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Langmuir 1995,11,408-410
Cryo-TEMEvidence: Sonication of Dihexadecyl Phosphate Does Not Produce Closed Bilayers with Smooth Curvature Leif Hammarstrom," Irena Velikian, Goran Karlsson, and Katarina Edwards Department of Physical Chemistry, Uppsala University, Box 532,S-75121 Uppsala, Sweden Received October 17, 1994. I n Final Form: December 6, 1994@ Dihexadecyl phosphate (DHP) is commonly used for formation of model membranes. In this report cryo-transmissionelectron microscopy (cryo-TEM)pictures are presented. They clearly show that after cooling t o room temperature, dispersions of DHP sonicated at 80 "C are dominated by open and folded bilayer fragments, rather than by vesicles with smooth curvature. Quantitative results of an earlier kinetic investigation of viologen reduction in DHP were reproduced using these dispersions. Since the late 1970s,a number of synthetic surfactants have been used for the construction of model membranes. One of the most commonly used is the negatively charged dihexadecyl phosphate (DHP).la It has generally been assumed that sonication of DHP above the gel-to-liquid crystalline transition temperature (Tm,around 65 "Clb) produces small, unilamellar vesicles, which remain stable a t room temperature. Such preparations have been used for the study of photoinduced energy2 and electron3 transfer, as well as membrane permeability and transmembrane transport proce~ses.~ In this report we present cryo-transmission electron microscopy (cryo-TEM) pictures which clearly show that after cooling to room temperature, sonicated dispersions of DHP are dominated by open and folded bilayer fragments, rather than vesicles with smooth curvature. Nevertheless, with these dispersions we were able to reproduce quantitative results of an earlier kinetic investigation of viologen reduction in DHP. Using natural phospholipids, we have performed a number of studies concerning surfactantkesicle interaction~ as~well as transmembrane electron transfer.6 In the latter studies, electrons were transferred via an amphiphilic viologen from an external reductant to a secondary electron acceptor in the vesicle interior. Since viologen was present only on the vesicle exterior, we could exclude interviologen electron exchange through the membrane, which had been claimed. Instead, the results lead us to propose a mechanism based on a ratedetermining disproportionation of two viologen radicals @Abstractpublished in Advance A C S Abstracts, February 1, 1995. (1)Fendler, J. H.Acc. Chem. Res. 1980,13,7, and references therein. (b) For sonicated DHP in water or noninteracting buffer at pH = 8, see refs 9d and 1lc. (2)(a) Tamai, N.; Yamazaki, T.; Yamazaki, I.; Mizuma, A.; Mataga, N. J . Phys. Chem. 1987,91,3503. (b) Kano, K.; Kawazumi, H.; Ogawa, T. J . Phys. Chem. 1981,85,2998. (3)A few recent examples include (a) Patterson, B. C.; Hurst, J. K. J.Phys. Chem. 1993,97,454.(b) Horvath, 0.; Fendler, J. H. J . Phys. Chem. 1992,96,9591. (c) Tricot, Y.-M.;Porat, Z.; Manassen, J. J.Phys. Chem. 1991,95,3242. (d) Chaabane, T. B.; Bernas, A,; Grand, D.; Hautecloque, S. J. Phys. Chem. 1987,91,6055.(e) Kevan, L.Radiat. Phys. Chem. 1992,39,333.(fJ Rafaeloff, R.;Maliyackel, A. C.; Grant, J . L.; Otvos, J. V.; Calvin; M. Now. J . Chim. 1986,10,613. (4)(a) Castaing, M.; Camplo, M.; Kraus, J.-L. Res. Commun. Chem. (b) Lymar, S.V.; Hurst, J. K. J . Am. Pathol. Pharmacol. 1993,81,131. Chem. SOC.1992, 114, 9498. (c) Roks, M. F. M.; Nolte, R. J. M. Macromolecules 1992,25,5398. (d)Ringsdorf, H.; Schlarb, B.; Tyminski, P. N.; O'Brien, D. F. Macromolecules 1988,21,671. (5) (a) Edwards, K.; Gustafsson; J.; Almgren, M.; Karlsson, G. J . Colloid Interface Sci. 1993,161,299. (b) Edwards, K.;Almgren, M. J . Colloid Interface Sci. 1991,147,1. (6)(a) Hammarstrom, L.;Almgren, M.; Norrby, T. J . Phys. Chem. 1992,96,5017. (b) Hammarstrom, L.;Almgren, M.; Lind, J.; Merenyi, G.; Norrby, T.; Akermark, B. J . Phys. Chem. 1993,97, 10083. (c) Hammarstrom,L.; Berglund, H.; Almgren, M. J . Phys. Chem. 1994,98, 9588.
