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Langmuir 1992,8, 2422-2426
Alteration of Permeability of Neutral and Electronegatively Charged Liposomes by Alkyl Sulfate Surfactants A. de la Maza,* J. L. Parra, and J. Sanchez Leal Instituto de Tecnologla Quimica y Textil, Comejo Superior de Investigaciones Cientificas (C.S.I.C.),Calle Jorge Girona, 18-26, 08034 Barcelona, Spain Received October 15, 1991. In Final Form: June 3, 1992 The partition coefficients have been determined for a series of anionic alkyl sulfate surfactants (alkyl chain lengths C10,C12, and C14) partitioning between the aqueous phase and the lipid bilayer of neutral or electronegativelycharged liposomes. The release of the fluorescent agent 6-carboxyfluoresceininduced by the surfactants was studied before these surfactants perturbed the bilayer architecture. Unilamellar liposomes formed by egg phosphatidylcholineplus or minus phosphatidic acid were used. From the results obtained we assume that there was a strong correlation between the partition coefficients and the ability of the different surfactants to modify the permeability of liposomes. Likewise, the results suggest that the hydrophobic interactions are the main forces involved in the alteration of the permeability of lipid bilayers although the electrostatic forces play a significant role in the modification of this parameter. Introduction Liposomes are currently considered to be potential tools for the controlled release of drugs, DNA transfer, or red blood cell substitutes.' Liposome tightness is impaired by their interaction with amphiphilic molecules, particularly when the vesicles are delivered into physiological fluids. Bearing in mind this range of liposomeapplications, it is important to consider the manner in which liposomal content is released in the presence of foreign molecules which may damage the phospholipid bilayers. Much has been written on the principles governing the interaction of Surfactants with phospholipid bilayers which lead to the breakdown of lamellar structures and the formation of surfactant-lipid mixed micelle^.^-^ However, several aspects involving the permeability alterations of liposomes at subsolubilizing concentrations have not been studied systematically. At these concentrations, surfactants incorporated into phospholipid bilayers cause changes in their physical properties.5 An obvious consequence of such perturbations could be a change in membrane permeability.6 At such concentrations it is generally accepted718that the equilibrium partition of the surfactant between the bilayer and the aqueous medium governs the incorporation of surfactants into the bilayer. The partition coefficient of surfactants defined as the relationship between both surfactant concentrations in the lipid bilayer per mole of lipid and in the aqueous surrounding medium can provide useful information about this distribution. In the present study, we investigated the partition coefficients of an alkyl sulfate series (with a specific alkyl chain length (210, (212, and C14), namely, sodium decyl, dodecyl, and tetradecyl sulfate (C10S04,C12S04, C14S04, respectively) in neutral or negatively charged unilamellar liposomes. For this purpose a series of experiments based (1) Gregoriadis, G. Liposome Technology, 4rd ed.; CRC Press: Boca Raton, FL, 1986; Vol. 111. (2) Helenius, A.; Simons, K. Biochim. Biophys. Acta 1975, 415, 29. (3) Lichtenberg, D.; Robson, R. J.; Dennis, E. A. Biochim. Biophys. Acta 1983, 737, 285. (4) Urbaneja, M. A.; Alonso, A.; Gonzalez-Mafias, J. M.; Gofii, F. M.; Partearroyo, M. A.; Tribout, M.; Paredes, S. Biochem. J. 1990,270,305.
( 5 ) Schubert, R.; Beyer, H.; Wolburg, H.; Schmidt, K. H. Biochemistry
1986.. 25.~5263. ~-, (6) De la Maza, A.; Sanchez Leal, J.; Parra, J. L.; Garcia, M. T. Ribosa, I. J. Am. Oil Chem. SOC. 1991,68,315. (7) Jackson, M. L.; Schmidt, C. F.; Lichtenberg, D.; Litman, B.; Albert, A. D. Biochemistry 1982,21, 4576. (8)Lichtenberg, D. Biochim. Biophys. Acta 1985, 821, 470.
