7522
Langmuir 1998, 14, 7522-7526
Electrophoretic Behavior of Stearylamine-Containing Liposomes E. Casals, M. Soler, M. Gallardo, and J. Estelrich* Department de Fisicoquı´mica, Facultat de Farma` cia, Universitat de Barcelona, Avda. Joan XXIII s/n, 08028-Barcelona, Catalonia, Spain Received April 17, 1998. In Final Form: October 19, 1998 When ζ potentials of liposomes formed by phosphatidylcholine (PC) and stearylamine (STE) at variable concentrations were compared with those corresponding to liposomes formed by phosphatidic acid (PA), a decline and a lack of linearity in their ζ potentials compared to the logarithm of ionic strength were found at concentrations of STE above 10% (molar ratio). Despite the fact that STE is fully protonated at the interval of pH used,2-8 ζ potentials were found to be dependent not only on the ionic strength of the medium, as predicted by the classical double-layer theory, but also on the pH. Determination of the STE distribution by spectrofluorometry following the labeling of STE with fluorescamine showed that STE seemed to be preferentially located in the outer monolayer. This apparent contradiction can be explained by the migration of STE molecules from the liposomal surface to the medium, where it is organized in the form of micelles with a diameter of about 2 nm. The presence of micelles in addition to liposomes involves a large adsorption-desorption equilibrium, which, in turn, is influenced by the variation in the electrostatic free energy of the double layer on the membrane. Thus, the surface charge density varies with the change in ionic strength and pH, and consequently, the electrophoretic behavior of STE liposomes differs from that of PA liposomes.
Introduction Liposomes are lipid structures used, among other applications, as a model of biological membranes, as a drug-delivery system, and as a system of well-defined physical characteristics in which colloidal behavior can be studied.1 The introduction of a charge on the lipid surface modifies the biological and physicochemical properties of liposomes, since electrostatic phenomena play a crucial role in many biological processes and the presence of a charge produces repulsive electrostatic interactions among the vesicles, compensating the van der Waals attractive forces and, therefore, increasing the stability of the vesicle dispersion. The control and prediction of the liposome stability are important when they are to be stored for a long time after preparation.2 The distribution of charged phospholipids between the inner and outer leaflets of the bilayer is another important characteristic. In biological membranes, the transbilayer lipid asymmetry is an essential feature, and in the liposomes, such asymmetry may lead to exocytosis or endocytosis, the major phenomena in cell traffic, by spontaneous breakdown of symmetry, which increases curvature and forces topological changes. The aim of this study is to monitor, by means of ζ potential measurements, the distribution of charged phospholipids in the membrane of liposomes obtained by the extrusion method, which bear different proportions of either positive or negative charge. Furthermore, from the results obtained, we attempt to explain the apparently anomalous electrophoretic behavior of stearylamine* To whom all communications should be addressed. Departament de Fisicoquı´mica, Universitat de Barcelona, Avda. Joan XXIII s/n, 08028-Barcelona, Catalonia, Spain. Telephone: +34-3-4024554. Telefax: +34-3-4021886. E-mail:
[email protected]. (1) Lasic, D. D. Liposomes: from Physics to applications; Elsevier: Amsterdam, 1993; Chapter 1. (2) Frøkjaer, S.; Hjorth, E. L.; Worts, O. In Optimization of Drug Delivery; Bundgaard, H., Bagger Hansen, A., Kofof, H., Eds.; Munksgaard: Copenhagen, 1982; pp 384-404.
