Langmuir 1993,9, 87&873
870
Notes Disintegration of Liposomes by Anionic Surfactants and Formation of Mixed Micelles A. de la Maza' and J. L. Parra Juez Znstituto de Tecnologiu Qutmica y Textil, Consejo Superior de Znvestigaciones Cienttficas (C.S.Z.C.), Departamento de Tecnologiu Qutmica, CfJorge Girona, 18-26, 08034 Barcelona, Spain Received May 28,1992. In Final Form: November 10,1992
Introduction Liposomes are lipid-water systems which have come into widespread use as a simplified model of biological membranes and delivery systems.'S2 The study of the process involved in liposome-surfactant solubilization has been of great value as this can provide useful information to better understand this complex p h e n o m e n ~ n . ~ ~ A significant contribution has been made by Lichtenberg,' who postulated that the minimum effective surfactant/phospholipid molar ratio producing solubilization depends on the surfactant critical micellar concentration and on the bilayer/ aqueous medium partition coefficients rather than on the nature of the surfactants. Accordingly, we carried out studies on the partition coefficients of different surfactants*?gincluding anionic alkyl sulphateslo in order to determine the main factors involved in the modifications of the permeability of bilayers by amphiphilic molecules. In the present work we seek to characterize the solubilizationof neutral and electricallycharged liposomes by an alkyl sulfate series (specific alkyl chain length (210, C12, and (214). In our case, lipid bilayers consisted of PC vesicles to which 9:l molar ratio of either phosphatidic acid (PA) or stearylamine (SA) was added when required to increase the negative or positive surface charge, respectively. Solubilization was assessed as a decrease in the turbidityl1J2of the liposome/surfactant systems. In order to evaluate these variations, three parameters were determined, namely, Resat,Resow, and Resolaccording to the nomenclature adopted by Li~htenberg.~J~ The results obtained will provide information on physicochemical ~
~~~
~~
factors involved ip the interactions of alkyl sulfates with liposomes and on how they affect vesicle solubilization.
Experimental Section Materials. Anionic surfactants CIO-SOI,Clz-SO4, and ClrSO4were obtained from Merck and further purified by a column chromatographic method.14 Phosphatidic acid (PA) and stearylamine (SA)werepurchasedfrom Sigmachemical Co. (St. Louis, MO). Phosphatidylcholine (PC) was purified from egg lecithin (Merck) according to the method of Singleton16and shown to be pure by thin-layer chromatography (TLC). Piperazine-l,4-bis(2-ethanesulfonicacid) (PIPES buffer) was obtained fromMerck. Polycarbonate membranes and membrane holders were purchased from Nucleopore (Pleasanton, CA).
Methods Liposome Preparation. Unilamellar liposome vesicles of a defined size (about 100 nm) were prepared by extrusion of large unilamellar vesicles previously obtained by the reverse phase evaporation, as described previously by Rigaud,lBJ' a method based on an early one described by Szokaand Papahadjopoulos.18 A lipidic film was formed by removing the organic solvent from a chloroform solution of lipids (PC or PCIPA, PCISA 9 1 molar ratio). The lipids were then redissolved in diethyl ether, and the PIPES buffer (20 mM PIPES with 110 mM NazS04 adjusted to pH 7.2) was added to the solution of lipids. Gentle sonication led to the formation of a WIO type emulsion. A viscous gel was formed after evaporating the ethyl ether under reduced pressure. The elimination of the final traces of the organic solvent transformed the gel into a liposome suspension. Unilamellar vesicles of 100 nm were obtained by extrusion of vesicle suspensions through 400-, 200-, and 100-nm polycarbonate membranes so as to achieve a uniform size distribution.19 Phosphorus Estimation. Phospholipid concentration of the liposomes was determined by the ascorbic acid spectrophotometric method for total phosphorus estimation.20 Particle Size Distribution of Liposome Preparations. Mean size and polydispersity of liposomes were determined using a Photon correlator spectrometer (Malvern Autosizer 4'700~PSI MV). The particle size distribution was determined by particle number measurements at 25 "C, lecture angle 90". Liposome Solubilization by Surfactants. The solubilization of liposomes by the surfactants leads to the disintegration of the lipid bilayers via mixed micelle formation.' This process can be monitored by measuring the variations in turbidity of the systems during solubilization.12 In order to evaluate these variations obtained, the effectivesurfactantlphospholipid molar ratio Re, in an aggregate (liposome or micelle) is defiied as foll0ws:l3
