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Influence of the alkyl chain length of alkyl glucosides on their ability to solubilize phosphatidylcholine liposomes
Colloids and Surfaces A: Physicochemical and Engineering Aspects 193 (2001) 221– 229 www.elsevier.com/locate/colsurfa
Influence of the alkyl chain length of alkyl glucosides on their ability to solubilize phosphatidylcholine liposomes O. Lo´pez, M. Co´cera, J.L. Parra, A. de la Maza * Departamento de Tecnologı´as de Tensioacti6os, Instituto de In6estigaciones Quı´micas y Ambientales de Barcelona (I.I.Q.A.B.), Consejo Superior de In6estigaciones Cientı´ficas (C.S.I.C.), Calle Jorge Girona 18 -26, 08034 Barcelona, Spain Received 8 December 2000; accepted 12 March 2001
Abstract The solubilizing alterations caused by a series of alkyl glucosides (alkyl chain length ranging from C8 to C12) in neutral and electrically charged phosphatidylcholine (PC) liposomes were investigated. The surfactant to phospholipid molar ratios (Re) and the bilayer/aqueous phase partition coefficients (K) were determined by monitoring the changes in the static light scattering (SLS) of the system during solubilization. Liposomes were formed by PC, to which phosphatidic acid (PA) or stearylamine (SA) was added when required to increase the negative or positive surface charge. The fact that at the two interaction levels investigated (100 and 0% of SLS of the surfactant/PC systems), the free surfactant concentration for each surfactant was always comparable to its critical micelle concentration (CMC) indicates that the liposome solubilization was mainly ruled by the formation of mixed micelles. The rise in the surfactant CMC (decrease in its alkyl chain length) led to an increase in the surfactant ability to saturate or solubilize liposomes and inversely in an abrupt decrease in its bilayer affinity, regardless of the electrical charge of liposomes. The overall balance of these opposite tendencies shows that the octyl glucoside showed the highest ability to saturate and solubilize liposomes (lowest Re values), whereas the dodecyl glucoside exhibited the highest degree of partitioning into liposomes or affinity with these bilayer structures (highest K values). The use of C9-Glu reduced approximately 2.5 times the concentration needed to saturate and solubilize 1.0 mM PC liposomes with respect to that needed for C8-Glu, regardless of the type of electrical charge present in bilayers. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Alkyl glucoside surfactants; Phosphatidylcholine liposomes; Surfactant/lipid molar ratios; Surfactant partition coefficients; Surfactant critical micelle concentrations; Static light-scattering
Abbre6iations: C8-Glu, octyl glucoside (n-octyl b-D-glucopyranoside); C9-Glu, nonyl glucoside (n-nonyl b-D-glucopyranoside); C10-Glu, decyl glucoside (n-decyl b-D-glucopyranoside); C11-Glu, undecyl glucoside (n-undecyl b-D-glucopyranoside); C12-Glu, dodecyl glucoside (n-dodecyl b-D-glucopyranoside); CMC, critical micellar concentration; K, bilayer/aqueous phase surfactant partition coefficient; KSAT, bilayer/aqueous phase surfactant partition coefficient for liposome saturation; KSOL, bilayer/aqueous phase surfactant partition coefficient for liposome solubilization; PC, phosphatidylcholine; PA, phosphatidic acid; PIPES, piperazine-1,4 bis(2-ethanesulphonic acid); PI, polydispersity index; Re, effective surfactant/lipid molar ratio; ReSAT, effective surfactant/ lipid molar ratio for liposome saturation; ReSOL, effective surfactant/lipid molar ratio for liposome solubilization; r2, regression coefficient; SLS, static light-scattering; SA, stearylamine; SW, surfactant concentration in the aqueous medium; SW,SAT, surfactant concentration in the aqueous medium for liposome saturation; SW,SOL, surfactant concentration in the aqueous medium for liposome solubilization; SB, surfactant concentration in the bilayers. * Corresponding author. Tel.: +34-93-4006161; fax: +34-93-2045904. 0927-7757/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 7 - 7 7 5 7 ( 0 1 ) 0 0 6 9 8 - 7
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1. Introduction A number of studies have been devoted to the understanding of the principles governing the interaction of different surfactants with simplified membrane models such as phospholipid or stratum corneum lipid bilayers [1– 9]. This interaction leads to the breakdown of lamellar structures and to the formation of lipid– surfactant mixed micelles. A significant contribution in this area has been made by Lichtenberg [10], who postulated that the effective surfactant to lipid molar ratio (Re) producing saturation and solubilization of liposomes depends on the surfactant critical micellar concentration (CMC) and on the bilayer/aqueous medium distribution coefficients of these amphiphilic compounds (K). One of the most commonly used amphiphilic compounds in membrane solubilization and reconstitution experiments is the nonionic surfactant octyl glucoside, which is believed to be a ‘mild’ surfactant with respect to its denaturing effect on proteins. This surfactant also shows a relatively high CMC, this characteristic being appropriate for bilayer reconstitution [11–17]. However, the use in these two processes of alkyl glucosides with higher alkyl chain lengths than that for the conventional octyl glucoside has been little investigated in spite of their excellent hydrolysis stability, improved ecological properties and lower toxicity as it has been demonstrated in ‘in vivo’ tests using Daphnia magna and Photobacterium phosphoreum [18,19]. We earlier studied the vesicle– micelle phase transitions involved in the interaction of the nonionic surfactants octyl glucoside and dodecyl maltoside with phosphatidylcholine (PC) unilamellar liposomes [20– 22]. We also investigated the sublytic activity of a series of alkyl glucosides (alkyl chain length ranging from C8 to C12) on PC liposomes [23]. In the present work we seek to extend these investigations by characterizing the Re and K parameters of a these nonionic surfactants when saturated and solubilized neutral and electrically charged PC liposomes (containing phosphatidic acid (PA) or stearylamine (SA) when required to increase the negative or positive surface charge). This study
will provide new information about the influence of the surfactant hydrophobic tail and the electrical charge of liposomes on the saturation and solubilization of these vesicles by the alkyl glucosides investigated in order to evaluate the influence of the hydrophobic and hydrophilic forces on this process.
2. Materials and methods Phosphatidylcholine (PC) was purified from egg lecithin (Merck, Darmstadt, Germany) according to the method of Singleton [24] and was shown to be pure by thin-layer chromatography (TLC). Phosphatidic acid (PA) from egg yolk lecithin and stearylamine (SA) were purchased from Sigma Chemicals Co (St Louis, MO). Lipids were stored in chloroform under nitrogen at − 20°C until use. The nonionic surfactants n-octyl b-D-glucopyranoside (C8-Glu), n-nonyl b-D-glucopyranoside (C9-Glu), n-decyl b-D-glucopyranoside (C10-Glu), n-undecyl b-D-glucopyranoside (C11-Glu) and ndodecyl b-D-glucopyranoside (C12-Glu) were purchased from Sigma Chemicals Co. (St. Louis, MO). Piperazine-1,4 bis(2-ethanesulphonic acid) (PIPES) was obtained from Merck. PIPES buffer was prepared as 10 mM PIPES containing 110 mM Na2SO4 and adjusted to pH 7.20 with NaOH. Polycarbonate membranes and membrane holders were purchased from Nucleopore (Pleasanton, CA).