containing liposomes in comparison with that of liposomes bearing phosphatidic acid. Materials and Methods Materials. Soybean phosphatidylcholine (Lipoid S-100) (PC) was purchased from Lipoid (Ludwigshafen, Germany). Stearylamine (STE), phosphatidic acid (PA), fluorescamine, and Triton X-100 were from Sigma (St. Louis, MO). Organic solvents (methanol, ethanol, chloroform) were obtained from Merck (Darmstadt, Germany) and used without purification. Acetone was of spectroscopic grade (Merck, Germany). Buffer solutions were prepared from a two-solution buffer system (solution A, 0.2 M anhydrous boric acid and 0.05 M citric acid monohydrate; solution B, 0.1 M trisodium phosphate dodecahydrate), as indicated elsewhere.3 Preparation of Multilamellar Liposomes (MLV). Phospholipids (PC alone or PC with STE or PA prepared at different molar ratios 10:0, 9.5:0.5, 9:1, 8:2, and 7:3) were dissolved in chloroform in a round-bottomed flask and dried in a rotary evaporator under reduced pressure at 50 °C to form a thin film on the flask. The film was hydrated with isotonic saline solution (150 mM NaCl) to give a lipid concentration of 10 µmol/mL. Multilamellar liposomes were formed by constant vortexing for 4 min on a vortex mixer and sonication in a bath for 4 min. Preparation of Extruded Liposomes. MLV were downsized to form unilamellar vesicles by extrusion at 50 °C in an Extruder device (Lipex Biomembranes, Canada) through polycarbonate membrane filters of variable pore size under nitrogen pressures of up to 55 × 105 N‚m-2.4 Liposomes were extruded sequentially through polycarbonate filters (0.8, 0.4, and 0.2 or 0.1 µm (Nucleopore, Cambridge, MA)) in order to obtain liposomes of a nominal size of 200 or 100 nm. Determination of STE. STE was determined spectrofluorometrically using fluorescamine as a labeling reagent.5 It was confirmed in advance that fluorescamine had no disruptive effect on the liposome structure. (3) Silberman, R. G. J. Chem. Educ. 1992, 69, A42. (4) Hope, M. J.; Bally, M. B.; Webb, G.; Cullis, P. R. Biochim. Biophys. Acta 1985, 812, 55. (5) Roy, M. T.; Gallardo, M.; Estelrich, J. Bioconjugate Chem. 1997, 8, 941.
10.1021/la980444x CCC: $15.00 © 1998 American Chemical Society Published on Web 12/05/1998
Stearylamine-Containing Liposomes Determination of STE in the Outer Vesicle Surface. At room temperature, aliquots (100-200 µL) of liposomes were diluted with 2.0 mL of borate buffer (pH 8.25). Fluorescamine solution (0.2 mL of a solution of 3 mg in 100 mL of acetone) was added, and the vesicle sample was shaken vigorously for 30 s. After 1 min, 2 mL of 1.6% Triton X-100 in borate buffer was added to the sample, followed by mixing. The resulting fluorescence was read within 2 h of the reaction by exciting the sample at 381 nm and measuring the radiation emitted at 471 nm. Determination of Total STE in the Vesicle. Aliquots (100200 µL) of liposomes were disrupted with 2.0 mL of 1.6% Triton X-100 in borate buffer (pH 8.25). Fluorescamine solution (0.2 mL) was added, and the vesicle sample was shaken vigorously for 30 s. After 1 min, 2 mL of borate buffer was added to the sample, followed by mixing. The samples were read as described above. Particle Size Analysis. The vesicle size distribution was determined by photon correlation spectroscopy with an Autosizer II spectrometer (Malvern Instruments, Malvern, U.K.) at 25 °C. The vesicles obtained by extrusion which afforded a unimodal vesicle distribution were sized by cumulant analysis.6 The sample, contained in a 5 mL glass cuvette, was placed in a thermally packed sample holder at 25 °C. The light source, a helium-neon laser of wavelength 632.8 nm and 5 mW, was focused onto the sample, and scattered light was detected at 90° to the incident beam by a photomultiplier tube, which was connected to a quantum photometer. Fluctuations in the scattered light intensity generated by the diffusion of vesicles in solution were analyzed, and the autocorrelation concentration was obtained via a Malvern 7032-N, 72-channel multibit correlator. The presence of micelles of STE was determined in a Malvern S4700 apparatus (Malvern Instruments, Malvern, U.K.). Samples were irradiated with an argon laser of 488 nm and 50 mW. The distribution of particles was measured by using the software CONTIN.7 Microelectrophoretic Mobility Measurements. Electrophoretic measurements were performed at 25 °C in a Zetasizer 4 (Malvern Instruments, Malvern, U.K.) based on the laserDoppler microelectrophoresis. Liposomes were diluted to a lipid concentration of 1 mg/mL with the medium (10 mM buffer, NaCl at different concentrations). The ζ potential of the liposomes was calculated from their electrophoretic mobility at 25 °C by means of the Henry correction of Smoluchowski’s equation8
µ)
2ζ f(κa) 3η
where µ is the particle electrophoretic mobility, is the dielectric constant, ζ is the ζ potential, η is the aqueous solution viscosity, and f(κa) is the Henry coefficient, which ranged from 1.353 to 1.488 depending on the vesicle radius (a) and on the ionic strength of the suspending solution; the dielectric constant and viscosity were taken to be 78 and 10-2 Pa‚s, respectively. The results are the average of four measurements at the stationary level. The standard deviation of the ζ potential values was less than 5% of the mean. Throughout these measurements, liposomes were diluted with the corresponding buffer to 1 µmol/mL. All experiments in this study were performed at least in triplicate.