on the measurement of the release of half the 6-carboxyfluorescein from the interior of liposome vesicles were carried out in order to determine the main factors involved in the alteration of the permeability of lipid bilayers by these surfactantsgJo and to establish a criterion for the evaluation of the activity of these surfactants in phospholipid vesicles. Materials Phosphatidylcholine (PC) was purified from egg lecithin (Merck) according to the method of Singleton et al.ll and was shown to be pure by thin-layer chromatography. Phosphatidic acid (PA) from egg yolk lecithin was purchased from Sigma Chemical Co. (St. Louis, MO). Both lipids were stored in chloroform under nitrogen at -20 "C until use. Anionic surfactantsCIOSOI,ClzSO,, and ClrSOr were obtained from Merck and further purified by a column chromatographic method.'* Piperazine-1,4-bis(2-ethanesulfonicacid) (PIPES buffer) obtained from Merck was prepared as 20 mM PIPES adjusted to pH 7.2 with NaOH, containing 110 mM NazS04. Polycarbonate membranes and membrane holders were purchased from Nucleopore (Pleasanton,CA). 6-Carboxyfluorescein (CF),was obtained from Eastman Kodak (Rochester,NY) and further purified by a column chromatographic method.13 Milli Q (Millipore)water was used in these experiments. Methods Preparation of Unilamellar Vesicles. Unilamellar vesicles of a defined size (100nm) were prepared by the extrusionof large unilamellar vesicles obtained by the reverse phase evaporation method14J5based on the method of Szoka and Papahadjopoulos.lB The required amounts of lipids (PC or PC/PA 9:land 8 2 molar ratio) in organic solution were mixed and dried in a round-bottom flask, evaporated under vacuum, and left overnight under reduced pressure in order to remove solvent traces. The lipids were then (9) Parker, C. A. Photoluminiscence of Solutions; Elsevier: New York, 1968; Chapter 4, p 303. (10) Weinstein, J. N.; Yoghikami, S.; Henkart, P.; Blumenthal, R.; Hagins, W. A. Science 1976,195,489. (11) Singleton, W. S.; Gray, M. S.; Brown, M. L.; White, J. L. J. Am. Oil Chem. SOC. 1965,42, 53. (12) Rosen, M. J. J. Colloid Interface Sci. 1981, 79, 587. (13) Ralston, E.; Hjelmeland, L. M.; Klausner, R. D.; Weinstein, J. N.; Blumenthal, R. Biochim. Biophys. Acta 1981,649, 133. (14) Rigaud, J. L.; Bluzat, A.; Buschlen, S. Physical Chemistry of Transmembrane Ion Motion; Elsevier: Amsterdam, 1983. (15) Rigaud, J. L.; Bluzat, A,; Buschlen, S. Biochem. Biophys. Res. ~. Commun.-1983, 111, 373. (16) Szoka, F.; Papahadjopoulos, D. Liposomes: Preparation and Characterization; Elsevier: Amsterdam, 1981; Chapter 3.
0743-7463/92/2408-2422$03.00/00 1992 American Chemical Society
Langmuir, Vol. 8, No. 10,1992 2423
Partition Coefficients of Surfactants in Liposomes redissolved in diethyl ether after which the PIPES buffer containing 10 mM CF was added to the etheral solution of phospholipids. Gentle sonication led to the formation of a water in oil (W/O) type emulsion. After the diethyl ether was evaporated under reduced pressure, a viscous gel was formed. The elimination of the final traces of the organic solvent transformed the gel into a liposome suspension. Unilamellar vesicles (of a uniform size distribution) were obtained by successive extrusion of vesicle suspensions through 800-,400-, 200-,and 100-nmpolycarbonatemembranes." Vesicles were freed of unencapsulated material by passage through Sephadex G-50 (Pharmacia, Uppsala, Sweden)) by column chromatography. All liposomes were allowed to equilibrate for at least 1 h at room temperature. The range of phospholipid concentration in liposome suspensions studied was 0.1-1.0 mM. Surface Tension Measurements and Critical Micelle Concentration Determinations. Surface tension values were measured by the ring methodla using a Lauda tensiometer 7201. The apparent values of surface tension were corrected using the Harkins-Jordan factors. The criticalmicelle concentrations (cmc) of alkyl sulfate surfactants in PIPES buffer were determined by plotting the equilibrium surface tension values versus log concentration. Quasielastic Light Scattering. The mean vesicle size and polydispersity of unilamellar liposome preparations were determined by photon correlation spectroscopy (Malvern Autosizer IIc). Samples were adjusted to an adequate concentration range with PIPES buffer. The measurements were made at 25 "C and a scattering angle of 90". Phosphorus Estimation. The phospholipid concentration of the liposome vesicles was determined by the ascorbic acid spectrophotometric method for total phosphorus e~timati0n.l~ Monitoring the Release of CF from Liposomes. Liposome suspensionscontaining a high concentration of CF in their interior aqueous compartments show little fluorescence. Upon release of CF from the interior to the bulk aqueous phase, the solution fluoresces s t r ~ n g l y .Permeability ~ changes of liposome bilayers induced by surfactants can therefore be determined quantitatively by monitoring any increase in the fluorescence.20s21Fluorescence measurements at 25 "C were performed on a Shimadzu RF-540 spectrofluorophotometer using an excitation wavelength of 495 nm and emission of 515.4 nm. The general procedure to assess the effects of surfactants on the release of liposomal contents consists of treating aliquots of liposomes in buffered medium (loaded with CF) with identical volumes of buffered solutions containing different surfactant concentrations. Afterward a measure of the proportion of the fluorescent dye released was carried out. The amount of released CF was calculated by means of the following equation2* % CF release = 4-10