2.1. Liposome preparation and characterization Unilamellar liposomes (lipid composition PC or PC/PA, PC/SA 9:1 molar ratio) of a defined size were prepared by extrusion of large unilamellar vesicles obtained earlier by a reverse phase evaporation method [25]. Vesicles of about 100 nm were obtained by extrusion through 800, 400, 200 and 100 nm polycarbonate membranes at 25°C using a thermobarrel extruder (Lipex, Biomembranes Inc. Vancouver, Canada). The final volumes of liposome suspensions were adjusted with PIPES buffer containing 110 mM Na2SO4 at pH 7.20 to provide a
O. Lo´ pez et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 193 (2001) 221–229
final lipid concentration ranging between 0.1 and 10.0 mM. Liposome preparations were then annealed at 25°C for 30 min and immediately treated with the alkyl glucosides tested. The quantitative analysis of lipid composition and concentration of liposomes after preparation was determined using thin-layer chromatography coupled to an automated flame ionization detection system (TLC-FID) (Iatroscan MK-5, Iatron Labrotary Tokyo, Japan) [26]. The vesicle size distribution and polydispersity index (PI) of liposomes after preparation was determined with dynamic light-scattering measurements using a photon correlator spectrometer (Malvern Autosizer 4700c PS/MV; Malvern, England). The studies were made by particle number measurement [27]. Sample was adjusted to the appropriate concentration range with PIPES buffer and the measurements were taken at 25°C at a reading angle of 90°.
2.2. Parameters in6ol6ed in the interaction of surfactants with PC liposomes In the analysis of the equilibrium partition model proposed by Schurtenberger [28] for bile salt/lecithin systems, Lichtenberg [10] and Almog et al. [12] have shown that for a mixing of lipids (at a lipid concentration L (mM)) and surfactant (at a concentration ST (mM)), in dilute aqueous media, the distribution of surfactant between lipid bilayers and aqueous media obeys a partition coefficient K, given (in mM − 1) by K=
SB [(L +SB)·SW]
(1)
where SB is the concentration of surfactant in the bilayers (mM) and SW is the surfactant concentration in the aqueous medium (mM). For L \ \ SB, the definition of K, as given by Schurtenberger, applies: K=
SB Re = (L·SW) SW
(2)
where Re is the effective molar ratio of surfac-
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tant 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 Lichtenberg [10] and Almog [12] for different surfactant lipid mixtures over wide ranges of Re values. Given that the lipid concentration range used in liposomes is similar to that used by Almog to test his equilibrium partition model, the K parameter has been determined using this equation. The solubilization of neutral and electrically charged PC liposomes was characterized by two parameters termed ReSAT and ReSOL, according to the nomenclature adopted by Lichtenberg [10] corresponding to the Re ratios at which the SLS of the system starts to decrease with respect to the original value and shows no further decrease. These parameters corresponded to the surfactant to lipid molar ratios at which the surfactant: (a) saturated liposomes; and (b) led to a complete solubilization of these structures. Liposomes were adjusted to the appropriate lipid concentration. Equal volumes of the appropriate surfactant solutions were added to the liposomes and the resulting mixtures were left to equilibrate for 24 h. This time was chosen as the optimum period needed to achieve a complete equilibrium surfactant/liposome for the lipid concentration range used [1,8]. SLS measurements were made with a spectrofluorophotometer Shimadzu RF-540 (Kioto Japan) with both monochromators adjusted to 500 nm at 25°C [20]. The assays were carried out in triplicate and the results given are the average of those obtained. The determination of Re and SW parameters was carried out on the basis of the linear dependence existing between the surfactant concentrations required to saturate and solubilize liposomes at the lipid concentration (L), which can be described by the equations: SSAT = SW,SAT + ReSAT·[L]
(4)
SSOL = SW,SOL + ReSOL·[L]
(5)
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where SSAT and SSOL are the total surfactant concentrations. The surfactant to lipid molar ratios ReSAT and ReSOL and the aqueous surfactant concentrations SW,SAT and SW,SOL are in each curve respectively the slope and the ordinate at the origin (zero phospholipid concentration). The KSAT and KSOL parameters (bilayer/aqueous phase surfactant partition coefficients for saturation and complete solubilization of liposomes) were determined from the Eq. (3).
3. Results and discussion The vesicle size distribution of liposomes after preparation varied little showing in all cases a similar value of about 100 nm (polydispersity index (PI) was always lower than 0.1) thereby indicating that the liposomes had a homogeneous size in all cases. The vesicle size after addition of equal volumes of PIPES buffer and equilibration for 24 h at 25°C showed always values similar to those obtained after preparation, with a slight rise in the PI (between 0.11 and 0.13). Hence, the liposomes investigated were reasonably stable in the absence of surfactants under the experimental conditions used. We reported the variation of the surface tensions of the surfactant solutions in PIPES buffer as a function of total surfactant concentration, as well as their CMC [25]. The increase in the alkyl chain length drastically reduced the surfactant CMC’s (Table 2) and slightly reduced the surface tensions in all cases (from 31 mN m − 1 for C8-Glu to 28.5 mN m − 1 for C12-Glu). Given that the surface tension at the CMC (kCMC) is used as one of the criteria of surface activity of the system, (the lower the kCMC, the higher the surface activity
[29,30]), we may assume that the longer the surfactant hydrophobic tail the higher its surface activity in the system.
3.1. Parameters in6ol6ed in the surfactant/liposomes interaction To determine the surfactant partition coefficients between bilayers and water, we first studied the validity of the equilibrium partition model proposed by Lichtenberg and Almog et al. [10,12] based on the Eq. (1) for the surfactants investigated. According to 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 demostrated the validity of this model for C8-Glu/ PC liposome systems in the range of lipid and surfactant concentration used in the present work [12]. To test the validity of the model for the alkyl glucosides tested using neutral and electrically charged PC liposomes, vesicles were mixed with varying sublytic surfactant concentrations (ST). The resultant surfactant-containing vesicles were then spun at 140 000× g at 25°C for 4 h to remove the vesicles [12]. No lipid was detected in the supernatants [26]. The surfactant concentration in the supernatants (SW) was determined by HPLC [31], and their concentration in the bilayers was calculated (SB = ST − SW). The results of the experiments in which SB and SW were measured (for each surfactant and type of liposomes in the same concentration range used to determine K) were plotted in terms of the dependence of L/SB on 1/SW. Straight lines were obtained for the
Table 1 Regression coefficients (r 2) of the straight lines corresponding to the dependence of L/SB on 1/SW for C8-Glu, C9-Glu, C10-Glu, C11-Glu, and C12-Glu
PC PC/PA PC/SA
C8-Glu (r 2)
C9-Glu (r 2)
C10-Glu (r 2)
C11-Glu (r 2)
C12-Glu (r 2)
0.991 0.990 0.992
0.992 0.991 0.991
0.989 0.990 0.993
0.994 0.991 0.991
0.990 0.992 0.989
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Fig. 1. Percentage change in static light-scattering (SLS) of pure PC liposomes, (PC concentration ranging from 1.0 to 10.0 mM), induced by the presence of increasing amounts of C8-Glu. Symbols: PC concentration (9) 1.0 mM; ( ) 3.0 mM; () 5.0 mM; ( ) 6.0 mM; () 8.0 mM; ( ) 10.0 mM.