Results and Discussion Extrusion by membranes with 100-nm nominal pores afforded liposomes of different average size according to the kind of charge. Negative liposomes presented a mean diameter of 108 ( 7 nm, while liposomes with stearylamine had diameters of 122 ( 11 nm. The polydispersity (this parameter ranges from 0 to 1; 0 refers to a monodispersed system and 1 refers to an extremely polydispersed system) of the positive vesicles was also higher (0.173 ( 0.023) than that obtained with phosphatidic acid-containing liposomes (0.100 ( 0.013). ζ potential values were obtained from microelectrophoretic measurements by means of the Henry form of (6) Koppel, D. E. J. Chem. Phys. 1972, 57, 4814. (7) Provencher, S. W. Makromol. Chem. 1979, 180, 201.
Langmuir, Vol. 14, No. 26, 1998 7523
Figure 1. Variation of the ζ potential of negatively charged liposomes with electrolyte concentration. Values are the average of three measurements carried out at the stationary level at pH 6.61. Error bars are approximated by symbol size. ([) PC; (O) PC:PA (95:5); (b) PC:PA (90:10); (2) PC:PA (80:20); (9) PC:PA (70:30).
the Smoluchowski equation. Figure 1 shows the plot of the ζ potential obtained in negatively charged liposomes at five concentration levels as a function of electrolyte concentration (from 0.01 to 0.16 M) at a constant pH of 6.61 (10 mM citrate-phosphate buffer). The ζ potential plotted as a function of ionic strength (I) was a straight line for any PA content. The increase in ζ potential for PA was between about -1 and -2 mV per mol % PA (depending on the ionic strength). A linear decrease in the absolute values of the ζ potential of liposomal suspensions with increasing electrolyte ionic strength in the form of a logarithm was also found for NaCl and other electrolytes.9 This linear dependency of ζ potential on the logarithm of ionic strength is predicted by the classical double-layer theory.10 On the basis of ζ potential measurements, Hauser et al.11 have concluded that PA incorporated in liquid-crystalline phosphatidylcholine bilayers is randomly distributed on the plane of the bilayer. Furthermore, the distribution of this negatively charged phospholipid between the two halves of the bilayer is uniform. However, this pattern was not reproduced with liposomes containing STE (Figure 2). This picture indicates a lack of linearity above 10% (molar ratio) of positive charge, while, below 10%, the relation between ζ potential and log I was linear (R2 g 0.998). In addition to the nonlinearity between ζ potential and ionic strength, absolute values obtained with STE liposomes were lower than that obtained with PA liposomes. As lipids (STE and PA) are not comparable either in size or in charge, we attempt to relate the ζ potential with the theoretical surface charge density. To calculate this parameter, we assumed the following: (a) liposomes were unilamellar and spherical vesicles; (b) lipids were distributed symmetrically in the bilayer; (c) the surface area of the lipid molecule was 70 Å2 for PC and PA and 25 Å2 for STE;12 (d) pKa1 and pKa2 of PA are 3.9 and 8.3, (8) Hunter, J. R. Zeta potential in Colloid Science; Academic Press: London, 1981; Chapter 3. (9) Carrio´n, F. J.; de la Maza, A.; Parra, J. L. J. Colloid Interface Sci. 1994, 164, 78. (10) Hunter, J. R. Zeta potential in Colloid Science; Academic Press: London, 1981; p 26. (11) Hauser, H.; Guyer, W.; Howell, K. Biochemistry 1979, 18, 3285. (12) Crommelin, D. J. A. J. Pharm. Sci. 1984, 73, 1559.
7524 Langmuir, Vol. 14, No. 26, 1998
Figure 2. Variation of the ζ potential of positively charged liposomes with electrolyte concentration. Values are the average of three measurements carried out at the stationary level at pH 6.61. Error bars are approximated by symbol size. ([) PC; (b) PC:STE (95:5); (O) PC:STE (90:10); (2) PC:STE (80:20); (9) PC:STE (70:30).
Figure 3. Variation of the ζ potential as a function of the theoretical surface density charge of both types of liposomes, both expressed in absolute values. Measurements were carried out at pH 6.61 and 0.04 M NaCl. Error bars are approximated by symbol size. (b) PC:PA; (0) PC:STE.
respectively,13 and pKa1 of STE is 9.5;14 and (e) the thickness of the bilayer is 4 nm. When ζ potential was plotted against the absolute values of the theoretical surface density charge, a nearly linear increase of the ζ potential with surface density charge was observed for negative liposomes, whereas a linear relationship was observed up to 20% STE liposomes, although the magnitude of the ζ potential was lower than that with PA liposomes. Figure 3 shows the behavior of both kinds of liposomes at an intermediate ionic strength (I ) 0.04). These results are consistent with two possibilities: a deviation of the ζ potential of lipid membranes from the predictions of the double-layer theory or an asymmetrical distribution of STE in the bilayer. The first case has been (13) Ptak, M.; Egret-Charlier, M.; Sanson, A.; Bouloussa, O. Biochim. Biophys. Acta 1980, 600, 387.