I , - Io
(1)
where lois the initial fluorescence intensity of the CF-loaded liposome suspension at 515.4 nm in the presence of any surfactant and I. is the fluorescence intensity at 515.4 nm after destroying the liposomes by addition of Triton X-100 (60 pL of 10% (v/v) aqueous Triton X solution to 2.0 mL of liposome suspension). It corresponds to the fluorescence intensity at the same wavelength measured 40 min after adding the surfactant solution to liposome suspensions. Partition Coefficients. In order to compare results obtained for different Surfactants, a simple parameter is required. Throughout this work, we have used a partition coefficient of (17) Szoka, F.; Olson, F.; Heath, T.;Vail, W.; Mayer, E.; Papahadjopoulos, D. Eiochim. Eiophys. Acta 1980,601, 559. (18) Lunkenheimer, K.; Wantke, D. Colloid Polym. Sci. 1981, 259, 354. (19) Rand,
M.C.; Greenberg, A. E.; Taraa, M. J. Standard Methods FortheExamimtionof Waterand Wastewater;American PublicHealth Association: Washington, 1976; p 466. (20) Patemostre, M.T.; Roux, M.; Rigaud, J. L. Biochemistry 1988, 27, 2668. (21) Ruiz, J.; Goiii, F. M.; Alonso, A. Eiochim. Eiophys. Acta 1988, 937. .~ 127. (22) Sunamoto, J.; Iwamoto, K.; Ikeda, H.; Furuse, K. Chem. Pharm. Bull. 1983,31, 4230.
surfactants between lipid bilayer and aqueous medium defined as1,8
where SW and S g are concentrations of surfactant in the aqueous medium and bilayer, respectively, for a system containing PL (mM phospholipids) and ST(mM total surfactant concentration). Defining the effective ratio of surfactant to phospholipid, Reff, as the ratio between surfactant concentration into bilayers (Se) and the total phospholipid concentration of liposomes (PL), it follows that (3)
Partition coefficients were determined experimentally plotting surfactant concentrations resulting in a half-maximal value of CF release versus liposomephospholipid concentration. A linear relationship is established which could be described by the equation
ST = Sw + Ref@)
(4)
where the effective surfactant to phospholipid molar ratio R,n and the aqueous concentration of surfactant SWare respectively the slope and the ordinate at the origin (zero phospholipid concentration).
Results and Discussion Particle Size Distribution of Liposome Preparations. The particle size of liposomes in the suspension in the range of starting phospholipid concentrations 0.1-1.0 mM varied little and showed similar values around 100 nm in all cases. In addition, the polydispersity index values were lower than 0.1, indicating that the size distribution was very homogeneous. Permeability Studies. It is known that, in lipid/ surfactant systems, complete equilibrium may take several hour^.^^,^^ However, a substantial part of the surfactant effect takes place 30 min after being added to the liposomes.21 In order to determine the time needed to obtain a constant rate of CF release of liposomes in the range of the phospholipid concentration investigated (0.165and 0.990 mM),a kinetic study of the interaction of liposomes with alkyl sulfates was carried out. For these testa neutral or electrically charged liposomes (PC:PA, 100, 91, and 8:2 molar ratios) were treated with C10S04, C12S04, and c14so4 at subsolubilizing concentrations, and subsequent changes in permeability were studied as a function of time. A simple example of C14S04 is shown in Figure 1. It can be seen that the permeability kinetics of neutral or negatively charged liposomes by C14S04 (surfactant concentration 0.1 mM) shows inhibition in the CF release which depends mainly on the negative charge of lipid bilayers, especially in the first stage of the process. This effect could be attributed to the electrostatic repulsion between the ionic head of surfactants and the negatively charged bilayers. In all cases approximately 40 min is required to obtain an equilibrium release at both low (graph A) and high (graph B) phospholipid concentrations. Similar resulta for Cl&04 and C&04 were also obtained. Likewise, Figure 2 shows the permeability kinetics of negatively charged liposomes (PC:PA 9:l molar ratio) caused by C1~S04(1.0mM), C12S04 (0.15 mM), and c14SO4 (0.15 mM) surfactants in the same range of the phospholipid concentration (0.1 and 1.0 mM). The ~
~~
~~~~
(23) Lichtenberg, D.; Zilberman, Y.; Greenzaid, P.; Zamir, S. Biochemistry 1979,18,3517. (24) Alonso, A.;Urbaneja, M. A.; Carmona, F. G.; Chovas,F.G.;G6mezFernhdez, J. C.; Goiii, F. M.Biochim. Biophys. Acta 1987,902, 237.