surfactants tested. The corresponding regression coefficients (r 2) for PC, PC/PA, and PC/SA liposomes are given in Table 1. Straight lines were dependent on L and intersected with the L/SB axis always at − 0.97 90.11, −0.96 9 0.13, and − 0.989 0.13 for PC, PC/PA, and PC/SA liposomes, respectively. Both the linearity of these dependences and the proximity of the intercept to −1 support the validity of this model to determine K for these surfactants/liposome systems. To determine the Re and SW parameters, a systematic investigation of SLS variations in neutral and charged PC liposomes caused by the addition of the surfactants tested was carried out for various lipid concentrations (depending on the solubility of each surfactant). The curves obtained for C8-Glu (PC conc ranging from 1.0 to 10.0 mM) are given in Fig. 1. The addition of surfactant led in all cases to an initial increase and a subsequent fall in the scattered intensity of the system until a low constant SLS value was achieved for complete bilayer solubilization via mixed micelle formation [10]. Surfactant concentrations producing 100% (SSAT) and 0% (SSOL) of
SLS were obtained for each lipid concentration by graphical methods. The arrows A and B (curve for PC liposomes, conc. 10.0 mM in Fig. 1) correspond to these two values. When plotting the surfactant concentrations thus obtained versus lipid concentration, curves were obtained in which an acceptable linear relationship was established in each case. The straight lines obtained corresponded to the Eq. (4) and Eq. (5) from which the Re and SW parameters were determined. The results obtained for neutral and charged PC liposomes for each surfactant tested including the regression coefficients (r 2) of the straight lines are given in Tables 2–4, respectively. Free surfactant concentrations (SW,SAT, SW,SOL) were always comparable to their CMC’s, regardless of the type of liposome used and the surfactant alkyl chain length. This indicates that the liposome solubilization was mainly ruled by formation of mixed micelles, in line with the results reported for the interaction of C8-Glu and dodecyl maltoside with PC liposomes [20–22]. These results extend to the alkyl glucosides studied the
226
C8-Glu C9-Glu C10-Glu C11-Glu C12-Glu a
CMC (mM)
SW,SAT (mM)
SW,SOL (mM)
ReSAT mole mole−1
ReSOL mole mole−1
KSAT (mM−1) KSOL (mM−1) r 2 (SAT)
r 2 (SOL)
18.0 5.6 1.80 0.58 0.18
17.8 5.6 1.75 0.57 0.17
18.3 5.65 1.81 0.59 0.18
1.3 1.4 1.6 2.0 2.8
3.6 3.9 4.5 5.0 6.0
0.03 0.10 0.35 1.17 4.33
0.990 0.998 0.997 0.993 0.994
0.04 0.14 0.45 1.41 4.76
0.992 0.997 0.996 0.999 0.992
The critical micelle concentrations of the surfactant tested is included. It also includes the regression coefficients (r 2) of the straight lines obtained.
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Table 2 Surfactant to lipid molar ratios (Re), partition coefficients (K), and surfactant concentrations in the aqueous medium (SW) resulting in the interaction (bilayer saturation and solubilization) of alkyl glucosides with neutral PC liposomesa
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generally admitted assumption that the free surfactant concentration (SW) must reach the CMC before solubilization starts to occur [10,20]. The Re values clearly increased from liposome saturation (ReSAT) to complete solubilization (ReSOL), regardless of the type of liposome used and the surfactant alkyl chain length. Given that the surfactant capacity to saturate or solubilize liposomes is inversely related to the Re values, the maximum activity at these two levels corresponded in all cases to the C8-Glu (lowest Re values), whereas the minimum corresponded to the C12-Glu (highest Re values). Thus, the lower the surfactant alkyl chain length the higher its ability to saturate or solubilize PC liposomes regardless of their positive or negative charge. Partition coefficients of the surfactants tested between the bilayers and water for saturation (KSAT) and complete liposome solubilization (KSOL) for neutral and electrically charged PC liposomes (Tables 2– 4) show that the C12-Glu molecules had the highest degree of partitioning into bilayers (maximum K values), whereas the C8-Glu showed the lowest (minimum K values). These findings could be correlated with the aforementioned higher surface activity of the alkyl glucosides with higher hydrophobic tails (lower kCMC values) [29,30]. It is noteworthy that the K values always increased from bilayer saturation to complete liposome solubilization regardless of the surfactant alkyl chain length. This means that the affinity of the surfactant molecules with the lipids building liposomes was greater in the complete bilayer solubilization (micellization process) than during the earlier step of bilayer saturation (for-
227
mation of mixed vesicles). This finding contrasts with the results reported by Ueno, who postulated that in the interacion of C8-Glu with PC liposomes K was independent of the surfactant concentration at low free surfactant concentrations [32]. The relationship between the KSAT and KSOL for each surfactant tested may be correlated with the dynamic surfactant/PC equilibrium existing between the transition steps from mixed vesicles to mixed micelles. Comparison of these two parameters in neutral and charged PC liposomes reveals that the higher the surfactant alkyl chain length the higher the quotient between both values (KSAT/KSOL) regardless of the positive or negative charge of liposomes. Hence, at the interaction level for bilayer saturation the degree of partitioning of surfactants into liposomes relatively increased with respect to that for complete liposome solubilization as the surfactant alkyl chain length rose. Hence, the increase in the length of the surfactant hydrophobic tail in addition to improve the partitioning of surfactant molecules into bilayers (increasing K values) also resulted in a relative decrease in their ability to be assocciated with the molecules building liposomes to form mixed micelles. Possibly, the first order phase transition from mixed vesicles into mixed micelles appears to be relatively hampered by the increasing surfactant hydrophobic tail. If the Re and K values for each surfactant tested in pure PC liposomes are plotted as a function of the surfactant CMC the graphs shown in Figs. 2 and 3 are obtained. A decrease in ReSAT and ReSOL occurred as the surfactant CMC rose
Table 3 Surfactant to lipid molar ratios (Re), partition coefficients (K), and surfactant concentrations in the aqueous medium (SW) resulting in the interaction (bilayer saturation and solubilization) of alkyl glucosides with PC/PA liposomesa
C8-Glu C9-Glu C10-Glu C11-Glu C12-Glu a
SW,SAT (mM) SW,SOL (mM) ReSAT mole mole−1
ReSOL mole mole−1
KSAT (mM−1)
KSOL (mM−1) r 2 (SAT)
r 2 (SOL)
18.0 5.6 1.77 0.58 0.18
3.7 3.9 4.6 5.1 6.1
0.03 0.11 0.35 1.17 4.13
0.04 0.14 0.43 1.42 4.52
0.992 0.993 0.996 0.998 0.994
18.4 5.7 1.9 0.59 0.19
1.4 1.5 1.6 2.1 2.9
It also included the regression coefficients (r 2) of the straight lines obtained.
0.991 0.995 0.992 0.993 0.996
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228
Table 4 Surfactant to lipid molar ratios (Re), partition coefficients (K), and surfactant concentrations in the aqueous medium (SW) resulting in the interaction (bilayer saturation and solubilization) of alkyl glucosides with PC/SA liposomesa
C8-Glu C9-Glu C10-Glu C11-Glu C12-Glu a
SW,SAT (mM) SW,SOL (mM) ReSAT mole mole−1
ReSOL mole mole−1
KSAT (mM−1)
KSOL (mM−1) r 2 (SAT)
r 2 (SOL)
18.0 5.6 1.78 0.58 0.19
3.7 4.0 4.5 5.0 6.2
0.03 0.10 0.35 1.15 3.91
0.04 0.14 0.44 1.38 4.53
0.992 0.991 0.993 0.994 0.998
18.3 5.7 1.86 0.60 0.19
1.4 1.6 1.7 2.0 2.9
0.994 0.994 0.992 0.989 0.990
It also included the regression coefficients (r 2) of the straight lines obtained.