(1)Papahadjopoulos, D. In Liposomes and Their Uses in Biology and Medicine. Ann. New York Acad. Sci. 1987,l-462. [total surfactant] - [surfactant monomer] (2)Ostro, M. J. Sci. Am. 1987,256,90. Re = (3)Paternostre, M. T.; Rous, M.; Rigaud, J. L. Biochemistry 1988,27, [total phospholipid] - [phospholipid monomer] 2668. (4)Almog, S.;Litman, B. J.; Wimley, W.; Cohen, J.; Wachtel, E. J.; The second term of the denominator is negligible due to the low Barenholz, Y.; Ben-Shaul, A.; Lichtenberg, D. Biochemistry 1990,29, solubility of phospholipids in water. The overall solubilization 4582. (5)Edwards, K.;Almgren, M. J. Colloid Interface Sci. 1991,147,1. (6)Inoue, T.; Yamahata, T.; Shimozawa, R. J. Colloid Interface Sci. (14)Rosen, M. J. J. Colloid Interface Sci. 1981,79,587. 1992,149,345. (15)Singleton, W. S.;Gray, M. S.; Brown, M. L.; White, J. L. J. Am. (7)Lichtenberg, D. Biochim. Biophys. Acta 1985,821,470. Oil Chem. SOC.1966,42,53. (8)De la Maza, A.;Sanchez Leal, J.; Parra, J. L.; Garcia, M. T.;Ribosa, (16)Rigaud, J. L.; Bluzat, A.; Buschlen, 5.In Physical Chemistry of I. J. Am. Oil Chem. Soc. 1991,68,315. Transmembranelon Motion; Spach, G., Ed.; Elsevier: Amsterdam, 1983; (9)De la Maza, A.;Parra, J. L.; Garcia, M. T.;Ribosa, I.; Sanchex Leal, pp 457-464. J. J. Colloid Interface Sci. 1992,148, 310. (17)Rigaud, J. L.; Bluzat, A,; Buschlen, S. Biochem. Biophys. Res. (10)De la Maza, A.; Parra, J. L.; Sanchez Leal, J. Langmuir 1992,8, Commun. 1983,111,373. 2422. (18)Szoka, F.;Papahadjopoulos, D. In Liposomes: Preparation and (11)Gofli,F.M.;Urbaneja,M.A.;Arrondo,J.L.R.;Alonso,A.;Durrani,Characterization; Knight, C. G., Ed.; Elsevier. North-Holland; AmsterA. A,; Chapman, D. Eur. J. Biochem. 1986,160,659. dam, 1981;Chapter 3. (12)Urbaneja, M. A.; Alonso, A.; Gonzalez-Maim, J. M.; Gofii, F. M.; (19)Szoka, F.;Olson, F.; Heath, T.; Vail, W.; Mayer, E.; PapahadPartearroyo, M. A.; Tribout, M.; Paredes, S. Biochem. J. 1990,270,305. jopoulos, D. Biochim. Biophys. Acta 1980,601,559. (13)Lichtenberg, D.; Robson, R. J.; Dennis, E. A. Biochim. Biophys. (20)Standard Methods, 14th ed.; American Public Health AsemiaActa 1983,737,285. tion: Washington, DC, 1976;pp 466-484.
0743-7463/93/2409-0870$04.00/00 1993 American Chemical Society
Notes
Langmuir, Vol. 9, No. 3, 1993 811
1
2
3
4
5
Phosphollpid In”
Surfactant [MI
Figure 1. Percentage change in turbidity of neutral liposomes (PC loo%),at bilayer lipid concentration ranging between 0.5 and 5.0 mM, versus C&04 surfactant concentrations. process can be characterized by three parameters termed Rewt, ReMac,and Resol, according to the nomenclature adopted by Lichtenberg7J3correspondingto the surfactant/lipid molar ratios at which turbidity starts to decrease, reaches 50% of the original value, and shows no further decrease. These parameters correspondedto the “Re”atwhichsurfactant (a)saturated liposomes, (b) resulted in a 50% solubilization of bilayers, and (c) d e f i e d as the minimal Re in mixed micelles once all the phospholipid is completely solubilized. The determination of these parameters can be carried out on the basis of the linear dependence existingbetween the surfactant concentrations required to achieve these parameters and the phospholipid concentration in liposomes. The equations describing the surfactant concentration needed to saturate the bilayer (eq l),solubilize 50% of liposomes (eq 2), or achieve the completesolubilizationof liposomesvia mixed micellesformation (eq 3) are given as
+ , ,S = S, + Re,,X(PL) s,, = s, + Re,,,X(PL) Seat= Sa Re,,X(PL)
. 1
. 2
3
. 4
5
Phospholipid [mM] 24
(1) (2)
(3) where the effective surfactant to phospholipid molar ratios (Re,,$, Reso$,,and Re,,) and the aqueous concentration of surfactants (Sa,Sb,and S,) are in each curve respectively the slope and the ordinate at the origin (zero phospholipid concentration). Liposome suspensions were adjusted to the adequate lipid concentration (from 1.0 to 10.0 mM). To these, equal volumes of the adequate surfactant solutions were added and the resulting mixtures were left to equilibrate for 24 h. Turbidity measurements were made at 25 OC with a Shimadzu RF-540 spectrofluorophotometer, with both monochromators adjusted at 500 nm. The assays were carried out in triplicate and the results given are the average of those obtained. Surface Tension and Critical Micellar Concentration Determinations. Surface tensions of surfactants in buffered medium were measured by the ring methodz1 using a K r h tensiometer (processor tensiometer K-12) which determines directlythe real surfacetension values at equilibrium. The critical micelle concentration values were determined by plotting the surface tension values versus surfactant concentration.