de la Maza et al.
2424 Langmuir, Vol. 8, No.10,1992
50.
40.
30.
20.
i o 20 50 a e 60 tlnw(mln1 CO 20 So 40 e atin*(minl A PI*SWY k . 0 . l aY B ~ i r ic ~iml.omY . Figure 1. Time curve of the release of CF trapped in neutral and negatively charged unilamellar vesicles (PC:PA molar ratio 10:0,9:1,and 82) caused by Cl4SO4 (surfactantconcentration 0.1 mM). The phospholipid concentrations of liposome were 0.1 mM (graph A) and 1.0 mM (graph B).
WRelllDlOl Trappid Dye
'1
XReIea of
A
'1
PF7
BO
B
Trapped pls
iK
20
io
io
20
30
40
50
60
time(mln1
io
20
M
40
50
aotlne(min)
A Phospholipid Cmc 0 1 mM 6 Phmpholipld Cone 1 OmM Figure 2. Time curve of the release of CF of negativelycharged liposomes (PC:PA molar ratio 9:l) caused by C&04 (1.0 mM), C&O, (0.15 mM), and Cl4SO4 (0.15 mM) surfactant. Phospholipid concentrations were 0.1 and 1.0 mM.
influenceof the hydrophobic interactions on the alteration of CF release of negatively charged liposomes is clearly observed. Thus, the presence of C14S01surfactant results in an increasing effect on CF release of liposomes,whereas the C1oSO4 surfactant shows a smaller capacity. Similar tendencies were observed when these surfactants were applied to both neutral or more negatively charged liposomes (PC:PA 8 2 molar ratio). Bearing in mind these results, we may assume that the electrostatic interactions between the alkyl sulfate surfactants and the negatively charged liposomes only modulate the permeability alterations caused by these surfactants in the lipid bilayer, the hydrophobic interaction being the main factor involved in the modification of these parameters. In order to determine the partition coefficient of surfactants between aqueous media and lipid bilayers, a systematicinvestigation of liposome permeability changes caused by the addition of surfactant to the neutral and negatively charged liposomes was carried out. To this end, changes in CF release from liposomes (lipid concentration from 0.1 to 1.0mM) versus surfactant concentration were determined 40 min after the surfactant addition at 25 "C. The CF release of liposome suspensions in the absence of Surfactants 40 min after preparation was negligible.
8
PC:PI k r r a t i o )
Figure 3. CF release caused by c&01(A),C&04 (B), and CUSO4 (C) surfactanta for neutral and electronegatively charged liposome suspensions (PC:PA molar ratios 100,91, and 82) at different phospholipid concentrations (e, 0.165 m M e, 0.330 mM; A, 0.495 mM A, 0.660 mM 0,0.852 mM; 0,0.990mM).