(or the alkyl chain length decreased). The Re fall was more pronounced at low CMC values for both parameters in all cases. The rise in the surfactant CMC also resulted in an abrupt fall in K, which was also specially noticeable at low CMC values (Fig. 3). Thus, the degree of partitioning of these surfactants into liposomes (or affinity with these structures) drastically decreased as the surfactant CMC increased. Similar results were obtained when plotting the Re and K values for PC/PA and PC/SA liposomes (results not shown). Hence, two opposite trends may be observed when comparing the variation of Re and K versus the surfactant CMC. The rise in the surfactant CMC resulted during the transition steps from mixed vesicles to mixed micelles in a progressive increase in the surfactant ability to saturate and solubilize liposomes and inversely in an abrupt decrease in the partitioning of these surfactants between the bilayers and the aqueous phase. Thus, for C12-Glu although a higher number of surfactant molecules were needed to saturate or solubilize liposomes, these molecules showed an increased affinity with these structures with respect to that exhibited by the more active C8-Glu. The overall balance of these two opposite tendencies leads to the conclusion that the C8-Glu was the more equilibrated surfactant in terms of hydrophilic-lipophilic balance, followed by the C9Glu, in order to obtain an improved interaction and, consequently, a maximum solubilizing effect on liposomes. It is noteworthy that the electrostatic interactions do not seem to play an important role in this process given that the presence of
negative or positive charges in liposomes only slightly increased and decreased the Re and K values, respectively (Tables 2–4). Hence, in general terms the resistance of charged liposomes to be saturated or solubilized by surfactants slightly increased with respect to neutral vesicles, whereas the surfactant affinity with these charged vesicles slightly decreased, these two effects being independent of the type of charge present in liposomes. From a practical viewpoint, comparison of the surfactant concentration (mM) needed to saturate
Fig. 2. Variation in surfactant to phospholipid molar ratios (Re) for the alkyl glucosides tested versus the surfactant CMC using PC unilamellar liposomes. Symbols: ( ) liposome saturation (ReSAT); () liposome solubilization (ReSOL).
O. Lo´ pez et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 193 (2001) 221–229
Fig. 3. Variation in bilayer/aqueous phase partition coefficients (K) for the alkyl glucosides tested versus the surfactant CMC using PC unilamellar liposomes. Symbols: ( ) liposome saturation (KSAT); () liposome solubilization (KSOL).
and solubilize liposomes for C8-Glu and C9-Glu surfactants and for 1.0 mM PC concentration reveals that when using C9-Glu a concentration approximately 2.5 times lower than that needed of C8-Glu was in all cases sufficient to produce the same effects in these bilayer structures. These findings open up new avenues in the application of this surfactant in solubilization of biological membranes, given its appropriate solubility in water, excellent hydrolysis stability and relatively high CMC, and may be considered as an interesting alternative with respect to the use of the conventional octyl glucoside.
Acknowledgements This work was supported by funds from D.G.I.C.Y.T. (Direccio´ n General de Investigacio´ n Cientı´fica y Te´ cnica), Spain.
References [1] M.A. Urbaneja, A. Alonso, J.M. Gonza´ lez-Man˜ as, F.M. Gon˜ i, M.A. Partearroyo, M. Tribout, S. Paredes, Biochem. J. 270 (1990) 305.
229
[2] T. Inoue, T. Yamahata, R. Shimozawa, J. Coll. Interf. Sci. 149 (1992) 345. [3] K. Edwards, M. Almgren, Langmuir 8 (1992) 824. [4] D.T. Downing, W. Abraham, B.K. Wegner, K.W. Willman, J.M. Marshall, Arch. Dermatol. Res. 285 (1993) 151. [5] U. Kragh-Hansen, M. le Marie, J.P. No¨ el, T. Gulik-Krzywicki, J.V. Møller, Biochemistry 32 (1993) 1648. [6] M.B. Ruiz, A. Prado, F.M. Gon˜ i, A. Alonso, Biochim. Biophys. Acta 1193 (1994) 301. [7] M. Silvander, G. Karlsson, K. Edwards, J. Coll. Interf. Sci. 179 (1996) 104. [8] M.A. Partearroyo, A. Alonso, F.M. Gon˜ i, M. Tribout, S. Paredes, J. Coll. Interf. Sci. 178 (1996) 156. [9] J. Cladera, J.L. Rigaud, J. Villaverde, M. Dun˜ ach, Eur. J. Biochem. 243 (1997) 798. [10] D. Lichtenberg, Biochim. Biophys. Acta. 821 (1985) 470. [11] P.K. Vinson, Y. Talmon, A. Walter, Biophys. J. 56 (1989) 669. [12] S. Almog, B.J. Litman, W. Wimley, J. Cohen, E.J. Wachtel, Y. Barenholz, A. Ben-Shaul, D. Lichtenberg, Biochemistry 29 (1990) 4582. [13] D.F. Cully, P.S. Paress, Mol. Pharmacol. 40 (1991) 326. [14] S.C.R. Lummis, I.L. Martin, Mol. Pharmacol. 41 (1992) 18. [15] N. Oku, J. Tsudera, K. Kurohani, S. Okada, Chem. Pharm. Bull. 44 (1996) 1928. [16] I.J. Tetlo, C.G. Bowsher, M.J. Emes, Biochem. J. 319 (1996) 717. [17] M. Seras, K. Edwards, M. Almgren, G. Carlson, M. Ollivon, S. Lesieur, Langmuir 12 (1996) 330. [18] M.T. Garcı´a, I. Ribosa, E. Campos, J. Sanchez Leal, Chemosphere 35 (1997) 545. [19] D. Balzer, Specialist Surfactants, in: Blackie Academic and Professional, Chapman and Hall Press, London, 1997. [20] A. de la Maza, J.L. Parra, Eur. J. Biochem. 226 (1994) 1029. [21] A. de la Maza, J.L. Parra, J. Am. Oil Chem. Soc. 73 (1996) 877. [22] A. de la Maza, J.L. Parra, Biophys. J. 72 (1997) 1668. [23] A. de la Maza, L. Coderch, P. Gonzalez, J.L. Parra, J. Control Release 52 (1998) 159. [24] W.S. Singleton, M.S. Gray, M.L. Brown, J.L. White, J. Am. Oil Chem. Soc. 42 (1965) 53. [25] A. de la Maza, J.L. Parra, J. Control Release 37 (1995) 33. [26] R.G. Ackman, C.A Mc. Leod, A.K. Banerjee, J. Planar Chromatogr. 3 (1990) 450. [27] A. de la Maza, A.M. Manich, L. Coderch, P Bosch, J.L. Parra, J. Coll. Surf. A 101 (1995) 9. [28] P. Schurtenberger, N. Mazer, W. Ka¨ nzig, J. Phys. Chem. 89 (1985) 1042. [29] M.J. Rosen, J. Am Oil Chem. Soc. 51 (1974) 461. [30] M.J. Rosen, J. Coll. Interf. Sci. 79 (1981) 587. [31] H. Seino, T. Uchibori, T. Nishitani, S. Inamasu, J. Am. Oil Chem. Soc. 61 (1984) 1761. [32] M. Ueno, Biochemistry 28 (1989) 5631.