Results and Discussion Stability of Liposome Preparations. The particle size distribution after preparation (lipid concentration from 1.0 to 10 mM) varied very little, showing in all cases a similar value around 100 nm. Moreover, the polydispersity index of liposomes after preparation was lower than 0.1, which indicated that the size distribution was very homogeneous. (21) Lunkenheimer, K.; Wantke, D. Colloid Polym. Sci. 1981, 259, 364.
. 0
J
0
I
2
3
4
5
Phospholipid [mM]
Figure 2. Plots the concentration of Clo-SOr, Clz-S04,and ClrSO4 Surfactants necessary to achieve the S, (A),, ,S (B),and Ssol(C)parameters for neutral liposomes versus bilayer lipid concentration.
Solubilization Studies. The liposome solubilization was studied by monitoring the variations in the turbidity of the surfactant/liposome systems as a function of surfactant concentration. Figure 1 shows, the solubilization curves of neutral liposomes (lipid concentration from 0.5 to 5.0 mM) due to the addition of different concentrations of C12-SO4. From these curves the surfactant concentration producing the saturation, the half solubilization, and the total solubilization of liposomes can be obtained by graphical methods. The mows A, B, and C (curve 5.0 mM lipid concentration) correspond respectively to these parameters, Le., the surfactant concentration at which turbidity starts to decrease (S,J, reaches 50%(Sw%),and shows no further decrease &,I). Plotting the surfactant concentrations previously obtained versus phospholipid concentration curves are obtained (Figure 2). An acceptable linear relationship is established in each case. The straight lines obtained correspond to the aforementioned equations 1,2, and 3,
872 Langmuir, Vol.9,No.3, 1993
Notes
Table I. Solubilizing Parameters of Neutral and Electronegatively Charged Liposomes (Lipid Bilayer Composition PC:PA 9 1 , PC/SA 9 1 Molar Ratio, and PC 100%). PC:PA (9:l) e a PC PC:SA (91) cmc mM S, sb S, Resat Reso., Resol r2 S, s b S, Re,, R e s o n c Re-1 rz Sa s b S, Remt Resor,RQ r2 cio-So4 2.40 2.39 2.41 2.41 2.52 3.29 4.17 0.991 2.40 2.41 2.46 2.40 0.50 0.49 0.51 0.52 1.18 1.99 2.84 0.992 0.50 0.51 0.53 1.10 ci&& ci4-so4 0.17 0.17 0.16 0.18 1.24 2.08 2.89 0.990 0.17 0.18 0.19 1.15
3.22 1.89 1.96
4.04 0.993 2.39 2.40 2.39 2.38 2.70 0.992 0.50 0.49 0.50 1.10 2.75 0.997 0.16 0.17 0.17 1.13
3.22 1.88 1.95
4.06 0.992 2.68 0.989 2.73 0.991
The cmc of the alkyl sulfate Surfactants tested and the regression coefficients of the straight lines of Figure 2 are also included.
7.