Figure 3showsthe values obtained when treating neutral and electronegatively charged liposomes with C1&04, C ~ Z S04, and C14S04surfactant. The results obtained for each surfactant investigated show similar tendencies in the range of the phospholipic concentration studied. The surfactant concentrationsresulting in a half-maximalvalue of CF release were obtained by graphs from these data. Figure 4 shows the half-maximalvalue of CF release for the studied surfactants represented versus phospholipid
Partition Coefficients of Surfactants in Liposomes
Langmuir, Vol. 8, No. 10, 1992 2425
Table I. Critical Midelle Concentration Values at 25 "C in PIPES Buffer, SW(Surfactant Concentration in Aqueous Medium), &fi (Effective Surfactant to Lipid Ratio), K (Partition Coefficient Values Resulting in 50% of CF Releare), and Regression Coefficienta of the Straight Linea of CloSO4, Clt804, and C&O, bilayer lipid composition cmc (PIPES) PCPA (100) PC:PA (91) PC:PA (82) (mM) Sw(mM) R ~ H K(mM-9 r2 Sw(mM) Rae K(mM-9 r2 Sw(mM) R.R K(mM-1) rz 1.257 1.116 1.12 0.998 1.192 1.265 1.06 0.987 1.294 cl@o4 2.4 1.270 0.98 0.994 0.083 0.253 3.04 0.989 0.089 0.253 2.84 0.990 0.094 0.255 2.71 ci&o4 0.60 0.988 0.038 0.140 3.68 0.992 0.042 0.145 0.17 3.45 0.990 0.046 0.152 Cl4SO4 3.30 0.990
Surfactant Concentration (mM)
Phospholipid Conc. (mM) .O
PO PA (molar rat io) Figure 4. Surfactantconcentrationsresultingin a half maximal value of CF releasefor CloS01, Cl~S04,and C14S04 versus phospholipid concentration,for neutral and electronegatively charged liposomes (PC:PA molar ratios, 10:0, 91,and 82).
concentration for neutral and electronegatively charged liposomes. A linear relationship was established in each investigated system. The straight lines obtained correspond to the aforementioned equation (4)
where the effective surfactant to phospholipid molar ratio Res and the aqueous concentration of surfactant SW are respectively the slope and the ordinate at the origin (zero phospholipid concentration). These results includingthe regression coefficients of the straight lines and the cmc values in the buffered medium are shown in Table I. It should be noted that all the SWvalues are in all cases smaller than the corresponding cmc values in the buffered medium. As the negative charge of liposomes increases, the SW shows increased values which attain the highest effect for C&O4 (PC:PA 8 2 molar ratio). In fact, the limiting Sw values in terms of partition coefficients must be always smaller than the corresponding cmc as a consequence of the favorable alternative of such surfactant to be incorporated into bilayers in front of the micelle formation. Given that SW represents the surfactant concentration in the aqueous medium which is needed to achieve the 50% of CF release (in equilibrium with the surfactant included in the lipid bilayer), these results suggest that
surfactant-liposome interactions are determined mainly by the action of surfactant monomers on the lipid bilayers, unlike the behavior of the surfactants in solubilization p r o ~ e s s ewhere s ~ ~ ~mixed micelle formation (including the phospholipid molecules) plays an important role. As regards the Reff values, it may be seen that the concentration of the surfactants in the liposomal bilayer increases in the series C14S04 to C12S04 to C&O4. The presence of PA in lipid bilayers results in a slight increase of these parameters, this increase being more significant for both Cl&04 and C&04 surfactants and negligible for C12S04. The quotient between Res and SW gives the partition coefficient of each surfactant between lipid bilayer and aqueous medium.718 From these data the surfactant which has a high K values is C14S04 (between 3.68 and 3.301, whereas C&O4 showslower values (between 0.98 and 1.12). The importance of the hydrocarbon chain length of alkyl sulfates with respect to the variation in the partition coefficient values can be established. Likewise, it should be noted that the partition coefficient values of surfactants are also slightly affected by the presence of negative charges in liposomes. The K values decrease as PA concentration in liposomes increases,which is inverse to what occurs with the R,ff and SW parameters. On comparison of the results of Figure 2 with the values given the Table I, a positive association between the
2426 Langmuir, Vol. 8, No. 10,1992
partition coefficient and the ability of the alkyl sulfate surfactants to modify the permeability of liposomes can be established. Inoue et alaz6demonstrate, when studying the incorporation of surfactant molecules, either charged or neutral phospholipid bilayer, by differential scanning calorimetry, that the main forces involved are hydrophobic in nature. Our results confirm the essential role of this hydrophobic effect on bilayer structures, which results in permeability alterations and partition coefficient changes. Electrostatic (26) Inoue, T.;Miyakawa,K.; Shimoulwa, R. Chem. Phys. Lipids 1986, 42, 261.
de la Maza et al.
interactions between the studied anionic alkyl sulfate surfactants and negatively charged liposomes could affect these interactions, especially in the early stages of the process where electrostatic repulsion could play a significant part. However, the partition coefficients of surfactants at the equilibrium are only slightly affected by the electrical nature of lipid bilayers.
Acknowledgment. We acknowledge the expert technical assistance of Mr. G. von Knorring. Registry No. CloSOdNa, 142-87-0; C&O,Na, 151-21-3;Clr-
SO,, 1191-50-0;6-carboxyfluorescein,3301-79-9.
