridine and purified chromatographically.12 The molecular weight of ceramide used was determined by low-resolution fast atom bombardment mass spectrometry.12 A molecular weight of 671 g was obtained for the majority compound of the ceramides type III used (Sigma). This value was similar to the molecular weight of ceramide 3 (667 g) calculated from the structure of this compound reported by P. W. Wertz,15 despite the fact that the ceramide type III used was a mixture of ceramides of different chain length (purity approx 99%). Hence, we used the molecular weight obtained to calculate the molarity of the lipid mixture investigated. Preparation and Characterization of SC Lipid Liposomes. We reported the formation of liposomes using a mixture of lipids modeling the SC composition,12 which was prepared following the method described by Wertz et al.5 Briefly, individual lipids were dissolved in chloroform/methanol 2:1. The solvent was removed with a stream of N2 and then under high vacuum. Aqueous lipid dispersions were prepared by suspension in PIPES buffer (30 min hydration) and sonication for 15 min at 65 °C until the suspensions become clear. Finally, vesicles of about 200 nm size were obtained by extrusion of these liposome suspensions through 800-200 nm polycarbonate membranes. The size of vesicles was determined using a photon correlator spectrometer (Malvern Autosizer 4700c PS/MV, Malvern, U.K.).16,17 The lipid composition and concentration of liposomes were determined using thin-layer chromatography coupled to an automated flame ionization detection system (TLC-FID, Iatroscan MK-5, Iatron Lab. Inc., Tokyo, Japan).18 To find out whether all the components of the lipid mixture formed liposomes, vesicular dispersions were analyzed for these lipids.18 The dispersions were then spun at 140 000g at 25 °C for 4 h to remove the vesicles.19 The supernatants were tested again for these (13) de la Maza, A.; Parra, J. L. Biophys. J. 1997, 72, 1668-1675. (14) Weinstein, J. N.; Ralston, E.; Leserman, L. D.; Klausner, R. D.; Dragsten, P.; Henkart, P.; Blumenthal, R. In Liposome Technology; Gregoriadis, G., Ed.; CRC Press: Boca Raton, FL, 1986; Vol. 3, pp 183204. (15) Wertz, P. W. In Liposome Dermatics (Griesbach Conference); Braun-Falco, O., Korting, H. C., Maibach, H., Eds.; Springer-Verlag: Berlin, 1992; pp 38-43. (16) de la Maza, A.; Parra, J. L. Langmuir 1996, 12, 6218-6223. (17) Lo´pez, O.; Co´cera, M.; Pons, R.; Azemar, N.; Lo´pez-Iglesias, C.; Wehrli, E.; Parra, J. L.; de la Maza, A. Langmuir 1999, 15, 4678-4681. (18) Ackman, R. G.; McLeod, C. A.; Banerjee, A. K. J. Planar Chromatogr.-Mod. TLC 1990, 3, 450-490. (19) Almog, S.; Litman, B. J.; Wimley, W.; Cohen, J.; Wachtel, E. J.; Barenholz, Y.; Ben-Shaul, A.; Lichtenberg, D. Biochemistry 1990, 29, 4582-4592.

10.1021/la010733c CCC: $22.00 © 2002 American Chemical Society Published on Web 12/06/2001

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Notes

components. No lipids were detected in any of the supernatants. The phase transition temperature of the lipid mixture forming liposomes was determined by 1H NMR showing a value of 55-56 °C.12 Parameters Involved in the Interaction of DM with SC Lipid Liposomes. In the analysis of the equilibrium partition model proposed by Schurtenberger et al.20 for bile salt/lecithin systems, Lichtenberg21 and Almog et al.19 have shown that for a mixing of lipids (lipid concn L (mM)) and surfactant in dilute aqueous media, the distribution of surfactant between lipid bilayers and water obeys a partition coefficient K, by

K ) SB/[(L + SB)SW]

(1)

where SB and SW are the surfactant concentration in the bilayers (mM) and in the aqueous medium (mM). For L . SB, the definition of K, as given by Schurtenberger, applies:

K ) SB/(LSW) ) Re/SW

(2)

where Re is the effective molar ratio of surfactant to lipid in the bilayers (Re ) SB/L). Under any other conditions, eq 2 has to be employed to define K; this yields