1
0
1
InCmc
Figure 3. Effective molar ratios Re ( R k c and Resod of CI0-SO4(a),c12-so4( O ) ,and C1&04 (0) surfactants versus In cmc of surfactants for neutral liposomes. where the Re parameters and the aqueous concentration of surfactant are respectively the slope and the ordinate at the origin (zero phospholipid concentration). Similar results were also obtained when treating electrically chargedliposomes (PUPAor PC/SA 9 1 molar ratio) with these surfactants under the same conditions (curves not shown). The parameters obtained including the regression coefficients of the straight lines (Figure 2) and the critical micelle concentration (cmc) values of the surfactant in PIPES buffer are shown in Table I. It should be pointed out that in the vast majority of cases, solubilization of electrically charged bilayers is virtually unaffected by the electriccharge. Onlynegatively charged bilayers appear to be slightly resistant to surfactant solubilization. The presence of stearylamine in the lipid bilayers (PC/SA 91 molar ratio) does not seem to affect the solubilizing parameters. On the other hand, the surfactant concentration in the aqueous medium corresponded approximately to the cmc of the surfactant in all cases regardless of the electrical charge of the liposomes. These results suggest that the surfactant-liposome solubilizationsare mainly determined by the formation of mixed micellesbetween the surfactant and the phospholipid molecules, unlike the behavior of the Surfactants in subsolubilizingprocesses in which the action of surfactant monomers plays a very important r0ie.8~9 In our opinion, the most striking result of this work is the observation that the Re parameters for C12-S04 show slightlysmder values than the C14-SO4surfactant, whereas the C1o-SO4 always show the higher values regardless of the electrical charge of the liposomes. The increased hydrophobic character of these surfactants due to the increase of their hydrocarbon chain length does not seem to be related to their solubilizing capacity of neutral or electrically charged liposomes specially for C12-SO4 surfactant.
50
1
1 2
E
5
m2o
50100200
50010~0
5000
Particle Size Distribution [nm] Figure 4. Mean vesicle size distribution of neutral liposomes (4
mM lipid concentration) treated with CIZ-SO, surfactant at different surfactant concentrations during the solubilizing process.
Graphs are obtained by plotting the Re parameters obtained for CIO-SO~, C12-SO4, and Clr-SO4 versus In cmc of surfactants for neutral liposomes (Figure 3). It may be seen that no linear relationship can be establishedbetween these solubilizing parameters and the surfactant cmc. Similar results were obtained when plotting these solubilizing parameters for electrically charged liposomes versus In cmc. Particle Size Distribution of Liposomes duringthe Solubilizing Processes. The variations in mean vesicle size distribution of neutral liposomes (4.0 mM phospholipid concentration) treated with the C12-SO4at different
Notes
concentrations are plotted in Figure 4. The five surfactant/ liposome systems studied correspond to the five points marked with a circle in Figure 1 which represent the following Re ratios: (1) 0.5; (2) 1.0; (3) 2.0; (4) 2.57; (5) 3.12. It should be noted that point 1shows a single distribution (mean vesicle size about 100 nm and polydispersity index 0.150). Similar tendency is observed in the sizedistribution curve corresponding to the point 2, in which the vesicle size was slightly increased (about 112 nm and polydispersityindex0.196). Curves correspondingto point3 show a binodal distribution with an increased polydispersity index (particle size about 100nm, and polydispersity index 0.384). It is noteworthy that a sharp distribution curve appears approximatelyat 35-40 nm which correspondsto a new particle size distribution. Curves corresponding to point 4 showed similar tendencies to those observed in point 3 with an increase in the low particle size distribution (mean particle size about 95 nm and polydispersity index 0.298). Finally the curves of point 5 again show a single distribution with amean particle size distribution of about 35 nm and polydispersity index 0.173. Regarding the results given in Figures 1 and 4 it is noteworthy that the solubilization of liposomes by alkyl sulfates leads to the progressive formation of a low particle size distribution which appears to be related to the decrease in the turbidity of the surfactant/liposome systemsduring
Langmuir, Vol. 9, No. 3,1993 873
the solubilizing process. The presence of these particles at the end of the process could be considered as an indication of the formation of surfactantiphospholipid mixed micelles. Similar results were obtained using the C10-SO4 and C14-SO4 surfactants in the same conditions. Lichtenberg and his co-workersin their review13express the necessity for the experimental data to correlate the surfactant cmc with ita solubilizing power. From our results, we conclude that, in all cases the Re parameters does not seem to be dependent to the cmc of these surfactants in the buffered working medium. Moreover, these parameters may be correlated with the biochemical and toxicological effects of these surfactants in "in vivo" and "in vitro" methods in which the C12-SO4 has been described as the most irritating.2sH The agreement between our results and those of toxicological studies suggeststhe future w e of liposomes as a biomimetic model which could become an alternative method to animal testing in surfactant studies.
Acknowledgment. We acknowledge the expert technical assistance of Mr. G. von Knorring. (22) Singer, E. J.; Pittz, In Surfactants in Cosmetics; Rieger, M. M., Eds.; Marcel Dekker, Inc.: New York, 1985; Vol 16, Chapter 6. (23) Kligman, A. M.; Woolding, J. Invest. Dermatol. 1967,49, 78. (24) Dugard, P. H.; Scheuplein, J. Invest. Dermatol. 1973,60, 26.