Influence of the alkyl chain length of alkyl glucosides on their ability to solubilize phosphatidylcholine liposomes O. Lo´pez, M. Co´cera, J.L. Parra, A. de la Maza * Departamento de Tecnologı´as de Tensioacti6os, Instituto de In6estigaciones Quı´micas y Ambientales de Barcelona (I.I.Q.A.B.), Consejo Superior de In6estigaciones Cientı´ficas (C.S.I.C.), Calle Jorge Girona 18 -26, 08034 Barcelona, Spain Received 8 December 2000; accepted 12 March 2001
Abstract The solubilizing alterations caused by a series of alkyl glucosides (alkyl chain length ranging from C8 to C12) in neutral and electrically charged phosphatidylcholine (PC) liposomes were investigated. The surfactant to phospholipid molar ratios (Re) and the bilayer/aqueous phase partition coefficients (K) were determined by monitoring the changes in the static light scattering (SLS) of the system during solubilization. Liposomes were formed by PC, to which phosphatidic acid (PA) or stearylamine (SA) was added when required to increase the negative or positive surface charge. The fact that at the two interaction levels investigated (100 and 0% of SLS of the surfactant/PC systems), the free surfactant concentration for each surfactant was always comparable to its critical micelle concentration (CMC) indicates that the liposome solubilization was mainly ruled by the formation of mixed micelles. The rise in the surfactant CMC (decrease in its alkyl chain length) led to an increase in the surfactant ability to saturate or solubilize liposomes and inversely in an abrupt decrease in its bilayer affinity, regardless of the electrical charge of liposomes. The overall balance of these opposite tendencies shows that the octyl glucoside showed the highest ability to saturate and solubilize liposomes (lowest Re values), whereas the dodecyl glucoside exhibited the highest degree of partitioning into liposomes or affinity with these bilayer structures (highest K values). The use of C9-Glu reduced approximately 2.5 times the concentration needed to saturate and solubilize 1.0 mM PC liposomes with respect to that needed for C8-Glu, regardless of the type of electrical charge present in bilayers. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Alkyl glucoside surfactants; Phosphatidylcholine liposomes; Surfactant/lipid molar ratios; Surfactant partition coefficients; Surfactant critical micelle concentrations; Static light-scattering
Abbre6iations: C8-Glu, octyl glucoside (n-octyl b-D-glucopyranoside); C9-Glu, nonyl glucoside (n-nonyl b-D-glucopyranoside); C10-Glu, decyl glucoside (n-decyl b-D-glucopyranoside); C11-Glu, undecyl glucoside (n-undecyl b-D-glucopyranoside); C12-Glu, dodecyl glucoside (n-dodecyl b-D-glucopyranoside); CMC, critical micellar concentration; K, bilayer/aqueous phase surfactant partition coefficient; KSAT, bilayer/aqueous phase surfactant partition coefficient for liposome saturation; KSOL, bilayer/aqueous phase surfactant partition coefficient for liposome solubilization; PC, phosphatidylcholine; PA, phosphatidic acid; PIPES, piperazine-1,4 bis(2-ethanesulphonic acid); PI, polydispersity index; Re, effective surfactant/lipid molar ratio; ReSAT, effective surfactant/ lipid molar ratio for liposome saturation; ReSOL, effective surfactant/lipid molar ratio for liposome solubilization; r2, regression coefficient; SLS, static light-scattering; SA, stearylamine; SW, surfactant concentration in the aqueous medium; SW,SAT, surfactant concentration in the aqueous medium for liposome saturation; SW,SOL, surfactant concentration in the aqueous medium for liposome solubilization; SB, surfactant concentration in the bilayers. * Corresponding author. Tel.: +34-93-4006161; fax: +34-93-2045904. 0927-7757/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 7 - 7 7 5 7 ( 0 1 ) 0 0 6 9 8 - 7
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1. Introduction A number of studies have been devoted to the understanding of the principles governing the interaction of different surfactants with simplified membrane models such as phospholipid or stratum corneum lipid bilayers [1– 9]. This interaction leads to the breakdown of lamellar structures and to the formation of lipid– surfactant mixed micelles. A significant contribution in this area has been made by Lichtenberg [10], who postulated that the effective surfactant to lipid molar ratio (Re) producing saturation and solubilization of liposomes depends on the surfactant critical micellar concentration (CMC) and on the bilayer/aqueous medium distribution coefficients of these amphiphilic compounds (K). One of the most commonly used amphiphilic compounds in membrane solubilization and reconstitution experiments is the nonionic surfactant octyl glucoside, which is believed to be a ‘mild’ surfactant with respect to its denaturing effect on proteins. This surfactant also shows a relatively high CMC, this characteristic being appropriate for bilayer reconstitution [11–17]. However, the use in these two processes of alkyl glucosides with higher alkyl chain lengths than that for the conventional octyl glucoside has been little investigated in spite of their excellent hydrolysis stability, improved ecological properties and lower toxicity as it has been demonstrated in ‘in vivo’ tests using Daphnia magna and Photobacterium phosphoreum [18,19]. We earlier studied the vesicle– micelle phase transitions involved in the interaction of the nonionic surfactants octyl glucoside and dodecyl maltoside with phosphatidylcholine (PC) unilamellar liposomes [20– 22]. We also investigated the sublytic activity of a series of alkyl glucosides (alkyl chain length ranging from C8 to C12) on PC liposomes [23]. In the present work we seek to extend these investigations by characterizing the Re and K parameters of a these nonionic surfactants when saturated and solubilized neutral and electrically charged PC liposomes (containing phosphatidic acid (PA) or stearylamine (SA) when required to increase the negative or positive surface charge). This study
will provide new information about the influence of the surfactant hydrophobic tail and the electrical charge of liposomes on the saturation and solubilization of these vesicles by the alkyl glucosides investigated in order to evaluate the influence of the hydrophobic and hydrophilic forces on this process.
2. Materials and methods Phosphatidylcholine (PC) was purified from egg lecithin (Merck, Darmstadt, Germany) according to the method of Singleton [24] and was shown to be pure by thin-layer chromatography (TLC). Phosphatidic acid (PA) from egg yolk lecithin and stearylamine (SA) were purchased from Sigma Chemicals Co (St Louis, MO). Lipids were stored in chloroform under nitrogen at − 20°C until use. The nonionic surfactants n-octyl b-D-glucopyranoside (C8-Glu), n-nonyl b-D-glucopyranoside (C9-Glu), n-decyl b-D-glucopyranoside (C10-Glu), n-undecyl b-D-glucopyranoside (C11-Glu) and ndodecyl b-D-glucopyranoside (C12-Glu) were purchased from Sigma Chemicals Co. (St. Louis, MO). Piperazine-1,4 bis(2-ethanesulphonic acid) (PIPES) was obtained from Merck. PIPES buffer was prepared as 10 mM PIPES containing 110 mM Na2SO4 and adjusted to pH 7.20 with NaOH. Polycarbonate membranes and membrane holders were purchased from Nucleopore (Pleasanton, CA).