0743-746319512411-0408$09.00/0
2c,v
-
c,vO
+ C,V2+
(1)
forming a doubly reduced, uncharged viologen (C,VO) that rapidly diffised through the membrane and was reoxidized by the acceptor ferricyanide. There was no detectable influence of conproportionation (reverse of eq 1). In a recent report by Lymar and Hurst,' the same system organization was used for an investigation with methylviologen ( M V ) in vesicles of DHP. The authors found that the observed reaction could be explained by the disproportionation mechanism also in this system. An additional mechanism involvingtransmembrane diffision of viologen radical (MV') was expressed under certain conditions. Interestingly, the experimental data presented suggest that the conproportionation reaction might influence the observed reaction a t high concentrations of W +In . order t o further examine this reaction step, we wanted to repeat our measurements with C,V using DHP vesicles. The DHP (acid form, Sigma) was added to 25 mM Tris (pH = 8.0) or Milli-Q water containing a stochiometric amount of NaOH (pH % 8.0 after sonication). At this pH the DHP was completely ionized. Thereafter the solution was heated to 80 "C (i.e. above Tm)and sonicated for 20 min (MSE Soniprep 150). The samples were prepared for cryo-TEM by the standard procedure.5a A small drop of sample solution was deposited on a HPF-grid and kept a t 25 "C within the controlled environment vitrification system (CEVS).8 After blotting, the sample was vitrified by plunging the grid into liquid ethane and thereafter transferred to a Zeiss EM902 electron microscope for examination. Figure l a shows a cryo-TEM micrograph of a sonicated DHP dispersion. The sample contains bilayer fragments together with irregular structures which probably represent folded and partially closed, crystalline bilayers. Note that the angular profiles with sharp edges are in conflict with the smooth curvature expected for a vesicle. For comparison we have included a micrograph of unilamellar vesicles (Figure lb). It is important to recognize that the micrographs are two-dimensional representations of the sample, so that the circumference of a spherical vesicle will appear as a perfect circle, i.e. with high and constant contrast around the edge where the electron beam passes most material. No such structures were seen in the DHP micrographs. Samples prepared in Milli-Q/ NaOH or Tris had the same general appearance. Varying (7)Lymar, S.V.;Hurst, J . K. J . Phys. Chem. 1994,98,989. (8) Bellare, J. R.; Davis, H. T.; Scriven, L. E.; Talmon, Y. J . Electron Microsc. Technol. 1988,10,87.
0 1995 American Chemical Society
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Langmuir, Vol. 11, No. 2, 1995 409
3 p""'\ n
20
40 120 200 Time ( s ) Figure 2. Reduction of CMV in sonicated DHP by dithionite as observed by stopped-flow: upper curve, CMV added after sonication; lower curve, CMV added before sonication. Conditions: [DHPI = 1mM, [CMV] = 20 pM, [ S 2 0 4 2 - l = 10 mM, [Tris] = 25 mM, pH = 8.0, T = 21 "C. 0
10
20
the sonication time (10-30 min) or sonication power did not improve the result, neither did 2 h incubation at 80 "C of the sonicated solution. Centrifugation ( 1h, 1OOOOOg) did reduce the size polydispersity, but the top fraction still contained small bilayer fragments and small irregular structures. Sonicated dispersions of DHP have generally been assumed to consist of small spherical or ellipsoidal vesi c l e ~ .However, ~ the methods used for structural characterization of these dispersions, i.e. light scattering and negative staining TEM, have not conclusivelyshown that this is the case. Determination of the size and shape of surfactant aggregates by use of light scattering is not simple, and the results p r e ~ e n t e d are ~ ~not - ~ in agreement. Negative staining TEM is known to induce artifacts due to the drying and staining procedures, particularly for hollow, water-containing structures such as vesicles. Freeze-fractureelectron microscopybenefits from a milder preparational method, and micrographs of DHP dispersions using this method have revealed irregular, roughly spherical features.gd In cryo-TEM, many of the artifacts
induced by other preparational methods are avoided, and the rapid cooling rate ensures that the structures remain in their original state. Evidently, it does not seem possible to prepare DHP vesicles by sonication, for use at room temperature. In order to check if we could still reproduce some previously reported detailed and quantitative results, we performed studies on cetyl-, methylviologen (CMV) reduction by dithionite in the DHP dispersions (25 mM Tris). Viologen did not change the appearanceof the micrographs. When CMV had been added after sonication, the time-resolved reduction could be fitted to two single-exponential functions. Both rate constants were proportional to [S2042-11/2 ( k l = 15 M-ll2 s-l, k2 = 3.8 M-l12 s-l), consistent with direct reduction by SOa-. The double-exponential reduction has been suggested to originate from binding of viologen at two different types of sites on the outside of the vesicles.1° When viologen was added before sonication,an additional, slower phase of reduction was observed (Figure 2). This phase has been attributed to transmembrane electron transfer from external viologen to viologen trapped inside vesicles.1° About 25% of the viologen reacted in this step, which could fairly well be described by adding a third single-exponentialfunction. These results, including the values of the rate constants, are in agreementwith earlier reports.1° We further found that the rate constant for the slowest step was proportional to the mole fraction of viologen (ks = 1 s-l (mole fraction)-l), as is the case in lecithin vesicles. The observation of what appears to be a transmembrane reduction ofviologen suggeststhat several of the structures observed by microscopy provide binding domains for CMV where the access of dithionite is restricted. The most straightforward explanation would be the existence of closed structures. There are several reports on entrapment of water-soluble solutes by DHP dispersions supporting this suggestion, even if the entrapment efficiency However, it is clear from the sometimes has been micrographs that a significant part of the DHP form nonclosed bilayer fragments. This has earlier been suggested by Carmona-Ribeiro,ll who has examined the properties of DHP dispersions quite extensively. She has further suggested that the packing of the bilayers is different for the fragments and the larger structures. This packing difference could possibly explain the reports1°J2
(9) (a)Mortara, R. A.; Quina, F. H.; Chaimovich, H. Biochem. Biophys. Res. Commun. 1978,81,1080. (b) Herrmann, U.; Fendler, J. H. Chem. Phys. Lett. 1979,64,270.(c)Tricot, Y.-M.; Furlong, D. N.; Sasse, W. H. F.; Daivis, P.;Snook, I.; Van Megen, W. J. J. Colloid Interface Sci. 1984, 97,380. (d) Humphry-Baker, R.; Thompson, D. H.; Lei, Y.; Hope, M. J.; Hurst, J. K. Langmuir 1991, 7 , 2592.