K ) Re/SW[1 + Re]

(3)

This approach is consistent with the experimental data offered by these authors for various surfactant/lipid systems over wide ranges of Re. Given that the lipid concentration range used 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 Re and K was carried out on the basis of the linear dependence existing between the surfactant concentrations required to achieve different levels of CF release and the lipid concentration (L), which can be described by the equation

ST ) SW + Re[L]

(4)

where ST is the total surfactant concentration and Re and SW are in each curve the slope and the ordinate at the origin (zero lipid concn), respectively. The permeability changes caused by DM in CF-containing SC liposomes were determined by monitoring the increase in the fluorescence intensity of the liposomes due to the CF released from the interior of vesicles to the bulk aqueous phase.12 Fluorescence measurements were made at 25 °C 60 min after the surfactant addition with the spectrofluorophotometer Shimadzu RF-540 (Kyoto Japan).13 The presence of DM did not affect the direct quenching of the spectrofluorophotometric CF signal.

Results and Discussion The characterization of the geometric properties of the SC liposomes demonstrated that these liposomes were formed by unilamellar vesicles in all cases.12 The vesicle size distribution of liposomes after preparation varied little (monomodal distribution of about 200 nm) and the polydispersity index (PI) was in all cases lower than 0.1, indicating a homogeneous size distribution in all cases. The vesicle size after the addition of equal volumes of PIPES buffer and equilibration for 60 min at 25 °C always showed values similar to those obtained after preparation, with a slight rise in PI (between 0.10 and 0.12). Hence, the liposomes appeared to be reasonably stable in the absence of DM under the experimental conditions used. Parameters Involved in the Interaction of DM with SC Lipid Liposomes. We first studied the validity of the equilibrium partition model proposed by Almog et al. and Lichtenberg based on eq 1 for DM. According to (20) Schurtenberger, P.; Mazer, N.; Ka¨nzig, W. J. Phys. Chem. 1985, 89, 1042-1049. (21) Lichtenberg, D. Biochim. Biophys. Acta 1985, 821, 470-478.

Figure 1. Time curves of the release of CF trapped into SC lipid liposomes at various lipid concentrations caused by the addition of 1.0 mM DM. Lipid concentration symbols: (b) 1.0 mM, (2) 2.0 mM, (4) 3.0 mM, and (9) 4.0 mM.

these authors, this equation may be expressed by L/SB ) (1/K)(1/SW) - 1. Hence, this validity requires a linear dependence between L/SB and 1/SW; this line should have a slope of 1/K, intersect with the L/SB axis at -1, and intersect with the 1/SW at K. These authors demonstrated the validity of this model for octyl glucoside/PC liposome systems in the range of lipid and surfactant concentrations used in the present work. To test the validity of the model for DM using SC liposomes, vesicles were mixed with varying sublytic DM concentrations (ST). The resultant surfactant-containing vesicles were then spun at 140 000g at 25 °C for 4 h to remove the vesicles.19 No lipids were detected in the supernatants.18 The DM concentration in the supernatants (SW) was determined by HPLC,22 and its concentration in bilayers was calculated (SB ) ST - SW). The SB and SW values thus obtained (in the same range of lipid and DM concentrations used to determine K) were plotted in terms of the dependence of L/SB on 1/SW. A straight line was obtained (r2 ) 0.991), which was dependent on L and intersected with the L/SB axis at -0.96 ( 0.11. Both the linearity of this dependence and the proximity of the intercept to -1 support the validity of this model to determine K for this system. To determine the time in which the leakage ceased, SC vesicles were treated with sublytic DM concentrations and changes in CF release were studied as a function of time. The CF release always showed a transient state of enhanced permeability of bilayers, in which about 40-60 min was needed to achieve the CF release plateaux. The longer times were associated with the higher CF release percentages (from 50 to 100%) as is shown in Figure 1. According to the concept of transient channels proposed by Schubert et al.,23 we assume that this biphasic behavior was due to the release of the encapsulated dye through holes, or channels, created in the membrane. The incorporation of surfactant monomers into membranes may directly induce the formation of hydrophilic pores or merely stabilize transient holes, in line with the reported interaction of DM with PC liposomes.13 The fact that in the case of SC liposomes longer times were needed to achieve the CF release plateaux may be due to the different gel-liquid crystal phase transition temperature of the lipids building these two liposomes affecting the positional (22) Seino, H.; Uchibori, T.; Nishitani, T.; Inamasu, S. J. Am. Oil Chem. Soc. 1984, 61, 1761-1765. (23) Schubert, R.; Beyer, K.; Wolburg, H.; Schmidt, K. H. Biochemistry 1986, 25, 5263-5269.

