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Langmuir 2000, 16, 8632-8642
Interactions between Nonionic Surfactants and Sterically Stabilized Phophatidyl Choline Liposomes† Markus Johnsson* and Katarina Edwards Department of Physical Chemistry, Uppsala University, Box 532, S-751 21 Uppsala, Sweden Received February 9, 2000. In Final Form: May 23, 2000 The interactions between sterically stabilized (EPC/PEG-lipid) liposomes and the nonionic surfactants octyl glucoside (OG) and octa(ethylene glycol) n-dodecyl monoether (C12E8) have been studied. Cryogenic transmission electron microscopy revealed that poly(ethylene glycol)-lipids (PEG-lipids) affected the OGmediated solubilization of the liposomes. Long-lived small bilayer disks were observed at OG concentrations saturating the bilayers. In addition, the macroscopic phase separation observed in EPC/OG systems, at OG concentrations such that the bilayers were close to being solubilized, was entirely inhibited by the inclusion of PEG-lipids in the membrane. OG was more efficient in destabilizing sterically stabilized liposomes than conventional ones, whereas for C12E8 the effective molar ratio of surfactant to lipid at bilayer saturation was essentially the same for both types of liposomes. Furthermore, the results reported illuminate the effect of PEG-lipids on, and the mechanism of, surfactant-induced growth of small unilamellar vesicles.
1. Introduction The general scheme for lipid bilayer dissolution by nonionic and ionic surfactants has been derived and discussed in a number of previous studies.1-5 In most cases, the so-called three-stage model1-5 has been shown to accurately describe the solubilization process. The model can be summarized as follows: The surfactants distribute between the bilayers and the aqueous phase up to a certain point where the membrane is assumed to be saturated with detergent. Further addition of surfactant induces the formation of mixed micelles. These have a constant composition and exist, over a certain concentration range, in equilibrium with bilayers of constant composition, i.e., surfactant saturated bilayers. At some point, depending on the nature of the surfactant and the lipid composition, all lamellar material is solubilized and the system enters the micellar one-phase region. The above model pertains to surfactant solubilization of conventional liposomes. The interactions between surfactants and sterically stabilized liposomes are, so far, not well documented, and the details of the solubilization process still remain to be revealed. Sterically stabilized liposomes are normally prepared by inclusion of lipidderivatized poly(ethylene glycol) (PEG-lipid) in the membrane. In contrast to conventional liposomes, they do not display any tendency to aggregate and flocculate during storage. This effect can be attributed to the polymer cloud surrounding the liposomes, which provides a steric barrier and thereby effectively increases interparticle repulsions. The phase behavior of lipid/PEG-lipid systems has been investigated by means of NMR,6 X-ray,6,7 and in dilute aqueous solution by cryogenic transmission electron microscopy (cryo-TEM).8 † Part of the Special Issue “Colloid Science Matured, Four Colloid Scientists Turn 60 at the Millennium”.
(1) Helenius, A.; Simons, K. Biochim. Biophys. Acta 1975, 415, 29. (2) Lichtenberg, D. Biochim. Biophys. Acta 1985, 821, 470. (3) Lasch, J. Biochim. Biophys. Acta 1995, 1241, 269. (4) Dennis, E. A. Arch. Biochem. Biophys. 1974, 165, 764. (5) Lichtenberg, D.; Robson, R. J.; Dennis, E. A. Biochim. Biophys. Acta 1983, 737, 285. (6) Kenworthy, A. K.; Simon, S. A.; McIntosh, T. J. Biophys. J. 1995, 68, 1903. (7) Hristova, K.; Kenworthy, A.; McIntosh, T. J. Macromolecules 1995, 28, 7693.
In the present study we have investigated and compared the phase and structural behavior in conventional and sterically stabilized egg phosphatidyl choline (EPC) liposome/nonionic surfactant systems. The surfactants studied are the widely used n-octyl-β-D-glucopyranoside (octyl glucoside) (OG) and octa(ethylene glycol) n-dodecyl monoether (C12E8). The phase boundaries were determined by means of turbidity and light scattering measurements. Structural investigations were carried out by means of cryo-TEM. In particular, we have focused our investigations on the effect of the PEG-lipids on the intermediate structures formed during the bilayer dissolution process. 2. Experimental Section 2.1. Materials. Egg phosphatidyl choline (EPC) of grade one was purchased from Lipid Products, Nutfield, England. The PEGlipid, with PEG of molar mass 2000 Da covalently attached via a carbamate linkage to 1,2-distearoyl phosphatidyl ethanolamine (DSPE-PEG(2000)), was obtained from Avanti Polar Lipids, Alabaster, AL. OG was bought from Fluka, Stockholm, Sweden. C12E8 was obtained from Sigma-Aldrich, Stockholm, Sweden. Lipids and surfactants were used as received. All other salts and reagents were of analytical grade and were used as received. 2.2. Preparation of Liposomes. EPC and EPC/PEG-lipid mixtures were prepared by dissolving the lipids in chloroform, removing the chloroform under a gentle stream of nitrogen, and evaporating the remaining chloroform under vacuum for at least 12 h. When PEG-lipids were included in the lipid mixture, the molar ratio of EPC to PEG-lipid was always 95:5. To the dry lipid films were added Hepes buffer (20 mM Hepes, 150 mM NaCl, pH 7.4, 0.01% NaN3), and the lipid mixtures were subjected to at least eight freeze-thaw cycles (including freezing in liquid nitrogen and thawing at 60 °C), whereafter the resulting dispersions were extruded (30 times) through polycarbonate filters (pore size 100 nm) mounted in a LiposoFast miniextruder from Avestin, Ottawa, Canada. The extrusion procedure typically produced unilamellar liposomes with a mean radius of about 50 nm as observed by cryo-TEM. Small unilamellar liposomes (vesicles) (SUVs) were prepared by sonication (1 h, 20 °C; Soniprep 150, MSE Scientific Instruments, Crawley, U.K.) of the lipid films in Hepes buffer, followed by filtration through a 0.2 µm filter (Sartorius Minisart). (8) Edwards, K.; Johnsson, M.; Karlsson, G.; Silvander, M. Biophys. J. 1997, 73, 258.