Casals et al.
Figure 4. Variation of the ζ potential of positively charged liposomes with electrolyte concentration. Values are the average of three measurements carried out at the stationary level at pH 2.32. Error bars are approximated by symbol size. ([) PC; (O) PC:STE (95:5); (b) PC:STE (90:10); (2) PC:STE (80:20); (9) PC:STE (70:30).
amply described,15,16 especially for lipid membranes.17-18 These authors concluded that such atypical behavior of charged colloids is especially evident at monovalent electrolyte concentrations < 0.01 M. The fact that the lowest electrolyte concentration used in this work was 0.01 M and that PA liposomes did not undergo this deviation seemed to support the second possibility. To see whether pH influenced the electrophoretic mobility of vesicles, ζ potentials of liposomes at the same compositions and at the same electrolyte conditions as those used at pH 6 were determined at pH 2, 4, and 8. Since the pKa of STE in phosphatidylcholine vesicles is 9.5,13 more than one unit lower than the normal pKa, approximately 10.7, we could expect that, at pH 2 and 4, the electrokinetic profile of pH 6 would be reproduced. At such pH values, the protonation of STE would be theoretically complete. Figures 4-6 show the results obtained at pH 2, 4, and 8, respectively. In the absence of positive lipid, PC liposomes were usually slightly negative. Below pH 4 the pure PC liposomes became positively charged, as expected. The ζ potentials of charged liposomes increased with decreasing pH. Linearity was only observed at the lowest STE concentration. Differences between ζ potentials obtained at pH 2, 4, and 6 could be due to a different degree of STE ionization or a pH-dependent modulation of the lipid distribution in the bilayer. These results and the considerations inferred from Figure 3 indicate a possible asymmetrical distribution of STE in the bilayer. To check this possibility, STE in the outermost layer and the total STE in liposomes were labeled by fluorescamine. This reacts with primary amines. The relationship of fluorescence intensities between the external and the total STE gave the percentage of external STE. STE seemed to be located predominantly in the outer monolayer of the (14) Quinn, P. J. The molecular biology of cell membranes; McMillan Press: London, 1976; p 69. (15) O’Brian, R. W.; White, L. R. J. Chem. Soc., Faraday Trans. 2 1978, 74, 1607. (16) Antonietti, M.; Vorwerg, L. Colloid Polym. Sci. 1997, 275, 883. (17) Egorova, E. M.; Dukhin, A. S.; Svetlova, I. E. Biochim. Biophys. Acta 1992, 102. (18) Egorova, E. M. Electrophoresis 1995, 16, 905.
Stearylamine-Containing Liposomes
Langmuir, Vol. 14, No. 26, 1998 7525
Figure 5. Variation of the ζ potential of positively charged liposomes with electrolyte concentration. Values are the average of three measurements carried out at the stationary level at pH 4.36. Error bars are approximated by symbol size. ([) PC; (O) PC:STE (95:5); (b) PC:STE (90:10); (2) PC:STE (80:20); (9) PC:STE (70:30).
Figure 6. Variation of the ζ potential of positively charged liposomes with electrolyte concentration. Values are the average of three measurements carried out at the stationary level at pH 8.68. Error bars are approximated by symbol size. ([) PC; (O) PC:STE (95:5); (b) PC:STE (90:10); (2) PC:STE (80:20); (9) PC:STE (70:30). Table 1. External Amine Expressed as Percentage Labeled by Fluorescamine PC/STE (molar ratio) external amine groupa (%)
9.5:0.5 76 ( 6
9:1 73 ( 3
8:2 72 ( 4
7:3 73 ( 5
a Values represent the mean and the corresponding standard deviation (n ) 3).
vesicle, although without any clear dependence of the content of the amino lipid on the percentage of external STE (Table 1). These results led to an apparent contradiction. On one hand, the ζ potential of STE liposomes did not fit the theoretical surface charge density calculated assuming a symmetrical distribution; on the other, if most of the charge was located on the outer surface, this would bring about ζ potential values above those predicted by a symmetrical distribution. However, STE liposomes showed lower ζ potentials.