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Figure 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 concentrations of DM. SC lipid concentration symbols: (O) 0.5 mM, (b) 1.0 mM, (2) 2.0 mM, (4) 3.0 mM, (9) 4.0 mM and (0) 5.0 mM. Table 1. Surfactant to Lipid Molar Ratios (Re), Normalized Partition Coefficients (K), and Surfactant Concentrations in the Aqueous Medium (SW) Resulting in the Subsolubilizing Interaction of DM with SC Lipid Liposomesa CF release %

SW [mM]

Re mol/mol

r2

K [mM-1]

10 20 30 40 50 60 70 80 90 100

0.008 0.011 0.019 0.025 0.032 0.037 0.043 0.050 0.053 0.065

0.23 0.41 0.49 0.56 0.64 0.70 0.76 0.82 0.88 0.97

0.994 0.991 0.993 0.991 0.996 0.992 0.995 0.997 0.991 0.996

23.37 26.45 17.31 14.35 12.21 11.12 10.05 9.01 8.83 7.57

a The regression coefficients (r2) of the straight lines obtained are included.

organization of lipid molecules, their polar heads and their mobility, and the formation and stabilization of the aforementioned holes. The spontaneous CF release in the absence of DM in this interval was negligible. Hence, changes in CF release were studied 60 min after the DM addition to liposomes at 25 °C. To determine Re and SW, a systematic study of the CF release changes due to the DM addition to liposomes (lipid concn ranging from 0.5 to 5.0 mM) was performed. The curves obtained are given in Figure 2. The DM concentrations resulting in different CF release percents were graphically obtained and plotted versus lipid concentration (results not shown). The r2 statistic (regression coefficients r2, Table 1) indicated that the straight lines obtained explained more than 98.9% of the DM concentration variability versus lipid concentration. Therefore, a good linear fit was established in each case. These findings confirm that the straight lines for eq 4 were appropriate to determine Re and SW. This method has also been demonstrated to be valid to study the interaction of various surfactants with SC lipid liposomes.17,24 The Re, K, and SW values obtained are given in Table 1. Different trends in the evolution of Re and K were observed as the CF release rose. Thus, whereas Re and SW progressively increased, the K values showed a maximum followed by an abrupt decrease up to the complete release of the encapsulated dye. The fact that SW always showed lower values than the DM critical (24) de la Maza, A.; Parra, J. L. Langmuir 1996, 12, 3393-3398.

Figure 3. Variation in the normalized bilayer/aqueous phase partition coefficient (K) versus the surfactant to SC lipid molar ratio (Re). (b) SC lipid liposomes, (O) PC liposomes.