2.1. Liposome preparation and characterization Unilamellar liposomes (lipid composition PC or PC/PA, PC/SA 9:1 molar ratio) of a defined size were prepared by extrusion of large unilamellar vesicles obtained earlier by a reverse phase evaporation method [25]. Vesicles of about 100 nm were obtained by extrusion through 800, 400, 200 and 100 nm polycarbonate membranes at 25°C using a thermobarrel extruder (Lipex, Biomembranes Inc. Vancouver, Canada). The final volumes of liposome suspensions were adjusted with PIPES buffer containing 110 mM Na2SO4 at pH 7.20 to provide a
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final lipid concentration ranging between 0.1 and 10.0 mM. Liposome preparations were then annealed at 25°C for 30 min and immediately treated with the alkyl glucosides tested. The quantitative analysis of lipid composition and concentration of liposomes after preparation was determined using thin-layer chromatography coupled to an automated flame ionization detection system (TLC-FID) (Iatroscan MK-5, Iatron Labrotary Tokyo, Japan) [26]. The vesicle size distribution and polydispersity index (PI) of liposomes after preparation was determined with dynamic light-scattering measurements using a photon correlator spectrometer (Malvern Autosizer 4700c PS/MV; Malvern, England). The studies were made by particle number measurement [27]. Sample was adjusted to the appropriate concentration range with PIPES buffer and the measurements were taken at 25°C at a reading angle of 90°.
2.2. Parameters in6ol6ed in the interaction of surfactants with PC liposomes In the analysis of the equilibrium partition model proposed by Schurtenberger [28] for bile salt/lecithin systems, Lichtenberg [10] and Almog et al. [12] have shown that for a mixing of lipids (at a lipid concentration L (mM)) and surfactant (at a concentration ST (mM)), in dilute aqueous media, the distribution of surfactant between lipid bilayers and aqueous media obeys a partition coefficient K, given (in mM − 1) by K=
SB [(L +SB)·SW]
(1)
where SB is the concentration of surfactant in the bilayers (mM) and SW is the surfactant concentration in the aqueous medium (mM). For L \ \ SB, the definition of K, as given by Schurtenberger, applies: K=
SB Re = (L·SW) SW
(2)
where Re is the effective molar ratio of surfac-
223
tant 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 Lichtenberg [10] and Almog [12] for different surfactant lipid mixtures over wide ranges of Re values. Given that the lipid concentration range used in liposomes is similar to that used by Almog to test his equilibrium partition model, the K parameter has been determined using this equation. The solubilization of neutral and electrically charged PC liposomes was characterized by two parameters termed ReSAT and ReSOL, according to the nomenclature adopted by Lichtenberg [10] corresponding to the Re ratios at which the SLS of the system starts to decrease with respect to the original value and shows no further decrease. These parameters corresponded to the surfactant to lipid molar ratios at which the surfactant: (a) saturated liposomes; and (b) led to a complete solubilization of these structures. Liposomes were adjusted to the appropriate lipid concentration. Equal volumes of the appropriate surfactant solutions were added to the liposomes and the resulting mixtures were left to equilibrate for 24 h. This time was chosen as the optimum period needed to achieve a complete equilibrium surfactant/liposome for the lipid concentration range used [1,8]. SLS measurements were made with a spectrofluorophotometer Shimadzu RF-540 (Kioto Japan) with both monochromators adjusted to 500 nm at 25°C [20]. The assays were carried out in triplicate and the results given are the average of those obtained. The determination of Re and SW parameters was carried out on the basis of the linear dependence existing between the surfactant concentrations required to saturate and solubilize liposomes at the lipid concentration (L), which can be described by the equations: SSAT = SW,SAT + ReSAT·[L]
(4)
SSOL = SW,SOL + ReSOL·[L]
(5)
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where SSAT and SSOL are the total surfactant concentrations. The surfactant to lipid molar ratios ReSAT and ReSOL and the aqueous surfactant concentrations SW,SAT and SW,SOL are in each curve respectively the slope and the ordinate at the origin (zero phospholipid concentration). The KSAT and KSOL parameters (bilayer/aqueous phase surfactant partition coefficients for saturation and complete solubilization of liposomes) were determined from the Eq. (3).
3. Results and discussion The vesicle size distribution of liposomes after preparation varied little showing in all cases a similar value of about 100 nm (polydispersity index (PI) was always lower than 0.1) thereby indicating that the liposomes had a homogeneous size in all cases. The vesicle size after addition of equal volumes of PIPES buffer and equilibration for 24 h at 25°C showed always values similar to those obtained after preparation, with a slight rise in the PI (between 0.11 and 0.13). Hence, the liposomes investigated were reasonably stable in the absence of surfactants under the experimental conditions used. We reported the variation of the surface tensions of the surfactant solutions in PIPES buffer as a function of total surfactant concentration, as well as their CMC [25]. The increase in the alkyl chain length drastically reduced the surfactant CMC’s (Table 2) and slightly reduced the surface tensions in all cases (from 31 mN m − 1 for C8-Glu to 28.5 mN m − 1 for C12-Glu). Given that the surface tension at the CMC (kCMC) is used as one of the criteria of surface activity of the system, (the lower the kCMC, the higher the surface activity
[29,30]), we may assume that the longer the surfactant hydrophobic tail the higher its surface activity in the system.
3.1. Parameters in6ol6ed in the surfactant/liposomes interaction To determine the surfactant partition coefficients between bilayers and water, we first studied the validity of the equilibrium partition model proposed by Lichtenberg and Almog et al. [10,12] based on the Eq. (1) for the surfactants investigated. According to 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 demostrated the validity of this model for C8-Glu/ PC liposome systems in the range of lipid and surfactant concentration used in the present work [12]. To test the validity of the model for the alkyl glucosides tested using neutral and electrically charged PC liposomes, vesicles were mixed with varying sublytic surfactant concentrations (ST). The resultant surfactant-containing vesicles were then spun at 140 000× g at 25°C for 4 h to remove the vesicles [12]. No lipid was detected in the supernatants [26]. The surfactant concentration in the supernatants (SW) was determined by HPLC [31], and their concentration in the bilayers was calculated (SB = ST − SW). The results of the experiments in which SB and SW were measured (for each surfactant and type of liposomes in the same concentration range used to determine K) were plotted in terms of the dependence of L/SB on 1/SW. Straight lines were obtained for the
Table 1 Regression coefficients (r 2) of the straight lines corresponding to the dependence of L/SB on 1/SW for C8-Glu, C9-Glu, C10-Glu, C11-Glu, and C12-Glu
PC PC/PA PC/SA
C8-Glu (r 2)
C9-Glu (r 2)
C10-Glu (r 2)
C11-Glu (r 2)
C12-Glu (r 2)
0.991 0.990 0.992
0.992 0.991 0.991
0.989 0.990 0.993
0.994 0.991 0.991
0.990 0.992 0.989
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225
Fig. 1. Percentage change in static light-scattering (SLS) of pure PC liposomes, (PC concentration ranging from 1.0 to 10.0 mM), induced by the presence of increasing amounts of C8-Glu. Symbols: PC concentration (9) 1.0 mM; ( ) 3.0 mM; () 5.0 mM; ( ) 6.0 mM; () 8.0 mM; ( ) 10.0 mM.
