Phospholipid vesicles from mixed micelles of egg yolk lecithin and a zwitterionic detergent (CHAPS)

LETTER TO THE EDITOR Phospholipid Vesicles from Mixed Micelles of Egg Yolk Lecithin and a Zwitterionic Detergent (CHAPS) Formation and properties of vesicles produced by the detergent depletion method from mixed micelles of egg yolk lecithin and a zwitterionic derivative of a trihydroxy bile salt (CHAPS) at molar ratios of detergent to lecithin between l0 and 1 were investigated. The detergent was removed by gel chromatography or dialysis. Dynamic light scattering, freeze fracture electron microscopy, and phase contrast optical microscopy were used for vesicle characterization. Since CHAPS molecules have no net charge, mixed micelles containing this detergent fuse more efficiently and produce larger vesicles compared to those produced from sodium cholate-lecithin mixed micelles. © 1989AcademicPress,Inc. Phospholipid vesicles are very important in many areas of scientific research and technology, and many methods for their preparation exist (1-4). One of the most useful techniques, especially in biochemistry for the reconstitution of membrane proteins, is the detergent depletion method (5-7). This consists of preparing first a detergent/phospholipid ( D / P L ) mixed micellar solution, then removing the detergent. Depending on the detergent used and experimental conditions, small or large unilamellar vesicles (SUV and LUV, respectively) are thus produced. Detergent molecules or ions are usually removed by dialysis or gel chromatography and therefore only detergents with relatively high critical micellization concentrations (CMC) may be used. These have either relatively low solubilizing power (octyl-glucoside) a n d / o r denature sensitive membrane proteins (bile salts, sodium dodecyl sulfate), the latter mostly because of their ionic character. To circumvent these deficiencies CHAPS, a novel detergent with a structure similar to bile salts but containing a zwitterionic polar group in the side chain, was recently synthesized (8). Studies of aqueous solutions of CHAPS (3-[3-cholamidopropyl-dimethyl ammonio]-l-propane sulfonate) indicate a CMC value of 6.5 m M (=0.0040 g / m l ) and aggregation numbers that progressively increase with detergent concentration from a mass-average value of 7 at twice the CMC to 22 at 20 times the CMC (J. M. Neugebauer and J. P. Kratohvil, manuscript in preparation). These values are not affected by the addition of NaCI (up to 0.6 M). Trihydroxy bile salts, sodium taurocholate and sodium cholate, structurally related to CHAPS, also exhibit a concentration-dependent self-association. However, aggregation numbers of these anionic detergents are considerably smaller than those of CHAPS, even in the presence of NaC1 ((9-11 ); J. M. Neugebauer and J. P. Kratohvil, unpublished results). Vesicles form from disk-like D / P L mixed micelles which fuse, bend, and ultimately close upon themselves (vesiculate) during a continuous removal of detergent ( 12, 13 ). Detergent ions or molecules are predominantly distributed

at the edges of disk-like micelles where they shield the exposed hydrocarbon chains of PL against the polar environment. It is known that the use of different detergents yields vesicles of different sizes. This was explained on a thermodynamic basis, involving mainly the curvature elasticity of the bilayer and effective interactions at the disk edges (12). Kinetic effects were also observed and qualitatively explained as a behavior of systems which were driven far from quasithermodynamic equilibrium conditions ( 13 ). Another possible parameter that influences the size of the vesicles produced is the ability of mixed micelles to fuse, which depends strongly on the characteristics of detergents used. Mixed micelles of sodium cholate (NaCh) and PL have negatively charged rims (14-16) and this probably reduces the collision frequency and fusion efficacy. To test this hypothesis we have used CHAPS, a detergent with a molecular structure similar to NaCh but without a net charge. One can expect that over the same time interval CHAPS/PL mixed micelles would fuse more readily than N a C h / P L micelles and therefore yield larger vesicles, EYL (Lipoid, Ludwigshafen, FRG, purity > 98% ) was used as received. NaCh and CHAPS (Calbiochem) were

TABLE I Diameters (nm) and Polydispersity Factor a (in brackets) as Determined by DLS of Vesicles Prepared by Removal of NaCh or CHAPS from Mixed Micelles at D/EYL = 2 by Gel Chromatography (GC) or by Dialysis

Na-cholate CHAPS

GC

Dialysis

30 [6] 208 [87]

43 [10] 290 [72]

a Polydispersity factor is defined as the standard deviation of a Gaussian distribution, i.e., half-width (in nm) of distribution at 63.2% of the curve height. 539

Journal of Colloid andlnterface Science, Vol. 133,No. 2, December1989

0021-9797/89 $3.00 Copyright© 1989by AcademicPress,Inc. All rightsof reproductionin any formreserved.