micelle concentration (0.125 mM13) confirms the generally admitted assumption that permeability alterations were mainly ruled by the action of surfactant monomers.21 Comparison of the Re and K values with those reported for the interaction of DM with PC liposomes13 shows that at the same interaction step (same CF release percent) both parameters always exhibited higher values. Given that the surfactant capacity to interact with liposomes is inversely related to the Re value, we assume that the DM activity was always less than that exhibited with PC liposomes. Hence, the SC lipid vesicles were more resistant to the surfactant perturbations than PC vesicles.13 However, the increased K values indicated that the affinity of DM molecules with SC liposomes was always higher than that for PC ones. Figure 3 shows the variation in K versus Re in the interaction of DM with SC liposomes (symbol b). A K maximum (26.45) was reached for Re ) 0.41. Increasing Re led to a fall in K up to Re ) 0.97, this decrease being more pronounced in the Re interval 0.41-0.64. Thus, the increase in Re resulted in two opposite effects on the bilayer/water DM partitioning. At low Re, K first rose possibly because only the outer vesicle leaflet was available for interaction with DM molecules, the binding of additional DM to the bilayer being hampered up to Re ) 0.64 (abrupt fall in K). The Re ) 0.41 may be correlated with the saturation of the outer vesicle leaflet by DM. Increasing Re values (from 0.64 to 0.97, lower decrease in K) led to an increased rate of flip-flop of the surfactant molecules (or permeabilization of the bilayers to DM), thus also making the inner monolayer available for interaction with added DM. These findings are in line with those reported by Ueno,27 who reported for two nonionic surfactants (octyl glucoside and octaethylene glycol monododecyl ether) that when the surfactants were incorporated into surfactant-free phospholipid vesicles, they became distributed only within the outer vesicle leaflet because of the slow flip-flop of these surfactants in the bilayer. In this sense, Kragh-Hansen et al. reported for DM slow flip-flop in phospholipid vesicles due to its marked hydrophilic properties, an appreciable amount of surfactant needing to be incorporated into liposomes for flipflop to start to occur.28 The aforementioned long time course required to reach a CF release plateau in this Re (25) Lasic, D. D. Liposomes from Lipids to Applications; Elsevier: Amsterdam, 1993; pp 56-62. (26) Co´cera, M.; Lo´pez, O.; Coderch, L.; Parra, J. L.; de la Maza, A. Colloids Surf., A 2001, 176, 167-176. (27) Ueno, M. Biochim. Biophys. Acta 1987, 904, 140-144. (28) Kragh-Hansen, U.; le Marie, M.; Møller, J. V. Biophys. J. 1998, 75, 2932-2946.

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interval (from 50 to 100% CF release) could be also correlated with this behavior. Figure 3 also shows the reported variation in K versus Re during the interaction of DM with PC liposomes13 (symbol O). Although both curves showed similar tendencies, in the case of SC liposomes the K maximum was higher than for PC vesicles (26.45 and 14.58, respectively), and was achieved at a higher Re value (0.41 and 0.28, respectively). Hence, more DM molecules were needed to saturate the outer vesicle leaflet of SC vesicles. These differences may be explained given the more hydrophilic nature of PC, which could facilitate the permeation of water and DM in PC vesicles either through the hydrophilic holes created on the polar heads or via formation of short-lived complexes of DM with PC polar heads and subsequent transfer through the bilayers via flip-flop.25 Furthermore, the different phase states of these liposomes at 25 °C (gel-like state for SC lipids and fluid liquid-crystalline state for PC) affecting the physical conditions for interaction of DM with these two liposomes should be taken into account. Comparison of the present Re and K values with those reported for the sublytic interaction of dodecyl glucoside (DG) with SC liposomes26 reveals that the Re values were always clearly lower, whereas the K values were higher in all cases. This increased DM activity and affinity with bilayers could be explained bearing in mind that its molecular structure appears to be more equilibrated in terms of hydrophilic-lipophilic balance (HLB) than that of the DG despite the identical length of their hydrophobic tails (according to Lin et al.,29 HLB values of 8.6 and 14.1 for DG and DM, respectively). In fact, the adsorption of the surfactant molecules and subsequent incorporation

Notes

into bilayers are correlated with the HLB of each surfactant as well as with the composition and physicochemical characteristics of the bilayer structure. Abbreviations SC PIPES CF Re K

stratum corneum piperazine-1,4 bis(2-ethanesulfonic acid) 5(6)-carboxyfluorescein effective surfactant/lipid molar ratio normalized bilayer/aqueous phase surfactant partition coefficient SW surfactant concentration in the aqueous medium surfactant concentration in the bilayers SB PC phosphatidylcholine Cer ceramides type III Chol cholesterol PA palmitic acid Chol-sulf cholesteryl sulfate DM dodecyl maltoside (n-dodecyl β-D-maltoside) TLC-FID thin-layer chromatography/automated flame ionization detection system PI polydispersity index regression coefficient r2

LA010733C (29) Lin, I. J.; Friend, J. P.; Zimmels, Y. J. Colloid Interface Sci. 1973, 45, 378-385.