surfactants tested. The corresponding regression coefficients (r 2) for PC, PC/PA, and PC/SA liposomes are given in Table 1. Straight lines were dependent on L and intersected with the L/SB axis always at − 0.97 90.11, −0.96 9 0.13, and − 0.989 0.13 for PC, PC/PA, and PC/SA liposomes, respectively. Both the linearity of these dependences and the proximity of the intercept to −1 support the validity of this model to determine K for these surfactants/liposome systems. To determine the Re and SW parameters, a systematic investigation of SLS variations in neutral and charged PC liposomes caused by the addition of the surfactants tested was carried out for various lipid concentrations (depending on the solubility of each surfactant). The curves obtained for C8-Glu (PC conc ranging from 1.0 to 10.0 mM) are given in Fig. 1. The addition of surfactant led in all cases to an initial increase and a subsequent fall in the scattered intensity of the system until a low constant SLS value was achieved for complete bilayer solubilization via mixed micelle formation [10]. Surfactant concentrations producing 100% (SSAT) and 0% (SSOL) of
SLS were obtained for each lipid concentration by graphical methods. The arrows A and B (curve for PC liposomes, conc. 10.0 mM in Fig. 1) correspond to these two values. When plotting the surfactant concentrations thus obtained versus lipid concentration, curves were obtained in which an acceptable linear relationship was established in each case. The straight lines obtained corresponded to the Eq. (4) and Eq. (5) from which the Re and SW parameters were determined. The results obtained for neutral and charged PC liposomes for each surfactant tested including the regression coefficients (r 2) of the straight lines are given in Tables 2–4, respectively. Free surfactant concentrations (SW,SAT, SW,SOL) were always comparable to their CMC’s, regardless of the type of liposome used and the surfactant alkyl chain length. This indicates that the liposome solubilization was mainly ruled by formation of mixed micelles, in line with the results reported for the interaction of C8-Glu and dodecyl maltoside with PC liposomes [20–22]. These results extend to the alkyl glucosides studied the
226
C8-Glu C9-Glu C10-Glu C11-Glu C12-Glu a
CMC (mM)
SW,SAT (mM)
SW,SOL (mM)
ReSAT mole mole−1
ReSOL mole mole−1
KSAT (mM−1) KSOL (mM−1) r 2 (SAT)
r 2 (SOL)
18.0 5.6 1.80 0.58 0.18
17.8 5.6 1.75 0.57 0.17
18.3 5.65 1.81 0.59 0.18
1.3 1.4 1.6 2.0 2.8
3.6 3.9 4.5 5.0 6.0
0.03 0.10 0.35 1.17 4.33
0.990 0.998 0.997 0.993 0.994
0.04 0.14 0.45 1.41 4.76
0.992 0.997 0.996 0.999 0.992
The critical micelle concentrations of the surfactant tested is included. It also includes the regression coefficients (r 2) of the straight lines obtained.
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Table 2 Surfactant to lipid molar ratios (Re), partition coefficients (K), and surfactant concentrations in the aqueous medium (SW) resulting in the interaction (bilayer saturation and solubilization) of alkyl glucosides with neutral PC liposomesa
O. Lo´ pez et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 193 (2001) 221–229
generally admitted assumption that the free surfactant concentration (SW) must reach the CMC before solubilization starts to occur [10,20]. The Re values clearly increased from liposome saturation (ReSAT) to complete solubilization (ReSOL), regardless of the type of liposome used and the surfactant alkyl chain length. Given that the surfactant capacity to saturate or solubilize liposomes is inversely related to the Re values, the maximum activity at these two levels corresponded in all cases to the C8-Glu (lowest Re values), whereas the minimum corresponded to the C12-Glu (highest Re values). Thus, the lower the surfactant alkyl chain length the higher its ability to saturate or solubilize PC liposomes regardless of their positive or negative charge. Partition coefficients of the surfactants tested between the bilayers and water for saturation (KSAT) and complete liposome solubilization (KSOL) for neutral and electrically charged PC liposomes (Tables 2– 4) show that the C12-Glu molecules had the highest degree of partitioning into bilayers (maximum K values), whereas the C8-Glu showed the lowest (minimum K values). These findings could be correlated with the aforementioned higher surface activity of the alkyl glucosides with higher hydrophobic tails (lower kCMC values) [29,30]. It is noteworthy that the K values always increased from bilayer saturation to complete liposome solubilization regardless of the surfactant alkyl chain length. This means that the affinity of the surfactant molecules with the lipids building liposomes was greater in the complete bilayer solubilization (micellization process) than during the earlier step of bilayer saturation (for-
227
mation of mixed vesicles). This finding contrasts with the results reported by Ueno, who postulated that in the interacion of C8-Glu with PC liposomes K was independent of the surfactant concentration at low free surfactant concentrations [32]. The relationship between the KSAT and KSOL for each surfactant tested may be correlated with the dynamic surfactant/PC equilibrium existing between the transition steps from mixed vesicles to mixed micelles. Comparison of these two parameters in neutral and charged PC liposomes reveals that the higher the surfactant alkyl chain length the higher the quotient between both values (KSAT/KSOL) regardless of the positive or negative charge of liposomes. Hence, at the interaction level for bilayer saturation the degree of partitioning of surfactants into liposomes relatively increased with respect to that for complete liposome solubilization as the surfactant alkyl chain length rose. Hence, the increase in the length of the surfactant hydrophobic tail in addition to improve the partitioning of surfactant molecules into bilayers (increasing K values) also resulted in a relative decrease in their ability to be assocciated with the molecules building liposomes to form mixed micelles. Possibly, the first order phase transition from mixed vesicles into mixed micelles appears to be relatively hampered by the increasing surfactant hydrophobic tail. If the Re and K values for each surfactant tested in pure PC liposomes are plotted as a function of the surfactant CMC the graphs shown in Figs. 2 and 3 are obtained. A decrease in ReSAT and ReSOL occurred as the surfactant CMC rose
Table 3 Surfactant to lipid molar ratios (Re), partition coefficients (K), and surfactant concentrations in the aqueous medium (SW) resulting in the interaction (bilayer saturation and solubilization) of alkyl glucosides with PC/PA liposomesa
C8-Glu C9-Glu C10-Glu C11-Glu C12-Glu a
SW,SAT (mM) SW,SOL (mM) ReSAT mole mole−1
ReSOL mole mole−1
KSAT (mM−1)
KSOL (mM−1) r 2 (SAT)
r 2 (SOL)
18.0 5.6 1.77 0.58 0.18
3.7 3.9 4.6 5.1 6.1
0.03 0.11 0.35 1.17 4.13
0.04 0.14 0.43 1.42 4.52
0.992 0.993 0.996 0.998 0.994
18.4 5.7 1.9 0.59 0.19
1.4 1.5 1.6 2.1 2.9
It also included the regression coefficients (r 2) of the straight lines obtained.
0.991 0.995 0.992 0.993 0.996
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228
Table 4 Surfactant to lipid molar ratios (Re), partition coefficients (K), and surfactant concentrations in the aqueous medium (SW) resulting in the interaction (bilayer saturation and solubilization) of alkyl glucosides with PC/SA liposomesa
C8-Glu C9-Glu C10-Glu C11-Glu C12-Glu a
SW,SAT (mM) SW,SOL (mM) ReSAT mole mole−1
ReSOL mole mole−1
KSAT (mM−1)
KSOL (mM−1) r 2 (SAT)
r 2 (SOL)
18.0 5.6 1.78 0.58 0.19
3.7 4.0 4.5 5.0 6.2
0.03 0.10 0.35 1.15 3.91
0.04 0.14 0.44 1.38 4.53
0.992 0.991 0.993 0.994 0.998
18.3 5.7 1.86 0.60 0.19
1.4 1.6 1.7 2.0 2.9
0.994 0.994 0.992 0.989 0.990
It also included the regression coefficients (r 2) of the straight lines obtained.