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FIG. i. Freeze fracture electron mlcrographs of several vesicle preparations. Bars indicate 100 nm. (a) and (b) NaCh vesicles prepared by dialysis: at two different magnifications; (c) CHAPS vesicles prepared by dialysis; (d) CHAPS vesicles prepared by gel filtration. Average diameters, as estimated by using 5 to 8 micrographs, are (a) 39, (b) 38, (c) 202, and (d) 240 nm, respectively. Diameters obtained from freeze fracture micrographs are slightly smaller than the ones obtained by DLS (Table I). This is probably due to the fact that some of the vesicles were not fractured through their middle. Of interest is the observation that some vesicles made from C H A P S / E Y L mixed micelles were not unilamellar. About 30% of them show two lamellae and in 1-2% of the vesicle population a third layer could be observed. At this point we do not know if this is a common property of vesicles of this size ( ~ 2 0 0 nm), if it can be attributed to some property of CHAPS, or to a complex phase behavior of this system such as coexistence of two (or even more ) different phases at low D/PL ratios. 540 Journal of Colloid andlnterface Science, Vol. 133, No. 2, December 1989

LETTER TO THE EDITOR

541

FIG. 1 - - C o n t i n u e d

purified according to the recently developed procedures (11, 17). Dry EYL films were hydrated with detergent solution in 0.1 M NaC1, 0.02% NAN3, pH 7.2 to a concentration of 5 mg E Y L / m l and at different D / P L molar

ratios ( 10 to 1). All mixed miceUar solutions were equilibrated by continuous shaking (minimal time 3 h) at 20°C. Clear solutions for D / P L > 1.75 were obtained. Detergent molecules were removed at 20°C either by gel chroma-

Journal of Colloid and Interface Science, Vol. 133,No. 2, December1989

542

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FIG. 1--Continued tography (GC) (Sephadex G50, 40 x .9 cm, flow rate 525 m l / h ) or by dialysis. Typically 0.5 ml of solution (5 mg EYL/ml, given D / P L ratio) was applied on the column or placed in a dialysis bag and dialyzed overnight against 1 liter of solvent. Journal of Colloid and Interface Science, Vol. 133, No. 2, D e c e m b e r 1989

The vesicles produced were investigated by dynamic light scattering (DLS: Nicomp 370 submicron particle sizer), freeze fracture EM, and phase contrast optical microscopy (OM). A few samples were run on a Sephacryl SI000 GC column and the obtained hydrodynamic di-

LETTER TO THE EDITOR

543

FIG. 1 - - C o n t i n u e d

ameters were in excellent agreement with the ones determined by DLS (data not shown). Table I shows the diameters of vesicles produced by removing NaCh and CHAPS from mixed micelles with

D / P L = 2 by GC and dialysis. The latter procedure yields for both detergents ~40% larger vesicles. The diameters obtained by DLS were in good agreement with freeze fracture data (Fig. 1). Journal of Colloid and Interface Science, Vol. 133,No. 2, December1989