(10) Thompson, D. H. P.; Barrette, W. C.; Hurst, J. K. J. Am. Chem. SOC.1987,109, 2003. ( l l ) ( a ) Carmona-Ribeiro, A. M. Chem. SOC.Rev. 1992, 209. (b) Carmona-Ribeiro,A. M.; Castuma, C. E.; Sesso, A.; Schreier, S. J . Phys. Chem. 1991,95,5361. (c) Carmona-Ribeiro, A. M. J. Colloid Interface Sci. 1990, 139, 343.
Figure 1. Cryo-TEM micrograph of (a)DHP sonicated at 80 "C in 25 mM Tris (pH = 8.0)and vitrified from 25 "C (the sample consists of bilayer fragments viewed edge-on (1)or face-on (2), together with larger, irregular structures (3)) and (b) lecithin with 8 mol % DHP sonicated at room temperature (unilamellar vesicles of varying size). The bars represent 100 nm.
410 Langmuir, Vol. 11, No. 2, 1995 of two-site binding of substrates in DHP dispersions. Furthermore, the great differences in reported average radii of DHP vesicle^^^-^ can be understood considering the observed heterogeneity in size and structure. It is not surprising that the DHP membranes have a discontinuous curvature when the temperature is far below T, and the membrane is in a crystalline rather than liquid-crystalline state. It has been shown by cryoTEM that vesicles of DMPC vitrified below T, exhibit an angular pr0fi1e.l~Engberts, Hoechstra, and co-workers14 have examined vesicles of a number of dialkyl phosphates, includingDHP, by i.a. electron microscopy. They reported that all surfactants formed vesicles above T, but that especially the ones with shorter chains (decyl or dodecyl) crystallized below that temperature. If vesicles are originally formed, it is interesting that they disintegrate into open fragments upon cooling, as observed in the present report. It has been claimed that bilayer fragments (12) (a) Almgren, M. J. Phys. Chem. 1981, 85, 3599. (b) Lukac, S. Photochem. Photobiol. 1982, 36, 13. (13)Chestnut, M. H.; Siegel, D. P.; Burns, J. L.; Talmon, Y. In Proceedingsofthe47thAnnualMeetingoftheElectronMicroscopySociety of America; Bailey, G . W., Ed.; San Francisco Press: San Francisco, CA, 1989. (14)(a)Wagenaar,L.;Rupert, L. A. M.; Engberts,J.B. F. N.;Hoekstra, D. J.O g . Chem. 1988, 54, 2638. (b) Fonteijn, T. A. A.; Hoekstra, D.; Engberts, J. B. F. N. Langmuir 1992, 8, 2437.
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are present also in sonicated cationic dioctadecyl dimethylammonium chloridehromide (DODACIDODAB)at room temperature (T, = 38 "C for DODAC).15 In conclusion, DHP is not suitable for preparation of small vesicles to be used at room temperature. The membranes formed are not like natural, liquid-crystalline membranes, but are highly irregular and contain kinks. Thus, their surface cannot be regarded as two-dimensional for use in surface kinetics, and studies of membrane permeability and transmembrane transport will be hampered by the heterogeneity of the membrane structure. The presence of fragments, without internal volumes, will further affect experiments on transmembrane processes since surface-bound reactants have to migrate to closed structures before transmembrane reactions may be observed. It is therefore not possible to draw detailed and quantitative conclusions on reactions studied in these dispersions. Acknowledgment. This work was supported by the Swedish Technical Research Council and the Swedish Natural Science Research Council. LA9408073 (15) (a) Kunitake, T.;Okahata, Y.; Yasunami, S. Chem. Lett. 1981, 1397. (b) Pansu, R. B.; Arrio, B.; Roncin, J.;Faure, J. J . Phys. Chem. 1990,94, 796.