(or the alkyl chain length decreased). The Re fall was more pronounced at low CMC values for both parameters in all cases. The rise in the surfactant CMC also resulted in an abrupt fall in K, which was also specially noticeable at low CMC values (Fig. 3). Thus, the degree of partitioning of these surfactants into liposomes (or affinity with these structures) drastically decreased as the surfactant CMC increased. Similar results were obtained when plotting the Re and K values for PC/PA and PC/SA liposomes (results not shown). Hence, two opposite trends may be observed when comparing the variation of Re and K versus the surfactant CMC. The rise in the surfactant CMC resulted during the transition steps from mixed vesicles to mixed micelles in a progressive increase in the surfactant ability to saturate and solubilize liposomes and inversely in an abrupt decrease in the partitioning of these surfactants between the bilayers and the aqueous phase. Thus, for C12-Glu although a higher number of surfactant molecules were needed to saturate or solubilize liposomes, these molecules showed an increased affinity with these structures with respect to that exhibited by the more active C8-Glu. The overall balance of these two opposite tendencies leads to the conclusion that the C8-Glu was the more equilibrated surfactant in terms of hydrophilic-lipophilic balance, followed by the C9Glu, in order to obtain an improved interaction and, consequently, a maximum solubilizing effect on liposomes. It is noteworthy that the electrostatic interactions do not seem to play an important role in this process given that the presence of
negative or positive charges in liposomes only slightly increased and decreased the Re and K values, respectively (Tables 2–4). Hence, in general terms the resistance of charged liposomes to be saturated or solubilized by surfactants slightly increased with respect to neutral vesicles, whereas the surfactant affinity with these charged vesicles slightly decreased, these two effects being independent of the type of charge present in liposomes. From a practical viewpoint, comparison of the surfactant concentration (mM) needed to saturate
Fig. 2. Variation in surfactant to phospholipid molar ratios (Re) for the alkyl glucosides tested versus the surfactant CMC using PC unilamellar liposomes. Symbols: ( ) liposome saturation (ReSAT); () liposome solubilization (ReSOL).
O. Lo´ pez et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 193 (2001) 221–229
Fig. 3. Variation in bilayer/aqueous phase partition coefficients (K) for the alkyl glucosides tested versus the surfactant CMC using PC unilamellar liposomes. Symbols: ( ) liposome saturation (KSAT); () liposome solubilization (KSOL).
and solubilize liposomes for C8-Glu and C9-Glu surfactants and for 1.0 mM PC concentration reveals that when using C9-Glu a concentration approximately 2.5 times lower than that needed of C8-Glu was in all cases sufficient to produce the same effects in these bilayer structures. These findings open up new avenues in the application of this surfactant in solubilization of biological membranes, given its appropriate solubility in water, excellent hydrolysis stability and relatively high CMC, and may be considered as an interesting alternative with respect to the use of the conventional octyl glucoside.
Acknowledgements This work was supported by funds from D.G.I.C.Y.T. (Direccio´ n General de Investigacio´ n Cientı´fica y Te´ cnica), Spain.
References [1] M.A. Urbaneja, A. Alonso, J.M. Gonza´ lez-Man˜ as, F.M. Gon˜ i, M.A. Partearroyo, M. Tribout, S. Paredes, Biochem. J. 270 (1990) 305.
229
[2] T. Inoue, T. Yamahata, R. Shimozawa, J. Coll. Interf. Sci. 149 (1992) 345. [3] K. Edwards, M. Almgren, Langmuir 8 (1992) 824. [4] D.T. Downing, W. Abraham, B.K. Wegner, K.W. Willman, J.M. Marshall, Arch. Dermatol. Res. 285 (1993) 151. [5] U. Kragh-Hansen, M. le Marie, J.P. No¨ el, T. Gulik-Krzywicki, J.V. Møller, Biochemistry 32 (1993) 1648. [6] M.B. Ruiz, A. Prado, F.M. Gon˜ i, A. Alonso, Biochim. Biophys. Acta 1193 (1994) 301. [7] M. Silvander, G. Karlsson, K. Edwards, J. Coll. Interf. Sci. 179 (1996) 104. [8] M.A. Partearroyo, A. Alonso, F.M. Gon˜ i, M. Tribout, S. Paredes, J. Coll. Interf. Sci. 178 (1996) 156. [9] J. Cladera, J.L. Rigaud, J. Villaverde, M. Dun˜ ach, Eur. J. Biochem. 243 (1997) 798. [10] D. Lichtenberg, Biochim. Biophys. Acta. 821 (1985) 470. [11] P.K. Vinson, Y. Talmon, A. Walter, Biophys. J. 56 (1989) 669. [12] S. Almog, B.J. Litman, W. Wimley, J. Cohen, E.J. Wachtel, Y. Barenholz, A. Ben-Shaul, D. Lichtenberg, Biochemistry 29 (1990) 4582. [13] D.F. Cully, P.S. Paress, Mol. Pharmacol. 40 (1991) 326. [14] S.C.R. Lummis, I.L. Martin, Mol. Pharmacol. 41 (1992) 18. [15] N. Oku, J. Tsudera, K. Kurohani, S. Okada, Chem. Pharm. Bull. 44 (1996) 1928. [16] I.J. Tetlo, C.G. Bowsher, M.J. Emes, Biochem. J. 319 (1996) 717. [17] M. Seras, K. Edwards, M. Almgren, G. Carlson, M. Ollivon, S. Lesieur, Langmuir 12 (1996) 330. [18] M.T. Garcı´a, I. Ribosa, E. Campos, J. Sanchez Leal, Chemosphere 35 (1997) 545. [19] D. Balzer, Specialist Surfactants, in: Blackie Academic and Professional, Chapman and Hall Press, London, 1997. [20] A. de la Maza, J.L. Parra, Eur. J. Biochem. 226 (1994) 1029. [21] A. de la Maza, J.L. Parra, J. Am. Oil Chem. Soc. 73 (1996) 877. [22] A. de la Maza, J.L. Parra, Biophys. J. 72 (1997) 1668. [23] A. de la Maza, L. Coderch, P. Gonzalez, J.L. Parra, J. Control Release 52 (1998) 159. [24] W.S. Singleton, M.S. Gray, M.L. Brown, J.L. White, J. Am. Oil Chem. Soc. 42 (1965) 53. [25] A. de la Maza, J.L. Parra, J. Control Release 37 (1995) 33. [26] R.G. Ackman, C.A Mc. Leod, A.K. Banerjee, J. Planar Chromatogr. 3 (1990) 450. [27] A. de la Maza, A.M. Manich, L. Coderch, P Bosch, J.L. Parra, J. Coll. Surf. A 101 (1995) 9. [28] P. Schurtenberger, N. Mazer, W. Ka¨ nzig, J. Phys. Chem. 89 (1985) 1042. [29] M.J. Rosen, J. Am Oil Chem. Soc. 51 (1974) 461. [30] M.J. Rosen, J. Coll. Interf. Sci. 79 (1981) 587. [31] H. Seino, T. Uchibori, T. Nishitani, S. Inamasu, J. Am. Oil Chem. Soc. 61 (1984) 1761. [32] M. Ueno, Biochemistry 28 (1989) 5631.