Figure 7. Size distribution of liposomes of PC/STE (80:20) extruded through a 200 nm membrane and further centrifuged. The peak of lowest size corresponds to STE micelles. The x-axis is expressed in logarithmic scale.
Hope and Cullis19 report that STE is completely sequestered into the inner monolayer in liposomes with acidic interiors. In contrast, our results were consistent with a distribution of STE predominantly in the outer monolayer, and such a predominance is also observed in an acidic medium. Since segregation of normal fatty acids in their protonated form was proposed by Hauser et al.,11 STE incorporated in phospholipid bilayers may be segregated. In this way, when we assay the content of STE in the outer layer, we might be determining the STE inserted into the outer monolayer as well as the STE present in the solution. Aliphatic monoamines are able to form micellar structures. The value of the critical micellar concentration (cmc) of STE determined by the du Nouy ring method was 67.6 µM.21 Such a value, compared with those for the most usual tensioactive agents, is extremely low. This signifies that STE is able to escape easily from the lipid bilayer and to protect its hydrocarbon chain from the hydrophilic environment; STE is organized in micelles. The presence of STE micelles in the liposomal preparation was checked by determining its particle distribution. For this purpose, liposomes with 20% STE and extruded through 200 nm were centrifuged at 100000g for 2 h, and the supernatant was analyzed by photon correlation spectroscopy using equipment with a more potent irradiation source and a higher resolution than those in the routine size determination. Figure 7 displays the two populations detected: one, corresponding to the remaining liposomes, was centered at 130 nm; the other with a peak of 2.2 ( 0.2 (analysis by intensity) can be attributed to STE micelles. To ensure that this peak corresponded to STE, a suspension of the amino lipid was prepared and after filtration through 0.22 µm membrane pores (Millipore, USA) was analyzed, and a unique peak of 2.1 ( 0.1 nm was found. Although the cmc of STE is relatively low, it is much higher than that of a phospholipid like PA (≈10-10 M). PA forms structures at very low concentrations, and as it is a double-chained lipid and its critical packing shape is a truncated cone, these structures are liposomes. STE, a single-chained lipid, with a cone as the critical packing shape, forms micelles. The presence of STE micelles in (19) Hope, M. J.; Cullis, P. R. J. Biol. Chem. 1987, 262, 4360. (20) Eastman, S. J.; Hope, M. J.; Cullis, P. R. Biochemistry 1991, 30, 1740. (21) Bass, G. E.; Powers, L. J.; Dillingham, E. O. J. Pharm. Sci. 1976, 65, 1525.
7526 Langmuir, Vol. 14, No. 26, 1998
the liposomal preparation means that the adsorptiondesorption equilibrium of STE between membrane and solution is important. The variation in the electrostatic free energy of the double layer on the membrane surface might influence the equilibrium constant of adsorption. This suggests that the surface charge density varies with the change in the salt condition in general; that is, the behavior of the surface potential in the case of STE must be different from that in the case of PA. The different behaviors of the ζ potential of a PA system and a STE system can be attributable to this. The presence of STE micelles suggests it is difficult to measure by spectrofluorometry using fluorescamine as a labeling reagent when determining the ratio of STE in the outer surface of liposomes to that of the inner liposomes. In this way, Table 1 presents an outer/inner ratio biased toward the outer surface, since the fluorescence intensity assumed as deriving from the external monolayer is actually originated by the STE present in this layer but also by the STE micelles and the monomer form present in the suspension. To overcome this difficulty, liposomes must be separated from the other STE-containing forms. This can be achieved by the minicolumn centrifugation technique22 using Sepharose 4B (Pharmacia, Uppsala, Sweden). This technique is rapid and suitable for those
Casals et al.
liposomal preparations which encapsulate permeant substances or with chemical species on the surface affected by an equilibrium of adsorption-desorption. On the basis of these results, we can conclude that, in a liposomal preparation which contains STE, liposomes coexist with STE micelles (they may also exist with the monomolecular form of STE). STE could be randomly distributed in the bilayer, but STE can undergo a rapid segregation into the medium. This fact is of paramount importance because STE has been extensively used to obtain cationic liposomes, and when a determined percentage of positive charge is desired, it is evident that such liposomes do not ensure the presence of the theoretical charge in the vesicle. Acknowledgment. Zetasizer 4 was afforded by Ministerio de Educacio´n y Ciencia (Spain) in the Programa Nacional de Medio Ambiente y Recursos Naturales (IN920210). The authors would like to thank Dr. Nu´ria Azemar (CID/CSIC, Barcelona) for the PCS measurements. LA980444X (22) Fry, D. W.; White, J. C.; Goldman, I. D. Anal. Biochem. 1978, 90, 809.