544

LETTER TO THE EDITOR

These results confirm the expected larger size of zwitterionic mixed micelles which must have higher fusogenic ability and therefore can grow/fuse to larger dimensions before they close upon themselves and form vesicles. The dialysis experiment is thought to be closer to equilibrium and the larger vesicles obtained by this procedure are not unexpected (Y. Nozaki and D. D. Lasic, submitted). Slow and fast removal of detergent (i.e., flow rates 5 and 30 ml/h for CHAPS/EYL = 5) on the GC column did not affect the size of vesicles produced. However, different starting D/EYL ratios did (Table II). Work aimed at a more thorough understanding of this process is underway. A tentative explanation follows. If the equilibrated mixed micellar solution at lower D/EYL ratio is run through a column, the mixed micelle-vesicle transformation is closer to quasiequilibrium than it is at higher D/EYL ratio. Consequently, the primary micelles which undergo fusion are larger at low D/EYL ratios. That this effect is not observed in dialysis experiments can be attributed to the following factors. The probability of mixed micelles fusing during chromatography is much smaller because of the dilution and spatial separation, regardless of shorter or longer passage times through the column. In addition, the inherent effect of long equilibration times of mixed miceUes, especially at lower D/EYL ratios, is superposed on these experimental parameters. In the 1:1 molar CHAPS/EYL dispersion, for instance, larger ( 1-3 #m) multilameUar aggregates are detected in phase contrast OM even after 2 days of mixing at 20°C. These aggregates are effectively filtered on the GC column and the vesicles produced are contaminated with only a few larger aggregates. In contrast, dialyzed samples at D/EYL > 1 (vesicle size similar to 254 nm for GC sample at D/EYL = l ) show practically no contamination with these aggregates; instead, at the resolution limit of OM (>/0.3 #m) many vesicles are observed exhibiting fast Brownian motion. In summary, this work shows the feasibility of using CHAPS to produce large, mostly unilamellar vesicles of controlled diameters. The vesicles produced are larger than vesicles produced from NaCh-EYL mixed micelles, as expected from the thermodynamic model of vesicle forTABLE II Diameters (nm) as Determined by DLS of Vesicles Obtained by Removal of CHAPS from CHAPS/EYL Mixed Micelles at Different Starting Molar Ratios D/EYLmolarratio

10

5

2.5

1

Gel chromatography Dialysis

73 280

86 283

134 303

254 555 a

Note. Concentration of EYL was 5 mg/ml (6.4 mM). a This value is affected by the presence of relatively few spherical and cylindrical multi- and oligolamellar aggregates (up to 10 #m in size), as observed by optical microscopy.

JournalofColloidandlnr,rface Science,Vol. 133,No. 2, December1989

mation (12). In addition, kinetic effects are also qualitatively explained. ACKNOWLEDGMENT We thank Dr. Martin C. Woodle from LTI for many helpful discussions. REFERENCES 1. Szoka, F. C., and Papahadjopoulos, D., Annu. Rev. Biophys. Bioeng. 9, 467 (1980). 2. Gregoriadis, G. (Ed.), "'Liposome Technology," Vol. 1. CRC Press, Boca Raton, FL, 1984. 3. Lichtenberg, D., and Barenholz, Y., in "Methods of Biochemical Analysis" (D. Glick, Ed.), Vol. 33, p. 337. Wiley, New York, 1988. 4. Woodle, M. C., and Papahadjopoulos, D., in "Methods in Enzymology" (S. and B. Fleischer, Eds.), Vol. 171, p. 193. Academic Press, New York, 1989. 5. Kagawa, Y., and Racker, E., J. Biol. Chem. 246, 5477 (1971). 6. Brunner, J., Skrabal, P., and Hauser, H., Biochim. Biophys. Acta 455, 322 (1976). 7. Weder, H. G., and Zumbuehl, O., in "Liposome Technology" (G. Gregoriadis, Ed.), Vol. I, p. 80. CRC Press, Boca Raton, FL, 1984. 8. Hjelmeland, L. M., Proc. Natl. Acad Sci., U.S.A. 77, 6368 (1980). 9. Kratohvil, J. P., Hsu, W. P., Jacobs, M. A., Aminabhavi, T. M., and Mukunoki, Y., ColloidPolym. Sci. 261, 781 (1983). 10. Kratohvil, J. P., Hepatology (Baltimore) 4, 85S ( 1984); Adv. Colloidlnterface Sci. 26, 131 (1986). 11. Hsu, W. P., Ph.D. thesis, Clarkson University, Potsdam, NY, 1985. 12. Lasic, D. D., Biochim. Biophys. Acta692, 501 (1982). 13. Lasic, D. D., Biochem. J. 256, 1 (1988). 14. Dervichian, D. G., Adv. Chem. Set 84, 78 (1968). 15. Small, D. M., Penket, S. A., and Chapman, D., Biochim. Biophys. Acta 176, 178 (1969). 16. Mazer, N. A., Benedek, G. B., and Carey, M. C., Biochemistry 19, 601 (1980). 17. Neugebauer, J. M., M.Sc. thesis, Clarkson University, Potsdam, NY 1985. DANILO D. LASIC FRANK J. MARTIN

Liposome Technology, Inc. 1050 Hamilton Court Menlo Park, California 94025 JUDITH M. NEUGEBAUER JOSIP P. KRATOHVlL

Department of Chemistry and Institute of Colloid and Surface Science Clarkson University Potsdam, New York 13676 Received June 2, 1989; accepted August 24, 1989