10.1021/la000190r CCC: $19.00 © 2000 American Chemical Society Published on Web 08/02/2000
Nonionic Surfactant and Liposome Interactions Ultrasonic irradiation of the lipid mixtures typically produced liposomes with a mean radius of about 15 nm. The lipid concentration of the dispersions was taken to be the gravimetrically determined concentration. The liposome dispersions were then diluted to the required concentrations. 2.3. Cryogenic Transmission Electron Microscopy. Electron microscopy investigations were performed with a Zeiss EM 902 A instrument operating at 80 kV. The procedure has been described elsewhere9,10 but consists, in short, of the following. Thin (10-500 nm) sample films were prepared by a blotting procedure performed under controlled temperature (25 °C) and humidity conditions within a custom-built environmental chamber. A drop of the sample solution was placed onto an electron microscopy copper-grid (EM-grid) coated with a perforated polymer film, and excess solution was thereafter removed by means of a filter paper, leaving a thin film of the solution on the EM-grid. Vitrification of the film was achieved by rapidly plunging the grid into liquid ethane held just above the freezing point. The vitrified sample was then transferred at low temperature to the microscope. The temperature was kept below 108 K during both the transfer and the viewing procedures in order to prevent sample perturbation and the formation of ice crystals. All samples investigated by cryo-TEM were incubated at 25 °C for at least 15 h and no longer than 48 h. 2.4. Determination of Critical Micellar Concentrations. The cmc’s of the surfactants were determined by a dye solubilization technique essentially as described by Alexandridis et al.11 Solutions of the surfactants were prepared by dissolving them in Hepes buffer and diluting to the desired concentrations. To these solutions, an aliquot of DPH (diphenylhexatriene) in methanol was added from a stock solution of 0.56 mM DPH in methanol. The solutions thus obtained contained a DPH concentration of 4 µM and a final concentration of methanol of 0.75% (v/v). The samples were incubated in the dark for at least 12 h at 25 °C, and the fluorescence intensity of the samples was then measured on a SPEX-fluorolog 1650 0.22-m double spectrophotometer (SPEX Industries Inc., Edison, NJ) with excitation wavelength set to 356 nm and detecting emitted light at 428 nm. The fluorescence intensity increases rapidly when DPH is dissolved in the hydrophobic interior of the micelles. 2.5. Turbidity Measurements. A Hewlett-Packard 8453 UV-visible spectrophotometer connected to a LAUDA RC6 CP thermostat set to 25 °C was used at a wavelength of 350 nm. The surfactants were added to the liposome dispersions from concentrated stock solutions by a Hamilton 25 µL syringe directly into the cuvette. The cuvette was turned upside down a couple of times and put into the thermostated cuvette holder. The turbidity of the resulting solution was measured after a period of about 5 min. In some cases, the turbidity was not constant when measured over a relatively short time scale. In those cases, the turbidity was taken after a prolonged incubation of about 20 min when the signal did not change significantly. Since the structural investigations by means of cryo-TEM were performed on samples equilibrated for more than 15 h, we also measured the turbidity of samples that were incubated for 15 h or more at 25 °C. 2.6. Light Scattering. The SPEX instrument, with excitation and emission wavelength set to 350 nm, was used to measure the scattered light at 90° from samples containing OG. The same procedure as described above for the continuous addition of surfactant was used. The light scattering experiments were a complement to the turbidity measurements and yielded essentially the same solubilization parameters as the turbidity measurements. The SPEX instrument was also used to measure the kinetics of surfactant-induced SUV growth. Approximately 5 mL of the SUV preparation was transferred to one of the driving syringes in a HI-TECH Rapid Kinetics Accessory, model SFA-11, fastmixing apparatus from HI-TECH Scientific Limited, Salisbury, (9) Bellare, J. R.; Davis, H. T.; Scriven, L. E.; Talmon, Y. J. Electron Microsc. Technol. 1988, 10, 87. (10) Dubochet, J.; Adrian, M.; Chang, J. J.; Homo, J. C.; Lepault, J.; McDowall, A. W.; Schultz, P. Q. Rev. Biophys. 1988, 21, 129. (11) Alexandridis, P.; Holzwarth, J. F.; Hatton, T. A. Macromolecules 1994, 27, 2414.
Langmuir, Vol. 16, No. 23, 2000 8633 England. The second driving syringe was filled with the appropriate surfactant solution in Hepes buffer. The liposome and surfactant solutions were rapidly mixed (
